Background

Identification of novel therapeutic targets is the first step in the drug development journey, and traditionally relies on deep understanding of the biology, a process which is lengthy and non-scalable. The advent of high-throughput screening and advanced machine learning methods enables rapidly uncovering novel therapeutic targets even where the biology is not yet well understood. This, in turn, allows the expansion of the universe of targets and even the reach of current compounds.

Methods

Using the CancerRx dataset of 987 human cell-lines screened across 346 compounds we train a GBDT to predict the sensitivity (IC50) of each cell-line to each drug. Each cell-line is represented as a vector of binary values (WT/MT) according to the genomic data of 299 cancer related genes. Once high accuracy of predicting the cell-line-drug IC50 is obtained we use SHAP values, which represent the extent of a feature's responsibility for a change in the model output, to obtain the genetic features which influence sensitivity the most. These features, specific mutations and combinations of mutations, represent potential therapeutic targets of each screened drug.

Results

We report here a validation set consisting of 8 drugs with well-known targets and/or biomarkers. In all cases we identify the correct drug-target without any use of the underlying biology or prior mechanistic knowledge. We then report additional genomic features the model predicts to be important for the sensitivity to specific drugs, representing potential targets which to date are unknown. Additionally, we identify specific genetic features that increase the resistance to specific compounds.

Conclusions

Using an unbiased GBDT-based ML algorithm on publicly available high throughput screening data we accurately identify known drug-targets as well as additional previously unknown candidates. These new findings can expand the patient population for whom a certain drug can provide benefit. Furthermore, due to the unbiased nature of the algorithm and the wide array of drugs and drug-targets used in the screen we identify additional potential drugs targets which are not being targeted by the current drugs.

Legal entity responsible for the study

The authors.

Funding

Fore Biotherapeutics.

Disclosure

I.K. Kifer, G. Tarcic: Financial Interests, Personal, Full or part-time Employment: NovellusDx. E. Goldfarb, M. Vidne: Financial Interests, Personal, Stocks/Shares: NovellusDx. All other authors have declared no conflicts of interest.

Background

ROS1-directed Tyrosine-Kinase-Inhibitors (TKIs) target activating fusions in the ROS1 proto-oncogene efficiently in non-small cell lung cancer (NSCLC) patients. Besides solvent-front mutations (SFMs) in resistance to targeted therapy, ROS1 aberrations other than fusions remain biologically unexplored. Driving on our thus far clinical investigations, we aimed at determining the functional impact and the potential to act as a drug target.

Methods

Tumor samples from NSCLC patients were screened with two next-generation sequencing (NGS) panels. Patients with activating ROS1 fusions, SFMs and benign Single-Nucleotide Polymorphisms (SNPs) were excluded using the gnomAD database. The Provean and PolyPhen-2 tools predicted the functional impact of the detected aberrations. ChimeraX was used for drug binding analysis.

Results

Of 8072 patients analyzed by NGS between 2018 and 2022, 110 (1.4%) patients harbored small-scale ROS1 aberrations. Our cohort consists of 113 aberrations leading to 95 (84.1%) missense, 12 (10.6%) truncating and 1 (0.1%) in-frame aberrations. In 10% of patients ROS1 aberration was mutually exclusive and most ROS1 aberrations were transitions (55%). Polyphen predicts 75.5% of aberrations to be ‘possibly’ (6.4%), respectively ‘probably damaging’ (68.1%, mean score 0.75, median score 0.999). Provean predicts 50% of aberrations to be ‘deleterious’. Polyphen-2 and Provean coherently predict 46.8% of aberrations to be ‘deleterious’ respectively ‘possibly/probably damaging’. Amid the tyrosine kinase domain almost all aberrations are predicted to be ‘probably damaging’ respectively ‘deleterious’ by both tools coherently and feature a higher-than-average score. Besides, no distinct molecular pattern occurs. The M2029T and R2083T missense mutations interfere with single hydrogen-bonds and Van-Der-Waals forces in the ROS1 drug-binding pocket between the respective residues and the TKIs crizotinib and lorlatinib. However, drug binding analysis revealed that the overall binding of the TKIs is not affected.

Conclusions

This evidence proves a functional and deleterious impact of specific aberrations and indicates a high potential to be targetable. We warrant further studies to elaborate these findings in vitro and in vivo.

Legal entity responsible for the study

M. Scheffler.

Funding

Has not received any funding.

Disclosure

L. Nogova: Financial Interests, Personal, Other, honoraria: Pfizer, Celgene, Novartis, Roche, Boehringer Ingelheim, Janssen Pharmaceuticals, Bristol Myers Squibb. D. Schaufler: Financial Interests, Personal, Other, honoraria: BMS, Boehringer Ingelheim, MSD, Novartis, Roche, Healthcare Consulting Cologne, AbbVie. R.N. Fischer: Financial Interests, Personal, Other, honoraria: BMS, MSD, Roche, Boehringer Ingelheim, AstraZeneca. R. Riedel: Financial Interests, Personal, Other, honoraria: Boehringer Ingelheim, Novartis. R. Büttner: Financial Interests, Personal, Other, consultant: Pfizer, Novartis. J. Wolf: Financial Interests, Personal, Other, honoraria: AbbVie, AstraZeneca, BMS, Boehringer Ingelheim, Chugai, Ignyta, Lilly, MSD, Novartis, Pfizer, Roche; Financial Interests, Personal, Other, consultant: AbbVie, AstraZeneca, BMS, Boehringer Ingelheim, Chugai, Ignyta, Lilly, MSD, Novartis, Pfizer, Roche. M. Scheffler: Financial Interests, Personal, Other, consultant: BMS, Boehringer Ingelheim, Takeda, Roche; Financial Interests, Personal, Other, travel funding: Boehringer Ingelheim, Mediolanum. All other authors have declared no conflicts of interest.

Background

The goal of lung cancer early detection is to identify the malignancy at the stage where surgical cure is possible, outcomes are superior, and treatment is less morbid. 5-Hydroxymethylcytosine (5hmC) signatures in circulating cell-free DNA (cfDNA) as diagnostic biomarkers have been examined in different types of cancer. However, little is known in the field of early lung cancer detection.

Methods

We utilized well established 5hmC-Seal method (5 ml plasma per patient) to map the 5hmC profiles in cfDNA from a cohort of 100 newly diagnosed early-stage lung adenocarcinoma (LUAD) patients and 90 healthy individuals. The differentially methylated regions (DhMRs) and differentially regulated 5hmC genes were identified, and then the functional enrichment analysis for genes with upregulated and downregulated 5hmC levels was performed. Using multiple deep learning methods, we separated samples into two groups (95 training samples, and 95 validation samples) for classifier model training and evaluation, which was used to assign a methylation score to the withheld samples. This process was repeated with 100 randomly selected training-test sets.

Results

We identfied 1,315 DhMRs including upregulated (n = 99) and downregulated (n = 1,216) regions in LUAD groups by comparing LUAD groups with control groups. Applying the variable selection procedure of the Elastic Net algorithm, we identified a nine-gene model. To evaluate the performance, the model training was repeated 100 times and received an average AUC of 0.969 (95%CI: 0.935-1) on training set and 0.936 (95%CI: 0.890-0.983) on validating set, revealing high sensitivity (86.0%) and specificity (91.1%).

Conclusions

We firstly discovered 5hmC-based biomarkers in circulating cfDNA of early-stage LUAD. It provides a foundation for effective future fluid-biopsy-based lung cancer screening.

Legal entity responsible for the study

Peking University Cancer Hospital and Institute.

Funding

National Key R&D Program of China.

Disclosure

All authors have declared no conflicts of interest.

Background

TRACERx is a multicentre, prospective study recruiting patients with early stage non small cell lung cancer. By leveraging longitudinal CT imaging, patterns and timing of metastatic spread and tumour growth rates can be tracked lesion by lesion. Circulating tumour DNA (ctDNA) in longitudinal plasma samples can be mapped to imaging timepoints to assess their role as a biomarker in monitoring cancer progression and the impact of therapy.

Methods

436 imaging scans were reviewed by two clinicians from 50 patients (median 7 scans per patient) with disease relapse post-surgical resection. Volumetric growth dynamics of individual metastatic lesions were tracked on serial images and tumour dimensions contoured using ITK-SNAP. ctDNA levels were detected using patient bespoke multiplex-PCR assay-panels based on tissue exome sequencing tracking 200-600 mutations per patient. For 22 patients we tracked ctDNA in 147 plasma samples (median 8 samples per patient) taken from before primary tumour resection to disease relapse and subsequent progression. Clonal mutations were used to track overall disease burden and subclonal mutation to track lesion-specific metastatic subclones.

Results

56% of patients had intrathoracic only relapse and 44% patients had extrathoracic relapse. Common sites of relapse were lung (30%), lymph node (30%), bone (12%) and brain (5%). ctDNA levels were overlayed with tumour growth rate plots demonstrating that ctDNA fraction does not always track with total volume. For two patients we assessed the subclonal composition of ctDNA, tracking lesions with specific subclones detected at autopsy. Most lesion-specific subclones were absent in ctDNA in both cases, despite encompassing a large fraction of total tumour volume on imaging, suggesting many metastatic lesions do not shed substantial amounts of ctDNA. In contrast subclones from specific sites of disease were overrepresented in ctDNA, suggesting a high rate of shedding.

Conclusions

Large-scale studies with longitudinal imaging, ctDNA tracking and clinical annotation, such as TRACERx, can map the pattern and pace of cancer progression adding further insight into the clinical disease course and mechanisms of spread.

Clinical trial identification

The TRACERx study (NCT01888601) is a prospective observational cohort study and PEACE (NCT03004755) is a national research autopsy programme, both approved by independent research ethics committees (13/LO/1546 and 13/LO/0972/AM05, respectively).

Legal entity responsible for the study

Cancer Research UK.

Funding

Cancer Research UK funded TRACERx, Invitae has funded ctDNA research.

Disclosure

A. Hackshaw: Financial Interests, Institutional, Advisory Board, Received fees as a member of Independent Data Monitoring Committee: Roche. C.Z. Abbosh: Financial Interests, Institutional, Invited Speaker: Novartis, Roche, AstraZeneca, BMS; Financial Interests, Institutional, Full or part-time Employment: AstraZeneca; Non-Financial Interests, Institutional, Other, patents issued to detect tumour recurrence (PCT/GB2017/053289): Patent; Non-Financial Interests, Institutional, Other, methods for lung cancer detection (PCT/US2017/028013): Patent; Non-Financial Interests, Institutional, Other, co-inventor to a patent application methods for tumour monitoring GB2114434.0: Patent. A.M. Frankell: Non-Financial Interests, Institutional, Other, co-inventor on a patent application to determine methods and systems for tumour monitoring (GB2114434.0): Patent. C. Swanton: Financial Interests, Personal, Invited Speaker, Activity took place in 2016: Pfizer, Celgene; Financial Interests, Personal, Invited Speaker, October 26th 2020: Novartis; Financial Interests, Personal, Invited Speaker: Roche/Ventana, BMS, AstraZeneca, MSD, Illumina, GlaxoSmithKline; Financial Interests, Personal, Advisory Board, AdBoard - November 12th, 2020: Amgen; Financial Interests, Personal, Advisory Board: Genentech, Sarah Canon Research Institute, Medicxi; Financial Interests, Personal, Advisory Board, Joined October 2020. Also have stock options: Bicycle Therapeutics; Financial Interests, Personal, Advisory Board, Member of the Science Management Committee. Also have stock options: GRAIL; Financial Interests, Personal, Other, Consultancy agreement: Roche Innovation Centre Shanghai; Financial Interests, Personal, Full or part-time Employment, Chief Clinician since October 2017: Cancer Research UK; Financial Interests, Personal, Ownership Interest, Co-Founder of Achilles Therapeutics. Also, have stock options in this company: Achilles Therapeutics; Financial Interests, Personal, Stocks/Shares, Stocks owned until June 2021: GRAIL, ApoGen Biotechnologies; Financial Interests, Personal, Stocks/Shares: Epic Biosciences, Bicycle Therapeutics; Financial Interests, Institutional, Research Grant, Funded RUBICON grant - October 2018 - April 2021: Bristol Myers Squibb; Financial Interests, Institutional, Research Grant, Collaboration in minimal residual disease sequencing technologies: Archer Dx Inc.; Financial Interests, Institutional, Research Grant: Pfizer, Ono Pharmaceutical, Boehringer Ingelheim; Financial Interests, Institutional, Invited Speaker, Chief Investigator for the MeRmaiD1 clinical trial and chair of the steering committee: AstraZeneca; Financial Interests, Institutional, Research Grant, Research Grants from 2015-2019: Roche-Ventana; Financial Interests, Personal, Other, Co-chief investigator: NHS-Galleri Clinical Trial; Non-Financial Interests, Personal, Principal Investigator, Chief Investigator for MeRmaiD1 clinical trial: AstraZeneca; Non-Financial Interests, Personal, Invited Speaker, From 2019: AACR; Non-Financial Interests, Personal, Other, Board of Directors: AACR; Non-Financial Interests, Personal, Advisory Role, EACR Advisory Council member: EACR. M. Jamal-Hanjani: Financial Interests, Institutional, Funding, CRUK Career Establishment Awardee: CRUK; Financial Interests, Institutional, Funding: IASLC, International Lung Cancer Foundation, Lung Cancer Research Foundation, Rosetrees Trust, UKI NETs, NIHR, NIHR UCLH Biomedical Research Centre; Financial Interests, Institutional, Advisory Board: Achilles Therapeutics; Financial Interests, Institutional, Invited Speaker: Astex Pharmaceuticals, Oslo Cancer Clusters; Non-Financial Interests, Institutional, Other, methods for lung cancer detection (PCT/US2017/028013): Patent. All other authors have declared no conflicts of interest.

Background

Lung cancer has the highest incidence and mortality among all cancers, with a 5-year survival rate of 15%. Presently, epidermal growth factor receptor (EGFR) mutation is the most common type of gene mutations detected in patients (pts) with advanced non-small-cell lung cancer, and EGFR is identified as the therapeutic target of EGFR tyrosine kinase inhibitors (TKIs). EGFR-TKIs have become the standard treatment. However, the most of the trials were developed in Asian countries, with a lack of data in western population.

Methods

This retrospective study aimed to review the medical records of EGFR- mutant advanced lung adenocarcinoma (LA) undergoing EGFR-TKIs treatment from 2015 to 2021, so as to examine the association of clinical factors with EGFR-TKI efficacy.

Results

Of the 53 stage IV LA pts enrolled in this study, 9 were treated with more than one TKI. The mean age was 74,2 years old, 32 pts were non-smoker females and 38 had ECOG-PS 0-1. The mutations del19 or 21 L861G were the most frequent (23 and 20 pts respectively). The 20 T790M mutation was detected in 6 pts. First-line TKI (Gefitinib, Afatinib, Erlotinib and Osimertinib) were used in 26 pts and prior chemotherapy was preferred in 29 pts. A 24% objective response rate (ORR) and 67% disease control rate were observed for EGFR-TKIs treatment in all lines. Higher ORR was seen in patients with 0/1 ECOG scores than those with 2 or greater scores (p=0.046). The subjects had a progression free survival (PFS) of 17,8 months (p<0,01; 95% CI:12.9-22.84) and an overall survival (OS) of 27,5 months (p<0.01; 95% CI: 18.27-36.823). The median PFS was longer in pts treat with first-line TKI (p=0.029). The multivariate analysis indicated that ECOG-PS (p=0.04) and bone metastasis (p=0.036) were independent prognostic factors for OS.

Conclusions

The EGFR-TKI therapy results in survival benefits for EGFR-mutant advanced LA patients. ECOG-PS and bone metastasis were independent prognostic factors for OS.

Legal entity responsible for the study

Hospital Professor Doutor Fernando Fonseca.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Non-small cell lung cancer (NSCLC) is one of the most common human cancers. In this regard, the search for new biomarkers for detecting NSCLC at early stages is relevant, and one of these markers may be the methylation of CpG-islands of miRNA genes. The aim of this work was to study the level of methylation of promoter CpG-islands of miRNA genes as promising NSCLC markers.

Methods

A representative set of 80 paired (tumour/normal) NSCLC samples was used in the study. The analysis of methylation was carried out using the method of quantitative methyl-specific PCR. Statistical analysis of methylation levels was performed using a non-parametric Mann-Whitney U-test. The optimal marker systems were selected based on the results of the ROC analysis. Mathematical packages IBM SPSS Statistics 22 are used.

Results

A representative set of 80 paired (tumour/normal) NSCLC samples was used in the study. The analysis of methylation was carried out using the method of quantitative methyl-specific PCR. Statistical analysis of methylation levels was performed using a non-parametric Mann-Whitney U-test. The optimal marker systems were selected based on the results of the ROC analysis. Mathematical packages IBM SPSS Statistics 22 are used. Results. The methylation status of 10 miRNA genes was studied, significant differences were established between methylation levels in tumour samples and histologically normal tissue of NSCLC patients for 8 genes: MIR-124A-1/3, MIR-125B-1, MIR-127, MIR-129-2, MIR-137, MIR-339 and MIR-375 (p‹0.001). Significant correlations were found between the methylation frequency of a number of miRNA genes and NSCLC progression: MIR-125B-1, MIR-137, MIR-124A-3 with cancer stage (p=0.001, 0.004 and 0.001, respectively) and MIR-125B-1 with metastasis (p=0.005). An effective marker system of 4 genes (MIR-125B-1, MIR-137, MIR-129-2, MIR-375) was determined by ROC analysis for the diagnosis of NSCLC at early stages with high clinical sensitivity (90%) and specificity (90 %); AUC value > 0.9.

Conclusions

Thus, new hypermethylated miRNA genes can be used as potential biomarkers for the early diagnosis of NSCLC.

Legal entity responsible for the study

The authors.

Funding

The study was supported by the state assignment of the Ministry of Science and Higher Education of the Russian Federation number FGFU-2022-0007.

Disclosure

All authors have declared no conflicts of interest.

Background

Tumor cells have a tendency to proliferate rapidly and can metastasize to other organs of the body. Respiratory carcinomas are among 3^rd predominant causes of cancer-related deaths in both genders around the globe. Type of epithelial lung cancer non-small cell lung carcinoma (NSCLC) constitutes 80% of all lung cancer types. The 5-year survival rate of NSCLC is about 16%. This Study was conducted to understand the chemoresistance through expression of proto-onco genes (AKT1, ALK), onco-suppressive (FBXW7, PTEN) genes and their correlation with miRNA 140-145 in NSCLC.

Methods

Bronchoscopy of lung cancer patients who were on different medications was conducted for collection of samples from regional health care facility under the guidelines of ethical review committee. Samples were subjected to histopathology and for further processing to carry out RNA extraction and gene expression studies through qRT-PCR/ gel-electrophoresis. The data was analyzed statistically through one-way ANOVA for analysis of variance and DMR.

Results

It has been revealed that the expression level of proto-onco ALK and AKT1 genes is significantly higher in lung cancer patients in comparison to normal ones (P<0.05). PTEN and FBXW7 are onco-suppressive genes, so these genes are downregulated after mutational changes in lung parenchyma of cancer patient samples (P<0.05). Correlation of PTEN and AKT1 genes revealed that upregulation of PTEN gene down express the AKT1 gene through PI3K/AKT/mTOR signaling pathway. Relatively higher expression of miRNA 140-145 disrupt the function of c-Myc and Cyclin-E proteins which is the thought to be the reason behind chemoresistance in NSCLC patients (P<0.05). Histopathological examination showed neoplastic cellular proliferation without infiltration into dermal areas in lung cancer tissue but in control, there is uniform cellular structure.

Conclusions

This study concludes that perturbation in miRNA 140-145 mediate post transcriptional activation of proto-onco genes through PI3k/ AKT pathway and hence contribute towards chemoresistance in 2nd-3rd grade NSCLC patients.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

The pathbreaking discovery of sotorasib as KRAS G12C inhibitor has revolutionized the prognostic outcome of KRAS mutated metastatic NSCLC. This is a single centre experience of KRAS mutated NSCLC in an Indian cohort, underscoring the unmet urgent need of sotorasib in this part of the world.

Methods

Among the 3899 NSCLC patients registered between 2016 to 2020, 163 patients were tested for KRAS alterations, of which 50 patients were confirmed to harbor a KRAS variant by molecular testing. Appropriate statistical analysis was done to compare G12C and non-G12C subtypes, including their survival outcomes.

Results

Median age was 63 years (range: 36-81years) with a preponderance of non-G12C mutations in both sexes. Smokers were higher in G12C group (16 vs 4 pts, p=0.09). G12C group showed lesser PDL1 expression (>1%) than non-G12C group (13 vs. 15 pts, p=0.09). Molecular analysis revealed 3 main types of KRAS mutations: G12C, G12V and G12D in 17 (34%), 9(18%) and 6 (12%) pts respectively. None of them had KRAS complex mutations. The co-mutations predominantly detected included TP53 in 7 (14%), STK11 in 3 (6%), FGFR4 in 4 (8%), PIK3CA, EGFR and ALK in 2 cases each, BRAF and CTNNB1 in 1 case each. Co-mutations were significantly more frequent in the non-G12C group (p<0.05). 36 patients received chemotherapy and rest were lost to follow up. The median PFS and OS of the entire cohort on chemotherapy were 5.4 months (mo) and 11.1 mo respectively. The median PFS and OS of G12C vs non-G12C groups were 6.4 mo (95% CI: 2.8-11.8) vs 3.8 mo (95%CI: 2.9-7.7) and 15.2 mo (95% CI: 9.8-17.2) vs 16.1 mo (95% CI: 10.7-19.3) respectively. With a median follow-up of 14.3 mo, both PFS and OS were not statistically significant between the groups, p=0.08 and 0.20 respectively.

Conclusions

This is by far the largest experience from India of KRAS mutated NSCLC, comparing G12C with other KRAS mutants. Despite limitations of retrospective nature, small sample size, heterogeneity in the cohort and missing data points, and limited molecular profiling this study is relevant in the light of approval of targeted therapy, sotorasib, which is currently unavailable in this country.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Circulating tumour cells (CTCs) could escape the host immune surveillance by altering the expression of immune regulatory molecules (PD-L1, PD-L2, CTLA-4, CD47) in cancer patients. Recent studies suggested that KRAS mutation might promote the immune escape of tumour cells by inducing the upregulation of these molecules. However, the regulatory effects of KRAS on the expression of CTLA-4 in patients with colorectal cancer (CRC) have yet to be explored. This study aimed to investigate KRAS and CTLA-4 mRNA expression profiling in CTCs from patients with CRC.

Methods

Peripheral blood from 50 CRC patients was collected to isolate CTCs using a negative selection (EasySep^TM) method. The presence of CTCs was confirmed by immunofluorescence staining using multiple tumor cell surface biomarkers (EpCAM, SNAIL-1, E-cadherin and MMP-9). The mRNA expressions of CTLA-4 and KRAS in CTCs were analysed by RT-qPCR. The association between the presence of CTCs and clinicopathological variables was evaluated.

Results

The cells, which are positive for at least one of the markers (EPCAM, SNAIL-1, E-Cadherin and MMP-9), and enlarged nucleus and cell size >8μm were considered as CTCs. Approximately 66% (n=33/50) of patients with CRC were CTC positive. The majority of patients with zero CTC or low CTC counts (˂10 CTCs) were diagnosed with early stages (stage I or II), while patients with higher CTCs counts (≥10 CTCs) were with advanced stages (stages III or IV) with CRC ((p < 0.0064). The presence of CTC were also significantly correlated with tumour type, size. The expressions of CTLA-4 and KRAS were higher in CTC positive groups compared to CTC negative in patients with CRC. Also, the expression levels of these two molecules were positively correlated to each other (Spearman’s rank test, r = 0. 0.6674; p<0.0001). Additionally, from clinical data analysis, we found that CTLA-4 gene expression weakly correlates with KRAS mutation (p < 0.060).

Conclusions

Our study demonstrated that KRAS gene activation might upregulate CTLA-4 expression in patients with CRC. Thus, understanding the role of the interactions between oncogenes and immune checkpoint molecules is essential to fully uncover the molecular mechanisms involved in immune escape by CTCs in patients with CRC.

Legal entity responsible for the study

The authors.

Funding

School of Medicine and Dentistry, Griffith University.

Disclosure

All authors have declared no conflicts of interest.

Background

The gene POU5F1 (also known as an OCT4) belonging to POU protein family is composed of 5 exons that can create many isoforms by alternative splicing. Most known isoform is OCT4A that is expressed in many types of stem cells. Its nuclear localization and presence of the DNA-binding domain enable it to regulate the expression of genes that are responsible for pluripotency and blocking of the differentiation of the cells. Other splicing products are numerous OCT4B isoforms (OCT4B-OCT4B5) that differ from OCT4A in the cell localization and in absence of the DNA-binding domain. Majority of OCT4 isoforms were discovered only recently and there have not been clear evidences about their role in the cells yet. The role of OCT4A, OCT4B and OCT4B1 in tumorigenesis have already been explored but without definite results. The aim of our research is the analysis of alternative splicing variants of the gene POU5F1 in association with the patient´s prognosis and their occurrence in the metastasis.

Methods

For this analysis we used fresh surgical specimens of the patients with colorectal cancer that were isolated from primary tumours (n=47) and from the metastasis (n=31). Subsequently, the total RNA was isolated and transcribed to cDNA. Through the quantitative PCR method using particular TaqMan assays for specific variants and with ΔΔ Ct method, we separately analyzed the relative gene expression of isoform A, isoforms B, but also all isoforms together.

Results

We found out that in tumour specimens isolated from primary tumours was the expression of all POU5F1 isoforms significantly lower than in adjacent non-tumour tissue. On the other hand, the significantly increased expression in metastatic tumor compared to primary tumours was observed only in experiment with probe specific for all POU5F1 isoforms whereas isoform A, neither isoform B were not reason of increased expression of POU5F1 in metastasis.

Conclusions

Our analysis indicate that alternative splicing play a pivotal role in metastatic colorectal cancer and whereas we exclude isoform A and isoforms B as a cause of increased expression of POU5F1 in metastasis, we consider as an important to identify responsible alternative transcript.

Legal entity responsible for the study

The authors.

Funding

VEGA 1/0269/22.

Disclosure

All authors have declared no conflicts of interest.

Background

The prognosis of advanced gall bladder carcinoma (GBC) remains poor despite the addition of newer targeted therapies. HER2 is a potential therapeutic target expressed in up to 20% GBC. We present an analysis of advanced or metastatic HER2-amplified or overexpressed GBC treated with trastuzumab and chemotherapy from an Indian cancer center.

Methods

Patients with advanced or metastatic GBC with immunohistochemical evidence of HER2 overexpression (IHC 3+) or HER2 amplification detected with FISH, treated with trastuzumab and first-line chemotherapy between 2020 and 2021 were included. Outcome measures were objective response rate (ORR), progression-free survival (PFS), and overall survival (OS).

Results

Twenty-six patients (age range, 44-71) with GBC were treated with trastuzumab and chemotherapy. Twenty patients (76.9%) had newly diagnosed advanced disease while six patients (23.1%) had recurred after prior curative treatment. Twenty-four patients (92.3%) received gemcitabine-cisplatin and two received (7.7%) gemcitabine-oxaliplatin as chemotherapy. Partial response was seen in 11 patients (42.3%) while 10 patients (38.5%) had stable disease (objective response rate 42.3%, overall response rate 80.8%). At a median follow-up of 15.7 months, median PFS was 9.7 months (95% CI, 9.3-10) and median OS was 11.9 months (95% CI, 11.2-12.5). Grade 2 ejection fraction drop was seen in 1 patient (3.8%). No adverse events of grade 3 or more were attributable to trastuzumab.

Conclusions

Trastuzumab added to chemotherapy for advanced GBC demonstrated better ORR, and PFS compared to historical control of chemotherapy alone, without any additional toxicities.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

The routine therapeutic landscape of metastatic CCC is still largely based on cytotoxic chemotherapy. This standard gets increasingly challenged with the advent of next generation sequencing (NGS) in routine practice and evolving trials of targeted therapies in CCC. However the value of comprehensive genetic profiling of CCC in actual routine clinical practice remains poorly characterized.

Methods

We performed a retrospective study at the clinical cancer center of Upper Austria (Tumorzentrum Oberösterreich) in 2018-2021. Tumor samples from 56 patients with advanced CCC underwent comprehensive genetic profiling. TruSight Tumor 170 Assay (Illumina), Archer FusionPlex Panel (ArcherDX), Oncomine Focus Assay (Thermo Fisher Scientific) were used for NGS analysis. Furthermore MSI status was determined by custom made Multiplex PCR-Based Methods. Immunhistochemistry (IHC) collected data on Her2neu and PDL-1expression.

Results

A potentially actionable pathogenic variant was identified in 32 patients (57%). Pathogenic variants were ranked according ESMO Scale for Clinical Actionability of molecular Targets (ESCAT). In 15 patients with ESCAT I-III criteria a molecular informed therapy was established targeting 8 FGFR alterations, 2 MSI high status, 1 Pi3kinase mutation, 2 BRCA1/2 mutations, 2 BRAF V600Emutations. In 13 of the 15 treated patients we could observe a favorable PFS2 (targeted therapy)/PFS1 (previous standard therapy) ratio >1,3, suggesting clinical benefit. Overall survival in patients receiving molecular matched therapy showed a positive trend compared to patients remaining on standard treatment protocol.

Conclusions

We could show that comprehensive molecular characterization of advanced CCC during routine clinical care leads to favorable PFS2/PFS1 ratios and probable positive overall survival. This underscores the recent ESMO recommendations for NGS reflex testing in advanced CCC.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Sensitive biomarkers for early cancer detection remain largely lacking. Circulating telomere DNA may help.

Methods

A case-control study with hospital hepatocellular carcinoma (HCC) cases was conducted to identify potential biomarkers for HCC detection (Discovery). We used next generation sequencing method for circulating cell-free DNA (ccfDNA) analysis. We modeled telomere and end sequence in circulation (Telecon) to detect HCC in Discovery. We validated Telecon in a prospective cohort among hepatitis B virus (HBV)-seropositive participants with biannual blood collections from 2012 till 2019, using nested case-control design (Validation).

Results

In Discovery, among short ccfDNA (25-60 nucleotides), telomere was more abundant in HCC patients than in controls (18.87-fold, P=6.4×10^-18). Telomere contributed 91% of the variation of the Telecon model, which distinguished HCC cases from controls completely (AUC=1.0). In Validation, among 18,185 participants, 2,893 were HBV-seropositive and developed 81 incident HCC cases (incidence rate 382 per 100,000 person-years). Telecon showed increasing detection performance using pre-diagnosis samples collected ≥4 years (AUC=0.538), 3-4 years (0.741), 2-3 years (0.742), 1-2 years (0.786), and 0-1 year (0.930) before diagnosis. Within one year before diagnosis and at a specificity of 98%, Telecon had a sensitivity of 68.2% (95% CI=52.4-81.4%) in detecting early HCC, yielding an estimated positive predict value of 15.2% among HBV-seropositive population. High Telecon was also associated with a higher risk of death among hospital HCC patients (hazard ratio 3.22, 95% CI=1.49-7.0), independent of tumor stage.

Conclusions

Circulating short telomere may effectively detect early and aggressive hepatocellular carcinoma in high-risk populations.

Legal entity responsible for the study

The authors.

Funding

China Scholarship Council (201808440263); Lau Grant (LC230003); Swedish Research Council (202001418).

Disclosure

Z. Zheng: Financial Interests, Personal, Royalties: ArcherDX; Financial Interests, Personal, Advisory Role: Helitec, GenEditBio. All other authors have declared no conflicts of interest.

Background

FusionPlex Dx (FP Dx) is a 2-part multimodal next-generation sequencing (NGS)-based in vitro diagnostic kitted device that detects structural alterations and oncogenic isoforms in RNA derived from formalin-fixed, paraffin-embedded (FFPE) samples from solid tumors using proprietary Anchored Multiplex PCR (AMP™) chemistry. FP Dx is approved as a companion diagnostic for five indications: METex14 skipping events, ALK, RET, ROS1 gene fusions to guide therapy in non-small cell lung cancer and NTRK1/2/3 gene fusions for all solid tumors. The assay also provides comprehensive genomic profiling for patients with solid tumors. This study summarizes the analytical performance of FP Dx.

Methods

FP Dx instructions for use were followed for all studies. Total nucleic acid was extracted from 1132 unique clinical FFPE specimens with positive or negative RNA alterations derived from ≥40 unique tumor types and 4 commercial reference FFPE samples generating a total of 3279 libraries. Libraries were sequenced on Illumina MiSeqDx and NextSeq 550Dx platforms. Pre-defined in-process control metrics were used for monitoring library preparation and sequencing, and only libraries meeting these metrics were included in the analysis (97.65%). Data were processed via Illumina sequencing software, R Analysis Suite Statistical Software, and Invitae Solid Tumor IVD Analysis.

Results

The limit of detection of FP Dx was measured to be 22 reads for structural alteration (ALK, RET, and ROS1 gene fusions), 26 – 35 reads for METex14 skipping events, and 31 reads for NTRK gene fusions. The accuracy of FP Dx was evaluated by comparison to Illumina TST-170 NGS, Oncomine Focus NGS, and Illumina TSO Comp assays. Concordance agreement results were >95%, demonstrating the accuracy of FP Dx. Overall agreement reproducibility results for NTRK, METex14, ALK, RET, and ROS1 alterations were ≥99.15%, and repeatability results were ≥92.50%, demonstrating consistent precision of FP Dx.

Conclusions

Collectively, these data support FP Dx’s ability to detect RNA structural alterations. The unique capabilities of the Assay chemistry and software allow for exhaustive characterization of Assay sensitivity for all targeted alterations, even without prior knowledge of gene fusion partners or breakpoints.

Legal entity responsible for the study

Invitae Corporation.

Funding

Invitae Corporation.

Disclosure

V. Haddad, N. Hohos, M. Stueve, A. Timm, A. Rao, H. Slocum, J. Lee: Other, Institutional, Full or part-time Employment: Invitae.

Background

The approach of targeted therapies has also evolved with a better understanding of cancer to achieve treatment outcomes accordingly. Cytochrome P450 (CYP) is one of these enzyme superfamilies that are of great pharmacogenomic interest due to its role in the biotransformation of several drugs, including anticancer drugs. These enzymes have the ability to metabolize procarcinogens and anticancer drugs, making them crucial in cancer etiology and treatment. Accordingly, the present study investigated the pharmacogenomics of Cytochrome P450 1A1, 3A4 & 3A5 genotypes in relation to treatment response (personalized medicine approach) in breast cancer cases receiving adjuvant tamoxifen.

Methods

Study participants included 300 women with breast cancer and a similar number of age-matched controls. CYP1A1, CYP3A4 & CYP3A5 genotypes were determined in genomic DNA by PCR-based RFLP. Follow-up was carried out to determine whether there was any correlation between the variants and treatment outcome. A Chi-square test and logistic regression models were used to investigate the association between genotype, use of concomitant CYP1A1, CYP3A4 & CYP3A5 inhibitors, and disease relapse rate.

Results

CYP1A1, CYP3A4 & CYP3A5 variant alleles were significantly more frequent in the cases than in the controls. Moreover, this study showed that the presence of inactive CYP1A1, CYP3A4 & CYP3A5 resulted in a decrease in the metabolic activation of anticancer agents, lowering the risk of toxicity but worsening the therapeutic response. A deeper understanding of CYP1A1, CYP3A4 & CYP3A5 could lead to more effective targeted therapies for breast cancer.

Conclusions

In spite of advances in treatment for breast cancer, an effective treatment outcome with no side effects remains a challenge in Asian countries. The study of pharmacogenomics is expected to help in the standardization of chemotherapy drugs and improve treatment outcomes in cancer patients carrying CYP1A1, CYP3A4 & CYP3A5 variant genotypes (precision medicine/targeted therapies).

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Previous studies have documented that both vitamin D deficiency and single nucleotide polymorphism (SNPs) of genes responsible for mediating downstream effects of melatonin with development of breast cancer. In India 70 percent of the population suffers from Vitamin D deficiency. There are no available reports on association of vitamin D and SNPs in Melatonin genes towards development of breast cancer in Indian population.

Methods

To this end, we evaluated 50 vitamin D-deficient breast cancer subjects and studied the gene expression patterns and SNPs of melatonin receptor genes (MTNR1a, MTNR1b) and Arylalkylamine N-acetyltransferase (AANAT). Age matched breast cancer patients without Vitamin D deficiency and age matched normal subjects (i.e without breast cancer served as control).

Results

Vitamin D-deficient studies demonstrated decreased expression MTNR1a, MTNR1b, and AANAT genes in vitamin D deficient breast cancer group as compared to subjects with normal vitamin D level (68% Vs 41%, p<0.001). Two two-staged analysis of genome-wide association (GWAS) showed association of MTNR1a, MTNR1b SNPs with cancer progression and metastasis in vitamin D deficient group. Interestingly the incidence of SNPs was 32% more in vitamin D-deficient breast cancer group when compared with the other groups.

Conclusions

Our pilot studies highlight the fact that both Vitamin D and products of melatonin have strong correlation with development of breast cancer and its invasiveness. Significant increase in SNPs of melatonin related genes under conditions of vitamin D insufficiency leads us to the contention that vitamin D and melatonin have additive effect in development of breast cancer. These experiments when repeated using large sample size coupled with animal studies in melatonin receptor knockout mice with breast cancer against vitamin D background can shed more light on the exact mechanism.

Legal entity responsible for the study

Raktim Mukherjee, Megha Dave, Vishnu Priya Veeraraghavan, Selvaraj Jayaraman.

Funding

Has not received any funding.

Disclosure

The author has declared no conflicts of interest.

Background

The advent of immune checkpoint inhibitors (ICIs) is underway to change the landscape of triple- negative breast cancer (TNBC) treatment. In this meta-analysis and position paper, we assess the role of atezolizumab and pembrolizumab in TNBC with the lens of precision medicine.

Methods

A systematic search of PubMed, Scopus, Web of Science and Google Scholar was conducted until July 15, 2022. Keywords were used as follows: triple-negative breast cancer, atezolizumab, pembrolizumab, biomarker, precision, chemotherapy.

Results

A total of 3 clinical trials (i. IMpassion 130, ii. IMpassion 131 and iii. KN355) were included. The objective response rate (ORR) was higher in the treatment arm (atezolizumab/pembrolizumab) (ORR=1.35) as compared to placebo. Moreover, the progression free survival (PFS) in the treatment arm had a positive effect size (Cohen’s d=1.5). TNBC is a known aggressive and heterogenous subtype of breast cancer that is largely associated with high recurrence rate and metastasis. While the clinical outcomes of treatment are discussed, the use of precision medicine to target TNBC patients is a work in progress. TNBC developmental therapy depends on biomarker-based and precise clinical trials. IMpassion 130 could not ascertain that these findings would extend to other chemoimmunotherapy combinations, but supported the addition of checkpoint inhibitors to standard chemotherapy in first line care of TNBC. IMpassion 131 enrolled patients through immune cell expression ≥1%, VENTANA PD-L1 (SP142) assay. KN355 classified participants based on prior treatments and chemotherapy status.

Conclusions

In the era of precision medicine, ICIs are largely being used in novel clinical trials for TNBC. Only one of the three trials tested patients with the immunohistochemical assay and reported immune expression. Prospective studies are required to further optimize care for TNBC and find the best available treatment response biomarkers and modalities in guiding breast cancer decision-making. It is also imperative to identify the subgroup of patients who would derive the greatest benefit with ICI and precision-medicine guided therapy.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Ovarian cancer (OC) is an aggressive tumour that is usually diagnosed in an advanced stage. The presence of somatic and/or germinal mutations in the genes BRCA1 and BRCA2 is known to be predictive of response to platinum agents and inhibitors of PARP (iPARP). Nevertheless, in recent years it has been shown that the efficacy can vary according to the specific mutation. In our community there is a high prevalence of patients (pts) carrying the BRCA1 c.211 A>G germline pathogenic variant, which is a founder mutation originated in the northwest of Spain. The aim of our study was to evaluate if these pts presented different outcomes when treated with first line platinum agents.

Methods

We performed a single-centre retrospective analysis of pts carrying somatic and/or germline pathogenic variants of BRCA1 and BRCA2 treated with platinum agents and iPARP between January 2014 and December 2021. Variants were detected with validated methods in tissue and/or blood samples. Data was extracted from electronic health records and analysed with SPSS software.

Results

We found 38 pts with the aforementioned characteristics. Median age upon diagnosis was 56.4 (range 40 – 82) years. The initial FIGO stage was IIIC in 57.9% and IV in 34.2% of the cases. The majority of pts had an ECOG PS of 1 (57.9%) or 0 (34.2%). 25 pts (65.8%) had a BRCA1 variant, and of those 12 pts carried the BRCA1 c.211 A>G variant. These pts presented a worse objective response rate (ORR) to first line platinum-based chemotherapy when compared to all the pts (58.3% vs 88.5%, p = 0.03) and to BRCA1 mutated pts (58.3% vs 84.6%, p = 0.14). Median progression free survival (PFS) to first line treatment was not significantly different among BRCA1 c.211 A>G variant carriers and the rest of the pts (27 vs 21 months, p = 0.44), and neither was overall survival (OS) (72 vs 91 m, p = 0.27).

Conclusions

We found that pts with BRCA1 c.211 A>G germline pathogenic variant treated in our centre had a worse response rate to platinum-based chemotherapy even thought PFS and OS were not significantly different. Further research should be carried out to test if this mutation conveys less sensitivity to DNA damaging agents.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Ovarian cancer (OC) is one of the most lethal malignancies in females. A biomarker with high sensitivity and specificity has yet not been discovered which can aid in early diagnosis. Mucin proteins (MUC) proteins have been considered promising diagnostic biomarkers in cancer. We analyzed MUC1 and MUC4 for their diagnostic role in OC.

Methods

We analyzed 31 cases of OC and 8 benign ovarian cysts. A pre-therapy whole blood sample was taken and serum levels of MUC1, MUC4 & MUC 16 (CA 125) were evaluated. Cut-off for MUC1 and MUC4 was set using ROC curve analysis at the highest sensitivity and specificity, 141.3ng/mL and 1.206ng/mL respectively. The values were compared with CA125 (established cutoff of 35 U/ml).

Results

Serum level of MUC1 was higher in 33/39 (84.6%) cases (28/31 in malignant cases, 3/8 in benign cases). However, the serum level of MUC4 was lower in 33/39 cases (26/31 in malignant cases, 5/8 in benign cases). CA125 was higher in 33/39 cases (28/31 in malignant cases, 4/8 in benign cases; table). When compared with histological variants of epithelial ovarian cancer, high levels of MUC1 were seen in HGSC, mucinous, endometrioid, and clear cell 16, 4, 3 & 2 respectively (p-value 0.567). For MUC4, a lower value serum level of MUC4 was associated with a diagnostic indicator of ovarian cancer (p= 0.9709). Higher serum level of CA125 was associated with OC (p=0.0025), Type 2 tumor (p= 0.0013), advanced stage (p= 0.0087) and presence of omental metastasis (p= 0.0311). The sensitivity and specificity of CA125 in our study were 90.32% and 62.5% respectively. The sensitivity and specificity of MUC1 in our study were 90.32% and 50% respectively. The sensitivity and specificity of MUC4 in our study were 83.87% and 37.5% respectively.

Table: 26P

Serum values in all cases including borderline and benign

Conclusions

The serum level of Mucin 1 was increased in ovarian cancer, while that of MUC4 was decreased in OC. A combined panel of mucin 1, MUC4, and ca125 can aid in a better diagnosis of OC.

Legal entity responsible for the study

AIIMS, New Delhi.

Funding

AIIMS, New Delhi.

Disclosure

All authors have declared no conflicts of interest.

Background

After the introduction of the molecular classification in EC guidelines, Next Generation Sequencing (NGS) analysis has become an essential tool for EC management. Molecular-driven therapies have also been recently tested, and some have obtained FDA approval. This retrospective cohort study aims to determine the clinical benefit rate (CBR) with the use of targeted therapies based on NGS in ECs patients.

Methods

After approval of the local Ethics Committee, a retrospective study was conducted on EC patients undergoing Foundation Medicine® testing at Fondazione Policlinico Universitario Agostino Gemelli IRCCS. All patients provided written informed consent. Formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens were analyzed by Foundation One® CDx, which detects 324 genes known to be drivers of solid tumors based on NGS assay. Efficacy outcomes were estimated by the Kaplan-Meier method and expressed as median with its 95% confidence interval. IBM-SPSS v.27.0 software was used for statistical analyses.

Results

Out of 35 tests performed, 11 patients received a targeted therapy based on actionable mutations detected with the NGS assays. All the 11 patients had been heavily pretreated (≥3 prior lines). One patient died because of COVID-19 and thus was excluded from the analysis. Out of the 10 patients included, targeted therapies showed an overall CBR of 80% in 8 patients (10% CR, 33.3% PR, 40% SD, and 20% PD). 7 patients were treated with a targeted agent belonging to the PI3K pathway, with 3 PR (42.9%), 3 SD (42.9%), and 1 PD (14.2%). 3 patients received PARP inhibitor treatment according to their HRD status, with 1 CR (33.3%), 1 SD (33.3%), and 1 PD (33.3%).

Conclusions

In our series, an outstanding CBR of 80% was achieved with the use of targeted therapy according to NGS assays in heavily pretreated patients (≥3 prior lines). Our finding underlines the importance of molecular-driven treatments and requires further investigation to confirm these results in terms of clinical benefit in a broader population. Besides, the use of molecular-driven treatments may avoid unnecessary exposure to potentially more toxic therapies and reduce the costs associated with inappropriate treatments.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

V. Salutari: Financial Interests, Personal and Institutional, Advisory Board: AstraZeneca, Clovis, GSK, Tesaro, MSD, Roche, PharmaMar, Eisai. G. Scambia: Financial Interests, Personal, Invited Speaker, Speaker: Johnson & Johnson, AstraZeneca & MSD, Olympus Europa, Baxter Healthcare, Intuitive Surgical Inc., GlaxoSmithKline; Financial Interests, Personal, Expert Testimony, Trainer: Covidien AG (Medtronic company); Financial Interests, Institutional, Invited Speaker, ‘IsoMSLN’ in Ovarian Cancer and Malignant Pleural Mesothelioma: Kiromic; Financial Interests, Institutional, Invited Speaker, Roll-over study for patients who have completed a previous cancer study with olaparib and who the investigator believes can benefit from continued treatment - ROSY-O: AstraZeneca; Financial Interests, Institutional, Invited Speaker, CATCH-R: Roll-over study to provide continuous access to clinical therapy with rucaparib: Clovis Oncology; Financial Interests, Institutional, Invited Speaker, Phase III, multicenter, placebo-controlled clinical study comparing chemo-immunotherapy (paclitaxel-carboplatin-oregovomab) versus chemotherapy (paclitaxel-carboplatin-placebo) in patients with advanced epithelial ovarian, tubal cancer of fallopian or peritoneal (FLORA-5): Oncoquest Pharmaceuticals Inc.; Financial Interests, Institutional, Invited Speaker, Phase IIb randomized, open-label, active comparator, parallel-group, multicenter study designed to evaluate the efficacy and safety of three different doses of the P2X3 receptor antagonist (BAY 1817080) versus placebo and Elagolix 150 mg in women with symptomatic endometriosis: Bayer AG; Financial Interests, Institutional, Invited Speaker, Usability of ITE transducers for sending electric fields for tumor treatment (TTFields): Novocure Ltd.; Financial Interests, Institutional, Invited Speaker, Phase III, multicentre, open-label extension trial to evaluate long-term safety and efficacy in patients with advanced cancers currently undergoing treatment or in follow-up in a pembrolizumab trial: Merck. D. Lorusso: Financial Interests, Personal, Advisory Board, Participation in Advisory Boards and Invited Speaker: GSK, Clovis Oncology, PharmaMar; Financial Interests, Personal, Advisory Board, Participation in Advisory Boards and Invited Speakers: AstraZeneca, MSD; Financial Interests, Personal, Other, Consultancy: PharmaMar, Amgen, AstraZeneca, Clovis Oncology, GSK, MSD, Immunogen, Genmab, Seagen; Financial Interests, Personal, Advisory Board, Participation in Advisory Boards: Merck Serono; Financial Interests, Personal, Advisory Board, Invited member of advisory board: Seagen, Immunogen, Genmab, Oncoinvest, Corcept, Sutro; Financial Interests, Institutional, Funding, Grant for founding academic trial: MSD, Clovis Oncology, PharmaMar; Financial Interests, Institutional, Funding, Grant for founding academic trial: GSK; Financial Interests, Institutional, Invited Speaker, ENGOT trial with institutional support for coordination: Clovis Oncology; Financial Interests, Institutional, Invited Speaker, ENGOT trial with institutional support for coordination: Genmab, MSD; Financial Interests, Institutional, Funding, Clinical trial/contracted research: AstraZeneca, Clovis Oncology, GSK, MSD, Seagen; Financial Interests, Institutional, Funding, Clinical trials/contracted research: Genmab, Immunogen, Incyte, Novartis, Roche; Non-Financial Interests, Personal, Principal Investigator, PI of several trials, no compensation received: GSK; Non-Financial Interests, Personal, Principal Investigator, PI of several trials. No personal compensation received: AstraZeneca, Genmab; Non-Financial Interests, Personal, Principal Investigator, PI in several trials. No personal compensation received: MSD; Non-Financial Interests, Personal, Principal Investigator, PI of clinical trial. No personal compensation received: Immunogen, Clovis, Incyte; Non-Financial Interests, Personal, Principal Investigator, PI of clinical trial. No personal compensation receive: Roche; Non-Financial Interests, Personal, Member, Board of Directors: GCIG. All other authors have declared no conflicts of interest.

Background

Recurrent medulloblastoma is a deleterious disease with a 5-year survival of less than 5% and no recognized standard treatment. In recent years, we have learned about the molecular characterization of the different types of medulloblastoma with four subgroups also appearing different subgroups, each with its oncogenic pathway activation. SHH activation is the hallmark of medulloblastoma group 2 and several molecules have been developed to block this signaling mechanism, including SMO inhibitors, that are approved for the treatment of basal cell carcinoma. The information that is known about SMO inhibitors in medulloblastoma comes from relapsed medulloblastoma trials without prior molecular selection of patients, so their actual efficacy might still be unknown.

Methods

We present a 19-year-old patient with a leptomeningeal relapse of group 2 delta medulloblastoma. Based in some sporadic response of selected patients in phase II trials, sonidegib 800 mg daily was initiated as compassionate use in a third line of treatment.

Results

After 2 months of treatment, MRI evaluation shows an almost complete response in intracranial disease and stabilization of spinal disease. In addition, the treatment was well tolerated.

Conclusions

Further molecular characterization of medulloblastoma could lead to better results in recurrent medulloblastoma, a disease for which there is currently no proven effective treatment.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

The aim of molecular tumor board (MTB) is to identify potential therapeutic strategies, based on genetic analysis, for patients (pts) not responding to standard therapies. All tumor types are eligible for MTB discussion and sarcomas are one of common target due to low number of standard and innovative treatments. Here we analyze the role of MTB in a sarcoma referral center.

Methods

We presented data from MTB including pts affected by soft tissue (STS) and bone sarcoma (BS) followed at Regina Elena National Cancer Institute in Rome and discussed from Dec 2019 to May 2022.

Results

We discussed 19 pts affected by STS (14 pts) and BS (5 pts). FoundationOne was performed in 74%, Archer FusionPlex Sarcoma Panel in 37%, DNA Focus Assay in 26%, Whole exome sequencing in 26%, Oncomine Comprehensive Assay Plus in 16%, Promega MSI PCR Testing Kit in 16% and immunohistochemistry for PD-L1 in 16% of pts. Techniques were chosen depending on the type of kit available, the cost, the alterations searched and the time to obtain results. Druggable targets were found in 11 pts: mTOR mutation (m), HGF amplification (amp), ATM splice site m, MET amp, KRAS m, CDK4 amp, MYC amp, PTCH1 m, PIK3CA m, MDM4 amp and PD-L1 overexpression. Three patients (16%) received precision therapy: Imatinib and everolimus for mTOR m in cordoma, Cabozantinib for HGF amp in osteosarcoma and Pembrolizumab in angiosarcoma with PD-L 1 >10%. Eight pts continued standard therapy due to maintenance of response (5 pts) or to absence of literature supporting target treatment (3 pts). Molecular analysis allowed reformulation of diagnosis for one patient due to the presence of EWSR1-CREB3L2 fusion, typical of low-grade fibromyxoid sarcoma, that led to a histology-based treatment choice. Four patients were addressed to best supportive care (21%) while 2 pts (10,5 %) died.

Conclusions

MTB could be an effective tool for decision-making in sarcoma, but the lack of literature data and drug access hinder treatment choice. Enrollment in clinical trials could lead to overcome the problem. Moreover, the timing for requesting molecular analyses, at diagnosis or at the end of standard therapies, needs to be defined, considering both the tumor heterogeneity and the delay in obtaining results and starting treatment.

Legal entity responsible for the study

Regina Elena National Cancer Institute.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Generally tissue samples from histopathology or open biopsy are used for cytogenetic analysis and only few studies have used tissue samples from Fine Needle aspiration for chromosomal analysis. To start neoadjuvant chemotherapy in bone sarcoma patients, we need final diagnosis. Open biopsy has its own disadvantages. FNAC and cytological examination can give valuable information about the tumor. Apart from immunocytochemistry and electron microscopic examination of aspirated tissue, chromosomal analysis has also become a tool in diagnosing bone sarcoma.

Methods

During the period of six years in our hospital from 2016 to 2022, we cytogenetically analyzed FNACs from 11 primary bone sarcomas (4 osteosarcomas and 7 Ewing sarcomas). Out of them one of the osteosarcomas showed abnormal, complex karyotypes seen in most highly-malignant osteosarcomas and Four Ewing's sarcoma aspirates displayed abnormal karyotypes; two of these had the characteristic 11;22 translocation, and in one of these cases molecular genetic analysis revealed the hybrid EWS/FLI1 transcript.

Results

Chromosomal analysis of FNACs from suspected osteosarcoma didn’t provide much information, few cases showed high-grade malignancy. But in Ewing's sarcomas, these were of great value in making the diagnosis. 11;22 translocation finding on chromosomal analysis was diagnostic and strongly supported the cytologic diagnosis in six cases of Ewing's sarcoma. Tissues obtained by FNAC was sufficient for cytologic, cytogenetic, and molecular genetic analysis in two tumors. Molecular genetic analysis in one tumor showed the classical EWS/FLI1 transcript in Ewing's sarcoma.

Conclusions

FNAC and cytological examination is of limited value in patients with osteosarcoma and cannot make precise diagnosis but they can give reliable information and support the diagnosis in cases of Ewing's Sarcomas and help in starting multimodality treatment for bone sarcoma patients at the earliest.

Clinical trial identification

CMCH1622.

Legal entity responsible for the study

CMCH.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Whilst liquid biopsies have emerged as a non-invasive and cost-effective alternative to tissue biopsies and are increasingly used in clinical practice, there is still uncertainty on how findings should be interpreted by treating oncologists. Here, we used liquid biopsies to characterise patients with advanced cancer and provide clinical interpretation and recommendations via the Genomic Tumour Advisory Board (GTAB) of the NHS South East Genomic Laboratory Hub.

Methods

Twenty seven patients with metastatic or unresectable disease were offered circulating tumour DNA (ctDNA) assay testing between November 2021 to January 2022 at St George’s University Hospital in London, UK. Written consent was obtained for all patients. ctDNA was isolated from peripheral blood using the FoundationOne® Liquid CDx assay that reports genomic alterations in 324 genes, microsatellite instability and blood tumour mutational burden. Assays were provided by Roche through a Familiarisation Programme. Results were reviewed by the GTAB to identify patients potentially eligible for NHS-approved clinical trials based on a genomic biomarker as well as those requiring referral to Cancer Genetics.

Results

Median age of patients was 54 years (29-85) and 63% were males. Tumour type varied and included cancers of the colorectum (40%), lung (15%), breast (15%), kidney (15%), penis (7%) and ovary (4%). Approximately 85% of the patients were previously treated with 33% having received more than two lines of treatment. ctDNA was not isolated in two patients (7%) likely due to low tumour burden. There was 100% concordance between standard molecular tissue testing and ctDNA results. Thirteen patients (48%) were eligible for at least one clinical trial based on a genomic biomarker. Three patients (11%) were referred to Cancer Genetics due to a potential germline mutation or young age and family history.

Conclusions

We found that 48% of heavily pre-treated cancer patients were eligible for a clinical trial based on a genomic biomarker. Our results support the benefits of molecular profiling for allocating patients to clinical trials, and importantly, highlight the need to support oncologists with clinical interpretation and recommendations of findings through a GTAB for subsequent patient management.

Legal entity responsible for the study

The authors.

Funding

Roche.

Disclosure

All authors have declared no conflicts of interest.

Background

Head and neck squamous cell carcinoma (HNSCC) is one of the leading causes of cancer as well as cancer related deaths. Most patients present in a locally advanced state which lead to significant morbidity and mortality. A biomarker for HNSCC has potential for earlier detection and better treatment of cases but it’s search has been elusive so far. In a previous analysis by investigators as well as at our centre, the levels of Prosaposin A (PSAP) were found to be differentially expressed in the serum of HNSCC patients when compared to age and sex matched healthy controls.

Methods

Multiple investigators have reported numerous probable biomarkers in HNSCC patients over the years but few have been tested in the clinical scenario. While differential expression of PSAP has led to it being tested as a biomarker in various sites, but no such study has been reported in HNSCC so far. Serum of biopsy proven HNSCC patients (n=91) were obtained before and after their treatment. Patients treated with both radical and palliative intent were included. Serum samples of a cohort of age and sex matched healthy volunteers (n=42) were also obtained. An ELISA assay was run to measure the PSAP levels in serum in ng/ml.

Results

Serum PSAP levels of cases at baseline (median: 2.762) were found to be significantly elevated when compared to healthy controls (median: 2.053) (p=0.0123). A Wilcoxan matched pairs signed rank analysis was run to evaluate ELISA values pre- and post-treatment. A significant decrease in PSAP levels (median: 1.610) were found irrespective of complete/partial response to treatment (p<0.0001); for patients having residual disease (n=62) (median: 2.622 vs 1.476) post treatment (p=0.0007) and for patients having complete clinical response (n=29) (median: 2.948 vs 2.359) post treatment (p=0.0075).

Conclusions

Serum PSAP levels were significantly elevated in HNSCC patients at baseline compared to healthy controls which decreased post treatment. We hereby propose serum PSAP as a possible biomarker in HNSCC patients. Further large-scale clinical studies are warranted to establish serum PSAP as a diagnostic/prognostic marker in HNSCC patients.

Legal entity responsible for the study

The authors.

Funding

Post Graduate Institute of Medical Education and Research.

Disclosure

All authors have declared no conflicts of interest.

Background

Inaccuracy in reporting methods and results in published literature is a major issue as it challenges not only the interpretation of conclusions but also the ability to accurately compare studies. The existing REMARK (REporting recommendations for tumour MARKer prognostic studies) guideline aims to improve the reporting of tumour biomarker prognostic studies. However, for generic translational cancer genomic studies no standard currently exists. We developed modified REMARK (m-REMARK) criteria to ensure transparency and standardized reporting of these kinds of studies.

Methods

Key elements to be addressed in the proposed checklist were identified by the proposing panel and ICGC ARGO members including experts in methodology, biostatistics, and preclinical, translational, and clinical cancer genomics. Several reporting guidelines produced for other types of medical research studies were reviewed. REMARK was used as a guide and was adapted to our purpose.

Results

A preliminary draft of m-REMARK was proposed in the context of a systematic review and meta-analysis recently published in the Journal of Clinical Oncology. Building on that work, the checklist is being re-discussed, revised, and improved by the ICGC ARGO network. For final approval, external revision by the REMARK community will be done.

Conclusions

After ICGC ARGO discussion, revision, and external approval, m-REMARK will be the new standard to be followed when designing and reporting translational cancer genomic studies. It will significantly impact on future cancer research and patient care.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

MATCH-R trial is an ongoing prospective clinico-biological study, whose main objective is to understand mechanisms of acquired resistance to specific cancer therapies and guide to design new treatment strategies to overcome resistance. Patients (pts) in case of progressive disease are required to undergo a tumor biopsy (bx) to perform extensive molecular profiling (WES and RNAseq).

Methods

This study aims to describe the characteristics of the population, targeted treatment and molecular profile found through tumor bx for each type of solid tumor. We collected data from medical records and molecular profile reports from tissue bx that underwent NGS from December 2014 to December 2021 within the MATCH-R trial (NCT02517892), focusing on the molecular target group (MTG), which is the population that received target therapy prior and/or after MATCH-R bx with >1% of tumor cells (TC).

Results

876 pts were enrolled of which 930 bx were obtained, (41 pts had more than one bx). 814 (87%) pts presented one bx with >1% viable TC. The percentage of TC was >30% among 568 bx; 11-30%: 211 bx, 1-10%: 77 bx; 0%: 72 bx. Within the MTG 269 pts were included; 174 pts (65%) were female with a median age of 57 (range 23-89). Most common cancer types were: lung [186, 69%], gastrointestinal [38, 14%], genitourinary [16, 6%], gynecological [10, 4%], endocrine [7, 3%] and breast [5, 22%]. The most frequently mutated/rearranged genes were EGFR (41%), FGFR 2-3 (15%), ALK (11%), BRAF (8%), KRAS (5%), ROS1 (4%), RET (3%), BRCA 1-2 (3%), MET (2.6%), HER2 (2%), NTKR (1.1%) and PIK3CA (0.7%). All pts received treatment for solid tumors with a median of 4 lines (range 1-13). The median number of target therapy lines received prior and after MATCH-R bx was 1 [1-6] and 1 [1-5] respectively. Median follow-up after MATCH-R bx was 10 months [2-87].

Conclusions

MATCH-R achieved an adequate tissue quality, at progressive disease, to perform extensive molecular profile. This study will provide valuable information that, through translational research, will allow us to understand the possible resistance mechanisms for the different treatments. Further updates will be provided.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

C. Baldini: Non-Financial Interests, Personal, Other: AstraZeneca, Bayer, BMS, Boehringer Ingelheim, GSK, MedImmune, Merck, NH TherAGuiX, Pfizer, Roche; Financial Interests, Personal, Other: GSK, BMS, AZ, Amgen, Sanofi, MSD travel accommodation; Non-Financial Interests, Personal, Principal Investigator: Principal/sub-Investigator of Clinical Trials for AbbVie, Adaptimmune, Adlai Nortye USA Inc., Aduro Biotech, Agios Pharmaceuticals, Amgen, Argen-X Bvba, Arno Therapeutics, Astex Pharmaceuticals, AstraZeneca Ab, Aveo, Basilea Pharmaceutica International L; Other, Personal, Funding: BMS Fundation; Other, Personal, Research Grant: from AstraZeneca, BMS, Boehringer Ingelheim, GSK, INCA, Janssen Cilag, Merck, Novartis, Pfizer, Roche, Sanofi. J. Soria: Financial Interests, Personal, Other: AstraZeneca and is now employed by Amgen; owns stock in AstraZeneca, Gritstone Bio, Relay Therapeutics; and serves on the Board of Directors for Hookipa Pharmaceuticals outside the submitted work. Giuseppe Curigliano reports a grant from Merck; Consul, Pfizer, Novartis, Seattle Genetics, Daiichi Sankyo. Y. Loriot: Financial Interests, Personal, Other: Astellas, Other, Personal, lectures, advisory boards AstraZeneca, Other, Personal, lectures, advisory boards BMS, Other, Personal, lectures, advisory boards Gilead, Advisory Board, Personal Janssen, Other, Personal, lectures, advisory boards Merck KGaA, A; Non-Financial Interests, Personal, Other: Incyte, Local PI, Institutional, Financial interest Janssen, Coordinating PI, Institutional, Financial interest Janssen, Steering Committee Member, Institutional, Financial interest Janssen, Local PI, Institutional, Financial interest Janssen, Research Gr. F. André: Financial Interests, Personal, Funding: Roche, AstraZeneca, Daiichi Sankyo, Pfizer, Novartis, Lilly; Financial Interests, Personal, Invited Speaker: Roche, AstraZeneca, Daiichi Sankyo, Pfizer, Novartis, Lilly; Financial Interests, Personal, Research Grant: Roche, AstraZeneca, Daiichi Sankyo, Pfizer, Novartis, Lilly. B. Besse: Financial Interests, Personal, Other: Sponsored Research at Gustave Roussy Cancer Center 4D Pharma, AbbVie, Amgen, Aptitude Health, AstraZeneca, BeiGene, Blueprint Medicines, Boehringer Ingelheim, Celgene, Cergentis, Chugai Pharmaceutical, Cristal Therapeutics, Daiichi Sankyo, Eli Lilly, Eisai. S. Ponce: Financial Interests, Personal, Other: Roche, MSD, AstraZeneca, Bristol Myers Squibb, PharmaMar, Boehringer Ingelheim, BMS. Expert testimony: Roche, MSD, AstraZeneca, Bristol Myers Squibb, PharmaMar, Boehringer Ingelheim, BMS. Travel, accommodations, expenses: MSD, Roche, BMS, AstraZeneca, Ph, RSD Pharma; Financial Interests, Personal, Invited Speaker: Bristol Myers Squibb. All other authors have declared no conflicts of interest.

Background

Accurate detection of Copy Number Variants (CNV) in homologous recombination repair (HRR) related genes is essential for HRR deficiency diagnostics and treatment administration such as PARP inhibitors. CNV detection from high-throughput sequencing (HTS) is expected to complete simultaneous comprehensive genetic markers evaluation, reduce cost and turnaround time and to increase sensitivity. It is still challenging to detect somatic CNV from small targeted panels that are widespread in clinical settings. The current aim: To evaluate clinical utility of somatic CNV detection from amplicon-based HTS panels.

Methods

High-throughput sequencing of 220 tumor samples (breast, ovarian, pancreatic and prostate) was performed with an AmpliSeq panel covering ATM, BRCA1, BRCA2 and CHEK2 genes (409 amplicons, 36kbs). Data from 160 cases passed quality control were analyzed for CNV. Following metrics were utilized for CNV detection: SNP allelic imbalance (more than 10% from hetero- or homozygosity), by-pool coverage drop (more than 20% from baseline), score of coverage consistency. For accurate SNP allelic imbalance estimation we implemented amplicon-based variant allele fraction (VAF) calculation ensuring reduction of amplicon dropout errors and PCR-enrichment artifacts. This allowed to increase concordance of VAF of detected SNPs.

Results

Over analyzed 160 samples, 7 samples displayed SNP allelic imbalance concordant with consistent coverage drop in BRCA1 (6 cases, 16% positive for BRCA1/2 mutations) and BRCA2 (1 case, 0% positive for BRCA1/2 mutations) genes. These samples were considered as putative for CNV. No cases with putative CNV in ATM or CHEK2, or with CNV in more than one gene were found. The observed number of positive samples corresponds to the level expected from literature data.

Conclusions

Integrative analysis of allelic imbalance and amplicon coverage from targeted high-throughput sequencing with small panels allows to detect samples putative for CNV for further validation with orthogonal methods. This allows to increase information yield from small gene panel sequencing and may lead for PARP inhibitors indication for additional 4% of patients with breast, ovariant, pancreatic and prostate cancer.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Cisplatin is used as first-line chemotherapy treatment for patients diagnosed with various types of malignancies, such as leukemia, lymphomas, breast, testicular, ovarian, head and neck, non-small lung, cervical cancers, and sarcomas. However, our recent study provided evince on the activation of DNA repair genes in p53 wild-type of breast, non-small lung and liver cancer, but not in mutated triple-negative breast cancer or p53 deficient myeloid leukemia.

Methods

RNA- and ChIP-Seq, cellular technics (toxicity, confocal immunostaining, western blot, real-time PCR) we found p53 wild-type interaction with EP300 at the promoters of PARP1, BRCA1 and RAD51.

Results

RNA-Seq data confirmed that pharmacological inhibition of ATM/ATR, EP300 and transient silencing of p53 prevent overexpression of genes contributing to nucleotide excision repair (GO:0006289), double-strand break repair via homologous recombination (GO:0000724) and nonhomologous end joining (GO:0006303) and DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest (GO:0006977). This was not observed in p53 single-nucleotide or deletion mutants. Since transcription of majority of genes involved in DNA repair is controlled by cell cycle progression via E2F activity, we paid attention to p53 interaction with chromatin upon cisplatin treatment. ChIP-Seq data confirmed the enrichment of approximately 1200 genomic locations, where only 7% overlapped with E2F1, but 22% with EP300 peaks. Prediction of target genes linked p53 enriched regions with genes assigned to nucleotide-excision repair and DNA damage recognition (GO:0000715). Cisplatin caused p53 co-localization with EP300 in the nuclei, but E2F1 silencing restrained the enrichment of both proteins on chromatin. Pharmacological inhibition of ATR/ATM, EP300 and p53 silencing considerably increased cisplatin cytotoxicity. The same effect of EP300 inhibitors on cisplatin cytotoxicity was observed in cisplatin-resistant p53 wild-type breast and non-small lung cancers.

Conclusions

ATM/ATR-p53/E2F1/EP300 pathway allows cells to increase resistance to cisplatin. Addition of EP300 inhibitors may improve cisplatin-based chemotherapies of p53 wild-type cancers and improve chemotherapy outcome.

Legal entity responsible for the study

The authors.

Funding

National Center for Research and Development (LIDER/22/0122/L-10/18/NCBR/2019) and Ministry of Education and Science (IDUB-60/2021).

Disclosure

All authors have declared no conflicts of interest.

Background

Growth and differentiation factor-15 (GDF-15) is a divergent member of the transforming growth factor β family associated with pathological conditions, including myocardial ischemia, autoimmune disease, and cancer. Studies on the role of GDF-15 in cancer are limited and controversial. Radioactive iodine (¹³¹I) ablation of the post-surgical thyroid remnants, as an immunomodulatory tool, is the standard treatment for differentiated thyroid carcinomas (DTC) and DTC associated with type 2 diabetes mellitus (DTC+T2DM). Given the aggressive nature of HER2-positive breast cancer, neoadjuvant dual anti-HER2 blockade (trastuzumab + pertuzumab) in combination with chemotherapy has been shown to improve clinical outcomes through immune system activation. We hypothesized that the predictive value of GDF-15 as a biomarker in DTC and HER2-positive breast cancer might plausibly be linked to its immune-regulatory function.

Methods

Peripheral venous blood was collected from 56 female patients with DTC (mean age 57.3±9.1 years) and 11 with DTC+T2DM (mean age 61.6±7.8 years) before and four days after ¹³¹I therapy and from 22 female patients presenting HER2-positive breast tumors (mean age 55.5±9.8 years) before and after neoadjuvant treatment. The serum levels of GDF-15 were measured by ELISA.

Results

GDF-15 concentration was higher in the HER2-positive breast tumors than in the DTC group (1522.4 pg/mL vs. 915.02 pg/mL, P < 0.001). Regarding the DTC study group, the association of DTC with T2DM led to higher GDF-15 serum levels (P < 0.001). A modulation in GDF-15 level was measured in response to HER2 inhibitors treatment (reduction by 18.8%). In the DTC and DTC+T2DM study groups, the ¹³¹I effect was illustrated by a GDF-15 significant increase (97.9% and 169.8%, respectively) in a dose-dependent fashion.

Conclusions

The described associations and mechanistic data suggest that GDF-15 might be a radiation-induced early biomarker in DTC patients. Furthermore, GDF-15 may act as a biomarker of response to action and synergism of trastuzumab and pertuzumab in targeting HER2-positive breast cancer. With a better understanding of the upstream cancer pathways reflected by GDF-15, new treatment targets may emerge.

Legal entity responsible for the study

The authors.

Funding

This work was supported by a grant from the Romanian Ministry of Education and Research, CCCDI - UEFISCDI, project number PN-III-P2-2.1-PED-2019-3313, within PNCDI III.

Disclosure

All authors have declared no conflicts of interest.

Background

Extracellular vesicles (EV) are nanometric lipid bilayer coated particles constitutively released from cells. EVs are a heterogeneous population and can be sampled as liquid biopsy. Beyond messengers between cells, EVs act as regulators and mediators of many processes underlying cancer evolution: inflammation, proliferation, invasion, immune modulation, angiogenesis, epithelial mesenchymal transition. The aim of the present research is to describe a new method to obtain high resolution images of individual EVs in patients with advanced cancer.

Methods

Plasma from 10 patients with advanced cancer enrolled in the institutional molecular profiling program STING (NCT04932525, sponsor: Gustave Roussy) was diluted in PBS 1:50 and loaded on slides for Single Molecule Localization Microscopy (SMLM) imaging. EVs were stained using a mix of anti-tetraspanin Ab (CD9+CD63+CD81) labelled with AF647 fluorophores to only select tetraspanin+ EVs for imaging and analysis. Single molecule imaging (dSTORM) was performed using Abbelight SMART-kit buffer on a SAFe360 Abbelight super-resolution module mounted on an Olympus Ix83 inverted microscope. Fluorophore labelled EVs were excited with the 640nm laser at 60% of nominal power over a ROI of 80*80 micrometers, by Abbelight Aster technology for homogeneous laser illumination; for each dataset 10000 frames were collected at 40 FPS, with two-three technical replicates per sample (RAW data). Single molecule localization in 3D was performed on the RAW data using Abbelight Neo Software, and localization clusters corresponding to labelled EVs were extracted using DBSCAN and K-Ripley clustering algorithms.

Results

High resolution images of EVs were obtained with SMLM. Number and size distribution of clusters were quantified for each Ab allowing to observe differences between patients samples. Thus, SMLM can be considered as an additional technique to detect and characterize individual EVs in clinical samples.

Conclusions

SMLM imaging with tetraspanin labeling is able to detect demonstrable differences in EV clusters quantity and size distributions in plasma from patients with advanced cancer. Additional investigations are ongoing to further develop the technique and make it applicable in clinical practice.

Legal entity responsible for the study

Gustave Roussy, Abbelight.

Funding

Abbelight.

Disclosure

M. Pagliuca: Financial Interests, Personal, Other, Travel expenses: Gilead, Pfizer. M. Triki: Financial Interests, Personal, Full or part-time Employment: Abbelight. C. Schietroma: Financial Interests, Personal, Full or part-time Employment: Abbelight. C. Butler: Financial Interests, Personal, Full or part-time Employment: Abbelight. B. Verret: Financial Interests, Personal, Other, Consultant fees, travel expenses: Lilly, Pfizer; Financial Interests, Personal, Other, Consultant fees: Netcancer, Pierre Fabre, Seagen, Gilead; Financial Interests, Institutional, Other, Consultant fees: Daiichi Sankyo, MSD; Financial Interests, Personal and Institutional, Other, Consultant fees, travel expenses: Novartis; Financial Interests, Personal, Other, Travel expenses: Accord Healthcare, Amgen. A. Italiano: Financial Interests, Personal, Research Grant, Research funding, Consulting fees: AstraZeneca, MSD, Merck; Financial Interests, Personal, Research Grant, Research funding, Consulting fees, Advisory board: Bayer, Roche; Financial Interests, Personal, Other, Research funding, Consulting fees: BMS; Financial Interests, Personal, Advisory Board, Research grants: Chugai; Financial Interests, Personal, Advisory Board: Daiichi Sankyo, Foundation Medicine, GSK, Springworks. D. Planchard: Financial Interests, Personal, Advisory Board: AstraZeneca, BMS, Merck, Novartis, Pfizer, Roche, Samsung, Celgene, AbbVie, Daiichi Sankyo, Janssen; Financial Interests, Personal, Invited Speaker: AstraZeneca, Novartis, Pfizer, priME Oncology, Peer CME, Samsung, AbbVie, Janssen; Non-Financial Interests, Personal, Principal Investigator, Institutional financial interests: AstraZeneca, BMS, Merck, Novartis, Pfizer, Roche, Daiichi Sankyo, Sanofi-Aventis, Pierre Fabre; Non-Financial Interests, Personal, Principal Investigator: AbbVie, Sanofi, Janssen. A. Bayle: Financial Interests, Personal, Advisory Board: Sanofi; Financial Interests, Personal and Institutional, Research Grant: AstraZeneca, BMS, Boehringer Ingelheim, GSK, INCA, Janssen Cilag, Merck, Pfizer, Roche, Sanofi; Non-Financial Interests, Personal, Other, Drug supplied: AstraZeneca, BMS, Boehringer Ingelheim, GSK, MedImmune, Merck, NH TherAGuiX, Pfizer, Roche; Financial Interests, Personal and Institutional, Other, Principal/sub-Investigator of Clinical Trials: AbbVie, Adaptimmune, Adlai Nortye USA Inc, Aduro Biotech, Agios Pharmaceuticals, Amgen, Astex Pharmaceuticals, AstraZeneca Ab, Aveo, Basilea Pharmaceutica International Ltd., Bayer Healthcare Ag, Bbb Technologies Bv, Beigene, BicycleTx Ltd., Blueprint Medi. F. André: Financial Interests, Institutional, Research Grant: AstraZeneca, Lilly, Novartis, Pfizer, Roche, Daiichi Sankyo; Other, Personal, Other, Founder: Pegacsy. S. Delaloge: Financial Interests, Institutional, Advisory Board: AstraZeneca, Novartis, Pierre Fabre, Orion, Sanofi, Rappta, Cellectis, Isis/Servier; Financial Interests, Institutional, Invited Speaker: Exact Sciences, Pfizer, Seagen, Lilly, AstraZeneca, MSD, Roche Genentech, BMS, Puma, AstraZeneca, Orion, Sanofi, Pfizer; Financial Interests, Institutional, Advisory Board, ad board: Besins Healthcare; Financial Interests, Institutional, Invited Speaker, ESMO symposium: Gilead; Financial Interests, Institutional, Advisory Board, scientific board: Elsan; Financial Interests, Institutional, Funding: GE; Financial Interests, Institutional, Invited Speaker, clinical research funding to my institution: Taiho; Non-Financial Interests, Personal, Invited Speaker, Société Française de Sénologie et Pathologie Mammaire: SFSPM; Non-Financial Interests, Personal, Principal Investigator, H2020 funding: European Commission.

Background

Multigene testing via NGS can detect multiple genomic alterations (GA). The use of current levels of evidence (LOE) requires understanding the functional consequences of the GA and an extensive literature search and have less practical value for narrowing down therapy recommendations if multiple GAs with the same LOE are found.

Methods

For each type of GA, depending on the gene and tumor type, a score from 1 to 10 was assigned independently by a group of biologists and oncologists; average scores were used for DB. Scores reflected the theoretical estimation of percentage of patients with biomarker that could be matched with such therapy, the efficacy of therapy based on expected benefit, quality of data, expert opinion; potential obstacles associated with access to therapy. To test the utility of the DB, we analyzed comprehensive molecular profiling (150+ gene NGS panels) (CMPR). GA were ranked using ESCAT and CRAC.

Results

A total of 134 genes were selected for 16 malignancies with 2 GA for each in average (234 GA in total). GA with scores 2-3 outnumbered GA with scores 9-10 (36 vs 2%). The majority (17 vs 13%) of genes have average scores of 2-2.5, 3.5-4, within one tumor type. To test CRAC practical value 208 CMPR (23.5% CRC, 16.3% PAAD, 11% BRCA, 49% - other) with 210 GA analyzed. 64 (31%) reports contained 79 GA of I-III ESCAT, 114 (55%) - 131 level IV GA. The highest scores reflected the highest LOE. No GA with the same LOE had the same score; GA IIIA and IV LOE had the largest range of scores (2-10 and 1-9). CRAC made it possible to identify additional targetable GA with scores 2-4 (45% were not present in CMPR).

Conclusions

Using CRAC scores to identify clinically significant GA proved to be a more comprehensive approach compared to ESCAT LOE (each LOE was represented by ⩾3 scores; each CMPR had BM with ⩾2 scores). CRAC available at crac.oncoatlas.ru.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Molecular diagnostics for the detection and characterization of genetic variants is based in the majority of cases on PCR technology. Since the primers and probes are designed according to the nucleotide sequence of the DNA region for the specific mutation, PCR technology can only be applied if the mutation is well known. In cases where the mutated region of a gene may have variable sequences, different between patients, a set of primers for each sequence variant is necessary, which involves a large volume of work. The nucleophosmin (NPM1) gene mutation is also included in this category where more than 24 variants of the gene are known of which 6 are more common.

Methods

The aim of our study was to establish a qPCR assay and a molecular kit (a set of primers) for detection and quantification of NPM1 gene mutations regardless of its sequence by using RNase H-dependent PCR (rhPCR). Several sets of rh-primers that show point mismatches in relation to some mutations have been designated and tested. The rh-primers carried a removable PCR blocker at the 3′ end. Only after the removal of this blocker by the RNaseH2 enzyme, the primers are expandable by DNA polymerase. qPCR was used to determine the extent to which the presence of these sequence mismatches could affect the H2-endonuclease activity of ribose digestion and PCR activation. The evaluation was carried out in a preclinical context, using synthetic fragments of NPM1 carrying mutations (AML mutation type A, B, D, I, K).

Results

After establishing the optimal combination for the primer sets as well as the reaction conditions, the sensitivity limit was tested in samples with the number of target copies that varied decreasingly: 1000000, 100000, 10000, 1000, 100, 10. It was observed that regardless of the nature of the mutation, the same set of primers can detect 10 copies of mutant NPM1.

Conclusions

The main advantage of this assay is that different sequence-variable NPM1 mutations can be detected and quantified with high specificity through a single rhPCR reaction. It is no longer necessary to design, synthesize and optimize primers and probes for each individual mutation. Our study demonstrates the potential use of rhPCR for monitoring patients with AML for minimum residual disease and recurrence risk.

Legal entity responsible for the study

Victor Babes Institute of Pathology.

Funding

National Program 1N/2019/PN19.29.01.03.

Disclosure

All authors have declared no conflicts of interest.

Background

Flow cytometry (FCM) is a co-criterion in Myelodysplastic Syndromes (MDS) diagnostics. The aims of the present study were (1) to develop a composite prognostic FCM-Score for OS in MDS ; (2) to asess whether a computational algorithm could improve the identification of aberrant expression and cell population frequencies and (3) to validate the accuracy of the prognostic FCM-Score for OS in MDS in an independent cohort.

Methods

FCM was performed in bone marrow (BM) of 399 patients cytomorphologically classified as MDS. Cell populations were identified. OS was assessed following univariate and multivariable Cox proportional hazards regression analysis using log-rank likelihood test to calculate FCM prognostic models. Kaplan Meier curves, and receiver operating characteristic curve (ROC) were used to test independent prognostic value of the models versus known diagnostic FCM-scores (Ogata-, FCSS-, iFS-score). T-REX pipeline was applied to check the feasibility of an unsupervised machine learning approach in identifying the FCM parameters. Validation of the prognostic score with best performance in an independent cohort of 110 MDS patients was performed.

Results

Prognostic FCM-scores were calculated based on the 9 FCM parameters with independent prognostic impact. FCM-scores, FCM-A: HR (95 %CI) 3.20 (2.15 - 4.48); FCM-B: 4.08 (2.54 - 6.55)), outperformed well known diagnostic scores (Ogata-score (2.00 (1.29 – 3.11))). FCM-scores allowed a better prognostic grading than IPSS-R (HR (95 %CI): 2.37 (1.61-3.49)). Kaplan Meier survival curves stratified by FCM-score A and B showed a highly significant overall survival benefit for patients with a low score (p<.0001) and FCM-A and FCM-B scores presented better discrimation capability than IPSS-R ((AUC): 0.69, and 0.71 vs. 0.62). T-REX pipeline was able to identify differences in expression of significant parameters between the low and high scoring patients. The validation of the OS prognostic score obtained presented good discrimination performance (c-stats 0.764; AUC 0.7463).

Conclusions

A promising novel prognostic score based on distinct FCM characteristics which could predict overall survival in MDS patients was presented.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

This study aimed to evaluate the predictive role of immune-related markers, BRCA1 germ-line status and BRCAness phenotype in BC patients undergoing NACT.

Methods

The study involved 70 BC patients with germline BRCA1 pathogenic alleles [49 triple-negative (TNBC) and 21 hormone receptor-positive (HR+)], 73 BRCA1 wild-type TNBCs and 130 BRCA1 wild-type/HR+ patients. The “inflamed” phenotype was identified by IHC on hematoxylin-eosin stained slides as an abundance of double-positive CD8/GZMB infiltrating lymphocytes (TILs) across the tumor. BRCAness was detected by multiplex PCR and fragment analysis. The expression of critical immune genes (PD1, PD-L1, LAG3, TAP2, IL10B, TGFB, HLA-E, HLA-G) was quantified using RT-PCR.

Results

The incidence of “inflamed” immune phenotype was significantly higher in BRCA1 mutation-driven vs. BRCA1 wild-type BCs regardless of tumor hormone receptor status [32% vs. 7%, p = 0.000]. Logistic regression showed that the abundance of CD8/GRMZ TILs was associated with up-regulation of immune checkpoint molecules PD1, PD-L1 and HLA-G [p = 0.07, 0.002 and 0.000, respectively]. Independent predictive markers of tumor response to NACT were “immune-inflamed” vs. “immune dessert” histological appearance [p = 0.014], BRCAness phenotype irrespective of BRCA1 germ-line status [p = 0.026], high expression of antigen presenting-related gene TAP2 [p = 0.08] and age at onset <39 years [p = 0.043].

Conclusions

Tumor immune phenotype is predictive for BC response to NACT in addition to BRCA-related markers.

Legal entity responsible for the study

The authors.

Funding

Russian Science Foundation, grant 22-15-00278.

Disclosure

All authors have declared no conflicts of interest.

Background

The prognosis of breast cancer in pre-menopausal women is very variable and dependent on the interactions of several biological factors. The androgen receptor (AR) could be among the prognostic variables related to survival although the data is scarce. The aim of the study was to examine the relationship between androgen receptor expression and survival outcomes in estrogen receptor (ER)-positive pre-menopausal invasive breast carcinoma patients.

Methods

We assessed the AR expression in ER-positive pre-menopausal invasive breast carcinomas and correlated this expression pattern with several clinical and pathologic parameters: tumor size, lymph node status, progesterone receptor (PgR) status, and human epidermal growth factor receptor type 2 (HER2) overexpression and evaluated the association of these parameters with survival using univariate analyses. Immunohistochemical analysis for AR, PgR, and HER2 were carried out and semiquantitative evaluation of staining was performed.

Results

AR expression was demonstrated in 61.44% of patients. No statistical difference was demonstrated in AR expression in relation to age, tumor size, lymph node status, PgR/HER2 status (p score = 0.758, 0.346, 0.604, 0.070, 0.48 respectively). AR expression was not an independent prognostic factor related to progression free survival and overall survival in ER-positive cancers.

Conclusions

AR expression was not associated with tumor size, ER/PgR/HER2 status. Although AR expression has prognostic significance in triple-negative breast cancer, it is not a prognostic marker in hormone-positive premenopausal breast cancer.

Legal entity responsible for the study

Hacettepe University Medical School Hospital.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

In many solid Notch pathway is deregulated, affecting most hallmarks in cancer. Notch pathway plays a key role in cellular differentiation and its dysregulations contributes to carcinogenesis. Notch gene expression is frequently altered in breast cancer (BC) and exists ample evidence of the role of Notch in tumor formation, progression and resistance to treatment in BC. A less explored function of Notch pathways in cancer is their role in leukocyte homing and activation. Understanding their role and relationship with immune infiltrates is an area of interest in cancer research.

Methods

The expression of four different Notch genes (Notch1, Notch2, Notch3 and Notch4) was explored in relation with A luminal BC patient outcome using transcriptomic data (Affymetrix dataset, exploratory cohort) and the METABRIC study (validation cohort). The TIMER online tool was used to explore the association of the identified notch and immune infiltration, and the TCGA and METABRIC studies to analyze the correlation between notch1 - 4 expression and genomic signatures of immune activation.

Results

We identified 2 individual genes called Notch1 and Notch2, which predict favorable prognosis in luminal A breast cancer. Their expression positively correlated with the presence of immune infiltrates within the tumor (dendritic cells, CD4+ T cells, neutrophils, CD8+ T cells and B cells), with markers of T cell activation and antigen presentation, and with gene signatures of immune surveillance (cytotoxic T lymphocyte activation and IFN gamma signature). By contrast Notch3 and Notch4 which predicted for detrimental outcome were not associated with any of these parameters.

Conclusions

Our analysis identifies a Notch signature composed of 2 genes with potential to recognize immune infiltrated and activated A luminal phenotype BC with favorable prognosis.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Anti-HER2 monoclonal antibodies improved the pathologic complete response (pCR) and prognostic of human epidermal growth factor receptor 2-positive (HER2+) breast cancer (BC). Antibiotics (ATB) and proton pump inhibitors (PPI) may affect the efficacy of cancer therapies, by modulating gut microbiota and consequently disturb the immune response. Here we aimed to evaluate the impact of ATB and PPI in HER2+ BC neoadjuvant treatment response.

Methods

We conducted a retrospective observational study in HER2+ BC patients, treated in our centre with neoadjuvant chemotherapy plus trastuzumab and pertuzumab, between July 2016 and March 2022. Achieving a pCR was compared regarding the use of ATB during the first 30 days, as well as the baseline use or initiation of PPI, in the first 30 days of the neoadjuvant scheme. We excluded: cancers of unknown primary site treated as BC; uncompleted neoadjuvant scheme; absence of pathologic assessment after treatment; additional treatments performed.

Results

Fifty-four female patients were included with a mean age of 52.9 years. Twenty-nine (53.7%) had BC staged as T2 and 30 (55.6%) had positive lymph nodes. Concerning pathological features: 75.0% had a Ki-67 score >30% and 72.2% had positive hormone receptors (HR+). A pCR was obtained in 29 (53.7%) patients and, of all sample, 6 (11.1%) took ATB and 27 (50.0%) PPI. All prescribed ATB were beta-lactams and the main motive was central venous catheter infection (n=2; 33.0%). Nor ATB (8.0 vs. 13.8%; p=0.675) neither PPI intake (56.0 vs. 44.8%; p=0.413) impacted the rates of pCR obtained. Furthermore, the association of ATB and PPI also did not affect the achievement of a pCR (4.0 vs. 10.3%; p=0.615). There was a tendency for non-achieving a pCR for HR+ BC (84.0 vs. 62.1%; p=0.073), while age, tumor size, lymph nodes status and Ki-67 did not correlate.

Conclusions

In our sample ATB and/or PPI intake was not associated with lower rates of pCR obtained after neoadjuvant HER2+ BC treatment. Future studies with long-term prognostic assessment are still needed.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

A. Teixeira: Financial Interests, Personal, Invited Speaker: Novartis. All other authors have declared no conflicts of interest.

Background

With the implementation of anti-HER2 therapies in clinical practice, the OS of patients with HER2(+) breast carcinoma significantly improves. The aim of this retrospective analysis is to asses the prognostic value of dynamic HER2 status conversion among the patients with metastatic disease, treated in our institution.

Methods

The data are obtained from the medical documentation of the patients with breast cancer in early, locally advanced and metastatic stage. Descriptive statistical analysis is used. As positive is defined the conversion from HER2 (-) to HER2 (+) status and as negative is defined the conversion from HER2 (+) to HER2 (-) receptor status. The HER2 status is determined by IHC or in situ hybridization method.

Results

From m.07.2006 to m.07.22 in our institution are treated 423 patients with BC. In 18 cases we found conversion in HR and/or HER2 receptor status. HER2 conversion is seen in 61% (11/18), respectively positive conversion in 6 pts, negative conversion in 3 pts and in 2 of the cases we observed positive and negative HER2 conversion in the course of treatment. The group of patients with HER2 conversion is heterogeneous. It includes women with locally advanced BC, treated with NACT (3/11), patients with synchronous (2/11) and metachronous (4/11) metastatic disease, where the receptor status has been reassessed in the course of treatment and women with resected oligometastatic disease (2/11), treated with adjuvant anti-HER2 therapy. The mean duration of anti-HER2 therapy among the patients with metastatic disease and positive HER2 conversion is 31.1 m. The mOS among the patients with HER2 conversion and metastatic disease is 80.1 m. For the patients with positive HER2 conversion the mOS is 90 m. and for the patients with negative HER2 conversion the mOS is 21 m. (p value=0.008).

Conclusions

The patients with metastatic BC and positive HER2 conversion have significantly higher mOS than the patients with metastatic disease and negative HER2 conversion (p<0.05). The mean duration of anti-HER2 therapy among the patients with metastatic disease and positive HER2 conversion is 31.1 m. which may contribute to the improved mOS in this population.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

In the last decades, the impact of patients’ eating and exercise habits on breast cancer (BC) management on one side, and inflammation on the other, have been deeply explored and proven. Here, we investigated whether unhealthy habits could correlate with bowel FDG uptake and the latter, in turn, with pathological Complete Response (pCR) to standard neoadjuvant chemotherapy (NAC).

Methods

The study included stage I-III BC patients undergoing NAC at IRCCS Humanitas Research Hospital, Humanitas Cancer Center, Rozzano, Italy. At baseline, patients fulfilled a survey concerning eating and lifestyle habits. In the absence of data on the effects of individual foods, the frequency of consumption of specific food items was aggregated for their inflammatory properties: alcohol and spirits as “pro-inflammatory drinks”, red and cured meats as “pro-inflammatory foods”, fruits and vegetables as “anti-inflammatory foods”. Before NAC, women performed a whole-body staging [18]F-FDG PET/CT scan. On PET/CT images, two regions of interest were designed on the area of highest uptake in the rectum-sigmoid district and in the colon, respectively, and radiotracer mean standardized uptake values (SUV_mean) were extracted.

Results

Data were recorded for 82 women (median age: 48 years), of whom 29 were diagnosed with triple-negative BC, 45 with a HER2-positive BC, 7 presented a Luminal B tumor, and 1 had a Luminal A BC. We found a positive correlation between colon SUV_mean and pro-inflammatory drinks (r = +0.33, p = 0.006) and foods (r = +0.25, p = 0.033) and a negative association with exercise frequency (r = -0.24, p = 0.04). A negative correlation was also observed between rectum SUV_mean and anti-inflammatory foods (r = -0.24, p = 0.027). Finally, colon SUV_mean was significantly lower in patients with pCR compared to those without pCR (p = 0.025).

Conclusions

Our study showed, for the first time, that bowel FDG uptake was affected by patients’ anti- and pro-inflammatory habits and that colon SUV_mean correlated with pCR. Moreover, our results suggest that PET scan may be an easy instrument for identifying patients with unhealthy lifestyle habits.

Legal entity responsible for the study

The authors.

Funding

IRCCS Humanitas Research Hospital (5X1000 funding).

Disclosure

All authors have declared no conflicts of interest.

Background

It is well established that cyclin D1 is an important component of estrogen regulation. We have previously reported that the transforming growth factor beta type I receptor (TβRI) is related to tamoxifen-sensitive tumors. In addition to TβRI, the type 2 receptor (TβRII) plays a key role in the regulation of TGF-β signaling.The aim of the present study was to explore the predictive value of cyclin D1/TβRII expression for the tamoxifen response in patients with hormone receptor-positive breast cancer.

Methods

Expression of cyclin D1 and TβRII by flow cytometry and their mRNA expression by quantitative real-time polymerase chain reaction was performed on tissue specimens from 60 women with hormone receptor-positive primary breast cancer who had received adjuvant tamoxifen treatment at a dose of 20 mg/day for at least 5 years. The primary end-point was progression-free survival (PFS).

Results

High mRNA expression of TβRII in tumor were significantly related to tamoxifen efficacy (р = 0.026). Co-expression analysis revealed that the population of cyclin D1-/TβRII+ was more prevalent in the tamoxifen-sensitive tumors (p=0.009). We showed a significant correlation between the TβRII and cyclin D1 expression (r = 0.39; p = 0.010). Low cyclin D1 expression as well as TβRII high expression tended to be closely associated with a better prognosis, although this difference was not significant (log-rank p = 0.08 and log-rank p = 0.09, respectively).

Conclusions

The findings of the present study indicate that cyclin D1-/TβRII+ cells may be involved in the tamoxifen response and are associated with sensitivity to this drug.

Legal entity responsible for the study

The authors.

Funding

The study was supported by the Russian Scientific Foundation, grant #19-75-30016.

Disclosure

All authors have declared no conflicts of interest.

Background

Targeting the innate immune system has attracted attention with the development of anti-CD32b antibodies. CD32b (FcγRIIB or FCGR2B) is involved in the phagocytosis of immune complexes and in the regulation of antibody production by B-cells. CD32b has been shown to perform unexpected activatory roles in both immune-signaling and monoclonal antibody (mAb) immunotherapy. Understanding their role and relationship with immune infiltrates is an area of interest in cancer research.

Methods

The KM Plotter online Tool (http://www.kmplot.com), a publicly available database, was used to study the relationship between the expression of CD32b gene from the validated immune signatures and patient clinical outcomes for BC. Patients were divided into two groups, high vs. low expression, based on the best cut-off values of gene expression (smallest p-value). The best cut-off values were determined by algorithms embedded in KM plotter. The OS KM plots are present with the hazard ratio (HR), the 95% confidence interval (CI) and log-rank p-value. The Tumor Immune Estimation Resource (TIMER) online tool was used to explore the association between the abundance of tumor immune infiltrates (B-cells, CD4+ T-cells, CD8+ T-cells, dendritic cells, macrophages, and neutrophils) and the expression of CD32b. The correlation graphics show the purity-corrected partial Spearman’s correlation and its statistical significance.

Results

We identified CD32b gene, which predict worse prognosis in HER2+ B luminal BC (OS: HR = 0.48 (0.31 -0.74), logrank P = 0.00083; RFS: 0.77 (0.62 – 0.95), logrank P = 0.016). Their expression positively correlated with the presence of immune infiltrates within the tumor CD4+ T cells (partial.cor =0.393, p = 2.29e-03), neutrophils (partial.cor = 0.476, p = 1.60e-04), CD8+ T cells (partial.cor = 0.285, p = 3.15e-02), dendritic cells (partial.cor = 0.639, p = 6.59e-08), macrophage (partial.cor = 0.639, p = 6.59e-08) and B cells (partial.cor = 0.125, p = 3.50e-01), with markers of T cell activation and antigen presentation.

Conclusions

High expression of CD32b is associated with worse prognosis in HER2+ B luminal BC. CD32b is a target for the future development of novel therapeutics.

Legal entity responsible for the study

The authors.

Funding

Cellex Foundation Institutional grant: Research facilities and equipment La Caixa Foundation Institutional grant: LCF/PR/CEO7/50610001.

Disclosure

All authors have declared no conflicts of interest.

Background

One of the most important prognostic factors that predict survival, and used for guiding the treatment plan in TNBC; is the axillary lymph nodes status. Recent studies have shown the impact of lymph node ratio (LNR) and number of negative lymph nodes (NLNs) on the prognosis of breast cancer patients besides, positive nodal affection (PLN). In our study, we try to deeply investigate the impact of LNR and the NLNs on both DFS and OS in TNBC patients as independent novel prognostic factors.

Methods

92 female patients TNBC, in Clinical Oncology department, Assuit University; Egypt. Older than 18 years, underwent (MRM) or (BCS) with axillary dissection from 2014 - 2019, they FU till 2021. LNR is defined as the number of positive lymph nodes divided by the total number of lymph nodes removed. SPSS version 26.0 used for descriptive statistics, Weka package tool used to discretize cut off value for LNR, NLNs. A multinomial logistic regression is constructed to ensure the ability of those discretized variables to predict survival. Primary end point was evaluating the relation between LNR and NLNs with DFS and OS & the secondary was a trial to estimate the number of NLNs that can be used as guidance to predict survival outcomes.

Results

Of 92 patients, The mean age was 49.45± SD11.75 & the median number of total LN removed was 12.3 ±SD 3.689, and, the median number of the PLN was 5±SD 4.751. MRM was the commonest done in 72 (78.3%). The most common T &N stage were: T2 in 59 (64.13%) & N2 in 27 (29.3%). Person correlation was significantly positive between DFS and NLNs (P = 0.001), while significantly negative LNR (P=0.002). The best LNR cut-off value by WEKA test was 0.19, while NLDs was 9. We found that Patients having LNR ≤ 0.19 or those with NLNs > 9 had longer DFS >24m (52%) (58%) respectively compared to those LNR >0.19 or NLNs ≤ 9. In addition, a multinominal regression test used to validate quality of them to predict DFI, concluding that having LNR≤ 0.19 and NLNs ≤ 9 associated with increase survival with P= 0.008 & 0.004 respectively. Regarding OS, it was found that both significantly affect OS (p = 0.006, 0.032) respectively.

Conclusions

New independent prognostic factors in TNBC are LNR and NLNs . Further multicentric researches are needed to standardize it, and a trial to be included in an international precision scoring model for survival.

Legal entity responsible for the study

Assuit Clinical Oncology Department, Faculty of Medicine.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Research on ovarian cancer (OC) is considerably neglected in Morocco. OVANORDEST (“OVAire dans le NORD-EST” (ovarian cancer in North-East of Morocco)) is a three-steps research project that aims to develop clinical research on this aggressive women's cancer for the first time in Morocco. This abstract is a biomarker analysis of affordable predictive and prognostic biomarkers based on systemic inflammatory response previously described in the hallmarks of cancer. This includes lymphocyte to monocyte (LMR), neutrophil to lymphocyte (NLR), and platelet to lymphocyte ratios (PLR) as available biomarkers for under-resourced settings to predict outcomes in OC.

Methods

We have first appraised all available meta-analyses that investigated the predictive value of these three biomarkers for overall survival using an umbrella systematic review (El Bairi et al. Front Oncol, 2021; PROSPERO: CRD42020201493). We further developed a real-world cohort of OC patients at the Mohammed VI University Hospital during the period 2005-2020. ROC (receiver operating characteristic) and Cox regression models were used to study the predictive impact of selected biomarkers.

Results

This retrospective study enrolled 250 cases of histologically confirmed epithelial OC with available data on the three biomarkers used. On univariable analysis, LMR, NLR, and PLR were all associated with overall survival (p <0.001). which was confirmed on multivariable analysis. In fact, patients with an LMR>3 had higher median overall survival (mOS) (31 vs. 23 months) (HR= 1.62; 95% CI: 1.00-2.6; p = 0.04) and those with an NLR<1.6 had also superior mOS (50 vs. 20 months) (HR= 2.04; 95% CI: 1.25-3.35; p = 0.0043). Similarly, OC patients with a PLR <134.2 had favourable mOS (49 vs.21 months; HR= 1.66; 95% CI: 1.07-2.58; p = 0.02).

Conclusions

These encouraging results on the prognostic value of peripheral immune response on OS were supportive of OVANORDEST-2 that will validate these findings using a prospective study to develop a prognostic nomogram.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

To determine the p53 gene suppressor and bcl-2 oncoprotein forecast role in non-epithelial ovarian tumor prognosis among child and adolescent patients.

Methods

Our search is based on immunohistochemical method results of 30 patients with non-epithelial ovarian tumors at I-IV stages, which were diagnosed and treated in oncogynecology and children oncology departments of national oncologic science center of Ministry of Public Health of Republic of Uzbekistan from 2010 till 2021.

Results

The analysis of immunohistochemical method results shows 6(20%) patients among which had been marked high expression bcl-2 oncoprotein, 9(30%) moderate express, 15(50%) low express. Analysis of p53 gene suppressor results shows 8(26,6%) patients among which had been marked high expression, 12(40%) moderate express, 10(33,3%) low express. The group of patients which high expression of p53 gene suppressor and bcl-2 oncoprotein took aggressive course of chemotherapy of scheme PVB or BEP (from 6 till 8 courses). The group patients of moderate and low expression of p53 gene suppressor and bcl-2 oncoprotein took moderately aggressive course of chemotherapy of scheme BEP or EP (from 4 till 6 courses). The high correlation between bcl-2 expression level increases and fast tumor growth, and therefore incurability of oncologic process among of patients had been revealed. Also the high probability of tumor recurrence was noticed. In the high-positive p53 gene suppressor rate group took place aggressive current of tumor process and these patients had early recurrence and metastases, which demanded recurrent aggressive chemotherapy.

Conclusions

p53 gene suppressor and bcl-2 oncoprotein expression in non-epithelial ovarian tumors among the child and adolescent patients is characterized with high and low rates, which enables to use this rate determination for given pathology currency prognosis identification.

Legal entity responsible for the study

The author.

Funding

Has not received any funding.

Disclosure

The author has declared no conflicts of interest.

Background

Leptomeningeal metastases (LM) have become increasingly common in NSCLC patients who harbor epidermal growth factor receptor (EGFR) mutation treated with EGFR-TKI and are correlated with inferior prognosis. Evidence in prior research demonstrated that EGFR amplification was more likely presented in advanced clinical stages and was associated with worse survival. However, whether the EGFR amplification is a prognostic marker in NSCLC-LM is still inconclusive.

Methods

This study enrolled patients diagnosed with NSCLC-LM from June 2019 to September 2021, who had received previous EGFR-TKI at Guangdong Sanjiu Brain Hospital. Cerebrospinal fluid (CSF) samples were collected and subjected to targeted next-generation sequencing (NGS) of 168 cancer-related genes. Clinical characteristics and overall survival (OS) were compared in patients with and without EGFR amplification.

Results

This study enrolled 53 NSCLC-LM patients, all of whom had EGFR mutations. TP53 and EGFR amplification are the two most frequent mutations in the study cohort, presenting 72% (38 of 53) and 40% (21 of 53), respectively. The rate of EGFR amplification was much higher at the time of leptomeningeal progression than at initial diagnosis (p<0.01). Karnoskfy performance status was poorer (p=0.021), and CSF pressure was higher (p=0.0067) in patients with than without EGFR amplification. A multivariable Cox proportional hazard regression model showed that EGFR amplification was an independent prognostic factor for poorer OS (8.3 vs 15 months; p=0.017). Median OS was shorter in NSCLC-LM patients with mutated than wild-type TP53, but the differences was not statistically significant (10 vs 17.3 months, p=0.184).

Conclusions

EGFR gene amplification could be a potential resistance mechanism to EGFR-TKI failure in NSCLC-LM and is associated with inferior clinical outcomes.

Legal entity responsible for the study

C. Zhou.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

The identification of patients most likely to benefit from ICI therapy - the so-called responders - has so far relied mainly on clinical trial data. The current methods to assess the risk-benefit of ICI treatments for individual patients are based on the Programmed Death Ligand 1 (PD-L1) expression level, which can be used to aid the clinician´s decision. BiomeOne® is a non-invasive, stool microbiome-based in vitro diagnostic test. Our aim was to compare the performance of BiomeOne® and PD-L1 in a cohort of NSCLC patients.

Methods

Stool samples from 38 stage III/IV NSCLC cancer patients (22 males, 16 females, 64.06 ± 9.37 years old) were taken prior to ICI treatment initiation with an at-home sampling collection kit (Norgen Biotek). Amplicon sequencing and bioinformatic analysis was performed, followed by BiomeOne® analysis, resulting in an output probability of response. Positive PD-L1 tests were all those > 1%. Best clinical response was assessed at the end of first line therapy. Specificity and sensitivity of each test was calculated for this cohort and used to assess the accuracy of each diagnostic test.

Results

Overall, the PD-L1 assay exhibited a sensitivity of 67.9% (95% confidence interval (CI) 50.6, 85.1) and a specificity of 50.0% (95% CI 19.0, 80.1) in predicting the response to ICI therapy. The positive predictive value (PPV) was 79.2% (95% CI 62.9, 95.4), while the negative predictive value (NPV) was 35.7% (95% CI 10.6, 60.8). The microbiome-based biomarker had an overall sensitivity of 78.6% (95% CI 63.3, 93.8) and a specificity of 50.0% (95% CI 19.0, 80.1). The PPV of BiomeOne® was 81.5% (95% CI 66.8, 96.1), while the NPV was 45.5% (95% CI 16.0, 74.9).

Conclusions

A microbiome-based biomarker (BiomeOne®) appears to have a higher sensitivity and PPV than the standard immunohistochemistry PD-L1 tests and may thus serve as an important companion diagnostic in the management of patients receiving immunotherapy.

Legal entity responsible for the study

The authors.

Funding

Biome Diagnostics GmbH.

Disclosure

B. Sladek, N. Gasche: Non-Financial Interests, Personal, Ownership Interest: Biome Diagnostics A. Valipour: Non-Financial Interests, Personal, Advisory Board: Biome Diagnostics. All other authors have declared no conflicts of interest.

Background

KRAS in one of the most frequently altered oncogenes in NSCLC. The prognostic role of the KRAS mutations in early-stage NSCLC is still unclear. In this study we aim to analyze the overall survival (OS) and the disease free survival (DFS) of patients with early stage NSCLC underwent lobectomy and systematic lymphadenectomy.

Methods

Patients who underwent lobectomy and systematic lymphadenectomy for clinical stage I-II lung adenocarcinoma and next-generation sequencing (NGS) were evaluated. Exclusion criteria were the neoadjuvant treatment, an incomplete resection and the presence of EGFR mutations. Mutations were classified as KRAS^wild-type, KRAS^G12C, or KRAS^other.

Results

A total of 257 patients were included in the study (Table). The KRAS mutation status was: 161 KRAS^{wild type}, 42 KRAS^g12c and 54 KRAS^other. All the patients reported a resection of at least 4 nodal stations and the median of resected lymph nodes was 16 (11-36). After pathological examination 200 patients confirmed the N0, 30 patients was N1 and the remainder N2. Adjuvant therapy was administered to 25 patients (9.7%). The KRAS^g12c group showed a non-significant worst 3-year OS compared to KRAS^other group (p=0.2). OS was not statistically significant different between patients with any mutation and patient with KRAS^{wild type}. However, the 3-year DFS of the KRAS^g12c group was substantially worse compared to the KRAS^other group (62.5% vs 74.3%; p=0.04). Table: 56P

Conclusions

Our results showed that in a homogeneous cohort of stage I-III lung adenocarcinoma after radical surgery, the group of KRAS^g12c mutation had a worst DFS compared to the KRAS^{wild type} and KRAS^other. without differences in terms of OS.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Head and neck squamous cell carcinoma (HNSCC) is a cancer which arises from oropharyngeal tissues. The endothelial growth factor receptor (EGFR) over expression promotes tumour aggressiveness, while CD8+ T cells have cytotoxic activity against tumour. In the present study, we hypothesized that prognostic impact of EGFR over expression may depend on enrichment of CD8+ T cells within the tumour micro-environment (TME) in patients with HNSCC.

Methods

Gene expression data of patients with HNSCC were selected from web-based gene survival analyzer Kaplan Meier Plotter (KM plotter) to demonstrate association between EGFR expression and long-term outcomes for increased and decreased amounts of CD8+ T cells in their TME. The 5-year overall survival rate was calculated in study cohorts which stratified by optimal threshold of EGFR expression level.

Results

A total of 445 patients with HNSCC were selected from an online KM plotter database and patients with increased and decreased amounts of CD8+ T cells in their TME were 267 and 178, respectively. The EGFR over expression was significantly associated with increased risk of mortality in both patients with increased (HR=1.62, 95% CI 1.12-2.35, p=0.010) and decreased (HR=1.71, 95% CI 1.08-2.70, p=0.021) amounts of CD8+ T cells in their TME. Median survival time and overall survival rate was significantly lower in patients with decreased amounts of CD8+ T cell in their TME (38 months for low expression cohort vs. 19 months for high expression cohort, p=0.021) (Figure 1A) than patients with increased amount of CD8+ T cells in their TME (56 months for low expression cohort vs. 31 months for high expression cohort, p=0.010).

Conclusions

Patients with HNSCC who had decreased amounts of CD8+ T cells in their TME have worse outcomes compared to their counterparts who had rich CD8+ T cells in their TME regardless of EGFR over expression. Therefore, concomitant immunotherapy using immune checkpoint inhibitors additional to EGFR targeted therapy may be beneficial for this high risk population.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Oral squamous cell carcinoma (OSCC) is one of the highest-ranking cancers among both genders in Pakistan. Obesity is linked to a much higher risk for developing multiple cancer types. Individuals with diabetes mellitus (DM) face an increased risk for developing oral cancer. Hence, the objective of this study was to identify the effect of obesity and DM on the prognosis of OSCC patients.

Methods

This retrospective cohort analysis was conducted on 193 patients diagnosed and treated for OSCC at Aga Khan University Hospital, Karachi, Pakistan. Patient information was obtained from hospital medical records. Obesity was defined as having a body-mass-index (BMI) of ≥25 kg/m² according to the WHO Asian cut-offs for BMI. Patient BMI was correlated with diabetes status, clinicopathological features and overall survival. Kaplan-Meier survival analysis was performed, along with univariate and multivariate cox regression analysis to test the effect of obesity and diabetes on overall survival.

Results

In a set of 193 patients, there were 148 males (76.7%) and 45 females (23.3%). The mean BMI was 24.4 (SD±5.25) and 42.7% of patients were found to be obese (≥25 BMI). 32 patients (16.6%) were diabetic. The risk of death was significantly higher in underweight patients (P=0.035) as compared to normal weight individuals. Diabetics had a higher mean BMI as compared to non-diabetics. However, DM was not a statistically valid predictor of survival.

Conclusions

Underweight OSCC patients were at a higher risk of death as compared to normal weight OSCC patients.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Worldwide, head and neck carcinomas represent the seventh most common cancer, with known risk factors such as alcohol consumption, use of various tobacco-based products, and infection with high-risk human papillomavirus (HR-HPV). Association with HPV infection has already been proven to represent an indicator of a better outcome for patients with oropharyngeal squamous cell carcinomas (SCC) only. This paper aimed to highlight the prognostic value of the HPV status in all head and neck squamous cell carcinomas (SCC), independent of the location, evaluated using both immunohistochemical (IHC) and molecular techniques, and to assess the p16 marker (clone 16P04, JC2) value for predicting HPV.

Methods

In this paper, 114 consecutive head and neck SCC cases were included. The association of HPV infection in these carcinomas was based on the IHC expression of p16 (clone 16P04, JC2) and PCR-based determination of HR-HPV, according to the indications of the 8^th edition of the American Joint Commission on Cancer and in cases with an inconclusive or equivocal IHC expression of p16.

Results

There were 20 females and 94 males with ages ranging from 32 to 86 years old. Most of the tumors were p16 negative (88.60%) and there were no statistically significant differences between HPV status and age (p=0.5714), gender (p=0.6973), or pT stage (p=0.5452). Kaplan-Meyer curves revealed a significant difference between the overall survival (OS) of the p16 positive SCC versus p16 negative cases (p=0.0472), independently of their location.

Conclusions

Although HPV status has a well-proved prognostic impact on oropharyngeal SCC, p16 expression should be evaluated for all head and neck SCC, because it seems to be an outcome predictor, independently of the location or stage. When IHC expression of p16 is inconclusive or equivocal, the HPV status should be established by PCR-based determination.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Oral squamous cell carcinoma (OSCC) remains the leading cause of cancer-related deaths worldwide. Despite extensive research on OSCC, the pathogenesis of OSCC is not fully understood. ALKBH1 is a mammalian RNA demethylase responsible for the demethylation of N6-methyladenosine (m6A) in RNA and numerous recent studies reported that ALKBH1 plays an important role in tumorigenesis and metastasis, but the role of ALKBH1 in OSCC is largely unknown.

Methods

In this study, we investigated the genetic alterations of the ALKBH1 gene in HNSCC and their association with clinicopathological features, including survival using openly available data from The Cancer Genome Atlas (TCGA). In addition, we have sequenced the complete ALKBH1 gene in 126 OSCC patients.

Results

We found that the genetic alterations of the ALKBH1 gene were significantly associated with clinicopathological features as well as TP53 mutation. Patients with mutation of the ALKBH1 gene had worse overall survival (OS). Truncating mutation of ALKBH1 gene predicted poor OS of OSCC patients. In addition, we identified several ALKBH1 missense mutations, including novel mutations in OSCC patients, and in silico analyses indicated that these mutations could be potentially pathogenic. Moreover, mutations in the ALKBH1 gene were also associated with nodal metastasis.

Conclusions

Our results suggest that ALKBH1 alterations were significantly associated with poor prognosis and may represent a new marker of prognosis in OSCC.

Legal entity responsible for the study

The authors.

Funding

Indian Council of Medical Research (Grant No. DHR-GIA, 2020-9530).

Disclosure

All authors have declared no conflicts of interest.

Background

Glioblastoma (GBM) is one of the deadliest cranial tumors occurring in adults. Various biomarkers have been tested for their significance in diagnosis, prognosis, and treatment of GBM. Some well-studied markers in GBM are Isocitrate dehydrogenase1 (IDH1), Murine double minute2 (MDM2), Epidermal Growth Factor Receptor (EGFR) and p53. The aim of this study was to investigate the protein expression of these markers in GBM patients of Pakistan.

Methods

A total of 51 surgically resected formalin-fixed paraffin-embedded specimens from patients diagnosed and treated at Aga Khan University Hospital were included in this study. Immunohistochemistry (IHC) for IDH1, MDM2, EGFR and p53 was performed using Dako EnVision System and respective monoclonal antibodies. Survival analysis was performed to check association of markers protein expression with prognosis in GBM patients.

Results

There were 36 males and 15 females in this study, with a median age of 48 at the time diagnosis. Overexpression of molecular markers was as follows: 55% for EGFR, 22% for p53, 79% for IDH1 and 85% for MDM2. We did observe that EGFR was significantly associated with increased age of our patients and with worse survival. Age >40 years was a predictor for worse prognosis as well.

Conclusions

EGFR overexpression and advanced age were worse prognostic indicators.

Legal entity responsible for the study

The authors.

Funding

Seed Money Grant, Aga Khan University, Karachi, Pakistan.

Disclosure

All authors have declared no conflicts of interest.

Background

Glioma is the most common primary tumor of the human central nervous system. It is characterized by fast growth, strong invasion and impossible to complete surgical resection. Despite advances in tumor diagnosis and treatment, including surgery, radiotherapy and chemotherapy, the median survival for WHO Grade II gliomas is 5 years, for WHO Grade III gliomas is 2-3 years, and for WHO Grade IV gliomas is 14.6 months. The study of glioma cell formation mechanism is of great significance for the treatment of glioma. S100A12 expression was found to be highly correlated with glioma progression. Hax-1 protein has a variety of biological functions, such as anti-apoptosis, regulation of cell migration and endocytosis, and participates in invasion, metastasis and tumorigenesis in different types of tumors. However, there are few studies on the regulatory mechanisms of S100A12 and HAX-1 in glioma cells, and their mechanisms of action have not been fully analyzed.

Methods

To explore the effect and mechanism of S100A12 and HAX-1 genes on proliferation, invasion and migration of glioma cells and Lay the foundation for the study of glioma treatment. In this study, we carried out immunohistochemical investigation of S100A12 and HAX-1 in 81 glioma tissues to determine their expressions in glioma cells, and evaluate the clinical significance of S100A12 and HAX-1 in glioma patients. Futher we knockdown the S100A12 and HAX-1 by shRNA, and evaluated cell proliferation, cell migration and cell apoptosis by MTT, colony formation assay, transwell assay, flow cytometry assay and western blot.

Results

We found that S100A12 and HAX-1 were upregulated in tissues of glioma patients and the expressions were correlated to WHO stage. Further, we found that knockdown S100A12 inhibits the proliferation, migration and invasion of glioma cells through regulating cell apoptosis and EMT.

Conclusions

Both S100A12 and HAX-1 play vital roles in glioma progression, and may be important regulatory molecules for biological behaviors of glioma cell lines.

Legal entity responsible for the study

Southern University of Science and Technology Hospital.

Funding

Has not received any funding.

Disclosure

The author has declared no conflicts of interest.

Background

Perioperative FLOT chemotherapy has improved the prognosis in patients with locally advanced resectable gastric cancer (GC). However, in 80% of cases, the tumor is resistant to the therapy, and the patients are exposed to unnecessary toxicity and delayed surgical treatment. The aim of this study was to identify molecular predictive markers of efficacy of perioperative FLOT chemotherapy in patients with locally advanced resectable GC.

Methods

The retrospective study included 185 patients. All tumor samples were assessed for HER2 and microsatellite instability (MSI) status. Among all cases there were 45 patients with locally advanced T_2-4N_1-2 M₀ GC, who underwent a total or subtotal gastrectomy with D2 lymphadenectomy and perioperative chemotherapy with FLOT. MSI detection was performed using mononucleotide markers (NR21, NR24, NR27, BAT25, BAT26) by fragment analysis. HER2 gene amplification testing was performed using fluorescent in situ hybridization. Also 19 patients were tested for copy number changes of the FGFR1, FGFR2, KRAS, MET, EGFR, CCND1, MYC genes using multiplex-ligation probe amplification analysis. The endpoints were progression-free survival (PFS) and objective response rate (ORR).

Results

MSI was detected in 4.8% (9/185) of GC cases. Prevalence of HER2 amplification was 7.5% (14/185). PFS in patients with HER2-positive GC, receiving perioperative chemotherapy with FLOT (4/45), was significantly lower than in patients with HER2-negative GC: the median was 156 and 317 days, respectively (p=0.0006). There was no correlation between the alteration and objective response (OR) (p=1.0). PFS in GC patients with KRAS amplification (3/19) was significantly lower comparing to patients without KRAS amplification: the median was 98 and 327 days, respectively (p<0.0001). There was no association between an increase in KRAS copy number and ORR (p=1.0). For MSI and other studied markers no statistically significant correlation with PFS and ORR was found (p>0.05).

Conclusions

The presence of HER2 and KRAS amplification have been shown as promising predictive markers of the treatment failure in patients treated with perioperative FLOT chemotherapy for locally advanced resectable GC.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Gastroesophageal adenocarcinoma (GEA) treatment has been revolutionized by the introduction of immune checkpoint inhibitors (CPIs) that, in combination with platinum-based chemotherapy as first line, have improved both progression-free survival (PFS) and overall survival (OS). However, there is an urgent need for the identification of predictive biomarkers of response, particularly for patients with a combined positive score (CPS)<5, where the benefit deriving form CPIs is less clear. We propose a novel immune classification for advanced GEA which in combination with CPS could improve patient selection for precision immunotherapy.

Methods

31 paraffin-embedded GEA tumor samples available for molecular analyses from metastatic, CPS<5 and chemotherapy naïve patients were collected from January 2004 to January 2019. A transcriptomic analysis using nCounter PanCancer Immune Profiling panel (Nanostring) was performed to study tumor microenvironment features. To validate the results, 23 fresh GEA biopsies were prospectively collected from January 2020 to September 2021 and characterized with RNA-seq analysis.

Results

Both Nanostring® and RNA-seq transcriptomic data were studied. We analyzed the cell types composing the immune infiltrate and their functional activity. This integrative study allowed the identification of four different groups across our patients. Among them, we identified a subgroup of patients whose tumors presented an enriched immune infiltrate (including dendritic cells, B cells, CD8+ T cells and macrophages) showing high immune function activity. Interestingly, these tumors expressed the most gene signatures related to immunomodulatory pathways –composed of genes such as PD-L1, PD-1, CTLA-4, TIGIT and LAG3– and immunotherapy response, highlighting they could potentially achieve the best benefit from CPIs, despite presenting CPS<5.

Conclusions

Our work allows the identification of an immune-enriched subtype of advanced GEA, characterized by high immune cell activity, which could potentially help achieve a more precise immunotherapy approach. Further validations are awaited.

Legal entity responsible for the study

The authors.

Funding

This work was supported by grants from the Carlos III Health Institute [grant number PI18/01508 and PI21/0693] to TF, [grant number PI18/01909 and PI21/00689] to AC and NT and [grant number PI21/00695] to CM. MCS was supported with a predoctoral grant by the Spanish Association against Cancer (AECC, Valencia-2019). VG was funded by a Joan Rodés contract JR21/00042 from the Carlos III Health Institute; FGV was founded by a post doc grant by Generalitat Valenciana Grant APOSTD/2021/168; NT was funded by Joan Rodés contract JR20/00005 from the Carlos III Health Institute. DR was funded by Joan Rodés contract JR16/00040 from the Carlos III Health Institute. JC was granted with a VLC Bioclinic grant 2021/257 from the University of Valencia. SZT was granted by a grant CA18/00042 contract for bioinformaticians from Carlos III. TF was supported by Joan Rodés contract JR17/00026 from the Carlos III Health Institute.

Disclosure

A. Cervantes: Financial Interests, Institutional, Advisory Board: Merck Serono, Amgen, Roche, Transgene, AnHeart Therapeutics; Financial Interests, Institutional, Invited Speaker: Amgen, Roche, Merck Serono, Foundation Medicine; Financial Interests, Personal, Other, Associate Editor: Annals of Oncology, ESMO Open; Financial Interests, Personal, Other, Editor: Cancer Treatment Reviews; Financial Interests, Institutional, Research Grant, Principal Investigator: Actuate Therapeutic, Amgen, Astellas Pharma, Beigene, Bayer, AstraZeneca, BMS, Amcure, FibroGen, Lilly, Genentech, MedImmune, Merck Serono, Novartis, Natera, MSD, Servier, Sierra Oncology, Adaptimmune, Takeda; Non-Financial Interests, Personal, Other, General and Scientific Director: INCLIVA Biomedical Research Institute. All other authors have declared no conflicts of interest.

Background

Neoadjuvant chemotherapy followed by surgery has been recommended for esophageal cancer (EC) patients. Though improved survival is obtained in patients with pathological complete response after multimodal therapy, still many patients do not respond to the therapy and show increased therapy induced complications. Therefore, identifying non-responders before starting the treatment is warranted to avoid unnecessary drug exposure. So far, no reliable biomarkers are available for prediction of response to neoadjuvant therapy in EC patients. We hypothesized that differential miRNA profiles may be exhibited by the EC patients who respond to neoadjuvant therapy versus non-responders. The present study aims to identify differentially expressed (DE) miRNAs that might have potential as biomarkers for predicting response to neoadjuvant therapy in EC.

Methods

Next generation sequencing (NGS) was performed to compare miRNA profiles of EC patients who respond to neoadjuvant therapy versus non-responder. Identification of targets of DE miRNAs followed by Ingenuity Pathway Analysis (IPA) was carried out to identify enriched pathways. Further, expression of 2 downregulated miRNAs, miR-335 and miR-107 and one upregulated miRNA, miR-99a was evaluated by qRT-PCR in clinical samples and their association with response was assessed. Next, we modulated the expression of miR-335 in esophageal cancer cell lines and its effect on EMT and PI3K signalling and cisplatin resistance was assessed.

Results

Out of 76 significantly DE miRNAs, 21 miRNAs were upregulated and 55 were down regulated. Ingenuity Pathway analysis indicated the significant enrichment of Xenobiotic metabolism, EMT signalling and PI3K/AKT signalling pathways in responders. Further, ectopic expression of miR-335 in EC cells led to decrease in EMT and PI3K signalling as well as resistance towards cisplatin in these cells.

Conclusions

The present study suggests association of miR-335, miR-107 and miR-99a with response to therapy in EC patients, however validation in larger cohorts is warranted.

Legal entity responsible for the study

The authors.

Funding

Indian Council of Medical Research, Govt. of India.

Disclosure

All authors have declared no conflicts of interest.

Background

Damage specific DNA binding protein 2 (DDB2) is originally implied in the recognition of ultraviolet-induced DNA damage and the initiation of nucleotide excision repair (NER) process. This protein also demonstrated dual roles in several cancers, acting either as an oncogene or a tumor suppressor gene depending on cancer localization. In this study, we investigated the unresolved role of DDB2 in pancreatic ductal adenocarcinoma (PDAC).

Methods

The expression level of DDB2 in pancreatic cancer tissues and its correlation with patient survival were evaluated using publicly available data. Two PDAC cell models with CRISPR-modified DDB2 expression were developed: DDB2 was repressed in DDB2-high T3M4 cells (T3M4 KO) while DDB2 was overexpressed in DDB2-low Capan-2 cells (Capan-2 ACT). Immunofluorescence and qPCR assays were used to investigate epithelial-to-mesenchymal transition (EMT) in these models. Migration and invasion properties of the cells were also determined using wound healing and transwell assays. Sensitivity to 5-fluorouracil (5-FU), oxaliplatin, irinotecan and gemcitabine were finally investigated by crystal violet assays.

Results

DDB2 expression level was reduced in PDAC tissues compared to normal ones and DDB2-low levels were correlated to shorter disease-free survival in PDAC patients. DDB2 knockout in T3M4 cells enhanced the transcription of SNAIL, ZEB1 and TWIST EMT transcription factors (EMT-TFs), increased the expression of the N-cadherin mesenchymal marker and decreased the levels of the E-cadherin epithelial marker. Conversely, DDB2 overexpression in Capan-2 cells increased E-cadherin levels and decreased N-cadherin levels. The migration and invasion properties were negatively correlated with DDB2 expression in both cell models. DDB2 has no effect on T3M4 cell sensitivity to chemotherapy but sensitized Capan-2 cells to 5-FU, oxaliplatin and gemcitabine.

Conclusions

Our study highlights the potential tumor-suppressive effects of DDB2 on PDAC progression. DDB2 could thus represent a promising therapeutic target and a prognosis and predictive biomarker in patients with PDAC.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

A 3-lipid signature has been recently proposed to predict for prognosis in pts with mCRPC. This study aimed at assessing the lipidomic profiles of pts with mCRPC to identify new prognostic and predictive biomarkers.

Methods

Plasma samples were collected from pts with mCRPC starting a 1st-line (1L) (n=29) and pretreated with >2 lines (>2L) (n=19). Lipids were extracted and analyzed with an untargeted lipidomic approach. T-test was applied to identify lipids differentially expressed. ROC curves and X-Tile were used to identify lipids’ threshold and to test association with overall survival (OS). Kaplan-Meier curves were constructed and Cox regression was used to adjust for prognostic variables.

Results

We identified and quantified a total of 907 plasma lipids. 68 lipid species were significantly dysregulated in >2L compared to 1L samples. 56 species were upregulated, and 12 were downregulated. >2L pts showed higher levels of acylcarnitine, diacylglycerols, phosphatidylethanolamine, triacylglycerols and ceramides (Cer). We tested the effect on OS of lipids included in the 3-lipid signature: Cer (d18:1/24:1), sphingomyelin (d18:2/16:0) and phosphatidylcholine (16:0/16:0). Only Cer (d18:1/24:1) was associated with OS, but without statistical significance in the multivariate model. Among upregulated lipids in >2L cohort, Cer (d18:1/18:0) (C18), Cer (d18:1/16:0), Cer (d18:2/18:0), Cer (d18:1/24:1) and Cer (d20:1/24:1) all showed proportional relative risk of death and significant association with OS in univariate models. However, only C18 retained significant association with OS after adjustment for basal PSA and line of treatment (HR: 3.26 [95% CI 1.37-7.76]). The association of C18 with OS was consistent in both subgroups 1L and >2L, separately analyzed. Biochemical response was only seen in 4/14 (28.6%) evaluable pts with high levels of C18.

Conclusions

Using a quantitative mass spectrometry approach, we characterized the lipidomic profile of highly pretreated mCRPC pts. We found that C18 is increased in these pts compared to therapy-naïve men, and significantly associated with OS, paving the way for further investigations on its prognostic and predictive value.

Legal entity responsible for the study

Ospedale Policlinico S. Martino.

Funding

Supported by Italian Ministry of Health funds (5×1000 and “Ricerca corrente”) of IRCCS Policlinico S. Martino, Genoa, Italy.

Disclosure

C. Cattrini: Financial Interests, Personal, Advisory Role: Janssen; Financial Interests, Personal, Other: Janssen, MSD. D.J. Pinato: Financial Interests, Personal, Advisory Role: Eisai, Mina Therapeutics, Roche, H3 Biomedicine, Da Volterra, AstraZeneca; Financial Interests, Personal, Speaker’s Bureau: Bayer, Viiv Healthcare, Falk Pharma, Roche; Financial Interests, Personal, Other: Bristol Myers Squibb, Bayer, MSD Oncology, Roche/Genentech, Bristol Meyers Squibb; Financial Interests, Personal, Funding: MSD Oncology, Bristol Meyers Squibb, GlaxoSmithKline. A. Gennari: Financial Interests, Personal, Speaker’s Bureau: Eisai Europe; Financial Interests, Personal, Other: Lilly, Roche, Eisai Europe, Novartis, Daiichi Sankyo Europe GmbH. All other authors have declared no conflicts of interest.

Background

Adequate clinical management of small renal tumors (SRTs, <4 cm) is important despite their slow growth and low metastatic potential. Surgical resection or nephrectomy remains the main treatment strategy. However, up to 30% of SRTs suspicious for malignancy by preoperative imaging reveal to be benign on final pathological examination after surgery. Moreover, most incidental tumors are detected in the elderly with comorbidities unsuitable for surgical treatment. Up to date, tumor growth rate is the only parameter determining aggressiveness of unresectable SRTs The study aimed at selection of methylated DNA biomarkers for non-invasive urine-based active surveillance of patients, diagnosed with SRTs.

Methods

Urine samples were collected at different time points from the 40 patients diagnosed with SRTs (N=163 in total), as well as 92 asymptomatic controls (ASC). Methylated DNA levels of six candidate biomarkers ZNF677, FBN2, PCDH8, TFAP2B, TAC1, and FLRT2 were evaluated by quantitative methylation-specific PCR. Statistical analysis was done by MedCalc, STATISTICA 8, and GraphPad Prism softwares.

Results

Methylated DNA levels of 4 out of 6 genes were significantly higher in the urine samples of SRTs compared to ASC and PCDH8 was characterized by the highest diagnostic potential (AUC = 0.69) with 63% of sensitivity and 74% of specificity. A combination of 2-3 genes showed slightly higher diagnostic values and the panel of PCDH8, TAC1 & FLRT2 (AUC = 0.73) was the most clinically promising. Moreover, methylated PCDH8 level was significantly correlated with tumor volume (Rs – 0.27; P = 0.006) among gradually growing SRTs, showing its potential for non-invasive monitoring of disease progression.

Conclusions

Urinary level of methylated PCDH8 is a promising biomarker for non-invasive diagnosis of SRTs and prognostic indicator for tumor growth kinetics.

Clinical trial identification

This work was funded by the 2014–2020 European Union Structural Funds according to the activity \"Intelligence. Joint science-business projects” grant No. J05-LVPA-K-04–0029.

Legal entity responsible for the study

S. Jarmalaite.

Funding

Thermo Pharma Baltic.

Disclosure

All authors have declared no conflicts of interest.

Background

Renal clear cell carcinoma (RCCC) is the most common subtype of kidney tumors, accounting for the majority of deaths from genitourinary cancers. Due to widespread use of ultrasound imaging, the majority of kidney tumors are detected at an early stage simultaneously resulting the increase of patient overtreatment. On the other hand, up to 30% of patients will develop the distant disease even after surgical tumor excision. Thus, there is a vital need for new, and non-invasive biomarkers providing valuable information about the disease presence, aggressiveness, and prognosis. The present study aimed at developing a gene-specific methylated DNA-based tool for the early non-invasive RCCC diagnosis and follow-up.

Methods

Genome-wide DNA methylation and gene expression profiling in 11 RCCC and paired non-cancerous renal tissues (NRT) was performed using Human DNA Methylation 1×244K and Gene Expression 8×60K Microarrays. Qualitative and quantitative methylation-specific PCR was applied for the validation of DNA methylation changes in 168 renal tissues and 307 urine samples.

Results

Abundant changes in DNA methylation and gene expression were observed in RCCC. Deregulated genes were enriched among biological processes related to tumor development and progression and ten such protein-coding genes were selected for further DNA methylation analysis. Significantly higher methylation frequencies for all genes were found in RCCC tissues compared to NRT (17-60% vs. 0-11%). The best diagnostic performance was demonstrated for a panel of six genes showing 85% sensitivity and 96% specificity. Moreover, higher methylation levels of selected six genes were detected in the urine of RCCC cases as compared to controls and the highest diagnostic power (AUC=0.78) was calculated for the panel of ZNF677 & PCDH8 with 78% sensitivity and 69% specificity. Moreover, the methylated status of ZNF677 & PCDH8 was identified as an independent highly prognostic tool for RCCC patients' overall survival with HR of 12.5.

Conclusions

To sum up, during this study a novel non-invasive and highly promising DNA methylation-based tool was developed for the detection of RCCC and patients‘ follow-up, which after further validation steps has a high potential for translation into clinical practice.

Legal entity responsible for the study

The authors.

Funding

This work was funded by the 2014-2020 European Union Structural Funds according to the activity “Intelligence. Joint science-business projects” grant No. J05-LVPA-K-04–0029.

Disclosure

All authors have declared no conflicts of interest.

Background

The multicenter INFINITY registry investigates biomarker-driven treatment and management of patients with advanced malignancies not eligible for standard therapy options within routine clinical care in Germany. PD-(L)1 antibodies are frequently used in different tumor types and several biomarkers have been proposed to identify patients most likely to benefit. However, there is still a great need to further understand mechanisms of resistance to these immunotherapies. This research project was designed to analyze the genomic tumor profile and immune cell infiltration of patients with clinical benefit from PD-(L)1 antibody treatment compared to those without.

Methods

We identified all patients who received PD-(L)1 antibodies (monotherapy or in combination) from the clinical database. We used a case-control design (clinical benefit versus no-clinical benefit) based on predefined criteria: clinical benefit was defined as treatment duration >182 days and no clinical benefit was defined as treatment duration 22-63 days. 24 tissue samples were retrieved from the INFINITY biobank, 12 samples each from patients with and without clinical benefit. Next generation sequencing was performed at a central Pathology Institute (University Hospital Basel) using the Oncomine Comprehensive Assay Plus panel (Thermo Fisher). If sufficient material was available, additional analyses by Cytometry by time of flight (CyTOF) were performed (University of Basel, Dept. of Biomedicine).

Results

Patient and disease characteristics including clinical outcome data, as well as in depth data on genomic profiling and immune cell infiltration comparing patients with vs without clinical benefit will be presented.

Conclusions

The biobank research project of the INFINITY registry aims to contribute to further understand mechanisms of resistance to PD-(L)1 blockade.

Legal entity responsible for the study

iOMEDICO AG.

Funding

Thermo Fisher Scientific.

Disclosure

P. Jermann: Financial Interests, Personal and Institutional, Invited Speaker: Thermo Fisher Scientific; Non-Financial Interests, Institutional, Research Grant, Reagent sponsoring: Thermo Fisher Scientific. H. Läubli: Non-Financial Interests, Personal and Institutional, Invited Speaker, and research support: Bristol Myers Squibb; Non-Financial Interests, Institutional, Invited Speaker: Merck Sharp & Dohme, Roche; Non-Financial Interests, Institutional, Invited Speaker, and research support: Novartis; Financial Interests, Personal, Advisory Board: GlycoEra AG, InterVenn; Financial Interests, Personal and Institutional, Advisory Board: Palleon Pharmaceutical; Non-Financial Interests, Institutional, Writing Engagements: Alector, Novocure; Financial Interests, Personal, Ownership Interest: Singenavir; Financial Interests, Personal and Institutional, Royalties: Memo Therapeutics. B. Kasenda: Financial Interests, Personal, Advisory Board: Astellas, Roche. N.W. Marschner: Financial Interests, Personal, Leadership Role: iOMEDICO; Financial Interests, Personal and Institutional, Advisory Role: MSD, Roche, Servier, Ipsen, Eisai, GSK, Beigene, AstraZeneca, Lilly, Pfizer, Novartis, Pierre Fabre, Seagen; Financial Interests, Personal, Other, Honoraria: MSD, Roche, Servier, Ipsen, Eisai, GSK, Beigene, AstraZeneca, Lilly, Pfizer, Novartis, Pierre Fabre, Seagen; Financial Interests, Institutional, Funding: MSD, Roche, Servier, Ipsen, Eisai, GSK, Beigene, AstraZeneca, Lilly, Pfizer, Novartis, Pierre Fabre, Seagen, Johnson & Johnson, Oncopeptides. All other authors have declared no conflicts of interest.

Background

Mitochondria are abundant organelles containing their own genome and involved in cellular energy production. Their role in various pathological situations such as cardiovascular disease, diabetes, nephropathy, neurodegenerative diseases or cancer has been widely described. Here we tested the role of mitochondrial DNA as a prognostic tool using different matrices: plasma, whole blood and tissue samples from patients with stage IV cancer.

Methods

A two-color panel for crystal digital PCR was designed. The first probe was localized on the mitochondrial NADH-Ubiquinone Oxidoreductase Chain 1 (MT-ND1) gene. The second probe was focused on the centromeric region of chromosome 17 (CEP17). Then, a MT-ND1/CEP17 copy number ratio (MCR) was calculated. Three different matrices were evaluated. 96 plasma samples were obtained from patients with stage IV non small cell lung cancers, 110 whole blood samples from different stage IV cancers (20 (18.2%) lung, 19 (17.3%) colon, 18 (16.4%) prostate, 17 (15.4%) urothelial and pancreatic, 10 (9.1%) gastric and 9 (8.2%) cholangiocarcinoma) and 99 metastatic tissue samples (21 (21.2%) lung, 17 (17.2%) prostate, colon and pancreas, 12 (12.1%) urothelial, 8 (8.1%) cholangiocarcinoma and 7 (7.0%) gastric cancer).

Results

Mean of MCR was variable between matrices: 19.9 (IQR: 9.0-23.5) for plasma samples, 94.5 (IQR: 78.5-108.9) for whole blood and 250.3 (IQR: 146.8-306.5) for metastatic tissues. Of note MCR also varied by metastatic site, with higher mean levels in liver metastases than in lung and lymph node metastases (330.3, 202.5, 170.1 respectively, p-value<0.001). MCR may be a useful prognostic tool for plasma and whole blood matrices, as patients with higher MCR had significantly longer median survival times compared to patients with lower MCR (HR = 3.71, 95% CI = 1.56-8.81, p=0.0029 and HR = 2.14, 95% CI = 1.08-4.25, p=0.029 for plasma and whole blood respectively). As the MCR is metastatic site-dependent, the prognostic value was not observed for tissue samples.

Conclusions

Our study reveals that MCR may be a useful prognostic biomarker for metastatic cancer in plasma or whole blood matrices but not in metastatic tissue.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Genomics has not yet been integrated into the clinical practice of Radiation Oncology. Liquid biopsy is a non-invasive tool based on the study of circulating tumor DNA (ctDNA), mainly applied to advanced cancer patients treated with chemotherapy. This work assesses the utility of liquid biopsy in early-stage/oligometastatic cancer patients undergoing stereotactic body radiation therapy (SBRT).

Methods

Next-generation sequencing (NGS) genomic panels were used to analyse germline, tissue biopsy and liquid biopsy samples from 24 cancer patients with lung lesions undergoing SBRT: 16 lung cancer primaries and 8 lung oligometastases from other primaries. Personalized tumor biomarkers were selected from NGS data for longitudinal ctDNA analysis in serial liquid biopsies collected from the patients during treatment and follow-up.

Results

Genomic panels revealed potential follow-up ctDNA biomarkers for all patients thanks to the integration of the different genomic tests which enabled to determine the origin of the variants identified, essential for successful biomarker selection; 1/3 of the genomic variants identified by the liquid biopsy panel were real tumor variants. Targeted ctDNA analysis showed that ctDNA detection before treatment was significatively associated with progression-free survival (12 months). In addition, 16 patients showed detectable levels of ctDNA during treatment, while no ctDNA signal was detected in 8 cases. In 14/16 ctDNA-positive patients, follow-up ctDNA signal was consistent with clinical status in both good responders and disease-progression cases. Liquid biopsy detected clinical relapse before imaging tests in 3/6 patients with progression. Besides, the genomic panels identified genetic alterations associated with potential targeted therapeutic alternatives (16/24 patients) and hereditary predisposition to cancer (2/24).

Conclusions

Genomics provides complementary and relevant information for patients undergoing SBRT. Liquid biopsy stands as a promising tool for tailoring SBRT treatments and surveillance protocols, as it can provide prognostic and predictive information.

Legal entity responsible for the study

The authors.

Funding

Fundación María Cristina Masaveu Peterson.

Disclosure

All authors have declared no conflicts of interest.

Background

Comprehensive molecular profiling (CMP) using next generation sequencing (NGS) panels is increasingly used to detect clinically relevant alterations and guide treatment decisions in patients with advanced cancers. As the clinical benefit of CMP is not clear, it raises practical and financial questions about the optimal type of panel used.

Methods

Retrospective analysis of NGS reports of patients with advanced solid tumors was performed. Profiling was accomplished using either a FDA-approved test system (FoundationMedicine, 324 genes) or laboratory developed test (LDT) (Solo Complex®, 411 genes). Each genomic alteration was ranked by its clinical actionability using ESMO Scale for Clinical Actionability of molecular Targets (ESCAT). We analyzed frequency of detected alterations, its actionability, tests price difference and rate of matched therapy recommendation.

Results

From 2019 to 2022 NGS reports of 235 patients were analyzed. Median age of patients was 57 years, 59,1% were female, 84,7% have already received at least one line of systemic therapy. There were 42,1% of patients with gastrointestinal tumors, 33,2% - non-small cell lung cancer and 6,8% - breast cancer. FDA-approved test was performed in 193 (82,1%) tumor samples, LDT - in 42 (17,9%) samples. Molecularly matched therapy was recommended to 16,7% and 17,1% respectively. Price of the FDT-approved test was 5800$ when LDT cost about 2300$ (cost difference 3500$). Frequency of alterations ranked as ESCAT tier 1-3 was 39% and 39% for both panels (p=0,8), ranked as tier 1-4 - 78% and 65% for FDA approved panel and LDT respectively (p=0,32). Detailed information on the number of molecular alterations of each tier per report is provided in the table. Table: 73P

Conclusions

Use of LDT showed comparable frequency of clinically relevant alterations detection and rate of molecularly matched therapy recommendations regarding FDA-approved test with lower profiling costs.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Wnt signalling is a critical developmental pathway and associated aberrant signaling drives oncogenesis including colorectal and bone cancer. β-catenin, a central protein forms a range of protein-protein interactions(PPIs), fundamental to driving the transcription of oncogenes inducing proliferation and tumour formation. To further elucidate these interactions, we have sought to i) identify inhibitors of β-catenin PPIs and ii) employ a proximity dependent labelling technique (MiniTurbo) to characterize novel PPIs.

Methods

MiniTurbo-Mutant Biotin ligase BirA is genetically fused to β-catenin and upon addition of Biotin, tagging is induced of proteins within 20nm in as quick as 10 minutes enabling streptavidin pull down and tagging to Mass spectrometry. CTNNB1 gene was successfully inserted into the Miniturbo plasmid construct using SLICE cloning. DH5alpha bacteria were transformed with plasmid and single colony selected with Ampicillin. Following plasmid purification, it was added with Retroviral components for viral transfection and production in HEK293 cells. U2OS adenocarcinoma cells transduced and selected using puromycin. Validated with western blot and immunofluorescence. 24-hour application of MSAB derivatives following induction of Miniturbo with Doxycycline. Streptavidin pulldown was carried out and samples were subjected to Mass Spectrometry following on-bead trypsin digestion. Proteomic analysis was carried out to elucidate novel PPI.

Results

-MSAB derivatives influence levels of β-catenin and Wnt target genes at protein and transcriptional level. -Successful introduction and selection of the miniturbo plasmid into U2OS cells. Western blots and immunofluorescence verification. -Novel Proteomic networks of Beta-catenin protein interactions in U2OS cells. The results illustrate the dynamic PPI network of β-catenin in a 2D cancer model and how those interactions are modulated in the presence of small molecule inhibitors for therapeutic indications.

Conclusions

Targeting β-catenin directly for Oncotherapy has been difficult with very little success. Further characterization and understanding of the dynamic β-catenin interactome may pave an avenue for addressing this challenge.

Legal entity responsible for the study

Ewing and Baud Lab Group/University of Southampton.

Funding

University of Southampton (50%)/Kerkut Trust Charitable organization (50%).

Disclosure

P. Skipp: Financial Interests, Institutional, Ownership Interest: TopMD Precision Medicine Ltd. All other authors have declared no conflicts of interest.

Background

Marine macroalgae Sargassum wightii is abundantly distributed on the southern coasts of Tamil Nadu, India. S. wightii was less explored for its medicinal properties. Although selenium nanoparticles (SeNPs) have been regarded as safer and efficient to enhance various biological functions, the agglomeration of SeNPs hinder its biological applicability. Hence, in the present study, SeNPs was surface functionalized with biologically active compounds present in S. wightii to produce SW-SeNPs which will not only enhance its biological availability but also will synergistically enhance its therapeutic property.

Methods

The biosynthesized SW-SeNPs was characterized using Transmission Electron Microscopy (TEM), Zeta potential analysis, Fourier Transform-Infrared spectroscopy (FT-IR) and X-ray diffraction (XRD). The anticancer effect of SW-SeNPs against A549 cells was determined. Various parameters such as cell viability, reactive oxygen species (ROS) production, DNA fragmentation, cell cycle progression and apoptosis were studied. Also, the level of p53, p-Akt and caspase-3 were determined using flow cytometry.

Results

SW-SeNPs was found to be in the range of 20 - 40 nm size with crystalline nature. The negative zeta potential of SW-SeNPs and FT-IR analysis confirmed the surface binding of bioactive components present in S. wightii in the SW-SeNPs. SW-SeNPs was studied for its anticancer potential against A549 cells. SW-SeNPs affected the A549 cells viability in dose-dependent manner. SW-SeNPs treatment increased the ROS generation and led to DNA fragmentation in A549 cells. Also, the cells were arrested in G2/M phase which ultimately induced apoptosis of the A549 cells through caspase-3 activation. The molecular mechanism of SW-SeNPs-induced A549 cells apoptosis was found to be through overproduced ROS-mediated activation of p53 and AKT signal pathways.

Conclusions

Altogether, the present study demonstrated the anticancer therapeutic effect of SW-SeNPs against lung cancer.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Oncogenic mutation p53 Y220C is the ninth most common mutation of p53 protein. The Y220C mutation creates an expanded surface pocket in the DNA-binding domain, rapidly unfolds and denatures under physiological conditions, which effectively cancels p53 signaling and leads to the development of a tumor. In this work the novel indazole derived compounds were tested. It was found that these molecules bind to the mutated p53 protein and stabilize its structure in vitro.

Methods

In this work the human breast carcinoma (MCF7 p53 wt, MCF7 p53 Y220C, MCF7 p53 -/-) and the human hepatocarcinoma (HUH7 p53 Y220C) cell lines were used. Cell viability was evaluated using colorimetric MTS test. The cells were treated with compounds at the concentration range of 0-200 μM. The obtained data were processed in GraphPad Prism. The expression of p53 target genes upon treatment with the compounds was also quantified using real-time quantitative PCR (RT-qPCR).

Results

Viability of cells incubated with the compounds were determined for cell lines with different p53 status. IC50 values for cells with wild-type p53 status (MCF7 p53 wt) and cells with TP53 knockout (MCF7 p53 -/-) could not be detected in the selected concentration range while in cells with mutated p53 (HUH7 p53 Y220C and MCF7 p53 Y220C) IC50 values were determined in the range of 32-55 μM. Expression of PUMA (BBC3) was found to be upregulated in HUH7 p53 Y220C and MCF7 p53 Y220C cells upon treatment with the compounds.

Conclusions

The results demonstrated that the compounds exert targeted effect on cells carrying p53 Y220C mutation. We established that the compounds selectively reduced the viability of p53 Y220C cell lines and upregulated transcription of p53 target genes associated with apoptosis in p53 Y220C-dependent manner. The work was funded by grant from the Russian Science Foundation 22-24-20034 and supported by the Kazan Federal University Strategic Academic Leadership Program (PRIORITY-2030).

Legal entity responsible for the study

Kazan Federal University.

Funding

The work was funded by grant from the Russian Science Foundation 22-24-20034 and supported by the Kazan Federal University Strategic Academic Leadership Program (PRIORITY-2030).

Disclosure

All authors have declared no conflicts of interest.

Background

The study of the role of oncosuppressors in tumor transformations and in the death of tumor cells is an important aspect in many areas of biochemistry. One of the most well-known tumor suppressors is the p53 protein. The function of the p53 protein is to regulate the expression of genes whose products lead to cell cycle arrest and apoptosis. Many types of cancer are associated with inactivation of the tumor suppressor p53 as a result of mutation. The p53^Y220C oncogenic mutation it creates an extended surface pocket in the DNA-binding domain. Therefore, the development of targeted drugs to modulate p53^Y220C activity is highly relevant. The aim of this work was to evaluate the effectiveness of (1H-pyrrol-1-yl)indazole derivatives as modulators of the activity of the mutant p53^Y220C..

Methods

In this work the following cell lines were used: human breast carcinoma (MCF7 p53^wt, MCF7 p53^-/-, MCF7 p53^Y220C) and human hepatocarcinoma (HUH7 p53^Y220C). Western blot analysis was performed to investigate the effect of indazole derivatives on p53 expression. Immunocytochemical analysis was performed to assess the ability of the studied compounds to induce refolding of the mutant p53^Y220C..

Results

According to the results of Western blot analysis, it was revealed that the compounds lead to an increase in the expression of the p53 protein in cell lines with mutant p53^Y220C. Immunocytochemical analysis of the HUH7 p53^Y220C cell line treated with compounds showed the ability of the studied compounds to induce refolding of the mutant p53^Y220C with the acquisition of a conformation corresponding to the wild-type protein.

Conclusions

Thus, the results showed that indazole derivatives have a specific effect on cells carrying the p53^Y220C mutation and stabilize the mutant protein in a conformation similar to the wild-type protein. The work was funded by the Russian Science Foundation grant 22-24-20034 and supported by the Strategic Academic Leadership Program of the Kazan Federal University (PRIORITY-2030).

Legal entity responsible for the study

Kazan Federal University.

Funding

Russian Science Foundation.

Disclosure

All authors have declared no conflicts of interest.

Background

The tumor suppressor p53 is inactivated by mutation in about half of all tumors, making mutant p53 a prime target for cancer therapy. Missense mutations R248W, R273H and R248Q are included in the five most frequent p53 mutations and in total lead to more than 630 000 new diagnosed cases of tumor diseases in the world every year. These mutations, located at or near the protein-DNA interface, lead to p53 inactivation by loss of direct p53-DNA interactions, and by causing conformational changes in the protein, resulting in lowering its stability. Using small molecule drugs to reactivate mutant p53 is a promising therapeutic approach for treating a wide variety of human malignancies. The aim of this research was to study the biological properties of derivatives of aminobenzothiazole considered as selective small-molecule stabilizers of the p53(R248W), p53(R273H) and p53(R248Q) mutants.

Methods

Mutant recombinant proteins were expressed in E. coli BL21 (DE3) pLysS and purified using affinity and size exclusion chromatography. Determination of the affinity of small molecule compounds for mutant proteins was performed by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). The cytotoxicity of compounds was assessed on cell lines containing mutant p53 using the MTS test. The biological activity of the compounds was studied using cytofluorimetric analysis of the cell cycle and programmed cell death, quantitative real-time PCR analysis of the expression of p53-dependent genes, and immunoblotting of intracellular protein level changes.

Results

Derivatives of aminobenzothiazole were found to stabilize p53 mutants in human cell lines, reactivating p53 transcriptional activity and the production of its target proteins. The compounds were more selective for p53-mutated than p53-wild type cells.

Conclusions

These studies will provide a more detailed understanding of the molecular mechanisms of reactivation of various forms of mutant p53, which, in turn, is of decisive importance in the development of new personalized anticancer drugs. The study was funded by RSF grant 22-24-20034 and strategically supported by Kazan Federal University Strategic Academic Leadership Program (PRIORITY-2030).

Legal entity responsible for the study

Kazan Federal University.

Funding

Russian Science Foundation.

Disclosure

All authors have declared no conflicts of interest.

Background

Network topology is a common tool in systems biology. The focus of our work were disordered proteins whose importance in signaling have long been discussed. A good tool for studying these regulatory proteins is to study triangles i.e., three-nodal signaling motifs where each node is related to the other two, which infers a closer connection between the nodes. Our research shows that oncotherapeutical targets and disordered proteins often form three-nodal motifs together. Our aim was to analyse of these motifs for better understanding the complex relationship of these proteins with oncotherapeutical targets.

Methods

The directed edges of three networks were extracted, and motifs were identified using the FANMOD program. For network analysis, Cytoscape and the Python NetworkX package were used. Then, disordered proteins were identified using the DisProt database, and CIViCmine were used for identifying biomarkers. Then, binary classification models were developed.

Results

We have shown that disordered proteins were more frequently in triangles and in regulatory motifs like unbalanced triangles and cycles. They form common motifs with known oncotherapeutical targets, where many disordered proteins are predictive biomarkers for the target (Human Cancer Signaling Network: 23%). Certain topological parameters are associated with predictive properties. Thus, we developed a machine learning model based on network topological data and biological characteristics of 110 target-disordered protein pairs, whose predictive properties couldbe identified from the literature . With this model we are able tocan predict potential predictive biomarker properties for the other identified 694 pairs in our networks.

Conclusions

We show based on network topology analysis that intrinsically disordered proteins have great potential as predictive biomarkers. Our results imply that investigation into these motifs may help to identify novel predictive biomarkers, which can be further studied individually and validated experimentally. As predictive biomarkers are vital for therapeutical decision making, developing a tool for predictive biomarker identification may have effect on the daily clinical practice.

Legal entity responsible for the study

The authors.

Funding

EFOP-3.6.3-VEKOP-16-2017-00009.

Disclosure

All authors have declared no conflicts of interest.

Background

The Kirsten rat sarcoma viral oncogene homolog (KRAS) belongs to the Ras protein family of small Guanosine-Triphosphate hydrolases (GTPases) that favors the activation of the mitogen-activated protein kinases (MAPK) pathway involved in cell survival and proliferation. KRAS mutations (mutKRAS) can initiate and maintain cancer progression, they are present in solid tumors (lung, colon and pancreas) and treatments remain difficult. The transcriptional co-activator (YAP1) and the transcription factor family Transcriptional enhanced associate domain 1-4 (TEAD1-4) are downstream effectors of the Hippo-YAP1 pathway that regulates cell proliferation, cell survival and cell migration. Several studies highlight the role of the Hippo-YAP1 pathway as bypass mechanism to MAPK pathway inactivation that leads to drug resistance. Here we investigate the effect of the dual inhibition of MAPK and Hippo-YAP1 signaling in cell lines driven by mutant KRAS.

Methods

We have looked at cell viability (CellTiter-Glo (CTG) assay), cell cycle progression (FUCCI system, FACS) and gene transcription (RNA-Seq) before and after the inhibition of either pathway alone or in combination.

Results

We observed that the dual inhibition of both pathways was beneficial for some of the tumor cell line tested. Cell lines responding to the dual inhibition of both pathways generally showed a strong cell growth inhibition effect. Mechanistically, we detected a dual cell cycle arrest at G0/G1 phase while decreasing G2/M phase (FACS analysis). These phenotypes were complemented by downregulated MYC and E2F target gene signatures (RNA-Seq) and reduced the abundance of Spindle Assembly Checkpoint (SAC) components (observed by proteomics and confirmed with capillarity).

Conclusions

In conclusion, the dual inhibition of MAPK and Hippo-YAP1 pathways can be beneficial for a subset of KRAS mutated cell lines. Further analyses will be required to better clarify the genetic and molecular context of this specific phenotypic response. Our data support the potential therapeutical impact of YAP1 inhibition for KRAS cancer patients. Key words: Hippo-YAP1, cancer resistance, mutKRAS, dual cell cycle arrest.

Legal entity responsible for the study

Sanofi.

Funding

Sanofi.

Disclosure

S.L. Tammaccaro, P. Prigent, J.C. Le Bail, O. Dos Santos, L. Dassencourt, P. Picard-Vernier, M. Eskandar, A. Buzy, J-C. Guillemot, Y. Veeranagouda, M. Didier, E. Spanakis, T. Kanno, M. Cesaroni, S. Mathieu, L. Canard, A.H. Casse, L. Debussche, J. Moll, I. Valtingojer: Financial Interests, Personal, Stocks/Shares, Sanofi's employee and/or shareholder: Sanofi.

Background

The search for anticancer therapy targets is the main task of oncology. One hopeful target is a membrane glycoprotein the sodium-dependent phosphate transporter NaPi2b overexpressed in ovarian carcinomas. NaPi2b is an antigen for the MX35 antibody which recognizes an epitope in the large extracellular domain (ECD) of the NaPi2b mostly in tumor but not in normal cells. The ECD contains 4 cysteines at positions 303, 322, 328, 350 and potential N-glycosylation sites at positions 295, 308, 313, 321, 335, 340. We formulated a hypothesis that the MX35 epitope of NaPi2b has a tumor-specific nature which availability for antibodies depends on disulfide bonds and N-glycosylation of the ECD. The aim of this work is to study the role of disulfide bonds and N-glycosylation in the recognition of the MX35 epitope of NaPi2b in intact ovarian cancer cells using confocal fluorescence microscopy.

Methods

We simulated the disulfide bonds reduction or partial deglycosylation of the ECD of NaPi2b by replacing cysteine at positions 303, 322, 328, 350 and asparagine at positions 295, 308, 313, 321, 335, 340 with alanine. Mutant forms were studied by confocal fluorescence microscopy of intact OVCAR8 ovarian cancer cells transfected by corresponding mutant constructs with subsequent assessment of the fluorescence intensity recognition of the mutant forms relative to the wild-type NaPi2b.

Results

The substitution of each cysteine at positions 303, 322, 328, 350 of NaPi2b results in a reduction of the MX35 epitope recognition by monoclonal antibodies. Substitution of the asparagine at position 313 increases the MX35 epitope recognition by monoclonal antibodies.

Conclusions

We showed that the recognition of the MX35 epitope of NaPi2b depends on the ECD conformation due to the presence of disulfide bonds between cysteines at positions 303, 322, 328, 350 and glycosylation of asparagine at position 313. Since malignant transformation of cells is accompanied by changes in the tumor microenvironment, including oxidative stress, we suggest this may lead to a rearrangement of disulfide bonds between cysteines and a change in the ECD conformation when the MX35 epitope becomes more accessible to antibodies in tumor cells, making it a potential tumor-specific epitope.

Legal entity responsible for the study

Research Laboratory \"Biomarker\", Institute of Fundamental Medicine and Biology, Kazan Federal University.

Funding

This work has been supported by Russian Science Foundation, Russia (project no. 20-14-00166) and the Kazan Federal University Strategic Academic Leadership Program (PRIORITY-2030).

Disclosure

All authors have declared no conflicts of interest.

Background

Microvesicles (MVs) are a subset of extracellular vesicles secreted by most body cells. They are being actively studied as cell-free biosimilar drugs. Many ways of inducing MVs are being investigated - natural (passive secretion, cell activation) and stressful (hypoxia, chemical impact, etc.).

Methods

The culture of T-lymphocytes was activated by CD3/CD28 antibodies with subsequent expansion. Some of the cells were reactivated. Cytochalasin B (CHB) was used to chemically induce MVs formation. Ultrasound (US) was used as a physical inductor. The resulting MVs were purified by differential centrifugation. The samples were studied by immunoblotting and nanoparticle tracking analysis (NTA).

Results

The NTA method was used to determine the concentration of MVs (for all samples, about 10⁸ particles/mL) and the dispersity of the particle size. The induction of MVs by activation with antibodies, treatment with CHB or US increased the monodispersity of the particles without affecting their average size. The application of US to activated cells led to a decrease in monodispersity and the appearance of a fraction of larger MVs (up to 700nm). As a result of Western blot analysis, an increase in the level of CD3 in samples of natural MVs, as well as induced by activation and US, was revealed compared with the cell control. In the samples obtained by US, an increase in the content of granzyme B was detected. In MVs induced by CHB, these proteins were not detected. With an equal amount of total protein, a decrease in the content of beta-actin was noted in the series cells>natural MVs>USinduction>CHB induction. This observation can be explained by a change in the cytoplasm/membrane ratio due to a difference in the size of cells and MVs (10μm and 0.1μm, respectively), as well as the mechanism of action of CHV (inhibition of actin filament synthesis).

Conclusions

All studied methods of induction, except for CHB, make it possible to obtain MVs carrying functionally significant molecules of T-lymphocytes. Further study the structure and functions of the MVs obtained by these methods is needed. The work was supported by the Russian Science Foundation grant 19-74-20026, with the support of the Strategic Academic Leadership Program of the Kazan (Volga Region) Federal University (PRIORITET- 2030).

Legal entity responsible for the study

Kazan Federal University.

Funding

The work was supported by the Russian Science Foundation grant 19-74-20026, with the support of the Strategic Academic Leadership Program of the Kazan (Volga Region) Federal University (PRIORITET- 2030).

Disclosure

All authors have declared no conflicts of interest.

Background

Human Vγ2Vδ2 T cells play important roles in microbial and tumor immunity by monitoring foreign- and self-prenyl pyrophosphate metabolites in isoprenoid biosynthesis. Accumulation of isoprenoid metabolites after bisphosphonate treatment allows Vγ2Vδ2 cells to recognize and kill tumors independently of their MHC expression or burden of non-synonymous mutations. Clinical trials with more than 400 patients show that adoptive immunotherapy with Vγ2Vδ2 T cells has few side effects but has resulted in only a few partial and complete remissions.

Methods

Ex vivo expansion for gamma delta T cells was done by pulsing with zoledronate in the presence of IL-2. Multicolor Flow Cytometry LSR II was used to assess the cell functionality by measuring cytokines expression (IFN-γ, and TNF-a). Human prostate cancer PC-3 cell line and NSG mice were used for the in vivo adoptive immunotherapy. PD-1 blockeds were used based on the approved clinical standards. Tumor size was assessed once weekly by external measurement of the longitudinal and transverse tumor diameter using a digital vernier caliper.

Results

We have tested Vγ2Vδ2 T cells for expression of inhibitory receptors and determined whether adding PD-1 checkpoint blockade to adoptively transferred Vγ2Vδ2 T cells enhances immunity to human PC-3 prostate tumors in an NSG mouse model. We find that Vγ2Vδ2 cells express PD-1, CTLA-4, LAG-3, and TIM-3 inhibitory receptors during the 14-day ex vivo expansion period, and PD-1, LAG-3, and TIM-3 upon subsequent stimulation by pamidronate-treated tumor cells. Expression of PD-L1 on PC-3 prostate cancer cells was increased by co-culture with activated Vγ2Vδ2 T cells. Importantly, anti-PD-1 mAb treatment enhanced Vγ2Vδ2 T cell immunity to PC-3 tumors in immunodeficient NSG mice, reducing tumor volume nearly to zero after 5 weeks of treatment.

Conclusions

These results demonstrate that PD-1 checkpoint blockade can enhance the effectiveness of adoptive immunotherapy with human γδ T cells in treating prostate tumors in a preclinical model. Our findings should provide new insights and perspectives to further increase the responsiveness of solid and less immunogenic tumors to immunotherapies.

Legal entity responsible for the study

The authors.

Funding

VA Healthcare.

Disclosure

All authors have declared no conflicts of interest.

Background

Neuroendocrine neoplasms (NENs) are a subgroup of poorly characterized rare tumors with challenging management. NENs are very heterogeneous and the identification of clinically useful biomarkers for patient stratification and treatment tailoring is urgently needed. This study proposes the use of near-patient models based on autologous extracellular matrix (aECM)-scaffolds and zebrafish (ZF) xenografts coupled with next generation sequencing techniques for the phenotype and genotype mapping of primary NEN cells. The aims are: to identify indolent versus aggressive behaviors, to define NEN sensitivity to drugs and to uncover the molecular signatures associated with these phenotypes.

Methods

Primary tumor cells isolated from surgical specimens of NEN patients were cultured in aECM scaffolds or injected into ZF embryos. In aECM scaffolds we evaluated cell morphology and neuroendocrine differentiation. In ZF we evaluated distant invasiveness and angiogenesis ability. For each primary culture, we performed transcriptome analysis of the tumor tissue compared to the healthy counterpart.

Results

Hitherto, we derived three primary cultures of neuroendocrine Merkel cell carcinoma (MCC) and one from grade 1 ileal NEN. The 3 MCC cultures into ZF embryos showed metastatic rates of 81%, 50% and 64%, indicative of tumor aggressiveness. For one MCC culture, angiogenic ability was also observed. The ileal cells were derived from both primary and metastatic lesions. Cells from the primary tumor showed a metastatic rate of 23%, while the ones from the metastasis of 79%. This result highlights the fidelity of ZF xenografting to the tumor behavior in the patient. RNA-seq was performed on Merkel's tumor specimens and bioinformatic analysis showed that ECM remodeling and regulation of apoptotic processes were the most enriched pathways.

Conclusions

We developed aECM scaffolds and ZF xenografts as personalized disease models integrated with multi-omics analysis. This study is expected to generate knowledge on NEN molecular signatures for disease aggressiveness and sensitivity to therapy. All identified markers will be correlated with clinical outcomes of enrolled patients, to validate their significance and clinical utility.

Clinical trial identification

IRSTB120.

Legal entity responsible for the study

IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) \"Dino Amadori\", Meldola, Italy IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) \"Dino Amadori\", Meldola, Italy.

Funding

Italian Ministry of Health.

Disclosure

All authors have declared no conflicts of interest.

Background

The deregulation of epigenetic mechanisms is considered the main agent in the development of GBM. One of the enzymes involved in epigenetic alternation and cancer development are (HDAC) by removal acetyl group from histone and creating condensed chromatin that suppresses transcription gene to histone and non-histone proteins involved in tumorigenesis. Histone deacetylase inhibitors (HADCi) are the most common therapeutic methods for many types of cancer treatments that enabled researchers to study and develop HDAC inhibitors. Mocetinostat, also known as MGCD0103 is an isotype-selective (HDACi) that selectively inhibits HDACs 1−3 and 11 in vitro.

Methods

To demonstrate the role of MGCD0103 to inhibit growth and induce apoptosis in glioblastoma cells. We used different concentrations of MGCD0103 (0.5,1.0,1,5,2.0,2.5) μM on two types of glioblastoma C6 and T98G.

Results

The study showed that MGCD0103 exhibited a potent inhibitory role in the treatment of glioblastoma cancer cells. The MGCD0103103 in a dose- and time-dependent manner are actively contributed to inhibiting cell proliferation, invasion, angiogenesis and induce apoptosis and differentiation through modulating in the molecular mechanism for many pathways inside of cells such as suppression of PI3K/AKT pathway and HDAC1 responsible of many biological processes in cancer initiation and progression, and activation intrinsic and extrinsic pathway involved in apoptosis by upregulating pro-apoptotic proteins BAX and downregulating anti-apoptotic proteins, Bid, Bcl2. Also, the mocetinostat increases the expression of the tumor suppressor gene and decreases the expression of the E2f1 transcription factor. In addition to MGCDO103 induced differentiation by activating differentiation marker GFAP and inhibiting differentiation marker (Id2, N-Myc).

Conclusions

The MGCD0103 is a promising anticancer agent that plays an important role treatment of glioblastoma cells.

Legal entity responsible for the study

F. Khathayer.

Funding

Has not received any funding.

Disclosure

The author has declared no conflicts of interest.

Background

Glioblastoma (GBM) is the most frequent and aggressive primary malignant brain tumor and PI3K/Akt signaling pathway is activated in almost 90% of cases. The aim of this study was to evaluate the capacity of natural compounds to potentiate the activity of temozolomide (TMZ) - the standard pharmacological drug for GBM.

Methods

U-87 MG (ATCC HTB-14) cells were treated with quercetin, resveratrol, curcumin and TMZ, alone or in combination of TMZ with either of them, for 30 minutes (for signaling protein phosphorylation) and 48 hours (for total signaling protein expression). LDH-based cytotoxicity and MTS assays were performed for dose selection. Signaling molecular patterns were studies by xMAP array technology. The effect of treatments was evaluated by assessing a 9-plex panel of total and phosphorylated signaling proteins.

Results

Based on the cytotoxicity testing, selected working concentrations were 30 μM for quercetin, 30 μM for resveratrol, 15 μM for curcumin and 50 μM temozolomide. We found strong increase in JNK Thr183/Tyr185, p38 Thr180/Tyr182 and STAT3 Ser727 phosphorylation and a moderate increase for ERK1/2 Thr185/Tyr187 phosphorylation after 30 minutes of treatment. Resveratrol treatment reduced Akt Ser473 phosphorylation, whereas the other treatments had no effect. After 48 hours treatment of U87 cells, the expression of most signaling proteins analyzed (CREB, JNK, ERK1/2, Akt and STAT3) was decreased upon treatment with natural products alone. When the natural compounds were combined with TMZ, an even stronger decrease in protein expression was observed: CREB fold-regulation 0.63-0.78), NFkB (fold-regulation 0.81-0.86) and p38 (fold-regulation 0.68-0.85).

Conclusions

Our results revealed that quercetin and resveratrol performed best in potentiating TMZ effects on signaling protein expression. Natural compounds could be used as therapeutic adjuvants in GBM therapy. Given the complexity of signaling pathways in GBM treatment approaches, further studies are required to unravel the mechanisms for precision therapy.

Legal entity responsible for the study

Victor Babes National Institute of Pathology.

Funding

Ministry of Research, Innovation and Digitization, project no. COP A 1.2.3., ID: P_40_197/2016, PN 19.29.01.04, PN 19.41.50.01, PED 382 and 31PFE/2021.

Disclosure

All authors have declared no conflicts of interest.

Background

High intratumor heterogeneity of glioblastoma (GBM) cells and their differential reactivity to chemotherapeutic drug temozolomide (TMZ) contribute to clonal selection of TMZ-resistant clones and the disease recurrence. Numerous molecules contribute to this process; however, an involvement of glycosylated macromolecules remains under investigated. To study their potential contribution to the resistance development, we investigated different molecular and functional characteristics of various GBM cells and their association with TMZ resistance in GBM model in vitro.

Methods

Six GBM cell lines (U343, LN215, U87, LN71, LN405, LN18) were characterised for TMZ resistance (IC₅₀), proliferation (growth curve, doubling time) and migration (wound healing assay) rates, clonogenicity. Transcriptional profiling of key proteoglycans (PGs) was performed by RT-PCR. Total content of glycosaminoglycans (GAGs) on the cells surface was determined by staining with dimethylmethylene blue. Content and localization of chondroitin sulfate (CS) were studied by immunocytostaining.

Results

All GBM cell lines were individual in terms of the studied parameters (migration, clonogenicity), expression of some PGs (CD44, chondroitin sulfate proteoglycan 4/NG2, heparan sulfate proteoglycan 2/perlecan, chondroitin sulfate proteoglycan 5, biglycan, glypican-1, syndecan-1, syndecan-3, lumican, phosphacan) and total GAG and CS content, while their proliferation rates were similar. The most TMZ-sensitive cell line was LN215 (IC50=32μM) and the most TMZ-resistant one was LN405 (IC50=319μM). TMZ-resistance was associated with the expression of hyaluronic-acid receptor CSPG CD44 (r=0,83; p<=0,05), a cell surface adhesion molecule, which is often overexpressed on cancer stem cells.

Conclusions

High CD44 expression is glioblastoma cells may help to identify TMZ-resistant patients and thereby optimise the treatment strategy for the patients and overcome TMZ-chemoresistance during GBM chemotherapy.

Legal entity responsible for the study

The authors.

Funding

This study was supported by the Government of Novosibirsk region (Grant number 11). A.V.S. was supported by a Scholarship of Russian Federation President for young scientists (SP-4000.2022.4).

Disclosure

All authors have declared no conflicts of interest.

Background

Glioblastoma multiforme (GBM) is one of the most aggressive primary brain tumors in adults with insufficient current treatment options and very poor prognosis. Aggressive behavior of GBM cells and their therapeutic resistance are based on complex biological features including changes in the microtubule cytoskeleton. Microtubule targeting belongs to one of the fundamental approaches to cancer treatment, and previously described overexpression of certain tubulin isoforms suggests possible benefit in the use of microtubule targeting agents, such as the repurposed compound flubendazole (FLU). This benzimidazole was previously shown to inhibit a range of solid tumor and leukemic cells with described effect on tubulin structure and polymerization. The aim of this study was to investigate the effect of FLU on microtubule polymerization in two GBM cell lines (A172 and T98G) and to examine the changes in microtubule appearance and posttranslational modifications after FLU treatment.

Methods

In FLU-exposed cells growing microtubule ends were detected via EB1/EB3-specific immunofluorescence staining. The changes in microtubules and their modifications were studied with fluorescent microscopy with subsequent image processing and analysis (ImageJ). The relative quantification of tubulin modifications was performed by Western blotting.

Results

FLU treatment reduced the abundance of EB1/EB3-labeled microtubule ends indicating inhibition of microtubule polymerization in a concentration-dependent manner. FLU also caused significant changes in the organization of microtubules and their posttranslational modifications.

Conclusions

Our results demonstrate that FLU exerts its effect on GBM cells by inhibiting microtubule polymerization, as well as inducing significant changes in the organization of microtubule network. Targeting the cytoskeleton with FLU could offer a strategic advantage for improving the outcome of GBM therapy and should be further investigated.

Legal entity responsible for the study

The authors.

Funding

This study was supported by Ministry of Health, Czech Republic, project No. NU20-03-00360.

Disclosure

All authors have declared no conflicts of interest.

Background

Glioblastoma multiforme (GBM) is one of the most common and aggressive primary brain tumors in adults with very poor prognosis. The treatment is complex and consists of the surgical resection followed by chemoradiotherapy using alkylating drug temozolomide (TMZ), which is problematic due to fast developing chemoresistance. Novel therapeutic approaches for GBM treatment are needed such as combination therapy where TMZ effect might be potentiated by other useful compound/s. The aim of this study was to investigate the effect of potential anticancer drug flubendazole (FLU) on TMZ treated GBM cell lines A172 and T98G and to verify potential improved efficiency of this combination towards GBM cells.

Methods

The effect of TMZ, FLU and their combination on GBM cell lines was evaluated by cell proliferation assay (WST-1) and changes in cellular morphology (phase contrast microscopy). Morphology and organization of microtubules in treated cells were examined by fluorescence microscopy. Selected tubulins as well as markers of STAT3 signaling and cell cycle regulation were determined by western blotting and IHC analysis. Quantification of TMZ and FLU in exposed cells was carried out by LC/MS analysis.

Results

In both tested cell lines, combination of TMZ + FLU showed a more significant inhibitory effect on the GBM cells proliferation as compared to single FLU and TMZ. Moreover, combined use of FLU and TMZ enhanced accumulation of both compounds inside the cells and produced significant changes in cell morphology and microtubule structure. In addition, in thus treated cells the expression and activation of STAT3 was inhibited resulting in a decrease in cdc2 and cyclin B1 levels, suggesting possible G2/M cell cycle arrest. Since STAT3 and its activated form were also found in clinical samples, it can be discussed as a relevant marker and possible target.

Conclusions

The use of FLU significantly improved TMZ effect in tested GBM cell lines. The employed drug combination markedly altered cellular microtubule network and STAT3 signaling pathway, resulting in cell cycle arrest at G2/M phase and subsequent mitotic catastrophe.

Editorial acknowledgement

This study was supported by Ministry of Health, Czech Republic, project No. NU20-03-00360.

Legal entity responsible for the study

The authors.

Funding

Ministry of Health, Czech Republic, project No. NU20-03-00360.

Disclosure

All authors have declared no conflicts of interest.

Background

3D tumor cell cultures are currently emerging as the novel standard for cytotoxicity testing as well as in vitro molecular studies, but various solutions are available to generate them. The aim of this study was to test whether there is one-fits-all solution to generate tumor spheroids for further studies.

Methods

We used three breast tumor cell lines (MCF-7, MDA-MB-231, MDA-MB-361) and one glioblastoma cell line (U87), as comparison for MDA-MB-361 which is a secondary (metastatic) brain tumor. Different hydrogels (Matrigel® Corning, TrueGel3D -1, -6,-7 with or w/o RGD adhesion peptide® Merck, and TrueGel3D® HTS Hydrogel Plates®Merck) were tested for spheroid formation, as well as ultra-low attachment surface 24 well-plates ®Corning (no gels). Cells were seeded in two concentrations (300 and 3000/cm²) and documented daily for the first 5 days, than weekly for spheroid formation. Viability was tested using dual fluorescein diacetate/propidium iodide stain.

Results

For higher cell concentrations, presence of spheroids can be documented as early as 5 days, but at least 7 days are recommended. Once formed, spheroids can be maintained more than 28 days, with twice a week cell medium change. Presence of organic molecules (basement membrane matrix or RGD peptide) in the gel impaired formation of tumor spheroids for aggressive cell lines (MDA-MB-261 and U87) and yielded mixed cultures (2D and spheroids) for the rest. Also, higher stiffness and the presence of a non-degradable cell linker prevented proliferation of cells and formation of spheroids, regardless of cell type. A crosslinker gradient, although favored formation of all tested breast cancer spheroids, interfered with formation of spheroids for U87 cells. Finally, ultra-low adherence plates allowed consistent formation of spheroids only for MCF-7 cells, whereas for the rest, irregular cell aggregates were observed at higher cell concentrations.

Conclusions

Dextran-based polymers can be used for tumor spheroid formation regardless of cell type, provided that no adhesion peptides are added.

Legal entity responsible for the study

Victor Babes National Institute of Pathology.

Funding

Ministry of Research, Innovation and Digitization, project no. COP A 1.2.3., ID: P_40_197/2016, PN 19.29.01.04 and 31PFE/2021, and CCCDI - UEFISCDI, project number PN-III-P2-2.1-PED-2019-3141, ctr. 382.

Disclosure

All authors have declared no conflicts of interest.

Background

Lung cancer is the leading cause of cancer-related deaths worldwide. KRAS is the most frequent mutated driver gen in non-small cell lung cancer (NSCLC). Nevertheless, KRAS has been recognized as undruggable for many years. KRASG12C inhibitors are being developed in clinical trials and have revealed promising results in lung cancer patients. While these therapies hold great promise, they face the same limitation as other targeted therapies, the therapeutic potential of these inhibitors can be impaired by resistance mechanisms. Deciphering resistance mechanisms to KRASG12Ci is of prime relevance to predict which patients may benefit from these therapies. And exploring resistance-overcoming therapeutic strategies is essential for improving precision oncology in KRAS-driven cancer.

Methods

We generated resistant cell lines to KRASG12C inhibitors, exposing cells to increasing concentrations of drugs until they were resistant to the drug. Once the resistant cell lines were generated, protein was extracted to characterize the resistant cell lines using phospho-arrays and Western Blot techniques. In addition, the DNA and RNA from parental and resistant cell lines were extracted and sequenced by NGS to look for transcriptomic and genomic differences. With all these data we identified some molecular alterations that may be responsible for the acquired resistance and explored some resistance-overcoming therapeutic strategies.

Results

We generated 11 Sotorasib- or Adagrasib-resistant cell lines and we characterized them at the proteomic, transcriptomic and genomic level. Using proteomic characterization, we observed differences in the expression and/or activation of some receptors, such as EGFR and FGFR, in many resistant cell lines. And using the transcriptomic and genomic data we found some alterations that also could explain the acquired resistances. Then, we explored some resistance-overcoming therapeutic strategies in vitro and in vivo, which have shown promising results.

Conclusions

1) We have found different mechanisms of acquired resistance to KRASG12Ci using proteomic, transcriptomic and genomic data. 2) We have found some effective resistance-overcoming therapeutic strategies in vitro and in vivo.

Legal entity responsible for the study

The authors.

Funding

Instituto de Salud Carlos III (PI19/00320).

Disclosure

L. Paz-Ares: Financial Interests, Personal, Advisory Board, Speaker fees: Roche, MSD, BMS, AZ, Lilly, PharmaMar, Beigene, Daiichi Sankyo, Medscape, PER; Financial Interests, Personal, Advisory Board: Merck Serono, Pfizer, Bayer, Amgen, Janssen, GSK, Novartis, Takeda, Sanofi, Mirati; Financial Interests, Personal, Other, Board member: Genomica, Altum sequencing; Financial Interests, Institutional, Invited Speaker: Daiichi Sankyo, AstraZeneca, Merck Sharp & Dohme Corp., BMS, Janssen-Cilag international NV, Novartis, Roche, Sanofi, Tesaro, Alkermes, Lilly, Takeda, Pfizer, PharmaMar; Financial Interests, Personal, Invited Speaker: Amgen; Financial Interests, Personal, Other, Member: AACR, ASCO, ESMO; Financial Interests, Personal, Other, Foundation Board Member: AECC; Financial Interests, Personal, Other, President. V CASEICA (Spanish Association of Cancer Research): ASEICA; Financial Interests, Personal, Other, Foundation president: ONCOSUR; Financial Interests, Personal, Other, member: Small Lung Cancer Group. All other authors have declared no conflicts of interest.

Background

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer characterized by late diagnosis and extensive metastasis. It is known to contain exclusively tumorigenic cancer stem cells (CSCs), which are resistant to chemotherapy like gemcitabine. We have previously shown that CD133+ CSCs contributes to disease progression and treatment failure in PDAC. In this study we demonstrate the therapeutic potential of novel dual BET/HDAC inhibitor TW09 to target CD133+ CSCs in a preclinical set-up.

Methods

shRNA mediated knockdown of cMyc was performed to delineate the downstream effectors and signaling pathways. Gene set enrichment analysis (GSEA) analysis was performed on TW09 and control treated pancreatic cancer cells to analyse CSC metabolism and growth. FACS sorting, qPCR, western blot and MTT assay characterized dose dependent effects on different patient-derived pancreatic cancer cell lines. Post TW09 treatment, sphere formation assay and CD133 surface staining determined the role on self-renewal capacity whereas, Annexin V and EdU staining delineated CSC specific signaling modulation. Additionally, organoid treatment using gemcitabine, TW09 and combination was performed to describe their therapeutic potential.

Results

GSEA revealed that the significantly enriched pathways involved in metabolism and OXPHOS were downregulated by TW09 in comparison to control. In addition, TW09 decreased gene expression of CSC, pluripotency and EMT related markers. TW09 negatively modulated CD133+ & ALDH+ population as well as sphere formation capacity confirming its strong CSC inhibiting property. CD133+ population specific loss of mitochondrial membrane potential, G2M arrest and apoptosis was also observed. Furthermore, organoid treatment uncovered that TW09 can sensitize resistant cells towards gemcitabine.

Conclusions

We elucidate here the deregulation of different molecular pathways involved in CSC maintenance, pluripotency and EMT using TW09 on primary PDAC cell lines. We also describe that TW09 shows cancer stem cell specific apoptosis and metabolic changes. We depict, in a preclinical set up, that TW09 has potent anti-tumor activity by selectively targeting CSCs and represents as a prospective therapeutic strategy for PDAC.

Legal entity responsible for the study

The authors.

Funding

Plasticity in Pancreatic Ductal Adenocarcinomas (PiPAC).

Disclosure

All authors have declared no conflicts of interest.

Background

Pancreatic ductal adenocarcinoma (PDAC) is one of the most metastatic malignancies worldwide. We previously identified a distinct subset of cancer stem cells within the invasive front of patient tumors called migrating cancer stem cells (miCSCs), characterized by CD133+CXCR4+ expression. It determines the metastatic phenotype of pancreatic cancer. Therefore, targeting CXCR4 represent a potential therapeutic approach to combat metastasis in PDAC.

Methods

We examined the effect of endogenous human peptides EPI-X4 and other derivatives thereof as CXCR4 antagonist on patient-derived primary pancreatic cancer cells and tumor-stroma crosstalk. We established these peptides as novel therapeutic strategy for combating metastatic activity of PDAC using combination therapy approaches and testing in vivo delivery system eg. peptide fatty-acid (FA) conjugates and silica nanoparticles (SiNP).

Results

Migration assay towards CXCR4 ligand CXCL12 showed that EPI-X4 and its derivatives (eg.JM#21) strongly inhibited migratory capacity in vitro. JM#21 was identified as the most potent EPI-X4 derivate. Western blot, gene expression and IF revealed that JM#21 increased Cadherin-1 expression by suppression of Snail1 via SHH pathway. Moreover, JM#21 suppressed CXCL12-induced self-renewal capacity of the tumor cells. Strikingly, JM#21 sensitized selected cell lines towards gemcitabine and paclitaxel. Furthermore, FA and SiNP combined JM#21 restricted miCSCs maintenance which was regulated via stellate cell secreted CXCL12. In serum conditions, both FA and SiNP combined JM#21 was stable and active, proving to be valuable in vivo delivery system.

Conclusions

In conclusion, our study reveals that targeting CXCR4 using human endogenous EPI-X4 derivates particularly JM#21 inhibits tumor-stroma crosstalk which is indispensable for miCSC maintenance. In mechanistic and preclinical set up, these peptides abrogate metastatic capacity of patient-derived pancreatic cancer cells by selective elimination of miCSCs. Moreover, tumor cells show increased susceptibility towards conventional treatment strategies enforcing EPI-X4 derivate as a novel combinatory therapy to treat metastatic pancreatic cancer.

Legal entity responsible for the study

The authors.

Funding

CRC 1279.

Disclosure

All authors have declared no conflicts of interest.

Background

Liver cancer has a poor prognosis predominantly resulting from tumour heterogeneity and limited treatment options. The two predominant types of liver cancer are Hepatocellular carcinoma (HCC) and Intrahepatic Cholangiocarcinoma (iCCA). CD44v6, a splice variant of the hyaluronic acid receptor CD44, is a marker for cancer stem cells, and is known to impact tumour formation and metastasis in several cancers. The expression of CD44v6 dramatically increases in liver epithelial cells upon carcinogen exposure. With this study, we aim to target CD44v6 in primary human liver carcinoma cell lines by using a small peptide inhibitor, and observe the effects of CD44v6 inhibition on migration, invasion and EMT.

Methods

CD44v6 expression was investigated in mouse and human liver and tumour tissue by immunohistochemistry. In addition, we used human HCC and CC cell lines to study the role of CD44v6 inhibition on cell migration, invasion and EMT.

Results

We observed CD44v6+ epithelial cells in premalignant livers of transgenic mouse models with chronic liver damage. The expression patterns of CD44v6+ cells differed in the mouse models, where the cells had a hepatocyte-like phenotype in some mouse models and a cholangiocyte-like phenotype in others. Deletion of Trp53 in the liver resulted in a significant increase in the number of CD44v6+ cells. CD44v6 expression was also increased in murine and human tumours compared to the liver, and coincided with Sox9 expression in murine tumours. Human liver cancer cell lines were analysed for CD44 expression and two CC cell lines were found to have high expression of CD44 (85-90%). Functional assays (clonogenic assay, wound healing assay and transwell migration assay) were performed using the CC cell lines. Treatment with AMC303 conferred a reduction in the migration potential of cells in CC cell lines. Inhibition of CD44v6 also resulted in a reduction in the colony formation potential of the cells, indicating a decrease in the stemness characteristics of the cells. A reduction in EMT in both 2D and 3D culture was also observed.

Conclusions

Inhibition of CD44v6 in liver cancer cell lines led to a reduction in chemotaxis, migration, EMT and the stemness characteristics of cells. In summary, CD44v6 appears to be a promising targetable candidate for liver carcinoma.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

Drugs targeting the epidermal growth factor receptor (EGFR), such as panitumumab and cetuximab, are standard of care for metastatic colorectal cancer (mCRC) patients with RAS/BRAF-wildtype tumors. Nevertheless, a fraction of these patients does not respond to them. There is a clinical need for an effective biomarker to further select patients for EGFR inhibition. A promising predictive biomarker is in vitro response testing using patient-derived organoids (PDOs).

Methods

Drug screens were performed by exposing 19 PDOs from 18 patients (1 patient with 2 PDOs) to a concentration range of panitumumab for 5 days. Drug sensitivity was measured by cell viability. Growth rate (GR) inhibition metrics were used for drug response curve fitting (area under the curve [GR_AUC], GR_max) to quantify PDO sensitivity. We compared in vitro sensitivity for all PDOs based on RAS/BRAF mutational status and sidedness. PDO sensitivity (n=6) was also compared to patient response (n=5), which was evaluated using % change in the sum of the size of target lesions on CT scans.

Results

PDOs derived from left-sided RAS/BRAF-wildtype tumors were more sensitive to panitumumab than rectum and right-sided RAS/BRAF-wildtype tumors and RAS/BRAF-mutant tumors (p < 0.05, Kruskal-Wallis test), concurrent with patient response in clinical trials. We observed varying PDO sensitivities to panitumumab for left-sided RAS/BRAF-wildtype tumors. For 6 PDOs receiving panitumumab, decrease in the size of target lesions was associated with PDO response in most patients (GR_AUC, GR_max). PDOs from patients previously exposed to chemotherapy did not show increased resistance to panitumumab.

Conclusions

We confirmed panitumumab sensitivity for PDOs from left-sided RAS/BRAF-wildtype tumors. PDOs from RAS/BRAF-wildtype rectum tumors were less sensitive to panitumumab compared to other left-sided tumors. Even within PDOs from left-sided RAS/BRAF-wildtype tumors, sensitivity to panitumumab varied. These results imply that PDOs may further aid in guiding EGFR inhibition compared to molecular pathology and sidedness only. We demonstrate that PDO response is associated with patient response to panitumumab, suggesting that PDOs can serve as a predictive biomarker for EGFR targeting treatment.

Clinical trial identification

ABR NL61668.041.17.

Legal entity responsible for the study

University Medical Center Utrecht (UMCU).

Funding

Foundation Hubrecht Organoid Technology.

Disclosure

All authors have declared no conflicts of interest.

Background

In the large intestine, the multipotent stem cells are located at the base of the crypt and differentiate into three main cell types: enterocytes, goblet cells, and enteroendocrine cells. Goblet cells’ main function is the synthesis and secretion of mucins. Genetic and epigenetic changes that provide survival advantages for stem or progenitor cells resulting in the deregulation of cellular differentiation are major causes of all carcinomas.

Methods

Our laboratory has a large collection of colorectal cancer (CRC) cell lines, well characterised in terms of gene expression and mutations. We analysed the presence of goblet cells in CRC cell lines using the genes Mucin 2 (MUC2) and Trefoil factor 3 (TFF3). The genes both at the mRNA level and at the protein level were investigated. The effects of various transcription factors were assessed by knockdown and overexpression techniques.

Results

We found that most of the cell lines are unable to produce goblet cells and that the number of MUC2 and TFF3-positive cells among the goblet cell positive cell lines was quite variable. While in the normal colon, MUC2 and TFF3 are always co-expressed, but that is not always the case in the CRC cell lines. MUC2-negative and TFF3-positive cell lines appear to reflect a novel interesting subset. The investigation of several transcription factors on goblet cell differentiation showed that downregulation of Atonal homologue 1 (ATOH1) had a dramatic effect on goblet cell production, while knocking down of SAM pointed domain ETS transcription factor (SPDEF), Caudal type homeobox 1 (CDX1), and 2 (CDX2) had a modest effect. Individually, none of these factors are sufficient to trigger the goblet cell differentiation.

Conclusions

As a conclusion, the percentage of goblet cells differs substantially between cell lines. Classification of the cell lines reveals an interesting major subset that has TFF3 expression without expressing MUC2. ATOH1, SPDEF, CDX1, and CDX2 had a significant effect on goblet cell differentiation, but on their own, they are not sufficient to induce the goblet cell differentiation. Understanding the mechanisms of goblet cell differentiation is important for advances in the prevention and treatment of CRC.

Legal entity responsible for the study

The authors.

Funding

1) Islamic Development Bank; 2) Institute of Molecular Biology and Biotechnologies, Azerbaijan National Academy of Sciences.

Disclosure

All authors have declared no conflicts of interest.

Background

Gastrointestinal stromal tumors (GIST) are the most frequent mesenchymal malignancies of the gastrointestinal tract. Pathogenesis of GIST is mostly associated with KIT tyrosine kinase gene mutations. Therefore, tyrosine kinases inhibitor imatinib mesylate (Gleevec) is the first-line GIST treatment. Nevertheless, GIST patients are facing secondary resistance to imatinib within 2 years of ongoing treatment. One of the possible mechanisms related to imatinib resistance is the FGF/FGFR autocrine loop, thus, FGF and FGFR might be prospective targets for imatinib-resistant GIST treatment. One of the possible approaches to identify the role of FGF/FGFR autocrine loop in both GISTs’ pathogenesis and imatinib sensitivity is the genome editing tool CRISPR/Cas9 used for target gene knock-out. Thereby the aim of the study was to generate imatinib resistant GIST cell line T1-IM-R expressing doxycycline-inducible endonuclease Cas9 which could be used as in vitro model for gene knockout studies in GIST.

Methods

Doxycycline-inducible endonuclease Cas9 gene transfer into imatinib resistant GIST cell line T1-IM-R was performed by lentiviral transduction using pCW-Cas9 plasmid (#50661, Addgene). Providing lentiviral transduction was successful, T1-IM-R cells were selected in the presence of 1 ug/ml puromycin and were used for further clonal selection. The level of Cas9 expression was detected by Western Blot analysis after 6 days of cultivation in the presence of 1 ug/ml doxycycline. The mouse monoclonal antibody against Cas9 (MA1-202, Invitrogen, USA) was used to detect endonuclease Cas9. The b-tubulin was used as loading control.

Results

Totally 32 clonal sublines were obtained after transduction of the T1-IM-R cell line by Cas9 gene containing lentiviral particles. Out of them 12 clonal sublines (T1-IM-R/Cas9) demonstrated higher levels of Cas9 expression. Clones T1-IM-R/Cas9-p4-E9 and T1-IM-R/Cas9-p4-C10 were selected for the further research.

Conclusions

Thus, we obtained 12 imatinib resistant GIST cell line T1-IM-R clones expressing Cas9 endonuclease. T1-IM-R/Cas9-p4-E9 and T1-IM-R/Cas9-p4-C10 clones might be used as the in vitro models for the further investigation of the effects of FGF/FGFR autocrine loop on the GIST imatinib sensitivity.

Legal entity responsible for the study

Research Laboratory \"Biomarker\", Institute of Fundamental Medicine and Biology, Kazan Federal University.

Funding

This paper has been supported by the Kazan Federal University Strategic Academic Leadership Program (PRIORITY-2030) (VS, DF, IL, DR, RK) and Russian Science Foundation (RSF) 20-15-00001 (PD, SB).

Disclosure

All authors have declared no conflicts of interest.

Background

Prostate cancer (PCa) is one of the main causes of death in men all over the world. To date, the emerging issue is the search for new diagnostic and prognostic biomarkers to distinguish patients with different risk types. Recent studies of PCa biomarkers have drawn attention to the remarkable heterogeneity of this tumor. Tumor heterogeneity (TH) is the main limitation of the ability to use biomarkers’ gene panels in clinics because of different cell subclones within one tumor.

Methods

Here we describe the relationship between tumor cells in a model of heterogeneous prostate cancer. Our model was based on direct and indirect co-cultivation of 2 prostate cell lines with aggressive (PC3-GFP) and indolent (22Rv1) phenotypes. To evaluate the tumor aggressiveness, we described the surface phenotype of adhesion molecules, measured the expression of genes related to metastasis in prostate cancer (Ai et al., 2017; Fan et al., 2018) and performed the motility tests.

Results

In experiments with direct co-cultivation of PC3 (aggressive) and 22Rv1 (indolent) cancer cells, we found a decrease of CD29 (integrin beta 1) on PC3 cells within 3 days, while the expression of other surface adhesion molecules (CD54, CD38, CD24 and CD44) was not altered, the surface phenotype of 22Rv1 did not change. Next, we assessed the expression of genes related to metastasis and showed that after 3 days of direct co-cultivation the expression of FLNC, AMACR, SNCG, HPN genes increases at least 2.5 times in 22Rv1, while HPN and FASN are upregulated in PC3 cells (for all measurements p<0.05, Mann-Whitney test). However, during indirect co-cultivation we did not detect the change in expression of metastasis-related genes neither in PC3 nor in 22Rv1. In experiments with a scratch assay, we showed that wound healing rate decreased by 8 times for PC3 and by 6 times for 22Rv1 compared to controls (p<0.05, Mann-Whitney test, n=3).

Conclusions

Thus, we conclude that the induction of an aggressive tumor phenotype is possible during only direct co-cultivation of cells with an indolent and aggressive tumor phenotype and is manifested in an increase in the expression of PCa biomarker genes associated with migration and metastasis, and describe the model of tumor phenotype induction for prostate cancer.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

PIM 1 and PI3K/mTOR pathways are frequently dysregulated in prostate cancer and may lead to decreased survival invasion and metastasis. Moreover, anti-tumour drug resistance has been associated with the interconnection of these pathways. Furthermore, current treatments exhibit issues with toxicity. Hence, these pathways were co-targeted with novel preclinical multikinase PIM/PI3K/mTOR inhibitor- AUM302, PI3K/mTOR inhibitor BEZ235 (Dactolisib) and PIM inhibitor, AZD-1208 in our laboratory using a cohort of cancer explants emanating from our PEOPLE: PatiEnt prOstate samPLes for rEsea ch study and our current SCREEN study. This cohort has a high Gleason grade score of ≥  8. Therefore, this study aims to assess the effect of the combination therapy on the transcriptional landscape of ex vivo prostate cancer models derived from prostate cancer patients.

Methods

Using the Nanostring GeoMX DSP technology, we aim to analyse the spatial transcriptomic profile of the co-targeted therapy treated ex vivo models to decipher the effects of heterogeneity on the co-targeted therapies' efficacy. Tissue microarrays of co-targeted treated twenty-five ex vivo 3mm cores derived from 4 patients will be analysed. Following RNA Scope analysis, morphology markers, including PAN CK positive and PAN CK negative, will be used to guide the selection of 270 regions of interest (ROI). ROI will be segmented and profiled using immunofluorescence. The morphological markers will define these segments into areas of illumination (AOIs) using a combination of the absence or presence of CD45 and pSTAT3. The AOIs will generate multiple expression profiles for the related ROI. We intend to use this flexible, high-dimensional spatial profiling to identify the spatial transcriptomic signatures and explore phosphorylation sites in cancer-targeted therapies.

Results

The spatial transcriptomics analysis of this study is in view.

Conclusions

Our findings will contribute to understanding how the spatial landscape of the tumour microenvironment enhances the efficacy of anti-tumour drugs and what subset of patients are more likely to benefit from such therapy.

Legal entity responsible for the study

Susan Heavey's Laboratory, Department of Targeted Interventions, University College London.

Funding

Prostate Cancer UK.

Disclosure

All authors have declared no conflicts of interest.

Background

The matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade multiple components of the extracellular matrix. The significant role of MMPs in tumor invasion, neoangiogenesis and metastasis presented it as ideal pharmacological targets for cancer therapy. The effects of ursolic acid (UA) on matrix metalloproteinase (MMP) gene expression, cell invasion and cell migration were investigated MDA MB- 231 cell line.

Methods

Cytotoxicity of UA towards cancer cells was evaluated by MTT assay. MDA MB- 231 cells were cultured for 48 hrs with different concentrations (0, 5, 15 and 25μM) of UA and mRNA expressions of MMP-1, MMP-3, MMP-7, MMP-9 and MMP- 11 were analyzed by real time PCR. Cell migration wound healing assay was done at 0, 6, 24, and 48 h periods for cell migration evaluation. To asses the cell invasion MDA MB-231 cancer cells were seeded on collagen based matrigel chamber and treated with 1, 5, 10 and 15μM UA, whereas untreated cells considered as control. After48 hrs cells invaded through matrigel quantified by colorimetric method.

Results

UA inhibited the proliferation of MDA MB -231 cells in a dose dependent manner and time dependent manner. Our results showed that UA significantly affect all MMPs except MMP-3 and 7. The expression of MMP 1, 3 and was reduced significantly 0.34, 0.11 and 0.68 fold in response to 25μM UA after 48hrs incubation in MDA MB-231 cells in comparison to control cells. Wound healing assay results exhibited that cells treated with 15μM UA significantly hindered the motility of these cells and 7.27% wound area remained unfilled after 48hrs. The cancer cells invasion was 89.75% at 25μM UA through matrigel after 48 hrs.

Conclusions

In conclusion, UA reduced the mRNA expressions of MMP-1, MMP-3 and MMP-9 in MDA-MB-231 cells. Ursolic acid also inhibited the invasion and migration of TNBC cells.

Legal entity responsible for the study

S. Rajoriya.

Funding

Indian Veterinary Research Institute, Bareilly U.P., India.

Disclosure

All authors have declared no conflicts of interest.

Background

After invasive ductal carcinoma, invasive lobular carcinoma (ILC) is the most frequent breast cancer and accounts for nearly 15% of all breast cancers (BC). Doxorubicin (DXR) is a very effective chemotherapeutic drug utilized to treat BC but induces drug resistance and severe side effects. Recently, liposomes have gained popularity as effective tumor microenvironment (TME) modulatory anticancer agents for various malignancies, including BC. This study propose a liposomal-DXR combination to produce an immune-responsive regulatory effect on competing endogenous RNAs (ceRNAs) through alteration in TME and investigate the immunological and anticancer impact of liposomal-DXR combination via regulation of ceRNAs and immune regulatory cytokines at BC cells.

Methods

MDA-MB cells were treated with varying concentration of liposomal-DXR combination and dose response curve was calculated. Total RNA was extracted and quantified by qRT-PCR and TNF-α ELISA kit. Computational target interaction was analyzed.

Results

Treatment resulted in a dose and time-dependent cell-viability and migration inhibition. Treatment altered the level of AKT1, ESR1, and ARID1A genes. Alteration in miR-17-5p compared to control cells indicated the alteration in ceRNAs network. TNF-α was found repressed in treated MDA-MB cells.

Conclusions

Present study propose the mechanism of liposomal-DXR as potential TME modulatory, immunoregulatory anti-ILC agent through altering ceRNAs circuit, AKT1, ESR1, and ARID1A genes, and immune regulatory TNF-α in BC cells.

Legal entity responsible for the study

The author.

Funding

Has not received any funding.

Disclosure

The author has declared no conflicts of interest.

Background

Previously, we established that mice of the C3H/Sn line, which multiply intensively, develop mammary gland tumors in 100% of cases before the age of 12 months. In virgin mice of this line, tumors occur in no more than 30%. During pregnancy, the level of steroids is higher and obviously this indicates a more intensive activation of mutated genes by steroid hormones than in virgin mice. The role of hormones at more distant stages of development of the body and organs has been revealed. The possibility of preventing the occurrence of tumors is considered in this work.

Methods

Mice of the C3H/Sn and BALB/c lines became pregnant at the same time. Synchronously born C3H/Sn and BALB/c mice were given to BALB/c females for nursing. Some of the mice were given per os approximately 1 μl of a solution of estradiol and corticosterone at the dose of 10^-9 M/L for 2 weeks.

Results

BALB/c mothers successfully nursed 15 C3H/Sn mice to puberty. After giving birth, the mice were divided into two groups: 1) intact - 7 pcs. 2) 8 pcs received a solution of estradiol and corticosterone (10-9 M/L) for 2 weeks once a day starting from the first day after delivery. No further action was taken on them. Adult mice were observed up to 3 generations. In 1 group of intact mice, mammary gland tumors were detected in 1 mouse out of 7. In the second group, in 6 out of 8. That is, treatment with estrogens in the early postnatal age leads to the manifestation of the effect in the distant future. It seems that the action of steroids can be biphasic: I — activation. II - is more strongly expressed during sexual maturation of animals, when the level of hormones increases. Such a mechanism of action in the case of unmutated genes ensures the development of mammary glands in children when they become adults. When mutated, it contributes to the development of cancer. Translation from laboratory experiments to the clinic can be based on the reverse scheme: strict limiting of the use of steroids with breast milk during the early postpartum period. Theoretically, no negative consequences are expected, except for breast hypoplasia.

Conclusions

The long-term effect of steroids in the early postnatal period on the development of mammary glands in the future state requires further study.

Legal entity responsible for the study

NAS Ukraine.

Funding

Has not received any funding.

Disclosure

The author has declared no conflicts of interest.

Background

The platinum and taxane drugs as a neoadjuvant and adjuvant chemotherapy are the most used for treatment of ovarian cancer (OC). In later stages of ovarian cancer, targeted therapy with monoclonal antibodies may be included in the treatment regimen. Sodium-dependent phosphate transporter NaPi2b is a perspective target for the ovarian cancer treatment with monoclonal antibodies since the overexpression of NaPi2b was found in about 90% of the cases of OC. Currently NaPi2b-specific therapeutic monoclonal antibodies XMT-1536 (NCT03319628) and XMT-1592 (NCT04396340) are successfully undergoing clinical trials for the treatment of ovarian and lung cancers. Previously it was shown that NaPi2b protein abundance decreased in OC tumors of patients after neoadjuvant therapy mainly including carboplatin, thus, the subsequent use the therapy of monoclonal antibodies might be not effective. Currently it is unknown which drug of the ovarian cancer neoadjuvant chemotherapeutic regimen is responsible for the decrease of the Napi2b protein abundance. Thereby, the aim of the research was to evaluate the effect of carboplatin on the NaPi2b mRNA and protein level in the OVCAR-4 ovarian cancer cell line.

Methods

The NaPi2b mRNA and protein level before and after carboplatin treatment of OVCAR-4 cells was determined by real-time PCR and Western-blotting respectively. The Statistical analysis was performed in GraphPad Prism software.

Results

Low abundance of NaPi2b protein was revealed in OVCAR-4 cells after the carboplatin treatment in IC25 and IC50 concentrations within 48 and 72 hours. At the same time NaPi2b mRNA level decreased after carboplatin treatment in IC25 and IC50 concentrations only within 72 hours. A weak correlation was observed between NaPi2b mRNA level and NaPi2b protein abundance in OVCAR-4 cells before and after the carboplatin treatment.

Conclusions

Thus, we revealed that carboplatin decreased the NaPi2b mRNA and protein abundance. The created model can be used to study the mechanisms underlying the regulation of NaPi2b gene expression. The NaPi2b level in ovarian cancer cells after neoadjuvant therapy may be a predictive marker for prescribing monoclonal antibodies therapy in patients with ovarian carcinoma.

Legal entity responsible for the study

Kazan Federal University.

Funding

This paper has been supported by the Kazan Federal University Strategic Academic Leadership Program (PRIORITY-2030).

Disclosure

All authors have declared no conflicts of interest.

Background

Chronic myeloid leukemia (CML) is a kind of blood cancer and most CML patients have associated with a chromosomal anomaly with the BCR-ABL fusion oncogene, which occurs as a result of translocation between the Abelson murine leukemia (ABL1) gene on chromosome 9 and breakpoint cluster region (BCR) gene on chromosome 22. Imatinib mesylate is the first small molecule developed to target the BCR-ABL fusion protein. Imatinib reduces BCR-ABL activity by binding to the inactive conformation of tyrosine kinases. Despite a high response rate in CML patients with imatinib therapy, almost one-third of patients still have an inadequate response to Imatinib. In other words, mutations in the imatinib-binding pocket or other regions of the BCR-ABL kinase result in various resistances to imatinib in CML patients. Therefore, there have revealed a need to develop a more potent new molecule with an imatinib function that is more resistant to mutations.

Methods

In this study, ABL kinase domain gene was purchased from Genscript Biotech. The gene was inserted to pET11a vector plasmid construct. The plasmid was transformed into E.coli, strain BL21 (Rosetta-2). Transformed E. coli were grown overnight on agar plates. The colonies were collected from agar plates and started large volume of culture in rich LB media. To be able to procure further purified protein, we were used Ni-NTA affinity chromatography. The purified protein solution was added to crystal screen conditions in Terasaki plates. Then, X-ray diffraction images were collected from the formed crystals in order to acquire the best 3D structure. These diffraction datas were collected from XtalCheck module (Rigaku Oxford Diffraction) at ambient temperature.

Results

We will have revealed the structures determined at ambient temperature and high resolution with the help of X-ray crystallography technique. Additionally, we will have re-evaluated structures and designed new target small molecule with approaching from the perspective of integrative structural biology.

Conclusions

The results propose that may developed a new generation of Imatinib that is more specific, high affinity and resistant to possible mutations to treat CML disease and improve patients’ lives.

Editorial acknowledgement

Hasan DeMirci, Department of Molecular Biology and Genetics, Koc University, Istanbul, Turkey; Koc University Isbank Center for Infectious Diseases (KUISCID), Istanbul, Turkey; Stanford PULSE Institute, SLAC National Laboratory, Menlo Park, CA, USA.

Legal entity responsible for the study

Koc University Structural Biology & Innovative Drug Development Center.

Funding

Koc University Structural Biology & Innovative Drug Development Center/ Deva pharmaceuticals Companies.

Disclosure

The author has declared no conflicts of interest.

Background

Tumour mutation burden (TMB) is a biomarker for cancer immune checkpoint blockade (ICB) response, and mutation signatures provide a “life history” of tumour evolution. Researchers have repurposed cancer gene panels (CGPs) to cheaply estimate these parameters. The low coverage of CGPs, however, may be problematic in the exploration of TMB as a biomarker for ICB. Restriction enzyme associated DNA sequencing (RADseq) uses restriction enzymes to achieve coverage at random loci throughout the genome. It was hypothesised that RADseq could recapitulate mutation signatures and estimate TMB in breast cancer samples.

Methods

In silico RADseq libraries were generated by scanning the human genome for restriction enzyme cut sites and filtering for regions with adjacent cut sites. The resulting fragments were overlapped with mutations from whole genome sequencing (WGS) of breast cancer genomes. RADseq libraries were evaluated in terms of the accuracy of TMB estimation and mutation profile recapitulation, and compared to a CGP used for TMB estimation in terms of these parameters. A mouse breast cancer cell line was transfected with APOBEC enzymes, which are known to cause mutations in breast cancer, and profiled using RADseq.

Results

Using cosine similarity (CS) to the WGS profile as a measure of mutation profile recapitulation quality, RADseq libraries were able to recapitulate WGS mutation signatures, while the CGP performed relatively poorly in this regard (e.g. enzyme_1 CS=0.90±0.06 vs CGP CS=0.28±0.19). The CGP had higher TMB estimation error than an enzyme library of comparable coverage (CGP range 0.0034-8.83 mutations/mb vs enzyme_2 range 0.0029-7.50, Wilcox p<0.001). Using RADseq, an increased TMB and the APOBEC mutation signature was detected in a mouse cell line transfected with APOBEC3A, both of which were absent in control cell lines.

Conclusions

CGP methods do not accurately capture “genome-wide” mutation parameters such as TMB and mutation signatures. This warrants investigation into whether these methods can be used in TMB estimation in ICB trials. By comparison, RADseq may offer a cheap, effective solution for TMB estimation. RADseq may also be of use to researchers seeking to study the evolution of mutation signatures in model systems, as we have demonstrated.

Legal entity responsible for the study

The authors.

Funding

Breast Cancer Foundation New Zealand.

Disclosure

All authors have declared no conflicts of interest.

Background

Copy number alterations (CNAs) are common genetic features in cancer with prognostic and therapeutic implications. At our institutional Molecular Tumor Board we introduced an ultra low-coverage Whole Genome Sequencing (ulcWGS, <0.5x coverage) to estimate numerical karyotype and CNAs. We performed this technique on 128 solid tumor samples from 2018 to 2022. Here we report 3 exemplary cases in which we detected potentially targetable CNAs via ulcWGS.

Methods

DNA was extracted from microdissected Formalin-Fixed, Paraffin-Embedded tissue slides. ulcWGS libraries were prepared using the NEBNext Ultra II Kit (New England Biolabs, Ipswich, MA, USA). Sequencing was performed on an Illumina MiSeq Instrument and raw reads were analyzed with a proprietary read-depth based software developed recently for calculated karyotyping of Acute Myeloid Leukemia. We used immunohistochemistry (IHC) and targeted sequencing (VariantPlex, ArcherDX/Invitae, Boulder, CO, USA) (VP) to validate CNAs.

Results

In patients reported in the table ulcWGS uncovered amplifications (amp) in FGFR1, ERBB2 and MDM2, respectively. In patient 1, IHC staining confirmed moderate to high (3+) membrane positivity for FGFR1, despite negativity of VP analysis. In patients 2 and 3, high-level amplifications were also confirmed via VP. Due to unavailability of clinical trials at the time of referral, just patient 2 received a matched therapy, particularly off-label trastuzumab deruxtecan (T-Dxd). At a 6-month follow-up, the patient showed a sustained stable disease on imaging and a tumor marker decrease.

Table: 106P

Patients’ characteristics

Conclusions

ulcWGS is a sensitive low throughput assay compatible with benchtop sequencing instruments. The method can provide clinicians with highly relevant information that valuably adds to panel-based analyses in the context of precision oncology.

Legal entity responsible for the study

The authors.

Funding

Philipps University Marburg University Hospital Gießen and Marburg, Campus Marburg.

Disclosure

F. Rodepeter: Financial Interests, Personal, Invited Speaker: AstraZeneca. C. Denkert: Financial Interests, Personal, Advisory Board: MSD Oncology, Daiichi Sankyo, Molecular Health, AstraZeneca, Roche, Lilly; Financial Interests, Personal, Invited Speaker: Merck, AstraZeneca, VmScope digital pathology software; Financial Interests, Personal, Ownership Interest, Cofounder and shareholder of Sividon Diagnostics until 2016: Sividon Diagnostic; Financial Interests, Institutional, Funding: Roche; Financial Interests, Institutional, Research Grant: Myriad. E. Mack: Financial Interests, Personal, Invited Speaker: Roche, Boehringer Ingelheim; Financial Interests, Personal, Advisory Board: Roche. All other authors have declared no conflicts of interest.