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Basic Science

15P - The analgesic compound palmitoylethanolamide reduces inflammation in human cardiomyocytes and vascular endothelial cells exposed to doxorubicin and anti-HER2 monoclonal antibody through PPAR-α and NLRP3-related pathways

Presentation Number
15P
Speakers
  • Simona Buccolo (Napoli, Italy)
Date
Sun, 11.09.2022

Abstract

Background

Palmitoylethanolamide (PEA) is an endogenous fatty acid mediator that is synthetized from membrane phospholipids by N-acyl phosphatidylethanolamine phospholipase D. It is a new analgesic drug with anti-inflammatory effects through the induction of PPAR-related pathways. We aimed to assess whether palmitoylethanolamide co-incubated during doxorubicin and trastuzumab, reduces anticancer drugs-related cardiotoxicity in cellular models.

Methods

Human vascular endothelial cells and cardiomyocytes were exposed to subclinical concentration of doxorubicin (at 100 and 200 nM) combined to trastuzumab ( at 100 and 200 nM) alone or in combination with a formulation composed by palmitoylethanolamide 500 nM for 48h. After the incubation period, we performed the following tests: determination of cell viability, through analysis of mitochondrial dehydrogenase activity, study of lipid peroxidation (quantifying cellular Malondialdehyde and 4-hydroxynonenal), intracellular Ca2+ homeostasis. Moreover, pro-inflammatory studied were also performed (activation of NLRP3 inflammasome; expression of peroxisome proliferator-activated receptor-α; mTORC1 Fox01/3a; p65/NF-κB and secretion of cytokines involved in cardiotoxicity (Interleukins 1β, 8, 6).

Results

Palmitoylethanolamide co-incubated with doxorubicin exerts vasculoprotective and cardioprotective effects, enhancing cell viability of 56.3-78.7 % compared to untreated cells (p<0,001 for all). Notably, PEA reduced significantly the cardiotoxicity through peroxisome proliferator-activated receptor-α –related pathways and NLRP3 inflammasome but without the involvement of calcium homeostasis. Several cytokines and chemokines were also reduced confirming its anti-inflammatory effect.

Conclusions

The present study demonstrates that palmitoylethanolamide protects against vasculotoxicity and cardiotoxicity of doxorubicin and trastuzumab by promoting an anti-inflammatory phenotype, representing a new therapeutic approach to resolve doxorubicin-induced vasculo-cardio toxicity and inflammation.

Legal entity responsible for the study

The authors.

Funding

Ricerca Corrente, Ministero della Salute.

Disclosure

All authors have declared no conflicts of interest.

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Basic Science

16P - Development of a 3D lung cancer model and evaluation of the efficiency of a human antibody directed against a novel target

Presentation Number
16P
Speakers
  • Eleana Hatzidaki (Florina, Greece)
Date
Sun, 11.09.2022

Abstract

Background

In-vitro cell models have been employed for the study of lung cancer progression and metastasis. Most often, these models are 2D cell cultures and are not representative of cancer complexity and cell interactions in vivo. Immune therapy, alone or in combination with chemo- or radio treatment is an attractive alternative for lung cancer management. We have designed a 3D model of lung cancer composed of bronchial epithelial, fibroblasts, epithelial, lung adenocarcinoma and immune cells grown in different layers in a matrigel.

Methods

Genes upregulated in the 3D culture were evaluated and an antibody against a specific over-expressed protein produced. The biological efficacy of the produced antibody on the expression of gene and various cell markers was evaluated. Expression of cell markers was determined using flow cytometry. Antibody was produced using immune cells cultured ex-vivo and activated against an immunogenic epitope of the protein-target. Antibody efficiency was calculated using gene and flow cytometry analysis.

Results

By genetic analysis, BMPR2 was one of the genes found over-expressed and was chosen as the antibody target. Lung cancer model was found to have increased expression of markers like Epcam (over expressed in lung cancer) and Notch (promotes tumor initiation). Analysis of the immune cell population only, showed an increased in the expression of CD25 (activated T and B cells), CD80/86 (APC), CD206 (macrophages), and also CTLA4 (down regulation of immune system). Incubation of the lung model with anti-BMPR2 antibody, decreased cell growth, increased immune marker expression (CD206) and decreased expression of genes involved in cancer progression (CD44, FOS, NRAS, ARAF).

Conclusions

We have developed a 3D cell culture model for lung cancer. We have identified a potential target for immunotherapy and developed and antibody against the specific target. It was found that addition of anti-BMPR2 antibody in our lung cancer model increased the expression of genes involved in immune activation and decreased genes involved in tumor progression. Our antibody could potentially be a novel therapeutic antibody for lung cancer treatment.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Basic Science

17P - Preclinical mouse model of palbociclib/fulvestrant resistance in hormone-receptor-positive breast cancer

Presentation Number
17P
Speakers
  • Yong Wha Moon (Seongnam, Korea, Republic of)
Date
Sun, 11.09.2022

Abstract

Background

Breast cancer is a leading cancer in global women cancer incidence. Although cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor combined with endocrine therapy is a highly effective therapy for hormone receptor (HR)-positive breast cancer, acquired resistance ultimately occurs in almost all cases. Preclinical in vivo model is essential to study mechanisms and to develop overcoming strategies of CDK4/6 inhibitor resistance.

Methods

We performed xenografting with HR-positive breast cancer cell line in nude mice. When the tumor size reached 50-100 mm3, palbociclib ± fulvestrant treatment was initiated. This resulted in an initial dose-dependent decrease in xenograft tumor size and subsequently acquired resistance occurred (>25% regrowth from maximal reduction) after 8-9 months. Mice were sacrificed and xenograft tumors were harvested. Using xenograft tumors, RNA microarray and whole exome sequencing (WES) were performed to find out resistance mechanisms of fulvestrant/palbociclib.

Results

We successfully established preclinical mouse model of palbociclib/fulvestrant resistance. RNA microarray revealed SNORA14B (-3.09X), TPT1(-2.33X), SNORA74A (-2.32X), SYNPO2 (2.12X), S100A7A (-2.10X), SNORD10(-1.64X), KIR2DL2 (-1.61X), PGM5 (-1.55X) as palbociclib-resistance genes. In addition, WES revealed ACLY, PRB4, and SMPD1 genes as palbociclib/fulvestrant combination-resistance genes. We also found fulvestrant resistance genes such as AGR3, ELOVL2, GFRA1, GREB1, IGF1R, IGKV1-17, NRIP1, PPM1K, TM4SF1 by WES. Those resistance genes are under further validation.

Conclusions

We established preclinical mouse model of palbociclib/fulvestrant resistance in HR-positive breast cancer. In addition we found out palbociclib/fulvestrant resistance-associated genes using this model. Our palbociclib/fulvestrant-resistance mouse model could be used for drug development to overcome CDK4/6 inhibitor and/or fulvestrant resistance.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Basic Science

18P - Adoptive cell therapy (ACT) with bispecific gamma delta TCR+ invariant TCR+ NKT-like cells for multiple myeloma (MM): Comparison of preconditioning (PC) on outcome

Presentation Number
18P
Speakers
  • Theresa A. Deisher (Seattle, United States of America)
Date
Sun, 11.09.2022

Abstract

Background

AVM0703 (AVM) is the subject of an actively enrolling US trial in relapsed/refractory (R/R) no-option Non-Hodgkin’s Lymphoma (NHL), with drug related adverse events limited to grades 1-3 and durable complete (CR) and partial responses (PR) to date. Acute supra-pharmacologic doses (>6 mg/kg) of AVM0703 mobilized endogenous bispecific gdTCR+ invTCR+ Natural Killer T-like cells (AVM_NKT) (PCT/US21/19773) in mice, humanized mice and clinical trial patients1. An ACT model was conducted to verify the direct tumor killing activity of AVM0703 induced novel immune cells (AVM_NKT) and to determine the effect of different PC on tumor killing of the ACT.

Methods

MOPC315 (Balb/c) MM cells were inoculated into the flank as single cell suspensions. When tumor volume reached ∼400mm3 well-established tumors, mice were PC’d with human equivalent dose (HED) cyclophosphamide-fludarabine (CyFlu HED 500/30 mg/mm2), with Placebo (PL) or with AVM0703 HED 18 mg/kg. ACT splenocytes (3.3M) from mice dosed with PL or AVM were iv injected 48 hours later (PL_ACT had 8,240 AVM_NKT and AVM_ACT had 150,000). The next day (13-16 hours later) the mice were euthanized and remaining live MOPC cells were measured in the tumor, spleen, bone marrow, thymus and blood by flow cytometry.

Results

PC was necessary for AVM_ACT effect since there was no difference in live MOPC cells between AVM_ACT versus PL_ACT in mice PC’d with PL. AVM PC was equivalent to CyFlu PC against MOPC in tumor and spleen when AVM_ACT were transferred. Neither AVM nor CyFlu PC (with AVM_ACT) reduced MOPC in blood, bone marrow or thymus compared to PL PC. Repeat daily AVM and CyFlu dosing for PC will be explored in subsequent studies.

PC Placebo AVM AVM CyFlu CyFlu
ACT AVM Placebo AVM Placebo AVM
* P<0.05 versus Placebo PC with Placebo ACT Tumor * * *
Spleen * * *
Blood
Bone marrow *
Thymus

Conclusions

AVM0703 could be a less-toxic PC agent before CarT or other ACT. 1 Blood 2021:138(S1) 4557

Legal entity responsible for the study

AVM Biotechnology, LLC.

Funding

AVM Biotechnology, LLC.

Disclosure

T.A. Deisher: Financial Interests, Personal, Officer, I am the Founder, CSO and a Board member of AVM Biotechnology, LLC: AVM Biotechnology, LLC; Financial Interests, Personal, Ownership Interest, I am the Founder of AVM Biotechnology, LLC and own 41% of the units: AVM Biotechnology, LLC. S.H. Sawas: Financial Interests, Institutional, Full or part-time Employment, Full-Time Employee at AVM Biotechnology since August 2020: AVM Biotechnology; Financial Interests, Personal, Ownership Interest, PIU Shares in the currently private company: AVM Biotechnology. All other authors have declared no conflicts of interest.

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Basic Science

19P - Gene expression profile (GEP) of extensive small-cell lung cancer (eSCLC) patients (pts) receiving first-line platinum-etoposide plus atezolizumab (PEA)

Presentation Number
19P
Speakers
  • Martina Lorenzi (Padova, Italy)
Date
Sun, 11.09.2022

Abstract

Background

First-line systemic treatment with PEA is a new standard of care for eSCLC. No predictive biomarkers for pts selection have been identified so far.

Methods

This is a single-center prospective translational study on eSCLC pts receiving first-line PEA, investigating the predictive value of circulating (cell-free DNA Next Generation Sequencing, microRNA profile and Telomerase Reverse Transcriptase -TERT- mRNA levels) and tissue (tumor microenvironment, TME; GEP and proteomics analyses) biomarkers. We report preliminary data of GEP on tumor samples in a training set of eSCLC pts. GEP was performed analyzing 770 immune/cancer-related genes by the Nanostring® PanCancer IO360 panel. Gene expression signatures were correlated with activity and efficacy endpoints. Survival measures were categorized according to median value.

Results

Twenty pts were included; median follow-up was 12.8 months. Overall response rate was 63%, median time to treatment failure (TTF), progression free (PFS) and overall survival (OS) were 5.0 (95%CI, 3.9-6.1), 5.0 (95%CI, 3.9-6.1) and 7.2 months (95%CI, 7.1 -7.4), respectively. A differential expression of genes encoded for costimulatory molecules and cytokines/chemokines was reported between responders and non-responders and in pts with longer versus shorter PFS. Responders showed upregulation of cell proliferation genes (p=0.037) and downregulation of the Notch signaling pathway (p=0.06). While no prognostic and predictive impact of the absolute CD8+ T-cells and cytotoxic T-cells signature scores was evidenced, lower cytotoxic T-cells/tumor-infiltrating lymphocytes (TILs) signature scores ratio (ssr) was associated with shorter PFS (p= 0.006), TTF (p=0.001) and OS (p=0.052). High T-cells/TILs ssr (p=0.02), mast cells/TILs (p=0.003) ssr and low macrophages/TILs ssr (p=0.04) were associated with better outcome.

Conclusions

We identify predictive and prognostic immune signatures in eSCLC receiving chemo-immunotherapy through GEP analysis. These data will be confirmed in a wider validation set of pts with a longer follow-up and complemented with TME and circulating biomarkers analyses.

Legal entity responsible for the study

University of Padua.

Funding

University of Padua.

Disclosure

L. Bonanno: Financial Interests, Personal, Advisory Board: AstraZeneca, MSD, BMS, Roche; Financial Interests, Personal, Invited Speaker: AstraZeneca, MSD, BMS, Roche; Financial Interests, Personal and Institutional, Invited Speaker: AstraZeneca. V. Guarneri: Financial Interests, Personal, Advisory Board: Roche, Eli Lilly, Novartis, MSD, Gilead; Financial Interests, Personal, Invited Speaker: Eli Lilly, Novartis; Financial Interests, Institutional, Invited Speaker: Eli Lilly, Roche, BMS, Novartis, AstraZeneca, MSD, Synton Biopharmaceuticals, Merck. G. Pasello: Financial Interests, Personal and Institutional, Advisory Board: AstraZeneca, BMS, Boehringer Ingelheim, Eli Lilly, MSD, Novartis, Pfizer, Roche, Takeda; Financial Interests, Institutional, Principal Investigator: Amgen, AstraZeneca, Roche, Eli Lilly, Novartis; Financial Interests, Institutional, Funding: AstraZeneca, Roche. All other authors have declared no conflicts of interest.

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Basic Science

20P - Liquid biopsy of early-stage lung cancer using extracellular vesicles

Presentation Number
20P
Speakers
  • Razelle Kurzrock (La Jolla, United States of America)
Date
Sun, 11.09.2022

Abstract

Background

Lung cancer is one of the most common and deadliest cancers with a 5-year survival rate of ∼ 20%. Approximately 70% of cases are detected when the disease is regional or distant and have lower survival rates than localized cancers. Improving the number of cases detected in a localized state could lead to better patient outcomes.

Methods

Non-small cell lung cancer (NSCLC) plasma samples were tested for protein biomarkers carried by extracellular vesicles (EVs) following isolation and analysis using multiplex ELISA. In total, 161 lung cancer cases (Stage I = 88, Stage II = 34, Stage III = 20, Stage IV = 19) and 683 control subjects (no known cancer diagnosis) were analyzed. The case cohort consisted of 41% smokers with a median age of 63 years and 42% female population while the control cohort consisted of 12% smokers with a median age of 58 years and 52% female population.

Results

A machine learning algorithm identified an initial set of 35 informative EV biomarkers for differentiation of NSCLC cases from controls. The ROC showed an AUC of 0.987 (95% CI: 0.981 – 0.993) with a sensitivity of 97% (CI: 93% - 99%) at a specificity of 92% (CI: 90% - 94%). When performance was broken down by stage, we found similar performance across all stages tested with sensitivities of 98%, 94%, 95% and 100% for stages I – IV, respectively. We then reduced the number of biomarkers used from 35 to 11 to identify the most informative, yielding a 92% sensitivity at 92% specificity. To better understand performance in a future independent validation, we performed an in-silico experiment randomly partitioning the dataset into ten training and validation sets (67%/33%). This showed a mean AUC of 0.982 (range 0.968-0.991), mean sensitivity of 95% (range 85% - 100%) and mean specificity of 92% (range 86% - 95%) across the ten held-out validation sets.

Conclusions

Our pilot study suggests a potential approach for detecting lung cancer at early stages, when treatment can be more effective, by using a liquid biopsy test based on EV biomarkers. Our results warrant further testing with independent datasets for biomarker and algorithm validation. Future planned studies will also evaluate the impact of controls with confounding disorders, such benign lung nodules, as well as COPD to better evaluate the best use in the clinical setting.

Legal entity responsible for the study

The authors.

Funding

Biological Dynamics.

Disclosure

J.P. Hinestrosa: Financial Interests, Personal, Full or part-time Employment: Biological Dynamics. J.M. Lewis, H. Balcer, G. Schroeder: Financial Interests, Institutional, Full or part-time Employment: Biological Dynamics. P. Billings: Financial Interests, Institutional, Member of the Board of Directors: Biological Dynamics. All other authors have declared no conflicts of interest.

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Basic Science

21P - Circulating free and extracellular vesicles-derived microRNA as prognostic biomarkers in resected early-stage non-small cell lung cancer

Presentation Number
21P
Speakers
  • Paola Ulivi (Meldola, Italy)
Date
Sun, 11.09.2022

Abstract

Background

Non-small cell lung cancer (NSCLC) is the primary cause of cancer-related death. About 20% of cases are diagnosed at an early stage, allowing an effective pulmonary resection but many patients experience disease recurrence. Biomarkers able to identify patients with a higher risk of relapse could be very useful. Circulating microRNAs (miRNAs), in both extracellular vesicles (EV)-free and EV-encapsulated forms, represent promising markers in this setting.

Methods

This is a prospective observational cohort study on early stage (IA-IIIA) NSCLC patients undergoing surgery at 3 different centers (IRST-IRCCS, VHIO and CHU de Strasburg). The main end-point was relapse within 2 years from surgery. A peripheral blood sample for plasma collection was taken from each patient pre-surgery. Circulating free and EV-derived miRNAs were extracted from plasma. MiRNA-specific libraries were prepared with the QIAseq miRNA Library Kit and sequenced on Illumina NextSeq 550. Data were normalized and analyzed for differential expression using the edgeR R statistical software package. The TMM normalization method was used. The Benjamini-Hochberg procedure was used to adjust for multiple testing.

Results

A total of 220 patients were enrolled. Median age was 68.5 (range 43.6-84.7) years. 99 (46.5%) patients had stage IA, 38 (17.8%) had stage IB, 9 (4.2%) had stage IIA, 27 (12.7%) had stage IIB and 40 (18.8%) had stage IIIA tumors. 168 (76%) had adenocarcinoma histology, 42 (19%) squamous cell carcinoma, and 12 (5%) had other tumor histotypes. The overall relapse rate was 25%, and a relapse rates of 10.5%, 16.7%, 11.1%, 38.5% and 48.6% were obtained for stage IA, IB, IIA, IIB and IIIA, respectively. Overall, 16 and 7 EV and EV-free miRNAs, respectively, resulted differently expressed in relapsed vs non-relapsed patients. 4 miRNAs (hsa-miR-182-5p, hsa-miR-183-5p, hsa-miR-1246 and hsa-miR-196b-5p) were significantly up-regulated in relapsed patients,in both EV and EV-free comparts.

Conclusions

This preliminary analysis revealed that both free and EV-derived miRNAs seem to have a role in the identification of patients with higher risk of relapse after surgery. Complete statistical analysis is ongoing and will be completed soon.

Editorial acknowledgement

This project was supported by the Italian Ministry of Health (IT-MoH) within ERANET TRANSCAN-2 Joint Transnational Call for Proposals 2016 (JTC 2016) on: "Minimally and non-invasive methods for early detection and/or progression of cancer" (ERP-2016-23671110).

Legal entity responsible for the study

Paola Ulivi.

Funding

Italian Ministry of Health ERP-2016-23671110.

Disclosure

All authors have declared no conflicts of interest.

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Basic Science

23P - MRE-seq based cancer screening for lung and colorectal cancer by deep learning analysis of cfDNA methylation pattern cancer screening

Presentation Number
23P
Speakers
  • Sun Hye Shin (Seoul, Korea, Republic of)
Date
Sun, 11.09.2022

Abstract

Background

Among various genomic features in the blood, methylation pattern could be useful not only in determining whether cancer is present, but in revealing its origin. The purposes of this prospective study were to evaluate the diagnostic performance of methylation sensitive restriction enzyme digestion followed by sequencing method (MRE-seq) using cell-free DNA (cfDNA) in the diagnosis of cancer and to investigate tissue-of-origin (TOO) using deep neural network (DNN).

Methods

We have developed a deep learning-based prediction model with the hypomethylation data obtained from MRE-seq. One hundred ninety-one cancer patients with stages I–IV (95 lung cancer, 96 colorectal cancer) and 126 non-cancer participants were enrolled in this study. DNA isolated from each sample was subjected to MRE-seq to analyze the difference in methylation patterns between cancer tissues and normal tissues.

Results

We achieved the overall area under the ROC curve (AUC) 0.978 with sensitivity 78.1% at specificity 99.2% for colorectal cancer, AUC 0.956 with sensitivity 66.3% at specificity 99.2% for lung cancer. In colorectal cancer, the sensitivity is 76.5% in stage I, 76.2% in stage II, 78.3% in stage III and 83.3% in stage IV at specificity 99.2%. In lung cancer, the sensitivity is 48.5% in stage I, 44.4% in stage II, 78.9% in stage III and 76.0%enl in stage IV at specificity 99.2 %. The true positive rate (TPR) of TOO model was 94.4% (85/90), 89.9% (80/89) for colorectal cancer and lung cancer respectively.

Conclusions

Global hypomethylation pattern obtained by MRE-seq was useful in the diagnosis of cancer with high accuracy and in the determination of TOO through DNN. The role of MRE-seq needs to be validate for various solid tumors in the future multicenter studies.

Clinical trial identification

NCT 04253509.

Legal entity responsible for the study

Eone Diagnomics Genome Center, Republic of Korea.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Basic Science

24P - Preliminary data on INHERITY LC: Germline mutations of a cohort of selected non-small cell lung cancer (NSCLC) patients

Presentation Number
24P
Speakers
  • Maria Zurera (Zaragoza, Spain)
Date
Sun, 11.09.2022

Abstract

Background

Almost 15% of NSCLCs are not attributable to smoking risk factor and genetic predisposition to this disease has not been broadly studied, especially in non-Asian population. INHERITY LC is a multicentric cohort aiming to explore germline mutations in patients with NSCLC and elucidate the role of hereditary as risk factor for NSCLC. We present the first exploratory data.

Methods

Using a 61-gene panel next generation sequencing (NGS) we explore the presence of germline mutations in a cohort of 54 Spanish patients with NSCLC. Blood samples were collected from May 2021 to February 2022. Selection criteria were: 1) familiar history of lung cancer (first-degree relative or two or more second-degree relatives with NSCLC) 2) age criteria and negative or low tobacco exposure (diagnose < 45 years or < 60 years with < 15 packs-year index) 3) presence of somatic actionable mutations in the tumor biopsy.

Results

Median age was 57 years; 23 male (43%), 31 female (57%); 46 adenocarcinoma (85%), 7 squamous (13%); 51 Caucasian (94%); 21 patients with first-degree relative (39%), 4 with two or more second-degree relatives (7%); 28 somatic actionable mutations (52%): 16 in EGFR (29%), 10 in ALK (18%), 2 in ROS-1 (4%). Germline variants were identified in 18 patients (33,3%). 4 of them pathogenic or likely pathogenic variants (7,5%): 1 pathogenic mutation in BRCA2; 3 variants likely-pathogenic (VLP) were found in XRCC2, BRIP1 and NBN genes. Variants of uncertain significance (VUS) were described in 14 patients in BRF1, POLD1, BRCA2, ATM, MRE11, CDH1, POLE, MSH2, PDGFRA, KIT, MLH1 and APC genes. ATM and BRCA2 were the most common reported genes on this series. Germline variants were identified in 33% of patients with a first-degree relative with NSCLC, in 32% of patients with somaticactionable mutations and in 18% of patients with diagnose of lung cancer under 45 years.

Conclusions

One third of patients with lung cancer family aggregation, absence of tobacco exposure, young age at diagnosis or presence of actionable mutations may have germline variants in genes involved in DNA repair mechanisms, which could have a role in NSCLC hereditary predisposition. INHERITY LC is ongoing and final results with a larger sample and multivariable studies are underway.

Legal entity responsible for the study

Asociación para la Investigación del Cáncer de Pulmón En Mujeres (ICAPEM).

Funding

ICAPEM.

Disclosure

All authors have declared no conflicts of interest.

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Basic Science

25P - Identification of novel kinase-activating fusions in non-small cell lung carcinomas (NSCLCs)

Presentation Number
25P
Speakers
  • Evgeny Imyanitov (Saint-Petersburg, Russian Federation)
Date
Sun, 11.09.2022

Abstract

Background

Gene fusions involving protein kinases are druggable targets characteristic for a subset of NSCLCs.

Methods

NGS analysis for 650 kinase genes was performed for 38 RNA lung adenocarcinomas obtained from young-onset patients (age range: 33-50 years; median age: 44 years; 22 males and 16 females). All these tumors were negative for activating events involving EGFR, ALK, ROS1, RET, MET, NTRK1/2/3, BRAF, HER2 and KRAS genes.

Results

Kinome RNAseq revealed 67 chimeric transcripts in 38 NSLC cases. Five of these chimeras were recurrent, being a result of large intrachromosomal rearrangements and observed in 2-19 tumors each; however, none of these translocations predicted for a protein product. The majority of aberrations were frameshift mutations. 18 fusion variants were in-frame, but only four rearrangements preserved the entire kinase domain within a chimeric transcript: BCR-PKHD1 t(22;6)(q11.23;p12.3), CLTC-RPS6KB1 17q23.1del0.2Mb, CDC42BPG-ATG2A 11q13.1del0.05Mb and MAPRE1-DGKB t(20;7)(q11.21;p21.2). By the time of the abstract submission, BCR-PKHD1 and recently reported CLIP1-LTK [Izumi et al., 2021, PMID 34819663] gene fusions were analyzed in 2754 NSCLCs, which were negative for all known actionable mutations, however no new instances of these translocations have been observed.

Conclusions

This study has not replicated the data on the frequent involvement of CLIP1-LTK fusions in NSCLCs, however identified a number of other kinase gene fusions deserving further investigation.

Legal entity responsible for the study

The authors.

Funding

Russian Science Foundation, grant 17-75-30027.

Disclosure

All authors have declared no conflicts of interest.

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Basic Science

26P - NOTCH1 driven metastasis in BRAF mutated colorectal cancer

Presentation Number
26P
Speakers
  • Mark White (Glasgow, United Kingdom)
Date
Sun, 11.09.2022

Abstract

Background

10% of colorectal cancers (CRC) will have a BRAF mutation (BM). This represents a poor prognostic group. We have developed a novel, metastatic genetically engineered mouse model (GEMM) of BM CRC and derived a prognostic RNA signature.

Methods

3 genes were targeted with the intestine-specific villinCreER: activating Braf V600E/+ (B), deletion of Trp53 fl/fl (P) and activating NOTCH1 intracellular domain Rosa 26N1icd/+ (N) to generate BP (n=29) and BPN (n=50) mice. Mice were induced and aged until clinical endpoint. Neutrophils were targeted in BPN mice with AZD5069 (CXCR2 inhibitor) +/- anti-PD1 antibody or vehicle control from 85 days post Cre induction (DPI). NOTCH1 Activation RNA-Seq Signature (NAS) was developed from BP and BPN murine tumour derived organoids along with previously published Kras G12D PN RNA-Seq data (Jackstadt Cancer Cell 2019) by differential expression analysis (R 4.0.2, limma 3.46) of N+ and N- organoids.

Results

NOTCH1 activation resulted in a larger tumour burden in BPN mice (BP=16mm2 vs BPN=36mm2 p<0.01) with more invasive tumours (T4 disease rate BP=21% vs BPN=61% p<0.01). Median overall survival (OS) was unaffected (190 DPI BP vs 163 DPI BPN p=0.30). 32/50 (64%) of BPN mice developed metastasis compared with 1/29 (3%) of BP mice. BPN metastasis included peritoneal disease (38%) and liver (12%). Endpoint BPN mice had neutrophilia (median 1.2 K/uL BP vs 4.7 K/uL BPN p<0.01). Therefore neutrophils were targeted with CXCR2i +/- anti PD1. 7/12 (58%) of vehicle control mice had metastasis compared to 4/11 (36%) with CXCR2i and 0/7 with CXCR2i + anti PD1 combination (Chi2 p=0.04). Translating the NAS to confident orthologous genes and applying to human CRC datasets, high NAS resulted in a shorter OS (HR=1.26, log rank p=0.01) and shorter RFS (HR=1.26, log rank p<0.01). There were no significant differences in NAS between KRAS mutational status but NAS was higher in BM tumours (t-test p<0.01).

Conclusions

NOTCH1 activation promotes metastasis in a Braf V600E GEMM of CRC. Metastasis is reduced by neutrophil inhibition which synergises with anti PD1 therapy, supporting an actionable phenotype. The NAS identifies a poor prognostic patient group and future work will see if neutrophil targeted therapy could benefit these patients.

Legal entity responsible for the study

The authors.

Funding

Cancer Research UK.

Disclosure

O.J. Sansom: Financial Interests, Institutional, Funding: Novartis, Cancer Research Technology, Redx. All other authors have declared no conflicts of interest.

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Basic Science

27P - Influence of single nucleotide polymorphisms (SNPs) in drug transporters ABCB1 and ABCG2 on intracerebral osimertinib efficacy in patients with non-small cell lung cancer

Presentation Number
27P
Speakers
  • Marijn G. Veerman (Rotterdam, Netherlands)
Date
Sun, 11.09.2022

Abstract

Background

Brain metastases (BM) are present in >30% of patients with metastatic epidermal growth factor receptor mutation-positive (EGFRm+) non-small cell lung cancer (NSCLC). The tyrosine kinase inhibitor (TKI) osimertinib (OSI) and its active metabolites are substrates of ABCB1 and ABCG2 and metabolized by CYP3A4. In this study, we investigated the relationships between the SNPs ABCB1 3435C>T, ABCG2 421C>A and 34G>A, and CYP3A4*22 and the progression and new occurrence of BM in EGFRm+ NSCLC patients treated with OSI.

Methods

This observational cohort study was performed in six Dutch hospitals from Nov 2014 until Oct 2021. For patients with baseline BM, the primary study endpoint was cerebral progression-free survival (CNS-PFS), c.q. time from OSI start to cerebral disease progression. For patients with no or unknown baseline BM, the primary endpoint was cerebral disease-free survival (CNS-DFS), c.q. time from OSI start to the occurrence of new BM. OSI stop for other causes was considered a competing risk. The relationships between SNPs and baseline characteristics with CNS-PFS and CNS-DFS were studied with competing-risks Cox regression. Variables with p<0.20 in the univariable analysis were included in a multivariable analysis with backward selection.

Results

From 571 included patients, 201 had baseline BM. The table shows the statistical significant associations with CNS-PFS and CNS-DFS.

CNS-PFS HR 95% CI p-value
Primary EGFR mutation (L858R vs exon 19 deletion) 2.06 1.34-3.16 0.001
Presence of T790M at baseline 0.60 0.40-0.89 0.011
Any prior treatment other than an EGFR-TKI 1.55 1.02-2.35 0.042
CNS-DFS HR 95% CI p-value
Primary EGFR mutation (L858R vs exon 19 deletion) 3.01 1.44-6.31 0.004
Haplotype ABCB1 3435C>T wild type or ABCG2 34G>A variant(present in 35%) 0.27 0.10-0.71 0.007
Performance status (grade >1 vs 0-1) 2.60 1.14-5.94 0.023
Age 0.97 0.94-1.00 0.040
Single ABCG2 421C>A variant(present in 21%) 2.17 1.03-4.58 0.042

Conclusions

In EGFRm+ NSCLC patients treated with OSI, the SNPs ABCG2 421C>A, ABCB1 3435C>T and ABCG2 34G>A can impact CNS-DFS, and not CNS-PFS. Genotyping these patients may guide monitoring strategies aimed at early detection of brain metastases.

Clinical trial identification

The Netherlands Trial Registry, Trial Number NL8914.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

M.G.D. Veerman: Financial Interests, Personal, Invited Speaker: Lilly. R.H. Mathijssen: Financial Interests, Personal, Invited Speaker: Novartis, Servier; Financial Interests, Personal, Other, Patent pending: Pamgene; Financial Interests, Institutional, Research Grant: Astellas, Bayer, Boehringer Ingelheim, Cristal Therapeutics, Pamgene, Pfizer, Novartis, Roche, Servier. All other authors have declared no conflicts of interest.

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