Next-generation sequencing (NGS) of cell-free tumor DNA (ctDNA) has great potential for liquid biopsy in cancer diagnostics and to identify patients with actionable genomic alteration. This study, a prospective longitudinal study, focused in a cohort of metastatic cancer patients without standard effective active antineoplastic medical treatment options to establish the rate of patients with actionable genomic alteration and the rate of patients accessing medical treatment. The final objective was to determine the clinical performance based on non-invasive tumor sequencing.
We collected plasma of 10 metastatic gastrointestinal patients with known status of the RAS genes and microsatellites instability in tumor tissue. CtDNA was extracted from plasma and genomic alterations were analyzed by Guardant 360 (Guardant Health, Biosequence, OncoDNA), a next generation sequencing panel. This panel consists of 73 cancer related genes and is able to identify different types of genomic alterations. Informed consent was obtained from all patients.
We were able to identify 78 somatic mutations in total resulting in a median number of eight somatic mutations per patient. The most common altered genes are well known tumor suppressor and oncogenes like TP53, APC, KRAS, MYC andEGFR. At least one actionable alteration in plasma cfDNA were detected in eight from the 10 patients (80%) but the proportion of patients for which a genomic identified recommended therapy was available to effectively initiate the treatment were only 37,5% (3/8). In these patients, the identification of alterations like c-MET amplification, FGFR1 amplification or PIK3CA c.1633G>A (p.E545K) mutation, involved in clinically actionable pathways, allowed the selection of a specific therapy. For the rest of cases the main causes of non-access to medical treatment associated with a specific mutation were, among others, the advanced pre-treated patient and clinical trial logistical access difficulties.
Our findings confirm the percentage of cases with potentially druggable aberrations is similar to other studies using this strategy and emphasizes their clinical value to identify candidate therapeutic targets.
Dr. Enrique Lastra, Hospital de Burgos.
Asociacion Caballeros Purísima Concepcion.
A. Rodrigo, A. Terradez: Employee of Biosequence-OncoDNA. I. Faull: Employee of Guardant Health. All other authors have declared no conflicts of interest.
Despite recent advances, early high-risk HER2-positive breast cancers (BC) have a risk of relapse close to 20%, with no validated prognostic biomarker. Circulating tumour DNA has been described to have a prognostic value for some early BC. Most published studies monitored mutations previously identified on tumour tissue. An alternate way to monitor plasma from patients with HER2-positive BC might be to look for ERBB2/HER2 amplification.
We selected 40 HER2-positive high-risk early BC (cT ≥ 2 and/or N+ and/or residual disease after neoadjuvant chemotherapy) included in our institutional BC-BIO-IPC 2009-005 prospective program (NCT01521676). Plasma samples were collected at diagnosis and cell-free DNA (cfDNA) was isolated using the Qiagen EZ1 ccfDNA Midi kits. We then used commercial kits (BioRad) for ERBB2 and TERT (reference gene), and looked for ERBB2 amplification by digital PCR (BioRad QX200).
After DNA isolation from 1.5 to 2ml of plasma, cfDNA was eluted in 55µl of buffer. The median cfDNA concentration was 0.08 ng/μl (range <0.05 - 0.306, Qubit), with 25 cases having a concentration below 0.1 ng/μl. Median concentration was 4.68 ng/μl for metastatic cases. After droplet generation, 25 samples had enough droplets to allow the reading and analysis of ERBB2 amplification results. Of these samples, 52% were amplified, the others being equivocal. None of these samples were classified as "non-ERBB2 amplified". All controls were accurately classified. Except for age (p = 0.05, T-test), identification of circulating ERBB2 amplification did not correlate to usual clinicopathological features, including survival (but relapse (12%) and deaths (8%) rates were low in this set). However, it tended to be correlated to cfDNA concentration (p = 0.06, T-test).
ERBB2 amplification detection in plasma is feasible and can be observed for more than 50% of patients with early high-risk HER2-positive BC. If confirmed, these results may lead to propose plasma ERBB2 amplification monitoring at diagnosis and during follow-up, to optimize the systemic management in case of persistence or increase of ERBB2 amplification during or after standard treatments.
NCT01521676.
Institut Paoli Calmettes.
Novartis.
R. Sabatier: This work has been partly funded by Novartis. All other authors have declared no conflicts of interest.
Endometrial cancer is the most common neoplasm of the female genital tract in Western Countries, with an incidence of 150000 new cases/year. Despite its high frequency, limited data are available about its molecular features. It is known that phospholipids play a key role in the cellular proliferation and dissemination in many human diseases. Some previous studies have demonstrated the importance of the lysophosphosphatidic acids’ (LPAs) metabolism and their signalling pathways in human endometrial cells. LPAs are produced via phospholipase A2 (PLA2) and phospholipase D (PLD), also known as autotaxin (ATX).
In the present work, we performed a prospective study involving 60 patients divided into three groups after a hysteroscopic guided biopsy: the first included 26 patients with a histological diagnosis of endometrial cancer; the second was made of 5 patients affected by histologically evidenced endometrial hyperplasia without atypia; the third was composed of 29 patients characterized by benign condition. All the groups underwent hysterectomy, with either open or laparoscopic surgery, and a second endometrial biopsy on the surgical specimen, in order to perform a quantitative real-time PCR using pre-designed ATX/PLD primers. Significant differences between groups were determined via unpaired two-way or one-way Student's t test and ANOVA. A P-value <0.05 was considered statistically significant.
From our data, we demonstrated a strong positive correlation between the gene expression of ATX in the surgical specimen and the endometrial cancers. Notably, we found other statistically significant results from the analysis of the population affected by endometrial cancer. In particular, the expression of autotaxin gene was higher in the endometrioid histotype, in case of type I endometrial cancer versus type II, in pre-menopausal women, in obese patients (BMI> 30) or in women with diabetes.
We could consider a possible role of ATX as a biomarker of endometrial cancer, mostly in endometrioid histotypes, reflecting the pathogenic conditions of type I endometrial cancer.
University of Bari, Italy.
Has not received any funding.
All authors have declared no conflicts of interest.
Gastric cancer (GC) as the fourth common cancer over the world and responsible for the second cause of cancer-related deaths, has high incidence rate in Iran and especially in its North-Western province, Ardabil. In this study, the circulating hTERT mRNA and DNA were studied in patients affected with GC in Ardabil.
Using conventional PCR and RT-PCR, Q-PCR and Q-RT-PCR, evaluating the circulating hTERT nucleic acids in the plasma samples of the 100 GC cases and the same number of sex-, age-, and residence place adjusted healthy volunteers, as the control group has been done.
The circulating hTERT mRNA level in patients was higher (40X) than the controls. In the case of circulating DNA, cases had higher difference (65X). These findings have been evaluated as significant difference. There were no significant relationships between hTERT level and age groups or adenocarcinoma type among cases.
The elevated hTERT mRNA/ DNA plasma level among patients indicates the accuracy of the proposed subjects. However, performing cohort studies on circulating hTERT mRNA is advised.
Ardabil University of Medical Sciences.
Has not received any funding.
All authors have declared no conflicts of interest.
More than 50% of melanomas harbor BRAF V600 mutations and can be treated with targeted therapy. Circulating tumor DNA (ctDNA) harbors the same genetic alterations as a tumor origin. The analysis of ctDNA provides diagnostic and prognostic information before, during treatment and at progression. For detection mutations in ctDNA droplet digital PCR (ddPCR) most often are used. This method is very analytically sensitive, but expensive and time-consuming.
We developed a biochip for simultaneous detection six somatic BRAF mutations (V600E [c.1799T>A], V600E [c.1799_1800delTGinsAA], V600M, V600K, V600R, V600D) in small amount of ctDNA. For preferable amplification of mutated over WT DNA we used nested LNA clamp PCR. PCR fragments were labeled via incorporation of Cy5-dTTP during the second round of PCR and hybridized with specific oligonucleotides immobilized on a biochip. Method allows detecting 0.05% mutated DNA in a background of WT DNA. We tested 35 ctDNA samples from melanoma patients. The ctDNA was isolated from 2-5 ml of plasma. For 10 patients plasma collected prior to surgical intervention. For 25 patients plasma collected during antitumor therapy. For all patients we knew BRAF status in tumor.
All ctDNA samples were tested by biochip assay and droplet digital PCR (in duplicate). For each assay we used 5 μl of ctDNA. The concordance of results was observed in 32/35 samples. In two cases mutations were detected only by biochip assay. These samples were collected prior surgical intervention and tumor DNA from these patients harbored coincident mutations. In one case mutation V600E was detected by biochip assay, but in ddPCR result we detected one positive drop in each repetition, but this isn’t enough to consider this sample V600E-positive. In general, ctDNA BRAF status strong correlated with tumor status and disease progression.
The biochip assay is a sensitive method for BRAF mutation detection in ctDNA. This method can be useful in diagnosis and treatment monitoring of melanoma patients. In addition, the biochip assay has the potential for multiplex analysis in contrast to ddPCR. It is very important for the study of small amounts of ctDNA.
This work was supported by Grant of the President of the Russian Federation (# MK-2519.2017.4).
Engelhardt Institute of Molecular Biology.
This work was supported by Grant of the President of the Russian Federation (# MK-2519.2017.4).
All authors have declared no conflicts of interest.
Circulating cell-free DNA (cfDNA) analysis can serve as a powerful diagnostic tool. However, most of the modern techniques used in oncology nowadays are based on expensive NGS procedures on the nuclear DNA. Here we propose a new approach for the development of cheap diagnostic tests based on cfDNA fragmentation profiles.
We used NGS data from cfDNA to obtain the coverage distribution by fragments of different lengths on healthy donors and patients with colorectal cancer. Based on fragments joint positioning we developed a method for selecting the characteristic and reference genome regions. The reference region was chosen as the area where the coverage levels have the greatest positive correlation between cancer samples and controls. The characteristic region is the region with the highest negative correlation between cancer patients and healthy individuals. Characteristic regions were chosen nearby the transcription start site (TSS) of the gene transcripts with the greatest expression difference between colorectal cancer samples and controls. In the end, we evaluated the results of the in silico qPCR in the selected regions to predict the diagnostic value of the characteristic and reference fragments.
We found several cfDNA biomarkers in the suppressor genes, among them KIF1B, LPAR1, APC, FN1, SEPT9. The results obtained from in silico qPCR on the selected loci coincided with the expected patterns.
The developed model for the detection of the characteristic cancer-specific loci based on the cfDNA analysis can be used as a design tool for the simple qPCR diagnostics systems.
Atlas Oncodiagnostics, LLS.
Atlas Oncodiagnostics, LLS.
All authors have declared no conflicts of interest.
Primary or acquired resistance to trastuzumab limits targeted treatment efficacy. Circulating tumor (ct)DNA is a potential surrogate to tissue that overcomes intratumoral heterogeneity with a minimal invasive approach. We assessed genomic alterations (GAs) of ctDNA and demonstrated molecular changes during trastuzumab therapy.
Prospective genomic profiling of longitudinal plasma samples from seven HER2-positive metastatic gastric cancer (GC) patients treated with trastuzumab-based chemotherapy, using the AVENIO ctDNA Surveillance targeted gene panel and specifically assessing ERBB2 and MET copy number variations (CNVs) status.
ctDNA sequencing data was available for four patients both at baseline and progression. At baseline, 75% of GAs identified were single nucleotide variations (SNVs), including common mutated genes such as TP53 (12.5%), PIK3CA (12.5%), KRAS (12.5%), and MET (12.5%). Twenty-five per cent were copy number variations (CNVs), specifically ERBB2 amplification. At progression, almost the majority of GAs were acquired (45.5%). MET amplification was the most frequently detected GA (26.7% of cases). ERBB2 amplification (13.3%), MET (13.3%), TP53 (6.7%), PIK3CA (6.7%), and ERBB2 (6.7%) mutations were other co-occurring GAs.
Follow-up ctDNA profiling during trastuzumab treatment identifies emergence of MET amplification and mutations, as a potential biomarker of resistance.
Research Ethics Committee of Hospital del Mar.
Roche.
All authors have declared no conflicts of interest.
Ovarian carcinomas (OvC) are the fifth cause of death by cancer in women and display a very poor prognosis (5-y survival of 30%). Genomic alterations involved in OvC can be identified only by invasive tumour sampling using surgical procedures or guided-biopsies, and are thus not easily monitored during treatment and follow-up. In the last years, circulating tumour DNA (ctDNA) has been explored in many fields for several tumour localizations. Concerning OvC, ctDNA detection is feasible and its correlation with response to treatment has been mentioned for relapsing disease. However, the capacity of ctDNA to be an early marker of relapse has not been described.
This prospective, national, multicentre, observational study (NCT03302884) is exploring the capacity of ctDNA to be an early marker of OvC recurrence after front-line treatments, i.e. to show significant modifications before clinical, radiological (RECIST v1.1) or biological (CA-125) diagnosis of relapse. CtDNA events are defined as the re-appearance of mutations non-detectable after treatment or an increase of mutant allele fraction (at least one log above the nadir). Tumour and germline DNA are collected to identify somatic mutations by panel-based NGS (Illumina NextSeq500). Patients are then classified according to their mutational profile with a specific DR subset of cases with DNA repair genes alterations. After cell-free DNA isolation, somatic mutations identified by tumour NGS are analysed using droplet digital PCR (BioRad QX200) to reach the highest level of sensitivity. Considering that the rate of ctDNA increase before diagnosis of relapse is significantly higher than the null value 0.70 (H0), assuming a true accuracy rate equal to 0.78 (H1), using a unilateral right-sided Z-score test, α = 5%, and β = 20%, the required sample size is 202, including 80 in the DR group. To fit the inclusion criteria, patients must be diagnosed with an ovarian, tubar or primitive peritoneal carcinoma without previous treatment and requiring (neo)adjuvant chemotherapy. The planned enrolment period is 2 years. Twenty patients have been included until June 2018 in the first opened centre.
NCT03302884.
Institut Paoli Calmettes.
AstraZeneca.
All authors have declared no conflicts of interest.
EGFR T790M mutation is associated with acquired resistance of lung cancer (LC) to first-generation TKIs. Several studies suggest that LCs often contain a small fraction of T790M-mutated cells even before the treatment, and the persistence of these mosaic clones renders poor tumor response to gefitinib or erlotinib.
We utilized a spectrum of methods to detect EGFR T790M allele in tumor and normal human tissues.
Allele-specific PCR (AS-PCR) analysis of treatment-naïve tumor samples revealed somatic T790M mutation in 3/394 (1%) LCs carrying TKI-sensitizing EGFR mutation, but in none of 334 LCs lacking EGFR exon 19 deletions (ex19del) or L858R substitutions and in none of 235 non-lung carcinomas. Cis-/trans- analysis of the ex19del and the T790M was performed on samples from two treatment-naïve LCs and revealed the location of both mutations in the same allele. In one of these tumors, T790M mutation was present only in a fraction of ex19del-containing cells, suggesting that the T790M was acquired later than the ex19del during natural tumor evolution. None of 920 LCs analyzed carried EGFR T790M in germ-line. Use of highly-sensitive and quantitative assays, such as ddPCR and NGS, produced high number of T790M-specific signals even in presumably T790M-negative DNA specimens. This background noise was evidently higher in degraded DNA isolated from formalin-fixed paraffin-embedded tissues as compared to high molecular weight DNA. Combination of AS-PCR, ddPCR and NGS revealed mosaic EGFR T790M allele only in 2 (3%) out of 68 LCs treated by the first-generation TKI. Both these tumors produced evident and durable response to gefitinib.
The occurrence of EGFR T790M mutation in treatment-naïve LC samples is significantly lower than reported in some previous studies. Interestingly, the emergence of EGFR T790M allele is linked to the presence of TKI-sensitizing mutations even in the absence of drug exposure, thus suggesting a biological interaction between these two genetic events. The detection of mosaic EGFR T790M mutations may be compromised by yet unresolved technical issues and may have limited clinical value.
N.N. Petrov Institute of Oncology.
AstraZeneca (Research Agreement with the N.N. Petrov Institute of Oncology); Russian Science Foundation (grant 17-75-30027).
All authors have declared no conflicts of interest.
Anemia not only impairs the quality of life of patients, but it leads to tumor hypoxia, resistance to therapy and reduces survival. Although recombinant Epo has revolutionized the treatment of anemia, clinical trials suggested a potential adverse effect of Epo on tumor recurrence and patient survival. Previously we identified altered EpoR mRNA transcripts in breast cancer cells lacking ligand binding domain exon 1-3 sequences in vitro. Our hypothesis is that altered expression of these proteins may be associated with unique clinicopathologic features.
The expression level of C-and N-terminal portion of EpoR were measured by quantitative RT-PCR in 157 retrospective cohort of archived breast cancers. In order to identify the variant EpoR mRNA forms we have established a SMART RACE PCR assay using EpoR specific primer sets producing large overlapping sequences. Total RNA was frozen sections of 20 primary breast cancers after the H&E diagnoses.The products of the SMART RACE and nested PCR reactions was subjected to sequence analysis.
Out of the 157 samples 95 (61%) showed more than 2-fold change between the expression levels of the C and N-terminal regions of EpoR by quantitative RT-PCR. Significant correlation was found between the expression levels of EpoR mRNA variants and lymphatic invasion of the tumors. Logistic regression model demonstrated the association of metastatic capacity and expression differences of extra and intracellular regions of EpoR in primary breast cancers (P < 0.05, greater than 2-fold change). Using a SMART RACE PCR assay a single EpoR mRNA species at ∼1500 kb, corresponding to the full length wild type EpoR mRNA, and several distinct bands at ∼1300 kb, ∼1000 kb and ∼800 kb, lacking exon 1-4 N-terminal ligand binding domain, were seen in the samples.
We showed that different variant forms of EPOR present not only in vitro but also in the individual breast tumor samples. These altered forms of EpoR may be responsible for the observed differences in the Epo responsiveness of EpoR bearing breast cancers.
Zsuzsa Rakosy.
OTKA PD 116996, 2015-2018.
The author has declared no conflicts of interest.
Trans-urethral resection of tumor (TURT) is the main treatment of non-muscle invasive urothelial carcinoma, but it is associated with high rate of recurrence and or progression, this arouses the need for adjuvant therapy. Topoisomerase II, KI67 and P53 are proliferation and cell cycle regulator markers that may predict tumor response to therapy. This study aimed to assess Top II, KI67 and P53 expression on clinical outcome and response to therapy of non-muscle invasive urothelial carcinoma.
Fifty cases of non-muscle invasive urothelial carcinoma were collected; Top II, KI67, and P53 expression were evaluated. Patients received treatment then recurrence was correlated with the expression of previous markers.
There was a significant association between high Top II score, P53 and Ki67 and high grade (P = 0.0001, 0.001, 0.0001), submucosal infiltration (P = 0.0001, 0.01) and recurrence (P = 0.01, 0.001, 0.001).
Top II, P53, and Ki-67 may predict response to therapy and the clinical outcome in non-muscle invasive urothelial carcinoma.
The authors.
Has not received any funding.
All authors have declared no conflicts of interest.
Cystectomy is the prime treatment of muscle-invasive urothelial carcinoma but it is associated with many complications and affects patients’ quality of life. Chemotherapy is an alternative modality, but it may not give the expected response. This arouses the need for markers that help to predict the response to chemotherapy. Her2-neu, Skp2 and HIF-1 regulate cell cycle progression, tumor adaptation to hypoxic environment and response to chemotherapy. This study aimed at evaluation of HER2-neu, SKP2 and HIF1 expression in muscle-invasive urothelial carcinoma and investigating the association between their expression and tumor response to chemotherapy.
One hundred specimens of non-metastatic muscle-invasive urothelial carcinoma were collected at the Pathology Department and the selected patients received the treatment and were followed up at the Clinical Oncology Department, Menoufia University. HER2-neu, SKP2 and HIF-1expression were evaluated using immunohistochemical techniques. The patients received chemotherapy followed by cystoscopic examination. Bladder biopsy was examined to determine tumor response.
A significant association was found between partial tumor response to chemotherapy and HER2-neu, SKP2 and HIF-1 positive expression (P = 0.004, 0.029 and 0.004,). Skp2 expression was significantly associated with low apoptotic count and high mitotic one (P = 0.008 and 0.01), while HIF-1 expression was significantly associated with necrosis (P = 0.008). A statistically significant association was found between Skp2 and HR2-neu expression (P = 0.018) and between SKP2 and HIF-1 expression as well (P = 0.013).
This study showed that HER2-neu, SKP2 and HIF-1expression can predict poor response to chemotherapy in muscle-invasive urothelial carcinoma and helps in selecting patients who will benefit from chemotherapy. In addition, targeted therapy against these markers can be effective in treatment.
The authors.
Has not received any funding.
All authors have declared no conflicts of interest.
Reactivation of interspersed repetitive sequences due to loss of methylation is associated with genome instability, one of the hallmarks in cancer. LINE-1 hypomethylation represents global methylation loss and has potential diagnostic and prognostic biomarkers in tumors. However, the correlation of LINE-1 hypomethylation with clinicopathological determinants and CpG island methylator phenotype (CIMP) in patients with liver tumors is not yet well defined.
We performed quantitative DNA methylation analysis in LINE-1, RASSF1A, and CCND2 genes using pyrosequencing in human hepatocellular carcinomas (HCC, n = 40), hepatocellular adenoma (HCA, n = 10), focal nodular hyperplasia (FNH, n = 5), and respective peritumoral liver tissues as well as healthy liver tissues (n = 5).
We found loss of LINE-1 DNA methylation only in HCCs and significantly correlated with poor survival (log rank test, p = 0.007). Inverse correlation was found between LINE-1 with RASSF1A DNA methylation levels (r2 = -0.47, p = 0.002). LINE-1 hypomethylation correlated with concurrent RASSF1/CCND2 hypermethylation (Fisher’s exact test, p = 0.02). LINE-1 hypomethylation and RASSF1A/CCND2 hypermethylation were not found in benign hepatocellular tumors (HCA and FNH) representing specific aberrations only in malignant liver transformation.
LINE-1 hypomethylation might serve as a future predictive biomarker to identify HCC patients with unfavorable overall survival and CIMP-phenotypes.
Medizinische Hochschule Hannover, Germany.
DFG Deutsche Forschunggemeinschaft.
All authors have declared no conflicts of interest.
One of the possible mechanisms of cisplatin resistance of tumors is a 6-phosphogluconate dehydrogenase (6-PGD) activation [R. Lin, 2015]. Investigation of the 6-PGD inhibition by selective small-molecule compound named Physcion is likely to be studied. However, there is a problem with selection of a less toxic solvent due to hydrophobicity of Physcion. This study was designed to optimize conditions and to study features of a combined action of cisplatin and Physcion on cell lines of pancreatic cancer AsPC-1 and lung carcinoma H1299.
For resazurin viability assay cells were cultured in 96-well plates (2000 cells/well) with 0-150 uM of Physcion in different solvents or in combination with cisplatin (0-128 uM). To detect a level of reactive oxygen species (ROS) by DCFDA (2’,7’–dichlorofluorescin diacetate) cells were cultured in 96-well plates (25000/well) with 0-150 uM of Physcion alone as well as in combination with cisplatin (1-16 uM).
There were no differences in cell viability between an action of solvents (DMSO and ethanol) and inhibitor in the range of 0-1000 uM of Physcion and 1-5% of solvents accordingly. Decreased cell viability in AsPC-1 by 35% and in H1299 by 40% was detected at 150 uM of Physcion dissolved in 3% ethyl acetate. Since ethyl acetate was not toxic for cells at this concentration it was used for further experiments. Combination of Physcion with cisplatin led to an increased cisplatin sensitivity of AsPC-1 cells in 1.4-1.6 times, H1299 cells in 1.9-2.2 times, as well as to an increase of ROS level in both cell lines in 1.3-1.7 times and 1.3-2.5 times, respectively. It was found that the level of ROS increased under Physcion treatment starting from 10 uM Physicion, while the decrease in viability began at 100 uM of Physcion.
As a result of the work it was shown that ethyl acetate was less toxic for cells. Combination of Physcion and cisplatin led to an increased cisplatin sensitivity of AsPC-1 and H1299 cells, as well as to an increased level of ROS. The obtained data is important for designing new schemes of chemotherapy and for further study of molecular mechanisms of resistance to chemotherapy.
Kazan Federal University.
The work was performed according to the Russian Government Program of Competitive Growth of Kazan Federal University.
All authors have declared no conflicts of interest.
Chronic lymphocytic leukemia is the most common adult leukemia and often presents as early-stage disease. It could be stable for many years with no or minimal intervention. CD62L is a one of the selectin family of adhesion molecules which plays an important role in the trafficking/homing of lymphocytes to the lymph node. The objective of this study was to evaluate CD62L expression in chronic lymphocytic leukemia, and its effect on survival of malignant B-cells.
This study was conducted on 35 patients of chronic lymphocytic leukemia and 15 age & sex matched healthy individuals as a control group. All patients were subjected to full history taking, clinical examination and laboratory investigations. CD62L/CD19 expression on lymphocytes was measured for all the study subjects using Flowcytometry technique before and after cell culture.
CD62L/CD19 expression increased minimally in control subjects at day 7 compared to day 0 (The mean ±SD 33.9±5.3 and 18.03±3.4 respectively) (P-Value < 0.001), while the diseased group showed marked elevation at day 7 than day 0 (The mean ±SD 90.7±6.4 and 36.9±17.2 respectively) (P-Value < 0.001). CD62L/CD19 expression in patients were higher than the control subjects at day 0 (The mean ±SD 36.9±17.2 and 18.03±3.4 respectively) and about three folds increase at day 7 (the mean ±SD 90.7±6.4 and 33.9±5.3 respectively) (P- Value < 0.001).
There is high expression of CD62L on lymphocytic cells in chronic lymphocytic leukemia patients associated with increased survival of malignant B-cells.
The authors.
Has not received any funding.
All authors have declared no conflicts of interest.
P53 polymorphism involves the substitution of an arginine for a proline at codon position 72. Many studies have investigated a genetic link between this variation and response to treatment in cancer. This study aimed to study p53 codon72 polymorphism in relation to cytogenetic response to imatinib treatment in patients with chronic myeloid leukemia (CML).
This study was conducted on 54 CML patients presented to Clinical Oncology Department, Menoufia University during the period from June 2013 and April 2015. They were classified according to their cytogenetic response to imatinib therapy into 40 CML patients cytogenetic responder to imatinib and 14 CML patients cytogenetic non responder to imatinib. Patients were genotyped for P53codon72 polymorphism using Polymerase Chain Reaction (PCR). Follow up of the patients should be done after 3, 6, 9, 12 and 18 months after diagnosis. And was done by CBC, conventional cytogenetic and FISH.
Age, gender, hematologic and cytogenetic response to imatinib in CML patients did not differ significantly among P53 codon72 genotypes (arg /arg, arg /pro and pro /pro) (P = 0.44, p = 0.45 and p = 0.11 respectively). P53 codon72 polymorphism did not significantly alter the risk to imatinib cytogenetic unresponsiveness (P = 0.9221).
It could be concluded that P53 codon72 polymorphism is not associated with imatinib unresponsiveness in CML.
The authors.
Has not received any funding.
All authors have declared no conflicts of interest.
The 100,000 Genomes Project aims to improve cancer care for NHS patients in the UK through personalised medicine. Our target is to return whole genome sequencing (WGS) results to clinicians in a clinically meaningful timescale to help with diagnosis and treatment choices for patient and in parallel to provide a research platform of genomic data linked to longitudinal clinical data. We demonstrate that WGA results for surgical resection or biopsy samples can be returned to clinicians within 2-3 weeks.
Currently our bioinformatics analysis of WGS includes somatic small variants and somatic structural variants, germline pertinent findings, mutational signatures and mutational burden. Work is underway to implement other genomic signatures for predicting therapeutic response for specific tumour types.
We present here an overview of clinical utility for reported outcomes. To date, bioinformatics reports for WGS, with links to potentially relevant therapies and UK clinical trials, have been produced for thousands of cancer patients in the UK. Our data suggests that WGS has the potential to affect patient management and facilitate an increase in recruitment to existing clinical trials. To iteratively develop a high-quality bioinformatics pipeline, we are obtaining feedback via the Interpretation Portal for Cancer (within the NHS) from Molecular Tumour Boards to improve our analysis. Further research analysis is being undertaken within the Research Embassy by Genomics England Clinical Interpretation Partnerships (
Future applications will include the utilisation of pan-genomic markers such as tumour mutational burden and genomic signatures to better stratify patients within the context of a clinical study.
Genomics England.
Government and NHSE.
All authors have declared no conflicts of interest.
As a mostly used epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), gefitinib significantly prolongs survival of lung adenocarcinoma patients with EGFR mutations. However, more than 10% of EGFR mutation-positive patients do not respond and a substantial fraction of responded patients progress after 8-12 months’ treatment. Identification of new biomarkers for EGFR-TKIs prognosis is vital. The objective of this study is to explore associations between MDM4 genetic variant and survival of lung adenocarcinoma patients treated with gefitinib.
384 patients with stage IIIB or IV lung adenocarcinoma were recruited between January 2009 and June 2013. Patients were treated with gefitinib orally at a daily dose of 250 mg as 1st-line monotherapy. MDM4 rs4245739 A>C genotypes were determined using MassArray system. Dual luciferase reporter gene assays evaluated the function of MDM4 rs4245739 genetic variant in lung adenocarcinoma cell lines A549 and H1299. The differences of patient clinical characteristics were calculated by student's t test or χ2 test. Survival differences were examined by log-rank test. Multivariate Cox regression analysis assessed prognostic factors for PFS or OS. Two-sided P < 0.05 indicated a significant difference.
Among 384 patients, EGFR mutations were positive in 181 patients (47.1%). Median progression-free survival (PFS) and overall survival (OS) for all patients with the rs4245739AC genotype were significantly longer than that of the AA carriers (PFS: 22.9vs.10.9 months, P < 0.001; OS: 27.3vs.16.5 months, P = 0.003). Notably, in the EGFR mutant subgroup, individuals with MDM4 rs4245739AC genotype showed 14.1 months prolonged PFS (28.8 months vs. 14.7 months; P = 0.022) and 12.2 months prolonged OS (31.4 months vs. 19.2 months; P = 0.047) compared to the AA group. Reporter gene assays showed that the rs4245739A allele leads to significantly increased MDM4 expression in lung adenocarcinoma cells compared to the C allele (P < 0.05).
MDM4 rs4245739 genotypes may act as prognostic biomarker for patients’ survival to gefitinib therapy and offer help to individualized treatment in lung adenocarcinoma patients with EGFR mutations.
Nasha Zhang.
Has not received any funding.
All authors have declared no conflicts of interest.
In recent years, parallel sequencing technologies have become integrated into daily clinical practice. Many institutions use amplicon-based approaches for the detection of somatic mutations. However, these assays do not routinely detect chromosomal aberrations or copy number changes (CNVs), which are still widely analysed by FISH and IHC, and struggle with de novo fusions. The development of new technologies to detect all therapeutically relevant genomic alterations in a single assay is an ongoing process. This study aimed to evaluate the TruSight Tumor 170 assay, a hybrid capture-based assay, for the simultaneous detection of somatic gene mutations, gene fusions and CNVs and its implementation into routine diagnostics.
Until now 24 of 48 tumour samples with known genetic aberrations were evaluated including three control samples. DNA and RNA were extracted from formalin-fixed, paraffin-embedded tissue. After DNA shearing, DNA and RNA libraries were prepared with the TruSight Tumor 170 assay (Illumina) according to the manufacturer’s instructions. Sequencing was performed on the NextSeq (Illumina). For data analysis, variant calling was performed using the TruSight Tumor 170 app on BaseSpace Sequence Hub (Illumina), and for variant interpretation BaseSpace Variant Interpreter (Illumina) and the Molecular Health Guide Software were used.
DNA and RNA were successfully extracted. 22 of 24 DNA libraries were analysable with DNA concentrations between 9 – 120 ng DNA. All 24 RNA libraries were analysable with RNA concentrations between 1 – 85 ng. All previously known exonic mutations and 1 high-level MET amplification were detected. Intronic variants and low-level MET amplifications were not detected. 9 of 13 fusions and splice variant were confirmed with the TruSight Tumor 170 assay. The remaining 4 aberrations were false positive by the previous methods.
The TruSight Tumor 170 assay works well even with very low DNA and RNA concentrations in comparison to other methods and can be used in a routine workflow. The number of samples will be increased to 48 samples to evaluate the assay further. Additionally, automated solutions for DNA/RNA extraction from same slide might be needed.
Carina Heydt.
Illumina.
K. Stecker: Molecular Health GmbH. J. Neumann: Illumina. All other authors have declared no conflicts of interest.
Organoids are 3D structures that recapitulate morphological, functional and genetic characteristics of the tissue of origin. Grown as organoids, primary cells can be kept in culture long-term without the requirement for immortalization or genetic alterations. In this study, we report the establishment of organoids derived of Head and Neck Squamous Cell Carcinomas (HNSCCs), which are tumors that arise from the squamous epithelium lining the oral cavity, pharynx and larynx. Overall five-year survival of patients diagnosed with this tumor is poor, and although many of the mutations detected in these tumors are therapeutically targetable, introduction of these treatment into the clinic has failed.
Organoids are established from HNSCC biopsies or surgical resections. Subsequently, the organoids are characterized using histology and DNA and RNA sequencing. In vitro, organoids are tested for sensitivity to a range of drugs.
Here we report the establishment of organoids derived of HNSCCs. These structures seem to recapitulate genetic and histological features of the tumors of which they are derived. Upon drug exposure in vitro, these organoids show differential responses to different therapies, including cisplatin, carboplatin and the anti-EGFR antibody cetuximab, which are currently used in the clinic. Our data suggests that this system might have the potential to guide personalized therapy in the future. To validate these findings, we are currently setting up a study to test the predictive potential of patient-derived organoid lines for the first line treatment of HNSCC patients. We will expose tumor-derived organoids to these therapies. Retrospectively, organoid responses will be compared to patient response. The results of this study will show if, in this patient group, organoids can serve as a tool to guide therapy choice.
This work suggests that HNSCC-derived organoids might have the potential to predict patient response, a hypothesis we will hope to validate by the study described above. Retrospectively, we will correlation patient outcome to in vitro organoid response. This will help us determine the potential of this system to predict therapy response of patients with HNSCC.
Hans Clevers research group, Hubrecht Institute.
Has not received any funding.
All authors have declared no conflicts of interest.
The 100,000 Genomes Project is a central element of NHS England’s Personalised Medicine Strategy, which aims to progress the move from a ‘one size fits all’ approach to patient treatment to more effective personalised therapies. The driver for this lies in the relative inefficacy of generic treatments; leading to at least 40% of patients receiving treatments that have no clinical benefit. This project is an NHS transformation initiative and aims to embed operational processes for Whole Genome Sequencing (WGS) of patient samples into routine clinical practice.
The Manchester Cancer Research Centre (MCRC) Biobank has been coordinating central sample collection in Greater Manchester since 2008. Historically, this activity has fallen outside of routine clinical practice, however integration of Biobanking activity and clinical pathways has enabled Manchester to deliver this national project. Steps taken include: Introducing methods in histopathology for routine frozen sample processing and tumour content assessment Roll-out of a shared patient tracker to facilitate recruitment A two-tier consent model enabling biobank consent at the time of surgery, followed by a more detailed genomic consent once sample eligibility has been confirmed Detailed data capture of sample ‘failure’ reasons to improve sample conversion rate Routine refrigeration of ‘out-of-hours’ specimens so that frozen tissue can be collected the following day Development of a ‘biopsy pathway’ to ensure learning can be main-streamed for real life clinical utility post project.
To date, Manchester have submitted ∼900 samples for Whole Genome Sequencing. Key achievements include: Samples submitted from 18 distinct tumour types Reduction of sample failure rates by > 10% Successful introduction of remote consenting model.
Delivery of the 100,000 Genomes Project in Manchester is a clear demonstration of holistic working between research infrastructure and clinical service, which will ultimately transform personalised medicine for cancer patients in England.
NHS England.
Has not received any funding.
All authors have declared no conflicts of interest.
ctDNA-based molecular profiling of advanced cancer patients with multi-gene next-generation sequencing (NGS) panels is rapidly gaining traction in clinical practice given faster results and more comprehensive stratification. However, clinical outcomes of patients with genomic alterations in plasma ctDNA by NGS panels remain poorly described. Here, we describe outcomes in advanced NSCLC patients with actionable alterations identified in plasma by InVisionFirst™-Lung.
A pooled-analysis across advanced NSCLC patients with actionable alterations detected by amplicon-based NGS (InVisionFirst) analysis was performed. Patients treated with matched targeted therapies evaluable for disease control at 3 months were collated for clinical outcomes analysis, based on disease stage and class of therapy. All patients provided written consent approved by the institutional ethics committee under which the studies were conducted.
Of 82 patients evaluable for outcome analyses, 71 patients (87%) had disease control at 3 months of therapy with a 61% response rate. Breakdown of patients by disease setting and by genomic alteration along with their 3-month disease control rate are shown in the table below.
Clinical outcomes in patients who have been treated with targeted therapy based on actionable alterations detected by amplicon-based NGS ctDNA analysis by InVisionFirst are consistent with those reported based on tissue profiling. ctDNA molecular profiling using InVisionFirst is an accurate and reliable tool for the detection of clinically relevant molecular alterations in advanced NSCLC patients.
Inivata Ltd.
Inivata Ltd.
C. Morris, E. Green: Inivata employee and shareholder. All other authors have declared no conflicts of interest. Summary of patient disease control at 3 months by actionable variantPrior therapy for advanced disease Genomic alteration N Number still on targeted therapy, no PD at 3 months % still on targeted therapy, no PD at 3 months Untreated for advanced disease All EGFR mutation Braf v600 mutation ALK / ROS1 fusion 9 6 2 1 7 5 1 1 78% 83% 50% 100% Prior cytotoxic chemotherapy for advanced disease but no targeted therapy All EGFR mutation Braf v600 mutation ALK / ROS1 fusion 18 9 2 7 16 8 1 7 89% 89% 50% 100% Prior therapy with targeted therapy All EGFR mutation (49 with T790) ALK / ROS1 fusion 55 49 6 48 42 6 87% 86% 100% Overall 82 71 87%
Gefitinib is currently one of the mostly used epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) recommended for treating non-small cell lung cancer. However, the factors that predict treatment prognosis and drug resistance to EGFR-TKIs remain elusive. The objective of this study is to exam the association between leukocyte relative telomere length (RTL) and prognosis or drug resistance of advanced lung adenocarcinoma to gefitinib treatment.
In this study, three hundred and sixty-nine patients with stage IIIB or IV lung adenocarcinoma were recruited between January 2009 and June 2013. All patients were treated with gefitinib orally at a daily dose of 250 mg as first-line monotherapy. Leukocyte RTL of each patient was measured using quantitative polymerase chain reaction (qPCR) protocol on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) and calculated according to Cawthon’s formula. Differences in patients’ characteristics were calculated by Pearson’s χ2 tests or Student’s t test. Cox proportional hazard regression analyses were used to calculate univariate and multivariate hazard ratios (HRs). Survival differences were examined using the log-rank test. Two-sided P < 0.05 indicated a significant difference.
Among 369 patients, EGFR mutations were positive in 181 patients (49.1%). Compared to long RTL, short leukocyte RTL was significantly associated with poor prognosis in all patients after gefitinib treatment (overall survival: 12.9 months vs. 17.8 months, P = 1.2 × 10-4; progression free survival: 7.8 months vs. 13.0 months, P = 0.043). Additionally, statistically significant association between short leukocyte RTL and shorten OS still existed among the EGFR mutant patients with gefitinib treatment (HR = 1.65, 95% CI = 1.28-2.12; P = 0.006). Besides EGFR mutation status, short RTL also contributed to significantly elevated risk of gefitinib primary resistance (HR = 1.50, 95% CI = 1.05-2.15, P = 0.027).
Our results highlight the potential of leukocyte RTL as a novel biomarker in advanced lung adenocarcinoma treated with EGFR-TKIs and the possibility of patient-tailored decisions based on leukocyte RTL.
Ming Yang.
Has not received any funding.
All authors have declared no conflicts of interest.
Acute myeloid leukemia (AML) is an "epigenetic" disease in the sense that the most common mutations in AML are somatic mutations in genes that regulate DNA methylation and demethylation. An aberrant DNA methylation distribution is a functional event in the process of leukemogenesis and a target of epigenetic therapy. Although epigenetic drugs (demethylating agents, histone deacetylase inhibitors) have been already used in the clinic for AML treatment, they do not provide point activation of the target region. Epigenetic variants of AML typical for children are particularly poorly understood.
To identify DNA methylation markers most common for any molecular subtype of pediatric acute myeloid leukemia (AML) we have applied a method of unbiased differential methylation screening of the genomes and designed multiplex methyl sensitive PCR system of DNA methylation markers belonging to the promoter regions of the genes CLDN7, CXCL14, GSG1L, EGFLAM, SOX8, and TMEM176A/176B.
The system has a sensitivity of 90% for determining the malignant process. We have studied bone marrow samples from 43 children with AML. All patients were treated by decitabine and all-trans retinoic acid in complex with standard chemotherapy (CT). Methylation index (MI) was 0.197 ± 0.181 for patients with a myeloblasts content less than 40%, and 0.514 ± 0.222 for patients with myeloblasts content more than 40% (p = 0.000736). Methylation of the CLDN7, GSGL1 and EGFLAM genes is absent in the group with low MI. Patients with the initial content of myeloblasts less than 40% demonstrate absence of methylation on the 15th day after the start of the CT. The average MI decreases in the group with the initially high content of myeloblasts due to decrease in the frequencies of methylation of the genes CXCL14, TMEM176A/176B, GSGL1 and SOX8. The 5-day course of demethylation therapy with decitabine is accompanied by an increase in the content of blast cells and an equalization of the methylation profile.
With the marker system developed it is possible to evaluate the malignant progression of blast cells, which are considered morphologically normal after CT, demonstrating at the same time the abnormal methylation profile of tumor cells.
Research Centre for Medical Genetics.
Council on grants of the President of the Russian Federation for state support of young Russian scientists and on state support of the leading scientific schools of the Russian Federation.
All authors have declared no conflicts of interest.
The genomic landscape of tumors is highly heterogeneous. We aim at developing a tumor-agnostic and treatments-specific gene expression signature that could facilitate treatment personalization in the context of massive drug development.
To this end, we developed PREDMED®, a tumor gene expression signature composed of multiple drug targets normalized with gene expression from reference tissues. For the clinical evaluation, the relation between the score of each drug targets in the signature and the effect of the targeted therapies, has been evaluated in different cohorts of patients. Bayesian statistics have been used in these studies to compute the probability of the relation between drug targets scores and outcome values.
In a pilot retrospective cohort of 11 patients with clear cell renal carcinomas (CCRCC) treated in first line with sunitinib (anti-VEGFR2) (UroCCR), VEGFR2 scores were positively associated to response (5 complete or partial responses, 6 progressions) under sunitinib with a probability of 98% (Bayesian logistic regression). In a second retrospective cohort of 37 patients with metastatic colorectal cancers (mCRC) treated in first line with bevacizumab (anti-VEGFA) associated to standard chemotherapy (Indivumed), VEGFA scores were positively associated to Progression-Free Survival with a probability of 94% (Bayesian logistic regression). In both studies, no relation between drug target expression and outcome values was found without PREDMED® (probability of a positive association: 50 % for CCRCC and 51% for mCRC) or with a partial normalization method (probability of a positive association: 36 % for CCRCC and 65% for mCRC), suggesting an advantage of using the PREDMED® normalization method.
These two preliminary studies of different tumor types for patients treated with anti-angiogenics, give encouraging signals for the use of a treatments-specific gene expression signature. We aim at further evaluating the generalizability of PREDMED® on other tumor types and targeted therapies, including immunotherapies.
Adaptherapy.
Adaptherapy.
Personal fees from Amgen, Celgene, Teva, Boehringer-Ingelheim, Menarini, Bayer, Lilly, MSD, outside the submitted work. L. Verlingue: Consulting for Adaptherapy. D. Malka: Personal fees and non-financial support from Roche, Sanofi-Aventis, Merck Serono; D. Bagnard: Scientific advisor for Adaptherapy, shareholder. All other authors have declared no conflicts of interest.
Diagnosis and treatment of Cancer of Unknown Origin (CUP) continues to be a challenge. In the era of personalized medicine, genomic profile by Next-Generation-Sequencing (NGS) in addition to immunohistochemistry (IHC) tests may complement clinical features improving diagnosis and detecting targetable mutations. In the present study we have analysed the clinical utility of the OncoDEEP® CUP platform for tissue of origin assessment and genotyping in a cohort of CUP patients.
We conducted this multicentric prospective exploratory study across 22 institutions (September 2017- March 2018). The cohort included 60 patients with histologically diagnosed metastatic CUP and available FFPE tumor samples. The molecular analysis was performed using the OncoDEEP® CUP platform, which includes a deep genomic tumor characterization as well as a panel of IHC tests.
Patient's median age was 60 years [24-80], 61.7% were females. At time of diagnosis,48% presented a single metastatic site whereas 52% has multimetastatic locations. In 10% of the cases, the analysis could not be completed due to insufficient tumor samples. In the remaining 54 samples, a probable primary origin was assessed in 43 patients (80%). Potentially actionable mutations were found in 36 cases (61%). Follow-up information has been obtained so far for 46 of the 60 patients (76%). Interestingly, in 37 patients (80%) the treatment was changed after performing the genomic study. From these, in 33 cases (72%) the therapeutical decisions were based in the results obtained from the study: one patient was included in a clinical trial (3%); 2 patients received immunotherapy (6%); and the rest (91%) received chemotherapy based on the results of the immunohistochemistry tests (not on the NGS results). 43% of the oncologists who participated in the study had not previously considered the therapy given. The evaluation of response is ongoing.
The genomic study OncoDEEP® CUP has identified new therapeutical options, both standard and innovative. The fact that no analysed patient has received targeted therapy based on NGS results for the moment shows the current barriers to accessing these treatments also in this kind of patients with limited options and bad prognosis.
Hospital General Universitario de Valencia.
Spanish Institute for Foreign Trade (ICEX).
S. Sauvage: Employee of OncoDNA. All other authors have declared no conflicts of interest.
STAMPEDE is a multi-arm, multi-stage (MAMS) platform protocol. It has recruited more than 10,000 patients since 2005. Hormone-sensitive metastatic or locally advanced prostate cancer patients starting androgen deprivation therapy (ADT) have been randomised between a number of experimental treatment pairings, including docetaxel or abiraterone, shown to improve overall survival. Funded by PCUK, the STRATOSPHERE consortium (Stratification for rational treatment-oncomarker pairings of STAMPEDE patients starting long-term hormone treatment) has established a framework to enable molecular analyses of archival tissue from these patients. We present the optimised work-flow and preliminary data on feasibility of next-generation sequencing (NGS) of FFPE archival samples.
Approximately 95% of patients participating in STAMPEDE have consented to donate their archival tissue for research. Retrospective tumour block acquisition started in March 2017 and is ongoing. To date, FFPE blocks from 1310 (35%) consenting patients randomised between ADT and abiraterone and docetaxel +/- zoledronic acid comparison arms (3909 total participants) have been retrieved from 64/112 STAMPEDE sites.
Morphological review has identified cancer in 86% of cores. DNA and RNA from alternate sections have been extracted using the Zymo Quick-DNA and Quick-RNA FFPE kits. The mean amount of DNA obtained per core was 23ng (range: 0-82ng) from a mean of 8 sections (range 2-14) at 10um thickness per block. DNA has been subjected to low-pass whole genome sequencing and analyses of the first 100 cores from participants recruited to the SOC arm is ongoing. RNA is being extracted from cores with >50% cellularity and >60% tumour content to assess transcriptomic changes and integrate with genomic data.
The STRATOSPHERE consortium is addressing the challenge of exploiting FFPE material with low-output, poor-quality nucleic acids from a clinically-important randomised cohort with long-term follow-up. Molecular data will be linked to clinical outcome to test differences in treatment effect by molecular sub-type.
University College London.
Prostate Cancer UK.
C. Gilson: Grant support from Clovis Oncology. N. James: Grant support, drug supplies and distribution, lecture fees, and advisory board fees from Astellas Pharma and Novartis, grant support, drug supplies and distribution, and lecture fees from Pfizer, grant support, drug supplies and distribution from Clovis Oncology, and grant support, discounted drug supplies, lecture fees, advisory board fees, and travel assistance from Sanofi-Aventis. N.W. Clarke: Receives consulting fees, fees for serving on an advisory board, and lecture fees from Janssen Pharmaceuticals. M. Sydes, M.K.B. Parmar: Grant support and drug supplies and distribution from Astellas Pharma, Clovis Oncology, Novartis, Pfizer, and Sanofi-Aventis. S. Chowdhury: Honoraria, lecture fees, and travel support from Janssen Pharmaceuticals. R.J. Jones: Lecture fees, fees for serving on an advisory board, and publication and writing support from Janssen and grant support, lecture fees, and fees for serving on an advisory board from Astellas Pharma. D. Berney: Has acted as advisor for AstraZeneca, Merck and Roche and has been paid a grant by Myriad genetics for validation of their Prolaris RNA based test for early prostate cancer. G. Attard: Consulting fees from Veridex, Novartis, and Millennium Pharmaceuticals, consulting fees, lecture fees, and travel support from Roche/Ventana Medical Systems and Astellas Pharma, consulting fees and travel support from Medivation, Abbott Laboratories, ESSA Pharma, and Bayer HealthCare Pharmaceuticals, lecture fees from Sanofi-Aventis and Takeda, grant support from AstraZeneca, Arno Therapeutics, and Innocrin Pharmaceuticals, and royalties for a patent on “17-substituted steroids useful in cancer treatment” (GB9305269) being licensed by Janssen Pharmaceuticals. All other authors have declared no conflicts of interest.
Molecular profiling using liquid biopsy ctDNA is rapidly gaining traction in routine clinical practice. However, there has been variable degree of accuracy and performance published to date and lack of prospective data on clinical outcomes for patients with actionable genomic alterations in ctDNA biopsies. Here, we describe the clinical validation and utility of InVision platform (InvisionFirst™-Lung, InvisionSeq™-Lung) in a large prospective cohort of advanced NSCLC patients.
We performed a prospective, single-centre, observational study enrolling advanced NSCLC patients including treatment-naïve, on TKI treatment or at progression. ctDNA molecular analysis was performed using amplicon-based NGS (InVision platform) and where available, in tissue by Sanger sequencing or sensitive validated allele-specific technique. Clinical validation was performed for core gene variants of EGFR Exons 18-21, BRAF V600, MET Exon 14, ERBB2 Ins 20, ALK & ROS1 fusions, KRAS and STK11. Patients treated with matched targeted therapies evaluable for disease control at 3 months were collated for outcome analyses.
Of 362 advanced NSCLC patients recruited, 172 were treatment-naïve, and 190 were pre-treated patients with known tissue molecular profile (EGFR, BRAF, ALK, ROS1). For clinically-relevant gene variants, concordance agreement between ctDNA and tumor tissue analysis was 95% with 75% sensitivity and 97% specificity. Of 58 evaluable patients analysed to date, 83% had disease control at 3 months of therapy, with response rate and median progression free survival of 61% and 5.7 months, respectively.
Our data endorses ctDNA molecular profiling using InVision as an accurate and reliable tool for the detection of clinically relevant molecular alterations in advanced NSCLC patients, with clinical outcomes consistent with expectations based on historical tissue profiling.
Gustave Roussy.
Inivata Ltd.
K. Howarth, V. Plagnol, C. Morris, E. Green: Inivata employee and shareholder. All other authors have declared no conflicts of interest.Key gene variants Tissue and Liquid Tissue only Liquid only No call PPV NPV Sensitivity Specificity Concordance EGFR Exons18-21 7 1 2 78 77.8 98.8 87.5 (0.473-0.997) 97.5 (0.913-0.997) 96.6 (0.904-0.993) BRAF V600E 1 2 0 75 NA 0.67 33.3 (0.084-0.957) 84-0.900 100 (0.952-100) 97.4 (0.910-0.997) ERBB2 Exon 20 2 0 0 49 100.0 100.0 100.0 (0.158-1) 100.0 (0.927-1) 100.0 (0.930-1) MET Exon14 1 2 0 45 100.0 95.8 33.3 (0.008-0.906) 100.0 (0.921-1) 95.8 (0.875-0.995) ALK fusion 8 5 NA NA 61.5 (0.316-0.987) NA NA ROS1 fusion 1 1 NA NA 50.0 (0.126-0.987) NA NA KRAS 22 3 7 56 75.9 95.0 88.0 (0.688-0.975) 88.9 (0.784-0.954) 88.64 (0.801-0.944) STK11 2 1 2 17 66.7 94.7 66.7 (0.094-0.992) 94.4 (0.727-0.999) 90.5 (0.696-0.988) Core gene variants 44 14 10 320 81.5 95.8 75.9 (0.628-0.861) 97.0 (0.945-0.985) 93.8 (0.909-0.960) Overall panel 64 27 36 1078 64.0 97.6 70.3 (0.598-0.795) 96.8 (0.955-0.977) 94.8 (0.934-0.960)
Advances in molecular oncology and cancer genetics in the last 15 years have defined many of the key driving oncogenes in human cancer. Despite these successes it is now apparent that tumour cells adapt and develop acquired resistance to these targeted inhibitors so that disease progresses within 5-7 months.
In the framework of this project, Gustave Roussy and XenTech are joining forces to develop a panel of patient-derived xenografts (PDXs) derived from biopsies collected from these patients at the stage of acquired resistance. These PDX models will be used to improve knowledge on the mechanisms underlying resistance to treatment and to evaluate response to new treatments. In this perspective, the development of 75 PDX-AR (Active Resistance) models is planned over 5 years. All the models are maintained under the same therapeutic pressure the parental tumor was submitted to at the time of biopsy, and will be subjected to extensive phenotypic and genotypic characterization. To favor successful xenograft establishment, the first two passages were performed without drug treatment, which was applied from the third passage on.
As of June 2018, 48 PDX models have been successfully obtained from 137 patients (global take rate =30%). The take rates are 21% for prostate cancer (12/57), 26% for lung adenocarcinoma (15/58), 100% for bladder cancer (9/9), 8% for cholangiocarcinoma (1/12), and 55% for tumor from other origin (11/20). With regard to resistance to targeted therapy, we obtained 9 baseline models, 9 models resistant to FGFR inhibitor, 4 resistant to ALK inhibitor, 7 resistant to 3rd generation EGFR inhibitor, 2 resistant to Enzalutamide (AR inhibitor). Preliminary analyses indicate that PDX mimic the clinical scenario observed in the matched patient (10/12 models recapitulate the pharmacological response of the original biopsy).
The generation of PDX models from patients who develop acquired resistance to targeted therapies is feasible. Preliminary analyses suggest that PDX models mimic the clinical scenario supporting the relevance of these models.
NCT02517892.
Gustave Roussy.
Lombard Odier.
All authors have declared no conflicts of interest.
As previous studies have reported, hypermutated CRC account for approximately 15%-17% among all CRC. The proportion and number of patients with hypermutated CRC cannot be underrated. Additionally, hypermutated patients’ options of therapy are different, as they have a greater potential benefit from immunotherapy.
We sequenced tumor mucosa of CRC patients with more than 24-month follow up data in our center and identified mutation profiles of the hypermutated CRC as the training dataset (ZJU). We obtained patients from The Cancer Genome Atlas (TCGA) as the validation dataset. Recurrently mutated genes were combined to calculate a compound score by summation of a Cox PH weighted sum of mutations. Patients with higher-than-median score were segregated as high-risk group. Outcomes were analyzed with Kaplan-Meier and Cox regression analyses using Python (3.6.0) and R (3.4.0).
We constructed a 4-gene signature (ACVR2A, APC, DOCK2 and POLE), training in 45 patients in ZJU and validating in 24 in TCGA. Patients of the high-risk group showed worse survival (HR = 8.62, P = 0.0010) and increased risk of recurrence (HR = 8.40, P = 0.0011, after adjusting age, sex, and TNM stage). We further compared our prognostic signature with other risk factors [including microsatellite instability (MSI) status, POLE driver mutation, BRAF p.V600E, tumor mutational burden and TNM staging], which proved our four-gene signature was better. The subgroup analysis suggested that our 4-gene signature would be more powerful in MSI CRC.
Our 4-gene signature is a better predictor of survival and recurrence for hypermutated CRC, especially for MSI CRC. This risk classifier may help us identify patients with poor prognosis but better responding to immunotherapy.
Has not received any funding.
All authors have declared no conflicts of interest.
KRAS mutation status is crucial in treatment decisions regarding the use of EGFR tyrosine kinase inhibitors in colorectal cancer (CRC). However, genetic testing is not available for some patients, either because tissue is limited and/or tests are not routinely offered. Hence, we aimed to build a nomogram based on clinical factors for the prediction of KRAS mutations in CRC.
Colorectal cancer patients who had their tumors genotyped for KRAS mutation at Fudan University Shanghai Cancer Center (FUSCC) were retrospectively analyzed. Variables of interest were integrated in a multivariate logistic regression model.
A total of 759 hospitalized patients were extracted from FUSCC database. KRAS mutation presented in 40.1% (309/759) cases. Multivariate logistic regression suggested that female (OR 1.47, 95%CI 1.06-2.04), mucinous histology (OR 2.04, 95%CI 1.28-3.25), right-sided tumor (OR 1.65, 95%CI 1.13-2.39) and high levels of preoperative CEA (OR 1.45, 95%CI 1.03-2.03), CA19-9 (OR 3.87, 95%CI 2.70-5.53) and albumin/globular protein (OR 2.02, 95%CI 1.33-3.06) were significantly correlated with KRAS mutation status. A nomogram was established and showed considerable discriminating accuracy (AUC 0.744, 95%CI 0.709-0.779) in this cohort. Patients with the highest score had 88.6% chance to bear a KRAS-mutant tumor. Subgroup analysis based on metastasis status revealed a sound applicability of the established nomogram both in metastatic (AUC 0.723, 95%CI 0.666-0.781) and non-metastatic (AUC 0.753, 95%CI 0.707-0.798) CRC.
Six simple and easy-to-collect characteristics defined a useful nomogram to predict KRAS status both in metastatic and non-metastatic CRC with great predictive accuracy.
Guoxiang Cai.
Has not received any funding.
All authors have declared no conflicts of interest.
5-fluorouracil (5-FU) is widely used in the therapy of solid tumors, including breast cancer (BC). Toxicity remains a major limitation in the clinical efficacy of 5-FU. Developed approximately 50 years ago, this competitive antagonist of uracil is still not exhaustively studied in terms of its mechanism of action, and the potential of molecular markers in predicting its efficacy and toxicity is not yet fully unravelled.
A modified Reduced Representation Bisulfite Sequencing genome-wide DNA methylation analysis assay, XmaI-RRBS, was applied to 170 BC samples obtained before chemotherapy, and to 10 normal breast tissue samples. Unsupervised hierarchical cluster analysis was used to discern intrinsic DNA methylation BC subtypes; clustering uncertainty was assessed with pvclust R package using bootstrap permutation approach.
In a DNA methylation BC subtype enriched in triple-negative breast cancer samples we have identified statistically significant enrichment with the samples non-methylated at the promoter region of the NME1 gene, compared to all other DNA methylation BC subtypes. Another finding was abnormal methylation of the DPYS gene in a DNA methylation BC subtype enriched in HER2 positive tumors.
Most studies of 5-FU resistance have focused on germline status of thymidylate synthase TYMS and DPYD genes, and 5-FU Toxicity and Chemotherapeutic Response Panels for the detection of germline variants in these genes are now widely available. It has also been shown that increased expression in tumor cells of some enzymes from the 5-FU metabolic pathway such as DPYD is correlated with resistance to 5-FU. Here we demonstrate differential methylation of the 5-FU drug pathway mediator genes DPYS and NME1 between the DNA methylation BC subtypes. DNA methylation is easily detected by methylation sensitive PCR and does not require RNA extraction. Thus, we suggest that the existing germline DNA tests may be supplemented with the tumour biopsy DNA methylation analysis in order to provide better prediction of 5-FU response and toxicity.
Research Centre for Medical Genetics.
Russian Science Foundation (project No.18-15-00430).
All authors have declared no conflicts of interest.
Targeted therapies have been developed this last decade and considerably improved PFS and OS of patients with NSCLC. Next-Generation Sequencing (NGS) is commonly used for the detection of actionable mutations in a panel of genes and FISH or IHC are the gold-standard assay for the detection of rearrangements of ALK, RET, ROS1, NTRK1 and MET. The aim of this study is to evaluate the suitability and reliability of NGS assay for the detection of rearrangements compared to the results obtained using FISH and IHC.
86 FFPE samples, primary tumors or micro biopsies of patients with NSCLC, were analyzed in 7 European labs for this study. All samples were qualified by a senior pathologist prior to analysis. DNA and RNA were extracted from FFPE samples using Qiagen AllPrep kit. TruSight™ Tumor 15 and INCa panel kits were used for DNA library preparation and Archer® FusionPlex® panel kit for RNA library preparation. Both libraries were finally pooled and analyzed using illumina MiSeq sequencing system. Data were finally analyzed using Variant Studio software or Biomedical Genomics Workbench and Ingenuity Variant Analysis for SNV or indel and Archer Analysis software for rearrangements. SNV, indel and rearrangements found for each sample were finally compared to FISH and IHC results and previous DNA sequencing data when available in the labs.
86 DNA and RNA libraries were sequenced. Among 86 samples 8 failed fusion quality control, due to too low amount or too degraded input RNA. No FISH and IHC results were available for 12 samples. Rearrangements were found for 28 samples, 22, 5 and 1 with an ALK, ROS1 and RET rearrangements respectively. Among 74 samples with previously known rearrangements, all samples showed full concordance with previous methods.
RNA based NGS is a suitable and reliable alternative to FISH and/or IHC for the detection of rearrangements of ALK, RET, ROS1, NTRK1 and MET genes for the theragnostic management of patients with NSCLC. Further investigations are needed to determine if an “all NGS strategy” is more likely cost-effective and faster than a “FISH and NGS strategy”.
Institut de Cancérologie de Lorraine.
Illumina.
A. Harlé: All reagents used in this study were provided free of charge by Illumina and Archer. I. Vogl: Works at Genolytic GmbH. All other authors have declared no conflicts of interest.
Colorectal cancer (CRC) is diagnosed too late due to the almost asymptomatic course of the disease. Many issues of diagnosis, prognosis and predicting of the treatment efficiency have not yet been resolved. The search and validation of new reliable molecular markers of CRC are still relevant. The aim of this study is to analyze the levels of anti-glycan antibodies in the sera of CRC patients and healthy donors.
Sera from 44 patients with CRC (15 with rectal cancer,16 with right-sided and 13 with left-sided colon cancer) and 53 healthy donors were analyzed using hydrogel glycan microarrays. Microarrays were manufactured according to the technology developed in EIMB RAS. Evaluation of expression levels of IgM antibodies to glycans was carried out using a fluorescent sandwich immunoassay. Statistical significance of the differences between groups was estimated according to the results of Kruskal-Wallis test.
The levels of IgM antibodies to the panel of immobilized glycans were measured in serum samples. It was shown that IgM antibody levels to 9 glycans - di-GalNAcβ, Manα1-4Manβ, 3'-sialyl-TF, 6-Su-6'-SiaLec, 3,6-SiaTn, SiaLea, Zymosan A, Zymosan, Lea - differed in healthy donors and in patients with rectal cancer (p < 0.01). Levels of IgM antibodies to di-GalNAcβ were found to be significantly different between patients with right-sided tumors and patients with rectal cancer, to 3,6-SiaTn - between patients with left-sided tumors and patients with rectal cancer, and to glycan Zymosan in all three groups of patients.
The levels of IgM antibodies to the 5 glycans - 3'-sialyl-TF, 6-Su-6'-SiaLec, 3,6-SiaTn, Sia6H (type2) and Zymosan A - differed between healthy donors and CRC patients with moderately differentiated adenocarcinomas (p < 0.01).
The level of IgM antibodies to glycan 3'-O-su-Lea was significantly different between patients with and without regional metastases (p < 0.01).
Median IgM antibody levels to the number of glycans vary between groups of patients with different tumor locations and with different tumor grades. Significant difference for 3'-O-su-Lea IgM antibodies among CRC patients indicates its possible role as a marker of regional metastases.
P. A. Hertsen Moscow Oncology Research Center - branch of FSBI NMRRC of the Ministry of Health of Russia, Moscow Russian Federation.
Program of fundamental research for state academies for the period 2013 - 2020 (subprogram #0103-2014-003).
All authors have declared no conflicts of interest.
Nearly half of acute myeloid leukemia (AML) patients show a normal karyotype, being the most heterogeneous group of patients. Because cytogenetic alterations are important prognostic factors, cryptic copy number alterations could explain some of the heterogeneity. Microarray-based comparative genomic hybridization (aCGH) has recently been used to study hematologic malignancies and has shown increased diagnostic yield, especially for cryptic findings.
Paired blood samples were collected from 12 AML patients before and after treatment induction during 2015-2017. Written informed consent was obtained at Hospital Manuel Uribe Angel (Envigado-Colombia) and Clínica Somer (Rionegro-Antioquia). The clinical records were reviewed to obtain important clinical variables. After DNA isolation, we used the CytoScan® 750K Array that enables the detection high resolution copy number across the genome as well as providing allelic imbalance information from single nucleotide polymorphisms (SNPs). This high density array contains greater than 750,000 markers for copy number and approximately 200,000 genotype-able SNPs which provide high resolution copy number, accurate breakpoint estimation, and loss of heterozygosity (LOH) detection.
A total of 53 copy number variations (CNV) were found. Most of the CNVs found in the patients before receiving the induction treatment (85%) remained after completing the induction phase. Only 15% of the alterations found disapeared after treatment. More than half of the alterations found are LOH (58%), followed by deletions (32%), and additions (8%). Chr X was the most affected (15%), most of which were LOH. The alterations found are described in detail in the table.
Induction treatment does not cause recovery of cytogenetic alterations in most cases. The perspective is to expand the cohort and make a correlation between the molecular findings and the outcomes of the patients, to define new markers related to prognosis and response to treatment.
Instituto Tecnológico Metropolitano.
Colciencias (Grant 669-2014).
All authors have declared no conflicts of interest.Cases Sex Karyotype Leukemia subtype Blasts(%) Outcome CNV found before treatment CNV found after treatment 5 F No data M3 85 CR Add (3q23) Remains Del (9p21.1-p22.1) Disapear LOH 11p11.2 Disapear LOH 19q13.42 Disapear LOH todo el cromosoma X Disapear 10 F No data M1M2 74 CR Del 13q12.2 Disapear Remains LOH X p11.22 Appears LOH 7q11.22 11 F 46,XX (6) and hypodiploid (4) M4 38 R Del 3 p21.31 Remains Del 5q23.3 Remains Del 7q11.22q36.1 Remains Del 10q11.21q23 Remains Del 11q13.4 Remains Del 12p13.2 Remains Del 12q12q24 Remains Del 16q24.1 Remains Del 17p13.1 Remains Del 17q11.2q12 Remains LOH Xp11.23 Remains LOH Xq11.1q13.1 Remains 12 F 46,XX (5), t(8;21 q22q22) (15) M2 20 CR LOH Xq11.1q26 Remains 13 M Normal Not classified 68 R LOH 1q21.3 Disapear Del 10q11.22 Remains 15 M 46,XY,add(3p?), -5(10), +15(2), +M(2)CP 10. M1M2 47 PR LOH 3p25.1 Remains Add 6q21 Remains Mon 7 Remains 17 M 45, X(-Y), t(8;21)(q22;q22)(9) M0 66 CR LOH 2p22 Remains LOH 12q13 Remains Add 16p12 Remains 18 F Normal Not classified 26 CR LOH 3p21 Remains Del 15q11 Remains LOH Xq22 Remains 19 F No data M0M1 65 CR LOH 1q42 Remains Del 13q14 Remains LOH 21q21 Disapear LOH Xq11q21 Remains S3 M Normal M4 91 R LOH 1q22 Remains LOH 3q26 Remains LOH 4q Remains LOH 5q31 Remains LOH 6p25 Remains LOH 6q25 Remains LOH 7q34 Remains LOH 10p15 Remains LOH 11q14 Remains LOH 14q23 Remains LOH 15q14 Remains LOH 18p11 Remains LOH 18q12 Remains LOH 22q13 Remains Appears Del 7p14 S4 M 46,XY,t(15;17) M3 96 CR Del 7p21 Remains Del 9p24 Remains LOH 10q23 Remains Add17q21 Disapear 6 M No data M3 60 Died Normal 14 F 46,XX,t(15;17) M3 No data Died Del 7q11.21 LOH 19p13.3 LOH Xp11.3 LOH Xq11.1q27.2 Add Xq26.3
In the era of precision oncology, tumor genomic information is incorporated in clinical decision making to personalize treatment strategies; however, drawing clinically relevant conclusions from tumor molecular profiles are not straightforward if more druggable drivers or targets are identified, more drugs are linked to the same genetic alteration or evidence are conflicting. Here, we present the analysis of 704 lung cancer profiles using our proprietary algorithm and the personalized treatment protocols established by the rule-based artificial intelligence software, the RealTime Oncology Treatment Calculator.
Lung cancer samples (469 adenocarcinomas, 115 squamous cell carcinomas, 57 small cell carcinomas, 63 other histology) were profiled using next generation sequencing of 50-58-600 cancer genes, fluorescent in situ hybridization, and immunohistochemistry. Variants were classified by the Molecular Treatment Calculator algorithm, which dynamically aggregates and ranks all relevant scientific and clinical evidence to find the most efficient drug for the best target of the strongest driver in the given tumor.
1008 different genetic variants were identified. 417 (41%) variants were classified as drivers and 49 (5%) as non-drivers. No direct evidence was available for 541 (54%) of the alterations, 460 (85%) of which were classified as drivers by the algorithm based on the aggregated evidence related to the cancer gene itself or other mutations in the same gene. 354 (50.3%) patients had more than one driver mutations, where the algorithmic ranking of drivers and related compounds was particularly warranted. The algorithm linked molecular targets to 799 (90%) of the driver variants.
Molecular treatment calculator algorithm is an efficient tool to classify tumor genetic variants and link them to molecular targets and drugs based on the curated, continuously expanding evidence database and the more than 20 000 rules of the RealTime Oncology Treatment Calculator. Calculator results represent optimal input for Molecular Tumor Boards to design personalized treatment strategies.
Oncompass Medicine Hungary Ltd.
National oncogenomic and precision oncotherapy program funded by the Hungarian Innovation Agency.
All authors have declared no conflicts of interest.
Preoperative differential diagnosis of benign and malignant thyroid tumors remains a challenge in many cases, thus complicating the optimal planning of patients’ treatment. We have recently demonstrated the possibility of discrimination between benign and malignant thyroid lesions using serum autoantibody biomarkers. Here we further assessed whether a protein commonly overexpressed in malignant thyroid tumors may elicit an autoantibody response that may be used for serum-based preoperative differential diagnosis of thyroid nodular lesions.
A protein cyclin D1 (CCND1) commonly used as a tissue biomarker of malignancy in thyroid lesions was produced as purified recombinant protein, and its serological reactivity was assessed using Western-blot analysis in a cohort of 70 patients with various thyroid tumors (classical papillary carcinomas (n = 25), follicular-patterned carcinomas (n = 15), borderline «uncertain malignant potential» tumors (n = 10), benign follicular adenomas (n = 20)) and 30 healthy volunteers.
Autoantibodies against cyclin D1 (CCND1) were found in 4/25 (16%) and 1/15 (7%) of patients with classical papillary and follicular-patterned carcinomas, respectively. None of patients with non-malignant tumors and healthy volunteers demonstrated any autoantibody response against CCND1 (p = 0.037 compared to classical variant papillary carcinomas).
Malignant but not benign thyroid neoplasms may trigger specific autoantibody response against cyclin D1. This finding is in line with the known expression pattern of CCND1 and its role as a key downstream effector of hyperactivated MAP-kinase cascade. The 100% specificity of anti-CCND1 autoantibodies in detection of cancer may allow the use of this candidate serum biomarker for preoperative diagnosis of thyroid lesions, particularly in conjunction with other potential biomarkers to compensate for a low diagnostic sensitivity. This work is supported by grant 16-15-10423 from Russian Science Foundation.
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences Engelhardt Institute of Molecular Biology, Russian Academy of Sciences Engelhardt Institute of Molecular Biology, Russian Academy of Sciences.
Russian Scientific Foundation.
All authors have declared no conflicts of interest.
Patient stratification, through the use of personalized medicine tools, is supposed to optimize patient care, reduce toxicities and increase the risk-benefit balance as well as decreasing healthcare costs. However, few studies have assessed the impact of Next Generation Sequencing (NGS) on clinical decision making. We evaluated whether information on mutational profiles modified clinical practice and care for patients with non-metastatic and metastatic cancer in an observational impact study.
This study was a multicentric observational decision impact analysis conducted in seven sequencing platforms and 117 hospitals working with these platforms. The platforms were certified by the French National Cancer Institute (INCa). All NGS analyses performed between October 2013 and September 2016 for adult patients treated for lung, colorectal cancer or melanoma, both metastatic and non-metastatic, were included. We excluded analyses on constitutional mutations, somatic mutations for other cancers or NGS analysis with exclusive research purpose. Patients’ pathways and referral patterns were obtained from NGS prescription forms and interviews with biomolecular biologists and clinicians. We extracted anonymized data on NGS results from the platforms.
1213 patients from more than 117 centres were analyzed. Even if panels used were relatively homogeneous - less than 20kb (97%) and commercial kits (80%) -, we observed significant variability among practices. Depending on the initial structure of care and on the platform to which it was related, patients benefited from an NGS analysis at diagnostic (2 platforms) or later on (5 platforms), of a review of the medical file by a multidisciplinary meeting or not and of an unequal access to medical innovation on the national territory.
We observed an important variability among practice on national territory. Thus patient equality of treatment is questionable. In a healthcare and economic perspective, important topics still need to be assessed to get a better understanding of the global impact of NGS. Among the outstanding questions: how can disutility of care, such as avoided complications and toxicities, limitation of diagnostic wavering, be evaluated?
INCa.
All authors have declared no conflicts of interest.
Recently in cancer research has shown that tumors are highly heterogeneous. Cancer developments are driven by specific cells-cancer stem cells (CSCs)-which are also responsible for metastases and drug resistance. Many studies have proved the significant role of miRNAs in controlling the Metastasis in CSCs. So, the present systematic analysis based on literature mining and bioinformatics approaches was performed to find the miRNAs in breast cancer.
After systematic analysis, we found miR-200c and miR-30c as regulators of metastasis in breast cancer. Then, using mammosphere formation in serum-free condition and colony formation assay were conducted to check the stemness properties of MDA-MB231 breast cancer cells. After RNA extraction of mammosphere and primary breast cancer tissue, real-time PCR analysis was performed for metastasis genes (CDH1/2, SNAI1, TWIST1/2 and ZEB1) and the prior selected miRNAs. Finally, differentially expressed genes and miRNAs in mammospheres have been determined and compared with seven malignant breast tissues.
Mammospheres demonstrated higher colony-forming ability and has higher expression of metastasis genes in comparison to their parental. Also, they have shown down regulated miR-200c and up regulated of miR-30c. Interestingly, 3 metastatic patients with grade II/III who received neo adjuvant therapy have higher expression of metastasis and have shown down regulated miR-200c and up regulated of miR-30c in comparison to primary breast normal tissue like mammospheres.
This study validates the use of breast cancer cell lines as models to elucidate the nature of BCSCs that may represent a novel target for therapy. Additionally, by correlating EMT genes with two miRNAs expression we suggest that simultaneous reduction of miR-200c and increasing in miR-30c could be an indicator for resistance to treatment or secondary metastasis.
Department of Stem Cells & Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology & Technology, ACECR, Tehran, Iran.
Department of Stem Cells & Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology & Technology, ACECR, Tehran, Iran.
All authors have declared no conflicts of interest.
AMPLICON deep sequencing to determine gene mutations (mut) and copy number alterations (CNA) helps to understand tumors' molecular biology and to personalize therapy. At Institut Català d’Oncologia l’Hospitalet it is used to find the best treatment options. We aim to analyze its efficiency.
We use AMPLICON sequencing to analyze genomic profile and CNA of unresectable or metastatic breast cancer tumors from selected patients (pts) with eligible criteria for clinical trials: 18-75 years, Performance Status (PS) ≤1, non-relevant comorbidity, organ preserved function and not heavily pretreated. We obtained the informed consent from all pts. An observational prospective analysis was carried out to evaluate pts and tumor characteristics, mut and CNA, target therapy (TT) and survival.
Since December 2017, 45 pts were tested. Median age at diagnosis of metastatic disease was 51 years (29-70) and at molecular screening 55 (29-72). Most patients had PS 0. In the metastatic setting, 32 pts (71%) had luminal breast tumors, 8 (18%) triple-negative and 5 (11%) HER2+. Median number of hormonotherapy lines at screening was 1 (0-5) and 1 chemotherapy line (0-5). Median follow up was 4.5 months. Only 2 pts died during this analysis. Overall, 30 mut were found. 23 pts (51%) had at least one mut. Almost 60% pts had 1 mut. Median number of mut was 1 (1-3). 49% pts had Wild Type (WT) tumors. The most prevalent mut were: 30% in PI3K, followed by 23% in ESR1, 13% in TP53, 7% in PTEN, 7% in FGRF4, and 3% in APC and in NOTCH. CNA was reported in 16 pts (35%). Pts with mut or CNA that could potentially benefit from TT were estimated in 13 pts (28%). 2 pts are currently under TT with PI3K inhibitors (inh) and 5 will be consider at progression for personalized treatment (PI3K inh and TAS 120).
Personalized therapy by sequencing tumors is an option for pts who could benefit from TT in clinical trials. In our analysis we found less mut or CNA than those described in literature, may be due to the different population. Potential benefit from TT was only considered in less than half of pts with any genomic alteration maybe because some mut are not targetable as ESR1 or p53 and lack of follow-up. Genomic sequencing can be a useful tool to achieve personalized therapy if used in selected pts but further evidence is needed.
Institut Català d\'Oncologia L\'Hospitalet.
Institut Català d\'Oncologia i Vall d\'Hebron Institut d\'Oncologia.
All authors have declared no conflicts of interest.
MOST (My Own Specific Therapy) is a prospective, randomized, open-label, adaptive phase II trial conducted in patients (pts) with progressive solid tumors (any subtype) after at least one prior therapy in advanced setting. This trial aims to evaluate the clinical benefit of therapies targeting molecular alterations identified in the patient’s tumor. NovellusDx’ technology measures the functional impact of alterations including Variants of Uncertain Significance (VUS), and TA efficacy based on NGS findings.
Pts were treated with sorafenib 400 mg BID based on KRAS/KDR, HRAS and BRAF (non-V600) oncogenic mutations/amplification. After 12 weeks of treatment (induction period), pts with stable disease were randomly assigned to continuation or interruption of sorafenib. NGS data from the treated pts were blindly analyzed by the functional assay: synthesized mutated genes and florescent reporter genes were transfected into live cells and scanned in a microscopy system. The specific pathways activation was evaluated by quantifying the subcellular localization of the reporter proteins. Sorafenib response prediction based on the assay was compared with the best change tumor size assessed in the MOST trial, and the maintenance treatment duration.
17 patients treated for at least 12 weeks with sorafenib were analyzed. Tumor genomic alterations that indicated the sorafenib treatment consisted in KRAS mutations (n = 9), KRAS amplifications (n = 3), HRAS mutation (n = 3), KDR amplification/mutation (n = 2). However, NGS data showed additional molecular aberrations. Of which, the functional impact and response to TA of 18 unique variants in 10 different genes was measured. The correlation between the assay-based response prediction and the variation in the tumor volume of the patients will be presented.
Multidisciplinary molecular tumor board recommends TA based on single actionable oncogenic mutations. Taking into account significant other genomic variants is crucial to accurately predict targeted agent efficacy.
NCT02029001.
Centre Léon Bérard.
Fundation ARC, INCa.
O. Zelichov, Z. Barbash, Y. Daitsh, L. Birnbaum, G. Tarcic: Full time employee of NovellusDx. All other authors have declared no conflicts of interest.
Cost-effectiveness is one of the major limitations for tumor molecular profiling along with a small fraction of patients who can benefit from precision oncology approach. Our experience of providing an affordable molecular profiling (AMP) proves that focused biomarker panels and specific selection of patients for testing are the possible ways to overcome those obstacles.
NGS assays were performed with AmpliSeq targeted panels. Data were analyzed with a proprietary pipeline. Apart from routine searching for nonsynonymous substitutions, NGS data were processed with custom-built algorithms for identifying amplifications, splice-affecting variants and evaluation of tumor mutational load. Within our concept of AMP in some cases, the following techniques were used to identify the additional biomarkers: MSI-assay, FISH (gene fusions), PD-L1 and others.
First 79 patients were analyzed. The most common cancer types were: breast cancer - 17, colorectal cancer – 12, lung cancer – 8 and rare tumor types. In 14 cases (15.1%), patients were denied molecular profiling due to there were conventional treatment options or extremely low chances to detect actionable alterations. The average total costs of the study were - 50704 rubles or less than 700 Euros per patient. In 22% of cases, there were found biomarkers of 2B, 3A/B, and 4 levels of evidence (according to OncoKB.org) since biomarkers 1 and 2A levels have almost always been studied in patients. Our proprietary algorithm detected actionable amplifications in NGS data from 2 patients. Pretreated patient with stage IV cervical cancer was indicated to afatinib treatment due to amplified EGFR. The treatment resulted in complete response and stable remission during next 6 months. The second case was metastatic colorectal cancer with amplification of FLT3 gene who demonstrated a partial clinical response to sorafenib. In both cases, all standard treatment options were exhausted.
Reasonable selection of biomarker panels helps to reduce costs and ensure the better clinical utility of routine tumor molecular profiling.
OncoAtlas, LLC.
Has not received any funding.
All authors have declared no conflicts of interest.
Triple-negative breast cancer (TNBC) subtype is associated with an adverse outcome among BC patients. Since Overall Survival (OS) and recurrence prediction are significant to define suitable treatment strategies, an identification of prognostic markers for Disease-Free Survival (DFS) and OS is relevant. Here we aimed to estimate the effects of previously identified predictors of DNA-damaging therapy POLR2L, RAD50, SLC34A2, SMARCA5 and WDHD1 on OS and DFS in TNBC.
Clinicopathological data of 1098 BC patients (TCGA Breast Invasive Carcinoma) and information of expression and copy number alterations (CNA) of selected genes were downloaded from the public cBioPortal site (
Among studied covariates, high mRNA expression of POLR2L (HR = 1.7; 95% CI: 1.16-2.48; p-value = 0.006) and RAD50 (HR = 1.9; 95% CI: 1.02-3.64; p-value = 0.043) genes and the progressive stage of disease (HR = 4.0; 95% CI: 2.08-7.68; p-value =3.15x10-5) were associated with worse OS of TNBC patients. Also high mRNA expression of POLR2L (HR = 1.9; 95% CI: 1.16-3.00; p-value = 0.01) gene and progressive stage of disease (HR = 3.379; 95% CI: 1.601-7.132; p-value = 0.001) were associated with increased risk of cancer relapse. All possible combinations of the examined genes and characteristics were tested to select a most powerful prognostic model. For OS (Log-rank test: p-value = 3.415x10-7) and DFS (Log-rank test: p-value = 3.331x10-5) the best multivariate Cox regression model included a combination of mRNA expression of POLR2L and RAD50 genes and tumor stage.
Our findings display that the mRNA expression of POLR2L and RAD50 genes both separately and in combination with a stage of disease influence on OS and DFS of TNBC patients. Thus, POLR2L and RAD50 could be considered as new potential prognostic markers of TNBC.
Research Laboratory “Biomarker”, IFMB KFU.
Program of Competitive Growth of Kazan Federal University.
All authors have declared no conflicts of interest.
From decades’ immortal cells have been used as a model system to study the mechanism of cancer, drug resistance and explore the potential efficacy of the anticancer drugs. However, the numerous study has been suggested that studying the immortal cancer has a backdrop to represent the drug resistance and heterogeneity of the tumor in the patients. So, Primary tumor cultures currently begin to constitute the golden standard to study the lacking things, which has to apply to study the mechanism of resistance and personalized therapy including in breast tumor.
Initially, 10 breast tumors with different subtypes were obtained from patients, with their informed consent. They were freshly taken biopsy samples. The patient biopsies underwent an enzymatic digestion with (3 mg /ml collagenase and 5unit/ml dispase). After enzymatic digestion cells were grown in normal complete media with different growth factors and ROCK inhibitor. All cell lines were maintained until their passage (p15-p20). The cells were characterized with different antibodies by flow cytometry, different gene expression and protein expression were assessed by PCR and western blot.
Here we found that, after characterization of the 10 samples, the most subpopulations have the breast cancer stem cells properties (CD44+/CD24low) as well as cancer-associated fibroblast (CAFs) characteristics with the positive expression of CD 140a. In the gene level, we found that the mesenchymal marker (fibronectin) and an oncofetal gene i.e. SALL4 are overexpressed in almost all cells except in Luminal B her2+ subtype, as compared to MCF 10a (a non-tumorigenic epithelial cell line). Further, we are impending to correlate the expression of metastatic and tumorigenic targets in those individual patients with cell proliferation, invasion, migration and drug efficacy.
The present study provided that the primary cell culture can be a better way to study the mechanism behind metastasis and drug resistance. It also contributes to the knowledge behind the cell heterogeneity and increases the drug’s efficacy. Primary tumor cell lines have the potential to improve the effectiveness of conventional clinical approaches to treat cancer of individual patients.
1) INCLIVA Biomedical Research Institute 2) CIBERONC.
CIBERONC.
All authors have declared no conflicts of interest.
Cancer-associated thrombosis (CAT) is one of the most threatening complications of cancer. Many questions are as yet unsolved in this field, from pathogenesis to clinical management. One of the most interesting issues is the role of oncogenes and oncosuppressors as link between carcinogenesis and CAT. There is much evidence on in vitro capacity of some oncogenes to enhance the clotting system, but less clinical evidence of association between cancer mutational status and risk of thromboembolic events. The aim of this study was to explore the relationship between mutational status of genes commonly analysed and risk of CAT.
We retrospectively evaluated molecular cancer features of all consecutive patients admitted to the National Cancer Institute’s Department in Milan between October 2016 and November 2017. Patients with previous thrombotic events and patients under anticoagulant therapy at cancer diagnosis were excluded. All molecular investigations requested by clinicians were recorded. Due to death as competing risk, the Fine and Gray proportional regression model was used to detect statistical association and estimate relative risk.
The resulting cohort consisted of 484 patients with solid tumors (most of all gastrointestinal - 47% -, lung - 18% - and breast - 15% - cancers). Molecular investigations were available for 375 (77%) patients, in particular Next Generation Sequencing (NGS) analysis for 148 (31%) patients. After a median follow up of 17 months, 118 patients (24%) exhibited clinical manifestations of thrombosis (i.e. deep vein thrombosis, pulmonary thromboembolism, splanchnic thrombosis, disseminated intravascular coagulation, arterial thrombosis) and 117 (24%) patients deceased without thrombotic events. Statistically relevant data were observed for TP53, KIT and SMAD4 mutational status (respectively, HR = 0.50, 4.30, 3.19; p = 0.04, 0.04 and 0.03).
This is the first study using large molecular panel gene analysis for thrombosis risk prediction. Mutational status of some genes was statistically associated to the risk of thrombosis. Due to methodological limits and low prevalence of mutations, prospective and controlled studies are required.
Fondazione IRCCS “Istituto Nazionale Tumori” - Milan.
Has not received any funding.
The author has declared no conflicts of interest.
The most important and well-established benefit of neoadjuvant therapy for breast cancer patients is increased breast conservation rate. However, in luminal A type breast cancer, the response to neoadjuvant chemotherapy (NCT) is not as good as other subtype of breast cancer, such as HER-2 or triple-negative breast cancer. In addition, with the advancement of multi gene assay tools for this subtype, adjuvant chemotherapy is not needed at all in significant proportion of this luminal A subtype. Through a selective neoadjuvant therapy, either chemotherapy or endocrine therapy using histopathologic markers and 70-gene assay (Mammaprint. Agendia inc.), we hypothesize that we could increase the breast conservation rate in lumnal A type breast cancer.
This study is a non-randomized, phase II, prospective study. The main inclusion criteria is women with stage I-IIIA, ER-positive, HER-2 negative breast cancer that tumor size is measurable. Breast conserving surgery (BCS) is not feasible considering the turmor size, location, and patient's breast size. Two surgeons in each institution will judge the feasibility of BCS. Main exclusion criteria is diffuse malignant microcalcifiation or multicentrica breast cancer. The conversion rate from BCS-ineligible to BCS-eligible with NCT was 35.8% in luminal A breast cancer in our previous study. We assumed that with the study regimen, the rate will be increased to 50.8% (15% increase). Given these estimates, under 10% type I error rate and 70% power, 220 patients in total will be enrolled from nine tertiary hospitals in Korea. All the patients initially will be tested with Mammaprint assay. When the Mammprint result is high risk, the patients will receive NCT. When the Mammprint result is low risk, the patients will receive neoadjuvant endocrine therapy. Postmenopausal women receive letrozole 2.5mg per day for 16 weeks. Premenopausal women receive leuprorelin every 4 weeks with letrozole for 16 weeks. The primary endpoint is conversion rate from BCS-ineligible to BCS-eligible of more than 50%. The secondary endpoint is actual breast conservation rate. pathologic complete response. clinical response rate, and disease-free survival.
Korean Breast Cancer Study Group (KBCSG).
Agendia Takeda Kwandong Pharmaceutical Co Ltd. Korea Shin Poong Pharm, Co. Ltd.
The author has declared no conflicts of interest.
The activating immune ligand, MICA, acts as a “kill me” signal through the NKG2D receptor expressed on natural killer (NK) cells that are key players in the fight against breast cancer (BC). Shedding of MICA during BC progression acts as a formidable barrier against NK cells' immune-surveillance. Recently, miR-20a was found to mediate immune escape through repressing MICA levels on BC cells. However, targeting miR-20a/MICA using natural compounds has seldomly been investigated. Vitexin, a flavone C-glycoside, showed potent anticancer properties. It was reported that acetylation of glycosides increases their cytotoxic activity, with an unknown impact on immunogenicity. Our group has successfully isolated 3'-O-acetylvitexin from Ocimum basilicum which showed potent cytotoxic effects against colon cancer cells but has never been investigated in BC. Our aim is to unravel the role of the immunogenic miR-20a/MICA axis in BC patients and its regulation by vitexin and 3'-O-acetylvitexin.
Breast tissues were collected from 26 BC patients. ER, PR and HER2 expression was quantified using immunohistochemistry. MDA-MB-231 TNBC cells and MCF-7 HR+ BC cells were treated with serial dilutions of vitexin and 3'-O-acetylvitexin. Their cytotoxic activities were assessed using MTT, colony forming and migration assays. Total RNA was extracted, reverse transcribed, then MICA and miR-20a were quantified using qRT-PCR.
miR-20a is upregulated in BC patients, while MICA was downregulated in MDA-MB-231 compared to MCF7 cells. Vitexin decreased MDA-MB-231 cellular viability and migration capacity. 3'-O-acetylvitexin resulted in a more pronounced dose-dependent repression of TNBC cellular viability, colonogenicity and migration capacity. Treatment with vitexin didn’t show any alteration in miR-20a but showed only 2 folds increase in MICA. However, 3'-O-acetylvitexin markedly decreased miR-20a with a concomitant increase in MICA by 12 folds.
3'-O-acetylvitexin displays more pronounced anticancer properties against TNBC through halting their progression and immune suppressive nature by modulating miR-20a/MICA axis. This highlights miR-20a/MICA axis as a potential therapeutic target in BC.
German University in Cairo (GUC).
Has not received any funding.
All authors have declared no conflicts of interest.
DNA double-strand break (DSB) is the most critical type of genotoxic stress. Clinical studies have revealed a link between genomic instability and response to anti-PD-1/PD-L1 therapy in cancer management. We investigated role of DBS repair and ATR/Chk1 DNA damage checkpoint in regulating PD-L1 expression and their use in therapy selection and study design.
Protein expression data proteins and phosphoproteins with major clinical outcome endpoints were obtained from The Cancer Genome Atlas project. A statistical correlation analysis was performed between the expression and distribution DBS repair and ATR/Chk1 DNA damage checkpoint pathway and PD-L1. Signaling network was also analysed for of therapeutic target identification.
The expression and distribution patterns of PD-L1 was measured in 7694 samples from 32 cancer type. Increased expression of PD-L1 was associated with higher tumor stage and grade. Analyses of the DNA damage ATR/Chk1 checkpoint signalling revealed strong correlation of PDL1 expression. PD-L1 expression in was upregulated in response to DSBs with strong correlation with MRE11 (correlation coefficient (r) =0.39, p < 0.01), RAD51 (r = 0.33, p < 0.01). This upregulation requires ATM/ATR/Chk1 kinases (Chk2_pt68; r = 0.36, p < 0.01 and Chk1_ps296; r = 0.33, p < 0.01). Interestingly Jab-1 expression was corelated with both Chk-1(r = 0.27, p < 0.01) and PD-L1 (r = 0.22, p < 0.01). We further investigate for the possible signaling mechanism for the correlation and found activation of oncogenic signaling in a cancer type specific manner. PI3K/AKT/mTOR/S6K, INFgamma/ JAK/STAT/IRF1 and Ras/BRAF/MEK/ERK were involved in mechanism of increased PD-L1 expression.
DSB-mediated immune activation is balanced by concomitant inhibitory signaling, via the checkpoint kinases ATM, ATR, and Chk1 drived PD-L1 expression in tumors. These observations have important clinical implications for therapy selection, particularly following progression on DNA damaging agents suggesting that PD-1/PD-L1 inhibitors may be a useful therapeutic strategy (with or without concurrent DNA damaging agents) for tumors.
Narender Kumar.
Has not received any funding.
All authors have declared no conflicts of interest.
In recent years, immunotherapy has shown remarkable success in the treatment of several cancers. Masking PDL-1 on cancer cellular surface or PD1 on T-cells may increase the lymphocyte activity against the tumor. Intracellular regulation of PDL-1 may thus be a promising therapeutic strategy. However, the mechanism regulating PDL-1 expression remains unclear. Moreover, there are evidences supporting the idea that deregulation of Hedgehog (Hh) signalling has a role in immunity and inflammation. Therefore, we investigated whether activation of the Hh signalling contributes to regulate PDL-1 or PD1 expression and whether its pharmacological modulation affects the anti-tumor function of activated lymphocytes.
We used a panel of human pancreatic and breast cancer cell lines, with different ER, PR and HER2 expression patterns. The effects induced by Hh inhibition, through the Smo-inhibitor NVP-LDE225, on signal transduction in cancer cells were investigated. We also tested how the overexpression of GLI1, tGLI1 and GLI3, main transcription factors in the Hh pathway, could affect the expression of PDL1.
Hh inhibition reduced the expression of PDL1 in all analysed cell lines. This effect is counteracted by upregulation of PDL-1, when GLI1 and, even more, tGLI1 are overexpressed. NVP-LDE225 treatment modulates the production of several secreted factors, such as secreted IL6-receptor which acts as an anti-inflammatory molecule.
Our results suggest that Hh pathway has a specific role in cancer immune evasion trough PDL-1 modulation. The inhibition of the Hh pathway could represent an interesting therapeutic approach in combination with anti-PD1 drugs.
Prof. Roberto Bianco.
Has not received any funding.
All authors have declared no conflicts of interest.
Despite the clinical success of immune checkpoint inhibitors in Breast Cancer (BC), yet, the incidence of poor clinical responses in some patients had recently appeared in the clinics suggesting the emergence of drug resistance. Recent research has shifted towards innate immunity highlighting the promise of natural killer cells (NKs) as a more directed immunotherapeutic tool. Yet, cancer cells can still evade immune recognition by NK cells by shedding NKG2D ligands. MICA and ULBP2 are among the most de-regulated NKG2D ligands in BC patients. Tumor microenvironment is also capable of dampening NKs cytotoxicity. Therefore, researchers hypothesize that a potential anti-cancer agent should disturb such triad: halt the oncogenic activity of BC cells, improve its immunogenic profile and alleviates the immune-suppressive microenvironment. miRNAs are suggested as multifunctional players tuning the expression of several targets simultaneously. Finding a novel miRNA that could efficiently halt such triad of oncogenicity was our main goal. miR-4317 is a novel miRNA that has seldomly been analyzed in oncology. Thus, our aim was to investigate the impact of miR-4317 on BC oncogenic and immunogenic profiles.
MDA-MB-231 and MCF7 cells were transfected using lipofection method. Functional analysis was performed using trypan blue, scratch, colony forming and invasion assays. Total RNA was extracted and quantified by qRT-PCR. Peripheral blood was collected from 50 healthy controls and NK cells were isolated using untouched negative selection method. Cytolytic activity of NK cells was assessed using LDH Assay.
Ectopic expression of miR-4317 in BC cells resulted in a marked repression of cellular viability, migration, invasion and clonogenicity of BC cells. miR-4317 resulted in a significant increase in the MICA and ULBP2 immune ligands on BC cell lines and alleviating the immune suppressive microenvironment. On the functional level, miR-4317 induced NK cells cytotoxicty upon co-culturing with BC cells.
miR-4317 potentiates NK cells mediated killing through improving immunogenic recognition ability of BC cells, re-modeling tumor microenvironment and hijacking BC hallmarks.
German University in Cairo.
Has not received any funding.
All authors have declared no conflicts of interest.
Incidence of Oropharyngeal Cancer varies greatly worldwide showing an increasing trend. This increasing trend in epidemiology of Oropharyngeal Cancer has been attributed to the infection by human papillomavirus [HPV]. In this study we aimed to determine the impact of the presence of HPV DNA and p16 in oropharyngeal cancer on response to treatment and toxicity in patients receiving radical chemo-radiotherapy at regional cancer center.
80 patients of squamous cell carcinoma of oropharynx were enrolled. HPV DNA and p16 status of all patients was evaluated using polymerase chain reaction and immunohistochemistry. The selected patients for this study were treated with concurrent chemoradiation therapy with weekly cisplatin.
Among 80 cases, 17cases (21.2%) shows positive results for p16 P16 was positive in 64.7 % cases of males and 35.3% cases of female on the other hand in p16 negative cases 84.1% cases were male while 15.9% cases were female. Overall, 83.8% of patients were tobacco users (smoking, n = 30 (44.8%); smokeless, n = 18 (26.9%), both, n = 19 (28.3%)). Tonsils (70%) is the most common site involved. Response of treatment was evaluated after 6 weeks of concurrent chemoradiation. Complete response was observed in 14 patients which were p16 positive and 47 patients which were p16 negative. Partial response in 3 (p16 positive) and 15 patients (p16 negativity). Stable disease was observed in 1 patient (p16 negative) and no cases with progression of disease was evaluated in response to treatment. Evaluation of toxicity was done and toxicity was graded in both HPV positive and HPV negative cases according to CTC. In both p16 positive and negative stomatitis (p = 0.75) was the most common adverse event. Oral stomatitis of Grade 1&2 (100% in p16 +ve and 96.8% in p16 -ve patients) and grade 3&4 (58.8% in p16+ and 49.20% in p16_ patients).
We concluded that patients with HPV positive and p16 positive OPSCC show better response to treatment. Risk of severe late toxic effect is low after treatment of oropharynx cancer as due to the escalation of therapy risk of late toxic effects are decreased. This may have implication in doing p16 routinely in clinical practice and implication while considering for treatment de-intensification strategies.
Priyanka Yadav, Surender Beniwal.
Indian Council of Medical Research, New Delhi, India.
All authors have declared no conflicts of interest.
Tumor mutational burden (TMB) correlates with response to immune checkpoint inhibitors (ICI) in advanced non-small-cell lung cancer (aNSCLC). We hypothesized that TP53 mutations could reflect TMB and predict ICI benefit.
TP53 mutations were assessed by next-generation sequencing in aNSCLC patients treated with programmed death-1 (PD-1) blockers. Clinical data, tumor programmed death ligand-1 (PD-L1) expression, and KRAS mutational status were collected. The primary endpoint was overall survival (OS).
In total, 72 patients (median [interquartile range] age: 61 [33-83] years) were included; 52 (72%) were male; 39 (54%) had performance status 0-1; 53 (74%) had adenocarcinoma; 20 (28%) received first-line ICI, 52 (72%) second line or more. In 65 patients with available data, 36 (55%) expressed PD-L1 in ≥ 50% of tumor cells, 20 (31%) in 1-49% of cells, and nine (14%) were PD-L1-negative. Non-synonymous TP53 mutations were observed in 41 (57%) and 25 (35%) harbored KRAS-mutated tumors. After a median follow-up of 15.2 months (95% confidence interval [CI] 10.3-17.4m), the median OS in the TP53-mutated group was 18.1 months (95% CI 6.6-not reached), vs. 8.1 months (95% CI 2.2-14.5, hazard ratio [HR]=0.48; 95% CI 0.25-0.95, p = 0.04) in the TP53-wild-type group. Median progression-free survival was significantly longer in TP53-mutated patients (4.5 months, 95% CI 2.8-18.1 versus 1.4, 95% CI 1.1-3.5; p = 0.03). Objective response rate (ORR) was higher in TP53 mutation patients (51.2% vs. 20.7%; p = 0.01). In multivariate analysis, TP53 mutations independently associated with longer OS (HR = 0.35, 95% CI 0.16-0.77, p = 0.009).
TP53-mutated status correlated with immunotherapy clinical benefit in aNSCLC.
Pr Gérard Zalcman.
Has not received any funding.
All authors have declared no conflicts of interest.
Computational method was developed to study how nucleus alterations would influence on cell cycle progression in cancer. It was verified that information retrieved from chromatin organisation areas and their heterogeneity can be used to infer nuclear morphological features. Diffuse large B-cell lymphoma (DBLC) is heterogeneous entity due to its morphologic alteration of cells. Numerous studies have attempted to further understand how nuclear morphology and expression antiapototic genes interfere on cell interaction and pathogenesis. Objective: To determine the immunohistochemical (bcl-6) molecular marker and morphological characteristics of nucleus (FISH probe BCL6) using digital pathology algorithm in patients with DBLC.
The study included data on 14 patients with diffuse large-cell B-cell lymphoma (mean age 50 ± 15 years) before therapy. TMA templates were prepared for BCL6 FISH break apart probe as well as for immunohistochemical (ihc) marker bcl-6. Fluorescence microscopy images obtained from tissue slides by histologic scanner. The number of stained pixels / area was quantified using computer quantitative ihc, nuclear and histopattern algorithms for each TMA core. The study of the nuclear morphology was pursued through the analysis of the first and second sets of DAPI staining images and the study of distribution green and red signals into cells. In this work, a semi-automatic bioinformatic application was implemented to manage the images sets and process them.
Concerning the nuclear morphological features evaluated (area, density of chromatin - eccentricity and solidity), the results demonstrated that the quantification of the nuclei area was the most meaningful parameter to disclose significant biological information as translocation of BCL6 gene. Besides it was associated with aneuplody and amplification of BCL6 genes and overexpression of bcl-6 protein. These nuclear morphological features revealed significant differences between cells with translocation of BCL6 and cells without translocations.
These findings support the hypothesis that morphology of nuclei are mediated by the rearrangement of the chromatin density and cytoskeleton interaction.
Anna Artemyeva.
Petrov Cancer Research Center.
All authors have declared no conflicts of interest.
CRPC is the lethal manifestation of the most frequent tumour in men. Despite of new target therapies emerged in last years patients will finally develop resistances that My be related to immune system alterations and it will lead to their death. Recent studies have shown that immunotherapy can be a critical therapeutic strategy. To identify how drugs that we use conventionally can modify the immune system at a microenvironment level is important to understand the biology of CRPC and the association with response/resistance to standard therapy, but also critical to the next future application of new immune therapeutic strategies in this setting.
This study is part of a multicentric National Biomarker Platform integrated by 56 centres initiated with the support of a Young Investigator Award from SOGUG and promoted by the Prostate Cancer Unit led by Dr. David Olmos at CNIO, that has enroled >900 patients. We establish a new prognostic factors explicative study using two prospective cohorts: CRPC patients treated with docetaxel (PROSTAC cohort) and abiraterone (AA, PROSABI cohort). Key inclusion criteria: a) histological confirmation of prostate cancer; b) documented criteria (PCWG) for CRPC; c) availability of tumour tissue; d) candidate for standard treatment with docetaxel or AA. Primary endpoint: to validate the prognostic value for overall survival (OS) of the expression signature (ES) in peripheral blood of 9 genes (Olmos et al, Lancet Oncol 2012) related to lymphocites B/T immunodeficiency in a 496 CRPC patients population treated with first line chemotherapy and AA. Secondary endpoints: a) to study the prognostic role for PFS of the ES; b) to compare its prognostic/predictive utility with other ES. Exploratory endpoints: a) to analyse an immunoprofile antibodies panel in peripheral blood in a subgroup of patients from this population and correlate those findings with the expression data in the primary tumour and the metastasis; b) explore new therapeutic strategies in TRAMP prostate cancer mouse models with different immune characteristics, with the global objective of identifying new clusters of patients candidates to immunotherapy that it is still a challenge.
We are grateful to all the patients, clinicians and research assistants involved in the PROCURE National Biomarkers Platform. We would like to acknowledge the lab team at CNIO and IBIMA. This work was supported by an unrestricted grant from “Fundación CRIS contra el cancer”, grants from “Fondo de Investigación Sanitaria, Instituto de Salud Carlos IIII” (PI17/008). During this work, NRL and RL were supported by grants from “Instituto de Salud Carlos III” (JR17/00007 to NRL and CM17-00221 to RL).
PROSTAC study: NCT02362620; PROSABI study: NCT02787837.
Hospital Univesitario La Princesa - CNIO Spanish National Cancer Research Centre.
This work was supported by an unrestricted grant from “Fundación CRIS contra el cancer”, grants from “Fondo de Investigación Sanitaria, Instituto de Salud Carlos IIII” (PI17/008).
N. Romero-Laorden, R. Lozano Mejorada: During this work, NRL and RL were supported by grants from “Instituto de Salud Carlos III” (JR17/00007 to NRL and CM17-00221 to RL). All authors have declared no conflicts of interest.
Metastatic renal-cell carcinoma (mRCC) is frequently fatal within months or a low number of years. Several life-extending drugs have been developed that can help alleviate symptoms, but treatment algorithms to arrive at which drugs, and in what order, are not yet defined. The immunotherapy checkpoint inhibitor nivolumab is the most commonly used second-line treatment, when first-line VEGF inhibition using a tyrosine kinase inhibitor (TKI) is no longer effective. However, in the pivotal trial, objective response rate to nivolumab in this setting was only 25%. Currently, there are no predictive biomarkers to identify patients with mRCC who will respond to nivolumab, nor is there data on the effect of first-line TKIs on patient’s likelihood of responding to second-line immunotherapy. Evaluating and correlating immune and molecular characteristics from blood and tumour may identify biomarkers that predict for immunotherapy response. Differences according to a particular TKI may also be identified; these variances could influence tolerance and response to second-line immunotherapy.
Fifty patients who have progressed on TKIs and plan to commence nivolumab will be recruited. Blood will be collected at time point 1 (Cycle1 Day 1 [C1D1] nivolumab) and time point 2 (after 12 weeks [+/- 7 days]). Tumour will also be sampled from a subset of these patients. Assays; FACS, human cytokines, ELISAS (VEGF, VEGFR1, VEGFR2, CD31), tumour IHC (CD3, CD4, CD8, CD25, FoxP3, CD68, PD-1, PD-L1, PD-L2), RNA seq and DNA analysis. Primary objective; to evaluate immunological signatures in patients with mRCC following disease progression on first-line TKI therapy with either pazopanib or sunitinib. Secondary objectives include; correlation of immunological profiles and molecular signatures at C1D1 with response to second-line checkpoint therapy, tolerance, progression-free survival and overall survival following second-line checkpoint therapy. Exploratory objectives include; change from baseline in blood based immune markers on nivolumab therapy, correlation of serum markers with tissue markers, correlation of angiogenic and immune markers with responses to nivolumab.
IRAS No. 227141.
The University of Manchester.
Novartis.
All authors have declared no conflicts of interest.
The mTOR pathway has a key role in regulating cell growth and is altered in 70% of cancers. Rapalogs have become standard of care in patients with metastatic breast, kidney and neuroendocrine cancers. Nevertheless, tumor escape occurs after several months in most patients, highlighting the need to understand the mechanisms of action and resistance. Thus, we aim to identify new target genes of rapalogs that could be used as biomarkers to predict the treatment efficacy, or as therapeutic targets, to overcome resistance.
Expression levels of target genes were evaluated in a panel of cancer cell lines by transcriptomic analyses, real-time qPCR and western blot. Transcriptional regulation was investigated by in silico analyses of the promoter region, gene reporter assays using site-directed mutagenesis, and ChIP. Stably modified cell lines were established by lentiviral transduction. The analysis of the putative interacting partners was performed by LC-MS/MS followed by functional classification using the bioinformatics tool DAVID. The interactions between proteins were investigated by co-IP. Splicing efficiency was assessed by double-gene reporter assay.
Using a panel of eight cancer cell lines, we showed that rapamycin down-regulates only one gene, named TRIB3, in common in all cell lines. These data were confirmed in vivo in patients treated with rapalogs for cancer. Transcriptional repression of TRIB3 by rapalogs was mediated by the interaction of FKBP3/LRRFIP1 with the promoter, suggesting an inhibitory effect of rapalogs independent of mTOR targeting. Furthermore, we identified TRIB3 as a component of the splicing machinery necessary for pre-mRNA splicing disturbance by rapalogs. In addition, we observed that, the overexpression of TRIB3, abolished the inhibitory effects of rapalogs on cancer cell viability.
These results identify TRIB3 as a new transcriptional target gene of rapalogs, and evidence it as a key component of spliceosome machinery. Thus, TRIB3 may be considered as a suitable biomarker to assess the efficacy of rapalogs treatment. Moreover, TRIB3 could also be suitable as a therapeutic target to synergize with rapalogs treatment.
Gustave Roussy.
INSERM, Gustave Roussy (taxe d’apprentissage), Opération Parrains Chercheurs, Odyssea, Dassault Foundation, Breast Cancer Research Foundation, and La Ligue contre le Cancer (Rhone-69, and Allier-03).
F. André: Research grant from Novartis. All other authors have declared no conflicts of interest.
First line therapies usually induce the longest progression free survival (PFS) in metastatic gastrointestinal cancers (GIC) as compared to subsequent lines of treatment. However, immunotherapy (IT) due to its mechanisms of action could influence sensitivity to conventional cancer therapy (CCT) after progression to IT and thereby, influence both tumor growth rate (TGR) and PFS. We have studied TGR and PFS before and after participation in phase I IT trials.
Patients enrolled in Phase I IT trials run at our institution between Jan 2012 and Sept 2017 with GIC including colorectal cancer (CRC), esophagogastric cancer (EGC) and pancreatic cancer (PC), treated with at least one line of CCT before and after IT failure were included in the analysis. Baseline characteristics were recorded. A ratio of PFS after/before IT (PFSaft/befIT) over 1.2 was considered clinically significant. TGR was calculated based on the formulas: TGR = 100 (exp(TG)−1), TG = 3 Log(Dt/D0)/t. Correlation between PFSaft/befIT and GRIm score was evaluated.
Nineteen patients met the inclusion criteria (17 CRC pt, 1 EGC pt, 1 PC pt). Table 1 shows baseline characteristics. Median PFS befIT and aftIT were 4.4 and 3.1 months, respectively. Seven of 19 patients presented a PFSaft/befIT rate over 1.2. TGRbef and TGRaft data were available from 9 patients; 1 patient (CRC) presented a decrease in TGR greater than 15% after IT failure. Patients experiencing a ratio of PFSaft/befIT over 1.2 tended to score better in GRIm prognostic classification (0-1: 100% vs 66%, (p = 0.09)).Characteristics N = 19 Male 11 Median (M) age at diagnosis (range) 54 (34-79) M lines prior to IT (range) 2 (1-5) Presence of liver disease (pre/IT/post) 9/10/13 CCT class (pre/post IT) Platinum derivatives Other alkylating agents (a) Antimetabolites Topoisomerase inhibitors (i) Antimicrotubules a Antiangiogenic a Signal transduction i Immunotherapy Others 4/8 0/1 15/11 12/4 1/1 11/9 2/2 4/0 3/3 Combined/monotherapy during IT 11/8 MSI/MSS/unknown 2/7/10
Our data suggest a better outcome on ensuing systemic therapies after IT. Further prospective investigations are needed to select the subset of patients who are more prone to a re-sensitization to CCT and to understand the mechanisms underlying.
Clínica Universidad de Navarra.
Has not received any funding.
I. Melero: Advisor: BMS, Roche, AstraZeneca, Genmab, Alligator, Tusk, Bioncotech, Merck-Serono. All other authors have declared no conflicts of interest.
The epidermal growth factor receptor (EGFR) is a therapeutic target in head and neck squamous cell carcinoma (HNSCC). Resistance to EGFR-targeted therapies such as cetuximab is a major clinical problem. This study aims to unravel resistance mechanisms by combining whole-exome sequencing (WES) and tumour kinase profiling. Based on the genetic and tumour kinase profile, new combination treatments can be designed to overcome therapy resistance.
Acquired cetuximab resistant HNSCC cell lines (SC263-R and SCC22b-R) were generated by chronically exposing initially sensitive cell lines to cetuximab. In parallel, control cell lines (SC263-PBS and SCC22b-PBS) were established by exposing these cells to the vehicle control. DNA samples were sequenced using Illumina’s HiSeq 1500 platform and variant calling was performed. Proteome Profiler Human Phospho-Kinase Antibody Array kit (R&D Systems) was used to determine the relative levels of protein kinase phosphorylation in sensitive and acquired resistant cell lines after cetuximab treatment.
Variant calling in cetuximab sensitive and acquired resistant cell lines demonstrated 10 and 1 novel single-nucleotide variants (SNVs) in SC263-R and SCC22b-R, respectively. Based on gene functions, these SNVs might be responsible for resistance to cetuximab treatment. Phospho-kinase analysis showed increased phosphorylation of Akt1/2/3 after cetuximab treatment in acquired resistant cells compared to sensitive cells. As such, combining cetuximab with an Akt1/2/3 inhibitor could be an interesting new combination treatment. Results obtained from WES and tumour kinase profiling will be further validated using Sanger sequencing and western blot, respectively.
This study demonstrated that resistance mechanisms for EGFR-targeted therapy can be identified through genetic and tumour kinase profiling. Our results showed that SNVs and increased Akt1/2/3 phosphorylation may lead to acquired cetuximab resistance. Personalised therapy using combinations of targeted therapies based on the molecular profile of the tumour may achieve the much-needed progress in HNSCC treatment.
University of Antwerp.
Stand Up to Cancer (the Flemish Cancer Society) and University of Antwerp.
All authors have declared no conflicts of interest.
Although knowledge of the molecular landscape in gynecologic tumors is increasing, the clinical impact of an extensive screening remains unclear. The aim of this monocentric study was to characterize molecular alterations in advanced gynecologic tumors, and to evaluate the clinical benefit of corresponding targeted agents.
Advanced gynecologic cancers were prospectively included in our molecular screening program. DNA and RNA extracted from tissue samples were analyzed by next generation sequencing, and when necessary, by comparative genomic hybridization array, polymerase chain reaction, fluorescence in situ hybridization and immunohistochemistry. Patients were enrolled, when possible, in early trials according to our molecular tumor board recommendations. We assessed clinical benefit with disease control rate (objective response and stable disease) and Growth Modulation Index (GMI defined as: time to progression under the early phase trial treatment/time to progression of the previous line of treatment) > 1.3.
245 patients (ovary, n = 135; endometrium, n = 57; cervix, n = 37; uterus, n = 12; vulva, n = 3; vagina, n = 1) were recruited from February 2013 to May 2017. 115 (47%) patients had at least one actionable alteration. 131 different molecular alterations were found. The most frequent actionable alterations were PIK3CA (n = 45), hormone receptor expression (n = 38), KRAS (n = 23), BRCA1 and BRCA2 (n = 18) and HER2 (n = 16). Median number of previous lines of treatment was 2. 47 patients received a treatment matched with tumor molecular profile. Main reasons for non-inclusion were non-progressive disease (n = 24, 51%) and general status deterioration or death (n = 13, 28%). Median progression-free survival was 12,1 weeks (95% CI 8,3-16). Median overall survival was 34,6 weeks (95% CI 46,9-104,6). Disease control rate was 38%, and 14 % of patients had a GMI > 1.3.
Molecular profiling for gynecologic tumors is feasible and detects molecular alterations with a positive clinical impact. The yield of this procedure could be improved by higher throughput sequencing techniques, wider access to genotype-matched treatments, and an earlier screening during the course of the disease.
Institut Bergonié.
Has not received any funding.
T. Grellety: Paid for consulting role for Novartis during the past 2 years. All other authors have declared no conflicts of interest.
BRCA1/2 germline mutations accompanied by somatic mutations in p53 associated genes are associated with increased risk of developing breast cancer (BC). Genes acting upstream of p53, or participating in growth arrest following DNA damage such as the BRCA1-interacting protein, ZNF350, may modify the risk of BC in women with mutant BRCA1/2 through mediating BRCA1-induced transcriptional repression of several tumor suppressor genes. As a potential BC susceptibility gene, single nucleotide polymorphisms (SNPs) may influence transcriptional repression of ZNF350 target genes and individuals’ BC risk. Moreover, rs2278414 (G/A) SNP in ZNF350 3’UTR was associated with BC onset in BRCA1/2 carriers (“A” allele hazard ratio= 2.47). We aimed at regulating rs2278414 SNP in ZNF350 by miRNAs in BC as it has never been investigated in such context.
In-silico analysis predicted miRNAs targeting ZNF350. Wild-type constructs containing “A” allele or ancestral “G” allele of rs2278414 SNP in ZNF350 3′UTR were designed. miR-target binding regions were inserted downstream to luciferase reporter gene in pmirGLO Luciferase microRNA Target Expression Vector. MDA-MB-231 BC cells were transfected with empty pmiRGLO vector (control), or constructs with “A” or “G” SNP, or a construct with mutant target region (deleted binding region to ensure site-specific binding). After 24h, cells were co-transfected with miR-150-5p. Relative luciferase activity was measured after 48h by Steady-Glo Luciferase Reporter Assay. Wound-healing and colony forming assays were performed for miR-150-5p in MDA-MB-231.
Using bioinformatics software (
The novel tumor suppressor miR-150-5p downregulates rs2278414 “A” allele in ZNF350 3’UTR thus decreasing BC risk.
German University in Cairo.
Has not received any funding.
All authors have declared no conflicts of interest.
Wnt/bβ-catenin-signaling pathway is the principal force of intestinal hemostasis and morphogenesis. Aberrant activity of this pathway is highly implicated in cancer development, particularly in colorectal cancer (CRC). Although Wnt/β-catenin pathway is a well-studied pathway, the precise molecular mechanisms involved in the nuclear interactions of β-catenin with DNA-binding transcription factors (other than TCF4) are still under debates. Human FLYWCH1 has been first characterized and identified in our lab as a novel transcription factor, which interacts with nuclear β-catenin and modulates its transcriptional activity (Muhammed et al., under publication).
FLYWCH1 differentially expressed between normal as well as between different stages of colorectal cancers. However, we have just started exploring the biological functions and mechanisms of FLYWCH1 in colon/ intestinal development, homeostasis and tumor formation.
Loss- and gain-of-function analysis, CRISPR-Cas9, RNA-Seq, Western blotting, Immunoprecipitation, qRT-PCR, and immunofluorescence techniques.
Endogenous β-catenin physically interacts with the C-terminal domain of FLYWCH1 protein in human CRC cell lines. FLYWCH1 overexpression negatively regulates the expression of some but not all bβ- catenin/TCF4 target genes in CRC cell lines. R-spondin1 synergizes with Wnt-3A in repressing of FLYWCH1 in CRC cell lines.
a) FLYWCH1 protein binds to the endogenous bβ-catenin protein while negatively regulates the transcription of a specific-subset of bβ-catenin /TCF target genes, regardless of Wnt signaling status. b) Activation of Wnt signaling forms a negative feedback loop during FLYWCH1 repressed β-catenin/TCF4 target gene in carcinogenesis. c) Our current and future studies with focuses on the in vivo role(s) of FLYWCH1 will provide insights on a novel molecular mechanism mediated by FLYWCH1 and the canonical Wnt/bβ-catenin pathway in CRC carcinogens, where may offer a new potential approach(s) and implications for future therapeutics.
University of Nottingham.
Saudi Government.
The author has declared no conflicts of interest.
Since 2010, when the Molecular Prescreening Program (MPP) was established at VHIO, we have adapted the techniques and procedures to improve the identification of genomic alterations in tumors from patients (pts) eligible to Early Clinical Trials (ECTs). Here we report the clinical utility of our program given the evolving molecular testing landscape and trends in drug development.
In the last 8 years, 5,775 formalin fixed paraffin embedded tumor samples were analyzed: from 2010 to 2014 we used a hotspot mutation panel (Sequenom, 20 oncogenes) plus FISH/IHC of selected markers; from 2015 to 2017 we moved to a custom amplicon-based NGS panel (MiSeq, 61 genes), a copy number (CN) panel (Nanostring, 44 genes) and a fusion + gene expression (exp) panel (Nanostring, 26 genes) with additional FISH/IHC in selected cases, including PDL1 and MSI status.
We found a stepwise increase in the MPP, from 207 pts in 2010 to 1168 pts in 2017. Most common tumors were colorectal, lung, breast, gynecologic, pancreatobiliary, head & neck and gastric. In the last 8 years, the number of ECTs increased tenfold: from 13 in 2010 (11 PI3K inhibitor [inh]) to 137 in 2017 (including 55 immunotherapy [IMT], 11 RAF/MEK/ERK inh, 9 FGFR inh, 8 PI3K inh, 6 EGFR/HER2 inh and 6 antibody-drug conjugates). Inclusion rate in ECTs with targeted agents reduced from 2016 to 2017 (228 to 133), while IMT trial recruitment increased (179 to 245). In 2017, 862 tumors had NGS, 708 tested with IHC, 267 CN panel and 342 fusion + exp assay. As a result of this molecular profiling, 116 pts (10%) were enrolled in ETCs with 18 diffent biomarker matches (5.5% mandatory, 4.5% enrichment) and 220 pts (19%) were recruited in unmatched trials.
The MPP at VHIO is constantly adapting to the needs of our ECTs portfolio. Biomarker matched trials evolved from a “few targets/ large populations” scenario to a complex situation with “many targets/ small populations”. Moreover, the substantial increase of IMT trials had a major impact in trial prioritization, and will guide clinical implementation of new markers currently under development, such as tumor mutational load and inflammatory gene exp signatures.
None
Vall d\'Hebron Institute of Oncology.
Has not received any funding.
All authors have declared no conflicts of interest.
The poly (ADP-ribose) polymerase (PARP) inhibitors appear to be a promising treatments strategy in BRCA-associated and/or sporadic triple-negative breast cancer (TNBC) due to the molecular heterogeneity of TNBC and the lack of defined molecular targets. However, there is no studies on the association between BMN 673 (talazoparib) efficacy, which is a novel and the most potent PARP1/2 inhibitor, and BRCA mutation status in TNBC. In the current study, we aimed to determine the cytotoxic and apoptotic effects of BMN 673 on TNBC cell lines according to BRCA mutational status.
HCC1937 [BRCA1 mutant (5382insC)] and MDA-MB-231 (BRCA1 wild-type, TNBC cell lines were treated with 0.01-10 nM concentration of BMN 673 for 6 and 12 days and the cytotoxic effect was determined by WST-1 assay. Apoptosis induction by BMN 673 treatment was evaluated by Annexin V and cell cycle analysis, as well as the morphologic changes of the apoptotic cells were observed by AO/EtBr dual staining.
BMN 673 considerably inhibited HCC1937 and MDA-MB-231 cell viability in dose and time-dependent manner. The cell viability of HCC1937 and MDA-MB-231 cells reduced by 34.9% and 47.5% in concentration of 10 nM BMN 673, respectively (p < 0.01). After 12 days exposure to BMN 673, a significant increase of total apoptotic cells was determined compared to the untreated control. The total percentage of apoptotic cells was 77.8% and 58.3% in HCC1937 and MDA-MB-231 cells treated with 10 nM of BMN 673, respectively (p < 0.01). After the HCC1937 and MDA-MB-231 cells were incubated with 10 nM of BMN 673, the percentage of cells in the G2/M phase significantly increased to 49.5% and 43.5%, respectively (p < 0.01). Additionally, a loss of membrane integrity and the condensation of nucleus were observed in these cells by AO/EtBr staining.
This study has suggested that BMN 673 demonstrates anti-cancer effects by inducing apoptosis in TNBC. However, BRCA mutated TNBC cells are far more sensitive to BMN 673 than to TNBC cells. Nevertheless, BMN 673 is greater potency than other PARP inhibitors at low nanomolar concentrations in TNBC cells.
The Scientific Research Projects Foundation of the Uludag University in Turkey.
This study was supported by a grant from the Scientific Research Projects Foundation (BAP) of the Uludag University of Turkey [Project No: BUAP(T)-2015/1].
All authors have declared no conflicts of interest.
Colorectal carcinomas (CRC) from the serrated route like serrated adenocarcinoma (SAC) and CC showing molecular features of microsatellite instability (hmMSI-H) share common features (preference for female gender, right side location, mucinous histology and altered CpG methylation patterns) but dramatically differ in terms of prognosis, development of immune response and treatment options. Despite this, to date no expression profiling comparison has been carried out to see which functions and genes may be responsible for such differences.
Molecular signatures of SAC and hmMSI-H were obtained by transcriptomic arrays and qPCR and immunohistochemistry (IHC) were used to validate differentially expressed genes at mRNA and protein level.
An over-representation of innate immunity functions (granulomonocytic recruitment, chemokine production, TLR signaling, antigen processing and presentation) were obtained from this comparison and ICAM1 was more expressed in hmMSI-H whereas two genes (CRCP and CXCL14) were more expressed in SAC. These array results were subsequently validated by qPCR and CXCL14 and ICAM1 by IHC.
Our findings show specific functions and genes which provide a better understanding of the role of the immune response in the serrated pathological route and may be of help in identifying particular molecular targets and future treatment options, especially in SAC which lacks molecular targeted therapy.
UCAM University (Murcia, Spain), Department of Pathology, Santa Lucía University Hospital (Cartagena, Spain).
Has not received any funding.
All authors have declared no conflicts of interest.
Circulating tumour DNA (ctDNA) can provide a minimally invasive liquid biopsy that may better capture tumour heterogeneity than archival biopsies. We report results of ctDNA and tumour molecular profiling from a gastrointestinal subset of patients recruited to the Tumour characterisation to Guide Experimental Targeted Therapy Trial (TARGET).
68 patients (pts) with metastatic small bowel cancer (SBC) or colorectal cancer (CRC) were recruited to TARGET between May 2015 and May 2018. Archival biopsies were analysed using a 24 gene NGS panel (Qiagen GeneRead DNAseq Targeted Panel V2) and in some cases a 322 gene NGS panel (Foundation Medicine). Upon recruitment pts underwent further testing with a plasma-based ctDNA NGS assay examining 653 genes. Patients were consented (optional) for pre-clinical model development from fresh blood (circulating tumour cells (CTCs)) and tumour biopsy samples.
The median age of pts was 56 (range 32-75). 48% (33/68) were colon tumours, 34% (23/68) rectal, 9% (6/68) rectosigmoid and 7% (5/68) small bowel. Pts had failed an average of 3 lines (range 1-5) of treatment prior to recruitment. 74% (50/68) of patients had both sufficient tumour DNA in their plasma, and archival biopsies for analysis. Sequencing of ctDNA detected all mutations reported in tumour in 76% (38/50) of pts. ctDNA analysis picked up additional mutations in 30% (15/50) of pts. The interval between collection of archival biopsies and blood tests (p = 0.300) did not affect detection of new mutations. 31% (6/19) of pts treated with anti-EGFR therapy developed recognised resistance mutations (KRAS and EGFR) in ctDNA on serial analysis. The most commonly detected mutations were TP53 (60%), KRAS (51%), PIK3CA (15%) and PTEN (3%). Other mutated genes included CTNNB1, BRAF, FGFR3 and ERRB2. Pre-clinical models (organoids and patient-derived xenografts (PDX)) were attempted from blood (CTCs) and/or tumour tissue in 29% (20/68) of pts. CTC organoid cultures were optimised and successful in 1/17 pts.
ctDNA may be used for routine molecular characterisation of metastatic SBC/CRC and results can be analysed to track the development of resistance.
Manchester Cancer Research Centre.
The Christie NHS Foundation Trust Manchester Cancer Research Institute.
All authors have declared no conflicts of interest.
FAT1 is a transmembrane protein helps in adhesion. FAT1 gene is localized at chromosome 4q35.2 encoding a 506KDa. The role of FAT1 in tumors is not fully characterized. Few reports suggest it behaves as a tumor suppressive and few reported it as an oncogenic. miRNAs are noncoding RNAs which bind to the 3’UTR of target mRNA and repress their expression. miR-221-3p/222-3p has been reported to have oncogenic role and targets tumor suppressors (e.g. CDKN1B, PTEN, PUMA etc.) in many cancers including GBM. Here, we have investigated the role of FAT1 gene in the regulation of miRNAs in glioblastoma.
In-silico examination of miR targets was done by target prediction software miRDB, TargetScan, miRTarBase. FAT1 knockdown was done using specific siRNA and mRNA expression analysis was done for FAT1 and miR targets by using gene specific primers and miR-221/222-3p using LNA-primers (locked nucleic acid) in GBM cell lines (U87MG, U373MG, A172 and LN229). Expression and correlation analysis of FAT1 and miR-221-3p was done in GBM tumor samples (n = 30).
We have detected increased expression of FAT1 and miRNAs (miR221-3p/miR222-3p) in different GBM cell lines (U87MG, U373MG, A172 & LN229). On FAT1 knockdown, using FAT1 specific siRNA we observed significantly decreased expression of miR-221/222-3p. In-silico analysis identified CDKN1B, CREBZF, PUMA and PTEN as potential targets of miR-221/222-3p. Furthermore, FAT1 knocked-down cells showed significantly increased expression of PUMA in all studied glioma cell lines. In order to ratify our in-vitro observation and its clinical application, we have done expression and correlation study in GBM tumor samples. We detected significant positive spearman correlation between FAT1 and miR-221-3p (r = 0.5669, p ≤ 0.0011) and negative correlation of FAT1 with PUMA (r= -0.5378, p ≤ 0.0022). These results suggest that FAT1 expression positively regulates the expression of miR-221-3p leading to downregulation of miR targets in GBM cell lines as well as in GBM tumors.
FAT1 plays an oncogenic role in glioma and miR-221/222-3p plays and important role. FAT1 may emerge as a target for therapeutic intervention. The exact mechanism involved in the oncogenesis by FAT1 is under study.
DST.
DST.
All authors have declared no conflicts of interest.
Breast cancer is a complex and heterogeneous disease where the patients present the same symptoms and suffer the same disease, but due to different genetic reasons. Breast cancer recurrence is one of the biggest morbidity causes in patients that suffer the disease, and 15-20% of them will develop triple-negative breast cancer tumors (TN). The mortality of patients harbouring TN tumors is higher comparing with other tumor subtypes, in part, due to the difficulty of getting personalized treatments, where chemotherapy is the standard treatment. These data show the need to look for new drug strategy, where clinical, genetics and molecular data could be integred and compared, in order to predict more accurate treatment responses. Here we study the role of the gene ATDC, previously related with radioresistance, in TN breast cancer cell lines treated with Doxorubicin.
To perform the experiments, knock-down of ATDC gene was performed in order to downregulate their expression. Modifyed and control cell lines treatment with Doxorubicin was done at different concentrations and at different times, measuring the number of viable cells by MTS assay and by flow cytometry, trying to observe possible changes when ATDC is not present. Finally, a possible relationship between ATDC and the phosphorylation levels of γ-H2AX was studied. Knocked-down and control cell lines were treated with Doxorubicin and the phosphorylation rate was analyzed by Western Blot.
Modifying the expression of ATDC in TN breast cancer cell lines who overexpress the gene, allowed us to observe differences in cell viability and apoptosis levels after Doxorubicin treatment. Those cells with lower levels of ATDC showed lower levels of viable cells and higher apoptosis, obtaining significant statistically differences between both groups. Furthermore, ATDC knock-down cell lines treated showed lower pohsporylation levels of γ-H2AX, comparing it with treated control cell lines.
ATDC expression is related with the sensitivity of a treatment based in Doxorubicin, due to their function in DNA damage protection. Is furhtermore related with γ-H2AX phosphorilation levels and their consecuent activation, allowing the activation of DNA repair system mechanisms.
Health Research Institute. INCLIVA.
Carlos III Health Institute. Spanish Government.
All authors have declared no conflicts of interest.
Lung cancer is the leading causes of cancer-related deaths globally. The most frequent histologic type of lung cancer is non–small-cell lung cancer (NSCLC), accounting for approximate 85%. NSCLC is notoriously aggressive, and tumors often undergo epithelial-mesenchymal transition (EMT), a process required for invasion and metastasis. The components that control this process are thus promising therapeutic targets. This study examines the role of Sonic Hedgehog (SHh)/Gli pathway signaling in the regulation of EMT in NSCLC.
Gli/EMT protein expression levels were examined by western blot in paired NSCLC patient tissues and NSCLC cell lines. Functional analyses were performed to investigate SHh/Gli signaling and EMT in NSCLC cell lines. MTS cell viability, luciferase reporter, qRT-PCR, and western blot assays were performed to analyze pathway activity, while wound healing and transwell assays were executed to measure cell migration and invasion. All experiments were conducted in triplicate, and subjected to two-sided student t-tests, ANOVA, and Scheffe's tests to determine significance when applicable.
Higher Gli1 expressions were detected in tumor samples than in paired normal tissues. Differential expression of EMT biomarkers and activation of p-AKT were observed in tumor tissues. N-Shh stimulation of cells significantly increased reporter activity in NSCLC cell lines, while Gli-i treatment of transfected cells showed less relative reporter activity. When subjected to both Gli-i and N-Shh treatment, NSCLC cell lines continued to demonstrate decreased Gli transcriptional activity. Gli inhibition is associated with decreased expression level of p-AKT, N-cadherin and Vimentin. Knockdown of both Gli1 and Gli2 showed decreased EMT, migrative and invasive ability. Cells stimulated by N-Shh demonstrated greater mobility. In addition, AKT-i treated cells also demonstrated inhibited EMT activity.
This study provides evidence for aberrant upregulation of the Gli signaling pathway and a strong association between expression of Gli versus AKT and EMT markers in NSCLC. Inhibition of Gli and AKT pathway activity may thus serve as a potential therapeutic strategy for the treatment of NSCLC patients.
Shanghai Chest Hospital.
National Natural Science Foundation.
All authors have declared no conflicts of interest.
microRNAs (miRNAs) emerge as a new step in the modulation of breast cancer response to treatment. The family of microRNAs 449 has been described as strong inducers of cell cycle arrest (including senescence) and apoptosis in distinct tumor types. The aim of this work is to analyse the miR-449 role in doxorubicin chemotherapy-based treatment of triple-negative breast cancer (TNBC).
Experiments were made on MDA-MB-231 and MDA-MB-231R (doxorubicin resistant) triple-negative breast cancer cells. Signature of miRs-449 and their target expression was measured by real time PCR and western blot. Functional and molecular studies of loss/gain of function and response to treatment were carried out trough transient transfection of miRNAs mimics/inhibitors. Functional studies included Cell Proliferation Studies (MTT/Colony Formation Assay), Viability, Apoptosis and Cell cycle (Flow Cytometry), Invasion and Metastasis (Wound healing/Migration-Invasion Chambers).
Through bioinformatic tools, we obtained the promoter sequence of the miR-449/CDC20B gene, emerging as a "hot spot", since it presented mutations, copy number variation (CVN), simple nucleotide polymorphisms (SNPs), and methylation. We also identified targets of this miRNAs family related to cancer pathways. Through in vitro studies, we obtained results of differential expression of miRs-449 between MDA-MB-231 and MDA-MB-231R treated with doxorubicin. In loss/gain of miR-449 function studies, we verified the modulation of some of their targets, and their impact on viability and on the cell cycle. Ex vivo studies have confirmed the positive correlation of the expression of the inducer factor of miRs-449, E2F1, with the sensitivity to doxorubicin treatment in triple-negative breast cancer patients. Using the KM Plotter-miRPower software, we have been able to correlate the overexpression of the CDC20B gene and the miR-449 with a higher probability of survival in breast cancer patients.
Results reveal for the first time the involvement of the miRNA-449 family in TNBC doxorubicin resistance, and suggest that regulation of its target genes, may be the mechanism behind their involvement.
INCLIVA Biomedical Research Institute.
CIBERONC / INCLIVA.
All authors have declared no conflicts of interest.
The products of TSC1 and TSC2 genes, hamartin and tuberin respectively, form a complex that is the natural inhibitor of mammalian target of rapamycin (mTOR). Mutations in these genes are associated with such diseases as tuberous sclerosis (TS) and lymphangioleiomyomatosis, for which the main pharmacologic treatment at present is everolimus, the mTOR kinase inhibitor. Benign tumors like renal angiomyolipoma and pancreatic neuroendocrine tumors (PNET) can occur in tuberous sclerosis in 70% and 10% patients, respectively, but the overwhelming majority of them is sporadic. Despite the benign character, these tumors can be life-threatening. At the present time, the pathogenesis of sporadic renal angiomyolipoma (sRA) and PNETs is largely unknown but is growing as a research topic. Referring to cases of the tumors that occur in tuberous sclerosis, we assumed that TSC1 and TSC2 genes might play a key role in the development of sRA and PNETs.
NGS was performed on 20 sRA and 12 PNETs (insulinomas) tumor samples, respectively.
Tumor sample 1st hit 2nd hit sRA-10 TSC2:c.1509_1510insGTCC: p.Q503fs – sRA-12 TSC2:c.G1832A:p.R611Q LOH 16p13.3 sRA-13 TSC2:c.204delA:p.A68fs – sRA-14 TSC2:c.C1372T:p.R458X TSC2:c.G3367C:p.G1123R sRA-16 TSC2:c.1454delT:p.I485fs TSC2:c.C5126G:p.P1709R sRA-17 TSC2:c.5171dupA:p.Q1724fs TSC2:c.A1793G:p.Y598C sRA-18 TSC2:c.C4713A:p.Y1571X – sRA-19 TSC2:c.G4829A:p.W1610X LOH 16p13 sRA-1 TSC2:c.C1111T:p.Q371X – sRA-2 TSC2:c.976-3_41del – sRA-3 TSC2:c.500delG:p.W167X – sRA-6 TSC2:c.629dupC:p.A210fs LOH 16p13.3 sRA-7 TSC2:c.1283delC:p.S428fs LOH 16p13.3 sRA-9 TSC2:c.G4289A:p.W1430X LOH 16p13.3 PNET-3 TSC2:c.600-4G>T TSC2:c.G4760A:p.C1587Y PNET-4 TSC2:c.649-1G>A TSC1:c.C1075T:p.P359S PNET-5 TSC2:c.G997A:p.V333M LOH 16p13.3 PNET-6 – LOH 16p13.3 PNET-8 TSC2:c.2838-1G>A TSC2:c.C2164T:p.L722F PNET-9 TSC2:c.C1306T:p.P436S LOH 16p13.3 PNET-10 TSC2:p.Q883X c.C2671T:p.H891Y PNET-11 TSC2:c.2355 + 1G>A LOH 16p13.3 PNET-12 TSC2 c.C2753T:p.S918F LOH 16p13.3
Mutations found in tuberous sclerosis complex genes in sporadic renal angiomyolipoma and insulinomas strongly suggest involvement of these genes in the development of these tumors. These findings assume the possibility of using non-invasive treatment, namely the use of such drugs as everolimus for the treatment of these diseases.
Research Centre for Medical Genetics.
The research was carried out within the state assignment.
All authors have declared no conflicts of interest.
Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) indicate the synthetically lethal effect in BRCA-defective breast cancers. BMN 673 has attracted considerable attention as the most potent PARPi with the highest PARP trapping efficiency. However, several mechanisms of PARPi resistance have been described in the literature. Solid lipid nanoparticles (SLNs) represent a promising new strategy to overcome resistance mechanisms due to unique features including their unique size and stability etc. In the present study, we aimed to determine a potential therapeutic effect of BMN 673 loaded SLNs formulation on homologous recombination (HR) in triple-negative breast cancer (TNBC).
Firstly, we produced BMN 673-SLNs by hot homogenization method. After characterization, the cytotoxic effect of BMN 673-SLNs (0.01-10 nM) on HCC1937BRCA1-/- and HCC1937-R (BMN 673 resistant) TNBC cell line was determined by WST-1 assay. Then, DNA damage, gene expression (PARP1, RAD51, H2AX) and western blot analysis were carried out to detect the effects of BMN 673-SLNs on HR mechanism.
The WST-1 results demonstrated that the viability of HCC1937 and HCC1937-R cells treated with 10 nM of BMN 673-SLNs for 12 days decreased to 29.5% and 34.9%, respectively (p < 0.05). The percentage of p-ATM, p-H2AX and double strand breaks-DNA (dsDNA) in HCC1937 cells was 2.5%, 16.5% and 59.9% at 10 nM of BMN 673-SLNs for 12 days, respectively. Additionally, the percentage of p-ATM, p-H2AX and dsDNA was found to be 2.2%, 9.0% and 71.2% in HCC1937-R cells, respectively. The expression level of H2AX (2.7-fold) and RAD51 (2.9-fold) were up-regulated whereas, PARP1 (-1.22-fold) mRNA expression was down-regulated in HCC1937 cells treated with 10 nM of BMN 673-SLNs for 12 days. Besides, we found that the H2AX, RAD51 and PARP1 expression levels were up-regulated 2.2-, 1.4- and 3.0-fold in HCC1937-R cells, respectively. These findings were supported by the results of western blot analysis.
SLNs could potentially provide therapeutic impact on HR mechanism and overcome drug-resistance for the treatment of TNBC. Thus, SLNs could provide suitable alternatives to existing treatment strategies after preclinical validation.
The Scientific Research Projects Foundation of the Uludag University of Turkey.
This study was supported by a grant from the Scientific Research Projects Foundation (BAP) of the Uludag University of Turkey [Project No: BUAP(T)-2015/1].
All authors have declared no conflicts of interest.
Melanoma progression is often characterized by mutations in the mitogen-activated protein kinase (MAPK) and phosphoinositol-3-kinase (PI3K) pathways. Understanding regulation of these pathways has led to the development of novel targeted therapies which show high response rates. However, many patients relapse with ensuing resistant disease. P63, a p53 homologue, carries a poorer prognosis when overexpressed. In keratinocytes, degradation could be regulated by two ubiquitin ligases, MDM2 and FBXW7. This project explored the expression of p63, MDM2 and FBXW7 in MAPK-inhibitor (MAPKi) sensitive and resistant melanoma cell lines.
Morphology change associated with the development of resistance was assessed by actin immunofluorescence experiments. Western blot and qRT-PCR enabled analysis of protein and RNA expression of p63, FBXW7 and MDM2, complemented by immunofluorescent staining and confocal microscopy data. Finally, a novel technique of flow cytometry enabled analysis of cell death in MAPKi-resistant melanoma cells treated with Nutlin-3A.
MAPKi resistance in melanoma cells is associated with increased p63 expression. Resistance is furthermore associated with reduced FBXW7 expression and enrichment of nuclear MDM2, suggesting a potential nuclear interaction between MDM2 and FBXW7. The resultant inactivation of FBXW7 explains the increased p63 expression upon MAPKi- resistance. Furthermore, by treating MAPKi-resistant cells with the MDM2-inhibitor Nutlin-3A, we were able to restore FBXW7 expression and promote p63-degradation, with resulting increased melanoma cell death.
This project identifies FBXW7 and MDM2 as regulators of p63 expression in MAPKi-resistant melanoma and proposes a possible role for Nutlin-3A in treating advanced MAPKi-resistant melanoma.
Queen Mary University of London (Blizzard Institute) - Cutaneous Research.
Has not received any funding.
All authors have declared no conflicts of interest.
Acute myeloid leukemia (AML) is a malignant clonal disorder characterized by mutations affecting myeloid differentiation, gene expression, and epigenetic profiles. Although treatment strategy depends on a genetic profile-based risk, the accuracy of this stratification system is highly variable. Changes in gene expression profiles of signaling pathways involved in hematopoietic development, such as Wnt/B-catenin, may contribute to the transformation, development, and maintenance of leukemic cells, and could be related to the clinical outcome.
qRT-PCR assays were designed to quantify mRNA levels of c-Myc, and B-catenin in blood samples from nine newly diagnosed AML patients before and after treatment. 18S ribosomal RNA was used to normalize gene expression. Statistical analysis was conducted in R statistical software. Relative expression rates (rERs) were implemented to quantify relative changes in gene expression levels after induction.
c-Myc and B-catenin expression decreased after induction treatment. Additionally, the rERs of both genes were linearly correlated (r = 0.937, p = 0.0002), and patients who has persistence of the disease after treatment have a higher rER -in both c-Myc and B-catenin-, compared to those who achieve complete remission (c-Myc: p = 0.0066; B-catenin: p = 0.04). However, c-Myc and B-catenin rERs were greatly variable in patients who partially responded to induction.
c-Myc and B-catenin are molecules from Wnt signaling pathway, related to activation of the molecular pathway. Patients who have a decrease in the expression of this genes are more likely to achieve complete remission than those who don't change the expression of this molecules. Further analyses are needed to determine the precise role of Wnt/B-catenin in AML therapy response.
Instituto Tecnológico Metropolitano.
Colciencias, grant 669-2014.
All authors have declared no conflicts of interest.
Lipid mediators of inflammation, leukotrienes, are involved in tumour development and progression. Leukotriene B4 receptors 1 and 2 (LTB4R and LTB4R2) have been suggested to regulate tumor progression by promoting cell proliferation, survival, migration and metastasis. LTB4R2 was reported to increase invasiveness of breast cancer (BC) cells through IL-8 (interleukin-8) pathway. The inhibitors of leukotriene receptors have been suggested for use in anti-cancer therapy.
We conducted Xma1-RRBS (Reduced Representation Bisulfite Sequencing) protocol for genome-wide DNA methylation assessment on 170 BC samples and 10 normal breast tissue samples as a reference material. Methylation status was determined by Bismark software, and identification of epigenetic BC molecular subtypes was carried out using hierarchical cluster analysis.
One of the BC epigenetic subtypes identified by genome-wide DNA methylation analysis appeared to be enriched with triple-negative breast cancer (TNBC) samples demonstrating LTB4R and LTB4R2 genes abnormal demethylation that is potent of initiating ectopic gene expression. Bisulfite Sanger sequencing has confirmed abnormal demethylation of the cytosine residues at positions +117, +121, +148, +184 and +186 relative to the LTB4R transcription start site.
By the present day no diagnostic panels to assess the methylation status of leukotriene B4 receptors have been published. Fine mapping of abnormal DNA methylation in the genomic region adjacent to LTB4R and LTB4R2 genes reported here allows development of a simple methylation sensitive PCR laboratory test that would identify tumours prone to leukotriene B4 receptors ectopic expression and thus potentially responsive to their inhibitors. An easy means to select such tumours promotes clinical trials aimed to improve TNBC individualized therapy by introducing the inhibitors of leukotriene receptors as anticancer agents.
Research Center for Medical Genetics RAMS.
Russian Science Foundation (project No.18-15-00430).
All authors have declared no conflicts of interest.
Glioblastoma multiforme (GBM) is an aggressive brain tumor. The median survival time is 14,5 months. GMB resistance to treatment is associated with cellular population of cancer stem cells (CSCs). Wnt-signaling pathway is recognized as a strategically important proliferation mechanism for all stem cells. This study compares the expression levels of Wnt-signaling pathway proteins in CD133+ and CD133- cells in the common pool of GBM.
Research is conducted with CD133+ and CD133- cells of U87MG cell line of human glioblastoma. High-performance liquid chromatography-mass spectrometry was used for proteome analysis. Mass spectrometry data were processed with MaxQuant (version 1.6.1.0) and Perseus (version 1.6.1) software, Max Planck Institute of Biochemistry (Germany). Biological processes, molecular functions, cells locations and protein pathways were annotated with a help of PubMed, PANTHER, Gene Ontology, KEGG and STRING v10 databases.
The statistically reliable changes in expression of 8 proteins from Wnt-signaling pathway (CACYBP, CSNK2A2, CSNK2B, CTNNB1, CUL1, RAC2, RHOA, RUVBL1) were discovered in CD133+cells of glioblastoma as opposed to CD133- cells of this glioblastoma cell line. We registered higher expression levels of nine proteins that positively influence the activity of Wnt-cascade. The increase in the expression of the cadherin complex components with underlying upregulation of casein kinases stimulated β-catenin degradation. Yet due to enhanced focal adhesion an increase of the β-catenin production was discovered. This may be either a mechanism of inducing stemness in the gliomaspheres, or one of the ways to incorporate the embryonic mechanisms of pluripotency in the glioblastoma cells.
Therefore, proteins of Wnt-signaling cascade are considered as a prospective target for regulating the activity of glioblastoma CSCs. Potentially, the pharmacological suppression of β-catenin would be able to disrupt the mechanisms of tumor cells interaction with extracellular matrix and local microenvironment. That can be an important step in the direction of prolonging the life of patients.
Far Eastern Federal University of Russia.
The study was funded by the Ministry of Education and Science of the Russia (grant no. 14.584.21.0027; ID: RFMEFI58417X0027.
All authors have declared no conflicts of interest.
Neurotrophic tyrosine receptor kinases (NTRK1-3) are a gene family encoding kinases involved in development and maturation of the central and peripheral nervous system. In cancer, fusion of one of these genes with various upstream partners leads to aberrant protein expression and unchecked proliferation. Identification of tumors driven by TRK fusions is clinically relevant because they can be targeted by highly selective small molecule inhibitors. Pan-TRK immunohistochemical (IHC) staining for aberrant expression of TRK proteins may be an important approach to identify tumors with TRK fusions when followed by confirmation. We describe the expression, both wild-type (WT) and fusion, of TRK proteins in 19 solid tumors.
An IHC staining protocol was developed using the pan-TRK (EPR17341) antibody in combination with VENTANA OptiView DAB IHC Detection kit, on the VENTANA BenchMark ULTRA platform. This protocol was tested on 3900 tissue samples, across 19 tumor types. A subset of IHC cases was designated as either WT or fused at the NTRK loci using in-situ hybridization (ISH). Staining patterns and percentages are reported, as is the rate of ISH positivity by tumor type.
The pan-TRK (EPR17341) IHC assay detected TRK protein expression across 271 samples in 16 solid tumor types. Tumors with ≥1% staining represented ∼ 7% of all tissues evaluated by IHC. TRK protein expression was negative in most solid tumors. However, in tumors of neuroendocrine/neural origin and in GIST, WT TRK expression ranged from ∼50-75%. The percentage of IHC positive tumors that went on to be ISH positive ranged from 0% in GIST to ∼60% in colorectal carcinoma, highlighting the differential utility of this stain by tumor histology.
In solid tumors with low WT TRK expression, this assay effectively identifies potential TRK fusions for confirmation. However, pan-TRK IHC is hampered in identifying potential fusions in neuroendocrine/neural tumors and GIST. Additional assay development and pathologist training is warranted to guide the interpretation of pan-TRK IHC staining.
Roche Tissue Diagnostics.
Loxo Oncology, Inc and Roche Tissue Diagnostics.
J. Feng, F. Hansen, L. Kivi, C. Kriegshauser, T.-S. Shen, P. Thorne-Nuzzo: Employee of Roche Tissue Diagnostics. K. Ebata, D. Morosini, B. Tuch, A.N. Sireci: Employee and shareholder of Loxo Oncology.
We explored antitumor activity of afatinib in vitro and in vivo in gastric cancer (GC). In addition, underlying antitumor and acquired resistant mechanisms of afatinib were also investigated.
Four GC cell lines were used to evaluate cell viability, cell cycle and apoptosis using cell proliferation assay and flow cytometry, respectively. Nine patient-derived xenograft (PDX) models were established to investigate the antitumor activity of afatinib in vivo. Immunohistochemistry and western blot were used to detect the expression level of ErbB family and downstream PI3K/AKT and MAPK pathway in cell lines and PDX models after afatinib treatment. In addition, one afatinib-resistant PDX model was established, and we detected the gene expression alterations between parental, sensitive and resistant PDX models via RNA sequencing and immunoblot. Furthermore, potential mechanisms of acquired resistance and corresponding reverse strategy were explored.
Afatinib significantly inhibited the proliferation of NCI-N87 cell line (IC50 = 2nM) and induced tumor growth regression or inhibition in EGFR-amplified/overexpressed and HER2-positive PDX models (TGI: 61.9% -116%, p < 0.05). However, in PDX models without ErbB family molecular variations, the anti-tumor activity of afatinib were weak (TGI: 25% -38%, p > 0.05). The effective treatment of afatinib induced cell cycle arrest at G1 (p < 0.05) and obvious apoptosis (p < 0.05) in NCI-N87 cells. Afatinib exerted its antitumor effect by inhibiting the phosphorylation of ErbB family and activation of PI3K/AKT and MAPK signaling pathway. We observed the overexpression and phosphorylation of EPHA2, and reactivation of MAPK pathway in the afatinib-resistant PDX model. The EPHA2 inhibitor ALW-II-41-37 could restore the sensitivity to afatinib by monotherapy (TGI: 60%, p < 0.001) or in combination with afatinib (TGI: 81%, p < 0.001).
Afatinib exerted selective antitumor activity against GC cell lines and PDX models via inducing cell apoptosis and cell cycle arrest at G1 phase, and suppressing phosphorylation of ErbB family, PI3K/AKT and MAPK signaling pathway. EPHA2 conferred the acquired resistance of afatinib against GC and the inhibition of EPHA2 could reverse the resistance of afatinib.
Zuhua Chen.
The National Key Research and Development Program of China.
All authors have declared no conflicts of interest.
The potential role of miRNA34a gene expression as biomarkers of colorectal cancer is not well known , the current study aimed to evaluate its diagnostic and prognostic value and its relationship with P53 gene expression, fate, stage, metastasis and overall survival of colorectal cancer.
This study was carried out on patients with colonic adenocarcinoma (group I), patients with begin colon polyp (group II) and controls (group III). All studied subjects were submitted to estimation of body mass index and laboratory investigations including determination of serum carbohydrate antigen 19-9 (CA19-9) and carcino-embryonic antigen (CEA) levels and Estimation of microRNA 34a and P53 gene expression by real time PCR.
The results of the present study showed statistically significant lower gene expression in colon cancer group compared to others. There was a significant negative relation between serum tumor markers (CEA and CA19-9) and micro RNA 34a gene expression in cancer patients. Also there was statistically significant positive relation between micro RNA 34a and P53 gene expression in both patients groups. The diagnostic accuracy of miRNA34a gene expression was both sensitive and specific for colon cancer compared to serum levels of both CEA and CA19.9. Micro RNA 34a and P53 gene expression had statistically significant relation with tumor stage and presence of metastases.
It can be concluded that the level of miRNA34a can be used to differentiate between colon cancers and begin adenomas. MiRNA34a gene expression has a main role in prognosis of colon cancer. MiRNA34a can be used as a therapeutic agent in colon cancer.
The authors.
Has not received any funding.
All authors have declared no conflicts of interest.
Combined hepatocellular-cholangiocarcinoma (cHCC-CC) is a very rare tumor. It has two different components (hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC)) in a single mass. Although several studies have determined genetic characteristics of cHCC-CC, Next-generation sequencing (NGS) data for comparing clonality of cHCC-CC are currently unavailable.
We selected four cHCC-CC cases and micro-dissected HCC, CC, and normal components separately from each case. DNA and RNA were isolated from each sample and sequenced by Oncomine Comprehensive Panel (OCP) interrogating 143 cancer genes using Ion S5 XL sequence platform. Genetic features of HCC and CC from each patient were compared.
All cases successfully produced NGS data. Two cases demonstrated different mutations between HCC and CC components (biclone) while two cases shared the same mutations between two components (monoclone). SNPs of TP53 (4/4) and PTEN (1/4) and gene amplifications of MET (1/4), c-MYC (1/4), and CDK6 (1/4) were found in CC component. In HCC component, SNPs of TP53 (3/4), PTEN (1/4) and CTNNB1 (1/4) and CCND1 amplification (1/4) were detected. Two biclonal cases showed histologically distinguished border between HCC and CC components with or without intermediate cell foci. Two monoclonal cases showed histologically ambiguous border between HCC and CC components with more intermingled pattern than biclonal cases.
We compared genetic compositions of HCC and CC components and matched clonality with histologic feature in cHCC-CC using targeted sequencing. Two (50%) of four cases had different clones between HCC and CC components with more distinguished histologic features than monoclonal cases. Considering such heterogeneity, partly sequencing is recommended for cHCC-CC, especially in those who have histologically distinguished HCC and CC components regardless of the presence of intermediate component.
Nara Yoon.
Has not received any funding.
All authors have declared no conflicts of interest.
Topoisomerase II-α is a molecular target of anthracyclines; several studies have suggested that topoisomerase II-α expression is related to response to anthracycline treatment. The objective of this study was to evaluate whether topoisomerase II-α overexpression predicts response to anthracycline treatment in locally advanced breast cancer patients.
This prospective study included 50 patients with primary non-metastatic locally advanced breast cancer according to American Joint Committee for Cancer Staging(T3-4;N0-3) treated between January 2012 and Jaune 2012 at Clinical Oncology Department, Tanta University Hospital. Topoisomerase II-α, HER2, estrogen receptor (ER), progesterone receptor (PR) expression and KI-67 were evaluated by immunohistochemistry in formalin-fixed, paraffin-embedded breast tumors from 50 patients presenting with locally advanced breast cancer.
Tumors from 50 patients, 45 (90%) showed topoisomerase II-α overexpression, patients 34 (68%) for ER positive, 32 (64%) for PR positive, and 10 (20%) for HER2 overexpression and 16 (32%) for high KI 67. Significant correlation between clinical and pathological response was observed with topo IIA, HER2 and KI-67: p values of ≤0.001, 0.005 and 0.015, respectively. 1. Responders: Clinical CR: 3 patients had co-expression of topo II and HER2, hormonal receptor negative and high KI-67. Clinical PR: 43 patients majority of them had topo IIA overexpression .fig(9-10) 2. Non responders: 4 (8%) patients all had negative (TOPOII/HER2), low KI-67 and 2 were hormone receptor positive, and another 2 were hormone receptor negative.
Our data support a correlation between topoisomerase II-α expression in locally advanced breast cancer patients and improved clinical benefit with neoadjuvant anthracycline-based therapy.
Has not received any funding.
The author has declared no conflicts of interest.
The long noncoding RNA (lncRNA) highly up-regulated in liver cancer (HULC) was identified as an oncogenic noncoding RNA that contributed to hepatocellular carcinoma (HCC) progression. Generally, lncRNAs have been shown to act as a sponge for microRNAs, hindering them from performing their actions. Recent study has shown that HULC sequester miR-186, meanwhile its expression was repressed by it. Our bioinformatics revealed that miR-186 targets HULC and the insulin-like growth factor 2-mRNA-binding protein-1 (IGF2BP1) which was shown to degrade HULC and to stabilize IGF1R in HCC. Therefore, we aimed at investigating the impact of miR-186 on IGF2BP1 and hence on HULC in liver cancer in attempt to unravel the underlying mechanism by which HULC enhances hepatocarcinogenesis.
A total of 18 HCC and 13 healthy liver biopsies were used. Screening and correlation analysis were done. miR-186 expression was forced in Huh-7 cell lines. miR-186, HULC and IGF2BP1 were quantified by qRT-PCR. Binding confirmation and functional analysis experiments were performed.
The expression level of HULC was positively correlated with IGF2BP1 expression in HCC tissues, where both were highly elevated compared to healthy controls. However, miR-186 was significantly downregulated in HCC tissues and negatively correlated with IGF2BP1 expression. Furthermore, miR-186 had shown considerable tumor suppressor activity in HCC. Upon forcing miR-186 expression in HCC cell lines, IGF2BP1 expression was repressed, while in contrast to what was previously mentioned by Wang Y et.al, HULC expression showed no change. In addition, miR-186 showed direct binding on IGF2BP1, meanwhile it was bound to HULC on one of its two predicted binding sites. Thus, the binding of miR-186 to HULC without altering its expression may indicate that HULC act as a sponge for miR-186, thereby relieving IGF2BP1 and promoting hepatocarcinogenesis.
HULC mediates its oncogenic action through sequestering the tumor suppressor miR-186, relieving its inhibitory effect on IGF2BP1 and thus promoting HCC progression. Finally, we suggest a novel lncRNA-microRNA-mRNA interaction to act as a novel target for halting HCC progression.
German University in Cairo.
Has not received any funding.
All authors have declared no conflicts of interest.
Mounting evidence demonstrated the potential of miR-506-3p to be employed in the diagnosis and treatment of a wide range of human malignancies due to its differential expression pattern and distinct biological roles. Despite the pivotal tumor suppressor role miR-506-3p plays in breast cancer (BC), it is frequently downregulated. Moreover, its role in governing cell cycle progression was not extensively studied in BC. Myc, E2F and Rb proteins are key players in cell cycle regulation. In BC, the CDK-RB-E2F axis is extensively deregulated by several genetic mutations. Additionally, the potent proto-oncogene Myc is highly expressed in BC. Thus, we aimed at uncovering the role miR-506-3p plays in cell cycle regulation in BC.
pMyc-TA-Luc, pE2F-TA-Luc and pRb-TA-Luc vectors have response elements or enhancer elements of cMyc, Rb, and E2F cell cycle regulatory proteins, respectively. MDA-MB-231 cells were transfected with the aforementioned cell cycle vectors as well as an empty pLuc vector containing an unspecific binding site (used as a control) (Clontech, Germany). After 24 hrs, cells were co-transfected with miR-506-3p. Some cells were exposed to the transfection reagent only (mock cells). Relative luciferase activity was measured after 48 hrs by Steady-Glo® Luciferase Reporter Assay. Unspecific (baseline) luminescence detected for empty pTAL-Luc vector was subtracted from all values. Functional characterization of miR-506-3p was performed in MDA-MB-231 triple-negative breast cancer (BC) cells and MCF-7 HR+ cells to investigate its impact on several hallmarks of cancer using MTT and wound-healing assays.
Ectopic expression of miR-506-3p led to a significant decrease in cMyc and E2F proteins with a concomitant increase in RB protein compared to mock cells. Since miR-506-3p has seldomly been investigated in BC, the performed functional analysis of miRNA-506-3p revealed a significant decrease in the cellular viability and migration capacity of both MDA-MB-231TNBC cells and in MCF-7 HR+ BC cells.
This study highlights a novel underlying mechanism of miR-506-3p as a tumor suppressor in BC through inhibiting cell cycle progression. This suggests its potential use in personalized BC therapy.
German University in Cairo (GUC).
Has not received any funding.
All authors have declared no conflicts of interest.
Hesperetin an aglycone extracted from citrus fruits, acts as a novel therapeutic agent in breast cancer (BC). TNBC patients showed the most heterogeneous molecular profiles portrayed with inferior overall survival. This highlights the emerging need of precision medicine to unravel the inter-wined molecular circuits in TNBC patients. Our group has uncovered the tumor suppressor activity of miR-486-5p in liver cancer and BC. However, its role in tuning the metastatic behaviour of TNBC cells remains to be unveiled. This study aims at investigating the interplay of hesperitin and miR-486-5p as a novel therapeutic approach towards achieving a personalized treatment code for TNBC patients.
Breast tissues were collected from 26 BC patients. ER, PR and HER-2 expression was quantified using immunohistochemistry. MDA-MB-231 TNBC cells were treated with serial dilutions of hesperitin. miR-486-5p loss and gain of function experiments was performed using lipofection technique. Functional analysis experiments were carried out such as MTT, colony forming and wound closure assays 48 hrs post-treatment. 5-Fluro uracil was used as a positive control. Total RNA extraction was performed followed by reverse transcription into cDNA which was quantified using qRT-PCR. Unpaired student’s t-test was performed using GraphPad 5.0.
miR-486-5p was down-regulated in 100% of BC patients. TNBC patients showed a selective diminished miR-486-5p levels compared to non-TNBC patients. Hesperetin markedly decreased MDA-MB-231 cellular viability and clonogenicity compared to vehicle control. miR-486-5p mimics repressed cellular viability, clonogenicity and metastatic potential of TNBC cells thus supporting its reported tumor suppressor activity. While anti-miR-486-5p resulted in an increase in TNBC hallmarks. Interestingly, a potential crosstalk between hesperitin and miR-486-5p was witnessed in MDA-MB-231 where hesperitin markedly increased miR-486-5p expression.
Hesperitin is a novel anti-neoplastic agent which mediates its anti-proliferative activity through inducing miR-486-5p levels thus efficiently harnessing TNBC progression and hijacking its metastatic potential.
German University in Cairo, GUC.
Has not received any funding.
All authors have declared no conflicts of interest.
Nivolumab and pembrolizumab that target the PD-1 immune check point have recently been approved in recurrent and/or metastatic head and neck squamous cell carcinoma (R/M HNSCC) patients who have failed platinum therapy. We aimed to evaluate the prognostic value of selected immune genes’ expression in a retrospective analysis of 96 untreated HNSCC patients.
We retrieved 96 HNSCC patients who underwent primary surgery at Institut Curie between 1990 and 2006. Real-time quantitative RT-PCR was used to analyze a panel of 46 genes classified in immunity genes and tissue specific genes. Univariate and multivariate analyses were performed to assess the prognostic value of overexpressed genes. This study was approved by the internal review board of Institut Curie.
Median age was 56 years [range: 35–78]. 80% of patients were men with a history of alcohol (70%) and/or tobacco consumption (73%). Twelve patients (12%) were HPV-positive. Primary tumor location was oral cavity (45%), oropharynx (21%), larynx (18%), and hypopharynx (17%). Patients had stage I, II, III, and IVa in 10%, 16%, 13%, and 61%, respectively. Median disease-free interval was 23 months [range: 0.7-185]. Most significantly overexpressed genes were OX40L (74%), TNFRSF9 (77%), and TNFRSF18 (74%) immunity genes, and CD80 (80%) and FOXP3 (62%) tissue specific genes. Median disease-free interval was 11 months in high OX40L mRNA level HNSCC patients versus 23 months in low OX40L mRNA level HNSCC patients (p = 0.0009).
OX40L overexpression was associated with a poor outcome in HNSCC patients treated with primary surgery.
Institut Curie.
Fondation ARC pour la recherche sur le cancer ICGEx.
All authors have declared no conflicts of interest.
Recent clinical data indicate a higher pathological complete response rate when platinum is added to neoadjuvant chemotherapy in triple-negative breast cancer (TNBC), and favorable outcomes have also been observed in metastatic TNBC patients treated with platinum-based regimen. BC associated with germline BRCA mutations display DNA double-strand break repair defects, which are thought to render them particularly sensitive to DNA repair targeting drugs such as PARP inhibitors and/or platinum analogs. In addition, BRCA1/2 genes promoter methylation, somatic mutation, as well as other genomic alterations in DNA repair genes may also drive homologous recombination-deficiency (HRD) and the so-called “BRCAness” phenotype. Yet, their association with sensitivity to platinum derivatives or PARP inhibitors in BC remains discussed.
A 75-year old women with rapidly progressive metastatic TNBC and clinical resistance to anthracylines, taxanes, capecitabine, pembrolizumab and eribulin was enrolled in a molecular profiling program (PERMED trial, NCT02342158). To identify clinically-actionable gene mutations and putative druggable pathways, a pretreatment biopsy of liver metastatic tissue was obtained and analyzed by whole genome array-CGH (aCGH) and targeted next-generation sequencing (NGS) of 559 cancer genes, along with germline sequencing. Gene copy number aberrations (CNAs) and somatic mutation status that could help therapeutic decision were examined.
No germline BRCA mutation was found. The aCGH analysis showed a genomic instability associated with a BRCAness profile and an HRD score of 19. The CNAs analysis showed a focal homozygous loss of RAD51B/RAD51L1 gene, while NGS identified a somatic mutation of BRCA1. Patient received carboplatin single-agent and developed a durable and almost complete response.
These data strongly suggest that carboplatin may be highly effective in advanced TNBC with no germline BRCA mutation but somatic genomic alterations disrupting DNA repair pathways, supporting precision medicine trials evaluating platinum derivatives in this setting.
Institut Paoli Calmettes.
Has not received any funding.
All authors have declared no conflicts of interest.
Recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) is an uncurable disease and is responsible of 4 000 deaths every year in France. Targeted therapies improve outcomes in several types of cancer and lead to development of the prescription of drugs according to the molecular features of the tumor, called “precision medicine”. The need to perform recent biopsy to perform those molecular analyses is a critical restriction and limits inclusion of patients into precision medicine programs.
To compare molecular profiles of primitive tumor and recurrence, we analyzed molecular alterations in primary HNSCC and paired local or distant recurrences from 31 patients using targeted next-generation sequencing (MiniSeq, Illumina), of 42 “cancer-associated” genes (Solid Tumor Solution, Sophia Genetics).
Primary tumors and recurrences harbored alterations common in HNSCC. The most frequent were TP53 mutations (76%), CDKN2A mutations (21%), PIK3CA mutations (16%), MET mutations (16%) and EGFR amplifications (8%). Twenty-six patients (83.9%) showed concordant molecular profiles between primary tumors and matched local or distant recurrences. Fifteen patients harbored alterations with potential therapeutic implications (mostly tyrosine kinase receptor genes or oncogenes activation) according to local Molecular Tumor Board criteria. Twenty-seven out of the 31 patients (87.1%) had concordant molecular profile between primary and recurrence when considering only these “druggable” alterations.
HNSCC primary tumors and metastases exhibit an intra-individual high genomic concordance. As these patients had limited therapeutic options, inclusion in precision medicine programs represents an important opportunity and should be allowed regardless of the origin of the sample, the primary tumor or its recurrence.
Centre Antoine Lacassagne.
Centre Antoine Lacassagne.
All authors have declared no conflicts of interest.
Selenium (Se) has been recognized as an essential element for animals and humans. In the late 1960s, it was first suggested that selenium might have anti-cancer properties. Selenium controls apoptosis in cell cycle. Cadmium (Cd) is a widely used heavy metal that affects human health through occupational and environmental exposure and has been reported as a cause of cancers such as lung, prostate and kidney. The previous studies on Ovarian and breast cancer have shown that cadmium increases telomerase activity and expression level of hTERT. In the present study, we find out the effect of sodium selenite and selenium l methionine and cadmium chloride on telomerase activity in adenocarcinoma cancer cell line (AGS).
The adenoma carcinoma gastric cancer cells (AGS) were cultured in RPMI1640 supplemented with 10 % fetal bovine serum (FBS) and 1% pen/strep. Concentrations of cadmium chloride, selenium l methionine and sodium selenite were added the adenocarcinoma gastric cancer cells (AGS). Finally, the telomerase activity was measured by telomeric repeat amplification protocol (TRAP) and qRT-TRAP assays.
Average telomerase activity (as threshold cycle (CT)) has been detected for three concentrations (5μM and 10μM and 20 μM) of the noted compounds. The results were 24.82±0.51, 25.31±1 and 25.63±1.32 for Sodium selenite, 24.56±0.25, 25.29±0.98 and 25.60±1.21 for Cadmium chloride and 24.49±0.18, 25.30±0.99 and 25.73±1.32 for Seleniul l methionine, respectively. Representing the signification decreasing telomerase activity at treated samples compared with the controls (p ≤ 0.05).
Decreasing in the telomerase activity which was caused by treating with Cadmium chloride, Selenium l methionine and Sodium selenite is in conflict with some previous findings indicating the increasing in the telomerase activity by treating Cadmium.
Ardabil University of Medical Sciences.
Ardabil University of Medical Sciences.
All authors have declared no conflicts of interest.
Gliomas are the most frequent CNS primary tumours, which account for up to 80 % of brain malignancies. Important diagnostic and prognostic factor, and recently significant WHO glioma classification marker as well, is mutational status of IDH1/2 genes. Moreover, search for another markers with predictive character is needed. Methylation status of MGMT gene promoter is one of relevant predictive markers, connected with therapeutic response to chemotherapy.
260 glioma samples were biopted or surgically resected at Neurosurgery Clinics within Moravian – Silesian Region, Czech Republic. DNA was extracted from native tissue samples. IDH1/2 gene mutation detection was performed with extension primer method – SnaPshot® assay. MGMT gene promoter methylation status was tested by nested methylation-specific PCR and real-time PCR, preceded by bisulfite conversion of DNA samples.
Analysis of 260 glioma samples was performed. Out of this, 218 samples were histopatologically diagnosed as high-grade (HG) gliomas and 42 samples were classified as low-grade (LG) gliomas. Median age of all patients was 60 years (range 24 – 82 years) at the time of diagnosis. 55 % of patients were men. IDH1 gene mutations were detected in 29, 2 % (76/260) of patients, MGMT gene promoter methylation was detected in 60, 8 % (158/260) of patients. IDH2 gene mutations were not detected. Median overall survival (mOS) was 9 months longer in patients with HG glioma and IDH1 gene mutation than in patients without mutation. If complete set of HG and LG gliomas was included, 11 months difference of mOS has been reached. Furthermore, statistically significant difference of mOS among the group of patients with IDH1 gene mutations and without mutations has been reached (p value < 0.001 and p < 0.001, respectively).
Observed relationship of IDH1 gene occurrence to median OS in patients with primary glioma was statistically significant. MGMT gene promoter methylation did not have any statistically significant influence to OS in observed HG glioma patients.
CGB Laborator a.s.
Has not received any funding.
All authors have declared no conflicts of interest.
Colorectal cancer (CRC) represents a relevant public health problem. Despite new therapeutic advances, prognosis of patients diagnosed with advanced disease is still poor. The identification of new markers involved in the mechanisms of invasiveness represents a priority in order to better understand cancer development and generate new therapeutic targets. We describe here the possible role of ZNF518B, a gene not yet well characterised, on tissue invasion. Human ZNF518B gene, which encodes a putative, zinc finger-containing transcription factor, yields two major alternative splicing isoforms.
Expression of ZNF518B was determined by RT-qPCR in several CRC cell lines and in a commercial cDNA array from CRC patients. To explore the mechanisms that relate the expression of the gene with tumourogenesis, the effects of silencing ZNF518B on the phenotype of human CRC cell lines has been studied. Among these experiments, we have performed apoptosis assays by flow cytometry, proliferation by MTT assays, invasion and migration assays using transwells and adhesion to type I collagen plates assays.
The analysis of a cDNA array from CRC patients showed that the expression of the whole gene and of its two major isoforms is significantly increased in tumour tissues when compared to normal mucosa. By the analysis in CRC cell lines, we have demonstrated that the gene is not involved in cell proliferation, but plays a significant role in cell migration and invasiveness. These data, together with the changes in the epithelial-to-mesenchymal transition markers, strongly suggest that ZNF518B plays a role in tumour cell dissemination.
ZNF518B might therefore be a candidate for invasiveness prognosis.
INCLIVA-Universidad de Valencia.
Has not received any funding.
All authors have declared no conflicts of interest.
An increasing number of molecularly targeted drugs are now available and, for some of these therapies, predictive biomarkers have already been identified. In particular, mutations in ERBB2 might represent an alternative mechanism for HER2 activation. They occur more frequently in HER2-negative (HER2-) tumors and seem to be good target for HER2 therapy. Furthermore, PI3KCA mutations showed to predict sensitivity to Fulvestrant, Buparlisib, Taselisib and resistance to Lapatinib. We evaluated the incidence of ERBB2 and PI3KCA mutations in 14 hormone receptor (HR)-positive BC and in their matched endocrine-resistant recurrences.
We evaluated a panel of genes including ERBB2 and PI3KCA, in FFPE tissues. We analysed 14 HR-positive BCs and their matched recurrences. All the relapses have been developed during an endocrine treatment.
4/14 pts were diagnosed with HER2+ BC, while 10/14 pts developed HER2- BC. Overall, we found 8 different mutations of ERBB2 in 9 samples: A356D, Q1206X, Q396X, Q393X, P523L, I654V, G1220C, 135 + 3G>T. We found 6 different mutations of PI3KCA in 10 samples: N345K, V344M, E542K, E545K, A1035V, H1047R. ERBB2 mutations were found in the 28.6% of primary tumors and in the 35.7% of relapsed sites. PI3KCA mutations were found in the 35.7% of primary tumors and in the 35.7% of relapsed sites. mOS in PI3KCA mutated pts was 112,8 months, while mOS in PI3KCA wild-type pts was 99. mOS in HER2+ and/or ERBB2 mutated pts was 115.6 months, while mOS in HER2- ERBB2 wild type pts was 97.5. mDFS in PI3KCA mutated pts was 41.2 months, while mDFS in PI3KCA wild-type pts was 28. mDFS in HER2+ and/or ERBB2 mutated pts was 46.4 months, while mDFS in HER2- wild-type pts was 28.5.
We found an overall detection rate of ERBB2 mutations higher than that described in literature, while PI3KCA mutations were in line with the literature. The identification of ERBB2 or PI3KCA mutation might justify a more targeted neo/adjuvant approach and might guide the subsequent treatment choices. According to the literature, pts with PI3KCA mutation showed better prognosis, while, contrary to the previous literature, in our study the majority of ERBB2 mutations occurred in HER2+ samples and HER2+ and/or ERBB2 mutated samples did not show worse outcomes.
Marta Venturelli.
Angela Serra Association.
All authors have declared no conflicts of interest.
Drugs targeting the RANK/RANK-ligand pathway may improve the outcome in early breast cancer, mainly due to prevention of bone metastasis. We aimed at exploring the value of RANK expression in primary breast cancer samples and its impact on the incidence of bone and visceral metastases.
RANK expression was assessed in samples of 79 ER-positive/HER2-negative early breast cancer by immunohistochemistry in tissue micro array (TMA) blocks. Correlation between high and low RANK expression was done with the clinicopathological factors using Fisher's exact test and with bone metastasis free survival (BMFS), visceral metastasis free survival (VMFS) and overall survival (OS) using Kaplan Meier and Cox regression tests.
Median age at diagnosis was 56 years (42-85 years). RANK was highly expressed in 27 patients (34.2%) while 52 patients (65.8%) had low expression. BMFS in patients with high RANK expression was poorer than those with low expression with an 8y BMFS of 54.9% in patients with high expression versus 83.9% in the RANK low expression group (HR = 2.93, 95%CI: 1.36 to 6.30, p = 0.006). RANK expression was similarly associated with more visceral metastases with 8y VMFS of 54.5% versus 83.8% respectively (HR = 3.20, 95%CI: 1.47 to 6.99, p = 0.004). Moreover, OS was worse in cases with high RANK expression with 8y OS of 70.0% versus 90.2% respectively (HR = 2.79, 95%CI: 1.20 to 6.49, p = 0.017). In the multivariate analysis, high RANK expression was still an independent predictor for bone metastasis (HR = 2.43, 95%CI: 1.10 to 5.37, p = 0.028) and visceral metastasis (HR = 2.69, 95%CI: 1.21 to 6.0, p = 0.016).
RANK expression in the primary ER+/HER2- breast cancer samples is associated with more bone and visceral metastasis denoting inherent aggressiveness of this subgroup of patients rather than organ tropism. Further research is still needed to confirm these results.
Centre Léon Bérard.
Has not received any funding.
All authors have declared no conflicts of interest.
Current standard chemoimmunotherapy achieves survival in almost 95% children with mature B-NHL. However, children with relapses still carry very poor prognosis.
Molecular profiling consisted of whole exome NGS, gene expression profiling and peripheral blood phosphoflow.
Both cases had identical clinical presentation and histology, progressed early during standard therapy and did not respond to intensive retrieval chemotherapy. In the first patient, a germ line variant of c.935C>G in the PI3K-delta subunit was found. Activation of PI3K downstream signaling pathway was detected using flow cytometry in patient´s peripheral blood lymphocytes. Increased levels of PI3K were confirmed also by gene expression profiling together with an increased expression of HR23B. Immunohistochemistry revealed strong expression of PD-1L. Therapy was changed to a single agent idelalisib window. In two weeks there was markedly decreased PI3K pathway activation in patient’s lymphocytes, but the disease still showed a radiological progression. During the time, ibrutinib, valproic acid, nivolumab and metronomic cyclophosphamide was added and local 21Gy radiation to the site of the abdominal relapse was used. Partial remission was documented and personalized immunotherapy with autologous dendritic cell-based vaccine was given. Residual tumor resected after 11 months did not show any viable tumor and treatment was gradually tapered down. His 3rd EFS 31 months on personalized treatment is the longest he ever experienced. The second patient died 11 months form diagnosis.
The first case was successfully treated with targeted and immune therapy that matched the molecular signature of the tumor, but in the second case, the therapy “missed” the target, because his molecular signature was not known at the time retrieval therapy. The findings suggest that molecular signatures among clinically and histologically similar patients could be unique, and a tissue biomarker-based customized therapy may be the best approach to address these poor prognosis patients.
Department of Pediatric Oncology, University Hospital, Brno.
AZV MZCR - 16-33209A, 16-34083A Masaryk University, Brno, Czech Republic - MUNI/A/1136/2017.
All authors have declared no conflicts of interest.
Pancreatic cancer (PDAC) is highly resistant to cytotoxic chemotherapy and several trials of molecularly targeted drugs have failed. However, some tumors with specific somatic mutations respond to targeted therapy and patients might profit from treatment stratified by genetic biomarker profiles.
We used MH Guide, a proprietary data tool, to annotate somatic mutation in PDAC patients who had received ≥ 1st line palliative therapy. A molecular tumor board (MTB) interpreted suggestions and matched consecutive therapies including off-lable use of any EMA-approved drug to biomarker profiles. Frequency of clinically relevant markers was the primary outcome. Secondary outcomes were choice of therapy, clinical outcomes and AE.
We enrolled 39 patients, analyzed 35 samples and had valid results from 31 tumors. The most common mutations were KRAS (n = 24), TP53 (n = 17), SMAD4 (n = 2), BRCA1/2 (n = 4; 3 germline), CDKN2A (n = 9), ATM (n = 6; 3 LOF), APC (n = 4), and MSH3 (n = 15, 1 germline). MH Guide suggested off-label treatment in 28 cases: PARP-inhibitors (n = 26), MEK/RAF inhibitors (n = 20), CDK inhibitors (n = 9), other kinase inhibitors (n = 3), mTOR inhibitors (n = 2). Common toxicity markers were: Platinum (XRCC1 n = 16; 5 homozygous, TPMT n = 1; MUTYH n = 3), paclitaxel (CYP2C8 n = 2), gemcitabine (CDA n = 4; 1homozygous), 5-FU (DPYD n = 1), doxorubicin (G6PD n = 1), docetaxel (GSTP1 n = 2), sunitinib (ABCG2 n = 2), irinotecan (ERCC2, n = 6; 5 homozygous, UGT1A1 n = 1). Three patients deceased before MTB decision and 8 prior to treatment, 5 patients were not eligible for active treatment. Seven patients received targeted treatment: Olaparib (n = 6, 1 still on treatment), palbociclib (n = 1) and remained median 84 days (range 18 – 114) on treatment. Four patients received cytotoxic chemotherapy according to MTB decision (among them one without positive markers) and in four cases cytotoxic therapy was used despite a molecularly targeted suggestion.
Unbiased evidence-based molecular stratification of pre-treated patients with PDAC is feasible in a clinical setting. Disease progression and tissue availability are major challenges. Conclusions on clinical outcomes are limited by the study size.
NCT02767700.
Karolinska University Hospital.
Molecular Health GmbH (Data analysis).
M. Kordes: Travel Grant from Molecular Health GmbH. M. Löhr: Consultant or advisory role: Molecular Health GmbH, Pharmcyte. Stock in Centogene AG. Horaria: Abbott, Mylan, Nordmark. Travel Grant: Abbott, Pharmcyte, Molecular Health GmbH. S. Kaduthanam, C. Hülsewig, K. Stecker, S. Brock: Employee of Molecular Health GmbH. J-E. Frödin: Travel grant: Amgen. All other authors have declared no conflicts of interest.
Molecular mechanisms responsible for resistance to targeted agents are no longer clear and need to be clarified. Many pathways have been stressed to be responsible for acquired resistance in HER2 amplified GC, among them, PI3K/AKT/mTOR plays a relevant role. NRF2 was recently identified as a possible mechanism implicated in resistance to chemotherapy.
OE19 and NCIN87, HER2+ GC cell lines were treated with increasing doses of Lapatinib (L) and Trastuzumab (T) to obtain resistant clones. These were isolated and characterized by performing mutational analysis and protein expression by Western blot (WB). Genome expression profile was done by ClariomS microarray. Inhibition of the altered pathways was evaluated using a panel of selected drugs. siRNAs were performed to characterise the role of the inhibition of selected proteins. An in vivo experiment was conducted to corroborate results. A retrospective cohort of HER2 amplified patients treated with T was analysed. An immunohistochemistry (IHC) analysis to evaluate the altered pathway detected in preclinical models was conducted.
L and T resistant clones were obtained. In resistant cells, protein expression underlined the activation of PI3K pathway and of its downstream effector RPS6 protein. Analysing microarray, it was possible to identify, the activation of a large number of genes regulated by NFR2. Its expression was confirmed by WB; NRF2 nuclear activation was detected by nuclear fractionating WB and immunofluorescence. A panel of target agents was used to evaluate NRF2 changes. It was possible to observe the its expression strongly decrease by using PI3K pathway inhibitors, suggesting a relation between this pathway and NRF2. To better clarify this phenomenon, siRNA of RPS6, that was detected to be activated in resistant cells, was performed and it was possible to observe a strong decrease of NRF2 expression and sensitivity to antiHER2 drugs was restored. IHC of both RPS6 and NRF2 were performed suggesting that the activation of both RPS6 and NRF2 related with less clinical benefit of T.
RPS6 through the activation of NRF2 should be considered a new mechanism responsible for antiHER2 drugs resistance in GC. Further investigations are needed.
INCLIVA Biomedical Research Institute.
INCLIVA Biomedical Research Institute.
All authors have declared no conflicts of interest.
Breast cancer is the most prevalent type of cancer among women worldwide, and also the leading cause of women death in developed countries. Among the different breast cancer subtypes, triple-negative breast cancer is the most aggressive one. Those patients can be treated with cytotoxic therapies, but the most of them develop resistance against those drugs. Due to this reason, it is important to study new therapeutic approaches to increase the efficacy of chemotherapy. Herein, microRNAs have a major role in tumoral cell regulation as well as in drug resistance. Our aim was to determine which microRNAs are differentially expressed by MDA-MB-231 cells and MDA-MB-231 cells with acquired resistance to doxorubicin.
A microRNA microarray (Genechip miRNA 4.0 Array, Affymetrix) was performed to compare microRNA expression between MDA-MB-231 and MDA-MB-231 with acquired resistance to doxorubicin. Studies of gain/loss of function of miR-99a were carried out in MDA-MB-231 cells by transient transfection of mimic and inhibitors. Then, those cells were treated with doxorubicin, paclitaxel and cyclophosphamide to establish the role of miR-99a in sensitivity or resistance to those drugs. Viability was measured by flow cytometry.
Among the differentially expressed microRNAs, we found that miR-99a-5p was downregulated in MDA-MB-231 resistant cells vs MDA-MB-231 wild type. We confirmed these results by PCR. The study of its involvement in resistance to different chemotherapies (doxorubicin and paclitaxel) showed that inhibition of miR-99a-5p in wild type cells increased resistance to doxorubicin and paclitaxel. Moreover, when we overexpressed this microRNA, the sensitivity to doxorubicin was increased but there were no changes in paclitaxel.
The present study provided that miR-99a-5p is involved in chemoresistance in triple-negative breast cancer. Nowadays we are performing microarray of mRNAs to find miR-99a-5p targets which are involved in the observed effects. The importance of this finding could be overcome the resistance to chemotherapy in triple-negative breast cancer patients, and can be a potential therapeutic target.
INCLIVA Biomedical Research Institute.
Has not received any funding.
All authors have declared no conflicts of interest.
Muscleblind-like 1 (MBNL1) has been deeply investigated because its implication in myotonic dystrophy. However, the role of RNA-binding proteins in breast cancer remains unknown. Recent findings suggest the role of MBNL1 as a suppressor of metastasis by stabilization of other genes in triple-negative breast cancer cell lines.
MDA-MB-231 (triple-negative) and MCF10a (non-tumoral negative control) breast cancer cell lines were selected. Silencing of MBNL1 was performed by siRNA (Termofischer) at 100nM and using Lipofectamine 2000 (Invitrogen). Effect of MBNL1 depletion on EMT markers was analized by RT-PCR and Western blot. 72 hours after MBNL1 siRNA transfection functional assays were performed. Effect on proliferation was measured by MTT and viability was measured by flow cytometry. Death cells were stained by propidium iodide and florescence was analized by FACS Fortessa. Changes of invasion and migration capacity were studied by transwell assays after 24h. Relation between MBNL1 expression and patients’ survival was studied by KM-Plotter software.
MBNL1 silencing upregulated gene and protein expression of epithelial-to-mesenchymal transition (EMT) markers in MDA-MB-231 cell line. Cell proliferation and viability decreased in MDA-MB-231 after siRNA transfection. Downregulation of this gene increased invasion but not migration capacity in triple-negative cell line by upregulation of pro-metastatic genes. In non-tumoral control cell line MBNL1 silencing had no effect on functional assays nor on EMT genes expression. Expression of MBNL1 in patients’ public databases showed that a higher expression significantly correlates with longer distant metastasis free survival and relapse free survival, as expected.
MBNL1 is involved on triple-negative breast cancer progression and metastasis through regulation of EMT markers. Regulation of MBNL1 could be a new strategy to control disease progression and avoid metastasis in breast cancer patients.
INCLIVA Reserach Institute.
INCLIVA Research Institute.
All authors have declared no conflicts of interest.
Myeloproliferative neoplasms (MPNs) are clonal hematological malignancies with an inherent tendency to progress to acute leukemia, after a variable period of time. Although the mechanisms involved in the disease transformation are still unclear, it´s known that transcription factor RUNX1 (AML1) is frequently deregulated in human leukemia, and recently, it has been described that the activity of RUNX1 together with CBF-β cofactor is regulated by the proteins p300 and HIPK2. In fact, HIPK2 phosphorylates both RUNX1 and p300, activating the whole transcriptional complex. Therefore, the study of the status of this complex seems to be interesting in order to understand the mechanisms involved in the leukemic transformation.
Our aim is to study the effect of different typical drugs in several NMPs cell lines and patient samples with differential expression of these genes and compare it with a context where the complex is downregulated. For this purpose, we knocked down some genes of the studied complex before the treatment and then performed different viability, apoptosis and cell cycle assays, to elucidate if the status of these genes could play an important role in the response to the different biochemical drugs tested, showing a new approach to these diseases and giving us new strategies to study the molecular changes that occur during leukemic transformation.
Here we show that the state of the Runx1/CBF-BETA/P300/Hipk2 can play a critical role for the progression of the Chronic Myeloproliferative Neoplasms.
The functional status of the components of the complex and its deregulation, could be playing an important role in the progression of leukemogenesis in the Chronic Myeloproliferative Neoplasms.
Instituto de Salud Carlos III. Ministery of Sciences. Govern of Spain.
Instituto de Salud Carlos III.
All authors have declared no conflicts of interest.
Cervical Cancer (CC) is the fourth most common cancer in women worldwide. Potential biomarkers in cancer can be identified by considering the changes in tumoral glycolysis. In this work, we investigated whether the expression of some glycolytic enzymes is associated with the overall and disease-free survival of patients with CC.
We analyzed the expression of 14 genes related with glycolysis (SLC2A1, ADPGK, ALDOA, EDARADD, ENO1, GAPDH, GPI, HK2, LDHA, PFKP, PGK1, PKM, TPI1P1, SLC9A1) in 67 CC, 10 high-grade cervical intraepithelial neoplasia (HG-CIN) and 17 healthy cervical epitheliums (HCE), as controls, with the Human Gene 1.0 ST Microarray (HG 1.0 ST). Validation of gene expression of LDHA and PFKP was performed by qRT-PCR and immunohistochemistry. The expression of LDHA and PFKP was correlated with 5-year survival of CC patients by the Kaplan-Meier method, and FIGO staging and the gene expression were included as covariates in a Cox proportional hazard model.
The amount of the mRNA transcribed from the 14 glycolytic genes was higher in CC tumors than in HG-CIN and HCE. Overexpression of genes TPI1P1, LDHA, PFKP, GAPDH, GPI, PGK1 was associated with poor survival, but only the overexpression of LDHA and PFKP genes was independent of the FIGO stage. The qRT-PCR assays confirmed that the overexpression of LDHA and PFKP were associated with poor overall survival (LDHA p= 0.004, PFKP p = 0.0043, Long-rang test) and disease-free survival (LDHA p = 0.001, PFKP p = 0.013, Long-rang test) in an independent fashion of the FIGO stage (LDHA p = 0.050, PFKP p = 0.029, Cox proportional-hazards regression). Interestingly, only the protein encoded by LDHA gene, analyzed by immunohistochemistry, showed a significant higher expression in recurrent than in non-recurrent tumors.
Our results indicate that the expression of LDHA and PFKP at the mRNA and protein levels may be good biomarkers for overall and disease-free survival, independent of FIGO clinical stage, and could be potential therapeutic target for CC.
Conacyt, Mexico.
Has not received any funding.
All authors have declared no conflicts of interest.
ROS1 hence became recognized as a distinct molecular target in NSCLC.Like ALK rearrangements ROS1 fusions are also found more in never smokers but define a distinct molecular subgroup with both rarely occurring together in same patient Crizotinib, a highly effective inhibitor of ROS1 kinase activity, is now FDA-approved for the treatment of patients with advanced ROS1-positive NSCLC .We report our experience in a tertiary cancer care centre in ROS-1 positive patients.
The present study was a retrospective analysis of a prospectively maintained lung cancer registry in whom the ROS1 mutation status was determined.Genetic Assessments for ROS1 and ALK rearrangements were identified by using FISH.All statistical analyses were carried out using the SPSS software version 22.0.
Between January 2015 and December 2017 there were 22 patients who were positive for ROS gene rearrangement. A total of 16 patients took Crizotinib either in first or second line.There were 13 male(60%) and 9 females(40%) in the cohort whose median age was 54 years.The median follow up in Crizotinib received arm was 322 days and 244 days in non-Crizotinib arm.Among the 16 patients who received Crizotinib, 2(12.5%) achieved (CR) as their best response and continue to remain in CR at follow up.13(81.2%) had (PR) as best response and of which on follow up 5 have progressed while 8 continue to maintain response.The median follow-up was 244 days.The estimated medianPFS for the entire cohort of patients was 4 months(95%CI:2.5–5.5 months).The estimated median PFS for the ROS mutant patients was significantly longer at 573 Days(95%CI:318-544 days) as compared to the estimated median PFS for ROS negative patients which was 79 days(95%CI:43-201 days),p=0.00.The estimated median OS for all patients was 13 months(95% CI:10.7–15.3months).The estimated median OS for ROS positive patients was not achieved (95%CI:2.4–25.6 months), while that for ROS negative patients was 79 days (95%CI:7.4–12.6months),p = 0.001.
ROS1 rearrangement with incidence of 4% of lung adenocarcinoma represents a important targetable driver mutation in Indian population. Crizotinib is effective treatment option with acceptable side effects in our population.
Tata Memorial Hospital.
Has not received any funding.
All authors have declared no conflicts of interest.
Cancer genomics research aims at investigation of biological pathways affected by mutations of these genes will help us to understand the determinants of cancer initiation, progression, and other biological functions. The sophisticated computational methods have been developed to facilitate the detection of cancer driver mutations and pathways. We made an attempt in this study to identify the potential candidate genes in the breast cancer carcinogenesis by computational integrative analysis. This approach is appropriated for identifying potentially useful diagnostic and prognostic biomarkers for therapeutic intervention of breast cancer.
Breast cancer tissue microarray datasets were retrieved from Gene Expression Omnibus database (GEO) and the upregulated and downregulated genes were analyzed. Gene ontology, pathway analysis and coexpression network patterns identification were performed.
A total of 1760 Differential Expressed Genes were identified, including 1083 upregulated and 677 downregulated genes. The Gene Ontology terms for molecular functions, biological processes, and cellular component were protein binding, small molecule metabolic process, and integral to membrane, respectively. The most significant pathway in BIOCARTA was DNA replication & repair and cell division regulation pathways. Protein to protein network analysis indicated that the significant hub genes including ARD1,AURKB, AIK2, AIM1, ARK2, AurB, IPL1, STK5, AIM-1, STK12, PPP1R48, aurkb-sv1, aurkb-sv2, CEP55, CT111, URCC6, C10orf3,MELK , HPK38,FOXA1,HNF3A, TCF3A,ARA, IGDA, IHG1, FKHL7, IRID1, RIEG3, FREAC3,ACKR1, CCBP1, CD234, WBCQ1 , PIR- FIGF and BAI1 associated protein 2(BAIAP2) . Gene coexpression network analysis identified 4 major modules,Ankyrin repeat domains,Forkhead box C1,Forkhead box A1 and Trefoil factor 3.
Our study concludes a comprehensive view of integrative analysis of gene expression patterns associated with the breast cancer onset and progression.
We acknowledge our affiliated institutions academic, administrative heads , faculties and other technical assistants.
M. Peer Mohammed.
Has not received any funding.
All authors have declared no conflicts of interest.
Telomerase is a ribonucleoprotein enzyme with reverse transcriptase activity, that more important in developmental processes including cell proliferation, differentiation, senescence and tumorigenesis. Selenium as a trace element for animals and human has been detected which can have anticancer properties, while cadmium (Cd) is a heavy metal in the natural environment and is very toxic. Purpose of this study was investigating the effects of Seleno-L-methionine, Sodium selenite and Cadmium chloride on the expression of telomerase activity in Chick embryo neural tube.
Using quantitative and qualitative methods RTQ- TRAP and TRAP assay, the effect of the noted elements on the telomerase activity of Chick embryo neural tube was studied.
Unlike expected, seleno-L-methionine increased telomerase activity. Cadmium chloride had the greatest effect on telomerase activity, while the effect of Sodium selenite was lower than two others. However, the increasing effect has been determined.
All three compounds at concentration of 5 μM and effect time of 6 hours had most impact on Chick embryo neural tube telomerase activity compared to the control groups. Cadmium chloride had greatest effect and sodium selenite were less effective.
Ardabil University of Medical Sciences.
Has not received any funding.
All authors have declared no conflicts of interest.
The vast majority of PARPi clinical trial experience in gynaecological cancer is for patients with platinum sensitive high grade serous ovarian carcinoma (HGSOC) and deleterious BRCA1/2 mutations. It is known that other homologous recombination deficiency (HRD) associated mutations exist eg RAD51, BRIP1 that can potentially sensitise to PARPi. Extremely limited clinical data exists on the use of PARPi in HGSOC with HRD mutations other than BRCA1/2; or in BRCA-mutated gynaecological cancers excluding HGSOC. We investigated the role of PARPi in a cohort of patients with non-BRCA and/or non-high grade serous (HGS) gynaecological cancer.
Retrospective case series from two tertiary referral centres.
Ten cases were identified. Non-HGS cohort included large cell neuroendocrine carcinoma (LCNEC) of the cervix, small cell ovarian cancer, low grade ovarian cancer (LGSOC) and endometrial carcinoma. Non-BRCA patients were affected by alterations of RAD51, ATM and EMSY. Median number of cycles of PARPi received was 2 (range 1-16). CA125 response was seen in 75% (6 of 8 cases with minimum three CA125 results) with a median reduction of 53.3%. Overall radiological response rate 28.6% (2 of 7 cases with minimum one restaging scan). Progression free survival (PFS) was greater for non-HGS BRCA mutant patients (median PFS 10.5m, range 2-15m), than for HGSOC non-BRCA patients (median PFS 1.2m, range 1-2m). Legend: Abbreviations as in text, germline (g), somatic (s).Site BRCA 1/2 Other muts Number cycles CA125 Response Radiological Response PFS Endometrial BRCA1 mut (g) 16 yes yes 15.5 LCNEC cervix BRCA2 VUS (s) 8 yes N 10.7 LGSOC BRCA2 VUS (g) 13 yes (<50%) yes (on treatment) 7.9 Small cell ovarian BRCA2 VUS (g) 2 no data N 2.2 HGSOC WT EMSY (s) 4 yes (<50%) N 2.7 HGSOC WT ATM (s) 2 N N 1.2 HGSOC WT RAD51L3 (s) 2 N N 1.0 HGSOC WT RAD51C (g) 2 N no data (on treatment) NA PP (as HGSOC) WT RAD51D (g) 1 yes (<50%) no data (on treatment) NA HGSOC WT RAD51C (g) 2 yes no data (on treatment) NA
In this series, deleterious BRCA mutation or variant of unknown significance (VUS) remains the single strongest predictor for response to PARPi irrespective of histology. It may be difficult to classify a BRCA mutation as deleterious or VUS in rarer gynaecological cancer populations. Non-BRCA mutations in HRD related genes eg RAD51/ATM may also sensitise to treatment with PARPi. Four patients remain on treatment and updated results will be presented.
Dr R Kristeleit.
Has not received any funding.
All authors have declared no conflicts of interest.
The epidemiology of ovarian cancer in Romania is not well characterized and only few limited regional data are available. Moreover, the BRCA status in patients with homologous recombination-deficient tumorsis not known and no data about dominant founder mutations exist. BRCA1/2 are tumor suppression genes critical to DNA repair through homologous recombination in response to DNA damage. Around 13% of high-grade serous ovarian cancers is attributable to germline BRCA mutations.
This is an observational, retrospective, national, multicentre study aiming to describe the prevalence of BRCA mutations in patients with platinum-sensitive high-grade serous ovarian, fallopian tube or primary peritoneal cancer. As the link between BRCA1/2 mutation and ovarian cancer is well known, the primary objective of this study is to describe the prevalence of germline BRCA mutations in Romania. Secondary objectives will explore the correlations between mutational status and demographic and clinical characteristics and identify any dominant founder mutations. Data from 234 patients tested between April 2016 –April 2017 in 47 oncology centers, will be analyzed. Data collection was made from the patient’s files, by investigators, as no databases exists in Romania. It is estimated that clinical study report will be finalized by September 2018. Variables explored: BRCA status, age, weight and/or BS, parity,personal history of breast cancer, family history of cancer, stage at diagnosis, ECOG performance status, number of platinum-based chemotherapy lines, progression free interval, smoking.
D133FR00135 Observational Study.
AstraZeneca.
AstraZeneca.
C.L. Cebotaru: Advisory board: Boehringer Ingelheim, Novartis, Pfizer; Lecture fees: AstraZeneca, BMS, Bayer, Ipsen, Astellas; Travel grants: Alvogen, Merck Serono, Boehringer Ingelheim; Educational grants: AstraZeneca. D.L. Stanculeanu: Grant/Research Support: Sandoz, Roche; Speaker’s Bureau: AstraZeneca, Astellas, Roche, BMS, Jonson and Jonson, Pfizer, Boehringer Ingelheim, Sandoz; consultant: AstraZeneca, Astellas, Roche, BMS, Pfizer, Boehringer Ingelheim, Sandoz. D.M. Median: Advisory role: Roche, Pfizer; Lecture fees: AstraZeneca, Teva, Roche, Sandoz; Travel grants: Alvogene, A&D Pharma; Educational grants: Pfizer, AstraZeneca, Roche; Institutional grants: Novartis, Lilly, Samsung Bioepis, Tesaro, Roche, Boehringer Ingelheim.
Plasminogen activator inhibitor-1 (PAI-1) is corelated with inflammation and tumorigenesis. We investigated the role of PAI-1 in tumor regulating immune component of tumor microenvironment.
Protein expression data proteins and phosphoproteins, tumor infiltrating immune cells with relative fraction of immune cell types and major clinical outcome endpoints were obtained from The Cancer Genome Atlas project. A statistical correlation analysis was performed between the expression and distribution of PAI-1, immune checkpoint expression, tumor infiltrating immune cells fraction and their distribution in each tumor. We used Cox proportional hazards model to evaluate the association of PAI-1 with clinical survival outcomes.
The expression and distribution patterns of PAI-1 was measured in 7858 samples from 32 cancer type. PAI-1 had an enrichment in the squamous cancers. Increased expression of PAI-1 was associated with higher tumor stage and grade. Analyses of the tumor microenvironment revealed unanticipated correlation of PAI-1 with immune checkpoint expression and infiltrating immune cells. PAI-1 expression was tightly correlated to TGF-beta response (corr=0.40; p < 0.01), tumor leucocyte fraction (corr=0.33; p < 0.01) and PD-L1 expression (corr=0.29; p < 0.01). PAI-1 was positively corelates with intra-tumor heterogeneity (p < 0.01), proliferation(p < 0.01), stroma fraction (p < 0.01) and macrophages (p < 0.01). PAI-1 was also negatively correlated with lymphocyte fraction, activate CD4 cells, T cells (FH, gamma-delta and CD8). These correlations were also found with individual cancer type. PAI-1 was associated with poor Overall survival (Hazards ratio (HR)= 1.4; p < 0.01), Progression free interval (HR = 1.3; p < 0.01), disease free interval (HR = 1.26; p < 0.01) and disease specific survival (HR = 1.42; p < 0.01). PAI-1 was also associated with poor clinical outcomes in individual cancer type.
PAI-1 is correlated with PD-L1 and influences the tumor progression by modulating tumor microenvironment. PAI-1 might be valuable target indicating opportunities for personalised combination therapy of cancer.
Narender Kumar.
Has not received any funding.
All authors have declared no conflicts of interest.
Therapies targeting immune checkpoints have recently shown promising activity in patients with lung adenocarcinoma (LAD). T-cell activation is controlled by the balance of co-stimulatory molecules and co-inhibitory molecules (immune checkpoint molecules).
A comprehensive analysis was performed for immune profiles of tumor microenvironment in LADs using cap analysis of gene expression (CAGE). CAGE is a method used to quantify promoter activities across the whole genome by determining the 5’ ends of capped RNA molecules with next-generation sequencing. Gene expressions of PD-L1, PD-L2, co-inhibitory molecules (CTLA-4, PD-1, TIM-3, BTLA, VISTA, and LAG-3), co-stimulatory molecules (CD28, OX40, GITR, CD317, CD27, and HVEM), and markers of immune cells (CD4, CD8, CD25, Foxp3, CD68, and CD204) were quantified using RNA extracted from frozen tumor tissue samples of 71 surgically resected LADs. The correlations between the expression of immunomodulatory molecules and the clinicopathologic features (histological grade, EGFR mutation status, smoking status, and overall survival) were analyzed.
LADs in this study were classified into two groups: tumors with high expression of almost all immunomodulatory targets (immunoreactive) and tumors with intermediate or low expression (non-immunoreactive). Co-stimulatory and co-inhibitory molecules were simultaneously highly expressed in immunoreactive tumors. The proportion of histologically high grade (acinar, papillary, solid or invasive mucinous) or EGFR mutation negative tumors was significantly higher than that of histologically low grade (AIS, MIA or lepidic) or EGFR mutation positive tumors in immunoreactive tumors. The prognosis of immunoreactive LADs was worse than that of non-immunoreactive LADs regardless of histological grade or EGFR mutation status (P = 0.002). High expression of CD137, TIM-3, and HVEM was an unfavorable prognostic factor.
LADs can be classified into immunogenic and non-immunogenic tumors. The prognosis of immunoreactive tumors was worse than that of non-immunoreactive tumors.
Juntendo University School of Medicine.
Grant-in-Aid for Scientific Research (C), Research Grant for RIKEN Omics Science Center from MEXT, and Research Grant to RIKEN Preventive Medicine and Diagnosis Innovation Program from MEXT.
All authors have declared no conflicts of interest.
Hepatocellular Carcinoma (HCC) has a generally poor prognosis, and molecular markers to improve early detection and predict outcomes are greatly needed. The aim of this study is to identify the recurrent genetic changes, elucidate their roles and discover new biomarkers for improving clinical management of HCC.
Western blotting and immunohistochemistry were performed to detect the expression level of GNMT. The tumor-suppressive effect of GNMT was determined by both in-vitro and in-vivo assays. Affymetrix cDNA microarray was used to identify the underlying molecular mechanism.
We find that the Glycine N-methyltransferase GNMT is less expressed in HCC, where it correlates with well overall survival. Elevating GNMT levels by various means was sufficient to inhibit HCC cell proliferation, clonogenicity, migration and invasion in vitro and tumorigenicity and distant metastasis in vivo Conversely, GNMT attenuation by siRNA elicited the opposite effects in HCC cells. RNA-sequence profiling analyses suggested that GNMT inhibits HCC oncogenesis and metastasis through various pathways, with deregulation of MAPK signaling figuring prominently.
Our results establish the significance of GNMT in inhibiting oncogenesis and metastasis in HCC by coordinating MAPK pathway control, with implications for its potential use as a diagnostic or prognostic biomarker and as a candidate therapeutic target in this disease setting.
Renji Hospital, School of Medicine, Shanghai Jiao Tong University.
National Natural Science Foundation of China.
All authors have declared no conflicts of interest.