Background

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.

Methods

We utilized a spectrum of methods to detect EGFR T790M allele in tumor and normal human tissues.

Results

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.

Conclusions

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.

Legal entity responsible for the study

N.N. Petrov Institute of Oncology.

Funding

AstraZeneca (Research Agreement with the N.N. Petrov Institute of Oncology); Russian Science Foundation (grant 17-75-30027).

Disclosure

All authors have declared no conflicts of interest.

Background

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.

Methods

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.

Results

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).

Conclusions

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.

Legal entity responsible for the study

Institut Català d\'Oncologia L\'Hospitalet.

Funding

Institut Català d\'Oncologia i Vall d\'Hebron Institut d\'Oncologia.

Disclosure

All authors have declared no conflicts of interest.

Background

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).

Methods

Firstly, we produced BMN 673-SLNs by hot homogenization method. After characterization, the cytotoxic effect of BMN 673-SLNs (0.01-10 nM) on HCC1937^BRCA1-/- 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.

Results

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.

Conclusions

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.

Legal entity responsible for the study

 The Scientific Research Projects Foundation of the Uludag University of Turkey.

Funding

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].

Disclosure

All authors have declared no conflicts of interest.

Background

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.

Trial Design

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.

Clinical trial identification

D133FR00135 Observational Study.

Legal entity responsible for the study

 AstraZeneca.

Funding

AstraZeneca.

Disclosure

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.

Background

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.

Methods

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.

Results

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.

Conclusions

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.

Legal entity responsible for the study

Instituto Tecnológico Metropolitano.

Funding

Colciencias (Grant 669-2014).

Disclosure

All authors have declared no conflicts of interest.Table: 38P

Background

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.

Methods

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).

Results

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.

Conclusions

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.

Legal entity responsible for the study

 DST.

Funding

DST.

Disclosure

All authors have declared no conflicts of interest.

Background

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.

Methods

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.

Results

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.

Conclusions

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.

Legal entity responsible for the study

 Conacyt, Mexico.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

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.

Methods

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.

Results

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.

Conclusions

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.

Legal entity responsible for the study

Ardabil University of Medical Sciences.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

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.

Methods

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).

Results

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.

Conclusions

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.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

Background

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.

Methods

Loss- and gain-of-function analysis, CRISPR-Cas9, RNA-Seq, Western blotting, Immunoprecipitation, qRT-PCR, and immunofluorescence techniques.

Results

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.

Conclusions

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.

Legal entity responsible for the study

 University of Nottingham.

Funding

Saudi Government.

Disclosure

The author has declared no conflicts of interest.