Tereza Halkova

MicroRNAs in Pancreatic Cancer: Involvement in Carcinogenesis and Potential Use for Diagnosis and Prognosis

Pancreatic cancer is one of the most fatal malignancies with increasing incidence and high mortality. Possibilities for early diagnosis are limited and there is currently no efficient therapy. Molecular markers that have been introduced into diagnosis and treatment of other solid tumors remain unreciprocated in this disease. Recent discoveries have shown that certain microRNAs (miRNAs) take part in fundamental molecular processes associated with pancreatic cancer initiation and progression including cell cycle, DNA repair, apoptosis, invasivity, and metastasis. The mechanism involves both positive and negative regulation of expression of protooncogenes and tumor suppressor genes. Various miRNAs are expressed at different levels among normal pancreatic tissue, chronic pancreatitis, and pancreatic cancer and may therefore serve as a tool to differentiate chronic pancreatitis from early stages of cancer. Other miRNAs can indicate the probable course of the disease or determine the survival prognosis. In addition, there is a growing interest directed at the understanding of miRNA-induced molecular mechanisms. The possibility of intervention in the molecular mechanisms of miRNAs regulation could begin a new generation of pancreatic cancer therapies. This review summarizes the recent reports describing functions of miRNAs in cellular processes underlying pancreatic cancerogenesis and their utility in diagnosis, survival prognosis, and therapy.

Polymorphisms in selected DNA repair genes and cell cycle regulating genes involved in the risk of papillary thyroid carcinoma.

BACKGROUND Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. In addition to causal somatic mutations in the BRAF gene and RET/PTC rearrangements, the contribution of single nucleotide polymorphisms (SNPs) in low-penetrance genes in the development of PTC has been proposed. METHODS Four SNPs in the XRCC1 (Arg399Gln, Arg280His, Arg194Trp and T-77C) and one SNP from each of three other genes participating in DNA repair pathways and/or cell cycle regulation (ATM Asp1853Asn, TP53 Arg72Pro, CDKN1B Val109Gly) were selected. The allelic and genotypic distributions of these variants as well as haplotypes of the XRCC1 were examined in 583 individuals comprising well-characterized cohorts of 209 PTC patients and 374 healthy volunteers. Correlations of polymorphism with clinical-pathological data and mutation status were performed. RESULTS XRCC1 T-77C polymorphism affects the genetic susceptibility for PTC development in men, the specific combination of XRCC1 haplotypes correlates with RET/PTC incidence, CDKN1B Val109Gly significantly influences the risk of developing PTC regardless of gender and in PTC cases, selected genotypes of TP53 Arg72Pro and ATM Asp1853Asn were significantly associated with monitored tumour characteristics. CONCLUSION It seems that SNPs in studied regulating genes contribute to the development of PTC and modify the tumour behaviour or characteristics.

RET Variants and Haplotype Analysis in a Cohort of Czech Patients with Hirschsprung Disease

Hirschsprung disease (HSCR) is a congenital aganglionosis of myenteric and submucosal plexuses in variable length of the intestine. This study investigated the influence and a possible modifying function of RET proto-oncogenes single nucleotide polymorphisms (SNPs) and haplotypes in the development and phenotype of the disease in Czech patients. Genotyping of 14 SNPs was performed using TaqMan Genotyping Assays and direct sequencing. The frequencies of SNPs and generated haplotypes were statistically evaluated using chi-square test and the association with the risk of HSCR was estimated by odds ratio. SNP analysis revealed significant differences in frequencies of 11 polymorphic RET variants between 162 HSCR patients and 205 unaffected controls. Particularly variant alleles of rs1864410, rs2435357, rs2506004 (intron 1), rs1800858 (exon 2), rs1800861 (exon 13), and rs2565200 (intron 19) were strongly associated with increased risk of HSCR (p<0.00000) and were over-represented in males vs. females. Conversely, variant alleles of rs1800860, rs1799939 and rs1800863 (exons 7, 11, 15) had a protective role. The haploblock comprising variants in intron 1 and exon 2 was constructed. It represented a high risk of HSCR, however, the influence of other variants was also found after pruning from effect of this haploblock. Clustering patients according to genotype status in haploblock revealed a strong co-segregation with several SNPs and pointed out the differences between long and short form of HSCR. This study involved a large number of SNPs along the entire RET proto-oncogene with demonstration of their risk/protective role also in haplotype and diplotype analysis in the Czech population. The influence of some variant alleles on the aggressiveness of the disease and their role in gender manifestation differences was found. These data contribute to worldwide knowledge of the genetics of HSCR.

A novel RET/PTC variant detected in a pediatric patient with papillary thyroid cancer without ionization history

Papillary thyroid carcinoma (PTC) is the most frequent type of thyroid cancer. Its development is often caused by the formation of RET/PTC fused genes. RET/PTC1 is the most prevalent form, where exon 1 of CCDC6 gene is fused with the intracellular portion of RET protooncogene starting with exon 12. We have discovered a novel RET/PTC1 variant which we have named RET/PTC1ex9 in metastatic PTC of 8-year-old boy. RET/PTC1ex9 detection was performed by real-time polymerase chain reaction with melting curve analysis and subsequent Sanger and next-generation sequencing. A fusion of exon 1 of CCDC6 with exon 9 of extracellular domain of RET followed by exon 12 of RET was revealed. This is the first RET/PTC variant among PTC cases that contain the extracellular part of RET. This observation could be probably explained by incorrect splicing of RET due to the somatic 32-bp deletion in exon-intron 11 boundary of RET.

Longitudinal molecular characterization of endoscopic specimens from colorectal lesions

AIM To compare molecular profiles of proximal colon, distal colon and rectum in large adenomas, early and late carcinomas. To assess feasibility of testing directed at molecular markers from this study in routine clinical practice. METHODS A prospective 3-year study has resulted in the acquisition of samples from 159 large adenomas and 138 carcinomas along with associated clinical parameters including localization, grade and histological type for adenomas and localization and stage for carcinomas. A complex molecular phenotyping has been performed using multiplex ligation-dependent probe amplification technique for the evaluation of CpG-island methylator phenotype (CIMP), PCR fragment analysis for detection of microsatellite instability and denaturing capillary electrophoresis for sensitive detection of somatic mutations in KRAS, BRAF, TP53 and APC genes. RESULTS Molecular types according to previously introduced Jass classification have been evaluated for large adenomas and early and late carcinomas. An increase in CIMP+ type, eventually accompanied with KRAS mutations, was notable between large adenomas and early carcinomas. As expected, the longitudinal observations revealed a correlation of the CIMP+/BRAF+ type with proximal location. CONCLUSION Prospective molecular classification of tissue specimens is feasible in routine endoscopy practice. Increased frequency of some molecular types corresponds to the developmental stages of colorectal tumors. As expected, a clear distinction is notable for tumors located in proximal colon supposedly arising from the serrated (methylation) pathway.

Prognostic Importance of Cell Cycle Regulators Cyclin D1 (CCND1) and Cyclin-Dependent Kinase Inhibitor 1B (CDKN1B/p27) in Sporadic Gastric Cancers

Background. Gastric cancer is known for a notable variety in the course of the disease. Clinical factors, such as tumor stage, grade, and localization, are key in patient survival. It is expected that molecular factors such as somatic mutations and gene amplifications are also underlying tumor biological behavior and may serve as factors for prognosis estimation. Aim. The purpose of this study was to examine gene amplifications from a panel of genes to uncover potential prognostic marker candidates. Methods. A panel of gene amplifications including 71 genes was tested by multiplex ligation-dependent probe amplification (MLPA) technique in 76 gastric cancer samples from a Caucasian population. The correlation of gene amplification status with patient survival was determined by the Kaplan-Meier method. Results. The amplification of two cell cycle regulators, CCND1 and CDKN1B, was identified to have a negative prognostic role. The medial survival of patients with gastric cancer displaying amplification compared to patients without amplification was 192 versus 725 days for CCND1 (P = 0.0012) and 165 versus 611 days for CDKN1B (P = 0.0098). Conclusion. Gene amplifications of CCND1 and CDKN1B are potential candidates to serve as prognostic markers for the stratification of patients based on the estimate of survival in the management of gastric cancer patients.

Abstract 4943: Detection of specific KRAS mutation type, Gly12Asp (GGT>GAT), in EUS-guided fine needle aspiration cytology (EUS-FNAC) identifies pancreatic cancer patients with poor prognosis

Background Although the disease progression of pancreatic ductal adenocarcinoma (PDAC) is very rapid with median survival typically around 3 to 6 months, there are rare cases of patients remaining on therapy for longer periods of time. Early estimation of survival prognosis would allow for rational decisions on complex therapy interventions, including radical surgery and robust systemic therapy regimens. Understandably, there is a great interest in finding prognostic markers that can be used for patient stratification. KRAS, often mutated in PDAC, has been a frequent subject of studies in molecular pancreatic cancer research. It was reported early on that KRAS mutation detected in pancreatic mass itself does not show any prognostic value (1). In this work we have investigated role of various KRAS mutation types on the prognosis of pancreatic cancer patients. Similar to other types of solid tumors it is expected that different KRAS mutations will result in slightly different clinical outcome (2,3) Methods A total of 118 pacients with PDAC clinically confirmed by EUS-FNAC were included in the study. DNA was extracted from FNAB slides following a standard cytology evaluation to ensure adequacy (viability, quantity) and to mark tumor cell fraction. Kaplan-Meier survival curves were calculated for individual KRAS mutation types. Results summary KRAS mutation was detected in 90% of specimes (106/ 118). All mutations were localized at exon 2, codon 12 with Gly12Asp (GGT>GAT) as the most frequent at 45% (48/106), followed by other types including Gly12Val (GGT>GTT) at 31% (33/106), Gly12Arg (GGT>CGT)at 17% (18/106), Gly12Cys (GGT/TGT) at 5% (5/106) and Gly12Ser (GGT/AGT) at 1% (1/106). Overall survival for patients with the Gly12Asp mutation was only 107 days, compared to 198 days for the Gly12Val, 286 days for Gly12Arg and 206 days for Gly12Cys (P = 0.01, long-rank test). Survival of patients with the Gly12Asp was less than a half when compared to all of the other mutation types combined (107 days vs. 218 days, P = 0.002, long-rank test). Conclusions The overall survival of PDAC patients correlates with the type of KRAS mutation present in the tumor. The most frequent Gly12Asp (GGT>GAT) mutation confers the worst prognosis resulting in a reduction of survival typically by 3 - 6 months. KRAS testing from EUS-FNAC slides presents a useful tool for stratification of PDAC patients. Supported by IGA MZ grant no. NT13638. Literature: 1) Salek C et al. Anticancer Res. 2009, 29, 1803-1810 2) Garassino MC, Ann Oncol, 2011, 22, 235-237 3) Fiala O et al., Cancer Genet., 2013, 206, 26-31. Citation Format: Marek Minarik, Tereza Halkova, Barbora Belsanova, Bohus Bunganic, Miroslav Zavoral, Lucie Benesova. Detection of specific KRAS mutation type, Gly12Asp (GGT>GAT), in EUS-guided fine needle aspiration cytology (EUS-FNAC) identifies pancreatic cancer patients with poor prognosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4943.

Abstract B09: Molecular phenotyping of colorectal tumors in clinical practice: Assignment of extended prognostic subtypes by direct testing of endoscopic specimens

Introduction: Since the pioneering work of Fearon and Vogelstein in 1990, detailed mechanisms of colorectal neoplasia have intensively been studied (1). Only recently, however, importance of molecular subtypes for prognosis has been confirmed on large cohorts (2,3). The so-called Jass classification is set to become an part of diagnostics complementary to the companion predictive genotyping (4). The diagnosis is mostly based on examination of tumor tissue from endoscopy. It was an intention of this work to evaluate feasibility of molecular phenotyping in addition to the standard testing performed from the same source material. Patients and Methods: A total of 145 carcinomas (105 fresh biopsies or 40 FFPE sections, all stages) have been acquired over a 3-year period. The molecular subtypes were assigned based upon results of CIMP/MSI/BRAF/RAS (5). To partition the most common type arising from traditional adenoma-carcinoma pathway, we have additionally examined MLH1 methylation and APC and TP53 mutations (6). The methodology was based on previously validated protocols including MS-MLPA for the evaluation of CIMP, PCR fragment analysis MSI and high-sensitive denaturing CE assay (DCE) for somatic mutations in RAS, BRAF, TP53 and APC genes. By extending Jass classification we recognize 6 molecular subtypes with distinct features: CIMP status > negative > TP53 > positive > Traditional CIMP (median prognosis) > positive > MLH1 ------------> negative > KRAS -----------------------> positive > Traditional CIN antiEGFR resistant (median prognosis) -----------------------> negative > APC> positive > Traditional CIN antiEGFR sensitive (median prognosis) ------------> positive > BRAF --------------------> negative> MSI > positive > Familial MSI (good prognosis) --------------------> positive > MSI --------------------------------> negative > Serrated CIMP (poor prognosis) --------------------------------> positive> Serrated CIMP+MSI (best prognosis) Results: A total of 105/105 (100%) of fresh tissue samples and 33/45 (73.3%) of FFPE samples provided high-quality DNA for subsequent completion of the complete molecular testing panel. The distribution of the six types was (i) 3x Serrated CIMP (ii) 9x Serrated CIMP+MSI; (iii) 2x Familial MSI, (iv) 26x Traditional CIN antiEGFR resistant (v) 12x Traditional CIN antiEGFR sensitive and (vi) 18x Traditional CIMP. The remaining 68 carcinomas were resulting from other combinations. Conclusions: Methodology as well as logistics of sample processing has been optimized for use at endoscopy unit. The distribution of Jass molecular subtypes corresponded to the larger foreign cohorts with the dominating contribution from the Traditional CIN/CIMP subtypes. Until CIMP/CIN/MSI panels covering all markers have been developed a multi-tier testing according to the above algorithm is the most cost efficient. Assignment of molecular subtypes by an optimized protocol is useable in clinical management of patients and feasible in routine practice. Supported by IGA Ministry of Health project No. NT 14383 Literature 1. Fearon ER, Vogelstein B. Cell. 1990 Jun 1;61(5):759-67. 2. Phipps AI et al. Gastroenterology 2015; 148: 77-87.e2 3. Sinicrope FA et al. Gastroenterology 2015; 148: 88-99. 4. Modest DP et al. Ann Oncol. 2016 Jun 29. pii: mdw261. [Epub ahead of print] 5. Jass JR. Int J Colorectal Dis 1999; 14: 194-200 6. Kim JH, Bae JM, Cho NY, Kang GH. Oncotarget 2016; Epub ahead of print Citation Format: Marek Minarik, Tereza Halkova, Petra Minarikova, Barbora Belsanova, Stepan Suchanek, Jiri Cyrany, Jan Bures, Miroslav Zavoral, Lucie Benesova. Molecular phenotyping of colorectal tumors in clinical practice: Assignment of extended prognostic subtypes by direct testing of endoscopic specimens. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B09.

Su2028 KRAS Mutation Assay on EUS FNA Specimens From Pacients With Pancreatic Mass

Differential diagnosis of solid pancreatic masses using EUS FNA is in 1015 % of cases still challenging. Promising method, which helps to distinguish between chronic pancreatitis and cancer, is point mutations of the proto-oncogene KRAS test. This method is not established in routine clinical practice yet.Objectives were the determination of the sensitivity of the KRAS assay using various kinds of samples of patients with pancreatic mass and testing the effect of the presence of KRAS mutations on the prognosis of survival. 147 patients underwent EUS-FNA examination of pancreatic mass, accompanied by blood sampling with subsequent separation of plasma for the detection of circulating tumor DNA. Part of biopsy sample was left native in a stabilizing solution and part as cytological smear. Samples (native aspirates, cytological smears, plasma) were examined for the presence of KRAS mutation by heteroduplex analysis, denaturing capillary electrophoresis.Among 147 patients with pancreatic masses, 118 were diagnosed as a cancer, 26 chronic pancreatitis, 3 neuroendocrine tumor. In total 147 native aspirates, 118 cytological smears and 94 plasma samples were examined. The highest sensitivity of KRAS mutation was reached in the group of pancreatic cancer patients using cytology, in which 90 % of KRAS mutation was detected (106/118 of the samples). When using the native cellular aspirates, mutation was detected in 78 % (92/118 samples), and examination of plasma was positive in 27 % (24/90 samples). In four patients with chronic pancreatitis KRAS mutations was detected, although none has been cytologically confirmed as a cancer. Two of these four patients were confirmed in the course of the disease as a cancer, one patient died because of alcoholic delirium and the last one was indicated for surgery recently.Examination of KRAS mutations can be performed in all patients undergoing EUS-FNA, with the cytology being the most reliable type of sample for genetic tests. KRAS examination would be reasonable to introduce into routine clinical practice in a group of patients with unclear differential diagnosis of chronic pancreatitis, especially in those with suspicion of cancer in inflammatory terrain.Kexwords: pancreatic cancer, chronic pancreatitis, KRAS mutation , EUS-FNA.

Abstract 3139: Liquid biopsy (ctDNA) testing in clinical management of solid cancers: 5-years of experience

Background: Detection and profiling of circulating tumor DNA (ctDNA) is an attractive tool for management of cancer patients, in particular for early detection of the relapse after surgery or monitoring of response to systemic therapy. The main advantage is minimal invasivity and applicability to a wide range of solid cancers. Methodologies are based on digital PCR with the limit of detection (LOD) below 0.01% mutated alleles, however, these often require significant amounts of input DNA (10s to 100s of ng). In the present work we demonstrate routine detection and clinical utility of ctDNA in a cohort of 423 patients covering a range of 5 different solid tumors. Methods: ctDNA is detected by somatic mutations found in primary tumor tissue by applying a specific mutation panel targeted to the tumor tape. Detection was done by denaturing capillary electrophoresis (input DNA at concentrations of 5 pg, LOD 0.03 - 1%). The cohort included samples from 257 colorectal cancer patients (CRC), 97 patients with ductal adenocarcinoma of the pancreas (PDAC), 32 patients with non-small cell lung cancer (NSCLC), 12 patients with gastric adenocarcinoma (GA) and 6 patients with head and neck cancers (HNC). A longitudinal monitoring of ctDNA levels prior to surgery, a week after surgery and at three-month follow-up intervals, was performed in 16 CRC patients. Overall ctDNA detection rate, radicality of resection, disease recurrence, tumor dynamics and survival prognosis were evaluated. Results: ctDNA rates were at 32% for NSCLC, 31% for CRC, 30% for GA, 25% for PDAC and 25% for HNC. When looking at a subgroup of patients in Stage IV of the disease the rates increased to 53% for NSCLC, 77% for CRC, 50% for GA, 46% for PDAC and 50% for HNC. 14 out 16 CRC patients with R0 resection remained ctDNA negative (88%), two patients dropped out of the study. Follow-up monitoring lead to detection of progression in 9 out of 13 patients (69%). In 3 patients (23%), ctDNA positivity preceded standard detection by CT scan. In 5 patients with follow-up exceeding 1 year (5 to 10 sample acquisitions over 15 to 28 month period) the ctDNA levels correlated with the clinical course of the disease (progression/stabilization/remission). There was a borderline statistically significance for prognostic role of ctDNA presence in PDAC patients with 140 days vs. 200 days (P = 0,0519, long-rank test) for ctDNA positive and negative, respectively. Conclusion: We have introduced a ctDNA method to routine management of 5 different solid cancers. Our results indicate clinical utility for resection radicality confirmation as well as early detection of disease progression and tumor dynamics in CRC patients. The same is applicable to approx. 50% of patients with advanced GA, NSCLC and HNC. CtDNA positivity may also indicate a negative prognosis for PDAC patients. Supported by IGA MZ grant no. NT 13660. Citation Format: Lucie Benesova, Barbora Belsanova, Tereza Halkova, Jiri Pudil, Bohus Bunganic, Milos Pesek, Bretislav Gal, Miroslav Zavoral, Miroslav Ryska, Marek Minarik. Liquid biopsy (ctDNA) testing in clinical management of solid cancers: 5-years of experience. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3139.