Petra Minarikova

Molecular biology of pancreatic cancer

In spite of continuous research efforts directed at early detection and treatment of pancreatic cancer, the outlook for patients affected by the disease remains dismal. With most cases still being diagnosed at advanced stages, no improvement in survival prognosis is achieved with current diagnostic imaging approaches. In the absence of a dominant precancerous condition, several risk factors have been identified including family history, chronic pancreatitis, smoking, diabetes mellitus, as well as certain genetic disorders such as hereditary pancreatitis, cystic fibrosis, familial atypical multiple mole melanoma, and Peutz-Jeghers and Lynch syndromes. Most pancreatic carcinomas, however, remain sporadic. Current progress in experimental molecular techniques has enabled detailed understanding of the molecular processes of pancreatic cancer development. According to the latest information, malignant pancreatic transformation involves multiple oncogenes and tumor-suppressor genes that are involved in a variety of signaling pathways. The most characteristic aberrations (somatic point mutations and allelic losses) affect oncogenes and tumor-suppressor genes within RAS, AKT and Wnt signaling, and have a key role in transcription and proliferation, as well as systems that regulate the cell cycle (SMAD/DPC, CDKN2A/p16) and apoptosis (TP53). Understanding of the underlying molecular mechanisms should promote development of new methodology for early diagnosis and facilitate improvement in current approaches for pancreatic cancer treatment.

Colorectal cancer screening: 20 years of development and recent progress

Colorectal cancer (CRC) is the second most common cancer in Europe and its incidence is steadily increasing. This trend could be reversed through timely secondary prevention (screening). In the last twenty years, CRC screening programs across Europe have experienced considerable improvements (fecal occult blood testing; transition from opportunistic to population based program settings). The Czech Republic is a typical example of a country with a long history of nationwide CRC screening programs in the face of very high CRC incidence and mortality rates. Each year, approximately 8000 people are diagnosed with CRC and some 4000 die from this malignancy. Twenty years ago, the first pilot studies on CRC screening led to the introduction of the opportunistic Czech National Colorectal Cancer Screening Program in 2000. Originally, this program was based on the guaiac fecal occult blood test (FOBT) offered by general practitioners, followed by colonoscopy in cases of FOBT positivity. The program has continuously evolved, namely with the implementation of immunochemical FOBTs and screening colonoscopy, as well as the involvement of gynecologists. Since the establishment of the Czech CRC Screening Registry in 2006, 2405850 FOBTs have been performed and 104565 preventive colonoscopies recorded within the screening program. The overall program expanded to cover 25.0% of the target population by 2011. However, stagnation in the annual number of performed FOBTs lately has led to switching to the option of a population-based program with personal invitation, which is currently being prepared.

Colorectal cancer prevention in the Czech Republic: time trends in performance indicators and current situation after 10 years of screening

The incidence and mortality of colorectal cancer (CRC) in the Czech Republic is significant. The National CRC Screening Program started in 2000 and was further enhanced in 2009. In 2010, the European Guidelines were introduced. The aim of the present trend study was to evaluate the quality of the Czech National Colorectal Cancer Screening Program using early performance and long-term impact indicators. The screening program has been assessed using data from three sources: the Czech National Cancer Registry, the Czech National Reference Centre, and the Czech CRC Screening Registry. The data were compared with a set of recommended quality control indicators. Between 2006 and 2010, a total of 1 881 299 fecal occult blood tests were performed, of which 87 397 were positive (4.6%). Until 2011, a total of 68 527 fecal occult blood test follow-up colonoscopies were performed. In addition, between 2009 and 2011, a total of 10 309 screening colonoscopies were performed. As a result, a total of 25 255 adenomas (32.0% rate) and 3379 CRCs (4.3% rate) were detected. A trend of cancer detection in earlier stages has been observed. The overall program coverage has increased to 22.7% of the target population in 2010. The majority of European guidelines’ quality indicators for nonpopulation-based programs were implemented in the Czech National CRC Screening program. An improvement in program management was accompanied by an increase in coverage as well as other performance indicators.

How significant is the association between metabolic syndrome and prevalence of colorectal neoplasia

The incidence and prevalence of metabolic syndrome (MS) and colorectal cancer (CRC) has been rising in developed countries. The association between these two diseases has been widely studied and reported. Less evidence is available about the relationship between MS and CRC precancerous lesions (adenomatous polyps, adenomas). The aim of this paper is to present an overview of our scientific understanding of that topic and its implication in clinical practice. One of the principal goals of current CRC secondary prevention efforts is to detect and remove the precancerous lesions in individuals with an average CRC risk to prevent the development of invasive cancer. MS is not currently considered a high-risk CRC factor and is therefore not included in the guidelines of organized screening programs. However, in light of growing scientific evidence, the approach to patients with MS should be changed. Metabolic risk factors for the development of adenomas and cancers are the same - obesity, impaired glucose tolerance, dyslipidemia, hypertension, cardiovascular diseases and diabetes mellitus type 2. Therefore, the key issue in the near future is the development of a simple scoring system, easy to use in clinical practice, which would identify individuals with high metabolic risk of colorectal neoplasia and would be used for individual CRC secondary prevention strategies. Currently, such scoring systems have been published based on Asian (Asia-Pacific Colorectal Screening Score; APCS) and Polish populations.

Monitoring of Circulating Tumor Cells by a Combination of Immunomagnetic Enrichment and RT-PCR in Colorectal Cancer Patients Undergoing Surgery

BACKGROUND The presence of circulating tumor cells (CTC) has been reported in patients with advanced colorectal cancer. Monitoring CTC (also known as a liquid-biopsy) has recently become the center of interest for low-invasive monitoring of cancer progression and predictive biomarkers testing. Along with high-cost technology and a complex methodology, a straightforward method based on magnetic beads enrichment followed by RT-PCR is set to allow for routine CTC analysis in colorectal cancer patients. OBJECTIVES The main purpose of this study was to evaluate the possibility of CTC detection in routine monitoring of patients starting before and continuing after surgery. MATERIAL AND METHODS The investigated group consisted of 30 patients mainly in advanced stages of colorectal cancer. In all patients, CTC detection was performed prior to surgery, in a subset of 14 patients additional sampling was done during and after surgery. In all cases, peripheral blood was processed using AdnaTest ColonCancer kit, which relies on enriching CTCs using EpCAM-functionalized magnetic beads and subsequently identifying tumorspecific CEA, EGFR and GA733-2 mRNA transcripts. RESULTS Out of all the tested samples, CTC were found in one patient suffering from advanced disease with lung and liver metastases. There, however, the positive finding was confirmed in 3 consecutive samples acquired before, during and shortly after palliative R2 resection. CONCLUSIONS The presence of CTC may be used to observe post-operative disease development. Due to the overall low CTC detection, further technology development may be necessary before its universal applicability to manage colorectal cancer patients.

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.

Circulating Free Tumor DNA in Patient Plasma is a Near-Perfect Marker for Metastatic Spread of Colorectal Cancer: A Study on 165 Patients Undergoing Surgical Treatment

Background: The occurence of circulating epithelial cells and cell-free tumor DNA (cfDNA) in peripheral blood of patients suffering from progressive forms of cancers has first been reported already in 1960s. With the great acceleration of genetic diagnostics by methods of polymerase chain reaction (PCR) the field of non-invasive examination of circulating molecular cancer biomarkers has recently been re-discovered. Aims: To investigate occurence of cfDNA as potential marker of regional and distant metastatic progression of colorectal cancer and to trace the source of cfDNA in patients undergoing surgery treatment at different levels of radicality. Methods: In a group of 165 patients in various stages of the disease tissue samples were initially acquired either as biopsies or resections. Samples were tested for a presence of the most frequent somatic colorectal cancer mutations within KRAS, APC, TP53, BRAF and PIK3CA genes. In addition, multiple plasma samples (n=789) were acquired over a time period covering (i) initial examination, (ii) immediately preceding a surgery (iv) postsurgery and (v) subsequent follow-up. Cell-free tumor DNA was traced in patient plasma by targeting mutations previously detected in tumor tissue. Results: Among patients who were positive for at least one of the detected somatic mutations in tumor tissue (102/165, 62%), cfDNA was present in 39 pre-surgery plasma samples. The frequency of cfDNA was correlated to the disease stage with 0% in Stages 0 and I, 9% in Stage II, 29% in Stage III and 94% in Stage IV. All cfDNA-positive patients (n=21) who underwent radical resection (R0) were free of cfDNA in subsequent testing of samples several days following surgery. All but 4 patients undergoing non-radical surgery, such as partial hepatic resections or paliative surgical treatment, remained cfDNA-positive also after the surgery. In some of the R0 patients, the cfDNA has re-appeared after a period of 18 22 months as a result of disease progression though local nodes or newly discovered metastases with continuous monitoring of the remainder of the group still in progress. Conclusion: Examination of a presence of cfDNA based on scanning plasma samples for presence of specific tumor mutations is a suitable tool for non-invasive monitoring of the disease progression as well as evaluation of surgery outcome. Supported by Czech Ministry of Health grant no. NS9809.

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.

Abstract 2406: Validation of a simple low-cost method to monitor ctDNA in patients with solid cancers

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Detection of circulation tumor DNA (ctDNA) in plasma has become a viable option for non-invasive monitoring of patients. Also termed “liquid biopsy” the approach is applicable for pre-diction of response and prediction of resistance to biological therapy (1, 2). Various techniques have been used for ctDNA detection, frequently employing clonal amplification on a digital PCR format (3) with limits of detection (LOD) below 0.01% of mutant alleles. However, these techniques suffer from high complexity, expensive instrumentation, and a considerable cost per sample. We hereby present a simple low-cost alternative that is implementable to routine ctDNA testing. Methods: A panel of PCR amplicons (106 - 174bp) was resolved by denaturing capillary electrophoresis (DCE) revealing minute presence of mutation specific hetero-duplexes. The final panel consisted of clinically relevant oncogenic mutations KRAS, NRAS, BRAF, PIK3CA and EGFR as well as cancer-related mutations in tumor suppressors TP53, APC and CTNNB1. A total of 299 patients was subsequently examined for presence of ctDNA in plasma including 194 with colorectal cancer (CRC), 26 with NSCLC and 79 with pancreatic cancer (PanC). CtDNA status was correlated to TNM stage and tumor markers (CEA and Ca19-9). In a subset of CRC patients (n = 20) the ctDNA was monitored in 2 - 6 month intervals and correlated to the therapy response. Results: The experimental LOD value was in the range between 0.03 - 1% for all tested mutations within the panel. A minimum input amount of DNA was 5 pg (0,005 ng).. The overall rate of ctDNA detection was 32% for CRC (stages I - IV), 31% for NSCLC (stages III - IV) and 27% for PanC (stages II - IV). The highest detection rate, 69%, was observed in Stage IV CRC patients. Comparison with tumor markers (TM) revealed 62% of cases positive for both TM and ctDNA and 13% TM-negative cases with ctDNA positivity. Post-operative absence or persistence of ctDNA was related to the radicality of the surgical treatment and the ctDNA levels were concordant with the response to adjuvant chemotherapy. In several patients a disease progression was signalized based on ctDNA even prior to actual clinical detection by CT imaging. Conclusion: DCE is a simple technique applicable for detection of ctDNA in cancer patients without a need for costly hardware/software equipment. The detection rates are 10 - 15% lower compared to the dedicated dPCR techniques, however, the method requires ca 100x less input DNA, the cost per patient is about 10-fold lower and the turnaround time per test is under 5 hours. Supported by the Czech Ministry of Health Grant 14383. Literature 1. Bettegowda C et al. Sci Transl Med. 2014,6(224):224ra24 2. Douillard JY et al. J Thorac Oncol. 2014, 9(9):1345-1353. 3. Benesova L et al. Anal Biochem 2013,433(2):227-234. Citation Format: Lucie Benesova, Barbora Belsanova, Petra Minarikova, Tereza Halkova, Jiri Pudil, Filip Pazdirek, Milos Pesek, Ondrej Fiala, Jiri Hoch, Miroslav Zavoral, Bohus Bunganic, Miroslav Levy, Ludmila Lipska, Lubos Petruzelka, Miroslav Ryska, Marek Minarik. Validation of a simple low-cost method to monitor ctDNA in patients with solid cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2406. doi:10.1158/1538-7445.AM2015-2406