Marek Minarik

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.

The dominant role of G12C over other KRAS mutation types in the negative prediction of efficacy of epidermal growth factor receptor tyrosine kinase inhibitors in non–small cell lung cancer

The role of KRAS mutations in molecular targeted therapy by epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC) has not been fully understood. The present investigation is aimed at an elucidation of the role of specific KRAS mutation types in predicting outcomes of patients with advanced NSCLC receiving EGFR-TKI therapy. Initially, 448 NSCLC patients were tested for the presence of KRAS mutations, to obtain frequencies of specific KRAS mutation types. Subsequently, the clinical outcome of treatment was evaluated in a subgroup of 38 KRAS-positive patients receiving EGFR-TKI therapy. KRAS mutations were detected in 69 of 448 patients (15.4%), mostly in smokers (17.86% vs. 5.8%, P = 0.0048), and appeared more frequently in adenocarcinomas than in squamous cell NSCLC or NSCLC that is not otherwise specified (21% vs. 6.99% vs. 4.4%, P = 0.0004). The most frequent type of KRAS mutation was G12C. The progression-free survival (PFS) was doubled in a group of non-G12C patients compared with that of the G12C group (9.0 wk vs. 4.3 wk, P = 0.009). The overall survival (OS) was not significantly different between non-G12C and G12C groups (12.1 wk vs. 9.3 wk, P = 0.068). The G12C KRAS mutation is a strong negative predictor for EGFR-TKI treatment, whereas other KRAS mutation types have not negatively predicted treatment efficacy compared with that for the wild-type KRAS genotype.

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.

Design of a fraction collector for capillary array electrophoresis.

This paper describes a prototype instrument for high‐throughput fraction collection with capillary array electrophoresis (CAE). The design of the system was based on a comprehensive collection approach, in which fractions from all capillaries were simultaneously collected in individual collection microwells in predefined time intervals. The location of the fractions in the microwells on the collection plate was determined by monitoring the individual zone velocities close to the end of each capillary. The collection microwell plate was fabricated from buffer‐saturated agarose gel, which maintained permanent electrical contact with the separation capillaries during the collection process. Since the collection gel plate consisted of over 90% water, liquid evaporation from the collection wells was minimized. A 12‐capillary array instrument was built with two‐point detection using a side illumination scheme. The collection performance was demonstrated by reinjection of selected fractions of a double‐stranded DNA (dsDNA) separation. The identity of collected DNA fragments was confirmed by PCR and sequencing.

Assembly of a large Y-STR haplotype database for the Czech population and investigation of its substructure

Twelve Y-chromosomal short tandem repeats (Y-STR) (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a, DYS385b, DYS437, DYS438, and DYS439) included in the PowerPlex Y Kit (Promega Corporation, Madison, USA) were studied for 1750 unrelated males living in 14 regions of the Czech Republic. A total of 1148 different haplotypes were found. The overall haplotype diversity (HD) was determined as 0.998. Analysis of Molecular Variance (AMOVA) reveals non-significant distances between regions concerning their haplotype distribution, thus allowing to use the whole sample as a representative reference database of the Czech Republic. Median network analysis shows a remarkable bipartite composition of the Czech haplotypes, falling in distinct clusters with Eastern and Western European roots.

Somatic TP53 mutation mosaicism in a patient with Li-Fraumeni syndrome.

We present a girl who developed adrenocortical adenoma at the age of 1 year and osteosarcoma at the age of 5 years. There was no history of cancer in her parents and their relatives. However, both tumors were typical for the Li–Fraumeni syndrome (LFS), and the patient met criteria for germline TP53 mutation testing. A mutation in codon 282 (Arg282Trp) was identified in her blood lymphocyte genomic DNA. The substitution was found in neither of her parents, which indicated a possibility of a de novo mutation. Unexpectedly, sequencing of the DNA of the patient repeatedly showed allelic imbalance in favor of the normal allele. This observation prompted us to investigate the putative somatic mosaicism in the patient consisting of normal cells and cells heterozygous for the mutation. The imbalance was also examined in two other non‐invasively sampled tissues, buccal cells, and cells from the urine sediment, and sequencing was confirmed with two other independent methods. While the findings in blood and the urine sediment were similar, in buccal cells both alleles were present in equal amounts. The allele ratio in lymphocytes was consistent with a mosaic where about 2/3 of cells carried two normal alleles and only 1/3 was heterozygous for the mutation. Despite the mosaicism the girl developed two early childhood tumors of mesodermal origin, and her phenotype was thus not milder than that of other germline TP53 mutation carriers. To our knowledge this is the first description of somatic mosaicism for a de novo TP53 mutation in LFS.

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.

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.

A novel high-resolution chipCE assay for rapid detection of EGFR gene mutations and amplifications in lung cancer therapy by a combination of fragment analysis, denaturing CE and MLPA

There is a growing interest in evaluating molecular markers as predictors of response to new generation of targeted cancer therapies. One of such areas is biological therapy targeting epidermal growth factor receptor gene (EGFR) in lung cancer. The testing of tumor tissue is focused on specific EGFR mutations and EGFR gene amplification, since tumors exhibiting positivity of either of the two marker types are highly sensitive towards the treatment. Although traditional methods of DNA sequencing and fluorescence in situ hybridization are still in use for the detection of EGFR mutations and gene amplification, respectively, there is a need for new dedicated techniques with the primary emphasis on simplicity, sensitivity, speed and cost effectiveness. The main purpose of this work was to integrate diverse assays for both EGFR tests onto a single platform to eliminate the need for different instruments and separate processing. We demonstrate a chip capillary electrophoresis (chipCE) application for EGFR mutation detection by a combination of fragment analysis and denaturing CE along with multiplex ligation‐dependent probe amplification (MLPA) for evaluation of EGFR amplification. All separations are carried out in denaturing sieving polymer on a modified Bioanalyzer 2100 chipCE instrument running at temperatures of up to 65°C. The main strength of the resulting high‐resolution chipCE application is in its simplicity, speed of analysis and minimal amount of sample required for complete testing of EGFR status. Such an approach could potentially fit medium throughput laboratories providing molecular pathology services for clinical oncologists with fast turnaround times and limited consumption of tissue material.

Skin rash as useful marker of erlotinib efficacy in NSCLC and its impact on clinical practice.

Erlotinib is an epidermal growth factor receptor tyrosine kinase inhibitor used in treatment of advanced NSCLC. Patients harboring EGFR or KRAS mutations represent minority of all patients in caucasian population and there is no available predictor for a predominant group of patients harboring the wild-type EGFR and wild-type KRAS genes. Skin rash is the most frequent manifestation of cutaneous toxicity of erlotinib. Rash is associated with a good therapeutic response. We aimed at the evaluation of rash as a predictor of therapeutic effect of erlotinib in patients harboring the wild-type EGFR and KRAS wild-type genes and to assess its possible usage in a clinical practice.Totally 184 patients with advanced stage NSCLC (IIIB, IV) harboring the wild-type EGFR and wild-type KRAS genes were analysed. Comparison of ORR, PFS and OS according to the occurrence of rash was performed. In order to assess the impact of rash in clinical practice it was conducted landmark analysis of the group of patients whose rash was observed during first month of treatment (n=124). Patients in whom progression was observed during the first month of treatment were excluded from the landmark analysis. The comparison of ORR was performed using Fishers exact test, visualization of survival was performed using Kaplan-Meier survival curves and the differences in survival were tested using the log-rank test. Median PFS in patients who were observed with rash during the treatment was 3.0 vs. 1.2 months in patients with no rash (p<0.001), median of OS in patients who were observed with rash during the treatment was 13.9 vs. 5.8 months in patients with no rash (p<0.001). ORR in patients who were observed with rash during the treatment was 17.4% vs. 3.3% in patients with no rash (p=0.001). Median of PFS after 1 month of treatment in patients who were observed with rash during the first month was 2.9 vs. 1.1 months in patients with no rash (p=0.027). Median of OS after 1 month of treatment in patients who were observed with rash during the first month was 13.8 vs. 9.9 months in patients with no rash (p=0.082). Rash is strongly associated with better survival and ORR in patients harboring wild-type EGFR and wild-type KRAS genes. Occurrence of rash during the first month of treatment is a useful predictor of better effect of erlotinib after one month of treatment. Patients who were not observed with rash during the first month of treatment are in high risk of progression. Optimization of the treatment of these patients can contribute restaging after two months of treatment, assessment of plasma levels of erlotinib and eventually attempt to dose escalation.