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Purpose: To investigate potential association between selected tumor markers and laboratory parameters (lactate dehydrogenase [LDH], neutrophils, hemoglobin, neutrophils, lymphocytes, C-reactive protein, albumin, carcinoembryonic antigen, and cytokeratin 19 fragment 21-1 [CYFRA 21-1]) and circulating tumor DNA (ctDNA) with survival in patients with advanced non-small cell lung cancer (NSCLC). Patients and Methods: The study encompassed 82 patients from a single center. All patients had (localy-) advanced adenocarcinomas. ctDNA was determined before starting therapy and at 6 weeks follow-up. Laboratory parameters were measured before each cycle of therapy and oncomarkers before starting the therapy as standard clinical practice. Mann-Whitney U test, Cox proportional hazards model, Fisher's exact test, and Kaplan-Meier survival estimation with Gehan-Wilcoxon test were used for statistical analysis of the corresponding variables. Results: We have confirmed predictive or prognostic significance for some of the selected laboratory markers and oncomarkers. Above all, we demonstrate a significant relationship between the levels of LDH and the oncomarker CYFRA 21-1 and the presence or absence of ctDNA at the time of diagnosis. We also demonstrate significantly lower CRP levels in patients within whom the ctDNA disappeared during treatment. A similar but statistically insignificant trend was observed for LDH. Conclusions: CYFRA 21-1, LDH and probably CRP correlate with ctDNA levels in NSCLC. Repeated measurement of these markers could thus help in early detection of disease progression in the same way as does ctDNA monitoring., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Observation of anticancer therapy effect by monitoring of minimal residual disease (MRD) is becoming an important tool in management of non-small cell lung cancer (NSCLC). The approach is based on periodic detection and quantification of tumor-specific somatic DNA mutation in circulating tumor DNA (ctDNA) extracted from patient plasma. For such repetitive testing, complex liquid-biopsy techniques relying on ultra-deep NGS sequencing are impractical. There are other, cost-effective, methods for ctDNA analysis, typically based on quantitative PCR or digital PCR, which are applicable for detecting specific individual mutations in hotspots. While such methods are routinely used in NSCLC therapy prediction, however, extension to cover broader spectrum of mutations (e.g., in tumor suppressor genes) is required for universal longitudinal MRD monitoring. Methods: For a set of tissue samples from 81 NSCLC patients we have applied a denaturing capillary electrophoresis (DCE) for initial detection of somatic mutations within 8 predesigned PCR amplicons covering oncogenes and tumor suppressor genes. Mutation-negative samples were then subjected to a large panel NGS sequencing. For each patient mutation found in tissue was then traced over time in ctDNA by DCE. Results: In total we have detected a somatic mutation in tissue of 63 patients. For those we have then prospectively analyzed ctDNA from collected plasma samples over a period of up to 2 years. The dynamics of ctDNA during the initial chemotherapy therapy cycles as well as in the long-term follow-up matched the clinically observed response. Conclusion: Detection and quantification of tumor-specific mutations in ctDNA represents a viable complement to MRD monitoring during therapy of NSCLC patients. The presented approach relying on initial tissue mutation detection by DCE combined with NGS and a subsequent ctDNA mutation testing by DCE only represents a cost-effective approach for its routine implementation., Competing Interests: MM is employed by Elphogene company. Elphogene is currently providing oncoMonitor liquid biopsy/ctDNA test, which is in part utilizing the DCE mutation detection technology presented in this work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Benesova, Ptackova, Halkova, Semyakina, Svaton, Fiala, Pesek and Minarik.)

Background/aim: Circulating tumour DNA (ctDNA) represents an emerging biomarker in non-small cell lung cancer (NSCLC). We focused on the combination of ctDNA and positron emission tomography/computed tomography (PET/CT) in the follow-up monitoring of advanced-stage NSCLC patients treated with chemotherapy., Patients and Methods: Eighty-four patients were enrolled in this study. 18 F-fluorodeoxyglucose PET/CT and ctDNA assessments were performed at baseline and after two cycles of chemotherapy (follow-up)., Results: There was a correlation of ctDNA with metabolic tumour volume (MTV), total lesion glycolysis (TLG), and iodine concentration (IC) at baseline (p=0.001, p=0.001, p=0.003) and at follow-up (p=0.006, p=0.002, p=0.001). The objective response was associated with follow-up ctDNA (p<0.001) and the change of all PET/CT parameters. ROC analyses showed that the combination of follow-up ctDNA with changes in SUVmax is very promising for the estimation of objective response and progression-free survival., Conclusion: The combination of ctDNA assessment with PET/CT is a promising approach for the follow-up monitoring of therapy response and prognosis estimation of advanced-stage NSCLC patients., (Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is a rare familial gastric cancer syndrome with an autosomal dominant pattern of inheritance. It is characterised by fundic gland polyposis of the gastric body and is associated with a significant risk of gastric adenocarcinoma. Unlike sporadic gastric cancer, Helicobacter pylori is usually absent in patients with GAPPS. This opposite-point finding has so far not been fully clarified. Prophylactic total gastrectomy is indicated in all cases of GAPPS with fundic gland polyposis and the presence of any dysplasia. If no dysplasia is found at histology, prophylactic gastrectomy is suggested at between 30 and 35 years of age, or at five years earlier than the age at which the youngest family member developed gastric cancer. Different phenotypes of GAPPS demand an individual approach to particular family members., Competing Interests: Declaration of competing interest None., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Introduction: Patients with locally advanced rectal cancer (LARC) are undergoing neoadjuvant chemoradiotherapy (NCRT) prior to surgery. Although in some patients the NCRT is known to prevent local recurrence, it is also accompanied by side effects. Accordingly, there is an unmet need to identify predictive markers allowing to identify non-responders to avoid its adverse effects. We monitored circulating tumor DNA (ctDNA) as a potential liquid biopsy-based biomarker. We have investigated ctDNA changes plasma during the early days of NCRT and its relationship to the overall therapy outcome. Methods and Patients: The studied cohort included 36 LARC patients (stage II or III) undergoing NCRT with subsequent surgical treatment. We have detected somatic mutations in tissue biopsies taken during endoscopic examination prior to the therapy. CtDNA was extracted from patient plasma samples prior to therapy and at the end of the first week. In order to optimize the analytical costs of liquid-biopsy testing, we have utilized a two-level approach in which first a low-cost detection method of denaturing capillary electrophoresis was used followed by examination of initially negative samples by a high-sensitivity BEAMING assay. The ctDNA was related to clinical parameters including tumor regression grade (TRG) and TNM tumor staging. Results: We have detected a somatic mutation in 33 out of 36 patients (91.7%). Seven patients (7/33, 21.2%) had ctDNA present prior to therapy. The ctDNA positivity before treatment reduced post-operative disease-free survival and overall survival by an average of 1.47 and 1.41 years, respectively ( p = 0.015, and p = 0.010). In all patients, ctDNA was strongly reduced or completely eliminated from plasma by the end of the first week of NCRT, with no correlation to any of the parameters analyzed. Conclusions: The baseline ctDNA presence represented a statistically significant negative prognostic biomarker for the overall patient survival. As ctDNA was reduced indiscriminately from circulation of all patients, dynamics during the first week of NCRT is not suited for predicting the outcome of LARC. However, the general effect of rapid ctDNA disappearance apparently occurring during the initial days of NCRT is noteworthy and should further be studied., (Copyright © 2020 Pazdirek, Minarik, Benesova, Halkova, Belsanova, Macek, Stepanek and Hoch.)

We compare two types of pancreatic carcinoma samples obtained by EUS-guided fine needle biopsy (EUS-FNB) in terms of the success rates and clinical validity of analysis of two most commonly investigated DNA/RNA pancreatic cancer markers, KRAS mutations and miR-21 expression. 118 patients with pancreatic ductal adenocarcinoma underwent EUS-FNB. The collected sample was divided, one part was stored in a stabilizing solution as native aspirate (EUS-FNA) and second part was processed into the cytological smear (EUS-FNC). DNA/RNA extraction was followed by analysis of KRAS mutations and miR-21 expression. For both sample types, the yields of DNA/RNA extraction and success rates of KRAS mutation and miRNA expression were evaluated. Finally, the resulting KRAS mutation frequency and miR-21 prognostic role were compared to literature data from tissue resections. The overall amount of isolated DNA/RNA from EUS-FNC was lower compared to the EUS-FNA, average yield 10 ng vs 147 ng for DNA and average yield 164 vs. 642 ng for RNA, but the success rates for KRAS and miR-21 analysis was 100% for both sample types. The KRAS-mutant detection frequency in EUS-FNC was 12% higher than in EUS-FNA (90 vs 78%). The prognostic role of miR-21 was confirmed in EUS-FNC (p = 0.02), but did not reach statistical significance in EUS-FNA (p = 0.06). Although both types of EUS-FNB samples are suitable for DNA/RNA extraction and subsequent DNA mutation and miRNA expression analysis, reliable results with clinical validity were only obtained for EUS-FNC.

Malignant transformation in gliomas is frequently supplemented by somatic mutations in isocitrate dehydrogenase 1 and isocitrate dehydrogenase 2 genes. It has recently emerged that mutations in these genes are associated with prolonged survival and should be used as prognostic factor in management of brain cancer patients. There are several approaches in use for the detection of isocitrate dehydrogenase 1 and 2 mutations; however, these often exhibit shortcomings such as convoluted protocols with long processing time, complex (and costly) dedicated fluorescent probes, and/or demand on amounts of input DNA. Therefore, a simple and rapid method would be highly desired. Here, we present development and validation of simple and reliable isocitrate dehydrogenase 1 and 2 mutation detection assay using denaturing capillary electrophoresis. The detection sensitivity in terms of the limiting mutated allele fraction detectable estimated from a series of dilution runs was 2.9%. The method was validated by comparing to results obtained by a widely accepted detection technique, the multiplex ligation-dependent probe amplification, on a set of 85 brain tumors. The concordance of both methods was 100%, but denaturing capillary electrophoresis assay required fivefold lower input of DNA (1 versus 5 μL of DNA at concentrations typically between 10 and 30 ng/μL)., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Erlotinib is a tyrosine kinase inhibitor targeting epidermal growth factor receptor (EGFR); it is used in the treatment of advanced non-small cell lung cancer (NSCLC). We focused on the role of serum concentration of erlotinib and its association with outcome and toxicity in patients with advanced NSCLC harbouring the wild-type EGFR gene or squamous histology., Patients and Methods: Clinical data of 122 patients were analyzed. Serum samples were collected within four weeks after the initiation of treatment., Results: There was no significant association of erlotinib concentration with PFS nor OS (p=0.352 and p=0.6393). Significant associations of erlotinib concentration with grade of skin rash and diarrhoea (p<0.0001 and p<0.0001) were found. Skin rash and diarrhoea were significantly associated with PFS (p=0.0338 and p=0.0001) and OS (p=0.0064 and p=0.0353)., Conclusion: Erlotinib concentration was not associated with outcome. Erlotinib concentration was associated with occurrence and severity of skin rash and diarrhoea; the outcome was associated with erlotinib toxicity., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

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.

Background and Aims: Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) has to date been recognized in only 8 families worldwide. Recently, different point mutations within the Ying Yang 1 (YY1) binding motif in promoter 1B of the APC gene were assigned as causal in 6 families with GAPPS., Methods: We diagnosed GAPPS across 3 generations in a Czech white family., Results: The proband's mother died of gastric cancer at 49 years of age. The proband died of gastric cancer at 56 years of age. All 3 of the proband's daughters inherited polyposis, involving exclusively the gastric fundus and body, with relative sparing of the lesser curve. The daughters have all been regularly surveyed endoscopically. Polyposis progressed rapidly with intestinal differentiated low-grade and high-grade dysplasia present on polypectomy specimens 5 years after the original diagnosis. On this basis, all 3 of the proband's daughters were scheduled for prophylactic total gastrectomy. Unfortunately, the middle daughter presented with generalized gastric adenocarcinoma and died at the age of 26 years. The other 2 daughters (aged 30 and 23 years) underwent total gastrectomy within 6 weeks of their sister's death; histology of surgical specimens showed gastric adenocarcinoma stage IA (pT1a, N0, M0) in both cases. Bi-directional Sanger sequencing of promoter 1B revealed a point mutation (c.-191 T>C) in all 3 daughters of the proband., Conclusions: Atypical endoscopic progression of the fundic gland polyposis, with the presence of dysplasia on polypectomy specimens and genetic testing with recently discovered mutations in promoter 1B of the APC gene might help clinicians to decide whether prophylactic gastrectomy should be performed., (Copyright © 2016 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.)

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.

Background: Serum lactate dehydrogenase (LDH) has been reported as a prognostic biomarker in malignant diseases. However, little is known on the dynamics of serum LDH levels during systemic treatment. We focused on the association of changes in serum LDH with outcome of patients with advanced-stage non-small cell lung cancer (NSCLC) treated with erlotinib., Patients and Methods: Clinical data of 309 patients were analyzed. Serum samples were collected within one week before initiation and after one month of treatment., Results: The change in serum LDH during the first month of erlotinib treatment was independently associated with disease control rate (p=0.006), progression-free survival (PFS) (p=0.010) and overall survival (OS) (p<0.001)., Conclusion: LDH is a commonly used serum biomarker, that is cheap and easy to detect. The results of our study suggest that the change in LDH serum level during the first month is a surrogate marker on the efficacy of erlotinib in patients with advanced NSCLC., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background/aim: The prognostic and predictive value of Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation in non-small cell lung cancer (NSCLC) is not well established. The present study aimed at the elucidation of the role of KRAS mutation in prediction of outcome of patients with advanced NSCLC receiving second- or third-line chemotherapy., Patients and Methods: The outcome of 127 patients with advanced NSCLC who recieved pemetrexed or docetaxel at second- or third-line therapy was retrospectively analyzed., Results: Progression-free survival was not significantly different between patients with KRAS mutation and those with wild-type KRAS. The results were the same even when taking into account the specific KRAS mutation. Overall survival was significantly longer for patients with wild-type KRAS vs. those with KRAS mutation (16.1 vs. 7.2 months, p=0,008). We observed shorter overall survival for those with G12C KRAS mutation vs. other KRAS mutations (median 10.3 vs. 6.4 months, p=0.011)., Conclusion: The presence of KRAS mutation (especially KRAS G12C mutation) correlated with adverse prognosis in patients treated with second- or third-line pemetrexed or docetaxel., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Molecular targeted therapy based on tyrosine kinase inhibitors (TKI), directed at epidermal growth factor receptor (EGFR) is one of the novel effective agents in management of advanced-stage of Non Small Cell Lung cancer (NSCLC). However several candidate predictors have been extensively studied, apart from activating EGFR gene mutations, no reliable biochemical or molecular predictors of response to erlotinib have been validated. The aim of our retrospective study was to evaluate the association of baseline serum albumin with outcomes in a large cohort of patients with advanced-stage NSCLC treated with erlotinib. Clinical data of 457 patients with locally-advanced (III B) or metastatic stage (IV) NSCLC treated with erlotinib were analysed. Serum samples were collected and the measurement was performed one day before the initiation of erlotinib treatment. Before the treatment initiation, low albumin was (<35 g/l) measured in 37 (8.1%) patients and normal albumin (≥ 35 g/l) was measured in 420 (91.9%). The median PFS and OS for patients with low serum albumin was 0.9 and 1.9 months compared to 1.9 and 11.4 months for patients with normal serum albumin (p=0.001 and p<0.001). The multivariate Cox proportional hazards model revealed that EGFR mutation status (HR=2.50; CI: 1.59-3.92; p<0.001) and pretreatment serum albumin (HR=1.73; CI: 1.21-2.47; p=0.003) were significant independent predictive factors for PFS, whereas EGFR mutation status (HR=3.14; CI: 1.70-5.81; p<0.001), stage (HR=1.48; CI: 1.09-2.02; p=0.013), ECOG PS (HR=1.77; CI: 1.37-2.29; p<0.001) and pretreatment serum albumin (HR=4.60; CI: 2.98-7.10; p<0.001) were significant independent predictive factors for OS. In conclusion, the results of present retrospective study indicate that pretreatment hypoalbuminemia is associated with poor outcome of NSCLC patients treated with erlotinib. Based on these results, measuement of serum albumin is an objective laboratory method feasible for estimation of prognosis of patients with advanced-stage NSCLC.

Background: Pemetrexed and erlotinib represent different agents commonly used for the second-line treatment of patients with advanced-stage non-small cell lung cancer (NSCLC)., Patients and Methods: We analyzed data of 137 patients with advanced-stage non-squamous NSCLC treated with pemetrexed or erlotinib in the second line. All patients harbored a wild-type epidermal growth factor receptor gene. Genetic testing was performed using a combination of denaturing capillary electrophoresis and direct Sanger sequencing., Results: overall response rate and disease control rate in patients treated with pemetrexed was 20.8% and 62.5% vs. 6.3% and 53.2% in patients treated with erlotinib (p=0.022; p=0.358). Median progression-free and overall survival in patients treated with pemetrexed was 1.6 and 11.3 months vs. 1.9 and 11.4 months in patients treated with erlotinib (p=0.470 and p=0.942, respectively). Erlotinib was associated with skin rash and diarrhea; pemetrexed was associated with hematological toxicity and fatigue., Conclusion: A similar efficacy and different, although well-tolerated, toxicity profile of both pemetrexed and erlotinib was shown., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

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.

Background: Tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) represent novel, effective tools in the management of advanced-stage non-small cell lung cancer (NSCLC). We aimed to evaluate the incidence and predictive role of EGFR gene amplification in patients with advanced-stage NSCLC treated with EGFR-TKIs., Patients and Methods: The study included 290 patients with advanced-stage (IIIB or IV) NSCLC. Multiplex ligation-dependent probe amplification (MLPA) and polymerase chain reaction (PCR) were used for detection of EGFR gene amplification and EGFR mutations, respectively., Results: EGFR amplification was detected in 26 (9.0%) patients. EGFR amplification was found more frequently in patients harboring the EGFR mutation (p<0.001). No significant corelation between EGFR gene amplification and survival was observed., Conclusion: EGFR gene amplification is associated with EGFR gene mutation. EGFR gene amplification is not a feasible predictive biomarker for treatment with EGFR-TKIs in patients with advanced-stage NSCLC., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Erlotinib is a low molecular weight tyrosine kinase inhibitor (TKI) directed at epidermal growth factor receptor (EGFR), widely used in the treatment of locally advanced or metastatic-stage non-small cell lung cancer (NSCLC). Although introduction of EGFR-TKIs have significantly extended survival of advanced-stage NSCLC patients, their efficacy in the entire patient population is relatively low. Aside from activating EGFR mutations, no reliable biochemical or molecular predictors of response to erlotinib have been established. The aim of our retrospective study was to evaluate the association of baseline serum levels of C-reactive protein (CRP) with outcomes in patients with advanced-stage NSCLC treated with erlotinib. We retrospectively analyzed clinical data of 595 patients with advanced-stage NSCLC (IIIB or IV) treated with erlotinib. Serum CRP was measured using an immunoturbidimetric method. High baseline levels of CRP (≥10 mg/l) were measured in 387 (65 %) patients, and normal levels (<10 mg/l) were measured in 208 (35 %) patients. The median progression-free survival (PFS) and overall survival (OS) for patients with high CRP was 1.8 and 7.7 compared to 2.8 and 14.4 months for patients with low CRP (p < 0.001 and p < 0.001). The multivariable Cox proportional hazards model revealed that CRP was significantly associated with PFS and also with OS (hazard ratio (HR) = 1.57, p < 0.001, and HR = 1.63, p < 0.001, respectively). In conclusion, the results of the conducted retrospective study suggest that high baseline level of CRP was independently associated with worse outcome of patients with advanced-stage NSCLC treated with erlotinib. CRP is a commonly used biomarker which is simple and easy to detect, and thus, it is feasible for the use in the routine clinical practice.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) represent novel effective agents approved for the treatment of patients with advanced-stage NSCLC. KRAS mutations have been reported as a negative prognostic and predictive factor in patients with NSCLC treated with EGFR-TKIs. Several studies have recently shown that statins can block tumour cell growth, invasion and metastatic potential. We analysed clinical data of 67 patients with locally advanced (IIIB) or metastatic stage (IV) NSCLC harbouring Kirsten rat sarcoma viral oncogene (KRAS) mutation treated with erlotinib or gefitinib. Twelve patients were treated with combination of EGFR-TKI and statin and 55 patients were treated with EGFR-TKI alone. Comparison of patients' survival (progression-free survival (PFS) and overall survival (OS)) according to the treatment used was performed using the Gehan-Wilcoxon test. The median of PFS and OS for patients treated with EGFR-TKI alone was 1.0 and 5.4 months compared to 2.0 and 14.0 months for patients treated with combination of EGFR-TKI and statin (p = 0.025, p = 0.130). In conclusion, the study results suggest significant improvement of PFS for patients treated with combination of statin and EGFR-TKI, and the difference in OS was not significant.

Objectives: A substantial proportion of patients with hypertrophic cardiomyopathy (HCM) do not have causative mutations in the genes for heart sarcomere. The purpose of this study was to evaluate the association between microRNA (miRNA) sequence variants and HCM., Methods: We performed genetic testing on 56 HCM patients who had previously been found to be negative for mutations in the 4 major genes for sarcomeric proteins. The coding and adjacent regions (120-220 nt) of selected miRNAs were analyzed for the presence of sequence variants. The testing was based on PCR amplification of DNA-encoding miRNAs and subsequent denaturing capillary electrophoresis., Results: A total of 3 different variants were detected in the 11 selected miRNAs. These included polymorphisms rs45489294 in miRNA 208b, rs13136737 in miRNA 367 and rs9989532 in miRNA 1-2. In the patient group, the most frequent polymorphism was in miRNA 208b (10 times) followed by miRNA 367 (7 times). Both polymorphisms were found to occur with similar frequencies in the group of healthy controls. The remaining detected variant was not present in the control group, but was not connected with the HCM phenotype in the children of the probands., Conclusion: Sequence variants in miRNAs of patients with HCM are not frequent and the contribution of these variants to the development of this disease was not demonstrated., (© 2015 S. Karger AG, Basel.)

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.

Background/aim: Tumor biomarkers are used for diagnostics and follow-up monitoring of patients with non-small cell lung cancer (NSCLC). We focused on the predictive role of neuron-specific enolase (NSE) and thymidine [corrected] kinase (TK) in patients with advanced-stage NSCLC treated with epidermal growth factor tyrosine kinase inhibitors (EGFR-TKIs)., Patients and Methods: In a total of 163 patients with advanced-stage (IIIB or IV) NSCLC treated with EGFR-TKIs (erlotinib or gefitinib), pre-treatment levels of NSE and TK were measured., Results: We observed significantly shorter progression-free (PFS) and overall survival (OS) in patients with high NSE levels (p=0.002; p=0.003) and also in those with high TK levels (p=0.026; p=0.020). The multivariate Cox proportional hazards model confirmed that high NSE is a strong independent predictive factor for short PFS (hazard ratio; HR=2.36; p=0.003)., Conclusion: High pre-treatment serum levels of NSE is an independent biomarker predicting poor outcome of patients with NSCLC treated with EGFR-TKIs., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: Tumor biomarkers are used for predicting therapy effect and prognosis of patients with non-small cell lung cancer (NSCLC). We focused on their potential role in prediction of the efficacy of erlotinib., Patients and Methods: In a total of 144 patients with advanced-stage (IIIB or IV) NSCLC treated with erlotinib, pre-treatment levels of soluble carcinoembryonic antigen (CEA) and cytokeratin markers in serum were measured., Results: The median progression-free and overall survival for patients with a high level of carcinoembryonic antigen (CEA) was 1.9 and 8.6 vs. 2.9 and 16.1 months for patients with low CEA (p=0.046 and p=0.116). The respective medians for patients with a high level of cytokeratin-19 fragment were 1.9 and 6.1 vs. 3.4 and 23.8 months for patients with the low cytokeratin-19 fragment (p<0.001 and p<0.001)., Conclusion: High pre-treatment serum levels of one or both biomarkers are associated with poor outcome of patients with NSCLC treated with erlotinib., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Erlotinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor used for the treatment of advanced stage non-small cell lung cancer (NSCLC). Clinical trials have shown high efficacy of erlotinib, particularly in patients harboring activating mutations in the EGFR gene. Here we describe the case of a 42-year-old female light smoker diagnosed with EGFR mutation-positive adenocarcinoma of the left lung with pleural metastases, pleural effusion and metastases to the thoracic vertebrae (stage IV, T4NXM1). Although the patient developed resistance to erlotinib in the first line, she achieved disease stabilization lasting for 13 months as a response to erlotinib retreatment after 6 cycles of second-line chemotherapy. In conclusion, retreatment with erlotinib in our patient was effective. Therefore it should be considered as a good treatment option for patients with NSCLC harboring EGFR mutation.

Background: Pemetrexed and erlotinib represent novel agents for the treatment of non-small cell lung cancer (NSCLC). The role of sequential treatment in NSCLC has not been elucidated yet. We compared the efficacy of second-line pemetrexed followed by third-line erlotinib (P-E) to treatment with the reverse sequence (E-P)., Patients and Methods: We analyzed data of 57 patients with advanced-stage (IIIB/IV) lung adenocarcinoma harboring wild-type epidermal growth factor receptor (EGFR) gene; 31 patients were treated with P-E and 26 patients with the E-P sequence., Results: The median progression-free survival (PFS) for patients treated with P-E was 3.6 months vs. 7.8 months for patients treated with E-P (p=0.029). The median overall survival (OS) for patients treated with P-E was 7.9 months vs. 26.3 months for patients treated with E-P (p=0.006)., Conclusion: The results proved a significant improvement of both PFS and OS for patients treated with the E-P sequence as compared to the P-E sequence.

Dual antiplatelet therapy is important treatment modality across the spectrum of coronary artery disease manifestations. However, a significant number of patients do not have a completely effective response to clopidogrel. This study assessed the impact of response after clopidogrel with Verify Now device on prognosis on patients undergoing coronary interventions. Consecutive patients following percutaneous coronary intervention were prospectively enrolled. A loading dose of 600 mg of clopidogrel was administered before or during PCI. Blood samples were drawn within 24 h after clopidogrel administration. The effect of clopidogrel was measured using VerifyNow. All patients were evaluated at 6 months. The primary end-point was the combination of death, MI and stroke. 378 patients (69.3 % men and 30.7 % women) were enrolled. The mean age was 67.2 ± 12.8 years, BMI 28.9 ± 17.7, and 116 patients had diabetes (30.7 %). During the 6-months follow-up 30 patients (7.94 %) experienced a monitored end-point: 12 patients (3.17 %) had MI; five patients (1.32 %) strokes and 15 patients (3.97 %) died. The remaining 248 patients (71.26 %) were end-point free. Factors associated with a poor prognosis were: leukocytes (OR 1.7 [1.2-2.4], p < 0.01), creatinine (OR 1.4 [1.1-2.5], p < 0.05) and at a borderline level the presence of AA allele of gene CYP2C19*2 (OR 2.5 [0.99-4.1], p = 0.052). The results using VerifyNow were similar between both groups (Group End-point: 208.5 ± 85.5, group No end-point 203.1 ± 91.3) and failed to show any prognostic value (OR 1.00 [0.992-1.007], p = 0.9). The measurement of clopidogrel efficacy using VerifyNow had no prognostic value for our unselected cohort of patients after PCI.

Background: Epidermal growth factor receptor (EGFR), Kirsten rat sarcoma viral oncogene (KRAS) and phosphatidylinositide-3-kinase catalytic subunit-alpha (PIK3CA) mutations are biomarkers used for the prediction of efficacy of EGFR tyrosine kinase inhibitors (EGFR-TKIs) in advanced non-small cell lung cancer (NSCLC)., Patients and Methods: In total, 223 patients with advanced-stage squamous cell NSCLC were tested; 179 patients were treated with EGFR-TKIs. Genetic testing was performed using a combination of denaturing capillary electrophoresis and direct Sanger sequencing., Results: EGFR mutations were detected in 7.2%; KRAS mutations in 7.4% and PIK3CA mutations in 3.8% of patients. No correlation of EGFR or PIK3CA mutation status with progression-free survival (PFS) (p=0.425; p=0.197), nor overall survival (OS) (p=0.673; p=0.687), was observed. KRAS mutations correlated with shorter OS (p=0.039), but not with PFS (p=0.120)., Conclusion: We did not observe any role of EGFR, KRAS, PIK3CA mutations in prediction of EGFR-TKIs efficacy in patients with advanced-stage squamous cell NSCLC.

Prognosis of solid cancers is generally more favorable if the disease is treated early and efficiently. A key to long cancer survival is in radical surgical therapy directed at the primary tumor followed by early detection of possible progression, with swift application of subsequent therapeutic intervention reducing the risk of disease generalization. The conventional follow-up care is based on regular observation of tumor markers in combination with computed tomography/endoscopic ultrasound/magnetic resonance/positron emission tomography imaging to monitor potential tumor progression. A recent development in methodologies allowing screening for a presence of cell-free DNA (cfDNA) brings a new viable tool in early detection and management of major cancers. It is believed that cfDNA is released from tumors primarily due to necrotization, whereas the origin of nontumorous cfDNA is mostly apoptotic. The process of cfDNA detection starts with proper collection and treatment of blood and isolation and storage of blood plasma. The next important steps include cfDNA extraction from plasma and its detection and/or quantification. To distinguish tumor cfDNA from nontumorous cfDNA, specific somatic DNA mutations, previously localized in the primary tumor tissue, are identified in the extracted cfDNA. Apart from conventional mutation detection approaches, several dedicated techniques have been presented to detect low levels of cfDNA in an excess of nontumorous (nonmutated) DNA, including real-time polymerase chain reaction (PCR), "BEAMing" (beads, emulsion, amplification, and magnetics), and denaturing capillary electrophoresis. Techniques to facilitate the mutant detection, such as mutant-enriched PCR and COLD-PCR (coamplification at lower denaturation temperature PCR), are also applicable. Finally, a number of newly developed miniaturized approaches, such as single-molecule sequencing, are promising for the future., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Molecular targeted therapy based on EGFR tyrosine kinase inhibitors (EGFR-TKI) is currently astate of the art option for management of advanced stage NSCLC. Activating EGFR mutations are preferable for a good treatment response to EGFR-TKI. The presented retrospective study evaluated a clinical observation of EGFR-TKI aiming at its efficacy and safety in comparison to a standard chemotherapy in the first-line treatment of advanced stage NSCLC. Total number of patients with advanced stage (IIIB, IV) EGFR mutation-positive NSCLC was 54 of which 23 were treated with EGFR-TKI and 31 patients with various chemotherapy regimens in the first line. The treatment efficacy was characterized in terms of disease control rate (DCR), progression-free survival (PFS) and overall survival (OS). The comparison of DCR was performed using Fisher's exact test and the differences in survival were tested using log-rank test. DCR for EGFR-TKI treatment was 95.6% vs. 70.9% for chemotherapy (p=0.032). Median of PFS in patients treated with EGFR-TKI was 7.2 months vs. 2.5 months in patients treated with chemotherapy (p<0.001). Median of OS was 14.5 months vs. 21.4 months (p=0.729). EGFR-TKI was associated with higher incidence of skin rash and diarrhoea; chemotherapy was associated with higher incidence of haematologic adverse events and nausea or vomiting. The analysis results showed a favourable DCR and PFS in patients treated with EGFR-TKI in the first line. The non-significant difference in OS could be attributed to a cross-over during the patient follow-up as well as the differences in performance status and age between both groups. EGFR-TKI is the optimal choice for the first-line treatment of EGFR mutation-positive NSCLC.

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 Fisher's 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.

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Molecular targeted therapy based on tyrosine kinase inhibitors, directed at the epidermal growth factor receptor (EGFR) is one of novel options for management of NSCLC. Erlotinib is EGFR tyrosine kinase inhibitor used for treatment of the advanced NSCLC. This presented study is focused on comparison of erlotinib and chemotherapy efficacy in the second line treatment of the advanced NSCLC. DCR and PFS became the primary endpoints.Total number of patients was 290. A group treated with chemotherapy in the second line consisted of 150 patients and a group treated with erlotinib in the second line consisted of 140 patients. Comparison of DCR was performed using Fisher's exact test, visualization of PFS was performed using Kaplan-Meier survival curves and differences were tested using the log-rank test. Genetic testing was performed using PCR direct sequencing. In the group treated with chemotherapy 2 CR, 23 PR and 51 SD were achieved vs. 5 CR, 10 PR and 55 SD in the group treated with erlotinib in the second line. DCR in patients treated with chemotherapy was 54.0% vs. 51.3% in patients without EGFR mutation treated with erlotinib (p=0.707); in patients harboring EGFR mutation, treated with erlotinib (n=9) outstanding results were achieved: 4 CR, 2 PR and 3 SD (not tested). Median of PFS in patients treated with chemotherapy was 2.1 months vs. 1.9 months in patients without EGFR mutation (p=0.879) vs. 8.4 months in patients harboring EGFR mutation treated with erlotinib (p=0.017). Results of analysis show that even patients without EGFR mutation are able to benefit from erlotinib treatment in the second line. The efficacy (DCR, PFS) of erlotinib in patients without EGFR mutation was comparable with chemotherapy. The treatment efficacy in a subgroup of patients harbouring EGFR mutation treated with erlotinib was significantly better than in patients without EGFR mutation.

Background: While efficient surgical treatment is the key to prolonged survival of patients with colorectal cancer, post-surgical follow-up is important for the early detection of relapsing disease or of disease progression. Current dispensarization, typically based on imaging CT, PET, MR, is frequently supported by the observation of tumour markers (CEA, CA19-9). Due to their limited sensitivity and selectivity, better tools for monitoring of the disease are desirable. Tumour cell-free DNA (cfDNA) has been recently demonstrated as a new promising molecular marker for observation and early detection of disease progression., Patients and Methods: We present results of post-surgical monitoring tumour cfDNA in the cases of seven patients suffering from advanced forms of CRC. We applied a mutation-based approach in which the total cfDNA was screened for a specific somatic mutation present in the primary tumour. We screened a panel of the most frequent somatic mutations covering the genes APC, KRAS, TP53, PIK3CA and BRAF. All patients were tested positive for tumour cfDNA prior to surgery. cfDNA was then evaluated within a week after surgery and subsequently in monthly intervals., Results: We present typical cases of colorectal cancer patients who underwent surgical treatment at different levels of radicality with or without adjuvant chemo/biotherapy. The tumour cfDNA status was found to be always closely correlated with the actual clinical status of the patient., Conclusion: The cfDNA appears to be a viable tool for the monitoring of the clinical progression of CRC in patients with cfDNA positivity prior to surgery.

Antiplatelet therapy is a cornerstone of cardiovascular treatment in patients with coronary artery disease and after myocardial infarction. Clopidogrel has become a popular antiplatelet agent due to its fast action and low frequency of adverse effects. Kinetics of clopidogrel metabolism is driven by enzymatic activity of the Cytochrome P450 system. Genotyping of CYP2C19 and CYP2C9 polymorphisms allows to identify slow metabolizers showing resistance to clopidogrel therapy. Today, a number of PCR-based techniques for single nucleotide polymorphism genotyping directed at clopidogrel resistance polymorphisms are in use. Here, we describe a new alternative genotyping approach combining the separation power of denaturing capillary electrophoresis with the analysis speed and ease of use of Bioanalyzer chipCE platform. Using an upgraded heater control, we present an optimization for allele separation of CYP2C19 I331V, CYP2C9 R144C, and CYP2C9 I359L polymorphisms employing run temperatures of up to 55°C. We demonstrate rapid and accessible approach to reproducible clopidogrel resistance with feasibility and low cost., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Hypertrophic cardiomyopathy (HCM) is a cardiovascular disease with autosomal dominant inheritance. It is caused by mutations in the genes coding for structural and/or regulatory proteins found in the sarcomere of cardiomyocytes. A group of genes, including the heavy chain of beta-myosin (MYH7), myosin binding protein C (MYBPC3), cardiac troponin I (TNNI3) and cardiac troponin T (TNNT2) are frequently affected by causal mutations. While exact mutation frequency data has been obtained for various populations, no screening has been reported for Central European populations., Patients and Methods: We performed a complete sequencing of MYH7, MYBPC3, TNNI3 and TNNT2 genes in 100 HCM patients., Results: We discovered mutations in a total of 40 patients (40%), including 4 patients with double mutations. A total of 35 different mutation types were detected, of which 17 were novel. The contributions from individual genes were: 24 mutations in MYBPC3 (54.5%), 14 in MYH7 (31.8%), 4 in TNNI3 (9%) and 2 mutations in TNNT2 (4.5%). We have observed a wide variability in disease manifestation across the different genes/mutation types. In addition, we have discovered differences in both frequency and distribution of mutations of the two most common genes (MYBPC3 and MYH7) compared to other populations., Conclusion: The most common gene responsible for HCM in our study population was MYBPC3, followed by MYH7, TNNI3 and TNNT2. Phenotypic heterogeneity, as well as the dissimilarity to other populations, prevents effective use of a pre-screening test, which would be directed at the most common mutation hotspots, in our population.

Background: DNA methylation is one of major factors in cancer progression. We observed multiple genes involved in cancer-related signaling and focused on patients with advanced non-small cell lung cancer (NSCLC) and evaluated methylation in relation to various clinical parameters., Patients and Methods: Thirty genes were examined in 121 NSCLC patients using the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) method. Correlations to gender, smoking status, tumor subtype, disease stage and EGFR/KRAS mutation status were performed by chi-square test., Results: 90% of tumors exhibited methylation of at least one gene. Most frequently methylated were cadherin-13 (CDH13), Ras associated domain-containing protein (RASSF1A), Wilms' tumor protein (WT1), adenomatous polyposis coli protein (APC), paired box protein Pax-5 (PAX5), estrogen receptor (ESR1), an inhibitor of cyclin-dependent kinase p15 (CDKN2B), paired box protein Pax-6 (PAX6), transcription factor GATA-5 (GATA5) and cell adhesion molecule 4 (IGSF4). Overall methylation (any gene) was increased in adenocarcinomas (p=0.0329), unrelated to gender or disease stage. Several genes exhibited variable methylation with gender (CDH13, p<0.001; GATA5, p=0.02; PAX6, p=0.01 and ESR1, p=0.03), smoking (CDH13, p=0.002), or epidermal growth factor receptor (EGFR) mutation status [Von Hippel-Lindau disease tumor supresor (VHL), p=0.001; CDKN2B, p=0.02; CDH13, p=0.02; APC, p=0.04 and ESR1, p=0.04]., Conclusion: Differences in gene methylation associated with gender, smoking and EGFR mutation suggest potential for prediction in relation to management of tyrosine kinase inhibitor therapy.

Case reports on the co-incidence of Kirsten rat sarcoma (KRAS) mutation and epidermal growth factor receptor (EGFR) amplification in patients with NSCLC are very rare. This combination is usually considered a negative prognostic factor, despite EGFR amplification alone having positive predictive value. The whole course of treatment of a patient with both EGFR amplification and KRAS mutation present is decribed. The patient in question was a smoker for whom both first- and second-line chemotherapy had been unsuccessful. In stage IV disease biological therapy was administered and proved highly beneficial. Today, 38 months since commencing the treatment, the patient still has no signs of progression and the therapy is still in progress.

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.

The presence of activating mutations within the tyrosine kinase domain of the epidermal growth factor receptor gene has been attributed to a positive response to biological therapy of lung cancer by small-molecular tyrosine kinase inhibitors, gefitinib and erlotinib. Among the two most significant mutation types are deletions in exon 19 and a single point substitution in exon 21 (termed L858R). The exon 19 deletions can readily be examined by fragment analysis, due to the characteristic length difference between the normal and mutated PCR product. Analysis of the L858R point mutation, however, presents a greater challenge. The current paper is aimed at developing a sensitive, yet simple, low-cost mutation detection assay directed at the L858R mutation using a method based on CE of heteroduplexes under partial denaturing conditions. We perform optimization of separation conditions on different commercial instruments including ones equipped with 8, 16 and 96 capillaries. We present normalized migration reproducibility in the range from 1 (8 and 16) to 5% (96) RSD. A reliable distinction of the R836R silent polymorphism from a potential presence of the L858R mutation is also demonstrated. In its implementation, the presented assay is just another application running on a conventional CE platform without the need of dedicated instrumentation.