Ethical principles for the usage and sharing of genomic data from researc.

The current significant development of human genome/exome sequencing in biomedical research is one of the important paths leading to personalized medicine. However, sequencing of human genetic information generates potentially sensitive and exploitable data, which leads to ethical, legal, and security issues. For this reason, it is necessary to follow several measures when working with these data, applying to their entire life cycle - i.e., acquisition, storage, processing, usage, sharing, archiving, and reuse. In addition, importance of good practice during the whole data life cycle is emphasized by current European trends towards open science and digital transformation. Therefore, the following recommendations have been developed, establishing principles for work with the whole human genome sequences or parts of it in research context. The recommendations are based on two documents published by the Global Alliance for Genomics and Health (GA4GH) and on foreign literature, thus summarizing recent relevant guidance on most aspects of working with human genomic data.

Circulating Tumor DNA correlates with Lactate Dehydrogenase, CYFRA 21-1, and CRP levels in patients with advanced NSCLC.

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.

Detection and Quantification of ctDNA for Longitudinal Monitoring of Treatment in Non-Small Cell Lung Cancer Patients Using a Universal Mutant Detection Assay by Denaturing Capillary Electrophoresis.

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.

Somatic Mutations in Exon 7 of the TP53 Gene in Index Colorectal Lesions Are Associated with the Early Occurrence of Metachronous Adenoma.

(1) Background: this prospective study was focused on detailed analysis of the mutation heterogeneity in colorectal lesions removed during baseline (index) colonoscopy to identify patients at high risk of early occurrence of metachronous adenomas. (2) Methods: a total of 120 patients after endoscopic therapy of advanced colorectal neoplasia size ≥10 mm (index lesion) with subsequent surveillance colonoscopy after 10-18 months were included. In total, 143 index lesions and 84 synchronous lesions in paraffin blocks were divided into up to 30 samples. In each of them, the detection of somatic mutations in 11 hot spot gene loci was performed. Statistical analysis to correlate the mutation profiles and the degree of heterogeneity of the lesions with the risk of metachronous adenoma occurrence was undertaken. (3) Results: mutation in exon 7 of the TP53 gene found in the index lesion significantly correlated with the early occurrence of metachronous adenoma (log-rank test p = 0.003, hazard ratio 2.73, 95% confidence interval 1.14-6.56). We did not find an association between the risk of metachronous adenomas and other markers monitored. (4) Conclusions: the findings of this study could lead to an adjustment of existing recommendations for surveillance colonoscopies in a specific group of patients with mutations in exon 7 of the TP53 gene in an index lesion, where a shortening of surveillance interval may be warranted.

Combination of Circulating Tumour DNA and 18F-FDG PET/CT for Precision Monitoring of Therapy Response in Patients With Advanced Non-small Cell Lung Cancer: A Prospective Study.

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. 18F-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.

Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) - A Helicobacter-opposite point.

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.

Monitoring of Early Changes of Circulating Tumor DNA in the Plasma of Rectal Cancer Patients Receiving Neoadjuvant Concomitant Chemoradiotherapy: Evaluation for Prognosis and Prediction of Therapeutic Response.

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.

Comparison of Native Aspirates and Cytological Smears Obtained by EUS-Guided Biopsies for Effective DNA/RNA Marker Testing in Pancreatic Cancer.

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.

Significance of postoperative follow-up of patients with metastatic colorectal cancer using circulating tumor DNA.

BACKGROUND: One of the most notable applications for circulating tumor DNA (ctDNA) detection in peripheral blood of patients with metastatic colorectal cancer (mCRC) is a long-term postoperative follow-up. Sometimes referred to as a "liquid (re)biopsy" it is a minimally invasive procedure and can be performed repeatedly at relatively short intervals (months or even weeks). The presence of the disease and the actual extent of the tumor burden (tumor mass) within the patient's body can be monitored. This is of particular importance, especially when evaluating radicality of surgical treatment as well as for early detection of disease progression or recurrence. AIM: To confirm the radicality of surgery using ctDNA and compare available methods for detection of recurrence in metastatic colorectal cancer. METHODS: A total of 47 patients with detected ctDNA and indications for resection of mCRC were enrolled in the multicenter study involving three surgical centers. Standard postoperative follow-ups using imaging techniques and the determination of tumor markers were supplemented by ctDNA sampling. In addition to the baseline ctDNA testing prior to surgery, a postoperative observation was conducted by evaluating ctDNA presence up to a week after surgery and subsequently at approximately three-month intervals. The presence of ctDNA was correlated with radicality of surgical treatment and the actual clinical status of the patient. RESULTS: Among the monitored patients, the R0 (curative) resection correlated with postoperative ctDNA negativity in 26 out of 28 cases of surgical procedures (26/28, 93%). In the remaining cases of R0 surgeries that displayed ctDNA, both patients were diagnosed with a recurrence of the disease after 6 months. In 7 patients who underwent an R1 resection, 4 ctDNA positivities (4/7, 57%) were detected after surgery and associated with the confirmation of early disease recurrence (after 3 to 7 months). All 15 patients (15/15, 100%) undergoing R2 resection remained constantly ctDNA positive during the entire follow-up period. In 22 cases of recurrence, ctDNA positivity was detected 22 times (22/22, 100%) compared to 16 positives (16/22, 73%) by imaging methods and 15 cases (15/22, 68%) of elevated tumor markers. CONCLUSION: ctDNA detection in patients with mCRC is a viable tool for early detection of disease recurrence as well as for confirmation of the radicality of surgical treatment.

Application of denaturing capillary electrophoresis for the detection of prognostic mutations in isocitrate dehydrogenase 1 and isocitrate dehydrogenase 2 genes in brain tumors.

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).

Serum Concentration of Erlotinib and its Correlation with Outcome and Toxicity in Patients with Advanced-stage NSCLC.

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.

[Patient with Three EGFR Mutations - Gradual Development of Resistance to Previous Targeted Treatment]. / Pacientka se tremi EGFR mutacemi - postupný rozvoj rezistence na predchozí cílenou lécbu.

BACKGROUND: Patients with sensitive EGFR mutations are already being treated with first and second generation tyrosine kinase inhibitors (TKIs). However, resistance to these drugs occurs over time, and over half of all cases is caused by a mutation (T790M) in the EGFR kinase domain. Osimertinib offers a new treatment option that overcomes this problem. Unfortunately, resistance to this drug also develops after several months of treatment and is caused by another mutation (C797S) in EGFR. CASE REPORT: Our case report provides evidence for the progressive development of EGFR-TKI resistance in a patient with a deletion of exon 19 in the EGFR gene. First, based on a mutation (T790M) identified after afatinib treatment and a subsequent mutation (C797S) mutation identified after osimertinib treatment. We mention overcoming this resistance (C797S) mutation by using 4th generation EGFR-TKI and other alternative procedures (chemotherapy, immunotherapy, and combinations of older EGFR-TKI generations). We also mention a rare case of peritoneal metastasis that occurred after previous treatment with osimertinib that we attempted to ameliorate by using erlotinib because the impaired condition of the patient did not allow treatment by chemotherapy. There are documented cases in which erlotinib has been successfully given to patients with peritoneal metastases and patients with the EGFR mutation C797S following progression to afatinib. This was not the case in our patient, probably because of the remaining EGFR mutation T790M. CONCLUSION: In our case report, erlotinib did not show efficacy after progression to osimetinib. Nowadays, chemotherapy is the only possible treatment in patients with good a performance status. The next-generation of TKIs are undergoing promising developments.Key words: EGFR - deletion on exon 19 - mutation T790M - mutation C797S - afatinib - osimertinibSubmitted: 12. 9. 2017Accepted: 12. 10. 2017 This project was supported by grant AZV 17-30 748A. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.

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.

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.

The first European family with gastric adenocarcinoma and proximal polyposis of the stomach: case report and review of the literature.

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.

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.

Change in Serum Lactate Dehydrogenase Is Associated with Outcome of Patients with Advanced-stage NSCLC Treated with Erlotinib.

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.

The Prognostic Role of KRAS Mutation in Patients with Advanced NSCLC Treated with Second- or Third-line Chemotherapy.

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.

[KRAS mutation assay on EUS-FNA specimens from pacients with pancreatic mass]. / Vysetrení mutace KRAS v EUS-FNA preparátech pacientu s tumorem pankreatu.

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

Pemetrexed Versus Erlotinib in the Second-line Treatment of Patients with Advanced-stage Non-squamous NSCLC Harboring Wild-type EGFR Gene.

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.