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.

Capillary electrophoresis, a method for the determination of nucleic acid ligands covalently attached to quantum dots representing a donor of Förster resonance energy transfer.

The synthesis and determination of the structure of a Förster resonance energy transfer probe intended for the detection of specific nucleic acid sequences are described here. The probe is based on the hybridization of oligonucleotide modified quantum dots with a fluorescently labeled nucleic acid sample resulting in changes of the fluorescence emission due to the energy transfer effect. The stoichiometry distribution of oligonucleotides conjugated to quantum dots was determined by capillary electrophoresis separation. The results indicate that one to four molecules of oligonucleotide are conjugated to the surface of a single nanoparticle. This conclusion is confirmed by the course of the dependence of Förster resonance energy transfer efficiency on the concentration of fluorescently labeled complementary single-stranded nucleic acid, showing saturation. While the energy transfer efficiency of the probe hybridized with complementary nucleic acid strands was 30%, negligible efficiency was observed with a noncomplementary strand.

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).

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.

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.

[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.

Variants in miRNA regulating cardiac growth are not a common cause of hypertrophic cardiomyopathy.

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.

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

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

Utility of cell-free tumour DNA for post-surgical follow-up of colorectal cancer patients.

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.

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

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

Multiplicity of EGFR and KRAS mutations in non-small cell lung cancer (NSCLC) patients treated with tyrosine kinase inhibitors.

BACKGROUND: Concurrent presence of EGFR and KRAS mutations in non-small cell lung cancer (NSCLC) patients is relatively rare and their appearance is believed to be mutually exclusive. Tumours harbouring KRAS mutation are perceived as not being capable of response to tyrosine kinase inhibitor (TKI) therapy. PATIENTS AND METHODS: This paper presents 5 case reports of patients with tumours harbouring multiple EGFR and/or KRAS mutations. There were 3 patients with EGFR mutations (2 x exon 19 deletions, 1 x L858R) combined with KRAS mutations (2 x Gly12Asp, 1 x Gly12Val), 1 patient with two EGFR mutations (exon 19 deletion + L858R) and 1 patient with two KRAS mutations (Ala11Pro + Gly12Val). RESULTS: All EGFR(+)/KRAS(+) patients had initially showed positive response to TKI treatment. The EGFR(+)/EGFR(+) patient has exhibited strong rash and good response with the best survival, while the KRAS(+)/KRAS(+) patient did not respond to TKI therapy. CONCLUSION: EGFR(+)/KRAS(+) combination does not necessarily pose a negative prediction. This is probably due to the multiclonal character of the tumour displaying partial response in the EGFR(+) subpopulation.

Denaturing capillary electrophoresis for automated detection of L858R mutation in exon 21 of the epidermal growth factor receptor gene in prediction of the outcome of lung cancer therapy.

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.

Dominance of EGFR and insignificant KRAS mutations in prediction of tyrosine-kinase therapy for NSCLC patients stratified by tumor subtype and smoking status.

BACKGROUND: Therapy by tyrosine kinase inhibitors (TKI) has become inevitable in treatment of advanced NSCLC. Mutations in EGFR and KRAS genes have been identified as the main potential predictive and prognostic factors. Here the clinical implications of EGFR/KRAS mutations in patients from two separate trials treated with gefitinib or erlotinib are analysed. PATIENTS AND METHODS: A total of 360 patients (269 gefitinib and 91 erlotinib) were evaluated. Mutations in EGFR (exon 19 and 21) and KRAS (codons 12 and 13) and their impact on response and survival with respect to tumor subtype and smoking status were assessed. RESULTS: Adenocarcinomas revealed 399 days to progression (TTP) and 548 days overall survival (OS) for EGFR mutated vs. 119 days to progression and 137 days survival for non-mutated, p<0.0001 (TTP) and p=0.0001 (OS). No EGFR effect was recorded for squamous cell tumors. For smoking status, both EGFR-mutated smokers and non-smokers profited from TKI therapy. Smokers: 243 vs. 122 days (mutated vs. non-mutated), p=0.0284 (TTP) and 244 vs. 126 days, p=0.0396 (OS); non-smokers: 390 vs. 71 days, p<0.0001, (TTP) and 548 vs. 135 days, p<0.0001 (OS). KRAS mutation in tumors did not result in a poorer prognosis in the subtype-selected groups, nor did it present as a negative factor in smokers. CONCLUSION: EGFR mutations possess statistical significance for a better therapy response and longer survival in all patients with adenocarcinomas (smokers as well as non-smokers). KRAS does not seem an "a priori" negative factor for TKI-based treatment of NSCLC.