Optimization of BRAF V600 Assay as A 2-Step Real-time PCR Protocol

The QClamp® BRAF Codon Specific Mutation Detection Kit is a real-time PCR assay for the detection of somatic mutations in codon 600 Valine at exon 15 in the BRAF gene which encodes the serine/threonine protein kinase, using purified DNA. The V600E mutation is the most common BRAF gene mutation found in human cancers. This mutation leads to production of a BRAF protein that is abnormally active, which disrupts regulation of cell growth and division. Mutations in this gene have been found in cancers, including non-Hodgkin lymphoma, colorectal cancer, malignant melanoma, papillary thyroid carcinoma, non-small-cell lung carcinoma, gastric cancer, and even prostate cancer. Currently, the established qPCR protocol for the QClamp® BRAF Mutation Detection Assay is comprised of a 4-step procedure: Denaturation, XNA Annealing, Primer Annealing and Extension. The purpose of this experiment was to test the feasibility of optimizing this assay to a more efficient and faster 2-step Real-time PCR which has just the Denaturation and the Primer Annealing/Extension steps. Optimization was attempted on both the ABI-QS5 and LC480 thermocycling instruments using parallel testing. The newly established 2-step thermocycling parameters were successfully tested and validated on the ABI-QS5 instrument. For the LC480, however, the experiment was not successful. This result might be due to the different platforms and technologies of the two instruments. Further research is needed to develop the mutational status scoring and acceptance criteria for clinical samples on the ABI-QS5, and to complete the development of the 2-step qPCR protocol individually on the LC480; and also to study the effects of factors such as temperature, ramp rates, PCR enzymes/master mix, primer/probes and XNA concentrations for both ABI-QS5, and the LC480

BRAF Mutations in Advanced Cancers: Clinical Characteristics and Outcomes

BACKGROUND: Oncogenic BRAF mutations have been found in diverse malignancies and activate RAF/MEK/ERK signaling, a critical pathway of tumorigenesis. We examined the clinical characteristics and outcomes of patients with mutant (mut) BRAF advanced cancer referred to phase 1 clinic. METHODS: We reviewed the records of 80 consecutive patients with mutBRAF advanced malignancies and 149 with wild-type (wt) BRAF (matched by tumor type) referred to the Clinical Center for Targeted Therapy and analyzed their outcome. RESULTS: Of 80 patients with mutBRAF advanced cancer, 56 had melanoma, 10 colorectal, 11 papillary thyroid, 2 ovarian and 1 esophageal cancer. Mutations in codon 600 were found in 77 patients (62, V600E; 13, V600K; 1, V600R; 1, unreported). Multivariate analysis showed less soft tissue (Odds ratio (OR) = 0.39, 95%CI: 0.20-0.77, P = 0.007), lung (OR = 0.38, 95%CI: 0.19-0.73, p = 0.004) and retroperitoneal metastases (OR = 0.34, 95%CI: 0.13-0.86, p = 0.024) and more brain metastases (OR = 2.05, 95%CI: 1.02-4.11, P = 0.043) in patients with mutBRAF versus wtBRAF. Comparing to the corresponding wtBRAF, mutBRAF melanoma patients had insignificant trend to longer median survival from diagnosis (131 vs. 78 months, p = 0.14), while mutBRAF colorectal cancer patients had an insignificant trend to shorter median survival from diagnosis (48 vs. 53 months, p = 0.22). In melanoma, V600K mutations in comparison to other BRAF mutations were associated with more frequent brain (75% vs. 36.3%, p = 0.02) and lung metastases (91.6% vs. 47.7%, p = 0.007), and shorter time from diagnosis to metastasis and to death (19 vs. 53 months, p = 0.046 and 78 vs. 322 months, p = 0.024 respectively). Treatment with RAF/MEK targeting agents (Hazard ratio (HR) = 0.16, 95%CI: 0.03-0.89, p = 0.037) and any decrease in tumor size after referral (HR = 0.07, 95%CI: 0.015-0.35, p = 0.001) correlated with longer survival in mutBRAF patients. CONCLUSIONS: BRAF appears to be a druggable mutation that also defines subgroups of patients with phenotypic overlap, albeit with differences that correlate with histology or site of mutation

A case of metastatic cecal cancer with mutation in the BRAF oncogene and poor survival

　A 79-year-old woman visited a previous hospital with a complaint of general fatigue. The patient was diagnosed with cecal cancer with multiple liver metastases and lymph node metastases on colonoscopy, abdominal ultrasonography and CT scan, and was referred to our division for treatment. Based on the diagnosis of non-curative colonic cancer, we planned to perform systematic chemotherapy after local surgical treatment. We performed an ileocecal resection, and the specimen showed poorly differentiated adenocarcinoma with mutation in the BRAF oncogene. After the surgical treatment, the tumor grew rapidly and the patient died from cancer on the 19th postoperative day without having the opportunity to undergo chemotherapy. 　Multiple genetic and epigenetic alterations in oncogenes and tumor suppressor genes are involved in the process of colorectal carcinogenesis. Some of the alterations have been identified as predictive and prognostic biomarkers. A mutation in the BRAF oncogene was reported to be associated with a very unfavorable prognosis in colorectal cancers. Some of the cases with rapid progression are suggested to have the BRAF oncogene mutation. According to our experience, chemotherapy before surgical treatment might improve the prognosis of cases with the BRAF mutation

PIK3CA Mutations Frequently Coexist with RAS and BRAF Mutations in Patients with Advanced Cancers

Oncogenic mutations of PIK3CA, RAS (KRAS, NRAS), and BRAF have been identified in various malignancies, and activate the PI3K/AKT/mTOR and RAS/RAF/MEK pathways, respectively. Both pathways are critical drivers of tumorigenesis.Tumor tissues from 504 patients with diverse cancers referred to the Clinical Center for Targeted Therapy at MD Anderson Cancer Center starting in October 2008 were analyzed for PIK3CA, RAS (KRAS, NRAS), and BRAF mutations using polymerase chain reaction-based DNA sequencing.PIK3CA mutations were found in 54 (11%) of 504 patients tested; KRAS in 69 (19%) of 367; NRAS in 19 (8%) of 225; and BRAF in 31 (9%) of 361 patients. PIK3CA mutations were most frequent in squamous cervical (5/14, 36%), uterine (7/28, 25%), breast (6/29, 21%), and colorectal cancers (18/105, 17%); KRAS in pancreatic (5/9, 56%), colorectal (49/97, 51%), and uterine cancers (3/20, 15%); NRAS in melanoma (12/40, 30%), and uterine cancer (2/11, 18%); BRAF in melanoma (23/52, 44%), and colorectal cancer (5/88, 6%). Regardless of histology, KRAS mutations were found in 38% of patients with PIK3CA mutations compared to 16% of patients with wild-type (wt)PIK3CA (p = 0.001). In total, RAS (KRAS, NRAS) or BRAF mutations were found in 47% of patients with PIK3CA mutations vs. 24% of patients wtPIK3CA (p = 0.001). PIK3CA mutations were found in 28% of patients with KRAS mutations compared to 10% with wtKRAS (p = 0.001) and in 20% of patients with RAS (KRAS, NRAS) or BRAF mutations compared to 8% with wtRAS (KRAS, NRAS) or wtBRAF (p = 0.001).PIK3CA, RAS (KRAS, NRAS), and BRAF mutations are frequent in diverse tumors. In a wide variety of tumors, PIK3CA mutations coexist with RAS (KRAS, NRAS) and BRAF mutations

Fluorescence virus-guided capturing system of human colorectal circulating tumour cells for non-invasive companion diagnostics

Background Molecular-based companion diagnostic tests are being used with increasing frequency to predict their clinical response to various drugs, particularly for molecularly targeted drugs. However, invasive procedures are typically required to obtain tissues for this analysis. Circulating tumour cells (CTCs) are novel biomarkers that can be used for the prediction of disease progression and are also important surrogate sources of cancer cells. Because current CTC detection strategies mainly depend on epithelial cell-surface markers, the presence of heterogeneous populations of CTCs with epithelial and/or mesenchymal characteristics may pose obstacles to the detection of CTCs. Methods We developed a new approach to capture live CTCs among millions of peripheral blood leukocytes using a green fluorescent protein (GFP)-expressing attenuated adenovirus, in which the telomerase promoter regulates viral replication (OBP-401, TelomeScan). Results Our biological capturing system can image epithelial and mesenchymal tumour cells with telomerase activities as GFP-positive cells. After sorting, direct sequencing or mutation-specific PCR can precisely detect different mutations in KRAS, BRAF and KIT genes in epithelial, mesenchymal or epithelial–mesenchymal transition-induced CTCs, and in clinical blood samples from patients with colorectal cancer. Conclusions This fluorescence virus-guided viable CTC capturing method provides a non-invasive alternative to tissue biopsy or surgical resection of primary tumours for companion diagnostics

Biochemical Properties of a Decoy Oligodeoxynucleotide Inhibitor of STAT3 Transcription Factor.

Cyclic STAT3 decoy (CS3D) is a second-generation, double-stranded oligodeoxynucleotide (ODN) that mimics a genomic response element for signal transducer and activator of transcription 3 (STAT3), an oncogenic transcription factor. CS3D competitively inhibits STAT3 binding to target gene promoters, resulting in decreased expression of proteins that promote cellular proliferation and survival. Previous studies have demonstrated antitumor activity of CS3D in preclinical models of solid tumors. However, prior to entering human clinical trials, the efficiency of generating the CS3D molecule and its stability in biological fluids should be determined. CS3D is synthesized as a single-stranded ODN and must have its free ends ligated to generate the final cyclic form. In this study, we report a ligation efficiency of nearly 95 percent. The ligated CS3D demonstrated a half-life of 7.9 h in human serum, indicating adequate stability for intravenous delivery. These results provide requisite biochemical characterization of CS3D that will inform upcoming clinical trials

Methylation array profiling of adult brain tumours: diagnostic outcomes in a large, single centre

The introduction of the classification of brain tumours based on their DNA methylation profile has significantly changed the diagnostic approach for cases with ambiguous histology, non-informative or contradictory molecular profiles or for entities where methylation profiling provides useful information for patient risk stratification, for example in medulloblastoma and ependymoma. We present our experience that combines a conventional molecular diagnostic approach with the complementary use of a DNA methylation-based classification tool, for adult brain tumours originating from local as well as national referrals. We report the frequency of IDH mutations in a large cohort of nearly 1550 patients, EGFR amplifications in almost 1900 IDH-wildtype glioblastomas, and histone mutations in 70 adult gliomas. We demonstrate how additional methylation-based classification has changed and improved our diagnostic approach. Of the 325 cases referred for methylome testing, 179 (56%) had a calibrated score of 0.84 and higher and were included in the evaluation. In these 179 samples, the diagnosis was changed in 45 (25%), refined in 86 (48%) and confirmed in 44 cases (25%). In addition, the methylation arrays contain copy number information that usefully complements the methylation profile. For example, EGFR amplification which is 95% concordant with our Real-Time PCR-based copy number assays. We propose here a diagnostic algorithm that integrates histology, conventional molecular tests and methylation arrays

Clinico-pathological characteristics of mucin-producing colorectal carcinoma

Colorectal carcinoma (CRC) is one of the most common malignancies worldwide with persistently high mortality. As a consequence, high demands are placed on research into this disease. The aim of this study was to evaluate the epidemiology of colorectal carcinoma in the Czech Republic in comparison with worldwide data. We focused on the evaluation of the latest findings in screening and diagnosis, investigated prognostic factors, and also focused on the therapeutic options of CRC. Furthermore, mucin-producing carcinomas are described in detail and mucin itself was confirmed as a risk factor for the prognosis of CRC patients. The experimental part of the study focused on gene mucin MUC13 and the MUC13 - miR-4647 axis, which is associated with adverse prognosis in CRC patients. Samples of CRC and samples of adjacent non-malignant mucosa tissue were used. The samples were collected from patients who underwent surgery at the General University Hospital in Prague, at the Thomayer University Hospital in Prague and at the University Hospital in Pilsen (Czech Republic). The collection of samples took place between 2011 and 2015. The total of 187 samples were collected from patients with sporadic colorectal cancer. The subjects of the study all provided information on their lifestyle habits, body mass index...Kolorektální karcinom (CRC) patří celosvětově k nejčastějším malignitám s přetrvávající vysokou mortalitou. Z toho důvodu jsou kladeny vysoké nároky na výzkum tohoto onemocnění. Cílem naší práce bylo zhodnocení epidemiologie CRC v České republice v porovnání se světem. Zaměřili jsme se na zhodnocení nejnovějších poznatků ve screeningu, diagnostice, zkoumali jsme prognostické faktory a v neposlední řadě terapeutické možnosti u CRC. Dále jsme podrobněji popsali karcinomy produkující mucin a samotný mucin, jako rizikový faktor pro prognózu pacientů s CRC. V experimentální části práce jsme se zabývali genem mucinu MUC13 a osou MUC13 - miR-4647, které jsou právě spojeny s horší prognózou u pacientů s CRC. V naší práci jsme použili vzorky CRC a okolní tkáně bez karcinomu odebraných od pacientů, kteří podstoupili chirurgický zákrok ve Všeobecné fakultní nemocnici v Praze, ve Fakultní Thomayerově nemocnici v Praze a ve Fakultní nemocnici v Plzni. Odběr proběhl v letech 2011 až 2015. Bylo odebráno 187 vzorků od pacientů se sporadickým kolorektálním karcinomem. Subjekty studie poskytly informace o svých životních návycích, body mass index (BMI), cukrovce a rodinném či osobním výskytu rakoviny v anamnéze. Pacienti s kolorektálním karcinomem s vyšší hladinou exprese genu MUC13 v nádorové tkáni měli horší...Chirurgická klinika 1. LF UK a FTNDepartment of Surgery First Faculty of Medicine Charles University and Thomayer University HospitalFirst Faculty of Medicine1. lékařská fakult

DIVERGENT ROLES OF THE TCF4 AND LEF1 WNT SIGNALING TRANSCRIPTION FACTORS IN COLON CANCER

The canonical WNT signaling pathway is necessary for guiding cell growth during embryonic development. In adults, WNT signaling maintains tissue stem cells and therefore plays an essential role in tissue homeostasis. In the colon, the WNT transcription factor, TCF4, is necessary for maintaining the intestinal stem cells. The initiating event in colon cancer is the aberrant activation of the WNT signaling pathway, which results in constitutive activity of TCF4. To determine how TCF4 influences colon cancer cell behavior, we silenced TCF7L2, the gene encoding TCF4, and used RNA sequencing and Hi-C to measure changes in transcription and nuclear structure in the SW480 colon cancer cell line. Loss of TCF4 resulted in A/B compartment switching, local chromatin reorganization, and a dramatic up-regulation in transcription. However, A/B compartment switching was not associated with changes in gene expression. We also found that loss of TCF4 resulted in the up-regulation of LEF1, another WNT transcription factor. Expressed LEF1 isoforms were found to be transcriptionally competent and over-compensated for WNT signaling activity upon loss of TCF4, suggesting a WNT-intrinsic feedback mechanism. Over-expression of LEF1 altered WNT signaling output to favor the expression of lymphoid genes, as opposed to a TCF4-based transcriptional program. ChIP-seq demonstrated that TCF4 and LEF1 bind distinct target genes, though they synergize to express MYC. TCF4 was found to bind the LEF1 promoter, indicative of direct repression, though LEF1 did not bind the TCF7L2 promoter. The CtBP1 protein, a known binding partner of TCF4, was found to be the most potent repressor of LEF1 expression. This demonstrates that despite the overall activation of WNT signaling in colon cancer, repressive functions of the WNT transcription factors are still intact, and the repression of LEF1 by TCF4 maintains a TCF4-centric transcriptional program in colon cancer cells

Expresión de genes relacionados con la angiogénesis como predictores de respuesta al tratamiento combinado (cirugía y quimioterapia) en el carcinoma de ovario seroso de alto grado

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid. Facultad de Medicina, Departamento de Medicina. Fecha de Lectura: 19-12-2018Ha sido financiado por un proyecto FIS (PI10/630) y por una beca recibida de la Fundación Mutua Madrileñ