Mutations In NRAS Genes Research Articles

Accurate detection of KRAS, NRAS and BRAF mutations in metastatic colorectal cancers by bridged nucleic acid-clamp real-time PCR

BackgroundPatients with metastatic colorectal cancer can benefit from anti-EGFR therapy, such as cetuximab and panitumumab. However, colorectal cancers harboring constitutive activating mutations in KRAS, NRAS and BRAF genes are not responsive to anti-EGFR therapy. To select patients for appropriate treatment, genetic testing of these three genes is routinely performed.MethodsWe applied bridged nucleic acid-clamp real-time PCR (BNA-clamp PCR) to detect somatic hotspot mutations in KRAS, NRAS and BRAF. PCR products from BNA-clamp PCR were subsequently analyzed Sanger sequencing. We then compared results with those from the PCR–reverse sequence-specific oligonucleotide probe (PCR-rSSO) method, which has been used as in vitro diagnostic test in Japan. To validate the mutation status, we also performed next generation sequencing using all samples.ResultsIn 50 formalin-fixed paraffin-embedded tissues, KRAS mutations were detected at frequencies of 50% (25/50) and 52% (26/50) by PCR-rSSO and BNA-clamp PCR with Sanger sequencing, respectively, and NRAS mutations were detected at 12% (6/50) and 12% (6/50) by PCR-rSSO and BNA-clamp PCR with Sanger sequencing, respectively. The concordance rate for detection of KRAS and NRAS mutations between the two was 94% (47/50). However, there were three discordant results. We validated these three discordant and 47 concordant results by next generation sequencing. All mutations identified by BNA-clamp PCR with Sanger sequencing were also identified by next generation sequencing. BNA-clamp PCR detected BRAF mutations in 6% (3/50) of tumor samples.ConclusionsOur results indicate that BNA-clamp PCR with Sanger sequencing detects somatic mutations in KRAS, NRAS and BRAF with high accuracy.

Mutational and Immunophenotypic Profiling of a Series of 8 Tubo-ovarian Carcinosarcomas Revealed a Monoclonal Origin of the Disease.

Carcinosarcomas are rare, highly aggressive neoplasms composed of a combination of carcinomatous and sarcomatous elements. These tumors represent a paradigmatic field for the study of intratumor heterogeneity. A series of 8 tubo-ovarian carcinosarcomas was characterized for the following: (i) immunohistochemical expression of MNF116, epithelial membrane antigen, vimentin, S100, chromogranin, synaptophysin, desmin, myogenin (MYF4), and p53; (ii) mutational profiling of KRAS, BRAF, PIK3CA, NRAS, TP53, and DICER1 genes. Heterologous differentiation was present in 6 of 8 tumors. Cytokeratin MNF116 and epithelial membrane antigen were positive in all the carcinomatous components and in 87.5% and 50.0% of the sarcomatous components, respectively. The sarcomatous components showed positive staining for vimentin in all cases. Two cases demonstrated positivity for neuroendocrine markers in their carcinomatous components. All rhabdomyosarcomas were positive for desmin and MYF-4. Chondrosarcomas were positive for S100. All but one tumor showed similar p53 immunoreactivity in both the carcinomatous and sarcomatous components, and one case showed cytoplasmic p53 expression. Three of 8 cases (37.5%) showed TP53 mutations, and, in 2 cases, the TP53 mutation was shared by both epithelial and mesenchymal components. DICER1 mutation was found in all components of one case. Mutations in KRAS, NRAS, BRAF, and PIK3CA genes were not found in the study cohort. Our results highlight the heterogeneity of ovarian carcinosarcomas at the phenotypic level. A common mutational signature was observed in both components in 3 of 4 informative tumors. More studies are required to dissect different levels of ovarian carcinosarcomas' heterogeneity in order to define the best therapeutic approaches to these aggressive neoplasms.

The combination of mutations in BRAF and NRAS genes within a one tumor in patients with cutaneous melanoma

The paper provides a clinical description and analysis of two melanoma cases with mutations in the BRAF and NRAS gene within one tumor. Until recently, it was believed that mutations in the BRAF and NRAS genes are exclusive and do not occur within the same tumor. Later it was revealed that these mutations can exist within one tumor, but this is still quite a rare phenomenon. Molecular genetic analysis of the tumor obtained from both patients showed a mutation in the 15 exon of the BRAF gene c.1799T>A (V600E) and exon 2 of the NRAS p.G13S gene (p.37 G>A). The expression level of non-mutant NRAS protein was different in both cases. In conclusion, so in one patient, despite the presence of a mutation in the NRAS gene, the level of expression of the non-mutant protein was absent, which can be related to possible posttranscriptional disorders in the regulation of gene expression.

Clinical Practice Use of Liquid Biopsy to Identify RAS/BRAF Mutations in Patients with Metastatic Colorectal Cancer (mCRC): A Single Institution Experience.

Tumor heterogeneity represents a possible cause of error in detecting predictive genetic alterations on tumor tissue and can be overcome by testing alterations in circulating tumor DNA (ctDNA) using liquid biopsy. We assessed 72 consecutive patients with a diagnosis of metastatic colorectal cancer (mCRC) using Idylla™ Biocartis, a fully automated platform that evaluates the most frequent mutations of KRAS, NRAS and BRAF genes. We correlated the results of liquid biopsy and standard tissue-based next generation sequencing (NGS) analyses to patient clinical features. The overall agreement was 81.94%. Concordance was 85.71% and 96.15% in treatment-naïve patients and in the patient subgroup with liver metastases, respectively. In liver metastases positive, treatment-naïve patients, sensitivity, specificity and positive predictive value (PPV) were 92.31%, 100% and 100%, respectively. Circulating mutational fraction (CMF) was significantly higher in patients with liver metastases and high carcinoembryonic antigen (CEA) levels. In a subgroup of patients pre-treated with anti-Epidermal Growth Factor Receptor (EGFR) agents, emerging KRAS mutations were evidenced in 33% of cases. Testing RAS/BRAF mutations on plasma using the Idylla™ Biocartis platform is feasible and reliable in mCRC patients in clinical practice.

Diffuse leptomeningeal enhancement in a patient with rapidly progressive dementia: Answer

Diffuse leptomeningeal enhancement in a patient with rapidly progressive dementia: Answer

Detection of NRAS G12D and NRAS G13C mutant genes among apparently healthy and haematologic malignant individuals in Federal Capital Territory, Nigeria

ABSTRACTRat Sarcoma gene mutations is an important aspect in the management of hematologic malignancies globally. Unfortunately, this is not the trend in West Africa, including Nigeria. This study was aimed at detecting NRAS G12D and NRAS G13C mutant genes among apparently healthy and haematologic malignant individuals, and to explore their association with some clinical and demographic factors as well as disease status and progression. A total of 200 cfDNAs, 100 each from haematologic malignant patients and blood donors, respectively, were analyzed for the presence of NRAS gene mutations in codons 12 and 13. These mutations were tested using multiplex allele-specific PCR (AS-PCR). The mutations were detected by selective amplification using mutation-specific synthetic oligonucleotides. NRAS G12D and NRAS G13C mutations were 20.0% and 10.0%, respectively. In 17.5% of the 100 haemapoietic cancer patients, NRAS G12D mutant genes were seen while 7.5% of NRAS G13C mutation was found. Both mutant genes were observed in five healthy blood donors each. This result confirms the existence of NRAS mutations in Nigerian haemapoietic cancer patients and the preponderance of G-A transitions over G-T transversions. Mutant NRAS genes were associated with the types and stages of cancer, highlighting probable connection between mutation and increased susceptibility as well as quick progression of hematologic malignancies in the population studied. The result also highlighted higher risk of susceptibility/progression associated with leukemia than other hematopoietic cancers. We recommend more studies on NRAS mutation specifically targeted at improved diagnosis and prognostic therapy. The role of RAS mutation should be explored in other aside blood cancers in the Nigerian population.

Relevance and clinicopathologic relationship of BRAF V600E, TERT and NRAS mutations for papillary thyroid carcinoma patients in Northwest China

BackgroundTo determine the relevance of the single or combination mutations of BRAF V600E, TERT, and NRAS genes and the clinicopathologic relationship in papillary thyroid cancer (PTC).MethodsPatients with PTC were enrolled into the study between February 2018 and April 2019. Based on the number of mutant genes, we classified the participants into single BRAF V600E mutation group, double mutations group and no mutation group. Single factor and multiple logistic regression analyses were applied to explore the independent factors. Review Manager 5.3 was used for meta-analysis to review the clinical efficacy of gene co-mutations.ResultsFinally, 483 patients were enrolled into the study and 419 (86.7%) of them harbored BRAF V600E mutation. TERT or NRAS mutation was likely to coexist with BRAF V600E mutation in PTC. BRAF V600E and NRAS promoter co-mutations was identified in 6 patients, with a prevalence of 1.2%. Prevalence of BRAF V600E and TERT coexistence in PTC was 2.1%. Significant differences were found among age, pathology, multifocality, bilateral lesions, lymph node metastasis, and 131I radiotherapy, P < 0.01. Multiple logistic regression analyses demonstrated that age [odds ratio (OR) = 1.044, 95% confidence interval (CI) = 1.013–1.076; P = 0.006], lymph node metastasis [OR = 0.094, 95% CI = 0.034–0.264; P < 0.001], 131I radiotherapy [OR = 7.628, 95% CI = 2.721–21.378; P < 0.001] were risk factors for BRAF V600E mutation. Besides, age [OR = 1.135, 95% CI = 1.069–1.205; P < 0.001], multiple leisions [OR = 4.128, 95% CI = 1.026–16.614; P = 0.046], pathology [OR = 3.954, 95% CI = 1.235–12.654; P = 0.021] were independent factors for combination mutations. Meta-analysis showed significant association of BRAF V600E+/TERT+ co-mutations with lymph node metastasis, multifocality, distant metastasis, tumor recurrence, extrathyroidal extension, and dead of disease.ConclusionsPrevalence of BRAF V600E mutation in Northwest China was higher than other areas. Age, multiple lesions, and pathology were independent factors for double mutation of BRAF V600E/TERT or BRAF V600E/NRAS. Coexistence of BRAF V600E and TERT promoter mutations was significantly correlated with poor outcome.

Frequency of mutations in BRAF, NRAS, and KIT in different populations and histological subtypes of melanoma: a systemic review.

The presence of mutations of BRAF, NRAS, and KIT genes is recognized as playing a role during carcinogenesis. Our study aims to evaluate and review other studies that present the frequency of mutations of BRAF, NRAS, and KIT genes for different populations, and analyse correlation to their clinical-pathological characteristics and to the demographics of melanoma. Thirty-two articles were selected from a collection of published literature studying 6299 patients. The parameters for correlation to different variables were calculated by odds ratio, for random and single effects. 38.5% of patients present BRAF gene mutations, 16.4% in NRAS, and 10% in KIT. Mutations of the BRAF gene were correlated to superficial spreading melanoma (odds ratio = 1.31), localization in the torso (odds ratio = 1.42) and presence of metastases. Mutations in NRAS were correlated to nodular melanoma (odds ratio = 1.57), localized in the limbs (odds ratio = 1.31). Mutations of the KIT gene were correlated to mucosal melanoma (odds ratio = 1.59). Populations in Brazil, the US, Sweden, Italian, and Australia were found to be correlated to mutations of BRAF and melanoma. Populations in Italy, Sweden, Spain, and the US were found to be correlated to mutations of NRAS. Populations in Japan, China, Turkey, Canada, and Russia were found to be correlated to mutations of KIT. Data correlated to the presence of melanoma and population type is due to the amount of studies performed across of globe.

PF247 CONSEQUENCES OF NRAS MUTATIONS IN PATIENTS WITH ACUTE MYELOID LEUKEMIA

Background:Acute myeloid leukemia (AML) is a malignancy with a high prevalence of NRAS mutations. Transforming point mutations, located dominantly at hotspot codons 12, 13 and 61 of NRAS gene, occur in 11 to 22% of patients. Mutations in NRAS gene stimulate signalling activity of RAS pathway, resulting in an increased cellular proliferation and reduced apoptosis. Prognostic significance of NRAS gene mutations in AML patients remains controversial.Aims:The aim of our work was to analyze the association of NRAS gene mutations in AML with patient characteristics and outcomes.Methods:A total of 297 consecutive AML (non‐acute promyelocyte leukemia) patients (median age 53 years; range 19–70) with curative therapy were analyzed by next generation sequencing (NGS) using ClearSeq AML panel (Agilent Technologies) on MiSeq and NextSeq (Illumina). The NRAS mutations detected by NGS with variant allele frequency (VAF) ≥1.0% were verified by NRAS StripAssay (ViennaLab). Only mutations scored as positive by both methods were included in further analyses. Overall survival (OS) was analyzed by log‐rank (Mantel‐Cox) test. The comparative statistical analyses were evaluated by t‐test and Fisher's exact test.Results:In total, 84 mutations were identified in 66/297 (22.2%) analyzed AML patients. Fifteen patients (22.7%) were carriers of multiple gene mutations (range 1–3 mutations per patient). The mutations were most frequently found in the codon G12 (44/84 mutations, 52.4%), the most frequent gene mutations were: G12D (26/84; 31.0% mutations), G13D (15/84; 17.9%) and G12S (14/84; 16.7%). Furthermore, rare mutation G60E was identified in one patient. The median VAF of NRAS mutations was 7.0% (range 1.0–53.4%). Frequency of co‐mutation with FLT3‐TKD reached statistical significance (p = 0.025) and a trend for co‐mutation with NPM1 (p = 0.082) and DNMT3A (p = 0.091). A trend for mutual exclusivity was observed for FLT3‐ITD mutations with allelic ratio above 0.5 (p = 0.063). Occurrence of NRAS mutations was associated with younger age (median 48 vs. 54 years, respectively, p = 0.012), and a trend towards higher white blood cell count (median 41.0 vs. 14.2, respectively, p = 0.069). There was no association with OS, blast percentage in BM or sex. The presence of multiple NRAS mutations was not associated with analyzed characteristics nor patient outcomes.Summary/Conclusion:Our study showed NRAS gene mutations detected by two independent methods in more than one fifth of AML patients with curative therapy. Most of them have low VAF, therefore suggesting their later occurrence during leukemogenesis and limited proliferative potential. Although approximately 5% of AML patients harbor multiple NRAS mutations, we were not able to identify the difference between patients with one or more NRAS mutations.Supported by Ministry of Health of the Czech Republic, grant nr. 15–25809A, project FNBr. 65269705 and project MUNI/A/1105/2018. All rights reserved.

PF265 GENE MUTATION PROFILE IN PATIENTS WITH ACUTE PROMYELOCYTIC LEUKEMIA (APL)

Background:Though survival rates have improved in majority of APL patients, some high‐risk or intermediate risk patients still encounter early mortality. The underlying genetic mechanism between the different risk groups remains incompletely understood, therefore bringing necessity to explore.Aims:The aim of this study was to identify mutations in a panel of 114 leukemia‐associated genes in newly diagnosed APL patients and to analyze the correlation between gene mutations and APL patients of different risk groups.Methods:Forty‐six newly diagnosed APL patients were enrolled between October 2015 and December 2018. Mutational assessment of 114 genes was performed using next‐generation sequencing approach. Induction treatment started with all‐trans retinoic acid (ATRA) and arsenic trioxide based double induction. Anthracycline was added when peripheral white blood cell (WBC) ≥10X109/L. Statistical comparisons were made between gene mutations and APL patients of different risk groups (high risk‐ HR, intermediate risk‐ IR and low risk‐ LR).Correlations between gene mutations and patients suffered from early death (ED, defined as patients who died during induction treatment) and patients reached complete remission (CR) were further investigated.Results:Of the 46 APL patients, there were 26.09% (12/46), 54.35% (25/46) and 19.57% (9/46) were HR, IR and LR patients, respectively. Six patients (13.04%, including 3 HR and 3 IR patients) died during induction treatment, while all of the other 40 patients achieved CR by the end of induction treatment. One or more mutations were detected in 91.30% of the patients (42/46). A total of 102 mutations in 52 genes were detected with the median number of gene mutations was 2 (range 0–6) and mutation rate ≥10%. Though it seemed that HR patients had the highest frequency of mutations (median number 3, range 1–5), LR patients had the lowest frequency of mutations (median number 1, range 0–4), and the median mutation number in IR patients was 2 (range 0–6), there were no significant difference between the three risk groups regarding the median gene mutation numbers (P&gt;0.05).There were 5 frequent mutations identified, including FLT3 (36.96%, 17/46), WT1 (15.22%, 7/46), FAT1 (13.04%, 6/46), ARIDIA (13.04%, 6/46), SETBP1 (8.70%, 4/46), etc. A total of 13 mutations in 12 genes including FLT3, WT1, NRAS, KRAS and NOTCH1 etc were detected in 6 ED patients. The median number of gene mutations in ED patients was 2 (range 1–4), which did not show difference with that in non‐ED patients (median number 2, range 0–6). Among these mutations, only NRAS gene mutations were detected in two ED patients (1 HR and 1 IR patients), which was the same mutation NRAS G12C. FLT3 gene mutations (including FLT3‐ITD and FLT3‐TKD) were the most frequently found in HR APL patients. 88.33% of HR patients(10/12) harbored FLT3 mutations, while 20% of IR patients (5/25) and 22.22% of LR patients (2/9) were found harbored FLT3 mutations(P &lt; 0.05). Among the 17 cases of FLT3 mutant patients, only one HR patient (5.88%, 1/17), who carried FLT3‐TKD D835Y mutation, died 3 days after induction treatment. Meanwhile, the less common WT1 gene mutations were all found in IR APL patients.Summary/Conclusion:HR APL patients had the highest frequency of gene mutations, however there were no significant difference between the three risk groups regarding the median mutation numbers. ED patients did not have more gene mutations compared with non‐ED patients. FLT3 gene mutations were the most frequently occurred mutations, especially in HR APL patients, though might have no relation with early mortality.

PF544 THE DIFFERENT MUTATIONAL LANDSCAPES OF A PANEL GENES BY NEXT GENERATION SEQUENCING IN 967 CASES OF MDS AND AML

Background: Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are myeloid disease with dysplasia characterized by multiple gene mutations in most cases. With advances in Next-generation sequencing techniques, the molecular landscapes in MDS or AML have been reported with further understanding the disease pathogenesis. As well known, MDS may end up in evolving into AML in most cases, especially the subtypes of MDS with excessive blasts. The driver mutation in MDS developing to AML is still open to study. The comparison of mutational landscapes between MDS and AML will provide evidence to better understand the underlying molecular mechanisms of the myeloid dysplasia evolution. Aims: To analyze the different mutational status of 22 genes commonly observed in MDS and AML. Methods: The bone marrow samples were collected from patients with AML (749 cases) and MDS (218 cases) with written consent from June 2017 to January 2019. Next generation sequencing of a panel of 22 genes was used to evaluate the mutations in MDS/AML patients. Results: 719/ 967(74.4%) cases with MDS/AML were detected at least one gene mutation. There were 274 gene mutations in 142/ 218 (65.1%) cases with MDS and 1224 gene mutations in 577/749(77.0%) cases with AML. Both the median mutations and the frequency of gene mutation between MDS and AML were significantly difference (p < 0.05). Compared to MDS, the mutation rate of CEBPA (11.10% vs 1.40%, p < 0.01), NRAS (14.00% vs 6.40%, p < 0.01), KIT (7.30% vs 0.00%,p < 0.01), NPM1 (17.80% vs 5.00%, p < 0.01), FLT3-ITD (15.40% vs 0.90%, p < 0.01), DNMT3A (10.90% vs 4.10%, p < 0.01), IDH2 (9.5% vs 2.8%, p < 0.01), and IDH1 (6.00% vs 1.40%, p < 0.01) genes were significantly higher in AML. While the mutation rate of ASXL1 (5.30% vs 14.7%, p < 0.01), U2AF1 (5.90% vs 22.00%, p < 0.01), RUNX1 (6.30% vs 10.60%, p < 0.05), SRSF2 (2.30% vs 6.00%, p < 0.05), EZH2 (0.50% vs 3.20%, p < 0.01), SETBP1 (0.70% vs 5.00%, p < 0.01), SF3B1 (2.50% vs 7.80%, p < 0.01), and CBL (0.80% vs 3.20%, p < 0.05) genes were significantly higher in MDS. There were no significant difference of the incidence in TET2, FLT3-TKD, JAK2, ETV6, TP53, PHF6 and ZRSR2 gene mutations. Considering the gene mutation increasing with age, the cases were divided into the young (age < 60 years old) and the old (age≥60 years old). In the young cases, the gene mutation profile was similar to the cohort with incidences of CEBPA, NRAS, KIT, NPM1, FLT3-ITD, DNMT3A, IDH1, plus TET2 gene mutations were significantly higher in AML (p < 0.05). The incidences of ASXL1, U2AF1, TP53, EZH2, SETBP1, and CBL gene mutations were significantly higher in MDS (p < 0.05), and the incidences of FLT3-TKD, JAK2, RUNX1, IDH2, ETV6, PHF6, SRSF2, SF3B1 and ZRSR2 gene mutations were not significantly different between AML and MDS cases (p>0.05). The old cohort showed a different gene mutation profile as compared to the young cohort with the incidences of CEBPA, NPM1, FLT3-ITD, and IDH2 gene mutations were significantly higher in AML (p < 0.01), and the incidences of U2AF1 and SF3B1 gene mutations were significantly higher in MDS. There were no significant differences of all the other gene mutations between AML and MDS cases in the old cohort. Summary/Conclusion: There are typical gene mutation patterns in MDS and AML cases. Some genes mutate in MDS and AML cases without differences and others mutate mainly in AML or MDS. The roles and significances of these gene mutation profiles warranted further investigations to understand the pathogenesis underlying MDS/AML.

Patterns of gene mutations in bile duct cancers: is it time to overcome the anatomical classification?

BackgroundTwo recent studies based on multi-omics data analysis identified distinct subtypes of bile-duct cancers (BDC) with important implications in terms of disease classification and patients' treatment. MethodsPatients with mutations in KRAS, NRAS, TP53, and ARID1A genes were classified in KRAS/TP53 group while patients with mutations in IDH1-2, BAP1, and PBRM1 were classified in IDH1-2/BAP1/PBRM1 group. The aim of this study was to define long-term outcomes among patients stratified by patterns of genes mutated. ResultsAmong 105 patients who underwent surgical resection for BDCs, 71 (68%) patients were classified in two groups based on patterns of genes mutated. While in IDH1-2/BAP1/PBRM1 group there were 58%, 22%, and 10% of patients with intrahepatic-cholangiocarcinoma (ICC), perihilar-cholangiocarcinoma (PHCC), and gallbladder cancer (GBC), in KRAS/TP53 group there were 42%, 78%, and 90% of patients with ICC, PHCC, and GBC (p = 0.003), respectively. Patients in IDH1-2/BAP1/PBRM1 group had a 5-year OS of 40% compared with 13% for KRAS/TP53 group (p = 0.032). In a multivariable model adjusted for margins, lymph-node status, microvascular invasion, and tumor grade, patients in KRAS/TP53 group had a 2.1-fold increased risk of death compared with patients in IDH1-2/BAP1/PBRM1 group (p = 0.028). ConclusionsGenetic data were able to overcome the clinical based staging system in predicting patients' prognosis.

Combined detection of KRAS, NRAS, BRAF and PIK3CA mutations in the plasma and tumor tissues of colorectal cancer patients

Objective: To analyze the concordance of KRAS, NRAS, BRAF and PIK3CA gene mutations detected in plasma and matched tumor tissues in colorectal cancer patients, in order to provide good evidences to support plasma could be a potential surrogate of tumor tissue for gene mutation test. Methods: One hundred and seventy-five cases of colorectal cancer were collected at the First Hospital of Jilin University, from October 2016 to October 2017.There were 101 males and 74 females, their ages ranged from 28 to 85 years,with median age of 59 years. The KRAS, NRAS, BRAF and PIK3CA gene mutations in the plasma and paired tumor specimens of all patients were detected by next generation sequencing. Results: The results of tissue samples test were gold standard. Comparison of the four genes showed that concordance rates between plasma and tissue samples were 81.1%(Kappa=0.543), 99.4%(Kappa=0.886), 99.4% (Kappa=0.886) and 97.7%(Kappa=0.714) respectively for KRAS, NRAS, BRAF and PIK3CA. The plasma detection rates of these genes were related to tumor stage(P=0.001), but not to gender(P=0.468) and age(P=1.000) of patients. Conclusions: The study shows a high concordance of KRAS, NRAS, BRAF and PIK3CA gene mutations in plasma against mutation status in tumor tissue. In colorectal cancer, tumor tissue remains the best specimen for gene detection. However, patients from tumor tissue specimens cannot be obtained, especially those with advanced metastases, plasma can be used instead of tissue to detect the mutation status of KRAS, NRAS, BRAF and PIK3CA to guide targeted therapy.

Prevalence of ras/mapk pathway mutation (KRAS, NRAS, and BRAF) in plasma cell myeloma at a single institute in Korea

Background & Aim Multiple myeloma (MM) is a hematologic malignancy characterized by neoplastic proliferation of clonal plasma cells in the bone marrow and its pathogenesis is not fully understood. Recent genomic studies have shown that the RAS/MAPK pathway (NRAS, KRAS, BRAF, FGFR3) is the most frequently mutated pathway in patients with MM, 40% to 55% of newly diagnosed patients with MM have driver mutations in NRAS or KRAS. About of patients with MM harbored the BRAF mutation at diagnosis, mostly on amino acid V600, increasing to 18% at relapse. To understand the prevalance the somatic mutation of the RAS/MAPK pathway (NRAS, KRAS, and BRAF) in MM, we performed the mutation analysis from 50 patients with MM. Methods, Results & Conclusion All bone marrow samples were from initial diagnostic specimens, evaluated by a hematologist at St Mary`s hospital, Seoul, between 2009 and 2012. The clinical and demographic characteristics of the 50 MM patients are summarized in Table 1. BRAF V600E was screened by allele-specific real-time PCR (AS-PCR). Using pyrosequencing, bone marrow were retrospectively analysed for RAS status that is, for mutations within KRAS and NRAS mutation. RAS mutations were detcted in 8(16.0%) of 50 patients, including 4 patients with KRAS mutation (2 patients; G12V, 2 patients; G13D), 4 patients with NRAS mutation (Q61R). Among 4 patients with KRAS mutaion, 1 patient had t(8;14) genetic aberrattion. Among 4 patients with NRAS Q61R mutaion, 3 patients had hyperdiploidy. The point mutations of NRAS and KRAS genes occurs at a rate of 20% of cases of MM. BRAF V600E mutation was detected in 8 (16.0%) of 50 MM patients. In these patients, 4 patients had IgA monoclonal protein and 3 patients had light chain (kappa) monoclonal protein, and 1 patient had IgG(kappa) monoclonal protein. In these patients 3 patients (37.5%, 3/8) had 1q gain, 3 patietns (37.5%, 3/8) had t(11;14), 1 patient (12.5%, 1/8) had t(8;14), and 1 patient (12.5%, 1/8) had add (14)(q32). Compared with BRAF wild type, those carrying the BRAF V600E mutation had higher frequency of IgA, and patients with mutated the BRAF V600E genotype had higher level of calcium (P = 0.0154) and beta-2 microglobulin (P=0.0491) (Table 2). BRAF and RAS mutation screening is important in the molecular diagnostics of MM and it provide a rationale for clinical development of targeted treatment in MM.

Clinical impact of testing for mutations and microRNAs in thyroid nodules.

BackgroundWe report results of a multicenter clinical experience study examining the likelihood of patients with indeterminate thyroid nodules to undergo surgery or have malignant outcome based on multiplatform combination mutation and microRNA testing (MPT).MethodsMPT assessed mutations in BRAF, HRAS, KRAS, NRAS, and PIK3CA genes, PAX8/PPARγ, RET/PTC1, and RET/PTC3 gene rearrangements, and the expression of 10 microRNAs. Baseline clinical information at the time of MPT and clinical follow‐up records were reviewed for 337 patients, of which 80% had negative MPT results. Kaplan Meier analysis for cumulative probability of survival without having a surgical procedure or malignant diagnosis over the course of patient follow‐up was determined for MPT results of 180 patients, among which only 14% had malignancy.ResultsA negative MPT result in nodules with Bethesda III or IV cytology (2009) conferred a high probability of non‐surgical treatment, with only 11% expected to undergo surgery and a high probability of survival without malignancy (92%) for up to 2 years follow up. A positive MPT result conferred a 57% probability of malignancy and was an independent risk factor for undergoing surgical treatment (Hazard Ratio [HR] 9.2, 95% confidence intervals 5.4‐15.9, P < .0001) and for malignancy (HR 13.4, 95% confidence intervals 4.8‐37.2, P < .0001). For nodules with weak driver mutations, positive microRNA test results supported high risk of cancer while negative results downgraded cancer risk.ConclusionMPT results are predictive of real‐world decisions to surgically treat indeterminate thyroid nodules, with those decisions being appropriately aligned with a patient's risk of malignancy over time.

A large collection of integrated genomically characterized patient-derived xenografts highlighting the heterogeneity of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) represents 10% of all breast cancers and is a very heterogeneous disease. Globally, women with TNBC have a poor prognosis, and the development of effective targeted therapies remains a real challenge. Patient-derived xenografts (PDX) are clinically relevant models that have emerged as important tools for the analysis of drug activity and predictive biomarker discovery. The purpose of this work was to analyze the molecular heterogeneity of a large panel of TNBC PDX (n = 61) in order to test targeted therapies and identify biomarkers of response. At the gene expression level, TNBC PDX represent all of the various TNBC subtypes identified by the Lehmann classification except for immunomodulatory subtype, which is underrepresented in PDX. NGS and copy number data showed a similar diversity of significantly mutated gene and somatic copy number alteration in PDX and the Cancer Genome Atlas TNBC patients. The genes most commonly altered were TP53 and oncogenes and tumor suppressors of the PI3K/AKT/mTOR and MAPK pathways. PDX showed similar morphology and immunohistochemistry markers to those of the original tumors. Efficacy experiments with PI3K and MAPK inhibitor monotherapy or combination therapy showed an antitumor activity in PDX carrying genomic mutations of PIK3CA and NRAS genes. TNBC PDX reproduce the molecular heterogeneity of TNBC patients. This large collection of PDX is a clinically relevant platform for drug testing, biomarker discovery and translational research.

NPM1 High Mutant Allele Burden is an Adverse Prognostic Factor for AML Patients with Mutated NPM1

To investigate the clinical features, accompanying gene mutation characteristics and prognostic factors of adult patients with acute myeloid leukemia with mutated NPM1 (NPM1+AML). Seventy-three patients with newly diagnosed adult NPM1+AML were selected. The mutations of 22 genes were detected by second generation sequencing and 43 fusion genes of AML were detected by real-time fluorescent quantitative PCR. The Kaplan-Meier survival curve and Cox multivariate regression analysis were used to study the prognostic factors. A total of 74 NPM1 site mutations were detected in 73 patients with NPM1+AML. The incidence rates were 92.0% L287fs, 2.7% Q289fs and W288fs, 1.4% L258fs and Q289H, among which 1 patient had 2 NPM1 mutations; the different mutation sites had no effect on the prognosis of NPM1+AML. The median value of NPM1 variant allele frequency (VAF) was 35.4% (1.8%-56.6%). Based on the uppermost quartile of 38.4%, the patients were classified as NPM1 VAF>38.4% (NPM1highAML) and NPM1 VAF≤38.4% (NPM1lowAML). Compared with NPM1lowAML, the early mortality rate was statistically significantly higher (33.3% vs 7.3%, P<0.05), and median EFS (148 d，95%CI 58-238 d vs 372 d，95%CI 264-480 d) (P<0.01) and median OS (179 d 95%CI 6-352 d vs 444 d) (P<0.01) were significantly shorter in NPM1high AML. A total of 126 accompanying gene mutation sites were detected in 87.7% of patients with NPM1+AML. The patients with NRAS gene mutation displayed a higher rate of complete remission (100% vs 58%) (P<0.05) and longer median OS (not reached to 320 d, 95%CI 150-490 d) (P<0.05). The 43 fusion genes were examined in 65 out of 73 cases of NPM1+AML, and in all the patients the fusion gene test was negative. Multivariate analysis showed that NPM1 VAF>38.4% was an independent prognostic factor for EFS (HR=3.1, 95% CI 1.6-6.4, P<0.01) and OS (HR=3.0, 95% CI 1.4-6.2, P<0.01). The NPM1 gene mutation in AML patients often is accompanied by other gene mutations, while the coexistence of fusion genes is rare; high NPM1 mutant allele burden is an independent prognostic factor for adult AML patients with mutated NPM1.

КЛИНИЧЕСКИЕ ОСОБЕННОСТИ ТЕЧЕНИЯ КОЛОТЕРАЛЬНОГО РАКА У ПАЦИЕНТОВ С МУТАЦИЯМИ EGFR-СИГНАЛЬНОГО ПУТИ

KRAS, NRAS, BRAF mutations are associated with an unfavorable prognosis for colorectal cancer (CRC). At the same time, there is no single point of view on disease development during adjuvant chemotherapy (ACT). Objective. The authors aimed at studying KRAS, NRAS and BRAF mutations in the tumor and their influence on the clinical characteristics of colorectal cancer development. Materials and Methods. Paraffin blocks of the primary CRC tumor (n=37) were used as the material for the study. Using genomic DNA, isolated from the primary tumor, real-time PCR was used to determine the most common mutations in CRC: KRAS gene (exon 2, codon region 12–13), NRAS gene (exon 3, codon region 61), B6F V600E gene. Results. The results of genotyping of DNA samples isolated from the primary CRC tumor paraffin blocks showed that BRAF gene mutations were detected in 8.2 % of cases, NRAS gene mutations were detected in 5.4 % of cases, and KRAS gene mutations were detected in 37.8 % of cases. The authors didn’t reveal any dependencies of the mutation distribution on patients’ gender and age. The examined mutations were more common in adenocarcinomas of high and moderate degrees of differentiation. The relapse-free period after ACT in patients with identified KRAS, NRAS, BRAF gene mutations is significantly less than in those without mutations. Conclusion. The findings suggest that EGFR signaling pathway mutations (KRAS, NRAS and BRAF) increase the risk of disease recurrence and are an unfavorable prognostic factor. Keywords: colorectal cancer, NRAS, KRAS, BRAF mutations, adjuvant chemotherapy.

Molecular biomarkers and histological parameters impact on survival and response to first- line systemic therapy of metastatic colorectal cancer patients.

BackgroundHistological parameters of primary tumour and nodal metastases are prognostic factors for survival of operable colorectal (CRC) patients, but not predictive for response rate of systemic therapy. KRAS mutations in codons 12 and 13 were first recognized as a predictive factor for resistance to anti-EGFR monoclonal antibodies. Not all patients with wild-type KRAS (wtKRAS) respond to anti-EGFR antibody treatment. Additional mechanisms of resistance may activate mutations of the other main EGFR effectors pathway, such as other mutations in RAS gene, mutations in P13K and PTEN expression.Patients and methodsIn the prospective study prognostic and predictive impact of histological parameters of primary tumour, KRAS and BRAF mutations on overall survival (OS) and objective response (OR) rate of metastatic CRC (mCRC) patients treated with 1st line systemic therapy were analysed. We additionally retrospectively analysed other mutations in RAS genes and their impact on survival and time to progression.ResultsFrom November 2010 to December 2012, we enrolled 154 patients in the study, 95 men and 59 women. Mutations in KRAS gene and V600E BRAF gene were found in 42% and in 3% of patients, respectively. Median OS of the patients with T1, T2 and T3 tumour was 65.4 months (95% CI, 55.7–75.6) while in patients with T4 tumour, lymphangiosis, vascular and perineural invasion it has not been reached yet. Median OS of the patients with G1, G2 and G3 of tumour differentiation was 65.6 (95% CI, 53.7–77.5) and 25.3 months (95% CI, 16.6–34.1), respectively. Median OS of the patients with stage N0, N1 and N2 was 65.6 (95% CI, 56.4–74.8) and 58.0 months (95% CI, 21.9–94.2), respectively. Median OS of wtKRAS and mutated KRAS patients was 56.5 (95% CI, 48.2–64.9) and 58 months (95% CI, 52.6–63.4), respectively. Median OS of mutated codon 12 and codon 13 patients was 57 (95% CI, 50.9–64.4) and 44 months (95% CI, 40.1–48.4), respectively. Median OS of wtBRAF and of mutated BRAF patients was 59.2 (95% CI, 52.5–65.9) and 27.6 months (95% CI, 12.6–42.5), respectively. wtKRAS significantly affected the response to the first systemic therapy (p = 0.028), while other parameters did not affected it, p= 0.07. In 14 patients (17%), additional mutations in NRAS gene, codon 61 and codon 146 were found. Median OS of wtNRAS, codon 61 and 146 patients was 67.1 months (50.3–67.6) while median OS of mutated NRAS patients has not been reached yet (p = 0.072). Median time to progression of wtNRAS, codon 61 and 146 patients was 11.7 months (10.4–14.5) while median time to progression of mutated NRAS patients was 7.9 months (6.1–11.0), (p = 0.025).ConclusionsMutated BRAF, N2 and G3 of primary tumour were poor prognostic factors for OS in mCRC patients. wtKRAS significantly affected the response to the first line systemic therapy. Histological parameters included in the analysis and mutated BRAF did not affect significantly the efficacy of 1st line systemic therapy in mCRC patients.

Phenotypic plasticity of metastatic melanoma

Introduction. Metastatic melanoma is characterized by clinical and morphological heterogeneity and plasticity. Rare cases of metastatic melanoma are known, that have no visual expression of melanocytic markers. Contradictory histology of such melanomas requires a differential diagnosis from morphologically similar non- melanocytic tumors, sarcoma or lymphoma. objective is to describe the extraordinary cases of metastatic melanomas with low expression of differentiation markers. material and methods. 15 melanoma cases with unusual clinical and morphological characteristics were included in the study. These tumors were examined by pathologist and cytologist, by immunohistochemical and FISH analyses, mutations in BRAF, NRAS and KIT genes were detected by PCR. results. Some primary tumors were amelanotic, therefore, they were difficult for differential tumor diagnosis. Most frequently, primary tumors were located on the back, shin or head. In 4 patients the primary focuses were not detected. Metastatic lesions were as large nodular tumors in soft tissues of the back or lower limb. Most tumors belong to mixed or spindle-cell histological types. Spindle-shaped cells were also revealed by cytological analysis. BRAF gene mutations were identified by genetic analysis in 27 % of tumors, NRAS and KIT gene mutations were not detected. In 4 cases FISH analysis was performed to detect EWSR1 gene rearrangements, but it did not confirm the diagnosis of sarcoma. conclusion. The results indicate the presence of a heterogeneous group of melanoma cases, which have a number of morphological and molecular features that bring them closer to sarcomas.