BRAF Mutation Testing Research Articles

1145P - Analysis of BRAF V600E mutation status – concordance of results from circulating tumor DNA and tissue-based testing and impact on prediction of the clinical course in patients undergoing BRAFi therapy

The current standard for determining eligibility of patients with metastatic melanoma for BRAF-targeted therapy is the tissue-based testing of BRAF mutations. As patients are rarely re-biopsied, the detection in blood in real-time might be advantageous reflecting the current mutational status of the patient's disease. Furthermore, metastatic melanoma lacks clinically efficient biomarkers. Here, genetic testing in plasma may represent a useful noninvasive biomarker for monitoring BRAF therapy. Blood samples from melanoma patients in two independent studies were subjected to plasma testing using the OncoBEAMTM BRAF V600E assay and tissue-based analysis using Sanger sequencing. In the first study, the tissue-based BRAF mutation status was compared with matched plasma samples from 74 patients diagnosed with progressive metastatic melanoma being either treatment-naïve or undergoing BRAFi therapy. In the second study, the mutational status in tissue and plasma samples from 131 patients of melanoma stage III or IV was compared in follow-up. Overall, results from BRAF plasma testing revealed a high degree of concordance with tissue testing in both studies with 89.2% (study 1) and 94% (study 2), respectively. In the second study 2 of 3 patients with negative OncoBEAM results in plasma, but with positive test in tumor tissue, did not respond to BRAFi therapy potentially due to a false positive result. 5 of 10 patients originally classified BRAF-negative in tumor, tested BRAF mutation-positive in plasma. In these cases, secondary malignancies were found to be responsible. Importantly, dynamic changes of BRAF mutant ctDNA over time correlated with the clinical course and response to treatment. Positivity of BRAF plasma testing was significantly earlier detectable than relapse using radio-imaging. Blood-based testing favorably compares with standard-of-care tissue-based BRAF mutation testing. Importantly, blood-based BRAF testing may be used to predict response to treatment and resistance prior to radio-imaging under BRAFi therapy thereby allowing for an improved treatment management.

Cost-Effectiveness Analysis of Different Genetic Testing Strategies for Lynch Syndrome in Taiwan.

Patients with Lynch syndrome (LS) have a significantly increased risk of developing colorectal cancer (CRC) and other cancers. Genetic screening for LS among patients with newly diagnosed CRC aims to identify mutations in the disease-causing genes (i.e., the DNA mismatch repair genes) in the patients, to offer genetic testing for relatives of the patients with the mutations, and then to provide early prevention for the relatives with the mutations. Several genetic tests are available for LS, such as DNA sequencing for MMR genes and tumor testing using microsatellite instability and immunohistochemical analyses. Cost-effectiveness analyses of different genetic testing strategies for LS have been performed in several studies from different countries such as the US and Germany. However, a cost-effectiveness analysis for the testing has not yet been performed in Taiwan. In this study, we evaluated the cost-effectiveness of four genetic testing strategies for LS described in previous studies, while population-specific parameters, such as the mutation rates of the DNA mismatch repair genes and treatment costs for CRC in Taiwan, were used. The incremental cost-effectiveness ratios based on discounted life years gained due to genetic screening were calculated for the strategies relative to no screening and to the previous strategy. Using the World Health Organization standard, which was defined based on Taiwan’s Gross Domestic Product per capita, the strategy based on immunohistochemistry as a genetic test followed by BRAF mutation testing was considered to be highly cost-effective relative to no screening. Our probabilistic sensitivity analysis results also suggest that the strategy has a probability of 0.939 of being cost-effective relative to no screening based on the commonly used threshold of $50,000 to determine cost-effectiveness. To the best of our knowledge, this is the first cost-effectiveness analysis for evaluating different genetic testing strategies for LS in Taiwan. The results will be informative for the government when considering offering screening for LS in patients newly diagnosed with CRC.

A UK feasibility and validation study of the VE1 monoclonal antibody immunohistochemistry stain for BRAF-V600E mutations in metastatic melanoma.

Background:Determining the BRAF mutation status of patients with advanced metastatic melanoma is essential in order to assess patients' eligibility for targeted BRAF inhibitor therapy. The aim of this study was to validate the utility of immunohistochemistry (IHC) to rapidly obtain the BRAF status in the UK cancer centre setting.Methods:All samples sent for molecular testing for detection of the BRAF mutation over a 26-month period were prospectively tested using the VE1 monoclonal antibody IHC stain.Results:Two-hundred and nineteen samples from 214 patients were identified. All patients were AJCC stage III/IV, except one. There was an overall 95.0% (208/219) concordance rate, with a sensitivity of 94.4% (84/89) and a specificity of 95.4% (124/130) when using genomic assays as the gold standard. Discordance resulted from the inability of the molecular technique to detect the V600E2 mutation and an inability of the IHC staining technique to detect non-V600E mutations. Molecular testing on smaller tumour deposits was also unreliable.Conclusions:IHC staining has good sensitivity and excellent specificity for BRAF V600E mutations. BRAF IHC can be incorporated into a BRAF mutation testing algorithm for UK cancer centres to as a feasible first-line assay and identify a subset of cases that require subsequent genomic testing. It has the additional major advantages of reduced cost and rapid turnaround time.

Molecular testing of different cytologic preparations in patients with advanced lung adenocarcinoma: which yields the best results?

This study constitutes the first systematic comparison of molecular results between different cytology preparations in patients with lung adenocarcinoma undergoing testing for EGFR, KRAS, and BRAF mutations. 115 archival cytology preparations (direct smears, ThinPrep preparations [TP], and cell blocks [CB]) from lung adenocarcinomas with known EGFR, KRAS, or BRAF mutations were tested and compared with clinical testing results. Results were compared between preparations and analyzed in relation to tumor purity and tumor cell content. 82 (77%) of 106 informative cases were concordant with clinical testing results. There was no significant difference in the concordance rate between CB, TP, air-dried smears, or alcohol-fixed smears (P=0.3803), nor between preparations with <25%, 25% to 50%, or >50% tumor purity (P=0.1147). Concordance rates were lower in preparations with ≤100 tumor cells (P = 0.0002). Smears, TP, and CB are all valid substrates for molecular testing. Although tumor purity did not significantly affect results, low tumor content showed poorer performance. Recording tumor purity and content is recommended.

Mutations of KRAS, NRAS, BRAF, EGFR, and PIK3CA genes in urachal carcinoma: Occurence and prognostic significance.

PurposeTargeted therapy represents an attractive alternative for rare tumors such as urachal carcinoma (UrC). The aim of this study was to assess the mutations of the most commonly affected 5 genes in the targetable EGFR-pathway in UrC and comapre their frequencies to those of found in urothelial and colorectal cancer.Materials and MethodsMutational hot-spots of selected genes were tested in 22 UrC samples by pyrosequencing. Mutational patterns were compared to those published for colorectal and urothelial cancers. Furthermore, we sought correlations between mutations and clinicopathological and follow-up data.ResultsWe found 11 mutations in 10 of 22 (45%) patients. The most frequently mutated gene was KRAS (27%) followed by BRAF (18%) and NRAS (5%), while no mutations were detected in the EGFR and PIK3CA genes. No correlation was found between the mutation status and clinicopathological parameters (Sheldon/Mayo stage, tumor grade, metastases). Furthermore, none of the mutations correlated with progression-free or overall survival.ConclusionsThe mutation pattern of UrC is more similar to colorectal than to urothelial cancer. However, the mutation characteristics of UrC seems to be unique suggesting that clinical decision-making for UrC cannot be simply adopted from urothelial or colorectal carcinoma. The high occurence of EGFR-pathway mutations warrants the testing for KRAS and BRAF mutations when considering anti-EGFR therapy in UrC.

BRAF Mutation Testing in Cell-Free DNA from the Plasma of Patients with Advanced Cancers Using a Rapid, Automated Molecular Diagnostics System

Cell-free (cf) DNA from plasma offers an easily obtainable material for BRAF mutation analysis for diagnostics and response monitoring. In this study, plasma-derived cfDNA samples from patients with progressing advanced cancers or malignant histiocytosis with known BRAF(V600) status from formalin-fixed paraffin-embedded (FFPE) tumors were tested using a prototype version of the Idylla BRAF Mutation Test, a fully integrated real-time PCR-based test with turnaround time about 90 minutes. Of 160 patients, BRAF(V600) mutations were detected in 62 (39%) archival FFPE tumor samples and 47 (29%) plasma cfDNA samples. The two methods had overall agreement in 141 patients [88%; κ, 0.74; SE, 0.06; 95% confidence interval (CI), 0.63-0.85]. Idylla had a sensitivity of 73% (95% CI, 0.60-0.83) and specificity of 98% (95% CI, 0.93-1.00). A higher percentage, but not concentration, of BRAF(V600) cfDNA in the wild-type background (>2% vs. ≤ 2%) was associated with shorter overall survival (OS; P = 0.005) and in patients with BRAF mutations in the tissue, who were receiving BRAF/MEK inhibitors, shorter time to treatment failure (TTF; P = 0.001). Longitudinal monitoring demonstrated that decreasing levels of BRAF(V600) cfDNA were associated with longer TTF (P = 0.045). In conclusion, testing for BRAF(V600) mutations in plasma cfDNA using the Idylla BRAF Mutation Test has acceptable concordance with standard testing of tumor tissue. A higher percentage of mutant BRAF(V600) in cfDNA corresponded with shorter OS and in patients receiving BRAF/MEK inhibitors also with shorter TTF. Mol Cancer Ther; 15(6); 1397-404. ©2016 AACR.

Microsatellite Instability in Colorectal Cancers by Race and Hispanic Ethnicity Group

Microsatellite instability (MSI) is found in nearly all Lynch syndrome colorectal cancers and in about half of serrated pathway cancers. Because serrated pathway cancer is more common, it produces about 80% of all microsatellite-unstable tumors. Current recommendations are to screen all colorectal cancers for MSI or failed expression of mismatch repair proteins. Positive tests for MSI or failure to express mismatch repair protein MLH1 should be followed by BRAF mutation tests or hypermethylation assays to …

Treatment pattern of systemic therapies in patients with metastatic melanoma and BRAF mutation test.

e21003Background: The National Comprehensive Cancer Network (NCCN) recommends systemic therapy, including target therapy, immunotherapy, and chemotherapy, for metastatic melanoma (MM). BRAF mutatio...

Diagnostic Accuracy of BRAF Immunohistochemistry in Colorectal Cancer: a Meta-Analysis and Diagnostic Test Accuracy Review.

The aim of this study was to evaluate the concordance between the BRAF (V600E) mutation test and immunohistochemistry (IHC) and to evaluate the diagnostic accuracy of BRAF IHC for colorectal cancer (CRC) through a systematic review, meta-analysis, and diagnostic test accuracy review. The current study included 1021 CRCs from eight eligible studies. The concordance rates were investigated between BRAF IHC and the mutation test. In addition, diagnostic test accuracy review was conducted and calculated using the value of area under curve (AUC) on the summary receiver operating characteristic (SROC) curve. The positive rate of BRAF IHC was 30.5% (range; 13.2-66.2%), and the BRAF mutation was found in 30.2% (range; 11.7-66.2%). The overall concordance rate between BRAF IHC and the mutation test was 0.944 (95% confidence interval (CI) 0.873-0.977). In the BRAF IHC-positive and -negative groups, the concordance rates between BRAF IHC and the mutation test were 0.895 (95% CI 0.800-0.945) and 0.956 (95% CI 0.878-0.985), respectively. The pooled sensitivity and specificity were 0.94 (95% CI 0.91-0.96) and 0.96 (95% CI 0.95-0.98), respectively. The diagnostic odds ratio was 272.86 (95% CI 46.11-1614.88), and the value of AUC on SROC curve was 0.9846. Taken together, our results suggest that BRAF IHC is strongly concordant with the BRAF mutation test and has high diagnostic accuracy in BRAF mutation analysis of CRCs. Further cumulative studies on detailed evaluation criteria are needed before application in daily practice.

Comprehensive Genomic Profiling Facilitates Implementation of the National Comprehensive Cancer Network Guidelines for Lung Cancer Biomarker Testing and Identifies Patients Who May Benefit From Enrollment in Mechanism-Driven Clinical Trials.

The National Comprehensive Cancer Network (NCCN) guidelines for patients with metastatic non-small cell lung cancer (NSCLC) recommend testing for EGFR, BRAF, ERBB2, and MET mutations; ALK, ROS1, and RET rearrangements; and MET amplification. We investigated the feasibility and utility of comprehensive genomic profiling (CGP), a hybrid capture-based next-generation sequencing (NGS) test, in clinical practice. CGP was performed to a mean coverage depth of 576× on 6,832 consecutive cases of NSCLC (2012-2015). Genomic alterations (GAs) (point mutations, small indels, copy number changes, and rearrangements) involving EGFR, ALK, BRAF, ERBB2, MET, ROS1, RET, and KRAS were recorded. We also evaluated lung adenocarcinoma (AD) cases without GAs, involving these eight genes. The median age of the patients was 64 years (range: 13-88 years) and 53% were female. Among the patients studied, 4,876 (71%) harbored at least one GA involving EGFR (20%), ALK (4.1%), BRAF (5.7%), ERBB2 (6.0%), MET (5.6%), ROS1 (1.5%), RET (2.4%), or KRAS (32%). In the remaining cohort of lung AD without these known drivers, 273 cancer-related genes were altered in at least 0.1% of cases, including STK11 (21%), NF1 (13%), MYC (9.8%), RICTOR (6.4%), PIK3CA (5.4%), CDK4 (4.3%), CCND1 (4.0%), BRCA2 (2.5%), NRAS (2.3%), BRCA1 (1.7%), MAP2K1 (1.2%), HRAS (0.7%), NTRK1 (0.7%), and NTRK3 (0.2%). CGP is practical and facilitates implementation of the NCCN guidelines for NSCLC by enabling simultaneous detection of GAs involving all seven driver oncogenes and KRAS. Furthermore, without additional tissue use or cost, CGP identifies patients with "pan-negative" lung AD who may benefit from enrollment in mechanism-driven clinical trials. National Comprehensive Cancer Network guidelines for patients with metastatic non-small cell lung cancer (NSCLC) recommend testing for several genomic alterations (GAs). The feasibility and utility of comprehensive genomic profiling were studied in NSCLC and in lung adenocarcinoma (AD) without GAs. Of patients with NSCLC, 71% harbored at least one GA to a gene listed in the guidelines or KRAS; 273 cancer-related genes were altered in at least 0.1% of the AD cases. Although logistical and administrative hurdles limit the widespread use of next-generation sequencing, the data confirm the feasibility and potential utility of comprehensive genomic profiling in clinical practice.

BRAF analysis before surgery for papillary thyroid carcinoma: correlation with clinicopathological features and prognosis in a single-institution prospective experience.

Risk stratification in patients with papillary thyroid carcinoma (PTC) currently relies on postoperative parameters. Testing for BRAF mutations preoperatively may serve as a novel tool for identifying PTC patients at risk of persistence/recurrence after surgery. The study involved 185 consecutive patients with a histological diagnosis of PTC and BRAF analysis performed on thyroid fine-needle aspiration biopsy (FNAB). We assessed BRAF status in FNAB specimens obtained before thyroidectomy for PTC, and examined its association with the clinicopathological characteristics identified postoperatively, and with outcome after a mean 55±15 months of follow-up. One hundred and fifteen of 185 (62%) PTCs carried a BRAF mutation. Univariate analysis showed that BRAF status correlated with the histological variant of PTC, cancer size, and stage at diagnosis, but not with gender, age, multifocality, or lymph node involvement. BRAF-mutated cases had a higher prevalence of persistent/recurrent disease by the end of the follow-up (11% vs. 8%), but this difference was not statistically significant. The Kaplan-Meier curve shows that among the patients with persistent/recurrent disease, BRAF-mutated patients needed a second treatment earlier than patients with BRAF wild-type, although the difference did not completely reach the statistical significance. Our study confirmed that preoperatively-identified BRAF mutation are associated with certain pathological features of PTC that correlate with prognosis. We speculate that it has a role in identifying PTCs that would generally be considered low-risk but that may reveal an aggressive behavior during their follow-up.

Outcomes of patients (pts) with BRAF mutated (BRAF MT) Colorectal Cancer (CRC): The Royal Marsden Experience.

644 Background: BRAF MT metastatic CRC (mCRC) is associated with a poor prognosis. For the first time, we report outcomes for BRAF MT mCRC at a single tertiary centre, compared to a matched control group, since introduction of routine somatic BRAF and RAS mutation testing. Methods: Pts with BRAF MT mCRC (diagnosed Oct 2010-Nov 2014) were compared to a matched group of BRAF wildtype (WT) pts treated in same time period who were randomly selected after matching for age, sex and stage. Demographic, tumour characteristic and treatment data were collected. Overall (OS) and progression-free survival (PFS) were calculated using the Kaplan-Meier method and comparisons made using χ² test or Cox regression. Results: Of 503 pts tested, 59 (11.7%) had BRAF MT tumours (16 early stage, 16 recurrent and 27 de novo metastatic). Median age (years) at diagnosis was 68 (27-85) compared with 70 (29-85) for BRAF WT pts (p = 0.995). Median OS for pts with mCRC was 18.2 months (m) for BRAF MT and 41.1m for BRAF WT pts (HR 2.73 P &lt; 0.01). For BRAF MT and BRAF WT pts with mCRC, median PFS on first-line treatment (1L) was 8.1m (n = 37) and 9.2m (n = 81) respectively (HR = 1.10 [P = 0.69]), PFS on 2L was 5.1m (n = 21) and 9.0m (n = 49) respectively (HR = 1.84 [P = 0.03]) while PFS on 3L was 1.7m (n = 10) and 6.6m (n = 20) respectively (HR = 2.75 [P = 0.02]). Fluoropyrimidine based doublet regimens were used in 94.6%, 85.7% and 20% of BRAF MT pts in 1L, 2L and 3L respectively compared with 87.2%, 92.5% and 52.4% in BRAF WT pts. Pts with localised disease had a recurrence rate of 50% (16/32) for pts with BRAF MT compared with 52.4% (33/63) for BRAF WT pts (p = 0.83). Conclusions: In this case-control study, poor OS of pts with BRAF MT mCRC is associated with reduced clinical benefit beyond 1L. Sequential doublet chemotherapy remains a reasonable option in these pts. The role of BRAF mutation in predicting recurrence of early CRC warrants further study. [Table: see text]

Role of gene expression profiling in defining indeterminate thyroid nodules in addition to BRAF analysis.

Fine-needle aspiration (FNA) is routinely used in the preoperative evaluation of thyroid nodules. However, 15% to 30% of aspirations yield indeterminate cytologic findings. Because the assessment of BRAF mutations seems to improve the diagnostic accuracy, this study evaluated BRAF mutations with Sanger sequencing and real-time methods in 650 consecutive thyroid aspirates. In addition, the expression of a large number of genes involved in basement membrane remodeling, extracellular matrix proteolysis, and cell adhesion was studied in both benign and malignant nodules to identify new diagnostic tools. In this prospective series, despite the use of a very sensitive BRAF mutational testing method, the frequency of a BRAF alteration being identified in indeterminate FNA samples was 3 of 68. Expression analysis revealed several genes that were differentially expressed between benign and malignant nodules (transforming growth factor, cadherin 1, collagen α1, catenin α1, integrin α3, and fibronectin 1 [FN1]), between follicular adenomas and follicular variant of papillary thyroid carcinoma (FN1, laminin γ1, integrin β2, connective tissue growth factor, catenin δ1, and integrin αV), and between BRAF-wild-type and BRAF-mutated papillary thyroid carcinomas (TIMP metallopeptidase inhibitor 1; catenin α1; secreted phosphoprotein 1; FN1; ADAM metallopeptidase with thrombospondin type 1 motif, 1; and selectin L). These data were partially confirmed with real-time polymerase chain reaction analysis and immunohistochemistry. When the cost/benefit ratio of the procedures was taken into account, BRAF mutational testing failed to increase diagnostic accuracy in cytologically indeterminate nodules. However, the additional analysis of the expression of specific molecular markers could have possible utility as a diagnostic tool, although further evidence based on a large series of samples is needed before definitive conclusions can be drawn. Cancer Cytopathol 2016;124:340-9. © 2015 American Cancer Society.

BRAF Mutation Testing and Metastatic Colorectal Cancer in the Community Setting: Is There an Urgent Need for More Education?

Patients with metastatic colorectal cancer (mCRC) with BRAF mutation (BRAF MT) generally have a poorer prognosis. BRAF MT may also have implications for treatment strategy. Despite this, inclusion of BRAF in routine molecular testing varies. Here we report the frequency of BRAF reporting in the South Australian (SA) mCRC registry reflecting community practice, together with the survival outcomes based on mutation status. The SA population-based mCRC registry was analysed to assess the number of patients where a BRAF MT result was available. The patient characteristics are reported and overall survival was analysed using the Kaplan-Meier method. Of the 3639 patients who have been entered in the registry, only 6.2% (227) have BRAF MT results available. Of the patients tested, the BRAF MT rate is 12.7%. The mutation rate was highest in rightsided primary; right colon 23 versus left colon 8.9% and rectum 7%. There was no significant difference in median age or male/female proportion. The median overall survival (mOS) for BRAF MT versus wild-type (WT) patients is 14.0 versus 32.9 months (p = 0.003). For patients who have chemotherapy (plus or minus surgery) the mOS is 14.6 months BRAF MT versus 36.1 months (p ≤ 0.001) WT. Liver or lung resection was performed on only 8% of the BRAF MT group versus 26.5% of the WT group. Results in a population setting confirm our understanding that BRAF MT is more frequently right sided and of lower frequency in rectal cancer. Survival is lower for patients with mCRC that have BRAF MT, regardless of the therapy. BRAF testing is currently performed infrequently in an Australian setting despite its importance as a significant prognostic factor, and the implications for alternate therapeutic approaches.

Cytogenetics in Hairy Cell Leukemia at Diagnosis and Relapse: The Rate of Therapy-Related Myeloid Malignancies Is Low

Cytogenetic analysis of a large cohort of hairy cell leukemia (HCL) patients (pts) has never been reported. The influence on outcome of cytogenetic abnormalities is unknown.We retrospectively reviewed records of pts with HCL treated at Memorial Sloan Kettering Cancer Center between January 1973 and December 2013. 501 pts were identified and 331 were confirmed to have a diagnosis of HCL by histopathology or immunophenotyping. 170 patients were excluded, either because their final diagnosis was an alternate disease or there was inaccessible clinical data. Descriptive statistics were used to evaluate the cohort.260/331 (79%) were men, overall median age at diagnosis was 53 years (range 20-84 years) and median follow-up was 61 months. 241 had classical HCL and 18 had variant disease based on absent expression of CD25 by immunophenotyping or immunohistochemistry with median diagnostic WBC of 3.2x10^9/L and 8.4x10^9/L respectively. BRAF mutation testing was performed infrequently (<10% pts). In 72 pts we were unable to review immunophenotyping data to confirm disease type. 64 pts never received treatment, 156 received one line and 111 received at least 2 lines of therapy during follow up. The most common initial treatment was cladribine (n=215, 80%). Assessment of bone marrow cytogenetics was available in 105 of 331 patients (32%) at diagnosis with 92 cases of classical HCL, 8 variant HCL and 5 with unconfirmed disease type. Karyotype was normal in 95 patients (90.5%) and abnormal in 10 (9.5%). Abnormal karyotype was more common in patients with variant than classical disease, seen in 3/8 versus 7/92 respectively (p=0.007). 9/10 with abnormal karyotype had multiple clones identified including at least one cytogenetically normal metaphase. Sex chromosome loss was present in 4/10 pts (3 with loss of Y and 1 female with loss of X) while loss of genetic material from chromosome 7 was the most common autosomal abnormality (3/10 patients). The presence of an abnormal karyotype at diagnosis did not worsen survival compared to patients with a normal karyotype with an estimated 5 year OS of 100% and 97%, respectively.30/111 pts (24 classical and 6 variant HCL) who received two or more lines of therapy had cytogenetics evaluated at some point during disease relapse and 17/30 had cytogenetic abnormalities. 6 of these had cytogenetics at diagnosis and 4/6 were normal. Pts with an abnormal karyotype received more lines of therapy prior to identification of a cytogenetic abnormality than those with normal cytogenetics with a median of 3 and 1 lines respectively (p=0.008). Pts with variant HCL were more likely to have abnormal cytogenetics at relapse (6/6) than those with classical disease (11/24) (p=0.017).Cytogenetic abnormalities of all evaluable pts with relapsed HCL were reviewed. The most common abnormalities seen at relapse were monosomies or partial chromosome deletions seen in 14/17 cases. The most common abnormalities were loss of material from chromosome 7 (6/17 pts) and chromosomes 13 and 17 (4/17 pts each). Multiple abnormalities were present in 11/17 pts. No abnormality appeared to be specific for classical or variant disease. In particular loss of material from chromosome 7 was seen in both disease types. Only 2 of the 331 pts developed a therapy-related acute myeloid leukemia (t-AML) both had classical HCL. The first developed t-AML with a complex monosomal karyotype after prior treatment with cladribine, pentostatin, fludarabine and rituximab. The second had t-AML with monosomy 7 following 4 treatments with cladribine. There were no cases of myelodysplasia.In summary we identified cytogenetic abnormalities in 10% of newly diagnosed HCL pts and found that this did not adversely affect survival. Pts with variant HCL are more likely to have abnormal cytogenetics at diagnosis and at disease relapse compared to those with classical disease. Cytogenetic changes appear to be more common in those who received more lines of cytotoxic chemotherapy. Loss of material from chromosome 7 is the most common cytogenetic change seen at diagnosis and at disease progression. The rate of therapy-related myeloid malignancies is low and is not increased in HCL variant. Identification of new molecular mutations arising during disease relapse may be insightful. DisclosuresPark:Actinium Pharmaceuticals, Inc.: Research Funding; Juno Therapeutics: Consultancy.

Pathology and prognostic factors of conjunctival melanoma

Conjunctival melanoma is a rare but potentially fatal disease. The 10-year melanoma mortality can be up to 30 %, recurrence rates after treatment up to 50 % and the overall incidence of metastasis is 26 %. Improved treatment options are needed to increase the tumor-free survival of affected patients. The aim of the study was to perform clinical and pathological staging using the TNM classification and to correlate the results with treatment modalities and recurrence rates. The study included a case series of 80 eyes from 80 patients (42 females and 38 males, age 28-90 years) with histopathologically proven conjunctival melanoma studied by reviewing medical records, pathology reports and color photographs. The main evaluated characteristics were demographic information, tumor size, thickness, pathological diagnosis, BRAF mutation testing, clinical and pathological staging, recurrence, metastasis and duration of follow-up (mean 48 months). The lesions predominantly involved the bulbar conjunctiva (60 %) and other sites that were less often involved were the palpebral conjunctiva (23 %), conjunctival fornix (22 %) and lacrimal caruncle (15 %). Of the tumors 36 % were TNM classified as pTis, 34 % as pT1, 20 % as pT2 (palpebral, fornix and caruncle) and 10 % as pT3. Local recurrences were noted in 36 % of the patients (18 % Tis, 26 % T1, 32 % T2 and 70 % T3) and regional and distant metastasis in 20 % of the patients (0 % Tis, 10 % T1, 15 % T2 and 60 % T3). In this study increasing T stages were more often associated with recurrences and metastasis. Future studies correlating the TNM staging with histopathological and genetic predictors may help to improve the management of patients with conjunctival melanoma.

Multi-center evaluation of the novel fully-automated PCR-based Idylla™ BRAF Mutation Test on formalin-fixed paraffin-embedded tissue of malignant melanoma

The advent of BRAF-targeted therapies led to increased survival in patients with metastatic melanomas harboring a BRAF V600 mutation (implicated in 46–48% of malignant melanomas). The Idylla™ System (Idylla™), i.e., the real-time-PCR-based Idylla™ BRAF Mutation Test performed on the fully-automated Idylla™ platform, enables detection of the most frequent BRAF V600 mutations (V600E/E2/D, V600K/R/M) in tumor material within approximately 90min and with 1% detection limit.Idylla™ performance was determined in a multi-center study by analyzing BRAF mutational status of 148 archival formalin-fixed paraffin-embedded (FFPE) tumor samples from malignant melanoma patients, and comparing Idylla™ results with assessments made by commercial or in-house routine diagnostic methods.Of the 148 samples analyzed, Idylla™ initially recorded 7 insufficient DNA input calls and 15 results discordant with routine method results. Further analysis learned that the quality of 8 samples was insufficient for Idylla™ testing, 1 sample had an invalid routine test result, and Idylla™ results were confirmed in 10 samples. Hence, Idylla™ identified all mutations present, including 7 not identified by routine methods.Idylla™ enables fully automated BRAF V600 testing directly on FFPE tumor tissue with increased sensitivity, ease-of-use, and much shorter turnaround time compared to existing diagnostic tests, making it a tool for rapid, simple and highly reliable analysis of therapeutically relevant BRAF mutations, in particular for diagnostic units without molecular expertise and infrastructure.

Abstract 2413: Rapid, automated BRAF mutation testing of cell-free DNA from plasma of patients with advanced cancers using the novel Idylla platform

Abstract Background: BRAF V600 mutations are prevalent in diverse cancers and can be targeted with BRAF inhibitors. Cell-free (cf) DNA from plasma offers an easily obtainable material for BRAF mutation analysis. Methods: cfDNA from plasma samples of patients with advanced cancers or malignant histiocytosis with known BRAF V600 status from formalin-fixed paraffin-embedded (FFPE) tumor samples, who progressed on systemic therapy was purified and 50-100ng were used for BRAF V600 mutations testing using the Idylla fully integrated real-time PCR-based platform (Biocartis, Mechelen, Belgium) with a quick turnaround time (&amp;lt; 60 minutes). Results were compared to mutation analysis of FFPE primary or metastatic tumor tissue obtained at different points of clinical care from a CLIA-certified laboratory. Results: cfDNA was extracted from plasma samples of 160 patients with advanced cancers (colorectal, n = 62; melanoma, n = 36; non-small cell lung, n = 13; breast, n = 10; thyroid, n = 10; appendiceal, n = 5; ovarian, n = 4; endometrial, n = 4; other cancers, n = 16). BRAF mutations were detected in 29% (47/160) of plasma samples and in 39% (62/160) of FFPE tumor samples, resulting in concordance in 141 cases (88%, kappa = 0.74, 95% confidence interval [CI] 0.63- 0.85) with sensitivity 73%, specificity 98%, positive and negative predictive value 96% and 85%, respectively. In addition, 35 (22%) patients had sequential plasma samples collection while on therapy including 9 of 17 patients, who had BRAF V600 mutation in FFPE but not in plasma at baseline. Of interest, in 3 of these 9 patients BRAF V600 mutation emerged in plasma later in the course of disease. If these 3 patients are included in the concordance analysis the overall agreement between testing modalities is 144 cases (90%, kappa 0.78, SE 0.05, 95% CI 0.68-0.88) with sensitivity 77%, specificity 98%, positive and predictive value 96% and 87%, respectively. In 15 of 19 discrepant cases identical plasma cfDNA samples were tested using an alternative cfDNA BRAF mutation PCR-based method (BEAMing, Sysmex Inostics, Baltimore, MD), which yielded 100% agreement with the Idylla. Longitudinally collected plasma samples were available in 16 patients with plasma and FFPE BRAF V600 mutations treated with predominantly BRAF targeting combinations. Changes in the amount of BRAF-mutant cfDNA corresponded with changes in serum tumor markers and disease burden visualized via imaging. Conclusions: Detecting BRAF V600 mutations in plasma cfDNA using the Idylla platform is a fast and noninvasive alternative to mutation testing of tumor tissue with an acceptable level of concordance and should be investigated further for testing and monitoring of BRAF mutation status in patients with cancer. Citation Format: Filip Janku, Helen J. Huang, Bart Claes, Gerald S. Falchook, Siqing Fu, Apostolia M. Tsimberidou, David S. Hong, Aung Naing, Jennifer J. Wheler, Sarina A. Piha-Paul, Daniel D. Karp, Vivek Subbiah, Ralph G. Zinner, Nishma Ramzanali, Rajyalakshmi Luthra, Sapna P. Patel, E. Scott Kopetz, Erwin Sablon, Geert Maertens, Razelle Kurzrock, Funda Meric-Bernstam. Rapid, automated BRAF mutation testing of cell-free DNA from plasma of patients with advanced cancers using the novel Idylla platform. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2413. doi:10.1158/1538-7445.AM2015-2413

Tumor rabdoide extrarrenal maligno de colon: presentación de 3 casos y revisión de la literatura

Malignant extrarenal rhabdoid tumour (MERT) of the colon is rare, with only 12 cases published to date. Clinically, they usually occur in elderly patients and present as large, aggressive tumours; their histopathology is that typical of rhabdoid tumours and their immunohistochemistry shows expression of vimentin and cytokeratines. We present 3 cases of colonic MERTs with features similar to previously published cases. One was possibly a metastasis of a renal cell carcinoma with rhabdoid features. We carried out a extensive immunohistological analysis of all 3 cases as well as testing for KRAS and BRAF mutations. Thus we could not only compare our findings with those of the other known cases but also make a differential diagnosis with primary colonic neoplasmas and metastases of similar appearance.

BRAF-mutated carcinomas among thyroid nodules with prior indeterminate FNA report: a systematic review and meta-analysis.

Several molecular analyses have been investigated for risk stratification of thyroid nodules, with a particular focus on the V600E mutation of the BRAF gene [BRAF(V600E)]. To date, there is no high-level evidence supporting or refuting a role for BRAF analysis in thyroid nodules with prior indeterminate cytology. To obtain more robust evidence, we reviewed and meta-analysed data from published articles. A comprehensive literature search of the PubMed/MEDLINE, Embase and Scopus databases was conducted using the terms 'BRAF', 'thyroid' and 'indeterminate'. The search was updated until March 2015, and references of the retrieved articles were also screened. Only original articles reporting BRAF mutation testing within nodules with indeterminate FNA were eligible for inclusion. The literature search revealed 82 articles, of which 8 were eligible for the study. Five studies were prospective and three retrospective. The majority of authors analysed BRAF mutations in FNA samples which were classified by the British or Bethesda system. Of the initial series of studies, a pooled number of 1361 cases were achieved of which 43 were BRAF mutated. Overall, the BRAF mutation rate was 4·6% (95% CI: 1-10·8%), ranging from 0 to 22·9%. When we included only histological series, 978 thyroid nodules were found. Of these, 245 were cancers. A very low rate of lesions with indeterminate cytology are BRAF mutated. Thus, the role of this biomarker to detect or exclude cancers in patients with such FNA reports is marginal and should be reconsidered in guidelines.