BRAF Point Mutations Research Articles

Abstract 2632: Clinical validation of circulating DNA analysis for the detection of point mutations and of the longitudinal metastatic colorectal patient follow up for detecting emergence of resistance to targeted therapy

Abstract Clinical validation of circulating DNA analysis as liquid biopsy for the detection of point mutations and of the longitudinal metastatic colorectal patient follow up for detecting emergence of resistance to targeted therapy We carried out two clinical studies in metastatic colorectal cancer patients (i) the clinical real-time evaluation of circulating tumor DNA (ctDNA) analysis for detecting point mutations in comparison with tumor tissue analysis before initiation of anti-EGFR therapy and (ii), the study of the emergence of associated resistance mutation in refractory patients under treatment. (i) We realized a real-time blinded prospective multicentric clinical study on 140 patients comparing KRAS (exon 2,3 and 4) and BRAF V600E mutations determination by plasma and tissue analysis carried out in the conditions of standard management care. On 121 patients where both analysis were made, 43% were found mutant by tumor tissue analysis while 57% were found KRAS mutant by ctDNA analysis. 7.2% of patients were found BRAF mutant by tumor tissue while 14.4% were determined mutant by ctDNA analysis. 13% of plasma samples carried multiple KRAS point mutations and 4% of plasma samples exhibited at least one KRAS point mutation combined to the presence of BRAFV600E point mutation. Median data turnaround time was 16 [3-273] days for tumor tissue while it was 2 [0.5-10] days for ctDNA analysis. CtDNA analysis appears much more sensitive than tissue analysis and may be more accurate in respect to the intra-tumor or inter-tumor heterogeneity and the clonal evolution dynamics. We present the first RAS and BRAF mutation distribution which takes into account the presence of multiple mutations in the same mCRC patient. (ii) In the other blinded clinical study, we retrospectively analyzed RAS (KRAS and NRAS) and BRAF mutations in serial plasma samples from 42 refractory mCRC patients to Folfox + cetuximab or dasatinib. 98% of the plasma were found mutant before or during treatment. 50% of KRAS mutant samples were missed by tumor tissue analysis before treatment. 4.8% of patients were found BRAF mutant by tumor tissue analysis while 7% were found mutant by ctDNA analysis. Longitudinal plasma analysis shows that 80% of initially WT patients acquire at least one RAS or BRAF mutation during treatment and that 38% of initially mutant patients acquire at least one newly KRAS or BRAF point mutation during treatment. CtDNA analysis allows tracking acquired resistance by studying the real-time clonal evolution of the tumor Patients may harbor mutations at very low frequency on ctDNA down to 0.01% before initiation or during treatment revealing the need of a high sensitive technique to detect mutant subclones. Our results indicate that plasma analysis could advantageously replace tumor tissue analysis and enables longitudinal examination of tumor molecular profiling. Citation Format: Alain R. Thierry, Safia El Messaoudi, Caroline Mollevi, Brice Pastor, Cynthia Sanchez, Jean-Luc Raoul, Rosine Guimbaud, Denis Pezet, Pascal Artru, Muriel Mathonnet, Christelle De La Fouchardiere, Christophe Borg, Eric Assenat, Olivier Bouche, Céline Gavoille, Scott Kopetz, Marc Ychou. Clinical validation of circulating DNA analysis for the detection of point mutations and of the longitudinal metastatic colorectal patient follow up for detecting emergence of resistance to targeted therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2632.

LG-47TYPE II RAF INHIBITORS INHIBIT BRAF MUTATIONS AND TRUNCATED FUSIONS IN PEDIATRIC LOW-GRADE GLIOMAS

LG-47. TYPE II RAF INHIBITORS INHIBIT BRAF MUTATIONS AND TRUNCATED FUSIONS IN PEDIATRIC LOW-GRADE GLIOMAS Mark W. Kieran1,2, Yu Sun1, Catherine Pilarz1, David Calligaris3, Emily J. Chadwick1, John A. Alberta1, Shakti H. Ramkissoon1,2, Lori A. Ramkissoon1, Veronica Matia Garcia1, Kim Wilkinson1, Michael Kane1, Liliana Goumnerova1,2, Susan N. Chi1,2, Peter Manley1,2, Karen D. Wright1,2, Nathalie Y. Agar1,3, Keith L. Ligon1,3, Rameen Beroukhim1,3, Pratiti Bandopadhayay1,2, Geoffrey Kannan1,2, Rosalind A. Segal1, Levi A. Garraway1, Nathanael S. Gray1, Michael Eck1, Charles D. Stiles1, and Sara J. Buhrlage1; Dana-Farber/Harvard Cancer Center, Boston, MA, USA; Boston Children’s Hospital/Harvard Medical School, Boston, MA, USA; BrighamandWomen’sHospital/HarvardMedical School,Boston,MA,USA PURPOSE: Abnormal signaling of the ras/raf pathway predominates in pediatric low-grade gliomas (PLGGs). Most (75%) non-NF1 low-grade gliomas have Ras-independent BRAF activation, either due to KIAA1549:BRAF truncated fusion duplication or V600E point mutation. While the latter BRAF oncoprotein functions as a monomer, the truncated fusion form requires dimerization. Type I BRAF V600E inhibitors approved for melanoma effectively abrogate monomeric signaling by targeting the “DFG-in” kinase conformation but paradoxically activate wild type RAF dimers. We therefore evaluated Type II RAF inhibitor activity (targeting the “DFG-out” kinase conformation). METHODS: We developed multiple pathway relevant model systems for different BRAF point mutations and KIAA1549 variants using neural progenitor cells from mouse embryos for in vitro, in vivo and mass spectrometry analysis. Additionally, we developed an organoid ex vivo avatar assay from direct patient samples to identify drugs inhibiting these targets. We subsequently treated a single patient with one of two identified compounds. RESULTS: Two different type II BRAF inhibitors capable of inhibiting downstream activation of pERK and tumor progression in vivo for multiple point mutations and BRAF truncated fusion forms were identified. One compound was active on authentic human PLGA cells ex vivo and showed excellent CNS penetration. CONCLUSIONS: Type II BRAF inhibitors possess a unique mechanism of action targeting the BRAF/MEK complex and inhibiting both BRAF point mutations and common structural abnormalities (KIAA1549) in PLGGs. These results have led to the development of a phase I/0/II trial. Neuro-Oncology 18:iii78–iii96, 2016. doi:10.1093/neuonc/now075.47 #The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Resistance of papillary thyroid cancer stem cells to chemotherapy.

Thyroid carcinoma is the most common endocrine neoplasm, with the highest mortality rate of all the endocrine cancers. Among the endocrine malignancies, ~80% are papillary thyroid carcinomas (PTCs). In the initiation and progression of this tumor, genetic alterations in the mitogen-associated protein kinase pathway, including RAS point mutations, RET/PTC oncogene rearrangements and BRAF point mutations, play an important role, particularly in deciding targeted therapy. In the present study, a small population of thyroid tumor cells, known as tumor spheres, were isolated and characterized from PTC surgical samples. These spheres can be expanded indefinitely in vitro and give rise to differentiated adherent cells when cultivated in differentiative conditions. The present study showed by reverse transcription-polymerase chain reaction and flow cytometric analysis that the undifferentiated PTC cells exhibited a characteristic antigen expression profile of adult progenitor/stem cells. The cells were more resistant to chemotherapeutics, including bortezomib, taxol, cisplatin, etoposide, doxorubicin and vincristine, than differentiated PTC cells and the majority possessed a quiescent status, as revealed by the various cell cycle characteristics and anti-apoptotic protein expression. Such advances in cancer thyroid stem cell biology may provide relevant information for future targeted therapies.

LG-44OCCURRENCE OF BRAF V600E MUTATION IN A PEDIATRIC BRAINSTEM GANGLIOGLIOMA WITH NEAR EXCLUSIVE GLIAL COMPONENT IN THE ORIGINAL BIOPSY

LG-44. OCCURRENCE OF BRAF V600E MUTATION IN A PEDIATRIC BRAINSTEM GANGLIOGLIOMA WITH NEAR EXCLUSIVE GLIAL COMPONENT IN THE ORIGINAL BIOPSY Alex Feldman, David Kelly, Alyssa Reddy, Jeffrey Blount, and Rong Li; University of Alabama at Birmingham, Birmingham, AL, USA A five-year-old male who initially presented with apneic episodes and morbid obesity underwent imaging studies, which revealed a dorsally exophytic medullary tumor in 2014. A biopsy showed a classic pilocytic astrocytoma (PA) with no apparent neoplastic neuronal component. Following completion of chemotherapy, the patient presented again in January of 2016 with worsening symptoms; the tumor was found to have grown in size with brainstem compression. Debulking was performed. Unexpectedly, the histology of this tumor showed a ganglioglioma (GG) with many clusters of enlarged, misshapen, dysmorphic-appearing and occasionally binucleated neurons. Furthermore, BRAF V600E mutations were identified in both prior and recurrent tumors. Recent publications have found that the BRAF V600E mutation is seen in 20-58% of all GGs. In particular, pediatric brainstem GGs appear to harbor this mutation at a rate greater than 50%. Few studies have investigated the distribution of BRAF aberrations among neuronal and glial components in GGs. Pilocytic astrocytomas often possess BRAF abnormalities, though fusion genes (i.e BRAF-KIAA1549) are considerably more common (60-80%) than the BRAF V600E missense mutations (only seen in 10-12% of pediatric brainstem PAs). This case highlights the presence of the BRAF point mutation in both glial and neuronal component of a pediatric brainstem GG, supporting the hypothesis that a glioneuronal lineage precursor is initially affected by the mutation. Additionally, the presence of the BRAF point mutation in a brainstem pilocytic astrocytoma should raise the possibility of a GG in which sampling artifact highlights a near exclusive glial component. Neuro-Oncology 18:iii78–iii96, 2016. doi:10.1093/neuonc/now075.44 #The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Whole Chromosome 7 Gain Predicts Higher Risk of Recurrence in Pediatric Pilocytic Astrocytomas Independently From KIAA1549-BRAF Fusion Status.

The most frequent genetic alteration identified in pediatric pilocytic astrocytomas and pilomyxoid variant is the KIAA1549-BRAF fusion, which typically results from a 2.0 Mb tandem duplication in chromosome band 7q34. Less frequent abnormalities include fusion genes,BRAF, FGFR, KRAS, and NF1 point mutations, and whole chromosome gains. To correlate genetic alterations with clinical course data, we retrospectively analyzed the tumors with pilocytic and pilomyxoid histology of a cohort of 116 pediatric patients, aged 5 months to 23 years. Gross total resection was associated with a decreased risk of recurrence (p = 0.001), supporting previous findings that complete tumor excision correlates with long-term and disease-free survival. We found no significant association between recurrence rate and the presence of the KIAA1549-BRAF fusion or BRAF mutation (p = 0.167). Interestingly, gain of whole chromosome 7 (WC7) was associated with a 4.7-fold increased risk of tumor recurrence, even after adjusting for surgical status (p = 0.025), and other genetic alterations. Using fluorescence in situ hybridization, we demonstrated that when WC7 gain accompanies the KIAA1549-BRAF fusion, the fusion likely arises first. This study highlights the utility of genetic studies for risk assessment of pilocytic and pilomyxoid astrocytomas, which may impact treatment selections.

Sensitive, specific, and modular KRAS, NRAS, and BRAF assays for simultaneous detection of 30 important point mutations in colorectal cancer specimens.

547 Background: Point mutations in KRAS, NRAS, and BRAF are implicated in the oncogenic RAS/RAF/MEK/ERK pathway and are prognostic and predictive markers in treating metastatic colorectal cancer patients. Here, we report the performance of three single tube highly multiplex and modular KRAS/NRAS/BRAF Assays capable of detecting 30 point mutations from FFPE-extracted DNA on the fully automated Modaplex System. Methods: Primers for multiplex KRAS/NRAS/BRAF Assays to detect 13, 13, and 4 point mutations for KRAS, NRAS, and BRAF genes, respectively, were designed using proprietary technology. Analytical sensitivity of each mutation was assessed using mutation specific ultramer oligos or Horizon Dx Mockblock specimens. Analytical specificity was determined on known wild-type FFPE DNAs. The assays were tested on DNA extracted from clinical FFPE specimens that were characterized by pyrosequencing. Modaplex data on these specimens were compared to pyrosequencing for concordance. Results: The modular Modaplex KRAS/NRAS/BRAF point mutation panels detect and differentiate 30 mutations in the KRAS, NRAS, and BRAF genes in three wells. Each assay requires just 5 µL of clinical sample extract (10-50ng DNA per assay) with a setup time of 15-30 minutes. The total run time including data analysis and setup is 4 hours. The kit includes an internal control to determine mutation status; and calibration controls to determine amplicon size. Analytical studies demonstrate the assays are sensitive (number of copies detected in one reaction), selective (mutant to wild-type ratio), and specific (relative to wild-type genomic DNA background). Nearly 110 clinical FFPE samples were tested, and the assays showed more than 95% concordance with pyrosequencing method (98% for BRAF, 97% for KRAS, and 100% for NRAS). Conclusions: Modaplex KRAS/NRAS/BRAF assays provide an accurate and sensitive method of mutation detection. These automated multiplexed assays to detect key mutations in three genes can be performed on a single platform facilitating clinical or translational research. *Modaplex KRAS/NRAS/BRAF Assays are for Research Use Only. Not for clinical diagnostic use.

A Targeted Q-PCR-Based Method for Point Mutation Testing by Analyzing Circulating DNA for Cancer Management Care.

Circulating cell-free DNA (cfDNA) is a valuable source of tumor material available with a simple blood sampling enabling a noninvasive quantitative and qualitative analysis of the tumor genome. cfDNA is released by tumor cells and exhibits the genetic and epigenetic alterations of the tumor of origin. Circulating cell-free DNA (cfDNA) analysis constitutes a hopeful approach to provide a noninvasive tumor molecular test for cancer patients. Based upon basic research on the origin and structure of cfDNA, new information on circulating cell-free DNA (cfDNA) structure, and specific determination of cfDNA fragmentation and size, we revisited Q-PCR-based method and recently developed a the allele-specific-Q-PCR-based method with blocker (termed as Intplex) which is the first multiplexed test for cfDNA. This technique, named Intplex(®) and based on a refined Q-PCR method, derived from critical observations made on the specific structure and size of cfDNA. It enables the simultaneous determination of five parameters: the cfDNA total concentration, the presence of a previously known point mutation, the mutant (tumor) cfDNA concentration (ctDNA), the proportion of mutant cfDNA, and the cfDNA fragmentation index. Intplex(®) has enabled the first clinical validation of ctDNA analysis in oncology by detecting KRAS and BRAF point mutations in mCRC patients and has demonstrated that a blood test could replace tumor section analysis for the detection of KRAS and BRAF mutations. The Intplex(®) test can be adapted to all mutations, genes, or cancers and enables rapid, highly sensitive, cost-effective, and repetitive analysis. As regards to the determination of mutations on cfDNA Intplex(®) is limited to the mutational status of known hotspot mutation; it is a "targeted approach." However, it offers the opportunity in detecting quantitatively and dynamically mutation and could constitute a noninvasive attractive tool potentially allowing diagnosis, prognosis, theranostics, therapeutic monitoring, and follow-up of cancer patients expanding the scope of personalized cancer medicine.

Clinical Relevance of Prognostic and Predictive Molecular Markers in Gliomas.

Sorting and grading of glial tumors by the WHO classification provide clinicians with guidance as to the predicted course of the disease and choice of treatment. Nonetheless, histologically identical tumors may have very different outcome and response to treatment. Molecular markers that carry both diagnostic and prognostic information add useful tools to traditional classification by redefining tumor subtypes within each WHO category. Therefore, molecular markers have become an integral part of tumor assessment in modern neuro-oncology and biomarker status now guides clinical decisions in some subtypes of gliomas. The routine assessment of IDH status improves histological diagnostic accuracy by differentiating diffuse glioma from reactive gliosis. It carries a favorable prognostic implication for all glial tumors and it is predictive for chemotherapeutic response in anaplastic oligodendrogliomas with codeletion of 1p/19q chromosomes. Glial tumors that contain chromosomal codeletion of 1p/19q are defined as tumors of oligodendroglial lineage and have favorable prognosis. MGMT promoter methylation is a favorable prognostic marker in astrocytic high-grade gliomas and it is predictive for chemotherapeutic response in anaplastic gliomas with wild-type IDH1/2 and in glioblastoma of the elderly. The clinical implication of other molecular markers of gliomas like mutations of EGFR and ATRX genes and BRAF fusion or point mutation is highlighted. The potential of molecular biomarker-based classification to guide future therapeutic approach is discussed and accentuated.

Analysis of somatic mutations in BRAF, CDKN2A/p16 and PI3KCA in patients with medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC), a neuroendocrine tumor originating from thyroid parafollicular cells, has been demonstrated to be associated with mutations in RET, HRAS, KRAS and NRAS. However, the role of other genes involved in the oncogenesis of neural crest tumors remains to be fully investigated in MTC. The current study aimed to investigate the presence of somatic mutations in BRAF, CDKN2A and PI3KCA in MTC, and to investigate the correlation with disease progression. DNA was isolated from paraffin‑embedded tumors and blood samples from patients with MTC, and the hotspot somatic mutations were sequenced. A total of 2 novel HRAS mutations, p.Asp33Asn and p.His94Tyr, and polymorphisms within the 3' untranslated region (UTR) of CDKN2A (rs11515 and rs3088440) were identified, however, no mutations were observed in other genes. It was suggested that somatic point mutations in BRAF, CDKN2A and PI3KCA do not participate in the oncogenesis of MTC. Further studies are required in order to clarify the contribution of the polymorphisms identified in the 3'UTR of CDKN2A in MTC.

Histopathological features of papillary thyroid carcinomas detected during four screening examinations of a Ukrainian-American cohort.

Background:There are limited data on the histopathology of papillary thyroid carcinomas (PTCs) diagnosed in irradiated populations. We evaluated the associations between iodine-131 dose and the histopathological characteristics of post-Chernobyl PTCs, the changes in these characteristics over time, and their associations with selected somatic mutations.Methods:This study included 115 PTCs diagnosed in a Ukrainian-American cohort (n=13 243) during prescreening and four successive thyroid screenings. Of these PTCs, 65 were subjected to somatic mutation profiling. All individuals were <18 years at the time of the Chernobyl accident and had direct thyroid radioactivity measurements. Statistical analyses included multivariate linear and logistic regression.Results:We identified a borderline significant linear-quadratic association (P=0.063) between iodine-131 dose and overall tumour invasiveness (presence of extrathyroidal extension, lymphatic/vascular invasion, and regional or distant metastases). Irrespective of dose, tumours with chromosomal rearrangements were more likely to have lymphatic/vascular invasion than tumours without chromosomal rearrangements (P=0.020) or tumours with BRAF or RAS point mutations (P=0.008). Controlling for age, there were significant time trends in decreasing tumour size (P<0.001), the extent of lymphatic/vascular invasion (P=0.005), and overall invasiveness (P=0.026).Conclusions:We determined that the invasive properties of PTCs that develop in iodine-131-exposed children may be associated with radiation dose. In addition, based on a subset of cases, tumours with chromosomal rearrangements appear to have a more invasive phenotype. The increase in small, less invasive PTCs over time is a consequence of repeated screening examinations.

Genetic Alterations in Differentiated Thyroid Cancer Patients with Acromegaly.

Acromegaly is associated with increased thyroid cancer risk. We aimed to analyze the frequency of point mutations of BRAF and RAS genes, and RET/PTC, PAX8/PPARγ gene rearrangements in patients with acromegaly having differentiated thyroid cancers (DTC) and their relation with clinical and histological features. 14 acromegalic patients (8 male, 6 female) with DTC were included. BRAF V600E and NRAS codon 61 point mutations, RET/PTC1, RET/PTC3, and PAX8/PPARγ gene rearrangements were analyzed in thyroidectomy specimens. We selected 14 non-acromegalic patients with DTC as a control group. 2 patients (14.3%) were detected to have positive BRAF V600E and 3 patients (21.4%) were detected to have NRAS codon 61 mutation. NRAS codon 61 was the most frequent genetic alteration. Patients with positive mutation had aggressive histologic features more frequently than patients without mutations. Comparison of the acromegalic and non-acromegalic patients with DTC revealed that BRAF V600E mutation was more frequent in non-acromegalic patients with DTC (14.2% vs. 64.3%, p=0.02). RET/PTC 1/ 3, PAX8/PPARγ gene rearrangements were not detected in any patient. None of the patients including the patients with positive point mutations had recurrence, and local and/or distant metastasis. NRAS codon 61 is the most frequent genetic alteration in this acromegaly series with DTC. Since acromegalic patients have lower prevalance of BRAF V600E mutation, BRAF V600E mutation may not be a causative factor in development of DTC in acromegaly. Despite the relation of BRAF V600E and NRAS codon 61 mutations with aggresive histopathologic features, their impact on tumor prognosis remains to be defined in acromegaly in further studies.

Abstract A1-43: Targeted amplicon-based next-generation sequencing of routine solid tumor specimens to detect clinically relevant somatic alterations

Abstract Background: Although precision medicine approaches have revolutionized oncology, widespread adoption requires robust, inexpensive approaches enabling the targeted assessment of all relevant alteration classes from routine tissue samples. Methods: Here we interrogated &amp;gt;7,000 cancer exomes and transcriptomes, along with &amp;gt;30,000 array based cancer genomes to identify recurrent somatic alterations (mutations, copy number alterations [CNAs] and gene fusions) across solid tumors. From this analysis, we developed and validated an integrated multiplexed PCR based Ion Torrent next generation sequencing panel (Oncomine Cancer Research Panel [OCP]) targeting the actionable somatic cancer genome optimized for 20ng of formalin-fixed, paraffin-embedded (FFPE) tissue isolated DNA/RNA. Results: We validated the OCP using FFPE cell line mixtures, as well as a prospective cohort of 104 FFPE tumor specimens sent for concurrent clinical molecular testing, with &amp;gt;97% sensitivity and specificity for the presence/absence of KRAS, EGFR, BRAF and ALK point mutations, indels or gene fusions in this molecular testing cohort. We also applied the OCP to 100 lung cancers, identifying known and novel alterations, including ALK and ROS1 gene fusions. Lastly, applying the OCP to 116 prostate cancers, including 50 previously treated samples, we recapitulated known molecular subtypes, observed distinct profiles according to previous treatment and obtained 100% concordance for isoform specific TMPRSS2:ERG gene fusion detection compared to qPCR. Additionally, OCP profiling supports a novel molecular subtype of prostate cancer defined by IDH1 R132 hotspot mutations and informed on resistance mechanisms in a pre- and post-treatment sample pair. Importantly, 44%, 35% and 9% of patients in the molecular testing, lung and prostate cancer cohorts, respectively, harbored additional alterations (beyond routine molecular testing) associated with FDA approved or NCCN guideline referenced therapies. Conclusions: Through analysis of both DNA and RNA to assess the actionable somatic cancer genome, the validated OCP panel may have utility in both clinical and research settings. Citation Format: Daniel H. Hovelson, Andrew S. McDaniel, Bryan Johnson, Andi K. Cani, Kate Rhodes, Paul D. Williams, Chia-Jen Liu, Santhoshi Bandla, Catherine S. Grasso, Michael J. Quist, Seth Sadis, Daniel R. Rhodes, Scott A. Tomlins. Targeted amplicon-based next-generation sequencing of routine solid tumor specimens to detect clinically relevant somatic alterations. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-43.

Abstract 4666: Revolutionizing clinical care using prospective, institution-wide tumor sequencing

Abstract Identification of predictive and prognostic biomarkers is central to clinical oncology. Use of targeted next generation sequencing (NGS) is increasing in molecular diagnostics labs, however the feasibility and impact of its routine application across all tumor types is largely untested. We launched an institution-wide effort to generate targeted NGS data (Oncopanel) for invasive tumors of all consenting patients. We hypothesized that this approach could replace traditional targeted testing, generate robust data on copy number alterations and structural variants, and provide novel patient-specific observations to facilitate clinical trial enrollment. Illumina NGS was performed on libraries prepared with Agilent SureSelect custom-designed hybrid capture of 4430 exons from 275 genes plus selected introns of 30 genes. Results were analyzed by an internally-developed computational pipeline for mutations, small insertions/deletions, copy number variation and rearrangements. Variants were characterized according to predictive, prognostic, or diagnostic actionability. Data is available for the first 4291 cases sequenced. Oncopanel succeeded in 96% of specimens with adequate DNA. In a subset analysis performed on the first 1000 cases, assay success ranged from 83-100% according to tumor type; breast carcinoma was most prone to failure (p&amp;lt;0.0001). Median number of mutations per case was 8 (range 0-205) and was lowest in endocrine malignancies and highest in skin malignancies. Three percent of tumors were hypermutated with mutational signatures revealing distinct pathogenic underpinnings, including prior temozolomide therapy, microsatellite instability, and UV exposure. Compared to clinical testing, Oncopanel showed 100% accuracy for detection of KRAS and BRAF point mutations in colon adenocarcinoma, EGFR exon 19 deletion mutations in lung adenocarcinoma, and for EGFR amplification in glioblastoma. Oncopanel was 97.5% sensitive and 87.5% specific for 1p19q deletion as compared to aCGH or FISH and was 80% sensitive and 100% specific as compared to ALK FISH and additionally detected 2 ALK rearranged tumors for which FISH failed. In the overall cohort, 26% of patients had an actionable variant (most commonly KRAS) and 39% had alterations with implications for clinical trial enrollment (most commonly in PI3K/PTEN/AKT pathway). TP53 was the most commonly altered gene overall and the most likely to be co-mutated with oncogenic drivers. High level amplifications were most common for EGFR, MDM2, CDK4, ERBB2, MYC, and CCND1. Two-copy deletions were most common for CDKN2A/B, followed by TP53 and PTEN. In several cases, Oncopanel data uncovered alterations that informed diagnosis and treatment of difficult-to-classify tumors. These efforts demonstrate that high quality, high throughput NGS data can be generated prospectively on an institutional level, thereby informing disease course and therapeutic options at an unprecedented scale. Citation Format: Lynette M. Sholl, Elizabeth Garcia, Yonghui Jia, Matthew Ducar, Bernard Fendler, Priyanka Shivdasani, Frank C. Kuo, Azra H. Ligon, Barrett J. Rollins, Neal I. Lindeman, Laura E. MacConaill. Revolutionizing clinical care using prospective, institution-wide tumor sequencing. [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 4666. doi:10.1158/1538-7445.AM2015-4666

Molecular analysis of residual ThinPrep material from thyroid FNAs increases diagnostic sensitivity.

Molecular testing for genetic alterations associated with malignancy is a potential triage approach for thyroid fine-needle aspiration (FNA) samples with indeterminate cytology. Because liquid-based FNA material allows for efficient RNA extraction, the authors analyzed residual material for common point mutations and rearrangements. Thyroid FNAs were classified according to The Bethesda System for Reporting Thyroid Cytopathology after routine ThinPrep slide preparation. Residual samples from malignant and indeterminate cases were submitted for molecular analysis, along with a random cohort of nondiagnostic and benign aspirates. Blinded analysis of BRAF and RAS point mutations and RET/PTC and PAX8/PPARγ rearrangements was correlated with subsequent follow-up. Adequate results were obtained in 402 of 597 cases (67%). Mutations or rearrangements were detected in 24 of 117 cytologically indeterminate specimens (21%) (17 RAS rearrangements, 6 BRAF rearrangements, and 1 PAX8/PPARγ rearrangement). BRAF mutations were preferentially associated with malignant cytologic diagnoses (22 of 42 cases; 52%), with less frequent detection in the suspicious for malignancy category (4 of 27 cases; 15%) and very low detection in all other categories (1%-4%). Surgical follow-up confirmed malignancy in all 21 BRAF-mutated cases, 42% of RAS-mutated cases (10 of 24 cases), and 37% of cases with no detected mutation (39 of 105 cases). Molecular analysis is feasible on residual ThinPrep material with the advantage of not requiring additional FNA procedures. The majority of BRAF mutations are identified in cases classified cytologically as malignant, and, to a lesser extent, as suspicious for malignancy. The usefulness of BRAF testing is limited by the low rate of BRAF-positive cases in other categories, thereby highlighting the need to identify other genetic drivers of clinically aggressive thyroid cancers.

Therapeutic Advances and Treatment Options in Metastatic Melanoma.

Over the past several years, management of advanced melanoma has been transformed by the development and approval of novel therapeutic approaches. Genetically targeted therapies are now effective treatment options for the approximately 50% of patients whose melanomas harbor activating point mutations in BRAF. Combination regimens of small-molecule inhibitors have been developed to delay the onset of acquired resistance. Specifically, combined BRAF and MEK inhibition improves response rates and survival compared with single-agent BRAF inhibitors and has now received regulatory approval. During the same time frame, excitement has surrounded the development of immunotherapy with checkpoint inhibitors. New immune checkpoint inhibitors blocking cytotoxic T lymphocyte antigen-4 (CTLA4) or programmed death-1 receptor/ligand (PD-1/PD-L1) improve patient outcomes by promoting an antitumor immune response. These agents have been associated with an increasing number of durable responses and are being developed in various combinations. In this review, we discuss the development of these targeted and immune therapies, review current patient management, and highlight future directions.

Role and complexity of next-generation sequencing in melanoma.

Role and complexity of next-generation sequencing in melanoma.

Análisis molecular del cáncer de colon esporádico

In Chile, colorectal cancer (CRC) is often diagnosed in late stages. Thus, surgical treatment must be complemented with chemotherapy. KRAS mutations and microsatellite instability have been detected in these tumors. However, the response to treatment in patients without KRAS mutations varies and requires a better understanding.To determine the frequency and distribution of somatic point mutations in KRAS, BRAF and PIK3CA genes and microsatellite instability status (MSI) in patients with colon cancer (CC).A prospective observational study of patients undergoing surgery for colon cancer. Tumor-derived DNA was analyzed by polymerase chain reaction (PCR) for the most frequent mutations of KRAS, BRAF and PIK3CA. PCR was also used to analyze MSI.Fifty-eight patients with sporadic CC were analyzed, 16 showed KRAS mutations (G12R, G12D, G12V, G13D) and out of the 42 patients that did not show any mutation, 10 had mutations in BRAF (V600E) and PIK3CA (E542K, E545D, E545K, Q546E, H1047R). BRAF mutations alone or in combination with PIK3CA mutations were observed in 27% of high MSI tumors and in 2% of tumors without instability (p < 0.049). A higher percentage of high MSI tumors were located in the right colon (p < 0.001), and showed BRAF mutation (p < 0.020).The highest percentage of high MSI and BRAF mutations was observed in the right colon. Therefore, this study suggests the presence of different molecular features between right and left colon tumors that should be considered when defining the therapeutic management.

Molecular diagnostics of fine needle aspiration for the presurgical screening of thyroid nodules.

“The incidence of thyroid cancer, the most common endocrine malignancy, is rising. The two most common types of thyroid cancer are papillary and follicular” thyroid carcinomas. “Fine-needle aspiration (FNA) of thyroid nodules” can permit to detect many genetic mutations and other molecular alterations, including RAS and BRAF point mutations, PAX8/peroxisome proliferator-activated receptor (PPAR)γ and “RET/PTC rearrangements, occurring in thyroid papillary and follicular carcinomas” (more than 70% of cases), which can be used successfully to improve the diagnosis “and the management of patients with thyroid nodules”. The most extensive experience has been accumulated with “the diagnostic use of BRAF mutation”, which is highly specific for malignancy. “Testing FNA samples for a panel of mutations” that typically includes RAS, BRAF, PAX8/PPARγ and RET/PTC could permit to achieve the biggest diagnostic impact. “The accuracy of cancer diagnosis in thyroid nodules could be improved significantly using these and other emerging molecular markers”.

Feinnadelzytologie von Schilddrüsenknoten: Molekulare Diagnostik in der klinischen Routine

Results for the detection of point mutations and rearrangements have thus far been obtained by fresh material of fine needle aspiration cytology (FNAC). After a first retrospective study we report on the diagnostic detection in routinely obtained, consecutive air-dried FNAC smears. RNA and DNA was extracted from 154 consecutive routine air-dried FNAC smears: 80 with microfollicular proliferation (MFP), 45 with follicular neoplasia (FN), 26 with the cytological diagnosis of papillary carcinomas (PTC) and 3 which were suspicious for malignancy. PAX8/PPARG and RET/PTC3 rearrangements were detected by qPCR, while BRAF and RAS point mutations were detected by pyrosequencing. Only 0.7 % and 5.3 % of the routine air-dried FNAC samples did not allow analysis of a point mutation or rearrangements, respectively. NRAS mutations could be detected in 7 MFP smears, and in one of FN and PTC samples, respectively. HRAS mutations were detected in one MPF and one FN sample. A KRAS mutation was only detected in one FN sample, whereas BRAF mutations were detected in 20 samples with PTC (but in no other sample). PAX8/PPARG was detected in 2 MFP samples, while RET/PTC was detected in only one MFP sample. In total, 13.8 % MFP-FNAC, 6.7 % FN-FNAC, and 80.8 % PTC-FNAC samples harbored a mutation. These results demonstrate that rearrangements and point mutations can be detected in routinely obtained air-dried FNAC samples.

Molecular Profiles of Papillary Thyroid Tumors Have Been Changing in the Last Decades: How Could We Explain It?

The molecular profile of thyroid tumors can be discovered today in at least 70–80% of cases. The most common alterations are point mutations of BRAF and RAS followed by RET/PTC rearrangements. Other less frequent oncogenes involved are PIK3CA, TP53, TSHR, PTEN, GNAS, and CTNNB1 (1), but taken together they are still not able to account for 100% of cases. According to the data reported and collected so far (http://www.sanger.ac.uk/genetics/CGP/), BRAFV600E point mutations are present in about 45% of papillary thyroid cancer (PTC), with a higher rate in classical and tall cell variants and a rather low prevalence in follicular variant (2). There are exceptions with higher prevalence of BRAFV600E in PTC found in areas of China with a very high iodine content in the drinking water (3) and PTC found in the volcanic area of Mount Etna in Sicily where the drinking water is particularly rich in several elements, such as boron, iron, manganese, and vanadium, often present in concentrations exceeding usual maximum limits (4). The observations of Jung et al (5), showing a high prevalence of BRAFV600E mutation in a series of PTC diagnosed in the United States over a 35-year period, from 1974 to 2009, are of great relevance in this regard, associated with a significant increase of its prevalence in the classical variant of PTC over these last decades. The first study that indicated an increase of the prevalence of BRAFV600E mutation was reported in 2005 in a UK series, and the authors hypothesized that some environmental/etiological agent(s) could be responsible for the changing molecular features of PTC over time (6). Similar results were reported 6 years later by Mathur et al (7) in a series of PTC patients reported from California, and the hypothesis in this study was that the increased prevalence of the BRAFV600E mutation could be responsible for the increasing rate of PTC. In 2012, we also reported a significant increasing rate of BRAFV600E mutation in an Italian multicenter study (8), and it was interesting to observe that the increase was similar when the analysis was separately performed in the four different groups of PTC patients (from Pisa, Milan, Perugia, and Catania). Because the reported series are really very heterogeneous from a geographical point of view, it is conceivable that the increased prevalence of BRAFV600E mutation is a worldwide phenomenon, making it plausible that only a common, or very similar, reason could explain this trend. It is known that higher iodine intake has been found to be associated with a higher incidence of PTC (9), and it is notable that programs of iodine prophylaxis appear to be associated with an increase in the prevalence of PTC and a decrease in follicular histology (follicular thyroid cancer [FTC]) (10–12). This evidence and the Chinese report (3) may raise the question of a link between iodine intake, BRAFV600E mutation, and the development of increased incidence of PTC, but up to now, no experimental evidence that iodine can induce somatic gene alterations in follicular cells has been reported. Nevertheless, it is known that the iodine radical (I●), iodinium ion (I ) and the hypoiodous acid intermediate (IO[IOH]) are commonly formed in the follicular cell and represent reactive elements that together or as an alternative to H2O2 can determine cell oxidative stress, which is required to create conditions for thyroid cell proliferation (13–15). Further experimental studies on this putative relationship between