Genomic aberrations frequently alter chromatin regulatory genes in chordoma.

Chordoma is a rare primary bone neoplasm that is resistant to standard chemotherapies. Despite aggressive surgical management, local recurrence and metastasis is not uncommon. To identify the specific genetic aberrations that play key roles in chordoma pathogenesis, we utilized a genome-wide high-resolution SNP-array and next generation sequencing (NGS)-based molecular profiling platform to study 24 patient samples with typical histopathologic features of chordoma. Matching normal tissues were available for 16 samples. SNP-array analysis revealed nonrandom copy number losses across the genome, frequently involving 3, 9p, 1p, 14, 10, and 13. In contrast, copy number gain is uncommon in chordomas. Two minimum deleted regions were observed on 3p within a ∼8 Mb segment at 3p21.1-p21.31, which overlaps SETD2, BAP1 and PBRM1. The minimum deleted region on 9p was mapped to CDKN2A locus at 9p21.3, and homozygous deletion of CDKN2A was detected in 5/22 chordomas (∼23%). NGS-based molecular profiling demonstrated an extremely low level of mutation rate in chordomas, with an average of 0.5 mutations per sample for the 16 cases with matched normal. When the mutated genes were grouped based on molecular functions, many of the mutation events (∼40%) were found in chromatin regulatory genes. The combined copy number and mutation profiling revealed that SETD2 is the single gene affected most frequently in chordomas, either by deletion or by mutations. Our study demonstrated that chordoma belongs to the C-class (copy number changes) tumors whose oncogenic signature is non-random multiple copy number losses across the genome and genomic aberrations frequently alter chromatin regulatory genes. © 2016 Wiley Periodicals, Inc.

Consistent copy number changes and recurrent PRKAR1A mutations distinguish Melanotic Schwannomas from Melanomas: SNP-array and next generation sequencing analysis.

Melanotic Schwannomas (MS) are rare tumors that share histological features with melanocytic tumors and schwannomas. However, their genetics are poorly understood. To elucidate the genetic characteristics of MS, we performed genome-wide studies in a series of cases. Twelve MS cases were available for the study. Genomic DNAs extracted from formalin-fixed paraffin embedded tumor tissues were subjected to copy number (CN) and allelic imbalance (AI) analysis by Single Nucleotide Polymorphism (SNP)-array and screened for mutations in coding exons of 341 key cancer-associated genes using a hybrid capture-based next-generation sequencing (NGS) assay. Sanger sequencing was used to further verify recurrent mutations detected by NGS study. SNP-array analysis revealed remarkably stereotypic chromosomal abnormalities in MS. Hypodiploidy was common, typically involving monosomies of chromosomes 1, 2, and 17. All 12 samples showed mutations in PRKAR1A gene, including 2 cases with 2 mutations each. The 14 mutations were scattered across PRKAR1A, and most were inactivating mutations. AI on 17q, presenting as loss of heterozygosity with or without CN losses, combined with a PRKAR1A mutation was observed in 9/12 MS cases. The remaining 3 cases included the two samples harboring two mutations in PRKAR1A. MS exhibits a stereotypic pattern of chromosomal losses. In contrast, melanomas are typically characterized by the presence of multiple CN aberrations, without demonstrable differences in the frequency of losses and gains. Inactivation of both alleles of PRKAR1A by "two hits" observed in almost all cases underscores the central role of PRKAR1A in the pathogenesis of this neoplasm. © 2015 Wiley Periodicals, Inc.

Sequencing of 279 cancer genes in ampullary carcinoma reveals trends relating to histologic subtypes and frequent amplification and overexpression of ERBB2 (HER2).

The biological relevance of histological subtyping of ampullary carcinoma into intestinal vs pancreaticobiliary types remains to be determined. In an effort to molecularly profile these subtypes of ampullary carcinomas, we conducted a two-phase study. In the discovery phase, we identified 18 pancreatobiliary-type ampullary carcinomas and 14 intestinal-type ampullary carcinomas using stringent pathologic criteria and performed next-generation sequencing targeting 279 cancer-associated genes on these tumors. Although the results showed overlapping of genomic alterations between the two subtypes, trends including more frequent KRAS alterations in pancreatobiliary-type ampullary carcinoma (61 vs 29%) and more frequent mutations in APC in intestinal-type ampullary carcinoma (43 vs 17%) were observed. Of the entire cohort of 32 tumors, the most frequently mutated gene was TP53 (n=17); the most frequently amplified gene was ERBB2 (n=5); and the most frequently deleted gene was CDKN2A (n=6). In the second phase of the study, we aimed at validating our observation on ERBB2 and assessed ERBB2 amplification and protein overexpression in a series of 100 ampullary carcinomas. We found that (1) gene amplification and immunohistochemical overexpression of ERBB2 occurred in 13% of all ampullary carcinomas, therefore providing a potential target for anti-HER2 therapy in these tumors; (2) amplification and immunohistochemical expression correlated in all cases, thus indicating that immunohistochemistry could be used to screen tumors; and (3) none of the 14 ERBB2-amplified tumors harbored any downstream driver mutations in KRAS/NRAS, whereas 56% of the cases negative for ERBB2 amplification did, an observation clinically pertinent as downstream mutations may cause primary resistance to inhibition of EGFR family members.

Clinical Experience of Cerebrospinal Fluid-Based Liquid Biopsy Demonstrates Superiority of Cell-Free DNA over Cell Pellet Genomic DNA for Molecular Profiling.

Cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) offers unique opportunities for genomic profiling of tumors involving the central nervous system but remains uncommonly used in clinical practice. We describe our clinical experience using cfDNA from CSF for routine molecular testing using Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets (targeting 468 cancer-related genes). In all, 148 cfDNA samples were assessed, comparing results of cfDNA versus genomic DNA (gDNA; gDNA from cell pellets) derived from the same CSF sample and the primary tumor. Of these, 71.6% (106/148) were successfully sequenced. Somatic alterations (mutations and fusions) were observed in 70.8% (75/106) of the samples; 97.3% (73/75) comprised variants confirming central nervous system involvement by a previously diagnosed tumor, 14.7% (11/75) had additional variants consistent with a therapy-related resistance mechanism, and 2.7% (2/75) had variants that independently diagnosed a new primary. Among samples with paired cfDNA and gDNA sequencing results, cfDNA was more frequently positive for at least one mutation [43.6% (55/126) versus 19.8% (25/126)] and harbored 1.6× more mutations (6.94 versus 4.65; P = 0.005), with higher mean variant allele fractions (41.1% versus 13.0%; P < 0.0001). Among mutation-positive cfDNAs, the corresponding gDNA was frequently negative (44.6%; 25/55) or failed sequencing (17.8%; 9/55). Routine molecular profiling of cfDNA is superior to gDNA from CSF, facilitating the capture of mutations at high variant allele frequency, even in the context of a negative cytology.

Enhanced specificity of clinical high-sensitivity tumor mutation profiling in cell-free DNA via paired normal sequencing using MSK-ACCESS.

Circulating cell-free DNA from blood plasma of cancer patients can be used to non-invasively interrogate somatic tumor alterations. Here we develop MSK-ACCESS (Memorial Sloan Kettering - Analysis of Circulating cfDNA to Examine Somatic Status), an NGS assay for detection of very low frequency somatic alterations in 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance of MSK-ACCESS, we report results from 681 prospective blood samples that underwent clinical analysis to guide patient management. Somatic alterations are detected in 73% of the samples, 56% of which have clinically actionable alterations. The utilization of matched normal sequencing allows retention of somatic alterations while removing over 10,000 germline and clonal hematopoiesis variants. Our experience illustrates the importance of analyzing matched normal samples when interpreting cfDNA results and highlights the importance of cfDNA as a genomic profiling source for cancer patients.

Reliable Clinical MLH1 Promoter Hypermethylation Assessment Using a High-Throughput Genome-Wide Methylation Array Platform.

Clinical testing for MLH1 promoter hypermethylation status is important in the workup of patients with MLH1-deficient colorectal and uterine carcinomas when evaluating patients for Lynch syndrome. Current assays use single gene-based methods to assess promoter hypermethylation. Herein, we describe the development and report the performance of a clinical assay for MLH1 promoter hypermethylation using the Infinium methylationEPIC (850k) bead-array platform. Using four cytosine-guanine dinucleotide (CpG) sites within the MLH1 gene promoter, a qualitative MLH1 promoter hypermethylation assay was developed and validated using 63 gastrointestinal and uterine carcinoma samples of known hypermethylation status based on a pyrosequencing reference test. The array-based method achieves clinically robust and reproducible results at an analytical sensitivity level of 8%. Of importance, the 850k array contains probes targeting >850,000 additional CpG sites across the genome, covering sites in most known genes as well as important enhancer regions provided by the Encyclopedia of DNA Elements and Functional Annotation of The Mammalian Genome projects. Thus, the testing modality presented may also be applied to determine the methylation status of other clinically relevant genes or regulatory regions, potentially providing a single laboratory testing workflow for all clinical methylation assays. Furthermore, the concomitant acquisition of genome-wide methylation information provides a workflow that seamlessly enables wider translational epigenetic research.

Genomic Profiling Identifies Association of IDH1/IDH2 Mutation with Longer Relapse-Free and Metastasis-Free Survival in High-Grade Chondrosarcoma.

PURPOSE: Chondrosarcomas are the second most common primary malignant bone tumors. Although histologic grade is the most important factor predicting the clinical outcome of chondrosarcoma, it is subject to interobserver variability. Isocitrate dehydrogenase 1 (IDH1) and IDH2 hotspot mutations were recently found to be frequently mutated in central chondrosarcomas. However, a few published articles have been controversial regarding the association between IDH1/IDH2 mutation status and clinical outcomes in chondrosarcomas. EXPERIMENTAL DESIGN: We performed hotspot sequencing of IDH1 and IDH2 genes in 89 central chondrosarcomas and targeted next-generation sequencing in 54 of them, and then correlated the IDH1/IDH2 mutation status with the patient's clinical outcome. RESULTS: Although no association was discovered between IDH mutation status and the patient's overall survival, IDH1/IDH2 mutation was found to be associated with longer relapse-free and metastasis-free survival in high-grade chondrosarcomas. Genomic profiling reveals TERT gene amplification and ATRX mutation, for the first time, in addition to TERT promoter mutation in a subset (6/30, 20%) of high-grade and dedifferentiated chondrosarcomas. These abnormalities in telomere genes are concurrent with IDH1/IDH2 mutation and with CDKN2A/2B deletion or TP53 mutation, suggesting a possible association and synergy among these genes in chondrosarcoma progression. We found 21% of patients with chondrosarcoma also had histories of second malignancies unrelated to cartilaginous tumors, suggesting possible unknown genetic susceptibility to chondrosarcoma. CONCLUSIONS: IDH1/IDH2 mutations are associated with longer relapse-free and metastasis-free survival in high-grade chondrosarcomas, and they tend to co-occur with TERT mutations and with CDKN2A/2B and TP53 alterations in a subset of high-grade chondrosarcomas.

Characterization of ozonated vegetable oils by spectroscopic and chromatographic methods.

In this work the effect of ozonation on olive oil, soybean oil, oleic-, linoleic- and linolenic acid was studied. The effects of ozonation time on the oils and acids were analyzed by 1H, 13C NMR. Further, the peroxide- and acid values, the viscosity and the molar mass were determined for pure and ozonated oils. The fatty chains in both ozonated oils showed a gradual decrease of unsaturation with the gradual increase of ozonation time. Reaction products were identified according to Criegee mechanism. The major product in the early stage of the reaction was ozonide. The disappearance of unsaturation and formation of ozonide was almost equal. Ozonation increased the peroxide and acid values for both oils, the increase being higher for soybean oil. After long ozonation times higher molar mass species, as well as low molar mass species were observed. These are interpreted as oligomeric ozonides and cross-ozonides, respectively.

Reliable Pan-Cancer Microsatellite Instability Assessment by Using Targeted Next-Generation Sequencing Data.

PURPOSE: Microsatellite instability (MSI)/mismatch repair (MMR) status is increasingly important in the management of patients with cancer to predict response to immune checkpoint inhibitors. We determined MSI status from large-panel clinical targeted next-generation sequencing (NGS) data across various solid cancer types. METHODS: The MSI statuses of 12,288 advanced solid cancers consecutively sequenced with Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets clinical NGS assay were inferred by using MSIsensor, a program that reports the percentage of unstable microsatellites as a score. Cutoff score determination and sensitivity/specificity were based on MSI polymerase chain reaction (PCR) and MMR immunohistochemistry. RESULTS: By using an MSIsensor score ≥ 10 to define MSI high (MSI-H), 83 (8%) of 996 colorectal cancers (CRCs) and 42 (16%) of 260 uterine endometrioid cancers (UECs) were MSI-H. Validation against MSI PCR and/or MMR immunohistochemistry performed for 138 (24 MSI-H, 114 microsatellite stable [MSS]) CRCs, and 40 (15 MSI-H, 25 MSS) UECs showed a concordance of 99.4%. MSIsensor also identified 68 MSI-H/MMR-deficient (MMR-D) non-CRC/UECs. Of 9,591 non-CRC/UEC tumors with MSS MSIsensor status, 456 (4.8%) had slightly elevated scores(≥3 and <10) of which 96.6% with available material were confirmed to be MSS by MSI PCR. MSI-H was also detected and confirmed in three non-CRC/UECs with low exonic mutation burden (< 20). MSIsensor correctly scored all 15 polymerase ε ultra-mutated cancers as negative for MSI. CONCLUSION: MSI status can be reliably inferred by MSIsensor from large-panel targeted NGS data. Concurrent MSI testing by NGS is resource efficient, is potentially more sensitive for MMR-D than MSI PCR, and allows identification of MSI-H across various cancers not typically screened, as highlighted by the finding that 35% (68 of 193) of all MSI-H tumors were non-CRC/ UEC.

Comprehensive detection of germline variants by MSK-IMPACT, a clinical diagnostic platform for solid tumor molecular oncology and concurrent cancer predisposition testing.

BACKGROUND: The growing number of Next Generation Sequencing (NGS) tests is transforming the routine clinical diagnosis of hereditary cancers. Identifying whether a cancer is the result of an underlying disease-causing mutation in a cancer predisposition gene is not only diagnostic for a cancer predisposition syndrome, but also has significant clinical implications in the clinical management of patients and their families. METHODS: Here, we evaluated the performance of MSK-IMPACT (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets) in detecting genetic alterations in 76 genes implicated in cancer predisposition syndromes. Output from hybridization-based capture was sequenced on an Illumina HiSeq 2500. A custom analysis pipeline was used to detect single nucleotide variants (SNVs), small insertions/deletions (indels) and copy number variants (CNVs). RESULTS: MSK-IMPACT detected all germline variants in a set of 233 unique patient DNA samples, previously confirmed by previous single gene testing. Reproducibility of variant calls was demonstrated using inter- and intra- run replicates. Moreover, in 16 samples, we identified additional pathogenic mutations other than those previously identified through a traditional gene-by-gene approach, including founder mutations in BRCA1, BRCA2, CHEK2 and APC, and truncating mutations in TP53, TSC2, ATM and VHL. CONCLUSIONS: This study highlights the importance of the NGS-based gene panel testing approach in comprehensively identifying germline variants contributing to cancer predisposition and simultaneous detection of somatic and germline alterations.

Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients.

Tumor molecular profiling is a fundamental component of precision oncology, enabling the identification of genomic alterations in genes and pathways that can be targeted therapeutically. The existence of recurrent targetable alterations across distinct histologically defined tumor types, coupled with an expanding portfolio of molecularly targeted therapies, demands flexible and comprehensive approaches to profile clinically relevant genes across the full spectrum of cancers. We established a large-scale, prospective clinical sequencing initiative using a comprehensive assay, MSK-IMPACT, through which we have compiled tumor and matched normal sequence data from a unique cohort of more than 10,000 patients with advanced cancer and available pathological and clinical annotations. Using these data, we identified clinically relevant somatic mutations, novel noncoding alterations, and mutational signatures that were shared by common and rare tumor types. Patients were enrolled on genomically matched clinical trials at a rate of 11%. To enable discovery of novel biomarkers and deeper investigation into rare alterations and tumor types, all results are publicly accessible.

GNAS Mutations in Fibrous Dysplasia: A Comparative Study of Standard Sequencing and Locked Nucleic Acid PCR Sequencing on Decalcified and Nondecalcified Formalin-fixed Paraffin-embedded Tissues.

It is well known that fibrous dysplasia (FD) is characterized by the presence of activating mutations involving G-nucleotide binding protein-α subunit (GNAS) involving codon R201 and rarely codon 227 with a mutation frequency between 45% and 93%. Herein, we investigate the sensitivity of detection of GNAS mutations in exons 8 and 9 using a standard and a highly sensitive locked nucleic acid polymerase chain reaction (LNA-PCR) sequencing in 52 cases of FD. In view of the recent report of GNAS mutations in a small number of low-grade osteosarcomas, we also tested in addition 12 cases of low-grade osteosarcomas. GNAS exon 8 mutations p.R201H (31%), p.R201C (15%), and p.R201S (2%) were identified in 50% of FD cases. LNA-PCR sequencing identified only 1 positive case within the mutation negative cases tested by standard PCR and Sanger sequencing. No mutations were identified in any of the low-grade osteosarcomas by standard and LNA-PCR sequencing. There was no association between age, site, size, specimen type, and mutational status. No exon 9 or codon 227 mutations were identified in any of tested cases. There was a significant difference in the sensitivity of the assay between decalcified and nondecalcified FDs (31% vs. 70%, P=0.002). LNA-PCR has no added value in enhancing detection sensitivity for GNAS mutations in FD. In addition to decalcification, innate somatic mosaicism contributes to the decreased sensitivity in mutation detection.

The molecular landscape of extraskeletal osteosarcoma: A clinicopathological and molecular biomarker study.

Extraskeletal osteosarcoma (ESOSA) is a rare soft tissue neoplasm representing <5% of osteosarcomas and <1% of all soft-tissue sarcomas. Herein, we investigate the clinicopathological and molecular features of ESOSA and explore potential parameters that may affect outcome. Thirty-two cases were retrieved and histomorphology was reviewed. Clinical history and follow-up were obtained through electronic record review. DNA from formalin-fixed paraffin-embedded (FFPE) tissue was extracted and processed from 27 cases. Genome-wide DNA copy number (CN) alterations and allelic imbalances were analyzed by single nucleotide polymorphism array using Affymetrix OncoScan FFPE Assay. Massive high-throughput deep parallel sequencing was performed using a customized panel targeting 410 cancer genes. Log rank, Fisher's exact test and Cox proportional hazards were used for statistical analysis. In this series of 32 patients (male n = 12, female n = 20), the average age was 66 years (19-93) and median follow up was 24 months (range 6-120 months). Frequent genomic alterations included CN losses in tumour suppressor genes including CDKN2A (70%), TP53 (56%) and RB1 (49%). Mutations affecting methylation/demethylation, chromatin remodeling and WNT/SHH pathways were identified in 40%, 27%, and 27%, respectively. PIK3CA and TERT promoter variant mutations were identified in 11% of the cases. Cases harbouring simultaneous TP53 and RB1 biallelic CN losses were associated with worse overall survival and local recurrence (p = 0.04, p = 0.02, respectively). CDKN2A losses and positive margins were also associated with worse overall survival (p = 0.002; p = 0.03, respectively). Our findings suggest that age above 60, positive margin status, simultaneous biallelic TP53 and RB1 losses and CDKN2A loss are associated with a worse outcome in ESOSA. Comparison between conventional paediatric osteosarcoma and ESOSA shows that, while both share genetic similarities, there are notable dissimilarities and mechanistic differences in the molecular pathways involved in ESOSA.

Recurrent, truncating SOX9 mutations are associated with SOX9 overexpression, KRAS mutation, and TP53 wild type status in colorectal carcinoma.

PURPOSE: The extent to which the developmental transcription factor SOX9 functions as an oncogene or tumor suppressor in colorectal carcinoma (CRC) is debatable. We aimed to clarify the effect of SOX9 mutations on SOX9 protein expression and their association with known molecular subtypes and clinical characteristics in advanced CRC. EXPERIMENTAL DESIGN: Next generation sequencing data (MSK-IMPACT) from CRC patients was used to interrogate SOX9, KRAS, NRAS, BRAF, TP53, APC, and PIK3CA. Mutant and wild type (WT) SOX9 cases underwent immunohistochemical (IHC) staining to assess protein expression. SOX9 allele-specific copy number was assessed by Affymetrix Oncoscan array. RESULTS: SOX9 was mutated in 38 of 353 (10.7%) CRC, of which 82% were frameshift or nonsense. Compared to SOX9 WT, SOX9 mutation was strongly associated with coexistent mutant KRAS (p=0.0001) and WT TP53 (p=0.0004). SOX9 was overexpressed in both SOX9 mutant and WT CRC. Among SOX9 mutants, the highest expression was noted for truncating exon 3 mutants (mean H scores 239±105 versus 147±119, p value=0.02). Further, SOX9 truncating mutants with loss of the WT allele demonstrated protein overexpression indicating the WT protein was not required for protein stabilization. CONCLUSIONS: SOX9 is overexpressed in CRC, including those with recurrent distal truncating mutations. The latter has structural similarity to the oncogenic isoform MiniSOX9, which is distally truncated due to aberrant splicing. This information suggests that truncated SOX9 has oncogenic features. SOX9 mutations are highly enriched in KRAS mutant and TP53 wild type CRC; and may provide a therapeutic target in approximately 11% of CRC.

AKT1 E17K in Colorectal Carcinoma Is Associated with BRAF V600E but Not MSI-H Status: A Clinicopathologic Comparison to PIK3CA Helical and Kinase Domain Mutants.

UNLABELLED: The PI3K/AKT/mTOR pathway is activated through multiple mechanisms in colorectal carcinoma. Here, the clinicopathologic and molecular features of AKT1 E17K-mutated colorectal carcinoma in comparison with PIK3CA-mutated colorectal carcinoma are described in detail. Interestingly, in comparison with PIK3CA mutants, AKT1 E17K was significantly associated with mucinous morphology and concurrent BRAF V600E mutation. Among PIK3CA mutants, exon 21 mutations were significantly associated with BRAF V600E mutation, MSI-H status, and poor differentiation, while exon 10 mutations were associated with KRAS/NRAS mutations. Three of four AKT1 mutants with data from both primary and metastatic lesions had concordant AKT1 mutation status in both. Both AKT1- and PIK3CA-mutant colorectal carcinoma demonstrated frequent loss of PTEN expression (38% and 34%, respectively) and similar rates of p-PRAS 40 expression (63% and 50%, respectively). Both patients with AKT1 E17K alone had primary resistance to cetuximab, whereas 7 of 8 patients with PIK3CA mutation alone experienced tumor shrinkage or stability with anti-EGFR therapy. These results demonstrate that AKT1 E17K mutation in advanced colorectal carcinoma is associated with mucinous morphology, PIK3CA wild-type status, and concurrent RAS/RAF mutations with similar pattern to PIK3CA exon 21 mutants. Thus, AKT1 E17K mutations contribute to primary resistance to cetuximab and serve as an actionable alteration. IMPLICATIONS: This first systematic study of AKT1 and PIK3CA hotspot mutations and their association with cetuximab resistance and BRAF V600E mutation has important ramifications for the development of personalized medicine, particularly in identifying patient candidates for PI3K or AKT inhibitors.

Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT): A Hybridization Capture-Based Next-Generation Sequencing Clinical Assay for Solid Tumor Molecular Oncology.

The identification of specific genetic alterations as key oncogenic drivers and the development of targeted therapies are together transforming clinical oncology and creating a pressing need for increased breadth and throughput of clinical genotyping. Next-generation sequencing assays allow the efficient and unbiased detection of clinically actionable mutations. To enable precision oncology in patients with solid tumors, we developed Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT), a hybridization capture-based next-generation sequencing assay for targeted deep sequencing of all exons and selected introns of 341 key cancer genes in formalin-fixed, paraffin-embedded tumors. Barcoded libraries from patient-matched tumor and normal samples were captured, sequenced, and subjected to a custom analysis pipeline to identify somatic mutations. Sensitivity, specificity, reproducibility of MSK-IMPACT were assessed through extensive analytical validation. We tested 284 tumor samples with previously known point mutations and insertions/deletions in 47 exons of 19 cancer genes. All known variants were accurately detected, and there was high reproducibility of inter- and intrarun replicates. The detection limit for low-frequency variants was approximately 2% for hotspot mutations and 5% for nonhotspot mutations. Copy number alterations and structural rearrangements were also reliably detected. MSK-IMPACT profiles oncogenic DNA alterations in clinical solid tumor samples with high accuracy and sensitivity. Paired analysis of tumors and patient-matched normal samples enables unambiguous detection of somatic mutations to guide treatment decisions.

Effect of supplementation with chokeberry juice on the inflammatory status and markers of iron metabolism in rowers.

BACKGROUND: The aim of this study was to analyze the effect of supplementation with chokeberry (Aronia melanocarpa) juice on the levels of pro-inflammatory cytokines, hepcidin, and selected markers of iron metabolism in rowers subjected to exhaustive exercise. METHODS: This double-blind study included 19 members of the Polish Rowing Team. The subjects were randomly assigned to the supplemented group (n = 10), receiving 150 mL of chokeberry juice for 8 weeks, or to the placebo group (n = 9). The participants performed a 2000-m test on a rowing ergometer at the beginning and at the end of the preparatory camp. Blood samples were obtained from the antecubital vein prior to each exercise test, one minute after completing the test, and after a 24-hour recovery period. The levels of hepcidin, interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), ferritin, iron, uric acid, and myoglobin were determined, as well as the total iron-binding capacity, unbound iron-binding capacity, and total antioxidant capacity (TAC). RESULTS: Post-exercise, there was a significant increase in IL-6 and a significant decrease in the TAC in both groups, prior to and after supplementation with chokeberry juice. At the end of the experiment, the supplemented athletes showed significantly lower post-exercise levels of TNF-alpha and significantly higher TACs and iron levels than the controls. CONCLUSION: Supplementation with chokeberry juice results in an increase in the antioxidant activity of plasma and contributes significantly to reducing the TNF-alpha level.