scholarly journals Characterizations of Gene Alterations in Melanoma Patients from Chinese Population

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Yi Luo ◽  
Zhenzhen Zhang ◽  
Jianfan Liu ◽  
Linqing Li ◽  
Xuezheng Xu ◽  
...  
Keyword(s):  
Braf Mutations ◽  
Driver Genes ◽  
Cancer Driver ◽  
Formalin Fixed Paraffin ◽  
Significant Difference ◽  
The Status ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Comprehensive Genomic Profiling ◽  
Gene Alterations

Melanoma is a human skin malignant tumor with high invasion and poor prognosis. The limited understanding of genomic alterations in melanomas in China impedes the diagnosis and therapeutic strategy selection. We conducted comprehensive genomic profiling of melanomas from 39 primary and metastatic formalin-fixed paraffin-embedded (FFPE) samples from 27 patients in China based on an NGS panel of 223 genes. No significant difference in gene alterations was found between primary and metastasis melanomas. The status of germline mutation, CNV, and somatic mutation in our cohort was quite different from that reported in Western populations. We further delineated the mutation patterns of 4 molecular subgroups (BRAF, RAS, NF1, and Triple-WT) of melanoma in our cohort. BRAF mutations were more frequently identified in melanomas without chromic sun-induced damage (non-CSD), while RAS mutations were more likely observed in acral melanomas. NF1 and Triple-WT subgroups were unbiased between melanomas arising in non-CSD and acral skin. BRAF, RAS, and NF1 mutations were significantly associated with lymph node metastasis or presence of ulceration, implying that these cancer driver genes were independent prognostic factors. In summary, our results suggest that mutational profiles of malignant melanomas in China are significantly different from Western countries, and both gene mutation and amplification play an important role in the development and progression of melanomas.

Utilization of somatic comprehensive genomic profiling (CGP) to identify patients (pts) with pancreatic cancer (PC) that harbor germline DNA damage repair (DDR) gene alterations.

2020 ◽  
Vol 38 (4_suppl) ◽  
pp. 760-760
Author(s):  
Matthew Lasowski ◽  
Samantha Stachowiak ◽  
Igli Arapi ◽  
Kulwinder Dua ◽  
Abdul H. Khan ◽  
...  
Keyword(s):  
Gene Mutations ◽  
Cytotoxic Agents ◽  
Variant Of Uncertain Significance ◽  
Damage Repair ◽  
Formalin Fixed Paraffin ◽  
Uncertain Significance ◽  
Formalin Fixed Paraffin Embedded ◽  
Comprehensive Genomic Profiling ◽  
Germline Testing ◽  
Gene Alterations

760 Background: Somatic and germline DDR gene alterations in PC have been postulated to positively predict response to DNA damaging cytotoxic agents. Due to the relatively high prevalence of germline DDR gene alterations, germline testing is recommended in all pts with PC. We examined whether somatic CGP can be used to reliably identify PC pts that merit germline testing. Methods: We retrospectively reviewed the electronic medical records of PC pts who underwent both somatic CGP (utilizing the Foundation One assay) and germline testing. DDR gene mutations were categorized as somatic-pathogenic, somatic-variant of uncertain significance (VUS), germline-pathogenic and germline-VUS. For somatic testing, DNA was extracted from formalin fixed paraffin embedded (FFPE) clinical specimens and CGP was done on hybrid-capture, adaptor ligation based libraries to a mean coverage depth of > 600 for up to 315 genes plus 47 introns from 19 genes frequently rearranged in cancer. Germline genetic testing was performed on submitted blood or saliva samples, utilizing commercial assays; next generation or Sanger sequencing of all coding regions and adjacent intronic nucleotides were performed. Results: Ninety-three pts had somatic CGP data, 51 (55%) pts had both somatic CGP and germline data available. Among the 51 pts with both germline and somatic data available, DDR gene alterations that were somatic-pathogenic, germline-pathogenic, somatic-VUS and germline-VUS were present in 7 (13.7%), 7 (13.7%), 23 (45.1%) and 16 (31.4%) pts, respectively. Of the 7 pts with somatic-pathogenic alterations, 5 (71%) had a concordant germline alteration and of the 7 pts with germline-pathogenic alterations, 5 (71%) had a concordant somatic alteration. Of the 23 pts with somatic-VUSs, 12 (52%) had a concordant germline VUS and of the 16 pts with germline-VUSs, 12 (75%) had a concordant somatic VUS. Conclusions: Both somatic and germline DDR gene alterations are common in PC pts. Despite the relatively high concordance rate between somatic and germline pathogenic DDR gene alterations, somatic CGP will miss approximately one fourth of the germline DDR gene alterations.

scholarly journals TruSight Oncology 500: Enabling Comprehensive Genomic Profiling and Biomarker Reporting with Targeted Sequencing

2020 ◽  
Author(s):  
Chen Zhao ◽  
Tingting Jiang ◽  
Jin Hyun Ju ◽  
Shile Zhang ◽  
Jenhan Tao ◽  
...  
Keyword(s):  
Error Rates ◽  
Cancer Therapies ◽  
Next Generation ◽  
Clinical Biomarkers ◽  
Base Specificity ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Comprehensive Genomic Profiling ◽  
Tumor Mutational Burden

AbstractBackgroundAs knowledge of mechanisms that drive the development of cancer grows, there has been corresponding growth in therapies specific to a mechanism. While these therapies show improvements in patient outcomes, they can be expensive and are effective only for a subset of patients. These treatments drive interest in research focused on the assignment of cancer therapies based on aberrations in individual genes or biomarkers that assess the broader mutational landscape, including microsatellite instability (MSI) and tumor mutational burden (TMB).MethodsHere we describe the TruSight™ Oncology 500 (TSO500; Research Use Only) bioinformatics workflow. This tumor-only approach leverages the next-generation sequencing-based assay TSO500 to enable high fidelity determination of DNA variants across 523 cancer-relevant genes, as well as MSI status and TMB in formalin-fixed paraffin-embedded (FFPE) samples.ResultsThe TSO500 bioinformatic workflow integrates unique molecular identifier (UMI)-based error correction and a dual approach variant filtering strategy that combines statistical modeling of error rates and database annotations to achieve detection of variants with allele frequency approaching 5% with 99.9998% per base specificity and 99% sensitivity in FFPE samples representing a variety of tumor types. TMB determined using the tumor-only workflow of TSO500 correlated well with tumor-normal (N =170, adjusted R2=0.9945) and whole-exome sequencing (N=108, adjusted R2=0.933). Similarly, MSI status determined by TSO500 showed agreement (N=106, 98% agreement) with a MSI-PCR assay.ConclusionTSO500 is an accurate tumor-only workflow that enables researchers to systematically characterize tumors and identify the next generation of clinical biomarkers.

scholarly journals Composite pheochromocytoma/paraganglioma-ganglioneuroma: analysis of SDH and ATRX status and identification of frequent HRAS and BRAF mutations

2021 ◽  
Author(s):  
Jingci Chen ◽  
Yan Wu ◽  
Pengyan Wang ◽  
Huanwen Wu ◽  
Anli Tong ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Mutation Rates ◽  
Potential Treatment ◽  
Braf Mutations ◽  
Genetic Changes ◽  
Formalin Fixed Paraffin ◽  
The Status ◽  
Formalin Fixed Paraffin Embedded ◽  
Composite Pheochromocytoma ◽  
Formalin Fixed

Introduction: Composite pheochromocytoma/paraganglioma (CP) is a rare neoplasm with most cases presented as single reports. Little is known about its pathogenesis and relationship with ordinary pheochromocytoma (PCC) or paraganglioma (PGL). Our study is aimed at analyzing the status of SDH and ATRX and identifying novel genetic changes in CP. Methods: 18 CP cases were collected. SDH and ATRX status was screened by immunohistochemistry. Targeted region sequencing (TRS) was successfully performed on formalin-fixed paraffin-embedded tissues in 2 cases within 3 years. Based on the TRS result, Sanger sequencing of BRAF and HRAS was performed in 15 cases (including the 2 cases with TRS performed), with 3 cases excluded due to the limited amount of tissue. Results: Histopathologically, all the cases were composite PCC/PGL-ganglioneuroma (GN). The GN components were either closely admixed or juxtaposed with the PCC/PGL component, with highly variable percentage (10-80%). All cases stained positive for SDHB and ATRX. HRAS and BRAF mutations were identified during TRS. In the subsequent Sanger sequencing, 20.0% (3/15) harbored BRAF mutations (K601E and K601N) and 46.7% (7/15) harbored HRAS mutations (Q61R, Q61L, G13R). The mutation rates were both significantly higher than reported in ordinary PCC/PGL. Conclusions: We demonstrated that composite PCC/PGL-GN might be a unique entity with frequent HRAS and BRAF mutations rather than genetic changes of SDH and ATRX. Our findings revealed the possible pathogenesis of composite PCC/PGL-GN and provided clues for potential treatment targets.

scholarly journals The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Michal Marczyk ◽  
Chunxiao Fu ◽  
Rosanna Lau ◽  
Lili Du ◽  
Alexander J. Trevarton ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Rna Sequencing ◽  
Rna Extraction ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
The Impact ◽  
Formalin Fixed

Abstract Background Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. Methods Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. Results Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63–0.66) and between technical replicates (median expression difference 0.13–0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31–0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91–0.96). Conclusions The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

scholarly journals Time and tumor type (primary or metastatic) do not influence the detection of BRAF/NRAS mutations in formalin fixed paraffin embedded samples from melanomas

2016 ◽  
Vol 54 (11) ◽  
Author(s):  
Miriam Potrony ◽  
Celia Badenas ◽  
Bénédicte Naerhuyzen ◽  
Paula Aguilera ◽  
Joan Anton Puig-Butille ◽  
...  
Keyword(s):  
Melanoma Patient ◽  
Primary Melanoma ◽  
Sentinel Lymph Nodes ◽  
Tumor Type ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Complete Sequencing ◽  
Formalin Fixed ◽  
Tumor Area

AbstractBackground:Methods:DNA was obtained from 144 FFPE samples (62 primary melanoma, 43 sentinel lymph nodes [SLN] and 39 metastasis).Results:Complete sequencing results were obtained from 75% (108/144) of the samples, and at least one gene was sequenced in 89% (128/144) of them.Conclusions:Preserving sufficient tumor area in FFPE blocks is important. It is necessary to keep the FFPE blocks, no matter their age, as they are necessary to decide the best treatment for the melanoma patient.

scholarly journals An Integrated Framework for Genome Analysis Reveals Numerous Previously Unrecognizable Structural Variants in Leukemia Patients’ Samples

2019 ◽  
Author(s):  
Jie Xu ◽  
Fan Song ◽  
Emily Schleicher ◽  
Christopher Pool ◽  
Darrin Bann ◽  
...  
Keyword(s):  
Genome Mapping ◽  
Large Fraction ◽  
Genomic Analysis ◽  
Structural Variants ◽  
Driver Genes ◽  
Pediatric Leukemia ◽  
Cis Acting ◽  
Cancer Driver ◽  
The Status ◽  
Intergenic Regions

AbstractWhile genomic analysis of tumors has stimulated major advances in cancer diagnosis, prognosis and treatment, current methods fail to identify a large fraction of somatic structural variants in tumors. We have applied a combination of whole genome sequencing and optical genome mapping to a number of adult and pediatric leukemia samples, which revealed in each of these samples a large number of structural variants not recognizable by current tools of genomic analyses. We developed computational methods to determine which of those variants likely arose as somatic mutations. The method identified 97% of the structural variants previously reported by karyotype analysis of these samples and revealed an additional fivefold more such somatic rearrangements. The method identified on average tens of previously unrecognizable inversions and duplications and hundreds of previously unrecognizable insertions and deletions. These structural variants recurrently affected a number of leukemia associated genes as well as cancer driver genes not previously associated with leukemia and genes not previously associated with cancer. A number of variants only affected intergenic regions but caused cis-acting alterations in expression of neighboring genes. Analysis of TCGA data indicates that the status of several of the recurrently mutated genes identified in this study significantly affect survival of AML patients. Our results suggest that current genomic analysis methods fail to identify a majority of structural variants in leukemia samples and this lacunae may hamper diagnostic and prognostic efforts.

scholarly journals Expression Concordance of 325 Novel RNA Biomarkers between Data Generated by NanoString nCounter and Affymetrix GeneChip

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Lucas Delmonico ◽  
Said Attiya ◽  
Joan W. Chen ◽  
John C. Obenauer ◽  
Edward C. Goodwin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Pearson Correlation ◽  
Gene Expression Profiles ◽  
Standard Operating Procedure ◽  
Driver Genes ◽  
Technology Platforms ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Rna Biomarkers

Background. With the development of new drug combinations and targeted treatments for multiple types of cancer, the ability to stratify categories of patient populations and to develop companion diagnostics has become increasingly important. A panel of 325 RNA biomarkers was selected based on cancer-related biological processes of healthy cells and gene expression changes over time during nonmalignant epithelial cell organization. This “cancer in reverse” approach resulted in a panel of biomarkers relevant for at least 7 cancer types, providing gene expression profiles representing key cellular signaling pathways beyond mutations in “driver genes.” Objective. To further investigate this biomarker panel, the objective of the current study is to (1) validate the assay reproducibility for the 325 RNA biomarkers and (2) compare gene expression profiles side by side using two technology platforms. Methods and Results. We have mapped the 325 RNA transcripts and in a custom NanoString nCounter expression panel to be compared to all potential probe sets in the Affymetrix Human Genome U133 Plus 2.0. The experiments were conducted with 10 unique biological formalin-fixed paraffin-embedded (FFPE) breast tumor samples. Each site extracted RNA from four sections of 10-micron thick FFPE tissue over three different days by two different operators using an optimized standard operating procedure and quality control criteria. Samples were analyzed using mas5 in BioConductor and NanoStringNorm in R. Pearson correlation showed reproducibility between sites for all 60 samples with r=0.995 for Affymetrix and r=0.999 for NanoString. Correlation in multiple days and multiple users was for Affymetrix r=0.962−0.999 and for NanoString r=0.982−0.991. Conclusion. The 325 RNA biomarkers showed reproducibility in two technology platforms with moderate to high concordance. Future directions include performing clinical validation studies and generating rationale for patient selection in clinical trials using the technically validated assay.

Results of first proficiency test for KRAS testing with formalin-fixed, paraffin-embedded cell lines in China

2014 ◽  
Vol 52 (12) ◽  
Author(s):  
Rui Zhang ◽  
Yanxi Han ◽  
Jie Huang ◽  
Liang Ma ◽  
Yulong Li ◽  
...  
Keyword(s):  
Cell Lines ◽  
Laboratory Testing ◽  
Proficiency Test ◽  
Cultured Cell ◽  
Proficiency Panel ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

AbstractLaboratory testing forArtificial FFPE samples were prepared from cultured cell lines to construct a proficiency panel of 10 samples covering eightThe percentages of mutant

scholarly journals Comparison of Whole Genome Amplification Methods for Analysis of DNA Extracted from Microdissected Early Breast Lesions in Formalin-Fixed Paraffin-Embedded Tissue

ISRN Oncology ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Nona Arneson ◽  
Juan Moreno ◽  
Vladimir Iakovlev ◽  
Arezou Ghazani ◽  
Keisha Warren ◽  
...  
Keyword(s):  
Comparative Genomic Hybridization ◽  
Whole Genome Amplification ◽  
Comparative Genomic ◽  
Whole Genome ◽  
Genomic Hybridization ◽  
Genome Amplification ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

To understand cancer progression, it is desirable to study the earliest stages of its development, which are often microscopic lesions. Array comparative genomic hybridization (aCGH) is a valuable high-throughput molecular approach for discovering DNA copy number changes; however, it requires a relatively large amount of DNA, which is difficult to obtain from microdissected lesions. Whole genome amplification (WGA) methods were developed to increase DNA quantity; however their reproducibility, fidelity, and suitability for formalin-fixed paraffin-embedded (FFPE) samples are questioned. Using aCGH analysis, we compared two widely used approaches for WGA: single cell comparative genomic hybridization protocol (SCOMP) and degenerate oligonucleotide primed PCR (DOP-PCR). Cancer cell line and microdissected FFPE breast cancer DNA samples were amplified by the two WGA methods and subjected to aCGH. The genomic profiles of amplified DNA were compared with those of non-amplified controls by four analytic methods and validated by quantitative PCR (Q-PCR). We found that SCOMP-amplified samples had close similarity to non-amplified controls with concordance rates close to those of reference tests, while DOP-amplified samples had a statistically significant amount of changes. SCOMP is able to amplify small amounts of DNA extracted from FFPE samples and provides quality of aCGH data similar to non-amplified samples.

Sign in / Sign up

Export Citation Format

Share Document