Towards standardization of next-generation sequencing of FFPE samples for clinical oncology: intrinsic obstacles and possible solutions

Иванов, М. В.; Лактионов, К. П.; Бредер, В. В.; Chernenko, Polina; Новикова, Е. И.; Telysheva, E N; Musienko, Sergey; Baranova, Ancha; Mileyko, Vladislav

doi:10.1186/s12967-017-1125-8

Cited by 29 publications

(24 citation statements)

References 89 publications

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“…In addition, another important observation was that the genetic and medication information regarding the detected variants in these clinical sequencing reports were also insufficient. In common with the reporting recommendations for formalin‐fixed paraffin‐embedded (FFPE) tissue samples , evidence‐based categorization of cancer‐related variants is of critical importance. However, only six laboratories (9.1%, 6/66) classified and reported the detected variants according to the level of evidence.…”

Section: Resultsmentioning

confidence: 99%

“…Because comprehensive clinical reports is the cornerstone for identifying best treatment strategies, clinical reports without appropriate clinical interpretations of ctDNA genotypes and with insufficient medication information may make it difficult for clinicians to decipher the data and take appropriate actions . Hence, in common with the reporting recommendations for FFPE tissue samples , evidence‐based categorization of cancer‐related variants and sufficient medication information is recommended to be well documented in the ctDNA analysis reports, although they are not mandatory requirement. Meanwhile, as the most commonly performed manual interpretation is prone to miss less well‐known treatment options or recent therapies, laboratories should place a great deal of importance should be placed on updating informative databases in a timely manner.…”

Section: Discussionmentioning

confidence: 99%

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From Somatic Variants Toward Precision Oncology: An Investigation of Reporting Practice for Next-Generation Sequencing-Based Circulating Tumor DNA Analysis

et al. 2019

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Background With the accelerated development of next‐generation sequencing (NGS), identified variants, and targeted therapies, clinicians who confront the complicated and multifarious genetic information may not effectively incorporate NGS‐based circulating tumor DNA (ctDNA) analysis into routine patient care. Consequently, standardized ctDNA testing reports are of vital importance. In an effort to guarantee high‐quality reporting performance, we conducted an investigation of the current detection and reporting practices for NGS‐based ctDNA analysis. Materials and Methods A set of simulated ctDNA samples with known variants at known allelic frequencies and a corresponding case scenario were distributed to 66 genetic testing laboratories for ctDNA analysis. Written reports were collected to evaluate the detection accuracy, reporting integrity, and information sufficiency using 21 predefined criteria. Results Current reporting practices for NGS‐based ctDNA analysis were found to be far from satisfactory, especially regarding testing interpretation and methodological details. Only 42.4% of laboratories reported the results in complete concordance with the expected results. Moreover, 74.2% of reports only listed aberrations with direct and well‐known treatment consequences for the tumor type in question. Genetic aberrations for which experimental agents and/or drug access programs are available may thus be overlooked. Furthermore, methodological details for the interpretation of results were missing from the majority of reports (87.9%). Conclusion This proof‐of‐principle study suggests that the capacity for accurate identification of variants, rational interpretation of genotypes, comprehensive recommendation of potential medications, and detailed description of methodologies need to be further improved before ctDNA analysis can be formally implemented in the clinic. Implications for Practice Accurate, comprehensive, and standardized clinical sequencing reports can help to translate complex genetic information into patient‐centered clinical decisions, thereby shepherding precision oncology into daily practice. However, standards, guidelines, and quality requirements for clinical reports of next‐generation sequencing (NGS)‐based circulating tumor DNA (ctDNA) analysis are currently absent. By using a set of simulated clinical ctDNA samples and a corresponding case scenario, current practices were evaluated to identify deficiencies in clinical sequencing reports of ctDNA analysis. The recommendations provided here may serve as a roadmap for the improved implementation of NGS‐based ctDNA analysis in the clinic.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

From Somatic Variants Toward Precision Oncology: An Investigation of Reporting Practice for Next-Generation Sequencing-Based Circulating Tumor DNA Analysis

et al. 2019

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“…We conclude that Nextera Flex library preparation might be a better option for a low input DNA quantity. FFPE processing can have an effect on variant calling results 31 . In our study, samples of fresh cells and cells processed with FFPE were called with Strelka2, SomaticSniper, and MuTect2.…”

Section: Effect Of Non-analytical and Analytical Factors On Mutation mentioning

confidence: 99%

“…FFPE is known to cause G>T/C>A artifacts as well 31 . Trimmomatic and BFC were investigated for their ability to detect these errors.…”

Section: Effect Of Non-analytical and Analytical Factors On Mutation mentioning

confidence: 99%

Achieving reproducibility and accuracy in cancer mutation detection with whole-genome and whole-exome sequencing

Xiao¹

2019

Preprint

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Clinical applications of precision oncology require accurate tests that can distinguish cancerspecific mutations from errors introduced at each step of next generation sequencing (NGS). For NGS to successfully improve patient lives, discriminating between true mutations and artifacts is crucial. To date, no study has addressed the effects of cross site reproducibility together with the potentially influential interactions between biological, technical, and computational factors on the accurate identification of variants.Here we systematically interrogated somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy. Different types of samples with varying input amount and tumor purity were processed using multiple library construction protocols. Whole-genome (WGS) and whole-exome sequencing (WES) were carried out at six sequencing centers followed by processing with nine bioinformatics pipelines to evaluate reproducibility. We identified artifacts of C>A mutations in WES due to sample and library processing and highlighted limitations of bioinformatics tools for artifact detection and removal.By examining the interactions and effects of NGS platforms, library preparation protocol, tumor content, read coverage, and bioinformatics processes concomitantly, here we recommend

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“…Next-generation sequencing (NGS) has been widely used in clinical molecular testing in recent years. Compared to conventional methods, NGS is able to detect multiple genetic alterations in a single assay, with higher sensitivity, fewer amounts of input DNA, shorter time, and lower cost (Ivanov et al, 2017). However, there are still many challenges faced in the molecular pathological laboratories, including optimization and familiarization of NGS testing, design and operation of bioinformatics pipeline, and interpretation and reporting of sequence variants.…”

Section: Introductionmentioning

confidence: 99%

Subjecting appropriate lung adenocarcinoma samples to next‐generation sequencing‐based molecular testing: challenges and possible solutions

Qiu

Ling

et al. 2018

Molecular Oncology

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Next‐generation sequencing (NGS) has recently been rapidly adopted in the molecular diagnosis of cancer, but it still faces some obstacles. In this study, 665 lung adenocarcinoma samples (558 TKI‐naive and 107 TKI‐relapsed samples) were interrogated using NGS, and the challenges and possible solutions of subjecting appropriate tissue samples to NGS testing were explored. The results showed that lower frequencies of HER2/BRAF/PIK3CA and acquired EGFR T790M mutations were observed in biopsy samples with <20% tumor cellularity than in those with ≥20%, but there were no significant differences in the frequencies of EGFR or KRAS mutations. Moreover, tumor heterogeneity was assessed by heterogeneity score (HS), which was calculated through multiplying by 2 the mutant allele frequency (MAF) of tumor cells. In TKI‐naive samples, intratumor heterogeneity could occur in EGFR,KRAS,HER2,BRAF, and PIK3CA mutant tumors, but the degree was variable. Higher EGFR, but lower BRAF and PIK3CA HS values were observed compared with KRAS HS. In TKI‐relapsed samples, analysis of concomitant sensitizing EGFR and T790M MAFs showed that intratumor heterogeneity was common in acquired EGFR T790M mutant tumors. The mutational status between primary and metastatic tumors was usually concordant, but KRAS,HER2, and PIK3CA HS were significantly higher in metastatic tumors than in primary tumors. Additionally, the discordance rate of mutational status in multifocal lung adenocarcinomas diagnosed as equivocal or multiple primary tumors was high. Together, our findings demonstrate that a comprehensive quality assessment is necessary during tissue process to mitigate the challenges of poor tumor cellularity, tumor heterogeneity, and multifocal clonally independent tumors.

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Towards standardization of next-generation sequencing of FFPE samples for clinical oncology: intrinsic obstacles and possible solutions

Cited by 29 publications

References 89 publications

From Somatic Variants Toward Precision Oncology: An Investigation of Reporting Practice for Next-Generation Sequencing-Based Circulating Tumor DNA Analysis

From Somatic Variants Toward Precision Oncology: An Investigation of Reporting Practice for Next-Generation Sequencing-Based Circulating Tumor DNA Analysis

Achieving reproducibility and accuracy in cancer mutation detection with whole-genome and whole-exome sequencing

Subjecting appropriate lung adenocarcinoma samples to next‐generation sequencing‐based molecular testing: challenges and possible solutions

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