Preanalytic Variables and Tissue Stewardship for Reliable Next-Generation Sequencing (NGS) Clinical Analysis

Abstract

it.An enduring goal of personalized medicine in cancer is the ability to identify patients who are likely to respond to specific therapies. Our growing understanding of the biology and molecular signatures of individual tumor types has facilitated the identification of predictive biomarkers and has led to an increasing number of diagnostic tests to be performed, often as serial and distinct assays on limited tumor specimens. The biomarker diagnostics field has been revolutionized by next-generation sequencing (NGS), which provides a comprehensive overview of the genomic profile of a tumor. Many preanalytic variables can influence the accuracy and reliability of NGS results. Standardization of preanalytic variables is, however, complicated by the plethora of specimen acquisition and processing methods. Variables across the tissue journey, including specimen acquisition, specimen fixation, and sectioning, as well as postfixation processing, such as nucleic acid extraction, library preparation, and choice of sequencing methods, are critical for the reliability of NGS analysis; thus, standardization would be beneficial. In this article, each step in the tissue journey is outlined, with specific focus on preanalytic variables that can influence NGS results. Practical considerations for standardization of these variables are provided to facilitate accurate, reliable, and reproducible NGS-based molecular characterization of tumors, ultimately informing diagnosis and guiding treatment.

Harmonization and Standardization of Panel-Based Tumor Mutational Burden Measurement: Real-World Results and Recommendations of the Quality in Pathology Study

“…16 There is a multitude of other wet-lab parameters, ranging from biological factors (e.g., tumor heterogeneity) and preanalytics (e.g., DNA quality) to sequencing (e.g., coverage) as well as bioinformatics parameters (e.g., germline subtraction) that can influence TMB scores. [22][23][24][25] Hence, as expected, absolute TMB values slightly varied. This scenario is not unknown to pathology in general and immune oncology response prediction in particular: just as for TMB, the established PD-L1 IHC assay for NSCLC quantifies a continuous variable in tumor cells ranging from 0% to 100% PD-L1 expressing cells, and several parameters, such as tumor heterogeneity and fixation, are known to influence PD-L1 scores.…”

Standardization of Sequencing Coverage Depth in NGS: Recommendation for Detection of Clonal and Subclonal Mutations in Cancer Diagnostics

“…Based on our experience in diagnostic NGS in haemato-oncology, we suggest to report at least following technical parameters: LOD, overall error of NGS assay (or at least sequencing error rate), the amount of DNA input, source, and quality of DNA, minimum coverage depth and the percentage of targeted bases sequenced at this minimum depth, total number of target reads covering variant region and number of reads supporting the variant. Special emphasis should be given to NGS standardization of the formalin-fixed paraffin-embedded (FFPE) samples (19, 20).…”

Are Molecular Alterations Linked to Genetic Instability Worth to Be Included as Biomarkers for Directing or Excluding Melanoma Patients to Immunotherapy?

“…Although the introduction into clinical practice of validated immuno-oncological biomarkers is currently limited by the heterogeneity of the types of specimens analyzed, because of the diversity of the used methodologies and the absence of a real sharing of the produced data, it is necessary to continue to support the efforts in conducting biomarker-driven trials (7). In recent years, multidisciplinary approaches have significantly increased the quest for an even more accurate molecular classification through the assessment of the mutational status in multiple oncogenes and tumor suppressor genes; in the immuno-oncological field, such efforts have already produced some approved tests (PD-L1 expression and microsatellite instability rates) and other advanced tests yet to be fully proven for efficacy (tumor mutation load, neoantigen pattern, intratumor T-cell infiltration rate) (5,(8)(9)(10).…”