Comparative Lesions Analysis Through a Targeted Sequencing Approach

Caterina Vicentini; Andrea Mafficini; Michele Simbolo; Matteo Fassan; Pietro Delfino; Rita  Teresa Lawlor; Borislav Rusev; Aldo Scarpa; Vincenzo Corbo

doi:10.3791/59844

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Method Article

Comparative Lesions Analysis Through a Targeted Sequencing Approach

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DOI:

10.3791/59844

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November 5th, 2019

Caterina Vicentini¹ , Andrea Mafficini¹ , Michele Simbolo² , Matteo Fassan³ , Pietro Delfino² , Rita Teresa Lawlor¹ , Borislav Rusev¹ , Aldo Scarpa¹^,² , Vincenzo Corbo¹^,²

¹ARC-Net Research Centre, University and Hospital Trust of Verona, ²Department of Diagnostics and Public Health, Section of Pathology, University and Hospital Trust of Verona, ³Department of Medicine (DIMED), Surgical Pathology and Cytopathology Unit, University of Padua

Summary

This article describes a method to identify clonal and subclonal alterations among different specimens from a given patient. Although the experiments described here focus on a specific tumor type, the approach is broadly applicable to other solid tumors.

Abstract

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective anti-tumor strategies. Although concerns are frequently raised due to differences in sample processing and depth of coverage, next-generation sequencing of solid tumors have unraveled a highly variable degree of ITH across tumor types. Capturing the genetic relatedness between primary and metastatic lesions through the identification of clonal and subclonal populations is critical to the design of therapies for advance-stage diseases. Here, we report a method for comparative lesions analysis that allows for the identification of clonal and subclonal populations among different specimens from the same patient. The experimental approach described here integrates three well-established approaches: histological analysis, high-coverage multi-lesion sequencing, and immunophenotypic analyses. In order to minimize the effects on the detection of subclonal events by inappropriate sample processing, we subjected tissues to careful pathological examination and neoplastic cell enrichment. Quality controlled DNA from neoplastic lesions and normal tissues was then subjected to high coverage sequencing, targeting the coding regions of 409 relevant cancer genes. While only looking at a limited genomic space, our approach enables evaluating the extent of heterogeneity among somatic alterations (single-nucleotide mutations and copy-number variations) in distinct lesions from a given patient. Through comparative analysis of sequencing data, we were able to distinguish clonal vs. subclonal alterations. The majority of ITH is often ascribed to passenger mutations; therefore, we also used immunohistochemistry to predict functional consequences of mutations. While this protocol has been applied to a specific tumor type, we anticipate that the methodology described here is broadly applicable to other solid tumor types.

Introduction

The advent of next generation sequencing (NGS) has revolutionized the way cancers are diagnosed and treated¹. NGS coupled to multiregional sequencing have exposed a high degree of intra-tumoral heterogeneity (ITH) in solid tumors², which explains in part the failure of targeted therapy due to the presence of subclones with differing drug sensitivity². An important challenge posed by genome-wide sequencing studies is the necessity to distinguish between passenger (i.e., neutral) and driver mutations in individual cancers³. Several studies have indeed shown ....