Integration of whole-exome and anchored PCR-based next generation sequencing significantly increases detection of actionable alterations in precision oncology

Abstract

Background: Frequency of clinically relevant mutations in solid tumors by targeted and whole-exome sequencing is ∼30%. Transcriptome analysis complements detection of actionable gene fusions in advanced cancer patients. Goal of this study was to determine the added value of anchored multiplex PCR (AMP)-based next-generation sequencing (NGS) assay to identify further potential drug targets, when coupled with whole-exome sequencing (WES).

Methods: Selected series of fifty-six samples from 55 patients enrolled in our precision medicine study were interrogated by WES and AMP-based NGS. RNA-seq was performed in 19 cases. Clinically relevant and actionable alterations detected by three methods were integrated and analyzed.

Results: AMP-based NGS detected 48 fusions in 31 samples (55.4%); 31.25% (15/48) were classified as targetable based on published literature. WES revealed 29 samples (51.8%) harbored targetable alterations. TMB-high and MSI-high status were observed in 12.7% and 1.8% of cases. RNA-seq from 19 samples identified 8 targetable fusions (42.1%), also captured by AMP-based NGS. When number of actionable fusions detected by AMP-based NGS were added to WES targetable alterations, 66.1% of samples had potential drug targets. When both WES and RNA-seq were analyzed, 57.8% of samples had targetable alterations.

Conclusions: This study highlights importance of an integrative genomic approach for precision oncology, including use of different NGS platforms with complementary features. Integrating RNA data (whole transcriptome or AMP-based NGS) significantly enhances detection of potential targets in cancer patients. In absence of fresh frozen tissue, AMP-based NGS is a robust method to detect actionable fusions using low-input RNA from archival tissue.

Keywords: Anchored multiplex PCR-based next-generation sequencing; Novel fusion; Oncogenic; RNA Sequencing; Whole-exome sequencing.

Fig. 1

Tumor types and corresponding biopsy sites of study cohort. 56 tumor samples from 55 patients and 14 different tumor origins underwent gene fusion assay in addition to whole-exome sequencing. Tumor types are listed on the left and the diagram illustrates anatomic sites (bold) and tumor origin (light gray).

Fig. 2

Integrative genomic landscape of whole-exome sequencing and actionable gene fusion detection.(A) CoMut plot representation of individual Tier1 mutations, copy number alterations by WES and fusions by AMP-based NGS in 56 tumor samples in the study cohort of 55 patients. Top: primary site, metastatic sites, gender and TMB/MSI profile. Left: individual gene alterations detected by WES and AMP-based assay. Bottom: Detection of targetable alteration by individual assays alone and in combination. (B) Percentage of molecular targets identified by individual NGS assays, alone and in combination. A significant increase in actionable genomic alterations is seen when results from both WES and AMP-based NGS are integrated.

Fig. 3

Selected known fusions detected in cancer patients enrolled in the Englander Institute for Precision Medicine.(A) Selected examples of known fusions detected by using AMP-based NGS are illustrated. Genes in red color are covered by the Archer^Ⓡ FusionPlex^Ⓡ Solid Tumor panel, and genes in black color were partners detected by the assay. The column on the left shows the protein domain structure of putative fusions and the vertical black line is the detected fusion breakpoint. The column on the right shows the exon structure of the same fusions. (B) Fluorescence in situ hybridization (FISH) fusion assay for TPM3-NTRK1 is positive in a case of colon cancer. In each nucleus the FISH assay demonstrates a yellow signal (superimposed red-green fused allele) and separate red and green signals (unfused allele) in represented nuclei. (C) FISH break-apart assay for FGFR3 is positive in a case of prostate adenocarcinoma. An FGFR3-MSI2 gene fusion was detected by Archer^Ⓡ FusionPlex^Ⓡ Solid Tumor. This FISH assay demonstrates a yellow signal (nontranslocated allele) and separate red (5′) and green (3′) signals in the translocated allele of FGFR3 in the representative nucleus (see alsoTable 1and Supplementary Tables).

Fig. 4

Schematic representation of one known fusion and two novel fusion partners. Coverage tracks with annotated breakpoints, genes partners, protein domains, gene transcripts and split reads involving (A)PIK3R3-MAST2,(B)NCOA4-RET and (C)EIF4H-ETV1. To further support the latter, (D) RNA-seq expression was compared between 2 ETV1 fusion-positive and 17 non-ETV1 fused samples (p = 0.012) (see also Supplementary Table 5).