Clinical cancer genomic profiling by three-platform sequencing of whole genome, whole exome and transcriptome

Abstract

To evaluate the potential of an integrated clinical test to detect diverse classes of somatic and germline mutations relevant to pediatric oncology, we performed three-platform whole-genome (WGS), whole exome (WES) and transcriptome (RNA-Seq) sequencing of tumors and normal tissue from 78 pediatric cancer patients in a CLIA-certified, CAP-accredited laboratory. Our analysis pipeline achieves high accuracy by cross-validating variants between sequencing types, thereby removing the need for confirmatory testing, and facilitates comprehensive reporting in a clinically-relevant timeframe. Three-platform sequencing has a positive predictive value of 97–99, 99, and 91% for somatic SNVs, indels and structural variations, respectively, based on independent experimental verification of 15,225 variants. We report 240 pathogenic variants across all cases, including 84 of 86 known from previous diagnostic testing (98% sensitivity). Combined WES and RNA-Seq, the current standard for precision oncology, achieved only 78% sensitivity. These results emphasize the critical need for incorporating WGS in pediatric oncology testing.

Feasibility of whole genome and transcriptome profiling in pediatric and young adult cancers

“…The clinical relevance of cWGTS findings that were not identified by clinical panel sequencing (MSK-IMPACT 4 , MSK-fusion 28 and panel testing of 88 cancer-predisposition genes 27 ) was determined by a multidisciplinary molecular tumor board. Consistent with recent studies 7 , 11 – 16 , cWGTS analyses identified at least one additional cancer-associated oncogenic variant in 54% of patients ( n = 62). Of these, 33 patients had one or more findings that were of direct clinical relevance, including 7 diagnostic (21%), 15 prognostic (45%), 5 therapy-informing (15%), 5 previously undescribed oncofusions (15%), and 6 germline (18%) biomarkers (Fig.…”

“…This allows us to annotate, validate, and prioritize SVs of diagnostic (e.g., MYB enhancer hijacking), prognostic (e.g., ATRX / TERT ), and therapeutic relevance (e.g., TP53 loss-of- function SVs). Consistent with recent literature for pediatric and rare cancers 7 , 15 , 16 , 58 , >50% of patients had additional findings of established biological or clinical significance. The majority of these findings were SVs in cancer genes, fusion genes, and genome-wide mutation signatures that targeted panels are not optimally designed to identify.…”

Feasibility of whole genome and transcriptome profiling in pediatric and young adult cancers

“…The clinical relevance of cWGTS findings that were not identified by clinical panel sequencing (MSK-IMPACT 4 , MSK-fusion 28 and panel testing of 88 cancer-predisposition genes 27 ) was determined by a multidisciplinary molecular tumor board. Consistent with recent studies 7 , 11 – 16 , cWGTS analyses identified at least one additional cancer-associated oncogenic variant in 54% of patients ( n = 62). Of these, 33 patients had one or more findings that were of direct clinical relevance, including 7 diagnostic (21%), 15 prognostic (45%), 5 therapy-informing (15%), 5 previously undescribed oncofusions (15%), and 6 germline (18%) biomarkers (Fig.…”

“…This allows us to annotate, validate, and prioritize SVs of diagnostic (e.g., MYB enhancer hijacking), prognostic (e.g., ATRX / TERT ), and therapeutic relevance (e.g., TP53 loss-of- function SVs). Consistent with recent literature for pediatric and rare cancers 7 , 15 , 16 , 58 , >50% of patients had additional findings of established biological or clinical significance. The majority of these findings were SVs in cancer genes, fusion genes, and genome-wide mutation signatures that targeted panels are not optimally designed to identify.…”

The value of comprehensive genomic sequencing to maximize the identification of clinically actionable alterations in advanced cancer patients: a case series

“…Our data emphasizes that tumor heterogeneity, and continual evolution of the genomic landscape contributes to disease progression. Therefore, our study joins other recently reported studies [ 53 , 54 ]. that suggests including a test that interrogates tumor/normal WES as well as the transcriptome can provide a more comprehensive assessment of a patients’ tumors identifying treatment options unrecognized in somatic only panel testing.…”

SJPedPanel: A pan-cancer gene panel for childhood malignancies

“…Using the previously published G4K study, SJPedPanel was found to cover 86% of the reported somatic markers, including SNV, Indel and SV. Similar findings were obtained by using real-time clinical sequencing samples 4,71 in which 91% of the reported clinically relevant variants were detected, including all SNVs, Indels and ITDs. At patient level, at least one variant was detected in 96% cases (median 3).…”

“…Consistent with the in-silico analysis [ Fig. 4a ], a comparison of SNV, Indel, SV and ITD variants (n=205 out of 389) discovered using three platform sequencing 4,71 (WGS, WES, RNASeq) indicated that SJPedPanel covers 88% of these variants whereas WES covers 78% [Additional file 3: Supplementary Fig. S9].…”

Feasibility of whole genome and transcriptome profiling in pediatric and young adult cancers

“…The clinical relevance of cWGTS findings that were not identified by clinical panel sequencing (MSK-IMPACT 4 , MSK-fusion 28 and panel testing of 88 cancer-predisposition genes 27 ) was determined by a multidisciplinary molecular tumor board. Consistent with recent studies 7 , 11 – 16 , cWGTS analyses identified at least one additional cancer-associated oncogenic variant in 54% of patients ( n = 62). Of these, 33 patients had one or more findings that were of direct clinical relevance, including 7 diagnostic (21%), 15 prognostic (45%), 5 therapy-informing (15%), 5 previously undescribed oncofusions (15%), and 6 germline (18%) biomarkers (Fig.…”

“…This allows us to annotate, validate, and prioritize SVs of diagnostic (e.g., MYB enhancer hijacking), prognostic (e.g., ATRX / TERT ), and therapeutic relevance (e.g., TP53 loss-of- function SVs). Consistent with recent literature for pediatric and rare cancers 7 , 15 , 16 , 58 , >50% of patients had additional findings of established biological or clinical significance. The majority of these findings were SVs in cancer genes, fusion genes, and genome-wide mutation signatures that targeted panels are not optimally designed to identify.…”

The value of comprehensive genomic sequencing to maximize the identification of clinically actionable alterations in advanced cancer patients: a case series

“…Our data emphasizes that tumor heterogeneity, and continual evolution of the genomic landscape contributes to disease progression. Therefore, our study joins other recently reported studies [ 53 , 54 ]. that suggests including a test that interrogates tumor/normal WES as well as the transcriptome can provide a more comprehensive assessment of a patients’ tumors identifying treatment options unrecognized in somatic only panel testing.…”

SJPedPanel: A pan-cancer gene panel for childhood malignancies

“…Using the previously published G4K study, SJPedPanel was found to cover 86% of the reported somatic markers, including SNV, Indel and SV. Similar findings were obtained by using real-time clinical sequencing samples 4,71 in which 91% of the reported clinically relevant variants were detected, including all SNVs, Indels and ITDs. At patient level, at least one variant was detected in 96% cases (median 3).…”

“…Consistent with the in-silico analysis [ Fig. 4a ], a comparison of SNV, Indel, SV and ITD variants (n=205 out of 389) discovered using three platform sequencing 4,71 (WGS, WES, RNASeq) indicated that SJPedPanel covers 88% of these variants whereas WES covers 78% [Additional file 3: Supplementary Fig. S9].…”

Feasibility of whole genome and transcriptome profiling in pediatric and young adult cancers

“…The clinical relevance of cWGTS findings that were not identified by clinical panel sequencing (MSK-IMPACT 4 , MSK-fusion 28 and panel testing of 88 cancer-predisposition genes 27 ) was determined by a multidisciplinary molecular tumor board. Consistent with recent studies 7 , 11 – 16 , cWGTS analyses identified at least one additional cancer-associated oncogenic variant in 54% of patients ( n = 62). Of these, 33 patients had one or more findings that were of direct clinical relevance, including 7 diagnostic (21%), 15 prognostic (45%), 5 therapy-informing (15%), 5 previously undescribed oncofusions (15%), and 6 germline (18%) biomarkers (Fig.…”

“…This allows us to annotate, validate, and prioritize SVs of diagnostic (e.g., MYB enhancer hijacking), prognostic (e.g., ATRX / TERT ), and therapeutic relevance (e.g., TP53 loss-of- function SVs). Consistent with recent literature for pediatric and rare cancers 7 , 15 , 16 , 58 , >50% of patients had additional findings of established biological or clinical significance. The majority of these findings were SVs in cancer genes, fusion genes, and genome-wide mutation signatures that targeted panels are not optimally designed to identify.…”

The value of comprehensive genomic sequencing to maximize the identification of clinically actionable alterations in advanced cancer patients: a case series

“…Our data emphasizes that tumor heterogeneity, and continual evolution of the genomic landscape contributes to disease progression. Therefore, our study joins other recently reported studies [ 53 , 54 ]. that suggests including a test that interrogates tumor/normal WES as well as the transcriptome can provide a more comprehensive assessment of a patients’ tumors identifying treatment options unrecognized in somatic only panel testing.…”

SJPedPanel: A pan-cancer gene panel for childhood malignancies

“…Using the previously published G4K study, SJPedPanel was found to cover 86% of the reported somatic markers, including SNV, Indel and SV. Similar findings were obtained by using real-time clinical sequencing samples 4,71 in which 91% of the reported clinically relevant variants were detected, including all SNVs, Indels and ITDs. At patient level, at least one variant was detected in 96% cases (median 3).…”

“…Consistent with the in-silico analysis [ Fig. 4a ], a comparison of SNV, Indel, SV and ITD variants (n=205 out of 389) discovered using three platform sequencing 4,71 (WGS, WES, RNASeq) indicated that SJPedPanel covers 88% of these variants whereas WES covers 78% [Additional file 3: Supplementary Fig. S9].…”