The development of next-generation sequencing (NGS)-based targeted gene panel assays has been instrumental in providing information to improve patient care by refining cancer diagnosis, stratifying patient risk, and informing therapeutic decisions.1–5 These assays were developed, however, primarily to identify clinical biomarkers commonly found in adult cancer sub-types.1,2,4 When compared to adult cancers, pediatric tumors have been shown to have distinct mutational profiles with much lower mutational burdens and an enrichment of structural variants. Thus, novel clinical profiling strategies are needed to address these unique considerations. Cancer whole genome and transcriptome sequencing (cWGTS) is a comprehensive platform that can assess the full spectrum of germline and somatically acquired mutations (including structural variants, copy number alterations, tumor mutational burden, and mutational signatures).6 While this methodology is increasingly recognized in the literature for potential clinical utility,7-13 the real-world implementation of this technology can be challenged by cost, complexity of laboratory and analytical workflows, and concerns regarding the sensitivity of low coverage whole genome sequencing (WGS) compared to high-depth NGS panels in detecting actionable mutations.3 In our manuscript, “Feasibility of whole genome and transcriptome profiling in pediatric and young adult cancers,” we demonstrate the feasibility and analytical validity, as well as explore the clinical utility of cWGTS in a cohort of pediatric, adolescent and young adult patients with rare solid tumors.
cWGTS Demonstrates Concordance with Clinically Validated NGS Panels
When comparing matched sequencing using cWGTS and CLIA-certified NGS targeted panels (MSK-IMPACT4), concordance of shared mutation calling was near 100%. As cWGTS was performed on fresh frozen tumors while targeted NGS assays were performed on formalin-fixed paraffin-embedded (FFPE) material, the vast majority of discordant calls were identified as subclonal mutations resulting from intratumoral heterogeneity.14 With regard to germline analysis by MSK-IMPACT15 and panel RNA assessment with MSK-Fusion16, cWGTS captured all relevant germline findings and fusions as reported by these methodologies. Importantly, fusion genes were supported by data in both WGS and RNA-seq, which offers the opportunity to orthogonally validate findings within a single workflow.
cWGTS Identifies Relevant and Novel Genomic Variants Missed on Targeted Panels
Similar to other studies7-13, 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. Additionally, we identified seven rare cancer gene alterations that were not identified by targeted NGS sequencing, including a disease defining mutation of KBTBD4 in a pineal parenchymal tumor of intermediate differentiation,17 a SETBP1 mutation in a germ cell tumor, and a SIX1 mutation in a Wilms tumor. The four other clinically relevant variants were germline in nature. Similarly, eight in-frame fusion genes were identified from WGS and RNA-seq in patients with no prior findings on clinical testing, five of which were novel. These included a t(2;6) (PAX3-FOXO3) translocation changing diagnosis to alveolar rhabdomyosarcoma from embryonal rhabdomyosarcoma (ERMS), a UACA-LTK fusion in a metastatic papillary thyroid carcinoma, and a pathognomonic SH3PXD2A-HTRA1 fusion establishing a diagnosis of schwannoma in a patient evaluated for relapsed stage IV neuroblastoma. Of potential therapeutic relevance, we identified an NTRK3-SLMAP fusion in a neuroblastoma patient which confers likely sensitivity to TRK inhibition.
cWGTS Provides Additional Insight Into Oncogenic Mechanisms in Pediatric Tumors
In addition to identifying novel mutations and fusions not otherwise reported on conventional NGS assays, cWGTS affords the opportunity to explore other disease defining alterations that are not yet addressed in current clinical testing modalities. These signals of interest include the detection of structural variants, the delineation of mutation signatures, detection of chromothripsis or whole genome duplication (WGD), cancer-associated viral sequences (i.e. EBV), estimation of telomere length, and gene expression signatures. We identified structural variants of prognostic relevance18 in neuroblastoma including events in TERT and ATRX, and recurrent structural variants in the tumor suppressor gene DLG2 in approximately 50% of osteosarcoma patients as well as 3/29 neuroblastoma patients. With the utilization of RNAseq data, we could more confidently assess the transcriptomic consequences of affected loci in structural variants (example: loss of TP53 RNA expression in the context of a TP53 structural variant). Global gene expression signatures were also used to cluster samples by tumor type, providing further opportunity to resolve a patient's diagnosis. Of the patients with RNAseq data available, an average of 18 gene expression biomarkers were identified per sample.
Global Assessment of Tumor Mutational Burden Has Demonstrable Clinical Utility
Compared to panel-based approaches which derive estimates of TMB, MSI and mutation signatures,19 WGS directly quantifies genome-wide mutation burden across all variant classes. Two pediatric patients (one with adrenocortical carcinoma, ACC, and one with clear cell carcinoma, CCC) had progressive on-treatment metastatic disease, no targetable biomarkers by conventional testing, and no further therapeutic options. cWGTS analyses revealed a profoundly rearranged genome in both cases scoring these two patients as the highest in fusion and SV burden in the cohort. The patient with ACC was treated with checkpoint blockade (nivolumab/ipilimumab), resulting in complete response after three cycles of therapy and is disease-free 26 months after therapy cessation, whereas the patient with CCC was treated with pembrolizumab, achieved a complete response after 6 cycles, and remains disease-free 10 months after therapy.
(A) (top) Circos plot for a tumor sample from a patient with metastatic ACC depicting high SV burden. (bottom) PET imaging shows resolution of a large pulmonary metastatic lesion following treatment with nivolumab and ipilimumab. (B) (top) Circos plot for a tumor sample from a patient with relapsed/refractory poorly differentiated CCC with high TMB and SV burden. (bottom) PET imaging shows resolution of multiple metastatic lesions following treatment with pembrolizumab (pembro).
Where do we go from here?
In this study, we demonstrate that cWGTS can be performed in a clinically relevant time frame to capture the full spectrum of cancer-associated genomic alterations that are frequently assessed using a diversity of targeted diagnostic assays, as well as additional analyses that are not yet available via clinical testing. This is of particular interest in pediatric tumors given that paucity of tissue can present challenges with platform prioritization for profiling. Several clinical hurdles still exist, including the requirement of fresh frozen tissue in this and previous cWGTS studies. Ongoing studies are underway to assess the feasibility and utility of cWGTS derived from FFPE tumor samples. Additionally, in this manuscript we perform an exploratory analysis of WGS on cell-free DNA (with matched fresh frozen samples) in a select subset of patients. We were able to perform de novo mutation calling in all variant classes, and in those samples with sufficient circulating tumor DNA content, we demonstrate good concordance with WGS results from tissue. This provides an alternative profiling strategy to explore in the context of limited tumor tissue.
A major criticism and barrier of utilizing WGS in a clinical context has been the complexity of various complementary workflows in order to ensure timely delivery and interpretation of genomic data. In this study, we execute an end-to-end sample-to-report turnaround time within 9 days, which is aligned to clinical needs for diagnosis and care decisions. Lastly, the question of utmost importance, is how do we confidently interpret and utilize the data generated to improve patient outcomes? The wealth of information gleaned requires the input of a multi-disciplinary team including pediatric oncologists, computational biologists, clinical geneticists, and molecular pathologists, among others. While we can use validated tools, such as OncoKB,21 to understand and categorize known variants, much of the emerging data, especially as relates to RNA expression and translation to clinical actionability, need to be explored further. In our manuscript, we describe how a single platform can provide a comprehensive understanding of a tumor’s mutational landscape (both somatic and germline) in a clinically relevant time frame. As we continue to optimize this workflow, we are now tasked to make cWGTS more broadly accessible to children and young adults with cancer. Moreover, we must further define and study the clinical utility of the comprehensive data generated through cWGTS through the conduct of genomically based therapeutic clinical trials.
*This blog post includes textual excerpts and figures from the referenced manuscript and represents the work of all authors
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