Molecular-guided therapy for the treatment of patients with relapsed and refractory childhood cancers: a Beat Childhood Cancer Research Consortium trial.

BACKGROUND: Children with relapsed central nervous system (CNS tumors), neuroblastoma, sarcomas, and other rare solid tumors face poor outcomes. This prospective clinical trial examined the feasibility of combining genomic and transcriptomic profiling of tumor samples with a molecular tumor board (MTB) approach to make real­time treatment decisions for children with relapsed/refractory solid tumors. METHODS: Subjects were divided into three strata: stratum 1-relapsed/refractory neuroblastoma; stratum 2-relapsed/refractory CNS tumors; and stratum 3-relapsed/refractory rare solid tumors. Tumor samples were sent for tumor/normal whole-exome (WES) and tumor whole-transcriptome (WTS) sequencing, and the genomic data were used in a multi-institutional MTB to make real­time treatment decisions. The MTB recommended plan allowed for a combination of up to 4 agents. Feasibility was measured by time to completion of genomic sequencing, MTB review and initiation of treatment. Response was assessed after every two cycles using Response Evaluation Criteria in Solid Tumors (RECIST). Patient clinical benefit was calculated by the sum of the CR, PR, SD, and NED subjects divided by the sum of complete response (CR), partial response (PR), stable disease (SD), no evidence of disease (NED), and progressive disease (PD) subjects. Grade 3 and higher related and unexpected adverse events (AEs) were tabulated for safety evaluation. RESULTS: A total of 186 eligible patients were enrolled with 144 evaluable for safety and 124 evaluable for response. The average number of days from biopsy to initiation of the MTB-recommended combination therapy was 38 days. Patient benefit was exhibited in 65% of all subjects, 67% of neuroblastoma subjects, 73% of CNS tumor subjects, and 60% of rare tumor subjects. There was little associated toxicity above that expected for the MGT drugs used during this trial, suggestive of the safety of utilizing this method of selecting combination targeted therapy. CONCLUSIONS: This trial demonstrated the feasibility, safety, and efficacy of a comprehensive sequencing model to guide personalized therapy for patients with any relapsed/refractory solid malignancy. Personalized therapy was well tolerated, and the clinical benefit rate of 65% in these heavily pretreated populations suggests that this treatment strategy could be an effective option for relapsed and refractory pediatric cancers. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02162732. Prospectively registered on June 11, 2014.

Genomic and Transcriptomic Analysis of Relapsed and Refractory Childhood Solid Tumors Reveals a Diverse Molecular Landscape and Mechanisms of Immune Evasion.

Children with treatment-refractory or relapsed (R/R) tumors face poor prognoses. As the genomic underpinnings driving R/R disease are not well defined, we describe here the genomic and transcriptomic landscapes of R/R solid tumors from 202 patients enrolled in Beat Childhood Cancer Consortium clinical trials. Tumor mutational burden (TMB) was elevated relative to untreated tumors at diagnosis, with one-third of tumors classified as having a pediatric high TMB. Prior chemotherapy exposure influenced the mutational landscape of these R/R tumors, with more than 40% of tumors demonstrating mutational signatures associated with platinum or temozolomide chemotherapy and two tumors showing treatment-associated hypermutation. Immunogenomic profiling found a heterogenous pattern of neoantigen and MHC class I expression and a general absence of immune infiltration. Transcriptional analysis and functional gene set enrichment analysis identified cross-pathology clusters associated with development, immune signaling, and cellular signaling pathways. While the landscapes of these R/R tumors reflected those of their corresponding untreated tumors at diagnosis, important exceptions were observed, suggestive of tumor evolution, treatment resistance mechanisms, and mutagenic etiologies of treatment. SIGNIFICANCE: Tumor heterogeneity, chemotherapy exposure, and tumor evolution contribute to the molecular profiles and increased mutational burden that occur in treatment-refractory and relapsed childhood solid tumors.

Improved methods for RNAseq-based alternative splicing analysis.

The robust detection of disease-associated splice events from RNAseq data is challenging due to the potential confounding effect of gene expression levels and the often limited number of patients with relevant RNAseq data. Here we present a novel statistical approach to splicing outlier detection and differential splicing analysis. Our approach tests for differences in the percentages of sequence reads representing local splice events. We describe a software package called Bisbee which can predict the protein-level effect of splice alterations, a key feature lacking in many other splicing analysis resources. We leverage Bisbee's prediction of protein level effects as a benchmark of its capabilities using matched sets of RNAseq and mass spectrometry data from normal tissues. Bisbee exhibits improved sensitivity and specificity over existing approaches and can be used to identify tissue-specific splice variants whose protein-level expression can be confirmed by mass spectrometry. We also applied Bisbee to assess evidence for a pathogenic splicing variant contributing to a rare disease and to identify tumor-specific splice isoforms associated with an oncogenic mutation. Bisbee was able to rediscover previously validated results in both of these cases and also identify common tumor-associated splice isoforms replicated in two independent melanoma datasets.

Temporospatial genomic profiling in glioblastoma identifies commonly altered core pathways underlying tumor progression.

BACKGROUND: Tumor heterogeneity underlies resistance and disease progression in glioblastoma (GBM), and tumors most commonly recur adjacent to the surgical resection margins in contrast non-enhancing (NE) regions. To date, no targeted therapies have meaningfully altered overall patient survival in the up-front setting. The aim of this study was to characterize intratumoral heterogeneity in recurrent GBM using bulk samples from primary resection and recurrent samples taken from contrast-enhancing (EN) and contrast NE regions. METHODS: Whole exome and RNA sequencing were performed on matched bulk primary and multiple recurrent EN and NE tumor samples from 16 GBM patients who received standard of care treatment alone or in combination with investigational clinical trial regimens. RESULTS: Private mutations emerge across multi-region sampling in recurrent tumors. Genomic clonal analysis revealed increased enrichment in gene alterations regulating the G2M checkpoint, Kras signaling, Wnt signaling, and DNA repair in recurrent disease. Subsequent functional studies identified augmented PI3K/AKT transcriptional and protein activity throughout progression, validated by phospho-protein levels. Moreover, a mesenchymal transcriptional signature was observed in recurrent EN regions, which differed from the proneural signature in recurrent NE regions. CONCLUSIONS: Subclonal populations observed within bulk resected primary GBMs transcriptionally evolve across tumor recurrence (EN and NE regions) and exhibit aberrant gene expression of common signaling pathways that persist despite standard or targeted therapy. Our findings provide evidence that there are both adaptive and clonally mediated dependencies of GBM on key pathways, such as the PI3K/AKT axis, for survival across recurrences.

Multiscale, multimodal analysis of tumor heterogeneity in IDH1 mutant vs wild-type diffuse gliomas.

Glioma is recognized to be a highly heterogeneous CNS malignancy, whose diverse cellular composition and cellular interactions have not been well characterized. To gain new clinical- and biological-insights into the genetically-bifurcated IDH1 mutant (mt) vs wildtype (wt) forms of glioma, we integrated data from protein, genomic and MR imaging from 20 treatment-naïve glioma cases and 16 recurrent GBM cases. Multiplexed immunofluorescence (MxIF) was used to generate single cell data for 43 protein markers representing all cancer hallmarks, Genomic sequencing (exome and RNA (normal and tumor) and magnetic resonance imaging (MRI) quantitative features (protocols were T1-post, FLAIR and ADC) from whole tumor, peritumoral edema and enhancing core vs equivalent normal region were also collected from patients. Based on MxIF analysis, 85,767 cells (glioma cases) and 56,304 cells (GBM cases) were used to generate cell-level data for 24 biomarkers. K-means clustering was used to generate 7 distinct groups of cells with divergent biomarker profiles and deconvolution was used to assign RNA data into three classes. Spatial and molecular heterogeneity metrics were generated for the cell data. All features were compared between IDH mt and IDHwt patients and were finally combined to provide a holistic/integrated comparison. Protein expression by hallmark was generally lower in the IDHmt vs wt patients. Molecular and spatial heterogeneity scores for angiogenesis and cell invasion also differed between IDHmt and wt gliomas irrespective of prior treatment and tumor grade; these differences also persisted in the MR imaging features of peritumoral edema and contrast enhancement volumes. A coherent picture of enhanced angiogenesis in IDHwt tumors was derived from multiple platforms (genomic, proteomic and imaging) and scales from individual proteins to cell clusters and heterogeneity, as well as bulk tumor RNA and imaging features. Longer overall survival for IDH1mt glioma patients may reflect mutation-driven alterations in cellular, molecular, and spatial heterogeneity which manifest in discernable radiological manifestations.

PD-1-Associated Gene Expression Signature of Neoadjuvant Trastuzumab-Treated Tumors Correlates with Patient Survival in HER2-Positive Breast Cancer.

The therapeutic HER2-targeting antibody trastuzumab has been shown to elicit tumor immune response in a subset of HER2-positive (HER2+) breast cancer. We performed genomic and immunohistochemical profiling of tumors from eight patients who have completed multiple rounds of neoadjuvant trastuzumabb to identify predictive biomarkers for trastuzumab-elicited tumor immune responses. Immunohistochemistry showed that all tumors had an activated tumor immune microenvironment positive for nuclear NF-κB/p65RelA, CD4, and CD8 T cell markers, but only four out of eight tumors were positive for the PD-1 immune checkpoint molecule, which is indicative of an exhausted immune environment. Exome sequencing showed no specific driver mutations correlating with PD-1 positivity. Hierarchical clustering of the RNA sequencing data revealed two distinct groups, of which Group 2 represented the PD-1 positive tumors. A gene expression signature that was derived from this clustering composed of 89 genes stratified HER2+ breast cancer patients in the TCGA dataset and it was named PD-1-Associated Gene Expression Signature in HER2+ Breast Cancer (PAGES-HBC). Patients with the Group 2 PAGES-HBC composition had significantly more favorable survival outcomes with mortality reduced by 83% (hazard ratio 0.17; 95% CI, 0.05 to 0.60; p = 0.011). Analysis of three longitudinal samples from a single patient showed that PAGES-HBC might be transiently induced by trastuzumab, independent of clonal tumor expansion over time. We conclude that PAGES-HBC could be further developed as a prognostic predictor of trastuzumab response in HER2+ breast cancer patients and be potentially used as an alternative biomarker for anti-PD-1 therapy trials.

Leveraging Spatial Variation in Tumor Purity for Improved Somatic Variant Calling of Archival Tumor Only Samples.

Archival tumor samples represent a rich resource of annotated specimens for translational genomics research. However, standard variant calling approaches require a matched normal sample from the same individual, which is often not available in the retrospective setting, making it difficult to distinguish between true somatic variants and individual-specific germline variants. Archival sections often contain adjacent normal tissue, but this tissue can include infiltrating tumor cells. As existing comparative somatic variant callers are designed to exclude variants present in the normal sample, a novel approach is required to leverage adjacent normal tissue with infiltrating tumor cells for somatic variant calling. Here we present lumosVar 2.0, a software package designed to jointly analyze multiple samples from the same patient, built upon our previous single sample tumor only variant caller lumosVar 1.0. The approach assumes that the allelic fraction of somatic variants and germline variants follow different patterns as tumor content and copy number state change. lumosVar 2.0 estimates allele specific copy number and tumor sample fractions from the data, and uses a to model to determine expected allelic fractions for somatic and germline variants and to classify variants accordingly. To evaluate the utility of lumosVar 2.0 to jointly call somatic variants with tumor and adjacent normal samples, we used a glioblastoma dataset with matched high and low tumor content and germline whole exome sequencing data (for true somatic variants) available for each patient. Both sensitivity and positive predictive value were improved when analyzing the high tumor and low tumor samples jointly compared to analyzing the samples individually or in-silico pooling of the two samples. Finally, we applied this approach to a set of breast and prostate archival tumor samples for which tumor blocks containing adjacent normal tissue were available for sequencing. Joint analysis using lumosVar 2.0 detected several variants, including known cancer hotspot mutations that were not detected by standard somatic variant calling tools using the adjacent tissue as presumed normal reference. Together, these results demonstrate the utility of leveraging paired tissue samples to improve somatic variant calling when a constitutional sample is not available.

Prospective Feasibility Trial for Genomics-Informed Treatment in Recurrent and Progressive Glioblastoma.

Purpose: Glioblastoma is an aggressive and molecularly heterogeneous cancer with few effective treatment options. We hypothesized that next-generation sequencing can be used to guide treatment recommendations within a clinically acceptable time frame following surgery for patients with recurrent glioblastoma.Experimental Design: We conducted a prospective genomics-informed feasibility trial in adults with recurrent and progressive glioblastoma. Following surgical resection, genome-wide tumor/normal exome sequencing and tumor RNA sequencing were performed to identify molecular targets for potential matched therapy. A multidisciplinary molecular tumor board issued treatment recommendations based on the genomic results, blood-brain barrier penetration of the indicated therapies, drug-drug interactions, and drug safety profiles. Feasibility of generating genomics-informed treatment recommendations within 35 days of surgery was assessed.Results: Of the 20 patients enrolled in the study, 16 patients had sufficient tumor tissue for analysis. Exome sequencing was completed for all patients, and RNA sequencing was completed for 14 patients. Treatment recommendations were provided within the study's feasibility time frame for 15 of 16 (94%) patients. Seven patients received treatment based on the tumor board recommendations. Two patients reached 12-month progression-free survival, both adhering to treatments based on the molecular profiling results. One patient remained on treatment and progression free 21 months after surgery, 3 times longer than the patient's previous time to progression. Analysis of matched nonenhancing tissue from 12 patients revealed overlapping as well as novel putatively actionable genomic alterations.Conclusions: Use of genome-wide molecular profiling is feasible and can be informative for guiding real-time, central nervous system-penetrant, genomics-informed treatment recommendations for patients with recurrent glioblastoma. Clin Cancer Res; 24(2); 295-305. ©2017 AACRSee related commentary by Wick and Kessler, p. 256.

A method to reduce ancestry related germline false positives in tumor only somatic variant calling.

BACKGROUND: Significant clinical and research applications are driving large scale adoption of individualized tumor sequencing in cancer in order to identify tumors-specific mutations. When a matched germline sample is available, somatic mutations may be identified using comparative callers. However, matched germline samples are frequently not available such as with archival tissues, which makes it difficult to distinguish somatic from germline variants. While population databases may be used to filter out known germline variants, recent studies have shown private germline variants result in an inflated false positive rate in unmatched tumor samples, and the number germline false positives in an individual may be related to ancestry. METHODS: First, we examined the relationship between the germline false positives and ancestry. Then we developed and implemented a tumor only caller (LumosVar) that leverages differences in allelic frequency between somatic and germline variants in impure tumors. We used simulated data to systematically examine how copy number alterations, tumor purity, and sequencing depth should affect the sensitivity of our caller. Finally, we evaluated the caller on real data. RESULTS: We find the germline false-positive rate is significantly higher for individuals of non-European Ancestry largely due to the limited diversity in public polymorphism databases and due to population-specific characteristics such as admixture or recent expansions. Our Bayesian tumor only caller (LumosVar) is able to greatly reduce false positives from private germline variants, and our sensitivity is similar to predictions based on simulated data. CONCLUSIONS: Taken together, our results suggest that studies of individuals of non-European ancestry would most benefit from our approach. However, high sensitivity requires sufficiently impure tumors and adequate sequencing depth. Even in impure tumors, there are copy number alterations that result in germline and somatic variants having similar allele frequencies, limiting the sensitivity of the approach. We believe our approach could greatly improve the analysis of archival samples in a research setting where the normal is not available.

Integrated genomic analyses reveal frequent TERT aberrations in acral melanoma.

Genomic analyses of cutaneous melanoma (CM) have yielded biological and therapeutic insights, but understanding of non-ultraviolet (UV)-derived CMs remains limited. Deeper analysis of acral lentiginous melanoma (ALM), a rare sun-shielded melanoma subtype associated with worse survival than CM, is needed to delineate non-UV oncogenic mechanisms. We thus performed comprehensive genomic and transcriptomic analysis of 34 ALM patients. Unlike CM, somatic alterations were dominated by structural variation and absence of UV-derived mutation signatures. Only 38% of patients demonstrated driver BRAF/NRAS/NF1 mutations. In contrast with CM, we observed PAK1 copy gains in 15% of patients, and somatic TERT translocations, copy gains, and missense and promoter mutations, or germline events, in 41% of patients. We further show that in vitro TERT inhibition has cytotoxic effects on primary ALM cells. These findings provide insight into the role of TERT in ALM tumorigenesis and reveal preliminary evidence that TERT inhibition represents a potential therapeutic strategy in ALM.