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Purpose: Patients ≥18 months of age with International Neuroblastoma Staging System (INSS) stage 3 unfavorable histology (UH), MYCN-nonamplified (MYCN-NA) tumors have favorable survival rates compared with other high-risk neuroblastoma populations. The impact of select clinical and biological factors on overall survival (OS) and event-free survival (EFS) were evaluated. Experimental Design: Patients enrolled on Children's Oncology Group (COG) A3973 (n = 34), ANBL0532 (n = 27), and/or biology protocol ANBL00B1 (n = 72) were analyzed. Tumors with available DNA (n = 65) and RNA (n = 42) were subjected to whole-exome sequencing (WES) and RNA sequencing. WES analyses and gene expression profiling were evaluated for their impact on survival. Multivariate analyses of EFS/OS using significant factors from univariate analyses were performed. Results: 5-year EFS/OS for patients treated with high-risk therapy on A3973 and ANBL0532 were 73.0% ± 8.1%/87.9% ± 5.9% and 61.4% ± 10.2%/73.0% ± 9.2%, respectively (P = 0.1286 and P = 0.2180). In the A3973/ANBL0532 cohort, patients with less than partial response (PR; n = 5) at end-induction had poor outcomes (5-year EFS/OS: 0%/20.0% ± 17.9%. Univariate analyses of WES data revealed that subjects whose tumors had chromosome 1p or 11q loss/LOH and chromosome 5 or 9 segmental chromosomal aberrations had inferior EFS compared with those with tumors without these aberrations. Multivariate analysis revealed that 11q loss/LOH was an independent predictor of inferior OS [HR, 3.116 (95% confidence interval, 1.034–9.389), P = 0.0435]. Conclusions: Patients ≥18 months of age at diagnosis who had tumors with UH and MYCN-NA INSS stage 3 neuroblastoma assigned to high-risk therapy had an 81.6% ± 5.3% 5-year OS. Less than PR to induction therapy and chromosome 11q loss/LOH are independent predictors of inferior outcome and identify patients who should be eligible for future high-risk clinical trials.

Triple-negative breast cancer (TNBC) is an aggressive disease that disproportionately affects African American (AA) women. Limited targeted therapeutic options exist for patients with TNBC. Here, we employ spatial transcriptomics to interrogate tissue from a racially diverse TNBC cohort to comprehensively annotate the transcriptional states of spatially resolved cellular populations. A total of 38,706 spatial features from a cohort of 28 sections from 14 patients were analyzed. Intratumoral analysis of spatial features from individual sections revealed heterogeneous transcriptional substructures. However, integrated analysis of all samples resulted in nine transcriptionally distinct clusters that mapped across all individual sections. Furthermore, novel use of join count analysis demonstrated nonrandom directional spatial dependencies of the transcriptionally defined shared clusters, supporting a conserved spatio-transcriptional architecture in TNBC. These findings were substantiated in an independent validation cohort comprising 17,861 spatial features representing 15 samples from 8 patients. Stratification of samples by race revealed race-associated differences in hypoxic tumor content and regions of immune-rich infiltrate. Overall, this study combined spatial and functional molecular analyses to define the tumor architecture of TNBC, with potential implications in understanding TNBC disparities. Significance: Spatial transcriptomics profiling of a diverse cohort of triple-negative breast cancers and innovative informatics approaches reveal a conserved cellular architecture across cancers and identify proportional differences in tumor cell composition by race.

Supplemental Figure S2 Outcome by Chromosome 9 Status. (A) EFS and (B) OS by Chromosome 9 status

XLSX file - 23K, Mapping statistics for genome sequencing of tumor and germline DNA from mTNBC

XLSX file - 45K, Genes containing breakpoint from translocation in mTNBC

XLSX file - 273K, Somatic genic focal copy number changes in mTNBC

XLSX file - 598K, edgeR differential expression results in mTNBC for select cancer related genes

XLSX file - 607K, Outlier differential expression results in mTNBC for select cancer related genes

XLSX file - 61K, List of somatic point mutations in mTNBC detected by NGS sequencing analysis

XLSX file - 42K, Ingenuity Canonical Pathways results for mTNBC based on edgeR differential expression results

XLSX file - 40K, Characteristics of mTNBC study population

PDF file - 1635K, PET/CT images of mTNBC2 breast lesion before and after treatment on combination MEK and AKT inhibitors

XLSX file - 48K, Transcriptome sequencing (RNA-seq) run statistics for mTNBC

PDF file - 135K, Validation of RB1 39bp homozygous deletion in mTNBC1

Supplemental Figure S3 Non-negative Matrix Factorization (NMF) Clustering of Tumor Specimens. (A) Clustering similarity matrix and (B) Silhouette plot of samples by cluster id. (C) Enrichment score for adrenergic (ADRN), mesenchymal (MES), and Schwann cell profile (SCP) gene signatures.

Purpose:Patients ≥18 months of age with International Neuroblastoma Staging System (INSS) stage 3 unfavorable histology (UH), MYCN-nonamplified (MYCN-NA) tumors have favorable survival rates compared with other high-risk neuroblastoma populations. The impact of select clinical and biological factors on overall survival (OS) and event-free survival (EFS) were evaluated.Experimental Design:Patients enrolled on Children's Oncology Group (COG) A3973 (n = 34), ANBL0532 (n = 27), and/or biology protocol ANBL00B1 (n = 72) were analyzed. Tumors with available DNA (n = 65) and RNA (n = 42) were subjected to whole-exome sequencing (WES) and RNA sequencing. WES analyses and gene expression profiling were evaluated for their impact on survival. Multivariate analyses of EFS/OS using significant factors from univariate analyses were performed.Results:5-year EFS/OS for patients treated with high-risk therapy on A3973 and ANBL0532 were 73.0% ± 8.1%/87.9% ± 5.9% and 61.4% ± 10.2%/73.0% ± 9.2%, respectively (P = 0.1286 and P = 0.2180). In the A3973/ANBL0532 cohort, patients with less than partial response (PR; n = 5) at end-induction had poor outcomes (5-year EFS/OS: 0%/20.0% ± 17.9%. Univariate analyses of WES data revealed that subjects whose tumors had chromosome 1p or 11q loss/LOH and chromosome 5 or 9 segmental chromosomal aberrations had inferior EFS compared with those with tumors without these aberrations. Multivariate analysis revealed that 11q loss/LOH was an independent predictor of inferior OS [HR, 3.116 (95% confidence interval, 1.034–9.389), P = 0.0435].Conclusions:Patients ≥18 months of age at diagnosis who had tumors with UH and MYCN-NA INSS stage 3 neuroblastoma assigned to high-risk therapy had an 81.6% ± 5.3% 5-year OS. Less than PR to induction therapy and chromosome 11q loss/LOH are independent predictors of inferior outcome and identify patients who should be eligible for future high-risk clinical trials.

Bipolar disorder is an often-severe mental health condition characterized by alternation between extreme mood states of mania and depression. Despite strong heritability and the recent identification of 64 common variant risk loci of small effect, pathophysiological mechanisms remain unknown. Here, we analyzed genome sequences from 41 multiply-affected pedigrees and identified variants in 741 genes with nominally significant linkage or association with bipolar disorder. These 741 genes overlapped known risk genes for neurodevelopmental disorders and clustered within gene networks enriched for synaptic and nuclear functions. The top variant in this analysis - prioritized by statistical association, predicted deleteriousness, and network centrality - was a missense variant in the gene encoding D-amino acid oxidase (DAOG131V). Heterologous expression of DAOG131V in human cells resulted in decreased DAO protein abundance and enzymatic activity. In a knock-in mouse model of DAOG131, DaoG130V/+, we similarly found decreased DAO protein abundance in hindbrain regions, as well as enhanced stress susceptibility and blunted behavioral responses to pharmacological inhibition of N-methyl-D-aspartate receptors (NMDARs). RNA sequencing of cerebellar tissue revealed that DaoG130V resulted in decreased expression of two gene networks that are enriched for synaptic functions and for genes expressed, respectively, in Purkinje neurons or granule neurons. These gene networks were also down-regulated in the cerebellum of patients with bipolar disorder compared to healthy controls and were enriched for additional rare variants associated with bipolar disorder risk. These findings implicate dysregulation of NMDAR signaling and of gene expression in cerebellar neurons in bipolar disorder pathophysiology and provide insight into its genetic architecture.

AKT S473 distribution based on ER expression measured by IHC for 31 lesions included of the discovery set. ER was measured in a binary scale (Panel A) and in continuous scale using the H-Score (Panel B).

Purpose: Little is known about the molecular signatures associated with specific metastatic sites in breast cancer. Using comprehensive multi-omic molecular profiling, we assessed whether alterations or activation of the PI3K–AKT–mTOR pathway is associated with specific sites of breast cancer metastasis.Experimental Design: Next-generation sequencing–based whole-exome sequencing was coupled with reverse-phase protein microarray (RPPA) functional signaling network analysis to explore the PI3K–AKT–mTOR axis in 32 pretreated breast cancer metastases. RPPA-based signaling data were further validated in an independent cohort of 154 metastatic lesions from breast cancer and 101 unmatched primary breast tumors. The proportion of cases with PI3K–AKT–mTOR genomic alterations or signaling network activation were compared between hepatic and nonhepatic lesions.Results: PIK3CA mutation and activation of AKT (S473) and p70S6K (T389) were detected more frequently among liver metastases than nonhepatic lesions (P < 0.01, P = 0.056, and P = 0.053, respectively). However, PIK3CA mutations alone were insufficient in predicting protein activation (P = 0.32 and P = 0.19 for activated AKT and p70S6K, respectively). RPPA analysis of an independent cohort of 154 tumors confirmed the relationship between pathway activation and hepatic metastasis [AKT (S473), mTOR (S2448), and 4EBP1 (S65); P < 0.01, P = 0.02, and P = 0.01, respectively]. Similar results were also seen between liver metastases and primary breast tumors [AKT (S473) P < 0.01, mTOR (S2448) P < 0.01, 4EBP1 (S65) P = 0.01]. This signature was lost when primary tumors were compared with all metastatic sites combined.Conclusions: Breast cancer patients with liver metastasis may represent a molecularly homogenized cohort with increased incidence of PIK3CA mutations and activation of the PI3K–AKT–mTOR signaling network. Clin Cancer Res; 23(16); 4919–28. ©2017 AACR.

Study Workflow Diagram. Key steps in the study are outlined, including the number of patients at each stage. BEV: bevacizumab.

Triple-negative breast cancer (TNBC) is an aggressive disease that disproportionately affects African American (AA) women. Limited targeted therapeutic options exist for patients with TNBC. Here, we employ spatial transcriptomics to interrogate tissue from a racially diverse TNBC cohort to comprehensively annotate the transcriptional states of spatially resolved cellular populations. A total of 38,706 spatial features from a cohort of 28 sections from 14 patients were analyzed. Intratumoral analysis of spatial features from individual sections revealed heterogeneous transcriptional substructures. However, integrated analysis of all samples resulted in nine transcriptionally distinct clusters that mapped across all individual sections. Furthermore, novel use of join count analysis demonstrated nonrandom directional spatial dependencies of the transcriptionally defined shared clusters, supporting a conserved spatio-transcriptional architecture in TNBC. These findings were substantiated in an independent validation cohort comprising 17,861 spatial features representing 15 samples from 8 patients. Stratification of samples by race revealed race-associated differences in hypoxic tumor content and regions of immune-rich infiltrate. Overall, this study combined spatial and functional molecular analyses to define the tumor architecture of TNBC, with potential implications in understanding TNBC disparities.Significance:Spatial transcriptomics profiling of a diverse cohort of triple-negative breast cancers and innovative informatics approaches reveal a conserved cellular architecture across cancers and identify proportional differences in tumor cell composition by race.

Supplementary material legend

Concordance between PIK3CA, AKT, and PTEN mutation status and AKT (S473) and p70S6 (T389) activation.

Supplementary figures and figure legends

List of Expression Datasets used for Survival Analysis

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 AACR.See related commentary by Wick and Kessler, p. 256

Case Demographics and Clinical Annotation

We propose a method to leverage spatial transcriptomics and bioinformatic analysis to identify sub-clonal loss of heterozygosity that could otherwise be missed by standard bulk sequencing in glioblastoma tumors. Glioblastomas are highly invasive and aggressive brain tumors with a low five-year survival rate of less than 10%. Current treatment strategies are not sufficient for long term disease outcome, and innovative approaches to analyze glioblastoma are imperative for future improved options. Many factors contribute to difficulty of treatment, though one critical consideration is the diffuse nature and known intratumoral heterogeneity of this cancer. It is important to determine LOH heterogeneity, as this genomic alteration is associated with copy number variation and specific classifications of gliomas. Loss of heterozygosity is an irreversible deletion of DNA that is a common alteration in cancer. Application of spatial transcriptomics can increase our understanding of genomic complexity across a tissue, and unsupervised graph clustering can computationally separate spatial regions with similar gene expression patterns. Our method investigates spatial clusters individually at known heterozygous SNP sites to identify evidence for LOH with Bayesian inference and a hidden Markov model. Our analysis centers on the balance of reference (A) and non-reference (B) spatial transcriptomics read counts at SNPs of interest. B allele frequency is the number of reads aligned to the alternative allele divided by the total number of reads counted at the SNP. At a normal two-copy state at a heterozygous site, B allele frequency is ideally 50 percent. LOH can be identified as this value deviates toward 0 or 100, and copy number alterations impact this frequency. Our method utilizes Bayes factor values, or likelihood ratios between two models, using allele counts to provide support for either LOH or a heterozygous event. For our analysis, spatial transcriptomics BAMs are processed with a graph clustering algorithm and split into cluster-specific BAMs. We calculate allele count coverage for each cluster at pre-selected heterozygous SNP positions identified through germline exome sequencing. Bayes factor values are then calculated at each SNP, resulting in evidence for LOH or a heterozygous event. Chromosomal regions are segmented by a hidden Markov approach. Cumulative metrics for each segment are evaluated to determine a “state” label of heterozygous, LOH, or undefined. We tested our method on three WHO grade 4 IDH wild-type EGFRvIII positive fresh frozen glioblastoma tissue samples. Sub-clonal heterogeneity was identified in two samples and confirmed by bulk exome sequencing, bulk RNA sequencing, and analysis of initial clinical reports. Citation Format: Michelle G. Webb, Frances Chow, Carmel McCullough, Bohan Zhang, Kyle Hurth, John Carpten, Gabriel Zada, David W. Craig. Identification of sub-clonal loss of heterozygosity (LOH) in glioblastomas analyzed with spatial transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3124.

Ovarian cancer (OvCa) is the deadliest gynecological malignancy in the United States. Surgery and chemotherapy are the primary treatments for OvCa, but 80% of late-stage OvCa patients experience chemo-resistant recurrence, which necessitates the development of new treatment strategies. The genomic diversity within a tumor and the interactions among various cell types within its microenvironment are considered key factors contributing to therapeutic efficacy. Specifically, women who harbor germline BRCA1 or BRCA2 mutations are at increased risk of developing high grade serous ovarian cancer (HGSC). To understand the genomic consequences, including somatic alterations, associated with defective DNA repair, tumor DNA and RNA was isolated from fresh frozen OCT embedded sections from 15 HGSC patients with BRCA1/2 mutation and 10 without any alterations in the BRCA genes (BRCA-wt). Whole exome sequencing (WES) and total RNA-seq was performed to determine the association of homologous recombination (HR) DNA repair gene defects with tumor mutation burden (TMB), neoantigen load (NL), and immunological assessment of tumor microenvironment. TMB was calculated from mutation rates of somatic WES. Germline exome analysis was used to validate inherited BRCA mutations. To identify the somatic mutations, somatic single nucleotide variants and InDels were detected using Strelka. TP53 mutations were most common with several other genes showing only modest mutation frequency. Mutation signatures were also detected with maftools and compared to the COSMIC mutational signature database. BRCA-mutated samples displayed a unique mutational signature associated with defects in HR DNA repair pathway. We also observed heterogeneity in copy number variation profiles amongst cases, as determined by the tool Sequenza. Furthermore, gene set enrichment analysis indicated differential enrichment of P53 pathway, MAPK pathway, and PTEN pathway, in addition to numerous pathways related to immune cell signaling and immune response. RNA-seq data was analyzed with CIBERSORTx and revealed different proportions of 22 immune effector cell types that accompany tumor cells within the HGSC microenvironment. Additional analyses are underway, including measuring differences in NL in BRCA-mutated versus BRCA-wt cases. This work will represent a significant advancement in our fundamental knowledge regarding the complexities of the TME and effective immunological responses in OvCa. Citation Format: Jing Qian, Lynda D. Roman, Seeta Rajpara, Monica Neuman, Varun Khetan, David W. Craig, Joseph Carlson, John D. Carpten. Assessing difference in the tumor and immune microenvironment of BRCA1/2 mutated versus BRCA wild type high grade serous ovarian cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6082.

Introduction: CRC is the second leading cause of cancer mortality in the United States. Clinical factors and molecular characteristics may impact therapeutics and prognosis. Scientific studies have uncovered significant aberrations, including critical genes and pathways. Despite these efforts, few studies have utilized similar sample sizes to TCGA, heterogeneous populations and integrative clinical data to compare their reported genomic changes, even though little is known about the tumor microenvironment and its significance for colorectal tumorigenesis. Methods: Clinical and DNA genomic data from 262 colorectal tumor/normal DNA samples were obtained for whole exome sequencing analysis from the Oncology Research Information Exchange Network and two colorectal tissue samples for Spatial Transcriptomics profiling from the USC colorectal cancer Moonshot project. Whole Exome Sequencing data analysis was used for identifying genetic alterations. Samples were stratified based on microsatellite instability, age group, anatomical subsites, and histological subtypes. Emphasis was given to comparing molecular characteristics among Hispanic/Latino patients since their reported higher mortality and cancer disparities. Mutation frequencies from our cohort were compared to those from the TCGA. An ongoing spatial transcriptomics analysis consists in processing two fresh-frozen tissue samples through the visium spatial gene expression library using standard short-read sequencing. Results: Tumor molecular profiling revealed 20 genes significantly mutated. We found differences in gene mutation frequencies among our samples, highlighting APC (60%), TP53 (56%), TTN (49%), and KRAS (44%) gene mutations. In comparison with TCGA data, in addition to the expected APC, TP53, PIK3CA, and KRAS mutations, we found frequent mutations in POLE and TTN. To assess the basis for the considerably different mutation rates, we evaluated MSI. Among the 262 tumors, 41 (16%) had high levels of MSI, 2 (1%) had low levels of MSI (MSI-L), and 215 (82%) were microsatellite stable (MSS). Cancer heterogeneity was reported among tumors with different characteristics such as age group, anatomical subsite, and histological subtypes. The ongoing spatial transcriptomics analysis that will be ready before the annual meeting will identify cellular populations and map immunological and inflammatory signals in the microenvironment. Conclusions: Our study reveals the impact of molecularly profiling tumors using considerable sample sizes and stratified samples in several subgroups. Our study adds important information about tumor heterogeneity and the tumor microenvironment in colorectal cancer by including our spatial transcriptomics data analysis. Citation Format: Enrique Velazquez Villarreal, Seeta Rajpara, Yuxin Jin, Jing Qian, Bohan Zhang, Brigette Waldrup, Donna Loza, Heinz-Josef Lenz, David W. Craig, John D. Carpten. Tumor molecular profiling and spatial transcriptomics to assess colorectal cancer heterogeneity and microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1515.