Validation of a Transcriptome-Based Assay for Classifying Cancers of Unknown Primary Origin.

INTRODUCTION: Cancers assume a variety of distinct histologies, and may originate from a myriad of sites including solid organs, hematopoietic cells, and connective tissue. Clinical decision-making based on consensus guidelines such as the National Comprehensive Cancer Network (NCCN) is often predicated on a specific histologic and anatomic diagnosis, supported by clinical features and pathologist interpretation of morphology and immunohistochemical (IHC) staining patterns. However, in patients with nonspecific morphologic and IHC findings-in addition to ambiguous clinical presentations such as recurrence versus new primary-a definitive diagnosis may not be possible, resulting in the patient being categorized as having a cancer of unknown primary (CUP). Therapeutic options and clinical outcomes are poor for patients with CUP, with a median survival of 8-11 months. METHODS: Here, we describe and validate the Tempus Tumor Origin (Tempus TO) assay, an RNA-sequencing-based machine learning classifier capable of discriminating between 68 clinically relevant cancer subtypes. Model accuracy was assessed using primary and/or metastatic samples with known subtype. RESULTS: We show that the Tempus TO model is 91% accurate when assessed on both a retrospectively held out cohort and a set of samples sequenced after model freeze that collectively contained 9210 total samples with known diagnoses. When evaluated on a cohort of CUPs, the model recapitulated established associations between genomic alterations and cancer subtype. DISCUSSION: Combining diagnostic prediction tests (e.g., Tempus TO) with sequencing-based variant reporting (e.g., Tempus xT) may expand therapeutic options for patients with cancers of unknown primary or uncertain histology.

Distinct genomic features in a retrospective cohort of mucosal, acral, and vulvovaginal melanomas.

BACKGROUND: Compared with sun-exposed melanomas, less is known regarding the pathogenesis of sun-protected melanomas. Sun-protected melanomas share many epidemiologic factors, but their genetic heterogeneity is not well studied. OBJECTIVE: We investigated the genomic profile of acral, mucosal, and vulvovaginal melanomas. We hypothesize that mucosal melanomas, recognized for their uniquely aggressive clinical behavior, have distinct genomic features. METHODS: We performed whole transcriptome messenger RNA and DNA (1711 genes) sequencing, messenger RNA expression profiling, tumor mutational burden, ultraviolet signature, and copy number variants analysis on 29 volar/digital acral, 7 mucosal, and 6 vulvovaginal melanomas. RESULTS: There was significant genetic heterogeneity, particularly in acral melanomas, with 36% having BRAF alterations, whereas other melanomas had none (P = .0159). Nonzero ultraviolet signatures were more frequent in acral melanomas, suggesting greater ultraviolet involvement. Mucosal melanomas formed a distinct group with increased expression of cell cycle and proliferation genes. Various targetable aberrations were identified, such as AURKA and ERBB2, in mucosal and acral melanomas, respectively. LIMITATIONS: The sample size was a small. CONCLUSION: There is significant genetic heterogeneity among sun-protected melanomas. Mucosal melanomas have upregulation in cell cycle and proliferation genes, which may explain their aggressive behavior. Ultraviolet radiation plays some role in a subset of acral but not other melanomas.

Validation of genomic and transcriptomic models of homologous recombination deficiency in a real-world pan-cancer cohort.

BACKGROUND: With the introduction of DNA-damaging therapies into standard of care cancer treatment, there is a growing need for predictive diagnostics assessing homologous recombination deficiency (HRD) status across tumor types. Following the strong clinical evidence for the utility of DNA-sequencing-based HRD testing in ovarian cancer, and growing evidence in breast cancer, we present analytical validation of the Tempus HRD-DNA test. We further developed, validated, and explored the Tempus HRD-RNA model, which uses gene expression data from 16,750 RNA-seq samples to predict HRD status from formalin-fixed paraffin-embedded tumor samples across numerous cancer types. METHODS: Genomic and transcriptomic profiling was performed using next-generation sequencing from Tempus xT, Tempus xO, Tempus xE, Tempus RS, and Tempus RS.v2 assays on 48,843 samples. Samples were labeled based on their BRCA1, BRCA2 and selected Homologous Recombination Repair pathway gene (CDK12, PALB2, RAD51B, RAD51C, RAD51D) mutational status to train and validate HRD-DNA, a genome-wide loss-of-heterozygosity biomarker, and HRD-RNA, a logistic regression model trained on gene expression. RESULTS: In a sample of 2058 breast and 1216 ovarian tumors, BRCA status was predicted by HRD-DNA with F1-scores of 0.98 and 0.96, respectively. Across an independent set of 1363 samples across solid tumor types, the HRD-RNA model was predictive of BRCA status in prostate, pancreatic, and non-small cell lung cancer, with F1-scores of 0.88, 0.69, and 0.62, respectively. CONCLUSIONS: We predict HRD-positive patients across many cancer types and believe both HRD models may generalize to other mechanisms of HRD outside of BRCA loss. HRD-RNA complements DNA-based HRD detection methods, especially for indications with low prevalence of BRCA alterations.

Genome-wide association and multi-omics studies identify MGMT as a novel risk gene for Alzheimer's disease among women.

INTRODUCTION: Variants in the tau gene (MAPT) region are associated with breast cancer in women and Alzheimer's disease (AD) among persons lacking apolipoprotein E ε4 (ε4-). METHODS: To identify novel genes associated with tau-related pathology, we conducted two genome-wide association studies (GWAS) for AD, one among 10,340 ε4- women in the Alzheimer's Disease Genetics Consortium (ADGC) and another in 31 members (22 women) of a consanguineous Hutterite kindred. RESULTS: We identified novel associations of AD with MGMT variants in the ADGC (rs12775171, odds ratio [OR] = 1.4, P = 4.9 × 10-8 ) and Hutterite (rs12256016 and rs2803456, OR = 2.0, P = 1.9 × 10-14 ) datasets. Multi-omics analyses showed that the most significant and largest number of associations among the single nucleotide polymorphisms (SNPs), DNA-methylated CpGs, MGMT expression, and AD-related neuropathological traits were observed among women. Furthermore, promoter capture Hi-C analyses revealed long-range interactions of the MGMT promoter with MGMT SNPs and CpG sites. DISCUSSION: These findings suggest that epigenetically regulated MGMT expression is involved in AD pathogenesis, especially in women.

A pan-cancer organoid platform for precision medicine.

Patient-derived tumor organoids (TOs) are emerging as high-fidelity models to study cancer biology and develop novel precision medicine therapeutics. However, utilizing TOs for systems-biology-based approaches has been limited by a lack of scalable and reproducible methods to develop and profile these models. We describe a robust pan-cancer TO platform with chemically defined media optimized on cultures acquired from over 1,000 patients. Crucially, we demonstrate tumor genetic and transcriptomic concordance utilizing this approach and further optimize defined minimal media for organoid initiation and propagation. Additionally, we demonstrate a neural-network-based high-throughput approach for label-free, light-microscopy-based drug assays capable of predicting patient-specific heterogeneity in drug responses with applicability across solid cancers. The pan-cancer platform, molecular data, and neural-network-based drug assay serve as resources to accelerate the broad implementation of organoid models in precision medicine research and personalized therapeutic profiling programs.

Real-world Evidence of Diagnostic Testing and Treatment Patterns in US Patients With Breast Cancer With Implications for Treatment Biomarkers From RNA Sequencing Data.

OBJECTIVE/BACKGROUND: We performed a retrospective analysis of longitudinal real-world data (RWD) from patients with breast cancer to replicate results from clinical studies and demonstrate the feasibility of generating real-world evidence. We also assessed the value of transcriptome profiling as a complementary tool for determining molecular subtypes. METHODS: De-identified, longitudinal data were analyzed after abstraction from records of patients with breast cancer in the United States (US) structured and stored in the Tempus database. Demographics, clinical characteristics, molecular subtype, treatment history, and survival outcomes were assessed according to strict qualitative criteria. RNA sequencing and clinical data were used to predict molecular subtypes and signaling pathway enrichment. RESULTS: The clinical abstraction cohort (n = 4000) mirrored the demographics and clinical characteristics of patients with breast cancer in the US, indicating feasibility for RWE generation. Among patients who were human epidermal growth factor receptor 2-positive (HER2+), 74.2% received anti-HER2 therapy, with ∼70% starting within 3 months of a positive test result. Most non-treated patients were early stage. In this RWD set, 31.7% of patients with HER2+ immunohistochemistry (IHC) had discordant fluorescence in situ hybridization results recorded. Among patients with multiple HER2 IHC results at diagnosis, 18.6% exhibited intra-test discordance. Through development of a whole-transcriptome model to predict IHC receptor status in the molecular sequenced cohort (n = 400), molecular subtypes were resolved for all patients (n = 36) with equivocal HER2 statuses from abstracted test results. Receptor-related signaling pathways were differentially enriched between clinical molecular subtypes. CONCLUSIONS: RWD in the Tempus database mirrors the overall population of patients with breast cancer in the US. These results suggest that real-time, RWD analyses are feasible in a large, highly heterogeneous database. Furthermore, molecular data may aid deficiencies and discrepancies observed from breast cancer RWD.

Associations of autozygosity with a broad range of human phenotypes.

In many species, the offspring of related parents suffer reduced reproductive success, a phenomenon known as inbreeding depression. In humans, the importance of this effect has remained unclear, partly because reproduction between close relatives is both rare and frequently associated with confounding social factors. Here, using genomic inbreeding coefficients (FROH) for >1.4 million individuals, we show that FROH is significantly associated (p < 0.0005) with apparently deleterious changes in 32 out of 100 traits analysed. These changes are associated with runs of homozygosity (ROH), but not with common variant homozygosity, suggesting that genetic variants associated with inbreeding depression are predominantly rare. The effect on fertility is striking: FROH equivalent to the offspring of first cousins is associated with a 55% decrease [95% CI 44-66%] in the odds of having children. Finally, the effects of FROH are confirmed within full-sibling pairs, where the variation in FROH is independent of all environmental confounding.

Integrated genomic profiling expands clinical options for patients with cancer.

Genomic analysis of paired tumor-normal samples and clinical data can be used to match patients to cancer therapies or clinical trials. We analyzed 500 patient samples across diverse tumor types using the Tempus xT platform by DNA-seq, RNA-seq and immunological biomarkers. The use of a tumor and germline dataset led to substantial improvements in mutation identification and a reduction in false-positive rates. RNA-seq enhanced gene fusion detection and cancer type classifications. With DNA-seq alone, 29.6% of patients matched to precision therapies supported by high levels of evidence or by well-powered studies. This proportion increased to 43.4% with the addition of RNA-seq and immunotherapy biomarker results. Combining these data with clinical criteria, 76.8% of patients were matched to at least one relevant clinical trial on the basis of biomarkers measured by the xT assay. These results indicate that extensive molecular profiling combined with clinical data identifies personalized therapies and clinical trials for a large proportion of patients with cancer and that paired tumor-normal plus transcriptome sequencing outperforms tumor-only DNA panel testing.

Genomic Fusions in Pigmented Spindle Cell Nevus of Reed.

Recent molecular studies of spitzoid neoplasms have identified mutually exclusive kinase fusions involving ROS1, ALK, RET, BRAF, NTRK1, MET, and NTRK3 as early initiating genomic events. Pigmented spindle cell nevus (PSCN) of Reed is a morphologic variant of Spitz and may be very diagnostically challenging, having histologic features concerning for melanoma. Their occurrence in younger patients, lack of association to sun exposure, and rapid early growth phase similar to Spitz nevi suggest fusions may also play a significant role in these lesions. However, to date, there is little data in the literature focused on the molecular characterization of PSCN of Reed with next-generation sequencing. We analyzed a total of 129 melanocytic neoplasms with RNA sequencing including 67 spitzoid neoplasms (10 Spitz nevi, 44 atypical Spitz tumors, 13 spitzoid melanomas) and 23 PSCN of Reed. Although only 2 of 67 (3.0%) of spitzoid lesions had NTRK3 fusions, 13 of 23 (57%) of PSCN of Reed harbored NTRK3 fusions with 5' partners ETV6 (12p13) in 2 cases and MYO5A (15q21) in 11 cases. NTRK3 fusions were confirmed with a fluorescent in situ hybridization break-apart probe. The presence of a NTRK3 fusion correlated with younger age (P=0.021) and adnexal extension (P=0.001). Other minor fusions identified in PSCN of Reed included MYO5A-MERTK (2), MYO5A-ROS1, MYO5A-RET, and ETV6-PITX3 leading to a total of 78% with fusions. Our study suggests that the majority of PSCN of Reed are the result of genomic fusions, and the most frequent and characteristic genomic aberration is an NTRK3 fusion.

A decade of research on the 17q12-21 asthma locus: Piecing together the puzzle.

Chromosome 17q12-21 remains the most highly replicated and significant asthma locus. Genotypes in the core region defined by the first genome-wide association study correlate with expression of 2 genes, ORM1-like 3 (ORMDL3) and gasdermin B (GSDMB), making these prime candidate asthma genes, although recent studies have implicated gasdermin A (GSDMA) distal to and post-GPI attachment to proteins 3 (PGAP3) proximal to the core region as independent loci. We review 10 years of studies on the 17q12-21 locus and suggest that genotype-specific risks for asthma at the proximal and distal loci are not specific to early-onset asthma and mediated by PGAP3, ORMDL3, and/or GSDMA expression. We propose that the weak and inconsistent associations of 17q single nucleotide polymorphisms with asthma in African Americans is due to the high frequency of some 17q alleles, the breakdown of linkage disequilibrium on African-derived chromosomes, and possibly different early-life asthma endotypes in these children. Finally, the inconsistent association between asthma and gene expression levels in blood or lung cells from older children and adults suggests that genotype effects may mediate asthma risk or protection during critical developmental windows and/or in response to relevant exposures in early life. Thus studies of young children and ethnically diverse populations are required to fully understand the relationship between genotype and asthma phenotype and the gene regulatory architecture at this locus.

Rare non-coding variants are associated with plasma lipid traits in a founder population.

Founder populations are ideally suited for studies on the clinical effects of alleles that are rare in general populations but occur at higher frequencies in these isolated populations. Whole genome sequencing in 98 Hutterites, a founder population of European descent, and subsequent imputation revealed 660,238 single nucleotide polymorphisms that are rare (<1%) or absent in European populations, but occur at frequencies >1% in the Hutterites. We examined the effects of these rare in European variants on plasma lipid levels in 828 Hutterites and applied a Bayesian hierarchical framework to prioritize potentially causal variants based on functional annotations. We identified two novel non-coding rare variants associated with LDL cholesterol (rs17242388 in LDLR) and HDL cholesterol (rs189679427 between GOT2 and APOOP5), and replicated previous associations of a splice variant in APOC3 (rs138326449) with triglycerides and HDL-C. All three variants are at well-replicated loci in GWAS but are independent from and have larger effect sizes than the known common variation in these regions. Candidate eQTL analyses in in LCLs in the Hutterites suggest that these rare non-coding variants are likely to mediate their effects on lipid traits by regulating gene expression.

Host genetic variation in mucosal immunity pathways influences the upper airway microbiome.

BACKGROUND: The degree to which host genetic variation can modulate microbial communities in humans remains an open question. Here, we performed a genetic mapping study of the microbiome in two accessible upper airway sites, the nasopharynx and the nasal vestibule, during two seasons in 144 adult members of a founder population of European decent. RESULTS: We estimated the relative abundances (RAs) of genus level bacteria from 16S rRNA gene sequences and examined associations with 148,653 genetic variants (linkage disequilibrium [LD] r 2 < 0.5) selected from among all common variants discovered in genome sequences in this population. We identified 37 microbiome quantitative trait loci (mbQTLs) that showed evidence of association with the RAs of 22 genera (q < 0.05) and were enriched for genes in mucosal immunity pathways. The most significant association was between the RA of Dermacoccus (phylum Actinobacteria) and a variant 8 kb upstream of TINCR (rs117042385; p = 1.61 × 10-8; q = 0.002), a long non-coding RNA that binds to peptidoglycan recognition protein 3 (PGLYRP3) mRNA, a gene encoding a known antimicrobial protein. A second association was between a missense variant in PGLYRP4 (rs3006458) and the RA of an unclassified genus of family Micrococcaceae (phylum Actinobacteria) (p = 5.10 × 10-7; q = 0.032). CONCLUSIONS: Our findings provide evidence of host genetic influences on upper airway microbial composition in humans and implicate mucosal immunity genes in this relationship.

Innate Immunity and Asthma Risk in Amish and Hutterite Farm Children.

BACKGROUND: The Amish and Hutterites are U.S. agricultural populations whose lifestyles are remarkably similar in many respects but whose farming practices, in particular, are distinct; the former follow traditional farming practices whereas the latter use industrialized farming practices. The populations also show striking disparities in the prevalence of asthma, and little is known about the immune responses underlying these disparities. METHODS: We studied environmental exposures, genetic ancestry, and immune profiles among 60 Amish and Hutterite children, measuring levels of allergens and endotoxins and assessing the microbiome composition of indoor dust samples. Whole blood was collected to measure serum IgE levels, cytokine responses, and gene expression, and peripheral-blood leukocytes were phenotyped with flow cytometry. The effects of dust extracts obtained from Amish and Hutterite homes on immune and airway responses were assessed in a murine model of experimental allergic asthma. RESULTS: Despite the similar genetic ancestries and lifestyles of Amish and Hutterite children, the prevalence of asthma and allergic sensitization was 4 and 6 times as low in the Amish, whereas median endotoxin levels in Amish house dust was 6.8 times as high. Differences in microbial composition were also observed in dust samples from Amish and Hutterite homes. Profound differences in the proportions, phenotypes, and functions of innate immune cells were also found between the two groups of children. In a mouse model of experimental allergic asthma, the intranasal instillation of dust extracts from Amish but not Hutterite homes significantly inhibited airway hyperreactivity and eosinophilia. These protective effects were abrogated in mice that were deficient in MyD88 and Trif, molecules that are critical in innate immune signaling. CONCLUSIONS: The results of our studies in humans and mice indicate that the Amish environment provides protection against asthma by engaging and shaping the innate immune response. (Funded by the National Institutes of Health and others.).

Genome-wide association study of recalcitrant atopic dermatitis in Korean children.

BACKGROUND: Atopic dermatitis (AD) is a heterogeneous chronic inflammatory skin disease. Most AD during infancy resolves during childhood, but moderate-to-severe AD with allergic sensitization is more likely to persist into adulthood and more often occurs with other allergic diseases. OBJECTIVE: We sought to find susceptibility loci by performing the first genome-wide association study (GWAS) of AD in Korean children with recalcitrant AD, which was defined as moderate-to-severe AD with allergic sensitization. METHODS: Our study included 246 children with recalcitrant AD and 551 adult control subjects with a negative history of both allergic disease and allergic sensitization. DNA from these subjects was genotyped; sets of common single nucleotide polymorphisms (SNPs) were imputed and used in the GWAS after quality control checks. RESULTS: SNPs at a region on 13q21.31 were associated with recalcitrant AD at a genome-wide threshold of significance (P < 2.0 × 10(-8)). These associated SNPs are more than 1 Mb from the closest gene, protocadherin (PCDH)9. SNPs at 4 additional loci had P values of less than 1 × 10(-6), including SNPs at or near the neuroblastoma amplified sequence (NBAS; 2p24.3), thymus-expressed molecule involved in selection (THEMIS; 6q22.33), GATA3 (10p14), and S-phase cyclin A-associated protein in the ER (SCAPER; 15q24.3) genes. Further analysis of total serum IgE levels suggested 13q21.31 might be primarily an IgE locus, and analyses of published data demonstrated that SNPs at the 15q24.3 region are expression quantitative trait loci for 2 nearby genes, ISL2 and proline-serine-threonine phosphatase interacting protein 1 (PSTPIP1), in immune cells. CONCLUSION: Our GWAS of recalcitrant AD identified new susceptibility regions containing genes involved in epithelial cell function and immune dysregulation, 2 key features of AD, and potentially extend our understanding of their role in pathogenesis.

Ethnic-specific associations of rare and low-frequency DNA sequence variants with asthma.

Common variants at many loci have been robustly associated with asthma but explain little of the overall genetic risk. Here we investigate the role of rare (<1%) and low-frequency (1-5%) variants using the Illumina HumanExome BeadChip array in 4,794 asthma cases, 4,707 non-asthmatic controls and 590 case-parent trios representing European Americans, African Americans/African Caribbeans and Latinos. Our study reveals one low-frequency missense mutation in the GRASP gene that is associated with asthma in the Latino sample (P=4.31 × 10(-6); OR=1.25; MAF=1.21%) and two genes harbouring functional variants that are associated with asthma in a gene-based analysis: GSDMB at the 17q12-21 asthma locus in the Latino and combined samples (P=7.81 × 10(-8) and 4.09 × 10(-8), respectively) and MTHFR in the African ancestry sample (P=1.72 × 10(-6)). Our results suggest that associations with rare and low-frequency variants are ethnic specific and not likely to explain a significant proportion of the 'missing heritability' of asthma.

Targeted enrichment of specific regions in the human genome by array hybridization.

While whole-genome resequencing remains expensive, genomic partitioning provides an affordable means of targeting sequence efforts towards regions of high interest. There are several competitive methods for targeted capture; these include molecular inversion probes, microdroplet-segregated multiplex PCR, and on-array or in-solution capture-by-hybridization. Enrichment of the human exome by array hybridization has been successfully applied to pinpoint the causative allele of Mendelian disorders. This protocol focuses on the application of Agilent 1 M arrays for capture-by-hybridization and sequencing on the Illumina platform, although the library preparation method may be adaptable to other vendors' array platforms and sequencing technologies.