Helio Costa

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a malignant primary cutaneous T-cell lymphoma that is challenging to distinguish from other neoplastic and reactive panniculitides. In an attempt to identify somatic variants in SPTCL that may be diagnostically or therapeutically relevant, we performed both exome sequencing on paired tumor-normal samples and targeted sequencing of hematolymphoid-malignancy–associated genes on tumor biopsies. Exome sequencing was performed on skin… Show more

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a malignant primary cutaneous T-cell lymphoma that is challenging to distinguish from other neoplastic and reactive panniculitides. In an attempt to identify somatic variants in SPTCL that may be diagnostically or therapeutically relevant, we performed both exome sequencing on paired tumor-normal samples and targeted sequencing of hematolymphoid-malignancy–associated genes on tumor biopsies. Exome sequencing was performed on skin biopsies from 4 cases of skin-limited SPTCL, 1 case of peripheral T-cell lymphoma, not otherwise specified with secondary involvement of the panniculus, and 2 cases of lupus panniculitis. This approach detected between 1 and 13 high-confidence somatic variants that were predicted to result in a protein alteration per case. Variants of interest identified include 1 missense mutation in ARID1B in 1 case of SPTCL. To detect variants that were present at a lower level, we used a more sensitive targeted panel to sequence 41 hematolymphoid-malignancy–associated genes. The targeted panel was applied to 2 of the biopsies that were evaluated by whole exome sequencing as well as 5 additional biopsies. Potentially pathogenic variants were identified in KMT2D and PLCG1 among others, but no gene was altered in >2 of the 7 cases sequenced. One variant that was notably absent from the cases sequences is RHOA G17V. Further work will be required to further elucidate the genetic abnormalities that lead to this rare lymphoma. Show less

African rainforests support exceptionally high biodiversity and host the world’s largest number of active hunter-gatherers. The genetic history of African rainforest hunter-gatherers and neighboring farmers is characterized by an ancient divergence more than 100,000 years ago, together with recent population collapses and expansions, respectively. While the demographic past of rainforest hunter-gatherers has been deeply characterized, important aspects of their history of genetic adaptation… Show more

African rainforests support exceptionally high biodiversity and host the world’s largest number of active hunter-gatherers. The genetic history of African rainforest hunter-gatherers and neighboring farmers is characterized by an ancient divergence more than 100,000 years ago, together with recent population collapses and expansions, respectively. While the demographic past of rainforest hunter-gatherers has been deeply characterized, important aspects of their history of genetic adaptation remain unclear. Here, we investigated how these groups have adapted—through classic selective sweeps, polygenic adaptation, and selection since admixture—to the challenging rainforest environments. To do so, we analyzed a combined dataset of 566 high-coverage exomes, including 266 newly generated exomes, from 14 populations of rainforest hunter-gatherers and farmers, together with 40 newly generated, low-coverage genomes. We find evidence for a strong, shared selective sweep among all hunter-gatherer groups in the regulatory region of TRPS1—primarily involved in morphological traits. We detect strong signals of polygenic adaptation for height and life history traits such as reproductive age; however, the latter appear to result from pervasive pleiotropy of height-associated genes. Furthermore, polygenic adaptation signals for functions related to responses of mast cells to allergens and microbes, the IL-2 signaling pathway, and host interactions with viruses support a history of pathogen-driven selection in the rainforest. Finally, we find that genes involved in heart and bone development and immune responses are enriched in both selection signals and local hunter-gatherer ancestry in admixed populations, suggesting that selection has maintained adaptive variation in the face of recent gene flow from farmers. Show less

The clinical management and therapy of many solid tumor malignancies depends on detection of
medically actionable or diagnostically relevant genetic variation. However, a principal challenge for
genetic assays from tumors is the fragmented and chemically damaged state of DNA in formalin-fixed,
paraffin-embedded (FFPE) samples. From highly fragmented DNA and RNA there is no current technology
for generating long-range DNA sequence data as is required to detect genomic structural… Show more

The clinical management and therapy of many solid tumor malignancies depends on detection of
medically actionable or diagnostically relevant genetic variation. However, a principal challenge for
genetic assays from tumors is the fragmented and chemically damaged state of DNA in formalin-fixed,
paraffin-embedded (FFPE) samples. From highly fragmented DNA and RNA there is no current technology
for generating long-range DNA sequence data as is required to detect genomic structural variation or
long-range genotype phasing. We have developed a high-throughput chromosome conformation capture approach for FFPE samples that we call Fix-C, which is similar in concept to Hi-C. Fix-C enables
structural variation detection from archival FFPE samples. This method was applied to 15 clinical
adenocarcinoma- and sarcoma-positive control specimens spanning a broad range of tumor purities. In
this panel, Fix-C analysis achieves a 90% concordance rate with fluorescence in situ hybridization assays, the current clinical gold standard. In addition, novel structural variation undetected by other
methods could be identified, and long-range chromatin configuration information recovered from these
FFPE samples harboring highly degraded DNA. This powerful approach will enable detailed resolution of
global genome rearrangement events during cancer progression from FFPE material and will inform the
development of targeted molecular diagnostic assays for patient care. Show less

Current regimens for the detection and surveillance of bladder cancer are invasive and have suboptimal sensitivity. Here, we present a novel high-throughput sequencing (HTS) method for detection of urine tumor DNA (utDNA) called utDNA CAPP-Seq (uCAPP-Seq) and apply it to 67 healthy adults and 118 patients with early-stage bladder cancer who had urine collected either prior to treatment or during surveillance. Using this targeted sequencing approach, we detected a median of 6 mutations per… Show more

Current regimens for the detection and surveillance of bladder cancer are invasive and have suboptimal sensitivity. Here, we present a novel high-throughput sequencing (HTS) method for detection of urine tumor DNA (utDNA) called utDNA CAPP-Seq (uCAPP-Seq) and apply it to 67 healthy adults and 118 patients with early-stage bladder cancer who had urine collected either prior to treatment or during surveillance. Using this targeted sequencing approach, we detected a median of 6 mutations per patient with bladder cancer and observed surprisingly frequent mutations of the PLEKHS1 promoter (46%), suggesting these mutations represent a useful biomarker for detection of bladder cancer. We detected utDNA pretreatment in 93% of cases using a tumor mutation–informed approach and in 84% when blinded to tumor mutation status, with 96% to 100% specificity. In the surveillance setting, we detected utDNA in 91% of patients who ultimately recurred, with utDNA detection preceding clinical progression in 92% of cases. uCAPP-Seq outperformed a commonly used ancillary test (UroVysion, P = 0.02) and cytology and cystoscopy combined (P ≤ 0.006), detecting 100% of bladder cancer cases detected by cytology and 82% that cytology missed. Our results indicate that uCAPP-Seq is a promising approach for early detection and surveillance of bladder cancer. Show less

The ClinGen PTEN Expert Panel was organized by the ClinGen Hereditary Cancer Clinical Domain Working Group to assemble clinicians, researchers, and molecular diagnosticians with PTEN expertise to develop specifications to the 2015 ACMG/AMP Sequence Variant Interpretation Guidelines for PTEN variant interpretation. We describe finalized PTEN-specific variant classification criteria and outcomes from pilot testing of 42 variants with benign/likely benign (BEN/LBEN), pathogenic/likely pathogenic… Show more

The ClinGen PTEN Expert Panel was organized by the ClinGen Hereditary Cancer Clinical Domain Working Group to assemble clinicians, researchers, and molecular diagnosticians with PTEN expertise to develop specifications to the 2015 ACMG/AMP Sequence Variant Interpretation Guidelines for PTEN variant interpretation. We describe finalized PTEN-specific variant classification criteria and outcomes from pilot testing of 42 variants with benign/likely benign (BEN/LBEN), pathogenic/likely pathogenic (PATH/LPATH), uncertain significance (VUS), and conflicting (CONF) ClinVar assertions. Utilizing these rules, classifications concordant with ClinVar assertions were achieved for 14/15 (93.3%) BEN/LBEN and 16/16 (100%) PATH/LPATH ClinVar consensus variants for an overall concordance of 96.8% (30/31). The variant where agreement was not reached was a synonymous variant near a splice donor with noncanonical sequence for which in silico models cannot predict the native site. Applying these rules to six VUS and five CONF variants, adding shared internal laboratory data enabled one VUS to be classified as LBEN and two CONF variants to be as classified as PATH and LPATH. This study highlights the benefit of gene-specific criteria and the value of sharing internal laboratory data for variant interpretation. Our PTEN-specific criteria and expertly reviewed assertions should prove helpful for laboratories and others curating PTEN variants. Show less

While PCR-based genotyping methods abound in molecular testing for lung cancer therapy, these approaches may not provide the robust sensitivity to detect accurate genotypes in a variable cancer genomic background. Here, we describe a study of a clinical tumor specimen containing a novel somatic single nucleotide variant that caused allele drop-out in EGFR L858R genotyping, resulting in a false-negative interpretation and impacting patient clinical management. We demonstrate that a subsequent… Show more

While PCR-based genotyping methods abound in molecular testing for lung cancer therapy, these approaches may not provide the robust sensitivity to detect accurate genotypes in a variable cancer genomic background. Here, we describe a study of a clinical tumor specimen containing a novel somatic single nucleotide variant that caused allele drop-out in EGFR L858R genotyping, resulting in a false-negative interpretation and impacting patient clinical management. We demonstrate that a subsequent unbiased next-generation sequencing approach correctly identified the driver mutation, and therefore may be more reliable for somatic variant detection. These findings magnify the potential pitfalls of PCR amplification-based approaches and stress the importance of unbiased and sensitive molecular testing strategies for therapeutic marker detection as molecular testing becomes the standard for determining clinical management of cancer patients. Show less

Genetic test misorders can adversely affect patient care. However, little is known about the types of misorders and the overall impact of a utilization management (UM) program on curbing misorders. This study aimed to identify different types of misorders and analyze the impact of a combined test review and consultative service on reducing misorders over time. Selected genetic tests were systematically reviewed between January and December 2015 at Stanford Health Care. Misorders were… Show more

Genetic test misorders can adversely affect patient care. However, little is known about the types of misorders and the overall impact of a utilization management (UM) program on curbing misorders. This study aimed to identify different types of misorders and analyze the impact of a combined test review and consultative service on reducing misorders over time. Selected genetic tests were systematically reviewed between January and December 2015 at Stanford Health Care. Misorders were categorized into five types: clerical errors, redundant testing, better alternatives, controversial, and uncategorized. Moreover, consultations were offered to help clinicians with test selection. Of the 629 molecular test orders reviewed, 13% were classified as misorders, and 7% were modified or canceled. Controversial misorders constitute the most common type (42%); however, unlike the other misorder types, they were negligibly affected by test review. Simultaneously, 71 consults were received. With the introduction of the UM program, genetic test misorders went from 22% at baseline to 3% at the end of the year. Our results show that the combined approach of test review and consultative service effectively reduced misorders over time and suggest that a UM program focused on eliminating misorders can positively influence health-care providers' behaviors.Genet Med advance online publication 26 January 2017. Show less

Identification of putative functional genetic mutations involved in cancer has been dramatically accelerated by developments in next generation sequencing technologies. However, analyzing an individual patient genome and interpreting mutation spectra to inform cancer origin and targeted treatment have been challenging. This study presents a framework interpreting a single patient’s genome and identifies a novel causal mutation in the phosphatase and tensin homolog (PTEN) tumor suppressor… Show more

Identification of putative functional genetic mutations involved in cancer has been dramatically accelerated by developments in next generation sequencing technologies. However, analyzing an individual patient genome and interpreting mutation spectra to inform cancer origin and targeted treatment have been challenging. This study presents a framework interpreting a single patient’s genome and identifies a novel causal mutation in the phosphatase and tensin homolog (PTEN) tumor suppressor. Through computational and experimental approaches, we demonstrate that this mutation causes PTEN to retain known tumor suppressor function while gaining protumor activity. This finding suggests a new role for PTEN and other tumor suppressor involvement in cancer formation and reveals the potential wealth of biological information currently underexploited by the lack of systematic approaches for cancer genome interpretation services. Show less

Large-scale sequencing efforts have documented extensive genetic variation within the human genome. However, our understanding of the origins, global distribution, and functional consequences of this variation is far from complete. While regulatory variation influencing gene expression has been studied within a handful of populations, the breadth of transcriptome differences across diverse human populations has not been systematically analyzed. To better understand the spectrum of gene… Show more

Large-scale sequencing efforts have documented extensive genetic variation within the human genome. However, our understanding of the origins, global distribution, and functional consequences of this variation is far from complete. While regulatory variation influencing gene expression has been studied within a handful of populations, the breadth of transcriptome differences across diverse human populations has not been systematically analyzed. To better understand the spectrum of gene expression variation, alternative splicing, and the population genetics of regulatory variation in humans, we have sequenced the genomes, exomes, and transcriptomes of cell lines derived from 45 individuals in the Human Genome Diversity Panel (HGDP). The populations sampled span the geographic breadth of human migration history and include Namibian San, Mbuti Pygmies of the Democratic Republic of Congo, Algerian Mozabites, Pathan of Pakistan, Cambodians of East Asia, Yakut of Siberia, and Mayans of Mexico. We discover that approximately 25.0% of the variation in gene expression found amongst individuals can be attributed to population differences. However, we find few genes that are systematically differentially expressed among populations. Of this population-specific variation, 75.5% is due to expression rather than splicing variability, and we find few genes with strong evidence for differential splicing across populations. Allelic expression analyses indicate that the cellular effects of common variants are shared across diverse populations. Together, these results provide a resource for studies analyzing functional differences across populations by estimating the degree of shared gene expression, alternative splicing, and regulatory genetics across populations from the broadest points of human migration history yet sampled. Show less