A clinical guide to hereditary cancer panel testing: evaluation of gene-specific cancer associations and sensitivity of genetic testing criteria in a cohort of 165,000 high-risk patients.

PURPOSE: Despite the rapid uptake of multigene panel testing (MGPT) for hereditary cancer predisposition, there is limited guidance surrounding indications for testing and genes to include. METHODS: To inform the clinical approach to hereditary cancer MGPT, we comprehensively evaluated 32 cancer predisposition genes by assessing phenotype-specific pathogenic variant (PV) frequencies, cancer risk associations, and performance of genetic testing criteria in a cohort of 165,000 patients referred for MGPT. RESULTS: We identified extensive genetic heterogeneity surrounding predisposition to cancer types commonly referred for germline testing (breast, ovarian, colorectal, uterine/endometrial, pancreatic, and melanoma). PV frequencies were highest among patients with ovarian cancer (13.8%) and lowest among patients with melanoma (8.1%). Fewer than half of PVs identified in patients meeting testing criteria for only BRCA1/2 or only Lynch syndrome occurred in the respective genes (33.1% and 46.2%). In addition, 5.8% of patients with PVs in BRCA1/2 and 26.9% of patients with PVs in Lynch syndrome genes did not meet respective testing criteria. CONCLUSION: Opportunities to improve upon identification of patients at risk for hereditary cancer predisposition include revising BRCA1/2 and Lynch syndrome testing criteria to include additional clinically actionable genes with overlapping phenotypes and relaxing testing criteria for associated cancers.

REVEL and BayesDel outperform other in silico meta-predictors for clinical variant classification.

Many in silico predictors of genetic variant pathogenicity have been previously developed, but there is currently no standard application of these algorithms for variant assessment. Using 4,094 ClinVar-curated missense variants in clinically actionable genes, we evaluated the accuracy and yield of benign and deleterious evidence in 5 in silico meta-predictors, as well as agreement of SIFT and PolyPhen2, and report the derived thresholds for the best performing predictor(s). REVEL and BayesDel outperformed all other meta-predictors (CADD, MetaSVM, Eigen), with higher positive predictive value, comparable negative predictive value, higher yield, and greater overall prediction performance. Agreement of SIFT and PolyPhen2 resulted in slightly higher yield but lower overall prediction performance than REVEL or BayesDel. Our results support the use of gene-level rather than generalized thresholds, when gene-level thresholds can be estimated. Our results also support the use of 2-sided thresholds, which allow for uncertainty, rather than a single, binary cut-point for assigning benign and deleterious evidence. The gene-level 2-sided thresholds we derived for REVEL or BayesDel can be used to assess in silico evidence for missense variants in accordance with current classification guidelines.

Genotype-phenotype associations among panel-based TP53+ subjects.

PURPOSE: Panel testing has led to the identification of TP53 pathogenic/likely pathogenic (P/LP) variant carriers (TP53+) who exhibit a broad range of phenotypes. We sought to evaluate and compare genotype-phenotype associations among TP53+ panel-ascertained subjects. METHODS: Between 2012 and 2017, 317 TP53+ subjects (279 females and 38 males) identified through panel testing at one testing laboratory were found to have evaluable clinical histories and molecular results. Subject cancer histories were obtained from test requisition forms. P/LP variants were categorized by type and were examined in relation to phenotype. RESULTS: Loss-of-function (LOF) variants were associated with the earliest age at first cancer, with a median age of 30.5 years (P = 0.014); increased frequency of a sarcoma diagnosis (P = 0.016); and more often meeting classic LFS testing and Chompret 2015 criteria (P = 0.004 and 0.002 respectively), as compared with dominant-negative missense, other missense, or miscellaneous (splice or in-frame deletion) P/LP variant categories. CONCLUSION: Loss-of-function variants were more often associated with characteristic LFS cancer histories than other variant categories in TP53+ carriers ascertained through multigene panel testing. These findings require validation in other TP53+ cohorts. Genetic counseling for panel-ascertained TP53+ individuals should reflect the dynamic expansion of the Li-Fraumeni syndrome phenotype.

Comprehensive Paired Tumor/Germline Testing for Lynch Syndrome: Bringing Resolution to the Diagnostic Process.

PURPOSE: The current diagnostic testing algorithm for Lynch syndrome (LS) is complex and often involves multiple follow-up germline and somatic tests. We aimed to describe the results of paired tumor/germline testing performed on a large cohort of patients with colorectal cancer (CRC) and endometrial cancer (EC) to better determine the utility of this novel testing methodology. MATERIALS AND METHODS: We retrospectively reviewed a consecutive series of patients with CRC and EC undergoing paired tumor/germline analysis of the LS genes at a clinical diagnostic laboratory (N = 702). Microsatellite instability, MLH1 promoter hypermethylation, and germline testing of additional genes were performed if ordered. Patients were assigned to one of five groups on the basis of prior tumor screening and germline testing outcomes. Results for each group are described. RESULTS: Overall results were informative regarding an LS diagnosis for 76.1% and 60.8% of patients with mismatch-repair-deficient (MMRd) CRC and EC without and with prior germline testing, respectively. LS germline mutations were identified in 24.8% of patients in the group without prior germline testing, and interestingly, in 9.5% of patients with previous germline testing; four of these were discordant with prior tumor screening. Upon excluding patients with MLH1 promoter hypermethylation and germline mutations, biallelic somatic inactivation was seen in approximately 50% of patients with MMRd tumors across groups. CONCLUSION: Paired testing identified a cause for MMRd tumors in 76% and 61% of patients without and with prior LS germline testing, respectively. Findings support inclusion of tumor sequencing as well as comprehensive LS germline testing in the LS testing algorithm. Paired testing offers a complete, convenient evaluation for LS with high diagnostic resolution.

A Bayesian framework for efficient and accurate variant prediction.

There is a growing need to develop variant prediction tools capable of assessing a wide spectrum of evidence. We present a Bayesian framework that involves aggregating pathogenicity data across multiple in silico scores on a gene-by-gene basis and multiple evidence statistics in both quantitative and qualitative forms, and performs 5-tiered variant classification based on the resulting probability credible interval. When evaluated in 1,161 missense variants, our gene-specific in silico model-based meta-predictor yielded an area under the curve (AUC) of 96.0% and outperformed all other in silico predictors. Multifactorial model analysis incorporating all available evidence yielded 99.7% AUC, with 22.8% predicted as variants of uncertain significance (VUS). Use of only 3 auto-computed evidence statistics yielded 98.6% AUC with 56.0% predicted as VUS, which represented sufficient accuracy to rapidly assign a significant portion of VUS to clinically meaningful classifications. Collectively, our findings support the use of this framework to conduct large-scale variant prioritization using in silico predictors followed by variant prediction and classification with a high degree of predictive accuracy.

TumorNext-Lynch-MMR: a comprehensive next generation sequencing assay for the detection of germline and somatic mutations in genes associated with mismatch repair deficiency and Lynch syndrome.

The current algorithm for Lynch syndrome diagnosis is highly complex with multiple steps which can result in an extended time to diagnosis while depleting precious tumor specimens. Here we describe the analytical validation of a custom probe-based NGS tumor panel, TumorNext-Lynch-MMR, which generates a comprehensive genetic profile of both germline and somatic mutations that can accelerate and streamline the time to diagnosis and preserve specimen. TumorNext-Lynch-MMR can detect single nucleotide variants, small insertions and deletions in 39 genes that are frequently mutated in Lynch syndrome and colorectal cancer. Moreover, the panel provides microsatellite instability status and detects loss of heterozygosity in the five Lynch genes; MSH2, MSH6, MLH1, PMS2 and EPCAM. Clinical cases are described that highlight the assays ability to differentiate between somatic and germline mutations, precisely classify variants and resolve discordant cases.

Somatic TP53 variants frequently confound germ-line testing results.

PURPOSE: Blood/saliva DNA is thought to represent the germ line in genetic cancer-risk assessment. Cases with pathogenic TP53 variants detected by multigene panel testing are often discordant with Li-Fraumeni syndrome, raising concern about misinterpretation of acquired aberrant clonal expansions (ACEs) with TP53 variants as germ-line results. METHODS: Pathogenic TP53 variants with abnormal next-generation sequencing metrics (e.g., decreased ratio (<25%) of mutant to wild-type allele, more than two detected alleles) were selected from a CLIA laboratory testing cohort. Alternate tissues and/or close relatives were tested to distinguish between ACE and germ-line status. Clinical data and Li-Fraumeni syndrome testing criteria were examined. RESULTS: Among 114,630 multigene panel tests and 1,454 TP53 gene-specific analyses, abnormal next-generation sequencing metrics were observed in 20% of 353 TP53-positive results, and ACE was confirmed for 91% of cases with ancillary materials, most of these due to clonal hematopoiesis. Only four met Chompret criteria. Individuals with ACE were older (50 years vs. 33.7; P = 0.02) and were identified more frequently in multigene panel tests (66/285; 23.2%) than in TP53 gene-specific tests (6/68; 8.8%, P = 0.005). CONCLUSION: ACE confounds germ-line diagnosis, may portend hematologic malignancy, and may provoke unwarranted clinical interventions. Ancillary testing to confirm germ-line status should precede Li-Fraumeni syndrome management.

Multigene Panel Testing Provides a New Perspective on Lynch Syndrome.

Purpose Most existing literature describes Lynch syndrome (LS) as a hereditary syndrome leading to high risks of colorectal cancer (CRC) and endometrial cancer mainly as a result of mutations in MLH1 and MSH2. Most of these studies were performed on cohorts with disease suggestive of hereditary CRC and population-based CRC and endometrial cancer cohorts, possibly biasing results. We aimed to describe a large cohort of mismatch repair (MMR) mutation carriers ascertained through multigene panel testing, evaluate their phenotype, and compare the results with those of previous studies. Methods We retrospectively reviewed clinical histories of patients who had multigene panel testing, including the MMR and EPCAM genes, between March 2012 and June 2015 (N = 34,981) and performed a series of statistical comparisons. Results Overall, MSH6 mutations were most frequent, followed by PMS2, MSH2, MLH1, and EPCAM mutations, respectively. Of 528 patients who had MMR mutations, 63 (11.9%) had breast cancer only and 144 (27.3%) had CRC only. When comparing those with breast cancer only to those with CRC only, MSH6 and PMS2 mutations were more frequent than MLH1 and MSH2 mutations ( P = 2.3 × 10-5). Of the 528 patients, 22.2% met BRCA1 and BRCA2 ( BRCA1/2) testing criteria and not LS criteria, and 5.1% met neither BRCA1/2 nor LS testing criteria. MSH6 and PMS2 mutations were more frequent than MLH1 and MSH2 mutations among patients who met BRCA1/2 testing criteria but did not meet LS testing criteria ( P = 4.3 × 10-7). Conclusion These results provide a new perspective on LS and suggest that individuals with MSH6 and PMS2 mutations may present with a hereditary breast and ovarian cancer phenotype. These data also highlight the limitations of current testing criteria in identifying these patients, as well as the need for further investigation of cancer risks in patients with MMR mutations.

Processed Pseudogene Confounding Deletion/Duplication Assays for SMAD4.

Mutations in SMAD4 have been associated with juvenile polyposis syndrome and combined juvenile polyposis/hereditary hemorrhagic telangiectasia syndrome. SMAD4 is part of the SMAD gene family. To date, there has been no report in the literature of a SMAD4 pseudogene. An unusual SMAD4 duplication pattern was seen in multiple patient samples using two different duplication/deletion platforms: multiplex ligation-dependent probe amplification and chromosomal microarray. Follow-up confirmatory testing included real-time quantitative PCR and sequencing of an exon/exon junction, all results leading to the conclusion of the existence of a processed pseudogene. Examination of clinical results from two laboratories found a frequency of 0.26% (12 in 4672 cases) for this processed pseudogene. This is the first report of the presence of a processed pseudogene for SMAD4. We believe that knowledge of its existence is important for accurate interpretation of clinical diagnostic test results and for new assay designs. This study also indicates how a processed pseudogene may confound quantitative results, dependent on placement of probes and/or primers in a particular assay design, potentially leading to both false-positive and false-negative results. We also found that the SMAD4 processed pseudogene affects next-generation sequencing results by confounding the alignment of the sequences, resulting in erroneous variant calls. We recommend Sanger sequencing confirmation for SMAD4 variants.

Clinical report of a 17q12 microdeletion with additionally unreported clinical features.

Copy number variations involving the 17q12 region have been associated with developmental and speech delay, autism, aggression, self-injury, biting and hitting, oppositional defiance, inappropriate language, and auditory hallucinations. We present a tall-appearing 17-year-old boy with marfanoid habitus, hypermobile joints, mild scoliosis, pectus deformity, widely spaced nipples, pes cavus, autism spectrum disorder, intellectual disability, and psychiatric manifestations including physical and verbal aggression, obsessive-compulsive behaviors, and oppositional defiance. An echocardiogram showed borderline increased aortic root size. An abdominal ultrasound revealed a small pancreas, mild splenomegaly with a 1.3 cm accessory splenule, and normal kidneys and liver. A testing panel for Marfan, aneurysm, and related disorders was negative. Subsequently, a 400 K array-based comparative genomic hybridization (aCGH) + SNP analysis was performed which identified a de novo suspected pathogenic deletion on chromosome 17q12 encompassing 28 genes. Despite the limited number of cases described in the literature with 17q12 rearrangements, our proband's phenotypic features both overlap and expand on previously reported cases. Since syndrome-specific DNA sequencing studies failed to provide an explanation for this patient's unusual habitus, we postulate that this case represents an expansion of the 17q12 microdeletion phenotype. Further analysis of the deleted interval is recommended for new genotype-phenotype correlations.

Utilization of multigene panels in hereditary cancer predisposition testing: analysis of more than 2,000 patients.

PURPOSE: The aim of this study was to determine the clinical and molecular characteristics of 2,079 patients who underwent hereditary cancer multigene panel testing. METHODS: Panels included comprehensive analysis of 14-22 cancer susceptibility genes (BRCA1 and BRCA2 not included), depending on the panel ordered (BreastNext, OvaNext, ColoNext, or CancerNext). Next-generation sequencing and deletion/duplication analyses were performed for all genes except EPCAM (deletion/duplication analysis only). Clinical histories of ColoNext patients harboring mutations in genes with well-established diagnostic criteria were assessed to determine whether diagnostic/testing criteria were met. RESULTS: Positive rates were defined as the proportion of patients with a pathogenic mutation/likely pathogenic variant(s) and were as follows: 7.4% for BreastNext, 7.2% for OvaNext, 9.2% for ColoNext, and 9.6% for CancerNext. Inconclusive results were found in 19.8% of BreastNext, 25.6% of OvaNext, 15.1% of ColoNext, and 23.5% of CancerNext tests. Based on information submitted by clinicians, 30% of ColoNext patients with mutations in genes with well-established diagnostic criteria did not meet corresponding criteria. CONCLUSION: Our data point to an important role for targeted multigene panels in diagnosing hereditary cancer predisposition, particularly for patients with clinical histories spanning several possible diagnoses and for patients with suspicious clinical histories not meeting diagnostic criteria for a specific hereditary cancer syndrome.

Short-term correction of arginase deficiency in a neonatal murine model with a helper-dependent adenoviral vector.

Neonatal gene therapy has the potential to ameliorate abnormalities before disease onset. Our gene knockout of arginase I (AI) deficiency is characterized by increasing hyperammonemia, neurological deterioration, and early death. We constructed a helper-dependent adenoviral vector (HDV) carrying AI and examined for correction of this defect. Neonates were administered 5 x 10(9) viral particles/g and analyzed for survival, arginase activity, and ammonia and amino acids levels. The life expectancy of arg(-/-) mice increased to 27 days while controls died at 14 days with hyperammonemia and in extremis. Death correlated with a decrease in viral DNA/RNA per cell as liver mass increased. Arginase assays demonstrated that vector-injected hepatocytes had ~20% activity of heterozygotes at 2 weeks of age. Hepatic arginine and ornithine in treated mice were similar to those of saline-injected heterozygotes at 2 weeks, whereas ammonia was normal. By 26 days, arginase activity in the treated arg(-/-) livers declined to <10%, and arginine and ornithine increased. Ammonia levels began increasing by day 25, suggesting the cause of death to be similar to that of uninjected arg(-/-) mice, albeit at a later time. These studies demonstrate that the AI deficient newborn mouse can be temporarily corrected and rescued using a HDV.

Loss of arginase I results in increased proliferation of neural stem cells.

Loss of arginase I (AI) results in a metabolic disorder characterized by growth retardation, increased mental impairment and spasticity, and potentially fatal hyperammonemia. This syndrome plus a growing body of evidence supports a role for arginase and arginine metabolites in normal neuronal development and function. Here we report our initial observations of the effects of AI loss on proliferation and differentiation of neural stem cells (NSCs) isolated from the germinal zones of embryonic and newborn AI knockout (KO) mice compared with heterozygous (HET) and wild-type (WT) control animals. By using both short and long-term proliferation assays (3 and 10 days, respectively), we found a 1.5-2-fold increase in the number of KO cells compared with WT. FACS analysis showed an increase in KO cells in the synthesis phase of the cell cycle vs. WT cells. After NSC differentiation, AI-deficient cells expressed beta-tubulin, SMI81 (SNAP25), glial fibrillary acidic protein, and CNPase, which are markers consistent with neurons, astrocytes, and oligodendrocytes. Many KO cells exhibited a more mature morphology and expressed mature neuronal markers that were decreased or not present in HET or WT cells. Limited, comparative expression array and quantitative RT-PCR analysis identified differences in the levels of several mRNAs encoding structural, signaling, and arginine metabolism proteins between KO and WT cells. The consequence of these changes may contribute to the differential phenotypes of KO vs. WT cells. It appears that AI may play an important and unanticipated role in growth and development of NSCs.

Farnesyltransferase inhibitors reverse altered growth and distribution of actin filaments in Tsc-deficient cells via inhibition of both rapamycin-sensitive and -insensitive pathways.

Farnesyltransferase inhibitors (FTI) have been developed as anticancer drugs and are currently being evaluated in clinical trials. In this study, we have examined the effects of FTIs on Tsc-null cells to gain insight into their effects on farnesylated Rheb GTPase. This protein is involved in the activation of mTOR/S6K signaling and is down-regulated by the Tsc1/Tsc2 complex. Both Tsc1(-/-) and Tsc2(-/-) mouse embryonic fibroblasts exhibit constitutive activation of S6K and grow in the absence of serum. Two different FTI compounds, the clinical compound BMS-214662 and the newly described BMS-225975, inhibit the constitutive activation of mTOR/S6K signaling and block serum-free growth of the Tsc-null mouse embryonic fibroblasts. We have also found that Tsc-null mouse embryonic fibroblasts grow under anchorage-independent conditions and that both FTI compounds inhibit this soft agar growth. These FTI effects are similar to those observed with rapamycin. Another interesting phenotype of Tsc-null mouse embryonic fibroblasts is that they are round and contain actin filaments predominantly at the cell periphery. The addition of FTIs, but not rapamycin, led to the reappearance of intracellular actin filaments and reduction of peripheral actin filaments. The ability of FTI to rearrange actin filaments seems to be largely mediated by the inhibition of Rheb protein, as induction of intracellular actin filaments by FTI was much less efficient in Tsc2-null cells expressing Rheb (M184L), a geranylgeranylated mutant Rheb that can bypass farnesylation. These results reveal that FTIs inhibit Rheb, causing two different effects in Tsc-deficient cells, one on growth and the other on actin filament distribution.

Loss of tuberous sclerosis complex 1 (Tsc1) expression results in increased Rheb/S6K pathway signaling important for astrocyte cell size regulation.

Individuals with tuberous sclerosis complex (TSC) develop central nervous system abnormalities that may reflect astrocyte dysfunction. In an effort to model astrocyte dysfunction in TSC, we generated mice lacking Tsc1 (hamartin) expression in astrocytes and demonstrated that Tsc1-null astrocytes exhibit abnormalities in contact inhibition growth arrest. In this study, we demonstrate that hamartin-deficient astrocytes are also defective in cell size regulation. We show that the increase in Tsc1-null astrocyte size is associated with increased activation of the S6-kinase pathway. In keeping with recent reports that the hamartin/tuberin complex may regulate Rheb and downstream S6K activation, we demonstrate that expression of either Rheb or S6K in primary astrocytes results in increased S6 pathway activation, and that inhibition of Rheb activity in Tsc1-deficient astrocytes using either pharmacologic or genetic strategies markedly reduces S6 activation. Collectively, these observations suggest that TSC inactivation in astrocytes results in defective cell size regulation associated with dysregulated Rheb/mTOR/S6K pathway activity.

Drosophila Rheb GTPase is required for cell cycle progression and cell growth.

Precise body and organ sizes in the adult animal are ensured by a range of signaling pathways. In a screen to identify genes affecting hindgut morphogenesis in Drosophila, we identified a P-element insertion in dRheb, a novel, highly conserved member of the Ras superfamily of G-proteins. Overexpression of dRheb in the developing fly (using the GAL4:UAS system) causes dramatic overgrowth of multiple tissues: in the wing, this is due to an increase in cell size; in cultured cells, dRheb overexpression results in accumulation of cells in S phase and an increase in cell size. Using a loss-of-function mutation we show that dRheb is required in the whole organism for viability (growth) and for the growth of individual cells. Inhibition of dRheb activity in cultured cells results in their arrest in G1 and a reduction in size. These data demonstrate that dRheb is required for both cell growth (increase in mass) and cell cycle progression; one explanation for this dual role would be that dRheb promotes cell cycle progression by affecting cell growth. Consistent with this interpretation, we find that flies with reduced dRheb activity are hypersensitive to rapamycin, an inhibitor of the growth regulator TOR. In cultured cells, the effect of overexpressing dRheb was blocked by the addition of rapamycin. These results imply that dRheb is involved in TOR signaling.

Identification of dominant negative mutants of Rheb GTPase and their use to implicate the involvement of human Rheb in the activation of p70S6K.

Rheb GTPases represent a unique family of the Ras superfamily of G-proteins. Studies on Rheb in Schizosaccharomyces pombe and Drosophila have shown that this small GTPase is essential and is involved in cell growth and cell cycle progression. The Drosophila studies also raised the possibility that Rheb is involved in the TOR/S6K signaling pathway. In this paper, we first report identification of dominant negative mutants of S. pombe Rheb (SpRheb). Screens of a randomly mutagenized SpRheb library yielded a mutant, SpRhebD60V, whose expression in S. pombe results in growth inhibition, G1 arrest, and induction of fnx1+, a gene whose expression is induced by the disruption of Rheb. Alteration of the Asp-60 residue to all possible amino acids by site-directed mutagenesis led to the identification of two particularly strong dominant negative mutants, D60I and D60K. Characterization of these dominant negative mutant proteins revealed that D60V and D60I exhibit preferential binding of GDP, while D60K lost the ability to bind both GTP and GDP. A possible use of the dominant negative mutants in the study of mammalian Rheb was explored by introducing dominant negative mutations into human Rheb. We show that transient expression of the wild type Rheb1 or Rheb2 causes activation of p70S6K, while expression of Rheb1D60K mutant results in inhibition of basal level activity of p70S6K. In addition, Rheb1D60K and Rheb1D60V mutants blocked nutrient- or serum-induced activation of p70S6K. This provides critical evidence that Rheb plays a role in the mTOR/S6K pathway in mammalian cells.