Discordance between germline genetic findings and abnormal tumor immunohistochemistry staining of mismatch repair proteins in individuals with suspected Lynch syndrome.

Background and Aims: Tumor immunohistochemical staining (IHC) of DNA mismatch repair (MMR) proteins is often used to guide germline genetic testing and variant classification for patients with suspected Lynch syndrome. This analysis examined the spectrum of germline findings in a cohort of individuals showing abnormal tumor IHC. Methods: We assessed individuals with reported abnormal IHC findings and referred for testing with a six-gene syndrome-specific panel (n=703). Pathogenic variants (PVs) and variants of uncertain significance (VUS) in MMR genes were designated expected/unexpected relative to IHC results. Results: The PV positive rate was 23.2% (163/703; 95% confidence interval [CI], 20.1%-26.5%); 8.0% (13/163; 95% CI, 4.3%-13.3%) of PV carriers had a PV in an unexpected MMR gene. Overall, 121 individuals carried VUS in MMR genes expected to be mutated based on IHC results. Based on independent evidence, in 47.1% (57/121; 95% CI, 38.0%-56.4%) of these individuals the VUSs were later reclassified as benign and in 14.0% (17/121; 95% CI, 8.4%-21.5%) of these individuals the VUSs were reclassified as pathogenic. Conclusions: Among patients with abnormal IHC findings, IHC-guided single-gene genetic testing may miss 8% of individuals with Lynch syndrome. In addition, in patients with VUS identified in MMR genes predicted to be mutated by IHC, extreme caution must be taken when the IHC results are considered in variant classification.

Impact of a Cancer Gene Variant Reclassification Program Over a 20-Year Period.

PURPOSE: Hereditary cancer genetic testing can inform personalized medical management for individuals at increased cancer risk. However, many variants in cancer predisposition genes are individually rare, and traditional tools may be insufficient to evaluate pathogenicity. This analysis presents data on variant classification and reclassification over a 20-year period. PATIENTS AND METHODS: This is a retrospective analysis of > 1.9 million individuals who received hereditary cancer genetic testing from a single clinical laboratory (March 1997 to December 2017). Variant classification included review of evidence from traditional tools (eg, population frequency databases, literature) and laboratory-developed tools (eg, novel statistical methods, in-house RNA analysis) by a multidisciplinary expert committee. Variants may have been reclassified more than once and with more than one line of evidence. RESULTS: In this time period, 62,842 unique variants were observed across 25 cancer predisposition genes, and 2,976 variants were reclassified. Overall, 82.1% of reclassification events were downgrades (eg, variant of uncertain significance [VUS] to benign), and 17.9% were upgrades (eg, VUS to pathogenic). Among reclassified variants, 82.8% were initially classified as VUS, and 47.5% were identified in ≤ 20 individuals (allele frequency ≤ 0.001%). Laboratory-developed tools were used in 72.3% of variant reclassification events, which affected > 600,000 individuals. More than 1.3 million patients were identified as carrying a variant that was reclassified within this 20-year time period. CONCLUSION: The variant classification program used by the laboratory evaluated here enabled the reclassification of variants that were individually rare. Laboratory-developed tools were a key component of this program and were used in the majority of reclassifications. This demonstrates the importance of using robust and novel tools to reclassify rare variants to appropriately inform personalized medical management.

Hereditary cancer testing challenges: assembling the analytical pieces to solve the patient clinical puzzle.

Expanded genetic test utilization to guide cancer management has driven the development of larger gene panels and greater diversity in the patient population pursuing testing, resulting in increased identification of atypical or technically challenging genetic findings. To ensure appropriate patient care, it is critical that genetic tests adequately identify and characterize these findings. We describe genetic testing challenges frequently encountered by our laboratory and the methodologies we employ to improve test accuracy for the identification and characterization of atypical genetic findings. While these findings may be individually rare, 15,745 (9%) individuals tested by our laboratory for hereditary cancer risk had an atypical genetic finding, highlighting the importance of employing highly accurate and comprehensive methods in clinical genetic testing.

Identification of pathogenic retrotransposon insertions in cancer predisposition genes.

Cancer risks have been previously reported for some retrotransposon element (RE) insertions; however, detection of these insertions is technically challenging and very few oncogenic RE insertions have been reported. Here we evaluate RE insertions identified during hereditary cancer genetic testing using a comprehensive testing strategy. Individuals who had single-syndrome or pan-cancer hereditary cancer genetic testing from February 2004 to March 2017 were included. RE insertions were identified using Sanger sequencing, Next Generation Sequencing, or multiplex quantitative PCR, and further characterized using targeted PCR and sequencing analysis. Personal cancer history, ancestry, and haplotype were evaluated. A total of 37 unique RE insertions were identified in 10 genes, affecting 211 individuals. BRCA2 accounted for 45.9% (17/37) of all unique RE insertions. Several RE insertions were detected with high frequency in populations of conserved ancestry wherein up to 100% of carriers shared a high degree of haplotype conservation, suggesting founder effects. Our comprehensive testing strategy resulted in a substantial increase in the number of reported oncogenic RE insertions, several of which may have possible founder effects. Collectively, these data show that the detection of RE insertions is an important component of hereditary cancer genetic testing and may be more prevalent than previously reported.

Classification of genetic variants in genes associated with Lynch syndrome using a clinical history weighting algorithm.

BACKGROUND: Lynch syndrome is a hereditary cancer syndrome associated with high risks of colorectal and endometrial cancer that is caused by pathogenic variants in the mismatch repair genes (MLH1, MSH2, MSH6, PMS2, EPCAM). Accurate classification of variants identified in these genes as pathogenic or benign enables informed medical management decisions. Previously, we developed a clinical History Weighting Algorithm (HWA) for the classification of variants of uncertain significance (VUSs) in BRCA1 and BRCA2. The BRCA1/2 HWA is based on the premise that pathogenic variants in these genes will be identified more often in individuals with strong personal and/or family histories of breast and/or ovarian cancer, while the identification of benign variants should be independent of cancer history. Here we report the development of a similar HWA to allow for classification of VUSs in genes associated with Lynch syndrome using data collected through both syndrome-specific and pan-cancer panel testing. METHODS: Upon completion of algorithm development, the HWA was tested using simulated variants constructed from 79,214 probands, as well as 379 true variants. Positive (PPV) and negative predictive values (NPV) were calculated on a per gene basis. RESULTS: 25,500 pathogenic and 50,500 benign simulated variants were analyzed using the HWA and the PPVs and NPVs for each gene were greater than 0.997 and 0.999, respectively. The HWA was also evaluated using 100 trials for each of the 379 true variants. PPVs of >0.998 and NPVs of >0.999 were obtained for all genes. CONCLUSIONS: We have developed and implemented a HWA to aid in the classification of VUSs in genes associated with Lynch syndrome. The work presented here demonstrates that this HWA is able to classify MLH1, MSH2, and MSH6 VUSs as either benign or pathogenic with high accuracy.

Identification of a Variety of Mutations in Cancer Predisposition Genes in Patients With Suspected Lynch Syndrome.

BACKGROUND & AIMS: Multigene panels are commercially available tools for hereditary cancer risk assessment that allow for next-generation sequencing of numerous genes in parallel. However, it is not clear if these panels offer advantages over traditional genetic testing. We investigated the number of cancer predisposition gene mutations identified by parallel sequencing in individuals with suspected Lynch syndrome. METHODS: We performed germline analysis with a 25-gene, next-generation sequencing panel using DNA from 1260 individuals who underwent clinical genetic testing for Lynch syndrome from 2012 through 2013. All patients had a history of Lynch syndrome-associated cancer and/or polyps. We classified all identified germline alterations for pathogenicity and calculated the frequencies of pathogenic mutations and variants of uncertain clinical significance (VUS). We also analyzed data on patients' personal and family history of cancer, including fulfillment of clinical guidelines for genetic testing. RESULTS: Of the 1260 patients, 1112 met National Comprehensive Cancer Network (NCCN) criteria for Lynch syndrome testing (88%; 95% confidence interval [CI], 86%-90%). Multigene panel testing identified 114 probands with Lynch syndrome mutations (9.0%; 95% CI, 7.6%-10.8%) and 71 with mutations in other cancer predisposition genes (5.6%; 95% CI, 4.4%-7.1%). Fifteen individuals had mutations in BRCA1 or BRCA2; 93% of these met the NCCN criteria for Lynch syndrome testing and 33% met NCCN criteria for BRCA1 and BRCA2 analysis (P = .0017). An additional 9 individuals carried mutations in other genes linked to high lifetime risks of cancer (5 had mutations in APC, 3 had bi-allelic mutations in MUTYH, and 1 had a mutation in STK11); all of these patients met NCCN criteria for Lynch syndrome testing. A total of 479 individuals had 1 or more VUS (38%; 95% CI, 35%-41%). CONCLUSIONS: In individuals with suspected Lynch syndrome, multigene panel testing identified high-penetrance mutations in cancer predisposition genes, many of which were unexpected based on patients' histories. Parallel sequencing also detected a high number of potentially uninformative germline findings, including VUS.

Development and validation of a new algorithm for the reclassification of genetic variants identified in the BRCA1 and BRCA2 genes.

BRCA1 and BRCA2 sequencing analysis detects variants of uncertain clinical significance in approximately 2 % of patients undergoing clinical diagnostic testing in our laboratory. The reclassification of these variants into either a pathogenic or benign clinical interpretation is critical for improved patient management. We developed a statistical variant reclassification tool based on the premise that probands with disease-causing mutations are expected to have more severe personal and family histories than those having benign variants. The algorithm was validated using simulated variants based on approximately 145,000 probands, as well as 286 BRCA1 and 303 BRCA2 true variants. Positive and negative predictive values of ≥99 % were obtained for each gene. Although the history weighting algorithm was not designed to detect alleles of lower penetrance, analysis of the hypomorphic mutations c.5096G>A (p.Arg1699Gln; BRCA1) and c.7878G>C (p.Trp2626Cys; BRCA2) indicated that the history weighting algorithm is able to identify some lower penetrance alleles. The history weighting algorithm is a powerful tool that accurately assigns actionable clinical classifications to variants of uncertain clinical significance. While being developed for reclassification of BRCA1 and BRCA2 variants, the history weighting algorithm is expected to be applicable to other cancer- and non-cancer-related genes.

Functional evaluation of BRCA2 variants mapping to the PALB2-binding and C-terminal DNA-binding domains using a mouse ES cell-based assay.

Single-nucleotide substitutions and small in-frame insertions or deletions identified in human breast cancer susceptibility genes BRCA1 and BRCA2 are frequently classified as variants of unknown clinical significance (VUS) due to the availability of very limited information about their functional consequences. Such variants can most reliably be classified as pathogenic or non-pathogenic based on the data of their co-segregation with breast cancer in affected families and/or their co-occurrence with a pathogenic mutation. Biological assays that examine the effect of variants on protein function can provide important information that can be used in conjunction with available familial data to determine the pathogenicity of VUS. In this report, we have used a previously described mouse embryonic stem (mES) cell-based functional assay to characterize eight BRCA2 VUS that affect highly conserved amino acid residues and map to the N-terminal PALB2-binding or the C-terminal DNA-binding domains. For several of these variants, very limited co-segregation information is available, making it difficult to determine their pathogenicity. Based on their ability to rescue the lethality of Brca2-deficient mES cells and their effect on sensitivity to DNA-damaging agents, homologous recombination and genomic integrity, we have classified these variants as pathogenic or non-pathogenic. In addition, we have used homology-based modeling as a predictive tool to assess the effect of some of these variants on the structural integrity of the C-terminal DNA-binding domain and also generated a knock-in mouse model to analyze the physiological significance of a residue reported to be essential for the interaction of BRCA2 with meiosis-specific recombinase, DMC1.

Viral endomyocardial infection is an independent predictor and potentially treatable risk factor for graft loss and coronary vasculopathy in pediatric cardiac transplant recipients.

OBJECTIVES: This study sought to evaluate the outcome and prevalence of viral endomyocardial infection after cardiac transplantation. BACKGROUND: Viral myocardial infection causes heart failure, but its role after cardiac transplantation is unclear. We hypothesized that viral infection of the cardiac allograft reduces graft survival. METHODS: Between June 1999 and November 2004, 94 pediatric cardiac transplant patients were screened for the presence of viral genome in serial endomyocardial biopsies (EMBs) using polymerase chain reaction (PCR) assays. Graft loss, advanced transplant coronary artery disease (TCAD), and acute rejection (AR) were compared in the PCR-positive (n = 37) and PCR-negative (n = 57) groups, using time-dependent Kaplan-Meier and Cox regression analyses. From November 2002 to November 2004, intravenous immunoglobulin therapy (IVIG) was administered to patients with PCR-positive EMBs. The outcomes of the IVIG-treated, PCR-positive patients (n = 20) were compared with IVIG-untreated, PCR-positive patients (n = 17). RESULTS: Viral genomes were detected in EMBs from 37 (39%) patients; parvovirus B19, adenovirus, and Epstein-Barr virus (EBV) were the most common. The PCR-positive group (n = 37, 25% graft loss at 2.4 years) had decreased graft survival (p < 0.001) compared with the PCR-negative group (n = 57, 25% graft loss at 8.7 years) and developed advanced TCAD prematurely (p = 0.001). The number of AR episodes was similar in both groups. On multivariate analysis, presence of viral genome was an independent risk factor for graft loss (relative risk: 4.2, p = 0.015). The time to advanced TCAD after becoming PCR-positive was longer in the IVIG-treated patients (p = 0.03) with a trend toward improved graft survival (p = 0.06). CONCLUSIONS: Viral endomyocardial infection is an independent predictor of graft loss in pediatric cardiac transplant recipients. This effect appears to be mediated through premature development of advanced TCAD. IVIG therapy in this subgroup may improve survival and merits further investigation.

Viral epidemiologic shift in inflammatory heart disease: the increasing involvement of parvovirus B19 in the myocardium of pediatric cardiac transplant patients.

BACKGROUND: Detection of viral genome in rejecting cardiac transplant patients has been reported, with coxsackievirus and adenovirus causing premature graft failure. Recently, parvovirus B19 (PVB19) genome in myocardial samples has been increasingly reported, but its role in cardiac pathology and effect on transplant graft survival are unknown. The objectives of this study were to determine if changes in the viruses identified in the myocardium represent an epidemiologic shift in viral myocardial disease and whether PVB19 adversely affects transplant graft survival. METHODS: From September 2002 to December 2005, nested polymerase chain reaction was used to evaluate endomyocardial biopsy specimens for 99 children (aged 3 weeks-18 years) with heart transplants for the presence of viral genome. Cellular rejection was assessed by histology of specimens. Transplant coronary artery disease (TCAD) was diagnosed by coronary angiography or histopathology. RESULTS: Specimens from 700 biopsies were evaluated from 99 patients; 121 specimens had viral genome, with 100 (82.6%) positive for PVB19, 24 for Epstein-Barr virus (EBV; 7 positive for PVB19 and EBV), 3 for CMV, and 1 for adenovirus. Presence of PVB19 genome did not correlate with rejection score, nor did a higher viral copy number. Early development of advanced TCAD (p < 0.001) occurred in 20 children with persistent PVB19 infection (> 6 months). CONCLUSIONS: PVB19 is currently the predominant virus detected in heart transplant surveillance biopsy specimens, possibly representing an epidemiologic shift. Cellular rejection does not correlate with the presence or quantity of PVB19 genome in the myocardium, but children with chronic PVB19 infection have increased risk for earlier TCAD, supporting the hypothesis that PVB19 negatively affects graft survival.

Mutations in the muscle LIM protein and alpha-actinin-2 genes in dilated cardiomyopathy and endocardial fibroelastosis.

Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality. Two genes have been identified for the X-linked forms (dystrophin and tafazzin), while mutations in multiple genes cause autosomal dominant DCM. Muscle LIM protein (MLP) is a member of the cysteine-rich protein (CRP) family and has been implicated in both myogenesis and sarcomere assembly. In the latter role, it binds zyxin and alpha-actinin, both of which are involved in actin organization. An MLP-deficient mouse has been described; these mice develop dilated cardiomyopathy and heart failure. Based upon these data, and the recent descriptions of mutations in MLP in patients with DCM or hypertrophic cardiomyopathy, we screened patients for mutations in the MLP and alpha-actinin-2 genes. We identified a patient with DCM and EFE, having a mutation in MLP with the residue lysine 69 substituted by arginine (K69R). This is within a highly conserved region adjacent to the first LIM domain involved in alpha-actinin binding. Analysis in cell culture systems demonstrated that the mutation abolishes the interaction between MLP and alpha-actinin-2 and the cellular localization of MLP was altered. In another individual with DCM, a W4R mutation was identified. However, this mutation did not segregate with disease in this family. In another patient with DCM, a Q9R mutation was identified in alpha-actinin-2. This mutation also disrupted the interaction with MLP and appeared to inhibit alpha-actinin function in cultured cells, in respect to the nuclear localization of actinin and the initiation of cellular differentiation.

Detection of viruses in myocardial tissues by polymerase chain reaction. evidence of adenovirus as a common cause of myocarditis in children and adults.

OBJECTIVES: The purpose of this study was to analyze cardiac tissue and blood for viral genomes using polymerase chain reaction (PCR) to define the common viral etiologies of myocarditis by age group. BACKGROUND: Enteroviruses are considered the most common cause of myocarditis at all ages. Diagnosis relies on viral cultures, serology, and cardiac histology, which lack sensitivity, as well as PCR. However, in many cases enteroviruses are not detected. METHODS: Cardiac samples were obtained for PCR analysis from patients with myocarditis (n = 624) and dilated cardiomyopathy (DCM) (n = 149). Patients were analyzed by age group, including neonates (n = 116), infants (n = 191), toddlers (n = 87), children (n = 110), adolescents (n = 92), and adults (n = 177). After nucleic acids had been extracted from an endomyocardial biopsy, an explant, or autopsy samples, PCR and reverse transcription PCR were performed to detect the genomic sequences of enterovirus, adenovirus, cytomegalovirus (CMV), herpes simplex virus (HSV), Epstein-Barr virus (EBV), parvovirus, respiratory syncytial virus (RSV), and influenza A virus. RESULTS: Viral genome was amplified (adenovirus = 142, enterovirus = 85, CMV = 18, parvovirus = 6, influenza A = 5, HSV = 5, EBV = 3, RSV = 1) from 239 (38%) of the 624 samples from myocarditis patients, including 26 patient samples in which dual infection was found. Virus was detected in 30 (20%) of 149 DCM patient samples; only adenovirus (n = 18) and enterovirus (n = 12) were detected. CONCLUSIONS: Polymerase chain reaction identified adenovirus as the most common virus in the myocardium of children and adults with myocarditis and DCM. Although enteroviruses are also found in these patients, they appear to be a less common cause of myocarditis than adenovirus.