Test Information Sheet

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1 Test Information Sheet GeneDx 207 Perry Parkway Gaithersburg, MD Phone: Fax: OncoGene Dx: High/Moderate Risk Panel Sequence Analysis and/or Exon Level Deletion/Duplication Testing of 23 Genes OncoGene Dx High/Moderate Risk Panel Gene List: APC BRCA2 EPCAM^ PALB2 SMAD4 ATM BPRIP1 MLH1 PMS2 STK11 BMPR1A CDH1 MSH2 PTEN TP53 BRCA1 CDKN2A MSH6 RAD51C VHL CHEK2 MUTYH RAD51D ^Next generation sequencing of this gene is not included in this panel. Clinical Features and Genetics: Cancer is a common disease affecting approximately 1 in 3 individuals in the U.S (SEER). While the majority of cancers are sporadic in nature, some families have hereditary forms of cancer that are associated with increased cancer risks compared with the general population. Approximately 5 10% of cancer cases are thought to be due to a hereditary predisposition. The features of a personal and/or family history of cancer that are suggestive of a hereditary cancer predisposition include: young ages at diagnosis, multiple primary cancers in a single individual, and several relatives affected with the same type of cancer or related cancers spanning multiple generations. For some of the well described hereditary conditions discussed below, clinical diagnostic criteria, based on personal medical history and family history, are available to help identify patients most likely to have a hereditary cancer syndrome. In many cases, however, patients do not meet the clinical diagnostic criteria or the criteria may overlap for multiple conditions, making it difficult to decide which genes should be tested and in what order. The OncoGeneDx High/Moderate Risk Panel offered at GeneDx includes analysis of 23 genes associated with hereditary predisposition to various cancers including the most well known hereditary cancer syndromes such as Hereditary Breast and Ovarian Cancer Syndrome (BRCA1, BRCA2) and Lynch Syndrome (MLH1, MSH2, MSH6, PMS2 and EPCAM) as well as moderately penetrant genes including ATM, BRIP1, CHEK2, RAD51C and RAD51D. Many of the genes on this panel are involved in the mismatch repair pathway, the Fanconi anemia pathway and/or play a role in DNA damage repair. All of the genes included on the OncoGeneDx High/Moderate Risk Panel are associated with dominantly inherited cancer risk with the exception of MUTYH associated polyposis which is recessively inherited. The level of cancer risk, relative to general population risk, that is associated with pathogenic variants in each of the genes is outlined in the attached table. With respect to medical management, all of the genes on the OncoGeneDx High/Moderate Risk Panel are discussed specifically in published guidelines such as the NCCN Genetic/Familial High Risk Assessment: Breast/Ovarian or Colorectal. Many of these dominantly associated genes are also associated with an autosomal recessive syndrome if an individual inherits two pathogenic variants in the same gene, one from each parent. For example, if both mother and father are carriers of pathogenic BRCA2 variants, each of their children would have a 25% chance to inherit both pathogenic variants, a 50% chance to inherit one of the pathogenic variants, and a 25% chance to inherit neither pathogenic variant. If a child inherited both pathogenic BRCA2 variants, they would have a rare condition called Fanconi anemia characterized by an increased risk for malignancy in children including leukemia and certain solid Information Sheet on High/Moderate Risk Panel Page 1 of 5 GeneDx Revision Date: 07/2016

2 tumors as well as physical abnormalities and bone marrow failure. All genes that have an associated recessive condition are noted in the attached table. Reason for referral: In addition to the features of hereditary cancer predisposition described above, a next generation sequencing panel for high/moderate risk genes may be especially helpful in cases in which: 1) The differential diagnosis includes various hereditary cancer syndromes. For example, if the family history consists of multiple cases of ovarian cancer, this may be associated with a breast/ovarian cancer syndrome such as BRCA1 or BRCA2 or Lynch syndrome (MLH1, MSH2, MSH6, PMS2, and EPCAM). Thus, the OncoGeneDx High/Moderate Risk Panel offers increased clinical sensitivity compared to testing only for the BRCA1/2 genes. Furthermore, panel testing is more cost effective than stepwise genetic testing (for example, ordering BRCA1/2 testing followed by additional genetic testing). 2) The family history includes a number of cancer cases, but they are of several different types. Therefore, the pattern does not seem to fit any one hereditary cancer syndrome in particular. 3) Some genetic testing has already been ordered due to a family history suggestive of a hereditary cancer predisposition, and results have been negative. OncoGeneDx High/Moderate Risk Panel includes five recently described, but well studied, cancer predisposition genes (ATM, BRIP1, CHEK2, RAD51C and RAD51D) in addition to genes associated with classic hereditary cancer syndromes, and may allow for detection of a causative pathogenic variant after initial testing is uninformative. Methods: Genomic DNA from the submitted specimen was enriched for the complete coding region and splice site junctions of the genes on the panel using a proprietary targeted capture system developed by GeneDx. For PTEN, nucleotides c. 700 through c in the promoter region are also sequenced. The products were sequenced on either an Illumina MiSeq or HiSeq instrument with 2x150 or 2x100 paired end reads, respectively. The sequence was aligned to reference sequences based on human genome build GRCh37/UCSC hg19. Capillary sequencing was used to confirm all variants with clinical or uncertain significance and to analyze regions with inadequate coverage by Next Generation sequencing. If present, apparently homozygous variants were confirmed using alternate primer pairs to significantly reduce the possibility of allele drop out. Concurrent deletion/duplication testing was performed for all of the genes on the panel using either exon level array CGH or MLPA. Confirmation of copy number changes was performed by MLPA, qpcr, or repeat acgh analysis. Data analysis was performed using gene specific filtering. The array was designed to detect most single exon deletions and duplications. For EPCAM, deletion/duplication analysis, but not sequencing, was performed. All sequence alterations are described according to the Human Genome Variation Society (HGVS) nomenclature guidelines. Benign and likely benign variants, if present, are not reported but are available upon request. The genes evaluated by this test are listed on the first page of the report. Test Performance: DNA sequencing will detect nucleotide substitutions and small insertions and deletions, while array CGH will detect exon level deletions and duplications. These methods are expected to be greater than 99% sensitive in detecting pathogenic variants identifiable by sequencing or array CGH. The likelihood of a false positive result is expected to be <1%. Technical Limitations: Neither sequencing nor exon level acgh can reliably detect mosaicism, and cannot detect chromosomal aberrations. Deletions involving more than 20bp and insertions involving more than 10bp are not reliably detected by the sequencing methodology, and deletions or duplications of less than 250bp are not reliably detected by array CGH. Regions of certain genes have inherent sequence properties that yield suboptimal data, potentially impairing accuracy of the results. For instance, sequence and deletion/duplication analysis of PMS2 and Information Sheet on High/Moderate Risk Panel Page 2 of 5 GeneDx Revision Date: 07/2016

3 CHEK2, among others, is complicated by the presence of pseudogenes or homologous sequences that involve multiple exons of these genes. In the absence of mrna/cdna studies, we cannot completely exclude the possibility of undetectable clinically significant variants in certain regions of these genes. Specifically, large deletions or duplications in PMS2 exons 2 5, 9, and will not be detectable due to the presence of pseudogenes. False negatives may also occur in the setting of bone marrow transplantation, recent blood transfusion, or suboptimal DNA quality. In individuals with active leukemia or lymphoma or with known chronic myeloid or lymphoid neoplasms (such as low grade MDS, CML, ET, P. vera, PMF, CLL), there is a possibility that testing of specimens containing leukocytes may detect an acquired somatic variant, resulting in a false positive result. In this situation, please contact one of our genetic counselors to discuss the utility of submitting an alternate specimen. Additionally, rare false negatives may occur when testing for a specific variant identified at a laboratory other than GeneDx if a positive control is not provided. Based on the specific array design and technology used, the reported coordinates of duplications and deletions at the exon or gene level can slightly differ among family members tested but, in general, relatives are expected to have the same copy number variant. The ability to detect genetic variants and naming conventions can differ among laboratories. Reporting of Results: Results will be interpreted and reported following recommendations of the American College of Medical Genetics as a guideline ( Variations detected by sequencing or deletion/duplication analysis will be analyzed and classified into the following categories based on current scientific knowledge. Our analysis includes a comprehensive assessment of the variation on a molecular and clinical level in order to determine its clinical significance and classification. Trained PhD analysts perform a detailed review of the variation on the molecular level, including exhaustive searches of gene and locus specific databases and the Human Gene Mutation Database (HGMD), and genetic counselors and clinical molecular geneticists carefully review literature reports. Pathogenic Variant Examples of variations that may be reported as pathogenic include frameshift variants and nonsense variants that are predicted to result in premature protein truncation or mrna decay, canonical splice site variants, and previously reported missense variants that are recognized as disease causing. Likely Pathogenic Variant Variations for which there is significant, but not conclusive, evidence supporting pathogenicity will be classified as Likely Pathogenic. Variant of Uncertain Significance Variations for which there is not sufficient evidence for classification will be classified as Variant of Uncertain Significance. Negative No variation of clinical or uncertain significance was detected. Any variation detected and classified as a likely benign or benign variant based on population data, review of the literature, Human Gene Mutation Database (HGMD), and appropriate locus specific databases will not be reported. Specimen Requirements and Shipping/Handling: Blood: Two EDTA (lavender top) tubes containing 4 ml each whole sterile blood Oral Rinse: Saliva collect in 30mL of mouthwash using our GeneDx collection kit Extracted DNA: >20 ug Buccal Swab: For family member testing only (excluding deletion/duplication family testing) Information Sheet on High/Moderate Risk Panel Page 3 of 5 GeneDx Revision Date: 07/2016

4 Test Codes and Turnaround Times Please contact us for price information: Test Code Description Turnaround Time B751 High/Moderate Risk Panel 3 weeks References Canto MI et al. International Cancer of the Pancreas Screening (CAPS) Consortium summit on the management of patients with increased risk for familial pancreatic cancer. Gut Mar;62(3): (PMID ) Frantzen C et al. Von Hippel Lindau Syndrome May 17 [Updated 2015 Aug 6]. In: Pagon RA, Adam MP, Ardinger HH, et al., editors. GeneReviews [Internet]. Seattle (WA): University of Washington, Seattle; Available from: National Cancer Institute at the National Institutes of Health. What you need to know about: cancer; risk factors. (URL: cancer) [February 2016 accessed]. NCCN Guidelines. Gastric Cancer. (URL: [February 2016 accessed]. NCCN Guidelines. Genetic/Familial High Risk Assessment: Breast and Ovarian. (URL: [February 2016 accessed]. NCCN Guidelines. Genetic/Familial High Risk Assessment: Colorectal. (URL: [February 2016 accessed]. Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute. SEER Cancer Statistics Review, : Lifetime Risk Tables (URL: [February 2016 accessed]. Gene Most commonly associated cancers Associated recessive syndrome Management Guidelines APC NM_ BMPR1A NM_ BRCA1 NM_ BRCA2 NM_ CDH1 NM_ CDKN2A NM_ NM_ EPCAM NM_ MLH1 NM_ MSH2 NM_ MSH6 NM_ MUTYH NM_ PALB2 NM_ PMS2 NM_ PTEN NM_ SMAD4 NM_ STK11 NM_ TP53 NM_ VHL NM_ ATM NM_ Colon, Small Bowel Colon, Gastric (if gastric polyps) High Risk Genes Breast, Ovarian, Pancreatic, Prostate, Endometrial serous carcinoma^ Breast, Ovarian, Pancreatic, Prostate, Endometrial serous carcinoma^ Gastric, Breast, Colon (signet ring)^ Fanconi anemia, CAPS #, NCCN Gastric Melanoma, Pancreatic CAPS # Colon, Endometrial, Ovarian^, Gastric^ Colon, Endometrial, Ovarian, Gastric, Pancreatic Colon, Endometrial, Ovarian, Gastric, Colon, Endometrial,Ovarian, Gastric^ Colon, Small Bowel, Endometrial serous carcinoma ^ MUTYH associated polyposis Breast, Pancreatic (possibly high risk)^ Fanconi anemia, CAPS # Colon, Endometrial, Ovarian^, Gastric^ Breast, Thyroid, Endometrial Colon, Gastric (if gastric polyps) Breast, Colon, Pancreatic, Gastric, Small Bowel, Endometrial Breast, Sarcoma, Brain, Hematologic malignancies, Adrenocortical, among others* Renal, Pancreatic neuroendocrine tumors, Hemangioblastoma, Pheochromocytoma Moderate Risk Genes,, CAPS # VHL GeneReviews (Frantzen 2000) Breast, Colon^, Ataxia telangiectasia Information Sheet on High/Moderate Risk Panel Page 4 of 5 GeneDx Revision Date: 07/2016

5 BRIP1 NM_ CHEK2 NM_ RAD51C NM_ RAD51D NM_ Breast^, Ovarian^ Fanconi anemia Breast, Prostate (possibly high risk)^, Colon^, Breast^, Ovarian^ Fanconi anemia Breast^, Ovarian^ Key Red font denotes significantly increased cancer risk. We consider significantly increased risk to be a relative risk of 4 or higher in relation to the general population risk. This translates to the following lifetime cancer risks: 50% breast cancer, 20% colon cancer, 11% endometrial cancer, 8% melanoma, 7% renal cancer, 6% pancreatic cancer, 6% ovarian cancer, 5% thyroid cancer, 4% gastric or small bowel cancer. Blue font denotes moderately increased cancer risk. We consider moderately increased risk to be a relative risk between 2 and 4 in relation to the general population risk. This translates to the following lifetime cancer risks: 24 49% breast cancer, 32 60% prostate cancer, 5 11% endometrial cancer, 3 6% pancreatic cancer. ^ Gene specific risk for this cancer type is not well defined. * High overall risk of cancer: 75% lifetime risk for males to develop cancer, nearly 100% risk for females. # CAPS International Cancer of the Pancreas Screening (CAPS) Consortium summit on the management of patients with increased risk for familial pancreatic cancer (Canto 2013). Information Sheet on High/Moderate Risk Panel Page 5 of 5 GeneDx Revision Date: 07/2016