Test Information Sheet

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1 Test Information Sheet GeneDx 207 Perry Parkway Gaithersburg, MD Phone: Fax: OncoGene Dx: Breast/Ovarian Cancer Panel Sequence Analysis and/or Exon Level Deletion/Duplication Testing of 20 Genes OncoGeneDx Breast/Ovarian Panel Gene List: ATM BRIP1 FANCC NBN RAD51C BARD1 CDH1 MLH1 PALB2 RAD51D BRCA1 CHEK2 MSH2 PMS2 TP53 BRCA2 EPCAM^ MSH6 PTEN XRCC2 ^Next generation sequencing of this gene is not included in this panel. Clinical Features and Genetics: In the general population, approximately 1 in 8 women (12%) will develop breast cancer in their lifetime, and 1 in 75 women (1.4%) will be diagnosed with ovarian cancer in their lifetime (SEER). Most cases of breast or ovarian cancers develop sporadically with no family history of the cancer. Individual risk factors and exposures, such as age, pregnancy history, menstrual history, benign breast disease, radiation exposure, and alcohol intake, are known to modify a woman s chance of developing these types of cancers. However, 5 10% of breast cancer cases and 15 20% of ovarian cancer cases are thought to be due to a hereditary predisposition. The features suggestive of a hereditary cancer predisposition include: young age at diagnosis, multiple primary cancers in a single individual, diagnosis of a cancer type that is not common in general population (such as ovarian cancer, male breast cancer, or pancreatic cancer), and several relatives affected with related cancers spanning multiple generations. Approximately 20 25% of familial breast cancer risk is thought to be attributed to pathogenic variants in the BRCA1 or BRCA2 genes (Easton 1999, Pharoah 2002, van der Groep 2011). The additional 18 genes on this panel may also account for a substantial proportion of hereditary breast and ovarian cancer cases. Many of these genes are involved in the pathway and/or play a role in DNA damage repair similar to the BRCA1 and BRCA2 genes. Newer genes that have been identified in families with breast and/or ovarian cancer have been included in the panel to make it as comprehensive as possible. These genes include BARD1, FANCC, NBN and XRCC2. The evidence available to date may be derived from a small number of patients with wide confidence intervals or is based upon an ethnic cohort with one specific variant. Accurate risk assessment may be complicated by the low penetrance of pathogenic variants in these genes and/or ascertainment bias. Since the cancer risks are not yet well defined, no consensus guidelines for medical management are available for these genes. All of the genes included on the OncoGeneDx Breast/Ovarian Cancer Panel are associated with dominantly inherited cancer risk. 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. Some of these genes are also associated with extremely rare autosomal recessive syndromes if an individual inherits two pathogenic variants in the same gene, one from each parent (biallelic pathogenic variants). 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, characterized by an increased risk for malignancy in children including leukemia and certain solid tumors as well as physical abnormalities and bone marrow failure. All genes that have an associated recessive condition are noted in the attached table. Information Sheet on Breast/Ovarian Cancer Panel Page 1 of 5 GeneDx Revision Date: 07/2016

2 With respect to medical management, many of the genes on the OncoGeneDx Breast/Ovarian Cancer Panel are discussed in the current versions of the NCCN guidelines NCCN Guidelines for Genetic/Familial High Risk Assessment or NCCN Guidelines for Colorectal Cancer Screening. The available medical management guidelines pertinent to each gene are listed in the attached table. Reason for referral: In addition to the features of hereditary cancer predisposition described above, a next generation sequencing panel for hereditary breast/ovarian cancer may be especially helpful in cases in which: 1) The family history is suggestive of a predisposition to hereditary breast and ovarian cancer. Although the BRCA1 and BRCA2 genes are thought to account for a significant proportion of such cases, there are several other genes that cause an increased risk of both types of cancer. The OncoGeneDx Breast and Ovarian Cancer panel includes analysis of the BRCA1 and BRCA2 genes as well as 18 other genes affecting breast and/or ovarian cancer risk. Thus, the OncoGeneDx Breast and Ovarian Cancer 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, if negative). 2) 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). 3) Prior genetic testing has been performed due to a family history suggestive of a hereditary cancer predisposition, and all results have been negative. OncoGeneDx includes newly identified cancer in addition to genes identified many years ago associated with classic hereditary cancer syndromes. 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. 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 PTEN, nucleotides c. 700 through c in the promoter region are also sequenced. 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 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, Information Sheet on Breast/Ovarian Cancer Panel Page 2 of 5 GeneDx Revision Date: 07/2016

3 potentially impairing accuracy of the results. For instance, sequence and deletion/duplication analysis of PMS2 and 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, previously reported missense variants that are recognized as disease causing and gross rearrangements. 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 collected in at least 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 Breast/Ovarian Cancer 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 B273 OncoGeneDx Breast/Ovarian Cancer 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 ) Easton DF. How many more breast cancer predisposition genes are there? Breast Can Res Aug;1(1): (PMID ) 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]. Pharoah PD et al. Polygenic susceptibility to breast cancer and implications for prevention. Nat Genet May;31(1):33 6. (PMID ) Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute. SEER Cancer Statistics Review, : Lifetime Risk Tables (URL: [February 2016 accessed]. van der Groep P, van der Wall E, and van Diest PJ. Pathology of hereditary breast cancer. Cell Oncol (Dordrecht) Apr;34(2): (PMID: ) Walsh T et al. Mutations in 12 genes for inherited ovarian, fallopian tube, and peritoneal carcinoma identified by massively parallel sequencing. Proc Natl Acad Sci Nov;108(44): (PMID: ) Information Sheet on Breast/Ovarian Cancer Panel Page 4 of 5 GeneDx Revision Date: 07/2016

5 Gene Most commonly associated cancers Associated recessive syndrome Management Guidelines BRCA1 NM_ BRCA2 NM_ CDH1 NM_ EPCAM NM_ MLH1 NM_ MSH2 NM_ MSH6 NM_ PALB2 NM_ PMS2 NM_ PTEN NM_ TP53 NM_ ATM NM_ BRIP1 NM_ CHEK2 NM_ RAD51C NM_ RAD51D NM_ BARD1 NM_ FANCC NM_ NBN NM_ XRCC2 NM_ Breast, Ovarian, Pancreatic, Prostate, Endometrial serous carcinoma^ Breast, Ovarian, Pancreatic, Prostate, Endometrial serous carcinoma^ Gastric, Breast, Colon (signet ring)^ Colon, Endometrial, Ovarian^, Gastric^ Colon, Endometrial, Ovarian, Gastric, Pancreatic Colon, Endometrial, Ovarian, Gastric, Colon, Endometrial, Ovarian, Gastric^ High Risk Genes, CAPS #, NCCN Gastric Breast, Pancreatic (possibly high risk)^, CAPS # Colon, Endometrial, Ovarian^, Gastric^ Breast, Thyroid, Endometrial Breast, Sarcoma, Brain, Hematologic malignancies, Adrenocortical, among others* Moderate Risk Genes Breast, Colon^, Ataxia telangiectasia Breast, Prostate (possibly high risk)^, Colon^ Breast^ Breast^, Melanoma^ Breast^, New Genes Nijmegen breakage syndrome, 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, 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 Breast/Ovarian Cancer Panel Page 5 of 5 GeneDx Revision Date: 07/2016