OvaNext

OvaNextTM is a next generation (next-gen) sequencing panel that simultaneously analyzes 23 genes that contribute to increased risk for breast, ovarian and/or uterine cancers.

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OvaNextTM is a next generation (next-gen) sequencing panel that simultaneously analyzes 23 genes that contribute to increased risk for breast, ovarian and/or uterine cancers.

Please note, all samples received starting October 18, 2013 for BreastNext and OvaNext will automatically include NF1 & RAD51D gene sequencing and deletion/duplication analyses at no additional cost. Additionally, Ambry will contact clinicians to discuss any clinically-significant NF1 & RAD51D incidental findings on all in-house samples.

Ambry utilizes next generation sequencing to offer a comprehensive genetic testing panel for hereditary gynecologic cancers (cancer of the breast, ovary and/or uterus), including BRCA1 and BRCA2. Genes on this panel include ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2,  EPCAM, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, STK11, and TP53. Full gene sequencing and analysis of all coding domains plus at least 5 bases into the 5’ and 3’ ends of all the introns and untranslated regions (5’UTR and 3’UTR) is performed for 22 of the 23 genes (excluding EPCAM). Gross deletion/duplication analysis is performed for all 23 genes. Specific-site analysis is available for individual gene mutations known to be in the family.

Disease Name 
Cancer, Ovarian, Breast Uterine
Hereditary Breast, Ovarian, and/or Uterine Cancer
Disease Information 

Ovarian cancer is the fifth most common cancer among women, affecting approximately 1 in 71 (1.4%) women in her lifetime.1 The NCI estimates that approximately 22,280 new cases of ovarian cancer will be diagnosed and 15,500 ovarian cancer deaths will occur in the U.S. in 2012.2  It is leading cause of death from gynecologic malignancy, characterized by advanced presentation with regional dissemination in the peritoneal cavity. Epithelial ovarian cancer is the most common form and arises as a result of genetic alterations sustained by the ovarian surface epithelium. 

Breast cancer is a disease in which cells in the breast become abnormal and multiply to form a malignant tumor. Breast cancer is the most common cancer in women in developed countries, affecting about 1 in 8 (~12.29%) women in her lifetime.1 The NCI estimates that there are approximately 227,000 new cases of female breast cancer and 2,200 new cases of male breast will diagnosed in the U.S. in 2012.2

Uterine cancer affects about 1 in 38 (2.61%)  women in her lifetime.1 The NCI estimates that approximately 47,130 new cases of uterine cancer will be diagnosed and 8,010 uterine cancer deaths will occur in the U.S. in 2012.

Hereditary breast cancers tend to occur earlier in life than non-inherited sporadic cases and are more likely to involve both breasts. Hereditary breast and ovarian cancers caused by mutations in the highly penetrant genes, BRCA1 and BRCA2, appear to be responsible for about 10-18% of total breast cancers and ovarian cancers.3,8,11 Additional genes have been discovered that contribute to the incidence of breast and ovarian cancers as well.3-6,8,11  Mutations in other genes, such as those associated with Lynch syndrome, can significantly increase the risk for uterine and ovarian cancer, while others, such as PTEN, can increase the risk for breast and uterine cancer. While BRCA1 and BRCA2 account for the majority of hereditary ovarian cancer, a significant proportion hereditary breast, ovarian and uterine cancer can be attributed to mutations in multiple other genes.

Since the best approach to genetic testing for individuals with these cancer types is not always clear, OvaNext test may be ideal.


Figure Legend: Estimated cancer risks for women in the general population (represented in gray) compared with women carrying a gene mutation on the OvaNext panel (represented in blue). (Based on available evidence. Estimates may change as more data emerges.)

OvaNext Panel Genes:

ATM is a gene classically associated with an autosomal recessive condition called ataxia-telangiectasia (AT). AT is an autosomal recessive disorder characterized by progressive cerebellar ataxia with onset between ages one and four, telangiectases of the conjunctavie, oculomotor apraxia, immune defects, and a predisposition to malignancy, particularly leukemia and lymphoma. Heterozygous carrier females also have an estimated 2-5 fold increased risk for breast cancer.Recent studies have also reported ATM germline mutations in individuals with familial pancreatic cancer. In one of these studies, ATM mutations were identified in 4.6% (4/87) of families with more than 3 affected members.35 The exact lifetime pancreatic cancer risk for ATM mutation carriers has not yet been established.

BRCA1 and BRCA2 are tumor suppressor genes. Mutations in these two highly penetrant genes increase the chance for cancer of the breast, ovaries and fallopian tubes, pancreas and prostate. Studies suggest female BRCA1 mutation carriers have between a 57-87% risk to develop breast cancer and between a 39-40% risk to develop ovarian cancer by age 70. Similarly male BRCA1 mutation carriers have a cumulative breast cancer risk of 1.2% by age 70.

Similar studies suggest female BRCA2 mutation carriers have between a 45-84% risk to develop breast cancer and between an 11-18% risk to develop ovarian cancer (including primary peritoneal and fallopian tube) by age 70. Male BRCA2 mutation carriers have up a 15% prostate cancer risk and a cumulative breast cancer risk of 6.8% by ages 65 and 70 respectively.

Furthermore, BRCA1/2 mutation carriers are at an increased risk for melanoma and cancer of the pancreas, gall bladder, bile duct and the stomach. Cancer risks are further modified by family history, reproductive choices, lifestyle and environmental factors and other genetic factors.

BARD1, BRIP1, MRE11A, NBN, RAD50, and RAD51C are genes involved in the Fanconi anemia (FA)–BRCA pathway, which is critical for DNA repair by homologous recombination and interact in vivo with BRCA1 and/or BRCA2.3,8-10 Mutations in these genes are estimated to confer up to a 4 fold increase in breast cancer risk, and mutations in each have been reported in at least 1 identified case of ovarian cancer to date.11

CHEK2 is a gene that receives signals from damaged DNA, transmitted to CHEK2 via ATM.  Known substrates of CHEK2 include BRCA1, BRCA2 and TP53, which have all been implicated in cellular processes responsible for the maintenance of genomic stability. Multiple studies indicate that mutations in the CHEK2 gene confer an increased risk of developing many types of cancer including breast, prostate, colon, thyroid, and kidney. Mutations are more likely to be found among women with bilateral versus those with unilateral breast cancers. A female carrier of a CHEK2 mutation has approximately a 2 fold increase in lifetime breast cancer risk and has a 1% risk per year of developing a second breast primary cancer. Ovarian cancer risk has also been suggested.11-15

CDH1 germline mutations have been associated with hereditary diffuse gastric cancer (HDGC) and lobular breast cancer in women. In one published study, the estimated cumulative risk of gastric cancer for CDH1 mutation carriers by age 80 years is 67% for men and 83% for women.16 HDGC patients typically present with diffuse-type gastric cancer with signet ring cells diffusely infiltrating the wall of the stomach and, at late stage, linitis plastica. An elevated risk of lobular breast cancer is also associated with HDGC,17 with an estimated lifetime breast cancer risk of 39-52%.

EPCAM, MLH1, MSH2, MSH6, and PMS2 are mismatch repair genes that have been associated with Lynch syndrome (HNPCC).  Lynch syndrome is estimated to cause 2-5% of all colon cancer. Lynch syndrome is associated with a significantly increased risk for colon cancer (60-80% lifetime risk), uterine/endometrial cancer (20-60% lifetime risk in women), stomach cancer (11-19% lifetime risk), and ovarian cancer (4-13% lifetime risk in women). Risk for cancer of the small intestine, hepatobiliary tract, upper urinary tract and brain are also elevated.18-20

MUTYH germline mutations are classically associated with an autosomal recessive form of hereditary polyposis. Clinical studies have shown that MUTYH mutations were detected in 33% and 57% of patients with clinical familial adenomatous polyposis (FAP)and attentuated familial adenomatous polyposis (AFAP), respectively, who are negative for mutations in the APC gene.21 Two common mutations, p.Y179C and p.G396D (originally designated as p.Y165C and p.G382D), have been reported as homozygous or compound heterozygous in about 70%-86% of MAP patients.22,23 Heterozygous mutations have also been associated with a 1.9 fold increased risk for breast cancer.24 In this series, characteristics of tumors and at of diagnosis in carriers with MUTYH variants were similar to those without MUTYH variants.21-24

NF1 mutations cause neurofibromatosis type 1 (NF1), an autosomal dominant disorder affecting multiple body systems. The most common neoplasms observed in individuals with NF1 include peripheral nerve sheath tumors, gastrointestinal stromal tumors (GIST), central nervous system gliomas, leukemias, paragangliomas (PGLs) and pheochromocytomas (PCCs), and breast cancer. Multiple population-based studies of have demonstrated a 3-5 fold increase in breast cancer risk for women with NF1, with the highest risks for those under 50 years of age. In addition, individuals with NF1 have an estimated lifetime risk for PGLs and PCCs of up to 7%.

PALB2 germline mutations have been associated with increased risk for pancreatic cancer, breast cancer, and fanconi-anemia complementation group N (FA-N). Familial pancreatic and/or breast cancer due to PALB2 mutations is inherited in an autosomal dominant pattern, while FA-N is an autosomal recessive condition. Females with a PALB2 mutation have a 2 to 4 fold increase in risk for breast cancer,25-27 and a recent report has suggested an increased risk for ovarian cancer as well.11 Recent studies have also identified PALB2 mutations in 1-3% of families with pancreatic cancer,36 however, the exact lifetime pancreatic cancer risk has not yet been established.

PTEN is a gene that has been associated with Cowden syndrome (CS), PTEN Hammartoma Tumor syndrome (PHTS), Bannayan-Riley-Ruvalcaba syndrome, Proteus syndrome and autism spectrum disorder. Cowden Syndrome is a multiple hamartoma syndrome with a high risk of developing tumors of the thyroid, breast, and endomentrium. Mucocutaneous lesions, thyroid abnormalities, fibrocystic disease, multiple uterine leiomyoma, and macrocephaly can also be seen. Affected individuals have a lifetime risk of up to 50% for breast cancer, 10% for thyroid cancer, and 5-10% for endometrial cancer. Over 90% of individuals with CS will express some clinical manifestation by their 20’s.28,29

RAD51D is a gene involved in the Fanconi anemia (FA)–BRCA pathway, which is critical for DNA repair by homologous recombination and interact in vivo with BRCA1 and/or BRCA2.  RAD51D germline mutations confer an increased susceptibility for breast and ovarian cancer. Relative cancer risk is estimated at 1.32 and 6.3 respectively. This translates to nearly a 10% lifetime risk for ovarian cancer. 

STK11germline mutations have been associated with Peutz-Jegher syndrome (PJS), an autosomal dominant disorder characterized by the development of gastrointestinal hamartomatous polyps and melanin hyperpigmentation of the skin and mucous membranes. Overall, individuals affected with PJS have a 57-81% risk of developing cancer by age of 70, with gastrointestinal/colon and breast cancers being the most common.30,31 Meta-analysis assessed the cancer risks of PJS and observed that STK11 mutation carriers ages 15-64 had a 36% risk of developing pancreatic cancer,38 while another study noted a risk of 11% for pancreatic cancer by 70 years.37

TP53 is a tumor suppressor gene that causes Li-Fraumeni syndrome (LFS)and Li-Fraumeni like (LFL) syndrome that can affect adults and children alike. An individual harboring a TP53 mutation has a 50% risk of developing cancer by age 30 and a lifetime cancer risk of up to 90%. The most common tumor types observed in LFS/LFL families include soft tissue and osteosarcomas, breast cancer, brain tumors (including astrocytomas, glioblastomas, medulloblastomas and choroid plexus carcinomas) and adrenocortical carcinoma (ACC); however, other cancers, including RCC, have been reported. Studies have shown that a small percentage of women who are negative for BRCA1 and BRCA2 negative are identified to have mutations in TP53.15,32,33 Multiple other cancer types have been observed in TP53 mutation carriers, including pancreatic cancer. 38-41

Testing Benefits & Indication 

Indications for Testing

Families with a combination of the cancers below and some common red flags for hereditary cancer in the family would be appropriate to consider for OvaNext testing.

  • Breast and ovarian cancer
  • 2 or more cases of ovarian cancer
  • Breast and uterine cancer
  • Uterine, ovarian, and/or colon cancer
  • Breast, ovarian or uterine cancer diagnosed at a young age (often <50 years)

Common Red Flags for Hereditary Cancer

  • Cancer diagnosed at a younger age than expected for the general population (</= 50 for most cancers)
  • Cancer diagnosed across generations and in multiple generations within a family
  • Individual with multiple  primary cancers (either in paired organs or in different organs)
  • A pattern of cancer in the family that is typical of a known cancer predisposition syndrome (for example colon and uterine cancer in Lynch syndrome or breast and pancreatic cancer with PALB2-related cancer)
     
Benefits of Testing
Knowing your patient has a genetic susceptibility for certain cancers can help with medical management. For example, this information can:
Modify breast cancer surveillance options and age of initial screening
 
  • Suggest specific risk-reduction measures (e.g. Considering prophylactic oopherectomy, or removal of the ovaries, after childbearing is complete for women with increased risk for breast/ ovarian cancer)
  • Clarify and stratify familial cancer risks, based on gene-specific cancer associations, such as risk for uterine, colon and ovarian cancer with mutations MLH1.
  • Offer treatment guidance (e.g. Avoidance of radiation-based treatment methods for individuals with a TP53 mutation)
  • Identify other at-risk family members
  • Provide guidance with new gene-specific treatment options and risk reduction measures as they emerge
Test Description 

The OvaNext Panel targets detection of mutations in 22 of the 23 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, STK11, and TP53) by next-generation sequencing or Sanger sequencing of all coding domains plus at least 5 bases into the 5’ and 3’ ends of all the introns and untranslated regions (5’UTR and 3’UTR). Genomic deoxyribonucleic acid (gDNA) is isolated from the patient’s specimen using a standardized kit and quantified by agarose gel electrophoresis. Sequence enrichment is carried out by incorporating the gDNA into microdroplets along with primer pairs designed to the target hereditary cancer gene coding exons followed by polymerase chain reaction (PCR) and next-generation sequencing. A secondary sequencing method is performed for any regions with insufficient read depth coverage for reliable heterozygous variant detection. Suspect variant calls other than those classified as "likely benign" or "benign" are verified by sanger sequencing in sense and antisense directions. Gene copy number analysis by a custom targeted microarray identifies gross deletions or duplications all 23 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, STK11, and TP53). If a deletion is detected in exons 13, 14, or 15 of PMS2, double stranded sequencing of the appropriate exon(s) of the pseudogene PMS2CL will be performed to determine if the deletion is located in the PMS2 gene or pseudogene. 

Mutation Detection Rate 

Analytical sensitivity for all genes is estimated to be greater than 99.97% of described mutations.

Specimen Requirements 

Blood: Collect 6-10cc blood in purple top EDTA tube (preferred) or yellow top citric acetate tube.

Storage: 2-8°C. Do not freeze.
Shipment: Room temperature for two-day delivery.
For transfusion patients: Wait at least two weeks after a packed cell or platelet transfusion and at least four weeks after a whole blood transfusion prior to blood draw

DNA: Collect 20μg of of DNA in TE (10mM Tris-Cl pH 8.0, 1mM EDTA); preferred at 200 ng/μl.
Quality: Please provide DNA OD 260:280 ratio (preferred 1.7-1.9) and send agarose picture with high molecular weight genomic DNA, if available.
Storage: -20°C.
Shipment: Shipment frozen on dry ice is preferred, or ship on ice.

Saliva: Collect 2 tubes with 2cc per tube in Oragene Self Collection container
Storage: At room temperature in sterile bag.
Shipment: Ship room temperature for two-day deliver

 

 

Billing Codes 
Test Code Technique
8830 OvaNext Gene Analysis

 

Turnaround Time 
Technique weeks
OvaNext Gene Analysis
(Ordered 2/1/14 or after)
8 - 12
OvaNext Gene Analysis
(Ordered before 2/1/14)
12 - 16

 

Specialty 
References 

1. http://seer.cancer.gov

2. http://www.cancer.gov

3. Pennington & Swisher. Hereditary ovarian cancer: beyond the usual suspects. Gyn Onc. 2012;124:347-353. [PMID: 22264603]

4. van der Groep & van del Wall. Pathology of hereditary breast cancer. Cell Oncol.  2011;34:71-88. [PMID: 21336636]

5. Walsh et al. Ten genes for inherited breast cancer. Cell Press.  2007:11;103-105. [PMID: 17292821]

6. Meindl et al. Hereditary breast and ovarian cancer: new genes, new treatments, new concepts. Dtsch Arztebl Int. 2011;108(19):323-330. [PMID: 21637635]

7. Renwick et al.,  ATM mutations that cause ataxia-telangiectasia are breast cancer susceptibility alleles. Nature Genetics. 2006;38(8):873-875. [PMID: 16832357]

8. Walsh et al., Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing. PNAS. 2010;107(28):12629-12633. [PMID: 16474176]

9. Heikkinen et al. RAD50 and NBS1 are breast cancer susceptibility genes associated with genomic instability. Carcinogenesis. 2006;27(8):1593-1599. [PMID: 16474176] 
 
10. Ghimenti et al., Germline mutations of the BRCA1-associated ring domain (BARD1) gene in breast and breast/ovarian families negative for BRCA1 and BRCA2 alterations. Genes, Chromosomes & Cancer. 2002;33:235-242. [PMID: 11807980]

11. Walsh et al., Mutations in 12 genes for inherited ovarian, fallopian tube, and peritoneal carcinoma identified by massively parallel sequencing. PNAS. 2011:108(44);10832-18037. [PMID: 22006311]

12. Bahassi, EM. et al., The checkpoint kinases Chk1 and Chk2 regulate the functional associations between hBRCA2 and Rad51 in response to DNA damage. Oncogene. 2008; 27, 3977–3985. [PMID: 18317453]

13. Orloff and Eng et al., Genetic and phenotypic heterogeneity in the PTEN hamartoma tumour syndrome. Oncogene. 2008; 27, 5387-5397. [PMID: 18794875]

14. Cybulski et al., CHEK2 is a multiorgan cancer susceptibility gene. Am J Hum Genet. 2004;75:1131–1135. [PMID: 15492928] 

15. Walsh et al., Spectrum of mutations in BRCA1, BRCA2, CHEK2, and TP53 in families at high risk of breast cancer. JAMA. 2006;295(12):1379-1388. [PMID: 16551709]

16. Narod SA., Testing for CHEK2 in the cancer genetics clinic: ready for prime time? Clin Genet. 2010;78:1-7. [PMID: 20597917]

17. Pharoah PD et al., Incidence of gastric cancer and breast cancer in CDH1 (E-cadherin) mutation carriers from hereditary diffuse gastric cancer families. Gastro. 2001;121:1348-1353. [PMID: 20597917]

18. Guilford P et al., Hereditary diffuse gastric cancer: translation of CDH1 germline mutations into clinical practice. Gastric Cancer. 2010;13:1-10 [PMID: 20373070]

19. Abdel-Rahman WM et al., The genetics of HNPCC: application to diagnosis and screening. Crit Rev Oncol Hematol. 2006;53:208-220. [PMID: 16434208]

20. Hedge MR & Roa BB. Genetic testing for hereditary nonpolyposis colorectal cancer (HNPCC). Curr Proto in Hum Genet. 2009;10.21(61):1-28. [PMID: 19360696]

21. Filipe B et al., APC or MUTYH mutations account for the majority of clinically well-characterized families with FAP and AFAP phenotype and patients with more than 30 adenomas. Clin Genet. 2009;76:242-255. [PMID: 19793053]

22. Sampson JR & Jones N., MUTYH-associated polyposis. Best Pract Res Clin Gastroenterol. 2009;23(2):209-18. [PMID: 19414147]

23. Barnetson RA et al., Germline mutation prevalence in the base excision repair gene, MYH, in patients with endometrial cancer. Clin Genet. 2007; 72:551-555. [PMID: 17956577]

24. Rennert et al. MutYH mutation carriers have increased breast cancer risk. Cancer. 2012 Apr 15;118(8):1989-93. [PMID: 21952991]

25. Jones, S. et al., Exomic sequencing identifies PALB2 as a pancreatic cancer susceptibility gene. Science. 2009;324(5924): p. 217. [PMID: 19264984]

26. Slater, E.P. et al., PALB2 mutations in European familial pancreatic cancer families. Clin Genet. 2010;78(5): p. 490-4. [PMID: 20412113]

27. Casadei S et al. Cancer Res, 2011;71(6):2222-2229. [PMID: 21285249]

28. Eng C. J Med Genet. Will the real Cowden syndrome please stand up: revised diagnostic criteria. 2000;37:828-830. [PMID: 11073535]

29. Starink TM et al., The Cowden syndrome: a clinical and genetic study in 21 patients. Clin Genet. 1986;29:222–233. [PMID: 3698331]

30. Lim W et al., Relative frequency and morphology of cancers in STK11 mutation carriers. Gastroenterology. 2004;126:1788-1794. [PMID: 15188174]

31. Hearle et al., Frequency and spectrum of cancers in the Peutz-Jeghers syndrome. Clin Cancer Res. 2006;12:3209-3215. [PMID: 16707622]

32. Birch JM et al., Prevalence and diversity of constitutional mutations in the p53 gene among 21 Li-Fraumeni families. Cancer Research. 1994;54: 1298-1304. [PMID: 8118819]

33. Olivier M et al., Li-Fraumeni and related syndromes: correlation between tumor type, family structure, and TP53 genotype. Cancer Research. 2003;63: 6643-6650. [PMID: 14583457]

34. Adapted from: http://www.nccn.org/professionals/physician_gls/f_guidelines.asp. Accessed Feb 28, 2012.  

35. Roberts, N., et al. (2011). ATM mutations in patients with hereditary pancreatic cancer. Cancer Discov, 2:41-46. [PMID: 22585167]

36. Tischkowitz, et al. (2009). Analysis of the gene coding for the BRCA2-interacting protein PALB2 in familial and sporadic pancreatic cancer. Gastroenterology, 137:1183–1186.

37. Hearle, et al. (2006). Frequency and spectrum of cancers in the Peutz-Jeghers syndrome. Clin Cancer Res, 12:3209-3215.

38. Giardiello, F., et al. (2000). Very high risk of cancer in familial Peutz-Jeghers syndrome. Gastroenterology, 119:1447-1453.

 

39 Lim, W., et al. (2004). Relative frequency and morphology of cancers in STK11 mutation carriers. Gastroenterology, 126:1788-1794. 

40. Hearle, et al. (2006). Frequency and spectrum of cancers in the Peutz-Jeghers syndrome. Clin Cancer Res, 12:3209-3215.

41. Giardiello, F., et al. (2000). Very high risk of cancer in familial Peutz-Jeghers syndrome. Gastroenterology, 119:1447-1453.