BreastNext

BreastNext is a next generation sequencing (NGS) panel that simultaneously analyzes 17 genes associated with increased risk for breast cancer, including BRCA1 and BRCA2.

PrintPrint

BreastNext is a next generation sequencing (NGS) panel that simultaneously analyzes 17 genes associated with increased risk for breast cancer, including BRCA1 and BRCA2.

Ambry utilizes next generation sequencing to offer a comprehensive hereditary breast cancer panel.  Genes on this panel include ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MRE11A, MUTYH, NBN, NF1, PALB2, PTEN, RAD50, RAD51C, RAD51D, and TP53. Full gene sequencing and gross deletion/duplication analysis is performed for all 17 genes. Specific-site analysis is available for individual gene mutations identified in a family.

Disease Name 
Breast Cancer
Hereditary Cancer
Hereditary Breast Ovarian Cancer (HBOC)
Disease Information 

Breast cancer is the most common cancer in women in developed countries, affecting about 1 in 8 (12.5%) women in their lifetime.[1]  The National Cancer Institute (NCI) estimates that approximately 232,670 new cases of female breast cancer and 2,360 new cases of male breast cancer will be diagnosed in the U.S. in 2014.[2]  The majority of breast cancers are sporadic, but 5-10% are due to inherited causes.   Hereditary breast cancers tend to occur earlier in life than non-inherited sporadic cases and are more likely to occur in both breasts. The highly penetrant genes, BRCA1 and BRCA2, appear to be responsible for around half of hereditary breast cancer.[3-5]  However, additional genes have been discovered that are associated with increased breast cancer risk as well.[3-7]   Mutations in the genes on the BreastNext panel can confer an estimated 20–87% lifetime risk for breast cancer.  Some of these genes have also been associated with increased risks for other cancers, such as pancreatic cancer with PALB2, ovarian cancer with BRCA1, BRCA2, RAD51C, and others, and sarcoma with TP53.[8-12]

BreastNext Panel Genes:
ATM is a gene classically associated with an autosomal recessive condition called ataxia telangiectasia (AT). AT is characterized by progressive cerebellar ataxia with onset between ages 1 and 4, telangiectases of the conjunctivae, oculomotor apraxia, immune defects, and a predisposition to malignancy, particularly leukemia and lymphoma. Women who carry ATM mutations also have an estimated 2-4 fold increased risk for breast cancer.[13]  Cancer risk estimates for male ATM mutation carriers are not currently available. Recent studies have also reported ATM germline mutations in individuals with familial pancreatic cancer. In one of these studies, ATM mutations were identified in 4/87 (4.6%) families with more than three affected members.[14]
 
BRCA1 and BRCA2 are tumor suppressor genes inherited in an autosomal dominant pattern. Mutations in these two highly penetrant genes increase the chance for cancer of the breast, ovaries (including primary peritoneal and fallopian tube), pancreas and prostate. Studies suggest female BRCA1 mutation carriers have a 57-87% lifetime risk to develop breast cancer and a 39-40% lifetime risk to develop ovarian cancer by age 70.[8-10, 15-17]  Male BRCA1 mutation carriers have a cumulative breast cancer lifetime risk of about 1.2% by age 70.[18, 19] Similar studies suggest female BRCA2 mutation carriers have a 45-84% lifetime risk to develop breast cancer and an 11-18% risk to develop ovarian cancer by age 70.[8-10, 20, 21]  Male BRCA2 mutation carriers have up a 15% lifetime prostate cancer risk and a cumulative lifetime breast cancer risk of 6.8% by ages 65 and 70 respectively.[18, 19, 21, 22]  BRCA1/2 mutation carriers may also be at an increased risk for melanoma, pancreatic cancer, and potentially other cancers.[23]  BRCA2 is also known as FANCD1.  Individuals who inherit a BRCA2/FANCD1 mutation from each parent may have a rare autosomal recessive condition called Fanconi-anemia type D1 (FA-D1), which affects multiple body systems.
 
BARD1, BRIP1, MRE11A, NBN, RAD50, RAD51C, and RAD51D are genes involved in the Fanconi anemia (FA)-BRCA pathway, critical for DNA repair by homologous recombination, and interact in vivo with BRCA1 and/or BRCA2.[4, 24, 25] Mutations in these genes are estimated to confer up to a three-fold increase in breast cancer risk, and mutations in each have been reported in at least one identified case of ovarian cancer to date.[11, 24, 26-29]  The ovarian cancer risk associated with mutations in RAD51C and RAD51D has been estimated to be 9% and 10%, respectively.[11, 28]   BRIP1, NBN, and RAD51C are each associated with a rare autosomal recessive disorder that affects multiple body systems. 

CHEK2 is a gene that receives signals from damaged DNA, transmitted via ATM. CHEK2 interacts in vivo with 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 CHEK2 confer an increased risk of developing many types of cancer including breast, colon, and other cancers. Mutations are more likely to be found among women with bilateral versus unilateral breast cancers. A female CHEK2 mutation carrier has approximately a two-fold increase in lifetime breast cancer risk, and has a 1% risk per year of developing a second breast primary cancer. Lifetime risks for other associated cancers are unknown. An increased risk for ovarian cancer has also been suggested.[27, 30-32]

CDH1 germline mutations are 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 was 67% for men and 83% for women.[33]  HDGC patients typically present with diffuse-type gastric cancer, with signet ring cells diffusely infiltrating the wall of the stomach and, at advanced stages, linitis plastica. An elevated risk of lobular breast cancer in women is also associated with HDGC, with an estimated lifetime breast cancer risk of 39-52%.[34]

MUTYH germline mutations are known to cause MUTYH-associated polyposis (MAP), an autosomal recessive condition predisposing to gastrointestinal polyposis and colorectal cancer. Individuals who carry two MUTYH mutations on different chromosomes (in trans) have an estimated lifetime colorectal cancer risk of up to 80%.[35]  In addition, some studies suggest that MUTYH mutations confer an increased risk to develop female breast cancer, this is estimated to be a  1.5-fold lifetime increased risk within the North African Jewish population. MUTYH mutations in the carrier state may also increase lifetime risks for cancers of the duodenum, stomach, and endometrium (females),[36-38] however, these data are limited and risks may vary between populations. Two common mutations in the Caucasian population, p.Y179C and p.G396D (originally designated as p.Y165C and p.G382D), account for the majority of pathogenic MUTYH alterations reported to date. Breast cancer risk estimates for male MUTYH mutation carriers are not currently available.

NF1 mutations cause neurofibromatosis type 1 (NF1), an autosomal dominant disorder affecting multiple body systems. It is characterized by multiple café-au-lait spots, axillary and inguinal freckling, multiple cutaneous neurofibromas, and Lisch nodules. 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 have demonstrated a 3 to 5-fold increase in lifetime breast cancer risk for women with NF1, with the highest risks for those less than 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 an increased lifetime risk for pancreatic cancer, breast cancer, and Fanconi-anemia type N (FA-N). Familial pancreatic and/or breast cancer due to PALB2 mutations is inherited in an autosomal dominant pattern, while FA-N is a rare autosomal recessive condition affecting multiple body systems. Females with a PALB2 mutation have a 2 to 4-fold increase in risk for breast cancer.[39, 40] A 2014 article concluded that in the context of a strong family history, mutations in PALB2 may be associated with up to a 58% risk of female breast cancer.  Without a family history, the risk for female breast cancer was estimated to be 33% (the difference attributed to genetic and/or environmental modifiers).[41]  Recent studies have identified PALB2 mutations in 1-3% of families with pancreatic cancer; however, the exact lifetime pancreatic cancer risk has not yet been established.[42, 43] Additionally, an increased risk for ovarian cancer has been suggested as well.[27]

PTEN is a gene associated with Cowden syndrome (CS), PTEN Hamartoma Tumor syndrome (PHTS), Bannayan-Riley-Ruvalcaba syndrome, Proteus syndrome and autism spectrum disorder. CS is a multiple hamartoma syndrome with a high risk of developing tumors of the thyroid, breast, and endometrium. Mucocutaneous lesions, thyroid abnormalities, fibrocystic disease, multiple uterine leiomyomata, 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 manifestations by their twenties.[44, 45]  Recent studies noted increased risks for renal cell cancer, colorectal cancer, and other cancers.[46, 47]  One study quotes up to a 31-fold increase in RCC risk for PTEN mutation carriers as compared to the general population.[48] 

TP53 is a tumor suppressor gene, and germline mutations within it are associated with Li-Fraumeni syndrome (LFS). An individual carrying a TP53 mutation has a 21-49% lifetime risk of developing cancer by age 30 and a lifetime cancer risk of 68-93%.[49]  The most common tumor types observed in LFS families include soft tissue and osteosarcomas, breast cancer, brain tumors (including astrocytomas, glioblastomas, medulloblastomas and choroid plexus carcinomas), and adrenocortical carcinoma (ACC); other cancers, including colorectal, gastric, ovarian, pancreatic, and renal, have also been reported.[12, 50]  Studies have shown that a small percentage of women with early onset breast cancer who do not carry BRCA1 and BRCA2 mutations are identified to have mutations in TP53.[32, 51, 52]
 
 
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 BreastNext testing.

  • Early-onset breast cancer (diagnosed ≤45 years of age)
  • Male breast cancer at any age
  • Breast and ovarian cancer in the same woman
  • Three or more cases of breast cancer*
  • Three or more cases of breast, ovarian, and/or pancreatic cancer*
  • Three or more cases of breast, uterine, and/or thyroid cancer*

Common Red Flags for Hereditary Cancer

  • Cancer diagnosed at a younger age than expected for the general population (≤ 50 years, for most cancers)
  • Cancer diagnosed across generations, and in multiple generations within a family, especially if diagnosed younger than average
  • 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 breast and pancreatic cancer with PALB2)

Benefits of Testing
Identifying patients with an inherited 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 oophorectomy, after childbearing is complete, for women with increased risk for breast/ ovarian cancer)
  • 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 

BreastNext analyzes 17 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MRE11A, MUTYH, NBN, NF1, PALB2, PTEN, RAD50, RAD51C, RAD51D, and TP53) by next generation sequencing or Sanger sequencing of all coding domains and well into the flanking 5’ and 3’ ends of all the introns and untranslated regions. In addition, sequencing of the promoter region is performed for PTEN (c.-1300 to c.-745).  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 targeted microarray identifies gross deletions and duplications in all 17 genes.

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 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

Turnaround Time 
TEST CODE TEST NAME TURNAROUND TIME (weeks)
8836 BreastNext (Ordered 11/3/14 or after) 2-4
  BreastNext (Ordered before 11/3/14) 8-12

 

 

Specialty 
References 

1. National Cancer Institute. Cancer Stat Fact Sheets.  Accessed October 22, 2014; Available from: http://seer.cancer.gov/.
2. National Cancer Institute.  Accessed October 22, 2014; Available from: http://www.cancer.gov/.
3. Castera, L., et al., Next-generation sequencing for the diagnosis of hereditary breast and ovarian cancer using genomic capture targeting multiple candidate genes. Eur J Hum Genet, 2014. 22(11): p. 1305-13.
4. Walsh, T., et al., Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing. Proc Natl Acad Sci U S A, 2010. 107(28): p. 12629-33.
5. van der Groep, P., E. van der Wall, and P.J. van Diest, Pathology of hereditary breast cancer. Cell Oncol (Dordr), 2011. 34(2): p. 71-88.
6. Walsh, T. and M.C. King, Ten genes for inherited breast cancer. Cancer Cell, 2007. 11(2): p. 103-5.
7. Meindl, A., et al., Hereditary breast and ovarian cancer: new genes, new treatments, new concepts. Dtsch Arztebl Int, 2011. 108(19): p. 323-30.
8. Antoniou, A., et al., Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case Series unselected for family history: a combined analysis of 22 studies. Am J Hum Genet, 2003. 72(5): p. 1117-30.
9. Chen, S. and G. Parmigiani, Meta-analysis of BRCA1 and BRCA2 penetrance. J Clin Oncol, 2007. 25(11): p. 1329-33.
10. Ford, D., et al., Genetic heterogeneity and penetrance analysis of the BRCA1 and BRCA2 genes in breast cancer families. The Breast Cancer Linkage Consortium. Am J Hum Genet, 1998. 62(3): p. 676-89.
11. Loveday, C., et al., Germline RAD51C mutations confer susceptibility to ovarian cancer. Nat Genet, 2012. 44(5): p. 475-6; author reply 476.
12. Olivier, M., et al., Li-Fraumeni and related syndromes: correlation between tumor type, family structure, and TP53 genotype. Cancer Res, 2003. 63(20): p. 6643-50.
13. Renwick, A., et al., ATM mutations that cause ataxia-telangiectasia are breast cancer susceptibility alleles. Nat Genet, 2006. 38(8): p. 873-5.
14. Roberts NJ, J.Y., Yu J, Kopelovich L, Petersen GM, Bondy ML, Steven Gallinger, Schwartz AG, Syngal S, Cote ML, Axilbund J, Schulick R, Ali SZ, Eshleman JR, Velculescu VE, Goggins M, Bert Vogelstein, Papadopoulos M, Hruban RH, Kinzler KW,  Klein AP, ATM Mutations in Patients with hereditary Pancreatic cancer. Cancer Discovery, 2011. 2(1): p. OF1-OF6.
15. Janavicius, R., Founder BRCA1/2 mutations in the Europe: implications for hereditary breast-ovarian cancer prevention and control. EPMA J, 2010. 1(3): p. 397-412.
16. Ferla, R., et al., Founder mutations in BRCA1 and BRCA2 genes. Ann Oncol, 2007. 18 Suppl 6: p. vi93-8.
17. Tulinius, H., et al., The effect of a single BRCA2 mutation on cancer in Iceland. J Med Genet, 2002. 39(7): p. 457-62.
18. Tai, Y.C., et al., Breast cancer risk among male BRCA1 and BRCA2 mutation carriers. J Natl Cancer Inst, 2007. 99(23): p. 1811-4.
19. Thompson, D., D.F. Easton, and C. Breast Cancer Linkage, Cancer Incidence in BRCA1 mutation carriers. J Natl Cancer Inst, 2002. 94(18): p. 1358-65.
20. Folkins, A.K. and T.A. Longacre, Hereditary gynaecological malignancies: advances in screening and treatment. Histopathology, 2013. 62(1): p. 2-30.
21. Shannon, K.M. and A. Chittenden, Genetic testing by cancer site: breast. Cancer J, 2012. 18(4): p. 310-9.
22. Kote-Jarai, Z., et al., BRCA2 is a moderate penetrance gene contributing to young-onset prostate cancer: implications for genetic testing in prostate cancer patients. Br J Cancer, 2011. 105(8): p. 1230-4.
23. van Asperen, C.J., et al., Cancer risks in BRCA2 families: estimates for sites other than breast and ovary. J Med Genet, 2005. 42(9): p. 711-9.
24. Damiola, F., et al., Rare key functional domain missense substitutions in MRE11A, RAD50, and NBN contribute to breast cancer susceptibility: results from a Breast Cancer Family Registry case-control mutation-screening study. Breast Cancer Res, 2014. 16(3): p. R58.
25. Pennington, K.P. and E.M. Swisher, Hereditary ovarian cancer: beyond the usual suspects. Gynecol Oncol, 2012. 124(2): p. 347-53.
26. Seal, S., et al., Truncating mutations in the Fanconi anemia J gene BRIP1 are low-penetrance breast cancer susceptibility alleles. Nat Genet, 2006. 38(11): p. 1239-41.
27. 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 U S A, 2011. 108(44): p. 18032-7.
28. Loveday, C., et al., Germline mutations in RAD51D confer susceptibility to ovarian cancer. Nat Genet, 2011. 43(9): p. 879-82.
29. Meindl, A., et al., Germline mutations in breast and ovarian cancer pedigrees establish RAD51C as a human cancer susceptibility gene. Nat Genet, 2010. 42(5): p. 410-4.
30. Bahassi, E.M., et al., The checkpoint kinases Chk1 and Chk2 regulate the functional associations between hBRCA2 and Rad51 in response to DNA damage. Oncogene, 2008. 27(28): p. 3977-85.
31. Cybulski, C., et al., CHEK2 is a multiorgan cancer susceptibility gene. Am J Hum Genet, 2004. 75(6): p. 1131-5.
32. Walsh, T., et al., Spectrum of mutations in BRCA1, BRCA2, CHEK2, and TP53 in families at high risk of breast cancer. Jama, 2006. 295(12): p. 1379-88.
33. Pharoah, P.D., et al., Incidence of gastric cancer and breast cancer in CDH1 (E-cadherin) mutation carriers from hereditary diffuse gastric cancer families. Gastroenterology, 2001. 121(6): p. 1348-53.
34. Guilford, P., B. Humar, and V. Blair, Hereditary diffuse gastric cancer: translation of CDH1 germline mutations into clinical practice. Gastric Cancer, 2010. 13(1): p. 1-10.