BRCAplus

BRCAplusTM is a next generation sequencing (NGS) panel that simultaneously analyzes 6 breast cancer susceptibility genes, all with published management guidelines.

PrintPrint

BRCAplusTM is a next generation sequencing (NGS) panel that simultaneously analyzes 6 breast cancer susceptibility genes, all with published management guidelines.

BRCAplus is a next generation sequencing panel of 6 genes associated with breast cancer (BRCA1, BRCA2, CDH1, PALB2, PTEN, and TP53). These six genes are associated with five hereditary cancer syndromes (hereditary breast and ovarian cancer or HBOC, hereditary diffuse gastric cancer, hereditary breast and pancreatic cancer, Cowden syndrome, and Li-Fraumeni syndrome) – all of which have published management guidelines. Identification of a mutation in one of these genes can help estimate cancer risk and guide treatment, screening, and/or prevention decisions for the patient. Family members can be tested for a familial mutation to determine if they have high risk (positive for the familial mutation) or average risk (negative for the familial mutation) for cancer.

Disease Name 
Hereditary Cancer
Hereditary Breast Ovarian Cancer (HBOC)
Breast Cancer
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 231,840 new cases of female breast cancer and 2,350 new cases of male breast cancer will be diagnosed in the U.S. in 2015.2 The majority of breast cancers are sporadic, but 5-10% are due to inherited causes. Hereditary breast cancer tends to occur earlier in life than non-inherited sporadic cases, and is 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 included in BRCAplus can confer an estimated 39–87% lifetime risk for breast cancer. These genes have also been associated with increased risks for other cancers, such as ovarian cancer with BRCA1 and BRCA2, thyroid cancer with PTEN, pancreatic cancer with PALB2, and sarcoma with TP53.8-12

BRCAplus Genes

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 70.8-10,13-15  Male BRCA1 mutation carriers have a cumulative breast cancer lifetime risk of about 1.2% by age 70.16,17 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 70.8-10,18,19  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.16,17,19,20  BRCA1/2 mutation carriers may also be at an increased risk for melanoma, pancreatic cancer, and potentially other cancers.21  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.

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.22 Patients with HDGC 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%.23

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.24-25  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).26  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.27-28 Additionally, an increased risk for ovarian cancer has been suggested as well.29

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.30-31  Recent studies noted increased risks for renal cell cancer, colorectal cancer, and other cancers.32-33  One study quotes up to a 31-fold increase in RCC risk for PTEN mutation carriers as compared to the general population.

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%.35  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.11, 36  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 TP53.37-39

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 BRCAplus 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, uterine, and/or thyroid cancer*
    *On the same side of the family

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 (e.g. breast and diffuse gastric cancer with CDH1 mutations, or breast and thyroid cancer with PTEN mutations) 

If increased risk of a hereditary cancer syndrome is suspected:

  • The American Society of Clinical Oncology (ASCO) recommends that genetic testing be offered to individuals with suspected inherited (genetic) cancer risk in situations where test results can be interpreted, and when they affect medical management of the patient. (J Clin Oncol., 2003)
  • The American Congress (formerly College) of Obstetricians and Gynecologists (ACOG) recommends referral to a specialist in cancer genetics or a healthcare provider with expertise in genetics for complete hereditary cancer risk assessment, which may lead to genetic testing.40

Establishing a molecular diagnosis can help guide preventive measures, direct surgical options and estimate personal and familial cancer risk. 

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)
  • Clarify and stratify familial cancer risks, based on gene-specific cancer associations, such as risk for uterine, thyroid, colorectal, and kidney cancer with PTEN mutations
  • 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 

BRCAplus analyzes 6 genes (BRCA1, BRCA2, CDH1, PALB2, PTEN, 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. 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 6 genes (BRCA1, BRCA2, CDH1, PALB2, PTEN, TP53).

Mutation Detection Rate 

BRCAplus can detect >99.9% of described mutations in the included genes, when present (analytic sensitivity).

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: Frozen on dry ice is preferred, or ship on ice

Saliva: Fill 1 tube with saliva up to black line (1cc of saliva) in Oragene Self Collection container. After tube is closed, 1cc of buffer will mix with saliva for a total volume of 2cc.
Storage: At room temperature in sterile bag
Shipment: Ship room temperature for two-day delivery

Turnaround Time 
TEST CODE TEST NAME TURNAROUND TIME (CALENDAR DAYS)
8836 BRCAplus  (Blood)  7-14
(Saliva)  9-16
Specialty 
Genes 
CDH1
PTEN
TP53
BRCA1
BRCA2
PALB2
Tests 
References 
  1. Surveillance, Epidemiology, and End Results Program. Cancer Stat Fact Sheets [April 29, 2015]. Available from http://seer.cancer.gov/.
  2. National Cancer Institute. Cancer Stat Fact Sheets.  Accessed October 22, 2014. Available from http://seer.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):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):12629-33.
  5. van der Groep P, et al. Pathology of hereditary breast cancer. Cell Oncol (Dordr). 2011. 34(2):71-88.
  6. Walsh T and King M-C. Ten genes for inherited breast cancer. Cancer Cell. 2007. 11(2):103-5.
  7. Meindl A, et al. Hereditary breast and ovarian cancer: new genes, new treatments, new concepts. Dtsch Arztebl Int. 2011. 108(19):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):1117-30.
  9. Chen S and Parmigiani G. Meta-analysis of BRCA1 and BRCA2 penetrance. J Clin Oncol. 2007. 25(11):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):676-89.
  11. Olivier M, et al. Li-Fraumeni and related syndromes: correlation between tumor type, family structure, and TP53 genotype. Cancer Res. 2003. 63(20):6643-50.
  12. Tan MH, et al. Lifetime cancer risks in individuals with germline PTEN mutations. Clin Cancer Res. 2012. 18(2):400-7.
  13. Janavicius R. Founder BRCA1/2 mutations in the Europe: implications for hereditary breast-ovarian cancer prevention and control. EPMA J. 2010. 1(3):397-412
  14. Ferla R, et al. Founder mutations in BRCA1 and BRCA2 genes. Ann Oncol. 2007. 18 Suppl 6:vi93-8.
  15. Tulinius H, et al. The effect of a single BRCA2 mutation on cancer in Iceland. J Med Genet. 2002. 39(7):457-62
  16. Tai YC, et al. Breast cancer risk among male BRCA1 and BRCA2 mutation carriers. J Natl Cancer Inst. 2007. 99(23):1811-4.
  17. Thompson D, et al. Breast cancer linkage, cancer incidence in BRCA1 mutation carriers. J Natl Cancer Inst. 2002. 94(18):1358-65.
  18. Folkins AK and Longacre TA. Hereditary gynaecological malignancies: advances in screening and treatment. Histopathology. 2013. 62(1):2-30.
  19. Shannon KM and Chittenden A. Genetic testing by cancer site: breast. Cancer J. 2012. 18(4):310-9.
  20. 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):1230-4.
  21. van Asperen CJ, et al. Cancer risks in BRCA2 families: estimates for sites other than breast and ovary. J Med Genet. 2005. 42(9):711-9.
  22. Pharoah PD, 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):1348-53.
  23. Guilford P, et al. Hereditary diffuse gastric cancer: translation of CDH1 germline mutations into clinical practice. Gastric Cancer. 2010. 13(1):1-10.
  24. Slater EP, et al. PALB2 mutations in European familial pancreatic cancer families. Clin Genet. 2010;78(5):490-4.
  25. Casadei S, et al. Contribution of inherited mutations in the BRCA2-interacting protein PALB2 to familial breast cancer. Cancer Res. 2011;71(6):2222-9.
  26. Antoniou AC, et al. Breast-cancer risk in families with mutations in PALB2. N Engl J Med. 2014;371(6):497-506.
  27. Tischkowitz MD, et al. Analysis of the gene coding for the BRCA2-interacting protein PALB2 in familial and sporadic pancreatic cancer. Gastroenterology. 2009;137(3):1183-6.
  28. Jones S, et al. Exomic sequencing identifies PALB2 as a pancreatic cancer susceptibility gene. Science. 2009;324(5924):217.
  29. 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):18032-7.
  30. Eng C. Will the real Cowden syndrome please stand up: revised diagnostic criteria. J Med Genet. 2000. 37(11):828-30.
  31. Starink TM, et al. The Cowden syndrome: a clinical and genetic study in 21 patients. Clin Genet. 1986. 29(3):222-33.
  32. Heald B, et al. Frequent gastrointestinal polyps and colorectal adenocarcinomas in a prospective series of PTEN mutation carriers. Gastroenterology. 2010. 139(6):1927-33.
  33. Tan MH, et al. Lifetime cancer risks in individuals with germline PTEN mutations. Clin Cancer Res. 2012;18(2):400-7.
  34. Mester JL, et al. Papillary renal cell carcinoma is associated with PTEN hamartoma tumor syndrome. Urology. 2012. 79(5):1187 e1-7.
  35. Hwang SJ, et al. Germline p53 mutations in a cohort with childhood sarcoma: sex differences in cancer risk. Am J Hum Genet. 2003. 72(4):975-83.
  36. Birch JM, et al. Prevalence and diversity of constitutional mutations in the p53 gene among 21 Li-Fraumeni families. Cancer Res. 1994. 54(5):1298-304.
  37. 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):1379-88.
  38. Gonzalez KD, et al. Beyond Li-Fraumeni syndrome: clinical characteristics of families with p53 germline mutations. J Clin Oncol. 2009. 27(8):1250-6.
  39. McCuaig JM, et al. Routine TP53 testing for breast cancer under age 30: ready for prime time? Fam Cancer. 2012. 11(4):607-13.
  40. American Congress of Obstetricians and Gynecologists Committee on Genetics. Committee Opinion No. 634: Hereditary cancer syndromes and risk assessment. Obstet Gynecol. June 2015. 125(6):1538-1543.