1. NGS panel - Genetic testing for breast cancer

Breast cancer

November 03, 2017

Clinical features

Breast cancer is the most common cancer among women and it is the third leading cause of cancer death 1. Approximately 1 in 8 women (12%) will develop breast cancer in their lifetime1. In year 2017, the estimated new cases and deaths were: 252,710 and 40,610 in USA. The risk factors include increasing age, inheritance susceptibility, alcohol intake, breast tissue density, estrogen, obesity, hormone etc.

Among several breast cancer risk genes, BRCA1 and BRCA2 are the most significant ones. Pathogenic variants in these two genes are asscociated with Hereditary breast and ovarian cancer (HBOC). HBOC should be suspected in individuals with a personal or family history of any of the following (based on the summarized data from Gene Reviews 1):

  • Breast cancer diagnosed at or before age 50 years
  • Ovarian cancer
  • Multiple primary breast cancers, in either one or both breasts
  • Male breast cancer
  • Triple-negative breast cancer (estrogen receptor-negative, progesterone receptor-negative, and HER2-negative)
  • The combination of pancreatic cancer and/or prostate cancer with breast cancer, and/or ovarian cancer
  • Breast cancer diagnosed at any age in an individual of Ashkenazi Jewish ancestry
  • Two or more relatives with breast cancer (at least one under age 50)
  • Three or more relatives with breast cancer at any age
  • A previously identified BRCA1 or BRCA2 pathogenic variant in the family

Breast cancer and/or HBOC susceptibility is a complex phenomenon, in which multiple genes may play a role. An estimated 5-10% of all breast ovarian cancers are directly attributed to inherited gene variants in the BRCA1 and BRCA2 genes1. About 46-87% of women (BRCA1) and 38-84% (BRCA2) of women with variants will develop breast ovarian cancer during their lifetimes 2-8.

Genetic changes in other, non-BRCA genes, are rarer but can also contribute to the risk of breast ovarian cancer (see Figure 1). All genes associated with breast cancer, depending on their penetrance, can be classified into the following three groups 9, 10, 11 (Figure 1):

  • Highly penetrant genes, including BRCA1, BRCA2, RAD51C, NBN, TP53, STK11, PTEN
  • Moderately penetrant genes, including CHEK2, PALB2, BRIP1, ATM, RAD50
  • Low penetrant genes, including FGFR2, CYP1A1, XRCC3, XRCC1, MAP3K1, TOX3, TGFB1, LSP1

Figure 2. Contribution of pathogenic variants in non BRCA1/2 genes to familial breast cancer, according to the large genetic study on 2134 BRCA1/BRCA2 negative women with familial breast cancer 13. 

The genes described as high penetrance breast cancer susceptibility genes are typically tumor suppressor genes that participate in cellular proliferation and thus in tumor growth 12. Each HBOC-associated gene may have a relatively small effect on breast cancer risk; however, in combination with other genetic loci and/or environmental factors, variants of this kind might significantly alter breast cancer risk 1. Individuals with heterozygous variants in tumor suppressor genes have an increased breast cancer risk of between 1.5-5 1,3. The lifetime risks of BRIP1 and PALB2 variants are associated with a 20-50% lifetime risk of breast cancer 12. Female relatives of individuals with ATM or CHEK2 variants have a 2-4 fold increased risk of breast cancer 1, 12. Today’s powerful, novel tools for large-scale sequencing and genetic testing give us more information about the genetic basis of breast ovarian cancer and its association with numerous moderate risk genes, such as MSH2, MSH6, PMS1, PMS2, RAD50, RAD51C, RAD51D, and XRCC211, 12.

Table 1: Overview of genes included in CENTOGENE’s breast cancer associated panels offered

Gene OMIM Chromosome Lifetime risk of specific gene mutation carriers
BRCA1 113705 17q12–21 For age >30 years: 3%1
For age >70 years: 85%1
BRCA2 600185 13q12-13 For age >30 years: 11.4%1
For age >60 years:16.2%1
ATM 607585 11q22.3 For age >30 years: 1
BARD1 601593 2q34-q35 Elevated1
BRIP1 605882 17q23.2 Lifetime risk: 3.4%1
For age >80 years: 8.3%1
CHEK2 604373 22q12.1 Lifetime risk: elevated1
MEN1 131100 11q13.1 2%-3%16
MLH1 120436 3p22.2 For age >70 years: 18,6%17, 12
MRE11A 600814 11q21 Elevated1, 11, 14
MSH2 609309 2p21-p16 For age >70 years: 11,2%12, 17
MSH6 600678 2p16.3 For age >70 years: 5,6%17, 12
MUTYH 604933 1p34.1 Elevated risk1, 12
NBN 602667 8q21 Lifetime risk: elevated1, 14
PALB2 610355 16p12.1 For age >46 years: 3.4%1
For age >85 years: 11.6%1
PMS1 600258 2q32.2 Elevated risk1
PMS2 600259 7p22.1 Elevated risk1, 12
RAD50 604040 5q31.1 Elevated risk1, 14
RAD51C 602774 17q25.1 Lifetime risk: 1%1
RAD51D 602954 17q12 Elevated risk1
XRCC2 600375 7q36.1 Elevated risk1, 10, 11, 14

Genetic testing of breast cancer-related genes may confirm a diagnosis of breast cancer and can help guide treatment and management decisions. Identification of a disease-causing variant would also guide testing and diagnosis of at-risk relatives. Early diagnostics of breast and/or ovarian cancer can increase the chances of detecting and localizing the cancer before it spreads. Furthermore, improvements in early detection and treatment of breast cancer have led to longer survival of breast cancer patients 1, 15. CENTOGENE has designed a special set of breast cancer related panels, specifically design to fit the affected family or individual with a family history of breast cancer.

The BRCA1/BRCA2 panel is designed for individuals with family history of breast/ovarian cancer with a previously identified pathogenic variant in the BRCA1 or BRCA2 genes. This panel includes MGS sequencing methods for the most common BRCA1/BRCA2 pathogenic single nucleotide variants as well as MLPA analysis for large deletion/duplications.

CENTOGENE has also designed a special Breast ovarian cancer panel which includes only those main highly penetrant genes aside from the BRCA1 and BRCA2 genes: CDH1, PTEN, STK11, TP53. The Breast ovarian cancer panel PLUS includes the moderately penetrant genes associated with breast cancer (ATM, BARD1, BRIP1, CHEK2, MEN1, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PMS1, PMS2, RAD50, RAD51C, RAD51D, XRCC2). This panel could be of great help to all patients with a history/family history of progressive breast cancer but without any known BRCA-associated identified pathogenic variants.

Finally, CENTOGENE designed the CentoBreast® panel, a comprehensive panel intended for genetic testing of hereditary breast-ovarian cancers caused by variants in those genes most commonly associated with breast-ovarian cancer. Using NGS bidirectional sequencing of the genes in the panel, including all exons, exon/intron boundaries and promoter regions, with additional validation of the sequencing results for every detected genetic variant, we can provide 100% variant detection reproducibility for Sanger sequencing.

The CentoBreast® panel includes all high and moderate risk genes associated with HBOC. Genetic testing for breast ovarian cancer improves our chances of preventing and treating at-risk individuals, enabling patients to reduce their cancer risk. It can give them a sense of relief regarding their future risk of cancer or allow them to participate in medical research that could, in the long run, help reduce deaths from hereditary breast and ovarian cancer.

National Comprehensive Cancer Network guidelines 15 suggest that women with a BRCA1/2 pathogenic variant, or other breast cancer-causing pathogenic variants, may wish to consider bilateral mastectomy as a primary surgical treatment of breast cancer because of their elevated rate of ipsilateral and contralateral breast cancer. Treatment of ovarian and other cancers in individuals with pathogenic variants in BRCA1/2 and other genes associated with breast cancer is similar to that of sporadic cancers.

For individuals affected with breast/ovarian cancer, a team of specialist, will discuss the treatment options and possible side effects. Common treatment options include surgery, radiation and chemotherapy, and hormone therapy. Every step of the therapy must be carefully planned, based on the personal medical history and conditions.

CENTOGENE offers a selection of breast/ovarian cancer panels (see Table 2), performed using highest quality NGS sequencing with maximal specificity and sensitivity of variant detections.

Table 2: The overview of CENTOGENE’s breast/ovarian cancer associated panels

Gene test Gene(s) name (OMIM, HGNC)
Breast ovarian cancer panel CDH1, PTEN, STK11, TP53
Breast ovarian cancer panel PLUS ATM, BARD1, BRIP1, CHEK2, MEN1, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PMS1, PMS2, RAD50, RAD51C, RAD51D, XRCC2
CentoBreast® panel ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD51C, STK11, TP53

Differential diagnosis

The differential diagnosis of breast cancer -related disorders – depending on the major symptoms in the initial case – includes the following diseases:

  • Fibroadenomas and cysts of breasts and ovarian benign cysts)
  • Breast lymphoma, and/or metastasis from other primary sites
  • Chronic or acute mastitis and other inflammatory cysts in breasts
  • Papilloma, ductal carcinoma, duct ectasia, and fibrocystic disease
  • Traumatic fat necrosis
  • Hyalinized fibroadenoma.

Testing strategy

CENTOGENE offers an advanced, fast and cost-effective strategy to test large NGS panels and diagnose complex phenotypes based on NGS technology. This approach offers an unparalleled advantage by reducing amplification/capture biases and providing sequencing of the entire gene with more uniform coverage.

To confirm/establish the diagnosis of hereditary breast/ovarian cancer, CENTOGENE offers the following testing strategy for breast cancer using NGS Panel Genomic targeted towards this specific phenotype:

Step 1: Next generation sequencing from a single filter card. The sequencing covers the entire gene (coding region, exon/intron boundaries, intronic and promoter) for all the genes included in the breast cancer panel. Copy Number Variants analysis derived from NGS data is also included.

Step 2: If no pathogenic variant is identified after analysis of the breast cancer panel, we further recommend continuing the bioinformatics analysis of the data using next generation sequencing to cover those genes which are either implicated in an overlapping phenotype or could be involved in a similar pathway but are not strongly clinically implicated based on the current information in literature.

Referral reasons

BRCA1/BRCA2-associated breast cancer panel testing is recommended for those individuals who have one of the following 1, 15:

  • A family member with a BRCA1/BRCA2 gene pathogenic variant
  • A personal history of breast cancer at age 45 or younger
  • A personal history of breast cancer at any age and a family member diagnosed with breast cancer at age 50 or younger
  • A personal history of breast cancer at any age and two or more family members diagnosed with breast, pancreatic and/or aggressive prostate cancer at any age
  • Ashkenazi Jewish heritage and a personal history of breast or pancreatic cancer
  • A personal history of triple negative breast cancer diagnosed at age 60 or younger
  • A personal or family history of ovarian cancer
  • A personal or family history of male breast cancer
  • A family member diagnosed with breast cancer at age 45 or younger

NCCN guidelines 15 recommend referral to a genetics expert (including Breast ovarian cancer panel, Breast ovarian cancer panel PLUS and CentoBreast®) for evaluation of breast cancer patients if they meet any of these criteria:

  • Anyone with a family history of one or more of the following:

    • A blood relative with a known variants in a gene which increases cancer risk
    • A blood relative with two or more primary breast cancers
    • Two or more relatives with breast cancer on the same side of the family, at least one who was diagnosed before age 50
    • A blood relative with ovarian cancer
    • A close blood relative with breast cancer before age 45
    • A blood relative with male breast cancer

  • Anyone of Ashkenazi Jewish ancestry with breast, ovarian, or pancreatic cancer at any age
  • Anyone with a cancer diagnosis and one or more of the following:

    • A blood relative with a known variants in a gene which increases cancer risk
    • Breast cancer at or before the age of 50
    • Triple-negative breast cancer at or before the age of 60
    • Ovarian, fallopian tube, or primary peritoneal cancer at any age
    • Male breast cancer at any age

  • Anyone with breast cancer at any age and one or more of the following:

    • A blood relative with a known variants in a gene which increases cancer risk
    • An primary breast cancer

  • Anyone with a personal or family history of three or more of the following, especially if any of the cases are diagnosed before age 50:

    • Pancreatic cancer
    • Prostate cancer
    • Melanoma
    • Sarcoma
    • Adrenal cancer
    • Brain tumors
    • Leukemia
    • Uterine cancer
    • Thyroid cancer
    • Kidney cancer
    • Diffuse gastric cancer
    • Colon cancer.

Test utility

Genetic testing with CENTOGENE breast cancer associated panels can contribute to an early diagnosis, improved choice of treatment, and genetic counselling for family members. At CENTOGENE we have analyzed thousands of breast-ovarian cancer patients samples from all over the world. This medical expertise, combined with the constant optimization of our internal processes and implementation of new diagnostic techniques, allows us to support you with attractive new diagnostics offers for genetic testing for breast/ovarian cancers.

Sequencing, deletion/duplication of breast cancer related genes should be performed in all individuals suspected of having breast cancer. In parallel, other genes reported to be related with this clinical phenotype should also be analyzed for the presence of pathogenic variants, due to the overlap in many clinical features caused by those particular genes. Confirmation of a clinical diagnosis through genetic testing can allow for genetic counseling and may direct medical management. Genetic counseling can provide a patient and/or family with the natural history of breast/ovarian cancer, identify at-risk family members, provide information about reproductive risks as well as preconception/prenatal options, and allow for appropriate referral for patient support and/or resources.