Next-Generation Sequencing Informs Genetic Testing in Breast Cancer

Targeted Therapies in OncologyMarch 2021
Pages: 114

As the understanding of genetic risk factors in breast cancer continues to grow, professional organizations have sought to provide specific recommendations for genetic testing that would prevent overtesting yet still diagnose as many mutations as possible in patients.

Susan M. Domchek, MD

Susan M. Domchek, MD

Over the past several years, the oncology community has witnessed a significant improvement in identifying genetic mutations associated with increased risk of breast malignancies.1 As the understanding of genetic risk factors in breast cancer continues to grow, professional organizations have sought to provide specific recommendations for genetic testing that would prevent overtesting yet still diagnose as many mutations as possible in patients.

“In general, the gold standard [for guidelines] is clinical utility,” said Susan M. Domchek, MD, the Basser Professor in Oncology at Penn Medicine in Philadelphia, executive director of the Basser Center for BRCA, and director of MacDonald Women’s Cancer Risk Evaluation Center. “We’re all looking at the same data, and it just comes down to perspective for how you interpret those data.”

Currently, controversy surrounds guideline recommendations for eligibility in genetic testing. The US Preventive Services Task Force and the National Comprehensive Cancer Network (NCCN) guidelines recommend genetic testing for patients with breast cancer and a family history associated with germline BRCA1/2 mutations or who have a personal history with an increased risk of BRCA1/2 mutations.1,2 Utilizing testing criteria recommended by the NCCN may not identify a small number of individuals who have no family history but carry pathogenic variants of high-risk breast cancer genes.

“The guidelines from the NCCN focus on the likelihood of finding genes where we have specific information [on] how to use that and how helpful it is to patients, for instance, [with] BRCA and other [high-penetrance] gene mutations,” said Domchek.

Conversely, the American Society of Breast Surgeons (ASBrS) recommends genetic testing for all patients with breast cancer.3 However, the paradigm shift from genetic testing in individuals with a family history to the testing of all patients with breast cancer, as recommended by ASBrS, may lead to unnecessary testing and the potential for harm.4 The optimal model for genetic testing in patients with breast cancer has not yet been identified, though some strategies have been proposed.

According to results of a recently published single-center, prospective study, population-based genetic testing in patients with breast cancer younger than 65 years may optimize the identification of germline pathogenic variants.5 The sensitivity and specificity of NCCN and ASBrS criteria for germline pathogenic variants were assessed. The primary analysis evaluated 9 established, actionable breast cancer predisposition genes (ATM, BRCA1/2, CDH1, CHEK2, NF1, PALB2, PTEN, and TP53). In the entire population evaluated (N=3907), 6.2% had germline pathogenic variants, with CHEK2, BRCA2, BRCA1, and ATM occurring at the highest frequency.

CHEK2 and ATM are challenging because they’re strongly modified by family history…so we view it as a risk factor,” Domchek said.

Approximately 48% of patients met NCCN guidelines’ criteria for genetic testing, whereas 52% of patients did not. Individuals who met NCCN criteria were more likely to have a pathogenic variant in 1 of the 9 genes, 9.0% versus 3.5%, compared with individuals who did not meet NCCN criteria (P<.001). The primary analysis revealed a sensitivity of 70.1% and a specificity of 53%. When the investigators modified the NCCN criteria to include patients who were 65 years old at the time of receiving a diagnosis and family history, the sensitivity increased to over 90% for pathogenic variants in 9 predisposition genes.5 Currently, the clinical utility in genetic testing in women over 65 years is unknown because the probability of detecting a germline pathogenic variant decreases with age.4

Variance of Unknown Significance

To improve upon a previous study by Yadav et al, Desai et al proposed to test individuals with breast cancer under 60 years.4 The sensitivity for detecting pathogenic variants decreased slightly when testing patients under 60 years compared with patients who were 65 years or younger, 98.1% versus 95.3% for BRCA1/2, respectively. The sensitivity for detection of pathogenic variants in 6 high-risk genes also decreased with age 60 years and under compared to 65 years and under, 94.8% versus 91%, respectively. For patients who received a breast cancer diagnosis who were over 60 years, the investigators concluded that using the NCCN family-based criteria would be appropriate. However, the investigators noted there might be an increased detection of variance of unknown significance (VUS) with this approach.

Next-Generation Sequencing

In addition to the controversy of patient selection for genetic testing, a lack of consensus exists regarding the number of genes that the testing panel should include. The introduction of next-generation sequencing has allowed genetic testing to become more accessible with a lower financial burden.6 There are over 170 breast cancer susceptibility variants identified based on the largest genome-wide association study.7

“Costs are coming down, but when people at low risk get genetic testing, we’re more likely to find things [such as] VUS, which are changes in the genetic code that we don’t understand, than we are to find something important. The risk-benefit ratio changes depending on [what] the risk is to begin with,” said Domchek.

A study using Surveillance, Epidemiology, and End Results (SEER) data evaluated 187,535 patients with breast cancer.8 However, only 25.2% of those patients had genetic testing analysis. This genetic testing demonstrated a prevalence of BRCA1/2 pathogenic variants in 5.2% of individuals, whereas other genetic variants (APC, CDH1, MLH1, MSH2, MSH6, NF1, PMS2, PTEN, RET, and TP53) were associated with an increased risk of breast cancer in 4.9% of individuals.

“We don’t do a great job of testing individuals who currently meet testing criteria, and we have a real problem with disparity. We want to make sure that people at the highest risk are being tested. That’s a clear priority,” stated Domchek.

Another study that evaluated the prevalence of germline pathogenic variants used a US-based consortium, Cancer Risk Estimates Related to Susceptibility Genes (CARRIERS).9 The analysis included 12 studies that were part of the population-based CARRIERS analysis with 32,247 case patients and 32,544 controls. There were 12 established breast cancer predisposition genes assessed (ATM, BARD1, BRCA1/2, CDH2, CHEK2, NF1, PALB2, PTEN, RAD51C, RAD51D, and TP53). The prevalence of germline pathogenic variants in the population-based analysis was 5.03% (95% CI, 4.79%-5.27%) in case patients and 1.33% (95% CI, 1.50%-1.78%) in controls (FIGURE).9 The highest rates of prevalence of pathogenic variants were seen with BRCA2, CHEK2, and BRCA1 genes.

Regarding the study, Domchek said, “If you look at 12 genes that are putatively associated with breast cancer, 5% of patients have mutations in those genes, but several of those [genes] are not clearly associated with risk.”

Results also determined pathogenic variants in BRCA1 (odds ratio [OR], 7.62; 95% CI,5.33-11.27) and BRCA2 (OR, 5.23; 95% CI, 4.09-6.77) were associated with high breast cancer risk. Moderate breast cancer risk was observed in pathogenic variants in PALB2 and CHEK2 (OR, 3.83; 95% CI, 2.68-5.63; and OR, 2.47; 95% CI, 2.02-3.05), respectively.

Furthermore, different pathogenic variants were associated with specific breast cancer subtypes. Pathogenic variants in BARD1, RAD51C, and RAD51D placed the individual at moderate risk for estrogen receptor (ER)– negative breast cancer and triple-negative breast cancer (TNBC). Conversely, pathogenic variants in ATM, CDH1, and CHEK2 were associated with ER-positive breast cancer. Additionally, there was a higher prevalence of pathogenic variants in BRCA1/2 and PALB2 observed with TNBC compared with ER-positive breast cancer, 8.13% versus 1.84%, respectively.

Breast Cancer Association Consortium

Another large study analyzed data from 44 studies in the Breast Cancer Association Consortium with 60,466 patients and 53,461 controls.10 For the purpose of population-based analysis, 48,826 patients and 50,703 controls were included from the total number of individuals in the study. Results showed pathogenic variants in 5 genes were associated with an increased risk of breast cancer (P <.0001). Additional pathogenic variants associated with high-risk breast cancer were found in BARD1, RAD51C, RAD51D, and TP53 (P <.05). There were also associations with pathogenic variants and different subtypes of breast cancer. As in the CARRIERS study, CHEK2 was associated with ER-positive compared with ER-negative breast cancer (OR, 2.67; 95% CI, 2.30-3.11; vs OR, 1.64; 95% CI, 1.25-2.16). Pathogenic variants in ATM were more frequently found in ER-positive than in ER-negative breast cancer (OR, 2.33; 95% CI, 1.87-2.91; vs OR, 1.01; 95% CI, 0.64-1.59). Additionally, there were more pathogenic variants (BARD1, BRCA1/2, PALB2, RAD51C, and RAD51D) associated with ER-negative compared with ER-positive breast cancer (P <.05).

The appropriate identification of patients and important pathogenic variants in genetic testing may lead to optimization of care in the age of precision medicine. The results from genetic testing may have implications for patients ranging from surgical, radiation, and targeted therapeutic intervention, as well as prevention strategies for patient family members.6 Patients with BRCA1/2 mutations with metastatic breast cancer may benefit from targeted therapy with PARP inhibitors.6,11

“In the early-stage [breast cancer] setting, we’re testing BRCA1/2 to talk about removal of the ovaries and the consideration for removal of the [contralateral] breast. The clearly actionable [genes] are BRCA1/2 and PALB2,” said Domchek.

A population-based cohort study evaluated pathogenic variants and clinical treatment pathways in patients with breast cancer using SEER registries in 20,568 individuals.11 Patients were nonexclusively stratified into 3 separate treatment subgroups including surgery, radiation, and chemotherapy. The results demonstrated an increased prevalence of bilateral mastectomy (n=15,126) and chemotherapy treatment (n= 8509) for individuals with pathogenic variants in BRCA1/2 or other genes (ATM, CDH1, CHEK2, NBN, NF1, PALB2, PTEN, and TP53). Individuals with BRCA1/2 and other genetic pathogenic variants were more likely to undergo bilateral mastectomy compared with those with VUS (OR, 5.52; 95% CI, 4.73-6.44; and OR, 2.41; 95% CI, 1.92-3.03; vs OR, 0.99; 95% CI, 0.89-1.1, respectively). Additionally, individuals with BRCA1/2 and other genetic pathogenic variants were more likely to receive chemotherapy treatment compared with those with VUS (OR, 1.76; 95% CI, 1.31-2.34; and OR, 1.27; 95% CI, 0.87-1.86; vs OR, 0.95; 95% CI, 0.81-1.11, respectively). There is currently no consensus on patient selection or gene selection.

“Our goal is to make sure that every candidate who is a good candidate for genetic testing gets tested regardless of their race, ethnicity, or socioeconomic status. Family history should be taken, and [patients] should know about the availability of testing and be counseled on their level of risk,” said Domchek. “This is the intersection among regulations, insurance coverage, and prior probability. I do think that genetic testing is only going to become more widespread, and our job is to make sure that patients understand what tests are being sent and the potential implications of the results.”


1. NCCN. Clinical Practice Guidelines in Oncology. Genetic/familial highrisk assessment: breast, ovarian, and pancreatic, version 2.2021. Accessed February 8, 2021.

2. BRCA-related cancer: risk assessment, genetic counseling, and genetic testing. US Preventive Services Task Force. August 20, 2019. Accessed February 8, 2020.

3. Manahan ER, Kuerer HM, Sebastian M, et al. Consensus guidelines on genetic testing for hereditary breast cancer from the American Society of Breast Surgeons. Ann Surg Oncol. 2019;26(10):3025-3031. doi:10.1245/ s10434-019-07549-8

4. Desai NV, Yadav S, Batalini F, Couch FJ, Tung NM. Germline genetic testing in breast cancer: rationale for the testing of all women diagnosed by the age of 60 years and for risk-based testing of those older than 60 years. Cancer. Published online November 4, 2020. doi:10.1002/cncr.33305

5. Yadav S, Hu C, Hart SN, et al. Evaluation of germline genetic testing criteria in a hospital-based series of women with breast cancer. J Clin Oncol. 2020;38(13):1409-1418. doi:10.1200/JCO.19.02190

6. Angeli D, Salvi S, Tedaldi G. Genetic predisposition to breast and ovarian cancers: how many and which genes to test? Int J Mol Sci. 2020;21(3):1128. doi:10.3390/ijms21031128

7. Zhang H, Ahearn TU, Lecarpentier J, et al. Genome-wide association study identifies 32 novel breast cancer susceptibility loci from overall and subtype-specific analyses. Nat Genet. 2020;52(6):572-581. doi:10.1038/ s41588-020-0609-2

8. Kurian AW, Morrow M, Katz S. Trends in genetic testing and results for women diagnosed with breast cancer or ovarian cancer, 2013-2017. Presented at: 2020 San Antonio Breast Cancer Symposium; December 8-12, 2020; virtual. Abstract PD10-01.

9. Hu C, Hart SN, Gnanaolivu R, et al. A population-based study of genes previously implicated in breast cancer. N Engl J Med. 2021;384(5):440- 451. doi:10.1056/NEJMoa2005936

10. Breast Cancer Association Consortium, Dorling L, Carvalho S, et al. Breast cancer risk genes - association analysis in more than 113,000 women. N Engl J Med. 2021;384(5):428-439. doi:10.1056/NEJMoa1913948

11. Kurian AW, Ward KC, Abrahamse P, et al. Association of germline genetic testing results with locoregional and systemic therapy in patients with breast cancer. JAMA Oncol. 2020;6(4):e196400. doi:10.1001/jamaoncol.2019.6400

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