The Perils of Single‐Site Genetic Testing for Hereditary Cancer Syndromes in the Era of Next‐Generation Sequencing

A challenge in counseling patients with a family history of hereditary cancer syndrome is deciding which genetic tests or panels to order. In some cases, expanded panels should be considered to provide a more complete assessment of a patient's genetic risk. The mutation discovered in the case reported in this article was in the ATM gene. The clinical significance of the mutation, potential therapeutic targets, and proper clinical management are discussed.

Abstract

A challenge in counseling patients with a family history suggesting a hereditary cancer syndrome is deciding which genetic tests or panels to order. In this article, we discuss the identification of multiple familial mutations through genetic counseling and panel testing. For patients meeting National Comprehensive Cancer Network criteria for clinical genetic testing, providers should consider expanded panels to provide a more complete assessment of one's genetic risk. The continued use of expanded panel testing in the clinical setting will help inform optimal management of cancer patients, as well as the management of their unaffected family members. The mutation discovered in this case was in the ATM gene. The clinical significance of the mutation, potential therapeutic targets, and proper clinical management are discussed.

Key Points.

• With single‐site genetic testing, there is the potential to miss hereditary genetic syndromes that can be managed clinically.

• Between 4% and 6% of hereditary breast and ovarian cancer syndromes are caused by genes other than BRCA1 and BRCA2.

• ATM is a DNA mismatch repair gene associated with double‐stranded DNA break repair and cell cycle checkpoint arrest.

• The risk of developing female breast cancer by age 50 and by age 80 in ATM heterozygotes is 9% and 17%–52%, respectively.

Patient Story

A 25‐year‐old white female with no pertinent past medical history presented to her obstetrician/gynecologist for an annual exam. She reported no health concerns and physical examination was unremarkable. A discussion of her extensive family history of cancer prompted a referral to the Ruth Paul Hereditary Cancer Program (RPHCP) at the George Washington University Cancer Center. She reported to the RPHCP in 2016 with a family history notable for a mother diagnosed with ductal carcinoma in situ and a BRCA1 Ashkenazi Jewish founder mutation at age 48, maternal grandmother diagnosed with breast cancer at age 61, paternal grandmother diagnosed with ovarian cancer at age 68, and a paternal grandfather diagnosed with colon cancer at age 38. Based on the pattern of malignancy and the known maternal familial mutation, the patient met the National Comprehensive Cancer Network (NCCN) guidelines for BRCA testing [1]. Additionally, because of the patient's paternal family history of malignancy, it was recommended that either the patient or her father undergo expanded panel testing. The patient's father underwent MyRisk testing at another institution. It revealed a PMS2 variant of uncertain significance (VUS) located at c.319C>T. With this information, the patient opted for BRCA testing only based on the known maternal mutation. A DNA sample was sent for Multisite 3 BRCAnalysis testing. The result was negative for the three Ashkenazi Jewish‐associated mutations in BRCA1 and BRCA2, including her mother's mutation.

In 2017, her father's PMS2 VUS was reclassified as a deleterious mutation. This decision was based on evidence that a proband with this PMS2 variant and a second PMS2 mutation developed constitutional mismatch repair deficiency (CMMRD). CMMRD occurs in patients with biallelic germline mutations in one of the mismatch repair genes: PMS2, MLH1, MSH2, or MSH6. In childhood, patients with this syndrome develop brain tumors, colorectal cancers, and hematologic malignancies [3]. Additionally, functional studies of PMS2 c.319C>T found mismatch repair deficiency, resulting in less than 20% relative repair efficiency [9]. As a result of this additional information, the patient reported back to RPHCP. RPHCP recommended PMS2 single‐site testing; however, her insurance would only cover expanded panel testing. The patient underwent MyRisk panel testing. She tested negative for her father's PMS2 mutation, but was found to be positive for a deleterious mutation in the ataxia telangiectasia mutated (ATM) gene located at c.5932G>T, one that had not been previously tested for in either parent.

The patient's younger sister also tested positive for the same ATM mutation and negative for both her mother's BRCA1 mutation and her father's PMS2 mutation. These results prompted the patient's mother to undergo expanded panel testing. She was found to carry the same ATM mutation located at c.5932G>T. The patient's older sister also underwent genetic testing and was found to carry the maternal ATM gene mutation and the paternal PMS2 mutation.

Germline Genotyping and Interpretation of Results

Germline genotype analysis was performed by Myriad Genetics, Inc. (Salt Lake City, UT) twice on the patient. Saliva samples were collected and mailed to the laboratory for polymerase chain reaction analysis of DNA extracted from the sample. The Multisite BRCAnalysis sequenced the patient's genome at the three sites associated with Ashkenazi Jewish founder mutations: BRCA1 c.68_69del, BRCA2 c.5946del, and most notably for her mother's mutation, BRCA1 c.5266dupC. The patient was negative for a pathogenic mutation at all sites, including the site of her mother's mutation. After her father's PMS2 VUS was reclassified to a pathogenic mutation, the patient returned to provide another saliva sample for MyRisk testing. This test analyzes 28 genes associated with an elevated risk for cancers such as colorectal, breast, and ovarian. The genes analyzed include the following: BRCA1, BRCA2, MLH1, MSH2, MSH6, PMS2, EPCAM, APC, MUTYH, CDKN2A, CDK4, TP53, PTEN, STK11, CDH1, BMPR1A, SMAD4, PALB2, CHEK2, ATM, NBN, BARD1, BRIP1, RAD51C, RAD51D, POLD1, POLE, and GREM. The test identified a pathogenic mutation in the ATM gene located at c.5932G>T. This mutation results in the premature truncation of the ATM protein at amino acid position 1978. This result indicated that the patient has a germline mutation rendering her heterozygous for function of the ATM protein. ATM and other genes associated with hereditary cancer syndromes are tumor suppressor genes or DNA mismatch repair genes. The protein products of these genes limit cell growth and proliferation and repair DNA mutations. The transformation of a cell requires the loss of function in both alleles for tumor suppressor and DNA mismatch repair genes. Patients with hereditary cancer syndromes have a higher incidence of cancer than the general population because the rate of somatic loss of a single allele is higher than the independent mutation of two alleles at the same locus [10]. Because this patient inherited a pathogenic mutation in the ATM gene, only one somatic mutation in her wild type ATM allele is required to develop cancer.

Functional Significance of ATM

ATM is located on chromosome 11q22‐23 and encodes a serine/threonine protein kinase that maintains genomic integrity. It plays an important role in double‐stranded DNA break (DSB) repair. The extensive network of cell signaling in DSB repair begins with MRE11‐RAD50‐NBS1 (MRN) complex sensing the DSB. The MRN complex recruits ATM to the site where it acts as a transducer: it recruits and cooperates with other sensor proteins such as 53BP1 and BRCA1, and it phosphorylates many downstream effector proteins [8]. ATM phosphorylates Chk1 and Chk2, which phosphorylate additional downstream proteins involved in cell‐cycle checkpoint arrest [8]. Phosphorylation of BRCA1 and RAD51 induces DNA repair through the homologous recombination (HR) pathway. Phosphorylation of p53 leads to apoptosis [8].

Studies have shown the risk of developing female breast cancer by age 50 and by age 80 in ATM heterozygotes is 9% and 17%–52%, respectively [11]. Some research suggests a higher lifetime risk of breast cancer in ATM missense mutations at 60% by age 80, compared with 30%–40% by age 80 for truncating mutations [11]. ATM mutations are also associated with an increased risk for pancreatic cancer; however, the exact risk is unknown. ATM homozygotes develop ataxia telangiectasia, a rare genetic disorder that causes immunodeficiency and progressive cerebellar neural degeneration in children between the ages of 1 and 5 years.

Multidisciplinary Hereditary Cancer Clinic

The patient was reviewed during a provider conference at the RPHCP's Multidisciplinary Clinic. They addressed the potential causes for the ATM mutation with a family history of a mother with a BRCA1 mutation and a father with a PMS2 mutation. The main considerations included (a) the mutation represents a de novo change in the ATM gene, (b) the ATM mutation indicates misattributed paternity, or (c) the ATM mutation was inherited from her mother, who had not yet undergone panel testing. She was then seen by an oncologist, a breast surgeon, and a primary care physician and was counseled based on the 2017 NCCN Guidelines for ATM, which include annual mammography and consideration of breast MRI starting at age 40 [1]. Although there is insufficient evidence for guidelines on pancreatic cancer screening, the patient was counseled to report any new familial pancreatic cancer diagnoses to the medical oncologist for consideration of possible pancreatic cancer screening strategies at that time. She was advised to meet with an RPHCP team member annually for review of recommendations related to her ATM mutation.

Implications for Clinical Practice and Potential Strategies to Target the Pathway

In heterozygous ATM mutation carriers and carriers of other moderate penetrance breast cancer susceptibility genes, it is important to follow patients closely with enhanced clinical breast awareness beginning at age 25 and to receive continued updates of family medical history. Beginning at age 40, ATM mutation carriers should undergo annual mammography and consider breast magnetic resonance imaging (MRI). ATM mutation carriers with strong family medical histories of breast cancer may consider risk‐reducing mastectomies with their providers. Additionally, 1% of the U.S. population is estimated as heterozygous for a mutation in the ATM gene [4]. Therefore, these patients must be counselled on the development of the autosomal recessive condition of ataxia telangiectasia in offspring and reproductive options [1].

If an ATM mutation carrier develops breast cancer, in vitro studies have indicated that cells with ATM mutations are susceptible to poly (ADP‐ribose) polymerase (PARP) inhibitors, similar to cells with BRCA1 and BRCA 2 mutations. PARP is an important mediator of the base excision repair (BER) pathway, which repairs single‐stranded DNA breaks. The inhibition of PARP in cells that are BRCA deficient results in a large number of mutations that would normally be repaired by HR [8]. Dysfunction of BER and HR is lethal to cells, proving PARP inhibition may be a successful treatment strategy in cancers with BRCA and/or ATM mutations. Another potential therapeutic target includes the ATR‐checkpoint kinase 1, a mediator of single‐stranded DNA repair [12]. Inhibition of ATR in cancer cell lines showed enhanced activity in those with ATM deficiency [12]. Further research in ATM‐deficient cell lines in patients with cancer needs to be initiated to determine if these promising preclinical results can be applied to therapeutic management.

Figure 1.

Family pedigree. This pedigree displays the patient's family history of cancer, age of diagnosis, and identified individual gene mutations.

Figure 2.

ATM function in the repair of double‐stranded DNA breaks. First, the MRN complex, composed of MRE11, RAD50, and NBS1, senses the DNA DSB. The MRN complex recruits ATM to the site where it acts as a transducer: it recruits and cooperates with other sensor proteins such as 53BP1 and BRCA1. ATM also phosphorylates downstream effector proteins. Phosphorylation of BRCA1 and RAD51 induces DNA repair through the homologous recombination pathway. Phosphorylation of Chk1 and Chk2, allows them to phosphorylate additional downstream proteins involves in cell‐cycle checkpoint arrest. Phosphorylation of p53 leads to apoptosis [2], [4].

Abbreviation: DSB, double‐stranded DNA break.

Patient Update

In the case of this 25‐year‐old newly identified ATM truncating mutation carrier, she is followed by a breast oncologist once yearly and continues contact with the RPHCP for updates regarding care guidelines for her mutation. Additional surveillance and risk reduction strategies will be offered as the patient ages and guidelines change. Her family members are seeking care from other hereditary cancer programs in proximity to where they live.

Conclusion

This case demonstrates the power of hereditary cancer screening with next‐generation sequencing. Before the development of panel testing, this patient and her family members would have only received testing for BRCA1/2 mutations. Upon receiving their negative result, they would be instructed to follow the screening guidelines recommended for the general population. Currently, it is common for patients and some providers to opt for single‐site testing, especially in the context of the identification of one positive pathogenic mutation in a family member. However, with single‐site testing, the possibility of a double mutation carrier in the family can be overlooked. In this case, if conventional knowledge was followed and single‐site testing was performed a second time, the familial ATM mutation would not have been identified. A study of 80,829 patients who underwent panel testing found that 7.1% had a single pathogenic mutation and 0.19% had multiple pathogenic mutations [5]. Although there are only a small percentage of identified double mutation carriers, they are more likely to be affected by cancer [5]. Therefore, it is important to determine double mutation carrier status to inform increased surveillance and guide treatment. With the increased use of expanded panel testing, it is essential to question patients with known familial mutations regarding the type of testing the familial mutation carriers received. This case also highlights the potential shortcomings of single‐site testing in the age of next‐generation sequencing.

The use of expanded panel testing in this case also allowed for the exploration of moderate‐risk breast cancer susceptibility genes. Knowing their mutation status, the patient and her sisters have awareness of their increased risk for breast cancer and will follow the screening guidelines for ATM mutation carriers as they continue to evolve. Studies have estimated the prevalence of mutations in genes other than BRCA1 and BRCA2 in DNA sequencing for hereditary breast and ovarian cancer screening to be 4%–6% [5]. These data suggest a significant number of individuals with breast cancer are affected by genes other than BRCA1 and BRCA2. For patients meeting NCCN criteria for clinical genetic testing, providers should consider expanded panels to provide a more complete assessment of one's genetic risk. The continued use of expanded panel testing in the clinical setting will inform optimal management of cancer patients, as well as the management of their unaffected family members.

Glossary of Genomic Terms and Nomenclature

Sequencing: determining the exact order of the bases in a strand of DNA

Common Types of Gene Mutations:

Missense mutation: change in one DNA base pair that results in the substitution of one amino acid for another in the protein made by a gene

Nonsense mutation: change in one DNA base pair that results in the substitution of one amino acid with a stop codon resulting in premature termination of the protein

Deletion: mutation resulting in loss of any number of nucleotides

Insertion: mutations resulting in gain of any number of nucleotide

Frame shift mutations: addition or loss of DNA bases changing a gene's reading frame, shifting the grouping of the bases forming codons, and thereby changing the code for amino acids; resulting protein is usually truncated and nonfunctional

Tumor suppressor gene: a type of gene that makes a protein called a tumor suppressor protein that helps control cell growth; mutations (changes in DNA) in tumor suppressor genes may lead to cancer

Germline genetic testing: DNA sequencing to evaluate inherited genetic changes in genes known to increase the risk of certain cancers

Author Contributions

Conception/design: Nicole Casasanta, Rebecca Kaltman

Provision of study material or patients: Nicole Casasanta, Elizabeth Stark, Rebecca Kaltman

Collection and/or assembly of data: Nicole Casasanta, Elizabeth Stark, Rebecca Kaltman

Data analysis and interpretation: Nicole Casasanta, Elizabeth Stark, Allison McHenry, Tara Biagi, Rebecca Kaltman

Manuscript writing: Nicole Casasanta, Elizabeth Stark, Allison McHenry, Tara Biagi, Rebecca Kaltman

Final approval of manuscript: Nicole Casasanta, Elizabeth Stark, Allison McHenry, Tara Biagi, Rebecca Kaltman

Disclosures

The authors indicated no financial relationships.