Lynch syndrome, also known as hereditary non-polyposis colorectal cancer (HNPCC), is one of the most common inherited cancer syndromes associated with a genetic predisposition to different cancer types, especially in the colon. Alterations in several genes involved in DNA mismatch repair (e.g., MLH1, MSH2, MSH6, PMS2 and EPCAM) have been linked to Lynch syndrome. Women with pathogenic mutations in these genes also have an increased risk of developing endometrial and ovarian cancers, while the risk of breast cancer is still controversial.
We report the case of a 29-year old woman, diagnosed with luminal A, invasive breast carcinoma. Due to the early onset of cancer and a grandfather diagnosed with cancer, a BRCA1/2 diagnostic test was performed and turned out negative. A multigene panel testing (Cancer Risk Test for 30 Hereditary Breast and Ovarian – HBOC – associated cancers) identified a pathogenic heterozygous variant in the PMS2 gene, compatible with a clinical diagnosis of Lynch syndrome. Since this mutated gene is associated with an increased risk of colorectal, uterine and ovarian cancers, a screening program for each of these types of cancers should be recommended along with a follow-up program for breast cancer. Individual and familial genetic counseling must be considered as recent data brings new information on PMS2 gene mutations and Lynch syndrome-related breast cancer risk; mutations in this gene are predictive molecular markers not only for colon and endometrial cancer but also for breast cancer. Multigene panel testing is an important molecular diagnostic tool for Hereditary Cancer Syndromes (HCS) and therefore for individual and familial oncologic management.
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2. Case Report
Lynch syndrome, also described as hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant inherited cancer susceptibility disorder with a prevalence of 1 in 370 to 1 in 3,000 individuals , .
It is caused by germline mutations in the DNA mismatch repair (MMR) genes: MLH1, MSH2, MSH6, PMS2 and EPCAM. Individuals carrying pathogenic germline mutations in these genes typically exhibit DNA microsatellite instability (MSI) and loss of MMR proteins expression and therefore have an increased risk for colon, rectum, endometrium, stomach, ovary, renal pelvis, ureter, brain and small bowel cancer , . Lynch syndrome-associated breast cancer was an issue initially raised by Lynch et al.,  several decades ago and since then, studies have provided evidence both for and against breast cancer being part of Lynch syndrome , , , , .
2. Case Report
We report the case of a 29-year old woman, referred to the Oncology clinic for a lump in her right breast. The ultrasound revealed a suspect lesion for breast malignancy and subsequent needle biopsy confirmed the diagnosis. The histopathological examination identified a luminal A (ER/PR positive, HER2 negative, Ki67=40%), cT2N1M0 stage IIb, invasive breast carcinoma. The patient underwent neoadjuvant chemotherapy, breast-conserving surgery and lymphadenectomy, followed by radiotherapy .
Breast cancer is rarely diagnosed before 40 years of age, so an early onset of the malignancy suggests a possible genetic susceptibility; therefore the patient was recommended for genetic counseling. After informed and written consent was obtained from the patient, germline mutations in BRCA1 and BRCA2 genes were analyzed using targeted sequencing (AmpliSeq™ BRCA Panel for Illumina®, Thermo Fisher Scientific, Inc., Waltham, MA, USA). The result of the test was negative as no pathogenic variants were identified in both BRCA genes.
Considering that the patient had a grandfather diagnosed with cancer at an early age and the BRCA negative status, an additional, more complex Hereditary Cancer Syndrome (HCS) test was indicated. Next Generation Sequencing (NGS) analysis, which detects both sequence variants and deletions-duplications, was performed for 30 genes involved in the development of breast, ovary, uterus, colon, stomach, pancreas, skin (melanoma) and prostate cancers (i.e., BRCA1, BRCA2, APC, ATM, BAP1, BARD1, BMPR1A, BRIP1, CDH1, CDKN2A, CDK4, CHEK2, EPCAM, GREM1, MLH1, MSH2, MSH6, MITF, MUTYH, NBN, PALB2, PMS2, POLD1, POLE, PTEN, RAD51C, RAD51D, SMAD4, STK11 and TP53 genes) using Cancer Risk Test (Gendia, Belgium). Coding exons of all genes were analyzed, with the following exceptions: for the PMS2 gene – exons 12-15 were not analyzed; for the EPCAM gene – only 3’ and deletions and duplications can be detected; for the GREM1 gene – only upstream regulatory region duplications can be detected; for the MITF1 gene – only position c.952 was analyzed; for the POLD1 geneʼ only position c.1433 was analyzed, and for the POLE gene – only position c.1270 was analyzed. Variants identified by NGS were later confirmed by Sanger sequencing. The reference sequence and exon numbering are according to the GenBank accession numbers, with the A of the ATG start codon in position 1.
The analysis identified a pathogenic heterozygous variant of the PMS2 gene: NM_000535.6 (PMS2):c.1076dupT (p.Leu359Phefs). This finding is compatible with the clinical diagnosis of Lynch syndrome and is associated with an increased risk for colorectal, uterine and ovarian cancer, and with a lower risk for renal and small bowel cancer. All risks are lower than the risks associated with other Lynch syndrome genes.
The PMS2 gene is a tumor suppressor gene, involved in DNA repair mechanism working together with the MLH1 gene to remove and repair DNA errors when signaled by the MSH2 and MSH6 genes. Heterozygous variants in the PMS2 gene are involved in Lynch syndrome and homozygous or compound heterozygous variants are involved in Constitutional Mismatch Repair (CMR) deficiency, which is associated with childhood cancers. The PMS2 c.1076dupT variant causes a frameshift defect resulting in a premature stop codon (PMS2: p.Leu359PhefsX6). The variant may result in a truncated protein or diminished mRNA due to mRNA decay and it is listed in the ClinVar database as pathogenic.
Due to this finding, a screening program for colorectal, uterine and ovarian cancer is recommended. The National Comprehensive Cancer Network (NCCN) has published practice guidelines for the management of individuals with Lynch syndrome . As heterozygous pathogenic PMS2 variants are associated with lower risks with other Lynch syndrome genes it is not completely clear what the best surveillance is for PMS2 variant carriers.
Following genetic counseling, the patient was recommended screening for colorectal cancer as PMS2 gene mutations have an 11-15% risk for developing colorectal tumors. Consequently, colonoscopy has to be performed every 3 years and optionally, if required, liquid biopsy testing (Circulating Tumor/CT-DNA tests). Another prophylactic measure would be chemoprevention using NSAIDs, so in this case, daily intake of Aspirin was recommended. As PMS2 gene mutations are associated with a 12-15% risk for uterine cancer by the age of 70 compared to the general risk of 1.7%, repeated transvaginal ultrasounds and endometrial biopsies are recommended. Hysterectomy might be suggested after childbearing is no longer needed. As PMS2-associated ovarian, renal pelvis and small bowel cancers risks are not precisely known, current protocols are not clear on screening recommendations for these types of cancers.
Repeated abdominal ultrasounds, evaluation of biomarkers such as CA125, and liquid biopsies may be indicated for ovarian cancer screening. When there is no further wish for children and certainly after menopause, surgical removal of the ovaries and fallopian tubes might be suggested. No further screening is recommended for other types of cancers , , .
Considering the diagnosis of breast cancer, bilateral breast cancer might occur, especially since recently it has been suggested that variants in the PMS2 gene are also associated with an increased risk of breast cancer. Therefore, the patient is recommended a mammogram every 6 months or MRI imaging, if necessary. Prophylactic bilateral mastectomy could also be considered, after subsequent surgical assessment.
Molecular studies based upon the hypothesis that MMR gene mutations might induce molecular instability in breast cancer tissue, both as drivers but also as accelerators of the malignant phenotype, support the theory that they could also increase the risk for breast cancer , , . It was demonstrated that MMR-deficient breast cancers were more likely to present poorly differentiated histology, a higher mitotic rate, peritumoral inflammatory response with lymphocytes, confluent necrosis, and more likely to be estrogen and progesterone receptor-negative than the MMR-proficient breast cancers .
In a recent study, researchers analyzed a database of more than 50,000 women who had undergone multi-gene hereditary cancer testing between 2013 and 2015. Interestingely, 423 women had a mutation in one of the four genes causing Lynch syndrome: MLH1, MSH2, MSH6 and PMS2. Further additional analyses revealed that women with pathogenic mutations in MSH6 and PMS2 genes had a two-fold higher risk of breast cancer compared to women in the general population. The conclusion of this study was that based on the incidence of cancer in the study population, 31 to 38% of women carriers for MSH6 and PMS2 gene mutations develop breast cancer, compared to 15% of women in the general population.
The women with mutations in MSH6 and PMS2 genes were more likely to meet BRCA1/2 criteria than Amsterdam criteria, and 11% had no personal or family history of the cancers traditionally associated with Lynch syndrome. This study has also important implications for cancer genetic testing and if further studies confirm the findings of this paper, it may become routine to offer testing for Lynch syndrome genes to families with a positive history of breast cancer, using multigene testing panel including Hereditary Breast Cancer and Lynch syndrome specific genes. Although it is still early to draw conclusions, another study supports the same hypothesis as it demonstrates that pathogenic variants in MSH6 or PMS2 genes are associated with a modest but statistically significant increased risk fo r breast cancer .
One of the most important and difficult tasks for the genetic counselors is to analyze and interpret a patient’s family history and oncopathogenic-linked variants. In cancer counseling, breast and ovarian familial cancers are most often associated to BRCA1/2 mutations, whereas colon and endometrial cancers are linked to Lynch syndrome and MMR genes. However, new studies suggests that pathogenic mutations in MSH6 and PMS2 genes, the Lynch syndrome genes, may also lead to increased risk for breast cancer and thus requiring additional screening measures for breast cancer. Unfortunately, current guidelines for management of MMR mutated carriers from the NCCN are indistinct, frequently evolving, and only include the most prominent of these breast cancer susceptibility genes.
As we learn more about the implications of more moderate-risk genes, the use of multigene panels will likely become routine testing, allowing a better identification of individuals at risk and will likely allow better predictions of cancer risks and therefore proper genetic counseling and preventive options.
In the absence of specific guidelines for non-BRCA genetic variants and to avoid possible “excessive” screening practices in the general population, the best option would be that women with PMS2 mutations or other Lynch syndrome-associated genes are provided with individualized management, by taking into account the medical, emotional and material benefits and disadvantages of breast cancer screening . In the next years, advances in genomics should be translated into a more accurate cancer risk estimates for non-carriers. As for carrier women, further studies will be needed to foster the development of interventions and tools that will allow for the optimal use of screening strategies by BRCA1/2-negative women in accordance with their genetic status and family history .
Currently, testing for Lynch syndrome genes is generally performed in case of positive personal or family history for colon or uterine cancer.
As new data suggests that MSH6 and PMS2 genes are associated with a two to three-fold increased risk of breast cancer compared to the general population, women with a personal or family history of breast cancer may want to consider multigene panel testing that includes the Lynch syndrome genes.
DRONCA Eleonora 
ACHIMAȘ CADARIU Patriciu 
MILITARU Mariela 
MARTIN Daniela 
CIUMĂRNEAN Lorena 
CĂTANĂ Andreea 
 Iuliu Hațieganu University of Medicine and Pharmacy, Cluj-Napoca (ROMANIA).
 Ioan Chiricuță Oncology Institute, Cluj-Napoca (ROMANIA).