Functions of Breast Cancer Predisposition Genes: Implications for Clinical Management

Approximately 5–10% of all breast cancer (BC) cases are caused by germline pathogenic variants (GPVs) in various cancer predisposition genes (CPGs). The most common contributors to hereditary BC are BRCA1 and BRCA2, which are associated with hereditary breast and ovarian cancer (HBOC). ATM, BARD1, CHEK2, PALB2, RAD51C, and RAD51D have also been recognized as CPGs with a high to moderate risk of BC. Primary and secondary cancer prevention strategies have been established for HBOC patients; however, optimal preventive strategies for most hereditary BCs have not yet been established. Most BC-associated CPGs participate in DNA damage repair pathways and cell cycle checkpoint mechanisms, and function jointly in such cascades; therefore, a fundamental understanding of the disease drivers in such cascades can facilitate the accurate estimation of the genetic risk of developing BC and the selection of appropriate preventive and therapeutic strategies to manage hereditary BCs. Herein, we review the functions of key BC-associated CPGs and strategies for the clinical management in individuals harboring the GPVs of such genes.     

Molecular Features and Clinical Management of Hereditary Pancreatic Cancer Syndromes and Familial Pancreatic Cancer

Hereditary pancreatic cancers are caused by several inherited genes. Familial pancreatic cancer is defined as pancreatic cancer arising in a patient with at least two first-degree relatives with pancreatic cancer in the absence of an identified genetic cause. Hereditary pancreatic cancer syndromes and familial pancreatic cancers account for about 10% of pancreatic cancer cases. Germline mutations in BRCA1, BRCA2, ATM, PALB2, CDKN2A, STK11, and TP53 and mismatch repair genes (MLH1, MSH2, MSH6, PMS2, and EPCAM) are among the well-known inherited susceptibility genes. Currently available targeted medications include poly (ADP-ribose) polymerase inhibitors (PARP) for cases with mutant BRCA and immune checkpoint inhibitors for cases with mismatch repair deficiency. Loss of heterozygosity of hereditary pancreatic cancer susceptibility genes such as BRCA1/2 plays a key role in carcinogenesis and sensitivity to PARP inhibitors. Signature 3 identified by whole genome sequencing is also associated with homologous recombination deficiency and sensitivity to targeted therapies. In this review, we summarize molecular features and treatments of hereditary pancreatic cancer syndromes and surveillance procedures for unaffected high-risk cases. We also review transgenic murine models to gain a better understanding of carcinogenesis in hereditary pancreatic cancer.     

Lynch Syndrome: Its Impact on Urothelial Carcinoma

Lynch syndrome, known as hereditary nonpolyposis colorectal cancer (HNPCC), is an autosomal-dominant familial cancer syndrome with an increased risk for urothelial cancer (UC). Mismatch repair (MMR) deficiency, due to pathogenic variants in MLH1, MSH2, MSH6, and PMS2, and microsatellite instability, are known for development of Lynch syndrome (LS) associated carcinogenesis. UC is the third most common cancer type in LS-associated tumors. The diversity of germline variants in the affected MMR genes and their following subsequent function loss might be responsible for the variation in cancer risk, suggesting an increased risk of developing UC in MSH2 mutation carriers. In this review, we will focus on LS-associated UC of the upper urinary tract (UUT) and bladder, their germline profiles, and outcomes compared to sporadic UC, the impact of genetic testing, as well as urological follow-up strategies in LS. In addition, we present a case of metastatic LS-associated UC of the UUT and bladder, achieving complete response during checkpoint inhibition since more than 2 years.     

Classification of MSH6 Variants of Uncertain Significance Using Functional Assays

Lynch syndrome (LS) is one of the most common hereditary cancer predisposition syndromes worldwide. Individuals with LS have a high risk of developing colorectal or endometrial cancer, as well as several other cancers. LS is caused by autosomal dominant pathogenic variants in one of the DNA mismatch repair (MMR) genes MLH1, MSH2, PMS2 or MSH6, and typically include truncating variants, such as frameshift, nonsense or splicing variants. However, a significant number of missense, intronic, or silent variants, or small in-frame insertions/deletions, are detected during genetic screening of the MMR genes. The clinical effects of these variants are often more difficult to predict, and a large fraction of these variants are classified as variants of uncertain significance (VUS). It is pivotal for the clinical management of LS patients to have a clear genetic diagnosis, since patients benefit widely from screening, preventive and personal therapeutic measures. Moreover, in families where a pathogenic variant is identified, testing can be offered to family members, where non-carriers can be spared frequent surveillance, while carriers can be included in cancer surveillance programs. It is therefore important to reclassify VUSs, and, in this regard, functional assays can provide insight into the effect of a variant on the protein or mRNA level. Here, we briefly describe the disorders that are related to MMR deficiency, as well as the structure and function of MSH6. Moreover, we review the functional assays that are used to examine VUS identified in MSH6 and discuss the results obtained in relation to the ACMG/AMP PS3/BS3 criterion. We also provide a compiled list of the MSH6 variants examined by these assays. Finally, we provide a future perspective on high-throughput functional analyses with specific emphasis on the MMR genes.     

Identification of Spliceogenic Variants beyond Canonical GT-AG Splice Sites in Hereditary Cancer Genes

Pathogenic/likely pathogenic variants in susceptibility genes that interrupt RNA splicing are a well-documented mechanism of hereditary cancer syndromes development. However, if RNA studies are not performed, most of the variants beyond the canonical GT-AG splice site are characterized as variants of uncertain significance (VUS). To decrease the VUS burden, we have bioinformatically evaluated all novel VUS detected in 732 consecutive patients tested in the routine genetic counseling process. Twelve VUS that were predicted to cause splicing defects were selected for mRNA analysis. Here, we report a functional characterization of 12 variants located beyond the first two intronic nucleotides using RNAseq in APC, ATM, FH, LZTR1, MSH6, PALB2, RAD51C, and TP53 genes. Based on the analysis of mRNA, we have successfully reclassified 50% of investigated variants. 25% of variants were downgraded to likely benign, whereas 25% were upgraded to likely pathogenic leading to improved clinical management of the patient and the family members.     

Homologous Recombination Deficiencies and Hereditary Tumors

Homologous recombination (HR) is a vital process for repairing DNA double-strand breaks. Germline variants in the HR pathway, comprising at least 10 genes, such as BRCA1, BRCA2, ATM, BARD1, BRIP1, CHEK2, NBS1(NBN), PALB2, RAD51C, and RAD51D, lead to inherited susceptibility to specific types of cancers, including those of the breast, ovaries, prostate, and pancreas. The penetrance of germline pathogenic variants of each gene varies, whereas all their associated protein products are indispensable for maintaining a high-fidelity DNA repair system by HR. The present review summarizes the basic molecular mechanisms and components that collectively play a role in maintaining genomic integrity against DNA double-strand damage and their clinical implications on each type of hereditary tumor.     

Detection of Germline Variants in 450 Breast/Ovarian Cancer Families with a Multi-Gene Panel Including Coding and Regulatory Regions

With the progress of sequencing technologies, an ever-increasing number of variants of unknown functional and clinical significance (VUS) have been identified in both coding and non-coding regions of the main Breast Cancer (BC) predisposition genes. The aim of this study is to identify a mutational profile of coding and intron-exon junction regions of 12 moderate penetrance genes (ATM, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53) in a cohort of 450 Italian patients with Hereditary Breast/Ovarian Cancer Syndrome, wild type for germline mutation in BRCA1/2 genes. The analysis was extended to 5′UTR and 3′UTR of all the genes listed above and to the BRCA1 and BRCA2 known regulatory regions in a subset of 120 patients. The screening was performed through NGS target resequencing on the Illumina platform MiSeq. 8.7% of the patients analyzed is carriers of class 5/4 coding variants in the ATM (3.6%), BRIP1 (1.6%), CHEK2 (1.8%), PALB2 (0.7%), RAD51C (0.4%), RAD51D (0.4%), and TP53 (0.2%) genes, while variants of uncertain pathological significance (VUSs)/class 3 were identified in 9.1% of the samples. In intron-exon junctions and in regulatory regions, variants were detected respectively in 5.1% and in 32.5% of the cases analyzed. The average age of disease onset of 44.4 in non-coding variant carriers is absolutely similar to the average age of disease onset in coding variant carriers for each proband’s group with the same cancer type. Furthermore, there is not a statistically significant difference in the proportion of cases with a tumor onset under age of 40 between the two groups, but the presence of multiple non-coding variants in the same patient may affect the aggressiveness of the tumor and it is worth underlining that 25% of patients with an aggressive tumor are carriers of a PTEN 3′UTR-variant. This data provides initial information on how important it might be to extend mutational screening to the regulatory regions in clinical practice.     

Characteristics of BRCA2 Mutated Prostate Cancer at Presentation

Genetic alterations of DNA repair genes, particularly BRCA2 in patients with prostate cancer, are associated with aggressive behavior of the disease. It has reached consensus that somatic and germline tests are necessary when treating advanced prostate cancer patients. Yet, it is unclear whether the mutations are associated with any presenting clinical features. We assessed the incidences and characteristics of BRCA2 mutated cancers by targeted sequencing in 126 sets of advanced prostate cancer tissue sequencing data. At the time of diagnosis, cT3/4, N1 and M1 stages were 107 (85%), 54 (43%) and 35 (28%) samples, respectively. BRCA2 alterations of clinical significance by AMP/ASCO/CAP criteria were found in 19 of 126 samples (15.1%). The BRCA2 mutated cancer did not differ in the distributions of TNM stage, Gleason grade group or histological subtype compared to BRCA2 wild-type cancers. Yet, they had higher tumor mutation burden, and higher frequency of ATM and BRCA1 mutations (44% vs. 10%, p = 0.002 and 21% vs. 4%, p = 0.018, respectively). Of the metastatic subgroup (M1, n = 34), mean PSA was significantly lower in BRCA2 mutated cancers than wild-type (p = 0.018). In the non-metastatic subgroup (M0, n = 64), PSA was not significantly different (p = 0.425). A similar trend was noted in multiple metastatic prostate cancer public datasets. We conclude that BRCA2 mutated metastatic prostate cancers may present in an advanced stage with relatively low PSA.     

Localization of MLH3 at the Centrosomes

Mutations in human DNA mismatch repair (MMR) genes are commonly associated with hereditary nonpolyposis colorectal cancer (HNPCC). MLH1 protein heterodimerizes with PMS2, PMS1, and MLH3 to form MutLα, MutLβ, and MutLγ, respectively. We reported recently stable expression of GFP-linked MLH3 in human cell lines. Monitoring these cell lines during the cell cycle using live cell imaging combined with confocal microscopy, we detected accumulation of MLH3 at the centrosomes. Fluorescence recovery after photobleaching (FRAP) revealed high mobility and fast exchange rates at the centrosomes as it has been reported for other DNA repair proteins. MLH3 may have a role in combination with other repair proteins in the control of centrosome numbers.     

Breast Cancer Predisposition Genes and Synthetic Lethality

BRCA1 and BRCA2 are tumor suppressor genes with pivotal roles in the development of breast and ovarian cancers. These genes are essential for DNA double-strand break repair via homologous recombination (HR), which is a virtually error-free DNA repair mechanism. Following BRCA1 or BRCA2 mutations, HR is compromised, forcing cells to adopt alternative error-prone repair pathways that often result in tumorigenesis. Synthetic lethality refers to cell death caused by simultaneous perturbations of two genes while change of any one of them alone is nonlethal. Therefore, synthetic lethality can be instrumental in identifying new therapeutic targets for BRCA1/2 mutations. PARP is an established synthetic lethal partner of the BRCA genes. Its role is imperative in the single-strand break DNA repair system. Recently, Olaparib (a PARP inhibitor) was approved for treatment of BRCA1/2 breast and ovarian cancer as the first successful synthetic lethality-based therapy, showing considerable success in the development of effective targeted cancer therapeutics. Nevertheless, the possibility of drug resistance to targeted cancer therapy based on synthetic lethality necessitates the development of additional therapeutic options. This literature review addresses cancer predisposition genes, including BRCA1, BRCA2, and PALB2, synthetic lethality in the context of DNA repair machinery, as well as available treatment options.     

Beyond BRCA: The Emerging Significance of DNA Damage Response and Personalized Treatment in Pancreatic and Prostate Cancer Patients

The BRCA1/2 germline and/or somatic pathogenic variants (PVs) are key players in the hereditary predisposition and therapeutic response for breast, ovarian and, more recently, pancreatic and prostate cancers. Aberrations in other genes involved in homologous recombination and DNA damage response (DDR) pathways are being investigated as promising targets in ongoing clinical trials. However, DDR genes are not routinely tested worldwide. Due to heterogeneity in cohort selection and dissimilar sequencing approaches across studies, neither the burden of PVs in DDR genes nor the prevalence of PVs in genes in common among pancreatic and prostate cancer can be easily quantified. We aim to contextualize these genes, altered in both pancreatic and prostate cancers, in the DDR process, to summarize their hereditary and somatic burden in different studies and harness their deficiency for cancer treatments in the context of currently ongoing clinical trials. We conclude that the inclusion of DDR genes, other than BRCA1/2, shared by both cancers considerably increases the detection rate of potentially actionable variants, which are triplicated in pancreatic and almost doubled in prostate cancer. Thus, DDR alterations are suitable targets for drug development and to improve the outcome in both pancreatic and prostate cancer patients. Importantly, this will increase the detection of germline pathogenic variants, thereby patient referral to genetic counseling.     

Ovarian Cancer: In Search of Better Marker Systems Based on DNA Repair Defects

Ovarian cancer is the fifth most common female cancer in the Western world, and the deadliest gynecological malignancy. The overall poor prognosis for ovarian cancer patients is a consequence of aggressive biological behavior and a lack of adequate diagnostic tools for early detection. In fact, approximately 70% of all patients with epithelial ovarian cancer are diagnosed at advanced tumor stages. These facts highlight a significant clinical need for reliable and accurate detection methods for ovarian cancer, especially for patients at high risk. Because CA125 has not achieved satisfactory sensitivity and specificity in detecting ovarian cancer, numerous efforts, including those based on single and combined molecule detection and “omics” approaches, have been made to identify new biomarkers. Intriguingly, more than 10% of all ovarian cancer cases are of familial origin. BRCA1 and BRCA2 germline mutations are the most common genetic defects underlying hereditary ovarian cancer, which is why ovarian cancer risk assessment in developed countries, aside from pedigree analysis, relies on genetic testing of BRCA1 and BRCA2. Because not only BRCA1 and BRCA2 but also other susceptibility genes are tightly linked with ovarian cancer-specific DNA repair defects, another possible approach for defining susceptibility might be patient cell-based functional testing, a concept for which support came from a recent case-control study. This principle would be applicable to risk assessment and the prediction of responsiveness to conventional regimens involving platinum-based drugs and targeted therapies involving poly (ADP-ribose) polymerase (PARP) inhibitors.     

Lynch Syndrome Identification in Saudi Cohort of Endometrial Cancer Patients Screened by Universal Approach

Lynch syndrome (LS) is the most common cause of inherited endometrial cancer (EC). The prevalence and molecular characteristic of LS in Middle Eastern women with EC have been underexplored. To evaluate the frequency of LS in a cohort of EC patients from Saudi Arabia, a total of 436 EC cases were screened utilizing immunohistochemistry (IHC), MLH1 promoter methylation analysis and next-generation sequencing technology. A total of 53 of 436 (12.2%) ECs were classified as DNA mismatch repair-deficient (dMMR). MLH1 promoter hypermethylation was detected in 30 ECs (6.9%). Three ECs (0.7%) were found to be LS harboring germline pathogenic variants (PVs)/likely pathogenic variants (LPVs): two in the MSH2 gene and one in the MSH6 gene. Three ECs (0.7%) were Lynch-like syndrome (LLS) carrying double somatic MSH2 PVs/LPVs. Seven cases were found to have variants of uncertain significance in cancer-related genes other than MMR genes. Our results indicate that LS prevalence is low among Saudi EC patients and LLS is as common as LS in this ethnicity. Our findings could help in better understanding of the prevalence and mutational spectrum of this syndrome in Saudi Arabia, which may help in defining best strategies for LS identification, prevention and genetic counseling for EC patients.     

Gene Mutation Profiles in Primary Diffuse Large B Cell Lymphoma of Central Nervous System: Next Generation Sequencing Analyses

The existence of a potential primary central nervous system lymphoma-specific genomic signature that differs from the systemic form of diffuse large B cell lymphoma (DLBCL) has been suggested, but is still controversial. We investigated 19 patients with primary DLBCL of central nervous system (DLBCL CNS) using the TruSeq Amplicon Cancer Panel (TSACP) for 48 cancer-related genes. Next generation sequencing (NGS) analyses have revealed that over 80% of potentially protein-changing mutations were located in eight genes (CTNNB1, PIK3CA, PTEN, ATM, KRAS, PTPN11, TP53 and JAK3), pointing to the potential role of these genes in lymphomagenesis. TP53 was the only gene harboring mutations in all 19 patients. In addition, the presence of mutated TP53 and ATM genes correlated with a higher total number of mutations in other analyzed genes. Furthermore, the presence of mutated ATM correlated with poorer event-free survival (EFS) (p = 0.036). The presence of the mutated SMO gene correlated with earlier disease relapse (p = 0.023), inferior event-free survival (p = 0.011) and overall survival (OS) (p = 0.017), while mutations in the PTEN gene were associated with inferior OS (p = 0.048). Our findings suggest that the TP53 and ATM genes could be involved in the molecular pathophysiology of primary DLBCL CNS, whereas mutations in the PTEN and SMO genes could affect survival regardless of the initial treatment approach.     

The Challenge of Diagnosing Constitutional Mismatch Repair Deficiency Syndrome in Brain Malignancies from Young Individuals

Biallelic germline mismatch repair (MMR) gene (MLH1, MSH2, MSH6, and PMS2) mutations are an extremely rare event that causes constitutional mismatch repair deficiency (CMMRD) syndrome. CMMRD is underdiagnosed and often debuts with pediatric malignant brain tumors. A high degree of clinical awareness of the CMMRD phenotype is needed to identify new cases. Immunohistochemical (IHC) assessment of MMR protein expression and analysis of microsatellite instability (MSI) are the first tools with which to initiate the study of this syndrome in solid malignancies. MMR IHC shows a hallmark pattern with absence of staining in both neoplastic and non-neoplastic cells for the biallelic mutated gene. However, MSI often fails in brain malignancies. The aim of this report is to draw attention to the peculiar IHC profile that characterizes CMMRD syndrome and to review the difficulties in reaching an accurate diagnosis by describing the case of two siblings with biallelic MSH6 germline mutations and brain tumors. Given the difficulties involved in early diagnosis of CMMRD we propose the use of the IHC of MMR proteins in all malignant brain tumors diagnosed in individuals younger than 25 years-old to facilitate the diagnosis of CMMRD and to select those neoplasms that will benefit from immunotherapy treatment.     

MicroRNA-mRNA Regulatory Network Mediates Activation of mTOR and VEGF Signaling in African American Prostate Cancer

African American (AA) men exhibit 1.6-fold higher prostate cancer (PCa) incidence and 2.4-fold higher mortality rates compared to European American (EA) men. In addition to socioeconomic factors, emerging evidence suggests that intrinsic biological differences may explain part of PCa disparities. In this study, we applied microRNA (miRNA)-driven bioinformatics to evaluate whether differential miRNA-mRNA regulatory networks play a role in promoting the AA PCa disparities. 10 differentially expressed miRNAs were imported to mirPath V.3 algorithm, leading to identification of 58 signaling pathways differentially regulated in AA PCa versus EA PCa. Among these pathways, we particularly focused on mTOR and VEGF signaling, where we identified 5 reciprocal miRNA-mRNA pairings: miR-34a-5p/HIF1A, miR-34a-5p/PIK3CB, miR-34a-5p/IGFBP2, miR-99b-5p/MTOR and miR-96-5p/MAPKAPK2 in AA PCa versus EA PCa. RT-qPCR validation confirmed that miR-34a-5p, miR-99b-5p and MAPKAPK2 were downregulated, while miR-96-5p, IGFBP2, HIF1A, PIK3CB and MTOR were upregulated in AA PCa versus EA PCa cells. Transfection of miRNA mimics/antagomir followed by RT-qPCR and Western blot analysis further verified that IGFBP2, HIF1A and PIK3CB are negatively regulated by miR-34a-5p, whereas MTOR and MAPKAPK2 are negatively regulated by miR-99b-5p and miR-96-5p, respectively, at mRNA and protein levels. Targeting reciprocal pairings by miR-34a-5p mimic, miR-99b-5p mimic or miR-96-5p antagomir downregulates HIF1α, PI3Kβ, mTOR, IGFBP2 but upregulates MAPKAPK2, subsequently reducing cell proliferation and sensitizing docetaxel-induced cytotoxicity in PCa cells. These results suggest that miRNA-mRNA regulatory network plays a critical role in AA PCa disparities, and targeting these core miRNA-mRNA pairings may reduce PCa aggressiveness and overcome the chemoresistance in AA patients.