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.     

Microsatellite Instability in Russian Patients with Colorectal Cancer

The aim of this study was to determine the characteristics of Russian patients with microsatellite instability (MSI) tumors. MSI in the tumor was determined in 514 patients with colon cancer using PCR and subsequent fragment analysis for five markers (NR21, NR24, BAT25, BAT26, and NR27). In the presence of microsatellite instability, the mismatch repair (MMR) system genes were examined using the NGS and MLPA methods to establish the diagnosis of Lynch syndrome. The overall frequency of MSI tumors was 15%: at stage I—19% (9/48), at stage II—21% (44/213), at stage III—16% (26/160), and at stage IV—2% (2/93). Patients with MSI tumors differed in the age of diagnosis, tumor localization, time of cancer recurrence, and stage of the disease. The overall and disease-free survival of patients whose tumors had MSI status was higher than that of patients with microsatellite-stable status, p = 0.04 and p = 0.02, respectively. Analysis of overall and disease-free survival of patients with Lynch syndrome and patients with sporadic colon cancer, but with MSI status, did not reveal significant differences, p = 0.52 and p = 0.24, respectively. The age of patients with Lynch syndrome was significantly younger than that of patients with sporadic colon cancer whose tumors had MSI status (p < 0.001).     

Liquid Biopsy as a Source of Nucleic Acid Biomarkers in the Diagnosis and Management of Lynch Syndrome

Lynch syndrome (LS) is an autosomal dominant inherited cancer predisposition disorder, which may manifest as colorectal cancer (CRC), endometrial cancer (EC) or other malignancies of the gastrointestinal and genitourinary tract as well as the skin and brain. Its genetic cause is a defect in one of the four key DNA mismatch repair (MMR) loci. Testing of patients at risk is currently based on the absence of MMR protein staining and detection of mutations in cancer tissue and the germline, microsatellite instability (MSI) and the hypermethylated state of the MLH1 promoter. If LS is shown to have caused CRC, lifetime follow-up with regular screening (most importantly, colonoscopy) is required. In recent years, DNA and RNA markers extracted from liquid biopsies have found some use in the clinical diagnosis of LS. They have the potential to greatly enhance the efficiency of the follow-up process by making it minimally invasive, reproducible, and time effective. Here, we review markers reported in the literature and their current clinical applications, and we comment on possible future directions.     

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.     

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.     

BAT26 Only Microsatellite Instability with High Tumor Mutation Burden—A Rare Entity Associated with PTEN Protein Loss and High PD-L1 Expression

Detecting microsatellite instability (MSI) in advanced cancers is crucial for clinical decision-making, as it helps in identifying patients with differential treatment responses and prognoses. BAT26 is a highly sensitive MSI marker that defines the mismatch repair (MMR) status with high sensitivity and specificity. However, isolated BAT26-only instability is rare and has not been previously reported. Of the 6476 cases tested using pentaplex MSI polymerase chain reaction, we identified two BAT26-only instability cases (0.03%) in this study. The case #1 patient was diagnosed with endometrial adenocarcinoma without MMR germline mutations. The endometrial tumor showed BAT26-only instability, partial loss of MLH1/PMS2 protein expression, and a high programmed cell death ligand 1 (PD-L1) combined positive score (CPS = 8). The tumor exhibited a somatic phosphatase and tensin homolog (PTEN) R303P missense mutation and loss of the PTEN protein. On a comprehensive cancer panel sequencing with ≥500 genes, the tumor showed an MSI score of 11.38% and high tumor mutation burden (TMB) (19.5 mt/mb). The case #2 patient was diagnosed with colorectal carcinoma with proficient MMR and PTEN protein loss without PTEN alteration, as well as a high PD-L1 CPS (CPS = 10). A pathogenic KRAS A146T mutation was detected with an MSI score of 3.36% and high TMB (13 mt/mb). In conclusion, BAT26-only instability is very rare and associated with PTEN protein loss, high TMB, and a high PD-L1 score. Our results suggest that patients with BAT26-only instability may show good responses to immunotherapy.     

Xeroderma Pigmentosum: Low Prevalence of Germline XPA Mutations in a Brazilian XP Population

Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder characterized by DNA repair defects that cause photophobia, sunlight-induced cancers, and neurodegeneration. Prevalence of germline mutations in the nucleotide excision repair gene XPA vary significantly in different populations. No Brazilian patients have been reported to carry a germline mutation in this gene. In this study, the germline mutational status of XPA was determined in Brazilian patients exhibiting major clinical features of XP syndrome. The study was conducted on 27 unrelated patients from select Brazilian families. A biallelic inactivating transition mutation c.619C>T (p.Arg207Ter) was identified in only one patient with a history of neurological impairment and mild skin abnormalities. These findings suggest that XP syndrome is rarely associated with inherited disease-causing XPA mutations in the Brazilian population. Additionally, this report demonstrates the effectiveness of genotype-phenotype correlation as a valuable tool to guide direct genetic screening.     

The Barley Chloroplast Mutator (cpm) Mutant: All Roads Lead to the Msh1 Gene

The barley chloroplast mutator (cpm) is a nuclear gene mutant that induces a wide spectrum of cytoplasmically inherited chlorophyll deficiencies. Plastome instability of cpm seedlings was determined by identification of a particular landscape of polymorphisms that suggests failures in a plastome mismatch repair (MMR) protein. In Arabidopsis, MSH genes encode proteins that are in charge of mismatch repair and have anti-recombination activity. In this work, barley homologs of these genes were identified, and their sequences were analyzed in control and cpm mutant seedlings. A substitution, leading to a premature stop codon and a truncated MSH1 protein, was identified in the Msh1 gene of cpm plants. The relationship between this mutation and the presence of chlorophyll deficiencies was established in progenies from crosses and backcrosses. These results strongly suggest that the mutation identified in the Msh1 gene of the cpm mutant is responsible for the observed plastome instabilities. Interestingly, comparison of mutant phenotypes and molecular changes induced by the barley cpm mutant with those of Arabidopsis MSH1 mutants revealed marked differences.     

Hereditary Prostate Cancer: Genes Related,Target Therapy and Prevention

Prostate cancer (PCa) is globally the second most diagnosed cancer type and the most common cause of cancer-related deaths in men. Family history of PCa, hereditary breast and ovarian cancer (HBOC) and Lynch syndromes (LS), are among the most important risk factors compared to age, race, ethnicity and environmental factors for PCa development. Hereditary prostate cancer (HPCa) has the highest heritability of any major cancer in men. The proportion of PCa attributable to hereditary factors has been estimated in the range of 5–15%. To date, the genes more consistently associated to HPCa susceptibility include mismatch repair (MMR) genes (MLH1, MSH2, MSH6, and PMS2) and homologous recombination genes (BRCA1/2, ATM, PALB2, CHEK2). Additional genes are also recommended to be integrated into specific research, including HOXB13, BRP1 and NSB1. Importantly, BRCA1/BRCA2 and ATM mutated patients potentially benefit from Poly (ADP-ribose) polymerase PARP inhibitors, through a mechanism of synthetic lethality, causing selective tumor cell cytotoxicity in cell lines. Moreover, the detection of germline alterations in MMR genes has therapeutic implications, as it may help to predict immunotherapy benefits. Here, we discuss the current knowledge of the genetic basis for inherited predisposition to PCa, the potential target therapy, and the role of active surveillance as a management strategy for patients with low-risk PCa. Finally, the current PCa guideline recommendations are reviewed.     

Heterogeneity of Mismatch Repair Status and Microsatellite Instability between Primary Tumour and Metastasis and Its Implications for Immunotherapy in Colorectal Cancers

Deficient mismatch repair system (dMMR)/microsatellite instability (MSI) is found in about 5% of metastatic colorectal cancers (mCRCs) with a major therapeutic impact for immune checkpoint inhibitor (ICI) use. We conducted a multicentre study including all consecutive patients with a dMMR/MSI mCRC. MSI status was determined using the Pentaplex panel and expression of the four MMR proteins was evaluated by immunohistochemistry (IHC). The primary endpoint was the rate of discordance of dMMR/MSI status between primary tumours and paired metastases. We included 99 patients with a dMMR/MSI primary CRC and 117 paired metastases. Only four discrepancies (3.4%) with a dMMR/MSI primary CRC and a pMMR/MSS metastasis were initially identified and reviewed by expert pathologists and molecular biologists. Two cases were false discrepancies due to human or technical errors. One discordant case could not be confirmed due to the low level of tumour cells. The last case had a confirmed discrepancy with a dMMR/MSI primary CRC and a pMMR/MSS peritoneal metastasis. Our study demonstrated a high concordance rate of dMMR/MSI status between primary CRCs and their metastases. The analysis of one sample, either from the primary tumour or metastasis, with consistent dMMR and MSI status seems to be sufficient prior to treatment with ICI.     

Microsatellite Instability: From the Implementation of the Detection to a Prognostic and Predictive Role in Cancers

Microsatellite instability (MSI) has been identified in several tumors arising from either germline or somatic aberration. The presence of MSI in cancer predicts the sensitivity to immune checkpoint inhibitors (ICIs), particularly PD1/PD-L1 inhibitors. To date, the predictive role of MSI is currently used in the selection of colorectal cancer patients for immunotherapy; moreover, the expansion of clinical trials into other cancer types may elucidate the predictive value of MSI for non-colorectal tumors. In clinical practice, several assays are used for MSI testing, including immunohistochemistry (IHC), polymerase chain reaction (PCR) and next-generation sequencing (NGS). In this review, we provide an overview of MSI in various cancer types, highlighting its potential predictive/prognostic role and the clinical trials performed. Finally, we focus on the comparison data between the different assays used to detect MSI in clinical practice.     

Responses of DNA Mismatch Repair Proteins to a Stable G-Quadruplex Embedded into a DNA Duplex Structure

DNA mismatch repair (MMR) plays a crucial role in the maintenance of genomic stability. The main MMR protein, MutS, was recently shown to recognize the G-quadruplex (G4) DNA structures, which, along with regulatory functions, have a negative impact on genome integrity. Here, we studied the effect of G4 on the DNA-binding activity of MutS from Rhodobacter sphaeroides (methyl-independent MMR) in comparison with MutS from Escherichia coli (methyl-directed MMR) and evaluated the influence of a G4 on the functioning of other proteins involved in the initial steps of MMR. For this purpose, a new DNA construct was designed containing a biologically relevant intramolecular stable G4 structure flanked by double-stranded regions with the set of DNA sites required for MMR initiation. The secondary structure of this model was examined using NMR spectroscopy, chemical probing, fluorescent indicators, circular dichroism, and UV spectroscopy. The results unambiguously showed that the d(GGGT)₄ motif, when embedded in a double-stranded context, adopts a G4 structure of a parallel topology. Despite strong binding affinities of MutS and MutL for a G4, the latter is not recognized by E. coli MMR as a signal for repair, but does not prevent MMR processing when a G4 and G/T mismatch are in close proximity.     

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.     

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.     

Serum Concentrations of TNF �� and Its Soluble Receptors in Patients with Adrenal Tumors Treated by Surgery

The peripheral blood levels of TNF α and its soluble receptors were studied in 39 patients with malignant and benign adrenal tumors treated by adrenalectomy. The concentrations of TNF α were significantly elevated in patients with malignant tumors of the adrenal cortex and in patients with Conn’s syndrome compared to control. In patients with non-functioning adenomas and pheochromocytomas, TNF α levels were similar to those detected in the control. In subjects with myelolipomas, the serum concentration of TNF α was lower compared to the control. After adrenalectomy, the levels of TNF α were decreased in patients with malignant tumors and in patients with Conn’s syndrome, nonfunctioniong adenomas and pheochromocytomas compared to the concentration before surgery. The serum concentrations of soluble receptors of TNF α did not differ among different patient groups and compared to the control. After adrenalectomy, the blood concentrations of TNF α R1 and TNF α R2 were decreased in patients with Conn’s syndrome. However, to confirm practicality of the evaluation of TNF α and its soluble receptors in differential diagnosis in patients with adrenal tumors, a larger study group is needed.     

A Case Report of Germline Compound Heterozygous Mutations in the BRCA1 Gene of an Ovarian and Breast Cancer Patient

The germline carrier of the BRCA1 pathogenic mutation has been well proven to confer an increased risk of breast and ovarian cancer. Despite BRCA1 biallelic pathogenic mutations being extremely rare, they have been reported to be embryonically lethal or to cause Fanconi anemia (FA). Here we describe a patient who was a 48-year-old female identified with biallelic pathogenic mutations of the BRCA1 gene, with no or very subtle FA-features. She was diagnosed with ovarian cancer and breast cancer at the ages of 43 and 44 and had a strong family history of breast and gynecological cancers.     

A Homozygous Loss-of-Function Mutation in MSH5 Abolishes MutSγ Axial Loading and Causes Meiotic Arrest in NOA-Affected Individuals

Non-obstructive azoospermia (NOA), characterized by spermatogenesis failure and the absence of sperm in ejaculation, is the most severe form of male infertility. However, the etiology and pathology between meiosis-associated monogenic alterations and human NOA remain largely unknown. A homozygous MSH5 mutation (c.1126del) was identified from two idiopathic NOA patients in the consanguineous family. This mutation led to the degradation of MSH5 mRNA and abolished chromosome axial localization of MutSγ in spermatocytes from the affected males. Chromosomal spreading analysis of the patient’s meiotic prophase I revealed that the meiosis progression was arrested at a zygotene-like stage with extensive failure of homologous synapsis and DSB repair. Therefore, our study demonstrates that the MSH5 c.1126del could cause meiotic recombination failure and lead to human infertility, improving the genetic diagnosis of NOA clinically. Furthermore, the study of human spermatocytes elucidates the meiosis defects caused by MSH5 variant, and reveals a conserved and indispensable role of MutSγ in human synapsis and meiotic recombination, which have not previously been well-described.     

How the Analysis of the Pathogenetic Variants of DDR Genes Will Change the Management of Prostate Cancer Patients

Herein, we analyze answers achieved, open questions, and future perspectives regarding the analysis of the pathogenetic variants (PV) of DNA damage response (and repair) (DDR) genes in prostate cancer (PC) patients. The incidence of PVs in homologous recombination repair (HRR) genes among men with metastatic PC varied between 11% and 33%, which was significantly higher than that in non-metastatic PC, and BRCA2 mutations were more frequent when compared to other DDR genes. The determination of the somatic or germline PVs of BRCA2 was able to define a tailored therapy using PARP inhibitors in metastatic castration-resistant prostate cancer (mCRPC) progression after first-line therapy, with significant improvements in the radiologic progression-free survival (rPFS) and overall survival (OS) rates. We propose testing all metastatic PC patients for somatic and germline HRR mutations. Somatic determination on the primary site or on historic paraffin preparations with a temporal distance of no longer than 5 years should be preferred over metastatic site biopsies. The prognostic use of DDR PVs will also be used in selected high-risk cases with non-metastatic stages to better arrange controls and therapeutic primary options. We anticipate that the use of poly-ADP-ribose polymerase (PARP) inhibitors in hormone-sensitive prostate cancer (HSPC) and in combination with androgen receptor signaling inhibitors (ARSI) will be new strategies.     

Germline POT1 Deregulation Can Predispose to Myeloid Malignancies in Childhood

While the shelterin complex guards and coordinates the mechanism of telomere regulation, deregulation of this process is tightly linked to malignant transformation and cancer. Here, we present the novel finding of a germline stop-gain variant (p.Q199*) in the shelterin complex gene POT1, which was identified in a child with acute myeloid leukemia. We show that the cells overexpressing the mutated POT1 display increased DNA damage and chromosomal instabilities compared to the wildtype counterpart. Protein and mRNA expression analyses in the primary patient cells further confirm that, physiologically, the variant leads to a nonfunctional POT1 allele in the patient. Subsequent telomere length measurements in the primary cells carrying heterozygous POT1 p.Q199* as well as POT1 knockdown AML cells revealed telomeric elongation as the main functional effect. These results show a connection between POT1 p.Q199* and telomeric dysregulation and highlight POT1 germline deficiency as a predisposition to myeloid malignancies in childhood.     

Integrative Genomic Tests in Clinical Oncology

Many clinical decisions in oncology practice rely on the presence or absence of an alteration in a single genetic locus, be it a pathogenic variant in a hereditary cancer gene or activating mutation in a drug target. In addition, there are integrative tests that produce continuous variables and evaluate complex characteristics of the entire tumor genome. Microsatellite instability (MSI) analysis identifies tumors with the accumulation of mutations in short repetitive nucleotide sequences. This procedure is utilized in Lynch syndrome diagnostic pipelines and for the selection of patients for immunotherapy. MSI analysis is well-established for colorectal malignancies, but its applications in other cancer types lack standardization and require additional research. Homologous repair deficiency (HRD) indicates tumor sensitivity to PARP inhibitors and some cytotoxic drugs. HRD-related “genomic scars” are manifested by a characteristic pattern of allelic imbalances, accumulation of deletions with flanking homology, and specific mutation signatures. The detection of the genetic consequences of HRD is particularly sophisticated and expensive, as it involves either whole genome sequencing (WGS) or the utilization of large next-generation sequencing (NGS) panels. Tumor mutation burden (TMB) can be determined by whole exome sequencing (WES) or middle-throughput NGS multigene testing. Although TMB is regarded as an agnostic indicator of tumor sensitivity to immunotherapy, the clinical utility of this test is proven only for a few cancer types.