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Objectives Frozen section (FS) is a technique widely used intraoperatively to render a preliminary histopathologic diagnosis, allowing for immediate decisions at the time of surgery. We aimed to investigate potential variations in tissue antigenicity induced by rapid freezing in a variety of gynecologic tumor samples. Methods A total of 177 FS and 177 non–frozen section (NFS) tissue slides were tested using a panel of immunostains commonly used in gynecologic pathology, including hormone receptors (estrogen receptor, progesterone receptor), HER2, mismatch repair proteins (MSH6, PMS2), programmed cell death 1 ligand 1 (PD-L1), p53, napsin A, and ɑ-methylacyl coenzyme-A racemase. Immunohistochemistry results were categorized as positive or negative, and positive cases were subsequently scored based on the distribution and intensity of the staining. Certain immunostains, such as HER2, PD-L1, and p53, were scored according to the established guidelines. Results The overall concordance between FS and NFS blocks was 87%; among the 13% of discrepant cases, most (10.7%) were classified as minor, with only quantitative differences without foreseeable clinical significance. In 2.3% of cases, there were major qualitative changes with potential impact on disease management. Conclusions We concluded that FS tissue blocks may, in most cases, safely be used for immunohistochemical studies because most discrepant cases showed only minor differences in staining, with no anticipated clinical significance. Nevertheless, for certain markers, including HER2, p53, and PMS2, a NFS block is preferred when that option is available. [ABSTRACT FROM AUTHOR]

Lynch syndrome, a hereditary cancer susceptibility syndrome, arises from pathogenic mutations in mismatch repair genes. This syndrome is strongly linked to colorectal and endometrial cancers, as well as an elevated risk for other cancers such as gastric, ovarian, renal pelvis/ureter, and prostate. Notably, Lynch syndrome is rarely associated with cholangiocarcinoma (CCA). In this case study, we present a unique instance of Lynch syndrome-related CCA resulting from a singular MSH6 mutation. Notably, our findings reveal discrepancies between immunohistochemistry (IHC) and microsatellite stability results compared to genetic testing outcomes. This discrepancy underscores the limitations of solely relying on IHC analysis and microsatellite stability testing for the detection of hereditary tumors, emphasizing the crucial role of genetic testing in such cases. This insight enhances our comprehension of the mechanisms involved in cancer development and underscores the significance of thorough analysis integrating immunohistochemistry and genetic testing for diagnosing Lynch syndrome-related cancers. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is the third most prevalent disease in the world, with an estimated 1.2 million new cases each year. Spontaneous CRCs account for around 70% of all CRCs, are caused by somatic mutations. Minor variations or single-nucleotide polymorphisms (SNPs) in oncogene or tumor-suppressor genes cause familial CRC. MSH2 and MSH6 genes are located on chromosome 2. These genes products are involved in the repair of DNA replication defects. If these proteins are changed, the replication errors are not rectified, resulting in damaged DNA leading to colorectal cancer. We employed a variety of computational methodologies to find nsSNPs that are harmful to the structure and function of the MSH6 protein and could be causing CRC in our study. SIFT, PROVEAN, Poly- Phen-2, PhD-SNP, and SNPs&GO were among the in silico methods used to do the computational research. According to the findings, mutations of G932Q, E1234Q, and F1104Q are important alterations in native MSH6 protein rs35717727 that may contribute to its dysfunction and, ultimately, disease. The study also provided three-dimensional structures of the native MSH6 protein and mutations. These nsSNPs should be considered as key target mutations in many disorders involving MSH6 dysfunction in future studies. This is the first thorough study to use in silico technologies to assess MSH6 gene variants, and it will be extremely useful in planning largescale investigations and developing precision medicines to treat disorders caused by these polymorphisms. Additionally, animal models of various autoimmune disorders with these mutations could aid in determining their precise involvement. [ABSTRACT FROM AUTHOR]

Objectives: To evaluate the relationships between adenine-thymine-rich interactive domain 1A (ARID1A) expression and the clinicopathologic features in T1 colorectal cancer (CRC) and to investigate whether the presence of ARID1A protein is related to genetic changes.Methods: We retrospectively studied 219 surgically resected T1 CRCs. ARID1A expression was assessed by immunohistochemical methods, and the correlation between ARID1A expression and clinicopathologic features was evaluated. The relationship between ARID1A expression and 409 cancer-related gene mutations was also evaluated using next-generation sequencing (NGS).Results: Immunohistochemical staining indicated negative ARID1A expression in 4.6%. Loss of ARID1A expression was significantly associated with younger age, lymphatic invasion, and lymph node metastasis (LNM). NGS showed that PKHD1, RNF213, and MSH6 mutations were more frequent in ARID1A-negative tumors, whereas KRAS mutations were more common in ARID1A-positive tumors.Conclusions: In T1 CRC, negative ARID1A expression was correlated with early onset, lymphatic invasion, and LNM. Mutations in some cancer-related genes were possibly related with ARID1A expression. [ABSTRACT FROM AUTHOR]

Osteosarcoma (OS) is one of the most common primary bone malignancies, with the survival rate of patients with OS remaining low. Therefore, we conducted this study to identify the potential role combination of both MSH6 gene silencing and cisplatin (DDP) plays in OS cell proliferation and apoptosis. Microarray‐based gene expression profiling was used to identify the differentially expressed genes (DEGs) in patients with OS, as well as microRNAs (miRNAs) that regulate the candidate gene. OS tissues from 67 patients with OS along with normal tissues from 24 amputee patients were collected for detection of the positive expression of mutS homolog 6 (MSH6) protein, mRNA, and protein expressions of c‐myc, cyclin D1, l‐2, B‐cell lymphoma 2 (Bcl‐2), Stathmin, proliferating cell nuclear antigen (PCNA), and Bcl‐2‐associated X (Bax). Moreover, after MSH6 silencing and DDP were treated on the selected human OS cell line MG63 with the highest expression of MSH6, cell viability, cell cycle distribution, and apoptosis were detected. The microarray analysis showed that MSH6 was upregulated in OS chip data. Furthermore, silencing MSH6 combined with DDP reduced expressions of c‐myc, cyclin D1, Bcl‐2, Stathmin, and PCNA, and elevated Bax expression, whereas inhibiting OS cell viability, impeding cell cycle distribution, and inducing apoptosis. In conclusion, our preliminary results indicated that the combination of MSH6 gene silencing coupled with DDP may have a better effect on the inhibition of OS cell proliferation and promote apoptosis, potentially providing targets for the OS treatment. The combination of MSH6 gene silencing coupled with cisplatin (DDP) may have a better effect on the inhibition of osteosarcoma (OS) cell proliferation and promote apoptosis, potentially providing targets for the OS treatment. [ABSTRACT FROM AUTHOR]

Lynch syndrome (LS), the most frequent form of hereditary colorectal cancer, involves mutations in mismatch repair genes. The aim of this study was to identify mutations in MSH6 from 97 subjects negative for mutations in MLH1 and MSH2. By direct sequencing, we identified 27 MSH6 variants, of which, nine were novel. To verify the pathogenicity of these novel variants, we performed in silico and segregation analyses. Three novel variants were predicted by in silico analysis as damaging mutations and segregated with the disease phenotype; while a novel frameshift deletion variant that was predicted to yield a premature stop codon did not segregate with the LS phenotype in three of four cases in the family. Interestingly, another frame-shift variant identified in this study, already described in the literature, also did not segregate with the LS phenotype in one of two affected subjects in the family. In all affected subjects of both families, no mutation was detected in other MMR genes. Therefore, it is expected that within these families, other genetic factors contribute to the disease either alone or in combination with MSH6 variants. We conclude that caution should be exercised in counseling for MSH6-associated LS family members. [ABSTRACT FROM AUTHOR]

Endometrial cancer in the lower uterine segment (LUS) is associated with Lynch syndrome with MLH1 or MSH2 germline mutation. Here, we report a case of carcinoma of the LUS diagnosed with Lynch syndrome based on MSH6 germline mutation in a 46-year-old woman with abnormal vaginal bleeding. She had had rectal cancer at age 39 with a family history of colon cancer (father, 75 years), pancreatic cancer (paternal grandmother, 74 years), and colon cancer (maternal grandmother, 85 years). Magnetic resonance imaging showed a tumor in the LUS. Endometrial biopsy revealed endometrioid adenocarcinoma G1. As her cancer history met the revised Bethesda criteria, we examined microsatellite instability and the result was negative, but loss of the MSH6 expression was detected by immunohistochemistry. Genetic testing revealed deleterious germline mutation of MSH6, which was compatible with Lynch syndrome. To our knowledge, this is the first case of endometrial carcinoma of the LUS with MSH6 germline mutation. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is the third most prevalent disease in the world, with an estimated 1.2 million new cases each year. Spontaneous CRCs account for around 70% of all CRCs, are caused by somatic mutations. Minor variations or single-nucleotide polymorphisms (SNPs) in oncogene or tumor-suppressor genes cause familial CRC. MSH2 and MSH6 genes are located on chromosome 2. These genes products are involved in the repair of DNA replication defects. If these proteins are changed, the replication errors are not rectified, resulting in damaged DNA leading to colorectal cancer. We employed a variety of computational methodologies to find nsSNPs that are harmful to the structure and function of the MSH6 protein and could be causing CRC in our study. SIFT, PROVEAN, Poly- Phen-2, PhD-SNP, and SNPs&GO were among the in silico methods used to do the computational research. According to the findings, mutations of G932Q, E1234Q, and F1104Q are important alterations in native MSH6 protein rs35717727 that may contribute to its dysfunction and, ultimately, disease. The study also provided three-dimensional structures of the native MSH6 protein and mutations. These nsSNPs should be considered as key target mutations in many disorders involving MSH6 dysfunction in future studies. This is the first thorough study to use in silico technologies to assess MSH6 gene variants, and it will be extremely useful in planning largescale investigations and developing precision medicines to treat disorders caused by these polymorphisms. Additionally, animal models of various autoimmune disorders with these mutations could aid in determining their precise involvement.

Background Colorectal cancer (CRC) keeps an upward incidence and rejuvenation trend in Kazakhstan, as well as worldwide, which is the motive for revising traditional risk factors and searching for cause-effective relationships at the genome level. Genetic nature of early-onset CRC is still unclear. The aim of this study was to determine the spectrum of cancer-related gene mutations among early-onset CRC patients. Methods The study included 125 CRC patients aged between 17 and 50 from Kazakhstan. DNAs were extracted from blood using the GeneJET Purification Kit. Next-generation sequencing (NGS) was done on the MiSeq using the TruSightCancer Kit. NGS data were analyzed by the MiSeq Reporter and Variant Studio. SIFT and PolyPhen-2 were used to predict potential pathogenic effects of missense variants on protein structure and function. Results We were able to detect 24 pathogenic/likely pathogenic mutations in 20 (16%) cases, out of which 8 were novel. Out of all mutations, five were detected in the APC gene, three in FANCI, three in BRCA2, two in BRCA1, two in MLH1, and one mutation each, in MSH6, MUTYH, BLM, NBN, ATM, BMPR1A, CHEK2, AIP, and DICER1 genes. The analysis of mutation type has revealed 10 frameshift mutations, 5 missense mutations, 5 stop-gain mutations, 1 in-frame deletion, and 3 mutations involving uncorrected splicing. All mutations were in the heterozygous state. Most of the mutations were not available in the 1000G, ESP6500 and ExAC databases. 5% were available in the dbSNP database, 29.1% in the COSMIC, and 38.4% in the ClinVar and/or LOVD. Pathogenic mutations in high penetrance CRC genes were higher in patients with family history of cancer (FHC) (21.1%, P = 0.0002) and in patients with primary multiple tumors (20.0 %, P = 0.0004) compared with patients without/unknown FHC (3.1%). Conclusions Molecular genetic study of CRC in young patients using NGS allows to identify CRC cases with syndromic and sporadic nature, to stratify the level of risk for a particular patient and his/her relatives, to influence on the diagnostic and therapeutic approaches and clinical examinations. The findings from this study show the diagnostic value of the detected pathogenic mutations in key CRC genes from the Kazakhstan population as promising biomarkers for CRC diagnosis in young people. Legal entity responsible for the study Institute of General Genetics and Cytology. Funding Ministry of Healthcare of the Republic of Kazakhstan. Disclosure All authors have declared no conflicts of interest. [ABSTRACT FROM AUTHOR]

Background The utility of next generation sequencing (NGS) is rapidly evolving within our medical practice with various applications. The 100K Genomes Project was established to sequence 100 000 genomes from NHS patients with cancer or rare diseases, and to date actionable findings have been found for approximately 50% of cancer cases. Data from the 100K Genomes Project were reviewed at Guy's Hospital Molecular Tumour Board (MTB). Methods Whole genome sequencing (WGS) was performed for the 100K Genomes Project and Genomics England using an Illumina platform. Results Between December 2016 and August 2018, 66 patients with colorectal cancer (CRC) were treated with surgery at GSTT. Median age was 68.5 (52-95). Majority were left sided (69%) vs right sided (30%) adenocarcinomas. TNM status was 1.5% / 47% / 37.9% / 13.6% for stage I/II/III/IV respectively. Analyses were performed in FFPE (35.8%) and frozen tissue (64.2%). Median estimated tumour purity was 53.5% (20-100%). Median tumour mutational burden (TMB) was 5.92 (2.94-173.26). Mutational signatures 6, 10 and 15 were found for 8 patients and were associated with high TMB. A total of 557 Domain 1 (actionable) variants (range 1-68) were identified for this cohort. After review in the MTB, at least one clinically relevant actionable variant was identified for 59.7% of the patients; KRAS 37.3%, NRAS 6%, BRAF 11.9%, TP53 70.1%, PIK3CA 35.8%, ATM 17.9%, RICTOR 14.9%, APC and PTEN 11.9% each and mTOR 8.6%. Somatic BRCA1 and BRCA2 variants were found in 7.6% and 12.1% respectively. Further validation was recommended for 9 patients (13.4%). DPYD alterations were identified in 11.9%, but none of the clinically relevant DPYD mutations related to 5FU toxicity were discovered for 2 of the patients that were tested. 9.4% were identified with potentially pathogenic germline variants (7.6% with RAD51D, RAD51C, MUTYH, MSH6 and PMS2 class 4 & 5 variants) and referral to Clinical Genetics was recommended. Conclusions GSTT MTB identified actionable mutations in more than half patients in the CRC cohort, out of whom 14% had alterations that made them eligible for clinical trials. Over 9% had potentially pathogenic germline variants. Our data from this small cohort resemble data in the literature from other MTBs. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest. [ABSTRACT FROM AUTHOR]

Background Mismatch repair deficiency (dMMR) / microsatellite instability-high (MSI-H) in cells lead to neoplastic development and progression, an important feature in Lynch syndrome. dMMR/MSI-H is a positive predictive marker for immune checkpoint inhibitors. Nivolumab is a PD-1 inhibitor, approved in the US for pts with dMMR/MSI-H colorectal cancers. Other than colorectal cancers, a subset of rare cancers also share this characteristic. Methods This is a phase 2, open-label, single arm study designed to investigate the efficacy and safety of nivolumab monotherapy in patients with advanced rare tumors with dMMR/MSI-H. This study is conducted as part of the MASTER KEY Project, which is a basket/umbrella trial including a registry study for rare cancers (UMIN000027552). dMMR was identified by a negative immunohistochemical staining of one of the following: MLH1, MSH2, MSH6, PMS2. Rare cancer was defined as annual incidence < 6 / 100,000 population. Other main inclusion criteria were: age ≥ 16, ECOG-PS of 0-1, unresectable lesion and have completed standard treatment, adequate organ function. Patients will receive intravenous nivolumab monotherapy of 240 mg every 2 weeks until disease progression, unacceptable toxicity, treatment discontinuation at the discretion of the investigator or patient, or death. Primary endpoint is response rate (central assessment), and secondary endpoints include progression free survival, overall survival, stable disease rate and toxicity. The trial will utilize a Bayesian hierarchical model enrolling 5 to 15 patients depending on the ongoing results as an adaptive design. Participating sites are National Cancer Center and Kyoto University Hospital, and will be expanding shortly. Clinical Trial Information JMA-IIA00344 [ABSTRACT FROM AUTHOR]

Gastroesophageal cancers, eg cancers of the stomach and esophagus, are a leading cause of cancer related death worldwide. Recent evidence suggests that these cancers are complex and heterogeneous diseases with emerging subtypes shown to influence response to treatment and survival. Immunotherapy is an advancing field and immune checkpoint inhibitors have become standard treatment options in numerous tumor types and is emerging therapy in gastroesophageal malignancies. Several immune checkpoint inhibitors have been examined in gastric and esophageal cancers, both as single agents and in combination with cytotoxic therapy. Tumors with deficiency in mismatch repair have demonstrated considerable activity with single agent immunotherapy. These tumors, typically characterized by loss or inactivation of DNA repair proteins MLH1, MSH2, MSH6, and PMS2, accumulate hundreds to thousands of mutations in the microsatellite regions of DNA during replication, mutations that would normally be repaired if the mismatch repair system were intact. These tumors are noted to have high lymphocytic infiltrates, and are primed for an anti-tumor immune response by host immunity, with high single agent immunotherapy activity of ∼50% response rates. However across several studies, particularly without patient selection or by selection based on PD-L1 expression alone, the response rates to anti-PD-1 or anti-PD-L1 therapy in gastric and esophageal cancer is marginal, ranging from 5-15%. The most compelling and transformative aspect of immunotherapy is that the few patients who do respond have durable responses, often greater than 1 year, generally with minimal side effects. Our challenge is to better select patients who will benefit from single agent immunotherapy. We will discuss the emerging data examining immunotherapy in upper gastrointestinal malignancies across first, second, and third line studies and the development of biomarkers to improve patient selection. [ABSTRACT FROM AUTHOR]