Tumor analysis of MMR genes in Lynch-like syndrome: Challenges associated with results interpretation.

BACKGROUND: Up to 70% of suspected Lynch syndrome patients harboring MMR deficient tumors lack identifiable germline pathogenic variants in MMR genes, being referred to as Lynch-like syndrome (LLS). Previous studies have reported biallelic somatic MMR inactivation in a variable range of LLS-associated tumors. Moreover, translating tumor testing results into patient management remains controversial. Our aim is to assess the challenges associated with the implementation of tumoral MMR gene testing in routine workflows. METHODS: Here, we present the clinical characterization of 229 LLS patients. MMR gene testing was performed in 39 available tumors, and results were analyzed using two variant allele frequency (VAF) thresholds (≥5% and ≥10%). RESULTS AND DISCUSSION: More biallelic somatic events were identified at VAF ≥ 5% than ≥10% (35.9% vs. 25.6%), although the rate of nonconcordant results regarding immunohistochemical pattern increased (30.8% vs. 20.5%). Interpretation difficulties question the current utility of the identification of MMR somatic hits in the diagnostic algorithm of suspected LS cases.

Inhibition of autophagy-related protein 7 enhances anti-tumor immune response and improves efficacy of immune checkpoint blockade in microsatellite instability colorectal cancer.

BACKGROUND: The efficacy of anti-PD-1 therapy is primarily hindered by the limited T-cell immune response rate and immune evasion capacity of tumor cells. Autophagy-related protein 7 (ATG7) plays an important role in autophagy and it has been linked to cancer. However, the role of ATG7 in the effect of immune checkpoint blockade (ICB) treatment on high microsatellite instability (MSI-H)/mismatch repair deficiency (dMMR) CRC is still poorly understood. METHODS: In this study, patients from the cancer genome altas (TCGA) COAD/READ cohorts were used to investigate the biological mechanism driving ATG7 development. Several assays were conducted including the colony formation, cell viability, qRT-PCR, western blot, immunofluorescence, flow cytometry, ELISA, immunohistochemistry staining and in vivo tumorigenicity tests. RESULTS: We found that ATG7 plays a crucial role in MSI-H CRC. Its knockdown decreased tumor growth and caused an infiltration of CD8+ T effector cells in vivo. ATG7 inhibition restored surface major histocompatibility complex I (MHC-I) levels, causing improved antigen presentation and anti-tumor T cell response by activating reactive oxygen species (ROS)/NF-κB pathway. Meanwhile, ATG7 inhibition also suppressed cholesterol accumulation and augmentation of anti-tumor immune responses. Combining ATG7 inhibition and statins improved the therapeutic benefit of anti-PD-1 in MSI-H CRC. Importantly, CRC patients with high expression of both ATG7 and recombinant 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) experienced worse prognosis compared to those with low ATG7 and HMGCR expression. CONCLUSIONS: Inhibition of ATG7 leads to upregulation of MHC-I expression, augments immune response and suppresses cholesterol accumulation. These findings demonstrate that ATG7 inhibition has therapeutic potential and application of statins can increase the sensitivity to immune checkpoint inhibitors.

Trans-lesion synthesis and mismatch repair pathway crosstalk defines chemoresistance and hypermutation mechanisms in glioblastoma.

Almost all Glioblastoma (GBM) are either intrinsically resistant to the chemotherapeutical drug temozolomide (TMZ) or acquire therapy-induced mutations that cause chemoresistance and recurrence. The genome maintenance mechanisms responsible for GBM chemoresistance and hypermutation are unknown. We show that the E3 ubiquitin ligase RAD18 (a proximal regulator of TLS) is activated in a Mismatch repair (MMR)-dependent manner in TMZ-treated GBM cells, promoting post-replicative gap-filling and survival. An unbiased CRISPR screen provides an aerial map of RAD18-interacting DNA damage response (DDR) pathways deployed by GBM to tolerate TMZ genotoxicity. Analysis of mutation signatures from TMZ-treated GBM reveals a role for RAD18 in error-free bypass of O6mG (the most toxic TMZ-induced lesion), and error-prone bypass of other TMZ-induced lesions. Our analyses of recurrent GBM patient samples establishes a correlation between low RAD18 expression and hypermutation. Taken together we define molecular underpinnings for the hallmark tumorigenic phenotypes of TMZ-treated GBM.

Mismatch repair protein deficiency and its implications on distant metastasis in colorectal cancer: A comprehensive analysis.

BACKGROUND: While previous studies have indicated variability in distant metastatic potential among different mismatch repair (MMR) states in colorectal cancer (CRC), their findings remain inconclusive, especially considering potential differences across various ethnic backgrounds. Furthermore, the gene regulatory networks and the underlying mechanisms responsible for these variances in metastatic potential across MMR states have yet to be elucidated. METHODS: We collected 2058 consecutive primary CRC samples from the South West of China and assessed the expression of MMR proteins (MLH1, MSH2, MSH6, and PMS2) using immunohistochemistry. To explore the inconsistencies between different MMR statuses and recurrence, we performed a meta-analysis. To delve deeper, we employed Weighted Gene Co-expression Network Analysis (WGCNA), ClueGo, and iRegulon, pinpointing gene expression networks and key regulatory molecules linked to metastasis and recurrence in CRC. Lastly, both univariate and multivariate Cox regression analyses were applied to determine the impact of core regulatory molecules on metastasis. RESULTS: Of the samples, 8.2% displayed deficient MMR (dMMR), with losses of MLH1 and PSM2 observed in 40.8% and 63.9%, respectively. A unique 24.3% isolated loss of PMS2 without concurrent metastasis was identified, a result that diverges from established literature. Additionally, our meta-analysis further solidifies the reduced recurrence likelihood in dMMR CRC samples compared to proficient MMR (pMMR). Two gene expression networks tied to distant metastasis and recurrence were identified, with a majority of metastasis-related genes located on chromosomes 8 and 18. An IRF1 positive feedback loop was discerned in the metastasis-related network, and IRF1 was identified as a predictive marker for both recurrence-free and distant metastasis-free survival across multiple datasets. CONCLUSION: Geographical and ethnic factors might influence peculiarities in MMR protein loss. Our findings also highlight new gene expression networks and crucial regulatory molecules in CRC metastasis, enhancing our comprehension of the mechanisms driving distant metastasis.

Long-Term Outcomes of dMMR/MSI-H Rectal Cancer Treated With Anti-PD-1-Based Immunotherapy as Curative-Intent Treatment.

BACKGROUND: Neoadjuvant anti-PD-1 therapy has shown encouraging efficacy in patients with deficient DNA mismatch repair (dMMR)/microsatellite instability-high (MSI-H) locally advanced rectal cancer (LARC), which suggests its potential as a curative-intent therapy and a promising treatment option for organ preservation. We aimed to investigate the long-term outcomes of patients with dMMR/MSI-H LARC who experienced clinical complete response (cCR) after anti-PD-1 therapy. METHODS: We retrospectively analyzed patients with dMMR/MSI-H LARC who achieved cCR and received nonoperative management following neoadjuvant anti-PD-1-based treatment from 4 Chinese medical centers. Patients were followed up for at least 1 year after they achieved cCR, their clinical data were collected, and survival outcomes were analyzed using the Kaplan-Meier method. RESULTS: A total of 24 patients who achieved cCR and received nonoperative management from March 2018 to May 2022 were included, with a median age of 51.0 years (range, 19.0-77.0 years). The median treatment course to reach cCR was 6.0 (range, 1.0-12.0). Fifteen patients (62.5%) continued their treatments after experiencing cCR, and the median treatment course was 17.0 (range, 3.0-36.0). No local regrowth or distant metastasis was observed in a median follow-up time of 29.1 months (range, 12.6-48.5 months) after cCR. The 3-year disease-free and overall survivals were both 100%. CONCLUSIONS: Patients with dMMR/MSI-H locally advanced or low-lying rectal cancer who achieved cCR following anti-PD-1-based therapy had promising long-term outcomes. A prospective clinical trial with a larger sample size is required to further validate these findings.

Gastroesophageal Adenocarcinomas With Defective Mismatch Repair: Current Knowledge and Clinical Management.

Esophageal, gastroesophageal junction, and gastric adenocarcinomas, referred to collectively as gastroesophageal adenocarcinomas (GEAs), are a major cause of global cancer-related mortality. Our increasing molecular understanding has led to the addition of biomarker-directed approaches to defined subgroups and has improved survival in selected patients, such as those with HER2 and Claudin18.2 overexpression. Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer, including GEA, but biomarkers beyond PD-L1 expression are lacking. Mismatch repair deficiency and/or high microsatellite instability (dMMR/MSI-H) is observed in 8% to 22% of nonmetastatic GEA, and 3% to 5% of patients with metastatic disease. dMMR/MSI-H tumors are associated with more favorable prognosis and significant benefit from ICIs, although some heterogeneity exists. The activity of ICIs in advanced dMMR/MSI-H cancer is seen across lines of therapy and should be recommended in the frontline setting. In patients with nonmetastatic dMMR/MSI-H cancer, increasing evidence suggests that perioperative and adjuvant chemotherapy may not provide benefit to the dMMR/MSI-H subgroup. The activity of perioperative chemotherapy-free immune checkpoint regimens in patients with nonmetastatic dMMR/MSI-H cancer is highly promising and underscores the need to identify this unique subgroup. We recommend MMR/MSI testing for all patients with GEA at diagnosis, and review the key rationale and clinical management implications for patient with dMMR/MSI-H tumors across disease stages.

Clinicopathological significance of microsatellite instability and immune escape mechanism in patients with gastric solid-type poorly differentiated adenocarcinoma.

BACKGROUND: In gastric solid-type poorly differentiated adenocarcinoma (PDA), the role of microsatellite instability and immune escape mechanism remains unclear. The current study aimed to elucidate the clinical significance of mismatch repair (MMR) status, genome profile, C-X-C motif chemokine receptor 2 (CXCR2) expression, and myeloid-derived suppressor cell (MDSC) infiltration in solid-type PDA. METHODS: In total, 102 primary solid-type PDA cases were retrieved, and classified into 46 deficient-MMR (dMMR) and 56 proficient-MMR (pMMR) cases based on immunohistochemistry (IHC) and polymerase chain reaction-based molecular testing results. The mRNA expression profiles (NanoString nCounter Assay) of stage-matched dMMR (n = 6) and pMMR (n = 6) cases were examined. The CXCR2 expression and MDSC infiltration (CD11b- and CD33-positive cells) were investigated via IHC in all solid-type PDA cases. RESULTS: mRNA analysis revealed several differentially expressed genes and differences in biological behavior between the dMMR (n = 46) and pMMR (n = 56) groups. In the multivariate analysis, the dMMR status was significantly associated with a longer disease-free survival (hazard ratio = 5.152, p = 0.002) and overall survival (OS) (hazard ratio = 5.050, p = 0.005). CXCR2-high expression was significantly correlated with a shorter OS in the dMMR group (p = 0.018). A high infiltration of CD11b- and CD33-positive cells was significantly correlated with a shorter OS in the pMMR group (p = 0.022, 0.016, respectively). CONCLUSIONS: dMMR status can be a useful prognostic predictor, and CXCR2 and MDSCs can be novel therapeutic targets in patients with solid-type PDA.

MRE11A: a novel negative regulator of human DNA mismatch repair.

BACKGROUND: DNA mismatch repair (MMR) is a highly conserved pathway that corrects DNA replication errors, the loss of which is attributed to the development of various types of cancers. Although well characterized, MMR factors remain to be identified. As a 3'-5' exonuclease and endonuclease, meiotic recombination 11 homolog A (MRE11A) is implicated in multiple DNA repair pathways. However, the role of MRE11A in MMR is unclear. METHODS: Initially, short-term and long-term survival assays were used to measure the cells' sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Meanwhile, the level of apoptosis was also determined by flow cytometry after MNNG treatment. Western blotting and immunofluorescence assays were used to evaluate the DNA damage within one cell cycle after MNNG treatment. Next, a GFP-heteroduplex repair assay and microsatellite stability test were used to measure the MMR activities in cells. To investigate the mechanisms, western blotting, the GFP-heteroduplex repair assay, and chromatin immunoprecipitation were used. RESULTS: We show that knockdown of MRE11A increased the sensitivity of HeLa cells to MNNG treatment, as well as the MNNG-induced DNA damage and apoptosis, implying a potential role of MRE11 in MMR. Moreover, we found that MRE11A was largely recruited to chromatin and negatively regulated the DNA damage signals within the first cell cycle after MNNG treatment. We also showed that knockdown of MRE11A increased, while overexpressing MRE11A decreased, MMR activity in HeLa cells, suggesting that MRE11A negatively regulates MMR activity. Furthermore, we show that recruitment of MRE11A to chromatin requires MLH1 and that MRE11A competes with PMS2 for binding to MLH1. This decreases PMS2 levels in whole cells and on chromatin, and consequently comprises MMR activity. CONCLUSIONS: Our findings reveal that MRE11A is a negative regulator of human MMR.

Clonal origin and genomic diversity in Lynch syndrome-associated endometrial cancer with multiple synchronous tumors: Identification of the pathogenicity of MLH1 p.L582H.

Lynch syndrome-associated endometrial cancer patients often present multiple synchronous tumors and this assessment can affect treatment strategies. We present a case of a 27-year-old woman with tumors in the uterine corpus, cervix, and ovaries who was diagnosed with endometrial cancer and exhibited cervical invasion and ovarian metastasis. Her family history suggested Lynch syndrome, and genetic testing identified a variant of uncertain significance, MLH1 p.L582H. We conducted immunohistochemical staining, microsatellite instability analysis, and Sanger sequencing for Lynch syndrome-associated cancers in three generations of the family and identified consistent MLH1 loss. Whole-exome sequencing for the corpus, cervical, and ovarian tumors of the proband identified a copy-neutral loss of heterozygosity (LOH) occurring at the MLH1 position in all tumors. This indicated that the germline variant and the copy-neutral LOH led to biallelic loss of MLH1 and was the cause of cancer initiation. All tumors shared a portion of somatic mutations with high mutant allele frequencies, suggesting a common clonal origin. There were no mutations shared only between the cervix and ovary samples. The profiles of mutant allele frequencies shared between the corpus and cervix or ovary indicated that two different subclones originating from the corpus independently metastasized to the cervix or ovary. Additionally, all tumors presented unique mutations in endometrial cancer-associated genes such as ARID1A and PIK3CA. In conclusion, we demonstrated clonal origin and genomic diversity in a Lynch syndrome-associated endometrial cancer, suggesting the importance of evaluating multiple sites in Lynch syndrome patients with synchronous tumors.

A multicenter study evaluating efficacy of immune checkpoint inhibitors in advanced non-colorectal digestive cancers with microsatellite instability.

BACKGROUND: One randomized phase III trial comparing chemotherapy (CT) with immune checkpoint inhibitors (ICI) has demonstrated significant efficacy of ICI in deficient DNA mismatch repair system/microsatellite instability-high (dMMR/MSI-H) metastatic colorectal cancer. However, few studies have compared ICI with CT in other advanced dMMR/MSI-H digestive tumors. METHODS: In this multicenter study, we included patients with advanced dMMR/MSI-H non-colorectal digestive tumors treated with chemotherapy and/or ICIs. Patients were divided retrospectively into two groups, a CT group and an immunotherapy (IO) group. The primary endpoint was progression-free survival (PFS). A propensity score approach using the inverse probability of treatment weighting (IPTW) method was applied to deal with potential differences between the two groups. RESULTS: 133 patients (45.1/27.1/27.8% with gastric/small bowel/other carcinomas) were included. The majority of patients received ICI in 1st (29.1%) or 2nd line (44.4%). The 24-month PFS rates were 7.9% in the CT group and 71.2% in the IO group. Using the IPTW method, IO treatment was associated with better PFS (HR=0.227; 95% CI 0.147-0.351; p < 0.0001). The overall response rate was 26.3% in the CT group versus 60.7% in the IO group (p < 0.001) with prolonged duration of disease control in the IO group (p < 0.001). In multivariable analysis, predictive factors of PFS for patients treated with IO were good performance status, absence of liver metastasis and prior primary tumor resection, whereas no association was found for the site of the primary tumor. CONCLUSIONS: In the absence of randomized trials, our study highlights the superior efficacy of ICI compared with standard-of-care therapy in patients with unresectable or metastatic dMMR/MSI-H non-colorectal digestive cancer, regardless of tumor type, with acceptable toxicity.

Imaging-guided prognostic score-based approach to assess the benefits of combotherapy versus monotherapy with immune checkpoint inhibitors in metastatic MSI-H colorectal cancer patients.

BACKGROUND: This retrospective study determined survival responses to immune checkpoint inhibitors (ICIs), comparing mono- (mono) and combo-immunotherapy (combo) in patients with microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC) by analyzing quantitative imaging data and clinical factors. METHODS: One hundred fifty patients were included from two centers and divided into training (n = 105) and validation (n = 45) cohorts. Radiologists manually annotated chest-abdomen-pelvis computed tomography and calculated tumor burden. Progression-free survival (PFS) was assessed, and variables were selected through Recursive Feature Elimination. Cutoff values were determined using maximally selected rank statistics to binarize features, forming a risk score with hazard ratio-derived weights. RESULTS: In total, 2258 lesions were annotated with excellent reproducibility. Key variables in the training cohort included: total tumor volume (cutoff: 73 cm3), lesion count (cutoff: 20), age (cutoff: 60) and the presence of peritoneal carcinomatosis. Their respective weights were 1.13, 0.96, 0.91, and 0.38, resulting in a risk score cutoff of 1.36. Low-score patients showed similar overall survival and PFS regardless of treatment, while those with a high-score had significantly worse survivals with mono vs combo (P = 0.004 and P = 0.0001). In the validation set, low-score patients exhibited no significant difference in overall survival and PFS with mono or combo. However, patients with a high-score had worse PFS with mono (P = 0.046). CONCLUSIONS: A score based on total tumor volume, lesion count, the presence of peritoneal carcinomatosis, and age can guide MSI-H mCRC treatment decisions, allowing oncologists to identify suitable candidates for mono and combo ICI therapies.

Real-time monitoring of replication errors' fate reveals the origin and dynamics of spontaneous mutations.

The efficiency of replication error repair is a critical factor governing the emergence of mutations. However, it has so far been impossible to study this efficiency at the level of individual cells and to investigate if it varies within isogenic cell populations. In addition, why some errors escape repair remains unknown. Here we apply a combination of fluorescent labelling of the Escherichia coli Mismatch Repair (MMR) complex, microfluidics, and time-lapse microscopy, to monitor in real-time the fate of >20000 replication errors. We show that i) many mutations result from errors that are detected by MMR but inefficiently repaired ii) this limited repair efficiency is due to a temporal constraint imposed by the transient nature of the DNA strand discrimination signal, a constraint that is likely conserved across organisms, and iii) repair capacity varies from cell to cell, resulting in a subpopulation of cells with higher mutation rate. Such variations could influence the fitness and adaptability of populations, accelerating for instance the emergence of antibiotic resistance.

Mismatch repair enzymes regulate telomere recombination in Saccharomycescerevisiae.

DNA mismatch repair (MMR) is a crucial mechanism that ensures chromosome stability and prevents the development of various human cancers. Apart from its role in correcting mismatches during DNA replication, MMR also plays a significant role in regulating recombination between non-identical sequences, a process known as homeologous recombination. Telomeres, the protective ends of eukaryotic chromosomes, possess sequences that are not perfectly homologous. While telomerase primarily maintains telomere length in the yeast Saccharomyces cerevisiae, recombination between telomeres becomes a major pathway for length maintenance in cells lacking telomerase. This study investigates the participation of MMR in telomere recombination. Our findings reveal that mutations in MMR genes activate type I recombination. Notably, among the MMR proteins, MutSα (Msh2 and Msh6) and MutLα (Mlh1 and Pms1) exerted the most pronounced effects on telomere recombination. We also found that yeast cells containing simple human telomeric TTAGGG DNA sequences preferentially utilize type II recombination to maintain their telomeres, highlighting the influence of the heterogeneous nature of yeast telomeric sequences on type II recombination. Furthermore, our observations indicate that MMR activity is indispensable for its impact on telomere recombination. Collectively, these results contribute to a more comprehensive understanding of the role of MMR in telomere recombination.

CDK-independent role of D-type cyclins in regulating DNA mismatch repair.

Although mismatch repair (MMR) is essential for correcting DNA replication errors, it can also recognize other lesions, such as oxidized bases. In G0 and G1, MMR is kept in check through unknown mechanisms as it is error-prone during these cell cycle phases. We show that in mammalian cells, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins inhibit the proteasomal degradation of p21, which competes with MMR proteins for binding to PCNA, thereby inhibiting MMR. The ability of D-type cyclins to limit MMR is CDK4- and CDK6-independent and is conserved in G0 and G1. At the G1/S transition, the timely, cullin-RING ubiquitin ligase (CRL)-dependent degradation of D-type cyclins and p21 enables MMR activity to efficiently repair DNA replication errors. Persistent expression of D-type cyclins during S-phase inhibits the binding of MMR proteins to PCNA, increases the mutational burden, and promotes microsatellite instability.

Mismatch repair status and surgical approach in apparent early-stage endometrial cancer.

OBJECTIVE: To test the hypothesis that mismatch repair (MMR) status (as an accurate surrogate marker for microsatellite stability) modifies the effect of surgical approach on oncological outcome for apparent early-stage endometrial cancer. METHODS: Observational data from a large prospective population study on endometrial cancer were analyzed using target trial methodology and doubly robust methods, including propensity score matching and adjusted regression analyses. Laparoscopy was compared with laparotomy, stratified by MMR status on outcomes of recurrence and site, and recurrence-free, overall, and disease-specific survival. RESULTS: After matching, there were 400 patients for analysis, with 200 in each treatment group. The mean age was 62 years and mean body mass index was 32 kg/m2. Most patients had early-stage disease (stage I n=362 (90%)) and endometrioid histology (n=363 (91%)). Adjuvant pelvic radiation was administered to 11%, adjuvant vaginal brachytherapy to 13% and adjuvant chemotherapy to 5% of patients. Five-year recurrence-free survival did not differ significantly between modes of surgery across the cohort (p=0.7) or within MMR strata (MMR-proficient p=0.9, MMR-deficient p=0.6). Similarly, there was no significant difference in overall or disease-specific survival by mode of surgery across the cohort or within MMR strata. There was no significant difference in the HR for recurrence for those treated with laparoscopy stratified by MMR status (MMR-proficient HR=0.99 (95% CI 0.28 to 3.58); MMR-deficient HR=0.83 (95% CI 0.24 to 2.87)), even when restricted to endometrioid subtype. CONCLUSION: In this study, there was no evidence of a difference in survival outcomes according to mode of surgery and MMR status.

Epigenetic MLH1 silencing concurs with mismatch repair deficiency in sporadic, naturally occurring colorectal cancer in rhesus macaques.

BACKGROUND: Naturally occurring colorectal cancers (CRC) in rhesus macaques share many features with their human counterparts and are useful models for cancer immunotherapy; but mechanistic data are lacking regarding the comparative molecular pathogenesis of these cancers. METHODS: We conducted state-of-the-art imaging including CT and PET, clinical assessments, and pathological review of 24 rhesus macaques with naturally occurring CRC. Additionally, we molecularly characterized these tumors utilizing immunohistochemistry (IHC), microsatellite instability assays, DNAseq, transcriptomics, and developed a DNA methylation-specific qPCR assay for MLH1, CACNA1G, CDKN2A, CRABP1, and NEUROG1, human markers for CpG island methylator phenotype (CIMP). We furthermore employed Monte-Carlo simulations to in-silico model alterations in DNA topology in transcription-factor binding site-rich promoter regions upon experimentally demonstrated DNA methylation. RESULTS: Similar cancer histology, progression patterns, and co-morbidities could be observed in rhesus as reported for human CRC patients. IHC identified loss of MLH1 and PMS2 in all cases, with functional microsatellite instability. DNA sequencing revealed the close genetic relatedness to human CRCs, including a similar mutational signature, chromosomal instability, and functionally-relevant mutations affecting KRAS (G12D), TP53 (R175H, R273*), APC, AMER1, ALK, and ARID1A. Interestingly, MLH1 mutations were rarely identified on a somatic or germline level. Transcriptomics not only corroborated the similarities of rhesus and human CRCs, but also demonstrated the significant downregulation of MLH1 but not MSH2, MSH6, or PMS2 in rhesus CRCs. Methylation-specific qPCR suggested CIMP-positivity in 9/16 rhesus CRCs, but all 16/16 exhibited significant MLH1 promoter hypermethylation. DNA hypermethylation was modelled to affect DNA topology, particularly propeller twist and roll profiles. Modelling the DNA topology of a transcription factor binding motif (TFAP2A) in the MLH1 promoter that overlapped with a methylation-specific probe, we observed significant differences in DNA topology upon experimentally shown DNA methylation. This suggests a role of transcription factor binding interference in epigenetic silencing of MLH1 in rhesus CRCs. CONCLUSIONS: These data indicate that epigenetic silencing suppresses MLH1 transcription, induces the loss of MLH1 protein, abrogates mismatch repair, and drives genomic instability in naturally occurring CRC in rhesus macaques. We consider this spontaneous, uninduced CRC in immunocompetent, treatment-naïve rhesus macaques to be a uniquely informative model for human CRC.

Plant-specific environmental and developmental signals regulate the mismatch repair protein MSH6 in Arabidopsis thaliana.

The DNA mismatch repair (MMR) is a postreplicative system that guarantees genomic stability by correcting mispaired and unpaired nucleotides. In eukaryotic nuclei, MMR is initiated by the binding of heterodimeric MutS homologue (MSH) complexes to the DNA error or lesion. Among these proteins, MSH2-MSH6 is the most abundant heterodimer. Even though the MMR mechanism and proteins are highly conserved throughout evolution, physiological differences between species can lead to different regulatory features. Here, we investigated how light, sugar, and/or hormones modulate Arabidopsis thaliana MSH6 expression pattern. We first characterized the promoter region of MSH6. Phylogenetic shadowing revealed three highly conserved regions. These regions were analyzed by the generation of deletion constructs of the MSH6 full-length promoter fused to the ß-glucuronidase (GUS) gene. Combined, our in silico and genetic analyses revealed that a 121-bp promoter fragment was necessary for MSH6 expression and contained potential cis-acting elements involved in light- and hormone-responsive gene expression. Accordingly, light exposure or sugar treatment of four-day old A. thaliana seedlings triggered an upregulation of MSH6 in shoot and root apical meristems. Appropriately, MSH6 was also induced by the stem cell inducer WUSCHEL. Further, the stimulatory effect of light was dependent on the presence of phyA. In addition, treatment of seedlings with auxin or cytokinin also caused an upregulation of MSH6 under darkness. Consistent with auxin signals, MSH6 expression was suppressed in the GATA23 RNAi line compared with the wild type. Our results provide evidence that endogenous factors and environmental signals controlling plant growth and development regulate the MSH6 protein in A. thaliana.

Comparison of standard mismatch repair deficiency and microsatellite instability tests in a large cancer series.

BACKGROUND: The tumor-agnostic indication of immune checkpoint inhibitors to treat cancers with mismatch repair deficiency (dMMR)/microsatellite instability (MSI) increased the demand for such tests beyond Lynch syndrome. International guideline recommendations accept immunohistochemistry (IHC) for dMMR or molecular techniques (PCR or NGS) for MSI status determinations considering the two tests are equal, although there are scattered reports contradicting to this presumption. MATERIALS AND METHODS: Here we have directly compared four protein MMR immunohistochemistry (IHC) to MSI Pentaplex PCR test in a large cancer patient cohort (n = 1306) of our diagnostic center where the two tests have been run parallel in 703 cases. RESULTS: In this study we have found a high discrepancy rate (19.3%) of the two tests which was independent of the tumor types. The MSI PCR sensitivity for MMR IHC status was found to be very low resulting in a relatively low positive and negative predicting values. As a consequence, the correlation of the two tests was low (kappa < 0.7). During analysis of the possible contributing factors of this poor performance, we have excluded low tumor percentage of the samples, but identified dMMR phenotypes (classic versus non-classic or unusual) as possible contributors. CONCLUSION: Although our cohort did not include samples with identified technical errors, our data strongly support previous reports that unidentified preanalytical factors might have the major influence on the poor performance of the MSI PCR and MMR IHC. Furthermore, the case is open whether the two test types are equally powerful predictive markers of immunotherapies.