An Analysis of Clinical, Surgical, Pathological and Molecular Characteristics of Endometrial Cancer According to Mismatch Repair Status. A Multidisciplinary Approach

Since 2016, our hospital has applied tumor testing with immunohistochemistry (IHC) in endometrial cancer in order to detect mutations of mismatch repair genes (MMR). All cases with MMR deficiency proteins expression are sent for genetic testing, except those with MLH1 protein deficiency, in which case genetic testing is performed if negative for promoter hypermethylation. The primary aim of this study was to investigate the ability of our algorithm to identify Lynch syndrome (LS). The Secondary aims were to investigate the relationship between MMR status and clinicopathological features and prognosis of primary endometrial cancer (EC). From January 2016 to December 2018, 239 patients with EC were retrospectively analyzed and subdivided according to MMR status. Patients were divided in three groups: MMR proficient, LS and Lynch-like cancer (LLC). LS was characterized by a lower age and BMI, more use of contraceptive and less use of hormonal replacement therapy, nulliparity and a trend versus a better prognosis. LLC appeared more related to MMR proficient than LS and exhibited a more aggressive behavior. Our multidisciplinary approach permitted a correct diagnosis of germline mutation in patients with newly diagnosis EC and it confirmed clinicopathologic and prognostic characteristics of LS.

CENP-A binding domains and recombination patterns in horse spermatocytes

Centromeres exert an inhibitory effect on meiotic recombination, but the possible contribution of satellite DNA to this “centromere effect” is under debate. In the horse, satellite DNA is present at all centromeres with the exception of the one from chromosome 11. This organization of centromeres allowed us to investigate the role of satellite DNA on recombination suppression in horse spermatocytes at the stage of pachytene. To this aim we analysed the distribution of the MLH1 protein, marker of recombination foci, relative to CENP-A, marker of centromeric function. We demonstrated that the satellite-less centromere of chromosome 11 causes crossover suppression, similarly to satellite-based centromeres. These results suggest that the centromere effect does not depend on satellite DNA. During this analysis, we observed a peculiar phenomenon: while, as expected, the centromere of the majority of meiotic bivalent chromosomes was labelled with a single immunofluorescence centromeric signal, double-spotted or extended signals were also detected. Their number varied from 0 to 7 in different cells. This observation can be explained by positional variation of the centromeric domain on the two homologs and/or misalignment of pericentromeric satellite DNA arrays during homolog pairing confirming the great plasticity of equine centromeres.

Mlh1 haploinsufficiency induces microsatellite instability specifically in intestine

Tumors of Lynch syndrome (LS) patients display high levels of microsatellite instability (MSI), which results from complete loss of DNA mismatch repair (MMR), in line with Knudson’s two-hit hypothesis. Why some organs, in particular those of the gastrointestinal (GI) tract, are especially prone to tumorigenesis in LS remains unknown. We hypothesized that MMR is haploinsufficient in certain tissues, compromising microsatellite stability in a tissue-specific manner before tumorigenesis. Using mouse genetics, we tested how levels of MLH1, a central MMR protein, affect microsatellite stability in vivo and whether elevated MSI is detectable prior to loss of MMR function and to neoplastic growth. We assayed MSI by sensitive single-molecule PCR in normal jejunum and spleen of 4- and 12-month old Mlh1+/+, Mlh1+/− and Mlh1−/− mice, accompanied by measurements of Mlh1 mRNA and MLH1 protein expression levels.While spleen MLH1 levels of Mlh1+/− mice were, as expected, approximately 50% compared to wildtype mice, MLH1 levels in jejunum varied substantially between individual Mlh1+/− mice and decreased with age. Apparently, Mlh1+/− mice with soma-wide Mlh1 promoter methylation were the most venerable to MLH1 expression level decrease in jejunum. MLH1 levels (prior to complete loss of the protein) inversely correlated with MSI severity in Mlh1+/− jejunum, while in spleens of the same mice, MLH1 levels and microsatellites remained stable. Thus, Mlh1 haploinsufficiency affects specifically the intestine where MMR levels are particularly labile, inducing MSI in normal cells long before neoplasia. A similar mechanism likely also operates in the human GI epithelium, and could explain the wide range in age of onset of LS-associated tumorigenesis.

Streamlining universal screening for lynch syndrome (LS): Towards improved yield of genetic counseling (GC).

503 Background: LS is largely underdiagnosed although Universal Screening (US) in colorectal cancer (CRC) patients through MisMatch Repair deficiency (MMR-d) testing is widely endorsed. Low adherence to guidelines among oncologists may be partly due to a lack of consensus on whether all MMR-d patients should be referred to GC/Genetic Testing (GT). As BRAF mutation rules out LS, we estimated the increased yield of LS diagnosis from GC /GT which could be obtained by selecting candidates for GC through BRAF testing. Methods: From 2011 to 2016, 1447 consecutive stage I-IV CRC surgical patients at a single institution, underwent immunohistochemistry (IHC) for LS using anti MLH1, MSH2, MSH6 and PMS2 antibodies. Oncologists were invited to refer all MMR-d patients to GC/GT. BRAFV600E testing was carried out only in case of MLH1 protein loss at IHC. Results: MMR-d was found in 194 patients (13%), with 171 showing loss of MLH1 expression (88%). Oncologists referred 27 (16%) to GC. Among the 21 who underwent GC, BRAF testing and GT, 9 were BRAF wild type (wt) (43%) and none had LS. Among the 23 MMR-d patients with loss of expression of MSH2, MSH6 or PMS2 (≠MLH1), oncologists referred 9 to GC (39%): 7 underwent GC / GT and 3 carried LS (43%) at GT. Median age was 76 years (range 30-97) in the MMR-d group, 78 (range 41-97) in the MLH1 group and 63 (range 30-86) in the ≠MLH1 group. Overall, LS was diagnosed in 3 of the 28 MMR-d patients (11%) who underwent GC /GT, possibly an underestimate due to the advanced median age of our MLH1 loss patients. Had we only offered GC to the 9 BRAF wt patients among the 21 with MLH1 loss, we could have avoided 12 (57%) of the GC sessions conducted, increasing the yield of LS diagnosis from 3/28 (11%) to 3/16 (19%) (75% increase). Conclusions: When US for LS is adopted, a GC referral rate reduction of 57% among MLH1 loss patients, and an overall increase in the yield of GC of about 75% can be obtained by testing for BRAF mutation before oncologist referral to GC rather than after. As multistep selection of patients by oncologists may be unfeasible, CRC pathology reports with combined MMR-d and BRAF testing (for MLH1 loss at IHC) and an ‘LS suspicion alert’ could improve oncologists’ awareness of LS and compliance with guidelines.

Microsatellite unstable gastrointestinal neuroendocrine carcinomas: a new clinicopathologic entity

Gastroenteropancreatic (GEP) neuroendocrine carcinomas (NECs) and mixed adenoneuroendocrine carcinomas (MANECs) are heterogeneous neoplasms characterized by poor outcome. Microsatellite instability (MSI) has recently been found in colorectal NECs showing a better prognosis than expected. However, the frequency of MSI in a large series of GEP-NEC/MANECs is still unknown. In this work, we investigated the incidence of MSI in GEP-NEC/MANECs and characterized their clinicopathologic and molecular features. MSI analysis and immunohistochemistry for mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2) were performed in 89 GEP-NEC/MANECs (six esophageal, 77 gastrointestinal, three pancreatic, and three of the gallbladder). Methylation of 34 genes was studied by methylation-specific multiplex ligation probe amplification. Mutation analysis of BRAF and KRAS was assessed by PCR-pyrosequencing analysis. MSI was observed in 11 NEC/MANECs (12.4%): seven intestinal and four gastric. All but two MSI-cases showed MLH1 methylation and loss of MLH1 protein. The remaining two MSI-cancers showed lack of MSH2 or PMS2 immunohistochemical expression. MSI-NEC/MANECs showed higher methylation levels than microsatellite stable NEC/MANECs (40.6% vs 20.2% methylated genes respectively, P<0.001). BRAF mutation was detected in six out of 88 cases (7%) and KRAS mutation was identified in 15 cases (17%). BRAF mutation was associated with MSI (P<0.0008), while KRAS status did not correlate with any clinicopathologic or molecular feature. Vascular invasion (P=0.0003) and MSI (P=0.0084) were identified as the only independent prognostic factors in multivariate analysis. We conclude that MSI identifies a subset of gastric and intestinal NEC/MANECs with distinct biology and better prognosis. MSI-NEC/MANECs resemble MSI-gastrointestinal adenocarcinomas for frequency, molecular profile and pathogenetic mechanisms.

Identifying BRAF mutations in colorectal cancer (CRC) by initial immunohistochemical (IHC) staining for mismatched protein expression as part of a Lynch syndrome screening program: A distinct clinicopathologic entity.

405 Background: Multiple genetic mutations resulting in different clinical phenotypes have been found in colorectal adenocarcinoma. Mutation in the BRAF oncogene is a key step in malignant transformation within the methylator pathway leading to colorectal cancer. There is little data regarding the identification of the subset of CRCs exhibiting mutations in the BRAF oncogene with initial IHC staining, as well as a paucity of information describing this unique subset of colorectal cancers Methods: Between 1/1/11 to 12/31/12, all newly diagnosed CRCs underwent IHC testing for MLH1, MSH2, MHS6, and PMS2 protein expression if sufficient pathologic material existed. If MLH1 protein expression was absent, BRAF V600E mutation analysis was preformed. Patient demographics, pathology and outcomes were examined through chart review. Statistical associations were determined by the TTest, Fisher’s exact probability test and Cox regression analysis of Kaplan-Meier curves. Results: 314 newly diagnosed CRC patients were identified and 278 (89%) underwent IHC staining (study population). 48 had absent MLH1 protein expression (15%) and underwent BRAF mutational analysis. 35/48 had the V600E mutation. Comparing the BRAF mutated group to the study population without BRAF mutation showed statistically significant differences. These differences included median age at diagnosis (78 vs. 65 years, p < 0.0001), female sex (77% vs. 51%, p = 0.0037), right colon location (77% vs. 42%, p = 0.0001) and significant mucinous component denoted pathologically (31% vs. 7%, p = 0.0001). There was no difference in stage of CRC at diagnosis between the groups. Survival rates at one year based on Kaplan Meier curves were 65% vs. 82% and results will be updated. Conclusions: BRAF mutations in CRC are associated with unique clinicopathologic characteristics and overall worse prognosis. This subgroup of CRC can be identified as part of a Lynch Screening program via ICH staining of the mismatch repair proteins MLH1, MSH2, MSH6, and PMS2.

Humans accumulate microsatellite instability with acquired loss of MLH1 protein in hematopoietic stem and progenitor cells as a function of age

Hematopoietic stem and progenitor cells (HPCs) are necessary for long-term survival. Genomic instability and persistent DNA damage may cause loss of adult stem cell function. The mismatch repair (MMR) pathway increases replication fidelity and defects have been implicated in malignant hematopoietic diseases. Little, however, is known about the role MMR pathway failure plays in the aging process of human HPCs. We hypothesized that loss of MMR occurs in HPCs as a process of human aging. We examined microsatellite instability and expression of the MMR genes MutL homologue 1 (MLH1) and MutS homologue 2 (MSH2) in HPCs and colony-forming cell-derived clones (CFCs) from human donors aged 0 to 86 years. CFCs from donors > 45 years had a greater frequency of microsatellite instability and CD34+ progenitors lacking MLH1 expression and protein than individuals ≤ 45 years. Loss of MSH2 did not correlate with age. Thus, a potentially early event in the normal human aging process is microsatellite instability accumulation in normal human HPCs associated with the loss of MLH1 protein expression.

Clinical Implications of Microsatellite Instability and MLH1 Gene Inactivation in Sporadic Insulinomas

Context: The molecular pathogenesis of sporadic insulinomas is unknown. There is a lack of biomarker to distinguish benign and malignant form of insulinoma. Objective: Our objective was to confirm the occurrence of microsatellite instability (MSI) in insulinomas, to identify alterations of mismatch repair (MMR) genes in the tumors, and to evaluate the possibility to distinguish benign and malignant insulinoma or to predict the clinical outcome of patients with these alterations. Design and Patients: We detected MSI and inactivation of MLH1 gene in 55 sporadic insulinomas by PCR, immunohistochemical staining, allelic typing, analysis of promoter methylation, and exon mutations. Their correlations with clinicopathological characteristics were analyzed with univariate and multivariate statistic analysis. Results: A high rate of MSI (MSI-H) was found in 33% of sporadic insulinomas. Reduced expression of mutL homolog 1 (MLH1) protein was observed in 36% of insulinomas and correlated with MSI-H (P = 0.008). Promoter methylation and loss of heterozygosity of MLH1 gene was found in 31 and 49% of insulinomas, respectively. Reduced expression of MLH1 and MSI-H were significantly associated with both tumor malignancy (P = 0.033 and P = 4.8 × 10−6, respectively) and incurable disease (P = 0.006 and P = 0.001, respectively). Conclusion: High frequency of MSI occurred in sporadic insulinomas. The silencing of MLH1 gene may partially contribute to the MSI-H in the tumors. Assessing MSI-H and expressions of MLH1 could be used to distinguish benign and malignant insulinomas and to predict the outcome of patients. Detecting of a high rate of microsatellite instability can be used to distinguish malignancy from benign, and predict clinical outcome of the sporadic insulinomas.