Loss Of MMR Protein Expression Research Articles

Abstract B042: Clinical and molecular characterization of Lynch syndrome-associated pancreas adenocarcinoma (PDAC)

Abstract Background: Lynch syndrome (LS) is a pan-cancer predisposition syndrome caused by pathogenic germline alterations (gPVs) in DNA mismatch repair genes (PMS2, MSH6, MLH1, MSH2, EPCAM). It is characterized by microsatellite instability (MSI)/deficient DNA mismatch repair (dMMR) of associated tumors, which predicts immune checkpoint blockade (ICB) response. Clinical and molecular features of LS-PDAC are not well defined. Herein we sought to describe clinical characteristics, MSI prevalence, and ICB response. Methods: Patients (pts) with LS-associated gPV and PDAC who had consented to IRB-approved protocol of matched tumor-normal sequencing (MSK-IMPACT from which MSIsensor status derivable) were included. MiMSI utilized to assess microsatellite stable (MSS) PDACs to account for low tumor content. MMR status defined with immunohistochemistry (IHC). Composite MSI adjudicated as either loss of MMR protein expression by IHC or MSI by MSISensor or MiMSI. FACETS algorithm assessed biallelic MMR gene alteration status. Mutational signature analysis performed on MSI tumors with deconstructSigs. Results: N=29 pts identified with LS-associated gPVs and PDAC: 10 MSH2 (34%), 9 MSH6 (31%), 7 PMS2 (24%), 2 MLH1 (7%), 1 EPCAM (3%). Majority white (24, 83%) and female (15, 52%). At diagnosis 62% pts early-stage disease (8 stage II, 10 stage III), 11 stage IV. Median age at diagnosis: 68 yrs (range 45-88). Eighteen (62%) had first-degree family history of LS spectrum cancers. Four pts had metachronous cancers (2 CRC, 1 uterine, 1 CRC and uterine). Orthogonal tumor testing feasible in 22 pts (76%). MiMSI (conducted for 16) reclassified 4 as MSI that were indeterminate by MSISensor; IHC (conducted for 15) classified 1/8 tumors as dMMR previously MSS on MSISensor. Whole cohort MSI status: 16 composite MSI (59%), 11 MSS (41%), and 2 MS unknown. gPVs in N= 16 with MSI tumors: 9 MSH2, 4 MSH6, 2 MLH1, and 1 PMS2; amongst N= 11 MSS tumors: 6 gMSH6, 4 gPMS2, and 1 EPCAM. Amongst 24 evaluable pts biallelic loss identified in 6 pts with MSI disease. The dominant mutational signature in 4/6 was Clock/aging and not MSI; 2 of these 4 harbored gMSH2, and 2/4 gMSH6. KRAS and TP53 mutations more common in sporadic MSS PDAC than LS-PDAC (p=0.001, p=0.005 respectively); KRAS G12R was enriched in LS PDAC vs sporadic PDAC (31.6% vs 15.3%, p=0.06). N= 13 (47%) received ICB, inclusive of 4 reclassified by MiMSI and 8 with metastatic disease. In evaluable pts with MSI disease, overall response rate 60% (2 CR, 4 PR, 4 SD). N= 1 MSS PDAC had POD as best response. Median ICB duration: 27.7 months (95% CI 9.0, NR). Conclusion: In this LS PDAC cohort we observed a higher proportion of MSH6, MSH2, PMS2 gPVs relative to other cohorts. LS-PDAC MSI arises frequently in association with gMSH2, gMLH1. MSI status determination with orthogonal testing is critical to appropriately select pts for ICB. Outcome to ICB in LS PDAC MSI comparable to other tumor types highlighting the importance of MSI as a predictive biomarker in PDAC. Citation Format: Emily Harrold, Catherine A. O'Connor, David Lin, Andrea Gazzo, Henry Walch, Megha Ranganathan, Amanda Catchings, Sarah Kane, Fergus Keane, Deborah M. Thurtle-Schmidt, Fiyinfolu Balogun, Anna Varghese, Kenneth Yu, David Kelsen, Alicia Latham, Britta Weigelt, Wungki Park, Zsofia Stadler, Eileen M. O'Reilly. Clinical and molecular characterization of Lynch syndrome-associated pancreas adenocarcinoma (PDAC) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B042.

Disparities in completion of genetic testing and counseling for Lynch syndrome in high-risk patients diagnosed with endometrial cancer (601)

Disparities in completion of genetic testing and counseling for Lynch syndrome in high-risk patients diagnosed with endometrial cancer (601)

Frequency and clinical features of deficient mismatch repair in ovarian clear cell and endometrioid carcinoma.

ObjectiveTo clarify the frequency of deficient mismatch repair (dMMR) in Japanese ovarian cancer patients, we examined microsatellite instability (MSI) status and immunohistochemistry (IHC) subtypes, including endometrioid carcinoma (EMC), clear cell carcinoma (CCC), or a mixture of both (Mix).MethodsWe registered 390 patients who were diagnosed with EMC/CCC/Mix between 2006 and 2015 and treated at seven participating facilities. For 339 patients confirmed eligible by the Central Pathological Review Board, MSI, IHC, and MutL homolog 1 methylation analyses were conducted. The tissues of patients with Lynch syndrome (LS)-related cancer histories, such as colorectal and endometrial cancer, were also investigated.ResultsMSI-high (MSI-H) status was observed in 2/217 CCC (0.9%), 10/115 EMC (8.7%), and 1/4 Mix (25%). Additionally, loss of MMR protein expression (LoE-MMR) was observed in 5/219 (2.3%), 16/115 (14.0%), and 1/4 (25%) patients with CCC, EMC, and Mix, respectively. Both MSI-H and LoE-MMR were found significantly more often in EMC (p<0.001). The median (range) ages of patients with MMR expression and LoE-MMR were 54 (30–90) and 46 (22–76) (p=0.002), respectively. In the multivariate analysis, advanced stage and histological type were identified as prognostic factors.ConclusionThe dMMR rate for EMC/CCC was similar to that reported in Western countries. In Japan, it is assumed that the dMMR frequency is higher because of the increased proportion of CCC.

Morphological predictors for microsatellite instability in urothelial carcinoma

IntroductionMicrosatellite instability occurs due to a series of mutations in the DNA pairing error repair (Mismatch repair; MMR) genes, which can affect germ cells as occurs in Lynch syndrome, whose patients are at high risk of developing multiple cancers. The loss of MMR protein is commonly determined by immunohistochemical studies. Although the relation between microsatellite instability and urothelial carcinomas has been widely studied, its evaluation is not currently performed in the analysis of urothelial carcinomas.MethodsIn this study, the microsatellite status of 139 urothelial carcinomas was analyzed and their clinicopathological characteristics were evaluated. We identified that 10.3% (13 patients) of urothelial carcinomas had loss of MMR protein expression (9 MLH1; 5 MSH2; 2 PMS2; 2 PSH6; n = 139).ResultsResults suggest that these tumors occur more frequently in males, are more frequently located in the bladder or ureters, and present a high tumor grade with a papillary histological pattern that does not infiltrate the lamina propria or, in the case of infiltrating tumors, that grows into perivesical tissues.ConclusionsWe identified patients with the aforementioned tumor characteristics as patients with a high probability of presenting loss of MMR protein expression, and consider that only these patients should undergo further immunohistochemical and molecular techniques for proper diagnosis. Therefore, we propose that the clinicopathological characteristics found in the present study could become possible markers to determine which cases should undergo additional tests.

Co-Occurrence of Familial Non-Medullary Thyroid Cancer (FNMTC) and Hereditary Non-Polyposis Colorectal Cancer (HNPCC) Associated Tumors-A Cohort Study.

Familial non-medullary thyroid cancer (FNMTC) is a form of endocrine malignancy exhibiting an autosomal dominant mode of inheritance with largely unknown germline molecular mechanism. Hereditary nonpolyposis colorectal cancer syndrome (HNPCC) is another hereditary autosomal dominant cancer syndrome which, if proven to be caused by germline mutations in mismatch repair genes (MMR)—MLHL, MSH2, MSH6, PMS2, and EPCAM—is called Lynch syndrome (LS). LS results in hereditary predisposition to a number of cancers, especially colorectal and endometrial cancers. Tumors in LS are characterized by microsatellite instability (MSI) and/or loss of MMR protein expression in immunohistochemistry (IHC). MSI is a rare event in thyroid cancer (TC), although it is known to occur in up to 2.5% of sporadic follicular TC cases. There are limited data on the role of germline MMR variants FNMTC. The goal of this study was to analyze the potential clinical and molecular association between HNPCC and FNMTC. We performed a cohort study analyzing the demographic, clinical, and pathologic data of 43 kindreds encompassing 383 participants (104 affected, 279 unaffected), aged 43.5 [7-99] years with FNMTC, and performed high-throughput whole-exome sequencing (WES) of peripheral blood DNA samples of selected 168 participants (54 affected by FNMTC and 114 unaffected). Total affected by thyroid cancer members per family ranged between 2 and 9 patients. FNMTC was more prevalent in women (68.3%) and characterized by a median tumor size of 1.0 [0.2-5.0] cm, multifocal growth in 44%, and gross extrathyroidal extension in 11.3%. Central neck lymph node metastases were found in 40.3% of patients at presentation, 12.9% presented with lateral neck lymph node metastases, and none had distant metastases. Family history screening revealed one Caucasian family meeting the clinical criteria for FNMTC and HNPCC, with five members affected by FNMTC and at least eight individuals reportedly unaffected by HNPCC-associated tumors. In addition, two family members were affected by melanoma. Genome Analysis Tool Kit (GATK) pipeline was used in variant analysis. Among 168 sequenced participants, a heterozygous missense variant in the MSH2 gene (rs373226409; c.2120G>A; p.Cys707Tyr) was detected exclusively in FNMTC- HNPCC- kindred. In this family, the sequencing was performed in one member affected by FNMTC, HPNCC-associated tumors and melanoma, one member affected solely by HNPCC-associated tumor, and one member with FNMTC only, as well as seven unaffected family members. The variant was present in all three affected adults, and in two unaffected children of the affected member, under the age of 18 years, and was absent in non-affected adults. This variant is predicted to be damaging/pathogenic in 17/20 in-silico models. However, immunostaining performed on the thyroid tumor tissue of two affected by FNMTC family members revealed intact nuclear expression of MSH2, and microsatellite stable status in both tumors that were tested. Although the MSH2 p.Cys707Tyr variant is rare with a minor allele frequency (MAF) of 0.00006 in Caucasians; it is more common in the South Asian population at 0.003 MAF. Therefore, the MSH2 variant observed in this family is unlikely to be an etiologic factor of thyroid cancer and a common genetic association between FNMTC and HNPCC has not yet been identified. This is the first report known to us on the co-occurrence of FNMTC and HNPCC. The co-occurrence of FNMTC and HNPCC-associated tumors is a rare event and although presented in a single family in our large FNMTC cohort, a common genetic background between the two comorbidities could not be established.

PATH-08. EVALUATION OF MISMATCH REPAIR GENE EXPRESSION BY IMMUNOHISTOCHEMISTRY MAY DETECT EARLY PHASE OF MMR DEFICIENCY IN RECURRENT GLIOMAS

Abstract BACKGROUND Mismatch Repair (MMR) Deficiency is common in recurrent gliomas from IDH-mutant and IDH-wild-type tumors. Emergence of MMR deficit is strongly associated with the chemotherapy using TMZ, and it is considered to be a key mechanism of acquiring resistance to TMZ. Several studies showed MMR protein loss detected by immunohistochemistry was well concordant with molecular genetics sequencing data, and sometimes even more reliable in detecting MMR functional deficit. Here we evaluated Mismatch Repair protein expression by immunohistrochemistry for the pairs of primary and recurrent gliomas. METHODS We investigated the expression of 4 MMR proteins (MLH1/MSH2/MSH6/PMS2) in 37 cases of paired primary-recurrent gliomas (7 grade II &amp; III astrocytomas, 16 grade II &amp; III oligodendrogliomas, and 14 glioblastomas). Clinical information including history of chemo-radiotherapy after primary surgery were collected retrospectively. RESULTS Except for one case of GBM, all primary tumors retained 4 MMR proteins. 10 of 37 recurrent tumors lost one or more MMR protein expression (4 GBM 3 astrocytoma, 3 oligodendroglioma), including one case in which primary tumor already had MSH2/MSH6 loss. 2 recurrent GBM lost MMR protein expression homogenously, while in other 8 tumors, the patterns of MMR protein loss were heterogenous. 8 of 10 tumors that lost MMR protein expression were recurrence after TMZ treatment. CONCLUSION Although the interpretation of the heterogenous loss of MMR protein expression is somewhat difficult, the immunohistochemical evaluation of MMR proteins appears to be feasible, and may be able to detect early phase of MMR deficiency and the rise of hypermutator phenotype.

Dedifferentiated Endometrioid Carcinomas with Neuroendocrine Differentiation: A Clinicopathological and Immunohistochemical Study of Three Cases.

PurposeTo investigate the relationship between dedifferentiated endometrioid carcinomas with neuroendocrine differentiation and mismatch repair deficiency.Patients and MethodsThe clinicopathological records and samples of three patients were retrieved from the Pathology Department of Zhejiang University’s School of Medicine Women’s Hospital.ResultsThe tumors comprised one dominant poorly differentiated component (60–90% of the neoplasm volume) and one well-differentiated glandular component. The poorly differentiated component showed solid sheets with organoid growth patterns and insular, trabecular and rosette/pseudorosette patterns. Large polygonal cells, vesicular nuclei, prominent nucleoli, and abundant eosinophilic cytoplasm were observed in the poorly differentiated area. All three cases were diffusely positive for p16 and for at least two of three neuroendocrine markers (chromogranin, synaptophysin, neural cell adhesion molecule (CD56)) in >10% of cancer cells. Loss of MMR protein expression was found in two patients: MLH1 and PSM2 in patient 2 and MSH2 and MSH 6 in patient 3. Abnormal P53 and SMARCB1 (INI1) expression was noted in patient 3. All three patients underwent total abdominal hysterectomy and bilateral salpingo-oophorectomy, and two received postoperative chemotherapy and/or radiation therapy. The patients survived disease-free for 60, 26 and 15 months, respectively.ConclusionDedifferentiated endometrioid carcinomas with neuroendocrine differentiation may be associated with mismatch repair deficiency and have an improved prognosis.

Patients with hMLH1 or/and hMSH2-deficient Metastatic Colorectal Cancer Are Associated with Reduced Levels of Vascular Endothelial Growth Factor-1 Expression and Higher Response Rate to Irinotecan-based Regimen.

The benefit of IFL (irinotecan, fluorouracil and leucovorin) regimen for metastatic colorectal cancer patients (mCRCs) with high levels of microsatellite instability (MSI-H) or loss of mismatch repair (dMMR) protein expression, is uncertain. This study investigated the association of tumour MMR-status and VEGF-1 expression with response to first-line IFL regimen in mCRCs. This prospective study analyzed patients diagnosed with mCRC between August 1st, 1998, and August 30th, 2003, at the Turku University Hospital, Finland. All patients received postoperative IFL regimen. Tumour expression of the MMR proteins, hMLH1 and hMSH2, and VEGF-1 expression were assessed by immunohistochemistry (IHC). Tumours with dMMR were those demonstrating loss of MMR protein expression, and tumours with high VEGF-1 expression were those showing moderate or strong cytoplasmic staining. The primary endpoint was the association between tumour hMLH1 or/and hMSH2-deficient and VEGF-1 expression; the relation between tumour MMR-status and IFL response rate was the secondary endpoint. Of the 67 mCRCs patients, 29 (43%) were hMLH1 or/and hMSH2-deficient and 15 (22%) were pMMR mCRCs. At diagnosis, patients with hMLH1 or/and hMSH2-deficient tumours expressed lower levels of VEGF-1 compared to pMMR tumour patients (p=0.01). More than half (n=17, 59%) of those with dMMR were chemosensitive to first-line IFL regimen, while just one-fifth (n=3, 20%) of those with pMMR were chemosensitive to the IFL regimen (p=0.045). Association between MMR-status and VEGF-1 expression predicts clinical outcome in mCRC patients.

Evaluating Mismatch Repair Deficiency in Pancreatic Adenocarcinoma: Challenges and Recommendations.

Purpose: Immune checkpoint inhibition has been shown to generate profound and durable responses in mismatch repair deficient (MMR-D) solid tumors and has elicited interest in detection tools and strategies to guide therapeutic decision-making. Herein we address questions on the appropriate screening, detection methods, patient selection, and initiation of therapy for MMR-D pancreatic ductal adenocarcinoma (PDAC) and assess the utility of next-generation sequencing (NGS) in providing additional prognostic and predictive information for MMR-D PDAC.Experimental Design: Archival and prospectively acquired samples and matched normal DNA from N = 833 PDAC cases were analyzed using a hybridization capture-based, NGS assay designed to perform targeted deep sequencing of all exons and selected introns of 341 to 468 cancer-associated genes. A computational program using NGS data derived the MSI status from the tumor-normal paired genome sequencing data. Available germline testing, IHC, and microsatellite instability (MSI) PCR results were reviewed to assess and confirm MMR-D and MSI status.Results: MMR-D in PDAC is a rare event among PDAC patients (7/833), occurring at a frequency of 0.8%. Loss of MMR protein expression by IHC, high mutational load, and elevated MSIsensor scores were correlated with MMR-D PDAC. All 7 MMR-D PDAC patients in the study were found to have Lynch syndrome. Four (57%) of the MMR-D patients treated with immune checkpoint blockade had treatment benefit (1 complete response, 2 partial responses, 1 stable disease).Conclusions: An integrated approach of germline testing and somatic analyses of tumor tissues in advanced PDAC using NGS may help guide future development of immune and molecularly directed therapies in PDAC patients. Clin Cancer Res; 24(6); 1326-36. ©2018 AACR.

Dedifferentiated endometrial carcinoma: A report of three cases and review of the literature.

Dedifferentiated endometrial carcinoma, which is defined microscopically as the co-existence of undifferentiated carcinoma and grade 1 or 2 endometrioid adenocarcinoma, is an aggressive type of cancer regardless of the percentage of undifferentiated components. It is reported that undifferentiated carcinoma comprises 9% of endometrial carcinoma. The percentage of dedifferentiated endometrial carcinoma has been hypothesized to be 40% of undifferentiated carcinoma. A precise pathological diagnosis is essential for defining the appropriate therapeutic approach and prognosis. Furthermore, since there is an association between dedifferentiated endometrial carcinoma and Lynch syndrome, it is important to identify the patient's genetic background. The current case report presents three cases of dedifferentiated endometrial carcinoma treated in our hospital. In immunohistochemical staining for DNA mismatch-repair (MMR) proteins in dedifferentiated endometrial carcinoma, the components of undifferentiated carcinoma demonstrated a loss of MMR protein expression, and it is suspected that there may be a germline mutation in these cases. Therefore, Lynch syndrome should be suspected and the appropriate genetic approaches in cases of dedifferentiated endometrial carcinoma should be considered.

Prognostic value of loss of MMR protein expression in endometrial carcinoma in African American and White women

Prognostic value of loss of MMR protein expression in endometrial carcinoma in African American and White women

Uncertainty in the Utility of Mismatch Repair Protein Expression measured by Immunohistochemistry in a Multicenter Population-based Study of Epithelial Ovarian Cancer

immunohistochemistry (IHC) for MMR protein expression. Although the utility of IHC has been demonstrated in colorectal cancer, its utility in ovarian cancer remains incompletely defined. We sought to evaluate the loss of protein expression of the MMR proteins (MLH1, MSH2 and MSH6) in three population-based samples of epithelial ovarian cancers. Methods: IHC staining was performed on full section slides or tissue microarray (TMA) slides for MLH1, MSH2, and MSH6 protein expression in paraffin-embedded tumor samples. Samples were considered to have loss of expression (LoE) if one or more proteins had an expression score of < 3 (on a scale of 9). Collection of demographic, clinical and family history information was attempted in all women. Results: Of 487 participants in whom IHC was completed, 147 were performed through full section slides and 340 were performed through TMA slides. In the overall sample, LoE of one or more of the MMR proteins was observed in 12.7%. Notably, the proportion of tumors with LoE was significantly higher in TMAs (17.9%) compared to full section slides (0.7%) (p <0.001). Finally, tumors with loss of MMR protein expression were more likely to be from individuals with tumors of non-serous histology (p=0.004). Conclusions: A substantial proportion of epithelial ovarian cancers have a loss of MMR protein expression. Protein expression results vary significantly by the tissue sampling methodology utilized, raising concerns about the clinical utility of this test for ovarian tumors.

Prevalence of the mismatch-repair-deficient phenotype in colonic adenomas arising in HNPCC patients: results of a 5-year follow-up study

In hereditary nonpolyposis colorectal cancer (HNPCC) syndrome, more than 90% of the carcinomas show microsatellite instability (MSI) due to a loss of mismatch repair (MMR) function. Although adenomas are very common in HNPCC and demonstrate an accelerated adenoma-carcinoma sequence, data about the prevalence and development of MSI in these early neoplastic lesions are lacking. To determine whether MSI and loss of MMR-protein expression are already present in early stages of tumorigenesis and could therefore be used as a screening tool to identify HNPCC patients before they develop an invasive carcinoma, we analyzed 71 adenomas of 36 HNPCC patients during a 5-year follow-up study. These 36 patients were part of a cohort of 122 HNPCC patients who were investigated at the Institute of Pathology, Klinikum Kassel, as part of the multicentric German HNPCC Consortium, which currently serves more than 2,880 registered families. The diagnosis of HNPCC was based either on the detection of a pathogenic germline mutation in the MSH2, MLH1, or MSH6 genes or in cases where a pathogenic mutation was not found; diagnosis of HNPCC was made, because all patients fulfilled the Amsterdam or Bethesda criteria and revealed a high degree of MSI (MSI-H) as well as loss of one of the MMR proteins by IHC in the cancer tissue. We found that most adenomas (58/71) were MSI-H and had loss of MMR-protein expression. Of the 71 adenomas, 3 were MSI-H with expression of all MMR proteins, and 3 out of 71 displayed loss of a MMR protein with the microsatellites being classified as microsatellite stable (MSS). However, 7 of the 31 adenomas that were located more than 5 cm away from the carcinoma revealed an MSS status (n=6) or low in MSI (n=1) and expressed all MMR proteins. In summary, a significant percentage of HNPCC-associated adenomas (7/31, 22.6%) developing at a distance of more than 5 cm from the corresponding carcinoma did not show the MSI-H MMR-deficient phenotype and expressed all MMR genes. To our knowledge, this is the first study that shows that in most HNPCC patients, the mutator pathway is already detectable in adenomas, but MMR-proficient adenomas can also be found. Therefore, screening for MMR deficiency should not be applied routinely in adenomas with the goal to identify HNPCC patients.

The contribution of the hereditary nonpolyposis colorectal cancer syndrome to the development of ovarian cancer

Objective. Ovarian cancer has one of the highest fractions of hereditary cases. The hereditary breast and ovarian cancer syndrome, primarily due to mutations in BRCA1 and BRCA2, is the main cause of heredity, but also the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome confers an increased risk of ovarian cancer. In order to clarify the contribution of HNPCC to the development of ovarian cancer, we collected data on family history of cancer and characterized MMR function in a consecutive series of 128 tumors unselected for age at diagnosis and previously characterized for BRCA gene mutations. Methods. Expression of the MMR proteins MLH1, PMS2, MSH2, and MSH6 was analyzed by immunohistochemistry using tissue microarray sections. Tumors with reduced staining or loss of staining were also analyzed for microsatellite instability (MSI). Results. Loss of MMR protein expression was identified in 3 ovarian cancers, all of which had a MSI-high phenotype. DNA sequence analysis revealed disease-causing germline mutations (deletions of exons 4–6 in MLH1 and a 1-nucleotide deletion in exon 5 of MSH6) in two patients diagnosed at ages 40 and 49 years, both of whom had family histories suggestive of HNPCC. The genetic defect in the third case, which was a 47-year old woman without knowledge about her family history with loss of MLH1/PMS2 expression in the tumor tissue, remains elusive. A family history suggestive of HNPCC was identified in an additional case, but this tumor showed normal, retained MMR protein expression and a microsatellite stable phenotype. Conclusions. About 2% of ovarian cancer is caused by germline mutations in the MMR-genes, a minor proportion as compared to the contribution of the BRCA-genes (11% in the present series). However, identification of HNPCC patients is important since it allows inclusion of high-risk individuals into control programs aimed at preventing the more frequent colorectal and endometrial cancers. Tumors within the HNPCC-spectrum should therefore be included when recording a family history of cancer among patients diagnosed with ovarian cancer.

Altered expression of the DNA mismatch repair proteins hMLH1 and hMSH2 in cutaneous dysplastic nevi and malignant melanoma

Molecular alterations in the mismatch repair system suggest that this mechanism may be important in the evolution of cutaneous melanoma. Our current study evaluated the expression of two mismatch repair proteins, hMLH1 and hMSH2, in dysplastic nevi (DN) and cutaneous melanoma (CM). Immunohistochemical staining of these proteins was performed on 55 CM and 30 DN specimens. The staining results were divided into three groups: negative, partially positive and strongly positive. Normal adjacent skin cells served as an internal control for positive immunostaining. Altered immunoreactivity of one of the proteins was found in four (13.4%) DN and seven (12.7%) CM. Lack of staining for hMLH1 was observed in two (6.7%) cases of DN and five (9.1%) cases of CM; staining for hMSH2 was absent in two (6.7%) of the DN and two (3.6%) of the CM specimens. Partially positive staining was found in 33.3% and 53.3% for hMLH1 and hMSH2, respectively, in DN, and in 54.5% and 69.1%, respectively, in CMM. Our study shows that complete or partial loss of MMR protein expression occurs in a subset of both DN and CM and may represent a distinct pathway in the development of some DN and CM.

Preoperative serum levels of the carcinoembryonic antigen in hereditary non-polyposis colorectal cancer compared to levels in sporadic colorectal cancer

Background: Carcinoembryonic antigen (CEA) serves as the most widely used and most cost-effective tumor marker in colorectal cancer for almost 30 years. Recent publications about serum CEA levels are based on patient groups without definite differentiation between hereditary and non-hereditary forms of colorectal cancer. Patients and methods: We compared preoperative CEA serum levels from 105 patients with hereditary non-polyposis colorectal cancer (HNPCC) and 107 patients with sporadic colorectal cancer including influences of age and Dukes stage. CEA values in cases of HNPCC were correlated to the findings of microsatellite analyses, mutation analyses of the MMR genes (MLH1, MSH2) and respective immunohistochemistries. Results: Thirty-three HNPCC patients (31%) and 37 patients with sporadic CRC (34%) revealed elevated CEA levels higher than 5 ng/ml. The mean preoperative CEA level in all Dukes stages of HNPCC patients was lower with 31.7 ± 180 ng/ml than in sporadic colorectal cancer with 68.3 ± 424 ng/ml, but without significance ( p = 0.72). HNPCC tumors with signs of de-differentiation (G3 and G4) revealed significantly higher CEA values with 62.2 ± 262 ng/ml in comparison to well-differentiated tumors (G1 and G2) with 5.0 ± 9.6 ng/ml ( p = 0.02). HNPCC patients with “classical characteristics” (high microsatellite instability (MSI), MMR gene mutation, loss of MMR protein expression) had lower preoperative CEA serum levels than those without equivalent genetic alterations, but without reaching statistical significance. CEA levels of HNPCC tumors increased significantly under occurrence of metastases with mean values of 170.3 ± 343 ( p < 0.02). Conclusions: Normal preoperative serum CEA levels do not have the same validity for all colorectal cancer patients. Low CEA levels in HNPCC patients could occur due to well-differentiated tumors and should be considered more critically than in sporadic CRC patients. Further studies including comparison of postoperative CEA development are necessary to elucidate the importance of these results.

Altered Expression of the Dna Mismatch Repair Proteins Hmlh1 and Hmsh2 in Cutaneous Dysplastic Nevi and Malignant Melanoma

Molecular alterations in the mismatch repair system suggest that this mechanism may be important in the evolution of cutaneous melanoma. Our current study evaluated the expression of two mismatch repair proteins, hMLH1 and hMSH2, in dysplastic nevi (DN) and cutaneous melanoma (CM). Immunohistochemical staining of these proteins was performed on 55 CM and 30 DN specimens. The staining results were divided into three groups: negative, partially positive and strongly positive. Normal adjacent skin cells served as an internal control for positive immunostaining. Altered immunoreactivity of one of the proteins was found in four (13.4%) DN and seven (12.7%) CM. Lack of staining for hMLH1 was observed in two (6.7%) cases of DN and five (9.1%) cases of CM; staining for hMSH2 was absent in two (6.7%) of the DN and two (3.6%) of the CM specimens. Partially positive staining was found in 33.3% and 53.3% for hMLH1 and hMSH2, respectively, in DN, and in 54.5% and 69.1%, respectively, in CMM. Our study shows that complete or partial loss of MMR protein expression occurs in a subset of both DN and CM and may represent a distinct pathway in the development of some DN and CM.

Immunohistochemical patterns in rectal cancer: application of tissue microarray with prognostic correlations.

We utilized the high-throughput tissue microarray method to characterize immunohistochemical expression patterns with correlations to prognosis in rectal cancer. Immunostaining for the markers Ki-67, Bcl-2, p53, EGFR, E-cadherin, beta-catenin, MLH1 and MSH2 was performed in 269 rectal cancers. Expression profiles were correlated to metastasis-free survival. Immunostaining revealed frequent upregulation and/or aberrant staining patterns for several of the markers, but Ki-67, p53, Bcl-2 and EGFR did not show any correlation to prognosis. However, reduced membranous staining for beta-catenin (p = 0.04), lack of cytoplasmic staining for beta-catenin (p = 0.04), reduced membranous staining for E-cadherin (p = 0.02) and lack of cytoplasmic staining for E-cadherin (p = 0.02) correlated with metastatic disease. Multivariate analysis including the factors Dukes' stage and tumor differentiation grade demonstrated increased risk of metastatic disease in tumors with lack of cytoplasmic staining for beta-catenin (HR = 3.1, p = 0.02), reduced membranous staining for beta-catenin (HR = 1.7, p = 0.06) and reduced membranous staining for E-cadherin (HR = 2.1, p = 0.06). Loss of MMR protein expression was confirmed to be a rare event in rectal cancer with loss of MLH1 staining in 3% and MSH2 in 1% of the tumors. The lack of prognostic information contributed by most of these markers suggests that single markers for prognosis may be of limited value in rectal cancer. However, altered expression of beta-catenin and E-cadherin correlated with metastatic disease, and these markers may have prognostic importance in rectal cancer.

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We thank Schulmann et al. for the valuable comments related to our article.1 We have rechecked the microsatellite instability (MSI) status and performed additional immunostainings in our material and would like to comment on the questions raised. The objective of our study was to evaluate the frequency of defective mismatch-repair (MMR) in a population-based series of small bowel carcinomas in the Southern Sweden health care region, 1989–1999 (Series I), and in patients who were diagnosed before age 60 (Series II). Because the paraffin-embedded tumor blocks were collected from different pathology departments, and the majority of the patients were diseased, data on family history of cancer were not available. For practical reasons, only tumor-containing blocks were available; therefore, we chose to use mononucleotide markers for the MSI analysis, because these markers can be interpreted without the need for matching normal tissue. However, we agree that an MSI classification based on the instability of BAT40 is suboptimal, although normal allelic size variations as large as those classified as unstable in our material are rare.2 Hence, we cannot rule out the possibility that these two tumors represent false-positive results. Because data on MSI and immunostaining in carcinoma of the small intestine are scarce, it is not known which markers detect MSI with the greatest sensitivity in this tumor type. The sensitivity of the MSI markers depends on the tumor type, as demonstrated, e.g., for colorectal carcinoma and endometrial carcinoma. Furthermore, even if all 4 tumors with low MSI (MSI-L; classified based on data from 3 MSI markers, of which only one was positive) in the population-based series had been excluded, only 7 of 11 tumors (65%) with high MSI (MSI-H) showed loss of MLH1 or MSH2. We do not believe that the MSI-H tumors with retained immunostaining represented false negative MLH1 staining; immunostaining was retained in the stromal components and in the tumor-infiltrating lymphocytes of all these tumors. We routinely perform MSI and MMR protein immunostaining to identify hereditary nonpolyposis colorectal cancer in patients with colorectal carcinoma and have previously experienced a high degree of correlation between the MSI and MMR protein immunostaining. The two tumors with microsatellite stability (MSS) that showed a loss of MLH1 staining likely represent false negative MSI tumors, and although it is a rare finding, loss of MMR protein expression in MSS tumors has been described by several authors and often has been associated with suboptimal archival tumor tissue.3 To characterize the genetic defect behind the MSI tumors with retained expression of MLH1 and MSH2, we have immunostained the 23 evaluable tumors (described in Table 1 of our recent article1) for the MMR protein MSH6. In the population-based series (Series I), one tumor (X42) showed loss of MSH6 staining, and one tumor (X115) among the young patients (Series II) also showed loss of MSH6 alone. In addition, loss of MSH6 was (as expected) observed in three tumors (X123, X134, and X139) with loss of MSH2 staining. Hence, after inclusion of MSH6 staining, loss of MMR protein staining was found in 7 of 15 MSI tumors in the population-based series and in 8 of 10 tumors among individuals younger than age 60 years. In summary, adding immunostaining for MSH6 indeed may explain an additional two discordant (MSI and retained immunostaining for MLH1 and MSH2) tumors. However, in the whole material retained staining for the MMR proteins MLH1, MSH2, and MSH6 still was present in 10 of 21 MSI tumors. Therefore, these findings suggest that other mechanisms may cause a subset of the MMR-defective carcinomas of the small bowel.