The Impact of MSI Phenotype by MLH1 Expression in Gastric Adenocarcinomas: Critical Analysis of the Brazilian Experience

Kleber Simões, MD, PhD, Carlos Eduardo Jacob, MD, PhD, Rui Manuel Reis, PhD, Bruno Zilberstein, MD, PhD, Suely Nonogaki, BSc, Rodrigo Albergaria Réssio, BSc, Alda Wakamatsu, MSc, Cláudio Bresciani, MD, PhD, Joaquim Jose Gama-Rodrigues, MD, PhD, Ivan Cecconello, MD, PhD, Adhemar Longatto-Filho, MSc, PhD, P.M.I.A.C., Venâncio Avancini Ferreira Alves, MD, PhD

Kleber Simões, Rodrigo Albergaria Réssio, Alda Wakamatsu, Adhemar Longatto-Filho, Venâncio Avancini Ferreira Alves, Laboratory of Medical Investigation (LIM) 14, Faculty of Medicine, University of São Paulo, São Paulo, Brazil
Rui Manuel Reis, Adhemar Longatto-Filho, Molecular Oncology Research Center, Barretos Cancer Hospital, Pio XII Foundation, Barretos, Brazil
Rui Manuel Reis, Adhemar Longatto-Filho, Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal; ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal
Suely Nonogaki, Division of Pathology, Adolfo Lutz Institute, São Paulo, Brazil
Carlos Eduardo Jacob, Bruno Zilberstein, Cláudio Bresciani, Joaquim Jose Gama-Rodrigues, Ivan Cecconello, Department of Gastroenterology, University of Sao Paulo School of Medicine, Brazil.

Conflict-of-interest statement: The authors declare that there is no conflict of interest regarding the publication of this paper.

Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http: //creativecommons.org/licenses/by-nc/4.0/

Correspondence to: Prof. Venâncio Avancini Ferreira Alves, M.D., PhD, Department of Pathology, University of São Paulo School of Medicine, Brazil.
Email: venancio@uol.com.br

Received: October 23, 2017
Revised: November 28, 2018
Accepted: December 2, 2018
Published online: December 21, 2018


BACKGROUND: Gastric cancer remains a clinical concern due to its high prevalence and mortality; consequently, efforts for comprehensive molecular subtyping, as related to morphology and prognosis, are mandatory.

MATERIALS AND METHODS: We correlated the immunohistochemical expression profile of a panel with two major mismatch-repair (MMR) enzymes MLH1 and MSH2 with clinicopathological features and prognosis in a series of 133 gastric cancers and compared with worldwide experience.

RESULTS: MMR negativity was found associated to clinicopathological features as proximal location, predominantly polypoid and fungating macroscopic types (Bormann´s type I and II), intestinal and tubular morphology according to Lauren´s and WHO systems respectively, expansible type according to Ming classification, heavy lymphoplasmacytic infiltrate and absent perineural infiltration. Loss of MLH1 immunoexpression was related to a beneficial impact on disease-specific survival; an independent prognostic impact at multivariate models was observed in cases with the loss of MLH1.

CONCLUSIONS: Taken together, these results validate the biological significance of immunohistochemical detection of MMR enzymes as a method to identify specific pathological features and to assess prognosis in gastric cancer.

Key words: Microsatellite instability; Mismatch repair enzymes; Gastric cancer; Pathology; Prognosis

© 2018 The Author(s). Published by ACT Publishing Group Ltd. All rights reserved.

Simões K, Jacob CE, Reis RM, Zilberstein B, Nonogaki S, Réssio RA, Wakamatsu A, Bresciani C, Gama-Rodrigues JJ, Cecconello I, Longatto-Filho A, Alves VAF. The Impact of MSI Phenotype by MLH1 Expression in Gastric Adenocarcinomas: Critical Analysis of the Brazilian Experience. Journal of Gastroenterology and Hepatology Research 2018; 7(6): 2767-2767 Available from: URL: http: //www.ghrnet.org/index.php/joghr/article/view/2180


Gastric cancer still carries high mortality rates and remains among the most important causes of cancer-related death worldwide, mainly in high incidence areas[1]. Data from USA pointed out 21,600 new cases and 10,990 deaths, annually[2]. Despite the progressive reduction in incidence in the last five decades and improvement on surgical and oncologic treatment, the worldwide incidence clearly demonstrate that Asia still remains as the main focus of gastric cancer burden, with circa 70% of the cases diagnosed globally; conversely, Central and South America have 7% of all gastric cancer reported[1]. However, mortality is Latin America, including Brazil, is proportionally high in comparison with other continents[1-3], which seriously indicate the necessity to improve gastric cancer management. Currently, several efforts are addressed to a better comprehension of molecular characteristics of gastric cancer subtyping, as related to morphology, in order to improve the histopathological classification of these tumors[4]. Moreover, the precise histopathological and molecular classification of the tumours represents critical variables for the efficiency of targeted therapies options[5]. Gastric carcinogenesis is related to a plethora of molecular alterations that involves, epigenetics alteration, genetic mutations and copy number aberrations of oncogenes, suppressor tumor genes, dietary related risk factors where and nitrates and high salt intakes, and infection agents, such as Helicobacter pylori (H. pylori )[6-11]. Among all these variables, the microsatellite instability (MSI) represented a genetic pathway, that is characterized by inactivation of DNA mismatch repair (MMR) genes, and consequently originates frameshift mutation in microsatellite regions leading to new alleles that are not found in the normal subjects[6,10]. The reports that explored the presence of MSI in gastric cancer are limited reports and showed opposing results concerning the associating MSI phenotype and patient survival[8]. This scenario, however, contrast with colorectal cancer (CRC), in which at least two apparently exclusive oncogenic phenotypes have already been well documented and their pathological characteristics and prognostic significance well characterized: chromosomal instability versus microsatellite instability (MSI) pathways, the latter related to methylation phenotype[9]. Chromosomal instability is characterized by significant structural genetic aberrations, such as loss of chromosomal arms, large deletions, insertions, translocations and rearrangements, commonly associated to the classical APC, kRAS and TP53 mutations, usually leading to a worse prognosis. The alternate molecular phenotype, MSI was initially identified as a phenotype of hereditary non-polyposis colorectal cancer syndrome-related tumors (HNPCC or Lynch syndrome); yet, MSI has also been detected in 15% to 20% of sporadic CRC. Although this subdivision is not yet well acknowledged in gastric cancer, rates from 5% to 50% of MSI have been reported in gastric carcinomas. For tumors of different organs, immunohistochemical assessment of MMR enzymes expression (MLH1, MSH2, MSH6 and PMS2) has been validated as a good predictive tool for MSI phenotype detection, and an alternative to more expensive molecular studies as the initial approach in research or diagnostic settings, including screening of tumors of patients fulfilling Amsterdan and/or Bethesda criteria for Lynch syndrome[11, 12]. Currently, validated and well-accepted technical recommendations about MMR status determination employing well-controlled and standardized immunohistochemical assays are avaliable[13-15]. However, herein we opted to analyze the expression of MLH1 enzyme that currently is accepted as one of the critical constituents of the MMR apparatus. The h-MLH1/PMS2 heterodimer was associated to the human MMR system and cancer. Genetic and epigenetic alterations occurring in MLH1 is one of the most important mechanisms that drive MMR alterations occurrence in MSI gastrointestinal carcinomas[16].


Patients and specimens

Paraffin blocks from 133 gastrectomy specimens treated between February 1993 to December 2002 at the Department of Gastroenterology, were retrieved from the files of Department of Pathology, Hospital das Clinicas, University of Sao Paulo, School of Medicine. Inclusion criteria for this study encompassed localized tumors, not showing distant metastatic disease at diagnosis, infiltrating at least muscularis propria (pT2a and higher according to TNM 2002/AJCC staging system), irrespective of lymph node status. All patients were treated according to a well-established surgical protocol (gastrectomy and D2 lymph node dissection) at the Department of Gastroenterology, Hospital das Clinicas, University of Sao Paulo, a national reference center[17]. Patients with familial history of gastric cancer were not included in this study. Clinical status and outpatient follow-up data of patients last updated was in July 2008.

Assessment of pathological variables

Four-micrometer histological sections obtained from original paraffin blocks and, after Haematoxylin-eosin staining, were submitted to histological review. Primary tumors were histologically classified according to World Health Organization[18] in tubular, tubule/papillary, signet-ring cell and mucinous categories. Lauren[19] and Ming´s[20] systems were also applied to primary tumors classification, stratifying lesions into diffuse or intestinal and infiltrating or expansile types respectively, the latter derived from evaluation of deepest tumoral edge. The maximum level of tumoral infiltration in gastric wall was determined as muscularis propria, subserosa, penetrating subserosa or direct invasion of adjacent structures. Lymphatic vascular and perineural invasion was assessed as non-detected or present. Pathological nodal status (pN) was determined by counting of histologically affected lymph nodes and classified as pN0, pN1, pN2 or pN3 according to AJCC/UICC TNM Staging system 1. Information about largest dimension of primary tumor was obtained from original surgical pathology reports, as well as its main location, classified as proximal (cardia, fundus and proximal corpus) or distal tumors (distal corpus, antrum and prepyloric region). Borrmann classification[21] was also achieved from the original macroscopic reports and the cases were grouped in polypoid/fungating (types I and II) or ulcerated/infiltrative/mixed categories (types III, IV and V). Peri/intratumoral inflammatory infiltrate was semi-quantified as absent, mild/moderate and intense whereas desmoplastic stromal response as absent, mild, moderate or intense. Final pathological TNM stage was also assessed.

Tissue Micro-Arrays

All haematoxylin-eosin stained slides were re-evaluated, allowing selection of primary tumor areas for Tissue Microarray (TMA) spotting as previously described[22]. The respective paraffin blocks were marked and TMA constructed using a 1.0 mm gauge for tissue cylinder extraction of donor and placement on receptor block, using a Manual Tissue Microarrayer from Beecher Instruments (model MTA1). Each individual case was sampled twice on receptor block as a strategy for overcoming representativity losses inherent to TMA method, as stated in papers describing validation of this technology[23-28]. TMA paraffin blocks were sectioned on a Leica microtome in three μm sections.


In situ detection of MMR enzymes was achieved through immunohistochemistry (IHC) employing primary monoclonal antibodies to MLH1 (clone G168-15, dilution 1: 100), MSH2 (clone G219-1129, dilution 1: 800), MSH6 (clone 44-MSH6, dilution 1: 100) and PMS2 (clone A16-4, dilution 1: 1.000), all purchased from BD Biosciences Pharmingen. For IHC assays, sectioned slides from TMA paraffin blocks were dewaxed, rehydrated and submitted to heat induced antigen retrieval in pH = 6.0 citrate buffer using a steamer for 30 minutes. Endogenous peroxidase was blocked using 6% H2O2 and sections from primary tumors overnightly incubated with primary antibodies at 4oC. Avidin-biotin free polymer-based amplification system (Novolink, Novocastra, UK) and diaminobenzidine as chromogen were used for development of reaction products. Positive controls represented by two cases of colorectal adenocarcinoma known to express all four MMR enzymes were used, as well as negative controls established by omitting the primary antibody incubation step, were used in every reaction. Lymphocytes, endothelial cells and, whenever present, non-neoplastic gastric foveolar cells served as internal positive controls. Representative spots of primary tumors placed on TMA were evaluated and scored as Lost (when no positive reaction was observed) or Preserved (when at least 10% of positive reaction was found), considering that at least some degree of well-defined nuclear immunostaining must be seen for a case to be regarded as positive. Positive internal controls (lymphocytes, endothelial cells or non-neoplastic mucosa) were found in a particular sample to validate the absence of neoplastic nuclear staining as a true negative result. Each MMR enzyme was individually analyzed, as well as all possible combination sets of negativities, with respective frequencies of each enzyme and combination set reported.

Validation of TMA findings in large sections All cases with TMA samples resulting negative were confirmed by validation of reaction in conventional large sections, with the same stringent criteria used for positive or negative assignment. Cases with negative result in TMA and positive in large section were scored as positive.

Statistical analysis

Clinicopathological variables were compared between MMR enzymes positive and negative groups, stratified according to MLH1 and MSH2 status in primary tumors (lost or preserved) using Pearson´s chi square test and considered significant at p < 0.05. To demonstrate influence of different clinicopathological variables, as well as primary tumor MMR enzymes status, on gastric cancer related mortality, univariate comparisons were made by Log-rank test (considered significant at p < 0.05 level). Cox multivariate proportion of hazards models were constructed to assess differential contributions of clinicopathological variables and MMR status of primary tumors on disease related mortality, with variables selected for inclusion in theses models based on their significance at univariate analysis (p < 0.05), as well as their independence in relation to each other.


Clinicopathological features

From 133 cases, 93 were male (69.9%) and 40 (30.1%) were female. Mean age was 60.1 + 12.8 years old (mean + SD) with a median of 61 years old. Mean primary tumor size was 5.9 + 3.0 cm, with a median of 5.5 cm. Proximal tumors comprising those aroused in cardia, fundus and proximal corpus represented 12.8% of lesions, with the remaining 87.2% aroused in distal corpus, antral and prepyloric regions. Macroscopic types according to Borrmann classification were distributed as follows, type I: 12 (9.0%), type II: 27 (20.3%), type III: 69 (51.9%), type IV: 18 (13.5%) and type V: seven (5.3%). Mean size of primary tumors was 5.9 + 2.1 cm (mean + SD), with a median of 5.5 cm. According to the Lauren histological classification, 83 cases (62.4%) were intestinal-type and 49 (36.8%) were diffuse-type. At World Health Organization (WHO) classification, tubular/tubule-papillary carcinoma comprised 77 cases (57.9%), mucinous type occurred in eight cases (6.0%) and signet-ring cell carcinoma type was assigned to 44 cases (33.1%).

According to Ming classification, 103 cases (77.4%) showed an infiltrating pattern, whereas 30 (22.6%) were found expansive. Histological evaluation of infiltrating level of primary tumor showed that in 58 cases (43.6%) carcinoma compromised muscularis propria, 74 cases (55.6%) invaded to subserosa connective tissue and in 1 case (0.8%) adjacent structures were compromised. Intratumoral lymphoplasmacytic infiltrate was absent in 8 cases (6.0%), mild to moderate in 93 (69.9%) and intense in 32 cases (24.1%). Desmoplastic stromal response was evaluated as absent to mild in 62 cases (46.6%), moderate in 49 (36.8%) and intense in 22 cases (16.6%). Lymphatic invasion was detected in 67 cases (50.4%) and blood vessel invasion in 23 cases (17.3%). Perineural infiltration was detected in 70 cases (52.6%). Lymph node status determined by pathological TNM stratified cases as follows: pN0 in 42 cases (31.6%), pN1 in 63 (47.4%), pN2 in 26 (19.5%) and pN3 in 2 (1.5%). Pathological staging according to TNM showed the following frequencies: Stage IB: 41 cases (30.8%), Stage II: 59 (44.4%), Stage IIIA: 29 (21.8%), Stage IIIB: 2 (1.5%) and Stage IV: 2 (1.5%). Kaplan-Meyer plots for the most significant clinico-pathological features at univariate analysis are demonstrated at Figure1.

Figure 1 Kaplan-Meyer plots for the most signifficant clinico-pathological features at univariate analysis (LogRank and p value in the inset).

MMR status

Despite inherent TMA methodological issues (spot physical or representatives losses), 116 cases were found adequate for MLH1 evaluation (87.2%). Thirty-one gastric adenocarcinoma cases showed loss of MLH1 expression (26.7%).

The MLH1 expression

MLH1 negative tumors showed a larger size (6.9 + 3.3 cm vs 5.6 + 2.7, p = 0.031) than the other groups, were more commonly located at proximal stomach (35.5% vs 7.1%, p < 0.001) and featured a predominantly polypoid and/or fungating Borrmann´s macroscopic types I and II (67.7% vs 18.8%, p < 0.001). Only four MLH1 negative tumors were classified as having diffuse/”signet-ring” cell morphology according to Lauren (12.9% vs 40.5%, p = 0.005) or WHO classification (p = 0.009). Ming´s expansile type was more commonly seen among MLH1 negative tumors when compared to positive ones (38.7% vs 18.8%, p = 0.027). Most MLH1 negative tumors invaded only muscularis propria (67.7%), whereas most MLH1 positive tumors invaded subserosa (62.4%, p = 0.011). Inflammatory infiltrate showed a trend to be more commonly qualified as intense in MLH1 negative tumors (41.9% vs 21.2%, p = 0.064). Table 1 resumes all clinicopathological findings and MLH1 expression.

Table 1 Correlation between clinico-pathological variables and MLH1 status of primary tumors.
Negative (n, %)Positive% (n, %)p
GenderMale22 (71%)59(70.2%)G3G8:G35
Female9 (29%)25 (29.8%)0.939
AgeMean +/- S.D.62.1+15.160,06+11.90.446
Primary tumor locationProximal (Cardia and Fundus)11 (35.5%)6 (7.1%)< 0.001
Distal (Corpus and Antrum)20 (64.5%)68 (92.9%)
Borrmann typeI and II21 (67.7%)16 (19%)< 0.001
III, IV and V10 (32.3%)68 (81%)
WHO histological typeTubular/Tubulo-papillary adenocarcinoma26 (83.9%)44 (52.4%)0.008
Mucinous adenocarcinoma2 (6.5%)4 (4.8%)
Signet-ring cell carcinoma2 (6.5%)32 (38.1%)
Other02 (2.4%)
Undifferentiated02 (2.4%)
Lauren histological typeIntestinal27 (87.1%)49 (59%)0.007
Difuse4 (12.9%)34 (41%)
Ming histological typeInfiltrative19 (61.3%)69 (82.1%)0.019
Expansile12 (38.7%)15 (17.9%)
Deepest infiltrative levelmuscularis propria21 (67.7%)30 (36.1%)0.01
subserosa10 (32.3%)52 (62.6%)
adjacent structures01 (1.2%)
Inflammatory infiltrateabsent2 (6.5%3 (3.6%)0.043
mild to moderate16 (51.6%)63 (75.9%)
accentuated13 (41.9%)17 (20.5%)
Lymphatic invasionnot detected16 (53.3%)36 (43.9%)0.4
present14 (46.7%)46 (56.1%)
Vascular invasionnot detected24 (85.7%)65 (78.3%)0.396
present4 (14.3%)18 (21.7%)
Perineural infiltrationnot detected17 (56.7%)34 (41.5%)0.199
present13 (43.3%)48 (58.5%)
Lymph node statusno lymph node metastasis10 (33.3%)25 (29.8%)0.187
metastases in 1 to 6 (pN1)18 (60%)38 (45.2%)
metastases in 7 to 15 (pN2)2 (6.7%)20 (23.8%)
metastases in more than 15 (pN3)01 (1.2%)
Pathological TNMStage IB10 (32.3%)25 (29.8%)0.285
Stage II18 (58.1%)35 (41.7%)
Stage IIIA3 (9.7%)21 (25%)
Stage IIIB01 (1.2%)
Stage IV02 (2.4%)

Survival analyses

Univariate survival analysis showed that increased gastric cancer related mortality was significantly related to male gender (LogRank: 5.11, p = 0.024), tumors greater than 5.5 cm (LogRank: 3.98, p = 0.046) and distally located (LogRank: 4.49, p 0.034), tumors showing a macroscopic Borrmann types III, IV or V (LogRank: 4.75, p = 0.029). Patients harboring tumors with a “signet-ring” or mucinous morphology according to WHO classification (LogRank: 8.61, p = 0.003), diffuse morphology according to Lauren´s classification (LogRank: 11.62, p = 0.003) and perineural infiltration (LogRank: 12.62, p < 0.001) followed a worse prognosis. Presence of lymph node metastases (LogRank: 10.08, p < 0.001) and TNM stages II and IIIA/IV in relation to stage IB (LogRank: 21.92, p < 0.001) were also significantly related to poorer survival. Age, Ming´s histological type, deepest level of neoplastic infiltration on gastric wall, inflammatory infiltrate and lymphovascular or venous invasion was not statistically related to survival in univariate analyses. The most significant values were seen for patients having MLH1 positive tumors (LogRank: 5.46, p = 0.019).

Cox hazard model analyses

The most significant prognostic variables observed in univariate analyses (gender, Lauren histological classification, perineural infiltration and TNM stage) were included in multivariate Cox hazard proportions models. When MLH1 was included in the Cox model with the variables mentioned, positive tumors had only a marginal association with survival (HR = 2.58, p = 0.053), with Lauren diffuse histological type and pathological TNM stages IIIA and IV showing independent values on survival (HR = 2.18, p = 0.032; HR = 3.68, p = 0.021 and HR = 56.88, p = 0.002 respectively).


Our data highlighted the prominent role of MMR immunohistochemistry evaluation in the clinical scenario of gastric cancer outcome. At least one MSI enzyme was tested negative in the casuistic of gastric carcinoma. An important survival benefit was observed when simultaneous loss of MLH1 and PMS2 (data not shown) expression was observed. Most of the genetic aberrations in gastric cancer have been classified as due to MSI or to loss of heterozigosity (LOH) phenotypes, which are almost mutually exclusive pathways of carcinogenesis[26-31]. Analysis of the length of PCR derived fragments of mono and/or dinucleotide markers has been used as the gold standard for genetic determination of MSI phenotype in gastric cancer as well as in neoplasms from other primary sites, mainly colorectal tumors, as a research or screening tool for patients with suspected familial cancer syndromes[10,32,33]. Molecular classification of MSI phenotype, low frequency MSI and high frequency MSI was proved to stratify sporadic cases of gastric cancer into subsets with preferential clinicopathological features such as tumor location, age, histological type and grade, pathological staging and prognosis[7,34-39]. More recently, immunohistochemistry has been successfully used to demonstrate defective mismatch repair enzyme (MMR) expression in cases with know MSI status, and is currently accepted as another gold standard methodology for MSI screening, mainly in colorectal and endometrial cancer[11,34,40,41,42-49]. Nevertheless, data characterizing MMR enzymes expression in sporadic gastric cancer are still lacking in literature, and its usefulness for the assessment of clinicopathological features, including prognosis, are yet infrequently explored[16,21,34,50,51]. In this context, our data fully characterized the expression of a panel of MMR enzymes in a series of sporadic gastric cancer cases employing a well-controlled immunohistochemistry protocol according to recent recommendations in literature, including antigen retrieval techniques and avidin-biotin free visualization systems[13,52]. In addition, correlations between the statuses of different MMRs related to clinicopathological features and prognosis were also explored.

Defective expression of at least one MMR enzyme was observed in 45 of 133 gastric cancer cases (33.6%), which is quite similar to the range of frequencies of microsatellite instability as determined by genetic approaches reported in literature[52,53]. However, the present study is original on assessing the expression profiles of MMR in gastric cancer and on showing that negativity of MMR enzymes occurs more commonly in association; with only 15 out of 45 cases with some defective MMR enzymes showed only one lost marker (33.3%). Moreover, 14 out of 45 (31.1%) and 7 out of 45 cases (15.5%) had three or all the four MMR enzymes tested negative, respectively.

Association between negative results for MMRs might rise a questioning whether technical issues, such as tissue preservation, problems with antigen retrieval or visualization system, could influence the false-negative results. Thus for, reviewing all cases with stringent criteria for interpretation of negativity[13], validation of TMA negative results in large sections from each case and the already mentioned employment of a well-controlled and literature validated immunohistochemistry procedures[49-52] fully corroborate our in situ findings.

Regarding association of immunohistochemical profile of MMR enzymes with clinical and pathological features, this study demonstrated that tumors located at cardia and fundus, predominantly polypoid and fungating macroscopic types (Borrmann´s types I and II) and intestinal histological type according to Lauren classification are associated with defective expression of MLH1 enzyme). Tubular and/or tubule-papillary histological phenotypes, the counterparts of intestinal type of Lauren´s classification, were significantly associated to loss of expression of MLH1.

Other important clinicopathological features related to MMR negativity were the expansile Ming´s histological type (MLH1, MSH2 and MSH6), tumoral infiltration restricted to muscularis propria (MLH1 and MSH2), absent perineural infiltration (MSH2, MSH6 and PMS2) and older patients at the time of diagnosis (PMS2). Heavy lymphocytic infiltration showed a trend to be associated with MLH1 and MSH6 loss of expression, as well as the absent pattern to mild desmoplastic stromal reaction (MSH6). These data not only represent a more comprehensive approach in relation to previously reported associations between MSI/MMR status and pathological features in gastric cancer, but also partially reproduce what is credited for colorectal cancer, strongly relating microsatellite phenotypes with more favorable pathological features affecting clinical outcome[29,55-59]. Moreover, prognostic impact of MMR immunohistochemistry was demonstrated with univariate disease-specific survival analyses of primary tumor status for MLH1, MSH6 and PMS2. Our data are in accordance with those reported for tumors of other sites, which immunohistochemical determination of MMR protein statuses was demonstrated to be a sensible and specific alternative for molecular analysis for microsatellite status determination, providing meaningful biological information to stratify patients in groups portending tumors with different pathological features as well as clinical outcomes as in colorectal, ovarian, urothelial, biliary and germinative malignant neoplasms[49,60-62]. Considering that mismatch-repair activity is functionally obtained through heterodimerization involving specific MMR enzymes in the so called MutSα (MLH1/PMS2) and MutLα (MSH2/MSH6) complexes[63,64], we have also explored associations between MMR protein profile and clinicopathological features with survival, as well as. The concurrent loss of expression of MLH1 and PMS2 resumed all associations reported with clinicopathological features when the analysis was undertaken for each isolated MMR enzyme. Moreover, Cox multivariate survival analyzes showed that concurrent loss of MLH1 and PMS2 was a significant and independent prognostic factor of improved survival, a fact revealed only when the two enzymes were analyzed in combination. The concurrent loss of expression of the pair MSH2/MSH6 was also related to clinicopathological features of better prognosis, but this pair was not found an independent prognostic factor in Cox multivariate models. Although MSI frequency in gastric cancer varies widely depending on studied group, from 7.1% in American patients of European descent up to 76% of intestinal phenotype gastric cancer in selected oriental populations[54,64,65], it is well known that microsatellite unstable phenotype is characterized by scattered genomic frame shift mutations at mono and/or dinucleotide repeats. Additionally, it is well characterized that increasing frequencies of microsatellite instability occur along progression through gastritis, metaplasia, dysplasia and neoplasia sequence[11,67,68] firstly stated by Correa[69], and is mechanistically explained by progressive loss of MLH1 and/or MSH2 protein expression mainly due to epigenetic silencing of gene expression secondary to CpG island methylation in the sporadic setting[32,70-72]. The better prognosis determinate by microsatellite analysis, unstable phenotype in gastric carcinomas has been reported[7,16,50] and reproduced at the immunohistochemical level in the present study, thus supporting the notion of an independent carcinogenic pathway[73], the resulting alterations in selected molecular pathways responsible for these are just recently being characterized[74]. However, as anticipated, the prognostic value of MSI still remains controversial; although, the pathologic TNM stage and MSI status have been continuously recognized as an independent prognostic factor for overall survival after curative surgery[75]. Frame shift mutations in repeating mono and/or dinucleotide sequences occurring in MSI phenotype were described in genes involved in different homeostatic molecular pathways, mainly those involved in cell proliferation and apoptosis, as well as inactivation of growth control imposed by TGFβ/TGFβRII pathway[34], and can explain the distinctive clinicopathological features and the better prognosis of our series of immunohistochemically detected MSI gastric cancers.

Recently, it was demonstrate an inverse association between the expression of the mismatch repair gene MLH1 and that of the base excision repair (BER) gene DNA polymerase β, which provided evidences that exist a crosstalk between MLH1 and Polβ that modulates the response to alkylation damage. This is important because Polβ/MLH1 can be considered for planning chemotherapeutic approaches for gastric cancer treatment.

In conclusion, in the present study, we characterized the expression profile of a panel of the four main MMR enzymes in a series of gastric carcinomas, employing properly standardized immunohistochemical detection of these antigens as a tool to detect cases presenting certain clinicopathological features. In addition, a survival benefit from MMR loss of expression was demonstrated, mainly when analysis was undertaken considering simultaneous loss of MLH1 and PMS2. These results taken together validate the biological significance of in situ immunohistochemical detection of MMR enzymes as a low cost and widespread available method to identify certain features and to assess prognosis in gastric cancer.


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