Microsatellite instability (MSI) characterizes colorectal carcinomas (CRCs) in hereditary nonpolyposis colorectal cancer (HNPCC) syndrome and a proportion of sporadic CRCs. These MSI+ CRCs share several clinicopathological features, including a reputation for better survival rates than MSI− cases and a pronounced stromal inflammatory reaction of still undefined nature. In the present study, the presence, spatial distribution, and activation status of infiltrating cytotoxic effectors were investigated comparatively in 18 MSI+ and 37 MSI− CRCs by immunohistochemistry. The frequency of apoptosis was also evaluated by morphology and in situ end-labeling. MSI+ cases carried significantly higher numbers of cytotoxic lymphocytes infiltrating within neoplastic epithelial structures, as shown by immunostaining for CD3 (15.1 ± 6.2 versus 4.6 ± 4.1, P < 0.001), CD8 (13 ± 6.4 versus 3.7 ± 3.8, P < 0.001), and TIA-1 (11.2 ± 6.5 versus1.9 ± 1.7, P < 0.001). These cytotoxic effectors were globally more activated in MSI+ than in MSI− tumors, as revealed by the expression of granzyme B (5.3 ± 4.5 versus 0.6 ± 1.3, P < 0.001). In MSI+ CRCs, the number of intraepithelial activated cytotoxic lymphocytes was significantly correlated with the proximal location of the tumor, a poorly differentiated phenotype, and the presence of peritumor lymphoid nodules. Multivariate analysis revealed that MSI was the major determinant of the presence of activated cytotoxic intraepithelial lymphocytes. Moreover, MSI+ CRCs also showed a significantly higher percentage of tumor cells undergoing apoptotic cell death (4.1 ± 2.1 versus 2.6 ± 1.1, P < 0.0001, by the TUNEL method), often located in close proximity of activated cytotoxic lymphocytes. These results are consistent with the presence of anti-tumor cytotoxic immune responses in most of MSI+ CRCs, a phenomenon that may at least in part contribute to the survival advantage ascribed to these patients. Microsatellite instability (MSI) characterizes colorectal carcinomas (CRCs) in hereditary nonpolyposis colorectal cancer (HNPCC) syndrome and a proportion of sporadic CRCs. These MSI+ CRCs share several clinicopathological features, including a reputation for better survival rates than MSI− cases and a pronounced stromal inflammatory reaction of still undefined nature. In the present study, the presence, spatial distribution, and activation status of infiltrating cytotoxic effectors were investigated comparatively in 18 MSI+ and 37 MSI− CRCs by immunohistochemistry. The frequency of apoptosis was also evaluated by morphology and in situ end-labeling. MSI+ cases carried significantly higher numbers of cytotoxic lymphocytes infiltrating within neoplastic epithelial structures, as shown by immunostaining for CD3 (15.1 ± 6.2 versus 4.6 ± 4.1, P < 0.001), CD8 (13 ± 6.4 versus 3.7 ± 3.8, P < 0.001), and TIA-1 (11.2 ± 6.5 versus1.9 ± 1.7, P < 0.001). These cytotoxic effectors were globally more activated in MSI+ than in MSI− tumors, as revealed by the expression of granzyme B (5.3 ± 4.5 versus 0.6 ± 1.3, P < 0.001). In MSI+ CRCs, the number of intraepithelial activated cytotoxic lymphocytes was significantly correlated with the proximal location of the tumor, a poorly differentiated phenotype, and the presence of peritumor lymphoid nodules. Multivariate analysis revealed that MSI was the major determinant of the presence of activated cytotoxic intraepithelial lymphocytes. Moreover, MSI+ CRCs also showed a significantly higher percentage of tumor cells undergoing apoptotic cell death (4.1 ± 2.1 versus 2.6 ± 1.1, P < 0.0001, by the TUNEL method), often located in close proximity of activated cytotoxic lymphocytes. These results are consistent with the presence of anti-tumor cytotoxic immune responses in most of MSI+ CRCs, a phenomenon that may at least in part contribute to the survival advantage ascribed to these patients. Most colorectal carcinomas (CRCs) in hereditary nonpolyposis colorectal cancer (HNPCC) syndrome1Aaltonen LA Peltomäki P Leach FS Sistonen P Pylkkänen L Mecklin JP Järvinen H Powell SM Jen J Hamilton SR Petersen GM Kinzler KW Vogelstein B de la Chapelle A Clues to the pathogenesis of familial colorectal cancer.Science. 1993; 260: 812-816Crossref PubMed Scopus (2597) Google Scholar, 2Wu C Akiyama Y Imai K Miyake S Nagasaki H Oto M Okabe S Iwama T Mitamura K Masumitsu H Nomizu T Baba S Maruyama K Yuasa Y DNA alterations in cells from hereditary non-polyposis colorectal cancer patients.Oncogene. 1994; 9: 991-994PubMed Google Scholar and a proportion (15% to 28%) of sporadic CRCs3Thibodeau SN Bren G Schaid D Microsatellite instability in cancer of the proximal colon.Science. 1993; 260: 816-819Crossref PubMed Scopus (2833) Google Scholar, 4Ionov Y Peinado MA Malkhosyan S Shibata D Perucho M Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis.Nature. 1993; 363: 558-561Crossref PubMed Scopus (2439) Google Scholar, 5Lothe RA Peltomäki P Meling GI Aaltonen LA Nyström Lahti M Pylkkänen L Heimdal K Andersen TI Moller P Rognum TO Fossa SD Haldorsen T Langmark F Brogger A de la Chapelle A Borresen AL Genomic instability in colorectal cancer: relationship to clinicopathological variables and family history.Cancer Res. 1993; 53: 5849-5852PubMed Google Scholar show a particular form of genetic instability, also termed microsatellite instability (MSI), which leads to the accumulation of deletion and insertion mutations at simple repeated sequences. This is the result of the failure to correct mistakes that may occur during DNA replication as a consequence of defects in the mismatch repair system.6Strand M Prolla TA Liskay RM Petes TD Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair.Nature. 1993; 365: 274-276Crossref PubMed Scopus (959) Google Scholar Five causative genes for HNPCC, namely, hMSH2, hMLH1, hPMS1, hPMS2, and hMSH6, have recently been identified as homologues of bacterial DNA mismatch repair genes (reviewed in 7Lynch HT Smyrk MD An update on Lynch syndrome.Curr Opin Oncol. 1998; 10: 349-356Crossref PubMed Scopus (49) Google Scholar). Sporadic CRCs with microsatellite instability (MSI+ cases) share some clinicopathological features with those occurring in HNPCC patients, such as the location in the proximal colon, the mucinous histotype, and poor differentiation.5Lothe RA Peltomäki P Meling GI Aaltonen LA Nyström Lahti M Pylkkänen L Heimdal K Andersen TI Moller P Rognum TO Fossa SD Haldorsen T Langmark F Brogger A de la Chapelle A Borresen AL Genomic instability in colorectal cancer: relationship to clinicopathological variables and family history.Cancer Res. 1993; 53: 5849-5852PubMed Google Scholar, 8Kim H Jen J Vogelstein B Hamilton SR Clinical and pathological characteristics of sporadic colorectal carcinomas with DNA replication errors in microsatellite sequences.Am J Pathol. 1994; 145: 148-156PubMed Google Scholar, 9Risio M Reato G di Celle PF Fizzotti M Rossini FP Foa R Microsatellite instability is associated with the histological features of the tumor in nonfamilial colorectal cancer.Cancer Res. 1996; 56: 5470-5474PubMed Google Scholar In particular, it has been reported that patients with HNPCC10Albano WA Recabaren JA Lynch HT Campbell AS Mailliard JA Organ CH Lynch JF Kimberling WJ Natural history of hereditary cancer of the breast and colon.Cancer. 1982; 50: 360-363Crossref PubMed Scopus (129) Google Scholar, 11Frei JV Hereditary nonpolyposis colorectal cancer (Lynch syndrome II): diploid malignancies with prolonged survival.Cancer. 1992; 69: 1108-1111Crossref PubMed Scopus (41) Google Scholar, 12Watson P Lin KM Rodriguez Bigas MA Smyrk T Lemon S Shashidharan M Franklin B Karr B Thorson A Lynch HT Colorectal carcinoma survival among hereditary nonpolyposis colorectal carcinoma family members.Cancer. 1998; 83: 259-266Crossref PubMed Scopus (205) Google Scholar, 13Kee F Collins BJ Patterson CC Prognosis in familial non-polyposis colorectal cancer.Gut. 1991; 32: 513-516Crossref PubMed Scopus (19) Google Scholar, 14Sankila R Aaltonen LA Järvinen HJ Mecklin JP Better survival rates in patients with MLH1-associated hereditary colorectal cancer.Gastroenterology. 1996; 110: 682-687Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar or MSI+ CRCs3Thibodeau SN Bren G Schaid D Microsatellite instability in cancer of the proximal colon.Science. 1993; 260: 816-819Crossref PubMed Scopus (2833) Google Scholar, 5Lothe RA Peltomäki P Meling GI Aaltonen LA Nyström Lahti M Pylkkänen L Heimdal K Andersen TI Moller P Rognum TO Fossa SD Haldorsen T Langmark F Brogger A de la Chapelle A Borresen AL Genomic instability in colorectal cancer: relationship to clinicopathological variables and family history.Cancer Res. 1993; 53: 5849-5852PubMed Google Scholar show apparently better survival rates than cases carrying MSI− tumors. One attractive hypothesis to explain this phenomenon is constituted by the possibility that, besides favoring the occurrence of mutations at genes critical for oncogenesis, the mutator phenotype may also increase the production of abnormal peptides able to elicit cytotoxic immune responses against tumor cells. Of interest, recent histopathological findings have indicated that MSI+ CRCs, either from HNPCC kindreds or sporadic cases, are characterized by the presence of a pronounced stromal inflammatory reaction.8Kim H Jen J Vogelstein B Hamilton SR Clinical and pathological characteristics of sporadic colorectal carcinomas with DNA replication errors in microsatellite sequences.Am J Pathol. 1994; 145: 148-156PubMed Google Scholar, 9Risio M Reato G di Celle PF Fizzotti M Rossini FP Foa R Microsatellite instability is associated with the histological features of the tumor in nonfamilial colorectal cancer.Cancer Res. 1996; 56: 5470-5474PubMed Google Scholar A similar lymphoid infiltrate is only infrequently observed in MSI− tumors.8Kim H Jen J Vogelstein B Hamilton SR Clinical and pathological characteristics of sporadic colorectal carcinomas with DNA replication errors in microsatellite sequences.Am J Pathol. 1994; 145: 148-156PubMed Google Scholar, 9Risio M Reato G di Celle PF Fizzotti M Rossini FP Foa R Microsatellite instability is associated with the histological features of the tumor in nonfamilial colorectal cancer.Cancer Res. 1996; 56: 5470-5474PubMed Google Scholar Nevertheless, only limited information is currently available on the presence and activation status of cytotoxic effectors in these tumors. Recent experimental data have brought considerable progress in our understanding of the molecular pathways involved in the process of cell killing mediated by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells.15Kägi D Ledermann B Bürki K Zinkernagel RM Hengartner H Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo.Annu Rev Immunol. 1996; 14: 207-232Crossref PubMed Scopus (537) Google Scholar In particular, it has been demonstrated that the granule exocytosis pathway is one of the main mechanisms of cellular cytotoxicity.15Kägi D Ledermann B Bürki K Zinkernagel RM Hengartner H Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo.Annu Rev Immunol. 1996; 14: 207-232Crossref PubMed Scopus (537) Google Scholar In this pathway, the recognition and tight binding of a susceptible target cell by a CTL or NK cell induces the release of electron-dense cytoplasmic granules by the effector cells. These secretory granules contain perforin, a protein that undergoes Ca2+-dependent polymerization on the target cell membrane forming a channel that allows the exocytosis of other granule constituents.16Liu CC Walsh CM Young JD Perforin: structure and function.Immunol Today. 1995; 16: 194-201Abstract Full Text PDF PubMed Scopus (330) Google Scholar These latter include granzymes,17Smyth MJ Trapani JA Granzymes: exogenous proteinases that induce target cell apoptosis.Immunol Today. 1995; 16: 202-206Abstract Full Text PDF PubMed Scopus (365) Google Scholar a family of neutral serine proteases, and the protein TIA-1,18Anderson P Nagler-Anderson C O'Brien C Levine H Watkins S Slayter HS Blue ML Schlossman SF A monoclonal antibody reactive with a 15-kd cytoplasmic granule-associated protein defines a subpopulation of CD8+ T lymphocytes.J Immunol. 1990; 144: 574-582PubMed Google Scholar, 19Tian Q Streuli M Saito H Schlossman SF Anderson P A polyadenylate binding protein localized to the granules of cytolytic lymphocytes induces DNA fragmentation in target cells.Cell. 1991; 67: 629-639Abstract Full Text PDF PubMed Scopus (341) Google Scholar which are critical for the induction of DNA fragmentation and apoptosis of the target cell. Expression of the TIA-1 protein is characteristic of cytotoxic cells, independently of their activation status, whereas perforin and granzymes are produced only by activated effectors.20Liu CC Rafii S Granelli-Piperno A Trapani JA Young JD Perforin and serine esterase gene expression in stimulated human T cells: kinetics, mitogen requirements and effects of cyclosporin A.J Exp Med. 1989; 170: 2105-2118Crossref PubMed Scopus (139) Google Scholar Recently, monoclonal antibodies specifically reacting with these granule proteins became available, thus allowing the identification of activated CTLs or NK cells in situ. The aim of the present investigation was to better define the nature of the lymphoid infiltrate that characterizes MSI+ CRCs. In particular, we sought to determine whether the presence, spatial distribution, and activation status of cytotoxic effectors infiltrating MSI+ CRCs could be compatible with a tumor-specific immune response. The frequency of apoptotic tumor cells was also comparatively analyzed in MSI+ and MSI− CRCs by in situ end-labeling. This study includes 55 primary CRCs from 55 patients (35 males and 20 females; mean age, 60.3 ± 13.9 and 61.2 ± 18.9 years, respectively). Twenty-eight carcinomas were located in the proximal colon (cecum, ascending colon, hepatic flexure, and transverse colon), whereas 27 had distal location (splenic flexure, descending colon, sigmoid, and rectum). Eleven tumors (seven proximal and four distal) were obtained from a series of CRC patients with a suspicion of genetic predisposition based on tumor family history and/or very early age of tumor onset (mean age, 38.5 ± 11.4 years). Five of them were identified as heterozygous carriers of constitutional pathogenetic MLH1 or MSH2 gene mutations.21Viel A Genuardi M Capozzi E Leonardi F Bellacosa A Paravatou Petsotas M Pomponi MG Fornasarig M Percesepe A Roncucci L Tamassia MG Benatti P Ponz de Leon M Valenti A Covino M Anti M Foletto M Boiocchi M Neri G Characterization of MSH2 and MLH1 mutations in Italian families with hereditary nonpolyposis colorectal cancer.Genes Chromosomes & Cancer. 1997; 18: 8-18Crossref PubMed Scopus (75) Google Scholar, 22Genuardi M Anti M Capozzi E Leonardi F Fornasarig M Novella E Bellacosa A Valenti A Gasbarrini G Roncucci L Benatti P Percesepe A: Ponz de Leon M Coco C De Paoli A Valentini M Boiocchi M Neri G Viel A MLH1 and MSH2 constitutional mutations in colorectal cancer families not meeting the standard criteria for hereditary nonpolyposis colorectal cancer.Int J Cancer. 1998; 75: 835-839Crossref PubMed Scopus (54) Google Scholar The other 44 cases were selected from a series of apparently sporadic CRC patients (mean age, 66.2 ± 11.1 years), with unavailable family history, and were enriched in right-sided tumors (21/44) to increase the probability of including MSI+ cases.8Kim H Jen J Vogelstein B Hamilton SR Clinical and pathological characteristics of sporadic colorectal carcinomas with DNA replication errors in microsatellite sequences.Am J Pathol. 1994; 145: 148-156PubMed Google Scholar Conventional histopathological parameters, including Duke's stage, grading, and histotype, were also considered. The presence of peritumor lymphoid nodules, also called Crohn's-like reaction,23Graham DM Appelman HD Crohn's-like lymphoid reaction and colorectal carcinoma: a potential histologic prognosticator.Mod Pathol. 1990; 3: 332-335PubMed Google Scholar was identified and scored as described by Graham and Appleman.23Graham DM Appelman HD Crohn's-like lymphoid reaction and colorectal carcinoma: a potential histologic prognosticator.Mod Pathol. 1990; 3: 332-335PubMed Google Scholar A CRC was defined as mucinous when more than 30% of the tumor area was composed of mucus lakes with interspersed neoplastic cells. Medullary carcinoma was classified as described by Rüschoff.24Rüschoff J Dietmaier W Lüttges J Seitz G Bocker T Zirngibl H Schlegel J Schackert HK Jauch KW Hofstaedter F Poorly differentiated colonic adenocarcinoma medullary type: clinical phenotypic and molecular characteristics.Am J Pathol. 1997; 150: 1815-1825PubMed Google Scholar Thirteen tumors were classified as mucinous whereas three CRCs were of the medullary type. DNA was extracted from peripheral blood lymphocytes and paraffin-embedded normal and tumor tissues and analyzed for evidence of genetic instability at a minimum of six of seven microsatellite loci, including tetra- (L-myc on 1p),25Mäkelä TP Hellsten E Vesa J Alitalo K Peltonen L An Alu variable polyA repeat polymorphism upstream of L-myc at 1p32.Hum Mol Genet. 1992; 1: 217Crossref PubMed Scopus (60) Google Scholar tri- (DM on 19p),26Wooster R Cleton Jansen AM Collins N Mangion J Cornelis RS Cooper CS Gusterson BA Ponder BA von Deimling A Wiestler OD Cornelisse CJ Devilee P Stratton MR Instability of short tandem repeats (microsatellites) in human cancers.Nature Genet. 1994; 6: 152-156Crossref PubMed Scopus (413) Google Scholar di- (D1S170 on 1p, CA21 on 2p, and D3S1611 on 3p),27Engelstein M Hudson TJ Lane JM Lee MK Leverone B Landes GM Peltonen L Weber JL Dracopoli NC A PCR-based linkage map of human chromosome 1.Genomics. 1993; 15: 251-258Crossref PubMed Scopus (43) Google Scholar, 28Hemminki A Peltomäki P Mecklin JP Järvinen H Salovaara R Nyström Lahti M de la Chapelle A Aaltonen LA Loss of the wild type MLH1 gene is a feature of hereditary nonpolyposis colorectal cancer.Nature Genet. 1994; 8: 405-410Crossref PubMed Scopus (282) Google Scholar and mono-nucleotide repeats (BAT-13 and BAT-26 on 2p).29Parsons R Myeroff LL Liu B Willson JK Markowitz SD Kinzler KW Vogelstein B Microsatellite instability and mutations of the transforming growth factor β type II receptor gene in colorectal cancer.Cancer Res. 1995; 55: 5548-5550PubMed Google Scholar Polymerase chain reaction was carried out in the presence of [α-33P]dATP (Amersham, Little Chalfont, UK) as described previously.30Viel A Dall'Agnese L Canzonieri V Sopracordevole F Capozzi E Carbone A Visentin MC Boiocchi M Suppressive role of the metastasis-related nm23–H1 gene in human ovarian carcinomas: association of high messenger RNA expression with lack of lymph node metastasis.Cancer Res. 1995; 55: 2645-2650PubMed Google Scholar Genetic instability was determined as mobility shift of 33P-labeled polymerase chain reaction products, by comparison between tumor and corresponding normal DNAs. If two or more markers were positive, tumors were considered as having high MSI and were defined as MSI+. Tumors with only one positive marker (low MSI) and CRCs with stable microsatellite sequences were defined as MSI−. Immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded material as previously described in detail.31Maestro R Gloghini A Doglioni C Piccinin S Vukosavljevic T Gasparotto D Carbone A Boiocchi M Human non-Hodgkin's lymphomas overexpress a wild-type form of p53 which is a functional transcriptional activator of the cyclin-dependent kinase inhibitor p21.Blood. 1997; 89: 2523-2528PubMed Google Scholar Briefly, sections were deparaffinized in xylene, rehydrated, washed in phosphate-buffered saline (PBS), and, for selected antigens, immersed in 0.01 mol/l citrate buffer, pH 6, or 0.1 mmol/l EGTA and microwaved for 5 minutes at 750 W three times. The sections were then kept for 15 minutes at room temperature (RT) before further PBS washing and immunostaining with a standard streptavidin-biotin-peroxidase procedure and diaminobenzidine (DAB) color reaction. Double-immunostaining procedures were performed on selected samples; the usual streptavidin-peroxidase procedure with brown DAB color reaction was followed by a second cycle of microwaving and a streptavidin-alkaline phosphatase immunostaining with Fast Red as chromogen. The following antibodies were used: anti-CD3 (polyclonal, 1:1000), anti-CD8 (clone cd8/144b, 1:25), and anti-β2-microglobulin (β2M) (polyclonal A072, 1:5000) obtained from DAKO (Glostrup, Denmark); anti-CD4 (clone IF6, 1:100) from Novocastra (Newcastle, UK); anti-granzyme B (GrB; clone GrB7, 1:20) from Monosan (Leiden, The Netherlands); anti-perforin (clone KM585 P1–8, 1:1000) from Kamiya (Nippon); anti-TIA-1 (1:1000) from Coulter Corp. (Hialeah, FL); anti-CD56 (clone 123C3.D5, 1:100) from Neomarker (Freemont, CA); and anti-HLA-ABC monomorphic with preference with HLA-B heavy chain (clone HC10, 1:800), a kind gift of Dr. S. Ferrone (Department of Microbiology and Immunology, New York Medical College, Valhalla, NY). A cocktail of the CAM5.2 (1:50; Becton and Dickinson, Milan, Italy) and the AE1 (1:200; Neomarker) antibodies was used for cytokeratin detection in double-labeling experiments. Nonspecific antibody binding was determined on sections incubated with the same concentration of an irrelevant antibody of the appropriate isotype. Normal tonsil and spleen were used as positive controls. Intratumor-intraepithelial CD3+, CD4+, CD8+, GrB+, perforin+, TIA-1+, and CD56+ cells were counted in 10 randomly selected areas of tumor at ×400 using a Leica DMB microscope. Fields were chosen to contain the maximal amount of neoplastic cells with minimal stroma or necrotic debris. The number of intratumor CD4+ lymphocytes was also evaluated at ×200 in the neoplastic stroma and at the advancing edge of the tumor and scored as follows: 0, <10 CD4+ lymphocytes; +, <50 CD4+ lymphocytes; and ++, >50 CD4+ lymphocytes. Quantification of the relative number of positive cells was performed in all cases by using a video-assisted measuring system (MicroImage, SC Casti Imaging, Italy). All cases were evaluated independently by two pathologists (C. Doglioni and M. Guidoboni). Counts of labeled cells by the two observers did not vary by more than 1%. Apoptotic cells were identified by conventional morphological criteria on hematoxylin and eosin sections. Moreover, the TUNEL method was also used to identify DNA fragmentation in situ by the protocol derived from Gavrieli32Gavrieli Y Sherman Y Ben Sasson SA Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.J Cell Biol. 1992; 119: 493-501Crossref PubMed Scopus (9190) Google Scholar as modified by Migheli.33Migheli A Cavalla P Marino S Schiffer D A study of apoptosis in normal and pathologic nervous tissue after in situ end-labeling of DNA strand breaks.J Neuropathol Exp Neurol. 1994; 53: 606-616Crossref PubMed Scopus (224) Google Scholar All reagents for TUNEL reaction were obtained from Boehringer (Mannheim, Germany). Briefly, rehydrated sections were digested with proteinase K (Sigma) at 20 μg/ml in Tris/EDTA (TE) for 20 minutes at RT. After washing in TE and quenching of peroxidase activity with 3% H2O2 in distilled water for 10 minutes, slides were immersed in TdT buffer (25 mmol/L Tris/HCl, 0.2 mol/L sodium cacodylate, 2.5 mmol/L cobalt chloride, pH 6.6) for 5 minutes and then incubated in TdT mixture composed of 1 part enzyme solution (TdT) and 9 parts label solution (fluorescein-dUTP), diluted 1:4 with TdT buffer, for 2 hours at 37°C. After washing with 2X SSC and Tris-buffered saline, slides were immersed in a solution of Tris-buffered saline/Triton with 2% bovine serum albumin for 15 minutes at RT, followed by incubation with Converter POD (peroxidase-labeled anti-fluorescein antiserum) for 30 minutes at RT. After washing, peroxidase activity was visualized with DAB color reaction. Slides were counterstained with Harris' hematoxylin, dehydrated, and mounted. Double labeling for GrB/TUNEL, CD8/TUNEL, and cytokeratin/TUNEL was performed on selected MSI+ CRCs by sequential TUNEL with DAB as chromogen giving a brown reaction product, followed by immunoalkaline phosphatase staining with a red (Fast Red) reaction product. For each case, the number of morphologically identifiable apoptotic cells and the TUNEL labeling index were determined by counting 1000 neoplastic cells in 10 consecutive fields at ×400, chosen randomly in non-necrotic areas of the tumors. Evaluation was performed with the same video-assisted method mentioned above. Possible relationships between immunophenotypic markers and clinicopathological parameters of CRCs were investigated by comparing mean cellular count values for different levels of clinicopathological variables. Data are expressed as mean ± SD. The Wilcoxon rank-sum test for unpaired data was performed for statistical evaluation of significant difference in the distribution of two groups. Mean counts for different immunophenotypic markers were also compared by analysis of covariance, introducing in the model age and gender. Variable frequency was compared using the χ2 and Fisher's exact tests. A stepwise regression discriminant analysis was performed to identify which of the clinical and histopathological variables discriminated between subjects, after adjusting for age and gender, using cellular count values as a dependent variable. Starting from a full model with all variables included, nonsignificant variables were progressively deleted with a step-down procedure based on a likelihood ratio test. R2, which represents the percentage of variability of each variable considered, was also given from the regression model. The Pearson's method for correlation analysis was used to compare TUNEL labeling index with the number of morphologically identifiable apoptotic cells and GrB+ cell counts, respectively. Overall, 18 of the 55 selected CRCs (33%) were classified as MSI+ and showed instability in two (six cases), three (three cases), four (four cases), five (four cases), and seven loci (one case). Microsatellite alterations at a single locus were found in only two samples. All loci investigated manifested replication errors, with variable frequencies of instability: 33% for L-myc, 31% for BAT26, 20% for BAT13 and D1S170, 14% for CA21, 13% for D3S1611, and 7% for DM. The observed mobility shifts resulted from both increases and decreases in fragment size. The MSI+ cases were also tested for the first panel of five microsatellite sequences previously proposed for the diagnosis of MSI in CRC.34Dietmaier W Wallinger S Bocker T Kullmann F Fishel R Rüschoff J Diagnostic microsatellite instability: definition and correlation with mismatch repair protein expression.Cancer Res. 1997; 57: 4749-4756PubMed Google Scholar High instability (≥40%) was confirmed in 17/18 MSI+ tumors, with the remaining one showing instability at 20% of the loci analyzed. These findings indicate that our panel of microsatellites and our MSI definition criteria were almost superimposable to those recently recommended for diagnostic use.34Dietmaier W Wallinger S Bocker T Kullmann F Fishel R Rüschoff J Diagnostic microsatellite instability: definition and correlation with mismatch repair protein expression.Cancer Res. 1997; 57: 4749-4756PubMed Google Scholar, 35Boland CR Thibodeau SN Hamilton SR Sidransky D Eshleman JR Burt RW Meltzer SJ Rodriguez-Bigas MA Fodde R Ranzani GN Srivastava S A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer.Cancer Res. 1998; 58: 5248-5257PubMed Google Scholar Five of the eighteen MSI+ tumors had developed in patients carrying germline MLH1 or MSH2 gene mutations. For the other 13 patients, the carrier status was excluded (n = 4), or not investigated (n = 9). The incidence of MSI differed substantially in relationship to some clinicopathological parameters (Table 1). In particular, the patients with MSI+ tumors were significantly younger than those with MSI− tumors (P = 0.004), although this was mainly due to to the presence of true and putative HNPCC cases in our series. In fact, with hereditary cases removed (nine MSI+; mean age, 39.6 ± 12.2 years, and two MSI−; mean age, 34 ± 7.1 years), there was no evidence that sporadic MSI+ CRCs (nine cases; mean age, 63.2 ± 14.8 years) occurred in patients significantly younger than sporadic MSI− tumors (35 cases; mean age, 66.9 ± 10.1). In the MSI+ group, there was also a large excess of cancers located in the proximal region (15/18), whereas only 13/37 MSI− tumors were right-sided (P < 0.001). Moreover, the frequency of MSI proved to be significantly higher in poorly differentiated (9/12) than in medium and well differentiated (9/43) cancers (P = 0.002). All but one MSI+ (17/18) and 25/37 MSI− tumors showed the presence of peritumor lymphoid nodules (P = 0.04). All of three of the CRCs of the medullary type were MSI+. No differences related to sex, tumor stage, or mucinous differentiation were found between the two groups. Immunohistochemical analysis showed that cases with total or partial loss of either β2M or HLA class I heavy chain were similarly distributed among MSI+ and MSI− tumors (Table 1).Table 1Distribution of Several Clinicopathological Characteristics of CRCs According to the Presence of MSITotalMSI− (n = 37)MSI+ (n = 18)p-valueAge (mean± SD)65.1± 12.151.4± 17.90.004*Wilcoxon rank sum test.Sex Males3526 (70.3%)9 (50.0%) Females2011 (29.7%)9 (50.0%)0.
