Evaluation of the MD Anderson tumor score for diffuse large B-cell lymphoma in the rituximab era.

OBJECTIVES: Diffuse large B-cell lymphoma (DLBCL) is an aggressive heterogeneous lymphoma with standard treatment. However, 30%-40% of patients still fail, so we should know which patients are candidates for alternative therapies. IPI is the main prognostic score but, in the rituximab era, it cannot identify a very high-risk (HR) subset. The MD Anderson Cancer Center reported a score in the prerituximab era exclusively considering tumor-related variables: Tumor Score (TS). We aim to validate TS in the rituximab era and to analyze its current potential role. METHODS: From GELTAMO DLBCL registry, we selected those patients homogeneously treated with R-CHOP (n = 1327). RESULTS: Five-years PFS and OS were 62% and 74%. All variables retained an independent prognostic role in the revised TS (R-TS), identifying four different risk groups, with 5-years PFS of 86%, 71%, 50%, and very HR (28%). With a further categorization of three variables of the original TS (Ann Arbor Stage, LDH and B2M), we generated a new index that allowed an improvement in HR assessment. CONCLUSIONS: (a) All variables of the original TS retain an independent prognostic role, and R-TS remains predictive in the rituximab era; (b) R-TS and additional categorization of LDH, B2M, and AA stage (enhanced TS) increased the ability to identify HR subsets.

New prognosis score including absolute lymphocyte/monocyte ratio, red blood cell distribution width and beta-2 microglobulin in patients with diffuse large B-cell lymphoma treated with R-CHOP: Spanish Lymphoma Group Experience (GELTAMO).

The International Prognostic Index (IPI) is the most widely used score for non-Hodgkin lymphoma but lacks the ability to identify a high-risk population in diffuse large B-cell lymphoma (DLBCL). Low absolute lymphocyte count and high monocytes have proved to be unfavourable factors. Red-cell distribution width (RDW) has been associated with inflammation and beta-2 microglobulin (B2M) with tumour load. The retrospective study included 992 patients with DLBCL treated with R-CHOP. In the multivariate analysis, age, Eastern Cooperative Oncology Group performance status (ECOG-PS), stage, bulky mass, B2M, RDW, and lymphocyte/monocyte ratio (LMR) were independently related to progression-free survival (PFS). A new prognosis score was generated with these variables including age categorized into three groups (0, 1, 2 points); ECOG ≥ 3-4 with two; stage III/IV, bulky mass, high B2M, LMR < 2·25 and RDW > 0·96 with one each; for a maximum of 9. This score could improve the discrimination of a very high-risk subgroup with five-year PFS and overall survival (OS) of 19% and 24% versus 45% and 59% of R (revised)-IPI respectively. This score also showed greater predictive ability than IPI. A new score is presented including complete blood cell count variables and B2M, which are readily available in real-life practice without additional tests. Compared to R-IPI, it shows a more precise high-risk assessment and risk discrimination for both PFS and OS.

Differential prognostic impact of GELTAMO-IPI in cell of origin subtypes of Diffuse Large B Cell Lymphoma as defined by the Hans algorithm.

The Grupo Español de Linfomas y Trasplantes de Médula Ósea International Prognostic Index (GELTAMO-IPI) stratifies four risk groups in diffuse large B cell lymphoma (DLBCL) patients treated with immunochaemotherapy: low (LR), low-intermediate (LIR), high-intermediate (HIR), and high (HR). The present study explores the effect of GELTAMO-IPI in the DLBCL subtypes defined by the immunohistochaemistry-based Hans algorithm, Germinal Centre B (GCB) and non-GCB. A multivariate Cox regression model including GELTAMO-IPI risk groups, cell of origin (COO) subtypes and their product was developed to evaluate interaction between the two variables. The COO subtype was available in 839 patients (380 GCB; 459 non-GCB) and both the GELTAMO-IPI and the COO subtype in 780 (353 GCB; 427 non-GCB). There were no differences in 5-year overall survival (OS) between the two subtypes. The Cox model revealed interaction between the GELTAMO-IPI risk groups and the COO subtypes (P = 0·005), indicating that GELTAMO-IPI has a different effect in the two subtypes. Three risk groups were stratified in both COO subtypes: in the GCB subtype, LR, LIR and the combined HIR+HR had 5-year OS of 100%, 75% and 52%, respectively. In the non-GCB subtype, LR, the combined LIR+HIR and HR had a 5-year OS of, 97%, 82% and 35% respectively. GELTAMO-IPI identifies a genuine poor outcome group of patients in the DLBCL non-GCB subtype.

Validation of the NCCN-IPI for diffuse large B-cell lymphoma (DLBCL): the addition of ß2 -microglobulin yields a more accurate GELTAMO-IPI.

The study included 1848 diffuse large B-cell lymphoma (DLBCL)patients treated with chemotherapy/rituximab. The aims were to validate the National Comprehensive Cancer Network International Prognostic Index (NCCN-IPI) and explore the effect of adding high Beta-2 microglobulin (ß2M), primary extranodal presentation and intense treatment to the NCCN-IPI variables in order to develop an improved index. Comparing survival curves, NCCN-IPI discriminated better than IPI, separating four risk groups with 5-year overall survival rates of 93%, 83%, 67% and 49%, but failing to identify a true high-risk population. For the second aim the series was split into training and validation cohorts: in the former the multivariate model identified age, lactate dehydrogenase, Eastern Cooperative Oncology Group performance status, Stage III-IV, and ß2M as independently significant, whereas the NCCN-IPI-selected extranodal sites, primary extranodal presentation and intense treatments were not. These results were confirmed in the validation cohort. The Grupo Español de Linfomas/Trasplante de Médula ósea (GELTAMO)-IPI developed here, with 7 points, significantly separated four risk groups (0, 1-3, 4 or ≥5 points) with 11%, 58%, 17% and 14% of patients, and 5-year overall survival rates of 93%, 79%, 66% and 39%, respectively. In the comparison GELTAMO IPI discriminated better than the NCCN-IPI. In conclusion, GELTAMO-IPI is more accurate than the NCCN-IPI and has statistical and practical advantages in that the better discrimination identifies an authentic high-risk group and is not influenced by primary extranodal presentation or treatments of different intensity.

Analysis of the mutational landscape of classic Hodgkin lymphoma identifies disease heterogeneity and potential therapeutic targets.

Defining the mutational landscape of classic Hodgkin lymphoma is still a major research goal. New targeted next-generation sequencing (NGS) techniques may identify pathogenic mechanisms and new therapeutic opportunities related to this disease. We describe the mutational profile of a series of 57 cHL cases, enriched in Hodgkin and Reed-Sternberg (HRS) cells. Overall, the results confirm the presence of strong genomic heterogeneity. However, several variants were consistently detected in genes related to relevant signaling pathways, such as GM-CSF/IL-3, CBP/EP300, JAK/STAT, NF-kappaB, and numerous variants of genes affecting the B-cell receptor (BCR) pathway, such as BTK, CARD11, BCL10, among others. This unexpectedly high prevalence of mutations affecting the BCR pathway suggests some requirement for active BCR signaling for cHL cell viability. Additionally, incubation of a panel of cHL cellular models with selective BTK inhibitors in vitro constrains cell proliferation and causes cell death. Our results indicate new pathogenic mechanisms and therapeutic opportunities in this disease.

Phase II trial of ofatumumab plus ESHAP (O-ESHAP) as salvage treatment for patients with relapsed or refractory classical Hodgkin lymphoma after first-line chemotherapy.

The management of recurrent/refractory (R/R) Hodgkin lymphoma (HL) remains challenging. Previously published data have shown some efficacy of rituximab in this setting. The purpose of this phase II trial was to investigate the activity of ofatumumab in combination with etoposide, steroids, cytarabine and cisplatin (O-ESHAP) in 62 patients with R/R classical HL. Treatment consisted of ESHAP plus ofatumumab 1000 mg on days 1 and 8 of the first cycle and day 1 of the second and third cycles. O-ESHAP was well tolerated with only 3% of patients requiring treatment discontinuation because of adverse events. Overall response rate was 73% (44% complete metabolic response). In multivariate analysis, early relapse (P < 0·001), bulky disease (P < 0·001) and B symptoms (P < 0·001) were the most important prognostic factors for response. No failures of stem cell mobilization were observed. The high response rate, particularly the complete metabolic response rate, the low toxicity profile, and the high mobilizing potential of the O-ESHAP regimen suggest that patients with R/R HL may benefit from this salvage regimen. However, with the encouraging results observed with other new therapeutic agents in HL, the O-ESHAP regimen could be restricted to patients failing these agents or to those with R/R nodular lymphocyte-predominant HL.

Zeb1 and Snail1 engage miR-200f transcriptional and epigenetic regulation during EMT.

Cell plasticity is emerging as a key regulator of tumor progression and metastasis. During carcinoma dissemination epithelial cells undergo epithelial to mesenchymal transition (EMT) processes characterized by the acquisition of migratory/invasive properties, while the reverse, mesenchymal to epithelial transition (MET) process, is also essential for metastasis outgrowth. Different transcription factors, called EMT-TFs, including Snail, bHLH and Zeb families are drivers of the EMT branch of epithelial plasticity, and can be post-transcriptionally downregulated by several miRNAs, as the miR-200 family. The specific or redundant role of different EMT-TFs and their functional interrelations are not fully understood. To study the interplay between different EMT-TFs, comprehensive gain and loss-of-function studies of Snail1, Snail2 and/or Zeb1 factors were performed in the prototypical MDCK cell model system. We here describe that Snail1 and Zeb1 are mutually required for EMT induction while continuous Snail1 and Snail2 expression, but not Zeb1, is needed for maintenance of the mesenchymal phenotype in MDCK cells. In this model system, EMT is coordinated by Snail1 and Zeb1 through transcriptional and epigenetic downregulation of the miR-200 family. Interestingly, Snail1 is involved in epigenetic CpG DNA methylation of the miR-200 loci, essential to maintain the mesenchymal phenotype. The present results thus define a novel functional interplay between Snail and Zeb EMT-TFs in miR-200 family regulation providing a molecular link to their previous involvement in the generation of EMT process in vivo.

Molecular profiling of circulating tumor cells links plasticity to the metastatic process in endometrial cancer.

BACKGROUND: About 20% of patients diagnosed with endometrial cancer (EC) are considered high-risk with unfavorable prognosis. In the framework of the European Network for Individualized Treatment in EC (ENITEC), we investigated the presence and phenotypic features of Circulating Tumor Cells (CTC) in high-risk EC patients. METHODS: CTC isolation was carried out in peripheral blood samples from 34 patients, ranging from Grade 3 Stage IB to Stage IV carcinomas and recurrences, and 27 healthy controls using two methodologies. Samples were subjected to EpCAM-based immunoisolation using the CELLection™ Epithelial Enrich kit (Invitrogen, Dynal) followed by RTqPCR analysis. The phenotypic determinants of endometrial CTC in terms of pathogenesis, hormone receptor pathways, stem cell markers and epithelial to mesenchymal transition (EMT) drivers were asked. Kruskal-Wallis analysis followed by Dunn's post-test was used for comparisons between groups. Statistical significance was set at p < 0.05. RESULTS: EpCAM-based immunoisolation positively detected CTC in high-risk endometrial cancer patients. CTC characterization indicated a remarkable plasticity phenotype defined by the expression of the EMT markers ETV5, NOTCH1, SNAI1, TGFB1, ZEB1 and ZEB2. In addition, the expression of ALDH and CD44 pointed to an association with stemness, while the expression of CTNNB1, STS, GDF15, RELA, RUNX1, BRAF and PIK3CA suggested potential therapeutic targets. We further recapitulated the EMT phenotype found in endometrial CTC through the up-regulation of ETV5 in an EC cell line, and validated in an animal model of systemic dissemination the propensity of these CTC in the accomplishment of metastasis. CONCLUSIONS: Our results associate the presence of CTC with high-risk EC. Gene-expression profiling characterized a CTC-plasticity phenotype with stemness and EMT features. We finally recapitulated this CTC-phenotype by over-expressing ETV5 in the EC cell line Hec1A and demonstrated an advantage in the promotion of metastasis in an in vivo mouse model of CTC dissemination and homing.

Role of microRNA in epithelial to mesenchymal transition and metastasis and clinical perspectives.

The microRNAs (miRNAs) are a class of small, 20-22 nucleotides in length, endogenously expressed noncoding RNAs that regulate multiple targets posttranscriptionally. Interestingly, miRNAs have emerged as regulators of most physiological and pathological processes, including metastatic tumor progression, in part by controlling a reversible process called epithelial-to-mesenchymal transition (EMT). The activation of EMT increases the migratory and invasive properties fundamental for tumor cell spread while activation of the reverse mesenchymal-to-epithelial transition is required for metastasis outgrowth. The EMT triggering leads to the activation of a core of transcription factors (EMT-TFs) - SNAIL1/SNAIL2, bHLH (E47, E2-2, and TWIST1/TWIST2), and ZEB1/ZEB2 - that act as E-cadherin repressors and, ultimately, coordinate EMT. Recent evidence indicates that several miRNAs regulate the expression of EMT-TFs or EMT-activating signaling pathways. Interestingly, some miRNAs and EMT-TFs form tightly interconnected negative feedback loops that control epithelial cell plasticity, providing self-reinforcing signals and robustness to maintain the epithelial or mesenchymal cell status. Among the most significant feedback loops, we focus on the ZEB/miR-200 and the SNAIL1/miR-34 networks that hold a clear impact in the regulation of the epithelial-mesenchymal state. Recent insights into the p53 modulation of the EMT-TF/miRNA loops and epigenetic regulatory mechanisms in the context of metastasis dissemination will also be discussed. Understanding the regulation of EMT by miRNAs opens new avenues for the diagnosis and prognosis of tumors and identifies potential therapeutic targets that might help to negatively impact on metastasis dissemination and increasing patient survival.

A multimarker panel for circulating tumor cells detection predicts patient outcome and therapy response in metastatic colorectal cancer.

Circulating tumor cells (CTCs), proposed as major players in cancer dissemination, have demonstrated clinical prognostic significance in several cancer types. However, their predictive value remains unclear. Here we evaluated the clinical utility of six CTC markers (tissue specific and epithelial to mesenchymal transition transcripts) both as prognostic and predictive tools in metastatic colorectal cancer (mCRC) patients. CTCs were immunoisolated from blood in 50 mCRC patients at baseline and at 4 and 16 weeks after treatment onset. Expression levels of GAPDH, VIL1, CLU, TIMP1, LOXL3 and ZEB2 were determined by qualitative polymerase chain reaction and normalized to the unspecific cell isolation marker CD45. At baseline, median progression-free survival (PFS) and overall survival (OS) for patients with high CTC markers were 6.3 and 12.7 months, respectively, versus 12.7 and 24.2 for patients with low CTC markers (PFS; p = 0.0003; OS; p = 0.044). Concerning response to therapy, PFS and OS for patients with increased CTC markers along treatment were, respectively, 6.6 and 13.1 months, compared with 12.7 and 24.3 for patients presenting CTC markers reduction (PFS; p = 0.004; OS; p = 0.007). Of note, CTC markers identified therapy-refractory patients not detected by standard image techniques. Patients with increased CTC markers along treatment, but classified as responders by computed tomography, showed significantly shorter survival times (PFS: 7.8 vs. 13.2; OS: 14.4 vs. 24.4; months). In conclusion, we have generated a CTC marker panel for prognosis evaluation and the identification of patients benefiting or not from therapy in mCRC. Our methodology efficiently classified patients earlier than routine computed tomography and from a minimally invasive liquid biopsy.

A core microRNA signature associated with inducers of the epithelial-to-mesenchymal transition.

Although it is becoming clear that certain miRNAs fulfil a fundamental role in the regulation of the epithelial-to-mesenchymal transition (EMT), a comprehensive study of the miRNAs associated with this process has yet to be performed. Here, we profiled the signature of miRNA expression in an in vitro model of EMT, ectopically expressing in MDCK cells one of seven EMT transcription factors (SNAI1, SNAI2, ZEB1, ZEB2, TWIST1, TWIST2 or E47) or the EMT inducer LOXL2. In this way, we identified a core subset of deregulated miRNAs that were further validated in vivo, studying endometrial carcinosarcoma (ECS), a tumour entity that represents an extreme example of phenotypic plasticity. Moreover, epigenetic silencing through DNA methylation of miRNA genes of the miR-200 family and miR-205 that are down-regulated during EMT was evident in both the in vitro (MDCK transfectants) and in vivo (ECS) models of EMT. The strong correlation between expression and DNA methylation suggests a major role for this epigenetic mark in the regulation of the miR-141-200c locus.

E47 and Id1 interplay in epithelial-mesenchymal transition.

E12/E47 proteins (encoded by E2A gene) are members of the class I basic helix-loop-helix (bHLH) transcription factors (also known as E proteins). E47 has been described as repressor of E-cadherin and inducer of epithelial-mesenchymal transition (EMT). We reported previously that EMT mediated by E47 in MDCK cells occurs with a concomitant overexpression of Id1 and Id3 proteins. Id proteins belong to class V of HLH factors that lack the basic domain; they dimerise with E proteins and prevent their DNA interaction, thus, acting as dominant negative of E proteins. Here, we show that E47 interacts with Id1 in E47 overexpressing MDCK cells that underwent a full EMT as well as in mesenchymal breast carcinoma and melanoma cell lines. By conducting chromatin immunoprecipitation assays we demonstrate that E47 binds directly to the endogenous E-cadherin promoter of mesenchymal MDCK-E47 cells in a complex devoid of Id1. Importantly, our data suggest that both E47 and Id1 are required to maintain the mesenchymal phenotype of MDCK-E47 cells. These data support the collaboration between E47 and Id1 in the maintenance of EMT by mechanisms independent of the dominant negative action of Id1 on E47 binding to E-cadherin promoter. Finally, the analysis of several N0 breast tumour series indicates that the expression of E47 and ID1 is significantly associated with the basal-like phenotype supporting the biological significance of the present findings.

Expression of Human MDGA1 Increases Cell Motility and Cell-Cell Adhesion and Reduces Adhesion to Extracellular Matrix Proteins in MDCK Cells.

Characterization of the novel human protein MDGA1 (MAM Domain containing Glycosylphosphatidylinositol Anchor-1) has been reported in our laboratory in the past few years. hMDGA1 is a glycoprotein containing 955 aminoacids (137 kDa) attached to the eukaryotic cell membrane by a GPI (Glycosylphosphatidylinositol) anchor and localized specifically into membrane microdomains known as lipid rafts. Moreover, MDGA1 protein contains structural features found in different types of cell adhesion molecules (CAMs) such as the presence of immunoglobulin domains and a MAM domain (Meprin, A5 protein, receptor protein-tyrosine phosphatase µ), suggesting a role of MDGA1 in cell migration and/or adhesion. In order to investigate this aim, stable MDCK cell lines expressing MDGA1 or the truncated proteins IgGPI (lacking the MAM domain) and MAMGPI (lacking Ig domains) were generated. Our results reveal that MDGA1 increases the ability of MDCK cells to migrate, as it contains both Ig and MAM domains which have been implicated in cell motility. In addition, cell adhesion to extracellular matrix proteins, mainly to collagen IV, is reduced by MDGA1 and the IgGPI and MAMGPI truncated proteins. Accordingly, silencing MDGA1 by siRNA revealed a significant increase in adhesion to collagen IV. Furthermore, MDGA1 expression, through the intrinsic properties of the MAM domain, increases cell-cell adhesion independently of the cell monolayer used, suggesting that MDGA1 mediates cell-cell adhesiveness in a heterophilic manner.

Expression of MMP-9 and TIMP-1 as prognostic markers in gastric carcinoma.

BACKGROUND/AIMS: The aim of the present work is to clarify the role of metalloproteinase-9 and its inhibitor in the evolution of gastric cancer after surgical resection. METHODOLOGY: We have studied 44 gastric cancer patients submitted to surgery. There were 13 proximal tumors, 16 located in the middle third and 15 in the distal one. Overall survival was 26% at 6 years. Metalloproteinase-9 and tissue inhibitor of metalloproteinase concentrations were investigated by means of ELISA in frozen samples of tumoral and normal gastric mucosa. RESULTS: Mean concentration of metalloproteinase-9 in tumoral tissue was 42 ng/mg of total protein, and this value was 6.9 times greater than the mean concentration in non-tumoral tissue. Cancer tissue also expressed higher levels of TIMP-1, 7.25 versus 4.39 ng/mg of protein. Higher levels of metalloproteinase expression in tumoral tissue, greater [metalloproteinase in tumor]/[metalloproteinase in non-tumor] ratio and greater [metalloproteinase]/[inhibitor] ratio in tumor cells, were all of them statistically related to a worse prognosis when T1 and T2 tumors were considered. CONCLUSIONS: The expression of metalloproteinase-9 or its inhibitor is related to a more aggressive phenotype of gastric cancer.

Biological and clinical significance of MMP-2, MMP-9, TIMP-1 and TIMP-2 in non-small cell lung cancer.

Our main aim consists of investigating the clinical usefulness of gelatinases and their tissue inhibitors in non-small cell lung cancer (NSCLC). Thus, we have analysed in 111 NSCLCs, levels and activity of MMP-2, MMP-9, TIMP-1 and TIMP-2, by Enzymoimmunoassay and Gelatine zymography, respectively. Our data revealed higher MMP-2 net activity in the NSCLC population analyzed in this study, this parameter showing a significant association with the TNM stage of tumours (P=0.002). Moreover, MMP-9 levels were significantly associated with poor clinical evolution of patients (P=0.02). Also, disease-free survival time was higher for patients whose tumours showed TIMP-1 increased levels (P=0.04). Of interest, Cox multivariate analysis revealed that TIMP-1 levels can be considered as an independent prognostic factor in NSCLC. Relative Risk (RR) to tumour relapse was more than two times lower for patients showing high TIMP-1 levels (RR=0.420, P=0.041). Therefore, according to our results, we conclude that MMP-9 and TIMP-1 levels of synthesis could be useful for the selection of patients with potentially unfavourable clinical evolution in order to establish adjuvant therapy protocols. Among these parameters, TIMP-1 level evaluation emerges as the main factor to predict the clinical outcome of patients.

Correlations of telomere length, telomerase activity, and telomeric-repeat binding factor 1 expression in colorectal carcinoma.

BACKGROUND: Telomere maintenance has been proposed as an essential step for tumor cell immortalization. The objectives of the current study were to investigate the mechanisms implicated in telomere length in colorectal carcinoma (CRC) and to evaluate the prognostic impact of telomere status. METHODS: Ninety-one colorectal carcinoma samples that were obtained from patients who underwent surgery were analyzed to investigate the factors related to telomere function. The authors studied telomerase activity, terminal restriction fragment (TRF) length, and telomeric-repeat binding factor (TRF1) expression and analyzed the prognostic implications of those factors. RESULTS: Most tumors (81.3%) displayed telomerase activity. Overall, telomeres in CRC specimens were significantly shorter compared with telomeres in normal, adjacent specimens (P=0.02). Moreover, tumors that demonstrated shortened telomeres displayed higher TRF1 levels than tumors without telomere shortening. In relation to patient prognosis, a significantly poor clinical course was observed in the group of patients who had tumors with longer telomeres (P=0.02), and this finding emerged as an independent prognostic factor in a Cox proportional hazards model (P=0.04; relative risk, 6.48). Among patients with tumors classified as telomerase-positive, telomere length ratios (the ratio of tumor tissues to normal tissues)

Impairment of stromelysin-1 transcriptional activity by promoter mutations in high microsatellite instability colorectal tumors.

Colorectal tumorigenesis is characterized by the sequential inactivation of a series of tumor suppressor genes (microsatellite-stable tumors) and genetic or epigenetic alterations in mismatch repair genes in nonpoliposic hereditary tumours and 13% to 15% of sporadic colorectal cancer [high microsatellite instability (MSI-H) tumors]. We hypothesized a molecular mechanism for MSI-H colorectal tumors related to matrix metalloproteinase 3 (MMP-3) promoter mutations, down-regulation of MMP-3 expression, and impairment of MMP-9 activation. We have now analyzed the 2.2-kb full MMP-3 promoter to assess the mutation distribution. The mutations found are restricted to the polymorphic region that includes the zinc-binding protein (ZBP-89) binding element. To show that these alterations were the cause of the low expression of this gene, we have generated three constructs with different MMP-3 promoters (wild type and two mutants) and we have expressed them in SW480 human colorectal cells. The basal transcriptional activity of wild-type MMP-3 promoter was much higher than the mutants activity. In addition, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transcriptional activity of wild-type MMP-3 promoter was 10-fold higher than the mutants activity. Dexamethasone inhibited the basal transcriptional activity of wild-type MMP-3 promoter and of the two mutants found in the MSI-H subgroup of colorectal tumors. Significantly, dexamethasone almost completely blunted the TPA-induced effect on wild-type MMP-3 promoter transcriptional activity and on the mutants, even below their basal activity. Our data show that mutations found in the polymorphic region of the MMP-3 promoter from MSI-H colorectal tumors impair its basal and induced transcriptional activity, which may contribute to their better clinical outcome.