Received date: August 23, 2013; Accepted date:October 15, 2013; Published date: October 25, 2013
Citation: Vageli DP, Papamichali R, Kambosioras K, Papandreou CN, Koukoulis GK (2013) Mismatch DNA Repair hMSH2, hMLH1, hMSH6 and hPMS2 mRNA Expression Profiles in Colorectal Carcinomas J Genet Syndr Gene Ther 4:191. doi:10.4172/2157-7412.1000191
Copyright: © 2013 Vageli DP, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Background: Mismatch repair (MMR) deficiency has been related with HNPCCs. So far, there is limited information on MMR mRNA profiles in sporadic colorectal carcinomas (CRCs). We previously showed that distinct MMR mRNA phenotypes were related to tumor stage and survival of patients with lung cancer or urinary bladder carcinomas.
Aim: The aim of this study was to quantify hMSH2, hMLH1, hMSH6 and hPMS2 mRNA levels, in CRCs and their adjacent normal tissues (ANTs), using accurate methodology, and to correlate MMR mRNA profiles with patient or tumor characteristics.
Materials and methods: We analyzed 31 fresh frozen tissue specimens of paired CRCs with their ANTs. We evaluated MMR mRNA profiles by a Q-real-time PCR, using hPBGD gene as reference control and creating a standard curve. The MMR mRNA levels were assigned as ratios MMR/hPBGD mRNAs. Relative expression of each MMR gene was given as ratios of CRCs/ANTs mRNA levels.
Results: All CRCs and their ANTs expressed low hPMS2 mRNA levels while a significant proportion of CRCs (73%) and their ANTs (82%) presented low hMSH2 mRNA levels. Analysis of relative expression patterns showed that hMSH6 and hMLH1 exhibited the highest percentages of reduction (53% and 45.5%, respectively). We found a correlation of transcriptional levels between hMSH2 and hMLH1, the crucial components of MMR mechanism and between their counterparts, hMSH6 and hPMS2, in CRCs of early stages, related to gender. On the contrary, CRCs of late stages revealed a correlation between reduced levels of hMSH2 and hMSH6, MutSa components, unrelated to gender but related to lymph node metastasis. Also, reduced hMSH2, hMSH6 and hMLH1 mRNA phenotypes correlated with advanced stage, and rectal localization.
Conclusion: In this study we demonstrated that MMR mRNA deficiency is a common event in sporadic CRCs. Specific profiles of MMR deficiency maybe related to tumor progression, especially in male patients.
Colorectal carcinomas; hMSH2; hMLH1; hMSH6; hPMS2; mRNA; Real time PCR; MMR phenotypes
Mismatch DNA repair (MMR) mechanism protect cells from replication errors and is important to genome stability [1-3]. Hereditary cancers, like Lynch syndrome, are linked with a deficiency of MMR mechanism [4-13]. Sporadic cancers have also been related with defective MMR mechanisms [14-24]. We have recently demonstrated that distinct MMR mRNA profiles are related to tumor staging and survival of patients with non-small cell lung carcinomas (NSCLCs) [16,17]. We have also showed dependence of hMSH2 and hMSH6 mRNA expression in urinary bladder carcinomas (UCCs) and revealed a correlation of hMSH6 reduction in UCCs (18). So far, there is a limited information on transcriptional levels of MMR genes in sporadic colorectal carcinomas (CRCs) [25,26].
In this study we quantified, with a precise Q-real time PCR method, the transcriptional levels of hMSH2, hMLH1, hMSH6 and hPMS2, MMR genes in CRCs and their paired adjacent normal tissues (ANTs) and we checked for correlations of MMR mRNA CRCs profiles with tumor or patients’ characteristics.
Fresh frozen colorectal tissue specimens consisting of paired tumor and their adjacent normal tissues (ANTs), were collected from 31 unselected patients, 17 male and 14 female with an age range 52- 92 years (median 74 years), who underwent surgery at the University Hospital of Larissa, Thessaly, Greece. The specimens were immersed immediately after surgery in RNA stabilizer solution (RNAlater®, Life Technologies) and they were preserved in -80°C, deep freezer, till RNA isolation. The localization of the tumor was rectum (11), right colon (10 cases), sigmoid (6) and left colon (1) while in three cases the exact localization was not available (Table 1). The histological review showed that all colorectal tumors of our collection were adenocarcinomas consisting of 1 well differentiated (WD), 24 moderately-differentiated (MD), 3 moderately to poorly differentiated (MD-PD) and 3 poorly differentiated (PD) tumors. Lymph node metastasis, nerve and vascular invasion was observed in 11/31 (35.5%), 18/31 (58%) and 13/31 (42%) out of total 31 of CRC tumors, respectively (Table 1).
|Case no./Age (years)/ sex||Histopathologic characteristics of tumors||hMSH2/ control mRNAa||hMLH1/ control mRNAa||hMSH6/ control mRNAa||hPMS2/ control mRNAa||Tumor/ANT MMR mRNA expressionb|
ANT, adjacent normal tissue; WD, well differentiated tumors; MD, moderately differentiated tumors; M/PD, moderately-poorly differentiated tumors; PD, poorly differentiated tumors; LN, lymph node metastasis; NF, nerve infiltration; VF, vessel infiltration; TL, tumor localization; n.a. not availiable; aRatios of mRNA expression; bRatios of tumor to ANT mRNA expression.
Table 1: Quantitative mRNA expression of MMR DNA repair genes in colorectal adenocaricnomas and their adjacent normal tissues.
Quantitative analysis of MMR mRNA expression
We used RNAeasy kit (Qiagen®, USA) for total RNA isolation and Super Script First-Strand Synthesis System (Invitrogen®, Life Technologies, UK) for cDNA synthesis, according the manufacturer’s instructions. The qPCR analysis of hMSH2, hMSH6, hPMS2 and hPBGD-control mRNA was performed using specific primers previously published (16,18) and probes for hMSH2: 5’-6FAMCATATAAGGCTTCTCCTGGC- BHQ1-3’, for hMSH6: 5’-6FAMCAGGAGCTTTTATCAATGGCTA- BHQ1-3’, for hPMS2: 5’-6FAMACTGCTCTTAACACAAGCGAGATGAAGAA- BHQ1-3’ and for hPBGD: 5’-6FAM-CCTCGTGCGGTTCCCTCTGCCTGA-BHQ1-3’, designed for Rotor Gene 6.1 instrument (CORBETT Research, Australia) using Platinum® Quantitative PCR Super Mix-UDQ (Invitrogen®, Life Technologies, UK) and annealing temperatures at 58°C for hMSH2 and hPBGD, at 54°C for hMSH6 and hPMS2. The qPCR analysis of hMLH1 was performed using QuantiFast Probe Assay SP kit (Qiagen®, USA) by applying in Rotor Gene 6.1 instrument, according the instructions. The quantitation of mRNAs was achieved creating a standard curve of serial dilutions of hPBGD copies, as previously described [16,27]. The mRNA expression of each MMR gene was expressed as a ratio of MMR mRNA to control hPBGD mRNAs (MMR/control mRNAs) and defined two major phenotypic groups, the reduced (r or p) for mRNA ratios <1 and the normal or elevated (R or P) for ratios ≥1, as previously described [16,18]. Additionally, the MMR gene expression of tumor samples was compared with that of the corresponding ANT samples. This value is indicated as relative mRNA expression of MMR genes between CRC tumors and ANTs (CRCs/ANT) of each patient (Table 1).
We used the paired Student’s t-test to compare ratios of hMSH2, hMLH1, hMSH6 and hPMS2 alterations between tumor and matched ANTs for different patient characteristics, including age, gender and clinical or histopathological parameters such as tumor location, grade, stage and lymph node metastasis, vascular invasion or perineurial invasion. The correlation between the mRNA expression ratios of hMSH2, hMLH1, hMSH6 and hPMS2 in CRCs and their ANTs for different patient and tumor characteristics was examined by Pearson test. The χ2 test was also used to examine the distribution of MMR mRNA phenotypes in tumor and ANT specimens at different tumor histological grades or stages or lymph node or nerve or vessel filtration or tumor location (Tables 2-4).
hMSH2 & hMLH1 & hMSH6 & hPMS2 mRNA quantification in CRCs and their ANTs
We evaluated hMSH2, hMLH1, hMSH6 and hPMS2 mRNA levels in primary CRCs and their corresponding ANTs relative to the reference hPBGD control gene by Q-RT-PCR. These data are summarized in Table 1 along with clinical and histopathological data.
|Characteristics||Relative copies of hMSH2 mRNA||Relative copies of hMLH1 mRNA||Relative copies of hMSH6 mRNA||Relative copies of hPMS2 mRNA||Tumor/ANT MMR mRNA gene expression|
|I||Â 5 (1WD+4MD)||0.0276c||0.0276||3.52||1.9||2.67q||3.7||0.0478||0.042||0.9739||0.773||0.626||0.847|
|Lymph node metastasis|
Mean age of the patients was 74years; ANT, Adjacent normal tissue; MMR, mismatch repair; WD, well differentiated tumors; MD, moderately differentiated tumors; M/PD, moderately-poorly differentiated tumors; PD, poorly differentiated tumors. ar=0.574765736, br=0.796820522, cr=0.99204665, dr=0.794450379, er=0.794045972, by Pearson test; correlation between hMSH2/hPBGD & hMLH1/hPBGD mRNA ratios; fr=-0.008816335, gr= -0.054661116, hr=-0.008369458, ir=-0.066294139, jr=0.893917164, kr=- 0.054872336, lr=0.892161159, by Pearson test; correlation between hMSH2/hPBGD & hMSH6/hPBGD mRNA ratios; mr=0.618906296, by Pearson test; correlation between hMSH2/hPBGD & hPMS2/hPBGD mRNA ratios; nr=0.819228481, or= 0.81809858, Pearson test; correlation between hMLH1/hPBGD & hPMS2/hPBGD mRNA ratios; pr=0.82653886, qr=0.99792412, rr=0.96520875, sr= -0.0030656, tr= 0.63185688, by Pearson test; correlation between hMSH6/hPBGD & hPMS2/hPBGD mRNA ratios; ur=0.999389, vr=0.999937, wr=-0.00904, xr= 0.999477, yr= 0.999475, by Pearson test; correlation between hMSH2 & hPMS2 mRNA ratios of Tumor/ANT; zr=0.644378, by Pearson test; correlation between hMSH2 & hMSH6 mRNA ratios of Tumor/ANT; Iar= 0.992066865, by Pearson test; correlation between hMSH6 & hPMS2 mRNA ratios of Tumor/ANT; ibP= 0.037994, icP=0.03214, by Studentâ€™s test, correlation between hMSH2/hPBGD ratios of Tumor/ANT
Table 2: Alterations in hMSH2, hMLH1, hMSH6 and hPMS2 mRNA levels between paired colorectal adenocarcinomas tumor and adjacent normal tissue samples relative to their clinical and histopathological parameters.
|MMR mRNA phenotype||CRCs (n) / observed phenotypic frequencies||Gender||Tumor Stage (n)||Invasion in||Tumor site||ANT (n) / observed phenotypic frequencies|
|r2||23 / 0.7667||12||11||2||11||10||12||11||7||16||10||13||8||8||3||1||3||22 / 0.7857|
|R2||7 / 0.2333||5||Â 2||2||4||Â 1||6||Â 1||5||Â 2||7||Â 0||2||3||2||0||0||6 / 0.2143|
|r1||12 / 0.3871||7||5||1||4||7||4||8||2||10||5||7||2||6||2||0||2||11 / 0.3548|
|R1||19 / 0.6129||10||9||4||11||4||15||4||11||Â 8||13||6||8||5||4||1||1||20 / 0.6452|
|r6||12 / 0.3871||7||5||0||9||6||5||7||4||Â 8||6||6||3||4||3||0||2||6 / 0.2000|
|R6||19 / 0.6129||10||9||5||6||5||14||5||9||10||12||7||7||7||3||1||1||24 / 0.8000|
|p2||31 / 1.0000||16||14||5||15||11||19||12||13||18||18||13||10||11||6||1||3||30 / 1.0000|
|P2||0 / 0.0000||0||Â 0||0||0||Â 0||0||Â 0||0||Â 0||0||Â 0||Â 0||Â 0||0||0||0||0 / 0.0000|
n, number of cases, r2, r1, r6, p2, MMR/control mRNA ratios <1; R2, R1, R6, P2, MMR mRNA ratios ≥1; M, male; F, female; N, no; Y, yes;, rt, right; rc, rectum; si, sigma; lf, left; n.a., not available.
Table 3: Distribution of individual hMSH2, hMLH1, hMSH6 & hPMS2 mRNA phenotypes in CRCs and their adjacent normal tissue (ANT).
All CRCs and their ANTs (100%) revealed low mRNA levels (MMR/ control mRNAs<1) of hPMS2 while a significant proportion of CRCs (73.3%) and their ANTs (82%) presented low mRNA levels of hMSH2. Also, a smaller proportion of CRCs (42% and 35.5%, respectively) and their ANTs (36% and 20%, respectively) exhibited low mRNA levels of hMLH1 and hMSH6.
|Lymph node metastasis|
*Four MMR phenotypes were defined by combination of reduced (r2, r1, r6 <1) or regular (R2, R1, R6≥1) hMSH2 , hMLH1 and hMSH6 mRNA levels relative to reference hPBGD mRNA control gene.
Table 4: Distribution of combined MMR mRNA phenotypes* in CRCs and their adjacent normal tissues (ANTs).
Statistical analysis by Pearson test, showed a correlation between mRNA expression ratios of hMSH2 and hMLH1, in CRCs (r=0.574765736) and ANTs (r=0.618906296), that was more intense in males (r=0.796820522), in stage I (r=0.99204665) or in stage II (r=0.794450379) tumors as well as in tumors without lymph node metastasis (r=0.794045972) (Table 2). Also, a significant correlation was observed between mRNA levels of hMSH6 and hPMS2 in tumors of female patients (r=0.82653886), in stage I (r=0.99792412) or stage II (r=0.96520875) tumors and in tumors without lymph node metastasis (r=0.63185688), (Table 2). Additionally, a correlation was observed between mRNA ratios of hMLH1 and hPMS2 and between hMSH2 and hMSH6, in stage III tumors (r=0.819228481 and r=0.893917164, respectively) and in tumors metastatic to lymph nodes (r=0.81809858 and r=0.892161159, respectively).
hMSH2 & hMLH1 & hMSH6 & hPMS2 mRNA relative expression
Calculation of CRCs/ANTs MMR mRNA ratios from quantification data (Table 1) revealed reduced mRNA expression ratios (≤0.8) of hMSH2, hMLH1, hMSH6 and hPMS2, with 41% (12/29), 45% (14/31), 53% (16/30) and 36.7% (11/30), respectively. As well as we observed overexpression (CRCs/ANTs ratios ≥1.8) of hMSH2, hMLH1, hMSH6 and/or hPMS2 in 41% (12/29), 32% (10/30), 36.7% (11/30) and 32% (10/30) of cases, respectively (Table 1).
There was a statistically significant correlation between relative expression levels (T/ANT) of hMSH2 and hPMS2 (r=0.999389), notable in male (r=0.999937), in stage II tumors (r=0.999477) and in tumors without lymph node metastasis (r=0.999475). However, a statistically significant correlation was observed between mRNA relative expression levels of hMSH2 and hMSH6 in tumors metastatic to lymph nodes (r=0.644378). Also, a correlation was observed between hMSH6 and hPMS2 relative expression in female (r= 0.992066865).
Notably, a statistically significant difference was observed between hMSH2 mRNA levels in CRCs tumors relative to their ANTs (P<0.04, by Student’s test) specifically in cases without lymph node metastasis (P<0.04, by Student’s test) (Table 2).
MMR phenotype sorting
We used the ratio of MMR mRNA expression relative to reference mRNA control (MMR/control gene mRNA levels), to adopt a functional unified assessment for our findings, as previously referred [17,19]. We classified our specimens in two major phenotypic groups, one with reduced (r) and the other with regular or elevated (R) ratios of expression (Materials & methods) and we subdivided our study group in eight phenotypic entities, r2 and R2 for hMSH2, r1 and R1 for hMLH1, r6 and R6 for hMSH6 and p2 and P2 for hPMS2, DNA repair system components or their combined phenotypes by descending MMR system activity (Table 3 and 4).
All CRCs and their ANTs showed p2 phenotype. The r2 was the most commonly founded in CRCs and their ANTs relative to r6 that was more often in CRCs relative to their ANTs (Table 2).
Specifically, r2 presented more often in stage II or stage III tumors relative to stage I (p=0.000) and it was more frequently observed in CRCs with lymph node metastasis, perineural or vascular invasion (p<0.009, p=0.000 and p=0.000, respectively) relative to cases without tumor invasion. Also, r2 was more frequent in CRCs located on the right colon or rectum compared to sigmoid colon tumors (p=0.000) (Figure 1 and 4).
Moreover, r6 was more common in CRCs compared to their ANTs (p<0.002, χ2-test), mainly in males (p=0.000). A significant difference was found between male and female patients (p<0.02; χ2-test). Also, r6 was more often in CRCs of stage II and III relative to I (p=0.000 and p<0.002, respectively) and CRCs of stage III relative to II (p<0.02). Additionally, the r6 was more common in CRCs with lymph node (p<0.004) or nerve (p<0.002) or vessel infiltration (p<0.0034) compared to their ANTs, with statistically significant difference between the number of cases showed nerve or vessel infiltration and those did not (p<0.0045 and p<0.01, respectively). In addition, r6 was more frequent in CRCs of rectum (p<0.000) relative to their ANTs with a statistically difference relative to sigmoid or right colon (p<0.03) (Table 3, Figure 2 and 4).
Additionally, r1 was observed commonly in ANTs of female patients relative to their CRCs (p=0.000), with a significant difference between female and male (p<0.009). However, r1 found commonly in stage III CRCs (p<0.0068) relative to their ANTs. A significant difference of r1 phenotype was found between stage III and stage II (p<0.0042) or I (p=0.000) as well as between stage II and I (p=0.000) cases. So, r1 was more frequently observed in CRCs with lymph node metastasis (p<0.002) relative to their ANTs, with a significant difference between number of cases found with lymph node or nerve or vessel infiltration (p=0.000, p=0000 and p=0.042, respectively) and those without tumor infiltration. In addition, r1 was more frequent in CRC tumors located to rectum relative to right colon (p=0.000) or to sigmoid (p=0.000) (Table 3, Figure 3 and 4).
Distribution of combined MMR mRNA phenotypes in CRCs and their ANTs
Table 4 presents the distribution of combined MMR mRNA in CRCs relative to patients and tumors’ characteristics. We found a statistically different distribution of r2r1, r2R1 and R2Rv phenotypes i) between CRCs and ANTs in male (p<0.03), ii) between male and female patients (p<0.03), iii) between CRCs and ANTs in stage III cases (p<0.026), iv) between stage III and stage II cases (p<0.023), v) between CRCs and their ANTs in cases with lymph node metastasis (p<0.02), vi) between cases with lymph node and without tumor metastasis (p<0.0014), vii) between CRCs and their ANTs of cases with perineurial invasion (p<0.01), viii) between cases with nerve and without nerve invasion (p=0.000) and ix) between cases presented vessel invasion relative to cases without vessel invasion (p=0.000). Specifically there was a significant different distribution of r2r1 and r2R1 phenotypes between CRCs and ANTs in cases located on rectum or sigmoid (r2r1) relative cases on right colon (r2R1) (p=0.000 and p=0.01, respectively).
The distribution of combined MMR mRNA phenotypes r2r6, r2R6 and R2R6 was statistically significant different i) between CRCs and ANTs in male (p=0.000), with a difference between male and female (p=0.000), ii) between CRCs and ANTs in stage III CRCs (p<0.027), with a difference between stage III and stage II (p=0.05) or between stage III and stage I (p=0.000) cases iii) between cases with node metastasis and cases without node infiltration (p<0.002), iv) between CRCs and their ANTs in cases with perineular invasion (p<0.04), v) between cases with perineural invasion compared to cases without perineural invasion (p=0.000) and v) between CRCs and ANTs in vessel filtrated cases (p=0.000), with a difference between vessel filtrated cases and non-vessel filtrated (p<0.005) (Table 4).
The distribution of r6r1, r6R1 and R6R1 was significant different between CRCs and ANTs in stage III cases (p<0.02), with a difference between Stage III and Stage II (p<0.002), and between node (p<0.0024), nerve (p<0.001), vessel (p=0.000) filtrated cases and cases without filtration (Table 4).
The aim of this study was to quantify the mRNA levels of hMSH2, hMLH1, hMSH6 and hPMS2, MMR genes in sporadic colorectal carcinomas and their ANTs, by Q-real time PCR and to correlate with clinical and histopathological data. So far, there is limited information on the transcriptional levels of the four major MMR DNA repair mechanism components, in non-hereditary CRCs and their ANTs [25,26]. Despite of the small number of patients  included in this study our results could be considered interesting and could give rise in a more extended investigation. We showed that the crucial components of MMR mechanism hMSH2 and hMLH1, and their counterparts hMSH6 and hPMS2 exhibited low mRNA expression profiles in a significant proportion of CRCs and their ANTs (58-100%). We also showed that mRNA expression correlated with tumor progression and tumor localization in the colon. Additionally, the transcription of the studied MMR genes was reduced in a significant proportion of CRCs (37-53%) relative to their paired ANTs indicating a possible mechanism of progressive genetic instability [1-3]. We have evaluated various MMR mRNA expression profiles and their relationship to tumor or patients’ characteristics.
We observed that CRCs of early histopathological stages (I-II), without lymph node metastasis, exhibited a correlation between the expression status of crucial MMR components, hMSH2 & hMLH1, and between their counterparts hMSH6 & hPMS2, maintained both in CRCs and in their ANTs . Notably, this correlation was reversed between male and female. Surprisingly, cases with lymph node metastasis, revealed a significant positive correlation of mRNA relative expression between hMSH2 and its counterpart hMSH6. Similarly, late stage (III) CRCs exhibited correlation between mRNA expression levels of hMLH1 and its counterpart hPMS2. Our results show a balanced transcriptional activation between the crucial MMR components hMSH2 & hMLH1 and between their counterpart’s hMSH6 and hPMS2, in CRCs of early stages related to gender. Surprisingly, later, an upcoming “unbalance” occurs reducing similarly the mRNA expression levels of MutLa or MutSa components in CRCs relative to their ANTs, that seem to related with tumor progression. Our observations could be significant, indicating a mechanism resulting to downregulation of hMSH2 and hMSH6 expression during tumor progression. The reduction of mRNA expression of MMR genes has been considered to be caused by gene deletions in hereditary cancers, like Lynch syndrome, or epigenetic modification of genes like methylation of hMLH1 in sporadic cancers or as recently it has been shown by specific miRNA regulation [28-34]. Cell biological studies have been shown the importance of retainance of MutLa components balance on cell cycle progression or apoptosis procedure, showing that the MutLa protein levels are essential to initiate apotosis and consequentely low expression levels lead to chemo-resistance [35,36].
Phenotypic sorting of our data revealed that the reduced r2 phenotype of crucial hMSH2, MMR mechanism component, was very common both in CRCs and their ANTs indicating a deficiency of MMR mechanism in epithelium of CRCs patients. Significantly, the reduced r6 phenotype of hMSH6, the counterpart of hMSH2, was more frequently observed in CRCs relative to their ANTS, supporting an affected MMR mechanism in CRC patients. Moreover, reduced (r2, r6 and r1) mRNA MMR phenotypes are related with tumor invasion, indicating their use as a tumor progressing index. We first observed that the reduced p2 phenotype of hPMS2 is a common finding in colorectal epithelium of patients with CRC. It is worthy to mention that hPMS2 low expression levels have been previously related with hereditary cancers with a late tumor onset . Here, we suggest that tumorigenesis in colon could be probably related with a molecular mechanism including decreased transcriptional activity of hPMS2.
Observing the combined phenotypic sorting of our data, we can summarize that reduced r2r1, r2r6 and r6r1 combined MMR phenotypes were related to advanced tumors (stage III) and gender. Specifically, r2r1 commonly shows a strong correlation with tumors presented with lymph or nerve or vessel invasion as well as r2r6 with nerve or vessel infiltration. In our previous study in lung cancer we suggested that r2r1 could be considered as a tumor progression index, while it has been correlated with worst prognosis in squamous cell lung carcinomas. Our data suggests that it could be an indicator of tumor progression in CRC and is in agreement with previous findings . Also, low MMR phenotypic profiles are correlated with male gender.
In conclusion, we presented for the first time a precise quantification of MMR mRNA levels, of hMSH2, hMLH1, hMSH6 and hPMS2, in small number of sporadic CRCs and their ANTs, correlated with clinical and histopathological data. Our findings indicate that tumoral epithelium of CRC patients of our group acquires MMR deficiency, during tumor progression. Distinct MMR mRNA profiles as low hMSH2, hMLH1 or hMSH6 mRNA levels (r2r1, r2r6 or r6r1) could be characterized as important indicators of lymph node metastasis and of perineural or vascular invasion. A different expression pattern was found in males relative to females with males showing MMR mRNA profiles related with tumor progressing. All CRCs and their ANTs of our cohort revealed low hPMS2 mRNA levels that were previously correlated with late tumor onset on hereditary colon cancers . Rectal localization was related with dysregulated MMR mRNA mechanism.