Resumen de Complejo regulador en el carcinoma de células renales: significado pronóstico de la expresión de MLH1, MSH2 y MSH6
INTRODUCTION.
Mismatch Repair (MMR) genes encode proteins that repair DNA mistakes produced during replication. Mutations on Mismatch Repair genes are related to accumulation of unstable microsatellite sequences and microsatellite instability (MSI). Lynch syndrome is known to be related to MSI, and the proteins of the MMR involved in the syndrome are MLH1, MSH2, MSH6 and PMS2. There are many studios about the role of MSI in endometrial cancer with different results; Black et als find that MSI is related to better disease-free and disease-specific survival; on the other hand, Zighelboim et als do not find relationship between MSI and prognosis. Carriers of an MMR gene mutation were at increased risk of some cancers; one of them is renal cell carcinoma.
MATERIAL AND METHODS.
We have analyzed 148 samples of renal cell carcinoma from patients operated with curative intention (radical or partial nefrectomy) from January 1989 to December 2006. The tumors were classified using World Helath Organization System TNM (2002) and the nuclear grade using Fuhrman grade; We have used tissue-array for immunohystoquemical study, and thanks to the use of monoclonal antibodies of mice or polyclonal antibodies of rabbit, we have determined the expression of proteins MLH1, MSH2 and MSH6. We have analyzed the expression of other molecular markers with the same technique: p53, ki67, Bcl2, CAIX, PTEN, pAKT, Survivin, MDM2, VEGF, Vimentin, E-Cadherin, alfa, beta and gammacathenin, P120, P-Cadherin, CD44. We have studied the relation between the expression of MLH1, MSH2 and MSH6 and survival and progression-free survival; we have studied the relationship between clinical and pathological variables and the expression of the other markers and survival and progression-free survival, and we have analyzed the effects that the proteins of the MMR have on the clinical and pathological variables and markers that have relationship with prognosis in the univariable study in terms of survival and progression-free survival (p < 0.1). For the statistical analyses we have used Chi square test, Fisher test, t-student; Survival curves were estimated using Kaplan-Meier method. Cox regression analysis was used in multivariable study. We did a stratified analyses for the study of the effects of the proteins of MMR in other variables. We used SPSS for all statistical analyses version 19.
RESULTS.
A total 148 samples of renal cell carcinoma were analyzed, from 145 patients operated between form January 1989 to December 2006 with curative intention (radical or partial nephrectomy) in our institution. The median follow-up was 93.45 months; the median age was 58.84 years; 96 were men and 52 women. 84 patients (56.8%) had not symptoms at diagnosis; 24.3% were T1a, 27.7%were T1b, 20% were T2 and 19% were T3a; incidence of T3b and T4 is very low (7% and 2.7%); with respect to nodal invasion, 93.2% had not nodal invasion, and 94.6% were M0 at diagnosis. The majority were clear cell carcinoma 73.6%, 15% were papillary carcinomas, 9% cromophobe carcinomas. 49% of the samples had nuclear differentiation grade II ( Fuhrman grade). The clinical and pathological features are detailed in table 1. 40% of the samples had positive expression of MLH1, 63% of MSH2 and 28% of MSH6. With respect to the expression of the other markers, 77% had negative expression of p53, 86% had negative expression of ki67, 67% had negative expression of Bcl2, 61% had positive expression of CAIX, 57% had more than 75% of nuclear PTEN expression and 56% had moderate expression; 73% had negative expression of pAkt; 40% had strong expression of cytoplasmatic survivin and 53% had positive expression of nuclear survivin. 21% had positive expression of MDM2; 78% had negative expression of VEGF; 49% had low expression of cytoplasmatic vimentine while 76% had negative expression of membranous vimentin; 77% had reduced E-cadherine expression; membranous P-Cadherine was positive in 27% and cytoplasmic expression was positive in 14%; membranous alfa-catenin was reduced in 75% and 81% had negative cytoplasmatic expression. Membranous beta-catenin was reduced in 35% and 88% had negative cytoplasmatic expression. Membranous gamma-catenin was reduced in 66% and 93% had negative cytoplasmatic expression. Membranous P120 was reduced in 47% and 89% had negative cytoplasmatic expression. 87% had reduced expression of CD44. We did not find relationship between the expression of MLH1, MSH2 and MSH6 and prognosis nor for overall survival rate neither for progression-free survival rate in univariate analyses( p: 067; 0.8 and 0.34 respectively for survival and p: 0.48; 0.87 and 0.8 respectively for progression free survival). With respect to clinical and pathological variables with prognostic implications in univariate analyses, we found that younger patients (<61 years), asymptomatic patients at diagnosis, T1-T2 stages, N0 stage, clear cell type (against papillary one) and low Furhrman grade (1/2) are associated to higher overall survival rate (p: 0.02, 0.02, <0.01, 0.004, 0.018 and <0.001 respectively), and younger patients (<61 years), T1-T2 stages, N0 stage and low Fuhrman grade (1/2) are associated to higher progression-free survival rate. With respect to the other markers related to prognosis, we found that negative expression of ki67, negative CAIX, positive cytoplasmatic PTEN, negative pAkt, positive nuclear survivin, negative VEGF, negative cytoplasmatic vimentin, preserved E-Cadherine, preserved membranous alfa-catenin and preserved membranous P120 were associated to higher overall survival rate (p: 0.02, 0.1, 0.016, 0.03, 0.07, 0.009, 0.07, 0.07, 0.1, 0.008), and negative ki67, negative CAIX, positive cytoplasmatic PTEN, negative pAkt, negative-low cytoplasmatic survivin, negative VEGF, negative cytoplasmatic vimentin, preserved E-Cadherine, preserved membranous beta-catenin and preserved P120 were associated to higher progression-free survival rate (p: 0.068, 0.065, 0.04, 0.1, 0.013, 0.79, 0.07, 0.05, 0.1, 0.001 respectively). In multivariate analyses, negative VEGF, preserved E-Cadherine and negative ki67 were independent factors in higher overall survival rate (p: 0.001, 0.031, 0.018 respectively) and positive cytoplasmatic survivin, preserved E-Cadherin and preserved P120 were independent factors in higher progression-free survival rate (p: 0.05, 0.05, 0.05 respectively). In the stratified analyses we found that MLH1 has a protective role for intense cytoplasmatic survivin in terms of overall survival rate (HR: 0.3; p: 0.05). MSH2 had a protective role in terms of overall survival rate for low Fuhrman grade (1/2) (HR 0.24; p: 0.078) and was a risk factor for <61 years (HR: 4.68; p: 0.075). MSH6 had a protective role in terms of overall survival rate for positive MDM2 (HR: 0.28; p: 0.099), positive CAIX (HR: 0.33; p: 0.045), positive Bcl2 (HR: 0.2; p: 0.086), preserved membranous beta-catenin (HR: 0.3; p: 0.074), and old patients (>61 years old) (HR: 0.22; p: 0.016), and it was a risk factor for high Fuhrman grade (3/4) (HR: 2.38; p: 0.1). On the other hand, MLH1 had a protective role in terms of progression-free survival for intense expression of cytoplasmatic survivin (HR: 0.34; p: 0.04) and it was a risk factor for negative expression of cytoplasmatic vimentin (HR: 1.73; p: 0.33). MSH2 had a protective role in terms of progression-free survival for positive p53 (HR: 0.049; p: 0.03) and for intense expression of cytoplasmatic survivin (HR: 0.24; p: 0.04), and it was a risk factor for positive cytoplasmatic Cadherin-P (HR: 5.09; p: 0.05). MSH6 was a risk factor in terms of progression-free survival for positive CAIX (HR: 5.24; p: 0.022) and for young patients (< 61 years old) (HR: 3.42; p: 0.03).
CONCLUSIONS.
1. Alterations of proteins MLH1, MSH2 and MSH6 are not a prognostic factor in terms of overall survival nor progression-free survival in renal cell carcinoma.
2. The expression of MLH1 is related to overall survival and progression-free survival for strong citoplasmatic survivin, and it is related only to progression-free survival for strong citoplasmatic vimentin.
3. The expression of MSH2 is related to overall survival for low grade tumours and positive MDM2, and it has a negative relationship with survival for young patients. MSH 2 is related to progression-free survival for positive p53, and strong citoplasmatic survivin and it has a negative relationship with progression-free survival for positive expression of citoplasmatic P Cadherin.
4. The expression of MSH6 is related to overall survival for old patients, MDM2, CAIX, Bcl2 and preserved membranous Betacatenin, and it has a negative relationship with survival for high grade tumours. MSH6 is related negatively with progression-free survival for young patients and negative expression of CAIX.
