Based on preclinical studies as well as translational studies using clinical specimens, microRNA expression levels have been suggested to be biomarkers for prognosis as well as treatment outcome in numerous malignancies (35
). Expression of seven candidate microRNAs was hypothesized to predict outcome of adjuvant chemotherapy in NSCLC, aiming to establish routine markers for selection of patients for adjuvant chemotherapy in order to improve treatment outcome.
We tested this hypothesis in a very large NSCLC cohort of 639 cases, randomized for treatment, using extensive quality and methodological control measures. We disposed of only one tumor section per case and made a selection of seven biologically relevant microRNAs to be analyzed for associations with clinicopathological covariates, prognosis and treatment outcome.
Interestingly, microRNA expression did correlate with histology for five out of seven microRNAs assessed: miR-21, miR-29b, miR-34a/b/c. Recently, a study on microRNA expression profiling was reported using 290 FFPE specimens from the EAGLE study, a population-based case control study in lung cancer (38
). A microRNA signature, including miR-29b and let-7a, was identified that strongly differentiated histological subtypes, i.e. adenocarcinoma and squamous cell carcinoma in male smoker patients. Interestingly, cigarette smoking intensity showed an inverse correlation with let-7 expression in female adenocarcinoma patients and a positive correlation with miR-21 expression in male squamous cell carcinoma patients. Furthermore, only in the group of male smokers with squamous cell carcinoma, a prognostic miR signature could be established, which included miR-34c-5p and miR-34a (38
). Unfortunately we did not dispose of smoking status data for IALT cases to confirm these relationships.
We found that positive expression of miR-21, miR-29b, miR-34a and miR-155 was associated with intense lymphoid infiltration, or presence of lymphatic invasion (miR-34a only). In this regard, it was previously shown that miR-21 expression is associated with an increased rate of lymph node metastasis in breast cancer and colorectal cancer patients (39
Overall, we were not able to show a prognostic value of any of the seven microRNAs in this patient cohort. Upregulated miR-21 expression has been previously associated with worse outcome in NSCLC cases (23
). However, the study by Markou et al.
consisted of few cases, 48, and it made use of matched tumor and normal tissues (24
). Consequently, results reported were based on the tumor tissue/ normal tissue (T/N) expression ratio. In contrast, for our main analysis we made use of tumor tissue expression values. Additionally, the study by Raponi et al.
made use of a microRNA array approach (23
). Another characteristic of our study was that we disposed only of FFPE tissue sections instead of frozen tumor specimens.
We did establish an association between let-7a expression and KRAS mutation status, albeit only in the multivariate analysis on the means and not the dichotomized expression values. The significance of this finding is unclear. So far it has been reported that the KRAS-LCS6 polymorphism results in upregulation of the KRAS gene and concomitant downregulation of let-7 (42
). However, this polymorphism was not found to be associated with KRAS mutations (43
We could not confirm an association between EGFR mutation status and miR-21 expression, as reported previously (22
Additionally, we could not show a predictive effect of any of the seven microRNAs on treatment outcome, which consisted of cisplatin-based adjuvant chemotherapy. Drugs combined with cisplatin among the 639 patients were either etoposide (54%), vinorelbine (33%), vinblastine (8%) or vindesine (6%) (8
). We previously showed that high microRNA-21 expression is related to fluoropyrimidine (5-FU) resistance in colorectal cancer as well as gemcitabine resistance in pancreatic cancer and the predictive value of microRNA expression may be chemotherapy and/ or tumor specific (21
As the prognostic and/ or predictive value of microRNAs might be dependent on the expression of more than one microRNA, we performed cluster analysis using standardized values of the 7 microRNAs. However, no combination of expression profiles of the seven microRNAs was found to be prognostic or predictive (data not shown). Finally, in situ
hybridization was performed using specimens from this cohort as well as specimens obtained from the University of Maryland. Treatment conditions for microRNA in situ
hybridization vary per probe and histology. Conditions had to be optimized using serial sections of an independent test cohort for which qRT-PCR data was available. For the IALT cases, a disadvantage was that we were completely blinded to origin of cases, specimens being collected from a worldwide multicenter study, with non standardized fixation and storage conditions. Fixation and procurement variations all affect the efficacy of in situ
hybridization to a much bigger extent than microRNA isolation and qRT-PCR (45
). Nevertheless, in standardized test cases as well as IALT-Bio cases, in situ
expression of microRNAs could be demonstrated.
In conclusion, many reports exist on microRNA expression profiling and the prognostic and/or treatment predictive impact of microRNA expression in NSCLC. However, many studies are limited by small sample size and by not being randomized. This study constitutes the largest ever group of NSCLC patients analyzed for the prognostic and predictive value of microRNA expression. The seven target microRNAs chosen for evaluation, miR-21, miR-29b, miR-34a/b/c, miR-155 and let-7a, were neither prognostic nor predictive in this patient cohort. Further studies, e.g. making use of a microRNA array approach, are warranted in NSCLC, in order to identify the prognostic or predictive value of expression of other microRNAs which were not included in this study.