So far, successful drug-specific predictive markers in NSCLC and in most malignancies include molecular phenotypes reflecting somatic genetic alterations; for example, small scale mutations or large scale structural gene changes. Regarding BRCA1, ERCC1, RRM1 and TYMS in NSCLC, however, differences in the “expression level” of the respective molecules are considered as markers, usually by referring to high vs. low expression. High vs. low is a qualitative description deriving from (semi)quantitative measurements in the case of q-PCR gene expression assessments. Undoubtedly, in order to understand high vs. low we need to refer to a standard. By using an external “normal” cell line RNA as a standard (as published before for ERCC1) , two of these genes, RRM1 and TYMS, appeared as very low expressed in tumors. However, even when coined as “normal”, cells in culture are, in fact, transformed; therefore, having acquired the ability of continuous division they inherently produce enzymes such as RRM1 and TYMS. In addition, cells in culture constitute a homogeneous system by definition. In comparison, cells in normal tissues comprise a heterogeneous environment and divide upon interaction with their surrounding cells. This explains the very low expression of RRM1 and TYMS in the normal lung tissue in comparison to the reference standard, as observed here.
Our approach was to use normal lung tissues located distally to tumors as a standard. Given the retrospective type of this study, it was difficult to find a large number of such normal samples with matched tumors. The limited number of samples in this reference group (12.5% of the total number of cases examined) may be considered as a drawback for the normal range of RQ values obtained in this study. However, with respect to the RQ values in the matched tumor samples at least, the group was considered representative for the remaining 87.5% of single tumor samples. The level of BRCA1, ERCC1, RRM1 and TYMS transcripts in NSCLC tumors of all histologic types, and especially SCC, was mostly within the normal range or increased. These observations are in line with previous reports for unchanged ERCC1  or increased ERCC1  and for increased TYMS mRNA expression [40, 41] in tumors vs. normal lung, whereby TYMS has also been associated with enhanced proliferation activity . Genetic or epigenetic BRCA1 silencing in NSCLC could not be inferred from our study, contrary to a previous report , since only two tumors in our series exhibited very low expression of this gene. RRM1 mRNA expression was found lower in NSCLC compared to the normal lung and the condition was attributed, at least in part, to LOH at 11p15.5 , which may also have accounted for the low RRM1 observed in 22% of tumors in this study. For BRCA1, ERCC1 and TYMS, expression below the normal range was encountered in <10% of the tumors examined. Overall, only in 11 cases (<4%) tumor B, E, R, T RQ values were lower than any normal ones, which would indicate absence of gene expression. Thus, although genetic / epigenetic changes may have been present in these cases, low expression of B, E, R, T indicative of gene pathology in NSCLC tumors could not be considered as a frequent event. In addition, considering that all mRNA markers strongly correlated with each other in tumors, our findings on mRNA expression of the two DNA-repair and the two DNA-replication genes in NSCLC reflect the expected patterns of tissues responding to increased demands for both these cell functions. Of note, our results are limited to gene expression, which does not preclude that for example, these particular DNA repair genes would be dysfunctional in NSCLC. BRCA1 and ERCC1 mRNA expression has as yet been addressed in a rather simplistic way in tissue studies with q-PCR, abolishing the possibility to investigate which mRNA variants are being produced from these genes. At least for ERCC1, detection of a particular splice variant might be important for the evaluation of platinum resistance, as has been reported for ovarian cancer cells .
In the noticeably heterogeneous patient cohort of the present study, none of the four markers was universally associated with OS and DFS in the adjuvant setting or PFS in the two different first-line settings. This finding was rather expected, since the prognostic value of the markers examined remains contradictory, as supported in recent meta-analyses for ERCC1, which also stress the need for standardization methods to assess gene expression by qPCR in tumors [27, 30]. The only independent predictors for OS, after adjusting the four mRNA markers for clinicopathologic parameters and treatment schemes, were disease stage (unfavorable if advanced) and histology type, meaning adenocarcinomas, which conferred a favorable outcome in comparison to squamous and large cell carcinomas. However, this latter finding must be considered as cohort specific, since the majority of patients in this study were chemo-naïve adenocarcinoma patients and received 1st line chemotherapy, where adenocarcinomas are the best responders among NSCLC . No interaction between histology type and BRCA1, ERCC1, RRM1, or TYMS transcript levels was observed in terms of outcome, despite the strong association of continuous RQ values with the same parameter. The relative expression of the four genes was similar in adeno- and large cell carcinomas but, in comparison to these, it was significantly increased in squamous cell carcinomas, in line with previously published data [4, 40]. Apparently, the expression of these genes did not predict for the outcome of adenocarcinoma patients in our series, in line with recent reports with immunohistochemistry for Ercc1 and Brca1 [44, 45].
The heterogeneous treatment regimens administered for advanced disease in this study constituted a very useful template to assess drug-specific interactions of BRCA1, ERCC1, RRM1, and TYMS, particularly with respect to patients’ PFS, an endpoint which is closely related to treatment efficacy although more prone to errors during assessment . It was interesting to observe the same treatment-related patterns of patient outcome for tumors with low and high expression of the markers examined, while tumors with normal expression of the same marker had different prognosis. Plausibly, the first approach for the interpretation of this finding would be to consider it an artifact due to the small number of tumors with low BRCA1, ERCC1, RRM1, and TYMS mRNA expression. However, this finding may also suggest that drug interactions, if any, with the molecules examined at the protein level may follow bell-shaped curves, as known for various biologic response models involving enzymes (e.g., concerning the cellular fate upon different levels of superoxide dismutase ) or drug efficiency . To prove this, functional studies in controlled systems with multicellular components are needed. It remains worthy considering, however, that the usually applied one-point cut-offs may mask the information needed for the evaluation of the herein investigated as well as for other mRNA markers.
In this context, normal ERCC1 transcript levels were associated with a favorable outcome in patients who received the platinum/taxanes doublet. Considering that normal ERCC1 mostly corresponds to low ERCC1 as addressed in the literature, this finding was in keeping with the reported platinum specificity of low ERCC1 [27, 30, 49]. However, a clear effect as would be expected for normal ERCC1 on patients treated with platinum-based regimens without taxanes was not observed in the present study.
Surprisingly, TYMS followed the same predictive pattern of significance as ERCC1 for platinum without taxanes, but an inverse pattern than ERCC1 for taxanes without platinum. For this molecule, significant results were obtained with respect to platinum (high TYMS – favorable) and taxanes (high TYMS – unfavorable), and less so for the combination of the two drugs (high TYMS – favorable). Since the sole independently significant interaction for patient outcome was TYMS with taxanes, patients with tumors expressing high TYMS mRNA, as compared to a normal lung tissue standard, may be spared receiving taxanes. This is new evidence, meriting clarification at the molecular level for the known microtubule stabilizers. In addition, there was an interaction of high TYMS with platinum as well, probably reflecting the increased efficacy of this drug in killing cells featuring active DNA replication. TYMS, an enzyme that is essential for DNA synthesis, has not yet been studied as a “platinum-target” molecule, as has been ERCC1 in the context of the nucleotide excision repair pathway. In NSCLC therapeutics, TYMS counts as an established target mostly being associated with the efficacy of the newer antifolate pemetrexed, which is a multi-targeted drug, nonetheless [50, 51]. Another observation from the present study concerns the widely used taxanes/platinum combination in advanced NSCLC patients. ERCC1 and TYMS were related in an opposing manner to patient outcome upon this treatment, with TYMS being more specific. It would be interesting to investigate the drug-specific predictive value of combined profiles of ERCC1 and TYMS in a larger series of patients with adequate statistical power.