Excision repair cross-complementation group 1 (ERCC1) gene expression analysis is currently used widely in the molecular diagnosis of cancer. According to numerous studies, ERCC1 gene expression correlates with overall survival and effectiveness of chemotherapy with platinum agents. However, the degree of this correlation differs among various studies, with certain authors reporting a complete lack of such a correlation. These contradictions may be attributed to a number of factors, including the heterogeneity of the tumor tissue. In this study, we attempted to assess the degree of genetic heterogeneity exhibited by tissue samples obtained from non-small-cell lung cancer (NSCLC) through the expression of the ERCC1 gene. This study included 25 samples of tumor tissue from patients with a morphologically confirmed NSCLC diagnosis. A total of three randomized sections of each specimen were used. The ERCC1 gene expression was assessed by quantitative polymerase chain reaction (qPCR) in the TaqMan format. When planning the experiment and analysis of qPCR data, the MIQE guidelines were taken into consideration. We established that the coefficient of variation of the relative level of ERCC1 gene expression in the majority of the samples exceeded 33% (P<0.05), indicating the significant heterogeneity of the sample. We also demonstrated that the degree of heterogeneity of the tumor tissue is largely dependent on disease stage.
Non-small-cell lung cancer (NSCLC) is currently the most common type of lung cancer (
Numerous studies suggest the application of platinum- over non-platinum-based therapy as first-line chemotherapy for NSCLC, as suggested by a large volume of data from clinical trials and meta-analyses (
One of the critical mechanisms of resistance to platinum-based chemotherapy is the damaged DNA repair mechanism. It is hypothesized that tumor cells with reduced DNA repair ability exhibit a higher sensitivity to treatment, leading to a better outcome following radiotherapy or chemotherapy, whereas an increased repair ability is associated with a poorer therapeutic response and increased tumor resistance (
The excision repair cross-complementation group 1 (ERCC1) gene, encoding the corresponding protein, is one of the key links in the chain of intracellular molecular processes responsible for the removal of platinum-induced DNA damage. The ERCC1 gene product belongs to the group of nucleotide excision repair enzymes, which are involved in DNA excision repair by nucleotide removal (
A significant volume of evidence has been accumulated, indicating that the survival time of NSCLC patients treated with gemcitabine + cisplatin was reduced through an increase in the ERCC1 gene expression level (
However, there is a considerable variation of the data currently available in the literature regarding the expression of ERCC1 and other molecular markers obtained through different (
Human tumors often display significant intratumor heterogeneity regarding various characteristics, including histology, gene expression, genotype and metastatic and proliferative potential. However, clonal genetic heterogeneity is considered one of the most interesting characteristics, the essence of which is the existence of alternative clones, namely histologically homogeneous tumor tissue expression profiles characterized by different molecular markers. This heterogeneity plays an important role in neoplasia, cancer progression and resistance to treatment (
Along with the histological heterogeneity of the investigated sample, intratumoral genetic heterogeneity caused by genome instability in tumors may complicate the molecular classification of tumors into clinically relevant subtypes (
Numerous studies have demonstrated the genetic heterogeneity of breast cancer tissue (
A total of 25 patients with invasive lung cancer underwent treatment between March, 2013 and February, 2014 at the N.N. Alexandrov National Cancer Center, Minsk, Belarus. The patients comprised 22 men and 3 women, with a median age of 59 years (range, 42–67 years). The diagnosis of stage 1–3a invasive lung cancer was determined according to the World Health Organization diagnostic criteria (2008) and confirmed by two pathologists. The patient sample included 12 patients with adenocarcinoma, 10 with squamous cell carcinoma and 10 with other types of NSCLC, according to the morphological characteristics of the tumor. None of the patients had received radiotherapy or chemotherapy prior to surgery.
The protocol of the present study was approved by the Institutional Review Board of the Alexandrov N.N. National Cancer Center for Oncology and Medical Radiology (ID no. 20080923) and all the patients provided written informed consent prior to their inclusion in the study.
All the tumor tissue samples were frozen in liquid nitrogen following resection and pathological examination. The samples were maintained for <6 months at −70°C. Approximately 20–30 mg of tumor tissue were used for the study. The samples were prepared using a scalpel and weighed on ice prior to placement in lysis solution. All the procedures on the tumor tissue samples and subsequent data analysis were conducted anonymously.
Total RNA was isolated from fresh-frozen NSCLC tissues using the RNAqueous-4PCR RNA isolation kit (Ambion, Inc., Austin, TX, USA) according to the manufacturer's protocol, without additional processing by DNase and proteinase. Tissue sample homogenization was performed by special polypropylene microplastics that were supplied with the kit. The degree of contamination of the RNA samples was determined by using agarose gel electrophoresis and by setting up a classical polymerase chain reaction (PCR) with specific oligonucleotide primers for the amplification of the GAPDH gene (data not shown). The concentration and purification of the total RNA fraction was evaluated using a Cary 50 spectrophotometer (Varian Australia Pty Ltd., Melbourne, Australia). RNA integrity was verified using agarose gel electrophoresis without denaturing conditions (data not shown). Tests for the presence of inhibitors of reverse transcription or quantitative PCR (qPCR) were conducted by preparing a series of serial dilutions of RNA samples prior to reverse transcription.
Reverse transcription was performed by using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol. A total of 1 mkg of the total RNA fraction was used per reaction (total volume, 20 µl). The kit working principle is based on using random oligohexamers. After the reverse transcription reaction, the samples were stored for <1 month at a temperature between −12 and −18°C.
qPCR was performed using the iQ5 detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and the Maxima HotStartTaq DNA Polymerase kit (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania). The total volume of the reaction mix was 25 µl. We used 50 ng cDNA, 0.5 µm of each oligonucleotide, including TaqMan probe for PCR (RNA18S5, 4319413E; ERCC1, Hs01012159_m1; Applied Biosystems, Warrington, UK), 0.2 mm of each dNTP, 50 mm KCl, 25 mm Tris-HCl, 2 mm MgCl2 and 1.25 IU HotStartTaq DNA polymerase in the reaction mix. The PCR conditions were as follows: 2 min at 50°C, 5 min at 95°C, followed by 45 cycles for 10 sec at 95°C and 60 sec at 60°C. Measurement of sample fluorescence was performed at the end of each amplification cycle using the FAM channel. ERCC1 gene expression was measured in triplicate and the fold-change of mRNA expression was calculated using the 2−ΔΔCt method, as described by Livak and Schmittgen (
Statistical analyses were performed using SPSS 23.0 software (SPSS, Inc., Chicago, IL, USA). The Spearman's rank correlation coefficient was used to investigate the correlation between ERCC1 gene expression and clinicopathological characteristics. The Mann-Whitney U test was used to determine the significance of the associations between different sections from the same sample. The Fisher transformation was used to determine the variety coefficient diversity. A two-tailed P<0.05 was considered to indicate a statistically significant difference.
In the present study, we measured the ERCC1 expression levels in 25 samples of human NSCLC. According to BestKeeper v 1.0 software (
Following normalization (ΔСр = Срtarget - Срreference), the values of the target CV in one sample within the three sections analysis were 1.32–48.1%, with a median of 5.045%. The differences between ERCC1 expression levels were normalized to internal control between sections from one sample, according to the Mann-Whitney U-test in 88% of the cases (22 patients) (P<0.01); the differences were not statistically significant in 22% of the cases (3 patients). Furthermore, all the values were analyzed according to the ERCC1 expression level obtained for normal lung tissue. ΔΔСр values (ΔСрtumor - ΔСрnormal) were calculated for each sample and the relative expression level of the target was calculated according to the 2−ΔΔСр equation. The CV of target gene expression for the three sections of the same sample ranged between 5.93 and 124.96%, with a median of 52.22% and a mean value of 53.16±32.77%. In addition, the CV value of the ERCC1 relative expression level in two samples was <10%, in another two samples it ranged between 10 and 20%, in three samples it ranged between 20 and 33% and 18 patients exhibited levels >33%.
A detailed data analysis demonstrated that the relative levels of ERCC1 gene expression was high in 12 and low in 6 samples. Overexpression of the target gene in all three sections was detected in only 5 cases. In addition, in 5 patients the level of expression was high in two of the three sections and the remaining 2 patients exhibited overexpression in only one of the three sections. Low levels of ERCC1 gene expression in two of the three sections were identified in 2 patients and in one section in 4 patients. Cases with low levels of ERCC1 gene expression in all three sections from one sample were not identified. The distribution of values of relative expression levels of ERCC1 gene is demonstrated in
There was no observed statistically significant correlation of the ERCC1 gene expression level with gender, age or histological type of tumor.
A recent study conducted by Sipos
Our results demonstrated that intratumoral ERCC1 and RNA18S5 expression in lung cancer tissue is heterogeneous, with minor changeability of variation series for Cp ERCC1 and RNA18S5 values in one patient (median <10%). The CV of the ERCC1 relative expression level directly used in the diagnosis in three sections from a single sample in the majority of the patients was >33% (P<0.01), reflecting the heterogeneity of this sample. Thus, for acquiring exact data on the relative expression of ERCC1, particularly in the field of border values, it is necessary to analyze several biopsies from one sample.
Following the MIQE recommendations (
According to the obtained results, we may select one of the possible approaches to calculate the required number of random sections. If we accept results from three sections as 100% reliable data, using ERCC1 gene expression data from one section allows for detection of 66.5% of overexpression and 57% of underexpression; and from two sections, 85.7 and 92%, respectively. Each section thereafter reduces the risk of false-negative results; however, successive sections increase the possibility of detecting a lower percentage of expression. A detailed analysis of the results of sampling shows that, at the first stage of disease, ∼57% of ERCC1 overexpression is detected in one section, but with two sections the detectability percentage may increase up to 87%. However, we may detect 66% overexpression from one section and 100% from two sections in patients at the second stage of the disease. We did not identify any statistically significant differences for the second stage of disease between two and three sections. Finally, in patients at the third stage of the disease, ERCC1 overexpression, if present, may be detected in 100% of the cases with a single section. Thus, we may be able to estimate the required number of biopsies necessary for detecting some minimum overexpression, i.e., 95%, but other studies must be priorly conducted, including several sections.
The increase in tumor homogeneity according to the ERCC1 expression level criterion from the first to the third stage may be explained by the higher histological homogeneity of the samples at the third stage (without normal tissue included in the analysis), as well as by the higher genetic homogeneity, ensued by the presence of a strongly dominant clone. Based on our data we may provide a possible explanation for the worse prognosis of the third stage in terms of molecular biology. Along with the ability to metastasise, larger tumor size and other pathogenetic factors, we may have to take into consideration the hiding of minor clones by dominant cell clones. Thus, during the early stage of the disease, a few histologically homogeneous populations of clones may be formed by mutations and the high proliferative activity of tumor cells. With disease progression, there may occur a generalization of a dominant (in condition of a specific organism) clone and hiding of minor clones. Following elimination of the dominant clone and a course of chemotherapy, minor clones may become dominant, as they are resistant to the administered drugs. This may result in early or late relapse and reduce the efficiency of chemotherapy.
We would like to thank Dr Vyacheslav Kurchin and Aleksey Kurchenkov at the N.N. Alexandrov National Cancer Center for providing expert technical assistance. This study was supported by the M13-012Lit grant for fundamental research program (2013–2014) from the Belarusian Republican Foundation for Fundamental Research, Belarus.
Distribution of values of the relative level of excision repair cross–complementation group 1 gene expression.
| Sections | ||||
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| Stage | Sample | 1 | 2 | 3 |
| 1 | 1 | + | + | – |
| 2 | + | + | + | |
| 3 | + | 0 | 0 | |
| 4 | – | 0 | – | |
| 5 | 0 | 0 | 0 | |
| 6 | + | 0 | + | |
| 7 | + | + | + | |
| 8 | 0 | 0 | 0 | |
| 9 | 0 | 0 | 0 | |
| 10 | 0 | 0 | + | |
| 11 | + | + | 0 | |
| 12 | 0 | 0 | 0 | |
| 2 | 13 | – | 0 | 0 |
| 14 | – | + | + | |
| 15 | 0 | 0 | 0 | |
| 16 | 0 | 0 | 0 | |
| 17 | + | – | + | |
| 18 | 0 | 0 | 0 | |
| 19 | + | + | + | |
| 3 | 20 | 0 | 0 | 0 |
| 21 | + | + | + | |
| 22 | + | + | + | |
| 23 | 0 | – | – | |
| 24 | 0 | 0 | 0 | |
| 25 | 0 | 0 | 0 | |
+, high level; –, low level; 0, normal.