Lung cancer is the leading cause of cancer-related deaths worldwide, with small-cell lung cancer (SCLC) representing approximately 15% of all lung cancer cases.1,2 SCLC represents one of the most aggressive human cancers, with early metastatic dissemination, initiated by cancer cell intravasation into blood, migration and consequent colonisation of sites distant to primary tumour. Despite some advances in therapeutic approaches, most of the advanced SCLC patients still die within the first year after diagnosis.3,4
SCLC is initially chemosensitive disease, with high response rates achieved with first-line chemotherapy regimens. However, majority of SCLC patients relapse within a few months and achieve only modest response rates to second-line chemotherapy, leading to poor survival rates. Platinum-based chemotherapeutic regimens with cisplatin or carboplatin still represent the only effective systemic therapy for SCLC patients. Unfortunately, still, no effective targeted therapy is available in clinical practice to treat SCLC.
Classical clinico-pathological characteristics (
In the last several years, growing body of evidence indicates that cancer stem-like cells (CSCs) behave as crucial actors in cancer development, progression and metastasis.8,9 CSCs have been identified in many human cancer types, including breast cancer10, prostate cancer11, pancreatic cancer12 and lung cancer.13 For detection and identification of lung CSCs several key regulators have been proposed, normally essential for maintenance of pluripotent state of embryonic stem cells and self-renewal of tissue-specific adult stem cells; these regulators include SRY (sex determining region Y)-box 2 (SOX2)14, homeobox protein NANOG (named after Celtic word Tír na nÓg meaning the land of the young)15 and octamer-binding transcription factor 4 (OCT4).16 Cancer stem cells seem to be enriched in tumours resistant to conventional systemic therapy and radiotherapy.17-19 Recent reports also suggest that SOX2, NANOG and OCT4 are potential diagnostic and prognostic markers in lung cancer.20-27 Moreover, as indicated by a recent publications28,29, SOX2 is a commonly activated tumour oncogene that activates ACT28 and EGFR29 signalling pathways in human cancers, altogether indicating its complex biological role in cell faith.
Recent studies mainly conduced in early stage non-small cell lung cancer (NSCLC) after radical surgical therapy correlated
The prognostic value of NANOG and OCT4 has only been evaluated in several retrospective studies with small number of NSCLC patients included.24-26,34-36 Elevated protein expression of both markers, NANOG24,25,34,35 or OCT424,26,36 in primary tumour was correlated with poor survival outcomes in early NSCLC patients treated with radical surgery. According to our knowledge there are no published data on prognostic or predictive value of NANOG or OCT4 expression in either blood or tumour tissue in SCLC patients.
Up to date, various studies have demonstrated that circulating cell-free tumour nucleic acids may reflect the same genetic characteristics as the primary tumour and are therefore attractive for non-invasive biomarkers determination especially during the course of diseases and in patients with no tumour tissue available.37 In lung cancer, previously mentioned study proposed circulating
The aim of our study was to evaluate the level of
The present study was conducted and is reported following recommendations for tumour marker prognostic studies (REMARK).40
50 consecutive patients with pathologically confirmed advanced SCLC, treated with first-line platinum or anthracycline-based chemotherapy and followed at University Clinic Golnik, Slovenia, between December 2009 and June 2013 were prospectively enrolled. For comparison, 50 volunteers with no clinical evidence of cancer disease were also included. Matching criteria were age and gender. 16 volunteers with other chronic pulmonary diseases (chronic obstructive pulmonary disease, asthma) were excluded from this study.
For
Patients included in this study were treated and followed according to the standard clinical practices in use at the time. All patients received first-line systemic therapy with cisplatin-etoposide or carboplatine-etoposide (PE) or cyclophosphamide–epirubicin–vincristine (CEV) chemotherapy. The dosing schedules, dose modifications and supportive therapy were offered according to the standard practice. The second-line chemotherapy including CEV or PE was offered at clinician’s discretion. Response to chemotherapy was evaluated according to the RECIST1.1 criteria41 at regular time intervals (every 2-3 months) using chest radiography or computerised tomography (CT) scans. Number of distant metastatic sites was defined as the number of the organs or organic systems involved in cancer disease.
Total RNA was isolated from whole blood using PAXgene Blood miRNA Kit (Qiagen) using the fully automated QIAcube system (Qiagen) to standardize the RNA isolation procedure. Total RNA quantity and purity were assessed using NanoDrop 2000 (ThermoScientific). After isolation and purification of total RNA from blood samples additional step including digestion of genomic DNA with DNaseI (ThermoScientific) was included. Reverse transcription reactions were performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems).
All reagents for RT-PCR were supplied by Applied Biosystems (USA). The expression of stem cell markers was detected by TaqMan RT-qPCR (ABI PRISM 7500 FAST Real-Time PCR System) using gene-specific primers–probe sets (SOX2: Hs01053049_s1, OCT4: Hs00999632_g1, NANOG: Hs02387400_g1) and TaqMan Universal PCR Master Mix II. All measurements were performed in triplicate and relative mRNA expression was determined by the ∆∆Ct method. GAPDH was used as endogenous control and pooled RNA from blood samples of healthy controls was used as a calibrator. All samples with threshold cycle ≥ 38.0 were considered as negative for
Median relative expression values of each analysed stem cell marker were compared between advanced SCLC patients and healthy controls using the Mann-Whitney U-test. The relationship between
Overall survival was defined as the period of time in months from the date of diagnosis to the date of death or last follow-up; the secondary endpoint PFS was defined as the period of time in months from the start of the first-line chemotherapy to the date of progression or death whichever occurred first. Survival probabilities, OS and PFS, were calculated by the Kaplan-Meier method and log-rank test was used to compare different categories, where optimal cut-off value between low and high expression level was set at the median mRNA expression level for each of the three markers analysed in SCLC patients. The independent prognostic value of each individual marker was tested in Cox regression model adjusted for gender, age, PS and the number of distant metastatic sites. A
The study was approved by the Slovenian National Medical Ethics Committee (approval number 135/07/09) before the enrolment of the SCLC patients and healthy controls. The informed consent was obtained before the start of the study from all subjects.
Demographic and treatment characteristics of 50 advanced SCLC patients and 34 healthy volunteers are listed in Table 1. At the time of diagnosis, median age of patients was 65 years (range 46-88 years), majority of the patients were male (35/50; 69%), and in good PS (PS ≤ 1 in 38/50; 76%). As first-line chemotherapy, the majority of patients received platinum-based chemotherapy (42/50; 84%). The second line chemotherapy was offered to 14/50 (28%) patients.
Characteristics of small-cell lung cancer (SCLC) patients and healthy volunteers
Age in years: median (range) | 65 (46-88) | 62 (47-78) |
Gender, | ||
Male | 34 (68) | 24 (71) |
Female | 16 (32) | 10 (29) |
PS East Cooperative Oncology Group performance status; CEV = cyclophosphamide-epirubicinvincristine; | ||
0–1 | 38(76) | |
≥ 2 | 12 (24) | |
Number of distant metastatic sites, | ||
< 3 | 34 (68) | |
≥ 3 | 16 (32) | |
Type of first-line chemotherapy, | ||
PE | 42 (84) | |
CEV | 8 (16) |
PE = platinum (cisplatin or carboplatin)-etoposide
As already mentioned in the methods section, cut-off value between low and high mRNA expression was set at the median expression level for the three markers analysed in SCLC patients. The associations between
Association between
bmedian mRNA expression levels for each of the three markers analysed were used to stratify patients as either | bmedian mRNA expression levels for each of the three markers analysed were used to stratify patients as either | bmedian mRNA expression levels for each of the three markers analysed were used to stratify patients as either | bmedian mRNA expression levels for each of the three markers analysed were used to stratify patients as either | bmedian mRNA expression levels for each of the three markers analysed were used to stratify patients as either | bmedian mRNA expression levels for each of the three markers analysed were used to stratify patients as either | ||||
---|---|---|---|---|---|---|---|---|---|
Age in years: median (range) | 63 (46–78) | 66 (47–88) | 0.301 Mann-Whitney U-test | 65 (47–79) | 65 (46–88) | 0.648 Mann-Whitney U-test | 64 (47–77) | 66 (46–88) | 0.466 Mann-Whitney U-test |
Gender, | 0.756 Fisher’s exact test | 0.762 Fisher’s exact test | |||||||
Male | 2 (35) | 22 (65) | 18 (53) | 16 (47) | 16 (47) | 18 (53) | 0.762 Fisher’s exact test | ||
Female | 7 (44) | 9 (56) | 7 (44) | 9 (56) | 9 (56) | 7 (44) | |||
PS East Cooperative Oncology Group performance status | 0.332 Fisher’s exact test | 0.742 Fisher’s exact test | 1.000 Fisher’s exact test | ||||||
0–1 | 16 (42) | 22 (58) | 20 (53) | 18 (47) | 19 (50) | 19 (50) | |||
≥ 2 | 3 (25) | 9 (75) | 5 (42) | 7 (58) | 6 (50) | 6 (50) | |||
Number of distant metastatic sites, | Fisher’s exact test | 0.762d | 1.000 Fisher’s exact test | ||||||
< 3 | 9 (26) | 25 (74) | 18 (53) | 16 (47) | 17 (50) | 17 (50) | |||
≥ 3 | 10 (62) | 6 (38) | 7 (44) | 9 (56) | 8 (50) | 8 (50 |
After the median follow-up of 8.5 months (range: 0.5-32.5 months) median PFS was 6.2 months and median OS was 8.4 months in 50 SCLC patients included into analysis.
The level of
Progression-free survival (PFS) after first-line chemotherapy and overall survival (OS) according to
All patients ( | 6.2 | 8.4 | ||||
| 7.4 | 0.377 | 9.9 | |||
| 5.6 | 1.988 (1.011-3.922) | 1.054 (0.938-1.183) | 7.5 | 2.370 (1.164-4.831) | 3.205 (1.536-6.711) |
| 5.7 | 0.221 | 0.299 | 7.4 | 0.347 | 0.376 |
| 6.5 | 0.693 (0.384-1.247) | 0.864 (0.656-1.139) | 8.7 | 0.750 (0.412-1.366) | 0.884 (0.674-1.161) |
| 5.6 | 0.156 | 0.227 | 7.8 | 0.251 | 0.416 |
| 7.3 | 0.652 (0.362-1.178) | 0.780 (0.521-1.167) | 9.9 | 0.810 (0.446-1.471) | 0.891 (0.673-1.178) |
95% CI = 95% confidence interval; HR = hazard ratio; MV = multivariate analysis adjusted for age, gender,
The present study aimed to compare
Our results indicate significantly higher
In contrast to SCLC, high SOX2 protein expression and
Detection of mRNA expression levels of selected biomarkers in whole blood is relatively new concept in lung cancer or any other human cancer that could be developed as an ancillary tool for disease screening and monitoring. Furthermore, it might represent an attractive approach for evaluating gene expression with no known underlying genetic mechanism affecting its expression. Moreover, detection of tumour specific DNA alterations42 and differential mRNA expression43-46 in primary tumours and/or circulating nucleic acids by high-throughput technologies (next-generation sequencing, microarrays) may provide a substantial advance in monitoring disease burden and treatment response in all human cancers.
The cut-off point for SOX2 positivity was set at the median value of
Our results indicate no difference in
The present study has several potential limitations, such as the small sample size that has an impact on statistical power of survival analysis and could therefore greatly limit the accuracy of the results. Furthermore, there are still methodological issues of biomarkers determination that should be appropriately resolved. The methods currently used for the evaluation of SOX2, NANOG or OCT4 expression in lung cancer patients differ greatly among the published reports. Studies conducted in lung cancer mainly used immunohistochemistry (IHC) for SOX2, NANOG and OCT4 protein expression and fluorescence in situ hybridisation (FISH) or quantitative PCR (qPCR) for
In conclusion, our prospective observational study found significantly higher mRNA expression levels of SOX2 in whole blood samples of advanced SCLC patients when compared to healthy controls. Equally importantly, a possible prognostic value of
Nevertheless, possible diagnostic and prognostic value of SOX2 still requires further evaluation in the frame of large-scale prospective trials. However, before embarking on large prospective clinical trials a proper standardization and validation of methodological approaches used for evaluation of selected biomarkers is necessary.