MARKERS ON OVERALL SURVIVAL IN SURGICALLY TREATED PATIENTS WITH NON SMALL CELL LUNG CANCER

Background/Aim. Non-Small Cell Lung Cancer (NSCLC) is one of the most common malignant tumors and a leading cause of cancer-related deaths. The aim of this study was to evaluate impact of biological markers on the overall survival rate in surgically treated NSCLC patients who received adjuvant chemotherapy and/or radiation therapy. Methods. This retrospective case series study was conducted at the Pulmonology Clinic and the Clinic for Chest Surgery, Military Medical Academy, Serbia. Patients with NSCLC were treated in the time period between 2008 and 2017. The survival analysis performed was based on immunohistological findings, histology type and tumor, node, metastasis (TNM) stages. Results. The mortality rate was higher in the adenocarcinoma patient group compared to the squamous cell carcinoma group, albeit without statistical significance (58.3% vs . 31.2%, respectively, p=0.175). Overall survival was lower in the adenocarcinoma patient group compared to the squamous cell carcinoma group (by approximately 750 days). Likewise, overall survival was lower in the adenocarcinoma patient group compared to the squamous cell carcinoma group for CD31 positive (p=0.029), p-63 positive (p=0.049), MMP-9 positive (p=0.032) and MMP-2 positive patients (p=0.016). Conclusion. Adenocarcinoma is a more aggressive cancer type in comparison to squamous cell carcinoma with a lower overall survival. Our research showed a poorer overall survival in the adenocarcinoma group compared to the squamous cell carcinoma group in CD31, p-63, MMP-9 and MMP-2 positive patients.


Introduction
Lung cancer is one of the most common malignant tumors and a leading cause of cancer-related deaths [1][2][3] . About 80% of all lung cancers are Non-Small Cell Lung Cancer (NSCLC), i.e. squamous cell carcinoma and adenocarcinoma 4,5 .
The NSCLC significantly decrease overall survival, life quality and working ability of patients, but increase direct and indirect medical cost 6 . Treatment options for patients with NSCLC consist of combined surgical treatment, radiation therapy and/or one of the chemotherapy treatment protocols based on the stage of illness, histology type and tumor marker findings and other parameters 6 .
Surgery represents a treatment of choice for patients with NSCLC stage I-IIIA according to tumor, node, metastasis (TNM) 8 th edition classification 7,8 . In addition to surgery, patients with resected NSCLC stage II-IIIA, who have a high risk of relapse, are treated with adjuvant chemotherapy and/or radiation therapy 6,9 . Patients with stage IIIB and IV NSCLC are generally treated with chemotherapy and radiation therapy. For NSCLC stage I and II, radiation therapy alone is considered effective only when surgical resection is not possible either due to limited pulmonary reserve or the presence of comorbidities 10 .
Histology and immunohistochemistry (IHC) analysis, as well as specific gene expression assessment, may become a predictive factor for response to chemotherapy in the future clinical research and patient treatment 11 . Expression of biomarkers (for example, HER-2, BCL-2, CD-31, p-63, BRAF, KRAS, etc.) can be tested at a protein level using IHC, while mRNA levels can be determined through reverse transcriptase PCR (RT-PCR)-based assays.
Therefore, these biomarkers are currently not in use in daily practice. Many biomarkers in lung cancer were point mutations and rearrangements in specific genes including EGFR, anaplastic lymphoma kinase, HER-2, BCL-2, CD-31, p-63, MMPs, BRAF, NUT, MET, ROS1, DDR2, FGFR1, KRAS, and PTEN 11 . These biomarkers might potentially provide additional information for clinical decision making.
The overall five-year survival rate for all lung cancer in stage with localized disease is about 52.2%, in stage with regional metastatic disease 25%, and in stage with distant metastatic disease 4% 12 .
The aim of this study is to evaluate impact of biological markers on the overall survival rate in surgically treated NSCLC patients, and after the adjuvant chemotherapy and/or radiation therapy.

Patient's data
This retrospective case series study of patients with NSCLC was designed as survival analysis based on immunohistological findings, histology type and TNM stages. Forty (40) NSCLC patients (17 females and 23 males; average age 59.22±8.31 years) were treated at the Pulmonology Clinic and Chest Surgery Clinic of the Military Medical Academy in Serbia, and were followed up over the period between 2008 and 2017.
Clinical files from all patients with clinically confirmed lung cancer admitted between 2010 and 2015 to the institutional healthcare network of the Military Medical Academy were accessed in both hard and electronic copies from the hospital registries. The following data were analyzed: demographic characteristics (age, gender), overall survival rate, immunohistological findings, histology type and TNM stages of NSCLC.

TNM Stage and Patients Treatment
A first step done in our hospital was to classify patients with NSCLC according to the TNM stages . T1N0M0 was classified as stage IA; T2N0M0 as stage IB; T1N1M0 as   stage IIA; T2N1M0 and T3N0M0 as stage IIB; and T3N1M0, T1N2M0, T2N2M0, T3N2M0 as stage IIIA. Stage IIIB was classified as T4 any N M0 and any T N3M0, whereas stage IV was classified as any T any N M1 13 .
A second step consisted of treating patients with NSCLC, according to their TNM stages. TNM stage I patients were treated only surgically. TNM stage IIA to IIIA patients were treated surgically and with adjuvant chemotherapy (etoposide and cisplatin --EP/PE protocol) and/or radiation therapy.
The above chemotherapy protocol was applied as follows: cisplatin at 60 mg/m 2 IV administered on day 1, plus etoposide at 120 mg/m 2 IV administered on days 1, 2 and 3, every 21 days for 4 cycles. Alternatively, cisplatin at 80 mg/m 2 IV was administered on day 1, plus etoposide at 100 mg/m 2 IV on days 1, 2 and 3, every 28 days for 4 cycles.
Radiotherapy was applied in patients with positive resection surface for malignancy and with N2 TNM stage 6 .

Histology and IHC
Following tumor excision, collected tissue was formalin-fixed and paraffin embedded (FFPE) as described below. Tissue slides were morphologically diagnosed at the Institute for Pathology and Forensic Medicine, and subsequently tested for a series of biomarkers using IHC standard protocol developed at the Laboratory for Immunohistochemistry and Electron Microscopy of the Institute for Medical Research.
Excised tissue was fixed in 5% neutral-buffered formalin, and processed in V.I.P.
Sakura apparatus for automatic fixation, dehydration and paraffin embedding. Tissue blocks were cut at 5-7 µm, and sections mounted on separate adherent chips (Super-Frost), and then dried at 56°C for one hour prior to staining.

Statistical Analyses
Continuous variables were presented as median with inter quartile range (IQR).
Categorical variables were reported as frequencies.
Differences between categorical variables were tested by Chi-square test, while significance of difference between continuous variables was tested by non-parametric Mann-Whitney U test. Overall survival estimates were calculated using the Kaplan-Meier method [mean (95% confidence interval -CI 95%)], and Log-rank (Mantel-Cox) test to assess differences between groups of NSCLC. A p-value of less than 0.05 was considered statistically significant.

Ethics Committee Approval
The study was conducted in accordance to the Declaration of Helsinki, and the protocol reviewed and approved on September 6, 2015 by the regional Ethics Committee of the Military Medical Academy.

Results
In our study, we analyzed 40   Overall survival of patients with adenocarcinoma and squamous cell carcinoma according to interval from surgery/ surgical resection to recurrence was presented in Table   2. Statistically significant difference was not observed between the groups. Cumulative survival was lower in the patient recurrence group with adenocarcinoma in comparison to the group with squamous cell carcinoma by approximately 810 days ( Figure 2).
Overall survival was estimated and compared among patients according to preoperative TNM stage in both patient groups (Table 3). There was no statistically significant difference in survival between patients in stages I, II and IIIA within the adenocarcinoma (p=0.060) and the squamous cell carcinoma group (p=0.970). However, overall survival between adenocarcinoma and squamous cell carcinoma according to initial TNM stage showed that patients with adenocarcinoma had lower statistically significant survival rate compared to the patients with squamous cell carcinoma in TNM stage IIIA (Log Rank (Mantel-Cox) test; p=0.007) (mean 374.4 days vs. 1586.4 days, respectively) ( Figure   3). No significant differences were observed between adenocarcinoma and squamous cell carcinoma by distribution of patients according to biological markers ( Table 4). The patients were most frequently positive for BCL-2, CD-31, p-63, MMP-9 and MMP-2, but rarely for HER-2 and MMP-14.
Overall survival was estimated and compared among NSCLC patients according to status of biological markers in two patient groups studied (Table 4)

Discussion
Non-small cell lung cancer is one of the major causes of cancer-related deaths 2 . To analyze survival in our NSCLC patients, we conducted a retrospective case-series study using the follow-up data from a time period between 2008 and 2017. Our study design aimed at assessing the overall survival in NSCLC patients according to specific biomarkers expression, TNM stage and histology type 14 .
In the operable NSCLC patients, adjuvant chemotherapy has been considered a standard modality of treatment following surgical resection of the tumor [14][15][16][17][18][19] . Besides, molecularly targeted therapy has significantly improved outcomes of patients with metastatic form of NSCLC 2, 18 . Nevertheless, for the majority of patients, platinum-based chemotherapy remains the gold standard treatment, and has led to significantly improved survival outcomes with approximately 10-11 months median survival 20 .
In our study, there were more male patients in the squamous cell carcinoma group, while female patients dominated the adenocarcinoma group. Men develop tracheal, bronchus and lung cancer more often compared to women (1/18 for men; 1/45 for women) 3 . The estimated numbers of lung cancer cases worldwide has increased by 44% in men and 76% in women since 1985 21 . The higher rate increase in women has been attributed to the fact that cigarette smoking in female population peaked two decades later than in male 21  Patients with adenocarcinoma had poorer prognosis compared to squamous cell carcinoma patients 24 . Similarly, in our study, the mortality rate was significantly higher in the adenocarcinoma group (58.3%) as opposed to squamous cell carcinoma group (31.2%).
According to the literature, generally, the five-year survival rate for all NSCLC patients in stage IA, IB, IIA and IIB is about 49%, 45%, 30% and 31% respectively 25 . This rate for NSCLC patients in stage IIIA and IIIB is about 14%, 5%, respectively 25 .
Overall survival of patients according to recurrence is of major significance 23 .
Recurrence rates reported following surgical cancer resection range from 30% to 75% 26 . The majority of recurrent tumors are distant, and more than 80% of recurrences occur within the first two years after resection. Cumulative survival was lower in our patient recurrence group with adenocarcinoma compared to the squamous cell carcinoma group by about 810 days. This is in line with our findings showing adenocarcinoma as a more aggressive cancer type than squamous cell carcinoma.
The complete resection of early stage NSCLC offers to patients high hopes for a successful therapeutic outcome. However, the recurrence rates post-resection remain high 27 .
For that reason, right from the beginning of therapy in NSCLC patients, a complete surgical removal needs to be ensured both macroscopically and microscopically. Often, occult micro- in NSCLC is of great significance 33, 34 . MMP-2 overexpression predicts a poor prognosis in early-stage NSCLC. This study shows that MMP-2 overexpression correlates with early cancer-related death. Other MMP subclasses are also associated with a degree of lung cancer aggressiveness. It is of note, that one systematic review suggests that MMP-2 expression has a poor prognostic significance of NSCLC patient's survival 37 .
MMP-9 has a role in extracellular matrix remodeling, increased cell proliferation, invasion/migration and angiogenesis 38 . Highly expressed MMP-9 correlates with shortened survival of NSCLC patients 33, 39 . MMP-9 expression is an independent prognostic marker for resected stage NSCLC. Thus, MMP-9 is a novel biomarker significantly and independently predicting worse prognosis of resected stage NSCLC. In a different study, tumor MMP-9 expression was associated with poor outcomes in adenocarcinoma, but not in squamous cell carcinoma patients 40 . MMP-9 expression was identified as an independent marker of relapse in completely resected lung adenocarcinoma.
In NSCLC patients, genetic aberrations of human epidermal growth factor-2 (HER-2) signaling pathway are associated with different sensitivity to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) 41 . This is a plausible mechanism and prognostic role of acquired resistance to the EGFR TKIs in EGFR-mutated tumors. Although in our study a vast majority of patients in both cancer groups were HER-2 negative, our positive adenocarcinoma patients showed lower survival rate compared to HER-2 negative adenocarcinoma patients. Gene amplification is a well-known mechanism of proto-oncogene activation and has been described in many human malignancies, including lung tumors.
However, HER-2 amplification seems far less common in NSCLC compared to other cancers. Recently, the predictive role of HER-2 overexpression has been more extensively studied with a purpose to identify anti-HER-2 agents applicable in NSCLC patients. Limitation of the study: Our study is limited by a low size effect and a retrospective character; the optimal management of lung cancer patients according to biomarkers needs to be determined by prospective clinical trials in large patient cohorts.

Conclusion
In conclusion, adenocarcinoma is more aggressive compared to squamous cell  Table 1 Overall survival in all patients with Lung Cancer according to biological markers tested      Kaplan-Meier analysissurvival curves in the patients with Non-Small Cell Lung Cancer (NSCLC) in IIIA TNM stage according to histology type (censored -alive at the end of the follow-up period) Table 4 Overall  Kaplan-Meier analysissurvival curves in positive p-63 patients with Non-Small Cell Lung Cancer (NSCLC) according to histology type (censored -alive at the end of the follow-up period)