Abstract

Background: In this study, we aimed to investigate the relationship between survival, tumor dimension, grade and stage in respect to transforming growth factor-?-activating kinase (TAK-1) extensity, severity and total score in patients undergoing resection for Stage 1B-2B non-small cell lung cancer.

Methods: Between January 2000 and December 2014, a total of 70 patients (64 males, 6 females; mean age: 63.4+9.6 years; range, 32 to 78 years) who underwent surgery with resectable non-small cell lung cancer in Stage 1-2b were included. The patients were divided into two groups as Group 1 (n=35) consisting of patients with squamous cell carcinoma and Group 2 (n=35) consisting of patients with adenocarcinoma. The control group consisted of 20 patients (Group 3) who underwent surgery due to non-cancer causes. The relationship between TAK-1 staining (extensity, severity, total scores) and grade, survival time, T factor, N factor, and chemotherapy administration was examined. Pathology specimens of the patients were evaluated for the degree of staining with TAK-1 primary antibody.

Results: There was a strong correlation between the tumor grade and TAK-1 primary antibody staining level, independently from histopathological type. A significant correlation was found between dimension, stage, and TAK-1 staining in patients with squamous cell carcinoma. No statistically significant difference was found in the other factors, except for grade factor, in patients with adenocarcinoma.

Conclusion: The current study provides precious information about the effects of TAK-1, in clinicopathological behavior and survival of malignant cells, particularly in common histopathological types of lung cancer. We believe that our data can be useful, particularly in evaluating the response to targeted therapies and the prognosis of the disease.

Angiogenesis plays an important role in the pathogenesis of non-small cell lung cancer (NSCLC). Increased lung tumor microvessel density is associated with the increased metastatic potential and decreased survival.[1-3]

Transforming growth factor (TGF)-β- activated kinase 1 (TAK-1), which is also found in angiogenesis, is one of the most recent molecules. It is a mitogen-activated protein kinase that is activated by the formation of a protein complex by various cytokines. It is a serine/threonine kinase and is associated with the progression of human cancer cells.[4] However, although there are many studies on other molecular markers in patients with lung cancer, very few studies have shown the relationship between TAK-1 positivity and prognosis.[5] In addition, in this study, the relationship between tumor development, progression and degree of invasion and TAK-1 in NSCLC patients was evaluated. However, the subtypes were not compared and difference of stage were not considered.[5]

In the present study, we aimed to investigate the relationship between histopathological grade, tumor size, pathological stage, and survival in terms of TAK-1 distribution, intensity, and total score in the pathology slides of the patients undergoing resection for Stage 1-2B squamous cell carcinoma (SCC) and adenocarcinoma (ADC).

Methods

This single-center, retrospective study was conducted at the Department of Thoracic Surgery, Faculty of Medicine of Ondokuz Mayıs University between January 2000 and December 2014. A total of 610 patients who underwent surgical resection for NSCLC were evaluated. Thoracic computed tomography (CT), positron emission tomography (PET)-CT, cranial magnetic resonance imaging (MRI), and bronchoscopy were performed in all of patients for preoperative diagnosis and staging. If necessary, transthoracic needle biopsy was also performed for the diagnosis. If the patient had mediastinopathic pathological lymph node, mediastinoscopy was performed before resection. Finally, a total of 70 patients (64 males, 6 females; mean age: 63.4+9.6 years; range, 32 to 78 years) who were evaluated as Stage I a,b-II a,b NSCLC and underwent anatomic resection were included. Twenty patients who underwent wedge resection for non-cancer reasons (spontaneous pneumothorax) were included in the control group. Exclusion criteria were as follows: patients who received any specific treatment such as chemotherapy and radiotherapy before surgery, synchronous or metachronous tumors, positive mediastinal lymphadenopathy, could not be performed anatomical resection, Stage IIIa and above disease, having any lung disease such as pneumonia and abscess, or having any life-limiting disease such as chronic liver disease, renal failure, severe arrhythmia, ischemic heart disease, and collagen tissue disease.

The patients were divided into three groups. Group 1 consisted of 35 patients diagnosed with SCC, Group 2 consisted of 35 patients diagnosed with ADC, and Group 3 (control group) consisted of 20 patients who underwent surgery due to non-cancer causes. All patients in Group 3 underwent wedge resection for recurrent spontaneous pneumothorax. Staging was performed both via radiographical examinations preoperatively and via histopathological examinations postoperatively. Pre- and postoperative tumor stage was determined according to 7th L ung C ancer Tumor, Node, Metastasis (TNM) classification and staging system.[6] Histopathological evaluations were performed based on the diagnostic criteria proposed by the 2015 edition of the World Health Organization (WHO) classification system.[7]

All of patients in Groups 1 and 2 were followed with thoracic CT, cranial MRI, and PET-CT at 3, 6 and 12 months annually for five years. Follow-up data were obtained using the electronic medical records of our hospital and by phone calls.

Immunohistochemical staining
All samples were fixed in 10% buffered formalin for 24 h. All sections obtained from blocks belonging to these specimens were examined and, then, blocks that best represented the invasive tumor were selected. Totally, 4-micron thick sections were obtained from these blocks, and these sections were studied immunohistochemically with TAK-1 primary antibody (anti-TAK 1 antibody, rabbit monoclonal antibody, ab196955, abcam) on the Ventana Benchmark XT2 (Roche Diagnostic, Solna, Sweden) automated staining instrument. The best dilution ratio was determined as 1/50 after various tests. As a positive control, the breast cancer specimen proposed by the company was used. Findings were evaluated using the Leica HMLB45 (Leica, Germany, 2000) light microscope.[8] The distribution and intensity of staining were scored semi-quantitatively by taking cytoplasmic staining into account. The staining distribution was classified as f ollows: 0 = between 0 and 10%, 1 = between 11 a nd 2 5%, 2 = b etween 2 6 a nd 5 0%, a nd 3 = m ore than 50%. The staining intensity was classified as follows: 0= no staining (Figure 1), 1= slight yellowish staining, 2= dark yellowish staining, and 3= brown staining. The scores for the distribution and intensity of staining were collected, and they were classified as f ollows: n egative ( 0) = b etween 0 a nd 2 , m ild (1) = between 3 and 4, and severe (2) = between 5 and 6. All pathological scorings were performed by two different pathologists.

Figure 1. No staining with TAK-1 on control patients (X400). TAK-1: Transforming growth factor (TGF)-Β-activated kinase 1.

Statistical analysis
Statistical analysis was performed using the SPSS version 11.0 software (SPSS Inc., Chicago, IL, USA). Continuous data were expressed in mean ± standard deviation (SD) or median (min-max), while categorical data were expressed in number and frequency. The assumptions of normality and of homogeneity of variances were checked by the Shapiro-Wilk test and the Levene's test. Then, the differences among the age and T measurements of the patients were assessed using the Student t-test according to the type of disease (SCC and ADC). The relationship between qualitative data presented in the cross-table and TAK-1 distribution, intensity and total score was assessed using the chi-square test and Cramer's V coefficient. A p value of < 0.05 was considered statistically significant.

Results

Baseline features of the patients are shown in Table 1.

Table 1. Overall characteristics of patients

Adenocarcinoma patient data for TAK-1
Pathological results for ADC patients showed that TAK-1 prevalence, intensity, and overall score were significantly different according to cancer grade (p=0.005). As the grade became higher, TAK-1 prevalence staining intensity and overall score of the tumor also increased (Table 2, Figure 2, 3a, b).

Table 2. TAK-1 distribution, intensity, overall score and grade for patients with ADC

Figure 2. Adenocarcinoma hematoxylin and eosin staining (H&E, x400).

Figure 3. (a) Score 3 staining with ADC-TAK-1 primary antibody (x400). (b) Score 6 staining with ADC-TAK-1 primary antibody (x400).
ADC: Adenocarcinoma; TAK-1: Transforming growth factor (TGF)-?-activated kinase 1. We found no correlation between survival and all variables of TAK-1 in ADC patients (p>0.05).

In addition, relationship between tobacco use, clinical and pathological stage, tumor size, mediastinal involvement, chemotherapy indication and TAK-1 staining properties were statistically non-significant (p>0.05).

Squamous cell carcinoma patient data for TAK-1
The results for SCC patients showed that TAK-1 distribution, intensity, and overall score were significantly different according to tumor grade. As the grade became higher, TAK-1 prevalence staining intensity and overall score of the tumor increased (Table 3, Figure 4, 5a, b).

Table 3. TAK-1 distribution, intensity, overall score and grade for patients with SCC

Figure 4. Squamous cell carcinoma hematoxylin and eosin staining (H&E, x400).

Figure 5. (a) Score 3 staining with SCC-TAK-1 primary antibody (x400). (b) Score 6 staining with SCC-TAK-1 primary antibody (x400).
SCC: Squamous cell carcinoma; TAK-1: Transforming growth factor (TGF)-?- activated kinase 1.

The TAK-1 distribution, intensity, and overall score values changed based on pathological stage subgroups (1A,1A,2A,2B). As the pathological stage increased, TAK-1 distribution, TAK-1 intensity and overall score increased (Table 4).

Table 4. TAK-1 Distribution, intensity, overall score and pathological stage for patients with SCC

The relationship between TAK-1 distribution values and T factor subgroups (T1A, T1B, T2A and T2B) were statistically non-significant, while TAK-1 intensity and overall score showed a correlation with an increasing T factor (p>0.05) (Table 5).

Table 5. Distribution, intensity, overall score of TAK-1 and T factor subgroups on patients with SCC

Relationship between tobacco use, N factor subgroups, chemotherapy indication and TAK-1 staining properties were statistically non-significant (p>0.05). While we found no correlation with survival and TAK-1 an increase in T subgroup and grade resulted in statistically significantly less survival years (Table 6).

Table 6. Distribution of grade, T factor and survival on patients with SCC

Discussion

The TAK-1 is a serine/threonine kinase that contains ligands of TNF-alpha, interleukin (IL)-1 and tall-like receptor (TLR) and is a key role in proinflammatory cytokine release.[9-12]

Tumor growth and metastasis development depend on the presence of the appropriate vascular source (angiogenesis). The mechanism of angiogenesis involves multiple, sequential or mutually independent pathways. The TAK-1, a molecule that is investigated to make any contribution to cancer development and progression, has been reported to play multiple roles in angiogenesis and cancer progression. There are studies in the literature showing that TAK-1 can play different roles in various cancers by acting with a tumor suppressor effect.[13] Despite all these studies, there are few studies in the literature investigating the relationship between TAK-1 and lung cancer.[5] Zhu et al.[5] found that TAK-1 expression in patients with NSCLC significantly increased tumor stages, metastasis rates and lymph node positivity, and survival was shorter in these patients compared to other patients. However, in this study, patients with NSCLC were evaluated in general, and subtype and stage were not specified specifically. In our study, TAK-1 levels were evaluated in patients who had SCC and ADC, separately. Our results support the literature. In addition, TAK-1 levels were found to be correlated with the tumor grade, pathological stage and T factor in SCC patients, whereas it was found to be positively correlated with tumor grade in patients with ADC. Furthermore, in our study, TAK-1 levels were not considered in only one parameter. The TAK-1 levels were evaluated histopathologically in terms of distribution, density, and overall score.

Activation of the TAK-1 signaling pathway enhances tumor development, invasion, and metastatic activities.[5,12,13] The TAK-1 has played an important role in the development of metastatic lesions in patients with breast, renal cell, and lung carcinomas. When TAK-1 was suppressed, there was a decrease in the bone-invading capacity and osteolysis of tumor cells in patients with breast cancer.[13,14] In the study of Zhu et al.,[5] prolonged TAK-1 activation in patients with lung cancer was effective in cancer progression, tumor development, and metastasis.[5] In our study, the slides were positively stained with TAK-1 in all patients with tumors regardless of grade, tumor size, and histopathological type. When the pathology slides of 20 patients, who were considered as the control group, undergoing surgery for benign lung diseases were examined, the TAK-1 was poorly stained at all variables. Both results of the literature and our study indicate that TAK-1 is strongly associated with tumorigenesis.

Futrhermore, TAK-1 has a major role in cigarette smoke-induced IL-8 release in airway smooth muscle cells and contributes to the development and progression of chronic obstructive lung disease and its associated lung cancer.[15,16] We found that there was no statistically significant relationship between smoking status and TAK-1 distribution, intensity, and total score in patients with lung cancer. However, considering all scores related to TAK-1, almost all parameters were closely associated with TAK-1 in patients with SCC. This suggests that smoking may potentiate the effects of TAK-1 positivity on tumorigenesis.

The TAK-1 positivity has been shown to have a positive effect on tumor growth rate. Cai et al.[17] reported that tumor growth rate was two-fold greater in TAK-1 positive ovarian cancer cells than in the control group. We found that there was no significant relationship between tumor size and TAK-1 distribution, intensity, and total score in patients with ADC. However, TAK-1 staining intensity increased, as the tumor size increased in patients with SCC. It was also observed that the mean survival time decreased as tumor size increased in patients with SCC. As a result, it may be possible to conclude that survival decreased as TAK-1 staining intensity increases, considering that TAK-1 staining intensity increases as the tumor size increases in patients with SCC. When TAK-1 staining distribution was assessed in patients with ADC, there was a decrease in the number of patients who lived longer than five years as TAK-1 staining distribution and intensity increased. Although this result was not statistically significant, we believe that the fact that the number of patients was small and advanced cancer patients were not included in this study may have led to a non-significant relationship between TAK-1 staining distribution and survival.

Histopathological evaluation of tumor cells in the literature showed that TAK-1 staining distribution and intensity of tumor cells increased progressively from low-grade ovarian cyst ADC to high-grade ovarian cyst ADC.[17] In our study, TAK-1 distribution, intensity, and total score were found to increase significantly with increasing tumor grade regardless of histopathological type. Moreover, while comparing tumor grade with survival time, no significant difference was observed in patients with ADC; however, survival time decreased significantly, as the tumor grade increased in patients with SCC. In addition, there was no statistically significant increase in patients with ADC, when the pathological stages of the patients were compared in terms of all TAK-1 variables. However, TAK-1 staining distribution and intensity increased, as the tumor stage progressed from Stage 1A to Stage 2B in patients with SCC.

The study of Carbone and Melis[18] showed that the tumor burden was reduced, the effect of chemotherapy was potentiated, and the survival rate increased thanks to genetic suppression of TAK-1 expression. While we found no correlation with survival and TAK-1, increase in T subgroup and grade resulted in statistically significantly less survival years. Although no significant difference between survival and TAK-1 was found in our study, it is thought that TAK -1 affects indirectly survivor through variables such as grade and T factor based on the relationship between T-factor and grade and TAK-1. In addition, it is believed that our results depend on the fact that our study was performed on early stage patients who had high survival rates.

In the literature, TAK-1 kinase selective inhibitors have been shown to increase anti-tumor activity of chemotherapeutics. Gene-targeted cancer therapy has given promising results in improving survival and quality of life in advanced and high-grade tumors. In recent years, there is a significant increase in the number of studies on targeted therapies in NSCLC and colon cancer.[18-20] Our study supports the data in the literature. Moreover, it is planned to examine both TAK-1 activity and the effects of chemotherapeutics on TAK-1 activity in further studies to be performed on advanced cancer patients who require postoperative adjuvant CT in our clinic. However, there was no significant difference in terms of TAK-1 distribution, intensity and total score in patients with N1 node-positive NSCLC who received chemotherapy after resection. We believe that this may be due to the fact that the cancer stages of the patients included in the study were low and they had no metastatic cancer.

While considering that an increase in the stage of lung cancer has a negative effect on five-year survival, TAK-1 staining distribution and intensity negatively affected survival rate in patients with SCC as in ovarian cancer and pancreatic cancer in the literature. Lam et al.[21] reported that TAK-1 caused an increase in the transition from normal cells to invasive cells by increasing free oxygen radicals in the SCC cells in the skin after subcutaneous injection. In our study, according to histopathological types of the tumor, no relationship was established in terms of TAK-1 distribution, intensity, and total score. Although there was a strong correlation with the levels of TAK-1 in patients with SCC, there was a significant relationship with only tumor grade in patients with ADC. Considering that TAK-1 is effective on angiogenesis of the tumor, this situation may be explained by the fact that ADCs usually use the lymphatic pathways for metastasis.

In conclusion, the TAK-1 has been one of the most popular targeted therapies in recent years. There are many in vitro studies on TAK-1. These studies have provided valuable information on the immunohistochemical effects of TAK-1. Therefore, we believe that our study would provide valuable information about the effects of TAK-1 on pathological clinical behavior and survival in cancer cells, particularly in the most common types of lung cancer. We consider that valuable information on targeted therapies, particularly in lung cancer, would be obtained thanks to future studies to be performed on this target molecule. Moreover, it is thought that it can provide very important information in the arrangement of the treatment plan, the identification of chemotheraphy, radiotherapy, chemoradiotherapy and targeted therapies, and the evaluation of the prognosis of the disease.

Ethics Committee Approval: The study protocol was approved by the Ondokuz Mayıs University Faculty of Medicine Clinical Research Ethics Committee (date: 28.11.2014, no: 2014/875PYO.TIP.1904.15.0.16). The study was conducted in accordance with the principles of the Declaration of Helsinki. Patient Consent for Publication: A written informed consent was obtained from each patient.

Data Sharing Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request.

Author Contributions: Conception or design of the experiment(s), or collection and analysis or interpretation of data, Drafting the manuscript or revising its intellectual content; and Approval of the final version of the manuscript to be published: P.S., Y.B.; Conception or design of the experiment(s), or collection and analysis or interpretation of data, drafting the manuscript or revising its intellectual content; and approval of the final version of the manuscript to be published: Y.S., A.B.

Conflict of Interest: The authors declared no conflicts of interest with respect to the authorship and/or publication of this article.

Funding: The authors received no financial support for the research and/or authorship of this article.

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