Premalignant Changes in the Bronchial Epithelium Are Prognostic Factors of Distant Metastasis in Non-Small Cell Lung Cancer Patients

Background The study assessed the possibility of dividing patients into groups based on the assessment of morphological changes in the epithelium of small-caliber bronchi located near the primary tumor in order to predict high and low risks of distant metastasis of non-small cell lung cancer. Methods In 171 patients with non-small cell lung cancer (T1-4N0-3M0) in small-caliber bronchi taken at a distance of 3–5 cm from the tumor, various variants of morphological changes in the bronchial epithelium (basal cell hyperplasia (BCH), squamous cell metaplasia (SM), and dysplasia (D)) were assessed. Long-term results of treatment, namely, distant metastasis, were assessed after 2 and 5 years. Results During the follow-up period, distant metastases were found in 35.1% (60/171) of patients. Most often, they were observed in patients of the high-risk group: BCH+SM−D− (51.6%, 40/95) and BCH−SM+D+ (54.4%, 6/11). Less often, distant metastases were observed in low-risk group patients: BCH+SM+D− (6.7%, 3/45) and BCH−SM−D− (10.0%, 2/20). Tumor size, grade, and stage were significant predictors of metastasis only in the high-risk group. The 5-year metastasis-free survival was better in the low-risk group of distant metastases. Conclusions Isolated BCH or dysplasia in small bronchi distant from foci of tumor is associated with a high-risk distant metastasis and less 5-year metastasis-free survival.


INTRODUCTION
The most common cause of cancer death in 2020 was lung cancer (1). High mortality is associated with the progression of the tumor process. Therefore, the search for various molecular biological markers involved in the mechanisms of distant metastasis in non-small cell lung cancer (NSCLC) remains relevant. The identification of patients with a high risk of tumor progression can be used to adequately prescribe adjuvant chemotherapy (AC) and to adjust its regimen in order to minimize adverse effects.
The most important factors associated with progression of NSCLC and predicting survival are tumor stage, histologic structure, grade, and biological aggressiveness (2)(3)(4)(5). However, these factors are not always effective in predicting the outcome of the tumor process. Our earlier study showed that different variants of the combination of morphological changes in the epithelium of small bronchi [basal cell hyperplasia (BCH), squamous cell metaplasia (SM), and dysplasia (D)], distant from foci of squamous cell carcinoma and lung adenocarcinoma, are associated with recurrence. The combination of BCH and SM is associated with high risk of recurrence of NSCLC regardless of the histologic type of tumor and neoadjuvant chemotherapy (NAC) (6,7). In this study, we considered the association of different variants of morphological changes of the respiratory epithelium of small bronchi adjacent to the tumor as risk factors for distant metastasis.

Patients
The study enrolled 171 patients with NSCLC (squamous cell carcinoma and adenocarcinoma, T 1-4 N 0-3 M 0 ) who were treated in the Cancer Research Institute, Tomsk NRMC, between 2005 and 2011. Patients were excluded if they were refused surgery and had an Eastern Cooperative Oncology Group (ECOG)/WHO performance score >2, small-cell lung cancer, associated severe diseases, and cardiovascular and pulmonary decompensation. Metastatic involvement was identified from the Local Cancer Register. The study was approved by the Institutional Review Board (IRB) (December 10, 2012; the number of approvals is 16).
The histologic diagnosis of lung cancer was made according to the International Association for the Study of Lung Cancer/ American Thoracic Society/European Respiratory Society (IASLC/ATS/ERS) lung adenocarcinoma classification (8) and the WHO criteria (9) and was confirmed by immunohistochemistry using a panel of antibodies: TTF-1 (clone 8G7G3/1, Dako), Napsin A (Rabbit Polyclonal, Cell Marque), and p63 (Rabbit Polyclonal, Leica) ( Figure 1).
Cancer stage was determined according to the TNM classification (10). The type of morphological lesions in the bronchial epithelium (BCH, SM, and D) of small bronchi (d = 0.5-2 mm), obtained at a distance of~3 cm from the tumor edge during surgery, was assessed as described earlier (11).

Statistical Analysis
The data were analyzed with the statistical software STATISTICA 12 (StatSoft, OK, USA) and GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). A multivariate logistic regression model was used to calculate odds ratios (OR) for type of bronchial lesions, histologic type, recurrence, type of therapy, gender, smoking status, grade, and stages T and N. p-Values for 2 × 2 tables were obtained by using Fisher's exact test. Survival was investigated with univariate and multivariate Cox regression models, yielding hazard ratios (HRs). This model adjusted for type of bronchial lesions, histologic type, recurrence, type of therapy, gender, smoking status, grade, and stages T and N. Metastasis-free survival (MFS) was calculated by the Kaplan-Meier method, and differences in survival curves among the groups were evaluated by the log rank test. p < 0.05 was considered statistically significant.

RESULTS
Over the entire follow-up period, distant metastases occurred in 35.1% (60/171) of patients with NSCLC. The clinical and pathological parameters of NSCLC patients depending on the presence or absence of distant metastasis are presented in Table 1.
The frequency of the stage T at diagnosis and the grade between the low-and high-risk groups were not significant (Fisher's exact test, p > 0.05) ( Figure 2).
This, as well as no correlation between stage T and grade (r 2 = 0.3448, p = 0.6998), may indicate the independence of three factors in the distant metastasis prognosis. The results presented in Table 3 allow us to compare the significance of three factors (risk groups, T, and grade) to determine the rate of developing distant metastases in NSCLC.
There is every reason to believe that the assignment of patients to high-and low-risk groups by the risk score system based on type of bronchial lesions is the most significant and independent prognostic factor of distant metastasis. Moreover, it is acceptable to believe that T3-4 and grade 3 are unfavorable factors only in the high-risk group of distant metastases.
We described the frequency of metastases depending on the cancer stage and the grade, separately for the low-and high-risk groups ( Figure 3).
The evaluation of the relationship of distant metastasis of NSCLC with the cancer stage showed that in BCH−SM−D− group single distant metastases occurred only in patients with stage IIIA, 16.7% (2/12) ( Figure 3A). In another low-risk group (BCH+SM+D−), metastases were in 11.1% (1/9) of patients with stage IIB, in 8.3% (1/12) of patients with IIIA, and in 9.09% (1/ 11) of patients with stage IIIB ( Figure 3B).
There were no metastases in the low-risk group of distant metastases in I-II stage during the observation period, while in the high-risk group of distant metastases (BCH+SM−D− and BCH−SM+D+), metastases occurred at any cancer stage ( Figures 3C, D). From Table 4, it follows that only the highrisk group of distant metastases is associated with the cancer stage (p = 0.0028).
The comparison of the rates of metastasis in cases with stage IIIA shows that at the same stage, the frequency of metastasis in the high-risk group is 20 times higher than in the low-risk group.

Survival Analyses
We explored the potential prognostic factors in NSCLC patients using univariate and multivariate Cox regression analyses. None of the investigated parameters in univariate and multivariate Cox analyses influenced 2-year MFS in NSCLC patients ( Table 5).
As shown in Table 6, the univariate Cox regression analysis revealed that the BCH+SM−D− type of bronchial lesions was significantly associated with poor 5-year MFS in patients with NSCLC.
The multivariate Cox regression analysis showed that the BCH+SM−D− type of bronchial lesions was an independent prognostic factor for the 5-year MFS. The Kaplan-Meier plots indicated that NSCLC patients with BCH+SM−D− exerted significantly worse survival than the patients with other type of bronchial lesions (p < 0.05; Figure 4).
The survival rates of NSCLC patients with different types of bronchial lesions who were alive for 5 years was 52.0% at BCH +SM−D−, 97.5% at BCH+SM+D−, 80.0% at BCH−SM+D+, and 100.0% at BCH−SM−D−.
Even more clearly, the association between the type of bronchial lesions and the frequency of distant metastasis was demonstrated when evaluating the curves of 5-year MFS in the low-and high-risk groups ( Figure 5).
The survival rates of the high-risk and low-risk groups of patients who were either diagnosed with NSCLC or who were alive at 5 years were 52.5% and 98.2%, respectively.

DISCUSSION
The results of the study indicate that the state of the epithelium in the small bronchi distant from the tumor can be considered as a factor that can be used to divide patients with NSCLC into groups of low and high risk of distant metastasis. The absence of changes in the epithelium (BCH−SM−D−) or the combination of BCH with squamous metaplasia (BCH+SM+D−) is associated with a low frequency of metastasis (10% or 6.7%, respectively). A high risk of distant metastases is associated with the isolated BCH (BCH+SM−D−) and SM combined with D of the respiratory epithelium (BCH−SM+D+).
Metastases in these groups were found in 51.6% and 54.5% of cases. The significance of the type of morphological premalignant changes in the epithelium of small bronchi is a predictor of the incidence of distant metastases, and the time of their clinical manifestation is also confirmed by the results of the Cox regression analyses. In the low-risk group of distant metastases, 5-year MFS was higher. It is noteworthy that the study demonstrated the significance of generally recognized factors in predicting distant metastasis: tumor size, grade, and stage of the process. However, an important innovation lies in the fact that these factors have a significant prognostic ability precisely in the high-risk group, stratified by type of bronchial lesions. It is known that microenvironment determines the invasiveness and ability of cells to intravasate, which is the first step in the metastatic process. This view explains the probability of a complex chain of cause-and-effect relationships between parenchymal-stromal relationships in small bronchi located near the tumor and the risk of distant metastasis of NSCLC.
The results of the study suggest that the variant of a combination of different types of morphological and molecular changes in the bronchial epithelium under conditions of chronic inflammation in the bronchi (in chronic bronchitis or NSCLC) is a stable condition reflecting the constitutive features of stromalparenchymal relationships during inflammation and the divergent nature of the progression of precancerous changes in the bronchial epithelium.
Previously, we analyzed the expression profiles of the BCH, SM, and D genes in small bronchi near the primary tumor in NSCLC. It was found that isolated BCH in the high-risk group of distant metastasis differs from the BCH combined with SM in the     to the tumor, which may have a constitutive nature, may be associated with different variants of parenchymal-stromal relations in carcinomas, which substantially determine the risk of distant metastasis.
Unfortunately, prior to this study, there was no concept of morphological changes in the epithelium of small bronchi as a factor associated with distant metastasis, and their prognostic significance in patients with NSCLC. Numerous studies are aimed      immune-inflammatory reactions in the tumor microenvironment, including their ability to increase the invasive and intravasation potential and the ability to metastasize. 8) The morphological type of precancerous changes in the bronchial epithelium of the bronchi adjacent to the tumor is a prognostic sign of the risk of metastasis.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by local ethics committee of the Cancer Research Institute, Tomsk NRMC, on December 10, 2012 (the number of approval is 16). The patients/participants provided their written informed consent to participate in this study.