Introduction
Lung cancer is the leading cause of cancer-related deaths worldwide, with an estimated 2.1 million new cancer cases and 1.8 million cancer-related deaths [1]. Lung cancer is broadly categorized into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) [2, 3]. Small cell lung cancer is an aggressive neuroendocrine tumour with early dissemination and poor prognosis [4]. The treatment protocols for both limited-stage and extensive-stage SCLC are concurrent chemotherapy and radiation therapy with platinum- based agents [2, 5]. Due to the high growth fraction of the disease, early dissemination with widespread metastases and early development of drug resistance, the treatment of SCLC remains dismal [5].
Programmed cell death ligand 1 (PD-L1) is a predictive biomarker for immunotherapy in several solid tumours, but its role in treating SCLC is not well defined [6]. The PD-1/PD-L1 pathway controls the induction and maintenance of immune tolerance within the tumour microenvironment [7]. Immune checkpoint inhibition has shown promising advances in cancer immunotherapy. Unlike NSCLC, no driver genes are yet identified that can be used for targeted therapy in SCLC. Immunotherapeutic agents and monoclonal antibodies that target PD-1/PD-L1 are currently being evaluated for their potential use in SCLC [8]. As per the 2018 recommendations of the International Association for the Study of Lung Cancer and the US Food and Drug Association guidelines in 2019, anti-PD-L1 immunotherapy can now be combined with chemo-radiotherapy in cases of SCLC [6, 9]. The current study was undertaken with the following objectives: to assess the frequency of PD-L1 expression in SCLC and to correlate the expression of PD-L1 with clinico-pathological parameters. As personalized, targeted therapy in SCLC is still in the evaluation phase, the study of the immunotherapeutic target PD-L1 may help define a new treatment strategy for SCLC [9].
Material and methods
The current study was a retrospective and prospective case series that included 100 cases of SCLC. The study protocol was approved by the Institutional Ethics Committee. Adequate clinical details were documented including age, sex, presenting clinical features, site/side of biopsy, pertinent radiological findings and tumour stage. The survival data for all the cases were obtained during clinical follow-up visits or by telephone. Formalin-fixed paraffin- embedded (FFPE) tissue specimens were evaluated for morphology in haematoxylin and eosin staining. All the cases with the presence of adequate tumour were assessed. The sections were evaluated for the presence or absence of tumour necrosis. The presence of immune cells around the tumour was also assessed in all the cases. The cases were diagnosed as SCLC with the aid of immunohistochemistry (IHC). The immunohistochemical panel used included pan-cytokeratin and neuroendocrine markers, namely chromogranin, synaptophysin, and insulinoma-associated protein 1 (INSM-1), along with thyroid transcription factor-1 (TTF-1). The testing for PD-L1 was done on FFPE tissue blocks using the rabbit monoclonal anti-PD-L1 antibody, clone SP263 on the Ventana Benchmark XT automated staining system (USA). A positive control (human placenta tissue) and negative control (by omitting the primary antibody) were run with every batch. The cases was analysed for the presence of membranous staining for PD-L1 in both tumour cells and immune cells. The percentage and staining intensity were assessed. The staining intensity was categorized between 1+ to 3+, wherein 3+ indicated staining identified at 10 magnification and 1+ indicated staining identified at 40 magnification. Based on the recommendations of the International Association for Study of Lung Cancer (ISALC), cases with more than 1% staining in either tumour cells or immune cells were categorized as positive. PD-L1 was also assessed using the cut-offs of ≥ 1%, ≥ 10%, ≥ 25% and ≥ 50%. The tumour proportion score and the immune proportion score (IPS) were determined. The combined positive score (CPS) that combines the PD-L1 expressing tumour cells and the PD-L1 positive immune cells was also determined.
Statistical analysis
Statistical software (SPSS for Windows, version 16.0) was used for the analysis. Categorical data are reported as numbers and percentages and continuous data as the median and range. The frequency of PD-L1 expression was assessed in both the tumour cells and immune cells. The tumour proportion score, IPS and CPS were calculated. The correlation of PD-L1 expression with the clinico-pathological features was evaluated using tests of significance, namely the Fisher exact test or c2 test or the unpaired Student t-test, and a p-value of < 0.05 was considered as significant while a p-value of < 0.01 was considered as highly significant. The survival data were analysed using the Kaplan-Meier estimator.
Results
Clinico-pathological parameters
The current study included 100 cases of SCLC wherein the age range of the patients was 24– 82 years. In the current study, the M : F ratio was 6.1 : 1. The most common clinical feature was cough, which was documented in 88% (n = 88) of cases, followed by breathlessness, present in 84% (n = 84) of cases. Among the included patients, 86% (n = 86) were either current or ex-smokers while 14% (n = 14) were non-smokers. Expression of synaptophysin was present in 90% of cases while chromogranin was positive in 89% of cases. Immunostaining for INSM-1 was performed in 31 cases and 93.5% of cases (n = 29) had positive nuclear staining. Based on the histological analysis, the presence of necrosis was documented in 40% of cases (Fig. 1). Regarding disease stage, 42% (n = 42) of cases were of stage T3, while stage T4 was present in 11% (n = 11) of cases and 35% (n = 35) cases had the nodal status of N3. The evidence of distant metastasis (M1) was documented in 29% of cases (n = 29). The survival duration varied 0.5– 12 months and 4 patients were alive during survival documentation. In the majority of cases the survival duration was within 6–12 months (Fig. 2, Table I).
PD-L1 expression
The PD-L1 expression was identified in 14% of cases (n = 14) using the cut-off of ≥ 1%. The tumour proportion score was 10% using the cut-off of ≥ 1%. Using the cut-off of ≥ 10%, ≥ 25% and ≥ 50%, the PD-L1 expression in the tumour cells was 7%, 4% and 3%, respectively. The intensity of staining varied from 1+ to 3+. The intensity of staining was 1+ in 30% (n = 3/10) of cases, 2+ in 40% (n = 4/10) of cases and 3+ in 30% (n = 3/10) of cases.
The presence of immune cells could be assessed in 92 cases. The immune proportion score was 9.78% (n = 9/92) of cases using the cut-off of ≥ 1%. Using the cut-off of ≥ 10% and ≥ 25%, PD-L1 expression was identified in the immune cells in 6.52% (n = 6/92) and 1.09% (n = 1/92) of cases. However, using the cut-off of ≥ 50%, PD-L1 expression was not identified in any case in the immune cells. The intensity of staining for PD-L1 in the immune cells varied from 2+ to 3+. In 77.78% (n = 7/9) of cases the intensity was 2+ while in 22.22% (n = 2/9) cases. Among the 14 cases that harboured PD-L1 expression, in 5 cases (35.7%), both tumour and immune cells were positive. In 5 cases (35.7%), PD-L1 expression was identified only in tumour cells and in 4 (28.6%) cases, only the immune cells expressed PD-L1.
The combined proportion score at the cut-off of ≥ 1% was 10% (Fig. 3).
Characteristics of PD-L1 positive cases (n = 14)
In the current study, 85.7% of cases with PD-L1 positivity were > 40 years of age, with 78.6% of cases being male patients. In the cases that expressed PD-L1, 64.3% of biopsies were from the endobronchial region, while 35.7% were from lung/intrathoracic masses. The most common clinical feature identified in the PD-L1 positive cases was cough, documented in 92.9% of cases (n = 13), followed by breathlessness in 85.7% of cases. All the patients with positive expression of PD-L1 were smokers. In the cases with PD-L1 expression, 57.1% had tumour stage T2. The nodal metastasis stage was N1 in 35.7% of cases. Distant metastasis was identified in 21.4% of cases. In the PD-L1 positive group, chromogranin expression (92.9%) was higher than synaptophysin expression (78.6%). TTF-1 was positive in 63.6% of cases and INSM-1 was positive in 75% of cases. In the PD-L1 positive group, necrosis on histology was identified in 71.4% of cases.
PD-L1 positive vs. PD-L1 negative cases – correlation of clinico-pathological parameters
In the PD-L1 positive group, 85.7% of cases were more than 40 years of age (p = 0.197), and 78.6% were male (p = 0.388). In the cases that expressed PD-L1, the most common clinical feature was cough, documented in 92.9% of cases (n = 13), followed by breathlessness, present in 85.7% of cases. All the patients who were positive for PD-L1 expression were smokers (p = 0.104). In the PD-L1 positive group, chromogranin expression (92.9%) was higher than synaptophysin expression (78.6%). In the PD-L1 positive group, the presence of necrosis on histology was identified in 71.4% of cases. This finding was statistically significant when compared to the PD-L1 negative group, with a p-value of 0.010. In the cases with PD-L1 expression, 57.1% had tumour stage T2 (p = 0.575). The nodal metastasis stage was N1 in 35.7% of cases (p = 0657); distant metastasis was identified in 21.4% of cases (p = 0.501).
The comparison of the survival outcome between the PD-L1 positive and the PD-L1 negative cases was not statistically significant (p = 0.458), indicating that expression of PD-L1 may not have any effect on survival (Table II).
Discussion
In the current study, PD-L1 expression was identified in 14% of cases using the cut-off of ≥ 1%. The tumour proportion score was 10% and the IPS was 9.78% using the cut-off of ≥ 1%. The recommended cut-offs for PD-L1 detection using the SP263 clone as per the ISAAC recommendations is staining in at least 25% of tumour cells for durvalumab therapy. In cases where nivolumab therapy is administered, the staining is sub-grouped as < 1%, 1%, 1–5%, 5–10% and 10% or greater [10]. The only study published from the Indian sub-continent, by Guleria et al., reported PD-L1 expression in 8.4% of cases. In cases of SCLC, the authors reported the positivity of PD-L1 as 2.9% in the tumour cells and 23.5% in the immune cells [11]. PD-L1 expression in SCLC is variable and the frequencies varied from as low as 5.8% to as high as 71.6% [12, 13]. Among the 14% of cases harbouring PD-L1 expression, 35.7% of both the tumour and immune cells were positive. In 35.7%, PD-L1 expression was identified only in the tumour cells and in 28.6% of cases, only the immune cells expressed PD-L1. In the study conducted by Wang et al. PD-L1 expression was identified in 45.3% of cases, including 5.7% of cases with PD-L1 expression in tumour cells, 28.9% of cases with PD-L1 expression in immune cells, and 10.7% of cases with PD-L1 positivity in both tumour and immune cells [14] (Table III).
In the present study, the intensity of staining varied from 1+ to 3+ in the tumour cells. The intensity of staining was 1+ in 30% of cases, 2+ in 40% of cases and 3+ in 30% (n = 3/10) of cases. The intensity of staining for PD-L1 in the immune cells varied from 2+ to 3+. In 77.78% of cases, the intensity was 2+ while in 22.22% of cases it was 3+. Yu et al. stated in their study that the staining intensity for PD-L1 was variable and varied from weak to strong. The majority of cases displayed a moderate intensity of staining [15].
The various commercially available IHC clones for PD-L1 include 5H1, E1L3N, E1J2J, SP142, 28–8, 22C3 and SP263. The analysis of staining intensity and percentage of cell staining (both tumour and immune cells) are variable for the various antibody clones. Amongst the various clones of PD-L1, SP263, 22C3 and 28–8 are validated for counting in tumour cells while the SP142 clone has been standardized for counting the immune cells (ICs). The current guidelines for assessing PD-L1 in tumour cells/ICs or both have not been universally standardized [30]. In the present study, the SP263 clone of PD-L1 was used to detect PD-L1 expression in SCLC. The SP263 clone stains the tumour cells better than the other clones; however, its efficacy to stain the ICs has not been validated [31]. Amongst the various studies published in the literature wherein PD-L1 expression was assessed in SCLC using the SP263 clone, the frequency of PD-L1 varied from 2.9 to 5% in the tumour cells [15–17].
In the present study, a comparison of the characteristics of the PD-L1 positive vs. the PD-L1 negative cases was performed. In the current study, 85.7% of patients with PD-L1 positivity were > 40 years of age, with 78.6% of patients being male. These findings indicate that PD-L1 positivity is likely in elderly males and is concordant with the findings of Wang et al. and Inamura et al. [14, 23]. In the study conducted by Yu et al. and Fan et al., the authors documented a female predominance in the cases with PD-L1 positivity [15, 27]. A comparison between the PD-L1 positive vs. the negative sub-groups regarding age and gender was not statistically significant, with p-values of 0.197 and 0.388, respectively. In the current study, cases that expressed PD-L1 included 64.3% of biopsies from the endobronchial region, while 35.7% were from lung/intrathoracic masses. Fan et al. reported PD-L1 expression to be significantly present in the central type of tumours that involved the segmental or more proximal bronchi compared to the peripheral tumours that involved the sub-segmental or more distal bronchi [27]. This finding may imply that PD-L1 expression is more frequent in tumours that involve the endobronchial or central regions. The preferential expression of PD-L1 in tumours of these regions may additionally affect the therapeutic response [15]. The most common clinical feature identified in the PD-L1 positive cases in the present study was cough, documented in 92.9% of cases, followed by breathlessness in 85.7% of cases. All the patients with positive PD-L1 expression were smokers. Smoking is a significant carcinogen responsible for the development of lung malignancies, in particular SCLC. Smoking leads to the development of multiple mutations and field carcinogenic effects. Mutations in p53, retinoblastoma and the KRAS gene play an important role in carcinogenesis [32–34]. In addition, the carcinogens in smoke possibly lead to activation of the PD-1/PD-L1 pathway. Smoking leads to activation of the inflammatory cascade with the secretion of cytokines in addition to activation of tumorigenesis [33]. This likely explains the high frequency of PD-L1 expression in association with smoking. This finding is in concordance with the results reported by Tsuruoka et al. [12]. However, a comparison of the PD-L1 positive vs. the PD-L1 negative categories regarding smoking was not statistically significant, with a p-value of 0.104. In the current study in the PD-L1 positive group, chromogranin expression (92.9%) was higher than synaptophysin expression (78.6%) and INSM-1 was positive in 75% of cases. This finding contrasts with the entire study group in which the expression of synaptophysin was higher than that of chromogranin. Chromogranin A is very specific but has limited sensitivity in the diagnosis of SCLC [35, 36]. This finding is concordant with the results of the study conducted by Yu et al. wherein the authors identified higher expression of chromogranin in the PD-L1 positive category, whereas the overall study group had a higher expression of synaptophysin [15]. TTF-1 was positive in 63.6% of cases in the PD-L1 positive category. The high expression of PD-L1 in the present study was in concordance with the findings of Yu et al., where the authors reported higher PD-L1 positivity in SCLCs expressing TTF-1 [15]. TTF-1 is the chief regulator for lung structure development and plays a role in neuroendocrine differentiation and tumour aggressiveness, particularly in cases of SCLC [37]. The high expression of PD-L1 in cases of SCLC that are TTF-1 positive may be due to the activation of oncogenic pathways mediated via TTF-1. The comparison of the PD-L1 positive vs. the PD-L1 negative groups in terms of TTF-1 expression was not statistically significant (p = 0.413).
In the present study, among the cases with PD-L1 expression, 57.1% of cases had a tumour stage of T2 followed by T3, which was present in 28.6% of cases. The nodal metastasis stage was N1 in 35.7% of cases and distant metastasis was identified in 21.4%. Clinical stages II and III together constituted 71.4% of the cases with PD-L1 positivity. In the study conducted by Inamura et al., 46% of cases with PD-L1 positivity had stage I disease [23]. Fan et al. reported the stage of the disease to be I/II in 77.8% of cases with PD-L1 expression, while distant metastasis was identified in 54.5% of cases [27]. In the study conducted by Wang et al. 63.6% of cases with PD-L1 expression had the clinical stage of I [14]. Compared to studies published in the literature, the cases with PD-L1 expression had a higher disease stage in the present study. In the PD-L1 positive group, the presence of necrosis on histology was identified in 71.4% of cases. A comparison with the PD-L1 negative subgroup in terms of necrosis was statistically significant with a p-value of 0.010. This finding is concordant with the study conducted by Wang et al., wherein the authors identified necrosis in 61.5% of tumours with PD-L1 expression [14]. In small cell lung cancer the tumours have a high mutation burden with a propensity of distant metastasis and an aggressive disease course. The occurrence of necrosis in histology implies that the tumour has a higher grade and poor differentiation. The presence of necrosis in cases with PD-L1 expression may imply that positivity is related to tumours with advanced disease stages [38].
The utility of PD-L1 as a prognostic marker in neuroendocrine tumours and SCLC is still not very clearly understood. The true prognostic potential of this immunotherapeutic marker is controversial [38].
Among the various studies published in the literature wherein PD-L1 expression has been assessed in neuroendocrine tumours and SCLCs, most studies have reported a better prognosis in tumours with PD-L1 positivity [12, 13, 21, 23, 24, 26]. Kim et al., Eichhorn et al. and Kasajima et al. concluded from their studies that PD-L1 expression in tumour cells was associated with poor prognosis; however, PD-L1 positivity in the immune cells was associated with longer survival and better prognosis [18–20]. Yu et al. and Fan et al. stated that tumours with PD-L1 positivity are generally associated with poor prognosis [15, 27].
In the present study, the survival analysis of the PD-L1 positive vs. the PD-L1 negative cases revealed no significant difference (p = 0.458). This finding is concordant with the conclusions derived from the studies conducted by Yoshimura et al., Oizumi et al. and Wang et al., wherein the authors did not find any significant association between PD-L1 expression and survival [14, 16, 17].
In the studies where PD-L1 expression was explicitly assessed in SCLCs using the SP263 clone, no significant difference in survival was documented by Yoshimura et al. and Oizumi et al., which was concordant with the findings of the present study [16, 17]. However, Yu et al. reported that cases of SCLCs with PD-L1 expression had the worst prognosis [15].
The present study is a novel attempt to study the frequency of PD-L1 in SCLC; however, the study has certain limitations. Firstly, the study sample included only cases of SCLCs; therefore, the expression of PD-L1 across the spectrum of neuroendocrine tumours could not be assessed. Secondly, all samples included in the study were small biopsy specimens. PD-L1 expression is associated with tumour heterogeneity; therefore, including only small biopsies fails to address this aspect accurately. However, most patients presented with advanced disease or metastasis in the current study, which are contraindications for resections. Finally, clinical data in response to treatment involving standard chemotherapy/radiotherapy or anti-PD-L1 therapy are not available. However, treatment response was not included in the objectives of the study.
Conclusions
In cases of SCLC, due to the high mutation burden of the tumour and aggressive clinical course, the treatment regime includes chemotherapy and radiotherapy. Immunotherapeutic drugs constitute a novel potential treatment modality, and combination therapy may serve as a path-breaking phenomenon in SCLC management.
The authors declare no conflict of interest.
References
1. Regzedmaa O, Zhang H, Liu H, et al. Immune checkpoint inhibitors for small cell lung cancer: opportunities and challenges. Onco Targets Ther 2019; 12: 4605-4620.
2.
Latimer KM, Mott TF. Lung cancer: diagnosis, treatment principles, and screening. Am Fam Physician 2015; 91: 250-256.
3.
Inamura K. Lung cancer: understanding its molecular pathology and the 2015 WHO Classification. Front Oncol 2017; 7: 193.
4.
George J, Lim JS, Jang SJ, et al. Comprehensive genomic profiles of small cell lung cancer. Nature 2015; 524: 47-53.
5.
Koinis F, Kotsakis A, Georgoulias V. Small cell lung cancer (SCLC): no treatment advances in recent years. Transl Lung Cancer Res 2016; 5: 39-50.
6.
Li Q, Yuan D, Ma C, et al. A new hope: the immunotherapy in small cell lung cancer. Neoplasma 2016; 63: 342-350.
7.
Gkika E, Benndorf M, Oerther B, et al. Immunohistochemistry and radiomic features for survival prediction in small cell lung cancer. Front Oncol 2020; 10: 1161.
8.
Han Y, Liu D, Li L. PD-1/PD-L1 pathway: current researches in cancer. Am J Cancer Res 2020; 10: 727-742.
9.
Lantuejoul S, Sound-Tsao M, Cooper WA, et al. PD-L1 testing for lung cancer in 2019: perspective from the IASLC pathology committee. J Thorac Oncol 2020; 15: 499-519.
10.
Tsao MS, Kerr KM, Dacic S, Yatabe Y, Hirsch FR. IASLC atlas of PD-L1 immunohistochemistry testing in lung cancer. Editorial Rx Press, North Fort Myers 2017.
11.
Guleria P, Kumar S, Malik PS, et al. PD-L1 expression in small cell and large cell neuroendocrine carcinomas of lung: an immunohistochemical study with review of literature. Pathol Oncol Res 2020; 26: 2363-2370.
12.
Tsuruoka K, Horinouchi H, Goto Y, et al. PD-L1 expression in neuroendocrine tumors of the lung. Lung Cancer 2017; 108: 115-120.
13.
Ishii H, Azuma K, Kawahara A, et al. Significance of programmed cell death-ligand 1 expression and its association with survival in patients with small cell lung cancer. J Thorac Oncol 2015; 10: 426-430.
14.
Wang H, Li Z, Dong B, et al. Prognostic significance of PD-L1 expression and CD8+ T cell infiltration in pulmonary neuroendocrine tumors. Diagn Pathol 2018; 13: 30.
15.
Yu S, Jia M, Li Y, et al. Differential Expression of PD-L1 in central and peripheral and TTF1-positive and -negative small-cell lung cancer. Front Med (Lausanne) 2021; 7: 621838.
16.
Yoshimura A, Yamada T, Miyagawa-Hayashino A, et al. Comparing three different anti-PD-L1 antibodies for immunohistochemical evaluation of small cell lung cancer. Lung Cancer 2019; 137: 108-112.
17.
Oizumi S, Yoh K, Matsumoto S, et al .Assessment of PD-L1 expression and oncogenic gene status in patients with small-cell lung cancer: Immuno-oncology biomarker study in LC-SCRUM-Japan. J Clin Oncol 2019; 37: 8558.
18.
Kim HS, Lee JH, Nam SJ, et al. Association of PD-L1 expression with tumor-infiltrating immune cells and mutation burden in high-grade neuroendocrine carcinoma of the lung. J Thorac Oncol 2018; 13: 636-648.
19.
Eichhorn F, Harms A, Warth A, et al. PD-L1 expression in large cell neuroendocrine carcinoma of the lung. Lung Cancer 2018; 118: 76-82.
20.
Kasajima A, Ishikawa Y, Iwata A, et al. Inflammation and PD-L1 expression in pulmonary neuroendocrine tumors. Endocr Relat Cancer 2018; 25: 339-350.
21.
Ohtaki Y, Kaira K, Atsumi J, et al. Prognostic significance of PD-L1 expression and tumor infiltrating lymphocytes in large cell neuroendocrine carcinoma of lung. Am J Transl Res 2018; 10: 3243-3253.
22.
Yasuda Y, Ozasa H, Kim YH. PD-L1 Expression in small cell lung cancer. J Thorac Oncol 2018; 13: e40-e41.
23.
Inamura K, Yokouchi Y, Kobayashi M, et al. Relationship of tumor PD-L1 (CD274) expression with lower mortality in lung high-grade neuroendocrine tumor. Cancer Med 2017; 6: 2347-2356.
24.
Miao L, Lu Y, Xu Y, et al. PD-L1 and c-MET expression and survival in patients with small cell lung cancer. Oncotarget 2017; 8: 53978-53988.
25.
Takada K, Toyokawa G, Okamoto T, et al. An Immunohistochemical analysis of PD-L1 protein expression in surgically resected small cell lung cancer using different antibodies and criteria. Anticancer Res 2016; 36: 3409-3412.
26.
Yu H, Batenchuk C, Badzio A, et al. PD-L1 expression by two complementary diagnostic assays and mRNA in situ hybridization in small cell lung cancer. J Thorac Oncol 2017; 12: 110-120.
27.
Fan Y, Ma K, Wang C, et al. Prognostic value of PD-L1 and PD-1 expression in pulmonary neuroendocrine tumors. Onco Targets Ther 2016; 9: 6075-6082.
28.
George J, Fernandez-Cuesta L, Walter, V et al. Comparative analysis of small cell lung cancer and other pulmonary neuroendocrine tumors. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16 –20; New Orleans, LA. Philadelphia (PA): AACR. Cancer Res 2016; 76: 122.
29.
Schultheis AM, Scheel AH, Ozretiæ L, et al. PD-L1 expression in small cell neuroendocrine carcinomas. Eur J Cancer 2015; 51: 421-426.
30.
Pacheco J, Bunn PA. Advancements in small-cell lung cancer: the changing landscape following IMpower-133. Clinical Lung Cancer 2019; 20: 148-160.
31.
Kintsler S, Cassataro MA, Drosch M, et al. Expression of programmed death ligand (PD-L1) in different tumors. Comparison of several current available antibody clones and antibody profiling. Ann Diagn Pathol 2019; 41: 24-37.
32.
Liao Y, Yin G, Wang X, et al. Identification of candidate genes associated with the pathogenesis of small cell lung cancer via integrated bioinformatics analysis. Oncol Lett 2019; 18: 3723-3733.
33.
Kadara H, Scheet P, Wistuba II, et al. Early events in the molecular pathogenesis of lung cancer. Cancer Prev Res (Phila) 2016; 9: 518-527.
34.
Walser T, Cui X, Yanagawa J, et al. Smoking and lung cancer: the role of inflammation. Proc Am Thorac Soc 2008; 5: 811-815.
35.
Thunnissen E, Borczuk AC, Flieder DB, et al. The use of immunohistochemistry improves the diagnosis of small cell lung cancer and its differential diagnosis. An International Reproducibility Study in a demanding set of cases. J Thorac Oncol 2017; 12: 334-346.
36.
Kyriakopoulos G, Mavroeidi V, Chatzellis E, et al. Histopathological, immunohistochemical, genetic and molecular markers of neuroendocrine neoplasms. Ann Transl Med 2018; 6: 252.
37.
Iida Y, Masuda S, Nakanishi Y, et al. Clinicopathological characteristics of thyroid transcription factor 1-negative small cell lung cancers. Hum Pathol 2018; 79: 127-134.
38.
Lu D, Ni Z, Liu X, et al. Beyond T cells: understanding the role of PD-1/PD-L1 in tumor-associated macrophages. J Immunol Res 2019; 4: 1919082.
