Research On The Corelation Between TMC Syndromes And Pathological Features Of Central Type Airway Non-small Cell Lung Cancer

1 BACKGROUNDLung cancer serious harms to human life and health, and is currently one of the first cancer causes of death, and the incidence rate is still rising. Primary central airway non-small-cell lung cancer refers to the primary non-small cell malignant epithelial tumors in the trachea, main bronchi and right middle section of bronchus. These caners were not well studied in the histologic structure, clinical features and TCM syndrome type distribution, compared with that primary in other parts of the lung. The uncertainty of this disease made it difficult for the treatment, both by Chinese medicine and western medicine. The early preliminary experimental results showed that salivary type carcinoma accounts for a high proportion in the central airway non-small cell lung caners, this not only caused great difficulties to the pathological diagnosis, but also brought new challenge for TCM syndrome research of the salivary type carcinoma. Therefore, it is necessary to do a comprehensive study for this group of tumor in histological analysis, differential diagnosis, TCM syndrome type analysis and chemotherapy and prognosis index, etc., so that to fully understand the characteristics and corelations between western medicine classification and TCM syndrome type, hope that will pave the foundation and provide theoretical baisis for a more targeted Chinese and western medicine treatment. This study was supported by Beijing University of Chinese Medicine Project for young teachers.2 OBJECTIVESA cohort study of non-small cell lung cancer from central type airway was subjected to classic pathology, Traditional Chinese Medicine Syndrome typing, immunohistochemistry and molecular pathology, to achieve the following objectives: (1) To clarify the clinical and pathological features of central airway non-small cell lung cancers, so that to provide guidance for selection of Western medicine treatments; (2) To demonstrate the distribution outline of TMC syndrome types in central airways non-small cell lung cancer patients, and to establish the interconnection between the TMC syndrome type and the pathological pattern, clinical features, tumor TNM staging, with the expectation that the results will be help for achieving the optimal therapy for the central airways non-small cell lung cancers by the combination treatment of both TMC and Western medicine; (3) To find a simple, efficient and optimized antibody panel to identify the histological types of central airway non-small cell lung cancers, including squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, myoepithelial carcinoma and salivary adenocarcinoma (not otherwise specified) etc.(4)To detect the expression of ERCC1, Beta-tubulin, which are assumed to be indicators for chemotherapy sensitivity and prognosis in central airway non-small cell lung cancer. The relationship between the positive rate and different types of TCM syndromes was explored; (5) To examine the ALK gene rearrangement events in central airway non-small cell lung cancer for different histological types, and to arouse the possibility of treatment by crizotinib and other targeted drugs in this group of patients.3 SUBJECTSThe patients with primary central airway non-small cell lung cancer at the clinical stage Ⅲ and stage Ⅳ were confirmed by pathological diagnosis; All the patients were hospitalized from June 2013 to May 2014 in Beijing Meitan General Hospital, Lung Cancer Center Treated with Minimally Invasive Interventional Pulmonology, a cohort of 109 cases were collected with their resected tumor specimens and clinical data. Ninety-one patients were male and eighteen females, the youngest 29 years old and the oldest 86 years old, with a median age of 62 years.4 METHODS and Research Project4.1 Research Project(1) To elucidate the histopathological pattern and theclinical characteristics of central airway non-small cell lung cancers; (2) To enlighten the distribution of TCM syndrome type and the correlation with pathologic histology and TNM staging in central airway non-small cell lung cancer. (3) To optimize the immunohistochemical antibody panel for the differential diagnosis of different histological types of central type airway non-small cell lung cancer; (4) To study the expression of ERCC1, Beta-tubulin in different histology patterns and the correlation with TCM syndrome types; (5) To explore the ALK gene rearrangements and the possibility of targeted therapy for these patients.4.2 Research Methods4.2.1 Clinical data collection and TCM syndrome type determinationDetailed Case Report Form was worked out by extensive literature review, basic data was obtained from patient’s medical records, together with in-depth interviews and physical examinations to determine the patient’s TCM syndrome type; All patients enrolled were confirmed with histopathological diagnosis and those who do not meet the requirements were excluded in this study. Staging of non-small cell lung cancers was carried out according to the 2009 AJCC TNM staging program,7th edition. TCM syndrome types were determined according to the Standard of Chinese medicine clinical research guidelines," 2002 edition.4.2.2 Tissue processing and pathological diagnosisSpecimens were fixed in neutral buffered formalin for 8 to 72 hours, then subjected to automatic dehydration machine, paraffin-embedded; Serial sections at a thickness of 4 microns were cut for HE staining and immunohistochemical staining; An automated tissue microarray instrument was used to make the tissue chips for immunohistochemical staining. All sections were carefully studied by two senior pathologists independently for pathological diagnosis and immunohistochemical scoring. The cases with disagreement in diagnosis or immunohistochemical scores were discussed and determined by joint consultation at a multi-headed microscope. Histological diagnosis of lung cancer was made according to standard WHO lung cancer classification (2004 edition), and the new 2011 IASLC/ATS/ERS classification of lung adenocarcinoma.4.2.3 Immunohistochemical staining and score criteria4.2.3.1 ALK-D5F3ALK-D5F3 was stained with working solution, by using the Ventana automated immunohistochemical staining machine. Positive was defined as the presence of a strong granular cytoplasmic staining of tumor cells, exclude known factors, including the lighter stippling dyeing of alveolar macrophage cytoplasm, nerve fibers and ganglion cells, infiltrating lymphocytes, necrosis and mucus; ALK-D5F3 negative control:no strong granular cytoplasmic staining of the tumor cells.4.2.3.2 p63p63 was stained with 1:200 dilution of original solution, by using the Ventana automated immunohistochemical staining machine. P63 positive staining was defined as a strong nuclei brown coloring. p63 positive expression was defined for the number of positive cells in ≥10% of all tumor cells. The pattern of P63-positive cells was observed with distinctive characteristics, divided into two types:diffuse type and the peripheral portion of the cancer nest,"edged type".4.2.3.3 p40p40 was stained with mouse monoclonal antibody,1:200 dilutions, using the Ventana automated immunohistochemical staining machine. p40 staining pattern was the same as p63, which was defined as nuclear coloring. The criteria of p40 positive expression was that there were≥10% positive cells compared with all the cancer cells. Distribution pattern of the positive cells was divided into two types:diffuse type and "edged type" which was surrounding the cancer nests.4.2.3.4 TTF-1TTF-1 was determined by mouse monoclonal antibody liquid,1:200 dilutions, using the Ventana automated immunohistochemical-staining machine. TTF-1 positive is defined as strong brown staining for nuclear.≥10% positive cells in all tumor cells were judged as positive expression, the other was negative.4.2.3.5 Ki-67Ki-67 was examined by antibody clone MIB-1 stock solution,1:200 dilution, using Ventana automatic immunohistochemical stainingmachine. Ki-67 positive was defined as nuclear staining, and was calculated by Ki-67 index, which was the percentage of positive cells compared with all tumor cells.4.2.3.6 SMASMA was stained by monoclonal antibody stock solution in 1:100 dilution, using the aforementioned manual immunohistochemical staining process. SMA showed positive staining in the cytoplasm with strong brown coloration. The pattern of positive was defined by its distribution characteristics, including diffuse type and "edged type" which was surrounded tumor cell nests.4.2.3.7 ERCC1ERCC1 staining was carried out with stocking solution, dilution of 1:300, using the aforementioned manual immunohistochemical staining process. Positive staining of ERCC1 is defined for nuclear coloration. ERCC1 expression is scored into four levels:no positive cells or<10% positive, a score of 0;>10% nuclear faint/barely perceptible staining, a score of 1+;> 10% tumor nuclei were moderate staining, a score of 2+;> 10% tumor nuclei were stained strongly positive, a score 3+. Two-tired criteria was used in this study:2+or 3+ fall into the category of positive,0 or 1+ the negative.4.2.3.8 Beta-tubulinBeta-tubulin was examined with mouse monoclonal antibody stock solution, dilution of 1:100, using the aforementioned manual immunohistochemical staining process. Positive staining was defined as cytoplasmic brown coloring. Beta-tubulin expression was scored in four levels, no cytoplasm coloring, a score of 0; weak/ barely perceptible stainingin<10% of tumor cells, a score of 1+;> 10% of tumor cells were weak to moderate coloring, a score of 2+;> 10% of tumor cells cytoplasm strong staining, a score of 3+; In this study, two classification criteria was adopted,0 or 1+negative,2+ or 3+ positive.4.2.4 ALK-FISHCommercially available ALK gene kit of color separation probe was employed for the detection according to the manufacturer’s instructions for staining. The 5’ALK probe labeling SpectrumGreen,3’ALK probe labeling SpectrumOrange. Paraffin sections of tissue microarray were subjected to the staining process. Assessments were made by pathologists under fluorescence microscope at 100X oil immersion objective lens. For each case at least 100 non-overlapping cells were assessed for hybridization signals. ALK-FISH positive defined as there were equal to or more than 15 cells showed red and green signal separation of fluorescent probes, and the red and green signal separation distance exceeds the diameter of the two signal size. If the number of positive cells was in between 5 to 15, another assessment of 100 cells should be counted.4.2.5 Statistical AnalysisSPSS 19.0 software was used for statistical analysis, using non-parametric chi-square test for count data and the t-test for measurement data. Rank sum test was used when there was heterogeneity in the square deviation.5 Results5.1 pathological and clinical characteristics of central airway non-small cell lung cancer5.1.1 The histological types of central airway non-small cell lung cancer comprised squamous cell carcinoma (69 cases, accounting for 63.3%), salivary type carcinoma (27 cases, accounting for 24.8%), adenocarcinoma (10 cases, accounting for 9.2%) and other 2 cases of adenosquamous carcinoma, sarcomatoid cancer cases. In the salivary type carcinoma, the most common type was adenoid cystic carcinoma, accounting for 55.6%, the second was myoepithelial carcinoma, accounting for 22.2%, salivary adenocarcinoma (not otherwise specified), accounted for 14.8%, in which myoepithelial carcinoma and salivary adenocarcinoma NOS is very rare primary malignancy in the lung.5.1.2 The clinical and pathological features showed that the elderly, male, smoking patients most commonly suffered from squamous cell carcinoma; while the young, female, non-smoking patients suffered from salivary type carcinoma most frequently.5.2 Distribution of TCM syndrome types in the central airway non-small cell lungcancer5.2.1 In the group of 109 cases of lung cancer, Qi Deficiency and Phlegm Dampness syndrome consisted 36 cases, Yin Deficiency and Heat Toxin syndrome consisted 12 cases, Deficiency of both Vital Energy and Yin consisted 40 cases and Qi and Blood Stasis syndrome consisted 21 cases; The distribution characteristics showed that the Deficiency of both Vital Energy and Yin syndrome was most common in squamous cell carcinoma and salivary type carcinoma, accounting for 41.4% and 40.7% respectively; Qi Deficiency and Phlegm Dampness syndrome was most common in adenocarcinoma, accounting for 50%.5.2.2 TCM syndrome types were closely related to age, to sex, to smoking history, and to Karnofsky score respectively. The elderly male patients with smoking history and Karnofsky score higher than 60 were mainly comprised Deficiency of both Vital Energy and Yin syndrome; whilst the younger female patients with non-smoking history and Karnofsky score less than 60 points were commonly consisted of Qi Deficiency and Phlegm Dampness syndrome.5.3 Differential diagnosis of immunohistochemistry and relations to TCM syndrome types5.3.1 p63There was significant difference (P<0.01) of p63 expression among different histological types. The positive expression rate was 100% in squamous cell carcinoma, 0% in adenocarcinoma, and 81.5% in salivary type carcinoma. Two patterns of p63 positive distribution were noted, diffuse/sparse type expression in squamous cell carcinoma, myoepithelial carcinoma and "edged type" in adenoid cystic carcinoma/mucoepidermoid carcinoma which surrounded the cancer nests. Diffuse type p63-positivity achieved 100% sensitivity and 82.5% specificity in the differential diagnosis of squamous cell carcinoma, while the "edge type" p63-positivity achieved 100% specificity and 100% sensitivity in the diagnosis of adenoid cystic carcinoma/mucoepidermoid carcinoma. There were markedly significant differences (P<0.01) between p63 expression rates and TCM syndrome types.5.3.2 p40There was markedly significant difference (P<0.01) between p40 expression and different histological types. The p40-positive expression rate was 100% in squamous cell carcinoma,0% in adenocarcinoma, and 77.8% in salivary type carcinoma. Two patterns of p40-positive distribution were noted, diffuse/sparse type in squamous cell carcinoma, myoepithelial carcinoma, and "edge type" in adenoid cystic carcinoma/mucoepidermoid carcinoma. Diffuse type p40-positive achieved 100% sensitivity and 90.0% specificity in the diagnosis of squamous cell carcinoma, while "edge type" p40-positive obtained 100% specificity and sensitivity in the diagnosis of adenoid cystic carcinoma and mucoepidermoid carcinoma. There were markedly significant differences (P<0.01) between p40 expression rates and TCM syndrome types.5.3.3 TTF-1TTF-1 expression rate is 0% in squamous cell carcinoma and 50% in adenocarcinoma. Two cases of salivary type carcinoma showed<10% scattered cells TTF-1 positivity, but did not meet the positive criteria. TTF-1 achieved 50% sensitivity and 100% specificity in the diagnosis of adenocarcinoma.5.3.4 SMANone of the squamous cell carcinoma and adenocarcinoma showed SMA expression. Myoepithelial carcinoma showed 100% diffuse expression, while the adenoid cystic carcinoma showed "edge type" positive rate of 93.3%. "edge type" SMA positive achieved 94.1% sensitivity and 100% specificity in diagnosis of adenoid cystic carcinoma/mucoepidermoid cancer.5.3.5 Ki-67There was markedly significant difference of Ki-67 index between the salivary type carcinoma and non-salivary carcinoma. Ki-67 index average of salivary type carcinoma was lower than those of non-salivary carcinoma.5.3.6 ERCC1 and Beta-tubulinThere was markedly significant difference (P<0.01) between ERCC1-positive rate and TCM syndrome type. The ERCC1 positive rate is higher in Qi Deficiency and Qi and Blood Stasis syndrome type compared with that in Yin Deficiency and Heat Toxin syndrome.5.3.7 Beta-tubulinThere was markedly significant difference (P<0.01) between Beta-tubulin-positive rate and histological type. The Beta-tubulin positive rate is higher in adenocarcinoma compared with that in salivary type carcinoma.5.4 ALK immunohistochemistry and ALK-FISH ALK was detected by comparison of both immunohistochemistry and FISH methods, and three cases of ALK-positive lung cancer were defined with 2 cases of adenocarcinoma (accounting for 20%) and one case of squamous cell carcinoma (accounting for 1.45%).6 Conclusions6.1 This study illustrates the unique clinical and pathological characteristics of the central airway non-small cell lung cancer, includes:(1) Patients are mainly composed of squamous cell carcinoma, salivary type carcinoma and adenocarcinoma histologically; (2) Salivary type carcinoma accounts for very high proportion of the malignancies; (3) Myoepithelial carcinoma and salivary adenocarcinoma NOS are rare types of lung primary; (4) The elderly, male, smoking patients are most likely suffering from squamous carcinoma, while the young, female, never smoking patients suffer from salivary type carcinoma more frequently.6.2 The correlation between TMC syndrome type distribution and clinical features has been clarified. Male, smoking, squamous cancer tends to be the Deficiency of both Vital Energy and Yin syndrome type, while female, never smoke, salivary type carcinoma/adenocarcinoma mainly consists the Qi Deficiency and Phlegm Dampness or Yin Deficiency and Heat Toxin syndrome.6.3 The study provides p40/TTF-1/SMA as an optimal combination of antibodies that can identify the histological types of central airway non-small cell lung cancer:(1) squamous cell carcinoma, p40 diffuse positive, TTF-1 and SMA negative; (2) adenocarcinoma:p40 negative, TTF-1 positive in 50%, SMA negative; (3) adenoid cystic carcinoma/mucoepidermoid carcinoma:p40 and SMA "edge type" positive, (-), TTF-1 negative, or individual scattered cells weak positive; (4) myoepithelial carcinoma:SMA positive, p40 partiallysparse positive, TTF-1 negative; (5) salivary type adenocarcinoma, NOS:p40, SMA, TTF-1 triple negative. The specificity of p40 for squamous carcinoma diagnosis is higher than that of p63; Ki-67 index of salivary type carcinoma of is lower than that of non-salivary types (squamous carcinoma and adenocarcinoma).6.4 ERCC1 expression rate is higher in Qi Deficiency and Phlegm Dampness/Qi and Blood Stasis syndrome than that in the Yin Deficiency and Heat Toxin syndrome; Beta-tubulin expression rate is the highest in adenocarcinoma, followed by squamous cell carcinomas, and the lowest in the salivary type carcinoma. The expression of ERCC1 and Beta-tubulin is help for choosing chemotherapy drugs and predicting patient prognosis.6.5 Some of the adenocarcinoma and squamous cell carcinoma from central airway primary are ALK gene rearrangement; these patients might be suitable to choose molecular targeted therapy. No ALK gene rearrangement is found in salivary type carcinoma in this group.7 INNOVATIONS7.1 It was the first time to conduct an in-depth study to the central airway non-small cell lung cancer in its histological type and the TCM distribution characteristics, so as to provide a theoretical basis for more targeted western and TCM treatment;7.2 The study found an optimal combination of antibodies for the differential diagnosis of central airway non-small cell lung cancer of various histologic type, and studied ERCC1, Beta-tubulin, ALK expression characteristics, these immune-markers and gene alterations might serve as a therapeutic targets and the optimal chemotherapeutic drug selections.