| BackgroundLung cancer is a worldwide malignant tumor with higher morbidity and mortality.Known as bronchial cancer due to its origin from bronchial epithelium,the cancer is the most common malignant tumor in the lower respiratory tract.Lung cancer is roughly divided into two major types in clinical pratcice recently:non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).Clinicians have developed different therapeutic strategies directed at different types of lung cancers.Among them,NSCLC is mainly composed of two histological types:adenocarcinoma and squamous cell carcinoma,accounting for about 80%of all primary lung cancers.Pulmonary adenocarcinoma,the most common histological type of lung cancer,accounts for almost 50%of NSCLC.According to a new classification system of lung cancers,which was proposed by the International Association for the Study of Lung Cancer,American Thoracic Society,and European Respiratory Society(IASLC/ATS/ERS)in 2011,pulmonary adenocarcinoma is classified into lepidic,acinar,papillary,micropapillary and solid patterns.In addition to the high morphological heterogeneity of pulmonary adenocarcinoma,the variants also include mucinous,colloid,fetal and intestinal adenocarcinoma.In recent years,with the development of medical molecular biology technology and precision medicine,we have made a great progress in treatment of lung cancer,especially the treatment and testing of targeting on pulmonary adenocarcinoma,which prolongs the survival of the patients.Meanwhile,researchers have been keeping their eyes on targeted treatment of various histological types.To investigate characteristics of molecular expression of various lung cancers and to explore the association with clinicopathologic characteristics and prognosis would be effective on precision treatment.Studies have shown that the histological subtypes of pulmonary adenocarcinoma are related to the overall survival rate of patients.Patients with micropapillary and solid type have a low survival and poor prognosis,but five-year free survival of pulmonary adenocarcinoma in situ with lepidic type is up to 100%.Moreover,mutation rates of epidermal growth factor receptor(EGFR)gene vary in different histological types,ranging 62%(insitu type),60%(microinvasive type),77%(lepidic type),49%(acinar type),50%(papillary type),43%(mircropapillary type ty),28%(solid type)and 0%(mucinous type).Besides,EGFR gene status is related to prognosis of the patients.Cases of EGFR gene mutation have a higher overall survival rate and their prognosis is satisfactory.Comparatively,prognosis of patients with wild-type EGFR gene is not so good.Thus,histological types of pulmonary adenocarcinoma have close relationship with EGFR gene status and patient's prognosis.However,morphology and immunohistochemical staining of pulmonary adenocarcinoma are similar to that of colorectal adenocarcinoma.Under microscope,its morphology showed adenoid,papillary or cribriform structures,but within glandular cavity,occasionally solid structure,high columnar cell,pseudostratified nucleus.necrosis and nuclear debris.At the same time,immunohistochemical staining should include an intestinal marker at least and/or pulmonary adenocarcinoma markers simultaneously.In view of the above similarities,pulmonary adenocarcinoma with intestinal differentiation is often mistaken in clinical practice.Considering the similarity,original gastrointestinal adenocarcinoma should be excluded at the beginning.Currently,due to tumors' heterogeneity,clinicians may realize this variant in some cases in clinical practice,but study on the molecular expression,clinicopathologic characteristics and the association with patient's prognosis is rare.We investigated the expression of CEA,Villin,CK20 and CDX2 in usual pulmonary adenocarcinoma and pulmonary adenocarcinoma with intestinal differentiation and tested EGFR gene status for the purpose of exploring the difference of intestinal marker expression between pulmonary adenocarcinoma with intestinal differentiation and usual pulmonary adenocarcinoma,association of intestinal marker expression with clinicopathologic characteristics and EGFR gene status in pulmonary adenocarcinoma with intestinal differentiation,association of intestinal marker expression,EGFR gene status and clinicopatho logic characteristics with prognosis in pulmonary adenocarcinoma with intestinal differentiation,difference of clinicopatho logic characteristics,EGFR gene status and prognosis between pulmonary adenocarcinoma with intestinal differentiation and usual pulmonary adenocarcinoma.For this reason,our research could serve as a guide to an extent for diagnosis and predicting prognosis of pulmonary adenocarcinoma,and as a theoretical basis for targeted gene theray and individualized treatment.Part I Expression of CEA,Villin,CK20 and CDX2 of intestinal molecules in pulmonary adenocarcinomaObjectiveTo observe the morphological features of usual pulmonary adenocarcinoma and pulmonary adenocarcinoma with intestinal differentiation under a light microscope respectively;to observe the distribution of CEA,Villin,CK20 and CDX2 expression in the types ofpulmonary adenocarcinoma;to analyze the difference.MethodsSixty surgical operation samples of patients with pulmonary adenocarcinoma were collected in the Department of Pathology,the second hospital of Shandong university during January,2009 to March,2015.All filed blocks were paraffin-embedded.Pulmonary adenocarcinoma was diagnosed by the pathological results,with lung-metastasis colorectal adenocarcinoma excluded by staining intestinal marker and doing relevant clinical examination.Each sample was put in 10%neutral formalin liquid and paraffin-embedded.Then,the blocks were cut into slices of 4?m and stained with hematoxylin-eosin(HE)before observed under a light microscope.Histological evaluation of each sample was performed by two experienced pathologists,according to a new classification proposed by IASLC/ATS/ERS in 2011.All cases were divided into two groups(each group,n=30),usual pulmonary adenocarcinoma(U group)and pulmonary adenocarcinoma with intestinal differentiation(I group).CEA,Villin,CK20 and CDX2 expression was examined by immunohistochemical staining PV-9000 two-step method,the positive cell number and staining intensity were observed under a microscope.The data were analyzed with SPSS18.0 software to work out the differential expression of CEA,Villin,CK20 and CDX2 in U group and I group respectively.Results1.Morphological structure of usual pulmonary adenocarcinoma included lepidic,acinar,papillary,micropapillary and solid patterns.Lepidic subtype showed monolayer tumor cells were arranged in wall attachment pattern;acinar subtype was composed of gland-like structure and showed infiltrating growth pattern;papillary subtype showed branched papillary structure with fibrous vascular axis,which was similar to thyroid papillary cancer;micropap illary subtype showed flower-like papillary structure with without fibrous vascular axis.The solid subtype indicated the cell characteristics of low differentiation tumor with or without necrosis.The cell properties and tumor structure of intestinal type differentiation,containing tall columnar epithelium with pseudostratified cell nuclear,and,sometimes showing a cribriform structure with nuclear debris necrosis,were similar to that of colorectal carcinoma.2.Positivity of Villin,CK20 and CDX2 expression in I group was higher than that in U group,there was statistical difference(P<0.0.5);but the positivity of CEA expression in U group was the same as in I group,there was no statistical difference(P>0.05).Conclusions1.Histology can be a basis for distinguishing pulmonary adenocarcinoma with intestinal differentiation from usual pulmonary adenocarcinoma,but immunohistochemical staining of relevant markers and clinical examination must be involved in distinguishing pulmonary adenocarcinoma with intestinal differentiation from metastatic colorectal adenocarcinoma.2.Pulmonary adenocarcinoma with intestinal differentiation has a characteristic molecular profile;CEA could not be a specific marker for diagnosing pulmonary adenocarcinoma with intestinal differentiation.Part ? Association of the expression of CEA,Villin,CK20 and CDX2 with clinicopathologic characteristics and EGFR gene status in pulmonary adenocarcinoma with intestinal differentiationObjectiveTo observe the expression of CEA,Villin,CK20 and CDX2 and test EGFR gene status in pulmonary adenocarcinoma with intestinal differentiation respectively;to investigate association of their expression with clinicopathologic characteristics and EGFR gene status.MethodsOn basis of the experimental results of Part I,thirty filed,paraffin-embedded samples of diagnosed pulmonary adenocarcinoma with intestinal differentiation were collected from Part I experiment.Expression of CEA,Villin,CK20 and CDX2 was examined by immunohistochemical staining PV-9000 two-step method,EGFR gene status was tested by fluorescence quantitative PCR(Polymerase Chain Reaction,PCR),the clinicopatho logic data of patients were collected from the Department of Pathology,the Second Hospital of Shandong University.Immunohistochemical staining results were evaluated by counting positive cell and observing staining intensity under a microscope.The data were analyzed with SPSS18.0 software,inclusive of(1)the association of CEA,Villin,CK20 and CDX2 expression with clinicopatho logic characteristics respectively;(2)the association of CEA,Villin,CK20 and CDX2 expression with EGFR gene status.Results1.The positivity of CEA expression varied from 85.7%to 100%in each group of clinicopathologic characteristics,suggesting no statistical difference(P>0.05);the positivity of Villin expression varied from 66.7%to 100%,indicating no statistical difference(P>0.05);the positivity of CK20 and CDX2 expression from 0%to 50%,indicating no statistical difference(P>0.05).Expression of CEA,Villin,CK20 and CDX2 were not associated with gender,age,smoking history,tumor size,lymph node metastasis and clinical stage.2.In EGFR gene mutation group,the positivity of CEA expression was 100%,in wild group,88.2%,suggesting no statistical difference(P>0.05);the positivity of Villin expression was 84.6%in EGFR gene mutation group,88.2%in wild group,suggesting no statistical difference(P>0.05);the positivity of CK20 expression was 46.2%in EGFR gene mutation group,17.6%in wild group,suggesting no statistical difference(P>0.05);the positivity of CDX2 expression was 8.0%in EGFR gene mutation group,29.4%in wild group,suggesting no statistical difference(P>0.05).Expression of CEA,Villin,CK20 and CDX2 were not associated with EGFR gene status.Conclusions1.Expression of intestinal marker possibly is not associated with clinicopathologic characteristics of pulmonary adenocarcinoma with intestinal differentiation.2.Expression of intestinal marker possibly is not associated with EGFR gene status in pulmonary adenocarcinoma with intestinal differentiationPart? Association of the expression of CEA,Villin,CK20 and CDX2,clinicopathologic characteristics and EGFR gene status with prognosis in pulmonary adenocarcinoma with intestinal differentiationObjectiveTo observe the expression of CEA,Villin,CK20 and CDX2 and test EGFR gene status in pulmonary adenocarcinoma with intestinal differentiation;to explore the association of their expression,clinicopathologic characteristics and EGFR gene status with patient's prognosis.MethodsBased on the experimental results of Part ?,thirty filed,paraffin-embedded samples of diagnosed pulmonary adenocarcinoma with intestinal differentiation were collected from the Part I experiment.Expression of CEA,Villin,CK20 and CDX2 was examined with immunohistochemical staining PV-9000 two-step method,EGFR gene status was tested with fluorescence quantitative PCR,the clinicopathologic material and follow-up data of patients were collected from the Department of Pathology and the Department of Medical Records,the Second Hospital of Shandong University.A 3-to-36-month follow-up started from the day of discharge.Immunohistochemical staining results were evaluated by counting positive cell and observing staining intensity under a microscope.The data were analyzed with SPSS18.0 software,inclusive of(1)the association of CEA,Villin,CK20 and CDX2 expression in pulmonary adenocarcinoma with intestinal differentiation with patient's prognosis;(2)the association of clinicopatholbgic characteristics in pulmonary adenocarcinoma with intestinal differentiation with patient's prognosis;(3)the association of EGFR gene status in pulmonary adenocarcinoma with intestinal differentiation with patient's prognosis.Results1.The gjroup of CK20 positive expression had a longer median survival than the group of CK20 negative expression,suggesting statistical difference(P<0.05);the median survival time of the group of CDX2 positive expression was shorter than that of the group of CDX2 negative expression,suggesting statistical difference(P<0.05);but the median survival time of group of CEA positive expression was 18 months,that of the group of CEA negative expression was 36 months,suggesting no statistical difference(P>0.05);the median survival time of the group of Villin positive expression was 24 months,that of the group of Villin negative expression was 10.5 months,suggesting no statistical difference(P>0.05).2.1n each group of clinicopathologic characteristics,the median survival time of patients with tumor size ?5cm was longer than that of the patients with tumor size>5cm,suggesting a significant difference(P<0.01),but no statistical difference was found in matching median survival time of patients with different gender,age,smoking history,lymph node metastasis and clinical stage(P>0.05).3.The median survival time of patients with EGFR gene mutation group was 20 months,but that of patients with wild-type EGFR gene group was 18 months,suggesting no statistical difference(P>0.05).Conclusions1.It could be concluded there was a relationship between the expression of CK20 and CDX2 and the prognosis of patients with pulmonary adenocarcinoma with intestinal differentiation.In contrast to a good prognosis of patients with CK20 positive expression,patients with CDX2 positive expression had a poor prognosis,but suggesting no relation between the expression of CEA and Villin and the prognosis of the patients.2.it could be concluded that tumor size might have relationshp with prognosis of the patients with pulmonary adenocarcinoma with intestinal differentiation,patients with tumor size<5cm have a good prognosis,but patients with tumor size>5cm have a poor prognosis.However,gender,age,smoking history,lymph node metastasis and clinical stage might have no relationship with prognosis of the patients with pulmonary adenocarcinoma with intestinal differentiation.3.It indicated there was no relation between EGFR gene status and prognosis of the patients with pulmonary adenocarcinoma with intestinal differentiation.Part IV Difference of clinicopathologic characteristics,EGFR gene status and prognosis between pulmonary adenocarcinoma with intestinal differentiation and usual pulmonary adenocarcinomaObjectiveTo observe the difference of clinicopathologic characteristics,EGFR gene status and prognosis between usual pulmonary adenocarcinoma and pulmonary adenocarcinoma with intestinal differentiation;to investigate whether cases of pulmonary adenocarcinoma with intestinal differentiation has a given clinicopatho logic characteristics,different EGFR gene status and prognosis or not.MethodsReferring to the experimental results of Part ? and those of morphologic and immunohistochemistry,pulmonary adenocarcinoma with intestinal differentiation(I group)and usual pulmonary adenocarcinoma(U group),sixty filed,paraffin-embedded samples used in Part ? experiment were divided into two groups.EGFR gene status was tested by fluorescence quantitative PCR,the clinicopatho logic materials and follow-up data of the patients were collected from the Department of Pathology and the Department of Medical Records,the Second Hospital of Shandong University.A 3-to-36-month follow-up started from the day of discharge.The data were analyzed with SPSS18.0 software,including(1)to compare the difference of clinicopatho logic characteristics between both groups;(2)to compare the difference of EGFR gene status between both groups:(3)to compare the difference of patient's prognosis between both groups.Results1.In each group of clinicopatho logic characteristics,female patients in I group accounted for 70%,male patients in U group 60%,suggesting statistical difference(P<0.05);the percentage of lymph node metastasis in I group was 93.3%,that in U group 73.3%,suggesting statistical difference(P<0.05);patients of clinical stage??? in I group accounted for 90%,patients of clinical stage ??? in U group 66.7%,suggesting statistical difference(P<0.05);the comparison of age,smoking history and tumor size between both groups indicated no difference(P>0.05).2.The percentage of EGFR gene mutation in I group was 43.3%,that in U group was 33.3%,there was no statistical difference(P>0.05).3.The median survival time of patients in I group was 21 months,that ofpatients in U group was 24 months,there was no statistical difference(P>0.05).Conclusions1.It indicates patients of pulmonary adenocarcinoma with intestinal differentiation are common among women,but patients of usual pulmonary adenocarcinoma are common among men;it could be speculated that pulmonary adenocarcinoma with intestinal differentiation was prone to lymph node metastasis;It could be concluded that patients of pulmonary adenocarcinoma with intestinal differentiation are at a higher clinical stage.But the agesmoking history and tumor size ofpatients of the two tpyes of tumors have no difference.2.It could be concluded that EGFR gene status of patients in usual pulmonary adenocarcinoma and pulmonary adenocarcinoma with intestinal differentiation might be same.3.it could be speculated patients with usual pulmonary adenocarcinoma and pulmonary adenocarcinoma with intestinal differentiation might have the same prognosis. |
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