Morphological Characterization And Molecular Profiling Of Malignant Pericardial Effusion In Patients With Pulmonary Adenocarcinoma

Chapter 1 Value of cell block combined with immunohistochemistry in the diagnosis of malignant pericardial effusion with pulmonary adenocarcinomaObjective: To explore the role of cell blocks combined with immunohistoche-mical examination in the diagnosis of malignant pericardial effusion,and provide accurate diagnosis for patients with pulmonary adenocarcinoma with pericardial metastasis.Methods: 41 cases of malignant pericardial effusion based cytology,cell blocks of HE staining and immunohistochemical staining by En Vision two step were retrospectively analysed,The different characteristics of cytology and histology were observed under microscope.while immunohistochemical method was used to detect the expression of an appropriate set of antibodies(TTF-1,Napsin A,CK7,p40,CK5/6,GCDFP-15,ER,Cg A,Villin,CK20,CDX-2,Calretinin,MC,CD20 and CD3),in the tumor cells of unknown origin.and the diagnositic value of TTF-1 and napsin A in pulmonary adenocarcinoma with pericardial metastasis was evaluated.Results: TCT showed heterogenous tumor cell population that were singlescatt-ered or displaying threedimensional clustering structure,and the cells showed pleomorphism,high nucleocytoplasmic ratio,nuclear membrane irregularities;Tumor cells in the cell block were concentrically and maintained their original cytological structure.Among 41 cases of malignant pericardial effusion,there were 29 cases caused by lung adenocarcinoma,2 cases of lung small cell carcinoma,1 cases of lung squamous cell carcinoma,4 cases of breast carcinoma,2 cases of lymphoma,2 case of gastrointestinal origin and 1 cases of mesothelioma.Pulmonary adenocarcinoma with pericardial metastasis showed positive expressions of TTF-1 and Napsin A was 89.7%and 82.8%.The combination detection of TTF-1 and Napsin A with a diagnostic sensitivity of 94.1% and a specificity of 100% for pulmonary adenocarcinoma.Conclusion: Liquid-based Cytology can clearly observe the structure and arrangement of tumour cells.The pericardial effusion cell blocks combined withimmunohistochemistry are useful to improve the accuracy of diagnosis,and helpful for the determination of classification and the primary site of tumor.Lung cancer is the most common metastatic tumor in malignant pericardial effusion,in which adenocarcinoma is the most common type.The combination detection of TTF-1 and Napsin A is helpful for the diagnosis of pericardial metastasis of pulmonary adenocarcinoma.Chapter 2 Morphological characterization and molecular profiling of malignant pericardial effusion in patients with pulmonary adenocarcinomaObjective: Malignant pericardial effusions(MPCEs)is a common complication observed in advanced pulmonary adenocarcinoma.In such cases,investigating molecular alterations can have significant therapeutic implication in determining anticancer drugs.To investigate the morphological and molecular profiles of MPCE in patients with pulmonary adenocarcinoma.Methods: Cytopathological and molecular profiles of 29 MPCE cases in patients with pulmonary adenocarcinoma were retrospectively analyzed.The control group consisted of 22 malignant pleural effusion(MPE)cases in patients with pulmonary adenocarcinoma.Clinicopathological features according to International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society(IASLC/ATS/ERS)international multidisciplinary classification of lung adenocarcinoma.ALK and c?MET expression was evaluated by fluorescence in situ hybridization(FISH).EGFR and KRAS mutations were detected by ARMS real?time polymerase chain reaction(RT?PCR).Results: MPCE was found to have micropapillary and solid pattern predominant with mucin secretion compared to acinar patterns,as seen in MPE(P=0.002).27 cases of MPCE(86.2%)and 21 cases of MPE(95.5%)underwent molecular analysis.Mutations in EGFR and KRAS,ALK rearrangement,and c?MET amplification were observed in MPCE and MPE with statistical differences(EGFR,55.6% vs 42.9%,P=0.383),(KRAS,7.4% vs 9.5%,P=0.792),(ALK,7.4% vs9.5%,P=0.792)and(c-MET,7.4% vs 0%,P=0.311).Additionally,two MPCE cases demonstrated EGFR-T790 M mutation and multiple insertions at L858.In all MPCE and MPE groups,the mutation rate of EGFR in Lepidic and micropapillary predominant was significantly higher than other subtypes(70.6% vs 38.7%,P =0.035).ALK was predominant in solid pattern with mucin secretion.There was a statistically significant difference compared with other subtypes(27.2% vs 2.7%,P =0.033).At the same time,no correlation was found between KRAS mutation and cMET amplification and histological subtypes.Conclusions: MPCE shows micropapillary and solid cytological patterns predominant with mucin secretion.MPCE are suitable to analyze oncogenic mutations and to develop targeted therapy for patients with pulmonary adenocarcinoma.Further molecular investigations may reveal novel molecular alterations.Pathologically characterized with lepidic and micropapillary subtypes had a high mutation rate of EGFR,and ALK rearrangement is most common in solid pattern predominant with mucin secretionChapter 3 Anglysis of molecular profiling in malignant pericardial effusion based on next generation sequencingObjective: The purpose of this study is to explore the difference of molecular profiling in malignant pericardial effusion by Next-generation sequencing and ARMS/FISH,To explore a more appropriate methods of gene mutations and guide clinical individualized therapy.Methods: Specimens of 27 cell wax block samples with MPCE we collected in papers part II,then NGS was used for high throughput gene detection of sample EGFR?KRAS?ALK?c-MET?BRAF?ERBB2?RET and ROS1 genes in these specimens,and compared with the results of ARMS/FISH detection in papers part II.Results: The concordance rate between gene status identified by NGS and ARMS/FISH in MPCE was 77.8%(21/27),a sensitivity of 100%,and a specificity of 83.3%.There was no significant difference in the positive rate of gene mutation between the two groups(EGFR,59.3 vs 55.6%,P=0.783),(KRAS,11.1% vs 7.4%,P=1.000),(ALK,3.7% vs 7.4%,P=1.000)and(c-MET,7.4% vs 7.4%,P=1.000).Inconsistency performance in 2 cases were detected single mutation by ARMS/FISH,whereas the NGS were double mutation.1cases were not detected mutation by ARMS,whereas the NGS were were detected.and the mutation sites were changed in 2 cases between two methods.Additionally,One case each of ERBB2 and BRAF mutations was detected by NGS,and the mutation abundance values of each gene were also displayed.Conclusions: NGS and ARMS/FISH are suitable for molecular profiling detection in MPCE,and the results on single gene detection are reliable and can replace each other.Compared of DNA point mutation by ARMS/FISH,the NGS detection range is wider,not only can detect for rare and coexisting mutations,but also indicate the mutation abundance.