Objective To investigate the role and primary mechanism of a novel fusion gene RELCH-RET in driving the malignant transformation of normal human bronchial epithelial(HBE)cells.Methods Based on retrospective clinical data from 456 non-small cell lung cancer(NSCLC)patients admitted in the Second Affiliated Hospital of Army Medical University from January 2019 to June 2022,a fusion gene,RELCH-RET,was identified as a research target.Three cell models were established:negative control(HBE VC,transfected with empty lentiviral vector),RET control(HBE RET,transfected with lentiviral overexpression vector of Flag-RET),and experimental group(HBE RELCH-RET,transfected with lentiviral overexpression vector of Flag-RELCH-RET).MTS assay and Transwell assay were used to detect cell proliferation and migratory and invasive abilities.In vivo tumorigenicity of the 3 cell models was assessed in 15 female non-obese diabetic/severe combined immunodeficiency(NOD/SCID)mice(SPF grade,4 weeks old,weighing 15.1±0.4 g)via subcutaneous xenograft experiments,with 5 animals in each group.Western blotting was employed to detect the autophosphorylation of RET(Y905)and the phosphorylation of downstream signaling proteins ERK1/2,EGFR(Y845)and STAT3(Y705).Dimerization and multimerization status of RELCH-RET were analyzed by chemical cross-linking(DTME treatment)in combination with Western blotting,with the reversibility being confirmed through de-cross-linking experiments.Results There were 3 cases carrying RELCH-RET fusion gene screened out from the 469 NSCLC patients.Compared with the HBE VC and HBE RET groups,the HBE RELCH-RET group exhibited significantly enhanced cell proliferation(P<0.01),and acquired migratory and invasive abilities(P<0.01),while the control groups did not demonstrate the abilities.In the mouse xenograft tumor model,HBE cells stably expressing RELCH-RET developed significant tumor nodules(P<0.001),whereas the control groups(empty vector and wild-type RET)failed to exhibit detectable tumor growth.Western blotting revealed that RELCH-RET could induce the autophosphorylation of the RET tyrosine residue(Y905)and significantly up-regulate the phosphorylation levels of ERK1/2,EGFR(Y845),and STAT3(Y705)proteins.Chemical cross-linking combined with Western blot analysis demonstrated that RELCH-RET formed a dimer(～170 kDa)in HBE cells,which is reversibly dissociated into monomers upon decross-linking treatment.Conclusion The novel fusion gene RELCH-RET,promotes ligand-independent dimerization/oligomerization,thereby mediating RET autophosphorylation,subsequently activates the downstream typical RET signaling pathway and ultimately drives the malignant transformation of normal HBE cells.

Objective To investigate the effect of endothelial cells in the perivascular niche on the stem cell-like characteristics of osteosarcoma cells and primarily explore the possible molecular mechanism.Methods A co-culture model was established in vitro using SV40T-human umbilical vein endothelial cells(HUVEC-T1)and osteosarcoma stem cells(OSCs)derived from the human osteosarcoma cell line 143B.Thus,there were 2 groups of cells,OSCs and co-cultured OSCs.The self-renewal capacity of OSCs between the 2 groups was assessed using a limiting dilution forming sphere assay.Flow cytometry and Western blotting were used to detect the expression of stem cell marker CD133 and stemness transcription factors SOX2 and NANOG.Fourteen female nude mice(4～6 weeks old,weighing 18～20 g)were randomly and equally divided into 2 groups of subcutaneous xenograft models:the control group(OSCs suspension)and the experimental group(OSCs+HUVEC-T1 mixed suspension).The tumor volume and mass were compared between the 2 xenograft groups.Immunofluorescence(IF)staining was used to verify the spatial proximity between endothelial cells and OSCs in vivo,while immunohistochemistry was employed to compare microvessel density(MVD)and CD133 expression level between the 2 groups.RNA-seq was performed to identify potential signaling pathways of endothelial cells affecting the stemness of OSCs.PCR and Western blotting were applied to confirm the RNA-seq findings.Exogenous protein treatment,IF staining,Western blotting and sphere formation assay were utilized to preliminarily validate the role of the identified pathway in regulating the stemness phenotype of OSCs.Results The in vitro co-culture model of HUVEC-T1 and OSCs was successfully established.Compared with the control group,the co-culture group exhibited significantly enhanced self-renewal ability of OSCs,laeger proportion of the stemness marker CD133+[(8.20±1.64)％vs(4.32±1.34)％,P<0.05],enhanced expression of CD117,SOX2 and NANOG(P<0.05),along with more sphere formation(P<0.05)and elevated SOX2/NANOG protein levels.The xenograft mice from the experimental group showed larger tumor volume(643.10±413.50 vs 247.90±93.66 mm3,P<0.05)and heavier tumor weight(0.52±0.27 vs 0.24±0.10 g,P<0.05)when compared the control group,correspondingly showing increased MVD(22.57±11.84 vs 11.43±5.38,P<0.05)and elevated CD133 expression(P<0.05).IF staining confirmed the adjacency of CD31-labeled endothelial cells and CD133-labeled OSCs in vivo.RNA-seq and functional experiments demonstrated that CXCR4 was highly expressed in co-cultured OSCs,CXCL12 was highly expressed in co-cultured endothelial cells,and exogenous CXCL12 promoted the sphere formation and expression levels of SOX2 and NANOG of OSCs(P<0.05).Conclusion Endothelial cells within the perivascular niche may promote the stemness phenotype of osteosarcoma cells via the CXCL12/CXCR4 pathway.

Objective To elucidate the effects of miR-616 on the malignant biological processes of osteosarcoma and to preliminarily explore its potential mechanisms.Methods In situ hybridization(ISH)was employed to analyze miR-616 expression in 11 paraffin-embedded osteosarcoma specimens collected in our department during January 2018 to December 2019.Quantitative real-time PCR(qRT-PCR)was used to compare the mRNA expression level of miR-616 in the osteoblast cell line hFOB1.19 and osteosarcoma cell lines 143B and HOS.Stable cell lines with miR-616 knockdown or overexpression were established via lentiviral transfection in 143B and HOS cells.Cell proliferation and apoptosis were detected by flow cytometry,while cell invasion and migration were assessed using Transwell and colony formation assays,respectively.To evaluate the effect of miR-616 on tumor growth in vivo,10 female nude mice(4 weeks old,weighing 18～20 g)were randomized into a control group and a miR-616 overexpression group.After the xenograft tumor model was constructed,the growth of subcutaneous tumors was monitored.Finally,next-generation sequencing and a dual-luciferase reporter assay were performed to identify the target genes of miR-616.Results ISH results showed that miR-616 expression was up-regulated in osteosarcoma tissues than adjacent tissues,and primarily localized in the cytoplasm.qRT-PCR confirmed that miR-616 level was significantly higher in 143B and HOS cells than hFOB1.19 cells(P<0.05).In vitro experiments revealed that miR-616 overexpression enhanced the proliferation,migration and invasion,while suppressing apoptosis in 143B and HOS cells(P<0.01).Conversely,miR-616 knockdown weakened these malignant phenotypes(P<0.05),with miR-616-3p showing a stronger effect on apoptosis than miR-616-5p.Animal experiments demonstrated that the tumor weight in the miR-616 overexpression group was significantly greater than that of the control group(98.00±17.22 vs 33.60±8.08 mg,P<0.01).Furthermore,KLF2 was identified and confirmed as a direct target of miR-616.Conclusion MiR-616 promotes malignant biological behaviors in osteosarcoma,and its expression level indicates that it may serve as a potential therapeutic target.

Objective:To investigate the pathological characteristics and the clinical significance of novel coronavirus (2019-nCoV)-infected pneumonia (termed by WHO as coronavirus disease 2019, COVID-19).Methods:Minimally invasive autopsies from lung, heart, kidney, spleen, bone marrow, liver, pancreas, stomach, intestine, thyroid and skin were performed on three patients died of novel coronavirus pneumonia in Chongqing, China. Hematoxylin and eosin staining (HE), transmission electron microcopy, and histochemical staining were performed to investigate the pathological changes of indicated organs or tissues. Immunohistochemical staining was conducted to evaluate the infiltration of immune cells as well as the expression of 2019-nCoV proteins. Real time PCR was carried out to detect the RNA of 2019-nCoV.Results:Various damages were observed in the alveolar structure, with minor serous exudation and fibrin exudation. Hyaline membrane formation was observed in some alveoli. The infiltrated immune cells in alveoli were majorly macrophages and monocytes. Moderate multinucleated giant cells, minimal lymphocytes, eosinophils and neutrophils were also observed. Most of infiltrated lymphocytes were CD4-positive T cells. Significant proliferation of type Ⅱ alveolar epithelia and focal desquamation of alveolar epithelia were also indicated. The blood vessels of alveolar septum were congested, edematous and widened, with modest infiltration of monocytes and lymphocytes. Hyaline thrombi were found in a minority of microvessels. Focal hemorrhage in lung tissue, organization of exudates in some alveolar cavities, and pulmonary interstitial fibrosis were observed. Part of the bronchial epithelia were exfoliated. Coronavirus particles in bronchial mucosal epithelia and type Ⅱ alveolar epithelia were observed under electron microscope. Immunohistochemical staining showed that part of the alveolar epithelia and macrophages were positive for 2019-nCoV antigen. Real time PCR analyses identified positive signals for 2019-nCoV nucleic acid. Decreased numbers of lymphocyte, cell degeneration and necrosis were observed in spleen. Furthermore, degeneration and necrosis of parenchymal cells, formation of hyaline thrombus in small vessels, and pathological changes of chronic diseases were observed in other organs and tissues, while no evidence of coronavirus infection was observed in these organs.Conclusions:The lungs from novel coronavirus pneumonia patients manifest significant pathological lesions, including the alveolar exudative inflammation and interstitial inflammation, alveolar epithelium proliferation and hyaline membrane formation. While the 2019-nCoV is mainly distributed in lung, the infection also involves in the damages of heart, vessels, liver, kidney and other organs. Further studies are warranted to investigate the mechanism underlying pathological changes of this disease.

Objective: To investigation HER2 status in gastric adenocarcinoma of Chinese and contributing factors to the HER2 expression. Methods: HER2 status of 40 842 gastric adenocarcinomas and clinical data were retrospectively collected from 23 hospitals dated from 2013 to 2016. The association between HER2 positivity and clinicopathologic features was analyzed. Results: Of the 40 842 patients the median age was 62 years, the male female ratio was 2.6∶1.0. The rate of HER2 positivity was 8.8% (3 577/40 842). HER2 expression was related to the tissue type, tumor location, Lauren classification and tumor differentiation (P values: 0.009, 0.001, <0.01 and <0.01, respectively). Different HER2 expression status was observed between primary and recurrent tumors in 7.6% (48/635) cases. The rates of HER2 positivity ranged from 2% to 10% among different institutions. The rates of HER2 FISH amplification were dramatically different among the 23 hospitals (0-100%) with an average rate of 10% (810/8 156) in patients with HER2 IHC 2+ . Conclusions HER2 expression is associated with clinicopathologic characteristics. HER2 re-assessment of tumor tissue and use of in situ hybridization techniques increase HER2 positivity. The current retrospective study should reflect the HER2 status in gastric adenocarcinoma of Chinese patients.

Objective To investigate the pathological feathers,diagnosis,differential diagnosis and prognosis of esophageal granular cell tumor. Methods The data of seven patients of esophageal granular cell tumor diagnosed from 2008 to 2014 in our hospital were analyzed. Seven cases of esophageal granular cell tumor were studied by hematoxylin-eosin and EliVision immunohistochemical staining,the clinical and pathological feathers were analyzed with review of the literatures. Results The pathological feathers of seven cases were very typical. The tumors located in the submucosa,and showed gray to grayish yellow,nodular,with no capsule. The size of these tumors ranged from 0. 4 ~1. 0 cm in diameter. Microscopically,the tumor cells were round,polygonal or spindle with small central nuclei and rich eosinophilic granules cytoplasm,the nucleolus could not be easily seen. Immunohistochemical stain showed that the tumor cells were positive for S-100,CD68 and NSE,but negative for SMA,Desmin,CK,CD34,CD117,DOG1,HMB45. The proportion of Ki-67 positive cells was very low. Histochemical stain showed that the tumor cells were positive for PAS. All 7 cases were benign,and the follow-up time was from 8 months to 6 years without recurrence in patients. Conclusion Granular cell tumor of esophagus is very rare in soft tissue tumors,most of which are benign and located in the middle and lower segment of esophagus. The tumor has a better prognosis. The definite diagnosis should depend on the pathological di-agnosis.

Purpose To analyze the clinical and pathological features of intracranial tumor in children. Methods 221 cases of in-tracranial tumors in children ( ages≤18 years) were retrospectively analyzed. Results The cases of intracranial tumor in children ac-counted for 7. 21% of total cases in our hospital during the same period. There is no documented gender bias. There were only 14 cases (6. 33%) with age less than 3 years old. The supratentorial tumors were 153 cases (69. 23%) and infratentorial tumors were 68 cases (30. 77%) . The most frequently affected sites were the cerebral hemispherse, sellar region, vermis and the fourth ventricle. There were 89 benign tumor and 132 malignant tumor in this series of cases. The most common five tumors were astrocytic tumors (30. 32%), embryonal tumors (19. 00%), craniopharyngiomas (11. 76%), ependymal tumors (8. 14%) and germ cell tumors (5. 88%). Conclusion The morbidity of intracranial tumors in children has increased in the recent years. The histological classifica-tion of intracranial tumors in children is multiple and it is essential to make a correct diagnosis.

The establishment of professional master degree in pathology is a new branch of the professional degree graduate education.It is an important measure to bring the clinical pathology into the teaching system of clinical medicine specialty in China.In response to a wave of education reform in the new century, our department has positively converted the traditional postgraduate training and teaching mode, innovated the teaching idea and content, and continued to improve the teaching method and means.On the basis of fully respecting students' subject consciousness, we have combined traditional and modern teaching means, made full use of modern education information technology, actively built and promoted pathology specialized degree graduate student research, teaching and clinical trinity management: the new training mode.Through the practice of bilingual, network, clinical problems and cases of teaching and combined with the leading edge forum of famous pathologists' research and the professional standardized training of clinical pathology, we have extensively carried out the education training, which is based on the theory, practice, research, and sub specialist guidance.

Objective To explore the clinicopathological and immunohistochemical features of wolffian adnexal tumour ( WAT) . Meth-ods The clinical and pathological features analysis and immunohistochemistry methods were utilized to study the histopathology features of a case Wolffian adnexal tumor. Results One case of unilateral WAT which located in broad ligament display a solid-cystic mass with pedicled and enveloped,its histology showed large and small tubulars,screen structure and solid zone. Its immunohistochemistry staining was positive for pan-cytokeratin,vimentin,CK18 and CD99,positive staining for α-inhibin in a little tumor cell,and negative for calretinin,CD10,EMA, ER,PR,Syn,CA15-3,CA19-9,Ki-67 index is less than 5%. PAS positive staining for the basement membrane around Gland like structure. Conclusion Wolffian adnexal tumour is diagnosised by its histopathological,immunohistochemical and it’ s distinctive location where Wolffi-an duct remnants are found.

Objective To analyze the clinicopathological characteristics, immunophenotypes and immu-noglobulin heavy chain gene rearrangement of gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Methods The clinical data of 35 patients with gastric MALT lymphoma who had been admitted to Southwest Hospital from January 1984 to June 2003 were retrospectively analyzed. The specimens of the gastric MALT lymphoma were obtained and their pathomorphological classifications were studied. The immunophenotypes and immunoglobulin heavy chain gene rearrangement were detected by immunohistochemistry and polymerase chain reaction, respectively. All the data were analyzed by chi-square test and Mann-Whitney U test. Results The main manifestations of patients with gastric MALT lymphoma were epigastric discomfort and abdominalgia. The percentage of patients with age≥40 accounted for 74% (26/35). The gastric MALT lyrnphoma has low differentia-tion in 32 patients, and high differentiation in 3 patients. There were 31 patients in Ⅰ E stage, 2 in Ⅱ E1 stage and 2 in Ⅳ stage. Pathological examination showed that gastric MALT lymphoma was composed of centrocyte-like cells and mononuclear-like B cells or lymphoplasma cells. Twenty-nine specimens were with lymphoepithelial lesions (83%), 2 with follicular colonization (6%), and 11 with plasma cells segregated beneath the surface epithelium (33%). All the 35 specimens were B cell immunotype. Restrictive expression of κ and λ light chains of the immunoglobulin was found in 19 cases, and Bcl-2 positive expression was found in 16 cases. High expres-sion of proliferating cell nuclear antigen was found in gastric MALT lymphoma with transformed blasts, which indicated that gastric MALT lymphoma with transformed blasts was more active in proliferation than those of low grade. A monoclonal pattern of immunoglobulin heavy chain gene rearrangement was detected in 33 patients (94%). Conclusions Digestive symptoms are the main manifestations of patients with gastric MALT lymphoma. The incidence of gastric MALT lymphoma increases with age. The final diagnosis of gastric MALT lymphoma depends on the pathological, immunohistochemical examination and laboratory test.