[Clinicopathological and molecular features of metaplastic thymoma].

Objective: To investigate the clinicopathological features, and molecular genetic alterations of metaplastic thymoma (MT). Methods: A total of ten MT cases, diagnosed from 2011 to 2021, were selected from the Department of Pathology of Jinling Hospital, Nanjing University Medical School, Nanjing, China for clinicopathological and immunohistochemical (IHC) examination and clinical follow-up. Fluorescence in situ hybridization (FISH), next-generation sequencing (NGS), and YAP1 C-terminus (YAP1-CT) IHC were performed to detect YAP1::MAML2 fusions. Results: There were four males and six females, ranging in age from 29 to 60 years (mean 50 years, median 54 years). Microscopically, all tumors showed a typical biphasic morphology consisting of epithelial components and gradually or abruptly transitioning spindle cell components. The two components were present in varying proportions in different cases. Immunophenotypically, the epithelial cells were diffusely positive for CKpan, CK5/6 and p63. The spindle cells were diffusely positive for vimentin and focally positive for EMA. TdT was negative in the background lymphocytes. Ki-67 proliferation index was less than 5%. YAP1 and MAML2 break-apart FISH analyses showed that all ten cases had narrow split signals with a distance of nearly 2 signal diameters and may be considered false-negative. Using YAP1::MAML2 fusion FISH assays, abnormal fusion signals were observed in all the ten cases. NGS demonstrated YAP1::MAML2 fusions in all eight cases with adequate nucleic acids; in two cases the fusions were detected by DNA sequencing and in eight cases by RNA sequencing. All ten cases of MT demonstrated loss of YAP1 C-terminal expression in epithelioid cells. Conclusions: MT is a rare and low-grade thymic tumor characterized by a biphasic pattern and YAP1::MAML2 fusions. Break-apart FISH assays may sometimes show false-negative results due to the proximity of YAP1 and MAML2, while YAP1 C-terminal IHC is a highly sensitive and specific marker for MT. Loss of YAP1 C-terminal expression can also be used to screen YAP1::MAML2 fusions for possible MT cases.

[Papillary renal neoplasm with reverse polarity: a clinicopathological analysis].

Objective: To study the clinical pathological characteristics, immunophenotype, molecular changes and prognosis of the papillary renal neoplasm with reverse polarity (PRNRP). Methods: Nine cases of PRNRP, diagnosed from 2013 to 2019, were retrieved from the Department of Pathology of Nanjing Jinling Hospital, Nanjing University School of Medicine. Histomorphology, immunophenotype and molecular genetics were analyzed with review of the literatures. Results: There were five male and four female patients, aged from 49 to 70 years, with an average age of 60.1 years. During a mean follow-up of 29 months, one patient died for other cause, and the others survived without disease. Microscopically, the tumor cells arranged in papillary structure with a fibrovascular core, the surface of which was covered with a single layer of cuboidal or columnar cells. The most prominent feature was that the tumor nuclei located at the top of the cytoplasm far from the basement membrane, and they were monotonous in size and arranged neatly with no or few nucleoli. Immunohistochemically, all nine cases of PRNRP showed diffuse positive expression of CK7 and E-cadherin, various degrees of P504s expression, and no expression of CD10 and CD117, with a Ki-67 index of 1%-3%. Unlike other papillary renal cell carcinoma, the nine cases of PRNRP all showed characteristic positive expression of GATA3. The fluorescence in situ hybridization assay showed that the majority of PRNRPs (8/9) did not have triploids on chromosomes 7 and 17. The sequencing of the KRAS gene confirmed the presence of a nonsense KRAS mutation in 8 of the 9 cases. Conclusions: PRNRP is a subtype of papillary renal cell carcinoma with characteristic morphological, immunophenotypic and molecular features, and indolent behaviors. More data are needed to define PRNRP as "carcinoma", and a definitive diagnosis of PRNRP is of great significance for proper treatment choice and accurate prognostication.

[Xp11 neoplasma with melanocytic differentiation: a clinicopathological analysis].

Objective: To investigate the clinical, histologic and immunophenotypic features, genetic alterations and prognosis of the rare Xp11 neoplasm with melanocytic differentiation. Methods: Twenty-one cases were selected from the Department of Pathology, Jingling Hospital, Nanjing University School of Medicine from May 2008 to May 2018. The clinicopathologic, immunohistochemical, molecular analysis and follow-up details were collected. Results: There were 7 males and 14 females, with their ages ranging from 4 to 57 years (mean 32.8 years). The tumors were located in kidney (11 cases), pelvis (three cases), and in pancreas, retroperitoneum, adrenal gland, small intestine, prostate, cervix and appendix (one case each). Microscopically, most tumors shared similar morphology such as purely nested or sheet-like architectures separated by a delicate vascular network, purely epithelioid cells with clear to granular eosinophilic cytoplasm, lacks of papillary structures, spindle cell or fat components, uniform round to oval nuclei with small visible nucleoli, and in most of them (16/21) melanin pigment. Immunohistochemically, all cases showed moderately (2+) or strongly (3+) positive staining for TFE3 and Cathepsin K. HMB45 and Melan A were focally expressed in three of 21 cases, while the remaining cases showed typically moderate(2+) or strong (3+) expression. None of the cases were immunoreactive for SMA, desmin, CKpan, S-100 and PAX8. All cases showed TFE3 rearrangement using fluorescence in-situ hybridization (FISH). Fusion FISH assays detected SFPQ-TFE3 gene fusion in 16 cases, NONO-TFE3 gene fusion in two, ASPL-TFE3 and MED15-TFE3 gene fusions in one case each. Polymerase chain reaction and direct sequencing detected SFPQ-TFE3 gene fusion in nine cases, NONO-TFE3 and MED15-TFE3 gene fusions in one case each. Clinical follow-up was available for 15 patients for 12 to 74 months. Six patients died of the disease; and three had recurrences and/or metastases. Six patients were alive with no evidence of disease after initial resection. Conclusions: Xp11 neoplasm with melanocytic differentiation has unique morphologic, immunophenotypic and genetic characteristics. The tumor is aggressive, and should be differentiated from Xp11 translocation RCC and perivascular epithelioid cell tumor.

[Detection of epidermal growth factor receptor gene mutations in different types of non-small cell lung cancer by droplet digital PCR and amplification refractory mutation system].

Objective: To compare amplification refractory mutation system(ARMS) and droplet digital PCR (ddPCR) in the detection of epidermal growth factor receptor (EGFR) gene mutations in patients with non-small cell lung cancer (NSCLC), and to investigate the clinical value of ddPCR. Methods: A total of 79 specimens of NSCLC, including 22 cases of cell block, 18 cases of surgical specimens, 12 cases of biopsy specimens and 27 cases of plasma samples, were analyzed for the mutation status of EGFR gene by ARMS and droplet digital PCR method. Results: In 18 cases of surgical specimens and 12 cases of biopsy specimens, the detection results by the two methods were identical with positive rates of 9/18 and 5/12, respectively. In 22 cases of effusion cell blocks, ARMS detected 19-del and L858R of EGFR gene in two cases, in which droplet digital PCR detected 19-del+ T790M mutations in one case and L858R+ T790M mutation in another. L858R mutation was detected by droplet digital PCR in one case but ARMS assay was negative. The remaining 19 cases were consistent by the two methods. In blood samples, the positive rate was 33.3%(9/27) by ARMS and 37.0%(10/27) by droplet digital PCR. Two cases showed L858R and 19-del+ T790M mutation by droplet digital PCR but ARMS assay detected only 19-del. The remaining 25 cases were consistent by the two methods. Conclusion: Droplet digital PCR method is more sensitive and accurate than ARMS for the detection of EGFR mutations in pleural fluid and blood samples, can be used in clinical test.

[Epidermal growth factor receptor (EGFR) mutation status before and after acquired resistance to EGFR tyrosine kinase inhibitors in patients with lung adenocarcinoma].

Objective: To investigate epidermal growth factor receptor (EGFR) mutation status in lung adenocarcinomas before and after acquiring resistance to EGFR tyrosine kinase inhibitors (TKIs) using ARMS method followed by further verification using droplet digital PCR technique. Methods: Twenty qualified patients were included, among them 13 were male and 7 were female patients. Before EGFR-TKIs treatment, 5 patients were EGFR wild-type by ARMS, and the other 15 patients had L858R or 19-del point mutations. The time to progression varied from 4 to 18 months. Mutation of exons 18, 19, 20 and 21 were detected by ARMS, and were verified by droplet digital PCR system method. Results: EGFR wild-type status was unchanged before and after acquired resistance to EGFR-TKIs in 5 lung adenocarcinoma patients. Alteration of EGFR mutation status occurred in 10 of the 15 patients with pre-treatment L858R or 19-del mutations. Among them, T790M mutation was found in 8 patients, L858R became G719X plus S768I mutation in one patient, and 19-del converted into wild-type in one other patient. Conclusions: T790M mutation is the primary type of EGFR mutation in lung adenocarcinomas with acquired resistance to EGFR-TKIs therapy. Acquired resistance to EGFR-TKIs dose not lead to the alteration of EGFR status in pre-treatment EGFR wild-type patients, but can alter EGFR mutation status in pre-treatment EGFR mutant patients.

[Molecular features of metanephric adenoma and their values in differential diagnosis].

Objective: To study the molecular features of metanephric adenoma (MA) and discuss their values in differential diagnosis. Methods: BRAF V600E immunohistochemistry (IHC) using the mutation-specific VE1 monoclonal antibody and Sanger sequencing of BRAF mutations were performed on 21 MAs, 16 epithelial-predominant Wilms tumors (e-WT) and 20 the solid variant of papillary renal cell carcinomas (s-PRCC) respectively. p16 protein was detected by IHC also. Fluorescence in situ hybridization (FISH) analyses using centromeric probes for chromosome 7 and 17 were performed on the three renal tumors in parallel. Results: Fourteen (14/21, 66.7%) of 21 MA cases demonstrated diffuse, moderate to strong cytoplasmic BRAF V600E IHC staining and the BRAF V600E protein expression was detected in 2 (2/16) of 16 e-WT cases for the first time, whereas all s-PRCCs were negative (P<0.05). All cases (including 14 MAs and 2 e-WTs) with diffuse, moderate to strong cytoplasmic BRAF V600E IHC staining were confirmed to harbor BRAF V600E missense mutations using Sanger sequencing, and no BRAF mutations were detected in cases with negative BRAF V600E protein expression. One case (1/21, 4.8%) showed trisomy of chromosome 7 alone, and another one (1/21, 4.8%) showed trisomy of chromosome 17 alone in 21 MAs. Two cases (2/16) of 16 e-WTs showed trisomy of chromosome 17 alone. In 20 s-PRCCs, trisomy of chromosomes 7 alone was reported in 2 cases (2/20), trisomy of chromosome 17 alone in 3 cases (3/20) and trisomy of chromosome 7 and 17 in 14 cases (14/20). The total positive rates of trisomy of chromosome 7 and/or 17 in MAs, e-WTs and s-PRCCs were 9.6% (2/21), 2/16 and 95.0% (19/20). p16 protein was positive in 81.0% (17/21) MAs, whereas the positive rates in e-WTs and s-PRCCs were 2/16 and 5.0% (1/20). Conclusions: Most MAs harbor BRAF V600E mutations, and MAs lack the gains of chromosome 7 and 17 that are characteristic of papillary renal cell carcinoma. These molecular features can be used to distinguish MA from its mimics. BRAF V600E IHC using the mutation-specific VE1 monoclonal antibody provides an effective method in BRAF V600E mutations detection of renal tumors. p16 is overexpressed in MA, and the finding suggests that the low proliferative rate of the tumor might be attributed to BRAF V600E-induced senescence mediated by p16.

CXCL2/MIF-CXCR2 signaling promotes the recruitment of myeloid-derived suppressor cells and is correlated with prognosis in bladder cancer.

The accumulation of myeloid-derived suppressor cells (MDSCs) has been observed in solid tumors and is correlated with tumor progression; however, the underlying mechanism is still poorly understood. In this study, we identified a mechanism by which tumor cells induce MDSC accumulation and expansion in the bladder cancer (BC) microenvironment via CXCL2/MIF-CXCR2 signaling. Elevated expression of CXCL2 and MIF and an increased number of CD33+ MDSCs were detected in BC tissues, and these increases were significantly associated with advanced disease stage and poor patient prognosis (P<0.01). A positive association was observed between CXCL2 or MIF expression and the number of tumor-infiltrating CD33+ MDSCs (P<0.01). Subsequently, we demonstrated that CD45+CD33+CD11b+HLA-DR- MDSCs from fresh BC tissues displayed high levels of suppressive molecules, including Arg1, iNOS, ROS, PDL-1 and P-STAT3, and stronger suppression of T-cell proliferation. Interestingly, these CD45+CD33+CD11b+HLA-DR- MDSCs exhibited increased CXCR2 expression compared with that in peripheral blood from BC patients or healthy controls (P<0.05). Chemotaxis assay revealed that bladder cancer cell line J82 induced MDSC migration via CXCL2/MIF-CXCR2 signaling in vitro. Mechanistic studies demonstrated that J82-induced MDSC trafficking and CXCR2 expression were associated with increased phosphorylation of p38, ERK and p65. Conversely, inhibition of the phosphorylation of p38, ERK or p65 decreased J82-induced MDSC trafficking and CXCR2 expression. CXCL2/MIF-stimulated activation of the mitogen-activated protein kinase and nuclear factor kappa B pathways in MDSCs was MyD88 dependent. Overall, our results identify the CXCL2/MIF-CXCR2 axis as an important mediator in MDSC recruitment and as predictors and potential therapeutic targets in BC patients.

[Application of BRAF V600E mutation-specific immunohistochemistry in diagnosis of gastrointestinal stromal tumors].

OBJECTIVE: To evaluate the utility of BRAF V600E allele-specific antibody in the diagnosis of gastrointestinal stromal tumors (GISTs). METHODS: BRAF V600E mutation-specific immunohistochemistry and BRAF sequencing were performed in 24 consecutive GISTs, including 14 cases of KIT or PDGFRA mutations and 10 cases of KIT/PDGFRA wild GISTs. RESULTS: GISTs of 11 men and 13 women with a mean age 54 years(range 29-75 years) were included with tumors arising from stomach (16 cases), small bowel (7 cases), and peritoneal cavity (1 case). Strong and diffuse cytoplasmic BRAF staining was noted in 4 of 24 cases (17%), while 1 of 24 cases (4%) showed weak staining, and 19 of 24 cases (79%) had no staining. The four cases with strong BRAF immunostain were confirmed to have BRAF mutations, including 3 cases in the stomach and 1 case in the small intestine. All tumors showed spindle cell morphology. Only one case had progressive disease. No BRAF mutations were detected in cases with weak or negative BRAF immunostain. CONCLUSION: BRAF V600E mutation-specific immunohistochemistry is a highly sensitive and specific marker for detecting BRAF-mutated GISTs.

[Three dimensional Finite Element Analysis of the Effects of Posts and Cores with Different Angles]

OBJECTIVE:The test is an analogue of the conditions that cast post and core can be made with different angles to get the core incline lingually or facially in clinic, so that the arrangement of the anterior teeth may be improved.METHODS:Analyse the stress distribution and concentration in the dentin of posts and cores with different angles by Ansys three dimensional finite element stress analysis method.RESULTS:The stress in root was positively related to the angles between the load and the axis of the tooth, the stress distributoin in dentin was related to the direction of the core but not related to the angles. The closer the distance from the load to cemento enamel junction,the greater the effect of the load on the stress in dentin.CONCLUSION:When the cast post and core with angle was used to rectify the facially malpositional maxillary anterior teeth, the core should not be inclined lingually excessively. It is important to assure the thickness of the facial and lingual canal wall during tooth preparing and to adjust the occlusion so that the root fracture can be avoided.

[The changes of chewing ability after orthognathic therapy: electromyographic analysis]

OBJECTIVE:The maxillo-facial skeletal deformity can only be corrected through orthognathics that consists of orthodontics and operation.There are various changes in aspect of the bite relationship between upper and lower dentition after orthognathic surgery.The change feature of the chewing efficiency of oral maxillary system can be detected through comparing studying of the contracti on force of chewing muscles before and after surgical operation.METHODS:All 6 patients in this research were diagnosed to be skeletal crossbite.Electromyogram was used to test the change of activity of superficial masseter,temporalis and digastrics.The examination was carried out 1 week before operation and 3 months after the operation. RESULTS:When mandibular is in its rest status,there is no significant difference in the change of muscular activities.when it is active,there is significant difference. CONCLUSION:The chewing ability of masticatory muscles is reduced after orthognathic surgery.The result indicated that there is a negative bite relationship between the upper and lower dentition,the occlusion relationship needs accurate adjustment for gaining stability of oral maxillary system.Therefore,orthodontics after operation is of vital importance.

[The relationship between loading on the TMJ and occlusal splints]

The primary purpose of this study is to estimate loading conditions on the TMJ with five different occlusal splint.We developed a model that simulated dynamic relationship between dental occlusion,masticatory muscles and TMJ.It is demonstrated that loading on the condyle increases whatever kinds of splints are used.The anterior splint brings about larger loading on the condyle than any other splint.When the ratio force of the muscle on the non pivot is1,left-side pivot split makes loading on left condyle decrease and right increase.It was change on both condyles when posterior teeth are missing including loading, contact and unilateral mastication.