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Objective:To identify genes interacting with the ubiquitin-conjugating enzyme E2S (UBE2S) in melanoma, and to explore the molecular mechanisms regulating the biological behavior of melanoma cells by UBE2S.Methods:Cultured A375 melanoma cells were divided into 2 groups to be transfected with LV-UBE2S-RNAi (14011-1) -containing lentivirus (gene knockdown group) and a negative control lentivirus CON053 (negative control group), respectively, and the UBE2S gene-knockdown cell line and negative control cell line were established. RNA was extracted from the two cell lines, purified, fragmented, and hybridized with GeneChip probes, followed by washing and dyeing, and then microarray data were obtained. Ingenuity pathway analysis (IPA) software was used to further analyze the gene expression profiling data to identify potential genes interacting with UBE2S. Western blot analysis was performed to verify the downstream molecules regulated by UBE2S. A two-tailed t test was used to screen for differentially expressed genes. Results:A total of 512 differentially expressed genes with a|fold change| > 2 and a P value < 0.05 were screened out between the gene knockdown group and negative control group, including 247 upregulated genes and 265 downregulated genes. IPA of differentially expressed genes revealed that interferon signaling and liver X receptor (LXR) /retinoid X receptor (RXR) activation pathways were significantly activated, while eukaryotic initiation factor-2 (EIF2) signaling and nuclear factor-κB (NF-κB) signaling pathways were inhibited. Among the upstream regulators, IFNA2 was predicted to be strongly activated, and NF-κB (complex) to be strongly inhibited. Based on the above bioinformatics analysis results, it was suggested that the UBE2S gene may exert some effects in melanoma cells by regulating the expression of IFITM1, STAT1, ISG15 and TNFRSF11B genes. Western blot analysis showed that IFITM1 protein was not expressed in A375 cells before and after the UBE2S gene knockdown. After the UBE2S gene knockdown, ISG15 protein expression was upregulated by 19.94 times, STAT1 protein expression was upregulated by 1.47 times, and TNFRSF11B protein expression was downregulated by 79.1%. Conclusion:UBE2S may interact with STAT1, ISG15 and TNFRSF11B to regulate the biological behavior of melanoma cells.
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Objective@#To evaluate the expression of p-AKT and p-mTOR, the key proteins in PI3K/AKT/mTOR pathway in pediatric Burkitt lymphoma (BL), and to investigate the clinical and prognostic significance.@*Methods@#Fifty-eight cases of pediatric BL and thirty cases of reactive hyperplastic lymphadenitis (RH) were collected at Children′s Hospital of Fudan University from September 2011 to July 2018. Paraffin sections of tissues were immune stained for p-AKT and p-mTOR, and the expression was assessed and correlated with the clinical features and prognosis.@*Results@#A total of 58 cases were diagnosed and 6 cases lost the follow-up. Of the remaining 52 BL patients including 43 males and 9 females, the median age was 5 years (range: 2 to 14 years). Regarding to the correlation between the two biomarkers, Spearman test showed that p-mTOR was positively associated with the expression of p-AKT (r=0.759, P<0.001). Of all BL patients, the positive rates of p-AKT and p-mTOR were 62.1% (36/58) and 60.3%(35/58) respectively, both significantly higher than control group (P=0.011, P=0.035 respectively). The presence of p-AKT was significantly associated with higher lactate dehydrogenase (LDH≥573 IU/L) level in patients of the disease (P=0.006), while p-mTOR was increased both in the higher LDH and lower ratio of albumin to globulin (A/G) group (P=0.006, P=0.034 respectively). Expression of p-AKT and p-mTOR did not show any statistical correlation with sex, age, St.jude stage, tumor size, B-symptom present or not, number of extra-nodal sites or international prognostic index (IPI) (P>0.05). Fifty-two patients had a median follow-up of 40 months (range: 5-87 months). Univariate analysis showed that p-AKT expression was significant in predicting both inferior OS (5-year estimate, 72.7% vs. 94.7%, χ2=4.123, P=0.042) and PFS (5-year estimate, 66.7% vs. 94.7%, χ2=5.822, P=0.016). The 5-year OS rate was 71.0% (22/31) for the p-mTOR positive cohort of patients compared to 95.2% (17/21) for p-mTOR negative group (χ2=4.881, P=0.027); however, there was no statistical significance in 5-year PFS rate (P>0.05). Especially, the 5-year OS and PFS rate of p-AKT/p-mTOR double-positive group were significantly lower than negative control group (including absence of single p-AKT or p-mTOR expression, and absence of both) (OS: 69.0% vs. 95.7%, χ2=6.285, P=0.012; PFS: 65.5% vs. 91.3%, χ2=5.405, P=0.020). The results of multivariate COX proportional risk regression analysis indicated that p-AKT/p-mTOR double-positive, higher LDH and IPI score 3-5 were independent prognostic factors for both OS and PFS, and the bulky tumor (>10 cm) for PFS of pediatric BL.@*Conclusion@#The expression of p-AKT and p-mTOR may be a potential reference for diagnosis and the independent prognostic indicators of pediatric BL.
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Objective@#To evaluate the expression of p-AKT and p-mTOR, the key proteins in PI3K/AKT/mTOR pathway in pediatric Burkitt lymphoma (BL), and to investigate the clinical and prognostic significance.@*Methods@#Fifty-eight cases of pediatric BL and thirty cases of reactive hyperplastic lymphadenitis (RH) were collected at Children′s Hospital of Fudan University from September 2011 to July 2018. Paraffin sections of tissues were immune stained for p-AKT and p-mTOR, and the expression was assessed and correlated with the clinical features and prognosis.@*Results@#A total of 58 cases were diagnosed and 6 cases lost the follow-up. Of the remaining 52 BL patients including 43 males and 9 females, the median age was 5 years (range: 2 to 14 years). Regarding to the correlation between the two biomarkers, Spearman test showed that p-mTOR was positively associated with the expression of p-AKT (r=0.759, P<0.001). Of all BL patients, the positive rates of p-AKT and p-mTOR were 62.1% (36/58) and 60.3%(35/58) respectively, both significantly higher than control group (P=0.011, P=0.035 respectively). The presence of p-AKT was significantly associated with higher lactate dehydrogenase (LDH≥573 IU/L) level in patients of the disease (P=0.006), while p-mTOR was increased both in the higher LDH and lower ratio of albumin to globulin (A/G) group (P=0.006, P=0.034 respectively). Expression of p-AKT and p-mTOR did not show any statistical correlation with sex, age, St.jude stage, tumor size, B-symptom present or not, number of extra-nodal sites or international prognostic index (IPI) (P>0.05). Fifty-two patients had a median follow-up of 40 months (range: 5-87 months). Univariate analysis showed that p-AKT expression was significant in predicting both inferior OS (5-year estimate, 72.7% vs. 94.7%, χ2=4.123, P=0.042) and PFS (5-year estimate, 66.7% vs. 94.7%, χ2=5.822, P=0.016). The 5-year OS rate was 71.0% (22/31) for the p-mTOR positive cohort of patients compared to 95.2% (17/21) for p-mTOR negative group (χ2=4.881, P=0.027); however, there was no statistical significance in 5-year PFS rate (P>0.05). Especially, the 5-year OS and PFS rate of p-AKT/p-mTOR double-positive group were significantly lower than negative control group (including absence of single p-AKT or p-mTOR expression, and absence of both) (OS: 69.0% vs. 95.7%, χ2=6.285, P=0.012; PFS: 65.5% vs. 91.3%, χ2=5.405, P=0.020). The results of multivariate COX proportional risk regression analysis indicated that p-AKT/p-mTOR double-positive, higher LDH and IPI score 3-5 were independent prognostic factors for both OS and PFS, and the bulky tumor (>10 cm) for PFS of pediatric BL.@*Conclusion@#The expression of p-AKT and p-mTOR may be a potential reference for diagnosis and the independent prognostic indicators of pediatric BL.
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Objective@#To investigate the clinicopathological features, diagnosis, differential diagnosis, treatment and prognosis of pediatric alveolar rhabdomyosarcoma (ARMS).@*Methods@#The clinical and pathological data of 25 pediatric ARMS from 2008 to 2018 in Children′s Hospital of Fudan University were collected. This histomorphology was assessed, and FOXO1 gene rearrangement was detected with FISH. The treatment details and outcome were analyzed.@*Results@#There were 13 males and 12 females, with ages range from 19 days to 14 years (median 6 years, mean 6.2 years). The ARMS were located in the limbs (13 cases), head and neck (4 cases), trunk (3 cases), abdominal cavity (3 cases), scrotum (1 case) and perianal region (1 case). The ARMS were classified histologically as classic group (18 cases), solid group (5 cases) and embryonic-alveolar mixed group (2 cases). The typical pathological characteristics were small dark round cells arranged in solid, glandular and papillary patterns. The tumor cells expressed ALK (D5F3) (21/25, 84.0%), muscle origin DES (23/25, 92.0%), myogenin (22/25, 88.0%), MYOD1 (19/25, 76.0%), and in some cases they also expressed neurogenic marker Syn (6/25, 24.0%). FOXO1 gene rearrangement was detected by FISH in 24/25 cases (96.0%).@*Conclusion@#Pediatric ARMS is rare and has unique clinicopathological characteristics, and needs to be differentiated from other common small round cell malignancies in children. ALK, DES, myogenin, MYOD1 immunohistochemistry and FOXO1 gene rearrangement are valuable aid in the diagnosis of ARMS.
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Objective@#To analyze the clinicopathological features, immunohistochemical (IHC) phentotype,diagnosis and differential diagnosis of infantile/congenital fibrosarcoma (IFS/CFS) with unusual histological features.@*Methods@#Five IFS/CFS at Children′s Hospital of Fudan University from March 2014 to July 2018 were analyzed for their diagnosis and differential diagnosis.@*Results@#Two cases were males, three cases were females. The clinical manifestation of IFS/CFS was a rapidly-growing and painless mass. There were no specific radiologic features. Histologically, the tumor cells are arranged in intersecting or sheet-like patterns. There were focal hemangioma-like areas in four cases. There were also focal areas of primitive asteroid, short-spindled, and oval tumor cells in three cases. IHC study showed the tumor cells diffusely expressed TLE1(2/5), Vimentin(5/5), and WT1(3/5), in a cytoplastic pattern; they focally expressed CD34(3/5), CD31(3/5), and α-SMA(2/5). Fluorescence in situ hybridization (FISH) detected break-apart positivity of ETV6 gene.@*Conclusions@#Hemangioma-like pattern, myxoid area, and TLE1 expression is very rare in IFS/CFS. Detection of ETV6 gene break-apart by FISH is very helpful in the diagnosis and differential diagnosis of IFS/CFS.
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Objective@#To evaluate the diagnostic value of a histologic scoring system in congenital biliary atresia and its prognostic relevance.@*Methods@#From January 2017 to June 2018 at Children′s Hospital of Fudan University, 172 wedge liver biopsy specimens were obtained from infants with neonatal cholestasis [119 patients with congenital biliary atresia (CBA) and 53 patients with non-obstructive cholestasis as control]. A pathologist, single-blinded to the final diagnosis, made the histological diagnosis individually based on an 8-feature (portal ductal proliferation, bile duct reaction, bile plugs in portal ductules, liver fibrosis, edema in portal region, cholestasis, inflammatory cells infiltration in portal region, and ductal plate malformation), 21-point scoring system.@*Results@#The main pathologic changes of biliary atresia were hepatocyte cholestasis, hyperplasia of bile ducts, fibrosis and infiltration of inflammatory cells in the portal area. There were significant difference in the degree of portal edema, bile duct hyperplasia and fibrosis between two groups (P<0.01). In addition, there were characteristic bile duct thrombosis in 97.5%(116/119) of the cases and abnormal development of bile duct plate in 9.2%(11/119) of the cases. Compared with non-CBA infant cholestasis group, the difference was statistically significant (P<0.05). The scoring system has high sensitivity, specificity (both 94.1%) and accuracy (94.3%) in the diagnosis of CBA. A score equal to or more than 11 points supported a diagnosis of CBA; whereas a score less than 11 points might suggest cholestasis. The degree of hepatic fibrosis and ductal plate malformation were related to prognosis.@*Conclusions@#The liver pathology scoring system (8-feature, 21-point) is more accurate in diagnosing CBA than previous methods, which may guide the clinicopathological diagnosis. This histological scoring system also helps to assess the prognosis of CBA.
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Objective To determine the expression of ubiquitin-conjugating enzyme E2S (UBE2S) in malignant melanoma (MM),and to evaluate its effect on the biological behavior of melanoma cells.Methods Immunohistochemical study was performed to determine the UBE2S expression in 128 primary MM tissue chips,64 metastatic MM tissue chips,16 non-tumor tissue chips (8 paralesional normal skin tissues and 8 normal epidermal tissues).Real-time quantitative RCR was conducted to determine the UBE2S mRNA expression in the melanoma cell lines A375,MUM-2B and MUM-2C.The melanoma cell lines A375 and MUM-2B were divided into 2 groups separately:interference group transfected with a lentiviral vector carrying UBE2S RNA interference sequence,and control group transfected with a lentiviral vector carrying control sequence.After 72 hours,real-time quantitative RCR was performed to determine the UBE2S mRNA expression in the melanoma cell lines A375 and MUM-2B.Caspase-3/7 activity in the groups was assessed by using kits,and cell apoptosis and cell cycle distribution were detected by flow cytometry.The effect of UBE2S knockdown on the migratory and invasive abilities of and N-cadherin expression in A375 cells were evaluated by Transwell assay and Western blot analysis respectively.Statistical analysis was carried out with SPSS 22.0 software by using independent sample t-test for the comparison of normally distributed data between two groups,chi-square test for enumeration data,MannWhitney U test for the comparison of non-normally distributed data,and Spearman's coefficient for assessment of the correlation of UBE2S expression with T staging of melanoma.Results UBE2S was highly expressed in 98 (51.0％) MM tissues,but lowly expressed in 16 non-tumor tissues,and the UBE2Sexpression rate significantly differed between the above two kinds of tissues (x2 =11.905,P ＜ 0.01).UBE2S expression was negatively correlated with T staging of melanoma (ρ =-0.210,P =0.043).The relative mRNA expression of UBE2S significantly differed among the A375,MUM-2B,and MUM-2C cells (F =817.228,P ＜ 0.01).After UBE2S knockdown,the caspase-3/7 activity was significantly up-regulated in the A375 interference group (t =17.572,P ＜ 0.01) and MUM-2B interference group (t =24.552,P ＜0.01) compared with the A375 and MUM-2B control groups respectively.Compared with the control group,the A375 interference group showed significantly increased proportion of A375 cells at G1 phase (t =7.365,P ＜ 0.01),decreased proportion at S phase (t =-9.190,P ＜ 0.01),and no change in the proportion of A375 cells at G2/M phase (t =-0.227,P ＞ 0.05).The MUM-2B interference group showed significantly increased proportions of MUM-2B cells at G1 (t =12.676,P ＜ 0.01) and G2/M phases (t =13.045,P ＜0.01),but significantly decreased proportion at S phase (t =-15.718,P ＜ 0.01) compared with the control group.Transwell assay revealed decreased migratory and invasive abilities of A375 cells in the interference group compared with the control group (t =-35.727,-125.000,P ＜ 0.05,＜ 0.01,respectively).Western blot analysis showed down-regulated expression of N-cadherin protein in A375 cells in the interference group compared with the control group.Conclusions UBE2S is over-expressed in melanoma tissues,whose expression is associated with the T staging of melanoma.Knockdown of UBE2S affects the apoptosis,cell cycle,migration and invasion of melanoma cells,and may promote the metastasis of MM cells by regulating N-cadherin expression.
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Objective@#To analyze the clinicopathologic characteristics of poorly-differentiated chordoma with INI1 loss in children and to discuss the differential diagnosis.@*Methods@#The clinical, radiological, histopathological profiles and molecular pathologic characteristics of two pediatric poorly differentiated chordoma cases with INI1 loss were reviewed.@*Results@#The patients were a girl and a boy. Both lesions involved the slope. Both patients were presented with progressive muscle weakness or neck pain. Radiological examination showed clivus bone destruction and compression of the brain stem and cervical spinal cord. Histologically, the tumor cells lacked typical organization and were associated with inflammatory cells infiltration. On high power field, the tumor cells were ovoid or fusiform with prominent atypia, vacuolated nuclei and prominent nucleoli. By immunohistochemistry, the tumor cells expressed cytokeratin, epithelial membrane antigen, brachyury and were negative for INI1. In both cases, INI1 gene deletion was detected by FISH.@*Conclusions@#Poorly-differentiated chordoma with INI1 loss mainly occurs in children. The morphology is different from classical chordoma.INI1 gene deletion is detectable by FISH. It can be distinguished from atypical teratoid/rhabdoid tumors and other neoplasms by the identification of nuclear brachyury expression. The loss of INI1 expression in poorly-differentiated chordoma might be associated with a poorly-differentiated morphology and an adverse prognosis.
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Laminin (LN) proteins are important components of extracellular matrix. These proteins regulate cell proliferation, differentiation, migration, and tissue repair. The LN family has 12 genes that encode 5 α, 4 β, and 3 γ proteins. LamininA5 (LAMA5) as an important gene can support pluripotent cell growth and have been widely studied. However, porcine LAMA5 is absent in all tested porcine genomic databases so far. In this study, we confirmed for the first time the existence of porcine LAMA5 through bioinformatics analysis, and verified this result by cDNA cloning and sequencing. To reveal the expression pattern of Laminin gene family, we detected the expression of Laminin genes in porcine tissues, somatic cells, and porcine induced pluripotent stem cells (piPSCs). The results showed that an alternative splicing variant of Laminin B1 (LAMB1-a) was found exclusively in all tested piPSCs. The expression of this alternative splicing variant is positively correlated with the pluripotent state of piPSCs. The above findings provide evidences and foundations for the father use of LN as extracellular matrix to facilitate the derivation and culture of porcine pluripotent stem cells.
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Oocyte-like cells (OLC) can be generated by stem cells after the induction and differentiation in vitro, and maturated when transplanted in vivo to improve the development potential. Human amniotic fluid stem cells (hAFSC) were cultured for 10 days in porcine follicle fluid (pFF) that was extracted from the medium follicle with high levels of hormones and Bmp 15 protein. After the induction, the cell aggregates showed the germ cell-like cells and produced the germ cell marker oct4, and triggered epigenetic changes with high expression of methylation transferase gene dnmt3b. The cell aggregates were packaged into porcine theca folliculi to form grafts, which were then transplanted into mouse renal capsule. After one month of transplantation, the morphology of OLC from a graft was not only similar to oocytes, but also expressed the germ cells markers (oct4, nanog, stella, ifitm3, dazl, nanos3, bmp15, and gd9). The results demonstrate that the in vivo differentiation model was useful for OLC development.
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Animals , Female , Humans , Mice , Amniotic Fluid , Cell Biology , Biomarkers , Cell Differentiation , Oocytes , Cell Biology , Ovarian Follicle , Stem Cells , Cell Biology , SwineABSTRACT
<p><b>OBJECTIVE</b>To study the clinicopathologic characteristics, immunophenotypes and differential diagnosis of primitive myxoid mesenchymal tumor of infancy (PMMTI).</p><p><b>METHODS</b>The clinical data, histological features and immunohistochemic results of 3 cases of PMMTI were reviewed.</p><p><b>RESULTS</b>There were 2 males and 1 female aged 4 years, 2 days and 3 months respectively. The tumor occurred in the head and neck (n = 2), and lumbar regions (n = 1).Histologically, they were composed of ovoid, short spindled to polygonal mesenchymal cells with less eosinophilic cytoplasm, or vacuolated cytoplasm. There was mild nuclear atypia with mitotic activity of 0-2/10 HPF.In most areas, the neoplastic cells showed a diffuse growth pattern, whereas in some areas, they formed a vaguely nodular pattern with peripheral collagenized stroma. They were embedded in a myxoid stroma that contained a rich delicate vascular network. Besides, small cyst-like spaces were also present in one case. The tumor cells expressed vimentin, but not alpha smooth muscle actin, desmin, myogenin, S-100 protein, CD34 and cytokeratin. The patients underwent surgery.One patient had local recurrences twice and died 2 years later. Compared with the primary tumor, the recurrent lesions exhibited increased cellularity, marked cellular atypia and mitotic activity (10/10 HPF). The other two patients remained well with no evidence of disease at last during follow-up.</p><p><b>CONCLUSIONS</b>PMMTI is a rare soft tissue tumor of infancy, composed of primitive mesenchymal cells and myxoid stroma.It occurs mainly in the somatic soft tissues of the trunk, head and neck region, and the extremities, and is characterized by a high rate of local recurrence if incompletely excised. Metastasis and tumor related death may occur, albeit very rarely.Increased awareness of this novel entity will help avoid misinterpreting the lesion as a variety of other infantile mesenchymal neoplasms, including congenital fibrosarcoma and lipoblastoma.</p>
Subject(s)
Child, Preschool , Female , Humans , Infant , Infant, Newborn , Male , Soft Tissue Neoplasms , Metabolism , Pathology , Vimentin , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To study the clinicopathologic characteristics of extrarenal malignant rhabdoid tumor (E-MRT) with emphasis on diagnosis and differential diagnosis.</p><p><b>METHODS</b>The clinical and pathologic data of 8 E-MRT cases were reviewed. The outcome was analyzed.</p><p><b>RESULTS</b>There were four males and four females. The age at presentation ranged from 3 days to 8 years (mean, 2.6 years; median, 3 years). The tumors were located in the extremities (n = 1), head and neck (n = 2), trunk (n = 2), cervical cord (n = 1), liver (n = 1) and retroperitoneum (n = 1). Histologically, the tumors were composed of a diffuse proliferation of rounded or polygonal cells with eccentric nuclei, prominent nucleoli, and glassy eosinophilic cytoplasm containing hyaline-like inclusion bodies, arranged in sheets and nests. Cellular atypia was easily observed and mitotic activity was high. Necrotic and hemorrhagic areas were abundant. On immunohistochemistry, the tumor cells expressed vimentin and epithelial marker such as EMA, AE1/AE3, and CAM5.2. The absence of INI1 protein expression was a distinctive feature. Follow-up of all eight cases revealed five deaths in one year and the other three were disease-free at last follow-up of one month, three months and seven months.</p><p><b>CONCLUSIONS</b>E-MRT is a rare and highly aggressive tumor of infancy and childhood. Recurrence and distant metastasis was common and the 5-year survival rate is low. Increased awareness of the clinocopathologic features and immunophenotypes of E-MRT is helpful for correct diagnosis and effective treatment.</p>