[L-DEP regimen salvage therapy for refractory primary hemophagocytic lymphohistiocytosis triggered by Epstein-Barr virus infection in 4 children].

Objective: To analyze the efficacy and safety of the L-DEP regimen (asparaginase, liposome doxorubicin, etoposide and methylprednisolone) as a salvage therapy for the refractory primary hemophagocytic lymphohistocytosis triggered by Epstein-Barr virus infection (EBV-pHLH) in children. Methods: In this retrospective case study, clinical and laboratory data before and after L-DEP regimen of 4 children diagnosed with EBV-pHLH in Beijing Children's hospital between January 2016 and June 2022 were collected, and the efficacy and safety of L-DEP regimen for the treatment of EBV-pHLH were analyzed. Results: Among 4 patients, there were 3 females and 1 male with the age ranged from 0.8 to 7.0 years. Two of them showed compound heterozygous mutations of PRF1, one with a heterozygous mutation of UNC13D, one homozygous mutation of ITK. Before the L-DEP therapy, all of them had anemia and a soaring level of soluble CD25, 3 patients had neutropenia and thrombopenia, 3 patients had a high level of ferritin, 3 patients had hypofibrinogenemia and 1 patient had hypertriglyceridemia. After receiving 1 or 2 cycles of L-DEP treatment, three achieved remission, including complete remission (1 case) and partial remission (2 cases), and the other one had no remission. The levels of blood cell counts, soluble CD25, triglyceride, fibrinogen and albumin were recovered gradually in 3 patients who got remission. All four patients underwent hematopoietic stem cell transplantation (HSCT) after L-DEP regimen, and three survived. All patients had no severe chemotherapy related complications. The main side effects were bone marrow suppression, infection and pancreatitis, which recovered after appropriate treatments, apart from one who died from severe infection after urgent HSCT. Conclusion: L-DEP regimen could be served as an effective and safe salvage treatment for refractory pediatric EBV-pHLH, and also provide an opportunity for patients to receive HSCT.

[Analysis of 9 cases of drug induced hypersensitivity syndrome related hemophagocytic lymphohistiocytosis].

Objective: To analyze the clinical features,treatment and prognosis of drug induced hypersensitivity syndrome related hemophagocytic lymphohistiocytosis (DIHS-HLH). Methods: This was a retrospective case study. Clinical characteristics, laboratory results, treatment and prognosis of 9 patients diagnosed with DIHS-HLH in Beijing Children's hospital between January 2020 and December 2022 were summarized. Kaplan-Meier survival analysis was used to calculate the overall survival rate. Results: Among all 9 cases, there were 6 males and 3 females, with the age ranged from 0.8 to 3.1 years. All patients had fever, rash, hepatomegaly and multiple lymph node enlargement. Other manifestations included splenomegaly (4 cases), pulmonary imaging abnormalities (6 cases), central nervous system symptoms (3 cases), and watery diarrhea (3 cases). Most patients showed high levels of soluble-CD25 (8 cases), hepatic dysfunction (7 cases) and hyperferritinemia (7 cases). Other laboratory abnormalities included hemophagocytosis in bone marrow (5 cases), hypofibrinogenemia (3 cases) and hypertriglyceridemia (2 cases). Ascending levels of interleukin (IL) 5, IL-8 and interferon-γ (IFN-γ) were detected in more than 6 patients. All patients received high dose intravenous immunoglobulin, corticosteroid and ruxolitinib, among which 4 patients were also treated with high dose methylprednisolone, 2 patients with etoposide and 2 patients with cyclosporin A. After following up for 0.2-38.6 months, 7 patients survived, and the 1-year overall survival rate was (78±14)%. Two patients who had no response to high dose immunoglobulin, methylprednisolone 2 mg/(kg·d) and ruxolitinib died. Watery diarrhea, increased levels of IL-5 and IL-8 and decreased IgM were more frequently in patients who did not survive. Conclusions: For children with fever, rash and a suspicious medication history, when complicated with hepatomegaly, impaired liver function and high levels of IL-5 and IL-8, DIHS-HLH should be considered. Once diagnosed with DIHS-HLH, suspicious drugs should be stopped immediately, and high dose intravenous immunoglobulin, corticosteroid and ruxolitinib could be used to control disease.

[Efficacy and safety of intermediate-dose cytarabine in the treatment of children with refractory high risk Langerhans cell histiocytosis].

Objective: To analyze the efficacy, safety, and long-term prognosis of intermediate-dose cytarabine (Ara-c) regimen in the treatment of children with refractory risk organ involvement Langerhans cell histiocytosis (LCH). Methods: Clinical data of 17 children with multisystem and risk organ involvement LCH who failed the first-line therapy and were treated with intermediate-dose Ara-c (250 mg/m2, twice daily) regimen in the Hematology Center, Beijing Children's Hospital from January 2013 to December 2016 were analyzed retrospectively. In addition to the basic treatment of vindesine and dexamethasone, the patients received two regimens: regimen A: the intermediate-dose Ara-c combined with cladribine and regimen B: the intermediate-dose Ara-c alone. The efficacy, safety and prognosis of the two regimens were analyzed. Results: Among all 17 patients, there were 11 males and 6 females, with the diagnosis age of 2.1 (1.6, 2.7) years. Ten children received regimen A, all of them achieved active disease-better (AD-B) after 8 courses of induction therapy. The disease activity scores (DAS) decreased from 5.5 (3.0, 9.0) to 1.0 (0, 2.3). Seven children received regimen B, and 6 of them achieved AD-B after 8 courses of induction therapy. The DAS decreased from 4.0 (2.0, 4.0) to 1.0 (0, 2.0). The follow-up time was 6.2 (4.9,7.2) and 5.2 (3.7,5.8) years in group A and B. The 5-year overall survival rate was 100.0% in both groups, and the 5-year event free survival rate was (88.9±10.5)% and (85.7±13.2)% in group A and B. Grade 3 or 4 myelosuppression was observed in 8 patients in group A and 2 patients in group B. Conclusions: The intermediate-dose Ara-c regimen (with or without cladribine) is effective and safe for patients with refractory high-risk LCH, with a good long-term prognosis.

[Interleukin-9 promotes the activation of hepatic stellate cells in mice infected with Schistosoma japonicum].

OBJECTIVE: To investigate the effects of interleukin-9 (IL-9) in the activation of hepatic stellate cells (HSCs) in mice infected with Schistosoma japonicum. METHODS: Primary HSCs were isolated from mice 7 weeks post-infection with S. japonicum using the in situ liver perfusion and density gradient centrifugation, and cultured in vitro. HSCs were randomly assigned to the PBS control group and IL-9 stimulation group (stimulation with 20 ng/mL IL-9). HSCs were harvested 48 h and 72 h poststimulation, and the expression of α-smooth muscle actin (α-SMA), type I collagen (Col I) and type III collagen (Col III) was determined in HSCs using Western blotting. RESULTS: Following stimulation with 20 ng/mL IL-9 for 48 h, the expression of α-SMA [(0.87 ± 0.02) vs. (0.69 ± 0.01); t = 17.39, P < 0.01], Col I [(0.74 ± 0.02) vs. (0.65 ± 0.01); t = 9.56, P < 0.01] and Col III [(0.94 ±0.04) vs. (0.75 ± 0.03); t = 6.15, P < 0.01] was significantly greater in HSCs in the IL-9 stimulation group than in the PBS control group. Following stimulation with 20 ng/mL IL-9 for 72 h, the expression of α-SMA was significantly greater in HSCs in the IL-9 stimulation group than in the PBS control group[(0.76 ± 0.02) vs. (0.58 ± 0.02); t = 12.52, P < 0.01]; however, there were no significant differences between the two groups in terms of Col I [(0.68 ± 0.02) vs. (0.66 ± 0.02); t = 1.15, P > 0.05] or Col III expression [(0.75 ± 0.01) vs. (0.72 ± 0.02); t = 2.22, P > 0.05]. CONCLUSIONS: IL-9 promotes the activation of HSCs in mice infected with S. japonicum.

[Efficacy and safety of modified hemophagocytic lymphohistiocytosis 04 regimen in Beijing Children's Hospital].

Objective: To evaluate the efficacy and safety of Beijing Children's Hospital (BCH) modified hemophagocytic lymphohistiocytosis (HLH) 04 regimen in the treatment of childhood HLH. Methods: A retrospective cohort study was conducted. From January 2016 to December 2017, 110 children with HLH who were treated with the modified HLH-04 regimen (replacing dexamethasone with methylprednisolone during the induction period, reducing the dose and frequency of etoposide, and not using cyclosporine except for autoimmune-related HLH) at the Hematology Oncology Center of Beijing Children's Hospital were selected as the modified group, while 102 children treated with the standard HLH-04 regimen from January 2012 to December 2015 were selected as the control group. The early remission rate, survival rate and adverse reactions of two groups were compared. Rank sum test and chi square test were used for comparison between groups. Results: The age of onset in the modified group was 1.9 (1.1, 3.5) years, with 65 males and 45 females. The age of onset in the control group was 2.0 (1.2, 4.6) years, with 47 males and 55 females. No significant difference was found in age and gender between 2 groups (both P>0.05). Except for fibrinogen (1.3 (1.0, 1.7) vs. 1.1 (0.8, 1.4) g/L, Z=-2.67, P=0.008) and natural killer cell activity (13.9 (13.4, 16.3) % vs.14.9 (12.0, 16.1) %, Z=-2.34, P=0.028), there were no statistically significant differences in etiology, disease duration, first clinical presentation, or laboratory tests between 2 groups (all P>0.05). At 2 months and 3 years, there were no statistically significant differences in overall survival between 2 groups (84.5% (93/110) vs.76.5% (78/102), 78.2% (86/110) vs. 67.6% (69/102), χ2=2.28, 3.07, P=0.131, 0.080). The first 3 weeks were the most common time for bone marrow suppression in the modified group, with a lower incidence than in the control group (47.3% (52/110) vs. 62.7% (64/102), χ2=5.11, P=0.024). The modified group had a lower rate of fungal infections than the control group (3.6% (4/110) vs. 13.7% (14/102), χ2=6.93, P=0.008). Compared with the control group, fewer children in the modified group died as a result of side effects from chemotherapy (8.0% (2/25) vs.30.3% (10/33), χ2=4.31, P=0.038). Conclusion: The BCH modified HLH-04 regimen reduced the intensity of chemotherapy, with overall efficacy no worse than the standard HLH-04 regimen, and significantly reduced the rate of chemotherapy-related myelosuppression, fungal infection and mortality.

[Ruxolitinib combined with liposomal doxorubicin, etoposide, methylprednisolone+/-PEG-asparaginase in treatment of relapsed/refractory pediatric hemophagocytic lymphohistiocytosis].

Objective: To investigate the efficacy and safety of ruxolitinib, liposomal doxorubicin, etoposide, methylprednisolone+/-PEG-asparaginase (RU-DEP+/-L) in the treatment of relapsed/refractory (R/R) pediatric hemophagocytic lymphohistiocytosis (HLH). Methods: The clinical data of R/R pediatric HLH, who accepted the RU-DEP+/-L regimen at Beijing Children's Hospital from January 2018 to December 2019 was retrospectively analyzed. Results: A total of 16 patients were included in this study, including 13 males and 3 females, aged[M(Q1,Q3)] 1 (1, 2) years at diagnosis. Thirteen patients were diagnosed with Epstein-Barr virus (EBV)-HLH, 2 with EBV-induced primary HLH, and 1 with unclear etiology, among which 3 patients were co-infected with CMV. After the first-line treatment, 11 patients had no response, and 5 patients relapsed after complete response. Nine patients received the RU-L-DEP regimen, and 7 patients received the RU-DEP regimen. The overall response rate and complete response of RU-DEP+/-L treatment were 10/16 and 3/16, respectively. The negative conversion rate of plasma EBV-DNA was 7/15. The median follow-up time was 35.1 (2.4, 40.7) months, and 9/16 patients were survival. The 3-year overall survival rate after RU-DEP+/-L treatment in response and accepted hematopoietic stem cell transplantation (HSCT) was higher than that without response and did not receive HSCT (P=0.048). Among the 16 patients, 9 had varying degrees of myelosuppression, and 13 had an infection. Conclusions: RU-DEP+/-L can be used as a salvage treatment in R/R pediatric HLH, which can provide a bridge to HSCT and play an important role in the control of HLH. The main adverse reactions are myelosuppression and infection, which can be tolerated.

[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.

[Anaplastic lymphoma kinase-translocation renal cell carcinoma: clinical and pathological analysis].

Objective: To investigate the clinicopathological features, molecular characteristics, differential diagnosis and prognosis of anaplastic lymphoma kinase (ALK)-translocation renal cell carcinoma. Methods: Two cases of ALK-translocation renal cell carcinoma diagnosed from January 2011 to December 2020 were retrospectively analyzed to characterize their morphological features, immunohistochemical expression and prognosis. Multiple molecular studies including fluorescence in situ hybridization (FISH), reverse transcriptase-polymerase chain reaction (RT-PCR), and next-generation sequencing were performed to characterize the genetic alterations. Results: Two patients included one male and one female, with 59 and 57 years old, respectively. Morphologically, case 1 resembled collecting duct carcinoma or renal medullary carcinoma, which demonstrated tubular, microcapsule and reticular structures, with a remarkable myxoid background and lymphocytes infiltration; case 2 resembled Xp11.2 translocation renal cell carcinoma or type 2 papillary renal cell carcinoma, which demonstrated tubular papillary and focal solid structures, with flocculent cytoplasm and many foamy histiocytes, but without myxoid background and lymphocytes infiltration. Immunohistochemistry showed strongly positive expression of ALK. CK7, E-cadherin, vimentin, PAX8 and CD10 showed various degrees of expression, and other antibodies were nonreactive. A variety of molecular assays showed definite ALK gene translocation, with rare VCL-ALK gene fusion (VCL exon and 16-ALK exon 20) in case 1, and EML4-ALK gene fusion (EML4 exon and 2-ALK exon 20) in case 2. Conclusions: ALK-translocation renal cell carcinoma is rare with various morphological features, and is easy to miss and misdiagnose. The characteristic ALK expression and molecular detection of ALK translocation are helpful for diagnosing this type of renal cell carcinoma.

[Expression of SS18-SSX fusion-specific antibody and SSX C-terminal antibody in synovial sarcoma and its diagnostic value].

Objective: To investigate the expression and diagnostic value of SS18-SSX fusion-specific antibody and SSX C-terminal antibody in synovial sarcoma (SS). Methods: Immunohistochemical (IHC) EnVision method was used to detect the expression of SS18-SSX fusion-specific antibody and SSX C-terminal antibody in 51 genetically confirmed cases of SS and 94 non-SS tumors diagnosed at Nanjing Jinling Hospital from August 2013 to December 2020. Results: IHC staining for SS18-SSX fusion-specific antibody revealed strongly diffuse nuclear staining in 48 of 51 (48/51, 94.1%) SS cases, whereas none of the 94 non-SS tumors showed any staining. IHC staining for SSX C-terminal antibody showed strongly diffuse nuclear staining in all 51 (51/51, 100%) SS cases; six of the 94 (6/94, 6.4%) non-SS tumors showed variable staining, including two cases each of leiomyosarcoma and fibrosarcoma, and one case each of malignant peripheral nerve sheath tumor and embryonal rhabdomyosarcoma. The sensitivity and specificity of SS18-SSX fusion-specific antibody in diagnosing SS were 94.1% and 100% and these of SSX C-terminal antibody were 100% and 93.6%, respectively. Conclusions: SS18-SSX fusion-specific antibody and SSX C-terminal antibody are highly sensitive and specific markers for SS. Immunohistochemistry using these antibodies may replace FISH or molecular genetic testing in most cases.

[Association between genotype and clinical phenotype in children with primary hemophagocytic lymphohistiocytosis].

Objective: To investigate the association between genotype and clinical phenotype in children with primary hemophagocytic lymphohistiocytosis (HLH). Methods: Clinical data of 38 children with primary HLH at Beijing Children's Hospital from November 2015 to October 2020 were analyzed retrospectively. According to whether destructive mutation site, familial HLH (FHL) or non-familial HLH, degranulation pathway and non-degranulation pathway were detected in them, these patients were divided into different groups. Clinical characteristics, laboratory tests and functional tests were analyzed in different groups. Wilcoxon test or chi-square test were used for comparison between groups. Results: Among 38 cases, there were 23 males and 15 females. The age of onset was 2.5 (0.1-13.7) years. PRF1 (13/38, 34%) and UNC13D (12/38, 32%) gene mutations had the highest detectable rate. Correlation analysis between genotypes and phenotypes suggested that patients in destructive mutation sites group (n=25) had a younger age of onset than that in non-destructive mutation sites group (n=13) (1.0 (0.1-9.0) vs. 4.0 (0.4-13.4) years, Z=-2.711, P=0.005). The incidence of central nervous system involvement and convulsion was higher in patients in familial HLH group (n=26) than that in non-familial HLH group (n=12)(62% (16/26) vs. 2/12, χ²=6.631, P=0.015; 54% (14/26) vs. 2/12, χ²=4.656, P=0.040). The levels of soluble CD25, bilirubin, interferon γ (IFN-γ) and interleukin 10 (IL-10) were higher in degranulation pathway impairment group than that in the non-degranulation pathway impairment group (38 444 (2 393-44 000) vs.15 304 (1 620-36 937) ng/L,Z=2.634,P=0.008; 23.5 (6.3-126.4) vs. 6.0 (3.6-31.0) µmol/L, Z=2.992, P=0.003; 20.7 (0-248.7) vs. 11.9 (2.6-21.0) ng/L, Z=2.156, P=0.031; 20.7 (4.3-2 500.0) vs. 11.8 (4.0-88.0) ng/L, Z=2.210, P=0.027). However, the levels of neutrophils, ferritin and lactate dehydrogenase (LDH) in degranulation pathway impairment group were lower in the non-degranulation pathway impairment group (0.5 (0.1-8.0)×109 vs. 1.0 (0.9-2.3)×109/L, Z=-3.197, P=0.001; 1 133 (78-10 452) vs. 3 048 (630-37 900) µg/L, Z=-2.407, P=0.016; 410 (188-1 254) vs. 599 (389-3 147) U/L, Z=-2.489, P=0.013). Conclusions: PRF1 and UNC13D gene mutations are most common in primary HLH. Patients with destructive gene mutations have a younger age of onset. Patients with familial HLH are more likely to have central nervous system involvement and convulsions. The levels of sCD25, bilirubin, IFN-γ and IL-10 are higher in the degranulation pathway patients.

MiR-340-5p mitigates the proliferation and activation of fibroblast in lung fibrosis by targeting TGF-ß/p38/ATF1 signaling pathway.

OBJECTIVE: Idiopathic pulmonary fibrosis (IPF) is associated with the occurrence and progress of the proliferation and activation of lung fibroblast. Recent studies have shown that microRNA-340-5p (miR-340-5p), a member of miRNA family, has modulated the skin fibroblast proliferation in scar formation disease. However, it is elusive whether miR-340-5p exert a pulmonary anti-fibrotic effect on IPF by moderating fibroblast bioactivity. The present study aimed to investigate the role of Adam10 in lung fibroblast regulation. MATERIALS AND METHODS: Human lung fibroblasts were carried out Transforming Growth factor-ß (TGF-ß) to stimulate proliferation and activation. MiR-340-5p mimics or inhibitor loaded in pcDNA3.1 aimed at overexpression or inhibition were respectively delivered to lung fibroblast using Lipofectamine 2000 for transfection. Then, siRNA-ATF1 (Activating transcription factor 1) was utilized to knockdown ATF1 expression in lung fibroblast after miR-340-5p overexpression and probed the role of ATF1 in lung fibroblast. Western blotting, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), Dual-Luciferase reporter system, Cell Counting Kit-8 (CCK-8) assay, scratch assay, and immunofluorescence were conducted to measure the alteration of miR-340-5p, ATF1, and fibrosis-relative level. RESULTS: We find that the expression of miR-340-5p markedly increases or decreases in fibroblast after mimics or inhibitor transfection respectively. Moreover, we demonstrate that the overexpression of miR-340-5p reduces the expression of ATF1 to prevent fibroblast activation and proliferation by targeting ATF1 and restrain MAPK/p38 pathway following TGF-ß stimuli. CONCLUSIONS: The above proved that the increased miR-340-5p ameliorates the activation and proliferation of lung fibroblast in fibrosis process via targeting ATF1 and MAPK/p38 pathway. Our research provides novel insight on the miRNA modulation of process of TGF-ß stimuli in lung fibroblast and verifies a potential target for the therapy of lung fibrosis in the future.

[Analysis of five cases of hepatitis associated aplastic anemia presenting with hemophagocytic lymphohistiocytosis at onset].

Objective: To discuss the clinical characteristics and management approaches to hepatitis associated aplastic anemia (HAAA) presenting as hemophagocytic lymphohistiocytosis (HLH) at onset. Methods: The clinical data and laboratory results of hospitalized 5 HAAA patients presenting as HLH at onset in Beijing Children's Hospital from January 2017 to May 2019 were analyzed retrospectively. Results: Among 5 cases, there were 4 males and 1 female. The age of onset was 6.0 (2.7-12.7) years. All patients presented with high fever, hepatomegaly, hepatic dysfunction (aspartate aminotransferase 1 716 (1 409-2 570) U/L, alanine aminotransferase 1 699 (937-2 540) U/L) at onset. After admission, the laboratory results showed pancytopenia (white blood cell 1.2 (0.6-6.7) ×10(9)/L, haemoglobin 94 (65-111) g/L, blood platelet 29 (10-41) ×10(9)/L), decreased fibrinogen (1.3 (1.1-2.5) g/L), significantly elevated triglyceride (4.0 (2.8-5.1) mmol/L), ferritin (1 766 (399-5 253) µg/L) and soluble CD25 (27 457 (9 625-44 000) ng/L). Hemophagocytosis was found in the bone marrow smears of all 5 patients. The diagnosis of acute hepatitis and HLH was confirmed. During the treatment of HLH, the blood cells remain below normal level and the further biopsy of bone marrow (iliac bone) indicated low myeloproliferation. After exclusion of congenital bone marrow failure syndromes and other pancytopenic diseases, HAAA was confirmed. After the diagnosis of HAAA, 1 patient received antithymocyte globulin (ATG) and cyclosporin treatment in our hospital, 1 patient received allogeneic stem cell transplantation (HSCT) in other hospital, 2 patients received ATG in other hospitals. Only 1 patient died of severe infection. Conclusions: HAAA can present as HLH at onset. It is mainly manifested by high fever, acute severe hepatitis, pancytopenia, elevated ferritin and hemophagocytosis in the bone marrow. The diagnosis of HAAA should be considered whenever cytopenia could not completely corrected while apparent improvement of HLH and hepatitis related complications were improved after immunosuppressive therapy. ATG or HSCT treatment should be performed as soon as the diagnosis of severe or transfusion dependent aplastic anemia is confirmed.

[Expression of H3.3 G34W mutant-specific antibody in giant cell tumors of bone and its diagnostic value].

Objective: To investigate the expression of H3.3 G34W mutant-specific antibody in giant cell tumors of bone (GCTB), and its value in the diagnosis of GCTB. Methods: Immunohistochemical (IHC) EnVision method was used to detect the expression of H3.3 G34W mutant-specific antibody and p63 in 83 GCTBs, 18 aneurysmal bone cysts, 23 chondroblastomas and 28 osteosarcomas diagnosed at Nanjing Jinling Hospital from June 2001 to April 2019. Results: Among the 83 cases of GCTB, 69 cases (69/83, 83.1%) expressed H3.3 G34W. H3.3 G34W expression was found exclusively in the mononuclear cell population with strong and diffuse nuclear staining. H3.3 G34W was expressed in 55 of 57 (96.5%) cases of GCTB in long bones, but only 14 of 26 (53.8%) cases of non-long bone GCTB. All recurrent (9/9)/metastatic GCTB (2/2), post-denosumab GCTB (3/3), primary malignant GCTB (3/3) and secondary malignant GCTB (5/5) also expressed H3.3 G34W. H3.3 G34W was negative in all aneurysmal bone cysts and chondroblastomas. H3.3 G34W was positive in 3 of 28(10.7%) cases of osteosarcomas, and giant cell-rich osteosarcoma(GCRO) was the only histological subtype of osteosarcoma that expressed H3.3 G34W. p63 was expressed in 71.1%(59/83) of GCTB, while the positive rates of p63 in aneurysmal bone cysts,chondroblastomas and osteosarcomas were 3/18, 43.5% (10/23) and 21.4% (6/28) respectively. The sensitivity and specificity of H3.3 G34W mutant-specific antibody in the diagnosis of GCTB were 83.1% and 95.7%. Conclusions: H3.3 G34W mutant-specific antibody is a highly sensitive and specific marker for GCTB and helpful for the diagnosis of GCTB and its variants. The limitation of this antibody is that as a mall number of GCTB harbor G34 mutation other than G34W, and thus that cannot be detected. The incidental expression of H3.3 G34W mutant protein in osteosarcoma could be a potential diagnostic dilemma, and the results of H3.3 G34W IHC staining needs careful interpretation.

[Eosinophilic solid and cystic renal cell carcinoma: clinicopathological analysis and molecular characterization].

Objective: To study the clinicopathological features, immunohistochemical phenotype, molecular changes, differential diagnosis and prognosis of eosinophilic solid and cystic renal cell carcinoma (ESC RCC). Methods: A total of 15 cases were selected from 2005 to 2019 at Nanjing Jinling Hospital,Nanjing University School of Medicine for clinicopathological and immunohistochemical analysis, 10 of which were subject to cancer-associated mutation analysis using targeted next-generation sequencing (NGS) panel. A literature review was also performed. Results: The patients' ages ranged from 15 to 68 years (mean, 33 years). The male-to-female ratio was 1.1∶1.0. During a mean follow-up of 22 months, none of the patients developed tumor recurrence, progression or metastasis. Histologically, the tumors typically demonstrated solid and cystic architectures and the neoplastic cells contained voluminous eosinophilic cytoplasm with prominent granular cytoplasmic stippling. Immunohistochemically, tumor cells in all cases were immunoreactive for CK20. Signal pathway related protein mTOR and S6 were positive in 14/15 and 6/15 cases, respectively. Cathepsin K, Melan A and HMB45 were at least focally positive in 12/15, 6/15 and 2/15 cases, respectively. CK7 and CD10 showed focal immunostain positivity in some cases, while TFE3, TFEB, CA9 and CD117 were negative in all cases. NGS demonstrated TSC1/TSC2 mutations in all tested cases (10/10). Conclusions: ESC RCC is a rare tumor that tends to occur in young patients with an indolent behavior. Diagnosis can be established by its distinct clinical and histopathologic findings, immunohistochemical phenotype and molecular genetics. The tumor may be considered as a new subtype of RCC.