Background and purpose:For patients with human epidermal growth factor receptor 2(HER2)-positive metastatic breast cancer,trastuzumab treatment can prolong the overall survival and significantly improve the prognosis of patients.However,the reference original research trastuzumab(Herceptin?)is more expensive.Biosimilars have comparable efficacy and safety profiles while increasing patient access to treatment.This clinical trial aimed to evaluate the efficacy,pharmacokinetics,safety and immunogenicity of the trastuzumab biosimilar AK-HER2 compared to trastuzumab(Herceptin?)in patients with HER2-positive metastatic breast cancer.Methods:This multi-center,randomised,double-blind phase Ⅲ clinical trial was conducted in 43 subcenters in China.This study complied with the research protocol,the ethical principles stated in the Declaration of Helsinki and the quality management standards for drug clinical trials.It was approved by the hospital's medical ethics committee.The clinical trial registration agency is the State Food and Drug Administration(clinical trial approval number:2015L04224;clinical trial registration number:CTR20170516).Written informed consent was obtained from subjects before enrollment.Enrolled patients were randomly assigned to the AK-HER2 group and the control group,respectively receiving AK-HER2 or trastuzumab(initial loading dose 8 mg/kg,maintenance dose 6 mg/kg,every 3 weeks as a treatment cycle,total treatment time is 16 cycles)in combination with docetaxel(75 mg/m2,treatment duration is at least 9 cycles).The primary endpoint of this clinical trial was the objective response rate(ORR9)between the AK-HER2 group and the control group in the 9th cycle.Secondary efficacy endpoints included ORR16,disease control rate(DCR),clinical benefit rate(CBR),progression-free survival(PFS)and 1-year survival rate.In this study,100 subjects(AK-HER2 group to control group=1:1)were randomly selected for blood sample collection after the 6th cycle of medication,The collection time points were 45 minutes after infusion(the end of administration),4,8,24,72,120,168,336,and 504 hours after the end of administration.After collection,blood samples were analyzed by PK parameter set(PKPS).Other evaluation parameters included safety and immunogenicity assessment.Results:A total of 550 patients with HER2-positive metastatic breast cancer were enrolled in this clinical trial between Sep.2017 and Mar.2021.In the AK-HER2 group(n=237),129 subjects in the experimental group achieved complete response(CR)or partial response(PR),and the ORR9 was 54.4%.There were 134 subjects in the control group(n=241)who achieved CR or PR,and the ORR9 was 55.6%.The ORR9 ratio between the AK-HER2 group and the control group was 97.9%[90%confidence interval(CI):85.4%-112.2%,P=0.784],which was not statistically significant.In all secondary efficacy endpoints,no statistically significant differences were observed between the two groups.We conducted a mean ratio analysis of pharmacokinetics(PK)parameters between the AK-HER2 group and the control group,and the results suggested that the pharmacokinetic characteristics of the two drugs are similar.The incidence of treatment emergent adverse event(TEAE)leading to drug reduction or suspension during trastuzumab treatment was 3.6%(10 cases)in the AK-HER2 group and 8.1%(22 cases)in the control group.There was statistically significant difference between the two groups(P=0.027).The incidence rate was significantly lower in the AK-HER2 group than in the control group,and there was no statistically significant difference among the other groups.The differences in the positive rates of anti-drug antibodies(ADA)and neutralizing antibodies(NAB)between groups were of no statistical significance(P=0.385 and P=0.752).Conclusion:In patients with HER2-positive metastatic breast cancer,AK-HER2 was comparable to the trastuzumab(Herceptin?)in terms of drug efficacy,pharmacokinetics,safety and immunogenicity.

Objective To investigate the influence of peripheral blood neutrophil-to-lymphocyte ratio (NLR),monocyte-to-lymphocyte ratio (MLR) and platelet-to-lymphocyte ratio (PLR) on the prognosis in the patients with multiple myeloma (MM).Methods A total of 159 newly diagnosed MM admitted and treated in the Affiliated Hospital of Guizhou Medical University from January 2019 to May 2023 were selected as the study subjects.The general clinical data,blood biochemical and marrow routine detection results before the in-itial treatment were collected.NLR,MLR and PLR were calculated.The univariate and multivariate Cox-re-gression model was adopted to analyze the influencing factors.The receiver operating characteristic (ROC) curve was used to analyze the predictive value.The Kaplan-Meier survival curve and Log-Rank test were used to conduct the survival analysis.Results The ROC curve showed that the critical values of NLR,MLR and PLR were 2.682,0.317 and 147.786 respectively.The patients were divided into the high/low NLR groups (n=61,n=98),high/low MLR group (n=76,n=83) and high/low PLR groups (n=59,n=100).The pro-portions of blood calcium<2.5 mmol/L and creatinine<177 μmmol/L in the low NLR group in the low NLR group were higher compared with the high NLR group (P<0.05);the blood calcium,creatinine and DS stage had statistical differences between the low MLR group and high MLR group (P<0.05);blood calcium had statistical difference between the low PLR group and high PLR group (P<0.05).After 3 treatment courses,the complete remission rate in the high NLR group,high MLR group and high PLR group was significantly lower than that in the corresponding low group (P<0.05).The multivariate Cox-regression analysis results showed that hemoglobin<100 g/L and high PLR were the independent risk factors affecting the progress free survival (PFS) stage in the patients with MM (P<0.05).The age>60 years old was the independent risk factors affecting the overall survival (OS) in the patients with MM (P<0.05).Conclusion NLR,MLR and PLR could serve as the assisted tool to evaluate the prognosis in the patients with MM.

[摘 要] 目的：构建靶向CD70分子的重组免疫毒素，通过表达、纯化制备PE38与抗CD70纳米抗体重组蛋白，体外抗肿瘤实验探究重组蛋白是否对高表达CD70分子的阳性肿瘤细胞具有杀伤活性。方法：通过基因工程手段，将CD70纳米抗体Nb 2B3基因片段通过一个连接子与pET21a-PE38基因片段相连，获得重组表达载体pET21a-Nb 2B3-PE38并转入BL21（DE3）感受态细胞中进行表达、纯化与鉴定。用间接ELISA及FACS法检测Nb 2B3-PE38与CD70分子的结合活性，MTT法检测Nb 2B3-PE38对高表达CD70分子的肾透明细胞癌786-O细胞的体外杀伤活性，Annexin Ⅴ-FITC/PI双染法检测Nb 2B3-PE38对786-O细胞凋亡的影响。结果：成功构建抗CD70纳米抗体重组免疫毒素Nb 2B3-PE38，纯化获得纯度>90%的重组蛋白，SDS-PAGE及WB检测结果表明目的蛋白正确表达，分子量为56 000。纯化后的Nb 2B3-PE38能与重组CD70抗原及786-O细胞表面的CD70分子特异性结合；25 µg/mL Nb 2B3-PE38即对786-O细胞产生极显著的杀伤作用（P<0.001），并且促进786-O细胞的细胞凋亡（P<0.01），其杀伤效应强于阳性对照顺铂（P<0.01）。结论：成功制备了特异性靶向CD70分子的免疫毒素Nb 2B3-PE38，其能够有效杀伤786-O细胞并诱导细胞凋亡且效果强于顺铂。

Objective:To determine the risk factors of drainage time longer than 1 day in patients with selective abdominal drainage after laparoscopic cholecystectomy.Methods:The clinical data related to patients with selective abdominal drainage undergoing laparoscopic cholecystectomy from November 2009 to November 2019 at Chinese PLA General Hospital were retrospectively analyzed. Of 233 patients enrolled into this study, there were 147 males and 86 females, with a median aged 59.0 (47.5, 65.5) years old. The patients were divided into drainage time 1 day group of 65 patients and longer than 1 day group of 168 patients according to postoperative drainage time. The baseline data and perioperative data were collected, the risk factors correlated with drainage time longer than 1 day were analyzed.Results:The drainage time was 1 in the 1 day group and 2~8 in another group. Among the 233 patients, there was one with biliary leakage and 14 patients had abdominal bleeding, all of them healed after 2~3 days. All of the 233 patients were recovered when discharged. Independent risk factors related to drainage time longer than 1 day include BMI≥28 kg/m 2 ( OR=3.443, 95% CI: 1.411-8.405, P=0.007), operation time ≥65 min ( OR=2.570, 95% CI: 1.310-5.045, P=0.006), thickness of gallbladder wall ≥0.5 cm ( OR=12.720, 95% CI: 1.350-5.478, P=0.005), postoperative stomachache ( OR=13.537, 95% CI: 1.685-108.748, P=0.014) and postoperative fever ( OR=8.156, 95% CI: 1.035-64.249, P=0.046). Conclusion:For patients undergoing selective abdominal drainage after laparoscopic cholecystectomy with BMI ≥28 kg/m 2, operation time ≥65 min, gallbladder wall thickness ≥0.5 cm, postoperative abdominal pain and fever, clinicians should appropriately prolong the drainage time to ensure medical safety.

Objective:To investigate the kinetic metrics of 68Ga-fibroblast activation protein inhibitor (FAPI)-04 in pancreatic cancers and normal organs by using total-body PET dynamic imaging. Methods:From December 2020 to December 2021, 68Ga-FAPI-04 total-body PET/CT dynamic imaging were performed on 6 pancreatic cancer patients (3 males, 3 females, median age 55.5 years) in Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University. Images were respectively analyzed. Manual delineations of volume of interests (VOIs) on multiple normal organs and pathological lesions were performed and time-to-activity curves (TACs) were generated. A reversible two-tissue compartment model (2TCM) was fitted for each tissue TAC. Rate constants including K1, k2, k3 and k4, and the total volume of distribution ( Vt) were obtained and compared by tissue types. Wilcoxon rank sum test and Spearman correlation analysis were used for data analysis. Results:Kinetic metrics varied significantly among normal organs and pancreatic cancer lesions ( z values: 2.00-1 240.00, all P<0.05). The highest K1 among lesions was observed in primary tumor (0.30 min -1), which was observed in the spleen (1.42 min -1) among normal organs. The highest k2 among lesions was observed in peritoneal metastases (0.24 min -1), which was observed in the spleen (2.59 min -1) among normal organs. Primary tumor showed the highest k3 of 0.17 min -1 among lesions, and the pancreas had the highest k3 of 0.16 min -1 among normal organs. Primary tumor had the highest k4 of 0.03 min -1 among lesions, and the heart, lungs, parotid glands had high k4(0.06 min -1) among normal organs. Vt were higher in pathological lesions compared to normal organs, with the highest in primary tumor (13.78 ml/cm 3). There were correlations between Vt in lesions and SUV mean( rs=0.86, P<0.001) or SUV max ( rs=0.77, P<0.001). Conclusion:The rate constants including K1, k2, k3 and k4, and Vt of 68Ga-FAPI-04 vary among normal organs and lesions.

Objective:To established a method for the detection of soluble programmed death ligand 1 (PD-L1) protein in serum based on the poly nanoantibody of lumazine synthase(LS).Methods:A dual nanobody-based sandwich ELISA was established with a competitive ELISA to screen nanobodies recognizing different epitopes of PD-L1 as paired antibodies. To improve sensitivity, PD-L1 nanobody P3C8 and lumazine synthase(LS) were fused, and nanobodies were obtained in polymeric forms as sPD-L1 protein captures, so as to develop an LS-displayed polymeric nanobody-based sandwich ELISA (LSNbs-ELISA) method to detect sPD-L1.Results:Compared with the Nbs-ELISA method, the LSNbs-ELISA method is approximately 11-fold more sensitive for sPD-L1 detection. The limit of detections (LODs) of Nbs-ELISA and LSNbs-ELISA for sPD-L1 in serum were 2.87 ng/ml and 0.255 ng/ml, respectively. Both assays were highly specific for the detection of sPD-L1 and did not react with structure-related proteins PD-1, CD27, CD70, CD137, and CD147 when spiked into the human serum.Conclusions:The Nbs-ELISA and LSNbs-ELISA assays both have high sensitivity and specificity for detecting sPD-L1 in serum and could have potential clinical applications.

【Objective】 To investigate the effects of RASSF6 gene on gastric cancer cells’ proliferation， autophagy， apoptosis， and sensitivity to oxaliplatin chemotherapy. 【Methods】 Gastric cancer BGC823 cells were cultured in vitro and divided into experimental control group （control group）， RASSF6 overexpression group （Oe group）， RASSF6 interference group， and lentivirus control group according to the expression effect of lentivirus gene. The changes in cell proliferation， cell cycle distribution， cell migration， autophagy， apoptosis and sensitivity to oxaliplatin in each group were detected， and the number of autophagy bodies in each group was detected by electron microscopy. Real-time PCR （qRT-PCR） and Western blotting were used to detect the expression levels of apoptosis- and autophagy-related genes in each group. 【Results】 Studies on the biological behavior of gastric cancer BGC823 cells induced by RASSF6 gene expression showed that compared with the control group， the percentage of G0/G1 phase cells in the Oe group increased， while the percentage of G2 and S phase cells decreased， with statistical significance （P<0.05）. The apoptosis rate was significantly increased （P<0.05）. The cell scratch assay showed that the scratch healing rate was significantly decreased （P<0.05）. Studies on the sensitivity of RASSF6 gene expression to oxaliplatin showed that compared with the drug group （L-OHP group）， the apoptosis rate of Oe+L-OHP group was increased significantly （P<0.05）. In the Oe+L-OHP group， the expression of anti-apoptotic protein Bcl-2 decreased， the expressions of Bax and Caspase-3 were increased； the expression of autophagosomes was increased； the expressions of Beclin-1 and P62 and the ratio of LC3-Ⅱ/LC3 were all increased （P<0.05）. 【Conclusion】 The RASSF6 gene plays a role in suppressing gastric cancer cell BGC823， which can increase the sensitivity to oxaliplatin chemotherapy by promoting apoptosis and autophagy.

Objective:To study the clinical manifestations, pathologic features, diagnosis and differential diagnosis, treatment and prognosis of lymphoplasmacytic lymphoma/Waldenstr?m′s macroglobulinemia (LPL/WM).Methods:Twenty-seven cases of LPL from January 2016 to December 2020 at Guangdong Provincial People′s Hospital were collected. The clinical data, histomorphology, immunophenotype, MYD88 L265P mutation, treatment and prognosis were analyzed retrospectively.Results:There were 19 males and 8 female patients, with median age of 63 years. The most common initial symptoms were fatigue related to anemia. Bone marrow was involved in all cases, lymphadenopathy was seen in 11 cases and splenomegaly in 10 cases. Monoclonal IgM type protein was detected in 25 cases, meeting the diagnostic criteria of WM. Microscopically, bone marrow and lymph nodes were infiltrated by small lymphocytes, plasmacytoid lymphocytes or plasma cells. The cells expressed pan B-cell markers and showed immunoglobulin light chain restriction. There was no expression of CD5, and low expression of CD23 and CD10; Ki-67 index was usually low. The positive rate of MYD88 L265P mutation was 73.9% (17/23). Most of the patients were treated with rituximab combined with alkylating agents, nucleoside analogues or immunomodulators, and the few patients with relapse or progression were treated with Ibutinib. During the 3-168 months′ follow-up period, recurrence or progression were seen in nine cases. Thrombocytopenia, elevated β2-microglobulin and high-risk group were associated with recurrence or progression of the disease ( P<0.05). The overall survival (OS) and progression-free survival (PFS) of the high-risk patients were significantly lower than those of the low-medium risk patients ( P<0.05). Conclusions:LPL/WM is an exclusive diagnosis; the detection of MYD88 L265P mutation has high diagnostic value, but it is not specific. These cases should be assessed comprehensively for their clinical manifestation, serum IgM protein level and immunophenotype. The overall prognosis of LPL/WM is good, but there are still a small number of high-risk patients with rapid progress, and so the symptomatic patients should be diagnosed accurately and treated in a timely manner.

Objective:To investigate the clinicopathological features, treatment and prognosis of fibrin-associated diffuse large B cell lymphoma (DLBCL) arising within concurrent atrial myxoma.Methods:Six cases of fibrin-associated DLBCL arising within concurrent atrial myxoma diagnosed at the Department of Pathology, Guangdong General Hospital, from 2006 to 2019 were included. The histology, immunophenotype, treatment and prognoses were analyzed.Results:The patients′ age ranged from 46 to 78 years (mean 59 years). There were 3 males and 3 females. The tumors were all discovered incidentally on histological examination of surgical pathology specimens excised for atrial myxoma. All patients appeared to have morphological features of DLBCL, B lineage immunophenotype, high proliferative index and latency type III of Epstein-Barr viral infection. They had complete tumor resections without adjuvant chemotherapy and were healthy at 5- to 120-month follow-ups.Conclusions:Fibrin-associated DLBCL arising within concurrent atrial myxoma is an unusual form of DLBCL associated with chronic inflammation, and its clinical outcome is indolent. The findings suggest that this type of lymphoma does not warrant excessive or unnecessary treatments after complete resection.

Objective:To investigate the optimal experimental conditions (including antigen retrieval time, antibody titers and antibody incubation time) for reliable detection of programmed death-ligand 1 (PD-L1) expression using PD-L1 (22C3) antibody concentrate, and to establish a laboratory developed test for PD-L1 detection.Methods:Using Dako PD-L1 IHC 22C3 pharmDX staining procedure and scoring guidelines as the standard reference (group A), the PD-L1 expression in 25 tissue specimens (including 15 lung cancer tissues, 5 tonsil tissues and 5 placenta tissues) was detected with Flex+/HRP detection kit (EnVision) under 8 different experimental conditions (groups B1 to B8). The staining results were then compared to those in group A.Results:In group B1, 3 tissue samples showed the percentages of PD-L1 positive tumor cells were similar to those in group A, while the percentages of PD-L1 positive tumor cells were lower than those in group A in the other samples. In group B7, two case showed a positive rate higher than that in group A that was also above the positive cut-off value, and the rest of the samples had a percentage of PD-L1 positive tumor cells slightly higher than that in group A, but still below the positive cut-off value. The staining results of group B8 were the closest to those of group A compared with the other groups. Although the percentages of PD-L1 positive tumor cells in the B2 to B6 groups were decreased in various degrees as compared with group A, they were still concordant with group A′s classification (positive vs. negative) and would not change the choice of clinical treatments.Conclusions:The experimental conditions are associated with detection rate of PD-L1 expression using 22C3 antibody. In the present study, the most-suitable alterative conditions in the PD-L1 detection using 22C3 antibody concentrate are those applied in the group B8 (including antigen retrieval in Dako PT Link tank at 97 ℃, pH 6.0 for 40 min and incubation with 22C3 antibodies (1∶100 dilution) at room temperature for 60 min, incubation with EnVision Flex+Linker at room temperature for 30 min, incubation with EnVision/HRP at room temperature for 30 min and DAB staining for 5 min), which could provide reliable results at minimum costs.