Analysis of risk factors for sentinel lymph node metastasis in patients with endometrial cancer.

OBJECTIVE: To investigate the risk factors of sentinel lymph node (SLN) metastasis in patients with endometrial carcinoma (EC), and to establish a risk nomogram model. METHODS: In this retrospective study, the clinical data of 79 EC patients who were treated in Zhumadian Central Hospital from January 2019 to January 2021 were analyzed. The patients were divided into SLN positive group and SLN negative group according to the occurrence of SLN metastasis. Univariate and multivariate analyses were performed to explore the factors affecting the occurrence of SLN metastasis in EC patients. The nomogram model predicting the risk of SLN metastasis in EC patients was constructed. The discrimination, accuracy and clinical benefit rate of the model were evaluated. RESULTS: Multivariate analysis showed that body mass index (BMI) ≥ 24 kg/m2, tumor diameter ≥ 2 cm, low differentiation, and cervical stromal involvement were risk factors for SLN metastasis in EC patients (P < 0.05). And the risk of SLN in EC patients increased with the increase in human epididymis protein 4 (HE4) level (P < 0.05). The constructed nomogram model was tested, and the area under the curve (AUC) of the model was 0.934 (95% CI: 0.878-0.979), the calibration curve obtained a Brier of 0.084. Decision curve results showed that 68 out of every 100 EC patients could benefit without compromising the interests of others, with a benefit rate of 68%. CONCLUSION: The occurrence of SLN in EC patients is related to their personal general characteristics, pathological characteristics, tumor markers, and other multi-dimensional indicators. The medical staff can evaluate the SLN risk of EC patients by combining multiple indicators.

Germline cytotoxic lymphocytes defective mutations in Chinese patients with lymphoma.

Certain patients with lymphoma may harbor mutations in perforin 1 (PRF1), unc-13 homolog D (UNC13D), syntaxin 11 (STX11), STXBP2 (syntaxin binding protein 2) or SH2 domain containing 1A (SH2D1A), which causes functional defects of cytotoxic lymphocytes. Data regarding the association between genetic defects and the development of lymphoma in Chinese patients are limited to date. In the present study, 90 patients with lymphoma were analyzed for UNC13D, PRF1, STXBP2, STX11, SH2D1A and X-linked inhibitor of apoptosis. Mutations were observed in 24 (26.67%) patients; 16 patients exhibited mutations in UNC13D, 7 exhibited PRF1 mutations, and 1 exhibited monoallelic mutation in STX11. UNC13D c.2588G>A/p.G863D mutation was detected in 9 patients (10.00%) and in 4/210 controls (1.90%). This mutation was predicted to be pathogenic and it predominantly existed in the Chinese population. These findings suggest that impaired cytotoxic machinery may represent a predisposing factor for the development of lymphoma. Furthermore, these data describe a distinct mutation spectrum in Chinese patients with lymphoma, whereby UNC13D is the most frequently mutated gene. In addition, these findings suggest UNC13D c.2588G>A mutation is a founder mutation in Chinese patients.

Doxorubicin-enriched, ALDH(br) mouse breast cancer stem cells are treatable to oncolytic herpes simplex virus type 1.

BACKGROUND: The primary objective of this study was to test whether oncolytic herpes simplex virus type 1 (HSV1) could eradicate chemoresistant cancer stem cells (CSCs). METHODS: The fluorescent aldefluor reagent-based technique was used to identify and isolate ALDH(br) cells as CSCs from the 4T1 murine breast cancer cell line. The presence of ALDH(br) 4T1 cells was also examined in 4T1 breast cancer transplanted in immune-competent syngeneic mice. RESULTS: Compared with ALDH(lo) cells, ALDH(br) cells had a markedly higher ability to form tumor spheres in vitro and a higher tumorigenic potential in vivo. ALDH(br) cells also exhibited increased doxorubicin resistance in vitro, which correlated with a selective increase in the percentage of ALDH(br) cells after doxorubicin treatment and an increased expression of P-glycoprotein (P-gp), a known chemoresistance factor. In contrast, oncolytic HSV1 was able to kill ALDH(br) cells in vitro and even more markedly in vivo. Furthermore, in in vivo studies, systemic administration of doxorubicin followed by intratumoral injection of oncolytic HSV1 resulted in much more significant suppression of tumor growth with increased median survival period compared with each treatment given alone (p<0.05). Though more CD8(+) T lymphocytes were induced by oncolytic HSV1, no significant specific T cell response against CSCs was detected in vivo. CONCLUSIONS: These results suggested that the use of oncolytic HSV1 following doxorubicin treatment may help eradicate residual chemoresistant CSCs in vivo.

[Construction of a new oncolytic virus oHSV2hGM-CSF and its anti-tumor effects].

OBJECTIVE: The aim of this study was to construct a new oncolytic virus oHSV2hGM-CSF and evaluate its oncolytic activity in vitro and in vivo in parallel with oHSV1hGM-CSF. METHODS: oHSV2hGM-CSF was a replication-competent, attenuated HSV2 based on the HG52 virus (an HSV2 strain). It was engineered to be specific for cancer by deletion of the viral genes ICP34.5 and ICP47 and insertion of the gene encoding hGM-CSF. To measure the in vitro killing effect of the virus, 15 human tumor cell lines (HeLa, Eca-109, PG, HepG2, SK/FU, CNE-2Z, PC-3, SK-OV3, A-549, 786-0, MCF-7, Hep-2, HT-29, SK-Mel-28, U87-MG) and mouse melanoma (B16R) cell line were seeded into 24-well plates and infected with viruses at MOI = 1 (multiplicity of infection, MOI), or left uninfected. The cells were harvested 24 and 48 hours post infection, and observed under the microscope. For animal studies, the oncolytic viruses were administered intratumorally (at 3-day interval) at a dose of 2.3 x 10(6) PFU (plaque forming unit, PFU) for three times when the tumor volume reached 7-8 mm3. The tumor volume was measured at 3-day intervals and animal survival was recorded. RESULTS: Both oHSV2hCM-CSFand oHSV1hGM-CSF induced widespread cytopathic effects at 24 h after infection. OHSV2hGM-CSF, by contrast, produced more plaques with a syncytial phenotype than oHSV1hGM-CSF. In the in vitro killing experiments for the cell lines HeLa, HepG2, SK-Mel-28, B16R and U87-MG, oHSV2hGM-CSF eradicated significantly more cells than oHSV1hGM-CSF under the same conditions. For the mouse experiments, it was observed that oHSV2hGM-CSF significantly inhibited the tumor growth. At 15 days after B16R tumor cells inoculation, the tumor volumes of the PBS, oHSV1hGCM-CSF and oHSV2hGM-CSF groups were (374.7 +/- 128.24) mm3, (128.23 +/- 45.32) mm3 (P < 0.05, vs. PBS group) or (10.06 +/- 5.1) mm3 (P < 0.01, vs. PBS group), respectively (mean +/- error). The long term therapeutic effect of oHSV2hGM-CSF on the B16R animal model was evaluated by recording animal survival over 110 days after tumor cells inoculation whereas all the mice in the PBS group died by day 22 (P < 0.01). The anti-tumor mechanism of the newly constructed oHSV2hGM-CSF against B16R cell tumor appeared to include the directly oncolytic activity and the induction of anti-tumor immunity to some degree. CONCLUSION: The findings of our study demonstrate that the newly constructed oHSV2hGM-CSF has potent anti-tumor activity in vitro to many tumor cell lines and in vive to the transplanted B16R tumor models.

[Generation of a herpes simplex virus-permissive mouse melanoma cell line B16RHSV].

OBJECTIVE: To generate an oncolytic herpes simplex virus (oHSV) permissive mouse melanoma cell line B16RHSV, preserving the tumorigenic ability in syngeneic mice. METHODS: The herpes simplex virus entry mediator (HVEM) gene was amplified by PCR from human melanoma cell line A375, and cloned into pGEM-T Easy vector for sequencing. The HVEM gene was then cloned into pcDNA3 vector to generate pcDNA3-HVEM for transfection of mouse melanoma cell line B16-F10 cells. After that, the putative transfected cells were selected in full growth medium containing G418. The HVEM-expressing cells were isolated by immunomagnetic bead separation. The mouse melanoma cell line expressing oHSV receptor-HVEM, designated as B16RHSV, was generated. The permissibility of B16RHSV cells to oHSV infection was examined with green fluorescence protein (GFP)-expressing oHSV (oHSVGFP). To investigate the tumorigenic ability of both cells in vivo, 2×10(5) cells in 100 µl were subcutaneously inoculated into the right flanks of C57/BL mice. RESULTS: In vitro, the B16RHSV mouse melanoma cells were shown by fluorescence microscopy capable of being infected by oHSVGFP. In vivo, the B16RHSV cells, like their wild type counterpart, grew to form melanoma in syngeneic mice. CONCLUSION: A herpes simplex virus-permissive mouse melanoma cell line was established. Its tumorigenicity remained unchanged.

[Identification of occult disseminated tumor cells by recombinant herpes simplex virus expressing GFP (HSV(GFP))].

OBJECTIVE: To develop a novel rapid protocol for the detection of occult disseminated tumor cells by a recombinant herpes simplex virus expressing GFP (HSV(GFP)). METHODS: Tumor cells of seven cell lines were exposed to HSV(GFP) and then examined for GFP expression by fluorescence microscopy. Various numbers of tumor cells (10, 100, 1000, 10 000) were mixed into 2 ml human whole blood, separated with lymphocytes separation medium, exposed to HSV(GFP), incubated at 37°C for 6 - 24 h and then counted for the number of green cells under the fluorescence microscope. Some clinical samples including peripheral blood, pleural effusion, ascites, spinal fluid from tumor-bearing patients were screened using this protocol in parallel with routine cytological examination. RESULTS: HSV(GFP) was able to infect all 7 tumor cell lines indicating that the HSV(GFP) can be used to detect different types of tumor cells. The detection sensitivity was 10 cancer cells in 2 ml whole blood. In the clinical samples, there were 4/15 positive by routine cytological examination but 11/15 positive by HSV(GFP), indicating a higher sensitivity of this new protocol. CONCLUSION: Recombinant herpes simplex virus-mediated green fluorescence is a simple and sensitive technique for the identification of occult disseminated cancer cells including circulating tumor cells (CTCs).