Patient Cranial Angle and Intrafractional Stability in CyberKnife Robotic Radiosurgery: A Retrospective Analysis.

Purpose: The aim of this study was to investigate whether variations in cranial angles and treatment accuracy during CyberKnife robotic radiosurgery necessitate adjustment of the margins of the planning target volume. Patients and Methods: Data from 66 patients receiving CyberKnife treatment for brain tumors were retrospectively analyzed. Patients were immobilized using a thermoplastic mask and headrest. The cranial angle was measured on planning CT and patients were divided into 2 groups: ≤10° (Group A) and >10° (Group B). Intrafractional motion was recorded using the CyberKnife tracking system over 50 min. Translational and rotational errors were compared between groups, and planning target volume margins were calculated. Results: In Group A, significant translational error differences were found along with the X-axis over time (P < .02). In Group B, significant differences occurred along with the Z-axis (P < .03). No significant rotational or 3-dimensional vector differences were found in either group. Group A had significantly lower Y-axis (P < .045) and roll axis (P < .005) errors compared to Group B. Estimated planning target volume margins in Group A were 0.56 mm (X), 0.46 mm (Y), and 0.47 mm (Z). In Group B, margins were 0.62 mm (X), 0.48 mm (Y), and 0.46 mm (Z). Margins covering 95% of intrafraction motion were 0.49 to 0.50 mm (X, Y, Z) and 0.69 mm (3-dimensional vector) for Group A, and 0.48 to 0.60 mm and 0.79 mm for Group B. With a 1-mm margin, complete coverage was achieved in Group A while 2.1% of vectors in Group B exceeded 1 mm. Conclusion: Adjusting cranial angle to ≤10° during thermoplastic mask molding provided better or similar intrafractional stability compared to >10°.

Prediction of recurrence-related factors for patients with early-stage cervical cancer following radical hysterectomy and adjuvant radiotherapy.

OBJECTIVE: To analyze recurrent factors in patients with clinical early-stage cervical cancer (ESCC) following hysterectomy and adjuvant radiotherapy. METHODS: We collected data from patients with ESCC, staged according to the 2009 Federation International of Gynecology and Obstetrics (FIGO) staging criteria, who underwent hysterectomy followed by adjuvant radiotherapy between 2012 and 2019. These patients were subsequently restaged using the 2018 FIGO criteria. Univariable and multivariable analyses, along with nomogram analyses, were conducted to explore factors associated with recurrence-free survival (RFS). RESULTS: A total of 310 patients met the inclusion criteria, with a median follow-up time of 46 months. Among them, 126 patients with ESCC were restaged to stage III C1 or III C2 after surgery due to lymph node metastasis (LNM) based on the 2018 FIGO staging criteria. Of these, 60 (19.3%) experienced relapse. The 1-, 3-, and 5-year RFS rates were 93.9%, 82.7%, and 79.3%, respectively. Multivariate analysis revealed that the number of positive lymph nodes (LNs), tumor diameter (TD) > 4 cm, and parametrial invasion (PI) were associated with recurrence. The nomogram indicated their predictive value for 3-year and 5-year RFS. Notably, the 5-year recurrence rate (RR) increased by 30.2% in patients with LNM, particularly those with ≥ 3 positive LNs (45.5%). Patients with stage III C2 exhibited a significantly higher RR than those with IIIC1 (56.5% vs. 24.3%, p < 0.001). The 5-year RFS for patients with TD > 4 cm was 65.8%, significantly lower than for those with TD ≤ 4 cm (88.2%). Subgroup analysis revealed higher 5-year RRs in patients with stage III C2 than that in patients with III-C1 (56.5% vs. 24.3%, p < 0.001), demonstrating a significant difference in the RFS survival curve. CONCLUSION: RR in patients with clinical ESCC after hysterectomy followed by adjuvant radiotherapy is correlated with the number of positive LNs, TD > 4 cm, and PI. Emphasis should be placed on the common high-risk factor of LNM association with recurrence after radical hysterectomy in ESCC.

Evaluation of the Position Error of Wearing Surgical Masks During Radiotherapy in Head and Neck Cancer Patients.

Purpose: Wearing a mask during the coronavirus disease 2019 epidemic (COVID-19) is a preventive way to reduce droplet and aerosol transmission. The purpose of this study was to evaluate the position error of wearing a surgical mask during radiotherapy in head and neck cancer patients. Patients and Methods: We collected and analyzed 2351 kV X-ray image records of 81 patients with head and neck cancer who underwent image-guided radiotherapy (IGRT). Patients with/without a surgical mask were divided into the head-neck (HN) mask group and head-neck-shoulder (HNS) mask group. The position error in the X (left-right), Y (superior-inferior), Z (anterior-posterior), 3D (three dimensional) vectors, as well as the pitch and yaw axes were compared between the four groups. Results: We found that patients wearing surgical masks in the HN mask group showed no significant differences in the mean position error of the different types of headrest (p>0.05). In the HNS mask group, only the type C headrest group showed significant differences (P < 0.05). The X axis values were -0.05±0.07 and -0.11± 0.01 cm (P = 0.04), and the pitch axis values were 0.34±0.29° and 0.83±0.08° (P = 0.01). Conclusion: The mean position error of most patients wearing surgical masks was not greater than patients without a surgical mask. Patients wearing while receiving treatment is a low-cost and easy-to-implement prevention method.

USP11 facilitates colorectal cancer proliferation and metastasis by regulating IGF2BP3 stability.

The abnormal expression of ubiquitin-specific protease 11 (USP11) is thought to be related to tumor development and progression; however, few studies have reported the biological function and clinical importance of USP11 in colorectal cancer (CRC). Therefore, it is necessary to further explore the role of USP11 in CRC. Immunohistochemical staining was used to explore the association between prognosis and USP11 expression in CRC. Cholecystokinin octapeptide (CCK-8), colony formation, transwell, and animal assays were used to study the abilities of proliferation, migration, and invasion in CRC cells. Co-immunoprecipitation assays, Western blotting, ubiquitination assays, and rescue experiments were performed to elucidate the interaction between USP11 and insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3). We verified that USP11 was overexpressed in CRC tissues and was associated with the depth of tumor invasion and metastasis. USP11 knockdown or overexpression could weaken or reinforce the abilities of proliferation, migration, and invasion in CRC cells in vivo or in vitro. IGF2BP3 was protected by USP11 from degradation via deubiquitination. The rescue experiments revealed that IGF2BP3 overexpression could effectively reverse the decrease in cell proliferation, migration, and invasion caused by USP11 knockdown. Therefore, USP11 might be involved in CRC tumorigenesis and development through a USP11-IGF2BP3 axis pathway, and USP11 overexpression might be a novel indicator for poor prognosis and a potential therapeutic target in CRC patients.

Using multivariate regression model with least absolute shrinkage and selection operator (LASSO) to predict the incidence of Xerostomia after intensity-modulated radiotherapy for head and neck cancer.

PURPOSE: The aim of this study was to develop a multivariate logistic regression model with least absolute shrinkage and selection operator (LASSO) to make valid predictions about the incidence of moderate-to-severe patient-rated xerostomia among head and neck cancer (HNC) patients treated with IMRT. METHODS AND MATERIALS: Quality of life questionnaire datasets from 206 patients with HNC were analyzed. The European Organization for Research and Treatment of Cancer QLQ-H&N35 and QLQ-C30 questionnaires were used as the endpoint evaluation. The primary endpoint (grade 3(+) xerostomia) was defined as moderate-to-severe xerostomia at 3 (XER3m) and 12 months (XER12m) after the completion of IMRT. Normal tissue complication probability (NTCP) models were developed. The optimal and suboptimal numbers of prognostic factors for a multivariate logistic regression model were determined using the LASSO with bootstrapping technique. Statistical analysis was performed using the scaled Brier score, Nagelkerke R(2), chi-squared test, Omnibus, Hosmer-Lemeshow test, and the AUC. RESULTS: Eight prognostic factors were selected by LASSO for the 3-month time point: Dmean-c, Dmean-i, age, financial status, T stage, AJCC stage, smoking, and education. Nine prognostic factors were selected for the 12-month time point: Dmean-i, education, Dmean-c, smoking, T stage, baseline xerostomia, alcohol abuse, family history, and node classification. In the selection of the suboptimal number of prognostic factors by LASSO, three suboptimal prognostic factors were fine-tuned by Hosmer-Lemeshow test and AUC, i.e., Dmean-c, Dmean-i, and age for the 3-month time point. Five suboptimal prognostic factors were also selected for the 12-month time point, i.e., Dmean-i, education, Dmean-c, smoking, and T stage. The overall performance for both time points of the NTCP model in terms of scaled Brier score, Omnibus, and Nagelkerke R(2) was satisfactory and corresponded well with the expected values. CONCLUSIONS: Multivariate NTCP models with LASSO can be used to predict patient-rated xerostomia after IMRT.

Growth-differentiation factor-8 (GDF-8) in the uterus: its identification and functional significance in the golden hamster.

Transforming growth factor-beta superfamily regulates many aspects of reproduction in the female. We identified a novel member of this family, growth-differentiation factor 8 (GDF-8) in the 72 h post coital uterine fluid of the golden hamster by proteomic techniques. Uterine GDF-8 mRNA decreased as pregnancy progressed while its active protein peaked at 72 h post coitus (hpc) and thereafter stayed at a lower level. At 72 hpc, the GDF-8 transcript was localized to the endometrial epithelium while its protein accumulated in the stroma. Exogenous GDF-8 slowed down proliferation of primary cultures of uterine smooth muscle cells (SMC) and endometrial epithelial cells (EEC). In addition, GDF-8 attenuated the release of LIF (leukaemia inhibiting factor) by EEC. As for the embryo in culture, GDF-8 promoted proliferation of the trophotoderm (TM) and hatching but discouraged attachment. Our study suggests that GDF-8 could regulate the behavior of preimplantation embryos and fine-tune the physiology of uterine environment during pregnancy.

Immunization with truncated sequence of Telomerase Reverse Transcriptase induces a specific antitumor response in vivo.

To select the MHC-I-binding epitope-rich sequence of mice telomerase reverse transcriptase (mTERT) and study the antitumor immune response induced by truncated TERT through mRNA-transfected dendritic cells (DCs) immunization in mice. The MHC-I-binding epitopes of TERT were predicted using bioinformatics software. The selected sequence of TERT (Truncated mTERT, TERT(t), mTERT cDNA 1776 bp-2942 bp encoding 584 aa-969 aa) was cloned from B16 mouse melanoma cells and inserted into pBluescriptIIKS(+) plasmid downstream of the T7 promoter. TERT(t) RNA was prepared through in vitro transcription. Bone marrow-derived DCs were electroporated with TERT(t) RNA and used to immunize syngeneic naïve mice. The quantity and cytotoxic activity of TERT-specific cytotoxic T lymphocytes (CTLs) in mice spleen were evaluated using IFN-gamma enzyme-linked immunospot (ELISPOT) and Lactate dehydrogenase release assay. The immunoprophylactic effects against TERT positive tumor induced by TERT(t) RNA transfected DC in vivo were evaluated through an immunized-challenged mouse model. TERT(t) was cloned and in vitro transcribed into TERT(t) mRNA. As shown in FCM analysis, the efficiency of DC electroporation is 35.1% (29.7-41.2%). After electroporation, a subtle increase of costimulator and MHC-II molecules were expressed on the cell surface. Immunization of TERT(t) mRNA transfected DCs induced IFN-gamma-secreting CTLs which manifested specific cytotoxic activity against TERT-positive target cells. In a cancer mouse model, vaccination of TERT(t) mRNA-transfected DCs suppressed the growth of TERT positive tumors (p=0.001) and prolong the survival time of tumor-bearing animals (p=0.029). TERT(t) evokes an antitumor immune response in vivo which is targeted to TERT. TERT(t) can be used as an antigeneic sequence to produce anti-TERT tumor vaccine.

Truncated TERT mRNA transfected dendritic cells evoke TERT specific antitumor response in vivo.

BACKGROUND/AIMS: To evaluate the antitumor immune response induced by truncated TERT (TERTt) mRNA transfected dendritic cells (DCs) in METHODOLOGY: Truncated mouse TERT sequence (according to mice telomerase reverse transcriptase mRNA 1776bp-2942bp) was cloned from B16 mice melanoma cells and inserted into pBluescript II KS(+) plasmid downstreaming of T7 promoter. The in vitro transcription was performed to prepare TERTt mRNA. The bone marrow-derived DCs isolated from BALB/c or C57B/L mice were electroporated with TERTt mRNA and recruited to immunize syngeneic naive mice respectively. The quantity and cytotoxic activity of tumor specific cytotoxic T lymphocytes (CTLs) in mice spleen were evaluated by using IFN-gamma enzyme-linked immunospot (ELIspot) and LDH release assay. The immunoprophylactic effects induced by TERTt mRNA transfected DC were evaluated in immunized-challenged mouse model. RESULTS: TERTt was cloned and transcripted into TERTt mRNA in vitro. TERTt mRNA transfected bone marrow-derived DCs were prepared. As shown by transfecting with EGFP mRNA, the DC transfected efficiency is 35.1% and there was a subtle increase of costimulator and MHC-II molecule expression after electroporation. Immunization with TERTt mRNA transfected DCs can induce TERTt and TERT-specific IFN-gamma secreting CTLs in the spleen of immunized mice. The splenocytes isolated from mice immunized with TERTt mRNA transfected DCs showed specific cytotoxic activity against TERTt and TERT-positive target cells. Using a syngeneic cancer mouse model, it was shown that TERTt mRNA transfected DCs vaccination can suppress the growth of TERT-positive tumor inoculation. CONCLUSIONS: TERTt mRNA transfected bone marrow-derived DCs can evoke antitumor immune response in vivo effectively and TERTt can serve as a universal tumor associated antigen to produce DC-based tumor vaccine.

Tumor cells with B7.1 and transmembrane anchored staphylococcal enterotoxin A generate effective antitumor immunity.

Staphylococcus enterotoxin A (SEA) stimulates T cells bearing certain TCR beta-chain variable regions, when bound to MHC-II molecules, and is a potent inducer of CTL activity and cytokines production. To decrease toxicity of SEA to the normal MHC-II(+) cells and to localize the immune response induced by SEA to the tumor site, my colleague previously genetically fused SEA with B7.1 transmembrane region (named as SEAtm) to make SEA express on the surface of tumor cells and tumor cells modified with SEAtm could induce efficient antitumor immunity in vitro. The tumor cell vaccines modified with multiple immune activators frequently elicited stronger antitumor immune responses than single-modified vaccines. In this study, we modified the tumor cell vaccine with B7.1 and SEAtm to improve efficiency in the application of SEA. First, SEAtm gene was subcloned from recombinant plasmid pLXSNSEP by PCR and murine B7.1 gene was cloned from splenocytes derived from C57BL/6 mice by RT-PCR. Then, the eukaryotic co-expression vector of SEA and murine B7.1 gene was constructed and named as pcDNA-BIS. B16 cell lines stably expressing SEA and/or B7.1 were established by screening with G418 after transfection and inactivated for the preparation of tumor cell vaccines to treat mice bearing established B16 tumors. The results indicated that the dual-modified tumor cell vaccine B16/B7.1+SEAtm (B16-BIS) elicited significantly stronger antitumor immune responses in vivo when compared with the single-modified tumor cell vaccines B16/B7.1 (B16-B7.1) and B16/SEAtm (B16-SEAtm), and supported the feasibility and effectiveness of the dual-modified tumor cell vaccine with superantigen and co-stimulatory molecule.

Heat shock protein 70/MAGE-3 fusion protein vaccine can enhance cellular and humoral immune responses to MAGE-3 in vivo.

MAGE-3, a member of melanoma antigen (MAGE) gene family, is recognized as an ideal candidate for tumor vaccine because it is expressed in a significant proportion of tumors of various histological types and can induce antigen-specific immune response in vivo. There is now substantial evidence that heat shock proteins HSPs isolated from cancer cells and virus-infected cells can be used as vaccines to produce cancer-specific or virus-specific immunity. In this research, we investigated whether M. tuberculosis HSP70 can be used as vehicle to elicit immune response to its accompanying MAGE-3 protein. A recombinant protein expression vector was constructed that permitted the production of fusion protein linking amino acids 195-314 of MAGE-3 to the C terminus of HSP70. We found that HSP70-MAGE-3 fusion protein can elicit stronger cellular and humoral immune responses against MAGE-3 expressing murine tumor than those elicited by MAGE-3 protein in vivo, which resulted in potent antitumor immunity against MAGE-3-expressing tumors. Covalent linkage of HSP70 to MAGE-3 was necessary to elicit immune response to MAGE-3. These results indicate that linkage of HSP70 to MAGE-3 enhanced immune responses to MAGE-3 in vivo and HSP70 can be exploited to enhance the cellular and humoral immune responses against any attached tumor-specific antigens.

Identification of HLA-A2-restricted CTL epitope encoded by the MAGE-n gene of human hepatocellular carcinoma.

BACKGROUND: Identification of the cytotoxic T lymphocytes (CTL) restricted epitopes of tumor antigens opens up possibilities of developing a new cancer vaccine. For the MAGE-n has been demonstrated closely associated with hepatocellular carcinoma (HCC) and HLA-A2.1 is found in over 50% of HCC patients in China, we aim at identifying MAGE-n-encoded peptide presented by HLA-A2.1. MATERIALS: A HLA-A2.1-restricted CTL epitope was identified by using an improved "reverse immunology" strategy: (a) computer-based epitope prediction from the amino acid sequence of MAGE-n antigen; (b) peptide-binding assay to determine the affinity of the predicted peptide with HLA-A2.1 molecule; (c) stimulation of primary T-cell response against the predicted peptides in vitro; and (d) testing of the induced CTLs toward HCC cells expressing MAGE-n antigen and HLA-A2.1. RESULTS: Of the five tested peptides, effectors induced by a peptide of MAGE-n at residue position 159-167(QLVFGIEVV) lysed HCC cells expressing both MAGE-n and HLA-A2.1. Our results indicated that peptide QLVFGIEVV was a new HLA-A2.1-restricted CTL epitope capable of inducing MAGE-n specific CTLs in vitro. CONCLUSIONS: Identification of the MAGE-n /HLA-A2.1 peptide QLVFGIEVV may facilitate peptide-based specific immunotherapy for HCC. The combination of epitope prediction, epitope reconstruction method and immunological methods can improve the efficiency and accuracy of CTL epitope studies.

[Establishment of mouse melanoma B16 cell lines co-expressing MAGE-1 and EGFP].

AIM: To construct the melanoma antigen-1(MAGE-1) eukaryotic expression plasmid and express MAGE-1 in mouse melanoma B16 cells. METHODS: The MAGE-1 gene was amplified by PCR and cloned into the eukaryotic expression vector pIRES2-EGFP to construct the pIRES2-EGFP-MAGE-1 plasmid. The plasmid was transfected into the B16 cells. The EGFP expression was detected under fluoroscent microscope and the MAGE-1 expression was detected by immunohistochemistry staining. RESULTS: The eukaryotic expression vector pIRES2-EGFP-MAGE-1 was constructed and transfected successfully into B16 cells, and the EGFP and MAGE-1 genes were co-expressed in the B16 cells. CONCLUSION: A mouse melanoma cell line B16 co-expressing MAGE-1 and EGFP genes has been established successfully, which lays the foundation for the research on application of MAGE-1 in the tumor immunotherapy.

Heat shock protein 70 / MAGE-1 tumor vaccine can enhance the potency of MAGE-1-specific cellular immune responses in vivo.

The cancer-testis antigen encoded by the MAGE-1 gene is an attractive antigen in tumor immunotherapy because it can be processed as a foreign antigen by the immune system and generate tumor-specific cellular immune response in vivo. However, increase of the potency of MAGE-1 DNA vaccines is still needed. The high degree of sequence homology and intrinsic immunogenicity of heat shock protein 70 (HSP70) have prompted the suggestion that HSP70 might have immunotherapeutic potential, as HSP70 purified from malignant and virally infected cells can transfer and deliver antigenic peptides to antigen-presenting cells to elicit peptide-specific immunity. In this research, we evaluated the enhancement of linkage of Mycobacterium tuberculosis HSP70 to MAGE-1 gene of the potency of antigen-specific immunity elicited by naked DNA vaccines. We found that vaccines containing MAGE-1-HSP70 fusion genes enhanced the frequency of MAGE-1-specific cytotoxic T cells in contract to vaccines containing the MAGE-1 gene alone. More importantly, the fusion converted a less effective DNA vaccine into one with significant potency against established MAGE-1-expressing tumors. These results indicate that linkage of HSP70 to MAGE-1 gene may greatly enhance the potency of DNA vaccines, and generate specific antitumor immunity against MAGE-1-expressing tumors.