PD-1 Inhibitor Aggravate Irradiation-Induced Myocardial Fibrosis by Regulating TGF-ß1/Smads Signaling Pathway via GSDMD-Mediated Pyroptosis.

Cancer therapy has entered a new era with the use of programmed cell death protein 1 (PD-1) immune checkpoint inhibitors. When combined with thoracic radiotherapy, it demonstrates synergistic anti-tumor effects and potentially worsens radiation-induced myocardial fibrosis (RIMF). RIMF is the final stage of radiation-induced heart disease (RIHD) and a potentially fatal clinical complication of chest radiotherapy. It is characterized by decreased ventricular elasticity and distensibility, which can result in decreased ejection fraction, heart failure, and even sudden cardiac death. Pyroptosis, a type of programmed cell death, is mediated by members of the gasdermin (GSDM) family and has been associated with numerous cardiac disorders. The effect of pyroptosis on myocardial fibrosis caused by a combination of radiotherapy and PD-1 inhibitors remains uncertain. In this study, a 6MV X-ray of 20 Gy for local heart irradiation was used in the RIHD mouse model. We noticed that PD-1 inhibitors aggravated radiation-induced cardiac dysfunction and RIMF, concurrently enhancing the presence of CD8+ T lymphocytes in the cardiac tissue. Additionally, our findings indicated that the combination of PD-1 inhibitor and thoracic radiation can stimulate caspase-1 to cleave GSDMD, thereby regulating pyroptosis and liberating interleukin-8 (IL-18). In the myocardium of mice, the manifestation of pyroptosis mediated by GSDMD is accompanied by the buildup of proteins associated with fibrosis, such as collagen I, transforming growth factor ß1 (TGF-ß1), interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), and tumor necrosis factor α (TNF-α). Moreover, it was discovered that TFG-ß1 induced the phosphorylation of Smad2/Smad3 when the cardiac underwent PD-1 inhibitor in conjunction with thoracic irradiation (IR). The findings of this research indicate that PD-1 inhibitor worsen RIMF in mice by triggering GSDMD-induced pyroptosis and influencing the TGF-ß1/Smads pathway. While using the caspase-1 inhibitor Z-YVAD-FMK, RIMF can be alleviated. Blocking GSDMD may be a viable strategy for managing myocardial fibrosis caused by the combination of PD-1 inhibitors and radiotherapy.

Coxsackie B virus-induced myocarditis in a patient with a history of lymphoma: A case report and review of literature.

INTRODUCTION: In rare occasions, coxsackievirus infections can cause serious illness, such as encephalitis and myocarditis. The immunotherapies of cancer could increase the risk of myocarditis, especially when applying immune checkpoint inhibitors. Herein, we report a rare case of Coxsackie B virus-induced myocarditis in a patient with a history of lymphoma. CASE PRESENTATION: A 32-year-old woman was admitted to the hospital with recurrent fever for more than 20 days, and she had a history of lymphoma. Before admission, the positron emission tomography/computed tomography result indicated that the patient had no tumor progression, and she was not considered the cancer-related fever upon arriving at our hospital. Patient's red blood cell, platelet count, and blood pressure were decreased. In addition, she had sinus bradycardia and 3 branch blocks, which was consistent with acute high lateral and anterior wall myocardial infarction. During hospitalization, the patient had recurrent arrhythmia, repeated sweating, poor mentation, dyspnea, and Coxsackie B virus were detected in patient's blood samples by pathogen-targeted next-generation sequencing. The creatine kinase, creatine kinase MB, and N-terminal pro-brain natriuretic peptide were persistently elevated. Consequently, the patient was diagnosed with viral myocarditis induced by Coxsackie B virus, and treated with acyclovir, gamma globulin combined with methylprednisolone shock therapy, trimetazidine, levosimendan, sildenan, continuous pump pressors with m-hydroxylamine, entecavir, adefovir, glutathione, pantoprazole, and low-molecular-weight heparin. Her symptoms worsened and died. CONCLUSION: We reported a case with a history of lymphoma presented with fever, myocardial injury, who was ultimately diagnosed with Coxsackie B virus-induced myocarditis. Moreover, pathogen-targeted next-generation sequencing indeed exhibited higher sensitivity compared to mNGS in detecting Coxsackie B virus.

Molecular recognition of ITIM/ITSM domains with SHP2 and their allosteric effect.

Src homology 2-domain-containing tyrosine phosphatase 2 (SHP2) is a non-receptor protein tyrosine phosphatase that is widely expressed in a variety of cells and regulates the immune response of T cells through the PD-1 pathway. However, the activation mechanism and allosteric effects of SHP2 remain unclear, hindering the development of small molecule inhibitors. For the first time, in this study, the complex structure formed by the intact PD-1 tail and SHP2 was modeled. The molecular recognition and conformational changes of inactive/active SHP2 versus ITIM/ITSM were compared based on prolonged MD simulations. The relative flexibility of the two SH2 domains during MD simulations contributes to the recruitment of ITIM/ITSM and supports the subsequent conformational change of SHP2. The binding free energy calculation shows that inactive SHP2 has a higher affinity for ITIM/ITSM than active SHP2, mainly because the former's N-SH2 refers to the α-state. In addition, a significant decrease in the contribution to the binding energy of certain residues (e.g., R32, S34, K35, T42, and K55) of conformationally transformed SHP2 contributes to the above result. These detailed changes during conformational transition will provide theoretical guidance for the molecular design of subsequent novel anticancer drugs.

Study on the allosteric activation mechanism of SHP2 via elastic network models and neural relational inference molecular dynamics simulation.

As a ubiquitous protein tyrosine phosphatase, SHP2 is involved in PD-1/PD-L1 mediated tumor immune escape and undergoes substantial conformational changes. Therefore, it is considered an ideal target for tumor intervention. However, the allosteric mechanisms of SHP2 binding PD-1 intracellular ITIM/ITSM phosphopeptides remain unclear, which greatly hinders the development of novel structure-based anticancer allosteric inhibitors. In this work, the open and closed structural models of SHP2 are first constructed based on this knowledge; next their motion modes are investigated via elastic network models such as the Gaussian network model (GNM), anisotropic network model (ANM) and adaptive anisotropic network model (aANM); and finally, a possible allosteric signaling pathway is proposed using a neural relational inference molecular dynamics (NRI-MD) simulation embedded with an artificial intelligence (AI) strategy. In GNM and ANM, the N-SH2, C-SH2 and PTP domains all exhibit distinct dynamics partitions, and the N-SH2/C-SH2 regions show a rigid rotation relative to PTP. According to a series of intermediate snapshots given by aANM, N-SH2 is first identified with pY223 specifically, inducing a D'E-loop to change from ß-sheets to random coils, and then, C-SH2 serves as a fulcrum to drive N-SH2 to rotate 110° completely away from the original active sites of PTP. Finally, a possible allosteric signaling-transfer path for SHP2, namely R220-R138-T108-R32, is proposed based on NRI-MD sampling. This work provides a possible allosteric mechanism of SHP2, which is helpful for the following design of novel allosteric inhibitors and is expected to be used in clinical synergies with PD-1 monoclonal antibody.

Large-scale Isolation of Exosomes Derived from NK Cells for Anti-tumor Therapy.

Exosomes are lipid bilayer-enclosed vesicles, actively secreted by cells, containing proteins, lipids, nucleic acids, and other substances with multiple biological functions after entering target cells. Exosomes derived from NK cells have been shown to have certain anti-tumor effects and potential applications as chemotherapy drug carriers. These developments have resulted in high demand for exosomes. Although there has been large-scale industrial preparation of exosomes, they are only for generally engineered cells such as HEK 293T. The large-scale preparation of specific cellular exosomes is still a major problem in laboratory studies. Therefore, in this study, we used tangential flow filtration (TFF) to concentrate the culture supernatants isolated from NK cells and isolated NK cell-derived exosomes (NK-Exo) by ultracentrifugation. Through a series of characterization and functional verification of NK-Exo, the characterization, phenotype, and anti-tumor activity of NK-Exo were verified. Our study provides a considerably time- and labor-saving protocol for the isolation of NK-Exo.

First-in-human phase I/Ib study of QL1706 (PSB205), a bifunctional PD1/CTLA4 dual blocker, in patients with advanced solid tumors.

BACKGROUND: QL1706 (PSB205) is a single bifunctional MabPair (a novel technical platform) product consisting of two engineered monoclonal antibodies (anti-PD-1 IgG4 and anti-CTLA-4 IgG1), with a shorter elimination half-life (t1/2) for CTLA-4. We report results from a phase I/Ib study of QL1706 in patients with advanced solid tumors who failed standard therapies. METHODS: In the phase I study, QL1706 was administered intravenously once every 3 weeks at one of five doses ranging from 0.3 to 10 mg/kg, and the maximum tolerated dose, recommended phase 2 dose (RP2D), safety, pharmacokinetics (PK), and pharmacodynamics (PD) of QL1706 were investigated. In the phase Ib study, QL1706 was administered at the RP2D intravenously every 3 weeks, and the preliminary efficacies in non-small cell lung cancer (NSCLC), nasopharyngeal carcinoma (NPC), cervical cancer (CC), and other solid tumors were evaluated. RESULTS: Between March 2020 and July 2021, 518 patients with advanced solid tumors were enrolled (phase I, n = 99; phase Ib, n = 419). For all patients, the three most common treatment-related adverse events (TRAEs) were rash (19.7%), hypothyroidism (13.5%), and pruritus (13.3%). The TRAEs and immune-related adverse events (irAEs) of grade ≥ 3 occurred in 16.0% and 8.1% of patients, respectively. In phase I, 2 of 6 patients in the 10mg/kg group experienced dose-limiting toxicities (DLTs) (grade 3 thrombocytopenia and grade 4 immune-mediated nephritis), so the maximum tolerated dose (MTD) was reached at 10 mg/kg. The RP2D was determined to be 5 mg/kg based on comprehensive analysis of tolerability, PK/PD, and efficacy. For all patients who received QL1706 at the RP2D, the objective response rate (ORR) and median duration of response were 16.9% (79/468) and 11.7 months (8.3-not reached [NR]), respectively; and the ORRs were 14.0% (17/121) in NSCLC, 24.5% (27/110) in NPC, 27.3% (15/55) in CC, 7.4% (2/27) in colorectal cancer, 23.1% (6/26) in small cell lung cancer. For immunotherapy-naive patients, QL1706 exhibited promising antitumor activities, especially in NSCLC, NPC, and CC, with ORRs of 24.2%, 38.7%, and 28.3%, respectively. CONCLUSIONS: QL1706 was well tolerated and demonstrated promising antitumor activity in solid tumors, especially in NSCLC, NPC, and CC patients. It is currently being evaluated in randomized phase II (NCT05576272, NCT05179317) and phase III (NCT05446883, NCT05487391) trials. Trial Registration ClinicalTrials.gov Identifier: NCT04296994 and NCT05171790.

Preliminary results of randomized phase II study of etoposide plus lobaplatin or etoposide plus cisplatin with concurrent thoracic radiotherapy in the treatment of limited-stage small cell lung cancer.

The purpose is to compare the clinical efficacy and toxicity of etoposide plus lobaplatin (EL) or etoposide plus cisplatin (EP) with concurrent thoracic radiotherapy during the treatment of limited-stage small cell lung cancer (LS-SCLC). Forty-two patients with LS-SCLC were randomly divided into EL ( n = 19) or EP ( n = 23) regimens combined with thoracic intensity-modulated radiotherapy. The primary endpoint was 1-year progression-free survival (PFS) rate. The 1-, 2-, and 3-year PFS rates in the EL and EP cohorts were 50.8, 38.1, and 12.7%; and 56.5, 43.5, and 29.0%, respectively ( P = 0.527), whereas the 1-, 2-, and 3-year overall survival (OS) rates were 72.2, 52.5, and 43.8%; and 73.9, 48.4, and 48.4%, respectively ( P = 0.923). The hematological toxicities were similar in two cohorts. However, gastrointestinal reactions were more severe in the EP group. The incidence of nausea and vomiting in EL and EP cohorts were 31.6% vs. 73.9% ( P = 0.006) and 20.1% vs. 60.9% ( P = 0.009), respectively. The two cohorts did not show ≥grade 4 radiation esophagitis and ≥grade 3 radiation pneumonitis. The incidence of acute radiation esophagitis in EL group was lower ( P = 0.038), both groups showed a similar incidence of radiation pneumonitis ( P = 1.000). EL or EP chemotherapy with concurrent thoracic radiotherapy showed similar PFS and OS. The EL group showed milder gastrointestinal toxicity and radiation esophagitis. Radiation pneumonitis and hematological toxicity were similar in the two regimens, which can be tolerated by patients.

Chondroitin polymerizing factor predicts a poor prognosis and promotes breast cancer progression via the upstream TGF-ß1/SMAD3 and JNK axis activation.

Aberrant composition of glycans in the tumor microenvironment (TME) contributes to tumor progression and metastasis. Chondroitin polymerizing factor (CHPF) is a glycosyltransferase that catalyzes the biosynthesis of chondroitin sulfate (CS). It is also correlated to transforming growth factor-ß1 (TGF-ß1) expression, a crucial mediator in the interaction of cancer cells with TME. In this study, we investigated the association of CHPF expression with the clinicopathological features of breast cancer (BRCA), as well the oncogenic effect and the underling mechanisms of CHPF upon BRCA cells. We found that CHPF expression is significantly increased in human BRCA tissues, and it is positively associated with TGF-ß expression (r = 0.7125). The high-expression of CHPF predicts a poor prognosis and is positively correlated with tumor mass, lymph node metastasis, clinical staging and HER-2 negative-expression. The mechanistic study revealed that it promotes BRCA cell proliferation, migration and invasion through TGF-ß1-induced SMAD3 and JNK activation in vitro, JNK (SP600125) or SMAD3 (SIS3) inhibitor can remove the promotion of CHPF upon cell proliferation, migration and invasion in MDA-MB-231 cells, which is derived from triple-negative breast cancer (TNBC). Collectively, our finding suggested CHPF may function as an oncogene and is highly expressed in human BRCA tissues. Pharmacological blockade of the upstream of JNK or SMAD3 signaling may provide a novel therapeutic target for refractory TNBC patients with CHPF abnormal high-expression.

The Effect of PD-1 Inhibitor Combined with Irradiation on HMGB1-Associated Inflammatory Cytokines and Myocardial Injury.

Purpose: To explore the effect of PD-1 inhibitors combined with irradiation on myocardial injury and the changes of HMGB1-associated inflammatory markers. Methods: Four groups of five mice were used, each groupformed by randomly dividing 20 mice (group A control; group B PD-1 inhibitors; group C Irradiation; group D PD-1 inhibitors+irradiation; n = 5 for each). The mice were treated with either PD-1 inhibitors or a 15 Gy dose of single heart irradiation, or both. Hematoxylin-eosin staining assessed the morphology and pathology of heart tissue; Masson staining assessed heart fibrosis; Tunel staining evaluated heart apoptosis; flow cytometry detected CD3+, CD4+, and CD8+ T lymphocytes in heart tissues; enzyme linked immunosorbent assay evaluated IL-1ß, IL-6, and TNF-ɑ of heart tissue; Western blot and quantitative real-time PCR (qPCR) detected the expression of protein and mRNA of HMGB1, TLR-4, and NF-κB p65 respectively. Results: The degree of heart injury, collagen volume fraction (CVF) and apoptotic index (AI) in groups B, C, and D were higher than group A, but the differences between the CVF and AI of group A and group B were not statistical significance (P>0.05). Similarly, the absolute counts and relative percentage of CD3+ and CD8+ T lymphocytes and the concentrations of IL-1ß, IL-6, and TNF-α in heart tissue with group D were significantly higher than the other groups (P<0.05). In addition, compared with group A, the expression of protein and mRNA of HMGB1 and NF-κB p65 in other groups were higher, and the differences between each group were statistically significant while TLR4 was not. In addition, interaction by PD-1 inhibitors and irradiation was found in inflammatory indicators, especially in the expression of the HMGB1 and CD8+ T lymphocytes. Conclusion: PD-1 inhibitors can increase the expression of HMGB1-associated inflammatory cytokines and aggravate radiation-induced myocardial injury.

Recombinant human endostatin combined with radiotherapy promotes cardiomyocyte apoptosis in rats via TGFß1/Smads/CTGF signaling pathway.

PURPOSE: The aim of the present study was to investigate the efficacy of recombinant human endostatin (ES) (rh-ES) combined with radiation on rat cardiomyocyte apoptosis and the regulatory mechanism of transforming growth factor beta1 (TGF-ß1)/Sma and Mad-related protein 3 (Smad3)/connective tissue growth factor (CTGF) signaling. METHOD: The primary cardiomyocytes were isolated from neonatal Sprague-Dawley rats for culture in vitro and divided into blank control group (without treatment), 10 Gy radiation + siTGF-ß1 siRNA (gene silencing) group, ES + siTGF-ß1 siRNA group, and 10 Gy radiation + ES + siTGF-ß1 siRNA group. Methyl thiazolyl tetrazolium assay was used to calculate the half-maximal inhibitory concentration (IC50) of rh-ES on cardiomyocytes. Adenoviral vector was constructed for virus packaging to silence TGF-ß1 expression in cardiomyocytes. Quantitative real-time polymerase chain reaction and Western blot were carried out to analyze TGF-ß1, Smad2, Smad3 and CTGF expression at both gene and protein levels. Flow cytometry and electron microscope were used to examine cell apoptosis. RESULTS: ES had a dose-dependent inhibitory effect on the proliferation of primary rat cardiomyocytes. ES combined with radiotherapy significantly inhibited cardiomyocyte proliferation and promoted cell apoptosis (P < 0.01). The gene and protein expression of TGF-ß1, Smad2, Smad3 and CTGF were significantly up-regulated in primary cardiomyocytes transfected with TGF-ß1 gene (P < 0.05). CONCLUSION: The combination therapy with rh-ES and radiation can promote cardiomyocyte apoptosis and aggravate myocardial cell damage via TGF-ß1/Smad3/CTGF signaling pathway.

Effect of PD-1 Inhibitor Combined with X-Ray Irradiation on the Inflammatory Microenvironment and Lung Tissue Injury in Mice.

PURPOSE: This study was designed to evaluate the effects of PD-1 inhibitor on lung tissue morphology and the immune system in a mouse model of radiation-induced lung injury (RILI) and to assess interactions between radiation therapy and PD-1 inhibition. METHODS: Twenty C57BL/6 mice were divided randomly into four groups of five mice each. Mice were treated with an anti-mouse PD-1 monoclonal antibody, whole thorax irradiation, both or neither. Lung tissue morphology and pathological changes were assessed by hematoxylin-eosin staining; lung fibrosis was assessed by Masson staining and analysis of hydroxyproline; CD3+, CD4+, and CD8+ T lymphocytes in lung tissues were detected immunohistochemically; and the concentrations of transforming growth factor-ß1 (TGF-ß1) and interleukin-6 (IL-6) in lung tissue were evaluated by cytokine multiplex analysis. RESULTS: Lung injury scores and indicators of pulmonary fibrosis were higher in mice administration whole thorax irradiation than in control mice. Inflammatory infiltrate scores, alveoli deformation scores, collagen volume fractions and hydroxyproline contents in lung tissues were all significantly higher in mice administered PD-1 inhibitor plus irradiation than in the other three groups. Similarly, the percentages of CD3+ and CD8+T cells and the concentrations of IL-6 and TGF-ß1 in lung tissue were significantly higher in mice treated with radiation and PD-1 inhibitor than in the other groups. However, PD-1 inhibitor and irradiation interacted significantly only in the elevation of TGF-ß1 level. CONCLUSION: Whole thorax X-ray irradiation in mice can cause pulmonary injury and fibrosis, which could be exacerbated by PD-1 inhibitors. Radiotherapy combined with PD-1 inhibitors may aggravate RILI by synergistically upregulating TGF-ß1 expression, thereby affecting the immune-inflammatory microenvironment in the lungs.

Safety and activity of WX-0593 (Iruplinalkib) in patients with ALK- or ROS1-rearranged advanced non-small cell lung cancer: a phase 1 dose-escalation and dose-expansion trial.

WX-0593 (Iruplinalkib) is a novel, highly selective oral ALK and ROS1 tyrosine kinase inhibitor (TKI). In this study, the safety, antitumor activity, and pharmacokinetics of WX-0593 were evaluated in advanced non-small cell lung cancer (NSCLC) patients with ALK or ROS1 rearrangement. In the dose-escalation phase and dose-expansion phase, patients were treated with WX-0593 until disease progression, unacceptable toxicity, or subject withdrawal. In the dose-escalation phase, the primary endpoints were maximum tolerated dose (MTD), dose-limiting toxicity (DLT), and safety assessed by investigators. In the dose-expansion phase, the primary endpoint was objective response rate (ORR) assessed by investigators. Between September 25, 2017 and October 15, 2018, a total of 153 patients received WX-0593 treatment. Two dose-limiting toxicities (DLTs) including one grade 3 QT interval prolonged and one grade 2 chronic heart failure were reported at the dose of 300 mg in one patient. MTD was not reached. Overall, 140 of the 152 (92%) patients experienced treatment-related adverse events (TRAEs) and 35 of the 152 (23%) patients had TRAEs ≥grade 3. The overall ORR was 59.3% (32 of 54) for the dose-escalation phase and 56.6% (56 of 99) for the dose-expansion phase. For patients who were ALK-rearranged and ALK TKI naive, the ORR were 81.0% (17 of 21) in the dose-escalation phase and 76.3% (29 of 38) in the dose-expansion phase, and for patients who previously received crizotinib as the only ALK TKI, the ORR were 38.1% (8 of 21) and 45.7% (21 of 46) for the two phases, respectively. For patients who were ROS1-rearranged, the ORR were 30.0% (3 of 10) in the dose-escalation phase and 44.4% (4 of 9) in the dose-expansion phase. WX-0593 showed favorable safety and promising antitumor activity in advanced NSCLC patients with ALK or ROS1 rearrangement.

A multicenter randomized trials to compare the bioequivalence and safety of a generic doxorubicin hydrochloride liposome injection with Caelyx ® in advanced breast cancer.

Purpose: To compare the pharmacokinetic (PK) bioequivalence (BE) and safety of a generic pegylated liposomal doxorubicin (PLD) formulation with the reference product Caelyx®. Methods: A multicenter, single-dose, open-label, randomized, two-way crossover study was conducted in patients with breast cancer. For each period, the patients were administered with the test or the reference PLD intravenously at a dose of 50 mg/m2. Cmax, AUC0-t and AUC0-∞ for free, and encapsulated doxorubicin (doxorubicin) and partial AUC (AUC0-48h, AUC48h-t) for encapsulated doxorubicin were evaluated in 17 blood samples taken predose, and increasing time intervals over the following 14 days in each period. A washout period of 28-35 days was observed before crossing over. Results: 48 patients were enrolled and randomised, of which 44 were included and analysed in bioequivalence set (BES). The 90% confidence intervals (CIs) of the geometric mean ratio (GMR) of Cmax, AUC0-t and AUC0-∞ for free doxorubicin and encapsulated doxorubicin all fall within the bioequivalent range of 80% to 125%. The 90% CIs of GMR of partial AUC (AUC0-48h, AUC48h-t) for encapsulated doxorubicin also fall within the bioequivalent range. 48 patients were all included in the safety set (SS). The incidence of treatment-emergent adverse events (TEAEs) related to T and R was 95.8% (46/48) and 97.8% (45/46) respectively. The highest incidence of TEAEs was various laboratory abnormalities. 2 patients withdrew due to T-drug-related AEs. Only one patient experienced serious adverse events and no death occurred in this study. There were no significant differences between the safety profiles of the generic formulation and Caelyx®. Conclusions: Bioequivalence between the test and the reference products was established for free and encapsulated doxorubicin. Clinical trial registration: http://www.chinadrugtrials.org.cn, identifier [CTR20210375].

NK cell-derived exosomes enhance the anti-tumor effects against ovarian cancer by delivering cisplatin and reactivating NK cell functions.

Exosomes are membranous vesicles actively secreted by almost all cells and they deliver certain intracellular molecules, including nucleic acids, proteins, and lipids, to target cells. They are also considered to be good carriers for drug delivery due to their biocompatibility, high permeability, low immunogenicity, and low toxicity. Exosomes from immune cells were also reported to have immunomodulatory activities. Herein we evaluated the application of exosomes derived from expanded natural killer cells (eNK-EXO) for the treatment of ovarian cancer (OC). We demonstrate that eNK-EXO express typical protein markers of natural killer (NK) cells, can be preferentially uptaken by SKOV3 cells, and display cytotoxicity against OC cells. Furthermore, eNK-EXO loaded with cisplatin could sensitize drug-resistant OC cells to the anti-proliferation effect of cisplatin. In addition, we show that eNK-EXO could activate NK cells from immunosuppressive tumor microenvironment, the mechanism of which is explored by transcriptional analysis. In summary, eNK-EXO exhibit anti-tumor activity against OC on its own, could be used to deliver cisplatin and enhance its cytotoxic effect against drug-resistant OC cells and also reverse the immunosuppression of NK cells, which may lead to great prospect of using eNK-EXO in the treatment of OC in the clinic. Our work also builds a strong foundation for further evaluation of eNK-EXO in other solid tumor therapies.

Genetic characteristics of a patient with multiple primary cancers: A case report.

BACKGROUND: Two or multiple primary malignant neoplasms (MPMNs) rarely occur in the same patient. It has been reported that MPMNs are easily misdiagnosed as the recurrence or metastasis of malignancies in clinical practice, affecting the choice of treatment for the patients, thereby resulting in the delay of optimal diagnosis. Next generation sequencing (NGS) can be used to distinguish between multiple primary lung cancers and intrapulmonary metastasis, and may distinguish the origin of tumours in different sites of the body. CASE SUMMARY: We report the case of 66-year-old woman who suffered from different malignant neoplasms in the rectum and esophageal and gastrointestinal tract. The first neoplasm rectal adenocarcinoma was diagnosed and removed in 2016. The second and third lesions were diagnosed with esophageal squamous-cell carcinoma (ESCC) and gastrointestinal stromal tumour (GIST), respectively, in 2019. Next-generation whole exome sequencing was performed on the tissue specimens of rectal carcinoma, esophageal cancer, GIST, and white blood cells to investigate the relationship between malignancies at different timeframe and determine whether the ESCC and GIST evolved from the rectal adenocarcinoma. Mutations including v-Ki-ras2-Kirsten rat sarcoma viral oncogene homolog, adenomatosis polyposis coli, and mothers against decapentaplegic homolog 4 were detected in rectal adenocarcinoma sample, mast/stem cell growth factor receptor was detected in GIST tissue, and lysine methyltransferase 2D was detected in ESCC specimen. Overall, ESCC and GIST were not genetically evolved from rectal adenocarcinoma, and this patient did not have a trunk driven clone. CONCLUSION: NGS is an effective tool to study clonal evolution of tumours and distinguish between MPMNs and intrapulmonary metastasis.

Reasonable Timing of Radiotherapy for Stage IV Non-Small-Cell Lung Cancer During Targeted Therapy Based on Tumour Volume Change.

PURPOSE: The aim of this study was to investigate the reasonable timing of radiotherapy for stage IV non-small-cell lung cancer (NSCLC) with EGFR-positive mutations during targeted therapy based on tumour volume change (TVC). PATIENTS AND METHODS: Simulation Computed Tomography Scan (SCTS) measurements were taken to test TVC in patients with stage IV NSCLC during targeted therapy at intervals of 10 days. The SCTS measurement was terminated when the tumour volume shrinkage rate in the latter simulation compared with the previous simulation was ≤5% or when the time after treatment was 90 days. Then, primary tumour radiotherapy was performed. Related parameters of the radiotherapy plan were compared between the implementation and simulation plans. RESULTS: Twenty-seven patients were enrolled in the analysis. After treatment, shrinkage of the primary tumour was observed in all patients, but the rate and speed were inconsistent. The average tumour volume decreased obviously within 40 days and was significantly different every 10 days (P ≤ 0.001). The average volume decreased slowly and tended to be stable (P>0.05) after 40 days. After the termination of SCTSs, 21 patients accepted primary tumour radiotherapy. No patients experienced grade 3+ acute radiation toxicity. The implementation radiotherapy plan was significantly better than that before treatment (all P<0.05) but not better than that on the 40th day after treatment (all P>0.05). CONCLUSIONS: To obtain a high radiation dose and control radiation toxicity, the 40th day after targeted therapy may be a reasonable time to start radiotherapy for stage IV NSCLC with EGFR-positive mutations. CLINICAL TRIAL REGISTRATION: https://www.clinicaltrials.gov/ct2/show/NCT03258671, identifier, NCT03258671.

Reovirus enhances cytotoxicity of natural killer cells against colorectal cancer via TLR3 pathway.

BACKGROUND: Cetuximab has been approved for use for first-line treatment of patients with wild-type KRAS metastatic colorectal cancer (CRC). However, treatment with cetuximab has shown limited efficacy as a CRC monotherapy. In addition, natural killer (NK) cell function is known to be severely attenuated in cancer patients. The goal of this study was to develop a new strategy to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by NK cells, in combination with cetuximab against CRC cells. METHODS: Ex vivo expanded NK cells were stimulated with reovirus, and reovirus-activated NK cells mediated ADCC assay were performed on CRC cells in combination with cetuximab. The synergistic antitumor effects of reovirus-activated NK cells and cetuximab were tested on DLD-1 tumor-bearing mice. Finally, Toll-like receptor 3 (TLR3) knockdown in NK cells, along with chemical blockade of TLR3/dsRNA complex, and inhibition of the TLR3 downstream signaling pathway, were performed to explore the mechanisms by which reovirus enhances NK cell cytotoxicity. RESULTS: We first confirmed that exposure of NK cells to reovirus enhanced their cytotoxicity in a dose-dependent manner.We then investigated whether reovirus-activated NK cells exposed to cetuximab-bound CRC cells exhibited greater anti-tumor efficacy than either monotherapy. Co-culture of CRC cell lines with reovirus-activated NK cells indicated that NK cytotoxicity was significantly higher in combination with cetuximab, regardless of KRAS mutation status or EGFR expression level. We also found that reovirus activation of NK cells, in conjunction with cetuximab, resulted in significantly stronger anti-tumor efficacy.Finally, TLR3 knockdown, inhibition of TLR3/dsRNA complex or TBK1/IKKε demonstrated that activation of NK cells by reovirus was dependent on TLR3 and its downstream signaling pathway. CONCLUSIONS: This study demonstrated that combination treatment of reovirus-activated NK cells with cetuximab synergistically enhances their anti-tumor cytotoxicity, suggesting a strong candidate strategy for clinical treatment of CRC.

Dose escalation and expansion (phase Ia/Ib) study of GLS-010, a recombinant fully human antiprogrammed death-1 monoclonal antibody for advanced solid tumors or lymphoma.

BACKGROUND: GLS-010, a novel engineered fully human immunoglobin G4 monoclonal antibody, can specially block the PD-1/PD-L1/2 axis and reactivate the antitumor immunity. AIM: This phase Ia/Ib study was carried out to evaluate the safety, recommended phase II dose (R2PD), and primary antitumor effects of GLS-010 in patients with advanced, refractory lymphoma and solid tumors. METHODS: In phase Ia study, patients with refractory solid tumors and lymphoma enrolled and received GLS-010 at a dose of 1, 4, or 10 mg/kg Q2W; 240 mg Q3W or Q2W. The primary objective was to assess the dose-limiting toxicity (DLT). In phase Ib study, doses were expanded in 9 specific tumors to ensure the R2PD and explore the efficacy. Tumor mutation burden level and PD-L1 expression were also assessed with whole-exome sequencing and immunohistochemistry (SP263), respectively. RESULTS: Up to April 18, 2020, a total of 289 patients (n = 24, phase Ia; n = 265, phase Ib) were enrolled. DLT was not observed in phase Ia part. The T1/2, CLss, and Vd were similar among all dose groups and different tumors. The most common treatment-emergent adverse events (TEAEs) were anemia, leukopenia, elevated alanine aminotransaminase/asparate aminotransferase (ALT/AST), and elevated bilirubin. And hypothyroidism was the most common immune-related adverse event (irAE). The incidence of grade ≥3 TEAE was 39.8%, while grade ≥3 irAE was only 4.5%. Based on safety studies, pharmacokinetics/pharmacodynamics, and preclinical data, 240-mg Q2W was recommended as the expansion dose. The overall objective response rate was 23.6%, with 10 patients achieving complete response. Patients with a high PD-L1 expression level (31.3% Versus. 13.7%, p = 0.012) or t-issue tumor mutation burden level (31.3% Versus. 5.6%, p = 0.009) showed a significantly better response. CONCLUSION: GLS-010 showed acceptable safety profile and favorable clinical response. The dose of 240 mg Q2W was an optimal recommended dose as monotherapy.

Prognostic Model to Predict Overall Survival for Metastatic Non-Small Cell Lung Cancer Patients Treated With Chemotherapy Combined With Concurrent Radiation Therapy to the Primary Tumor: Analysis From Two Prospective Studies.

PURPOSE: The role of radiotherapy, in addition to chemotherapy, has not been thoroughly determined in metastatic non-small cell lung cancer (NSCLC). The purpose of the study was to investigate the prognostic factors and to establish a model for the prediction of overall survival (OS) in metastatic NSCLC patients who received chemotherapy combined with the radiation therapy to the primary tumor. METHODS: The study retrospectively reviewed 243 patients with metastatic NSCLC in two prospective studies. A prognostic model was established based on the results of the Cox regression analysis. RESULTS: Multivariate analysis showed that being male, Karnofsky Performance Status score < 80, the number of chemotherapy cycles <4, hemoglobin level ≤120 g/L, the count of neutrophils greater than 5.8 ×109/L, and the count of platelets greater than 220 ×109/L independently predicted worse OS. According to the number of risk factors, patients were further divided into one of three risk groups: those having ≤ 2 risk factors were scored as the low-risk group, those having 3 risk factors were scored as the moderate-risk group, and those having ≥ 4 risk factors were scored as the high-risk group. In the low-risk group, 1-year OS is 67.7%, 2-year OS is 32.1%, and 3-year OS is 19.3%; in the moderate-risk group, 1-year OS is 59.6%, 2-year OS is 18.0%, and 3-year OS is 7.9%; the corresponding OS rates for the high-risk group were 26.2%, 7.9%, and 0% (P<0.001) respectively. CONCLUSION: Metastatic NSCLC patients treated with chemotherapy in combination with thoracic radiation may be classified as low-risk, moderate-risk, or high-risk group using six independent prognostic factors. This prognostic model may help design the study and develop the plans of individualized treatment.

Simultaneous integrated boost of intensity-modulated radiation therapy to Stage II-III non-small cell lung cancer with metastatic lymph nodes.

Local tumor failure remains a major problem after radiation-based nonsurgical treatment for unresectable locally advanced nonsmall cell lung cancer (NSCLC）and inoperable stage II NSCLC. The aim of this study was to evaluate the feasibility of simultaneous integrated boost of intensity-modulated radiation therapy (SIB-IMRT) to stage II-III NSCLC with metastatic lymph nodes (ChiCTR 2000029304). Patients were diagnosed by pathology or PET-CT. PTV was divided into two parts as follows, the PTV of primary tumor (PTVp) and the PTV of metastatic lymph nodes (PTVn). The radiotherapy doses were simultaneously prescripted 78 Gy (BED = 101.48 Gy) for PTVp and 60-65 Gy (BED = 73.6-81.25 Gy) for PTVn, 26f/5.2 weeks. Response was scored according to WHO criteria. Radiotherapy toxicity was scored according to RTOG criteria. Hematology and gastrointestinal toxicity were scored according to CTCAE1.0 criteria. A total of 20 patients were enrolled. Seventeen patients were diagnosed by pathology and three patients were diagnosed by PET-CT. All patients were treated with SIB-IMRT. The objective response rate (ORR) was 90%, with CR 25%, PR 65%, NC 10%, and PD 0%. Although radiotherapy toxicity was common, there were no grade ≥3, with radiation pneumonitis (10 cases), esophagitis (17 cases), and dermatitis (12 cases). The local control rates at 1, 3, and 5 years were 85%, 75%, and 70%, respectively. The overall survival（OS）and local progression-free survival (LPFS) rates at 1, 3, and 5 years were 90%, 42.6%, and 35.5% and 84.4%, 35.5%, and 28.4%, respectively. SIB-IMRT can significantly improve ORR and survival for stage II-III NSCLC with metastatic lymph nodes, with high safety, and satisfactory efficacy. However, due to the limitation of small sample, these findings are needed to confirm by future trials with a larger sample size.