Identification of RNF213 as a Potential Suppressor of Local Invasion in Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a lethal cancer with poor survival especially when it spreads. The histopathology of its rare intraductal papillary neoplasm of the bile duct type (IPNB) characteristically shows cancer cells originating within the confined bile duct space. These cells eventually invade and infiltrate the nearby liver tissues, making it a good model to study the mechanism of local invasion, which is the earliest step of metastasis. To discover potential suppressor genes of local invasion in ICC, we analyzed the somatic mutation profiles and performed clonal evolution analyses of the 11 pairs of macrodissected locally invasive IPNB tissues (LI-IPNB) and IPNB tissues without local invasion from the same patients. We identified a protein-truncating variant in an E3 ubiquitin ligase, RNF213 (c.6967C>T; p.Gln2323X; chr17: 78,319,102 [hg19], exon 29), as the most common protein-truncating variant event in LI-IPNB samples (4/11 patients). Knockdown of RNF213 in HuCCT1 and YSCCC cells showed increased migration and invasion, and reduced vasculogenic mimicry but maintained normal proliferation. Transcriptomic analysis of the RNF213-knockdown vs control cells was then performed in the HuCCT1, YSCCC, and KKU-100 cells. Gene ontology enrichment analysis of the common differentially expressed genes revealed significantly altered cytokine and oxidoreductase-oxidizing metal ion activities, as confirmed by Western blotting. Gene Set Enrichment Analysis identified the most enriched pathways being oxidative phosphorylation, fatty acid metabolism, reactive oxygen species, adipogenesis, and angiogenesis. In sum, loss-of-function mutation of RNF213 is a common genetic alteration in LI-IPNB tissues. RNF213 knockdown leads to increased migration and invasion of ICC cells, potentially through malfunctions of the pathways related to inflammation and energy metabolisms.

Pharmacogenetic and clinical risk factors for bevacizumab-related gastrointestinal hemorrhage in prostate cancer patients treated on CALGB 90401 (Alliance).

The objective of this study was to discover clinical and pharmacogenetic factors associated with bevacizumab-related gastrointestinal hemorrhage in Cancer and Leukemia Group B (Alliance) 90401. Patients with metastatic castration-resistant prostate cancer received docetaxel and prednisone ± bevacizumab. Patients were genotyped using Illumina HumanHap610-Quad and assessed using cause-specific risk for association between single nucleotide polymorphisms (SNPs) and gastrointestinal hemorrhage. In 1008 patients, grade 2 or higher gastrointestinal hemorrhage occurred in 9.5% and 3.8% of bevacizumab (n = 503) and placebo (n = 505) treated patients, respectively. Bevacizumab (P < 0.001) and age (P = 0.002) were associated with gastrointestinal hemorrhage. In 616 genetically estimated Europeans (n = 314 bevacizumab and n = 302 placebo treated patients), grade 2 or higher gastrointestinal hemorrhage occurred in 9.6% and 2.0% of patients, respectively. One SNP (rs1478947; HR 6.26; 95% CI 3.19-12.28; P = 9.40 × 10-8) surpassed Bonferroni-corrected significance. Grade 2 or higher gastrointestinal hemorrhage rate was 33.3% and 6.2% in bevacizumab-treated patients with the AA/AG and GG genotypes, versus 2.9% and 1.9% in the placebo arm, respectively. Prospective validation of these findings and functional analyses are needed to better understand the genetic contribution to treatment-related gastrointestinal hemorrhage.

HJURP is a cell-cycle-dependent maintenance and deposition factor of CENP-A at centromeres.

The histone H3 variant CenH3, called CENP-A in humans, is central in centromeric chromatin to ensure proper chromosome segregation. In the absence of an underlying DNA sequence, it is still unclear how CENP-A deposition at centromeres is determined. Here, we purified non-nucleosomal CENP-A complexes to identify direct CENP-A partners involved in such a mechanism and identified HJURP. HJURP was not detected in H3.1- or H3.3-containing complexes, indicating its specificity for CENP-A. HJURP centromeric localization is cell cycle regulated, and its transient appearance at the centromere coincides precisely with the proposed time window for new CENP-A deposition. Furthermore, HJURP downregulation leads to a major reduction in CENP-A at centromeres and impairs deposition of newly synthesized CENP-A, causing mitotic defects. We conclude that HJURP is a key factor for CENP-A deposition and maintenance at centromeres.

Head-to-head comparison of three chelates reveals DOTAGA promising for 225 Ac labeling of anti-FZD10 antibody OTSA101.

To select the most suitable chelate for 225 Ac radiolabeling of the anti-FZD10 antibody OTSA101, we directly compared three chelates: S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA), 2,2',2″-(10-(1-carboxy-4-((4-isothiocyanatobenzyl)amino)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (p-SCN-Bn-DOTAGA), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DO3A-NHS-ester). We evaluated the binding affinity of the chelate-conjugated OTSA101 antibodies, as well as the labeling efficiency and stability in murine serum of 225 Ac-labeled OTSA101 as in vitro properties. The biodistribution, intratumoral distribution, absorbed doses, and therapeutic effects of the chelate-conjugated OTSA101 antibodies were assessed in the synovial sarcoma mouse model SYO-1. Of the three conjugates, DOTAGA conjugation had the smallest impact on the binding affinity (p < 0.01). The labeling efficiencies of DOTAGA-OTSA101 and DO3A-OTSA101 were 1.8-fold higher than that of DOTA-OTSA101 (p < 0.01). The stabilities were similar between 225 Ac-labeled DOTA-OTSA101, DOTAGA-OTSA101, and DO3A-OTSA101in serum at 37 and 4°C. The dosimetric analysis based on the biodistribution revealed significantly higher tumor-absorbed doses by 225 Ac-labeled DOTA-OTSA101 and DOTAGA-OTSA101 compared with 225 Ac-DO3A-OTSA101 (p < 0.05). 225 Ac-DOTAGA-OTSA101 exhibited the highest tumor-to-bone marrow ratio, with bone marrow being the dose-limiting tissue. The therapeutic and adverse effects were not significantly different between the three conjugates. Our findings indicate that among the three evaluated chelates, DOTAGA appears to be the most promising chelate to produce 225 Ac-labeled OTSA101 with high binding affinity and high radiochemical yields while providing high absorbed doses to tumors and limited absorbed doses to bone marrow.

Hypoxia-Induced CD36 Expression in Gastric Cancer Cells Promotes Peritoneal Metastasis via Fatty Acid Uptake.

BACKGROUND: The lipid scavenger receptor cluster of differentiation 36 (CD36) has been shown to have a pro-metastatic function in several cancers. Adipose tissue, a favorable site for peritoneal metastasis (PM) from gastric cancer (GC), promotes this process by providing free fatty acids (FFAs); however, the role of CD36 in PM progression from GC remains to be elucidated. MATERIALS AND METHODS: We evaluated CD36 expression in the GC cells under various conditions. CD36 overexpressing (CD36OE) MKN45 cells were prepared and their migration and invasive properties were assessed. A PM mouse model was used to investigate the biological effects of palmitic acid (PA) and CD36. Furthermore, we examined the clinical role of CD36 expression in 82 human PM samples by immunohistochemical staining. RESULTS: Hypoxia markedly increased CD36 expression in GC cells. In normoxia, only CD36OE MKN45 cells treated with PA showed an increase in migration and invasion abilities. An increased expression of active Rac1 and Cdc42 was observed, which decreased following etomoxir treatment. Conversely, hypoxia increased those capacities of both vector and CD36OE MKN45 cells. In a mouse model transplanted with CD36OE MKN45 cells, more peritoneal tumors were observed in the high-fat diet group than those in the normal diet group. In clinical samples, 80% of PM lesions expressed CD36, consistent with hypoxic regions, indicating a significant association with prognosis. CONCLUSION: Our findings indicate that a hypoxia in the peritoneal cavity induces CD36 expression in GC cells, which contributes to PM through the uptake of FFAs.

Pseudomonas otitidis bacteremia in an immunocompromised patient with cellulitis: case report and literature review.

BACKGROUND: Pseudomonas otitidis belongs to the genus Pseudomonas and causes various infections, including ear, skin, and soft tissue infections. P. otitidis has a unique susceptibility profile, being susceptible to penicillins and cephalosporins but resistant to carbapenems, due to the production of the metallo-ß-lactamase called POM-1. This revealed genetic similarities with Pseudomonas aeruginosa, which can sometimes lead to misidentification. CASE PRESENTATION: We report the case of a 70-year-old Japanese male who developed cellulitis and bacteremia during chemotherapy for multiple myeloma. He was initially treated with meropenem, but blood culture later revealed gram-negative bacilli identified as P. otitidis using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Carbapenem resistance was predicted from previous reports; therefore, we switched to dual therapy with levofloxacin and cefepime, and favorable treatment results were obtained. CONCLUSION: This is the first reported case of P. otitidis cellulitis and bacteremia in an immunocompromised patient. Carbapenems are typically used in immunocompromised patients and P. otitidis is often resistant to it. However, its biochemical properties are similar to those of Pseudomonas aeruginosa; therefore, its accurate identification is critical. In the present study, we rapidly identified P. otitidis using MALDI-TOF MS and switched from carbapenems to an appropriate antimicrobial therapy, resulting in a successful outcome.

Randomized phase I/II study of vascular endothelial growth factor receptor peptide vaccines for patients with hepatocellular carcinoma.

AIM: We evaluated the safety and efficacy of vascular endothelial growth factor receptor (VEGFR)-targeted peptide vaccines for the immunization of patients with unresectable hepatocellular carcinoma (HCC) who had responded to transarterial chemoembolization. METHODS: Twenty-two patients were randomized 1:1 to receive VEGFR-targeted peptides or placebo. The primary end-point was the safety assessment of the immunization. The secondary end-points were evaluation of immunological responses and clinical outcomes. RESULTS: No severe adverse events were induced by the study agents. Among the 12 patients in the vaccine group, a VEGFR1-specific cytotoxic T lymphocyte (CTL) response was induced in eight (66.7%) patients and a VEGFR2-specific CTL response was induced in 10 (83.3%). The median progression-free survival (PFS) and overall survival (OS) rates were 4.8 and 52.0 months, respectively, in the vaccine group, and 2.7 and 21.8 months, respectively, in the placebo group. No statistically significant differences were found between the two groups (PFS p = 0.925, OS p = 0.190). When divided into two groups according to immunoreactivity, the median PFS of patients with and without a strong immune response to VEGFR1 were 7.4 and 2.7 months, and that to VEGFR2 were 10.6 and 2.7 months, respectively; there were significant differences according to the immune response. CONCLUSIONS: Immunotherapy with peptide vaccines targeting VEGFR1 and VEGFR2 was well tolerated with no serious adverse events. It also effectively induced peptide-specific CTLs in patients with unresectable HCC.

Treatment satisfaction with molidustat in CKD-related anemia in non-dialysis patients: a post-hoc analysis of two clinical trials.

BACKGROUND: Erythropoiesis-stimulating agents (ESAs) are the standard treatment for patients with renal anemia to increase hemoglobin (Hb) levels and reduce the need for blood transfusions. However, treatments targeting high Hb levels require high doses of ESAs administered intravenously, which is associated with an elevated risk of adverse cardiovascular events. Furthermore, there have been some problems such as hemoglobin variability and low achievement of target hemoglobin due to the shorter half-lives of ESAs. Consequently, erythropoietin-promoting medications, such as hypoxia-inducible factor-prolyl hydroxylase (HIF-PH) inhibitors, have been developed. This study aimed to evaluate changes in the Treatment Satisfaction Questionnaire for Medicine version II (TSQM-II) domain scores relative to baseline in each trial, to assess patient satisfaction with molidustat versus darbepoetin alfa. METHODS: This post-hoc analysis of two clinical trials compared treatment satisfaction with an HIF-PH inhibitor, molidustat, versus a standard ESA, darbepoetin alfa, as part of therapy in patients with non-dialysis chronic kidney disease (CKD) and renal anemia. RESULTS: Exploratory outcome data using the TSQM-II showed that both arms in both trials had enhanced treatment satisfaction over the course of the study period, as well as improvements in most TSQM-II domains at week 24 of treatment. Molidustat was associated with convenience domain scores at multiple time points depending on the trial. More patients were highly satisfied with the convenience of molidustat than that of darbepoetin alfa. Patients treated with molidustat had increased global satisfaction domain scores compared with those treated with darbepoetin alfa; however, the differences in global satisfaction domain scores were not significant. CONCLUSION: These patient-reported satisfaction outcomes support the use of molidustat as a patient-centered treatment option for CKD-related anemia. REGISTRATION OF CLINICAL TRIALS: ClinicalTrials.gov Identifier: NCT03350321 (November 22, 2017). CLINICALTRIALS: gov Identifier: NCT03350347 (November 22, 2017).

CHOROIDAL VASCULAR DENSITY IN DIABETIC RETINOPATHY ASSESSED WITH SWEPT-SOURCE OPTICAL COHERENCE TOMOGRAPHY.

PURPOSE: We aimed to assess choroidal vascularity by diabetic retinopathy (DR) stage using the choroidal vascular density (CVD) obtained from swept-source optical coherence tomography en-face images. METHODS: This prospective, cross-sectional, multicenter study included patients from Niigata City General Hospital and Saiseikai Niigata Hospital between October 2016 and October 2017. Choroidal vascular density was obtained by binarizing swept-source optical coherence tomography en-face images of patients with diabetes and those with DR, patients without DR, and healthy age-matched volunteers. RESULTS: Patients were allocated to the healthy control (n = 28), no DR (n = 23), nonproliferative DR (NPDR) without diabetic macular edema (DME) (n = 50), NPDR + DME (n = 38), and proliferative DR (PDR) or any previous treatment with panretinal photocoagulation (n = 26) groups. Investigation of the choriocapillaris slab level indicated that the no DR group had significantly high CVD values ( P < 0.05), and the PDR groups had significantly low CVD values ( P < 0.01). Investigation of the large choroidal vessel level indicated that the NPDR + DME and PDR groups had significantly lower CVD values than the control group ( P < 0.05 and P < 0.01, respectively). CONCLUSION: We found that at the choriocapillaris slab level, the no DR group had a higher CVD and the NPDR with DME and PDR groups had a lower CVD than the control group. At the level of the large choroidal vessels, the NPDR with DME and PDR groups had a lower CVD than the control group. There were significant differences in choroidal vasculature found using CVD obtained from swept-source optical coherence tomography en-face images of patients with diabetes and DR.

[Significance of Inflammatory/Nutritional Index in Pathological Stage â ¡-â ¢ Colorectal Cancer].

The aim of this study was to evaluate the inflammatory/nutritional index in patients with colorectal cancer. A total of 600 patients with pStage Ⅱ-Ⅲ colorectal cancer who underwent radical resection at our hospital between January 2008 and September 2022 were retrospectively reviewed. Onodera's prognostic nutritional index(OPNI), CRP-to-albumin ratio, modified Glasgow prognostic score, neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, lymphocyte-to-monocyte ratio were measured preoperatively. Clinical and pathological data were assessed using univariate and multivariate analysis to determine prognostic factors for overall survival(OS), relapse-free survival(RFS)and post-relapse overall survival (PROS). Moreover, these patients were divided into high and low groups based on OPNI, these survival outcome for OS, RFS and PROS were assessed using Kaplan-Meier analysis with the logrank test. In multivariate analysis, the independent prognostic factors were gender, age, OPNI, histological type, pStage for OS, gender, OPNI, venous invasion and pStage for RFS, and OPNI, histological type and resection of recurrent site for PROS. In Kaplan-Meier analysis, patients in the low OPNI group had significant poor prognosis for OS, RFS and PROS. OPNI is a useful prognostic factor in colorectal cancer.

FZD10-targeted α-radioimmunotherapy with 225 Ac-labeled OTSA101 achieves complete remission in a synovial sarcoma model.

Synovial sarcomas are rare tumors arising in adolescents and young adults. The prognosis for advanced disease is poor, with an overall survival of 12-18 months. Frizzled homolog 10 (FZD10) is overexpressed in most synovial sarcomas, making it a promising therapeutic target. The results of a phase 1 trial of ß-radioimmunotherapy (RIT) with the 90 Y-labeled anti-FZD10 antibody OTSA101 revealed a need for improved efficacy. The present study evaluated the potential of α-RIT with OTSA101 labeled with the α-emitter 225 Ac. Competitive inhibition and cell binding assays showed that specific binding of 225 Ac-labeled OTSA101 to SYO-1 synovial sarcoma cells was comparable to that of the imaging agent 111 In-labeled OTSA101. Biodistribution studies showed high uptake in SYO-1 tumors and low uptake in normal organs, except for blood. Dosimetric studies showed that the biologically effective dose (BED) of 225 Ac-labeled OTSA101 for tumors was 7.8 Bd higher than that of 90 Y-labeled OTSA101. 90 Y- and 225 Ac-labeled OTSA101 decreased tumor volume and prolonged survival. 225 Ac-labeled OTSA101 achieved a complete response in 60% of mice, and no recurrence was observed. 225 Ac-labeled OTSA101 induced a larger amount of necrosis and apoptosis than 90 Y-labeled OTSA101, although the cell proliferation decrease was comparable. The BED for normal organs and tissues was tolerable; no treatment-related mortality or obvious toxicity, except for temporary body weight loss, was observed. 225 Ac-labeled OTSA101 provided a high BED for tumors and achieved a 60% complete response in the synovial sarcoma mouse model SYO-1. RIT with 225 Ac-labeled OTSA101 is a promising therapeutic option for synovial sarcoma.

Serial circulating tumor DNA monitoring of CDK4/6 inhibitors response in metastatic breast cancer.

Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) significantly improve progression-free survival and have become the standard therapy for estrogen receptor-positive/human epidermal growth factor receptor 2-negative metastatic breast cancer patients. Treatment surveillance by radiological imaging has some limitations in detection and repeated biopsy genomic profiling is not clinically feasible. Serial circulating tumor DNA (ctDNA) analysis may provide insights into treatment response. Here we performed serial ctDNA analysis (n = 178) on 33 patients. Serial ctDNA analysis identified disease progression with sensitivity of 75% and specificity of 92%. In eight of 12 patients (61%) responding to CDK4/6i who eventually developed progressive disease, serial sampling every 3 or 6 months captured the initial rise of ctDNA with an average lead time of 3 months. In three of eight patients that did not respond to CDK4/6i (progressive disease at first radiological assessment, 3 months), biweekly sequencing within the first cycle of CDK4/6i treatment (1 month) detected sustained ctDNA levels (≥0.2% variant allele frequency), with lead time of 2 months. Serial ctDNA analysis tracked RECIST response, including clinically challenging scenarios (bone metastases or small-sized target lesions), as well as detecting acquired genetic alterations linked to CDK4/6i resistance in the G1 to S transition phase. Circulating tumor DNA analysis was more sensitive than carcinoembryonic antigen or cancer antigen 15-3 serum tumor markers at monitoring tumor response to CDK4/6i treatment. Our findings indicated the possible clinical utility of serial ctDNA analysis for earlier progressive disease detection and real-time monitoring of CDK4/6i response.

Two polymorphic gene loci associated with treprostinil dose in pulmonary arterial hypertension.

OBJECTIVE: Prostacyclin infusion for pulmonary arterial hypertension (PAH) is an effective therapy with varied dosing requirements and clinical response. The major aim of this study was to determine new biologically-based predictors of prostacyclin treatment response heterogeneity. METHODS: Ninety-eight patients with hemodynamically defined PAH at two academic medical centers volunteered for registry studies. A stable dose of treprostinil was the quantitative phenotype for the genome-wide association study (GWAS). Candidate genes with the largest effect sizes and strongest statistical associations were further characterized with in silico and in-vitro assays to confirm mechanistic hypotheses. The clinical significance of these candidate predictors was assessed for mechanistically consistent physiologic effects in an independent cohort of patients. RESULTS: GWAS identified three loci for association with P < 10-6. All three loci had clinically significant effect sizes. Specific single-nucleotide polymorphisms (SNPs) at two of the loci: rs11078738 in phosphoribosylformylglycinamidine synthase and rs10023113 in CAMK2D encoded sequence changes with clear predicted consequences. Production of the primary mediator of prostacyclin-induced vasodilation, cyclic AMP, was reduced in human cell lines by the missense variant rs11078738 (p.L621P). Located in the promoter of CAMK2D, the allele of rs10023113 associated with a higher treprostinil dose has higher ventricular transcription of CAMK2δ. At initial diagnostic catheterization in a separate cohort of patients, the same allele of rs10023113 was associated with elevated right mean atrial and ventricular diastolic pressures. CONCLUSIONS: The quantitative phenotype of stable treprostinil dose identified two gene loci associated with pharmacodynamic response and right ventricular function in PAH worth further investigation.

Phase II Adjuvant Cancer-specific Vaccine Therapy for Esophageal Cancer Patients Curatively Resected After Preoperative Therapy With Pathologically Positive Nodes; Possible Significance of Tumor Immune Microenvironment in its Clinical Effects.

OBJECTIVES: To elucidate the efficacy of adjuvant vaccine monotherapy using 3 Human Leukocyte Antigen (HLA)-A∗24-restricted tumor-specific peptide antigens for ESCC, upregulated lung cancer 10, cell division cycle associated 1, and KH domain-containing protein overexpressed in cancer 1. SUMMARY OF BACKGROUND DATA: ESCC patients with pathologically positive nodes (pN(+)) have a high risk for postoperative recurrence, despite curative resection after preoperative therapy. Subclinical micrometastases are an appropriate target for cancer vaccine. METHODS: This is a non-randomized prospective phase II clinical trial (UMIN000003557). ESCC patients curatively resected after preoperative therapy with pN(+) were allocated into the control and vaccine groups (CG and VG) according to the HLA-A status. One mg each of three epitope peptides was postoperatively injected 10 times weekly followed by 10 times biweekly to the VG. The primary and secondary endpoints were relapse-free survival (RFS) and esophageal cancer-specific survival (ECSS), respectively. RESULTS: Thirty were in the CG and 33 in the VG. No significant difference was observed in RFS between the CG and VG (5-year RFS: 32.5% vs 45.3%), but the recurrence rate significantly decreased with the number of peptides which induced antigen-specific cytotoxic T lymphocytes. The VG showed a significantly higher 5-year ECSS than the CG (60.0% vs 32.4%, P = 0.045) and this difference was more prominent in patients with CD8+ and programmed death-ligand 1 double negative tumor (68.0% vs 17.7%, P = 0.010). CONCLUSIONS: Our cancer peptide vaccine might improve the survival of ESCC patients, which is warranted to be verified in the phase III randomized controlled study.

Lymphocytes in tumor-draining lymph nodes co-cultured with autologous tumor cells for adoptive cell therapy.

BACKGROUND: Tumor-draining lymph nodes (TDLNs) are primary sites, where anti-tumor lymphocytes are primed to tumor-specific antigens and play pivotal roles in immune responses against tumors. Although adoptive cell therapy (ACT) using lymphocytes isolated from TDLNs were reported, characterization of immune activity of lymphocytes in TDLNs to tumor cells was not comprehensively performed. Here, we demonstrate TDLNs to have very high potential as cell sources for immunotherapy. METHODS: Lymphocytes from TDLNs resected during surgical operation were cultured with autologous-tumor cells for 2 weeks and evaluated tumor-reactivity by IFNγ ELISPOT assay. We investigated the commonality of T cell receptor (TCR) clonotypes expanded by the co-culture with tumor cells with those of tumor infiltrating lymphocytes (TILs). RESULTS: We found that that TCR clonotypes of PD-1-expressing CD8+ T cells in lymph nodes commonly shared with those of TILs in primary tumors and lymphocytes having tumor-reactivity and TCR clonotypes shared with TILs could be induced from non-metastatic lymph nodes when they were co-cultured with autologous tumor cells. CONCLUSION: Our results imply that tumor-reactive effector T cells were present even in pathologically non-metastatic lymph nodes and could be expanded in vitro in the presence of autologous tumor cells and possibly be applied for ACT.

Identification of cytotoxic T cells and their T cell receptor sequences targeting COVID-19 using MHC class I-binding peptides.

Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) was first reported in China in December 2019, various variants have been identified in different areas of the world such as United Kingdom (alpha), South Africa (beta and omicron), Brazil (gamma), and India (delta). Some of SARS-CoV-2 variants, each of which is characterized by a unique mutation(s) in spike protein, are concerned due to their high infectivity and the capability to escape from neutralizing antibodies elicited by vaccinations. To identify peptide epitopes that are derived from SARS-CoV-2 viral proteins and possibly induce CD8+ T cell immunity, we investigated SARS-CoV-2-derived peptides that are likely to bind to major histocompatibility complex (MHC) class I molecules. We identified a total of 15 peptides that bind to human leukocyte antigen (HLA)-A*24:02, HLA-A*02:01, or HLA-A*02:06, and possibly induce cytotoxic T lymphocytes (CTLs); thirteen of them corresponded to ORF1ab polyprotein, one peptide to spike protein and the remaining one to membrane glycoprotein. CD8+ T cells that recognize these peptides were detected in peripheral blood samples in three individuals recovered from COVID-19 as well as non-infected individuals. Since most of these peptides are commonly conserved among other coronaviruses including SARS-CoV and/or MERS-CoV, these might be useful to maintain T cell responses to coronaviruses that are pandemic at present and will become the future threat. We could define pairs of TRA and TRB sequences of nine CTL clones that recognize SARS-CoV-2-derived peptides. We might use these SARS-CoV-2-derived peptide-reactive TCR sequences for investigating the history of SARS-CoV-2 infection.

Contribution of pre-existing neoantigen-specific T cells to a durable complete response after tumor-pulsed dendritic cell vaccine plus nivolumab therapy in a patient with metastatic salivary duct carcinoma.

Although immune checkpoint inhibitors (ICIs) have emerged as new therapeutic options for refractory cancer, they are only effective in select patients. Tumor antigen-pulsed dendritic cell (DC) vaccine therapy activates tumor-specific cytotoxic T lymphocytes, making it an important immunotherapeutic strategy. Salivary ductal carcinoma (SDC) carries a poor prognosis, including poor long-term survival after metastasis or recurrence. In this study, we reported a case of refractory metastatic SDC that was treated with a tumor lysate-pulsed DC vaccine followed by a single injection of low-dose nivolumab, and a durable complete response was achieved. We retrospectively analyzed the immunological factors that contributed to these long-lasting clinical effects. First, we performed neoantigen analysis using resected metastatic tumor specimens obtained before treatment. We found that the tumor had 256 non-synonymous mutations and 669 class I high-affinity binding neoantigen peptides. Using synthetic neoantigen peptides and ELISpot analysis, we found that peripheral blood mononuclear leukocytes cryopreserved before treatment contained pre-existing neoantigen-specific T cells, and the cells obtained after treatment exhibited greater reactivity to neoantigens than those obtained before treatment. Our results collectively suggest that the rapid and long-lasting effect of this combination therapy in our patient may have resulted from the presence of pre-existing neoantigen-specific T cells and stimulation and expansion of those cells following tumor lysate-pulsed DC vaccine and ICI therapy.

Crosstalk between cancer-associated fibroblasts and immune cells in peritoneal metastasis: inhibition in the migration of M2 macrophages and mast cells by Tranilast.

BACKGROUND: The role of tumor-stroma interactions in tumor immune microenvironment (TME) is attracting attention. We have previously reported that cancer-associated fibroblasts (CAFs) contribute to the progression of peritoneal metastasis (PM) in gastric cancer (GC), and M2 macrophages and mast cells also contribute to TME of PM. To elucidate the role of CAFs in TME, we established an immunocompetent mouse PM model with fibrosis, which reflects clinical features of TME. However, the involvement of CAFs in the immunosuppressive microenvironment remains unclear. In this study, we investigated the efficacy of Tranilast at modifying this immune tolerance by suppressing CAFs. METHODS: The interaction between mouse myofibroblast cell line LmcMF and mouse GC cell line YTN16 on M2 macrophage migration was investigated, and the inhibitory effect of Tranilast was examined in vitro. Using C57BL/6J mouse PM model established using YTN16 with co-inoculation of LmcMF, TME of resected PM treated with or without Tranilast was analyzed by immunohistochemistry. RESULTS: The addition of YTN16 cell-conditioned medium to LmcMF cells enhanced CXCL12 expression and stimulated M2 macrophage migration, whereas Tranilast inhibited the migration ability of M2 macrophages by suppressing CXCL12 secretion from LmcMF. In PM model, Tranilast inhibited tumor growth and fibrosis, M2 macrophage, and mast cell infiltration and significantly promoted CD8 + lymphocyte infiltration into the tumor, leading to apoptosis of cancer cells by an immune response. CONCLUSION: Tranilast improved the immunosuppressive microenvironment by inhibiting CAF function in a mouse PM model. Tranilast is thus a promising candidate for the treatment of PM.

Tumor-Informed Approach Improved ctDNA Detection Rate in Resected Pancreatic Cancer.

Pancreatic cancer is one of the cancers with very poor prognosis; there is an urgent need to identify novel biomarkers to improve its clinical outcomes. Circulating tumor DNA (ctDNA) from liquid biopsy has arisen as a promising biomarker for cancer detection and surveillance. However, it is known that the ctDNA detection rate in resected pancreatic cancer is low compared with other types of cancer. In this study, we collected paired tumor and plasma samples from 145 pancreatic cancer patients. Plasma samples were collected from 71 patients of treatment-naïve status and from 74 patients after neoadjuvant therapy (NAT). Genomic profiling of tumor DNA and plasma samples was conducted using targeted next-generation sequencing (NGS). Somatic mutations were detected in 85% (123/145) of tumors. ctDNA was detected in 39% (28/71) and 31% (23/74) of treatment-naïve and after-NAT groups, respectively, without referring to the information of tumor profiles. With a tumor-informed approach (TIA), ctDNA detection rate improved to 56% (40/71) and 36% (27/74) in treatment-naïve and after-NAT groups, respectively, with the detection rate significantly improved (p = 0.0165) among the treatment-naïve group compared to the after-NAT group. Cases who had detectable plasma ctDNA concordant to the corresponding tumor showed significantly shorter recurrence-free survival (RFS) (p = 0.0010). We demonstrated that TIA improves ctDNA detection rate in pancreatic cancer, and that ctDNA could be a potential prognostic biomarker for recurrence risk prediction.

Metabolic changes induced by TGF-ß1 via reduced expression of phosphatidylserine decarboxylase during myofibroblast transition.

Metabolic alteration is increasingly recognized as an important pathogenic process that underlies fibrosis across many organ types, and metabolically targeted therapies could become important strategies for reducing fibrosis. In present study, target enzymes that are involved in changes in phospholipid metabolism during fibroblast-to-myofibroblast transition induced by transforming growth factor beta 1 (TGF-ß1) were examined. Different amounts of phospholipids were found in the 2 groups. In response to TGF-ß1 stimulation, 17 lipids decreased and 17 increased. The latter included the phospholipids phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE). Furthermore, among the rate-limiting enzymes that regulate these phospholipids, phosphatidylserine decarboxylase (PISD), which controls conversion of PS to PE and is localized in mitochondria, decreased in response to TGF-ß1. Knockdown of PISD alone without TGF-ß1 stimulation increased expression of α-smooth muscle actin mRNA and production of total collagen. Taken together, these results indicate that PISD is involved in the mechanism of fibrogenesis by regulating phospholipid metabolism.