Altered mucosal immunity in HIV-positive colon adenoma: decreased CD4+ T cell infiltration is correlated with nadir but not current CD4+ T cell blood counts.

BACKGROUND: People living with HIV (PLWH) face greater risks of developing non-AIDS-defining cancers (NADCs) than the general population; however, the underlying mechanisms remain elusive. The tumor microenvironment plays a significant role in the carcinogenesis of colorectal cancer (CRC), an NADC. We studied this carcinogenesis in PLWH by determining inflammatory phenotypes and assessing PD-1/PD-L1 expression in premalignant CRC stages of colon adenomas in HIV-positive and HIV-negative patients. METHODS: We obtained polyp specimens from 22 HIV-positive and 61 HIV-negative participants treated with colonoscopy and polyp excision. We analyzed adenomas from 33 HIV-positive and 99 HIV-negative patients by immunohistochemistry using anti-CD4, anti-CD8, anti-FoxP3, and anti-CD163 antibodies. Additionally, we analyzed the expression levels of immune checkpoint proteins. We also evaluated the correlation between cell infiltration and blood cell counts. RESULTS: HIV-positive participants had fewer infiltrating CD4+ T cells than HIV-negative participants (p = 0.0016). However, no statistical differences were observed in infiltrating CD8+ and FoxP3+ T cells and CD163+ macrophages. Moreover, epithelial cells did not express PD-1 or PD-L1. Notably, CD4+ T cell infiltration correlated with nadir blood CD4+ T cell counts (p <  0.05) but not with current blood CD4+ T cell counts. CONCLUSION: Immune surveillance dysfunction owing to decreased CD4+ T cell infiltration in colon adenomas might be involved in colon carcinogenesis in HIV-positive individuals. Collectively, since the nadir blood CD4+ T cell count is strongly correlated with CD4+ T cell infiltration, it could facilitate efficient follow-up and enable treatment strategies for HIV-positive patients with colon adenomas.

Clinical Trial of a Cancer Vaccine Targeting VEGF and KIF20A in Advanced Biliary Tract Cancer.

BACKGROUND: As the prognosis of biliary tract cancer (BTC) is extremely poor and treatment options are limited, new treatment modalities are urgently needed. We designed a phase II clinical trial to investigate the immune responses and clinical benefits of OCV-C01, an HLA-A*24:02-restricted three-peptide cancer vaccine targeting VEGFR1, VEGFR2, and KIF20A. PATIENTS AND METHODS: Participants were patients with advanced BTC who had unresectable tumours and were refractory to standard chemotherapy. OCV-C01 was injected weekly until the discontinuance criteria were met. RESULTS: Six participants, including four patients positive for HLA-A*24:02, were enrolled in this study for assessment of efficacy. Four out of six patients exhibited vaccine-specific T-cell responses to one or more of three antigens. Log-rank tests revealed that vaccine-specific T cell responses contributed significantly to overall survival. CONCLUSION: The cancer vaccine had positive effects on survival, indicating that this approach warrants further clinical studies.

Gastrointestinal lesion in adult-onset Langerhans cell histiocytosis.

BACKGROUND: Langerhans cell histiocytosis (LCH) is a rare disease primarily occurring in children, and commonly involves the bone and skin; gastrointestinal tract involvement is notably rare. The incidence and significance of gastrointestinal lesions in adult LCH are unclear; thus, we aimed to investigate adult Japanese cases of LCH and clarify the features of gastrointestinal involvement. METHODS: We gathered clinical information on 43 Japanese cases of adult LCH and analyzed patient backgrounds, affected organs, features of the gastrointestinal lesions, and the clinical courses. RESULTS: Thirteen patients underwent endoscopic examinations: an upper gastrointestinal endoscopy alone in 5, lower gastrointestinal endoscopy alone in 3, and both in 5 patients. A gastric lesion (one case), colonic lesion (one case), and both gastric and rectal lesions (one case) were detected. The three cases of gastrointestinal involvement also exhibited nongastrointestinal multisystem LCH lesions and showed no gastrointestinal symptoms or increased uptake on positron emission tomography. Endoscopy revealed small erosions without specific features; histological examinations were required for diagnosis. These three cases were treated with chemotherapy, comprising vinblastine/prednisolone, methotrexate, and daily 6-mercaptopurine, for 36 weeks; in two cases, the clinical condition remained stable for several years post-treatment. One case showed recurrence 1 year 7 months after treatment, and chemotherapy was re-administered. No case with single-system disease exhibited gastrointestinal involvement. CONCLUSIONS: Although gastrointestinal LCH lesions are rare, they were more common than expected in our cases of multisystem LCH. However, these lesions were relatively small and did not affect the patients' clinical courses.

Embolization coil migration in the stomach and spontaneous excretion: a case report and review of the literature.

Embolization coil migration to the gastrointestinal tract is a rare complication. This report describes our experience of coil migration in the stomach and spontaneous excretion. A 77-year-old man, who was diagnosed with esophageal squamous cell carcinoma with multiple lymph node metastases, had a bleeding left gastric artery and splenic artery pseudoaneurysm associated with an abdominal lymph node mass, that was treated by coil embolization, after which the coil migrated into the stomach. Because there were no complications such as active bleeding or peritonitis, our patient was followed carefully, and excretion of the coil was documented. No standard management exists for migrated coils. Conservative treatment is an option, as in this case.

Safe Use of Nivolumab in a Patient with Epipharyngeal Carcinoma and Preexisting Ulcerative Colitis: A Histologically Proven Case Report.

Nivolumab, an antibody against human programmed cell death 1 (PD-1), enhances pre-existing immune responses and has antitumor activity. However, it may also cause undesirable immune-related adverse events (irAEs), such as anti-PD-1-related colitis. In addition, Nivolumab can worsen pre-existing autoimmune diseases. Ulcerative colitis (UC) is a chronic inflammatory disease of the colon. Its exact cause is unknown, but it may involve the dysregulation of the mucosal immune response. Thus, it is of great interest whether nivolumab can affect UC activity. This is the first report of a patient with epipharyngeal carcinoma and ulcerative colitis who was confirmed to have been safely treated with nivolumab based on autopsy findings.

A pilot study to establish human T-cell leukemia virus type 1 (HTLV-1) carrier model using common marmoset (Callithrix jacchus).

BACKGROUND: For the diagnosis and treatment of adult T-cell leukemia/lymphoma (ATLL) caused by human T-lymphotropic virus type 1 (HTLV-1) are required therapeutic modalities urgently. Non-human primate models for ATLL would provide a valuable information for clinical studies. We did a pilot study to establish an ATLL non-human primate model using common marmosets (Callithrix jacchus). METHODS: We inoculated HTLV-1-producing MT-2 cells into 9-month-old marmosets, either intraperitoneally or intravenously. We next administrated MT-2 cells into 13-month-old marmosets under cyclosporine A (CsA) treatment to promote infection. HTLV-1 infection was determined by measuring HTLV-1 antibody titer in the common marmosets. RESULTS: The HTLV-1 antibody titer increased in the intraperitoneally inoculated marmoset with or without CsA treatment, and it kept over five 5 years though proviral copy number (proviral load, PVL) remained low throughout the study. CONCLUSION: We obtained HTLV-1 asymptomatic carriers of common marmosets by inoculating MT-2 cells.

Non-transmissible MV Vector with Segmented RNA Genome Establishes Different Types of iPSCs from Hematopoietic Cells.

Recent advances in gene therapy technologies have enabled the treatment of congenital disorders and cancers and facilitated the development of innovative methods, including induced pluripotent stem cell (iPSC) production and genome editing. We recently developed a novel non-transmissible and non-integrating measles virus (MV) vector capable of transferring multiple genes simultaneously into a wide range of cells through the CD46 and CD150 receptors. The MV vector expresses four genes for iPSC generation and the GFP gene for a period of time sufficient to establish iPSCs from human fibroblasts as well as peripheral blood T cells. The transgenes were expressed differentially depending on their gene order in the vector. Human hematopoietic stem/progenitor cells were directly and efficiently reprogrammed to naive-like cells that could proliferate and differentiate into primed iPSCs by the same method used to establish primed iPSCs from other cell types. The novel MV vector has several advantages for establishing iPSCs and potential future applications in gene therapy.

Extremely Low Organ Toxicity and Strong Antitumor Activity of miR-34-Regulated Oncolytic Coxsackievirus B3.

Oncolytic virotherapies have emerged as new modalities for cancer treatment. We previously reported that coxsackievirus B3 (CVB3) is a novel oncolytic virus (OV) with a strong ability to lyse human non-small cell lung cancer cells; however, its non-specific toxicity against normal cells remains to be resolved. To improve its safety profile, microRNA target sequences complementary to miR-34a/c, which is expressed preferentially in normal cells, were inserted into the 5' UTR or 3' UTR of the CVB3 genome. In the presence of miR-34a/c, the gene-modified CVB3 could not replicate in normal cells. We also found that the pathogenicity of CVB3 was reduced to a greater extent by targeting miR-34a than miR-34c; in addition, it was more effective to insert the target sequences into the 3' UTR rather than the 5' UTR of the viral genome. Ultimately, we developed a double-miR-34a targeting virus (53a-CVB) by inserting miR-34a targets in both the 5' UTR and 3' UTR of the virus. 53a-CVB was minimally toxic to cells in normal tissue, but maintained nearly its full oncolytic activity in mice xenografted with human lung cancer. 53a-CVB is the first miR-34-regulated OV and represents a promising platform for the development of safe and effective anti-cancer therapies.

A Novel Combination Therapy for Human Oxaliplatin-resistant Colorectal Cancer Using Oxaliplatin and Coxsackievirus A11.

BACKGROUND: Colorectal cancer (CRC) is a major cause of morbidity and mortality throughout the world. It is the third most common cancer worldwide and the fourth most common cause of cancer-related death. FOLFOX, a combination of leucovorin calcium, fluorouracil, and oxaliplatin, is the first-line chemotherapy for stage III and stage IV CRC. However, patients with FOLFOX-resistant CRC have a poor prognosis. In recent years, virochemotherapy has been proposed as a potential treatment for chemotherapy-resistant cancer. MATERIALS AND METHODS: Through our first screening assay, we found that coxsackievirus A11 (CVA11) displayed potent oncolytic activities. We tested whether coxsackievirus A11 (CVA11) has oncolytic activity in human CRC cells in vitro and in vivo. We also examined whether pretreatment of oxaliplatin-resistant CRC cells with oxaliplatin enhances the oncolytic activity of CVA11. RESULTS: We found that CVA11 was potently oncolytic against the oxaliplatin-sensitive Caco-2 cell line, but had little effect on the oxaliplatin-resistant line WiDr. However, pretreatment of WiDr cells with oxaliplatin enhanced the oncolytic activity of CVA11, and the combination therapy was more cytotoxic than either oxaliplatin treatment or CVA11 infection alone. Furthermore, growth of subcutaneous WiDr tumors in a xenograft model was significantly lower in mice treated with oxaliplatin followed by intratumoral CVA11 injection compared with mice receiving either treatment alone. CONCLUSION: Oxaliplatin pretreatment sensitized oxaliplatin-resistant CRC cells to lysis by CVA11 infection in vitro and in vivo. Taken together, these findings identify a novel potential chemovirotherapeutic modality for the treatment of oxaliplatin-resistant human CRC.

A phase I clinical trial of RNF43 peptide-related immune cell therapy combined with low-dose cyclophosphamide in patients with advanced solid tumors.

The objective of this study was to investigate the safety and the tolerability of combined cellular immunotherapy with low-dose cyclophosphamide (CPA) in patients with advanced solid tumors. This study targeted a novel tumor-associated antigen, ring finger protein 43 (RNF43). Eligible patients were resistant to standard therapy, HLA-A*24:02- or A*02:01-positive and exhibiting high RNF43 expression in their tumor cells. They were administered 300 mg/m2 CPA followed by autologous lymphocytes, preliminarily cultured with autologous RNF43 peptide-pulsed dendritic cells (DCs), RNF43 peptide-pulsed DCs and systemic low dose interleukin-2. The primary endpoint was safety whereas the secondary endpoint was immunological and clinical response to treatment. Ten patients, in total, were enrolled in this trial. Primarily, no adverse events greater than Grade 3 were observed. Six out of 10 patients showed stable disease (SD) on day 49, while 4 other patients showed progressive disease. In addition, one patient with SD exhibited a partial response after the second trial. The frequency of regulatory T cells (Tregs) in patients with SD significantly decreased after CPA administration. The ratio of interferon-γ-producing, tumor-reactive CD8+ T cells increased with time in patients with SD. We successfully showed that the combination of immune cell therapy and CPA was safe, might induce tumor-specific immune responses and clinical efficacy, and was accompanied by a decreased ratio of Tregs in patients with RNF43-positive advanced solid tumors.

A novel, polymer-coated oncolytic measles virus overcomes immune suppression and induces robust antitumor activity.

Although various therapies are available to treat cancers, including surgery, chemotherapy, and radiotherapy, cancer has been the leading cause of death in Japan for the last 30 years, and new therapeutic modalities are urgently needed. As a new modality, there has recently been great interest in oncolytic virotherapy, with measles virus being a candidate virus expected to show strong antitumor effects. The efficacy of virotherapy, however, was strongly limited by the host immune response in previous clinical trials. To enhance and prolong the antitumor activity of virotherapy, we combined the use of two newly developed tools: the genetically engineered measles virus (MV-NPL) and the multilayer virus-coating method of layer-by-layer deposition of ionic polymers. We compared the oncolytic effects of this polymer-coated MV-NPL with the naked MV-NPL, both in vitro and in vivo. In the presence of anti-MV neutralizing antibodies, the polymer-coated virus showed more enhanced oncolytic activity than did the naked MV-NPL in vitro. We also examined antitumor activities in virus-treated mice. Complement-dependent cytotoxicity and antitumor activities were higher in mice treated with polymer-coated MV-NPL than in mice treated with the naked virus. This novel, polymer-coated MV-NPL is promising for clinical cancer therapy in the future.

Phase I clinical trial of a five-peptide cancer vaccine combined with cyclophosphamide in advanced solid tumors.

We designed a phase I trial to investigate the safety, immune responses and clinical benefits of a five-peptide cancer vaccine in combination with chemotherapy. Study subjects were patients positive for HLA-A2402 with locally advanced, metastatic, and/or recurrent gastrointestinal, lung or cervical cancer. Eighteen patients including nine cases of colorectal cancer were treated with escalating doses of cyclophosphamide 4days before vaccination. Five HLA-A2402-restricted, tumor-associated antigen (TAA) epitope peptides from KOC1, TTK, URLC10, DEPDC1 and MPHOSPH1 were injected weekly for 4weeks. Treatment was well tolerated without any adverse events above grade 3. Analysis of peripheral blood lymphocytes showed that the number of regulatory T cells dropped from baseline after administration of cyclophosphamide and confirmed that TAA-specific T cell responses were associated significantly with longer overall survival. This phase I clinical trial demonstrated safety and promising immune responses that correlated with vaccine-induced T-cell responses. Therefore, this approach warrants further clinical studies.

TLR7 ligand augments GM-CSF-initiated antitumor immunity through activation of plasmacytoid dendritic cells.

Vaccination with irradiated granulocyte macrophage colony-stimulating factor (GM-CSF)-transduced autologous tumor cells (GVAX) has been shown to induce therapeutic antitumor immunity. However, its effectiveness is limited. We therefore attempted to improve the antitumor effect by identifying little-known key pathways in GM-CSF-sensitized dendritic cells (GM-DC) in tumor-draining lymph nodes (TDLN). We initially confirmed that syngeneic mice subcutaneously injected with poorly immunogenic Lewis lung carcinoma (LLC) cells transduced with Sendai virus encoding GM-CSF (LLC/SeV/GM) remarkably rejected the tumor growth. Using cDNA microarrays, we found that expression levels of type I interferon (IFN)-related genes, predominantly expressed in plasmacytoid DCs (pDC), were significantly upregulated in TDLN-derived GM-DCs and focused on pDCs. Indeed, mouse experiments demonstrated that the effective induction of GM-CSF-induced antitumor immunity observed in immunocompetent mice treated with LLC/SeV/GM cells was significantly attenuated when pDC-depleted or IFNα receptor knockout (IFNAR(-/-)) mice were used. Importantly, in both LLC and CT26 colon cancer-bearing mice, the combinational use of imiquimod with autologous GVAX therapy overcame the refractoriness to GVAX monotherapy accompanied by tolerability. Mechanistically, mice treated with the combined vaccination displayed increased expression levels of CD86, CD9, and Siglec-H, which correlate with an antitumor phenotype, in pDCs, but decreased the ratio of CD4(+)CD25(+)FoxP3(+) regulatory T cells in TDLNs. Collectively, these findings indicate that the additional use of imiquimod to activate pDCs with type I IFN production, as a positive regulator of T-cell priming, could enhance the immunologic antitumor effects of GVAX therapy, shedding promising light on the understanding and treatment of GM-CSF-based cancer immunotherapy.

Absence of LTB4/BLT1 axis facilitates generation of mouse GM-CSF-induced long-lasting antitumor immunologic memory by enhancing innate and adaptive immune systems.

BLT1 is a high-affinity receptor for leukotriene B4 (LTB4) that is a potent lipid chemoattractant for myeloid leukocytes. The role of LTB4/BLT1 axis in tumor immunology, including cytokine-based tumor vaccine, however, remains unknown. We here demonstrated that BLT1-deficient mice rejected subcutaneous tumor challenge of GM-CSF gene-transduced WEHI3B (WGM) leukemia cells (KO/WGM) and elicited robust antitumor responses against second tumor challenge with WEHI3B cells. During GM-CSF-induced tumor regression, the defective LTB4/BLT1 signaling significantly reduced tumor-infiltrating myeloid-derived suppressor cells, increased the maturation status of dendritic cells in tumor tissues, enhanced their CD4(+) T-cell stimulation capacity and migration rate of dendritic cells that had phagocytosed tumor-associated antigens into tumor-draining lymph nodes, suggesting a positive impact on GM-CSF-sensitized innate immunity. Furthermore, KO/WGM mice displayed activated adaptive immunity by attenuating regulatory CD4(+) T subsets and increasing numbers of Th17 and memory CD44(hi)CD4(+) T subsets, both of which elicited superior antitumor effects as evidenced by adoptive cell transfer. In vivo depletion assays also revealed that CD4(+) T cells were the main effectors of the persistent antitumor immunity. Our data collectively underscore a negative role of LTB4/BLT1 signaling in effective generation and maintenance of GM-CSF-induced antitumor memory CD4(+) T cells.

Influence of Epstein-Barr virus infection in adult T-cell leukemia.

The involvement of adult T-cell leukemia (ATL) cells in organs such as the skin and lymph nodes is observed in about 50% of cases of ATL. Epstein-Barr virus (EBV) infection has often been observed in the clinical course of ATL. In this study, we established two B-cell lines from peripheral blood of patients with ATL. EBV DNA, proviral DNA for HTLV-1 and Tax mRNA were detected in both lines. As part of the characterization of these cells, an enhanced expression of intercellular adhesion molecule-1 (ICAM-1) (CD54) or ICAM-3 (ICAM-3) (CD50), lymphocyte function-1 (LFA-1) (CD11a/CD18), and Mac-1 (CD11b/CD18) was observed. To investigate the role of the interaction of these viruses, we transfected EBV and/or HTLV-1 into a healthy donor's lymphocytes, an EBV-infected B cell line, Raji, and a HTLV-1 negative T-cell line, Jurkat. Enhanced expression of adhesion molecules was also observed in double transfectants (EBV and HTLV-1). In the clinical course of ATL, LMP-1, EBNA-2, CD50 and CD54 were detected in lymph nodes and skin specimens by immunohistochemical staining. Furthermore, high levels of interleukin-4 (IL-4) were detected in these cell lines and transfectants. The results indicated that coinfection with HTLV-1 and EBV may induce aggressive organ involvement through the enhanced expression of adhesion molecules via IL-4 signaling. A new mechanism of ATL involvement is discussed.