Gut microbiota and clinical response to immune checkpoint inhibitor therapy in patients with advanced cancer.

BACKGROUND: There is currently no well-accepted consensus on the association between gut microbiota and the response to treatment of immune checkpoint inhibitors (ICIs) in patients with advanced cancer. METHODS: Fecal samples were collected before ICI treatment. Gut microbiota was analyzed using 16 S ribosomal RNA sequencing. We investigated the relationship between the α-diversity of fecal microbiota and patients' clinical outcomes. Microbiota profiles from patients and healthy controls were determined. Pre-treatment serum was examined by cytokine array. RESULTS: We analyzed 74 patients, including 42 with melanoma, 8 with kidney cancer, 13 with lung cancer, and 11 with other cancers. Combination therapy of anti-PD1 and anti-CTLA-4 was used in 14 patients, and monotherapy in the rest. Clinical benefit was observed in 35 (47.3 %) cases, including 2 complete responses, 16 partial responses, and 17 stable diseases according to RECIST criteria. No significant difference in α-diversity was found between the benefiter and non-benefiter groups. However, patients with α-diversity within the range of our healthy control had a significantly longer median overall survival (18.9 months), compared to the abnormal group (8.2 months) (p = 0.041, hazard ratio = 0.546) for all patients. The microbiota composition of the benefiters was similar to that of healthy individuals. Furthermore, specific bacteria, such as Prevotella copri and Faecalibacterium prausnitzii, were associated with a favorable outcome. We also observed that serum IL-18 before treatment was significantly lower in the benefiters, compared to non-benefiters. CONCLUSIONS: The α-diversity of gut microbiota is positively correlated with more prolonged overall survival in cancer patients following ICI therapy.

RAB38 is a potential prognostic factor for tumor recurrence in non-small cell lung cancer.

Ras-related protein Rab-38 (RAB38) is a member of the Ras small G protein family that regulates intracellular vesicular trafficking. Although the expression of RAB38 is reportedly deregulated in several types of cancer, its role in tumor biology remains to be elucidated. In the present study, the expression of RAB38 was analyzed in tumor specimens from patients with non-small cell lung cancer (NSCLC) with tumor recurrence within 4 years (Group R), and those remaining disease-free following initial surgery (Group NR), by reverse transcription-semi-quantitative PCR and subsequent semi-quantification using ImageJ v4.0 software. The results revealed that the expression of RAB38 in Group R and NR specimens was positively associated with tumor recurrence; a high expression level was also associated with poor survival rate in these patients. Using NSCLC cell lines, it was demonstrated that tumor cells with mutations in the active epidermal growth factor receptor (EGFR) gene expressed higher levels of RAB38 compared with those with the wild-type gene by reverse transcription-PCR and western blot analysis. Furthermore, following specific RAB38 gene knockdown by short hairpin RNA transfection, EGFR mutants exhibited markedly reduced invasiveness when compared with cells transfected with empty vector controls by Matrigel Transwell assays. These results suggest that RAB38 is an important prognostic factor in NSCLC, and may serve a critical role in NSCLC-associated tumor metastasis.

Autocrine CCL5 promotes tumor progression in esophageal squamous cell carcinoma in vitro.

The pro-tumoral effects of CCL5 have been identified in numerous cancer types. We successfully cultivated 4 esophageal squamous cell carcinoma (ESCC) cell lines, including TWES-1, TWES-3 and a pair of cell lines derived from primary lesion (TWES-4PT) and metastatic lymph node (TWES-4LN) of the same patient. Whole genome screening showed that TWES-4LN expressed higher levels of CCL5 compared to that of TWES-4PT; quantification of protein secretion displayed comparable results, suggesting that CCL5 could be associated with lymph node metastasis in ESCC. CCL5 knockdown by siRNA significantly reduced basal growth rate, tumor migration and invasiveness in the paired cell lines; whereas this treatment induced cell apoptosis in TWES-1 and TWES-3. CCR5 antagonist maraviroc significantly inhibited tumor migration and invasion in the paired cell lines without affecting tumor growth. Collectively, these results suggest that CCL5 autocrine loop may promote ESCC progression; targeting the CCL5/CCR5 axis could be a potential therapeutic strategy for this deadly disease.

Identification of low-molecular-weight vitellogenin 1 (Vg1)-like proteins as nucleotide excision repair (NER) factors in developing zebrafish (Danio rerio) using a transcription-based DNA repair assay.

Nucleotide excision repair (NER) removes helix-distorting DNA lesions such as UV-induced pyrimidine dimers and cisplatin-induced strand crosslinking. Our earlier studies have identified low-molecular-weight proteins homologous to the 150-kDa vitellogenin 1 (Vg1) as UV-damaged DNA-binding factors expressed in developing zebrafish (Danio rerio). This present study explored if Vg1-like proteins also participated in NER in zebrafish. Immunoblot analysis of affinity-captured 12 h post-fertilization (hpf) zebrafish extract proteins showed a transient binding of a 30-kDa Vg1-like polypeptide to UV-damaged DNA. A transcription-based in vitro repair assay revealed a significant up-regulation of UVC or cisplatin-suppressed transcriptional activity of a marker cDNA driven by a SP6 RNA polymerase-regulated promotor after incubating the damaged plasmid with the extracts of 12 hpf embryos or 96 hpf larvae. The up-regulation of UV or cisplatin-suppressed transcription was abolished in the presence of a monoclonal anti-zebrafish Vg1 antibody. The differential sensitivity of UV-induced repair in 12 and 96 hpf zebrafish extracts to exogenous ATP suggested a development-dependent expression of Vg1-like NER factors. A T4 endonuclease V digestion assay showed no inhibition of the anti-Vg1 antibody on the excision of UV-induced cyclobutane pyrimidine dimers. Our results identified the participation of Vg1-like factors in NER in developing zebrafish, and these factors may function at post-incison steps of NER.

A comparison of immunohistochemical and molecular methods used for analyzing the BRAF V600E gene mutation in malignant melanoma in Taiwan.

AIMS: The BRAF V600 mutation has been shown to be clinically meaningful in terms of both the prognosis and sensitivity of BRAF inhibitors in patients with metastatic melanoma. Recently, a BRAF V600E mutation-specific antibody, VE1, was generated for the detection of tumors bearing BRAF V600E mutations. To determine the clinical value of immunohistochemical testing, we compared the prevalence of mutant BRAF detected by VE1 with direct sequencing results. METHODS: Paraffin-embedded, formalin-fixed melanoma biopsies were analyzed for the BRAF mutation status by immunohistochemistry with the VE1 antibody. Sanger sequencing was applied to verify the immunohistochemical results. RESULTS: A total of 73 melanoma cases with tumor samples from primary lymph nodes and metastatic sites were selected for this study. Direct sequencing demonstrated that 18 of 73 cases (24.6%) harbored the BRAF V600 mutation: 17 with V600E and one with V600K. All 18 tumors shown to harbor the BRAF V600E/K mutations were VE1-positive. One additional case was false-positive for VE1. The sensitivity and specificity of VE1 was 100% (18/18) and 98% (54/55), respectively. The overall concordance between the immunohistochemical method and direct sequencing was excellent (98.6%). CONCLUSIONS: Our findings demonstrate that immunohistochemical analysis using VE1 constitutes a highly sensitive test for the detection of BRAF mutations and suggest that this cost-effective method is suitable as a rapid diagnostic approach complementary to molecular testing.

Epstein-Barr virus-infected cell line TCC36B derived from B lymphocytes infiltrating renal pelvis urothelial carcinoma.

UNLABELLED: This study reports an initial analysis of an EBV-infected B cell line (TCC36B), established from an urothelial carcinoma (UC) lesion of the renal pelvis. MATERIALS AND METHODS: Cytofluorometric and G-banding analyses were performed for phenotyping and cytogenetics. PCR was used to detect EBV DNA, and sequence analysis to investigate mutations and deletions of the latent membrane protein (LMP)-1 gene of EBV. RESULTS: TCC36B cells proliferated in vitro and showed positivity for surface CD19, CD20, HLA-DR and IgG(λ), indicating that they belong to B-cells. Cytogenetic analysis showed 46,XX with a unique clonal abnormality of dup(2)(p13p25). EBV DNA was detected in TCC36B cells. Sequence analysis revealed a 30-bp deletion and 7 point mutations on the LMP-1 gene in TCC36B cells. CONCLUSION: These results suggest the involvement of an EBV variant in the pathogenesis of UC. This cell line should thus facilitate further investigations on the aetiological role of EBV in urothelial cancer.

Characterization of the response of dendritic cells and regulatory T cells to tumor antigens in patients with renal cell carcinoma.

BACKGROUND: This study characterized dendritic cells (DCs), regulatory T cells (Tregs) and the immune responses to tumor antigens in renal cell carcinoma (RCC) patients. METHODS: Thirty patients with RCC and five healthy donors were studied. DCs were generated from the adherent cells among peripheral blood mononuclear cells (PBMCs), then cultured in medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for 7 days. The phenotypes of the DCs and Tregs were analyzed by flow cytometry. A mixed lymphocyte reaction (MLR) was performed to assess the functioning of the DCs and Tregs. A cytotoxic assay was performed to measure the antigen presentation ability of the DCs from the RCC patients (RCC-DCs). These DCs were pretreated with TNF-alpha (TNF-DCs) or tumor lysate (TuLy-DCs) on the 3rd day of DC culture. RESULTS: The RCC-DCs expressed significantly less CD40 (p = 0.03) and CD80 (p = 0.007) upon TNF-alpha cultivation than the DCs from healthy donors. Theperipheral Tregs during stage I disease were significantly less (p = 0.032) than during stages II-IV. The RCC-DCs were as efficient as DCs from healthy donors (p = 0.83) when stimulating the proliferation of allogeneic T cells; however, these RCC-DCs were less efficient when stimulating autologous T cells than allogeneic T cells (p = 0.023). Tregs inhibited autologous T cell proliferation rather than allogeneic T cell proliferation in response to TuLy-DCs stimulation. Prostaglandin E(2) did not increase the ability of immature DCs to stimulate T cell proliferation. CONCLUSIONS: Patients with RCC have less potent anti-tumor immune responses.

Immunotherapy with dendritic cells pulsed by autologous dactinomycin-induced melanoma apoptotic bodies for patients with malignant melanoma.

The primary goal of this study was to evaluate the efficacy of immunotherapy for patients with metastatic melanoma with autologous melanoma apoptotic bodies (MAB)-pulsed dendritic cells (DCs). Accessible tumors from eligible patients with refractory metastatic melanoma were surgically removed and processed for primary culture. The autologous tumor cells were treated with dactinomycin to obtain MAB. To generate DCs, adherent peripheral blood mononuclear cells were cultured in complete medium containing granulocyte macrophage-colony stimulating factor and interleukin-4. MAB-pulsed DCs were given either intradermally (i.d.) or intravenously. Patients were immunized at monthly intervals and boosted with keyhole limpet hemocyanin (KLH) and MAB 2 weeks post-vaccination, with a maximum of four cycles. Of the 10 patients enrolled in this trial, nine were treated with MAB-pulsed DCs; two were given intravenous vaccinations and the other seven were i.d. injected. Mild tenderness in the draining lymph nodes lasting for less than 48 h and enlargement of the draining lymph nodes were noted in all seven i.d. cases. Treatment-related grade 3-4 toxicity, neutropenia, skin ulceration, tumor growth at the injection site, and sepsis were not observed in any of the patients. Delayed-type hypersensitivity to KLH was observed in all patients, whereas no delayed-type hypersensitivity to autologous tumor antigens was observed. One patient achieved partial response with reduction in lung metastatic tumor mass, and a presence of vesicles in the post-vaccination KLH response. Two patients had stable disease for more than 24 months; one was still alive at the time of submission of this report, the other eventually developed multiple metastases. MAB-pulsed DC immunotherapy is well tolerated in patients with malignant melanoma; however, its efficacy is only modest. Combination with other modalities is required to enhance DC-based immunotherapy.

Bisphosphonate zoledronic acid enhances the inhibitory effects of gefitinib on EGFR-mutated non-small cell lung carcinoma cells.

Patients with non-small cell lung carcinoma (NSCLC) bearing epidermal growth factor receptor (EGFR) gene mutations are good responders to gefitinib (Iressa), an EGFR tyrosine kinase inhibitor (EGFR-TKI), yet these patients may eventually develop acquired resistance to all available EGFR-TKIs. Nitrogen-containing bisphosphonates (N-BPs) are inhibitors of farnesyl diphosphate (FPP) synthase as well as chelators of divalent cations. This study was undertaken to examine if the N-BP zoledronic acid (zoledronate) possessing antitumor activity could enhance the antitumor effect of gefitinib on the HCC827 NSCLC cell line expressing mutated EGFR. Both gefitinib and zoledronate were cytotoxic to HCC827 cells when treated alone. Combined treatment with gefitinib (0.025 microM) that induced G0/G1 arrest and zoledronate (50 microM) that caused S/G2/M accumulation generated an additive induction in cell cytotoxicity, sub-G1 cell population, and apoptosis. Gefitinib suppressed EGF-activated phosphorylation of ERK1/2 and Akt, while zoledronate seemed to impose its pharmacological effect independent of ERK1/2 and Akt phosphorylation. The volumes of xenografted tumors in nude mice co-administered with gefitinib (1 mg/kg/day, five days a week, p.o.) and zoledronate (10 microg/kg, twice weekly, i.p.) were significantly smaller than those of tumors in mice treated with gefitinib alone at the last stage of a 6-week in vivo study. Severe peri-tumoral fat loss frequently observed in gefitinib-treated mice disappeared in mice receiving the combined treatment. Hence, combined treatment of gefitinib with zoledronate may form a basis to develop a more effective and less toxic therapy for NSCLC with EGFR gene mutations.

Clodronate alleviates cachexia and prolongs survival in nude mice xenografted with an anaplastic thyroid carcinoma cell line.

Cancer cachexia is one of the most common manifestations of advanced malignant disease and is frequently associated with decreased survival. Previously, we reported the establishment of a new anaplastic thyroid carcinoma cell line, Thena, and its mouse xenograft, Thena-Nu, which induced cachexia in athymic nude mice. Subsequent studies showed that the addition of clodronate to Thena-Nu cultures reduced cell proliferation as well as cytokine production in a dose- and time-dependent manner. Weekly administration of clodronate induced tumor cytostasis, attenuation of cachexia, as well as prolongation of survival in Thena-Nu-bearing mice. Reduced serum interleukin 6, tumor necrosis factor-alpha, and granulocyte colony stimulating factor levels were detected, whereas, serum leukemia inhibitory factor levels were not reduced. Liver necrosis, observed in tumor-bearing mice, was also improved following clodronate treatment. Discontinuation of clodronate treatment, however, resulted in progressive tumor growth and weight loss. Our results demonstrated that clodronate could exert therapeutic efficacy on amelioration of cancer cachexia in the hosts. Nevertheless, this study also points out that a longer period of treatment is required to maintain these effects.