The influence of microbial dysbiosis on immunochemotherapy-related efficacy and safety in diffuse large B-cell lymphoma.

The gut microbiome influences cancer development and the efficacy and safety of chemotherapy but little is known about its effects on lymphoma. We obtained stool samples from treatment-naive, newly diagnosed patients with diffuse large B-cell lymphoma (DLBCL) (n = 189). We first performed 16S ribosomal RNA gene sequencing (n = 158) and then conducted whole-genome shotgun sequencing on additional samples (n = 106). We compared the microbiome data from these patients with data from healthy controls and assessed whether microbiome characteristics were associated with treatment outcomes. The alpha diversity was significantly lower in patients with DLBCL than in healthy controls (P < .001), and the microbial composition differed significantly between the groups (P < .001). The abundance of the Enterobacteriaceae family belonging to the Proteobacteria phylum was markedly higher in patients than in healthy controls. Functional analysis of the microbiome revealed an association with opportunistic pathogenesis through type 1 pili, biofilm formation, and antibiotics resistance. Enterobacteriaceae members were significantly enriched in patients who experienced febrile neutropenia and in those who experienced relapse or progression (P < .001). Interestingly, greater abundance of Enterobacteriaceae correlated with shorter progression-free survival (P = .007). The cytokine profiles of patients whose microbiome was enriched with Enterobacteriaceae were significantly associated with interleukin 6 (P = .035) and interferon gamma (P = .045) levels. In summary, patients with DLBCL exhibited gut microbial dysbiosis. The abundance of Enterobacteriaceae correlated with treatment outcomes and febrile neutropenia. Further study is required to elucidate the origin and role of gut dysbiosis in DLBCL.

Avelumab for the treatment of relapsed or refractory extranodal NK/T-cell lymphoma: an open-label phase 2 study.

This study aimed to assess the efficacy and safety of treatment with avelumab, an anti-programmed death ligand 1 (PD-L1) antibody, in patients with relapsed or refractory extranodal natural killer/T-cell lymphoma (ENKTL). In this phase 2 trial, 21 patients with relapsed or refractory ENKTL were treated with 10 mg/kg of avelumab on days 1 and 15 of a 28-day cycle. The primary end point was the complete response (CR) rate based on the best response. Targeted sequencing and immunohistochemistry were performed using pretreatment tumor tissue, and blood samples were drawn before and after treatment for measurement of cytokines and soluble programmed cell death protein 1 (PD1), PD-L1, and PD-L2. The CR rate was 24% (5 of 21), and the overall response rate was 38% (8 of 21). Although nonresponders showed early progression, 5 responders currently continue to receive treatment and have maintained their response. Most treatment-related adverse events were grade 1 or 2; no grade 4 adverse events were observed. Treatment responses did not correlate with mutation profiles, tumor mutation burden, serum levels of cytokines, or soluble PD1/PD-L1 and PD-L2. However, the response to avelumab was significantly associated with the expression of PD-L1 by tumor tissue (P = .001). Therefore, all patients achieving CR showed high PD-L1 expression, and their tumor subtyping based on PD-L1 expression correlated with treatment response. In summary, avelumab showed single-agent activity in a subset of patients with relapsed or refractory ENKTL. The assessment of PD-L1 expression on tumor cells might be helpful for identifying responders to avelumab. This trial was registered at www.clinicaltrials.gov as #NCT03439501.

Pre-treatment serum IL-10 predicts the risk of secondary central nervous system involvement in patients with diffuse large B-cell lymphoma.

BACKGROUND: As diffuse large B-cell lymphoma (DLBCL) is a very heterogeneous group of lymphomas, much effort has gone in trying to identify patients with increased risk for early death or secondary central nervous system (CNS) involvement. To better predict their outcomes, we measured the levels of various cytokines in serum samples of patients with DLBCL and analyzed their clinical outcomes. METHODS: We measured the levels of seven serum cytokines at diagnosis in 313 DLBCL patients who were treated with R-CHOP. Their impact on clinical outcomes, including time to secondary CNS involvement and the 3-year overall survival (OS) rate, were analyzed. RESULTS: The median age was 56 years (range, 16-86 years), and 177 patients (57%) were men. Secondary CNS involvement was found in 5.4% (16/294) cases, and time to secondary CNS involvement was significantly short in patients with elevated interleukin (IL)-10 (p = 0.012). With the 3-year OS rate of the whole cohort being 73.6%, serum levels of several cytokines, such as CCL3 > 4.0 pg/mL (54.3% vs. 76.1%, p = 0.001), CCL5 > 450 pg/mL (57.0% vs. 78.1%, p < 0.001), any expression of IL-6 (59.3% vs. 76.6%, p = 0.001), and any expression of IL-10 (68.2% vs. 84.5%, p = 0.001), showed prognostic impact. Higher expressions of these cytokines were associated with worse manifestations of clinical prognostic factors. CONCLUSIONS: Our study revealed that some cytokines impact OS and secondary CNS involvement. Future studies are required to elucidate how these findings can be incorporated to the conventional prognostic factors for more tailored approaches.

Serum levels of soluble programmed death-ligand 1 (sPD-L1) in patients with primary central nervous system diffuse large B-cell lymphoma.

BACKGROUND: The interaction of programmed death-1 protein (PD-1) and programmed death-1 ligand (PD-L1) produces immunosuppressive activity, protecting tumor cells from anti-tumor immunity and possibly releasing soluble PD-L1 (sPD-L1) from PD-L1 expressing tumor cells. Therefore, we measured serum levels of sPD-L1 in patients with primary central nervous system lymphoma (PCNSL) and explored its clinical implications. METHODS: Sixty-eight patients with newly diagnosed PCNSL had diffuse large B-cell lymphoma and were treated with high-dose methotrexate-containing chemotherapy. The measurement of sPD-L1 and cytokines was performed using serum samples archived at diagnosis, and the tissue expression of PD-L1 was also analyzed from archived paraffin-embedded tissue blocks. Disease relapse, progression-free survival (PFS), and overall survival (OS) were analyzed according to the extent of sPD-L1 in serum and PD-L1 in tissue. RESULTS: The median level of serum sPD-L1 (0.429 ng/mL) was higher than in healthy control patients (0.364 ng/mL). The occurrence of relapse was more frequent in the high sPD-L1 (78%) than the low sPD-L1 group (50%), though the groups did not have different clinical or pathological characteristics at diagnosis. As a result, the OS and PFS for the high sPD-L1 group were significantly lower than those in the low group. PD-L1-positive tumor cells were found in 35 patients (67%), and the extent of PD-L1-postive tumor cells was positively associated with serum sPD-L1 levels (r = 0.299, P = 0.031). Among the 34 cytokines analyzed, only the serum level of IL-7 correlated with the serum level of sPD-L1 (r = 0.521, P < 0.001). CONCLUSIONS: Serum levels of sPD-L1 could reflect the expression of PD-L1 in PCNSL tumor cells and predict patient survival outcomes. Therefore, sPD-L1 in serum could be a feasible biomarker for determining a risk-adapted treatment strategy for PCNSL patients. TRIAL REGISTRATION: The study population was patients who were diagnosed with PCNSL between January 2009 and February 2017 and registered for our prospective cohort studies after providing written informed consent (ClinicalTrials.gov: NCT00822731 [date of registration - January 14, 2009] and NCT01877109 [date of registration - June 13, 2013]).

Profiles of serum cytokines and their clinical implications in patients with peripheral T-cell lymphoma.

To better predict the outcomes of patients with peripheral T-cell lymphoma (PTCL), we measured the levels of various cytokines in serum samples from patients with PTCL and analyzed their clinical outcomes. We measured 34 cytokines in samples from 121 PTCL patients (55 PTCL-not otherwise specified (NOS), 44 angioimmunoblastic T-cell lymphoma (AITL), and 22 ALK- anaplastic large cell lymphoma) at diagnosis. Their impact on clinical outcomes, including overall survival and complete response rate, were analyzed with other clinical variables. The median age of patients was 58 years (range, 20-85 years) and 81 patients (66.9%) were male. The median overall survival among all patients was 56.1 months (95% CI 21.4-90.8) and median progression-free survival was 19.3 months (95% CI 12.3-26.3). Patients with AITL were more likely to express higher levels of serum cytokines, and 7 cytokines showed mean levels that were significantly higher than those in other subtypes. In this subgroup, IL-10 higher than 3.8 pg/mL was associated with adverse outcomes. In patients with ALK- anaplastic large cell lymphoma, 9 cytokines showed a prognostic impact, with higher levels of interferon γ, interleukin (IL)-8, IL-10, IL-17, IL-23, IP-10, monocyte chemoattractant protein-1, macrophage inflammatory protein-1ß, and RANTES negatively affecting clinical outcomes. In PTCL-NOS, patients with elevated levels of interferon γ, IL-7, and IL-23 showed poor outcomes. The current analysis demonstrated different cytokine profiles according to histologic subtype, which revealed the heterogeneity of PTCL. In addition, cytokine levels can be used as prognostic markers and may be useful for therapeutic applications in PTCL patients.

Vitamin D deficiency is associated with inferior survival of patients with extranodal natural killer/T-cell lymphoma.

Vitamin D deficiency is a common health issue; however, the effect of vitamin D deficiency on the survival of T-cell lymphoma is still not clear. We evaluated the impact of serum vitamin D level of patients with peripheral T-cell lymphoma (PTCL) and extranodal natural killer/T-cell lymphoma (ENKTL) on survival outcome. Pretreatment levels of 25-hydroxyvitamin D [25(OH)D] and inflammatory cytokines were measured in serum samples that were archived at diagnosis, and we evaluated their association with survival in newly diagnosed patients with PTCL (n = 137) and ENKTL (n = 114) at a university-based hospital in Korea. An independent cohort from Rui Jin Hospital (Shanghai, China) was used for validation. The median 25(OH)D serum level was 12.0 ng/mL (1.3-60.0 ng/mL), and 40% had less than 10 ng/mL, which was defined as vitamin D deficiency. Median serum 25(OH)D levels were similar between PTCL (11.5 ng/mL) and ENKTL (12.9 ng/mL); however, vitamin D deficiency was associated with inferior survival in ENKTL but not with PTCL. The independent validation cohort (n = 115) also showed a significant association of vitamin D deficiency with poor survival in ENKTL. The 25(OH)D level had an inverse relation with inflammatory cytokines; this association had a negative effect only on survival of ENKTL, and not on PTCL. In conclusion, vitamin D deficiency was associated with inferior survival outcome of patients with ENKTL.

Macrophage inflammatory protein 1 alpha (MIP-1α) may be associated with poor outcome in patients with extranodal NK/T-cell lymphoma.

The macrophage inflammatory protein 1α (MIP-1α) is anticipated to have a role in extranodal natural killer (NK)/T-cell lymphoma (ENKTL) because the expression of MIP-1α is related to Epstein-Barr virus (EBV) latency in EBV-related non-Hodgkin lymphoma cells. Thus, we measured the serum level of MIP-1α in 69 patients with ENKTL using frozen serum samples that were archived at diagnosis. As serum level of MIP-1α was not detectable in 19 patients (range: 0-24.37 pg/mL), patients were dichotomized into positive (n = 50) and negative (n = 19) MIP-1α groups according to the presence of detectable level of MIP-1α in serum. MIP-1α-positive group showed a significantly poor overall survival (OS) in comparison with the MIP-1α-negative group (p = 0.004). In the subgroup analysis, the positivity of MIP-1α was significantly associated with OS in patients with stage IIIE/IV and a detectable level of EBV DNA (p = 0.002 and 0.032, respectively). Multivariate analysis also showed that the positivity of MIP-1α was independently associated with worse OS together with bone marrow involvement (p = 0.002). An in vitro study with patient-derived ENKTL tumour cells showed the expression of CCR1 and CCR5 on the surface of tumour cells (28% and 14%, respectively) , and the addition of MIP-1α to the culture media of tumour cells increased cell growth supporting the negative impact of MIP-1α on the prognosis of ENKTL patients. In conclusion, serum levels of MIP-1α could predict survival outcomes in patients with ENKTL. Therefore, MIP-1α should be considered for prognostication and a potential therapeutic target. Copyright © 2016 John Wiley & Sons, Ltd.

Serum level of CXCL10 is associated with inflammatory prognostic biomarkers in patients with diffuse large B-cell lymphoma.

Inflammatory biomarkers, such as the neutrophil to lymphocyte ratio (NLR), lymphocyte to monocyte ratio (LMR), and Glasgow Prognostic Score (GPS) have been proposed to predict prognosis in diffuse large B-cell lymphoma (DLBCL). C-X-C motif ligand 10 (CXCL10) is a chemokine released from inflammatory cells in the tumor microenvironment and is known to promote tumor cell migration and invasion. In this study, we investigated the clinical impact of pretreatment serum level of CXCL10 on the prognostic value of inflammatory biomarkers in 313 patients with DLBCL who were enrolled into a prospective cohort study. Serum level of CXCL10 was measured in archived pretreatment frozen samples. The high CXCL10 (>median value) group was significantly associated with high tumor burden status, including advanced stage (III-IV), elevated serum lactic dehydrogenase, and a higher risk International Prognostic Index. Progression-free survival of the high CXCL10 group was significantly worse than that of the low CXCL10 group, and secondary central nervous system involvement was more frequent in the high CXCL10 group. High CXCL10 was associated with high C-reactive protein level (r = 0.246), low albumin level (r = -0.289), low absolute lymphocyte count (r = -0.185), and risk stratification according to NLR, LMR, and GPS. C-X-C motif ligand 10 promoted cell migration of patient-derived cells and several DLBCL cell lines. However, the prognostic value of high CXCL10 was lost in the multivariate analyses. Nevertheless, we suggest serum CXCL10 may have clinical value if it can be more easily assessed because of its contribution to the prognostic value of NLR, LMR, and GPS in DLBCL.

Oncogenic Role of BOLL in Colorectal Cancer.

BACKGROUND: Boule-like RNA-binding protein (BOLL protein) is the progenitor of the Deleted in Azoospermia (DAZ) gene family. To date, previous studies have focused on the reproductive function of BOLL. While we were identifying new DNA methylation biomarkers for colorectal cancer (CRC), we found that BOLL protein was overexpressed in CRC. AIM: The aim of this study was to determine the role of BOLL in CRC by epigenetic and functional studies in vivo and in vitro. METHODS: BOLL promoter methylation and expression were determined by MethyLight, RT-PCR, Western blot, and immunohistochemistry. The functional role of BOLL in CRC was evaluated by cell proliferation, colony formation, migration and invasion, cell cycle status, and tumor growth in a xenograft model. RESULTS: BOLL promoter methylation was enhanced in CRC tissues compared with normal colorectal tissues [97/124 (78 %) vs. 2/124 (2 %)]. However, the mean immunoreactivity score of CRC tissues and paired adjacent normal tissues was 8.15 ± 0.18 (SD) and 3.35 ± 0.19 (SD), respectively (p < 0.01). No significant association was observed between immunoreactivity score and clinicopathological parameters, including age, gender, tumor size, tumor differentiation, and tumor node metastasis stage. Expression of BOLL in CRC cell lines significantly enhanced cell proliferation (p < 0.01), colony formation (p < 0.01), and migration (p < 0.01). In BOLL-expressing cells, the percentage of cells in S-phase of the cell cycle was significantly increased. Tumor volume in BOLL xenografted mice was significantly enhanced after subcutaneous implantation (p < 0.01). CONCLUSIONS: Our study demonstrated an oncogenic role of BOLL in CRC despite tumor-specific promoter hypermethylation.

Alcohol dehydrogenase, iron containing, 1 promoter hypermethylation associated with colorectal cancer differentiation.

BACKGROUND: The aberrant methylation of CpG islands in the promoter is associated with colorectal cancer (CRC) carcinogenesis. In our previous study, the promoter of alcohol dehydrogenase, iron containing, 1 (ADHFE1) was most highly methylated in CRC compared to normal colorectal mucosa. In this study, we examined the expression and function of the ADHFE1 in CRC. METHODS: We examined the promoter methylation and mRNA expression of ADHFE1 with 5-aza-2'-deoxycytidine (5-Aza-2-dC) in 12 CRC cell lines, 124 paired CRC and adjacent normal mucosa, and 59 advanced adenomas. To confirm methylation of ADHFE1, we performed bisulfite genomic sequencing in 3 CRC cell lines, 6 paired CRC and adjacent normal mucosa. ADHFE1 protein expression was studied using western blot and immunohistochemistry, respectively in the 36 and 243 paired CRC and adjacent normal tissue. We transfected the DLD-1 with pcDNA3.1 vector containing ADHFE1 and examined the expression of differentiation marker, such as ALP, CEA and Cdx2. We examined the ADHFE1 expression at distinct developmental stages in mouse embryos. RESULTS: The ADHFE1 promoter was hypermethylated in all CRC cell lines, 81.8% in CRCs, and 84.7% in advanced adenomas, with reciprocal change by 5-Aza-2-dC. The expression of ADHFE1 mRNA was down-regulated in all CRC cell lines and 96.3% in CRC tissues. The expression of ADHFE1 protein was down-regulated in 91.7% of CRC tissues. In the immunohistochemistry, normal epithelial cells at the crypt top showed very strong ADHFE1 expression, whereas they were much weaker at the crypt base. In CRC, the good differentiation was significantly associated with high ADHFE1 expression. The activity of differentiation marker, such as ALP and CEA, was higher in pcDNA3.1-ADHFE1 transfected CRC cells with consistent correlation with ADHFE1 protein than control. In mouse embryos, ADHFE1 in the large intestine was the first detected at E15.5. At E18.5, ADHFE1 was predominantly expressed in the top of the mature crypt epithelium. CONCLUSIONS: It showed that the hypermethylation of ADHFE1 promoter in CRC is concordance with down-regulation of ADHFE1 mRNA and ADHFE1 protein. ADHFE1 has an important role of differentiation in CRC, as well as normal colorectal mucosa and embryonic developmental processes.

The impact of sMICA/sMICB on immunochemotherapy outcomes in newly diagnosed diffuse large B-cell lymphoma.

Introduction: Soluble MHC class I-related chain A (sMICA) and B (sMICB) play a critical role tumor evolution and poor prognosis through an immune evasion mechanism. Thus, this study determines the interaction between sMICA/sMICB and the tumor immune environment in newly diagnosed diffuse large B-cell lymphoma (ND-DLBCL). Methods: We analyzed sMICA/sMICB, cytokine in serum, and macrophage polarization analysis in tissue samples before the first chemotherapy administration. This research was performed to investigate the correlation between sMICA/sMICB expression and treatment outcomes as well as their influence on the immune system within ND-DLBCL. Results: Of the 262 patients, 47.3% (n = 124) presented stage III or IV at diagnosis and 50.8% (n = 133) had a high International Prognostic Index (IPI ≥ 3). The patients with high (p = 0.034 and 0.004), elevated lactate dehydrogenase (p = 0.002 and 0.030), advanced stage (p = 0.003 and 0.012), and higher IPI risk (p = 0.009, and 0.032) correlated with the detection of sMICA or sMICB. The median progression-free survival (PFS) of patients with sMICA (p = 0.006) or sMICB (p =0.032) was inferior. Among the patients with advanced-stage or high IPI, those with sMICA or sMICB presented an inferior PFS and OS compared to those without. TNF-a, a pro-inflammatory cytokine, showed statistical significance with detected sMICA (p = 0.035) or sMICB (p = 0.044). Among anti-inflammatory cytokines, IL-1RA (P-value = 0.013) and IL-10 (p = 0.005) were associated with detecting sMICB, but not sMICA. In tissue samples, sMICA or sMICB detection did not correlate with the CD68/CD163 ratio. Discussion: Conclusively, the identification of sMICA/sMICB presented unfavorable immunochemotherapy outcomes, and it was assumed that sMICA or sMICB and various cytokines interact, but the relationship with macrophage differentiation is unclear. Therefore, further research is needed to determine the relationship between sMICA/sMICB and tumor microenvironment in DLBCL.

Circulating Tumor DNA-Based Genotyping and Monitoring for Predicting Disease Relapses of Patients with Peripheral T-Cell Lymphomas.

PURPOSE: Plasma circulating tumor DNA (ctDNA) could reflect the genetic alterations present in tumor tissues. However, there is little information about the clinical relevance of cell-free DNA genotyping in peripheral T-cell lymphoma (PTCL). MATERIALS AND METHODS: After targeted sequencing plasma cell-free DNA of patients with various subtypes of PTCL (n=94), we analyzed the mutation profiles of plasma ctDNA samples and their predictive value of dynamic ctDNA monitoring for treatment outcomes. RESULTS: Plasma ctDNA mutations were detected in 53 patients (56%, 53/94), and the detection rate of somatic mutations was highest in angioimmunoblastic T-cell lymphoma (24/31, 77%) and PTCL, not otherwise specified (18/29, 62.1%). Somatic mutations were detected in 51 of 66 genes that were sequenced, including the following top 10 ranked genes: RHOA, CREBBP, KMT2D, TP53, IDH2, ALK, MEF2B, SOCS1, CARD11, and KRAS. In the longitudinal assessment of ctDNA mutation, the difference in ctDNA mutation volume after treatment showed a significant correlation with disease relapse or progression. Thus, a ≥ 1.5-log decrease in genome equivalent (GE) between baseline and the end of treatment showed a significant association with better survival outcomes than a < 1.5-log decrease in GE. CONCLUSION: Our results suggest the clinical relevance of plasma ctDNA analysis in patients with PTCL. However, our findings should be validated by a subsequent study with a larger study population and using a broader gene panel.

Exosomal and Soluble Programed Death-Ligand 1 (PD-L1) Predicts Responses to Pembrolizumab in Patients with Extranodal NK/T-Cell Lymphoma.

Soluble and exosomal programed death-ligand 1 (PD-L1) can be upregulated in extranodal natural killer/T-cell lymphoma (ENKTL). However, its clinical role in predicting outcomes after pembrolizumab treatment has yet to be studied in ENKTL patients. We investigated the association between pre-treatment soluble and exosomal PD-L1 and outcomes in ENKTL patients who received pembrolizumab as a salvage treatment. The production of soluble and exosomal PD-L1 was analyzed in vitro using an etoposide-resistant ENKTL cell line. Serum levels of soluble and exosomal PD-L1 were measured in patients with relapsed or refractory ENKTL prior to treatment with pembrolizumab. Relapsed or refractory ENKTL patients who received pembrolizumab as a salvage therapy between May 2017 and March 2021 were analyzed at our institute. Soluble and exosomal PD-L1 was significantly higher in serum samples of relapsed or refractory ENKTL patients compared with healthy controls, which is consistent with increased production of soluble and exosomal PD-L1 in an etoposide-resistant ENKTL cell line (SNK6R), which was found to show increased expression of soluble and exosomal PD-L1. Serum-soluble PD-L1 levels were significantly correlated with exosomal PD-L1, and were significantly lower in responders to pembrolizumab compared with non-responders. Longitudinal analysis after pembrolizumab also revealed a relationship between PD-L1 levels and responses. Treatment outcomes and overall survival after pembrolizumab were significantly better in patients with low soluble and exosomal PD-L1. In conclusion, soluble and exosomal PD-L1 can predict responses to pembrolizumab in ENKTL patients, making it a useful pre-treatment biomarker for ENKTL patients receiving pembrolizumab.

Clinical relevance of serum-derived exosomal messenger RNA sequencing in patients with non-Hodgkin lymphoma.

Background: The clinical utility of mRNA cargo in exosomes is unclear, although exosomes have potential as non-invasive biomarkers. This study aimed to investigate the feasibility of exosomal mRNA sequencing for monitoring disease status and predicting outcomes in non-Hodgkin lymphoma (NHL) patients. Methods: Exosomes were isolated from archived serum samples of 33 patients with NHL who were registered into our prospective cohort: diffuse large B-cell lymphoma (DLBCL, n = 17), intravascular B-cell lymphoma (IVL, n = 1), primary mediastinal large B-cell lymphoma (PMBL, n = 4), follicular lymphoma (FL, n = 3), mantle cell lymphoma (MCL, n = 3), and extranodal NK/T-cell lymphoma (ENKTL, n = 5). Exosomal mRNA sequencing was performed, and its concordance with clinical course was analyzed and compared with those of circulating tumor DNA (ctDNA) mutations. Results: Exosomal mRNA sequencing was performed successfully in 26 cases (79%, 26/33), whereas the remaining seven cases were not completed due to their small amount of RNA. The exosomal mRNA sequencing of DLBCL showed gene expression profiles consistent with activated B-cell-like and germinal center type. The longitudinal assessment of exosomal mRNA sequencing results in accordance with the clinical course showed that the post-treatment changes of exosomal mRNA expression were more consistent with treatment outcome than were those of ctDNA mutations. In particular, the exosomal mRNA expression of genes such as BCL2 and BCL6 was increased at the time of disease progression in DLBCL and FL patients. Conclusions: This study demonstrated the feasibility of exosomal mRNA expression profiles as a biomarker for NHL patients. Our results might provide the rationale for studies to explore the potential of exosomal mRNA as a biomarker in NHL patients.

Epigenomic analysis of aberrantly methylated genes in colorectal cancer identifies genes commonly affected by epigenetic alterations.

BACKGROUND: Determination of the profile of genes that are commonly methylated aberrantly in colorectal cancer (CRC) will have substantial value for diagnostic and therapeutic applications. However, there is limited knowledge of the DNA methylation pattern in CRC. MATERIALS AND METHODS: We analyzed the methylation profile of 27,578 CpG sites spanning more than 14,000 genes in CRC and in the adjacent normal mucosa with bead-chip array-based technology. RESULTS: We identified 621 CpG sites located in promoter regions and CpG islands that were greatly hypermethylated in CRC compared to normal mucosa. The genes on chromosome 18 showed promoter hypermethylation most frequently. According to gene ontology analysis, the most common biologically relevant class of genes affected by methylation was the class associated with the cadherin signaling pathway. Compared to the genome-wide expression array, mRNA expression was more likely to be downregulated in the genes demonstrating promoter hypermethylation, even though this was not statistically significant. We validated ten CpG sites that were hypermethylated (ADHFE1, BOLL, SLC6A15, ADAMTS5, TFPI2, EYA4, NPY, TWIST1, LAMA1, GAS7) and 2 CpG sites showing hypomethylation (MAEL, SFT2D3) in CRC compared to the normal mucosa in the array studies using pyrosequencing. The methylation status measured by pyrosequencing was consistent with the methylation array data. CONCLUSIONS: Methylation profiling based on bead-chip arrays is an effective method for screening aberrantly methylated genes in CRC. In addition, we identified novel methylated genes that are candidate diagnostic or prognostic markers for CRC.

Exosomal miR-1305 in the oncogenic activity of hypoxic multiple myeloma cells: a biomarker for predicting prognosis.

Background: Exosomes have emerged as important mediators of tumor progression, and a prognostic role for serum exosomal miRNAs has been suggested in multiple myeloma (MM). Given the association of hypoxia with tumor aggressiveness, including cancer stem cell-like phenotypes, we explored exosomal miRNAs from MM cells under hypoxic conditions and analyzed their diverse roles both in promoting oncogenic activity and in predicting prognosis. Methods: The human MM cell line, RPMI 8226, was cultured under hypoxic conditions and their exosome production and exosomal miRNA profiles were compared with those of normoxic parental cells. The survival outcome of myeloma patients was compared using serum levels of exosomal miRNAs, and the effects of exosomal miRNAs on the target genes of MM cells and adjacent immune cells were analyzed. Results: Increased expression of stem cell markers and exosome production were observed in hypoxic MM cells. Exosome miRNA analysis identified a higher expression of miR-1305 in exosomes isolated from hypoxic MM cells than in those of normoxic parental cells. The overall survival of patients with high exosomal miR-1305 was poorer than it was in patients with low exosomal miR-1305. In hypoxic MM cells, an increase of exosomal miR-1305 led to a decrease of cellular miR-1305 and increased expression of the miR-1305 target genes, MDM2, IGF1 and FGF2 resulted in the promotion of oncogenic activity of MM. Exosomal miR-1305 was also transferred from MM cells to macrophages, and miR-1305-transferred macrophages showed tumor-promoting, M2-macrophage phenotypes. Conclusions: Exosome-mediated secretion of miR-1305 in MM cells promoted oncogenic activity of hypoxic MM cells and high serum levels of exosomal miR-1305.

Isolation of Small Extracellular Vesicles From Human Serum Using a Combination of Ultracentrifugation With Polymer-Based Precipitation.

Methods for reproducibly isolating and enriching small extracellular vesicles (EVs) from blood are essential for clinical utilization of small EVs in cancer patients. We combined ultracentrifugation (UC) with polymer-based precipitation (ExoQuick [EQ] or Total Exosome Isolation [TEI] kit) to isolate small EVs (diameter, 30-150 nm) from the serum of breast cancer patients. We compared the performance of four cycles of UC (UC4x) with that of two cycles of UC followed by enrichment using the EQ (UC2x→EQ) or TEI (UC2x→TEI) kits. The mean concentration of small EVs isolated from 1 mL of serum using UC2x→EQ (139.0±29.1 µg) and UC2x→TEI (140.4±5.0 µg) did not differ from that obtained using UC4x (141.8±26.9 µg). The mean number of EV particles obtained using UC4x was 29.2±9.9×109 per mL of serum, whereas UC2x→EQ and UC2x→TEI yielded higher numbers of EVs (50.7±17.0×109 and 59.3±20.6×109, respectively). Concentrations of EV microRNAs, including miR-21 and miR-155, did not differ between the three methods. In conclusion, performing UC prior to the use of polymer-based precipitation kits could be feasible for isolating small EVs from human serum in large sample-based translational researches.

Serum-Derived Exosomal MicroRNA Profiles Can Predict Poor Survival Outcomes in Patients with Extranodal Natural Killer/T-Cell Lymphoma.

Exosomes containing microRNAs (miRNAs) might have utility as biomarkers to predict the risk of treatment failure in extranodal NK/T-cell lymphoma (ENKTL) because exosomal cargo miRNAs could reflect tumor aggressiveness. We analyzed the exosomal miRNAs of patients in favorable (n = 22) and poor outcome (n = 23) groups in a training cohort. Then, using the Nanostring nCounter® microRNA array, we compared them with miRNAs identified in human NK/T lymphoma (NKTL) cell line-derived exosomes to develop exosomal miRNA profiles. We validated the prognostic value of serum exosomal miRNA profiles with an independent cohort (n = 85) and analyzed their association with treatment resistance using etoposide-resistant cell lines. A comparison of the top 20 upregulated miRNAs in the training cohort with poor outcomes with 16 miRNAs that were upregulated in both NKTL cell lines, identified five candidate miRNAs (miR-320e, miR-4454, miR-222-3p, miR-21-5p, and miR-25-3p). Among these, increased levels of exosomal miR-4454, miR-21-5p, and miR-320e were associated with poor overall survival in the validation cohort. Increased levels were also found in relapsed patients post-treatment. These three miRNAs were overexpressed in NKTL cell lines that were resistant to etoposide. Furthermore, transfection of NKTL cell lines with miR-21-5p and miR-320e induced an increase in expression of the proinflammatory cytokines such as macrophage inflammatory protein 1 alpha. These studies show that serum levels of exosomal miR-21-5p, miR-320e, and miR-4454 are increased in ENKTL patients with poor prognosis. Upregulation of these exosomal miRNAs in treatment-resistant cell lines suggests they have a role as biomarkers for the identification of ENKTL patients at high risk of treatment failure.

STAT6 activation by Toxoplasma gondii infection induces the expression of Th2 C-C chemokine ligands and B clade serine protease inhibitors in macrophage.

Toxoplasma gondii provoked rapid and sustained nuclear translocation of signal transducer and activator of transcription (STAT) 6, a central mediator of interleukin (IL)-4, in macrophage-differentiated human acute monocytic leukemia cells without exogenous IL-4 in western blot and immunofluorescence assay. Phosphorylation of STAT6 occurred immediately after the entry of T. gondii and only by live tachyzoites, not by killed or soluble extract. It was impeded by Janus kinase (JAK) 3 inhibitor and small interfering RNA (siRNA) of STAT6. It induced expression of IL-4 responsive genes such as IL-4R, CD40, and CD23. It also mediated expression of two large clusters of C-C chemokine ligands (CCLs) and serine protease inhibitors (SERPINs) in microarray of T. gondii-infected macrophages. CCL1, 2, 8, 13, and 22 and SPINT2, SERPINB3, B4, and B13 were increased by the infection and inhibited by the treatment of JAK3 inhibitor and siRNA-mediated STAT6 silencing, which suggested the expression was governed by STAT6 activation. Secreting those CCLs, T. gondii-infected macrophages may attract more monocytes and Th2 cells of CCR3 and CCR4 to enrich the Th2 environment nearby the infected macrophages, and induced SERPINs may participate in protection from intracellular damages produced by activated lysosomal enzymes and in the inhibition of caspase activity to block the apoptosis. This suggests that T. gondii exploits cytokine cross-regulation through STAT6 activation to obviate various toxoplasmacidal reactions by interferon-gamma.