美山 貴彦

BACKGROUND: Distinguishing between central nervous system lymphoma (CNSL) and CNS infectious and/or demyelinating diseases, although clinically important, is sometimes difficult even using imaging strategies and conventional cerebrospinal fluid (CSF) analyses. To determine whether detection of genetic mutations enables differentiation between these diseases and the early detection of CNSL, we performed mutational analysis using CSF liquid biopsy technique. METHODS: In this study, we extracted cell-free DNA from the CSF (CSF-cfDNA) of CNSL (N = 10), CNS infectious disease (N = 10), and demyelinating disease (N = 10) patients, and performed quantitative mutational analysis by droplet-digital PCR. Conventional analyses were also performed using peripheral blood and CSF to confirm the characteristics of each disease. RESULTS: Blood hemoglobin and albumin levels were significantly lower in CNSL than CNS infectious and demyelinating diseases, CSF cell counts were significantly higher in infectious diseases than CNSL and demyelinating diseases, and CSF-cfDNA concentrations were significantly higher in infectious diseases than CNSL and demyelinating diseases. Mutation analysis using CSF-cfDNA detected MYD88L265P and CD79Y196 mutations in 60% of CNSLs each, with either mutation detected in 80% of cases. Mutual existence of both mutations was identified in 40% of cases. These mutations were not detected in either infectious or demyelinating diseases, and the sensitivity and specificity of detecting either MYD88/CD79B mutations in CNSL were 80% and 100%, respectively. In the four cases biopsied, the median time from collecting CSF with the detected mutations to definitive diagnosis by conventional methods was 22.5 days (range, 18-93 days). CONCLUSIONS: These results suggest that mutation analysis using CSF-cfDNA might be useful for differentiating CNSL from CNS infectious/demyelinating diseases and for early detection of CNSL, even in cases where brain biopsy is difficult to perform.

Not all patients with cancer and severe neutropenia develop fever, and the fecal microbiome may play a role. In a single-center study of patients undergoing hematopoietic cell transplant (n = 119), the fecal microbiome was characterized at onset of severe neutropenia. A total of 63 patients (53%) developed a subsequent fever, and their fecal microbiome displayed increased relative abundances of Akkermansia muciniphila, a species of mucin-degrading bacteria (P = 0.006, corrected for multiple comparisons). Two therapies that induce neutropenia, irradiation and melphalan, similarly expanded A. muciniphila and additionally thinned the colonic mucus layer in mice. Caloric restriction of unirradiated mice also expanded A. muciniphila and thinned the colonic mucus layer. Antibiotic treatment to eradicate A. muciniphila before caloric restriction preserved colonic mucus, whereas A. muciniphila reintroduction restored mucus thinning. Caloric restriction of unirradiated mice raised colonic luminal pH and reduced acetate, propionate, and butyrate. Culturing A. muciniphila in vitro with propionate reduced utilization of mucin as well as of fucose. Treating irradiated mice with an antibiotic targeting A. muciniphila or propionate preserved the mucus layer, suppressed translocation of flagellin, reduced inflammatory cytokines in the colon, and improved thermoregulation. These results suggest that diet, metabolites, and colonic mucus link the microbiome to neutropenic fever and may guide future microbiome-based preventive strategies.

The intestinal microbiota is an important modulator of graft-versus-host disease (GVHD), which often complicates allogeneic hematopoietic stem cell transplantation (allo-HSCT). Broad-spectrum antibiotics such as carbapenems increase the risk for intestinal GVHD, but mechanisms are not well understood. In this study, we found that treatment with meropenem, a commonly used carbapenem, aggravates colonic GVHD in mice via the expansion of Bacteroides thetaiotaomicron (BT). BT has a broad ability to degrade dietary polysaccharides and host mucin glycans. BT in meropenem-treated allogeneic mice demonstrated upregulated expression of enzymes involved in the degradation of mucin glycans. These mice also had thinning of the colonic mucus layer and decreased levels of xylose in colonic luminal contents. Interestingly, oral xylose supplementation significantly prevented thinning of the colonic mucus layer in meropenem-treated mice. Specific nutritional supplementation strategies, including xylose supplementation, may combat antibiotic-mediated microbiome injury to reduce the risk for intestinal GVHD in allo-HSCT patients.

OBJECTIVES: Optimal selection of pretransplant conditioning is crucially vital for improving survival and quality-of-life of patients who receive allogeneic hematopoietic cell transplantation (allo-HCT), particularly in those with high-risk diseases. In this study, we evaluated the efficacy and safety of recently-developed reduced-toxicity myeloablative regimen that combines fludarabine, intravenous busulfan, and melphalan (FBM). METHODS: We conducted a single-center retrospective analysis of 39 patients (23 with myeloid neoplasms and 16 with lymphoid neoplasms), with a median age of 50 (range, 17-68) years, who underwent their first allo-HCT using the FBM regimen. Graft types were bone marrow in 11, peripheral blood in 11, and cord blood in 17 patients. Cyclosporine- or tacrolimus-based graft-versus-host disease (GVHD) prophylaxis was administered. The primary end point of the study was the overall survival rate at 2-year after transplantation. RESULTS: After a median follow-up of 910 days for the surviving patients, 2-year overall survival was 62% for the entire cohort; 73% in the low-to-intermediate-risk group and 44% in the high-to-very high-risk group classified by the refined CIBMTR Disease Risk Index. Cumulative incidences of engraftment, grade II-IV acute GVHD, chronic GVHD, relapse, and non-relapse mortality were 95%, 56%, 56%, 31%, and 17%, respectively. CONCLUSION: These results suggest that our FBM regimen can be applied to allo-HCT using various graft types and yields acceptable outcomes with relatively low non-relapse mortality in both myeloid and lymphoid neoplasms. Also, we observed a promising survival in the group of patients with high-risk diseases, warranting more accumulation of patients and longer follow-up.

Severe leukopenia, thrombocytopenia, and bi-cytopenia due to nivolumab have been reported. In this report, we present the first case of nivolumab-induced severe pancytopenia in a patient with lung adenocarcinoma. A 56-year-old Japanese man with lung adenocarcinoma received nivolumab therapy as second-line treatment. After 3 cycles of this therapy, although computed tomography (CT) showed a reduced tumor size, laboratory findings revealed pancytopenia and a bone marrow biopsy showed a severely hypoplastic marrow. The pancytopenia was diagnosed as an adverse effect of nivolumab; filgrastim (75 μg/day), steroid-pulse therapy (intravenous methylprednisolone: 500 mg/day), and subsequently intravenous prednisolone (50 mg/day) were administered. Furthermore, intravenous administration of immunoglobulins was also performed. However, these treatments were ineffective. He was further diagnosed with fungal pneumonia and a catheter-related bloodstream infection. Anti-bacterial chemotherapy was administered. Two months after hospitalization, the neutrophil count improved to 1000/μL, but multiple red blood cell and platelet transfusions were needed. Therefore, further chemotherapy for lung adenocarcinoma could not be initiated, and the patient died due to progression of lung cancer 118 days after the onset of pancytopenia. The possibility of severe pancytopenia as an immune-related adverse event should be considered as a mandatory prerequisite for nivolumab therapy.

The human immune system is a fine network consisted of the innumerable numbers of functional cells that balance the immunity and tolerance against various endogenous and environmental challenges. Although advances in modern immunology have revealed a role of many unique immune cell subsets, technologies that enable us to capture the whole landscape of immune responses against specific antigens have been not available to date. Acquired immunity against various microorganisms including host microbiome is principally founded on T cell and B cell populations, each of which expresses antigen-specific receptors that define a unique clonotype. Over the past several years, high-throughput next-generation sequencing has been developed as a powerful tool to profile T- and B-cell receptor repertoires in a given individual at the single-cell level. Sophisticated immuno-bioinformatic analyses by use of this innovative methodology have been already implemented in clinical development of antibody engineering, vaccine design, and cellular immunotherapy. In this article, we aim to discuss the possible application of high-throughput immune receptor sequencing in the field of nutritional and intestinal immunology. Although there are still unsolved caveats, this emerging technology combined with single-cell transcriptomics/proteomics provides a critical tool to unveil the previously unrecognized principle of host-microbiome immune homeostasis. Accumulation of such knowledge will lead to the development of effective ways for personalized immune modulation through deeper understanding of the mechanisms by which the intestinal environment affects our immune ecosystem.

To expand our knowledge of the ontogeny of the T-cell receptor (TCR) repertoire of antigen-specific T-cell subsets, we combined next-generation deep sequencing and single-cell multiplex clonotype analysis to evaluate the diversity and frequency of paired TCRs, their functions and whether clonotypic TCRs are shared among different individuals. Using an HLA-A*02-restricted cytomegalovirus (CMV) pp65-derived immunogenic peptide, we found that the more dominant pp65-specific TCR clonotypes in the blood of healthy donors have higher binding affinities for the CMV peptide and arise from clonotypes that are highly shared among individuals. Interestingly, these highly shared HLA-A*02-restricted CMV-specific TCRs were detected in a CMV-seronegative individual as well as in HLA-A*02-negative donors albeit at lower frequency. More intriguingly, these shared TCR clonotypes were abundant in the stem memory T-cell subset, and TCR diversity of the stem memory T-cell repertoire was significantly lower than in the central memory and effector memory T-cell repertoires. These results suggest that the stem memory T-cell subset may serve as a reservoir of highly shared and highly functional memory T-cells.

Polycomb repressive complex 2 (PRC2) participates in transcriptional repression through methylation of histone H3K27. The WD-repeat protein embryonic ectoderm development (EED) is a non-catalytic but an essential component of PRC2 and its mutations were identified in hematopoietic malignancies. To clarify the role(s) of EED in adult hematopoiesis and leukemogenesis, we generated Eed conditional knockout (Eed(Δ/Δ)) mice. Eed(Δ/Δ) mice died in a short period with rapid decrease of hematopoietic cells. Hematopoietic stem/progenitor cells (HSPCs) were markedly decreased with impaired bone marrow (BM) repopulation ability. Cell cycle analysis of HSPCs demonstrated increased S-phase fraction coupled with suppressed G0/G1 entry. Genes encoding cell adhesion molecules are significantly enriched in Eed(Δ/Δ) HSPCs, and consistently, Eed(Δ/Δ) HSPCs exhibited increased attachment to a major extracellular matrix component, fibronectin. Thus, EED deficiency increases proliferation on one side but promotes quiescence possibly by enhanced adhesion to the hematopoietic niche on the other, and these conflicting events would lead to abnormal differentiation and functional defect of Eed(Δ/Δ) HSPCs. In addition, Eed haploinsufficiency induced hematopoietic dysplasia, and Eed heterozygous mice were susceptible to malignant transformation and developed leukemia in cooperation with Evi1 overexpression. Our results demonstrated differentiation stage-specific and dose-dependent roles of EED in normal hematopoiesis and leukemogenesis.

Plasmablastic lymphoma is a rare and aggressive malignancy strongly associated with HIV infection. The refractory/relapsed disease rate is high, and the survival rate is characteristically poor. There are no satisfactory salvage regimens for relapsed cases. We successfully performed autologous stem cell transplantation using a regimen consisting of MCNU (ranimustine), etoposide, cytarabine, and melphalan in a Japanese patient with relapsed AIDS-related plasmablastic lymphoma of the oral cavity. Highly active antiretroviral therapy continued during the therapy. Therapy-related toxicity was tolerable, and a total of 40 Gy of irradiation was administered after autologous stem cell transplantation. The patient has remained in complete remission for 16 months since transplantation.