ALBERT

Description: Structural chromosomal abnormalities and their break-points were characterized in 17 patients with multiple myeloma (MM) and 4 with plasma cell leukemia by banding. Chromosome 14q32 translocations with a variety of partners were detected in 13 patients, and a variant translocation t(8;22)(q24.1;q11) was detected in 1. Three recurrent 14q32 translocations have been identified: t(6;14)(p21.1;q32.3) occurring in 3 cases, and t(11;14)(q13;q32.3) and t(14;18) (q32.3;q21.3) each occurring in 2 cases. Translocations t(1;14)(q21;q32.3), t(3;14)(p11;q32),t(7;14)(q11.2;q32.3), and t(11;14)(q23;q32.3) were found in each patient, whereas in the remaining 2 patients, partner chromosomes could not be determined. The band 19p13.3 was newly delineated as a recurrent breakpoint involved in translocations in MM. Chromosomes 1 and 6 were also commonly involved in structural abnormalities (14 and 10 patients, respectively), although no particular bands were noted. However, the short arm of chromosome 1 was preferentially involved in deletion, suggesting a certain antioncogene on 1p associated with the development of myeloma. In addition; fluorescence in situ hybridization was successfully applied to determine the nature of the structural abnormalities in a patient with t(8;22) translocation. The present findings suggest that there may be subsets of 14q32 translocations specific to MM.

Description: Several leukocyte populations normally reside in mouse skin, including Langerhans cells and γδ T cells in the epidermis and macrophage and mast cells in the dermis. Interestingly, these skin resident leukocytes are frequently identified within or around hair follicles (HFs), which are known to contain stem cell populations that can generate the epidermal architecture or give rise to the melanocyte lineage. Thus, we reasoned that HFs might serve as a local reservoir of the resident leukocyte populations in the skin. When vibrissal follicles of adult mice were cultured in the presence of stem cell factor (SCF), interleukin 3 (IL-3), IL-7, granulocyte-macrophage colony-stimulating factor, and Flt3 ligand, CD45+/lineage–/c-kit+/FcϵRI+ cells became detectable on the outgrowing fibroblasts in 10 days and expanded progressively thereafter. These HF-derived leukocytes showed characteristic features of connective tissue-type mast cells, including proliferative responsiveness to SCF, metachromatic granules, mRNA expression for mast cell proteases-1, -4, -5, and -6, and histamine release on ligation of surface IgE or stimulation with substance P or compound 48/80. These results, together with our findings that HFs contain c-kit+ cells and produce SCF mRNA and protein, suggest that HFs provide a unique microenvironment for local development of mast cells.

Description: Introduction Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for various hematologic malignancies. Infertility associated with ovarian failure is a serious late complication for female survivors of allo-HSCT. While the role of pretransplant conditioning regimen has been well appreciated, it remains to be elucidated whether GVHD could be causally related to female infertility. We have addressed this issue in a mouse model of non-irradiated bone marrow transplantation (BMT) to avoid devastating effects of irradiation on the ovary. Method Female B6D2F1 (H-2b/d) mice (10 week old) were injected with 80 × 106 splenocytes from allogeneic B6 (H-2b) or syngeneic B6D2F1 donors on day 0. To track migration of donor T cells, CAG-EGFP B6 mice were utilized. Morphometric and immunohistochemical assessments were performed on ovarian sections harvested from recipients on day +21 to evaluate the integrity of ovarian architecture and inflammatory changes. To examine ovary functions, recipients were injected with pregnant mare’s serum gonadotropin (PMSG) on day +19 and human chorionic gonadotropin (hCG) on day +21 to induce ovulation and then oocytes were collected. To evaluate fertility of the recipients, recipients were mated with B6D2F1 males after BMT and mating was repeated 6 times until day +150, and newborns were enumerated. Result In this model, GVHD occurred early after BMT at a peak around day +21 particularly in the gut and liver. Histological examination of the ovaries from allogeneic recipients on day +21 demonstrated GFP+ donor T cells infiltrating in the layer of granulosa cells of mature follicles beyond the basement membrane, cleaved-caspase 3+ apoptotic granulose cells surrounded by lymphocytes (satellitosis), and the destruction of PAS+ basal membrane, whereas there was neither donor T cell infiltration nor pathological changes of the ovaries in syngeneic animals (p = 0.002, Table). Majority of T cells infiltrating in the ovary was CD8+ T cells. In experiments of forced ovulation, numbers of oocytes were significantly less in allogeneic animals than in syngeneic animals (p = 0.043, Table), indicating an impairment of ovary functions. When allogeneic recipients were mated to healthy male mice, numbers of both delivery and newborns per litter decreased, whereas syngeneic recipients remain to be fully fertile. As a result, total numbers of newborns delivered by day +150 after BMT were significantly less in allogeneic animals than in syngeneic animals (p〈 0.001, Table). Administration of 10 mg/kg of prednisolone from day 0 to day +20 significantly reduced ovarian GVHD and restored numbers of oocytes in allogeneic animals to the levels of syngeneic animals. Conclusion Our results demonstrate for the first time that GVHD targets ovary and induces impairment of ovary functions and infertility; and therefore, control of GVHD as well as protection of the ovary from the conditioning is important to prevent female fertility after allo-HSCT. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points Both immature and mature neutrophils differentiate into a previously unrecognized hybrid population when cultured with GM-CSF. The resulting hybrids exhibit dual phenotype and functionality of both neutrophils and dendritic cells.

Description: [Introduction] Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell lymphoma (PTCL) with a dismal prognosis. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment in ATL patients. Mogamulizumab, a humanized anti-CC chemokine receptor 4 (CCR4) monoclonal antibody, is a novel immunotherapeutic agent, effective in treating patients with PTCL such as ATL, PTCL-not specified, and cutaneous T-cell lymphoma. However, in allo-HSCT setting, we should be careful to use mogamulizumab because CCR4 is expressed in regulatory T cells: The mogamulizumab treatment may accelerate GVHD by eradicating regulatory T cells in allo-HSCT patients. Here, we retrospectively analyzed the effect of mogamulizumab on GVHD development in ATL patients treated with mogamulizumab prior to allo-HSCT. [Patients and Methods] Data from the Fukuoka Bone Marrow Transplantation Group were retrospectively analyzed after the approval of mogamulizumab use in Japan. [Results] A total of 24 patients with ATL received mogamulizumab prior to allo-HSCT between April 2012 and April 2015 in our group. The median age at allo-HSCT was 58.5 years (range, 32-72). The median intervals from the last administration of mogamulizumab to allo-HSCT were 25 days (range, 9-126). The median total dose of mogamulizumab was 3 mg/kg (range, 1-8 mg/kg). After treatment with mogamulizumab, 18 patients (75%) had achieved in remission (CR in 4 patients and PR in 14) at allo-HSCT. Ten patients received unrelated bone marrow, 5 received related peripheral blood, and 9 received cord blood as stem cell sources. Eleven patients were treated with full-intensity conditioning and 13 received reduced-intensity conditioning. Graft-versus-host disease (GVHD) prophylaxis consisted of calcineurin inhibitors (cyclosporine or tacrolimus) with short-term methotrexate in 14 patients and mycophenolate mofetil in 9. The cumulative incidence (CI) of acute GVHD at 100 days was 66.6% in grade 2-4 and 33.3% in grade 3-4. The involved organs of acute GVHD were skin in 14 patients, gut in 10, and liver in 4. Among 14 patients who developed grade 2-4 acute GVHD, 5 had severe fluid retention such as pleural effusion or ascites associated with GVHD. Chronic GVHD was observed in 6 patients, and 5 of them were extensive disease. The CI of transplant-related mortality (TRM) and relapse at 1-year were 53.2% (95%CI, 29.3-72.3%) and 29.6% (95%CI, 12.6-48.9%), respectively. The leading cause of death was GVHD (n = 7). The 1-year overall survival and progression-free survival were 19.2% (95%CI, 5.7-38.8%) and 17.2% (95%CI, 4.9-35.7%), respectively. [Discussion] Use of mogamulizumab prior to transplantation in allo-HSCT patients has a merit to decrease the burden of ATL cells. However, it was associated with an increase of TRM due to severe GVHD. Although most of ATL patients achieved better disease status at allo-HSCT through mogamulizumab and the survival rate was expected to be 50% based on the previous data, the survival in the present study was ~20%. These data suggest that mogamulizumab administered before transplantation may have retained until an early phase of post-transplantation, and the donor or host-derived regulatory T cells might be eliminated, allowing the GVHD T-cell clone to expand. Since mogalizumab is a potent anti-ATL agent, we need to develop new treatment protocols integrating mogalizumab at a suitable dose or administration timing, to minimize the unwanted GVHD development in future studies. Disclosures Akashi: Asahi Kasei: Research Funding, Speakers Bureau; Shionogi: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Chugai: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Consultancy, Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding, Speakers Bureau.

Description: Abstract 244 Bacterial infection is a serious complication of bone marrow transplantation (BMT). Intestinal GVHD, in particular, significantly enhances the risk for Gram-negative septicemia. The majority of the intestinal microbiome of the hosts consists of non-cultivable obligate anaerobes, while the Gram-negative bacteria such as Escherichia coli (E. coli) make up a small proportion of the microflora. Thus, it remains unclear why Gram-negative septicemia is dominant in intestinal GVHD, while the role of systemic immunosuppression and use of antibiotics is well-appreciated. We evaluated gut flora changes in the course of GVHD in mouse models of BMT without giving antibiotic or immunosuppresive drugs. Lethally irradiated B6D2F1 (H-2b/d) or B6C3F1 (H-2b/k) mice were injected with 5 × 106 T-cell depleted BM alone (non-GVHD controls) or with 2 × 106 T cells (GVHD group) from MHC-mismatched B6 (H-2b) donors on day 0. Intestinal microflora was identified by using terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA gene libraries constructed from each sample of gut contents. It consisted of approximately 80% of obligate anaerobe, and 20% of anaerobe such as Lactobacilli and Clostridia with very few E. coli before BMT. After BMT, this diversity of gut flora was preserved in non-GVHD controls (Table). In contrast, GVHD mice showed a marked increase of E. coli with a significant decrease in the members of obligate anaerobe. This was associated with dissemination of E. coli to the mesenteric lymph nodes (mLNs) and liver, with elevated serum levels of lipopolysaccharide (LPS). Such a loss of diversity of gut flora with a flora shift towards E. coli was significantly associated with morbidity and mortality of GVHD. Numbers of T-RFLP peaks that indicate diversity of intestinal flora were inversely correlated with GVHD clinical scores (p

Description: Partner sites of 14q32 translocations found in B-cell malignancies were detected by fluorescence in situ hybridization (FISH) using yeast artificial chromosome (YAC) clones, Y20 and Y6, containing the human Ig heavy chain (IgH) gene locus. Y20 spans a 160-kb upstream and 40-kb downstream region of the JH segments on chromosome band 14q32.33. Y6 is 300-kb upstream of Y20, and spans a further 320-kb telomeric region. The human DNA sequences amplified by Alu polymerase chain reaction of the YAC clones were used as probes for FISH to study six patients with non-Hodgkin's lymphoma (NHL), one patient with acute lymphoblastic leukemia, and one cell line FR4 established from a plasmacytoma. Three telomeric YAC clones each specific for 3q, 8q, and 18q were also used to further characterize 14q32 translocations. The IgH YACs were successfully applied to detect cytogenetically invisible subtelomeric translocation of the IgH gene locus to each partner site in t(14;18), t(8;14), and t(14;19), and to identify t(3;14) (q27;q32.33) in three patients with 14q32 translocation of unknown origin. Furthermore, complex translocations involving more than three chromosomes were detected in an NHL patient with t(8;14), and t(3;12), and in the FR4 with der(14)t(8;14), der(8)dic(1;8), and del(1)(q21). The technique would be a useful tool in elucidating the mechanisms of a 14q32 translocation in B-cell malignancies.

Description: Crypt base intestinal stem cells (ISCs) marked by Lgr5 and Olfm4 maintain the intestinal epithelium, and Paneth cells (PCs) provide an epithelial niche for ISCs in the small bowel. ISCs are reduced during gastrointestinal (GI) GVHD, but the precise mechanisms including the role of niche injury are unknown. Additionally, the specific effects of Interferon-γ (IFNγ) on intestinal epithelium in GVHD remain ill-defined. We evaluated kinetics of ISC loss by histology after allogeneic (allo) BMT using Lgr5-LacZ reporter mice. In both MHC- and miHA-mismatched models (LP〉B6 and B6〉BDF1), ISC numbers quickly recovered from pretransplant TBI conditioning in recipients of T-cell-depleted (TCD) BMT by day +10, but ISCs failed to recover in recipients of allo T cells. T-cell-induced ISC reduction was functionally validated by genetic marking of stem cell progeny and by culturing intestinal organoids from crypts isolated post-BMT. Similar to the kinetics of ISC loss, ISC-dependent organoid-forming capacity was impaired in recipients of allo T cells compared with TCD BMT recipients on day +10 (p

Description: Allogeneic hematopoietic stem cell transplantation (SCT) is a curative therapy for various hematologic disorders. Graft-versus-host disease (GVHD) and infections are the major complications of SCT, and their close relationship has been suggested. In this study, we evaluated a link between 2 complications in mouse models. The intestinal microbial communities are actively regulated by Paneth cells through their secretion of antimicrobial peptides, α-defensins. We discovered that Paneth cells are targeted by GVHD, resulting in marked reduction in the expression of α-defensins, which selectively kill noncommensals, while preserving commensals. Molecular profiling of intestinal microbial communities showed loss of physiologic diversity among the microflora and the overwhelming expansion of otherwise rare bacteria Escherichia coli, which caused septicemia. These changes occurred only in mice with GVHD, independently on conditioning-induced intestinal injury, and there was a significant correlation between alteration in the intestinal microbiota and GVHD severity. Oral administration of polymyxin B inhibited outgrowth of E coli and ameliorated GVHD. These results reveal the novel mechanism responsible for shift in the gut flora from commensals toward the widespread prevalence of pathogens and the previously unrecognized association between GVHD and infection after allogeneic SCT.