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Multicentric Castleman disease (MCD) is a heterogeneous lymphoproliferative disorder. It is characterized by inflammatory symptoms, and interleukin (IL)-6 contributes to the disease pathogenesis. Human herpesvirus 8 (HHV-8) often drives hypercytokinemia in MCD, although the etiology of HHV-8-negative MCD is idiopathic (iMCD). A distinct subtype of iMCD that shares a constellation of clinical features including thrombocytopenia (T), anasarca (A), fever (F), reticulin fibrosis (R), and organomegaly (O) has been reported as TAFRO-iMCD, however the differences in cytokine profiles between TAFRO-iMCD and iMCD have not been established. We retrospectively compared levels of serum interferon γ-induced protein 10 kDa (IP-10), platelet-derived growth factor (PDGF)-AA, interleukin (IL)-10, and other cytokines between 11 cases of TAFRO-iMCD, 6 cases of plasma cell type iMCD, and 21 healthy controls. During flare-ups, patients with TAFRO-iMCD had significantly higher serum IP-10 and tended to have lower PDGF-AA levels than the other 2 groups. In addition, serum IL-10, IL-23, and vascular endothelial growth factor-A were elevated in both TAFRO-iMCD and iMCD. Elevated serum IP-10 is associated with inflammatory diseases including infectious diseases. There was a strong correlation between high serum IP-10 and the presence of TAFRO-iMCD, suggesting that IP-10 might be involved in the pathogenesis of TAFRO-iMCD.

Chronic graft-versus-host disease (GVHD) is a major cause of late death and morbidity after allogeneic hematopoietic cell transplantation, but its pathogenesis remains unclear. We investigated the role of the programmed death-1 (PD-1) pathway in chronic GVHD using a well-defined mouse model of B10.D2 (H-2d) donor to BALB/c (H-2d) recipients. PD-1 expression on allogeneic donor T cells was upregulated continuously in chronic GVHD development, whereas PD-L1 expression in host tissues was transiently upregulated and declined to basal levels in the late posttransplant period. Blockade of the PD-1 pathway by anti–PD-1, anti–PD-L1, or anti–PD-L2 mAbs exacerbated clinical and pathologic chronic GVHD. Chimeric mice revealed that PD-L1 expression in host tissues suppressed expansion of IL-17+IFN-γ+ T cells, and that PD-L1 expression on hematopoietic cells plays a role in the development of regulatory T cells only during the early transplantation period but does not affect the severity of chronic GVHD. Administration of the synthetic retinoid Am80 overcame the IL-17+IFN-γ+ T cell expansion caused by PD-L1 deficiency, resulting in reduced chronic GVHD damage in PD-L1−/− recipients. Stimulation of the PD-1 pathway also alleviated chronic GVHD. These results suggest that the PD-1 pathway contributes to the suppression of Th17/Th1-mediated chronic GVHD and may represent a new target for the prevention or treatment of chronic GVHD.

Bronchiolitis obliterans (BO) is a devastating complication of allogeneic hematopoietic stem cell transplantation (HSCT). We retrospectively studied 465 patients who underwent HSCT in the Okayama BMT Group between 2000 and 2009, and describe the detailed clinical features of 13 patients with BO. The 5-year cumulative incidence of BO was 3.43 %. The median time from transplantation to onset of BO was 15 months. In seven of the 13 patients, the primary symptom was only cough, indicating that cough was an important initial symptom for early diagnosis. The median duration from the onset of BO to the requirement of O2 supplementation was 13 months and the main cause of death was respiratory failure. History of chronic graft-versus-host disease was a significant risk factor. Furthermore, female recipients were at greater risk of BO than male recipients; however, no other previously reported risk factors were detected. It is currently difficult to prevent BO on the basis of the reported risk factors. A novel strategy for the early diagnosis and treatment of BO is required.

Chronic graft-versus-host disease (GVHD) remains a major late complication of allogeneic bone marrow transplantation (BMT). In a previous study, impaired thymic negative selection of the recipients permitted the emergence of pathogenic T cells that cause chronic GVHD using MHC class II-deficient (H2-Ab1 KO) B6 into C3H model and CD4+ T cells isolated from chronic GVHD mice caused chronic GVHD when administered into the secondary recipients. In this study, we evaluated the kinetics of regulatory T cell (Treg) reconstitution in wild type B6 into C3H model. After myeloablative conditioning, host Tregs disappeared rapidly, followed by expansion of Tregs derived from the donor splenic T cell inoculum. However, the donor splenic T cell–derived Treg pool contracted gradually and was almost completely replaced by newly generated donor bone marrow (BM)-derived Tregs in the late post-transplantation period. Next, we compared the effects of cyclosporine (CSA) and mammalian target of rapamycin (mTOR) inhibitors on Treg reconstitution. Administration of CSA significantly impaired Treg reconstitution in the spleen and thymus. In contrast, BM-derived Treg reconstitution was not impaired in mTOR inhibitor-treated mice. Histopathological examination indicated that mice treated with CSA, but not mTOR inhibitors, showed pathogenic features of chronic GVHD on day 120. Mice treated with CSA until day 60, but not mTOR inhibitors, developed severe chronic GVHD followed by adoptive transfer of the pathogenic CD4+ T cells isolated from H2-Ab1 KO into C3H model. These findings indicated that long-term use of CSA impairs reconstitution of BM-derived Tregs and increases the liability to chronic GVHD. The choice of immunosuppression, such as calcineurin inhibitor-free GVHD prophylaxis with mTOR inhibitor, may have important implications for the control of chronic GVHD after BMT.

We describe a rare case of recurrent Stenotrophomonas maltophilia bacteremia in a previously healthy 45-year-old man. The infection was caused by osteomyelitis at the site of an iliac crest bone graft harvest. A genetic analysis using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) revealed that the blood isolates and pathogens obtained from the surgical wound were identical. Initial treatment with levofloxacin and cefozopran was ineffective, but the patient's infection was successfully treated by long-term administration of latamoxef and trimethoprim-sulfamethoxazole. The present case suggests that attention should be given to the possibility of S. maltophilia infection in any situations.

We herein describe a case of Wuchereria bancrofti infection in a previously healthy 37-year-old Nepalese man. The patient presented with a history of milky urine with subsequent acute urinary retention lasting for a few days. The presence of microfilariae was confirmed on both peripheral blood and urine smears obtained at midnight. He was conservatively treated with diethylcarbamazine combined with doxycycline. Filariasis was previously endemic in southern parts of Japan, although it has been eradicated. Clinicians should remember filariasis as a potential etiology of urinary retention, especially in cases that may be associated with imported infectious disease.

High-dose chemotherapy supported by autologous stem cell support/transplantation (HDT/ASCT) has been a standard of care over the last two decades in patients with relapsed or refractory(R/R) diffuse large B-cell lymphoma (DLBCL), which is sensitive to salvage chemotherapy. HDT/ASCT for high-risk DLBCL in upfront setting remains controversial, so it is not recommended for clinical practice. Various promising salvage chemotherapy regimens have been reported in phase 2 studies; however, two large randomized phase 3 studies showed similar efficacy of R-ICE vs. R-DHAP and R-GDP vs. R-DHAP. Since the registry data shows feasibility and efficacy of HDT/ASCT in elderly R/R DLBCL patients, older age (> 65 years) itself is not a contraindication for HDT/ASCT. Rituximab maintenance failed to demonstrate a significant benefit compared with observation only after HDT/ASCT. While sensitive R/R DLBCL might be cured by HDT/ASCT even in third-line therapy, the prognosis of insensitive R/R DLBCL is extremely poor. Further study to establish treatment strategies for high-risk patients defined by prognostic factors or biomarkers, and insensitive patients is warranted.

An 82-year-old Japanese man presented with a gastric involvement of peripheral T-cell lymphoma, not otherwise specified. Although gastrointestinal lesions were not detected on computed tomography, oesophagogastroduodenoscopy revealed a slight elevation of the gastric mucosa, with changes in mucosal colour and the presence of abnormal microvessels. This led to the prompt detection of gastric involvement in lymphoma. This case highlights the usefulness of detailed observation of the gastric mucosa for the endoscopic detection of gastric involvement of peripheral T-cell lymphoma.

We report a 34-year-old Japanese female with the vascular type of Ehlers-Danlos syndrome. She had thin translucent skin, extensive bruising, toe joint hypermobility, left lower extremity varicose veins, and chronic wrist, knee and ankle joint pain. She also had dizziness caused by autonomic dysfunction. Magnetic resonance angiography showed tortuous vertebral and basilar arteries, mild left carotid canal bulging, and right anterior tibial artery hypoplasia. Electron microscopic examinations of a skin biopsy revealed extremely dilated rough endoplasmic reticulum in dermal fibroblasts and wide variability of individual collagen fibril diameters. A molecular analysis using a conventional total RNA method and a high-resolution melting curve analysis using genomic DNA revealed a novel missense mutation within exon 48 of the COL3A1 gene, c.3428G>A, leading to p.Gly1143Glu.

The prognosis for patients with advanced or refractory extranodal natural killer (NK)/T-cell lymphoma, nasal type (ENKL) is extremely poor. Thus, allogeneic stem cell transplantation (allo-HSCT) should be considered for this disease. However, reports of allo-HSCT for ENKL are limited because of the rarity of the disease. Here, we describe the clinical course of 12 cases of advanced and refractory ENKL treated with allo-HSCT, including five cases with cord blood transplant. With a median follow-up of 13 months (range, 1-168 months), seven patients are alive in remission, five have died, and one treatment-related death occurred. All patients with disease progression at transplant died of disease progression, whereas seven of eight patients with a complete or partial response are long-term survivors. Allo-HSCT is a feasible and promising consolidation therapy for advanced and relapsed ENKL. The disease status before allo-HSCT is well associated with general outcome, and thus induction treatment is very important for this disease.

Continuous cell division is a hallmark of cancer and cell-cycle regulators therefore represent relevant target molecules for tumor therapy. Among these targets the cyclins are of particular interest as they are overexpressed in various tumor entities with little expression in normal tissue. Here we review evidence that these molecules are recognized by the immune system, summarize why cyclins A, B and D in particular appear to be interesting targets for active and passive immunotherapy, and discuss whether the entire family could be an interesting novel class of tumor antigens for cancer treatment and prevention.

For patients with malignant hematological diseases that relapse after allogeneic hematopoietic stem cell transplantation (HSCT), a second HSCT is thought to be a curative option. A second donor is usually searched for due to HLA discrepancy between the graft and the host. However, it is unclear whether HLA discrepancy between the graft and the first donor has an impact on the outcome of second HSCT. To address this issue, we focused on second HSCT patients who had received first HSCT from an HLA-mismatched (MM) donor and compared the effects of HLA discrepancy between graft and first donor with those between graft and host. We retrospectively analyzed 646 patients receiving second HSCT between 1994 and 2016 after an initial HLA-MM transplantation. With regard to the graft versus host results, the one-allele mismatch (1 MM) group (relative risk [RR], 1.88; 95% confidence interval [CI], 0.79-4.45; p=0.163) and more than one-allele mismatch group (≥ 2 MM) (RR, 1.84; 95% CI, 0.75-4.51; p=0.182) had higher risks of grade III-IV acute GVHD compared to the HLA-matched (0 MM) group, although the results were not significant. In contrast, almost no difference in risk of acute GVHD was found among the 0, 1, and ≥ 2 MM group with respect to graft vs. first donor. Furthermore, with regard to graft vs. host, the ≥ 2 MM group showed a significantly higher risk of treatment-related mortality (TRM) (RR, 1.90; 95% CI, 1.04-3.50; p=0.038) compared to the 0 MM group. In contrast, the risk of relapse was slightly lower in the ≥ 2 MM group (RR, 068; 95% CI, 0.44-1.06; p=0.086). Consequently, no significant difference in OS was found among the three groups. In the analysis of each HLA allele MM, B allele MM between graft and host was associated with an increased risk of grade III-IV acute GVHD (RR 2.87; 95% CI, 1.42-5.79; p=0.003) and DR allele MM between graft and host was associated with a lower risk of relapse (RR, 0.75; 95% CI, 0.58-0.95; p=0.018) and higher risk of TRM (RR, 1.44; 95% CI, 1.03-2.00; p=0.033). In contrast, with regard to graft vs. first donor, there were no significant differences in relapse, TRM, or OS among the three groups, and also analysis of each HLA allele MM indicated no associations with relapse, TRM, or OS. These findings suggested that HLA discrepancy between graft and host, rather than between graft and first donor may induce transplant-related immunological responses in second HSCT leading to an increase in TRM. In conclusion, HLA-MM donor is an option after initial HLA-MM transplantation, however, TRM remains a challenge, particularly with a ≥ 2 MM donor regarding to graft versus host. In this study, the biological effects of HLA discrepancy between the graft and the first donor on the outcome appeared negligible, and our findings shed light on the role of nonhematopoietic APCs on transplant-related immunological responses. Figure. Figure. Disclosures Nakamae: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Research Funding. Ichinohe:Mundipharma: Honoraria; Eisai Co.: Research Funding; Ono Pharmaceutical Co.: Research Funding; Alexion Pharmaceuticals: Honoraria; Zenyaku Kogyo Co.: Research Funding; Kyowa Hakko Kirin Co.: Research Funding; CSL Behring: Research Funding; Novartis.: Honoraria; Takeda Pharmaceutical Co.: Research Funding; Taiho Pharmaceutical Co.: Research Funding; Chugai Pharmaceutical Co.: Research Funding; Astellas Pharma: Research Funding; Repertoire Genesis Inc.: Research Funding; Sumitomo Dainippon Pharma Co.: Research Funding; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; JCR Pharmaceuticals: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Pfizer: Research Funding; Nippon Shinyaku Co.: Research Funding; MSD: Research Funding; Otsuka Pharmaceutical Co.: Research Funding. Kanda:Ono: Consultancy, Honoraria, Research Funding; Shionogi: Consultancy, Honoraria, Research Funding; Nippon-Shinyaku: Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Kyowa-Hakko Kirin: Consultancy, Honoraria, Research Funding; Taiho: Research Funding; Pfizer: Research Funding; Taisho-Toyama: Research Funding; MSD: Research Funding; Novartis: Research Funding; Tanabe-Mitsubishi: Research Funding; Chugai: Consultancy, Honoraria, Research Funding; Otsuka: Research Funding; Dainippon-Sumitomo: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Eisai: Consultancy, Honoraria, Research Funding; Sanofi: Research Funding; CSL Behring: Research Funding; Asahi-Kasei: Research Funding; Celgene: Consultancy, Honoraria; Mochida: Consultancy, Honoraria; Alexion: Consultancy, Honoraria; Takara-bio: Consultancy, Honoraria.

The rapid development of genomics and proteomics has accelerated the discovery of antigens that play a role in host-tumor interaction and can be potentially targeted in tumor immunotherapy. Several independent approaches to characterize such antigens and identify the relevant epitopes have been developed. However, the detection, expansion, and characterization of antigen-specific T cells are essential steps common to all strategies. Efficient identification of epitopes, in particular in a preclinical setting, is often hampered by lack of significant numbers of antigen presenting cells at sufficient purity that readily expand low-frequency T-cell precursors. Using the cylins as a model family of self-tumor antigens, we show that CD40-activated primary human B cells can be used very efficiently to identify novel epitopes and characterize such tumor antigen-specific T cells.

Purpose: Cyclin D1, a key cell cycle regulator, is overexpressed in multiple types of cancer. Such tumor-associated genes may be useful targets for cancer immunotherapy. Nevertheless, it had previously been suggested that efficient T cells recognizing cyclin D1-derived epitopes are absent from the repertoire because of thymic deletion. We attempted to induce autologous CTL from healthy donors and patients with cyclin D1-overexpressing tumors using a highly efficient T-cell expansion system based on CD40-activated B cells as antigen-presenting cells.Experimental Design: Cyclin D1-derived, HLA-A*0201–restricted epitopes were predicted by multiple computer algorithms, screened in HLA-A2-binding assays, and used for T-cell stimulation. The generated CTL lines and clones were analyzed by IFN-γ enzyme-linked immunosorbent spot assay or cytolysis assay.Results: After screening, at least two naturally processed and presented HLA-A*0201–binding cyclin D1 epitopes were identified. CTL specific for these epitopes could be successfully generated from HLA-A2+ donors. T cells efficiently recognized target cells pulsed with the cognate peptide and cyclin D1-expressing tumor cell lines in an HLA-A*0201–restricted manner. More importantly, HLA-A*0201–matched, primary cyclin D1+ tumor cells were efficiently recognized by cyclin D1-specific CTL. These CTL could be generated from patients with mantle cell lymphoma and cyclin D1+ colon cancer.Conclusions: These results underscore that cyclin D1 needs to be considered as a target for broad-based antitumor immunotherapy.

The risks of secondary lung cancer in patients with early stage non-small and small cell lung cancers are estimated to be 1-2% and 2-10% per patient per year, respectively. Surprisingly, the incidence of second primary cancer in locally advanced non-small cell lung cancer at 10 years, following cisplatin-based chemotherapy with concurrent radiotherapy, increases to 61%. Those patients, on the road to being cured, cannot overlook the possibility of developing a second primary cancer. We developed a second primary lung cancer model using cisplatin as a carcinogen in A/J mice to screen for chemopreventive agents for a second malignancy. In the primary lung tumour model, 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), benzo(a)pyrene (BaP), urethane induces specific K-ras mutations in codon 12, codon 12, and codon 61, respectively, in the A/J mice. In this study, we investigated the mechanisms of carcinogenicity by cisplatin in the A/J mice. In the cisplatin-induced tumours, we found no K-ras codon 12 mutation, which is the major mutation induced by NNK or BaP. K-ras gene mutations in codon 13 and codon 61 were found in one tumour (4%) and five tumours (17.8%), respectively. These findings suggest that cisplatin is partially related to K-ras codon 61 mutations, and that the mechanism of carcinogenicity by cisplatin is different from that by NNK or BaP.

The oncogenic mechanisms underlying acute lymphoblastic leukemia (ALL) in adolescents and young adults (AYA; 15-39 years old) remain largely elusive. Here we have searched for new oncogenes in AYA-ALL by performing RNA-seq analysis of Philadelphia chromosome (Ph)-negative AYA-ALL specimens (n = 73) with the use of a next-generation sequencer. Interestingly, insertion of D4Z4 repeats containing the DUX4 gene into the IGH locus was frequently identified in B cell AYA-ALL, leading to a high level of expression of DUX4 protein with an aberrant C terminus. A transplantation assay in mice demonstrated that expression of DUX4-IGH in pro-B cells was capable of generating B cell leukemia in vivo. DUX4 fusions were preferentially detected in the AYA generation. Our data thus show that DUX4 can become an oncogenic driver as a result of somatic chromosomal rearrangements and that AYA-ALL may be a clinical entity distinct from ALL at other ages.

Multicentric Castleman disease (MCD) describes a heterogeneous group of disorders involving systemic inflammation, characteristic lymph node histopathology, and multi-organ dysfunction because of pathologic hypercytokinemia. Whereas Human Herpes Virus-8 (HHV-8) drives the hypercytokinemia in a cohort of immunocompromised patients, the etiology of HHV-8-negative MCD is idiopathic (iMCD). Recently, a limited series of iMCD cases in Japan sharing a constellation of clinical features, including thrombocytopenia (T), anasarca (A), fever (F), reticulin fibrosis (R), and organomegaly (O) has been described as TAFRO syndrome. Herein, we report clinicopathological findings on 25 patients (14 males and 11 females; 23 Japanese-born and two US-born), the largest TAFRO syndrome case series, including the first report of cases from the USA. The median age of onset was 50 years old (range: 23-72). The frequency of each feature was as follows: thrombocytopenia (21/25), anasarca (24/25), fever (21/25), organomegaly (25/25), and reticulin fibrosis (13/16). These patients frequently demonstrated abdominal pain, elevated serum alkaline phosphatase levels, and acute kidney failure. Surprisingly, none of the cases demonstrated marked hypergammoglobulinemia, which is frequently reported in iMCD. Lymph node biopsies revealed atrophic germinal centers with enlarged nuclei of endothelial cells and proliferation of endothelial venules in interfollicular zone. 23 of 25 cases were treated initially with corticosteroids; 12 patients responded poorly and required further therapy. Three patients died during the observation period (median: 9 months) because of disease progression or infections. TAFRO syndrome is a unique subtype of iMCD that demonstrates characteristic clinicopathological findings. Further study to clarify prognosis, pathophysiology, and appropriate treatment is needed.

There has been a growing interest in the use of B cells for cancer vaccines, since they have yielded promising results in preclinical animal models. Contrary to dendritic cells (DCs), we know little about the migration behavior of B cells in vivo. Therefore, we investigated the interactions between CD40-activated B (CD40B) cells and cytotoxic T cells in vitro and the migration behavior of CD40B cells in vivo. Dynamic interactions of human antigen-presenting cells (APCs) and T cells were observed by time-lapse video microscopy. The migratory and chemoattractant potential of CD40B cells was analyzed in vitro and in vivo using flow cytometry, standard transwell migration assays, and imaging of fluorescently labeled murine CD40B cells. Murine CD40B cells show migratory features similar to human CD40B cells. They express important lymph node homing receptors which were functional and induced chemotaxis of T cells in vitro. Striking differences were observed with regard to interactions of human APCs with T cells. CD40B cells differ from DCs by displaying a rapid migratory pattern undergoing highly dynamic, short-lived and sequential interactions with T cells. In vivo, CD40B cells are home to the secondary lymphoid organs where they accumulate in the B cell zone before traveling to the B/T cell boundary. Moreover, intravenous (i.v.) administration of murine CD40B cells induced an antigen-specific cytotoxic T cell response. Taken together, this data show that CD40B cells home secondary lymphoid organs where they physically interact with T cells to induce antigen-specific T cell responses, thus underscoring their potential as cellular adjuvant for cancer immunotherapy.

BACKGROUND Donor lymphocyte infusion (DLI) is used for treatment of hematologic malignancy relapse or mixed chimerism after allogeneic hematopoietic stem cell transplantation. Although graft‐versus‐host disease is well recognized as one of the adverse effects of DLI, there are limited reports on noninfectious pulmonary complications (NIPCs) after DLI. CASE REPORT A 55‐year‐old woman with acute myeloid leukemia received DLI for conversion from recipient predominant to complete donor chimerism on Day +193 after allogeneic HSCT. Eight weeks later, she complained of dyspnea with fever; chest computed tomography revealed diffuse, bilateral, ground glass opacity and reticular appearance. She was diagnosed as having NIPC based on serum and bronchoalveolar lavage fluid (BALF) findings. She was successfully treated with prednisolone (PSL) and completely recovered. DISCUSSION We analyzed the cell profile from the BALF and 27 cytokines and chemokines in the serum using the Bio‐Plex platform. The cells consisted of recipient predominant macrophages and T cells. The serum cytokine and chemokine profile showed significant elevation of interleukin (IL)−1β, IL‐6, IL‐8, tumor necrosis factor‐α, macrophage inflammatory protein (MIP)−1α, and MIP‐1β, which declined with the improvement of symptoms after initiation of PSL treatment. CONCLUSION Inflammatory effectors by recipient cells, rather than allogeneic responses by donor cells, played an important role in the pathogenesis of NIPCs after DLI in the present case.

About 50% of cervical cancers are associated with human papillomavirus type 16 (HPV-16), and since the HPV-16 E6 and E7 oncoproteins are constitutively expressed in the tumor cells, they are attractive targets for cytotoxic T lymphocyte (CTL)-mediated immunotherapy. Nevertheless, only a limited number of HPV-16 E6 epitopes have been identified to date. Using reverse immunological methods, we have generated a CTL clone against the HPV-16 E6(49-57) epitope restricted by HLA-A*2402, which is the most common allele in Japan and relatively frequent worldwide, capable of lysing 293T cells transduced with HLA-A*2402 and HPV-16 E6. Although it was unable to recognize the SiHa cervical cancer cell line positive for HPV-16 and HLA-A*2402, the cells became susceptible to lysis when transduced with E6-E7 genes, which was unexpectedly offset by pretreatment with interferon (IFN)-gamma alone. Interestingly, however, combined pretreatment with a proteasome inhibitor, bortezomib and IFN-gamma fully restored CTL-mediated lysis of the original SiHa cells. Furthermore, such intervention of 2 of 4 other cervical cancer cell lines expressing HPV-16 E6 and HLA-A*2402 was found to induce IFN-gamma production by specific CTLs. Tetramer analysis further revealed that induction of E6(49-57)-specific T cells was possible in 5 of 7 patients with HPV-16-positive high grade cervical intraepithelial neoplasia or cervical cancer by in vitro stimulation with E6(49-57) peptide. Thus, these findings together indicate that E6(49-57) is a candidate epitope for immunotherapy and immunological monitoring of such patients.

We report a case of POEMS syndrome in a 20-year-old patient diagnosed after visiting an eye clinic with a chief complaint of reduced visual acuity. A male university student aged 20 years was referred to our department complaining of blurred vision in both eyes that had persisted for 1 month. He also noted headache, nausea, and paresthesia in the lower extremities around the same time. The visual acuity of his right and left eye was 20/40 and 20/20, respectively. Optic disc edema and serous retinal detachment were present. Brain magnetic resonance imaging showed no intracranial abnormalities, while elevated cerebrospinal fluid pressure, reduced nerve conduction velocity in both lower extremities, hepatosplenomegaly, M proteinemia, high blood VEGF levels, osteoblastic and osteolytic changes in the spine, and atypical plasma cells in bone lesions were noted. From the above findings, the patient was diagnosed with POEMS syndrome. He received high-dose dexamethasone, thalidomide, and radiotherapy on the sacral mass, followed by high-dose melphalan with autologous stem-cell support, and showed subsequent systemic and ophthalmologic improvement. Here, we report the youngest case ever of POEMS syndrome with ocular manifestation. If patients have optic disc edema in both eyes with no intracranial space-occupying lesion, POEMS syndrome should be considered in differential diagnosis, regardless of age.

The thymus leukemia antigens (TL) belong to the MHC class Ib family and can be recognized by CD8-dependent or -independent cytotoxic T lymphocytes (CTL) showing TL, but not H-2, restriction. We previously reported that the CTL epitope is TAP independent and in the present study we further characterize the recognition mechanism of CD8-dependent TL-specific TCRalphabeta CTL. We first prepared empty TL tetramers by way of peptide-independent folding with recombinant proteins produced in an Escherichia coli expression system, and showed that TL-specific CTL recognized TL without putative TL-associated peptide and/or post-translational modifications of TL by mammalian and insect cells. We next prepared transfectants expressing various chimeric TL molecules with mouse or human MHC class I as well as chimeric TL tetramers with recombinant proteins produced by insect cells, and demonstrated that chimeric TL whose alpha3 domain was replaced by that of H-2K(b), but not of HLA-A2, was sufficient for binding and activation of TL-specific CTL. These results indicate that TL-specific CTL recognize predominantly their alpha1/alpha2 domain as an epitope(s) and that the binding activity to the murine CD8 of the alpha3 domain of H-2K(b) is sufficient to induce their CTL activity, although it is known to be weaker than that of TL, but stronger than that of HLA. The results taken together indicate that CD8-dependent TL-specific TCRalphabeta CTL recognize an epitope(s) of the alpha1/alpha2 domain of TL free from antigenic molecules, and that CD8 plays an important role in stable interactions between TL and their corresponding TCR.

We report the identification of two novel minor histocompatibility antigens (mHAgs), encoded by two separate single nucleotide polymorphisms on a single gene, BCL2A1, and restricted by human histocompatibility leukocyte antigen (HLA)-A*2402 (the most common HLA-A allele in Japanese) and B*4403, respectively. Two cytotoxic T lymphocyte (CTL) clones specific for these mHAgs were first isolated from two distinct recipients after hematopoietic cell transplantation. Both clones lyse only normal and malignant cells within the hematopoietic lineage. To localize the gene encoding the mHAgs, two-point linkage analysis was performed on the CTL lytic patterns of restricting HLA-transfected B lymphoblastoid cell lines obtained from Centre d'Etude du Polymorphisme Humain. Both CTL clones showed a completely identical lytic pattern for 4 pedigrees and the gene was localized within a 3.6-cM interval of 15q24.3–25.1 region that encodes at least 46 genes. Of those, only BCL2A1 has been reported to be expressed in hematopoietic cells and possess three nonsynonymous nucleotide changes. Minigene transfection and epitope reconstitution assays with synthetic peptides identified both HLA-A*2402– and B*4403-restricted mHAg epitopes to be encoded by distinct polymorphisms within BCL2A1.

Background. T cells specific for minor histocompatibility antigens (mHAgs) play a major role in both graft-versus-host disease and the graft-versus-tumor effect after HLA-identical bone marrow transplantation (BMT). However, characterization of individual T-cell responses to mHAgs is difficult and has generally involved extensive screening of T-cell clones isolated from bulk T-cell cultures generated from BMT recipients. In this report, we describe a new approach that permits both direct visualization of CD8 + T-cell responses to mHAgs and cloning of T cells reacting with mHAgs presented by individual HLA alleles of interest. Methods and Results. Panels of Epstein-Barr virus-transformed B-cell lines (B-LCL) expressing retrovirally transduced HLA cDNA were used as stimulator cells in an enzyme-linked immunospot (ELISPOT) assay to identify CD8 + T cells reacting with mHAgs in cultures generated from postBMT recipient peripheral blood. T cells specific for mHAgs presented by selected HLA alleles could then be captured and cloned using an interferon-y secretion assay and magnetic bead selection. A majority of T-cell clones thus isolated exhibited cytolytic activity against the same HLA-transfected B-cell lines used for the ELISPOT assay. Conclusion. The ELISPOT assay was useful for identification of the HLA alleles presenting mHAgs recognized by individual T-cell lines. This approach for isolating mHAgs-specific CD8 + T-cell clones should assist in characterizing responses restricted by HLA alleles of interest, which are common in a certain ethnic group.

We report here three cases of peripheral T-cell lymphoma unspecified (PTCL-US), which presented with bone marrow infiltration and hepatosplenomegaly and were successfully treated with high-dose chemotherapy (HDCT) and autologous peripheral blood stem cell transplantation (auto-PBSCT). The patients were all characterized by cytokine-induced symptoms such as fever, anasarca, cytopenia, poor general condition, and disseminated intravascular coagulation syndrome. Laboratory data showed extremely high levels of soluble interleukin-2 receptor, beta(2)-microglobulin, and ferritin. All three patients were negative for anti-adult T-cell leukemia antibody. In one patient, hemophagocytosis was revealed by a histological examination of the bone marrow. The International Prognostic Index was high for all three patients, and they all achieved complete remission after the intensive chemotherapy for remission induction. During complete remission, they were treated with HDCT [modified interleukin-converting enzyme regimen] followed by auto-PBSCT. The recovery of hematopoiesis after auto-PBSCT was prompt and sustained engraftment was obtained. No serious adverse effects other than myelosuppression were noted. One patient died due to cerebrovascular disease without relapse 18 months after auto-PBSCT. The other two patients are still alive and have not suffered from relapse. Our observations suggest that auto-PBSCT following HDCT may be an effective and safe therapeutic modality for high-risk PTCL-US patients characterized by hepatosplenomegaly and cytokine-induced syndrome.

Analysis of HLA restriction specificity is one of the important steps in characterizing T cell clones. This usually requires either a panel of HLA-typed cells or HLA cDNA transfectants. Although preparation of HLA cDNA transfectants is laborious, utilization of transfectants is advantageous when a suitable panel is not available due to linkage disequilibrium or rarity of the HLA allele of interest. In this report, we describe an efficient and rapid HLA cloning and expression system. Three sets of PCR primers specific for HLA-A, B and C loci were designed by extensively sequencing 5'- and 3'- untranslated regions of HLA class I genes. The PCR-amplified products were introduced into modified Phoenix retrovirus vectors containing a puromycin resistant gene under the control of a LTR promotor. Gibbon ape leukemia virus (GALV)-pseudotyped retrovirus was produced and infected into B-lymphoid cell lines. Following expansion in selection media, more than 80% of cells expressed transduced HLA at a comparable level to that normally expressed. These results indicate that locus-specific PCR cloning and utilization of GALV-pseudotyped retroviral vector can be an effective and relatively efficient tool for constructing a panel of different HLA transfectants.

We describe a rare case of recurrent Stenotrophomonas maltophilia bacteremia in a previously healthy 45-year-old man. The infection was caused by osteomyelitis at the site of an iliac crest bone graft harvest. A genetic analysis using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) revealed that the blood isolates and pathogens obtained from the surgical wound were identical. Initial treatment with levofloxacin and cefozopran was ineffective, but the patient's infection was successfully treated by long-term administration of latamoxef and trimethoprim-sulfamethoxazole. The present case suggests that attention should be given to the possibility of S. maltophilia infection in any situations.

Pulmonary complications in patients with hematological malignancies are often caused by infection but are sometimes associated with an underlying disease such as organizing pneumonia (OP). Here, we report a case of life-threatening steroid-resistant OP associated with myelodysplastic syndrome (MDS) and successfully performed allogeneic hematopoietic cell transplantation (HSCT). A 33-year-old female with refractory anemia with excess blasts-1 that had progressed from refractory anemia with ringed sideroblasts and concomitant Sweet’s syndrome was admitted. Multiple pulmonary infiltrates were revealed on a chest computed tomography scan, which progressively worsened even after chemotherapy and corticosteroid therapy. No evidence of infection was observed in bronchoalveolar lavage fluid. A histological examination of a transbronchial lung biopsy specimen showed lymphocyte invasion with fibrosis, indicating that the pulmonary infiltrates were OP associated with MDS. Before transplantation, she suffered from respiratory failure and required oxygen supplementation. She developed idiopathic pneumonitis syndrome on day 61 that responded well to corticosteroid therapy, and the OP pulmonary infiltrates improved gradually after HSCT, She was discharged on day 104 and is well without recurrence of OP or MDS 2 years after HSCT.

Background In the course of graft-versus-host disease (GVHD) or diseases that histologically mimic GVHD (e.g. toxic epidermal necrolysis, Stevens–Johnson syndrome), it is known that epidermal Langerhans cells (LCs) are depleted from the epidermis. However, the mechanism and significance of LC depletion is not well known. Objectives To investigate the numerical, morphological and phenotypic changes in LCs and apoptosis of LCs in the course of GVHD using a non-irradiated mouse GVHD model. Methods BALB/c nu/nu mice and C57BL/6 mice were used as recipients and donors, respectively. Recipient mice were injected with T-cell-enriched donor spleen cells. Skin samples were harvested at various times after the inoculation. The numerical and morphological changes were examined by an immunofluorescence study of epidermal sheets. Apoptosis was studied by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling method and flow cytometric analysis using annexin V. Phenotypic change was studied by flow cytometric analysis of epidermal cell suspensions. The mixed epidermal cell lymphocyte reaction (MELR) was performed to examine functional changes in the epidermal cells. Results Five days after inoculation, a graft-versus-host reaction occurred. Epidermal LCs began to decrease from the sixth day. On the fifth day, the LCs became larger and had prominent dendrites. Immediately before the LCs began to decrease, many LCs became round in shape, with scanty dendrites. LC apoptosis was not observed in the epidermis either on the fifth or seventh day. Phenotypically, the expression of CD40, CD80, CD86 and major histocompatibility complex class II antigen on the LCs was upregulated on the fifth and seventh day. Epidermal cells from GVHD mice showed an increased allostimulatory capacity in the secondary MELR. Conclusions These results suggest that at early GVHD onset, most LCs may not undergo apoptosis in the epidermis but are phenotypically activated, resulting in further activation of alloreactive T cells and aggravation of the disease.

Although high-dose chemotherapy with autologous peripheral blood stem cell transplantation (autoPBSCT) has been shown or confirmed to be an effective treatment for high-risk and relapsed non-Hodgkin's lymphoma (NHL), relapse after autoPBSCT remains a serious problem. In a clinical trial to overcome relapse, we adopted a treatment plan in which PBSCs purified in vitro to CD34+ cells to deplete tumor cells (CD34+ autoPBSCT), total body irradiation (TBI) of 1200 cGy, and melphalan, 180 mg/m2, were used as a preconditioning regimen. Eighteen patients with relapsed or high-risk NHL participated in the study. This study compared the incidence of complications following CD34+ autoPBSCT preconditioned with the TBI regimen (n = 10): the TBI group; CD34+ autoPBSCT with the non-TBI regimen (n = 8): the non-TBI group; and unselected autoPBSCT with the non-TBI regimen (n = 19): the unselected autoPBSCT control group. After day 30 posttransplantation, 6 of 10 patients treated with the TBI regimen developed 11 infectious complications in total, compared with only 1 of 8 patients treated with the non-TBI regimen and 4 of 19 patients given unselected autoPBSCT. Two fatal complications occurred in the TBI group, but none occurred in the other 2 groups. The CD4+ lymphocyte count at 1 month posttransplantation was significantly lower in the TBI group than in the unselected autoPBSCT group. These findings suggest that the addition of TBI to the preconditioning regimen for CD34+ autoPBSCT is associated with an increased incidence of severe infectious complications after transplantation.

Introduction: Hematological complete remission (CR) is the evident prognostic factor of allogeneic hematopoietic stem cell transplantation (HSCT) in patients with Acute Myeloid Leukemia (AML). CR with incomplete blood cell count recovery (CRi) after induction therapy was identified as independent prognostic factor for inferior long-term outcomes in patients with AML achieving remission (Chen et al, JCO 2015). There is a paucity of data regarding the impact of response (CR vs. CRi) prior to allogeneic HSCT. Here we examined whether CRi provide prognostic information on the transplant outcomes. Methods: We retrospectively analyzed 73 consecutive adults with AML who received first allogeneic HSCT between 2008 and 2015. All clinical data were collected from medical records. CRi were defined CR with absolute neutrophil count < 1,000/mm3 and/or platelet count < 100,000/mm3. CR and CRi were confirmed just prior to allogeneic HSCT in bone marrow and peripheral blood. Cytogenetic risk group was assigned based on CIBMTR criteria. Chemotherapy regimens prior to allogeneic HSCT defined as follows; standard-dose (cytarabine plus anthracycline), high-dose (cytarabine at individual dose ≥ 1g/m2 with or without other drugs), low-dose (azacitidine or low dose cytarabine). Categorical and continuous variables were analyzed using Fisher's exact test and Mann-Whitney U test, respectively. Overall survival (OS) and relapse free survival (RFS) were estimated by Kaplan-Meiyer method. Cumulative incidence of relapse (CIR), non-relapse mortality (NRM), engraftment rate and cumulative infectious events were calculated using Grayfs method. Multivariate analysis was performed using the Fine-gray proportional hazard regression model for NRM and cumulative infectious events. Results: A total of 48 (66%) were in CR and 25 (34%) in CRi. The characteristics of the study population, donors and transplants stratified by blood count recovery pre-HSCT are summarized in Table 1. There were several statistically differences between patients in CR and CRi. More patients in CR had longer time from final chemotherapy to HSCT, lower HCT-CI and a higher proportion of major ABO mismatch donor. Median follow-up from allogeneic HSCT was 18 months (range, 1 to 80 months) in the patients still alive. Patients in CR and CRi had a similar 2-year OS (63% vs. 60%, p = 0.29), 2-year RFS (58% vs. 54%, p = 0.42) and 2-year CIR (30% vs. 19%, p = 0.64). 2-year NRM was significantly higher for patients in CRi (17% vs. 34%, p = 0.046) (Figure 1). Ten patients in CRi (40%) died after allogeneic HSCT. One patient died from relapse of AML. Causes of NRM were infection (n=2), veno-occlusive disease (n=3), respiratory failure (n=3) and chronic graft-versus-host disease (n=1). The median time of neutrophil engraftment was 15 days for patients in CR (range, 10 to 25 days) and 18 days for patients in CRi (range, 11 to 49 days). Engraftment rate of neutrophil for patients in CR and CRi was 96% and 92%, respectively. 2 patients in CRi died before neutrophil engraftment. Engraftment rate of neutrophil within 30 days after allogeneic HSCT was significantly lower for patients in CRi (96% vs 84%, p = 0.037). The median time of platelet engraftment was 22 days for patients in CR (range, 10 to 77 days) and 26 days for patients in CRi (range, 12 to 164 days). Engraftment rate of platelet for patients in CR and CRi was 92% and 80%, respectively. 3 patients in CRi died before platelet engraftment. Engraftment rate of platelet within 60 days after allogeneic HSCT was lower for patients in CRi (85% vs. 65%, p = 0.093). To assess the effect of delayed engraftment, we examined infectious events, including bacteria, fungus and virus, within 60 days after allogeneic HSCT. Significantly more patients in CRi had higher cumulative infectious events (46% vs. 72%, p = 0.0086) (Figure 2). There was no bleeding event within 60 days after allogeneic HSCT. Multivariate analysis demonstrated that CRi was an independent risk factor of early infection after allogeneic HSCT (hazard ratio: HR 2.65, 95% CI: 1.37-5.08, p = 0.0037) without a difference in NRM (HR 1.21, 95% CI: 0.26-5.59, p = 0.81). Conclusion: Our data, although retrospectively collected, show that incomplete blood count prior to allogeneic HSCT is a predictable marker of early infection after HSCT in patients with AML. This suggests that we need to develop prophylactic strategies for early infection after allogeneic HSCT based on risk assessments. Disclosures Maeda: Mundipharma KK: Research Funding.

Introduction: The creatinine clearance rate (Ccr) is a more accurate indicator of renal function than serum creatinine. There remains a paucity date of prognostic value of mild to severe renal impairment calculated by Ccr for patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). Herein, we evaluated the transplant outcome of patients with mild to severe renal dysfunction and compared to those without renal dysfunction. Patients and methods: A total of 186 patients (118 male and 68 female; median age, 50 years; range 15-70 years) with hematological malignancies who underwent first allo-HSCT in our institution between 2008 and 2015 were retrospectively collected from medical records. Transplant outcomes were compared with regard to renal function before allo-HSCT. The threshold of renal dysfunction was 60 ml/min (Ccr < 60) calculated by Ccr. Overall survival (OS) was estimated by Kaplan-Meiyer. Engraftment rate, cumulative incidence of relapse (CIR), non-relapse mortality (NRM), acute graft-versus-host disease (aGVHD), chronic GVHD and acute kidney injury (AKI) within 100 days after alo-HSCT were calculated using Grayfs method. Factors associated with significance (p < 0.1) on univariate analyses were subjected to multivariate analysis. Multivariate analysis was performed using the Cox proportional hazards regression model for OS and Fine-gray proportional hazard regression model for NRM and CIR. Results: Among the 186 patients, renal dysfunction (Ccr < 60) was observed in 30 patients (16%) before conditioning regimen. We observed no significant difference between Ccr < 60 group and Ccr ≥ 60 group in terms of clinical characteristics including gender, HLA disparity, stem cell source, disease risk, comorbidity, performance status at allo-HSCT and GVHD prophylaxis. However, compared to Ccr ≥ 60 group, patients with Ccr < 60 group were significantly older (p = 0.0008), lower estimated GFR (p < 0.0001), higher serum creatinine (p < 0.0001), more cases diagnosed non-Hodgkin lymphoma (p = 0.0003) and more cases received reduced intensity conditioning regimen (p = 0.002). All but one patient with CCr < 60 group achieved neutrophil engraftment median 15 days (range, 9-24) after transplantation (engraftment rate 97%) and 1 patient died before engraftment. Regarding the transplantation outcome, no significant difference was observed in OS, CIR, NRM, aGVHD, cGVHD and AKI after allo-HSCT between 2 groups (Ccr < 60 vs. CCr ≥ 60; 2-year OS, 45% vs. 49%, p = 0.6, 2-year CIR, 41% vs. 30%, p = 0.3, 2-year NRM, 23% vs. 26%, p = 0.6, day-100 AKI, 57% vs. 63%, p = 0.6, Figure 1). Multivariate analysis demonstrated that Ccr < 60 before allo-HSCT was not an independent prognostic factor for OS, CIR and NRM (Table 1). To investigate the impact of mild to severe renal failure on the transplant outcome, we next explored the clinical results of 30 patients with renal failure more in detail. Of them, 13 patients (43%) had estimated GFR < 60 ml/min/1.73m2 and only 5 patients (17%) had a serum creatinine level > 1.2mg/dl. Two patients (7%) developed end-stage renal failure requiring dialysis. Eighteen patients (60%) died at a median 194.5 (range, 15-859) days after allo-HSCT. Relapse of the primary disease was the leading cause of death (n=9) and cause of NRM were infection (n=3), veno-occlusive disease (n=2), respiratory failure (n=2), cGVHD (n=1) and central nervous system complication (n=1). No patients died from renal failure. All patients with severe renal dysfunction defined as Ccr < 40ml/min (Ccr; 13.4 ml/min, 36 ml/min and 36 ml/min) died 17, 15 and 72 days after allo-HSCT, respectively. Conclusion: Our cohorts with total 186 patients underwent allo-HSCT showed that mild renal dysfunction defined as Ccr < 60 ml/min before allo-HSCT did not affect adversely for transplant outcomes. In line with previous reports, Ccr could detect renal impairment that could not identified by GFR estimated from serum creatinin. On the other hand, no patients with severe renal dysfunction could survive after allo-HSCT. To our knowledge, the current study includes the largest number of patients with Ccr < 60ml/min. However, our small experience clearly desires larger series patients for evaluating the real impact of mild to severe renal dysfunction before transplantation. Disclosures No relevant conflicts of interest to declare.

The number of elderly patients with diffuse large B cell lymphoma (DLBCL) continues to increase but the data regarding autologous stem cell transplantation (ASCT) for elderly patients are limited. We analyzed 484 patients, ages 60 years or over, diagnosed with relapsed/refractory DLBCL who received ASCT from 1993 to 2010 in the Japan Society for Hematopoietic Cell Transplantation database. Median age was 64 years (range, 60 to 78). To evaluate the impact of age at ASCT, patients were classified into 3 groups: those between the ages of 60 to 64, 65 to 69, and 70 years or over. Overall nonrelapse mortality (NRM) at day 100, 1 year, and 2 years was 4.1%, 5.9% and 7.7%, respectively. NRM did not significantly differ among age groups (P = .60). Two-year progression-free survival (PFS) and overall survival (OS) were 48% and 58%, respectively. PFS and OS were significantly longer in patients 60 to 64 years old; however, the survival rate was acceptable even in those 70 or over, with a 2-year OS of 46%. ASCT is feasible in selected elderly patients and age alone should not be a contraindication for ASCT. Eligibility should be individualized and identification of a subset of elderly patients at high risk of treatment-related morbidity or mortality warrants investigation.

Recently, atypical Castleman's disease (CD) was reported in Japan. This disease is considered as TAFRO syndrome or non-idiopathic plasmacytic lymphadenopathy (IPL), a constellation of clinical symptoms, namely, thrombocytopenia, anasarca, fever, reticulin fibrosis, and organomegaly without hyper-γ-globulinemia. Histopathologically, this disease is similar to hyaline vascular (HV)-type CD. Here, we present a 43-year-old Japanese woman meeting the clinical criteria of TAFRO syndrome who was successfully treated with combined corticosteroid therapy. She showed a rapidly progressive course of thrombocytopenia, systemic lymphadenopathy, fever, anasarca, and increase in acute inflammatory proteins without hyper-γ-globulinemia. Lymph node biopsy was performed and revealed HV-type CD without human herpes virus 8 infection, which was clinicopathologically compatible with non-IPL. The association of these atypical features with well-known multicentric Castleman's disease (MCD), namely, HV-type histology with systemic lymphadenopathy, marked thrombocytopenia even with a high level of interleukin-6, and increased acute inflammatory proteins without hyper-γ-globulinemia, suggests that TAFRO syndrome as presented in our case is a novel entity, which may have been diagnosed as MCD in the past. To define this novel entity more clearly and to demonstrate its etiology, further nationwide surveys of this syndrome and MCD are needed.

Nat. Genet. 48, 569–574 (2016); published online 28 March 2016; corrected after print 29 August 2016 In the version of this article initially published, the following statement was not included in the Online Methods: “The results published here are in part based upon data generated by the TARGET initiative managed by the National Cancer Institute (NCI).

We report a 34-year-old Japanese female with the vascular type of Ehlers-Danlos syndrome. She had thin translucent skin, extensive bruising, toe joint hypermobility, left lower extremity varicose veins, and chronic wrist, knee and ankle joint pain. She also had dizziness caused by autonomic dysfunction. Magnetic resonance angiography showed tortuous vertebral and basilar arteries, mild left carotid canal bulging, and right anterior tibial artery hypoplasia. Electron microscopic examinations of a skin biopsy revealed extremely dilated rough endoplasmic reticulum in dermal fibroblasts and wide variability of individual collagen fibril diameters. A molecular analysis using a conventional total RNA method and a high-resolution melting curve analysis using genomic DNA revealed a novel missense mutation within exon 48 of the COL3A1 gene, c.3428GA, leading to p.Gly1143Glu.

Sonography-guided cutting needle biopsy for the diagnosis of malignant lymphoma has recently come into wide use. However, surgery is sometimes unavoidable for the diagnosis of malignant lymphoma, particularly for low-grade malignant lymphoma such as extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue, because cutting needle biopsy offers limited diagnostic accuracy for low-grade malignant lymphoma. Of course, unnecessary invasive procedures like open biopsy should be avoided wherever possible, given the cosmetic problems and burden on the patient. We tried to diagnose malignant lymphoma using the combination of cutting needle biopsy, flow cytometry and polymerase chain reaction to identify monoclonal rearrangement of immunoglobulin heavy chain genes. We have used this method in two cases in whom malignant lymphoma was suspected in the head and neck region, allowing diagnosis of mucosa-associated lymphoid tissue lymphoma in both cases. One case involved a 23-year-old woman with mucosa-associated lymphoid tissue lymphoma in the parotid glands, and the other involved a 77-year-old man with mucosa-associated lymphoid tissue lymphoma in the thyroid. The combination of cutting needle biopsy, flow cytometry and immunoglobulin heavy chain gene rearrangement testing might offer a useful alternative to open biopsy for the diagnosis of mucosa-associated lymphoid tissue lymphoma. We recommend this procedure, particularly for young women or patients with poor performance status in whom malignant lymphoma is suspected.

SummaryCellular adjuvants such as dendritic cells (DC) are in the focus of tumour immunotherapy. In DC-vaccine trials, induction of tumour antigen-specific immunity is observed frequently and well-documented clinical responses have been reported. However, the overall response rate is less than 3%, therefore alternative strategies are being investigated. CD40-activated B cells (CD40-B) have been characterized previously as an interesting alternative because they present antigen efficiently and can be expanded by several logs from small amounts of peripheral blood. To determine the central technical challenges of cell-based vaccines we performed a single-patient analysis of 502 patients from DC-based tumour vaccine trials and identified at least three factors contributing to their limited efficiency: (1) lack of cell numbers; (2) lack of documented purity thus high contamination of bystander cells; and (3) lack of quality control and thus heterogeneous or unknown expression of important surface molecules such as major histocompatibility complex (MHC) and chemokine receptors. Based on these findings we re-evaluated the CD40-B approach in cancer patients. Here, we show that proliferation of B cells from cancer patients is equivalent to that observed in healthy donors. Purity is always > 90% after 2 weeks and remains stable for several weeks. They have comparable antigen-presenting capability determined phenotypically and by allogeneic mixed lymphocyte reaction. Expression of CCR7 and CD62L was detected in all samples and B cells migrated towards the relevant homing chemokines. Taken together, CD40-B cells from cancer patients can be expanded in virtually unlimited numbers at high purity and full function concerning antigen-presentation and migratory properties.

The calcineurin inhibitors (CIs) cyclosporine A and tacrolimus are essential for graft-versus-host disease prophylaxis but are associated with adverse effects, including neurotoxicity. We report a case of irreversible CI-induced neuropathic pain following allogeneic hematopoietic stem cell transplantation. The patient developed dysesthesia, electric shock-like pain, and severe itching followed by intractable analgesic-resistant pain in the lower extremities. There were no abnormal radiographic findings, and there was no improvement with a reduction of CI dosage or with administration of a calcium channel blocker. These clinical findings are similar to but inconsistent with CI-induced musculoskeletal pain syndromes previously reported in organ transplantation.

Background and Objectives: Thrombotic microangiopathy (TMA) after hematopoietic stem cell transplantation (SCT) is a devastating complication of SCT and lacks optimal managements with a high mortality. Its pathogenesis is not well understood but suggested that endothelial toxicity caused by chemoradiotherapy, infections, immunosuppressive drugs, graft-versus-host-disease (GVHD) and infections have been thought to play important roles. Recently recombinant human soluble thrombomodulin (rTM) was reported to be a safe and efficient treatment of disseminated intravascular coagulation for hematological malignancies. rTM which contains active extracellular domain of thrombomodulin, inactivates coagulation and the thrombin-rTM complexes activates protein C to activated protein C (APC). APC in activates factors Va and VIIIa with protein S, and subsequently lead to further thrombin formation. And, also rTM possesses cytoprotective effects against calcineurin inhibitor induced endothelial cell damage, anti-inflammatory activity such as decrease of high-mobility group box 1 protein, IL-6 and TNF-a. Hence we compared the efficacy of rTM with TMA to other conventional treatments. Patients and Methods: Of 223 patients who had received SCT in our institute from January 2009 to March 2014, 16 patients diagnosed as TMA after allogeneic stem cell transplantation were identified from database. Diagnosis of TMA was based on the Bone Marrow Transplant Clinical Trials Network (BMT-CTN). Patients treated with rTM as a first-line therapy were categorized as rTM group and treated with other therapies as a first-line therapy were as control group. Results: TMA occurrence was total 16 cases (7.2%). rTM group had 9 patients and control group had 7 patients. rTM group had male dominant (M/F 8/1 vs. 1/6) compared to control group. Both group showed similar age, disease distribution (rTM; 2 Leukemia, 5 Lymphoma and 2 ATLL vs. control; 3 Leukemia, 2 CML-BC and 2 ML), disease risk (rTM; 2 standard (1-2 CR or MDS) and 7 high vs. control; 7 high), hematopoietic cell transplantation specific comorbidity index, type of donor source (rTM; 5 related PBSC and 4 unrelated BM vs. control; 4 related PBSC and 3 unrelated BM), HLA compatibility (rTM; 4 haplo-identical donor vs. control; 3 haplo), conditioning regimen (rTM; 8 reduced intensity vs. control; 5 reduced intensity), GVHD prophylaxis (rTM; 5 CyA/Tac and short MTX, 4 Tac/ATGmPSL vs. control; 5 CyA/Tac and short MTX, 2 Tac/ATGmPSL) and year of transplant. Median onset of TMA in rTM group and control group were on day 24.5 (16-274) and day 72 (7-338) (p=0.13) post SCT, respectively. Compared with control group, rTM group revealed significantly superior overall survival after the onset of TMA (Figure1, p=0.043). Seven patients (77.8%) of rTM group recovered from TMA, while no patients recovered in control group. Acute GVHD were occurred in 5 cases of rTM group and in 4 cases of control group (p=0.29). Grade II to IV acute GVHD were occurred in 3 of rTM group and in 4 cases of control groups (Figure 2, P=0.66). Leading causes of death were 4 GVHD of 5 cases in rTM group and 3 TMA of 7 cases in control group. Both group had no lethal hemorrhagic event. Conclusion; The use of rTM in TMA improved overall survival after the onset of TMA in this retrospective small number analysis. rTM reduced TMA mortality but GVHD was still a major cause of death in TMA survivors. Larger prospective studies are warranted to fully evaluate the efficacy of rTM. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Chronic graft-versus-host disease (GVHD) remains a major cause of late death and morbidity following allogeneic hematopoietic cell transplantation. Recently, in addition to Th2 cells, Th1 and Th17 cells have been shown to contribute to chronic GVHD progression. IL-12 induces Th1 cells and IL-23 plays a role in stabilizing and/or amplifying Th17 cells as well as in inducing IFN-γ/IL-17 double-producing cells. Because monoclonal antibody (mAb) targeting the p40 subunit common to both IL-12 and IL-23 can inhibit both IL-12 and IL-23 receptor-mediated signaling, we investigated the effects of anti-p40 mAb on a well-defined chronic GVHD mice model: B10.D2 (H-2d)→Balb/c (H-2d). Sublethally irradiated BALB/c mice were transplanted with 2×106 spleen T cells and 8×106 TCD-BM cells from B10.D2 mice. Full donor chimerism was recognized. Anti-p40 mAb was injected peritoneally on every third day from day 0 of BMT. We found that anti-p40 mAb significantly ameliorated the clinical score compared with the controls (P < 0.05). Histopathological examination of the skin on day 28 showed significantly reduced chronic GVHD damage in anti-p40 mAb-treated animals (2.8 ± 0.4 vs. 6.0± 0.3, P < 0.01). Anti-p40 mAb was injected intraperitoneally to mice from day 15 of BMT, when mice had just developed clinical signs of chronic GVHD, and anti-p40 mAb significantly improved the clinical scores (P < 0.05). Cells isolated from PLNs were harvested on day 28 after BMT and analyzed for cytokine expression. Intracellular staining revealed that IFN γ single positive (IL-17-) and IFN γ/IL-17 double-positive cells were suppressed in anti-p40 mAb-treated allogeneic recipients compared with control recipients (38±9% vs. 58±8%, P=0.1) (1.5±0.2% vs. 4.0±0.4%, P=0.0003). The cytokine levels of IFN γ and IL-17 were also decreased in serum from anti-p40 mAb-treated allogeneic recipients (IFN γ; 10.0±0.6 pg/ml vs. 35±7 pg/ml, P=0.03, IL-17; 2.8 pg/ml vs. 7.5±2 pg/ml, P=0.2). Since IFN γ/IL-17 double-positive cells are enriched in the target organs of several autoimmune disease models, it has been suggested that these double producers are particularly pathogenic in tissue inflammation and autoimmunity. These double-positive cells show higher expression of T-bet and lower expression of ROR γt than IL-17 single-positive T cells. Therefore, we examined ROR γt and T-bet expression in donor IL-17+ CD4+ T cells isolated from PLNs harvested on day 28 after BMT. Anti-p40 mAb-treated recipients displayed marginally higher ROR γt expression than control-treated allogeneic recipients (4.7±0.6% vs. 3.2±0.5%, P = 0.08). By contrast, anti-p40 mAb-treated recipients showed significantly lower T-bet expression than controls (0.77±0.2% vs. 1.6±0.3%, P = 0.03). This reduction in T-bet expression was associated with IL-22 production by T cell from anti-p40 mAb-treated recipients (42±18 pg/ml vs. 110±17 pg/ml, P = 0.03). The levels of IL-22 were also decreased in serum from anti-p40 mAb-treated allogeneic recipients 28 days after BMT (19±5 pg/ml vs. 206±78 pg/ml, P = 0.04). These results suggested that anti-p40 mAb attenuated chronic GVHD via suppression of IFN-γ/IL-17-producing cells. Moduration of the IL-12/IL-23 pathway may represent a new strategy for the treatment of chronic GVHD and anti-p40 which is clinically available as ustekinumab, might be promising therapeutic agents for chronic GVHD. Disclosures No relevant conflicts of interest to declare.

Apart from their role as antigen presenting cells, human peripheral blood monocyte and CD34+ cell-derived dendritic cells (DC), have been demonstrated to exert cytotoxicity against some tumor cells, and their tumoricidal activity can be enhanced by some stimili. However, there have been no reports concerning the tumoricidal activity of human cord blood dendritic cells (CBDC). In this article, we report that human cord blood monocyte-derived DC acquire the ability to kill hematological tumor cells, after activation with lipopolysaccharide (LPS) or γ-interferon (IFN-γ), associated with the enhanced TNF-α-related apoptosis-inducing ligand (TRAIL) expression in CBDC cytoplasm. The CD14-positive cells collected from cord blood were induced to CBDC in vitro. After activation with IFN-γ for 12 h, CBDC exhibited cytotoxicity against HL60 and Jurkat cells, while activation with LPS induced cytotoxicity against Daudi and Jurkat cells. However, both LPS- and IFN-γ-stimulated CBDC showed no cytotoxic activity against normal CD14-negative cord blood mononuclear cells. The formation of umbilical cord hematopoietic progenitor colonies, identified as burst-forming unit-erythroid and colony-forming unit granulocyte-macrophage, was not inhibited by stimulated or unstimulated CBDC. IFN-γ or LPS stimulation enhanced intracellular but not cellular surface TRAIL, and neither intracellular nor cellular surface tumor necrosing factor-α and Fas Ligand as analyzed by flow cytometry. Our results show that activated CBDC can serve as cytotoxic cells against hematological tumor cells without damaging the normal hematopoietic progenitor cells. (Cancer Sci 2005; 96: 127–133)