ALBERT

Description: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) using cord blood or haploidentical donor is a promising alternative option for patients who can not find an HLA-matched donor. However, HLA-mismatch allo-HSCT may be complicated by graft-versus-host disease (GVHD), a major cause of non-relapse mortality mediated by alloreactive T cells. Infusion of anti-thymocyte globulin (ATG) is used both as a treatment for and as a prophylactic against GVHD but ATG, like other immunosuppressive regimens, reacts against cells without distinguishing between donor and recipient cells. ATG is polyclonal and causes many side effects such as allergic reaction and increasing susceptibility to infection due to repression of T cells. Our ultimate goal is to develop a novel therapeutic approach to GVHD with anti-HLA monoclonal antibody specifically targeting the mismatched HLA molecule of donor origin to transiently ablate alloreactive T cells. To generate allele-specific anti-HLA antibody (ASHmAb), we established a rapid and efficient strategy using two different HLA-transgenic mouse strains, HLA-A24 and HLA-A2 and immunized them with HLA-A* 02:01 and HLA-A* 24:02 tetramers loaded with cytomegalovirus PP65 peptide, respectively. Antibodies were screened for HLA allele-specific complement-dependent cytotoxicity in vitro. We successfully established three killing ASHmAbs (kASHmAbs) against HLA-A* 02:01, -A* 02:01/–A* 03:01, and -A* 23:01/-A* 24:02. In vivo cytotoxicity and specificity of a kASHmAb against HLA-A* 02:01 (A* 02:01-kASHmAb) was examined in detail using a xenogeneic GVHD mouse model. To induce fatal GVHD, non-irradiated NOD/Shi-scid/IL-2Rγnull (NOG) mice were injected with either HLA-A2 or HLA-A26 positive human peripheral blood mononuclear cells (PBMCs) from healthy donors (i.e. HLA-A2 mice or HLA-A26 mice, respectively). Administration of A* 02:01-kASHmAb promoted the survival of HLA-A2 mice to 100%, with a mean survival of more than 6 months. In contrast, administration of A* 02:01-kASHmAb to nonreactive HLA was not effective; all HLA-A26 mice died within 2 months (p

Description: Recent genetic studies have revealed frequent and specific pathway mutations involving multiple components of the RNA splicing machinery in myelodysplasia. Among these, SRSF2 mutations are more prevalent in CMML subtype and are associated with poor prognosis. Mutations showed a prominent hotspot involving proline 95, causing either P95H, P95L, or P95 conversion. Comprehensive analysis in our large cohort of MDS revealed that SRSF2 mutations showed a significant trend to coexist with TET2, STAG2, ASXL1 and RUNX1 mutations, while being mutually exclusive with EZH2 mutations. On the other hand, the molecular mechanism by which SRSF2 mutations lead to myelodysplasia remains largely unknown. 　To elucidate the role of SRSF2 mutations in the development of myelodysplasia, we generated a heterozygous conditional knock-in mouse model of Srsf2 P95H mutation and crossed them with Vav1-Cre transgenic mice. Srsf2 P95H mutant mice exhibited macrocytic anemia, otherwise no significant changes in total peripheral blood (PB) cell counts compared to wild-type mice at 8-15 weeks after birth. There was no significant difference in lineage composition as well as blood cell morphology between wild-type and mutant mice in both bone marrow (BM) and PB. Flow cytometry of BM cells showed significant decrease of the number of hematopoietic stem cells (HSCs) and multipotent progenitor cells defined as Lin-Sca-1+Kit+ (LSK) fractions in Srsf2 P95H mice compared to wild-type mice. On the other hand, there were no significant differences in the number of more differentiated progenitor cells including common myeloid progenitors (CMPs), granulocyte/macrophage lineage-restricted progenitors (GMPs), megakaryocyte/erythrocyte lineage-restricted progenitors (MEPs), and common lymphoid progenitors (CLPs) between Srsf2 P95H and wild-type mice. These observations suggested that heterozygous Srsf2 mutation led to deregulation of hematopoietic stem cells, which however, is not sufficient for the development of MDS. 　We next performed noncompetitive transplantation experiments to assess the cell intrinsic effects of Srsf2 P95H mutations. In PB, decreased white blood cell counts and progressive anemia were observed in mutant mice, which were evident as early as one month after transplantation. Cytological analysis of PB revealed morphological abnormalities in mice reconstituted with Srsf2 mutated cells, including hypersegmentation in neutrophils and dysplasia in the erythroid lineage. Srsf2 mutant-reconstituted mice showed normo-to-hypercellular marrow, where abnormal megakaryocyte distribution adjacent to trabecular bone and erythroid dysplasia was observed. Flow cytometrical analysis revealed decreased numbers of HSCs, LSK fractions and CMPs, whereas there was no significant change in the number of MEPs, GMPs and CLPs in BM. The BM erythroid progenitors were decreased in mutant-reconstituted mice, whereas the mutant mice showed splenic erythropoiesis with increased erythroid progenitors, suggesting the presence of extramedullary hematopoiesis, which was not seen in wild-type Srsf2 transduced mice. These observations suggested that the Srsf2 mutation led to ineffective hematopoiesis and morphological abnormalities, which seemed to recapitulate the phenotype of MDS. 　Subsequently, we assessed the reconstitution capacity of whole BM cells from Srsf2 mutant mice in competitive transplantation experiments. The donor chimerism of Srsf2 P95H-derived cells in PB was significantly lower than that of wild-type cells. At 4 months post transplantation, the chimerism of Srsf2 P95H-derived cells was remarkably lower than that of wild-type cells in the fractions of HSCs, MPPs, CMPs, MEPs, GMPs and CLPs in BM. Furthermore, the reduced donor chimerism for Srsf2 P95H mutants was recapitulated in secondary transplantation experiments. 　In summary, our results demonstrated that heterozygous P95H mutation of Srsf2 led to deregulation of hematopoietic stem cells that was evident from reduced competitive repopulation and impaired hematopoietic differentiation. Whereas mice reconstituted with Srsf2 mutant BM cells developed MDS-like phenotype in non-competitive transplantation setting, Srsf2 mutation by itself does not seem to be sufficient to develop MDS without transplantation, raising the possibility that an additional genetic and/or epigenetic events was required for overt MDS phenotype. Disclosures No relevant conflicts of interest to declare.

Description: Pyothorax-associated lymphoma (PAL) is a lymphoproliferative disorder developing in the pleural cavity after a long-standing history of pyothorax. The pathogenesis, clinical features and optimal treatment have not been clarified. To investigate clinicopathological features of pyothorax-associated lymphoma (PAL), we examined medical records of 98 patients (88 males and 10 females) with PAL at a median age of 70 (range, 51–86). Seventy-nine patients had a history of artificial pneumothorax. Median interval between diagnosis and artificial pneumothorax was 43 years (range, 19–64). At diagnosis, performance status was 0–1 (n=56) and 2–4 (n=42). Clinical stages were I (n=42), II (n=26), III (n=8) and IV (n=22). In situ hybridization using a probe for EBV-encoded RNA-1 demonstrated the presence of the EBV genome in the nucleus of tumor cells in 28 of 29 (88%) patients. Immunohistochemical studies revealed that 12 of 14 (86%) and 11 of 17 (65%) were positive for EBNA-2 and LMP-1, respectively. Seventeen were treated supportively. The other 81 received aggressive treatments; chemotherapy (n=52), radiotherapy (n=7), surgery (n=4) and combination (n=18). Response rates to chemotherapy, radiotherapy, and chemoradiotherapy were 56%, 71%, and 83%, respectively. Five-year overall survival was 0.35 (95% confidence interval, 24–45%). The 5-year overall survival rates were 0.47 (95% CI, 0.30– 0.63), 0.35 (95% CI, 0.16– 0.54) and 0.15 (95% CI, 0.04–0.33) in patients with stages I, II and I-IV disease, respectively (Figure 1). Overall survival are different among the three groups (p

Description: [Background] Patients treated with immunosuppressive drugs for autoimmune diseases may suffer "other iatrogenic immunodeficiency-associated lymphoproliferative disorders" (OIIA-LPD). Some OIIA-LPDs regress after drug withdrawal but others need cytotoxic chemotherapy. However, the factors associated with a response to drug cessation remain unknown. [Methods] We collected clinical data on OIIA-LPD diagnosed between 2009 and 2018 at the University of Tsukuba Hospital and Toranomon Hospital in Japan. Clinicopathological features were retrospectively analyzed. The probability of overall survival (OS) and progression-free survival (PFS) was calculated using the Kaplan-Meier method. OS was defined as the time between the date of diagnosis of OIIA-LPD and the date of death or last follow-up. PFS was defined as the time from the date of diagnosis of OIIA-LPD to the date of commencing chemotherapy or the date of death or last follow up. [Results] Fifty-four cases of OIIA-LPD were identified, of which 25 were diagnosed at the University of Tsukuba Hospital and 29 at Toranomon Hospital. The male to female ratio was 1:3.5 and the median age at diagnosis was 70 years (range, 35-85). Thirty-four patients (63%) had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Most patients (n=50 [93%]) had rheumatoid arthritis (RA). The remaining 4 patients had systemic lupus erythematosus (SLE), myasthenia gravis (MG), polymyalgia rheumatica (PMR), and polymyositis (PM), respectively. Methotrexate (MTX), tacrolimus, and biological agents were used in 49, 17, and 16 cases, respectively. Histological diagnoses of OIIA-LPD type were diffuse large B-cell lymphoma (DLBCL) (n=24), composite lymphoma of DLBCL and follicular lymphoma (FL) (n=1), Hodgkin lymphoma (HL) (n=18), MALT lymphoma (n=2), peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) (n=2), angioimmunoblastic T-cell lymphoma (AITL) (n=1), B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and classic HL (n=1), extranodal NK/T-cell lymphoma (ENKTL) (n=1), Burkitt lymphoma (n=1), and not specified (n=3). Epstein-Barr virus (EBV) was detected in 61% of cases tested (31/51) by in situ hybridization for EBER. At diagnosis of OIIA-LPD, 44 cases (81%) were under MTX treatment. MTX was discontinued in all of these, after which spontaneous regression was observed in 27 (61%). There was no clinical response in 5 patients (11%) within 2 months of MTX cessation, and 12 patients (27%) received chemotherapy without confirming whether stopping MTX would have been sufficient intervention. In 27 cases with spontaneous regression after cessation of MTX, 18 achieved complete remission without chemotherapy, but 9 needed chemotherapy due to re-initiation of LPD. Regarding the histological diagnosis, spontaneous regression was observed in OIIA-LPD patients with DLBCL in 12 of 24 cases (50%) and those with HL in 8 of 18 (44%). After achieving spontaneous regression, patients with DLBCL (10/12 cases, 83%) did not require any form of chemotherapy more often than those with HL (4/8 cases, 50%). Neither EBV positivity nor histology were associated with spontaneous regression. With a median follow-up of 26.1 months (range, 1-120.7), 2-year OS and PFS was 91.9% and 30.1%, respectively. The 2-year PFS of DLBCL and HL was 33.1% and 17.5%, respectively (p=0.533). Only four patients died due to the progression of OIIA-LPD. [Conclusion] Fifty-four cases of OIIA-LPD were retrospectively analyzed. Spontaneous regression after MTX discontinuation was observed in approximately 60% of these patients. No factors associated with response to drug cessation were associated with histological features or EBV positivity, but patients with DLBCL remained in complete remission more frequently than those with HL. Despite the fact that 2-year PFS is low, our retrospective analyses revealed favorable OS of OIIA-LPD because chemotherapy resulted in a good response regardless of whether MTX discontinuation was effective. Disclosures No relevant conflicts of interest to declare.

Description: Key Points Blood-specific expression of the Srsf2 P95H mutant results in decreased stem/progenitor cell numbers and a reduced repopulation capacity. Srsf2 P95H mutation by itself is not sufficient to develop MDS but contributes to the MDS phenotype in transplantation settings.

Description: Although allogeneic hematopoietic stem cell transplantation has recently been applied to patients with myelofibrosis with reproducible engraftment and resolution of marrow fibrosis, no data describe the outcomes of umbilical cord blood transplantation. We describe 14 patients with primary (n = 1) and secondary myelofibrosis (n = 13) who underwent reduced-intensity umbilical cord blood transplantation. Conditioning regimens included fludarabine and graft-versus-host disease prophylaxis composed cyclosporine/tacrolimus alone (n = 6) or a combination of tacrolimus and mycophenolate mofetil (n = 8). Thirteen patients achieved neutrophil engraftment at a median of 23 days. The cumulative incidence of neutrophil and platelet engraftment was 92.9% at day 60 and 42.9% at day 100, respectively. Posttransplantation chimerism analysis showed full donor type in all patients at a median of 14 days. The use of umbilical cord blood could be feasible even for patients with severe marrow fibrosis, from the viewpoint of donor cell engraftment.

Description: Backgrounds Angioimmunoblastic T-cell lymphoma (AITL) is a distinct subtype of T-cell lymphoma, characterized by generalized lymphadenopathy and frequent autoimmune-like manifestations. The diagnosis of AITL is sometimes challenging for hematopathologists, because the tumor cell content is generally low and relatively large reactive lymphocytes are confused as tumor cells. Possibly because of the low tumor cell frequency, clonal rearrangement of T-cell receptor gene is undetectable in 30% of the cases. We identified recurrent mutations in RHOA at c.G50T, predicting to generate p.G17V in 70% of AITL and PTCL-NOS harboring AITL features, which implies diagnostic properties for AITL (M S-Y and SC, unpublished). Purpose To establish a novel cost-effective method to diagnose AITL, we performed allele-specific realtime PCR to detect RHOA G17V mutation. Methods Genomic DNA was extracted from 119 AITL and PTCL-NOS samples, which include 47 periodate-lysine-paraformaldehyde (PLP)-fixed, 12 formalin-fixed-paraffin- embedded (FFPE) and 60 frozen tissues. Forty-one out of 60 genomic DNA samples, purified from frozen tissue, were amplified by RepliG kit (QIAGEN). Allele-specific primers for RHOA G17V mutant and wild-type sequences were designed by Wangkumhang's algorithm. The [mut] and [WT] values were individually measured by realtime PCR using each primer set, and the [mut]/([mut]+[WT]) values were calculated. Mutant allele frequencies were determined by amplicon-based deep sequencing using MiSeq. Results The [mut] values were distributed from 1.5×10-7 to 7.6×10-2, and the [WT] values were from 7.9×10-5 to 1.3×10-2. The [mut]/[mut]+[WT] values were distributed from 1.9×10-4 to 8.5×10-1. We set a cut-off value to determine existence of mutation as 2% for MiSeq, and 1.3×10-2 for the [mut]/([mut]+[WT]) value. Then we compared these two methods to detect RHOA G17V mutation. Forty-three cases were positive for the RHOA mutation in this study cohort by MiSeq, including 32 AITL and 11 PTCL-NOS cases. The [mut]/([mut]+[WT]) values of DNA from FFPE samples tend to be lower than those from other samples, and 4 out of 12 FFPE samples determined as mutation-positive by MiSeq were not detected by our allele-specific realtime PCR. We therefore excluded the FFPE samples and analyzed the data of 107 DNA samples, purified from PLP-fixed and frozen tissues. Rank correlation coefficient was 0.753. Sensitivity was 97.4%, and specificity was 97.1%. Positive concordance rate was 94.9%, and negative concordance rate was 98.6%. Conclusions We established a method to detect RHOA G17V hotspot mutation for AITL. It is expected to be highly accurate and cost effective. Disclosures: No relevant conflicts of interest to declare.

Description: Background EBV-associated post-transplant lymphoproliferative disorder (EBV-PTLD) is uncommon but one of serious complications after allogeneic stem cell transplantation (SCT). However, there has been little literature published on clinicopathological feature of it. Method We retrospectively investigated 825 cases of allogeneic SCT (unrelated bone marrow (uBM) 159, related peripheral blood (rPB) 94, cord blood (CB) 572) at Toranomon Hospital between January 2006 to November 2012. EBV-PTLD was defined as histologically confirmed Epstein-Barr virus (EBV) positive lymphoproliferative disorder developed after allogeneic SCT. Results We identified 12 cases of EBV-PTLD in our cohort. Cumulative incidence of EBV-PTLD at 2 years was 1.5%. Median time from allogeneic SCT to the diagnosis of EBV-PTLD was 6.4 (2.5-26) months. Eight patients were male and median age at the diagnosis of EBV-PTLD was 58.5 years (28-66). Underlying diseases were acute myeloid leukemia (n=5), myelodysplastic syndrome (1), acute lymphoblastic leukemia (2), adult T cell leukemia-lymphoma (1), aplastic anemia (2) and chronic myeloid leukemia in blastic phase (1). Conditioning regimens were fludarabine-based (n=10) and TBI/CY (n=2). None had an antithymocyte globulin-containing regimen. Donor sources were uBM (n=1) and CB (11). EBV serology before allogeneic SCT was tested in 11 and all were positive. Patients who received CB showed higher incidence of EBV-PTLD compared to those in non-CB cohort (2.2% vs 0.6%, p 〈 0.01), although patients characteristics was different between them. One patient developed PTLD after third allogeneic SCT. All but one patients had a history of acute graft-versus-host disease (aGVHD) of grade I (n=2), grade II (6) and grade III (3), respectively. Chronic GVHD was observed in 6 patients. Eight patients were on immunosuppressive therapy (IST) with calcineurin inhibitors and/or steroids when EBV-PTLD was suspected for the first time. Although lymphadenopathy was detected by CT scan in 7 patients, surface lymph nodes were swollen in only 3 patients. Initial manifestations were fever in 8, and diarrhea in 5 patients. EBV-PTLD was diagnosed from lymph nodes (n=3), skin (3), bone marrow (2), stomach/duodenum (1), colon (2), and lung (1), respectively. Histological feature was monomorphic (n=4), polymorphic (2), early lesion (4), and unknown (2), respectively. LMP1 and EBNA2 was positive in 40% (4/10) and 30% (3/10), suggesting latency status of I in 50% (6/12), II in 8.3% (1/12), III in 25% (3/12), and unknown in 16.7% (2/12). EBV DNA of 100 copies/microL or above was detected in peripheral blood in all of the EBV-PTLD cases. The treatment for EBV-PTLD were rituximab alone (n=3), rituximab plus reduction of IST (6), rituximab plus cytotoxic chemotherapy (1), and observation alone(2). Although 8 patients achieved response, 2 patients suffered a relapse of EBV-PTLD. With a median follow up of 27.5 (4.1-47.7) months, 2-year overall survival was 46.9% after diagnosis of EBV-PTLD. Eight patients died and 4 are alive without relapse of EBV-PTLD. Causes of death were EBV-PTLD (n=2), relapse of underlying disease(3), infectious disease(2), and aGVHD(1). Conclusion Twelve cases of EBV-PTLD were identified in 825 transplants. Cumulative incidence of EBV-PTLD at 2 years was 1.5%. The incidence of EBV-PTLD after CB transplant was higher than that after non CB transplant, although we have to take into consideration that patients feature was different between them. Response to rituximab and/or reduction of IST was observed in 8 patients with a 2-year overall survival rate of 46.9%. Patients with prior aGVHD and with longer duration of immunosuppressive therapy may have an increased risk of developing EBV-PTLD. Since the initial manifestations were often equivocal, and survival rate after diagnosis is not high, having EBV-PTLD in mind as one of the possibilities is critical for prompt diagnosis. Disclosures: No relevant conflicts of interest to declare.