Abstract: Introduction: Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell lymphoma caused by human T-cell lymphotropic virus type I. In younger patients, allogeneic hematopoietic stem cell transplantation (HSCT) has been shown to improve prognosis through a graft-versus-ATL (GvATL) effect. On the other hand, elderly patients with ATL are often not amenable to HSCT, and the prognosis is extremely poor with existing chemotherapy. The most widely used chemotherapy for ATL, CHOP (combination of cyclophosphamide-doxorubicin-vincristine-prednisone) and similar chemotherapy, have shown a complete response (CR) rate of about 20% and a long-term prognosis of less than 10%. In a phase II study of mogamulizumab, a humanized defucosylated anti-C-C chemokine receptor 4 monoclonal antibody, in concurrent combination with chemotherapy (mLSG15), the primary endpoint of CR was 52% in the mLSG15 + mogamulizumab group compared to 33% in the mLSG15 alone group. However, no improvement was observed in overall survival (OS) or progression-free survival (PFS). In addition, mLSG15 is a potent chemotherapy and not feasible in majority of elderly patients. Considering the possibility that mogamulizumab may unlock the tumor immune evasion mechanism by removing regulatory T cells and exert an immunological effect like GvATL, we investigated its usefulness as an immunological consolidation therapy following chemotherapy, with the aim of suppressing recurrence. Methods: New-onset treatment-naive ATL patients who were not eligible to HSCT received 3 courses of CHOP (combination of cyclophosphamide div 750 mg/m 2/day1, doxorubicin iv 50 mg/m 2/day1, vincristine iv 1.4 mg/m 2/day1 [max. 2.0 mg/body, and prednisone po/iv 100 mg/body/day1-5) at 21-day intervals, and then the first course of mogamulizumab (1 mg/kg for 8 doses at 2-week intervals) was initiated between the start of the third course of CHOP-21 and day 42. The primary endpoint was OS at months 12. Secondary endpoints were proportion of subjects with PFS, OS, overall response rate (ORR), organ-specific response rate, CR rate and adverse events (AEs) at month 12. Results: A total of 24 patients were enrolled and the median age was 75.5 years (range, 64-85). The median follow-up period was 11.0 (3.0-30.6) months. Five subjects discontinued study treatment before mogamulizumab administration attributable to insufficient effect (n=2), continuation deemed inappropriate (n=2), and start of the next course extended (n=1). ORR was 87.5%. Organ-specific response rates for peripheral blood, other than peripheral blood, target lesion, and skin lesion were 83.3%, 87.5%, 79.2%, and 66.7%, respectively. OS and PFS rates in the full analysis set (FAS) at months 12 were 52.6 (30.9-70.4)% and 26.6 (10.9-45.3)%, respectively. The median survival time (MST) and 80% confidence interval (CI) for OS was 12.1 (5.0 -21 .0) months (Figure 1). Since the lower limit of the 80% CI was below the threshold of 6 months, this treatment was not considered to be effective. However, the point estimate of MST was 12.1 months, which was equivalent to the expected value. One possible reason for the failure to reach statistical significance could be attributable to occurrence of events in the early stage of the protocol regimen. In other words, patients who did not respond to the preceding chemotherapy did not benefit from mogamulizumab, and only patients who managed to overcome chemotherapy benefited from the immune effects of mogamulizumab, resulting in long-term survival (Figure 1). In the long-term follow-up, the MST (95% CI) of the FAS was 12.4 (4.9-21.0) months, and the median OS (95% CI) at 24 months was 25.1 (9.6-44.1) %. AEs were observed in 16 subjects, of which serious AEs (grade 4/5) were observed in 5 subjects (acute respiratory distress syndrome, ventricular fibrillation, heart failure, hyponatremia, and pneumonia). Largely, AEs were resolved/recovered in 14 subjects including 3 subjects with serious AE; 2 subjects resulted in death, which were not considered treatment-related. Conclusions: Mogamulizumab as an immunological consolidation therapy after CHOP 21 might be one of the treatment options for transplant-ineligible elderly patients with previously-untreated ATL, despite not clearly showing improved OS. Further studies are warranted to examine the dosing timing of mogamulizumab to obtain its maximum benefit for prolonged survival. Figure 1 Figure 1. Disclosures Kato: Abbvie: Consultancy, Research Funding; AstraZeneca: Consultancy; Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Chugai: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Dainippon-Sumitomo: Honoraria; Eisai: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Kyowa Kirin: Honoraria, Research Funding; MSD: Honoraria; Mundi: Honoraria; Novartis: Consultancy, Research Funding; Ono: Honoraria, Research Funding. Suzuki: Kyowa-kirin: Honoraria, Research Funding, Speakers Bureau; Chugai: Honoraria, Research Funding, Speakers Bureau; Taiho: Research Funding; Ohtsuka: Honoraria, Research Funding; Takeda: Research Funding, Speakers Bureau; Shionogi: Research Funding; Eisai: Honoraria, Research Funding; Bristol-Meyer Squib: Honoraria; MSD: Honoraria; Janssen: Honoraria; Abbvie: Speakers Bureau; Meiji Seika: Honoraria, Speakers Bureau; Ohtsuka: Honoraria. Akashi: Sumitomo Dainippon Pharma: Consultancy; Kyowa Kirin: Consultancy, Research Funding; Celgene: Research Funding; Astellas Pharma: Research Funding; Shionogi: Research Funding; Asahi Kasei Pharma: Research Funding; Chugai: Research Funding; Bristol-Myers Squibb: Research Funding.

Abstract: Background: Human herpes virus-6 (HHV6)-associated limbic encephalitis/myelitis is rare but life-threatening nervous system complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT). When the allo-HSCT recipients present encephalitis-associated manifestations such as short-term memory dysfunction, disorientation, consciousness disturbance and seizures, it is essential to immediately perform polymerase chain reaction (PCR) analysis for detection of HHV6 viral DNA in the cerebrospinal fluid (CSF) for the definite diagnosis. However, if reactivation of HHV6 is confined to the spinal cord but not cerebrum, these patients lack encephalitis-like symptoms but manifest only sensory nerves-related symptoms such as severe unexplained pain, dysesthesia and prutitus, leading to the delayed examination of HHV6 DNA detection in CSF. As a result, the patients with HHV6-associated myelitis can be sometimes misdiagnosed as having calcineurin inhibitor (CI)-induced pain syndrome (CIPS), since such manifestations are commonly observed in these two syndromes. In this study, we evaluate incidence and clinical features of HHV6 encephalitis/myelitis and CIPS after allo-HSCT. Method: We retrospectively reviewed the medical records of 435 patients who underwent allo-HSCT between 2002 and 2014 in our facility. HHV6-associated encephalitis/myelitis was directly proved when HHV6 DNA was detected by PCR in CSF. For those patients who were unable to undergo lumbar puncture because of severe thrombocytopenia or a deteriorated general condition, we diagnosed HHV6-associated encephalitis/myelitis if they satisfied more than 2 of the following 3 criteria: (1) typical clinical manifestations as described above; (2) detection of HHV6 DNA in peripheral blood; or (3) limbic encephalopathy based on the selective involvement of the medial temporal lobe on magnetic resonance imaging (MRI). CIPS was diagnosed using three factors: (a) typical clinical manifestations including unexplained severe pain and cutaneous pruritus without skin rash; and (b) not detection of HHV6 DNA in CSF; or (c) abnormal findings at X-ray, MRI, or bone scintigraphy at joint of knee and foot. Results: Twenty-five patients were diagnosed as having HHV-6 encephalitis/myelitis with a cumulative incidence of 5.7%. Median onset was on day 19 after transplantation. HHV-6 encephalitis/myelitis was documented in 15 of 99 cord blood transplant (CBT) recipients (15.2%) and 10 of 336 recipients (3.0%) transplanted with bone marrow or peripheral blood stem cells. This result suggests that a higher incidence of HHV-6 encephalitis/myelitis occurring in CBT recipients. Four patients manifested typical symptoms at the onset of HHV6-associated encephalitis. However, 11 patients presented with dysesthesia and pruritus, described as typical manifestations of patients with CIPS, and the remaining 10 showed both symptoms. Six of 11 patients with CIPS-like symptom also exhibited dysautonomia (bladder and rectal disturbance and sinus tachycardia) and/or abdominal pain. Positive results for brain MRI scans (limbic encephalitis) were observed in 9 of 14 patients (64.3%) who developed encephalitis-type symptoms, which were not found in the 11 patients who had CIPS-like symptoms; none presenting with CIPS-like symptoms had positive results for spinal MRI as well. On the other hand, 8 patients (1.8%) were diagnosed as having CIPS on the 5 to 91 days (madian 22 days) posttransplant. For graft-versus-host disease (GVHD) prophylaxis, 2 patients received cyclosporine, and 6 received tacrolimus. CI concentrations in these patients were maintained within the target ranges at onset of the pain. Abnormal findings at X-ray and MRI were not observed in all patients, but only one patient showed abnormal findings at joint of hand, finger and ankle in bone scintigraphy. For the treatment of CIPS, in 7 patients dosage of CI was reduced, whereas in one CI was switched into another one. Clinical symptoms in all of these patients were improved and exacerbation of GVHD was not seen. Conclusion: Detection of HHV6 DNA in CSF is crucial to make a differential diagnosis of HHV6 myelitis and CIPS. Transplantation physicians should be aware that CIPS-like dysesthesia and pruritus might be early manifestations to suggest the reactivation of HHV6, especially for patients who develop myelitis. 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.

Abstract: Abstract 522 Hemophagocytic lymphohistiocytosis (HLH) is a syndrome characterized by hemophagocytosis in the bone marrow with systemic inflammatory reactions. We have reported that the inflammatory cytokinemia including interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) might play a key role in activating macrophages to induce HLH (Akashi et al. Br J Haematol, 1994). Recent studies have shown that the engulfment of blood cells by macrophages is regulated in part by the interaction of CD47 and its ligand, signal regulatory protein alpha (SIRPA). Blocking the CD47 and SIRPA interaction by using anti-CD47 monoclonal antibodies can induce engulfment of blood cells, suggesting that the SIRPA signaling provide “don't eat me signals” to prevent macrophages from digesting self blood cells. These data led us to hypothesize that HLH is caused by the disruption of self-recognition by macrophages through impairment of the CD47-SIRPA system. To test this hypothesis, we evaluated the expression level of CD47 in bone marrow cells in 24 patients with HLH. Interestingly, the expression of CD47 was significantly downregulated (by ∼2-fold reduction in average at the protein level) in the CD34+CD38− hematopoietic stem cell (HSC) fraction in HLH patients, whereas that of the CD34+CD38+ progenitor and other mature blood cell fractions was unchanged. We then purified the CD34+CD38− HSCs and CD34+CD38+ progenitor cells from HLH patients and normal controls, co-cultured them with human macrophages in the presence of IFN-gamma and lipopolysaccharide, and evaluated the percentage of phagocyting macrophages. The numbers of phagocyting macrophages were significantly higher in cultures of CD34+CD38− HSCs from HLH patients, as compared to those in cultures of normal HSCs and of progenitor populations, suggesting that the expression level of CD47 inversely correlated with the efficiency of hemophagocytosis. Furthermore, normal HSCs but not CD34+CD38+ progenitors or other mature blood cells down-regulated CD47 in vitro in response to IFN-gamma, TNF-alpha, and IL-6, suggesting that these cytokines plays a critical role in down-regulation of CD47 especially in HSCs. In contrast, the expression level of SIRPA did not differ in myeloid cells between HLH patients and normal controls, and mutations of SIRPA were not found in HLH patients. Thus, in HLH, inflammatory cytokines down-regulate CD47 selectively in HSCs, resulting in the engulfment of HSCs by macrophages. Our data strongly suggest that the cytokine deregulation can lead to HLH in human, through disruption of self-recognition guided by the CD47-SIRPA system at the HSC stage. Disclosures: No relevant conflicts of interest to declare.

Abstract: [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.

Abstract: Introduction: Tyrosine kinase inhibitors (TKIs) have dramatically improved outcome of patients with chronic myeloid leukemia (CML) and Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) with lesser incidence of serious adverse events. Recently, cases with fatal pulmonary hypertension (PH) have been sporadically documented in association with dasatinib treatment. French group reported incidence of PH diagnosed by cardiac catheterization as 0.45% (13 of 2900 patients) in the symptomatic patients treated with dasatinib. In contrast, a Korean group prospectively evaluated PH by non-invasive echocardiography in CML patients treated with dasatinib, and demonstrated 8 of 66 patients (12.1%) exhibited a significant increase in right ventricular systolic pressure, indicating subclinical PH might be more common event in dasatinib-treated patients. In this study, we prospectively examined the patients treated with three TKIs by echocardiography to clarify incidence of clinical and subclinical PH as well as factors associated with PH. Patients and Methods: Total of 108 patients (99 with CML, 9 with Ph+ ALL) receiving TKIs in our institutions were enrolled to this study. Forty-one patients have been on treatment with dasatinib (38%), 37 with imatinib (34%) and 30 with nilotinib (28%). Patients underwent echocardiography to evaluate values of tricuspid regurgitation pressure gradient (TRPG), which relates to severity of PH. Patients with higher values of TRPG than the upper limit (30mmHg) was suspected of PH by European Society of Cardiology criteria. Results: Among 108 patients, median age was 63 years old, and median duration of TKIs treatment was 26.5 months. Median daily dosage was 100 mg for dasatinib, 300 mg for imatinib, 600 mg for nilotinib groups, respectively (Table 1). In imatinib group, patients' age was significantly higher, and duration of treatment was also longer than those of the 2nd generation TKIs. Echocardiography revealed mean values of TRPG as 23.2, 23.2 and 23.1 mmHg in dasatinib, imatinib and nilotinib groups, respectively. There was no significant difference in TRPG values among 3 groups (p=0.99). We also found no relationship between TRPG values and duration of TKIs treatment in each group. Interestingly, we detected a significant inverse correlation between daily dosage of imatinib and TRPG values (p=0.012, Figure 1), while such relationship was not observed in dasatinib and nilotinib groups (p=0.68 and p=0.49). TRPG values higher than 30 mmHg were documented in 13 of 108 patients (12.0%); 5 of 41 (12.2%) in dasatinib group, 4 of 37 (10.8%) in imatinib group, and 4 of 30 (13.3%) in nilotinib group (p=0.95). Discussion: PH is characterized by proliferation of pulmonary vascular smooth muscle cells (SMCs). Recent reports showed that imatinib suppresses abnormal proliferation of SMCs through inhibiting platelet-derived growth factor receptors (PDGFR), resulting in improvement of PH in animal models. Clinical studies in symptomatic PH patients reported that imatinib considerably improved pulmonary hemodynamics. Of note, in our study, dosage of imatinib was significantly correlated with lower values of TRPG, suggesting imatinib possibly decreases TRPG values in a dose-dependent manner. This finding strongly supports the reports indicating imatinib as a therapeutic agent of PH. In contrast, in vitro studies have shown that dasatinib has stronger potential to inhibit PDGFR compared to imatinib; nevertheless, onsets of PH have been reported in dasanitib-treated patients, but not with imatinib nor nilotinib. Our study demonstrated the incidence of TRPG elevation as 12.0% in dasatinib group, which was consistent with Korean report (12.1%). However, there was no significant difference in TRPG values among 3 groups, indicating no apparent evidence which dasatinib treatment might be specifically associated with occurrence of PH. These results suggested that subclinical PH might be more common than expected. Careful follow-ups with echocardiography are necessary for the patients under any TKI treatments. Table 1. Patient characteristics Dasatinib group (n=41) Imatinib group (n=37) Nilotinib group (n=30) p Median age 55(17-77) 68(22-92) 62.5(24-85) 0.0004 Median dosage (mg/day) 100(18-100) 300(100-600) 600(300-800) Mean months from start of TKI 68.5(2-287) 104.8(2-228) 61.3(3-153) 0.007 Mean TRPG(mmHg) 23.2(9-40) 23.2(8-46) 23.1(7-35) 0.995 Disclosures No relevant conflicts of interest to declare.

Abstract: Hemophagocytic lymphohistiocytosis (HLH) is characterized by deregulated engulfment of hematopoietic stem cells (HSCs) by BM macrophages, which are activated presumably by systemic inflammatory hypercytokinemia. In the present study, we show that the pathogenesis of HLH involves impairment of the antiphagocytic system operated by an interaction between surface CD47 and signal regulatory protein α (SIRPA). In HLH patients, changes in expression levels and HLH-specific polymorphism of SIRPA were not found. In contrast, the expression of surface CD47 was down-regulated specifically in HSCs in association with exacerbation of HLH, but not in healthy subjects. The number of BM HSCs in HLH patients was reduced to approximately 20% of that of healthy controls and macrophages from normal donors aggressively engulfed HSCs purified from HLH patients, but not those from healthy controls in vitro. Furthermore, in response to inflammatory cytokines, normal HSCs, but not progenitors or mature blood cells, down-regulated CD47 sufficiently to be engulfed by macrophages. The expression of prophagocytic calreticulin was kept suppressed at the HSC stage in both HLH patients and healthy controls, even in the presence of inflammatory cytokines. These data suggest that the CD47-SIRPA antiphagocytic system plays a key role in the maintenance of HSCs and that its disruption by HSC-specific CD47 down-regulation might be critical for HLH development.

Abstract: Eosinophils play an important role in the pathogenesis of allergic reactions or chronic inflammatory diseases by releasing various types of cytokines and chemical mediators. Recently, we have identified murine eosinophil-committed progenitors (mEoPs) in mouse bone marrow. The expression of receptor for IL-5, a critical cytokine for proliferation and differentiation of eosinophils, was a key marker to isolate mEoPs: mEoP was IL-5Ra+Lineage(lin)-CD34+c-Kitlow population in murine bone marrow (J Exp Med.201, 1891ndash;7, 2005). Here we report that EoPs are prospectively isolatable also in human bone marrow. We analyzed the expression of human IL-5Ra in human stem and progenitor populations, and found that a fraction of common myeloid progenitor (CMP; lin-CD34+CD38+CD45RA-IL-3Ra+) population expressed hIL-5Ra on their surface by using anti-human IL-5Ra monoclonal antibodies. IL-5Ra protein and mRNA were undetectable in hematopoietic stem cells (HSCs; lin-CD34+CD38-), common lymphoid progenitors (CLPs; lin-CD34+CD38+CD10+), megakaryocyte/erythrocyte progenitors (MEPs; lin-CD34+CD38+CD45RA-IL-3Ra-), or granulocyte/monocyte progenitors(GMPs; lin-CD34+CD38+CD45RA+IL-3Ra+) by FACS and RT-PCR, respectively. The IL-5Ra+ cells within the CMP fraction constituted only ~0.04% of steady-state bone marrow mononuclear cells, and gave rise only to pure eosinophil colonies. Thus we termed this population as human EoP (hEoP). Both HSCs and the IL-5Ra- fraction of CMPs gave rise to IL-5Ra+ hEoPs in vitro in the presence of IL-3 and GM-CSF, while MEPs or GMPs never generated hEoPs, indicating that human eosinophil pathway diverges at the CMP stage, and that the eosinophil potential was lost at the GMP or MEP stage. Accordingly, the human eosinophil pathway is different from that in murine hematopoiesis where mEoPs develop from the GMP stage. Strikingly, the number of hEoPs in the bone marrow of patients with hypereosinophilic syndrome was significantly (~4-fold) increased as compared to that in normal bone marrow, suggesting that hEoP represents a critical stage for eosinophilia in vivo. Thus, the hEoP is an attractive candidate for therapeutic target in eosinophil-related allergic and inflammatory disorders. This population might also be very useful to study the molecular mechanism of human eosinophil development.

Abstract: Mcl-1 (myeloid cell leukaemia-1) is an anti-apoptotic member of the Bcl-2 family protein, originally identified through the analysis of differentiating myeloid cells. We have recently reported that disruption of murine MCL-1 in adult murine hematopoiesis resulted in a complete deficiency of hematopoietic cells in the bone marrow, and that MCL-1 expression was activated in response to activation of c-Kit tyrosine kinase receptor signaling (Science307;1101, 2005). Here we propose that human MCL-1 also plays a critical role in maintenance of normal and malignant human hematopoiesis. The analysis of FACS-sorted human HSCs and myeloid progenitors demonstrated that, as in case of murine hematopoiesis, HSCs expressed hMCL-1 at the highest level, and that its expression gradually downregulated in committed myeloid progenitors such as CMPs and GMPs. We also found that unlike murine long-term HSCs that express c-Kit but not FLT3 receptor tyrosine kinase, human long-term HSCs express both of these receptors. Ligation of FLT3 receptor in purified human HSCs immediately induced the expression of MCL-1, suggesting that FLT3 supports human HSC survival through stimulating MCL-1 expression. We then evaluated whether hMCL-1 is expressed in leukemic stem cells. Thirty patients with acute myeloid leukemia (AML) were enrolled in this study. Within the immature CD34+ blast population, the CD34+CD38− fraction expressed higher levels of MCL-1 than those in more differentiated CD34+CD38+ fraction. In most cases, CD34+CD38− AML cells expressed 2 to 10-fold higher levels of MCL-1 transcripts as compared to normal HSCs. Interestingly, the group of samples expressed highest levels of MCL-1 constituted mostly of FLT3/internal tandem duplications (ITDs) positive AML, suggesting that constitutive activation of FLT3 signaling by FLT3 mutations might induce overexpression of MCL-1. A high level expression of MCL-1 was also found in MOLM-13 and MV4-11, FLT3/ITD positive AML cell lines. Treatment of MOLM-13 and MV4-11 with the small molecule tyrosine kinase inhibitor PKC 412, resulted in induction of apoptosis, which was associated with decreased expression of MCL-1 transcripts and proteins. Based on these data, MCL-1 might play a critical role in maintenance of normal and malignant HSCs in human at least through FLT3 signaling. Our data also suggest that constitutive activation of FLT3 signaling by FLT3 mutation might contribute to leukemic transformation through enforcing survival of AML stem cells.

Abstract: Two types of activating FLT3 mutations have been described in AML. FLT3/ITDs can be detected in 20% to 30% of patients with AML. Point mutations of FLT3/D835 have been described in 7% of adult AML patients. Activating FLT3 mutations has been associated with the leukocytosis and poor prognosis. We retrospectively analyzed the significance of FLT3 mutations in AML patients of normal karyotype treated with autologous peripheral blood stem cell transplantation (auto-PBSCT). METHOD: We evaluated 34 consecutive patients with first CR AML of normal karyotype who received myeloablative therapy and auto-PBSCT to analyze clinical features and outcomes between patients with and without FLT3/ITDs and FLT3/D835. There were 16 males and 18 females with a median age of 41.5 years ranging 15–74 years. Cytogenetic G-banding analysis was performed with standard method. The distribution of morphologic types of AML according to the FAB classification was as follows; one patient was M0, twelve M1, eight M2, seven M4 and six M5. The pre-transplant conditioning regimen was G-CSF combined with the high-dose chemotherapy consisting of busulfan (16 mg/kg), etoposide (40 mg/kg) and Ara-C (3 g/m2x4) (BEA regimen). DNA PCR assay was used to detect FLT3/ITDs. All abnormal longer products by agarose gel electrophoresis were subsequently sequenced. DNA PCR assay followed by direct sequence were used to detect FLT3/D835. RESULT: FLT3/ITDs were detected in 8 of 34 patients (23.5 %). FLT3 D835 mutation was detected in 2 of 34 patients (5.9%). To define clinical differences between patients with and without FLT3/ITDs, clinical variables at the diagnosis were compared. WBC (p=0.013), LDH (p=0.0147), the percentage of PB and BM blasts (p=0.0422 and P=0.0021) were significantly higher in the FLT3/ITDs patients. Other clinical parameters such as age, sex, FAB classification, Hb, Plt, remission induction therapy, interval from diagnosis to auto-PBSCT, number of infused CD34+ cells and hematological recovery after auto-PBSCT are not associated with the presence or absence of FLT3/ITDs. We analyzed the clinical significance of FLT3/ITDs mutations. OS and DFS was similar in patients with or without FLT3/ITDs (5 years OS, 71.4% vs 78.9%, p=0.5746; 5 years DFS, 72.9% vs 68.6%, p=0.9273 by the log-rank test). DISCUSSION: As far as we know, this is the first report to describe the significance of FLT3 mutations in AML patients of normal karyotype treated with auto-PBSCT. We show that FLT3 mutations have no prognostic impact in autotransplanted AML 1CR patients of normal karyotype. Our data suggest dose escalation of chemotherapy may conquer the poor prognostic implications of FLT3 mutations. The prognostic significance of activating FLT3 mutations in AML patients with normal karyotype should be evaluated in relation with post-remission therapeutic modalities.

Abstract: Relapse of acute myeloid leukemia (AML) is a critical problem in clinics. Particularly, the prognosis of patients who relapsed after allogeneic stem cell transplantation (allo-SCT) is still poor. Self-renewing leukemic stem cells (LSCs), which are mainly enriched within CD34+CD38- fraction, cause re-growth of leukemia and eventually lead to recurrence. Therefore, evaluation of residual LSCs is crucial to estimate the efficacy of therapies including allo-SCT. As we previously described, T-cell immunoglobulin mucin-3 (TIM-3) is a useful marker for discrimination of normal and malignant HSCs. We, therefore, prospectively evaluated the frequencies of LSCs at multiple time points and assessed the validity of TIM-3 as a minimal residual disease (MRD) marker in the setting of allo-SCT. 50 AML patients who had been detected CD34+CD38-TIM-3+ LSCs in BM sample at least once before SCT were analyzed. All 50 patients underwent allo-SCT at Fukuoka Blood and Marrow Transplantation Group (FBMTG)-related hospitals from July 2015 to May 2018. 43 out of 50 patients achieved complete donor chimerism confirmed by short tandem repeat PCR (STR-PCR) at the time of engraftment (day 15-36), and these 43 patients was prospectively evaluated the frequencies of residual CD34+CD38-TIM-3+ LSCs using multi-color flow cytometry. Median age of the evaluated 43 AML patients at SCT was 44.8 years (28-67). 26 patients underwent SCT on disease and 17 patients at hematological CR. Relapse-free survival (RFS) was calculated from the time of SCT until last follow-up or documentation of relapse. In each timing (at diagnosis, relapse, engraftment, etc.), we tracked the frequencies of TIM-3+ cells within CD34+CD38- fraction in BM. Of note, the proportion of CD34+CD38- fraction was 0.016 [0.0056 - 0.0267] % of BMMNC at engraftment. Then, according to the frequency of TIM-3+ cells within CD34+CD38- fraction, we classified patients into 3 groups; 'high' ( 〉 90 % of CD34+CD38- cells were positive for TIM-3, n=5), 'intermediate' (61-90 % of CD34+CD38- cells were positive for TIM-3, n=11), and 'low' ( 〈 60 % of CD34+CD38- cells were positive for TIM-3, n=27). This classification revealed that the recurrence rate within the observation period (median 334.4 days) was 100 % (5 of 5) in 'high' group, 45.4 % (5 of 11) in 'intermediate' group and 14.8 % (4 of 27) in 'low' group. Relapse-free survival was 83 (72-92) days in 'high' group, 368 (56-869) days in 'intermediate' group and 482.6 (45-1029) days in 'low' group, respectively (p 〈 0.01) (Figure 1). Of note, all of 5 patients in 'high' group exhibited hematological relapse within 100 days. Thus, our new classification using TIM-3 as LSC-specific marker could isolate the patients at high risk for early relapse in allo-SCT settings even if they were considered as hematological CR and maintained complete donor chimerism assessed by clinically available methods (three-color FACS, STR-PCR, FLT3-ITD status and fusion gene specific qPCR) at the same timepoint. It suggests that monitoring of LSCs using TIM-3 should be a more sensitive and versatile strategy to predict early relapse of AML than aberrant surface markers monitoring by multi-color FACS because conventional FACS did not detect MRD population at engraftment while our strategy detected (we could detect LSCs (CD34+CD38-TIM-3+ fraction) within BMMNC in 0.0053 [0 - 0.012] %), and, the cases whose aberrant markers could be traced were only 28 cases of 43 in this study. We also confirmed that the identical driver mutations were detected at both the initial diagnosis and relapse using whole exome sequencing (WES) of purified LSCs. As a typical example, WES detected CEBPA mutation (c.11dupG, 72.7 % and 50.0 %) and WT1 mutation (c.1091_1092insTTGTACGGTC, 41.9 % and 39.5 %) in a single patient. Additionally, we also validated that purified TIM-3+ LSCs at engraftment harbored the identical mutations by amplicon sequencing. It indicates that CD34+CD38-TIM-3+ cells should represent LSCs throughout the clinical course, from diagnosis to relapse. In summary, TIM-3 expression represents the clones of functional LSCs are involved in relapse. Evaluation of TIM-3+ LSCs by multi-color FACS should be a highly sensitive strategy to predict relapse in clinical allo-SCT settings, and might enable us to appropriately intervene to overcome the poor clinical outcomes of AML. Figure 1. Figure 1. Disclosures Akashi: Taiho Pharmaceutical: Research Funding; Novartis pharma: Research Funding; Pfizer: Research Funding; Kyowa Hakko Kirin: Research Funding, Speakers Bureau; Eisai: Research Funding; Celgene: Research Funding, Speakers Bureau; Astellas Pharma: Research Funding; sanofi: Research Funding; MSD: Research Funding; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Chugai Pharma: Research Funding; Eli Lilly Japan: Research Funding; Ono Pharmaceutical: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi-kasei: Research Funding.