[Kidney transplantation for end-stage renal disease after third allogeneic hematopoietic stem cell transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia].

An 18-year-old man underwent allogenic bone marrow transplantation (BMT) for Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL). Ph+ALL relapsed 3 months after the first BMT, and the patient underwent a second BMT. However, Ph+ALL relapsed 4 months after the second BMT, and he received a haploidentical peripheral blood stem cell transplantation (haplo-PBSCT) from his father. Molecular complete remission was confirmed 29 days after haplo-PBSCT. However, the patient needed dialysis for end-stage renal disease due to thrombotic microangiopathy 3 years and 2 months after haplo-PBSCT. He received a kidney transplantation from his father 7 years and 10 months after haplo-PBSCT, and got off dialysis after the kidney transplantation. Immunosuppressive therapy with methylprednisolone, tacrolimus, and mycophenolate mofetil was started for kidney transplantation, but the dose of immunosuppressive agents was reduced successfully without rejection soon after kidney transplantation. The patient has maintained long-term remission since the haplo-PBSCT, and his kidney function was restored by the kidney transplantation from his father.

Optimizing Treatment Options for Newly Diagnosed Acute Myeloid Leukemia in Older Patients with Comorbidities.

Traditionally, the goal of AML therapy has been to induce remission through intensive chemotherapy, maintain long-term remission using consolidation therapy, and achieve higher rates of a cure by allogeneic transplantation in patients with a poor prognosis. However, for the elderly patients and those with comorbidities, the toxicity often surpasses the therapeutic benefits of intensive chemotherapy. Consequently, low-intensity therapies, such as the combination of a hypomethylating agent with venetoclax, have emerged as promising treatment options for elderly patients. Given the rise of low-intensity therapies as the leading treatment option for the elderly, it is increasingly important to consider patients' age and comorbidities when selecting a treatment option. The recently proposed comorbidity-based risk stratification for AML allows prognosis stratification not only in patients undergoing intensive chemotherapy, but also in those receiving low-intensity chemotherapy. Optimizing treatment intensity based on such risk stratification is anticipated to balance treatment efficacy and safety, and will ultimately improve the life expectancy for patients with AML.

Importance of TKI treatment duration in treatment-free remission of chronic myeloid leukemia: results of the D-FREE study.

Treatment-free remission (TFR) is a new goal for patients with chronic myeloid leukemia in chronic phase (CML-CP) with a sustained deep molecular response (DMR) to treatment with tyrosine kinase inhibitors (TKIs). However, optimal conditions for successful TFR in patients treated with second-generation (2G)-TKIs are not fully defined. In this D-FREE study, treatment discontinuation was attempted in newly diagnosed CML-CP patients treated with the 2G-TKI dasatinib who achieved BCR-ABL1 levels of ≤ 0.0032% (MR4.5) on the international scale (BCR-ABL1IS) and maintained these levels for exactly 1 year. Of the 173 patients who received dasatinib induction therapy for up to 2 years, 123 completed and 60 (48.8%) reached MR 4.5. Among the first 21 patients who maintained MR4.5 for 1 year and discontinued dasatinib, 17 experienced molecular relapse defined as loss of major molecular response (BCR-ABL1IS > 0.1%) confirmed once, or loss of MR4 (BCR-ABL1IS > 0.01%) confirmed on 2 consecutive assessments. The estimated molecular relapse-free survival rate was 16.7% at 12 months. This study was prematurely terminated according to the protocol's safety monitoring criteria. The conclusion was that sustained DMR for just 1 year is insufficient for TFR in CML-CP patients receiving dasatinib for less than a total of 3 years of treatment.

[Aggressive B-cell lymphoma with IGH/MYC, IGH/BCL2, and IGH/CCND1 translocations].

A 70-year-old man was admitted to our hospital due to fever, lymphadenopathy, and leukocytosis. White blood cell count was 22,700/µl with 92% blastoid cells. Bone marrow examination revealed abnormal lymphoid cell expansion. Abnormal cells expressed surface CD5 (dim), CD10, CD19, CD20, CD23 (dim) antigens, and kappa immunoglobulin light chains. Cytogenetic analysis of bone marrow cells at the time of diagnosis showed t (11:14) (q13;q32), t (14;18) (q32;q21), and t (8;14;18) (q24;q32;q21). Fluorescence in situ hybridization analyses of bone marrow identified translocations of IGH/MYC, IGH/BCL2, and IGH/CCND1. The patient was diagnosed with aggressive B-cell lymphoma with IGH/MYC, IGH/BCL2, and IGH/CCND1 translocation and was treated with various chemotherapies including R-CHOP, R-ESHAP, DA-EPOCH-R, R-hyper-CVAD, and radiotherapy. However, the lymphoma recurred after every chemotherapy session. Finally, he died after 6 months after first admission. Double-hit lymphoma/triple-hit lymphoma has previously been reported to present with an aggressive clinical course. In the present case, co-existence of IGH/CCND1, IGH/MYC, and IGH/BCL2 is very rare. Further clinical and biological investigations are necessary to establish an optimal treatment strategy.

Acute promyelocytic leukemia presenting as recurrent spinal myeloid sarcomas 3 years before developing leukemia: A case report with review of literature.

The de novo myeloid sarcoma (MS) type of acute promyelocytic leukemia (APL) is rare, and clinical features may differ from extramedullary diseases in advanced APL. Many cases occur as a spinal tumor, and some occur in the absence of bone-marrow diseases or coagulation abnormalities. Fluorescence in situ hybridization analysis of MS tissue is useful for accurate diagnosis, even in preserved tissue.

Mechanisms for mTORC1 activation and synergistic induction of apoptosis by ruxolitinib and BH3 mimetics or autophagy inhibitors in JAK2-V617F-expressing leukemic cells including newly established PVTL-2.

The activated JAK2-V617F mutant is very frequently found in myeloproliferative neoplasms (MPNs), and its inhibitor ruxolitinib has been in clinical use, albeit with limited efficacies. Here, we examine the signaling mechanisms from JAK2-V617F and responses to ruxolitinib in JAK2-V617F-positive leukemic cell lines, including PVTL-2, newly established from a patient with post-MPN secondary acute myeloid leukemia, and the widely used model cell line HEL. We have found that ruxolitinib downregulated the mTORC1/S6K/4EBP1 pathway at least partly through inhibition of the STAT5/Pim-2 pathway with concomitant downregulation of c-Myc, MCL-1, and BCL-xL as well as induction of autophagy in these cells. Ruxolitinib very efficiently inhibited proliferation but only modestly induced apoptosis. However, inhibition of BCL-xL/BCL-2 by the BH3 mimetics ABT-737 and navitoclax or BCL-xL by A-1331852 induced caspase-dependent apoptosis involving activation of Bak and Bax synergistically with ruxolitinib in HEL cells. On the other hand, the putative pan-BH3 mimetic obatoclax as well as chloroquine and bafilomycin A1 inhibited autophagy at its late stage and induced apoptosis in PVTL-2 cells synergistically with ruxolitinib. The present study suggests that autophagy as well as the anti-apoptotic BCL-2 family members, regulated at least partly by the mTORC1 pathway downstream of STAT5/Pim-2, protects JAK2-V617F-positive leukemic cells from ruxolitinib-induced apoptosis depending on cell types and may contribute to development of new strategies against JAK2-V617F-positive neoplasms.

Molecular mechanisms for enhancement of stromal cell-derived factor 1-induced chemotaxis by platelet endothelial cell adhesion molecule 1 (PECAM-1).

Platelet endothelial cell adhesion molecule 1 (PECAM-1) is a cell adhesion protein involved in the regulation of cell adhesion and migration. Interestingly, several PECAM-1-deficient hematopoietic cells exhibit impaired chemotactic responses to stromal cell-derived factor 1 (SDF-1), a chemokine essential for B lymphopoiesis and bone marrow myelopoiesis. However, whether PECAM-1 is involved in SDF-1-regulated chemotaxis is unknown. We report here that SDF-1 induces tyrosine phosphorylation of PECAM-1 at its immunoreceptor tyrosine-based inhibition motifs in several hematopoietic cell lines via the Src family kinase Lyn, Bruton's tyrosine kinase, and JAK2 and that inhibition of these kinases reduced chemotaxis. Overexpression and knockdown of PECAM-1 enhanced and down-regulated, respectively, SDF-1-induced Gαi-dependent activation of the PI3K/Akt/mTORC1 pathway and small GTPase Rap1 in hematopoietic 32Dcl3 cells, and these changes in activation correlated with chemotaxis. Furthermore, pharmacological or genetic inhibition of the PI3K/Akt/mTORC1 pathway or Rap1, respectively, revealed that these pathways are independently activated and required for SDF-1-induced chemotaxis. When coexpressed in 293T cells, PECAM-1 physically associated with the SDF-1 receptor CXCR4. Moreover, PECAM-1 overexpression and knockdown reduced and enhanced SDF-1-induced endocytosis of CXCR4, respectively. Furthermore, when expressed in 32Dcl3 cells, an endocytosis-defective CXCR4 mutant, CXCR4-S324A/S325A, could activate the PI3K/Akt/mTORC1 pathway as well as Rap1 and induce chemotaxis in a manner similar to PECAM-1 overexpression. These findings suggest that PECAM-1 enhances SDF-1-induced chemotaxis by augmenting and prolonging activation of the PI3K/Akt/mTORC1 pathway and Rap1 and that PECAM-1, at least partly, exerts its activity by inhibiting SDF-1-induced internalization of CXCR4.

Atypical chronic myeloid leukemia with isochromosome (X)(p10): A case report.

Atypical chronic myeloid leukemia (aCML) is a rare subtype of myelodysplastic/myeloproliferative neoplasm (MDS/MPN). Although recurrent chromosomal and genetic abnormalities are frequently observed in aCML, none are specific to this type of leukemia. The present study reported a case of aCML associated with i(X)(p10), a rare recurrent chromosomal abnormality of hematological malignancy. A 40-year-old female was referred to the Tokyo Medical and Dental University Hospital (Tokyo, Japan) due to slight leukocytosis and anemia. A bone marrow aspiration revealed 4% blasts and granulocytic hyperplasia with dysplasia. A G-banded cytogenetic analysis of the bone marrow cells revealed 46, X, isochromosome X(iX)(p10) in all metaphases. The percentage of the neutrophil precursors promyelocytes, myelocytes and metamyelocytes in the peripheral blood was >10% throughout the clinical course of the patient, which resulted in a diagnosis of atypical chronic myeloid leukemia. Treatment with hydroxycarbamide was not able to effectively alleviate leukocytosis, and the disease progressed with the appearance of an additional cytogenetic abnormality, t(10;17)(p13;q21). Subsequently, the patient underwent allogeneic stem cell transplantation from a sibling donor, and subsequent cytogenetic analysis revealed a normal karyotype with full donor chimerism. The isodicentric X(idicX)(q13) mutation is a similar abnormality to i(X)(p10) and may result in a loss of the X-inactive specific transcript gene located at Xq13.2, the deletion of which has been previously reported to result in the development of MDS/MPN in mice. In addition, i(X)(p10) was identified as the sole chromosomal abnormality at the diagnosis of aCML in the case of the present study, which is similar to patients from previous studies of other hematological malignancies and supports the hypothesis that i(X)(p10) may have served a primary role in the leukemogenesis of aCML.

Acute myeloid leukemia with t(3;21)(q26.2;q22) developing following low-dose methotrexate therapy for rheumatoid arthritis and expressing two AML1/MDS1/EVI1 fusion proteins: A case report.

The t(3;21)(q26.2;q22) translocation is a rare chromosomal abnormality exhibited almost exclusively in therapy-related myelodysplastic syndrome/acute myeloid leukemia (t-MDS/AML) or in the blastic crisis phase of chronic myelogenous leukemia, which results in the fusion of the runt related transcription factor 1 (RUNX1, also called AML1) gene at 21q22 to the myelodysplasia syndrome 1 (MDS1)-ecotropic virus integration site 1 (EVI1) complex locus (MECOM) at 3q26.2, generating various fusion transcripts, including AML1/MDS1/EVI1 (AME). The present study examined the case of an 84-year-old Japanese woman who developed t-MDS/AML with t(3;21)(q26.2;q22) subsequent to receiving low-dose methotrexate (MTX) treatment for rheumatoid arthritis. Following treatment with MTX for 6 years, the patient developed anemia and neutropenia, and MTX was discontinued. A total of 3 years later, the patient was diagnosed with MDS with t(3;21)(q26.2;q22) and del (5q), which progressed rapidly to AML within 3 months. The patients was subsequently treated with azacitidine and cytarabine chemotherapy, but succumbed to the disease 6 months after diagnosis. Sequencing analysis of the nested reverse transcription-PCR products from the leukemic cells revealed the expression of two types of alternatively-spliced AME transcripts with or without RUNX1 exon 6 sequences. Western blot analysis of the leukemic cells of the patient additionally revealed that the corresponding AME fusion protein products were expressed at high levels, and that these cells also prominently expressed CCAAT/enhancer-binding protein α, the repression of which has been reported to be involved in leukemogenesis mediated by AME. To the best of our knowledge, the case discussed in the present study represents the first report of MDS/AML with t(3;21)(q26.2;q22) developing following low-dose MTX therapy for rheumatoid arthritis. Nonetheless, the clinical and molecular features of the patient in the present study were representative of those patients who typically develop this disease following exposure to chemotherapy or radiotherapy for primary malignancy, which implicates MTX in the pathogenesis of t-MDS/AML. Moreover, we confirmed the expression of two AME fusion proteins for the first time in primary leukemic cells and analyzed several cellular factors implicated in AME-mediated leukemogenesis to gain some insight into its molecular mechanisms.

Bence-Jones Protein λ-type Multiple Myeloma Patient Withdrawn from Maintenance Hemodialysis after Long-term Bortezomib and Dexamethasone Therapy.

The effectiveness of bortezomib treatment for multiple myeloma (MM) is well established. However, the protocol by which maintenance therapy using bortezomib should be continued for myeloma patients requiring regular hemodialysis remains to be established. We herein report a case of MM with severe renal insufficiency requiring hemodialysis for nearly 30 months which was finally withdrawn from renal replacement therapy during monthly maintenance treatment with bortezomib and dexamethasone for two years. The details of this case are essential for establishing clinical guidelines for applying intermittent low-frequency bortezomib therapy in dialysis-dependent myeloma patients.

[Refractory primary myeloid sarcoma of the breast with MLL-AF9 rearrangement].

A 28-year-old woman presented with a right breast mass and axillary lymphadenopathy. Biopsy of the breast mass revealed myeloid sarcoma (MS) staining positive for CD4, CD13, CD33, and CD68/KP-1. Bone marrow aspiration revealed leukemic cell infiltration (9%). Leukemic cells possessed cytogenetic abnormalities of +8 and t(9;11)(p22;q23) with +22 (lymph node only), and molecular analyses confirmed the MLL-AF9 fusion gene. After induction chemotherapy and 2(nd) consolidation therapy, complete remission was maintained. However, during consolidation radiotherapy for the breast mass, the disease progressed in both the breast and bone marrow. She received re-induction therapy and proceeded to allogeneic stem cell transplantation. However, the disease relapsed in the breast soon after transplantation, and she died from disease progression. Trisomy 8 and the MLL-AF9 fusion gene have been reported in cases with MS in the breast. Trisomy 22 found additionally and exclusively in the extramedullary lesion implies extramedullary progression of MS from the medullary site of origin and may have been associated with the distinctive therapy resistance of these lesions in our case.

Impact of prior azacitidine on the outcome of allogeneic hematopoietic transplantation for myelodysplastic syndrome.

To clarify the clinical impact of prior use of azacitidine (AZA) on outcomes of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for myelodysplastic syndrome (MDS), we retrospectively reviewed the clinical outcomes of 15 MDS patients who were treated with AZA before allo-HSCT (AZA group). We compared the outcomes of these 15 patients with 52 MDS patients who were solely given the best supportive care (BSC) before allo-HSCT (BSC group). Although patients in the AZA group were older with higher International Prognostic Scoring System (IPSS) scores compared to patients in the BSC group, no significant differences were found between the two groups in overall survival (OS), disease-free survival (DFS), cumulative incidence of relapse (CIR) or non-relapse mortality. However, in patients with a higher IPSS score (Int-2/High), pre-transplant AZA may provide better OS and DFS and lower CIR. Acute graft-versus-host disease rates were similar between the two groups. These results should be reassuring to patients with high-risk MDS receiving AZA before allo-HSCT.

FLT3-ITD confers resistance to the PI3K/Akt pathway inhibitors by protecting the mTOR/4EBP1/Mcl-1 pathway through STAT5 activation in acute myeloid leukemia.

FLT3-ITD and FLT3-TKD are the most frequent tyrosine kinase mutations in acute myeloid leukemia (AML), with the former associated with poor prognosis. Here, we show that the PI3K inhibitor GDC-0941 or the Akt inhibitor MK-2206 induced apoptosis through the mitochondria-mediated intrinsic pathway more efficiently in hematopoietic 32D cells driven by FLT3-TKD (32D/TKD) than FLT3-ITD (32D/ITD), which robustly activated STAT5. The resistance to GDC-0941 and MK-2206 was gained by expression of the constitutively activated STAT5 mutant STAT5A1*6 in 32D/TKD cells, while it was abrogated by the STAT5 inhibitor pimozide in 32D/ITD cells or FLT3-ITD-expressing human leukemic MV4-11 cells. GDC-0941 or MK-2206 induced dephosphorylation of 4EBP1 more conspicuously in 32D/TKD than in 32D/ITD, which was prevented or augmented by STAT5A1*6 or pimozide, respectively, and correlated with downregulation of the eIF4E/eIF4G complex formation and Mcl-1 expression. Furthermore, exogenous expression of Mcl-1 endowed resistance to GDC-0941 and MK-2206 on 32D/TKD cells. Finally, it was confirmed in primary AML cells with FLT3-ITD that pimozide enhanced 4EBP1 dephosphorylation and Mcl-1 downregulation to augment cytotoxicity of GDC-0941. These data suggest that the robust STAT5 activation by FLT3-ITD protects cells treated with the PI3K/Akt pathway inhibitors from apoptosis by maintaining Mcl-1 expression through the mTORC1/4EBP1/eIF4E pathway.

Acquired factor X deficiency associated with atypical AL-amyloidosis.

We herein describe the case of a 77-year-old woman with acquired factor X deficiency that was likely caused by atypical amyloidosis. The patient developed severe gastrointestinal bleeding as a result of a significant decrease of factor X activity. Neither proteinuria nor diarrhea was observed as an initial manifestation. Although a bone marrow examination revealed direct fast scarlet-positive extracellular deposits, they did not exhibit red-to-green dichroism under polarized light. Immunofluorescence microscopy showed that the fibrillar proteins were positive for CD138 but negative for ß2-microglobulin or amyloid A antibodies. These atypical pathological features of immunoglobulin light chain-amyloidosis in this patient might be related to its unique clinical presentation.

The predictive value for pulmonary infection by area over the neutrophil curve (D-index) in patients who underwent reduced intensity hematopoietic stem cell transplantation.

We evaluated the predictive value of the D-index for pulmonary infection in the early phase of reduced intensity stem cell transplantation (RIST). Out of 68 patients, ten patients developed a pulmonary infection within 100 days after RIST. Both the D-index and the cD-index were higher in the patients with pulmonary infection than in the control group (P = 0.009, P = 0.042, respectively). The best sensitivity and specificity, calculated with receiver operating characteristic curves, showed that the D-index was superior to the duration of neutropenia in predicting pulmonary infection. We also evaluated the utility of a cumulative D-index until 21 days after RIST (D21-index). The D21-index was higher in the patients with pulmonary infection (P = 0.047). The cutoff value of the D21-index was lower than that of the D-index (8650 vs. 11000) with comparable sensitivity and specificity. Our results demonstrate that the D21-index, as well as the D-index, are useful tools for the prediction of pulmonary infection in RIST.

Proliferation and survival signaling from both Jak2-V617F and Lyn involving GSK3 and mTOR/p70S6K/4EBP1 in PVTL-1 cell line newly established from acute myeloid leukemia transformed from polycythemia vera.

The gain of function mutation JAK2-V617F is very frequently found in myeloproliferative neoplasms (MPNs) and is strongly implicated in pathogenesis of these and other hematological malignancies. Here we report establishment of a new leukemia cell line, PVTL-1, homozygous for JAK2-V617F from a 73-year-old female patient with acute myeloid leukemia (AML) transformed from MPN. PVTL-1 is positive for CD7, CD13, CD33, CD34, CD117, HLA-DR, and MPO, and has complex karyotypic abnormalities, 44,XX,-5q,-7,-8,add(11)(p11.2),add(11)(q23),-16,+21,-22,+mar1. Sequence analysis of JAK2 revealed only the mutated allele coding for Jak2-V617F. Proliferation of PVTL-1 was inhibited and apoptosis was induced by the pan-Jak inhibitor Jak inhibitor-1 (JakI-1) or dasatinib, which inhibits the Src family kinases as well as BCR/ABL. Consistently, the Src family kinase Lyn was constitutively activated with phosphorylation of Y396 in the activation loop, which was inhibited by dasatinib but not by JakI-1. Further analyses with JakI-1 and dasatinib indicated that Jak2-V617F phosphorylated STAT5 and SHP2 while Lyn phosphorylated SHP1, SHP2, Gab-2, c-Cbl, and CrkL to induce the SHP2/Gab2 and c-Cbl/CrkL complex formation. In addition, JakI-1 and dasatinib inactivated the mTOR/p70S6K/4EBP1 pathway and reduced the inhibitory phosphorylation of GSK3 in PVTL-1 cells, which correlated with their effects on proliferation and survival of these cells. Furthermore, inhibition of GSK3 by its inhibitor SB216763 mitigated apoptosis induced by dasatinib but not by JakI-1. Together, these data suggest that apoptosis may be suppressed in PVTL-1 cells through inactivation of GSK3 by Lyn as well as Jak2-V617F and additionally through activation of STAT5 by Jak2-V617F. It is also speculated that activation of the mTOR/p70S6K/4EBP1 pathway may mediate proliferation signaling from Jak2-V617F and Lyn. PVTL-1 cells may provide a valuable model system to elucidate the molecular mechanisms involved in evolution of Jak2-V617F-expressing MPN to AML and to develop novel therapies against this intractable condition.

Clinical outcome of hematopoietic stem cell transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL): experience from a single institution.

To identify factors affecting transplant outcome, data from 65 Philadelphia Chromosome-positive acute lymphoblastic leukemia (Ph + ALL) patients who had undergone allogeneic hematopoietic transplantation (allo-HSCT) in our institution were analyzed. The probability of OS (overall survival) and DFS (disease free-survival) at 3 years after allo-HSCT was 40.1% and 38%, respectively. Multivariate analysis showed that gender and disease status (p = 0.0059, p = 0.0039, respectively) were significant factors for OS. Among 51 patients with CR (complete remission), multivariate analysis showed that the factors associated with OS included gender (p = 0.014), number of white blood cell at diagnosis (p = 0.025), and the source of stem cells (bone marrow versus cord blood; BM stem cell source was associated with favorable OS, p = 0.042). Twenty-one patients relapsed after allo-HSCT with a median of 207 days (range, 19-1,324 days). The estimated cumulative incidence of relapse at 3 years was 39.4%. Patients with CR showed a lower relapse rate at 3 years (34.2%) when compared with patients with non-CR (62.7%). Among 21 patients, eight patients received imatinib-based chemotherapy and 13 received chemotherapy without imatinib before HSCT. In terms of treatment after relapse, seven patients received chemotherapy with imatinib and 13 received chemotherapy without imatinib. Five patients underwent a second HSCT. One patient survived, and 20 patients died. In this study, disease status at time of allo-HSCT had a significant impact on OS, DFS, and relapse. Imatinib administration given before allo-HSCT was not associated with favorable outcome. Second-generation tyrosine kinase inhibitors may be more suitable candidates for Ph + ALL before and after allo-HSCT.