Multiple Myeloma Presenting with Autoimmune Autonomic Ganglionopathy.

Autoimmune autonomic ganglionopathy is an autonomic disorder that occurs as a symptom of paraneoplastic neurological syndrome. To date, there have been no reports on multiple myeloma with autoimmune autonomic ganglionopathy. A 37-year-old Japanese woman suffered from orthostatic hypotension was diagnosed with multiple myeloma (IgG kappa type), and a serological examination revealed the presence of anti-ganglionic nicotinic acetylcholine receptor (anti-gAChR) antibodies. She was treated for multiple myeloma, as a result, the autonomic disturbance improved and her anti-gAChR antibody titer decreased to undetectable levels, despite the fact that she only achieved a partial remission of multiple myeloma. Treatment for multiple myeloma may improve autoimmune autonomic ganglionopathy.

Dose-Modified Ifosfamide, Epirubicin, and Etoposide is a Safe and Effective Salvage Therapy with High Peripheral Blood Stem Cell Mobilization Capacity for Poorly Mobilized Hodgkin's Lymphoma and Non-Hodgkin's Lymphoma Patients.

A dose modified ifosfamide, epirubicin, and etoposide (IVE) regimen was prospectively assessed for its efficacy in mobilizing peripheral blood stem cells for autologous transplantation. Two patients with Hodgkin's lymphoma and two with non-Hodgkin's lymphoma who were undergoing stem cell therapy were studied. All patients had a history of multiple treatments with insufficient stem cell mobilization. The dose modified IVE regimen consisted of ifosfamide 3 g/m(2) intravenously (IV) administered on days 1-2 in combination with epirubicin 50 mg/m(2) IV on day 1 and etoposide 200 mg/m(2) (100 mg/m(2) in two patients with complete remission) IV on days 1-3. The ifosfamide dosage was reduced to two-thirds of the original protocol. A substantial high yield of CD34(+) cells was achieved when patients were treated with a dose-modified IVE regimen, compared with that during the previous regimen (two with the ifosfamide, carboplatin, and etoposide [ICE] regimen, one with high-dose cyclophosphamide and one with the original IVE regimen). Two patients who had refractory and residual disease received a 200 mg/m(2) dose of etoposide, which resulted in tumor reduction (one patient with complete remission and one with further reduction in tumor size). After the IVE regimen, all four patients had a sufficient yield of CD34(+) cells in total, which was available for stem cell transplantation. Hematological and non-hematological toxicities were comparable in all regimens. This single-center prospective study demonstrated that the dose-modified IVE regimen can be used as a safe treatment with high mobilizing efficacy in heavily pretreated lymphoma patients.

Impact of age on outcomes of allogeneic hematopoietic stem cell transplantation with reduced intensity conditioning in elderly patients with acute myeloid leukemia.

Previous studies have repeatedly reported that increasing age is a significant risk factor for worse outcomes after allogeneic hematopoietic stem cell transplantation (allo-HSCT) among patients with acute myeloid leukemia (AML). However, more recent studies reported conflicting results regarding the association between age and outcomes in elderly patients. Therefore, we conducted a large-scale, nationwide retrospective study to examine the impact of age on outcomes of allo-HSCT with reduced intensity conditioning (RIC) for AML patients who were older than 50 years. Of the 757 patients, 89 patients (11.8%) were 50-54, 249 patients (32.9%) were 55-59, 301 patients (39.8%) were 60-64 and 118 patients (15.6%) were ≥65 years old. The 3-year overall survival (OS) (47.8, 45.2, 37.9, and 36.6% for patients aged 50-54, 55-59, 60-64, and ≥65 years, respectively, P = 0.24) and nonrelapse mortality (NRM) (24.0, 22.8, 29.2, and 27.6% for patients aged 50-54, 55-59, 60-64, and ≥65 years, respectively, P = 0.49) were not significantly different among the four age groups. Multivariate analysis revealed that increased age had no significant effect on OS or NRM after adjusting for covariates. These results suggested that advanced patient age is not a contraindication for RIC allo-HSCT in elderly AML patients.

A single high-resolution HLA mismatch has a similar adverse impact on the outcome of related hematopoietic stem cell transplantation as a single low-resolution HLA mismatch.

The relative importance of the resolution level of HLA typing has not been fully defined for related donor transplantation. To address this question, we retrospectively evaluated patients who underwent a first related hematopoietic stem cell transplantation (HSCT) from 2000 to 2011 from an HLA high-resolution matched (MRD, n = 2,244), high-resolution 1 locus-mismatched (HR-MMRD, n = 116), or low-resolution 1 locus-mismatched related donor (LR-MMRD, n = 396) in the graft-versus-host direction at three loci (HLA A, B, and DRB1) using the database of the Japan Society for Hematopoietic Cell Transplantation. The median age was 40 years (0-74). The median follow-up duration of surviving patients was 950 days. Although the cumulative incidences of grade III-IV acute graft-versus-host disease (GVHD) in the HR-MMRD and LR-MMRD groups were significantly higher than those in the MRD group (HR-MMRD 19.8%, LR-MMRD 20.4%, and MRD 9.5%), there was no statistically significant difference between the HR-MMRD and LR-MMRD groups (P = 0.65). Although both HR-MMRD and LR-MMRD were significantly associated with an increased risk of non-relapse mortality and a worse overall survival, there was no statistically significant difference between the HR-MMRD and LR-MMRD groups. In conclusion, LR-MM and HR-MM have a similar adverse impact on the outcome in related HSCT.

Crizotinib resistance in acute myeloid leukemia with inv(2)(p23q13)/RAN binding protein 2 (RANBP2) anaplastic lymphoma kinase (ALK) fusion and monosomy 7.

This is the first report on the development of a p.G1269A mutation within the kinase domain (KD) of ALK after crizotinib treatment in RANBP2-ALK acute myeloid leukemia (AML). An elderly woman with AML with an inv(2)(p23q13)/RANBP2-ALK and monosomy 7 was treated with crizotinib. After a short-term hematological response and the restoration of normal hematopoiesis, she experienced a relapse of AML. Fluorescence in situ hybridization using the ALK break-apart probe confirmed the inv(2)(p23q13), while G-banded karyotyping revealed the deletion of a segment of the short arm of chromosome 1 [del(1)(p13p22)] after crizotinib therapy. The ALK gene carried a heterozygous mutation at the nucleotide position g.716751G>C within exon 25, causing the p.G1269A amino acid substitution within the ALK-KD. Reverse transcriptase PCR revealed that the mutated ALK allele was selectively transcribed and the mutation occurred in the ALK allele rearranged with RANBP2. As both the del(1)(p13p22) at the cytogenetic level and p.G1269A at the nucleotide level newly appeared after crizotinib treatment, it is likely that they were secondarily acquired alterations involved in crizotinib resistance. Although secondary genetic abnormalities in ALK are most frequently described in non-small cell lung cancers harboring an ALK alteration, this report suggests that an ALK-KD mutation can occur independently of the tumor cell type or fusion partner after crizotinib treatment.

inv(2)(p23q13)/RAN-binding protein 2 (RANBP2)-ALK fusion gene in myeloid leukemia that developed in an elderly woman.

A 75-year-old woman presented with marked leukocytosis; the white cell count was 143.6 × 10³/µL with 38.6 % monocytes and 13.6 % immature granulocytes, including blasts. Bone marrow (BM) aspirate smears showed >90 % cellularity with hyperplasia of myeloid-lineage cells, 14.6 % monocytes, and 32.1 % blasts. The granulocyte series showed a range of dysplastic morphologies. The rate of peroxidase positivity was 51.5 %. CD36+ cells with monocytic differentiation comprised 64.6 % mononuclear cells. Metaphase spreads obtained from the BM revealed an aneuploid karyotype with -7 and a submetacentric marker chromosome derived from chromosome 2, which was determined to be inv(2)(p23q13) by fluorescence in situ hybridization using the Vysis ALK probe. RAN-binding protein 2 (RANBP2)-ALK fusion mRNA was confirmed by reverse transcriptase-mediated polymerase chain reaction and nucleotide sequencing. High-sensitivity anti-ALK immunohistochemistry of a BM biopsy specimen demonstrated nuclear membrane staining of leukemia cells. As the leukemia showed features of chronic myelomonocytic leukemia, the patient was treated with standard daunorubicin-cytarabine followed by azacitidine, leading to the durable suppression of leukemia progression. These findings suggest that inv(2)(p23q13)/RABBP2-ALK defines a small subset of myeloid leukemia characterized by differentiation to monocytes and sharing features of myelodysplastic syndrome/myeloproliferative neoplasm.

Short- and long-term effects of rituximab for the treatment of thrombotic thrombocytopenic purpura: four case reports.

We report four cases of thrombotic thrombocytopenic purpura (TTP) successfully treated with rituximab in combination with plasma exchange and other immunosuppressive agents. All four cases fulfilled the diagnostic criteria of TTP with severe deficiencies in ADAMTS13 activity and a detectable anti-ADAMTS13 inhibitor. Four weekly doses of 375 mg/m(2) rituximab were initiated on day 3-29 of presentation as a salvage treatment for relapsing/refractory disease in three patients and as a first-line treatment in one. Resolution of clinical symptoms and hematological abnormalities occurred as early as the second dose and, after the completion of treatment, all four patients achieved complete response (CR). They are currently free from relapse and the duration of CR has been 13-72 months. During the treatment course, the level of ADAMTS13 activity and the titer of the inhibitor correlated well with resolution or exacerbation of the disease. This report suggests that rituximab exhibits short- and long-term favorable effects for the treatment of TTP and that a severe ADAMTS13 deficiency and ADAMTS13 inhibitor positivity may support early administration of rituximab in both acute/refractory and relapsing cases.

Outbreak of pandemic 2009 influenza A/H1N1 infection in the hematology ward: fatal clinical outcome of hematopoietic stem cell transplant recipients and emergence of the H275Y neuraminidase mutation.

We report an outbreak of pandemic 2009 influenza A/H1N1 virus (2009 H1N1) infection that occurred in the hematology ward of our institution during the 2010-2011 influenza season. A total of seven hospitalized patients with hematologic tumors, including five recipients of hematopoietic stem cell transplantation (HSCT), successively developed rapid influenza detection test (RIDT)-positive influenza A; four patients had laboratory-confirmed 2009 H1N1 infection. Three HSCT recipients required mechanical ventilation support and two were admitted to the intensive care unit; they died of progressive respiratory failure despite receiving available anti-viral drugs. We implemented outbreak-control measures including transferal of RIDT-positive patients to a single-patient room and chemoprophylaxis with oseltamivir. We note that the H275Y neuraminidase mutation was detected in respiratory specimens from three patients, who were administered therapeutic or prophylactic dosages of oseltamivir. The present report demonstrates that the nosocomial 2009 H1N1 outbreak in the hematology ward led to fatal clinical outcomes and the emergence of a resistant virus at a markedly high rate.

Reappraisal of BCL3 as a molecular marker of anaplastic large cell lymphoma.

The BCL3 gene was initially discovered through its involvement in a recurring translocation, t(14;19)(q32;q13), which is found in some patients with B-cell chronic lymphocytic leukemia (B-CLL). The translocation leads to the juxtaposition of BCL3 to the immunoglobulin heavy chain gene locus, resulting in high-level expression of the BCL3 transcript. The Bcl-3 protein includes 7 tandem copies of the ankyrin repeat element in the central domain, a structure that is characteristic of the IkappaB family of inhibitors of the nuclear factor kappaB transcription factors. Anaplastic large cell lymphoma (ALCL) is a subtype of aggressive non-Hodgkin's lymphoma that is characterized by expression of CD30 and the NPM/ALK chimeric protein, which is generated by t(2;5)(p23;q35). We compared the gene expression profiles of ALCL with those of another CD30+ neoplasm, Hodgkin's disease (HD), and found that BCL3 is expressed at higher levels in ALCL than in HD. A comparison by real-time polymerase chain reaction assay revealed that t(2;5)+ ALCL expresses a high level of BCL3 messenger RNA relative to the levels expressed in other hematologic tumors, and the level in ALCL is comparable to or even higher than that in t(14;19)+ B-CLL. An immunohistochemical analysis of ALCL tumor tissues showed that the lymphoma cells exhibited strong nuclear staining by a monoclonal antibody against Bcl-3. We suggest that Bcl-3 sequestrates the (p50)2 homodimer to the nucleus and that the kappaB sites are occupied by the (p50)2/Bcl-3 ternary complex. Future studies should identify the relationships among the 3 independent molecules (ie, NPM/ALK, CD30, and Bcl-3) that are activated in t(2;5)+ ALCL.

Comparison of gene expression profiles of lymphoma cell lines from transformed follicular lymphoma, Burkitt's lymphoma and de novo diffuse large B-cell lymphoma.

To determine the specific gene expression in B-cell lymphoma subtypes, we compared expression profiles of cell lines from transformed follicular lymphoma (tFL), Epstein-Barr virus-negative (EBV(-)) Burkitt's lymphoma (BL) and EBV(+)BL. Complementary DNAs were synthesized from these cell lines and hybridized with the Atlas Human 1.2 Array membrane. Hierarchical clustering analysis based upon the levels of 43 genes highlighted characteristic expression patterns of the 3 lymphoma subtypes. Genes expressed at higher levels in tFL than EBV(-)BL and EBV(+)BL included calcium/calmodulin-dependent protein kinase I (CAMK1) and mitogen-activated protein kinase 10 (MAPK10). EBV(-)BL was characterized by high-level expression of amyloid beta precursor protein (APP), heat shock 27 kD protein 1 (HSPB1) and mothers against decapentaplegic homolog 1 (MADH1). Gardner-Rasheed feline sarcoma viral oncogene homolog (FGR) was the most significant gene to delineate EBV(+)BL. A subtype prediction algorithm using 34 genes correctly classified 22 (92%) of 24 lymphomas into FL/tFL, EBV(-)BL or EBV(+)BL. By comparison with normal reference B-cell materials, the expression patterns of the selected genes were characteristic of lymphomas. We extended the clustering analysis to cell lines from de novo diffuse large B-cell lymphoma (DLBCL). The DLBCL cell lines were either separated from the former 3 lymphoma subtypes or segregated with EBV(+)BL, possibly reflecting variable genetic abnormalities. The associations of CAMK1 with tFL, APP and MADH1 with EBV(-)BL, FGR with EBV(+)BL, and BCL2 with tFL and DLBCL were confirmed by real-time quantitative reverse transcriptase-mediated polymerase chain reaction assays. This study has provided new molecular markers, expressions of which are closely associated with B-cell lymphoma subtypes.

High-level expression of BCL3 differentiates t(2;5)(p23;q35)-positive anaplastic large cell lymphoma from Hodgkin disease.

Anaplastic large cell lymphoma (ALCL) with t(2;5)(p23;q35) and Hodgkin disease (HD) share many cellular features, including expression of CD30. We compared gene expression profiles of 4 ALCL (Karpas 299, SU-DHL-1, DEL, SR-786) and 3 HD cell lines and found that BCL3, which encodes a nuclear protein belonging to the I kappa B family of inhibitors of nuclear factor-kappa B (NF-kappa B) transcriptional factors, was expressed at higher levels in ALCL than HD. Northern and Western blotting analyses confirmed the high-level expression of BCL3 in ALCL at both mRNA and protein levels. We established a real-time reverse transcriptase-mediated polymerase chain reaction assay to measure the BCL3 mRNA level and found a predominant level of BCL3 expression in t(2;5)(+) ALCL; the levels of cell lines and clinical materials were comparable to or higher than that of a B-cell chronic lymphocytic leukemia carrying t(14;19)(q32;q13). Southern blotting and fluorescence in situ hybridization disclosed that the BCL3 gene copies were amplified in SU-DHL-1, whereas Karpas 299 carried 4 BCL3 gene loci. The BCL3 gene contains 2 cytosine-guanine dinucleotide (CpG) islands, and the intragenic 3' CpG was entirely demethylated in SU-DHL-1 and DEL. In contrast to HD, in which NF-kappa B was constitutively activated, ALCL cells consistently showed (p50)(2) homodimer binding activity on electrophoretic mobility shift assay. It is suggested that the high-level nuclear Bcl-3 sequesters the (p50)(2) homodimer to the nucleus, which may account for the contradictory effect of CD30 stimulation on ALCL and HD. We propose that BCL3 is overexpressed by genetic and epigenetic modifications, potentially contributing to the development of t(2;5)(+) ALCL.

Characterization of t(3;6)(q27;p21) breakpoints in B-cell non-Hodgkin's lymphoma and construction of the histone H4/BCL6 fusion gene, leading to altered expression of Bcl-6.

A recurrent translocation, t(3;6)(q27;p21), in non-Hodgkin's lymphoma results in fusion of BCL6 with a particular histone H4 gene on 6p21. We cloned five H4/BCL6 junctions from both der(3) and der(6) chromosomes. The breakpoints on H4 were distributed within the single exon or close to the terminal palindrome, and those on BCL6 were localized within or close to the translocation hypercluster. Deletions or duplications of variable numbers of nucleotides were identified at the junctions. A total of eight single nucleotide alterations were introduced into the translocation/mutation cluster of BCL6, whereas four single nucleotide substitutions were identified within a 360-bp region of H4. Thus, the somatic hypermutation mechanism is likely to target H4, resulting in a predisposition to the development of translocation with BCL6. Lymphoma cells carrying H4/BCL6 produced fusion transcripts containing both H4 and BCL6 messages; however, the cells expressed only moderate levels of BCL6 mRNA. We constructed expression plasmids that mimicked the H4/BCL6 fusion gene and transiently introduced them into COS-7 cells. H4/BCL6-transfected cells expressed markedly higher levels of Bcl-6 protein than cells transfected with a plasmid carrying BCL6 driven by its normal promoter and displayed bright nuclear staining with a characteristic punctate pattern with an anti-Bcl-6 antibody. Deletion analyses revealed that the high-level Bcl-6 expression was promoted by the H4 regulatory sequences. The levels of expression of activating transcription factor 3, prefoldin 4, and retinoblastoma-binding protein 7 significantly increased in accordance with that of BCL6, suggesting that Bcl-6 may act as a transcriptional activator. Our study suggested that t(3;6)(q27;p21) leads to BCL6 overexpression; however, the high-level BCL6 expression may not be required to maintain the malignant phenotype of lymphoma cells.

The gene for interleukin-21 receptor is the partner of BCL6 in t(3;16)(q27;p11), which is recurrently observed in diffuse large B-cell lymphoma.

BCL6 translocation affecting the chromosomal band 3q27 can involve a number of non-immunoglobulin (non-IG) gene loci as partners. We report here that the gene for interleukin-21 receptor (IL-21R) is the partner of BCL6 in t(3;16)(q27;p11) translocation. The two breakpoints on 16p11 of a lymphoma cell line YM and case no. 1012 with diffuse large B-cell lymphoma, both of which carried t(3;16), were localized within the 27-kb intron 1 of IL-21R. As a result of t(3;16), the promoter region of IL-21R was substituted for the regulatory sequences of BCL6 in the same transcriptional orientation. Reverse transcriptase-mediated polymerase chain reaction revealed chimeric mRNA consisting of two non-coding exons 1a/1b of IL-21R and coding exons of BCL6 in both lymphoma cells. Fluorescence in situ chromosomal hybridization of YM metaphase cells revealed fusion signals that contained both the BCL6 and IL-21R sequences on the der(3)t(3;16) chromosome. IL-21R was actively transcribed in YM cells, while BCL6 that was under the control of the IL-21R promoter was only moderately expressed at the mRNA and protein level. We constructed expression plasmid of BCL6 that followed the promoter sequences of IL-21R. COS-7 cells transiently transfected with the plasmid expressed high level Bcl-6 protein and displayed nuclear staining with a characteristic punctate pattern by immunofluorescence, indicating that expression of BCL6 can be enhanced by t(3;16). This study added to the list of non-IG partners of BCL6 translocations a new class of gene, i.e. cytokine receptor gene, the expression of which is closely associated with lymphoid cells.