Development and evaluation of a rapid one-step high sensitivity real-time quantitative PCR system for minor BCR-ABL (e1a2) test in Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL).

OBJECTIVE: Minimal residual disease assessment of BCR-ABL messenger ribonucleic acid levels is crucial in Philadelphia chromosome-positive acute lymphoblastic leukemia for prognosis and treatment planning. However, accurately quantifying minor BCR-ABL transcripts, which comprise 70% of Philadelphia chromosome-positive acute lymphoblastic leukemia cases, lacks a national-approved method. METHODS: We developed the "Otsuka" minor BCR-ABLmessenger ribonucleic acid assay kit with exceptional precision (0.00151%). Minor BCR-ABL messenger ribonucleic acid levels were analyzed in 175 adults, 36 children with acute lymphoblastic leukemia and 25 healthy individuals to evaluate the kit's performance. RESULTS: The "Otsuka" kit showed high concordance with a commonly used chimeric gene screening method, indicating reliable detection of positive cases. Quantitative results demonstrated a robust correlation with both a laboratory-developed test and a diagnostic research product. The "Otsuka" kit performs comparably or even surpass to conventional products, providing valuable insights into Philadelphia chromosome-positive acute lymphoblastic leukemia pathology. CONCLUSIONS: The 'Otsuka" minor BCR-ABL messenger ribonucleic acid assay kit exhibits excellent performance in quantifying minor BCR-ABL transcripts in Philadelphia chromosome-positive acute lymphoblastic leukemia patients. Our results align well with established screening methods and show a strong correlation with laboratory-developed tests and diagnostic research products. The "Otsuka" kit holds great promise as a valuable tool for understanding Philadelphia chromosome-positive acute lymphoblastic leukemia pathology and guiding effective treatment strategies.

Rapid reduction in BCR-ABL1 transcript predicts deep molecular response in dasatinib-treated chronic-phase chronic myeloid leukaemia patients.

OBJECTIVES: We conducted a phase-II study to evaluate the efficacy and safety of dasatinib in patients newly diagnosed with chronic-phase chronic myeloid leukaemia (CML-CP) in Japan (IMIDAS PART 2 study). METHODS: Seventy-nine patients were administered 100 mg dasatinib once daily. We examined pretreatment and post-treatment influences of various factors. The BCR-ABL1 international scale (IS), halving time (HT) and reduction rate of BCR-ABL1 transcript within the initial 1 or 3 months of therapy (RR-BCR-ABL11m,3m ) were the post-treatment factors investigated to predict the molecular response. RESULTS: The estimated major molecular response (MMR), molecular response 4.0 (MR4.0) and molecular response 4.5 (MR4.5) rates were 77.2%, 49.4% and 35.4%, respectively, at 12 months. Grade 3/4 non-haematologic adverse events were infrequent. Multivariate analysis showed that age >65 years was significantly correlated with MR4.0 and MR4.5 (deep molecular response: DMR) at 12 months. All post-treatment factors at 3 months predicted DMR by univariate analysis. However, RR-BCR-ABL13m was the only significant landmark for predicting DMR by multivariate analysis. CONCLUSIONS: Primary treatment of CML-CP with dasatinib enabled early achievement of MMR and DMR, particularly in elderly patients, with high safety. Furthermore, RR-BCR-ABL13m was found to be a more useful predictor of DMR than HT-BCR-ABL1 and BCR-ABL1 IS.

Successful haploidentical hematopoietic stem cell transplantation for paroxysmal nocturnal hemoglobinuria with severe pancytopenia developed after long-term aplastic anemia treatment.

Haploidentical hematopoietic stem cell transplantation (HSCT) with posttransplant cyclophosphamide is an alternative treatment for aplastic anemia-paroxysmal nocturnal hemoglobinuria (PNH) syndrome with poor prognostic factors. Ravulizumab treatment for PNH before HSCT might have a beneficial effect.

Report of phase I and II trials of melphalan, prednisolone, and thalidomide triplet combination therapy versus melphalan and prednisolone doublet combination therapy in Japanese patients with newly diagnosed multiple myeloma ineligible for autologous stem cell transplantation.

We conducted a phase I study to determine the recommended dose of thalidomide combined with melphalan plus prednisolone (MPT) and a phase II study evaluating the efficacy and safety of this MPT regimen in transplant-ineligible Japanese patients with untreated multiple myeloma. The recommended dose was determined to be 100 mg/day in the phase I study. In the phase II, randomized, double-blind, parallel-group study, patients were allocated to either MPT (n = 52) or MP (n = 51), with 21 and 29 patients completing the study, respectively. Overall response rate, the primary endpoint, was significantly higher in the MPT [40.4% (21/52 patients), 95% confidence interval (CI) 27.0-54.9%] than in the MP [19.6% (10/51 patients), 95% CI 9.8-33.1%] group (P = 0.022). Time to response was also significantly shorter in the MPT group. Incidences of hematological toxicities were similar in the two groups, suggesting that addition of thalidomide did not increase hematological toxicity. Although incidences of some non-hematological toxicities tended to be higher in the MPT group, the low incidence of ≥ Grade 3 toxicities suggests that MPT therapy was well tolerated. These results support the safety and efficacy of MPT therapy in untreated Japanese multiple myeloma patients.

Rapid diagnosis of mixed phenotype acute leukemia after identifying a blood histogram abnormality.

A 38-year-old woman was suffering from back, right arm, and ankle joint pain, and visited our emergency department. Upon admission, the white blood cell (WBC) count was high (11,700/µL), and low numbers of red blood cells (2.21 × 106/µL) and platelets (PLTs) (42,000/µL) were observed. A PLT histogram showed an abnormally shaped peak at around 20-30 fL, suggesting the presence of giant PLTs or PLT aggregation. The WBC histogram showed abnormal elevation at 35 fL and around 100 fL, suggesting abnormal cells including nucleated red blood cells. A peripheral blood smear was prepared, and morphology was examined. As a result, blasts (4%) including many orthochromatic erythroblasts (48/100 WBCs) were observed. Acute leukemia was suspected, and the patient was transferred the next day to a hospital with a hematology department. Bone marrow aspiration revealed that 99% of cells were blasts positive for B lymphoid lineage markers and myeloperoxidase. The patient was diagnosed with mixed phenotype lineage acute leukemia, treated immediately, and achieved remission. Thus, careful observation of histogram abnormalities of an automatic blood cell analyzer is important for rapid diagnosis of acute leukemia.

Evaluation of the safety and efficacy of recombinant soluble thrombomodulin for patients with disseminated intravascular coagulation associated with acute leukemia: multicenter prospective study by the Tohoku Hematology Forum.

It has been suggested that use of recombinant soluble thrombomodulin (rTM) is superior to conventional drugs in treatment of disseminated intravascular coagulation (DIC) complicating acute leukemia. However, its safety and efficacy have not been fully examined in prospective studies. Here, we performed a multicenter prospective study to examine outcomes of rTM treatment for DIC in patients with acute leukemia. Of 33 patients registered in this study, 13 had acute myeloid leukemia (AML), three had acute lymphoblastic leukemia (ALL), and 17 had acute promyelocytic leukemia (APL). The cumulative rates of DIC resolution at day 7 and day 35 were 56 and 81% in AML/ALL and 53 and 77% in APL, respectively. The median time from the initiation of rTM to DIC resolution was 4 days in AML/ALL and 6 days in APL patients. Adverse events related to hemorrhage occurred in two AML/ALL patients (13%) and three APL patients (18%). Of these, one AML/ALL patient died with intracranial hemorrhage, and two APL patients died with intracranial hemorrhage and pulmonary hemorrhage. These results suggest that rTM may improve the survival of acute leukemia patients with DIC by inhibiting early death related to hemorrhagic events, as reported previously.

Switching to nilotinib in patients with chronic myeloid leukemia in chronic phase with molecular suboptimal response to frontline imatinib: SENSOR final results and BIM polymorphism substudy.

Optimal management of patients with chronic myeloid leukemia in chronic phase with suboptimal molecular response (MR) to frontline imatinib is undefined. We report final results from SENSOR, which evaluated efficacy/safety of nilotinib in this setting. A substudy assessed whether BIM polymorphisms impacted response to nilotinib. In this single-arm, multicenter study, Japanese patients with suboptimal MR per European LeukemiaNet 2009 criteria (complete cytogenetic response, but not major MR [MMR]) after ≥18 months of frontline imatinib received nilotinib 400mg twice daily for 24 months. MR, BCR-ABL1 mutations/variants, and BIM polymorphisms were evaluated in a central laboratory. Primary endpoint was the MMR rate at 12 months (null hypothesis of 40%). Of 45 patients (median exposure, 22.08 months), 39 completed the study and six discontinued. At 12 and 24 months, 51.1% (95% CI, 35.8%-66.3%) and 66.7% (95% CI, 51.0%-80.0%) achieved MMR, respectively. Cumulative MMR incidence by 24 months was 75.6%. Of 40 patients analyzed, 10 of 12 (83.3%) with and 17 of 28 (60.7%) without BIM polymorphisms achieved MMR at 24 months. The safety profile was manageable with dose reductions and interruptions. Nilotinib provided clinical benefit for patients with suboptimal response to imatinib, and BIM polymorphisms did not influence MMR achievement. ClinicalTrials.gov: NCT01043874.

[Second transplant from the same donor without conditioning for bone marrow aplasia after non-myeloablative hematopoietic stem cell transplantation for chronic myeloid leukemia].

We describe a 56-year-old woman with chronic myeloid leukemia (CML) and a past history of stroke, who underwent nonmyeloablative hematopoietic stem cell transplantation (NST) with conditioning consisting of fludarabine and cyclophosphamide. The regimen related toxicity was minimal and patient did not require transfusions of any blood products nor did she have any infections after the NST Since mixed chimerism was observed in both lymphocytes (70% were donor type) and granulocytes (none were donor type) at 56 days after NST, donor lymphocyte infusion (DLI) was performed on day 68 and then immunosuppressant therapy was discontinued. DLI resulted in graft versus leukemia (GVL) effect, causing pancytopenia and bone marrow aplasia. A second hematopoietic stem cell transplantation was performed without conditioning on day 157, and complete donor type hematopoiesis and molecular remission of CML were achieved. Although engraftment of donor hematopoietic stem cells was not obtained after the first transplantation, donor lymphocytes were engrafted by nonmyeloablative conditioning and immunosuppression. That is, the same result might have been achieved even if the patient had received transfusion of only donor lymphocyte subsets in the first step. Based on this case report, a potential cell therapy is proposed composed of the preceding donor lymphocyte infusion alone, which induces GVL effects, and subsequent donor hematopoietic stem cell transplantation.

Diffuse large B cell lymphoma of the cranial vault: two case reports.

Malignant lymphoma of the cranial vault is a rare entity and the tumor growth patterns are not well understood. Here we report two cases of malignant lymphoma involving the scalp and epidural space with slight changes in the intervening skull. A 63-year-old woman presented with a scalp mass in her right frontal area. Computed tomography (CT) and magnetic resonance (MR) imaging demonstrated mass lesions in the scalp and epidural space with slight osteolytic changes in the intervening skull. She underwent resection of the lesions. A 53-year-old man presented with a mass in his right frontal area. CT and MR imaging showed mass lesions in the scalp and epidural space without changes in the skull. He underwent resection of the lesions. The histopathological diagnosis was diffuse large B cell lymphoma in both cases. Microscopic examination of the intervening skull found that the bone marrow was diffusely replaced by lymphoma cells, and lymphoma cells extended to the extra- or intra-cranial space along the emissary veins without destruction of the cortical or trabecular bone. These histopathological findings explain the radiological findings and provide the clues to elucidate the mechanism of extension of cranial vault lymphoma.

Primary marginal zone lymphoma of the thymus accompanied by chromosomal anomaly 46,X,dup(X)(p11p22).

We report a case of primary marginal zone lymphoma in the thymus of a 34-year-old woman. She was initially suspected of having a mediastinal plasmacytoma because of the presence of dominantly proliferating plasmacytic cells in a small fragment obtained by thoracoscopic biopsy, and an elevated level of serum monoclonal IgA. However, histology of the tissue obtained by a subsequent open surgical biopsy revealed diffuse proliferation of atypical monocytoid B-lymphocyte-like cells, which showed prominent plasmacytic differentiation and a close association with thymic epithelial cells consistent with the histology of a marginal zone lymphoma of the thymus. These lymphoma cells were positive for CD19, CD20, IgA, and kappa, and negative for CD5, CD10, and other T/NK-cell and myelomonocyte antigens. Both G-banded and spectral karyotyping analyses revealed the lymphoma cells carried a chromosomal anomaly, 46,X,dup(X)(p11p22). Although large cell type B-cell lymphoma in the thymus (mediastinal diffuse large B-cell lymphoma), which is categorized as a definite subtype in revised European-American classification of lymphoid neoplasms and the new World Health Organization classification, is not infrequent, primary marginal zone lymphoma of the thymus is extremely rare. To our knowledge, this is the first case report of primary marginal zone lymphoma of the thymus with a detailed chromosomal analysis.

Cardiac surgery in a patient with multiple myeloma combined with renal amyloidosis.

We present a case of a 62-year-old woman who underwent mitral valve and aortic valve replacement owing to infective endocarditis. Previously, the patient had been diagnosed with renal amyloidosis and multiple myeloma. She underwent chemotherapy and autologous peripheral blood stem cell transplantation and has achieved nearly complete remission. The patient's postoperative course was almost uneventful, and she was discharged on the 22nd postoperative day. This is the first case report about cardiac surgery for the patient with multiple myeloma combined with renal amyloidosis.

Altered expression of retinoblastoma protein-interacting zinc finger gene, RIZ, in human leukaemia.

The retinoblastoma protein-interacting zinc finger gene (RIZ), a member of the nuclear protein methyltransferase superfamily, is characterized by the presence of the N-terminal PR domain. The RIZ gene encodes for two proteins, RIZ1 and RIZ2. While RIZ1 contains the PR (PRDI-BF1 and RIZ homologous) domain, RIZ2 lacks it. RIZ gene expression is altered in a variety of human cancers and RIZ1 is now considered to be a candidate tumour suppressor. To investigate the role of RIZ in leukaemogenesis, we analysed the differential expression of RIZ1 and RIZ2 by quantitative real-time reverse-transcription polymerase chain reaction assay. Our results showed that the expression of RIZ1 was significantly decreased in leukaemia cell lines (14 out of 17, 82%) and in patients with acute myeloblastic leukaemia (eight out of 14, 57%). In contrast, RIZ2 expression was increased in patients with acute lymphoblastic leukaemia (eight out of 11, 73%), compared with normal bone marrow cells. These findings indicate that suppression of RIZ1 expression or enhancement of RIZ2 expression may have an important role in leukaemogenesis.

Nucleotide alteration of retinoblastoma protein-interacting zinc finger gene, RIZ, in human leukemia.

The retinoblastoma protein-interacting zinc finger gene (RIZ) is a zinc-finger type DNA binding protein and is postulated as a member of the nuclear protein-methyltransferase superfamily. RIZ gene encodes for two proteins, RIZ1 and RIZ2. While RIZ1 contains the N-terminal PR (PRDI-BF1 and RIZ homologous)-domain, RIZ2 lacks it. RIZ1 is now considered as a tumor suppressor. We analyzed nucleotide alteration of RIZ gene in human leukemia. The results revealed a single nucleotide polymorphism (SNP), T1704 to A, near the conserved Rb-binding domain, leading to an amino acid change, Asp283 to Glu. Interestingly, 17 of 21 leukemia cell lines are homozygous for the T1704 allele whereas only 2 of 20 normal subjects are homozygous for the allele. In addition, one base pair deletion in the poly (A)9 tract in the coding region near the C-terminal zinc-fingers was identified, resulting in frameshift, in 1 out of 17 leukemia cell lines, but no mutation in samples from 15 patients with acute lymphoblastic leukemia (ALL) and 6 patients with adult T cell leukemia (ATL). In the PR or SH3 (src homology 3) domain of the RIZ gene, no mutation was found. These findings suggest that RIZ may be a possible target of structural alteration leading to leukemia.

Quantitative analysis of a MDR1 transcript for prediction of drug resistance in acute leukemia.

BACKGROUND: Assessing the drug resistance of leukemic cells is important for treatment of leukemia. We developed a quantitative reverse transcription (RT)-PCR method for multidrug resistance 1 (MDR1) and multidrug resistance-related protein 1 (MRP1) transcripts to evaluate drug resistance, and applied it to clinical samples. METHODS: The cutoffs for copy numbers of MDR1 and MRP1 transcripts were defined based on copy numbers in healthy bone marrow mononuclear cells. To confirm that the cutoffs reflected biological resistance, we established vincristine (VCR)-resistant K562 sublines that showed various degrees of drug resistance and examined the correlation between the copy numbers of these transcripts and the biological resistance of these clones. In addition, we compared the sensitivity and specificity of quantitative RT-PCR to a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometric (FCM) analysis. RESULTS: The defined cutoff for copy numbers of MDR1 transcripts corresponded with the degree of biological resistance of VCR-resistant K562 sublines. Clinical study revealed that the concentrations of MDR1 mRNA in all relapsed patients with acute myelogenous leukemia (AML) were above the cutoff. Moreover, both AML and acute lymphoblastic leukemia patients with high MDR1 mRNA expression at diagnosis tended to show a low remission rate and short remission periods. No association was observed between the amounts of MRP1 transcripts and clinical outcomes. The specificity and sensitivity of quantitative RT-PCR for MDR1 were superior to the MTT assay and FCM analysis. CONCLUSION: These results suggest the efficacy of this quantitative analysis of MDR1 transcripts for the prediction of clinical drug resistance in acute leukemia.

T-cell variant of classical Hodgkin's lymphoma with nodal and cutaneous manifestations demonstrated by single-cell polymerase chain reaction.

The atypical cells of CD30(+) cutaneous lymphoproliferative disorders (CD30CLD) are commonly of T-cell origin and frequently have a similar morphology as Hodgkin or Reed-Sternberg cells of Hodgkin's lymphoma (HL). HL is one of the tumors associated with CD30CLD. Although most studies support a B-cell derivation of the tumor cells in HL, recently a few cases of classical HL with T-cell genotype have been reported. We report a patient who presented with CD30CLD whose lymph nodes showed classical HL of mixed cellularity subtype at presentation. By single-cell PCR, the same clonal gene rearrangements of the T cell receptor-beta gene locus could be assigned to the CD30(+) and CD15(+) cells of both skin and lymph node. In a lymph node biopsy specimen taken in relapse after several courses of chemotherapy, the CD30(+) tumor cells were abundant. The T cell-derived tumor cells displayed aberrant expression of the Pax-5 gene in all specimens. A common clonal origin of both CD30CLD and HL of the lymph node in the patient presented here suggests that HL with T-cell genotype exists in association with CD30CLD as well as in sporadic cases and may share clonal origin with the skin tumor.

Detection of clone-specific immunoglobulin heavy chain genes in the bone marrow of B-cell-lineage lymphoma after treatment.

In order to determine the appropriate treatment of malignant lymphoma, it is important to know the degree to which extra-nodal invasion of lymphoma cells has occurred. We amplified complementarity-determining region (CDR) III genes in 64% of lymph node samples at the onset or relapse of B-cell-lineage non-Hodgkin's lymphoma (NHL) in 22 patients. By using a clone-specific CDR III probe in each patient, we were able to detect minimal residual disease (MRD) of lymphoma cells in the bone marrow and/or blood in 9 out of 14 cases (64.2%) at the onset of the disease or relapse, whereas abnormal cells in the bone marrow and/or blood were identified by routine morphological analysis in only 4 out of 22 cases (18.2%). This indicates that extranodal invasion of malignant cells may be common in patients with NHL. In some cases, the clone-specific CDR III gene was still expressed in the samples of bone marrow and/or peripheral blood even after chemotherapy, when other markers associated with NHL were no longer expressed. Five out of six cases in this group had a worse outcome associated with NHL. On the other hand, most of the cases whose clone-specific CDR III gene was no longer expressed in the bone marrow and/or in circulation after treatment had a relatively fair prognosis. These results indicate that the detection at molecular level of MRD in extranodal organs may prove useful as a predictor of prognosis for NHL.

The absolute number of peripheral blood CD34+ cells predicts a timing for apheresis and progenitor cell yield in patients with hematologic malignancies and solid tumors.

Retrospective analysis was conducted in 51 autologous peripheral blood progenitor cell (PBPC) collections using the Spectra AutoPBSC System from patients with hematologic malignancies and solid tumors to study the predictive value of CD34+ cell counts in the peripheral blood for the yield of CD34+ cells in the apheresis product. The correlation coefficients for CD34+ cells microL(-1) of peripheral blood with CD34+ cell yield (x 10(6) kg(-1) of body weight and x 10(5) kg(-1) of body weight L(-1) of blood processed) were 0.903 and 0.778 (n=51 collections), respectively. Products collected from patients with CD34+ cell counts below 15 microL(-1) in the peripheral blood contained a median of 0.49 x 10(6) CD34+ cells kg(-1) (range: 0.05-2.55), whereas those with CD34+ cell counts more than 15 microL(-1) contained a median of 3.72 x 10(6) CD34+ cells kg(-1) (range: 1.06-37.57). From these results, a number of at least 15 CD34+ cells microL(-1) in the peripheral blood ensured a minimum yield of 1 x 10(6) CD34+ cells kg(-1) as obtained by a single apheresis procedure. The number of CD34+ cells in the peripheral blood can be used as a good predictor for timing of apheresis and estimating PBPC yield. With regard to our results, apheresis with a possibly poor efficiency should be avoided because the collection procedure is time-consuming and expensive.