Correlation between alterations in blood flow of malignant lymphomas after induction chemotherapies and clinical outcomes: a pilot study utilising contrast-enhanced ultrasonography for early interim evaluation of lymphoma treatment.

AIM: To clarify the utility of contrast-enhanced ultrasonography (CEUS) for interim evaluation of response to chemotherapy in lymphoma treatment. MATERIALS AND METHODS: CEUS was performed both before (day 0) and after the treatment (7 and/or 14 days), and a time-intensity curve was obtained. The patients were divided into two groups (complete remission [CR] group and non-CR group) according to the results of conventional response evaluation, and peak enhancement (PE), time to peak enhancement, perfusion index (PI), the total area under the curve during wash-in (AUC-in), and the total AUC were compared between the groups. RESULTS: Among 27 patients with various types of lymphoma, the median change ratio of PE and PI at day 7 evaluation were significantly different between the CR group and the non-CR group (0.81 versus 1.39, p=0.017 for PE and 0.92 versus 2.09, p=0.010 for PI). The change ratio of PE < 1.09 (specificity: 86%; sensitivity, 88%) and PI < 1.65 (specificity: 86%; sensitivity: 94%) distinguished CR from non-CR. Patients who achieved a PE change ratio <1.09 or a PI change ratio <1.65 had significantly better estimated progression-free survival (p<0.001). CONCLUSION: The present study demonstrated that changes in tumour perfusion parameters evaluated with CEUS at 1 week after the treatment initiation were significantly different between lymphoma patients in CR group and non-CR group. Alterations in perfusion parameters evaluated via CEUS could impact the prognosis of lymphoma patients.

Prognostic factors influencing clinical outcome of allogeneic hematopoietic stem cell transplantation following imatinib-based therapy in BCR-ABL-positive ALL.

We investigated prognostic factors for the clinical outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) following imatinib-based therapy. Among 100 adult patients who were prospectively enrolled in the JALSG Ph+ALL202 study, 97 patients obtained complete remission (CR) by imatinib-combined chemotherapy, among whom 60 underwent allo-HSCT in their first CR. The probabilities of overall survival (OS) and disease-free survival (DFS) at 3 years after HSCT were 64% (95% CI, 49-76) and 58% (95% CI, 43-70), respectively. Prognostic factor analysis revealed that the major BCR-ABL transcript was the only unfavorable predictor for OS and DFS after HSCT by both univariate (HR, 3.67 (95% CI 1.49-9.08); P=0.005 and HR, 6.25 (95% CI, 1.88-20.8); P=0.003, respectively) and multivariate analyses (HR, 3.20 (95% CI, 1.21-8.50); P=0.019 and HR, 6.92 (95% CI, 2.09-22.9); P=0.002, respectively). Minimal residual disease status at the time of HSCT had a significant influence on relapse rate (P=0.015). Further study of the BCR-ABL subtype for the clinical impact on outcome of allo-HSCT in Ph+ALL is warranted.

Pre-transplant imatinib-based therapy improves the outcome of allogeneic hematopoietic stem cell transplantation for BCR-ABL-positive acute lymphoblastic leukemia.

A high complete remission (CR) rate has been reported in newly diagnosed Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) following imatinib-based therapy. However, the overall effect of imatinib on the outcomes of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is undetermined. Between 2002 and 2005, 100 newly diagnosed adult patients with Ph+ALL were registered to a phase II study of imatinib-combined chemotherapy (Japan Adult Leukemia Study Group Ph+ALL202 study) and 97 patients achieved CR. We compared clinical outcomes of 51 patients who received allo-HSCT in their first CR (imatinib cohort) with those of 122 historical control patients in the pre-imatinib era (pre-imatinib cohort). The probability of overall survival at 3 years after allo-HSCT was 65% (95% confidence interval (CI), 49-78%) for the imatinib cohort and 44% (95% CI, 35-52%) for the pre-imatinib cohort. Multivariate analysis confirmed that this difference was statistically significant (adjusted hazard ratio, 0.44, P=0.005). Favorable outcomes of the imatinib cohort were also observed for disease-free survival (P=0.007) and relapse (P=0.002), but not for non-relapse mortality (P=0.265). Imatinib-based therapy is a potentially useful strategy for newly diagnosed patients with Ph+ALL, not only providing them more chance to receive allo-HSCT, but also improving the outcome of allo-HSCT.

Durable molecular complete remission induced by low-dose imatinib plus low-dose interferon alpha in a patient with chronic myelogenous leukaemia.

A 50-year-old male was diagnosed with chronic myelogenous leukaemia (CML) in chronic phase in March 2000. He was treated initially with hydroxyurea, administered orally. This was changed to interferon alpha (IFN) 5 million units (5 MIU) subcutaneously daily in May 2000; complete cytogenetic response was achieved 11 months later. IFN dosage was reduced to 5 MIU, alternate days, in June 2001 and a cytogenetic relapse occurred 3 months later. Since April 2002, he has received IFN 5 MIU three times weekly in combination with imatinib 200 mg/day. The Philadelphia chromosome disappeared from his peripheral blood cells in July 2002 and a complete molecular response was achieved in January 2003. Serial molecular studies between January 2004 and January 2005 showed no detectable major BCR/ABL chimeric transcript. Grade 2 neutropenia and grade 1 non-haematological adverse effects have been observed. This case report suggests the combination of low-dose imatinib and IFN would be tolerable and effective for CML patients in chronic phase.

Pure red cell aplasia and myelofibrosis in B-cell neoplasm.

We describe an unusual case of B-cell neoplasm accompanied by pure red cell aplasia (PRCA) and myelofibrosis in a 67-year-old male presenting with severe anaemia. A few unclassified, myeloperoxidase-negative blastoid cells were seen on bone marrow aspiration, and erythroid cell hypoplasia and myelofibrosis on bone marrow biopsy. An autoimmune PRCA was suspected, as serum CH50, C3 and C4 levels were consistently low. Ciclosporin was effective in treating the anaemia, but anaemia returned when the drug was discontinued. Thirteen months later, the patient was admitted with pleural effusion and ascites that contained monoclonal CD19+ CD20+ immature blast cells with a complex karyotype, thought to be neoplastic B-cells. The unclassified blastoid cells seen earlier may therefore have been from the same origin. The patient deteriorated rapidly and died. Only one case of non-Hodgkin's lymphoma with PRCA and myelofibrosis has been reported previously. We discuss the possibility that dysregulated T-cells induced by neoplastic B-cells may have given rise to concomitant PRCA and myelofibrosis.

WT1 expression level and clinical factors in multiple myeloma.

Although Wilm's Tuomor gene (WT1) was first identified as a tumor suppressor gene for Wilm's tumor, WT1 overexpression has been detected in different malignant cell types including leukemia. Increased expression of WT1 in acute leukemia is potentially used as a marker of minimal residual disease. However, the significance of the gene for multiple myeloma is still not clear. To determine the clinical relevance of WT1 expression in multiple myeloma, we examined the association of clinical parameters and WT1 expression in bone marrow for 17 newly diagnosed multiple myeloma patients. WT1 was assessed by real-time quantitative polymerase chain reaction (RQ-PCR) and calculated standardized WT1 expression level per 100 plasma cells in the bone marrow specimen as "corrected WT1". The expression of standardized WT1 and corrected WT1 in myeloma was 59 to 1,600 copies/microg RNA and 0.05 to 406.3 copies/microg RNA/100 plasma cells, respectively, lower than in leukemia. WT1 transcripts increased when clinical factors worsen, including the stage, amount of M protein, Hb, platelet count, blood urea nitrogen (BUN), creatinine, serum alkaline phosphatase (ALP), calcium, beta2-microglobulin, thymidine kinase activity (TK), and C-reactive protein (CRP). In conclusion, the expression level of WT1 could be an additional marker to the standard parameters considered in risk assessment for multiple myeloma.

Proton-number fluctuation as a signal of the QCD critical end point.

We argue that the event-by-event fluctuation of the proton number is a meaningful and promising observable for the purpose of detecting the QCD critical end point in heavy-ion collision experiments. The long range fluctuation of the order parameter induces a characteristic correlation between protons which can be measured. The proton fluctuation also manifests itself as anomalous enhancement of charge fluctuations near the end point, which might be already seen in existing data.

Role of tumor suppressor genes in the development of adult T cell leukemia/lymphoma (ATLL).

Adult T cell leukemia/lymphoma (ATLL) is one of the peripheral T cell malignant neoplasms strongly associated with human T cell leukemia virus type-I (HTLV-I). Although the viral transactivating protein Tax has been proposed to play a critical role in leukemogeneis as shown by its transforming activity in various experimental systems, additional cellular events are required for the development of ATLL. One of the genetic events in ATLL is inactivation of tumor suppressor genes. Among many candidates for tumor suppressor genes, the main genetic events have been reported to center around the cyclin-dependent kinase inhibitors ((CDKIs) p15INK4A, p16INK4B, p18INK4C, p19INK4D, p21WAF1, p27KIP1, and p57KIP2), p53 and Rb genes; all of them play a major regulatory role during G1 to S transition in the cell cycle. Acute/lymphomatous ATLL has frequent alterations of p15 (20%) and p16 (28-67%), while chronic/smoldering ATLL has fewer abnormalities of p15 (0-13%) and p16 (5-26%). Most of these changes are deletion of the genes; fewer samples have mutations. ATLL patients with deleted p15 and/or p16 genes have significantly shorter survival than those individuals with both genes preserved. Although genetic alterations of p18, p19, p21, p27 have rarely been reported, inactivation of these genes may contribute to the development of ATLL because low expression levels of these genes seem to mark ATLL. The p53 gene is mutated in 10-50% of acute/lymphomatous ATLL. Functional impairment of the p53 protein, even if the gene has wild-type sequences, has been suggested in HTLV-I infected cells. Each of these genetic events are mainly found in acute/lymphomatous ATLL, suggesting that alterations of these genes may be associated with transformation to an aggressive phenotype. The Rb tumor suppressor gene is infrequently structurally altered, but one half of ATLL cases have lost expression of this key protein. Notably, alterations of one of the CDKIs, p53 and Rb genes appear to obviate the need for inactivation of other genes in the same pathway. A novel tumor suppressor gene on chromosome 6q may also have a critical role in the pathogenesis of ATLL. Taken together, tumor suppressor genes are frequently altered in acute/lymphomatous ATLL and their alteration is probably the driving force fueling the transition from chronic/smoldering to acute/lymphomatous ATLL.

Analysis of 20-year follow-up study of LVP regimen for adult acute lymphoblastic leukemia.

In an attempt to develop a new intensive chemotherapy for adults with untreated acute lymphoblastic leukemia (ALL), 3 sequential programs were designed for 62 patients (age range, 15 to 74 years; median age, 32 years) consisting of the LVP-79 (1979-1984, 27 patients), LVP-85 (1984-1986, 14 patients), and LVP-87 (1987-1989, 21 patients) regimens. The influence of clinical and biologic characteristics on the patient outcome was also examined. L-asparaginase (L-asp), vincristine, and prednisolone, defined collectively as LVP, were administered for induction chemotherapy in all protocols. After achieving complete remission (CR), patients underwent 2 years of multi-agent consolidation, intensification, and maintenance therapy consisting of various combinations. No significant differences were noted between the 3 groups regarding CR rate or survival. In total, 47 of 62 patients (75.8%) achieved CR. The median overall survival (OS) and median CR durations were 550 days and 341 days, respectively. Overall, the estimated survival rate at 20 years was 18.1%. The disease-free survival rate at 20 years was 26.2%. According to univariate analysis, the most favorable pretreatment characteristic for achieving CR was age. A younger age (<40 years of age), platelet count >30 x 10(9)/L, having L1 morphology (French-American-British [FAB]classification subtype), female sex, and the absence of chromosomal abnormalities also helped improve survival rate. According to multivariate analysis, presence of Ph chromosome was found to be a major influencing factor for OS. Although higher doses of L-asp were administered than those used in previous studies, the adverse effect of L-asp was rarely identified. Therefore, it should be considered one of the key drugs for treatment of adult ALL. Further strategies still need to be developed to obtain better survival in adult ALL.

Comparison of statistical power between 2 * 2 allele frequency and allele positivity tables in case-control studies of complex disease genes.

In case-control studies of complex disease genes, allele frequencies or allele positivities at candidate loci or markers are compared between cases and controls. Although 2 x 2 contingency tables based on allele frequency and allele positivity are generally used to perform simple statistical tests (e.g. a comparison of two proportions and a chi2 test), little is known about the difference in power between the two tables. In this study, we investigated the number of subjects required to obtain a power of 1-beta with a significance level of alpha for the allele frequency and allele positivity tables. A large difference in the required number of subjects was found between the two tables. Allele positivity tables were suitable for the detection of susceptibility alleles showing a dominant mode of inheritance (MOI). On the other hand, allele frequency tables were suitable for the identification of susceptibility alleles showing a recessive MOI or a multiplicative MOI. In the case of an additive MOI, a suitable table was determined by combining the frequency of the susceptibility allele and the penetrance. These results imply that there are cases in which true association is detected based on one contingency table and is not detected based on another. A simulation analysis revealed that the type I error rate was not much inflated under the null hypothesis of no association, even when a statistical test was performed twice using both allele frequency and allele positivity tables. In contrast, under the alternative hypothesis, the loss of power was marked when a test was performed once using an unsuitable table. In conclusion, statistical tests should be performed using both tables, without adjustment of multiplicity, in case-control studies of complex disease genes when the study objective is exploratory.

Leukaemia inhibitory factor and interleukin 6 inhibit secretion of prolactin and growth hormone by rat pituitary MtT/SM cells.

The rat pituitary cell line, MtT/SM, has the characteristics of somatomammotrophs. The cells secrete both prolactin (PRL) and growth hormone (GH). We examined the effects of cytokines such as leukaemia inhibitory factor (LIF), interleukin 6 (IL-6), oncostatin M and interleukin 11 on the secretion of these hormones by the cells. These cytokines stimulate proliferation of the cells and inhibit the secretion of PRL by 70-80% and that of GH by 50%. They induce tyrosine phosphorylation of STAT3 in the cells. The cells containing PRL or GH decreased at 48 h after treatment of the cells with LIF or IL-6. These results suggest that the LIF/IL-6 family of cytokines inhibits the functions of mammotrophs and somatotrophs in the pituitary gland.

[Evaluation of hematopoiesis by granulocyte elastase after hematopoietic stem cell transplantation].

To evaluate hematopoietic restoration after hematopoietic stem cell transplantation (HSCT), we sequentially monitored the post-HSCT level of granulocyte elastase (GE), a sensitive parameter of qualitative and quantitative changes in granulopoiesis. We compared it with routinely used hematopoietic recovery indices, such as leukocyte and reticulocyte count. We also compared levels of GE in the clinical administration of different colony stimulating factors (granulocyte-colony stimulating factor (G-CSF) versus macrophage-colony stimulating factor (M-CSF) and in different types of hematopoietic stem cell transplantation (allogeneic bone marrow transplantation (BMT) versus autologus peripheral blood stem cell transplantation (PBSCT)). Days to first increase of GE after HSCT were 3.0 and 2.3 days earlier than increase of leukocytes in allo-BMT and auto-PBSCT, respectively. Recovery of highly fluorescent reticulocytes and monocytes were later than recovery of GE. These results indicated that granulopoiesis after transplantation started before the increase in peripheral leucocyte count, and that GE was the earliest indicator of hematopoietic recovery. On the basis of GE level, M-CSF had the same stimulating effect on granulopoiesis as G-CSF. The nadir of GE in PBSCT was significantly higher than that in BMT, indicating continuous granulopoiesis in PBSCT. From these results, we concluded that measurements of GE can be used for the clinical evaluation of myelosuppression by different conditioning regimens as well as of granulopoiesis induced by various cytokines.

[Analysis of pulmonary function in leukemia patients after bone marrow transplantation: effects of prior chemotherapy].

To evaluate the effects of prior chemotherapy on pulmonary function after bone marrow transplantation(BMT), pulmonary function tests were performed prior to and after BMT on 7 acute leukemia (AL) and 13 chronic myelogenous leukemia (CML) patients given with CY (60 mg/kg x 2 days), total body irradiation (3 Gy x 4 days, 10 cGy/min), and CyA plus short-term MTX. No patient had graft-versus-host disease or lung complications. Pulmonary function after BMT did not deteriorate in the AL patients; however, both %Vital Capacity(%VC) and DL/VA decreased significantly in the CML patients (%VC before BMT: 112.1 +/- 11.5%, after BMT: 93.7 +/- 9.4%; DL/VA before BMT: 79.2 +/- 14.6%, after BMT: 54.1 +/- 10.6%). Although prior regimens of busulfan (BU) or interferon (IFN) were equal risk factors for decreased %VC after BMT, decreases in DV/VA were more significant in CML patients who received IFN. CML patients, especially those who have received BU or IFN, should be carefully monitored for pulmonary function to prevent respiratory failure after BMT.

Mutations of the E2F4 gene in hematological malignancies having microsatellite instability.

Mutations of coding repeats within the E2F4, TGF-betaRII, BAX, IGFIIR, and hMSH3 are critical targets of microsatellite instability (MSI) in many kinds of cancers. We analyzed 9 childhood acute lymphoblastic leukemia (ALL) samples, 5 acute myelocytic leukemia (AML) samples, and 10 adult T-cell leukemia (ATL) samples having MSI to determine whether they had mutations of the E2F4, TGF-betaRII, BAX, IGFIIR, and hMSH3 genes. Frameshift mutations were found at trinucleotide repeats within a coding exon of the E2F4 gene in 2 of 10 (20%) ATL samples and 1 of 9 (11%) childhood ALL samples. No mutations were found in the TGF-betaRII, BAX, IGFIIR, and hMSH3 genes. E2F4 is a transcription factor that influences the cell-cycle progression. These results suggest that mutations of the E2F4 gene, presumably caused by an abnormality of one of the DNA repair genes, may play an important role in development of ATL and childhood ALL. (Blood. 2000;95:1509-1510)

Possible association of human leucocyte antigen DR1 with delayed sleep phase syndrome.

The study investigated the human leucocyte antigen (HLA), types A, B and DR, of 42 patients with delayed sleep phase syndrome (DSPS) and compared the frequencies of the antigens with those in 117 healthy controls. The comparison revealed that the gene frequencies and positivities of HLA-A, -B and -DR, except for DR1, had no significant differences between the patients and controls. The frequency of HLA-DR1 was increased in the DSPS patients as compared with that in the healthy controls (P = 0.0069 in positivity). Although the corrected P-value (0.069) for multiple comparisons almost reached the significance level, the results indicated a possible association of the HLA-DR1 antigen with DSPS. This study suggests that there are genetic predispositions to DSPS.

HLA genes and haplotypes in Ryukyuans suggest recent gene flow to the Okinawa Islands.

Polymorphism of HLA genes was investigated in a population sample of Ryukyuans living on the main island of Okinawa (n = 197), in the southwestern islands of Japan. Serological typing was applied to class I loci (HLA-A, -B, and -C) and to HLA-DRB1; nucleotide sequence-level typing was performed using PCR microtiter plate hybridization and PCR single-strand conformation polymorphism methods. Ryukyuans showed a higher frequency of DRB1*0405 and lower frequencies of DRB1*1502 and DRB1*1302 compared with Hondo Japanese living on main islands. Principal components and phylogenetic analyses of 12 East Asian populations, including Ryukyuans, were performed based on the allele frequencies of HLA-A, -B, and -DRB1. In the principal components analysis 3 Japanese populations (Ryukyuans, Hondo Japanese, and Ainu) formed a cluster and showed the highest affinity to 2 Korean populations. In the phylogenetic tree Ryukyuans and Ainu were neighbors, but the genetic distance between them was larger than the distances between Ryukyuans and Hondo Japanese and between Ryukyuans and Korean populations. The geographic cline of the predominant haplotype in Ryukyuans, A*24-B*54-DRB1*0405, suggests that an ancestral population possessing A*24-B*54-DRB1*0405 moved into the Okinawa Islands after the divergence of Ryukyuans from the Ainu. Such a recent gene flow, probably from South China to the Okinawa Islands, is considered the major cause of difference in genetic characteristics between Ryukyuans and the Ainu.

Identification of the gene variations in human CD22.

CD22, a member of the immunoglobulin superfamily, is a B-cell transmembrane glycoprotein that acts as an accessory-signaling component of the B-cell antigen receptor (BCR). Recent evidence indicating the role of CD22 as a negative regulator of BCR signal transduction prompted us to test the possibility that genetic variations of human CD22 may be associated with autoimmune diseases. In this study, variation screening of the entire CD22 coding region was performed, and possible association with rheumatic diseases was tested, using the genomic DNA from 207 healthy Japanese individuals, 68 patients with systemic lupus erythematosus (SLE), and 119 patients with rheumatoid arthritis (RA). Through the variation screening, seven non-synonymous and four synonymous substitutions were identified. In addition, single base substitutions were found in two introns flanking exon-intron junctions. Among these variations, Q152E substitution within the second extracellular domain was observed with a marginally higher frequency in the patients with SLE (3/68, 4.4%) than that in healthy individuals (1/207, 0.5%) (P=0.048. SLE vs healthy individuals), although this difference was no longer significant after correction for the number of comparisons (Pc=0.62). No significant association was observed between any of the variations and RA. These findings indicate that a number of genetic variants are present in CD22, and suggest that CD22 could be considered a candidate for the susceptibility genes to autoimmune diseases.

Ovarian cancer: loss of heterozygosity frequently occurs in the ATM gene, but structural alterations do not occur in this gene.

Ataxia-telangiectasia is a multisystem recessive disease characterized clinically by cerebellar ataxia, oculocutaneous telangiectasias, immunodeficiency, sensitivity to radiomimetic agents and an increased predisposition to cancer. This pleiotropic disorder is caused by mutations in the ATM gene, which is located at the human chromosomal region 11q23. Loss of heterozygosity (LOH) at 11q22-q23 is a frequent event in ovarian cancer, suggesting the presence of a tumor suppressor gene in this region. We have found that LOH in the ATM gene occurred in 44% of informative cases in a series of 22 primary ovarian tumors. LOH of this region occurred at the same frequency during the advanced stages (III-IV; 3/9, 33%) as in the early stages (I-II; 4/13, 31%) of ovarian cancer. To investigate the role of ATM in ovarian cancer, we used a PCR-based single-strand conformation polymorphism assay for mutation detection of the entire coding sequence of the ATM gene (65 exons) in 22 ovarian tumors. No somatic alterations of the ATM gene were found in these ovarian cancer samples including those with LOH present in the ATM gene. Our study has identified a region (11q23) which probably contains a frequently altered tumor suppressor gene in ovarian cancer, and this gene does not appear to involve the coding sequences of the ATM gene.

Detailed deletion mapping of the long arm of chromosome 6 in adult T-cell leukemia.

Previously, we have found that the loss of heterozygosity (LOH) was frequently observed on chromosome 6q in acute/lymphoma-type adult T-cell leukemia (ATL), suggesting a putative tumor-suppressor gene for ATL may be present on chromosome 6q. To further define a region containing this gene, we performed fine-scale deletional mapping of chromosome 6q in 22 acute/lymphomatous ATL samples using 24 highly informative microsatellite markers. LOH was found in 9 samples (40. 9%) at 1 or more of the loci examined. Of the 9 samples, 8 shared the same smallest commonly deleted region flanked by D6S1652 and D6S1644 (6q15-21). The genetic distance between these two loci is approximately 4 cM. These results suggest that a putative tumor-suppressor gene on chromosome 6q15-21 probably plays a very important role in the evolution of acute/lymphomatous ATL. Our map provides key information toward cloning the gene.