Kohji Ezaki

A new type of plasma prekallikrein deficiency associated with homozygosity for Gly104Arg and Asn124Ser in apple domain 2 of the heavy-chain region.

Three Japanese patients demonstrated plasma prekallikrein (PK) deficiency (PKD) after an examination of the proband family line named ‘PKD Seki’. A molecular genetic analysis of these PK genes showed homozygous amino acid substitutions Gly104Arg and Asn124Ser in exon 5, which encodes part of the apple domain 2 (A2) of the heavy chain. This is the first case involving substitutions in the heavy chain of the PK gene which affected blood coagulation. Because the apple domains of PK bind to the C‐terminal domain (D6H) of high‐molecular weight kininogen (HMWK), the two substitutions in A2 may therefore be the main cause of PKD Seki. We subsequently investigated the effects of amino acid substitutions in A2 to elucidate the binding activity of PK to HMWK using mutant A2 proteins produced in Escherichia coli. We clearly demonstrated that the Gly104Arg‐substitution with the Asn124Ser‐substitution in A2 reduce the binding activity of A2 to HMWK. PKD Seki is the first significant case to show the amino acid substitutions in the A2 affecting the binding capacity of PK with HMWK. Our findings therefore suggest that the binding of PK to HMWK may play a crucial role in the first step of blood coagulation.

Weekly Administration of Epoetin Beta for Chemotherapy-induced Anemia in Cancer Patients: Results of a Multicenter, Phase III, Randomized, Double-blind, Placebo-controlled Study

OBJECTIVE The efficacy and safety of weekly administration of epoetin beta (EPO) for chemotherapy-induced anemia (CIA) patients was evaluated. METHODS One hundred and twenty-two patients with lung cancer or malignant lymphoma undergoing chemotherapy were randomized to the EPO 36 000 IU group or the placebo group. Hematological response and red blood cell (RBC) transfusion requirement were assessed. Quality of life (QOL) was assessed using the Functional Assessment of Cancer Therapy-Anemia (FACT-An) questionnaire. RESULTS Mean change in hemoglobin level with EPO increased significantly over placebo (1.4 +/- 1.9 g/dl versus -0.8 +/- 1.5 g/dl; P < 0.001). The proportion of patients with change in hemoglobin level > or =2.0 g/dl was higher for EPO than those for placebo (P < 0.001). After 4 weeks of administration, the proportion of RBC transfusion or hemoglobin level <8.0 g/dl was significantly lower for EPO than those for placebo (P = 0.046). The changes in the FACT-An total Fatigue Subscale Score (FSS) were less deteriorated with EPO than those with placebo. Progressive disease (PD) did not influence the change in hemoglobin level but there was less decrease in FSS in non-PD patients. No significant differences in adverse events were observed. Thrombovascular events and pure red cell aplasia related to EPO were not observed. Retrospective analysis of survival showing the hazard ratio of EPO to placebo was 0.94. CONCLUSION Weekly administration of EPO 36 000 IU significantly increased hemoglobin level and ameliorated the decline of QOL in CIA patients over the 8-week administration period.

A combination trial of human lymphoblastoid interferon and bestrabucil (KM2210) for adult T-cell leukemia-lymphoma.

Both human lymphoblastoid interferon (HLBI) and bestrabucil, the conjugate of chlorambucil and β‐estradiol, have antitumor activity against adult T‐cell leukemia‐lymphoma (ATLL). Because an in vitro study showed that these two agents combined had a synergistic antiproliferative effect on MOLT‐4 and WI‐38VA13 cell lines, the authors evaluated the clinical efficacy of this combination in a pilot study with a poor‐risk group of ATLL patients. The patients were treated daily with 6 × 106 IU of HLBI subcutaneously and 100 mg of bestrabucil orally. In patients with lymphoma‐type ATLL or hypercalcemia, prednisolone also was given daily. Of 12 patients suitable for evaluation, nine had partial responses, one had a minor response, and two had no response. All five patients with skin infiltration and both patients with hypercalcemia responded. A history of prior chemotherapy did not affect the response rate. The time to clinical response was 3 to 16 days (median, 11 days) after initiation of treatment. The response duration was 4 to 108+ weeks (median, 9 weeks), but all patients except one relapsed, even during continuing treatment. No serious side effects were observed. Although the response rate with this combination treatment was high, the response duration was short, and other treatments would have to be added to achieve control of this aggressive disease.

Proliferative effects of several hematopoietic growth factors on acute myelogenous leukemia cells and correlation with treatment outcome

The response of human acute myelogenous leukemia (AML) cells to four different hematopoietic growth factors (granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin-1 β (IL-1β), interleukin-3 (IL-3), and stem cell factor (SCF)) and the relationship of the proliferative response of the AML cells to treatment outcome were studied. Proliferative responses were analyzed in 79 patients with de novo AML and 19 patients with AML arising from myelodysplastic syndrome (MDS). In de novo AML, a positive proliferative response (stimulation index >2) was seen in 65 to 75% of cases. AML cells arising from MDS had a much higher incidence of proliferative response to each growth factor (79 to 90%) and a much higher level of 3H-TdR incorporation. The relationship to treatment outcome was evaluated in 79 patients with de novo AML. The patients whose leukemic cells had a positive proliferative response to any growth factor, especially IL-3 and SCF, had a poorer outcome, ie a lower complete remission (CR) rate, shorter CR duration, and shorter survival. The outcome was particularly poor in patients whose leukemic cells had proliferative responses to all four or any of the growth factors, compared to patients whose leukemic cells had no response. This increased response may be a marker of poor prognosis in patients with AML.

Alternating non‐cross‐resistant chemotherapy for non‐Hodgkin's lymphoma of intermediate‐grade and high‐grade malignancy. A pilot study

Thirty‐two patients with advanced non‐Hodgkins lymphoma (NHL) with aggressive histologic findings were treated with cyclophosphamide, doxorubicin, methotrexate with leucovorin rescue, bleomycin, vincristine, etoposide, ifosfamide, and prednisolone (CAMBO‐VIP), in which presumably non‐cross‐resistant myelosuppressive and nonmyelosuppressive agents were administered during alternate weeks for 12 weeks. To ensure the high‐dose intensity of the protocol, dose reduction and delay in treatment were minimized. Three patients were treated inadequately. Twenty‐six (89.7%) of 29 evaluable patients had a complete response, and three had a good partial response. Relapse occurred in four patients, with a median follow‐up of 29 months. The actuarial overall survival and disease‐free survival were estimated to be 87.6% and 75.9%, respectively. The CAMBO‐VIP treatment was well tolerated; myelosuppression was severe but transient and caused no serious infections. Side effects that affected dose intensity were oral ulceration, occurring in 28 patients, and blister formation under the thickened skin of palms and/or soles, followed by desquamation (5 patients). Hepatic toxicity was generally mild to moderate; it was severe in one patient. A 12‐week regimen of CAMBO‐VIP was effective for advanced NHL with aggressive histologic findings.

Interleukin-1β (IL-1β) and acute leukemia: In vitro proliferative response to IL-1β, IL-1β content of leukemic cells and treatment outcome

We evaluated the in vitro proliferative response to exogenous IL-1β in terms of tritiated thymidine (3H-TdR) incorporation in leukemic cells obtained from 119 patients with various types of acute leukemia. The content of IL-1β in leukemic cells was measured by enzyme-amplified sensitivity immunoassay. We observed a significant proliferative response to exogenous IL-1β in leukemic cells from 2766 patients with de novo AML, 129 patients with ALL, 23 patients with AUL, 812 patients with AML arising from MDS, 47 patients with myeloid crisis of CML, and 04 patients with lymphoid crisis of CML. Proliferation was marked in myeloid leukemic cells of a more premature stem cell origin. There were no significant differences in proliferative responses among the different FAB classes of de novo AML. The IL-1β content of leukemic cells was low in patients with lymphoid leukemia, but there was no significant difference among the various types of myeloid leukemia. There was no correlation between the proliferative response to exogenous IL-1β and the IL-1β content of leukemic cells. When we correlated the proliferative response to exogenous IL-1β with treatment outcome in patients with de novo AML, we found the rate of complete remission (CR) to be lower in those with a high proliferative response. We noted a longer duration of CR (p = 0.07) and of survival (p < 0.05) in patients with a low proliferative response. Thus, a high proliferative response to IL-1β in the cells of AML patients may indicate a poor prognosis.

Increased blood cell destruction during vigorous regeneration of bone marrow after intensive chemotherapy for non-Hodgkin lymphoma

WE HAVE reported a new high dose regimen for the treatment of non-Hodgkin lymphoma (NHL), CAMBO-VIP[ 11, consisting of four myelosuppressive drugs (doxorubicin, cyclophosphamide, etoposide and ifosfamide) and four non-myelosuppressive drugs (vincristine, methotrexate with leucovorin rescue, bleomycin, and prednisolone), administered during alternate weeks for a total period of 12 weeks. We obtained a high response rate and prolonged disease-free survival with this regimen. The treatment was well tolerated: myelosuppression was severe but transient and caused no serious infectious complications. However, we noticed transient elevation of serum lactate dehydrgenase (LDH) level in some patients at or shortly after the completion of CAMBO-VIP treatment. 18 of 36 patients who were treated with this regimen showed LDH level over 1.5 times normal values, and 6 of them displayed over 3-fold normal values. LDH elevation was not associated with liver function abnormality as demonstrated by elevation of transaminases or total bilirubin. All of these patients were in complete or partial response with no evidence of tumour progression. Therefore, some other factors must be considered as the cause of LDH elevation. Klimo et a[.[21 reported similar elevation of serum LDH of unknown aetiology after completion of MACOP-B treatment for NHL. Elevation of serum LDH in our patients consisted of increase in isoxymes LDHr and LDH2. Interestingly, serum haptoglobin was undetectable in all 6 patients who were examined at the time of LDH elevation. Reticulocytosis and leukoerythroblastosis in peripheral blood were also observed in all of these 6 and other patients. In one particular patient, as many as 166 erythroblasts per 100 white blood cells were counted. These abnormalities, including LDH elevation, returned to normal relatively rapidly, usually within 2-3 weeks. These abnormalities suggest transient excessive blood cell destruction which might be associated with vigorous recovery of haemopoiesis following chemotherapy-induced myelosuppression. Haemopoiesis may become temporarily defective as a result of rapid cell proliferation, to such an extent that a proportion of newly formed cells are prematurely destroyed in the bone marrow or soon after they appear in the peripheral blood. Granulocyte or granulocyte-macrophage colony stimulating factors may augment these abnormalities, when they are used to expedite the recovery of bone marrow function following chemotherapy-induced suppression.

Intensive sequential post-induction therapy for adults with acute myelogenous leukemia in first remission: Long-term follow-up and results

We designed a post-induction therapy including intensive sequential therapy with non-cross-resistant drugs in an effort to prolong disease-free survival (DFS) for adults with acute myelogenous leukemia. Forty-five patients entered this study and 33 of 35 patients entering complete remission received the post-induction therapy. With a median follow-up for survivors of 3.5 years from complete remission, the actuarial 5-year DFS was 46% +/- 19% (95% confidence interval). The five-year DFS for patients over 45 years of age was comparable to that for patients under 45 years of age (50% +/- 26% vs 47% +/- 28%). Furthermore, the actuarial 5-year DFS for patients who required two courses of induction therapy was comparable to that for patients who required only one course of induction therapy (45% +/- 29% vs 50% +/- 25%). The toxicity of post-induction therapy was tolerable and no patients died during complete remission.

Factor VII deficiency: a double heterozygote of an Arg402Stop with a deletion of the C-terminal five amino acids and a Thr359Met

To the Editor: Factor VII (FVII) is a vitamin K-dependent zymogen of a serine protease that initiates blood coagulation (1). Factor VII is known to be present at low concentrations in plasma (0.5 mg ⁄L) and it has the shortest half-life (3– 4 h) among the known coagulation factors (2). The complete amino acid sequence has been determined by an analysis of the full-length cDNA sequence (3). The mature FVII protein, with molecular mass of about 50 kDa, consists of 406 amino acid residues, which is secreted from liver cells into blood stream as a single-chain zymogen. The mature FVII is converted auto-catalytically into its active form; factor VIIa. The structure of the genomic DNA of human FVII contains nine exons separated by eight introns and spans over 12.8 kb (4). Hereditary FVII deficiency is a rare hemorrhagic disorder that is transmitted in an autosomal recessive manner. A great number of FVII deficiencies have so far been analyzed at the molecular genetics level (5– 7). The patient was 35-yr-old Japanese female (Fig. 1). Laboratory data showed: RBC: 3.90 · 10 ⁄L, Hb: 123 g ⁄L, WBC: 6.80 · 10 ⁄L, PBC: 226 · 10 ⁄L, PT: 17.0 s (10.8–12.8 s in normal), APTT: 32.8 s (25.0–35.0 s in normal) and fibrinogen: 2.9 g ⁄L. Her plasma procoagulant activity of factor VII (FVII: C) (8) was markedly reduced (less than 3% of normal), while that of factors II, IX and X were normal, which confirmed the diagnosis of an isolated factor VII deficiency (Table 1). Her antigen level of FVII (FVII: Ag) was also greatly reduced to less than 1% (Table 1). Her family members (Fig. 1) were also studied after consent was obtained and the results are summarized in Table 1. Her parents were not consanguineous. No bleeding tendency was known for other family members. APTT was essentially within the normal range in all family members examined, as was PT except the proband (Table 1). The FVII: C and FVII: Ag levels of other family members were about half of normal plasma except for subject III-3 in whom it was normal. The FVII genes of the seven family members were analyzed to allow the haplotypic assignments to be made (9, 10). The proband’s genomic DNA had a C to T transition at nucleotide 11642 in exon 8 that resulted in a nonsense mutation Arg402Stop and a C to T transition at nucleotide 11514 in exon 8 that resulted in a missense mutation Thr359Met. These two mutations existed independently in a different site of a different allele of FVII gene. The number of nucleotides correlated to that of the human FVII genomic DNA (GenBank accession no.: J02933). Accordingly, the proband was suspected to have FVII deficiency caused by a double heterozygote of an Arg402Stop and a Thr359Met. The nucleotide sequences of exon 8 of other family members were analyzed and summarized in Table 1. The genotypes of the grand mother (subject I-1), the elder sister (subject II-1), the two daughters of the elder sister (subjects III-1 and III-2) and the daughter of the proband (subject III-4) were heterozygous for a Thr359Met, whereas the third daughter of the elder sister (subject III3) was genetically normal. Her father was not available for this study, however, he might well have had an Figure 1 A pedigree of the coagulation factor VII (FVII)-deficient family. Subject II-2 (shown by the arrows and the closed circle) represents the proband. The open square (male) or circles (female) represent normal level of clotting activity. Individuals heterozygous for the mutations are indicated by dots in the symbols. The phenotypes of the family members were determined based on biochemical methods. The numbers beneath the family individuals indicate the plasma FVII: C and FVII: Ag (%), respectively. NT: not tested. doi:10.1111/j.1600-0609.2009.01219.x European Journal of Haematology ISSN 0902-4441

Lysis of autologous tumor cells by large granular lymphocytes in patients with acute leukemia in complete remission: Correlation between lytic activity and clinical outcome

In order to evaluate the effect of specific immune response on prognosis in acute leukemia, we investigated the correlation between the lysis of autologous tumor cells (ATC) by lymphocytes and prognosis. Peripheral mononuclear cells (PMC) from most patients with acute leukemia in complete remission (CR) do not exhibit cytotoxic activity against fresh-frozen ATC, although they have adequate cytotoxic activity against K562 cells. When the large granular lymphocyte (LGL) fraction was used in this study, we observed lysis of ATC in 17 (43.6%) of 39 patients with acute leukemia (12 (42.9%) of the 28 patients with acute myelogenous leukemia (AML) and 5 (45.5%) of the 11 patients with acute lymphocytic leukemia (ALL)). With regard to prognosis, the lytic activity of the LGL fraction did not reflect the duration of CR. The median CR duration in AML patients was 13 months for the lysis-positive group and 11 months for the lysis-negative group. No significant correlation was also found between lytic activity of the LGL fraction and overall survival in each patient. However, the lysis-positive group tended to have a longer survival, the median overall survival being 48 months for the lysis-positive group vs 12 months for the lysis-negative group. The prolonging of overall survival in the lysis-positive group was attributed to a high rate of induction of second remissions in this group. Long-term patient survival in the two groups did not differ.