Rearrangements in the human T-cell-receptor alpha-chain locus in patients with adult T-cell leukemia carrying translocations involving chromosome 14q11.

We describe 5 cases of adult T-cell leukemia (ATL) carrying translocations at chromosome 14q11, where the genes for alpha- and delta-chains of the T-cell receptor (TCR alpha/delta) reside (Croce et al., 1985; Isobe et al., 1988). Since the TCR alpha/delta locus is the region where several types of chromosome translocations occur in T-cell tumors, rearrangements of the TCR alpha/delta locus in those ATL cases were studied as a first step to characterize these translocations at the molecular level. For this purpose we have generated an extensive series of probes to define the specificity and the diversity of rearrangement occurring at the widely spanned J alpha-C alpha locus and the complex D delta-J delta-C delta-V delta 2 locus. Using a set of probes, we have found the deletion of the TCR delta locus in all ATL cases, and at least 2 rearrangements in the J delta locus in each case of ATL. It is possible that translocations in the TCR alpha locus may be involved in ATL.

Cytogenetic studies of non-lymphocytic neoplasias using hematopoietic growth factors: GM-CSF provides pronounced mitotic increase and improvement of the quality of banded chromosomes compared to G-CSF.

Thirty patients with a variety of non-lymphocytic neoplasias were studied cytogenetically using short-term liquid cultures of bone marrow or peripheral blood cells with or without either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage CSF (GM-CSF). The final concentration of these growth factors was 10 ng/ml and the duration of culture was 48 h, since these provided optimal increases in mitotic index (MI). In GM-CSF-stimulated culture, 23 out of the 30 patients (77%) had a significantly (p < 0.05) higher MI than that in unstimulated culture, whereas only five (17%) did so in G-CSF-stimulated culture. The quality of banded chromosomes was considerably good in 17 out of the 30 patients (57%) with GM-CSF, whereas it was so only in two (7%) and three (10%) patients with no CSF and G-CSF, respectively. Of the 30 patients, 27 (90%) had the same chromosomal findings with G-CSF/GM-CSF as without CSF, but the remaining three (10%) showed a remarkable change after stimulation. Culture for 72 h with G-CSF or GM-CSF disclosed a minor abnormal clone which was undetected in 24 and 48 h cultures in a patient with myelofibrosis with myeloid metaplasia. Thus, compared with G-CSF, adding GM-CSF to cell culture may be useful for cytogenetic studies of non-lymphocytic neoplasias. The chromosomal findings may, however, be changed by these growth factors for some patients.

Clinical significance of beta 2-microglobulin in serum of adult T cell leukemia.

To clarify the clinical and biological significance of beta 2-microglobulin (beta 2-M) in serum of adult T cell leukemia (ATL) associated with human lymphotropic virus type-I (HTLV-I), beta 2-M was measured in 52 patients with ATL (acute ATL, 35 patients; lymphoma ATL, two patients; chronic ATL, 12 patients; smoldering ATL, three patients), and it was compared with serum lactic dehydrogenase (LDH). Statistical analysis disclosed a correlation between beta 2-M level and the percentage of abnormal lymphocytes (P < 0.05) and platelet count (P < 0.01). There was a correlation between LDH and platelet count (P < 0.01), and a tendency of correlation between LDH and the percentage of abnormal lymphocytes (P < 0.15). Significant difference was present in beta 2-M as well as LDH between acute ATL and chronic ATL (P < 0.01), and between acute ATL and smoldering ATL (P < 0.01). We also investigated a significant inverse correlation between beta 2-M level as well as LDH level and the length of survival after the initial diagnosis (P < 0.01). Thus, the beta 2-M level may indicate the aggressiveness of ATL cells and predict the length of survival.

Increased levels of interleukin-6 (IL-6) in serum and spontaneous in vitro production of IL-6 by lymph node mononuclear cells of patients with angio-immunoblastic lymphadenopathy with dysproteinemia (AILD), and clinical effectiveness of cyclosporin A.

Serum levels of cytokines and in vitro cytokine production by lymph node mononuclear cells (LNMC) were studied in four patients with angio-immunoblastic lymphadenopathy with dysproteinemia (AILD) or AILD-type T cell lymphoma. An increased level of serum interleukin-6 (IL-6) was detected on initial diagnosis in both of two patients examined. Spontaneous production of IL-6 by LNMC was detected in all four patients studied. Immunosuppressive therapy with cyclosporin A (CsA) was attempted in a 68-year-old man, who was refractory to intensive combination chemotherapy. The increased level of IL-6 in this patient decreased to normal within 3 weeks of CsA administration and the patient became symptom-free. One and a half months later, the IL-6 level gradually increased along with clinical exacerbation. We also measured serum levels of IL-1 alpha, IL-2, IL-4, IFN-alpha, gamma and TNF-alpha in parallel with IL-6, but these factors were only sporadically detected. IL-6 production by LNMC was stimulated by IL-2 but inhibited by CsA. These observations suggest that IL-6 is one of the important cytokines to be involved in the pathophysiology of AILD and CsA is a useful reagent for relieving symptoms.

Morphological subtyping of acute myeloid leukemia with maturation (AML-M2): homogeneous pink-colored cytoplasm of mature neutrophils is most characteristic of AML-M2 with t(8;21).

Morphologic and cytochemical features of 30 acute myeloid leukemia subtype M2 (AML-M2) patients with t(8;21) were compared with those of 50 AML-M2 patients without t(8;21). It was disclosed that irregular nuclear shape, Auer bodies, and at least 90% myeloperoxidase positivity in blast cells, and pseudo-Pelger-Huët anomaly of the nuclei and homogeneous pink-colored cytoplasm of mature neutrophils were observed in 90-100% of the t(8;21)+ patients. The percentages of patients showing these features were significantly (P < 0.01) lower in the t(8;21)- group. Among these morphological features, homogeneous pink-colored cytoplasm of mature neutrophils is most characteristic of t(8;21)+ AML-M2, because it was seen in 90% of the t(8;21)+ patients but in only 2% of the t(8;21)- patients. Conversely, pale-colored cytoplasm without any granules in mature neutrophils or dyserythropoietic features was observed in 84% of the t(8;21)- patients, but in none of the t(8;21)+ patients. These data suggest that it is possible to subtype AML-M2 patients morphologically by the recognition of homogeneous pink-colored or pale-colored cytoplasm of mature neutrophils and dyserythropoietic features. Thus, the morphologic subtyping of AML-M2 can be utilized alone or in combination with chromosomal or molecular subtyping for biological and clinical studies of AML with maturation.

No parental origin bias for the rearranged chromosomes in myeloid leukemias associated with t(9;22), t(8;21) and t(15;17).

We investigated parental origin of rearranged chromosomes 9 and 22 (9q + and 22q -) in five patients with Ph-positive chronic myeloid leukemia (CML) using the C-banding and silver-staining methods of nucleolus organizer regions, respectively; of rearranged chromosome 21 (21q +) in seven patients with t(8;21)-positive acute myeloid leukemia (AML); and of rearranged chromosome 15 (15q +) in six patients with t(15;17)-positive AML. It was found that these rearranged chromosomes can be of either paternal or maternal origin. Although the number of patients examined was small, these results indicate that the genes rearranged as a result of these chromosome translocations (ABL, BCR, AML-1 and PML) are not genomically imprinted.

Clinical significance of tissue polypeptide antigen in serum of acute nonlymphocytic leukemia.

Tissue polypeptide antigen (TPA) in serum was measured at diagnosis in 27 patients with acute nonlymphocytic leukemia (ANLL) (1 FAB M0, 1 M1, 10 M2, 7 M3, 5 M4, 1 M5, 1 M6 and 1 MU). Statistical analysis disclosed a close correlation of TPA level with age (P < 0.01), hemoglobin level (P < 0.05), therapeutic response (P < 0.01) and the length of survival after the initial diagnosis (P < 0.02). A significant difference in TPA level was present between patients with complete remission and those with poor response. To our knowledge, this is the first report to prove a correlation of TPA level with therapeutic response and the length of survival in ANLL.

Acute nonlymphocytic leukemia with normal karyotype. Is its in vivo drug susceptibility age-dependent?

Nineteen patients with acute nonlymphocytic leukemia (ANLL) de novo who had no detectable chromosomal abnormalities received intensive remission induction chemotherapy. After the first chemotherapy cycle, ten of 14 (71%) patients aged 60 years or less entered complete remission (CR) whereas none of five patients aged more than 65 years attained CR. On the other hand, leukemia showed a drug resistance in three (21%) of the former patients and in four (80%) of the latter patients. One patient in each group died during induction. Generally, older ANLL patients have an inferior CR rate after chemotherapy and a poor prognosis. This has been explained by the facts that the risk of induction death increases and that specific chromosomal abnormalities associated with poor prognosis are considerably more frequent in older patients. Our data may indicate, however, that in ANLL with normal karyotype older patients show a low initial response rate because of drug resistance.

Inversion(10)(q11q24) in a case of adult T-cell leukemia.

We report a case of adult T-cell leukemia (ATL) with complex chromosome abnormalities including an inversion (10)(q11q24) in a 64-year-old man. Although some abnormalities of chromosome 10 have been seen in ATL and other lymphoid neoplasias, inv(10)(q11q24) has previously been reported only in a case of T-cell chronic lymphocytic leukemia. Recent studies have revealed a rearrangement of a novel homeobox-containing gene called TCL-3 or HOX11 on 10q24 in T-cell acute lymphoblastic leukemia with the specific chromosome translocation t(10;14)(q24;q11), and thus the significance of 10q24 aberrations in leukemogenesis is indicated. We suggest that, despite the rarity of this anomaly, inv(10) (q11q24) may be a new chromosome inversion related to T-cell neoplasia and that the 10q24 anomaly may be an important cytogenetic clue for the elucidation of the pathogenesis of some peripheral T-cell neoplasias.

Appearance time of leukemic cells with t(8;21) in bone marrow.

A 30-year-old man was referred because of slight leukocytosis. The hematological findings, including those of the bone marrow, showed no evidence of leukemia. The level of neutrophil alkaline phosphatase (NAP) in the peripheral blood was normal, as were the chromosomes from bone marrow cells. Fifteen months later, the disease was diagnosed as M2 (according to the French-American-British classification) showing a t(8;21)(q22;q22) and a low NAP level as two markers of M2 cells. This is probably the first case of acute leukemia in which the cytogenetic analysis was performed before and after the appearance of a specific chromosome abnormality.

High rate of chromosomal abnormalities in HTLV-I-infected T-cell colonies derived from prodromal phase of adult T-cell leukemia: a study of IL-2-stimulated colony formation in methylcellulose.

To detect chromosomal abnormalities in prodromal phase of adult T-cell leukemia (ATL), we established a clonal culture method for human T-lymphotropic virus type I (HTLV-I) infected T-cells in methylcellulose containing recombinant human interleukin 2 (rhIL-2). We tried to analyze chromosomes of 187 colonies (4, 23, 69, 74, and 17, from HTLV-I-uninfected normal T-cells, HTLV-I-Infected normal T-cells, HTLV-I carriers, smoldering ATL, and chronic ATL, respectively), using chromosomal banding methods. In the prodromal group, 53% of colonies (84/160) (36/69, 37/74, 11/17 in HTLV-I carriers, smoldering ATLs, and chronic ATL, respectively) had chromosomal abnormal clones. In HTLV-I carriers, multiple clones with simple chromosomal abnormalities were observed. In more progressed chronic ATL, more complex chromosomal abnormalities were detected, and specific colonies were selected. Thus, colonies in the prodromal phase of ATL are characterized by cytogenetical clonal evolution and clonal changes.

Cytogenetic studies of human T-cell leukemia virus type I carriers. A family study.

Recently, the chromosome 14q11 anomaly has been reported to be specific to adult T-cell leukemia (ATL), and this anomaly has also been confirmed in the preleukemic state of adult T-cell leukemia (pre-ATL) patients. Because the cytogenetic abnormality at the stage of human T-cell leukemia virus type I (HTLV-I) carrier remains uncertain, we performed cytogenetic studies of lymphocytes stimulated with phytohemagglutinin in three HTLV-I carriers and three non-HTLV-I carriers in an ATL family. As a result, in three HTLV-I carriers, four of 311 cells examined (1.3%) had chromosome 14q11 anomaly. However, in three non-HTLV-I carriers, none of 260 cells examined had chromosome 14q11 anomaly. These results suggest that chromosome 14q11 anomaly is already present at the stage of HTLV-I carrier and seems to be an important cytogenetic clue to the pathogenesis of ATL.

Therapeutic and prognostic value of chromosomal AP classification at the blastic phase of Ph-positive chronic myeloid leukemia: comparison of data from Nagasaki University, Japan and Roswell Park Memorial Institute, U.S.A.

To assess parameters of therapeutic response and survival after the onset of the blastic phase (BP) in 47 patients with Philadelphia chromosome (Ph)-positive chronic myeloid leukemia (CML), a number of clinical hematologic, and cytogenetic data at the BP were evaluated. Among the eleven parameters examined, only the chromosomal findings correlated with the therapeutic response and survival after onset of the BP. The patients were divided into three groups on the basis of the chromosome findings in the bone marrow: one, with only a Ph (PP), another with two different clones, i.e., one clone with Ph only and another with additional karyotypic changes (AP), and a third group with only abnormal clones containing karyotypic abnormalities in addition to the Ph (AA). The number of patients with AA was 12, 18 with AP, and 17 with PP. The results were as follows: (1) The percentage of patients with a good therapeutic response was 25% (3/12) in AA, 22% (4/18) in AP, and 76% (13/17) in PP; (2) The median survival after the onset of the BP was 1.5 mo for AA, 2.4 mo for AP, and 7.3 mo for PP. Statistically, there was a significant difference between PP and the other two groups (p < 0.05). These data were reevaluated and compared to those of 64 patients with Ph-positive CML in Roswell Park Memorial Institute (RPMI) who had been reported earlier in 1983.

Increased plasma M-CSF concentration in patients with adult T cell leukemia: clinical correlation.

Plasma concentration of M-CSF was measured in 35 patients with adult T cell leukemia (ATL), using a radioimmunoassay (RIA). ATL patients showed elevated levels of plasma M-CSF concentration when compared with healthy adult volunteers. Higher M-CSF levels were observed in acute ATL patients than in patients with chronic or smouldering ATL (P < 0.0001). There was a significant positive correlation of M-CSF concentration with serum lactic dehydrogenase (LDL) level, a reliable marker for assessing the grade of malignancy in ATL (P = 0.0003). There was, however, no correlation of M-CSF concentration with total counts of peripheral blood ATL cells, neutrophils or monocytes, or with serum calcium levels. Although there was a significant positive correlation of M-CSF concentration with body temperature (P = 0.003), there was not a significant correlation of M-CSF concentration with C-reactive protein (CRP), a protein indicative of the severity of inflammation (P = 0.063). These results indicate that plasma M-CSF concentration reflects the disease activity of ATL, and can thus serve as a marker in the clinical subclassification of ATL patients.

Analytical electron microscopy of Bacterionema matruchotii calcification.

The morphology and elemental analyses of calcification occurring in Bacterionema matruchotii were studied by analytical electron microscopy. Early electron densities were found associated with metachromatic granules and cell membranes. Amorphous calcium phosphate appeared before apatite was detected.

Formation of Bacterionema matruchotii protoplast.

Protoplast formation with Bacterionema matruchotii was studied turbidimetrically and microscopically. The protoplasts were inducible during culture by addition of glycine, lysozyme and Ficoll to a medium containing Tween-80 and polyethylene glycol.

Study of the effective dose of a topical antifungal agent, omoconazole nitrate, on the basis of percutaneous pharmacokinetics in guinea-pigs and mice.

The clinically useful optimum dose of omoconazole nitrate, a topical antifungal agent, has been examined by analysing the percutaneous pharmacokinetics of the drug to assess its pharmacological activity in an in-vivo study. Creams containing omoconazole nitrate were prepared on a pilot basis. The therapeutic effect of the omoconazole nitrate creams was examined in an in-vivo pharmacological dermatophytosis infection model in guinea-pigs. Creams containing 0.25% or higher concentrations of omoconazole nitrate resulted in significant inhibition compared with no treatment and with vehicle-treated controls. In the mycological examination no growth of dermatophytes was observed for creams containing 1% or higher concentrations. In an in-vitro hairless mouse skin-permeability test a non-linear least squares program based on a fast inverse Laplace transform algorithm was used to calculate the partition and diffusion parameters of omoconazole nitrate in the stratum corneum and viable epidermis. The time-course of drug concentrations in the skin of the guinea-pig, estimated on the basis of these parameters, led to predictions that percutaneous drug concentrations on the guinea-pig would require 10 or more days to reach equilibrium in the skin; that drug concentrations in the corneum-viable epidermis border, where dermatophytes are considered to grow, would exceed the minimum effective concentration when 0.1% higher concentration creams were used; and that for binding to keratin drug concentrations would reach the practical minimum effective concentration when creams containing 0.5% or more omoconazole nitrate were used. These results show that partition and diffusion parameters obtained from in-vitro skin permeation studies can be used to predict in-vivo percutaneous pharmacokinetics and to estimate therapeutically effective concentrations.

[In vitro antifungal activity of omoconazole nitrate, a novel imidazone antimycotic drug, against clinical isolates from patients with cutaneous mycosis].

In vitro antifungal activities of omoconazole nitrate (OMZ), a novel antifungal imidazole antimycotic drug, are examined against clinical isolates obtained from patients with cutaneous mycosis and its activity was compared with that of bifonazole (BFZ). The clinical isolates tested were 70 of dermatophytes including Trichophyton rubrum (47 isolates), T. mentagrophytes (22 isolates), Microsporum gypseum (1 isolate), and 27 isolates of Candida albicans. MIC values of OMZ to dermatophytes distributed in a range of < or = 0.04 to 0.63 microgram/ml were similar to those of BFZ (< or = 0.04 to 1.25 micrograms/ml). MIC values of OMZ to C. albicans were in a range of 0.16 to 2.5 micrograms/ml indicating that OMZ had more potent activities than BFZ (1.25 to 5 micrograms/ml). These results showed that in vitro antifungal activities of OMZ against clinical isolates of dermatophytes and C. albicans were greater than or similar to those of BFZ.

[Various factors influencing in vitro antifungal activities of omoconazole nitrate (OMZ), a new imidazole antimycotic].

Effects of various factors on in vitro antifungal activities of omoconazole nitrate (OMZ) were investigated with bifonazole (BFZ) as the reference drug using an agar dilution method and a broth dilution method. Composition of the culture medium significantly altered the activity as determined by minimum inhibitory concentration (MIC) of OMZ; OMZ exhibited a greater anti-Candida activity (smaller MIC values) on casitone agar (CA) than on Sabouraud dextrose agar (SDA). Anti-Candida activity of OMZ was the highest in pH 5 medium, but it was lowered by an increase of inoculum size, a prolongation of the incubation period and the addition of calf serum to the medium. Anti-Trichophyton activity of OMZ was not influenced with these factors except for the addition of calf serum. The activity of OMZ was fungicidal against Candida at pH 5.0 and against Trichophyton at pH 5.0 and 6.6. The geometric mean MIC of OMZ was lower than that of BFZ against C. albicans freshly-isolated on acid CA.

[In vitro antifungal activity of omoconazole nitrate, a new imidazole antimycotic].

In vitro antifungal activities of omoconazole nitrate (OMZ), a new topical imidazole antifungal agent, were studied against 364 strains of 22 genera and 47 species of stock cultures of a wide range of medically important fungi. The test was carried out using the agar dilution method with bifonazole (BFZ) as the reference drug. OMZ exhibited a broad spectrum of antifungal activities. Most of yeasts including Malassezia, dimorphic fungi, non-pigmented hyphomycetes and dermatophytes were almost equally or more greatly susceptible to OMZ than to BFZ, although the MIC values of these drugs against Candida and probably some other fungi were influenced differently by testing media. Comparable susceptibility to OMZ were observed for fresh clinical isolates of dermatophytes and Candida albicans. OMZ was also active against Malassezia. These results have encouraged us to proceed in vivo studies using animal models of cutaneous mycoses.