[A case of multidrug-resistant pulmonary tuberculosis].

A 44-year-old woman with multidrug-resistant pulmonary tuberculosis was admitted to our hospital in August 1998. She had been treated with the anti-tuberculosis agents isoniazid (INH), rifampicin (RFP), pyrazinamide and streptomycin (SM) for two months. However, tubercule bacilli found in a sputum culture on admission showed resistance to INH, RFP and SM, and so these agents were replaced with kanamycin (KM), ethionamide, cycloserine and levofloxacin. Unfortunately, the bacilli persisted in the sputum smears, and the patient complained of prolonged pain in the sites of intramuscular injection of KM. In January 1999, inhalation of KM was begun, resulting in the disappearance of the bacilli from the sputum and in improvements in chest radiographs. Inhalation of KM could be an effective therapy, with fewer adverse effects, in cases of multidrug-resistant pulmonary tuberculosis.

Multi-clonal expansion of unique human T-lymphotropic virus type-I-infected T cells with high growth potential in response to interleukin-2 in prodromal phase of adult T cell leukemia.

We established a simple IL-2-dependent colony-forming assay for T cells infected with human T-lymphotropic virus type-I (HTLV-I). IL-2-dependent cell lines were subsequently established by expanding individual colonies in liquid cultures. Lymphocyte-rich fractions were prepared from 31 HTLV-I carriers, 12 patients with smoldering ATL, 11 chronic ATL, 12 crisis ATL and 10 acute ATL. Primary colonies of CD4+ p19+ T cells were formed in all cases of carriers, smoldering and chronic ATL, and in 10 of 12 crisis cases. In contrast, no colony was formed from cells of patients with acute ATL. The rate of establishment of cell lines in HTLV-I carriers was significantly lower than that in patients of prodromal phase ATL. Cell lines established from cells of three prodromal cases were clonally identical to the parent ATL cells, while others had clonally distinct cell lines. Our results indicated the presence of four components of HTLV-I-infected T cells: (1) normal carrier T cells capable of forming colonies but not cell lines; (2) pre-malignant T cells capable of forming colonies as well as cell lines; (3) malignant T cells capable of forming colonies as well as cell lines; (4) fully malignant T cells unresponsive to IL-2. Our results suggest the presence of a multiclonal expansion of unique T cells in the prodromal phase of ATL, which have a high growth potential in response to IL-2. The coexistence of multiclonality with a dominant ATL clone may be closely related to the underlying pathology in HTLV-I leukemogenesis.

Establishment and characterization of a new erythropoietin-dependent acute myeloid leukemia cell line, AS-E2.

We have established an erythropoietin-dependent human leukemia cell line, AS-E2, from a patient with acute myeloid leukemia. These cells have many characteristics of late erythroid progenitor cells, they are positive for CD36, Glycophorin A, and CD71 but negative for CD41, and positive for benzidine and PAS staining. These cells express GATA-1 and have low affinity erythropoietin (EPO) receptor on their surface. Interestingly, AS-E2 cells are strictly dependent on EPO for their growth and survival; other cytokines including GM-CSF, stem cell factor, or IL-3 cannot support the growth of this cell line. These features are similar to late erythroid lineage cells, like normal BFU-E or CFU-E, and we have demonstrated that EPO stimulation induces the tyrosine phosphorylation of several proteins in AS-E2 cells including the EPO receptor and JAK2 kinase. This new cell line is a useful reagent to study biological and molecular events during the late stages of erythropoiesis, and to understand transforming events in human erythroid cells.

Frequent hepatic involvement in adult T cell leukemia: comparison with non-Hodgkin's lymphoma.

We examined 111 patients with acute type- or lymphoma type-adult T-cell leukemia (ATL) and compared them with 106 patients with non-Hodgkin's lymphoma (NHL). In addition to skin involvement and hypercalcemia which are already known to be frequent in ATL, ATL patients showed an higher incidence of hepatic involvement. There was more frequent palpable hepatomegaly, higher total bilirubin, GOT, GPT, lactate dehydrogenase (LDH), and alkaline phosphatase values in ATL than in NHL patients (p < 0.0001). Among 36 autopsied liver samples, invasion of ATL cells was confirmed in 22 cases. ATL patients with impaired hepatic function showed shorter survival times than patients without hepatic dysfunction. Moreover, ATL patients showed a worse performance status (PS), a higher incidence of lytic bone lesions, lower total protein (TP) and serum albumin levels than NHL patients. This invasive characters of ATL cells and consequent impaired general condition seemed to be factors affecting the poor prognosis recorded in ATL.

Integration patterns of HTLV-I provirus in relation to the clinical course of ATL: frequent clonal change at crisis from indolent disease.

We examined human T-lymphotropic virus type I (HTLV-I) DNA integration in 68 patients with adult T-cell leukemia/ lymphoma (ATL) by Southern blotting using EcoRI, which does not cut within the 9 kb of the genome and probes for pX and gag-pol region of HTLV-I. We detected defective proviral integration as a monoclonal band of various sizes with the pX but not with the gag-pol probe, or a monoclonal band of less than 9 kb with the pX probe, in 20 patients (29.4%). These were designated defective (D) type. With both probes, a single band greater than 9 kb was detected in 34 (50.0%), designated complete (C) type, and two or more bands greater than 9 kb, were designated multiple (M) type, in 14 (20.6%). Advanced age, a high LDH value, and hypercalcemia were more frequent in D type patients. The median survival time (MST) was 6.8, 24.4, and 33.3 months, for D, C, and M types, respectively (log rank P = .006). Among 52 sequentially examined patients, the HTLV-I integration patterns changed in 4 (7.5%). In three of these four, the rearrangements of the T-cell receptor (TCR)b gene concomitantly changed, suggesting the appearance of a new ATL clone. Another patient had the same rearrangement of the TCRb gene, indicating clonal evolution. The HTLV-I integration pattern changed at crisis from indolent to aggressive ATL in three patients. These findings suggested that the HTLV-I integration patterns have clinical implications in ATL pathophysiology. In contrast to the clonal evolution characteristic of the multistep carcinogenesis of most human malignancies, the frequent clonal change of ATL at crisis is a peculiar phenomenon, probably reflecting the emergence of multiple premalignant clones in viral leukemogenesis as suggested in Epstein-Barr virus associated lymphomagenesis in the immunocompromised host.

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.

CD5-expressing B-cell lymphomas/leukemias: relatively high frequency of CD5+ B-cell lymphomas with an overall poor prognosis in Nagasaki Japan.

To characterize CD5+ B-cell neoplasms in Japan, where chronic lymphocytic leukemia (CLL) is rare and of different subtypes in comparison with Western countries, we collected 58 cases of CD5+ B-cell lymphomas/leukemias and analyzed their clinicopathologic features. According to the French-American-British (FAB) and standard histologic classification, the cases corresponded to small lymphocytic lymphoma (SLL, group I; n = 22, consisting of CLL, n = 10, CLL/PL, n = 3, and CLLmixed, n = 7); intermediate differentiated lymphoma/mantle cell lymphoma (IDL/MCL, group II, n = 18); and others with CD5-positive lymphomas (group III, n = 18). The CD5+ B-cell lymphomas showed morphologic and prognostic variability among the three groups. The clinical and immunophenotypic features were remarkably consistent in leukemic disease being seen in 73% of all cases, splenomegaly in 63%, and intense CD19, CD20, surface membrane immunogobulin M (SmIgM) or SmIgM and SmIgD, light-chain expression, and no CD10 expression. The median survival time of groups I, II, and III was 7.8, 3.3, and 0.8 years, respectively. These findings suggest that CD5 antigens may serve as valid markers for the prognosis and clinical features of B-cell lymphomas and that CD5+ B-cell lymphomas with an overall poor prognosis occurs at a relatively high frequency in Japan. This also suggests that a combination of immunophenotypic and morphologic features is of value for characterizing CD5+ B-cell neoplasms.

[Phenotypical diagnosis of Japanese chronic lymphocytic leukemia--an international cooperative study based on the French-American-British classification of mature B-cell leukemias].

To better understand the accuracy in the diagnosis of chronic lymphocytic leukemia (CLL) and characterize the clinical features of CLL in Japan, where the disease is extremely rare, an international cooperative study was conducted by hematologists between Japan and UK. Blood and bone marrow films from 36 patients with a possible-diagnosis of CLL were referred to two laboratories of Nagasaki University Hospital (Nagasaki) and Royal Marsden Hospital (London). According to the FAB criteria, typical CLL 16 and CLL/PL 2, a subtype of atypical CLL, were completely accordant in diagnosis. However, phenotypical diagnosis of CLL mixed, the other of atypical CLL, and intermediate lymphocytic leukemia (ILL) in leukemic phase often gave inconsistent results. Especially, 8 cases of atypical CLL designated as likely CLL were equivocal between CLL and ILL, suggesting clinical feature more close to typical CLL than ILL. This indicates the presence of a relatively high incidence of atypical CLL in Japan which dose not exactly fit with the FAB Criteria. Finally, we would like to emphasize that an international cooperative study allows improvement of accuracy in diagnosis and better understanding of the disease entity of lymphoid malignancies, having on ethnically different morbidity.

Usefulness of immunocytochemistry for phenotypical analysis of acute leukemia; improved fixation procedure and comparative study with flow cytometry.

We investigated the phenotypes of blast cells of 53 patients with acute leukemia by a modified streptavidin-biotin alkaline phosphatase (SAB-AP) labeling technique, using a panel of monoclonal antibodies [MoAb; anti-CD11b, CD13, CD14, CD33, CD34, CD41, CD3, CD7, CD10, CD19, anti-HLA-DR, and anti-myeloperoxidase (MPO)]. The selection of an optimal fixative solution for each antigen from five options of various combinations of formalin, acetone, methanol, and/or ethanol, successfully conserved cell morphology and improved specific reaction compared with the conventional methods which used a single fixative for multiple antigens. We compared the SAB-AP results with those obtained by flow cytometry (FCM) for surface markers in each case. High concordance rates for both positive and negative results were observed for each marker. However, positive reaction for some markers (anti-CD13, CD14, CD33, and CD34) were often noted only in the cytoplasm by the SAB-AP method, indicating that combination of these two methods is essential for the precise immunophenotyping of poorly differentiated leukemia cells.

Morphological and immunophenotypical characterization of Japanese B-cell lymphocytic leukemia.

We have analyzed the clinical and laboratory features of 42 patients with B-cell leukemia. Based on the FAB criteria, the cases were classified in 3 groups: I) typical CLL 15, II) atypical CLL 9 which included 6 cases with large cells, and III) B-cell lymphoma in leukemic phase 18. Cases diagnosed as typical CLL (group I) had similar features to those seen in CLL patients from Western countries. The morphology and markers in cases from group III corresponded to B-cell lymphoma in leukemic phase. On the other hand, group II included 3 cases classified as atypical CLL according to FAB criteria. 1 CLL/PL and 2 mixed CLL and 6 cases with rather distinct features, namely: 1) lymphocytosis (42 +/- 41 x 10(9)/l in average) with large mature-looking lymphocytes with abundant cytoplasm: 2) an immunological profile consistent with CLL but, in addition with the consistent expression of CD38; 3) absence of a monoclonal band in the serum and 4) a clinical course and prognosis similar to CLL. Our findings suggest the existence of a B-cell disorder in Japan very close to CLL but distinct from typical and atypical CLL as seen in Western countries. Further studies would clarify whether such an entity is exclusively confined to Japan having a distinct natural history.

Established IL-2-dependent double-negative (CD4- CD8-) TCR alpha beta/CD3+ ATL cells: induction of CD4 expression.

We established IL-2-dependent T cells from an adult T-cell leukaemia (ATL) patient whose leukaemic cells changed from CD4 single-positive in the initial phase to double-negative (CD4- CD8-) at the time of exacerbation. The cells termed SO-4 were of ATL cell origin and showed the double-negative TCR alpha beta/CD3+ T-cell phenotype. SO-4 cells acquired CD4 antigen expression following stimulation with concanavalin A (ConA) or immobilized anti-CD3 antibody. The induction was inhibited by herbimycin A, an inhibitor of protein tyrosine kinase (PTK) activity. No CD4 mRNA was detectable in unstimulated SO-4 cells but a 3.0 kb signal specific for CD4 mRNA was detected after stimulation. These findings indicate that SO-4 cells return to their original phenotype (CD4 single-positive) by stimulation involving PTK. The results indicate that there is a pathway of phenotypic cycling between CD4 single-positive and double-negative T cells.

Significance of soluble interleukin-2 receptor levels for evaluation of the progression of adult T-cell leukemia.

BACKGROUND: The authors conducted a survey of a large cohort of patients with adult T-cell leukemia (ATL) and a group of human T-cell leukemia virus type 1 (HTLV-1) carriers to elucidate whether measurements of soluble interleukin-2 receptor (sIL-2R) levels are indicative of ATL tumor burden and correlate with clinical progression. METHODS: Using a sandwich enzyme immunoassay, the authors determined sIL-2R in the serum of 135 patients with ATL diagnosed and subclassified according to the Japan Lymphoma Study Group criteria and in the serum of healthy HTLV-1 seropositive persons. Also included were patients in the preleukemic state of ATL (pre-ATL), which is characterized by only slight blood changes but does not fit the diagnostic criteria of ATL. In the five subjects who finally advanced to overt ATL, the authors prospectively performed serial measurements of the receptor. RESULTS: Serial measurements of sIL-2R levels taken until overt ATL developed showed that these levels in the initial samples were higher than those of control subjects, even when subjects were asymptomatic or in the pre-ATL state. The serial levels of the five subjects gradually increased despite being in a clinically stable condition, finally reaching markedly high levels at the time ATL became overt. The mean sIL-2R levels of the smoldering, chronic, acute, and lymphoma subtypes of ATL were 1680 U/ml, 6680 U/ml, 45,940 U/ml, and 34,620 U/ml, respectively (P < 0.01). The sIL-2R levels of each subtype at the time of diagnosis were more correlated with tumor burden, malignant behavior, and prognosis than lactate dehydrogenase (LDH) levels. In the low, moderate, and high sIL-2R subgroups, the median survival time and percent survival probability at 2 years was 30.2 months (46.0%), 16.5 months (25.0%), and 7.7 months (15.3%), respectively. CONCLUSIONS: Serial measurements of sIL-2R levels are of clinical importance because changes of the levels correlate with disease progression, especially in early phase of ATL. The data suggest that sIL-2R may be more useful than LDH. In addition, emphasis may be placed on sIL-2R as an indicator of ATL progression status and prognosis for survival. The value of this marker in clinical practice should be confirmed prospectively.

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.

DNA aneuploidy of adult T-cell leukemia cells.

To clarify the biological characteristics of adult T-cell leukemia (ATL), immunophenotyping and DNA aneuploid analysis were performed in 72 ATL cases, using flow cytometric techniques. DNA aneuploidy was found in 45 cases (62.5%); the DNA index ranged from 1.03 to 2.16 (mean: 1.24). The incidence of aneuploidy in smoldering, chronic, acute, and lymphoma ATL subtypes was 20.0%, 46.6%, 76.3%, and 77.8%, respectively. The aneuploid patients had a greater tumor burden (adenopathy, hepatosplenomegaly, and leukocytosis with ATL cells), a higher level of serum LDH, and a higher incidence of hypercalcemia, compared with the diploid group. Further, unusual aberrant immunophenotypes were identified predominantly in the aneuploid group. Patients with aneuploidy had a 7.6 month median survival time (MST) with a 2 year survival rate of 24.6%, significantly worse than in the patients with diploidy, whose MST was 25.4 months with a 2 year survival of 60.1%. In some aneuploid patients, the disease often progresses from a static to an aggressive form. Thus, the determination of aneuploidy and unusual immunophenotype should be useful for detecting clinical behavior and for monitoring ATL patients, particularly in regard to such progression.

Human T-lymphotropic virus type-I influence on hepatotropic virus infections and the subsequent development of hepatocellular carcinoma.

We studied the serologic status, with respect to hepatitis B virus (HBV), hepatitis C virus (HCV), and human T-lymphotropic virus type-I (HTLV-I) of blood donors in an area endemic for adult T-cell leukemia (ATL). Similarly, we studied the serologic status of hepatocellular carcinoma (HCC) patients from the same district. In the donors, the incidence of serological positivity for these viruses was 0.95, 1.23, and 3.75%, respectively. There was a positive correlation between the presence of anti-HCV and serological HTLV-I positive status in these subjects (1.9 vs. 1.1%) for those with HTLV-I negative status, implying high susceptibility for HCV infection among HTLV-I carriers. Fifty-nine percent of HCC patients were positive for anti-HCV and twenty-six percent of those were simultaneously positive for the antibody to HTLV-I. HCC patients infected with HTLV-I were younger than patients not so infected (61.5 +/- 8.8 vs. 64.8 +/- 8.4 years, p < 0.05). These observations suggest the possibility that HTLV-I could be one of the factors that promote the development of HCC caused by hepatotropic viruses.

Unusual morphological features of adult T-cell leukemia cells with aberrant immunophenotype.

We describe 4 cases of adult T-cell leukemia (ATL) with unusual morphology and aberrant immunophenotype. All patients were Japanese and born in the Nagasaki district, an area endemic for HTLV-I. Peripheral blood and/or bone marrow films revealed bizarre giant cells with and without large nucleoli; the cells were 5 to 6 times the diameter of erythrocytes, resembling Hodgkin's cells. Some peripheral blood cells were morphologically similar to prototypic ATL cells, while many other cells in the bone marrow showed unusual morphology. Furthermore, leukemic cells had aberrant immunophenotypes such as the CD8-positive type in patients 1 and 2, the CD4-.CD8- double-negative type in patient 3, and the CD5 antigen defect in patient 4. All patients had marked elevations of the serum calcium and LDH and organomegaly, while all had a short survival. Anti-HTLV-I antibodies and provirus DNA monoclonality were demonstrated in all patients. The results suggested that the unusual morphology and aberrant ATL cell immunophenotype may be indicative of a high grade malignant behaviour of ATL.

Characteristics of chemotherapy-induced clinical remission in long survivors with aggressive adult T-cell leukemia/lymphoma.

The acute and lymphoma types of adult T-cell leukemia/lymphoma (ATL) usually have a very poor prognosis, although some patients achieve long survival after chemotherapy. A total of 114 patients with these aggressive types of ATL were newly diagnosed at our institution from 1975 to 1989. By multivariate analysis, poor performance status and high serum creatine levels were associated with shortened survival. With combination chemotherapy, 20 patients achieved complete remission (CR), 53 achieved partial remission (PR) and 35 showed no response. Fifteen of the CR or PR patients survived for more than two years and all other patients survived for less than two years. As compared with short survivors (< 2 years) after remission, long survivors (> or = 2 years) after remission had a higher CR/PR ratio, a longer time until remission and a higher doxorubicin dose to achieve remission. Death due to causes other than the primary disease occurred in 18% of short survivors after remission and in 11.2% of nonresponders, but in none of the long survivors. Long survivors with acute ATL included 6 patients with CR and 5 patients with PR. All four lymphoma type ATL long survivors achieved CR. Monoclonal integration of HTLV-I provirus was detected in the peripheral blood mononuclear cells of all 3 PR long survivors with acute ATL studied, but was not detected in all 4 CR cases studied at remission. The minimum CD4/CD8 ratio of peripheral mononuclear cells at remission was < 1.0 in all acute ATL long survivors with CR, and was > 1.0 in all acute ATL long survivors with PR. Three out of six acute ATL long survivors with CR developed suspected viral infection just before achieving CR. Our findings show that in aggressive ATL the characteristics of remission are heterogeneous even among long survivors.

Phenotypic diversity and prognosis of adult T-cell leukemia.

We examined phenotypically 107 patients with adult T-cell leukemia (ATL), using a panel of monoclonal antibodies, in order to clarify the occurrence of aberrant phenotypes, and to determine the correlation between phenotypic diversity and prognosis. The incidence of the typical (CD4+.CD8-) phenotype, the double-negative (CD4-.CD8-), the double-positive (CD4+.CD8+), and the CD8-positive (CD4-.CD8+) phenotypes was 81%, 7%, 7%, and 4%, respectively. The median survival time (MST) for all patients was 10.0 months with 17% survival at 2 years. The patients with typical phenotypes had a 10.2 month MST with 20% survival at 2 years, significantly better than the patients with the unusual phenotypes whose MST were 4.9, 7.8, and 2.6 months, respectively, for the double-negative, double-positive, and CD8-positive phenotypes. Lack of antigens reactive with CD2, CD3, CD5, and WT31 monoclonal antibody panels was one factor in bad prognosis, but the presence of CD4 and CD8 antigen abnormalities was much more significant.

Cohort study of hepatotropic virus and human T lymphotropic virus type-I infections in an area endemic for adult T cell leukemia.

We tested for antibodies to hepatitis B virus (HBV), hepatitis C virus (HCV), and human T lymphotropic virus type-I (HTLV-I) in 629 normal inhabitants of an adult T cell leukemia (ATL) endemic area and in patients with ATL, HTLV-I associated myelopathy (HAM), and hepatocellular carcinoma (HCC) from the same district. The prevalence of serological positivity for each virus was 28.0, 6.4, and 32.6%, respectively, among the 629 inhabitants. There was a positive association between the presence of anti-HCV and serological HTLV-I positive or negative status of these subjects (9.3% vs 5.0%). Conversely, there was no correlation between HBV and HTLV-I serologic prevalence. Only inhabitants positive for anti-HCV showed significantly high serum aminotransferase levels. The levels were not affected by superimposed HTLV-I infection among anti-HCV positives. Fifty three percent of HCC patients were positive for anti-HCV; 35% of whom were simultaneously positive for antibody to HTLV-I. On the other hand, only 2 ATL patients (4.2%) and 2 HAM patients (7.7%) had anti-HCV. These findings suggest that high serum aminotransferase levels are mainly caused by HCV infection and persons with HCV and HTLV-I double infections are at a high risk for the development of HCC but not ATL or HAM.