Isoprinosine and Imuthiol, two potentially active compounds in patients with AIDS-related complex symptoms.

Isoprinosine and Imuthiol are immunomodulators with a unique effect on T-cells. The possibility of using them in treating patients with acquired immunodeficiency syndrome related complex (ARC) was initially examined regarding their in vitro effects on peripheral blood mononuclear cells. In six ARC patients Isoprinosine (100 micrograms/ml) and Imuthiol (10 pg/ml) induced in vitro an early chromatin activation as measured by nuclear refringency test and potentiated phytohemagglutinin (5 micrograms/ml) in the same 20-min assay in the absence of fetal calf serum. In all patients an early phytohemagglutinin induced chromatin dispersion was observed with a dose related response before interleukin 2 production can occur. Isoprinosine and Imuthiol increased significantly both the percentage and the absolute number of T4+ cells when peripheral blood mononuclear cells were incubated for 4 days in RPMI supplemented with 10% fetal calf serum. No changes in T8+ cells were noted. Three homosexual ARC patients were then treated p.o. with Imuthiol (5-10 mg/kg/week) for 4 to 6 months. Without any deleterious effect a clinical improvement (in terms of adenopathy and opportunistic infection regression) and restoration of the response to recall antigens were observed in all three patients. One patient with less than 500 T4+ lymphocytes/mm3 exhibited a complete restoration of OKT profiles. In such patients clinical and immunological effects of Isoprinosine have already been reported by others. Altogether these preliminary results indicate that more data should be obtained on the effects of these two agents in ARC patients.

High incidence of Hox11L2 expression in children with T-ALL.

The orphan homeobox gene HOX11L2 was previously found to be transcriptionally activated as a result of the t(5;14)(q35;q32) translocation in three T-ALL cases. We now tested by RT-PCR Hox11L2 expression in 23 consecutive cases of T-ALL (15 children aged 0.8-14 years, eight adults aged 17-55 years) and as control 13 B-ALL patients from a single institution. Hox11L2 expression was undetectable in all patients with B-ALL, nor in adults with T-ALL. Nine children (60% of the cases), all boys, expressed Hox11L2. Blast cells from most of the latter patients carried surface CD1a, CD10 and not CD34 antigens, in contrast to the other children. FISH, M-FISH and IPM-FISH analysis failed to detect a t(5;14)(q35;q32) in one of them, which suggests a possible distinct genetic mechanism in Hox11L2 expression induction. Hence, Hox11L2 expression seems to be the most frequent abnormality in childhood T-ALL to date, comparable to the t(12;21) in child B-ALL.

Surface markers in adult acute myeloblastic leukemia: correlation of CD19+, CD34+ and CD14+/DR--phenotypes with shorter survival. Groupe d'Etude Immunologique des Leucémies (GEIL).

The immunophenotype of blast cells was investigated in a multicentric study of 154 adult acute myeloblastic leukemias (AMLs). A panel of 27 monoclonal antibodies (MoAbs) was tested in indirect immunofluorescence. Expression of CD14 (UCHM1), CD19 (SB4), CD36 (OKM5), and HLA-DR were associated with higher mean leucocyte counts. CD14 expression correlated with low hemoglobin level and the absence of CD33 (MY9) with low platelet counts. Extramedullary disease was associated with CD16 (Leu11b) and HLA-DR antigen positivity. The study of relationships between surface markers and FAB criteria confirmed the predominant expression of CD14 in the M5 sub-group (p less than 0.000001) and the association of CD19 and CD36 with monocytic M4/M5 subgroups (respectively p less than 0.00002 and p less than 0.000001). All patients received an induction therapy including an anthracycline and cytarabine. The median follow-up was 13 months. The achievement of complete remission (CR) was inversely correlated with CD34 (B13C5) and CD19 expression: CR was obtained in 31 of 59 (53%) CD34-positive AML versus 64 of 75 (85%) CD34-negative cases (p less than 0.0001) and 11 of 24 (46%) CD19-positive versus 95 of 122 (78%) CD19-negative cases (p less than 0.01). In univariate analysis, a longer survival was associated with CD33 expression and the combined phenotypes CD36+/CD19- and CD16+/CD14-. Conversely, the CD18(IOT18)+/CDw65(VIM2)- phenotype was related to shorter survival. The expression of CD19, of CD34, and of the combined phenotype CD14+/DR--correlated with shorter survival as demonstrated both in univariate and multivariate analysis (p less than 0.03 in each case in multivariate analysis).

Immunological classification of acute myeloblastic leukemias: relevance to patient outcome.

Immunophenotyping is a major tool to assign acute leukemia blast cells to the myeloid lineage. However, because of the large heterogeneity of myeloid-related lineages, no clinically relevant immunological classification of acute myeloblastic leukemia (AML) has been devised so far. To attempt at formulating such a classification, we analyzed the pattern of expression of selected antigens, on blast cells collected at AML diagnosis. Patients were eligible if they had a first diagnosis of de novo AML and a sufficient number of blast cells for proper immunophenotyping. The relative expression of CD7, CD13, CD14, CD15, CD33, CD34, CD35, CD36, CD65, CD117, and HLA-DR were analyzed by cytometry in a test series of 176 consecutive AML cases. Statistical tools of clusterization allowed to remove antigens with overlapping distribution, leading us to propose an AML classification that was validated in a second AML cohort of 733 patients. We identified five AML subsets (MA to ME) based on the expression of seven antigens within four groups (CD13/CD33/CD117, CD7, CD35/CD36, CD15).-MA and MB-AML have exclusively myeloid features with seldom extramedullary disease and rare expression of lymphoid antigens. No cases of acute promyelocytic leukemia (APL) were observed within MB AML. MC AML have either myeloid or erythroblastic features. MD AML have more frequently high WBC counts than other subsets, which were related to the expression of CD35/CD36 and CD14 and to monoblastic differentiation. ME AML lack CD13, CD33, and CD117 but display signs of terminal myeloid differentiation. Specific independent prognostic factors were related to poor overall survival in each immunological subset: CD34+ (P<3 x 10(-4)) in MA AML, CD7+ in MB AML, non-APL cases (P<0.03) in MC AML, CD34+ (P<0.002) and CD14+ (P<0.03) in MD AML, CD14+ in ME AML (P<0.01). The inclusion of seven key markers in the immunophenotyping of AML allows a stratification into clinically relevant subsets with individual prognostic factors, which should be considered to define high-risk AML populations.

Translocation t(5;14)(q35;q32) in three cases of childhood T cell acute lymphoblastic leukemia: a new recurring and cryptic abnormality.

We report three cases of T-ALL in which conventional cytogenetic analysis yielded normal karyotypes, but for which a new M-FISH technique (IPM-FISH) was able to detect a translocation. For these patients this technique highlighted a new, recurring and cryptic translocation t(5;14)(q35;q32) in childhood T-ALL which might be phenotypically restricted. The most innovative part of this technique is the use of interspersed polymerase chain reaction (IRS-PCR) painting probes that show an R-band pattern simultaneous with the combinatorial labeling. Contrary to the DOP-PCR, IRS-PCR-derived probes provide stronger hybridization signals at the telomeric ends that potentially increase the possibility of detecting cryptic translocations. All the IPM-FISH findings were validated by FISH with whole chromosome painting and unique sequence probes. These results demonstrate the efficient use of IPM-FISH as an improved, single-step method for the identification of cryptic chromosomal abnormalities. This new IPM-FISH technique is a good tool to display cryptic chromosomal abnormalities.

Characteristics of pro-T ALL subgroups: comparison with late T-ALL. The Groupe d'Etude Immunologique des Leucémies.

A group of 30 acute lymphoblastic leukemias (ALL) with the early pro-T phenotype CD7+/cCD3+/CD1-/CD3-/CD4-/CD8-were identified among 103 newly diagnosed ALL with T-lineage markers (T-ALL). Pro T-ALL was more often observed in adults, and showed a lower incidence of hyperleukocytosis than more mature T-ALL. Mediastinal masses and polar acid phosphatase positivity in blast cells were however observed with the same frequency in pro T-ALL and late T-ALL, and rearrangements of both T-cell receptor (TCR) beta and gamma genes were observed in half the pro T-ALL cases tested. The expression of CD34, DR, and myeloid (My) markers was significantly more frequent in pro T-ALL than in late T-ALL, and these three features were strongly linked. TCR gene rearrangements were two to three times more frequent in CD34- and My-pro T-ALL. However, both CD34+ and My+ pro T-ALL showed an incidence of mediastinal masses and polar acid phosphatase positivity similar to this observed in CD34- and My- cases. This supports the assumption that both types of ALL indeed are engaged in the T-lineage, and confirms intracytoplasmic cCD3 as the earliest marker for this lineage. Moreover, CD34 appears to persist up to an early stage of T-cell maturation, where the cells retain myeloid potentiality. Loss of CD34 correlates with TCR-beta gene rearrangement and definitive commitment to the T lineage. Event-free survival analysis suggested a poorer outcome for pro T-ALL in adult patients.

Modification of topoisomerase genes copy number in newly diagnosed childhood acute lymphoblastic leukemia.

Topoisomerase genes were analyzed at both DNA and RNA levels in 25 cases of newly diagnosed childhood acute lymphoblastic leukemia (ALL). The results of molecular analysis were compared to risk group classification of children in order to identify molecular characteristics associated with response to therapy. At diagnosis, allelic imbalance at topo-isomerase IIalpha (TOP2A) gene locus was found in 75% of informative cases whereas topoisomerase I and IIbeta gene loci are altered in none or only one case, respectively. By semi-quantitative Polymerase chain reaction, we found a 2.5 to 8-fold TOP2A gene amplification in 72% of the children, which was correlated to gene overexpression in every case. These results show that TOP2A gene amplification is a frequent event in ALL at diagnosis. Interestingly, we also identified a small population of children that do not present TOP2A gene amplification or gene overexpression and who are significantly associated with very high risk classified patients showing glucocorticoid resistance. In conclusion, characterization of TOP2A gene status in childhood ALL at diagnosis provides useful complementary information for risk assessment.

Latent acute promyelocytic leukemia t(15;17)(q22;q12-21) and sarcoidosis: long-term cohabitation.

The association of sarcoidosis with hematological malignancies is a well-known phenomenon. To our knowledge, we report the first case involving sarcoidosis and acute promyelocytic leukemia (APL) t(15;17)(q22;q12-21). The major interest lies in the chronology of the two diseases: the APL demonstrated an unusual smoldering evolution, suggesting that pre-existing sarcoidosis may have a non-fortuitous immunological impact on leukemic clone proliferation.

New human MHC class I antigens segregating with HLA-A antigens detected on some lymphocyte subpopulations.

Recent genetic studies of the murine chromosome 17 have demonstrated that many genes encode class I antigens, most of which are still not detected serologically; most of these genes belong to the Tla region. Five human alloantisera were selected from 383 female sera and were further studied using a panel of peripheral blood lymphocytes (PBL), B lymphocytes (BL), and PHA activated lymphocytes (PHA-L) from the same blood donors. After intensive platelet absorption, the five sera still reacted positively by a complement-dependent cytotoxicity technique with PHA-L, but negatively with PBL and BL. The antigens detected by these antibodies segregated with an HLA-A allele and were assumed to belong to the class I antigen series as they could be blocked by a turkey anti-beta 2 microglobulin serum. They were found on some lymphocyte populations: PHA-L, common acute lymphoblastic leukemia (cALL) cells, and (preliminary results) a small subpopulation of PBL cells (mostly NK cells), but were not found on chronic lymphocytic leukemia (CLL) cells, T and early T acute lymphoblastic as well as myeloblastic leukemia cells. Kinetic studies showed that several hours of culture with PHA were necessary for the antigen to be expressed. These results show that the antigens described do not belong to the classic HLA antigen series but could be considered to belong to the human Qa-like antigens or to be the human counterpart to the second murine H-2K locus antigens.

A new DR14 allele (DRB1*1411) containing a short DR11 sequence and its haplotypic association.

In the present work, we describe a new DR14 allele. Sequencing of its second DRB1 exon showed it to be DRB1*1404 from codon numbers 9 to 56 and 61 to 86, and DRB1*11 from codons 57 to 60 inclusive.

Decreased membrane "fluidity" of T lymphocytes from untreated patients with Hodgkin's disease.

Plasma membrane "fluidity" of peripheral blood T lymphocytes from untreated patients with Hodgkin's disease (HD) and healthy controls was studied using the fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4(trimethylamino)phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). In 13 consecutive patients a significant increase of T lymphocyte plasma membrane microviscosity was observed with both DPH and TMA-DPH. These alterations seemed unrelated to the cholesterol (Chol) and phospholipid (PL) content of HD T lymphocytes since the Chol/PL ratio was comparable in both HD and control cells. Since prostaglandin E2 (PGE2) from monocytic origin has been claimed to be responsible for the impairment of cell-mediated immunity (CMI) associated with HD, we studied the effect of exogeneously added PGE2 (0.1 microM) on control subjects T lymphocyte membrane "fluidity". Using the fluorescent probe DPH and the spin labelled fatty acid probe 16 NMS for electron paramagnetic resonance study, we observed a PGE2-induced fluidization of control T lymphocyte membranes which is specifically located in the inner part of the plasma membrane, whereas the plasma membrane surface seemed unaffected by PGE2 as judged by the TMA-DPH probe. Thus, PGE2 does not appear to be responsible for the alterations of T lymphocyte membranes observed in HD. Intrinsic alterations and/or other mediators might be involved.

Membrane markers, karyotypic abnormalities, ultrastructure and functional properties of lymphocytes in a case of 'D-cell' chronic lymphatic leukemia.

D cells are lymphocytes bearing both receptors for the third complement component and the ability to form spontaneous rosettes with SRBC. We report the case of a patient with a D-cell chronic lymphatic leukemia who presented a long evolution without treatment and whose leukemic cell characteristics have been extensively studied. Cytogenetic analysis showed numerous karyotypic abnormalities among leukemic cells; all metaphases were hypodiploid and arranged in four different clones; seven marker chromosomes were present. The cells were found to bear human T-cell specific antigen, the T helper/inducer phenotype, HLA-A and HLA-B determinants, but no HLA-DR antigens. They displayed a high proliferative response to PHA and Con A, no response to PWM stimulation, and possibly the capacity of allogeneic stimulation in the mixed lymphocyte culture system. Assays for cell-mediated cytotoxicity in the CML system, and for K and NK activities were negative.

PreB1 (CD10-) acute lymphoblastic leukemia: immunophenotypic and genomic characteristics, clinical features and outcome in 38 adults and 26 children. The Groupe dEtude Immunologique des Leucémies.

The less differentiated stage (CD10-) of B-lineage acute lymphoblastic leukaemia (ALL) described as preB1-ALL in the GEIL nomenclature, accounts for less than 10% of ALL. It is classically considered to be associated with translocation (4;11)(q21;q23), and to have a poor prognosis. We report an extensive immunophenotypic, genomic and clinical study of a series of 64 preB-1 ALL patients, representing 6.3% of a cohort of consecutive ALLs. The engagement of preB1-ALL cells in the B-lineage was confirmed by their B-lineage score, equal to or higher than 2. In addition, more than 90% of the cases tested showed rearranged IGH genes. Translocation (4;11) was the most frequent karyotypic anomaly seen, but only accounted for 24% of the preB1-ALL cases tested. Expression of the myeloid associated antigen CD15 was also found with high incidence in this subset. Clinical and biological features at presentation showed more significant differences between preB1- and T-ALL than between preB1- and preB2-ALL (CD10+). However, outcome characteristics of the 22 children with preB1-ALL confirmed the worse prognosis of this entity.

T-cell repertoire of normal, rejected, and pathological corneas: phenotype and function.

The specific immune mechanisms of corneal graft rejection are not completely understood. Recently, the technique of growing T-cell lines from rejected allografts using recombinant IL-2 has enabled the cells involved in allograft rejection to be recognized. In the present study, this method was applied for the extraction and propagation of T lymphocytes from rejected, normal, and diseased corneas. T-cell lines could successfully be grown from rejected and normal corneas, but not from corneas with keratoconus or pseudophakic bullous keratopathy. The phenotypic repertoire of the grown cells was studied by FACS scan analysis. Rejected corneas were invaded by a mixture of activated CD4+ and CD8+ T-cell lines, with one population being predominant. In normal corneas only activated CD8+ cells with cytotoxic function were cultured. No cells were obtained from diseased corneas. The in vitro function of cell lines was assessed by primed lymphocyte testing. The present study shows that the technique of propagating invading T-cell lines from organ grafts can be applied to human corneas, offering a new approach to understanding the immunological mechanisms occurring inside this immune tissue.

Immunomodulating effects of a short-term oral treatment with C 1821 in untreated cancer patients: a controlled study.

C 1821 is a purified glycoprotein extract from Klebsiella pneumoniae serotype 2 with a molecular weight of about 350,000. It enhances immune responses in animals when given orally and the oral route of administration is devoided of any toxicity even in humans. The present controlled trial showed that C 1821 given per os at the single daily dose of 4 mg for 14 days in untreated cancer patients (mostly lymphomas) significantly enhanced delayed cutaneous hypersensitivity to recall antigens using the Multitest system (7 antigens). It also increased basal levels of lymphocyte cAMP and particularly of cGMP which were decreased in these patients. When incubated in vitro with lymphocytes from either normal controls or patients, C 1821 showed a dose-dependent stimulation of cAMP synthesis which was more pronounced in patients than in controls.

[Diagnosis of myeloid hematologic malignancies: contributions of the 2001 World Health Organization (WHO) classification]. / Diagnostic des hémopathies malignes myéloïdes: apports de la classification OMS 2001.

Although the French-American-British (FAB) morphologic classification of myeloid malignancies has been accepted for many years, the important advances in cytogenetic, immunophenotype and genetic fields needed to be integrated in an updated approach using all the available informations. Thus, the World Health Organization (WHO) Classification of haematopoietic malignancies not only incorporates morphology, immunophenotype, cytogenetic and molecular features, but also ensures to be clinically useful. This collaborative project has begun in 1995 and has been published in 2001. The proposed WHO classification is less a disruption with regard to the FAB classification than an updated revision where morphology is always important. These review emphasises the modifications proposed in the new WHO classification concerning the myeloid disorders.

[Expression of HLA-DP antigens on normal and leukemic blood cells]. / Expression des antigènes HLA-DP sur les cellules sanguines normales et leucémiques.

HLA-DP allodeterminant elicit weak cellular and humoral immune responses. This study was undertaken to find out if the low immunogenicity of HLA-DP could be accounted for by a low level of expression on peripheral blood mononuclear cells. Double staining experiments and flow cytometry were performed in 28 healthy individuals, HLA-DP was found to be as expressed as DR and DQ on B lymphocytes. Furthermore, a DP+/DR- lymphocyte sub-population was identified and characterized as freshly activated T lymphocytes.

[Role of allogeneic transplantation of bone marrow in juvenile chronic myelomonocytic leukemia]. / Place de la greffe allogénique de moelle osseuse dans la leucémie myélomonocytaire chronique juvénile.

Two boys with clinical and haematological evidence of juvenile chronic myelomonocytic leukaemia had no chromosomal anomaly. In addition, one presented with an unbalanced Epstein-Barr virus serology, and the other with xantholeukaemia. Allogenic bone marrow transplantation was performed in the first boy after an 18-month period during which treatment with 6-mercaptopurine, intensive chemotherapy and splenectomy had failed. Conditioning included cyclophosphamide, high-dose cytarabine and whole-body irradiation. There was no complication, and 16 months after transplantation the patient was in complete remission. The second boy received a bone marrow transplant on the 6th month of the disease, after failure of 6-mercaptopurine. Conditioning included etoposide, busulfan and cyclophosphamide. On the 35th post-transplantation day the child had severe pancytopenia and his spleen remained enlarged. A second transplantation was performed after treatment with melphalan and whole-body irradiation. Twelve months later, the patient was in complete remission. The indications and modalities of allogenic bone marrow transplantation in juvenile chronic myelomonocytic leukaemia and the value of pre-transplantation splenectomy are discussed.

[Primary cutaneous non T non B CD4+ CD56+ leukemia (2 cases): an original anatomoclinical syndrome]. / Leucémie aleucémique non T non B CD4+ CD56+ (2 cas). Une entité anatomoclinique originale.

INTRODUCTION: Non T non B CD4+ CD56+ leukemia is often revealed by cutaneous lesions. We report 2 patients with this disorder who had characteristic anatomoclinical findings. CASE REPORTS: An 81 year-old female and a 75 year-old man presented with erythematous macules which increased in number and progressed to infiltrated plaques and nodules. The lesions became ecchymotic, but the patients remained in good general condition and blood count as well as bone marrow examination were unremarkable. A cutaneous biopsy revealed a lymphomatous mononuclear cell infiltrate. The cells expressed CD4 and CD56, but not CD3. The cutaneous lesions preceded for 10 and 9 months respectively the appearance of overt leukemia and medullar involvement. At this stage, the patients deceased rapidly from their leukemia. DISCUSSION: This is an original anatomoclinical syndrome. The histopathologist must pay attention to the unusual CD4+ and CD3- immunophenotype and search for CD56 expression. The malignant cell responsible for this type of leukemia is now individualized and corresponds to a type II dendritic cell precursor.