Michel Bernier

Identification of natural killer cell receptor phenotypes associated with leukemia.

Natural killer (NK) cells play a key role in defense against tumor cells that have the capacity to downregulate human leukocyte antigen (HLA) class I expression. It has been reported that leukemic cells can have downregulated expression of HLA class I molecules. The polymorphic nature of NK cell receptor (NKR) genes generates diverse repertoires in the human population, which display specificity in the innate immune response. In the present study, 11 KIR and two CD94/NKG2 receptors were genotyped by PCR-SSP in 96 leukemic patients and 148 healthy Caucasians. Here, we report a significant increased frequency of the more inhibitory AB killer cell immunoglobulin-like receptor (KIR) phenotype in leukemic patients compared to the controls (31.1% in healthy controls vs 51.0% in leukemic patients, Pc=0.002), which is related to the high prevalence of the inhibitory KIR2DL2 in this population (Pc=0.007). Moreover, two specific KIR phenotypes AB1 and AB9, including all inhibitory KIRs, were significantly associated with leukemic patients. Our study suggests that an important percentage of leukemic patients express a KIR phenotype in favor of escape from NK cell immunity.

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 × 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.

Acute myeloid leukaemia M0: haematological immunophenotypic and cytogenetic characteristics and their prognostic significance: an analysis in 241 patients.

Haematological, immunophenotypic and cytogenetic characteristics were analysed in 241 patients with acute myeloid leukaemia (AML) M0, including 58 children. Children < 3 years and adults between 60 and 70 years of age were most frequently affected. Immunophenotyping showed a heterogeneous phenotype. Anti‐myeloperoxidase was positive in about half of the patients. Cytogenetic data were available from 129 (54%) patients. A normal karyotype was found in only 24%. Most of the abnormalities were unbalanced and the chromosomes 5, 7, 8 and 11 were the most frequently affected. Survival data were available from 152 treated patients (63%). The median overall survival for all patients was 10 months, 20 months for children (n = 36), 10 months for the young adult group (n = 50) and 7 months for the elderly patients (n = 66) (P = 0·09). Karyotype was not a prognostic factor influencing survival. AML M0 shows the immunological characteristics of early progenitor cells, but the expression of the different markers and cytogenetic abnormalities is heterogeneous. The prognosis is poor compared with other de novo AML and similar to that of AML with multilineage dysplasia or AML following myelodysplastic syndromes.

Adhesion to Bone Marrow Stroma Inhibits Apoptosis of Chronic Lymphocytic Leukemia Cells

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of monoclonal long-lived B cells which are apparently resistant to normal apoptotic regulation. Since bone marrow stromal cells play an essential role in B lymphopoiesis, we have investigated whether stromal cells influence B-CLL cell survival. Our results indicate that intimate contact with stromal cells reduces B-CLL cell apoptosis and prevents the loss of bcl-2 protein expression. Binding of B-CLL cells to stromal cells requires simultaneous action of beta1 and beta2 integrins. The interaction between B-CLL cells and other cell types seems important for their survival and may represent an important mechanism underlying accumulation of malignant cells in B-CLL patients.

Immunophenotypic patterns and cytogenetic anomalies in acute non-lymphoblastic leukemia subtypes: a prospective study of 432 patients.

This study prospectively analysed the relationships between immunophenotypic and cytogenetic features of blast cells in 432 acute non-lymphoblastic leukemias (ANLL) at presentation. An abnormal karyotype was detected in 232 cases (54%). These abnormalities were related to immunophenotypic markers as detected using a consensual panel of monoclonal antibodies allowing lineage assignment and investigation of myeloid marker expression on blast cells. In univariate analysis, CD9, CD10, CD15, CD34 and TdT expression appeared significantly associated with chromosomal anomalies. Multivariate analysis identified CD34 and CD9 expression as independently predictive of the presence of at least one cytogenetic abnormality (P < 10−4 and P < 0.03, respectively). significant associations between immunophenotypic and karyotypic features were observed both within individual fab subgroups and independently from morphological criteria. specific features were seen in five anll entities: m0 or m1/b lineage antigen positivity/t(9;22) or del(11)(q23); m2/cd13−/t(8;21); M4/CD13+, CD34+, CD36+/inv(16); M4 or M5/lack of B lineage antigen/del(11)(q23) or t(9;11). More practically, and although the relationships demonstrated only represent a fraction of homogeneous immunophenotypic subgroups, identification of such immunophenotypic features should prompt careful karyotypic examination, eventually using molecular biology analysis on non-growing cells.

Heterogenous response of B lymphocytes to transforming growth factor-beta in B-cell chronic lymphocytic leukaemia: correlation with the expression of TGF-beta receptors

We investigated the potential role of transforming growth factor‐beta (TGF‐β) on spontaneous and cytokine‐induced proliferation of B‐cell chronic lymphocytic leukaemia (B‐CLL) cells in vitro. Purified B lymphocytes from 21 B‐CLL patients were cultured for 5 d in the presence of medium alone, IL‐2 and/or IL‐10, in the presence or absence of TGF‐β, and proliferation was measured by 3H‐thymidine incorporation. TGF‐β inhibited B‐cell proliferation in the majority of patients (15/21) but no inhibition was detected in 6/21 patients whatever the type of stimulant used. Addition of neutralizing antibodies to TGF‐β increased spontaneous and cytokine‐induced proliferation; this effect was dose dependent and specific because addition of an irrelevant chicken IgG had no effect on B‐CLL proliferation. In resistant patients, neutralizing antibodies to TGF‐β did not increase the proliferation. The expression of TGF‐β receptors on B‐CLL cells was significantly lower than the one observed on normal CD5+ B lymphocytes for which the sensitivity to TGF‐β inhibition was more marked than in CLL. In addition, we found a strong correlation between the response of leukaemic B cells to TGF‐β inhibitory action and the expression of TGF‐β receptors on these cells. In summary, TGF‐β appears to function in CLL as a negative regulator of B lymphocytes but loss of responsiveness to this factor accompanied by a decrease of TGF‐β receptor expression, might provide a selective advantage to B‐CLL lymphocytes.

Early induction of apoptosis in B-chronic lymphocytic leukaemia cells by hydroxychloroquine: activation of caspase-3 and no protection by survival factors

We have investigated the effect of hydroxychloroquine (HCQ), an anti‐rheumatic drug, on malignant B cells from 20 patients with B‐chronic lymphocytic leukaemia (B‐CLL). HCQ induced a decrease in cell viability in a dose‐ and time‐dependent manner. The mean IC50 was 32 ± 7 μg/ml (range, 10‐75 μg/ml) for 24 h of exposure. This cytotoxic effect was owing to apoptosis, as demonstrated by morphological changes, annexin V binding capacity and DNA fragmentation (28 ± 4% of apoptotic cells as early as 5 h post incubation, increasing to 82 ± 4% at 18 h post treatment). The apoptosis was associated with caspase‐3 activation because the cleavage and activity of caspase‐3 were increased by HCQ. The amount of bcl‐2 protein was reduced during apoptosis, evidenced using quantitative flow cytometry. As early as 1 h post‐HCQ treatment, a reduction of the mitochondrial transmembrane potential was measured by 3,3’‐dihexyloxacarbocyanine iodide. Interestingly, the HCQ effect was not affected by exposure to interleukin‐4 or co‐culture with bone marrow stromal cells. Our observations suggest that HCQ may offer a new therapeutic tool in the treatment of B‐CLL patients.

TGF-beta activity and expression of its receptors in B-cell chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in Western countries and results from the accumulation of B-lymphocytes which are functionally abnormal and predominantly non-cycling in vivo. Consequently, it is important to understand why B-CLL cells accumulate in GO phase. Since TGF-beta is an important negative regulator of the immune system, a loss of responsiveness to this factor might provide a selective advantage to B-CLL cells. Here we review data on the role of TGF-beta in B-CLL. We show that the B-CLL cell response to TGF-beta signals is abnormal in vitro (inhibition of proliferation and induction of apoptosis). This lack of response of B-CLL cells to TGF-beta inhibition appears to be accompanied by a decrease or a loss of TGF-beta receptor expression.

Myeloid and Lymphoid Recovery Following Allogeneic Bone Marrow Transplantation: A Comparative Study between Related, Unrelated Bone Marrow and Allogeneic Peripheral Stem Cell Transplantation

We studied myeloid and lymphoid recovery during a period of 12 months following HLA matched allogeneic bone marrow transplantation (BMT) in 15 patients. Patients were divided into three groups. Each group contained 5 patients according to the source of hematopoietic stem cell transplantation (HST): 1) related bone marrow transplantation (BMT), 2) allogeneic peripheral blood stem cell transplantation (PBSCT) and 3) matched unrelated donor transplantation (MUD). The rate and pattern of recovery of granulocytes, lymphocytes (T-cell subsets, B-cells, NK cells, subsets of CD45) were studied by cell counting and flow cytometry. Our results suggest faster recovery of PMN after PBSCT. Higher CD4 cell counts observed in the PBSCT group may have an impact on a lower incidence of opportunistic infections. Chronic GvHD mediated GvL effect seems to be more important in blood stem cell transplanted patients and this may have an influence on disease free survival.

Immunological definition of acute minimally differentiated myeloid leukemia (MO) and acute undifferentiated leukemia (AUL).

Immunophenotyping has become an important tool in the diagnosis of acute leukemia for several reasons. Indeed the use of a standardized panel of monoclonal antibodies (MoAb) to B and T cells, and myeloid cells, as well as non lineage restricted antigens, permits allocation of more than 98% of acute leukemia to their respective lineage. In ALL, immunophenotyping has established a basis for precise and biologically oriented classification of the disease which may be of prognostic importance. In AML immunological markers are particularly important for identification of acute leukemia with minimal myeloid, erythroblastic or megakaryoblastic differentiation. Immunological markers also allow the identification of acute leukemias with minimal or aberrant marker expression, acute biphenotypic leukemia in which single cells coexpress different lineage associated markers and acute bilineage leukemia where there are two separate blast cell populations (usually lymphoid and myeloid). There is sometimes confusion in the literature about the definition of acute unclassifiable and acute undifferentiated leukemia. This is mainly due to misinterpretation of phenotypic data or to the lack of relevant lineage specific markers in these studies, especially for the detection of cytoplasmic antigens. Indeed, it is important to stress that in hematopoietic precursors, antigens detected by monoclonal antibodies first appear in the cytoplasm during early differentiation and are only expressed on the membrane later. This has been demonstrated not only for the T lineage (Cy CD3), the B lineage (CyCD22) but also for the myeloid lineage (CyCD13).(ABSTRACT TRUNCATED AT 250 WORDS)