G. Dighiero

Chronic Lymphocytic Leukaemia: Proposals for a Revised Prognostic Staging System

In the past the clinical course of chronic lymphocytic leukaemia (CLL) has been considered to be highly variable primarily due to a lack of understanding of prognostic factors. In the mid-1’370s Rai et al (1975) developed a simple staging system based on easily obtainable clinical and haematological data. The validity of the Rai clinical staging classification to predict the expected duration of survival of CLL patients has been confirmed by a number of subsequent publications (Phillips et al, 1977; Binet et al, 1977; Dighiero et al, 1979; Santoro et al, 1979; Rundles & Moore, 1978; Montserrat et al, 1977). This staging system brought a new perspective to clinical investigations in CLL by allowing the study of groups of patients of similar life expectancy in more comparable therapeutic trials. With use, certain limitations of the staging system have been recognized. The major inadequacies that have emerged requiring a revision of the system include: (1) the use of too many clinical groups (stages) making the design of useful therapeutic trials more difficult; and (2) a lack of consideration for the role of isolated organomegaly (e.g. splenomegaly or hepatomegaly) without lymphadenopathy in estimating the clinical prognosis of patients. An international workshop on CLL met in Paris in November 1979 and data on 295 patients were presented (Binet et al, 1981). In a second meeting in Montreal in August 1980 data on a larger number of patients were pooled. The outcome of a multivariatc analysis of more than 900 patients demonstrated three patterns of survival curves and the analysis resulted in a proposal for a new staging classification of CLL. Patients with anaemia (Hb < 10 g/dl) and/or thrombocytopenia ( < 100 x 10y/l) were found to have the poorest prognosis and were to be called group C. The prognosis of the remaining patients (approximately 80% of all the patients) was found to depend on the number of clinically relevant areas involved. Clinical enlargement of the spleen, the liver, and of lymph nodes in the cervical, axillary and inguinal regions constituted * Sponsored by the French National Cancer League and the National Leukemia Association, Inc. (U.S.A.). Participants: A. AUQUIER, Paris, France; J. BENNETT, Rochester, New York, U.S.A.; J.-L. BINET, Paris, France; K. BREMER, Essen, Germany; D. CATOVSKY, London, England; P. CHANDRA, Upton, New York, U.S.A.; C. CHASTANG, Paris, France; E. P. CRONKITE, Upton, New York, U.S.A.; G. DIGHIERO, Paris, France; D. A. G. GALTON, London, England; M. M. HANSEN, Copenhagen, Denmark; A. HUANG, Durham, N. Carolina, U.S.A.; C. JACQUILLAT, Paris, France; E. MONTSERRAT, Barcelona, Spain; H. PIGUET, Rouen, France; K. R . RAI, New Hyde Park, New York, U.S.A.; C. ROZMAN, Barcelona, Spain; W. RUNDLES, Durham, N. Carolina, U.S.A.; A. SAWITSKY, New Hyde Park, New York, U.S.A.; P. STRYCKMANS, Brussells, Belgium; C. SULTAN, Paris, France; M. WEIL, Paris, France. Correspondence: Dr A. Sawitsky, Long Island Jewish-Hillside Medical Center,. New Hyde Park, New York 11042, U.S.A.

Small RNAs analysis in CLL reveals a deregulation of miRNA expression and novel miRNA candidates of putative relevance in CLL pathogenesis.

MicroRNAs (miRNAs) are a novel class of small noncoding RNA molecules that regulate gene expression by inducing degradation or translational inhibition of target mRNAs. There are more than 500 miRNA genes reported in the human genome, constituting one of the largest classes of regulatory genes. Increasing experimental evidence supports the idea of aberrant miRNA expression in cancer pathogenesis. We analyzed the pattern of miRNA expression in chronic lymphocytic leukemia (CLL) cells and our results showed a global reduction in miRNA expression levels in CLL cells associated to a consistent underexpression of miR-181a, let-7a and miR-30d. We observed overexpression of miR-155 and a set of five miRNAs that are differentially expressed between patients with different clinical outcomes. Five novel miRNA candidates cloned from leukemic cells are reported. Surprisingly, predicted mRNA targets for these novel miRNA revealed a high proportion of targets located in a small region of chromosome 1, which is frequently altered in human cancer. Additionally, several targets were shared by at least two of miRNA candidates. Predicted targets included several genes recently described as tumor suppressors. These data could afford new avenues for exploring innovative pathways in CLL biology and therapy.

Critical self-epitopes are key to the understanding of self-tolerance and autoimmunity

Abstract Our ideas of self-tolerance emerged from the horror autotoxicus concept of Ehrlich and blood group compatibility rules of Landsteiner, which influenced the clonal deletion hypothesis, and challenged the concept of autoimmunity propagated by Metchnikoff and his students. Normal individuals produce autoantibodies against self-antigens, although autoantibodies against a few critical self-epitopes have not been detected. Dighiero and Rose discuss critical and non-critical self-epitopes, key to the understanding of self-tolerance and autoimmunity.

Predictive value of serum thymidine kinase level for Ig-V mutational status in B-CLL.

In B-CLL IgVH genes mutational status is a major prognostic factor. Since sequencing of IgVH genes is not available in most laboratories, an easily performed surrogate assay is desirable. To identify the best surrogate assay, and to better discriminate prognostic subgroups we analyzed clinical and biological data from 58 typical CLL cases. A higher serum thymidine kinase level (>15 U/l) proved to be a strong predictor of mutational status, and the only independent one among the studied parameters. To further identify prognostic subgroups, cluster analysis was employed on 38 cases on which all data were available, which segregated two groups including 25 and 13 patients, respectively. These two clusters differed by their proliferative potential and appeared to discriminate patients with very different clinical course and outcome. s-TK was strikingly different among these two clusters, suggesting that s-TK level could be used routinely to identify patients at risk of progression.

High expression of AID and active class switch recombination might account for a more aggressive disease in unmutated CLL patients: link with an activated microenvironment in CLL disease

Interaction of chronic lymphocytic leukemia (CLL) B cells with tissue microenvironment has been suggested to favor disease progression by promoting malignant B-cell growth. Previous work has shown expression in peripheral blood (PB) of CLL B cells of activation-induced cytidine deaminase (AID) among CLL patients with an unmutated (UM) profile of immunoglobulin genes and with ongoing class switch recombination (CSR) process. Because AID expression results from interaction with activated tissue microenvironment, we speculated whether the small subset with ongoing CSR is responsible for high levels of AID expression and could be derived from this particular microenvironment. In this work, we quantified AID expression and ongoing CSR in PB of 50 CLL patients and characterized the expression of different molecules related to microenvironment interaction. Our results show that among UM patients (1) high AID expression is restricted to the subpopulation of tumoral cells ongoing CSR; (2) this small subset expresses high levels of proliferation, antiapoptotic and progression markers (Ki-67, c-myc, Bcl-2, CD49d, and CCL3/4 chemokines). Overall, this work outlines the importance of a cellular subset in PB of UM CLL patients with a poor clinical outcome, high AID levels, and ongoing CSR, whose presence might be a hallmark of a recent contact with the microenvironment.

Evaluation of residual disease in B-cell chronic lymphocytic leukemia patients in clinical and bone-marrow remission using CD5-CD19 markers and PCR study of gene rearrangements.

We evaluated minimal residual disease (MRD) in 23 CD5 + B-chronic lymphocytic leukemia (CLL) patients who achieved clinico-hematological remission confirmed by bone-marrow biopsy. MRD was evaluated by dual marker analysis flow-cytometry using CD5 and CD19 markers, and by the study of Ig heavy chain gene rearrangements using the fast polymerase chain reaction (PCR). According to our laboratory conditions patients were considered to be in complete phenotypic remission when total CD19+ cells were < 25% and the ratio of CD5 + CD19 + /CD19 + cells was < 25%. According to these strict criteria only 9 of the 23 patients were in complete phenotypic remission. In order to evaluate the sensitivity of the above method, PCR analysis of the configuration of the Ig heavy chain gene region was performed in 12 of these patients. Five of 7 patients in complete phenotypic remission retained a detectable monoclonal rearrangement of the Ig heavy chain gene. For the remaining 5 patients in partial phenotypic remission, only one failed to show a monoclonal band and this is probably explained by the presence of an unusual gene rearrangement. In conclusion, this study suggests that PCR is more sensitive than dual marker flow-cytometry for evaluation of residual disease and that it is indeed possible to achieve complete remission at the molecular level, in B-CLL. Nevertheless, we suggest a word of caution as this was a retrospective study, and samples were not assessed before treatment. Thus the possibility that apparent molecular remission might correspond to unusual gene rearrangements cannot be completely excluded in these cases.

Natural killer (NK) cell activity during HIV infection: a decrease in NK activity is observed at the clonal level and is not restored after in vitro long-term culture of NK cells.

NK cell activity is impaired in HIV‐infected patients. The mechanisms behind the altered NK functions are not clear, and conflicting data concerning NK and antibody‐dependent cellular cytotoxicity (ADCC) activity have been reported. In order to investigate whether this impairment is also observed at the clonal level and whether it is related to a defect at the target cell binding and/or the post‐binding level, we evaluated highly purified NK cell lines and cloned NK cells obtained from 22 HIV‐infected patients at different stages of disease and compared them with normal controls for their ability to: (i) kill K‐562 and U‐937 cell lines using a 51Cr release assay; (ii) bind and kill K‐562 and U‐937 cells at the single cell binding level; (iii) release NK cytotoxic factor (NKCF), tumour necrosis factor‐alpha (TNF‐α) and interferon‐gamma (IFN‐γ); (iv) kill anti‐IgM preincubated Daudi cell line (ADCC activity). This study with cloned NK cells or NK cell lines from HIV‐infected individuals showed: (i) a decrease in their lytic capability against target cell lines; (ii) a low ability to form conjugates with K‐562 and U‐937 cell lines with respect to controls; (iii) a decreased ability to kill bound target cells; (iv) low levels of released NKCF, TNF‐α and IFN‐γ after incubation with U‐937 cells. Taken together, these findings suggest that the impaired NK cell function during HIV infection is also observed at the clonal level and is related to defects both at the target and post‐binding levels. However, the precise mechanisms remain to be determined. The inability to restore normal NK activity after long‐term culture in the presence of high levels of recombinant IL‐2 is in agreement with the hypothesis of a ‘general anergic process’ during HIV infection.

Sera with high levels of anti-smooth muscle and anti-mitochondrial antibodies frequently bind to cytoskeleton proteins.

Using ELISA methods, 54 sera from chronic active hepatitis (CAH) patients displaying high levels of anti‐smooth muscle antibodies (SMA) and 18 sera from primary biliary cirrhosis (PBC) patients with high levels of anti‐M2 antibodies were examined for the presence of high antibody levels against actin, tubulin, myosin, tropomyosin, troponin. vimenlin and desmin. Our results showed that: (i) in CAH with high SMA activity, increased antibody levels were found in 51‐9% of sera for actin, 31.5% for myosin, 35.2% for tubulin, 34.0% for tropomyosin, 11.3% for troponin. 22.6% for vimentin and 43.4% for desmin, compared with natural antibody levels in 21 normal sera; (ii) Similar high levels of these antibodies were found in the case of PBC; (iii) in most cases, sera simultaneously bound to several antigens of the panel; and (iv) approximately 26% of the CAH sera were found to be negative with the seven antigens examined while 22% were reacted with a cytoskeleton protein (CP) other than actin. These results indicate that current opinion associating SMA with anti‐actin activity in CAH is confirmed for only 50% of cases and that although a good correlation between SMA and anti‐CP antibodies can be obtained, there is still a significant percentage of SMA for which the putative antigen recognized needs to be determined.

Do CLL B cells correspond to naive or memory B-lymphocytes? Evidence for an active Ig switch unrelated to phenotype expression and Ig mutational pattern in B-CLL cells

Recent work suggests that chronic lymphocytic leukemia (B-CLL) expressing unmutated immunoglobulin V genes could correspond to the proliferation of naive B cells whereas those expressing mutated genes, may correspond to the proliferation of post-germinal center B cells. Current data from gene profiling expression have failed to demonstrate a clear-cut distinction between these two forms of B-CLL disease. In the present study, we have investigated the complete VH nucleotide sequence and the presence of RNA transcripts from different CH domains in 25 B-CLL patients. Our results demonstrate that: (1) expression of IgD is not related to the mutational frequency and activation of the isotype switch pathway; (2) isotype switch, leading to simultaneous expression at the transcriptional and protein level of IgM, IgD, IgG and IgA, occurs in a small percentage of patients, and (3) different mechanisms such as VDJ duplication and trans-splicing or RNA splicing of long nuclear transcript, could be involved in isotype switch. Our results highlight the difficulty in assigning a normal counterpart to B-CLL cells and raise the possibility that a different B cell development pathway, independent from classical germinal centers, might exist in B-CLL.

Impaired proliferative capacity and abnormal cytokine profile of naive and memory CD4 T cells from HIV-seropositive patients.

Purified naive and memory CD4 T cells from healthy donors, HIV+ asymptomatic carriers and AIDS patients were examined for their proliferative activity and their pattern of cytokine secretion (IL‐4, IL‐6, interferon‐gamma (IFN‐γ) and tumour necrosis factor‐alpha (TNF‐α)) upon stimulation with phytohaemagglutinin (PHA), phorbol myristate acetate (PMA) and cross‐linked anti‐CD3 MoAb, in the presence of recombinant IL‐2 (rIL‐2). We found a decrease in the proliferative capacity of naive CD4 T cells following stimulation with PHA and PMA, and a sharp decline in this response upon cross‐linked anti‐CD3 stimulation in both subsets, although it predominated in the naive subpopulation. In AIDS patients, less pronounced impairment of thymidine uptake by the naive subset was found upon PHA and cross‐linked anti‐CD3 MoAb stimulation. In addition, an altered secretion pattern of the different cytokines was observed, consisting of abnormal secretion of IL‐6 by both naive and memory cells, an abnormal pattern of IFN‐γ secretion and frequent loss of detectable lL‐4 production by HIV patients. These abnormalities were even more pronounced in AIDS patients than in the asymptomatic carriers. Overall, our results extend previous reports indicating functional impairment of memory CD4 subsets in HIV+ subjects by showing that this impairment involves naive CD4 T cells.