Leukemia Research Vol. 16. No. 6/7. pp. 717 720. 1992, Printed in Great Britain.

0145-2126/92 $5.00 + .00 Pergamon Press Ltd

MEETING REPORT 5th I N T E R N A T I O N A L WORKSHOP ON CHRONIC LYMPHOCYTIC LEUKEMIA E. MONTSERRAT,J. L. BINET, D. CATOVSKY,G. DIGHIERO,R. P. GALE, K. R. RAI and C. ROZMAN Sitges (Barcelona), Spain, 26-28 April 1991

(Received 15 October 1991. Accepted 14 November 1991) Abstract--The International Workshop on CLL (IWCLL) met in Sitges (Barcelona), Spain, from 26 to 28 April 1991. Biology, clinical aspects and treatment of chronic lymphocytic leukemia (CLL) were reviewed by 60 investigators from 14 countries.* This is a brief summary of the highlights of the meeting reported in detail elsewhere.t

Key words: CLL, International workshop

BIOLOGY

igaris-Cappio (Turin, Italy), T. De France (Paris, France), G. Gahrton and G. Juliusson (Stockholm, Sweden), and B. Barlogie (Arkansas, U.S.A.). The major consideration was B-cell development, This is a complex dynamic process: about 109 B cells are produced daily. Most (75-90%) die, probably in the bone marrow and also in peripheral lymphoid tissues. Mechanisms responsible for selecting B cells are incompletely defined but are likely to involve both negative and positive factors. Although the early phases of B-cell development were previously thought to be antigen-independent, this may not be so. Programmed cell death (apoptosis) is likely to be important in determining the fate of B cells. A recent model of CLL involves transgenic mice with the BCL-2 transgene, a proto-oncogene implicated in follicular lymphomas with the t(14;18) translocation. B cells in these mice are immortalized leading to their accumulation in lymphoid tissues. Most mice die of an autoimmune disorder resembling systemic lupus erithemathosus (SLE). This is of interest in view of the increased incidence of autoantibodies in CLL. Of note, B cells immortalized in these mice are not the CD5+ B cells typical of CLL. Similarly, BCL-2 activation appears absent in CLL in humans (vide infra). A major issue in B-cell development is the role of cytokines including lymphokines and other growth regulating factors. Many of these molecules (e.g. IL2, IL-4, and IL-6) have direct or indirect (e.g. ILl or tumor necrosis factor) effects on normal and malignant B cells. These factors often interact. CLL B cells appear to release some of these constitutively.

THE first session focused on the biology of normal and C L L B cells. Speakers included M. Cooper (Alabama, U.S.A.), R. Foa (Turin, Italy), F. Cal* The 5th International Workshop on CLL (IWCLL) was held in Sitges (Barcelona), Spain, 26-28 April 1991, organized, on behalf of the IWCLL, by C. Rozman and E. Montserrat. Participants at this meeting were: G. Bandini (Italy), B. Barlogie (U.S.A.), A. Berrebi (Israel), J. L. Binet (France), V. A. Boussiotis (Greece), G. Brittinger (Germany), F. Caligaris-Cappio (Italy), D. Catovsky (U.K.), C. Chastang (France), B. Cheson (U.S.A.), M. Cooper (U.S.A.), T. De France (France), G. De Rossi (Italy), G. Dighiero (France), R. Foa (Italy), K. Foon (U.S.A.), R. P. Gale (U.S.A.), G. Gahrton (Sweden), C. H. Geisler (Denmark), A. Guarini (Italy), M. R. Grever (U.S.A.), T. Hamblin (U.K.), M. Hansen (Denmark), G. Haughton (U.S.A.), B. Jaksic (Croatia), G. Juliusson (Sweden), N. Kay (U.S.A.), M. J. Keating (U.S.A.), E. Kimby (Sweden), T. J. Kipps (U.S.A.), K. Lennert (Germany), E. Matutes (U.K.), H. Merle-Beral (France), M. Michallet (France), S. Molica (Italy), E. Montserrat (Spain), S. Mulligan (Australia), S. O'Brien (U.S.A.), A. Orfao (Spain), D. Oscier (U.K.), G. A. Pangalis (Greece), L. Piro (U.S.A.), A. Polliack (Israel), K. R. Rai (U.S.A.), J. C. Reverter (Spain), S. Richards (U.K.), A. A. Rimm (U.S.A.), C. Rozman (Spain), M. Rovira (Spain), A. Sawitsky (U.S.A.), J. Soler (Spain), D. Tassies (Spain), S. Tura (Italy), A. Urbano-Ispizua (Spain), T. Vallespl (Spain), N. Villamor (Spain), N. Vifiolas (Spain), B. Vitale (Croatia), S. Woessner (Spain), H. W. L. Ziegler-Heitbrock (Germany). t Leukemia & Lymphoma (1992) 5 (Suppl. 1), 1-188. Correspondence to: Prof. E. Montserrat, Postgraduate School of Hematology "Farreras Valenti", Hospital Clinic, Villarroel, 170, 08036 Barcelona, Spain. 717

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Because these cells also express growth factors receptors, the possibility of an autocrine stimulation of CLL has been raised. Microenvironmental (stromal) cells may also be involved in the response of normal and B cells to interleukins. These cells can release additional growth factors or presenting lymphokines or other factors to B cells or their progenitors at short range. Whether interleukins and other growth factors are important in causing or perpetuating CLL is unknown. Most studies are still in a descriptive phase. Extensive growth factor abnormalities are described in CLL B cells; it is uncertain if these are a cause or a consequence of the disease. The normal counterpart of the CD5 CLL B cell and activation pathways for normal and CLL B cells were also discussed. CD5 CLL B cells differ from their normal counterparts in several aspects including regulation of CD5 and BCL2 expression (vide supra) and response to IL-2 and IL-4 after in vitro activation with anti-Ig antibodies (an in vitro model of antigendependent activation) or antibodies to CD40 (an alternative, antigen-independent activation pathway). It is possible to release CLL B cells from their apparent block in maturation by combined antiIg and IL-2 or by anti-CD40 and IL-4. These data indicate plasticity of the CLL phenotype. This probably reflects the fact that CLL arises from transformation of a progenitor B cell rather than a more mature counterpart. For example, cells with both pre-B cells and plasma cells phenotype are detected in typical cases of CLL by using anti-idiotype antibodies or probes specific for Ig-gene rearrangements, and are therefore related to the CLL clone. These data question whether the CLL phenotype does reflect where the leukemic transformation occurs. Extensive cytogenetic data from patients with CLL indicate frequent trisomy 12, structural abnormalities involving chromosome 12q13 to q22, chromosome 13q14, and chromosome 14 are also common. These abnormalities have different prognostic implications, multiple and complex abnormalities being associated with poor outcome. In contrast to other leukemias, there is little evidence of cytogenetic clonal evolution in CLL. Restriction fragment length polymorphism (RFLP) and in situ hybridization analyses indicate frequent rearrangement or deletion of the retinoblastoma (RB) tumor suppressor gene. The meaning of this, however, is unclear since there is rarely homozygous inactivation or expression of an abnormal RB protein. BCL-2 is only infrequently rearranged in CLL. However, sensitive analyses suggested a greater frequency than would be suspected based on cytogenetic data. The second session focused on studies of Ig variable (V) gene expression, antibody activity of mouse

and human CD5 B-cell related malignancies, and phenotype variation in CLL B cells. Speakers included G. Haughton (Chapell Hill, U.S.A.), T. Kipps (San Diego, U.S.A.), G. Dighiero (Paris, France), and H. Merle-B6ral (Paris, France). Murine lymphomas which commonly arise from Ly 1+ (CD5+) B cells are an interesting model of CLL and other B-cell neoplasias. These tumors appear later in life and their frequency is increased by transferring syngeneic lymphoid cells from old to young animals. As these lymphomas are not detected until late in life, these experiments indicate that there is an early commitment to neoplastic development. Interestingly, these lymphomas frequently express cross-reactive idiotypes and restricted Ig VH and Ig VL genes. In mice, the repertoire of CD5+ B at birth and before antigen exposure approximates random selection. However, during the first few weeks of postnatal life, this repertoire is driven by predominantly type 2 thymus independent antigens upon an antigen selection process. The results is that 5-20~ of normal adult CD5+ B cells and mouse lymphomas bind to phosphatidyl choline. These cells express a very restricted pattern of V genes (VH11 generally paired to VK9 and VH12 generally paired to VK4). Another interesting feature is that CLL B lymphocytes frequently express a restricted set of genes revealed by the frequency with which membrane IgM from CLL B lymphocytes display binding to the Fc portion of IgG. Also, the G6 cross-reactive idiotype is frequently shared by CLL B cells, confirming a restricted use of VH genes. Interestingly, whereas CLL B cells expressing G6 idiotype display considerable structural similarities in CDR3 regions, normal G6 expressing B cells show substantial variation. These results suggest that there is a non-stoichastic IgV gene rearrangement in B-CLL. By using the heterohybridomas derived from CLL B cells it is possible to demonstrate that these cells are frequently committed to produce natural autoantibodies. The high frequency of this autoantibody activity among CLL B lymphocytes favors the idea that they are expressing a restricted set of genes. In summary, these data suggest that B-cell malignancies frequently occur within the autoreactive B-cell repertoire. The activation of this repertoire through a continuous challenge by self-antigens could create suitable conditions for malignant transformation. Finally, the phenotypic expression of myeiomonocytic and activation markers in CLL was also discussed. A proportion of CLL cases display either myelomonocytic (CDlla, C D l l c , CD14) or activation (CD25, CD38, CD71) antigens. The meaning of this is unknown.

Chronic lymphocyticleukemia CLINICAL ASPECTS Speakers in this session included D. Catovsky (London, U.K.), K. Lennert (Kiel, Germany), G. Brittinger (Essen, Germany), G. Pangalis (Athens, Greece), and E. Montserrat (Barcelona, Spain). The classification of CLL and related disorders was reviewed. Confusion still occurs within B-cell disorders which resemble B-CLL. The most common ones are the leukemic phase of low- or intermediategrade non-Hodgkin's lymphomas, chiefly follicular lymphomas, intermediate or mantle-zone lymphoma, and the so-called splenic lymphoma with villous lymphocytes. The distinct immunophenotype help to make the correct diagnosis in most instances. As a rule, non-Hodgkin's lymphomas in leukemic phase express strong SmIg, FMC7 and mCD22. In contrast, B-CLL has weak SmIg and do not express FMC7 or mCD22. Nevertheless, problems still arise because CLL has a degree of heterogeneity and may undergo transformation with a mixture of large and small cells or with increased number of prolymphocytes, a feature of prognostic importance. Another issue was the different histologic pictures of CLL and lymphoplasmacytic/cytoid immunocytoma (LP-IC) according to the Kiel classification. Three histologic subtypes are described in CLL (diffuse, pseudofollicular, and tumor forming) and LPIC (lymphoplasmacytoid, lymphoplasmacytic, and polymorphic). In contrast with CLL, EP-IC shows, besides a mixed picture of plasma cells and lymphocytes, other more subtle morphological differences, not only surface IgM/D but also IgG and rarely IgA, or heavy chains only; moreover, LP-IC shows more often remnants of lymphoid follicles recognizable from the network of follicular dendritic cells. PAS-positive intranuclear inclusions as well as increased mast cells, and hemosiderosis can be found in some cases of LP-IC but not in CLL. From the clinical point of view, there are also some differences, CLL displaying bone marrow infiltration and peripheral blood lymphocytosis, more frequently than LP-IC; also, the prognosis seems to be poorer for patients with LP-IC. Clearly, more experience on lymph node histology in CLL and LP-IC should accumulate to ascertain if these constitute separate entities rather than a continuous spectrum of the same process. The concept of 'smoldering' (or stable) CLL was also reviewed. There was agreement in that patients in early stage (Binet's A), Hb levels above 12 or 13 g/ dl, blood lymphocyte counts less than 30 x 109/1, and moderate bone marrow infiltration have a lifeexpectancy not different from controls. The only controversy in defining 'smoldering' CLL is how to evaluate bone marrow infiltration (biopsy or aspir-

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ate) and whether lymphocyte doubling time should be included or not among the criteria. Some patients may be classified differently if aspirate rather than biopsy is used to evaluate bone marrow infiltration. Doubling time, on the other hand, is a very useful dynamic parameter to assess the pace of the disease. Whatever the criteria used, however, there are approximately 10% of patients with early CLL who progress and that cannot be identified by the current criteria. Prospective studies are needed, particularly to ascertain whether patients with 'active' early disease (e.g. not fulfilling 'smoldering' criteria) may gain some benefit from being treated before a change to a more advanced stage is noticed. The natural history and prognosis of CLL in younger adults was also discussed. There are no differences in presenting features in young and old patients. Median survival of younger patients with CLL is 12 years vs 5 in older persons. Well-known prognostic factors (clinical stages, bone marrow histology, blood lymphocyte counts, and doubling time) discriminate groups with different survival. This is of interest when planning treatment (vide

infra). TREATMENT Speakers in this session included C. Chastang (Paris, France), M. M. Hansen (Copenhagen, Denmark), E. Kimby (Stockholm, Sweden), E. Montserrat, D. Catovsky, M. Keating (Houston, U.S.A.), M. Michallet (Grenoble, France), G. Pangalis and V. Boussiotis (Athens, Greece), L. Piro (La Jolla, U.S.A.), G. de Rossi (Rome, Italy), B. Jaksic (Zagreb, Croatia), K. Rai (New York, U.S.A.), S. Richards (Oxford, U.K.), A. Rimm (Medical College Wisconsin, U.S.A.), and J. L. Binet (Paris, France). Although chlorambucil and prednisone still are the basis for most treatment comparisons, promising new drugs and treatment strategies were discussed. The major issue was which patients should be treated and how. There was agreement that patients with CLL in early stage should not be treated unless they progress. Trials by the French, British (MRC), and IGIC (Vienna) groups have shown that early therapy with chlorambucil for stage A CLL produce no survival advantage over no treatment and even might be harmful. The reasons for this are unclear. Possibilities include increased numbers of epithelial neoplasms and disease progression following an initial response. These studies draw attention to the subtle and still undefined mechanisms by which the disease remains stable in a large proportion of stage A patients. There might be a subgroup of stage A patients (namely, those not fulfilling 'smoldering'

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criteria) (vide supra) who could gain some benefit from being treated early, but this remains unproved. Whereas there is no doubt that patients with advanced stage need to be treated, what is the best treatment is controversial. An important question is the role of anthracyclines. Although initial results from the French Cooperative Group indicated a beneficial effect of a modified CHOP regimen (cyclophosphamide, low-dose doxorubicin, vincristine, and prednisone) in stage C patients, these results require confirmation. Whilst complete remissions (CRs) are significantly higher with CHOP (with either low- or high-doxorubicin doses) this is not associated with a survival advantage in all studies. One of the striking findings of the French CLL 80 trial was the statistically significant survival difference in stage C patients treated with CHOP as compared to those receiving COP (median, 62 vs 22 months). This difference may be unduly large owing to a poor result in the COP arm. For example, in other studies median survival of stage C patients treated without anthracyclines is about 50 months. These results are an improvement on the median survivals for stage C (Rai stages III and IV) reported in the late 1970s and early 1980s, which ranged between 19 and 24 months. In the French CLL 85 trial there was no survival advantage for stage B patients treated with CHOP vs those treated with intermittent chlorambucil + prednisone, although the follow-up is still short. Whether there is some biological feature accounting for the different response rate of CLL patients according to their stage is not known. Clearly, more studies are needed. The uncertainties over anthracyclines may be resolved by larger and simpler trials and/or by meta-analysis. Two new drugs are fludarabine and 2-chlorodeoxyadenosine (2-CDA). Fludarabine is a highly promising agent for CLL therapy. The complete response (CR) rate in previously treated patients is about 40% and in those previously untreated, about 65%. Median remission duration is about 1.5-2 years, with one third of relapsing patients responding to retreatment. There are several ongoing randomized studies to further define the place of fludarabine in the treatment strategy of patients with CLL. Results with 2-CDA were also discussed. Almost one half of patients with refractory and advanced disease seem to respond to this agent, although responses are transient. Myelosuppression (specially, thrombocytopenia), and some immunosuppression, are the main side-effects. 2-CDA seems to be an

important new agent for refractory CLL which has to be further investigated. There is renewed interest in the possible role of alpha interferon (o:IFN) in CLL. Some studies suggest that odFN could be useful when given either simultaneously or sequentially with chlorambucil. However, o~IFN could be more effective when the tumor burden has been reduced by chemotherapy. Trials testing the effect of cdFN in the long-term control of the disease once the remission is achieved seem reasonable. Experience with allogeneic bone marrow transplantation (BMT) in young patients with CLL continues to accumulate slowly. The number of reported transplants is 26. Eleven patients are alive and in remission 5-48 months after transplant. Although there are insufficient data for critical analyses, allogenic transplants offer the possibility for complete remission and possibly cure in some young persons with CLL. This is important in view of the incurability of CLL with other approaches. However, the considerable early treatment mortality must be considered in the context of prognostic factors and the relatively long survival of young patients with CLL (vide supra). No data were presented on autotransplants in CLL. This approach, however, is currently being explored in some centers, particularly now that CRs can be obtained in CLL with CHOP or fludarabine. SUMMARY In the past few years important advances have been made in our understanding of the biology and natural history of CLL; also, new strategies and agents offer promise in the treatment of this form of leukemia. Now, patients can be treated on a more rationale basis and with real prospects for a sustained control of their disease, and improved quality of life. Hopefully, progresses in CLL will continue to accumulate in the coming years.

Acknowledgements--The 5th International Workshop on CLL was organized thanks to generous grants provided by: Berlex Laboratories (U.S.A.), Bristol-Myers S.A.E. (Spain), Cilag International, Menarini Diagn6sticos, S.A. (Spain), Schering Plough International, and Fondo de InvestigacionesSanitarias de la Seguridad Social, and Divisi6 de Ci~neies de la Salut from Barcelona University. Thanks are due to Ana Roncal6s, Maria-Jos6 S~inchezMelero, Carmen Torres, Cristina Montserrat, and Begofia Aranda for their secretarial support and assistance.