Acute myeloid leukaemia with 8p11 (MYST3) rearrangement: an integrated cytologic, cytogenetic and molecular study by the groupe francophone de cytogénétique hématologique.

Thirty cases of acute myeloid leukaemia (AML) with MYST histone acetyltransferase 3 (MYST3) rearrangement were collected in a retrospective study from 14 centres in France and Belgium. The mean age at diagnosis was 59.4 years and 67% of the patients were females. Most cases (77%) were secondary to solid cancer (57%), haematological malignancy (35%) or both (8%), and appeared 25 months after the primary disease. Clinically, cutaneous localization and disseminated intravascular coagulation were present in 30 and 40% of the cases, respectively. AMLs were myelomonocytic (7%) or monocytic (93%), with erythrophagocytosis (75%) and cytoplasmic vacuoles (75%). Immunophenotype showed no particularity compared with monocytic leukaemia without MYST3 abnormality. Twenty-eight cases carried t(8;16)(p11;p13) with MYST3-CREBBP fusion, one case carried a variant t(8;22)(p11;q13) and one case carried a t(8;19)(p11;q13). Type I (MYST3 exon 16-CREBBP exon 3) was the most frequent MYST3-CREBBP fusion transcript (65%). MYST3 rearrangement was associated with a poor prognosis, as 50% of patients deceased during the first 10 months. All those particular clinical, cytologic, cytogenetic, molecular and prognostic characteristics of AML with MYST3 rearrangement may have allowed an individualization into the World Health Organization classification.

Ontology-based lymphocyte population description using mathematical morphology on colour blood images.

Despite modern technologies (immunophenotyping, molecular probing, etc.) cytomorphologic examination of stained peripheral blood smears by microscopy remains the main way of diagnosis in a large variety of diseases (e.g. leukaemic disorders). Using tools from mathematical morphology for processing peripheral blood colour images, we have developed an image-based approach, to provide an objective and understandable description of lymphocyte populations according to a specifically designed ontology. This ontology-based framework needs a conceptualisation of the problem from a morphological viewpoint, the introduction of an adapted language, the generation of representative image databases, the development of image processing and data classification algorithms to automate the procedure and the validation of the system by human expertise. In this paper we present the main concepts, algorithms and some results to illustrate the high-performance of the approach. The aim of our work is to reconcile the automatisation with the medical expertise, so that they can reinforce each other.

Non-Hodgkin's lymphomas with Burkitt-like cells are associated with c-Myc amplification and poor prognosis.

Out of 344 patients with newly diagnosed non-Hodgkin's lymphoma (NHL), this study identified 16 patients presenting Burkitt-like cells (BLCs) after cytological and/or histological review. Conventional cytogenetic analysis showed at diagnosis complex chromosomal abnormalities in 13 cases and a normal karyotype in three cases. However, neither t(8;14)(q24;q32) nor the variants t(2;8)(p12;q24) or t(8;22)(q24;q11) was detected. FISH studies showed c-MYC amplification in all cases with four to more than seven copies in 10 - 77% metaphase or inter-phase cells. This study did not observe any gene fusion signal for c-MYC/IgH excluding a t(8;14) translocation and partial tri or polysomy of chromosome 8. It also excluded in that cases a break apart for the c-MYC locus. This study also never detected IgL/c-MYC, IgK/c-MYC or X-c-MYC. The BLCs were present whatever the lymphoma sub-type: follicular lymphoma (FL) was diagnosed in six out of 16 patients, mantle cell lymphoma (MCL) in four out of 16 patients, marginal zone lymphoma (MZL) in two out of 16 patients and diffuse large B-cell lymphomas (DLBCL) in three out of 16 patients. One additional patient presented a T-cell lymphoma. The clinical course was aggressive with a poor prognosis, as death occurred in nine patients, within 6 months after diagnosis for eight of them. These data could suggest a sub-group of NHL patients (15 B-NHL, 1 T-NHL) have been identified with a poor prognosis characterized by the association of Burkitt-like cells and c-MYC amplification without t(8;14)(q24;q32) or its variants. The possibility that this profile may represent a distinct morphologic NHL sub-set remains to be determined on a large cohort of patients.

Cytogenetic study of 75 erythroleukemias.

Chromosomal abnormalities of erythroleukemia (EL) are often described as complex and unspecific. A retrospective study of 75 EL defined following the WHO classification was performed by the Groupe Francophone de Cytogénétique Hématologique (GFCH) in order to reexamine the cytogenetics of this infrequent leukemia subtype. Clonal chromosomal abnormalities were found in 57 patients (76%), distributed in 4 subgroups according to their ploidy status: pseudodiploid (16%), hypodiploid (47%), hyperdiploid (19%), and 18% mixed cases associating 2 different clones (hypodiploid+hyperdiploid) or (pseudodiploid+hyperdiploid). Complex rearrangements and hypodiploid chromosome number were widely dominant (50%). Partial or entire monosomies represented 56% of abnormalities. Chromosomes 5 and 7 were the most frequently involved (41 and 33 times, respectively), followed by chromosomes 8, 16, and 21 (19 times each). Unbalanced abnormalities were more frequent than balanced. All these kinds of abnormalities were observed in de novo as well as in secondary EL. Four out of 7 cases of "pure erythroid" leukemia were associated with a BCR-ABL fusion. Lastly, no chromosome abnormality specific to EL could be established. However, the large overlap of chromosomal abnormality patterns of EL (pure erythroid form excepted) and refractory anemia with excess of blasts in transformation (RAEB-t) favors the hypothesis of similarities between these 2 hematologic disorders.

[Reproductibility of the morphological identification of schisocytes and evaluation of non observer-dependent methods]. / Schizocytes : reproductibilité du diagnostic morphologique et intérêt de méthodes observateur-indépendantes.

Schistocytes are red blood cell fragments observed on a blood smear. They are a mark of mechanical haemolytic anaemias whose group of the thrombotic micro-angiopathies requires an urgent treatment. The detection of the schistocytes and sometimes their quantification are thus of primary importance. To evaluate this search for schistocytes, several surveys of practice were carried out (1999-2003) including pictures of blood fields (identification of schistocytes among abnormal red blood cells) near biologists of variable level of specialization. The aim was to try to lead to a consensus, in particular for the morphological criterias of identification. Our results indicated that: 1) the biologists are badly sensitized with the importance of this research and the consequences of their response for the diagnosis; 2) the morphological identification of the schistocytes is difficult with an important variability of the criteria according to the observers. An investigation overviewed by the French Group of Cellular Hematology (Delphi method) allowed the development of a morphological consensus (fragments of triangular/crescent/helmet forms with rectilinear zone testifying to the zone of break). In order to cancel the observer-dependent identification of the schistocytes, a software of morphometric analysis (Q-WIN, Leica) was developed for sorting, starting from digitalized microscopic fields, the fragmented - among the normal - red blood cells. The results appeared encouraging, but not yet optimized. An automated analyzer (Bayer ADVIA 120) was also evaluated for the measurement of the schizocytes ("fragmented red blood cells" parameter). The moderate over-estimation of the real schizocytes (+ 0,4%) encouraged to observe the clinical value of the fragmented red blood cells detection in a group of patients that undergone a bone-marrow transplantation. The predictive value of the test (98%) was satisfactory.

Chromosomal instability and ATR amplification gene in patients with persistent and polyclonal B-cell lymphocytosis (PPBL).

Forty-three patients with persistent and polyclonal B-cell lymphocytosis (PPBL) were studied. The PPBL diagnosis was based on the presence of a polyclonal lymphocytosis and the detection of binucleated lymphoid cells on peripheral blood examination. In order to define the cytogenetic profile in these patients, conventional cytogenetic analysis and fluorescence in situ hybridization were performed at diagnosis in all patients and also at follow-up in 10 patients. When excluding + i(3q) and PCC, chromosomal instability is a common occurrence in PPBL and is characterized by other independent clonal abnormalities, del(6q), + der(8) or + 8, polyploid karyotype, structural changes, aneuploidy and/or non clonal chromosomal aberrations with either loss or more frequently gain of chromosomes. These data show the presence of a chromosomal instability in 67.5% of PPBL patients. Finally, ATR amplification was detected by hybridization with BAC probe 26217 in + i(3q) positive metaphase cells. No ATR deletion was observed in the + i(3q) negative B-cells. As the natural history of PPBL remains unclear, it is necessary to diagnose PPBL patients and useful to recommend a careful and continued long follow-up in all PPBL patients.

Report of 34 patients with clonal chromosomal abnormalities in Philadelphia-negative cells during imatinib treatment of Philadelphia-positive chronic myeloid leukemia.

Imatinib mesylate (Gleevec), an inhibitor of the BCR-ABL tyrosine kinase, was introduced recently into the therapy of chronic myeloid leukemia (CML). Several cases of emergence of clonal chromosomal abnormalities after therapy with imatinib have been reported, but their incidence, etiology and prognosis remain to be clarified. We report here a large series of 34 CML patients treated with imatinib who developed Philadelphia (Ph)-negative clones. Among 1001 patients with Ph-positive CML treated with imatinib, 34 (3.4%) developed clonal chromosomal abnormalities in Ph-negative cells. Three patients were treated with imatinib up-front. The most common cytogenetic abnormalities were trisomy 8 and monosomy 7 in twelve and seven patients, respectively. In 15 patients, fluorescent in situ hybridization with specific probes was performed in materials archived before the initiation of imatinib. The Ph-negative clone was related to previous therapy in three patients, and represented a minor pre-existing clone that expanded after the eradication of Ph-positive cells with imatinib in two others. However, in 11 patients, the new clonal chromosomal abnormalities were not detected and imatinib may have had a direct effect. No myelodysplasia was found in our cohort. With a median follow-up of 24 months, one patient showed CML acceleration and two relapsed.

[Role of the biologist in the study of schistocytes]. / Rôle du biologiste confronté à la recherche de schizocytes.

The appearance of schistocytes in a peripheral blood film is considered to be an important diagnostic marker for thrombotic microangiopathy. However, the morphological analysis of schistocytes remains uneasy. To determine practice patterns in the biological management of schistocytosis, the French Group of Cellular Hematology from the French Society of Hematology conducted a survey on the approach of the diagnosis of microangiopathy. A guideline is proposed in order to cancel the substantial variation among biologists.

[New diagnostic approaches in malignant B-cell lymphomas]. / Nouvelles approches diagnostiques des lymphomes malins B.

The new WHO classification of B-cell neoplasms published in 2001 gathers leukaemia and lymphoma. This classification highlights the stage of differentiation of the tumour cell. It groups B-cell neoplasms morphology, histology (nodular or diffuse pattern, cell morphology), immunophenotypes, cytogenetic, molecular abnormalities and clinical data. Recent advances of the normal B cell differentiation understanding and the development of new types of analysis have shown that mature B-cell neoplasms appear to recapitulate stages of normal B-cell differentiation, leading to such a new classification.

[Persistent polyclonal B-cell lymphocytosis]. / Lymphocytose polyclonale avec lymphocytes sanguins binucléés.

Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare and recently described entity. PPBL is diagnosed predominantly but not exclusively in women, usually smokers and is characterized by a moderate, chronic and absolute lymphocytosis (> 4 x 109/L). Peripheral blood examination show in all cases atypical binucleated lymphocytes. A polyclonal serum IgM is also associated and HLA-DR7 expression is present in most cases. The B cells are polyclonal with kappa and lambda light-chain expression. No clonal rearrangement of immunoglobulin heavy chain genes is observed. Finally, +i(3q) is a recurrent chromosomal abnormality and detected in 77% of cases with premature chromosome condensation in 50% and both abnormalities in 41% of cases. The benign clinical course of PPBL and lack of biological evolution in the majority of cases suggest that recognition is so important that aggressive therapy could be avoided.

Morphometric and colorimetric analysis of peripheral blood smears lymphocytes in B-cell disorders: proposal for a scoring system.

Distinguishing leukemic phases of B-cell disorders in peripheral blood smears is well recognized to be difficult in some cases since it depends on subtle and subjective criteria. In order to quantify cytological features and to assess objective descriptions, a morphometric analysis was performed on 83 peripheral blood smears of B-cells disorders (n = 77) and healthy donors (n = 6). Using standardized May-Grunwald Giemsa staining, standardized image acquisition system and well defined microscopic fields, we have analyzed lymphoid cells, measuring morphometric and color parameters. By combining seven relevant morphometric criteria (the nuclear shape, the cellular shape and area, the nucleo-cytoplasmic ratio, the nuclear red/blue ratio, the cytoplasmic green/blue ratio and the proportion of cells with nucleolus), we have established a score that could range from a minimum of -3 (large B-CLL type) to a maximum of +8 (large MCL type): negative scores corresponds to different types of B-CLL (n = 30), including "atypical B-CLL" (n = 6), the score zero correspond to healthy donors (n = 6) used as baseline, the positive score values correspond to +1 for Follicular lymphoma (n = 2), +3 for Splenic Lymphoma with Villous Lymphocytes (n = 12), +4 for Hairy Cell Leukemia (n = 7), for Hairy Cell Leukemia-variant (n = 2), +6 for B-prolymphocytic leukemia (n = 6) and +7 and +8 for most Mantle Cell Lymphoma (n = 18). Testing T-cell disorders samples (n = 10) using the same protocol, the profile is different and cannot be confused with B-cell diseases. Our scoring system indicates that measurement of some common morphologic features in standardized conditions provides objective criteria to characterize those diseases and might be helpful for diagnosis.

A multicentric study of 41 cases of B-prolymphocytic leukemia: two evolutive forms.

B-prolymphocytic leukemia (B-PLL) is an infrequent disease with a poor prognosis. We present the clinical and biological features of 41 patients. Median age was 67 years [42-89] and male-female sex ratio was 2.4. The immunophenotyping revealed B-cell phenotype, with a high level expression of surface IgM and/or IgD in all cases, FMC7+ in 76 % of cases and CD5+ in 67%. Marked spontaneous in-vitro apoptosis was observed in most cases tested (n = 12). The median overall survival time was 5 years and the event-free survival time was 37 months. As detected by univariate and multivariate analysis, the only variables associated with a poor prognosis were advanced age and anemia. No significant difference was observed between de novo PLL (n = 27) and prolymphocytoid transformation of chronic lymphocytic leukemia (n = 14). Two groups of patients were individualized according to their clinical course: patients who died within one year of diagnosis (n = 14) and patients who had a prolonged survival (n = 23) without any treatment in some cases. The comparison between the 2 groups showed that they differed in age (p = 0.01) and anemia (p = 0.02). We also observed that the patients with p53 mutations had a worse clinical outcome. Taken together these data confirm that B-PLL should be regarded as a distinct form of chronic lymphoproliferative disorder and suggest the existence of two patterns of clinical evolution.

A new morphologic classification system for acute promyelocytic leukemia distinguishes cases with underlying PLZF/RARA gene rearrangements.

Acute promyelocytic leukemia (APL) is typified by the t(15;17) translocation, which leads to the formation of the PML/RARA fusion gene and predicts a beneficial response to retinoids. However, approximately 10% of all APL cases lack the classic t(15;17). This group includes (1) cases with cryptic PML/RARA gene rearrangements and t(5;17) that leads to the NPM/RARA fusion gene, which are retinoid-responsive, and (2) cases with t(11;17)(q23;q21) that are associated with the PLZF/RARA fusion gene, which are retinoid-resistant. A key issue is how to rapidly distinguish subtypes of APL that demand distinct treatment approaches. To address this issue, a European workshop was held in Monza, Italy, during June 1997, and a morphologic, immunophenotypic, cytogenetic, and molecular review was undertaken in 60 cases of APL lacking t(15;17). This process led to the development of a novel morphologic classification system that takes into account the major nuclear and cytoplasmic features of APL. There were no major differences observed in morphology or immunophenotype between cases with the classic t(15;17) and those with the cryptic PML/RARA gene rearrangements. Auer rods were absent in the t(5;17) case expressing NPM/RARA. Interestingly, this classification system distinguished 9 cases with t(11;17)(q23;q21) and, in addition, successfully identified 2 cases lacking t(11;17), which were subsequently shown to have underlying PLZF/RARA fusions. The PLZF/RARA cases were characterized by a predominance of blasts with regular nuclei, an increased number of Pelger-like cells, and by expression of CD56 in 4 of 6 cases tested. Use of this classification system, combined with an analysis for CD56 expression, should allow early recognition of APL cases requiring tailored molecular investigations. (Blood. 2000;96:1287-1296)

Characterization of acute promyelocytic leukemia cases lacking the classic t(15;17): results of the European Working Party. Groupe Français de Cytogénétique Hématologique, Groupe de Français d'Hematologie Cellulaire, UK Cancer Cytogenetics Group and BIOMED 1 European Community-Concerted Action "Molecular Cytogenetic Diagnosis in Haematological Malignancies".

Acute promyelocytic leukemia (APL) is typified by the t(15;17), generating the PML-RAR alpha fusion and predicting a beneficial response to retinoids. However, a sizeable minority of APL cases lack the classic t(15;17), prompting the establishment of the European Working Party to further characterize this group. Such cases were referred to a workshop held in Monza, Italy and subjected to morphologic, cytogenetic, and molecular review, yielding 60 evaluable patients. In the majority (42 of 60), molecular analyses revealed underlying PML/RAR alpha rearrangements due to insertions (28 of 42) or more complex mechanisms, including 3-way and simple variant translocations (14 of 42). Metaphase fluorescence in situ hybridization (FISH) demonstrated that insertions most commonly led to formation of the PML-RAR alpha fusion gene on 15q. In 11 of 60 workshop patients, PLZF/RAR alpha rearrangements were identified, including 2 patients lacking the t(11;17)(q23;q21). In one case with a normal karyotype, FISH analysis revealed insertion of RAR alpha into 11q23, and PLZF-RAR alpha was the sole fusion gene formed. Two patients were found to have t(5;17), one with a diffuse nuclear NPM staining pattern and with NPM-RAR alpha and RAR alpha-NPM transcripts detected. In the other with an unbalanced der(5)t(5;17)(q13;q21) and a nucleolar NPM localization pattern, an NPM/RAR alpha rearrangement was excluded, and FISH revealed deletion of one RAR alpha allele. In the remaining 5 workshop patients, no evidence was found for a rearrangement of RAR alpha, indicating that in rare instances, alternative mechanisms could mediate the differentiation block that typifies this disease. This study highlights the importance of combining morphologic, cytogenetic, and molecular analyses for optimal management of APL patients and better understanding of the pathogenesis of the disease. (Blood. 2000;96:1297-1308)

Hypergranular promyelocytic leukemia: correlation between morphology and chromosomal translocations including t(15;17) and t(11;17).

The FAB group has reviewed 32 cases of promyelocytic leukemia and variant forms. By utilizing published criteria the ability to make a correct diagnosis by morphology with molecular genetic confirmation and to eliminate cases that did not have the PML/RARalpha rearrangement was excellent.

Lymphoma classification--from controversy to consensus: the R.E.A.L. and WHO Classification of lymphoid neoplasms.

BACKGROUND: Controversy in lymphoma classification dates back to the first attempts to formulate such classifications. Over the years, much of this controversy arose from the assumption that there had to be a single guiding principle--a 'gold standard'--for classification, and from the existence of multiple different classifications. DESIGN: The International Lymphoma Study Group (I.L.S.G.) developed a consensus list of lymphoid neoplasms, which was published in 1994 as the 'Revised European-American Classification of Lymphoid Neoplasms' (R.E.A.L.). The classification is based on the principle that a classification is a list of 'real' disease entities, which are defined by a combination of morphology, immunophenotype, genetic features, and clinical features. The relative importance of each of these features varies among diseases, and there is no one 'gold standard'. In some tumors morphology is paramount, in others it is immunophenotype, a specific genetic abnormality, or clinical features. An international study of 1300 patients, supported by the San Salvatore Foundation, was conducted to determine whether the R.E.A.L. Classification could be used by expert pathologists and had clinical relevance. Since 1995, the European Association of Pathologists (EAHP) and the Society for Hematopathology (SH) have been developing a new World Health Organization (WHO) Classification of hematologic malignancies, using an updated R.E.A.L. Classification for lymphomas and applying the principles of the R.E.A.L. Classification to myeloid and histiocytic neoplasms. A Clinical Advisory Committee (CAC) was formed to ensure that the WHO Classification will be useful to clinicians. RESULTS: The International Lymphoma Study showed that the R.E.A.L. Classification could be used by pathologists, with inter-observer reproducibility better than for other classifications (> 85%). Immunophenotyping was helpful in some diagnoses, but not required for many others. New entities not specifically recognized in the Working Formulation accounted for 27% of the cases. Diseases that would have been lumped together as 'low grade' or 'intermediate/high grade' in the Working Formulation showed marked differences in survival, confirming that they need to be treated as distinct entities. Clinical features such as the International Prognostic Index were also important in determining patient outcome. The WHO Clinical Advisory Committee concluded that clinical groupings of lymphoid neoplasms was neither necessary nor desirable. Patient treatment is determined by the specific type of lymphoma, with the addition of grade within the tumor type, if applicable, and clinical prognostic factors such as the International Prognostic Index (IPI). CONCLUSIONS: The experience of developing the WHO Classification has produced a new and existing degree of cooperation and communication between oncologists and pathologists from around the world, which should facilitate progress in the understanding and treatment of hematologic malignancies.

The World Health Organization classification of hematological malignancies report of the Clinical Advisory Committee Meeting, Airlie House, Virginia, November 1997.

Since 1995, the European Association of Pathologists and the Society for Hematopathology have been developing a new World Health Organization (WHO) classification of hematologic malignancies. The classification includes lymphoid, myeloid, histiocytic, and mast cell neoplasms. The WHO project involves 10 committees of pathologists, who have developed lists and definitions of disease entities. A Clinical Advisory Committee of international hematologists and oncologists was formed to ensure that the classification will be useful to clinicians. A meeting was held in November 1997 to discuss clinical issues related to the classification. The WHO has adopted the Revised European-American Classification of Lymphoid Neoplasms, published in 1994 by the International Lymphoma Study Group, as the classification of lymphoid neoplasms. This approach to classification is based on the principle that a classification is a list of "real" disease entities, which are defined by a combination of morphology, immunophenotype, genetic features, and clinical features. The relative importance of each of these features varies among diseases, and there is no one "gold standard." The WHO classification has applied the principles of the Revised European-American Classification of Lymphoid Neoplasms to myeloid and histiocytic neoplasms. The classification of myeloid neoplasms recognizes distinct entities defined by a combination of morphology and cytogenetic abnormalities. The Clinical Advisory Committee meeting, which was organized around a series of clinical questions, was able to reach a consensus on most of the questions posed. The questions and the consensus are discussed in detail in this article. Among other things, the Clinical Advisory Committee concluded that clinical grouping of lymphoid neoplasms was neither necessary nor desirable. Patient treatment is determined by the specific type of lymphoma, with the addition of grade within the tumor type, if applicable, and clinical prognostic factors such as the international prognostic index. The experience of developing the WHO classification has produced a new and exciting degree of cooperation and communication between oncologists and pathologists from around the world. This should facilitate progress in the understanding and treatment of hematologic malignancies.

The World Health Organization classification of neoplastic diseases of the haematopoietic and lymphoid tissues: Report of the Clinical Advisory Committee Meeting, Airlie House, Virginia, November 1997.

INTRODUCTION: Since 1995, the European Association of Pathologists (EAHP) and the Society for Hematopathology (SH) have been developing a new World Health Organization (WHO) classification of haematological malignancies. The classification includes lymphoid, myeloid, histiocytic and mast cell neoplasms. DESIGN: The WHO project involves 10 committees of pathologists, who have developed lists and definitions of disease entities. A Clinical Advisory Committee (CAC) of international haematologists and oncologists was formed to ensure that the classification will be useful to clinicians. A meeting was held in November 1997 to discuss clinical issues related to the classification. RESULTS: The WHO has adopted the 'Revised European-American Classification of Lymphoid Neoplasms' (REAL), published in 1994 by the International Lymphoma Study Group (ILSG), as the classification of lymphoid neoplasms. This approach to classification is based on the principle that a classification is a list of 'real' disease entities, which are defined by a combination of morphology, immunophenotype, genetic features and clinical features. The relative importance of each of these features varies among diseases, and there is no one 'gold standard'. The WHO classification has applied the principles of the REAL classification to myeloid and histiocytic neoplasms. The classification of myeloid neoplasms recognizes distinct entities defined by a combination of morphology and cytogenetic abnormalities. The CAC meeting, which was organized around a series of clinical questions, was able to reach a consensus on most of the questions posed. The questions and the consensus are discussed in detail below. Among other things, the CAC concluded that clinical groupings of lymphoid neoplasms was neither necessary nor desirable. Patient treatment is determined by the specific type of lymphoma, with the addition of grade within the tumour type, if applicable, and clinical prognostic factors such as the international prognostic index (IPI). CONCLUSION: The experience of developing the WHO classification has produced a new and exciting degree of cooperation and communication between oncologists and pathologists from around the world, which should facilitate progress in the understanding and treatment of haematological malignancies.

The World Health Organization classification of neoplasms of the hematopoietic and lymphoid tissues: report of the Clinical Advisory Committee meeting--Airlie House, Virginia, November, 1997.

INTRODUCTION: Since 1995, the European Association of Pathologists and the Society for Hematopathology have been developing a new World Health Organization (WHO) classification of hematologic malignancies. The classification includes lymphoid, myeloid, histiocytic, and mast cell neoplasms. MATERIALS AND METHODS: The WHO project involves ten committees of pathologists, who have developed lists and definitions of disease entities. A Clinical Advisory Committee (CAC) of international hematologists and oncologists was formed to ensure that the classification will be useful to clinicians. A meeting was held in November 1997 to discuss clinical issues related to the classification. RESULTS: WHO has adopted the 'Revised European-American Classification of Lymphoid Neoplasms' (REAL), published in 1994 by the International Lymphoma Study Group (ILSG), as the classification of lymphoid neoplasms. This approach to classification is based on the principle that a classification is a list of 'real' disease entities, which are defined by a combination of morphology, immunophenotype, genetic features, and clinical features. The relative importance of each of these features varies among diseases, and there is no one 'gold standard'. The WHO classification has applied the principles of the REAL classification to myeloid and histiocytic neoplasms. The classification of myeloid neoplasms recognizes distinct entities defined by a combination of morphology and cytogenetic abnormalities. The CAC meeting, which was organized around a series of clinical questions, was able to reach a consensus on most of the questions posed. The questions and the consensus are discussed in detail below. Among other things, the CAC concluded that clinical groupings of lymphoid neoplasms were neither necessary nor desirable. Patient treatment is determined by the specific type of lymphoma, with the addition of grade within the tumor type, if applicable, and clinical prognostic factors such as the international prognostic index (IPI). CONCLUSION: The experience of developing the WHO classification has produced a new and exciting degree of cooperation and communication between oncologists and pathologists from around the world, which should facilitate progress in the understanding and treatment of hematologic malignancies.

Five years follow-up after 2-chloro deoxyadenosine treatment in thirty patients with hairy cell leukemia: evaluation of minimal residual disease and CD4+ lymphocytopenia after treatment.

Between March 1992 and August 1993, thirty patients with hairy cell leukemia (HCL) were treated in a single institution with 2-chlorodeoxyadenosine (2-CdA) for one course (N=27) or two courses at six month interval (N=3). Sixteen patients were previously untreated, 14 had been treated with alpha interferon (alpha IFN) (N=5), alpha IFN and splenectomy (N=8) and splenectomy, alpha IFN and Deoxycoformycin (N=1). Overall results in 29 evaluable patients were: 25 CR (86%), 3 PR (10%), one failure. The three PR patients relapsed after 18, 24 months and five years. Two were retreated successfully. Two CR patients relapsed after five years. Careful clinical survey, sequential bone marrow biopsies (BMB) with DBA44 immunostaining for assessment of response and detection of residual disease and serially evaluation of lymphocyte subsets counts were performed. Results of bone marrow biopsies study show 1) a progressive reduction in hairy cell infiltration during the first six months after therapy and not after that indicating that the best moment for the evaluation of response may be the sixth month, 2) the persistence of a very small number of DBA44+ cells (80% of BMB). There was a correlation between the presence of > 5% DBA44 positive cells on 6th month BMB and relapse. 60% had an absolute CD4+ lymphocyte count less than 0.2 10(9)/l at least on one examination after treatment. CD4+ lymphocyte level persisted less than baseline level in 8/18 patients tested after four and/or five years. Lymphopenia was less marked in splenectomized patients: 7/7 splenectomized patients tested have recovered a CD4+ lymphocyte count equal to pretherapy level compared to 3/11 non splenectomized patients (p: 0.004). Three opportunistic infections were observed early (first 6 months) after 2CdA therapy: pneumocystis pneumonia, retinitis due to toxoplasma in the patient who failed and legionella pneumonia in a patient retreated after relapse. Two patients developed a second carcinoma 6 and 12 months after therapy. Five patients died, three from a cause unrelated to HCL, one from HCL and one from infection while in second CR. At five years, overall survival is 83% and progression free survival is 66%. Our study shows 1) long-lasting response in the majority of patients after 2-CdA, 2) a correlation between persistent minimal residual disease detected with DBA44 immunostaining and occurrence of relapse and 3) a profound and persistent CD4+ lymphopenia more marked in non splenectomized patients.