A novel biomarker score for the screening and management of patients with plasma cell proliferative disorders.

OBJECTIVE: Monoclonal plasma cell proliferative disorders comprise a wide spectrum of diseases associated to clonal B-cell expansion. Serum protein electrophoretic profile (SPEP) and circulating free light chains (FLCs) levels are the mainstay of diseases management. Recently, soluble (s) Syndecan-1 (SDC1, CD138) produced by myeloma plasma cells has been suggested in the monitoring and follow-up of patients with myeloma. The aim of our study is to evaluate sCD138 in addition with FLCs and SPEP for the screening of patients with different evolutive disease pathways. PATIENTS AND METHODS: Sera from 73 patients with monoclonal gammopathy of undetermined significance (MGUS), 120 smoldering and 42 multiple myeloma (SMM and MM, respectively), 70 HCV-related mixed cryoglobulinemia (MC), 35 B-cell non-Hodgkin's lymphoma (B-NHL) and sera from 50 healthy donors (HD), were tested for sCD138, FLCs (assessed by means of ELISA and turbidimetric assay, respectively) and electrophoresis pattern (performed on Capillarys system) for the generation of a novel biomarker score (BS). RESULTS: Our results were grouped according to the two main lines of disease progression (vs. MM or B-NHL): in one group we found BS mean values of 0.2, 3.4, 5.3, 7.1 for HD, MGUS, SMM and MM, respectively; in the other group of 0.2, 4.4, 6.7 for HD, MC and B-NHL. CONCLUSIONS: We showed that BS mean values follow the ingravescence disease status towards the two main lines of progression to cancerous conditions; it could represent an additional useful tool in the management of screening and/or follow-up.

High dose of idarubicin-based regimen for diffuse large cell AIDS-related non-Hodgkin's lymphoma patients: a pilot study.

BACKGROUND AND OBJECTIVES: Intensive chemotherapy (CHT) in AIDS-related non-Hodgkin's lymphoma (AIDS-NHL patients) is a vexing problem. Our purpose was to evaluate the feasibility of a high dose idarubicin (HD-IDA)-based regimen in diffuse large cell (DLC) AIDS-NHL patients. DESIGN AND METHODS: Fourteen stage I-IV untreated DLC AIDS-NHL patients with a performance status <3 and no prior AIDS-related diseases received CIOD: cyclophosphamide, HD-IDA (25 mg/m2 in 8 patients, 20 mg/m2 in 6 patients) vincristine and dexamethasone plus granulocyte colony-stimulating factor (G-CSF) and prophylaxis against infections. The outcomes measured were: rate of response, disease-free survival (DFS), overall survival (OS) and the impact of chemotherapy on immunologic and virological parameters. RESULTS: Complete response was achieved in 13/14 cases (response rate: 93%). The median time of response and survival was 33 (range 5-79) and 35.5 (range 6-84) months, respectively. At 60 months the DFS and OS were 71% and 44%, respectively. CIOD with idarubicin 20 mg/m2 was better tolerated than that with 25 mg/m2 and was administered with a higher mean average-relative-dose-intensity (95.38+/-7% vs 83.35+/-15.59%, p=0.0001). Opportunistic infections were more frequent in patients with a baseline CD4 <100 than those with >100 cells/microL (4/5 vs 1/9: p=0.0229). After 3 CIOD courses the mean CD4 cells/microL was significantly lower (p=0.001) and the mean HIV.1 RNA load was significantly higher (p=0.045) than at baseline. INTERPRETATION AND CONCLUSIONS: The proposed chemotherapeutic regimen for AIDS-related non-Hodgkin's lymphoma is feasible in an outpatient setting in selected patients with relatively well-preserved immune function.

Molecular remission following high-dose hydroxyurea and fludarabine plus cytarabine in a patient with simultaneous acute myeloid leukemia and low-grade lymphoma.

The occurrence of acute myeloid leukemia (AML) as a secondary tumor has been frequently reported in patients who received various chemotherapy regimens for hematologic malignancies wile the concomitant development of chronic lymphoproliferative diseases (CLD) and AML in previously untreated patients is extremely rare. We report a case with an apparently spontaneous occurrence of AML and non Hodgkin low-grade lymphoma diagnosed by immunological, cytogenetical and molecular analyses. In particular genetic studies allowed to identify the coexistence of a clonal lymphoid population and a myeloid blast component characterized by inv(16) marker and CBFbeta-MYH11 gene fusion. Complete remission of AML and the CLD was obtained following high doses of hydroxyurea and two consolidation cycles of fludarabine plus intermediate dose cytarabine.

Unmanipulated peripheral blood stem cell autograft in chronic lymphocytic leukemia: clinical findings and biological monitoring.

BACKGROUND AND OBJECTIVES: To investigate the feasibility of peripheral blood stem cell (PBSC) transplantion in patients with high-risk chronic lymphocytic leukemia (CLL) in remission after fludarabine therapy, the clinical impact of minimal residual disease (MRD) monitoring and the immunologic reconstitution after transplantation. DESIGN AND METHODS: Twenty CLL patients, in clinical complete remission (CR) after fludarabine, were offered an unmanipulated PBSC transplant and were longitudinally monitored for MRD and immunologic reconstitution. RESULTS: Due to unsatisfactory PBSC collection, 4 patients received bone marrow cells. All patients engrafted. Two patients died, one due to infection and one because of another neoplasia. Thirteen patients are at present in clinical CR after a median follow-up of 17 months and 18 patients are alive with a survival probability of 0.87 (+/-0.04) at 52 months after transplant. Fifteen patients had a molecular remission. Three of them showed a molecular relapse 16-28 months after autograft, followed by a clinical relapse 10-16 months later. Three of the four patients who remained persistently rearranged could be revaluated over time and showed an immunologic relapse 11-26 months after transplant; two of these had a clinical relapse 12 and 7 months later. A marked and persistent impairment of both the B- and T-immunologic compartments was recorded in the horizontal follow-up. INTERPRETATION AND CONCLUSIONS: Unmanipulated PBSC autograft is a feasible procedure that produces prolonged molecular remissions in high-risk CLL patients. Persistence or reappearance of a molecular signal after engraftment is predictive of subsequent immunologic and clinical CLL recurrence. The long -lasting impairment of the host immune repertoire after fludarabine followed by autograft has to be taken into account in the patients' management.

Acute myeloid leukemia and lung cancer occurring in a chronic lymphocytic leukemia patient treated with fludarabine and autologous peripheral blood stem-cell transplantation.

An increased incidence of different malignancies associated to chronic lymphocytic leukemia (CLL) has been reported. The association of CLL and acute leukemia is a rare event described in < 1% of CLL, the type of acute leukemia being either from the lymphoid or more often from the myeloid lineage. The coexistence of acute myeloid leukemia (AML) and CLL in the same patient has been occasionally reported. Most of these cases have been associated with the administration of chemotherapy or radiotherapy for CLL, suggesting that the former may be a secondary leukemia. On the other hand, CLL could precede, but could also be diagnosed at the same, or delayed time as AML, suggesting the presence of other leukemogenic factors. We describe the exceptional development of AML and lung cancer in a patient with previously diagnosed CLL in minimal residual disease status after fludarabine treatment followed by autologous peripheral blood stem-cell transplantation.

Chronic myeloid leukemia cells resistant to interferon-alpha lack STAT1 expression.

INTRODUCTION: Interferon-alpha (IFN) plays a role in the management of different neoplasias, particularly those of hematological origin. The mechanisms of action of IFN are still poorly understood and the individual response is unpredictable. In the present study, the pattern of intracellular gene expression following in vitro and in vivo exposure of chronic myeloid leukemia (CML) cells to IFN was evaluated and correlated with the response to in vivo treatment with IFN. MATERIALS AND METHODS: CML patients in different phases of the disease were studied. The pattern of expression of two IFN-inducible proteins involved in IFN-mediated biological activities, the p91 and p84 proteins (STAT1alpha and STAT1beta), components of the IFN-stimulated gene factor 3 (ISGF3) complex and the enzyme 2'-5' oligoadenylate synthetase (2'-5' OASE) were investigated by Western blot in peripheral blood mononuclear cells stimulated or not in vitro by IFN. RESULTS AND CONCLUSIONS: In 6/9 patients evaluated before starting treatment, STAT1 was expressed either constitutively or after in vitro stimulation by IFN. In three cases, STAT1 remained negative even after in vitro activation. The pattern of protein expression correlated with the subsequent hematological response to prolonged in vivo IFN administration: the presence of STAT1 being associated with the clinical response to IFN and the absence and non-inducibility of STAT1 with resistance to IFN. This was further substantiated by studies carried out in ten patients analyzed at the time of a documented clinico-hematological response or resistance to the in vivo administration of IFN. Finally, in order to establish whether the pattern of response to IFN treatment could be predicted at diagnosis, cells cyropreserved at diagnosis from patients with a documented complete response, confirmed also by cytogenetic negativity, or resistance, were studied. While complete responders proved STAT1 positive, none of the four resistant cases ever expressed STAT1. The expression of 2'-5' OASE did not correlate with the clinical response to IFN. This study documents the pivotal role of STAT1 in the in vitro and in vivo responses of CML cells to IFN. The constitutive or induced presence or absence of STAT1 shows a predictive correlation with the response or resistance to treatment with IFN and could be utilized to identify, at diagnosis, resistant patients who may be spared an expensive and unnecessary prolonged IFN administration.

Interferon-alpha and bcr-abl antisense oligodeoxynucleotides in combination enhance the antileukemic effect and the adherence of CML progenitors to preformed stroma.

We have studied the in vitro effect of IFN-alpha and bcr-abl antisense oligodeoxynucleotides (As ODN) alone and in combination with the aim of enhancing the antileukemic activity of the two single agents and evaluating whether the two agents in combination might restore the adherence capacity of chronic myeloid leukemia (CML) progenitors to preformed stroma. We have also correlated the increased adhesion found after in vitro treatment with the expression of adhesion molecules on leukemic progenitors. Incubation of the BV173 cell line with escalating doses of IFN-alpha (100-10000 U/ml) showed a colony growth inhibition between 10 and 30%. IFN-alpha and junction-specific As ODN in combination showed a greater antiproliferative effect compared to that observed with the two agents used alone. In particular, As ODN at a concentration of 40 microg/ml in combination with IFN-alpha at 100 and 1000 U/ml showed a greater inhibitory effect compared to that obtained with IFN-alpha only. Addition of As ODN to IFN-alpha at 10000 U/ml did not result in a greater BV173 inhibition. In a further set of experiments, primary cells from 16 CML patients at diagnosis were incubated with 40 microg/ml of J-spec As ODN, several control ODNs and IFN-alpha at 1000 U/ml alone and in combination. A significantly greater elimination of CML progenitors was found after treatment with the combination of IFN-alpha and J-spec As ODN, compared to any other treatment group, confirmed also by a more marked effect on p210 expression. The deficient adhesion of CML progenitors on human preformed stroma was restored at levels similar to that of normal bone marrow cells after treatment with IFN-alpha and/or J-spec As ODN, while the phenotypic analysis showed that the combined treatment increased significantly the expression of CD49b and CD62L on CML CD34+ cells. However, when the expression of adhesion molecules was blocked with specific monoclonal antibodies, only CD49d (expressed on more than 90% of CML CD34+ cells) appeared to influence the functional activity of adhesion molecules. In conclusion, IFN-alpha and bcr-abl As ODN in combination exert a marked in vitro antileukemic activity and could be a useful approach for in vitro purging of CML cells prior to autologous transplantation.

Monitoring of CD34+ cells during leukapheresis allows a single, successful collection of hemopoietic progenitors in patients with low numbers of circulating stem cells.

We have studied a total of 188 patients with hematological malignancies, submitted to mobilization therapy with G-CSF associated or not with chemotherapy in order to: (1) establish the lower limit of circulating progenitor cells that allows the collection of 2 x 10(6) CD34+ cells/kg by a single leukapheresis, utilizing the instrument set on standard parameters; (2) evaluate whether the number and quality of CD34+ cells collected remain stable during leukapheresis; and (3) collect a sufficient number of circulating CD34+ cells by a single procedure in patients in whom such an approach would have been insufficient to reach the target with the instrument set on standard parameters. The retrospective analysis conducted in 85 patients showed that 19 circulating CD34+ cells/microl represented the cut-off number capable of discriminating between patients who will require one or more apheresis to collect 2 x 10(6) CD34+ cells/kg. The validity of this value was prospectively confirmed in 70 subsequent patients. Based on in vitro results that showed the stability in the number of CD34+ cells, the proportion of different CD34+ cell subpopulations and the clonogenic capacity of the stem cell compartment during leukapheresis both in the blood of the patients and in samples taken directly from the instrument, we have adapted the blood volume to be processed in 33 patients with <19 PB CD34+ cells/microl. Stem cell collection was monitored during the leukapheresis and the procedure was prolonged for a time period estimated to be sufficient to reach the target number of CD34+ cells with a single procedure. The median increment of the total blood volume processed, calculated from the volume set automatically by the instrument was 25.2%, with a median of 3.3-fold total blood volume processed. In all cases, a sufficient CD34+ cell collection was completed in a single procedure. After autograft, the pattern of blood reconstitution was similar to that of all the other patients.

Clinical characteristics and outcome of young chronic lymphocytic leukemia patients: a single institution study of 204 cases.

A retrospective analysis on chronic lymphocytic leukemia (CLL) patients 55 years of age) were observed. At diagnosis, younger and older patients displayed a similar distribution of clinical features, except for a significantly higher male/female ratio in younger patients (2.85 v 1. 29; P <.0001). Both groups showed an elevated rate of second primary cancers (8.3% v 10.7%), whereas the occurrence of Richter's syndrome was significantly higher in younger patients (5.9% v 1.2%; P <. 00001). Younger and older patients showed a similar overall median survival probability (10 years) but were characterized by a different distribution of causes of deaths: CLL unrelated deaths and second primary malignancies predominated in the older age group, whereas the direct effects of leukemia were prevalent in the younger age group. Although younger and older patients displayed a similar survival, the evaluation of the relative survival rates showed that the disease had a greater adverse effect on the expected survival probability of the younger population. Multivariate analysis showed that for young CLL patients only dynamic parameters, such as lymphocyte doubling time and other signs of active disease, were the independent factors that significantly influenced survival probability (P =.00001). A prolonged clinico-hematologic follow-up allowed us to identify two subsets of young CLL patients with a different prognostic outcome: a group of patients (40%) with long-lasting stable disease without treatment and an actuarial survival probability of 94% at 12 years from diagnosis and another group (60%) with progressive disease and a median survival probability of 5 years after therapy. For the latter patients, the therapeutic effect of innovative therapies with curative intents needs to be investigated in prospective, comparative clinical trials.

Evaluation of marrow and blood haemopoietic progenitors in chronic lymphocytic leukaemia before and after chemotherapy.

We have evaluated the number and differentiation pattern of CD34+ cells, as well as the CFU-GM, BFU-E and CFU-GEMM progenitors from the blood (PB) and marrow (BM) of 53 chronic lymphocytic leukaemia (CLL) patients. Twenty-four patients were untreated and 29 were studied at 2 months from the last course of fludarabine or chlorambucil; 6 patients, studied after fludarabine therapy, were further evaluated after mobilization with cyclophosphamide and G-CSF. PB of untreated patients showed a median number of CD34+ cells, CFU-GM, BFU-E and CFU-GEMM/10(5) seeded cells and per litre of PB similar to those of normal controls. No differences were also found in the number of clonogenic progenitors/10(5) cells in patients studied before and after therapy, while significantly fewer BFU-E/l of PB were found after fludarabine. The number of circulating CD34+ cells/l of PB was significantly lower in patients treated with fludarabine or chlorambucil compared to untreated patients. BM growth was significantly reduced in untreated CLL patients compared to healthy donors. Treatment with fludarabine or chlorambucil restored BM progenitors at levels similar to those of normal controls; this effect did not occur for CFU-GM in patients treated with fludarabine. Three-colour fluorescence analysis demonstrated a differentiation pattern of CD34+ cells, with a greater expression of CD13 and CD33 after treatment with fludarabine compared to untreated patients and normal controls. In 4 patients previously treated with fludarabine who underwent a successful cyclophosphamide and G-CSF mobilization therapy, 4x10(6) CD34+ cells/kg were collected. These 4 patients showed a notable increase of CD34+ cells and of clonogenic cells in the PB, but a marked decrease of BM progenitor cells. The 2 patients who failed CD34+ cell mobilization had a reduced CFU-GM growth both in the PB and in the BM. Taken together, these studies indicate that residual haemopoietic progenitors are present in untreated CLL patients and that stem cell mobilization and collection can be carried out following fludarabine treatment.

p53 expression in B-cell chronic lymphocytic leukemia: a marker of disease progression and poor prognosis.

We have analyzed by immunocytochemistry (ICC) the frequency of p53 protein expression in 181 cases of B-cell chronic lymphocytic leukemia (CLL) followed at a single institution to assess the relationship between p53 and the clinical and morphological features of the disease, as well as the possible involvement of this protein in the pathogenesis of the more aggressive forms of CLL. The overall frequency of p53 protein positivity in CLL was 15% (27 of 181 cases). There were no significant differences in age, sex, absolute lymphocyte count, or lymphocyte doubling time between p53-positive and -negative patients. By contrast, p53-positive patients had a significantly higher percentage of prolymphocytes (P = .002) and a significantly lower percentage of residual CD3-positive T lymphocytes (P = .0001). No correlation was found between the percentage of p53-positive cells and the percentage of cells in cycle assessed by the monoclonal antibody Ki-67. When the percentage of p53 positivity was correlated with the clinical stage of the disease, the proportion of p53-positive cases increased significantly from Binet's stage A (8 of 108; 7.4%), to stage B (12 of 49; 24.4%) and C (7 of 24; 29.2%) (P = .002). p53 positivity correlated also with the phase of the disease, showing a low expression at diagnosis (8 of 112; 7.1%) and a significantly higher expression in patients studied during the course of the disease (7 of 35; 20%) and, to a further extent, with disease progression (12 of 34; 35.3%) (P = .0001). The association of p53 protein expression with mutations in the gene was confirmed by direct sequence of the entire cDNA in 15 of the 17 ICC positive cases tested (88%). A significantly shorter treatment-free interval from diagnosis (P = .003) and a poorer response to therapy (P = .007) was observed in p53-positive compared with p53-negative patients. Overall survival from the time of diagnosis, as well as from the time of p53 protein analysis, was significantly shorter in patients with p53 protein expression (P = .03 and .0001, respectively). Moreover, in multivariate analysis, p53 expression and stage C were independently associated with a short survival. The results of this study indicate that in CLL the expression of the p53 protein, analyzed by a simple and reliable immunocytochemical method, is strongly associated with p53 gene mutations, a morphological variant (CLL with >10% prolymphocytes), advanced clinical stage, progressive disease, poor response to therapy, and short survival.

Immunocytochemical diagnosis of acute promyelocytic leukemia (M3) with the monoclonal antibody PG-M3 (anti-PML).

Acute promyelocytic leukemia (APL) is characterized by a reciprocal 15; 17 chromosomal translocation, which fuses the promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARalpha) genes, leading to the expression of the PML/RARalpha fusion oncoprotein. Immunocytochemical labeling of the wild-type PML protein with the PG-M3 monoclonal antibody (MoAb) directed against the amino terminal portion of the human PML gene product, produces a characteristic nuclear speckled pattern that is due to localization of the protein into discrete dots (5 to 20 per nucleus), named PML nuclear bodies. The architecture of PML nuclear bodies appears to be disrupted in APL cells that bear the t(15; 17), thus resulting in a change of the nuclear staining pattern from speckled (wild-type PML protein) to microgranular (PML-RARalpha fusion protein). To assess whether the PG-M3 MoAb could assist in the diagnosis of APL (M3), bone marrow and/or peripheral blood samples from 100 cases of acute nonlymphoid leukemias of different subtypes were blindly immunostained with the PG-M3 MoAb, using the immunoalkaline phosphatase (APAAP) or immunofluorescence technique as detection system. Notably, the abnormal (micropunctate) pattern of the PML/RARalpha fusion protein (usually >/=50 small granules/per nucleus) was observed in APL (M3) samples, but not in other types of acute nonlymphoid leukemias. Immunocytochemical labeling with PG-M3 was particularly useful in the diagnosis of microgranular variant of APL (M3V) (three cases misdiagnosed as M4 and M5), and also to exclude a morphologic misdiagnosis of APL (six of 78 cases). In all cases investigated, immunocytochemical results were in agreement with those of reverse transcription-polymerase chain reaction (RT-PCR) for PML/RARalpha. Because the epitope identified by PG-M3 is located in the aminoterminal portion of PML (AA 37 to 51), the antibody was suitable for recognizing APL cases characterized by breakpoint occurring at different sites of PML (bcr 1, bcr 2 and bcr 3). In conclusion, immunocytochemical labeling with PG-M3 represents a rapid, sensitive, and highly-specific test for the diagnosis of APL that bears the t(15; 17). This should allow an easy and correct diagnosis of this subtype of acute leukemia to any laboratory provided with a minimal equipment for immunocytochemistry work.

Immunophenotype of adult and childhood acute lymphoblastic leukemia: changes at first relapse and clinico-prognostic implications.

The immunologic features of leukemic cells at the time of 1st hematologic relapse were compared to those obtained at initial diagnosis in 128 patients (69 children and 59 adults) with acute lymphoblastic leukemia (ALL) treated at a single institution. An immunophenotypic change was observed in 59 cases (46%), more frequently in T (20/25) than in B (39/103) lineage ALL (80 vs 38%, P=0.0008), but with a similar incidence in adults and children. Of these cases, 34 (24 B- and 10 T-ALL) changed at relapse their intralineage subgroup affiliation, although no complete shift from B to T lineage ALL, or vice versa, was observed. The myeloid antigens CD13 and/or CD33 were frequently lost (2/5 cases) or acquired (12/123 cases) at relapse. In 21 cases, the immunophenotype at relapse was more undifferentiated than at diagnosis, while it was more differentiated in 13 cases. Initial treatment intensity or preceding treatment with teniposide did not affect the phenotypic profile at relapse. Complete response (CR) rate to salvage therapy and event-free survival were not influenced by the immunophenotypic shifts, nor by the presence, at relapse, of leukemic cells expressing the myeloid antigens CD13 and/or CD33. Univariate analysis suggested that prognosis after relapse was dependent on the duration of 1st CR, patients' age and immunophenotype at the time of diagnosis, with a worse outcome for patients with T lineage ALL and for patients with the less differentiated subgroup of B lineage ALL (CD19+ and CD10-). Multivariate analysis showed that only two factors, duration of 1st CR and grade of immunologic differentiation at diagnosis, have independent prognostic value in relapsed ALL.

Ultrastructural investigation of circulating villous lymphoid cells: a tool in the differential diagnosis of splenic lymphoma with villous lymphocytes.

Ultrastructural examination of circulating lymphoid cells was performed in three cases of splenic lymphoma with circulating lymphocytes (SLVL) in order to define morphological features helpful to distinguish this lymphoma from hairy cell leukemia (HCL). The samples for ultrastructural investigation were obtained by Ficoll sedimentation from peripheral blood and routinely processed for electron microscopy. The ultrastructural features examined were: morphology of villi, morphology of nuclei, presence of nucleoli, distribution of heterochromatin, type of cytoplasmic organelles, presence of specific intracytoplasmic structures such as the ribosome-lamella complex, lysosome-like bodies and perinuclear microfibrils. Our results and a careful review of the literature seemed to confirm that SLVL has electron microscopic features typical enough to be relevant in the differential diagnosis with HCL.

Diagnostic relevance of peripheral blood immunocytochemistry in hairy cell leukaemia.

AIMS--(1) To assess the diagnostic relevance of peripheral blood immunocytochemistry in hairy cell leukaemia (HCL); (2) to compare the immunostaining of bone marrow biopsy specimens with bone marrow and peripheral blood cytospins; (3) to evaluate the sensitivity of the different markers used; (4) to identify the ultrastructural localisation of DBA.44 in HCL variant. METHODS--Immunoenzymatic staining procedures, immunoperoxidase and immunoalkaline phosphatase, were used with a panel of monoclonal antibodies directed to HCL associated antigens. Ultrastructural immunostaining was performed using colloidal gold conjugated antibodies. RESULTS--HCL showed strong cytoplasmic reactivity for CD22, CD25, CD103, DBA.44, kappa, or lambda light chains. Peripheral blood diagnostic hairy cells were found in all the cases with absolute counts ranging from 0.11 x 10(9)/l up to 6.4 x 10(9)/l and values increasing with the size of the spleen. A median of 36.5% of leukaemic cells was found in bone marrow aspirates and 70% in bone marrow trephine specimens. The monoclonal antibodies CD22 and DBA.44 showed the highest and the lowest percentage of positive hairy cells, respectively; this difference was statistically significant (p = 0.0025). Ultrastructural immunolabelling with DBA.44 showed a cytoplasmic membrane localisation of the antigen in one case of HCL variant. CONCLUSIONS--(1) Immunocytochemistry is a useful technique which enhances the accuracy of diagnosis in HCL; (2) peripheral blood immunocytochemistry is recommended because it highlights hairy cells in all cases; (3) CD22 appears to be the most sensitive of the markers tested; (4) ultrastructural analysis is a useful tool in selected cases of HCL variant.

Monoclonal antibody Ki-67 identifies B and T cells in cycle in chronic lymphocytic leukemia: correlation with disease activity.

Ki-67 is a monoclonal antibody that recognises a nuclear antigen expressed during most phases of the cell cycle. We have analysed, by immunocytochemistry, the frequency, morphology, and clinical significance of Ki-67+ cells in 108 patients with B-cell chronic lymphocytic leukemia (CLL). Because in normal peripheral blood Ki-67+ cells are mainly T lymphocytes, we have also investigated, by double immunoenzymatic staining, the proportion of Ki-67+ T cells (Ki-67/CD3+) in CLL. Four groups of patients were identified: (i) 47 with stage A, (ii) 32 with stages B + C, (iii) 24 with greater than 10% of circulating prolymphocytes (CLL/PL) and (iv) five with Richter's syndrome. Within stage A CLL, two groups were considered: A' (Hb greater than or equal to 12 g/dl and lymphocytes less than 30 + 10(9)/l) and A" (Hb less than 12 g/dl or lymphocytes greater than or equal to 30 x 10(9)/l). The percentage and absolute number of Ki-67+ leukemic cells was found to increase with the stage of the disease and correlate with the proportion of prolymphocytes. On the other hand, the proportion of Ki-67+ T cells (CD3+) was significantly higher in patients with CLL stage A' (29.3 +/- 4.5), which includes patients with long-standing, stable disease, than in CLL stage A" (9.5 +/- 3.3), B + C (7.1 +/- 4.6), and CLL/PL (6.4 +/- 2.8). Ki-67 seems to identify patients with more aggressive forms of CLL, such as CLL/mu 2PL with more than 10% Ki-67+ cells (25% of the cases) and Richter's syndrome, in which all the large lymphoma cells are Ki-67+. Long-term follow-up will establish whether Ki-67 is a good prognostic marker and can predict disease outcome.