E. Matutes

Subcutaneous CAMPATH-1H in fludarabine-resistant/relapsed chronic lymphocytic and B-prolymphocytic leukaemia

Seven patients with B‐cell leukaemia — six with chronic lymphocytic leukaemia (CLL) and one with B‐prolymphocytic leukaemia (B‐PLL) — were treated with CAMPATH‐1H , a genetically reshaped CD52 monoclonal antibody, administered subcutaneously (s.c.) three times a week for 6–12 weeks. Four were resistant to, and three had had a short partial remission (PR) following, fludarabine chemotherapy. The patient with B‐PLL achieved complete remission and three patients with CLL attained PR; two of the latter were retreated. The three remaining patients were non‐responders. Three patients were transfusion‐dependent before CAMPATH and all three became transfusion‐independent after treatment. The overall median survival from starting CAMPATH‐1H was 11 months. Three patients reactivated cytomegalovirus (CMV) during the course of treatment, and two were treated with, and responded to, ganciclovir.

Cytogenetic studies in splenic lymphoma with villous lymphocytes

Summary. We report the cytogenetic findings on 31 cases of splenic lymphoma with villous lymphocytes (SLVL). TPA stimulated cells from peripheral blood (28 cases), spleen (two cases) and lymph node (one case) with SLVL have been analysed. A clonal chromosome abnormality was found in 27/31 patients (87%): this was identified as a simple abnormality in 12 cases and a complex one in 15. Four recurring abnormalities were seen: t(11:14) (q13:q32)in five patients, deletions or translocations involving 7q in seven patients. iso 17q in four patients and translocations involving 2p11 in four patients. The high frequency of clonal chromosome abnormalities in SLVL contrasts with the usually benign clinical course of this disease.

Atypical lymphocyte morphology: An adverse prognostic factor for disease progression in stage A CLL independent of trisomy 12

We studied 270 patients with Binet stage A chronic lymphocytic leukaemia looking for adverse prognostic factors. In a multivariate analysis the following features were found to be risk factors for disease progression: atypical lymphocyte morphology (defined as either > 10% prolymphocytes or > 15% lymphocytes with cleaved nuclei or lymphoplasmacytoid cells); more than two karyotypic abnormalities; lymphocyte count > 30 × 109/l; lymphocyte doubling time < 1 year; enlargement of one or more lymph node groups.

Long‐term follow‐up of patients with hairy cell leukaemia after treatment with pentostatin or cladribine

We report the long‐term follow‐up results on two groups of patients with hairy cell leukaemia (HCL) treated with either pentostatin (deoxycoformycin) or cladribine (2‐chlorodeoxyadenosine). 165 HCL patients received treatment with pentostatin (between 1986 and 1994), and 45 were treated with cladribine (between 1992 and 1997). Age and sex characteristics were similar in the two groups. 38 patients in the pentostatin group and 12 in the cladribine group were previously untreated. 22 patients in the cladribine group had received prior treatment with pentostatin; four were resistant, 17 had relapsed following partial (four) or complete (13) responses, and one was not evaluable for response. The response rates were the same in the two groups: 82% complete response (CR), 15% partial response (PR) for pentostatin and 84% CR, 16% PR for cladribine. Relapse rates were 24% for pentostatin and 29% for cladribine after median follow‐up of 71 and 45 months respectively. At 45 months, however, the relapse rate for pentostatin was only 9.7%. We found a statistically significant difference in the disease‐free interval (DFI) between the two groups suggesting that patients may relapse more quickly after cladribine. The majority of relapsed patients achieved second remissions following further therapy with either pentostatin or cladribine, with no evidence of cross resistance between the two agents. The 5‐year survival for all patients was 97% and treatment‐ related toxicity was low. We conclude that both pentostatin and cladribine induce durable remissions in the majority of HCL patients. Longer follow‐up is required to establish whether some patients are cured as there is no plateau in DFI, and which of these two agents may be the treatment of choice.

Adhesion receptors on peripheral blood leukemic B cells. A comparative study on B cell chronic lymphocytic leukemia and related lymphoma/leukemias

The expression of a series of adhesion receptors: L-selectins (CD62L): Leu-8, several integrins (LFA-1: CD11a/CD18, VLA-4: CD49d/CD29 and VLA-5: CD49e/CD29), ICAM-1(CD54) and the ‘homing receptor’ (CD44) were investigated by a dual color flow cytometry in 56 cases of B cell disorders namely, 39 chronic lymphocytic leukemias (CLL), four hairy cell leukemia (HCL), seven splenic lymphoma with villous lymphocytes (SLVL) and six other non-Hodgkin’s lymphoma (NHL). The functional activity of L-selectins was assessed with L-selectin ligand analogs (polyphosphomonester core polysaccharide: PPME and fucoidin). Leukemic B cells were identified with phycoerythrin-conjugated monoclonal antibodies (McAbs) anti-CD19, anti-kappa/lambda investigated simultaneously for the expression of adhesion receptors estimated with fluorescein-isothiocyanate (FITC) conjugated McAbs. The percentage of leukemic cells expressing L-selectins (Leu-8) was high in CLL (52% of positive cases) and integrin expression (LFA-1, VLA-4, 5) was low (19 and 33%, respectively), while a reverse pattern, low Leu-8 (17%), and a high VLA-4 (77%), was observed in non-CLL cases. The expression of LFA-1 α-chain was variable in non-CLL cases, and the LFA-1 heterodimer was expressed on most clonal B cell in NHLs (92%). LFA-1 α-chain was detected on cells from only one HCL case, while β2 integrin was regularly expressed on hairy cells. VLA-5 integrin was found on a relatively small number (26%) of mature B cell leukemias. A remarkable finding was the detection of ICAM-1 in all CLL cases albeit the number of positive cells was significantly lower (P < 0.05) compared to non-CLL cases. CD44 was expressed on a high number of neoplastic cells in all the investigated categories. There was no correlation between the expression of the adhesion molecules and clinical and laboratory parameters except for CD18 which was expressed on a significantly (P < 0.05) higher number of leukemic cells in CLL with more advanced stages. This study demonstrates that even closely related B cell leukemia/lymphomas have a certain well defined and strictly variable adhesion profile which is characteristic of the disease entity and therefore, the adhesion profile may offer additional information useful for differential diagnosis and study of disease pathogenesis.

Detection of minimal residual disease in B-lineage acute lymphoblastic leukaemia by quantitative flow cytometry

The clinical significance of detecting minimal residual disease (MRD) in B‐lineage acute lymphoblastic leukaemia (ALL) was evaluated by quantitative flow cytometry using a combination of TdT with CD10 and CD19. 53 patients with B‐cell precursor ALL were followed during and after completion of treatment (median follow‐up 23 months). Nine patients relapsed and MRD had been detected in six of them, 5–15 weeks before relapse despite morphological complete remission. 43 patients remain in clinical remission and in none of these was MRD detected. Disease‐free survival based on the detection of MRD by flow cytometry showed a statistically significant difference between both groups (P < 0.0001). The absence of MRD correlates with a low relapse rate, whereas the presence of MRD predicted early relapse. This study has shown that flow cytometry can improve the morphologic assessment of bone marrow (BM) remission status in B‐lineage ALL. The finding of < 5% blasts in BM aspirates did not correlate with ‘true’ remission in a proportion of cases as residual leukaemic blasts were detected by flow cytometry in nine samples from six patients. On the other hand, the presence of > 5% blasts assessed by morphology was not necessarily a feature of relapse in five patients as these cells were shown to have a phenotype identical to normal TdT‐negative B‐cell precursors. Quantitative flow cytometry was more informative than conventional morphology to assess remission status and showed a strong correlation with clinical outcome. This methodology is useful to define MRD in the majority of patients with B‐lineage ALL and should be tested in prospective clinical trials.

The role of an anti‐myeloperoxidase antibody in the diagnosis and classification of acute leukaemia: a comparison with light and electron microscopy cytochemistry

Summary. The enzyme myeloperoxidase (MPO) is the hallmark of the myeloid lineage. We have analysed the presence of MPO in blasts from 180 cases of acute leukaemia (103 acute myeloid leukaemia (AML) and 77 acute lymphoid leukaemia (ALL) by means of monoclonal antibodies anti‐MPO and immunocytochemistry (alkaline phosphatase anti‐alkaline phosphatase method). The aim of the study was to investigate the specificity and sensitivity of this marker compared with MPO cytochemistry by light (LM) and electron microscopy (EM), and with the expression of myeloid antigens. Anti‐MPO was positive (> 3% blasts) in all but one of the 90 AML positive by LM cytochemistry. Of 13 AML cases negative by MPO cytochemistry, six showed 3.10% blasts reactive with anti‐MPO and were also positive with antibodies to CD13 arid/or CD33. The presence of MPO was confirmed in four of these by EM. The overall positivity of anti‐MPO in AML was 92%. Anti‐MPO was negative in all but two ALL (6% and 8% positive blasts). The blasts in these two cases were also GD13, CD33 and MPO positive by EM; both were thus reclassified as biphenotypic. Another two ALL reinterpreted as biphenotypic were negative by MPO cytochemistry and anti‐MPO but were MPO positive by EM and with CD13 and/or CD33. We conclude that anti‐MPO is a sensitive and specific early marker of myeloid blasts and should be incorporated in the routine immunophenotyping of acute leukaemia.

Mycosis fungoides and Sezary syndrome are not associated with HTLV-I infection: an international study

Association between mycosis fungoides (MF), its leukaemic variant Sezary syndrome (SS) and the human T‐cell lymphotropic virus type‐I (HTLV‐I) has been controversial, with the reported incidence of infection varying between 0% and nearly 100%. We studied 127 patients (85 MF, 28 SS, five Sezary cell leukaemia, four lymphomatoid papulosis, and five unspecified cutaneous T‐cell lymphomas (CTCL)) originating from Europe (France, Spain, U.K., Portugal) or from U.S.A. (California) for the presence of HTLV‐I infection markers. HTLV‐I and ‐II serology were performed on 78 patients using standard immunological methods. Reverse transcriptase (RT) assay was also performed in 26 cases using an RT‐PCR‐based method of high sensitivity. Molecular analyses were performed on 215 DNA samples (121 from fresh PBMCs, 26 from PBMCs after short‐term culture and 68 from skin lesions) by PCR amplification using HTLV‐I and ‐II gag, pol, env, pX and LTR specific primers.

Disease Features in Acute Myeloid Leukemia with t(8;21)(q22;q22). Influence of Age, Secondary Karyotype Abnormalities, CD19 Status, and Extramedullar Leukemia on Survival

Over a period of 14 years, 50 patients (12 children and 38 adults) of whom 46 had acute myeloid leukemia (AML) and 4 had myelodysplastic syndrome characterized by the t(8;21)(q22;q22) translocation were referred to the Royal Marsden Hospital. The clinico-pathological features of these cases were analyzed to determine the influence of age, secondary karyotype abnormalities, and expression of the lymphoid marker CD 19 on event free survival, and presence of extramedullary leukemia on overall survival. They were treated with a variety of chemotherapy protocols and some had bone marrow transplantation. There appeared to be no difference in survival between children (age >17 years) and adults (age >16 years). Out of the 50 cases, 16 (32%) had the (8;21) translocation alone, 17 (34 %) had additional loss of a sex chromosome and the remaining 17 (34%) had other karyotype abnormalities of which deletion or translocation of the long arms of a #9 was most common (observed in 8 of the 17 patients). The karyotype groups had a significant impact on survival, the group with loss of a sex chromosome having a poorer outcome and the group with abnormalities of chromosome 9 having a better outcome. CD19 positivity was seen in 21 of the 33 cases (63%) in whom it was measured compared to 11% observed in controls with AML without a t(8;21). CD19 status did not exert any influence on event free survival. Extramedullary leukemia (EML) occurred in 5 of the 50 cases (10%). In one patient it was observed at diagnosis but in the others it presented concurrent with bone marrow relapse. The overall survival of patients with EML was worse than that of the other patients but did not achieve statistical significance and was probably adversely affected by other factors.

Frequent deletions at 11q23 and 13q14 in B cell prolymphocytic leukemia (B-PLL)

Deletions of the long arm of chromosomes 11 and 13 are the most frequent structural chromosome aberrations in various types of lymphoproliferative disorders. However, these regions have not been studied so far in B cell prolymphocytic leukemia (B-PLL). We have investigated the incidence of 13q deletions in 18 B-PLL cases by fluorescence in situ hybridization (FISH), using molecular probes for the RB1 and D13S25 loci. Chromosome 11q deletions were evaluated by FISH using the yeast artificial chromosome (YAC) clone 755b11 from the chromosome 11q22.3-q23.1 region, which has been previously shown to be deleted in 20% of cases of chronic lymphocytic leukemia. Chromosome 11q23 deletions were found in 7/18 (39%) cases of B-PLL. Monoallelic loss of RB1, D13S25 and BRCA2 was present in 10/18 (55%), 6/18 (33%) and 3/18 (16%) of the cases, respectively. All the cases with D13S25 and BRCA2 deletion showed RB1 loss. Deletions of 13q14 and 11q23 are frequent chromosome aberrations in B-PLL and, in contrast to CLL, there is a preferential loss of RB1 with respect to the D13S25 locus suggesting that allelic loss of the RB1 gene may play a role in the pathogenesis of B-PLL.