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In chronic-phase chronic myeloid leukaemia (CP-CML), residual BCR-ABL1+ leukaemia stem cells are responsible for disease persistence despite TKI. Based on in vitro data, CHOICES (CHlorOquine and Imatinib Combination to Eliminate Stem cells) was an international, randomised phase II trial designed to study the safety and efficacy of imatinib (IM) and hydroxychloroquine (HCQ) compared with IM alone in CP-CML patients in major cytogenetic remission with residual disease detectable by qPCR. Sixty-two patients were randomly assigned to either arm. Treatment ‘successes’ was the primary end point, defined as ≥0.5 log reduction in 12-month qPCR level from trial entry. Selected secondary study end points were 24-month treatment ‘successes’, molecular response and progression at 12 and 24 months, comparison of IM levels, and achievement of blood HCQ levels >2000 ng/ml. At 12 months, there was no difference in ‘success’ rate (p= 0.58); MMR was achieved in 80% (IM) vs 92% (IM/HCQ) (p= 0.21). At 24 months, the ‘success’ rate was 20.8% higher with IM/HCQ (p= 0.059). No patients progressed. Seventeen serious adverse events, including four serious adverse reactions, were reported; diarrhoea occurred more frequently with combination. IM/HCQ is tolerable in CP-CML, with modest improvement in qPCR levels at 12 and 24 months, suggesting autophagy inhibition maybe of clinical value in CP-CML.

Le mode de financement des spécialités relevant du dispositif pérenne post-autorisation temporaire d’utilisation (ATU) et les exigences pour l’obtention du remboursement des spécialités en sus des groupes homogènes de séjours (GHS) évoluent. Cette situation amène les équipes pharmaceutiques en charge de leur gestion à s’adapter. Ainsi, nous nous interrogeons sur les moyens à mettre en œuvre pour assurer celle-ci et son impact.

•La conciliation est le socle de la sécurisation des traitements médicamenteux.•Les ressources humaines nécessaires à cette activité sont cependant limitées.•Le préparateur en pharmacie hospitalière est une ressource clé dans ce processus.•Ce dernier estime que sa place est légitime en tant qu’acteur de la pharmacie clinique.•Son rôle dans la conciliation doit être formalisé et structuré en France.

L’hirudothérapie présente un intérêt en chirurgie réparatrice et reconstructive pour éviter la congestion veineuse des lambeaux. Les sangsues Hirudo medicinalissont porteuses d’Aeromonasspp.Des infections par ces bactéries entraînant la perte du lambeau ont été décrites. Il n’existe actuellement aucun consensus national de bon usage des sangsues en établissement de santé.

L’équipe pharmaceutique procède à des audits réguliers des conditions de stockage et de détention des médicaments dans les unités de soins (US) pour s’assurer du respect des bonnes pratiques et du référentiel réglementaire. Notre étude propose d’évaluer la mise en place de ces audits dans notre établissement et d’envisager des axes d’améliorations.

Le comité de retour d’expérience (CREX) est une démarche collective permettant d’analyser des évènements indésirables (EI). Un CREX a été mis en place à la pharmacie à usage intérieur de notre centre hospitalier universitaire depuis 2008. Devant la réapparition d’EI déjà traités en CREX, un bilan d’activité ainsi qu’un suivi des actions correctives (AC) sur le long terme est nécessaire.

Study design: An experimental nonrandomized controlled study on tumor tissue samples. Objective: Estimation of the efficacy of thermablation using radiofrequency to treat metastases of the vertebrae. Summary of background data: Radiofrequency thermablation is one of the management techniques for vertebral metastatic lesions, which is developed and widely used in recent years. Materials: 12 metastatic lesions in 11 patients had undergone surgical intralesional excision and stabilization of the spine. During the surgery, tumor tissue samples were collected pre- and post-thremablation for analyses through histological and ultrastructural studies. However, presently there have been no reports on the evaluation of the tumor tissue necrosis, which should be one of the cogent proofs for the efficacy of this technique. Results: The total analyzed tissues under the light microscope showed 75% of necrosis, and went up to 83% necrosis under the electron microscope. This was mainly due to incomplete necrosis in the renal carcinoma metastases. Conclusions: Thermablation with radiofrequency treatments seems to effectively cause necrosis of the tumor tissues in nearly all the cases, as shown by the histological and ultrastructural studies.

The state of T‐cell receptor beta‐chain gene rearrangement in human T‐cell leukaemias has been analysed. All forms of leukaemia tested (T‐CLL, ALL, PLL, Sezary syndrome and ATL) exhibit rearrangements of C beta genes confirming the clonality of these neoplasias. However we find no evidence for common gene rearrangements nor for restricted rearrangement patterns within this type of neoplasia. We find evidence of T‐cells with C beta 1 and C beta 2 rearrangements, sometimes associated with Igh JH rearrangements, but several cases of T‐cell leukaemia with a marker inversion of chromosome 14 (q11;q32) do not have Igh JH rearrangements. The results suggest that TCR beta gene rearrangement occurs early in T‐cell ontogeny but that this rearrangement is most often irrelevant to leukaemogenesis.

Two distinct types of T-cell receptors (TCR), designated alpha beta and gamma delta, have been identified on the surface of T cells. In the adult, T cells bearing the gamma delta TCR are a minority and they have the phenotype CD3+, CD4-, CD8-/+. By using appropriate probes, rearrangements of the TCR alpha, beta, and gamma genes have been extensively investigated in a variety of lymphoproliferative disorders. Because the TCR delta gene has been cloned only recently, no comparable information exists with respect to this in human leukemias. We report the analysis of the TCR delta gene configuration in 21 T-cell acute and chronic leukemias, 40 B-cell leukemias, 4 acute myeloid leukemias of difficult classification, and 12 normal controls. The TCR delta genes were structurally modified in all T-cell disorders and in germ-line configuration in all controls and all but one case of non-T-cell leukemias tested. In one case of T-chronic lymphocytic leukemia (CD3+, CD4-, CD8+) we found rearrangement and expression of TCR gamma and delta (but not alpha and beta), suggesting that leukemic transformation took place in a cell bearing a TCR gamma delta rather than a TCR alpha beta. In two cases of pre-T-acute lymphoblastic leukemia, only delta was rearranged out of the three TCR genes tested. This finding is in keeping with the suggestion that the TCR delta gene might be the first to rearrange in T cell ontogeny, and that its mode of rearrangement may play a role in the subsequent choice of the cell between production of a TCR alpha beta or gamma delta. Thus, TCR delta chain gene analysis can provide novel information of the clonal nature of T-cell disorders, particularly if the analysis of the beta and gamma genes has not been helpful.

beta and gamma T cell receptor (TCR) gene configuration was studied in 12 patients with large granular lymphocyte T cell leukemia (LGL- leukemia). Both genes were found rearranged in ten cases. In the remaining two patients TCR beta was found in germline configuration. In one of them rearrangement of T cell-rearranging gene gamma (TRG gamma) and a gamma mRNA were demonstrated. We suggest that in this patient the leukemic T cells arose from one of the rare T cells bearing a gamma- delta rather than an alpha-beta TCR heterodimeric molecule. In the other patient several discrete TRG gamma rearrangements were detected. Because her leukemic cells were shown to be monoclonal on the grounds of their karyotype, we suggest that her leukemia originated before any rearrangement had taken place. The combined use of TCR beta and TRG gamma probes provides new information on the origin and clonal expansion of lymphoid cells in LGL-leukemia.

Rearrangement of the immunoglobulin heavy chain (IgH) gene can be utilized as a marker of clonality in a number of B-lineage lymphoproliferative disorders including acute lymphoblastic leukaemia (ALL). We have used a PCR technique involving a panel of amplimers for the 6 different Variable (VH) region families and for a consensus sequence of the Joining (JH) segment to detect clonal IgH rearrangements in the peripheral blood (PB) and/or bone marrow (BM) of 28 patients (17 children and 11 adults) with B-lineage ALL at presentation (20 patients) or with overt relapse (8 patients). The age range of the patients was 2––65 years (mean 15.7 years). Follow up remission BM samples were analysed in 22 patients during and after therapy (2––7 samples per patient), 1––50 months after presentation or relapse. In 1 relapsed case, previously stored complete remission (CR) samples were analysed retrospectively.Clonal IgH chain rearrangements were detected by PCR in 90% of patients studied initially. The 2 VH region families most commonly used were the large VH3 family (65%) and the smaller more JH-proximal VH4 family (22%). More than one VH clone was detectable in 25% of the cases. A gene ““fingerprinting”” modification of a previously described method was applied to the detection of minimal residual disease (MRD) in follow up BM samples with a sensitivity of 10−−3to 10−−4. In 8 of 14 patients remaining in complete remission (CR) during the time of study, all PCR analyses on BM samples in the first 6 months were negative, in some cases as early as 2 weeks post-induction therapy, and a further patient reverted from being PCR positive in the first month after the commencement of therapy to sustained PCR negativity. One adult remains in CR at 50 months after presentation and has been PCR negative at 2 time points after cessation of maintenance therapy (30 and 50 months).Eight patients relapsed in the study period comprising 6 BM and 2 isolated CNS relapses. In 4 cases of BM relapse occurring within 7 months of the start of therapy, all BM remission samples tested in this period were PCR positive. In 2 other patients BM samples tested 7 and 2 months respectively prior to relapse were PCR negative. In the 2 patients with isolated CNS relapse, PCR of BM samples from 2 and 10 months before the relapse were negative. Results thus far suggest that early and sustained loss of PCR positivity after commencement of therapy, particularly in children may be associated with a low risk of early relapse and this early negative PCR result may prove to be predictive for a better prognosis in this group of ALL patients. Further long term analyses of a larger number of appropriately stratified patients will be required to determine the ultimate significance of these studies, the frequency of clonal evolution and the relevant contributions of various treatment phases including transplantation at eliminating the disease.

An unusual case of prolymphocytic leukemia of the B cell type (B-PLL) in a 79-year-old patient is reported. The clinical and cytomorphological features of the disease were typical of B-PLL, but membrane and cytoplasmic immunoglobulins (Ig) could not be demonstrated by immunofluorescence techniques; 3% to 4% of the cells were shown to have IgG kappa in the cytoplasm by a more sensitive immunoperoxidase method. The cells were unreactive with a panel of monoclonal antibodies against T cell antigens but they were positive with B cell lineage reagents: FMC4, anti-HLA-Dr determinants; FMC7, which reacts with most B-PLL; anti-B1 and anti-B4, which react with most B cell leukemias. Analysis of Ig genes at the DNA level demonstrated that both heavy-chain alleles and one Kchain allele were rearranged, confirming that the patient's cells were of B lineage. Chromosome analysis revealed a consistent abnormality, t(17;21)(p11;p11), in all cells and, in addition, a 14q+ marker in 10% of the cells. This study highlights the value of DNA analysis techniques for the characterization of neoplastic B cells. The low rate of expression of Ig genes, despite their rearrangement, suggests that a specific transcriptional or posttranscriptional defect must exist in these cells.

An unusual case of prolymphocytic leukemia of the B cell type (B-PLL) in a 79-year-old patient is reported. The clinical and cytomorphological features of the disease were typical of B-PLL, but membrane and cytoplasmic immunoglobulins (Ig) could not be demonstrated by immunofluorescence techniques; 3% to 4% of the cells were shown to have IgG kappa in the cytoplasm by a more sensitive immunoperoxidase method. The cells were unreactive with a panel of monoclonal antibodies against T cell antigens but they were positive with B cell lineage reagents: FMC4, anti-HLA-Dr determinants; FMC7, which reacts with most B-PLL; anti-B1 and anti-B4, which react with most B cell leukemias. Analysis of Ig genes at the DNA level demonstrated that both heavy- chain alleles and one kappa chain allele were rearranged, confirming that the patient's cells were of B lineage. Chromosome analysis revealed a consistent abnormality, t(17;21)(p11;p11), in all cells and, in addition, a 14q+ marker in 10% of the cells. This study highlights the value of DNA analysis techniques for the characterization of neoplastic B cells. The low rate of expression of Ig genes, despite their rearrangement, suggests that a specific transcriptional or posttranscriptional defect must exist in these cells.

Chromosome 13q deletion is among the most common cytogenetic abnormalities in chronic lymphocytic leukaemia (CLL). We investigated the 13q14.3 deletion in 44 CLL patients by Southern blotting following purification of clonal B CLL cells to >90%. Two sets of probes were used to investigate the site of clonal deletion, the D13S25 and D13S319 markers (at 13q14.3) and probes for exons 11 and 26-27 of the BRCA2 gene (at 13q12). Homozygous and heterozygous deletion at the 13q14.3 region was found in five and 17 patients, respectively. Despite the recent report of the BRCA2 gene involvement in >80% of CLL patients, we failed to detect a single case of homozygous or heterozygous deletion involving the 13q12 region. Our data support previous findings that the 13q14.3, and not the 13q12 region, is the major site of candidate tumour suppressor gene(s) in CLL.

A chromosomal translocation in a T-cell leukemia involving the short arm of human chromosome 11 at band 11p15 disrupts the rhombotin gene. This gene encodes a protein with duplicated cysteine-rich regions called LIM domains, which show homology to zinc-binding proteins and to iron-sulfur centers of ferredoxins. Two homologues of the rhombotin gene have now been isolated. One of these, designated Rhom-2, is located on human chromosome 11 at band 11p13, where a cluster of T-cell leukemia-specific translocations occur; all translocation breakpoints at 11p13 are upstream of the Rhom-2 gene. Human and mouse Rhom-2 are highly conserved and, like rhombotin, encode two tandem cysteine-rich LIM domains. Rhom-2 mRNA is expressed in early mouse development in central nervous system, lung, kidney, liver, and spleen but only very low levels occur in thymus. The other gene, designated Rhom-3, is not on chromosome 11 but also retains homology to the LIM domain of rhombotin. Since the Rhom-2 gene is such a common site of chromosomal damage in T-cell tumors, the consistency of translocations near the rhombotin gene was further examined. A second translocation adjacent to rhombotin was found and at the same position as in the previous example. Therefore, chromosome bands 11p15 (rhombotin) and 11p13 (Rhom-2) are consistent sites of chromosome translocation in T-cell leukemia, with the 11p15 target more rarely involved. The results define the rhombotin gene family as a class of T-cell oncogenes with duplicated cysteine-rich LIM domains.

Two distinct types of T-cell receptors (TCR), designated αβ and γδ, have been identified on the surface of T cells. In the adult, T cells bearing the 76 TCR are a minority and they have the phenotype CD3+, CD4-, CD8-/ +. By using appropriate probes, rearrangements of the TCR α, β, and 7 genes have been extensively investigated in a variety of lymphoproliferative disorders. Because the TCR δ gene has been cloned only recently, no comparable information exists with respect to this in human leukemias. We report the analysis of the TCR δ gene configuration in 21 T-cell acute and chronic leukemias, 40 B-cell leukemias, 4 acute myeloid leukemias, 2 leukemias of difficult classification, and 12 normal controls. The TCR δ genes were structurally modified in all T-cell disorders and in germ-line configuration in all controls and all but one case of non-T-cell leukemias tested. In one case of T-chronic lymphocytic leukemia (CD3+, CD4-, CD8+) we found rearrangement and expression of TCR γ and δ (but not α and β). suggesting that leukemic transformation took place in a cell bearing a TCR γδ rather than a TCR αβ. In two cases of pre-T-acute lymphoblastic leukemia, only δ was rearranged out of the three TCR genes tested. This finding is in keeping with the suggestion that the TCR δ gene might be the first to rearrange in T cell ontogeny, and that its mode of rearrangement may play a role in the subsequent choice of the cell between production of a TCR αβ or γδ. Thus, TCR δ chain gene analysis can provide novel information of the clonal nature of T-cell disorders, particularly if the analysis of the β and γ genes has not been helpful.

The state of T‐cell receptor beta‐chain gene rearrangement in human T‐cell leukaemias has been analysed. All forms of leukaemia tested (T‐CLL, ALL, PLL, Sezary syndrome and ATL) exhibit rearrangements of C beta genes confirming the clonality of these neoplasias. However we find no evidence for common gene rearrangements nor for restricted rearrangement patterns within this type of neoplasia. We find evidence of T‐cells with C beta 1 and C beta 2 rearrangements, sometimes associated with Igh JH rearrangements, but several cases of T‐cell leukaemia with a marker inversion of chromosome 14 (q11;q32) do not have Igh JH rearrangements. The results suggest that TCR beta gene rearrangement occurs early in T‐cell ontogeny but that this rearrangement is most often irrelevant to leukaemogenesis.

β and  γ T cell receptor (TCR) gene configuration was studied in 12 patients with large granular lymphocyte T cell leukemia (LGL-leukemia). Both genes were found rearranged in ten cases. In the remaining two patients TCR  β was found in germline configuration. In one of them rearrangement of T cell-rearranging gene γ(trgγ)and a γmRNA were demonstrated. We suggest that in this patient the leukemic T cells arose from one of the rare T cells bearing a γ–δ rather than an  α-β TCR heterodimericmolecule. In the other patient several discrete TRG γ rearrangements were detected. Because her leukemic cells were shown to be monoclonal on the grounds of their karyotype, we suggest that her leukemia originated before any rearrangement had taken place. The combined use of TCR β and TRG 7 probes provides new information on the origin and clonal expansion of lymphoid cells in LGL-leukemia.© 1988 by Grune&Stratton, Inc. 0006-4971/88/7102-0008S3.00/0

The t(17;19)(q21-q22;p13.3) in acute lymphoblastic leukemia results in creation of an E2A-HLF fusion protein with structural and functional properties of a chimeric transcription factor. Two types of genomic rearrangements underlie fusion of E2A with HLF. In type I rearrangements, an insertion that codes for a portion of the chimera not found in either wild type protein occurs between E2A exon 13- and HLF exon 4-encoded sequences. This insertion is derived from a cryptic exon created at the junction between chromosomes 17 and 19, and includes intronic portions of both E2A and HLF with intervening nontemplated N nucleotides. Type II rearrangements arise from more 5′ breakpoints in E2A and result in fusion cDNAs with E2A exon 12 spliced directly to HLF exon 4. Analysis of the genomic structure of HLF shows that these different modes of protein fusion result from selective constraints to maintain the proper HLF reading frame, because a direct E2A exon 13 to HLF exon 4 splice would lead to translation of a truncated E2A protein lacking any contribution from HLF. These features underscore the requirement for DNA binding and/or dimerization conferred by the bZIP portion of the E2A-HLF chimera in t(17;19)-ALL.

A proportion of haematological malignancies over express the multidrug resistance (MDR) efflux proteins - P-glycoprotein (P-gp), multidrug resistance protein (MRP1) and lung resistance proteins (LRP) that confer resistance to a number of structurally unrelated cytotoxic drugs. The clinical significance of the observed over expression of P-gp in B-CLL regardless of stage or treatment of the disease is unclear, although a protective effect on cell survival has been reported. Recent evidence suggest that a sub-group of B-CLL exist with an aggressive clinical course that is often resistant to treatment. B-CLL cells from this subgroup of patients have unmutated immunoglobulin heavy chain variable region (IgVH) and express zeta-associated protein (ZAP-70). We analysed the association between the expression of MDR proteins and the prognosis indicator ZAP-70 in cells from 46 B-CLL patients (30M/16F, 33 stage A, 7 B, 6 C) for (i) ZAP-70 expression using an antibody (clone 2F3.2)-Alexa Fluor 488 conjugate by flow cytometry and expressed as a ratio of B to T-cell mean cell fluorescence, (ii) the expression of P-gp, MRP1 and LRP protein by flow cytometry using specific antibodies and expressed as MCF ratio to isotype controls, (iii) flow cytometric analysis of functional activity of P-gp expressed as MCF ratio in the presence and absence of verapamil, a known inhibitor of P-gp, (iv) IC50 (μg/ml) for cytotoxic drugs 2-chlorodeoxyadenosine (CdA), chlorambucil (Chl) and fludarabine (FdR) using the cytotoxicity MTT assay and (v) IgVH mutational status by sequence analysis of FR1/JH polymerase chain reaction products in 29/46 (63%) of patients. 13/46 (28%) patients were ZAP-70+ve and the remaining 33/46 (72%) were ZAP-70−ve. P-gp expression (median 1.59 in both groups p=0.85) and P-gp functional activity (median 2.01 vs 1.84 p=0.77) were not statistically different between ZAP-70+ve and ZAP-70−ve groups. There was no significant difference in the expressions of MRP1 (median 2.77 vs 3.48 p=0.31) or LRP (median 4.87 vs 5.18 p=0.56) between ZAP-70+ve and ZAP-70−ve patients. The IC50 levels for CdA, Chl and FdR between ZAP+ve (median 0.04, 6.4 & 0.54mg/ml respectively) and ZAP−ve groups (median 0.065, 12.15 & 0.72 respectively) were not statistically significant (p=0.67, 0.45, 0.36 respectively). 9 ZAP-70+ve and 15 ZAP-70−ve patients were sequentially investigated over a median period of 3 years for expression of P-gp, MRP1, LRP and P-gp function. There were no significant changes in P-gp expression and P-gp function between presentation and follow samples over this period. However, a significant increase in expression of MRP1 (median at presentation 1.65 and follow up 5.23 p= 0.0001) and LRP (median at presentation 3.92 and follow up 5.84 p= 0.0002 respectively) were observed for the ZAP-70−ve group. IgVH mutation analysis was concordant with ZAP-70 expression in 6 patients with poor prognosis (ZAP-70+ve/IgVH unmutated) and in 16 with good prognosis (ZAP-70−ve/IgVH mutated). Statistical analysis of MDR protein expression and IC50 in this smaller cohort of patients provided data similar to that in the main group. MDR proteins, their activity and in vitro drug sensitivity appear not to significantly contribute to the poor prognosis subset of B-CLL identified by their ZAP-70+ve/IgVH unmutated status. The significance of increased MRP1 and LRP expression in the good prognosis ZAP−ve group is unclear.

B-Chronic lymphocytic leukemia (B-CLL) patients whose malignant cells harbour unmutated immunoglobulin heavy chain variable region (IgVH) genes or express the zeta-associated protein tyrosine kinase ZAP-70 show a worse prognosis than do patients with mutated IgVH genes or ZAP-70−ve expression. The inability of malignant cells to activate the pro-apoptotic p53 pathway in response to ionizing radiation (IR) also correlates with a poor prognosis. We studied ZAP-70 expression and IgVH mutation status in 161 patients with B-CLL in order to determine the degree of concordance between these two prognostic criteria (M104/F57, wbc 2.44–576x109/l lymphocytes 0.56–287x109/l). We also studied the functional status of the p53 pathway and the apoptotic response to ionizing radiation in cells from a subset of patients from both prognostic categories. A human ZAP-70 antibody (clone 2F3-2) was conjugated to the Alexa Fluor 488 dye using a zenon mouse IgG labelling kit and used for a FACS based assay. FACS results were expressed as a ratio of B-cell mean cell fluorescence to T-cell mean cell fluorescence with a cut off at > 0.75 identifying a ZAP-70+ve sub-group. IgVH mutational status was studied by sequence analysis of FR1/JH polymerase chain reaction products. The ability of 5Gy ionizing radiation to augment levels of p53 and its transcriptional target p21CIP1 was quantified by western blot analysis. Cleavage of the caspase 3 target poly(ADP ribose) polymerase (PARP) was used as a measure of apoptosis induction. ZAP-70+ve expression was observed in 25% (41/161) of the samples with a median ratio of 0.85 (range 0.76–1.46) while the remaining 120 samples were ZAP-70−ve, with a median ratio of 0.56 (range 0.19–0.73). IgVH mutation status was analysed in 92 of these patients. Assignment of prognostic category by both criteria was concordant in 72/92 (78.2%) of the cases of which 54/92 (58.6%) were ZAP−ve/IgVH mutated (good prognosis) and 18/92 (19.5%) were ZAP+ve/IgVH unmutated (poor prognosis) patients. The remaining 21.7% were discordant, ie., either ZAP+ve/IgVH mutated (5.4%) or ZAP−ve/IgVH unmutated (16.3%). Isolates from 5/6 ZAP+ve/IgVH unmutated patients upregulated p53 in response to IR but nevertheless failed to initiate PARP cleavage, suggestive of a block in the apoptotic pathway distal to p53 induction. In 9 ZAP−ve/IgVH mutated isolates studied, 7 induced p53, p21 and PARP cleavage following IR. In conclusion, this large cohort of CLL patients demonstrated a good correlation between ZAP-70 expression and IgVH mutational status in identifying a poor prognosis sub-group. However, this prognostic category, as defined by both IgVH mutation status and ZAP-70 expression failed in some cases to predict the ability of B-CLL cells to induce an apoptotic response to DNA damage in vitro. Induction of the p53 pathway was not always sufficient to secure an apoptotic response, especially in the poor prognosis group. A combination of ZAP-70 and IgVH analysis with a functional assay for DNA damage-induced apoptosis will identify individuals in either prognostic category who are unlikely to respond to conventional cytotoxic drugs. Alternative therapeutic strategies independent of DNA damage-inducing agents may be of value in the treatment of these patients.

B-Chronic lymphocytic leukemia (B-CLL) patients whose malignant cells harbour unmutated immunoglobulin heavy chain variable region (IgVH) genes or express the zeta-associated protein tyrosine kinase ZAP-70 show a worse prognosis than do patients with mutated IgVHgenes or ZAP-70−veexpression. The inability of malignant cells to activate the pro-apoptotic p53 pathway in response to ionizing radiation (IR) also correlates with a poor prognosis. We studied ZAP-70 expression and IgVHmutation status in 161 patients with B-CLL in order to determine the degree of concordance between these two prognostic criteria (M104/F57, wbc 2.44–576x109/l lymphocytes 0.56–287x109/l). We also studied the functional status of the p53 pathway and the apoptotic response to ionizing radiation in cells from a subset of patients from both prognostic categories. A human ZAP-70 antibody (clone 2F3-2) was conjugated to the Alexa Fluor 488 dye using a zenon mouse IgG labelling kit and used for a FACS based assay. FACS results were expressed as a ratio of B-cell mean cell fluorescence to T-cell mean cell fluorescence with a cut off at > 0.75 identifying a ZAP-70+vesub-group. IgVHmutational status was studied by sequence analysis of FR1/JHpolymerase chain reaction products. The ability of 5Gy ionizing radiation to augment levels of p53 and its transcriptional target p21CIP1was quantified by western blot analysis. Cleavage of the caspase 3 target poly(ADP ribose) polymerase (PARP) was used as a measure of apoptosis induction. ZAP-70+veexpression was observed in 25% (41/161) of the samples with a median ratio of 0.85 (range 0.76–1.46) while the remaining 120 samples were ZAP-70−ve, with a median ratio of 0.56 (range 0.19–0.73). IgVHmutation status was analysed in 92 of these patients. Assignment of prognostic category by both criteria was concordant in 72/92 (78.2%) of the cases of which 54/92 (58.6%) were ZAP−ve/IgVHmutated (good prognosis) and 18/92 (19.5%) were ZAP+ve/IgVHunmutated (poor prognosis) patients. The remaining 21.7% were discordant, ie., either ZAP+ve/IgVHmutated (5.4%) or ZAP−ve/IgVHunmutated (16.3%). Isolates from 5/6 ZAP+ve/IgVHunmutated patients upregulated p53 in response to IR but nevertheless failed to initiate PARP cleavage, suggestive of a block in the apoptotic pathway distal to p53 induction. In 9 ZAP−ve/IgVHmutated isolates studied, 7 induced p53, p21 and PARP cleavage following IR. In conclusion, this large cohort of CLL patients demonstrated a good correlation between ZAP-70 expression and IgVHmutational status in identifying a poor prognosis sub-group. However, this prognostic category, as defined by both IgVHmutation status and ZAP-70 expression failed in some cases to predict the ability of B-CLL cells to induce an apoptotic response to DNA damage in vitro. Induction of the p53 pathway was not always sufficient to secure an apoptotic response, especially in the poor prognosis group. A combination of ZAP-70 and IgVHanalysis with a functional assay for DNA damage-induced apoptosis will identify individuals in either prognostic category who are unlikely to respond to conventional cytotoxic drugs. Alternative therapeutic strategies independent of DNA damage-inducing agents may be of value in the treatment of these patients.

A proportion of haematological malignancies over express the multidrug resistance (MDR) efflux proteins - P-glycoprotein (P-gp), multidrug resistance protein (MRP1) and lung resistance proteins (LRP) that confer resistance to a number of structurally unrelated cytotoxic drugs. The clinical significance of the observed over expression of P-gp in B-CLL regardless of stage or treatment of the disease is unclear, although a protective effect on cell survival has been reported. Recent evidence suggest that a sub-group of B-CLL exist with an aggressive clinical course that is often resistant to treatment. B-CLL cells from this subgroup of patients have unmutated immunoglobulin heavy chain variable region (IgVH) and express zeta-associated protein (ZAP-70). We analysed the association between the expression of MDR proteins and the prognosis indicator ZAP-70 in cells from 46 B-CLL patients (30M/16F, 33 stage A, 7 B, 6 C) for (i) ZAP-70 expression using an antibody (clone 2F3.2)-Alexa Fluor 488 conjugate by flow cytometry and expressed as a ratio of B to T-cell mean cell fluorescence, (ii) the expression of P-gp, MRP1 and LRP protein by flow cytometry using specific antibodies and expressed as MCF ratio to isotype controls, (iii) flow cytometric analysis of functional activity of P-gp expressed as MCF ratio in the presence and absence of verapamil, a known inhibitor of P-gp, (iv) IC50(μg/ml) for cytotoxic drugs 2-chlorodeoxyadenosine (CdA), chlorambucil (Chl) and fludarabine (FdR) using the cytotoxicity MTT assay and (v) IgVHmutational status by sequence analysis of FR1/JHpolymerase chain reaction products in 29/46 (63%) of patients. 13/46 (28%) patients were ZAP-70+veand the remaining 33/46 (72%) were ZAP-70−ve. P-gp expression (median 1.59 in both groups p=0.85) and P-gp functional activity (median 2.01 vs 1.84 p=0.77) were not statistically different between ZAP-70+veand ZAP-70−vegroups. There was no significant difference in the expressions of MRP1 (median 2.77 vs 3.48 p=0.31) or LRP (median 4.87 vs 5.18 p=0.56) between ZAP-70+veand ZAP-70−vepatients. The IC50levels for CdA, Chl and FdR between ZAP+ve(median 0.04, 6.4 & 0.54mg/ml respectively) and ZAP−vegroups (median 0.065, 12.15 & 0.72 respectively) were not statistically significant (p=0.67, 0.45, 0.36 respectively). 9 ZAP-70+veand 15 ZAP-70−vepatients were sequentially investigated over a median period of 3 years for expression of P-gp, MRP1, LRP and P-gp function. There were no significant changes in P-gp expression and P-gp function between presentation and follow samples over this period. However, a significant increase in expression of MRP1 (median at presentation 1.65 and follow up 5.23 p= 0.0001) and LRP (median at presentation 3.92 and follow up 5.84 p= 0.0002 respectively) were observed for the ZAP-70−vegroup. IgVHmutation analysis was concordant with ZAP-70 expression in 6 patients with poor prognosis (ZAP-70+ve/IgVHunmutated) and in 16 with good prognosis (ZAP-70−ve/IgVHmutated). Statistical analysis of MDR protein expression and IC50in this smaller cohort of patients provided data similar to that in the main group. MDR proteins, their activity and in vitro drug sensitivity appear not to significantly contribute to the poor prognosis subset of B-CLL identified by their ZAP-70+ve/IgVHunmutated status. The significance of increased MRP1 and LRP expression in the good prognosis ZAP−vegroup is unclear.