Mutant N-RAS induces erythroid lineage dysplasia in human CD34+ cells.

RAS mutations arise at high frequency (20-40%) in both acute myeloid leukemia and myelodysplastic syndrome (which is considered to be a manifestation of preleukemic disease). In each case, mutations arise predominantly at the N-RAS locus. These observations suggest a fundamental role for this oncogene in leukemogenesis. However, despite its obvious significance, little is known of how this key oncogene may subvert the process of hematopoiesis in human cells. Using CD34+ progenitor cells, we have modeled the preleukemic state by infecting these cells with amphotropic retrovirus expressing mutant N-RAS together with the selectable marker gene lacZ. Expression of the lacZ gene product, beta-galactosidase, allows direct identification and study of N-RAS-expressing cells by incubating infected cultures with a fluorogenic substrate for beta-galactosidase, which gives rise to a fluorescent signal within the infected cells. By using multiparameter flow cytometry, we have studied the ability of CD34+ cells expressing mutant N-RAS to undergo erythroid differentiation induced by erythropoietin. By this means, we have found that erythroid progenitor cells expressing mutant N-RAS exhibit a proliferative defect resulting in an increased cell doubling time and a decrease in the proportion of cells in S + G2M phase of the cell cycle. This is linked to a slowing in the rate of differentiation as determined by comparative cell-surface marker analysis and ultimate failure of the differentiation program at the late-erythroblast stage of development. The dyserythropoiesis was also linked to an increased tendency of the RAS-expressing cells to undergo programmed cell death during their differentiation program. This erythroid lineage dysplasia recapitulates one of the most common features of myelodysplastic syndrome, and for the first time provides a causative link between mutational activation of N-RAS and the pathogenesis of preleukemia.

Expression of CD33 is a predictive factor for effect of gemtuzumab ozogamicin at different doses in adult acute myeloid leukaemia.

It remains unclear in adult acute myeloid leukaemia (AML) whether leukaemic expression of CD33, the target antigen for gemtuzumab ozogamicin (GO), adds prognostic information on GO effectiveness at different doses. CD33 expression quantified in 1583 patients recruited to UK-NCRI-AML17 (younger adults) and UK-NCRI-AML16 (older adults) trials was correlated with clinical outcomes and benefit from GO including a dose randomisation. CD33 expression associated with genetic subgroups, including lower levels in both adverse karyotype and core-binding factor (CBF)-AML, but was not independently prognostic. When comparing GO versus no GO (n=393, CBF-AMLs excluded) by stratified subgroup-adjusted analysis, patients with lowest quartile (Q1) %CD33-positivity had no benefit from GO (relapse risk, HR 2.41 (1.27-4.56), P=0.009 for trend; overall survival, HR 1.52 (0.92-2.52)). However, from the dose randomisation (NCRI-AML17, n=464, CBF-AMLs included), 6 mg/m2 GO only had a relapse benefit without increased early mortality in CD33-low (Q1) patients (relapse risk HR 0.64 (0.36-1.12) versus 1.70 (0.99-2.92) for CD33-high, P=0.007 for trend). Thus CD33 expression is a predictive factor for GO effect in adult AML; although GO does not appear to benefit the non-CBF AML patients with lowest CD33 expression a higher GO dose may be more effective for CD33-low but not CD33-high younger adults.

A comparison of clofarabine with ara-C, each in combination with daunorubicin as induction treatment in older patients with acute myeloid leukaemia.

The study was designed to compare clofarabine plus daunorubicin vs daunorubicin/ara-C in older patients with acute myeloid leukaemia (AML) or high-risk myelodysplastic syndrome (MDS). Eight hundred and six untreated patients in the UK NCRI AML16 trial with AML/high-risk MDS (median age, 67 years; range 56-84) and normal serum creatinine were randomised to two courses of induction chemotherapy with either daunorubicin/ara-C (DA) or daunorubicin/clofarabine (DClo). Patients were also included in additional randomisations; ± one dose of gemtuzumab ozogamicin in course 1; 2v3 courses and ± azacitidine maintenance. The primary end point was overall survival. The overall response rate was 69% (complete remission (CR) 60%; CRi 9%), with no difference between DA (71%) and DClo (66%). There was no difference in 30-/60-day mortality or toxicity: significantly more supportive care was required in the DA arm even though platelet and neutrophil recovery was significantly slower with DClo. There were no differences in cumulative incidence of relapse (74% vs 68%; hazard ratio (HR) 0.93 (0.77-1.14), P=0.5); survival from relapse (7% vs 9%; HR 0.96 (0.77-1.19), P=0.7); relapse-free (31% vs 32%; HR 1.02 (0.83-1.24), P=0.9) or overall survival (23% vs 22%; HR 1.08 (0.93-1.26), P=0.3). Clofarabine 20 mg/m2 given for 5 days with daunorubicin is not superior to ara-C+daunorubicin as induction for older patients with AML/high-risk MDS.

Characteristics and outcome of patients with therapy-related acute promyelocytic leukemia front-line treated with or without arsenic trioxide.

Therapy-related acute promyelocytic leukemia (t-APL) is relatively rare, with limited data on outcome after treatment with arsenic trioxide (ATO) compared to standard intensive chemotherapy (CTX). We evaluated 103 adult t-APL patients undergoing treatment with all-trans retinoic acid (ATRA) alone (n=7) or in combination with ATO (n=24), CTX (n=53), or both (n=19). Complete remissions were achieved after induction therapy in 57% with ATRA, 100% with ATO/ATRA, 78% with CTX/ATRA, and 95% with CTX/ATO/ATRA. Early death rates were 43% for ATRA, 0% for ATO/ATRA, 12% for CTX/ATRA and 5% for CTX/ATO/ATRA. Three patients relapsed, two developed therapy-related acute myeloid leukemia and 13 died in remission including seven patients with recurrence of the prior malignancy. Median follow-up for survival was 3.7 years. None of the patients treated with ATRA alone survived beyond one year. Event-free survival was significantly higher after ATO-based therapy (95%, 95% CI, 82-99%) as compared to CTX/ATRA (78%, 95% CI, 64-87%; P=0.042), if deaths due to recurrence of the prior malignancy were censored. The estimated 2-year overall survival in intensively treated patients was 88% (95% CI, 80-93%) without difference according to treatment (P=0.47). ATO when added to ATRA or CTX/ATRA is feasible and leads to better outcomes as compared to CTX/ATRA in t-APL.

Mutant RAS selectively promotes sensitivity of myeloid leukemia cells to apoptosis by a protein kinase C-dependent process.

RAS mutations arise at high frequency in human malignancy and have been shown to play a role in the disruption of both normal differentiation and proliferation. In addition, RAS influences a number of intracellular signaling pathways, which impinge on proteins that regulate programmed cell death. In this study, we have examined whether this oncogene can influence the activation of the apoptotic process induced by a range of therapeutic agents used to treat leukemia, and we have identified the downstream targets of RAS mediating the observed changes in sensitivity. Using myeloid leukemia cells (P39) retrovirally transduced with mutant H-RAS, we found that the influence of this oncogene was highly dependent on the inducer used: whereas RAS had no significant effect on spontaneous apoptosis or on the response to the cytotoxic drugs (doxorubicin or 1-beta-arabinofuranosylcytosine), P39-RAS cells showed a strongly augmented response to all-trans-retinoic acid (ATRA) in both the induction of apoptosis and differentiation. Because, under some circumstances, RAF has been associated with promoting apoptosis, we examined whether the activation of this kinase by mutant RAS could be responsible for the augmented response to ATRA. However, constitutive activation of RAF did not alter the apoptotic sensitivity of these cells, making it unlikely that RAS promotes apoptosis by stimulating this kinase. Nor did we find that BCL-2 was differentially down-regulated in P39-RAS cells. Rather, we found that the activation of protein kinase C (PKC) by low-dose phorbol ester could almost entirely recapitulate transformation by RAS, in terms of promoting both apoptosis and differentiation after treatment with ATRA. Moreover, the RAS-induced phenotype could be completely abolished by a specific inhibition of PKC under conditions that had no effect on the response of control cells. In conclusion, we have shown that mutant RAS promotes differentiation-associated cell death in P39 cells by stimulating the activity of PKC, which is itself an important regulator of myeloid differentiation. PKC activation, in turn, powerfully synergizes with the PKC-independent action of ATRA. This work identifies a possible explanation for the ability of this oncogene to promote myeloid differentiation of hematopoietic cells. Clinically, it raises the possibility that although leukemias expressing mutant RAS may not show an altered response to cytotoxic agents, they may show enhanced sensitivity to differentiation therapy with ATRA.

Mapping of breakpoints, and relationship to BCR-ABL RNA expression, in Philadelphia-chromosome-positive chronic myeloid leukaemia patients with a breakpoint around exon 14 (b3) of the BCR gene.

The BCR gene, on chromosome 22, is involved in the Philadelphia (Ph1) chromosome which is a characteristic cytogenetic marker of chronic myeloid leukaemia (CML). Breakpoints in CML occur within the M-bcr region (5.8 kb) which encompasses exons 12-15 (b1-b4), and the M-bcr can be conveniently divided into five zones by restriction mapping. One of these zones (3) contains exon b3 which can be either present or absent from the hybrid mRNA, even if it is present in the chimaeric gene. We have mapped the breakpoints around BCR exon b3 and related this to the type of RNA splice site expressed, in CML patients at diagnosis. Breakpoints within zone 3 were restriction mapped to one of six sub-zones and the site related to the type of RNA splice site. Two clusters of breakpoints within zone 3 were observed. One cluster was located around exon b3 and often resulted in deletion of exon b3 from the chimaeric gene. The majority of this cluster expressed b2-a2 spliced RNA, usually as a consequence of a deletion removing exon b3. The second cluster occurred within two sub-zones that spanned an Alu sequence, and 90% of this cluster exhibited b3-a2 spliced RNA. Furthermore, a greater number of patients had entered blast crisis if the RNA contained BCR exon b3 (8 of 10 patients), compared to those with b2-a2 spliced RNA (3 of 12 patients). The high degree of heterogeneity in the site of the breakpoint within zone 3 of the M-bcr, combined with the type of BCR-ABL hybrid mRNA expressed, further implicates BCR exon b3 in the pathogenesis of CML.

Methylation of the major breakpoint cluster region (M-bcr) in Philadelphia-positive CML.

It has previously been shown that a cluster of HpaII sites with the potential to be methylated exist around exon b3 of the M-bcr region involved in the formation of the Philadelphia chromosome in chronic myeloid leukemia (CML). The degree of hypermethylation of these sites can be directly correlated with the percentage of immature cells, whilst progressive hypomethylation occurs during the maturation of the granulocyte lineage. We have examined samples obtained from CML patients at diagnosis, during chronic phase, and blast crisis to examine the degree of methylation of this region in the non-rearranged BCR gene and the rearranged BCR-ABL gene. A low degree of methylation of the non-rearranged gene, similar to that observed in normal individuals, was observed in diagnosis and chronic phase samples. Increased methylation was observed during blast crisis indicative of the presence of immature cells in the samples. In contrast, a significantly lower degree of methylation was observed in the rearranged BCR-ABL gene at the onset of blast crisis. Division of the samples into those patients who had lost exon b3 during the formation of the BCR/ABL gene and those that had retained exon b3 produced differing patterns of methylation during disease progression. The former group, who also expressed a b2-a2 mRNA, showed an increase in methylation of the non-rearranged BCR gene prior to and during blast crisis, with a inverse decrease in the methylation of the BCR/ABL gene. Those patients who had retained exon b3, and expressed a b3-a2 mRNA, showed no change in the extent of methylation of the BCR/ABL gene but did exhibit an increase in methylation of the BCR gene during blast crisis. The consequence of the differing degree of methylation during disease progression could affect, to some extent, the specificity of protein binding or RNA expression.

Two new polymorphisms but no mutations of the KIT gene in patients with myelodysplasia at positions corresponding to human FMS and murine W locus mutational hot spots.

The KIT proto-oncogene encodes a tyrosine kinase receptor which plays a critical role in haemopoiesis. We have screened genomic DNA from bone marrow mononuclear cells of 46 patients with myelodysplasia (MDS) for mutations/deletions of exons 6, 13, 17, and 21 of the KIT gene (stem cell factor receptor) using polymerase chain reaction (PCR), polyacrylamide gel electrophoresis, and autoradiography to detect single-stranded conformational polymorphisms (SSCP). These exons include positions analogous to those mutated in the FMS gene (colony-stimulating factor-1 receptor) in myelodysplastic syndrome (MDS) and mutated/deleted in the Dominant White Spotting mouse (W locus) which results in macrocytic anaemia. Two different gel running conditions were used for each exon. Polymorphisms were identified only at 4 degrees C in exon 17 (three out of 44 MDS samples and two of 21 DNA samples from normal subjects), and in the non-coding region of exon 21 (five out of 34 MDS samples and seven out of 19 normals). Direct sequencing identified a G to A base change at nucleotide 3169 within exon 21, and a C to T change at position 2415 in exon 17. No conformational changes suggestive of mutations or deletions have been found to date, although we cannot rule out low frequency clonal abnormalities undetectable by our method, which has a sensitivity in our hands of approximately 5%. Polymorphisms occur frequently in the KIT gene. Together with this study, a total of five have been described.

Amplification and sequencing of genomic breakpoints located within the M-bcr region by Vectorette-mediated polymerase chain reaction.

The polymerase chain reaction (PCR) cannot be used to amplify the breakpoint in the chimaeric BCR-ABL gene in CML and acute leukaemias due to the large variation in the sites of breakpoint in the BCR gene (within a 5.8 kb region) and in the ABL gene (within a 150 kb region). The disease state is usually monitored using RNA-PCR to monitor abnormal transcripts. We have used a new modification of the PCR to amplify breakpoints within zone 3 of the M-bcr. A synthetic oligonucleotide linker, the Vectorette, is ligated to restriction digested DNA, and amplification is carried out between primers for a known target sequence and the Vectorette linker. Three Philadelphia chromosome Ph1-positive CML patients with breakpoints within the ALU region of zone 3 have been amplified and the sequence immediately around the breakpoint determined. The breaks occurred within 70 bp and two were only 14 bp apart. The Vectorette-PCR technique has the potential to rapidly identify and sequence breakpoints, and will enable the design of patient-specific primers to monitor disease progression, particularly following bone marrow transplantation.

A C-terminal FMS mutation in a patient with B-cell malignancy.

The FMS proto-oncogene encodes a polypeptide growth factor receptor expressed on the cell surface of monocytes and B lymphocytes within the haematological system. Mutations of the FMS gene at codons 301 and 969 have been detected in a number of haematological disorders. Mutations at these codons are thought to be important in the pathogenesis of leukaemia in cells expressing a mutant receptor. Following our finding that the colony stimulating factor-1 receptor (CSF-1R) was expressed on B cells, we have assessed DNA from 17 patients with B-cell chronic lymphocytic leukaemia (CLL), 15 with acute lymphoblastic leukaemias (ALL), two samples from patients with B-cell non-Hodgkin's lymphoma (B-NHL), and 20 haematologically normal individuals for the presence of C-terminal mutations of the FMS gene. Using single stranded conformational polymorphism analysis (SSCP), a single band shift was detected resulting from a nucleotide insertion at codon 965 in the DNA isolated from a patient with B-NHL. These results indicate that mutations of the FMS gene in this region are rare in B-cell malignancy but may contribute to the pathogenesis of leukaemias and lymphomas in a small subset of patients. However, the presence of other mutations not detected using this type of analysis cannot be excluded.

Autologous MHC-dependent leukaemia-reactive T lymphocytes in a patient with chronic myeloid leukaemia.

We have investigated the presence of T cells capable of recognizing autologous leukaemia cells in a patient with CML. We demonstrated that these cells were anergic to the leukaemia cells. They were estimated to be present at a frequency of 1:4000 T cells in the peripheral blood. However, following incubation with high dose rIL-2, these effector cells were able to proliferate, secrete TH1 cytokines and lyse target cells upon challenging with fresh autologous leukaemia cells. Both CD4 and CD8 T cells were involved in the proliferative responses. Moreover, the immune response was blocked by monomorphic anti-HLA antibodies, suggesting that the MHC molecules are needed by the T cells for leukaemia cell/antigen recognition. This work therefore indicates a potential reservoir of effector cells in CML patients which may be exploited for the modulation of leukaemia cell growth. It also provides evidence for a distinct T cell population capable of mediating GVL responses.

Further evidence that the site of the breakpoint in the major breakpoint cluster region (M-bcr) may be a prognostic indicator.

A break in chromosome 22 within the major breakpoint cluster region (M-bcr) is a characteristic of Philadelphia chromosome-positive CML. We have determined the zone of the breakpoint in 80 chronic myelogenous leukemia (CML) patients and have confirmed our previous observation that a relationship does exist between the subregion of the breakpoint within the M-bcr and the average length of the chronic phase of the disease. Patients with a 3' breakpoint have a statistically shorter chronic phase (25 months) than patients with a 5' break (55 months). Thus, a molecular analysis of the M-bcr may provide a prognostically useful indicator of the probable length of the chronic phase, although the underlying mechanism of blast transformation, and the role (if any) of the hybrid phl-abl mRNA, is still unclear.

Aberrant expression of p21RAS but not p120GAP is a common feature of myelodysplasia.

The RAS gene family encodes signal transducing proteins which are involved in the regulation of cell growth and differentiation. Constitutively 'activating' point mutations of RAS have been detected at high frequency in preleukaemia and acute leukaemia, however, the distribution of expression of p21RAS in normal or preleukaemic primary haematopoietic cells has not been studied. We have examined the expression of p21RAS and its negative regulator/downstream effector, p120GAP, in combination with lineage-specific cluster of differentiation markers in normal and preleukaemic myeloid bone marrow cells using flow cytometry. Normal bone marrow was characterized by low and uniform levels of p21RAS expression throughout all lineages analysed. In contrast, 28% (9/32) of patients with myelodysplastic syndrome (MDS) over-expressed p21RAS. In three of these patients a single over-expressing peak of p21RAS expression was observed, with no evidence of a population exhibiting expression within the normal range. In 6/32 MDS patients over-expression of p21RAS was observed only in a subpopulation of the myeloid cells. Follow-up samples were analysed in three of these six patients; over-expression was confirmed in each patient and in two patients a relative expansion of the over-expressing cell population was observed. Eight out of nine of the patients with aberrant p21RAS expression were diagnosed with low-risk MDS. From 21 MDS patients screened for p120GAP expression, no reduction or loss of expression was observed, however, one AML patient demonstrated a heterogeneous pattern of expression. p120GAP expression was lower (P < 0.05) in the AML group than in normals. The results of the study suggest that over-expression of the RAS gene product, p21RAS, may represent an alternative or additional mechanism of activation of the RAS signalling pathway and that this may play a role in leukaemogenesis, however, there is no evidence from this study that loss of p120GAP expression is a feature of myelodysplasia.

Nature of HLA-associated predisposition to childhood acute lymphoblastic leukemia.

A molecular analysis was carried out in 63 sequentially diagnosed childhood acute lymphoblastic leukemia (ALL) patients and 1011 controls to investigate the homozygosity rate for HLA-DR53. HLA-DR53 is associated with acute myeloblastic leukemia at the protein level, and our previous study has shown its association with early-onset chronic myeloid leukemia only in homozygous form at the DNA level. In the present study, the homozygosity rates for DR53 were 17.5 and 13.6% in patients and controls, respectively. Ten of the 11 homozygous patients were boys. In the common ALL group (n = 40), all seven DR53 homozygous patients were boys, and among 19 girls this genotype was not observed (P = 0.006). For males, homozygosity for DR53 revealed a relative risk (RR) of 3.29 (P = 0.008) for common ALL. Five of the 11 relapsed patients were homozygous for DR53. Heterozygous frequencies for HLA-DR53 were not different between patients and controls. Homozygosity for DR53 was associated with a very high relapse rate (45.5 vs 7.7%, P = 0.002, RR = 9.1). These results extended our findings in chronic myeloid leukemia and showed the recessive nature and the male predominance of the interactive HLA influence on the development of childhood leukemia. Molecular mimicry of an HLA-DR53 epitope by oncogenic (retro)viruses or putative susceptibility genes in linkage disequilibrium with HLA-DR53 may be responsible for this association.

Non-dysplastic myelodysplasia?

Patients successfully treated for a malignancy with cytotoxic therapy have an increased risk of developing secondary myelodysplasia (MDS) and acute myeloid leukemia (AML). We report a patient in remission from Hodgkin's disease (HD) who remains hematologically normal 4 years after combination chemotherapy, but who has biological and genetic abnormalities characteristic of myelodysplasia. X-inactivation analysis using a 5' phosphoglycerate kinase (PGK) probe demonstrates polyclonal hematopoiesis, but cytogenetic analysis reveals a clonal population with a minority of metaphases having a 7q-deletion. NRAS mutations are not detectable 1 year after treatment, but are present in two separate clones (at codons 12 and 15) analyzed by single-stranded conformational polymorphism (SSCP), followed by cloning and sequencing 4 years after treatment. The presence of an activated NRAS with the same codon 12 mutation was independently confirmed by the nude mouse tumorigenicity assay. In vitro peripheral blood granulocyte-macrophage colony-forming units (CFU-GM) have changed from normal to undetectable levels while erythroid burst forming units (BFU-E) were significantly reduced on two occasions during the period of observation. These abnormalities are characteristic of MDS. Continued clinical follow-up will determine whether these evolving genetic and biological abnormalities pre-date the onset of clinical and morphological features of MDS.

Expression of AML1-ETO in human myelomonocytic cells selectively inhibits granulocytic differentiation and promotes their self-renewal.

The t(8;21) translocation is one of the most frequent translocations in acute myeloid leukaemia (AML), giving rise to the AML1-ETO fusion protein (or RUNX1-CBF2T1). This abnormality is associated with myelocytic leukaemia with dysplastic granulopoiesis. Here, we demonstrate that when expressed in a normal human (CD34(+)) progenitor population, AML1-ETO selectively inhibits granulocyte colony formation but not monocyte colony formation. In bulk liquid culture, we found that though AML1-ETO transiently inhibited the proliferation of CD34(+) cells, it promoted long-term growth of myeloid cells for more than 80 days, suggesting that differentiation was inhibited. In support of this, cultures expressing AML1-ETO demonstrated enhanced retention of colony-forming capacity. Phenotypic examination of AML1-ETO cultures revealed a defect in granulocytic differentiation in terms of retention of CD34(+) cells within the culture and delayed CD11b upregulation. Morphologically, granulocyte terminal differentiation in AML1-ETO-expressing cells was inhibited by 83+/-5%, giving rise to a build-up of early to intermediate granulocytes that exhibited a number of morphological features associated with t(8;21) leukaemias. In contrast, AML1-ETO had little or no effect on monocytic differentiation. Taken together, these results suggest that expression of AML1-ETO selectively inhibits the differentiation of granulocytic cells and promoted extensive self-renewal, supporting a causal role for t(8;21) translocations in leukaemogenesis.

Prevention of graft-versus-host disease by ex vivo T cell depletion: reduction in graft failure with augmented total body irradiation.

Graft-versus-host disease prevention was attempted in 35 consecutive patients with hematological malignancy who received bone marrow from an HLA match sibling donor who was depleted of T cells ex vivo. Five of the first 8 patients who received cyclophosphamide 60 mg/kg on 2 consecutive days followed by fractionated total body irradiation (TBI) (6 x 2 Gy) had graft failure. The subsequent 27 patients had received an extra fraction of TBI (7 x 2 Gy), and only one failed to have stable engraftment. There were no differences in nucleated cell dose, granulocyte-macrophage colony-forming units, or T cell numbers given to the two groups. Neutrophil but not platelet regeneration of those patients who successfully grafted was slower than in a group of historical controls receiving unmanipulated marrow. Significant graft-versus-host disease was prevented with no increase in relapse rate. We suggest that engraftment can be reliably achieved by augmenting the TBI conditioning in recipients of T cell-depleted matched allogeneic bone marrow.

Autologous bone marrow transplantation with monoclonal antibody purged marrow for high risk acute lymphoblastic leukemia.

Fifty-four patients with acute lymphoblastic leukemia (ALL: 1 relapse, 21 high risk first complete remission (CR 1), 29 second CR (CR 2), and 3 third CR (CR 3) were treated by autologous bone marrow transplantation at three centers. Before storage, the marrows were purged ex vivo with appropriate MAbs RFAL3 (CD10), SB4 (CD19), and RFT2 (CD7), with rabbit serum as the source of complement. All patients received total body irradiation either 750 cGy (middose 15 cGy/min) as a single fraction or 6 x 200 cGy over 3 days (midline dose 16 cGy/min) with lung shielding from 1,100 cGy. The patients who received 750 cGy also received cyclophosphamide or the same drug combined with ara-C or prednisone, teniposide, vincristine, ara-C, and dauno-rubicin. Patients receiving 200 cGy x 6 also received either cyclophosphamide, melphalan, or ara-C and cyclophosphamide. Three patients died of post transplantation complications (interstitial pneumonia, hepatitis B liver necrosis, or encephalitis). This gives a procedure related mortality of 5%. Nonfatal complications were 10 cases of septicemia, 4 interstitial pneumonia, 2 interstitial nephritis, 1 veno-occlusive disease (VOD), and 1 case of hemolytic uremic syndrome. The patient autografted in relapse died of relapse within 2 months. In CR 1 6 or 21 patients have had a relapse, and the actuarial leukemia free survival from CR is 65% (median follow-up 16 months). In CR 2-3 18 of 32 patients have relapsed, and the actuarial leukemia free survival is 31% (median follow-up 18.5 months) from CR. Twelve patients have achieved an inversion, (i.e., present CR longer than previous CR), with a further seven with the potential to achieve inversion. We conclude that ABMT in high risk ALL has a low procedure related mortality (5%), and there are few other complications. The in vitro purging with MAbs had no adverse effect on bone marrow reconstitution, but this study was not designed to demonstrate its antileukemic efficacy. The actuarial leukemia free survival time in the present study for patients with high risk CR 1 and the inversions in CF 2-3 are promising and indicate a potential beneficial effect of ABMT.

Oral 9-cis retinoic acid (Alitretinoin) in the treatment of myelodysplastic syndromes: results from a pilot study.

A multicenter phase II study was initiated to investigate the efficacy, toxicity and tolerability of an oral regimen of 9-cis retinoic acid (9CRA) as a differentiation-inducing agent stimulating both retinoic acid receptor (RAR) and retinoic X receptor (RXR). Thirty patients with myelodysplastic syndromes (MDS) were enrolled into the study. The MDS subtypes were distributed as follows: 14 refractory anaemia (RA), four refractory anaemia with ringed sideroblasts (RARS), and 12 refractory anaemia with excess blasts (RAEB). The age ranged from 40 to 81 years (median 70). None of these had previously received treatment for MDS other than supportive therapy. 9CRA (Alitretinoin capsules, kindly provided by Allergan-Ligand Retinoid Therapeutics) was given daily at 60 mg/m2 p.o. for 1 week, followed by an intra-patient escalation to 100 mg/m2 during the second week, up to a maximum of 140 mg/m2. The planned treatment duration was 48 weeks. Twenty-five were available for assessment. One patient (4%) with RA achieved complete hematological remission. Four (16%), two with RA, two with RAEB, had minor responses resulting in decreased transfusion requirements or increased neutrophils. Thus, the overall response rate was 20% in evaluable patients with MDS and 17% in the study group on an intention-to-treat basis. The most frequent side-effects included headache (77%), dry skin (57%), arthralgias (30%), and rash (23%). In conclusion, although modest responses were noted in this study, the treatment tolerability was suboptimal. It is conceivable that a lower dosage schedule may be efficacious and better tolerated so enabling prolonged exposure which may be required to induce a differentiation effect.

Distribution of leukemia, lymphoma, and allied disease in parts of Great Britain: analysis by administrative districts and simulations of adjacencies. Leukaemia Research Fund 1984 Data Collection Group.

An analysis is presented of the distribution of cases of leukemia and allied disorders occurring in 151 administrative districts from England, Wales, and Scotland during 1984. The age-adjusted rates for certain conditions present an unusual pattern highlighting excessively high and low rates in parts of the country, some of which share contiguous boundaries. In particular, high rates for non-Hodgkin's lymphoma are found in rural Yorkshire districts, whereas leukemias and primary polycythemias are much more common in the Midland districts.