The impact of dose escalation and resistance modulation in older patients with acute myeloid leukaemia and high risk myelodysplastic syndrome: the results of the LRF AML14 trial.:the results of the LRF AML14 trial

The acute myeloid leukaemia (AML)14 trial addressed four therapeutic questions in patients predominantly aged over 60 years with AML and High Risk Myelodysplastic Syndrome: (i) Daunorubicin 50 mg/m(2) vs. 35 mg/m(2); (ii) Cytarabine 200 mg/m(2) vs. 400 mg/m(2) in two courses of DA induction; (iii) for part of the trial, patients allocated Daunorubicin 35 mg/m(2) were also randomized to receive, or not, the multidrug resistance modulator PSC-833 in a 1:1:1 randomization; and (iv) a total of three versus four courses of treatment. A total of 1273 patients were recruited. The response rate was 62% (complete remission 54%, complete remission without platelet/neutrophil recovery 8%); 5-year survival was 12%. No benefits were observed in either dose escalation randomization, or from a fourth course of treatment. There was a trend for inferior response in the PSC-833 arm due to deaths in induction. Multivariable analysis identified cytogenetics, presenting white blood count, age and secondary disease as the main predictors of outcome. Although patients with high Pgp expression and function had worse response and survival, this was not an independent prognostic factor, and was not modified by PSC-833. In conclusion, these four interventions have not improved outcomes in older patients. New agents need to be explored and novel trial designs are required to maximise prospects of achieving timely progress.

European development of clofarabine as treatment for older patients with acute myeloid leukemia considered unsuitable for intensive chemotherapy.

PURPOSE:
Treatment options for older patients with acute myeloid leukemia (AML) who are not considered suitable for intensive chemotherapy are limited. We assessed the second-generation purine nucleoside analog, clofarabine, in two similar phase II studies in this group of patients.
PATIENTS AND METHODS:
Two consecutive studies, UWCM-001 and BIOV-121, recruited untreated older patients with AML to receive up to four or six 5-day courses of clofarabine. Patients in UWCM-001 were either older than 70 years or 60 to 69 years of age with poor performance status (WHO > 2) or with cardiac comorbidity. Patients in BIOV-121 were >or= 65 years of age and deemed unsuitable for intensive chemotherapy.
RESULTS:
A total of 106 patients were treated in the two monotherapy studies. Median age was 71 years (range, 60 to 84 years), 30% had adverse-risk cytogenetics, and 36% had a WHO performance score >or= 2. Forty-eight percent had a complete response (32% complete remission, 16% complete remission with incomplete peripheral blood count recovery), and 18% died within 30 days. Interestingly, response and overall survival were not inferior in the adverse cytogenetic risk group. The safety profile of clofarabine in these elderly patients with AML who were unsuitable for intensive chemotherapy was manageable and typical of a cytotoxic agent in patients with acute leukemia. Patients had similar prognostic characteristics to matched patients treated with low-dose cytarabine in the United Kingdom AML14 trial, but had significantly superior response and overall survival.
CONCLUSION:
Clofarabine is active and generally well tolerated in this patient group. It is worthy of further evaluation in comparative trials and might be of particular use in patients with adverse cytogenetics.

Philadelphia-Negative Classical Myeloproliferative Neoplasms: Critical Concepts and Management Recommendations From European LeukemiaNet

We present a review of critical concepts and produce recommendations on the management of Philadelphia-negative classical myeloproliferative neoplasms, including monitoring, response definition, first-and second-line therapy, and therapy for special issues. Key questions were selected according the criterion of clinical relevance. Statements were produced using a Delphi process, and two consensus conferences involving a panel of 21 experts appointed by the European LeukemiaNet (ELN) were convened. Patients with polycythemia vera (PV) and essential thrombocythemia (ET) should be defined as high risk if age is greater than 60 years or there is a history of previous thrombosis. Risk stratification in primary myelofibrosis (PMF) should start with the International Prognostic Scoring System (IPSS) for newly diagnosed patients and dynamic IPSS for patients being seen during their disease course, with the addition of cytogenetics evaluation and transfusion status. High-risk patients with PV should be managed with phlebotomy, low-dose aspirin, and cytoreduction, with either hydroxyurea or interferon at any age. High-risk patients with ET should be managed with cytoreduction, using hydroxyurea at any age. Monitoring response in PV and ET should use the ELN clinicohematologic criteria. Corticosteroids, androgens, erythropoiesis-stimulating agents, and immunomodulators are recommended to treat anemia of PMF, whereas hydroxyurea is the first-line treatment of PMF-associated splenomegaly. Indications for splenectomy include symptomatic portal hypertension, drug-refractory painful splenomegaly, and frequent RBC transfusions. The risk of allogeneic stem-cell transplantation-related complications is justified in transplantation-eligible patients whose median survival time is expected to be less than 5 years.

The incidences of trisomy 8, trisomy 9 and D20S108 deletion in polycythaemia vera: an analysis of blood granulocytes using interphase fluorescence in situ hybridization:an analysis of blood granulocytes using interphase fluorescence in situ hybridization

We have used interphase fluorescence in situ hybridization (IFISH) to detect trisomy 8, trisomy 9 and 20q deletion in circulating granulocytes from patients with polycythaemia vera (PV). Out of 64 PV patients, 15 (23%) exhibited an abnormality. Two patients had trisomy 9, three had trisomy 8 and 10 patients had hemizygous deletion of D20S108 (a locus in the 20q common deleted region). Aberrant nuclei ranged from 10% to 80% in these 15 cases. There was no correlation between the presence of a marker and sex, age, interval between presentation and IFISH analysis, neutrophil or platelet count or therapy. Conventional marrow cytogenetic karyotype results were available in 23 cases and there was concurrence between these and blood IFISH in 16 cases (13 normal and three with 20q/D20S108 deletion by both methods). Three patients with D20S108 deletion by IFISH were normal by previous marrow cytogenetic testing and four cases with 20q deletion by previous marrow cytogenetics had normal blood granulocytes according to IFISH. Thus, we confirm that trisomies 8 and 9 and deletion of 20q are diagnostically useful markers of PV. IFISH analysis of blood granulocytes is a practical method for detecting these markers, but as an adjunct to, not as a substitute for, conventional marrow cytogenetics.

Differential TERT promoter methylation and response to 5-aza-2'-deoxycytidine in acute myeloid leukemia cell lines:TERT expression, telomerase activity, telomere length, and cell death

The catalytic subunit of human telomerase (TERT) is highly expressed in cancer cells, and correlates with complex cytogenetics and disease severity in acute myeloid leukemia (AML). The TERT promoter is situated within a large CpG island, suggesting that expression is methylation-sensitive. Studies suggest a correlation between hypermethylation and TERT overexpression. We investigated the relationship between TERT promoter methylation and expression and telomerase activity in human leukemia and lymphoma cell lines. DAC-induced demethylation and cell death were observed in all three cell lines, as well as telomere shortening in HL-60 cells. DAC treatment reduced TERT expression and telomerase activity in OCI/AML3 and HL-60 cells, but not in U937 cells. Control U937 cells expressed lower levels of TERT mRNA, carried a highly methylated TERT core promoter, and proved more resistant to DAC-induced repression of TERT expression and cell death. AML patients had significantly lower methylation levels at several CpGs than "well elderly" individuals. This study, the first to investigate the relationship between TERT methylation and telomerase activity in leukemia cells, demonstrated a differential methylation pattern and response to DAC in three AML cell lines. We suggest that, although DAC treatment reduces TERT expression and telomerase activity, this is unlikely to occur via direct demethylation of the TERT promoter. However, further investigations on the regions spanning CpGs 7-12 and 14-16 may reveal valuable information regarding transcriptional regulation of TERT.

Evaluation of mixed chimerism by in vitro amplification of dinucleotide repeat sequences using the polymerase chain reaction

The influence of mixed hematopoietic chimerism (MC) after allogeneic bone marrow transplantation remains unknown. Increasingly sensitive detection methods have shown that MC occurs frequently. We report a highly sensitive novel method to assess MC based on the polymerase chain reaction (PCR). Simple dinucleotide repeat sequences called microsatellites have been found to vary in their repeat number between individuals. We use this variation to type donor-recipient pairs following allogeneic BMT. A panel of seven microsatellites was used to distinguish between donor and recipient cells of 32 transplants. Informative microsatellites were subsequently used to assess MC after BMT in this group of patients. Seventeen of the 32 transplants involved a donor of opposite sex; hence, cytogenetics and Y chromosome-specific PCR were also used as an index of chimerism in these patients. MC was detected in bone marrow aspirates and peripheral blood in 18 of 32 patients (56%) by PCR. In several cases, only stored slide material was available for analysis but PCR of microsatellites or Y chromosomal material could be used successfully to assess the origin of cells in this archival material. Cytogenetic analysis was possible in 17 patients and MC was detected in three patients. Twelve patients received T-cell-depleted marrow and showed a high incidence of MC as revealed by PCR (greater than 80%). Twenty patients received unmanipulated marrow, and while the incidence of MC was lower (44%), this was a high percentage when compared with other studies. Once MC was detected, the percentages of recipient cells tended to increase. However, in patients exhibiting MC who subsequently relapsed, this increase was relatively sudden. The overall level of recipient cells in the group of MC patients who subsequently relapsed was higher than in those who exhibited stable MC. Thus, while the occurrence of MC was not indicative of a poor prognosis per se, sudden increases in the proportions of recipient cells may be a prelude to graft rejection or relapse.

Utility of sarcoma-specific fusion gene analysis in paraffin-embedded material for routine diagnosis at a specialist centre.

AIMS: Diagnosis of soft tissue sarcomas can be difficult. It can be aided by detection of specific genetic aberrations in many cases. This study assessed the utility of a molecular genetics/cytogenetics service as part of the routine diagnostic service at the Royal Marsden Hospital. METHODS: A retrospective audit was performed over a 15-month period to evaluate the diagnostic usefulness for soft tissue sarcomas with translocations of fluorescence in situ hybridisation (FISH) and reverse-transcriptase PCR (RT-PCR) in paraffin-embedded (PE) material. Results were compared with histology, and evaluated. RESULTS: Molecular investigations were performed on PE material in 158 samples (total 194 RT-PCR and 174 FISH tests), of which 85 were referral cases. Synovial sarcoma, Ewing sarcoma and low-grade fibromyxoid sarcoma were the most commonly tested tumours. Myxoid liposarcoma showed the best histological and molecular concordance, and alveolar rhabdomyosarcoma showed the best agreement between methods. FISH had a higher sensitivity for detecting tumours (73%, compared with 59% for RT-PCR) with a better success rate than RT-PCR, although the latter was specific in identifying the partner gene for each fusion. In particular, referral blocks in which methods of tissue fixation and processing were not certain resulted in higher RT-PCR failure rates. CONCLUSIONS: FISH and RT-PCR on PE tissue are practical and effective ancillary tools in the diagnosis of soft tissue sarcomas. They are useful in confirming doubtful histological diagnoses and excluding malignant diagnoses. PCR is less sensitive than FISH, and the use of both techniques is optimal for maximising the detection rate of translocation-positive sarcomas.

Abnormalities on 1q and 7q are associated with poor outcome in sporadic Burkitt's lymphoma. A cytogenetic and comparative genomic hybridization study.

Comparative genomic hybridization (CGH) studies have demonstrated a high incidence of chromosomal imbalances in non-Hodgkin's lymphoma. However, the information on the genomic imbalances in Burkitt's Lymphoma (BL) is scanty. Conventional cytogenetics was performed in 34 cases, and long-distance PCR for t(8;14) was performed in 18 cases. A total of 170 changes were present with a median of four changes per case (range 1-22). Gains of chromosomal material (143) were more frequent than amplifications (5) or losses (22). The most frequent aberrations were gains on chromosomes 12q (26%), Xq (22%), 22q (20%), 20q (17%) and 9q (15%). Losses predominantly involved chromosomes 13q (17%) and 4q (9%). High-level amplifications were present in the regions 1q23-31 (three cases), 6p12-p25 and 8p22-p23. Upon comparing BL vs Burkitt's cell leukemia (BCL), the latter had more changes (mean 4.3 +/- 2.2) than BL (mean 2.7 +/- 3.2). In addition, BCL cases showed more frequently gains on 8q, 9q, 14q, 20q, and 20q, 9q, 8q and 14q, as well as losses on 13q and 4q. Concerning outcome, the presence of abnormalities on 1q (ascertained either by cytogenetics or by CGH), and imbalances on 7q (P=0.01) were associated with a short survival.