The use of real-time quantitative polymerase chain reaction and comparative genomic hybridization to identify amplification of the REL gene in follicular lymphoma.

Using comparative genomic hybridization (CGH), aberrations in DNA copy number were studied before and after transformation of follicular lymphoma to diffuse large B-cell lymphoma in six patients (15 lymph node biopsies in total). The most common and also the most discrete and intense amplification occurring in four out of 15 biopsies from three different patients was of 2p13-16. Using real-time quantitative polymerase chain reaction (RQ-PCR), REL amplification was found to be implicated at this locus. This technique also identified amplified REL in a further two biopsies, presumably below the detection level of CGH. REL amplification was quantified by comparing it, in most cases, with three endogenous reference genes, albumin, beta2-microglobulin and CD8alpha, that lie close to REL on 2p. There was no correlation apparent between 2p13-16 amplification or REL amplification and transformation. This study shows the usefulness of coupling CGH, for detecting recurring abnormalities, with the real-time PCR technique for rapid gene dosage quantification and confirms that the REL gene is a potential candidate in the pathogenesis of a particular subset of follicular lymphomas.

A phase II study to evaluate the combination of fludarabine, mitoxantrone and dexamethasone (FMD) in patients with follicular lymphoma.

BACKGROUND: 'Molecular response' is being investigated as a therapeutic goal in follicular lymphoma (FL). High response rates in FL with the fludarabine combination 'FMD' have been associated with 'molecular remission'. A phase II study of FMD in FL was therefore conducted. PATIENTS AND METHODS: Fifty-four patients, ten of whom were newly diagnosed received FMD. Forty-four percent of the previously treated patients had 'chemoresistant' disease. Treatment comprised: fludarabine 25 mg/m2 days 1-3, mitoxantrone 10 mg/m2 day 1, and dexamethasone 20 mg days 1-5. Blood/bone marrow was collected for quantitation of t(14;18) by 'real-time' PCR. RESULTS: The overall response rate was 37 of 54 (69%), complete responses being seen in 11 patients (20%), with no difference between newly diagnosed and the previously treated patients. However, the response rate in 'chemosensitive' relapse was 84% compared to 44% in patients in whom the last prior regimen had failed. Molecular responses were seen in 17 of 25 and PCR negativity in 8 of 25, although molecular and clinical responses did not always correlate. Toxicity was moderate, 19 patients required admission. However, in 6 of 12 patients, subsequent G-CSF mobilised stem cell harvests failed. CONCLUSIONS: FMD was well tolerated but with a lower than expected response rate. Molecular responses were seen in the majority of responding patients however, 'molecular remission' was rare.

Factors which predict unsuccessful mobilisation of peripheral blood progenitor cells following G-CSF alone in patients with non-Hodgkin's lymphoma.

INTRODUCTION: High-dose therapy with haematopoietic progenitor cell support has increasingly been utilised for patients with haematological malignancies. Peripheral blood is the stem cell source of choice, however, various mobilisation strategies are used by different centres. PATIENTS AND METHODS: Over a 2-year period, 52 patients with non-Hodgkin's lymphoma (median age 47 years, range 16-64 years) underwent peripheral blood progenitor cell mobilisation using G-CSF alone (16 microg/kg/day). The harvest was considered successful if > or =1 x 10(6) CD34(+) cells/kg were collected by leukapheresis. The histological subtypes of non-Hodgkin's lymphoma comprised: follicular (24 patients), diffuse large B-cell (14 patients), lymphoplasmacytoid (four patients), mantle cell (three patients), lymphoblastic lymphoma (one patient) and small lymphocytic lymphoma/chronic lymphocytic leukaemia (six patients). The median interval from diagnosis of non-Hodgkin's lymphoma to mobilisation was 27 months (range 2 months to 17 years). The median number of prior treatment episodes was 2 (range 1-5); 26 patients had received fludarabine alone or in combination. At the time of peripheral blood progenitor cell mobilisation, 20 patients were in 1st remission and 32 were in > or =2nd remission; 30 patients were in partial remission and 22 were in complete remission; the bone marrow was involved in nine patients. RESULTS: Peripheral blood progenitor cell mobilisation/harvest was unsuccessful in 19 out of 52 (37%) patients (mobilisation: 18, harvest: 1). The factors associated with unsuccessful mobilisation or harvest were: prior fludarabine therapy (P=0.002), bone marrow involvement at diagnosis (P=0.002), bone marrow involvement anytime prior to mobilisation (P=0.02), histological diagnosis of follicular, mantle cell, or lymphoplasmacytoid lymphoma, or small lymphocytic lymphoma/chronic lymphocytic leukaemia (P=0.03) and female gender (P=0.04). CONCLUSION: Although peripheral blood progenitor cells can be successfully mobilised and harvested from the majority of patients with non-Hodgkin's lymphoma after treatment with G-CSF alone, the latter is unsuccessful in approximately one-third of patients. These factors should be taken into account when patients are being considered for high-dose treatment.

A Phase I/II Study of a 72-h Continuous Infusion of Etoposide in Advanced Soft Tissue Sarcoma.

Purpose. The study was performed to assess the antitumour activity and toxicity of a 72-h continuous infusion of single-agent etoposide as second-line treatment for patients with locally advanced or metastatic soft tissue sarcoma (STS), following reports of substantial activity using this schedule of etoposide administration as first-line treatment in combination with ifosfamide.Patients/method. This was an open phase I/II trial performed at a single institution in patients with metastatic or locally advanced STS who had failed first-line treatment with doxorubicin + ifosfamide combination chemotherapy or, less commonly, single-agent treatment with doxorubicin or ifosfamide. Etoposide was given as a continuous intravenous infusion over 72 h. The starting dose level was 200 mg m(-2) day(-1) x 3 escalating in 10% steps in cohorts of three patients until dose-limiting toxicity was encountered.Results. Seventeen patients were treated, median age 47 years (range 26-71 years). No responses were seen in 16 assessable patients despite etoposide levels in the cotoxic range. The steady-state plasma concentration exceeded 8 mug ml-(1) in all patients and in patients treated at >/= 600 mg m -(2) the mean steady-state level was 14.4 mug ml -(1). The median event-free survival was 6 weeks (95% confidence interval (CI) 3.31-8.69) and the overall survival 16 weeks (95% CI 9.28-22.72). The maximum tolerated dose in this pretreated patient group was 200 mg mm(-2) day(-1) x 3. The dose-limiting toxicity was myelosuppression.Discussion. Etoposide given by 72-h infusion is inactive as second-line chemotherapy in STS. It is associated with significant toxicity when given in these doses, in this patient group.