A novel prognostic model in myeloma based on co-segregating adverse FISH lesions and the ISS: analysis of patients treated in the MRC Myeloma IX trial.

The association of genetic lesions detected by fluorescence in situ hybridization (FISH) with survival was analyzed in 1069 patients with newly presenting myeloma treated in the Medical Research Council Myeloma IX trial, with the aim of identifying patients associated with the worst prognosis. A comprehensive FISH panel was performed, and the lesions associated with short progression-free survival and overall survival (OS) in multivariate analysis were +1q21, del(17p13) and an adverse immunoglobulin heavy chain gene (IGH) translocation group incorporating t(4;14), t(14;16) and t(14;20). These lesions frequently co-segregated, and there was an association between the accumulation of these adverse FISH lesions and a progressive impairment of survival. This observation was used to define a series of risk groups based on number of adverse lesions. Taking this approach, we defined a favorable risk group by the absence of adverse genetic lesions, an intermediate group with one adverse lesion and a high-risk group defined by the co-segregation of >1 adverse lesion. This genetic grouping was independent of the International Staging System (ISS) and so was integrated with the ISS to identify an ultra-high-risk group defined by ISS II or III and >1 adverse lesion. This group constituted 13.8% of patients and was associated with a median OS of 19.4 months.

Assessment of fludarabine plus cyclophosphamide for patients with chronic lymphocytic leukaemia (the LRF CLL4 Trial): a randomised controlled trial.

BACKGROUND: Previous studies of patients with chronic lymphocytic leukaemia reported high response rates to fludarabine combined with cyclophosphamide. We aimed to establish whether this treatment combination provided greater survival benefit than did chlorambucil or fludarabine. METHODS: 777 patients with chronic lymphocytic leukaemia requiring treatment were randomly assigned to fludarabine (n=194) or fludarabine plus cyclophosphamide (196) for six courses, or chlorambucil (387) for 12 courses. The primary endpoint was overall survival, with secondary endpoints of response rates, progression-free survival, toxic effects, and quality of life. Analysis was by intention to treat. This study is registered as an International Standard Randomised Controlled Trial, number NCT 58585610. FINDINGS: There was no significant difference in overall survival between patients given fludarabine plus cyclophosphamide, fludarabine, or chlorambucil. Complete and overall response rates were better with fludarabine plus cyclophosphamide than with fludarabine (complete response rate 38%vs 15%, respectively; overall response rate 94%vs 80%, respectively; p<0.0001 for both comparisons), which were in turn better than with chlorambucil (complete response rate 7%, overall response rate 72%; p=0.006 and 0.04, respectively). Progression-free survival at 5 years was significantly better with fludarabine plus cyclophosphamide (36%) than with fludarabine (10%) or chlorambucil (10%; p<0.00005). Fludarabine plus cyclophosphamide was the best combination for all ages, including patients older than 70 years, and in prognostic groups defined by immunoglobulin heavy chain gene (V(H)) mutation status and cytogenetics, which were tested in 533 and 579 cases, respectively. Patients had more neutropenia and days in hospital with fludarabine plus cyclophosphamide, or fludarabine, than with chlorambucil. There was less haemolytic anaemia with fludarabine plus cyclophosphamide (5%) than with fludarabine (11%) or chlorambucil (12%). Quality of life was better for responders, but preliminary analyses showed no significant difference between treatments. A meta-analysis of these data and those of two published phase III trials showed a consistent benefit for the fludarabine plus cyclophosphamide regimen in terms of progression-free survival. INTERPRETATION: Fludarabine plus cyclophosphamide should now become the standard treatment for chronic lymphocytic leukaemia and the basis for new protocols that incorporate monoclonal antibodies.

Haplotypes in the tumour necrosis factor region and myeloma.

This study described the haplotypic structure across a region of chromosome 6 including the tumour necrosis factor (TNF) gene, and investigated its influence on the aetiology of myeloma. A total of 181 myeloma cases from the Medical Research Council Myeloma VII trial and 233 controls from the Leukaemia Research Fund Case Control Study of Adult Acute Leukaemia were included in the analysis. Genotyping by induced heteroduplex generator analysis was carried out for single nucleotide polymorphisms (SNP) located at positions -1031, -863, -857, -308 and -238 of the 5' promoter region of TNF-alpha gene, and 252 in the LT-alpha gene; and five microsatellites, TNFa, b, c, d and e. Haplotypes were inferred statistically using the phase algorithm. A limited diversity of haplotypes was observed, with the majority of variation described by 12 frequent haplotypes. Detailed characterization of the haplotype did not provide greater determination of disease risk beyond that described by the TNF-alpha-308 SNP. Some evidence was provided for a decreased risk of myeloma associated with the TNF-alpha-308 variant allele A, odds ratio, 0.57; 95% confidence interval, 0.38-0.86. The results of this study did not support our starting hypothesis; that high producer haplotypes at the TNF locus are associated with an increased risk of developing myeloma.

Serum free light chains for monitoring multiple myeloma.

Monoclonal immunoglobulin free light chains (FLC) are found in the serum and urine of patients with a number of B-cell proliferative disorders, including multiple myeloma. Automated immunoassays, which can measure FLC in serum, are useful for the diagnosis and monitoring of light chain (AL) amyloidosis, Bence Jones myeloma and non-secretory myeloma patients. We report the results of a study investigating the utility of serum FLC measurements in myeloma patients producing monoclonal intact immunoglobulin proteins. FLC concentrations were measured in presentation sera from 493 multiple myeloma patients with monoclonal, intact immunoglobulin proteins. Serial samples were assayed from 17 of these patients and the FLC measurements were compared with other disease markers. Serum FLC concentrations were abnormal in 96% of patients at presentation. FLC concentrations fell more rapidly in response to treatment than intact immunoglobulin G (IgG) and showed greater concordance with serum beta2 microglobulin concentrations and bone marrow plasma cell assessments. It was concluded that serum FLC assays could be used to follow the disease course in nearly all multiple myeloma patients. In addition, because of their short serum half-life, changes in serum FLC concentrations provide a rapid indication of the response to treatment.

The development of targeted chemotherapy for CNS lymphoma-a pilot study of the IDARAM regimen.

PURPOSE: We have developed and evaluated a CNS-targeted chemotherapy regimen based on the pharmacokinetic properties of the individual drugs in the combination. PATIENTS AND METHODS: In a twin-track study, 16 patients with secondary CNS lymphoma (SCNSL) and 8 with primary CNS lymphoma (PCNSL) were treated with IDARAM which comprised idarubicin 10 mg/m(2) i.v., days 1 and 2; dexamethasone 100 mg, 12-h infusion, days 1, 2 and 3; cytosine arabinoside (ARA-C) 1.0 g/m(2), 1-h infusion, days 1 and 2; methotrexate 2.0 g/m(2), 6-h infusion, day 3 (with folinic acid rescue); and cytosine arabinoside 70 mg plus methotrexate 12 mg, intrathecally, days 1 and 8. Two cycles were delivered at 3-weekly intervals. After response assessment, patients received adjuvant cranial radiotherapy (40 Gy over 20 fractions). RESULTS: The series comprised 24 patients, 11 male and 13 female. Their median age was 53 years (range 21 to 73 years). Grade 4 neutropenia and thrombocytopenia occurred in the majority of patients treated. Of the eight PCNSL patients, seven achieved complete remission (CR). Four remained in CR at the time of this report with a median duration of follow-up of 25 months (range 11 to 42 months). Of the 16 SCNSL patients, 12 achieved CR. Seven patients remained in CR at the time of this report with a median duration of follow-up of 24 months (range 18 to 57 months). CONCLUSION: This study suggests that IDARAM is an effective regimen in both PCNSL and SCNSL and is suitable for further development and evaluation.

Colonic dendritic cells, intestinal inflammation, and T cell-mediated bone destruction are modulated by recombinant osteoprotegerin.

Autoimmune associated bone disease and intestinal inflammation are closely linked with deregulation and hyperactivation of autoreactive CD4 T cells. How these T cells are activated and mediate disease is not clear. Here we show that in the Interleukin 2-deficient mouse model of autoimmunity spontaneous osteopenia and colitis are caused by increased production of the ligand for receptor activator of NFkappaB (RANKL). RANKL acting via its receptor, receptor activator of NFkappaB (RANK), increases bone turnover and promotes intestinal dendritic cell (DC) survival in vivo. Modulation of RANKL-RANK interactions with exogenous recombinant osteoprotegerin (Fc-OPG) reverses skeletal abnormalities and reduces colitis by decreasing colonic DC numbers. This study identifies a common causal link between bone disease and intestinal inflammation and establishes the importance of DC in mediating colonic inflammation in vivo.

An UK myeloma forum phase II study of thalidomide; long term follow-up and recommendations for treatment.

Myeloma remains incurable with conventional treatment in the vast majority of patients. The introduction of thalidomide in 1999 for the treatment of relapsed disease offers the opportunity to treat patients who have developed myelotoxicity or who are refractory to conventional chemotherapy. The optimal schedule remains unresolved and only two studies have reported long term follow-up data. We report a phase II low dose escalation study of thalidomide with long term follow-up showing overall survival (OS) of 19 months and progression free survival (PFS) of 14 months. In addition we report on the side effects and toxicity and give recommendations for the use of thalidomide in the relapsed setting based upon these findings.

A randomised multicentre trial of modified CHOP versus MCOP in patients aged 65 years and over with aggressive non-Hodgkin's lymphoma.

BACKGROUND: The aim of this study was to determine in a randomised trial whether there is any significant difference in toxicity between modified CHOP and MCOP chemotherapy in elderly patients with aggressive non-Hodgkin's lymphoma (NHL) and to determine whether this reduced dose chemotherapy can be administered with full dose intensity, low toxicity and produce acceptable survival. PATIENTS AND METHODS: Between 1993 and 2000, 155 eligible patients were randomised into this trial mainly from three centres (Nottingham, Birmingham and Leeds, UK). The patients were newly diagnosed with aggressive NHL and had a median age of 74 years (range 65-91 years). Ninety-six patients (62%) had bulky stage I or II disease; 59 patients (38%) had either stage III or IV disease; 77% had one or more extranodal sites involved at presentation; and 31% showed B symptoms. Seventy-seven patients were randomised to receive six cycles of modified CHOP (cyclophosphamide 600 mg/m(2) i.v., doxorubicin 30 mg/m(2) i.v., vincristine 1 mg i.v. all on day 1 with prednisolone 20 mg bd for days 1-5) every 21 days and 78 patients to MCOP (mitozantrone 10 mg/m(2) i.v. substituted for doxorubicin). Growth factors were not used routinely. After completion of chemotherapy, 39 patients received involved field radiotherapy (35-40 Gy) in 20 fractions. RESULTS: One hundred and one patients (65%) completed all six cycles of chemotherapy. The median course dose intensity was 97%. The median follow-up for 53 surviving patients was 51 months. The median survival was 19 months (95% confidence interval 10-36 months) with an actuarial survival of 47% at 2 years and 42% at 3 years (CHOP versus MCOP, P = 0.79). There was no significant difference in any of the toxicities experienced with either CHOP or MCOP, except for white cell count (46 patients on MCOP and 27 patients on CHOP had grade 3 or 4 toxicity, P = 0.002) and red cell transfusion (37 patients, MCOP; 17 patients, CHOP; P = 0.001). Grade 3 or 4 neutropenia was documented in 75 patients (50%). One patient died from toxicity whilst in remission and seven patients died with septicaemia and persistent NHL. CONCLUSION: This multicentre randomised trial provides further information on the dose intensity achievable with CHOP or MCOP regimens in elderly patients (median age 74 years) with aggressive NHL. These dose-reduced regimens can be given with nearly 100% dose intensity with 65% of patients completing all the treatment. Survival is comparable to that observed with the more intensive regimens given in this age group.

Waldenström macroglobulinemia. Development of diagnostic criteria and identification of prognostic factors.

To establish whether a combination of morphologic and immunophenotypic criteria could be developed to more precisely define Waldenström macroglobulinemia (WM) and prognostic factors, we retrospectively assessed the clinical and laboratory features of 111 cases of WM. Bone marrow infiltration by small lymphocytes was documented in each case; and diffuse, interstitial, nodular, and paratrabecular patterns of infiltration were documented in 58%, 32%, 6%, and 4% of cases, respectively. Ninety percent were characterized by a surface immunoglobulin-positive, CD19+CD20+CD5-CD10-CD23- immunophenotype. The median overall survival from diagnosis was 60 months; univariate analysis revealed the following adverse prognostic factors: older than 60 years, performance status more than 1, platelet count less than 100 x 10(3)/microL (< 100 x 10(9)/L), pancytopenia, and diffuse bone marrow infiltration. Associated median survival was 40, 38, 46, 28, and 59 months, respectively. Multivariate analysis revealed age, performance status, and platelet count as prognostically significant, but stratification of patients according to the International Prognostic Index had limited value. We suggest defining WM by the following criteria: IgM monoclonal gammopathy; bone marrow infiltration by small lymphocytes, plasmacytoid cells, and plasma cells in a diffuse, interstitial, or nodular pattern; and a surface immunoglobulin-positive, CD19+CD20+CD5-CD10-CD23- immunophenotype.

In multiple myeloma, only a single stage of neoplastic plasma cell differentiation can be identified by VLA-5 and CD45 expression.

The nature of the proliferating fraction in myeloma is still not known and understanding the characteristics of this fraction is central to the development of effective novel therapies. However, myeloma plasma cells typically show a very low rate of proliferation and this complicates accurate analysis. Although the level of CD45 and/or VLA-5 has been reported to identify proliferating 'precursor' plasma cells, there are discrepancies between these studies. We have therefore used a rigorous sequential gating strategy to simultaneously analyse cycle status and immunophenotype with respect to CD45, VLA-5 and a range of other integrin molecules. In 11 presentation myeloma patients, the proliferative fraction was distributed evenly between CD45+ and CD45- cells, however, cycling plasma cells were consistently VLA-5-. There was close correlation between the expression of VLA-5 and a range of other integrin molecules (CD11a, CD11c, CD103), as well as the immunoglobulin-associated molecules CD79a/b (Spearman, n = 10, P < 0.0001). In short-term culture, cells that were initially VLA-5-showed increasing VLA-5 expression with time. However, simultaneous analysis of the DNA-binding dye 7-amino-actinomycin D demonstrated that this was not as a result of differentiation, as VLA-5+ plasma cells were all non-viable. This was confirmed in freshly explanted plasma cells from nine patients. Discrete stages of plasma cell differentiation could not be distinguished by the level of CD45 or VLA-5 expression. The results indicate that there is a single stage of plasma cell differentiation, with the phenotype CD38+CD138+VLA-5-. These findings support the hypothesis that neoplastic bone marrow plasma cells represent an independent, self-replenishing population.

The impact of attaining a minimal disease state after high-dose melphalan and autologous transplantation for multiple myeloma.

Initial studies with high-dose therapy (HDT) in myeloma suggest some beneficial effects of attaining a complete response (CR); however, the effect on survival is difficult to assess owing to inconsistencies in the definition of response between studies. We have analysed 96 newly diagnosed patients aged less than 65 years who received HDT and assessed the effect of response on survival using electrophoresis, immunofixation and fluorescent IgH polymerase chain reaction (PCR) to define CR. Patients received induction chemotherapy with C-VAMP (adriamycin, vincristine, methylprednisolone, cyclophosphamide) followed by melphalan 200 mg/m2 and reinfusion of peripheral blood stem cells. There was a high response to C-VAMP [CR = 24%, partial response (PR) = 64%], with all but one patient improving the depth of response after HDT (CR = 69%, PR = 31%). The progression-free survival (PFS) and overall survival (OS) were excellent at a median of 46.4 months and 72+ months. There was a trend towards an improved PFS in patients with an immunofixation-negative CR compared with patients with a PR (49.4 months, 41.14 months; P = 0.26). This was not evident when electrophoresis was used to define CR. The method used to define CR did not impact on the overall survival and fluorescent IgH PCR failed to add any additional prognostic information. This study supports the widespread use of the European Bone Marrow Transplantation group (EBMT) response criteria and suggests that immunofixation should be performed on all patients who become electrophoresis negative.

The interleukin-6 receptor alpha-chain (CD126) is expressed by neoplastic but not normal plasma cells.

Interleukin-6 (IL-6) is reported to be central to the pathogenesis of myeloma, inducing proliferation and inhibiting apoptosis in neoplastic plasma cells. Therefore, abrogating IL-6 signaling is of therapeutic interest, particularly with the development of humanized anti-IL-6 receptor (IL-6R) antibodies. The use of such antibodies clinically requires an understanding of IL-6R expression on neoplastic cells, particularly in the cycling fraction. IL-6R expression levels were determined on plasma cells from patients with myeloma (n = 93) and with monoclonal gammopathy of undetermined significance (MGUS) or plasmacytoma (n = 66) and compared with the levels found on normal plasma cells (n = 11). In addition, 4-color flow cytometry was used to assess the differential expression by stage of differentiation and cell cycle status of the neoplastic plasma cells. IL-6R alpha chain (CD126) was not detectable in normal plasma cells, but was expressed in approximately 90% of patients with myeloma. In all groups, the expression levels showed a normal distribution. In patients with MGUS or plasmacytoma, neoplastic plasma cells expressed significantly higher levels of CD126 compared with phenotypically normal plasma cells from the same marrow. VLA-5(-) "immature" plasma cells showed the highest levels of CD126 expression, but "mature" VLA-5(+) myeloma plasma cells also overexpressed CD126 when compared with normal subjects. This study demonstrates that CD126 expression is restricted to neoplastic plasma cells, with little or no detectable expression by normal cells. Stromal cells in the bone marrow microenvironment do not induce the overexpression because neoplastic cells express higher levels of CD126 than normal plasma cells from the same bone marrow in individuals with MGUS. (Blood. 2000;96:3880-3886)

Positive and negative selection to reduce tumour contamination in peripheral blood stem cell harvests.

Peripheral blood progenitor cells used during high dose treatments for malignancy may be contaminated with tumour cells that could later contribute to recurrence. CD34+ selected harvests still contain tumour cells and an additional negative selection may be capable of reducing this contamination. We have assessed a two-stage technique in which a CD34+ selection is followed by a tumour specific depletion stage using a B cell or breast cancer specific antibody panel. Initial small-scale selections on 11 patients with NHL and breast cancer showed that cell loss was greatest following the CD34+ selection with a median yield of 38.8 per cent (range 17. 2-56.4 per cent). The addition of the depletion stage resulted in a minimal loss of CD34+ cells with a yield for this step of 94.2 per cent (range 77.5-99.3 per cent). Clinical scale selections were performed on seven patients with CLL and a median of 2.8x10(6)/kg CD34+ cells (range 1.5-6.1x10(6)/kg) were collected. Cell recovery was 53.3 per cent following CD34+ selection and 76.9 per cent following the tumour specific depletion stage, resulting in a final product containing a median of 1.0x10(6)/kg CD34+ cells (range 0. 55-2.0x10(6)/kg). All unmanipulated harvests were heavily contaminated with tumour cells (median contamination 10.2 per cent, range 2.0-83.1 per cent) as measured by flow cytometry and a median 4.7 log (range 3-5 log) tumour cell purge was produced following two-stage selection. Six of the patients have received cells manipulated in this way with median engraftment times of neutrophils>0.5x10(9)/l=16 days (range 13-20 days) and platelets>20x10(9)/l=16.5 days (range 11-42 days). At a median follow-up of 25 months, these transplanted patients remain well and in molecular complete remission.

High-producer haplotypes of tumor necrosis factor alpha and lymphotoxin alpha are associated with an increased risk of myeloma and have an improved progression-free survival after treatment.

PURPOSE: To determine the effect of polymorphic variations in the tumor necrosis factor alpha (TNFalpha) and lymphotoxin alpha (LTalpha) genes on the predisposition to myeloma and the effect of these polymorphisms on response to treatment and overall survival. PATIENTS AND METHODS: Genotype distribution was determined in 63 patients with monoclonal gammopathy of uncertain significance (MGUS) and 198 patients with myeloma and compared with that in 250 age- and sex-matched population-based controls. The effect on treatment response and survival was determined in 171 myeloma patients treated with either conventional or high-dose chemotherapy. RESULTS: Comparison of the extended TNFalpha/LTalpha haplotype in the myeloma cases and controls showed a significant excess of high-producer alleles in the cases. The double heterozygotes TNF1/2 and LT10.5/5.5 were present in 35.8% of cases but in only 18% of the controls; this presence was associated with a significant increased risk of myeloma (odds ratio, 2.05; 95% confidence interval, 1.26 to 3.35). A similar odds ratio was seen in the MGUS cases, suggesting that this genotype is associated with the initiation of plasma-cell disorders rather than the progression of MGUS to myeloma. The median overall survival time of myeloma patients was 53.8 months and showed no difference with regard to TNFalpha/LTalpha polymorphic status. A trend toward an improved progression-free survival was apparent in cases with a high-producer haplotype, although this effect was seen only in patients receiving high-dose chemotherapy. CONCLUSION: Individuals with polymorphisms associated with a high production of TNFalpha/LTalpha are at a significantly increased risk of developing MGUS and myeloma. The impact of polymorphic status on overall survival is minimal, although there is a trend toward an increased progression-free survival in the high-producer group.

FLUDAP: salvage chemotherapy for relapsed/refractory aggressive non-Hodgkin's lymphoma.

The aim of this study was to investigate the combination of fludarabine phosphate, dexamethasone, cytosine arabinoside and cis-platinum (FLUDAP) in the treatment of patients with relapsed/refractory aggressive non-Hodgkin's lymphoma (NHL). This regimen comprises: dexamethasone 100 mg/d continuous infusion (cont. inf.) d1-3; cytosine arabinoside (ara-C) 1 g/m2/d cont. inf. d 2,3; fludarabine phosphate 30 mg/m2 short inf. 4hr prior to each 24hr ara-C inf.; cis-platinum 50 mg/m2 4hr inf. at the start of each 24hr ara-C inf. G-CSF (lenograstim, Granocyte) is given at 263 microg s.c. daily from day 7 until the neutrophil count reaches 1.0x10(9)/l. The regimen repeats at 21 day intervals. A total of 33 patients were registered (median age 47 years; 24 males, 9 females); the majority (73%) were refractory to their previous treatment and most had advanced disease by Ann Arbor stage. Thirteen (39%) of the 33 enrolled patients (52% of the 25 fully evaluable patients who received at least 2 courses of FLUDAP) responded to treatment. A maximum response of complete remission was achieved in 5 patients, good partial remission in 3, and partial remission in 5. Twelve patients went on to successful stem cell supported intensification therapy. Median survival times were higher in the responding patients, and in those patients transplanted post-FLUDAP. The toxicity associated with the FLUDAP regimen was generally predictable; frequently reported severe events included haematological toxicity and infection. In conclusion, the FLUDAP regimen shows promise as a salvage regimen and increases the available therapeutic options in the treatment of recurrent/refractory aggressive NHL.

Clinicopathological correlates of IgM paraproteinemias.

IgM paraproteinemia is considered to be the major defining feature of Waldenström's macroglobulinemia (WM), but it may also occur in other B-cell lymphoproliferative disorders. In this study we have reviewed the final pathological diagnosis of 106 patients with IgM paraproteinemia investigated in our laboratories between April 1993 and May 1999. In 22 of the 106 patients (20.8%), there was no clinical or laboratory evidence of an underlying lymphoproliferative disorder, and a diagnosis of monoclonal gammopathy of undetermined significance (MGUS) was therefore made. In 60 cases (56.6%), a diagnosis of WM was made, while in the remaining 24 patients, the final diagnosis was chronic lymphocytic leukemia (n = 10), diffuse large B-cell lymphoma (n = 5), extranodal marginal-zone lymphoma (n = 3), follicular lymphoma (n = 3), and mantle-cell lymphoma (n = 3). The median paraprotein concentration in patients with WM, MGUS, and "other" lymphoproliferative disorders was 13 g/L (range, 2-54), 6 g/L (range, 3-30), and 4.5 g/L (range, 3-61), respectively. It is clear that IgM paraproteins are demonstrable in all subtypes of peripheral B-cell disorders and, although paraprotein concentrations are generally higher in WM, there is considerable overlap. Immunophenotypic criteria are therefore essential for the accurate diagnosis of WM.