Matthew W. Jenner

A compendium of myeloma-associated chromosomal copy number abnormalities and their prognostic value

To obtain a comprehensive genomic profile of presenting multiple myeloma cases we performed high-resolution single nucleotide polymorphism mapping array analysis in 114 samples alongside 258 samples analyzed by U133 Plus 2.0 expression array (Affymetrix). We examined DNA copy number alterations and loss of heterozygosity (LOH) to define the spectrum of minimally deleted regions in which relevant genes of interest can be found. The most frequent deletions are located at 1p (30%), 6q (33%), 8p (25%), 12p (15%), 13q (59%), 14q (39%), 16q (35%), 17p (7%), 20 (12%), and 22 (18%). In addition, copy number-neutral LOH, or uniparental disomy, was also prevalent on 1q (8%), 16q (9%), and X (20%), and was associated with regions of gain and loss. Based on fluorescence in situ hybridization and expression quartile analysis, genes of prognostic importance were found to be located at 1p (FAF1, CDKN2C), 1q (ANP32E), and 17p (TP53). In addition, we identified common homozygously deleted genes that have functions relevant to myeloma biology. Taken together, these analyses indicate that the crucial pathways in myeloma pathogenesis include the nuclear factor-κB pathway, apoptosis, cell-cycle regulation, Wnt signaling, and histone modifications. This study was registered at http://isrctn.org as ISRCTN68454111.

Lenalidomide (Revlimid), in combination with cyclophosphamide and dexamethasone (RCD), is an effective and tolerated regimen for myeloma patients

Lenalidomide (Revlimid) is an oral analogue of thalidomide that lacks both the neurotoxic and teratogenic side effects associated with the parent drug and, in laboratory studies, has more potent antitumour and immunomodulatory activity. In vitro studies suggested that its action may be synergistic with a number of conventional chemotherapeutic agents and clinical studies have shown it to be effective when combined with dexamethasone for the treatment of relapsed/refractory myeloma (Richardson et al, 2002, 2006; Dimopoulos et al, 2005; Rajkumar et al, 2005; Weber et al, 2006). Based on the in vitro data, we postulated that the addition of an alkylating agent to lenalidomide and dexamethasone could increase response rates; therefore, we conducted a retrospective analysis to assess the efficacy and toxicity profile when lenalidomide was used in combination with cyclophosphamide and dexamethasone for patients with relapsed refractory disease. Multiply relapsed/refractory patients were given lenalidomide 25 mg p.o. days 1–21, cyclophosphamide 500 mg p.o. days 1, 8, 15 and 21, and dexamethasone 40 mg p.o. days 1–4 and 12–15, of a 28 day cycle for a maximum of 9 cycles. Toxicity profiles and response were assessed every 4 weeks. Twenty-one patients were included with a median age of 59 years (range: 34–76 years). All were heavily pretreated with a median of four previous lines of therapy (range: 1–8): 14 patients had received high-dose melphalan, 21 patients thalidomide, 17 patients bortezomib and two had undergone an allogeneic bone marrow transplantation. The median time from diagnosis to treatment initiation was 54 months (range: 11–122 months). To date, 93 complete courses of therapy have been given to 21 patients with median number of five courses (range: 1–8). The side effect profile was highly predictable with the neutrophil count falling below 0.5 · 10/l in eight patients (38%). By the end of the third cycle of treatment, this required a dose reduction or withdrawal of cyclophosphamide in 10 patients (48%), and a dose reduction of lenalidomide in five patients (24%). Granulocyte colony-stimulating factor was required in 12 patients (57%). Despite this, i.v. antibiotics for neutropenic fever were only required in six patients (29%). In contrast to other immunomodulatory drugs, the side effect profile of the combination was highly tolerable with no patient experiencing sedation, constipation or worsening of peripheral neuropathy. Deep vein thrombosis occurred in three patients (14%) with a heavy myeloma load, which is similar to the rates seen with the lenalidomide and dexamethasone combination (Dimopoulos et al, 2005; Weber et al, 2006). Response to therapy using the European Group for Blood and Marrow Transplantation criteria (Blade et al, 1998) was seen in 15 of 20 assessable patients [one complete response (CR), three very good partial response (PR), nine PR and two minimal response], resulting an overall response rate (CR + PR) of 65%. The median time to response was prompt at 31 days (range: 15– 68), which is in contrast to what would be expected with a VAD (dexamethasone, doxorubicin, vincristine)-type regimen in this setting. This prompt response resulted in no difference in response rates between those patients who required a dose reduction in therapy compared with those who tolerated fulldose treatment as all patients received full-dose therapy for their first treatment course. To date, only two patients have discontinued therapy because of a failure to respond to therapy; however, one unusual case discontinued therapy as a consequence of hepato-toxicity although the relationship to lenalidamide remains uncertain. With a median follow-up of 8 months (range: 4–15), there were no treatment-related deaths, but five patients have died from progressive/relapsed myeloma with the median time to progression of 171 days. In summary, the combination of RCD is effective in heavily pretreated myeloma patients and has manageable toxicities. The toxicity profile of RCD seems better than with similar combinations using melphalan based on the currently available datasets. The observed overall response rate of 65% appears superior to lenalidomide alone (17–23%) or in combination with dexamethasone (48–51%) (Dimopoulos et al, 2005; Richardson et al, 2006; Weber et al, 2006). In view of the frequent yet tolerable neutropenia, we would suggest that further studies using this combination should be carried out with less frequent cyclophosphamide dosing, which may be optimal especially in the elderly population.

Homozygous Deletion Mapping in Myeloma Samples Identifies Genes and an Expression Signature Relevant to Pathogenesis and Outcome

Purpose: Myeloma is a clonal malignancy of plasma cells. Poor-prognosis risk is currently identified by clinical and cytogenetic features. However, these indicators do not capture all prognostic information. Gene expression analysis can be used to identify poor-prognosis patients and this can be improved by combination with information about DNA-level changes. Experimental Design: Using single nucleotide polymorphism–based gene mapping in combination with global gene expression analysis, we have identified homozygous deletions in genes and networks that are relevant to myeloma pathogenesis and outcome. Results: We identified 170 genes with homozygous deletions and corresponding loss of expression. Deletion within the “cell death” network was overrepresented and cases with these deletions had impaired overall survival. From further analysis of these events, we have generated an expression-based signature associated with shorter survival in 258 patients and confirmed this signature in data from two independent groups totaling 800 patients. We defined a gene expression signature of 97 cell death genes that reflects prognosis and confirmed this in two independent data sets. Conclusions: We developed a simple 6-gene expression signature from the 97-gene signature that can be used to identify poor-prognosis myeloma in the clinical environment. This signature could form the basis of future trials aimed at improving the outcome of poor-prognosis myeloma. Clin Cancer Res; 16(6); 1856–64

MMSET deregulation affects cell cycle progression and adhesion regulons in t(4;14) myeloma plasma cells

The t(4;14) translocation leads to the simultaneous overexpression of two genes, FGFR3 and MMSET, in myeloma plasma cells. The findings of this study suggest that MMSET is implicated in the pathogenesis of the t(4;14) translocation. Background The recurrent immunoglobulin translocation, t(4;14)(p16;q32) occurs in 15% of multiple myeloma patients and is associated with poor prognosis, through an unknown mechanism. The t(4;14) up-regulates fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain (MMSET) genes. The involvement of MMSET in the pathogenesis of t(4;14) multiple myeloma and the mechanism or genes deregulated by MMSET upregulation are still unclear. Design and Methods The expression of MMSET was analyzed using a novel antibody. The involvement of MMSET in t(4;14) myelomagenesis was assessed by small interfering RNA mediated knockdown combined with several biological assays. In addition, the differential gene expression of MMSET-induced knockdown was analyzed with expression microarrays. MMSET gene targets in primary patient material was analyzed by expression microarrays. Results We found that MMSET isoforms are expressed in multiple myeloma cell lines, being exclusively up-regulated in t(4;14)-positive cells. Suppression of MMSET expression affected cell proliferation by both decreasing cell viability and cell cycle progression of cells with the t(4;14) translocation. These findings were associated with reduced expression of genes involved in the regulation of cell cycle progression (e.g. CCND2, CCNG1, BRCA1, AURKA and CHEK1), apoptosis (CASP1, CASP4 and FOXO3A) and cell adhesion (ADAM9 and DSG2). Furthermore, we identified genes involved in the latter processes that were differentially expressed in t(4;14) multiple myeloma patient samples. Conclusions In conclusion, dysregulation of MMSET affects the expression of several genes involved in the regulation of cell cycle progression, cell adhesion and survival.

Deletions of CDKN2C in Multiple Myeloma: Biological and Clinical Implications

Purpose: Deletions of chromosome 1 have been described in 7% to 40% of cases of myeloma with inconsistent clinical consequences. CDKN2C at 1p32.3 has been identified in myeloma cell lines as the potential target of the deletion. We tested the clinical impact of 1p deletion and used high-resolution techniques to define the role of CDKN2C in primary patient material. Experimental Design: We analyzed 515 cases of monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and newly diagnosed multiple myeloma using fluorescence in situ hybridization (FISH) for deletions of CDKN2C. In 78 myeloma cases, we carried out Affymetrix single nucleotide polymorphism mapping and U133 Plus 2.0 expression arrays. In addition, we did mutation, methylation, and Western blotting analysis. Results: By FISH we identified deletion of 1p32.3 (CDKN2C) in 3 of 66 MGUS (4.5%), 4 of 39 SMM (10.3%), and 55 of 369 multiple myeloma cases (15%). We examined the impact of copy number change at CDKN2C on overall survival (OS), and found that the cases with either hemizygous or homozygous deletion of CDKN2C had a worse OS compared with cases that were intact at this region (22 months versus 38 months; P = 0.003). Using gene mapping we identified three homozygous deletions at 1p32.3, containing CDKN2C, all of which lacked expression of CDKN2C. Cases with homozygous deletions of CDKN2C were the most proliferative myelomas, defined by an expression-based proliferation index, consistent with its biological function as a cyclin-dependent kinase inhibitor. Conclusions: Our results suggest that deletions of CDKN2C are important in the progression and clinical outcome of myeloma.

The addition of cyclophosphamide to lenalidomide and dexamethasone in multiply relapsed/refractory myeloma patients; a phase I/II study

We report the results of a Phase I/II dose escalation study to determine the maximum tolerated dose (MTD) of cyclophosphamide when combined with lenalidomide and dexamethasone in relapsed/refractory myeloma. Thirty‐one patients were enrolled in cohorts of 3, at five dose levels of cyclophosphamide to a maximum of 700 mg on days 1 and 8 of a 28‐d cycle. Patients received lenalidomide 25 mg days 1–21 and dexamethasone 20 mg orally days 1–4 and 8–11. The MTD was 600 mg cyclophosphamide, days 1 and 8. Grade 3/4 haematological complications occurred in 26% of patients, grade 3/4 infection in 3% (both at 700 mg cyclophosphamide), with thromboembolic complications in 6% of patients. Overall complete response (CR) rate was 29%, very good partial response rate 7% and partial response rate 45% giving an overall response rate of 81%. After 21 months median follow‐up, projected 2‐year progression‐free survival was 56%, with 80% overall survival at 30 months. Ten further patients were treated at MTD with a 40% CR rate. No dose reductions for any study drugs or deaths occurred during cycles 1–9. Lenalidomide, cyclophosphamide and dexamethasone is a safe, effective combination in relapsed myeloma inducing a high response rate, warranting further investigation in phase III trials.

The combination of cyclophosphomide, thalidomide and dexamethasone is an effective alternative to cyclophosphamide-vincristine-doxorubicin-methylprednisolone as induction chemotherapy prior to autologous transplantation for multiple myeloma: a case-matched analysis

A retrospective case-matched study was conducted to compare the oral regimen CTD (cyclophosphamide – thalidomide – dexamethasone) and infusional CVAMP (cyclophosphamide – vincristine – doxorubicin – methylprednisolone) as induction therapy followed by autologous peripheral blood stem-cell transplantation (PBSCT) for newly diagnosed multiple myeloma patients. The response rate after three cycles of treatment was statistically higher with CTD (n = 27) compared to CVAMP (n = 27) (89% vs. 56%, P = 0.016). Toxicity studies showed more neutropenia (grade 3/4) (4% vs. 60%, P = 0.0002) with CVAMP and more thrombotic episodes with CTD (11% vs. 4%). CTD may emerge as the superior induction regimen prior to PBSCT, in terms of high efficacy and better tolerability.

Use of a biosimilar granulocyte colony-stimulating factor for peripheral blood stem cell mobilization: an analysis of mobilization and engraftment

Peripheral blood haematopoietic progenitor cell mobilization has become a standard procedure prior to autologous stem cell transplantation. Biosimilar granulocyte colony‐stimulating factors (GCSF) have recently been awarded European Union (EU) licences for stem cell mobilization but data for their use in this context remain limited. The biosimilar GCSF, Ratiograstim® (Ratiopharm, Ulm, Germany) was granted an EU licence in September 2008 and incorporated into clinical practice in the Wessex Blood and Marrow Transplantation Programme in December 2008. Data were retrospectively collected for 154 consecutive patients undergoing peripheral blood stem cell harvest between January 2009 and December 2011 using the biosimilar GCSF. 131 consecutive patients from the preceding 3 years, who had received Neupogen®, were used as a control. We analysed both parameters relevant to stem cell collection and engraftment data, where patients proceeded to transplantation. We found no statistically significant difference between the two groups when comparing CD34 predictors, total number of CD34+ stem cells collected, number of days required for collection, or for time to engraftment. This is, to our knowledge, the largest direct comparison of a biosimilar GCSF with originator GCSF for stem cell mobilization. The use of biosimilar GCSF can produce a significant cost saving, allowing investment in other areas of stem cell transplantation.

Updates to the guidelines for the diagnosis and management of multiple myeloma

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Second malignancies in the context of lenalidomide treatment: an analysis of 2732 myeloma patients enrolled to the Myeloma XI trial.

We have carried out the largest randomised trial to date of newly diagnosed myeloma patients, in which lenalidomide has been used as an induction and maintenance treatment option and here report its impact on second primary malignancy (SPM) incidence and pathology. After review, 104 SPMs were confirmed in 96 of 2732 trial patients. The cumulative incidence of SPM was 0.7% (95% confidence interval (CI) 0.4–1.0%), 2.3% (95% CI 1.6–2.7%) and 3.8% (95% CI 2.9–4.6%) at 1, 2 and 3 years, respectively. Patients receiving maintenance lenalidomide had a significantly higher SPM incidence overall (P=0.011). Age is a risk factor with the highest SPM incidence observed in transplant non-eligible patients aged >74 years receiving lenalidomide maintenance. The 3-year cumulative incidence in this group was 17.3% (95% CI 8.2–26.4%), compared with 6.5% (95% CI 0.2–12.9%) in observation only patients (P=0.049). There was a low overall incidence of haematological SPM (0.5%). The higher SPM incidence in patients receiving lenalidomide maintenance therapy, especially in advanced age, warrants ongoing monitoring although the benefit on survival is likely to outweigh risk.