ALBERT

Description: In this double-blind, placebo-controlled study, 363 patients with untreated multiple myeloma were randomized to receive either melphalan-prednisone and thalidomide (MPT) or melphalan-prednisone and placebo (MP). The dose of melphalan was 0.25 mg/kg and prednisone was 100 mg given daily for 4 days every 6 weeks until plateau phase. The dose of thalidomide/placebo was escalated to 400 mg daily until plateau phase and thereafter reduced to 200 mg daily until progression. A total of 357 patients were analyzed. Partial response was 34% and 33%, and very good partial response or better was 23% and 7% in the MPT and MP arms, respectively (P 〈 .001). There was no significant difference in progression-free or overall survival, with median survival being 29 months in the MPT arm and 32 months in the MP arm. Most quality of life outcomes improved equally in both arms, apart from constipation, which was markedly increased in the MPT arm. Constipation, neuropathy, nonneuropathy neurologic toxicity, and skin reactions were significantly more frequent in the MPT arm. The number of thromboembolic events was equal in the 2 treatment arms. In conclusion, MPT had a significant antimyeloma effect, but this did not translate into improved survival. This trial was registered at www.clinicaltrials.gov as #NCT00218855.

Description: The etiology of the osteolytic bone disease (OBD) in multiple myeloma (MM) is not fully understood but seems to involve interactions between myeloma cells and the bone marrow microenvironment. Over-expression of the osteoclast activation cytokine, receptor activator of nuclear factor-κB ligand (RANKL), and decreased expression of the inhibitor of RANKL, called osteoprotegerin (OPG), is believed to be important. Macrophage inflammatory proteins are also reported to play a role in osteoclast activation. Recently, the role of osteoblast inactivation in OBD has come into focus, especially the importance of over expression of dickkopf 1 (DKK1), an inhibitor of the Wnt signalling pathway that induces osteoblastogenesis and osteoblast activation. Micro array profiling studies in MM have also introduced other candidate genes. Besides DKK1, over expression of CKS2, PSME2 and DHFR have been reported. We examined 9 candidate genes involved in OBD: RANKL, RANK, OPG, MIP1α, PTHrp, DKK1, CKS2, PSME2 and DHFR. Bone marrow (BM) aspirates from 170 newly diagnosed, un-treated MM patients and from 9 healthy volunteers were collected over a period of 12 years. Median age in the MM group was 62 years (33–83 years), male/ female ratio 1:1.25. 62 % of the patients had advanced disease at diagnosis (Durie Salmon stage III), 30 % of the patients had no, 28% limited, and 42 % advanced bone lesions. The immune phenotype of the myeloma cells were characterised by flowcytometry and afterwards used in fluorescence activated cell sorting (FACS) of the myeloma cells using a FACS Aria (Becton Dickinson immune systems) to ≥98% purity. For global amplification 100 PCs were FACS-sorted directly to PCR tubes, followed by global reverse transcription (RT)-PCR, generating a cDNA archive. By using a polyadenylating step we generated a 5′-oligo(dT)-transcript-poly(A)-3′ cDNA, that is finally amplified by PCR using a sequence independent X-(dT) 24 primer. Gene expression of the analysed genes and a housekeeping gene (β-actin) were performed by real-time quantitative polymerase chain reaction (RQ-PCR), using specific designed oligo primers and Taqman probes. Only over-expression of DKK1 in highly purified plasma cells was correlated to the degree of OBD in the studied population. We found expression of DKK1 in myeloma cells in about 42 % (71/170) of the samples. Expression of DKK1 correlated (p

Description: Background: Somatic driver mutations in hematopoietic cells may lead to clonal hematopoiesis of indeterminate potential (CHIP). In patients with lymphoma CHIP has been associated with increased risk of therapy-related myeloid neoplasms (tMN) and inferior survival after autologous stem cell transplantation as demonstrated in a large single center study and in a case-control study (Gibson CJ et al., JCO 2017 and Berger G et al., Blood 2018). Here, we investigated the clinical impact of clonal hematopoiesis in a nation-wide population-based cohort of Danish lymphoma patients undergoing autologous transplant with prospective data from four national patient registries. Methods: Patients with lymphoma who had undergone leukapheresis at all danish transplant centers from 2000 to 2012 were identified. DNA and RNA was extracted from mobilized peripheral blood products. Targeted sequencing of all samples was performed using an Illumina TruSeq Custom Amplicon panel (Illumina, San Diego, CA, USA) designed to cover 〉95% of mutations associated with CHIP (ASXL1, ASXL2, BCOR, BRCC3, CBL, CREBBP, DNMT3A, ETV6, GNB1, IDH1, IDH2, JAK2, KRAS, NRAS, PPM1D, RAD21, SF3B1, SRSF2, TET2, TP53). To allow detection of low-level mutations and secure variant calling, unique molecular identifiers (UMI's) were used. Filtering of variants was done by stringent criteria consistent with earlier studies. Assessment of mutations was performed blinded to the patients' clinical data. Prospective clinical patient data was obtained for all patients from four national registries, including the Danish Lymphoma Registry (diagnosis, involvement, lymphoma treatment, relapse and death), the Danish National Patient Registry (hospital admission diagnoses and treatments), the Danish Cancer Registry (primary and secondary cancer diagnoses) and the Danish Pathology Database (histopathological examinations and diagnoses), respectively. Results: Samples from 574 patients were included. The median age was 55.5 years (IQR: 45.3 - 62.2) and the median follow-up time for survivors was 9.2 years (IQR: 7.1 - 11.2). The lymphoma subtypes were typical of patients selected for autologous transplantation; diffuse large B-cell lymphoma (191 pts), follicular lymphoma (102 pts), mantle cell lymphoma (88 pts), Hodgkin's lymphoma (80 pts), peripheral T-cell lymphoma (77 pts) and other histologies (36 pts). Of the 574 patients analyzed, 191 (33.3%) of the patients had somatic mutations meeting CHIP criteria (total mutations called=210). The most commonly mutated genes were DNMT3A (n=59, 28%), TET2 (n=48, 23%), PPM1D (n=34, 16%), ASXL1 (n=21, 10%) and TP53 (n=18, 8%). As expected CHIP mutations were more frequent in patients above 60 years (p=0.002). Prevalence of CHIP was associated with an inferior overall survival (p=0.004) and event-free survival (p=0.03). It was also associated with increased risk of biopsy-confirmed tMN (p=0.03) and higher probability of receiving blood transfusions after autologous transplant (p=0.027). Especially patients with mutations in DNA damage response genes PPM1D and TP53 (found in 48 pts, 8.3%) had a significantly increased risk of adverse outcomes. Both overall survival and event-free survival were significantly poorer with the presence of DNA damage pathway mutations (p

Description: Abstract 4983 Background: Full length Hepatocyte growth Factor (HGF) (consisting of four “kringle”-domains) is known to be produced by multiple myeloma plasma cells (MM PC) in vivo and HGF is among the 70 most up regulated genes in MM. Elevated serum levels of HGF are known to be an adverse prognostic factor and recently, MM PC expression of the HGF receptor cMET has been shown to be an adverse prognostic factor. Functionally, HGF inhibits osteoblastogenesis in vitro and promotes migration of MM PC. So far, studies on HGF and its impact in MM have focused on measuring full-length or all isoforms of HGF expression. However, naturally occurring shorter isoforms of HGF (known as NK1 and NK2) are known to work as partial inhibitors of full-length HGF (Otsuka et al, Mol and Cell Biol, 2000). We examined the HGF isoforms and cMET expression at the mRNA level in isolated MM PC of 〉150 newly diagnosed patients with MM, 18 MGUS patients and 8 healthy volunteers (HV) and associated it to overall survival (OS) and degree of osteolytic bone disease (OBD). Methods: Aberrant MM PCs (CD38++/CD19-/CD45-/i/CD56-/+/++) were sorted directly into PCR tubes by fluorescence activated cell sorting (FACS) using a FACS Aria (BDIS). In all cases a PC-purity above 98% was obtained. A cDNA archive was generated by global reverse transcription. By using a polyadenylating step 5x-oligo(dT)-transcript-poly(A)-3xcDNA were generated and finally amplified by PCR using a sequence independent X-(dT)24 primer. The conditions of the reverse transcriptase reaction is designed to limit the size of the first strand cDNA to 300–700 bases, which leads to a more uniform and unbiased amplification. Isoform specific primers were designed using the Primer Express program and experimentally tested. The HGF version covered full-length HGF (transcript variant 1 and 3), HGF2 covered the 2 kringle domain versions (transcript variant 2 and 4) and HGF5 covered the 1 kringle domain version (transcript variant 5). cMET only exists in one isoform. Quantitative PCR was performed using β-actin as internal reference gene, using the δCt method. Determination of positivity or negativity was made from a cut-off-value at 10E-05. OBD was evaluated by standard radiographic methods. The MM patients were treated with either high-dose melphalan with ASCT or melphalan-prednisone according to age recommendations. Results: At least one of the HGF isoforms were found to be expressed in PCxs from 43% of MM patients compared to 32% of MGUS patients, and 0% of HV. Full length HGF was expressed in 17 %, HGF2 in 29%, and HGF5 in 26% of the MM patients. Expression of any HGF variant was associated to an adverse OS (p=0.04) (Fig. 1). The quantitative expression of the transcript variant 5 associated negatively to OS (p=0.02), while expression of the other specific isoforms showed no association to OS. Thus, somehow surprisingly, MM PC expression of the shorter isoform of HGF (HGF5) was more predictive of poor prognosis. A tendency towards elevated expression of full-length HGF in patients with no OBD compared to limited or advanced was observed but did not reach statistically significance (p=0.13). Pre-liminary analysis of cMET expression data showed no correlation between MM PC cMet expression and degree of OBD. Further data on cMet will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

Description: Previous investigations have shown that the novel hydroxamate PXD101 potently inhibits the enzymatic activity of histone deacetylase (HDAC) at nanomolar concentration, and the growth of cancer cell lines derived from solid malignancies at low micromolar potency. We have now examined and present results showing the activity of PXD101 on 〉 20 cell lines corresponding to a variety of haematological cancer types including acute T-cell leukaemia, cutaneous T-cell lymphoma, anaplastic large T-cell lymphoma, mantle cell lymphoma, Burkitt’s lymphoma, diffuse large-cell lymphoma, plasma cell leukaemia and multiple myeloma (MM). Our data show that PXD101 strongly inhibits the growth of these cancer types at submicromolar potency. In addition, we will present in vitro data demonstrating that PXD101 efficiently inhibits the growth of doxorubicin-resistant leukemic cells, thereby supporting the possibility that this compound may be effective on drug-resistant haematological cancers. Finally, we will present results demonstrating that PXD101 used in combination with Velcade mediates greater growth-inhibition than does either drug used as monotherapy on a multiple myeloma cell line. These promising preclinical findings, taken together with previous results demonstrating the activity of PXD101 in a murine leukaemia animal model, support the evaluation of PXD101 for the treatment of haematological malignancies in a clinical setting. To this end, a Phase I trial using PXD101 in patients with advanced hematological tumors refractory to standard therapy was initiated. PXD101 is administered as a 30 minute i.v. infusion on days 1 to 5 of a 21 day cycle. Dose levels 600 mg/m2/d and 900 mg/m2/d have been examined (3 patients each) and recruitment to the dose level 1000 mg/m2/d is ongoing (5 patients to date). Patient characteristics are 8 males and 3 females with median age of 67 (range 58 – 73). Median number of prior therapies is 5 (range 4 – 7). Of the 11 patients enrolled to date, diagnoses were: MM (2), Non-Hodgkin’s lymphoma (5), CLL (4). Eleven patients have been treated with 1–8 cycles of therapy. Adverse events related to PXD101 treatment have mainly been grade 1–2. The most frequent adverse events were nausea (12/23 cycles), fatigue (9/23 treatment cycles), vomiting (8/23 cycles) and diarrhoea (7/23 cycles). Potential antineoplastic activity has been observed. One patient with MM had a possible tumor lysis syndrome with decrease in M-component, increasing urate and serum creatinine requiring dialysis. In addition, one patient with Richter transformation of CLL refractory to other treatments has now been in stable disease for 〉 6 months. We continue to accrue patients in this study, and updated data will be presented.