Introduction: Even though the majority of newly diagnosed multiple myeloma (NDMM) patients has benefited significantly from the introduction of novel agents, it remains an unmet need to identify the best upfront treatment strategy for high-risk disease. Risk-adapted trial designs (e.g., the OPTIMUM/MUKnine trial, NCT03188172, and the GMMG-CONCEPT trial, NCT03104842) aim to answer this question, which in turn drive research efforts to improve risk assessment in NDMM. Recently, our group has published two novel prognostic tools for NDMM: (I) The Second Revision of the International Staging System (R2-ISS), which identifies four different risk categories by combining conventional prognostic markers (D’Agostino et al. – J Clin Oncol 2022). (II) Plasma Cell Leukemia-like (PCL-like) status, which identifies high-risk disease by combining tumor transcriptomic data of 54 genes reflecting high levels of circulating tumor cells (Hofste op Bruinink et al. – J Clin Oncol 2022). Since both tools may detect different types of aggressive disease, we hypothesized that combining PCL-like status with the R2-ISS classification would improve prognostic accuracy in NDMM. Methods: Baseline characteristics including serum markers and high-risk cytogenetic aberrations were collected from NDMM patients enrolled in the HOVON-65/GMMG-HD4 (EudraCT 2004-000944-26), HOVON-87/NMSG-18 (EudraCT 2007-004007-34), EMN02/HO95 (EudraCT 2009-017903-28) and MMRF CoMMpass studies (NCT01454297) and used to determine the R2-ISS classification. PCL-like status was calculated from transcriptomic profiles of CD138-enriched bone marrow tumor cells. Hazard ratios (HRs) for PCL-like status, R2-ISS classification or a combination thereof were estimated using a Cox proportional hazards model stratified by study cohort and corrected for age ≤ 65 years. Results: For 865 NDMM patients of known age, both the R2-ISS classification and PCL-like status could be determined, with a median follow-up time of 55 months. 18% were classified as R2-ISS low (R2-ISS I), 30% as R2-ISS low-intermediate (R2-ISS II), 43% as R2-ISS intermediate-high (R2-ISS III) and 9% as R2-ISS high (R2-ISS IV); 10% as PCL-like MM and 90% as intramedullary MM (i-MM). In univariate analyses, PCL-like status was associated with both an inferior PFS (HR, 1.8; 95% confidence interval (CI), 1.4 to 2.3; P < 0.0001) and OS (HR, 2.4; 95% CI, 1.8 to 3.3; P < 0.0001). This also applied to the R2-ISS classification when comparing R2-ISS IV to R2-ISS I, II and III (HR for PFS, 2.0; 95% CI, 1.5 to 2.6; P < 0.0001 and HR for OS, 3.0; 95% CI, 2.2 to 4.2; P < 0.0001). PCL-like status retained its prognostic significance in a multivariate model when combined with the R2-ISS classification, both in terms of PFS (HR, 1.6; 95% CI, 1.2 to 2.1; P = 0.0004) and OS (HR, 2.1; 95% CI, 1.6 to 2.9; P < 0.0001). Of note, 2% of patients classified both as PCL-like MM and R2-ISS IV, translating into a median PFS of 12 months and a median OS of 15 months. Patients with PCL-like MM and R2-ISS III (5%) had a comparable median OS to those with i-MM and R2-ISS IV (7%) (47 and 38 months, respectively). Validation is planned in other European Myeloma Network trial cohorts. Conclusions: 1. PCL-like status combined with the R2-ISS classification improves prognostic accuracy in NDMM. 2. The presence of both PCL-like MM and R2-ISS IV may confer exceptionally high risk. 3. Patients with PCL-like MM and R2-ISS III have a comparable OS to those with i-MM and R2-ISS IV.

The randomized, open-label, phase III trial HOVON-65/GMMG-HD4 was designed to evaluate the efficacy of bortezomib prior to HDM for response and progression-free survival (PFS) in patients with newly diagnosed MM. The trial was performed in 75 referral centers in the Netherlands and Belgium (HOVON group) and Germany (GMMG group). Patients with Salmon & Durie (SD) stage II or III, age 18–65 years inclusive, were randomly assigned to 3 cycles of VAD (vincristine 0.4 mg, adriamycine 9 mg/m2 days 1–4, dexamethasone 40 mg days 1–4, 9–12, and 17–20) or PAD (bortezomib 1.3 mg/m2 days 1,4,8,11, adriamycine 9 mg/m2 days 1–4, dexamethasone 40 mg days 1–4, 9–12, and 17–20). No thrombosis prophylaxis was given. Stem cells were mobilized using the CAD regimen, including cyclophosphamide 1000 mg/m2 iv day 1, and G-CSF. After induction therapy, all patients were to receive 1 or 2 cycles of high-dose melphalan (HDM) 200 mg/m2 with autologous stem cell rescue followed by maintenance with thalidomide 50 mg daily (VAD arm) or bortezomib, 1.3 mg/m2 once every 2 weeks (PAD arm) for 2 years. Between May 4, 2005 and May 16, 2008, 833 patients were randomized. After the trial was closed, we here report the planned interim analysis data on response after induction and HDM-1 of the initial 150 (75 per arm) randomized patients. The data of the initial 300 registered patients (150 per arm) will be available by November 1, 2008 and presented. The 2 randomization arms were equal for SD stage of disease, ISS stage, and distribution of chromosomal abnormalities. 134 patients (89%) completed PAD/VAD and 130 patients (87%) completed HDM-1, with no difference between the treatment arms. Full dose bortezomib could be administered in 95 % (PAD1), 79 % (PAD2) and 85 % (PAD3) of patients. Successful stem cell apheresis was achieved in all 132 patients who received CAD. Adverse events CTC grade 2–4 during PAD vs VAD included neurologic or polyneuropathy (PNP) 38% vs 21 %, constitutional symptoms 30 % vs 24 %. PNP of CTC grade 1–4 was more frequent in the PAD arm (p=0.01), while DVT/pulmonary embolism was diagnosed in 10 % during VAD and 6 % during PAD. Responses were assessed according to EBMT criteria including VGPR after PAD/VAD, after HDM-1 and best response on protocol treatment. Complete Response (CR), Very Good Partial Response (VGPR) and Partial Response (PR) in both arms were compared by logistic regression (table 1)

Multiple Myeloma (MM) is characterized by the presence of a monoclonal protein, immunodeficiency, anemia, renal failure and bone lesions. New agents like Bortezomib (Bor) and Thalidomide (Thal) have shown efficacy in 40% of patients with relapsed/refractory MM, and up to 75% in first-line treatment. Classical prognostic classifications such as serum b2-microglobulin, albumin and chromosomal aberrations, have insufficient predictive power in estimating long-term outcome with these targeted agents. We have performed gene expression profiling (GEP) in newly diagnosed MM patients who were included in a large multicenter, prospective phase III trial (HOVON65) comparing Bor with standard induction prior to high dose therapy (HDT). The aim of this profiling was to gain new insights in the pathogenesis of MM and to design a prognostic index for treatment based on molecular profiling. In 258 patients, CD138 magnetic cell selected (MACS) myeloma plasma cells (PC) obtained at diagnosis with PC purity > 80% were used for RNA extraction and applied to the Affymetrix GeneChip U133 plus 2.0 arrays. Data from the gene expression arrays were pre-processed using GCRMA (Bioconductor). Unsupervised gene clustering (Omniviz) using a correlation visualisation matrix could determine the clusters based on the top variably expressed genes, and differential gene expression within these clusters could be determined (BRB-array tools, p<0.001, FDR<1%). Enriched genes and pathways were determined using Ingenuity systems®. Based on GEP, the translocations t(4;14), t(14;16), t(14;20) or t(11;14) could be shown to drive the clusters using 1.25% of the most variable genes (675 genes). Increasing the variable genes within the analysis (2.5%, 5%, 10% or 20%) identifies 10 different clusters of myeloma, with the translocations being distributed differentially. This could indicate that other factors, besides the chromosomal aberrations have an important role in biologically driving the clustering of myeloma GEP. Using 10% of most variable genes, 4 out of 10 clusters were predominantly driven by genes involved in the translocations; for example: up-regulation of MMSET/FGFR3 (cluster 1), MAF downstream targets (cluster 9), CCND1/MS4A1/VPREB3 (cluster 5) and INHBE (cluster 10). Three of the larger clusters show an expression of genes involved in protein biosynthesis and/or interferon signalling; one of which showed expression of XRCC4, FRZB and HGF (cluster 4), one showing expression of IFIT27, IFIT3 and TRAIL (cluster 6) and the third showed expression of EGFR and CHES1 (cluster 8). Another cluster was identified with a high NF-κB index (cluster 3), in which NF-κB transcriptional activity is calculated from the mean expression level of four probe sets corresponding to CD74, IL2RG, and TNFAIP3 (2×) as described by Keats et al. 2 out of 10 clusters, each containing 6 samples, were shown to be non-descriptive (clusters 2 and 7). When combining GEP with clinical symptoms at presentation, 2 clusters (4 & 6) were identified which had a high frequency of trisomies, IgG isotype and a significantly higher incidence of bone lesions. In addition, clusters 1, 8, 9 and 10 presented more frequently with renal failure, thrombocytopenia and anemia, and clusters 5, 9 and 10 showed less bone involvement. These data indicate that GEP may identify clusters of patients, who have a distinct clinical expression of organ impairment (ROTI). In conclusion, we describe molecular subgroups of MM showing distinct gene expression signatures in combination with specific chromosomal aberrations and clinical characteristics. Awaiting the clinical analysis of the trial, the response and survival data of the molecular subgroups will be included in the analysis.

Disclosure: No relevant conflicts of interest to declare.

Leukemia is the most common type of cancer in pediatric patients (pts). Treatment options for Philadelphia-chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML) include: risk-adapted chemotherapy, imatinib (im) and stem cell transplant (SCT). Relapsed leukemia has few therapeutic options. Dasatinib (SPRYCEL®, formerly BMS-354825) is a novel, oral, multi-targeted kinase inhibitor of BCR-ABL, KIT, and SRC kinases that was recently granted approval for adults with CML, and Ph+ ALL with resistance or intolerance to prior therapy. CA180018 is the first trial evaluating dasatinib in pediatric patients pts and Ph+ and in Ph- leukemias. CA180018 is a phase I/II dose-finding study of dasatinib in pts aged 1–20 with CML or relapsed Ph+ ALL resistant or intolerant to im, or Ph- ALL or AML in ≥2nd relapse performed in collaboration with 12 centers (6 countries) from the ITCC Consortium. Preliminary data are available on the first 15 pts treated from March-July 2006 (2 chronic phase (CP) CML, 7 Ph+ ALL, 1 accelerated phase (AP) CML, 3 Ph- ALL, and 2 Ph- AML) at a starting dose of 60 mg/m2 daily (course = 3 weeks). Intra-patient dose escalation was allowed for lack of initial response, and a 3+3 design for maximum tolerated dose (MTD) determination. Hematologic, cytogenetic, and molecular responses, as well as plasma and cerebrospinal fluid (CSF) pharmacokinetic (PK) analysis and BCR-ABL mutational analysis are ongoing. Median age was 11 yrs (range 4–17), median time from diagnosis of leukemia was 19.5 months (range 1.4–89.9). Prior therapy included chemotherapy, im, and stem cell transplant. Median duration on study is 0.69 month (range 0.03–3.45). Intra-patient dose escalation to 80 or 100 mg/m2 occurred in 7 pts. Six pts remain on study. There have been 7 responders: 4 complete hematologic responses (CHR): 1 CP CML, 1 AP CML, 2 Ph+ ALL; 5 cytogenetic responses (CyR): 2 Ph+ ALL complete CyR (CCyR), 1 AP CML CCyR, 1 CP CML partial CyR (PCyR), 1 Ph+ ALL minor CyR who never achieved a CHR, and then progressed; and 2 Ph+ ALL pts with CNS disease who cleared the CSF of leukemic blasts with single agent dasatinib. Preliminary PK in 7 pts showed rapid absorption with a median Tmax of 1.0 h, and mean terminal phase half-life (SD) of 2.7 (1.1) h. There is one dose-limiting toxicity (DLT): grade 4 anaphylactic shock which occurred 5 hours after the first dose. Dasatinib has otherwise been well tolerated up to 100 mg/m2 with toxicities including grade 3/4 thrombocytopenia, and sepsis. Nine pts are off study: 8 for progressive disease (2 Ph+ ALL pts with a resistant T315I BCR-ABL mutation), and 1 DLT. These preliminary results provide evidence supporting the safety and efficacy of dasatinib in pediatric pts with relapsed or refractory leukemia. An updated analysis including further enrollment, safety data, PK, cytogenetic and molecular responses, and mutational analyses will be presented.

Older patients (pts) have a high incidence of P-glycoprotein (P-gp) positive AML blast cells. P-gp acts as drug efflux pump for a variety of agents including daunorubicin (DNR). The P-gp inhibitor PSC-833 can inhibit the function of P-gp in AML cells and restore sensitivity to DNR. Combining PSC-833 with DNR may improve response rate and survival in AML pts. However, previous intervention studies have produced conflicting results. From 1997 to 1999 we conducted a multi-center phase 3 study in pts of older age with previously untreated AML, in which we prospectively investigated the effect of PSC-833 combined with standard antileukemic treatment in relation to the flowcytometric P-gp status at diagnosis. Pts were randomized to receive two cycles of DNR (45 mg/m2, days 1-3) and Ara-C (200 mg/m2, 7 days) or the same regimen with a lower dose of DNR (35 mg/m2, days 1–3) plus PSC-833 (2 mg/kg loading dose followed by 10 mg/kg/24h for 72 hours). Pts who attained a complete remission (CR) were to receive a common consolidation cycle of Ara-C (1 g/m2, days 1–4), mitoxantrone (6 mg/m2, days 1–4) and etoposide (80 mg/m2, days 1–4).

Telomeres, the ends of linear chromosomes, have a critical role in protection against chromosome end-to-end fusion. Telomeres shorten in every cell division due to the end replication problem. Telomerase is a reverse transcriptase that adds telomeric DNA repeats to the ultimate chromosome end. In vitro models of long-term fibroblast cultures have identified two sequential mortality stages, senescence (M1) and crisis (M2). Senescence can be bypassed by loss of p53 or Rb function, whereas escape from crisis can only be achieved by activating a telomere maintenance mechanism, mostly telomerase reactivation. Cells that bypass senescence (M1) did not reactivate telomerase, resulting in further telomere shortening to a critical telomere length upon reaching crisis (M2). In these models, critical telomere shortening induces extensive chromosome instability, most likely via chromosome end-to-end fusions. Dicentric chromosomes lead to anaphase breakage-fusion-bridges resulting in multiple chromosomal aberrations. To investigate whether similar mechanisms may be involved in the development of genetic instability in human cancer, we studied telomere length and expression of critical telomeric proteins (TRF2 and POT1) in acute myeloid leukemia (AML) patients. AML is a good model for these studies since distinct subgroups of AML are characterized by either exchanges along chromosome arms (translocation or inversion), or by a complex karyotype with multiple chromosome aberrations. Groups were age-matched. Telomere length was studied in metaphase arrested leukemic cells using quantitative fluorescence in situ hybridization (Q-FISH) using a telomere-specific probe. Subsequently, metaphase spreads were hybridized with a leukemia-specific probe to confirm leukemic origin of each metaphase. Telomeres were significantly shorter in AML samples with multiple chromosomal abnormalities in comparison to AML samples with a reciprocal translocation/inversion or no abnormalities (mean±SEM=16±1.7 AFU, n=12 versus 29±4.3 AFU, n=18; p=0.015). Interestingly, telomerase activity level is significantly higher in AML samples with multiple chromosomal abnormalities, compared to AML samples with a reciprocal translocation or inversion (mean±SEM=330±95, n=11 versus 70±21, n=13; p=0.02). Expression levels of telomeric proteins TRF2 and POT1 were similar in these AML groups. Our observations suggest that, consistent with previous in vitro models in fibroblasts, critical telomere shortening may have a role in the development of genetic instability in human AML. Critically short telomeres in association with high levels of telomerase activity suggest that AML cells with multiple chromosomal abnormalities have bypassed crisis (M2). The longer telomeres and low levels of telomerase activity in AML cells with a reciprocal translocation or inversion suggest that they originate from an earlier stage, preceding crisis. Consequently, telomere length modulation may have a role in cancer prevention.

We here report the clinical, cytogenetic, fluorescence in situ hybridization (FISH), and Southern blot data on 14 patients with a myeloid malignancy and structural aberration of chromosome band 11q23 associated with overrepresentation or amplification of the MLL gene. The number of copies of MLL varied from three (two cases) to a cluster consisting of multiple hybridization spots. Together with previous reports, available data indicate that amplification of 11q23/MLL is a recurrent genetic change in myeloid malignancy. It affects mainly elderly patients and is often associated with dysplastic bone marrow changes or with complex karyotypic aberrations, suggestive of genotoxic exposure. It is associated with a poor prognosis. In addition, FISH analysis of nine cases with additional 11q probes showed that the overrepresented chromosomal region is generally not restricted to MLL, and Southern blot analysis indicated that amplification does not involve a rearranged copy of this gene. The significance of MLL amplification and the mechanisms by which it could play a role in leukemogenesis and/or disease progression remain to be elucidated.