ALBERT

Description: Key Points The intensified standard-of-care regimens for younger patients with MCL do not overcome the deleterious effects of TP53 mutations. MCLs with TP53 mutations should be considered for alternative frontline treatment.

Description: INTRODUCTION: During the past decades, the outcome of Mantle Cell Lymphoma (MCL) treatment has improved substantially in younger patients. In a recent update of the Nordic MCL2 trial we show very long response durations after a median follow-up of 11.4 years, but we also observe a continuous pattern of relapses even after 10 years of remission.(Eskelund et al, 2016) The course of this disease remains very heterogeneous, and with the current prognostic indexes we are unable to identify patients who might be cured by the current standard-of-care and others who would possibly benefit from alternative frontline approaches. Recently, next-generation sequencing (NGS) studies have explored the mutational landscape of MCL, however, in inhomogeneous and diversely treated cohorts or with short follow-up. Still, TP53 and NOTCH1/2 mutations have been shown to be prognostic markers. In our current study we examine the prognostic impact of aberrations in the most frequently mutated genes in MCL in a homogenously and optimally treated patient cohort, with a long-term follow-up. MATERIAL AND METHODS: Freshly frozen DNA from diagnostic bone marrow samples from patients included in two prospective Nordic trials, MCL2 and MCL3 were analysed. In both trials patients received intensified first line induction therapy with alternating courses of R-CHOP and R-HD-Cytarabine and consolidation with high-dose therapy and ASCT. All patients signed an informed consent.(Geisler et al, 2008; Kolstad et al, 2014). NGS was performed using the Ion Torrent Technology. A targeted panel of 8 genes frequently mutated in MCL was constructed on the basis of previous NGS studies.(Bea et al, 2013; Zhang et al, 2014) The panel included all coding regions of the following genes: ATM, CCND1, TP53, KMT2D, NOTCH1, NOTCH2, WHSC1 and BIRC3. Cut-off for calling a mutation was set to a variant allele frequency 〉3%. Mean depth was 〉1500X in all patients. RESULTS: So far, we have mutational data from 72 patients. Patients were previously untreated and 1 mutation (2-4). Mutations were distributed as follows: ATM 15 (21%), KMT2D 11 (15%), WHSC1 7 (10%), TP53 6 (8%), CCND1 5 (7%), NOTCH2 4 (6%), NOTCH1 3 (4%), BIRC3 2 (3%). In univariate analyses, mutations in TP53 were highly predictive of an inferior outcome (median OS and PFS were 14 and 10 months, respectively; p1 mutations. In multivariate analyses, the only prognostic significant mutations were TP53 (p150 patients from the combined MCL2 and MCL3 cohort. Disclosures Jerkeman: Amgen: Research Funding; Gilead: Research Funding; Janssen: Research Funding; Celgene: Research Funding; Mundipharma: Research Funding. Geisler:Roche: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Sanofi: Consultancy.

Description: INTRODUCTION: The addition of high-dose cytarabine to mantle cell lymphoma (MCL) treatment regimens has significantly prolonged survival of patient subgroups, but relapses are common and are usually associated with treatment resistance. High-dose cytarabine is effective due to the improved retention of ara-CTP by target cells, but likewise toxic, causing mainly hematological side effects. Thus, understanding the molecular mechanism(s) responsible for resistance and to identify predictive markers for resistance and/or sensitizing agents would be of great clinical value. In an attempt to elucidate those mechanisms and to create a tool for drug discovery investigations, we established a unique and molecularly reproducible cytarabine resistant model from the Z138 MCL cell line. Using molecular profiling, we confirm that down-regulation of the deoxycytidine kinase (dCK) protein is key to development of resistance. The MCL resistance model was carefully characterized by screening with annotated compound libraries focused on (i) chemotherapeutics to identify potential cross-resistance and/or sensitivity, and (ii) epigenetic pathways to investigate sensitivity, but also to select individual candidates for sensitization of cytarabine resistant cells. Furthermore, we investigated the hypothesis that the levels of dCK at diagnosis can be used to predict cytarabine resistance through measurement of event-free survival using the Nordic MCL 2/3 cohort, where patients are treated with a combinatorial protocol including high-dose cytarabine. MATERIAL AND METHODS: The first resistant sub-clone defined as Z138 Cytarabine Resistant (Z138-CytR) was established by continuous exposure of wild type Z138 Cytarabine Naïve Sensitive cells (Z138-CytNS) to increasing concentrations (0.005 - 0.3 µM) of cytarabine. Using this model, we could identify the approximate time to resistance development, and utilize this information for developing a novel highly reproducible time-controlled cytarabine resistant model. Molecular changes were investigated by protein and gene expression analyses. Utilizing drug libraries, the cell model was further used to identify substances with growth reducing effect on cytarabine resistant cells. RESULTS AND CONCLUSION: Gene expression profiling revealed that major transcriptional changes occur during the initial phase of adaptation to cellular growth in cytarabine containing media, and only few genes are deregulated upon development of resistance. Instead, resistance to cytarabine was shown to be mediated by down-regulation of the dCK protein, responsible for activation of nucleoside analogue prodrugs. Consequently, cytarabine resistant cells showed cross-resistance to other nucleoside analogues including gemcitabine, cladribine and fludarabine. Of major importance, using drug libraries, we identify substances with growth reducing effect on cytarabine resistant cells. Further investigations are needed to pinpoint compounds that can prevent the down-regulation, or possibly restore dCK protein levels. The possibility to predict cytarabine resistance in diagnostic samples was assessed, but analysis show that the majority of patients have moderate to high expression of dCK at diagnosis, corresponding well to the initial successful response to cytarabine-containing treatment protocols. Disclosures Geisler: Roche: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Sanofi: Consultancy. Jerkeman:Celgene: Research Funding; Gilead: Research Funding; Janssen: Research Funding; Amgen: Research Funding; Mundipharma: Research Funding.