The Bruton's Tyrosine Kinase (BTK)-inhibitor ibrutinib is highly active in mantle cell lymphoma (MCL) but may inhibit response to anti-CD20 antibody as previously shown in CLL models. We investigated ...how antibody-dependent cellular cytotoxicity (ADCC) induced by type I/II anti-CD20 antibodies was affected by treatment with ibrutinib in MCL. Furthermore, we investigated if lenalidomide, a potential sensitizer to anti-CD20 treatment, could prevent an inhibitory effect of ibrutinib.
Anti-CD20 (rituximab/obinutuzumab) opsonized MCL cell lines were co-cultured with ibrutinib (± lenalidomide)-exposed effector cells, and analyzed for evaluation of cell death.
Cell death induced by rituximab was reduced with 75% at 0.5 µM ibrutinib and with 52% at 0.1 µM ibrutinib when induced by obinutuzumab, even by addition of lenalidomide. Moreover, obinutuzumab was associated with higher rate of cell death compared to rituximab.
Ibrutinib negatively affects anti-CD20 induced cell death in MCL, not reversed by lenalidomide. Explorations of sequential administration and selective BTK-inhibitors may reveal the optimal combination of novel agents in MCL.
The addition of high-dose cytarabine to the treatment of mantle cell lymphoma (MCL) has significantly prolonged survival of patients, but relapses are common and are normally associated with ...increased resistance. To elucidate the mechanisms responsible for cytarabine resistance, 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.
Effects of different substances on cytarabine-sensitive and resistant cells were evaluated by assessment of cell proliferation using methyl-14C-thymidine incorporation and molecular changes were investigated by protein and gene expression analyses.
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 key genes, including SPIB, are deregulated upon the later development of resistance. Resistance was shown to be mediated by down-regulation of the deoxycytidine kinase (dCK) protein, responsible for activation of nucleoside analogue prodrugs. This key event, emphasized by cross-resistance to other nucleoside analogues, did not only effect resistance but also levels of SPIB and NF-κB, as assessed through forced overexpression in resistant cells. Thus, for the first time we show that regulation of drug resistance through prevention of conversion of pro-drug into active drug are closely linked to increased proliferation and resistance to apoptosis in MCL. Using drug libraries, we identify several substances with growth reducing effect on cytarabine resistant cells. We further hypothesized that co-treatment with bortezomib could prevent resistance development. This was confirmed and show that the dCK levels are retained upon co-treatment, indicating a clinical use for bortezomib treatment in combination with cytarabine to avoid development of resistance. The possibility to predict cytarabine resistance in diagnostic samples was assessed, but analysis show that a majority of patients have moderate to high expression of dCK at diagnosis, corresponding well to the initial clinical response to cytarabine treatment.
We show that cytarabine resistance potentially can be avoided or at least delayed through co-treatment with bortezomib, and that down-regulation of dCK and up-regulation of SPIB and NF-κB are the main molecular events driving cytarabine resistance development.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Despite recent advances in lymphoma treatment, mantle cell lymphoma (MCL) remains incurable, and we are still unable to identify patients who will not benefit from the current standard of care. Here, ...we explore the prognostic value of recurrent genetic aberrations in diagnostic bone marrow (BM) specimens from 183 younger patients with MCL from the Nordic MCL2 and MCL3 trials, which represent current standard-of-care regimens. In the univariate model, mutations of TP53 (11%) and NOTCH1 (4%), and deletions of TP53 (16%) and CDKN2A (20%), were significantly associated with inferior outcomes (together with MIPI, MIPI-c, blastoid morphology, and Ki67 > 30%); however, in multivariate analyses, only TP53 mutations (HR, 6.2; P < .0001) retained prognostic impact for overall survival (OS), whereas TP53 mutations (HR, 6.9; P < .0001) and MIPI-c high-risk (HR, 2.6; P = .003) had independent prognostic impact on time to relapse. TP53-mutated cases had a dismal outcome, with a median OS of 1.8 years, and 50% relapsed at 1.0 years, compared to a median OS of 12.7 years for TP53-unmutated cases (P < .0001). TP53 mutations were significantly associated with Ki67 > 30%, blastoid morphology, MIPI high-risk, and inferior responses to both induction- and high-dose chemotherapy. In conclusion, we show that TP53 mutations identify a phenotypically distinct and highly aggressive form of MCL with poor or no response to regimens including cytarabine, rituximab, and autologous stem-cell transplant (ASCT). We suggest patients with MCL should be stratified according to TP53 status, and that patients with TP53 mutations should be considered for experimental frontline trials exploring novel agents.
•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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Survival for patients diagnosed with mantle cell lymphoma (MCL) has improved drastically in recent years. However, patients carrying mutations in tumour protein p53 (TP53) do not benefit from modern ...chemotherapy-based treatments and have poor prognosis. Thus, there is a clinical need to identify missense mutations through routine analysis to enable patient stratification. Sequencing is not widely implemented in clinical practice for MCL, and immunohistochemistry (IHC) is a feasible alternative to identify high-risk patients. The aim of the present study was to investigate the accuracy of p53 as a tool to identify patients withTP53missense mutations and the prognostic impact of overexpression and mutations in a Swedish population-based cohort. In total, 317 cases were investigated using IHC and 255 cases were sequenced, enabling analysis of p53 andTP53status among 137 cases divided over the two-cohort investigated. The accuracy of predicting missense mutations from protein expression was 82%, with sensitivity at 82% and specificity at 100% in paired samples. We further show the impact of p53 expression andTP53mutations on survival (hazard ratio of 3 center dot 1 in univariate analysis for both), and the association to risk factors, such as high MCL International Prognostic Index, blastoid morphology and proliferation, in a population-based setting.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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Background: Although survival rates in Mantle cell lymphoma (MCL) have improved, by addition of the anti-CD20-monoclonal antibody (anti-CD20-mAb) rituximab, the disease is still regarded incurable ...and novel combinations are required to improve outcome. Ibrutinib, a BTK-inhibitor, has shown activity in MCL and is currently combined with anti-CD20 therapy in clinical trials. However, preclinical data on CLL has shown reduced effect of anti-CD20-mAb when combined with ibrutinib in vitro, possibly due to reduced activation of NK cells. (Da Roit et al., 2015) Consequently, we investigated how ibrutinib affects the cytotoxic effect of anti-CD20-mAb on MCL cell lines in vitro. We also investigated if the addition of lenalidomide, a potential sensitizer of anti-CD20-ab would overcome an inhibitory effect of ibrutinib.
Methods: PBMC from healthy donors were pretreated with ibrutinib 0/0.01/0.05/0.1/1/5 µM, 1h, 37°C before incubation with anti-CD20-opsonized (rituximab/obinutuzumab, 1µM, 20 min, 37°C), CFSE-labelled MCL cell lines (Jeko-1 and REC), o/n, 37°C. Ratio eff: target 100:1. On day 2, samples were stained with 7-AAD and analyzed in flow cytometry (iQue screener plus®). Rate of cell death was calculated from rate of 7-AAD out of CFSE-positive cells compared to control (PBMC + anti-CD20-mAb+target cells). In secondary experiments, PBMC was incubated with lenalidomide (0/0.01/0.05/1 µM, 20 min, 37°C) before the addition of ibrutinib 1 µM and subsequent proceeding according to protocol as described above.
Results: A significant lower rate of cell death compared to control could be observed in Jeko-1 with rituximab and ibrutinib (figure 1a) at 0.5µM: (25 ±5.56%, p=0.0227), 1 µM (20±3.03%, p=0.0241) and 5 µM (21±1.65% , p=0.0123) and with obinutuzumab and ibrutinib (figure 2) at 0.1 µM (48±0.26%,p=0.0032), 1 µM (17±0.58%, p=0,0045) and 5 µM (11±1.09%, p=0.0078). Further, obinutuzumab was associated with enhanced cell death compared to rituximab in Jeko-1, (149±100% vs 31±25.4%, p=0.0296). For REC, a significant lower cell death was observed at 5µM ibrutinib(27±2.7%), p=00012) in series with rituximab. In series with obinutuzumab, cell death was significant lower at 0.5 µM (86 ±0.99%, p= 0.0437) ibrutinbi but not for higher concentrations. Lenalidomide did not affect the rate of cell death (data not shown).
Conclusion: Our study shows that pretreatment of PBMC with ibrutinib negatively affected the cytotoxic effect of anti-CD20-mAb rituximab and obinutuzumab on MCL in vitro. Further, obinutuzumab was associated with enhanced cytotoxicity compared to rituximab. The addition of lenalidomide, a potential synergizer of anti-CD20-ab did not overcome the inhibitory effect of ibrutinib. Further studies, i.e. of sequential administration, may reveal how to optimize the combination of ibrutinib with anti-CD20-ab to improve outcome in MCL patients.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Summary
Survival for patients diagnosed with mantle cell lymphoma (MCL) has improved drastically in recent years. However, patients carrying mutations in tumour protein p53 (TP53) do not benefit from ...modern chemotherapy‐based treatments and have poor prognosis. Thus, there is a clinical need to identify missense mutations through routine analysis to enable patient stratification. Sequencing is not widely implemented in clinical practice for MCL, and immunohistochemistry (IHC) is a feasible alternative to identify high‐risk patients. The aim of the present study was to investigate the accuracy of p53 as a tool to identify patients with TP53 missense mutations and the prognostic impact of overexpression and mutations in a Swedish population‐based cohort. In total, 317 cases were investigated using IHC and 255 cases were sequenced, enabling analysis of p53 and TP53 status among 137 cases divided over the two‐cohort investigated. The accuracy of predicting missense mutations from protein expression was 82%, with sensitivity at 82% and specificity at 100% in paired samples. We further show the impact of p53 expression and TP53 mutations on survival (hazard ratio of 3·1 in univariate analysis for both), and the association to risk factors, such as high MCL International Prognostic Index, blastoid morphology and proliferation, in a population‐based setting.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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.
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.
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.
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
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.
So far, we have mutational data from 72 patients. Patients were previously untreated and <66 years (median 58, range 37-65). Fifty-three percent were either MIPI intermediate or high risk. Eighty-five percent of patients had bone marrow involvement at diagnosis. After a median follow-up of 11.6 years, median overall (OS) and progression-free survival (PFS) of all 72 patients were 12.4 and 9.8 years, respectively.
Of the 72 patients, mutations were detected in 33 cases. Twenty-one patients carried 1 mutation and 12 patients had >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; p<0.0001 for both outcomes) (Fig). Likewise, patients with either NOTCH1 or NOTCH2 mutations displayed worse OS (median OS 8.1 years, p=0.023), and there was a trend towards inferior PFS (median PFS 4.5years, p=0.087). No other mutations predicted better or worse outcomes, nor did carrying any one or >1 mutations. In multivariate analyses, the only prognostic significant mutations were TP53 (p<0.0001, HR=15.9) and NOTCH1 (p=0.012, HR=5.3).
TP53 mutations were associated with increased Ki67 expression, median 30% (range 20-80%), higher risk MIPI/MIPI-B/MIPI-C, and 4 out of 6 patients had blastoid morphology at diagnosis. All 6 TP53 mutations were found in the DNA-binding domain, and 5 were missense mutations while the last was a frameshift mutation. Patients with missense mutations all died within 3 years (range 6-34 months) due to relapsing or progressive disease, while PFS and OS of the latter patient was 4.6 years and 8.4 years, respectively.
We found no predictors for late relapses.
Here we evaluate the prognostic impact of mutations in eight genes that are commonly involved in MCL in a cohort of 72 younger patients who received intensified induction therapy and ASCT in the Nordic MCL2 trial. In line with previous reports, we demonstrate a negative prognostic impact of TP53 and NOTCH1/2 mutations; however, in multivariate analyses only TP53 and NOTCH1 mutations held significance. Missense mutations in the DNA binding domain of TP53 seem to predict an exceedingly poor short term outcome. At the annual meeting, we will present mutational analyses and long term follow up of >150 patients from the combined MCL2 and MCL3 cohort.
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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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Food components modify the risk of cancer at a large number of sites but the mechanism of action is unknown. In the present investigation, we studied the effect of the peptide lactoferricin derived ...from bovine milk lactoferrin on human colon cancer CaCo-2 cells. The cells were either untreated or treated with 2.0, 0.2, or 0.02 μM lactoferricin. Cell cycle kinetics were investigated with a bromodeoxyuridine DNA flow cytometric method. The results show that lactoferricin treatment slightly but significantly prolonged the S phase of the cell cycle. Lactoferricin treatment lowered the level of cyclin E1, a protein involved in the regulation of genes required for G1/S transition and consequently for efficient S phase progression. The slight prolongation of the S phase resulted in a reduction of cell proliferation, which became more apparent after a long treatment time.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Treatment of Caco-2 cells with the peptide lactoferricin4–14, results in reduction of the growth rate by prolongation of the S phase of the cell cycle. Lactoferricin1–25 is formed in the gut by ...cleavage from lactoferrin and the bioactive amino acids are found within lactoferricin4–14. Our hypothesis is that the reduction of the rate of S phase progression may result in increased DNA repair. To test this hypothesis, Caco-2 cells were subjected to UV light that caused DNA lesions and then the cells were grown in the absence or presence of 2.0μM lactoferricin4–14. Evaluation of DNA strand breaks using the comet assay showed that lactoferricin4–14 treatment indeed resulted in a reduction of comets showing damaged DNA. In the search for a mechanism, we have investigated the levels of several proteins involved in cell cycle regulation, DNA replication, and apoptosis using Western blot. Lactoferricin4–14 treatment resulted in an increased expression of flap endonuclease-1 pointing to increased DNA synthesis activity. Lactoferricin4–14 treatment decreased the expression of the proapoptotic protein B-cell lymphoma 2-associated X protein (or Bax), indicating decreased cell death. As we have found previously, lactoferricin4–14 treatment reduced the expression of cyclin E involved in the G1/S transition. Immunofluorescence microscopy showed that a lower γ-H2AX expression in lactoferricin4–14-treated cells, pointing to more efficient DNA repair. Thus, altogether our data show that lactoferricin4–14 treatment has beneficial effects.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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