Littérature scientifique sur le sujet « Peripheral neuropathy, murine models, bortezomib, multiple myeloma »

Abstract Abstract 4936 Despite recent advances in the treatment of MM, the disease remains incurable and many of the most effective, newer combination therapies are accompanied by significant side effects that have a major negative impact on the patient's quality of life. Pegylated liposomal doxorubicin (PLD) and bortezomib have shown anti-MM efficacy in the laboratory and for the treatment of previously treated MM patients, leading to FDA approval for patients who have failed one prior therapy. Using our severe combined immunodeficiency-hu murine models of human MM, we have previously demonstrated that lower doses of PLD administered daily are more effective and better tolerated than higher amounts given weekly. Moreover, the combination of bortezomib and dexamethasone has been shown to be effective for previously untreated MM patients. Prior studies by our group have shown that combining chemotherapy including PLD with bortezomib administered at 1.0 mg/m2 on days 1, 4, 8, and 11 of a 28-day cycle rather than the standard 1.3 mg/m2 on the same days of a 21-day schedule is effective for MM patients with relapsed or refractory disease and associated with a reduction in the incidence and severity of peripheral neuropathy. Thus, we conducted a single-arm multi-center phase II study for previously untreated MM patients to evaluate the combination of intravenously administered dexamethasone, bortezomib and PLD (DVD). The treatment consisted of intravenous administration of 40 mg dexamethasone followed by 1.0 mg/m2 bortezomib and finally 5.0 mg/m2 PLD on days 1, 4, 8, and 11 of a 28-day cycle. Patients were treated to a maximum response plus two additional cycles or completed a maximum of eight cycles of therapy without disease progression. To date, 22 (of 35 planned) patients have been enrolled with a median age of 64 years (range, 42-79 years). The majority of those on study (68 %) were diagnosed with International Staging System II or III MM. Four patients are too early to assess for response. To date, among the 18 evaluable patients, 16 (89%) have shown objective responses to the DVD regimen, including 2 complete responses (11%), 8 partial responses (44%) and 6 minimal responses (33%). The other 2 patients (11%) had stable disease, with one of these subjects showing a continuing reduction in M-protein after 2 cycles of therapy to date. Thus, disease control was achieved in all patients. To date, no patient has shown progressive disease after 2+ - 12+ months of follow-up. Six patients experienced grade 3 adverse events and one patient with a prior history of pulmonary interstitial fibrosis developed a grade 4 toxicity (shortness of breath). Grade 3 adverse events in three of the six patients were judged not to be related to the study treatment. The most common grade 3 adverse event was reversible neutropenia (n=2). To date, only 2 patients (9%) have developed peripheral neuropathy (grade 1). Notably, there have been no cases of stomatitis or hand-foot syndrome. Thus, these results suggest that the DVD regimen using a modified schedule and doses of the combination of intravenous dexamethasone, bortezomib and PLD is a well tolerated treatment that produces high response rates for previously untreated patients with multiple myeloma. Disclosures Berenson: Millennium Pharmaceuticals: Consultancy, Research Funding, Speakers Bureau; Centocor Ortho Biotech: Consultancy, Speakers Bureau. Hilger:Millennium Pharmaceutcals: Employment.

e20552 Background: The survival of patients with multiple myeloma has improved dramatically since the introduction of proteasome inhibitors such as bortezomib, which can have the adverse effect of peripheral neuropathy. This study retrospectively examines causes of chronic pain in myeloma patients and the modalities and duration of treatments used for pain control. Methods: Rush University Medical Center multiple myeloma patients who were diagnosed and treated between 2000-2019 were included. Outcome measures were abstracted from the medical record and included: classes of pain medication used, duration of treatment, prevalence of peripheral neuropathy symptoms, and use of adjunct treatment modalities. Descriptive statistical models including Chi-square and Fisher’s exact test were used for categorical variable analysis. Results: In all, 134 patients were included, of which 75.4% (N = 101) patients received at least one cycle of bortezomib. 42.5% (N = 57) patients were seen in palliative clinic. 72.9% (N = 97) reported bone pain symptoms. A total of 73.7% (N = 98) patients experienced peripheral neuropathy symptoms. 86.1% (N = 87) of patients who received bortezomib reported neuropathy, as compared to 34.4% (N = 11) of patients who did not have bortezomib therapy (OR 11.8, p < 0.0001). 66.4% (N = 67) patients who received bortezomib took anticonvulsants as compared to 31.3% (N = 10) of those who did not receive bortezomib (OR 4.3, p < 0.0005). Patients were on anticonvulsant therapy for a mean of 32.6 months (SD = 26.7) with no significant difference in the bortezomib group. 79.7% (N = 106) patients took opioid medications. Norco was the most commonly used opioid (N = 55) and average duration of use was 36.6 months (SD = 34). 18.8% (N = 25) took antidepressant medications such as TCAs or SNRIs for pain, and all of these patients received bortezomib therapy (p = 0.0003). 36.6% (N = 45) of patients received radiation and 16.5% (N = 22) underwent kyphoplasty. 70.7% (N = 94) patients attended at least one physical therapy session. There was no statistically significant difference in radiation or kyphoplasty utilization between patients who had received bortezomib and those who had not, but there was a significant difference in physical therapy (OR 4.9, p < 0.0001). Conclusions: Patients who received bortezomib as part of their myeloma treatment were more likely to experience peripheral neuropathy and required anticonvulsant therapy more frequently. It is important to better understand and define the health burden of chronic pain and use of pain medications in patients with multiple myeloma.

Abstract Pharmacological inhibition of the proteasome with bortezomib (BTZ) has translated into an improved clinical outcome in patients with multiple myeloma and mantle cell lymphoma. Despite the observed clinical activity, BTZ anti-tumor activity in B-cell lymphoma has been partially hindered by treatment-related toxicities (peripheral neuropathy) preventing further dose escalation and emergence of acquired resistance. To further develop therapeutic strategies targeting the proteasome system, we studied the anti-tumor activity and mechanisms-of-action of ixazomib (MLN2238), a reversible proteasome inhibitor, in pre-clinical lymphoma models. Previously we demonstrated that ixazomib is active in various lymphoma pre-clinical in vitro models and that is capable of inducing cancer cell death in a caspase-independent manner. To further explore the effects of ixazomib, we investigated its anti-tumor activity in murine lymphoma models and investigated the mechanisms responsible for cell death observed in our pre-clinical models. For in vivo studies, 6-8 week old severe combined immunodeficiency (SCID) mice were inoculated via tail vein injection (iv) with mantle cell lymphoma Granta cells (day 0) and assigned to observation, ixazomib (iv) (at 6mg/kg/dose on days +1, 4, 8, 11, 15 and 18) or BTZ (ip) (at 0.8mg/kg/dose on days +1, 4, 8, 11, 15, and 18). Differences in survival (measured as the time to limb paralysis development) were evaluated by log-rank test across treatment arms. In addition, we studied the role of p21 in the anti-tumor activity of ixazomib. A panel of rituximab-sensitive (RSCL) and -resistant cell lines (RRCL) was exposed to ixazomib. Changes in cell cycle distribution and expression levels of key cell cycle regulatory proteins were evaluated by Western blotting and flow cytometry respectively. To further define the role of p21 in ixazomib activity, transient p21 knock down was achieved using electroporation with a pooled p21 siRNA. Down regulation of p21 was confirmed by Western blotting. Following transient p21 knock down, RSCL or RRCL were exposed to ixazomib and changes in cell viability were determined using the cell titer glo assay. Finally, RSCL and RRCL were exposed to ixazomib (10nM) +/- the cell cycle inhibitor roscovitine (10nM) and viability was determined by measuring changes in ATP content. As single agent, ixazomib prolonged the survival of Granta-bearing SCID mice when compared to control or BTZ (median=24 vs. 27 vs. 35 days; P = 0.012). In addition, in vitro exposure of lymphoma cell lines to ixazomib resulted in p21 and cell cycle arrest in G1 (RSCL) or G2/M (RRCL). Transient knock down of p21 rescued both Raji RSCL and RRCL from the cytotoxic effects of ixazomib when compared to controls. Moreover, in vitro exposure of RSCL to ixazomib in the presence to roscovitine resulted in synergistic effects on cell viability. Together our data suggests that ixazomib is more effective than BTZ in controlling mantle cell lymphoma growth in vivo. In addition, MLN2238 anti-tumor activity appears to be mediated partially by the stabilization of p21. (Ixazomib was obtained from Millennium Pharmaceuticals, Inc. Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund.) Disclosures: Czuczman: Genetech, Onyx, Celgene, Astellas, Millennium, Mundipharma: Advisory Committees Other.

Abstract Bortezomib (VELCADE®) is a potent inhibitor of the proteasome which exerts its antimyeloma effect in part by blocking the activation of NF-κB. As NF-κB is critical for lymphocyte development and survival, there is great interest in harnessing the potential immunomodulatory effects of bortezomib. In murine hematopoietic transplantation models, bortezomib inhibits in vitro mixed lymphocyte responses and promotes the apoptosis of alloreactive T cells protecting against acute graft-versus-host disease. However, no data exists on the in vivo effects of bortezomib on human T cells. To characterize the effects of bortezomib on immune function, we profiled peripheral blood lymphocytes subsets and T cell associated cytokines in 39 patients with multiple myeloma. Two cycles of bortezomib 1.3 mg/m2 were administered by intravenous infusion on days 1, 4, 8, and 11 of a 21-day treatment cycle. The patients had received prior induction chemotherapy and would proceed to autologous transplant following treatment with bortezomib. Study population consists of 23 male and 16 female patients with the median age of 56 years (range 38–69). Myeloma characteristics at diagnosis were as follows (number of patients): IgG (28), IgA (10), light chain only (1), with stage I (1), II (12), or stage III (26) disease. Peripheral blood was collected at baseline (cycle 1, day 1) and at one week after the last dose of bortezomib (cycle 2, day 18) and analyzed for lymphocyte subsets by standard multicolor flow cytometry. Th1 and Th2 serum cytokines were measured at the same timepoints using a multiplexed cytometric bead array (BD Biosciences). Following treatment with bortezomib, no significant changes were detected in either Th1 or Th2 serum cytokine levels: IL-2 (p=0.116), TNF-alpha (p=0.854), IFN-gamma (p=0.070), IL-4 (p=0.240), IL-6 (0.236), IL-10 (0.151) as analyzed by Wilcoxon signed ranks test. Analysis of lymphocyte subsets using a paired student’s T-test demonstrated a 38% decrease in CD56+ NK cells (p=0.02) and a 26% increase in CD4/CD8 ratio (p=0.0006) which appears to be secondary to a decrease in CD8+ cytotoxic T-cells (p=0.054). (Table 1.) In conclusion, we observe an alteration of lymphocyte subsets following only two cycles of bortezomib. Further analysis of the effects of long term treatment with bortezomib is warranted. These studies may provide insights into the role of bortezomib as an immunomodulatory agent. Peripheral Blood Lymphocyte Subsets Pre-bortezomib (/mm 3 ) Post-bortezomib(/mm 3 ) Difference(/mm 3 ) P-value CD2 1446 1259 −187 0.085 CD3 1273 1160 −113 0.28 CD4 842 802 −40 0.54 CD8 412 337 −75 0.055 CD19 90 94 4 0.86 CD20 87 95 8 0.78 CD56 206 148 −58 0.022 CD4/CD8 ratio 2.53 3.19 0.66 0.0006

Abstract Proteasome inhibition is a validated strategy for therapy of multiple myeloma, but this disease remains challenging as relapses are common, and often associated with increasing chemoresistance. Moreover, nonspecific proteasome inhibitors such as bortezomib can induce peripheral neuropathy and other toxicities that may compromise the ability to deliver therapy at full doses, thereby decreasing efficacy. One novel approach may be to target the immunoproteasome, a proteasomal variant found predominantly in cells of hematopoietic origin that differs from the constitutive proteasome found in most other cell types. Using purified preparations of constitutive and immunoproteasomes, we screened a rationally designed series of peptidyl-aldehydes and identified several with relative specificity for the immunoproteasome. The most potent immunoproteasome-specific inhibitor, IPSI-001, preferentially targeted the β1i subunit of the immunoproteasome in vitro and in cellulo in a dose-dependent manner. This agent induced accumulation of ubiquitin-protein conjugates, proapoptotic proteins, and activated caspase-mediated apoptosis. IPSI-001 potently inhibited proliferation in myeloma patient samples and other hematologic malignancies. Importantly, IPSI-001 was able to overcome conventional and novel drug resistance, including resistance to bortezomib. These findings provide a rationale for the translation of IPSIs to the clinic, where they may provide antimyeloma activity with greater specificity and less toxicity than current inhibitors.

In this study, we demonstrate expression and examined the biologic sequelae of PI3K/p110δ signaling in multiple myeloma (MM). Knockdown of p110δ by small interfering RNA caused significant inhibition of MM cell growth. Similarly, p110δ specific small molecule inhibitor CAL-101 triggered cytotoxicity against LB and INA-6 MM cell lines and patient MM cells, associated with inhibition of Akt phosphorylation. In contrast, CAL-101 did not inhibit survival of normal peripheral blood mononuclear cells. CAL-101 overcame MM cell growth conferred by interleukin-6, insulin-like growth factor-1, and bone marrow stromal cell coculture. Interestingly, inhibition of p110δ potently induced autophagy. The in vivo inhibition of p110δ with IC488743 was evaluated in 2 murine xenograft models of human MM: SCID mice bearing human MM cells subcutaneously and the SCID-hu model, in which human MM cells are injected within a human bone chip implanted subcutaneously in SCID mice. IC488743 significantly inhibited tumor growth and prolonged host survival in both models. Finally, combined CAL-101 with bortezomib induced synergistic cytotoxicity against MM cells. Our studies therefore show that PI3K/p110δ is a novel therapeutic target in MM and provide the basis for clinical evaluation of CAL-101 to improve patient outcome in MM.

Abstract Abstract 3832 Poster Board III-768 Introduction ALA is an antioxidant often used in the management of peripheral neuropathy (PN) for patients with multiple myeloma (MM). A clinical trial evaluating ALA in diabetic neuropathy showed this drug to be effective for patients with both somatic and autonomic neuropathies. It also normalized the endoneural blood flow, reduced oxidative stress and improved vascular dysfunction. Bortezomib (Velcade®), the first-in-class proteasome inhibitor (PI), which is approved for the treatment of patients with MM, may cause PN. As a result, patients are often treated empirically with ALA. In this study, we investigated whether ALA has any impact on the anti-MM effects of bortezomib. Methods First, cells from the MM cell lines RPMI8226 and MM1S (1×105 cells per 100μl) were treated with ALA alone to determine whether ALA had any effects on their growth as determined with an MTS assay. MM cells were plated in a 96-well plate using serum-free media. The cells were treated with either media alone or ALA at concentrations ranging from 1 to 1000 μM for 48 hours. The acidity of ALA at these doses was determined and if the pH was less than 7, we neutralized it using NaOH. Second, we measured the proliferation of cells exposed to bortezomib alone and combinations of a fixed concentration of bortezomib and escalating concentrations of ALA. Results The exposure of cells to ALA alone had a stimulatory effect on the growth of both MM cell lines in vitro. ALA alone at 1000 μM resulted in an increase in cell viability of MM1S cells by approximately 10% when compared to the control group. ALA alone also stimulated the growth of RPMI8226 cells but at much lower concentrations than observed for MM1S. Compared to untreated cells, there was an increase in cell viability with ALA at concentrations as low as 1 μM and a concentration dependent increase at concentrations of 1, 10, 100, and 1000 μM in RPMI8226 cells. At the highest concentration (1000 μM) of ALA, cell viability increased 150% when compared to RPMI8226 cells incubated with media alone. Next, we evaluated the effect of ALA on bortezomib's anti-MM activity. As a single agent, bortezomib reduced MM1S (20 nM) and RPMI8226 (5 nM) cell viability by 93% and 70% respectively. When ALA was added at a clinically achievable concentration (100 μM) to bortezomib (RPMI8226, 5 nM; MM1S, 20 nM), a reduction in the anti-MM effects of bortezomib on these cell lines was observed when compared to bortezomib treatment alone. Compared to bortezomib alone, the combination of ALA plus bortezomib doubled cell viability (increased RPMI8226 and MM1S cell viability from 32.5% to 65% and 7.5% to 15%, respectively). These negative effects of ALA on bortezomib's anti-MM activity were consistently observed in multiple experiments involving both of these cell lines evaluating concentrations of ALA ranging from 100 to 1000 μM and bortezomib ranging from 5 to 20 nM. Conclusions Our data suggest that ALA has the potential to antagonize the anti-MM effects of bortezomib based on our in vitro results using MM cell lines. Thus, it is possible that ALA could negatively impact the therapeutic benefit of bortezomib for MM patients and this requires further study especially if ALA is accepted as an intervention in bortezomib-related neuropathy. We are currently completing studies evaluating primary MM patients' tumor cells in vitro and our human MM xenograft models in vivo to further validate this impact of ALA on bortezomib's anti-MM activity and whether changes in treatment schedule of these drugs may prevent this inhibitory effect from occurring. In addition, because part of bortezomib's anti-tumor effects are related to reactive oxygen species (ROS) levels, we are evaluating whether the inhibitory effects of ALA on this PI may be overcome by increasing intracellular ROS levels. Disclosures: Hilger: Millennium Pharmaceutcals: Employment. Berenson:Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Abstract Background: BCL-2 and MCL-1 promote multiple myeloma (MM) cell survival. Venetoclax (VEN) is a potent, selective, orally bioavailable small-molecular inhibitor of BCL-2. When combined with bortezomib, which can inhibit MCL-1, VEN can enhance the activity of bortezomib in MM cell lines and xenograft models. Methods: In this Phase 1b, open label, dose escalation study, patients with relapsed/refractory (R/R) MM received daily VEN (50 - 1200 mg per designated dose cohort) with bortezomib and dexamethasone. The objectives of the study were to assess the safety, pharmacokinetics, maximum tolerated dose, recommended phase 2 dose (R2PD), and efficacy (objective response rate [ORR], time to progression [TTP], and duration of response [DoR]) of combination therapy in this patient population. Results: As of 01July2016, 66 patients were enrolled, with 54 in the dose escalation cohort and 12 in the safety expansion at R2PD of 800 mg. The median age was 64 years and 39 (59%) were ISS stage II/III. The median number of prior therapies was 3 (range: 1 - 13), and 21 (32%) were refractory to prior bortezomib, 37 (56%) were refractory to prior lenalidomide, and 41 (62%) had prior stem cell transplant. Forty-three (65%) patients discontinued the study for the following primary reasons: 33 related to disease progression, 5 due to AEs (1 each: respiratory failure and cardiac failure, lung adenocarcinoma, sepsis, renal impairment, and Guillain-Barre syndrome; none were considered by the investigator as related to VEN), 2 withdrew consent, and 3 for other reasons not specified. Adverse events (AEs) were reported in 65 (99%) patients, with common AEs in ≥20% of patients being diarrhea (41%), thrombocytopenia (39%), constipation (38%), nausea (36%), insomnia (32%), peripheral neuropathy (30%), peripheral edema (29%), anemia (27%), peripheral sensory neuropathy (27%), dyspnea (24%), fatigue (24%), and asthenia (24%). Grade 3/4 AEs in ≥10% of patients included thrombocytopenia (29%), anemia (15%) and neutropenia (14%). Serious AEs in ≥2 patients were febrile neutropenia, thrombocytopenia, cardiac failure, pyrexia, influenza, lower respiratory tract infection, pneumonia, sepsis, acute kidney injury, respiratory failure, embolism, and hypotension. One dose-limiting toxicity of lower abdominal pain was reported for a patient who received 1200 mg VEN. Five deaths were reported during the study, 4 due to disease progression and 1 due to respiratory syncytial virus infection (not considered by the investigator as related to VEN). After co-administration with bortezomib and dexamethasone, dose-normalized VEN exposure at steady-state appeared to be within the exposure range observed with VEN monotherapy in patients with MM. The ORR for all evaluable patients was 68% (44/65) and 26 (40%) achieved very good partial response (VGPR) or better (3 stringent complete response [sCR], 8 CR, 15 VGPR) (Figure). For all patients, median DoR was 8.8 months (95% CI: 7.2, 15.8) and TTP was 8.6 months (95% CI: 5.7, 10.2), with a median follow up of 4.9 months (range: .03 - 26.7). High ORR of 89% was seen in patients who were non-refractory to prior bortezomib (39/45) or who had 1 - 3 prior therapies (31/35). In 31 patients who were non-refractory to bortezomib and had 1 - 3 prior therapies had ORR of 94% (29/31), 68% (21/31) with VGPR or better; median DoR was 10.6 months and TTP was 11.3 months for this subgroup. Moreover, patients who were bortezomib naïve and had 1 - 3 prior lines of therapy had ORR of 100% (12/12), and median DoR was 15.8 months and TTP was 17.1 months. In patients who were non-refractory to prior bortezomib but who were refractory to lenalidomide, the ORR was 86% (19/22) as compared with 91% (20/22) in those non-refractory to lenalidomide. Clinical responses were comparable in patients with t(11;14) MM (ORR, 78% [7/9]) and without t(11;14) MM (ORR, 66% [37/56]). In the t(11;14) group, 3 patients were bortezomib-refractory, and 2 of them achieved a PR as best response. Also, 4 patients had more than 3 prior lines, with 3 of them achieving PR. Conclusions: VEN in combination with bortezomib and dexamethasone has an acceptable safety profile in patients with R/R MM. Efficacy results, including 68% ORR in all patients and 94% ORR in patients not refractory to bortezomib and who received 1 - 3 prior lines of therapy, indicates promising efficacy of this novel combination and supports the ongoing Phase 3 trial with this regimen in patients with R/R MM. Figure. Figure. Disclosures Moreau: Janssen: Honoraria, Speakers Bureau; Novartis: Honoraria; Takeda: Honoraria; Celgene: Honoraria; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria. Roberts:Genentech: Patents & Royalties: Employee of Walter and Eliza Hall Institute of Medical Research which receives milestone payments related to venetoclax; AbbVie: Research Funding; Servier: Research Funding; Janssen: Research Funding; Genentech: Research Funding. Agarwal:AbbVie: Honoraria, Research Funding; Millennium: Consultancy; Amgen: Consultancy; Janssen: Speakers Bureau; Onyx: Speakers Bureau; Celgene: Speakers Bureau. Facon:Amgen: Consultancy, Speakers Bureau; Millenium/Takeda: Consultancy; Bristol: Consultancy; Janssen: Consultancy, Speakers Bureau; Novartis: Consultancy; Celgene: Consultancy, Speakers Bureau; Karyopharm: Consultancy. Kumar:Glycomimetics: Consultancy; AbbVie: Research Funding; Janssen: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; BMS: Consultancy; Celgene: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Sanofi: Consultancy, Research Funding; Kesios: Consultancy; Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Noxxon Pharma: Consultancy, Research Funding. Touzeau:AbbVie: Research Funding. Cordero:AbbVie: Employment. Ross:AbbVie: Employment, Equity Ownership. Munasinghe:AbbVie: Employment. Jia:AbbVie: Employment. Salem:AbbVie: Employment. Leverson:AbbVie: Employment, Other: Shareholder in AbbVie. Maciag:AbbVie: Employment. Verdugo:AbbVie: Employment, Other: may own stock. Harrison:Janssen Cilag: Research Funding, Speakers Bureau; AbbVie: Research Funding.

Abstract Abstract 3875 Poster Board III-811 We investigated genotyping of 172 candidate gene loci (2016 SNPs) related to hereditary neuropathy, energy production and fast axonal transport, nociception and pain transmission, mitochondria, neurogenesis, neuroprotection, immune function, and bortezomib (Velcade®) mechanism of action. Our aim was to identify predictive classifiers for peripheral neuropathy (PN) in multiple myeloma (MM) following treatment with bortezomib in the phase 3 VISTA trial of bortezomib plus melphalan–prednisone (VMP, N=344) vs MP (N=338) in previously untreated MM patients ineligible for high-dose therapy (median age 71 years). We collected whole blood samples from 323 patients who provided consent for this analysis, from 99/151 participating clinical centers. Samples were transferred to a central laboratory for DNA extraction. After quality control for DNA integrity and quantity, DNA from 139 patients on the VMP arm (VISTA PGx subset) was included in this case-control candidate gene study. A custom array was developed by Illumina (San Diego, CA) for this analysis. Patient characteristics of the VISTA PGx subset were similar to those of the overall population. Of the PGx subset, 51.8% had reported PN events (36.0% grade ≥2). We sought to confirm VISTA findings in an independent cohort of patients in the IFM2005-01 phase 3 study of bortezomib–dexamethasone (Vel/Dex) vs VAD in previously untreated MM patients aged ≤65 years eligible for high-dose therapy (median age 57 years); 215 samples were collected from consenting patients on the Vel/Dex arm, of whom 46% had reported PN events (23.7% grade ≥2) including peripheral neuropathies, dysesthesia, and paresthesia. The VISTA analysis included cases and matched controls with result correlations to clinical adverse event descriptions of “peripheral sensory neuropathy”, “peripheral neuropathy NEC”, and/or “neuralgia”, or any occurrence of these three adverse events. Multiple covariates were included in single-marker association analyses using logistic regression in SAS (PROC LOGISTIC, SAS, v9.1) under three different genotypic models (additive, dominant, recessive) followed by FDR correction. No SNPs had significant associations with the terms above. However, analysis for time to onset of PN using Cox regression and the log rank test in SAS (PROC PHREG PROC TPHREG and PROC LIFETEST, SAS v9.1) using the same models and multiple testing corrections methods identified two SNPs significantly associated with shorter time to onset of any PN (rs4553808; CTLA4; FDR=0.002) and time to onset of grade ≥2 PN (rs1474642; PSMB1; FDR =0.014). Both of these associations were found in the recessive model so that 8.3% of patients with PN following bortezomib treatment have the homozygous recessive genotype of CTLA4 and 6% of patients with grade ≥2 PN have the recessive PSMB1 genotype. Both of these associations were also significant under Bonferroni's correction method. Two additional SNPs were identified that had significant association with grade ≥2 PN (rs12568757; CTSS, FDR=0.027) and with grade ≥3 PN (rs11974610; GJE1; FDR=0.041) but these were not significant under Bonferroni's method. These results suggest an immune-related influence for PN development involving genes associated with immune function (CTLA4, CTSS) and reflexive coupling within Schwann cells (GJE1). Mutations in the target (PSMB1) subunit suggest an influence with drug binding as well. When we investigated associations with these SNPs in the IFM2005-01 dataset, no significant associations were found in the analysis of time to onset of PN following multiple testing corrections; however, rs4553808 (CTLA4) had the same trend of association with time to onset of grade ≥2 PN in the recessive model (p=0.105). The differences in association were likely due to differences between the VISTA and IFM2005-01 study populations (e.g. neuropathy status at entry, rate of onset of PN, age, and baseline characteristics including β2-microglobulin, albumin, and diabetes status). This is the first report of an association between genes associated with immune function (e.g. CTLA4) and time to onset of bortezomib-associated PN. Although the clinical utility of this study is limited due to the low number of genes investigated and the exploratory nature of this analysis, further genome-wide studies may result in a predictive classifier for bortezomib-associated PN applicable to the clinic. This work is currently ongoing. Disclosures: Ricci: Johnson & Johnson: Employment, Equity Ownership. Favis:Johnson & Johnson: Employment, Equity Ownership, Research Funding. Sun:Johnson & Johnson: Employment. van de Velde:Johnson & Johnson: Employment, Equity Ownership. Broderick:Johnson & Johnson: Employment. Meyers:Johnson & Johnson: Employment, Equity Ownership. Harousseau:Janssen Cilag: Honoraria; Celgene: Honoraria. Richardson:Millennium Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Johnson and Johnson: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Keryx: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. San Miguel:Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees; OrthoBiotech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract Background Appropriate animal models for hematological malignancies are highly attractive, because they allow the study of the tumor biology and underlying disease mechanisms. They also constitute a major prerequisite for rapid bench-to-bedside translation of investigational anticancer therapies. To validate our multiple myeloma patient (pt)-derived xenograft (MM PDX) model (Schueler et al, Expert Opin Biol Ther, 2013), we systematically analyzed a panel of MM PDX with regard to their sensitivity towards standard of care treatment and compared these data with the pts' clinical outcome. Methods Bone marrow (BM) cells of 11 MM pts were implanted intratibialy (i.t.) into 103 NOD/Shi-scid/IL-2Rγnull (NOG) mice (n= 6-18 / pt sample). Mice were treated according to pts' therapy with VCD (Bortezomib, Cyclophosphamide, Dexamethasone), or to evaluate additional treatment options with Rd (Lenalidomide, Dexamethasone). Tumor growth and antitumoral activity in mice were assessed in tumor-bearing mice and compared to untreated control mice as well as to pts' response. Tumor growth in the mouse model was monitored by whole-body fluorescence-based in-vivo-imaging (IVI) using CF750-labeled α-HLA ABC antibody before and during treatment as well as 24h after last treatment cycle as described (Schueler J. PLOSone 2013). Mock-injected animals served as negative controls. Engraftment of human MM cells in murine organs was confirmed by flow cytometry and patho-histological analyses (immunostaining) at the end of the study. Results The pt cohort included a typical MM clientele for referral centers, with a median age of 75 years (range 56-85), median BM infiltration of 80% (20-90), and high- and standard-risk cytogenetics in 5 and 6 pts, respectively. All pts had advanced disease with Durie&Salmon stage III and active/symptomatic MM. All pts received VCD after diagnosis and BM sampling. MM cell engraftment could reliably be determined from experimental day 10 on in all 11 MM PDX models, at all assessed sites, namely within the BM, spleen and peripheral blood (PB) of recipient mice. Individual pt samples displayed distinct tumor growth patterns in vivo. Fluorescence intensity of engrafted murine organs ranged from 2- to 15-fold compared to mock injected control mice. Mean IVI signals in BM of recipient mice were 10-fold higher as compared to spleen signals, qualifying the BM niche as the preferred homing localization of pts' MM cells. Of note, both injected and non-injected BM sites were infiltrated by MM cells 10 days after tumor cell injection. Engraftment of human MM cells in the respective murine organs was confirmed by flow cytometry (HLA ABC, CD138, CD38) and histology and verified MM engraftment via both methods, confirming prior reports (Schüler PLOSone 2013; Groen Blood 2012;120:e9-16, Overdijk MAbs. 2015;7:311-21). The murine engraftment capacity was independent of MM type, disease stage, BM infiltration and cytogenetics of the donor pt. VCD was applied to 9 different MM PDX models and induced partial remission (PR; defined as at least 50% reduction of murine tumor load in BM, spleen and/or PB) in 5 out of 9 tested MM PDX models, whereas 2 cases each showed stable disease (SD) or progression (PD). The response rates in the mouse avatars mirrored the clinical outcome of the respective MM pts in 8/9 cases; only one MM pt showed serological and clinical PR, whereas the corresponding mice displayed SD. Rd induced PR in 1 and PD in a second MM PDX model, underlining the feasibility of MM PDX for drug screening approaches. Conclusions Due to the complex tumor biology, murine models of MM are still challenging. Our data support the preclinical rationale to use i.t.-injected NOG mice, since they closely resemble clinical MM with respect to symptoms, disseminated disease sites and response to anticancer treatment. Possible applications for the MM mouse avatars include development of new anticancer drugs as well as definition of biomarker strategies and selection of treatment options for individual pts with relapsed/refractory MM. The data of our preclinical study may serve as a useful future strategy to guide treatment decisions in refractory pts. The suitability as a drug development tool will be additionally determined performing treatment experiments with novel agents, e.g. elotuzumab or daratumumab. Disclosures Schueler: Oncotest GmbH: Employment. Klingner:Oncotest GmbH: Employment.

In the last 20 years several in vivo rat models of peripheral neuropathy induced by antineoplastic drugs clarified the phatophysiological mechanisms involved in peripheral neurotoxicity. Moreover these models allowed to identify neuroprotection strategies for preventing sensory nerve and dorsal root ganglia (DRG) neurons damages. Only a few cancer cell lines is able to induce the development of cancer in immunocompetent rats, therefore these models do not represent the best way to investigate, at the same time, the antineoplastic activity and the neurotoxic effects of these drugs; by contrast, murine models are widely employed in oncological studies and can be useful for this purpose. During the Ph.D Programme in Neuroscience, the aim of this projecct has been the neurophysiologic, histophatologic and behavioural characterization of 4 murine models of peripheral neuropathy induced by the proteasome inhibitor bortezomib. The neurotoxic side effects of several antineoplastic agents are among the main reason for tretament modification or their limitation as antitumor therapy. The mechanisms underline the onset of peripheral neurophaties are still not clear, even though it is accepted that the neurotoxicity is dose- and drug-dependent. Bortezomib is a proteasome inhibitor and it is used as an efficient chemotherapeutic agent in the tretament of multiple myeloma. In 2003 the Food and Drug Administration (FDA) approved the use of bortezomib for the treatment of advanced multiple myeloma, then in 2008 the drug was promoted as a first-line chemotherapeutic agent for the treatment of myeloma. However patients treated with bortezomib develop a peripheral neuropathy, often combined with neuropathic pain. This project consist of 3 consecutive phases: (A.1) The development of 4 in vivo murine models of bortezomib-induced peripheral neuropathy. Aim: to evaluate the neuropathologic mechanisms involved in bortezomib-induced peripheral neuropathy in two immunocompetent murine strains (Balb/c and CD1) and in two immunodeficient murine strains (Hola Hsd NuNu and Scid). _Aim of work and summary 12 (A.2) The development of a multiple myeloma murine model in association with bortezomib treatment. Aim: to evaluate, at the same time, the antineoplastic activity and the neurotoxic effect of bortezomib in Scid mice. (B) The localization of Ca2+ transporters Calbindin and Parvalbumin in DRG neurons (respectively markers of small size neurons and big size neurons). Aim: verify the distribution of different DRG neurons subpopulations (small size, medium size and big size neurons) to quantify the damage induced by the treatment. Female Balb/c, CD1, Hola Hsd NuNu and Scid mice (initial body weight 19-21 gr.) were treated with bortezomib to develop and to study the neuropathologic mechanisms of the peripheral neuropathy (A.1). Balb/c mice were administered with bortezomib twice a week for 4 weeks at the dose of 0,8 mg/Kg; CD1 and Hola Hsd NuNu were treated with bortezomib 0,8 mg/Kg twice a week for 6 weeks while Scid mice were administered with bortezomib 1 mg/Kg once a week for 5 weeks. During the treatment period we evaluated both general toxicity parameters, like mortality and body weight changes, and neurotoxicity parameters, including the sensory nerve conduction velocity (NCV) in the caudal and digital nerve, the morphological and morphometrical analysis of DRG neurons and sciatic nerve and the assessment of neuropathic pain by behavioural dynamic test. Bortezomib has been well tolerated in all 4 strains: Balb/c and Scid mice treated with the compound showed a statistically significant decrease in body weight compared to the control ones (respectively of 10,65 and 8,17%); on the contrary CD1 mice showed an increase in body weight even though not comparable to the control ones (reduction of 7,9% respect to control mice) and Hola Hsd NuNu mice revealed an increase in body weight similar to control ones. All 4 strains disclosed different neurophisiological alterations. Bortezomib caused a clear reduction both in caudal and in digital NCV. Balb/c showed a decrease in caudal NCV of 27,3% and in digital NCV of 17,9% (compared to control ones). CD1 mice showed a reduction in caudal NCV of 20,3% and in digital NCV 26,9%; Hola Hsd NuNu mice bared a decrease in caudal NCV of 15,2% and in digital NCV of 13,5%; Scid mice showed a reduction in caudal NCV of 21,35% and in digital NCV of 23,83%. _Aim of work and summary 13 The morphological analysis highlighted alterations of DRG neurons and satellite cells induced by bortezomib; moreover the compound caused a severe and frequent degeneration of myelinic fibers. The morphometrical analysis of DRG neurons showed the increase in size of soma, nucleus and nucleolus in Balb/c treated mice respect to control ones. In CD1 mice only the nucleolus was increased in size, while in Hola Hsd NuNu and Scid mice bortezomib caused an increase in both of soma and nucleolus size. Furthermore Bortezomib induced a statistically significant increase of g-ratio (the ratio of the inner axonal diameter to the total outer diameter) and a trend decrease of myelin thickness. Dynamic test highlighted the onset of mechanical allodynia (a pain due to a stimulus which does not normally provoke pain). CD1 e Scid mice showed a statistically significant reduction of the answer to the mechanical stimulus respect to control ones (respectively of 29,1% and 22,1%); on the contrary in Balb/c mice and Hola Hsd NuNu bortezomib caused a not statistically significant decrease of answer (respectively 21,5% and 20,4%). Female Scid mice (initial body weight 19-21 gr.) were injected with 1x106 RPMI 8226 cells (human cellular line of multiple myeloma) and half of these animals were treated with bortezomib 1 mg/Kg once a week for 5 weeks (A.2) to evaluate, at the same time, the antineoplastic activity and the neurotoxic effect of the compound. Also in this phase, parameters of general toxicity and of neurotoxicity were evaluated. Moreover we monitored the tumor increase. Bortezomib has been well tolerated in all groups: mice injected with RPMI 8226 cells revealed an increase in body weight similar to control ones, while mice injected with RPMI 8226 cells and treated with the compound showed a decrease in body weight between the second and the fourth administration that recovered until the end of treatment. Two groups disclosed different neurophisiological alterations. Bortezomib caused a relevant decrease both in caudal (27,1% respect to control animals) and in digital NCV (19,1% respect to control animals) while multiple myeloma reduced only the digital NCV (14,21% respect to control animals). The morphological analysis showed alterations of DRG neurons and satellite cells induced by multiple myeloma that were confirmed in animals treated with bortezomib; moreover multiple myeloma caused a frequent degeneration of myelinic fibers that increased in animals treated with bortezomib. _Aim of work and summary 14 The morphometrical analysis of DRG neurons showed the decrease in size of soma, nucleus and nucleolus in mice with multiple myeloma, while bortezomib caused an increase in size of soma and nucleolus. The morphometrical analysis of sciatic nerve showed the involvement of multiple myeloma in the increase of g-ratio and in the decrease of myelin thickness. The treatment with bortezomib highlighted these results. Dynamic test demonstrated the onset of mechanical allodynia only in mice with multiple myeloma treated with bortezomib: the decrease in the answer to the stimulus is statistically significant in comparison to control ones (14,53%) but it is not relevant if compared to animals with multiple myeloma (9,6%). Furthermore, the evaluation of tumor size demonstrated that bortezomib has an efficient antineoplastic role starting from the first week of treatment. Since the morphometrical analysis of DRG neurons showed the increase in size of soma, nucleus and nucleolus of animals treated with bortezomib, we decided to measure the damage induced by the compound proving the distribution of different neuronal subpopulations (small, medium and big neurons) (B). Unexpectedly Calbindin and Parvalbumin labelled neurons with similar somatic areas both in control animals (respectively 478,1 ± 210,5 µm2 e 451,5 ± 193.5 µm2) and in treated mice (respectively 368,2 ± 144,1 µm2 e 342,6 ± 136 µm2). A difference between the labelling of control animals respect to treated ones is clear, particularly if we consider the percentage of parvalbumin labelling: indeed the DRG neurons of control animals treated represent the 33,15% of all cells while the DRG neurons of treated animals are the 15,36% of all neurons. The characterization of in vivo murine models of bortezomib-induced peripheral neuropathy of this study is relevant since it could represent a starting point for evaluating the anticancer activity of the compound and, at the same time, its neurotoxicity. Furthermore these models could be useful to test the neuroprotective action of new experimental agents and to evaluate the non-interference between the chemotherapeutic agent and the neuroprotectant agents.