Multiple myeloma (MM) is a hematologic malignancy characterized by clonal proliferation of plasma cells and overproduction of monoclonal immunoglobins. Treatment with melphalan is currently standard of care for younger and fit patients when followed by hematopoietic stem cell transplantation (HSCT), and in transplant ineligible patients when used in combination regimens. It has been previously shown that changes in the p53 pathway are associated with melphalan efficacy, but the regulatory role of the p14ARF-MDM2-p53 axis has yet to be fully explored. Recently, a non-coding RNA, ANRIL (antisense non-coding RNA in the INK4-ARF locus) has been shown to negatively regulate the transcription of the entire INK4-ARF locus and simultaneously modulate the p53 and pRb pathways. Moreover, some single nucleotide polymorphisms (SNPs) in ANRIL have previously been associated with susceptibility to several malignancies. Here we investigated select ANRIL SNPs in DNA from patient-derived peripheral blood mononuclear cells obtained from 108 MM patients treated with high-dose melphalan followed by HSCT. Our results show that the rs2151280 (CàT) SNP in ANRIL was associated with worse progression-free survival (TC/CC vs TT: HR = 0.53, 95%CI, [0.26, 1.07], P = 0.07; adjusted HR = 0.39, 95%CI, [0.18, 0.84], P = 0.016), and the TT variant had higher ANRIL expression and lower p15, p14ARF, and p16 expression compared to the TC/CC variants. Our results indicate that ANRIL may be involved in melphalan-mediated apoptosis via down-regulating p14ARF and subsequent p53, and that the rs2151280 polymorphism may be a potential prognostic biomarker for relapse in melphalan-treated MM patients.

High dose melphalan followed by autologous stem cell transplantation remains standard of care for eligible patients with multiple myeloma, but disease response and toxicity, including severe mucositis, varies among patients. Our randomized trial investigated duration of cryotherapy (2 and 6 hours) for reduction of mucositis prevalence and severity and explored factors associated with variability in pharmacokinetics and outcomes from melphalan therapy. The results demonstrate 2-hour is at least as effective as 6-hour cryotherapy in decreasing severe mucositis. From a population pharmacokinetic model, we identified fat free mass, hematocrit, and creatinine clearance were significant covariates, as had been reported previously. Furthermore, we observed the rs4240803 SLC7A5 polymorphism was significantly associated with pharmacokinetic variability, and pharmacokinetics was associated with both mucositis and neutropenia. However, melphalan exposure was not associated with progression-free or overall survival in our dataset. These findings contribute to ongoing efforts to personalize melphalan dosing in transplant patients.

High‐dose melphalan (HDM) is part of the conditioning regimen in patients with multiple myeloma (MM) receiving autologous stem cell transplantation (ASCT). However, individual sensitivity to melphalan varies, and many patients experience severe toxicities. Prolonged severe neutropenia is one of the most severe toxicities and contributes to potentially life‐threatening infections and failure of ASCT. Granulocyte‐colony stimulating factor (G‐CSF) is given to stimulate neutrophil proliferation after melphalan administration. The aim of this study was to develop a population pharmacokinetic/pharmacodynamic (PK/PD) model capable of predicting neutrophil kinetics in individual patients with MM undergoing ASCT with high‐dose melphalan and G‐CSF administration. The extended PK/PD model incorporated several covariates, including G‐CSF regimen, stem cell dose, hematocrit, sex, creatinine clearance, p53 fold change, and race. The resulting model explained portions of interindividual variability in melphalan exposure, therapeutic effect, and feedback regulation of G‐CSF on neutrophils, thus enabling simulation of various doses and prediction of neutropenia duration.

Background/Aim: SLC7A5 is recognized as the major mediator of melphalan uptake into multiple myeloma (MM) cells; however, its contribution to the inter-patient variability of melphalan efficacy and toxicity is yet to be well elucidated. This study aimed to investigate the impact of a single nucleotide polymorphism (SNP) rs4240803 in SLC7A5 on the gene expression, ex vivo sensitivity to melphalan, and clinical outcomes in MM patients who were undergoing autologous stem cell transplantation with high-dose melphalan. Materials and Methods: Peripheral blood mononuclear cells (PBMC) were collected from 108 MM patients prior to melphalan therapy. Clinical data were also collected from these patients following melphalan therapy. Results: rs4240803 was associated with elevated expression of SLC7A5 mRNA, higher ex vivo sensitivity to melphalan in PBMCs, and positive 90-day response in these patients (p=0.047, 0.10, 0.049, respectively). Conclusion: rs4240803 impacted the expression of SLC7A5, thus contributing to the clinical response of MM patients to melphalan therapy.

Introduction: For fit multiple myeloma (MM) patients, autologous hematopoietic stem cell transplant (HSCT) is standard of care as part of first line therapy, demonstrating longer progression-free survival when compared to upfront bortezomib, lenalidomide, and dexamethasone (IFM/DFCI 2009, ASH 2015). The use of granulocyte colony stimulating factor (G-CSF) after HSCT accelerates time to neutrophil recovery by 1 - 6 days when compared with control (Klumpp TR et al, JCO, 1995 & Schmitz N et al, BMT 2004). The American Society of Clinical Oncology guidelines recommend that G-CSF should be initiated 1-5 days after administration of high-dose chemotherapy and should be continued until the absolute neutrophil count (ANC) is 2000-3000/mm3 (Smith TJ, JCO, 2015). We have evaluated the role of G-CSF starting day +1, day +5, and day +7 post-transplant in three sequential cohorts of MM patients focusing on the duration of severe neutropenia (rather than the time to neutrophil engraftment), infections, and mucositis. Methods: As part of changes in the standard of care institutional protocols for autologous HSCT of myeloma patients at Ohio State University, three sequential cohorts of myeloma patients were identified that received G-CSF daily post-transplant until ANC>1500/mm3 or WBC>5/mm3. Two hundred twenty-six (226) patients received G-CSF on day +1 (n=43), day +5 (n=78), and day +7 (n=105) from May 2012 to August 2015. The majority of the patients received levofloxacin, acyclovir and fluconazole as prophylaxis. We evaluated the duration of severe neutropenia (ANC<500), the onset of bacteremia, WHO grade 2-4 mucositis, and the time to biochemical progression as part of an IRB approved protocol (NCT01653106). Results:The cohort of patients receiving G-CSF on day +5 had a shorter median follow up (455 days versus 979 days in the day +1 and 1355 days in the day +7 cohorts), since the patients were enrolled at a later timepoint. No statistically significant difference was noted in terms of age, gender, cytogenetic or ISS stage distribution, number of infused cells per kg, and melphalan dose among the three cohorts of patients. The duration of severe neutropenia was significantly increased for patients receiving G-CSF on day +7 (mean 153.4 hrs, SD 36.9) compared to those receiving G-CSF on day +1 (mean: 104.8 hrs, SD 19.4; p-value <0.0001) or day +5 (mean: 120.5 hrs, SD 27.4; p-value <0.0001). There was also a statistically significant difference between the duration of severe neutropenia for G-CSF day +1 or day +5 (p-value 0.013). The duration of neutropenia was not influenced by gender, International Staging System or R-ISS at diagnosis. Moreover, no statistically significant difference was noted in terms of duration of severe neutropenia when patients were subdivided based on the number of stem cell infused (< or > of 5 x 106 cells). Patients who received G-CSF on day +1 or day +5 had a significantly decreased incidence of grade 2 or higher mucositis (4.65% vs 10.3%) for day +1 and day +5 versus 21.9% for day +7 (fisher-test, p = 0.014 and 0.0255). Similarly, the incidence of bacteremia was decreased in those patients treated with G-CSF on days +1 and +5 versus day +7 (21.8%, 23.2%, and 33.3% for those treated on days +1, +5, and +7, respectively; p = 0.09). The group of patients who received G-CSF on day +5 had a longer length of stay, with two patients with prolonged hospitalization (mean 17.7 days, SD 5.6 days, range 12-49) when compared to the other groups (day +1 group mean 13.88 days, SD 2.8 days, range 10-21 versus day +7 group mean 15.2 days, SD 2.7 days, range 11-21 days; Two-tailed t-test p = 0.012). Lastly, our data indicates a trend, though not statistically significant, toward a longer time to biochemical progression in patients who received G-CSF on day +1 when compared to the other two groups. Conclusion: We conclude that starting G-CSF injections the day after stem cell infusion (day +1) decreases the duration of severe neutropenia, infections, and mucositis in comparison to day +5 and day +7 in patients receiving autologous transplant for MM. A prospective trial would definitively test this, but a multi-institution meta-analysis via CIBMTR would be more cost-effective, acknowledging that differing anti-infective prophylaxis regimens would prevent effective between institution comparisons of infection. Disclosures No relevant conflicts of interest to declare.

Autologous stem cell transplant (ASCT) with high‐dose melphalan (HDM) is the standard treatment for fit multiple myeloma (MM) patients. It is generally believed that some DNA repair proteins impact the activity to repair melphalan‐induced DNA damage, thus potentially contributing to the patient's clinical response. However, knowledge of these proteins is limited. In the current study, we investigated the roles of XRCC1, a protein involved in base excision repair and single‐strand break repair, in melphalan response in MM cells. Small interfering RNA knockdown of XRCC1 significantly increased the accumulation of melphalan‐induced DNA damage in MM cells and sensitized them to melphalan treatment, indicating that genetic variation in XRCC1 may impact response to melphalan treatment. We then evaluated the association between an XRCC1 variant with reduced activity, rs25487 (R399Q), and clinical outcomes of 108 MM patients with melphalan therapy. Our results showed that XRCC1 rs25487 was associated with prolonged progression‐free survival (PFS) in MM patients. The adjusted hazard ratio for PFS between patients carrying rs25487 AA/AG and GG was 0.42 (95% confidence interval: 0.25, 0.84, P = .014). Taken together, these results indicate that XRCC1 is involved in the repair of melphalan‐induced DNA damage and XRCC1 rs25487 variant with impaired DNA repair function influences the clinical responses of HDM in MM patients.

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Introduction: Melphalan is an interstrand cross-link (ICL)-inducing agent and, in the setting of autologous stem cell transplantation for multiple myeloma, is one of the most effective treatments, providing 30 months of disease stability on average, but with a dramatic progression free survival (PFS) range of 6 months to 12 years. While 200 mg/m2 is the standard dose, there is extensive interpatient variability, and individual melphalan sensitivity with the ability to repair double-stand breaks in primary myeloma cells mirrored by a similar efficiency in peripheral blood mononuclear cells (PBMCs) (Gkotzamanidou M et al, Leukemia and BJC, 2013 and 2014). Our hypothesis is that our PK model will predict > 85% of interpatient variability, AUC achieved as well as measurements of DNA damage ex vivo will correlate with mucositis and duration of neutropenia, and we will be able to create an integrated model to personalize melphalan dosing to maximize myeloma cell killing while minimizing toxicities. Methods: We enrolled 146 patients on a prospective trial using a block randomization scheme based on fat-free mass, calculated creatinine clearance, and hemoglobin as known factors affecting melphalan disposition. Plasma was collected from all patients to assess melphalan pharmacokinetics, and PBMCs were collected to assess ex vivosensitivity to melphalan therapy, including p53 gene expression and WST-1-based cytotoxicity. DNA from PBMCs was collected to assess the presence of SLC7A5 polymorphisms associated with melphalan-induced enteritis (Giglia JL et al, BBMT, 2014). PK/PD modeling and identification of covariates contributing to observed variability in melphalan disposition and outcomes is being achieved using a nonlinear mixed effects approach Results: Melphalan has been quantified in samples from 119 patients, and this data was used in population modeling. Estimated PK parameters (CV%) for the model were ӨCL=0.445 L/min (33.6%), ӨV1=19.4 L (36.6%), ӨQ=0.392 L/min (34.5%) and ӨV2=17.4 L (36.9%). Creatinine clearance (normalized to 70 kg), hematocrit, fat free mass on CL, sex on V1, and body surface area on Q were chosen for the final covariate model. IC50value (CV%=31.2%), and the baseline of p53 mRNA level (47.3%) and viability (20.1%) in donor’s PBMCs were variable. Response of p53 mRNA expression and viability in donor’s PBMCs were dose-dependent. PD modeling, and covariate and SCLA5 polymorphism analyses are underway. Conclusions: This study represents the largest and most comprehensive for identifying variables associated with melphalan pharmacokinetics and pharmacologic activity. An integrated PK/PD model that incorporates PK/PD and other predictive factors form the basis of a prospective randomized clinical trial to validate that the model reduces toxicities while prolonging PFS. Disclosures Hofmeister: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millenium: Honoraria, Research Funding; ARNO therapeutics: Research Funding; Onyx: Membership on an entity's Board of Directors or advisory committees.