Myeloma Survival Research Articles

Trends in survival from myeloma, 1990\u20132015: a competing risks analysis

BackgroundMyeloma survival has greatly increased over past decades. We investigated trends in survival over time in New Zealand by age, ethnicity, and geography and thus examined potential inequalities among these population subgroups.MethodsFrom data supplied by the New Zealand Ministry of Health, all new diagnoses of multiple myeloma (ICD-10 code C90) between 1990 and 2016 were extracted, as well as their matched mortality data. Cox’s proportional hazards regression and competing risks regression were used to estimate multivariable survival functions.ResultsBetween 1 January 1990 and 1 December 2015, 6642 myeloma cases were registered by the New Zealand Cancer Registry. Although survival from myeloma increased substantially from 1990–1994 to 2010–2015, 5-year survival was still only about 60% in 2010–2015. The greatest improvement in survival was for people aged 60–69 years at diagnosis. Using Cox’s proportional hazards regression, Māori showed an increased risk of myeloma death but this was predominantly due to differences in competing risks among ethnic groups. Competing risks analysis found the greatest improvement in myeloma survival in Pacific Islanders, and in 2010–2015 Māori had better survival than other ethnicities. Myeloma survival improved significantly over time in all regional health authorities but in all time periods the Central and Southern regions had significantly poorer survival than the Midland region.ConclusionsImprovements in myeloma survival have been unequal across subgroups and regions in New Zealand. Detailed information about utilization of chemotherapeutic agents and transplantation in New Zealand is not available. This information, as well as more detailed hematological data, is essential to further explore the relationships and reasons for differing myeloma survival in population subgroups of New Zealand.

Abstract LB217: ROBO1 promotes homing, dissemination, and survival of multiple myeloma within the bone marrow microenvironment

Abstract Introduction: The bone marrow (BM) niche promotes multiple myeloma (MM) growth, survival and drug resistance. Therapies targeting both cancer cells and the microenvironment are highly effective. We were interested in identifying novel signaling pathways supporting MM pathogenesis through MM-BM crosstalk. The transmembrane receptor Roundabout 1 (ROBO1) plays a role in growth and dissemination of solid tumors, however its function in MM is unknown. Material and Methods: We analyzed ROBO1 expression in cell lines and primary samples via western blot, immunohistochemistry (IHC) and gene expression profiling. We used short hairpin RNA and CRISPR-Cas9 for ROBO1 knock down (KD) and knock out (KO), respectively. For protein structure-function and rescue studies, we stably expressed full-length (FL) or mutant ROBO1 devoid of extracellular (Cyt) or intracellular domain (DeltaCyt), including patient-derived G674* truncation, with a C-terminus FLAG tag. We used a 3D hydrogel encapsulation system to study proliferation; FLAG immunoprecipitation (IP) followed by mass spectrometry to identify ROBO1 interacting partners; and immunofluorescence to detect ROBO1 localization. To study tumor growth in vivo, we performed PET-CT of mice inoculated subcutaneously or intramedullary with WT or ROBO1 KO MM cells and retrieved tumors for RNA sequencing. To study dissemination and homing, KO and FL addback MM cells were injected intravenously in SCID mice. Mice were monitored for development of tumors or hindlimb paralysis and femora/tumors harvested once mice reached endpoint. Results: ROBO1 is highly expressed in MM cell lines and primary cells but low/absent in normal plasma cells and other hematologic cancer cell lines. ROBO1 KD is specifically cytotoxic for MM cells and ROBO1 KO decreases proliferation, a phenotype fully rescued by FL ROBO1. Compared to WT, ROBO1 KO significantly decreases intramedullary (mean tumor volume (MTV): 1323 vs 457 mm3, p value= 0.02) and extramedullary (MTV: 2684 vs 823 mm3, p value= 0.001) tumor growth in vivo. We further discovered that ROBO1 KO decreases adhesion of MM to BM endothelial and BMSC, which is fully rescued by FL ROBO1. In a disseminated mouse model, ROBO1 KO cells generate bone plasmacytoma with reduced BM invasion, as compared to the extensive BM infiltration observed with ROBO1 FL cells. Consistently, in primary samples from patients, we detected ROBO1 expression only in 1 out of 10 solitary plasmacytoma (dim staining) as compared to 14 out of 14 MM bone marrow biopsy samples tested (11 strong, 3 dim, p value= 0.0001). Mechanistically, we show for the first time that ROBO1 C-terminus is cleaved in a ligand-independent fashion; translocates to the nucleus; and is necessary and sufficient to rescue ROBO1 KO proliferative defect. Viceversa, mutants lacking the cytoplasmic domain, including the G674* truncation, act dominantly negative. Interactomic and RNA sequencing studies point to a previously unknown function of ROBO1 in RNA processing, setting the bases for future studies. Conclusions: We show for the first time that ROBO1 is necessary for MM growth and homing to the BM. Cleaved ROBO1 cytosolic domain translocates to the nucleus and is necessary and sufficient to rescue ROBO1 KO proliferative defect, possibly by participating in RNA processing. These data suggest that ROBO1 C-terminus may be a novel molecular target in MM. Citation Format: Giada Bianchi, Peter G. Czarnecki, Matthew Ho, Aldo M. Roccaro, Antonio Sacco, Yawara Kawano, Annamaria Gulla, Anil Aktas Samur, Tianzeng Cheng, Kenneth Wen, Yu-Tzu Tai, Maria Moscvin, Xinchen Wu, Gulden Camci-Unal, Matteo Claudio Da VIa, Niccolo Bolli, Ruben D. Carrasco, Irene M. Ghobrial, Kenneth C. Anderson. ROBO1 promotes homing, dissemination, and survival of multiple myeloma within the bone marrow microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB217.

Abstract 2628: Racial differences in the impact of socioeconomic status on cancer-specific survival in multiple myeloma

Abstract Background: Multiple myeloma (MM) incidence and outcomes differ across racial/ethnic groups in the United States. Interactions between socioeconomic status (SES) with ethnicity in MM incidence and survival outcomes are not well understood. Our objective was to evaluate disparities in cancer-specific survival of patients diagnosed with MM by race/ethnicity. Methods: We conducted a population-based retrospective cohort study of patients ages 20+ years diagnosed with MM between 2000 and 2015 using Surveillance, Epidemiology and End Results, Census Tract-level SES and Rurality Database. SES was defined using the National Cancer Institute's time-dependent composite score developed by Yost et al. (2001). Yost index quintiles were where the 1st and 5th quintiles representing the lowest and highest SES categories respectively. Cumulative incidence functions were used to analyze cancer-specific survival across strata racial/ethnic and SES and equality of functions was determined using Gray's test. Subdistribution hazard ratios (SHR) and 95% confidence intervals (CI) were calculated using the Fine and Gray regression models adjusted for age, sex, year of diagnosis, marital status, insurance status, and treatment with chemotherapy. Race-specific risk estimates were stratified by age (<65 and 65+ years). Results: Overall, 58,095 MM patients were included in our analysis among whom 63.0% were non-Hispanic White, 19.5% were Black, 0.3% were American Indian/Alaskan Native, 5.8% were Asian/Pacific Islander and 11.4% were Hispanic. Compared to White MM patients (median age 69 years), Black (64 years), American Indian/Alaskan Native (64 years), Asian/Pacific Islander (67 years) and Hispanic (64 years) patients were younger on average. A higher proportion of Black (42.8%) and Hispanic (27.9%) MM patients were in the lowest SES quintile compared to White (10.6%), American Indian/Alaskan Native (15.2%), and Asian/Pacific Islander (8.9%) MM patients. Cumulative incidence functions for cancer-specific survival were significantly different across SES quintiles (P < 0.0001) and racial/ethnic groups (P < 0.0001). Overall, MM patients in the lowest SES quintile had a significantly increased risk of MM-specific mortality (SHR: 1.28, 95% CI 1.21-1.36) compared to patients in the highest quintile. Risk estimates comparing the lowest to the highest quintile of SES were higher among Black (SHR 1.39, 95% CI 1.08-1.77), Hispanic (SHR: 1.78, 95% CI 1.21-2.63), and White (SHR 1.44, 95% CI 1.23-1.69) MM patients ages <65 years at diagnosis. Conclusion: Low SES level is independently associated with poor MM-specific survival. However, the impacts of SES on MM-specific survival differ by race/ethnicity and age with the greatest increased risk observed in younger Black, Hispanic, and White patients. Citation Format: Huiwen Deng, Ashwini Zolekar, Hsiao-Ching Huang, Mary H. Smart, Colin C. Hubbard, Brian C. Chiu, Pritesh R. Patel, Karen Sweiss, Gregory S. Calip. Racial differences in the impact of socioeconomic status on cancer-specific survival in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2628.

A Novel Phthalimide-Derivative, TC11, Induced G2/M Arrest and Apoptosis of High-Risk Myeloma Cells By Interacting with Nucleophosmin1 and α-Tubulin, but Not By Binding to Celeblon

Background and Purpose:Emergence of novel drugs including immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide and pomalidomide have markedly improved the survival of multiple myeloma (MM) patients. However, IMiDs have substantial limitation in the treatment of MM; little effect on MM patients with high-risk cytogenetic changes and their teratogenicity. Recently it was reported that IMiDs showed anti-tumor activity as well as teratogenicity by directly binding to cereblon (CRBN).We have previously reported that a novel phthalimide-derivative, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3-dione (TC11), induced apoptosis of high-risk MM cells in vivo and also showed anti-osteoclastogenic activity. At the same time, we observed abnormal α-tubulin fragmentation in TC11-treated MM cell (Matsushita et. al. PLoS One 2015). We have also demonstrated that TC11 bound to nucleophosmin 1(NPM1) and α-tubulin using in vitro virus (IVV) method (Shiheido et. al. PLoS One 2012). Interestingly, lenalidomide also bound to NPM1. The purpose of this study is to clarify molecular mechanisms of anti-myeloma effects of TC11.Results and Discussion:(1) Effect of TC11 is CRBN-independent:Recently it was reported that lenalidomide bound to CRBN, resulting in degradation of its substrates, IKZF1/3 and CK1α. In this study, we found that lenalidomide caused degradation of IKZF1/3 and CK1α in the high-risk MM cell line, KMS34. However, TC11 didn't down-regulate CRBN or its substrates, IKZF1/3 and CK1α. Taken together with the result of Biacore assay, TC11 induced MM cell death via CRBN-independent pathway.(2) Cell cycle arrest by TC11 vs. lenalidomide:Then we tried to elucidate molecular mechanisms of TC11 by the microarray analysis of KMS21, which is highly sensitive to TC11 and lenalidomide. Microarray Gene Set Enrichment Analysis (GSEA) indicated that TC11 upregulated the gene set of G2/M checkpoint, however lenalidomide did not. These results were also confirmed by analyzing the DNA contents of MM cells using flow cytometry and by western blot analysis of cell cycle regulators, phosphorylated cdc2 and p21. These data suggest that TC11 induced apoptosis of MM cells by distinct molecular mechanism from lenalidomide probably by regulating G2/M cell cycle. TP53 gene regulates G1/S cell cycle and occasionally deleted in high-risk MM cells. However, TC11 is able to induce apoptosis of TP53 -deleted high-risk MM cells by causing G2/M arrest.(3) Inhibition of tubulin assembly by TC11:Since we have previously showed that TC11 bound to α-tubulin by IVV method, we speculated that TC11 affected tubulin polymerization or depolymerization. Treatment of MM cells with TC11 significantly reduced polymerized fraction of α-tubulin, indicating TC11 inhibited tubulin polymerization. In contrast, lenalidomide did not inhibit either tubulin polymerization or depolymerization. In this regard, the mechanism of TC11 is different from that of lenalidomide.(4) Inhibition of oligomerization of NPM1 by TC11:We have also showed that TC11 bound to NPM1 by IVV method (Shiheido et. al. PLoS One 2012). It has been reported that oligomerized NPM1 had pleiotropic biological functions. Thus we examined whether TC11 affected NPM1 oligomerization using blue native polyacrylamide gel electrophoresis. As a result, TC11 treatment significantly reduced the amount of oligomerized NPM1 fraction. At the same time, we found that TC11 significantly increased in phosphorylation of NPM1 at Ser4, Thr95 and Thr199. It has been reported that phosphorylation of NPM1 inhibited its oligomerization (Mitrea et. al. Proc Natl Acad Sci U S A 2014). Thus, we speculated that TC11-induced phosphorylation resulted in inhibition of oligomerization of NPM1.Conclusion:It was shown that TC11 bound to α-tubulin and NPM1, but not to CRBN. TC11 inhibited tubulin assembly as well as NPM1 oligomerization, leading to G2/M arrest and mitotic catastrophe. These novel molecular events by TC11 induced apoptosis of TP53 -deleted high-risk MM cells. [Display omitted] DisclosuresYonemura:IDAC Theranostics Inc.: Employment. Tabata:IDAC Theranostics Inc.: Employment. Yanagawa:IDAC Theranostics Inc.: Employment. Hattori:Cosmic Corporation Co., Ltd.: Research Funding; Merck Sharp & Dohme: Research Funding; Takeda Pharmaceutical Company Limited.: Research Funding.

Development of a Prognostic Model That Predicts 3- and 5-Year Overall Survival in Multiple Myeloma Using the Connect® MM Patient Registry

Introduction: Novel therapies have improved the median overall survival (OS) for newly diagnosed multiple myeloma (NDMM). In an unselected population of NDMM patients, the estimated rates of 5-year OS were 77%, 53%, and 19% for revised international staging system (R-ISS) Stage I, II, and III, respectively (Kastritis, 2017). However, the improvements have not been uniform, and prognosis varies considerably based on several baseline characteristics. Because of this clinical heterogeneity it is helpful to have a clear idea of risk factors involved in OS of patients with NDMM. Connect MM is the first and largest US-based, multicenter, prospective observational registry of patients with NDMM. Using data from the Connect MM registry, an unselected patient population reflective of the heterogeneity seen in routine clinical practice, we developed a prognostic tool to identify factors predictive of 3- and 5-year OS.Methods: The Connect MM registry is designed to describe baseline characteristics, patterns of care, and outcomes for NDMM in clinical practice. Patients were enrolled in Cohort 1 (n = 1493) between Sept 2009 and Dec 2011. Enrollment for Cohort 2 (n = 1518) began Dec 2012 and was completed April 2016. Data cut-off was July 7, 2016. Multivariate analyses of patient- and disease-related characteristics were conducted using Cohort 1 data to identify key predictors of OS. A series of univariate Cox regression models were used to identify variables significantly associated with 3-year and 5-year OS in patients ≤75 years and >75 years, as 75 years was determined to be the most discriminating age cut point, using Connect MM data, in identifying independent factors significantly associated with 3-year and 5-year OS. Prediction matrices for OS were constructed from the Cox model. Models were evaluated using the concordance (C)-index, a goodness of fitness measure. Internal validation of the logistic model in predicting the probability of survival was performed on data from Cohort 1. External validation of the model was performed on data from Cohort 2 and the Celgene-sponsored randomized phase III NDMM trials, MM-015 (N = 459) and FIRST (N = 1623).Results: Characteristics associated with late survival identified by multivariate analyses (P < 0.05) were: age, EQ-5D mobility, del(17p), ISS stage, platelet count, solitary plasmacytoma, Eastern Cooperative Oncology Group performance status, history of diabetes, creatinine category, and triplet therapy use (Figure). The C-index results were robust and consistent in the external validations: Connect MM Cohort 2 (C-statistic = 0.698), MM-015 (C-statistic = 0.649) and MM-020 (C-statistic = 0.647), thereby confirming model validity.Conclusions: This is the first assessment of long-term survival using data from the Connect MM patient registry. Our study highlighted factors known to impact OS, including, mobility, del(17p) and ISS stage. We have developed a model that allows for robust prediction of long-term survival in patients with NDMM based on a variety of baseline characteristics. For clinicians, identifying patients with an increased likelihood of longer survival may be a useful tool to identify patient specific risk factors to better inform treatment decisions. [Display omitted] DisclosuresTerebelo:Celgene: Consultancy; Janssen: Speakers Bureau; Takeda: Speakers Bureau; Pharmacyclics: Speakers Bureau. Abonour:Celgene: Other: Steering committee, Research Funding; Takeda: Other: Steering committee, Research Funding; Prothena: Research Funding. Gasparetto:Janssen, BMS, Celgene, Takeda: Honoraria; Janssen, BMS, Celgene: Other: Travel, accommodations, or other expenses paid or reimbursed; Celgene: Research Funding; Janssen, BMS, Celgene: Consultancy. Toomey:Celgene: Consultancy; Myriad Genetics: Speakers Bureau; Dava Oncology: Other: Travel. Durie:Takeda: Consultancy; Janssen: Consultancy. Hardin:Celgene: Consultancy. Jagannath:Celgene: Consultancy; Merck: Consultancy; Novartis: Consultancy; Bristol-Meyers Squibb: Consultancy; Medicom: Speakers Bureau; MMRF: Speakers Bureau. Wagner:EveryFit: Consultancy; Gilead: Consultancy; Janssen: Consultancy. Narang:Celgene: Consultancy, Speakers Bureau; Janssen: Speakers Bureau. Srinivasan:Celgene: Employment. Yue:Celgene: Employment. Zafar:Celgene: Employment. Kitali:Celgene: Employment. Agarwal:Celgene Corporation: Employment. Rifkin:Amgen: Consultancy; Boehringer Ingelheim: Consultancy; Celgene: Consultancy; EMD Serono: Consultancy; Sandoz: Consultancy; Takeda: Consultancy; McKesson: Other: Stock.

Initial Phase 2 Results of Ibrutinib Combined with Carfilzomib/Dexamethasone in Patients with Relapsed/Refractory Multiple Myeloma

Initial Phase 2 Results of Ibrutinib Combined with Carfilzomib/Dexamethasone in Patients with Relapsed/Refractory Multiple Myeloma

Infections and Immune System Impairment in Multiple Myeloma: Increasing Frequency of Serious Complications in the “Novel Agents Era”— a Retrospective Real Life Analysis

New treatments options introduced during recent decades have improved the survival of multiple myeloma(MM)patients.Managing the complications of the disease and its treatment, such as infections become more important as MM-patients survive longer. Infections are a significant cause of morbidity and a leading cause of death in MM-patients.Effect of novel therapies on the risk of infection remains to be established and new trials on prophylactic measures are needed.These advances have transformed myeloma into a chronic condition, with multiple relapses resulting in cumulative immunosuppression and higher risk of infection. In addition to the immunodeficiency related to myeloma, the type of anti-myeloma therapy used also plays a role in the development of infectious events.We aimed to study the development of severe infectious events of grade 3-4 requiring often hospitalization in a group of patients under treatment at diagnosis as well as at relapse. We evaluated the impact of the type of anti-myeloma therapy and of the disease features on patients' infectious complications in the context of novel agents.We retrospectively reviewed data from 195 myeloma patients diagnosed from 1999 to 2016 in order to assess type and outcome of infections (Table 1). 7 of these were life-threatening (3 viral interstitial pneumonias and 4 gram negative sepsis)and 103 have resulted in discontinuation of therapy(respectively 73 bacterial, 10 fungal,18 viral and 2 parasitic-infectious-complications). We focus on time of occurrence and number of prior therapeutic lines and on disease biological aggressiveness.Our aim was to define risk-factors and to organize an effective antimicrobial prophylaxis strategy in our cohort of patients.In our analysis 30 patients presented a FUO(fever of unknown origin)without isolation of pathogens.They were 19 elderly-patients and 11 young in a neutropenic phase after chemotherapy. They showed defervescence following antibiotic therapy with piperacillin-tazobactam.165 patients presented infectious complications with a well-known etiology (Figure 1): 100 bacterial, 45 viral, 15 fungal and 5 parasitic infections. Pathogens isolated were:Candida-Albicans followed by C.Parapsylosis and Aspergillus-Flavus among fungal infections,E.Coli,Klebsiella-Pneumoniae and Pseudomonas-aeruginosa between bacterial complications,CMV,HSV,HZV in viral manifestations and Leishmanias among parasitic-events.Advanced age is a meaningful risk-factor togheter with biologically aggressiveness and relapse condition. The majority of patients were older than 65 years (125 as 76%) in a relapse setting (125 as 77%).Kind of anti-myeloma therapy used also plays a role in the development of infection. Specifically the predominant part of patients developing fungal infections(13 as 89%)showed neutropenia after chemotherapy or previous therapy with Imids. The majority of patients with viral infections(39 as 87%) presented lymphopenia and previous therapies bortezomib-based.Bacterial infections have shown mostly prevalent in neutropenic-phases (88 as 88%) usually in relapse phases (78 as 78%)or in hypogammaglobulinemic patients(68 as 68%).Finally most of parasitic infections have been shown after high doses steroid treatment and with more than 2 of therapeutic lines(100%).Severe infections represent a significant comorbidity in MM, often underestimated, in all phases of the disease expecially in refractory/relapsed patients.Immunoglobulin-replacement-therapy or antibiotic-prophylaxis may possible have a protective-role in high risk old patients with high ISS stage and aggressive disease. Variety of factors underlies susceptibility to infections including defects of innate and adaptive-immunity(neutropenia,hypogammaglobulinemia).Need for antimicrobial-prophylaxis depends on risk for and seriousness of infections.Based on our experience,in the first three-months of therapy with IMIDs we now begin prophylactic antibacterial and antifungal therapy (quinolone and fluconazole).All patients treated to date(30 in total)did not require therapy-discontinuation.High-risk-patients should receive antimicrobial-prophylaxis and trials on prophylactic-measures are needed.Key to the management of infection is the understanding of the specific risk factors and periods in order to perform a risk adjusted prophylactic and treatment strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Overall Survival of Transplant Eligible Patients with Newly Diagnosed Multiple Myeloma: Comparative Effectiveness Analysis of Modern Induction Regimens on Outcome

Aims: Overall survival (OS) of multiple myeloma (MM) has improved remarkably over time, with a recent study reporting a 4-year OS rate of approximately 81.5% with or without upfront autologous stem cell transplantation (ASCT) in patients receiving modern therapy (Attal M, et al. N Engl J Med 2017; 376:1311-1320). However, these results may overestimate the current OS of MM in this population, since patients enrolled in clinical trials represent a highly selected group, and may not reflect outcomes in a more generalizable population of MM patients. In order to remove the selection bias inherent in clinical trial enrollment, we studied the outcome of all newly diagnosed MM patients eligible for ASCT seen at the Mayo Clinic during the time period corresponding to the IFM trial. We also performed a comparative effectiveness analysis of bortezomib, lenalidomide, dexamethasone (VRD) (the regimen used in the IFM trial) and other induction regimens used during this period at our institution.Methods: We reviewed electronic medical records to identify all patients with MM 65 years of age and younger who were seen at the Mayo Clinic between January 1, 2010 and August 31, 2015, and had a stem cell harvest performed within 12 months of initial diagnosis. We excluded patients with baseline serum creatinine >2gm/dL. Data on baseline characteristics, cytogenetics, initial therapy, ASCT, date of progression, and relevant prognostic variables were analyzed. OS was estimated using Kaplan Meier method. Differences between survival curves were tested for statistical significance using the two-sided log-rank test.Results: 488 patients (267 men and 221 women) were studied. The estimated median follow-up duration was 46 months. Median age was 57.8 years (range, 32.8-64.9). Initial therapy consisted of VRD (n=126); bortezomib, cyclophosphamide, dexamethasone (VCD)(n=138); lenalidomide, dexamethasone (Rd)(n=142); bortezomib, dexamethasone (n=36); ixazomib, cyclophosphamide, dexamethasone (ICD) (n=15); carfilzomib, lenalidomide, dexamethasone (KRD)(n=8); ixazomib, lenalidomide, dexamethasone (IRD)(n=3); and other regimens (n=20). The median OS has not been reached. The 4 year OS rate of the whole cohort was 80.6%; 5 year OS rate was 74.1%. FISH results for mSMART risk stratification were available in 369 patients; 270 were standard risk (73.1%); 48 intermediate risk (13%); and 51 high risk (13.8%). The 4 year OS rates for standard, intermediate, and high risk MM were 83.7%, 58.2%, and 69.8%, respectively (P=0.008). The 4 year OS rate for the 3 most commonly used regimens VRD, VCD, and Rd were 77.4%, 78.6%, and 85.2%, respectively (P=0.14). However, the proportion of patients treated with VRD, VCD, and RD who had high risk disease was different; 32.7%, 9.4%, and 5.8%, respectively. Similarly, the proportion of patients treated with VRD, VCD, and RD who had intermediate risk disease was 11.2%, 18.7%, and 2.9%, respectively. Adjusting for mSMART risk, P value for the comparison of OS between the 3 regimens remained non-significant, P=0.21. OS was then compared after pooling similar regimens; the 4 year OS rates for VRD/IRD/KRD (IMid/PI combination)(n=137), VCD/ICD (PI/Alkylator combination)(n=153), and RD/VD (doublet regimen)(n=178) were 78.4%, 78.6%, and 82.4%, respectively, P=0.61. As expected, the use of regimens varied by risk stratification. The 4 year survival rates for the 3 regimens stratified by mSMART risk classification are shown in the Table. Among patients with high risk MM (n=50), 34 received IMiD/PI, 10 PI/Alkylator, and 6 doublet. Similarly, among patients with intermediate risk MM (n=44), 14 received IMiD/PI, 24 PI/Alkylator, and 6 doublet.Conclusions: The 4 year OS rate of consecutive MM patients 65 years of age and younger eligible for ASCT treated at a single institution was 80.6%, and is comparable to results achieved in a contemporaneous randomized trial. This is a benchmark for this patient population; benchmark 4 year OS rates for standard, intermediate, and high risk MM are 83.7%, 58.2%, and 69.8%, respectively. Outcome of high risk MM has improved considerably during this time period. Comparative effectiveness analysis shows near identical outcomes (83-84% 4-year OS rate) in standard risk patients receiving common MM regimens. [Display omitted] DisclosuresKumar:Skyline: Honoraria; Celgene, Millennium, BMS, Onyx, Janssen, Noxxon, AbbVie, Amgen, Merck, Oncopeptides, Skyline Diagnostics, Takeda: Consultancy; Celgene, Millennium/Takeda, Onyx, AbbVie, Janssen, Sanofi, Novartis, Amgen, Genentech, Merck, Oncopeptides, Roche, Skyline Diagnostics: Research Funding. Gertz:Celgene, Novartis, Smith-Kline, Prothena, Ionis, Amgen: Honoraria; Millennium: Consultancy, Honoraria. Dispenzieri:Celgene, Millenium, Pfizer, Janssen: Research Funding. Dingli:Karyopharm Therapeutics: Research Funding; Alexion Pharmaceuticals: Consultancy; Millenium: Consultancy; Takeda: Consultancy; Janssen: Consultancy. Russell:Vyriad: Equity Ownership; Imanis Life Sciences: Equity Ownership. Kapoor:Takeda, Celgene and Amgen: Research Funding.

Copper-64-Labeled Daratumumab As a PET Imaging Tracer for Multiple Myeloma: Potential Use for Minimal Residual Disease Detection

Introduction : Minimal residual disease (MRD) is known to be an independent predictor of progression free survival (PFS) in multiple myeloma (MM). Methods of MRD detection include flow cytometry, PCR based sequencing, and imaging. However, irrespective of the sensitivity of MRD detection, techniques based on bone marrow biopsies cannot account for a “patchy,” non-homogenous bone marrow infiltration. Positron emission tomography (PET) coupled with computed tomography (CT) using 18fluoro-deoxyglucose as the radiotracer (18FDG-PET) is a commonly used imaging method that is much more sensitive than traditional radiographs, but it has several limitations; for one, lesions with a low metabolic rate may result in false negatives. Also, diffuse tracer uptake by normal marrow can obscure focal MM lesions. Daratumumab (Dara), a human anti-CD38 IgG1 (ĸ subclass) antibody against the highly expressed plasma cell receptor CD38, has been recently approved by the FDA for the treatment of MM. Based on the idea that Dara can selectively target MM cells irrespective of their metabolism, our group labeled Dara with the positron-emitting isotope copper-64 (64Cu-DOTA-Dara) in order to generate a novel imaging agent that can detect MM cells with high specificity. Our group already safely used 64Cu-labeled trastuzumab to detect HER2-positive tissue in breast cancer patients (Mortimer JE et. al. 2014), and here we provide the first pre-clinical evidence that 64Cu-labeled antibodies can be considered powerful imaging tools for MM detection.Methods :Commercially availableDara was conjugated with the NHS active ester of DOTA (Macrocyclics). DOTA-conjugated antibody was incubated with 64Cu, chased with 1 mM diethylenetriamine pentaacetic acid (DTPA), and purified on a preparative size-exclusion column. The immunoreactivity of 64Cu-DOTA-Dara was also performed using purified CD38-Fc (Chimerigen Laboratories) in an HPLC size shift assay. To assess whether 64Cu-DOTA-Dara can localize to MM cells in vivo, 5 x 106 human MM1S GFP+/Luc+ cells were injected intravenously into 6 NOD-SCID mice. At 14 days, when the bioluminescence signal in the bones of the mice were detected, mice were injected with 64Cu-DOTA-Dara (50 µCi, 10 µg), and PET imaging was performed at 21 and 44 hr after the injection (Figure 1A). Cancer free mice were used as controls (Figure 1B). 18F-FDG PET/CT scans in the same group of mice were also performed. Biodistribution studies and histological analysis were also conducted.Results : 64Cu-DOTA labeling did not affect Dara immunoreactivity against the antigen in the HPLC size shift assay. The biodistribution studies show that 64Cu-DOTA-Dara was highly detectable in the femurs, tibias, sternum and skull, known sites of MM cell invasion, which were negative in cancer-free control mice. The radionuclide was also highly detected in the lung, liver, and spleen, relative to controls. In contrast, the majority of 64Cu-DOTA-Dara remained in the blood of the control mice. Moreover, PET images show that 64Cu-DOTA-Dara PET/CT displays a higher resolution and specificity in detecting MM cells in the bones not only compared to 18F-FDG PET/CT, but even compared to bioluminescence signals from the MM cell line. To assess the specificity of 64Cu-Dara signal compared to 18F-FDG, studies using step sections in histological analysis are in process.Conclusions : Our data support the use of 64Cu-DOTA-Dara as a novel sensitive imaging agent to detect MM. The relatively short half life of 64Cu (12.7 hours) limits potential marrow toxicity and is sufficiently long for PET/CT imaging up to 48 hours after administration of the radionuclide. A pilot study assessing the safety and feasibility of 64Cu-DOTA-Dara imaging in human subjects is under way. [Display omitted] DisclosuresKeats:Amgen: Research Funding. Krishnan:Takeda: Speakers Bureau; Onyx: Speakers Bureau; Sutro: Consultancy; Celgene: Consultancy, Equity Ownership, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

Real-World and Clinical Trial Data in Relapsed/Refractory Multiple Myeloma (RRMM): Evaluating Treatment Duration and Comparing Effectiveness and Efficacy

Real-World and Clinical Trial Data in Relapsed/Refractory Multiple Myeloma (RRMM): Evaluating Treatment Duration and Comparing Effectiveness and Efficacy

Hypoxia-Inducible KDM3A Addiction in Multiple Myeloma

Background: In the bone marrow (BM) microenvironment, there is a myeloma cell fraction that has acquired chemo-resistance via epigenetic gene expression change. Hypoxic stress is an important factor constituting the BM microenvironment. Recently, we demonstrated that hypoxia-induced microRNA-210 suppressed a ribosomal RNA demethylase, leading to downregulation of IRF4, which is an essential factor in myeloma oncogenesis (Ikeda et al., Cancer Sci 2017). However, myeloma cells showed an anti-apoptotic phenotype without IRF4 under hypoxia, suggesting that there might be another anti-apoptosis factors.Methods: To determine new anti-apoptosis factor(s) under hypoxia, we conducted gene expression analysis on multiple myeloma (MM) samples (n=4) and cell lines (RPMI-8226, KMS-11, KMS-12-BM, MM.1S, and U266) that cultured under hypoxia (1% O2) for 48 hours. Then, we identify hypoxia-induced gene(s) and conducted functional analysis of the gene(s).Results: We found that significantly upregulation of glycolytic genes and histone demethylases. Among these, upregulation of H3K9 demethylases, KDM3A and KDM7A were found in all examined cells. We examined a western blot analysis for KDM3A and KDM7A in normoxia- and hypoxia-subjected myeloma cell lines and found that KDM3A showed a stronger increase in hypoxia than that of KDM7A. This result suggested that KDM3A has more potent function in chronic hypoxia than KDM7A does. We confirmed significant upregulation of KDM3A in 15 MM samples cultured in hypoxia (1% O2) for 48 hours by q-RT-PCR. Expression of KDM3A was HIF-1α dependent but not HIF-2α. We found that expression of KDM3A and HIF1A showed a positive correlation in myeloma patient samples. Apoptosis was increased in KDM3A-knockdowned myeloma cells under chronic hypoxia (1% O2) for 72-96 hours. However the target of KDM3A is reported to be IRF4, KDM3A accumulates while IRF4 is suppressed under hypoxia. Therefore we examined the new target gene of KDM3A under hypoxia. We found that the most candidate target of KDM3A under hypoxia was MALAT1. This long non-coding RNA (lncRNA) induces the expression of glycolytic genes such as SLC2A1, PFKFB3, and PFKFB4 via accumulation of HIF-1α. Although IRF4 is crucial factor for myeloma survival under normoxia, it may be switched by HIF-1α-KDM3A-MALAT1 loop under hypoxia (Figure). Activation of this loop contribute to acquiring anti-apoptotic phenotype under hypoxia via upregulation of glycolytic genes.Conclusion: We conclude KDM3A is promising as a therapeutic target molecule for myeloma cells adapted to the BM microenvironment. Development of KDM3A inhibitor is desired. [Display omitted] DisclosuresIkeda:TaNeDS (Daiichi Sankyo): Research Funding; Kyowa Kirin: Research Funding; Fujimoto: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Otsuka: Research Funding; Toyama Kagaku: Research Funding; Chugai: Research Funding; Asahi Kasei: Research Funding; Eisai: Research Funding. Kitadate:Fujimoto: Research Funding; Kyowa Kirin: Research Funding; Eisai: Research Funding; Pfizer: Research Funding; Toyama kagaku: Research Funding; Otsuka: Research Funding; Chugai: Research Funding; Asahi Kasei: Research Funding; Novartis: Research Funding. Abe:Kyowa Kirin: Research Funding; Fujimoto: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Otsuka: Research Funding; Toyama Kagaku: Research Funding; Chugai: Research Funding; Asahi Kasei: Research Funding; Eisai: Research Funding. Tagawa:TaNeDS (Daiichi Sankyo): Research Funding.

Next generation proteomics with drug sensitivity screening identifies sub-clones informing therapeutic and drug development strategies for multiple myeloma patients

With the introduction of novel therapeutic agents, survival in Multiple Myeloma (MM) has increased in recent years. However, drug-resistant clones inevitably arise and lead to disease progression and death. The current International Myeloma Working Group response criteria are broad and make it difficult to clearly designate resistant and responsive patients thereby hampering proteo-genomic analysis for informative biomarkers for sensitivity. In this proof-of-concept study we addressed these challenges by combining an ex-vivo drug sensitivity testing platform with state-of-the-art proteomics analysis. 35 CD138-purified MM samples were taken from patients with newly diagnosed or relapsed MM and exposed to therapeutic agents from five therapeutic drug classes including Bortezomib, Quizinostat, Lenalidomide, Navitoclax and PF-04691502. Comparative proteomic analysis using liquid chromatography-mass spectrometry objectively determined the most and least sensitive patient groups. Using this approach several proteins of biological significance were identified in each drug class. In three of the five classes focal adhesion-related proteins predicted low sensitivity, suggesting that targeting this pathway could modulate cell adhesion mediated drug resistance. Using Receiver Operating Characteristic curve analysis, strong predictive power for the specificity and sensitivity of these potential biomarkers was identified. This approach has the potential to yield predictive theranostic protein panels that can inform therapeutic decision making.

Improved survival in multiple myeloma during the 2005–2009 and 2010–2014 periods

Improved survival in multiple myeloma during the 2005–2009 and 2010–2014 periods

How to Treat High-Risk Myeloma at Diagnosis and Relapse.

Survival in multiple myeloma has improved greatly during the past 2 decades, but this change has primarily benefited patients who have standard-risk disease. Patients with high-risk disease remain a challenge to diagnose and treat. To improve their clinical outcomes, it is imperative to develop tools to readily identify them and to provide them with the most effective available treatments. The most widely used stratification system, the revised International Staging System, incorporates serum β-2 microglobulin, albumin, lactate dehydrogenase, and high-risk chromosomal abnormalities [del(17p), t(4;14), and t(14;16)]. Recent updates have included mutational status and chromosome 1q abnormalities. Plasma cell leukemia, extramedullary disease, circulating plasma cells, renal failure, and frailty are also associated with poor outcome. The treatment approach for a newly diagnosed patient with high-risk multiple myeloma should include induction therapy, autologous stem cell transplantation if appropriate, and maintenance therapy. Triplet therapy with a proteasome inhibitor, immunomodulatory drug, and steroid, with or without an anti-CD38 antibody, should be considered for induction, along with a proteasome inhibitor and/or immunomodulatory drug for maintenance. Aiming for a deep and sustained response is important. Similar principles apply at relapse, with close monitoring of response, especially extramedullary disease and active management of side effects, so that patients can continue therapy and benefit from treatment. Immune-based therapies, including autologous CAR T-cell-based therapies and bispecific antibodies, show promising activity in relapsed disease and are being actively explored in earlier disease settings. As the prognosis for high-risk disease remains poor, the future goal for this patient group is to develop specific clinical trials to explore novel approaches and therapies efficiently.

5-Hydroxymethylcytosines in circulating cell-free DNA and overall survival in patients with multiple myeloma.

8032 Background: The epigenetic mark 5-methylcytosines (5mC) have been associated with poor prognosis and survival in multiple myeloma (MM), but the prognostic role of 5-hydroxymethylcytosines (5hmC) as marks of tissue-specific enhancers generated from 5mC through active demethylation is unknown. We showed that 5hmC can be profiled in circulating cell-free DNA (cfDNA) and is associated with relapse/death in another lymphoproliferative disorder diffuse large B-cell lymphoma. To date, no study has investigated genome-wide 5hmC profiles in cfDNA for its prognostic significance in MM. Methods: A total of 354 newly diagnosed MM patients at the University of Chicago Medical Center were prospectively enrolled between 2010-2017. Blood samples were collected at the time of diagnosis. Patients were followed through 31 December 2020 (avg. follow-up = 77.8 mths). We collected baseline clinical, laboratory, and cytogenetic data from electronic medical records. Vital status was ascertained in 351 of the 354 patients (deaths = 73) using the National Death Index. We profiled genome-wide 5hmC in cfDNA using the 5hmC-Seal and next-generation sequencing. The 5hmC-Seal data were mapped to the human genome reference (hg19) and annotated to gene bodies. Overall survival (OS) was defined as time from diagnosis until death from any cause. We used Cox proportional hazards model and the elastic net regularization to identify genes with modified 5hmC levels that are associated with OS. Patients were randomly divided into a training set (n = 264) and testing set (n = 87). Results: The median age at diagnosis was 61.8 years and 47% (n = 165) were males. We used the differential 5hmC enrichment levels of a preliminary four-gene marker panel (i.e., YPEL1, VIPR2, PLAC8L1, and CYP2D6) to compute a weighted prognostic score (wp-score). In the training set (deaths = 55), MM patients with high wp-score had worse OS (Hazard Ratio [HR] = 2.2, 95% Confidence Interval [CI]: 1.3-3.9; p = 0.004). The same trend was observed in the testing set (deaths = 18) (HR = 3.5, 95% CI: 1.1-10.6; p = 0.02). The 5hmC-based wp-score remained significantly associated with OS after controlling for standard prognostic factors, suggesting that 5hmC-based wp-score for this 4-gene panel is an independent prognostic factor for MM. We also explored population-specific 5hmC and wp-score. We found that 5hmC profiles in cfDNA differ between Blacks (n = 117) and Whites (n = 234). In addition, 5hmC marker genes associated with OS differ between Blacks (13 genes) and Whites (20 genes). Conclusions: Our results suggest that 5hmC in cfDNA at the time of diagnosis correlate with OS in MM and the 5hmC marker genes associated with OS differ between Blacks and Whites. These findings suggest that a plasma-derived cfDNA 5hmC-modified gene panel holds promise as a noninvasive approach for predicting prognosis in MM and may be integrated in clinical practice to improve precision care.

Second primary malignancies (SPM) in African American (AA) and white patients with multiple myeloma in the National Veterans Affairs (VA) healthcare system.

10507 Background: In the recent decade, novel therapies have led to significant improvements in overall survival (OS) in symptomatic multiple myeloma (MM). With this increase in OS, there is an increase in the incidence of second primary malignancies (SPM) in patients with MM. This is related to multiple factors; we hypothesize that racial disparities also play a role. There is a paucity of studies with large, high quality datasets evaluating racial disparities in SPMs in MM. Our goal is to explore patterns of incidence of SPMs in US Veterans with MM, focusing on racial disparities. Methods: We conducted a retrospective cohort study based on electronic health record (EHR) and cancer registry data recorded in the VA’s Corporate Data Warehouse (CDW) between January 1, 1999 and January 1, 2018. We compared incidence of SPMs (defined as malignancies occurring after the diagnosis and treatment initiation of MM) using the standardized incidence rate ratio (IRR). Univariate and multivariable analyses were performed, using a Cox model adjusting for age, race, gender, treatment era, smoking status, and stage at MM diagnosis. Results: There were 8467 Veterans (97.4% male) with MM identified with self-reported race: White, 5029 (59.4%); AA, 2178 (25.7%); and other or unknown, 1260 (14.9%). At 7.5 years of follow up, 430 (5.1%) MM patients developed SPM while 8037 did not. 982 had received HSCT (Hematopoietic Stem cell transplant) of whom 65 (6.62%), a significantly higher proportion developed SPM versus those without HSCT (n = 7485) of whom 365 (4.87%) developed SPM (p = 0.024). Among those Veterans who had SPM 380 (88.3%) had solid tumors, 50 (11.6 %) had hematological malignancies. Of 88.3% with solid SPM the distribution was: prostate cancer, 113 (29.7%); digestive tract cancers, 63 (16.6%); lung cancer, 55 (14.4%); and GU (bladder/renal/testicular), 48(12.6%). The cumulative incidence of developing SPM increased steadily over time for the duration of this study period of 7.5 years from diagnosis. The median age of diagnosis for Whites was 68.2 years and for AA was 64.3 years, (p &lt; 0.001) demonstrating that MM occurred earlier in AA. The hazard ratio for AA versus whites to develop SPMs was 1.21 (0.975-1.505) (p = 0.0823). However, the risk of prostate cancer was significantly higher in AA with an adjusted hazard ratio of 1.81 (1.196-2.739) (p = 0.005). No racial disparities were observed in the incidence of other types of SPMs. Conclusions: Our study suggests that the risk of SPMs does not plateau over a patient’s lifetime. HSCT was found to be an independent predictor of SPM, while smoking and agent orange were not. Our cohort is one of the largest groups of AA with MM in published literature. AA did not have a higher incidence of SPMs overall; but had a significantly higher incidence of prostate cancer than whites. We hope to develop an individualized predictive model for SPM in patients with MM.

Effect of the Amount of Autologous Hematopoietic Stem Cells on Survival and Engraftment in Multiple Myeloma

Background: Autologous stem cell transplantation (ASCT) is currently a gold standard treatment for eligible multiple myeloma (MM) patients. The recommended dose of CD34+ hematopoietic progenitor cells (HPCs) for adequate engraftment is above 2 × 106 cells/kg. Objectives: This study aimed to evaluate the relationship between the number of CD34+ HPCs and the survival in MM patients who underwent ASCT in the Hematology Department of Abdurrahman Yurtaslan Ankara Oncology Training and Research Hospital, Ankara, Turkey. Materials and Methods: The statistical population of this consisted of 200 MM patients who underwent ASCT within 2009-2019. The clinical characteristics of the patients, disease status pre-SCT, number of infused CD34+ cells, neutrophil, and platelet engraftment days were recorded. The patients were divided into two groups, based on whether the re-infused CD34+ HPCs dose was &lt; 5 × 106 cells/kg (Group 1) or ≥ 5 × 106 cells/kg (Group 2). The groups were compared in terms of engraftment and overall survival (OS) times. Results: A total of 200 patients were included in our study. Group 1 (n=125) included patients with &lt; 5 × 106 cells/kg CD34+ HPC re-infusion, and Group 2 (n=75) consisted of patients with ≥ 5 × 106cells/kg CD34+ HPC re-infusion. The patients’ median age scores in Group 1 and Group 2 were 57 (25-71) and 56 (33-72) years, respectively. The median follow-up period was 33 months (6-130). The median OS of all patients was 71 months (95% confidence interval, 59.1-82.9). The median neutrophil and platelet engraftment times were similar between the groups (P=0.4 and P=0.4, respectively). In both groups, the median OS time was 71 months (P=0.8), which was similar. Conclusion: The greater number of CD34+ HPCs re-infusion for ASCT after high dose melphalan chemotherapy in MM patients did not affect platelet and neutrophil engraftment time and OS; therefore, this amount of reinfusion was not required.

Circular RNA_0000190 and its target microRNA-767-5p are dysregulated, and they are related to risk stratification as well as prognosis in multiple myeloma patients.

This study aimed to explore the correlation between circular RNA_0000190 (circ_0000190) and microRNA-767-5p (miR-767-5p), and their correlations with biochemical indices, risk stratification, treatment response, and survival in multiple myeloma (MM) patients. Bone marrow (BM) plasma cells of 86 MM patients (during standard diagnostic procedures) and 30 healthy donors (HDs) (examination of the eligibility for BM transplantation) were obtained, among which circ_0000190 and miR-767-5p expressions were detected using reverse transcription quantitative polymerase chain reaction. In MM patients, Durie-Salmon stage and International Staging System (ISS) stage were assessed. Clinical responses (including complete response (CR) and objective response rate (ORR)) were assessed. The progression-free survival (PFS) and overall survival (OS) were calculated. Circ_0000190 was decreased, but miR-767-5p was increased in MM patients compared with HDs. Circ_0000190 was negatively correlated with miR-767-5p in both HDs and MM patients. In MM patients, circ_0000190 was negatively correlated with ISS stage, serum creatinine, beta-2-microglobulin, and lactate dehydrogenase but was positively correlated with albumin. Whereas an opposite trend in miR-767-5p was observed. Regarding clinical response, circ_0000190 had the value for predicting increased ORR, while miR-767-5p had the value for predicting decreased CR and ORR. Circ_0000190 high expression was correlated with better PFS and OS, while miR-767-5p high expression was correlated with worse PFS and OS. Multivariate Cox's analyses revealed circ_0000190 high expression as an independent factor predicting better OS. Circ_0000190 and its target miR-767-5p are dysregulated, and they are related to risk stratification and prognosis in MM patients.

Current approaches to management of high-risk multiple myeloma.

The median overall survival in multiple myeloma is rapidly approaching 10 years; however, in nearly a fifth of patients the prognosis remains poor. Therefore, the modern-day management of myeloma patients should be individualized, with a more intense and continuous approach in these high-risk patients. This includes first-line treatment based on multi-drug combinations employing the most effective drug combinations, upfront autologous stem cell transplantation (in eligible patients with tandem transplantation being a consideration), and maintenance based on proteasome inhibitor-based combinations. This paper reviews the results of recent retrospective analyses and clinical trials, but also gives a glance into the future by presenting the ongoing trials.

ROBO1 Promotes Homing, Dissemination, and Survival of Multiple Myeloma within the Bone Marrow Microenvironment.

The bone marrow (BM) microenvironment actively promotes multiple myeloma (MM) pathogenesis and therapies targeting both cancer cells and the niche are highly effective. We were interested in identifying novel signaling pathways supporting MM-BM crosstalk. Mutations in the transmembrane receptor Roundabout 1 (ROBO1) were recently identified in MM patients, however their functional consequences are uncertain. Through protein structure-function studies, we discovered that ROBO1 is necessary for MM adhesion to BM stromal and endothelial cells and ROBO1 knock out (KO) compromises BM homing and engraftment in a disseminated mouse model. ROBO1 KO significantly decreases MM proliferation in vitro and intra- and extramedullary tumor growth, in vivo. Mechanistically, ROBO1 C-terminus is cleaved in a ligand-independent fashion and is sufficient to promote MM proliferation. Viceversa, mutants lacking the cytoplasmic domain, including the human-derived G674* truncation, act dominantly negative. Interactomic and RNA sequencing studies suggest ROBO1 may be involved in RNA processing, supporting further studies.