The Impact of CD34+ Cell Collection Yields for Autologous Transplant on Survival Outcomes in Multiple Myeloma.

INTRODUCTION: According to previous data, higher yields of stem-cells collected to support autologous transplantation may predict for improved outcomes. We aimed to assess the association between high stem-cells collection and survival outcomes in multiple myeloma (MM) MATERIALS AND METHODS: We reviewed all patients who underwent autologous transplantation for MM at our center over a 10-year period, and initially used a predefined threshold of 8 × 106/kg used in previous studies. RESULTS: Six hundred twenty-one patients were analyzed. Higher mobilization did not correlate with favorable outcomes post-transplant. The most efficient mobilizers, collecting ≥8 × 106/kg (n = 478) achieved a shorter median progression-free survival (PFS) of 24.1m versus 34.5m in patients collecting 4.5 to 8 × 106/kg (n = 129). A small group (n = 14) collecting ≤4.5 × 106/kg but minimum of 2 × 106/kg to support autologous transplantation exhibited the worst outcomes (median PFS 11.4m). Further analysis of potential confounders identified greater use of bortezomib induction in the lower mobilizers, however, sensitivity analysis in patients receiving bortezomib revealed similar results- worst outcomes to the most efficient mobilizers. CONCLUSION: Although bortezomib is not considered stem-cell toxic, it may be associated with lower stem cell collection yields. Bortezomib's efficacy at induction may partially explain the improved outcomes, however, other factors may be involved, and are discussed. We can conclude that with our large cohort and long follow-up, high stem-cell mobilization does not appear to predict for a long-term survival advantage.

Single-cell profiling of multiple myeloma reveals molecular response to FGFR3 inhibitor despite clinical progression.

Genomic characterization of cancer has enabled identification of numerous molecular targets, which has led to significant advances in personalized medicine. However, with few exceptions, precision medicine approaches in the plasma cell malignancy multiple myeloma (MM) have had limited success, likely owing to the subclonal nature of molecular targets in this disease. Targeted therapies against FGFR3 have been under development for the past decade in the hopes of targeting aberrant FGFR3 activity in MM. FGFR3 activation results from the recurrent transforming event of t(4;14) found in ∼15% of MM patients, as well as secondary FGFR3 mutations in this subgroup. To evaluate the effectiveness of targeting FGFR3 in MM, we undertook a phase 2 clinical trial evaluating the small-molecule FGFR1-4 inhibitor, erdafitinib, in relapsed/refractory myeloma patients with or without FGFR3 mutations (NCT02952573). Herein, we report on a single t(4;14) patient enrolled on this study who was identified to have a subclonal FGFR3 stop-loss deletion. Although this individual eventually progressed on study and succumbed to their disease, the intended molecular response was revealed through an extensive molecular characterization of the patient's tumor at baseline and on treatment using single-cell genomics. We identified elimination of the FGFR3-mutant subclone after treatment and expansion of a preexisting clone with loss of Chromosome 17p. Altogether, our study highlights the utility of single-cell genomics in targeted trials as they can reveal molecular mechanisms that underlie sensitivity and resistance. This in turn can guide more personalized and targeted therapeutic approaches, including those that involve FGFR3-targeting therapies.

Post Salvage Therapy Autologous Transplant for Relapsed Myeloma, Ongoing Relevance within Modern Treatment Paradigms?

BACKGROUND: Salvage transplant has been historically considered effective therapy for myeloma patients relapsing after first transplant, if they achieved adequate remission duration. However, the efficacy of novel agent combinations has called this paradigm into question. MATERIALS AND METHODS: We performed a retrospective analysis in a homogeneously treated cohort of 106 patients undergoing ASCT2 at our institution, all of whom received novel agent-based chemotherapy (immunomodulatory agent [IMiD] and/or proteasome inhibitor [PI]) for both induction and relapse. As an exploratory objective we assessed whether predictive thresholds of progression free survival post first transplant (ASCT1) for benefit post ASCT2 vary with use of IMiD maintenance post ASCT1. RESULTS: The overall response rate (ORR) was 98% post-ASCT2 and treatment-related mortality (TRM) was low at 1.8%. With a median follow-up of 26 months (range 0.5-85) from ASCT2, median overall survival (OS) is estimated at 80 months (95% CI: ≥ 49-months) and median progression-free survival after ASCT2 (PFS2) at 24 months (95% CI 19-39). PFS post first transplant (PFS1) at >/= 50 months was associated with improved OS. Predictors of PFS2 included PFS1 ≤42 months and progression on IMiD-based maintenance post- ASCT1. CONCLUSION: ASCT2 continues to offer acceptable outcomes for most patients treated within modern day treatment paradigms, with longer PFS after ASCT1 and IMiD non-refractory disease being associated with improved outcomes.

Kinetics of response to first- and second-line therapies in multiple myeloma: Assessment by both M-spikes and light chains.

OBJECTIVES: The prognostic value of kinetics of response to multiple myeloma (MM) therapy is controversial. We aimed to expand the knowledge on this topic by reviewing the kinetics of response to both first- and second-line MM therapy, utilizing a homogeneously treated cohort and analyzing separately both M-spike and light chain (LC) responses for each patient. METHODS: We reviewed all patients who received first-line cyclophosphamide, bortezomib and dexamethasone induction followed by autologous transplant with melphalan and lenalidomide maintenance in our center between 2007 and 2019. RESULTS: Analyzing 360 patients, we observed no correlation between response kinetics to first- versus second-line therapy at the individual patient level. Time to best response to first-line therapy was not a predictor of outcome; however, longer time to best response was highly predictive of a favorable outcome in the second-line setting, independent of other factors. Patients with IgA-MM cleared their M-spike faster than IgG-MM, probably reflecting different half-lives of these isotypes rather than disease biology, as the clearance of LC in both subtypes was similar. CONCLUSIONS: Analyzing both M-spike and LC responses in a homogenously treated cohort, we identified important insights regarding the prognostic value of kinetic patterns. Prospective analysis may shed more light on unsolved questions.

sciCNV: high-throughput paired profiling of transcriptomes and DNA copy number variations at single-cell resolution.

Chromosome copy number variations (CNVs) are a near-universal feature of cancer; however, their individual effects on cellular function are often incompletely understood. Single-cell ribonucleic acid (RNA) sequencing (scRNA-seq) might be leveraged to reveal the function of intra-clonal CNVs; however, it cannot directly link cellular gene expression to CNVs. Here, we report a high-throughput scRNA-seq analysis pipeline that provides paired CNV profiles and transcriptomes for single cells, enabling exploration of the effects of CNVs on cellular programs. RTAM1 and -2 normalization methods are described, and are shown to improve transcriptome alignment between cells, increasing the sensitivity of scRNA-seq for CNV detection. We also report single-cell inferred chromosomal copy number variation (sciCNV), a tool for inferring single-cell CNVs from scRNA-seq at 19-46 Mb resolution. Comparison of sciCNV with existing RNA-based CNV methods reveals useful advances in sensitivity and specificity. Using sciCNV, we demonstrate that scRNA-seq can be used to examine the cellular effects of cancer CNVs. As an example, sciCNV is used to identify subclonal multiple myeloma (MM) cells with +8q22-24. Studies of the gene expression of intra-clonal MM cells with and without the CNV demonstrate that +8q22-24 upregulates MYC and MYC-target genes, messenger RNA processing and protein synthesis, which is consistent with established models. In conclusion, we provide new tools for scRNA-seq that enable paired profiling of the CNVs and transcriptomes of single cells, facilitating rapid and accurate deconstruction of the effects of cancer CNVs on cellular programming.

Fixed duration vs. prolonged duration treatment after first line therapy in patients with systemic light chain amyloidosis.

BACKGROUND: The main objective of treatment in systemic light chain amyloidosis (AL amyloidosis) is to achieve the best hematological response. Deeper responses are associated with better organ responses and survival. In this study, we analysed the efficacy of prolonged duration treatment after first line in patients with AL amyloidosis. METHODS: Retrospective analysis that included patients older than 18 years with AL amyloidosis. We excluded patients with more than 30% marrow plasmacytosis or concurrent multiple myeloma. Two cohorts identified accordingly if they received or not prolonged treatment after the first line. Survival analysis regarding progression free survival (PFS) and overall survival (OS) estimated with Kaplan-Meier and comparisons between groups with log-rank. RESULTS: Thirty-eight patients were included in the analysis with a median age of 55 years. Twenty-one patients received prolonged duration treatment and 17 did not. In the prolonged duration group, after a median duration of 12 months, the median PFS was 58.8 months. In the fixed duration treatment group, PFS was 30.6 months. The difference was significant with p = .0045 favouring prolonged duration treatment. Organ response was sustained for a longer period in the prolonged duration treatment group. For OS, the difference was not significant. CONCLUSIONS: Prolonged duration treatment in patients with systemic light chain amyloidosis correlated with better PFS and deeper organ responses. Prospective studies are needed to analyse this further.

Design, Synthesis, and Characterization of 4-Aminoquinazolines as Potent Inhibitors of the G Protein-Coupled Receptor Kinase 6 (GRK6) for the Treatment of Multiple Myeloma.

Both previous and additional genetic knockdown studies reported herein implicate G protein-coupled receptor kinase 6 (GRK6) as a critical kinase required for the survival of multiple myeloma (MM) cells. Therefore, we sought to develop a small molecule GRK6 inhibitor as an MM therapeutic. From a focused library of known kinase inhibitors, we identified two hits with moderate biochemical potencies against GRK6. From these hits, we developed potent (IC50 < 10 nM) analogues with selectivity against off-target kinases. Further optimization led to the discovery of an analogue (18) with an IC50 value of 6 nM against GRK6 and selectivity against a panel of 85 kinases. Compound 18 has potent cellular target engagement and antiproliferative activity against MM cells and is synergistic with bortezomib. In summary, we demonstrate that targeting GRK6 with small molecule inhibitors represents a promising approach for MM and identify 18 as a novel, potent, and selective GRK6 inhibitor.

Combined EZH2 Inhibition and IKAROS Degradation Leads to Enhanced Antitumor Activity in Diffuse Large B-cell Lymphoma.

PURPOSE: The efficacy of EZH2 inhibition has been modest in the initial clinical exploration of diffuse large B-cell lymphoma (DLBCL), yet EZH2 inhibitors are well tolerated. Herein, we aimed to uncover genetic and pharmacologic opportunities to enhance the clinical efficacy of EZH2 inhibitors in DLBCL. EXPERIMENTAL DESIGN: We conducted a genome-wide sensitizing CRISPR/Cas9 screen with tazemetostat, a catalytic inhibitor of EZH2. The sensitizing effect of IKZF1 loss of function was then validated and leveraged for combination treatment with lenalidomide. RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing analyses were performed to elucidate transcriptomic and epigenetic changes underlying synergy. RESULTS: We identified IKZF1 knockout as the top candidate for sensitizing DLBCL cells to tazemetostat. Treating cells with tazemetostat and lenalidomide, an immunomodulatory drug that selectively degrades IKAROS and AIOLOS, phenocopied the effects of the CRISPR/Cas9 screen. The combined drug treatment triggered either cell-cycle arrest or apoptosis in a broad range of DLBCL cell lines, regardless of EZH2 mutational status. Cell-line-based xenografts also showed slower tumor growth and prolonged survival in the combination treatment group. RNA-seq analysis revealed strong upregulation of interferon signaling and antiviral immune response signatures. Gene expression of key immune response factors such as IRF7 and DDX58 were induced in cells treated with lenalidomide and tazemetostat, with a concomitant increase of H3K27 acetylation at their promoters. Furthermore, transcriptome analysis demonstrated derepression of endogenous retroviruses after combination treatment. CONCLUSIONS: Our data underscore the synergistic interplay between IKAROS degradation and EZH2 inhibition on modulating epigenetic changes and ultimately enhancing antitumor effects in DLBCL.

A descriptive cost-analysis of MYX.1/MCRN003, a phase 2 clinical trial evaluating high-dose weekly carfilzomib, cyclophosphamide, and dexamethasone in relapsed and refractory multiple myeloma.

BACKGROUND: The prevalence of multiple myeloma is increasing and there is a need to evaluate escalating therapy costs (Canadian Cancer Statistics A, 2020). The MYX.1 phase II trial showed that high-dose weekly carfilzomib, cyclophosphamide, and dexamethasone (wKCD) is efficacious in relapsed and refractory disease. We conducted a descriptive cost analysis, from the perspective of the Canadian public healthcare system, using trial data. METHODS: The primary outcome was the mean total cost per patient. Resource utilization data were collected from all 75 trial patients over a trial time horizon. Costs are presented in Canadian dollars (2020). RESULTS: The cost of treatment was calculated from the time of patient (pt) enrollment until the second data lock. The mean total cost was $203 336.08/pt (range $17 891.27-$505 583.55) Canadian dollars (CAD, where 1 CAD = 0.67 Euro (EUR)) and $14 081.45/pt per cycle. The median number of cycles was 15. The predominant cost driver was the cost of chemotherapy accounting for an average of $179 332.78/pt or $12 419.17/pt per cycle. Carfilzomib acquisition accounted for the majority of chemotherapy costs - $162 471.65/pt or $11 251.50/pt per cycle. Fifty-six percent (56%) of patients had at least one hospitalization during the trial period with an average cost of $12 657.86 per hospitalization. Three patients developed thrombotic microangiopathy (TMA) with an average cost of $18 863.32/pt including the cost of hospitalizations and therapeutic plasma exchange. CONCLUSIONS: High-dose wKCD is an active triplet regimen for relapsed and refractory multiple myeloma (RRMM) associated with reduced total cost compared with twice-weekly carfilzomib-based regimens.

An open-label, pharmacokinetic study of lenalidomide and dexamethasone therapy in previously untreated multiple myeloma (MM) patients with various degrees of renal impairment - validation of official dosing guidelines.

Lenalidomide is a backbone agent in the treatment of multiple myeloma, but dose adjustment is required for those with renal impairment (RI). We evaluated the pharmacokinetics (PK) and safety of lenalidomide and dexamethasone as frontline pre-transplant induction, with doses adjusted at start of each cycle based on creatinine clearance, as per the official dosing guidelines. After 4 cycles, PK studies showed that patients with moderate RI (30 ≤ CrCl < 60 mL/min) receiving 10 mg dosing may be under-dosed and those with severe RI (CrCl <30ml/min) appeared appropriately dosed initially, but sustained significant decreases in maximum serum concentration (Cmax) after repeated dosing, due to rapid clinical improvement and enhanced drug clearance. PK drug monitoring during cycle 1 may facilitate appropriate and timely dose adjustments. Adverse events rates did not vary based on severity of RI. No patient discontinued lenalidomide for toxicity. This supports the feasibility and safety of frontline lenalidomide in transplant-eligible patients with RI.

Toxicity and survival outcomes of autologous stem cell transplant in multiple myeloma patients with renal insufficiency: an institutional comparison between two eras.

Autologous stem cell transplant (ASCT) is a feasible treatment option for multiple myeloma (MM) patients with renal insufficiency; however, these patients tend to experience higher rates of drug toxicity and transplant-related mortality (TRM) during ASCT. Recent adoption of bortezomib-based induction regimens and dose reduction of melphalan during conditioning may improve outcomes in this population. In this single center retrospective study, we compared the toxicity and survival outcomes of 96 MM patients with renal insufficiency undergoing ASCT between two eras: 1998-2007 and 2008-2016. The proportion of dialysis dependent patients was similar in both groups (49 and 45%). We found no TRM in those transplanted more recently as compared with 13% in the older era of ASCT. There were significantly more high grade (grades 3-4) toxicities in the older era of ASCT including high grade electrolyte abnormalities, mucositis, delirium, and bleeding. Patients transplanted more recently had significantly higher overall response rate (ORR) as well as deeper responses to ASCT (≥VGPR in 79% vs 39%). Progression-free survival (PFS) was prolonged by 26 months in the more recent era compared with the older era. Overall, improvements in treatment regimens have resulted in reduced TRM and toxicities for patients with renal insufficiency undergoing ASCT.

Chromosomal Instability and mTORC1 Activation through PTEN Loss Contribute to Proteotoxic Stress in Ovarian Carcinoma.

High-grade serous ovarian carcinoma commonly arises from fallopian tube secretory epithelium and is characterized by a high level of chromosomal instability. To model the acquisition of aneuploidy during early carcinogenesis, chromosome missegregation was induced in immortalized tubal epithelial cells, which proved acutely detrimental to cellular fitness. The phenotype was characterized by accumulation of misfolded proteins, activation of the unfolded protein response (UPR), decreased protein synthesis, and enhanced vulnerability to proteasome inhibition. However, chromosome missegregation also resulted in heightened transformation potential, assessed by colony formation in soft agar. Ovarian cancer cells retained intrinsic sensitivity to proteasome inhibitors under adherent culture conditions, but acquired resistance as spheroids (recapitulating their native configuration in ascites) by downregulating protein synthesis via mTORC1 suppression. Loss of PTEN drove constitutive mTORC1 activity, enhanced proteotoxic stress, as evidenced by UPR induction, and resensitized tumor spheroids to proteasome inhibition both in vitro and in vivo. In cohorts of primary ovarian carcinomas, mTORC1 and UPR signaling pathways were closely associated. These results implicate attenuation of protein synthesis as a protective mechanism in tumor spheroids, which may explain the overall poor response to bortezomib in clinical trials of patients with advanced ovarian cancer. However, patients with PTEN-deficient tumors may represent a subpopulation potentially amenable to treatment with proteasome inhibitors or other therapeutic agents that disrupt protein homeostasis. SIGNIFICANCE: Chromosome instability and protein synthesis are important factors that determine the efficacy of proteotoxic stress-inducing agents, such as proteasome inhibitors, in the treatment of ovarian cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/21/5536/F1.large.jpg.

Addition of Cyclophosphamide "On Demand" to Lenalidomide and Corticosteroids in Patients With Relapsed/Refractory Multiple Myeloma-A Retrospective Review of a Single-center Experience.

INTRODUCTION: The combination of lenalidomide and dexamethasone (Len-Dex) is an established regimen for patients with relapsed or refractory myeloma. To prolong the benefit of this effective regimen, the Myeloma Program at Princess Margaret Cancer Centre has routinely added a third agent, oral cyclophosphamide (Cy) given weekly, to Len-Dex at disease progression. PATIENTS AND METHODS: In the present report, we describe the cases of 53 patients who had received Len-Dex-Cy for a minimum of 4 weeks from January 2007 to December 2014 after progression with Len-Dex alone. The dose of added Cy ranged from 250 to 500 mg weekly. The median number of previous regimens, including Len-Dex, was 2 (range, 2-4); 80% of patients had undergone previous autologous stem cell transplantation. RESULTS: The overall rate of response equal to or greater than a partial response was 34%, and clinical benefit (stable disease or better) was observed in 87% of the patients. The median duration of Len-Dex-Cy therapy was 6.9 months (range, 0.9-55.1 months). The median progression-free survival was 6.1 months (range, 4.2-8.1 months) from the addition of Cy and 24.1 months (range, 15.9-32.0 months) from start of Len-Dex. CONCLUSION: The addition of Cy for patients with myeloma developing progression with Len-Dex was an inexpensive option with manageable toxicity that resulted in a clinically meaningful extension of disease control.

Patient-Derived Xenografts for Prognostication and Personalized Treatment for Head and Neck Squamous Cell Carcinoma.

Overall survival remains very poor for patients diagnosed as having head and neck squamous cell carcinoma (HNSCC). Identification of additional biomarkers and novel therapeutic strategies are important for improving patient outcomes. Patient-derived xenografts (PDXs), generated by implanting fresh tumor tissue directly from patients into immunodeficient mice, recapitulate many of the features of their corresponding clinical cancers, including histopathological and molecular profiles. Using a large collection of PDX models of HNSCC, we demonstrate that rapid engraftment into immunocompromised mice is highly prognostic and show that genomic deregulation of the G1/S checkpoint pathway correlates with engraftment. Furthermore, CCND1 and CDKN2A genomic alterations are predictive of response to the CDK4and CDK6 inhibitor abemaciclib. Overall, our study supports the pursuit of CDK4 and CDK6 inhibitors as a therapeutic strategy for a substantial proportion of HNSCC patients and demonstrates the potential of using PDX models to identify targeted therapies that will benefit patients who have the poorest outcomes.

Single-center Experience in Treating Patients With t(4;14) Multiple Myeloma With and Without Planned Frontline Autologous Stem Cell Transplantation.

BACKGROUND: Translocation t(4;14) has traditionally been classified as a high-risk cytogenetic feature in patients with multiple myeloma with shortened progression-free (PFS) and overall survival (OS) despite initial response to treatment. Recent data have shown an improved long-term survival in these patients treated with novel agents, such as bortezomib. PATIENTS AND METHODS: We conducted a retrospective study on our patients with t(4;14) multiple myeloma treated with bortezomib-based induction between July 1, 2006 and June 30, 2014 to assess the real-world outcomes of these patients in a tertiary center. RESULTS: Among the 75 patients analyzed, the median PFS was 33.5 months, and the median OS was 69.6 months after a median follow-up of 41 months. Even in the era of novel agents, patients who received frontline autologous stem cell transplant had a better PFS than those who received chemotherapy alone (median PFS, 24.2 months vs. 41.5 months; P = .01). Hypercalcemia at the time of presentation was found to be a significant predictor of progression (hazard ratio [HR], 10.1; 95% confidence interval [CI], 4.0-26.0) and death (HR, 9.4; 95% CI, 3.2-27.8), and co-harboring of del(17p) by fluorescent in situ hybridization with t(4;14) was associated with a significantly inferior OS (HR, 4.0; 95% CI, 1.4-11.4). CONCLUSION: Even in the era of novel agents, t(4;14) remains a negative prognostic marker. Frontline autologous stem cell transplant remains as an essential tool when treating these high-risk patients, but further prospective randomized studies are needed to determine the most effective strategy for this patient group.