Anderson, Kenneth C.

AbstractThe phase 3 FIRST trial demonstrated significant improvement in progression‐free survival (PFS) and overall survival (OS) with an immune‐stimulatory agent, lenalidomide, in combination with low‐dose dexamethasone until disease progression (Rd continuous) vs melphalan +prednisone + thalidomide (MPT) in transplant‐ineligible patients with newly diagnosed multiple myeloma (NDMM). Rd continuous similarly extended PFS vs fixed‐duration Rd for 18 cycles (Rd18). Outcomes in the Canadian/US subgroup (104 patients per arm) are reported in this analysis. Rd continuous demonstrated a significant improvement in PFS vs MPT (median, 29.3 vs 20.2 months; HR, 0.69 [95% CI, 0.49‐0.97]; p = 0.03326) and an improvement vs Rd18 (median, 21.9 months). Median OS was 56.9 vs 46.8 months with Rd continuous vs MPT (p = 0.15346) and 59.5 months with Rd18. The overall response rate was higher with Rd continuous and Rd18 (78.8% and 79.8%) vs MPT (65.4%). In the 49.0%, 52.9%, and 29.8% of patients with at least very good partial response in the Rd continuous, Rd18, and MPT arms, respectively, the median PFS was 56.0, 30.9, and 40.2 months, respectively. The most common grade 3/4 treatment‐emergent adverse events were neutropenia (28.4%, 30.1%, and 52.0%), anemia (23.5%, 21.4%, and 23.5%), and infections (37.3%, 30.1%, and 24.5%) with Rd continuous, Rd18, and MPT, respectively. These results were consistent with those in the intent‐to‐treat population, confirming the benefit of Rd continuous vs MPT in the Canadian/US subgroup and supporting the role of Rd continuous as a standard of care for transplant‐ineligible patients with NDMM.

Abstract Background: Persons of African ancestry (AA) experience a 1.5-2-fold risk of multiple myeloma (MM) compared to persons of European ancestry (EA). We assembled a set of MM patients with self-reported AA in order to evaluate the contribution of genetics to etiology in this high-risk group. Methods: Here we present the results of a meta-analysis of two GWAS in 1,813 cases and 8,871 controls of AA. We also conducted an admixture mapping scan to identify risk alleles associated with local ancestry, fine-mapped the 23 known susceptibility loci to find markers that could better capture MM risk in individuals of AA, and constructed a polygenic risk score (PRS) to assess the aggregated effect of known MM risk alleles. Finally, we conducted an eQTL analysis measuring gene expression in those genes harboring a risk variant in malignant plasma cells from 292 of the patients from a single site. Results: In GWAS analysis, we identified two suggestive novel loci located at 9p24.3 and 9p13.1 at P<1 × 10-6, but no genome-wide significant association was noted. In admixture mapping, we observed a genome-wide significant inverse association between local AA at 2p24.1-23.1 and MM risk in AA individuals. 20 of the 23 known EA risk variants showed directional consistency and 9 replicated at P<0.05 in AA individuals. In eight regions, we identified markers that better capture MM risk in persons of AA. AA individuals with a PRS in the top 10% had a 1.82-fold (95%CI: 1.56, 2.11) increased MM risk compared to those with average risk (25-75%). The strongest functional association was between the risk allele for variant rs56219066 at 5q15 and lower ELL2 expression (P= 5.1 × 10–12). Conclusion: Our study shows that common genetic variation contributes to MM risk individuals of AA. This abstract is also being presented as Poster C040. Citation Format: Zhaohui Du, Niels Weinhold, Gregory Chi Song, Kristen A. Rand, David J. Van Den Berg, Amie E. Hwang, Xin Sheng, Victor Hom, Sikander Ailawadhi, Ajay K. Nooka, Seema Singhal, Karen Pawlish, Edward Peters, Cathryn Bock, Ann Mohrbacher, Alexander Stram, Sonja I. Berndt, William J. Blot, Graham Casey, Victoria L. Stevens, Rick Kittles, Phyllis J. Goodman, W. Ryan Diver, Anselm Hennis, Barbara Nemesure, Eric A. Klein, Benjamin A. Rybicki, Janet L. Stanford, John S. Witte, Lisa Signorello, Esther M. John, Leslie Bernstein, Antoinette Stroup, Owen W. Stephens, Maurizio Zangari, Frits Van Rhee, Andrew Olshan, Wei Zheng, Jennifer J. Hu, Regina Ziegler, Sarah J. Nyante, Sue Ann Ingles, Michael Press, John David Carpten, Stephen Chanock, Jayesh Mehta, Graham A Colditz, Jeffrey Wolf, Thomas G. Martin, Michael Tomasson, Mark A. Fiala, Howard Terebelo, Nalini Janakiraman, Laurence Kolonel, Kenneth C. Anderson, Loic Le Marchand, Daniel Auclair, Brian C.-H. Chiu, Elad Ziv, Daniel Stram, Ravi Vij, Leon Bernal-Mizrachi, Gareth J. Morgan, Jeffrey A. Zonder, Carol Ann Huff, Sagar Lonial, Robert Z. Orlowski, David V. Conti, Christopher A. Haiman, Wendy Cozen. A meta-analysis of genome-wide association study and eQTL analysis of multiple myeloma among African Americans [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr PR05.

8599 Background: MM survival varies enormously from few months to more than a decade. Baseline prognostic factors (PF) have been identified that are linked to survival outcomes, especially B2M and albumin. MM survival plots are typically characterized by 3 segments of slopes with decreasing steepness, suggesting that different PF may govern these time segments. Methods: We therefore examined, in S9321, PF that were associated with overall survival (OS) from baseline (BL) and from 3 subsequent landmarks 3, 5, and 7 years later (LM3, LM5, LM7). With a median follow-up of 9 years, OS and EFS were virtually identical in transplant and standard therapy arms so that the data were pooled. Results: Median OS and EFS for 817 eligible patients are 48 and 22 months. The BL model identified age (>60yr), hemoglobin (<10g/gL), platelet count (<130,000/uL), albumin (<3.5g/dL), B2M (>3.5g/dL), calcium (>10mg/dL), creatinine (>2mg/dL), LDH (>190U/L), performance status (PS, >1), IL-6 level (>140mg/L) and plasma cell labeling index (PCLI, >1%) as being univariately associated with short OS, while age (p=0.002), platelet count (p=0.04), calcium (p<0.001), B2M (p=0.002), LDH (p=0.010) and PCLI (p<0.001) retained significance in multivariate analysis. For the LM3 analyses, age (p=0.006) and B2M (p=0.009) were independently associated with poor OS, whereas PCLI was the only PF associated with LM5 (p=0.026) and age with LM7 (p<0.001). While associated univariately with OS from BL, CRP, platelet count, hemoglobin, albumin, calcium, creatinine, LDH, IL6 and PS all failed to affect OS from later LM times. Conclusions: Whereas the initial 3 years of OS are governed by a combination of features related to disease aggressiveness (LDH, calcium, albumin, CRP), tumor burden (hemoglobin, platelet count) and host tolerance (PS, creatinine), B2M and PCLI as reflectors of tumor burden and proliferation have sustained long-term OS implications. The age factor figures in partly due to death from other causes. In an era of ever-improving survival outcomes, such LM-based PF analysis may reveal trial-specific differences and thus provide guidance toward individualizing therapy. [Table: see text]

e14640 Background: MicroRNAs (miRNAs) are non-coding RNAs that act as negative regulators of gene expression. They have been described to play roles in solid tumors and hematologic malignancies. However the role of miRNAs in multiple myeloma (MM) has not been yet fully described. Methods: We performed miRNA-profiling of primary bone marrow-derived CD138+ MM cells, compared to their normal cellular counterparts and validated data by qRT-PCR. In vitro and in vivo functional studies were performed on miRNA-15a- and -16–1-precursors-transfected MM cells. Effect of miRNA-15a and -16–1 on signaling cascades have been evaluated by western blot and immunofluorescence. NF-kB activity has been studied using the Active Motif TransAM kit. In vivo MM cell growth has been evaluated by either using an in vivo imaging model or bioluminescence. Angiogenesis has been studied both in vitro and in vivo using the chorioallantoic membrane model. Results: We identified a MM-specific miRNA signature characterized by down-expression of miRNA-15a, -16–1 and over-expression of miRNA-222/-221/-382/-181a/-181b (P<0.01). Data were confirmed by qRT-PCR on matched samples. Predicted targets for the decreased miRNAs in MM patients included pro-angiogenic cytokines, oncogenes, cell cycle regulators, NFkB activators. Conversely, predicted targets for the increased miRNAs included cell cycle inhibitors, suppressors of cytokine signaling, and pro-apoptotic factors. We demonstrated that miRNA-15a and - 16–1 regulate proliferation and growth of MM cells. Indeed, transfected cells showed decreased DNA synthesis; decreased cyclinD1/cyclinD3/CDC25a/pRb protein expression; phase G1 cell cycle arrest. Moreover, transfected cells showed inhibition of NFkB pathway as shown by reduced p65-/p50-/p52-NFkB activities; downregulation of p-p65/p50/p52 nuclear protein level; upregulation of cttosolic phospho-IkB; and inhibited translocation of p-p65 from the cytolplasm to the nucleus. Similarly, inhibition of MM cell growth was confirmed in vivo; and anti-angiogenic properties of miRNA-15a and -16–1 were demonstrated both in vitro and in vivo. Conclusions: These data indicate that miRNAs play a pivotal role in the biology of MM; providing the basis for the development of new miRNA-based targeted therapies in this disease. No significant financial relationships to disclose.

PURPOSE Smoldering multiple myeloma (SMM) is a precursor condition of multiple myeloma (MM) with a 10% annual risk of progression. Various prognostic models exist for risk stratification; however, those are based on solely clinical metrics. The discovery of genomic alterations that underlie disease progression to MM could improve current risk models. METHODS We used next-generation sequencing to study 214 patients with SMM. We performed whole-exome sequencing on 166 tumors, including 5 with serial samples, and deep targeted sequencing on 48 tumors. RESULTS We observed that most of the genetic alterations necessary for progression have already been acquired by the diagnosis of SMM. Particularly, we found that alterations of the mitogen-activated protein kinase pathway ( KRAS and NRAS single nucleotide variants [SNVs]), the DNA repair pathway (deletion 17p, TP53, and ATM SNVs), and MYC (translocations or copy number variations) were all independent risk factors of progression after accounting for clinical risk staging. We validated these findings in an external SMM cohort by showing that patients who have any of these three features have a higher risk of progressing to MM. Moreover, APOBEC associated mutations were enriched in patients who progressed and were associated with a shorter time to progression in our cohort. CONCLUSION SMM is a genetically mature entity whereby most driver genetic alterations have already occurred, which suggests the existence of a right-skewed model of genetic evolution from monoclonal gammopathy of undetermined significance to MM. We identified and externally validated genomic predictors of progression that could distinguish patients at high risk of progression to MM and, thus, improve on the precision of current clinical models.

Abstract BAFF is a member of the tumor necrosis factor (TNF) family and plays a role in B cell survival including MM cells. We have previously established a role for BAFF in localization and survival of MM cells in the bone marrow (BM) microenvironment. (Cancer Res2006; 66(13): 6675–82). Here we validate the role of BAFF in MM patients and demonstrate the in vivo activity of anti-BAFF antibody in a SCID-hu MM model. We first performed gene expression profiling (GEP) on CD 138+ plasma cells isolated from 90 MM patients and 11 healthy controls using the Affymetrix U133A arrays. GEP analysis demonstrated increased BCMA expression (p&lt;0.0001, student T test) on newly diagnosed and relapsed MM versus normal plasma cells. Flow cytometry performed on MM patient cells demonstrated the presence of all 3 receptors on CD 138+ cells. ELISA assays confirmed increased plasma BAFF levels in 51 MM plasma (mean: 1049 pg/ml; range: 176–4252 pg/ml) compared to 11 normal donors (mean: 461pg/ml; range: 317–652pg/ml) [p&lt;0.001]. To understand the functional significance that BAFF might play in the biology of MM, we studied the effects of recombinant BAFF (rh-BAFF) on MM cells directly and in the context of its BM microenvironment. Our data demonstrate that rh-BAFF confers a survival advantage to MM cells and protects them against dexamethasone-induced cytotoxicity. Importantly, anti-apoptotic proteins Bcl2 and XIAP were upregulated, as were growth and survival signals belonging to the AKT and MAPKinase pathways. Because of the survival advantage conferred by BAFF on MM cells we evaluated the use of a clinical grade-neutralizing antibody to BAFF. To evaluate the in vivo activity of anti-Baff in MM we used the SCID-hu model, where MM cells grow in the context of human BM microenvironment. A cohort of SCID-hu mice bearing INA-6 MM cells, were treated i.p. weekly with anti-BAFF neutralizing antibody (10 mg/kg, n=3) or control isotype (10 mg/kg, n=3), respectively, for four weeks. Serum levels of soluble human IL-6 receptor (shuIL-6R) released by MM cells into the murine serum were monitored as a measure of MM growth. At the end of treatment we observed a significant (p=0.048) reduction in shuIL-6R level. This translated into a survival advantage of 3.1 weeks in the anti-BAFF treated animals versus the control group (p= 0.02). We also evaluated in vivo effects of anti-BAFF on the bone compartment by radiographic analysis and tartrate-resistant acid phosphatase (TRAP) staining of human bone implants. Our results demonstrate a decrease in radiologically evident lytic lesions in the anti-BAFF treated animals. This was accompanied by a significant decrease in TRAP + osteoclasts in bone sections from treated mice when compared to control mice. Our data therefore suggests that anti-BAFF antibody may impact MM cell growth directly and/or via effects on the BM microenvironment by impacting bone resorption. Taken together, these data show a role for BAFF mediating MM cell survival and its in vivo anti-tumor activities provide a preclinical rational for its clinical evaluation in MM.

Abstract BAFF is a member of the tumor necrosis factor (TNF) family and is critical for the maintenance and homeostasis of normal B-cell development. Importantly, BAFF promotes the generation of rapidly dividing immunoglobulin secreting plasmablasts from activated memory B cells by enhancing their survival. Given that MM is a cancer of plasma cells and that the signaling cascades implicated in receptor ligand interactions of BAFF are crucial in MM cell biology, we hypothesized that this cytokine may play a critical role in MM cell development, survival, and proliferation. We performed gene expression profiling (GEP) on CD 138+ plasma cells isolated from 90 MM patients (45 newly diagnosed and 45 relapsed) and 11 healthy controls using the Affymetrix U133A arrays. Our data demonstrates increased expression of transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA), 2 receptors used by BAFF to exert its effects. Our data also shows an increased expression of a proliferation-inducing ligand (APRIL), another member of the TNF family with homology to BAFF. Expression levels of BAFF and BAFF-R could not be determined because of lack of these probe sets on the Affymetrix U133A arrays. GEP analysis shows increased BCMA expression (p&lt;0.0001, student T test) on newly diagnosed and relapsed MM versus normal plasma cells. Flow cytometry on MM cell lines demonstrated a differential expression of the three receptors of BAFF, with BCMA present on most cell lines but BAFF-R expressed at low levels only on LR5 cells and DOX40 MM cells. In contrast, flow cytometry performed on MM patient cells demonstrated the presence of all 3 receptors on CD 138+ cells. ELISA assays performed on 30 MM sera demonstrated a mean BAFF level of 618 pg/ml (range: 128–2126pg/ml) versus 235pg/ml (range: 158–326pg/ml) in 7 normal donor sera. Fifty six% (17/30) of MM patients had BAFF levels in excess of the highest value noted in normals. To understand the role BAFF might play in the biology of MM, we studied the effects of recombinant BAFF (rh-BAFF) on MM cells directly and in the context of its bone marrow microenvironment. (abstract # 554746) rh-BAFF conferred a survival advantage to MM cells and protected them against dexamethasone-induced cytotoxicity. Importantly, anti-apoptotic proteins Bcl2 and Mcl-1 were upregulated, as were growth and survival signals belonging to the JAK/STAT and MAPKinase pathways. Conversely, neutralizing antibody to BAFF blocked, at least in part, blocked the upregulation of anti-apoptotic proteins with associated growth and survival, confirming that these effects were due to BAFF. Importantly, all of these signals were downregulated even in the presence of bone marrow stromal cells (BMSCs). These data therefore show a role for BAFF mediating MM cell survival and provide the framework for inhibiting BAFF, either alone or in combination with dexamethasone, to improve patient outcome in MM.

Abstract Introduction: We investigated tumor gene expression profiling (PGx) to identify predictive classifiers of response and elucidate the mechanism of efficacy of proteasome inhibition in multiple myeloma (MM) by bortezomib, a first-in-class proteasome inhibitor. Analysis of 44 samples from the SUMMIT phase 2 trial identified predictive classifiers of response in patients (pts) with refractory MM (ASH 2002, Abstr 1519). We now report on the PGx analysis of the SUMMIT-derived classifier in an independent sample set from the APEX phase 3 trial, a multicenter randomized study of 669 pts that demonstrated the superiority of bortezomib to high-dose dex for relapsed MM following 1 to 3 prior therapies (Richardson NEJM 2005). As well, we report on a new classifier of response built by combining samples from SUMMIT and MLN34101-040 (040) (a study of bortezomib in pts who failed dex in APEX or were not eligible for APEX) and tested in the APEX samples. Methods: 93 participating clinical centers collected pretreatment bone marrow aspirates and enriched tumor cells on site before centralized RNA extraction and microarray analysis. Approximately 85% of pts consented to whole-genome analysis. Quality control for RNA integrity and quantity, microarray data quality, and tumor cell purity resulted in 129 patient samples from APEX evaluable for PGx; 67 were from the bortezomib arm and 62 from the dex arm. 48 samples were evaluable from 040. Patient characteristics of this subset were similar to those of the overall study population. Predictive classifiers were identified and tested via separate training and test datasets. Results: Consistent with the SUMMIT analysis, the APEX and 040 datasets exhibit biologic features expected of MM, including the overexpression of translocation target genes such as cyclin D1, MAF, and MMSET/FGFR3. Classifiers built using only the SUMMIT samples predict the outcome of pts in the APEX bortezomib arm with an overall accuracy of 51% (sensitivity [Sn] = 49%, specificity [Sp] = 55%; P = .81) and in the dex arm with an overall accuracy of 56% (Sn = 64%, Sp = 50%; P = .31). If the classifier was built using data from both the SUMMIT and 040 trials (87 total samples), the prediction of response in the APEX bortezomib arm was statistically significant (66% accuracy, Sn = 89%, Sp = 42%; P = .006). The same classifier only predicts the response of dex-treated pts with an overall accuracy of 46% (Sn = 72%, Sp = 28%; P = .88), suggesting specificity for bortezomib. Many of the overexpressed genes associated with bortezomib insensitivity relate to protein synthesis (EIF4B, RPL5) and RNA binding (SFRS10, CPSF6). Future Directions: This work highlights practical and scientific issues that inform future efforts to implement PGx in clinical trials. Predictive accuracy may be influenced by the clinical end point, limited sample size, and/or technical considerations associated with distinct trials and genomic data. The influence of study differences (relating to pts’ time from diagnosis, number of prior therapies, molecular subtypes, or hybridization characteristics) on the ability to build classifiers and identify biologic pathways related to response are being examined; these and potential insights on drug mechanisms will be presented.

Abstract Ribonucleotide reductase is the rate-limiting enzyme of deoxynucleoside triphosphate synthesis and is therefore an excellent target for cancer chemotherapy. Inhibition of ribonucleotide reductase results in inhibition of DNA synthesis and has anti-neoplastic effects. Several ribonucleotide reductase inhibitors (RRIs) are already in clinical practice, including Hydroxyurea and Gemcitabine. Here we examine the anti-MM activity of a novel RRI, Didox. Our data shows that Didox has potent cytotoxicity against MM cells in 48-hour cultures. Apoptosis induced in MM cell lines by Didox (200mmol/l), evidenced by increased sub-G1 cell fraction, is caspase dependent, confirmed by blocking experiments with Z-VAD-FMK. Unlike other RRIs which mainly target the pyrimidine metabolism pathway (hydroxyurea and gemcitabine), didox targets both the purine and pyrimidine metabolism pathways in MM, as demonstrated by transcriptional profiling using the Affymetrix U133A 2.0 gene chip. Specifically, our data shows a ≥ 2 fold downregulation of genes in these pathways as early as 12 hours after exposure to Didox. Importantly, ribonucleotide reductase (RR) M1 component transcript was downregulated, associated with downregulation of this enzyme by Western Blot analysis, and inhibition of DNA synthesis. Furthermore, genes involved in DNA repair mechanisms were also downregulated after exposure to Didox, demonstrated by heirachial clustering of gene chip data and further validation by western blotting. Specifically RAD 51 homologue, which is involved in nucleotide excision repair and recombination repair, was downregulated in MM cells both transcriptionally as well as at the protein level. This was accompanied by downregulation of the BCL family protein including Bcl-2, Bclxl, and XIAP both at the transcriptional and post transcriptional levels. Since Didox acts on MM cells by inhibiting DNA synthesis and repair, combination studies with melphalan, an alkylating agent commonly used in MM, were next performed. Our data shows a strong in vitro synergism, with combination indices of &lt;0.7 by the Chou Talahay method. When combined with DNA damaging agents like doxorubicin, additive cytotoxicity was noted. These studies therefore provide the preclinical rationale for evaluation of Didox, alone and in combination with alkylating agents and anthracyclines, to improve patient outcome in MM.

Abstract INTRODUCTION: Previous studies have demonstrated the clinical activity of the mTOR inhibitor RAD001 in low- grade lymphomas. Our preclinical studies demonstrated activity of mTOR inhibitors in Waldenstrom Macroglobulinemia (WM) cell lines and patient samples. This phase II study aimed to determine safety and activity of the oral mTOR inhibitor RAD001 (Novartis Pharmaceutical, MA) in patients with relapsed or refractory WM. METHODS: Patients who had at least one previous therapy for WM, and who had symptomatic relapsed or refractory disease were eligible. NCI CTCAE v3.0 was used for toxicity assessment. All patients received daily RAD001 at 10 mg. A cycle was considered 28 days. Patients were allowed to stay on therapy until progression of disease or excessive toxicity. This study was conducted in a collaborative effort between Dana Farber Cancer Institute (DFCI) and Mayo Clinic College of Medicine. Here, we report the data on the patients accrued at DFCI. RESULTS: 19 pts (15 men and 4 women) have been treated to date. All patients had symptomatic disease and required therapy. The median number of lines of prior treatment was 3 (range 1 – 5) including included rituximab, nucleoside analogues (fludarabine or 2-CDA), combination chemotherapy (e.g. CHOP, CVP), chloramucil, and bortezomib. The median IgM at baseline was 3330 mg/dL (range 1010– 7410). The median follow on RAD001 was 8 months (range 3 – 22 months). Eighteen pts are currently evaluable for response. Best responses to RAD001 after 2 cycles using IgM monoclonal protein were as follows: partial remission in 8 (44%), minimal response in 5 (28%). Progressive disease occurred in 4 (22%) and stable disease occurred in 1 (6%). The overall response rate (PR+MR) was 72%. The median duration of response has not been reached (3–22+ months). Patients tolerated therapy well without significant toxicities. Grade 3 and 4 toxicities included grade 4 thrombocytopenia in 1 patient, grade 3 pneumonia in 1 patient, grade 3 hyperglycemia in 1 patient and grade 3 mucositis in 1 patient. Other adverse events of grade 2 or lower included nail cracking, mucositis, diarrhea, and fatigue. Attributable toxicities otherwise proved manageable with appropriate supportive care, and RAD001 was generally well tolerated. One patient enrolled on the study withdrew consent and changed to hospice care within 3 weeks of therapy, and passed away due to disease progression. CONCLUSIONS: The use of the oral RAD001 single agent RAD001 in patients with relapsed or refractory WM was welltolerated and demonstrated significant activity achieving an overall response rate in 72% of patients. Future studies of combination of this agent with rituximab and bortezomib are currently being planned.