A Phase I Trial of Single-Agent Reolysin in Patients with Relapsed Multiple Myeloma

This article is featured in Highlights of This Issue, p. 5861

Reovirus serotype 3–dearing strain is a naturally occurring, ubiquitous, nonenveloped human reovirus with a genome that consists of 10 segments of double-stranded RNA (1). Community-acquired reovirus infection in humans is generally mild and limited to the upper respiratory and gastrointestinal tract. Reovirus has been shown to preferentially replicate in malignant cells with transformed RAS pathways (2–6) and mediates antitumor activity via induction of apoptosis, direct cytolysis, and activation of a tumor-directed immune response. The specificity of reovirus for cancer cells makes Reolysin, its intravenous form, an attractive drug.

Early-phase clinical trials in patients with metastatic solid tumors have shown that single-agent Reolysin is safe and well tolerated (7–9). No dose-limiting toxicities (DLT) were experienced, and the most severe side effects (grade 3 or 4) included fatigue, flu-like symptoms, neutropenia, lymphopenia, and hyponatremia. In these intravenous monotherapy trials, up to 45% of patients had evidence of at least stable disease, but only one patient in the 72 total enrolled achieved a partial response. Phase Ib clinical trials combined chemotherapeutic agents with Reolysin (e.g., gemcitabine, carboplatin, docetaxel, and paclitaxel) in melanoma, head and neck, prostate, ovarian, non–small cell lung, and pancreatic cancers (10–13). These trials confirmed the safety of Reolysin in combination with other agents and demonstrated an improvement in clinical response with the addition of a chemotherapeutic stressor (Supplementary Table S1). The mechanisms underlying this improved clinical efficacy are unclear; however, chemotherapy may enhance productive reoviral infection and thereby increase malignant cell apoptosis (14).

Preclinical evidence has demonstrated that Reolysin induces multiple myeloma cell oncolysis (15, 16) alone and in combination with the proteasome inhibitor (PI) bortezomib, and showed that the combination synergistically upregulated endoplasmic reticulum (ER) stress-related genes and proapoptotic genes associated with ER stress (BH3 containing proapoptotic bcl-2 family members, NOXA, BIM, and PUMA). Thirukkumaran and colleagues (17) showed that the autophagosome marker, LC3-II, was greatly increased following treatment with reovirus; results suggesting that reovirus promoted autophagic cell death via ER stress-induced activation of the Akt-mTOR signaling pathway. Collectively, these studies provided preclinical justification for the use of Reolysin in patients with multiple myeloma.

Reolysin has not previously been infused into patients with relapsed hematologic malignancies. We embarked on a phase I trial of Reolysin in relapsed multiple myeloma patients to demonstrate safety and tolerability. Secondary objectives included response assessment, measurement of neutralizing anti-reoviral antibody (NARA) titers, and staining for productive reoviral infection within patient myeloma cells. In vitro and correlative analyses of multiple myeloma cell lines and patient samples were undertaken to evaluate potential markers of multiple myeloma cell sensitivity to reovirus.

RPMI-8226 and NCI-H929 multiple myeloma cell lines were obtained from ATCC. OPM2 cells were a kind gift from Michael Kuehl (NIH, Bethesda, MD). Multiple myeloma cell lines were maintained in RPMI-1640 media supplemented with 10% FBS in a humidified incubator 37°C with 5% CO₂. Reolysin used for preclinical studies were a gift from Dr. Matt Coffey (Oncolytics Biotech Inc.)

The antibody to reovirus capsid protein was a gift from Dr. Matt Coffey (Oncolytics Biotech Inc). The following antibodies were used in this study: antibody to reovirus capsid protein (compliments of Dr. Matt Coffey of Oncolytics Biotech, Inc.), caspase-3 (1:33, antigen retrieval, Abcam), p38 (1:250, antigen retrieval, Abcam), junctional adhesion molecule 1 (JAM-1), and cancer upregulating gene 2 (CUG2). The viral RNA in situ hybridization (ISH) protocol has been previously published (14, 18, 19). In brief, after digestion in protease, the tissue and reoviral RNA probes (locked nucleic acid modified 5′ digoxigenin tagged, Exiqon) were coincubated at 60°C for 5 minutes, then hybridized for 2 to 15 hours at 37°C. After a wash in 0.1× SSC and 2% BSA at 50°C for 10 minutes, the reoviral RNA–probe complex was visualized via NBT/BCIP (Roche) due to the action of the alkaline phosphatase conjugation to antidigoxigenin antibody. Negative controls included myeloma cases not exposed to reovirus and omission of the probe; myeloma cell lines either infected or sham infected with reovirus served as additional controls.

Optimal detection of JAM-1 and CUG2 by IHC was determined using the Leica Bond Max (dilution of 1:150 with pretreatment in antigen retrieval solution 2 for 30 minutes at 95°C). Positive controls included malignant cell lines with high sensitivity to reoviral infection. The CUG2 and JAM-1 antibodies were commercially obtained from Abcam.

Patient serum was collected at baseline and weekly for 3 weeks during the first cycle of treatment. Dilutions of patient serum were treated with a 1:1000 dilution dose of reovirus [Oncolytics; 2.53 × 10¹⁰ 50% tissue culture infective dose (TCID₅₀)/mL] known to cause 80% cell death of L929 mouse cells. The serum and virus mixture were coincubated for 2 hours to allow any antibodies in the serum to neutralize the virus before culturing with L929 cells as previously described (20). Cell survival was measured by MTT assay (ATCC) after 72 hours. Goat serum (Lampire Biological Laboratory) was used as a positive control for the NARA assay. NARA endpoint titer was expressed as the last dilution where any neutralization occurred before reovirus-only treated L929 cells (20% survival). NARA titer assay posttest trend analysis was conducted utilizing GraphPad Prism 6 software.

The Ohio State University Cancer Institutional Review Board (Columbus, OH) approved the phase I study, and informed consent was obtained from all enrolled patients (www.clinicaltrials.gov, NCT01533194). Patients with relapsed and refractory myeloma according to the International Myeloma Working Group (IMWG) diagnostic criteria for symptomatic myeloma were enrolled (21). Patients must have received prior lenalidomide and bortezomib therapy, progressed on or within 60 days of the most recent therapy, and had an Eastern Cooperative Oncology Group (ECOG) performance score ≤ 2 or Karnofsky Performance Status ≥ 60%. Prior autologous and allogeneic transplantations were permitted.

Patients were required to have measurable disease defined as serum monoclonal protein ≥ 500 mg/dL, > 200 mg of monoclonal protein in a 24-hour urine sample, or serum immunoglobulin-free light chain ≥ 100 mg/L with an abnormal kappa to lambda-free light chain ratio. Adequate organ and marrow function was required with absolute neutrophil count ≥ 1,000/μL, platelet count ≥ 50,000/μL, total bilirubin < 1.5 mg/dL, and AST and ALT ≤ 5× the institutional upper limit of normal. There was no serum creatinine requirement. Exclusion criteria included congestive heart failure with a LVEF < 50% at the time of screening.

This was an open-label, dose-escalating, single-center phase I trial of Reolysin (serotype 3 – replication competent dearing strain) in which Reolysin was provided by Oncolytics Biotech Inc. and distributed by the Pharmaceutical Management Branch of the National Cancer Institute (Bethesda, MD).

Reolysin was administered intravenously over 60 minutes on days 1 to 5 every 28 days. Bone marrow biopsies were obtained at screening and cycle 1 day 8. Peripheral blood was obtained for correlative analyses on days 1, 8, 15, and between days 22 and 28 of cycle 1. Females of childbearing potential underwent pregnancy testing at the time of screening and patients with a history of congestive heart failure underwent evaluation of cardiac function with an echocardiogram or MUGA scan at screening. The study was designed to enroll an initial cohort of 3 patients at 3 × 10⁹ TCID₅₀/day, and assuming no DLTs, an additional 9 patients would be treated at 3 × 10¹⁰ TCID₅₀/day. These dose levels were chosen based on tolerability in previous trials in patients with advanced solid tumors. Patients were scheduled to continue treatment for up to 12 cycles or until the time of disease progression or intolerance of therapy.

The Common Terminology Criteria for Adverse Events version 3.0 (CTCAE v. 3) was used to grade toxicities. A DLT was defined as one of the following occurring during the first cycle of therapy that was determined to be possibly, probably, and definitely related to single-agent Reolysin: ANC <500/μL lasting 5 days or more or grade 4 thrombocytopenia (platelet count <25,000/μL) with bleeding; LVEF < 50% in patients that underwent screening echocardiography or development of grade ≥ 2 heart failure in any patient; any grade 3–4 nonhematologic adverse event, laboratory abnormalities not correctable within 24 hours, or nausea or vomiting that does not resolve to grade 2 or less within 24 hours and leads to missing > 1 day of study drug; or liver function abnormalities that do not resolve to < grade 1 within 5 days and lead to missing > 1 day of study drug. For grade 3–4 nonhematologic toxicities, grade 4 neutropenia or thrombocytopenia, or grade 2 cardiac toxicity, all remaining Reolysin doses in that cycle were held.

Although response was not the primary endpoint of this trial, patients with measurable disease were assessed by standard IMWG Uniform response criterion (21) with assessment of myeloma proteins once monthly. If a patient had more than one monoclonal protein spike in the serum (or urine) then the monoclonal protein followed for assessing response was the larger one (relative to normal) meeting IMWG criteria for being measurable.

Twelve refractory patients were enrolled and demographics are summarized in Table 1. Eight patients were male, 11 were Caucasian, the median age was 61 years (range 48–77), and the median ISS stage at the time of enrollment was 2 (range 1–3). The median β-2 microglobulin was 3.2 mg/L (range 1.9–28.9) and 5 patients had a baseline creatinine < 1.0 mg/dL. All patients had received prior treatment with lenalidomide and bortezomib. The median number of prior therapies at enrollment was 5 (range 1–10) and the median number of lines of therapy was 3.5 (range 1–9) as defined by the IMWG.

Three patients were treated at dose level 3 × 10⁹ TCID₅₀ and 9 patients were treated at dose level 3 × 10¹⁰ TCID₅₀. Treatment was well tolerated and no DLTs were experienced at either dose level. The top ten worst grade adverse events are listed in Supplementary Fig. S1. Grade 3 toxicities were minimal and included 3 patients with neutropenia, 2 with leukopenia, and one each with thrombocytopenia and hypophosphatemia. Grade 2 toxicities included leukopenia, anemia, neutropenia, thrombocytopenia, and myalgias. Other grade 1 toxicities included increased aspartate aminotransferase and alkaline phosphatase, lymphopenia, and nausea. All grade 2 and 3 toxicities occurred in patients treated at dose level 2. Five different patients had evidence of grade 2 and/or 3 hematologic toxicities reported as possibly or probably related to Reolysin. One patient had pancytopenia on day 4 of cycle 1. Resolution was evident by day 11 of cycle 1, and this patient had an associated increase of serum lambda-free light chain from 1,435 to 3,000 mg/L over the course of cycle 1. A second patient had a decrease of the neutrophil and total white blood cell count evident on day 8 of cycle 1. These adverse events were associated with an initial 10% decrease of the m-protein, but the patient rapidly progressed and received only one cycle of therapy. A third patient had evidence of decreased neutrophils, total white blood cell count, and platelets on day 4 of cycle 1. All cytopenias resolved by day 23 of cycle 1 and were associated with a minimal decrease of the serum lambda-free light chains during cycle 1. Ultimately, this patient received a total of two cycles of treatment. The fourth patient had stable disease over the course of five cycles of treatment and did not have evidence of cytopenias during cycle 1, but developed grade 3 decreases of the neutrophil and total white blood cell count during cycle 2. These toxicities were evident on day 4 of cycle 2, and resolved by the start of cycle 3 (see Supplementary Table S2).

IHC analysis of sensitive (RPMI-8226), intermediately resistant (H929), and resistant (OPM2) multiple myeloma cell lines indicated two primary findings: (i) reoviral RNA was present in all cell lines and increased with higher multiplicities of infection (MOI) and (ii) viral capsid protein was evident in all cell lines, but to a greater degree in sensitive cells treated at higher MOI (Fig. 1A–C). Furthermore, these findings indicate that resistance to reovirus may be defined by a lack of viral replication or viral capsid protein expression. IHC analysis was preferred to qRT-PCR in this study for three primary reasons: (i) IHC specifically identified active viral RNA and/or protein inside of the multiple myeloma cells, whereas qRT-PCR would be unable to differentiate the location of viral RNA, whether it be on the cell surface or inside the cell; (ii) ISH methods allowed for coexpression analyses, such that we were able to correlate the detection of reovirus based on cell phenotype (e.g., CD138⁺); and (iii) ISH allowed, in part, to determine the relative degree of viral genome and/or proliferation in infected cells.

Seven patients had adequate tissue samples for both pre- and posttreatment laboratory evaluation (Table 2). Reoviral RNA was found in post-Reolysin bone marrow biopsies in all 7 evaluable patients, and viral protein was found in 5 of the 6 evaluable patient samples. Results confirmed reoviral cell entry as evidenced by positive viral RNA staining in 20% to 100% of myeloma cells (Fig. 1D). There was no association (P = 0.22) between the percentage of myeloma cells positive for viral RNA and the number of cycles of therapy patients received. Reoviral RNA localized to myeloma cells (Fig. 2A and B). Of the evaluable patient samples, reoviral capsid protein expression and caspase-3 staining were minimal, indicating nonproductive infection and limited induction of apoptosis (table 2). Importantly, caspase-3 expression paralleled reoviral capsid protein expression. Expression of p38 was used as an indicator of RAS pathway activation and multiple myeloma cell permissiveness to reoviral infection (22). Of the evaluable patient samples, p38 expression before treatment was evident in a mean of 72% (range 19%–99%) of myeloma cells (defined by CD138 coexpression), and was 94% and 99% in patients experiencing stable disease for 8 and 4 cycles of treatment, respectively.

JAM-1 mediates clathrin-dependent viral internalization and CUG2 has been shown to inhibit the phosphorylation of PKR, so we analyzed their respective roles in reoviral permissiveness in multiple myeloma cells. IHC analysis of JAM-1 and CUG2 in sensitive (RPMI-8226), intermediately resistant (H929), and resistant (OPM2) multiple myeloma cell lines indicated that JAM-1 was expressed in sensitive cell lines only, whereas CUG2 was expressed in all cell lines (Fig. 3A). CUG2 expression was lowest in resistant cell lines and highest in sensitive cell lines. These findings highlighted the possibility that JAM-1 and CUG2 may be markers of reovirus sensitivity in multiple myeloma cells.

Patient samples were analyzed for coexpression of JAM-1/CD138 and CUG2/CD138. Low-null JAM-1 expression was evident in all evaluable patient samples, suggesting that decreased tropism of the virus for multiple myeloma cells is associated with decreased viral efficiency in vivo. CUG2 was evident in two of the evaluable patient samples. One patient had evidence of CUG2 expression in the majority of multiple myeloma cells pre- and posttreatment (Fig. 3B). This patient progressed after two cycles of treatment and had relatively high p38 expression (98%) on pretreatment sample analysis. Analysis of pre- and posttreatment samples from the patient experiencing the longest duration of stable disease indicated only scattered CUG2 expression. Pretreatment analysis revealed that this patient also had relatively high p38 expression (94%).

Evaluation of the anti-Reolysin immunologic response revealed that all patients had NARA titers in their serum posttreatment. These data indicate that each patient developed neutralizing antibodies against reovirus (Fig. 4). Trend analysis indicated a significant increase (P = 0.001) in NARA titers from pretreatment to week 3. Evaluating the 3 patients with the longest duration of stable disease (4, 5, and 8 months) suggested higher week 1 and 2 endpoint titers in the patient receiving eight cycles of treatment. These data suggest that NARA antibodies may be indicative of a beneficial antitumor immune response mediated by the activation of a cross-reactive T-cell population.

All twelve enrolled patients were eligible for response assessment. A total of 6 patients (50%) received more than one cycle of treatment, the median cycles of therapy was 1.5, and the best response was stable disease. One patient received 3 × 10⁹ TCID₅₀ and had minimal evidence of apoptosis (caspase-3) on the posttreatment bone marrow biopsy. This patient completed eight cycles of treatment and experienced stable disease for 8 months. Significantly, this patient was also noted to have a progressive increase of NARA and a concomitant decrease in the monoclonal protein during cycle 1. Two other patients receiving 3 × 10¹⁰ TCID₅₀ had stable disease and were each treated for four and five cycles, both had stable monoclonal proteins during cycle 1, but caspase-3 staining was not determined on posttreatment bone marrow samples. During cycle 1, a total of 5 patients had decreased myeloma proteins (range 6%–22%, mean 14%), 3 had minimal increases (6, 7, and 12%), and the remaining 4 had evidence of grossly progressive disease.

The purpose of this study was to establish the safety and tolerability of Reolysin in patients with relapsed or refractory multiple myeloma. We have successfully shown that two dose levels of Reolysin (3 × 10⁹ TCID₅₀/day and 3 × 10¹⁰ TCID₅₀/day) were well tolerated. No DLTs were experienced and grade 3 toxicities were limited to neutropenia, leukopenia, thrombocytopenia, and hypophosphatemia.

Single-agent Reolysin was not associated with any objective responses in relapsed and refractory multiple myeloma patients. Six of the 12 (50%) patients in this study received more than one cycle of therapy and 3 patients received 4, 5, and 8 cycles of treatment. The longest duration of stable disease was 8 months. This patient was treated with the 3 × 10⁹ TCID₅₀/day dose, had evidence of reoviral capsid protein expression in CD138⁺ cells, apoptosis on the posttreatment bone marrow biopsy (Table 2), and a 13% decrease in the monoclonal protein during cycle 1.

We propose that the lack of objective clinical response in our population resulted from a combination of potential factors, including (i) an inability to overcome inherent viral resistance for productive cytolysis, (ii) limited production of a viral-mediated antitumor immune response, and/or (iii) inadequate viral dosing.

As seen in our patient population (Fig. 1D), and supported by our in vitro analyses of sensitive, intermediately resistant, and resistant multiple myeloma cell lines (Fig. 1A–C), the presence of intracellular viral genome but not reoviral capsid protein suggested resistance to active viral proliferation. Of the myriad of factors that could mediate this resistance, we evaluated the roles of cell entry and RAS pathway activation.

Preclinical models suggest that reoviral cell entry is initiated by the engagement of the outer viral capsid filamentous attachment protein σ1 with JAM-1 and a cell-surface carbohydrate. Once attached, β1-integrin–mediated viral internalization occurs via clathrin-dependent endocytosis. Following internalization, acid-dependent cysteine proteases in the endocytic compartment remove the outer capsid protein σ3, exposing a viral membrane-penetration protein that is responsible for piercing the endosomal membrane and delivering transcriptionally active virus into the cytoplasm (23).

Whether these fundamental principles accurately define the in vivo mechanisms of cell entry and viral proliferation in multiple myeloma cells is unknown. To elucidate the importance of JAM-1, we conducted in vitro analyses of sensitive, intermediately resistant, and resistant cell lines, and correlative analyses of patient samples. In vitro analyses confirmed that JAM-1 expression was positively associated with multiple myeloma cell sensitivity (Fig. 3A). However, IHC analysis of patient samples revealed that JAM-1 was not expressed on myeloma cells. Considering that viral RNA and not protein was evident in patient samples, we propose that reovirus may have passively, albeit selectively, entered the myeloma cells by bypassing JAM-1. This hypothesis assumes that reovirus binding to the sialic acid and JAM-1 receptors is necessary for viral uncoating and subsequent replication. Future strategies using therapeutics that upregulate JAM-1 will likely elucidate whether active clathrin-dependent reoviral cell entry is necessary for viral proliferation, viral-mediated apoptosis, and subsequent clinical benefit.

Reovirus has been shown to preferentially replicate and induce apoptosis in cells with a constitutively activated RAS pathway (22), potentially via EGFR, N- and/or K-RAS mutations. In multiple myeloma, the proportion of RAS mutations increases with disease progression, and 46% to 81% of patients with relapsed disease have been found to have N- or K-RAS mutations (24, 25). Recently, a study evaluating the prevalence of RAS mutations in multiple myeloma patient samples from 133 patients demonstrated N- and K-RAS mutations in similar proportions, and patients with N-RAS–mutated tumors, in comparison with those with wild-type tumors, were less sensitive to bortezomib (25). The mechanisms underlying this decreased bortezomib sensitivity of N-RAS–mutated tumors require further investigation. Regardless, these findings are instrumental in confirming the equivocal prevalence of N- and K-RAS mutations in multiple myeloma.

The role of a constitutively activated RAS pathway in multiple myeloma patients treated with Reolysin was explored in our trial. Numerous studies suggest that reovirus inhibits double-stranded RNA-activated protein kinase (PKR) autophosphorylation in RAS-mutated cells, an effect that permits viral replication to occur (2–6). Others suggest that RAS transformation enhances numerous steps in the viral life cycle, including proteolytic disassembly, degree of infectivity, and apoptosis-mediated viral release (26). We evaluated p38 MAPK as a marker of RAS-pathway activation in our relapsed population (22) and found no obvious association between p38, length of disease stability, or the amount of viral protein present in malignant cells (Table 2). These findings suggest that measurement of p38 is not a marker for reoviral sensitivity, intracellular replicative activity, or efficacy in patients with multiple myeloma.

Cancer upregulated gene 2 (CUG2) is a novel oncogene that is overexpressed in numerous malignancies. Like RAS mutations, CUG2 expression is associated with inactivation of PKR and activation of the RAS/p38 MAPK signaling pathway (27). We evaluated the role of CUG2 in vitro and in patient samples. Preclinical analysis of sensitive, intermediately resistant, and resistant multiple myeloma and melanoma cell lines indicated that CUG2 was more highly expressed in sensitive cell lines (Fig. 3A). In these models, increased viral load and concomitant exposure to chemotherapy increased CUG2 expression, viral proliferation, and apoptosis (Fig. 3C). These results suggest that in vitro, CUG2 expression enhances reovirus sensitivity and cytolysis. Analysis of patient samples revealed that 2 of the 7 evaluable patients had evidence of CUG2 expression. One patient had significant pre- and posttreatment CUG2 expression in multiple myeloma cells (Fig. 3B), viral genome in 100% of cells, elevated pretreatment p38 expression (98%), minimal reovirus capsid protein and apoptotic activity, and received two cycles of treatment (patient C). In the other patient, CUG2 expression was scattered, 36% of cells contained viral genome, pretreatment p38 expression was high (94%), reoviral capsid protein, and apoptosis were minimal, and the patient received eight cycles of treatment (patient A). Collectively, these results suggest that CUG2 expression may be an indicator of RAS/p38 pathway activation and entry of the virus into multiple myeloma cells, but not productive infection or clinical response.

Natural killer (NK) and T-cell–mediated killing has a prominent role in reovirus-mediated antimultiple myeloma activity (28, 29). Dendritic cells (DC) have the ability to directly respond to reoviral RNA via pattern recognition receptors (PRR) in a viral replication independent manner (30). DC activation by reovirus induces the upregulation of numerous proinflammatory cytokines including IFNs that function, in part, to increase NK cell activation and recruitment. Extensive cross-talk between DCs and NK cells via IFN subsequently acts to generate a tumor-reactive cytotoxic T-cell response; one that is likely strengthened by production of viral- and NK cell-mediated tumor-associated antigens (28). Numerous patients in our trial had experienced stable disease that was not associated with significant viral proliferation or apoptosis. This finding suggests that disease stability may have resulted from immune-mediated cytotoxicity.

Virus clearance may be mediated by NARA, but the degree to which its activity limits reovirus-mediated antimultiple myeloma activity is unknown. Evidence from the REO 005 trial indicates that antitumor activity persisted despite a 250-fold median increase in NARA titers (20), likely because the virus “hitchhikes” on peripheral blood mononuclear cells and platelets (31). In our trial, a significant increase in NARA titers was evident over the first cycle of treatment (Fig. 4). Though our findings are not conclusive, it is unlikely that NARA is able to clear a significant proportion of infused reovirus to preclude antitumor activity in patients with myeloma, and in fact, may be instrumental in activating a beneficial antitumor immune response.

Use of viral oncolytics as cytolytic therapy is gaining momentum. Recently, Russel and colleagues reported on 2 patients with refractory multiple myeloma that were treated with 10¹¹ TCID₅₀ MV-NIS, an engineered measles virus (32). Initial infusion resulted in hypotension, tachycardia, and high fever in both patients, but each responded to treatment and one achieved complete remission lasting for over 9 months. In fact, as this was a dose-escalation trial starting at 10⁶ TCID₅₀, no objective responses were noted until the administration of 10¹¹ TCID₅₀, results suggesting that treatment at or near the MTD is necessary to achieve objective responses. Reolysin as monotherapy or combined with chemotherapy has not reached the MTD with individual doses up to 3 × 10¹⁰ TCID₅₀ for 5 consecutive days with cycles repeated every 21 to 28 days (total cycle dose of 1.5 × 10¹²). Currently, the NCIC Clinical Trials Group is conducting a 4-arm phase II trial (www.clinicaltrials.gov - NCT01708993) and this trial is the first to evaluate higher individual doses of Reolysin at 4.5 × 10¹⁰ TCID₅₀, days 1 to 3 every 3 weeks, total cycle dose 1.35 × 10¹¹ TCID₅₀. Considering the dose–response relationship evident with other viral oncolytics (33–36), higher individual doses of Reolysin may be necessary to induce clinical benefit in patients with multiple myeloma.

Clearly, further study will be necessary to define a role for Reolysin treatment in multiple myeloma. Our trial, however, shows that Reolysin is well tolerated and associated with disease stability, though the mechanisms underlying this activity remain unclear. Importantly, our study lays the groundwork for future clinical trials that will further examine the role for Reolysin in treatment refractory multiple myeloma patients, either as a single agent or in combination.

No potential conflicts of interest were disclosed.

The content is solely the responsibility of the authors and does not represent the official views of the National Cancer Institute or the NIH.

Conception and design: G.B. Lesinski, C.C. Hofmeister

Development of methodology: G.J. Nuovo, G.B. Lesinski, C.C. Hofmeister

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): G.J. Nuovo, A. Stiff, G.B. Lesinski, D.M. Benson Jr, C.C. Hofmeister

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D.W. Sborov, G.J. Nuovo, A. Stiff, T.A. Mace, G.B. Lesinski, M.R. Grever, C.C. Hofmeister

Writing, review, and/or revision of the manuscript: D.W. Sborov, G.J. Nuovo, A. Stiff, T.A. Mace, G.B. Lesinski, D.M. Benson Jr, Y.A. Efebera, A.E. Rosko, F. Pichiorri, M.R. Grever, C.C. Hofmeister

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D.W. Sborov

Study supervision: G.B. Lesinski, C.C. Hofmeister

Other (enrolled patients): Y.A. Efebera

This work was supported by the generous contributions of the Multiple Myeloma Opportunities for Research and Education (MMORE) and National Cancer Institute of the NIH under Award Number U01CA076576 (to PI M.R. Grever). D.W. Sborov was supported under Award Number T32CA165998 (PI Miguel Villalona and Steven Devine). G.J. Nuovo and G.B. Lesinski received research funding from Oncolytics Biotech Inc.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.