Greiner, Suzanne

Abstract Multiple myeloma is a highly radiosensitive malignancy but, at the present time, radionuclide-based interventions have no proven place in disease management. Bone-seeking radionuclides such as 153-Sm-EDTMP and 166-Ho-DOTMP are promising agents for systemic delivery of ionizing radiation to sites of myeloma disease activity, but they are associated with significant myelosuppression at therapeutically effective doses and have therefore been used only in the context of myeloma stem cell transplantation protocols. We previously reported that the proteasome inhibitor PS-341 potently and selectively sensitizes myeloma cell lines and primary myeloma cells to the lethal effects of ionizing X-irradiation (Goel et al, Exp Hematol. 33, 784, 2005). To determine whether PS-341 is equally effective in sensitizing myeloma cells to ionizing beta-radiation and to extend our initial observations to an in vivo model, we combined PS-341 with the bone-seeking radionuclide 153-Sm-EDTMP. In vitro clonogenic assays were performed using a panel of myeloma cell lines and demonstrated synergistic killing following co-treatment with PS-341 and 153-Sm-EDTMP. Using the orthotopic, syngeneic 5TGM1 myeloma model, the median survivals of mice treated with saline, two doses of PS-341 (0.5 mg/kg), or a single non-myeloablative dose of 153-Sm-EDTMP (22.5 MBq) were 21, 22 and 28 days, respectively. In contrast, mice treated with combination therapy comprising two doses of PS-341 (0.5 mg/kg), one day prior to and one day following 153-Sm-EDTMP (22.5 MBq) showed a greatly prolonged median survival of 49 days. Correlative studies indicated that, compared to single-agent therapy, combination treatment with PS-341 and 153-Sm-EDTMP rapidly reduced the clonogenicity of bone-marrow resident 5TGM1 cells, slowed the elevation of serum myeloma-associated paraprotein levels, and was associated with longer term preservation of bone mineral density. The myelotoxicity of single agent and combination therapies was evaluated by comparing peripheral blood cell counts in each of the treatment groups, and by clonogenicity assays of hematopoietic progenitors isolated form normal mice receiving identical treatment regimens. Treatment with 153-Sm-EDTMP led to significant, but transient, myelosuppression which did not differ between animals treated with 153-Sm-EDTMP alone versus those treated with the combination of PS-341 plus 153-Sm-EDTMP. In summary, PS-341 is a potent in vivo radiosensitizer that greatly enhances the therapeutic potency, without increasing myelotoxicity, of skeletal targeted radiotherapy in the syngeneic, orthotopic 5TGM1 myeloma model. Based on these findings, we propose to conduct a phase I clinical trial at Mayo Clinic to evaluate the combination of PS-341 plus non-myeloablative skeletal targeted radiotherapy (using 153-Sm-EDTMP) in patients with advanced or treatment-refractory multiple myeloma.

Disease monitoring in nonalcoholic steatohepatitis (NASH) is limited by absence of noninvasive biomarkers of disease regression or progression. We aimed to examine the role of multiparametric three‐dimensional magnetic resonance elastography (3D‐MRE) and magnetic resonance imaging proton density fat fraction (MRI‐PDFF) in the detection of NASH regression after interventions. This is a single‐center prospective clinical trial of 40 patients who underwent bariatric surgery. Imaging and liver biopsies were obtained at baseline and 1 year after surgery. The imaging protocol consisted of multifrequency 3D‐MRE to determine the shear stiffness at 60 Hz and damping ratio at 40 Hz, and MRI‐PDFF to measure the fat fraction. A logistic regression model including these three parameters was previously found to correlate with NASH. We assessed the model performance in the detection of NASH resolution after surgery by comparing the image‐predicted change in NAFLD activity score (delta NAS) to the histologic changes. A total of 38 patients (median age 43, 87% female, 30 of 38 with NAS ≥ 1, and 13 of 38 with NASH) had complete data at 1 year. The NAS decreased in all subjects with NAS ≥ 1 at index biopsy, and NASH resolved in all 13. There was a strong correlation between the predicted delta NAS by imaging and the delta NAS by histology (r = 0.73, P < 0.001). The strength of correlation between histology and the predicted delta NAS using single conventional parameters, such as the fat fraction by MRI‐PDFF or shear stiffness at 60 Hz by MRE, was r = 0.69 (P < 0.001) and r = 0.43 (P = 0.009), respectively. Conclusion: Multiparametric 3D‐MRE and MRI‐PDFF can detect histologic changes of NASH resolution after bariatric surgery. Studies in a nonbariatric setting are needed to confirm the performance as a composite noninvasive biomarker for longitudinal NASH monitoring.

Abstract Background: MV-NIS is an Edmonston-lineage measles virus that expresses the human sodium-iodide symporter (hNIS). The virus is oncolytic, and its activity can be monitored by noninvasive imaging of radioiodine uptake by hNIS. The receptor is CD46, a membrane regulator of complement activation that is overexpressed on myeloma cells. Methods: Formal studies were done in collaboration with the RAID program to assess the efficacy, biodistribution and safety of MV-NIS in transgenic, measles susceptible mice and in non-human primates. The protocol therapy for the animal studies was modeled on the planned human clinical trial. Mouse efficacy studies were done in irradiated 6-week old female CB17 ICR SCID mice implanted subcutaneously with human KAS-6/1 cells. Mouse toxicity & biodistribution were done in 184 6–8 week IfnarKO x CD46 Ge mice treated with 105–107 TCID50 MV-NIS. Squirrel monkey toxicity studies were done in 12 measles-naïve adults with 108 TCID50. The clinical trial is a 3 patient/cohort dose escalation study which allows for 1 dose of MV-NIS/patient. Once MTD of MV-NIS is reached, subsequent cohorts will be treated with CTX 10 mg/kg 2 days prior to MV-NIS at MTD/100. Immune response to measles is tested pre & post therapy. PK & biodistribution are tested by measurements of MV-N in blood, urine, & gargle samples and by serial nuclear imaging, respectively. BM myeloma cells are tested for CD46 expression and MV infectivity. Results: Preclinical studies: The minimum effective dose in the mouse model was 4 x 106 TCID50 /kg. The MTD for MV-NIS with CTX was not reached in the mouse study. Pre-treatment with CTX changed the biodistribution of MV-NIS infected cells and the kinetics of virus elimination from infected tissues in mice and monkeys. In monkeys, MV-N mRNA was detected at day 1 in the buccal cavity with levels declining on day 2, then surging on day 8, suggesting viral replication. There were no unscheduled deaths or treatment related lesions on necropsy. Patients: Three patients were treated with a dose of 106 TCID50 (Table). No DLT was observed. Two patients had MV-N amplified from their gargle specimen. Accrual to dose level 2 has begun. Conclusions: IV administration of MV-NIS is feasible. The virus is capable of traveling to a distant site and replicating before being cleared by the immune system. Patient 2 Patient 2 Patient 3 *copies per microgram RNA of cells derived from gargle sample at each time point: day +3, +8, +15, +22, and +29; no MV-N detected in urine or blood Anti-measles neutralizing Ab titer, pre-Rx / post Rx 1:10 / 1:160 < 1:10 / 1:20 < 1:10 / 1:10 AEs possibly related Gr 2 plts Gr 1 plts, cough, fever, rigors Gr 2 Incr aPTT CD46 copies/cell CD138+ / CD138- BMPC 79,771 / 10,322 140,187 / 10,145 28,258 / 11,228 MV-N QRT-PCR: d3/ d8/ d15/ d22/ d29 (cp/ug RNA)* −/ −/ −/ nd / nd −/ −/ 16,440 / 1405 / − 123-I uptake on SPECT No Uptake day 8 No

The lack of reliable, noninvasive methods to diagnose early nonalcoholic steatohepatitis (NASH) is a major unmet need. We aimed to determine the diagnostic accuracy of three‐dimensional magnetic resonance elastography (3D‐MRE), with shear stiffness measured at 60 Hz, damping ratio at 40 Hz, and magnetic resonance imaging proton density fat fraction (MRI‐PDFF) in the detection of NASH in individuals undergoing bariatric surgery. Obese adults at risk for NASH were enrolled between 2015 and 2017 (prospective cohort, n = 88) and 2010 and 2013 (retrospective cohort, n = 87). The imaging protocol consisted of multifrequency 3D‐MRE (mf3D‐MRE) with shear waves delivered at different frequencies to explore parameters that best correlated with histologic NASH, and MRI‐PDFF to estimate steatosis. The prospective cohort was used to establish the optimal mf3D‐MRE technical parameters for NASH detection. The two cohorts were then combined to derive predictive models of NASH and disease activity by nonalcoholic fatty liver disease activity score (NAS) using the three imaging parameters that correlated with NASH. A total of 175 patients (median age 45, 81% women, and 81 [46%] with histologic NASH) were used for model derivation. From the complex shear modulus output generated by mf3D‐MRE, the damping ratio at 40 Hz and shear stiffness at 60 Hz best correlated with NASH. The fat fraction obtained from MRI‐PDFF correlated with steatosis ( P < 0.05 for all). These three parameters were fit into a logistic regression model that predicted NASH with cross‐validated area under the receiver operating characteristic curve (AUROC) = 0.73, sensitivity = 0.67, specificity = 0.80, positive predictive value = 0.73 and negative predictive value = 0.74, and disease activity by NAS with cross‐validated AUROC = 0.82. Conclusion: The mf3D‐MRE allows identification of imaging parameters that predict early NASH and disease activity. This imaging biomarker represents a promising alternative to liver biopsy for NASH diagnosis and monitoring. The results provide motivation for further studies in nonbariatric cohorts.

Abstract Bowel obstruction is common sequelae of advanced ovarian cancer. Reports suggest that from 5.5 percent to 51 percent of ovarian cancer patients with Stage III or Stage IV disease develop a malignant bowel obstruction. During the course of the disease, one-third of all ovarian cancer patients also will develop pleural effusions, and three-quarters of these contain malignant cells. Such complications adversely affect both survival and quality of life. Our murine primary patient ovarian cancer intraperitoneal tumorgrafts (Avatars) maintain high fidelity of genetic aberrations from the original patient tumor, thus representing a clinically relevant model system. Expression microarray data of Avatars with preferential metastasis to the bowel has revealed upregulation of IGF2. To determine if IGF2 overexpression was sufficient for metastasis, four patient Avatars were transfected with IGF2/Gluc. In order to minimize the potential for genetic drift sometimes seen with in vitro culture, pre-transplantation tumors were minced and incubated with lentiviruses (Control vector or IGF-2/Gluc expression vector) ex-in vivo for a short time (2 hours), then directly transplanted intraperitoneally into immunodeficient mice (five or six mice in each Avatar model). Total 32 animals were used in this study. Stable transfection and overexpression of human IGF-2 was achieved and maintained after three passages as determined by ELISA on tumor tissue lysates. Only one out of 32 mice did not engraft tumor after three months, and tumor taking rate was 96.875%, similar to our previous passage successful rate at 98% and indicate that our ex-in vivo transfection method and overexpression of human IGF-2 did not substantially affect the tumor taking rate. By using Xenogen IVIS200 imaging system (Xenogen Corp.), pleural fluid was observed in all the Avatar mice with tumors overexpressing IGF-2 or not, and the retrieved pleural fluids were positive for malignant cells. However higher pleural nodules forming rate was more common in mice with IGF-2 overexpressing tumors 77% (n=9) vs 40% (n=6). In model PH003/IGF-2, the tumor had 100% bowel metastasis (n=9) compare to 60% in the control group (n=6). Taken together, our Avatar models not only effectively recapitulate ovarian cancer abdominal peritoneum metastasis, but also can mimic advanced ovarian cancer with malignant pleural fluid. The IGF-2 overexpression enhanced metastatic potential relative to control tumor. These results also indicate that the ex-in vivo transfection method developed in this study may be a viable approach for biological research and therapies. This work is funded by the Mayo Ovarian SPORE (CA136393). Citation Format: Xiaonan Hou, Marc A Becker, Saravut J Weroha, Kristina A Butler, Suzanne M Greiner, Paul Haluksa. Overexpression of IGF-2 in primary patient ovarian cancer tumorgrafts increases bowel and thoracic metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1196. doi:10.1158/1538-7445.AM2014-1196

Multiple myeloma is a radiosensitive malignancy that is currently incurable. Here, we generated a novel recombinant vesicular stomatitis virus [VSV(Δ51)-NIS] that has a deletion of methionine 51 in the matrix protein and expresses the human sodium iodide symporter (NIS) gene. VSV(Δ51)-NIS showed specific oncolytic activity against myeloma cell lines and primary myeloma cells and was able to replicate to high titers in myeloma cells in vitro. Iodide uptake assays showed accumulation of radioactive iodide in VSV(Δ51)-NIS–infected myeloma cells that was specific to the function of the NIS transgene. In bg/nd/xid mice with established subcutaneous myeloma tumors, administration of VSV(Δ51)-NIS resulted in high intratumoral virus replication and tumor regression. VSV-associated neurotoxicity was not observed. Intratumoral spread of the infection was monitored noninvasively by serial gamma camera imaging of 123I-iodide biodistribution. Dosimetry calculations based on these images pointed to the feasibility of combination radiovirotherapy with VSV(Δ51)-NIS plus 131I. Immunocompetent mice with syngeneic 5TGM1 myeloma tumors (either subcutaneous or orthotopic) showed significant enhancements of tumor regression and survival when VSV(Δ51)-NIS was combined with 131I. These results show that VSV(Δ51)-NIS is a safe oncolytic agent with significant therapeutic potential in multiple myeloma.

Multiple myeloma is a highly radiosensitive skeletal malignancy, but bone-seeking radionuclides have not yet found their place in disease management. We previously reported that the proteasome inhibitor PS-341 selectively sensitizes myeloma cells to the lethal effects of ionizing radiation. To extend these observations to an in vivo model, we combined PS-341 with the bone-seeking radionuclide 153-Sm-EDTMP. In vitro clonogenic assays demonstrated synergistic killing of myeloma cells exposed to both PS-341 and 153-Sm-EDTMP. Using the orthotopic, syngeneic 5TGM1 myeloma model, the median survivals of mice treated with saline, 2 doses of PS-341 (0.5 mg/kg), or a single nonmyeloablative dose of 153-Sm-EDTMP (22.5 MBq) were 21, 22, and 28 days, respectively. In contrast, mice treated with combination therapy comprising 2 doses of PS-341 (0.5 mg/kg), 1 day prior to and 1 day following 153-Sm-EDTMP (22.5 MBq) showed a significantly prolonged median survival of 49 days (P < .001). In addition to prolonged survival, this treatment combination yielded reduced clonogenicity of bone marrow–resident 5TGM1 cells, reduced serum myeloma–associated paraprotein levels, and better preservation of bone mineral density. Myelosuppression, determined by peripheral blood cell counts and clonogenicity assays of hematopoietic progenitors, did not differ between animals treated with 153-Sm-EDTMP alone versus those treated with the combination of PS-341 plus 153-Sm-EDTMP. PS-341 is a potent, selective in vivo radiosensitizer that may substantially affect the efficacy of skeletal-targeted radiotherapy in multiple myeloma.