Matthew J. O'Shaughnessy

Hematopoietic reconstitution by multipotent adult progenitor cells: precursors to long-term hematopoietic stem cells

For decades, in vitro expansion of transplantable hematopoietic stem cells (HSCs) has been an elusive goal. Here, we demonstrate that multipotent adult progenitor cells (MAPCs), isolated from green fluorescent protein (GFP)-transgenic mice and expanded in vitro for >40–80 population doublings, are capable of multilineage hematopoietic engraftment of immunodeficient mice. Among MAPC-derived GFP+CD45.2+ cells in the bone marrow of engrafted mice, HSCs were present that could radioprotect and reconstitute multilineage hematopoiesis in secondary and tertiary recipients, as well as myeloid and lymphoid hematopoietic progenitor subsets and functional GFP+ MAPC-derived lymphocytes that were functional. Although hematopoietic contribution by MAPCs was comparable to control KTLS HSCs, approximately 103-fold more MAPCs were required for efficient engraftment. Because GFP+ host-derived CD45.1+ cells were not observed, fusion is not likely to account for the generation of HSCs by MAPCs.

Multipotent adult progenitor cells can suppress graft-versus-host disease via prostaglandin E2 synthesis and only if localized to sites of allopriming

Multipotent adult progenitor cells (MAPCs) are nonhematopoietic stem cells capable of giving rise to a broad range of tissue cells. As such, MAPCs hold promise for tissue injury repair after transplant. In vitro, MAPCs potently suppressed allogeneic T-cell activation and proliferation in a dose-dependent, cell contact-independent, and T-regulatory cell-independent manner. Suppression occurred primarily through prostaglandin E(2) synthesis in MAPCs, which resulted in decreased proinflammatory cytokine production. When given systemically, MAPCs did not home to sites of allopriming and did not suppress graft-versus-host disease (GVHD). To ensure that MAPCs would colocalize with donor T cells, MAPCs were injected directly into the spleen at bone marrow transplantation. MAPCs limited donor T-cell proliferation and GVHD-induced injury via prostaglandin E(2) synthesis in vivo. Moreover, MAPCs altered the balance away from positive and toward inhibitory costimulatory pathway expression in splenic T cells and antigen-presenting cells. These findings are the first to describe the immunosuppressive capacity and mechanism of MAPC-induced suppression of T-cell alloresponses and illustrate the requirement for MAPC colocalization to sites of initial donor T-cell activation for GVHD inhibition. Such data have implications for the use of allogeneic MAPCs and possibly other immunomodulatory nonhematopoietic stem cells for preventing GVHD in the clinic.

Ex vivo Inhibition of NF-κB Signaling in Alloreactive T-cells Prevents Graft-versus-host Disease

The ex vivo induction of alloantigen‐specific hyporesponsiveness by costimulatory pathway blockade or exposure to immunoregulatory cytokines has been shown to inhibit proliferation, IL‐2 production, and the graft‐versus‐host disease (GVHD) capacity of adoptively transferred T‐cells. We hypothesized that inhibition of the intracellular NF‐κB pathway in alloreactive T‐cells, which is critical for T‐cell activation events including IL‐2 transcription, could lead to alloantigen hyporesponsiveness and loss of GVHD capacity. We demonstrate that treatment of mixed lymphocyte reaction (MLR) cultures with PS1145, a potent inhibitor of NF‐κB activation, can induce T‐cell hyporesponsiveness to alloantigen in primary and secondary responses while preserving in vitro responses to potent mitogenic stimulation. GVHD lethality in recipients of ex vivo PS1145‐treated cells was profoundly inhibited. Parking of control or PS1145‐treated MLR cells in syngeneic Rag−/− recipients resulted in intact contact hypersensitivity (CHS) responses. However, GVHD lethality capacity also was restored, suggesting that lymphopenic expansion uncoupled alloantigen hyporesponsiveness. These results indicate that the NF‐κB pathway is a critical regulator of alloresponses and provide a novel small molecule inhibitor based approach that is effective in preventing early posttransplant GVHD lethality but that also permits donor T‐cell responses to recover after a period of lymphopenic expansion.

Outgrowth of CD4low/negCD25+ T Cells with Suppressor Function in CD4+CD25+ T Cell Cultures upon Polyclonal Stimulation Ex Vivo

CD4+CD25+ regulatory T cells (Tregs) play an essential role in controlling autoimmunity and allograft rejection. Several ex vivo activation and expansion protocols have been developed to amplify cell numbers and suppressor function of murine and human Tregs. We demonstrate in this study that ex vivo activation and expansion of murine Tregs resulted in an enrichment of a CD4low/negCD25+ T cell population that was more than 20-fold more potent than expanded conventional Tregs in suppressing an in vitro CD4+CD25− T cell response to allo-Ag. The generation of CD4low/negCD25+ T cells was independent of the presence of Tregs in the culture, and suppressor function was acquired only after activation and expansion. CD4low/negCD25+ T cells expressed either an αβ or γδ TCR, had an activated phenotype, and did not express the transcription factor FoxP3. Despite expressing the cell surface Ags lymphocyte activation gene-3 (CD223) and CD103, neither was essential for suppressor cell function. Suppression by CD4low/negCD25+ T cells was prevented by a semipermeable membrane and was independent of IL-10 and TGF-β. In summary, we describe in this study CD4low/negCD25+ FoxP3neg T cells with highly potent suppressor cell function derived from cultures of an enriched population of CD4+CD25+ T cells that may contribute to the suppressor activity of ex vivo expanded bone fide Tregs.

Drug development for noncastrate prostate cancer in a changed therapeutic landscape

The unprecedented progress in the treatment of metastatic castration-resistant prostate cancer is only beginning to be realized in patients with noncastrate disease. This slow progress in part reflects the use of trial objectives focused on time-to-event end points, such as time to metastasis and overall survival, which require long follow-up durations and large sample sizes, and has been further delayed by the use of approved therapies that are effective at the time of progression. Our central hypotheses are that progress can be accelerated, and that outcomes can be improved by shifting trial objectives to response measures occurring early that solely reflect the effects of the treatment. To test these hypotheses, a continuously enrolling multi-arm, multi-stage randomized trial design, analogous to that used in the STAMPEDE trial, has been developed. Eligibility is focused on patients with incurable disease or those with a high risk of death with any form of monotherapy alone. The primary objective is to eliminate all disease using a multimodality treatment strategy. End points include pathological complete response and an undetectable level of serum prostate-specific antigen, with recovery of serum testosterone levels. Both are binary, objective, and provide an early, quantitative indication of efficacy.

PD24-11 EVALUATION OF A MULTIMODAL STRATEGY TO ACCELERATE DRUG EVALUATIONS IN EARLY STAGE METASTATIC PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: The paradigm of first testing systemic treatments in advanced disease followed by development in earlier disease states and finally large-scale trials evaluating whether the approach, in combination with local therapy, can prevent or delay the time-to-event measures of disease progression or death in patients with 00high-risk00 tumors is no longer practical now that 6 lifeprolonging systemic therapies in metastatic castration-resistant prostate cancer are available. Our objective was to evaluate a multimodal treatment platform and a short term endpoint of treatment efficacy as a new strategy to rapidly evaluate and prioritize regimens for large-scale phase 3 testing. METHODS: We conducted a pilot study of twenty men with oligometastatic M1a (extrapelvic nodal disease) or M1b (bone disease) at diagnosis. All sites of disease were treated using a multimodal approach that included androgen deprivation (ADT), radical prostatectomy plus pelvic lymphadenectomy (retroperitoneal lymphadenectomy in the presence of clinically positive retroperitoneal nodes), and stereotactic body radiotherapy to osseous disease and/or the primary site. ADT was discontinued in responding patients. Outcomes of each treatment were assessed sequentially. The primary endpoint of 00no evidence of disease00 (NED) was defined by an undetectable PSA (<0.05 ng/mL) with noncastrate levels of testosterone at 20 months (>150 ng/dL). RESULTS: Each treatment modality contributed to the outcome: 95% of the cohort achieved an undetectable PSA with multimodal treatment, including 25% of patients after ADT alone and an additional 50% and 20% after surgery and radiotherapy, respectively. Overall, 20% of patients (95% confidence interval 3-38%) achieved the primary endpoint, which persisted for 5, 6, 27+, and 46+ months. All patients meeting the primary endpoint had been classified with M1b disease at presentation. CONCLUSIONS: Treatment directed at all sites can eliminate detectable disease in selected patients with newly diagnosed metastatic prostate cancer. A multimodal treatment strategy inclusive of the NED endpoint for patients who present with disease that is beyond the limits of curability by any single modality should be considered to enable the evaluation of new approaches in order to prioritize large-scale testing in early stages of advanced disease.

PD33-03 TREATMENT OF THE PRIMARY TUMOR WITH VASCULAR-TARGETED PHOTODYNAMIC THERAPY (VTP) OVERCOMES RESISTANCE TO SYSTEMIC PD-1/PD-L1 INHIBITION: EFFECTS ON PRIMARY TUMOR CONTROL AND PREVENTION OF LUNG METASTASIS IN A PRE-CLINICAL RCC MODEL

genomic landscape in clear cell renal cell carcinoma (ccRCC). We propose here a novel strategy of capturing variant alleles present in different tumor regions by sampling multiple tumor sites and combining all DNA to be sequenced as one sample. METHODS: Ex vivo core needle biopsies were obtained from six geographically different regions of five primary renal tumors that were resected via partial or radical nephrectomy at a single institution from 4/2014 to 7/2014. All patients had signed informed consents for tissue utilization, and our institutional review board had approved the study. DNA was extracted using standard techniques. Each tumor region was sequenced individually (R1-R6) and also combined together and sequenced as pooled sample. Tumor DNA and matched normal DNA were profiled for genomic alterations in 341 key cancer-associated genes using our custom, deep sequencing MSK-IMPACT assay (Integrated Mutation Profiling of Actionable Cancer Targets). RESULTS: There were 40 samples sequenced: 5 patients x (6 regions þ 1 pool þ 1 normal), and the average sequence coverage is 905x (range 509x e 1920x). 5/5 tumors were clear cell RCC and 4/5 tumors were stage pT3 or higher. The median tumor size was 6.0 cm (IQR 4.2 e 7.5 cm). All five tumors had a VHL mutation present, with each one being present in all six regions and the pooled sample. Other genes including BAP1, KDM5C, TSC1, TP53, TRAF7, and ERBB2 exhibited various degrees of regional heterogeneity. All variant alleles detected in a single tumor region were also detected in that tumor’s pooled DNA sample (Figure 1). CONCLUSIONS: We present here a feasible, cost effective strategy for accurately profiling a renal tumor’s genetic landscape while overcoming the obstacle of intratumor heterogeneity. By pooling DNA from multiple tumor regions and performing ultra deep sequencing on this combined sample, we were able to detect every mutation that was captured when sequencing each region individually.

78 INTENSITY-MODULATED RADIOTHERAPY REPLACES 3-DIMENSIONAL EXTERNAL BEAM AS THE PREFERRED RADIOTHERAPY MODALITY IN PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: Intensity-modulated radiotherapy (IMRT) has been shown to improve local disease control while minimizing adverse effects, compared to 3-dimensional external beam radiotherapy (3-D EBRT). The uptake of IMRT in common clinical practice has not been previously described. Our objectives were to use the Surveillance, Epidemiology, and End Resultse Medicare (SEERMedicare) dataset to (1) analyze time trends in the type of radiotherapy (RT) used for primary local treatment for prostate cancer and (2) identify disease characteristics associated with trends in the type of RT used. METHODS: From the linked SEER-Medicare dataset (inclusive of years 2000-2005), we identified cases of incident non-metastatic prostate cancer managed with radiation within 12 months of diagnosis. Medicare claims for IMRT, 3-D EBRT, brachytherapy (BT) and any combination thereof were identified. The frequency of each RT strategy was documented over time and then stratified by patient demographic and tumor characteristics and compared by chi-square. Tumor characteristics were categorized as low risk (T1, WHO grade 1 or 2), intermediate risk (T2, WHO grade 1 or 2) and high risk ( T3 or WHO grade 3). RESULTS: 42,151 patients who received primary radiotherapy for prostate cancer were identified. The rate of IMRT use significantly increased from 0 to 46.4% (P 0.0001) of all patients undergoing RT, whereas EBRT use declined from 55.1% to 9.2% (P 0.0001) over the same interval. BT EBRT combination therapy decreased from 21.7% to 6.3% (P 0.0001) whereas BT IMRT combination increased from 0% to 12.9% (P 0.0001). BT use as monotherapy peaked in 2002 at 43.5% but decreased over the subsequent three years to 25.2%. When the results were stratified by tumor risk, similar trends were seen. BT is more common for low and intermediate risk prostate cancer. CONCLUSIONS: Our population-based study using SEERMedicare data shows that IMRT has largely replaced EBRT as the preferred radiotherapy for localized prostate cancer, regardless of risk category. Factors contributing to the increased use of IMRT include the possibility of delivering higher doses of radiation more safely and more favorable reimbursement.