Maria C. Russell

CD40‐Specific Costimulation Blockade Enhances Neonatal Porcine Islet Survival in Nonhuman Primates

The widespread clinical implementation of alloislet transplantation as therapy for type 1 diabetes has been hindered by the lack of suitable islet donors. Pig‐to‐human islet xenotransplantation is one strategy with potential to alleviate this shortage. Long‐term survival of porcine islets has been achieved using CD154‐specific antibodies to interrupt the CD40/CD154 costimulation pathway; however, CD154‐specific antibodies seem unlikely candidates for clinical translation. An alternative strategy for CD40/CD154 pathway interruption is use of CD40‐specific antibodies. Herein, we evaluate the ability of a chimeric CD40‐specific monoclonal antibody (Chi220) to protect islet xenografts. Neonatal porcine islets (∼50 000 IEQ/kg) were transplanted intraportally into pancreatectomized diabetic macaques. Immunosuppression consisted of induction therapy with Chi220 and the IL‐2 receptor‐specific antibody basiliximab, and maintenance therapy with sirolimus and the B7‐specific fusion protein belatacept. Chi220 effectively promoted xenoislet engraftment and survival, with five of six treated recipients achieving insulin‐independent normoglycemia (median rejection‐free survival 59 days; mean 90.8 days, maximum 203 days). No thromboembolic phenomena were observed. CD40 represents a promising alternative to CD154 as a therapeutic target, and the efficacy of CD40‐specific antibodies in islet xenotransplantation warrants further investigation.

LFA-1–specific therapy prolongs allograft survival in rhesus macaques

Outcomes in transplantation have been limited by suboptimal long-term graft survival and toxicities associated with current immunosuppressive approaches. T cell costimulation blockade has shown promise as an alternative strategy to avoid the side effects of conventional immunosuppressive therapies, but targeting CD28-mediated costimulation alone has proven insufficient to prevent graft rejection in primates. Donor-specific memory T (TM) cells have been implicated in costimulation blockade-resistant transplant rejection, due to their enhanced effector function and decreased reliance on costimulatory signaling. Thus, we have tested a potential strategy to overcome TM cell-driven rejection by targeting molecules preferentially expressed on these cells, such as the adhesion molecule lymphocyte function-associated antigen 1 (LFA-1). Here, we show that short-term treatment (i.e., induction therapy) with the LFA-1-specific antibody TS-1/22 in combination with either basiliximab (an IL-2Rα-specific mAb) and sirolimus (a mammalian target of rapamycin inhibitor) or belatacept (a high-affinity variant of the CD28 costimulation-blocker CTLA4Ig) prolonged islet allograft survival in nonhuman primates relative to control treatments. Moreover, TS-1/22 masked LFA-1 on TM cells in vivo and inhibited the generation of alloproliferative and cytokine-producing effector T cells that expressed high levels of LFA-1 in vitro. These results support the use of LFA-1-specific induction therapy to neutralize costimulation blockade-resistant populations of T cells and further evaluation of LFA-1-specific therapeutics for use in transplantation.

Nondepleting Anti‐CD40‐Based Therapy Prolongs Allograft Survival in Nonhuman Primates

Costimulation blockade of the CD40/CD154 pathway has been effective at preventing allograft rejection in numerous transplantation models. This strategy has largely depended on mAbs directed against CD154, limiting the potential for translation due to its association with thromboembolic events. Though targeting CD40 as an alternative to CD154 has been successful at preventing allograft rejection in preclinical models, there have been no reports on the effects of CD40‐specific agents in human transplant recipients. This delay in clinical translation may in part be explained by the presence of cellular depletion with many CD40‐specific mAbs. As such, the optimal biologic properties of CD40‐directed immunotherapy remain to be determined. In this report, we have characterized 3A8, a human CD40‐specific mAb and evaluated its efficacy in a rhesus macaque model of islet cell transplantation. Despite partially agonistic properties and the inability to block CD40 binding of soluble CD154 (sCD154) in vitro, 3A8‐based therapy markedly prolonged islet allograft survival without depleting B cells. Our results indicate that the allograft‐protective effects of CD40‐directed costimulation blockade do not require sCD154 blockade, complete antagonism or cellular depletion, and serve to support and guide the continued development of CD40‐specific agents for clinical translation.

Development of a Prognostic Genetic Signature to Predict the Metastatic Risk Associated with Cutaneous Melanoma

Purpose: The development of a genetic signature for the identification of high-risk cutaneous melanoma tumors would provide a valuable prognostic tool with value for stage I and II patients who represent a remarkably heterogeneous group with a 3% to 55% chance of disease progression and death 5 years from diagnosis. Experimental Design: A prognostic 28-gene signature was identified by analysis of microarray expression data. Primary cutaneous melanoma tumor tissue was evaluated by RT-PCR for expression of the signature, and radial basis machine (RBM) modeling was performed to predict risk of metastasis. Results: RBM analysis of cutaneous melanoma tumor gene expression reports low risk (class 1) or high risk (class 2) of metastasis. Metastatic risk was predicted with high accuracy in development (ROC = 0.93) and validation (ROC = 0.91) cohorts of primary cutaneous melanoma tumor tissue. Kaplan–Meier analysis indicated that the 5-year disease-free survival (DFS) rates in the development set were 100% and 38% for predicted classes 1 and 2 cases, respectively (P < 0.0001). DFS rates for the validation set were 97% and 31% for predicted classes 1 and 2 cases, respectively (P < 0.0001). Gene expression profile (GEP), American Joint Committee on Cancer stage, Breslow thickness, ulceration, and age were independent predictors of metastatic risk according to Cox regression analysis. Conclusions: The GEP signature accurately predicts metastasis risk in a multicenter cohort of primary cutaneous melanoma tumors. Preliminary Cox regression analysis indicates that the signature is an independent predictor of metastasis risk in the cohort presented. Clin Cancer Res; 21(1); 175–83. ©2015 AACR.

CD40 Blockade Combines with CTLA4Ig and Sirolimus To Produce Mixed Chimerism in an MHC-defined Rhesus Macaque Transplant Model

In murine models, T‐cell costimulation blockade of the CD28:B7 and CD154:CD40 pathways synergistically promotes immune tolerance after transplantation. While CD28 blockade has been successfully translated to the clinic, translation of blockade of the CD154:CD40 pathway has been less successful, in large part due to thromboembolic complications associated with anti‐CD154 antibodies. Translation of CD40 blockade has also been slow, in part due to the fact that synergy between CD40 blockade and CD28 blockade had not yet been demonstrated in either primate models or humans. Here we show that a novel, nondepleting CD40 monoclonal antibody, 3A8, can combine with combined CTLA4Ig and sirolimus in a well‐established primate bone marrow chimerism‐induction model. Prolonged engraftment required the presence of all three agents during maintenance therapy, and resulted in graft acceptance for the duration of immunosuppressive treatment, with rejection resulting upon immunosuppression withdrawal. Flow cytometric analysis revealed that upregulation of CD95 expression on both CD4+ and CD8+ T cells correlated with rejection, suggesting that CD95 may be a robust biomarker of graft loss. These results are the first to demonstrate prolonged chimerism in primates treated with CD28/mTOR blockade and nondepletional CD40 blockade, and support further investigation of combined costimulation blockade targeting the CD28 and CD40 pathways.

Gene expression profiling for molecular staging of cutaneous melanoma in patients undergoing sentinel lymph node biopsy.

BACKGROUND A gene expression profile (GEP) test able to accurately identify risk of metastasis for patients with cutaneous melanoma has been clinically validated. OBJECTIVE We aimed for assessment of the prognostic accuracy of GEP and sentinel lymph node biopsy (SLNB) tests, independently and in combination, in a multicenter cohort of 217 patients. METHODS Reverse transcription polymerase chain reaction (RT-PCR) was performed to assess the expression of 31 genes from primary melanoma tumors, and SLNB outcome was determined from clinical data. Prognostic accuracy of each test was determined using Kaplan-Meier and Cox regression analysis of disease-free, distant metastasis-free, and overall survivals. RESULTS GEP outcome was a more significant and better predictor of each end point in univariate and multivariate regression analysis, compared with SLNB (P < .0001 for all). In combination with SLNB, GEP improved prognostication. For patients with a GEP high-risk outcome and a negative SLNB result, Kaplan-Meier 5-year disease-free, distant metastasis-free, and overall survivals were 35%, 49%, and 54%, respectively. LIMITATIONS Within the SLNB-negative cohort of patients, overall risk of metastatic events was higher (∼30%) than commonly found in the general population of patients with melanoma. CONCLUSIONS In this study cohort, GEP was an objective tool that accurately predicted metastatic risk in SLNB-eligible patients.

An MHC-defined primate model reveals significant rejection of bone marrow after mixed-chimerism induction despite full MHC matching

In murine models, mixed hematopoietic chimerism induction leads to robust immune tolerance. However, translation to primates and to patients has been difficult. In this study, we used a novel MHC‐defined rhesus macaque model to examine the impact of MHC matching on the stability of costimulation blockade‐/sirolimus‐mediated chimerism, and to probe possible mechanisms of bone marrow rejection after nonmyeloablative transplant. Using busulfan‐based pretransplant preparation and maintenance immunosuppression with sirolimus, as well as CD28 and CD154 blockade, all recipients demonstrated donor engraftment after transplant. However, the mixed chimerism that resulted was compartmentalized, with recipients demonstrating significantly higher whole blood chimerism compared to T cell chimerism. Thus, the vast majority of T cells presenting posttransplant were recipient—rather than donor‐derived. Surprisingly, even in MHC‐matched transplants, rejection of donor hematopoiesis predominated after immunosuppression withdrawal. Weaning of immunosuppression was associated with a surge of antigen‐experienced T cells, and transplant rejection was associated with the acquisition of donor‐directed T cell alloreactivity. These results suggest that a reservoir of alloreactive cells was present despite prior costimulation blockade and sirolimus, and that the postimmunosuppression lymphocytic rebound may have lead to a phenotypic shift in these recipient T cells towards an activated, antigen‐experienced phenotype, and ultimately, to transplant rejection.

CTLA4Ig Prevents Alloantibody Formation Following Nonhuman Primate Islet Transplantation Using the CD40-Specific Antibody 3A8

Islet transplantation to treat type 1 diabetes has been limited in part by toxicities of current immunosuppression and recipient humoral sensitization. Blockade of the CD28/CD80/86 and CD40/CD154 pathways has shown promise to remedy both these limitations, but translation has been hampered by difficulties in translating CD154‐directed therapies. Prior CD40‐directed regimens have led to prolonged islet survival, but fail to prevent humoral allosensitization. We therefore evaluated the addition of CTLA4Ig to a CD40 blockade‐based regimen in nonhuman primate (NHP) alloislet transplantation. Diabetic rhesus macaques were transplanted allogeneic islets using the CD40‐specific antibody 3A8, basiliximab induction, and sirolimus with or without CTLA4Ig maintenance therapy. Allograft survival was determined by fasting blood glucose levels and flow cytometric techniques were used to test for donor‐specific antibody (DSA) formation. CTLA4Ig plus 3A8, basiliximab and sirolimus was well tolerated and induced long‐term islet allograft survival. The addition of CTLA4Ig prevented DSA formation, but did not facilitate withdrawal of the 3A8‐based regimen. Thus, CTLA4Ig combines with a CD40‐specific regimen to prevent DSA formation in NHPs, and offers a potentially translatable calcineurin inhibitor‐free protocol inclusive of a single investigational agent for use in clinical islet transplantation without relying upon CD154 blockade.

A novel risk-adjusted nomogram for rectal cancer surgery outcomes.

IMPORTANCE The circumferential resection margin is the primary determinant of local recurrence and a major factor in survival in rectal cancer. Neither chemotherapy nor chemoradiation compensates for a margin positive for cancer. OBJECTIVE To identify treatment-related factors associated with hospital margin-positive resection and to develop a tool that could be used by individual hospitals to assess their outcomes based on their unique mix of patient and tumor characteristics. DESIGN Retrospective review of the National Cancer Data Base, 1998-2007. SETTINGS Community and academic/research hospitals. PARTICIPANTS Individuals with histologically confirmed localized rectal/rectosigmoid adenocarcinoma. EXPOSURE All individuals underwent radical resection for rectal cancer with or without neoadjuvant therapy. MAIN OUTCOMES AND MEASURES Rate of margin positivity determined and adjusted for patient- and tumor-related factors to calculate expected margin positivity per hospital. An observed to expected ratio was calculated based on patient- and tumor-related factors to identify treatment-associated variation. RESULTS The overall margin-positive resection rate was 5.2%. Patients with margins positive for cancer were more likely to be older, male, and African American; not have private insurance; and have their cancer diagnosed later in the study period. Associated tumor-related factors include rectal location, higher American Joint Committee on Cancer stage, signet/mucinous histology, and poor/undifferentiated grade. Among hospitals that were significantly low outliers, 47% were comprehensive community hospitals, and 43.9% were academic/research hospitals; of those that were significantly high outliers, 52.3% were comprehensive community hospitals, and 17.8% were academic/research hospitals. High-volume centers made up 80% of significantly low outlier hospitals and 17% of significantly high outlier hospitals. The rates of chemotherapy and radiation were similar, but low outlier hospitals gave more neoadjuvant radiation (26.3% vs 17%). CONCLUSIONS AND RELEVANCE After adjustment for patient- and tumor-related factors, we identified both low and high outlier hospitals for margin positivity at resection, as well as potentially modifiable risk factors. The nomogram created in this model allows for the evaluation of observed and expected event rates for individual hospitals, providing a hospital self-assessment tool for identifying targets for improvement.

Effect of Perioperative Transfusion on Recurrence and Survival after Gastric Cancer Resection: A 7-Institution Analysis of 765 Patients from the US Gastric Cancer Collaborative

BACKGROUND The prognostic effect of perioperative blood transfusion on recurrence and survival in patients undergoing resection of gastric adenocarcinoma (GAC) remains controversial. STUDY DESIGN All patients who underwent resection for GAC from 2000 to 2012 at the 7 institutions of the US Gastric Cancer Collaborative were identified. The effect of transfusion on recurrence-free (RFS) and overall survival (OS) in the context of adverse clinicopathologic variables was examined by univariate and multivariate regression analyses. RESULTS Of 965 patients, 765 underwent curative intent R0 resection. Median follow-up was 44 months; 30-day mortalities were excluded. Median estimated blood loss (EBL) was 200 mL, and 168 patients (22%) received perioperative allogeneic blood transfusions. Transfused patients were less likely to receive adjuvant therapy (44% vs 56%; p = 0.01). Transfusion was associated with significantly decreased median RFS (13.5 vs 37.2 months, p < 0.001). Median OS was similarly decreased in patients receiving transfusions (18.6 vs 49.8 months, p < 0.001). On multivariate analysis, transfusion remained an independent risk factor for decreased RFS (hazard ratio [HR] 1.63; 95% CI 1.13 to 2.37; p = 0.010) and decreased OS (HR 1.79; 95% CI 1.21 to 2.67; p = 0.004), regardless of EBL or need for splenectomy. Timing (intraoperative vs postoperative) and volume of transfusion did not alter the negative prognostic effect of transfusion on survival. CONCLUSIONS Perioperative allogeneic blood transfusion is associated with decreased RFS and OS after resection of gastric cancer, independent of adverse clinicopathologic factors. This supports the judicious use of perioperative transfusion during resection of gastric cancer.