N. Joseph Espat

Phase I Hepatic Immunotherapy for Metastases study of intra-arterial chimeric antigen receptor modified T cell therapy for CEA+ liver metastases

Purpose: Chimeric antigen receptor–modified T cells (CAR-T) have demonstrated encouraging results in early-phase clinical trials. Successful adaptation of CAR-T technology for CEA-expressing adenocarcinoma liver metastases, a major cause of death in patients with gastrointestinal cancers, has yet to be achieved. We sought to test intrahepatic delivery of anti-CEA CAR-T through percutaneous hepatic artery infusions (HAIs). Experimental Design: We conducted a phase I trial to test HAI of CAR-T in patients with CEA+ liver metastases. Six patients completed the protocol, and 3 received anti-CEA CAR-T HAIs alone in dose-escalation fashion (108, 109, and 1010 cells). We treated an additional 3 patients with the maximum planned CAR-T HAI dose (1010 cells × 3) along with systemic IL2 support. Results: Four patients had more than 10 liver metastases, and patients received a mean of 2.5 lines of conventional systemic therapy before enrollment. No patient suffered a grade 3 or 4 adverse event related to the CAR-T HAIs. One patient remains alive with stable disease at 23 months following CAR-T HAI, and 5 patients died of progressive disease. Among the patients in the cohort that received systemic IL2 support, CEA levels decreased 37% (range, 19%–48%) from baseline. Biopsies demonstrated an increase in liver metastasis necrosis or fibrosis in 4 of 6 patients. Elevated serum IFNγ levels correlated with IL2 administration and CEA decreases. Conclusions: We have demonstrated the safety of anti-CEA CAR-T HAIs with encouraging signals of clinical activity in a heavily pretreated population with large tumor burdens. Further clinical testing of CAR-T HAIs for liver metastases is warranted. Clin Cancer Res; 21(14); 3149–59. ©2015 AACR.

Liver myeloid-derived suppressor cells expand in response to liver metastases in mice and inhibit the anti-tumor efficacy of anti-CEA CAR-T

Chimeric antigen receptor-modified T cell (CAR-T) technology, a promising immunotherapeutic tool, has not been applied specifically to treat liver metastases (LM). While CAR-T delivery to LM can be optimized by regional intrahepatic infusion, we propose that liver CD11b+Gr-1+ myeloid-derived suppressor cells (L-MDSC) will inhibit the efficacy of CAR-T in the intrahepatic space. We studied anti-CEA CAR-T in a murine model of CEA+ LM and identified mechanisms through which L-MDSC expand and inhibit CAR-T function. We established CEA+ LM in mice and studied purified L-MDSC and responses to treatment with intrahepatic anti-CEA CAR-T infusions. L-MDSC expanded threefold in response to LM, and their expansion was dependent on GM-CSF, which was produced by tumor cells. L-MDSC utilized PD-L1 to suppress anti-tumor responses through engagement of PD-1 on CAR-T. GM-CSF, in cooperation with STAT3, promoted L-MDSC PD-L1 expression. CAR-T efficacy was rescued when mice received CAR-T in combination with MDSC depletion, GM-CSF neutralization to prevent MDSC expansion, or PD-L1 blockade. As L-MDSC suppressed anti-CEA CAR-T, infusion of anti-CEA CAR-T in tandem with agents targeting L-MDSC is a rational strategy for future clinical trials.

Obstructive Jaundice Expands Intrahepatic Regulatory T Cells, Which Impair Liver T Lymphocyte Function but Modulate Liver Cholestasis and Fibrosis

Although obstructive jaundice has been associated with a predisposition toward infections, the effects of bile duct ligation (BDL) on bulk intrahepatic T cells have not been clearly defined. The aim of this study was to determine the consequences of BDL on liver T cell phenotype and function. After BDL in mice, we found that bulk liver T cells were less responsive to allogeneic or syngeneic Ag-loaded dendritic cells. Spleen T cell function was not affected, and the viability of liver T cells was preserved. BDL expanded the number of CD4+CD25+Foxp3+ regulatory T cells (Treg), which were anergic to direct CD3 stimulation and mediated T cell suppression in vitro. Adoptively transferred CD4+CD25− T cells were converted into Treg within the liver after BDL. In vivo depletion of Treg after BDL restored bulk liver T cell function but exacerbated the degrees of inflammatory cytokine production, cholestasis, and hepatic fibrosis. Thus, BDL expands liver Treg, which reduce the function of bulk intrahepatic T cells yet limit liver injury.

T cell infiltrate and outcome following resection of intermediate-grade primary neuroendocrine tumours and liver metastases.

BACKGROUNDnTumour-infiltrating lymphocytes (TILs) have been shown to predict survival in numerous malignancies. The importance of TILs in primary pancreatic neuroendocrine tumours (NETs) and NET liver metastases (NETLMs) has not been defined.nnnMETHODSnWe identified 87 patients with NETs and 39 with NETLMs who had undergone resection. Immunohistochemistry was performed to determine TIL counts. Recurrence-free survival (RFS) and overall survival (OS) were determined using the log-rank test.nnnRESULTSnThe median follow-up time was 62 months in NET patients and 48 months in NETLM patients. Vascular invasion and histologic grade were the only independent predictors of outcome for NETs and NETLMs, respectively. Analysis of intermediate-grade NETs indicated that a dense T cell (CD3+) infiltrate was associated with a median RFS of 128 months compared with 61 months for those with low levels of intratumoral T cells (P= 0.05, univariate analysis). Examination of NETLMs revealed that a low level of infiltrating regulatory T cells (Treg, FoxP3+) was a predictor of prolonged survival (P < 0.01, univariate analysis).nnnCONCLUSIONSnA robust T cell infiltrate is associated with improved RFS following resection of intermediate-grade NETs, whereas the presence of more Treg correlated with shorter OS after treatment of NETLMs. Further study of the immune response to intermediate-grade NETs and NETLMs is warranted.

Biliary obstruction results in PD-1-dependent liver T cell dysfunction and acute inflammation mediated by Th17 cells and neutrophils

Biliary obstruction is a common clinical problem that is associated with intrahepatic inflammation and impaired immunity. PD‐1 is well known to mediate T cell dysfunction but has been reported to promote and attenuate acute inflammation in various injury models. With the use of a well‐established murine model of BDL, we studied the effects of intrahepatic PD‐1 expression on LTC function, inflammation, and cholestasis. Following BDL, PD‐1 expression increased significantly among LTCs. Increased PD‐1 expression following BDL was associated with decreased LTC proliferation and less IFN‐γ production. Elimination of PD‐1 expression resulted in significantly improved proliferative capacity among LTC following BDL, in addition to a more immunostimulatory cytokine profile. Not only was LTC function rescued in PD‐1−/− mice, but also, the degrees of biliary cell injury, cholestasis, and inflammation were diminished significantly compared with WT animals following BDL. PD‐1‐mediated acute inflammation following BDL was associated with expansions of intrahepatic neutrophil and Th17 cell populations, with the latter dependent on IL‐6. PD‐1 blockade represents an attractive strategy for reversing intrahepatic immunosuppression while limiting inflammatory liver damage.

Anti-KIT designer T cells for the treatment of gastrointestinal stromal tumor

BackgroundImatinib mesylate is an effective treatment for metastatic gastrointestinal stromal tumor (GIST). However, most patients eventually develop resistance and there are few other treatment options. Immunotherapy using genetically modified or designer T cells (dTc) has gained increased attention for several malignancies in recent years. The aims of this study were to develop and test novel anti-KIT dTc engineered to target GIST cells.MethodsHuman anti-KIT dTc were created by retroviral transduction with novel chimeric immune receptors (CIR). The gene for stem cell factor (SCF), the natural ligand for KIT, was cloned into 1st generation (SCF-CD3ζ, 1st gen) and 2nd generation (SCF-CD28-CD3ζ, 2nd gen) CIR constructs. In vitro dTc proliferation and tumoricidal capacity in the presence of KIT+ tumor cells were measured. In vivo assessment of dTc anti-tumor efficacy was performed by treating immunodeficient mice harboring subcutaneous GIST xenografts with dTc tail vein infusions.ResultsWe successfully produced the 1st and 2nd gen anti-KIT CIR and transduced murine and human T cells. Average transduction efficiencies for human 1st and 2nd gen dTc were 50% and 42%. When co-cultured with KIT+ tumor cells, both 1st and 2nd gen dTc proliferated and produced IFNγ. Human anti-KIT dTc were efficient at lysing GIST in vitro compared to untransduced T cells. In mice with established GIST xenografts, treatment with either 1st or 2nd gen human anti-KIT dTc led to significant reductions in tumor growth rates.ConclusionsWe have constructed a novel anti-KIT CIR for production of dTc that possess specific activity against KIT+ GIST in vitro and in vivo. Further studies are warranted to evaluate the therapeutic potential and safety of anti-KIT dTc.

The Effects of Sorafenib on Liver Regeneration in a Model of Partial Hepatectomy

BACKGROUNDnSorafenib is currently approved for advanced hepatocellular carcinoma (HCC) and is presently being studied as an adjuvant treatment for HCC following resection. The effects of sorafenib on liver regeneration have not been clearly defined. Our objective was to identify the effects of sorafenib on liver regeneration in a murine partial hepatectomy (PH) model.nnnMATERIALS AND METHODSnWe performed PH in C57Bl/6 mice treated with a range of sorafenib doses with assessments at several time points. Liver sinusoidal endothelial cells (LSEC) and hepatocyte DNA synthesis and proliferation were assessed with 5-bromo-2-deoxyuridine (BrdU) and Ki67 by flow cytometry and immunohistochemistry.nnnRESULTSnTreatment with sorafenib did not result in any deaths following PH. When we measured BrdU uptake to assess DNA synthesis, there was a statistically significant increase at 48 h post-PH for nonfibrotic LSEC following treatment with 60 mg/kg of sorafenib. However, BrdU and Ki67 staining among LSEC and hepatocytes was not significantly affected by sorafenib at any of the other doses or time points. BrdU and Ki67 flow cytometry data correlated with immunohistochemistry findings and postoperative liver weights.nnnCONCLUSIONnIn a murine PH model, sorafenib did not alter the repair response of normal or fibrotic livers following PH as measured by changes in liver weight, DNA synthesis, and cellular proliferation. These findings suggest sorafenib administered following hepatic resection does not impair liver regeneration.

Feasibility of purely laparoscopic resection of locally advanced rectal cancer in obese patients.

BackgroundTotally laparoscopic (without hand-assist) resection for rectal cancer continues to evolve, and both obesity and locally advanced disease are perceived to add to the complexity of these procedures. There is a paucity of data on the impact of obesity on perioperative and oncologic outcomes for totally-laparoscopic rectal cancer resection (TLRR) for locally advanced disease.MethodsIn order to identify potential limitations of TLRR, a single-institution database was queried and identified 26 patients that underwent TLRR for locally advanced rectal cancers (T3/T4) over a three-year period. Patients were classified as normal-weight (NW, body mass index (BMI)=18.5 to 24.9kg/m2), overweight (OW, BMI=25 to 29.9kg/m2) and obese (OB, BMI >/= 30kg/m2). Perioperative outcomes, lymph node harvest and margin status were assessed.ResultsSeven patients were classified as NW (26.9%), 12 as OW (46.2%) and 7 as OB (26.9%). Age, tumor stage, gender and American Society of Anesthesiologists (ASA) scores were similar. OB had more co-morbidities (median 3.0, range 0.0 to 5.0 vs. 2.0, range 0.0 to 3.0 for NW and 1.0, range 0.0 to 3.0 for OW). Five patients had tumors <5cm from anal verge (NW=2; OW=1; OB=2). A median of 19.0, range 9.0 to 32.0; 20.0, range 9.0 to 46.0 and 19.0, range 15.0 to 31.0 lymph nodes were retrieved in the NW, OW and OB, respectively (Not Significant (NS)). Median node ratios for NW, OW and OB were 0.32, 0.13 and 0.00, respectively. All groups had negative proximal and distal margins. Radial margins were negative for 100% of NW, 83.3% of OW and 85.7% of OB (NS). Conversion rates were 14.3% for NW, 16.7% for OW & 0% for OB (NS). NW, OW and OB had complication rates of 28.3%, 33.3% and 14.3%, respectively. Median operative time, median estimated blood loss and median length of hospital stay were similar for all groups.ConclusionThe perceived limitation that obesity would have on TLRR was not demonstrated by the analyzed data. Although our findings are limited by the modest sized cohort, the results suggest that it is reasonable to offer TLRR to obese patients with rectal cancer.

Thermal tumor ablation therapy for colorectal cancer hepatic metastasis

Surgical resection for colorectal hepatic metastases (CRHM) is the preferred treatment for suitable candidates, and the only potentially curative modality. However, due to various limitations, the majority of patients with CRHM are not candidates for liver resection. In recent years, there has been an increasing interest in the role of thermal tumor ablation (TTA) as a component of combined resection-ablation strategies, staged hepatic resections, or as standalone adjunct treatment for patients with CRHM. Thus, ablative approaches have expanded the group of patients with CRHM that may benefit from liver-directed treatment strategies.

Liver metastases induce reversible hepatic B cell dysfunction mediated by Gr-1+CD11b+ myeloid cells

LM escape immune surveillance, in part, as a result of the expansion of CD11b+MC, which alter the intrahepatic microenvironment to promote tumor tolerance. HBC make up a significant proportion of liver lymphocytes and appear to delay tumor progression; however, their significance in the setting of LM is poorly defined. Therefore, we characterized HBC and HBC/CD11b+MC interactions using a murine model of LM. Tumor‐bearing livers showed a trend toward elevated absolute numbers of CD19+ HBC. A significant increase in the frequency of IgMloIgDhi mature HBC was observed in mice with LM compared with normal mice. HBC derived from tumor‐bearing mice demonstrated increased proliferation in response to TLR and BCR stimulation ex vivo compared with HBC from normal livers. HBC from tumor‐bearing livers exhibited significant down‐regulation of CD80 and were impaired in inducing CD4+ T cell proliferation ex vivo. We implicated hepatic CD11b+MC as mediators of CD80 down‐modulation on HBC ex vivo via a CD11b‐dependent mechanism that required cell‐to‐cell contact and STAT3 activity. Therefore, CD11b+MC may compromise the ability of HBC to promote T cell activation in the setting of LM as a result of diminished expression of CD80. Cross‐talk between CD11b+MC and HBC may be an important component of LM‐induced immunosuppression.