Low-Dose Cyclophosphamide Induces Antitumor T-Cell Responses, which Associate with Survival in Metastatic Colorectal Cancer.

Purpose: Anticancer T-cell responses can control tumors, but immunosuppressive mechanisms in vivo prevent their function. The role of regulatory T cells (Tregs) in metastatic colorectal cancer is unclear. We have previously shown depletion of Tregs enhances colorectal cancer-specific effector T-cell responses. Low-dose cyclophosphamide targets Tregs in animal models and some human studies; however, the effect of cyclophosphamide in metastatic colorectal cancer is unknown.Experimental Design: Fifty-five patients with metastatic colorectal cancer were enrolled in a phase I/II trial and randomly assigned to receive 2-week-long courses of low-dose (50 mg twice a day) cyclophosphamide or not. The absolute number, phenotype, and antitumor function of peripheral blood-derived lymphocyte subsets were monitored throughout treatment, as well as during 18-month follow-up.Results: Initially, cyclophosphamide reduced proliferation in all lymphocyte subsets; however, a rapid mobilization of effector T cells overcame this decrease, leading to increased absolute T-cell numbers. In contrast, a reduction in proportional and absolute Treg, B-cell, and NK-cell numbers occurred. The expansion and subsequent activation of effector T cells was focused on tumor-specific T cells, producing both granzyme B and IFNγ. Cyclophosphamide-treated patients demonstrating the most enhanced IFNγ+ tumor-specific T-cell responses exhibited a significant delay in tumor progression [HR = 0.29; 95% confidence interval (CI), 0.12-0.69; P = 0.0047), compared with nonresponders and no-treatment controls.Conclusions: Cyclophosphamide-induced Treg depletion is mirrored by a striking boost in antitumor immunity. This study provides the first direct evidence of the benefit of naturally primed T cells in patients with metastatic colorectal cancer. Our results also support the concept that nonmutated self-antigens may act as useful targets for immunotherapies. Clin Cancer Res; 23(22); 6771-80. ©2017 AACR.

Effect of Modified Vaccinia Ankara-5T4 and Low-Dose Cyclophosphamide on Antitumor Immunity in Metastatic Colorectal Cancer: A Randomized Clinical Trial.

IMPORTANCE: The success of immunotherapy with checkpoint inhibitors is not replicated in most cases of colorectal cancer; therefore, different strategies are urgently required. The oncofetal antigen 5T4 is expressed in more than 90% of cases of metastatic colorectal cancer (mCRC). Preliminary data using modified vaccinia Ankara-5T4 (MVA-5T4) in mCRC demonstrated that it safely induced serologic and T-cell responses. OBJECTIVE: To determine whether antitumor immunity in mCRC could be increased using MVA-5T4, metronomic low-dose cyclophosphamide, or a combination of both treatments. DESIGN, SETTING, AND PARTICIPANTS: In this randomized clinical trial, 55 patients with inoperable mCRC and prior stable disease after standard chemotherapy were enrolled at a single center and randomized to watch and wait (n = 9), cyclophosphamide treatment only (n = 9), MVA-5T4 only (n = 19), and a combination of MVA-5T4 and cyclophosphamide (n = 18). Patients were enrolled and treated from July 9, 2012, through February 8, 2016, and follow-up was completed on December 13, 2016. Data were analyzed based on intention to treat. INTERVENTIONS: Patients randomized to a cyclophosphamide group received 50 mg twice daily on treatment days 1 to 7 and 15 to 21. Patients randomized to a MVA-5T4 group received an intramuscular injection at a dose of 1 × 109 50% tissue culture infectious dose on treatment days 22, 36, 50, 64, 78, and 106. MAIN OUTCOMES AND MEASURES: The predefined primary end point was the magnitude of anti-5T4 immune responses (5T4-specific T-cell and antibody levels) generated at treatment week 7. Secondary end points included analysis of the kinetics of anti-5T4 responses, progression-free survival (PFS), and overall survival (OS). RESULTS: Fifty-two patients (38 men and 14 women; mean [SD] age, 64.2 [10.1] years) were included in the study analysis. The 5T4-specific antibody immune responses were significantly increased in the MVA-5T4 (83.41 [36.09] relative units [RU]; P = .02) and combination treatment (65.81 [16.68] RU; P = .002) groups compared with no treatment (20.09 [7.20] RU). Cyclophosphamide depleted regulatory T cells in 24 of 27 patients receiving MVA-5T4, independently prolonging PFS (5.0 vs 2.5 months; hazard ratio [HR], 0.48; 95% CI, 0.21-1.11; P = .09). MVA-5T4 doubled baseline anti-5T4 responses in 16 of 35 patients, resulting in significantly prolonged PFS (5.6 vs 2.4 months; HR, 0.21; 95% CI, 0.09-0.47; P < .001) and OS (20.0 vs 10.3 months; HR, 0.32; 95% CI, 0.14-0.74; P = .008). No grade 3 or 4 adverse events were observed. CONCLUSIONS AND RELEVANCE: This initial randomized clinical immunotherapy study demonstrates a significant survival benefit in mCRC. Prior depletion of regulatory T cells by cyclophosphamide did not increase immune responses generated by MVA-5T4 vaccination; however, cyclophosphamide and MVA-5T4 each independently induced beneficial antitumor immune responses, resulting in prolonged survival without toxic effects. Larger clinical trials are planned to further validate these data. TRIAL REGISTRATION: isrctn.org Identifier: ISRCTN54669986.

Fibrofatty Band-Associated Small Bowel Obstruction After Marathon Running.

We report a 57-year-old man who developed subacute small bowel obstruction after running a marathon. A fibrofatty band was identified restricting the terminal ileum upon subsequent imaging. Surgical division of the band resulted in complete resolution of the patient's symptoms. Fibrofatty bands are embryonic remnants of the vitellointestinal duct and have not previously been reported to cause small bowel obstruction at the terminal ileum. We discuss the origin of remnant fibrofatty bands and physiological impact of running a marathon upon the gastrointestinal tract that would have contributed to development of subacute small bowel obstruction.

Tracking the kinetics of intrahepatic immune responses by repeated fine needle aspiration of the liver.

Liver disease is an increasing global health burden. The final sequalae of cirrhosis, liver failure and hepatocellular carcinoma are often the result of inflammation driven by intrahepatic lymphocytes. Accurate assessment of organ-specific diseases ideally employs tissue sampling though this is rarely performed. Here we report our experiences of utilising repeated fine needle aspirations (FNAs) to assess liver-derived leukocytes. In 88 patient samples, we obtained a mean of 36,959 lymphocytes from each FNA-derived biopsy (SD 22,319 cells, range 5034-91,242 cells) measured by flow cytometry. This quick technique required minimal analgesia compared to liver biopsy (p = 0.03); was well tolerated and safe, and hence repeated sampling up to 3 times within a week was feasible. We detail the technique to rapidly derive a single cell suspension suitable for multiparameter flow cytometry analysis. Finally we illustrate the importance of organ-derived sampling by showing that natural killer (NK) cells from FNA samples have a markedly altered phenotype compared to those assessed in peripheral blood. In combination these data validate FNA as a powerful and well-tolerated method of sampling intrahepatic lymphocytes to study the immunology of acute and chronic liver diseases.

The paradox of NKp46+ natural killer cells: drivers of severe hepatitis C virus-induced pathology but in-vivo resistance to interferon α treatment.

OBJECTIVE: There is evidence that natural killer (NK) cells help control persistent viral infections including hepatitis C virus (HCV). The phenotype and function of blood and intrahepatic NK cells, in steady state and after interferon (IFN) α treatment has not been fully elucidated. DESIGN: We performed a comparison of NK cells derived from blood and intrahepatic compartments in multiple paired samples from patients with a variety of chronic liver diseases. Furthermore, we obtained serial paired samples from an average of five time points in HCV patients treated with IFNα. RESULTS: Liver NK cells demonstrate a distinct activated phenotype compared to blood manifested as downregulation of the NK cell activation receptors CD16, NKG2D, and NKp30; with increased spontaneous degranulation and IFN production. In contrast, NKp46 expression was not downregulated. Indeed, NKp46-rich NK populations were the most activated, correlating closely with the severity of liver inflammation. Following initiation of IFNα treatment there was a significant increase in the proportion of intrahepatic NK cells at days 1 and 3. NKp46-rich NK populations demonstrated no reserve activation capacity with IFNα treatment and were associated with poor viral control on treatment and treatment failure. CONCLUSIONS: NKp46 marks out pathologically activated NK cells, which may result from a loss of homeostatic control of activating receptor expression in HCV. Paradoxically these pathological NK cells do not appear to be involved in viral control in IFNα-treated individuals and, indeed, predict slower rates of viral clearance.

Escalating regulation of 5T4-specific IFN-γ+ CD4+ T cells distinguishes colorectal cancer patients from healthy controls and provides a target for in vivo therapy.

The relationship between the adaptive CD4+ T cell response and human cancer is unclear. The oncofetal antigen 5T4 is expressed on many human carcinomas, including colorectal cancer (CRC) cells, but has limited expression on normal tissues. We previously identified anti-5T4 CD4+ T cells in a proportion of CRC patients, and we extended this study to examine whether the quality or quantity of the T cell response reflects tumor stage. An overlapping peptide library spanning 5T4 was used as a target to enumerate cognate IFN-γ+CD4+ T-cells (measured as spot forming cells [SFC]/105 cultured T cells) in peripheral blood-derived lymphocytes following a 12-day in vitro culture period comparing patients pre-operatively (n = 27) to healthy controls (n = 17). Robust 5T4-specific T cell responses were present in 100% of healthy donors. There was a steady loss of T cell responses with advancing tumors with a significant negative correlation from stage I to III (P = 0.008). The predictability of the decline meant < 200 SFC/105 was only found in subjects with stage III CRC. The mechanism of loss of T cell response is independent of HLA-DR type or patient age, but does correspond to increases in Foxp3+ regulatory T cells (Tregs). Using low-dose cyclophosphamide to reduce the proportion of Tregs in vivo resulted in increased anti-5T4 T cell responses in CRC patients. The selective loss of 5T4-specific IFN-γ+CD4+ T cell responses implies a link between tumor stage and antitumor Th1 effector function; depleting Tregs can enhance such responses.

Rapid early innate control of hepatitis C virus during IFN-α treatment compromises adaptive CD4+ T-cell immunity.

The ability to control HCV with IFN-α-based treatments provides an opportunity in humans to study how the rate of viral clearance in vivo impinges on the development of antiviral responses. Ex vivo (IFN-γ-producing) and cultured antiviral CD4(+) T cells, serum cytokines, and viral loads were measured repeatedly in a cohort of chronically HCV-infected subjects (n = 33) receiving IFN-α. Rapid control of virus indicated by an increased calculated rate of virus clearance, occurred in those subjects demonstrating absent/minimal T-cell responses (p < 0.0006). Surprisingly, in subjects who demonstrated the most robust T-cell responses (and reduced serum IL-10), there was actually a reduced rate of early virus clearance. A subsequent analysis of NK-cell function in available subjects (n = 8) revealed an inverse correlation between pretreatment NK-cell expression of NKp46 and the potential to upregulate cytotoxic function on exposure to IFN-α (p < 0.004), as well as the subsequent measured rate of viral clearance (p = 0.045). Thus, the CD4(+) T-cell response during IFN-α treatment appears to be shaped by the rate of innate virus suppression. These data suggest that individuals who respond most effectively to immune intervention may be most in need of subsequent vaccination to prevent reinfection.