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Rapid assessment of therapy for advanced cancer, especially immunotherapy (IO), is critical. For advanced cancers most therapies, even targeted/personalized ones fail. Even worse, IO has been reported to cause more than doubling in the cancer growth rate (defined as hyper-progressive disease or HPD) in 5-40% (typically ~20%) of patients. Early identification of HPD, or any inactive therapy, and switching to an effective therapy can be lifesaving. Though a consensus has not emerged, there are several reports that describe sensitization to chemotherapy following failed immunotherapy, this suggests that, at least in some cases, switching results in dramatic benefit. Due to inflammatory infiltrates causing “pseudoprogression” (false readings of cancer progression) early during treatment with IO, ineffective IO is often continued for 4-6 months before imaging can definitively say the IO is failing. During this time progressive, potentially fatal cancer growth, as well as medical and financial side effects can occur. The cost of this ineffective or counterproductive IO is typically $10,000 to over $20,000 a month. There are numerous reports of highly sensitive mutation based, or methylation sequence based, cfDNA signatures and methods. Unfortunately, they are inherently difficult tests which typically cost thousands of dollars and do not report the results to clinicians for 1 to 2 weeks. They have not been adopted for routine use to monitor on every visit basis and allow near real-time adjustment of therapy. Rapid detection of failure can lead to lifesaving therapy changes; detection of benefit can enhance compliance and improve clinical outcomes. Previously a research lab style solid gold electrode, requiring artful polishing and reuse, has been reported to be selectively sensitive to cancer DNA due to ctDNA’s altered pattern of methylation. Electrochemical detection of the binding of cfDNA, following a 10-minute incubation, has been published. Details of the dramatically revised and optimized binding methodology are in the methods section of this poster. Compared to previous methods an over 2-fold increase in cfDNA binding is shown. Cancer samples not distinguishable from normal with the previous method show statistically significant detection with the new method. Dramatically increased ability to separate cancer from normal DNA is shown. The new method uses inexpensive, clinically and POC suitable, disposable electrodes; it is highly sensitive to clinical samples. 5 µL of a 10 pg/ul sample provides reproducible readings. cfDNA from normal persons generates readings significantly different than from those with cancer (p < .01). Patient time courses illustrate the correlation of the electrode response with cancer burden and suggests this is a monitoring tool that should be compared to, and may have benefits over, previously approved monitoring tests for advanced cancer such as carcinoembryonic antigen (CEA). Citation Format: Floyd Taub, Matt Russo, Shannon Tunney, Suzin Wright, Chuck Henry. A simplified, potentially point-of-care (POC), electrode method detects changes in the amount of cfDNA/ctDNA and evaluates the response of advanced cancer patients to therapy. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P049.

Beta-alethine (BA) is a small molecule drug that has demonstrated impressive anti-tumor immune responses against melanoma, particularly when combine with low dose anti-PD1. Here we report on the dose of BA that optimally enhances anti-PD1, the efficacy of BA combined with various immune checkpoint inhibitors (ICIs) in a murine bladder cancer and we propose a potential mechanism of action of BA to explain its synergistic effect. We tested 3 different doses of BA that were logs apart (30 mg/kg, 30 ug/kg, and 600 ng/kg) either alone or in combination with low dose (10 ug per mouse for a total of 3 doses 3 days apart) anti-PD1 in the DBA mouse Cloudman melanoma mouse model. The lowest dose of BA (600 ng/kg) in combination with anti-PD1 resulted in cures in 33% of the mice when treatment was initiated when tumors were of substantial size (average size of 30 mm2). Previous work starting with smaller tumor sizes (average 20 mm2) resulted in an 80% cure rate when a low dose of BA was used in combination with low dose anti-PD1. Analysis of blood from treated animals demonstrated that the combination led to decreased expression of multiple checkpoint molecules on both T cells and myeloid cells and to a decrease in tumor promoting inflammatory macrophages. We further demonstrated efficacy of combining BA with other ICI therapies in a s.c. MBT2 murine bladder cancer study in C3H mice. In these studies, we saw that BA was synergistic with a-PD1 at all doses tested (30 mg/kg, 30 ug/kg and 600 ng/kg weekly s.c.). The effect was seen even when only 4 weekly doses of BA were given. Interestingly, tumor growth increased when BA was discontinued, a phenomenon observed in previous compassionate use veterinary studies. Synergy with OX40 and CTLA4, were also seen, but further studies of dose and schedule are required for optimization. Our results demonstrate that a low dose of BA is optimal to enhance the efficacy of anti-PD1 and that this observed combinatorial effect is conserved in at least two different tumor types in different mouse strains. Based on analysis of T and myeloid cells in treated mice, we hypothesize that BA works via inducing pleiotropic changes that result in downregulation of multiple immune checkpoint molecules on T cells and on myeloid cells making it less feasible for these cells to use other pathways of immune suppression as compared to single agent anti-PD1 therapy and thus increase potential for cures. Citation Format: Floyd Taub, Suzin Wright, Amanda Guth. Enhancement of checkpoint inhibitor therapy by beta-alethine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-145.

Previously we reported that the combination of anti-PD1 and β-alethine completely stopped cancer growth in a syngeneic mouse melanoma model even under conditions when neither of the drugs had statistically significant effects as a single agent. In order to further examine this dramatic result, we extended the studies, evaluated immune parameters and tested if long-term immunity to re-challenge occurred. Surprisingly, three low doses of anti-PD1, spread over 9 days, were sufficient to allow concurrent and continuing weekly dosing of β-alethine to not only halt tumor growth, but to reverse it. The established melanoma regressed over the subsequent 4-6 weeks. Even more striking was that the short course of anti-PD1, which failed to reduce cancer growth as a single therapy, altered the immune system such that subsequent treatment with β-alethine was effective. β-alethine therapy, beginning eight days after the conclusion of anti-PD1 therapy, when tumors averaged 40 mm2, was sufficient to cause at least stabilization of cancer in all animals and complete response (no palpable tumor on repeated measurements) in the majority of animals. Statistical comparisons of all animals receiving combination therapy (either concurrently or sequentially) with controls resulted in significant differences using ANOVA for tumor size (p=0.005) or X2 tests for tumor presence (p Citation Format: Floyd E. Taub, Suzin Wright, Amanda Guth. Combination checkpoint inhibitor therapy: Anti-PD1 and Beta-alethine lead to complete responses of melanoma in a syngeneic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-191. doi:10.1158/1538-7445.AM2017-LB-191