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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

A series of studies have shown that a purified cell regulatory sialoglycopeptide (CeReS) that arrests cell division and induces cellular differentiation is fully capable of functionally interacting with target insect and mammalian cells in the microgravity environment. Data from several shuttle missions suggest that the signal transduction events that are known to be associated with CeReS action function as well in microgravity as in ground‐based experiments. The molecular events known to be associated with CeReS include an ability to interfere with Ca2+ metabolism, the subsequent alkalinization of cell cytosol, and the inhibition of the phosphorylation of the nuclear protein product encoded by the retinoblastoma (RB) gene. The ability of CeReS to function in microgravity opens a wide variety of applications in space life sciences.

Human neural cell aggregate cultures were prepared from dissociated fetal brain tissue and maintained in rotation culture. After 35 days in culture, aggregates had the histologic appearance of dense, immature, neural cells in a tightly packed neuropil. Electron microscopy revealed ultrastructural features suggestive of immature neurons and neuroglia. In addition, neuron-specific enolase and glial fibrillary acidic protein associated with radial glial cells were detected within the aggregates by immunoperoxidase staining. When infected with a laboratory-adapted strain of cytomegalovirus (CMV), [AD169], cells containing large, bizarre, nuclei and CMV-induced intranuclear inclusion bodies were dispersed throughout the aggregates at 16 days postinfection. In situ hybridization using a CMV-specific DNA probe and electron microscopy confirmed the presence of virus sequences as well as virus particles at histologic sites of cytopathology. In sharp contrast, aggregate cultures infected with a CMV strain recovered from the retina of an acquired immune deficiency syndrome (AIDS) patient with CMV retinitis and encephalitis displayed distinct foci of cytopathology at 23 days postinfection, a pattern not observed in CMV [AD169]-infected aggregates. Our findings suggest that human neural cell aggregates represent a a promising multicellular non-neoplastic culture system in which to study the replication of human neurotropic viruses within neural tissue.

THE INCIDENCE of and mortality from cervical cancer in the United States have decreased dramatically over the past 40 years, in part because of early diagnosis and treatment of cervical cancer precursor lesions. The success of cervical cytological screening has served as a model for early diagnosis of other types of cancer. Although numerous studies have shown that lack of cytological screening is a major risk factor for the development of invasive cervical cancer, 1-3 it is important to emphasize that none of the screening, diagnostic, or therapeutic techniques used in medicine are perfect. Accordingly, a few women will develop cervical cancer despite adherence to accepted screening protocols . In addition, problems inherent with sampling, interpretation, and effective clinical follow-up preclude total prevention of cervical cancer. In recent years it has become evident that the cost and morbidity associated with the detection and treatment of low-grade cervical lesions have escalated, probably

An improved procedure is presented for the binding to filter paper and subsequent purification of DNA from plasmid-containing bacterial colonies. The procedure includes treatments with NaOH, enzymatic digestion, and organic solvent extraction of the filter-bound DNA. This method allows isolation of DNA in a reusable form from thousands of colonies in several hours. Double-labeling experiments with [ 3 H]thymidine and [ 14 C]proline indicated that (i) during purification the DNA:protein ratio is increased several hundredfold; (ii) little or no DNA is lost during the procedure; (iii) the resultant purified DNA is tenaciously bound to the paper. Thus, the final filter-bound DNA allows multiple sequential hybridizations of different probes to one filter.

Peripheral blood lymphocytes from normal individuals and patients with autoimmune abnormalities such as insulin-dependent diabetes mellitus and thyroiditis were infected with Epstein-Barr virus, and the culture supernatants were tested for autoantibodies that reacted with normal tissues. Between 58 and 86% of Epstein-Barr virus-transformed cultures produced immunoglobulin M antibodies, and between 9 and 24% of the transformed cultures produced immunoglobulin G antibodies that reacted with normal tissues. Ten Epstein-Barr virus-transformed clones secreting human immunoglobulin M monoclonal autoantibodies were isolated. Four of these monoclonal autoantibodies were studied in depth and found to react with antigens in multiple organs, including thyroid, pancreas, stomach, smooth muscle, and nerves. It is concluded that Epstein-Barr virus can trigger the production of autoantibodies without infecting the target cells to which the autoantibodies are directed.

Simultaneous EEG recordings were taken from the superior colliculus and the hippocampus in awake, freely moving albino rats. A simple method for scoring the synchronization-desynchronization shifts in each of the two structures allowed for a quantitative assessment of the congruence in activity between the two recordings. Intrarater and interrater reliability measurements were high, indicating that the method may be useful. It is also inexpensive particularly when long time periods of EEG activity require analysis. The congruence between the hippocampus and superior colliculus EEG recordings is of interest and, to our knowledge, has not been reported previously.

An increased prevalence of Type 1 (insulin-dependent) diabetes has been reported in patients with congenital rubella. Rubella virus multiplies in the pancreas, and we have hypothesized that studies of children with congenital rubella would be of great importance in following the development of Type 1 diabetes in a defined, susceptible population. Two hundred and forty-one children with congenital rubella (mean age 17.4 +/- 0.3 years; 65% black and hispanic) have been evaluated, 30 of whom already have diabetes and 17 of whom have borderline glucose tolerance. In these latter two groups, HLA-DR3 is significantly increased and HLA-DR2 significantly decreased. Pancreatic islet cell cytotoxic surface antibodies are found in 20% of the total congenital rubella population, including in more than 50% in the time period before they develop diabetes and are not related to any specific HLA type. In addition, anti-microsomal and anti-thyroglobulin antibodies are found in 34% of this population. The data demonstrate that Type 1 diabetes developing in congenital rubella patients has the genetic and immunological features of classical Type 1 diabetes, namely the presence of HLA-DR3, the absence of HLA-DR2, islet cell surface antibodies before decompensation and an increased prevalence of anti-thyroid antibodies. Patients with non-diabetic congenital rubella represent an easily identifiable group in whom other immunological factors associated with Type 1 diabetes can be elucidated and possibly modified.

Human monoclonal antibodies that react with normal tissues (autoantibodies) have recently been prepared from peripheral blood lymphocytes (PBLs) of patients with insulin-dependent diabetes mellitus (IDDM) and thyroiditis. This chapter describes the methods by which these monoclonal autoantibodies have been obtained and evaluates their reactivity with normal tissue antigens. In addition, the capacity of these monoclonal autoantibodies to react with antigens in multiple organs is described.

An increased incidence of insulin-dependent diabetes mellitus (IDDM) has been reported in patients with congenital rubella syndrome (CRS). Thus, studies of children with CRS would be of great importance in following the development of IDDM in a susceptible population. A total of 242 children with CRS, 30 of whom already have diabetes (mean age, 17.4 +/- 0.3 years) have been evaluated. In this latter group, the frequency of HLA DR3 is significantly increased and that of HLA DR2 significantly decreased. While pancreatic islet cell cytotoxic or surface antibodies (ICSA) are found in 20.2% of the total population of patients with CRS, they are present in 50%-80% of patients with glucose abnormalities. In all but five of the ICSA-positive patients, glucose abnormalities are currently present. In addition, glucose intolerance is found in greater than 50% of the DR3-positive nondiabetic patients with CRS evaluated to date. The data demonstrate that patients with CRS at risk for IDDM have the same genetic and immunologic features seen in classic IDDM, namely the presence of HLA DR3 and the absence of HLA DR2 and the high prevalence of ICSA before decompensation.