NCI Clinical and Translational Exploratory/Developmental Studies

Hereditary diffuse gastric cancer syndrome (HDGC) is a hereditary condition associated with increased risk of diffuse gastric cancer and lobular breast cancer. While HDGC is classically caused by germline mutations in the CDH1 gene, recent evidence has identified the CTNNA1 gene, coding for α-E-catenin, as a new putative HDGC risk gene. We previously showed that 12% and 67% of individuals with a CTNNA1 loss-of-function (LOF) variant identified on multigene panel testing had a personal history of diffuse gastric cancer or breast cancer respectively. However, given limitations of previously collected data the extent and magnitude of the cancer risks associated with LOF variants in CTNNA1 remain uncertain at this time. Elucidating accurate cancer risk estimates for CTNNA1 LOF variant carriers is critical to allow for proper cancer risk management of this affected cohort as well as their family members. Our preliminary data demonstrates that CTNNA1 LOF variant carriers can be successfully recruited as study participants, enabling collection of detailed personal and family history with creation of three-generation pedigrees that can be used for cancer penetrance analyses. Furthermore, we show that CTNNA1 LOF variant carriers may have differing variant-specific cancer risks, with potentially reduced gastric cancer risk associated with C-terminal LOF variants. Finally, we demonstrate that patient-derived gastric organoids, including from CTNNA1 LOF variant carriers, can be successfully established. Taken together we hypothesize that CTNNA1 LOF variant-specific cancer risks can be established through a combination of improved cancer penetrance estimates and patient-derived gastric organoid models. We will investigate this hypothesis by first defining the cancer penetrance of CTNNA1 LOF variant carriers through the prospective CTNNA1 Family Expansion (CAFÉ) Study, enabling collection of detailed personal and family medical history, with subsequent creation of three-generation pedigrees that will be utilized for cancer penetrance analysis. Secondly, we will determine CTNNA1 variant-specific gastric tumorigenesis using patientderived gastric organoids, which are invaluable tools for recapitulating gastric cancer development. We will utilize these patient-derived gastric organoids to test the carcinogenic potential of different CTNNA1 LOF variants. Together, the study results from this proposal will be critical for establishing variant-specific cancer risks for CTNNA1 LOF variants, which will ultimately help inform cancer risk management decisions for these affected patients and their families.

Ultraviolet radiation (UVR) exposure has long been linked to melanoma development, with clear correlations seen between latitude and the incidence of melanoma, particularly in individuals with fair skin and a poor ability to tan. As such, sporadic cutaneous melanoma has one of the highest mutational burdens of all cancers. The high mutational burden following UVR exposure is associated with increased neoantigen load and greater immune recognition. These observations suggest that the melanocytes that initiate melanoma survive UVR induced stress/cell damage and then avoid immune recognition. Unrelated studies in our lab focused on BRAF inhibitor resistance in melanoma identified a stress-activated epigenetic program mediated through histone deacetylase (HDAC)-8 that allowed escape from both cell death and immune recognition through transcriptional reprogramming. The objective of this proposal is to develop novel melanoma prevention strategies that target the epigenetic programs required for UVR-damaged melanocytes to avoid cell death and immune recognition. We will test the hypothesis that UVR-induced HDAC8 activation allows melanocytes to avoid cell death and immune attack, allowing cells with pathogenic mutations to survive and later develop into melanoma. This proposal is conceptually innovative in seeking to define the role of HDAC8 in the epigenetic reprogramming of melanocytes that experience UVR-induced stress and DNA damage. We will further address the role of immune evasion in the development of melanoma precursor lesions and will use unique mouse models, single cell RNA-Seq, and multi-Omics approaches to identify core pathways and regulators involved in the persistence of UVR-damaged melanocytes that constitute the precursor lesions for melanoma development. In Aim 1, we will define how HDAC8 modulates the transcriptional profile of melanocytes following UV-irradiation and will determine how this contributes to increased cell survival and reduced immunogenicity. We will then use our recently developed HDAC8 mouse model to demonstrate the role for HDAC8-induction in melanoma development and will perform single cell RNA-Seq and immune profiling to characterize how HDAC8 activation in melanocytes modulates the immune microenvironment of the skin. In Aim 2, we will determine whether inhibition of HDAC8 using genetic silencing or small molecule HDAC8 inhibitors reverses the survival and immune escape transcriptional programs that are activated in melanocytes following UVR exposure. In vivo studies will evaluate whether HDAC8 inhibitors or silencing of HDAC8 in mouse skin melanocytes are sufficient to delay the initiation of melanoma development in our BRAF mutant/PTEN-silenced melanoma model, and the role of the immune system in this response. At completion of this work we expect to have demonstrated the proof-of-principle for the development of HDAC8 inhibitors as a novel melanoma prevention strategy.

Esophageal adenocarcinoma (EAC) is a major cause of cancer morbidity and mortality in the US, affecting nearly 20,000 people each year. The known epidemiological factors associated with EAC and its precursor Barrett’s esophagus (BE) gastroesophageal reflux disease, smoking, and obesity - do not completely explain why EAC has continued to increase in incidence over the past four decades. Elucidating the full repertoire of factors that modulate the initiation and progression of EAC will advance our understanding of EAC tumorigenesis and impact clinical management of patients with BE. Emerging evidence suggests microbiome alterations, potentially due to antibiotics or diet changes, are possible causative factors for BE and EAC. Exploratory studies have identified microbiome differences between BE and EAC patients and healthy study subjects, which suggests dysbiotic microbiomes or the presence of absence of specific oncogenic microbiome species in the lower esophagus may mediate the initiation of BE and/or progression of BE to EAC. We propose to use state-of-the-art computational methods to identify and characterize both candidate dysbiotic esophageal microbiomes and candidate oncogenic microbiome species in an existing, well-characterized cohort of patients with BE who either progressed to EAC (n=40) or remained non-progressing and stable with benign disease (n=40). The patients from this case-control study have previously been evaluated by whole genome sequencing of biopsies obtained from two locations in the esophagus at each of two time points. Using an analysis pipeline developed by Dr. Sam Minot of our group, we will identify the different microbiome species present in these biopsies from the whole genome sequencing data. By comparing the microbiome species present in either non-progressing patients, progressing patients, or in both, we will be able to determine which microbiome species are associated with BE in general and which are indicative of patients who progress to EAC. We will validate our findings using microbiome specific digital droplet PCR analysis and in-situ hybridization. The results of this exploratory study will inform development of a larger, prospective study examining the direct role of the microbiome in the development of EAC. The ultimate clinical impact of this study would be identification of an easily assayed target for determining cancer progression risk in patients with BE, as well as suggesting means of modulating EAC risk through elimination of the oncogenic microbe(s) or introduction of tumor suppressive microbe(s) through antibiotic or probiotic treatments.

With over 15.5 million cancer survivors today in the United States, increased attention is being given to quality of life after cancer treatment. Cancer-related fatigue (CRF), a persistent, subjective sense of physical, emotional, and/or cognitive exhaustion related to cancer or its treatment that is not proportional to recent activity, is the most common, and one of the most devastating symptoms among patients with cancer. CRF can persist for months or years after cancer therapy is completed. Prostate cancer (PCa) is the most prevalent cancer in men and has the highest Medicare expenditure of any cancer that affects older men. Modern tailored treatment regimens have resulted in longer life expectancy, which lead to treatment related morbidity and adverse side effects, including psychological distress, pain and CRF. This R21 will investigate the therapeutic and biological effects of Swedish Massage Therapy (SMT) for CRF in PCa and is an extension of our completed study in breast cancer survivors with CRF demonstrating that SMT produces clinically and statistically significant improvements in fatigue and quality of life (QOL). A homogenous group of PCa survivors with CRF (n=15 per group) will be randomized to one of three conditions: 6 weeks of once-weekly SMT, 6 weeks of once-weekly Light Touch (LT), or a 6 week wait list control (WLC) condition. Subjects that complete SMT or LT will be followed up for persistent changes in CRF at 6 and 12 weeks after the last study visit. We hypothesize that SMT will have a clinically meaningful benefit in improving fatigue, mood, and QOL in PCa patients, and that this will correlate with favorable changes in physiological parameters that may underlie CRF and potentially impact disease progression. Primary Aim: To conduct a preliminary study to determine whether a 6-week SMT intervention decreases CRF among PCa survivors who have received both radiation and androgen deprivation therapy. Secondary Aim: To determine whether the hypothesized decrease in CRF with SMT is associated with modulation of specific immune system factors underlying chronic inflammation. Exploratory Aims: 1) To determine whether SMT improves self-reported fatigue, QOL, depression, and anxiety significantly more than LT or WLC, as assessed (respectively) by the Patient-Reported Outcomes Measurement System (PROMIS) Fatigue Scale, the Quality of Life, Enjoyment, and Satisfaction Questionnaire – short form (Q-LES-Q), the Quick Inventory of Depressive Symptoms – Self-Report (QIDS-SR16), and the GAD-7. 2) To gather preliminary data about the sustained actions of 6 weeks of SMT versus LT on CRF, by measuring MFI and PROMIS Fatigue Scale scores at 6 weeks and 12 weeks after the intervention. This research will provide preliminary data demonstrating that SMT is an effective intervention for mitigating CRF in a group of PCa survivors, that this effect is mediated by changes in key pro- and anti-inflammatory cytokines, and that SMT improves QOL and mood symptoms.

Chemotherapy-induced peripheral neuropathy (CIPN) is a severe dose-limiting toxicity that affects over 65% of patients receiving platinum-based chemotherapy for gastrointestinal cancers. Not only does CIPN increase mortality by limiting the dose of chemotherapy, CIPN affects walking, writing, eating, and dressing via numbness, tingling, pain, cold sensitivity, and cramping in the hands and feet. There are no FDA-approved treatments for CIPN because we need a greater understanding of the pathophysiology of CIPN and mechanisms of action of promising treatments for CIPN. In addition, most research on CIPN in humans has focused on taxane-based chemotherapy in patients with breast cancer. Here, we are focusing on platinum-based chemotherapy in patients with gastrointestinal cancers because taxane- and platinum-induced CIPN are both very common but exhibit distinct signs, symptoms, and mechanisms of toxicities, and may respond differently to a given treatment. This project is motivated by an innovative perspective that CIPN symptoms are not due simply to peripheral nerve damage, but rather that CIPN symptoms are worsened by damage to the interoceptive brain system—which processes sensations from the body—as well as systemic inflammation. This is a Phase II randomized clinical trial to assess the effects of 12 weeks of exercise during platinum chemotherapy on CIPN, interoception, and inflammation in 60 patients with gastrointestinal cancer. The specific aims of this project are to assess the effects of exercise vs. nutrition education (control) on: (1) patient reported CIPN, (2) clinical assessments of CIPN (tactile sensitivity and cold-induced pain), (3) interoception (functional connectivity in the interoceptive brain system and self-report), and (4) inflammation (serum inflammatory cytokines). This is the first study to examine the effects of exercise during platinum chemotherapy on CIPN and to examine roles of interoception and inflammation in the treatment of platinum-induced CIPN. Results from this study will inform an R01 application to more definitively assess this theory of CIPN and exercise. If this work is successful, we will have identified an effective intervention (exercise) for alleviating CIPN and we will have a better understanding of how exercise alleviates CIPN (i.e., interoception and inflammation). We believe this knowledge can be applied to improve prediction, tracking, sub-typing, and treatment of CIPN using exercise or other interventions that affect these pathways. This will ultimately help alleviate the burden of chemotherapy on treating patients with cancer.

Chronic kidney disease (CKD), defined as Stages 3-5, including end stage renal disease (ESRD)) at the time of cancer diagnosis is believed to influence cancer outcomes, but the base of evidence for such effects is limited. We seek in this proposal to extend the understanding of CKD as a risk factor for cancer mortality, and to quantify the development of incident CKD in patients treated with specific potentially nephrotoxic cancer regimens. In particular, cisplatin, carboplatin, capecitabine, irinotecan, and etoposide are components of standard cancer treatment protocols, and treated individuals have had an increased CKD risk in some cancers, but the risk following standard lung or colorectal cancer treatment is unknown. Approximately 15% of adults in the United States are estimated to be affected with CKD as of 2020. There is a pressing need to understand the risks of CKD incidence, progression and mortality in the setting of cancer treatment. We will utilize a large, population-based dataset, that of the Surveillance Epidemiology End Results (SEER) program of the National Cancer Institute, linked to Medicare (SEER-Medicare), to address question of import to prognosis in both CKD and cancer. Surprisingly little of the existing literature concerns long-term follow-up. Such evidence could be crucial to reduce nephrotoxicity, acute kidney injury, and ensure that dose-limiting side effects of antineoplastic agents do not prevent effective therapy of the cancer of interest. We will analyze claims data to evaluate whether those with CKD are at greater risk of cancer or CKD-related mortality after cancer treatment, and whether particular therapies might increase the risk of CKD incidence. These analyses should greatly heighten awareness of CKD at diagnosis and that which develops during therapy, leading to additional monitoring, personalized therapy, and potential survival benefits for patients with cancer and CKD alike.

Pregnancy imposes remarkable changes to the breast; 1- during pregnancy, impressive cellular proliferation and differentiation of the ductal tree is observed, 2- during lactation, the ultimate function of breast and 3- during post-lactation involution that is associated with tissue remodeling and inflammation. Interestingly both the proliferation during pregnancy and the inflammation during involution engage signaling pathways associated with cancer and indeed, the risk of breast cancer increases following pregnancy. However, extended lactation reduces that risk but the molecular mechanism of the protective effect of breastfeeding remains unknown. Based on our preliminary data, we postulate that the ovarian-derived hormone stanniocalcin 1 (STC1) is the potential mediator of the protective effect of breastfeeding. STC 1 is a secreted hormone produced mainly by the ovaries during lactation. It has been found to act as an inhibitor of the protease PAPP-A, Pregnancy-Associated Plasma Protein A. PAPP-A is a secreted protease that promotes proliferation through IGF signaling by degrading IGFBP5 during involution, promotes immune evasion and we reported takes advantage of the microenvironment of involution to promote metastasis. PAPP-A is frequently overexpressed in the breast cancers and we have generated the first mouse model of PAPP-A driven mammary tumors. Importantly for this application, we found that long lactation prevents the formation of PAPP-A-driven tumors. Our hypothesis is that ovarian-derived STC1 produced during lactation reach the mammary gland through the circulation, saturates and inhibits PAPP-A and is the mechanism underlying the protective effect of long lactation. We present preliminary data that incubation of PAPP-A with serum taken from lactating females, but not from non-lactating females, inhibits its ability to degrade IGFBP-5. Further, depletion of STC1 from lactating -female serum blocks this effect. The goal of this exploratory award is to test if transfusions of blood from lactating donors into nonlactating recipients during post-partum involution, can mimic the protective effect of lactation in these recipient females. In order to test this possibility, we propose the following aims; Specific aim 1: Characterize potential fluctuations in the serum concentration and activity of STC1 throughout lactation and determine if transfusions of blood from lactating females can mimic the protective effect of long lactation in wild-type non-lactating mice. Specific aim 2: Testing the protective effect of ovarian STC1 against PAPP-A driven mammary tumors. In this aim, we will perform the same experiment described in aim 1 but in the MMTV-PAPP-A transgenic mice with two exceptions. First, since this model is much more aggressive than normal involution without prior lactation, we will use a more intense scheduling of transfusions. Second, since 75% of PAPP-A transgenic mice develop postpartum mammary tumors when females do not lactate, we will monitor tumor formation.

Lung cancer is the leading cause of cancer-related death in the United States. Overall, patients diagnosed with early-stage lung cancer have a much better prognosis. Cigarette smoking is the major cause of lung cancer and screening for lung cancer is currently recommended for high-risk current and former smokers. Compared with nonsmokers, smokers have an almost 30-fold increased risk of developing lung cancer. Unfortunately, despite significant efforts, early detection of lung cancer remains sub-optimal with conventional approaches suffering from high false-positive rates or limited sensitivity. Improved understanding of the early events underlying smoking-related lung cancer development is crucial to the identification of new biomarkers and targets for early detection and prevention. Further, new methods that detect those early events in lung cancer development in a non-invasive and cost-effective manner with high sensitivity and specificity are urgently needed. Recent advances in super-resolution microscopy revolutionize the field of optical microscopy and offer a new ability to visualize molecular structure at nanometer resolution that is invisible under a conventional light microscope. We propose an innovative approach to adapt super-resolution microscopy to improve the early detection of lung cancer. Our approach is built upon our recent discovery that chromatin folding becomes gradually fragmented in early lung carcinogenesis, even when cells still appear normal under conventional light microscope. Our ultimate goal is to detect such nanoscale chromatin “misfolding” in bronchial cells present in sputum to improve early detection of lung cancer. In this project, we will first establish disrupted nanoscale chromatin folding as an early event in lung carcinogenesis utilizing a mouse model of carcinogen-induced lung adenocarcinoma as well as existing well-annotated human lung tissue specimens (Aim 1). In addition, we will evaluate the feasibility of super-resolution imaging of nanoscale chromatin “misfolding” for early detection of lung cancer using existing sputum samples and data from the Pittsburgh Lung Screening Study, a communitybased research cohort of current and ex-smokers, screened with low-dose computed tomography and followed for lung cancer (Aim 2).

Precancerous lesions of pancreatic ductal adenocarcinoma (PDAC) called pancreatic intraductal papillary mucinous neoplasms (IPMN) can be detected radiographically, but monitoring and identifying patients who can benefit from surgical intervention before the development of malignant tumor has been an immense challenge in the management of patients with IPMN. Here we propose to investigate the feasibility of repurposing FDAapproved Bazedoxifene as a chemopreventive therapy for patients with IPMN in preclinical models, which may complement and improve the current care for these patients who are at high-risk for developing PDAC. Using unbiased computational and chemical screens, we have previously identified Bazedoxifene as a novel IL-6 signaling antagonist that can directly bind to GP130, a common component of IL-6 receptor complex. IL6/STAT3 is a vital oncogenic signaling axis that promotes pancreatic tumorigenesis. Elevated IL-6 level is associated with inflammation, aging, and poor prognosis and metastasis in pancreatic cancer patients. More recently, IL-6 secretion stimulated by IL-1-NFkB-JAK/STAT signaling is implicated in activating tumorpromoting inflammatory cancer associated fibroblast cells (iCAF). While inhibition of IL-6 and STAT3 to suppress PDAC has been pursued previously, here we propose to explore the uncharted efficacy of Bazedoxifene (a well-tolerated FDA-approved medication that is prescribed for osteoporosis prevention and can be taken orally) as a chemopreventive measure for patients who are identified as high-risk for developing PDAC, specifically those with detectable premalignant IPMN. To test our novel hypothesis that repurposing Bazedoxifene for chemoprevention would block IPMN progression to PDAC via inhibition of IL-6 signaling, we will 1) use 3D organoids derived from our unique mouse model for IPMN and from surgically resected IPMNs from patients to evaluate the functional and molecular impacts of Bazedoxifene as a chemopreventive agent on epithelial cells (Aim 1); 2) use our IPMN mouse model and orthotopically implanted murine and human IPMN 3D organoids to investigate the efficacy of Bazedoxifene as a chemopreventive therapy in vivo (Aims 1 & 2); 3) to investigate if Bazedoxifene also affects the stromal components, specifically the activation of quiescent fibroblast cells and the interconvertibility between CAF subtypes in vitro and in vivo (Aims 1 & 2). And lastly 4) since our knowledge of IL-6, metabolic alterations, CAF subtypes in pancreatic tumorigenesis has been gathered majorly from premalignant PanIN and invasive PDAC, to address this gap in knowledge, we will also compare and contrast IPMN organoids with PanIN and PDAC organoids to advance our understanding of the understudied IPMN (Aims 1 & 2). The success of our application will be transformative to clinical care of patients with IPMN and may be applicable to other known high-risk groups (i.e. patients with chronic pancreatitis, familial mutations, diabetes) for pancreatic cancer.

Objectives: Loss-of-function (LOF) mutations in succinate dehydrogenase subunit (SDHx) genes result in SDH-deficiency and increase the lifetime risk for developing a number of cancers, including GI stromal tumors, paraganglioma, pheochromocytoma, and renal cell carcinoma. In patients with a known LOF/pathogenic germline SDHx mutation, genetic counseling (to screen other family members) and enhanced cancer screening procedures are indicated. When SDH-deficient tumors are detected at early stages, they are often curable. However, 3.2% of the population carry germline SDHx missense variants of unknown significance (VUS, not known to be pathogenic or benign). Therefore, we currently do not have the ability to identify many patients at risk and screen them appropriately because of our lack of knowledge of the functional consequences of germline SDHx VUS. Plan: This proposal will functionally test SDHA VUS utilizing a deep mutational scanning (DMS) approach in a novel SDHA-deficient cell line that we have generated. We will identify all LOF SDHA variants by pairing DMS with a negative metabolic selection method developed in our laboratory. To determine if this approach extends to test VUS of the other SDHx genes (SDHB, C, D), we will generate knockout models of these individual genes and asses negative metabolic selection in each cell line. Human models, which can provide strong evidence to be used for clinical variant classification will be generated and validated for testing individual variants. Methods: To address the large number of SDHA variants seen in the population, we will create libraries of all possible SDHA amino-acid variants by saturation mutagenesis and use this library to complement our SDHAdeficient cell line. We will select against SDHA LOF variants by growing the libraries under metabolite-depleted conditions requiring fully functional SDH (-pyruvate, -aspartic acid). Following deep sequencing, depletion analysis will be performed. Functional interpretations (functionally normal or LOF) will be made for each variant by comparing calculated effect scores with those of nonsense/ synonymous controls (Aim 1). To fill the current void of SDH-deficient cell lines, we will use CRISPR/Cas9 to generate SDHB-, SDHC-, and SDHD-knockout cell lines and validate them. Negative selection using metabolite-depleted medium will be applied to these novel models to determine their utility for future DMS experiments (Aim 2). Clinical Relevance: Our best chance of a cancer cure lies in early detection in patients with SDH-deficient tumors, thus the critical medical need lies in classifying SDHx genetic variants to enable identification and subsequent screening of at-risk patients.

) Insomnia and fatigue commonly occur during and/or after cancer treatment. While variable by diagnosis and stage of illness, 30-60% of cancer patients endorse difficulty initiating and/or maintaining sleep and 50-90% endorse fatigue. Despite these high prevalence rates, and the substantial overlap in occurrence, most research related to cancer-related fatigue (CRF) has assessed this pervasive and persistent symptom in a manner that does not take into account sleep quality and quantity. This is, in part, due to the definition of CRF as “pathological levels of persistent physical, emotional, and/or cognitive ‘enervation’ that appear not to be relieved, or improved by, rest or sleep”. To date only a few studies have evaluated whether behavioral treatments such as Cognitive Behavioral Therapy for Insomnia (CBT-I) can provide some relief to patients who are experiencing CRF. The effect sizes for CBT-I outcomes, in the context of cancer, have been found to be comparable to those observed in “primary insomnia”. The findings with respect to CBT-I effects on CRF have been more modest. The smaller effects of CBT-I on CRF may be due to a variety of factors, but one simple possibility is that CBT-I is under-dosed for patients with cancer. In other words, subjects with cancer may require more sessions to achieve sleep durations that can affect daytime fatigue. Accordingly, we propose to evaluate the association between CBT-I dose (Low dose [4 or 8 sessions] or High Dose [10 or 12 sessions]) and changes in sleep duration and CRF. The study will enroll 70 subjects to obtain an analysis sample of 60 subjects. Subjects will be diagnosed with breast cancer and will be between the ages of 25-85 (inclusive). In order to allow age to be an analyzable factor, recruitment will be stratified so that the following cohorts are equally populated (i.e., 25-45, 45-65, 65-85). The focus of the R21 study will be on feasibility. The primary outcomes will be related to recruitment, subject adherence, treatment acceptability, and subject retention. Descriptive data will also be obtained to examine whether high dose CBT-I (i.e., larger improvements in sleep efficiency and/or sleep duration) positively affects CRF. The study will be undertaken in collaboration with our program post doc (Dr. Muench) as she transitions to Jr. Faculty and pursues this program of research on a more independent basis. The study collaborators will also include: Dr. Knashawn Morales (statistics); Dr. Sheila Garland (psycho-oncology & sleep); and Dr. Donn Posner (CBT-I).

Chemotherapy-induced peripheral neuropathy (CIPN) is a severe dose-limiting toxicity that affects over 65% of the roughly 250,000 patients per year receiving neurotoxic chemotherapy (e.g., taxane-, platinum-, vinca alkaloid-based), which is widely used for breast, prostate, lung, colorectal, and blood cancers among others. Not only does CIPN increase mortality by limiting the dose of chemotherapy, CIPN affects walking, writing, eating, and dressing via numbness, tingling, pain, cold sensitivity, and cramping in the hands and feet. There are no FDA-approved treatments for CIPN because we need a greater understanding of the pathophysiology of CIPN and mechanisms of action of promising treatments for CIPN. To date, most research on CIPN pathophysiology has focused on peripheral sensory nerve damage. This project is motivated by an innovative perspective that CIPN symptoms are not due simply to peripheral nerve damage, but rather that CIPN symptoms are worsened by damage to the interoceptive brain system (i.e., the brain’s reaction to peripheral nerve damage). The long-term vision for this project is mechanism-based CIPN sub-typing and mechanismbased treatments for CIPN informed by brain fMRI, as has been done in other brain-mediated conditions such as chronic pain and depression. This is a Phase II randomized clinical trial to assess CIPN and fMRI brain connectivity in the interoceptive brain system in 66 patients with CIPN from neurotoxic chemotherapy before and after 6 weeks of exercise vs. usual care. This study will be included as part of the clinical trial URCC 19075. The specific aims of this project are (1) to obtain preliminary estimates of the relationships between CIPN and functional connectivity in the interoceptive brain system in patients with CIPN, and (2) to obtain preliminary estimates of the effects of exercise vs. no exercise on both CIPN severity and functional connectivity in the interoceptive brain system in patients with CIPN. This is the first study to examine the interoceptive brain system in the pathophysiology and treatment of CIPN. Results from this study will inform an R01 application to more definitively assess this theory of CIPN and exercise. If this work is successful, it will reveal a novel objective biomarker of CIPN—functional connectivity in the interoceptive brain system—to improve prediction, tracking, sub-typing, and treatment of CIPN using exercise or other interventions that affect this brain system. This will ultimately help alleviate the burden of chemotherapy on treating patients with cancer. Chair: Ian Kleckner

An estimated one in five children in the United States has cancer. Fortunately, the 5-year overall survival rate has improved over the years and is currently more than 80% for many types of cancer, thanks to advances in chemotherapy. Unfortunately, chemotherapy drugs have adverse effects such as peripheral neuropathy (socalled chemotherapy-induced peripheral neuropathy, CIPN). CIPN is a debilitating condition that negatively affects the quality of life of affected patients. The drugs used to treat CIPN, such as opioids, antiepileptics, and antidepressants, are often ineffective, and/or have profound adverse effects, such as constipation, sedation, addiction, respiratory depression, and even death. Furthermore, nonpharmacologic treatments such as transcutaneous electrical nerve stimulation (TENS), acupuncture, and yoga have shown mixed results. Importantly, most of the procedures have been tested in adults with CIPN; much less research has been done in adolescent and young adult (AYA) cancer patients. Consequently, the management of CIPN in AYA patients with cancer remains a challenge. Thus, this exploratory R21 proposal addresses the unmet need in treating AYAs with CIPN by introducing a potentially effective noninvasive electrocutaneous nerve stimulation technique called scrambler therapy (ST), which is different from TENS. Given that the clinical presentation of CIPN in AYAs is similar to that of older adults, and with supporting evidence from a recently published small study from Italy, we believe that ST is a promising novel treatment option for patients of all ages with CIPN. Thus, it is worth investigating this novel treatment further. Our central hypothesis is that ST significantly reduces pain and improves neuropathy, physical function, and quality of life (QoL) of AYA cancer patients with CIPN. To test this hypothesis, we propose a prospective, randomized, wait-list controlled pilot study with two specific aims: Specific Aim 1: To evaluate the efficacy and safety of ST for CIPN in AYA cancer patients, and Specific Aim 2: To evaluate the impact of ST on physical functioning and quality of life (QoL) in AYA cancer patients. The overall objective of this proposal is to show that ST may be a valuable alternative to pharmacologic management of CIPN that can effectively improve pain and neuropathy and improve QoL for AYA cancer patients with CIPN. We expect to see significant improvement in pain and other symptom burden of CIPN in AYA patients. Finally, this pilot study is not a mechanistic study. Instead, it will focus on patient-reported outcomes, physical functioning, and QoL to demonstrate effectiveness. As such, pain will be our primary outcome measure, whereas physical performance and QoL will be our secondary outcome measures. The long-term goal is to use ST safely and effectively for all types of intractable neuropathic pain, which if successful, will result in changes in the clinic.

Despite the advent of prophylactic vaccines to prevent infection with oncogenic human papillomaviruses (HPVs), the incidence of cancers caused by HPV remains high, both in the US and world-wide. Intraepithelial HPV cancer precursors present an opportunity for cancer interception: they are relatively accessible and clinically indolent, making it possible to carry out window-ofopportunity, proof-of-concept treatment trials without compromising standard of care. However, while trials testing therapeutic HPV vaccines to treat CIN2/3, the precursor to HPV cervical cancers, have shown partial success, their focus has been upon generating HPV-specific immune responses. HPV oncogenes driving transformation present rational antigenic targets, yet little is known about mechanisms of histologic regression. The projects we propose will focus on the lesion itself. There is an increasing appreciation of the role of microbes at the gut and other niches in cancer, and evidence of dysbiosis in the context of CIN. Currently, our understanding of the functional contributions of tumor microbiomes to tissue susceptibility to treatment is incomplete, in part owing to difficulty in obtaining longitudinal data from human interventional trials. We will analyze subject-matched, clinically annotated specimens collected before, during, and after treatment, from a novel interventional clinical trial testing treatment of CIN2/3 with a repurposed, topical formulation of artesunate, a plant-derived compound used as part of frontline treatment for acute malaria. (NCT02354534) The clinical outcomes from were highly impactful; histologic regression (HR) occurred in 19/28 (68%) of treated subjects -- more than twice the expected rate of spontaneous regression observed over the same timeframe. We will now build on this promising clinical data to determine tissue-based biomarkers of response to ART, as well as phenotypes of treatment resistance, with the intent of developing next-generation interventions to treat CIN and HPV-related malignancies at other anatomic sites. We will capitalize on a unique extant biorepository, and an outstanding team of expert scientists to study constituents of the tumor microenvironment (TME), including tumor and immune cells, microbes, and metabolites, in subject-matched samples of responders vs non-responders. Insights gained will inform strategies to overcome ART treatment resistance. Finally, this work will begin to address an enormous unmet clinical need for accessible treatment options for incipient HPV cancers, which now are either excisional or ablative, and require sequential access to health care infrastructure. An inexpensive, self-administered, non-surgical treatment without cold chain requirements would be transformative.

Although lung cancer mortality has improved in recent years, it remains the leading cancer killer worldwide. Low dose computed tomography (CT) scan screening in high-risk patients has shown improved cancer-specific survival, however, it carries >95% of false-positive rate, a subsequent risk of unnecessary invasive diagnostic biopsy, and repeated radiation exposure. Liquid biopsy assays have been developed to overcome these challenges and to enhance the diagnostic accuracy of CT-screened lung nodules. Among many biomarkers, tumor-derived exosomes (TEXs) have emerged as potent cancer biomarkers. We have discovered a new exosomal marker, Thomsen-Friedenreich glycoantigen (TF-Ag-α; Galβ1-3GalNAc alpha), as a potential biomarker for lung cancer early detection. TF-Ag-α is a unique target expressed on the cell surface of about 84% lung cancers (both nonsmall cell and small cell lung cancer) and other carcinomas (such as breast cancer, colon cancer, etc), but not on normal tissues. Currently there are no reports about TEX TF-Ag-α and its roles in the screening and early detection of lung cancer and other cancers. In this project, we select lung cancer as the disease model and aim to demonstrate the clinical utility of TEX TF-Ag-α in cancer liquid biopsy. To transform our discovery to clinical applications, we have developed a monoclonal antibody, JAA-F11, with high specificity to TF-Ag-α and a surface plasmon resonance (SPR)-based liquid biopsy assay to measure the levels of exosomal TF-Ag-α in blood. We have demonstrated, for the first time, that exosomes carry TF-Ag-α. More importantly, we successfully detected exosomal TF-Ag-α in as low as 10uL serum samples from both early stage and late stage nonsmall cell lung cancer (NSCLC) and small cell lung cancer (SCLC) patients, but little signals from those of normal controls. Based on our promising pilot data, we hypothesize that exosomal TF-Ag-α is a potent biomarker for lung cancer screening and early detection. In this project, we propose to (1) further develop the liquid biopsy assay and comprehensively characterize its sensing performances including analytical sensitivity (limit of detection, limit of quantitation), specificity, linear range, and repeatability; (2) demonstrate the diagnostic values of exosomal TF-Ag-α in lung cancer screening and early detection using both cell-derived exosomes and serum samples from well-defined cohorts of patients including lung cancer patients (both NSCLC and SCLC), individuals with benign pulmonary nodules at high risk of lung cancer and normal controls with no pulmonary nodules (total n=200). We aim to develop an effective cancer specific biomarker and an accurate liquid biopsy assay to complement the diagnosis of pulmonary nodules detected during low dose CT screening and to enhance the accuracy of low dose CT.

Preventive services have been emphasized as essential components of the medical care system. However, the disparities in cancer incidence and mortality rates experienced by vulnerable populations are evident in rates of screening for colorectal cancer (CRC). While community health centers can play an important role in addressing these disparities, their current operations are not set up to ensure that every eligible patient receives timely CRC screening. The current patient self-test relies on a fecal immunochemical test that is plagued with low rates of positive early cancer identification and difficulty in excluding non-neoplastic causes of intestinal bleed. Until a low-cost, simple, and highly accurate diagnostic method is developed, screening rates in minorities, uninsured, and low-income populations will likely remain low. The long-term goal of this project is to improve the accessibility of CRC screening through the development of a new, on-demand diagnostic approach that has potential to enable self-testing at home followed by signal development and diagnosis after sending the test to a central facility (for example by mail). The objective of the current R21 application is to pre-validate a set of five CRC cancer antigen biomarkers through their multiplexed detection on a 3D paper-based microfluidic device in CRC patients as well as to test the possibility of using a self-regenerative photo-catalyst to amplify mass spectrometry (MS) signals for early CRC detection. The test is designed to be stable enabling storage under ambient conditions, a condition critical for successful remote sampling. The test is also rapid enough to enable point-ofcare testing on a portable mass spectrometer. Although the proposed detection strategy is based on immunoassay, the use of a photoredox catalyst to amplify MS signal is novel. Traditional immunoassay tests use colorimetric detection via enzyme amplification, necessitating both cold storage and analysis of the once-initiated colorimetric signal within a specified time to ensure the validity of the test. This research program will follow three specific aims: (1) using 356 CRC patient biorepository samples to validate the five selected biomarkers CEA, CA199, CA242, CA125, CA153 and to optimize their multiplexed detection via paper-based immunoassay, (2) investigation and selection of a photosystem for mass spectrometry signal amplification, and (3) using an independent set of patient samples collected in the field to validate proposed method through the development of a prototype 3D paper-based microfluidic device for CRC detection in whole blood samples. The project is innovative because it combines new levels of simplicity and practicality, modest levels of cost, and a centralized detection strategy, which will redefine the breadth of application and performance/cost ratio for accurate CRC detection in underserved communities. The proposed research is significant because it has potential to improve cancer care among all populations, irrespective of their race, geographic location, or income.

Radiation-induced dysphagia is a devastating complication of chemoradiation treatment for head and neck cancer. Deficits in oral and pharyngeal movement during swallowing are the most prevalent cause of radiationinduced dysphagia. The adverse effects of these swallowing problems can lead to long-term dietary restrictions, malnutrition, and placement of a feeding tube to prevent aspiration. Recent evidence in other areas of the body demonstrate that radiation can damage peripheral nerves resulting in changes in motor function. However, the neural mechanisms underlying radiation-induced dysphagia are unknown. An understanding of the pathophysiology of radiation-induced dysphagia is needed to develop more effective therapeutic targets aimed at preserving post chemoradiation swallowing function. Swallowing is a coordinated activity controlled by a neural pattern-generating circuitry in the brainstem that relies heavily on sensory information. Nociceptors are a subset of sensory neurons that are sensitized by tissue injury. When nociceptor sensory axons are damaged, they trigger protective responses that can drive changes in neural control leading to disturbances in coordinated motor output. We propose that nociceptor activity interfering with swallowing function may be another potential mechanism at play after chemoradiation muscle injury. In the proposed study, we will characterize how oropharyngeal swallowing is affected by chemoradiation and determine whether injury of sensory neurons can contribute to dysphagia post-treatment. We hypothesize that chemoradiation-induced axon injury is associated with changes in oral and pharyngeal swallowing kinematics after treatment. This research has two specific aims that are strongly supported by preliminary data. In Aim 1 we will determine the effect of chemoradiation to the mylohyoid muscle on the movement of the oral and pharyngeal structures during swallowing. Kinematic analysis and force measures will be used to quantify functional deficits. In Aim 2 we will determine the nerve injury/stress-like response induced in trigeminal sensory neurons following chemoradiation to the mylohyoid muscle. We will identify sensory neurons in the trigeminal ganglion projecting from the mylohyoid and measure their expression of injury/stress-induced markers. We will also test alternative mechanisms and develop a predictive model to quantify the complication risk of treatment to tissue and behavioral outcomes. The proposed experiments will establish the feasibility of a novel neural-based mechanism underlying radiation-induced dysphagia and define specific points of swallowing dysfunction after chemoradiation in the rat that will serve as targets for assessing future treatments.

Abstarct Individuals with Li-Fraumeni syndrome (LFS) are predisposed to a wide spectrum of cancer types at a relatively early age. Despite that LFS is linked to germline mutations of p53, these aberrations alone cannot explain the complex phenotype of the syndrome. The observation that female mutation carriers have higher cancer risk than males points to a role of sex steroid signaling in affecting disease penetrance. Further, breast cancer whose etiology relates to aberrant hormonal signaling is the most common malignancy in LFS. However, the effects of irregular hormone signaling on breast tumorigenesis in LFS have not been explored. Estrogen is essential for proper development and function of breast tissue. It regulates proliferation and differentiation of epithelial cells in adolescence and pregnancy. Because cells in development are susceptible to malignant transformation, it is well accepted that the proliferative action of estrogen is coordinated with an increase in genome surveillance. Two receptors (ERα and ERβ) mediate the effects of estrogen in mammary tissue. While ERα is responsible for the proliferative activity of the hormone, ERβ promotes differentiation and regulates DNA damage response which let us to propose that this subtype is employed by estrogen to ensure genome integrity. In support of this role, our findings demonstrate that ERβ enhances the activity of p53 in response to genotoxic stress and loss of synergistic tumor suppressor function as a result of combined inactivation in mammary gland results in earlyonset breast tumors in mice. In addition to wild-type form, we have seen that ERβ binds to mutant p53 to inhibit its oncogenic function. Since LFS tissues are often heterozygous for mutant p53, we hypothesize that ERβ coordinates with both alleles of p53 and other pathways to maintain genome stability and, therefore, dysregulation of ERβ signaling in LFS mammary tissue results in malignant transformation and increased incidence of breast cancer. Our proposed research will: 1) investigate whether ERβ affects breast tumorigenesis in LFS, 2) determine effects of ERβ agonists on penetrance in LFS and 3) elucidate the mechanism of ERβ action. To test whether abnormal ERβ signaling affects breast cancer in LFS, we will analyze virgin and pregnant mice with germline point p53 mutations that mimic human LFS phenotypes and ubiquitous and mammary glandspecific inactivation of ERβ. We will also assess ERβ expression in human LFS tissues and identify associations with the age of tumor onset (Aim 1). In addition, we will treat LFS mice with ERβ agonists that are in clinical trials for other diseases expecting these compounds to potentiate its tumor suppressor activity and prevent breast cancer and its associated mortality (Aim 2). Further, we will delineate the mechanisms of ERβ action by analyzing LFS tissues for pathways that are associated with the phenotype of the syndrome (Aim 3). Determining the effects of estrogen signaling on LFS tissues will lead through the development of novel estrogen responsive biomarkers to a better model to predict cancer risk in the syndrome and assist the evaluation of endocrine compounds as a novel chemoprevention regimen to reduce cancer incidence and its associated mortality.