Purpose:This study investigates the impact of customized yoga practice on mitigation of side effects, quality of life and longevity in cancer survivors in Kannur, Kerala, India. Method: An observational study was conducted among 323 cancer survivors, categorized into four groups: breast, head and neck, gynaecological, and other types. Participants were trained in yoga postures customized to the needs unique to different cancer types and the post-treatment sequelae, and followed up from 2006 to 2024. The outcomes were recorded using a subjective improvement scale of 0-10 to assess the positive effects of yoga on the reduction of symptoms, enhancement of QOL at baseline, 0-2, 3-6, and 7+ years Results: Findings indicate that consistent yoga practice significantly increased survival rates and reduced disability. Survivors of Breast cancer showed the most pronounced benefits, followed by head and neck. Gynaecological survivors demonstrated moderate improvement in early years, with marked increases after 7+ years. Regular yoga practice enhances resilience and coping ability, contributing to long-term well-being and longevity. Conclusion:Yoga improves longevity, quality of life, and reduces treatment-related side effects in survivors. This study's findings confirm yoga as a holistic, low-risk intervention that enhances physical and psychological well-being, daily functioning, and overall quality of life. Notably, despite challenges in tracking long-term survivors, our sample size of 323 (2.31% of the State of Kerala's estimated 14,000 survivors in the 2000s) is substantial, underscoring the study's significance and reliability.

Background: There is a huge gap in Breast Cancer survival globally with the death rate being significantly higher in developing countries due to taboo, ignorance, lack of awareness, sociocultural, economic, and environmental barriers, leading to inaccessibility to healthcare facilities, proper guidance, and treatment management. Healthcare providers in rural India regularly see women coming in an advanced stage and in Assam, Northeast (NE) India, the situation is grimmer. This study is based on an analysis of the database of door-to-door Breast cancer screening of the rural villages in Assam from February 2020 to April 2024, reporting the Breast Cancer Screening status, disparities leading to late detection and death, and the success of Breast Cancer Hub’s Grassroots approach, providing sustainable solutions. Methods: In this study, we report the data from February 2020 to April 2024 (N=22,278), of in-person Door-to-Door Breast Cancer Screening by Breast Cancer Hub (BCH), a nonprofit organization in the remote 18 villages in Assam, India. BCH-generated questionnaire on health and lifestyle, family history, demography, socio-economic condition and other determining conditions. The data was incorporated from notebook used by BCH teams in the field, into Excel sheets. A complete descriptive statistics is used for understanding the data. For goodness of fit test, chi square is used. All the analysis have been compiled with the help of SPSS version 21. Results: This is the pioneering study, as BCH is the first group to conduct door-to-door Breast Cancer Screening in N=22,278 villagers (8085 families), in the underprivileged rural Seventeen villages and One Tea Garden Estate in Assam. From our evaluation, we confirm that the awareness of Breast Cancer Symptoms was <1% among the villagers. The Breast Self-Exam was performed by 0% of the populace. No participant (0%) underwent Clinical Breast Exam. Women 40 years and above, never performed screening mammograms (0%) or ultrasounds (0%) but only visited the hospital when the symptoms were extremely severe eventually getting diagnosed at a late stage leading to death. We investigated the socio-economic condition and huge barrier to care due to financial constraints, villagers living on a daily wage and health always took the last priority as they fall below the poverty line. In addition, the lifestyle is influenced by the tremendous intake of tobacco in all forms, especially adding tobacco, betel nut, slaked lime, and other ingredients to the betel leaves and chewing in the form of Paan. We encountered 76 suspicious cases, that needed further evaluation and screening. But, during screening, the team faced challenges, as the villagers could not financially afford to travel to cancer diagnosis-equipped hospitals which are extremely far away, leading them to succumb to faith healers and eventually death. The family members avoided taking patients to hospitals as it would be a full-day affair and they couldn’t miss their daily wage, as food is their priority. Also, they were not aware of government healthcare cards for the Below Poverty Line population. Therefore, BCH created a sustainable framework, by teaching every member Breast Self-Examination (BSE), providing BSE card in local language, accompanying suspicious cases to hospitals, providing transport, generating villagers’ income certificates and health cards, helping with hospital registration, patient communication with healthcare professionals, providing Aid for diagnosis and treatment not covered under the government schemes, dropping patients home with follow-ups, counseling, support, taking the same patients back to the hospital on the next appointments, assisting with the comprehensive treatment process, case by case. Discussion: BCH is the trendsetter to execute this framework in the untapped villages in India, revolutionizing the Breast cancer scenario, and driving impactful changes in Breast Cancer survival. Citation Format: Lopamudra Das Roy. Door-to-door Breast Cancer Screening in 22,278 populaces from Feb. 2020-April 2024: Breast Cancer Hub's Trendsetting Grassroots Sustainable Solutions, overcoming the Disparity, & Challenges in the Rural Remote Villages in Poverty, of Assam, India [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr PS10-05.

10550 Background: Cervical Cancer is still significant cause of mortality and morbidity among females in India. Various methods of cancer screening are currently practiced for cervical cancer prevention including human papilloma virus (HPV) screening, pap smear, visual inspection with Lugol’s iodine (VILI), visual inspection with acetic acid (VIA) etc. The success of any cancer screening method is based on the feasibility of large-scale implementation at community level and the scientific validity of the screening test. VIA/VILI has been proven to prevent mortality in cervical cancer. This study is based on analysis of prospectively maintained database of community based cervical cancer screening using VIA/VILI in northern Kerala from 2002 to 2020. Methods: Retrospective analysis of prospective database of cervical cancer screening from 2002 to 2020 done by Malabar Cancer Care Society in Kerala, a non-governmental organization with support from governmental bodies like ISRO. Data was collected into excel sheets and were rechecked. The data on demography, type of screening, detection of precancerous and cancerous lesions was analyzed. Descriptive statistics were used to analyze the data using SPSS Version 20.0. Results: A total of 48008 individuals were screened over 18 years. 55% were premenopausal. 33% were fully asymptomatic and remaining 66% had white discharge per vaginum (25%), bleeding per vaginum (14%) or combination of symptoms (25%). 18% had abnormal per speculum findings (bleeds on touch, discharge etc.) 86% had unhealthy cervix.26% (12650) had VIA/VILI and 74% (35658) had pap smear. The pap smear findings were normal (41%), atrophic (17%), inflammatory (40%) and 2% had pre-cancerous/ cancerous changes. Of these lesions, 46% were HSIL, 36% were LSIL, 5% ASUS and 13% were squamous cell carcinoma (52 cases). Screen positive rate was 325 (0.005%) out of 48008. Treatment of precancerous lesions was by Cryotherapy (54%), LEEP (10%) and Surgery (36%). Treatment of invasive carcinoma was by surgery (91%) and radiation (85%). Referral to regional cancer center was done in 26% patients. The screen positive rate shows decreasing trend over the last two decades. Conclusions: Cervical Cancer screening in large scale in low resource setting is feasible in India by both pap smear and VIA/VILI with community based interventions led by NGO with support from governmental bodies. This model can be considered ideal for India and other low middle income countries with logistic difficulties for large scale HPV based screening.

Antibody-derived chimeric antigen receptor (CAR) T cell therapy has achieved gratifying breakthrough in hematologic malignancies but has shown limited success in solid tumor immunotherapy. Monoclonal antibody, TAB004, specifically recognizes the aberrantly glycosylated tumor form of MUC1 (tMUC1) in all subtypes of breast cancer including 95% of triple-negative breast cancer (TNBC) while sparing recognition of normal tissue MUC1. We transduced human T cells with MUC28z, a chimeric antigen receptor comprising of the scFv of TAB004 coupled to CD28 and CD3ζ. MUC28z was well-expressed on the surface of engineered activated human T cells. MUC28z CAR T cells demonstrated significant target-specific cytotoxicity against a panel of human TNBC cells. Upon recognition of tMUC1 on TNBC cells, MUC28z CAR T cells increased production of Granzyme B, IFN-γ and other Th1 type cytokines and chemokines. A single dose of MUC28z CAR T cells significantly reduced TNBC tumor growth in a xenograft model. Thus, MUC28z CAR T cells have high therapeutic potential against tMUC1-positive TNBC tumors with minimal damage to normal breast epithelial cells.

Antibody-derived Chimeric Antigen Receptor (CAR) T cells have great success in reduction of liquid form of tumor, but not solid tumor. Our novel antibody TAB004 can specifically recognize the tumor form of MUC1 (tMUC1) while sparing the normal MUC1 in several subtypes of breast cancers including >95% of triple negative breast cancer (TNBC). In vivo delivery of TAB004 specifically accumulates in the xenograft TNBC tumor only. Therefore, we hypothesize that TNBC can be specifically targeted with TAB004-derived tMUC1-specific CAR T cells. A panel of 45 human breast cancer cell lines were tested for tMUC1 level, and 11 out of 13 TNBC cell lines showed higher frequency of tMUC1 expression compared to that on normal cells. We engineered a 2nd generation human CAR using TAB004 that was coupled to the CD28-CD3zeta, named as MUC28z CAR. MUC28z was well expressed on activated human T cells. Compared to their mock control, MUC28z CAR T cells retained higher CD25 and CD11c, particularly on CD8 T cells. The susceptibility of TNBC cells to MUC28z CAR T cell cytolysis was dramatic while still corresponding to their tMUC1 level. Only the tMUC1-specific MUC28z CAR T cells expressed and released large amount of IFN-gamma after antigen recognition on TNBC cells. However, IFN-gamma was only involved in the tumor killing by MUC28z CAR T cells at lower E:T ratio. The in vivo treatment of TNBC tumor by MUC28z CAR T cells dramatically controlled HCC70 TNBC tumor growth in a xenograft model. Taken together, our TAB004-derived MUC28z CAR T cells are very potent at killing TNBC tumor cells both in vitro and in vivo, which is very promising to provide a safe, effective and tumor antigen-specific novel immunotherapeutic intervention. Citation Format: Ru Zhou, Mahboubeh Yazdanifar, Lopamudra Das Roy, John Maher, Pinku Mukherjee. Tumor MUC1 glycoprotein-highly specific CAR T cells control triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2305.

In recent years, vaccines against tumor antigens have shown potential for combating invasive cancers, including primary tumors and metastatic lesions. This is particularly pertinent for breast cancer, which is the second-leading cause of cancer-related death in women. MUC1 is a glycoprotein that is normally expressed on glandular epithelium, but is overexpressed and under-glycosylated in most human cancers, including the majority of breast cancers. This under-glycosylation exposes the MUC1 protein core on the tumor-associated form of the protein. We have previously shown that a vaccine consisting of MUC1 core peptides stimulates a tumor-specific immune response. However, this immune response is dampened by the immunosuppressive microenvironment within breast tumors. Thus, in the present study, we investigated the effectiveness of MUC1 vaccination in combination with four different drugs that inhibit different components of the COX pathway: indomethacin (COX-1 and COX-2 inhibitor), celecoxib (COX-2 inhibitor), 1-methyl tryptophan (indoleamine 2,3 dioxygenase inhibitor), and AH6809 (prostaglandin E2 receptor antagonist). These treatment regimens were explored for the treatment of orthotopic MUC1-expressing breast tumors in mice transgenic for human MUC1. We found that the combination of vaccine and indomethacin resulted in a significant reduction in tumor burden. Indomethacin did not increase tumor-specific immune responses over vaccine alone, but rather appeared to reduce the proliferation and increase apoptosis of tumor cells, thus rendering them susceptible to immune cell killing.

Screening for breast cancer has predominantly been done using mammography. Unfortunately, mammograms miss 50% cancers in women with dense breast tissue. Multi-modal screenings offer the best chance of enhancing breast cancer screening effectiveness. We evaluated the use of TAB004, an antibody that recognizes the tumor form of the glycoprotein MUC1 (tMUC1), to aid early detection of breast cancer. Our experimental approach was to follow tMUC1 from the tissue into circulation. We found that 95% of human breast cancer tissues across all subtypes stained positive for TAB004. In breast cancer cell lines, we showed that the amount of tMUC1 released from tumor cells is proportional to the cell's tMUC1 expression level. Finally, we showed that TAB004 can be used to assess circulating tMUC1 levels, which when monitored in the context of cancer immunoediting, can aid earlier diagnosis of breast cancer regardless of breast tissue density. In a blinded pilot study with banked serial samples, tMUC1 levels increased significantly up to 2 years before diagnosis. Inclusion of tMUC1 monitoring as part of a multi-modal screening strategy may lead to earlier stage diagnosis of women whose cancers are missed by mammography.

Abstract Antibody-derived Chimeric Antigen Receptor (CAR) T cells have great success in reduction of liquid form of tumor, but not solid tumor. Our novel antibody TAB 004 can specifically recognize the tumor form of MUC1 (tMUC1) while sparing the normal MUC1 in several subtypes of breast cancers including >95% of triple negative breast cancer (TNBC). In vivo delivery of TAB 004 specifically accumulates in the xenograft TNBC tumor only. Therefore, we hypothesize that TNBC can be specifically targeted with TAB 004-derived tMUC1-specific CAR T cells. A panel of 45 human breast cancer cell lines were tested for tMUC1 level, and 11 out of 13 TNBC cell lines showed higher frequency of tMUC1 expression compared to that on normal cells. We engineered a 2nd generation human CAR using TAB 004 that was coupled to the CD28-CD3zeta. CAR was well expressed on activated human primary CD8 and CD4 T cells. Compared to their mock control, CAR-T cells expressed higher CD25 and CD11c, particularly on CD8 T cells. Those CAR-T cells specifically recognized the tMUC1 antigen and formed tight synapse with tMUC1high tumor cells in vitro. The susceptibility of TNBC cells to CAR-T cell cytolysis was dramatic while still corresponding to their tMUC1 level. Only the tMUC1-specific CAR-T cells expressed and released large amount of IFN-gamma after antigen recognition on TNBC cells. However, IFN-gamma was not directly involved in the tumor killing by CAR-T cells. The in vivo treatment of TNBC tumor by CAR-T cells is ongoing. Taken together, our TAB 004-originated CAR-T cells are very potent at killing TNBC tumor cells in a tMUC1-highly specific manner, which is very promising to provide a safe, effective and tumor antigen-specific immunotherapeutic intervention.

e15751 Background: APC that has progressed after treatment with gemcitabine has a dismal prognosis and novel treatment approaches are needed. Reg is a potent oral inhibitor of VEGFR 1-3, PDGFR, TIE-2, FGFR-1, KIT, and the RAF kinases, and has activity in pancreatic xenograft models. We tested the activity of Reg in patients (pts) with refractory APC. Methods: This single arm, single center phase II study evaluated Reg (120mg/d, for 21 days, followed by 7-day break, with escalation to 160mg after the 1st cycle if tolerated) in pts with metastatic pancreatic cancer whose disease had progressed after at least one prior line of therapy and treatment with gemcitabine. Pts underwent radiographic evaluation every 2 cycles. The primary endpoint was 16-wk PFS. Kaplan Meier techniques were used to estimate PFS and OS. Serum tumor MUC1 antigen (tMUC1) concentrations were measured at baseline, end of cycle 1, and off-treatment using the TAB 004 antibody (Agkura Personal Score blood test, OncoTAb, Inc). Relative change of tMUC1 from baseline to end of cycle 1 was compared between those with and without 16-wk disease control. Results: 20 pts were enrolled into the study. Median age = 65 (47-79), and 80% (16/20) had 2 or more prior lines of therapy for advanced disease. Landmark 16 wk PFS = 10% (2/20), crossing a predefined futility boundary to demonstrate 20% improvement over historical controls with BSC. ORR was 5% (1/20), and DCR at 8 wks was 20% (4/20). Median PFS was 6.1 wks (95% CI: 2.9 – 7.1), and median OS was 9.4 wks (95% CI: 8.1 – 17.0). 10% of pts (2/20) had protocol defined Reg dose escalation to 160mg, and 30% (6/20) had dose reduction to 80mg. The most frequent grade 3-4 adverse events included hyponatremia (35%), fatigue (20%), and hypoalbuminemia (20%). Baseline tMUC1 varied substantially, mean 69.6ug/mL, median 51.3ug/mL (10.8 – 310.5). Serum tMUC1 decreased by a mean of 20.8% in the pts with disease control at 16 wks (n = 2), but increased by 65% (mean) in pts with disease progression or death within 16 wks (n = 13, p = 0.048). Conclusions: Reg has minimal activity as a single agent in pts with heavily treated APC. Serum tMUC1 levels measured by TAB 004 antibody may be a novel tumor marker in this disease. Different treatment approaches are needed. Clinical trial information: NCT02080260.

Screening for breast cancer has predominantly been done using mammography. Unfortunately, mammograms miss 50% cancers in women with dense breast tissue. Multi-modal screenings offer the best chance of enhancing breast cancer screening effectiveness. We evaluated the use of TAB004, an antibody that recognizes the tumor form of the glycoprotein MUC1 (tMUC1), to aid early detection of breast cancer. Our experimental approach was to follow tMUC1 from the tissue into circulation. We found that 95% of human breast cancer tissues across all subtypes stained positive for TAB004. In breast cancer cell lines, we showed that the amount of tMUC1 released from tumor cells is proportional to the cell’s tMUC1 expression level. Finally, we showed that TAB004 can be used to assess circulating tMUC1 levels, which when monitored in the context of cancer immunoediting, can aid earlier diagnosis of breast cancer regardless of breast tissue density. In a blinded pilot study with banked serial samples, tMUC1 levels increased significantly up to 2 years before diagnosis. Inclusion of tMUC1 monitoring as part of a multi-modal screening strategy may lead to earlier stage diagnosis of women whose cancers are missed by mammography.

Abstract In 2016, Pancreatic Cancer (PC) has moved to 3rd leading cause of cancer-related deaths in the USA with 94% dying within 5 years of diagnosis. 90% of PC is Pancreatic Ductal Adenocarcinoma (PDA), of which 80% of PDA overexpress tumor associated Mucin-1 (tMUC1), a membrane bound glycoprotein that is hypoglycosylated. Overexpression of tMUC1 is associated with metastasis and poor prognosis. However the mechanism remains obscure. Transforming growth factor-β (TGF-β) plays a role in poor prognosis. TGF-β is a cytokine with dual functionality. Within normal cells and early carcinogenesis, TGF-β functions as a tumor suppressor and induces apoptosis. This effect is mediated by activation of the canonical Smad pathway via engagement of TGF-β Receptor 1 (TGF-βRI). However during later stages of cancer, TGF-β becomes a tumor promoter and stimulates epithelial to mesenchymal transition, migration, and invasion thus enhancing metastasis. This TGF-β effect is mediated by the noncanonical Erk1/2 pathway. We have recently shown correlation between tMUC1 and TGF-β within an exogenous tMUC1 model of PDA. Therefore, it is hypothesized that the mechanism between the two newly connected pathways exists in PDA tumors with high endogenous tMUC1 levels. We propose that the tyrosine kinases present in the cytoplasmic tail of tMUC1 are intermediary between the two pathways, thus leading to enhanced metastasis. First the secretion levels of TGF-β1 were assessed in a panel of 13 PDA cell lines with variable expression levels of tMUC1 by ELISA. The effects of TGF-β1 to induce apoptosis versus invasiveness on a variety of tMUC1 high and tMUC1 low PDA cell lines were determined. The effects of tMUC1 knockdown were evaluated in tMUC1 high cell lines via siRNA. We studied the role of tyrosine kinases and other protein complexes in mediating the interactions between tMUC1 and TGF-β via proteomics analysis. Finally, we evaluated the anti-tumor efficacy of neutralizing TGF-β1 in vivo in high and low tMUC1-expressing PDA tumors. In tMUC1 high PDA cells, TGF-β1 is secreted less when compared to tMUC1 low PDA cells, allowing for the more resistant cells to diminish TGF-β mediated effects endogenously. It is shown that with TGF-β1 treatment, tMUC1 high cells are more invasive, resist apoptosis, and activate Erk pathway, while tMUC1 low cells activate SMAD pathway. However, tMUC1 knockdown via siRNA does not affect TGF-β receptor levels. When studying the tyrosine kinases, c-Src becomes more phosphorylated in the presence of TGF-β1 in tMUC1 high cells, while in tMUC1 low cells c-Src becomes more deactivated in the presence of TGF-β1. Finally, within the mouse model, tMUC1-high PDA tumors respond to the neutralization of TGF-β1 by decreasing tumor size, while having no statistical effect on tMUC1-low PDA tumors. tMUC1 expression is influential in TGF-β function in an endogenous model. Neutralizing TGF-β in tMUC1 high expressing tumors can be considered as a possible treatment. This has high clinical significance for patients with PDA. Citation Format: Priyanka Grover, Monica D. Nye, Mahboubeh Yazdanifar, Mohammad Ahmad, Ru Zhou, Lopamudra Das Roy, Kajal Grover, Shu-ta Wu, Sritama Nath, Pinku Mukherjee. MUC1 regulates TGFβ function in pancreatic cancer [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 321. doi:10.1158/1538-7445.AM2017-321

Rationale and Background: Immunotherapy of cancer has gained much attention in the past decade with the development of immune checkpoint inhibitors and chimeric antigen receptor (CAR) technology that can activate and redirect patient T cells to kill tumors that over-express a specific antigen. CARs are fusion receptors that are comprised of an antibody-derived single-chain variable fragment (scFv) coupled via hinge and transmembrane elements to a T cell signaling and co-stimulatory domain. This technology is in its early stages of development and has not been fully exploited for the treatment of metastatic epithelial cancers. We focus our studies on Pancreatic Ductal Adenocarcinomas (PDAC) and Triple Negative Breast Cancer (TNBC). However, if successful, the studies will be applicable to other epithelial tumors. For CAR-T cells to work, and to avoid off target toxicities, both the target antigen and the antibody recognizing the target have to be highly specific. The challenge is that there are few such antigen-antibody combinations for solid tumors. We have recently developed a novel patented antibody (designated TAB 004) that specifically recognizes ONLY the tumor-associated form of MUC1 (tMUC1) but not the normal form of MUC1 (nMUC1) in several subtypes of breast cancers including TNBC and in PDAC. We show compelling data that TAB004 recognizes tMUC1 in >90% of human TNBCs and 85% of PDAC but spares all normal epithelial tissues. The antigenic isoform that TAB004 recognizes is completely hidden in normal epithelia making it extremely safe for development of CAR-T cells. Hypothesis: TNBC and PDAC can be specifically targeted with the tMUC1-CAR-engineered T cells, whilst sparing normal organs. Methodology and Results: We have engineered several TAB-specific CAR constructs using the scFv fragment of TAB 004. Six constructs are developed, 3 for human T cells and 3 for mouse T cells: 1. TAB- CD28-CD3zeta (2nd generation CAR), 2. TAB-CD28-41BB-CD3zeta (3rd generation CAR), and 3. TAB-CD28-OX40-CD3zeta (3rd generation CAR). Data shows that we can successfully engineer human T cells to express the TAB-CAR on their surface and that these engineered T cells can bind specifically to tumor cells expressing the unique tMUC1 epitope, become activated, and effectively kill the tumor cells. We show that these engineered T cells only minimally bind and kill normal epithelial cell lines. However, some of the cell lines are more resistant than others. We are therefore conducting combination therapy with various drugs that are known to enhance immune based therapies including checkpoint inhibitors, COX-2 inhibitors, cyclophosphamide and others. We may also have data to show that the engineered TAB-CAR T cells kill tMUC1-expressing TNBC and PDAC cells in vivo. We will show data that TAB-CAR-T cell kill human PDAC and TNBC cells in vitro and propose to conduct the same in vivo xenograft model of human PDAC and metastatic TNBC. In the following months, we will test if TAB-CAR-T cell retards tumor growth in an orthotopic syngeneic mouse model of PDAC and metastatic BC in human MUC1.Tg immune competent mice. We further propose to demonstrate that the TAB-CAR-T cell can mediate apoptosis in the immune competent KCM mice (KC X human MUC1.Tg mice) that develop spontaneous PDAC and the MMT mice (PyVMT X human MUC1.Tg mice) that develop spontaneous mammary gland tumors. Both models mimic the human disease progression and express human MUC1 in a tissue specific manner. This is important since all normal epithelia in these mice express the nMUC1 except the tumors that expresses the target, tMUC1. Impact: If successful, this project will have a major impact and accelerate progress toward a clinical trial for PDAC and metastatic TNBC. This will be the first attempt to test the efficacy of a CAR-T cell in an immune competent, human MUC1.Tg mouse model that develops spontaneous tumors within the appropriate stromal and hormonal microenvironment. Citation Format: Pinku Mukherjee, Ru Zhou, Mahboubeh Yazdanifar, Das Roy Lopamudra. Development and future of CAR T cell therapy for pancreatic ductal adenocarcinoma and triple negative breast cancer [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr A37.

We report that Mucin1 (MUC1), a transmembrane glycoprotein that is overexpressed in >80% of pancreatic ductal adenocarcinoma (PDA), induced a pro-angiogenic tumor microenvironment by increasing the levels of neuropilin-1 (NRP1, a co-receptor of vascular endothelial growth factor (VEGF)) and its ligand VEGF. Expression of tumor-associated MUC1 (tMUC1) positively correlated with NRP1 levels in human and mouse PDA. Further, tMUC1hi PDA cells secreted high levels of VEGF and expressed high levels of VEGF receptor 2 (VEGFR2) and its phosphorylated forms as compared with tMUC1low/null PDA. This enabled the tMUC1hi/NRP1hi PDA cells to (a) induce endothelial cell tube formation, (b) generate long ectopic blood vessels and (c) enhance distant metastasis in a zebrafish xenograft model. Concurrently, the proteins associated with epithelial-to-mesenchymal transition, N-cadherin and Vimentin, were highly induced in these tMUC1/NRP1hi PDA cells. Hence, blocking signaling via the NRP1–VEGF axis significantly reduced tube formation, new vessel generation and metastasis induced by tMUC1hi PDA cells. Finally, we show that blocking the interaction between VEGF165 and NRP1 with a NRP1 antagonist significantly reduced VEGFR signaling and PDA tumor growth in vivo. Taken together, our data suggest a novel molecular mechanism by which tMUC1 may modulate NRP1-dependent VEGFR signaling in PDA cells.

BACKGROUND: Earlier detection of transformed cells using target-specific imaging techniques holds great promise. We have developed TAB 004, a monoclonal antibody highly specific to a protein sequence accessible in the tumor form of MUC1 (tMUC1). We present data assessing both the specificity and sensitivity of TAB 004 in vitro and in genetically engineered mice in vivo. METHODS: Polyoma Middle T Antigen mice were crossed to the human MUC1.Tg mice to generate MMT mice. In MMT mice, mammary gland hyperplasia is observed between 6 and 10 weeks of age that progresses to ductal carcinoma in situ by 12 to 14 weeks and adenocarcinoma by 18 to 24 weeks. Approximately 40% of these mice develop metastasis to the lung and other organs with a tumor evolution that closely mimics human breast cancer progression. Tumor progression was monitored in MMT mice (from ages 8 to 22 weeks) by in vivo imaging following retro-orbital injections of the TAB 004 conjugated to indocyanine green (TAB-ICG). At euthanasia, mammary gland tumors and normal epithelial tissues were collected for further analyses. RESULTS: In vivo imaging following TAB-ICG injection permitted significantly earlier detection of tumors compared with physical examination. Furthermore, TAB-ICG administration in MMT mice enabled the detection of lung metastases while sparing recognition of normal epithelia. CONCLUSIONS: The data highlight the specificity and the sensitivity of the TAB 004 antibody in differentiating normal versus tumor form of MUC1 and its utility as a targeted imaging agent for early detection, tumor monitoring response, as well as potential clinical use for targeted drug delivery.

Abstract Background: Breast cancer (BC) remains the second leading cause of cancer-related deaths for women in the United States and is recognized to be a heterogeneous disease. Advances in technologies such as whole genome sequencing are leading the way to precision medicine and the leading researchers are envisioning personalized therapies in the not too distant future. However, given the diversity of cancer cell populations, that remains a challenging task at best. The tumor form of MUC1 (designated tMUC1), a transmembrane glycoprotein, is aberrantly glycosylated and overexpressed in ∼95% of BC. We have developed an antibody (TAB004) that specifically recognizes tMUC1 across all major subtypes of BC and importantly does not recognize normal breast epithelia. This is a significant development in light of the challenges faced in treating triple negative BC. Methods: A panel of thirty BC cell lines was obtained from ATCC. The following techniques were used to assess the specificity of TAB 004 to the major subtypes based on ER, PR and Her2 expression: 1) Flow cytometry to quantify membrane bound expression of tMUC1 using Cy7-conjugated TAB004; 2) Western blotting to detect molecular weight patterns of tMUC1 in whole cell lysate; 3) A TAB004 based GMP-grade ELISA kit to measure shed tMUC1 in the supernatant and 4) In vivo imaging of tumors in mice using TAB 004 conjugated to Indocyanine Green (ICG). Specificity and sensitivity was further confirmed using primary human serum and tissue samples from all major BC subtypes obtained from bio-repositories at Duke University Cancer Center, Fox Chase Cancer Center and Carolinas Health Care System. Shed tMUC1 in serum samples were tested using the TAB 004 ELISA kit and tissue sections were analyzed using immunohistochemical (IHC) staining with TAB 004 conjugated to HRP. Results: 1) Flow cytometry data shows that TAB 004 recognized tMUC1 on all major BC subtypes: 25 out of 30 BC cell lines tested had higher expression than a normal epithelial breast cell line; 2) Western blotting also detected tMUC1 on all BC subtypes with distinct molecular weight patterns; 3) ELISA showed high levels of shed tMUC1 by most BC cells and correlated with bound/cytoplasmic levels. 4) In vivo imaging shows clear localization of TAB004-ICG to the tumors expressing tMUC1. Primary human breast cancer patient data shows that shed tMUC1 was detected in the serum obtained from all major BC subtypes and showed statistically significant differentiation from normal/benign. IHC results show strong tMUC1 expression in malignant tissue with excellent differentiation from adjacent normal tissue. Conclusion: TAB004 antibody's extraordinary specificity across major BC subtypes has been confirmed with flow cytometry, western blotting, ELISA and Immunohistochemistry. A number of clinical applications are under development: (a) An ELISA test as a supplement to mammography for the early detection of BC in women with dense breasts; (b) serum monitoring during treatment and to detect disease recurrence; and, (c) targeted antibody-drug/antibody-imaging agent based therapies and imaging modalities particularly for triple negative BC. Citation Format: Das Roy L, Zhou R, Dillon L, Moore LJ, Puri R, Marks JR, Lyerly HK, Mukherjee P. A monoclonal antibody with exceptional specificity across major breast cancer subtypes. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-09-16.

e22153 Background: Breast cancer (BC) remains the second leading cause of cancer-related deaths for women in the US. Mammography has improved early detection, but can miss up to 25 percent of BC since mammogram sensitivity is less than 50% in women with dense breast tissue. BC is often detected in these women when a lump is discovered and the cancer is late stage. There is a critical need for an accurate diagnostic for these women. The tumor form of MUC1 (designated TMUC1),a transmembrane glycoprotein, is aberrantly glycosylated and overexpressed in ~95% of BC. We have developed an antibody (TAB004), which specifically recognizes TMUC1. Methods: 1) Using TAB004, a GMP-grade ELISA kit was used to test circulating levels of TMUC1 in sera of patients. We have tested ~250 patient sera (includes normal, benign, and malignant) collected from Duke University Cancer Center, National Cancer Institute (NCI), Early detection research network (EDRN) reference set, and Fox Chase Biorepositories. The samples represented the major BC subtypes (Luminal A, Luminal B, Triple Negative, and HER2 type). 2) Expression of bound TMUC1 on BC tissue was determined with Immunohistochemistry (IHC) using TAB004. 3) We quantified surface expression of TMUC1 using Cy5-conjugated TAB004 and flow cytometry on a panel of over 30 human BC cell lines that represented the major subtypes. 4) Finally, TAB004 conjugated to a fluorophore was used to image human BC tumors in vivo in mice. Results: 1) TMUC1 ELISA results show statistically significant differentiation between normal/benign and malignant sera distributions (p = .000 to .056 from the various institutions). 2) IHC results show strong TMUC1 expression in malignant tissue sparing normal tissue. 3) Flow cytometry data shows that all BC subtypes expressed TMUC1. 4) In vivo imaging shows localization of TAB004-fluorophore to the tumor. Conclusions: A unique tumor specific antibody based serum ELISA can be used for the early detection of breast cancer in women with dense breast tissue. Clinical adoption of the TAB004 based serum ELISA in conjunction with mammography will lead to early detection of BC in these women.

14 Background: Earlier detection of abnormal cells using target-specific techniques holds great promise. One of those targets Mucin-1 (MUC1) is expressed as a hypo-glycosylated form (i.e., tMUC1) in 95% of breast tumors and plays a crucial role in cancer progression. We have developed Tab004, a monoclonal antibody highly specific to a protein sequence accessible in tMUC1. Here we present data assessing the specificity and sensitivity of Tab004 in vitro and in genetically engineered mice in vivo overtime. Methods: We have developed human MUC1 Tg mice that were bred to mice that carry the oncogene, polyoma middle T antigen driven by the MMTV promoter. These mice designated MMT develop mammary gland tumors spontaneously and expresses the human form of tMUC1. PyVMT and C57Bl/6 served as controls. Mice develop mammary gland hyperplasia between 8-10 weeks of age that progresses to ductal carcinoma in situ by 12-14 weeks and adenocarcinoma by 18-24 weeks. Approximately 40% of the mice develop metastasis to the lung and other organs. The tumor progression appropriately mimics the human disease. MMT mice (n = 20) were injected twice monthly retro-orbitally with 12.5ug (100uL) of Tab004-conjugated to indocyanine green (ICG) and imaged thereafter from 8 to 22 weeks of age. Fluorescence was assessed 4 and 24 hrs post injection using the IVIS system. At euthanasia, tissue was collected for further analyses. Further, human breast tumor and normal mammary epithelial tissues were evaluated by immunohistochemical staining. Results: Tab004 specifically recognizes tMUC1 and not normal MUC1. In MMT mice, ICG-conjugated Tab004 allowed early detection of tumors in vivo sparing recognition of normal mammary epithelia in the C57BL/6 mice or in the PyV MT tumors. Detection with ICG-conjugated Tab004 allowed monitoring of tumor progression overtime. Importantly, ICG-conjugated Tab004 permitted significantly earlier detection than physical examination. Conclusions: The data highlight the specificity and the sensitivity of Tab004 in detecting tMUC1 in vitro, in situ and in relevant murine models in vivo. Thus, Tab004 will have significant clinical relevance for development as a targeted imaging agent and in the future for targeted drug delivery.

Objective Eighty percent of pancreatic ductal adenocarcinomas (PDAs) overexpress mucin 1 (MUC1), a transmembrane mucin glycoprotein. MUC1(high) PDA patients also express high levels of cyclooxygenase 2 (COX-2) and show poor prognosis. The cytoplasmic tail of MUC1 (MUC1-CT) partakes in oncogenic signaling, resulting in accelerated cancer progression. Our aim was to understand the regulation of Cox-2 expression by MUC1.Methods Levels of COX-2 and MUC1 were determined in MUC1(-/-), MUC1(low), and MUC1(high) PDA cells and tumors using reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemistry. Proliferative and invasive potential was assessed using MTT and Boyden chamber assays. Chromatin immunoprecipitation was performed to evaluate binding of MUC1-CT to the promoter of COX-2 gene.Results Significantly higher levels of COX-2 mRNA and protein were detected in MUC1(high) versus MUC1(low/null) cells, which were recapitulated in vivo. In addition, deletion of MUC1 gene and transient knockdown of MUC1 led to decreased COX-2 level. Also, MUC1-CT associated with the COX-2 promoter at approximate to 1000 base pairs upstream of the transcription start site, the same gene locus where nuclear factor B p65 associates with the COX-2 promoter.Conclusions Data supports a novel regulation of COX-2 gene by MUC1 in PDA, the intervention of which may lead to a better therapeutic targeting in PDA patients.

BACKGROUND:IL-17A is a pro-inflammatory cytokine that is normally associated with autoimmune arthritis and other pro-inflammatory conditions. Recently, IL-17A has emerged as a critical factor in enhancing breast cancer (BC)-associated metastases. We generated immune competent arthritic mouse models that develop spontaneous BC-associated bone and lung metastasis. Using these models, we have previously shown that neutralization of IL-17A resulted in significant reduction in metastasis. However, the underlying mechanism/s remains unknown.METHODS:We have utilized two previously published mouse models for this study: 1) the pro-arthritic mouse model (designated SKG) injected with metastatic BC cell line (4T1) in the mammary fat pad, and 2) the PyV MT mice that develop spontaneous mammary gland tumors injected with type II collagen to induce autoimmune arthritis. Mice were treated with anti-IL-17A neutralizing antibody and monitored for metastasis and assessed for pro-inflammatory cytokines and chemokines associated with BC-associated metastasis.RESULTS:We first corroborate our previous finding that in vivo neutralization of IL-17A significantly reduced metastasis to the bones and lungs in both models. Next, we report that treatment with anti-IL17A antibody significantly reduced the expression of a key chemokine, CXCL12 (also known as stromal derived factor-1 (SDF - 1)) in the bones and lungs of treated mice. CXCL12 is a ligand for CXCR4 (expressed on BC cells) and their interaction is known to be critical for metastasis. Interestingly, levels of CXCR4 in the tumor remained unchanged with treatment. Consequently, protein lysates derived from the bones and lungs of treated mice were significantly less chemotactic for the BC cells than lysates from untreated mice; and addition of exogenous SDF-1 to the lysates from treated mice completely restored BC cell migration. In addition, cytokines such as IL-6 and M-CSF were significantly reduced in the lung and bone lysates following treatment. The data presented suggests that systemic neutralization of IL-17A can block the CXCR4/SDF-1 signaling pathway by reducing the expression of SDF-1 in the metastatic niches and significantly reducing metastasis in both mouse models.CONCLUSION:In our model, neutralization of IL-17A regulates SDF-1 expression in the metastatic niches either directly or indirectly via reducing levels of IL-6 and M-CSF.