Abstract: Evaluation of new technologies requires rigorous methods to provide unbiased estimates of the performance and so inform future clinical practice. We review evidence on DNA cytometry reported earlier in this journal and point to the standards for reporting of diagnostic accuracy as a metric against which this article can be evaluated. The cross-sectional nature of the data and incomplete reporting limit the clinical utility of the study. With application of improved reporting standards for diagnostic tests and improved design and evaluation of new technologies for screening, we may better inform practices to improve clinical outcomes and population health. Clin Cancer Res; 17(22); 6971–2. ©2011 AACR.

Abstract: Kruppel-like factor 9 (KLF9) is a member of the KLF family of transcription factors. Previous studies report significant KLF member involvement in breast cancer estrogen response, notably KLF4 [1, 2] and KLF5 [3, 4] . KLF9 has been shown in uterine cancer cells to function as an estrogen-dependent regulator of the estrogen response pathway [5]. We aim to evaluate the role of KLF9 in breast cancer cell estrogen response. We have performed studies demonstrating that KLF9 exhibits an early-activated estrogen response. Five sites have been identified upstream of the KLF9 gene that interact with estrogen receptor alpha (ERα) [6-8] ; we observe ERα enrichment at three of these sites that is estradiol-dependent. Though KLF9 has been shown to act as an essential element in estrogen response in the uterus, KLF9 response in breast cancer cells has yet to be characterized. To study the role of KLF9 in estrogen-mediated responses in transcriptional and proliferative activity, we have manipulated the level of KLF9 expression in MCF-7 breast cancer cells. These cells have been shown to exhibit a clear transcriptional response to estradiol in luciferase reporter assays. We have optimized RNAi conditions to achieve significant knockdown of the KLF9 gene in MCF-7 cells. Additionally, we have produced and tested a KLF9 overexpression vector construct that significantly upregulates expression of KLF9 in this cell line. These tools will be used to more extensively characterize the role of KLF9 in MCF7 cell estrogen-stimulated transcription and proliferation. We are measuring transcriptional response to estrogen signaling with luciferase reporter assays and estrogen stimulated cell proliferation with BrdU proliferation assays. Elucidating KLF9 involvement in E2-mediated breast cancer cell signaling and response, therefore, is an important component of understanding of the regulatory mechanisms behind estrogen response in breast cancer. 1. Akaogi K et al. (2009). Oncogene 28(32): 2894. 2. Quintana AM et al. (2011). BMC Cancer 11: 30. 3. Guo P et al. (2010). International Journal of Cancer 126(1): 81. 4. Zhao KW et al. (2011). Biochemical Journal 437(2): 323. 5. Velarde MC et al. (2007). Molecular Endocrinology 21(12): 2988. 6. Carroll JS et al. (2006). Nature Genetics 38(11): 1289. 7. Hua S et al. (2008). Molecular Systems Biology 4:188. 8. Fullwood MJ et al. (2009). Nature 462(7269): 58. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 67. doi:1538-7445.AM2012-67

Abstract: Double heterozygosity is an extremely rare occurrence in hereditary breast and ovarian cancer syndrome (HBOC [MIM 604370; MIM 612555]) where two pathogenic variants, one in BRCA1 and one in BRCA2, are found in an individual. To date, only a few case reports and case series have been reported in the literature (1-3). Furthermore, little is known about the clinical characteristics, family history, and tumor histology in these patients. In this study, we utilized targeted gene testing with next-generation sequencing (NGS) technology in an early-onset metastatic breast cancer patient from France. We evaluated germline variants using Pathway Genomics' BRCATrueTM NGS test, which analyzes variants covering all exons and exon flanking regions in both the BRCA1 and BRCA2 genes. All variant calls were determined after alignment and mapping to the GRCh37/hg19 reference genome. Variant calls were confirmed by Sanger sequencing. In this patient, a c.1016dupA (p.V340GfsX6) frameshift variant was found in BRCA1 along with a c.6814delA (p.R2272EfsX8) frameshift variant in BRCA2. Both frameshift variants are predicted to truncate the BRCA proteins. The BRCA1 c.1016dupA variant is considered a Norwegian founder mutation but has also been observed in individuals who are of French-Canadian, French, Italian or Dutch ancestry (4-7). The BRCA2 c.6814delA (p.R2272Efs*8) pathogenic variant, also known as 7042delA, is predicted to truncate the BRCA2 protein and has been identified in individuals with a personal or family history of breast and/or ovarian cancer (8,9). To the best of our knowledge, the combination of these two pathogenic variants in an individual has not been previously reported. In a clinical diagnostic setting, the possibility of double heterozygosity of pathogenic variants in more than one susceptibility gene should be considered, especially in patients with early-onset metastatic cancers. Furthermore, genetic testing and genetic counseling should also be indicated for high-risk family members. 1. Heidemann, S. et al. (2012) Breast cancer research and treatment 134, 1229-1239 2. Lavie, O., et al. (2011) Annals of oncology : official journal of the European Society for Medical Oncology / ESMO 22, 964-966 3. Nomizu, T., et al. (2012). Breast cancer 4. Andersen, T. I., Borresen, A. L., and Moller, P. (1996) American journal of human genetics 59, 486-487 5. Caputo, S., et al. (2012) Nucleic acids research 40, D992-1002 6. Dorum, A., et al. (1999). American journal of human genetics 65, 671-679 7. Simard, J., et al. (1994). Nature genetics 8, 392-398 8. Novakovic, S., et al. (2012) International journal of oncology 41, 1619-1627 9. Tea, M. K., et al. (2014) Maturitas 77, 68-72. Citation Format: Curtit E, Meynard G, Villanueva C, Mansi L, Chaix M, Vilalta A, Kuo JZ, Villa M, Neidich J, Tomar A, Arianpour A, Lebahar P, Pivot X. Double heterozygosity for BRCA1 and BRCA2 pathogenic variants in a French metastatic breast cancer patient. [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 P2-09-10.

Abstract: Current therapy for malignant brain tumors, such as glioblastoma multiforme (GBM), is insufficient, with nearly universal recurrence. Available drug therapies are unsuccessful because they fail to penetrate through the region of the brain containing tumor cells and they fail to kill the cells most responsible for tumor development and therapy resistance, brain cancer stem cells (BCSCs). To address these challenges, we combined two advances in technology: 1) brain-penetrating polymeric nanoparticles that can be loaded with drugs and are optimized for intracranial convection-enhanced delivery (CED); and 2) re-purposed, FDA-approved compounds, which were identified through library screening to target BCSCs. Using fluorescence imaging, positron emission tomography (PET), and magnetic resonance imaging (MRI), we demonstrate that brain-penetrating nanoparticles can be delivered intracranially to large volumes in both rat and pig. We identified several FDA-approved agents that potently inhibit proliferation and self-renewal of BCSCs. When loaded into brain-penetrating nanoparticles and administered by CED, one of these agents dramatically increased survival in rats bearing BCSC-derived xenografts. This new approach to controlled delivery in the brain should have a significant impact on treatment of GBM and suggests new routes for drug and gene delivery to treat other diseases of the CNS. Bibliography 1. Sawyer AJ, Saucier-Sawyer J, Booth C, Liu J, Patel T, Piepmeier JM, and Saltzman WM. Convection-enhanced delivery of camptothecin-loaded polymer nanoparticles for treatment of intracranial tumors, Drug Delivery and Translational Research 1:34-42 (2011). PMCID: PMC3117592 2. Zhou J, Patel TR, Sirianni RW, Strohbehn G, Zheng MQ, Duong N, Schafbauer T, Huttner AJ, Huang Y, Carson RE, Zhang Y, Sullivan DJ, Piepmeier JM, and Saltzman WM. Highly penetrative nanocarriers loaded with drugs targeted to resistant cells improve treatment of glioblastoma, Proceedings of the National Academy of Sciences, 110 (29): 11751-11756 (2013). PMCID: PMC3718184 3. Deng Y, Saucier-Sawyer JK, Hoimes CJ, Zhang J, Seo YE, Andrejecsk JW, and Saltzman WM. The effect of hyperbranched polyglycerol coatings on drug delivery using degradable polymer nanoparticles. Biomaterials 35:6595-6602 (2014). PMCID: PMC4062180. 4. Strohbehn G, Coman D, Han L, Ragheb RT, Fahmy TM, Huttner AJ, Hyder F, Piepmeier JM, Saltzman WM, and Zhou J. Imaging the delivery of brain-penetrating PLGA nanoparticles in the brain using magnetic resonance, Journal of Neuro-Oncology 121:441-449 (2014). PMCID: PMC4323763. 5. Cheng CJ, Bahal R, Babar IA, Pincus Z, Barrera F, Liu C, Svoronos A, Braddock DT, Glazer PM, Engelman DM, Saltzman WM, and Slack FJ. MicroRNA silencing for cancer therapy targeted to the tumor microenvironment, Nature 518:107-110 (2015). PMCID: PMC4367962. 6. Cheng CJ, Tietjen GT, Saucier-Sawyer JK, and Saltzman WM. A holistic approach to targeting disease with polymer nanoparticles, Nature Reviews Drug Discovery 14:239-247 (2015). PMCID: PMC4451203. 7. Saucier-Sawyer JK, Seo Y, Gaudin A, Sawyer AJ, Deng Y, Quijano E, Huttner A, and Saltzman WM. Distribution of nanoparticles after convection-enhanced delivery (CED) in the brain, Journal of Controlled Release 232:103-112 (2016). [PCMID: in process] Citation Format: Mark Saltzman. Convection-enhanced delivery of nanomaterials for glioma. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr IA15.

Abstract: Determining what drives conversion of non-lethal to lethal breast cancers is of utmost importance for understanding and blocking tumor metastasis. We have identified semaphorin 7a (Sem7a) as a potent driver of human breast cancer progression, suggesting it may represent a novel target to inhibit metastasis. Sem7a is a GPI membrane anchored protein that promotes attachment and spreading in multiple cell types1,2; previous reports have identified a role for Sem7a in mouse mammary tumorigenesis, including promotion of tumor growth and EMT, as well as induction of angiogenic cytokine production by macrophages and metastatic outgrowth in the lung3-5. However, Sem7a has not been investigated in human breast tumors, so we utilized publicly available microarray datasets and found that SEMA7A gene expression is increased in ~45% of all breast cancers. Further, we found SEMA7A upregulation was associated with poor prognosis, which is consistent with a role in metastasis. Specifically, breast cancer patients with high expression of SEMA7A are at risk for early recurrence and have decreased overall and distant metastasis-free survival rates. We used in vitro and in vivo approaches to assess the role of SEM7a in human breast tumor progression. In vivo, we show that shRNA mediated silencing of Sem7a significantly slows tumor growth, delays tumor progression from in situ to locally invasive disease, and decreases tumor associated lymphangiogenesis. Furthermore, having previously shown that collagen I and COX-2 drive tumor progression in our breast cancer models6,7, we identified a relationship between COX-2 and Sem7a expression at the mRNA and protein levels. Finally, using three- dimensional spheroid and tube formation assays, we show that semaphorin 7a promotes tumor cell invasion on collagen, and lymphangiogenesis, via activation of the β1-integrin receptor. Our results indicate that Sem7a may be novel target for blocking multiple aspects of breast tumor progression that lead to metastasis. References: 1. Pasterkamp, R.J., Peschon, J.J., Spriggs, M.K. & Kolodkin, A.L. Semaphorin 7A promotes axon outgrowth through integrins and MAPKs. Nature 424, 398-405 (2003). 2. Scott, G.A., McClelland, L.A. & Fricke, A.F. Semaphorin 7a promotes spreading and dendricity in human melanocytes through beta1-integrins. The Journal of investigative dermatology 128, 151-161 (2008). 3. Allegra, M., et al. Semaphorin-7a reverses the ERF-induced inhibition of EMT in Ras-dependent mouse mammary epithelial cells. Molecular biology of the cell 23, 3873-3881 (2012). 4. Garcia-Areas, R., et al. Semaphorin7A promotes tumor growth and exerts a pro-angiogenic effect in macrophages of mammary tumor-bearing mice. Frontiers in physiology 5, 17 (2014). 5. Ma, B., et al. Role of chitinase 3-like-1 and semaphorin 7a in pulmonary melanoma metastasis. Cancer research 75, 487-496 (2015). 6. Lyons, T.R., et al. Cyclooxygenase-2-dependent lymphangiogenesis promotes nodal metastasis of postpartum breast cancer. The Journal of clinical investigation (2014). 7. Lyons, T.R., et al. Postpartum mammary gland involution drives progression of ductal carcinoma in situ through collagen and COX-2. Nature medicine 17, 1109-1115 (2011). Citation Format: Sarah Black, Andrew Nelson, Natalia Gurule, Bernard Futscher, Traci Lyons. Silencing of semaphorin 7a attenuates breast tumor progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr PR10.

Abstract: A solid tumor is like an aberrant organ - comprised of cancer cells and a variety of host cells embedded in an extra-cellular matrix - nourished by blood vessels and drained by lymphatic vessels. Using an array of imaging technologies and animal models, we have shown that blood and lymphatic vessels as well as matrix associated with tumors are abnormal and these abnormalities can create a hostile tumor microenvironment (e.g., hypoxia, high interstitial fluid pressure). These abnormalities can fuel tumor progression as well as prevent treatments from reaching and attacking tumor cells (Nature 2011). In my presentation, I will discuss two emerging strategies to “normalize” the tumor microenvironment to enhance the outcome of radiation, chemo- and immuno-therapies. Vascular normalization hypothesis: After realizing that the abnormal structure and function of tumor vessels is a result of the imbalance between endogenous pro- and anti-angiogenic molecules, we proposed a novel hypothesis: Anti-angiogenic therapy can transiently “normalize” the abnormal tumor vasculature, resulting in more efficient delivery of drugs and oxygen to cancer cells. We also hypothesized that chemo-, immuno- and/or radiation therapy given during this “window of normalization” is likely to yield the best outcome for combination therapy (Nature Medicine 2001; Science 2005). This hypothesis offered a potential explanation for why drugs, such as bevacizumab (whose goal is to destroy tumor vessels) improve the outcome of chemotherapeutics (that require blood vessels for drug delivery), and more importantly, offered guidelines to improve such combination therapies. We first tested this hypothesis in a variety of pre-clinical models (PNAS 1996; 1998; Nature 2002; Cancer Research, 2004; Cancer Cell, 2004). Our work demonstrated that blockade of VEGF signaling or upregulation of thrombospondin transiently prunes the immature and leaky vessels of tumors in mice and actively remodels the remaining vasculature so that it more closely resembles the normal vasculature. This “normalized” vasculature is characterized by less leaky, less dilated, and less tortuous vessels, with a more normal basement membrane and greater vessel coverage by pericytes. These morphological changes are accompanied by functional changes: decreased interstitial fluid pressure, decreased tumor hypoxia, and improved penetration of drugs in these tumors. The outcome of combination therapy was found to be synergistic when the cytotoxic therapy was given during the normalization window (Cancer Cell 2004). We then dissected the molecular and cellular mechanisms of vascular normalization (Cancer Cell, 2004). We discovered that an increase in angiopoietin 1 expression contributes to the increased pericyte coverage and an increase in MMP activity contributes to the basement membrane normalization. We further showed that the kinetics of vascular normalization determines the outcome of combined antiangiogenic and radiation therapy (Cancer Cell, 2004). After careful and rigorous characterization of tumor vasculature in pre-clinical models, in collaboration with medical, surgical and radiation oncologists, we evaluated the molecular, structural and functional changes in the vasculature of rectal carcinomas in patients receiving bevacizumab with radiation and chemotherapy. This study provided the first glimpse of how anti-angiogenic therapy might work in patients (Nature Medicine, 2004), and supported our pre-clinical findings on vascular normalization. In collaboration with neuro-oncologists and neuro-radiologists, we also demonstrated vascular normalization in recurrent glioblastoma in patients receiving an oral pan-VEGF receptors tyrosine kinase inhibitor - cediranib - and discovered that the extent of normalization correlates with both progression-free and overall survival (Cancer Cell 2007; Cancer Research 2009). More crucially, patients whose tumor blood flow went up - and not down - after cediranib treatment survived longer (Cancer Research 2012). Our pre-clinical and clinical findings on normalization have been confirmed by a number of laboratories worldwide, and represent a paradigm shift for the field of anti-angiogenesis therapy (Physiological Reviews 2011). Vascular normalization offers unprecedented opportunities to improve treatment of various vascular disorders - besides cancer - that afflict millions of people worldwide, including wet age-related macular degeneration - a leading cause of blindness. Indeed, our team has demonstrated that vascular normalization using bevacizumab can reverse chronic progressive hearing loss in patients with schwannomas (neurofibromatosis type 2) (New England Journal of Medicine, 2009). Matrix normalization hypothesis: In parallel, our laboratory discovered that cancer cells co-opt the host stromal cells into producing pro- and antiangiogenic cytokines and extra-cellular matrix. By revealing that stromal cells are active participants rather than passive bystanders in tumor progression and therapeutic response (Cell 1998; Nature Medicine 1999, 2001; Nature 2002), we proposed these cells as a novel target for cancer therapy (Nature Medicine 2003). We also discovered that both cancer cells and stromal cells compress tumor vessels, and depleting these cells can open blood vessels (Nature 2004). By imaging collagen in vivo (Nature Medicine 2003) and directly measuring movement of drugs in tumors in vivo (Nature Medicine 2004), we discovered that collagen matrix produced by stromal cells can compress blood vessels and impede drug delivery. More importantly, the hormone relaxin and the FDA-approved anti-hypertensive drugs that target stromal cells can “normalize” the tumor microenvironment by reorganizing the collagen matrix (PNAS 2011). These findings offer new hope for improving delivery and efficacy of therapeutics in highly fibrotic tumors (e.g., desmoplastic breast cancer, pancreatic cancer). Citation Format: {Authors}. {Abstract title} [abstract] . In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr PL02-01. doi:1538-7445.AM2012-PL02-01

Abstract: Finding the right balance between cancer prevention and early detection is essential for an efficient anti-cancer strategy. Yet, the underlying biological question “how much cancer risk results from chance, how much from environmental factors’’ continues to be controversial. Recently, the “bad luck’’ hypothesis was strengthened based on observing a tight correlation between a tissues cancer risk and its number of stem cell divisions [1]. This interpretation was later challenged by pointing out that certain even drastic changes of environmental risk factors would not affect theat correlation [2] . Based on this argument they derived an estimate of the upper bound of environmental contributions to canceriogenesis. Here we argue that the argument made in [2] did not fully appreciate that typically several mutations are requited before cancer initiation occurs [3] . We show that by incorporating this aspect into the analysis the correlation between the number of stem cell divisions and cancer risk would respond to changes of environmental factors. We re-analyze the data in [1] to derive a slightly more stringent upper bound of the environmental risk contributions for a number of cancers than the ones obtained in [2] . [1] C. Tomasetti, B. Vogelstein, Science 347(6217), 78-81 (2015) [2] S. Wu, S.Powers et al., Nature 529, 43-47 (2015) [3] C. Nordling, British Journal of Cancer 7(1), 68-72 (1953) Citation Format: Sven Bilke, Paul S. Meltzer. Environment or accident? A more stringent bound on environmental contributions to cancerogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-291.

Abstract: The interaction between the c-MET tyrosine kinase receptor (RTK) on the surface of cancer cells with its ligand, hepatocyte growth factor (HGF) secreted from surrounding tumor stroma, results in paracrine signal transduction in cancer cells to promote growth, invasion, angiogenesis and metastases. c-MET gene amplification/over-expression, or activating mutations, frequently function as oncogenic driver in several types of cancers. c-MET is thus considered a key target(1) for both TKIs or biologics. However, lack of suitable animal models has represented an obstacle for the development of new cMET inhibitors. While patient derived xenografts (PDXs) mirror patients’ histopathological/genetic profiles(2-6), they usually lose human stroma, the source of HGF; moreover the HGF originated from murine stroma is incapable of triggering a paracrine signaling because of lack of cross reactivity with the human c-MET. Some PDX grow independent of paracrine mechanisms because of their constitutive c-MET activation by the presence of activating mutations, gene amplification or autocrine signaling, and can be used to evaluate the efficacy of TKIs(5, 7, 8). However they are not suitable to assess biologics, e.g. antibodies disrupting receptor-ligand interaction. Alternatively it is possible to grow tumors in hu-HGF-transgenic immunocompromised mice (humanization) where an artificial paracrine signaling is created. Interestingly, we have identified a unique HCC-PDX, LI0801, which maintains human stroma throughout passages, as demonstrated by immunochemistry staining for human vimentine and human HGF, both markers of stromal components(7, 8). The mechanisms of human stroma maintenance in this PDXremain unclear. We showed that LI0801 responds to different TKIs(7, 8). In addition we evaluated the efficacy of an anti-human c-MET monoclonal antibody in reducing tumor burden in LI0801, and we were able to demonstrate great tumor response to anti-human c-MET antibody, which is significantly more effective than crizotinib. To the best of our knowledge, LI0801 is the first paracrine c-MET PDX reported, and represents an important model to study paracrine cMET/HGF signaling and to evaluate new c-MET inhibitors, both TKIs, and biologics. References 1. Comoglio PM, et al. Nat Rev Drug Discov. 2008;7:504-16. 2. Ding L, et al. Nature. 2010;464:999-1005. 3.Marangoni E, et al. A new model of patient tumor-derived breast cancer xenografts for preclinical assays. Clin Cancer Res. 2007;13:3989-98. 4.Chen D, Oncotarget. 2015. 5. Yang M, et al. International journal of cancer Journal international du cancer. 2013;132:E74-84. 6. Zhang L, et al. A subset of gastric cancers with EGFR amplification and overexpression respond to cetuximab therapy. Sci Rep. 2013;3:2992. 7. Bladt F, et al. Cancers (Basel). 2014;6:1736-52. 8. Chen D. AACR Annual Meeting 2012. 2012;Poster. Citation Format: Dawei Chen, Ziyong Sun, Likun Zhang, Jie Cai, Davy X. Ouyang, Jean-Pierre Wery, Henry Li. Paracrine c-MET/HGF HCC PDX: evaluation of a biologics targeting c-MET. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 747.

Abstract: Molecular classification of breast cancers into receptor-based subtypes, including estrogen positive or triple negative breast cancers, establish targets that guide individualized treatment for patients (Journal of clinical oncology:27, 1153-1154, 2009). However, drug resistance observed for breast cancer raises the challegens for the breast cancer treatment (CA: a cancer journal for clinicians 59, 303-313, 2009). Targeting the death receptor-5 (DR-5) to induce breast cancer apoptosis is a promising strategy for breast cancer treatment (Immunity 3, 673-682, 1995, Breast Cancer Res Treat 113, 217-230, 2009). TRA-8 is a DR-5 specific agonistic antibody (Nature medicine 7, 954-960, 2001), and TRA-8 can induce apoptosis in breast cancer cells (Clinical cancer research: 9, 3731-3741, 2003). TRA-8 signal DR-5-mediated apoptotic signal via formation of the death inducing signaling complex (DISC) at the DR-5 death domain (Immunity 12, 599-609, 2000). Calmodulin (CaM) is a mediator of calcium signaling and has been shown to play a role in regulating DR-5 mediated apoptosis (Biochem Cell Biol 87, 919-926, 2009). Understanding the mechanism of DR-5-CaM interaction and the role of CaM-DR-5 binding in regulating DR-5 mediated DISC formation, may unveil CaM-DR-5 binding as a potential target to modulate DR-5 mediated apoptosis. In this study we characterize an interaction between CaM and DR-5 and the role of CaM-DR-5 binding in DR-5-mediated DISC formation in MCF-7 and MDA-MB-231 breast cancers. Complex-immunoprecipitation of DR-5 from MCF-7 and MDA-MB-231 lysates demonstrated endogenous CaM and DR-5 form a complex. Native PAGE of recombinant 6xHis-SUMO-CaM incubated with MCF-7 and MDA-MB-231 lysates showed the formation of a CaM and DR-5 complex. CaM pull down of DR-5 from MCF-7 and MDA-MB231 lysates in the presence of 1 mM Ca2+ or 2 mM EGTA showed CaM/DR-5 interaction is calcium dependent. TRA-8 activation of DR-5 resulted in CaM recruitment into the DR-5 mediated DISC. Additionally, Either EGTA or CaM antagonist trifluopherazine (TFP) attenuated DR-5 mediated DISC formation in MCF-7 and MDA-MB-231 breast cancers. We present evidence for the Ca2+ dependent CaM and DR-5 interaction and a role of CaM-DR-5 binding in regulating DR-5 mediated DISC formation in breast cancer cells. Results from this study provide the basis for the further characterization of CaM-DR5 interactions and its important role in cellular activities, which could lead to novel strategies for effectively regulating DR-5-mediated apoptosis in breast cancer. Citation Format: Romone M. Fancy, Hong Wang, Tong Zhou, Yuhua Song. Calmodulin binding to DR-5 and the role of CaM-DR-5 binding in DR-5-mediated DISC formation in breast cancer. [abstract]. In: Pro ceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2282. doi:10.1158/1538-7445.AM2014-2282

Abstract: Characterization of the tumor microenvironment (TME) is expected to be critical in informing prognosis and guiding response prediction1,2. Recent studies have classified the TME based on tumor infiltrating leukocytes (TILs), programmed death-ligand 1 (PDL1) expression, and the nature of inflammatory responses1,3. Tumor studies involving transcript profiling and IHC have begun to inform the diversity and complexity of the tumor-infiltrated immune compartment (TIIC) by defining aspects of cellular composition, spatial distribution, and function. The use of flow cytometry allows the elucidation of an array of immune cell subtypes across entire samples. This study characterizes immune cell population subsets and tumor cells in four cancer types (breast, head and neck, colorectal, and gall bladder) from patients using multi-color flow cytometry. A good correlation was observed (r= 0.89, p=0.003) between tumor content, as evaluated by a pathologist (Hematoxylin and Eosin staining), and tumor content (EpCAM/panCK[7/8]+ cells) as estimated by flow cytometry (for all cancers excepting head and neck). Using pathology scoring we divided the samples into hi gh-content ( & gt;10% tumor) and low-content ( & lt;10% tumor) samples. Samples with high tumor content were further examined by comparing the immune population in the tissue with their matched peripheral blood mononuclear cells (PBMC). We observed significantly higher proportions of T-cells, T-regulatory cells (Tregs) and exhausted T-cells (PD-1+ T-cells) in TIIC in comparison to its matched PBMC, while a lower proportion of B-cells and NK cells was noted. Also, samples with high tumor content had a significantly increased proportion of CD8 T-cells, Tregs and NK cells and a lower proportion of effector CD4 (CD4+Foxp3-) T-cells in comparison to the samples with low tumor content. The results from this study demonstrate the differences in the peripheral immune repertoire from the TIIC, reaffirming the need to robustly characterize the local TME. Additionally, the similarity in Treg and exhausted T-cell profiles across the indications examined may support a broad utility for therapeutics that can successfully target immune exhaustion or suppression. References: 1. Marshall, Henry, et al. "Immuno-oncology: emerging targets and combination therapies." Frontiers in oncology 8 (2018): 315. 2. Li, Xiaotong, et al. "Immune profiling of pre-and post-treatment breast cancer tissues from the SWOG S0800 neoadjuvant trial." Journal for immunotherapy of cancer 7.1 (2019): 88. 3. Binnewies, Mikhail, et al. "Understanding the tumor immune microenvironment (TIME) for effective therapy." Nature medicine 24.5 (2018): 541-550. Citation Format: Nandini Pal Basak, Shivani Vignesh, Sampratha Bhagyashekar, Manimaran Manickam, Debika Datta, Anupam Nath, Misti Jain, Jason Wall. Flow cytometric characterization of the tumor-infiltrating immune compartment: Significant differences in the tumor immune repertoire versus peripheral blood [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4990.