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The ability to monitor response to therapy and disease progression in metastatic breast cancer (MBC) patients is a major step in patient management. Imaging is the method of choice for the assessment of disease status and the monitoring of disease progression. However, this approach remains expensive, expose patients to radiation and thus is mainly performed every 2-3 months. During this time interval, the disease may progress significantly on ineffective treatment and the patient may present treatment related toxicities due to the inability to detect progression at earlier times. Circulating levels of tumor associated biomarkers such as CA15-3 and CEA are often determined to track disease status of MBC. However, even though they can provide information about disease progression, they do not always provide a reliable measure of response to therapy. The monitoring of disease status and progression through the measurement of drivers of disease should provide an alternative and complementary approach to existing strategies in order to better to monitor the disease status and enable proactive management of MBC patients. Progranulin also called Glycoprotein 88kDa (PGRN/GP88) is an autocrine growth factor overexpressed in breast cancer. Biological studies have established GP88 as a critical player in breast tumorigenesis. GP88 overexpression is associated with the malignant phenotype, estrogen independence, increased proliferation, survival, and drug resistance. High PGRN/GP88 tumor expression measured by immunohistochemistry in invasive ductal carcinoma is an independent prognostic marker associated with increased risk of recurrence and mortality. Clinical studies have demonstrated that GP88 circulating levels as measured by enzyme immunoassay are elevated in breast cancer patients, compared to healthy individuals. In MBC patients, circulating GP88 levels correlate with overall survival. These facts are supportive of the hypothesis that the measurement of circulating GP88 levels in MBC patients can serve as an additional biomarker to monitor MBC disease status and be predictive to outcome. A prospective study was established is to identify whether there is a statistically significant change in serum GP88 levels associated with time to progression of breast cancer as measured by RECIST 1.1 criteria in MBC patients. With the assumptions that patients will provide a baseline and four follow up visits and that 20% of the visits record a disease progression, time to progression. Taking the plausible and clinically relevant performance to be 75% sensitivity and 46% false positive, a sample of ninety patients would give 85% power. Under IRB approved protocols at the University of Maryland Greenebaum Comprehensive Cancer Center and at two Baltimore Medstar Health Facilities, a total of 103 female breast cancer patients with measurable or evaluable metastatic disease will be consented and enrolled. The patients have been re-staged within 4 weeks and will continue or begin new therapy. Currently, we have enrolled sixty-five subjects at the three facilities. In addition to standard laboratory assessment and radiographic imaging/staging every 2-3 months on study, blood samples will be collected from each patient. The samples are stored at -70C until evaluated for GP88 using a GP88 enzyme linked immunoassay. We will analyze the GP88 serum level in correlation with survival and with disease status determined as responder, stable or progressing based on the RECIST criteria. This study is supported by grant R44CA210817 from the National Cancer Institute to Ginette Serrero Principal Investigator. Citation Format: Ginette Serrero, Paula Rosenblatt, Nancy Tait, Barbara Rector, Jennifer A. Latteri, Binbin Yue, Katherine Tkaczuk. On-going prospective study to measure serum progranulin/GP88 levels in metastatic breast cancer patients in association with disease status [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-16-01.

The human epidermal growth factor receptor (HER) family of receptors plays a significant role in the pathogenesis of several human cancers. Among them, HER2 is overexpressed in several cancers. In particular, overexpression with or without gene amplification occurs in 15-30% of breast cancers and this has both prognostic and predictive implications. HER2 overexpression and amplification is associated with shorter disease-free and overall survivals and with resistance to certain hormonal agents as well as increased risk of metastasis to the brain. Two types of treatment targeting HER2 biological activity have been developed: anti-Her2 monoclonal antibodies and small molecule tyrosine kinase inhibitors such as lapatinib and neratinib targeting HER2 and EGFR and Afatinib targeting all HER family members. Trastuzumab is a humanized anti-HER2 monoclonal antibody that binds to domain IV of HER2 extracellular segment and blocks its signaling. It is the first therapeutic antibody targeting Her2 overexpression approved by the FDA. It is administered in combination with standard of care chemotherapy. More recently, since it was shown that Trastuzumab was an internalizing antibody, two antibody drug conjugates using trastuzumab have been approved and used in the standard of care: Ado-Trastuzumab-Emtansine (Kadcyla) consisting of trastuzumab conjugated to the drug mertansine DM1 and fam-trastuzumab-deruxtecan-nhki (Enhertu). Enhertu is another antibody drug conjugate using Trastuzumab to deliver a topoisomerase inhibitor deruxtecan. Our laboratory has been focused in developing fully human internalizing anti-Her2 antibodies that compete or not with trastuzumab for binding to Her2. These antibodies have been developed by immunizing fully human mice with recombinant Her2 protein. Human Ab producing Tc mice (TC-mAb mice) stably maintain a mouse-derived engineered chromosome containing the entire human Ig heavy and kappa chain loci in a mouse Ig knockout background. After production of hybridoma secreting fully human immunoglobulins, the screening process included inhibition of binding of trastuzumab to Her2 by enzyme linked immunoassay and by Octet epitope binning as well as internalization assay. Several internalizing antibodies with Kd ranging from 10-9 M to 10-12 M were selected. They were stratified by their ability to compete or not with trastuzumab. They were further characterized for their ability to deliver a cytotoxic payload in Her2 overexpressing cells as well as for their efficacy to inhibit proliferation, signaling and migration of Her2 overexpressing breast cancer cells. Data related to these antibodies will be presented here. In conclusion, the use of fully human TC mice in our laboratory represents an attractive approach to develop fully human monoclonal antibodies to high value cancer targets that can by-pass the need for humanization and affinity maturation of antibodies. Citation Format: Ginette Serrero, Jianping Dong, Binbin Yue, Udaya Yerramalla, Jun Hayashi. Characterization of fully human internalizing anti-HER2 monoclonal antibodies that compete with Trastuzumab for binding to HER2 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-19-01.

Progranulin (PGRN/GP88) is an 88 kDa glycoprotein characterized by seven and a half double cysteine rich repeats in the granulin-epithelin family. PGRN/GP88 is an autocrine biological driver of tumorigenesis, survival & drug resistance in several cancers including breast, ovarian, multiple myeloma, prostate cancers, non-small cell lung carcinoma (NSCLCA) & digestive cancers. PGRN/GP88 tissue expression is an independent prognostic factor of recurrence while elevated serum PGRN/GP88 level in metastatic breast, lung & prostate cancer patients is associated with poor outcomes such as progression & shortened survival. An anti-human PGRN/GP88 monoclonal antibody inhibiting PGRN/GP88 action has been developed & expressed as recombinant antibody in CHO cells. Activities including pharmacology, manufacturing, formulation & GLP toxicology studies have been carried out. The IND application has been cleared by the Food and Drug Administration to proceed with the first-in-human AG01 clinical study in adult patients ((pts) with advanced solid tumors. We present an ongoing First in Human Phase 1A (dose escalation,1+(3+3) & IB (expansion cohorts) study of AG01 in pts advanced solid tumor malignancies (1A) with 4 expansion cohorts (1B) in pts with advanced Triple Negative Breast Cancer (TNBC), Hormone Resistant ER+/Her2- BC, advanced NSCLC & mesothelioma. Study Design: This is an open-label, dose escalation study of AG01 antibody administered intravenously (IV) over 90 minutes every 14 days +/- 1 day (1A), followed by 4 predefined expansion cohorts, which will be treated at the RP2D determined in the phase 1A of this study. In the 1A part, initially accelerated titration design will be utilized to guide dose progression & estimation of the maximum tolerated dose (MTD and/or maximum administered dose (MAD). In the 1A portion of the study pts with advanced relapsed/refractory solid tumor malignancies who failed 1 or more standard of care (SOC) therapies (tx) or for whom no SOC tx exists will be accrued. The primary objective of the 1A part is to determine the MTD and/or MAD of AG01. Secondary objectives are to determine the RP2D, assess the safety/tolerability, the pharmacokinetics (PKs) & immunogenicity of AG01 & the preliminary anti-tumor activity of AG01 via RECIST 1.1. The exploratory objectives are to determine PGRN/GP88 expression in tumor tissue and PGRN/GP88 blood levels using A&G’s ELISA test. In the 1B Cohort Expansion phase, 4 separate cohorts of pts with PGRN/GP88 tissue expression of 1+, 2+, 3+ by IHC will be enrolled. Cohort 1- TNBC: ER and/or PR < 1% by IHC, HER2 < 3+ by IHC and/or FISH negative, pts must have failed 1 or more SOC tx for metastatic BC. Cohort 2- Hormone-resistant BC: ER and/or PR >1%, HER2 < 3+ by IHC and/or FISH negative, failed 1 or more prior hormonal tx (HT) or HT/CD4/6 kinase inhibitor tx or other targeted tx. Cohort 3- NSCLCA: metastatic/recurrent NSCLCA failed 2 or more SOC tx. Cohort 4 -Mesothelioma- failed 1 or more SOC tx for metastatic/recurrent mesothelioma or not a candidate for SOC tx. The primary objective of 1B part is to evaluate the antitumor efficacy of AG01 by overall response rate (ORR) defined as completed response (CR), partial response (PR), stable disease >=24 weeks (SD) (CR+PR+ SD) based on RECIST v1.1 in the 4 cohorts with each cohort assessed separately for response. Secondary objectives are to evaluate progression free survival (PFS), duration of response (DOR) & overall survival (OS) of pts in cohorts 1-4, & to evaluate the ORR, DOR & PFS based on GP88 tissue expression, to further characterize the PKs & the safety/tolerability of AG01. Exploratory objectives in 1B part will assess AG01 effect on circulating PGRN/GP88 levels in plasma or other potential biomarkers in the 4 cohorts. The estimated study sample is approximately 77 pts; 17 pts for the 1A part & 60 pts for 1B part. The study is open to accrual at the UMGCCC. Supported by NCI grants R44CA224718 and R44CA162629 Citation Format: KATHERINE TKACZUK, Paula Rosenblatt, Ranee Mehra, Katherine Scilla, Nancy Tait, Binbin Yue, Ginette Serrero. First in Human Study of AG01 a chimerized monoclonal antibody to Progranulin/Glycoprotein 88 (GP88) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-13-01.

Background: Triple negative breast cancer (TNBC) is characterized by invasiveness and short survival. Identifying novel TNBC targeted therapies to potentiate standard of care (SOC) therapy, is an unmet need. Progranulin (PGRN/GP88) is a biological driver of tumorigenesis, survival, and drug resistance in several cancers including breast cancer (BC). PGRN/GP88 tissue expression is an independent prognostic factor of recurrence while elevated serum PGRN/GP88 level is associated with poor outcomes such as progression of disease and shortened survival. Since PGRN/GP88 expression is elevated in 30% TNBC, we investigated the effect of inhibiting PGRN/GP88 effect on the proliferation and tumor growth of triple negative breast cancer cells. Methods: For this purpose, we have developed a neutralizing anti-human PGRN/GP88 monoclonal antibody AG01 and examined its effect on the proliferation, migration, signaling pathway activation and biomarker expression of two TNBC cell lines MDA-MB-231 and HS578-T expressing PGRN/GP88, both in vitro and in vivo. Results: The inhibition of PGRN/GP88 action by AG01 treatment reduced proliferation and migration in a dose-and time-dependent fashion in MDA-MB-231 and HS578-T cells. Western blot analysis showed decreased expression of phosphorylated protein kinases p-Src, p-AKT and p-ERK involved in proliferation and survival upon AG01 treatment for both cell lines. Transwell assay showed that AG01 treatment inhibited migration and invasion in a dose-dependent fashion. Microarray analysis of several oncoproteins in cells treated with AG01 or control antibody demonstrated the inhibition of the expression of several markers of migration and angiogenesis. In vivo xenograft studies with MDA-MB-231 cells injected in athymic nude mice showed that AG01 treatment inhibited tumor growth as well as Ki67 expression, mitotic index and microvessel counts when compared to antibody control treated mice. In vivo dose response of AG01 in MDA-MB-231 tumor bearing mice will be provided. Conclusion: TNBC is a disease with poor prognosis in need of novel targeted therapeutic solutions. PGRN/GP88 represents a therapeutic target for TNBC with two companion diagnostics (tissue test and ELISA to measure GP88 circulating levels). Blocking PGRN/GP88 with AG01 antibody treatment will provide novel targeted therapeutic option for TNBC which could address the issue of toxicity, and unresponsiveness associated with SOC. This work is supported by a grant CA 224718 from the National Cancer Institute to GS. Citation Format: Ginette Serrero, Rupa Guha, Jianping Dong, Binbin Yue. Anti-progranulin (GP88) antibody AG01 inhibitory effect on the growth of triple negative breast cancer cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-17-10.

The ability to detect metastatic breast cancer (MBC) & readily monitor response to therapy is an important goal in the management of breast cancer (BC). Imaging is used for the assessment & monitoring of disease progression. However, this is expensive & represents a late marker of disease status. The disease may progress significantly on ineffective treatment & the patient may have treatment related toxicities due to the lack of knowledge of the progression at earlier times. Circulating tumor associated biomarkers CA15-3 & CEA can be used to track disease status of MBC, however, they are not always a reliable measure of disease progression. The monitoring of disease progression through measurement of drivers of disease may better reflect the disease status & enable proactive management sooner. GP88 (granulin/epithelin precursor or progranulin) has been identified as a driver of tumorigenesis & disease progression through its signaling as an autocrine growth & survival factor for several cancers including BC1. We hypothesized that measurement of circulating serum GP88 levels can serve as a biomarker to monitor MBC disease status & be prognostic to outcome 2. Clinical studies have demonstrated circulating levels of GP88 statistically differ in healthy volunteers, early BC patients (pts), & pts with metastatic disease & levels correlate with overall survival2,3. The primary aim of this prospective study is to identify whether there is a statistically significant change in serum GP88 levels associated with response to treatment & time to progression of BC as measured by RECIST 1.1 criteria in MBC pts. Under an IRB approved protocol at the University of Maryland Greenbaum Comprehensive Cancer Center, a total of 103 female pts with measurable or evaluable metastatic disease who have been re-staged in within 4 weeks & will continue or begin new therapy & meet the additional criteria will be consented & enrolled. Currently, we have enrolled 23 pts. In addition to standard of care laboratory assessment & radiographic imaging/staging every 2-3 months, blood samples will be collected from each patient. The samples will be stored at -70C until tested for GP88 using a GP88 enzyme linked immunoassay. We will analyze the serial GP88 serum levels & correlate with treatment response using RECIST 1.1 criteria (stable, progression, response), time to progression & overall survival. We assume that pts will provide a baseline & 4 follow up visits/GP-88 levels & that 20% of the visits will record a disease progression & time to progression. The probable & clinically relevant GP-88 test performance will then have 75% sensitivity & 46% false positivity with 85% power in a sample of 90 pts. References:1. Serrero, G. (2003) Autocrine growth factor revisited. PC-Cell derived growth factor (PCDGF) a critical player in breast cancer tumor genesis. Biochem. Biophysics. Res. Commun. 308, 409-413. 2 Tkaczuk, K.R., et al., Increasing Circulating Level of the Survival Factor GP88 (Progranulin) in the Serum of Breast Cancer Patients When Compared to Healthy Subjects. Breast Cancer (auckl), 2011. 5: p. 155-62 3. Tkaczuk, K. H. R., D. Hawkins, B. Yue, D. Hicks, N. Tait and G. Serrero (2020). "Association of Serum Progranulin Levels with Disease Progression, Therapy Response and Survival in Patients with Metastatic Breast Cancer." Clin Breast Cancer 20(3): 220-227. Citation Format: Katherine HR Tkaczuk, Paula Rosenbatt, Sarah Sulkowski, Nancy Tait, Binbin Yue, Ginette Serrero. Glycoprotein 88 (GP-88) serum levels as a marker of response to therapy in patients with metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT1-05-01.

Progranulin (PGRN/GP88) is an 88 kDa glycoprotein characterized by seven and a half double cysteine rich repeats which is the largest member of the granulin-epithelin protein family. PGRN/GP88 has been demonstrated as a biological driver of tumorigenesis, survival, and drug resistance in several cancers including breast cancer (BC), lung prostate, ovarian and digestive cancers. PGRN/GP88 tissue expression is an independent prognostic factor of recurrence in breast, lung cancers while elevated serum PGRN/GP88 level in metastatic breast, lung, ovarian and prostate cancer patients. Elevated PGRN/GP88 levels are associated with poor outcomes such as progression and shortened survival. An anti-human PGRN/GP88 monoclonal antibody able to inhibit PGRN/GP88 action in vitro and in vivo has been developed, chimerized and expressed in CHO cells. All IND enabling activities including pharmacology, GMP manufacturing, formulation and GLP toxicology studies have been conducted. The IND application has been filed and cleared by the Food and Drug Administration. A first-in-human, first-in-class phase 1 safety and efficacy clinical study of AG01 in patients with solid tumors and advanced disease with special focus on patients with breast, lung and ovarian cancers has been initiated and is on-going at the University of Maryland Greenebaum Cancer Center. The trial is registered as NCT05627960 to clinicaltrials.gov site. Supported by grants R44CA162629 and R44CA224718 from the National Cancer Institute to GS. Citation Format: Katherine R. Tkaczuk, Paula Rosenblatt, Ranee Mehra, Katherine A. Scilla, Nancy Tait, Katerra Caple, Binbin Yue, Ginette Serrero. On-going phase 1A clinical trial for AG01 an first-in-class anti-progranulin (GP88) monoclonal antibody in patients with advanced malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT240.

BACKGROUND: Triple negative breast cancer (TNBC) is characterized by invasiveness and short survival. Identifying novel TNBC-targeted therapies, to potentiate standard of care (SOC) therapy, is an unmet need. Progranulin (PGRN/GP88) is a biological driver of tumorigenesis, survival, and drug resistance in several cancers including breast cancer (BC). PGRN/GP88 tissue expression is an independent prognostic factor of recurrence while elevated serum PGRN/GP88 level is associated with poor outcomes. Since PGRN/GP88 expression is elevated in 30% TNBC, we investigated the involvement of progranulin on TNBC. METHODS: The effect of inhibiting PGRN/GP88 expression in TNBC cells by siRNA was investigated. The effects of a neutralizing anti-human PGRN/GP88 monoclonal antibody AG01 on the proliferation and migration of two TNBC cell lines expressing PGRN/GP88 were then examined in vitro and in vivo. RESULTS: Inhibition of GP88 expression by siRNA and AG01 treatment to block PGRN/GP88 action reduced proliferation and migration in a dose-dependent fashion in MDA-MB-231 and HS578-T cells. Western blot analysis showed decreased expression of phosphorylated protein kinases p-Src, p-AKT, and p-ERK upon AG01 treatment, as well as inhibition of tumor growth and Ki67 expression in vivo. CONCLUSION: PGRN/GP88 represents a therapeutic target with companion diagnostics. Blocking PGRN/GP88 with antibody treatment may provide novel-targeted solutions in TNBC treatment which could eventually address the issue of toxicity and unresponsiveness associated with SOC.

Background Progranulin (GP88) is a critical player in breast tumorigenesis. GP88 tumor expression is associated with increased recurrence and mortality, whereas GP88 circulating levels are elevated in patients with breast cancer compared with healthy individuals. We examined here the correlation between serum GP88 levels in patients with metastatic breast cancer (MBC) with overall survival and disease status determined as response to therapy or progression of disease. Patients and Methods An institutional review board (IRB)-approved study prospectively enrolled 101 patients with MBC at the University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer Center. GP88 serum levels were correlated with patients’ disease status determined by Response Evaluation Criteria In Solid Tumors (RECIST) 1.1 criteria and survival outcomes by Kaplan-Meier analysis and log rank statistics. Results Patients’ survival was stratified by serum GP88 level. Patients with serum GP88 55 ng/mL. Examination of GP88 serum levels in association with disease status showed a statistically significant association between serum GP88 levels and disease progression or response to therapy while CA15-3 level was only associated to progression. Conclusion The association of serum GP88 level with survival and disease status suggests the potential of using the serum GP88 test for monitoring disease status in patients with MBC. Measurement of serum GP88 levels in patients with MBC may have clinical value as a cost-effective adjunct to the management of patients with MBC with imaging.

Investigation of the role of progranulin/GP88 on the proliferation and survival of a wide variety of cells has been steadily increasing. Several human diseases stem from progranulin dysregulation either through its overexpression in cancer or its absence as in the case of null mutations in some form of frontotemporal dementia. The present review focuses on the role of progranulin/GP88 in cancer development, progression, and drug resistance. Various aspects of progranulin identification, biology, and signaling pathways will be described. Information will be provided about its direct role as an autocrine growth and survival factor and its paracrine effect as a systemic factor as well as via interaction with extracellular matrix proteins and with components of the tumor microenvironment to influence drug resistance, migration, angiogenesis, inflammation, and immune modulation. This chapter will also describe studies examining progranulin/GP88 tumor tissue expression as well as circulating level as a prognostic factor for several cancers. Due to the wealth of publications in progranulin, this review does not attempt to be exhaustive but rather provide a thread to lead the readers toward more in-depth exploration of this fascinating and unique protein.

Background Progranulin (GP88) is an 88-kDa glycoprotein growth factor with important biological effects in tumorigenesis and tumour survival. We investigated the usefulness of measuring serum GP88 concentrations as a predictive biomarker for hepatocellular carcinoma in patients with viral hepatitis C after treatment with direct-acting antiviral agents. Methods We measured the serum GP88 concentrations by using a sandwich enzyme-linked immunoassay from 67 healthy control subjects and 29 patients (20 patients who did not develop hepatocellular carcinoma and 9 patients who developed hepatocellular carcinoma after treatment) with viral hepatitis C after treatment with asunaprevir and daclatasvir. Results The serum GP88 concentrations of patients with chronic hepatitis C prior to antiviral treatment were significantly higher than those of healthy control subjects. After antiviral treatment, the serum GP88 concentrations of patients who eventually developed hepatocellular carcinoma were significantly higher than those who did not develop hepatocellular carcinoma. The changes in the serum GP88 concentrations before and after treatment in patients who developed hepatocellular carcinoma were significantly lower than those in patients who did not develop hepatocellular carcinoma. The cumulative incidence of hepatocellular carcinoma was significantly higher in either patients with high serum GP88 concentrations after treatment or those with small changes of serum GP88 concentrations pre- and post-treatment. Conclusions Sustained high concentrations of serum GP88 in patients treated with direct-acting antiviral agents are correlated with the risk of developing hepatocellular carcinoma.

Imaging is the method of choice for monitoring of disease response to therapy during and post-treatment in metastatic breast cancer (MBC) patients. However, imaging has limited sensitivity for real-time monitoring so clinicians use circulating tumor markers such as CA15-3, as surrogates for monitoring changes in disease, albeit with some limitations. Measuring tumor markers associated with the biologically driven tumor processes of development, proliferation and survival would add significantly to current disease information available and would improve the clinical management of this underserved patient population. Biological studies demonstrated that the 88kDa glycoprotein Progranulin (GP88) is a critical driver of breast cancer (BC) cell proliferation, survival, invasiveness and drug resistance. GP88 is expressed in tumor tissue and not in normal breast tissue and is secreted in the circulation of BC patients. Studies demonstrated that high tumor GP88 expression is prognostic for recurrence and that BC patients had a statistically elevated GP88 serum level compared to healthy controls. We hypothesized that changes in serum GP88 levels correlate with changes in disease state as defined by RECIST 1.1. In this current study, we examined whether GP88 and CA15-3 serum levels were correlated to disease progression and response to therapy. Additionally, we compared the information provided by the GP88 /CA15-3 combination in disease progression or response to therapy. 101 stage 4 MBC patients undergoing Standard of Care therapy were consented and enrolled under an IRB approved protocol at the University of Maryland Greenebaum Comprehensive Cancer Center. Patient demographics together with clinical and disease characteristics were collected as part of the study. Blood samples were drawn from each patient at specific times at follow-up visits during and post-therapy and tested for GP88 using A&G's GP88 enzyme linked immunoassay. Samples were tested for CA15-3 at the University's laboratory. Disease status was monitored and assessed by RECIST1.1 criteria and such assessment entered into the database. Statistical analyses were carried out using the serum GP88 and CA15-3 results to test each for association with contemporaneous RECIST assessment of disease progression (PD) or response (R). We determined that contemporaneous GP88 is significantly associated with PD (p 0.0101) and R (p 0.0194) while CA-15-3 is associated with PD (p We conclude that circulating levels of GP88/PGRN in MBC patients are correlated with both disease progression and response to therapy and that monitoring circulating GP88/PGRN levels would be complementary to CA15-3 determination and imaging to provide valuable insight into real-time monitoring of the disease status of MBC patients. Citation Format: Serrero G, Hawkins DM, Hicks DJ, Tait N, Yue B, Tkaczuk KR. Circulating progranulin (GP88) level is associated with both response to therapy and progression of disease in metastatic breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-12-10.

Antibody-drug conjugates (ADC) are effective antibody-based therapeutics for hematopoietic and lymphoid tumors. However, there is need to identify new targets for ADCs, particularly for solid tumors and cancers with unmet needs. From a hybridoma library developed against cancer cells, we selected the mouse monoclonal antibody 33B7, which was able to bind to, and internalize, cancer cell lines. This antibody was used for identification of the target by immunoprecipitation and mass spectrometric analysis, followed by target validation. After target validation, 33B7 binding and target positivity were tested by flow cytometry and western blot analysis in several cancer cell lines. The ability of 33B7 conjugated to saporin to inhibit in vitro proliferation of PTFRN positive cell lines was investigated, as well as the 33B7 ADC in vivo effect on tumor growth in athymic mice. All flow cytometry and in vitro internalization assays were analyzed for statistical significance using a Welsh’s T-test. Animal studies were analyzed using Two-Way Analysis of Variance (ANOVA) utilizing post-hoc Bonferroni analysis, and/or Mixed Effects analysis. The 33B7 cell surface target was identified as Prostaglandin F2 Receptor Negative Regulator (PTGFRN), a transmembrane protein in the Tetraspanin family. This target was confirmed by showing that PTGFRN-expressing cells bound and internalized 33B7, compared to PTGFRN negative cells. Cells able to bind 33B7 were PTGFRN-positive by Western blot analysis. In vitro treatment PTGFRN-positive cancer cell lines with the 33B7-saporin ADC inhibited their proliferation in a dose-dependent fashion. 33B7 conjugated to saporin was also able to block tumor growth in vivo in mouse xenografts when compared to a control ADC. These findings show that screening antibody libraries for internalizing antibodies in cancer cell lines is a good approach to identify new cancer targets for ADC development. These results suggest PTGFRN is a possible therapeutic target via antibody-based approach for certain cancers.

Progranulin is an autocrine growth factor that promotes proliferation, migration, invasion, and chemoresistance of various cancer cells. These mechanisms mainly depend on the protein kinase B (Akt)/mechanistic target of rapamycin (mTOR) pathway. Recent studies have shown that patients with hematopoietic cancer have elevated serum progranulin levels. Thus, the current study aimed to investigate the role of progranulin in hematopoietic cancer cells and how it modulates their proliferation. Both knockdown of progranulin and progranulin neutralizing antibody treatment inhibited proliferation in several human hematopoietic cancer cell lines. Moreover, progranulin depletion not only decreases the phosphorylation level of the Akt/mTOR pathway but also, surprisingly, increases the expression of transforming growth factor-beta (TGF-β) and phosphorylation of mothers against decapentaplegic homolog 2 (SMAD2) in Kasumi-1 cell. Furthermore, LY2109761, an inhibitor of TGF-β receptor type I/II kinase, and TGF-β neutralizing antibody blocked the inhibition of proliferation induced by progranulin depletion. These data provide new insights that progranulin alters cell proliferation via the TGF-β axis and progranulin could be a new therapeutic target for hematopoietic cancers., Hematologic neoplasms, Progranulin, Mechanistic target of rapamycin complex 1, Transforming growth factor beta.

Background & aims Progranulin (PGRN) is known to promote tumorigenesis and proliferation of several types of cancer cells. However, little is known about the clinicopathological features of patients with gastrointestinal stromal tumors (GISTs) with regard to PGRN expression. Methods A retrospective analysis was performed on patients with GISTs who underwent curative surgical resection between 2007 and 2017. PGRN expression was evaluated by immunohistochemical (IHC) analysis and semi-quantitatively categorized (no expression, 0; weak, 1+; moderate, 2+; strong, 3+). Tumors with a staining intensity of 2+ or 3+ were considered high PGRN expression. Results Fifty-four patients were analyzed; 31 patients (57%) were male. The median age at surgery was 60 years (range, 33–79), and the most common primary site was the stomach (67%). Thirty-five patients (65%) had spindle histology; 42 patients (78%) were separated as a high-risk group according to the modified National Institutes of Health (NIH) classification. High PGRN-expressing tumors were observed in 27 patients (50%), had more epithelioid/mixed histology (68% vs. 32%; p = 0.046), and KIT exon 11 mutations (76% vs. 24%; p = 0.037). Patients with high PGRN-expressing tumors had a worse recurrence-free survival (RFS) (36% of 5-year RFS) compared to those with low PGRN-expressing tumors (96%; p60 years) were independent prognostic factors for poor RFS. Conclusions High PGRN-expressing GISTs showed more epithelioid/mixed histology and KIT exon 11 mutations. PGRN overexpression was significantly associated with poor RFS in patients with GISTs who underwent curative resection.

Prostate cancer (PCa) is the second most common cancer, causing morbidity and mortality among men world-wide. The expression of the androgen receptor (AR) and its splice variants is a crucial factor of prostate cancer biology that has not been comprehensively studied in PCa tumors. The aim of this study was to characterize the protein expression of the AR and its splice variant, AR-V7, and their subcellular distributions in PCa by immunohistochemistry and to correlate the results to the clinicopathological data and prognosis. Immunohistochemical staining for AR and AR-V7 was performed on a tissue microarray (TMA) with specimens from 410 PCa patients using an immunoreactive score (IRS) or only the percentage of AR-V7 staining in cytoplasmic granules. Nuclear or cytoplasmic AR staining was not associated with prognosis. AR-V7 staining was only occasionally observed in the nucleus. However, AR-V7 staining in the cytoplasm or in cytoplasmic granules was associated with relapse-free survival (RFS). AR-V7 staining of the cytoplasm was associated with a shorter RFS, whereas AR-V7 staining of cytoplasmic granules was associated with a longer RFS. In a multivariate Cox&rsquo, s regression analysis, only negative (&lt, 5%) AR-V7 staining of cytoplasmic granules remained an independent prognostic factor for RFS (HR = 5.3, p = 0.006). In a further subgroup analysis by multivariate Cox&rsquo, s regression analysis, AR-V7 was an independent prognostic factor in the following groups: age &le, 65 (HR = 9.7, p = 0.029), negative CK20 staining (HR = 7.0, p = 0.008), and positive perineural invasion (HR = 3.7, p = 0.034). Altogether, AR-V7 protein in granular cytoplasmic structures is an independent prognostic factor for RFS in PCa patients.

Progranulin (PGRN) has been characterized as an autocrine growth and survival factor and is known to stimulate tumorigenesis and proliferation of several types of cancer cell. However, little is known about the prognostic role of PGRN in colorectal cancer (CRC). A retrospective analysis was performed for patients with colorectal cancer who underwent curative resection between May 2013 and June 2015. PGRN expression in tumor cells was semi-quantitatively categorized (no expression, 0; weak/focal, 1+; moderate/focal or diffuse, 2+; strong/diffuse, 3+), and high expression was considered for tumors graded ≥2+ staining intensity. A total of 109 patients (28 stage I, 32 stage II, and 49 stage III) were analyzed. Thirty-eight patients (35%) had tumors with high PGRN expression, and there was a trend of elevated pre-operative CEA and CA19–9 levels in patients with high PGRN-expressing tumors compared to those with low PGRN-expressing tumors (CEA, 49% vs. 21%; CA19–9, 21% vs. 7%). The 3–year recurrence-free survival (3Y–RFS) and overall survival rates were 83.7% (95% CI, 76.8–90.6) and 96.0% (95% CI, 92.3–99.7), respectively. Patients with high PGRN-expressing tumors had a worse rate of 3Y–RFS (66.8%) compared to those with low PGRN-expressing tumors (92.4%; p = 0.010). Multivariate analysis showed that high PGRN expression, age (>66 years), stage (III), and perineural invasion (+) were independent prognostic factors associated with poor RFS after adjusting for confounding factors including sex, MSI, tumor location, KRAS, and lympho-vascular invasion. PGRN overexpression was significantly associated with poor RFS in patients with CRC who have undergone curative resection.

Thomas Greither,1,* Kersten Fischer,2,* Gerit Theil,2 Marios Marcou,1,2 Hans-Juergen Holzhausen,3 Katrin Weigelt,4 Ginette Serrero,5,6 David Hicks,5 Binbin Yue,5 Paolo Fornara,2 Bernd Wullich,4 Helge Taubert,4 Sven Wach,4 Verena Lieb4 1Center for Reproductive Medicine and Andrology, Martin Luther University Halle-Wittenberg, Halle, Germany; 2Department of Urology, Martin Luther University Halle-Wittenberg, Halle, Germany; 3Department of Pathology, Martin Luther University Halle-Wittenberg, Halle, Germany; 4Department of Urology and Pediatric Urology, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany; 5A&G Pharmaceutical Inc., Columbia, Maryland, USA; 6Program in Oncology, University of Maryland Greenebaum Comprehensive Cancer Center, Baltimore, Maryland, USA *These authors contributed equally to this work Background: GP88/Progranulin is a well-recognized cell growth promoter in different cancers, and elevated serum GP88 levels have been described as negative prognostic factor in breast cancer. However, serum levels in prostate cancer (PCa) patients have not yet been studied. Material and Methods: We analyzed serum GP88 levels by enzyme immunosorbent assay and correlated them with clinicopathological parameters in PCa patients. PCa patients were separated into two groups based on the serum GP88 median level (low ≤44.56 ng/mL or high >44.56 ng/mL) and according to their median age (younger ≤66 years or elder patients >66 years). Results: Low serum GP88 levels were more often detected in younger patients and high levels in elder patients (P=0.018; Fisher’s exact test). PCa patients were separated into three groups, Gleason score (GS) ≤6; GS=7; and GS≥8. In receiver operating characteristic analyses, we could distinguish GS≤6 from GS=7 [area under the curve (AUC): 0.646; P=0.018] and GS≤6 from GS≥8 (AUC: 0.629; P=0.048) but not GS=7 from GS≥8. For survival analysis, GP88 levels were separated into two groups by an optimized cutoff value of 36.92 ng/mL. Using this GP88 stratification, all PCa patients and younger patients with a low serum GP88 level had a significantly better overall survival compared with patients with higher serum GP88 levels (log-rank test P=0.010 and P=0.024). Conclusion: Serum GP88 levels are significantly different depending on age and GS, and they are associated with the prognosis of PCa patients. Keywords: GP88, progranulin, prostate cancer, Gleason score

Assessing central nervous system (CNS) involvement in patients with lymphoma or carcinoma is important in determining therapy and prognosis. Progranulin (PGRN) is a secreted glycosylated protein with roles in cancer growth and survival; it is highly expressed in aggressive cancer cell lines and specimens from many cancer types. We examined PRGN levels by Enzyme Immuno-Assay (EIA) in cerebrospinal fluid (CSF) samples from 230 patients, including 18 with lymphoma [12 with CNS metastasis (CNS+); 6 without CNS metastasis (CNS-)], 21 with carcinomas (10 CNS+; 11 CNS-), and 191 control patients with non-cancer neurological diseases, and compared PRGN levels among these disease groups. Median CSF PGRN levels in the CNS+ lymphoma group were significantly higher than in the CNS- lymphoma and control non-cancer groups; and were also significantly higher in the CNS+ carcinoma group than in the CNS- carcinoma and control groups, except for patients with infectious neurological disorders. Receiver operating characteristic curve analyses revealed that CSF PGRN levels distinguished CNS+ lymphoma from CNS- lymphoma and non-cancer neurological diseases [area under curve (AUC): 0.969]; and distinguished CNS+ carcinomas from CNS- carcinomas and non-cancer neurological diseases (AUC: 0.918). We report here, for the first time, that CSF PGRN levels are higher in patients with CNS+ lymphoma and carcinomas compared to corresponding CNS- diseases. This would imply that measuring CSF PGRN levels could be used to monitor CNS+ lymphoma and metastasis.

Introduction: Antibody-drug conjugates (ADC) are effective antibody-based therapeutics for a growing number of cancers, particularly hematopoietic and lymphoid tumors. There is need to identify new targets for ADCs, particularly for cancers with unmet needs. Methods: Our strategy has been to develop hybridoma libraries developed against cancer cells surface proteins and select antibody able to bind and internalize in cancer cell lines. Selected antibody can then be used for identification of the cancer cell surface target by immunoprecipitation (IP) and mass spectrometric analysis (Mass-spec), followed by target validation. After target validation, binding and target positivity can be tested by flow cytometry and western blot analysis on several cancer cell lines. The in vitro and in vivo efficacy of the antibody conjugated to a cytotoxic payload as ADC can then be evaluated with the target positive cancer cell lines.Results: Using the strategy described above, we have selected a monoclonal antibody named 33B7 able to bind and internalize in several cancer cell lines. The cell surface target binding 33B7 was identified by IP/Mass-spec as Prostaglandin F2 Receptor Negative Regulator (PTGFRN), a transmembrane protein in the Tetraspanin family. This target was then validated by showing that cells transfected with PTGFRN cDNA bound and internalized the antibody. Screening by Flow bidning and by western blot identified several human cancer cells lines expressing PTGFRN. In vitro treatment of these cell lines with the 33B7-saporin ADC inhibited their proliferation in a dose-dependent fashion. 33B7 conjugated to saporin was also able to block tumor growth in vivo in mouse xenografts when compared to a control ADC. Conclusion: These findings show that screening antibody libraries for internalizing antibodies in cancer cell lines is a good approach to identify new cancer targets for ADC development. These results suggest PTGFRN is a possible therapeutic target via antibody-based approach for certain cancers. The elucidation of PTGFRN role in cancer cells, particularly in possibly mediating metastasis is presently being carried out. Citation Format: Jorge Marquez, Jianping Dong, Chun Dong, Ginette Serrero. Prostaglandin F2 Receptor Negative Regulator (PTGFRN) is a novel target to inhibit tumor growth via antibody drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB116.

GP88 (progranulin; PGRN) is a secreted 88kDa glycosylated protein, with important functions, including inflammation and tumorigenesis. We assessed the significance of GP88 expression in survival outcomes of patients with malignant lymphoma (ML).Serum samples from 254 previously untreated ML patients were examined to measure GP88 concentrations using a sandwich human GP88 ELISA kit. Immunohistochemical analyses were performed to examine GP88 tumor tissue expression.The median serum GP88 concentration of ML patients was 91.3ng/ml, and was significantly higher than that of the control group (median, 57.7ng/ml) (p0.0001). Association between GP88 serum concentrations and overall survival (OS) was examined in patients with diffuse large B cell lymphoma (DLBCL) who had been stratified based on their serum GP88 concentrations. Kaplan Meier survival analysis showed that patients with serum GP88 concentrations of ≤116 and116ng/ml, had 5-y OS rates of 70% and 50%, respectively (p=0.02). The immunohistochemical analyses of GP88 tumor expression revealed that DLBCL patients had lymphoma cells that were positive for GP88.High serum GP88 concentrations are associated with poor prognosis in patients with DLBCL.

Biological therapy with companion diagnostic such as the combination of anti-Her-2 therapy (Herceptin™) and measurement of Her-2 expression in breast tumors (HercepTest™) has proven successful in Oncology. Screening targets that have both diagnostic and therapeutic applications (theranostic targets) at the discovery stage should provide the best strategy for the development of novel targeted therapies with companion diag- nostics in Oncology. This strategy is at the basis of the product pipeline developed by AG Pharmaceutical. To identify theranostic targets, AG has developed a biological screen for functional drivers of tumorigenesis abnormally expressed in cancer tissues when compared to normal tissue counterparts. The advantage of this strategy is that identified targets will combine from the start, diagnostic and therapeutic applications. AG has identified a secreted 88 kDa autocrine growth and survival factor, GP88 (progranulin) overex- pressed in cancer tissue and has demonstrated its critical role in the biological process of breast cancer devel- opment, invasiveness, survival and drug resistance. A protein/antibody-based pipeline consisting of prod- ucts able to detect GP88 in tissues, in circulation and to block GP88 was developed at AG. This pipeline combines a neutralizing anti-GP88 monoclonal antibody therapeutic with two companion diagnostic tissue (IHC) and serum (EIA) tests. Diagnostic kits for measurement of GP88 in tissue and serum of cancer pa- tients have been validated in clinical studies. Preclinical and pathological studies support the importance of diagnostic and therapeutic products and the potential of GP88 targeted therapy with companion diagnostic in the clinical settings for several cancer indications such as anti-estrogen resistant breast cancer. [Review].

Background: The Nottingham Prognostic Index (NPI), which includes nodal status, tumor size and histological grade was established to provide predictive value information on post-surgery survival for primary breast cancer patients. Attempts to improve NPI's performance have included addition of other biomarker expression and morphological features such as vascular invasion. In the present study, we investigated whether expression of the autocrine growth and survival factor GP88 (progranulin), known to be overexpressed in breast cancer, whereas it is negative in normal mammary tissue, would improve NPI's predictive value. Methods: We examined the tumor tissue GP88 expression by immunohistochemistry (IHC) in formalin fixed paraffin embedded tissue sections from 508 cases of estrogen receptor positive (ER+) invasive ductal carcinoma (IDC) with known clinical outcomes (disease-free and overall survivals) and with known NPI. GP88 IHC tumor tissue expression was determined using an anti-GP88 antibody (clone 6B3) developed in our laboratory. GP88 expression was scored (0, 1+, 2+, 3+) by two board certified pathologists and classified into two IHC score groups of GP88 < 3+ (0, 1+, 2+) and GP88 = 3+. The correlation between GP88 scoring, NPI and disease-free (DFS) and overall survival (OS) outcomes was then examined by Kaplan Meier analysis, Cox proportional Hazard (CPH) ratio and Pearson's C2 test. Results: Kaplan-Meier survival graphs categorized by NPI scores (< 3.4, 3.4-5.4, and >5.4) and by GP88 expression (< 3+ and 3+) showed that for each NPI subgroup, patients with GP88 IHC score of 3+ had a worse disease-free survival (DFS) and overall survival (OS) than patients within the same NPI subgroup with tumors that had GP88 IHC score < 3+. When adjusted for NPI, high GP88 score was highly significantly associated with recurrence with a hazard ratio of 3.30 (95% CI 2.12 to 5.14). Conclusions: The data suggest that measuring GP88 tumor tissue expression by IHC at time of diagnosis for breast cancer patients with primary ER+ IDC could provide recurrence prediction and survival information complementary to that provided by the determination of NPI alone and thus may be useful for risk management of patients diagnosed with breast cancer. Citation Format: Serrero G, Hawkins DM, Bejarano PA, Ioffe O, Tkaczuk KR, Elliott RE, Head JF, Phillips J, Godwin AK, Weaver J, Hicks D, Yue B. Improvement in risk predictive value of Nottingham prognostic index by determining GP88 tumor tissue expression for estrogen receptor positive breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-03-06.

Prostate cancer, is still a major cause of morbidity and mortality among men worldwide. The expression of the survival and proliferation factor progranulin (GP88) has not yet been comprehensively studied in PCa tumors. The aim of this study was to characterize GP88 protein expression in PCa by immunohistochemistry and to correlate the findings to the clinico-pathological data and prognosis. Immunohistochemical staining for GP88 was performed by TMA with samples from 442 PCa patients using an immunoreactive score (IRS). Altogether, 233 cases (52.7%) with negative GP88 staining (IRS Citation Format: Helge W. Taubert, Amer Abdulrahman, Markus Eckstein, Rudolf Jung, Juan Guzman, Katrin Weigelt, Ginette Serrero, Binbin Yue, Carol Geppert, Robert Stöhr, Arndt Hartmann, Bernd Wullich, Sven Wach. Progranulin (GP88) is an independent prognostic factor for prostate cancer patients [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 4327.

The enzyme-linked immunosorbent assay (ELISA) is a well-established methodology for detection of analytes in various biological fluids. The assay described herein has been validated for the detection of PGRN/GP88/GEP in blood (serum/ plasma), urine and cerebrospinal fluid (CSF), and synovial fluid and may also be used for breast milk, ductal lavage, nipple aspirates, and saliva. The ability to measure circulating levels of PGRN/GP88/GEP has proven to have clinical utility for several human diseases such as cancer where changes of PGRN/GP88/GEP can be determined as a mean to monitor disease status or response to therapy. In the case of frontotemporal dementia (FTD), the ability to measure PGRN/GP88/GEP levels in plasma and cerebrospinal fluid may be useful in distinguishing PGRN mutation carriers among FTD populations at large. The assay used is a sandwich ELISA where a highly specific antihuman PGRN/GP88/GEP monoclonal antibody is employed as a capture antibody coated on 96-well microplates. After contact with serum (or other bodily fluid), unbound material is washed away before application of another PGRN/GP88/GEP detecting antibody which in turn is detected by a horseradish peroxidase (HRP) conjugated antibody. After further washing to remove all unbound HRP, a substrate (TMB) is added, and after approximately 6 min, a color is developed and can be read as optical density at 620 nm (or 450 nm if using HCL as a stop solution) in a microplate reader. The test described herein is capable of measuring very low levels of PGRN/GP88/GEP such as 0.2 ng/mL as found in CSF of certain FTD patients. Additionally, we have demonstrated the potential clinical utility of measuring the changes of PGRN/GP88/GEP blood levels in cancer patients undergoing therapy.

Immunohistochemistry is a well characterized and robust staining technique for the identification and localization of proteins in biological tissues. The following methodology describes the process for identifying progranulin (PGRN/GP88/GEP) in the cellular cytoplasm of target tissues such as tumor biopsies. Such tissue is collected from cancer patients and stabilized using formalin-fixed paraffin embedding after which 4-5 μm sections of the tissue are mounted on positively charged microscope slides. After deparaffinization using ethanol, the tissue is processed through an antigen retrieval process using citric acid at pH 6.4. A monoclonal antibody (Clone AG03) against human PGRN/GP88/GEP is then contacted with the tissue before a horseradish peroxidase (HRP) conjugated antibody is employed to detect AG03 bound to PGRN/GP88/GEP. Subsequent application of the HRP substrate results in color generation. Using a light microscope, a trained user can "score" the percentage of cells stained and intensity of such staining. Scoring ranges from 0 (no staining) to 3+ (strong cytoplasmic staining in10% tumor cells). When scoring is correlated to patient outcome, we have demonstrated that a patient with tumor PGRN/GP88/GEP staining of "3+" has a fivefold increased risk of disease recurrence within 5 years.

Author(s): Cooper, Yonatan A; Nachun, Daniel; Dokuru, Deepika; Yang, Zhongan; Karydas, Anna M; Serrero, Ginette; Yue, Binbin; Alzheimer's Disease Neuroimaging Initiative; Boxer, Adam L; Miller, Bruce L; Coppola, Giovanni | Abstract: ObjectiveChanges in progranulin (GRN) expression have been hypothesized to alter risk for Alzheimer's disease (AD). We investigated the relationship between GRN expression in peripheral blood and clinical diagnosis of AD and mild cognitive impairment (MCI).MethodsPeripheral blood progranulin gene expression was measured, using microarrays from Alzheimer's (n = 186), MCI (n = 118), and control (n = 204) subjects from the University of California San Francisco Memory and Aging Center (UCSF-MAC) and two independent published series (AddNeuroMed and ADNI). GRN gene expression was correlated with clinical, demographic, and genetic data, including APOE haplotype and the GRN rs5848 single-nucleotide polymorphism. Finally, we assessed progranulin protein levels, using enzyme-linked immunosorbent assay, and methylation status using methylation microarrays.ResultsWe observed an increase in blood progranulin gene expression and a decrease in GRN promoter methylation in males (P = 0.007). Progranulin expression was 13% higher in AD and MCI patients compared with controls in the UCSF-MAC cohort (F2,505 = 10.41, P = 3.72*10-5). This finding was replicated in the AddNeuroMed (F2,271 = 17.9, P = 4.83*10-8) but not the ADNI series. The rs5848 SNP (T-allele) predicted decreased blood progranulin gene expression (P = 0.03). The APOE4 haplotype was positively associated with progranulin expression independent of diagnosis (P = 0.04). Finally, we did not identify differences in plasma progranulin protein levels or gene methylation between diagnostic categories.InterpretationProgranulin mRNA is elevated in peripheral blood of patients with AD and MCI and its expression is associated with numerous genetic and demographic factors. These data suggest a role in the pathogenesis of neurodegenerative dementias besides frontotemporal dementia.

The enzyme-linked immunosorbent assay (ELISA) is a well-established methodology for detection of analytes in various biological fluids. The assay described herein has been validated for the detection of PGRN/GP88/GEP in blood (serum/ plasma), urine and cerebrospinal fluid (CSF), and synovial fluid and may also be used for breast milk, ductal lavage, nipple aspirates, and saliva. The ability to measure circulating levels of PGRN/GP88/GEP has proven to have clinical utility for several human diseases such as cancer where changes of PGRN/GP88/GEP can be determined as a mean to monitor disease status or response to therapy. In the case of frontotemporal dementia (FTD), the ability to measure PGRN/GP88/GEP levels in plasma and cerebrospinal fluid may be useful in distinguishing PGRN mutation carriers among FTD populations at large. The assay used is a sandwich ELISA where a highly specific antihuman PGRN/GP88/GEP monoclonal antibody is employed as a capture antibody coated on 96-well microplates. After contact with serum (or other bodily fluid), unbound material is washed away before application of another PGRN/GP88/GEP detecting antibody which in turn is detected by a horseradish peroxidase (HRP) conjugated antibody. After further washing to remove all unbound HRP, a substrate (TMB) is added, and after approximately 6 min, a color is developed and can be read as optical density at 620 nm (or 450 nm if using HCL as a stop solution) in a microplate reader. The test described herein is capable of measuring very low levels of PGRN/GP88/GEP such as 0.2 ng/mL as found in CSF of certain FTD patients. Additionally, we have demonstrated the potential clinical utility of measuring the changes of PGRN/GP88/GEP blood levels in cancer patients undergoing therapy.

Immunohistochemistry is a well characterized and robust staining technique for the identification and localization of proteins in biological tissues. The following methodology describes the process for identifying progranulin (PGRN/GP88/GEP) in the cellular cytoplasm of target tissues such as tumor biopsies. Such tissue is collected from cancer patients and stabilized using formalin-fixed paraffin embedding after which 4-5 μm sections of the tissue are mounted on positively charged microscope slides. After deparaffinization using ethanol, the tissue is processed through an antigen retrieval process using citric acid at pH 6.4. A monoclonal antibody (Clone AG03) against human PGRN/GP88/GEP is then contacted with the tissue before a horseradish peroxidase (HRP) conjugated antibody is employed to detect AG03 bound to PGRN/GP88/GEP. Subsequent application of the HRP substrate results in color generation. Using a light microscope, a trained user can "score" the percentage of cells stained and intensity of such staining. Scoring ranges from 0 (no staining) to 3+ (strong cytoplasmic staining in >10% tumor cells). When scoring is correlated to patient outcome, we have demonstrated that a patient with tumor PGRN/GP88/GEP staining of "3+" has a fivefold increased risk of disease recurrence within 5 years.

GP88 (Progranulin; PGRN) is a secreted glycosylated protein with important functions in several processes, including immune response and cancer growth. Recent reports have shown that PGRN is a therapeutic target for rheumatoid arthritis (RA) because of its capability to bind with tumor necrosis factor receptor (TNFR). However, the serum PGRN level in RA patients has not been investigated. We used enzyme-linked immunosorbent assay (ELISA) to quantify the serum levels of PGRN in 417 healthy subjects, 56 patients with RA and 31 patients with osteoarthritis (OA). In RA patients, we also measured the serum TNF-α and sTNFR concentration. Immunohistochemical staining of PGRN was performed using synovectomy tissue of RA patients. The serum PGRN normal range was established as 40.1 ± 8.7 ng/ml. PGRN levels were not influenced by sex or age. A significant increase in serum PGRN levels was observed in RA (50.2 ± 11.1 ng/ml) and OA (45.4 ± 6.6 ng/ml) groups compared to those in age-matched healthy controls (40.4 ± 9.9 ng/ml) (p

The 88 kDa glycoprotein known as GP88, Progranulin or PC cell derived growth factor is an autocrine growth factor with a unique cysteine rich motif that is over expressed in breast cancer whereas it is negative in normal mammary epithelial cells. It has been shown to play a major role in estrogen independence, tamoxifen resistance and tumorigenesis of breast cancer cells. In the present study, we investigated the effect of GP88 overexpression on the response of the human breast cancer MCF-7 cells to the pure estrogen receptor antagonist fulvestrant (ICI 182,780). While fulvestrant effectively inhibited cell proliferation of empty vector transfected cells, it had no inhibitory effect on the proliferation of GP88 overexpressing breast cancer cells. Mouse xenograft experiments in athymic ovariectomized nude mice showed that GP88 over expressing cells were fulvestrant resistant in vivo in contrast to low GP88 expressing cells. We show that the ability of fulvestrant to induce apoptosis determined by measuring cleavage of poly (ADP-ribose) polymerase was inhibited by GP88. Anti-apoptotic activity of GP88 was associated with sustained expression of bcl-2 and bcl-xL after fulvestrant treatment. In contrast, fulvestrant was still able to inhibit the ability of estrogen to stimulate ERE-luciferase reporter gene activity as well as vEGF expression in GP88 over expressing MCF-7 cells similarly to control MCF-7 cells. Collectively, our data suggest that GP88 prevents apoptosis induced by faslodex and contributes to antiestrogen resistance in human breast cancer.

Mesothelioma is an aggressive but rare form of cancer with a poor prognosis. 1-year survival is ~40% and 5-year survival remains in single digits. Treatment involves a combination of surgery, chemotherapy, and radiation therapy. Mesothelioma is a disease with unmet medical needs and deserving development of novel targeted therapies. Our laboratory is focused on developing novel antibodies useful in treating rare cancers such as mesothelioma. We have raised a mouse antibody library against human cancer cell surface proteins (CSPs). Antibodies were screened against several cancer cells including mesothelioma to identify antibodies specific for cancer CSP as druggable candidates. Screening identified antibodies that bound to and internalized into cancer cells. Such candidates were further characterized. One such antibody (AG02) was identified as having superior binding and internalization characteristics to the other antibodies. The therapeutic potential of AG02 was established as follows; 1) the cell surface protein named CSP-1 against which AG02 bound on the cell surface of selected cancer cells was identified by immunoprecipitation followed by mass spectrometry. Identified CSP-1 was demonstrated as being associated with metastatic potential thus emphasizing its potential as an important target for development of an anti-cancer agent; 2) We demonstrated that upon binding to CSP-1, AG02 antibody internalized the CSP-1 target; 3) We confirmed that AG02 antibody was first-in-class as no other internalizing antibody to this CSP-1 had been developed; 4) Since AG02 is an internalizing antibody, we developed ADC by conjugating AG02 to a generic cytotoxic drug saporin, routinely used as a cost-effective payload in ADCs for proof-of-concept (POC) study. Our POC AG02ADC was tested against a panel of cancer cells and found to bind strongly to CSPs in several cancers with orphan status and unmet needs including mesothelioma. In vitro and in vivo studies established that: (1) AG02 conjugated to saporin at 10nM was able to kill cells expressing CSP while it did not kill non-CSP-1 expressing cells, natural or transfected. (2) AG02-ADC acted in a dose dependent fashion, (3) when tested against different know cancer cell types, AG02-ADC killed >50% of cancer cells while a non-specific mouse IgG-ADC or Drug alone had no effect. (4) Using AG02-ADC treatment in nude mice, we demonstrated tumor growth inhibition >50% in several xenografts. While saporin was used in these studies as payload and shows efficacy, we expect more potent drugs such as MMAE or DM1 to have considerably more efficacy. In summary, we have demonstrated that: AG02 is specific for a CSP-1 overexpressed in certain cancers and involved in metastasis; AG02 is internalized in CSP-1 expressing natural cancer cells; AG02-ADC kills CSP-1 expressing cancer cells. Targeting mesothelioma may enable AG02-ADC to benefit of the FDA’s Orphan Drug Program as it nears drug and clinical development. Citation Format: Jorge Marquez, Chun Dong, Jianping Dong, Binbin Yue, Ginette Serrero. Novel antibody drug conjugate to inhibit mesothelioma tumor growth [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 210.

57 Background: As an autocrine growth factor, progranulin (PGRN) has been known to stimulate tumorigenesis and proliferation of several types of cancer cells. However, little is known about the clinicopathological features of patients with gastrointestinal stromal tumor (GIST) according to the PGRN expression. Methods: A retrospective analysis was performed for patients with GIST who underwent curative surgical resection between March 2007 and March 2018. PGRN expression in tumor cells was evaluated by immunohistochemical (IHC) analysis and semi-quantitatively categorized (no expression, 0; weak, 1+; moderate, 2+; strong, 3+). High expression was considered for tumors graded 2+ or 3+ staining intensity. Results: A total of 54 patients were analyzed, and 31 patients (57%) were male. Median age at surgery was 60 years (range, 33-79), and the most common primary site was stomach (36 patients, 67%). Spindle histology was observed in 35 patients (65%). Median tumor size was 8 cm (range, 2-47), and low mitotic count (≤ 5/50 HPF) was observed in 18 patients (33%). According to the modified NIH classification, 42 patients (78%) were grouped into high risk. With IHC evaluation, KIT and CD34 expression were observed in 51 patients (94%) and 40 patients (80%), respectively, and 27 patients (50%) had high PGRN-expressing tumors. Among the 34 patients whose tumors were genotyped, 25 patients (74%) had an exon 11 mutation, and D842V was observed in 3 patients (9%). According to the PGRN expression, high PGRN-expressing tumors had more epithelioid/mixed histology (68% vs. 32%; p = 0.046), and KIT exon 11 mutation (76% vs. 24%; p = 0.037). Regarding other clinicopathological characters, such as sex, age at surgery, primary site, size, mitotic count, risk classification, and KIT/CD34 expression, there was no significant difference between high PGRN-expressing tumors and low PGRN-expressing tumors. Conclusions: High PGRN-expressing GISTs had more epithelioid/mixed histology and more KIT exon 11 mutation in GIST patients who underwent curative surgical resection.

Background: In the US, the majority of BC is diagnosed by screening XRAY mammography (MM) but up to ∼20% of BC are undetected by MM. The development of reliable, blood-based BC screening test to increase the sensitivity and specificity of currently existing BC screening methods such as MM. Rationale: GP88 is expressed & secreted by BC cells & is not expressed by normal mammary epithelial cells. Two retrospective randomized multi-site trials (one training study & one validating study with about 300 cases each) have demonstrated that elevated GP88 expression in ER+ IDC was statistically correlated with a 4-fold increase in the risk of 5-yr disease recurrence. Multivariate analysis showed that GP88 as a risk predictor was independent from PR expression, tumor size, grade, lymph node status & disease stage. The quantitative GP88 EIA developed to determine the amount of GP88 in biological fluids was developed at A&G. The assay is highly specific for GP88 & both sensitive & linear over a wide dynamic range, i.e. detection of GP88 concentrations from 0.1 to 20ng/ml. A baseline (28.4 ± 5 ng/ml) was established for healthy volunteers (HV). In BC pts a statistically significant increase of serum GP88 was seen for early stage pts (40.7 ± 16 ng/ml; p=0.007). Additionally, a stratification of pts according to their clinical outcomes shows that pts having no evidence of disease (NED) have serum GP88 levels within the range of HV. These data suggest that pts with breast tumors express & secrete high levels of GP88. Objectives: 1. To determine prospectively GP-88 blood levels in HV at average risk of developing BC who are undergoing screening MM & in women with biopsy-confirmed BC. 2. To establish the statistical distribution of GP88 serum levels in these women by baseline BIRAD classification (1-6). 3. To determine if baseline GP88 level is predictive of change in BIRADS classification from baseline to 12-mos follow-up. 4. To determine if baseline GP88 level is predictive of the appearance of BC at 12 mos follow-up in HV who were cancer-free at baseline. Inclusion Criteria: Female, aged >=40 yrs old, presenting to UM Breast Center for screening MM or for diagnostic MM or diagnostic workup and/or biopsy due to BIRADS 0 MM at an outside facility Study procedures: Serum levels of GP88 in subjects with average BC risk factors will be measured prospectively at baseline; 3-6 mos & 6-12 mos. & correlated with BIRADS reading of the screening MM, BIRADS 1 or 2 (benign), BIRADS 3 (short term MM follow-up) & BIRADS 4 or 5 (suspicious and tissue diagnosis needed immediately). GP88 blood level will be correlated with pathologic results of breast biopsies performed on subjects with suspicious BIRADS (4 & 5) MM & final pathologically confirmed diagnosis of BC as BIRADS 6. Statistical Considerations The total number of pts will be 725 & screened up to 1400 subjects. Study is UM IRB approved & just started accruing. Funding is provided by Maryland Industry Partnership Grant (MIPS)& Avon Grant No. 02-2013-018. Contact: Ntait@umm.edu. Citation Format: Katherine HR Tkaczuk, Cristina Campassi, Susan Kesmodel, Emily Bellavance, John Olson, Elizabeth Nichols, Steven J Feigenberg, Binbin Yue, David Hicks, Ginette Serrero. A prospective study of glycoprotein 88 GP-88 blood test in healthy women with Gail model risk

Background: GP88 (Progranulin, acrogranin) is an 88kDa glycoprotein overexpressed in breast tumors and involved in their proliferation and survival. Biological studies have shown that GP88 in ER+ breast cancer cells was associated with estrogen independence and resistance to anti-estrogen therapies and aromatase inhibitors. In addition, GP88 stimulated migration, invasion and angiogenesis, hallmarks of metastasis. Pathological studies have demonstrated that GP88 was preferentially expressed in invasive ductal carcinoma while lobular carcinoma was mostly negative. No expression was found in normal mammary tissue. Two retrospective studies totaling 530 cases of ER+ formalin-fixed paraffin-embedded invasive ductal carcinoma (IDC) established that high GP88 expression was associated with a 4-fold increase in risk of recurrence and 2.5-fold increase in death when compared to low GP88 scores. Multivariate analysis showed that GP88 remains a predictor of recurrence even when adjusted for prognostic factors such as tumor size, grade, stage, lymph node status and age. High level of GP88 was also found to be elevated in the serum of breast cancer patients when compared to healthy individuals. Since GP88 tumor tissue expression appears as a prognostic factor for ER+ breast cancer, we proposed to investigate whether GP88 tumor tissue expression would correlated to Oncotype Dx® Recurrence Score which is now for early stage ER+ node negative breast cancer patients. Methods: Eight five cases from women ages 37–77 with ER+ invasive mammary carcinoma from three different institutions and with an available Oncotype Dx recurrence score were selected with approval from each site's IRB. GP88 expression was determined by immunohistochemistry (IHC) using the anti-human GP88 6B3 monoclonal antibody (A&G) on a Ventana automated staining platform. For all cases examined, GP88 IHC scores (0, 1+, 2+, 3+) were compared to routine clinicopathologic factors (tumor size, grade, and stage), PR, HER2/neu and Ki67 expression (by image analysis) and to their Oncotype DX® recurrence score (Genomic Health). The associations of GP88 with the parameters described above were assessed by t test or ANOVA. Results: The GP88 tissue expression correlated with Oncotype Dx Recurrence score (p < 0.03) as well as with Ki-67 index (p < 0.004). Age, HER2/neu and PR status did not correlate with GP88 expression. Details of these finding will be presented here. Conclusion: This study shows that GP88, an important tumor aggressiveness indicator, significantly correlates with Oncotype DX score and confirms that GP88 correlates with other tumor clinicopathologic parameters and prognostic markers. Further studies are underway to determine whether GP88 in combination with routine markers, particularly Ki67, could be used instead of Oncotype DX Assay to predict outcome and provide information for the management of ER+ breast cancer patients. This study is supported by grant 02–2010-010 from the Avon Foundation for Women. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-40.

We examined progranulin (PGRN) levels in cerebrospinal fluid (CSF) samples during the acute phase in 15 patients with neuromyelitis optica spectrum disorders (NMOSD) and compared the results with those from 17 patients with multiple sclerosis (MS), 30 patients with other inflammatory neurological diseases (OIND), and 20 non-inflammatory controls (NIC). CSF PGRN levels of NMOSD patients were significantly higher than those of MS patients and NICs. These levels correlated with CSF interleukin-6 levels, CSF cell counts, CSF protein levels, improvements in the Expanded Disability Status Scale score, and affected total spinal cord lesion length in the NMOSD patients.

Lipid droplet (LD), a multi-functional organelle, is often found to associate with other cellular membranous structures and vary in size in a given cell, which may be related to their functional diversity. Here we established a method to separate LD subpopulations from isolated CHO K2 LDs into three different size categories. The subpopulation with smallest LDs was nearly free of ER and other membranous structures while those with larger LDs contained intact ER. These distinct subpopulations of LDs differed in their protein composition and ability to recruit proteins. This method was also applicable to LDs obtained from other sources, such as Huh7 cells, mouse liver and brown adipose tissue, et al. We developed an in vitro assay requiring only isolated LDs, Coenzyme A and ATP to drive lipid synthesis. The LD subpopulation nearly depleted of ER was able to incorporate fatty acids into triacylglycerol and phospholipids. Together, our data demonstrate that LDs in a given cell are heterogeneous in size and function and suggest that LDs are one of cellular lipid synthetic organelles.

Background The Nottingham Prognostic Index (NPI), which combines numerical values for nodal status, tumor size and histological grade, is used in the standard of care to provide predictive value information on post-surgery survival for patients with primary breast cancer. Attempts to improve the performance of the NPI algorithm have been carried out by testing the inclusion of other biomarker expression and morphological features such as vascular invasion. In the present study, we investigated whether expression of the autocrine growth and survival factor GP88 (progranulin), known to be overexpressed in breast cancer, would improve NPI’s predictive value. Methods We examined by immunohistochemistry (IHC) the GP88 expression in 508 cases of estrogen receptor positive invasive ductal carcinoma with known clinical outcomes and for which NPI had been determined. GP88 IHC expression was scored by two board certified pathologists and classified into two score groups of GP88 5.4) and GP88 expression showed that for patients within the same NPI subgroup, patients having tumors with a high GP88 expression (GP88 IHC score of 3+) had a worse DFS than patients with tumors that had a low GP88 expression (GP88 IHC score

Imaging technologies are the methods of choice in the standard of care (SOC) to monitor therapy response in metastatic breast cancer (MBC) patients. However, such methods are expensive and have limited sensitivity to detect disease response in a timely manner. Measurement of the circulating tumor markers such as CA15-3, CA27.29 and CEA has provided additional minimally invasive methods in disease management of MBC patients. While useful, they have limitations in providing clinicians with a reliable insight into real-time disease monitoring. Understanding of real-time biological processes may provide better biomarkers of the disease state and thus aid real-time clinical management of MBC patients by identifying circulating disease associated biomarkers. Thus, addition of such new circulating biomarkers may improve the management of MBC patients. We have characterized a target biomarker that would fit these criteria, the 88kDa glycoprotein Progranulin (GP88). GP88 is expressed in tumor tissue and not in normal mammary tissue counterpart and is secreted in the circulation of BC patients. Biological studies have established GP88 as one of the critical drivers for breast cancer cell proliferation, survival, invasiveness and drug resistance. Clinical studies have demonstrated that elevated GP88 tumor levels were prognostic for recurrence and that breast cancer patients had a statistically elevated GP88 serum level than healthy individuals. Using tissue and serum tests to detect and quantify GP88 could provide an new strategies for identifying patients at high risk of recurrence and monitoring disease progression in BC patients undergoing therapy. In the present study, we examined whether GP88 serum levels were elevated in MBC patients and whether GP88 serum levels were correlated to patient overall survival. Under an IRB approved protocol, 92 MBC patients that met the inclusion criteria and were undergoing therapy at the UMGCCC Breast Clinic were consented and enrolled. Clinical and disease characteristics along with serum CA15-3 values were collected as part of the study. Serum samples were collected from each patient during therapy and subsequently the patients were monitored. The serum was stored at -80C until tested for GP88 using a GP88 enzyme linked immunoassay developed in our laboratory. Statistical analysis using Kaplan-Meier functions established whether there was a correlation between GP88 serum level and overall survival in MBC patients. By analyzing the KM plots at different GP88 cut points, we identified two populations with distinct survival characteristics. When examined more thoroughly the difference in overall survival of patients with 60ng/ml was statistically significant (P=0.0002). Correlation analysis of serum GP88 and CA15-3 were performed and will be presented. We conclude that circulating levels of GP88 in MBC patients are correlated with overall survival. It would appear that patients that can be managed to have a GP88 below 60ng/ml will survive longer. Thus measuring circulating GP88 levels would provide additional information to that available in today's SOC for monitoring. This valuable insight into real-time disease status will assist clinicians in patient management. Citation Format: Serrero G, Hawkins DM, Yue B, Hicks D, Tait N, Tkaczuk KR. Determination of a serum progranulin (GP88/PGRN) level associated with overall survival in metastatic breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-02-12.

Purpose: GP88 (progranulin) is a critical player of breast tumorigenesis for estrogen receptor positive (ER+) breast cancer. Pathological studies showed that GP88 was expressed in invasive ductal carcinoma (IDC), but not in normal mammary tissue, benign lesions or lobular carcinoma. The present study examines GP88 prognostic significance in association with recurrence risk for patients with ER+ IDC. Patients and Methods: Two retrospective multi-site clinical studies examined GP88 expression by immunohistochemistry (IHC) analysis in paraffin-embedded tumor tissues in correlation with patients’ survival outcomes. The training study established a GP88 cut-off value associated with decreased disease-free (DFS) and overall (OS) survivals. The validation study verified the GP88 cut-off value and compared GP88 prognostic information with other prognostic factors in multivariate analysis. Results: GP88 expression is associated with a statistically significant increase in recurrence risk for ER+ IDC patients. The training study established that GP88 3+ score by IHC analysis was associated with decreased DFS (p=0.0004) and OS (p=0.0036). The independent validation study verified that GP88 3+ score for the high risk group and demonstrated that GP88 3+ score was associated with a 5.9-fold higher hazard of disease recurrence and a 2.5-fold higher mortality hazard compared to patients with tumor GP88 Conclusion: Our training and validation studies demonstrate that the survival factor GP88 is a prognostic biomarker, predictive of recurrence risk and increased mortality for ER+ IDC patients, independent from other prognostic factors. These results provide support for measuring GP88 tissue expression for newly diagnosed early stage breast cancer patients. This work was supported by grants R43CA124179, and U01CA113916 from the National Cancer Institute, grants 07-2007-064 and 02-2010-010 from the Avon Foundation for Women. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-32.