5062 Background: Guideline recommended genetic testing in advanced prostate cancer (PC) is underutilized and not always accepted by patients (pts). We assessed attitudes and decisional conflict (DC) surrounding genetic testing associated with subsequent test completion, and differences between white and nonwhite pts. Methods: Eligibility for this prospective single institution study included pts with N1 or M1 PC who had not yet completed genetic testing. Upon informed consent, pts were given a 24-question survey using a Likert scale of 0 (strongly agree) to 4 (strongly disagree) to assess attitudes toward genetic testing including a validated assessment of DC. DC and DC subscores (range 0-100, with 100 being highest DC) were calculated from subsets of survey responses. Self-identified race was obtained from the EMR. Two-group comparisons between white and nonwhite pts, and between those who completed genetic testing and who did not, were conducted with SAS v9.4 software using Fisher’s exact test for categorical variables and Wilcoxon rank sum test for Likert scale survey questions and DC score variables. Results: Of 42 enrolled pts (21 white, 17 black, 1 Asian, 3 declined), 22 (52.4%) completed genetic testing. Compared to white pts, nonwhite pts expressed more concern about test result privacy (mean = 1.72 v 2.95, p = 0.002), test results being used for non-healthcare purposes (1.78 v 3.00, p = 0.003), and trying unproven treatments (1.72 v 2.67, p = 0.01). Nonwhite pts felt more external pressure in decision-making compared to white pts (0.67 v 0.29, p = 0.04). No significant differences were appreciated in completion of testing, DC, or any subscore between racial groups. Compared to pts who did not complete testing, those who completed testing were more likely to report they knew which options were available (0.73. v 1.25, p = 0.05), knew the benefits of each option (0.77 v 1.30, p = 0.04), knew the risk and side effects of each option (0.95 v 1.50, p = 0.05), were clear about which benefits matter most to themselves (0.73 v 1.37, p = 0.02), and were clear about the best choice for themselves (0.73 v 1.35, p = 0.02). DC (28.59 v 18.11, p = 0.03) was higher in pts who did not complete testing, along with uncertainty (31.25 v 19.32, p = 0.02) and informed (31.25 v 20.45, p = 0.03) subscores. No differences were seen in values clarity, support, or effective decision subscores. Conclusions: In our study, nonwhite pts expressed greater concern about privacy, data misuse, and trying unproven treatments. Those who did not complete testing had more DC with greater uncertainty about knowledge and decision making. These findings will help direct targeted interventions to increase knowledge, trust, and decisional certainty about genetic testing in pts with advanced PC. Ongoing studies will assess the impact of these interventions on rates of testing completion at our institution.

Supplementary Table S3: Baseline Tumor pFGFR3 IHC Intensity

Supplementary Table S2: Cystectomy Pathologic Staging

Results of myogenesis and myopathy pathway analyses of skeletal muscle

Results of cytokine array analyses of plasma samples

Introduction: Somatic and germline testing are now recommended for patients with advanced prostate cancer. Though referrals for genetic testing have increased since 2017 along with developments in National Comprehensive Cancer Network guidelines, genomic profiling in select cancers remains low, with a reported 50% to 80% of eligible patients not completing testing. This study aims to quantify race-associated disparities in both referrals to genetic counseling and completion of genetic testing in patients with advanced prostate cancer. Methods: Henry Ford Health’s electronic medical record (Epic) was queried to identify patients diagnosed with stage III or stage IV prostate cancer between Q1 2017 and Q2 2022. Demographics, site of referral, completion of counseling referrals, and completion of somatic and/or germline testing were identified. Incidence and completion of referral were calculated. Population comparisons were performed with Chi-squared testing. Results: Out of 4,505 unique patients diagnosed with prostate cancer, 919 patients were diagnosed with stage III prostate cancer and 468 patients were diagnosed with stage IV prostate cancer. Black patients had a higher incidence of stage IV versus stage III cancer compared to non-Hispanic whites (NHWs) (31.84% versus 24.92%, P-Value = 0.0042). In stage IV prostate cancer, Black patients were more likely to receive referrals to genetic counseling compared to NHWs (32.2% versus 21.1%, P-Value = 0.011). Black patients were more likely to have a referral placed from the main campus (downtown cancer institute) versus community clinics (suburban) compared to white patients (80.0% versus 50.8%, P-Value = 0.0018). There were no statistically significant differences in completion of testing between the downtown and suburban campuses (67.1% and 61.9%), completion of referral between Black and White patients (48.8% and 43.3%), completion of any genetic testing (66.7% and 64.2%), or completion of germline testing (51.1% and 58.2%). Patients with stage IV prostate cancer who completed their genetic counseling referral were more likely to complete a form of genetic testing (P-Value <0.00001) compared to those who did not present to a genetic counselor. Conclusion and Discussion: Black patients were more likely to present with stage IV disease and more likely to receive a referral to genetic counseling. Though testing completion rates were not significantly different between Black and White patients, overall referral, completion of referral, and testing completion rates remain low in the entire population. Increased indications for testing provide an opportunity for improved referral rates. The statistically significant increase in completed testing by patients who completed their genetic counseling referral reflects both the importance of counseling for optimizing care and stresses the implication that there are significant barriers to patient access that are worth studying further. Additional assessment is underway to better understand both provider- and patient-based barriers to genetic testing. Citation Format: Kyle McElyea, James Purtell, Mohammed Baseer, Avery Ralston, Maria Jamil, Brigid Jacob, Clara Hwang. Assessment of disparities in completion of genetic testing in patients with advanced prostate cancer [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr A066.

Supplementary Figure Legends

Tumor incidence and growth patterns in PyMT+ mice with and without DMAPT treatment

This file contains a detailed description of the synthesis as well as the characterization of lead compounds PG-S3-001, PG-S3-002, and PG-S3-003. Results pertaining to the interaction of PG-S3-001 with relevant kinases is also included; Table 1. Results of Kinome Screen for Kinases involved in Regulation of STAT3.

Supplementary Table S1: All-Grade Toxicity

Pathways analyses using DAVID of differentially expressed genes under different conditions and in different cell types. (XLSX 139Â kb)

The majority of estrogen receptor-positive (ERα+) breast cancers respond to endocrine therapies. However, resistance to endocrine therapies is common in 30% of cases, which may be due to altered ERα signaling and/or enhanced plasticity of cancer cells leading to breast cancer subtype conversion. The mechanisms leading to enhanced plasticity of ERα-positive cancer cells are unknown. We used short hairpin (sh)RNA and/or the CRISPR/Cas9 system to knockdown the expression of the dependence receptor UNC5A in ERα+ MCF7 and T-47D cell lines. RNA-seq, quantitative reverse transcription polymerase chain reaction, chromatin immunoprecipitation, and Western blotting were used to measure the effect of UNC5A knockdown on basal and estradiol (E2)-regulated gene expression. Mammosphere assay, flow cytometry, and immunofluorescence were used to determine the role of UNC5A in restricting plasticity. Xenograft models were used to measure the effect of UNC5A knockdown on tumor growth and metastasis. Tissue microarray and immunohistochemistry were utilized to determine the prognostic value of UNC5A in breast cancer. Log-rank test, one-way, and two-way analysis of variance (ANOVA) were used for statistical analyses. Knockdown of the E2-inducible UNC5A resulted in altered basal gene expression affecting plasma membrane integrity and ERα signaling, as evident from ligand-independent activity of ERα, altered turnover of phosphorylated ERα, unique E2-dependent expression of genes effecting histone demethylase activity, enhanced upregulation of E2-inducible genes such as BCL2, and E2-independent tumorigenesis accompanied by multiorgan metastases. UNC5A depletion led to the appearance of a luminal/basal hybrid phenotype supported by elevated expression of basal/stem cell-enriched ∆Np63, CD44, CD49f, epidermal growth factor receptor (EGFR), and the lymphatic vessel permeability factor NTN4, but lower expression of luminal/alveolar differentiation-associated ELF5 while maintaining functional ERα. In addition, UNC5A-depleted cells acquired bipotent luminal progenitor characteristics based on KRT14+/KRT19+ and CD49f+/EpCAM+ phenotype. Consistent with in vitro results, UNC5A expression negatively correlated with EGFR expression in breast tumors, and lower expression of UNC5A, particularly in ERα+/PR+/HER2− tumors, was associated with poor outcome. These studies reveal an unexpected role of the axon guidance receptor UNC5A in fine-tuning ERα and EGFR signaling and the luminal progenitor status of hormone-sensitive breast cancers. Furthermore, UNC5A knockdown cells provide an ideal model system to investigate metastasis of ERα+ breast cancers.

Abstract Radical mastectomies are progressively becoming a surgery of the past. Women today are increasingly opting for lumpectomies, a less invasive treatment option. Clinical data has shown no difference in survival or clinical outcomes between the two surgical groups in early stages of breast cancer. There is, however, an undesired outcome yet to be adequately addressed. Lumpectomies have, in some cases, failed to remove marginal malignant tumor cells left as a product from surgery. Following surgery, tumor biopsies are analyzed for marginal tumor cells. Biopsies are cut in, fixed, processed in a tissue processor, embedded into a paraffin block, and stained with H&E. After processing, presence of marginal cancerous tissue is determined by a pathologist. This process takes 3-5 days, ultimately requiring the patient to undergo reoperation if a positive margin is discovered. Presently, the reoperation rate is 20-30%. A device capable of imaging removed tissue to determine remaining marginal tumor during surgery would greatly reduce the reoperation rate. Multiple intraoperative imaging tools existing or are emerging for breast tumor margin assessment. Current devices fail to meet acceptable clinical specifications due to either long procedure time (15 mins+), or low sensitivity (~70%), and low specificity (~68%). An unmet need exists in developing an intraoperative margin assessment device that is rapid, sensitive, capable of measuring the entire tissue surface, and images a depth of 2mm+. The MarginPAT device presents a multi-modal photoacoustic/ultrasound imaging system for rapid and highly sensitive breast cancer margin assessment. After surgical removal of tumor, tissue is inserted, via cartridge, into MarginPAT for imaging. An automatic scan (<3 mins) provides 3D images of the excised tissue, providing margin status. Such immediate feedback allows the surgeon to re-operate immediately or allow the patient to return home based on the marginal results from MarginPAT. Our preliminary study on 40 patient samples, showed 93% sensitivity and 90% specificity in margin assessment by precise localization of adipose tissue using PA imaging and further RF spectrum analysis of ultrasound signaling. Other than the superior sensitivity and specificity, the unique features of MarginPAT include deep tissue sensing (>3 mm) and high surface scanning speed (20 cm2/per min). These achieved specifications meet the needs of intraoperative margin assessment, which are expected to surpass similar platforms. More importantly, MarginPAT supports conventional ultrasound imaging, allowing for use in ultrasound-guided wire localization. These multimodality functions allow MarginPAT to be utilized in multiple fields of breast cancer diseases, another advantage over other platforms. In conclusion, current clinical results show through both verification and validation that MarginPAT competently performs in the operating room. Citation Format: Kyle McElyea, George Sandusky, Rui Li, Lu Lan, Ji-Xin Cheng, Linda K. Han, Pu Wang. Intraoperative assessment of breast tumor margins using multimodal photoacoustic tomography (MarginPAT) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1873. doi:10.1158/1538-7445.AM2017-1873

Abstract Purpose: To assess the clinical and pharmacodynamic activity of dovitinib in a treatment-resistant, molecularly enriched non–muscle-invasive urothelial carcinoma of the bladder (NMIUC) population. Experimental Design: A multi-site pilot phase II trial was conducted. Key eligibility criteria included the following: Bacillus Calmette-Guerin (BCG)-unresponsive NMIUC (>2 prior intravesical regimens) with increased phosphorylated FGFR3 (pFGFR3) expression by centrally analyzed immunohistochemistry (IHC+) or FGFR3 mutations (Mut+) assessed in a CLIA-licensed laboratory. Patients received oral dovitinib 500 mg daily (5 days on/2 days off). The primary endpoint was 6-month TURBT-confirmed complete response (CR) rate. Results: Between 11/2013 and 10/2014, 13 patients enrolled (10 IHC+ Mut−, 3 IHC+ Mut+). Accrual ended prematurely due to cessation of dovitinib clinical development. Demographics included the following: median age 70 years; 85% male; carcinoma in situ (CIS; 3 patients), Ta/T1 (8 patients), and Ta/T1 + CIS (2 patients); median prior regimens 3. Toxicity was frequent with all patients experiencing at least one grade 3–4 event. Six-month CR rate was 8% (0% in IHC+ Mut−; 33% in IHC+ Mut+). The primary endpoint was not met. Pharmacodynamically active (94–5,812 nmol/L) dovitinib concentrations in urothelial tissue were observed in all evaluable patients. Reductions in pFGFR3 IHC staining were observed post-dovitinib treatment. Conclusions: Dovitinib consistently achieved biologically active concentrations within the urothelium and demonstrated pharmacodynamic pFGFR3 inhibition. These results support systemic administration as a viable approach to clinical trials in patients with NMIUC. Long-term dovitinib administration was not feasible due to frequent toxicity. Absent clinical activity suggests that patient selection by pFGFR3 IHC alone does not enrich for response to FGFR3 kinase inhibitors in urothelial carcinoma. Clin Cancer Res; 23(12); 3003–11. ©2016 AACR.

Metastasis of cancer cells to distant organ systems is a complex process that is initiated with the programming of cells in the primary tumor. The formation of distant metastatic foci is correlated with poor prognosis and limited effective treatment options. We and others have correlated Mouse double minute 2 (Mdm2) with metastasis; however, the mechanisms involved have not been elucidated. Here, it is reported that shRNA-mediated silencing of Mdm2 inhibits epithelial-mesenchymal transition (EMT) and cell migration. In vivo analysis demonstrates that silencing Mdm2 in both post-EMT and basal/triple-negative breast cancers resulted in decreased primary tumor vasculature, circulating tumor cells, and metastatic lung foci. Combined, these results demonstrate the importance of Mdm2 in orchestrating the initial stages of migration and metastasis.Implication: Mdm2 is the major factor in the initiation of metastasis. Mol Cancer Res; 15(11); 1598-607. ©2017 AACR.

Abstract Breast cancer progression is associated with systemic effects, including functional limitations and sarcopenia without the appearance of overt cachexia. Autocrine/paracrine actions of cytokines/chemokines produced by cancer cells mediate cancer progression and functional limitations. The cytokine-inducible transcription factor NF-κB could be central to this process, as it displays oncogenic functions and is integral to the Pax7:MyoD:Pgc-1β:miR-486 myogenesis axis. We tested this possibility using the MMTV-PyMT transgenic mammary tumor model and the NF-κB inhibitor dimethylaminoparthenolide (DMAPT). We observed deteriorating physical and functional conditions in PyMT+ mice with disease progression. Compared with wild-type mice, tumor-bearing PyMT+ mice showed decreased fat mass, impaired rotarod performance, and reduced grip strength as well as increased extracellular matrix (ECM) deposition in muscle. Contrary to acute cachexia models described in the literature, mammary tumor progression was associated with reduction in skeletal muscle stem/satellite-specific transcription factor Pax7. Additionally, we observed tumor-induced reduction in Pgc-1β in muscle, which controls mitochondrial biogenesis. DMAPT treatment starting at 6 to 8 weeks age prior to mammary tumor occurrence delayed mammary tumor onset and tumor growth rates without affecting metastasis. DMAPT overcame cancer-induced functional limitations and improved survival, which was accompanied with restoration of Pax7, Pgc-1β, and mitochondria levels and reduced ECM levels in skeletal muscles. In addition, DMAPT restored circulating levels of 6 out of 13 cancer-associated cytokines/chemokines changes to levels seen in healthy animals. These results reveal a pharmacological approach for overcoming cancer-induced functional limitations, and the above-noted cancer/drug-induced changes in muscle gene expression could be utilized as biomarkers of functional limitations. Mol Cancer Ther; 16(12); 2747–58. ©2017 AACR.

4526 Background: FGFR3 aberrations are common in low grade non-muscle invasive bladder cancer (NMIBC) tumors but decrease in frequency in metastatic UC. This multisite single-arm trial assessed the clinical and pharmacodynamic activity of dovitinib (an oral FGFR3/VEGFR2 inhibitor) in a treatment resistant, molecularly enriched NMIBC population. Methods: Patients (pts) with BCG refractory NMIBC despite at least 2 prior intravesical therapy regimens with increased tumor FGFR3 expression by centrally analyzed immunohistochemistry (IHC+) or FGFR3 mutations (Mut+) assessed in a CLIA-certified lab were eligible. Pts received dovitinib 500 mg once daily (5 days on / 2 days off) on q28d cycles. The primary endpoint was 6-month TURBT-confirmed complete response (CR) rate. Results: Between 11/2013 and 10/2014, 13 pts enrolled (10 IHC+ Mut-, 3 IHC+ Mut+). Demographics included: median age 70 years; 85% male; CIS (3 pts), Ta/T1 (8 pts), and Ta/T1 + CIS (2 pts); median prior regimens 3; median time from last treatment 6 months. Pts received a median of 4 dovitinib cycles. Dose reductions were required in 10 pts (77%). Treatment related grade 3/4 events included: fatigue, hypertension, hypertriglyceridemia (2 pts, 15% each); hepatotoxicity, stomatitis, rash (1 pt, 8% each). 6-month complete response rate was 8% (0% in IHC+ Mut-; 33% in IHC+ Mut+). The primary endpoint was not met. One pt remains a CR at 15+ months. One pt (8%) developed muscle-invasion on study. Pharmacodynamically active (94-5812 nM) dovitinib concentrations in urothelial tissue were observed in all 9 PK-evaluable pts. Reductions in pFGFR3 IHC staining were observed post-dovitinib treatment. A concordant urine FGFR3 mutation was noted in 1 of the 3 Mut+ pts. Conclusions: Oral dovitinib consistently achieved biologically active concentrations within the urothelium and demonstrated pharmacodynamic FGFR3 inhibition. Long-term administration of dovitinib was not feasible due to frequent treatment related toxicity. Absent clinical activity in FGFR3 IHC+ Mut- pts suggests that this should not be an entry criterion for future trials targeting FGFR3 in UC. Clinical trial information: NCT01732107.

AbstractConstitutively activated STAT3 protein has been found to be a key regulator of pancreatic cancer and a target for molecular therapeutic intervention. In this study, PG-S3-001, a small molecule derived from the SH-4-54 class of STAT3 inhibitors, was found to inhibit patient-derived pancreatic cancer cell proliferation in vitro and in vivo in the low micromolar range. PG-S3-001 binds the STAT3 protein potently, Kd = 324 nmol/L by surface plasmon resonance, and showed no effect in a kinome screen (>100 cancer-relevant kinases). In vitro studies demonstrated potent cell killing as well as inhibition of STAT3 activation in pancreatic cancer cells. To better model the tumor and its microenvironment, we utilized three-dimensional (3D) cultures of patient-derived pancreatic cancer cells in the absence and presence of cancer-associated fibroblasts (CAF). In this coculture model, inhibition of tumor growth is maintained following STAT3 inhibition in the presence of CAFs. Confocal microscopy was used to verify tumor cell death following treatment of 3D cocultures with PG-S3-001. The 3D model was predictive of in vivo efficacy as significant tumor growth inhibition was observed upon administration of PG-S3-001. These studies showed that the inhibition of STAT3 was able to impact the survival of tumor cells in a relevant 3D model, as well as in a xenograft model using patient-derived cells. Mol Cancer Ther; 15(5); 794–805. ©2016 AACR.

Abstract High levels of apurinic/apyrimidinic endonuclease/redox factor 1 (APE1/Ref-1 or Ref-1) expression have been reported in numerous cancers such that Ref-1 is an emerging target in a variety of cancer types. Ref-1 is a dual function protein with both DNA repair activity as well as redox activity. Ref-1 is responsible for the repair of baseless sites in DNA caused by alkylation and oxidative DNA damage as well as regulating several transcription factors including HIF-1a, NFkB, AP-1, and STAT3. Using siRNA to knockdown Ref-1 in pancreatic ductal adenocarcinoma (PDAC) cells (MIA-PaCa-2), we quantitated Ref-1 expression following transfection with scrambled control and Ref-1 siRNA using Indica Lab's HALO CytoNuclear software. Knockdown of greater than 85% greatly decreases the proliferation of PDAC cells, and supports Ref-1 as a target in pancreatic cancer. To assess the efficacy of targeting Ref-1 in vivo, a small molecule Ref-1 redox inhibitor, APX3330, was used in combination therapy with a PDAC standard of care agent, Gemcitabine, in a PDAC xenograft mouse model. In this study, NSG mice were treated with: Gemcitabine (35mg/kg), APX3330 (12.5, 25, and 50mg/kg), and combinations of APX3330 and Gemcitabine (12.5, 25, and 50mg/kg), as well as a vehicle control group. At termination of the study, tumors were harvested, tissue sections prepared, stained for H&E, and immunostained for Ki67 and CD31. Slides were then imaged via Aperio's ScanScope. Immunostains were quantitated to predict the effectiveness of the combination treatment in a PDAC in vivo model. IHC slides from treated tumors were quantified using Aperio's ImageScope software to determine the percent of cell proliferation and angiogenesis in the various treatment groups. These preclinical studies, demonstrated additive effects of combining APX3330 with Gemcitabine to reduce pancreatic tumor volumes and cell proliferation. Significantly decreased tumor volumes in the combination treatments of APX3330 with Gemcitabine were found compared to each agent alone. All treatments, single or combination yielded tumor volumes significantly different from the vehicle control. A statistically significant decrease in cell proliferation determined from Ki67 staining was found amongst the 12.5 and 50 mg/kg doses of APX3330, all three combination groups, and in the Gemcitabine group compared to vehicle control. There was a trend toward, yet not statistically significant, increased anti-angiogenic effects in the 12.5 and 25 mg/kg dose groups of APX3330 alone compared to all other treatment groups. This data continues to demonstrate a single agent response of pancreatic tumor cells in a preclinical model using APX3330, but more importantly, a novel combination effect when APX3330 is combined with Gemcitabine in a xenograft model, both via tumor volume and cell proliferation marker, Ki67 supporting the use of APX3330 in combination with Gemcitabine in PDAC patients. Citation Format: Kyle C. McElyea, Max H. Jacobsen, Max Schmidt, Huiwen Cheng, Mark R. Kelley, George E. Sandusky, Melissa L. Fishel. Efficacy study of APX3330, a Ref-1 redox inhibitor, and Gemcitabine in a mouse pancreatic ductal adenocarcinoma model. [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 5183.