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Purpose: The recent approval of anti-programmed death-ligand 1 immunotherapy in combination with nab-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor immune microenvironment (TIME). Experimental Design: We examined immune-related gene expression patterns before and after neoadjuvant chemotherapy (NAC) in a series of 83 breast tumors, including 44 TNBCs, from patients with residual disease (RD). Changes in gene expression patterns in the TIME were tested for association with recurrence-free (RFS) and overall survival (OS). In addition, we sought to characterize the systemic effects of NAC through single-cell analysis (RNAseq and cytokine secretion) of programmed death-1–high (PD-1HI) CD8+ peripheral T cells and examination of a cytolytic gene signature in whole blood. Results: In non-TNBC, no change in expression of any single gene was associated with RFS or OS, while in TNBC upregulation of multiple immune-related genes and gene sets were associated with improved long-term outcome. High cytotoxic T-cell signatures present in the peripheral blood of patients with breast cancer at surgery were associated with persistent disease and recurrence, suggesting active antitumor immunity that may indicate ongoing disease burden. Conclusions: We have characterized the effects of NAC on the TIME, finding that TNBC is uniquely sensitive to the immunologic effects of NAC, and local increases in immune genes/sets are associated with improved outcomes. However, expression of cytotoxic genes in the peripheral blood, as opposed to the TIME, may be a minimally invasive biomarker of persistent micrometastatic disease ultimately leading to recurrence.

BACKGROUND: Among patients diagnosed with non-muscle invasive bladder cancer (NMIBC), 30% to 70% experience recurrences within 6 to 12 years of diagnosis. The need to screen for these events every 3 to 6 months and ultimately annually by cystoscopy makes bladder cancer one of the most expensive malignancies to manage. OBJECTIVE: The purpose of this study was to identify reproducible prognostic microRNAs in resected non-muscle invasive bladder tumor tissue that are predictive of the recurrent tumor phenotype as potential biomarkers and molecular therapeutic targets. METHODS: Two independent cohorts of NMIBC patients were analyzed using a biomarker discovery and validation approach, respectively. RESULTS: miRNA Let-7f-5p showed the strongest association with recurrence across both cohorts. Let-7f-5p levels in urine and plasma were both found to be significantly correlated with levels in tumor tissue. We assessed the therapeutic potential of targeting Lin28, a negative regulator of Let-7f-5p, with small-molecule inhibitor C1632. Lin28 inhibition significantly increased levels of Let-7f-5p expression and led to significant inhibition of viability and migration of HTB-2 cells. CONCLUSIONS: We have identified Let-7f-5p as a miRNA biomarker of recurrence in NMIBC tumors. We further demonstrate that targeting Lin28, a negative regulator of Let-7f-5p, represents a novel potential therapeutic opportunity in NMIBC.

Purpose: Despite adjuvant endocrine therapy for patients with estrogen receptor alpha (ER)-positive breast cancer, dormant residual disease can persist for years and eventually cause tumor recurrence. We sought to deduce mechanisms underlying the persistence of dormant cancer cells to identify therapeutic strategies. Experimental Design: Mimicking the aromatase inhibitor–induced depletion of estrogen levels used to treat patients, we developed preclinical models of dormancy in ER+ breast cancer induced by estrogen withdrawal in mice. We analyzed tumor xenografts and cultured cancer cells for molecular and cellular responses to estrogen withdrawal and drug treatments. Publicly available clinical breast tumor gene expression datasets were analyzed for responses to neoadjuvant endocrine therapy. Results: Dormant breast cancer cells exhibited upregulated 5′ adenosine monophosphate-activated protein kinase (AMPK) levels and activity, and upregulated fatty acid oxidation. While the antidiabetes AMPK-activating drug metformin slowed the estrogen-driven growth of cells and tumors, metformin promoted the persistence of estrogen-deprived cells and tumors through increased mitochondrial respiration driven by fatty acid oxidation. Pharmacologic or genetic inhibition of AMPK or fatty acid oxidation promoted clearance of dormant residual disease, while dietary fat increased tumor cell survival. Conclusions: AMPK has context-dependent effects in cancer, cautioning against the widespread use of an AMPK activator across disease settings. The development of therapeutics targeting fat metabolism is warranted in ER+ breast cancer.

PURPOSE: Circulating tumor DNA in plasma may present a minimally invasive opportunity to identify tumor-derived mutations to inform selection of targeted therapies for individual patients, particularly in cases of oligometastatic disease where biopsy of multiple tumors is impractical. To assess the utility of plasma DNA as a “liquid biopsy” for precision oncology, we tested whether sequencing of plasma DNA is a reliable surrogate for sequencing of tumor DNA to identify targetable genetic alterations. METHODS: Blood and biopsies of 1–3 tumors were obtained from 4 evaluable patients with advanced breast cancer. One patient provided samples from an additional 7 tumors post-mortem. DNA extracted from plasma, tumor tissues, and buffy coat of blood were used for probe-directed capture of all exons in 149 cancer-related genes and massively parallel sequencing. Somatic mutations in DNA from plasma and tumors were identified by comparison to buffy coat DNA. RESULTS: Sequencing of plasma DNA identified 27.94 +/− 11.81% (mean +/− SD) of mutations detected in a tumor(s) from the same patient; such mutations tended to be present at high allelic frequency. The majority of mutations found in plasma DNA were not found in tumor samples. Mutations were also found in plasma that matched clinically undetectable tumors found post-mortem. CONCLUSIONS: The incomplete overlap of genetic alteration profiles of plasma and tumors warrants caution in the sole reliance of plasma DNA to identify therapeutically targetable alterations in patients, and indicates that analysis of plasma DNA complements, but does not replace, tumor DNA profiling. TRIAL REGISTRATION: Subjects were prospectively enrolled in trial NCT01836640 (registered April 22, 2013).

The recent approval of anti-PD-L1 immunotherapy in combination with nAB-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor-immune microenvironment (TIME). Patients with TNBC are routinely treated with neoadjuvant chemotherapy (NAC). Stromal tumor-infiltrating lymphocytes (sTILs) in the pre-treatment diagnostic biopsy are predictive of pathologic complete response (pCR). In patients with residual disease (RD) at surgery, sTILs confer good prognosis. However, the effect of chemotherapy on sTILs and how it influences the TIME are poorly understood. We examined immune-gene expression patterns before and after NAC in a series of 83 breast tumors, including 44 TNBCs, from patients with RD. sTILs were enumerated by standardized guidelines. Gene expression patterns were tested for association with recurrence-free (RFS) and overall survival (OS). T cell receptor sequencing (TCRseq) was performed on a subset (n=15) of tumors. In 4 patients undergoing NAC, PD-1-high and -negative CD8+ peripheral blood mononuclear cells (PBMCs) were profiled using single-cell RNAseq and multiplexed cytokine secretion assays. Post-NAC sTILs (≥30%) were only predictive of outcome (RFS p=0.019; OS p=0.05) in TNBC patients, but not in non-TNBC patients (RFS p=0.28; OS p=0.78) confirming that the prognostic capacity of sTILs is confined to TNBC. Pre-NAC sTILs were not predictive of outcome in either group, likely due to exclusion of patients experiencing pCR. The change in sTILs during NAC did not prognosticate outcome in TNBC, suggesting that in the post-NAC setting, only the most proximal measurement of sTILs is meaningful. However, these results did suggest that NAC alters the TIME. To examine the interplay among NAC, the TIME, and clinical outcomes, we tested the change in expression of 770 immune-related genes during NAC in univariate cox-proportional hazards models. In non-TNBC, no change in expression of any single gene was associated with RFS or OS at a false-discovery rate (FDR) of 10%. In TNBC, individual changes in 12 genes and 204 genes were identified as associated with RFS and OS, respectively (FDR Citation Format: Justin M Balko, Mellissa Nixon, Paula I Gonzalez-Ericsson, Mark A Pilkinton, Wyatt J McDonnell, Violeta Sanchez, Susan R Opalenik, Sherene Loi, Brent Rexer, Vandana Abramson, Valerie Jansen, Simon Mallal, Jonathan D Marotti, Kevin Shee, Todd W Miller, Melinda E Sanders, Ingrid A Mayer, Roberto Salgado. Immunologic correlates of long-term outcome in the residual disease of triple-negative breast cancer after neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-15.

PURPOSE Patients and providers often lack clinical decision tools to enable effective shared decision making. This is especially true in the rapidly changing therapeutic landscape of metastatic kidney cancer. Using the International Metastatic Renal Cell Carcinoma Database Consortium (IMDC) criteria, a validated risk prediction tool for patients with metastatic renal cell carcinoma, we created and user-tested a novel interactive visualization for clinical use. METHODS An interactive visualization depicting IMDC criteria was created, with the final version including data for more than 4,500 patients. Usability testing was performed with nonmedical lay-users and medical oncology fellow physicians. Subjects used the tool to calculate median survival times based on IMDC criteria. User confidence was surveyed. An iterative user feedback implementation cycle was completed and informed revision of the tool. RESULTS The tool is available at CloViz—IMDC. Initially, 400 lay-users and 15 physicians completed clinical scenarios and surveys. Cumulative accuracy across scenarios was higher for physicians than lay-users (84% v 74%; P = .03). Eighty-three percent of lay-users and 87% of physicians thought the tool became intuitive with use. Sixty-eight percent of lay-users wanted to use the tool clinically compared with 87% of physicians. After revisions, the updated tool was user-tested with 100 lay-users and 15 physicians. Physicians, but not lay-users, showed significant improvement in accuracy in the updated version of the tool (90% v 67%; P = .008). Seventy-two percent of lay-users and 93% of physicians wanted to use the updated tool in a clinical setting. CONCLUSION A graphical method of interacting with a validated nomogram provides prognosis results that can be used by nonmedical lay-users and physicians, and has the potential for expanded use across many clinical conditions.

Estrogens have been shown to elicit anticancer effects against estrogen receptor α (ER)-positive breast cancer. We sought to determine the mechanism underlying the therapeutic response. Response to 17β-estradiol was assessed in ER+ breast cancer models with resistance to estrogen deprivation: WHIM16 patient-derived xenografts, C7-2-HI and C4-HI murine mammary adenocarcinomas, and long-term estrogen-deprived MCF-7 cells. As another means to reactivate ER, the anti-estrogen fulvestrant was withdrawn from fulvestrant-resistant MCF-7 cells. Transcriptional, growth, apoptosis, and molecular alterations in response to ER reactivation were measured. 17β-estradiol treatment and fulvestrant withdrawal induced transcriptional activation of ER, and cells adapted to estrogen deprivation or fulvestrant were hypersensitive to 17β-estradiol. ER transcriptional response was followed by an unfolded protein response and apoptosis. Such apoptosis was dependent upon the unfolded protein response, p53, and JNK signaling. Anticancer effects were most pronounced in models exhibiting genomic amplification of the gene encoding ER (ESR1), suggesting that engagement of ER at high levels is cytotoxic. These data indicate that long-term adaptation to estrogen deprivation or ER inhibition alters sensitivity to ER reactivation. In such adapted cells, 17β-estradiol treatment and anti-estrogen withdrawal hyperactivate ER, which drives an unfolded protein response and subsequent growth inhibition and apoptosis. 17β-estradiol treatment should be considered as a therapeutic option for anti-estrogen-resistant disease, particularly in patients with tumors harboring ESR1 amplification or ER overexpression. Furthermore, therapeutic strategies that enhance an unfolded protein response may increase the therapeutic effects of ER reactivation.

Historically, ductal carcinoma in situ (DCIS) of the breast has been managed aggressively with surgery and radiotherapy because of a risk of progression to invasive ductal carcinoma. However, this treatment paradigm has been challenged by overtreatment concerns and evidence that suggests that DCIS can be stratified according to risk of recurrence or risk of progression to invasive disease. Traditional methods of risk stratification include histologic grade and hormone receptor status. Recent technological advancements have enabled an era of precision medicine, where DCIS can be molecularly analyzed by tools, such as next-generation DNA and RNA sequencing, to identify molecular biomarkers for risk stratification. These findings have led to the development of tools such as the Oncotype DX Breast DCIS Score, a gene expression–based assay with the potential to prevent overtreatment in low-risk disease.

Robotic surgical skill development is central to training in urology as well as other surgical disciplines. Vesicourethral anastomosis (VUA) in robotic prostatectomy is a challenging task for novices due to delicate tissue and difficult suturing angles. Commercially available, realistic training models are limited. Here, we describe the development and validation of a 3D-printed model of the VUA for ex vivo training using the da Vinci Surgical System. Models of the bladder and urethra were created using 3D-printing technology based on estimations of average in vivo anatomy. 10 surgical residents without prior robotics training were enrolled in the study: 5 residents received structured virtual reality (VR) training on the da Vinci Skills Simulator ("trained"), while the other 5 did not ("untrained"). 4 faculty robotic surgeons trained in robotic urologic oncology ("experts") were also enrolled. Mean (range) completion percentage was 20% (10-30%), 54% (40-70%), and 96% (85-100%) by the untrained, trained, and expert groups, respectively. Anastomosis integrity was rated as excellent (as opposed to moderate or poor) in 40%, 60%, and 100% of untrained, trained, and expert groups, respectively. Face validity (realism) was rated as 8 of 10 on average by the expert surgeons, each of whom rated the model as a superior training tool to digital VR trainers. Content validity (usefulness) was rated as 10 of 10 by all participants. This is the first reported 3D-printed ex vivo trainer for VUA in robotic prostatectomy validated for use in robotic simulation. The addition of 3D-printed ex vivo training to existing digital simulation technologies may augment and improve robotic surgical education in the future.

Objective To rank percentages of underrepresented residents in surgical subspecialties and understand the experience of mentees and mentors who participated in the inaugural University of California, San Francisco Urology UnderRepresented Trainees Entering Residency (UReTER) Mentorship Program for Black, Indigenous, and/or LatinX medical students applying into urology. Methods Medical student mentees across the country were recruited via social media and email listservs. Demographic information and photos of mentors were presented on the UReTER website. Medical students could choose a mentor, and once matched, both parties were notified. A survey was emailed to all participants on Urology Match Day 2021. Result The 2018 –2019 ACGME Databook showed underrepresented minority residents made up 7.6% of urology residents, lagging behind neurosurgery, vascular surgery, general surgery, and obstetrics and gynecology. 71 mentees and 101 mentors volunteered for the UReTER Mentorship Program (71 mentor-mentee couplets). Overall response rate was 51% [33 mentors and 32 mentees]. Of mentees who completed the survey, 16 (47%) participated in the 2021 Urology Match; 15 (94%) matched and 6 (38%) felt that UReTER helped them match. Conclusion Feedback on this pilot program was very positive including a high match rate among those who participated. Future changes to the program include expanded student outreach, increased structure, broadened mentor network. The implementation of a low-cost program to increase underrepresented applicants into Urology has great potential to increase representation and improve the field. This program can and should be replicated in all subspecialties.

To determine if older men with Gleason grade group (GG) 1 prostate cancer have a higher risk of having adverse pathology at radical prostatectomy after initially being managed with active surveillance (AS).A total of 365 patients with GG1 prostate cancer initially managed with AS followed by delayed radical prostatectomy were identified. The primary outcome was adverse pathology after delayed radical prostatectomy in the men that were65 years vs. men ≥65 years at the initiation of AS. Adverse pathology was defined as GG ≥3 or pT3 or pN1. Multivariable Cox proportional hazards regression models were used to calculate risk of adverse pathological findings at radical prostatectomy by age group.At diagnosis, there were no significant differences in median prostate specific antigen density, percent positive biopsy cores, multiparametric magnetic resonance imaging (mpMRI) results or composite genomic classifier scores (derived from three commercially available genomic tests) between the two age groups. Men ≥65 years had more adverse pathology at radical prostatectomy (59.2% vs. 44.1%, P0.01) and lower rates of biopsy upgrade-free survival and adverse pathology-free survival (log-rank P0.01). On multivariable analysis age ≥65 years (Hazard Ratio (HR) 2.21, 95% Confidence Interval (CI) 1.57, 3.12) was associated with adverse pathology at radical prostatectomy. In separate multivariable analyses done for each age group, mpMRI (HR 3.33, 95% CI 1.01, 10.95) was predictor of adverse pathology in the group ≥65 years.Older patients might require closer monitoring on AS and additional testing such as mpMRI might improve their risk stratification.

Primary hyperoxaluria 1 (PH1) is a devastating condition involving recurrent urolithiasis, early end-stage renal disease and multisystemic deposition of calcium oxalate crystals. Treatment options for PH1 are limited, inevitably requiring transplantation, usually combined kidney and liver transplant. Here we report successful compassionate use of Nedosiran, an RNA interference targeting lactate dehydrogenase, in an index patient. Monthly Nedosiran injections led to dramatically decreased plasma oxalate levels, decreased frequency of weekly hemodialysis sessions from 6 to 3, and deferral of combined kidney and liver transplant. Nedosiran represents a novel and impactful potential therapeutic for PH1 patients with end-stage renal disease.

Lessons learned Fulvestrant is a selective estrogen receptor (ER)-downregulating antiestrogen that blocks ER transcriptional activity and is approved for ER-positive breast cancer. Fulvestrant also induces accumulation of insoluble ER and activates an unfolded protein response; proteasome inhibitors have been shown to enhance these effects in preclinical models. Background Fulvestrant is a selective estrogen receptor (ER)-downregulating antiestrogen that blocks ER transcriptional activity and is approved for ER-positive (+) breast cancer. Fulvestrant also induces accumulation of insoluble ER and activates an unfolded protein response; proteasome inhibitors have been shown to enhance these effects in preclinical models. Methods This is a single-center phase Ib study with a 3+3 design of fulvestrant and the proteasome inhibitor ixazomib (MLN9708) in patients with advanced ER+ breast cancer that was progressing on fulvestrant. A dose-escalation design allowed establishment of the ixazomib maximum tolerated dose (MTD). Secondary objectives included progression-free survival, pharmacokinetics, and tumor molecular analyses. Results Among nine evaluable subjects, treatment was well-tolerated without dose-limiting toxicities The MTD of ixazomib was 4 mg in combination with fulvestrant. Plasma concentrations of the active form of ixazomib (MLN2238) in the 4-mg dose cohort had a median (range) maximal concentration (Cmax ) of 155 (122-171) ng/mL, time of maximal concentration (Tmax ) of 1 (1-1.5) hour, terminal elimination half-life of 66.6 (57.3-102.6) hour after initial dose, and area under the curve (AUC) of 5,025 (4,160-5,345) ng*h/mL. One partial response was observed, and median progression-free survival was 51 days (range, 47-137). Conclusion This drug combination has a favorable safety profile and antitumor activity in patients with fulvestrant-resistant advanced ER+ breast cancer that justifies future testing.

Recent molecular characterization of gliomas has uncovered somatic gene variation and DNA methylation changes that are associated with etiology, prognosis, and therapeutic response. Here we describe genomic profiling of gliomas assessed for associations between genetic mutations and patient outcomes, including overall survival (OS) and recurrence-free survival (RFS). Mutations in a 50-gene cancer panel, 1p19q co-deletion, and MGMT promoter methylation (MGMT methylation) status were obtained from tumor tissue of 293 glioma patients. Multivariable regression models for overall survival (OS) and recurrence-free survival (RFS) were constructed for MGMT methylation, 1p19q co-deletion, and gene mutations controlling for age, treatment status, and WHO grade. Mutational profiles of gliomas significantly differed based on WHO Grade, such as high prevalence of BRAF V600E, IDH1, and PTEN mutations in WHO Grade I, II/III, and IV tumors, respectively. In multivariate regression analysis, MGMT methylation and IDH1 mutations were significantly associated with improved OS (HR = 0.44, p = 0.0004 and HR = 0.21, p = 0.007, respectively), while FLT3 and TP53 mutations were significantly associated with poorer OS (HR = 19.46, p

INTRODUCTION AND OBJECTIVE:50% to 75% of patients with non-muscle invasive bladder cancer (NMIBC) experience recurrences within 6 to 12 years of diagnosis, and the cost of screening makes bladder c...

PurposeDespite adjuvant anti-estrogen therapy for patients with estrogen receptor alpha (ER)-positive breast cancer, dormant residual disease can persist for years and eventually cause tumor recurrence. We sought to deduce mechanisms underlying the persistence of dormant cancer cells to identify therapeutic strategies.Experimental DesignMimicking the aromatase inhibitor-induced depletion of estrogen levels used to treat patients, we developed preclinical models of dormancy in ER+ breast cancer induced by estrogen withdrawal in mice. We analyzed tumor xenografts and cultured cancer cells for molecular and cellular responses to estrogen withdrawal and drug treatments. Publicly available clinical breast tumor gene expression datasets were analyzed for responses to neoadjuvant anti-estrogen therapy.ResultsDormant breast cancer cells exhibited upregulated 5’ adenosine monophosphate-activated protein kinase (AMPK) levels and activity, and upregulated fatty acid oxidation. While the anti-diabetes AMPK-activating drug metformin slowed the estrogen-driven growth of cells and tumors, metformin promoted the persistence of estrogen-deprived cells and tumors through increased mitochondrial respiration driven by fatty acid oxidation. Pharmacologic or genetic inhibition of AMPK or fatty acid oxidation promoted clearance of dormant residual disease, while dietary fat increased tumor cell survival.ConclusionsAMPK has context-dependent effects in cancer, cautioning against the widespread use of an AMPK activator across disease settings. The development of therapeutics targeting fat metabolism is warranted in ER+ breast cancer.Statement of Translational RelevanceDormant cancer cells that survive adjuvant therapy can ultimately give rise to recurrent/advanced tumors that frequently develop resistance to all approved therapies. Patients with early-stage estrogen receptor alpha (ER)-positive breast cancer are typically treated with surgical resection followed by ≥5 years of adjuvant anti-estrogen therapy that neutralizes ER and suppresses, but often does not eliminate, tumor-initiating cells. Estrogen withdrawal, which mimics aromatase inhibitor therapy, induced activation of the metabolic sensor 5’ adenosine monophosphate-activated protein kinase (AMPK) and upregulated fatty acid oxidation (FAO) in preclinical models. Treatment with the anti-diabetes AMPK-activating drug metformin or high dietary fat intake promoted survival of dormant ER+ breast cancer cells, while anti-anginal drugs that inhibit FAO induced clearance of dormant tumor cells. These findings caution against using AMPK modulators with anti-estrogens in patients with ER+ breast cancer, and warrant testing of FAO inhibitors as anti-cancer agents in combination with anti-estrogens.

Despite the success of approved systemic therapies for estrogen receptor α (ER)-positive breast cancer, drug resistance remains common. We hypothesized that secreted factors from the human tumor microenvironment could modulate drug resistance. We previously screened a library of 297 recombinant-secreted microenvironmental proteins for the ability to confer resistance to the anti-estrogen fulvestrant in 2 ER(+) breast cancer cell lines. Herein, we considered whether factors that enhanced drug sensitivity could be repurposed as therapeutics and provide leads for drug development. Screening data revealed bone morphogenic protein (BMP)4 as a factor that inhibited cell growth and synergized with approved anti-estrogens and cyclin-dependent kinase 4/6 inhibitors (CDK4/6(i)). BMP4-mediated growth inhibition was dependent on type I receptor activin receptor-like kinase (ALK)3-dependent phosphorylation (P) of mothers against decapentaplegic homolog (SMAD/P-SMAD)1 and 5, which could be reversed by BMP receptor inhibitors and ALK3 knockdown. The primary effect of BMP4 on cell fate was cell-cycle arrest, in which RNA sequencing, immunoblot analysis, and RNA interference revealed to be dependent on p21(WAF1/Cip1) upregulation. BMP4 also enhanced sensitivity to approved inhibitors of mammalian target of rapamycin complex 1 and CDK4/6 via ALK3-mediated P-SMAD1/5 and p21 upregulation in anti-estrogen-resistant cells. Patients bearing primary ER(+) breast tumors, exhibiting a transcriptomic signature of BMP4 signaling, had improved disease outcome following adjuvant treatment with anti-estrogen therapy, independently of age, tumor grade, and tumor stage. Furthermore, a transcriptomic signature of BMP4 signaling was predictive of an improved biologic response to the CDK4/6(i) palbociclib, in combination with an aromatase inhibitor in primary tumors. These findings highlight BMP4 and its downstream pathway activation as a therapeutic opportunity in ER(+) breast cancer.—Shee, K., Jiang, A., Varn, F. S., Liu, S., Traphagen, N. A., Owens, P., Ma, C. X., Hoog, J., Cheng, C., Golub, T. R., Straussman, R., Miller, T. W. Cytokine sensitivity screening highlights BMP4 pathway signaling as a therapeutic opportunity in ER(+) breast cancer.

Despite the clinical success of anti-estrogen therapies, phosphatidylinositol 3-kinase inhibitors (PI3Ki), and mechanistic target of rapamycin complex I inhibitors (mTORC1i) for the treatment of patients with ER+ breast cancer, disease recurrence and progression are common. We found that a tumor transcriptional profile reflecting high stromal fibroblast content was associated with poor outcome in 3 cohorts of patients with ER+ breast cancer. We hypothesized that individual factors in the tumor microenvironment (TME) significantly contribute to drug resistance. To test this hypothesis, we screened 297 recombinant secreted proteins for ability to confer resistance to the anti-estrogen fulvestrant in MCF-7 and T47D ER+ breast cancer cells. Screen results were validated, and expansion screening included the anti-estrogen tamoxifen, the PI3Ki pictilisib, and the mTORC1i everolimus in 4 cell lines. To identify hits are most likely to be relevant to ER+ breast cancer, a bioinformatics filter was developed utilizing gene and protein expression in human tissues relevant to the TMEs of ER+ breast cancer. After filtering, the top screening hit was fibroblast growth factor 2 (FGF2), which confers resistance to anti-estrogens, PI3Ki, and mTORC1i, and is highly expressed in tissues and cell types associated with ER+ breast cancer. FGF2 did not rescue cells from the CDK4/6i palbociclib or the DNA-damaging agent doxorubicin, demonstrating pathway selectivity in the rescue phenotype. FGF2 rescued cells from anti-estrogen-, PI3Ki-, and mTORC1i-induced apoptosis and cell cycle arrest via activation of FGFR signaling through FRS2a, MEK1/2, ERK1/2, and downstream upregulation of cyclin D1 and degradation of Bim. FGF2-mediated anti-cancer effects were abrogated by co-treatment with the FGF2-neutralizing antibody GAL-F2, the pan-FGFR inhibitor PD-173074, the MEK inhibitor trametinib, or palbociclib. Cell cycle- and apoptosis-specific effects of FGF2 were abrogated by RNAi targeting cyclin D1 and Bim, respectively. We generated a transcriptional signature of FGF2 response by RNA-seq of fulvestrant-treated MCF-7 and T47D cells treated +/- FGF2. In 3 cohorts of patients with ER+ breast cancer, a signature of FGF2 signaling was significantly associated with poor prognosis and predictive of anti-estrogen resistance, including in a multivariate analysis including age, tumor grade, tumor stage, and FGFR amplification status. Finally, the therapeutic potential of targeting FGF2 was confirmed in 3 mouse models of ER+ breast cancer: 1) FGF2 rescue MCF-7 xenografts from fulvestrant; 2) GAL-F2 synergized with fulvestrant to suppress growth of 59-2-HI murine mammary adenocarcinomas that recruit FGF2-secreting stroma; 3) GAL-F2 synergized with fulvestrant to induce regression of HCI-003 patient-derived xenografts. Therapeutic effects coincided with increased tumor cell apoptosis and decreased proliferation, but not changes in tumor vasculature. These findings warrant consideration of FGF2 as a novel therapeutic target in ER+ breast cancer. Citation Format: Shee K, Hinds JW, Yang W, Hampsch RA, Patel K, Varn FS, Cheng C, Jenkins NP, Kettenbach AN, Demidenko E, Owens P, Lanari C, Faber AC, Golub TR, Straussman R, Miller TW. A microenvironment secretome screen reveals FGF2 as a mediator of resistance to anti-estrogens and PI3K/mTOR pathway inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-08.

Introduction Rates of prostate specific antigen screening and prostate cancer diagnoses have declined following controversial changes to prostate cancer screening guidelines by the USPSTF (U.S. Preventive Services Task Force) in 2011 and 2012 advising against all prostate specific antigen screening. To understand whether uneven media coverage of the guidelines may have influenced shifts in clinical decision making, this study analyzes news reports about the USPSTF and AUA (American Urological Association) prostate cancer screening guidelines. Methods Two national databases were queried for news articles published within 30 days of the release of each guideline. Media analysis included consequences of screening, accuracy of guideline descriptions, discussion of primary evidence and neutrality of headlines. Thematic changes between the preliminary and final USPSTF guidelines were compared. Results Of the 45 news articles in the sample 24 were about the preliminary USPSTF guidelines, 17 about the final USPSTF guidelines and 4 about the revised AUA guidelines. More than 80% of the articles discussed adverse consequences of prostate specific antigen screening while a minority accurately summarized the recommendations or discussed limitations of the evidence. Significantly more articles about the final vs preliminary USPSTF guidelines had headlines opposing screening or suggested urologists’ financial gains from prostate specific antigen tests (p Conclusions The USPSTF guidelines were more extensively reported in the news than the AUA guidelines. Articles frequently emphasized the negative aspects of screening and did not discuss limitations of the evidence. These findings may have influenced patients’ decisions underlying declines in prostate specific antigen screening since 2011.

Introduction The literature on perioperative pain control and management in inflatable penile prosthesis placement is not firmly established. Because inflatable penile prosthesis placement is an elective procedure, pain can be one of the many issues that influence patient decision making. Pain control also presents a unique challenge to providers in an era of widespread opiate abuse. Aim To review published data on pain management before, during, and after penile prosthetic surgery. Methods Peer-reviewed literature and conference abstracts were analyzed for all relevant publications related to this issue. Results The past several decades have seen a shift from general to local anesthesia for penile prosthetic surgery. This has been well characterized and is seen as successful with different local anesthetic options and techniques. To date, only one study has provided follow-up for longer than 1 week regarding postoperative pain management for prosthetic surgery. Conclusion Perioperative pain management for the patient receiving a penile prosthetic is well characterized; postoperative pain management is not. Although periprocedural local anesthesia has been well described for penile prosthesis surgery, a standardized postoperative pain management plan does not exist. This review highlights the need for further characterization of postoperative pain and the subsequent development of an algorithmic approach for management. Reinstatler L, Shee K, Gross MS. Pain Management in Penile Prosthetic Surgery: A Review of the Literature. Sex Med Rev 2018;6:162–169.

Growing evidence suggests that microbes can influence the efficacy of cancer therapies. By studying colon cancer models, we found that bacteria can metabolize the chemotherapeutic drug gemcitabine (2′,2′-difluorodeoxycytidine) into its inactive form, 2′,2′-difluorodeoxyuridine. Metabolism was dependent on the expression of a long isoform of the bacterial enzyme cytidine deaminase (CDDL), seen primarily in Gammaproteobacteria. In a colon cancer mouse model, gemcitabine resistance was induced by intra-tumor Gammaproteobacteria, dependent on bacterial CDDL expression, and abrogated by co-treatment with the antibiotic ciprofloxacin. Gemcitabine is commonly used to treat pancreatic ductal adenocarcinoma (PDAC), and we hypothesized that intra-tumor bacteria might contribute to drug resistance of these tumors. Consistent with this possibility, we found that of the 113 human PDACs that were tested, 86 (76%) were positive for bacteria, mainly Gammaproteobacteria.

Despite the clinical efficacy of anthracycline agents such as doxorubicin, dose-limiting cardiac toxicities significantly limit their long-term use. Here, we present the case of a 33-year-old female patient with extensive metastatic ER+/PR+/HER2– mucinous adenocarcinoma of the breast, who was started on doxorubicin/cyclophosphamide therapy after progressing on paclitaxel and ovarian suppressor goserelin with aromatase inhibitor exemestane. The patient was comanaged by cardiology, who carefully monitored measures of cardiac function, including EKGs, serial echocardiograms, and profiling of lipids, troponin, and pro-BNP every 2 months. The patient was treated with the cardioprotective agent dexrazoxane, and changes in cardiac markers [e.g. decreases in ejection fraction (EF)] were immediately addressed by therapeutic intervention with the ACE inhibitor lisinopril and beta-blocker metoprolol. The patient had a complete response to doxorubicin therapy, with a cumulative dose of 1,350 mg/m2, which is significantly above the recommended limits, and to our knowledge, the highest dose reported in literature. Two and a half years after the last doxorubicin cycle, the patient is asymptomatic with no cardiotoxicity and an excellent quality of life. This case highlights the importance of careful monitoring and management of doxorubicin-mediated cardiotoxicity, and that higher cumulative doses of anthracyclines can be considered in patients with ongoing clinical benefit.

INTRODUCTION AND OBJECTIVES:The Human Papilloma Virus (HPV) is a recognized carcinogenic factor. Previous studies have shown a possible association between HPV and bladder cancer, however HPV serol...

The patient is a 72-year-old female who presents with new onset jaundice. The patient has a past medical history significant for right-sided estrogen receptor (ER)-positive and left-sided ER-negative breast cancers in 2005 and 2009, respectively, and recent 1-year history of ER-positive right-sided breast cancer with bone and brain metastases. CT scan and endoscopic ultrasound (EUS) revealed a new 2 cm mass in the head of the pancreas, leading to EUS-guided fine-needle aspiration of the lesion. Pathologic workup revealed adenocarcinoma with signet-ring cells, representing either metastatic breast or primary pancreatic cancer. Immunohistochemistry and molecular diagnostic workup identified positive GATA-binding protein 3 (GATA3) immunoreactivity and a mutation in Erb-B2 receptor tyrosine kinase 2 (ERBB2), also known as human epidermal growth factor receptor 2 (HER2). Here, we review the diagnostic markers commonly used to differentiate metastatic breast vs primary pancreatic adenocarcinoma, and discuss the challenges of utilizing GATA3 immunoreactivity and ERBB2 mutations for diagnosis.

Precision medicine requires that a patient's tumor be accurately genotyped to identify a potentially effective targeted therapy. However, genotyping a tumor in patients with oligometastatic disease is complicated by the potential for intratumor and intertumor heterogeneity, and the requirement for sufficient tumor tissue obtained by invasive biopsy for genetic profiling. We sought to determine whether circulating tumor DNA in plasma provides a surrogate for solid tumor biopsy, and captures the genetic heterogeneity of tumors in patients with metastatic breast cancer. We hypothesized that genetic mutations detected in plasma DNA are reflective of the genetic mutations present in all tumors within a patient. Eight patients with advanced/metastatic breast cancer have thus far been enrolled in an ongoing clinical study (NCT01836640). Tumor specimens from two (n=4) or three (n=4) tumor sites and blood were obtained with one month. Blood was separated into plasma and buffy coat fractions. DNA extracted from tissue, buffy coat, and plasma samples was used for massively parallel DNA sequencing using the Ion Proton platform with a custom TargetSeq capture probe set covering all exons of 196 genes (4.1 Mb). All tumor and buffy coat samples, and plasma samples from three patients have thus far been analyzed. Tumor mutations were identified by comparison to buffy coat DNA sequences. We achieved sequencing coverage of ∼100-fold for tumor and buffy coat DNA samples, and ∼1,000-fold for plasma DNA samples. In Patient #1, we obtained 14 tumor nodules from a mastectomy specimen and used 3 nodules for DNA sequencing; Among the 73 point mutations detected in DNA from at least one tumor nodule, 29 mutations (40%) were detected in plasma DNA, and 10 mutations were found in plasma but not in tumors. In Patient #5, we analyzed bilateral breast tumors and a brain metastasis; among 151 mutations detected in at least one tumor, 80 (53%) were found in plasma, and an additional 18 mutations were found in plasma but not tumors; mutations specific to the brain tumor were less likely to be found in plasma; interestingly, the bilateral breast tumors showed genetic and histologic similarity, and so were likely derived from a single clone. Patient #6 had only one lung metastasis evaluable by DNA sequencing; 64/125 (51%) tumor-derived mutations were detected in plasma, and an additional 26 mutations were found in plasma but not the tumor. Preliminary ResultsMutationsTumorPlasma (Plasma only)TotalPlasma concordance with tumorPlasma concordance with totalTumor concordance with totalPatient #17329 (10)8339.7%46.9%87.9%Patient #515180 (18)16952.9%57.9%89.3%Patient #612564 (26)15151.2%59.6%82.8% These data suggest that, although challenging to get multiple biopsies for comparison, plasma is a promising surrogate for solid tumor biopsy to identify potentially targetable mutations. However, the ability of plasma DNA to genetically reflect all tumors in a patient with oligometastatic disease remains to be clarified through further analysis. Citation Format: Chamberlin MD, Shee K, Varn FS, Bean JR, Marotti JD, Gui J, Gemery JM, Barth RJ, Rosenkranz KM, Tsapakos MJ, McNulty NJ, Cheng C, Miller TW. Plasma DNA as a surrogate for tumor biopsy to identify genetic alterations in patients with metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-09-20.

Despite adjuvant anti-estrogen therapy for patients with ER+ breast cancer, clinically dormant residual disease can persist for years and eventually cause tumor recurrence. Mimicking the aromatase inhibitor-induced depletion of estrogen levels used to treat patients, we developed clinically relevant xenograft models of dormancy induced by estrogen withdrawal. Dormant breast cancer cells exhibited upregulated AMPK levels and activity. Metformin is an AMPK-activating drug approved to treat diabetes that is being tested in many ongoing clinical trials for treatment of cancer. We found that metformin promoted survival of dormant ER+ breast cancer cells through increased mitochondrial respiration driven by fatty acid oxidation. Pharmacologic or genetic inhibition of AMPK or fatty acid oxidation promoted clearance of dormant residual disease, while dietary fat increased tumor cell survival. These findings indicate that AMPK has context-dependent effects in cancer, caution against the widespread use of an AMPK activator across disease settings, and warrant the development of cancer therapeutics targeting fat metabolism. Citation Format: Riley Hampsch, Nicole Traphagen, Jason Wells, Charlotte McCleery, Jennifer Fields, Kevin Shee, Lloye Dillon, Darcy Pooler, Lionel Lewis, Eugene Demidenko, Yina Huang, Jonathan Marotti, William Kinlaw, Todd Wayne Miller. AMPK activation by metformin promotes survival of dormant ER+ breast cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A02. doi:10.1158/1535-7163.TARG-19-LB-A02

To examine patterns of crowdsourcing on the social media platform Twitter among urologists. Urologists’ public Twitter accounts were reviewed for original posts seeking clinical advice or feedback, and associated reply posts, before and after the 140-character-limit expansion in 2017. Predictors of responses to crowdsourcing requests were determined using multivariable regression. When patient data were posted, we noted whether consent was documented. A total of 276 posts in 23 crowdsourcing requests prior to character-limit expansion were analyzed. Reasons for crowdsourcing included requesting solutions to a clinical dilemma (82 posts, 30%); advice seeking about a surgical plan (77 posts, 28%); surveying colleagues’ experiences with a new product (64 posts, 23%); and soliciting feedback about a proposed course of action (53 posts, 19%). Recent completion of training (as a proxy for inexperience) did not appear to disproportionately motivate crowdsourcing; authors’ median time in practice was 7 years, and authors practicing for ≤ 7 years initiated 57% of requests. 22 (96%) crowdsourcing requests received ≥ 1 reply. Of 15 requests about a specific patient, eight included imaging, but only one cited patient consent. A second analysis of 184 posts in 17 crowdsourcing requests initiated after character-limit expansion demonstrated significantly more authors replying per request (P = 0.01), but no change in the frequency of patient-specific crowdsourcing or citation of consent. Urologists are leveraging Twitter for crowdsourcing clinical guidance and experiential knowledge. Nearly all requests were answered, suggesting low barriers to entry for novice users. Even after character-limit expansion, dissemination of potentially identifiable patient data remains a concern.

p53 is an important tumor-suppressor protein that is mutated in more than 50% of cancers. Strategies for restoring normal p53 function are complicated by the oncogenic properties of mutant p53 and have not met with clinical success. To counteract mutant p53 activity, a variety of drugs with the potential to reconvert mutant p53 to an active wildtype form have been developed. However, these drugs are associated with various negative effects such as cellular toxicity, nonspecific binding to other proteins, and inability to induce a wildtype p53 response in cancer tissue. Here, we report on the effects of a curcumin analog, HO-3867, on p53 activity in cancer cells from different origins. We found that HO-3867 covalently binds to mutant p53, initiates a wildtype p53-like anticancer genetic response, is exclusively cytotoxic toward cancer cells, and exhibits high anticancer efficacy in tumor models. In conclusion, HO-3867 is a p53 mutant–reactivating drug with high clinical anticancer potential.

Activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway has been implicated in anti-estrogen resistance in breast cancer. We tested the therapeutic potential of the novel PI3K/mTOR dual inhibitor P7170 in a panel of anti-estrogen-sensitive and anti-estrogen-resistant models of ER+ breast cancer. Estrogen receptor-positive (ER+) breast cancer cells were treated ±P7170. Fresh cores from primary ER+/HER2- tumors from two patients were treated ±P7170 ex vivo. Mice bearing breast cancer xenografts were randomized to treatment with vehicle, fulvestrant, P7170, or combinations, and tumor volumes were measured. Tissues and cells were analyzed for markers of pathway activity, cell viability, and apoptosis. In cell lines, P7170 exhibited IC50 values in the range of 0.9-7 nM and induced apoptosis. P7170 potently inhibited mTOR activity (≤ 25 nM) and inhibited PI3K at higher concentrations (≥ 200 nM). P7170 completely inhibited MCF-7 tumor growth, significantly inhibited growth of fulvestrant-resistant T47D tumors, and suppressed tumor cell proliferation but did not induce apoptosis. While P7170 inhibits PI3K and mTOR in ER+/HER2- human breast cancer cells and tumors ex vivo, in vivo data indicate that the primary mechanism of P7170 anti-tumor action is inhibition of mTOR and cell proliferation. P7170 is a novel agent worthy of further investigation for the treatment of ER+ breast cancer.

Hyperactivation of the PI3K pathway has been implicated in resistance to antiestrogen therapies in estrogen receptor α (ER)–positive breast cancer, prompting the development of therapeutic strategies to inhibit this pathway. Autophagy has tumor-promoting and -suppressing roles and has been broadly implicated in resistance to anticancer therapies, including antiestrogens. Chloroquine (CQ) is an antimalarial and amebicidal drug that inhibits autophagy in mammalian cells and human tumors. Herein, we observed that CQ inhibited proliferation and autophagy in ER(+) breast cancer cells. PI3K inhibition with GDC-0941 (pictilisib) induced autophagy. Inhibition of autophagy using CQ or RNA interference potentiated PI3K inhibitor-induced apoptosis. Combined inhibition of PI3K and autophagy effectively induced mitochondrial membrane depolarization, which required the BH3-only proapoptotic proteins Bim and PUMA. Treatment with GDC-0941, CQ, or the combination, significantly suppressed the growth of ER(+) breast cancer xenografts in mice. In an antiestrogen–resistant xenograft model, GDC-0941 synergized with CQ to provide partial, but durable, tumor regression. These findings warrant clinical evaluation of therapeutic strategies to target ER, PI3K, and autophagy for the treatment of ER(+) breast cancer.—Yang, W., Hosford, S. R., Traphagen, N. A., Shee, K., Demidenko, E., Liu, S., Miller, T. W. Autophagy promotes escape from phosphatidylinositol 3-kinase inhibition in estrogen receptor–positive breast cancer.

Tumor microenvironment (TME) cytokine screening revealed FGF2 as a clinically relevant mechanism of resistance to anti-estrogens, mTORC1 inhibition, and PI3K inhibition in ER+ breast cancer. Shee et al. highlight an underdeveloped aspect of precision oncology: treating patients according to their TME constitution., Drug resistance to approved systemic therapies in estrogen receptor–positive (ER+) breast cancer remains common. We hypothesized that factors present in the human tumor microenvironment (TME) drive drug resistance. Screening of a library of recombinant secreted microenvironmental proteins revealed fibroblast growth factor 2 (FGF2) as a potent mediator of resistance to anti-estrogens, mTORC1 inhibition, and phosphatidylinositol 3-kinase inhibition in ER+ breast cancer. Phosphoproteomic analyses identified ERK1/2 as a major output of FGF2 signaling via FGF receptors (FGFRs), with consequent up-regulation of Cyclin D1 and down-regulation of Bim as mediators of drug resistance. FGF2-driven drug resistance in anti-estrogen–sensitive and –resistant models, including patient-derived xenografts, was reverted by neutralizing FGF2 or FGFRs. A transcriptomic signature of FGF2 signaling in primary tumors predicted shorter recurrence-free survival independently of age, grade, stage, and FGFR amplification status. These findings delineate FGF2 signaling as a ligand-based drug resistance mechanism and highlights an underdeveloped aspect of precision oncology: characterizing and treating patients according to their TME constitution.

BackgroundPrecision oncology seeks to integrate multiple layers of data from a patient's cancer to effectively tailor therapy. Conventional chemotherapies are sometimes effective but accompanied by adverse events, warranting the identification of a biomarker of chemosensitivity.ObjectiveIdentify an mRNA biomarker that predicts chemosensitivity across solid tumor subtypes.MethodsWe performed a pan-solid tumor analysis integrating gene expression and drug sensitivity profiles from 3 cancer cell line datasets to identify transcripts correlated with sensitivity to a panel of chemotherapeutics. We then tested the ability of an mRNA biomarker to predictive clinical outcomes in cohorts of patients with breast, lung, or ovarian cancer.ResultsExpression levels of several mRNA transcripts were significantly correlated with sensitivity or resistance chemotherapeutics in cancer cell line datasets. The only mRNA transcript significantly correlated with sensitization to multiple classes of DNA-damaging chemotherapeutics in all 3 cell line datasets was encoded by Schlafen Family Member 11 (SLFN11). Analyses of multiple breast, lung, and ovarian cancer patient cohorts treated with chemotherapy confirmed SLFN11 mRNA expression as a predictive biomarker of longer overall survival and improved tumor response.ConclusionsTumor SLFN11 mRNA expression is a biomarker of sensitivity to an array of DNA-damaging chemotherapeutics across solid tumor subtypes.

362 Background: The Human Papilloma Virus (HPV) is a recognized carcinogenic factor. Previous studies have shown a possible association between HPV and bladder cancer, however HPV serology has not been reported. In this study, we assessed the correlation between bladder cancer and HPV positive serology. Methods: Using NHANES, a large nationally-representative population-based survey, we gathered clinical and demographic data on all patients with a diagnosis of bladder cancer over from 2007-2010 and analyzed their HPV serology status. We assessed the effects of positive serologic results for HPV18, HPV16, HPV11, and HPV6 on the rates of bladder cancer diagnosis. Results: Over the four-year period there was a weighted population of 155,650,000 Americans. The prevalence of HPV positive serology was as follows: HPV18–5.5%, HPV16–13.2%, HPV11–6.4%, HPV6–17.4%. There were 443,948 bladder cancer cases. Among cases there was a higher prevalence of male gender (80% vs. 53%, p = 0.0056), Caucasian race (93% vs. 66%, p = 0.001), and HPV6 positive serology (18.1% vs. 17.6%, p = 0.0005). There was no difference among the other three HPV types. On logistic regression controlling for race, gender, and smoking status, increasing body weight (OR 1.01, p < 0.0001) and HPV6 positivity (OR 1.9, p < 0.0001) were associated with increased odds of bladder cancer diagnosis while HPV11 positivity (OR 0.23, p < 0.0001) was associated with a decreased odds of bladder cancer diagnosis. Conclusions: Prior research has shown that there is a relationship between HPV and bladder cancer, but this relationship has previously been incompletely defined. In this large, nationally representative database, we describe a significant association of HPV6 positive serology with a diagnosis of bladder cancer. This finding is striking and might affect future investigation into the pathophysiologic basis of this relationship.

Precision oncology requires delivering the right drug to the right patient at the right time, but “time” is rarely studied preclinically before a drug enters a particular clinical setting. Despite showing efficacy against recurrent/metastatic solid tumors, drugs sometimes fail to prevent tumor recurrence when given (neo)adjuvantly. The biology and microenvironments of overt tumors likely differ substantially from dormant cancer cells. The development of preclinical models to investigate clinically dormant disease will increase understanding of residual tumor cell biology and enable the development of therapeutics for rational adjuvant trials. Despite treatment with adjuvant anti-estrogen therapies, ˜30% of patients with ER+ breast cancer (BC) experience recurrence. In contrast to other BC subtypes, ER+ relapses occur late, often appearing years to decades after initial diagnosis and treatment. This delay suggests that ER+ BC cells can undergo extended periods of clinical dormancy. We developed novel, clinically relevant xenograft models of dormancy in ER+ BC. Low systemic levels of estrogens in mice can be further suppressed by ovariectomy, mimicking the effects of aromatase inhibitor (AI)-induced estrogen deprivation (ED) therapy. In ovariectomized mice, luciferase-labeled MCF-7, HCC-1428, HCC-1500, and MDA-MB-415 cells, as well as the HCI-017 PDX model, formed palpable orthotopic tumors upon 17b-estradiol supplementation. ED induced rapid tumor regression and decreased bioluminescent signal. However, a small proportion of ER+ BC cells survived ED for >1 year in a clinically dormant, growth-suppressed state. This residual cell population stabilized after ˜90 days of ED, as evidenced by stabilization of bioluminescent signal. Transcriptional and immunohistochemical analyses revealed significant upregulation of AMP-activated protein kinase (AMPK)-alpha-2 levels and activity in clinically dormant tumor cells compared to estrogen-driven or acutely ED xenografts. Dormant tumor cells were dependent upon AMPK activity for survival, as short-term pharmacologic inhibition of AMPK reduced residual bioluminescent signal. Metformin is an AMPK-activating drug approved for the treatment of diabetes. Metformin is currently being tested as an anti-cancer agent in many clinical trials at various points in the disease progression of diverse cancer types. In our models of clinically dormant ER+ BC, AMPK activation via metformin slowed ED-induced tumor regression, promoted residual tumor cell survival, and caused earlier tumor regrowth. In vitro studies indicated that metformin promotes cell survival during ED by enhancing fatty acid β-oxidation. Conversely, metformin treatment slowed estrogen-driven tumor growth, in agreement with prior observations that metformin slows growth of various tumor subtypes. These findings suggest that AMPK activation may be efficacious against growing tumors, but deleterious when used in combination with drugs that suppress tumor growth and induce regression. More broadly, this work highlights the issue that the time in a disease course needs to be considered when testing potential anti-cancer agents such as AMPK modulators. Citation Format: Miller TW, Hampsch RA, McCleery CF, Wells JD, Fields JL, Dillon LM, Shee K. Timing provides context for the paradoxical effects of AMPK activation in ER+ breast cancer: Suppressing growing tumors, but promoting dormant tumor cell survival and recurrence [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 P5-04-08.

Patients with rare tumors may lack approved treatments and clinical trial access. Although each rare tumor is uncommon, cumulatively they account for approximately 25% of cancers. We recently initiated a Rare Tumor Clinic that emphasized a precision medicine strategy.We investigated the first 40 patients presenting at the Rare Tumor Clinic. Next-generation sequencing (NGS) of tissue and plasma-derived, circulating-tumor DNA (ctDNA), and protein markers were assessed.Median age was 58 years (range, 31-78 years); 70% (28/40) were women; median number of previous systemic therapies was 2 (range 0-7). The most common diagnoses were sarcoma (Identifying genomic and protein markers in patients with rare/ultrarare tumors was feasible. When therapies were matched,50% of patients attained SD ≥6 months, PR, or CR. Further precision medicine clinical investigations focusing on rare and ultrarare tumors are urgently needed.Although rare tumors are infrequent by definition, when all subtypes of rare cancers are combined, they account for approximately 25% of adult malignancies. However, patients with rare tumors may lack approved treatments and clinical trial access. This paper describes an institutional a Rare Tumor Clinic focused on a precision medicine strategy. Performing genomics and protein analyses was feasible amongst patients with rare cancers. Over 50% of patients attained SD ≥6 months, PR, or CR when they received matched therapy (genomically targeted and/or immunotherapy). Further studies investigating the efficacy of the precision therapy approach among rare tumors are warranted.

Background: Circulating tumor DNA in plasma may present a minimally invasive approach to identify tumor-derived mutations that could be used to inform the selection of targeted therapies for individual patients, particularly in cases of metastatic disease where biopsy is often difficult. We hypothesized that plasma DNA will genetically reflect DNA derived from multiple tumors in patients with metastatic breast cancer. To test this hypothesis and assess the utility of plasma DNA obtained as a “liquid biopsy” for precision medicine, we sought to determine whether massively parallel sequencing of plasma DNA is a reliable surrogate for sequencing of DNA from tissue biopsies in patients with metastatic breast cancer. Methods: Blood samples were obtained from 7 patients with multiple advanced breast cancer lesions (recurrent breast and metastatic tumors), and tumor specimens were obtained thereafter by biopsy or surgical excision. DNA extracted from plasma, buffy coat of blood, and tumor tissues was used for probe-directed capture of all exons in 196 genes followed by massively parallel sequencing with an average coverage of 3000x for plasma DNA. Tumor and plasma DNA sequences were bioinformatically compared to buffy coat controls, and high-confidence somatic mutations were called. One patient with extensive metastatic disease was evaluated in further detail to study the contribution of different tumors to the overall plasma DNA pool. In this patient, 9 metastatic tumors were sampled in an axillary lymph node, heart, kidney (2), liver, omentum (3), and ovary by biopsy or at autopsy. Results: Mutations were found in plasma that were represented in one or more tumors in each patient. Three classes of mutations were discovered: 1) mutations overlapping between both plasma and tumors (e.g., TP53 p.R273C and SRC p.E527K); 2) mutations found in plasma but not tumors (e.g., AKT p.E17K and multiple known and novel ESR1 mutations); 3) mutations found in tumors but not plasma (e.g., PIK3CA p.H1047R, p.D350G, and p.N345K). The presence of mutations in each of these classes was validated in plasma and/or tumors using mutation-specific droplet digital PCR (ddPCR). In the patient with extensive metastatic disease, DNA sequencing revealed heterogeneity of tumor contribution to plasma DNA, with some tumors better represented than others. No correlation was found between tumor size (measured by CT scan) and mutational burden in plasma. Interestingly, a significant correlation was found between blood perfusion to the organ where the tumor resides and mutational burden in plasma, with the greatest tumor contribution coming from the heart metastasis (Pearson's r = 0.835, p=0.039). Conclusions: Plasma DNA sequencing adds a layer of depth to sequencing analysis of tumor biopsy samples, and serves to both confirm tumor-derived mutations as well as detect new mutations. However, plasma DNA profiling does not comprehensively reflect the mutational profiles of tumors in patients with metastatic breast cancer, and thus is unlikely to serve as a surrogate for tumor biopsy as a source of DNA for genetic profiling. Furthermore, plasma DNA contains many mutations not found in tumors, which will confound treatment decision-making and precision medicine.Background: Circulating tumor DNA in plasma may present a minimally invasive approach to identify tumor-derived mutations that could be used to inform the selection of targeted therapies for individual patients, particularly in cases of metastatic disease where biopsy is often difficult. We hypothesized that plasma DNA will genetically reflect DNA derived from multiple tumors in patients with metastatic breast cancer. To test this hypothesis and assess the utility of plasma DNA obtained as a “liquid biopsy” for precision medicine, we sought to determine whether massively parallel sequencing of plasma DNA is a reliable surrogate for sequencing of DNA from tissue biopsies in patients with metastatic breast cancer. Methods: Blood samples were obtained from 7 patients with multiple advanced breast cancer lesions (recurrent breast and metastatic tumors), and tumor specimens were obtained thereafter by biopsy or surgical excision. DNA extracted from plasma, buffy coat of blood, and tumor tissues was used for probe-directed capture of all exons in 196 genes followed by massively parallel sequencing with an average coverage of 3000x for plasma DNA. Tumor and plasma DNA sequences were bioinformatically compared to buffy coat controls, and high-confidence somatic mutations were called. One patient with extensive metastatic disease was evaluated in further detail to study the contribution of different tumors to the overall plasma DNA pool. In this patient, 9 metastatic tumors were sampled in an axillary lymph node, heart, kidney (2), liver, omentum (3), and ovary by biopsy or at autopsy. Results: Mutations were found in plasma that were represented in one or more tumors in each patient. Three classes of mutations were discovered: 1) mutations overlapping between both plasma and tumors (e.g., TP53 p.R273C and SRC p.E527K); 2) mutations found in plasma but not tumors (e.g., AKT p.E17K and multiple known and novel ESR1 mutations); 3) mutations found in tumors but not plasma (e.g., PIK3CA p.H1047R, p.D350G, and p.N345K). The presence of mutations in each of these classes was validated in plasma and/or tumors using mutation-specific droplet digital PCR (ddPCR). In the patient with extensive metastatic disease, DNA sequencing revealed heterogeneity of tumor contribution to plasma DNA, with some tumors better represented than others. No correlation was found between tumor size (measured by CT scan) and mutational burden in plasma. Interestingly, a significant correlation was found between blood perfusion to the organ where the tumor resides and mutational burden in plasma, with the greatest tumor contribution coming from the heart metastasis (Pearson's r = 0.835, p=0.039). Conclusions: Plasma DNA sequencing adds a layer of depth to sequencing analysis of tumor biopsy samples, and serves to both confirm tumor-derived mutations as well as detect new mutations. However, plasma DNA profiling does not comprehensively reflect the mutational profiles of tumors in patients with metastatic breast cancer, and thus is unlikely to serve as a surrogate for tumor biopsy as a source of DNA for genetic profiling. Furthermore, plasma DNA contains many mutations not found in tumors, which will confound treatment decision-making and precision medicine. Citation Format: Shee K, Chamberlin MD, Varn FS, Bean JR, Marotti JD, Wells WA, Trask HW, Hamilton JS, West RJ, Kaufman PA, Schwartz GN, Gemery JM, McNulty NJ, Tsapakos MJ, Barth RJ, Arrick BA, Gui J, Cheng C, Miller TW. Broken promise of liquid biopsy: Plasma DNA does not accurately reflect tumor DNA in metastatic breast cancer [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 P6-07-03.

Tissue growth and regeneration are autonomous, stem-cell-mediated processes in which stem cells within the organ self-renew and differentiate to create new cells, leading to new tissue. The processes of growth and regeneration require communication and interplay between neighboring cells. In particular, cell competition, which is a process in which viable cells are actively eliminated by more competitive cells, has been increasingly implicated to play an important role. Here, we discuss the existing literature regarding the current landscape of cell competition, including classical pathways and models, fitness fingerprint mechanisms, and immune system mechanisms of cell competition. We further discuss the clinical relevance of cell competition in the physiological processes of tissue growth and regeneration, highlighting studies in clinically important disease models, including oncological, neurological, and cardiovascular diseases.

As crucial regulators of cell motility, survival, and proliferation, Rac GTPases (Rac1/2/3) have been implicated in cancer. We previously found that P-REX1, a guanine nucleotide exchange factor that activates Rac GTPases, forms a PI3K-driven positive feedback loop to promote activation of Rac enzymes, receptor tyrosine kinases, PI3K/AKT/mTOR, and MEK/ERK in ER+ breast cancer cells. Importantly, inhibition of Rac GTPases with a small molecule (EHT1864) simultaneously suppressed activation of both the PI3K/AKT/mTOR and MEK/ERK pathways. While co-targeting of the PI3K/AKT/mTOR and MEK/ERK pathways with drug combinations has anti-tumor activity in preclinical models and is being tested in ongoing clinical trials, targeting Rac as a common upstream signaling node may be a more efficient means of simultaneously targeting these two oncogenic pathways. In breast cancer cell lines, treatment with EHT1864 decreased activation of AKT, mTOR, p70S6K, and S6 in a dose-dependent manner. Pulldown of activated Rac from cell lysates revealed that GTP-bound Rac1 and/or Rac3 bind MEK1/2, ERK1/2, p70S6K, S6, Raptor, Rictor, and mTOR. Temporal analysis indicated that EHT1864 inhibits phosphorylation of p70S6K (an mTORC1 substrate) before AKT is inhibited, suggesting that Rac may directly activate p70S6K and/or mTORC1. Mining of sensitivity data from ∼700 cell lines to a panel of 138 drugs (COSMIC/GDSC database) revealed that the growth-suppressive effects of EHT1864 correlate most strongly with growth-suppressive patterns induced by a p70S6K inhibitor, supporting the notion that Rac directly activates p70S6K. Additionally, EHT1864 treatment dose-dependently decreased phosphorylation of ERK1/2 and MEK1/2, and induced apoptosis in breast cancer cell lines. In a panel of 16 breast cancer lines, cells with activating mutations in PIK3CA (encodes the p110-alpha subunit of PI3K) exhibit increased EHT1864 sensitivity. Pharmacokinetic analysis of EHT1864 (100 mg/kg, i.p.) in plasma in NSG mice revealed that drug was present at >50 uM for 1 h after administration. In mice bearing s.c. ER+/HER2+ BT-474 breast cancer xenografts, EHT1864 (100 mg/kg BID) significantly slowed tumor growth compared to vehicle control. Thus, therapeutically targeting Rac could be an effective means of reducing cancer cell proliferation and survival by simultaneously suppressing both the PI3K/AKT/mTOR and MEK/ERK signaling pathways in ER+ and HER2+ breast cancers. Citation Format: Hampsch RA, Shee K, Miller TW. Therapeutic targeting of Rac GTPases in ER+ and HER2+ breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-01.

Drug resistance presents a challenge to the treatment of cancer patients. Many studies have focused on cell-autonomous mechanisms of drug resistance. By contrast, we proposed that the tumour micro-environment confers innate resistance to therapy. Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anticancer drugs. We found that stroma-mediated resistance is common, particularly to targeted agents. We characterized further the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibitors because most patients with this type of cancer show some degree of innate resistance. Proteomic analysis showed that stromal cell secretion of hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI(3)K)-AKT signalling pathways, and immediate resistance to RAF inhibition. Immunohistochemistry experiments confirmed stromal cell expression of HGF in patients with BRAF-mutant melanoma and showed a significant correlation between HGF expression by stromal cells and innate resistance to RAF inhibitor treatment. Dual inhibition of RAF and either HGF or MET resulted in reversal of drug resistance, suggesting RAF plus HGF or MET inhibitory combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. More generally, this study indicates that the systematic dissection of interactions between tumours and their micro-environment can uncover important mechanisms underlying drug resistance.

Drug resistance remains a vexing problem in the treatment of cancer patients. While many studies have focused on cell autonomous mechanisms of drug resistance, we hypothesized that the tumor microenvironment may confer innate resistance to therapy. We employed a novel co-culture assay to systematically assay the ability of 23 stromal cell types to influence the sensitivity of 45 cancer cell lines to 35 anti-cancer drugs testing more than 15,000 cancer-stroma-drug combinations. We found that stroma-mediated resistance is surprisingly common - particularly to targeted chemotherapies. We decided to further characterize the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibition because most patients exhibit some degree of innate resistance. Proteomic analysis showed that stromal secretion of the growth factor hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the MAPK and PI3K/AKT pathways, and immediate resistance to RAF inhibition. Immunohistochemistry confirmed tumor stromal HGF expression in 11 out of 15 patients with BRAF-mutant melanoma. Among these patients, only one experienced a complete response, and that patient was negative for stromal HGF, consistent with our prediction of HGF-mediated RAF-inhibitor resistance. In contrast, of 6 evaluable patients with stable disease 4 had particularly high levels of stromal HGF expression. Dual inhibition of RAF and MET in BRAF-mutant melanoma cell lines resulted in reversal of drug resistance, suggesting RAF/MET combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. Interestingly, the activation of MET in these cell lines was cell autonomous, caused, for example, by autocrine secretion of HGF and resulting in complete resistance to RAF inhibitors even in the absence of stromal cells. Treatment with a MET inhibitor re-sensitized these cell lines to RAF inhibitors suggesting the same RAF/MET combination therapy for non-melanoma BRAF mutant cancers as well. More generally, these studies indicate that the systematic dissection of tumor-microenvironment interactions may reveal important mechanisms underlying drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4837. doi:1538-7445.AM2012-4837