ERF mutations reveal a balance of ETS factors controlling prostate oncogenesis.

Half of all prostate cancers are caused by the TMPRSS2-ERG gene-fusion, which enables androgens to drive expression of the normally silent E26 transformation-specific (ETS) transcription factor ERG in prostate cells. Recent genomic landscape studies of such cancers have reported recurrent point mutations and focal deletions of another ETS member, the ETS2 repressor factor ERF. Here we show these ERF mutations cause decreased protein stability and mostly occur in tumours without ERG upregulation. ERF loss recapitulates the morphological and phenotypic features of ERG gain in normal mouse prostate cells, including expansion of the androgen receptor transcriptional repertoire, and ERF has tumour suppressor activity in the same genetic background of Pten loss that yields oncogenic activity by ERG. In the more common scenario of ERG upregulation, chromatin immunoprecipitation followed by sequencing indicates that ERG inhibits the ability of ERF to bind DNA at consensus ETS sites both in normal and in cancerous prostate cells. Consistent with a competition model, ERF overexpression blocks ERG-dependent tumour growth, and ERF loss rescues TMPRSS2-ERG-positive prostate cancer cells from ERG dependency. Collectively, these data provide evidence that the oncogenicity of ERG is mediated, in part, by competition with ERF and they raise the larger question of whether other gain-of-function oncogenic transcription factors might also inactivate endogenous tumour suppressors.

CDK9-mediated transcription elongation is required for MYC addiction in hepatocellular carcinoma.

One-year survival rates for newly diagnosed hepatocellular carcinoma (HCC) are <50%, and unresectable HCC carries a dismal prognosis owing to its aggressiveness and the undruggable nature of its main genetic drivers. By screening a custom library of shRNAs directed toward known drug targets in a genetically defined Myc-driven HCC model, we identified cyclin-dependent kinase 9 (Cdk9) as required for disease maintenance. Pharmacological or shRNA-mediated CDK9 inhibition led to robust anti-tumor effects that correlated with MYC expression levels and depended on the role that both CDK9 and MYC exert in transcription elongation. Our results establish CDK9 inhibition as a therapeutic strategy for MYC-overexpressing liver tumors and highlight the relevance of transcription elongation in the addiction of cancer cells to MYC.

Applying 89Zr-Transferrin To Study the Pharmacology of Inhibitors to BET Bromodomain Containing Proteins.

Chromatin modifying proteins are attractive drug targets in oncology, given the fundamental reliance of cancer on altered transcriptional activity. Multiple transcription factors can be impacted downstream of primary target inhibition, thus making it challenging to understand the driving mechanism of action of pharmacologic inhibition of chromatin modifying proteins. This in turn makes it difficult to identify biomarkers predictive of response and pharmacodynamic tools to optimize drug dosing. In this report, we show that (89)Zr-transferrin, an imaging tool we developed to measure MYC activity in cancer, can be used to identify cancer models that respond to broad spectrum inhibitors of transcription primarily due to MYC inhibition. As a proof of concept, we studied inhibitors of BET bromodomain containing proteins, as they can impart antitumor effects in a MYC dependent or independent fashion. In vitro, we show that transferrin receptor biology is inhibited in multiple MYC positive models of prostate cancer and double hit lymphoma when MYC biology is impacted. Moreover, we show that bromodomain inhibition in one lymphoma model results in transferrin receptor expression changes large enough to be quantified with (89)Zr-transferrin and positron emission tomography (PET) in vivo. Collectively, these data further underscore the diagnostic utility of the relationship between MYC and transferrin in oncology, and provide the rationale to incorporate transferrin-based PET into early clinical trials with bromodomain inhibitors for the treatment of solid tumors.

Cabozantinib resolves bone scans in tumor-naïve mice harboring skeletal injuries.

The receptor tyrosine kinase inhibitor cabozantinib (XL184, BMS-907351 Cometriq) has displayed impressive clinical activity against several indications, culminating in its recent approval for medullary thyroid cancer. Among malignancies with tropism for the bone (prostate, breast), one striking feature of early clinical reports about this drug has been the rapid and complete resolution of bone scans, a phenomenon almost never observed even among therapies already shown to confer survival benefit. In castration-resistant prostate cancer, not all conventional response indicators change as dramatically posttreatment, raising the possibility that cabozantinib may impair the ability of bone-seeking radionuclides to integrate within the remodeling bone. To test this hypothesis, we surgically induced bone remodeling via physical insult in non-tumor-bearing mice and performed 18F-sodium fluoride (18F-NaF) positron emission tomographic (PET) and technetium 99m-methylene diphosphonate (99mTc-MDP) single-photon emission computed tomographic (SPECT) scans pre- and posttreatment with cabozantinib and related inhibitors. A consistent reduction in the accumulation of either radiotracer at the site of bone remodeling was observed in animals treated with cabozantinib. Given that cabozantinib is known to inhibit several receptor tyrosine kinases, we drugged animals with various permutations of more selective inhibitors to attempt to refine the molecular basis of bone scan resolution. Neither the vascular endothelial growth factor receptor (VEGFR) inhibitor axitinib, the MET inhibitor crizotinib, nor the combination was capable of inhibiting 18F-NaF accumulation at known bioactive doses. In summary, although the mechanism by which cabozantinib suppresses radionuclide incorporation into foci undergoing bone remodeling remains unknown, that this phenomenon occurs in tumor-naïve models indicates that caution should be exercised in interpreting the clinical significance of this event.

Utility of FDG-PET in clinical neuroendocrine prostate cancer.

BACKGROUND: Fluorodeoxyglucose (FDG) positron emission tomography (PET) has well-characterized limitations in prostate adenocarcinoma (PCA). However, data assessing the utility of PET in neuroendocrine prostate cancer (NEPC) is limited to isolated case reports. Herein, we describe the first case series to assess the utility of FDG-PET in NEPC. METHODS: Inclusion criteria consisted of clinically progressive metastatic PCA in the setting of a chromogranin-A levels >1.5× the upper limit of normal, and ≥1 FDG-PET scan after the diagnosis of NEPC, which yielded 23 patients. All metastatic lesions on CT, PET, and bone scan were read by two independent physicians. RESULTS: Five hundred ninety two unique lesions were identified across all imaging modalities, 510 were bone metastases, and 82 were soft tissue metastases. Of bone lesions, 22.2%, 92.7%, and 77.6% were detected by PET, CT, and bone scan, respectively. Of soft tissue lesions, 95.1% and 97.5% were detected by PET and CT, respectively. Stratified by the median survival from NEPC diagnosis, patients who survived <2.2 versus ≥2.2 years had more PET avid bone (8 vs. 2, P = 0.06) and soft tissue lesions (7 vs. 1, P = 0.01), and higher average SUVmax of bone (5.49 vs. 3.40, P = 0.04) and soft tissue lesions (8.02 vs. 3.90, P = 0.0002). CONCLUSIONS: In patients with clinical NEPC, we demonstrate that FDG-PET has clinical utility in the detection of metastatic disease. In addition to detection, PET allows for treatment response to determine tumor viability. With novel therapies on the horizon to treat NEPC, consideration to investigate the use of FDG-PET to monitor response is warranted.

Pairwise comparison of 89Zr- and 124I-labeled cG250 based on positron emission tomography imaging and nonlinear immunokinetic modeling: in vivo carbonic anhydrase IX receptor binding and internalization in mouse xenografts of clear-cell renal cell carcinoma.

PURPOSE: The PET tracer, (124)I-cG250, directed against carbonic anhydrase IX (CAIX) shows promise for presurgical diagnosis of clear-cell renal cell carcinoma (ccRCC) (Divgi et al. in Lancet Oncol 8:304-310, 2007; Divgi et al. in J Clin Oncol 31:187-194, 2013). The radiometal (89)Zr, however, may offer advantages as a surrogate PET nuclide over (124)I in terms of greater tumor uptake and retention (Rice et al. in Semin Nucl Med 41:265-282, 2011). We have developed a nonlinear immunokinetic model to facilitate a quantitative comparison of absolute uptake and antibody turnover between (124)I-cG250 and (89)Zr-cG250 using a human ccRCC xenograft tumor model in mice. We believe that this unique model better relates quantitative imaging data to the salient biological features of tumor antibody-antigen binding and turnover. METHODS: We conducted experiments with (89)Zr-cG250 and (124)I-cG250 using a human ccRCC cell line (SK-RC-38) to characterize the binding affinity and internalization kinetics of the two tracers in vitro. Serial PET imaging was performed in mice bearing subcutaneous ccRCC tumors to simultaneously detect and quantify time-dependent tumor uptake in vivo. Using the known specific activities of the two tracers, the equilibrium rates of antibody internalization and turnover in the tumors were derived from the PET images using nonlinear compartmental modeling. RESULTS: The two tracers demonstrated virtually identical tumor cell binding and internalization but showed markedly different retentions in vitro. Superior PET images were obtained using (89)Zr-cG250, owing to the more prolonged trapping of the radiolabel in the tumor and simultaneous washout from normal tissues. Estimates of cG250/CAIX complex turnover were 1.35 - 5.51 × 10(12) molecules per hour per gram of tumor (20 % of receptors internalized per hour), and the ratio of (124)I/(89)Zr atoms released per unit time by tumor was 17.5. CONCLUSION: Pairwise evaluation of (89)Zr-cG250 and (124)I-cG250 provided the basis for a nonlinear immunokinetic model which yielded quantitative information about the binding and internalization of radioantibody bound to CAIX on tumor cells in vivo. (89)Zr-cG250 is likely to provide high-quality PET images and may be a useful tool to quantify CAIX/cG250 receptor turnover and cG250-accessible antigen density noninvasively in humans.

Noninvasive Imaging of PSMA in prostate tumors with (89)Zr-Labeled huJ591 engineered antibody fragments: the faster alternatives.

Engineered antibody fragments offer faster delivery with retained tumor specificity and rapid clearance from nontumor tissues. Here, we demonstrate that positron emission tomography (PET) based detection of prostate specific membrane antigen (PSMA) in prostatic tumor models using engineered bivalent antibodies built on single chain fragments (scFv) derived from the intact antibody, huJ591, offers similar tumor delineating properties but with the advantage of rapid targeting and imaging. (89)Zr-radiolabeled huJ591 scFv (dimeric scFv-CH3; (89)Zr-Mb) and cysteine diabodies (dimeric scFv; (89)Zr-Cys-Db) demonstrated internalization and similar Kds (∼2 nM) compared to (89)Zr-huJ591 in PSMA(+) cells. Tissue distribution assays established the specificities of both (89)Zr-Mb and (89)Zr-Cys-Db for PSMA(+) xenografts (6.2 ± 2.5% ID/g and 10.2 ± 3.4% ID/g at 12 h p.i. respectively), while minimal accumulation in PSMA(-) tumors was observed. From the PET images, (89)Zr-Mb and (89)Zr-Cys-Db exhibited faster blood clearance than the parent huJ591 while tumor-to-muscle ratios for all probes show comparable values across all time points. Ex vivo autoradiography and histology assessed the distribution of the probes within the tumor. Imaging PSMA-expressing prostate tumors with smaller antibody fragments offers rapid tumor accumulation and accelerated clearance; hence, shortened wait periods between tracer administration and high-contrast tumor imaging and lower dose-related toxicity are potentially realized.

Results of photon radiotherapy for unresectable salivary gland tumors: is neutron radiotherapy's local control superior?

BACKGROUND: The results of RTOG-MRC randomized trial of photon (n=15) versus neutron (n=17) therapy in the 1980's reported an improved local control (LC) with neutron radiotherapy for unresectable salivary gland tumors. Due to increased severe toxicity with neutron radiotherapy and the paucity of neutron-therapy centers, we analyzed our institution's results of photon radiotherapy for unresectable salivary gland tumors. PATIENTS AND METHODS: From 1990 to 2009, 27 patients with unresectable salivary gland cancer underwent definitive photon radiotherapy at our institution. Nodal involvement on presentation was found in 9 patients. Median dose of radiotherapy was 70 Gy. Chemotherapy was given to 18 patients, most being platinum-based regimens. Local control (LC), locoregional control (LRC), distant metastasis-free survival (DMFS), overall survival (OS), and toxicity outcomes were assessed. RESULTS: With a median follow-up of 52.4 months, the 2/5-year actuarial LC was 69% (95%CI ± 21.0%)/55% (± 24.2%), LRC was 65% (± 21.4%)/47% (± 21.6%), and DMFS was 71% (± 21.8%)/51% (± 22.8%), respectively using competing risk analysis. The median OS was 25.7 months, and the 2/5-year OS rates were 50% (± 19.0%)/29% (± 16.6%), respectively. Higher histologic grade was significant for an increased rate of DM (intermediate grade vs. low grade, p=0.04, HR 7.93; high grade vs. low grade, p=0.01, HR 13.50). Thirteen (48%) patient's experienced acute grade 3 toxicity. Late grade 3 toxicity occurred in three (11%) patients. CONCLUSIONS: Our data compares favorably to neutron radiotherapy with fewer late complications. Photon radiotherapy is an acceptable alternative to neutron radiotherapy in patients who present with unresectable salivary gland tumors.

Orthopedic Training in the United States A Continuously Evolving Process.

Orthopedic surgery in the United States has gone through many changes over the past few centuries. Starting with a small sect of subspecialized surgeons, advances in technology and surgical skills have paralleled the growth of the specialty. To keep up with demand, the training of orthopedic surgeons has undergone many iterations. From apprenticeships to the current residency model, the field has always adapted to ensure the constant production of well-trained surgeons to take care of the growing orthopedic needs in the population. In order to guarantee this, many regulatory committees have been formed over the years to help guide the regulation and certification of orthopedic training programs. With current day residents facing new challenges, the specialty continues to adapt the way it trains its future.

A Novel Machine Learning Predictive Tool Assessing Outpatient or Inpatient Designation for Medicare Patients Undergoing Total Hip Arthroplasty.

BACKGROUND: The Centers for Medicare and Medicaid Services removed total hip arthroplasty (THA) from the inpatient-only list. This has created significant confusion regarding which patients qualify for an inpatient designation. The purpose of this study is to develop and validate a novel predictive tool for preoperatively objectively determining "outpatient" vs "inpatient" status for THA in the Medicare population. METHODS: A cohort of Medicare patients undergoing primary THA between January 2017 and September 2019 was retrospectively reviewed. A machine learning model was trained using 80% of the THA patients, and the remaining 20% was used for testing the model performance in terms of accuracy and the average area under the receiver operating characteristic curve. Feature importance was obtained for each feature used in the model. RESULTS: One thousand ninety-one patients had outpatient stays, and 318 qualified for inpatient designation. Significant associations were demonstrated between inpatient designations and the following: higher BMI, increased patient age, better preoperative functional scores, higher American Society of Anesthesiologist Physical Status Classification, higher Modified Frailty Index, higher Charlson Comorbidity Index, female gender, and numerous comorbidities. The XGBoost model for predicting an inpatient or outpatient stay was 78.7% accurate with the area under the receiver operating characteristic curve to be 81.5%. CONCLUSIONS: Using readily available key baseline characteristics, functional scores and comorbidities, this machine-learning model accurately predicts an "outpatient" or "inpatient" stay after THA in the Medicare population. BMI, age, functional scores, and American Society of Anesthesiologist Physical Status Classification had the highest influence on this predictive model.

Internalization of secreted antigen-targeted antibodies by the neonatal Fc receptor for precision imaging of the androgen receptor axis.

Targeting the androgen receptor (AR) pathway prolongs survival in patients with prostate cancer, but resistance rapidly develops. Understanding this resistance is confounded by a lack of noninvasive means to assess AR activity in vivo. We report intracellular accumulation of a secreted antigen-targeted antibody (SATA) that can be used to characterize disease, guide therapy, and monitor response. AR-regulated human kallikrein-related peptidase 2 (free hK2) is a prostate tissue-specific antigen produced in prostate cancer and androgen-stimulated breast cancer cells. Fluorescent and radio conjugates of 11B6, an antibody targeting free hK2, are internalized and noninvasively report AR pathway activity in metastatic and genetically engineered models of cancer development and treatment. Uptake is mediated by a mechanism involving the neonatal Fc receptor. Humanized 11B6, which has undergone toxicological tests in nonhuman primates, has the potential to improve patient management in these cancers. Furthermore, cell-specific SATA uptake may have a broader use for molecularly guided diagnosis and therapy in other cancers.

Androgen Receptor Upregulation Mediates Radioresistance after Ionizing Radiation.

Clinical trials have established the benefit of androgen deprivation therapy (ADT) combined with radiotherapy in prostate cancer. ADT sensitizes prostate cancer to radiotherapy-induced death at least in part through inhibition of DNA repair machinery, but for unknown reasons, adjuvant ADT provides further survival benefits. Here, we show that androgen receptor (AR) expression and activity are durably upregulated following radiotherapy in multiple human prostate cancer models in vitro and in vivo. Moreover, the degree of AR upregulation correlates with survival in vitro and time to tumor progression in animal models. We also provide evidence of AR pathway upregulation, measured by a rise in serum levels of AR-regulated hK2 protein, in nearly 20% of patients after radiotherapy. Furthermore, these men were three-fold more likely to experience subsequent biochemical failure. Collectively, these data demonstrate that radiotherapy can upregulate AR signaling after therapy to an extent that negatively affects disease progression and/or survival.

Annotating STEAP1 regulation in prostate cancer with 89Zr immuno-PET.

UNLABELLED: Antibodies and antibody-drug conjugates targeting the cell surface protein 6 transmembrane epithelial antigen of prostate 1 (STEAP1) are in early clinical development for the treatment of castration-resistant prostate cancer (PCa). In general, antigen expression directly affects the bioactivity of therapeutic antibodies, and the biologic regulation of STEAP1 is unusually complicated in PCa. Paradoxically, STEAP1 can be induced or repressed by the androgen receptor (AR) in different human PCa models, while also expressed in AR-null PCa. Consequently, there is an urgent need to translate diagnostic strategies to establish which regulatory mechanism predominates in patients to situate the appropriate therapy within standard of care therapies inhibiting AR. METHODS: To this end, we prepared and evaluated (89)Zr-labeled MSTP2109A ((89)Zr-2109A), a radiotracer for PET derived from a fully humanized monoclonal antibody to STEAP1 in preclinical PCa models. RESULTS: (89)Zr-2109A specifically localized to the STEAP1-positive human PCa models CWR22Pc, 22Rv1, and PC3. Moreover, (89)Zr-2109A sensitively measured treatment-induced changes (∼66% decline) in STEAP1 expression in CWR22PC in vitro and in vivo, a model we showed to express STEAP1 in an AR-dependent manner. CONCLUSION: These findings highlight the ability of immuno-PET with (89)Zr-2109A to detect acute changes in STEAP1 expression and argue for an expansion of ongoing efforts to image PCa patients with (89)Zr-2109A to maximize the clinical benefit associated with antibodies or antibody-drug conjugates to STEAP1.

Non-invasive mapping of deep-tissue lymph nodes in live animals using a multimodal PET/MRI nanoparticle.

The invasion status of tumour-draining lymph nodes (LNs) is a critical indicator of cancer stage and is important for treatment planning. Clinicians currently use planar scintigraphy and single-photon emission computed tomography (SPECT) with (99m)Tc-radiocolloid to guide biopsy and resection of LNs. However, emerging multimodality approaches such as positron emission tomography combined with magnetic resonance imaging (PET/MRI) detect sites of disease with higher sensitivity and accuracy. Here we present a multimodal nanoparticle, (89)Zr-ferumoxytol, for the enhanced detection of LNs with PET/MRI. For genuine translational potential, we leverage a clinical iron oxide formulation, altered with minimal modification for radiolabelling. Axillary drainage in naive mice and from healthy and tumour-bearing prostates was investigated. We demonstrate that (89)Zr-ferumoxytol can be used for high-resolution tomographic studies of lymphatic drainage in preclinical disease models. This nanoparticle platform has significant translational potential to improve preoperative planning for nodal resection and tumour staging.

Imaging tumor burden in the brain with 89Zr-transferrin.

UNLABELLED: A noninvasive technology that indiscriminately detects tumor tissue in the brain could substantially enhance the management of primary or metastatic brain tumors. Although the documented molecular heterogeneity of diseases that initiate or eventually deposit in the brain may preclude identifying a single smoking-gun molecular biomarker, many classes of brain tumors are generally avid for transferrin. Therefore, we reasoned that applying a radiolabeled derivative of transferrin ((89)Zr-labeled transferrin) may be an effective strategy to more thoroughly identify tumor tissue in the brain, regardless of the tumor's genetic background. METHODS: Transferrin was radiolabeled with (89)Zr, and its properties with respect to human models of glioblastoma multiforme were studied in vivo. RESULTS: In this report, we show proof of concept that (89)Zr-labeled transferrin ((89)Zr-transferrin) localizes to genetically diverse models of glioblastoma multiforme in vivo. Moreover, we demonstrate that (89)Zr-transferrin can detect an orthotopic lesion with exceptional contrast. Finally, the tumor-to-brain contrast conferred by (89)Zr-transferrin vastly exceeded that observed with (18)F-FDG, currently the most widely used radiotracer to assess tumor burden in the brain. CONCLUSION: The results from this study suggest that (89)Zr-transferrin could be a broadly applicable tool for identifying and monitoring tumors in the brain, with realistic potential for near-term clinical translation.