Impact of pre-analytical variables on deep learning accuracy in histopathology.

AIMS: Machine learning (ML) binary classification in diagnostic histopathology is an area of intense investigation. Several assumptions, including training image quality/format and the number of training images required, appear to be similar in many studies irrespective of the paucity of supporting evidence. We empirically compared training image file type, training set size, and two common convolutional neural networks (CNNs) using transfer learning (ResNet50 and SqueezeNet). METHODS AND RESULTS: Thirty haematoxylin and eosin (H&E)-stained slides with carcinoma or normal tissue from three tissue types (breast, colon, and prostate) were photographed, generating 3000 partially overlapping images (1000 per tissue type). These lossless Portable Networks Graphics (PNGs) images were converted to lossy Joint Photographic Experts Group (JPG) images. Tissue type-specific binary classification ML models were developed by the use of all PNG or JPG images, and repeated with a subset of 500, 200, 100, 50, 30 and 10 images. Eleven models were generated for each tissue type, at each quantity of training images, for each file type, and for each CNN, resulting in 924 models. Internal accuracies and generalisation accuracies were compared. There was no meaningful significant difference in accuracies between PNG and JPG models. Models trained with more images did not invariably perform better. ResNet50 typically outperformed SqueezeNet. Models were generalisable within a tissue type but not across tissue types. CONCLUSIONS: Lossy JPG images were not inferior to lossless PNG images in our models. Large numbers of unique H&E-stained slides were not required for training optimal ML models. This reinforces the need for an evidence-based approach to best practices for histopathological ML.

Probabilistic modeling of personalized drug combinations from integrated chemical screen and molecular data in sarcoma.

BACKGROUND: Cancer patients with advanced disease routinely exhaust available clinical regimens and lack actionable genomic medicine results, leaving a large patient population without effective treatments options when their disease inevitably progresses. To address the unmet clinical need for evidence-based therapy assignment when standard clinical approaches have failed, we have developed a probabilistic computational modeling approach which integrates molecular sequencing data with functional assay data to develop patient-specific combination cancer treatments. METHODS: Tissue taken from a murine model of alveolar rhabdomyosarcoma was used to perform single agent drug screening and DNA/RNA sequencing experiments; results integrated via our computational modeling approach identified a synergistic personalized two-drug combination. Cells derived from the primary murine tumor were allografted into mouse models and used to validate the personalized two-drug combination. Computational modeling of single agent drug screening and RNA sequencing of multiple heterogenous sites from a single patient's epithelioid sarcoma identified a personalized two-drug combination effective across all tumor regions. The heterogeneity-consensus combination was validated in a xenograft model derived from the patient's primary tumor. Cell cultures derived from human and canine undifferentiated pleomorphic sarcoma were assayed by drug screen; computational modeling identified a resistance-abrogating two-drug combination common to both cell cultures. This combination was validated in vitro via a cell regrowth assay. RESULTS: Our computational modeling approach addresses three major challenges in personalized cancer therapy: synergistic drug combination predictions (validated in vitro and in vivo in a genetically engineered murine cancer model), identification of unifying therapeutic targets to overcome intra-tumor heterogeneity (validated in vivo in a human cancer xenograft), and mitigation of cancer cell resistance and rewiring mechanisms (validated in vitro in a human and canine cancer model). CONCLUSIONS: These proof-of-concept studies support the use of an integrative functional approach to personalized combination therapy prediction for the population of high-risk cancer patients lacking viable clinical options and without actionable DNA sequencing-based therapy.

Synaptophysin-Ki67 double stain: a novel technique that improves interobserver agreement in the grading of well-differentiated gastrointestinal neuroendocrine tumors.

A common problem in the assessment of Ki67 proliferative index in well-differentiated gastrointestinal neuroendocrine tumors is distinguishing tumor from non-tumor. This is because background stromal lymphocytes, entrapped non-neoplastic glands, and the delicate vascular network characteristic of neuroendocrine tumors frequently contain a subset of proliferating cells. Furthermore, in small biopsies, crush and cautery artifact can alter the morphologic appearance of tumor cells, making the Ki67 proliferative index more difficult to assess. To address these issues, we developed a synaptophysin-Ki67 double stain using a commercially available immunohistochemistry kit, allowing simultaneous visualization of tumor and proliferating nuclei. To test this method, three gastrointestinal pathologists individually graded 50 gastrointestinal neuroendocrine tumors first using synaptophysin-Ki67 double-stained slides and then, after a washout period, using Ki67-only stained slides (along with routine hematoxylin- and eosin-stained slides). Interobserver agreement on Ki67 proliferative index was moderate using the Ki67-only stained slides (intraclass correlation 0.51, 95% confidence interval: 0.35-0.66) and improved using the synaptophysin-Ki67 double stain (intraclass correlation 0.79, 95% confidence interval: 0.69-0.86). Similarly, interobserver agreement on tumor grade was fair with Ki67-only stained slides (κ=0.39, P<0.001) and improved with the double stain (κ=0.58, P<0.001). Analysis of individual pathologists' scores revealed that fewer total number of tumor cells counted correlated with higher grade designation and appeared to contribute to grade discordance. In tumors cited as particularly challenging to assess by the pathologists, three of four tumors were grade discordant with the Ki67-only stain, whereas all four tumors were grade concordant with the synaptophysin-Ki67 stain. The synaptophysin-Ki67 double stain is the first technique to address specifically the histomorphologic challenges of evaluating Ki67 proliferative index in well-differentiated gastrointestinal neuroendocrine tumors. Although further validation is needed, this study provides evidence that the synaptophysin-Ki67 double stain can improve interobserver agreement.

Lin28 promotes growth of prostate cancer cells and activates the androgen receptor.

Prostate cancer (CaP) progresses to a castration-resistant state assisted by multifold molecular changes, most of which involve activation of the androgen receptor (AR). Having previously demonstrated the importance of the Lin28/let-7/Myc axis in CaP, we tested the hypothesis that Lin28 is overexpressed in CaP and that it activates AR and promotes growth of CaP cells. We analyzed human clinical CaP samples for the expression of Lin28 by quantitative real-time RT-PCR, Western blot analysis, and IHC. Growth characteristics of CaP cell lines transiently and stably expressing Lin28 were examined. The clonogenic ability of CaP cells expressing Lin28 was determined by colony formation and soft agar assays. Increase in expression of AR and subsequent increase in transcription of AR-target genes were analyzed by quantitative real-time RT-PCR, luciferase assays, and ELISA. LNCaP cells stably expressing Lin28 were injected into nude mice, and tumorigenesis was monitored. We found that Lin28 is overexpressed in clinical CaP compared to benign prostates. Overexpression of Lin28 enhanced, while down-regulation reduced, growth of CaP cells. Lin28 enhanced the ability of CaP cells to form colonies in anchorage-dependent and anchorage-independent conditions. LNCaP cells stably expressing Lin28 exhibited significantly higher tumorigenic ability in vivo. Lin28 induced expression of the AR and its target genes such as PSA and NKX3.1. Collectively, our findings demonstrate a novel role for Lin28 in CaP development and activation of the AR axis.

Optical molecular imaging detects changes in extracellular pH with the development of head and neck cancer.

Noninvasive localized measurement of extracellular pH in cancer tissues can have a significant impact on the management of cancer. Despite its significance, there are limited approaches for rapid and noninvasive measurement of local pH in a clinical environment. In this study, we demonstrate the potential of noninvasive topical delivery of Alexa-647 labeled pHLIP (pH responsive peptide conjugated with Alexa Fluor(®) 647) to image changes in extracellular pH associated with head and neck squamous cell carcinoma using widefield and high resolution imaging. We report a series of preclinical analyses to evaluate the optical contrast achieved after topical delivery of Alexa-647 labeled pHLIP in intact fresh human tissue specimens using widefield and high-resolution fluorescence imaging. Using topical delivery, Alexa-647 labeled pHLIP can be rapidly delivered throughout the epithelium of intact tissues with a depth exceeding 700 µm. Following labeling with Alexa-647 labeled pHLIP, the mean fluorescent contrast increased four to eight fold higher in clinically abnormal tissues as compared to paired clinically normal biopsies. Furthermore, the imaging approach showed significant differences in fluorescence contrast between the cancer and the normal biopsies across diverse patients and different anatomical sites (unpaired comparison). The fluorescence contrast differences between clinically abnormal and normal tissues were in agreement with the pathologic evaluation. Topical application of fluorescently labeled pHLIP can detect and differentiate normal from cancerous tissues using both widefield and high resolution imaging. This technology will provide an effective tool to assess tumor margins during surgery and improve detection and prognosis of head and neck cancer.

Ultrasound backscatter microscopy for imaging of oral carcinoma.

OBJECTIVES: Ultrasound backscatter microscopy (UBM), or ultrasound biomicroscopy, is a noninvasive, label-free, and ionizing radiation-free technique allowing high-resolution 3-dimensional structural imaging. The goal of this study was to evaluate UBM for resolving anatomic features associated with squamous cell carcinoma of the oral cavity. METHODS: The study was conducted in a hamster buccal pouch model. A carcinogen was topically applied to cheeks of 14 golden Syrian hamsters. Six additional hamsters served as healthy controls. A high-frequency (41 MHz, 6-mm focal depth, lateral and axial resolutions of 65 and 37 µm, respectively) UBM system was used for scanning the oral cavity after 14 weeks of carcinogen application. Histologic analyses were conducted on scanned regions. RESULTS: The histologic structure of buccal tissue and microvasculature networks could be visualized from the UBM images. Epithelial and mucosal hypertrophy and neoplastic changes were identified in animals subjected to the carcinogen. In animals with invasive squamous cell carcinoma, lesion development and destruction of the structural integrity of tissue layers were noted. CONCLUSIONS: In this pilot study, UBM generated sufficient contrast for morphologic features associated with oral carcinoma compared to healthy tissue. This modality may present a practical technique for detection of oral neoplasms that is potentially translatable to humans.

Improved clinical outcomes with multi-modality therapy for sinonasal undifferentiated carcinoma of the head and neck.

OBJECTIVE: To examine outcomes among patients treated for sinonasal undifferentiated carcinoma (SNUC) of the head and neck. STUDY DESIGN: Retrospective review. METHODS: The records of 16 consecutive patients with newly diagnosed, non-metastatic SNUC were analyzed. Initial treatment consisted of: surgery alone (6 patients), surgery with post-operative chemoradiotherapy (4 patients), and primary radiation therapy with concurrent chemotherapy (6 patients). RESULTS: The median survival for patients treated by surgery followed by postoperative chemoradiotherapy was 30 months compared to 7 months and 9 months for patients treated by surgery alone and upfront chemoradiotherapy, respectively (p=0.20). The 2-year locoregional control was 18% for patients treated with upfront chemoradiotherapy, 37% for patients treated with surgery alone, and 78% for patients treated with surgery plus chemoradiotherapy (p=0.49). CONCLUSION: While the potential role of selection bias must be considered, multi-modality therapy using surgery and post-operative chemoradiotherapy yielded the most favorable outcomes for SNUC and should be recommended whenever feasible.

Co-clinical Modeling of the Activity of the BET Inhibitor Mivebresib (ABBV-075) in AML.

BACKGROUND/AIM: The therapeutic potential of bromodomain and extra-terminal motif (BET) inhibitors in hematological cancers has been well established in preclinical and early-stage clinical trials, although as of yet, no BETtargeting agent has achieved approval. To add insight into potential response to mivebresib (ABBV-075), a broadspectrum BET inhibitor, co-clinical modeling of individual patient biopsies was conducted in the context of a Phase I trial in acute myeloid leukemia (AML). MATERIALS AND METHODS: Co-clinical modeling involves taking the patient's biopsy and implanting it in mice with limited passage so that it closely retains the original characteristics of the malignancy and allows comparisons of response between animal model and clinical data. Procedures were developed, initially with neonate NOD/Shi-scid-IL2rγnull (NOG) mice and then optimized with juvenile NOG-EXL as host mice, eventually resulting in a robust rate of engraftment (16 out of 26, 62%). RESULTS: Results from the co-clinical AML patient-derived xenograft (PDX) modeling (6 with >60% inhibition of bone marrow blasts) were consistent with the equivalent clinical data from patients receiving mivebresib in monotherapy, and in combination with venetoclax. The modeling system also demonstrated the activity of a novel BD2-selective BET inhibitor (ABBV-744) in the preclinical AML setting. Both agents were also highly effective in inhibiting blast counts in the spleen (10/10 and 5/6 models, respectively). CONCLUSION: These findings confirm the validity of the model system in the co-clinical setting, establish highly relevant in vivo models for the discovery of cancer therapy, and indicate the therapeutic value of BET inhibitors for AML and, potentially, myelofibrosis treatment.

A Genomically and Clinically Annotated Patient-Derived Xenograft Resource for Preclinical Research in Non-Small Cell Lung Cancer.

Patient-derived xenograft (PDX) models are an effective preclinical in vivo platform for testing the efficacy of novel drugs and drug combinations for cancer therapeutics. Here we describe a repository of 79 genomically and clinically annotated lung cancer PDXs available from The Jackson Laboratory that have been extensively characterized for histopathologic features, mutational profiles, gene expression, and copy-number aberrations. Most of the PDXs are models of non-small cell lung cancer (NSCLC), including 37 lung adenocarcinoma (LUAD) and 33 lung squamous cell carcinoma (LUSC) models. Other lung cancer models in the repository include four small cell carcinomas, two large cell neuroendocrine carcinomas, two adenosquamous carcinomas, and one pleomorphic carcinoma. Models with both de novo and acquired resistance to targeted therapies with tyrosine kinase inhibitors are available in the collection. The genomic profiles of the LUAD and LUSC PDX models are consistent with those observed in patient tumors from The Cancer Genome Atlas and previously characterized gene expression-based molecular subtypes. Clinically relevant mutations identified in the original patient tumors were confirmed in engrafted PDX tumors. Treatment studies performed in a subset of the models recapitulated the responses expected on the basis of the observed genomic profiles. These models therefore serve as a valuable preclinical platform for translational cancer research. SIGNIFICANCE: Patient-derived xenografts of lung cancer retain key features observed in the originating patient tumors and show expected responses to treatment with standard-of-care agents, providing experimentally tractable and reproducible models for preclinical investigations.

Clinical emergence of neurometastatic merkel cell carcinoma: a surgical case series and literature review.

Merkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine neoplasm of possible viral origin and is known for its aggressive behavior. The incidence of MCC has increased in the last 15 years. Merkel cell carcinoma has the potential to metastasize, but rarely involves the central nervous system. Herein, we report three consecutive surgical cases of MCC presenting at a single institution within 1 year. We used intracavitary BCNU wafers (Gliadel(®)) in two cases. Pathological features, including CK20 positivity, consistent with MCC, were present in all cases. We found 33 published cases of MCC with CNS involvement. We suggest that the incidence of neurometastatic MCC may be increasing, parallel to the increasing incidence of primary MCC. We propose a role for intracavitary BCNU wafers in the treatment of intra-axial neurometastatic MCC.

Novel Patient Metastatic Pleural Effusion-Derived Xenograft Model of Renal Medullary Carcinoma Demonstrates Therapeutic Efficacy of Sunitinib.

BACKGROUND: Renal medullary carcinoma (RMC) is a rare but aggressive tumor often complicated by early lung metastasis with few treatment options and very poor outcomes. There are currently no verified RMC patient-derived xenograft (PDX) mouse models established from metastatic pleural effusion (PE) available to study RMC and evaluate new therapeutic options. METHODS: Renal tumor tissue and malignant PE cells from an RMC patient were successfully engrafted into 20 NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. We evaluated the histopathological similarity of the renal tumor and PE PDXs with the original patient renal tumor and PE, respectively. We then evaluated the molecular integrity of the renal tumor PDXs between passages, as well as the PE PDX compared to two generations of renal tumor PDXs, by microarray analysis. The therapeutic efficacy of sunitinib and temsirolimus was tested in a serially-transplanted generation of 27 PE PDX mice. RESULTS: The pathologic characteristics of the patient renal tumor and patient PE were retained in the PDXs. Gene expression profiling revealed high concordance between the two generations of renal tumor PDXs (RMC-P0 vs. RMC-P1, r=0.865), as well as between the first generation PE PDX and each generation of the renal tumor PDX (PE-P0 vs. RMC-P0, r=0.919 and PE-P0 vs. RMC-P1, r=0.843). A low number (626) of differentially-expressed genes (DEGs) was seen between the first generation PE PDX and the first generation renal tumor PDX. In the PE-P1 xenograft, sunitinib significantly reduced tumor growth (p<0.001) and prolonged survival (p=0.004) compared to the vehicle control. CONCLUSIONS: A metastatic PE-derived RMC PDX model was established and shown to maintain histologic features of the patient cancer. Molecular integrity of the PDX models was well maintained between renal tumor and PE PDX as well as between two successive renal tumor PDX generations. Using the PE PDX model, sunitinib demonstrated therapeutic efficacy for RMC. This model can serve as a foundation for future mechanistic and therapeutic studies for primary and metastatic RMC.

Intraoperative Margin Assessment in Oral and Oropharyngeal Cancer Using Label-Free Fluorescence Lifetime Imaging and Machine Learning.

OBJECTIVE: To demonstrate the diagnostic ability of label-free, point-scanning, fiber-based Fluorescence Lifetime Imaging (FLIm) as a means of intraoperative guidance during oral and oropharyngeal cancer removal surgery. METHODS: FLIm point-measurements acquired from 53 patients (n = 67893 pre-resection in vivo, n = 89695 post-resection ex vivo) undergoing oral or oropharyngeal cancer removal surgery were used for analysis. Discrimination of healthy tissue and cancer was investigated using various FLIm-derived parameter sets and classifiers (Support Vector Machine, Random Forests, CNN). Classifier output for the acquired set of point-measurements was visualized through an interpolation-based approach to generate a probabilistic heatmap of cancer within the surgical field. Classifier output for dysplasia at the resection margins was also investigated. RESULTS: Statistically significant change (P 0.01) between healthy and cancer was observed in vivo for the acquired FLIm signal parameters (e.g., average lifetime) linked with metabolic activity. Superior classification was achieved at the tissue region level using the Random Forests method (ROC-AUC: 0.88). Classifier output for dysplasia (% probability of cancer) was observed to lie between that of cancer and healthy tissue, highlighting FLIm's ability to distinguish various conditions. CONCLUSION: The developed approach demonstrates the potential of FLIm for fast, reliable intraoperative margin assessment without the need for contrast agents. SIGNIFICANCE: Fiber-based FLIm has the potential to be used as a diagnostic tool during cancer resection surgery, including Transoral Robotic Surgery (TORS), helping ensure complete resections and improve the survival rate of oral and oropharyngeal cancer patients.

Congenital presentation of synchronous Atypical Teratoid Rhabdoid Tumor and Malignant Rhabdoid Tumor of the urinary bladder in a term infant.

Atypical teratoid/rhabdoid tumor (AT/RT) is a rare central nervous system (CNS) tumor diagnosed primarily in infants and usually portends a poor prognosis. Despite being the most common embryonal tumor in children less than 1 year old, diagnosis is difficult to make based on clinical findings or imaging alone. A complete diagnosis of AT/RT requires identification of loss of integrase interactor 1 (INI1) protein or the SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily b, member 1 (SMARCB1) gene, in its most common presentation. Moreover, their presentation with other primary rhabdoid tumors in the body raises significant suspicion for rhabdoid tumor predisposition syndrome (RTPS). We report a case of a one-month-old infant admitted for worsening emesis and failure to thrive, who was later found to have brain and bladder masses on radiologic imaging. Autopsy with subsequent immunoprofile and molecular testing were crucial in establishing the absence of INI1 nuclear expression and possible homozygous deletion of SMARCB1 in the urinary bladder tumor tissue. Sequencing of the peripheral blood demonstrated probable single copy loss at the SMARCB1 locus. The constellation of findings in tumor and peripheral blood sequencing suggested the possibility of germline single copy SMARCB1 loss, followed by somatic loss of the remaining SMARCB1 allele due to copy neutral loss-of-heterozygosity. Such a sequence of genetic events has been described in malignant rhabdoid tumors (MRT). Dedicated germline testing of this patient's family members could yield answers as to whether rhabdoid tumor predisposition syndrome will continue to have implications for the patient's family.

Deep Functional and Molecular Characterization of a High-Risk Undifferentiated Pleomorphic Sarcoma.

Nonrhabdomyosarcoma soft-tissue sarcomas (STSs) are a class of 50+ cancers arising in muscle and soft tissues of children, adolescents, and adults. Rarity of each subtype often precludes subtype-specific preclinical research, leaving many STS patients with limited treatment options should frontline therapy be insufficient. When clinical options are exhausted, personalized therapy assignment approaches may help direct patient care. Here, we report the results of an adult female STS patient with relapsed undifferentiated pleomorphic sarcoma (UPS) who self-drove exploration of a wide array of personalized Clinical Laboratory Improvement Amendments (CLIAs) level and research-level diagnostics, including state of the art genomic, proteomic, ex vivo live cell chemosensitivity testing, a patient-derived xenograft model, and immunoscoring. Her therapeutic choices were also diverse, including neoadjuvant chemotherapy, radiation therapy, and surgeries. Adjuvant and recurrence strategies included off-label and natural medicines, several immunotherapies, and N-of-1 approaches. Identified treatment options, especially those validated during the in vivo study, were not introduced into the course of clinical treatment but did provide plausible treatment regimens based on FDA-approved clinical agents.

Educational and Career Development Outcomes Among Undergraduate Summer Research Interns: A Pipeline for Pathology, Laboratory Medicine, and Biomedical Science.

Nurturing undergraduate students' interest in careers in science, technology, engineering, and medicine is important to developing the future health-care workforce. Summer research internships provide experiential learning that is important to sustaining students' interest in science, technology, engineering, and medicine careers and inspiring higher educational goals. The Edmondson Summer Research Internship is a mentored program for undergraduate students in University of California Davis Health's Department of Pathology and Laboratory Medicine. To evaluate intern satisfaction, perceptions on the program's influence on their career development, and higher educational outcomes, 102 former interns from a 15-year period were invited to participate in an online survey. Responses were received by 58 (57%) of 102 respondents. Not all respondents answered every question. Overall satisfaction was very high/high in 55 (95%) of 58. Ninety-three percent (54/58) strongly agreed/agreed that the internship was an important part of their career development. Almost all who applied to career/professional opportunities strongly agree/agreed that they perceived the internship to be advantageous (96%, 46/48). Forty-four percent (25/57) received additional education after completing their undergraduate degree, with 25% (14/57) receiving a doctoral degree. Few reported prior experience with a clinical laboratory (8/48, 17%), pathologist (10/48, 21%), or clinical laboratory scientist (12/48, 25%). Based on their internship experience, 55% (32/58) strongly agree/agreed that they positively considered pathology or laboratory medicine as a career choice. The Edmondson Summer Research Internship is seen as important to higher educational goals and career development, increases exposure to pathology and laboratory medicine, and demonstrates the value of hosting a mentored research program for undergraduates.

Fluorescence lifetime imaging for intraoperative cancer delineation in transoral robotic surgery.

This study evaluates the potential for fluorescence lifetime imaging (FLIm) to enhance intraoperative decisionmaking during robotic-assisted surgery of oropharyngeal cancer. Using a custom built FLIm instrument integrated with the da Vinci robotic surgical platform, we first demonstrate that cancer in epithelial tissue diagnosed by histopathology can be differentiated from surrounding healthy epithelial tissue imaged in vivo prior to cancer resection and ex vivo on the excised specimen. Second, we study the fluorescence properties of tissue imaged in vivo at surgical resection margins (tumor bed). Fluorescence lifetimes and spectral intensity ratios were calculated for three spectral channels, producing a set of six FLIm parameters. Current results from 10 patients undergoing TORS procedures demonstrate that healthy epithelium can be resolved from cancer (P < .001) for at least one FLIm parameter. We also showed that a multiparameter linear discriminant analysis approach provides superior discrimination to individual FLIm parameters for tissue imaged both in vivo and ex vivo. Overall, this study highlights the potential for FLIm to be developed into a diagnostic tool for clinical cancer applications of the oropharynx. This technique could help to circumvent the issues posed by the lack of tactile feedback associated with robotic surgical platforms to better enable cancer delineation.

Prolonged androgen receptor loading onto chromatin and the efficient recruitment of p160 coactivators contribute to androgen-independent growth of prostate cancer cells.

BACKGROUND: Growth of most ablation-resistant prostate cancers (CaPs) is dependent on androgen receptor (AR) activity in chromatin, but cancer cells in these tumors have acquired altered AR activation. It is unclear how the aberrantly activated AR loads onto regulatory regions of AR-targeted genes. The purpose of this study was to assess the AR chromatin loading in an androgen-depleted environment. METHODS: The expression of PSA in androgen-resistant CaP cells was determined using RT-PCR and Western blot analysis. In order to investigate the binding of the AR to the PSA gene regulatory regions, chromatin immunoprecipitation (ChIP) was performed in the androgen-independent cds2 cell line in the presence or absence of androgens. In addition, we examined the involvement of p160 coactivators in the chromatin loading of the AR. RESULTS: It was found that constitutive activation of PSA expression was the result of sustained occupancy by the AR at the regulatory region of this gene. This stable AR loading was not blocked by the AR antagonist bicalutamide. Furthermore, androgen-resistant CaP cells highly expressed both AR and the p160 coactivators and the AR was able to recruit TIF2. Downregulation of TIF2 using short hairpin RNA disrupted the AR loading to the PSA enhancer and subsequently inhibited AR activity. CONCLUSION: Prolonged AR localization to the regulatory regions of AR targeted genes and the recruitment of p160 coactivators are a potential mechanism leading to androgen-independent activation of the AR. Disruption of AR chromatin loading could therefore become an important therapeutic target for this disease.

Interleukin-17 receptor-like gene is a novel antiapoptotic gene highly expressed in androgen-independent prostate cancer.

We have recently identified a new gene, interleukin-17 receptor-like (IL-17RL), which is expressed in normal prostate and prostate cancer. This investigation is focused on the role of IL-17RL in prostate cancer. We found that IL-17RL was expressed at significantly higher levels in several androgen-independent prostate cancer cell lines (PC3, DU145, cds1, cds2, and cds3) and tumors compared with the androgen-dependent cell lines (LNCaP and MLC-SV40) and tumors. In an in vivo model of human prostate tumor growth in nude mice (CWR22 xenograft model), IL-17RL expression in tumors was induced by androgen deprivation. The relapsed androgen-independent tumors expressed higher levels of IL-17RL compared with the androgen-dependent tumors. Overexpression of IL-17RL in tumor necrosis factor alpha (TNFalpha)-sensitive LNCaP cells inhibited TNFalpha-induced apoptosis by blocking activation of caspase-3 downstream to caspase-2 and caspase-8. Reciprocally, knocking down IL-17RL expression by small interfering RNA induced apoptosis in all the prostate cancer cell lines studied. Taken together, these results show that IL-17RL is a novel antiapoptotic gene, which may confer partially the property of androgen-independent growth of prostate cancer by promoting cell survival. Thus, IL-17RL is a potential therapeutic target in the treatment of prostate cancer.

XPO1 inhibition by selinexor induces potent cytotoxicity against high grade bladder malignancies.

Treatment options for high grade urothelial cancers are limited and have remained largely unchanged for several decades. Selinexor (KPT-330), a first in class small molecule that inhibits the nuclear export protein XPO1, has shown efficacy as a single agent treatment for numerous different malignancies, but its efficacy in limiting bladder malignancies has not been tested. In this study we assessed selinexor-dependent cytotoxicity in several bladder tumor cells and report that selinexor effectively reduced XPO1 expression and limited cell viability in a dose dependent manner. The decrease in cell viability was due to an induction of apoptosis and cell cycle arrest. These results were recapitulated in in vivo studies where selinexor decreased tumor growth. Tumors treated with selinexor expressed lower levels of XPO1, cyclin A, cyclin B, and CDK2 and increased levels of RB and CDK inhibitor p27, a result that is consistent with growth arrest. Cells expressing wildtype RB, a potent tumor suppressor that promotes growth arrest and apoptosis, were most susceptible to selinexor. Cell fractionation and immunofluorescence studies showed that selinexor treatment increased nuclear RB levels and mechanistic studies revealed that RB ablation curtailed the response to the drug. Conversely, limiting CDK4/6 dependent RB phosphorylation by palbociclib was additive with selinexor in reducing bladder tumor cell viability, confirming that RB activity has a role in the response to XPO1 inhibition. These results provide a rationale for XPO1 inhibition as a novel strategy for the treatment of bladder malignancies.