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Expansion of a single repetitive DNA sequence, termed a tandem repeat (TR), is known to cause more than 50 diseases1,2. However, repeat expansions are often not explored beyond neurological and neurodegenerative disorders. In some cancers, mutations accumulate in short tracts of TRs, a phenomenon termed microsatellite instability; however, larger repeat expansions have not been systematically analysed in cancer3–8. Here we identified TR expansions in 2,622 cancer genomes spanning 29 cancer types. In seven cancer types, we found 160 recurrent repeat expansions (rREs), most of which (155/160) were subtype specific. We found that rREs were non-uniformly distributed in the genome with enrichment near candidate cis-regulatory elements, suggesting a potential role in gene regulation. One rRE, a GAAA-repeat expansion, located near a regulatory element in the first intron of UGT2B7 was detected in 34% of renal cell carcinoma samples and was validated by long-read DNA sequencing. Moreover, in preliminary experiments, treating cells that harbour this rRE with a GAAA-targeting molecule led to a dose-dependent decrease in cell proliferation. Overall, our results suggest that rREs may be an important but unexplored source of genetic variation in human cancer, and we provide a comprehensive catalogue for further study.

Expansion of a single repetitive DNA sequence, termed a tandem repeat (TR), is known to cause more than 50 diseases. However, repeat expansions are often not explored beyond neurological and neurodegenerative disorders. In some cancers, mutations accumulate in short tracts of TRs (STRs), a phenomenon termed microsatellite instability (MSI); however larger repeat expansions have not been systematically analyzed in cancer. Here, we identified TR expansions in 2,622 cancer genomes, spanning 29 cancer types. In 7 cancer types, we found 160 recurrent repeat expansions (rREs); most of these (155/160) were subtype specific. We found that rREs were non-uniformly distributed in the genome with an enrichment near candidate cis-regulatory elements, suggesting a role in gene regulation. One rRE located near a regulatory element in the first intron of UGT2B7 was detected in 34% of renal cell carcinoma samples and was validated by long-read DNA sequencing. Moreover, targeting cells harboring this rRE with a rationally designed, sequence-specific DNA binder led to a dose-dependent decrease in cell proliferation. Overall, our results demonstrate that rREs are an important but unexplored source of genetic variation in human cancers, and we provide a comprehensive catalog for further study.

Loss of the von Hippel–Lindau (VHL) tumor suppressor is a hallmark feature of renal clear cell carcinoma. VHL inactivation results in the constitutive activation of the hypoxia-inducible factors (HIFs) HIF-1 and HIF-2 and their downstream targets, including the proangiogenic factors VEGF and PDGF. However, antiangiogenic agents and HIF-2 inhibitors have limited efficacy in cancer therapy due to the development of resistance. Here we employed an innovative computational platform, Mining of Synthetic Lethals (MiSL), to identify synthetic lethal interactions with the loss of VHL through analysis of primary tumor genomic and transcriptomic data. Using this approach, we identified a synthetic lethal interaction between VHL and the m6A RNA demethylase FTO in renal cell carcinoma. MiSL identified FTO as a synthetic lethal partner of VHL because deletions of FTO are mutually exclusive with VHL loss in pan cancer datasets. Moreover, FTO expression is increased in VHL-deficient ccRCC tumors compared to normal adjacent tissue. Genetic inactivation of FTO using multiple orthogonal approaches revealed that FTO inhibition selectively reduces the growth and survival of VHL-deficient cells in vitro and in vivo. Notably, FTO inhibition reduced the survival of both HIF wild type and HIF-deficient tumors, identifying FTO as an HIF-independent vulnerability of VHL-deficient cancers. Integrated analysis of transcriptome-wide m6A-seq and mRNA-seq analysis identified the glutamine transporter SLC1A5 as an FTO target that promotes metabolic reprogramming and survival of VHL-deficient ccRCC cells. These findings identify FTO as a potential HIF-independent therapeutic target for the treatment of VHL-deficient renal cell carcinoma.

To personalize treatment for renal cell carcinoma (RCC), it would be ideal to confirm the activity of druggable protein pathways within individual tumors. We have developed a high-resolution nanoimmunoassay (NIA) to measure protein activity with high precision in scant specimens (eg, fine needle aspirates [FNAs]). Here, we used NIA to determine whether protein activation varied in different regions of RCC tumors. Since most RCC therapies target angiogenesis by inhibiting the vascular endothelial growth factor (VEGF) receptor, we quantified phosphorylation of extracellular signal-regulated kinase (ERK), a downstream effector of the VEGF signaling pathway. In 90 ex vivo FNA biopsies sampled from multiple regions of 38 primary clear cell RCC tumors, ERK phosphorylation differed among patients. In contrast, within individual patients, we found limited intratumoral heterogeneity of ERK phosphorylation. Our results suggest that measuring ERK in a single FNA may be representative of ERK activity in different regions of the same tumor. As diagnostic and therapeutic protein biomarkers are being sought, NIA measurements of protein signaling may increase the clinical utility of renal mass biopsy and allow for the application of precision oncology for patients with localized and advanced RCC. Patient summary In this report, we applied a new approach to measure the activity of extracellular signal-regulated kinase (ERK), a key cancer signaling protein, in different areas within kidney cancers. We found that ERK activity varied between patients, but that different regions within individual kidney tumors showed similar ERK activity. This suggests that a single biopsy of renal cell carcinoma may be sufficient to measure protein signaling activity to aid in precision oncology approaches.

Significance Tumors are often infiltrated by T lymphocytes recognizing either self- or mutated antigens but are generally inactive. Self-specific CD8+ T cells are particular characteristics of tumor types with lower tumor mutation burden and poorer outcomes. Unlike foreign-specific T cells, they have been curiously resistant to stimulation with cognate antigens. We developed an innate immunity-stimulating nanoparticle to activate tumor-infiltrating CD8+ T cells recognizing both self- and neoantigens in a potent yet safe manner. This resulted in effective tumor growth inhibition or elimination in two murine tumor models and the activation of endogenous T cells in patient-derived tumor organoids across three cancer types. This strategy represents a promising pathway for broadly effective cancer immunotherapy., Tumors are often infiltrated by T lymphocytes recognizing either self- or mutated antigens but are generally inactive, although they often show signs of prior clonal expansion. Hypothesizing that this may be due to peripheral tolerance, we formulated nanoparticles containing innate immune stimulants that we found were sufficient to activate self-specific CD8+ T cells and injected them into two different mouse tumor models, B16F10 and MC38. These nanoparticles robustly activated and/or expanded antigen-specific CD8+ tumor-infiltrating T cells, along with a decrease in regulatory CD4+ T cells and an increase in Interleukin-17 producers, resulting in significant tumor growth retardation or elimination and the establishment of immune memory in surviving mice. Furthermore, nanoparticles with modification of stimulating human T cells enabled the robust activation of endogenous T cells in patient-derived tumor organoids. These results indicate that breaking peripheral tolerance without regard to the antigen specificity creates a promising pathway for cancer immunotherapy.

BACKGROUND: Prognostic models for patients with metastatic renal cell carcinoma (mRCC) include select laboratory values. These models have important limitations, including reliance on a limited array of laboratory tests, and use of dichotomous (“high-low”) cutoffs. We applied a Laboratory-Wide Association Study (LWAS) framework to systematically evaluate common clinical laboratory results associated with survival for patients diagnosed with mRCC. METHODS: We used laboratory data for 3,385 patients diagnosed with mRCC from 2002 to 2017. We developed a LWAS framework, to examine the association with 53 common clinical laboratory tests results (641,712 measurements) and overall survival. We employed false-discovery rate to test the association of multiple laboratory tests with survival, and validated these results using 3 separate cohorts to generate a standardized hazard ratio (sHR), reported for a 1 standard deviation unit change in each laboratory test. RESULTS: The LWAS approach confirmed the association of laboratory values currently used in prognostic models with survival, including calcium (HR 1.35, 95%CI 1.24–1.48), leukocyte count (HR 1.40, 95%CI 1.30–1.51), platelet count (HR 1.36, 95%CI 1.27–1.51), and hemoglobin (HR 0.79, 95%CI 0.72–0.86). Use of these tests as continuous variables improved model performance. LWAS also identified acute phase reactants associated with survival not typically included in prognostic models, including serum albumin (HR 0.66, 95%CI 0.61–0.72), ferritin (HR 1.25, 95%CI 1.08–1.45), alkaline phosphatase (HR 1.31, 95%CI 1.23–1.40), and C-reactive protein (HR 1.70, 95%CI 1.14–2.53). CONCLUSIONS: Routinely measured laboratory tests can refine current prognostic models, facilitate comparisons across clinical trial cohorts, and match patients with specific systemic therapies. Published by Elsevier Inc.

PURPOSE A challenge in the diagnosis of renal cell carcinoma (RCC) is to distinguish chromophobe RCC (chRCC) from benign renal oncocytoma, because these tumor types are histologically and morphologically similar, yet they require different clinical management. Molecular biomarkers could provide a way of distinguishing oncocytoma from chRCC, which could prevent unnecessary treatment of oncocytoma. Such biomarkers could also be applied to preoperative biopsy specimens such as needle core biopsy specimens, to avoid unnecessary surgery of oncocytoma. METHODS We profiled DNA methylation in fresh-frozen oncocytoma and chRCC tumors and adjacent normal tissue and used machine learning to identify a signature of differentially methylated cytosine-phosphate-guanine sites (CpGs) that robustly distinguish oncocytoma from chRCC. RESULTS Unsupervised clustering of Stanford and preexisting RCC data from The Cancer Genome Atlas (TCGA) revealed that of all RCC subtypes, oncocytoma is most similar to chRCC. Unexpectedly, however, oncocytoma features more extensive, overall abnormal methylation than does chRCC. We identified 79 CpGs with large methylation differences between oncocytoma and chRCC. A diagnostic model trained on 30 CpGs could distinguish oncocytoma from chRCC in 10-fold cross-validation (area under the receiver operating curve [AUC], 0.96 (95% CI, 0.88 to 1.00)) and could distinguish TCGA chRCCs from an independent set of oncocytomas from a previous study (AUC, 0.87). This signature also separated oncocytoma from other RCC subtypes and normal tissue, revealing it as a standalone diagnostic biomarker for oncocytoma. CONCLUSION This CpG signature could be developed as a clinical biomarker to support differential diagnosis of oncocytoma and chRCC in surgical samples. With improved biopsy techniques, this signature could be applied to preoperative biopsy specimens.

Loss of the von Hippel–Lindau (VHL) tumor suppressor is a hallmark feature of renal clear cell carcinoma. VHL inactivation results in the constitutive activation of the hypoxia-inducible factors (HIFs) HIF-1 and HIF-2 and their downstream targets, including the proangiogenic factors VEGF and PDGF. However, antiangiogenic agents and HIF-2 inhibitors have limited efficacy in cancer therapy due to the development of resistance. Here we employed an innovative computational platform, Mining of Synthetic Lethals (MiSL), to identify synthetic lethal interactions with the loss of VHL through analysis of primary tumor genomic and transcriptomic data. Using this approach, we identified a synthetic lethal interaction between VHL and the m(6)A RNA demethylase FTO in renal cell carcinoma. MiSL identified FTO as a synthetic lethal partner of VHL because deletions of FTO are mutually exclusive with VHL loss in pan cancer datasets. Moreover, FTO expression is increased in VHL-deficient ccRCC tumors compared to normal adjacent tissue. Genetic inactivation of FTO using multiple orthogonal approaches revealed that FTO inhibition selectively reduces the growth and survival of VHL-deficient cells in vitro and in vivo. Notably, FTO inhibition reduced the survival of both HIF wild type and HIF-deficient tumors, identifying FTO as an HIF-independent vulnerability of VHL-deficient cancers. Integrated analysis of transcriptome-wide m(6)A-seq and mRNA-seq analysis identified the glutamine transporter SLC1A5 as an FTO target that promotes metabolic reprogramming and survival of VHL-deficient ccRCC cells. These findings identify FTO as a potential HIF-independent therapeutic target for the treatment of VHL-deficient renal cell carcinoma.

We have developed the Peralta Stone Extraction System to increase the safety of ureteral stone extraction. The device combines a nitinol stone basket and low-pressure balloon into a single device. After visualization, the stone is captured in the tipless nitinol basket and enveloped by a low-pressure balloon. We tested the performance of device prototypes in a porcine model using stone mimics with diameters ranging from 4.2 to 6.2 mm. Stones extracted with the device required less force when compared with stones in a standard ureteral stone basket. The force reduction was most pronounced for stones greater than 4.2 mm in diameter, and when traversing a ureteral stenosis model. In conclusion, a combination stone basket and balloon device may provide a new and safer way to extract ureteral stones.

Computed tomography (CT) perfusion is a novel imaging method to determine tumor perfusion using a low-dose CT technique to measure iodine concentration at multiple time points. We determined if early changes in perfusion differ between primary renal tumors and metastatic tumor sites in patients with renal cell carcinoma (RCC) receiving targeted anti-angiogenic therapy. A total of 10 patients with advanced RCC underwent a CT perfusion scan at treatment baseline and at one week after initiating treatment. Perfusion measurements included blood volume (BV), blood flow (BF), and flow extraction product (FEP) in a total of 13 lesions (six primary RCC tumors, seven RCC metastases). Changes between baseline and week 1 were compared between tumor locations: primary kidney tumors vs metastases. Metastatic lesions had a greater decrease in BF (average BF difference &plusmn, standard deviation (SD): &minus, 75.0 mL/100 mL/min &plusmn, 81) compared to primary kidney masses (&minus, 25.5 mL/100 mL/min &plusmn, 35). Metastatic tumors had a wider variation of change in BF, BV and FEP measures compared to primary renal tumors. Tumor diameters showed little change after one week, but early perfusion changes are evident, especially in metastatic lesions compared to primary lesions. Future studies are needed to determine if these changes can predict which patients are benefiting from targeted therapy.

Overdiagnosis and overtreatment refer to the detection and treatment of conditions that would not ultimately affect an individual's health. With increasing detection of small renal masses there is growing awareness of the overdiagnosis and overtreatment of these tumors, supported by studies showing that 15-30% of nephrectomy specimens are pathologically benign, and that many small renal cell carcinomas are indolent. The harms of overdiagnosis and overtreatment are numerous, including psychosocial stressors and renal morbidity, in addition to unnecessary surgical complications. A greater understanding of the potential harms of overdiagnosis and overtreatment is crucial as clinicians focus on optimizing patient selection for renal mass biopsy, active surveillance protocols, and minimally invasive surgery. PATIENT SUMMARY: In this mini-review we discuss the issues of overdiagnosis and overtreatment in patients with kidney cancer. We enumerate the risks of overdiagnosis and overtreatment, and examine the next steps towards preventing these harms.

In vitro cancer cultures, including three-dimensional organoids, typically contain exclusively neoplastic epithelium but require artificial reconstitution to recapitulate the tumor microenvironment (TME). The co-culture of primary tumor epithelia with endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has been particularly elusive. Here, an air-liquid interface (ALI) method propagated patient-derived organoids (PDOs) from >100 human biopsies or mouse tumors in syngeneic immunocompetent hosts as tumor epithelia with native embedded immune cells (T, B, NK, macrophages). Robust droplet-based, single-cell simultaneous determination of gene expression and immune repertoire indicated that PDO TILs accurately preserved the original tumor T cell receptor (TCR) spectrum. Crucially, human and murine PDOs successfully modeled immune checkpoint blockade (ICB) with anti-PD-1- and/or anti-PD-L1 expanding and activating tumor antigen-specific TILs and eliciting tumor cytotoxicity. Organoid-based propagation of primary tumor epithelium en bloc with endogenous immune stroma should enable immuno-oncology investigations within the TME and facilitate personalized immunotherapy testing.

Bladder cancer has the highest recurrence rate of all cancers due in part to inadequate transurethral resection. Inadequate resection is caused by the inability of cystoscopes to detect invisible lesions during the resection procedure. To improve detection and resection of nonmuscle invasive bladder cancer, we quantified the ability of a surface-enhanced Raman nanoparticle and endoscope system to classify bladder tissue as normal or cancerous. Both antibody-based (active) and tissue permeability-based (passive) targeting mechanisms were evaluated by topically applying nanoparticles to ex vivo human bladder tissue samples. Multiplexed molecular imaging of CD47 and Carbonic Anhydrase 9 tumor proteins gave a receiver operating characteristic area under the curve (ROC AUC of 0.93 (0.75, 1.00). Furthermore, passively targeted nanoparticles enabled tissue classification with an ROC AUC of 0.93 (0.73, 1.00). Passively targeted nanoparticles penetrated 5-fold deeper and bound to tumor tissue at 3.3-fold higher concentrations in cancer compared to normal bladder urothelium, suggesting the existence of an enhanced surface permeability and retention effect in human bladder cancer.

PubMed: 30740513, Neutrophils have a critical role in regulating the immune system. The immune system is compromised during chemotherapy, increasing infection risks and imposing a need for regular monitoring of neutrophil counts. Although commercial hematology analyzers are currently used in clinical practice for neutrophil counts, they are only available in clinics and hospitals, use large blood volumes, and are not available at the point of care (POC). Additionally, phlebotomy and blood processing require trained personnel, where patients are often admitted to hospitals when the infections are at late stage due to lack of frequent monitoring. Here, a reliable method is presented that selectively captures and quantifies white blood cells (WBCs) and neutrophils from a finger prick volume of whole blood by integrating microfluidics with high-resolution imaging algorithms. The platform is compact, portable, and easy to use. It captures and quantifies WBCs and neutrophils with high efficiency (> 95%) and specificity (> 95%) with an overall 4.2% bias compared to standard testing. The results from a small cohort of patients (N = 11 healthy, N = 5 lung and kidney cancer) present a unique disposable cell counter, demonstrating the ability of this tool to monitor neutrophil and WBC counts within clinical or in resource-constrained environments.

Introduction Surgical management of penile cancer depends on accurate margin assessment and staging. Advanced optical imaging technologies may improve penile biopsy and organ-sparing treatment. We evaluated the feasibility of confocal laser endomicroscopy for intraoperative assessment of benign and malignant penile tissue. Patients and methods With institutional review board approval, 11 patients were recruited, 9 with suspected penile cancer, and 2 healthy controls. Confocal laser endomicroscopy using a 2.6-mm fiber-optic probe was performed at 1 or 2 procedures on all subjects, for 13 imaging procedures. Fluorescein was administered intravenously approximately 3 minutes prior to imaging for contrast. Video sequences from in vivo (n = 12) and ex vivo (n = 6) imaging were obtained of normal glans, suspicious lesions, and surgical margins. Images were processed, annotated, characterized, and correlated with standard hematoxylin and eosin histopathology. Results No adverse events related to imaging were reported. Distinguishing features of benign and malignant penile tissue could be identified by confocal laser endomicroscopy. Normal skin had cells of uniform size and shape, with distinct cytoplasmic membranes consistent with squamous epithelium. Malignant lesions were characterized by disorganized, crowded cells of various size and shape, lack of distinct cytoplasmic membranes, and hazy, moth-eaten appearance. The transition from normal to abnormal squamous epithelium could be identified. Conclusions We report the initial feasibility of intraoperative confocal laser endomicroscopy for penile cancer optical biopsy. Pending further evaluation, confocal laser endomicroscopy could serve as an adjunct or replacement to conventional frozen section pathology for management of penile cancer.

A combination of optical imaging technologies with cancer-specific molecular imaging agents is a potentially powerful strategy to improve cancer detection and enable image-guided surgery. Bladder cancer is primarily managed endoscopically by white light cystoscopy with suboptimal diagnostic accuracy. Emerging optical imaging technologies hold great potential for improved diagnostic accuracy but lack imaging agents for molecular specificity. Using fluorescently labeled CD47 antibody (anti-CD47) as molecular imaging agent, we demonstrated consistent identification of bladder cancer with clinical grade fluorescence imaging systems, confocal endomicroscopy, and blue light cystoscopy in fresh surgically removed human bladders. With blue light cystoscopy, the sensitivity and specificity for CD47-targeted imaging were 82.9 and 90.5%, respectively. We detected variants of bladder cancers, which are diagnostic challenges, including carcinoma in situ, residual carcinoma in tumor resection bed, recurrent carcinoma following prior intravesical immunotherapy with Bacillus Calmette-Guerin (BCG), and excluded cancer from benign but suspicious-appearing mucosa. CD47-targeted molecular imaging could improve diagnosis and resection thoroughness for bladder cancer.

Many important experiments in cancer research are initiated with cell line data analysis due to the ease of accessibility and utilization. Recently, the ability to capture and characterize circulating tumor cells (CTCs) has become more prevalent in the research setting. This ability to detect, isolate, and analyze CTCs allows us to directly compare specific protein expression levels found in patient CTCs to cell lines. In this study, we use immunocytochemistry to compare the protein expression levels of total cytokeratin (CK) and androgen receptor (AR) in CTCs and cell lines from patients with prostate cancer to determine what translational insights might be gained through the use of cell line data. A non-enrichment CTC detection assay enables us to compare cytometric features and relative expression levels of CK and AR by indirect immunofluorescence from prostate cancer patients against the prostate cancer cell line LNCaP. We measured physical characteristics of these two groups and observed significant differences in cell size, fluorescence intensity, and nuclear to cytoplasmic (N/C) ratio. We hope that these experiments will initiate a foundation to allow cell line data to be compared against characteristics of primary cells from patients.

Novel agents that inhibit targets in the hypoxia and MTOR pathways can achieve response rates of 30-60% in renal cell carcinoma (RCC); however, biomarkers have not yet been identified to measure degree of target inhibition and mechanism of biologic response in individual patients. We developed the use of a high-throughput, nanoscale immunoassay (NIA, Nanopro1000 Instrument, Protein Simple) to profile hypoxia pathways and downstream signaling in serially sampled clinical specimens from patients with RCC. NIA uses charge-based separation to distinguish multiple charged modifications of individual proteins, and measure relative ratios of individual unphosphorylated and phosphorylated isoforms. We first developed and optimized assays to measure therapeutic targets and proteins in hypoxia, proliferation and metabolic pathways (including CaIX, HIF, VEGFR, ERK, AKT, MYC, glutaminase) to analyze frozen surgical specimens, fine needle aspirates (FNA's) and blood PBMC specimens from patients with RCC. Next, we analyzed more than 200 FNA's from solid tumors, comparing RCC with paired adjacent non-tumor tissue and other epithelial malignancies. Basal MAPK signaling across the samples ranged across 3 logs for both normal and tumor tissue, demonstrating the difficulty in setting a threshold predictive of response based on a single pre-treatment measurement. To test whether analysis of serial specimens could detect changes, we prospectively collected and analyzed blood peripheral mononuclear cells from 20 RCC patients before and during treatment with standard targeted therapies including MTOR and tyrosine kinase inhibitors. We report for the first time that targeted inhibitors can preferentially inhibit or activate specific protein phospho-isoforms in patients with RCC. Our studies demonstrate that rapid and quantitative nanoproteomic profiling in very small amounts of serially sampled clinical specimen may accelerate translational studies for predictive proteomic biomarkers of response to targeted therapy. Citation Format: Alice C. Fan, John Leppert, Joanna E. Liliental, Liwen Xu, Thomas J. Metzner, Emelyn Shroff, Alia Yaghi, Christine Yost, James D. Brooks, Lauren C. Harshman, Chiara Sabatti, Sandhya Srinivas, Dean W. Felsher. Nano-scale proteomic profiles of response to targeted therapy in patients with RCC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2283. doi:10.1158/1538-7445.AM2013-2283

Background: The role of cleaved caspase-3 as an integral player in the apoptotic process of mammalian cells has prompted the investigation of an assay to detect cleaved caspase-3 in circulating tumor cells (CTCs) as one way to assess CTC viability. Methods: Using a previously developed non-enrichment based assay, nucleated blood cells and CTCs are adhered to a single slide. An antibody for cleaved caspase-3 was incorporated into the assay in addition to the existing antibodies for cytokeratin (to identify tumor cells) and CD45 (to identify white blood cells), as well as a nuclear stain (DAPI). The modified assay was initially tested on cultured MCF 10a cells spiked into normal donor blood. Before spiking, apoptosis was induced in these cells using staurosporine. Following validation of the assay in cell lines, the assay was performed using blood samples from 9 different breast cancer patients, as well as 3 different normal donor samples. Results: CTCs were detected in the patient samples as single cells, as well as clusters. The results of the assay showed that only 1 out of 896 detected CTCs was positive for cleaved caspase-3 (0.11%). Of these CTCs, 134 of them were found to be in clusters, and all 134 were negative for cleaved caspase-3, indicating that whether in clusters or as single cells, the vast majority of CTC's detected with this assay are not undergoing irreversible apoptotic processes via cleaved caspase-3. The results also showed that 0.40% of white blood cells in healthy donors expressed cleaved caspase-3, compared to 0.51% of the white cells in cancer patients, indicating that healthy donors and cancer patients express cleaved caspase-3 in their white blood cells at nearly the same rate. Conclusions: The morphologic characterization used in this platform to select healthy-appearing cells identifies a population of circulating tumor cells with virtually no evidence of caspase-related ongoing apoptosis, a finding that supports the morphologic impression of viability. 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 3619. doi:1538-7445.AM2012-3619

194 Background: We have established a fluid phase biopsy approach that identifies CTCs which preserves cytologic features in high-definition (HD) for diagnostic pathology without using immune or surface receptor-based enrichment. HD-CTCs identified with this approach can be used for enumeration and molecular characterization. Methods: Blood was collected from metastatic prostate cancer patients and normal donors in Cyto-Chex tubes (Streck, Omaha, NE) as part of IRB approved protocols at each site. Following erythrocyte lysis, 3 million nucleated cells were deposited on a glass slide. Samples were incubated with a pan-cytokeratin (CK), CD45, and androgen receptor (AR) antibodies and counter-stained with DAPI. LNCaP cells were spiked into normal blood. Images were obtained with a fluorescent scanning microscope and analyzed with a computer algorithm. Candidate HD-CTCs were subsequently verified by expert readers. Slides were re-imaged for quantitative analysis using at a fixed exposure and gain. Results: A total of 227 CTCs from ten patients were compared to 20 LNCaP cells. The median (range) HD-CTCs in this cohort was: 9 (1-62) cells/ml. The mean ± standard deviation measurements in HD-CTCs were observed: CK intensity 60.4±154; total cell area 89.0 ± 53.8 µm2; nuclear area 61.1 ± 36.0 µm2. LNCaP cells spiked into normal blood gave the following values: CK intensity 1166+/−306; total cell area 143 ± 48.1 µm2; nuclear area 63.1 ± 18.6 µm2. CTCs were additionally classified as either AR positive (AR+) or AR negative (AR−). 37 of the 227 (16.3%) HD-CTCs were AR+. The average CK intensity was significantly higher in AR+ versus AR− cells at 174.23 and 39.86, respectively (p Conclusions: We find a positive association between AR and CK expression on a per cell basis. Further, we find AR is expressed at comparable levels in CTCs from patients and human prostate cancer cells in culture. The HD-CTC based approach may be used for enumeration and molecular interrogation of CTCs in patients with prostate cancer.

Hematologic spread of carcinoma results in incurable metastasis; yet, the basic characteristics and travel mechanisms of cancer cells in the bloodstream are unknown. We have established a fluid phase biopsy approach that identifies circulating tumor cells (CTCs) without using surface protein-based enrichment and presents them in sufficiently high definition (HD) to satisfy diagnostic pathology image quality requirements. This 'HD-CTC' assay finds5 HD-CTCs mL(-1) of blood in 80% of patients with metastatic prostate cancer (n = 20), in 70% of patients with metastatic breast cancer (n = 30), in 50% of patients with metastatic pancreatic cancer (n = 18), and in 0% of normal controls (n = 15). Additionally, it finds HD-CTC clusters ranging from 2 HD-CTCs to greater than 30 HD-CTCs in the majority of these cancer patients. This initial validation of an enrichment-free assay demonstrates our ability to identify significant numbers of HD-CTCs in a majority of patients with prostate, breast and pancreatic cancers.

Circulating tumor cell (CTC) counts are an established prognostic marker in metastatic prostate, breast, and colorectal cancer. However, there is little knowledge about CTC incidence rates and morphologic heterogeneity in non-small cell lung cancer (NSCLC) and their correlation to disease progression and usability for clinical staging. Here, we present a fluid phase biopsy approach that identifies CTCs without surface receptor-based enrichment, and presents them in sufficiently high definition (HD) to satisfy diagnostic pathology image quality requirements. The HD-CTC assay will be used to monitor HD-CTC counts and morphology in early stage, locally advanced, and metastatic NSCLC patient over time. 150 newly diagnosed NSCLC patients are being enrolled for this project. At the time of diagnosis 50 of the patients will have early stage cancers (stage I-II); 50 patients will have locally advanced (stage III) cancers, and 50 will be patients presenting with metastatic (stage IV) disease. Blood samples from each patient will be obtained at the time of diagnosis and 6 months later. We present here an interim data set that shows an incidence rate of 78% at baseline with a median CTC count of 19.5/ml of blood (range 0-441.6). Our preliminary data demonstrates that, despite stringent morphologic inclusion criteria for the definition of HD-CTCs, our HD-CTC assay shows high sensitivity in the detection of lung cancer CTCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4151. doi:10.1158/1538-7445.AM2011-4151