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Liquid biopsies such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) have great potential to serve as prognostic and predictive biomarkers in urologic cancers. The possibility of using liquid biopsies for real-time noninvasive and dynamic monitoring of response to therapy has been an active area of investigation. In this brief review, we outline the evidence for the potential clinical utility of CTC and ctDNA analyses in prostate, urothelial, and renal cancers.

PDF file - 110K, Supplemental Tables. (S1) Clinical characteristics and CTC data for metastatic prostate cancer patients treated with hormonal therapy. (S2) Time course data for metastatic prostate cancer patients treated with hormonal therapy and analyzed for AR signaling in CTCs.

Supplementary Table S1. Genes tested for Prostate CTC digital PCR Assay; Supplementary Table S2. Clinical information for prostate cancer patients and healthy donor subjects; Supplementary Table S3. Clinical information for patients enrolled on prospective trial of first-line abiraterone for mCRPC; Supplementary Table S4. Clinical information for patients with localized prostate cancer in radical prostatectomy study; Supplementary Table S5. Droplet digital PCR primers used in this study.

All supplementary tables

Supplementary Figure S1. (A) Schematic of d-CTC assay. Whole blood is processed using the CTC-iChip, resulting in a bulk product highly enriched for intact CTCs (labeled green) but also contains WBCs (labeled red) and RBCs (unlabeled); Supplementary Figure S2. Graphs showing droplet digital PCR signal for each gene in 12 metastatic prostate cancer patients compared to 34 healthy male control subjects; Supplementary Figure S3. (A, B) Graphs of relationships between Prostate CTCM Score and serum PSA, and CTC droplet digital PCR KLK3 signal and serum PSA at the pre-treatment time point; Supplementary Figure S4. (A) Kaplan-Meier curves for radiographic progression-free survival (R-PFS) by CTC HOXB13 status at pretreatment; Supplementary Figure S5. (A) Heatmap of droplet digital PCR CTC signal after whole transcriptome amplification for blood samples from healthy donor controls arranged by sex and age, and from patients with clinically localized prostate cancer, arranged by D'Amico Risk Group; Supplementary Figure S6. (A) Box plots showing pre-operative leave-one-out cross validated (LOOCV) CTCL Score in clinically localized prostate cancer patients according to microscopic SVI or pelvic LN involvement identified at the time of radical prostatectomy

All supplementary figures

Methods describing microfluidic CTC enrichment, RNA extraction, amplification and detection, single cell and bulk RNA sequencing

PDF file - 49K, Supplemental Methods for (a) single molecule sequencing and AR transcriptional signature, (b) HB CTC-chip fabrication, (c) HB CTC-chip blood processing, and (d) automated fluorescence microscopy.

Supplemental Tables

Supplemental figures and box plots

Purpose: The androgen receptor (AR) mRNA splice variant AR-V7 has emerged as a predictive biomarker for response to AR-targeted therapies. There are currently no commercially available assays to detect AR splice variants. The branched chain RNA in situ hybridization (ISH) platform enables the highly sensitive detection of RNA transcripts in formalin-fixed, paraffin-embedded (FFPE) tissues.Experimental design: We designed a branched chain RNA ISH probe to target the unique cryptic exon CE3 of AR-V7 using multiple tiling probes. This automated ISH assay was applied to tumor tissue from two distinct clinical cohorts that we hypothesized would differ in AR-V7 status.Results: We detected AR-V7 in all tumor samples from men with metastatic castration-resistant prostate cancer with tissue obtained after disease progression despite at least one subsequent line of hormonal therapy (abiraterone, enzalutamide, or bicalutamide; n = 12). We detected AR-V7 in just one tumor from men who had undergone prostatectomy for localized adenocarcinoma (n = 30; Gleason 4 + 5 = 9 in the AR-V7–positive sample). Given the apparent distinction between the above groups by AR-V7 signal, we analyzed pretreatment AR-V7 status as a predictive and prognostic biomarker in men with treatment-naïve metastatic disease. Patients with metastases but without detectable AR-V7 RNA at baseline had significantly longer overall survival (log-rank P = 0.044) and a trend toward superior progression-free survival (log-rank P = 0.055).Conclusions: Within an institutional cohort, the RNA ISH assay identified AR-V7 within FFPE tissue and may have prognostic value in metastatic castration-sensitive prostate cancer. These preliminary findings warrant further study in larger cohorts. Clin Cancer Res; 23(2); 363–9. ©2016 AACR.

Purpose:Neuroendocrine (NE) bladder carcinoma is a rare and aggressive variant. Molecular subtyping studies have found that 5% to 15% of muscle-invasive bladder cancer (MIBC) have transcriptomic patterns consistent with NE bladder cancer in the absence of NE histology. The clinical implications of this NE-like subtype have not been explored in depth.Experimental Design:Transcriptome-wide expression profiles were generated for MIBC collected from 7 institutions and clinical-use of Decipher Bladder. Using unsupervised clustering, we generated a clustering solution on a prospective training cohort (PTC; n = 175), developed single-sample classifiers to predict NE tumors, and evaluated the resultant models on a testing radical cystectomy (RC) cohort (n = 225). A random forest model was finalized and applied to 5 validation cohorts (n = 1302). Uni- and multivariable survival analyses were used to characterize clinical outcomes.Results:In the training cohort (PTC), hierarchical clustering using an 84-gene panel showed a cluster of 8 patients (4.6%) with highly heterogeneous expression of NE markers in the absence of basal or luminal marker expression. NE-like tumors were identified in 1% to 6.6% of cases in validation cohorts. Patients with NE-like tumors had significantly worse 1-year progression-free survival (65% NE-like vs. 82% overall; P = 0.046) and, after adjusting for clinical and pathologic factors, had a 6.4-fold increased risk of all-cause mortality (P = 0.001). IHC confirmed the neuronal character of these tumors.Conclusions:A single-patient classifier was developed that identifies patients with histologic urothelial cancer harboring a NE transcriptomic profile. These tumors represent a high-risk subgroup of MIBC, which may require different treatment.

Control cell line validations.

Supplementary Figure Legend

Muscle-invasive bladder cancer can be treated with either radical cystectomy or bladder preservation approaches, and there is a need for reliable biomarkers to guide the optimal choice of therapy. The recent elucidation of the genomic landscape and biological drivers of bladder cancer has enabled the identification of tumor molecular features that may be helpful in driving clinical decision-making. Here, we summarize recent efforts to develop molecular biomarkers that could be leveraged to guide therapeutic decisions, post-treatment monitoring, and the optimal use of bladder preservation approaches for the effective treatment of muscle-invasive bladder cancer.

Cancer-associated alterations in DNA methylation include hypermethylation of focal CpG islands within promoters, as well as hypomethylation of large stretches of gene-poor chromosomal domains, so called Partially Methylated Domains (PMDs). While CpG island hypermethylation is associated with silencing of tumor suppressor genes, the functional consequences of hypomethylation are uncertain. PMDs may be cause or consequence of cancer cell proliferation, and they have been associated with transcriptional de-repression of repetitive sequences, as well as histone modification-triggered silencing of rare protein encoding genes within these domains. In studying Circulating Tumor Cells (CTCs) isolated from blood specimens of patients with prostate cancer, we mapped PMDs at the single cell level, identifying a set of 40 “core PMDs” that are shared across cancer cells, within and across different individuals, as well as in prostate cancer cell lines. By analyzing single nuclei from micro-dissected early-stage prostate tumors, we find that hypomethylation of these core PMDs occurs early during prostate tumorigenesis. Core PMDs are highly enriched for immune-related genes, many of which are co-located within a single chromosomal locus targeted for hypomethylation-associated silencing. Nested between adjacent PMDs, we also identify domains that have retained DNA methylation (Preserved Methylation Islands; PMIs), which have sharply demarcated borders, spanning one or a small number of genes. In contrast to PMDs, PMIs are enriched for cell proliferation-associated genes. Together, these observations suggest that hypomethylation-associated silencing of immune-related genes, with sparing of adjacent proliferation-associated genes, is an early event in prostate cancer development. These large-scale chromosomal modifications are detectable within CTCs and may present diagnostic opportunities. Citation Format: Daniel A. Haber, Hongshan Guo, Ben S. Wittner, Joanna A Vuille, Michael S. Lawrence, Chin-Lee Wu, Mehmet Toner, David T. Miyamoto, Shyamala Maheswaran. Hypomethylation of immune-related loci contribute to prostate tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr IA010.

Cancer therapy often results in heterogeneous responses in different metastatic lesions in the same patient. Inter- and intratumor heterogeneity in signaling within various tumor compartments and its impact on therapy are not well characterized due to the limited sensitivity of single-cell proteomic approaches. To overcome this barrier, we applied single-cell mass cytometry with a customized 26-antibody panel to PTEN-deleted orthotopic prostate cancer xenograft models to measure the evolution of kinase activities in different tumor compartments during metastasis or drug treatment. Compared with primary tumors and circulating tumor cells (CTC), bone metastases, but not lung and liver metastases, exhibited elevated PI3K/mTOR signaling and overexpressed receptor tyrosine kinases (RTK) including c-MET protein. Suppression of c-MET impaired tumor growth in the bone. Intratumoral heterogeneity within tumor compartments also arose from highly proliferative EpCAM-high epithelial cells with increased PI3K and mTOR kinase activities coexisting with poorly proliferating EpCAM-low mesenchymal populations with reduced kinase activities; these findings were recapitulated in epithelial and mesenchymal CTC populations in patients with metastatic prostate and breast cancer. Increased kinase activity in EpCAM-high cells rendered them more sensitive to PI3K/mTOR inhibition, and drug-resistant EpCAM-low populations with reduced kinase activity emerged over time. Taken together, single-cell proteomics indicate that microenvironment- and cell state–dependent activation of kinase networks create heterogeneity and differential drug sensitivity among and within tumor populations across different sites, defining a new paradigm of drug responses to kinase inhibitors. Significance: Single-cell mass cytometry analyses provide insights into the differences in kinase activities across tumor compartments and cell states, which contribute to heterogeneous responses to targeted therapies.

Muscle-invasive bladder cancer (MIBC) is a sex-biased cancer with a higher incidence in men but worse outcomes in women. The root cause behind these observations remains unclear. To investigate whether sex-specific tumor biology could explain the differences in clinical behavior of MIBC, we analyzed the transcriptome profiles from transurethral resected bladder tumors of 1000 patients. Female tumors expressed higher levels of basal- and immune-associated genes, while male tumors expressed higher levels of luminal markers. Using molecular subtyping, we found that the rates of the basal/squamous subtype were higher in females than in males. Males were enriched with tumors of the luminal papillary (LumP) and neuroendocrine-like subtypes. Male MIBC tumors had higher androgen response activity across all luminal subtypes and male patients with LumP tumors were younger. Taken together, these data confirm differences in molecular subtypes based on sex. The role of the androgen response pathway in explaining subtype differences between men and women should be studied further. PATIENT SUMMARY: We explored the sex-specific biology of bladder cancer in 1000 patients and found that women had more aggressive cancer with higher immune activity. Men tended toward less aggressive tumors that showed male hormone signaling, suggesting that male hormones may influence the type of bladder cancer that a patient develops.

ImportanceBladder-preserving trimodality therapy can be an effective alternative to radical cystectomy for treatment of muscle-invasive bladder cancer (MIBC), but biomarkers are needed to guide optimal patient selection. The DNA repair protein MRE11 is a candidate response biomarker that has not been validated in prospective cohorts using standardized measurement approaches.ObjectiveTo evaluate MRE11 expression as a prognostic biomarker in MIBC patients receiving trimodality therapy using automated quantitative image analysis.Design, Setting, and ParticipantsThis prognostic study analyzed patients with MIBC pooled from 6 prospective phase I/II, II, or III trials of trimodality therapy (Radiation Therapy Oncology Group [RTOG] 8802, 8903, 9506, 9706, 9906, and 0233) across 37 participating institutions in North America from 1988 to 2007. Eligible patients had nonmetastatic MIBC and were enrolled in 1 of the 6 trimodality therapy clinical trials. Analyses were completed August 2020.ExposuresTrimodality therapy with transurethral bladder tumor resection and cisplatin-based chemoradiation therapy.Main Outcomes and MeasuresMRE11 expression and association with disease-specific (bladder cancer) mortality (DSM), defined as death from bladder cancer. Pretreatment tumor tissues were processed for immunofluorescence with anti-MRE11 antibody and analyzed using automated quantitative image analysis to calculate a normalized score for MRE11 based on nuclear-to-cytoplasmic (NC) signal ratio.ResultsOf 465 patients from 6 trials, 168 patients had available tissue, of which 135 were analyzable for MRE11 expression (median age of 65 years [minimum-maximum, 34-90 years]; 111 [82.2%] men). Median (minimum-maximum) follow-up for alive patients was 5.0 (0.6-11.7) years. Median (Q1-Q3) MRE11 NC signal ratio was 2.41 (1.49-3.34). Patients with an MRE11 NC ratio above 1.49 (ie, above first quartile) had a significantly lower DSM (HR, 0.50; 95% CI, 0.26-0.93; P = .03). The 4-year DSM was 41.0% (95% CI, 23.2%-58.0%) for patients with an MRE11 NC signal ratio of 1.49 or lower vs 21.0% (95% CI, 13.4%-29.8%) for a ratio above 1.49. MRE11 NC signal ratio was not significantly associated with overall survival (HR, 0.84; 95% CI, 0.49-1.44).Conclusions and RelevanceHigher MRE11 NC signal ratios were associated with better DSM after trimodality therapy. Lower MRE11 NC signal ratios identified a poor prognosis subgroup that may benefit from intensification of therapy.

Purpose Conventional rectal spacers (nonI-SPs) are low-contrast on computed tomography (CT), often necessitating magnetic resonance imaging for accurate delineation. A new formulation of spacers (I-SPs) incorporates iodine to improve radiopacity and CT visualization. We characterized placement, stability, and plan quality of I-SPs compared to nonI-SPs. Methods and Materials Patients with intact prostate cancer (n = 50) treated with I-SPs and photons were compared to randomly selected patients (n = 50) with nonI-SPs (photon or proton therapy). The I-SP was contoured on the planning CT and cone beam CTs at 3 timepoints: first, middle, and final treatment (n = 200 scans). I-SPs Hounsfield units (HU), volume, surface area (SA), centroid position relative to prostate centroid, and distance between prostate/rectum centroids were compared on the planning CTs between each cohort. I-SP changes were evaluated on cone beam CTs over courses of treatment. Dosimetric evaluations of plan quality and robustness were performed. I-SP was tested in a phantom to characterize its relative linear stopping power for protons. Results I-SPs yielded a distinct visible contrast on planning CTs compared to nonI-SPs (HU 138 vs 12, P .1). Dosimetric analysis concluded there were no significant changes in plan quality or robustness for I-SPs compared to nonI-SPs. The I-SP relative linear stopping power was 1.018, necessitating HU override for proton planning. Conclusions I-SPs provide a manifest CT contrast, allowing for delineation on planning CT alone with no magnetic resonance imaging necessary. I-SPs radiopacity, size, and relative position remained stable over courses of treatment from 28 to 44 fractions. No changes in plan quality or robustness were seen comparing I-SPs and nonI-SPs.

Context Genomic stratification can impact prostate cancer (PC) care through diagnostic, prognostic, and predictive biomarkers that aid in clinical decision-making. The temporal and spatial genomic heterogeneity of PC together with the challenges of acquiring metastatic tissue biopsies hinder implementation of tissue-based molecular profiling in routine clinical practice. Blood-based liquid biopsies are an attractive, minimally invasive alternative. Objective To review the clinical value of blood-based liquid biopsy assays in PC and identify potential applications to accelerate the development of precision medicine. Evidence acquisition A systematic review of PubMed/MEDLINE was performed to identify relevant literature on blood-based circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and extracellular vesicles (EVs) in PC. Evidence synthesis Liquid biopsy has emerged as a practical tool to profile tumor dynamics over time, elucidating features that evolve (genome, epigenome, transcriptome, and proteome) with tumor progression. Liquid biopsy tests encompass analysis of DNA, RNA, and proteins that can be detected in CTCs, ctDNA, or EVs. Blood-based liquid biopsies have demonstrated promise in the context of localized tumors (diagnostic signatures, risk stratification, and disease monitoring) and advanced disease (response/resistance biomarkers and prognostic markers). Conclusions Liquid biopsies have value as a source of prognostic, predictive, and response biomarkers in PC. Most clinical applications have been developed in the advanced metastatic setting, where CTC and ctDNA yields are significantly higher. However, standardization of assays and analytical/clinical validation is necessary prior to clinical implementation. Patient summary Traces of tumors can be isolated from blood samples from patients with prostate cancer either as whole cells or as DNA fragments. These traces provide information on tumor features. These minimally invasive tests can guide diagnosis and treatment selection.

Background Bladder-sparing trimodality therapy (TMT) is an alternative to radical cystectomy (RC) for muscle-invasive bladder cancer (MIBC), and biomarkers to inform therapy selection are needed. Objective To evaluate the prognostic value of immune and stromal signatures in MIBC treated with TMT. Design, setting, and participants We used a clinical-grade platform to perform transcriptome-wide gene expression profiling of primary tumors from 136 MIBC patients treated with TMT at a single institution. We observed 60 overall survival events at 5 yr, and median follow-up time for patients without an event was 5.0 yr (interquartile range 3.1, 5.0). Expression data from another cohort of 223 MIBC patients treated with neoadjuvant chemotherapy (NAC) and RC were also analyzed. Outcome measurements and statistical analysis Molecular subtype, immune, and stromal signatures were evaluated for associations with disease-specific survival (DSS) and overall survival (OS) in TMT patients, and in patients treated with NAC and RC. Results and limitations Gene expression profiling of TMT cases identified luminal (N = 40), luminal-infiltrated (N = 26), basal (N = 54), and claudin-low (N = 16) subtypes. Signatures of T-cell activation and interferon gamma signaling were associated with improved DSS in the TMT cohort (hazard ratio 0.30 [0.14–0.65], p = 0.002 for T cells), but not in the NAC and RC cohort. Conversely, a stromal signature was associated with worse DSS in the NAC and RC cohort (p = 0.006), but not in the TMT cohort. This study is limited by its retrospective nature. Conclusions Higher immune infiltration in MIBC is associated with improved DSS after TMT, whereas higher stromal infiltration is associated with shorter DSS after NAC and RC. Additional studies should be conducted to determine whether gene expression profiling can predict treatment response. Patient summary We used gene expression profiling to study the association between tumor microenvironment and outcomes following bladder preservation therapy for invasive bladder cancer. We found that outcomes varied with immune and stromal signatures within the tumor. We conclude that gene expression profiling has potential to guide treatment decisions in bladder cancer.

Significance The documented efficacy of COX-2 inhibitors in cancer chemoprevention and in suppression of metastasis is predominantly attributed to inflammatory responses, whereas their effects on tumor-stromal interaction are poorly understood. Through single-cell transcriptome analyses in an immune-compromised mouse xenograft model and in vitro reconstitution experiments, we uncover a tumor-stromal paracrine pathway in which secretion by tumor cells of the COX-2 product prostaglandin E2 induces prolactin production by stromal cells, which activates signaling in disseminated tumor cells with upregulated prolactin receptor expression. Analysis of multiple human cancers confirms differential tumor and stromal cell expression of COX-2, prolactin, and prolactin receptor. Together, these findings may provide novel biomarkers to inform the selective application of COX-2 inhibitors and point to additional targets for suppressing metastasis recurrence., Tumor-stromal communication within the microenvironment contributes to initiation of metastasis and may present a therapeutic opportunity. Using serial single-cell RNA sequencing in an orthotopic mouse prostate cancer model, we find up-regulation of prolactin receptor as cancer cells that have disseminated to the lungs expand into micrometastases. Secretion of the ligand prolactin by adjacent lung stromal cells is induced by tumor cell production of the COX-2 synthetic product prostaglandin E2 (PGE2). PGE2 treatment of fibroblasts activates the orphan nuclear receptor NR4A (Nur77), with prolactin as a major transcriptional target for the NR4A-retinoid X receptor (RXR) heterodimer. Ectopic expression of prolactin receptor in mouse cancer cells enhances micrometastasis, while treatment with the COX-2 inhibitor celecoxib abrogates prolactin secretion by fibroblasts and reduces tumor initiation. Across multiple human cancers, COX-2, prolactin, and prolactin receptor show consistent differential expression in tumor and stromal compartments. Such paracrine cross-talk may thus contribute to the documented efficacy of COX-2 inhibitors in cancer suppression.

Purpose: Neuroendocrine (NE) bladder carcinoma is a rare and aggressive variant. Molecular subtyping studies have found that 5% to 15% of muscle-invasive bladder cancer (MIBC) have transcriptomic patterns consistent with NE bladder cancer in the absence of NE histology. The clinical implications of this NE-like subtype have not been explored in depth. Experimental Design: Transcriptome-wide expression profiles were generated for MIBC collected from 7 institutions and clinical-use of Decipher Bladder. Using unsupervised clustering, we generated a clustering solution on a prospective training cohort (PTC; n = 175), developed single-sample classifiers to predict NE tumors, and evaluated the resultant models on a testing radical cystectomy (RC) cohort (n = 225). A random forest model was finalized and applied to 5 validation cohorts (n = 1302). Uni- and multivariable survival analyses were used to characterize clinical outcomes. Results: In the training cohort (PTC), hierarchical clustering using an 84-gene panel showed a cluster of 8 patients (4.6%) with highly heterogeneous expression of NE markers in the absence of basal or luminal marker expression. NE-like tumors were identified in 1% to 6.6% of cases in validation cohorts. Patients with NE-like tumors had significantly worse 1-year progression-free survival (65% NE-like vs. 82% overall; P = 0.046) and, after adjusting for clinical and pathologic factors, had a 6.4-fold increased risk of all-cause mortality (P = 0.001). IHC confirmed the neuronal character of these tumors. Conclusions: A single-patient classifier was developed that identifies patients with histologic urothelial cancer harboring a NE transcriptomic profile. These tumors represent a high-risk subgroup of MIBC, which may require different treatment.

PURPOSE/OBJECTIVE(S) To evaluate a convolutional neural network auto-contouring (AC) model created for treatment planning in patients receiving radiotherapy for localized prostate cancer after insertion of a radiopaque rectal spacer hydrogel. MATERIALS/METHODS The deep learning model, trained by 125 patients, auto contours target volumes (prostate and proximal seminal vesicles), OARs (bladder, rectum, femoral heads and penile bulb) and radiopaque rectal spacers. ACs were evaluated against MD manual contours (MCs) submitted for treatment planning. ACs were not available for MD review while creating MCs. Individual volumes as well as composite volumes (overall performance) were qualitatively evaluated by a radiation oncologist using a 1 (minor discrepancy, little to no dose-volume impact), 2 (moderate discrepancy or editable with substantial efficiency gain), 3 (significant discrepancy or editable with meaningful efficiency gain), and 4 (rejected due to gross error or editable without efficiency gain) scoring scale. Quantitative evaluation of geometric differences was performed with mean distance to agreement (MDA) and Dice Similarity Coefficient (DSC) for each volume. RESULTS A total of 68 cases were evaluated by quantitative and qualitative metrics (Table 1). MCs were performed by 4 experienced MDs. Average composite score of 2.22 (SD 0.73) indicates substantial to meaningful efficiency gain in contouring process. Composite scores for 100% of cases evaluated indicate a meaningful efficiency gain in the contouring process, with 60% of composite scores indicating only minor to moderate discrepancy between contours. Prostate and SV AC average scores were above 2. Mean prostate MDA and DSC were 1.71 and 0.85, respectively, both within tolerance range recommended by AAPM TG 132. CONCLUSION The model can accurately auto contour target volumes, OARs and spacer with meaningful to substantial efficiency gain, with a majority of target volumes qualitatively evaluated by a physician as having only mild to moderate discrepancies. Quantitative analysis shows contours for most OARs have only minor geometric differences unlikely to have significant dose-volume impacts. Further analysis with comparison of treatment plans generated from ACs and MCs is warranted.

Summary Although muscle-invasive bladder cancer is commonly treated with radical cystectomy, a standard alternative is bladder preservation therapy, consisting of maximum transurethral bladder tumour resection followed by radiotherapy with concurrent chemotherapy. Although no successfully completed randomised comparisons are available, the two treatment paradigms seem to have similar long-term outcomes; however, clinicopathologic parameters can be insufficient to provide clear guidance in the selection of one treatment over the other. Recent advances in the molecular understanding of bladder cancer have led to the identification of new predictive biomarkers that ultimately might help guide the tailored selection of therapy on the basis of the intrinsic biology of the tumour. In this Review, we discuss the existing evidence for molecular alterations and genomic signatures as prognostic or predictive biomarkers for bladder preservation therapy. If validated in prospective clinical trials, such biomarkers could enable the identification of subgroups of patients who are more likely to benefit from one treatment over another, and guide the use of combination therapies that include other modalities, such as immunotherapy, which might act synergistically with radiotherapy.

The multiplicity of new therapies for breast cancer presents a challenge for treatment selection. We describe a 17-gene digital signature of breast circulating tumor cell (CTC)–derived transcripts enriched from blood, enabling high-sensitivity early monitoring of response. In a prospective cohort of localized breast cancer, an elevated CTC score after three cycles of neoadjuvant therapy is associated with residual disease at surgery (P = 0.047). In a second prospective cohort with metastatic breast cancer, baseline CTC score correlates with overall survival (P = 0.02), as does persistent CTC signal after 4 weeks of treatment (P = 0.01). In the subset with estrogen receptor (ER)–positive disease, failure to suppress ER signaling within CTCs after 3 weeks of endocrine therapy predicts early progression (P = 0.008). Drug-refractory ER signaling within CTCs overlaps partially with presence of ESR1 mutations, pointing to diverse mechanisms of acquired endocrine drug resistance. Thus, CTC-derived digital RNA signatures enable noninvasive pharmacodynamic measurements to inform therapy in breast cancer. Significance: Digital analysis of RNA from CTCs interrogates treatment responses of both localized and metastatic breast cancer. Quantifying CTC-derived ER signaling during treatment identifies patients failing to respond to ER suppression despite having functional ESR1. Thus, noninvasive scoring of CTC-RNA signatures may help guide therapeutic choices in localized and advanced breast cancer. Cancer Discov; 8(10); 1286–99. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1195

Cancer therapy often results in heterogeneous responses in different metastatic lesions in the same patient. Inter- and intra-tumor heterogeneity in proteomic signaling within the various tumor compartments and its impact on therapy are not well characterized due to the limited sensitivity of single cell proteomic approaches. To overcome this barrier, we applied single cell mass cytometry with a customized 29-antibody panel [against cell states, receptor tyrosine kinases (RTK) and phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR)-, mitogen-activated protein kinase (MAPK)-, and cytokine-signaling] to PTEN-deleted orthotopic prostate cancer xenograft models to measure the evolution of kinase activities in different tumor compartments during metastasis and upon drug treatment. Compared with primary tumors and circulating tumor cells (CTCs), bone metastases but not lung and liver metastases exhibited elevated PI3K/mTOR signaling and RTKs including c-Met protein, which, when suppressed, impaired tumor growth in the bone. Intra-tumoral heterogeneity within tumor compartments also arises from highly proliferative EpCAMhigh epithelial cells with increased PI3K and mTOR kinase activities co-existing with poorly proliferating EpCAMlow mesenchymal populations with reduced kinase activities, findings recapitulated in epithelial and mesenchymal CTC populations in metastatic prostate and breast cancer patients. Increased kinase activity in EpCAMhigh cells rendered them more sensitive to PI3K/mTOR inhibition and drug resistant EpCAMlow populations with reduced kinase activity emerged over time. Taken together, single cell proteomics identified microenvironment- and cell state-dependent activation of kinase networks creating heterogeneity and differential drug sensitivity among and within tumor populations across different sites, defining a new paradigm of drug responses to kinase inhibitors.

Introduction: Wnt signaling is important for normal development, cell proliferation, and cell differentiation. However, aberrations in the pathway can lead to tumorigenesis and cancer progression. Recent genome-wide studies have demonstrated the frequent occurrence of Wnt pathway alterations in prostate cancer. Although alterations in the canonical Wnt pathway in prostate cancer may have an impact on prognosis, recent studies suggest that the noncanonical Wnt pathway also plays an important role in disease progression and treatment resistance.Areas covered: We review the literature with regard to the potential prognostic significance of noncanonical Wnt signaling in prostate cancer. After a brief overview of the canonical and noncanonical Wnt pathways, we discuss the preclinical and clinical evidence for activation of Wnt signaling in prostate cancer. We focus on clinical evidence for noncanonical Wnt pathway components to serve as potential prognostic biomarkers.Expert opinion: Although many therapeutic options are available for men with prostate cancer, there remains an unmet need for prognostic and predictive biomarkers to precisely guide clinical management. Early evidence suggests that components of the noncanonical Wnt pathway may serve as prognostic biomarkers. However, prospective validation studies are necessary before these biomarkers can be routinely applied in the clinic.

Background Erythroderma is a severe manifestation of pemphigus foliaceus (PF), a blistering disease mediated by IgG autoantibodies against desmoglein 1. Increasing evidence supports the contribution of angiogenic mediators in the pathogenesis of erythroderma. Objective To evaluate the in situ expression of vascular endothelial growth factor (VEGF) and endoglin in patients with PF with erythroderma. Methods Formalin-fixed paraffin-embedded skin samples obtained from patients with erythrodermic PF (n = 19; 12 patients with endemic PF), non-erythrodermic PF (n = 17), pemphigus vulgaris (PV; n = 10), psoriasis (n = 10) and healthy individuals (HI; n = 10) were processed in an automated immunohistochemistry platform utilizing anti-VEGF and anti-endoglin as primary antibodies. Reactivity was evaluated both manually (0 = negative; 1+ = mild; 2+ = intense) and through an automated microvessel analysis algorithm. Results Vascular endothelial growth factor expression in erythrodermic PF was higher than in non-erythrodermic PF (P = 0.034) and in HI (P = 0.004), and similar to psoriasis (P = 0.667) and PV (P = 0.667). In non-erythrodermic PF, VEGF positivity was similar to HI (P = 0.247), and lower than psoriasis (P = 0.049) and PV (P = 0.049). Both erythrodermic and non-erythrodermic PF presented similar endoglin expression (P = 0.700). In addition, endoglin positivity during erythrodermic PF was similar to psoriasis (P = 0.133) and lower than PV (P = 0.0009). Increased expression of in situVEGF suggests that healing processes are triggered in response to tissue damage led by autoantibodies in PF, especially during erythroderma. Reduced endoglin positivity suggests that an unbalanced angiogenesis may occur during erythrodermic PF. Further studies may help to confirm if the regulation of VEGF and endoglin expression in patients with PF can contribute to control the healing process and enable disease remission. Conclusion Overexpression of VEGF in erythrodermic PF as well as in PV and psoriasis points out a dysregulated repair process in severe forms of these diseases and suggests VEGF and endoglin could act as prognostic markers and future therapeutic targets to enable proper healing in PF.

Blood-based biomarkers are critical in metastatic prostate cancer, where characteristic bone metastases are not readily sampled, and they may enable risk stratification in localized disease. We established a sensitive and high-throughput strategy for analyzing prostate circulating tumor cells (CTC) using microfluidic cell enrichment followed by digital quantitation of prostate-derived transcripts. In a prospective study of 27 patients with metastatic castration-resistant prostate cancer treated with first-line abiraterone, pretreatment elevation of the digital CTCM score identifies a high-risk population with poor overall survival (HR = 6.0; P = 0.01) and short radiographic progression-free survival (HR = 3.2; P = 0.046). Expression of HOXB13 in CTCs identifies 6 of 6 patients with ≤12-month survival, with a subset also expressing the ARV7 splice variant. In a second cohort of 34 men with localized prostate cancer, an elevated preoperative CTCL score predicts microscopic dissemination to seminal vesicles and/or lymph nodes (P < 0.001). Thus, digital quantitation of CTC-specific transcripts enables noninvasive monitoring that may guide treatment selection in both metastatic and localized prostate cancer. Significance: There is an unmet need for biomarkers to guide prostate cancer therapies, for curative treatment of localized cancer and for application of molecularly targeted agents in metastatic disease. Digital quantitation of prostate CTC-derived transcripts in blood specimens is predictive of abiraterone response in metastatic cancer and of early dissemination in localized cancer. Cancer Discov; 8(3); 288–303. ©2018 AACR. See related commentary by Heitzer and Speicher, p. 269. This article is highlighted in the In This Issue feature, p. 253

Advances in our molecular understanding of cancer biology have paved the way to an expanding compendium of molecularly-targeted therapies, accompanied by the urgent need for biomarkers that enable the precise selection of the most appropriate therapies for individual cancer patients. Circulating biomarkers such as circulating tumor cells (CTCs) are poised to fill this need, since they are "liquid biopsies" that can be performed non-invasively and serially, and may capture the spectrum of spatial and temporal tumor heterogeneity better than conventional tissue biopsies. Increasing evidence suggests that moving beyond the enumeration of CTCs towards more sophisticated molecular analyses can provide actionable data that may predict and potentially improve clinical outcomes. In this review, we discuss the potential of molecular CTC analyses to serve as prognostic and predictive biomarkers to guide cancer therapy and early cancer detection. As technologies to capture and analyze CTCs continue to increase in sophistication, we anticipate that the potential clinical applications of CTCs will grow exponentially in the coming years.

Significance Identifying predictive biomarkers of therapeutic response for melanoma patients treated with immune checkpoint inhibitors is a major challenge. By combining microfluidic enrichment for melanoma circulating tumor cells (CTCs) together with RNA-based droplet digital PCR quantitation, we have established a highly sensitive and robust platform for noninvasive, blood-based monitoring of tumor burden. Serial monitoring of melanoma patients treated with immune checkpoint inhibitors shows rapid changes in CTC score, which precede standard clinical assessment and are highly predictive of long-term clinical outcome. Early on-treatment digital monitoring of CTC dynamics may thus help identify patients likely to benefit from immune checkpoint inhibition therapy., A subset of patients with metastatic melanoma have sustained remissions following treatment with immune checkpoint inhibitors. However, analyses of pretreatment tumor biopsies for markers predictive of response, including PD-1 ligand (PD-L1) expression and mutational burden, are insufficiently precise to guide treatment selection, and clinical radiographic evidence of response on therapy may be delayed, leading to some patients receiving potentially ineffective but toxic therapy. Here, we developed a molecular signature of melanoma circulating tumor cells (CTCs) to quantify early tumor response using blood-based monitoring. A quantitative 19-gene digital RNA signature (CTC score) applied to microfluidically enriched CTCs robustly distinguishes melanoma cells, within a background of blood cells in reconstituted and in patient-derived (n = 42) blood specimens. In a prospective cohort of 49 patients treated with immune checkpoint inhibitors, a decrease in CTC score within 7 weeks of therapy correlates with marked improvement in progression-free survival [hazard ratio (HR), 0.17; P = 0.008] and overall survival (HR, 0.12; P = 0.04). Thus, digital quantitation of melanoma CTC-derived transcripts enables serial noninvasive monitoring of tumor burden, supporting the rational application of immune checkpoint inhibition therapies.

Purpose/objective(s) Radiotherapy (RT) with or without androgen deprivation therapy (ADT) plays a key role in the salvage treatment of prostate cancer recurrent after prostatectomy. However, not all patients benefit from salvage therapy, and there is an unmet need for biomarkers to distinguish responders from non-responders. Prostate cancer depends on androgen receptor (AR) signaling, and expression of AR splice variants (ARVs) that enable androgen-independent AR signaling is associated with resistance to ADT in the metastatic setting. Recent in vitro data suggest that ARVs also mediate DNA repair after irradiation, suggesting that ARV expression may also be a biomarker of resistance to RT. However, the landscape of ARV expression in primary prostate cancer and its effect on treatment outcomes remain unexplored. Here, we hypothesized that ARVs may be detectable in primary prostate cancer and furthermore may modulate response to salvage RT+ADT. Materials/methods We retrospectively identified 46 prostate cancer patients treated with prostatectomy followed by salvage RT+ ADT at a single institution from 1995 to 2012. Median age at salvage was 64.5 years. The indication for salvage therapy was biochemical failure after an undetectable post-operative PSA in 72%, gross local recurrence in 17%, and persistently elevated PSA after surgery in 11%. Median RT dose was 64.8 Gy, and all patients received concurrent ADT. We comprehensively interrogated the landscape of ARVs by performing ultra-deep targeted RNA-seq of archival formalin-fixed paraffin-embedded prostatectomy samples. Using a custom library of > 3000 primers spanning all AR exons and introns, we evaluated 21 native splice junction sites and 20 splice variants with a mean depth of coverage of > 5000x. We tested for association between splice variant expression and clinical outcomes using the log-rank test and Cox proportional hazards model. Results In total, 76% of patients had one or more detectable AR splice variants. The most commonly detected variants were AR-45 (exon 1b-2) in 41%, AR-V9 (exon 3-cryptic exon (CE) 5) in 20%, and AR-V7 (exon 3-CE3) in 13%. At a median follow-up of 33.8 months, biochemical progression-free survival (BPFS) at 3 years was 60%, distant metastasis-free survival was 90%, and overall survival was 100%. Among detected splice variants, only AR-V7 was associated with differential clinical outcomes, with a median BPFS of 10.9 months in AR-V7 positive vs 73.4 months in AR-V7 negative patients (P = 0.0020, HR 5.23, 95% CI 1.62-16.87). Conclusion Using a custom ultra-deep targeted RNA-Seq approach, we provide among the first comprehensive descriptions of the AR splice variant landscape in primary prostate cancer. Moreover, we show that detectable AR-V7 expression in prostatectomy specimens was associated with inferior outcomes following subsequent salvage RT+ADT, suggesting for the first time that AR-V7 may modulate outcomes for localized as well as metastatic disease.

Precise rare-cell technologies require the blood to be processed immediately or be stabilized with fixatives. Such restrictions limit the translation of circulating tumor cell (CTC)-based liquid biopsy assays that provide accurate molecular data in guiding clinical decisions. Here we describe a method to preserve whole blood in its minimally altered state by combining hypothermic preservation with targeted strategies that counter cooling-induced platelet activation. Using this method, whole blood preserved for up to 72 h can be readily processed for microfluidic sorting without compromising CTC yield and viability. The tumor cells retain high-quality intact RNA suitable for single-cell RT-qPCR as well as RNA-Seq, enabling the reliable detection of cancer-specific transcripts including the androgen-receptor splice variant 7 in a cohort of prostate cancer patients with an overall concordance of 92% between fresh and preserved blood. This work will serve as a springboard for the dissemination of diverse blood-based diagnostics., The current FDA-approved whole blood stabilization method for circulating tumor cell (CTC) isolation suffers from RNA degradation. Here the authors combine hypothermic preservation and antiplatelet strategies to stabilize whole blood up to 72 h without compromising CTC yield and RNA integrity.

The rapidly growing array of therapeutic options in cancer requires informative biomarkers to guide the rational selection and precision application of appropriate therapies. Circulating biomarkers such as circulating tumor cells have immense potential as noninvasive, serial ‘liquid biopsies’ that may be more representative of the complete spectrum of a patient’s individual malignancy than spatially and temporally restricted tumor biopsies. In this review, we discuss the current state-of-the-art in the isolation and molecular characterization of circulating tumor cells as well as their utility in a wide range of clinical applications such as prognostics, treatment monitoring and identification of novel therapeutic targets and resistance mechanisms to enable real-time adjustments in the clinical management of cancer.

The recent expansion of therapeutic options for the treatment of metastatic prostate cancer highlights the need for precision medicine approaches to enable the rational selection of appropriate therapies for individual patients. In this context, circulating biomarkers in the peripheral blood are attractive as readily accessible tools for predicting and monitoring therapeutic response. In the case of circulating tumor cells and circulating tumor DNA, they may also serve as a noninvasive means of assessing molecular aberrations in tumors at multiple time points before and during therapy. These so-called "liquid biopsies" can provide a snapshot view of tumor molecular architecture and may enable clinicians to monitor the molecular status of tumors as they evolve during treatment, thus allowing for individualized precision therapeutic decisions for patients over time. In this review, we outline recent progress in the field of circulating biomarkers in metastatic prostate cancer and evaluate their potential for enabling this vision of real-time precision medicine.

Recent advances in microfluidic approaches have enabled the efficient isolation and detailed molecular characterization of circulating tumor cells (CTCs) in the peripheral blood of patients with cancer. Single-cell molecular analyses of CTCs reveal a tremendous degree of intracellular heterogeneity in CTC populations, reflective of heterogeneity across different patients as well as the underlying heterogeneity of tumors within each individual patient. These studies have enabled the identification of heterogeneous drug resistance mechanisms that can coexist in treatment refractory tumors. CTC analyses also enable serial noninvasive monitoring in patients and can capture the emergence of tumor heterogeneity over time, whether due to tumor evolution through genetic instability or through cellular plasticity. The presence and extent of intratumoral heterogeneity as revealed through the study of CTCs have important clinical implications for understanding and predicting the development of treatment resistance in a variety of solid tumors and for formulating appropriate therapeutic strategies in the effective treatment of cancer.

The standard definitive treatment options for muscle-invasive bladder cancer are radical cystectomy or bladder-sparing trimodality therapy (TMT), consisting of maximal transurethral resection of bladder tumor followed by chemoradiation therapy. Although multiple series show these two treatment approaches have similar overall long-term outcomes, recent studies suggest that molecular biomarkers may identify subgroups of patients who may preferentially benefit from one type of therapy over the other. Such biomarkers include tumor-intrinsic genes involved in DNA repair and signal transduction, which confer sensitivity or resistance to radiation therapy based on the intrinsic biology of the tumor. Emerging biomarkers also include molecular signatures of the tumor microenvironment, suggesting an important role for tumor-extrinsic factors in governing therapeutic sensitivity or resistance. This presentation will review the evidence and rationale underlying potential prognostic and predictive molecular biomarkers in bladder preservation therapy. A specific focus will be on our recent gene expression profiling of 136 muscle-invasive bladder cancer patients treated with bladder-sparing TMT. Our analysis showed that higher immune infiltration was associated with improved disease-specific survival after TMT, whereas higher stromal infiltration was associated with shorter disease-specific survival in a comparison cohort treated with radical cystectomy after neoadjuvant chemotherapy. These findings require validation studies in additional cohorts, which are currently ongoing. An additional focus of the presentation will be on emerging liquid biopsy biomarkers of minimal residual disease after bladder cancer therapy. The standard follow-up for patients after bladder-sparing TMT includes cystoscopies with biopsies and imaging studies at regular intervals. Liquid biopsy approaches such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) may enable noninvasive monitoring of response and detection of minimal residual disease after therapy, with a substantial lead time over conventional follow-up approaches including imaging. If validated in prospective clinical trials, molecular biomarkers may guide the tailored selection of appropriate treatments for individual patients with muscle-invasive bladder cancer, as well as enable noninvasive monitoring of patients after therapy. Such biomarkers also have potential to guide the use of synergistic combinations of immunotherapy with chemoradiation therapy for bladder cancer. Citation Format: David T. Miyamoto. Molecular biomarkers of chemoradiation response in bladder preservation therapy [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr IA25.