Bram De Laere

Efficacy of Cabazitaxel in Castration-resistant Prostate Cancer Is Independent of the Presence of AR-V7 in Circulating Tumor Cells.

BACKGROUND Androgen receptor splice variant 7 (AR-V7) in circulating tumor cells (CTCs) from patients with metastatic castration-resistant prostate cancer (mCRPC) was recently demonstrated to be associated with resistance to abiraterone and enzalutamide. Cabazitaxel might, however, remain effective in AR-V7-positive patients. OBJECTIVE To investigate the association between AR-V7 expression in CTCs and resistance to cabazitaxel. DESIGN, SETTING, AND PARTICIPANTS We selected patients with mCRPC from the multicenter, randomized, phase 2, randomized, open-label, multicenter study in mCRPC on the pharmacodynamic effects of budesonide on cabazitaxel (Jevtana) (CABARESC). Before the start of the first and third cabazitaxel cycle, CTCs were enumerated using the CellSearch System. In patients with ≥10 CTCs in 7.5 ml blood at baseline, the expression of AR-V7 was assessed by quantitative polymerase chain reaction. OUTCOME MEASURES AND STATISTICAL ANALYSIS The primary end point was the association between the AR-V7 status and the CTC response rate (decrease to fewer than five CTCs in 7.5 ml blood during treatment). Secondary end points were the prostate-specific antigen (PSA) response rate (RR) and overall survival (OS). Analyses were performed using chi-square and log-rank tests. RESULTS AND LIMITATIONS AR-V7 was detected in 16 of 29 patients (55%) with ≥10 CTCs and was more frequently found in abiraterone pretreated patients (5 of 5 [100%] treated vs 7 of 20 [35%] untreated; p=0.009). We found no differences in CTC and PSA RRs. The presence of AR-V7 in CTCs was not associated with progression-free survival (hazard ratio [HR]: 0.8; 95% confidence interval [CI], 0.4-1.8) or overall survival (HR 1.6; 95% CI, 0.6-4.4). CONCLUSIONS The response to cabazitaxel seems to be independent of the AR-V7 status of CTCs from mCRPC patients. Consequently, cabazitaxel might be a valid treatment option for patients with AR-V7-positive CTCs. PATIENT SUMMARY Tools are needed to select specific treatments for specific patients at specific times. The presence of the gene AR-V7 in CTCs has been associated with resistance to anti-androgen receptor treatments. We investigated whether this holds true for cabazitaxel, but we found cabazitaxel to be effective independent of the presence of AR-V7.

Comprehensive Profiling of the Androgen Receptor in Liquid Biopsies from Castration-resistant Prostate Cancer Reveals Novel Intra-AR Structural Variation and Splice Variant Expression Patterns

BACKGROUND Expression of the androgen receptor splice variant 7 (AR-V7) is associated with poor response to second-line endocrine therapy in castration-resistant prostate cancer (CRPC). However, a large fraction of nonresponding patients are AR-V7-negative. OBJECTIVE To investigate if a comprehensive liquid biopsy-based AR profile may improve patient stratification in the context of second-line endocrine therapy. DESIGN, SETTING, AND PARTICIPANTS Peripheral blood was collected from patients with CRPC (n=30) before initiation of a new line of systemic therapy. We performed profiling of circulating tumour DNA via low-pass whole-genome sequencing and targeted sequencing of the entire AR gene, including introns. Targeted RNA sequencing was performed on enriched circulating tumour cell fractions to assess the expression levels of seven AR splice variants (ARVs). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Somatic AR variations, including copy-number alterations, structural variations, and point mutations, were combined with ARV expression patterns and correlated to clinicopathologic parameters. RESULTS AND LIMITATIONS Collectively, any AR perturbation, including ARV, was detected in 25/30 patients. Surprisingly, intra-AR structural variation was present in 15/30 patients, of whom 14 expressed ARVs. The majority of ARV-positive patients expressed multiple ARVs, with AR-V3 the most abundantly expressed. The presence of any ARV was associated with progression-free survival after second-line endocrine treatment (hazard ratio 4.53, 95% confidence interval 1.424-14.41; p=0.0105). Six out of 17 poor responders were AR-V7-negative, but four carried other AR perturbations. CONCLUSIONS Comprehensive AR profiling, which is feasible using liquid biopsies, is necessary to increase our understanding of the mechanisms underpinning resistance to endocrine treatment. PATIENT SUMMARY Alterations in the androgen receptor are associated with endocrine treatment outcomes. This study demonstrates that it is possible to identify different types of alterations via simple blood draws. Follow-up studies are needed to determine the effect of such alterations on hormonal therapy.

Evaluation and consequences of heterogeneity in the circulating tumor cell compartment

A growing understanding of the molecular biology of cancer and the identification of specific aberrations driving cancer evolution have led to the development of various targeted agents. Therapeutic decisions concerning these drugs are often guided by single biopsies of the primary tumor. Yet, it is well known that tumors can exhibit significant heterogeneity and change over time as a result of selective pressure. Circulating tumor cells (CTCs) are shed from various tumor sites and are thought to represent the molecular landscape of a patients overall tumor burden. Moreover, a minimal-invasive liquid biopsy facilitates monitoring of clonal evolution during therapy pressure and disease progression in real-time. While more information becomes available regarding heterogeneity among CTCs, comparison between these studies is needed. In this review, we focus on the genomic and transcriptional heterogeneity found in the CTC compartment, and its significance for clinical decision making.

Differential impact of RB status on E2F1 reprogramming in human cancer

The tumor suppressor protein retinoblastoma (RB) is mechanistically linked to suppression of transcription factor E2F1-mediated cell cycle regulation. For multiple tumor types, loss of RB function is associated with poor clinical outcome. RB action is abrogated either by direct depletion or through inactivation of RB function; however, the basis for this selectivity is unknown. Here, analysis of tumor samples and cell-free DNA from patients with advanced prostate cancer showed that direct RB loss was the preferred pathway of disruption in human disease. While RB loss was associated with lethal disease, RB-deficient tumors had no proliferative advantage and exhibited downstream effects distinct from cell cycle control. Mechanistically, RB loss led to E2F1 cistrome expansion and different binding specificity, alterations distinct from those observed after functional RB inactivation. Additionally, identification of protumorigenic transcriptional networks specific to RB loss that were validated in clinical samples demonstrated the ability of RB loss to differentially reprogram E2F1 in human cancers. Together, these findings not only identify tumor-suppressive functions of RB that are distinct from cell cycle control, but also demonstrate that the molecular consequence of RB loss is distinct from RB inactivation. Thus, these studies provide insight into how RB loss promotes disease progression, and identify new nodes for therapeutic intervention.

Cell-free DNA profiling of metastatic prostate cancer reveals microsatellite instability, structural rearrangements and clonal hematopoiesis

Background There are multiple existing and emerging therapeutic avenues for metastatic prostate cancer, with a common denominator, which is the need for predictive biomarkers. Circulating tumor DNA (ctDNA) has the potential to cost-efficiently accelerate precision medicine trials to improve clinical efficacy and diminish costs and toxicity. However, comprehensive ctDNA profiling in metastatic prostate cancer to date has been limited. Methods A combination of targeted- and low-pass whole genome sequencing was performed on plasma cell-free DNA and matched white blood cell germline DNA in 364 blood samples from 217 metastatic prostate cancer patients. Results ctDNA was detected in 85.9% of baseline samples, correlated to line of therapy and was mirrored by circulating tumor cell enumeration of synchronous blood samples. Comprehensive profiling of the androgen receptor (AR) revealed a continuous increase in the fraction of patients with intra-AR structural variation, from 15.4% during first line mCRPC therapy to 45.2% in fourth line, indicating a continuous evolution of AR during the course of the disease. Patients displayed frequent alterations in DNA repair deficiency genes (18.0%). Additionally, the microsatellite instability phenotype was identified in 3.81% of eligible samples (≥0.1 ctDNA fraction). Sequencing of non-repetitive intronic- and exonic regions of PTEN, RB1 and TP53 detected biallelic inactivation in 47.5%, 20.3% and 44.1% of samples with ≥0.2 ctDNA fraction, respectively. Only one patient carried a clonal high-impact variant without a detectable second hit. Intronic high-impact structural variation was twice as common as exonic mutations in PTEN and RB1. Finally, 14.6% of patients presented false positive variants due to clonal hematopoiesis, commonly ignored in commercially available assays. Conclusions ctDNA profiles appear to mirror the genomic landscape of metastatic prostate cancer tissue and may cost-efficiently provide somatic information in clinical trials designed to identify predictive biomarkers. However, intronic sequencing of the interrogated tumor suppressors challenge the ubiquitous focus on coding regions and is vital, together with profiling of synchronous white blood cells, to minimize erroneous assignments which in turn may confound results and impede true associations in clinical trials.

Circulating tumor cells and survival in abiraterone- and enzalutamide-treated patients with castration-resistant prostate cancer

The outcome to treatment administered to patients with metastatic castration‐resistant prostate cancer (mCRPC) greatly differs between individuals, underlining the need for biomarkers guiding treatment decision making.

TP53 outperforms other androgen receptor biomarkers to predict abiraterone or enzalutamide outcome in metastatic castration-resistant prostate cancer

Purpose: To infer the prognostic value of simultaneous androgen receptor (AR) and TP53 profiling in liquid biopsies from patients with metastatic castration-resistant prostate cancer (mCRPC) starting a new line of AR signaling inhibitors (ARSi). Experimental Design: Between March 2014 and April 2017, we recruited patients with mCRPC (n = 168) prior to ARSi in a cohort study encompassing 10 European centers. Blood samples were collected for comprehensive profiling of CellSearch-enriched circulating tumor cells (CTC) and circulating tumor DNA (ctDNA). Targeted CTC RNA sequencing (RNA-seq) allowed the detection of eight AR splice variants (ARV). Low-pass whole-genome and targeted gene-body sequencing of AR and TP53 was applied to identify amplifications, loss of heterozygosity, mutations, and structural rearrangements in ctDNA. Clinical or radiologic progression-free survival (PFS) was estimated by Kaplan–Meier analysis, and independent associations were determined using multivariable Cox regression models. Results: Overall, no single AR perturbation remained associated with adverse prognosis after multivariable analysis. Instead, tumor burden estimates (CTC counts, ctDNA fraction, and visceral metastases) were significantly associated with PFS. TP53 inactivation harbored independent prognostic value [HR 1.88; 95% confidence interval (CI), 1.18–3.00; P = 0.008], and outperformed ARV expression and detection of genomic AR alterations. Using Cox coefficient analysis of clinical parameters and TP53 status, we identified three prognostic groups with differing PFS estimates (median, 14.7 vs. 7.51 vs. 2.62 months; P < 0.0001), which was validated in an independent mCRPC cohort (n = 202) starting first-line ARSi (median, 14.3 vs. 6.39 vs. 2.23 months; P < 0.0001). Conclusions: In an all-comer cohort, tumor burden estimates and TP53 outperform any AR perturbation to infer prognosis. See related commentary by Rebello et al., p. 1699

Abstract IA03: Differential impact of RB pathway status on E2F1 reprogramming and disease progression in human prostate cancer

The retinoblastoma tumor suppressor (RB) is mechanistically linked to suppression of E2F1-mediated cell cycle regulation. Abrogation of RB function is associated with poor clinical outcome across various tumor types, which frequently elicit a preference for either RB depletion or functional inactivation, yet the basis for selectivity is unknown. Here, examination of RB pathway alterations in advanced prostate cancer revealed that cyclin dependent kinase (CDK)/cyclin/CDKi alterations are infrequent, and identify RB loss as the major mechanism of pathway disruption in human disease. Furthermore, RB status was readily traced through cell-free DNA analyses in human specimens, thus identifying new ways to assign RB status in the clinical setting. Strikingly, RB depletion in human disease was not associated with a higher Ki67 index, indicating a role for the RB/E2F1 pathway in regulating processes distinct from cell cycle control and associated with lethal-stage disease. Subsequent mechanistic investigation utilized isogenic prostate cancer models, wherein RB could be differentially inactivated through depletion or through hormone-induced, CDK-mediated phosphorylation. Unbiased molecular interrogation uncovered a novel E2F1 cistrome and downstream engagement of transcriptional networks exclusively observed after RB loss, with binding specificity divergent from canonically described E2F1 binding patterns. Additionally, E2F1 cistrome alterations elicited by RB depletion were seen to be distinct from those after phosphorylation-induced RB functional inactivation, providing needed insight into the basis of selectivity for RB loss versus CDK-mediated inactivation observed in human disease. Analyses of human CRPC tumor samples further underscored the clinical relevance of RB loss-induced gene expression programs, which were significantly correlated with reprogrammed E2F1 binding identified herein. Taken together, the studies presented are the first to identify the consequences of RB loss, demonstrating molecular distinction from RB inactivation and illustrating the clinical relevance of RB loss-induced E2F rewiring. Citation Format: Christopher McNair, Kexin Xu, Amy C. Mandigo, Matteo Benelli, Benjamin Leiby, Daniel Rodrigues, Johan Lindberg, Henrik Gronberg, Bram De Laere, Luc Dirix, Tapio Visakorpi, Fugen Li, Felix Y. Feng, Johann de Bono, Francesca Demichelis, Mark A Rubin, Myles Brown, Karen E. Knudsen. Differential impact of RB pathway status on E2F1 reprogramming and disease progression in human prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr IA03.

Abstract B040: Differential impact of RB status on E2F1 reprogramming in human cancer

Recent examination of advanced prostate cancer (PCa) has suggested a major mechanism of progression to castration-resistant disease (CRPC) to be loss of the retinoblastoma (RB) protein. Along with its critical role in controlling cell cycle progression, RB is known to have important tumor-suppressor functions, and has been shown in PCa to be lost exclusively in late-stage disease. Additionally, loss of RB has been shown to correlate with increased E2F1 transcript and protein expression, via E2F-dependent mechanisms. Despite the vital role RB loss has been shown to play in this fatal stage of disease, the molecular underpinnings remain undefined. Thus, in order to elucidate these CRPC specific alterations, the current study utilizes isogenic models of RB loss in combination with genome-wide binding and transcriptional studies. Data presented herein demonstrate that loss of RB is frequent in CRPC, and represents the main mechanism of RB pathways disruption in PCa as detected through analyses of tumor samples and cell-free DNA. However, this phenomenon is not correlated with changes in proliferative indices, suggesting a role for RB loss outside of canonical cell cycle control. Further, RB loss induces significant genome-wide transcriptional alterations, including upregulation in Myc, E2F, and DNA-repair related pathways. Additionally, loss of RB significantly expands E2F1 binding capacity in castrate conditions, while largely maintaining the RB-intact E2F1 cistrome. Strikingly, while the current RB/E2F1 paradigm suggests that E2F1 exclusively occupies promoter regions of DNA in order to regulate transcriptional changes, RB loss induces marked reprogramming of E2F1 occupied regions, with a distinct increase in enhancer-bound E2F1. Further, motif analyses suggest divergence away from canonical E2F1 binding motifs after RB loss, specifically in regions of expanded E2F1 binding, and additionally suggest likely interaction of novel E2F1 cofactors under RB loss conditions. Interestingly, changes in E2F1 binding capacity after RB loss were seen to be distinct from those detected after androgen-induced RB inactivation, suggesting that the molecular alterations underlying RB loss are discrete from those resulting from functional inactivation. With respect to putative mechanism, it is of note that chromatin accessibility was not significantly altered to sufficiently explain the widespread changes in E2F1 cistrome, regardless of RB status, suggesting a mechanism outside simple opportunistic E2F1 binding after RB loss. Finally, interrogation of a CRPC patient tumor cohort showed predictive capacity for an “Expanded E2F1 Signature,” resulting from genes exhibiting gained E2F1 binding and differential expression after RB loss, in predicting loss of RB in patient samples, and indicating a novel E2F1-driven set of targets vital for CRPC transition in human disease. Together, these data present the first insight into E2F1 activity resulting from RB loss, and the role these changes play in progression to CRPC. Citation Format: Christopher McNair, Kexin Xu, Amy C. Mandigo, Matteo Benelli, Benjamin Leiby, Daniel Rodrigues, Johan Lindberg, Henrik Gronberg, Mateus Crespo, Bram De Laere, Luc Dirix, Tapio Visakorpi, Fugen Li, Felix Y. Feng, Johann de Bono, Francesca Demichelis, Mark A. Rubin, Myles Brown, Karen E. Knudsen. Differential impact of RB status on E2F1 reprogramming in human cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B040.

Patients with metastatic hormone receptor-positive breast cancer express PIK3CA oncogene mutational heterogeneity in circulating tumor cells

We present a single-cell application to determine PIK3CA mutations in CTCs, which uncovered the degree of intra-patient heterogeneity in patients with metastatic hormone receptor-positive breast cancer (HR+ MBC) and high CTC count (>10 CTCs/7.5mL). Using CellSearch and DEPArray we isolated circulating tumor cells (CTCs) and white blood cells (WBCs) from peripheral blood and sequenced PIK3CA exons 9 and 20 by targeted amplicon sequencing. Comparative analysis between the primary tumor (PT, n=27 patients), circulating cell-free DNA (cfDNA, n=31 patients), single (n=146 CTCs) and pools (n=70 CTC suspensions, ranging 5-120 cells/suspension) of CTCs from 26 patients and metastases/DTCs (n=11 patients) was performed. Mutations were frequent in PT (15/27 (55.5%)) and showed slight and substantial agreement with cfDNA (n=21; kappa=0.14) and CTCs (n=22; kappa=0.6733), respectively. A wild-type genotype in WBCs indicates a high specificity. Inter-compartmental concordance was observed in 13/18 (72.2%) patients and temporal heterogeneity in 4/18 patients (22.2%). CTC analysis reveals both mutational homoand heterogeneity with cases showing the presence of different mutant and wild-type CTC subpopulations. Additionally, unique double-mutated CTCs were detected in 8/26 patients (30.7%). The developed liquid biopsy provides an insight into inter and intra-patient PIK3CA mutational heterogeneity in patients with HR+ MBC, paving the way towards the application of a more personalized medicine. Introduction Breast cancer is a heterogeneous disease, where specific genetic alterations can be associated with response or resistance to a given therapy [1,2]. The PI3K-Akt-mTor pathway is a validated target in the treatment of breast cancer [3,4]. The catalytic subunit of the phosphoinositide-3 kinase (PIK3CA) is mutated in up to 25% of patients with breast cancer, with mutation frequencies rising to 40% in the hormone receptor-positive (HR+) subgroups [5,6]. Hotspot mutations in helical and kinase domain results in constitutively enzymatic activity, enhanced tumorigenicity and the capability to form heterogeneous tumors [7-9]. Genetic discordances between PTs and the metastasis have been repeatedly observed, emphasizing the need for real-time cancer genoand phenotyping. Since the sequential assessment of biomarkers in metastases is not routinely done, a real-time ‘liquid biopsy’ with the molecular characterization of circulating tumor cells (CTCs) may constitute an alternative approach [10,11]. In both localized and advanced breast cancer the enumeration of CTCs has demonstrated clinical validity with respect to progression-free and overall survival [12]. In addition, the molecular characterization of these cells may assist in the identification of relevant biomarkers [13-15]. Assessment of the inter-patient heterogeneity allows for a useful stratification of patients in histological, molecular and functional subtypes, but is not able to predict the prevailing driving mechanisms of disease progression in an individual patient at any particular point in time. The heterogeneous nature of cancer can be demonstrated by divergence and subtle differences of the molecular profiles between early and advanced disease [16,17]. These observations may be in part explained by the emergence of different tumor cell subpopulations within the primary and/or metastatic sites [18,19]. Clinically, this subclonal development whilst receiving treatment was translated into disease progression and is defined as drug resistance. Therefore, the molecular characterization of the progressing tumor burden and the longitudinal follow-up are an essential prerequisite for personalized medicine to become a reality. This emphasizes the attractiveness of a liquid biopsy using CTCs as real-time input material, which may infer the underlying PT and/or metastases [20,21]. Performing single cell sequencing allows the detection of rare subpopulations, the assessment of the level of genetic heterogeneity and its clonal evolution during therapy [22-24]. Within this study we utilized the previously reported methodology to obtain single CTCs by di-electrophoresis [25] and subjected these samples to a PIK3CA mutational analysis by massive parallel sequencing (MPS). We have assessed the degree of mutational *Correspondence to: Bram De Laere, Oosterveldlaan 24, B-2610 Wilrijk, Belgium, Tel: +32 (0)3 443 3637; Fax: +32 (0)3 443 3036; E-mail: bramdelaere@gmail.com, Bram.DeLaere@uantwerpen.be