Development and initial clinical correlation of a DNA methylation-based blood test for prostate cancer.

BACKGROUND: One of the principle limitations for more precise management of advanced prostate cancer is the lack of accurate biomarkers allowing estimation of tumor burden, ongoing assessment of progression, and response to treatment. Although prostate-specific antigen (PSA) performs modestly, nonsecreting cancers including those with early castrate-resistance warrant investigation of other predictive biomarkers. The objectives of these studies were to develop and perform initial validation of a circulating tumor DNA (ctDNA) methylation assay. METHODS: Methylation DETection of Circulating Tumor DNA (mDETECT) is a highly multiplexed targeted sequencing DNA methylation-based ctDNA blood test that captures the vast majority of prostate cancer phenotypes due to a careful development process that ensures that each probe region is methylated in at least 50% of all methylation-based subtypes and is not methylated in normal tissues. Next-generation sequencing of targeted polymerase chain reaction (PCR) products whose amplification is biased towards methylated DNA ensures the specificity of the assay by identifying multiple tumor-specific methylated CpG residues in each read. RESULTS: The final test is comprised of 46 PCR probes to 40 regions. It is relatively resistant to contaminating normal DNA and as a result functions in both serum and plasma samples. The assay was initially validated in a variety of prostate cancer cell lines to ensure specificity. Using a small number of longitudinal samples from prostate cancer patients initiating androgen deprivation therapy, the ability of mDETECT to track tumor burden was assessed compared with PSA. The mDETECT test signal generally paralleled that of PSA increasing and decreasing commensurate with tumor evolution in these patients. In two cases it appeared to anticipate clinical progression by a number of months compared to PSA and in a PSA nonproducing case, it was able to track tumor progression. CONCLUSIONS: mDETECT offers a promising tool for the assessment of prostate cancer burden based on the sensitive detection of prostate-specific ctDNA and requires further validation.

Opposing roles for mammary epithelial-specific PPARγ signaling and activation during breast tumour progression.

BACKGROUND: Among women worldwide, breast cancer is the most commonly diagnosed cancer, and the second leading cause of cancer-related deaths. Improved understanding of breast tumourigenesis may facilitate the development of more effective therapies. Peroxisome proliferator-activated receptor (PPAR)γ is a transcription factor that regulates genes involved in insulin sensitivity and adipogenesis. Previously, we showed, using 7,12-dimethylbenz [a] anthracene (DMBA)-treated haploinsufficient PPARγ mice, that PPARγ suppresses breast tumour progression; however, the PPARγ expressing cell types and mechanisms involved remain to be clarified. Here, the role of PPARγ expression and activation in mammary epithelial cells (MG) with respect to DMBA-mediated breast tumourigenesis was investigated. METHODS: PPARγ MG knockout (PPARγ-MG KO) mice and their congenic, wild-type controls (PPARγ-WT) were treated once a week for six weeks by oral gavage with 1 mg DMBA dissolved in corn oil and maintained on a normal chow diet. At week 7, mice were randomly divided into those maintained on a normal chow diet (DMBA Only; PPARγ-WT: n = 25 and PPARγ-MG KO: n = 39) or those receiving a diet supplemented with the PPARγ ligand, rosiglitazone (ROSI, 4 mg/kg/day) (DMBA + ROSI; PPARγ-WT: n = 34 and PPARγ-MG KO: n = 17) for the duration of the 25-week study. RESULTS: Compared to DMBA Only-treated PPARγ-WTs, both breast tumour susceptibility and serum levels of proinflammatory and chemotactic cytokines, namely IL-4, eotaxin, GM-CSF, IFN-γ, and MIP-1α, were decreased among PPARγ-MG KOs. Cotreatment with ROSI significantly reduced breast tumour progression among PPARγ-WTs, correlating with increased BRCA1 and decreased VEGF and COX-2 protein expression levels in breast tumours; whereas, surprisingly DMBA + ROSI-treated PPARγ-MG KOs showed increased breast tumourigenesis, correlating with activation of COX-2. CONCLUSION: These novel data suggest MG-specific PPARγ expression and signaling is critical during breast tumourigenesis, and may serve as a strong candidate predictive biomarker for response of breast cancer patients to the use of therapeutic strategies that include PPARγ ligands.

Loss of PPARγ expression in mammary secretory epithelial cells creates a pro-breast tumorigenic environment.

Breast cancer is the leading cause of new cancer diagnoses among women. Using peroxisome proliferator-activated receptor (PPAR)γ((+/-)) mice, we showed normal expression of PPARγ was critical to stop 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast tumorigenesis. PPARγ is expressed in many breast cell types including mammary secretory epithelial (MSE) cells. MSEs proliferate as required during pregnancy, and undergo apoptosis or reversible transdifferentiation during involution once lactation is complete. Thus, MSE-specific loss of PPARγ was hypothesized to enhance DMBA-mediated breast tumorigenesis. To test this, MSE cell-specific PPARγ knockout (PPARγ-MSE KO) and control (PPARγ-WT) mice were generated, mated and allowed to nurse for three days. One week after involution, dams were treated with DMBA to initiate breast tumors, and randomized on week 7 to continue receiving a normal chow diet (DMBA Only: PPARγ-WT, n = 15; PPARγ-MSE KO, n = 25) or one supplemented with a PPARγ activating drug (DMBA + ROSI: PPARγ-WT, n = 17; PPARγ-MSE KO, n = 24), and monitored for changes in breast tumor outcomes. PPARγ-MSE KOs had significantly lower overall survival and decreased mammary tumor latency as compared to PPARγ-WT controls. PPARγ activation significantly reduced DMBA-mediated malignant mammary tumor volumes irrespective of genotype. MSE-specific PPARγ loss resulted in decreased mammary gland expression of PTEN and Bax, increased superoxide anion production, and elevated serum eotaxin and RANTES, creating a protumorigenic environment. Moreover, PPARγ activation in MSEs delayed mammary tumor growth in part by down-regulating Cox-1, Cox-2 and cyclin D1. Collectively, these studies highlight a protective role of MSE-specific PPARγ during breast tumorigenesis, and support a novel chemotherapeutic role of PPARγ activation in breast cancer.

Stromal adipocyte PPARγ protects against breast tumorigenesis.

Peroxisome proliferator-activated receptor (PPAR)γ regulates the expression of genes essential for fat storage, primarily through its activity in adipocytes. It also has a role in carcinogenesis. PPARγ normally stops the in vivo progression of 7,12-dimethylbenz[a]anthracene (DMBA)-mediated breast tumours as revealed with PPARγ haploinsufficient mice. Since many cell types associated with the mammary gland express PPARγ, each with unique signal patterns, this study aimed to define which tissues are required for PPARγ-dependent antitumour effects. Accordingly, adipocyte-specific PPARγ knockout (PPARγ-A KO) mice and their wild-type (PPARγ-WT) controls were generated, and treated with DMBA for 6 weeks to initiate breast tumorigenesis. On week 7, mice were randomized to continue on normal chow diet or one supplemented with rosiglitazone (ROSI), and followed for 25 weeks for tumour outcomes. In PPARγ-A KO versus PPARγ-WT mice, malignant mammary tumour incidence was significantly higher and mammary tumour latency was decreased. DMBA + ROSI treatment reduced average mammary tumour volumes by 50%. Gene expression analyses of mammary glands by quantitative real-time polymerase chain reaction and immunofluorescence indicated that untreated PPARγ-A KOs had significantly decreased BRCA1 expression in mammary stromal adipocytes. Compared with PPARγ-WT mice, serum leptin levels in PPARγ-A KOs were also significantly higher throughout the study. Together, these data are the first to suggest that in vivo PPARγ expression in mammary stromal adipocytes attenuates breast tumorigenesis through BRCA1 upregulation and decreased leptin secretion. This study supports a protective effect of activating PPARγ as a novel chemopreventive therapy for breast cancer.

Growth rates and outcomes of observed large renal masses.

INTRODUCTION: The natural history of small renal masses has been well defined, leading to the recommendation of active surveillance in some patients with limited life expectancy. However, this information is less clear for large renal masses (LRM), leading to ambiguity for management in the older, comorbid patient. The objective of this study was to define the natural history, including the growth rate and metastatic risk, of LRM in order to better counsel patients regarding active surveillance. METHODS: This was a retrospective review of patients with solid renal masses >4 cm that had repeated imaging identified from an institutional imaging database. Patient comorbidities and outcomes were obtained through retrospective chart analysis. Outcomes assessed included tumour growth and metastatic rates, as well as cancer-specific (CSS) and overall survival (OS) usimg Kaplan-Meier methodology. RESULTS: We identified 69 patients between 2005 and 2016 who met the inclusion criteria. Mean age at study entry was 75.5 years; mean tumour maximal dimension at study entry was 5.6 cm. CSS was 83% and OS 63% for patients presenting without metastasis, with a mean followup of 57.5 months. The mean growth rate of those that developed metastasis during followup (n=15) was 0.98 cm/year (95% confidence interval [CI] 0.33-1.63) as compared to those that did not develop metastasis (n=46), with a growth rate of 0.67 cm/year (95% CI 0.34-1) (non-significant). Seven patients had evidence of metastasis at the baseline imaging of their LRM and had subsequent growth rate of 1.47 cm/year (95% CI 0.37-2.57) (non-significant) CONCLUSIONS: Compared to small renal masses, LRM are associated with higher metastasis rates and lower CSS and more rapid growth rates. Selection criteria for recommending observation of LRM in older, comorbid patients should be more conservative than for small renal masses.