Early Detection and Investigation of Extracellular Vesicles Biomarkers in Breast Cancer.

Breast cancer (BC) is the most commonly diagnosed malignant tumor in women worldwide, and the leading cause of cancer death in the female population. The percentage of patients experiencing poor prognosis along with the risk of developing metastasis remains high, also affecting the resistance to current main therapies. Cancer progression and metastatic development are no longer due entirely to their intrinsic characteristics, but also regulated by signals derived from cells of the tumor microenvironment. Extracellular vesicles (EVs) packed with DNA, RNA, and proteins, are the most attractive targets for both diagnostic and therapeutic applications, and represent a decisive challenge as liquid biopsy-based markers. Here we performed a study based on a multiplexed phenotyping flow cytometric approach to characterize BC-derived EVs from BC patients and cell lines, through the detection of multiple antigens. Our data reveal the expression of EVs-related biomarkers derived from BC patient plasma and cell line supernatants, suggesting that EVs could be exploited for characterizing and monitoring disease progression.

Detection and Investigation of Extracellular Vesicles in Serum and Urine Supernatant of Prostate Cancer Patients.

Prostate Cancer (PCa) is one of the most frequently identified urological cancers. PCa patients are often over-diagnosed due to still not highly specific diagnostic methods. The need for more accurate diagnostic tools to prevent overestimated diagnosis and unnecessary treatment of patients with non-malignant conditions is clear, and new markers and methods are strongly desirable. Extracellular vesicles (EVs) hold great promises as liquid biopsy-based markers. Despite the biological and technical issues present in their detection and study, these particles can be found highly abundantly in the biofluid and encompass a wealth of macromolecules that have been reported to be related to many physiological and pathological processes, including cancer onset, metastasis spreading, and treatment resistance. The present study aims to perform a technical feasibility study to develop a new workflow for investigating EVs from several biological sources. Serum and urinary supernatant EVs of PCa, benign prostatic hyperplasia (BPH) patients, and healthy donors were isolated and investigated by a fast, easily performable, and cost-effective cytofluorimetric approach for a multiplex detection of 37 EV-antigens. We also observed significant alterations in serum and urinary supernatant EVs potentially related to BPH and PCa, suggesting a potential clinical application of this workflow.

Urinary Cell-Free DNA Integrity Analysis.

Urine cell-free DNA is an important source of diagnostic markers for different diseases, especially for cancer. It could be important to achieve the urine cell-free DNA integrity to establish its provenience from cancer cells or dead inflammatory cells for necrosis in urine or from normal cells with the purpose to use it as an early diagnostic tool for urological cancers or other diseases. Here we describe a simple, noninvasive approach from urine collection to DNA integrity analysis using real-time PCR.

Analysis of Copy Number Variation in Urine: c-Myc Evaluation Using a Real-Time PCR Approach.

Urine cell-free DNA has been shown as an informative noninvasive source of biomarkers for a number of diseases, especially for urological cancers. Starting from the hypothesis that the gain of c-Myc gene is a frequent aberration in several cancer types, including prostate cancer, we analyzed c-Myc copy number variation in urine, studying a little case series of prostate cancer patients, to test its feasibility. Here we report a general protocol that may be considered to analyze gene copy number variation in the urine cell-free fraction.

Urinary Exosomes in Prostate Cancer.

The analysis of liquid biopsy as a source of diagnostic, prognostic, and predictive biomarkers is still object of the main research in the prostate cancer field. Many advantages, such as less invasiveness compared to plasma or serum analysis and the rich content, confer to urine a role as an interesting fluid to be analysed especially in urological diseases. Here we report a workflow focused on profile, concentration, and protein surface characterization of EVs from urinary supernatant.

Genome-wide plasma DNA methylation features of metastatic prostate cancer.

Tumor DNA circulates in the plasma of cancer patients admixed with DNA from noncancerous cells. The genomic landscape of plasma DNA has been characterized in metastatic castration-resistant prostate cancer (mCRPC) but the plasma methylome has not been extensively explored. Here, we performed next-generation sequencing (NGS) on plasma DNA with and without bisulfite treatment from mCRPC patients receiving either abiraterone or enzalutamide in the pre- or post-chemotherapy setting. Principal component analysis on the mCRPC plasma methylome indicated that the main contributor to methylation variance (principal component one, or PC1) was strongly correlated with genomically determined tumor fraction (r = -0.96; P < 10-8) and characterized by hypermethylation of targets of the polycomb repressor complex 2 components. Further deconvolution of the PC1 top-correlated segments revealed that these segments are comprised of methylation patterns specific to either prostate cancer or prostate normal epithelium. To extract information specific to an individual's cancer, we then focused on an orthogonal methylation signature, which revealed enrichment for androgen receptor binding sequences and hypomethylation of these segments associated with AR copy number gain. Individuals harboring this methylation pattern had a more aggressive clinical course. Plasma methylome analysis can accurately quantitate tumor fraction and identify distinct biologically relevant mCRPC phenotypes.

Genetic Predisposition to Breast and Ovarian Cancers: How Many and Which Genes to Test?

Breast and ovarian cancers are some of the most common tumors in females, and the genetic predisposition is emerging as one of the key risk factors in the development of these two malignancies. BRCA1 and BRCA2 are the best-known genes associated with hereditary breast and ovarian cancer. However, recent advances in molecular techniques, Next-Generation Sequencing in particular, have led to the identification of many new genes involved in the predisposition to breast and/or ovarian cancer, with different penetrance estimates. TP53, PTEN, STK11, and CDH1 have been identified as high penetrance genes for the risk of breast/ovarian cancers. Besides them, PALB2, BRIP1, ATM, CHEK2, BARD1, NBN, NF1, RAD51C, RAD51D and mismatch repair genes have been recognized as moderate and low penetrance genes, along with other genes encoding proteins involved in the same pathways, possibly associated with breast/ovarian cancer risk. In this review, we summarize the past and more recent findings in the field of cancer predisposition genes, with insights into the role of the encoded proteins and the associated genetic disorders. Furthermore, we discuss the possible clinical utility of genetic testing in terms of prevention protocols and therapeutic approaches.

Androgen receptor in breast cancer: A wolf in sheep's clothing? A lesson from prostate cancer.

The possibility that a receptor for androgen is expressed in Breast Cancer (BC) is fascinating given that the tumor is predominantly estrogen-dependent. The androgen receptor (AR) is emerging as a new marker and a potential new therapeutic target in the treatment of BC patients. The recent availability of selective AR inhibitors (e.g. bicalutamide, enzalutamide, apalutamide) approved for the treatment of prostate cancer has opened up the possibility to use them in BC patients whose tumors express AR. However, AR appears to have various functions according to the BC subtype, e.g. ER-positive or triple negative BC and the patient prognosis is different on the basis of the presence or absence of estrogen and progesterone receptors. Moreover, a different AR expression was seen according to the various ethnicities. Of note, in population at low economical income, the availability of anti-AR compounds at low cost could open the possibility to treat AR-positive triple negative BC that are highly present in these populations. Up to now, AR detection is not routinely performed in BC. The standardization of AR detection methods could render AR an easily detectable marker in primary BC and metastatic samples. Nevertheless, the overall concordance of 60% of AR expression in primary tumor and metastasis implies that a clinician who need the AR value to give anti-AR therapy should have the data on both the tumor materials. Following the comprehensive studies on prostate cancer the possibility to test AR on liquid biopsies suggest the use of this biomarker for a real-time disease monitoring. Finally, considering the possibility to treat patients with immune checkpoint inhibitors there is the need to know the relation between microenvironment and AR in BC.

Know your enemy: Genetics, aging, exposomic and inflammation in the war against triple negative breast cancer.

Triple negative breast cancer (TNBC) is one of the most biologically aggressive and very often lethal breast disease. It is one of the most puzzling women malignancies, and it currently appears not to be a good candidate to a standardized, unanimously accepted and sufficiently active therapeutic strategy. Fast proliferating and poorly differentiated, it is histopathologically heterogeneous, and even more ambiguous at the molecular level, offering few recurrent actionable targets to the clinicians. It is a formidable and vicious enemy that requires a huge investigational effort to find its vital weak spots. Here, we provide a broad review of "old but gold" biological aspects that taken together may help in finding new TNBC management strategies. A better and updated knowledge of the origins, war-like tactics, refueling mechanisms and escape routes of TNBC, will help in moving the decisive steps towards its final defeat.

Plasma AR status and cabazitaxel in heavily treated metastatic castration-resistant prostate cancer.

BACKGROUND: Plasma androgen receptor (AR) copy number status has been identified as a potential biomarker of response in patients with metastatic castration-resistant prostate cancer (mCRPC) receiving docetaxel or the AR-targeted therapies abiraterone or enzalutamide. However, the relevance of plasma AR status in the context of cabazitaxel therapy is unknown. PATIENTS AND METHODS: Between September 2011 and January 2018, pretherapy plasma samples were collected from 155 patients treated with second- or third-line cabazitaxel at standard or reduced dose in different biomarker protocols. Droplet digital polymerase chain reaction was used to identify plasma AR gain and normal samples. The primary objective was to evaluate associations of plasma AR status with treatment outcome. In an exploratory analysis, a comparison between plasma AR and treatment type was investigated by incorporating updated data from our prior study of 85 post-docetaxel patients receiving abiraterone or enzalutamide. RESULTS: We observed a shorter median overall survival (OS) and progression-free survival (PFS) in AR-gained compared to AR-normal patients (OS 10.5 versus 14.1 months, hazard ratio (HR) = 1.44, 95% confidence interval [CI] 0.98-2.13, P = 0.064 and PFS 4.0 versus 5.0 months, HR = 1.47, 95% CI 1.05-2.07, P = 0.024). In patients with mCRPC receiving second-line therapies, a significant treatment interaction was observed between plasma AR and cabazitaxel versus AR-directed therapies for OS (P = 0.041) but not PFS (P = 0.244). In an exploratory analysis, AR-gained patients treated with initial reduced dose of cabazitaxel had a significantly shorter median OS (7.3 versus 11.5 months, HR = 1.95, 95% CI 1.13-3.38, P = 0.016) and PFS (2.7 versus 5.0 months, HR = 2.27, 95% CI 1.39-3.71, P = 0.001). CONCLUSION: Plasma AR status has a potential clinical utility in patients being considered for cabazitaxel. Validation of these findings in prospective trials is warranted.

Plasma Androgen Receptor and Docetaxel for Metastatic Castration-resistant Prostate Cancer.

Plasma androgen receptor (AR) gain identifies metastatic castration-resistant prostate cancer (mCRPC) patients with worse outcome on abiraterone/enzalutamide, but its relevance in the context of taxane chemotherapy is unknown. We aimed to evaluate whether docetaxel is active regardless of plasma AR and to perform an exploratory analysis to compare docetaxel with abiraterone/enzalutamide. This multi-institutional study was a pooled analysis of AR status, determined by droplet digital polymerase chain reaction, on pretreatment plasma samples. We evaluated associations between plasma AR and overall/progression-free survival (OS/PFS) and prostate-specific antigen (PSA) response rate in 163 docetaxel-treated patients. OS was significantly shorter in case of AR gain (hazard ratio [HR]=1.61, 95% confidence interval [CI]=1.08-2.39, p=0.018), but not PFS (HR=1.04, 95% CI 0.74-1.46, p=0.8) or PSA response (odds ratio=1.14, 95% CI=0.65-1.99, p=0.7). We investigated the interaction between plasma AR and treatment type after incorporating updated data from our prior study of 73 chemotherapy-naïve, abiraterone/enzalutamide-treated patients, with data from 115 first-line docetaxel patients. In an exploratory analysis of mCRPC patients receiving first-line therapies, a significant interaction was observed between plasma AR and docetaxel versus abiraterone/enzalutamide for OS (HR=0.16, 95% CI=0.06-0.46, p<0.001) and PFS (HR=0.31, 95% CI=0.12-0.80, p=0.02). Specifically, we reported a significant difference for OS favoring abiraterone/enzalutamide for AR-normal patients (HR=1.93, 95% CI=1.19-3.12, p=0.008) and a suggestion favoring docetaxel for AR-gained patients (HR=0.53, 95% CI=0.24-1.16, p=0.11). These data suggest that AR-normal patients should receive abiraterone/enzalutamide and AR-gained could benefit from docetaxel. This treatment selection merits prospective evaluation in a randomized trial. PATIENT SUMMARY: We investigated whether plasma androgen receptor (AR) predicted outcome in metastatic castration-resistant prostate cancer (mCRPC) patients treated with docetaxel, and we performed an exploratory analysis in patients treated with docetaxel or AR-directed drugs as first-line mCRPC therapy. We showed that plasma AR normal favored hormonal treatment, whilst plasma AR-gained patients may have had a longer response to docetaxel, suggesting that plasma AR status could be a useful treatment selection biomarker.

Studying Copy Number Variations in Cell-Free DNA: The Example of AR in Prostate Cancer.

Serum and plasma cell-free DNA (cfDNA) has been shown as an informative noninvasive source of biomarkers for different diseases, including cancer. Starting from the hypothesis that the gain of androgen receptor (AR) gene is a frequent aberration in advanced prostate cancer patients, we analyzed it in cfDNA as a potential predictive biomarker of specific treatments. Here we report a general protocol that may be considered to analyze gene copy number variations in serum or plasma fluids.

From cfDNA to Sequencing: Workflows and Potentials.

Cell-free DNA (cfDNA) is acquiring increasingly importance in oncologic clinical practice, mostly due to its role in predicting the onset of therapy resistance by following the mutation status changes of patients. In this field, high-sensitivity methods like next-generation sequencing (NGS) could help to accurately detect somatic mutations at low frequency. Here, we report some advantages and limitations of NGS approaches for cfDNA mutation analyses with the aim of choosing the most suitable in terms of sensitivity, specificity, data output, costs, and time work.

Urinary Cell-Free DNA: Potential and Applications.

Urine could be a convenient source of biomarkers for different diseases and clinical applications, mostly for cancer diagnosis, prognosis, treatment monitoring, and prenatal diagnosis. The ultra-noninvasive sampling and the possibility to analyze large volume are the main undisputed advantages of urine-based protocols. Recent and comprehensive studies showed that urinary cell-free DNA (ucfDNA) is informative to identify the genomic signature of patients, resulting in a huge tool to track the tumor evolution and for personalized medicine in urological and non-urological cancer.In this chapter, we reported the main published evidences on ucfDNA, with the aim at discussing its promising and translatable role in clinical practices.

Urinary Cell-Free DNA: Isolation, Quantification, and Quality Assessment.

Urine cell-free DNA is an important source of diagnostic markers for different diseases (e.g., cancer and prenatal diagnosis). It is important to achieve a simple and fast protocol to maximize the recovery of DNA from urine supernatant and to assess its quality. Here we describe a simple approach from urine collection to DNA quality assessment for downstream analyses.

Plasma androgen receptor and serum chromogranin A in advanced prostate cancer.

Recently, mixed forms between adenocarcinoma and neuroendocrine prostate cancer (NEPC) have emerged that are characterized by persistent androgen receptor (AR)-signalling and elevated chromogranin A (CgA) levels. The main aim of this study was to analyze castration-resistant prostate cancer (CRPC) patients treated with abiraterone or enzalutamide, assessing progression-free/overall survival (PFS/OS) in association with circulating AR and CgA. AR aberrations were analyzed by droplet digital PCR in pre-treatment plasma samples collected from two biomarker protocols [197 patients from a retrospective study (REC 2192/2013) and 59 from a prospective trial (REC 6798/2015)]. We subdivided patients into three groups according to CgA by receiver-operating characteristic (ROC) curves. In the primary cohort, plasma AR gain and mutations (p.L702H/p.T878A) were detected in 78 (39.6%) and 16 (8.1%) patients, respectively. We observed a significantly worse PFS/OS in patients with higher-CgA than in patients with normal-CgA, especially those with no AR-aberrations. Multivariable analysis showed AR gain, higher-CgA and LDH levels as independent predictors of PFS [hazard ratio (HR) = 2.16, 95% confidence interval (95% CI) 1.50-3.12, p < 0.0001, HR = 1.73, 95% CI 1.06-2.84, p = 0.026, and HR = 2.13, 95% CI 1.45-3.13, p = 0.0001, respectively) and OS (HR = 1.72, 95% CI 1.15-2.57, p = 0.008, HR = 3.63, 95% CI 2.13-6.20, p < 0.0001, and HR = 2.31, 95% CI 1.54-3.48, p < 0.0001, respectively). These data were confirmed in the secondary cohort. Pre-treatment CgA detection could be useful to identify these mixed tumors and would seem to have a prognostic role, especially in AR-normal patients. This association needs further evaluation in larger prospective cohorts.

Carcinosarcoma of the prostate: case report with molecular and histological characterization.

BACKGROUND:: We report a case of prostatic carcinosarcoma, a rare variant of prostatic cancer, which is composed of a mixture of epithelial and mesenchymal components with a generally poor outcome. AIMS AND METHODS:: We aim to identify molecular alterations, in particular copy number variations of AR and c -MYC genes, methylation and expression of glutathione S-transferase P1 (GSTP1), programmed death-ligand 1 (PD-L1), AR, and phosphorylated AR expression. RESULTS:: We found a distinct molecular pattern between adenocarcinoma and carcinosarcoma, which was characterized by high AR copy number variation gain; positive expression of PD-L1, AR, and phosphorylated AR; low espression of GSTP1 in epithelial component. The sarcomatoid component had a lower gain of the AR gene, and no expression of PD-L1, AR, phosphorylated AR, or GSTP1. Both components had a gain of c-MYC copy number variation. CONCLUSIONS:: Our findings suggest that carcinosarcoma has specific molecular characteristics that could be indicative for early diagnosis and treatment selection.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR.

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Association among metabolic syndrome, inflammation, and survival in prostate cancer.

BACKGROUND: Metabolic syndrome (MS) and inflammation (INF) alterations are among the factors involved in cancer progression. The study aimed to assess the relationship between MS and INF and its effect on progression-free/overall survival (PFS/OS) in metastatic castration-resistant prostate cancer (mCRPC) treaed with abiraterone or enzalutamide. METHODS: We, retrospectively, evaluated patients with mCRPC in 7 Italian Institutes between March 2011 and October 2016. MS was defined by modified adult treatment panel-III criteria. INF was characterized by at least one of these criteria: neutrophil to lymphocyte ratio ≥ 3, elevated erythrocyte sedimentation rate or C-reactive protein. RESULTS: Eighty-three of 551 (15.1%) patients met MS criteria at baseline and 34 (6.2%) during treatment. MS patients (MS+) presented a greater INF profile compared to MS- (P<0.0001). Median PFS was 3.7 for MS+ vs. 8.7 months for MS- (hazard ratio [HR] = 2.77; 95% CI: 2.12-3.61; P<0.0001). Median OS was 6.9 and 19 months in MS+ and MS-, respectively (HR = 3.43; 95% CI: 2.56-4.58; P<0.0001). We also demonstrated INF led to shorter PFS and OS (4.5 vs. 8.5 months, HR = 1.48, 95% CI: 1.15-1.90, P = 0.002, and 11.2 vs. 18.8 months, HR =1.66, 95% CI: 1.26-2.18, P = 0.0003, respectively). The combination of MS with INF provided the identification of high-risk prognostic group (MS+/INF+ vs. MS-/INF-) with worse PFS (3.7 vs. 9 months, HR = 2.7, 95% CI: 1.88-3.89, P<0.0001) and OS (6.3 vs. 20.4 months, HR = 4.04, 95% CI: 2.75-5.93, P<0.0001). Multivariable analysis confirmed that MS was independently associated with PFS (HR = 2.07; 95% CI: 1.03-4.18; P = 0.041) and OS (HR = 4.87; 95% CI: 2.36-10.03; P<0.0001). The absence of INF as an independent predictor of survival underlined the correlation between MS/INF. CONCLUSIONS: Pretreatment identification of MS and INF alterations might represent an available and easy tool for better prognostication of patients with mCRPC. A prospective evaluation is warranted.

Publisher Correction: Transcribed ultraconserved region 339 promotes carcinogenesis by modulating tumor suppressor microRNAs.

In the originally published version of this Article, the positions of the final two authors in the author list were inadvertently inverted during the production process. This error has now been corrected in both the PDF and HTML versions of the Article.