In the last years, circulating miRNAs have emerged as a new class of promising cancer biomarkers. Independent studies have shown the feasibility of using these small RNAs as tools for the diagnosis and prognosis of different types of malignancies as well as for predicting and possibly monitoring treatment response. However, despite an initial enthusiasm for their possible clinical application, widespread inconsistencies have been observed among the studies, and miRNA-based tools still represent the object of research within clinical diagnostic or treatment protocols. The poor overlap of results could be explained, at least in part, by preanalytical and analytical variables and donor-related factors that could generate artefacts, impairing an accurate quantification of circulating miRNAs. In fact, critical issues are represented by nonuniform sample choice, handling, and processing, as well as by blood cell contamination in sample preparation and lack of consensus for data normalization. In this review, we address the potential technical biases and individual-related parameters that can influence circulating miRNA studies' outcome. The exciting potential of circulating miRNAs as cancer biomarkers could confer an important advance in the disease management, but their clinical significance might not be proven without a global consensus of procedures and standardized protocols for their accurate detection.

Fenretinide [N-(4-hydroxyphenyl)-retinamide (4HPR)] is a synthetic retinoid with antitumor activity that induces apoptosis in various types of cancer cell. We showed previously that 4HPR upregulates the proapoptotic gene placental bone morphogenetic protein (PLAB), which is a mediator of 4HPR-induced apoptosis in ovarian cancer cells. Here, we investigated the signaling cascade involving PLAB that mediates the apoptotic effect. In 4HPR-sensitive ovarian cancer cells, 4HPR-induced reactive oxygen species (ROS) are involved in PLAB upregulation and apoptosis, both events abrogated by the antioxidants vitamin C and butylated hydroxyanisole. We analyzed the expression and activation of endoplasmic reticulum (ER) stress-associated molecules and show that 4HPR-induced ER stress is a consequence of ROS generation. Salubrinal, an ER stress inhibitor, abrogated 4HPR-induced PLAB upregulation and protected the cells from apoptosis. Downstream of ROS generation and ER stress, 4HPR activated c-Jun N-terminal kinase (JNK), which was inhibited by vitamin C and salubrinal. The JNK inhibitor SP600125 reduced 4HPR-induced PLAB upregulation, by decreasing PLAB mRNA half-life, and protected the cells from apoptosis. These data indicate that 4HPR-induced PLAB upregulation occurs downstream of a signaling cascade involving ROS generation, ER stress induction and JNK activation and that these steps are mediators of 4HPR-induced apoptosis.

Purpose: To investigate the potential of circulating-miRNAs (ct-miRNA) as noninvasive biomarkers to predict the efficacy of single/dual HER2-targeted therapy in the NeoALTTO study. Experimental Design: Patients with plasma samples at baseline (T0) and/or after 2 weeks (T1) of treatment were randomized into training (n ¼ 183) and testing (n ¼ 246) sets. RT-PCR-based high-throughput miRNA profiling was employed in the training set. After normalization, ct-miRNAs associated with pathologic complete response (pCR) were identified by univariate analysis. Multivariate logistic regression models were implemented to generate treatment-specific signatures at T0 and T1, which were evaluated by RT-PCR in the testing set. Event-free survival (EFS) according to ct-miRNA signatures was estimated by Kaplan-Meier method and Cox regression model. Results: In the training set, starting from 51 ct-miRNAs associated with pCR, six signatures with statistically significant predictive capability in terms of area under the ROC curve (AUC) were identified. Four signatures were confirmed in the testing set: lapatinib at T0 and T1 [AUC 0.86; 95% confidence interval (CI), 0.73-0.98 and 0.71 (0.55-0.86)], respectively; trastuzumab at T1 (0.81; 0.70-0.92); lapatinib þ trastuzumab at T1 (0.67; 0.51-0.83). These signatures were confirmed predictive after adjusting for known variables, including estrogen receptor status. ct-miRNA signatures failed to correlate with EFS. However, the levels of ct-miR-140-5p, included in the trastuzumab signature, were associated with EFS (HR 0.43; 95% CI, 0.22-0.84). Conclusions: ct-miRNAs discriminate patients with and without pCR after neoadjuvant lapatinib-and/or trastuzumab-based therapy. ct-miRNAs at week two could be valuable to identify patients responsive to trastuzumab, to avoid unnecessary combination with other anti-HER2 agents, and finally to assist deescalating treatment strategies.

We developed a simple, robust, sensitive, cost-effective, spectrophotometrically-based system to identify hemolyzed plasma/serum specimens. The procedure requires only 2 μl of sample, thus representing a useful tool for research studies and an essential pre-analytical quality control for an optimal biobanking of liquid biopsies.

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