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(NSCLCs). Several tyrosine kinase inhibitors (TKIs) have shown high efficacy in patients whose tumors harbour a ROS1 fusion. However, the limited availability of preclinical models of ROS1-positive NSCLC hinders the discovery of new drugs and the understanding of the mechanisms underlying drug resistance and strategies to overcome it. Methods: The ADK-VR2 cell line was derived from the pleural effusion of a treatment-naïve NSCLC patient bearing SDC4-ROS1 gene fusion. The sensitivity of ADK-VR2 and its crizotinib-resistant clone ADK-VR2 AG143 (selected in 3D culture in the presence of crizotinib) to different TKIs was tested in vitro, in both 2D and 3D conditions. Tumorigenic and metastatic ability was assessed in highly immunodeficient mice. In addition, crizotinib efficacy on ADK-VR2 was evaluated in vivo. Results: 2D-growth of ADK-VR2 cells was partially inhibited by crizotinib. On the contrary, the treatment with other TKIs, such as lorlatinib, entrectinib and DS-6051b, did not result in cell growth inhibition. TKIs showed dramatically different efficacy on ADK-VR2 cells, depending on the cell culture conditions. In 3D culture, ADK-VR2 growth was indeed almost totally inhibited by lorlatinib and DS-6051b. The clone ADK VR2 AG143 showed higher resistance to crizotinib treatment in vitro, compared to its parental cell line, in both 2D and 3D cultures. Similarly to ADK-VR2, ADK-VR2 AG143 growth was strongly inhibited by lorlatinib in 3D conditions. Nevertheless, ADK-VR2 AG143 sphere formation was less affected by TKIs treatment, compared to the parental cell line. In vivo experiments highlighted the high tumorigenic and metastatic ability of ADK-VR2 cell line, which, once injected in immunodeficient mice, gave rise to both spontaneous and experimental lung metastases while the crizotinib-resistant clone ADK-VR2 AG143 showed a slower growth in vivo. In addition, ADK-VR2 tumor growth was significantly reduced but not eradicated by crizotinib treatment. Conclusions: The ADK-VR2 cell line is a promising NSCLC preclinical model for the study

Cancer cells reprogram their metabolism to support their growth. Since the discovery of the Warburg effect, several other metabolic alterations and metabolites have been described in cancer cells, including lactate, glutamine and lipid metabolism reprogramming. Together these alterations provide rapidly dividing tumor cells with metabolic intermediates needed for nucleotide, protein and fatty acid biosynthesis. MicroRNAs are a class of small non-coding RNAs involved in the regulation of virtually all biological pathways. Altered microRNA expression patterns are associated with the onset and development of several diseases, including cancer. Tumor suppressor microRNAs targeting molecules involved in tumor metabolism are frequently downregulated in cancers. Therefore, microRNAs can serve as potential tumor biomarkers and also represent interesting therapeutic targets. This review summarizes recent findings about microRNAs involved in the regulation of tumor metabolism.

ARID1A belongs to a class of chromatin regulatory proteins that function by maintaining accessibility at most promoters and enhancers, thereby regulating gene expression. The high frequency of ARID1A alterations in human cancers has highlighted its significance in tumorigenesis. The precise role of ARID1A in cancer is highly variable since ARID1A alterations can have a tumor suppressive or oncogenic role, depending on the tumor type and context. ARID1A is mutated in about 10% of all tumor types including endometrial, bladder, gastric, liver, biliopancreatic cancer, some ovarian cancer subtypes, and the extremely aggressive cancers of unknown primary. Its loss is generally associated with disease progression more often than onset. In some cancers, ARID1A loss is associated with worse prognostic features, thus supporting a major tumor suppressive role. However, some exceptions have been reported. Thus, the association of ARID1A genetic alterations with patient prognosis is controversial. However, ARID1A loss of function is considered conducive for the use of inhibitory drugs which are based on synthetic lethality mechanisms. In this review we summarize the current knowledge on the role of ARID1A as tumor suppressor or oncogene in different tumor types and discuss the strategies for treating ARID1A mutated cancers.

Background In the last years, several efforts have been made to classify colorectal cancer (CRC) into well-defined molecular subgroups, representing the intrinsic inter-patient heterogeneity, known as Consensus Molecular Subtypes (CMSs). Methods In this work, we performed a meta-analysis of CRC patients stratified into four CMSs. We identified a negative correlation between a high level of anaplastic lymphoma kinase (ALK) expression and relapse-free survival, exclusively in CMS1 subtype. Stemming from this observation, we tested cell lines, patient-derived organoids and mice with potent ALK inhibitors, already approved for clinical use. Results ALK interception strongly inhibits cell proliferation already at nanomolar doses, specifically in CMS1 cell lines, while no effect was found in CMS2/3/4 groups. Furthermore, in vivo imaging identified a role for ALK in the dynamic formation of 3D tumor spheroids. Consistently, ALK appeares constitutively phosphorylated in CMS1, and it signals mainly through the AKT axis. Mechanistically, we found that CMS1 cells display several copies of ALKAL2 ligand and ALK-mRNAs, suggesting an autocrine loop mediated by ALKAL2 in the activation of ALK pathway, responsible for the invasive phenotype. Consequently, disruption of ALK axis mediates the pro-apoptotic action of CMS1 cell lines, both in 2D and 3D and enhanced cell-cell adhesion and e-cadherin organization. In agreement with all these findings, the ALK signature encompassing 65 genes statistically associated with worse relapse-free survival in CMS1 subtype. Finally, as a proof of concept, the efficacy of ALK inhibition was demonstrated in both patient-derived organoids and in tumor xenografts in vivo. Conclusions Collectively, these findings suggest that ALK targeting may represent an attractive therapy for CRC, and CMS classification may provide a useful tool to identify patients who could benefit from this treatment. These findings offer rationale and pharmacological strategies for the treatment of CMS1 CRC.

The molecular background of hepatocellular carcinoma (HCC) is highly heterogeneous, and biomarkers predicting response to treatments are an unmet clinical need. We investigated miR-30e-3p contribution to HCC phenotype and response to sorafenib, as well as the mutual modulation of TP53/MDM2 pathway, in HCC tissues and preclinical models. MiR-30e-3p was downregulated in human and rat HCCs, and its downregulation associated with TP53 mutations. TP53 contributed to miR-30e-3p biogenesis, and MDM2 was identified among its target genes, establishing an miR-30e-3p/TP53/MDM2 feedforward loop and accounting for miR-30e-3p dual role based on TP53 status. EpCAM, PTEN, and p27 were demonstrated as miR-30e-3p additional targets mediating its contribution to stemness and malignant features. In a preliminary cohort of patients with HCC treated with sorafenib, increased miR-30e-3p circulating levels predicted the development of resistance. In conclusion, molecular background dictates miR-30e-3p dual behavior in HCC. Mdm2 targeting plays a predominant tumor suppressor function in wild-type TP53 contexts, whereas other targets such as PTEN, p27, and EpCAM gain relevance and mediate miR-30e-3p oncogenic role in nonfunctional TP53 backgrounds. Increased circulating levels of miR-30e-3p predict the development of sorafenib resistance in a preliminary series of patients with HCC and deserve future investigations. Significance: The dual role of miR-30e-3p in HCC clarifies how the molecular context dictates the tumor suppressor or oncogenic function played by miRNAs.

The expression of non–coding RNAs (ncRNAs) is dysregulated in human cancers. The transcribed ultraconserved regions (T-UCRs) express long ncRNAs involved in human carcinogenesis. T-UCRs are non-coding genomic sequence that are 100% conserved across humans, rats and mice. Conservation of genomic sequences across species intrinsically implies an essential functional role and so we considered the expression of T-UCRs in lung cancer. Using a custom microarray we analyzed the global expression of T-UCRs. Among these T-UCRs, the greatest variation was observed for antisense ultraconserved element 83 (uc.83-), which was upregulated in human lung cancer tissues compared with adjacent non cancerous tissues. Even though uc.83- is located within the long intergenic non-protein coding RNA 1876 (LINC01876) gene, we found that the transcribed uc.83- is expressed independently of LINC01876 and was cloned as a 1143-bp RNA gene. In this study, functional analysis confirmed important effects of uc.83- on genes involved in cell growth of human cells. siRNA against uc.83- decreased the growth of lung cancer cells while the upregulation through a vector overexpressing the uc.83- RNA increased cell proliferation. We also show the oncogenic function of uc.83- is mediated by the phosphorylation of AKT and ERK 1/2, two important biomarkers of lung cancer cell proliferation. Based on our findings, inhibition against uc.83- could be a future therapeutic treatment for NSCLC to achieve simultaneous blockade of pathways involved in lung carcinogenesis.

Simple Summary For the first time, neuronal features are described in blastic plasmacytoid dendritic cell neoplasm (BPDCN) by a complex array of molecular techniques, including microRNA and gene expression profiling, RNA and Chromatin immunoprecipitation sequencing, and immunohistochemistry. The discovery of unexpected neural features in BPDCN may change our vision of this disease, leading to the designing of a new BPDCN cell model and to re-thinking the relations occurring between BPDCN and nervous system. The observed findings contribute to explaining the extreme tumor aggressiveness and also to propose novel therapeutic targets. In view of this, the identification, in this work of new potential neural metastatic inducers might open the way to therapeutic approaches for BPDCN patients based on the use of anti-neurogenic agents. Abstract Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive hematologic malignancy originating from plasmacytoid dendritic cells (pDCs). The microRNA expression profile of BPDCN was compared to that of normal pDCs and the impact of miRNA dysregulation on the BPDCN transcriptional program was assessed. MiRNA and gene expression profiling data were integrated to obtain the BPDCN miRNA-regulatory network. The biological process mainly dysregulated by this network was predicted to be neurogenesis, a phenomenon raising growing interest in solid tumors. Neurogenesis was explored in BPDCN by querying different molecular sources (RNA sequencing, Chromatin immunoprecipitation-sequencing, and immunohistochemistry). It was shown that BPDCN cells upregulated neural mitogen genes possibly critical for tumor dissemination, expressed neuronal progenitor markers involved in cell migration, exchanged acetylcholine neurotransmitter, and overexpressed multiple neural receptors that may stimulate tumor proliferation, migration and cross-talk with the nervous system. Most neural genes upregulated in BPDCN are currently investigated as therapeutic targets.

Human hepatocellular carcinoma (HCC) is the most frequent primary tumor of the liver and the third cause of cancer-related deaths. The multikinase inhibitor sorafenib is a systemic drug for unresectable HCC. The identification of molecular biomarkers for the early diagnosis of HCC and responsiveness to treatment are needed. In this work, we performed an exploratory study to investigate the longitudinal levels of cell-free long ncRNA GAS5 and microRNAs miR-126-3p and -23b-3p in a cohort of 7 patients during the period of treatment with sorafenib. We used qPCR to measure the amounts of GAS5 and miR-126-3p and droplet digital PCR (ddPCR) to measure the levels of miR-23b-3p. Patients treated with sorafenib displayed variable levels of GAS5, miR-126-3p and miR-23b-3p at different time-points of follow-up. miR-23b-3p was further measured by ddPCR in 37 healthy individuals and 25 untreated HCC patients. The amount of miR-23b-3p in the plasma of untreated HCC patients was significantly downregulated if compared to healthy individuals. The ROC curve analysis underlined its diagnostic relevance. In conclusion, our results highlight a potential clinical significance of circulating miR-23b-3p and an exploratory observation on the longitudinal plasmatic levels of GAS5, miR-126-3p and miR-23b-3p during sorafenib treatment.

The incidence of cutaneous melanoma (CM) has increased in the past few decades. The biology of melanoma is characterized by a complex interaction between genetic, environmental and phenotypic factors. A greater understanding of the molecular mechanisms that promote melanoma cell growth and dissemination is crucial to improve diagnosis, prognostication, and treatment of CM. Both small and long non‐coding RNAs (lncRNAs) have been identified to play a role in melanoma biology; microRNA and lncRNA expression is altered in transformed melanocytes and this in turn has functional effects on cell proliferation, apoptosis, invasion, metastasis, and immune response. Moreover, specific dysregulated ncRNAs were shown to have a diagnostic or prognostic role in melanoma and to drive the establishment of drug resistance. Here, we review the current literature on small and lncRNAs with a role in melanoma, with the aim of putting into some order this complex jigsaw puzzle.

Metastasis is responsible for the majority of cancer‐related deaths. Particularly, challenging is the management of metastatic cancer of unknown primary site (CUP), whose tissue of origin (TOO) remains undetermined even after extensive investigations and whose therapy is rather unspecific and poorly effective. Molecular approaches to identify the most probable TOO of CUPs can overcome some of these issues. In this study, we applied a predetermined set of 89 microRNAs (miRNAs) to infer the TOO of 53 metastatic cancers of unknown or uncertain origin. The miRNA expression was assessed with droplet digital PCR in 159 samples, including primary tumors from 17 tumor classes (reference set) and metastases of known and unknown origin (test set). We combined two different statistical models for class prediction to obtain the most probable TOOs: the nearest shrunken centroids approach of Prediction Analysis of Microarrays (PAMR) and the least absolute shrinkage and selection operator (LASSO) models. The molecular test was successful for all formalin‐fixed paraffin‐embedded samples and provided a TOO identification within 1 week from the biopsy procedure. The most frequently predicted origins were gastrointestinal, pancreas, breast, lung, and bile duct. The assay was applied also to multiple metastases from the same CUP, collected from different metastatic sites: The predictions showed a strong agreement, intrinsically validating our assay. The final CUPs' TOO prediction was compared with the clinicopathological hypothesis of primary site. Moreover, a panel of 13 miRNAs proved to have prognostic value and be associated with overall survival in CUP patients. Our study demonstrated that miRNA expression profiling in CUP samples could be employed as diagnostic and prognostic test. Our molecular analysis can be performed on request, concomitantly with standard diagnostic workup and in association with genetic profiling, to offer valuable indications about the possible primary site, thereby supporting treatment decisions., Cancer of unknown primary site (CUP) patients suffer the burden of a metastatic disease whose site of origin is unidentifiable, even after intensive clinical investigations. The lack of a recognized primary site limits patients' treatment options. In this manuscript, we present a molecular assay, based on digital miRNA profiling, that allowed the prediction of the primary site of 53 CUPs.

Simple Summary Patients with cancer of unknown primary site suffer the burden of an uncertain disease, which is characterized by the impossibility to identify the tissue where the tumor has originated. The identification of the primary site of a tumor is of great importance for the patient to have access to site-specific treatments and be enrolled in clinical trials. Therefore, patients with cancer of unknown primary have reduced therapeutic opportunities and poor prognosis. Advancements have been made in the molecular characterization of this tumor, which could be used to infer the tumor site-of-origin and thus broaden the diagnostic outcome. Moreover, we describe here the novel therapeutic opportunities that are based on the genetic and immunophenotypic characterization of the tumor, and thus independent from the tumor type, which could provide most benefit to patients with cancer of unknown primary. Abstract Distant metastases are the main cause of cancer-related deaths in patients with advanced tumors. A standard diagnostic workup usually contains the identification of the tissue-of-origin of metastatic tumors, although under certain circumstances, it remains elusive. This disease setting is defined as cancer of unknown primary (CUP). Accounting for approximately 3–5% of all cancer diagnoses, CUPs are characterized by an aggressive clinical behavior and represent a real therapeutic challenge. The lack of determination of a tissue of origin precludes CUP patients from specific evidence-based therapeutic options or access to clinical trial, which significantly impacts their life expectancy. In the era of precision medicine, it is essential to characterize CUP molecular features, including the expression profile of non-coding RNAs, to improve our understanding of CUP biology and identify novel therapeutic strategies. This review article sheds light on this enigmatic disease by summarizing the current knowledge on CUPs focusing on recent discoveries and emerging diagnostic strategies.

BACKGROUND: The incidence of cutaneous melanoma (cm) has increased in the last decades. germline mutations in the high-penetrance melanoma susceptibility gene CDKN2A (Cyclin-dependent kinase inhibitor 2a) are associated with a younger age at diagnosis and an increased risk to develop pancreatic cancer. METHODS: We retrospectively analyzed the data of patients with prior diagnosis of cm referring to our service from January 2005 to may 2017. the aim was to investigate the rate of multiple cms (mPm), assessing their clinical/pathological features. moreover, the genetic tests of patients who had undergone CDKN2A/CDKN2B, CDK4 and MITF screening were evaluated. RESULTS: One hundred fifteen patients (9.26%) were diagnosed with MPMs: 70 males (60.87%) and 45 women (39.13%). 75 patients (43 males and 32 females) underwent genetic screening for germline mutations. The screening revealed that 4/75 patients (5.33%) were carriers of the non-synonymous missense variation c.442g>a (p.ala148thr) in CDKN2A exon 2 in heterozygosis, 3 of whom had at least one in-situ melanoma. In 1 patient (1.33%) we detected the variation c.249C>A, p.His83Gln in CDKN2A exon 2 in heterozygosis and in 1 patient (1.33%) the mutation c.952g>a (p.glu318lys) in MITF gene was found. CONCLUSIONS: This study confirms the need for a full body skin examination and a prolonged surveillance in patients affected by cM, as MPMs were detected in up to 10% of total cases in our series and synchronous lesions in 1/5. Moreover, it reflects the great variability of cM high-susceptibility genes mutational status within the Italian territory. Patients carrying c.952g>a (p.glu318lys) MITF mutation have a higher risk to develop a nodular cm.

Non-small cell lung cancer (NSCLC) remains a problem worldwide due to its rapid progression and low rate of response to treatment. The heterogeneity of these tumors observed in histopathology exam but also in the mutational status and gene expression pattern makes this malignancy difficult to treat in clinic. The present study investigated the effect of miR-21 and let-7 family members as prognostic biomarkers in NSCLC patients based on the results published in different studies regarding this subject until March 2019. The analysis revealed that these two transcripts are steady biomarkers for prediction of patient outcome or survival. Upregulated expression of miR-21 is associated with poor outcome of patients with NSCLC [HR = 1.87, 95% CI = (1.41, 2.47), p < 0.001]. The analysis regarding let-7 family, specifically let-7a/b/e/f, revealed that downregulated expression of these transcripts predicts poor outcome for NSCLC patients [HR = 2.61, 95% CI = (1.58, 4.30), p < 0.001]. Besides, the reliability of these microRNAs is reflected in the fact that their prognostic significance is constant given the different sample types (tissue, FFPE tissue, serum, serum/plasma or exosomes) used in the selected studies.

not present

Breslow thickness (BT) is the most important histopathologic factor for primary melanoma staging. BT determines the margins for wide local excision whether sentinel lymph node biopsy should be performed and subsequent melanoma staging, and patient management. The correct determination of a 0.8-mm cutoff in melanoma is important for pathologists because discrepancies leading to a change in stage can have significant clinical implications, including incorrect and/or inappropriate prognostic information, investigation, management, and follow-up. Difficulties in BT determination are mostly represented by the presence of regression or melanoma associated with a pre-existing nevus. This study aimed at investigating a molecular parameter, namely microRNA (miRNA) expression, in reference to BT assessment. Melanoma cell proliferation is influenced by miRNA dysregulation. Indeed, some miRNAs sustain and induce proliferative signals or repress growth-suppressive pathways, thereby promoting melanoma carcinogenesis. To identify the miRNAs correlating with BT, we analyzed our global miRNA expression data of 20 thin melanomas and identified two potential candidates, miR-21-5p and miR-146a-5p. We assessed the expression of these two specific miRNAs in 90 archive formalin-fixed and paraffin-embedded samples of superficially spreading melanomas (SSMs) and 25 nodular melanomas from two independent cohorts and correlated the individual and combined miRNA expression with BT and other tumor characteristics. The individually normalized expression of miR-21-5p and miR-146a-5p showed a highly significant and linear correlation with BT in SSM, and their combined expression value was more strongly correlated (Pearson’s r = 0.799, 95% CI = 0.71–0.86) than their individual expressions. This correlation was not significant in nodular melanoma. In SSM, we observed that the combined miRNA expression above or below 1.5 was significantly associated with overall survival and successfully identified all patients with relapsing SSM. We concluded that the combined assessment of miR-21-5p and miR-146a-5p expression in superficially spreading melanoma, in association with BT measurement, could aid pathologists in SSM staging.

Patients with occult primary cancer or cancer of unknown primary (CUP) carry the double burden of an aggressive disease and reduced access to therapies, which are typically tumor-type oriented. Moreover, experimental in-vitro and in-vivo models summing up CUP features and heterogeneity are currently unavailable. We derived and expanded two CUP cell lines, and corresponding patient-derived xenografts (PDXs), from ascites tumor cells. CUP cell lines, one spontaneously growing as organoid, and PDXs were subjected to histological, immune-phenotypical, molecular and genomic (CNV and target sequencing) characterization to show their ability to recapitulate the original tumor. MicroRNA profile and genomic information were used to identify a possible primary site and patient-specific therapeutic approaches. Data on CUP most frequently altered genes were used to design a custom CUP-specific, 92-gene panel for target NGS analysis with Sure Select XS technology (Agilent Technology). Drug-screening assays were performed in vitro and in vivo on two models targeting the most promising actionable pathways. We obtained unexpected and promising results on drug combinations efficacy. This study brings personalized therapy closer to CUP patients and paves the way to future applications of personalized medicine for metastatic patients with adverse prognosis. This work was supported by grants from the Italian Association for Cancer Research (AIRC) to MF (IG 18464). Citation Format: Noemi Laprovitera, Claudia Fumarola, Elisa Porcellini, Stefano Cairo, Giulia Gallerani, Francesco Gelsomino, Irene Salamon, Maria Naddeo, Andrea Ardizzoni, Andrea Cavazzoni, Manuela Ferracin. Cancer of Unknown Primary: novel therapeutic opportunities from patient-derived cell cultures and in vivo models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3011.

Purpose: The oncogenic miR-155 is upregulated in many human cancers, and its expression is increased in more aggressive and therapy-resistant tumors, but the molecular mechanisms underlying miR-155-induced therapy resistance are not fully understood. The main objectives of this study were to determine the role of miR-155 in resistance to chemotherapy and to evaluate anti-miR-155 treatment to chemosensitize tumors. Experimental Design: We performed in vitro studies on cell lines to investigate the role of miR-155 in therapy resistance. To assess the effects of miR-155 inhibition on chemoresistance, we used an in vivo orthotopic lung cancer model of athymic nude mice, which we treated with anti-miR-155 alone or in combination with chemotherapy. To analyze the association of miR-155 expression and the combination of miR-155 and TP53 expression with cancer survival, we studied 956 patients with lung cancer, chronic lymphocytic leukemia, and acute lymphoblastic leukemia. Results: We demonstrate that miR-155 induces resistance to multiple chemotherapeutic agents in vitro, and that downregulation of miR-155 successfully resensitizes tumors to chemotherapy in vivo. We show that anti-miR-155-DOPC can be considered non-toxic in vivo. We further demonstrate that miR-155 and TP53 are linked in a negative feedback mechanism and that a combination of high expression of miR-155 and low expression of TP53 is significantly associated with shorter survival in lung cancer. Conclusions: Our findings support the existence of an miR-155/TP53 feedback loop, which is involved in resistance to chemotherapy and which can be specifically targeted to overcome drug resistance, an important cause of cancer-related death. Clin Cancer Res; 23(11); 2891–904. ©2016 AACR.

Clonal evolution of chronic lymphocytic leukemia (CLL) often follows chemotherapy and is associated with adverse outcome, but also occurs in untreated patients, in which case its predictive role is debated. We investigated whether the selection and expansion of CLL clone(s) precede an aggressive disease shift. We found that clonal evolution occurs in all CLL patients, irrespective of the clinical outcome, but is faster during disease progression. In particular, changes in the frequency of nucleotide variants (NVs) in specific CLL-related genes may represent an indicator of poor clinical outcome.

// Ilaria Bononi 1, * , Manola Comar 2, * , Andrea Puozzo 1, * , Mariarita Stendardo 3 , Piera Boschetto 3 , Sara Orecchia 4 , Roberta Libener 4 , Roberto Guaschino 5 , Silvia Pietrobon 1 , Manuela Ferracin 6 , Massimo Negrini 1, 7 , Fernanda Martini 1 , Massimo Bovenzi 8 , Mauro Tognon 1 1 Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, School of Medicine, University of Ferrara, Ferrara, Italy 2 Institute for Maternal and Child Health - IRCCS “Burlo-Garofolo”– Trieste, University of Trieste, Trieste, Italy 3 Department of Medical Sciences, School of Medicine, University of Ferrara, Ferrara, Italy 4 Mesothelioma BioBank, Pathology Unit and City Hospital, Alessandria, Italy 5 Transfusion Medicine, City Hospital, Alessandria, Italy 6 Department of Experimental, Diagnostic and Specialty Medicine – DIMES, University of Bologna, Bologna, Italy 7 Laboratory for Technologies of Advances Therapies (LTTA), University of Ferrara, Ferrara, Italy 8 Clinical Unit of Occupational Medicine, Department of Medical Sciences, School of Medicine, University of Trieste, Trieste, Italy * These authors have contributed equally to this work Correspondence to: Mauro Tognon, email: tgm@unife.it Fernanda Martini, email: mrf@unife.it Keywords: microRNA, mesothelioma, biomarker, asbestos, worker Received: June 07, 2016 Accepted: September 16, 2016 Published: October 03, 2016 ABSTRACT Malignant pleural mesothelioma (MPM), a fatal cancer, is an occupational disease mostly affecting workers ex-exposed to asbestos fibers. The asbestos, a cancerogenic mineral of different chemical composition, was widely employed in western Countries in industrial manufactures of different types. MPM may arise after a long latency period, up to five decades. MPM is resistant to conventional chemo- and radio-therapies. Altogether, these data indicate that the identification of new and specific markers are of a paramount importance for an early diagnosis and treatment of MPM. In recent years, microRNAs expression was found dysregulated in patients, both in cancer cells and sera, affected by tumors of different histotypes, including MPM. Cell and circulanting microRNAs, found to be dysregulated in this neoplasia, were proposed as new biomarkers. It has been reported that circulating microRNAs are stable in biological fluids and could be employed as potential MPM biomarkers. In this investigation, circulating microRNAs (miR) from serum samples of MPM patients and workers ex-exposed to asbestos fibers (WEA) and healthy subjects (HS) were comparatively analyzed by microarray and RT-qPCR technologies. Our results allowed (i) to select MiR-3665, an endogenous stable microRNA, as the internal control to quantify in our analyses circulating miRNAs; to detect (ii) miR-197-3p, miR-1281 and miR 32-3p up-regulated in MPM compared to HS; (iii) miR-197-3p and miR-32-3p up-regulated in MPM compared to WEA; (iv) miR-1281 up-regulated in both MPM and WEA compared to HS. In conclusion, three circulating up-regulated microRNAs, i.e. miR-197-3p, miR-1281 and miR-32-3p are proposed as potential new MPM biomarkers.

The levels of expression of O6-methylguanine-DNA methyltransferase (MGMT) are relevant in predicting the response to the alkylating chemotherapy in patients affected by glioblastoma. MGMT promoter methylation and the published MGMT regulating microRNAs (miRNAs) do not completely explain the expression pattern of MGMT in clinical glioblastoma specimens. Here we used a genome-wide microarray-based approach to identify MGMT regulating miRNAs. Our screen unveiled three novel MGMT regulating miRNAs, miR-127-3p, miR-409-3p, and miR-124-3p, in addition to the previously identified miR-181d-5p. Transfection of these three novel miRNAs into the T98G glioblastoma cell line suppressed MGMT mRNA and protein expression. However, their MGMT- suppressive effects are 30-50% relative that seen with miR-181d-5p transfection. In silico analyses of The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) revealed that miR-181d-5p is the only miRNA that consistently exhibited inverse correlation with MGMT mRNA expression. However, statistical models incorporating both miR-181d-5p and miR-409-3p expression better predict MGMT expression relative to models involving either miRNA alone. Our results confirmed miR-181d-5p as the key MGMT-regulating miRNA. Other MGMT regulating miRNAs, including the miR-409-3p identified in this report, modify the effect of miR-181d-5p on MGMT expression. MGMT expression is, thus, regulated by cooperative interaction between key MGMT-regulating miRNAs.

Background Cancer of Unknown Primary is a rare syndrome of metastatic cancers for which the primary site cannot be recognized after detailed physical examinations, blood analyses, imaging and immunohistochemical (IHC) testing. CUPs make up 3-5% of newly diagnosed cancers worldwide and represent an important diagnostic and clinical problem. Treatment failure to empirical chemotherapy is common, resulting in progressive disease and poor prognosis. We and others proposed that a molecular inference of the tissue of origin could be feasible for most CUP tumors (Ferracin et al. J Pathol, 2011), thus giving access to more effective therapeutic agents and potentially extending CUP patient life-expectancy. Methods We optimized a cancer-type classifier based on microRNA (miRNA) expression for the prediction of CUP primary tumor site (Laprovitera et al., Front Oncol. 2018). Specifically, we designed a molecular assay to quantify the absolute copy number of 89 miRNAs in formalin-fixed paraffin-embedded (FFPE) samples using EvaGreen-based Droplet Digital PCR (BioRad). We tested 153 samples from three groups: primary tumors (N=95), metastases of known origin (N=10) and cancers of unknown origin (N=48). CUP diagnosis was obtained after a detailed histo-pathological investigation as per CUP diagnostic guidelines (NCCN occult primary v.2-2019 and ESMO guidelines). The study was approved by the local ethical committee. Results We investigated a pre-determined set of 89 miRNAs to infer the site-of origin of metastatic cancers of unknown or uncertain primary. Primary tumors from 16 different sites (lung, pancreas, liver, bile duct, kidney, bowel, testis, ovary, endometrium, stomach, bladder, breast, prostate, melanoma, gastrointestinal neuroendocrine, head and neck) were tested for absolute miRNA expression and used to train our classifier (Tibshirani et al. PNAS, 2002). We used the metastases of known origin as a test-set for our molecular prediction. We applied the shrunken centroids approach as predictive algorithm to obtain the most probable CUP site(s)-of- origin. Our results were judged against the hypothesized primary site suggested by standard diagnostic workup and pathological assessment. The molecular test was successfully applied to all CUP samples and provided a site-of-origin identification (with a probability > 50%) for all samples, potentially within a one-week time frame from sample inclusion. Conclusions Our study demonstrated that miRNA expression profiling in CUP samples have the potential to be employed in a clinical setting. Our molecular analysis can be performed on-request, concomitantly with the standard diagnostic workup and in association with genetic profiling, to offer valuable indication about the possible primary site thereby supporting treatment decisions. This work was supported by grants from the Italian Association for Cancer Research (AIRC) to MF (IG 18464). Citation Format: Noemi Laprovitera, Elisa Porcellini, Mattia Riefolo, Elisabetta Broseghini, Ingrid Garajova, Silvia Sabbioni, Martin Pichler, Andrea Ardizzoni, Antonia D'Errico, Manuela Ferracin. Cancer of unknown primary site-of-origin: An enigma ready to be miR-solved [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5432.

Background Blastic plasmacytoid dendritic cell neoplasm (BPDCN) and myeloid sarcoma (MS) are two extremely rare and aggressive hematological diseases. Both malignancies most commonly arise with skin lesions with or without extramedullary organ involvement before leukemic dissemination. Given its rarity and less known biology, in some cases with defective/ambiguous phenotype distinguish between these two entities may be challenging for pathologists and clinicians.1 Can the machine learning predictive model help to solve this diagnostic question? Here we performed the first study of microRNA (miRNA) profiling of BPDCN and MS in order to 1) Discover new molecular features selectively driving BPDCN respect to MS 2) Develop a machine learning based-prediction tool useful for discriminating BPDCN and MS. Methods We performed miRNA profiling (NanoString Technologies) of cutaneous biopsies of 16 BPDCN and 23 MS cases. Using Supervised Analysis, we identified 49 miRNAs differentially expressed that were randomly validated by qRT-PCR and next interrogated by functional enrichment analysis. Finally, a machine learning algorithm based on Linear Discriminant Analysis2 was applied to identify candidate miRNAs able to discriminate BPDCN from MS cases. Results In line with the overlapping clinical features of BPDCN and MS, the molecular profiling of these two diseases was proved to be extremely similar. Unsupervised Analysis well demonstrated that the miRNA profiles of the two malignancies are closely related and indeed, BPDCN and MS cases cluster together. When a Supervised Analysis was applied, we identified a set of 49 miRNAs differentially expressed in BPDCN respect to MS, 25 down- and 24 up-regulated. Of relevance, down-regulated miRNAs were predicted to be markedly involved in the apoptosis regulation of BPDCN. Machine learning predictive model identified a set of 12 miRNAs (5 up and 7 down) able to discriminate cutaneous BPDCN from MS. Conclusion This is the first miRNA profiling study in BPDCN and MS that showed how strongly these two diseases overlap at molecular level. Despite their similarity, BPDCN cases displayed a set of miRNAs significantly down-regulated when compared to MS, with possible dysregulation of cell death pathway. Of practical interest, we designed a machine learning predictive model based on the expression of 12 miRNAs, which alone may be applied to distinguish between the two hematological diseases. This tool, if validated in a larger set of cases, may help to differentiate BPDCN and MS in cases with defective/ambiguous phenotype. References 1. Weltgesundheitsorganisation. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Revised 4th edition. (Swerdlow SH, Campo E, Harris NL, et al., eds.). Lyon: International Agency for Research on Cancer; 2017. 2. Laginestra MA, Piccaluga PP, Fuligni F, et al. Pathogenetic and diagnostic significance of microRNA deregulation in peripheral T-cell lymphoma not otherwise specified. Blood Cancer J. 2014;4:259. doi:10.1038/bcj.2014. Citation Format: Maria Rosaria Sapienza, Fabio Fuligni, Federica Melle, Valentina Tabanelli, Valentina Indio, Alessandro Pileri, Lorenzo Cerroni, Francesco Bacci, Giovanna Motta, Maria Antonella Laginestra, Saveria Mazzara, Luciano Cascione, Alessandro Laganà, Claudio Agostinelli, Manuela Ferracin, Elena Sabattini, Carlo Croce, Stefano Pileri. Development of a miRNA-based prediction tool to discriminate cutaneous blastic plasmacytoid dendritic cell neoplasm from cutaneous myeloid sarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1417.

Background Malignant cutaneous melanoma is a potentially lethal form of skin cancer whose worldwide incidence has been increasing constantly over the past decades. A fraction of all cutaneous melanoma patients (up to 8%) develop multiple melanomas during their lifetime, usually at a young age and within 3 years from the first tumor/diagnosis, a condition known as multiple primary melanoma (MPM). Patients affected by multiple primary melanoma could have a genetically determined susceptibility, though germline mutations in hereditary melanoma genes are rarely detected in these patients. Therefore, a better characterization of MPM pathogenesis and biological features is of the outmost importance. To characterize the biology of multiple-primary cutaneous melanoma, we performed a global microRNA (miRNA) profile by small-RNA sequencing of single and multiple primary melanomas, including familial melanoma and multiple tumors from the same patient. Material and methods RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissues of 3 benign nevi (BN), 9 single cutaneous melanomas (CM), 35 MPMs. We assessed the overall miRNA profile by small-RNA sequencing (Illumina). Sequencing data were analyzed using Genespring GX software (Agilent Technologies). The differentially expressed miRNAs were validated in all samples by qRT-PCR using miRCURY LNA assays (Qiagen). Melanoma isomiRs were identified as described in Loheret al. (PMID: 25229428). Pathway enrichment analysis of the differentially expressed miRNAs and their targets was performed using MetaCore software (Clarivate Analytics). Results MPM miRNA profiling revealed that MPMs with and without family history have a similar microRNA profile, which is different from CM and BN. Indeed, 22 miRNAs were differentially expressed in MPM compared to single cutaneous melanoma, including key miRNAs involved in epithelial-mesenchymal transition (EMT). Moreover, the comparison between the first and second tumor from the same patient revealed that the second tumor's miRNA profile was different. Consequently, ten miRNAs were validated in a larger cohort of single and MPM samples (N=29). From our global miRNA analysis, we also identified some melanoma-specific miRNA isoforms (isomiRs) that showed a differential expression in our dataset and TCGA melanoma samples. We analyzed the pathways specifically regulated by the differentially expressed miRNAs and their network of target genes and the contribution of specific isomiRs to target regulation and MPM development. Conclusion Overall, our study demonstrated a more differentiated and less metastasis-prone status of MPM compared to single primary tumors and provided insights into miRNA/isomiR contribution to multiple melanoma molecular pathogenesis. Citation Format: Elisabetta Broseghini, Elisa Porcellini, Mattia Riefolo, Martina Lambertini, Giorgio Durante, Eric Londin, Annalisa Patrizi, Emi Dika, Manuela Ferracin. Unraveling the role of microRNAs in multiple primary melanoma pathogenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4833.

Background Cancer of unknown primary (CUP) is a heterogeneous group of metastatic cancers whose primary site cannot be determined after standard clinical and pathological evaluation. CUP patients are generally treated with empirical chemotherapy and have poor prognosis. As reported in recent studies, CUPs present an average of 4 genetic alterations per tumor and these genetic alterations can be detected in circulating tumor DNA (Kato 2017). Thus, a potential improvement in CUP outcomes could derive from targeted therapies directed toward actionable mutations, and Circulating tumor cells (CTCs) can provide valuable information. Here, we describe a case of a 51-yr-old Caucasian female (Pt#71) with metastatic CUP. CTCs were detectable in her blood and analyzed for genetic alterations. Methods Using CellSearch and DEPArray NxT, n=3 CTCs and n=1 leukocyte as a control were isolated from patient's peripheral blood. Single-cell whole genome amplification was obtained with Ampli1 WGA Kit and libraries were generated with Ampli1 OncoSeek Panel and Ampli1 LowPass kits. In parallel, formalin-fixed paraffin embedded (FFPE) tissue derived from a lymph node biopsy was analyzed with two different methods. One curl was sent for comprehensive genomic profile to FoundationOne assay testing (Roche). From another curl, DNA was extracted and directly processed with DEPArray LibPrep and DEPArray OncoSeek Panel kits. After sequencing on Illumina MiSeq platform, raw data were analyzed on MSBiosuite platform (Menarini Silicon Biosystems). Results CTCs genome-wide characterization allowed the detection of sub-chromosomal losses, including a LOH region comprising the APC gene, and patterns of extensive gains. Moreover, amplification signals were detected in correspondence of FGFR2 and CCNE1 genes. Targeted sequencing on both CTCs and bulk tumor DNA confirmed the FGFR2 amplification in all the samples and detected a somatic variant in APC gene (APC:p.T1556Nfs*3). FoundationOne assay on FFPE biopsy reported the same amplification on FGFR2 and CCNE1 genes, along with a somatic variant in ARID1A gene (ARID1A:p.R1276*), a gene not present in the OncoSeek Panel. On the other hand, APC somatic variant was not identified by FoundationOne, probably due to contamination by normal cells and/or tumor heterogeneity. Conclusions The comprehensive genomic profile of tumor FFPE tissue and CTCs was highly overlapping and allowed the characterization of genetic alterations in this CUP case, revealing potentially actionable mutations and copy number alterations. The advantage of isolating and analyzing single CTCs is clearly shown by the non-invasive procedure combined with a precise detection of druggable alterations due to cell purity. Citation Format: Elisa Porcellini, Francesco Gelsomino, Noemi Laprovitera, Mattia Riefolo, Marianna Garonzi, Paola Tononi, Francesca Fontana, Antonia D'Errico, Maria Pantaleo, Nicolò Manaresi, Andrea Ardizzoni, Manuela Ferracin. Isolation and genetic characterization of circulating tumor cells from cancer of unknown primary [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5361.

Archival formalin-fixed paraffin-embedded (FFPE) tissues represent an extraordinary source of smallRNAs, including microRNAs (miRNAs). Contrary to other RNA molecules, miRNAs are stable, nuclease-resistant and quantifiable even in low quality samples. The accurate assessment of miRNA levels in archival samples is of great interest for many pathological conditions, including cancer. In human tumors, microRNA expression is type-specific and can be used as diagnostic, prognostic or response-to-treatment biomarker. In this study, we provide a method for multiple miRNA quantification in 96-well plates, using EvaGreen-based droplet digital PCR technology and miRCURY LNA miRNA assays. This approach allows the absolute quantification of a customizable panel of miRNAs at the same time and under identical experimental conditions, to be used for diagnostic or prognostic applications.

Long non-coding RNAs (lncRNAs) are ncRNAs more than 200 nucleotides long that participate to a wide range of biological functions. However, their role in cancer is poorly known. By using an NGS-based approach we analyzed the intragenic and poliA-lncRNAs in hepatocellular carcinoma (HCC) and we assayed the relationships between their deregulated expression and clinical-pathological characteristics. The expression profile of lncRNAs was studied in a discovery series of 28 HCC and matched cirrhosis and was validated in an independent cohort of 32 HCC patients both in tissue and serum. The correlation between lncRNA expression and clinical-pathological variables, EMT markers and putative sponged microRNAs level were investigated. Functional experiments were performed in HCC-derived cell lines to clarify the role of selected lncRNAs in HCC. A panel of deregulated lncRNAs differentiated HCC from cirrhotic tissue. CASC9 and LUCAT1 were up-regulated in a subset of HCC-derived cell lines and in half of HCCs which displayed a lower recurrence after surgery. LUCAT1 and CASC9 silencing increased cell motility and invasion capability in HCC cells and influenced the EMT phenotype. LUCAT1 was demonstrated to directly sponge the onco-miR-181d-5p. Both LUCAT1 and CASC9 were secreted in exosomes, and higher circulating CASC9 levels were associated with tumor size and HCC recurrence after surgery, suggesting its potential usage as putative non-invasive prognostic biomarker of recurrence.

// Jitka Mlcochova 1 , Petra Faltejskova-Vychytilova 1, 2 , Manuela Ferracin 3 , Barbara Zagatti 3 , Lenka Radova 1 , Marek Svoboda 2 , Radim Nemecek 2 , Stanislav John 4 , Igor Kiss 2 , Rostislav Vyzula 2 , Massimo Negrini 3 , Ondrej Slaby 1, 2 1 Central European Institute of Technology, Masaryk University, Brno, Czech Republic 2 Masaryk Memorial Cancer Institute, Department of Comprehensive Cancer Care, Masaryk University, Faculty of Medicine, Brno, Czech Republic 3 Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy 4 Department of Oncology and Radiotherapy, Faculty Hospital Hradec Kralove, Hradec Kralove, Czech Republic Correspondence to: Ondrej Slaby, e-mail: on.slaby@gmail.com Massimo Negrini, e-mail: ngm@unife.it Keywords: microRNA, metastatic colorectal cancer, EGFR, cetuximab, panitumumab Received: May 17, 2015 Accepted: October 08, 2015 Published: October 20, 2015 ABSTRACT The aim of our study was to investigate whether microRNAs (miRNAs) could serve as predictive biomarkers to anti-EGFR therapy (cetuximab, panitumumab) in patients with RAS wild-type (wt-RAS) metastatic colorectal cancer (mCRC). Historical cohort of 93 patients with mCRC (2006–2009) was included and further divided into exploratory and validation cohorts. MiRNAs expression profiling was performed on the exploratory cohort of 41 wt-KRAS mCRC patients treated with cetuximab to identify miRNAs associated with time to progression (TTP). The validation was performed on two independent cohorts: 28 patients of wt-RAS mCRC treated with cetuximab and 24 patients of wt-RAS mCRC treated with panitumumab. We identified 9 miRNAs with significantly different expression between responders and non-responders to cetuximab therapy ( P ≤ 0.01). These 9 miRNAs were further evaluated in two independent cohorts of patients and miR-31-3p ( P < 0.001) and miR-31-5p ( P < 0.001) were successfully confirmed as strongly associated with TTP in wt-RAS mCRC patients treated with cetuximab but not panitumumab. When evaluated on the complete cohort of cetuximab patients ( N = 69), miR-31-3p (HR, 5.10; 95% CI, 2.52–10.32; P < 0.001) and miR-31-5p (HR, 4.80; 95% CI, 2.50–9.24; P < 0.001) were correlated with TTP on the comparable level of significance. There was no difference in miR-31-5p/3p expression levels in RAS mutated and wild-type tumor samples. MiR-31-5p/3p are promising predictive biomarkers of cetuximab response in wt-RAS mCRC patients.

In this study we investigated specific biological and clinical features associated with chronic lymphocytic leukemia (CLL) patients carrying stereotyped BCR subset #4 (IGHV4-34) among a prospective cohort of 462 CLL/MBL patients in early stage (Binet A). All subset #4 patients (n = 16) were characterized by the IGHV mutated gene configuration, and absence of unfavorable cytogenetic lesions, NOTCH1 or SF3B1 mutations. Gene and miRNA expression profiling evidenced that the leukemic cells of subset #4 cases showed significant downregulation of WDFY4, MF2A and upregulation of PDGFA, FGFR1 and TFEC gene transcripts, as well as the upregulation of miR-497 and miR-29c. The transfection of miR-497 mimic in primary leukemic CLL cells induced a downregulation of BCL2, a known validated target of this miRNA. Our data identify biological characteristics associated with subset #4 patients, providing further evidence for the putative role of BCR in shaping the features of the tumor cells in CLL.

A cancer cell is the final product of a complex mixture of genetic, epigenetic and epitranscriptomic alterations, whose final interplay contribute to cancer onset and progression. This is specifically true for colorectal cancer, a tumor with a strong epigenetic component, which acts earlier than any other genetic alteration in promoting cancer cell malignant transformation. The pattern of progressive, and usually subtype-specific, DNA and histone modifications that occur in colorectal cancer has been extensively studied in the last decade, providing plenty of data to explore. For this tumor, it became recently evident that also RNA modifications play a relevant role in the activation of oncogenes or repression of tumor suppressor genes. In this review we provide a brief overview of all epigenetic and epitranscriptomic changes that have been found associated to colorectal cancer till now. We explore the impact of these alterations in cancer prognosis and response to treatment and discuss their potential use as cancer biomarkers.

High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecologic neoplasm, with five-year survival rate below 30%. Early disease detection is of utmost importance to improve HGSOC cure rate. Sera from 168 HGSOC patients and 65 healthy controls were gathered together from two independent collections and stratified into a training set, for miRNA marker identification, and a validation set, for data validation. An innovative statistical approach for microarray data normalization was developed to identify differentially expressed miRNAs. Signature validation in both the training and validation sets was performed by quantitative Real Time PCR (RT-qPCR). In both the training and validation sets, miR-1246, miR-595 and miR-2278 emerged significantly over expressed in the sera of HGSOC patients compared to healthy controls. Receiver Operating Characteristic curve analysis revealed miR-1246 as the best diagnostic biomarker, with a sensitivity of 87%, a specificity of 77% and an accuracy of 84%. This study is the first step in the identification of circulating miRNAs with diagnostic relevance for HGSOC. According to its specificity and sensitivity, circulating miR-1246 levels are worthy to be further investigated as potential diagnostic biomarker for HGSOC.

The onset and selection of resistant clones during cancer treatment with chemotherapy or targeted therapy is a major issue in the clinical management of metastatic colorectal cancer patients. It is possible that a more personalized treatment selection, using reliable response-to-therapy predictive biomarkers, could lead to an improvement in the success rate of the proposed therapies. Although the process of biomarker selection and validation could be a long one, requiring solid statistics, large cohorts and multicentric validations, non-coding RNAs (ncRNAs) and in particular microRNAs, proved to be extremely promising in this field. Here we summarize some of the main studies correlating specific ncRNAs with sensitivity/resistance to chemotherapy, anti-VEGF therapy, anti-EGFR therapy and immunotherapy in colorectal cancer (CRC).

Purpose: Despite its indolent nature, chronic lymphocytic leukemia (CLL) remains an incurable disease. To establish the potential pathogenic role of miRNAs, the identification of deregulated miRNAs in CLL is crucial. Experimental Design: We analyzed the expression of 723 mature miRNAs in 217 early-stage CLL cases and in various different normal B-cell subpopulations from tonsils and peripheral blood. Results: Our analyses indicated that CLL cells exhibited a miRNA expression pattern that was most similar to the subsets of antigen-experienced and marginal zone–like B cells. These normal subpopulations were used as reference to identify differentially expressed miRNAs in comparison with CLL. Differences related to the expression of 25 miRNAs were found to be independent from IGHV mutation status or cytogenetic aberrations. These differences, confirmed in an independent validation set, led to a novel comprehensive description of miRNAs potentially involved in CLL. We also identified miRNAs whose expression was distinctive of cases with mutated versus unmutated IGHV genes or cases with 13q, 11q, and 17p deletions and trisomy 12. Finally, analysis of clinical data in relation to miRNA expression revealed that miR26a, miR532-3p, miR146-5p, and miR29c* were strongly associated with progression-free survival. Conclusion: This study provides novel information on miRNAs expressed by CLL and normal B-cell subtypes, with implication on the cell of origin of CLL. In addition, our findings indicate a number of deregulated miRNAs in CLL, which may play a pathogenic role and promote disease progression. Collectively, this information can be used for developing miRNA-based therapeutic strategies in CLL. Clin Cancer Res; 20(15); 4141–53. ©2014 AACR.

Background: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an extremely rare and aggressive hematological disease, deriving from the malignant transformation of plasmacytoid, alpha-interferon producing dendritic cells (pDCs). Recent studies shed new light on BPDCN genomic and trascriptomic alterations, but the microRNA (miRNA) profile is still largely unknown. We analyzed the miRNA expression profile of BPDCN patients to enhance our understanding of the molecular mechanisms driving this disease and to identify new potential diagnostic and therapeutic biomarkers. Methods: We performed the miRNA profiling (NanoString Technologies) of 26 BPDCN patients and 4 normal pDCs samples as controls. To better investigate the functional consequences of miRNAs dysregulation, 9 BPDCN patients, were also analyzed at the transcriptomic level. We applied a Moderated t-test to find genes and miRNAs differentially expressed between BPDCNs and pDCs. The most deregulated miRNAs and mRNAs were validated by qRT-PCR and immunohistochemistry and analyzed by miRNET, a bioinformatic tool for statistical analysis and functional interpretation of miRNA and mRNA data. Results: We found that, according to the supervised clustering analysis of miRNAs, tumor samples display a molecular signature well distinct from their normal counterpart. Indeed, BPDCN patients expressed a set of 175 miRNAs significantly deregulated and potentially involved in essential biological processes and therefore in malignant transformation. Thus, to evaluate the impact of these miRNAs on the BPDCN transcriptome, miRNAs and mRNAs expression profiles were analyzed by miRNET tool. Thanks to this integrative approach, we uncovered the most relevant miRNA-mRNA networks in BPDCN setting and in particular we identified 9 up-regulated hub miRNAs, targeting multiple genes and synergistically inter-connected: hsa-mir-93-5p, hsa-mir-106b-5p, hsa-mir-19b-3p, hsa-mir-19a-3p, hsa-mir-21-5p, hsa-mir-181a-5p, hsa-mir-25-3p, hsa-mir-155-5p, hsa-mir-17-3p (in order of relevance). These 9 hub miRNAs regulate the expression of genes already described as relevant in BPDCN patients (ex. TCF4, RHOA, EP300) and, according to functional enrichment analysis, that could aberrantly interfere with TLR signaling, protein translation and DNA transcription regulation. Of interest, most of these hub miRNAs are classified as oncomir (OncoMir Cancer Database) and, if validated in an extended number of cases, promising targets for anti-miRNA based therapy. In conclusion, we identified a panel of miRNAs that regulate relevant cancer-related pathways and can be also used as new potential biomarkers and therapeutic targets in BPDCN. Citation Format: Maria Rosaria Sapienza, Manuela Ferracin, Fabio Fuligni, Federica Melle, Giovanna Motta, Maria Antonella Laginestra, Maura Rossi, Luciano Cascione, Alessandro Laganà, Claudio Agostinelli, Elena Sabattini, Alessandro Pileri, Carlo Maria Croce, Stefano Aldo Pileri. Integrative analysis of microRNAs in blastic plasmacytoid dendritic cell neoplasm [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1805.

Background: Programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1) inhibitors represent novel therapeutic options for advanced non-small cell lung cancer (NSCLC). However, approximately 50% of patients do not benefit from therapy and experience rapid disease progression. PD-L1 expression is the only approved biomarker of benefit to anti-PD-1/PD-L1 therapy. However, its weakness has been evidenced in many studies. More recently, tumor mutational burden (TMB) has proved to be a suitable biomarker, but its calculation is difficult to obtain for all patients. Methods: We tested specific NSCLC genetic alterations as potential immunotherapy biomarkers. Tumor DNA was obtained from advanced NSCLC patients treated with anti-PD-1 monoclonal antibody nivolumab ( n = 44) or pembrolizumab ( n = 3). The mutational status of 22 genes was assessed by targeted next-generation sequencing and the association with survival was tested in uni- and multivariate models. The association between gene mutations and clinical benefit was also investigated. Results: The most frequently mutated genes were TP53 (49%), KRAS (43%), ERBB2 (13%), SMAD4 (13%), DDR2 (13%), STK11 (9%), ERBB4 (6%), EGFR (6%), BRAF (6%), and MET (6%). We confirmed that KRASmut patients have a better response to PD-1 inhibitors, showing a longer progression-free survival (PFS) and overall survival (OS) than KRASwt patients. In addition, we observed that patients with ERBB-family mutations, including EGFR, ERBB2, and ERBB4 all failed to respond to PD-1 antibodies, independently of KRAS status. Conclusions: This study suggests that the analysis of KRAS and ERBB-family gene mutational status is valuable when assessing the clinical practice for the selection of NSCLC patients to treat with PD-1 inhibitors.

// Laura Lupini 1, * , Felice Pepe 2, 8, * , Manuela Ferracin 3 , Chiara Braconi 4 , Elisa Callegari 1 , Sara Pagotto 2, 8 , Riccardo Spizzo 5 , Barbara Zagatti 1 , Paola Lanuti 6 , Francesca Fornari 7 , Reza Ghasemi 2 , Renato Mariani-Costantini 2, 8 , Luigi Bolondi 7 , Laura Gramantieri 7 , George A. Calin 9 , Silvia Sabbioni 10 , Rosa Visone 2, 8 , Angelo Veronese 2, 8 , Massimo Negrini 1 1 Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy 2 Unit of General Pathology, Aging Research Center (Ce.S.I.), G. d’Annunzio University Foundation, Chieti, Italy 3 Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy 4 Division of Cancer Therapeutics, Institute of Cancer Research, London, UK 5 Division of Experimental Oncology 2 CRO, Aviano, Italy 6 Department of Medicine and Aging Science, G. d’Annunzio University, Chieti, Italy 7 S.Orsola-Malpighi University Hospital, Bologna, Italy 8 Department of Medical, Oral and Biotechnological Sciences, G. d’Annunzio University, Chieti, Italy 9 Department of Experimental Therapeutics, MD Anderson Medical Centre, Houston, TX, USA 10 Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy * These authors have contributed equally to this work Correspondence to: Angelo Veronese, email: a.veronese@unich.it Massimo Negrini, email: ngm@unife.it Keywords: TP53, hsa-miR-145-5p, hsa-miR-483-3p, HCC, PUMA Received: October 15, 2015 Accepted: April 10, 2016 Published: April 22, 2016 ABSTRACT The miR-145-5p, which induces TP53-dependent apoptosis, is down-regulated in several tumors, including hepatocellular carcinomas (HCCs), but some HCCs show physiological expression of this miR. Here we demonstrate that in HCC cells carrying wild-type TP53 the steady activation of the miR-145 signaling selects clones resistant to apoptosis via up-regulation of the oncogenic miR-483-3p. Expression of the miR-145-5p and of the miR-483-3p correlated negatively in non-neoplastic liver (n=41; ρ=-0.342, P=0.028), but positively in HCCs (n=21; ρ=0.791, P

Identification of factors to detect chemotherapy-resistant tumours at diagnosis is a first priority for risk-adapted therapy in the oncology of children and young adults, where more individualized, effective, and less toxic treatments are highly desirable. In this study, we analysed the miRNAs discriminating Ewing's sarcoma (EWS) patients with different clinical outcomes in order to identify new indicators of prognosis. miRNA expression was investigated in 49 primary EWSs by using the Agilent human miRNA microarray v.2 and/or qRT-PCR. Statistical power of the samples studied for miRNA expression was verified, indicating adequate sample size. Microarray analysis defined a signature of five miRNAs (miR-34a, miR-23a, miR-92a, miR-490-3p, and miR-130b) as an independent predictor of risk for disease progression and survival. Validation analysis in the extended sample set indicated that both miR-34a and miR-490-3p achieved sufficient statistical power to predict prognosis. Results were particularly robust for miR-34a, which appeared associated with either event-free or overall survival and emerged as a significant predictor also after multivariate analysis. Patients with the highest expression of miR-34a did not experience adverse events in 5 years; in contrast, patients with the lowest expression recurred within 2 years. High expression of miR34a can be detected also in paraffin-embedded tissues by in situ hybridization, thus contributing to an easy routine evaluation of this miRNA. Functional analysis of miR-34a in EWS cell lines indicated that when miR-34a expression was enforced, cells were less proliferative, less malignant, and sensitized to doxorubicin and vincristine. Expression of miR-34a could be increased in p53wt cells by treatment with nutlin-3a. Accordingly, nutlin-3a synergizes with doxorubicin. Overall, our data indicate that miR-34a expression is a strong predictor of outcome in EWS. Restoration of miR-34a activity may be useful to decrease malignancy and increase tumour sensitivity to current drugs, so sparing excessive long-term toxicity to EWS patients.

Cancer of unknown primary (CUP) represents a common and important clinical problem. There is evidence that most CUPs are metastases of carcinomas whose primary site cannot be recognized. Driven by the hypothesis that the knowledge of primary cancer could improve patient’s prognosis, we investigated microRNA expression profiling as a tool for identifying the tissue of origin of metastases. We assessed microRNA expression from 101 formalin-fixed, paraffin-embedded (FFPE) samples from primary cancers and metastasis samples by using a microarray platform. Forty samples representing ten different cancer types were used for defining a cancer-type-specific microRNA signature, which was used for predicting primary sites of metastatic cancers. A 47-miRNA signature was identified and used to estimate tissue-of-origin probabilities for each sample. Overall, accuracy reached 100% for primary cancers and 78% for metastases in our cohort of samples. When the signature was applied to an independent published dataset of 170 samples, accuracy remained high: correct prediction was found within the first two options in 86% of the metastasis cases (first prediction was correct in 68% of cases). This signature was also applied to predict 16 CUPs. In this group, first predictions exhibited probabilities higher than 90% in most of the cases. These results establish that FFPE samples can be used to reveal the tissue of origin of metastatic cancers by using microRNA expression profiling and suggest that the approach, if applied, could provide strong indications for CUPs, whose correct diagnosis is presently undefined.

Background Malignant pleural mesothelioma (MPM) is a rare but aggressive asbestos-related cancer that develops by mesothelial cell transformation. At present, there are no effective therapies for MPM. Great efforts have been made in finding specific markers/mechanisms for MPM onset, including studies into microRNAs (miRNAs). Recent studies have shown the differential expression of mature miRNAs in several human cancers, suggesting their potential role as oncogenes or tumor suppressor genes. Methods In this study, we investigated miRNAs profile in five human normal pleural mesothelial short-term cell cultures (HMCs) and five MPMs, with microarray approach. These results were confirmed by real-time quantitative reverse-transcriptase polymerase chain reaction and Western blotting. Results A comparative analysis of miRNA expression in MPM and HMCs was carried out. Microarray profiling showed different miRNA expression between MPM and HMCs. Specifically, members of the oncomiRNA miR 17-92 cluster and its paralogs, namely miR 17-5p, 18a, 19b, 20a, 20b, 25, 92, 106a, 106b , were markedly upregulated. Besides, in our investigation, additional miRNAs, such as miR-7, miR-182, miR-214, and miR-497 were found to be dysregulated in MPM. Conclusions These data are in agreement with results that have previously been reported on dysregulated miRNAs for other solid human tumors. Moreover, in our investigation, additional miRNAs were found to be dysregulated in MPM. Interestingly, gene products that regulate the cell cycle are targets and predicted targets for these miRNAs. Our data suggest that specific miRNAs could be key players in MPM development/progression. In addition, some of these miRNAs may represent MPM markers and potential targets for new therapeutic approaches.

Background Ewing sarcoma (EWS) is an aggressive childhood bone tumor with still highly unmet clinical needs. The tumor is characterized by the fusion oncoprotein EWS-FLI1 and the high expression of the membrane glycoprotein CD99. CD99 has been found to be released by EWS cells linked to exosomes (EXOs), small vesicles able to influence cellular behavior and surrounding microenvironment by transferring their contents into nearby or distant recipient cells. The delivery of EXOs devoid of CD99 was sufficient to induce neural differentiation in recipient EWS cells through miR- 34a-dependent inhibition of Notch-NF-kB signaling, thus indicating a possible therapeutic potential. Aims The study has the ambition to: 1. Evaluate the capabilities of EXOs released by Ewing's sarcoma, to influence cancer growth and/or metastasis through the horizontal transfer of their cargos. 2. Characterize EXOs secreted by CD99-silenced EWS cells in terms of miRNAs and protein and evaluate their general antimetastatic function, if any, taking advantage of their capabilities to be transferred into a broad panel of different Ewing's sarcoma cell lines, as recipients. Results In the present study we identified the role of Ewing sarcoma-derived EXOs as mediators of signals involved in cancer growth, metastases and differentiation. In particular, we analyzed the ability of EXOs, expressing or not CD99, to modulate the phenotype of EWS cells. Delivery of EXOs from CD99-silenced cells was sufficient to induce neural differentiation in recipient EWS cells by neurite outgrowth and increased expression of β-III tubulin marker of neural differentiation accompanied in vitro by a significant reduction of proliferation, invasion and chemotaxis convey the same phenotype obtained by stable CD99 silencing. Moreover, microarray analysis revealed a signature of 56 miRNAs differentially expressed in EXOs derived from CD99-silenced cells compared to EWS-derived EXOs. We focused our attention on miR-214 and miR-199. Those miRNAs are involved in the regulation of CD99 itself like, EWS-FLI1 or CD99-mediated pathways related to cytoskeleton and membrane organization. As a next step we searched for any tumorigenic effect possibly associated with miR-199 and mir-214. The restored expression of miR-199 and miR-214 in two Ewing Sarcoma cell lines was accompanied by a significant reduction of proliferation, invasion and chemotaxis in vitro. The outcomes deriving from miR-199 and miR-214 enforced expression were also evaluated on capability of forming foci in agar semisolid medium. Results showed a significant decrease of the number of foci in both cell lines. Conclusions Exosomes deprived of CD99 can alter the balance of important cellular components necessary for the metastatic process of EWS cells, thus representing a novel exciting therapeutic possibility for the design of novel therapy against metastatic EWS. AIRC IG18451 to KS; IG18815 to AC. Citation Format: Alessandra De Feo, Marika Sciandra, Federica Felicetti, Manuela Ferracin, Alessandra Carè, Katia Scotlandi. Exosome-mediated transfer of sh-CD99 is sufficient to modulate cell differentiation in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3549.

Osteosarcoma (OS) and Ewing's sarcoma (EWS) are the two most common pediatric solid tumors, after brain tumors. Multimodal treatments have significantly improved prognosis in localized disease but outcome is still poor in metastatic patients, for whom therapeutic options are often inadequate. Preclinical drug testing to identify promising treatment options that match the molecular make-up of these tumors is hampered by the lack of appropriate and molecularly well-characterized patient-derived models. To address this need, a panel of patient-derived- xenografts (PDXs) was established by subcutaneous implantation of fresh, surgically resected OS and EWS tumors in NSG mice. Tumors were re-transplanted to next mice generations and fragments were collected for histopathological and molecular characterization. A model was considered established after observing stable histological and molecular features for at least three passages. To evaluate the similarity of the model with primary tumor, we performed a global gene expression profiling and tissue microarrays (TMA), to assess tumor specific biomarkers on tissues from OS/EWS tumors and their PDXs (1st and 3rd passage). Moreover, we verified the feasibility of these models for preclinical drug testing. We implanted 61 OS and 29 EWS samples: 14/38 (37%) primary OS and 9/23 (39%) OS lung metastases successfully engrafted; while among EWS, 5/26 (19%) primary samples and 1/3 (33%) metastases were established. Comparison between patient samples and PDXs, highlighted that histology and genetic characteristics of PDXs were stable and maintained over passages. In particular, correlative analysis between OS and EWS samples and their PDXs was extremely high (Pearson's r range r=0.94-0.96), while patient-derived primary cultures displayed reduced correlation with human samples (r=0.90-0.93), indicating that in vitro adaptation superimpose molecular alterations that create genetic diversion from original tumors. No significant differentially expressed gene profile was observed from the comparison between EWS samples and PDXs (fold change > 2, adjusted p Citation Format: Maria Cristina Manara, Giordano Nicoletti, Manuela Ferracin, Camilla Cristalli, Alberto Righi, Mauro Magnani, Michela Pasello, Piero Picci, Patrizia Nanni, Pier-Luigi Lollini, Katia Scotlandi. Collection of patient-derived xenografts (PDX) to study the biology and therapy of bone sarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2079.

Adjuvant therapy is a systemic treatment administered after primary tumor resection to reduce the risk of relapse and death in colorectal cancer (CRC) patients. Generally, adjuvant treatment is recommended for stage III and ‘high-risk' stage II CRC (NCCN guidelines). Nonetheless, adjuvant treatment is still debated for stage IIA (T3N0) patients, whose estimated recurrence rate is 15-20% in the absence of any further therapy after resection of the primary tumor. With the aim to find additional prognostic biomarkers that could improve current patient selection, we analyzed the global methylation profile of a discovery group of 10 relapsing/non-relapsing stage IIA colon cancer samples, without any other known risk factor (low-risk), by Illumina Epic 850K array. We identified a panel of 25 Illumina probes that were able to predict recurrence in chemotherapy-naïve patients. Then, we assessed the methylation of the most predictive Cytosine-Guanine (CpG) dinucleotides in 107 stage IIA colon cancer patients from two different cohorts, by EpiTYPER technology. We validated several CpGs as differentially methylated in relapsing vs. non-relapsing CRCs (p Citation Format: Sara Pagotto, Elisa Porcellini, Maria G. Bacalini, Diletta Sarti, Ingrid Garajova, Elisabetta Broseghini, Noemi Laprovitera, Francesco Vasuri, Paolo Garagnani, Renato Mariani-Costantini, Michele Basso, Rosa Visone, Luigi M. Larocca, Manuela Ferracin, Angelo Veronese. Epigenetic biomarkers of prognosis in stage IIA colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3313.

A transcriptome-wide approach reveals the key contribution of NFI-A in promoting erythroid differentiation of human CD34 + progenitors and CML cells

Deregulated cell proliferation and apoptosis play a major role in hepatocellular carcinoma (HCC). MicroRNAs participate in the modulation of key molecules linked to hepatocarcinogenesis. Purpose: This study aims to investigate the role of miR-221 in the modulation of Bmf, a proapoptotic BH3-only protein, and to characterize miR-221 contribution to hepatocarcinogenesis through modulation of apoptosis. Experimental Design: Transfection of miR-221 and anti-miR-221 in HCC-derived cell lines and luciferase reporter assay were used to assess Bmf as a target of miR-221. Modulation of miR-221 and Bmf expression contributed to characterize their role in anoikis. Primary HCC tissues were analyzed to assess the clinical relevance of in vitro findings. Results: Enforced miR-221 expression caused Bmf down-regulation, whereas anti-miR-221 induced its up-regulation. A luciferase reporter assay confirmed Bmf as a target of miR-221. Following matrix detachment, miR-221 silencing led to increased apoptotic cell death. The analysis of HCC tissues revealed an inverse correlation between miR-221 and Bmf expression and a direct correlation between Bmf and activated caspase-3, as a marker of apoptosis. High miR-221 levels were associated with tumor multifocality and reduced time to recurrence after surgery. Conclusions: Our results indicate that miR-221, by targeting Bmf, inhibits apoptosis. Moreover, in HCC, miR-221 overexpression is associated with a more aggressive phenotype. These findings, together with the previously reported modulation of CDKN1B/p27 and CDKN1C/p57, show that miR-221 simultaneously affects multiple pro-oncogenic pathways and suggest miR-221 as a potential target for nonconventional treatment against HCC. (Clin Cancer Res 2009;15(16):5073–81)

Objectives Micro-RNAs are a group of small noncoding RNAs with modulator activity of gene expression. Recently, micro-RNA genes were found abnormally expressed in several types of cancers. To study the role of the micro-RNAs in human kidney and bladder cancer, we analyzed the expression profile of 245 micro-RNAs in kidney and bladder primary tumors. Methods and materials A total of 27 kidney specimens (20 carcinomas, 4 benign renal tumors, and 3 normal parenchyma) and 27 bladder specimens (25 urothelial carcinomas and 2 normal mucosa) were included in the study. Total RNA was used for hybridization on an oligonucleotide microchip for micro-RNA profiling developed in our laboratories. This microchip contains 368 probes in triplicate, corresponding to 245 human and mouse micro-RNA genes. Results A set of 4 human micro-RNAs (miR-28, miR-185, miR-27, and let-7f-2) were found significantly up-regulated in renal cell carcinoma (P < 0.05) compared to normal kidney. Human micro-RNAs miR-223, miR-26b, miR-221, miR-103-1, miR-185, miR-23b, miR-203, miR-17-5p, miR-23a, and miR-205 were significantly up-regulated in bladder cancers (P < 0.05) compared to normal bladder mucosa. Of the kidney cancers studied, there was no differential micro-RNA expression across various stages, whereas with increasing tumor-nodes-metastasis staging in bladder cancer, miR-26b showed a moderate decreasing trend (P = 0.082). Conclusions Our results show that different micro-RNAs are deregulated in kidney and bladder cancer, suggesting the involvement of these genes in the development and progression of these malignancies. Further studies are needed to clarify the role of micro-RNAs in neoplastic transformation and to test the potential clinical usefulness of micro-RNAs microarrays as diagnostic and prognostic tool.

The hypothesis to use microRNAs (miRNAs) circulating in the blood as cancer biomarkers was formulated some years ago based on promising initial results. After some exciting discoveries, however, it became evident that the accurate quantification of cell-free miRNAs was more challenging than expected. Difficulties were linked to the strong impact that many, if not all, pre- and post- analytical variables have on the final results. In this study, we used currently available high-throughput technologies to identify miRNAs present in plasma and serum of patients with breast, colorectal, lung, thyroid and melanoma tumors, and healthy controls. Then, we assessed the absolute level of nine different miRNAs (miR-320a, miR-21-5p, miR-378a-3p, miR-181a-5p, miR-3156-5p, miR-2110, miR-125a-5p, miR-425-5p, miR-766-3p) in 207 samples from healthy controls and cancer patients using droplet digital PCR (ddPCR) technology. We identified miRNAs specifically modulated in one or more cancer types, according to tissue source. The significant reduction of miR-181a-5p levels in breast cancer patients serum was further validated using two independent cohorts, one from Italy (n = 70) and one from US (n = 90), with AUC 0.66 and 0.73 respectively. This study finally powers the use of cell-free miRNAs as cancer biomarkers and propose miR-181a-5p as a diagnostic breast cancer biomarker. The hypothesis to use microRNAs (miRNAs) circulating in the blood as cancer biomarkers was formulated some years ago based on promising initial results. After some exciting discoveries, however, it became evident that the accurate quantification of cell-free miRNAs was more challenging than expected. Difficulties were linked to the strong impact that many, if not all, pre- and post-analytical variables have on the final results. In this study, we used currently available high-throughput technologies to identify miRNAs present in plasma and serum of patients with breast, colorectal, lung, thyroid and melanoma tumors, and healthy controls. Then, we assessed the absolute level of nine different miRNAs (miR-320a, miR-21-5p, miR-378a-3p, miR-181a-5p, miR-3156-5p, miR-2110, miR-125a-5p, miR-425-5p, miR-766-3p) in 207 samples from healthy controls and cancer patients using droplet digital PCR (ddPCR) technology. We identified miRNAs specifically modulated in one or more cancer types, according to tissue source. The significant reduction of miR-181a-5p levels in breast cancer patients serum was further validated using two independent cohorts, one from Italy (n = 70) and one from US (n = 90), with AUC 0.66 and 0.73 respectively. This study finally powers the use of cell-free miRNAs as cancer biomarkers and propose miR-181a-5p as a diagnostic breast cancer biomarker.

Droplet digital PCR (ddPCR) has been successfully used with TaqMan assays to assess gene expression through the quantification of mRNA and miRNA. Recently, a new ddPCR system that can also run DNA-binding dye-based assays has been developed but it has not yet been tested for miRNA. We tested and compared the feasibility of quantifying miRNA with the new QX200 Droplet Digital PCR system when used with EvaGreen dye– and TaqMan probe–based assays. RNA from plasma and serum of 28 patients with cancer and healthy persons was reverse-transcribed and quantified for two circulating miRNAs and one added exogenous miRNA, with both EvaGreen dye–based miRCURY LNA miRNA assays and TaqMan assays. Amplification and detection of target miRNAs were performed on the QX200 ddPCR system. Conditions required to run miRCURY LNA miRNA assays were optimized. The EvaGreen-based assay was precise, reproducible over a range of concentrations of four orders of magnitude, and sensitive, detecting a target miRNA at levels down to 1 copy/μL. When this assay was compared with TaqMan assays, high concordance was obtained for two endogenous miRNAs in serum and plasma (Pearson r > 0.90). EvaGreen dye–based and TaqMan probe–based assays can be equally used with the ddPCR system to quantify circulating miRNAs in human plasma and serum. This study establishes the basis for using EvaGreen dye–based assays on a ddPCR system for quantifying circulating miRNA biomarkers and potentially other low-abundance RNA biomarkers in human biofluids. See all the articles in this CEBP Focus section, “Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology.” Cancer Epidemiol Biomarkers Prev; 23(12); 2638–42. ©2014 AACR.