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Over the last decade, the development of novel and high penetrance genomic approaches to analyze biological samples has provided very new insights in the comprehension of the molecular biology and genetics of tumors. The use of these techniques, consisting of exome sequencing, transcriptome, miRNome, chromosome alteration, genome, and epigenome analysis, has also been successfully applied to adrenocortical carcinoma (ACC). In fact, the analysis of large cohorts of patients allowed the stratification of ACC with different patterns of molecular alterations, associated with different outcomes, thus providing a novel molecular classification of the malignancy to be associated with the classical pathological analysis. Improving our knowledge about ACC molecular features will result not only in a better diagnostic and prognostic accuracy, but also in the identification of more specific therapeutic targets for the development of more effective pharmacological anti-cancer approaches. In particular, the specific molecular alteration profiles identified in ACC may represent targetable events by the use of already developed or newly designed drugs enabling a better and more efficacious management of the ACC patient in the context of new frontiers of personalized precision medicine.

L’ultimo decennio ha visto lo sviluppo esponenziale della genomica, ossia dell’insieme di tecniche di analisi molecolare dei campioni biologici. Tale approccio molecolare ha rivoluzionato la biologia e la genetica anche del carcinoma della corteccia surrenalica (ACC). L’applicazione di tali tecniche all’analisi di ampie casistiche di ACC ha portato all’identificazione di alterazioni nell’espressione genica, nello stato di metilazione, nella struttura cromosomica, dotate di valore prognostico e predittivo, permettendo di formulare una classificazione molecolare della malattia. Tale classificazione potrebbe rivestire un ruolo rilevante non solo per migliorare l’accuratezza della prognosi, ma soprattutto per identificare nuovi bersagli terapeutici utilizzabili per la cura di questo tumore, che ha ad oggi limitati e poco efficaci approcci terapeutici farmacologici.

In recent years it has been recognized that clinical translation of novel therapeutic strategies for patients with adrenocortical carcinoma (ACC) often fails. These disappointing results indicate that the currently utilized tumor models only poorly reflect relevant pathophysiology and, thereby, do not predict clinical applicability of novel pharmacological approaches. However, also the development of new preclinical ACC models has remained a challenge with only one human cell line (NCI-H295R) and one recently established human pediatric xenograft model (SJ-ACC3) being available for this highly heterogeneous malignancy. Our current study furthermore reveals a poor reproducibility of therapeutic action between different clones of the most commonly used tumor model NCI-H295R. In an attempt to broaden the current preclinical armamentarium, we aimed at the development of patient-individual tumor models. During these studies, one xenograft (MUC-1) displayed marked engraftment and sustained tumor growth. MUC-1 tumor analysis revealed highly vascularized, proliferating and SF-1 positive xenografts. In a next step, we characterized all currently available human tumor models for ACC for Ki67, SF-1 and EGF-receptor status in comparison with MUC-1-xenografts. In addition, we established a primary culture, which is now viable over 31 passages with sustained nuclear SF-1 and cytoplasmic 3βHSD immuno-positivity. Subsequent investigation of therapeutic responsiveness upon treatment with the current systemic gold standard EDP-M (etoposide, doxorubicin, cisplatin and mitotane) demonstrated maintenance of the clinically observed drug resistance for MUC-1 exclusively. In summary, we provide evidence for a novel patient-derived tumor model with the potential to improve clinical prediction of novel therapeutic strategies for patients with ACC.

// Giada Poli 1, * , Giulia Cantini 1, * , Roberta Armignacco 1, * , Rossella Fucci 1 , Raffaella Santi 2 , Letizia Canu 1 , Gabriella Nesi 2 , Massimo Mannelli 1 , Michaela Luconi 1 1 Endocrinology Unit, Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Florence Italy 2 Division of Pathological Anatomy, Department of Surgery and Translational Medicine, University of Florence, Florence Italy * These authors contributed equally to this work Correspondence to: Michaela Luconi, email: michaela.luconi@unifi.it Keywords: metformin, H295R, IGF-1R, apoptosis, tumor proliferation Received: March 24, 2016 Accepted: June 09, 2016 Published: July 06, 2016 ABSTRACT Adrenocortical carcinoma (ACC) is a rare heterogeneous malignancy with poor prognosis. Since radical surgery is the only available treatment, more specific and effective drugs are urgently required. The anti-diabetic drug metformin has been associated with a decreased cancer prevalence and mortality in several solid tumors, prompting its possible use for ACC treatment. This paper evaluates the in vitro and in vivo anti-cancer effects of metformin using the ACC cell model H295R. Metformin treatment significantly reduces cell viability and proliferation in a dose- and time-dependent manner and associates with a significant inhibition of ERK1/2 and mTOR phosphorylation/activation, as well as with stimulation of AMPK activity. Metformin also triggers the apoptotic pathway, shown by the decreased expression of Bcl-2 and HSP27, HSP60 and HSP70, and enhanced membrane exposure of annexin V, resulting in activation of caspase-3 apoptotic effector. Metformin interferes with the proliferative autocrine loop of IGF2/IGF-1R, which supports adrenal cancer growth. Finally, in the ACC xenograft mouse model, obtained by subcutaneous injection of H295R cells, metformin intraperitoneal administration inhibits tumor growth, confirmed by the significant reduction of Ki67%. Our data suggest that metformin inhibits H295R cell growth both in vitro and in vivo . Further preclinical studies are necessary to validate the potential anti-cancer effect of metformin in patients affected by ACC.

The adrenal gland is a multiendocrine organ with a steroidogenic mesenchymal cortex and an inner catecholamine-producing medulla of neuroendocrine origin. After embryonic development, this plastic organ undergoes a functional postnatal remodeling. Elucidating these complex processes is pivotal for understanding the early bases of functional endocrine disorders and tumors affecting the mature gland. We developed an in vitro human adrenal cell model derived from fetal adrenal specimens at different gestational ages, consisting of neuroendocrine and cortical components and expressing the zona and functional markers of the original fetal organ. These cortical and neuroendocrine progenitor cells retain in vitro an intrinsic gestational-age-related differentiation and functional program. In vitro these cells spontaneously form 3-dimensional structure organoids with a structure similar to the fetal gland. The organoids show morphofunctional features and adrenal steroidogenic factor, steroid acute regulatory, cytochrome-P450-17A1, dosage-sensitive, sex-reversal, adrenal hypoplasia-critical region on chromosome X protein , NOTCH1, and nephroblastoma overexpressed/cysteine-rich protein 61/connective tissue growth factor/nephroblastoma overexpressed gene-3; stem (BMI1, nestin); and chromaffin (chromogranin A, tyrosine hydroxylase) markers similar to those of the populations of origin. This in vitro human adrenal system represents a unique but preliminar model for investigating the pathophysiological processes underlying physiologic adrenal remodeling and pathologic alterations involved in organ hypo- and hyperplasia and cancer.-Poli, G., Sarchielli, E., Guasti, D., Benvenuti, S., Ballerini, L., Mazzanti, B., Armignacco, R., Cantini, G., Lulli, M., Chortis, V., Arlt, W., Romagnoli, P., Vannelli, G. B., Mannelli, M., Luconi, M. Human fetal adrenal cells retain age-related stem- and endocrine-differentiation potential in culture.

Metabolic interplay between the tumor microenvironment and cancer cells is a potential target for novel anti-cancer approaches. Among stromal components, adipocytes and adipose precursors have been shown to actively participate in tumor progression in several solid malignancies. In adrenocortical carcinoma (ACC), a rare endocrine neoplasia with a poor prognosis, cancer cells often infiltrate the fat mass surrounding the adrenal organ, enabling possible crosstalk with the adipose cells. Here, by using an in vitro co-culture system, we show that the interaction between adipose-derived stem cells (ASCs) and the adrenocortical cancer cell line H295R leads to metabolic and functional reprogramming of both cell types: cancer cells limit differentiation and increase proliferation of ASCs, which in turn support tumor growth and invasion. This effect associates with a shift from the paracrine cancer-promoting IGF2 axis towards an ASC-associated leptin axis, along with a shift in the SDF-1 axis towards CXCR7 expression in H295R cells. In conclusion, our findings suggest that adipose precursors, as pivotal components of the ACC microenvironment, promote cancer cell reprogramming and invasion, opening new perspectives for the development of more effective therapeutic approaches.

Context Novel tumor markers are urgently needed to better stratify adrenocortical cancer (ACC) patients and improve therapies for this aggressive neoplasm. Objective To assess the diagnostic and prognostic value of the actin-bundling protein fascin-1 (FSCN1) in adrenocortical tumors. Design, Setting and Participants A local series of 37 malignant/37 benign adrenocortical tumors at Careggi University Hospital and two independent validation ACC cohorts (Cochin, TCGA) from the European Network for the Study of Adrenal Tumors were studied. Main Outcome Measures FSCN1 expression was quantified by immunohistochemistry, Western blot and quantitative RT-PCR in ACC specimens; overall and disease-free survival associated with FSCN1 expression were assessed by Kaplan-Meier analysis and compared with that of Ki67 labeling index and tumor stage. Results Despite the low diagnostic power, in the Florence ACC series, FSCN1 immunohistochemical detection appeared as an independent prognostic factor, also refining results obtained with staging and Ki67 labeling index. The robust prognostic power of FSCN1 levels was further confirmed in two independent ACC cohorts. A positive correlation was found between FSCN1 and steroidogenic factor-1 (SF-1), with a substantially higher expression of both factors in ACCs at advanced stages and with at least one of the three Weiss score parameters associated with invasiveness. Moreover, we demonstrated FSCN1 role in promoting cell invasion in a human ACC cell line only in the case of increased SF-1 dosage. Conclusions These findings show that FSCN1 is a novel independent prognostic marker in ACC and may serve as a potential therapeutic target to block tumor spread.

// Francesca Salvianti 1, * , Letizia Canu 1, * , Giada Poli 1 , Roberta Armignacco 1 , Cristian Scatena 2 , Giulia Cantini 1 , Alessandra Di Franco 1 , Stefania Gelmini 1 , Tonino Ercolino 1 , Mario Pazzagli 1 , Gabriella Nesi 3 , Massimo Mannelli 1 , Pamela Pinzani 1, * and Michaela Luconi 1, * 1 Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Florence, Italy 2 Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy 3 Department of Surgery and Translational Medicine, University of Florence, Florence, Italy * These authors have equally contributed to the work Correspondence to: Michaela Luconi, email: michaela.luconi@unifi.it Keywords: adrenal carcinoma, miRNA, liquid biopsy, rare tumor, quantitative real time RT-PCR Received: February 27, 2017 Accepted: April 19, 2017 Published: July 10, 2017 ABSTRACT Adrenocortical cancer (ACC) is a rare aggressive malignancy. Recent ACC integrated genomics analysis contributed to redefine the risk groups on molecular basis, including tumor microRNAs (miRs), detectable also in the bloodstream. We developed a quantitative real-time (RT) assay for the measurement of miR483 and miR483-5p absolute levels in plasma samples. miR483/miR483-5p levels were evaluated in plasma samples of 27 patients with ACC before surgery and at follow-up. Statistically significant differences in miR483-5p and miR483 levels were found between stage 1/2 and stage 3/4 ACCs in pre-surgery and post-surgery samples. ROC curve analysis of miR483–5p levels gave a prediction of the clinical stage (accuracy 0.917±0.084), with the best cut-off value of 0.221 ng/ml, prognosticating overall and recurrence-free survival. In a multivariate Cox analysis (HR 16.2, 95%CI[1.39-188.6, P

Bone modulates testis function through osteocalcin (OCN) production. This paper assesses the association between serum OCN and androgen production recovery in morbidly obese males at 9 months after bariatric surgery.A cohort of n=103 obese males with mean±s.d. body mass index (BMI) 47.7±8.2 kg m(-2), age 42±11 years, consisting of n=76 patients undergoing gastric bypass and n=27 in the waiting list for surgery.At 9 months from surgery, a significant increase was observed in mean±s.d. total OCN (tOCN=10.4±10.3 ng ml(-1), P0.001) and undercarboxylated OCN (ucOCN=5.4±3.7 ng ml(-1), P0.001), total testosterone (TT, 5.6±6.5 nM, P0.001) and calculated free testosterone (cFT, 0.035±0.133 nM, P0.006), sex hormone binding globulin (SHBG, 21.2±16.7 nM, P0.001) and decrease in estradiol (E2, -30.1±51.9 pM, P0.001) levels only in operated patients, with a significant reduction in BMI (24%) and waist (20%). A positive correlation existed between tOCN and ucOCN (age-adjustment (age-adj.): β=0.692, P0.001) and their variations (age-adj.: β=0.629, P0.001) after surgery. Multivariate analysis in operated patients showed a significant positive association between variations in tOCN and TT (age-adj.: β=0.289, P=0.012), SHBG (age-adj.: β=0.326, P=0.005) but not with cFT variation. tOCN, but not luteinizing hormone (LH) variation was the only significant predictive factor of cFT recovery in the hypogonadal (TT12 nM) operated subjects even after age- and BMI-adjustment (adj.: β=0.582, P0.05). cFT improvement was significantly higher when considering operated patients with tOCN increase (0.045±0.123 vs -0.02±0.118 nM, P=0.015), hypogonadism (0.059±0.111 vs -0.059±0.138 nM, P=0.002) and younger than 35 years (0.102±0.108 vs -0.019±0.123 nM, P=0.009).OCN recovery observed after bariatric surgery is significantly associated with cFT improvement independently of BMI variation and age in hypogonadal morbidly obese males.

At present, mitotane (MTT) represents the first-line pharmacological approach for the treatment of advanced adrenocortical carcinoma (ACC). Despite clear evidence that the drug can reduce the clinical signs of steroid excess in secreting ACC, the mechanism mediating the possible toxic effect of MTT on tumor cells still remains obscure. This study investigated the intracellular events underlying the toxic effect of MTT by studying qualitative and quantitative alterations in mitochondrial morphology and functions in human adrenocortical cancer cell lines, H295R and SW13. Increasing concentrations of MTT resulted in rapid intracellular accumulation and conversion of the drug. Cytostatic and cytotoxic effects were evident at doses corresponding to the therapeutic window (30–50 μM) through an apoptotic mechanism involving caspase 3/7. Electron microscopic analysis of cell mitochondria displayed MTT-induced dose- and time-dependent alterations in the morphology of the organelle. These alterations were characterized by a marked swelling and a decrease in the number of respiratory cristae, accompanied by a significant depolarization of the mitochondrial membrane potential, finally leading to the disruption of the organelle. A drastic reduction of oxygen consumption was observed due to mitochondrial membrane damage, which was accompanied by a decrease in the levels of VDAC1 integral membrane channel. These findings contribute to better understand the intracellular mechanism of action of MTT in ACC cells, showing that its cytotoxic effect seems to be mainly mediated by an apoptotic process activated by the disruption of mitochondria.

Besides their well-known anti-diabetic effects, the peroxisome-proliferator-activated receptor gamma (PPARgamma) thiazolidinedione ligands (TZD) have been suggested to also display anti-inflammatory properties. The receptor role in mediating such effects is far from being elucidated. Here, we demonstrated that PPARgamma is necessary for TZD to interfere with TNFalpha and IFNgamma inflammatory activity in human endothelial cells. Different PPARgamma ligands similarly inhibit cytokinic stimulation of IFNgamma-inducible-protein-of-10-kDa (IP10) secretion in a dose-dependent manner and prevent the induced phosphorylation/activation of extracellular-signaling-regulated-kinases (ERK1/2). To further confirm the PPARgamma role in mediating both rapid and long term anti-inflammatory effects of its ligands, we evaluated RGZ inhibitory action in PPARgamma-silenced and -overexpressing cells. PPARgamma-silencing results in a reversion of RGZ inhibitory activity on cyto/chemokine secretions and rapid ERK phosphorylation. Conversely, receptor overexpression significantly increases RGZ inhibitory activity. Finally, PPARgamma-overexpression results in a reduction of ERK1/2 phosphorylation and inflammatory secretions in response to TNFalpha and IFNgamma even in the absence of RGZ, suggesting a restraining effect controlled by endogenous ligands. In conclusion, our data provide the first evidence that PPARgamma is involved in the anti-inflammatory action of TZD in endothelial cells, not only by modulating cyto/chemokine secretions but also by restraining ERK activation through a novel rapid nongenomic mechanism.

PPAR-γ is a member of the nuclear hormone receptor superfamily of transcription factors, whose thiazolidinedione ligands (TZD) have been recently demonstrated to also possess anticancer properties in addition to their well-known insulin-sensitizer and glucose/lipid regulation activity. In this minireview, we summarize the current knowledge on PPAR-γ in normal and tumoral corticotropic pituitary and adrenal cells. The receptor expression has been shown in ACTH-secreting cells in both normal and adenomal pituitary as well as in normal and tumor adrenal cortex. Preclinical studies conducted both in vitro on tumor cells and in vivo on xenograft tumor models obtained by subcutaneous injection of cancer cells have evidenced the anticancer properties of TZD, in particular rosiglitazone (RGZ) and pioglitazone (PIO). In both pituitary and adrenocortical cancer, RGZ treatment results in inhibition of cell proliferation, through G0/G1 cell-cycle arrest and induction of cell apoptosis, leading to significant inhibition of tumor growth in the xenograft tumor models. In addition, since RGZ can reduce ACTH and corticosterone secretion in mouse corticotropic pituitary tumors, both RGZ and PIO have been used in the treatment of Cushing’s disease with variable but generally unsatisfactory results. Discrepancies in the antitumor effects of TZD observed between successful preclinical and unsuccessful clinical studies may be particularly due to differences in treatment duration and doses used.

Adrenocortical carcinoma (ACC) is a rare malignancy, the prognosis of which is mainly dependent on stage at diagnosis. The identification of disease-associated markers for early diagnosis and drug monitoring is mandatory. Circulating tumor cells (CTCs) are released into the bloodstream from primary tumor/metastasis. CTC detection in blood samples may have enormous potential for assisting in the diagnosis of malignancy, estimating prognosis, and monitoring the disease.The aim of the study was to investigate the presence of CTCs in blood samples of patients with ACC or benign adrenocortical adenoma (ACA).We conducted the study at a university hospital.CTC analysis was performed in blood samples from 14 ACC patients and 10 ACA patients. CTCs were isolated on the basis of cell size by filtration through ScreenCell devices, followed by identification according to validated morphometric criteria and immunocytochemistry.We measured the difference in CTC detection between ACC and ACA.CTCs were detected in all ACC samples, but not in ACA samples. Immunocytochemistry confirmed the adrenocortical origin. When ACC patients were stratified according to the median value of tumor diameter and metastatic condition, a statistically significant difference was found in the number of CTCs detected after surgery. A significant correlation between the number of CTCs in postsurgical samples and clinical parameters was found for tumor diameter alone.Our findings provide the first evidence for adrenocortical tumors that CTCs may represent a useful marker to support differential diagnosis between ACC and ACA. The correlation with some clinical parameters suggests a possible relevance of CTC analysis for prognosis and noninvasive monitoring of disease progression and drug response.

Metabolic pathologies mainly originate from adipose tissue (AT) dysfunctions. AT differences are associated with fat-depot anatomic distribution in subcutaneous (SAT) and visceral omental (VAT) pads. We address the question whether the functional differences between the two compartments may be present early in the adipose stem cell (ASC) instead of being restricted to the mature adipocytes. Using a specific human ASC model, we evaluated proliferation/differentiation of ASC from abdominal SAT-(S-ASC) and VAT-(V-ASC) paired biopsies in parallel as well as the electrophysiological properties and functional activity of ASC and their in vitro-derived adipocytes. A dramatic difference in proliferation and adipogenic potential was observed between the two ASC populations, S-ASC having a growth rate and adipogenic potential significantly higher than V-ASC and giving rise to more functional and better organized adipocytes. To our knowledge, this is the first comprehensive electrophysiological analysis of ASC and derived-adipocytes, showing electrophysiological properties, such as membrane potential, capacitance and K(+)-current parameters which confirm the better functionality of S-ASC and their derived adipocytes. We document the greater ability of S-ASC-derived adipocytes to secrete adiponectin and their reduced susceptibility to lipolysis. These features may account for the metabolic differences observed between the SAT and VAT. Our findings suggest that VAT and SAT functional differences originate at the level of the adult ASC which maintains a memory of its fat pad of origin. Such stem cell differences may account for differential adipose depot susceptibility to the development of metabolic dysfunction and may represent a suitable target for specific therapeutic approaches.

Background Fifty to sixty percent of patients with essential thrombocythemia harbor the JAK2V617F mutation. The impact of this mutation on clinical phenotype is still debated. The aim of this study was to evaluate possible correlations between JAK2V617F mutant allele burden and both clinical presentation and hematologic abnormalities in essential thrombocythemia patients.Design and Methods In this single-center retrospective study, JAK2V617F allele load was measured by sensitive quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in the granulocytes of 260 patients diagnosed as having essential thrombocythemia according to WHO criteria.Results Median V617F allele burden in patients with the mutation (n=165, 63.4%) was 24%, ranging from 1% to 87%; an allele burden greater than 51% was found in 5% of the patients. Older patients presented progressively higher percentages of the V617F allele. Signs of stimulated erythropoiesis and myelopoiesis, as well as higher PRV-1 levels, were found in patients with the mutation, but no linear correlation with load of mutant allele could be ascertained; on the other hand, the frequency of patients with erythropoietin-independent erythroid colonies progressively increased depending on mutant allele load. Splenomegaly and microvessel symptoms were significantly more represented among patients with greater than 50% and 25% JAK2V617F allele burden, respectively. Increasing mutant allele load correlated with higher frequency of arterial thrombosis at diagnosis, as confirmed also in multivariate analysis; the relative risk was 3.0 (95% CI 1.3–6.8; p=0.01) in patients having a greater than 25% mutant allele burden.Conclusions The JAK2V617F mutant allele burden contributes to determining the clinical phenotype in patients with essential thrombocythemia.

Rosiglitazone (RGZ), a thiazolidinedione ligand of the peroxisome proliferator-activated receptor (PPAR)-γ, has been recently described as possessing antitumoral properties. We investigated RGZ effect on cell proliferation in two cell line models (SW13 and H295R) of human adrenocortical carcinoma (ACC) and its interaction with the signaling pathways of the activated IGF-I receptor (IGF-IR). We demonstrate a high expression of IGF-IR in the two cell lines and in ACC. Cell proliferation is stimulated by IGF-I in a dose- and time-dependent manner and is inhibited by RGZ. The analysis of the main intracellular signaling pathways downstream of the activated IGF-IR, phosphatidyl inositol 3-kinase (PI3K)-Akt, and extracellular signal-regulated kinase (ERK1/2) cascades reveals that RGZ rapidly interferes with the Akt and ERK1/2 phosphorylation/activation which mediates IGF-I stimulated proliferation. In conclusion, our results suggest that RGZ exerts an inhibitory effect on human ACC cell proliferation by interfering with the PI3K/Akt and ERK1/2 signaling pathways downstream of the activated IGF-IR.

To the editor: A JAK2 V617F mutation is harbored by most patients with polycythemia vera (PV) and has pathogenetic[1][1] and diagnostic[2][2] relevance. We used this molecular marker to trace origin of disease in a case of PV manifested 5 years after autologous transplantation for acute myeloid

Objective Expression profiling of microRNA (miRNA) was performed in granulocytes isolated from patients with primary myelofibrosis (PMF), with the aim of identifying abnormally expressed miRNAs in comparison with normal subjects or patients with polycythemia vera (PV) or essential thrombocythemia (ET). Patients and Methods Using stem loop–primed reverse transcription and TaqMan quantitative real-time polymerase chain reaction, the expression of 156 mature miRNAs was evaluated using pooled granulocytes from PMF patients, either wild-type or JAK2 617V>F mutant with >51% allele burden, and control subjects. Differentially expressed miRNAs were then validated on additional control and PMF samples, and also on PV or ET granulocytes. Results There was a global downregulation of miRNA expression in PMF granulocytes; 60 miRNAs, of 128 called present , displayed differential expression compared to normal samples. Twelve miRNAs, which had been selected based on statistically different expression level, were finally validated. In PMF granulocytes, levels of miR-31 , -150 , and -95 were significantly lower, while those of miR-190 significantly greater, than control and PV or ET samples; on the other hand, miR-34a , -342 , -326 , -105 , -149 , and -147 were similarly reduced in patients with PMF, PV, or ET compared to controls. Increased expression of miR-182 and -183 correlated with JAK2 617V>F allele burden. Three in silico–predicted putative target genes ( DTR , HMGA2 , and MYB ), showed deregulated expression in PMF granulocytes that correlated with expression level of regulatory miRNA. Conclusions A defined miRNA profile distinguishes PMF granulocytes from those of normal subjects and, partially, also from PV or ET patients.

Abnormal mobilization of CD34+ hematopoietic stem/progenitor cells in peripheral blood (PB) characterizes patients (pts) with Primary Myelofibrosis (PMF), and is attributed to both disruption of CXCR4/SDF-1 axis by a bone marrow (BM) proteolytic environment (Xu M, Blood 2005) and reduced CXCR4 expression on CD34+ cells (Rosti V, BCMD 2007) due to a transcriptional defect (Guglielmelli P, Stem Cells 2007). CXCR4 expression on CD34+ cells was inversely related to their number in PB and associated with advanced disease. We aimed at addressing potential mechanisms of reduced CXCR4 transcription in PMF CD34+ cells. To rule out cytokine-induced transcriptional regulation, CD34+ cells from controls and PMF pts were exposed to a range of SDF-1, G-CSF, TGF-beta and Interferon-gamma concentrations, and CXCR4 protein and mRNA level were concurrently measured by flow cytometry and quantitative PCR (RTQ-PCR), respectively. SDF-1 at 500 ng/mL for 24 hrs reduced CXCR4+/CD34+ cells to less than 10% basal value while CXCR4 mRNA level was unchanged, suggesting ligand-induced receptor internalization but no effect on gene transcription. Other cytokines tested were ineffective. Since abnormal methylation of CXCR4 has been implicated in reduced CXCR4 expression in pancreatic cancer cells (Sato N, Canc Biol Ther 2005), we evaluated methylation pattern of five CpG islands in CXCR4 promoter in PMF CD34+ cells and in HEL, K562 and HL-60 cell lines as control, using both methylation-specific PCR and bisulphite sequencing. A correlation was found between CXCR4 expression and extent of CpG island methylation in cell lines: K562 had 8±6% CXCR4+ cells and all five CpG islands were methylated while in HL-60, that showed bright CXCR4 expression in 90% of the cells, the promoter was in a completely un-methylated state; in HEL cell line, that expressed CXCR4 in 37±10% of cells, partial methylation of CpG island 1 and 5 was observed. On the other hand, CXCR4 promoter CpG island 1 hypermethylation was found in CD34+ cells of 15 PMF pts, and in a minority of them also of CpG island 4 or 5. The effect of hypomethylating agent 5-azacytydine (5-AZA; 1 uM up to 72 hrs) was evaluated in CD34+ cells from 10 PMF pts; starting from 12 hr incubation, significantly increased proportion (from 3- to greater than 10-fold basal level) of CD34+ cells expressed CXCR4, and a three- to five-fold induction of CXCR4 RNA by RTQ-PCR was detectable at 8–12hr. Similar effects were observed after 5-AZA treatment of HEL, while in K562 cells changes in CXCR4 expression were minimal. Concurrent incubation with 5-AZA and HDAC inhibitor SAHA induced effects similar to hypomethylating agent alone. In both PMF CD34+ and HEL cells treated with 5-AZA, hypermethylation of CpG 1 was almost completely reverted at 24–48 hr. Finally, 5-AZA treated CD34+ cells showed enhanced migration in response to SDF-1. These data point to abnormal methylation of CXCR4 promoter as a mechanims for reduced CXCR4 expression in PMF CD34+ cells, possibly contributing to their constitutive migration in PB. Additionally, owing demonstration of an effect of 5-AZA combined with HDAC inhibitor TSA on the proportion of JAK2617V>F PMF mutant clones in culture (Shi J, Canc Res 2007), they represent a proof-of-principle for exploring use of hypomethylating agents in PMF.