Claudia Augello

Inhibitors of apoptosis proteins (IAPs) expression and their prognostic significance in hepatocellular carcinoma

BackgroundSimilarly to other tumor types, an imbalance between unrestrained cell proliferation and impaired apoptosis appears to be a major unfavorable feature of hepatocellular carcinoma (HCC). The members of IAP family are key regulators of apoptosis, cytokinesis and signal transduction. IAP survival action is antagonized by specific binding of Smac/DIABLO and XAF1. This study aimed to investigate the gene and protein expression pattern of IAP family members and their antagonists in a series of human HCCs and to assess their clinical significance.MethodsRelative quantification of IAPs and their antagonist genes was assessed by quantitative Real Time RT-PCR (qPCR) in 80 patients who underwent surgical resection for HCC. The expression ratios of XIAP/XAF1 and of XIAP/Smac were also evaluated. Survivin, XIAP and XAF1 protein expression were investigated by immunohistochemistry. Correlations between mRNA levels, protein expression and clinicopathological features were assessed. Follow-up data were available for 69 HCC patients. The overall survival analysis was estimated according to the Kaplan-Meier method.ResultsSurvivin and Livin/ML-IAP mRNAs were significantly over-expressed in cancer tissues compared to non-neoplastic counterparts. Although Survivin immunoreactivity did not correlate with qPCR data, a significant relation was found between higher Survivin mRNA level and tumor stage, tumor grade and vascular invasion.The mRNA ratio XIAP/XAF1 was significantly higher in HCCs than in cirrhotic tissues. Moreover, high XIAP/XAF1 ratio was an indicator of poor prognosis when overall survival was estimated and elevated XIAP immunoreactivity was significantly associated with shorter survival.ConclusionOur study demonstrates that alterations in the expression of IAP family members, including Survivin and Livin/ML-IAP, are frequent in HCCs. Of interest, we could determine that an imbalance in XIAP/XAF1 mRNA expression levels correlated to overall patient survival, and that high XIAP immunoreactivity was a poor prognostic factor.

MicroRNA profiling of hepatocarcinogenesis identifies C19MC cluster as a novel prognostic biomarker in hepatocellular carcinoma

Progressive hepatocarcinogenesis is a stepwise process that drives liver transformation. However, the molecular mechanisms of early liver transformation are far from clear. A role for microRNAs (miRNA) as diagnostic and prognostic factors in human tumours, including hepatocellular carcinoma (HCC), is promising. We aimed to identify novel miRNA as biomarkers for differential diagnosis and predictors of disease progression.

miR-296 regulation of a cell polarity–cell plasticity module controls tumor progression

The expression of small, non-coding RNA or microRNAs (miR), is frequently deregulated in human cancer, but how these pathways affect disease progression is still largely elusive. Here, we report on a miR, miR-296, which is progressively lost during tumor progression and correlates with metastatic disease in colorectal, breast, lung, gastric, parathyroid, liver and bile ducts cancers. Functionally, miR-296 controls a global cell motility gene signature in epithelial cells by transcriptionally repressing the cell polarity–cell plasticity module, Scribble (Scrib). In turn, loss of miR-296 causes aberrantly increased and mislocalized Scrib in human tumors, resulting in exaggerated random cell migration and tumor cell invasiveness. Re-expression of miR-296 in MDA-MB231 cells inhibits tumor growth in vivo. Finally, miR-296 or Scrib levels predict tumor relapse in hepatocellular carcinoma patients. These data identify miR-296 as a global repressor of tumorigenicity and uncover a previously unexplored exploitation of Scrib in tumor progression in humans.

MicroRNA-425-3p predicts response to sorafenib therapy in patients with hepatocellular carcinoma

Sorafenib is the standard of care in advanced hepatocellular carcinoma (HCC), however no criteria have been established to select patients likely to benefit from this therapy. In this study, we evaluated the predictive role of microRNAs (miRNAs) in this setting of patients.

TP53 and p16INK4A, but not H-KI-Ras, are involved in tumorigenesis and progression of pleomorphic adenomas.

The putative role of TP53 and p16INK4A tumor suppressor genes and Ras oncogenes in the development and progression of salivary gland neoplasias was studied in 28 cases of pleomorphic adenomas (PA), 4 cases of cystic adenocarcinomas, and 1 case of carcinoma ex‐PA. Genetic and epigenetic alterations in the above genes were analyzed by Polymerase Chain Reaction/Single Strand Conformational Polymorphism (PCR/SSCP) and sequencing and by Methylation Specific‐PCR (MS‐PCR). Mutations in TP53 were found in 14% (4/28) of PAs and in 60% (3/5) of carcinomas. Mutations in H‐Ras and K‐Ras were identified in 4% (1/28) and 7% (2/28) of PAs, respectively. Only 20% (1/5) of carcinomas screened displayed mutations in K‐Ras. p16INK4A promoter hypermethylation was found in 14% (4/28) of PAs and 100% (5/5) carcinomas. All genetic and epigenetic alterations were detected exclusively in the epithelial and transitional tumor components, and were absent in the mesenchymal parts. Our analysis suggests that TP53 mutations and p16INK4A promoter methylation, but not alterations in the H‐Ras and K‐Ras genes, might be involved in the malignant progression of PA into carcinoma.

Quantitative DNA methylation analysis improves epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is a rare disorder characterized by overgrowth and predisposition to embryonal tumors. BWS is caused by various epigenetic and/or genetic alterations that dysregulate the imprinted genes on chromosome region 11p15.5. Molecular analysis is required to reinforce the clinical diagnosis of BWS and to identify BWS patients with cancer susceptibility. This is particularly crucial prenatally because most signs of BWS cannot be recognized in utero. We established a reliable molecular assay by pyrosequencing to quantitatively evaluate the methylation profiles of ICR1 and ICR2. We explored epigenotype-phenotype correlations in 19 patients that fulfilled the clinical diagnostic criteria for BWS, 22 patients with suspected BWS, and three fetuses with omphalocele. Abnormal methylation was observed in one prenatal case and 19 postnatal cases, including seven suspected BWS. Seven cases showed ICR1 hypermethylation, five cases showed ICR2 hypomethylation, and eight cases showed abnormal methylation of ICR1 and ICR2 indicating paternal uniparental disomy (UPD). More cases of ICR1 alterations and UPD were found than expected. This is likely due to the sensitivity of this approach, which can detect slight deviations in methylation from normal levels. There was a significant correlation (p < 0.001) between the percentage of ICR1 methylation and BWS features: severe hypermethylation (range: 75–86%) was associated with macroglossia, macrosomia, and visceromegaly, whereas mild hypermethylation (range: 55–59%) was associated with umbilical hernia and diastasis recti. Evaluation of ICR1 and ICR2 methylation by pyrosequencing in BWS can improve epigenotype-phenotype correlations, detection of methylation alterations in suspected cases, and identification of UPD.

miR-494-3p is a novel tumor driver of lung carcinogenesis

Lung cancer is the leading cause of tumor-related death worldwide and more efforts are needed to elucidate lung carcinogenesis. Here we investigated the expression of 641 miRNAs in lung tumorigenesis in a K-Ras(+/LSLG12Vgeo);RERTn(ert/ert) mouse model and 113 human tumors. The conserved miRNA cluster on chromosome 12qF1 was significantly and progressively upregulated during murine lung carcinogenesis. In particular, miR-494-3p expression was correlated with lung cancer progression in mice and with worse survival in lung cancer patients. Mechanistically ectopic expression of miR-494-3p in A549 lung cancer cells boosted the tumor-initiating population enhanced cancer cell motility, and increased the expression of stem cell-related genes. Importantly, miR-494-3p improved the ability of A549 cells to grow and metastasize in vivo, modulating NOTCH1 and PTEN/PI3K/AKT signaling. Overall, these data identify miR-494-3p as a key factor in lung cancer onset and progression and possible therapeutic target.

Molecular analyses in the diagnosis of myeloproliferative neoplasm-related splanchnic vein thrombosis

Dear Editor, Myeloproliferative neoplasms (MPNs) are characterized by proliferation of mature blood elements, progressive stromal alterations and/or evolution to acute myeloid leukemia. Significant morbidity and mortality are due to an increased risk of thrombosis, either arterial or venous [1]. Clinical manifestations may vary widely; in particular, venous thrombosis can occur not only in common sites, but also in unusual sites, including cerebral [2] or splanchnic district (SVT), the latter showing a prevalence ranging between 1 and 23 %. As the occurrence of SVT can be related to MPNs in about half of the cases, the search for JAK2 V617F mutation should be performed in all SVT patients, even in the presence of normal blood cell counts [3, 4]. However, this mutation is not detectable in all MPN patients, rendering this biomarker far from being an optimal diagnostic test in SVT-related MPN cases. Regarding MPL, its mutations have been documented in a significantly lower number of SVT patients [5, 6]. Finally, after the description of calreticulin (CALR) mutations [7, 8], Turon et al. [9] and Haslam et al. [10] have recently evaluated their incidence in two cohorts of SVT patients of different etiologies, but they detected them in only 1.9% and in 0% of all cases, respectively. To gain further insights, we retrospectively evaluated a consecutive series of 29 patients from our hospital between 1979 and 2013 with a diagnosis of MPN-related SVT. All our cases were evaluated for JAK2 V617F and MPL mutations. In 27 patients out of 29, JAK2 V617F mutation was detected, and we also evaluated JAK2 allele burden: it varied widely and the median value was 27 % (range 4.8– 97 %). The two JAK2-negative patients were evaluated for MPL mutations, and one of them carried a rare mutant variant, W515K. In the other patient, CALR was subsequently evaluated but did not carry any mutation. All these features are reported in Table 1. These molecular data are in line with previous findings in the literature and stress the critical role of searching for JAK2 V617F mutation in all SVT patients [3, 4]. However, this diagnostic test alone is not always enough: indeed, two patients of ours did not carry this mutation. As a consequence, in JAK2-negative cases, the next step should be to search for CALR and, if negative, also for MPL mutations, even though in the literature the latter have been reported in only 3.4 % of SVT cases [5, 6, 10]. Furthermore, our data show that JAK2 allele burden varies widely in MPN-related SVT patients, thus not representing a useful tool to distinguish between the different BCR-ABL1negative MPNs. Regarding CALR mutations, in the only A. Iurlo (*) :D. Cattaneo : E. Fermo :A. Cortelezzi Oncohematology Division, IRCCS Ca’ Granda – Maggiore Policlinico Hospital Foundation and University of Milan, Milan, Italy e-mail: aiurlo@policlinico.mi.it

MicroRNA as potential biomarker in HCV-associated diffuse large B-cell lymphoma

Aims To identify molecular characteristics to hepatitis C virus (HCV)-associated diffuse large B-cell lymphoma (DLBCL) through a comprehensive miRNAs expression profiling. Methods In this study, miRNA profiles were obtained from 37 patients with DLBCLs and 60 patients with reactive lymph nodes, equally distributed according to HCV presence. Germinal centres, from reactive lymph nodes were used as controls. Clinical features at presentation were available for all patients. Results A set of 52 miRNAs define a signature for HCV-associated DLBCL. Importantly, decreased expression of miR-138-5p and increased expression of miR-147a, miR-147b and miR-511-5p in HCV DLBCL was found to be a poor prognostic factor for HCV-positive DLBCL patients. Conclusions These data reveal molecular differences in diffuse DLBCL patients according to HCV presence, potentially useful as novel prognostic or therapeutic biomarkers.

PDGFB hypomethylation is a favourable prognostic biomarker in primary myelofibrosis.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterised by the clonal proliferation of the haematopoietic precursors together with the progressive development of bone marrow fibrosis. This stromal alteration is an important clinical issue and specific prognostic markers are not currently available. In bone marrow biopsies from 58 PMF patients, we explored the methylation pattern of genes encoding cytokines involved in the stromal reaction, namely platelet-derived growth factor-beta (PDGFB), transforming growth factor-beta (TGFB) and basic fibroblast growth factor (FGF2). We also evaluated the methylation profile of the Long Interspersed Nucleotide Element 1 (LINE-1). PDGFB, FGF2 and LINE-1, but not TGFB, were significantly differently methylated in PMF compared to controls. Significantly, PDGFB hypomethylation (<16%) was correlated with a favourable PMF prognosis (grade of marrow fibrosis, p=0.03; International Prognostic Scoring Systems p=0.01 and Dynamic International Prognostic Scoring Systems, p=0.02). Although the basis of the association of PDGFB hypomethylation with favourable prognosis remains to be clarified, we speculate that hypomethylation in PMF could represent the effect of acquired somatic mutations in genes involved in epigenetic regulation of the genome.