Francesca Simonato

Therapeutic targeting of myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) represent a subset of myeloid cells that expand under pathological conditions, such as cancer development, acute and chronic infections, trauma, bone marrow transplantations, and some autoimmune diseases. MDSCs mediate a negative regulation of the immune response by affecting different T lymphocyte subsets. Potential mechanisms, which underlie this inhibitory activity range from those requiring direct cell-to-cell contact with others, more indirect, and mediated by the modification of the microenvironment. Pharmacological inhibition of MDSC suppressive pathways is a promising strategy to overcome disease-induced immune defects, which might be a key step in enhancing the effectiveness of immune-based therapies.

Molecular Typing of Lung Adenocarcinoma on Cytological Samples Using a Multigene Next Generation Sequencing Panel

Identification of driver mutations in lung adenocarcinoma has led to development of targeted agents that are already approved for clinical use or are in clinical trials. Therefore, the number of biomarkers that will be needed to assess is expected to rapidly increase. This calls for the implementation of methods probing the mutational status of multiple genes for inoperable cases, for which limited cytological or bioptic material is available. Cytology specimens from 38 lung adenocarcinomas were subjected to the simultaneous assessment of 504 mutational hotspots of 22 lung cancer-associated genes using 10 nanograms of DNA and Ion Torrent PGM next-generation sequencing. Thirty-six cases were successfully sequenced (95%). In 24/36 cases (67%) at least one mutated gene was observed, including EGFR, KRAS, PIK3CA, BRAF, TP53, PTEN, MET, SMAD4, FGFR3, STK11, MAP2K1. EGFR and KRAS mutations, respectively found in 6/36 (16%) and 10/36 (28%) cases, were mutually exclusive. Nine samples (25%) showed concurrent alterations in different genes. The next-generation sequencing test used is superior to current standard methodologies, as it interrogates multiple genes and requires limited amounts of DNA. Its applicability to routine cytology samples might allow a significant increase in the fraction of lung cancer patients eligible for personalized therapy.

Laterally transferred elements and high pressure adaptation in Photobacterium profundum strains.

BackgroundOceans cover approximately 70% of the Earths surface with an average depth of 3800 m and a pressure of 38 MPa, thus a large part of the biosphere is occupied by high pressure environments. Piezophilic (pressure-loving) organisms are adapted to deep-sea life and grow optimally at pressures higher than 0.1 MPa. To better understand high pressure adaptation from a genomic point of view three different Photobacterium profundum strains were compared. Using the sequenced piezophile P. profundum strain SS9 as a reference, microarray technology was used to identify the genomic regions missing in two other strains: a pressure adapted strain (named DSJ4) and a pressure-sensitive strain (named 3TCK). Finally, the transcriptome of SS9 grown under different pressure (28 MPa; 45 MPa) and temperature (4°C; 16°C) conditions was analyzed taking into consideration the differentially expressed genes belonging to the flexible gene pool.ResultsThese studies indicated the presence of a large flexible gene pool in SS9 characterized by various horizontally acquired elements. This was verified by extensive analysis of GC content, codon usage and genomic signature of the SS9 genome. 171 open reading frames (ORFs) were found to be specifically absent or highly divergent in the piezosensitive strain, but present in the two piezophilic strains. Among these genes, six were found to also be up-regulated by high pressure.ConclusionThese data provide information on horizontal gene flow in the deep sea, provide additional details of P. profundum genome expression patterns and suggest genes which could perform critical functions for abyssal survival, including perhaps high pressure growth.

Transcription factors in myeloid-derived suppressor cell recruitment and function.

In normal hematopoiesis, differentiation and maturation of cell populations belonging to various lineages are tightly regulated by the interaction of many transcription factors. The relative numbers of different myeloid cells depends on their proliferative/apoptotic rate, while their identity relates to their recruitment to the sites of action and the expression of specific genes regulating their function. Under pathological conditions, as during chronic inflammation and cancer development, an aberrant hematopoiesis occurs, with the consequent expansion of myeloid-derived suppressor cells (MDSCs). These cells have distinctive properties that determine their ability to tune down the immune system by principally inactivating CD8(+) T cells. Understanding the molecular networks regulating the phenotypic and functional determination of MDSCs is essential to identify potential therapeutic targets to revert immune deregulation in cancer.

The miR-17-92 microRNA cluster: a novel diagnostic tool in large B-cell malignancies.

Diffuse large B-cell lymphoma (DLBCL) can present as de novo or can arise through the transformation of many indolent lymphomas, including follicular lymphoma (FL). The morphological differentiation between germinal center-DLBCL (GC-DLBCL) and high-grade (grade 3) FL could be challenging; the accurate sub-classification of large B-cell lymphomas is mandatory in order to select the most appropriate among the new-targeted therapies. Recent expression profiling studies reported microRNAs (miRNAs) (and miR-17-92 cluster, in particular) as useful tools in differentiating DLBCL and FL. However, these preliminary results are based on cell line-derived data or did not consider grade 3 FL cases. To investigate this point, 36 cases of GC-DLBCL and 18 cases of grade 3 non-transforming FL were considered. All diagnoses were based on the World Health Organization criteria and were confirmed by clinical, histological, and immunohistochemical data. Six members of the miR-17-92 cluster (ie, miR-18b, miR-19b, miR-20a, miR-92, miR-93, and miR-106a) and two control miRNAs (ie, miR-150 and miR-210) were quantified by quantitative reverse transcription-polymerase chain reaction. All the considered miR-17-92 cluster miRNAs were significantly overexpressed in GC-DLBCL, being miR-20a and miR-106a the most dysregulated (P<0.001). Receiver operating characteristics (ROCs) analysis was used to find the optimal cut-off in distinguishing the two histotypes. The ROC estimated thresholds for miR-18b, miR-19b, miR-20a, miR-92, and miR-106a displayed a sensitivity level higher than 0.80 in achieving the GC-DLBCL diagnosis. The classification tree built on the six thresholds allowed the correct identification of 35/36 GC-DLBCL (97.2%). Profiling the miR-17-92 cluster is a promising investigative method for differentiating GC-DLBCL from high-grade FL. Subject to the validation of these findings in further larger studies; miR-17-92 cluster could represent a reliable, standardizable diagnostic tool for the sub-classification of large B-cell lymphoid neoplasm.

Next-Generation Sequencing of Lung Cancer EGFR Exons 18-21 Allows Effective Molecular Diagnosis of Small Routine Samples (Cytology and Biopsy)

Selection of lung cancer patients for therapy with tyrosine kinase inhibitors directed at EGFR requires the identification of specific EGFR mutations. In most patients with advanced, inoperable lung carcinoma limited tumor samples often represent the only material available for both histologic typing and molecular analysis. We defined a next generation sequencing protocol targeted to EGFR exons 18-21 suitable for the routine diagnosis of such clinical samples. The protocol was validated in an unselected series of 80 small biopsies (n=14) and cytology (n=66) specimens representative of the material ordinarily submitted for diagnostic evaluation to three referral medical centers in Italy. Specimens were systematically evaluated for tumor cell number and proportion relative to non-neoplastic cells. They were analyzed in batches of 100-150 amplicons per run, reaching an analytical sensitivity of 1% and obtaining an adequate number of reads, to cover all exons on all samples analyzed. Next generation sequencing was compared with Sanger sequencing. The latter identified 15 EGFR mutations in 14/80 cases (17.5%) but did not detected mutations when the proportion of neoplastic cells was below 40%. Next generation sequencing identified 31 EGFR mutations in 24/80 cases (30.0%). Mutations were detected with a proportion of neoplastic cells as low as 5%. All mutations identified by the Sanger method were confirmed. In 6 cases next generation sequencing identified exon 19 deletions or the L858R mutation not seen after Sanger sequencing, allowing the patient to be treated with tyrosine kinase inhibitors. In one additional case the R831H mutation associated with treatment resistance was identified in an EGFR wild type tumor after Sanger sequencing. Next generation sequencing is robust, cost-effective and greatly improves the detection of EGFR mutations. Its use should be promoted for the clinical diagnosis of mutations in specimens with unfavorable tumor cell content.

Programmed cell death 4 and microRNA 21 inverse expression is maintained in cells and exosomes from ovarian serous carcinoma effusions

Ovarian serous carcinoma (OSC) is a fatal gynecologic malignancy usually presenting with bilateral localization and malignant peritoneal effusion. Programmed cell death 4 (PDCD4) is a tumor suppressor gene whose expression is directly controlled by microRNA‐21 (miR‐21). Exosomes are small cell‐derived vesicles that participate in intercellular communication, delivering their cargo of molecules to specific cells. Exosomes are involved in several physiological and pathological processes including oncogenesis, immunomodulation, angiogenesis, and metastasis. The current study analyzed the expression of PDCD4 and miR‐21 in resected OSC specimens and in cells and exosomes from OSC peritoneal effusions.

GPER-1 and estrogen receptor-β ligands modulate aldosterone synthesis.

Fertile women have lower blood pressure and cardiovascular risk than age-matched men, which suggests that estrogens exert cardiovascular protective effects. However, whether 17 β-estradiol (E2) blunts aldosterone secretion, and thereby affects the gender dimorphism of blood pressure, is unknown. We therefore sought for the estrogen receptor (ER) subtypes in human adrenocortical tissues ex vivo by performing gene and protein expression studies. We also investigated the effect of E2 on aldosterone synthesis and the involved receptors through in vitro functional experiments in the adrenocortical cells HAC15. We found that in the human adrenal cortex and aldosterone-producing adenoma cells, the most expressed ERs were the ERβ and the G protein-coupled receptor-1 (GPER-1), respectively. After selective ERβ blockade, E2 (10 nmol/L) markedly increased both the expression of aldosterone synthase and the production of aldosterone (+5- to 7-fold vs baseline, P < .001). Under the same condition, the GPER-1 receptor agonist 1-[4-(6-bromo-benzo (1, 3)dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c] quinolin-8-yl]-ethanone (G-1) (10 nmol/L) mimicked this effect, which was abrogated by cotreatment with either the GPER-1 receptor antagonist (3aS*,4R*,9bR*)-4-(6-Bro-mo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinoline (G-15), or a selective protein kinase A inhibitor 8-Bromo-2-monobutyryladenosine-3,5-cyclic mono-phosphorothioate, Rp-isomer. Silencing of the ERβ significantly raised aldosterone synthase expression and aldosterone production. Conversely, silencing of the GPER-1 lowered aldosterone synthase gene and protein expression. Moreover, it blunted the stimulatory effect of E2 on aldosterone synthase that was seen during ERβ blockade. These results support the conclusion that in humans, E2 inhibits aldosterone synthesis by acting via ERβ. Pharmacologic disinhibition of ERβ unmasks a potent secretagogue effect of E2 that involves GPER-1 and protein kinase A signaling.

A 4-MicroRNA Signature Can Discriminate Primary Lymphomas From Anaplastic Carcinomas in Thyroid Cytology Smears

Anaplastic thyroid carcinoma (ATC) and primary thyroid lymphoma (PTL) are uncommon tumors of the thyroid gland with several overlapping clinical and pathologic features that may render their differentiation difficult in fine‐needle aspiration (FNA) cytology. MicroRNA (miRNA) signatures have been recently reported as useful diagnostic tools applied to cytology specimens.

Fine needle aspiration of non-small cell lung cancer: current state and future perspective.

A. Fassina, R. Cappellesso, F. Simonato, C. Lanza, A. Marzari and M. Fassan