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Despite the significant achievements in the diagnosis and treatment of metastatic breast cancer (MBC), this condition remains substantially an incurable disease. In recent years, several clinical studies have aimed to identify novel molecular targets, therapeutic strategies, and predictive biomarkers to improve the outcome of women with MBC. Overall, ~40% of hormone receptor (HR) + /HER2 - MBC cases harbor alterations affecting the (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. This pathway is a major target in oncogenesis, as it regulates growth, proliferation, cell survival, and angiogenesis. Lately, the pharmacologic targeting of PIK3CA in HR + /HER2 - MBC has shown significant benefits after the occurrence of endocrine therapy resistance. The orally available α-selective PIK3CA inhibitor, alpelisib, has been approved in this setting. To perform an optimal patients' selection for this drug, it is crucial to adopt a tailored methodology. Clinically relevant PIK3CA alterations may be detected in several biospecimens (e.g. tissue samples and liquid biopsy) using different techniques (e.g. real-time PCR and next-generation sequencing). In this study, we provide an overview of the role of PIK3CA in breast cancer and of the characterization of its mutational status for appropriate clinical management., Competing Interests: NF has received honoraria for consulting, advisory role, and/or speaker bureau from Merck Sharp & Dohme (MSD), Boehringer Ingelheim, and Novartis. UM received personal fees (for service in the speaker bureau and as an advisor) from Boehringer Ingelheim, AstraZeneca, Roche, MSD, Amgen, Merck, Eli Lilly, Thermofisher, and Diaceutics. MF received honoraria for consulting, advisory role, speaker bureau, and/or research funding from Astellas Pharma, QED Therapeutics, Diaceutics, Tesaro, Roche, Eli Lilly, and Novartis. CM received honoraria for consulting from Roche, Bayer, AstraZeneca, and Daiichi Sankyo. SB received honoraria for consulting/advisory role/speaker bureau from AstraZeneca, Novartis, Bayer, and Diaceutics. SZ received research funding from AstraZeneca. CC received honoraria for consulting/advisory role/speaker bureau from Novartis, Eli-Lilly, Pfizer, and Roche. PV received honoraria from AstraZeneca, BMS, Celgene, Eli-Lilly, GSK, Incyte, MSD, Novartis, Pfizer, and Teva and research funds from Novartis and Pfizer. AD received honoraria for advisory board roles from Bayer, Roche, Merck, PharmaMar, and Novartis Oncology. EM received honoraria for consulting, advisory role, and/or speaker bureau from Novartis, Roche, and Shire. GV received honoraria for consulting, advisory role, speaker bureau, travel, accommodation, expenses, and/or research funding from MSD Oncology, Pfizer, Dako, Roche/Genetech, Astellas Pharma, Novartis, Bayer, Daiichi Sankyo, Menarini, Ventana Medical Systems Dako/Agilent Technologies, Cepheid, and Celgene. The handling editor declared a shared affiliation, though no other collaboration, with one of the authors UM., (Copyright © 2021 Fusco, Malapelle, Fassan, Marchiò, Buglioni, Zupo, Criscitiello, Vigneri, Dei Tos, Maiorano and Viale.)

Chronic lymphocytic leukaemia (CLL) is characterised by a heterogeneous clinical course. Such heterogeneity is associated with a number of markers, including TP53 gene inactivation. While TP53 gene alterations determine resistance to chemotherapy, it is not clear whether they can influence early disease progression. To clarify this issue, TP53 mutations and deletions of the corresponding locus [del(17p)] were evaluated in 469 cases from the O-CLL1 observational study that recruited a cohort of clinically and molecularly characterised Binet stage A patients. Twenty-four cases harboured somatic TP53 mutations [accompanied by del(17p) in 9 cases], 2 patients had del(17p) only, and 5 patients had TP53 germ-line variants. While del(17p) with or without TP53 mutations was capable of significantly predicting the time to first treatment, a reliable measure of disease progression, TP53 mutations were not. This was true for cases with high or low variant allele frequency. The lack of predictive ability was independent of the functional features of the mutant P53 protein in terms of transactivation and dominant negative potential. TP53 mutations alone were more frequent in patients with mutated IGHV genes, whereas del(17p) was associated with the presence of adverse prognostic factors, including CD38 positivity, unmutated-IGHV gene status, and NOTCH1 mutations.

Molecular characterization of Circulating Tumor Cells (CTCs) is still challenging, despite attempts to minimize the drawbacks of Whole Genome Amplification (WGA). In this paper, we propose a Next-Generation Sequencing (NGS) optimized protocol based on molecular tagging technology, in order to detect CTCs mutations while skipping the WGA step. MDA-MB-231 and MCF-7 cell lines, as well as leukocytes, were sorted into pools (2-5 cells) using a DEPArray™ system and were employed to set up the overall NGS procedure. A substantial reduction of reagent volume for the preparation of libraries was performed, in order to fit the limited DNA templates directly derived from cell lysates. Known variants in TP53 , KRAS , and PIK3CA genes were detected in almost all the cell line pools (35/37 pools, 94.6%). No additional alterations, other than those which were expected, were found in all tested pools and no mutations were detected in leukocytes. The translational value of the optimized NGS workflow is confirmed by sequencing CTCs pools isolated from eight breast cancer patients and through the successful detection of variants. In conclusion, this study shows that the proposed NGS molecular tagging approach is technically feasible and, compared to traditional NGS approaches, has the advantage of filtering out the artifacts generated during library amplification, allowing for the reliable detection of mutations and, thus, making it highly promising for clinical use.

Observational/retrospective studies indicate that prostaglandin-endoperoxide synthase-2 (PTGS2) inhibitors could positively affect colorectal cancer (CRC) patients' survival after diagnosis. To obtain an acceptable cost/benefit balance, the inclusion of PTGS2 inhibitors in the adjuvant setting needs a selective criterion. We quantified the 72 kDa, CRC-associated, glycosylated form of PTGS2 in 100 frozen CRC specimens and evaluated PTGS2 localization by IHC in the same tumors, scoring tumor epithelial-derived and stroma-derived fractions. We also investigated the involvement of interleukin-1 beta (IL1β) in PTGS2 induction, both in vitro and in CRC lysates. Finally, we used overall survival (OS) as a criterion for patient selection. Glycosylated PTGS2 can be quantified with high sensibility in tissue lysates, but the expression in both tumor and stromal cells limits its use for predictive purposes. Immunohistochemistry (IHC) analysis indicates that stromal PTGS2 expression could exert a protective role on patient OS. Stromal PTGS2 was prevalently expressed by cancer-associated fibroblasts exerting a barrier function near the gut lumen, and it apparently favored the antitumor M1 macrophage population. IL1β was directly linked to gPTGS2 expression both in vitro and in tumors, but its activity was apparently prevalent on the stromal cell population. We suggest that stromal PTGS2 could exert a positive effect on patients OS when expressed in the luminal area of the tumor.

We report a case of an elderly woman presenting with a huge cervical mass invading the tracheal lumen. Diagnosed as invasive poorly differentiated thyroid cancer, after an endotracheal biopsy, stenting and radiotherapy, it was judged eligible for total thyroidectomy, but surgery was delayed due to pulmonary thromboembolism. The patient was therefore treated with lenvatinib with a neoadjuvant intent until hemodynamic stability was obtained. Thyroidectomy and radioiodine therapy were then performed and the postdose scan revealed an area of modest uptake in the anterior part of the neck. The patient is now in a good clinical status and she continues her follow-up program without any adjuvant therapy.

Richter syndrome is the name given to the transformation of the most frequent type of leukemia, chronic lymphocytic leukemia, into an aggressive lymphoma. Patients with Richter syndrome have limited response to therapies and dismal survival. The underlying mechanisms of transformation are insufficiently understood and there is a major lack of knowledge regarding the roles of microRNA that have already proven to be causative for most cases of chronic lymphocytic leukemia. Here, by using four types of genomic platforms and independent sets of patients from three institutions, we identified microRNA involved in the transformation of chronic lymphocytic leukemia to Richter syndrome. The expression signature is composed of miR-21, miR-150, miR-146b and miR-181b, with confirmed targets significantly enriched in pathways involved in cancer, immunity and inflammation. In addition, we demonstrated that genomic alterations may account for microRNA deregulation in a subset of cases of Richter syndrome. Furthermore, network analysis showed that Richter transformation leads to a complete rearrangement, resulting in a highly connected microRNA network. Functionally, ectopic overexpression of miR-21 increased proliferation of malignant B cells in multiple assays, while miR-150 and miR-26a were downregulated in a chronic lymphocytic leukemia xenogeneic mouse transplantation model. Together, our results suggest that Richter transformation is associated with significant expression and genomic loci alterations of microRNA involved in both malignancy and immunity., (Copyright© 2019 Ferrata Storti Foundation.)

Background: The demonstration of EGFR T790M gene mutation in plasma is crucial to assess the eligibility of Non Small Cell Lung Cancer (NSCLC) patients, who have acquired resistance to first or second generation Tyrosine Kinase Inhibitors (TKIs), to receive a subsequent treatment with osimertinib. Since circulating tumor DNA (ctDNA) is present in very low amounts in plasma, high sensitive and specific methods are required for molecular analysis. Improving sensitivity of T790M mutation detection in plasma ctDNA enables a larger number of NSCLC patients to receive the appropriate therapy without any further invasive procedure., Methods: A tag-based next generation sequencing (NGS) platform capable of tagging rare circulating tumor DNA alleles was employed in this study for the identification of T790M mutation in 42 post-TKI NSCLC patients., Results: Compared to Real Time PCR, tag-based NGS improved the T790M detection rate (42.85% versus 21.4%, respectively), especially in those cases with a low median mutation abundance (i.e. 0.24, range 0.07-0.78). Moreover, the tag-based NGS identified EGFR activating mutations more efficiently than Real Time PCR (85.7% versus 61.9% detection rate, respectively), particularly of the L858R variant type (0.06-0.75 mutation abundance range). Patients in whom the T790M mutation was detected in plasma, achieved an objective response to osimertinib (9/14, 64.28%)., Conclusions: Tag-based NGS represents an accurate and sensitive tool in a clinical setting for non-invasive assessment and monitoring of T790M variant in NSCLC patients.

Splenectomy in addition to immunotherapy with rituximab can provide quick and sometimes durable disease control in patients with splenic marginal zone lymphoma (SMZL). However, systemic chemotherapy is ultimately required in many cases. The BRISMA (Bendamustine-rituximab as first-line treatment of splenic marginal zone lymphoma)/IELSG (International Extranodal Lymphoma Study Group)36 trial is an open-label, single arm phase II study designed by the IELSG in cooperation with the Fondazione Italiana Linfomi and the lymphoma Study Association according to Simon's two-stage method. The primary endpoint was complete response rate. Fifty-six patients with SMZL diagnosis confirmed on central revision were treated with bendamustine (90 mg/m 2  days 1, 2) and rituximab (375 mg/m 2  day 1) every 28 days for six cycles (B-R). The overall response and CR rates were 91% and 73%, respectively. Duration of response, progression-free survival and overall survival at 3 years were 93% (95% confidence interval [CI] 81-98), 90% (95% CI 77-96) and 96% (95% CI 84-98), respectively. Toxicity was mostly haematological. Neutropenia grade ≥3 was recorded in 43% of patients; infections and febrile neutropenia in 5·4% and 3·6%. Overall, 14 patients (25%) experienced serious adverse events. Five patients (9%) went off-study because of toxicity and one patient died from infection. In conclusion, B-R resulted in a very effective first-line regimen for SMZL. Based on the results achieved in the BRISMA trial, B-R should be considered when a chemotherapy combination with rituximab is deemed necessary for symptomatic SMZL patients., (© 2018 British Society for Haematology and John Wiley & Sons Ltd.)

In order to investigate the role of microRNAs in the pathogenesis of different B-cell lymhoma subtypes, we have applied an array-based assay to a series of 76 mixed non-Hodgkin B-cell lymphomas, including Burkitt's lymphoma (BL), diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, mantle cell lymphoma (MCL) and follicular lymphoma. Lymphomas clustered according to histological subtypes, driven by two miRNA clusters (the miR-29 family and the miR-17-92 cluster). Since the two miRNA clusters are known to be MYC-regulated, we investigated whether this would be supported in MYC-driven experimental models, and found that this signature separated BL cell lines and a MYC -translocated MCL cell lines from normal germinal center B-cells and other B-cell populations. Similar results were also reproduced in tissue samples comparing BL and reactive lymph node samples. The same series was then quantitatively analyzed for MYC expression by immunohistochemistry and MYC protein levels were compared with corresponding miRNA signatures. A specific metric was developed to summarize the levels of MYC-related microRNAs and the corresponding protein levels. We found that MYC-related signatures are directly related to MYC protein expression across the whole spectrum of B-cells and B-cell lymphoma, suggesting that the MYC-responsive machinery shows predominantly quantitative, rather than qualitative, modifications in B-cell lymphoma. Novel MYC-related miRNAs were also discovered by this approach. Finally, network analysis found that in BL MYC-related differentially expressed miRNAs could control, either positively or negatively, a limited number of hub proteins, including BCL2, CDK6, MYB, ZEB1, CTNNB1, BAX and XBP1., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflict of interest.

First line drug treatment of follicular lymphoma (FL) patients is followed by a highly variable disease-free time before relapse in about one third of patients. No molecular marker is able to predict efficiently the risk of relapse. We investigated the expression profile of microRNAs (miRNAs) by microarrays and of the tumor microenvironment by immunohistochemistry in 26 FLs and 12 reactive lymph nodes (rLN) as reference. Twenty-nine miRNAs were differentially expressed in FLs compared to rLNs and some of them discriminated grade 1 from 3a FLs. Both FLs and rLNs displayed molecular heterogeneity. FLs grouped into two clusters mostly driven by the tumor T-cell content. Among 21 drug-treated FL patients with an average follow-up of 13.5 years, eight cases relapsed. Twenty-six miRNAs discriminated between relapsed and non-relapsed FLs. Ten miRNAs also correlated with Foxp3 + cells number. Notably, Foxp3 + cells were significantly less in relapsed patients and lower Foxp3 + cell number associated with shorter time-to-relapse. Foxp3 + cells did not co-expressed follicular helper T-cell markers and were therefore classified as regulatory T cells rather than follicular regulatory T-cells. These findings introduce new knowledge about the relationship between miRNA alterations and infiltrating immune cells and show that Foxp3 + cells might be predictive of disease relapse., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Although the progression of chronic lymphocytic leukemia (CLL) requires the cooperation of the microenvironment, the exact cellular and molecular mechanisms involved are still unclear. We investigated the interleukin (IL)-23 receptor (IL-23R)/IL-23 axis and found that circulating cells from early-stage CLL patients with shorter time-to-treatment, but not of those with a more benign course, expressed a defective form of the IL-23R complex lacking the IL-12Rβ1 chain. However, cells from both patient groups expressed the complete IL-23R complex in tissue infiltrates and could be induced to express the IL-12Rβ1 chain when cocultured with activated T cells or CD40L + cells. CLL cells activated in vitro in this context produced IL-23, a finding that, together with the presence of IL-23 in CLL lymphoid tissues, suggests the existence of an autocrine/paracrine loop inducing CLL cell proliferation. Interference with the IL-23R/IL-23 axis using an anti-IL-23p19 antibody proved effective in controlling disease onset and expansion in xenografted mice, suggesting potential therapeutic strategies., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Chronic lymphocytic leukemia (CLL) clones are characterized by loss of a critical region in 13q14.3, (del(13)(q14)) involving the microRNA (miRNA) cluster miR-15a and miR-16-1. We have investigated the effects of replacement of miR-15a and miR-16-1. CLL cells transfected with these miRNA mimics exhibited a decrease in cell viability in vitro and impaired capacity for engraftment and growth in NOD/Shi-scid,γcnull (NSG) mice. No synergistic effects were observed when the two miRNA mimics were combined. The phenomena were not restricted to CLL with the del(13)(q14) lesion. Similar effects induced by miRNA mimics were seen in cells with additional chromosomal abnormalities with the exception of certain CLL clones harboring TP53 alterations. Administration of miRNA mimics to NSG mice previously engrafted with CLL clones resulted in substantial tumor regression. CLL cell transfection with miR-15a and miR-16-1-specific inhibitors resulted in increased cell viability in vitro and in an enhanced capacity of the engrafted cells to grow in NSG mice generating larger splenic nodules. These data demonstrate that the strong control by miR-15a and miR-16-1 on CLL clonal expansion is exerted also at the level of full-blown leukemia and provide indications for a miRNA-based therapeutic strategy.

The cytology of 130 indeterminate nodules (Thy 3) was retrospectively reviewed according to the British Thyroid Association 2014 classification. Nodules were divided into Thy 3a (atypical features) and Thy 3f (follicular lesion) categories. Histology was available as a reference for 97 nodules. Pre-surgical evaluations comprised biochemical tests, color-Doppler ultrasonography (US), semi-quantitative elastography-US (USE), contrast-enhanced US (CEUS), and mutation analysis from cytological slides. Thyroid malignancy was the final diagnosis for 19% of surgically-treated nodules. No statistically significant difference in the risk of malignancy was found between Thy 3a (26%) and Thy 3f (14%) nodules. Histology of the Thy 3a and Thy 3f nodules showed a higher incidence of Hurtle cell adenomas in Thy 3f (29%) than in Thy 3a (3%) nodules (P=0.01). The only pre-surgical difference concerned the BRAF V600E mutation, which was positive in some Thy 3a but not in any Thy 3f nodules (P=0.04). Receiver-operating characteristic (ROC) analysis was used to obtain cut-off values from US (score), USE (ELX 2/1 strain index), and CEUS (time-to-peak index and peak index) data. The cut-off values were similar for Thy 3a and Thy 3f nodules. Data showed that malignancy can be suspected if the US score is >2, ELX 1/2 strain index >1, time-to-peak index >1, and peak index <1. In a sub-group of 24 revised nodules (12 Thy 3a and 12 Thy 3f) with histology as a reference, the diagnostic power of cumulative pre-surgical analysis by means of US, USE, and CEUS showed high positive and negative predictive values (83% and 100%, respectively) for the presence of malignancy in Thy 3a and Thy 3f nodules. In conclusion, in our series of revised Thy 3 nodules, malignancy was low and displayed no significant differences between Thy 3a and Thy 3f categories. The use of cut-offs based on histology as a reference could reduce surgery. Our data support the conviction that, in mutation-negative Thy 3a and Thy 3f nodules, observation should be the first choice when not all instrumental results are suspect.

In the late B cell differentiation stages, miRNAs expression modifications promoting or inhibiting key pathways are only partially defined. We isolated 29 CD19+ human B cell samples at different stages of differentiation: B cells from peripheral blood; naïve, germinal center (GC) and subepithelial (SE) B cells from tonsils. SE cells were further split in activated and resting B cell. The miRNA expression profile of these B cells was assessed by microarray analysis and selected miRNAs were validated by quantitative RT-PCR and in situ hybridization on normal tonsils. The comparison of all samples showed changes in 107 miRNAs in total. Among 48 miRNAs differentially expressed in naïve, GC and SE cells, we identified 8 miRNAs: mir-323, mir-138, mir-9*, mir-211, mir-149, mir-373, mir-135a and mir-184, strictly specific to follicular cells that had never been implicated before in late stages of B cell development. Moreover, we unveiled 34 miRNAs able to discriminate between CD5- activated B cells and resting B cells. The miRNAs profile of CD5- resting B cells showed a higher similarity to naïve CD5+ than CD5- activated B cells. Finally, network analysis on shortest paths connecting gene targets suggested ZEB1 and TP53 as key miRNA targets during the follicular differentiation pathway. These data confirm and extend our knowledge on the miRNAs-related regulatory pathways involved in the late B cell maturation.

The present study describes the case of a 45-year-old man diagnosed with metastatic lung adenocarcinoma, which harbored a deletion within exon 19 of the epidermal growth factor receptor ( EGFR ) gene. The patient was subsequently treated with gefitinib (250 mg/day orally from May 2013 to March 2014), but developed acquired resistance to the drug following 11 months of treatment. Tumor burden molecular analysis was performed on a tumor rebiopsy and plasma sample, and histological analysis was also performed on the tumor rebiopsy. A small cell transformation retaining the original EGFR mutation was detected in the tumor rebiopsy, while the T790M mutation together with the activating ex19del mutation were identified only in the plasma sample. The patient was treated with cytotoxic chemotherapy (off-label schedule with epirubicin 80 mg/mq and paclitaxel 160 mg/mq every 21 days for 6 cycles) and radiation (50.4 Gy administered in 28 fractions of 1.8 Gy once daily for 5.5 weeks) specific for small cell lung cancer, and may also have benefitted from treatment with a third generation T790M-specific EGFR-TKI. To better describe the mechanisms of resistance to TKI inhibitors and to optimize therapeutic regimens, the simultaneous analysis of tumor biopsies and circulating tumor DNA should be considered.

Oncogene-targeted therapies based on mutated BRAF- and/or MEK-specific inhibitors have been developed for melanoma treatment. Although these drugs induce tumor regression in a high percentage of patients, clinical responses are frequently limited in time and tumors often recur. Recent studies suggested that the combination of BRAF/MEK inhibition with immunotherapy could represent a promising strategy for the cure of melanoma. NK cells are suitable effectors for tumor immunotherapy. Here we show that PLX4032 (a mutant BRAFV600 inhibitor) had no effect on the functional properties of NK cells cultured in the presence of IL-2 or IL-15. In contrast, PD0325901 (a MEK inhibitor) induced the down-regulation of the main activating NK receptors and inhibited NK cell function. Importantly, PD0325901 did not affect the anti-tumor activity of NK cells that had been exposed to a combination of IL-15 and IL-18. In addition, both PLX4032 and PD0325901 did not exert any inhibitory effect on in vitro IL-2 or IL-15 pre-activated NK cells.Our data may provide a rationale for future clinical protocols that combine IL-15/IL-18 cytokine administration with MEK inhibitors. In addition, they suggest that oncogene-targeting drugs are compatible with NK-based adoptive therapy., Competing Interests: CONFLICTS OF INTERESTS The authors declare that they have no conflicts of interest.

Objective: We performed an external and multicentric validation of the predictive value of abdominal computed tomography (aCT) on time to first treatment (TTFT) in early stage chronic lymphocytic leukemia (CLL) patients., Methods: aCT was performed at diagnosis in 181 Rai 0 patients enrolled in the O-CLL1-GISL trial (clinicaltrial.gov ID:NCT00917549)., Results: Fifty-five patients showed an abnormal aCT. Patients with an abnormal aCT showed a significantly shorter TTFT than those with normal aCT (P < 0.0001). At multivariate analysis, aCT (P = 0.011), β-2 microglobulin (P = 0.019), and CD38 expression (P = 0.047) correlated with TTFT. Following IWCLL 2008 criteria, 112 (61.9%) cases remained at Rai 0, while 69 (38.1%) satisfied the criteria of clinical monoclonal B-cell lymphocytosis (cMBL). Reclassified Rai 0 patients with an abnormal aCT showed a significantly shorter TTFT than those with a normal aCT (P < 0.0001). At multivariate analysis, only aCT (P = 0.011) correlated with TTFT. Eleven cMBL cases (15.9%) showed an abnormal aCT and were reclassified as small lymphocytic lymphomas (SLL); nonetheless, TTFT was similar for cMBLs and SLLs., Conclusion: Our results confirm the ability of the abnormal aCT to predict progression in early stage cases., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Several factors support CLL cell survival in the microenvironment. Under different experimental conditions, IL21 can either induce apoptosis or promote CLL cell survival. To investigate mechanisms involved in the effects of IL21, we studied the ability of IL21 to modulate gene and miRNA expressions in CD40-activated CLL cells. IL21 was a major regulator of chemokine production in CLL cells and it modulated the expression of genes involved in cell movement, metabolism, survival and apoptosis. In particular, IL21 down-regulated the expression of the chemokine genes CCL4, CCL3, CCL3L1, CCL17, and CCL2, while it up-regulated the Th1-related CXCL9 and CXCL10. In addition, IL21 down-regulated the expression of genes encoding signaling molecules, such as CD40, DDR1 and PIK3CD. IL21 modulated a similar set of genes in CLL and normal B-cells (e.g. chemokine genes), whereas other genes, including MYC, TNF, E2F1, EGR2 and GAS-6, were regulated only in CLL cells. An integrated analysis of the miRNome and gene expression indicated that several miRNAs were under IL21 control and these could, in turn, influence the expression of potential target genes. We focused on hsa-miR-663b predicted to down-regulate several relevant genes. Transfection of hsa-miR-663b or its specific antagonist showed that this miRNA regulated CCL17, DDR1, PIK3CD and CD40 gene expression. Our data indicated that IL21 modulates the expression of genes mediating the crosstalk between CLL cells and their microenvironment and miRNAs may take part in this process.

Interleukin (IL)-31A binds to an heterodimer composed of IL-31 receptor A (IL-31RA) and Oncostatin M Receptor (OSMR). The IL-31/IL-31R complex is involved in the pathogenesis of various skin diseases, including cutaneous T-cell lymphoma. No information is available on the relations between the IL-31/IL-31R complex and B-cell lymphoma. Here we have addressed this issue in follicular lymphoma (FL), a prototypic germinal center(GC)-derived B-cell malignancy. IL-31 enhanced primary FL cell proliferation through IL-31R-driven signal transducer and activator of transcription factor 1/3 (STAT1/3), extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt phosphorylation. In contrast, GC B cells did not signal to IL-31 in spite of IL-31R expression. GC B cells expressed predominantly the inhibitory short IL-31RA isoform, whereas FL cells expressed predominantly the long signaling isoform. Moreover, GC B cells lacked expression of other IL-31RA isoforms potentially involved in the signaling pathway. IL-31 protein expression was significantly higher in surface membrane than in cytosol of both FL and GC B cells. IL-31 was detected in plasma membrane microvesicles from both cell types but not released in soluble form in culture supernatants. IL-31 and IL-31RA expression was higher in lymph nodes from FL patients with grade IIIa compared with grade I/II, suggesting a paracrine and/or autocrine role of IL-31/IL-31RA complex in tumor progression through microvesicle shedding.

Two decades after the discovery of the first animal microRNA (miRNA), the number of miRNAs in animal genomes remains a vexing question. Here, we report findings from analyzing 1,323 short RNA sequencing samples (RNA-seq) from 13 different human tissue types. Using stringent thresholding criteria, we identified 3,707 statistically significant novel mature miRNAs at a false discovery rate of ≤ 0.05 arising from 3,494 novel precursors; 91.5% of these novel miRNAs were identified independently in 10 or more of the processed samples. Analysis of these novel miRNAs revealed tissue-specific dependencies and a commensurate low Jaccard similarity index in intertissue comparisons. Of these novel miRNAs, 1,657 (45%) were identified in 43 datasets that were generated by cross-linking followed by Argonaute immunoprecipitation and sequencing (Ago CLIP-seq) and represented 3 of the 13 tissues, indicating that these miRNAs are active in the RNA interference pathway. Moreover, experimental investigation through stem-loop PCR of a random collection of newly discovered miRNAs in 12 cell lines representing 5 tissues confirmed their presence and tissue dependence. Among the newly identified miRNAs are many novel miRNA clusters, new members of known miRNA clusters, previously unreported products from uncharacterized arms of miRNA precursors, and previously unrecognized paralogues of functionally important miRNA families (e.g., miR-15/107). Examination of the sequence conservation across vertebrate and invertebrate organisms showed 56.7% of the newly discovered miRNAs to be human-specific whereas the majority (94.4%) are primate lineage-specific. Our findings suggest that the repertoire of human miRNAs is far more extensive than currently represented by public repositories and that there is a significant number of lineage- and/or tissue-specific miRNAs that are uncharacterized.

Background. Molecular diagnostics has offered new techniques for searching for mutations in thyroid indeterminate lesions. The study's aim was to evaluate the BRAF mutations' incidence in an Italian regional population. Subjects and Methods. 70 Caucasian patients born in Liguria with indeterminate or suspicious cytological diagnoses. Results. A BRAF gene mutation was successfully analyzed in 56/70 patients. The mutation was BRAF V600E in 12/56 cases (21%) and BRAF K601E in 2/56 (4%). Of the BRAF mutated samples on cytological diagnosis (14/56 cases), 2/14 cases (14%) were benign on final histology and 12/14 (86%) were malignant. All BRAF-mutated cases on cytology that were found to be benign on histological examination carried the K601E mutation. Of the nonmutated BRAF cases (42/56, 75%) which were later found to be malignant on definitive histology, 5 cases were follicular carcinomas (36%), 3 cases were incidentally found to be papillary microcarcinomas (22%), 2 were cases papillary carcinomas (14%), 1 was case follicular variant of papillary carcinoma (7%), 1 was case medullary carcinoma (7%), 1 case was Hurtle cell tumor (7%), and 1 case was combined cell carcinoma and papillary oncocytic carcinoma (7%). Conclusions. The presence of the BRAF V600E mutation may suggest a more aggressive surgical approach. BRAF K601E mutation did not correlate with malignancy indexes.

After removal of an ovarian mass in a 43-year-old woman, a struma ovarii was diagnosed. Within this teratoma, a papillary thyroid cancer was found. The tumor was negative for BRAF, NRAS, KRAS, PIK3CA and c-KIT mutations on molecular analysis. Thyroid function and morphology were normal. Thyroidectomy, L-T4 TSH-suppressive therapy and rhTSH-induced radioiodine ablation were performed. So far, the follow-up has been favorable. This is the first case of thyroid cancer in a struma ovarii in which mutations of PIK3CA exons 9 and 20, and c-KIT exons 9, 11 and 13 have been evaluated and the third in which ablation has been performed under rhTSH. The prognosis of patients with thyroid cancer in a struma ovarii is generally poor. In our patient, as in those who undergo ablative radioactive iodine therapy, this was not the case.

The 2014 OECI Oncology Days was held at the 'Prof. Dr. Ion Chiricuta' Oncology Institute in Cluj, Romania, from 12 to 13 June. The focus of this year's gathering was on developments in personalised medicine and other treatment advances which have made the cost of cancer care too high for many regions throughout Europe.

A risk score based on three biological features (CD38, ZAP-70, and IGHV mutational status) was previously developed to predict progression-free survival (PFS) in untreated Binet A CLL patients. Here we perform a score validation analysis in a prospective and independent cohort of patients. Biological markers (CD38, ZAP-70, and IGHV mutational status) and gene expression profiles (GEP) of leukemic cells from CLL patients included in a prospective multicenter observational study (O-CLL1-GISL protocol, clinicaltrial.gov ID:NCT00917549) were used to assess the value and reproducibility of this score. To date, 468 Binet A patients were classified as low- (0 positive marker), intermediate- (1 positive marker), or high-risk (2 or 3 positive markers) using the progression risk score. The 3-year PFS probability was 91.7%, 82.9%, and 57.4% for low-, intermediate-, and high-risk (P < 0.0001) cases, respectively. These values were similar to those found in the original cohort. At Cox multivariate analysis, Rai stage, absolute lymphocyte count, progression risk score, and β-2 microglobulin maintained an independent prognostic impact on PFS. This score remained a predictor of progression when analysis was limited to 371 Rai 0 cases (P < 0.0001). Finally, the cells from the different CLL risk groups showed differences in their gene expression patterns. These results confirm the ability of this progression risk score to predict PFS among Binet A patients. The utility of the score was also extended by demonstrating that it retains prognostic value when applied exclusively to Rai 0 patients. Specific transcriptional patterns were significantly associated with risk groups., (© 2014 Wiley Periodicals, Inc.)

Hepatocyte growth factor, produced by stromal and follicular dendritic cells, and present at high concentrations in the sera of patients with chronic lymphocytic leukemia, prolongs the survival of leukemic B cells by interacting with their receptor, c-MET. It is, however, unknown whether hepatocyte growth factor influences microenvironmental cells, such as nurse-like cells, which deliver survival signals to the leukemic clone. We evaluated the expression of c-MET on nurse-like cells and monocytes from patients with chronic lymphocytic leukemia and searched for phenotypic/functional features supposed to be influenced by the hepatocyte growth factor/c-MET interaction. c-MET is expressed at high levels on nurse-like cells and at significantly higher levels than normal on monocytes from patients. Moreover, the hepatocyte growth factor/c-MET interaction activates STAT3(TYR705) phosphorylation in nurse-like cells. Indoleamine 2,3-dioxygenase, an enzyme modulating T-cell proliferation and induced on normal monocytes after hepatocyte growth factor treatment, was detected together with interleukin-10 on nurse-like cells, and on freshly-prepared patients' monocytes. Immunohistochemical/immunostaining analyses demonstrated the presence of c-MET(+) and indoleamine 2,3-dioxygenase(+) cells in lymph node biopsies, co-expressed with CD68 and vimentin. Furthermore nurse-like cells and chronic lymphocytic monocytes significantly inhibited T-cell proliferation, prevented by anti-transforming growth factor beta and interleukin-10 antibodies and indoleamine 2,3-dioxygenase inhibitors, and supported CD4(+)CD25(high+)/FOXP3(+) T regulatory cell expansion. We suggest that nurse-like cells display features of immunosuppressive type 2 macrophages: higher hepatocyte growth factor levels, produced by leukemic or other microenvironmental surrounding cells, may cooperate to induce M2 polarization. Hepatocyte growth factor may thus have a dual pathophysiological role: directly through enhancement of survival of the leukemic clone and indirectly by favoring T-cell immunosuppression., (Copyright© Ferrata Storti Foundation.)

Reactive oxygen species (ROS) are metabolism by-products that may act as signaling molecules to sustain tumor growth. Antioxidants have been used to impair cancer cell survival. Our goal was to determine the mechanisms involved in the response to antioxidants of a human cell culture (PT4) containing glioblastoma (GBM) tumorigenic initiating cells (TICs). ROS production in the absence or presence of N-acetyl-L-cysteine (NAC), tiron, and trolox was evaluated by flow cytometry (FCM). The effects of these antioxidants on cell survival and apoptosis were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) and FCM. The biological processes modulated by these drugs were determined by oligonucleotide microarray gene expression profiling. Our results showed that NAC, tiron and trolox impaired PT4 cell survival, had minor effects on ROS levels and caused wide deregulation of cell cycle genes. Furthermore, tiron and trolox caused inhibition of cell survival in two additional cell cultures containing TICs, FO-1 and MM1, established from a melanoma and a mesothelioma patient, respectively. NAC, instead, impaired survival of the MM1 cells but not of the FO-1 cells. However, when used in combination, NAC enhanced the inhibitory effect of PLX4032 (BRAF V600E inhibitor) and Gefitinib (EGFR inhibitor), on FO-1 and PT4 cell survival. Collectively, NAC, tiron and trolox modulated gene expression and impaired the growth of cultures containing TICs primarily by inhibiting cell cycle progression.

Background: Uveal melanoma is the most frequent primary tumour of the eye. It is molecularly clearly distinct from cutaneous melanoma and shows a different pattern of driver mutations. The influence of sunlight ultraviolet (UV) exposure on the aetiology of uveal melanoma is a matter of debate. The recent identification of driver mutations in the promoter of the telomerase reverse transcriptase (TERT) gene with UV-induced cytidine-to-thymidine transitions in cutaneous melanoma prompted us to investigate whether these mutations also occur in uveal melanoma., Methods: We analysed 50 cases of uveal melanoma obtained from enucleation surgery for mutations in the genes GNAQ, GNA11, BAP1, SF3B1, EIFAX1 and TERT, measured gene expression using microarrays and analysed gene copy numbers by SNP arrays., Results: We detected a TERT mutation in only one case of a 57-year-old white male patient with clinical and histopathological features typical for uveal melanoma. The tumour showed mutations in GNA11 and EIF1AX that are typical for uveal melanoma and absent from cutaneous melanoma. No mutations were detected in GNAQ, BAP1 and SF3B1 that are frequently mutated in uveal melanoma. Both copies of chromosome 3 were retained. Several tumours among which the one carrying the TERT promoter mutation showed elevated TERT expression. Consistent with previous reports, GNAQ is inversely associated with chromosome 3 monosomy and metastasis. BAP1 mutations are significantly associated with chromosome 3 monosomy but not with relapse., Conclusion: These data indicate that TERT mutations are rare in uveal melanoma. No conclusion can be drawn on their potential influence on tumour progression.

Introduction: Mycosis Fungoides (MF) is a rare malignant T-cell lymphoma, involving mainly the skin. In 50%-75% of cases, it can involve organs other than skin, with a 11%-14% Central Nervous System involvement (CNS)., Case Report: A 82-year-old woman presented to our Department with a 15-years history of MF with skin lesions. Neurological examination showed dysarthria and a left facio-brachial-crural hemiparesis. A CT scan showed a right fronto-rolandic lesion. A MRI, including DWI, confirmed the presence of the "neoplastic" lesion with slight hemorrhagic component and leptomeningeal contrast enhancement. Molecular TCR rearrangement test by PCR analysis was performed on skin biopsy, showed the presence of a single peak which fits with a monoclonal TCRG gene rearrangement (size 67). Molecular TCR test was also performed on the cerebrospinal fluid (CSF), which confirmed the presence of lymphocyte clone T g/ more expressed with the same size of that observed in the skin biopsy A total body CT scan did not show any lymphnodal or extranodal disease. The patient died after ten days., Conclusion: MF usually occurs in the context of advanced and often histologically transformed cutaneous disease. Isolated CNS involvement is remarkably rare. This case highlights the need for regular neurologic follow-up after the diagnosis of MF, in particular when features that suggest risk of disease progression are present. Furthermore, the analysis of the skin biopsy and above all of CSF by PCR technique, based on our experience, should always be executed in MF patients with signs or symptoms suggesting CNS involvement.

Total body computed tomography (TB-CT) scan is not mandatory in the diagnostic/staging algorithm of chronic lymphocytic leukemia (CLL). The aim of this study was to determine the value and prognostic significance of TB-CT scan in early stage CLL patients. Baseline TB-CT scan was performed in 240 Binet stage A CLL patients (179 Rai low- and 61 Rai intermediate-risk) included in a prospective multicenter observational study (clinicaltrial.gov ID:NCT00917549). The cohort included 69 clinical monoclonal B lymphocytosis (cMBLs). Patients were restaged considering only radiological data. Following TB-CT scans, 20% of cases reclassified as radiologic Binet (r-Binet) stage B. r-Binet B patients showed a higher incidence of unfavorable cytogenetic abnormalities (P = 0.027), as well as a shorter PFS (P = 0.001). At multivariate analysis, r-Binet stage [HR = 2.48; P = 0.004] and IGHV mutational status [HR = 3.01; P = 0.002] retained an independent predictive value for PFS. Among 179 Rai low-risk cases, 100 were redefined as r-Rai intermediate-risk based upon TB-CT scan data, showing a higher rate of cases with higher ZAP-70 (P = 0.033) and CD38 expression (P = 0.029) and β2-microglobulin levels (P < 0.0001), as well as a shorter PFS than those with r-Rai low-risk (P = 0.008). r-Rai stage [HR = 2.78; P = 0.046] and IGHV mutational status [HR = 4.25; P = 0.009] retained a significant predictive value for PFS at multivariate analysis. Forty-two percent of cMBL patients were reclassified as r-small lymphocytic lymphomas (r-SLLs) by TB-CT scan. TB-CT scan appears to provide relevant information in early stage CLL related to the potential and the timing of patients to progress towards the more advanced disease stages., (Copyright © 2013 Wiley Periodicals, Inc.)

Metastatic colorectal cancer (mCRC) is frequently characterized by the presence of mutations of the KRAS oncogene, which are generally associated with a poor response to treatment with anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibodies. With the methods currently used, a case is classified as KRAS-mutated when approximately 20% of the cells bear an activating KRAS mutation. These considerations raise the question of whether cells with a mutated KRAS can be found in mCRC cases classified as KRAS wild-type when more sensitive methods are used. In addition, the issue arises of whether these mCRC cases with low proportion of KRAS-mutated cells could account at least in part for the therapeutic failure of anti-EGFR therapies that occur in 40-60% of cases classified as KRAS wild type. In this study, we compared the classical assays with a very sensitive test, a locked nucleic acid (LNA) polymerase chain reaction (PCR), capable of detecting KRAS-mutated alleles at extremely low frequency (detection sensitivity limit 0.25% mutated DNA/wild-type DNA). By analyzing a cohort of 213 mCRC patients for KRAS mutations, we found a 20.6% discordance between the sequencing/TheraScreen methods and the LNA-PCR. Indeed, 44 mCRC patients initially considered KRAS wild type were reclassified as KRAS mutated by using the LNA-PCR test. These patients were more numerous among individuals displaying a clinical failure to anti-EGFR therapies. Failure to respond to these biological treatments occurred even in the absence of mutations in other EGFR pathway components such as BRAF.