Proposal of a Panel of Genes Identified by miRNA Profiling as Candidate Prognostic Biomarkers in Lung Carcinoids.

AIM: To validate the prognostic role of a panel of genes previously uncovered by our group to be specific targets of miRNAs differentially expressed in lung carcinoids with aggressive pathological features. METHODS: Four genes, namely, cyclic AMP response element binding protein-1 (CREBP1), activin A receptor type 2B (ACVR2B), LIM homeobox 2 (LHX2), and Krüppel-like factor 12 (KLF12), were identified in a previous study by our group using in silico analysis to be regulated by 3 miRNAs (miR-409-3p, miR-409-5p, and miR-431-5p) that were shown to be downregulated in aggressive lung carcinoids. These genes were analyzed using real-time PCR in a cohort of 102 lung carcinoids. Fifty high-grade lung carcinomas served as control group. Their expression was correlated with the expression of miR-409-3p, miR-409-5p, and miR-431-5p and with clinical pathological parameters and disease-free survival. RESULTS: The expression of all but CREBP1 gene was significantly different between lung carcinoids and high-grade neuroendocrine carcinomas. ACVR2B and LHX2 were significantly inversely correlated with miR-409-3p and miR-409-5p. High levels of ACVR2B and LHX2 were significantly associated with atypical histotype, high tumor grade, and higher proliferation Ki-67 index (all p < 0.05). Low levels of KLF12 were significantly associated with the presence of necrosis and positive nodal status (all p < 0.05). Finally, low KLF12 expression was associated with shorter disease-free survival in lung carcinoids as a whole and in atypical carcinoids, only (all p < 0.001). CONCLUSIONS: ACVR2B, LHX2, and KFL12 are novel potential biomarkers associated with aggressive features in lung carcinoids.

Multiple Assays to Determine Methylguanine-Methyltransferase Status in Lung Carcinoids and Correlation with Clinical and Pathological Features.

BACKGROUND: O6-methylguanine-methyltransferase (MGMT) is a key enzyme for the DNA repair machinery strongly associated with response to alkylating agents in different tumors. Data on its expression and related clinical impact in neuroendocrine tumors are limited to the gastro-entero-pancreatic system, with controversial results in terms of prognostic or predictive value. In lung carcinoids, although clinical efficacy of alkylating agents has been shown in small studies, very few data to date are available on MGMT status. OBJECTIVE: To assess MGMT status in lung carcinoids using multiple assays and to compare data with major clinical and pathological features. METHODS: A retrospective series of 95 lung carcinoids and 51 control cases of high-grade neuroendocrine lung carcinomas was analyzed for MGMT promoter methylation, MGMT gene expression, and MGMT protein expression using pyrosequencing, quantitative real-time PCR, and immunohistochemistry, respectively. RESULTS: MGMT protein expression was inversely correlated with MGMT promoter methylation and positively with MGMT gene expression. MGMT promoter methylation progressively increased from carcinoids to high-grade carcinomas. In the carcinoid group, decreased MGMT gene expression was significantly associated with aggressive features (atypical histotype, grade G2, larger tumor size, higher T stage, and positive nodal status) but not with survival. MGMT promoter methylation was associated with lower stage and negative nodal status. CONCLUSIONS: Our study investigated MGMT status in a large series of lung carcinoids in the attempt to move forward a rational use of alkylating agents in these tumors. Interestingly, low MGMT gene expression defines a subgroup of lung carcinoids with aggressive features.

Detailed genomic characterization identifies high heterogeneity and histotype-specific genomic profiles in adrenocortical carcinomas.

Molecular characterization of adrenocortical carcinoma has been recently established, but the correlation between molecular profiles and clinical and pathological characteristics is still poorly defined with no data available about genetic heterogeneity along disease progression. In this scenario, a detailed molecular profile was correlated with clinical and pathological characteristics in adrenocortical carcinoma patients to identify potentially novel biomarkers. Targeted next-generation sequencing and copy number variation analyses for 18 most frequently altered genes in adrenocortical carcinoma were assessed on 62 adult cases (including 10 with matched primary and metastatic/recurrence samples) and results correlated with major clinical and pathological characteristics of tumors. A total of 433 somatic deleterious genetic alterations (328 gene mutations and 105 copy number variations) were identified in 57/62 cases, five resulted wild type for all genes tested. TERT, CDK4, ZNRF3,and RB1 were altered in more than 30% of cases. Among histological variants genotypes were significantly different. Lowest mutation burden was found in the oncocytic type (p = 0.006), whereas the highest with a prevalence of RB1 (p = 0.001) and CDK4 (p = 0.002) was found in the conventional and myxoid ones, respectively. None of the 10 cases with matched samples showed a stable genotype along tumor progression, although allelic frequencies or percentages of altered nuclei at fluorescence in situ hybridization were in most cases similar among different tumor samples for genes that were stable along tumor progression. Among individual genes, an altered p53/Rb1 pathway was the strongest adverse molecular signature, being associated with high Ki-67 index, high tumor stage, aggressive disease status, and shorter disease-free survival. The genomic signature in adrenocortical carcinoma is changing along tumor progression and is associated with specific clinical and pathological features, including histological variant and prognosis.

Identification of MicroRNAs Differentially Expressed in Lung Carcinoid Subtypes and Progression.

AIM: To extensively explore microRNA expression profiles in lung carcinoids in correlation with clinical and pathological features. METHODS: A PCR-based array was employed in the screening phase to analyze 752 microRNAs in a discovery set of 12 lung carcinoids, including 6 typical (3 with lymph node metastasis) and 6 atypical (3 with lymph node metastasis). The results were validated by means of real-time PCR in 37 carcinoids, including 22 typical (4 with lymph node metastasis) and 15 atypical (7 with lymph node metastasis), and 19 high-grade neuroendocrine carcinomas. RESULTS: Unsupervised cluster analysis segregated the pilot cases into 3 distinct groups. Twenty-four microRNAs were differentially regulated in atypical versus typical carcinoids, and 29 in metastatic versus nonmetastatic cases. Eleven microRNAs were selected for validation. All but 1 were significantly different among lung neuroendocrine tumor histotypes. Moreover, 5 (miR-129-5p, miR-409-3p, miR-409-5p, miR-185 and miR-497) were significantly upregulated in typical compared to atypical carcinoids. MiR-409-3p, miR-409-5p and miR-431-5p were also significantly downregulated in carcinoids metastatic to the lymph nodes. Predictive in silico analysis of specific target genes showed that these 3 latter microRNAs linked to metastatic potential are implicated in several cellular functions and highlighted several novel genes which may be worth exploring. CONCLUSIONS: Our findings demonstrate that lung carcinoids have distinct microRNA expression profiles as compared to high-grade neuroendocrine carcinomas and that specific microRNAs might have potential implications as diagnostic tools or clinical biomarkers.

Differential expression of HER2, STAT3, SOX2, IFI16 and cell cycle markers during HPV-related head and neck carcinogenesis.

The aim of this study was to analyze protein and gene expression of HER2 in 224 head and neck precancerous and malignant lesions by immunohistochemistry and FISH analysis. In parallel, expression of pStat3, Sox2, IFI16 and p16, Ki67 was evaluated. Immunohistochemical analysis was assessed on formalin-fixed paraffin-embedded (FFPE) tissue specimens. A combined method for HPV detection consisting of p16 immunostaining and two PCR probes was applied. HER2 gene status was evaluated by FISH analysis. HPV DNA was detected in 24% of cases with predominant HPV16 genotype. HPV-positive lesions had higher HER2, pStat3 and within carcinoma group, and higher IFI16 expression compared to the HPV-negative group (Fig. 1A-B-C). A strong positive correlation between Sox2 and proliferative activity was observed, whereas IFI16 expression displayed a negative relationship with Sox2 and Ki67 activity. The most striking result was higher pStat3 expression in HPV-positive lesions and its strong positive correlation with IFI16 expression. The presence of HPV may induce upregulation of HER2/neu, pStat3 and IFI16. High levels and a strong positive correlation between pStat3 and IFI16 suggest their synergistic pro-apoptotic effects in HPV-positive lesions.

Expression and pharmacological inhibition of thymidylate synthase and Src kinase in nonsmall cell lung cancer.

The combination of cytotoxic chemotherapy with signaling pathway inhibitors represents a potential strategy to improve the treatment of nonsmall cell lung cancer (NSCLC). Thymidylate synthase (TS) is an enzyme essential for DNA synthesis, and its overexpression has been associated with the reduced sensitivity to antifolate agents. Src is a tyrosine kinase that modulates the cytotoxicity of cancer cells after drug treatment, and in vitro data indicate that its inhibition could revert the resistance to TS-inhibiting drugs. Our study investigated the significance of TS and Src expression in NSCLC tissues, and the effects of their pharmacological inhibition in cell lines. In tumor and normal tissues from 94 resected NSCLC patients, TS and Src transcript levels were found positively correlated (R(S) = 0.66), associated with patients smoking history and overall survival. At multivariate analysis, TS gene expression was an independent prognostic factor (relative risk (RR) = 1.78, from 1.16 to 2.72; p < 0.01). Immunohistochemical detection in tumor specimens confirmed that Src kinase activation, evaluated by phospho-specific antibody, was associated to a higher TS expression. In cell lines, dasatinib, a Src-inhibiting agent, synergistically enhanced pemetrexed-cytotoxicity of A549 cells, as evaluated by MTT and apoptosis assays. The biological explanation for this interaction was based on the upregulation of TS messenger RNA and protein levels induced by pemetrexed, which was significantly prevented by dasatinib cotreatment. The data of our study suggest that TS and Src may belong to a common pathway that bears prognostic significance in NSCLC, and that Src represents a potential target to improve the efficacy of TS-inhibiting agents.

Tissue Expression and Pharmacological In Vitro Analyses of mTOR and SSTR Pathways in Adrenocortical Carcinoma.

New therapies for advanced adrenocortical carcinoma (ACC) are urgently needed, as the majority of the patients experience a rapid and inexorable progression despite surgery and adjuvant mitotane. In vitro data suggest that somatostatin receptors (SSTRs) and mTOR pathway might represent reasonable targets for novel therapies, being involved in functionality and growth of ACC cells. However, in vitro analysis of combination treatments targeting both mTOR and SSTR as compared to mitotane are poorly explored in ACC. This study aimed to investigate in vitro the effects on cell growth of pasireotide, everolimus, and mitotane, alone or combined, in the two ACC cell lines H295R and SW13 (mitotane sensitive and resistant, respectively). Moreover, the tissue expression of mTOR pathway molecules and SSTR (types 1-5) was assessed in 58 ACCs. In both cell lines, only everolimus induced a significant inhibition of cell growth. Conversely, the combinations among mitotane, pasireotide, and everolimus produced antagonistic effects on mitotane-induced growth inhibition on H295R cell line. A heterogeneous profile of mTOR-related molecules and SSTR expression was observed in ACC samples, being the mTOR pathway found activated in approximately 30% of cases. In conclusion, our data suggest caution in designing combinations of mitotane with other drugs potentially active in ACC, such as mTOR inhibitors or somatostatin analogs.

BRCA1-Associated Protein 1 (BAP1) Immunohistochemical Expression as a Diagnostic Tool in Malignant Pleural Mesothelioma Classification: A Large Retrospective Study.

INTRODUCTION: Malignant pleural mesothelioma (MPM) is a highly aggressive disease with limited therapeutic options. Histological subtype remains among the most reliable prognostic factors, because the epithelioid subtype associated with the best prognosis and the sarcomatoid subtype with the worst. The biphasic subtype has an intermediate prognosis, but its definitive histological diagnosis may be challenging owing to the difficulty of assessing the neoplastic nature of the stromal component. Recent data identified BRCA1-associated protein 1 gene (BAP1) as one of the most frequently mutated genes in MPM. Immunohistochemical testing for BRCA1-associated protein 1 (BAP1) has been proposed to be predictive for the detection of BAP1 mutation in neoplastic cells. The aim of the present study was to define the diagnostic usefulness of immunohistochemical determination of BAP1 in MPM, with clinicopathological correlation. METHODS: A series of 143 MPMs were investigated for BAP1 protein expression in correlation with clinical and pathological data, including with a newly proposed nuclear grade. A pilot series of 20 selected cases were also investigated for BAP1 mutational status. RESULTS: Negative nuclear staining for BAP1 occurred in 62% of MPMs (including 27% with a cytoplasmic pattern) and was significantly associated with the presence of BAP1 mutation, epithelioid subtype, and a better prognosis. In a subgroup of cases, the pattern of expression of BAP1 in stromal cells supported their distinction as reactive versus neoplastic, thus helping achieve the correct classification of biphasic histological subtype. CONCLUSIONS: We showed that BAP1 protein determination is a diagnostic tool to correctly distinguish biphasic MPM from epithelial subtypes with an atypical/activated reactive stroma and an independent prognostic parameter in MPM.

Mesothelioma families without inheritance of a BAP1 predisposing mutation.

Familial malignant mesothelioma clusters are ideal candidates to explore BAP1 genomic status as a predisposing risk factor. We report data on BAP1 analysis in four families with multiple mesothelioma cases to investigate possible BAP1 alterations associated with an inherited cancer syndrome. We also recorded family history of cancer and assessed asbestos exposure. By genomic direct sequencing, we found no evidence of a BAP1 germline mutation in tumor DNA samples (one mesothelioma per family: n = 3 epithelioid; n = 1 biphasic). On the other hand, we identified a novel BAP1 somatic alteration (c.329_335delinsTC) in exon 5 (n = 1 biphasic), and we hypothesized the occurrence of somatic inactivating events not identifiable by sequencing in the other cases (n = 3 epithelioid), as demonstrated by the loss of nuclear BAP1 immunostaining. History of other cancers was in sites not typical of the BAP1 cancer syndrome. Asbestos exposure was occupational (n = 2 clusters), household (n = 1), and unknown (n = 1). These family units without inheritance of a BAP1 predisposing mutation expand the number of unmutated germline BAP1 families with multiple mesothelioma cases. This suggests that besides the exposure to asbestos other currently unknown genetic or epigenetic factors may be responsible for the high incidence of mesothelioma in BAP1-unmutated families.

Androgen deprivation modulates gene expression profile along prostate cancer progression.

Androgen deprivation therapy (ADT) is the standard of care for metastatic prostate cancer and initially induces tumor regression, but invariably results in castration-resistant prostate cancer through various mechanisms, incompletely discovered. Our aim was to analyze the dynamic modulation, determined by ADT, of the expression of selected genes involved in the pathogenesis and progression of prostate cancer (TMPRSS2:ERG, WNT11, SPINK1, CHGA, AR, and SPDEF) using real-time polymerase chain reaction in a series of 59 surgical samples of prostate carcinomas, including 37 cases preoperatively treated with ADT and 22 untreated cases, and in 43 corresponding biopsies. The same genes were analyzed in androgen-deprived and control LNCaP cells. Three genes were significantly up-modulated (WNT11 and AR) or down-modulated (SPDEF) in patients treated with ADT versus untreated cases, as well as in androgen-deprived LNCaP cells. The effect of ADT on CHGA gene up-modulation was almost exclusively detected in cases positive for the TMPRSS2:ERG fusion. The correlation between biopsy and surgical samples was poor for most of the tested genes. Gene expression analysis of separate tumor areas from the same patient showed an extremely heterogeneous profile in the 6 tested cases (all untreated). In conclusion, our results strengthened the implication of ADT in promoting a prostate cancer aggressive phenotype and identified potential biomarkers, with special reference to the TMPRSS2:ERG fusion, which might favor the development of neuroendocrine differentiation in hormone-treated patients. However, intratumoral heterogeneity limits the use of gene expression analysis as a potential prognostic or predictive biomarker in patients treated with ADT.

mTOR pathway activation in multiple myeloma cell lines and primary tumour cells: pomalidomide enhances cytoplasmic-nuclear shuttling of mTOR protein.

mTOR is a protein kinase that plays a central role in regulating critical cellular processes. We evaluated the activation and cellular localization of the mTOR pathway in multiple myeloma (MM) and analyzed the role of pomalidomide in regulating mTOR. By immunohistochemistry cytoplasmic p-mTOR stained positive in 57 out 101 (57.6%) cases with a nuclear p-mTOR localization in 14 out 101 samples (13.8%). In the 70 MM samples analyzed for the entire pathway, p-mTOR expression significantly correlated with p-AKT, p-P70S6K, and p-4E-BP1 suggesting that the AKT/mTOR pathway is activated in a subset of MM patients. Immunofluorescence assays demonstrated that mTOR protein is distributed throughout the cytoplasm and the nucleus at baseline in MM cell lines and in plasma cells of 13 MM patients and that pomalidomide facilitated the shift of the mTOR protein in the nucleus. By western blotting, treatment with pomalidomide increased nuclear mTOR and p-mTOR expression levels in the nucleus with a concomitant decrease of the cytoplasmic fractions while does not seem to affect significantly AKT phosphorylation status. In MM cells the anti-myeloma activity of pomalidomide may be mediated by the regulation of the mTOR pathway.

Impact of pregnancy on prognosis of differentiated thyroid cancer: clinical and molecular features.

OBJECTIVE: Differentiated thyroid cancer (DTC) commonly occurs in women of child-bearing age and represents the second most frequent tumor diagnosed during pregnancy only behind breast cancer. It is possible that associated physiological changes could favor tumor development and growth. However, few data are available about the outcome of DTC related to pregnancy, leading to conflicting results. METHODS: Among the study population, 340 patients with DTC <45 years old were retrospectively studied. Patients were divided into three groups according to the time of tumor diagnosis in respect of pregnancy. Group 1, diagnosis of DTC at least 2 years after delivery; group 2, diagnosis during pregnancy or within the second year after delivery; and group 3, nulliparous patients at the time of diagnosis. We evaluated clinical outcome and immunohistochemical expression of estrogen receptor α (ERα), ERß, progesterone receptor, and aromatase. We also analyzed the gene expression of NIS (SLC5A5) and the prevalence of BRAF(V600E) mutations. RESULTS: Persistence/recurrence of disease was significantly higher in group 2 patients than control groups (P=0.023). No significant differences were observed in other clinical parameters. Furthermore, no differences among the groups were recorded about ER pattern, NIS expression, and BRAF mutations. CONCLUSIONS: Persistence/recurrence of DTC is significantly higher in pregnant patients, suggesting that pregnancy could really exert a negative prognostic role in patients with DTC. The underlying mechanisms are not yet clarified and further studies are required. Our results suggest that a more careful follow-up is needed when diagnosis of DTC occurs during pregnancy or shortly after.

MicroRNA expression patterns in adrenocortical carcinoma variants and clinical pathologic correlations.

Several microRNAs (miRNAs) were shown to be deregulated in adrenocortical carcinoma (ACC) as compared with adenoma, but a detailed assessment of their expression in its histologic variants and correlation with clinicopathologic characteristics has not been performed, so far. Our aim was to assess the expression of 5 selected miRNAs (IGF2 gene-related miR-483-3p and 5p and hypoxia-induced miR-210, miR-195, and miR-1974) in a series of 51 ACCs (35 classical, 6 myxoid, and 10 oncocytic) as compared with clinical and pathologic features and immunohistochemical expression of prognostic markers, including steroidogenic factor 1, p53, ß-catenin, and glucose transporter 1. Oncocytic carcinomas had a reduced expression of miR-483-3p (P = .0325), miR-483-5p (P = .0175), and miR-210 (P = .0366), as compared with other histotypes. Overexpression of miR-210 was associated with the presence of necrosis (P = .0035), high Ki-67 index (P = .0013), and high glucose transporter 1 expression (P = .0043), whereas an inverse correlation with mitotic rate was observed in cases with high miR-493-3p (P = .0191) and miR-1974 (P = .0017) expression. High miR-1974 was also associated with low Ki-67 (P = .0312) and European Network for the Study of Adrenal Tumors stage (P = .0082) and negative p53 (P = .0013). At univariate analysis myxoid/classic histotype (P = .026), high miR-210 (P = .0465), high steroidogenic factor 1 protein (P = .0017), high Ki-67 (P = .0066), and high mitotic index (P = .0006) were significantly associated the shorter overall survival, the latter being the sole independent prognostic factor at multivariate analysis (P = .017). In conclusion, (a) miR-483-3p, miR-483-5p, and miR-210 are differentially expressed in ACC variants, and (b) high miR-210 is associated with clinicopathologic parameters of aggressiveness and a poor prognosis.

Impact of non-small-cell lung cancer-not otherwise specified immunophenotyping on treatment outcome.

INTRODUCTION: The vast majority of non-small-cell lung cancers (NSCLCs) presents as advanced disease, and histological diagnosis is widely based on small samples. The differential activity and toxicity profile of new cytotoxic and molecular-targeted therapies according to histotypes requires a precise subtyping of NSCLC. Immunohistochemistry (IHC) contributes to define the most probable histotype; however, the real impact of IHC characterization of NSCLC-not otherwise specified (NOS) in terms of outcome is not well established. METHODS: A large series of 224 advanced "nonsquamous" NSCLC diagnosed on small biopsy or cytological samples and homogeneously treated was retrospectively selected, all having adequate follow-up data available. Reviewed diagnoses resulted into two groups: adenocarcinoma (ADC) and NSCLC-NOS. The latter was further characterized by IHC (TTF-1, Napsin-A, p40, and Desmocollin-3) -identify a possible, most probable differentiation lineage. RESULTS: Sixty-seven percentage of cases were classified as ADC based on morphological examination only ("morphological ADC") and 33% as NSCLC-NOS. IHC profiling of NSCLC-NOS identified 43.2% of cases with an ADC immunophenotype ("NSCLC favor ADC"), 10.8% with a phenotype favoring squamous lineage, and 46% lacking differentiation features. Survival curves confirmed no difference in terms of outcome between the morphological ADC and the NSCLC favor ADC groups, while a significantly poorer outcome was found in the "null" group in terms of best response, progression-free survival or overall survival (OS). CONCLUSION: Tumors with an IHC profile ADC-like had an OS comparable with that of morphological ADCs. These findings support the use of IHC to optimize lung cancer histological typing and therapy.

Diagnostic and prognostic role of steroidogenic factor 1 in adrenocortical carcinoma: a validation study focusing on clinical and pathologic correlates.

The pathologic characterization of adrenocortical cancer is still problematic for several reasons, including the identification of novel markers of diagnostic or prognostic relevance. Among them, steroidogenic factor 1 deserves major interest because of its potential usefulness as a marker of adrenocortical derivation and of biological aggressiveness. Our aim was to validate its prognostic relevance in a large series of adrenocortical cancer, comparing the performance of 2 different commercial antibodies and investigating its expression in adrenocortical cancer variants and in comparison with clinical and pathologic features. Seventy-five (including 53 classical, 10 myxoid, and 12 oncocytic) adrenocortical cancer cases were included in tissue microarrays and analyzed for the immunohistochemical expression of steroidogenic factor 1 using 2 commercial antibodies, 1 polyclonal and 1 monoclonal (N1665). Nuclear steroidogenic factor 1 staining was assessed using a semiquantitative score and correlated with adrenocortical cancer type and clinical pathologic characteristics. A weak but significant correlation was found comparing the 2 antibodies with a positive rate of 88% and 58% using the monoclonal and polyclonal antibodies, respectively. High steroidogenic factor 1 expression with the N1665 antibody was positively correlated with high mitotic count, high Ki-67 index, and high European Network for the Study of Adrenal Tumors (ENSAT) stage and negatively associated with loss of functionality and presence of oncocytic features. Moreover, high steroidogenic factor 1 expression with this same antibody was significantly associated at univariate analysis with a decreased survival, together with high Ki-67 and mitotic indexes, with a trend to significance confirmed by multivariate analysis, thus supporting the detection of steroidogenic factor 1 using the N1665 antibody as a novel prognostic marker in adrenocortical cancer.