Identification of functional pathways and molecular signatures in neuroendocrine neoplasms by multi-omics analysis.

BACKGROUND: Neuroendocrine neoplasms (NENs) represent a heterogeneous class of rare tumors with increasing incidence. They are characterized by the ability to secrete peptide hormones and biogenic amines but other reliable biomarkers are lacking, making diagnosis and identification of the primary site very challenging. While in some NENs, such as the pancreatic ones, next generation sequencing technologies allowed the identification of new molecular hallmarks, our knowledge of the molecular profile of NENs from other anatomical sites is still poor. METHODS: Starting from the concept that NENs from different organs may be clinically and genetically correlated, we applied a multi-omics approach by combining multigene panel testing, CGH-array, transcriptome and miRNome profiling and computational analyses, with the aim to highlight common molecular and functional signatures of gastroenteropancreatic (GEP)-NENs and medullary thyroid carcinomas (MTCs) that could aid diagnosis, prognosis and therapy. RESULTS: By comparing genomic and transcriptional profiles, ATM-dependent signaling emerged among the most significant pathways at multiple levels, involving gene variations and miRNA-mediated regulation, thus representing a novel putative druggable pathway in these cancer types. Moreover, a set of circulating miRNAs was also selected as possible diagnostic/prognostic biomarkers useful for clinical management of NENs. CONCLUSIONS: These findings depict a complex molecular and functional landscape of NENs, shedding light on novel therapeutic targets and disease biomarkers to be exploited.

Interaction Proteomics Identifies ERbeta Association with Chromatin Repressive Complexes to Inhibit Cholesterol Biosynthesis and Exert An Oncosuppressive Role in Triple-negative Breast Cancer.

Triple-negative breast cancer (TNBC) is characterized by poor response to therapy and low overall patient survival. Recently, Estrogen Receptor beta (ERß) has been found to be expressed in a fraction of TNBCs where, because of its oncosuppressive actions on the genome, it represents a potential therapeutic target, provided a better understanding of its actions in these tumors becomes available. To this end, the cell lines Hs 578T, MDA-MB-468 and HCC1806, representing the claudin-low, basal-like 1 and 2 TNBC molecular subtypes respectively, were engineered to express ERß under the control of a Tetracycline-inducible promoter and used to investigate the effects of this transcription factor on gene activity. The antiproliferative effects of ERß in these cells were confirmed by multiple functional approaches, including transcriptome profiling and global mapping of receptor binding sites in the genome, that revealed direct negative regulation by ERß of genes, encoding for key components of cellular pathways associated to TNBC aggressiveness representing novel therapeutic targets such as angiogenesis, invasion, metastasis and cholesterol biosynthesis. Supporting these results, interaction proteomics by immunoprecipitation coupled to nano LC-MS/MS mass spectrometry revealed ERß association with several potential nuclear protein partners, including key components of regulatory complexes known to control chromatin remodeling, transcriptional and post-transcriptional gene regulation and RNA splicing. Among these, ERß association with the Polycomb Repressor Complexes 1 and 2 (PRC1/2), known for their central role in gene regulation in cancer cells, was confirmed in all three TNBC subtypes investigated, suggesting its occurrence independently from the cellular context. These results demonstrate a significant impact of ERß in TNBC genome activity mediated by its cooperation with regulatory multiprotein chromatin remodeling complexes, providing novel ground to devise new strategies for the treatment of these diseases based on ligands affecting the activity of this nuclear receptor or some of its protein partners.

The Role of lncRNAs in Rare Tumors with a Focus on HOX Transcript Antisense RNA (HOTAIR).

Rare cancers are identified as those with an annual incidence of fewer than 6 per 100,000 persons and includes both epithelial and stromal tumors from different anatomical areas. The advancement of analytical methods has produced an accurate molecular characterization of most human cancers, suggesting a "molecular classification" that has allowed the establishment of increasingly personalized therapeutic strategies. However, the limited availability of rare cancer samples has resulted in very few therapeutic options for these tumors, often leading to poor prognosis. Long non coding RNAs (lncRNAs) are a class of non-coding RNAs mostly involved in tumor progression and drug response. In particular, the lncRNA HOX transcript antisense RNA (HOTAIR) represents an emergent diagnostic, prognostic and predictive biomarker in many human cancers. The aim of this review is to highlight the role of HOTAIR in rare cancers, proposing it as a new biomarker usable in the management of these tumors.

Aberrant Expression of Long Non Coding RNA HOTAIR and De-Regulation of the Paralogous 13 HOX Genes Are Strongly Associated with Aggressive Behavior of Gastro-Entero-Pancreatic Neuroendocrine Tumors.

Gastro-entero-pancreatic neuroendocrine neoplasms (GEP-NENs) are rare diseases occurring in the gastrointestinal tract and pancreas. They are characterized by the loss of epithelial tubular gland elements, and by the increased expression of neuroendocrine markers. GEP-NENs are subdivided into two histo-pathological types, gastro-entero-pancreatic neuroendocrine tumors (GEP-NETs) and gastro-entero-pancreatic neuroendocrine carcinomas (GEP-NECs). According to WHO 2017 and 2019 classification criteria are graded and staged in four categories, NET-G1, NET-G2, NET-G3, and NEC-G3. The molecular characterization of these tumors can be fundamental for the identification of new diagnostic, prognostic and predictive biomarkers. The main purpose of this study was to analyze the expression of the paralogous 13 HOX genes, normally involved in embryogenic development and frequently deregulated in human cancers, and of the HOX regulating lncRNA HOTAIR in GEP-NENs. The expression of HOX genes is gradually lost in the transition from GEP NET G1 to NET/NEC G3 tumors, while HOTAIR expression, inversely correlated with HOX genes expression and weakly expressed in low-grade GEP NENs, becomes aberrant in NET G3 and NEC G3 categories. Our data highlights their potential role in the molecular stratification of GEP-NENs by suggesting new prognostic markers and potential therapeutic targets.

Noncoding RNAs as circulating biomarkers in osteosarcoma patients.

Noncoding RNAs (ncRNAs) identify a large family of RNAs that do not encode proteins and represent an important group of tumor biomarkers, directly involved in the process of tumor pathogenesis and progression. Many of them have also been identified in biological fluids of patients with cancer, especially blood, suggesting their role as an emerging class of circulating biomarkers. Many ncRNAs, both miRNAs and lncRNAs, are deregulated in sarcoma tissues, with the most consistent data in osteosarcomas. In patients with osteosarcoma, the role of ncRNAs as circulating biomarkers is taking enormous value, above all for their ability to vary expression levels during disease progression and in response to therapy. In this mini-review, we summarize the main studies supporting the role of circulating ncRNAs in monitoring disease status in patients with osteosarcoma.

LncRNA HOTAIR in Tumor Microenvironment: What Role?

lncRNAs participate in many cellular processes, including regulation of gene expression at the transcriptional and post-transcriptional levels. In addition, many lncRNAs can contribute to the development of different human diseases including cancer. The tumor microenvironment (TME) plays an important role during tumor growth and metastatic progression, and most of these lncRNAs have a key function in TME intracellular signaling. Among the numerous identified lncRNAs, several experimental evidences have shown the fundamental role of the lncRNA HOTAIR in carcinogenesis, also highlighting its use as a circulating biomarker. In this review we described the contribution of HOTAIR in the TME modulation, highlighting its relation with cellular and non-cellular components during tumor evolution and progression.

Noncoding RNAs within the HOX gene network in tumor pathogenesis and progression.

HOX genes are involved with normal development, cell identity, cell differentiation, cell metabolism, apoptosis, autophagy as well as with diseases such as tumor pathogenesis and progression. In particular, the genes belonging to HOX paralogous 13 seem to carry out a relevant role in both tumor development and disease progression. In recent years, several noncoding RNAs (ncRNA) sequences have been identified in HOX loci, including long noncoding RNA (lncRNA) and microRNA (miRNA), highly conserved during evolution. Many studies have shown that specific intergenic ncRNAs in HOX loci could directly modulate HOX genes expression in normal and pathological conditions. In the present review we attempt to describe the role of these ncRNAs, through the regulation of the HOX gene network, in normal cell biology, and, with particular emphasis, in diseases such as in cancer pathogenesis and progression.

Posterior HOX genes and HOTAIR expression in the proximal and distal colon cancer pathogenesis.

BACKGROUND: Increasing evidences showed that the location of the primary tumor on the right (proximal) or left (distal) side of the colon have a prognostic/predictive value for colon cancer patients. However, the understanding of the molecular mechanisms that contribute to the pathogenesis in different location of colon is still unclear. Probably an important role could be played by genes that control the spatial-temporal development of bodily structures, such as HOX genes. METHODS: The main purpose of this study was to analyze the expression of the paralogous 13 HOX genes and of the HOX regulating lncRNA HOTAIR in distal and proximal CRC cases. We have carried out a Tissue Micro Array with left and right CRC samples associated with all clinical-pathological parameters of patients. The expression of HOX genes was evaluated by immunohistochemistry and the staining of HOTAIR was performed by in situ hybridization using a specifically designed LNA probe. RESULTS: All paralogous 13 HOX genes and HOTAIR are silent in normal tissue and expressed in CRC samples. HOXB13, HOXC13 and HOTAIR showed a statistical association with lymph nodes metastasis (p value = 0.003, p value = 0.05, p value = 0.04). HOXB13, HOXC13 and lncRNA HOTAIR are overexpressed in right CRCs samples (p value < 0 and p value = 0.021). HOTAIR is also strongly correlated with HOXB13 (p value = 0.02) and HOXC13 (p value = 0.042) expression. CONCLUSIONS: Our data highlighted an important role of posterior HOX genes in colorectal cancer carcinogenesis. Specifically, the aberrant expression of the HOXB13, HOXC13 and HOTAIR in proximal colon cancers could add an important dowel in understanding molecular mechanisms related to tumor pathogenesis in this location.

Sex Hormone Receptors in Benign and Malignant Salivary Gland Tumors: Prognostic and Predictive Role.

The role of sex hormone receptors in human cancer development and progression has been well documented in numerous studies, as has the success of sex hormone antagonists in the biological therapy of many human tumors. In salivary gland tumors (SGTs), little and conflicting information about the role of the estrogen receptor alpha (ERα), progesterone receptor (PgR) and androgen receptor (AR) has been described and in most cases the use of sex hormone antagonists is not contemplated in clinical practice. In this study, we analyzed a panel of sex hormone receptors that have not been widely investigated in SGTs-ERα, PgR, AR, but also ERß and GPR30-to define their expression pattern and their prognostic and predictive value in a case series of 69 benign and malignant SGTs. We showed the aberrant expression of AR in mucoepidermoid and oncocytic carcinoma, a strong relation between cytoplasmic ERß expression and tumor grade, and a strong correlation between nuclear GPR30 expression and disease-free survival (DFS) of SGT patients.

Integrative Histologic and Bioinformatics Analysis of BIRC5/Survivin Expression in Oral Squamous Cell Carcinoma.

Survivin is a well-known protein involved in the inhibition of apoptosis in many different cancer types. The aim of this study was to perform an integrated bioinformatic and histologic analysis in order to study the expression and prognostic role of Survivin and its related gene BIRC5 in oral cancer. Publicly available databases were accessed via Gene Expression Omnibus and Oncomine, in addition raw data from The Cancer Genome Atlas (TCGA) were also obtained in order to analyze the rate of gene mutation, expression and methylation in patients with oral squamous cells carcinoma (OSCC). Immunohistochemistry (IHC) was also performed in order to evaluate the nuclear and cytoplasmic expression of Survivin and their correlation with cell proliferation in samples from OSCC patients. Results of this study revealed that Survivin is rarely mutated in OSCC samples and upregulated when compared to non-cancerous tissue. A negative correlation between the methylation of the island cg25986496 and BIRC5 mRNA expression was detected from TCGA data. IHC staining revealed that cytoplasmic (and not nuclear) expression of Survivin is associated with poor overall survival in OSCC patients, while the nuclear expression correlates with higher proliferation rate. In addition, data from TCGA database revealed that BIRC5 gene expression is an independent prognostic factor for OSCC patients.

HOTAIR role in melanoma progression and its identification in the blood of patients with advanced disease.

The molecular mechanisms responsible for the metastatic progression of melanoma have not been fully defined yet. We have recently shown that an important role in this process is certainly played by HOX genes, whose regulation is under control of particular non-coding RNAs, some of which are present within the HOX locus. HOTAIR is the most studied among them, whose aberrant expression is associated with the metastatic progression of many malignancies. The aim of this study was to verify the role played by HOTAIR in metastatic progression of melanoma and to evaluate the circulating levels of HOTAIR in the blood of patients with metastatic melanoma. A series of melanocytic lesions were selected to evaluate the potential changes in the expression of HOTAIR during the evolution of the disease through in situ and molecular approaches. None of the benign melanocytic lesions showed the presence of HOTAIR. The staining of HOTAIR resulted very weak in the primary pT1 lesions, while it was very strong in all pairs of primary tissues and corresponding metastases. Surprisingly, we found the presence of HOTAIR in some intratumoral lymphocytes, while this positivity decreased in lymphocyte component further away from the tumor. HOTAIR was also detected in the serum of selected metastatic patients. These data allowed us to speculate on the fundamental role played by HOTAIR in tumor evolution of melanoma. Its presence in intratumoral lymphocytes might suggest that its involvement in the modulation of tumor microenvironment and the detection in the serum could be used in the management of melanoma patients.

Hedgehog signalling pathway orchestrates angiogenesis in triple-negative breast cancers.

BACKGROUND: Several evidences suggest a marked angiogenic dependency in triple-negative breast cancer (TNBC) tumorigenesis and a potential sensitivity to anti-angiogenic agents. Herein, the putative role of Hedgehog (Hh) pathway in regulating TNBC-dependent angiogenesis was investigated. METHODS: Expression and regulation of the Hh pathway transcription factor glioma-associated oncogene homolog1 protein (GLI1) were studied on the endothelial compartment and on TNBC-initiated angiogenesis. To evaluate the translational relevance of our findings, the combination of paclitaxel with the Smo inhibitor NVP-LDE225 was tested in TNBC xenografted mice. RESULTS: Tissue microarray analysis on 200 TNBC patients showed GLI1 overexpression paired with vascular endothelial growth factor receptor 2 (VEGFR2) expression. In vitro, Hh pathway promotes TNBC progression in an autocrine manner, regulating the VEGF/VEGFR2 loop on cancer cell surface, and in a paracrine manner, orchestrating tumour vascularisation. These effects were counteracted by Smo pharmacological inhibition. In TNBC xenografted mice, scheduling NVP-LDE225 rather than bevacizumab provided a better sustained inhibition of TNBC cells proliferation and endothelial cells organisation. CONCLUSIONS: This study identifies the Hh pathway as one of the main regulators of tumour angiogenesis in TNBC, thus suggesting Hh inhibition as a potential new anti-angiogenic therapeutic option to be clinically investigated in GLI1 overexpressing TNBC patients.

COX-2 expression positively correlates with PD-L1 expression in human melanoma cells.

BACKGROUND: The resistance to PD-1/PD-L1 inhibitors for the treatment of melanoma have prompted investigators to implement novel clinical trials which combine immunotherapy with different treatment modalities. Moreover is also important to investigate the mechanisms which regulate the dynamic expression of PD-L1 on tumor cells and PD-1 on T cells in order to identify predictive biomarkers of response. COX-2 is currently investigated as a major player of tumor progression in several type of malignancies including melanoma. In the present study we investigated the potential relationship between COX-2 and PD-L1 expression in melanoma. METHODS: Tumor samples obtained from primary melanoma lesions and not matched lymph node metastases were analyzed for both PD-L1 and COX-2 expression by IHC analysis. Status of BRAF and NRAS mutations was analyzed by sequencing and PCR. Co-localization of PD-L1 and COX-2 expression was analyzed by double fluorescence staining. Lastly the BRAFV600E A375 and NRASQ61R SK-MEL-2 melanoma cell lines were used to evaluate the effect of COX-2 inhibition by celecoxib on expression of PD-L1 in vitro. RESULTS: BRAFV600E/V600K and NRASQ61R/Q61L were detected in 57.8 and 8.9% of the metastatic lesions, and in 65.9 and 6.8% of the primary tumors, respectively. PD-L1 and COX-2 expression were heterogeneously expressed in both primary melanoma lesions and not matched lymph node metastases. A significantly lower number of PD-L1 negative lesions was found in primary tumors as compared to not matched metastatic lesions (P = 0.002). COX-2 expression significantly correlated with PD-L1 expression in both primary (P = 0.001) and not matched metastatic (P = 0.048) lesions. Furthermore, in melanoma tumors, cancer cells expressing a higher levels of COX-2 also co-expressed a higher level of PD-L1. Lastly, inhibition of COX-2 activity by celecoxib down-regulated the expression of PD-L1 in both BRAFV600E A375 and NRASQ61R SK-MEL-2 melanoma cell lines. CONCLUSIONS: COX-2 expression correlates with and modulates PD-L1 expression in melanoma cells. These findings have clinical relevance since they provide a rationale to implement novel clinical trials to test COX-2 inhibition as a potential treatment to prevent melanoma progression and immune evasion as well as to enhance the anti-tumor activity of PD-1/PD-L1 based immunotherapy for the treatment of melanoma patients with or without BRAF/NRAS mutations.

Analytical Criticalities Associated to Different Immunological Methods for Serum Free Light Chain Detection in Plasma Cell Dyscrasias: A Description of Particular Clinical Cases.

Current criteria for differential diagnosis of multiple myeloma (MM), Monoclonal gammopathy of undetermined significance (MGUS), and smoldering multiple myeloma (SMM) are included in the 2003 guidelines by the International Myeloma Working Group (IMWG). An updated version was then published in 2014, highlighting the importance of serum free light chain (sFLC) detection, as well as the κ/λ ratio as excellent indicators of clonality. At present, two commercial assays for sFLC quantification are available: the Freelite™ assay and the N-Latex assay. The first was developed by The Binding Site based on a mixture of polyclonal antibodies directed against a variety of FLC epitopes. It may be run on a wide range of nephelometers, as well as on turbidimeters. The second method was developed by Siemens and runs exclusively on Siemens instruments. It employs a probe mixture of mouse monoclonal antibodies. The aim of our study was to evaluate sFLC measurement and calculated κ/λ ratio in 85 patients with monoclonal gammopathies (MGs) in order to compare methods. We demonstrated that there is only a moderate concordance between the two FLC assays. In particular, in one case, we observed no qualitative alterations of the serum protein pattern, and in the absence of a Freelite™ assay, sFLC measurement would not have been possible to highlight the increase of λ FLC.

Programmed Death Ligand 1 (PD-L1) Tumor Expression Is Associated with a Better Prognosis and Diabetic Disease in Triple Negative Breast Cancer Patients.

Triple Negative Breast Cancers (TNBC) subtype is an aggressive disease with poor clinical outcome. The only treatment available is surgery followed by chemotherapy or radiotherapy. Programmed death-ligand 1 (PD-L1) is a trans-membrane protein expressed on a wide variety of cells including immune cells, epithelial and vascular endothelial cells. Recently, PD-1/PD-L1 pathway signaling was described as an adaptive immune resistance mechanism enacted by the tumor cells to evade the immune response. Its presence on tumor cell membranes, acquired for this reason, through time, is an important prognostic value. However, data available in the literature about PD-L1 immunohistochemical expression in breast cancer are often discordant and not uniform, probably for the use of different antibodies clones and the high molecular heterogeneity of the different tumor types. The absence of target therapies, in particular for TNBC, has shifted the clinical attention mainly on the role of PD-L1 in this subtype of breast cancer. In this study, we evaluated tumor and TIL (tumor infiltrating lymphocytes) PDL-1 expression in a series of TNBC, included in Tissue Micro Arrays (TMAs), to define its real prognostic value, optimizing immunohistochemistry method with an "approved for diagnostic assay" antibody. PD-L1 expression directly correlated with proliferation index (Ki-67), glycemia, the presence of diabetes and indirectly with menopausal status, presence of lymph node metastasis and relapse. The analysis of Kaplan-Meier showed that an increased PD-L1 expression was strongly associated with better disease-free survival (DFS) but not correlated with overall survival (OS). Our data confirmed that PD-L1 could be an important marker for prognostic stratification and for planning immune checkpoint inhibitors therapies in patients with TNBC.

PD-L1 Immunohistochemical Detection in Tumor Cells and Tumor Microenvironment: Main Considerations on the Use of Tissue Micro Arrays.

PD-1/PD-L1 (programmed death 1/programmed death ligand 1) pathway plays a critical role in immune escape of tumor cells. Recent studies have described that PD-L1 is heterogeneously expressed in various types of cancer, although its prognostic/predictive value is still uncertain. These problems are partly due to a not well defined operating protocol for its detection by immunohistochemistry, but also because most of the studies conducted on large case series were made by Tissue Micro Array (TMA). We are going to discuss this latter point, to highlight that TMA must be set up in an appropriate manner, especially for some markers, such as PD-L1, which, besides being poorly expressed in tumor cells, can be expressed by cells of the tumor microenvironment.

Variability in Immunohistochemical Detection of Programmed Death Ligand 1 (PD-L1) in Cancer Tissue Types.

In normal cell physiology, programmed death 1 (PD-1) and its ligand, PD-L1, play an immunoregulatory role in T-cell activation, tolerance, and immune-mediated tissue damage. The PD-1/PD-L1 pathway also plays a critical role in immune escape of tumor cells and has been demonstrated to correlate with a poor prognosis of patients with several types of cancer. However, recent reports have revealed that the immunohistochemical (IHC) expression of the PD-L1 in tumor cells is not uniform for the use of different antibodies clones, with variable specificity, often doubtful topographical localization, and with a score not uniquely defined. The purpose of this study was to analyze the IHC expression of PD-L1 on a large series of several human tumors to correctly define its staining in different tumor tissues.

Coexistence of granular cell tumor and invasive ductal breast cancer in contralateral breasts: a case report.

Granular cell tumor (GCT) is a benign tumor of the breast that can mimic, on breast imaging, invasive carcinomas. Biological evolution of mammary GCT is unknown, especially if it is associated with an invasive carcinoma in the same or contralateral breast. This report details the morphological features of these synchronous lesions highlighting their biological characteristics and suggesting an appropriate follow up.

The role of HOTAIR in the modulation of resistance to anticancer therapy.

Leading anti-tumour therapeutic strategies typically involve surgery and radiotherapy for locally advanced (non-metastatic) cancers, while hormone therapy, chemotherapy, and molecular targeted therapy are the current treatment options for metastatic cancer. Despite the initially high sensitivity rate to anticancer therapies, a large number of patients develop resistance, leading to a poor prognosis. The mechanisms related to drug resistance are highly complex, and long non-coding RNAs appear to play a crucial role in these processes. Among these, the lncRNA homeobox transcript antisense intergenic RNA (HOTAIR), widely implicated in cancer initiation and progression, likewise plays a significant role in anticancer drug resistance. It can modulate cell activities such as proliferation, apoptosis, hypoxia, autophagy, as well as epithelial-mesenchymal transition, thereby contributing to the development of resistant tumour cells. In this manuscript, we describe different mechanisms of antitumor drug resistance in which HOTAIR is involved and suggest its potential as a therapeutic predictive biomarker for the management of cancer patients.

Endocrine nuclear receptors and long non­coding RNAs reciprocal regulation in cancer (Review).

Nuclear receptors (NRs) are transcriptional regulators involved in different aspects of normal cell physiology. Their deregulation is associated with aberrant expression, gene mutations and/or epigenetic alterations that can be related to the pathogenesis of various human diseases, and especially in cancer. In particular, a complex genomic network involved in the development and progression of NR­mediated cancer has been highlighted. Advanced genomic technologies have made it possible to understand that the expression of any particular NR in a given cancer subtype is only one component of a larger transcriptional machinery that is controlled by multiple associated NRs and transcription factors. Additionally, their ability to regulate and to be regulated by molecules of non­coding RNAs, microRNAs as well as long non­coding RNAs, is opening new scenarios for understanding the role of NRs in cancer initiation and progression. In the present review, the authors aimed to outline the reciprocal interactions that exist between the main NRs and long non­coding RNAs in different tumor diseases, to suggest new diagnostic biomarkers as well as therapeutic strategies for these tumors.