The Impact of Inadequate Exposure to Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors on the Development of Resistance in Non-Small-Cell Lung Cancer Cells.

Epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer (NSCLC) patients treated with EGFR-tyrosine kinase inhibitors (TKIs) inevitably develop resistance through several biological mechanisms. However, little is known on the molecular mechanisms underlying acquired resistance to suboptimal EGFR-TKI doses, due to pharmacodynamics leading to inadequate drug exposure. To evaluate the effects of suboptimal EGFR-TKI exposure on resistance in NSCLC, we obtained HCC827 and PC9 cell lines resistant to suboptimal fixed and intermittent doses of gefitinib and compared them to cells exposed to higher doses of the drug. We analyzed the differences in terms of EGFR signaling activation and the expression of epithelial-mesenchymal transition (EMT) markers, whole transcriptomes byRNA sequencing, and cell motility. We observed that the exposure to low doses of gefitinib more frequently induced a partial EMT associated with an induced migratory ability, and an enhanced transcription of cancer stem cell markers, particularly in the HCC827 gefitinib-resistant cells. Finally, the HCC827 gefitinib-resistant cells showed increased secretion of the EMT inducer transforming growth factor (TGF)-ß1, whose inhibition was able to partially restore gefitinib sensitivity. These data provide evidence that different levels of exposure to EGFR-TKIs in tumor masses might promote different mechanisms of acquired resistance.

CDK9-55 guides the anaphase-promoting complex/cyclosome (APC/C) in choosing the DNA repair pathway choice.

DNA double-strand breaks (DSBs) contribute to genome instability, a key feature of cancer. DSBs are mainly repaired by homologous recombination (HR) and non-homologous end-joining (NHEJ). We investigated the role of an isoform of the multifunctional cyclin-dependent kinase 9, CDK9-55, in DNA repair, by generating CDK9-55-knockout HeLa clones (through CRISPR-Cas9), which showed potential HR dysfunction. A phosphoproteomic screening in these clones treated with camptothecin revealed that CDC23 (cell division cycle 23), a component of the E3-ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome), is a new substrate of CDK9-55, with S588 being its putative phosphorylation site. Mutated non-phosphorylatable CDC23(S588A) affected the repair pathway choice by impairing HR and favouring error-prone NHEJ. This CDK9 role should be considered when designing CDK-inhibitor-based cancer therapies.

Lysosome purinergic receptor P2X4 regulates neoangiogenesis induced by microvesicles from sarcoma patients.

The tumor microenvironment modulates cancer growth. Extracellular vesicles (EVs) have been identified as key mediators of intercellular communication, but their role in tumor growth is largely unexplored. Here, we demonstrate that EVs from sarcoma patients promote neoangiogenesis via a purinergic X receptor 4 (P2XR4) -dependent mechanism in vitro and in vivo. Using a proteomic approach, we analyzed the protein content of plasma EVs and identified critical activated pathways in human umbilical vein endothelial cells (HUVECs) and human progenitor hematopoietic cells (CD34+). We then showed that vessel formation was due to rapid mitochondrial activation, intracellular Ca2+ mobilization, increased extracellular ATP, and trafficking of the lysosomal P2XR4 to the cell membrane, which is required for cell motility and formation of stable branching vascular networks. Cell membrane translocation of P2XR4 was induced by proteins and chemokines contained in EVs (e.g. Del-1 and SDF-1). Del-1 was found expressed in many EVs from sarcoma tumors and several tumor types. P2XR4 blockade reduced EVs-induced vessels in angioreactors, as well as intratumor vascularization in mouse xenografts. Together, these findings identify P2XR4 as a key mediator of EVs-induced tumor angiogenesis via a signaling mediated by mitochondria-lysosome-sensing response in endothelial cells, and indicate a novel target for therapeutic interventions.

Conditioned medium of primary lung cancer cells induces EMT in A549 lung cancer cell line by TGF-ß1 and miRNA21 cooperation.

The epithelial-mesenchymal transition (EMT) plays a key role in tumor progression, drug resistance and metastasis. Recently, numerous microRNA (miRNA) have been described to regulate EMT in tumor progression. In this study, we found that conditioned medium from the LC212 non-small-cell lung cancer (NSCLC) cell line (LC212-CM) induces morphological changes and overexpression of Vimentin, CD90, SMAD 2/3, SLUG and TWIST in A549 NSCLC cells, consistent with a mesenchymal phenotype. To identify the soluble mediators in LC212-CM involved in this phenomenon, we performed miRNA profiling and TGF-ß1 quantification. We found that LC212-CM contains high levels of TGF-ß1 as well as different secreted miRNAs. We focused our attention on Homo sapiens-microRNA21 (hsa-miR21), one of most relevant miRNA associated with lung cancer progression, metastasis and EMT. An hsa-miR21 antagomiR was able to prevent the LC212-CM-induced EMT phenotype in A549 cells. Furthermore, we found that TGF-ß1 and hsa-miR21 cooperate in the induction of EMT in A549 cells. Intriguingly, TGF-ß1 was found to induce hsa-miR21 expression in A549 cell, thus suggesting that the hsa-miR21 mediates at least in part the pro-EMT effects of TGF-ß1. In conclusion, hsa-miR21 and TGF-ß1 are involved in autocrine and paracrine circuits that regulate the EMT status of lung cancer cells.

Programmed Death Ligand 1 (PD-L1) Expression in Primary Angiosarcoma.

Angiosarcomas are rare malignant endothelial-cell tumors of vascular or lymphatic origin, and are among the most aggressive subtypes of soft-tissue sarcomas. The prognosis is poor and treatment is challenging in many cases. PD-1/PD-L1 pathway plays a critical role in immune escape of tumor cells. Recent studies described that PD-L1 is widely expressed in various types of cancer, providing the basis for the development of PD1/PD-L1 antibodies as anti-cancer immunotherapy. Despite the well-known potential of PD-L1 as prognostic and predictive biomarker, only few studies described its IHC expression in cancer subtypes for the extreme difficulty in developing standard protocol with the different antibody clones available. We analyzed the IHC expression of PD-L1 on a series of angiosarcomas at different body location, showing its aberrant expression in about 66% of samples with no relation with prognosis. Our study allowed us to correctly define PD-L1 staining in angiosarcoma tumor tissues with final purpose to stratify patients for immune checkpoint inhibitors therapies.

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.

Richter Syndrome With Plasmablastic Lymphoma at Primary Diagnosis: A Case Report With a Review of the Literature.

Richter syndrome (RS) is considered as the rare development of an aggressive lymphoid malignancy in a preexisting small lymphocytic lymphoma/chronic lymphocytic leukemia. The most common aggressive lymphoma developing in this setting is diffuse large B-cell lymphoma, but classical Hodgkin lymphoma and other much rarer entities such as prolymphocytic lymphoma and dendritic cell sarcoma are also described, most frequently in the progression of the disease over time. A clonal relation between the 2 neoplastic proliferations can be frequently found, whereas clonally unrelated cases are commonly considered as independent tumors, probably due to a variable combination of multiple causes, responsible independently for the 2 neoplasms. RS with plasmablastic lymphoma is reported very rarely, during the clinical course of the small lymphocytic lymphoma/chronic lymphocytic leukemia. Herein, an unusual case of RS with the coexistence of plasmablastic lymphoma and B-small lymphocytic lymphoma in the same lymph node at the time of first diagnosis is described.

LncRNA HOTAIR as Prognostic Circulating Marker and Potential Therapeutic Target in Patients with Tumor Diseases.

In the recent years the importance of the role played by non-coding RNA on the regulation of gene expression was increased by numerous studies. The research mainly focused on small ncRNAs, such as miRNAs, while the functions of long non-coding RNA (lncRNA) have been much less studied. lncRNAs can be transcribed from intergenic, intragenic or specific chromosomal regions. Compared to miRNAs, lncRNAs have a complex secondary and tertiary structure which allows to bind proteins, RNA, DNA and to carry out their regulatory functions. Several studies showed that extracellular ncRNAs can circulate in the blood of both healthy and diseased patients. Most of the circulating ncRNAs are included in lipid or lipoprotein vesicles, such as apoptotic bodies, macrovesicles or exosomes, in which they are highly stable. The presence of circulating ncRNAs in the blood of cancer patients versus normal subjects suggested the possibility that these molecules may represent new diagnostic markers. HOTAIR is a HOX transcript antisense RNA, located in the HOXC locus, able to repress transcription in the posterior region of the HOXD locus. HOTAIR has been involved in the evolution of several primary tumors, wherein increase of HOTAIR expression has endorsed invasion and metastasis. In this review, we describe the experimental evidences on the potential role as circulating marker of lncRNA HOTAIR.

Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis.

Ultraconserved regions (UCRs) have been shown to originate non-coding RNA transcripts (T-UCRs) that have different expression profiles and play functional roles in the pathophysiology of multiple cancers. The relevance of these functions to the pathogenesis of bladder cancer (BlCa) is speculative. To elucidate this relevance, we first used genome-wide profiling to evaluate the expression of T-UCRs in BlCa tissues. Analysis of two datasets comprising normal bladder tissues and BlCa specimens with a custom T-UCR microarray identified ultraconserved RNA (uc.) 8+ as the most upregulated T-UCR in BlCa tissues, although its expression was lower than in pericancerous bladder tissues. These results were confirmed on BlCa tissues by real-time PCR and by in situ hybridization. Although uc.8+ is located within intron 1 of CASZ1, a zinc-finger transcription factor, the transcribed non-coding RNA encoding uc.8+ is expressed independently of CASZ1. In vitro experiments evaluating the effects of uc.8+ silencing, showed significantly decreased capacities for cancer cell invasion, migration, and proliferation. From this, we proposed and validated a model of interaction in which uc.8+ shuttles from the nucleus to the cytoplasm of BlCa cells, interacts with microRNA (miR)-596, and cooperates in the promotion and development of BlCa. Using computational analysis, we investigated the miR-binding domain accessibility, as determined by base-pairing interactions within the uc.8+ predicted secondary structure, RNA binding affinity, and RNA species abundance in bladder tissues and showed that uc.8+ is a natural decoy for miR-596. Thus uc.8+ upregulation results in increased expression of MMP9, increasing the invasive potential of BlCa cells. These interactions between evolutionarily conserved regions of DNA suggest that natural selection has preserved this potentially regulatory layer that uses RNA to modulate miR levels, opening up the possibility for development of useful markers for early diagnosis and prognosis as well as for development of new RNA-based cancer therapies.

Immune-phenotypical markers for the differential diagnosis of melanocytic lesions.

For specific subsets of melanocytic proliferations, there are morphologic limitations in the histological diagnosis, especially for borderline melanocytic tumors. In particular, Spitzoid proliferations can be difficult to diagnose. For these reasons, in the last years, clinic research has focused attention on discovery of new diagnostic markers. Published gene expression and proteomic profiling data indicate new candidate molecules involved in melanoma pathogenesis, and useful in differential diagnosis of difficult melanocytic lesions. Recently, the diagnostic power of galectin-3 was demonstrated in series of melanocytic lesions, with a strong increasing of expression in malignant lesions compared with benign lesions. Similarly, the accumulation of Collagen XVII antibody was detected in vertical melanoma fronts and associated with invasive phenotype. Moreover, overexpression of cyclin D1 and p21 was detected in Spitz nevi compared with non-spitzoid melanomas; Ki-67 appears highly expressed in deep areas of non-spitzoid melanomas. In this review, we overview of the main molecular markers that a useful tool for the differential diagnosis of benign, borderline and malignant melanocytic lesions, related to their biological behavior, useful also for predicting the evolution of the disease.

Atypical amplification of chromosome region 22q12 in melanoma: A case report.

Morphological, ultrastructural and immunohistochemical characteristics of clear cell sarcoma (CCS) of the soft tissue frequently overlap with those of malignant melanoma. Thus, the differential diagnosis between the two lesions represents an important diagnostic dilemma. However, a number of genetic factors can be used to differentiate the two tumors; in particular, the t(12;22)(q13;q12) chromosomal translocation is typical of CCS, resulting in fusion of the EWSR1 gene on chromosome 22q12 and the ATF1 gene on chromosome 12q13. The detection of this molecular alteration has proved useful in the differential diagnosis of the two lesions. The present study reports the case of a 71-year-old male patient with a suspicious lymph node mass. Immunohistochemical analysis of the lesion indicated a diagnosis of metastatic melanoma, however, cytogenetic analysis using fluorescence in situ hybridization was additionally performed to investigate the chromosomal rearrangements of the 22q12 region and completely exclude the possibility of CCS. The current case did not demonstrate the presence of the translocation, supporting the diagnosis of melanoma. However, a clear orange amplification signal was observed relative to an ~500-kb region adjacent to the EWSR1 gene in the centromeric direction of chromosome 22q12. To the best of our knowledge, this is the first description of a 22q12 chromosomal alteration in melanoma. Furthermore, despite the presence of numerous genes in this region, their amplification has not previously been associated with the pathogenesis of melanoma.

Borderline Brenner tumor of the ovary: a case report with immunohistochemical and molecular study.

BACKGROUND: Borderline Brenner tumor of the ovary is a rare entity characterized by papillary structures with a fibro-vascular core, covered by a transitional epithelium, and by the absence of stromal infiltration. It is associated, by definition, with a benign component of Brenner tumor. CASE: We report a case of a 68-year-old woman, with a right ovarian mass, whose morphology and immuno-profile were consistent with the diagnosis of a borderline Brenner tumor. Immunohistochemistry carried out on selected markers may help to formulate the diagnosis, more than the molecular analyses.

SPARC/osteonectin is involved in metastatic process to the lung during melanoma progression.

The existence of a "metastasis gene signature" that predisposes primary breast cancer cells to metastasize to the lungs has been recently highlighted by gene expression profiling studies. The combination of genes responsible for this process includes genes encoding several metalloproteinases as well as the gene encoding SPARC (secreted protein acidic and rich in cysteine)/osteonectin. SPARC is involved in normal tissue remodeling as it regulates the deposition of extracellular matrix, but also plays a role in neoplastic transformation. Aberrant SPARC expression has been detected both in stromal cells associated with cancer and in cancer cells. The main aim of this study was to investigate whether or not SPARC might be involved in directing metastasis of other types of cancer to the lung. We constructed a tissue microarray containing lung metastases from a variety of primary tumors in different organs and used immunohistochemistry to assess SPARC expression. We found SPARC overexpressed mainly in lung metastases from melanoma. We then assessed the expression of SPARC mRNA and protein in metastatic melanoma from different anatomic sites and in their corresponding primary tumors, and found that it is overexpressed in lung metastases. Our data strongly support the hypothesis that SPARC is involved in directing melanoma metastases specifically to the lung, which underpins its potential as prognostic marker and novel target for specific therapy.

Molecular strategies for detecting chromosomal translocations in soft tissue tumors (review).

Approximately one third of soft tissue tumors are characterized by chromosomal aberrations, in particular, translocations and amplifications, which appear to be highly specific. The identification of fusion transcripts not only supports the diagnosis, but provides the basis for the development of novel therapeutic strategies aimed at blocking the aberrant activity of chimeric proteins. Molecular biology, and in particular, cytogenetic and qualitative and quantitative polymerase chain reaction technologies, allow with high efficiency and specificity, the determination of specific fusion transcripts resulting from chromosomal translocations, as well as the analysis of gene amplifications. In this review, various molecular techniques that allow the identification of translocations and consequent fusion transcripts generated are discussed in the broad spectrum of soft tissue tumors.

IMRT-SIB with concurrent and neo-adjuvant platinum-based chemotherapy for locally advanced head and neck squamous cell cancer: analysis of clinical outcomes in a retrospective series of a single institution.

AIMS AND BACKGROUND: To evaluate results of an intensity-modulated radiotherapy with simultaneous integrated boost schedule with concurrent and neo-adjuvant platinum-based chemotherapy for the definitive treatment of locally advanced head and neck cancer in a retrospective series. METHODS AND STUDY DESIGN: Between May 2007 and February 2010, 28 consecutive patients with locally advanced head and neck cancer (stage II, 11%; III, 18%; IV, 71%) received intensity-modulated radiotherapy with simultaneous integrated boost with concurrent and neoadjuvant (20/28 patients) chemotherapy, at 1.8 G/die to 54 Gy to the elective volume and 66 Gy (2.2 Gy/die) to the tumor volume. Acute and late toxicities were scored according to RTOG/EORTC. A quality of life questionnaire for late xerostomia was also administered. Locoregional control and overall survival were estimated using Kaplan-Meier analysis. RESULTS: Median follow-up was 50 months, there was no grade 4 acute/late toxicity. Major acute toxicities were grade 2+ mucositis, 79%; grade 2+ xerostomia, 54%; grade 2+ dysphagia, 86%; 54% of patients required parenteral nutrition. The most relevant late reaction was grade 1 xerostomia (64%), which gradually recovered with time. A linear correlation between the RTOG/EORTC scale and the quality of life questionnaire value (P = 0.0120, r2 = 0.2641) was found, receiver operating characteristic analysis (ROC) confirmed sensitivity of the quality of life questionnaire to define grade 2 late salivary toxicity (P = 0.019). Five-year actuarial locoregional control and overall survival were 81% ± 7.7 SE and 82% ± 7.3 SE, respectively. CONCLUSIONS: A prospective trial of the intensity-modulated radiotherapy with simultaneous integrated boost schedule tested in this retrospective series with concurrent and neoadjuvant chemotherapy seems warranted in order to establish this approach as a standard regimen of intensity-modulated radiotherapy with simultaneous integrated boost chemoradiation.

Hyperexpression of HOXC13, located in the 12q13 chromosomal region, in well­differentiated and dedifferentiated human liposarcomas.

Liposarcoma (LPS) is the most common soft tissue neoplasm in adults and is characterized by neoplastic adipocyte proliferation. Some subtypes of LPSs show aberrations involving the chromosome 12. The most frequent are t(12;16) (q13;p11) present in more than 90% of myxoid LPSs and 12q13-15 amplification in well-differentiated and dedifferentiated LPSs. In this region, there are important oncogenes such as CHOP (DDIT3), GLI, MDM2, CDK4, SAS, HMGA2, but also the HOXC locus, involved in development and tumor progression. In this study, we evaluated the expression of HOXC13, included in this chromosomal region, in a series of adipocytic tumors. We included 18 well-differentiated, 4 dedifferentiated, 11 myxoid and 6 pleomorphic LPSs as well as 13 lipomas in a tissue microarray. We evaluated the HOXC13 protein and gene expression by immunohistochemistry and quantitative PCR. Amplification/translocation of the 12q13-15 region was verified by FISH. Immunohistochemical HOXC13 overexpression was observed in all well-differentiated and dedifferentiated LPSs, all characterized by the chromosome 12q13-15 amplification, and confirmed by quantitative PCR analysis. In conclusion, our data show a deregulation of the HOXC13 marker in well­differentiated and dedifferentiated LPSs, possibly related to 12q13-15 chromosomal amplification.

MYC chromosomal aberration in differential diagnosis between Burkitt and other aggressive lymphomas.

Myc oncogenetic deregulation is abundantly described in several solid human cancer and lymphomas. Particularly, Burkitt's lymphoma belongs to the family of B Non Hodgkin aggressive lymphomas. Although it is morphologically characterized, immunophenotypic and cytogenetic diagnosis remains complex. In 2008, the WHO has introduced a new diagnostic class of aggressive B-cell lymphomas with features intermediate between BL and DLBCL. This diagnostic class represents a temporary container of aggressive B-cell lymphomas, not completely belonging to the BL and DLBCL categories. The importance of establishing a correct diagnosis would allow a better prognostic classification and a better therapeutic approach. In this review, we summarize the main diagnostic approaches necessary for appropriate diagnoses and we emphasize the importance of cytogenetic analysis of the oncogene Myc in the histopathological diagnosis and the prognostic/predictive stratification. In this contest, Myc represents the more involved gene in the development of these lymphomas. Therefore, we analyze the genetic aberrations causing its over-expression and the concomitant deregulation of molecular pathways related to it. We also propose a FISH approach useful in the diagnosis of these lymphomas.