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The Special Issue in Cancers, “The Biological and Clinical Aspects of Merkel Cell Carcinoma”, walks the avid reader through the interesting and sometimes even mysterious facets of Merkel cell carcinoma (MCC), starting at its carcinogenesis to also cover innovative treatment options [...]

Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer where Merkel cell Polyomavirus (MCPyV) contributes to the pathogenesis. In an adjuvant setting, radiotherapy (RT) is believed to give a survival benefit. The prognostic impact of sex related to MCPyV-status and adjuvant RT were analyzed in patients referred to Karolinska University Hospital. Data were collected from 113 patients&rsquo, hospital records and MCPyV analyses were made in 54 patients (48%). We found a significantly better overall survival (OS) for women compared to men and a significant difference in OS in patients receiving adjuvant RT. Furthermore, we found that men with virus negative MCC have an increased risk for earlier death (HR 3.6). This indicates that MCPyV positive and negative MCC act as two different diseases, and it might be due to different mechanism in the immune response between male and female patients. This could have significance in tailoring treatment and follow-up in MCC patients in the future.

Ovarian germ cell tumors (OGCTs) and sex cord stromal tumors (SCSTs) are rare gynecologic tumors that are derived from germ and stromal cells, respectively. Unlike their epithelial counterparts, molecular pathogenesis of these tumor types is still poorly understood. Here, we characterized microRNA (miRNA) expression profiles of 9 OGCTs (2 malignant and 7 benign) and 3 SCSTs using small RNA sequencing. We observed significant miRNA expression variations among the three tumor groups. To further demonstrate the biological relevance of our findings, we selected 12 miRNAs for validation in an extended cohort of 16 OGCTs (9 benign and 7 malignant) and 7 SCSTs by reverse transcription-quantitative polymerase chain reaction. Higher expression of miR‑373‑3p, miR‑372‑3p and miR‑302c‑3p and lower expression of miR‑199a‑5p, miR‑214‑5p and miR‑202‑3p were reproducibly observed in malignant OGCTs as compared to benign OGCTs or SCSTs. Comparing with benign OGCTs, miR‑202c‑3p and miR‑513c‑5p were more abundant in SCSTs. Additionally, we examined Beclin 1 (BECN1), a target of miR‑199a‑5p, in the clinical samples using western blot analysis. Our results show that BECN1 expression was higher in malignant OGCTs than benign OGCTs, which is concordant with their lower miR‑199a‑5p expression. This study suggests that these miRNAs may have potential value as tumor markers and implications for further understanding the molecular basis of these tumor types.

High-risk human papillomavirus (HPV) testing is a recommended triage approach for females with atypical squamous cells of undetermined significance (ASCUS), but due to its poor specificity this approach is not recommended for patients with low-grade squamous intraepithelial lesions (LSIL). The objective of the current study was to determine microRNA (miR)-205 expression levels in liquid-based cytology (LBC) samples, and evaluate their ability to predict cervical intraepithelial neoplasia grade 2/3 or worse (CIN2/3+) in females with minor cytological abnormalities. LBC samples were obtained from patients attending the Swedish Cervical Cancer Screening Program. The Mann-Whitney U test, one-way analysis of variance, Kruskal-Wallis test, Spearman rank order correlation analysis, and Pearson's χ2 test were used to assess the results. Accuracy analyses indicated that high miR-205 expression had a significantly higher specificity to high-risk HPV testing, and a sensitivity similar to that of high-risk HPV testing to predict CIN2+ and CIN3+ in women with LSIL, but not those with high-grade squamous intraepithelial lesions. Although further research is required for females with LSIL, miR-205 expression in LBC samples may be a novel triage marker for, or a beneficial supplement to high-risk-HPV testing in these patients.

miR-223-3p is deregulated in several tumor types and plays an important role in tumorigenesis and progression. However, its role in the pathogenesis of testicular germ cell tumor (TGCT) remains uncharacterized. We previously demonstrated that miR-223-3p expression was increased in TGCTs compared with normal testes (NT), suggesting that miR-223-3p may have an oncogenic role in TGCT. Using published dataset and The Cancer Genome Atlas database, we validated higher miR-223-3p expression in TGCTs than NT, and found a negative correlation between miR-223-3p and FBXW7 mRNA expression levels. Using both gain- and loss-of-function experiments, we show that miR-223-3p regulates FBXW7 protein expression, cell growth and apoptosis in TGCT cell lines. Additionally, we demonstrate that ectopic expression of the full-length coding sequence of FBXW7 could rescue the cell growth and apoptotic effects mediated by miR-223-3p. Our findings suggest an oncogenic role for miR-223-3p in TGCT, which promotes cell growth and inhibits apoptosis through repression of FBXW7.

Viruses can inhibit host autophagy through multiple mechanisms, and evasion of autophagy plays an important role in immune suppression and viral oncogenesis. Merkel cell polyomavirus (MCPyV) T‐antigens are expressed and involved in the pathogenesis of a large proportion of Merkel cell carcinoma (MCC). Yet, how MCPyV induces tumorigenesis is not fully understood. Herein, we show that MCPyV T‐antigens induce miR‐375, miR‐30a‐3p and miR‐30a‐5p expressions, which target multiple key genes involved in autophagy, including ATG7, SQSTM1 (p62) and BECN1. In MCC tumors, low expression of ATG7 and p62 are associated with MCPyV‐positive tumors. Ectopic expression of MCPyV small T‐antigen and truncated large T‐antigen (LT), but not the wild‐type LT, resulted in autophagy suppression, suggesting the importance of autophagy evasion in MCPyV‐mediated tumorigenesis. Torin‐1 treatment induced cell death, which was attenuated by autophagy inhibitor, but not pan‐caspase inhibitor, suggesting a potential role of autophagy in promoting cell death in MCC. Conceptually, our study shows that MCPyV oncoproteins suppress autophagy to protect cancer cells from cell death, which contribute to a better understanding of MCPyV‐mediated tumorigenesis and potential MCC treatment., What's new? About four‐fifths of Merkel cell carcinomas harbor the Merkel cell polyomavirus (MCPyV) genome, though mutations in viral T antigens generally render MCPyV replication‐deficient. Here, the authors describe a network by which MCPyV oncoproteins and viral‐regulated miRNAs hijack autophagy machinery in MCC. In cells, expression of MCPyV T‐antigens induced miR‐375, miR‐30a‐3p, and miR‐30a‐5p expression. Subsequent experiments validated these miRNAs as autophagy regulators that specifically target the genes ATG7, SQSTM1, and BECN1. Autophagy suppression protected MCC cell survival. This novel insight into the role of MCPyV and autophagy regulation in MCC may be relevant in the generation of future therapeutic strategies.

MicroRNA-375 (miR-375) is deregulated in multiple tumor types and regulates important targets involved in tumorigenesis and metastasis. This miRNA is highly expressed in Merkel cell carcinoma (MCC) compared to normal skin and other non-MCC skin cancers, and its expression is high in Merkel cell polyomavirus (MCPyV)-positive (MCPyV+) and low in MCPyV-negative (MCPyV&minus, ) MCC tumors. In this study, we characterized the function and target of miR-375 in MCPyV+ and MCPyV&minus, MCC cell lines. Ectopic expression of miR-375 in MCPyV&minus, MCC cells resulted in decreased cell proliferation and migration, as well as increased cell apoptosis and cell cycle arrest. However, in MCPyV+ MCC cells, inhibition of miR-375 expression reduced cell growth and induced apoptosis. Additionally, the expression of lactate dehydrogenase B (LDHB), a known target of miR-375, was inversely correlated with miR-375. Silencing of LDHB reduced cell growth in MCPyV&minus, cell lines, while its silencing in MCPyV+ cell lines rescued the cell growth effect mediated by miR-375 inhibition. Together, our results suggest dual roles of miR-375 and LDHB in MCPyV and non-MCPyV-associated MCCs. We propose that LDHB could be a therapeutic target in MCC and different strategies should be applied in virus- and non-virus-associated MCCs.

The use of imatinib mesylate has greatly improved the clinical outcome for gastrointestinal stromal tumor (GIST) patients. However, imatinib resistance is still a major clinical challenge, and the molecular mechanisms are not fully understood. We have previously shown that miR-125a-5p and its mRNA target PTPN18 modulate imatinib response in GIST cells. Herein, we evaluated phosphorylated FAK (pFAK) as a candidate downstream target of PTPN18 and the possible association of this regulation with imatinib resistance in GIST. FAK and pFAK expressions were evaluated in GIST882 cells transfected with short hairpin RNA or short interfering RNA targeting PTPN18 or miR-125a-5p mimic, imatinib-resistant GIST882R subclones and clinical samples using Western blot analyses. FAK phosphorylation was blocked using the FAK inhibitor 14 (FAKi) and the effects on cell viability and apoptosis were evaluated using WST-1 assay and cleaved PARP expression. Clinical associations of FAK and pFAK expression with imatinib resistance, KIT mutation and patient outcome were assessed by Fisher's exact test or log-rank test. Over-expression of miR-125a-5p and silencing of PTPN18 increased pFAK, but not FAK, expression in GIST cells. Higher pFAK expression was observed in the GIST882R subclones with acquired imatinib resistance compared to their imatinib-sensitive parental cells. Treatment with FAKi in imatinib-resistant GIST882R cells reduced cell viability and increased apoptosis upon imatinib treatment. Additionally, FAKi could rescue the imatinib resistance effect mediated by miR-125a-5p over-expression. In clinical samples, high FAK and pFAK expressions were associated with KIT mutation status, and high FAK expression was also associated with metastasis in GIST. Higher pFAK was found in cases with shorter overall survival. Our findings highlight an important role for miR-125a-5p regulation and its downstream target pFAK for imatinib resistance in GIST. pFAK and FAK may have prognostic values in GIST.

Background: Gastrointestinal stromal tumour (GIST) is mainly initialised by receptor tyrosine kinase gene mutations. Although the tyrosine kinase inhibitor imatinib mesylate considerably improved the outcome of patients, imatinib resistance still remains a major therapeutic challenge in GIST therapy. Herein we evaluated the clinical impact of microRNAs in imatinib-treated GISTs. Methods: The expression levels of microRNAs were quantified using microarray and RT–qPCR in GIST specimens from patients treated with neoadjuvant imatinib. The functional roles of miR-125a-5p and PTPN18 were evaluated in GIST cells. PTPN18 expression was quantified by western blotting in GIST samples. Results: We showed that overexpression levels of miR-125a-5p and miR-107 were associated with imatinib resistance in GIST specimens. Functionally, miR-125a-5p expression modulated imatinib sensitivity in GIST882 cells with a homozygous KIT mutation but not in GIST48 cells with double KIT mutations. Overexpression of miR-125a-5p suppressed PTPN18 expression, and silencing of PTPN18 expression increased cell viability in GIST882 cells upon imatinib treatment. PTPN18 protein levels were significantly lower in the imatinib-resistant GISTs and inversely correlated with miR-125a-5p. Furthermore, several microRNAs were significantly associated with metastasis, KIT mutational status and survival. Conclusions: Our findings highlight a novel functional role of miR-125a-5p on imatinib response through PTPN18 regulation in GIST.

DOG1, a Ca(2+)-activated Cl(-) channel (CaCC), was identified in 2004 to be robustly expressed in gastrointestinal stromal tumors (GIST). It was rapidly included as a tumor marker in routine diagnostics, but the functional role remained unknown. CaCCs are important regulators of normal physiological functions, but also implicated in tumorigenesis, cancer progression, metastasis, cell migration, apoptosis, proliferation and viability in several malignancies. We therefore investigated whether DOG1 plays a role in the three latter in GIST by utilizing in vitro cell model systems. Confocal microscopy identified different subcellular localizations of DOG1 in imatinib-sensitive and imatinib-resistant cells. Electrophysiological studies confirmed that DOG1-specific pharmacological agents possess potent activating and inhibiting properties. Proliferation assays showed small effects up to 72 h, and flow cytometric analysis of adherent cells with 7-AAD/Annexin V detected no pharmacological effects on viable GIST cells. However, inhibition of DOG1 conveyed pro-apoptotic effects among early apoptotic imatinib-resistant cells. In conclusion, DOG1 generates Cl(-) currents in GIST that can be regulated pharmacologically, with small effects on cell viability and proliferation in vitro. Inhibition of DOG1 might act pro-apoptotic on some early apoptotic GIST cell populations. Further studies are warranted to fully illuminate the function of DOG1 and its potential as therapeutic target.

Wig-1, also known as ZMAT3, is a p53 target gene that encodes an RNA-binding zinc-finger protein involved in the regulation of mRNA stability through binding to AU-rich elements (AREs). We have used microarray analysis to identify novel Wig-1 target mRNAs. We identified 2447 transcripts with >fourfold differential expression between Wig-1 and control small interfering (si)RNA-treated HCT116 cells. Several p53 target genes were among the deregulated transcripts. We found that Wig-1 regulates FAS and 14-3-3σ mRNA independently of p53. We show that Wig-1 binds to FAS mRNA 3′-UTR and decreases its stability through an ARE in the 3′-UTR. Depletion of Wig-1 was associated with increased cell death and reduced cell cycle arrest upon DNA damage. Our results suggest a role of Wig-1 as a survival factor that directs the p53 stress response toward cell cycle arrest rather than apoptosis through the regulation of FAS and 14-3-3σ mRNA levels.

e21047 Background: Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer. The pathogenesis is linked to the immune system and Merkel cell polyoma virus (MCPyV) is present in about 80% of MCC. Reports have shown that patients with a virus-negative MCC have a worse prognosis. Adjuvant radiotherapy given after surgery has previously been shown to give a survival benefit in retrospective data. Our aim is to analyze clinical variables and their prognostic impact in a Swedish cohort. Methods: All patients with confirmed diagnosis of MCC referred to Karolinska University Hospital in Sweden from 1989-2018 were included and retrospective data was collected. Results: 113 patients were included. Median age at operation was 75.4 years (range 19-99); 64 (57%) were female. Treatment included surgery with (n = 46) or without adjuvant radiotherapy (RT). 43% of female patients and 37% of male patients received RT. Median overall survival (OS) was 2,6 years (range 0,1-25,8), for female patients 3,4 years (range 0,1-25,8) and for male patients 1,8 years (range 0,1-19,0). Median OS in surgery group was 1,8 years (range 0,4-25,8). With adjuvant RT median OS improved with 1,7 years (p = 0,0001) to 3,5 years (range 0,2-22,1), for female patients 4,0 years (range 0,2-22,1) and for male patients 2,0 years (range 0,3-8,8). MCPyV status was available in 53 patients. 74% were MCPyV positive, 26% negative. MCPyV gave no difference in OS, however when gender was added in to multivariate analysis, female patients with virus negative disease had better outcome than virus negative males (p = 0.03). In virus positive MCC there was no difference in OS between female and male patients. Conclusions: Our data confirms the positive impact of adjuvant radiotherapy on survival and illustrates the difference in outcome between female and male patients, with women having a better outcome. In the MCPyV group the tendency is that men with virus negative MCC has the worst outcome. Our findings indicate that MCPyV positive and negative MCC act as two different diseases and it also raises questions if there is a difference in the disease itself or the immune response towards MCC between male and female patients.

The prognosis of non-small cell lung cancer (NSCLC) is poor, since it has often metastasized to distant organs by the time of diagnosis. Therefore, biomarkers predicting metastasis are crucial. miRNAs play important roles in the regulation of different tumor cell processes, including metastasis. We recently showed that miRNA-214 is linked to a radioresistant phenotype of NSCLC. miRNA-214 has been linked to metastasis in other tumor types. Therefore, we examined the role of miRNA-214 in the metastatic potential of NSCLC. We showed that downregulation of miRNA-214 increased invasive potential, and conversely, overexpression of miRNA-214 decreased invasiveness of NSCLC cells in vitro. Gene expression and bioinformatic analyses of NSCLC cells with ablated miRNA-214, identified a number of metastasis-related target genes, including pregnancy-associated plasma protein A (PAPP-A), alpha protein kinase 2 (ALPK2), cyclin-dependent kinase 6 (CDK6) and tumor necrosis-factor alpha-induced protein 3 (TNFAIP3). These were validated on mRNA and protein level to be regulated by miRNA-214. Through immunoprecipitation we showed that only ALPK2 is directly regulated by miRNA-214. We also examined the protein expression of these four genes in NSCLC tumors with respect to metastatic potential. These results showed that NSCLC tumors express these proteins at moderate-high levels in the nucleus, cytoplasm and/or plasma membrane although with no significant correlation to the overall survival or the metastatic potential of the patients. However, we also showed that the membrane-localized PAPP-A had a higher expression level compared to the cytoplasm-localized. In conclusion, we show that low miRNA-214 expression is linked to a higher invasive potential of NSCLC cells.

Deregulation of microRNA (miRNA) expression in adrenocortical carcinomas (ACCs) has been documented to have diagnostic, prognostic, as well as functional implications. Here, we evaluated the mRNA expression ofDROSHA,DGCR8,DICER(DICER1),TARBP2, andPRKRA, the core components in the miRNA biogenesis pathway, in a cohort of 73 adrenocortical tumors (including 43 adenomas and 30 carcinomas) and nine normal adrenal cortices using a RT-qPCR approach. Our results show a significant over-expression ofTARBP2,DICER, andDROSHAin the carcinomas compared with adenomas or adrenal cortices (PTARBP2, but notDICERorDROSHA, is a strong molecular predictor to discriminate between adenomas and carcinomas. Functionally, we showed that inhibition of TARBP2 expression in human NCI-H295R ACC cells resulted in a decreased cell proliferation and induction of apoptosis. TARBP2 over-expression was not related to gene mutations; however, copy number gain of theTARBP2gene was observed in 57% of the carcinomas analyzed. In addition, we identified thatmiR-195andmiR-497could directly regulate TARBP2 and DICER expression in ACC cells. This is the first study to demonstrate the deregulation of miRNA-processing factors in adrenocortical tumors and to show the clinical and biological impact of TARBP2 over-expression in this tumor type.

Gastrointestinal stromal tumors (GISTs) are thought to originate from the electrically active pacemaker cells of the gastrointestinal tract. Despite the presence of synaptic-like vesicles and proteins involved in cell secretion it remains unclear whether GIST cells possess regulated release mechanisms. The GIST tumor cell line GIST882 was used as a model cell system, and stimulus-release coupling was investigated by confocal microscopy of cytoplasmic free Ca(2+) concentration ([Ca(2+)]i), flow cytometry, and luminometric measurements of extracellular ATP. We demonstrate that GIST cells have an intact intracellular Ca(2+)-signaling pathway that regulates ATP release. Cell viability and cell membrane integrity was preserved, excluding ATP leakage due to cell death and suggesting active ATP release. The stimulus-secretion signal transduction is at least partly dependent on Ca(2+) influx since exclusion of extracellular Ca(2+) diminishes the ATP release. We conclude that measurements of ATP release in GISTs may be a useful tool for dissecting the signal transduction pathway, mapping exocytotic components, and possibly for the development and evaluation of drugs. Additionally, release of ATP from GISTs may have importance for tumor tissue homeostasis and immune surveillance escape.

Malignant cutaneous melanoma is a highly aggressive form of skin cancer. Despite improvements in early melanoma diagnosis, the 5-year survival rate remains low in advanced disease. Therefore, novel biomarkers are urgently needed to devise new means of detection and treatment. In this study, we aimed to improve our understanding of microRNA (miRNA) deregulation in melanoma development and their impact on patient survival. Global miRNA expression profiles of a set of melanoma lymph node metastases, melanoma cell lines, and melanocyte cultures were determined using Agilent array. Deregulated miRNAs were evaluated in relation with clinical characteristics, patient survival, and mutational status for BRAF and NRAS. Several miRNAs were differentially expressed between melanocytes and melanomas as well as melanoma cell lines. In melanomas, miR-193a, miR-338, and miR-565 were underexpressed in cases with a BRAF mutation. Furthermore, low expression of miR-191 and high expression of miR-193b were associated with poor melanoma-specific survival. In conclusion, our findings show miRNA dysregulation in malignant melanoma and its relation to established molecular backgrounds of BRAF and NRAS oncogenic mutations. The identification of an miRNA classifier for poor survival may lead to the development of miRNA detection as a complementary prognostic tool in clinical practice.

Introduction Background and aim Merkel cell carcinoma (MCC) is an aggressive type of skin cancer. About 80% of MCCs harbour integrated Merkel cell polyomavirus (MCV) genome with a mutation in the large T (LT) gene. MCV T-antigens are required for neoplastic transformation and maintenance of cell growth. however the molecular mechanism by which the virus induces tumorigenesis remains unclear. In this study we aimed to identify and characterise functional role of MCV-regulated microRNAs in MCC. Material and methods We did silencing or ectopic expression of MCV T-antigens to identify specific miRNAs regulated by MCV T-antigens by RT-qPCR. The involvement of MCV T-antigens and miRNAs in autophagy was evaluated using LC3-II conversion, mRFP-EGFP-LC3 reporter and/or transmission electron microscopy. The targets of miRNAs were verified by western blot analysis and luciferase reporter assays. We also look for the DICER expression by western blot. Results and discussions We identified specific miRNAs regulated by MCV T-antigens. Using both gain- and loss-of-function experiments, we showed that MCV T-antigens and MCV-regulated miRNAs (miR-375, miR-30a-5p and miR-30a-3p) could regulate autophagy. We further demonstrated that miR-375 could directly regulate ATG7 and SQSTM1, while both miR-30a-3p and miR-30a-5p could target BECN1. We also identified the MCV T-antigens regulates these miRNAs through post-transcriptionally by regulating DICER expression in MCV positive MCC. Conclusion We provide evidence that MCV T-antigens regulate miRNAs and autophagy in MCC. MCV T-antigens increase miRNA expressions through pots-transcriptional regulation, and several of these miRNAs can directly target multiple autophagy genes that lead to suppression of autophagy in MCC.

e21601Background: Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer. The pathogenesis is linked to the immune system and immunocompromised patients have an increased r...

The discovery of gene fusion mutations, particularly in leukemia, has consistently identified new cancer pathways and led to molecular diagnostic assays and molecular-targeted chemotherapies for cancer patients. Here, we report our discovery of a novel CREB3L2-PPARγ fusion mutation in thyroid carcinoma with t(3;7)(p25;q34), showing that a family of somatic PPARγ fusion mutations exist in thyroid cancer. The CREB3L2-PPARγ fusion encodes a CREB3L2-PPARγ fusion protein that is composed of the transactivation domain of CREB3L2 and all functional domains of PPARγ1. CREB3L2-PPARγ was detected in

The diagnosis of follicular thyroid carcinoma (FTC) in the absence of metastasis can only be established postoperatively. Moreover, high-risk FTCs are often not identifiable at the time of diagnosis. In this study, we aimed to identify transcriptional markers of malignancy and high-risk disease in follicular thyroid tumors. The expression levels of 26 potential markers of malignancy were determined in a panel of 75 follicular thyroid tumors by a TaqMan quantitative RT-PCR approach. Logistic regression analysis (LRA) was used for gene selection and generation of diagnostic and prognostic algorithms. An algorithm based on the expression levels of five genes (TERT, TFF3, PPARγ, CITED1, and EGR2) could effectively predict high-risk disease with a specificity of 98.5%. The metastatic potential could be predicted in all four cases with apparently benign or minimally invasive (MI) disease at the time of diagnosis, but poor long-term outcome. In addition, a second model was produced by implementing two genes (TERT and TFF3), which was able to distinguish adenomas from de facto carcinomas. When this model was tested in an independent series of atypical adenomas (AFTA) and MI-FTCs, 16 out of 17 AFTAs were classified as ‘benign’, while MI-FTCs with vascular invasion (sometimes referred to as ‘moderately invasive’) and/or large tumor size tended to classify in the ‘malignant’ group. The reported models can be the foundation for the development of reliable preoperative diagnostic and prognostic tests that can guide the therapeutic approach of follicular thyroid neoplasms with indeterminate cytology.

Gastrointestinal stromal tumor (GIST) is the most commonly diagnosed mesenchymal tumor in the gastrointestinal tract. This tumor type is driven by gain-of-function mutations in receptor tyrosine kinases (such as KIT, PDGFRA, and BRAF) or loss-of-function mutations in succinate dehydrogenase complex subunit genes (SDHx). Molecular studies on GIST have improved our understanding of the biology of the disease and have led to the use of targeted therapy approach, such as imatinib for KIT/PDGFRA-mutated GIST. Recently, microRNAs have emerged as important regulators of KIT expression, cancer cell behavior, and imatinib response in GIST. This chapter aims to provide an overview on current understanding of the biological roles of microRNAs in GIST and possible implications in prognosis and therapeutic response.

The Ras effector NORE1A (RASSF5A) is a putative tumor suppressor and is inactivated in several human cancers. NORE1A has not been studied in thyroid cancer.The objective of this study was to investigate whether NORE1A is involved in follicular thyroid cancer (FTC) development.We analyzed NORE1A expression in 25 FTCs, eight follicular thyroid adenomas, and seven normal thyroid tissues by TaqMan quantitative RT-PCR. The results were evaluated in relation to RASSF1A expression, RAS mutations, and PAX8-PPARgamma fusions assessed in the same material. NORE1A promoter methylation was assessed by the combined bisulfite restriction endonuclease assay.Although the NORE1A mRNA levels of the majority of the tumors were similar to those in the normal controls, the cases harboring a PAX8-PPARgamma translocation (n = 6) exhibited dramatically reduced NORE1A expression (P0.001). In contrast, RAS mutations (n = 5) and NORE1A down-regulation were mutually exclusive. A significant reduction in the expression of the NORE1A homolog and the bona fide tumor suppressor gene RASSF1A was observed, but with weak correlation to the respective NORE1A values. No NORE1A promoter methylation was detected in the 32 thyroid tumors analyzed.Our experiments demonstrate the suppression of NORE1A, a known Ras effector, in PAX8-PPARgamma carrying FTCs.

Purpose: Ezrin is a cytoskeleton linker protein that is actively involved in regulating the growth and metastatic capacity of cancer cells. It has recently been reported to be involved in dissemination of pediatric soft tissue sarcoma (STS).Experimental Design: To further evaluate the prognostic value of ezrin in STS progression, we screened 50 primary STSs of high malignancy grade using immunohistochemistry. At the initial surgery, all patients were without local or distant metastasis. The expression was then compared with the outcome during follow-up for at least 4 years or until the patients' death.Results: Twenty-five of the 50 STSs analyzed (50%) showed ezrin immunoreactivity in the membrane and cytoplasm of the tumor cells. A significant association was shown between positive expressions of ezrin and death in disease as well as overall survival (P = 0.014 and 0.007, respectively). Similarly, ezrin expression was significantly associated with development of distant metastasis during follow-up (P = 0.031), also excluding locally recurrent disease (P = 0.049). The relative abundance of metastasis in ezrin-positive cases was observed both over time and irrespective of time. In comparison with clinical, histopathologic, and genetic characteristics of the STSs, ezrin expression was found to correlate significantly with an infiltrative growth pattern outside the tumor capsule as well as with copy number gain of chromosomal region 9cen-q22.Conclusion: Our findings suggest that ezrin immunoreactivity could be valuable as an additional prognostic marker in highly malignant STSs and support a causative role of ezrin in STS tumor dissemination.

The demonstration of the PAX8-PPAR(gamma) fusion oncogene in a subset of follicular thyroid tumors provides a new and promising starting point to dissect the molecular genetic events involved in the development of this tumor form. In the present study, we compared the gene expression profiles of follicular thyroid carcinomas (FTCs) bearing a PAX8-PPAR(gamma) fusion against FTCs that lack this fusion. Using unsupervised clustering and multidimensional scaling analyses, we show that FTCs possessing a PAX8-PPAR(gamma) fusion have a highly uniform and distinct gene expression signature that clearly distinguishes them from FTCs without the fusion. The PAX8-PPAR(gamma)(+) FTCs grouped in a defined cluster, where highly ranked genes were mostly associated with signal transduction, cell growth and translation control. Notably, a large number of ribosomal protein and translation-associated genes were concurrently underexpressed in the FTCs with the fusion. Taken together, our findings further support that follicular carcinomas with a PAX8-PPAR(gamma) rearrangement constitute a distinct biological entity. The current data represent one step to elucidate the molecular pathways in the development of FTCs with the specific PAX8-PPAR(gamma) fusion.

Deregulation of microRNAs (miRNAs) contributes to the development and progression of many cancer types; however, their functions in the pathogenesis of testicular germ cell tumor (TGCT) remain unclear. Here, we determined miRNA expression profiles of TGCTs and normal testes using small RNA sequencing, and identified several deregulated miRNAs in TGCTs, including the miR-506~514 cluster. In functional studies in vitro we demonstrated that miR-514a-3p induced apoptosis through direct regulation of the paternally expressed gene 3 (PEG3), and ectopically expressed PEG3 could rescue the apoptotic effect of miR-514a-3p overexpression. Silencing of PEG3 or miR-514a-3p overexpression reduced nuclear accumulation of p50 and NF-κB reporter activity. Furthermore, PEG3 was co-immunoprecipitated with tumor necrosis factor receptor-associated factor 2 (TRAF2) in TGCT cell lysates. We propose a model of PEG3-mediated activation of NF-κB in TGCT. Loss of miR-514a-3p expression in TGCT increases PEG3 expression that recruits TRAF2 and activates the NF-kappa B pathway, which protects germ cells from apoptosis. Importantly, we observed strong expression of PEG3 and nuclear p50 in the majority of TGCTs (83% and 78%, respectively). In conclusion, our study describes a novel function for miR-514a-3p in TGCT and highlights an unrecognized mechanism of PEG3 regulation and NF-κB activation in TGCT.

Altered expression of specific microRNAs (miRNAs) has been observed in human cervical cancer. However, the biological functions of many of these miRNAs are yet to be discovered. We previously showed that miR-944 is significantly more abundant in cervical cancer tissues than their normal counterparts. In this study, we investigated the functions and targets of miR-944 in human cervical cancer cells. MiR-944 is located in the intron of the tumor protein p63 (TP63) gene, which is frequently overexpressed in cervical carcinomas. Using gain- and loss-of-function experiments in vitro, we demonstrate that miR-944 promotes cell proliferation, migration and invasion, but has no effect on apoptosis, in human cervical cancer cells. To identify the targets of miR-944, we performed photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation followed by deep sequencing. Among the candidate targets, we validated HECW2 (HECT domain ligase W2) and S100PBP (S100P binding protein) as direct targets of miR-944 using luciferase reporter assays and western blot analysis. Our findings reveal novel functions and targets of miR-944 in human cervical cancer cells, which may provide new insights of its role in cervical carcinogenesis. What's new? While miR-944 has been shown to be associated with tumor development and progression in several tumor types, its functions and targets remain undetermined. This study stands out as the first report of miR-944 functions and targets in human cancer. The authors demonstrate that miR-944 functions as an oncogene in human cervical cancer cells by promoting cell proliferation, migration, and invasion. In addition, they identified and validated HECW2 and S100PBP as direct targets of miR-944 in human cervical cancer cells. The findings provide new insights into the biological roles of miR-944 in human cervical cancer cells.

Prostate cancer cell lines have been widely used as model systems characterizing pathogenetic, functional, and therapeutic aspects of prostate cancer development. However, their chromosomal compositions are poorly characterized. In this study, five prostate cancer cell lines-TSU-Pr1, JCA-1, NCI-H660, ALVA-31, and PPC-1-were investigated by G-banding, comparative genomic hybridization (CGH), and spectral karyotyping. The results were combined with our previous findings in the prostate cancer cell lines PC-3, DU145, and LNCaP. By comparative genomic hybridization (CGH), the most frequent losses were observed at 13q, 8p, 9p, and 4q, whereas gains were most commonly seen at 8q, 10q, and 18p. The composite karyotypes were characterized by multiple numerical and structural chromosomal aberrations. Recurrent breakpoints at 5q11, 8p11, and 10q22 were observed to participate in deletion and translocation events in five of the cell lines, suggesting the importance of tumor suppressor and/or oncogenes in these regions. ALVA-31 and PPC-1 shared nine identical derivative chromosomes, two of which have also been detected in PC-3. In addition, the identification of the same homozygous deletion at D10S541 and of an identical TP53 gene mutation in all three cell lines suggests a common origin of these cell lines.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm in the gastrointestinal tract. In most GISTs, the underlying mechanism is a gain-of-function mutation in the KIT or the PDGFRA gene. Imatinib is a tyrosine kinase inhibitor that specifically blocks the intracellular ATP-binding sites of these receptors. A correlation exists between plasma levels of imatinib and progression-free survival, but it is not known whether the plasma concentration correlates with the intracellular drug concentration. We determined intracellular imatinib levels in two GIST cell lines: the imatinib-sensitive GIST882 and the imatinib-resistant GIST48. After exposing the GIST cells to imatinib, the intracellular concentrations were evaluated using LC-MS (TOF). The concentration of imatinib in clinical samples from three patients was also determined to assess the validity and reliability of the method in the clinical setting. Determination of imatinib uptake fits within detection levels and values are highly reproducible. The GIST48 cells showed significantly lower imatinib uptake compared with GIST882 in therapeutic doses, indicating a possible difference in uptake mechanisms. Furthermore, imatinib accumulated in the tumor tissues and showed intratumoral regional differences. These data show, for the first time, a feasible and reproducible technique to measure intracellular imatinib levels in experimental and clinical settings. The difference in the intracellular imatinib concentration between the cell lines and clinical samples indicates that drug transporters may contribute toward resistance mechanisms in GIST cells. This highlights the importance of further clinical studies to quantify drug transporter expression and measure intracellular imatinib levels in GIST patients.

Tumor cytogenetic analysis from 27 patients with breast cancer diagnosed at the Singapore General Hospital revealed complex karyotypic aberrations in 12 cases. The study group comprised 25 women and 2 men, ranging in age from 33 to 78 years (median 52 years). Ethnic distribution consisted of 22 Chinese, 3 Malaysian, and 2 Indian patients. Pathologic assessment disclosed 24 invasive ductal, 2 invasive mucinous, and 1 mixed invasive mucinous and ductal carcinomas. Histologic grading showed 3 grade 1, 10 grade 2, and 12 grade 3 tumors; 2 cancers were not graded, because they had been subjected to prior chemotherapy. Tumor sizes ranged from 1.5 to 10 cm (median 3 cm). Eleven cases were axillary node negative, whereas the remaining 16 node-positive cancers affected as many as 3 nodes in 8 cases and 4 or more nodes in another 8. Twenty cases demonstrated estrogen-receptor positivity, and 8 cases progesterone-receptor positivity. The spectrum of cytogenetic abnormalities involved chromosomes 1, 3, 6, 7, 8, 11, 16, and 17 and ranged from gains and deletions of both long and short arms, trisomy, monosomy, and other rearrangements. There was a trend toward the presence of karyotypic abnormalities in tumors of higher grade.

Alterations of 3p are the most frequently observed changes in follicular thyroid carcinomas. Loss of 3p25-pter has been speculated to be a critical event in the malignant transformation of a subset of thyroid follicular neoplasms. The present report describes a minimally invasive follicular thyroid carcinoma (FTC) with a balanced t(3;7)(p25;q34) and dic(15;22)(p11;p11) as the only abnormalities. The alterations were present in all metaphases analyzed and were demonstrated by G-banding, spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH). This study represents the second case of FTC where 3p25 is involved in a balanced translocation. The findings support the existence of a gene locus in this region which is involved in the tumorigenesis of thyroid carcinoma.

MicroRNAs (miRNAs) are short non-coding RNA regulators that control gene expression mainly through post-transcriptional silencing. We previously identified miR-205 in a signature for human cervical cancer using a deep sequencing approach. In this study, we confirmed that miR-205 expression was frequently higher in human cervical cancer than their matched normal tissue samples. Functionally, we demonstrate that miR-205 promotes cell proliferation and migration in human cervical cancer cells. To further understand the biological roles of miR-205, we performed in vivo crosslinking and Argonaute 2 immunoprecipitation of miRNA ribonucleoprotein complexes followed by microarray analysis (CLIP-Chip) to identify its potential mRNA targets. Applying CLIP-Chip on gain- and loss-of-function experiments, we identified a set of transcripts as potential targets of miR-205. Several targets are functionally involved in cellular proliferation and migration. Two of them, CYR61 and CTGF, were further validated by Western blot analysis and quantification of mRNA enrichment in the Ago2 immunoprecipitates using qRT-PCR. Furthermore, both CYR61 and CTGF were downregulated in cervical cancer tissues. In summary, our findings reveal novel functional roles and targets of miR-205 in human cervical cancer, which may provide new insights about its role in cervical carcinogenesis and its potential value for clinical diagnosis.

Adrenocortical carcinoma (ACC) is an aggressive tumor showing frequent metastatic spread and poor survival. Although recent genome-wide studies of ACC have contributed to our understanding of the disease, major challenges remain for both diagnostic and prognostic assessments. The aim of this study was to identify specific microRNAs (miRNAs) associated with malignancy and survival of ACC patients. miRNA expression profiles were determined in a series of ACC, adenoma, and normal cortices using microarray. A subset of miRNAs showed distinct expression patterns in the ACC compared with adrenal cortices and adenomas. Among others, miR-483-3p, miR-483-5p, miR-210, and miR-21 were found overexpressed, while miR-195, miR-497, and miR-1974 were underexpressed in ACC. Inhibition of miR-483-3p or miR-483-5p and overexpression of miR-195 or miR-497 reduced cell proliferation in human NCI-H295R ACC cells. In addition, downregulation of miR-483-3p, but not miR-483-5p, and increased expression of miR-195 or miR-497 led to significant induction of cell death. Protein expression of p53 upregulated modulator of apoptosis (PUMA), a potential target of miR-483-3p, was significantly decreased in ACC, and inversely correlated with miR-483-3p expression. In addition, high expression of miR-503, miR-1202, and miR-1275 were found significantly associated with shorter overall survival among patients with ACC (P values: 0.006, 0.005, and 0.042 respectively). In summary, we identified additional miRNAs associated with ACC, elucidated the functional role of four miRNAs in the pathogenesis of ACC cells, demonstrated the potential involvement of the pro-apoptotic factor PUMA (a miR-483-3p target) in adrenocortical tumors, and found novel miRNAs associated with survival in ACC.

Colorectal cancer (CRC) is one of the most common and deadly forms of cancer. Despite improved treatment modalities, post-operative recurrence and metastasis remain the major problems for extending patient survival after surgery. This highlights the need to search for biomarkers for prognostication and treatment stratification of colorectal cancer patients. In this study, we applied the SYBR-green quantitative PCR-based array approach to screen for differentially expressed miRNAs between patients with short (

Archived formalin-fixed, paraffin-embedded human tumors are widely available and represent a unique source of morphologically defined material. Formalin-fixed, paraffin-embedded tissue is known to contain a wealth of molecular information in the form of microRNAs (miRNAs), which could be correlated with clinical outcome for improved prognostication and/or treatment response. miRNAs are endogenous, noncoding RNAs ( approximately 22 nucleotides) and may function as tumor suppressors or oncogenes. A reliable, robust methodology is needed to take full advantage of archived human cancers, especially for those where fresh-frozen tumor banks are unavailable, for example, malignant melanoma. To this end, we applied a simple-to-use protocol for extracting total RNA from various formalin-fixed, paraffin-embedded specimens (colon, liver, prostate, thyroid, uterus, and skin), optimized for small RNA recovery. Using a "poison primer" strategy (ie, primer silencing), we blocked the amplification of ribosomal RNA, enabling the successful sequencing of 17 novel and 53 known miRNAs (including small RNAs) from 10-year-old archived normal skin, cutaneous scalp melanoma, and sentinel lymph nodes (both negative and positive for metastasis) excised from a 52-year-old man. The cloning incidence provided an estimation of the level of specific miRNA expression, which was confirmed by Northern analysis and quantitative real-time polymerase chain reaction. This methodology can therefore be used to facilitate miRNA discovery from archived human cancers.

MicroRNAs (miRNAs) are approximately 22 nucleotide-long noncoding RNAs involved in several biological processes including development, differentiation and proliferation. Recent studies suggest that knowledge of miRNA expression patterns in cancer may have substantial value for diagnostic and prognostic determinations as well as for eventual therapeutic intervention. We performed comprehensive analysis of miRNA expression profiles of 27 sarcomas, 5 normal smooth muscle and 2 normal skeletal muscle tissues using microarray technology and/or small RNA cloning approaches. The miRNA expression profiles are distinct among the tumor types as demonstrated by an unsupervised hierarchical clustering, and unique miRNA expression signatures were identified in each tumor class. Remarkably, the miRNA expression patterns suggested that two of the sarcomas had been misdiagnosed and this was confirmed by reevaluation of the tumors using histopathologic and molecular analyses. Using the cloning approach, we also identified 31 novel miRNAs or other small RNA effectors in the sarcomas and normal skeletal muscle tissues examined. Our data show that different histological types of sarcoma have distinct miRNA expression patterns, reflecting the apparent lineage and differentiation status of the tumors. The identification of unique miRNA signatures in each tumor type may indicate their role in tumorigenesis and may aid in diagnosis of soft tissue sarcomas.

Recent studies suggest that knowledge of differential expression of microRNAs (miRNA) in cancer may have substantial diagnostic and prognostic value. Here, we use a direct sequencing method to characterize the profiles of miRNAs and other small RNA segments for six human cervical carcinoma cell lines and five normal cervical samples. Of 166 miRNAs expressed in normal cervix and cancer cell lines, we observed significant expression variation of six miRNAs between the two groups. To further show the biological relevance of our findings, we examined the expression level of two significantly varying miRNAs in a panel of 29 matched pairs of human cervical cancer and normal cervical samples. Reduced expression of miR-143 and increased expression of miR-21 were reproducibly displayed in cancer samples, suggesting the potential value of these miRNAs as tumor markers. In addition to the known miRNAs, we found a number of novel miRNAs and an additional set of small RNAs that do not meet miRNA criteria. [Cancer Res 2007;67(13):6031–43]

In this study we present two novel anaplastic thyroid carcinoma (ATC) lines (HTh 104 and HTh 112) and further characterize six frequently used ATC lines (HTh 7, HTh 74, HTh 83, C 643, KAT-4, and SW 1736). Three of the lines carried a heterozygous BRAF mutation V600E, which is in line with reports of BRAF mutations in primary ATC and papillary thyroid cancer. Several nonrandom breakpoints were identified by spectral karyotyping (SKY) and G-banding in these lines including the novel 1p36 and 17q24-25 as well as 3p21-22 and 15q26 that are also implicated in well-differentiated thyroid cancers. Comparative genomic hybridization showed frequent gain of 20q, including the UBCH10 gene in 20q13.12, which was further confirmed by array-comparative genomic hybridization and fluorescence in situ hybridization analyses. Our results concur with previous studies in both primary tumors and cell lines, indicating that gain of chromosome 20 is important in the pathogenesis of ATC and/or progression of differentiated thyroid cancers to ATC.

A case of Waldenstrom macroglobulinemia with Burkitt-type t(8;14)(q24.1;q32) and total and partial trisomy 3 is reported. This is an unusual combination of chromosomal abnormalities in Waldenstrom macroglobulinemia.

Multiple endocrine neoplasia (MEN) is defined as concurrent neoplasia or hyperplasia in more than one endocrine gland. MEN is well known in humans and has also been reported in small animals. We report on a dog family of a mixed breed with Alaskan malamute as a major influence, where three members developed thyroid carcinomas and another dog had clinical signs mimicking the other three but without a confirmed diagnosis. The age of onset of the tumour was between 96-109 months. Clinical, biochemical and immunohistochemical examinations revealed that the affected individuals typically demonstrated symptoms including calcitonin positive thyroid cancer, hypothyroidism and chronic dermatitis. In addition, elevated serum calcium and multinodular adrenocortical hyperplasia were demonstrated in a single member. The diagnosis observed is similar to the familial form of medullary thyroid carcinoma (FMTC) in human. This is the first report of FMTC in dog. Up to 95% of FMTC and MEN2 is known to be caused by activating mutations in the RET gene. The dog Ret gene was analysed as a candidate in this pedigree. The complete dog Ret genomic sequence was predicted in silico. The lack of demonstratable Ret mutation suggests the involvement of alternative predisposing mutation in this pedigree. The unique occurrence of familial MTC makes this potentially an important model in further defining the genetic basis of MTC.

Soft tissue sarcomas (STSs) arising in the abdominal cavity constitute a group of aggressive tumours, typically of very large size and with a high recurrence rate in the affected patients. While some distinct genetic etiologies have been described, the genetic background of this tumour group is not well characterised. Here we have assessed gross chromosomal alterations in a series of such tumours obtained from 26 patients. CGH alterations were found in tumours from 23 of the patients (88%), the most frequent being loss of 13q21 (46%) and gain of 17p and/or q (46%). Furthermore, mutations of C-KIT exon 11 were demonstrated in five tumours from four patients, and the two myxoid liposarcomas exhibited a translocation t(12;16)(q13;p11). From the pattern of chromosomal alterations detected, a genetic progression of events was clearly evident in the tumours. Taken together with analysis of subsequent relapses from the same patients, the common CGH alteration +12q13 was suggested to be a relatively early event in the genetic progression, similar to t(12;16)(q13;p11) and C-KIT mutations. Moreover, -1p21-22, -13q21, -14q, -Xp22, +9q34, +17p, +17q, and +20q13 would all represent relative later events. The most consistent alteration was loss of 13q, that was found to target the 13q14-21 and 13q34 regions as determined by CGH and Southern blot analyses. Loss of 13q was identified independently of +12q13 and C-KIT mutation and the patient's sex, and was observed in all common subtypes of STS, suggesting that it is a general and late event in the genetic progression. The findings provide a starting point for further dissection of the target genes involved in development of STSs in the abdominal cavity.

By positional cloning, we identified two breakpoint-spanning genes in a familial clear cell renal cell carcinoma (CCRCC)-associated t(1;3)(q32.1;q13.3): LSAMP and NORE1 (RASSF1 homolog). Both genes are downregulated in 9 of 9 RCC cell lines. While the NORE1A promoter predominantly presents partial methylation in 6 of the cell lines and 17/53 (32%) primary tumors, the LSAMP promoter is completely methylated in 5 of 9 cell lines and in 14/53 (26%) sporadic and 4 familial CCRCCs. Expression of LSAMP and NORE1A proteins in CCRCC cell lines inhibited cell proliferation. These characteristics indicate that LSAMP and NORE1A may represent new candidate tumor suppressors for CCRCC.

In this study, we characterized the chromosomal composition of an intra-abdominal soft tissue sarcoma diagnosed as a malignant fibrous histiocytoma (MFH). By applying a combination of spectral karyotyping, G-banding, and comparative genomic hybridization (CGH), this case was shown to carry large chromosome markers with material mainly from chromosomes 6 and 8. Further characterization of this unique tumor revealed high-level amplifications at the 6q21 approximately q23, 8p21 approximately pter, 8q24 approximately qter, and 12q13 approximately q21 regions. Using array CGH, these amplified regions were found to include MASL1 in 8p, as well s MDM2 and CDK4 in 12q, which have been shown to be amplified in MFH. Similarly, gains of 6q and 8q have also been seen in MFH. In conclusion, our study demonstrates the occurrence of large chromosome markers in MFH and suggests that the regions 6q21 approximately q23, 8p21 approximately pter, 8q24 approximately qter, and 12q13 approximately q21 might harbor oncogenes that could play a role in MFH's tumorigenesis. In addition, gain of 12q13 approximately q21, which is typical of well-differentiated liposarcoma, may also occur in MFH, supporting the previously suggested overlap in genetic etiologies between these two tumor types.

Hodgkin lymphoma (HL) is a malignant lymphoma composed of a minority of neoplastic cells—the Hodgkin and Reed–Sternberg (HRS) cells—and a majority of nonneoplastic inflammatory cells. The low proportion of tumor cells makes genetic studies of primary neoplasia difficult. Therefore, established HL-derived cell lines are commonly used as model systems. Here we have characterized the chromosomal composition of four such cell lines: L-540, DEV, HDLM-2, and CO. Using spectral karyotyping (SKY), reversed DAPI banding, and comparative genomic hybridization (CGH), the karyotypes were characterized and previously unidentified marker chromosomes were resolved. The karyotype for CO was incompatible with the original description but showed striking similarities with the T-ALL-derived cell line CCRF-CEM, suggesting that CO had been cross-contaminated and overgrown prior to arrival at our laboratory. Multiple numerical and structural abnormalities were identified in DEV and HDLM-2, as well as in L-540. Refined composed karyotypes are suggested for the cell lines studied, to be used as references for further studies of lymphoma.

The goals of this study were to evaluate the potential of detecting cryptic amplification and deletion of cancer-related genes using array-based comparative genomic hybridization (CGH), and to identify candidate cancer genes by combined parallel analyses of copy number and gene expression profiles in nasopharyngeal carcinoma (NPC) cell lines. We established global DNA copy number and mRNA expression profiles on human NPC cell lines using a high-density cDNA microarray. The DNA copy number alterations detected by array CGH were compared to the DNA copy number variations identified by metaphase CGH. A cryptic amplification at 3q26 was detected by array CGH, which was not found by metaphase CGH. By amplicon mapping and parallel analyses of DNA copy number and mRNA expression levels, we identified several candidates which could be important mediators in tumor formation or progression. Taken together, the combination of copy number and gene expression profiling using cDNA microarrays provides an improved strategy for gene discovery in human cancer.

By using comparative genomic hybridization (CGH), we characterized the genetic profiles of 36 VHL-related pheochromocytomas. We then compared the results with those of sporadic and MEN 2-related pheochromocytomas. In 36 VHL-related tumors, loss of chromosome 3 and chromosome 11 were found in 34 tumors (94%) and 31 tumors (86%), respectively. There was significant concordance of deletions in chromosomes 3 and 11 (Kappa=0.64, P=0.0095), suggesting that they are involved in two different but necessary and complementary genetic pathways. The loss of chromosome 11 appeared to be specific for VHL-related pheochromocytoma as it was not present in any of the 10 VHL-related CNS hemangioblastomas studied and was significantly less common when compared with (a) sporadic pheochromocytomas from previously published results (13%; P=

The chromosomal translocation t(X;18)(p11.2;q11.2) is tightly linked to the tumorigenesis of synovial sarcoma. Through this translation the SYT gene on chromosome 18 is fused with a testis/cancer antigen gene on the X chromosome, generating either a SYT-SSX1, SYT-SSX2, or less often a SYT-SSX4 fusion gene. It has been anticipated that the individual synovial sarcoma carries only one of these variants, however, in this study we demonstrated that SYT-SSX1 and SYT-SSX2 co-exist in a significant proportion of the cases. From 121 SYT-SSX positive primary tumors, co-expression of SYT-SSX1 and SYT-SSX2 was seen in 12 cases (10%), which were characterized in further detail both at the RNA, DNA and chromosomal level. In all 12 cases the SYT-SSX1 and SYT-SSX2 fusions resulted in identical SYT-SSX fusion transcripts. However, at the genomic level the translocations were different, and most likely occurred between variable intronic sites in the target genes. By interphase FISH analyses of 10 cases SYT-SSX2 translocations were found to be the most abundant in all but one of the cases, in which SYT-SSX1 was predominating. The findings reveal a new heterogenous feature of synovial sarcoma, accounting for approximately 10% of all cases, which may shed light on the molecular genetic mechanisms behind translocations in general, and on the etiology of synovial sarcoma in particular.

Small cell lung cancer (SCLC) is a frequently occurring, highly aggressive tumor with a generally poor clinical outcome. In order to approach the genetic mechanisms behind the tumor progression, a general screen for DNA copy number alterations was performed using comparative genomic hybridization (CGH). In the series of 23 cases analyzed, CGH alterations were frequently detected ranging from 9 to 22 abnormalities in the individual tumors. The most frequent losses were detected on chromosome arms 3p (23/23), 13q14-21 (23/23), 4p (20/23), 4q (20/23), and 2q22-24 (18/23), while gains preferentially involved chromosome arms 19p (18/23), 19q (17/23), 1p31-35 (15/23), 17q22-25 (11/23), and 5p14-15.3 (9/23). In addition, high level amplification at chromosome arms 1p32-33 and 2p22-24 were found in three and two cases, respectively. Candidate genes for these amplifications include the l-MYC (1p32) and n-MYC (2p24.1) oncogenes, which have been previously found to be overexpressed in SCLC. Taken together, the findings demonstrate a high level of chromosomal instability in SCLC, which is well in agreement with the highly malignant phenotype of this tumor type. Subchromosomal regions involved in gains and losses were delineated and the amplifications of 1p32-33 and 2p22-24 were demonstrated, thus providing starting points for the exact characterization of molecular events involved in SCLC tumor progression.

Soft tissue sarcomas constitute a heterogeneous group of malignant tumors of mesenchymal origin, the classification of which may present a diagnostic challenge. We present here the cytological, histopathological, immunohistochemical, and cytogenetic findings of an unusual case of a highly aggressive sarcoma. Based on the morphology and the immunohistochemical profile, this primitive tumor and its metastases could not be conclusively classified as any of the defined subtypes of sarcomas, although the findings were suggestive of a variant of rhabdomyosarcoma. Cytogenetic characterization using G-banding, SKY, FISH, and CGH revealed almost identical chromosomal compositions of the primary tumor and the metastasis. The hypertetraploid karyotype was characterized by numerical imbalances as well as by an unbalanced translocation t(1;19)(q12;q13.2), which has not been previously reported.