[The role of SF3B1 mutations in EVI1-rearranged myeloid neoplasms].

In acute myeloid leukemia (AML), EVI1 rearrangement represented by inv(3)(q21q26) or t(3;3)(q21;q26) causes EVI1 overexpression via structural rearrangement of an enhancer, and confers poor prognosis. My colleagues and I performed a mutational analysis of EVI1-rearranged myeloid neoplasms and identified SF3B1, a core RNA splicing factor, as the most commonly co-mutated gene. Indeed, latent leukemia development in transgenic mice bearing the humanized inv(3)(q21q26) allele was significantly accelerated by co-occurrence of Sf3b1 mutation. Intriguingly, we found that this SF3B1 mutant induced mis-splicing of EVI1 itself, which generated an aberrant EVI1 isoform with in-frame insertion of 6 amino acids near the DNA-binding domain of EVI1. This aberrant EVI1 isoform exhibited DNA-binding activity different from wild-type EVI1 and significantly enhanced the self-renewal capacity of murine hematopoietic stem cells. We also identified the cryptic branch point and exonic splicing enhancer required for this EVI1 mis-splicing induced by the SF3B1 mutant. These data provide a basis for further elucidation of the molecular mechanism and potential therapeutic candidates for EVI1-rearranged AML.

Aberrant EVI1 splicing contributes to EVI1-rearranged leukemia.

Detailed genomic and epigenomic analyses of MECOM (the MDS1 and EVI1 complex locus) have revealed that inversion or translocation of chromosome 3 drives inv(3)/t(3;3) myeloid leukemias via structural rearrangement of an enhancer that upregulates transcription of EVI1. Here, we identify a novel, previously unannotated oncogenic RNA-splicing derived isoform of EVI1 that is frequently present in inv(3)/t(3;3) acute myeloid leukemia (AML) and directly contributes to leukemic transformation. This EVI1 isoform is generated by oncogenic mutations in the core RNA splicing factor SF3B1, which is mutated in >30% of inv(3)/t(3;3) myeloid neoplasm patients and thereby represents the single most commonly cooccurring genomic alteration in inv(3)/t(3;3) patients. SF3B1 mutations are statistically uniquely enriched in inv(3)/t(3;3) myeloid neoplasm patients and patient-derived cell lines compared with other forms of AML and promote mis-splicing of EVI1 generating an in-frame insertion of 6 amino acids at the 3' end of the second zinc finger domain of EVI1. Expression of this EVI1 splice variant enhanced the self-renewal of hematopoietic stem cells, and introduction of mutant SF3B1 in mice bearing the humanized inv(3)(q21q26) allele resulted in generation of this novel EVI1 isoform in mice and hastened leukemogenesis in vivo. The mutant SF3B1 spliceosome depends upon an exonic splicing enhancer within EVI1 exon 13 to promote usage of a cryptic branch point and aberrant 3' splice site within intron 12 resulting in the generation of this isoform. These data provide a mechanistic basis for the frequent cooccurrence of SF3B1 mutations as well as new insights into the pathogenesis of myeloid leukemias harboring inv(3)/t(3;3).

CRISPRi links COVID-19 GWAS loci to LZTFL1 and RAVER1.

BACKGROUND: To identify host genetic variants (SNPs) associated with COVID-19 disease severity, a number of genome-wide association studies (GWAS) have been conducted. Since most of the identified variants are located at non-coding regions, such variants are presumed to affect the expression of neighbouring genes, thereby influencing COVID-19 disease severity. However, it remains largely unknown which genes are influenced by such COVID-19 GWAS loci. METHODS: CRISPRi (interference)-mediated gene expression analysis was performed to identify genes functionally regulated by COVID-19 GWAS loci by targeting regions near the loci (SNPs) in lung epithelial cell lines. The expression of CRISPRi-identified genes was investigated using COVID-19-contracted human and monkey lung single-nucleus/cell (sn/sc) RNA-seq datasets. FINDINGS: CRISPRi analysis indicated that a region near rs11385942 at chromosome 3p21.31 (locus of highest significance with COVID-19 disease severity at intron 5 of LZTFL1) significantly affected the expression of LZTFL1 (P<0.05), an airway cilia regulator. A region near rs74956615 at chromosome 19p13.2 (locus located at the 3' untranslated exonic region of RAVER1), which is associated with critical illness in COVID-19, affected the expression of RAVER1 (P<0.05), a coactivator of MDA5 (IFIH1), which induces antiviral response genes, including ICAM1. The sn/scRNA-seq datasets indicated that the MDA5/RAVER1-ICAM1 pathway was activated in lung epithelial cells of COVID-19-resistant monkeys but not those of COVID-19-succumbed humans. INTERPRETATION: Patients with risk alleles of rs11385942 and rs74956615 may be susceptible to critical illness in COVID-19 in part through weakened airway viral clearance via LZTFL1-mediated ciliogenesis and diminished antiviral immune response via the MDA5/RAVER1 pathway, respectively. FUNDING: NIH.

Human chromosome 3p21.3 carries TERT transcriptional regulators in pancreatic cancer.

Frequent loss of heterozygosity (LOH) on the short arm of human chromosome 3 (3p) region has been found in pancreatic cancer (PC), which suggests the likely presence of tumor suppressor genes in this region. However, the functional significance of LOH in this region in the development of PC has not been clearly defined. The human telomerase reverse transcriptase gene (hTERT) contributes to unlimited proliferative and tumorigenicity of malignant tumors. We previously demonstrated that hTERT expression was suppressed by the introduction of human chromosome 3 in several cancer cell lines. To examine the functional role of putative TERT suppressor genes on chromosome 3 in PC, we introduced an intact human chromosome 3 into the human PK9 and murine LTPA PC cell lines using microcell-mediated chromosome transfer. PK9 microcell hybrids with an introduced human chromosome 3 showed significant morphological changes and rapid growth arrest. Intriguingly, microcell hybrid clones of LTPA cells with an introduced human chromosome 3 (LTPA#3) showed suppression of mTert transcription, cell proliferation, and invasion compared with LTPA#4 cells containing human chromosome 4 and parental LTPA cells. Additionally, the promoter activity of mTert was downregulated in LTPA#3. Furthermore, we confirmed that TERT regulatory gene(s) are present in the 3p21.3 region by transfer of truncated chromosomes at arbitrary regions. These results provide important information on the functional significance of the LOH at 3p for development and progression of PC.

Inherited unbalanced reciprocal translocation with 3q duplication and 5p deletion in a foetus revealed by cell-free foetal DNA (cffDNA) testing: a case report.

BACKGROUND: Since 2011, screening maternal blood for cell-free foetal DNA (cffDNA) fragments has offered a robust clinical tool to classify pregnancy as low or high-risk for Down, Edwards, and Patau syndromes. With recent advances in molecular biology and improvements in data analysis algorithms, the screening's scope of analysis continues to expand. Indeed, screening now encompassess additional conditions, including aneuploidies for sex chromosomes, microdeletions and microduplications, rare autosomal trisomies, and, more recently, segmental deletions and duplications called copy number variations (CNVs). Yet, the ability to detect CNVs creates a new challenge for cffDNA analysis in couples in which one member carries a structural rearrangement such as a translocation or inversion. CASE PRESENTATION: We report a segmental duplication of the long arm of chromosome 3 and a segmental deletion of the short arm of chromosome 5 detected by cffDNA analysis in a 25-year-old pregnant woman. The blood sample was sequenced on a NextSeq 550 (Illumina) using the VeriSeq NIPT Solution v1 assay. G-band karyotyping in amniotic fluid only detected an abnormality in chromosome 5. Next-generation sequencing in amniocytes confirmed both abnormalities and identified breakpoints in 3q26.32q29 and 5p13.3p15. The foetus died at 21 weeks of gestation due to multiple abnormalities, and later G-band karyotyping in the parents revealed that the father was a carrier of a balanced reciprocal translocation [46,XY,t(3;5)(q26.2;p13)]. Maternal karyotype appeared normal. CONCLUSION: This case provides evidence that extended cffDNA can detect, in addition to aneuploidies for whole chromosomes, large segmental aneuploidies. In some cases, this may indicate the presence of chromosomal rearrangements in a parent. Such abnormalities are outside the scope of standard cffDNA analysis targeting chromosomes 13, 18, 21, X, and Y, potentially leading to undiagnosed congenital conditions.

Exploring the role of BAFF as biomarker in the detection of uveal melanoma metastases.

PURPOSE: While B-cell activating factor (BAFF) was identified to promote the invasion in other malignancies, its role in the progression of uveal melanoma (UM) still remains unexplored. Here, we analysed the serum level of BAFF in UM patients with regard to its significance as biomarker for the metastases. METHODS: In this retrospective study, serum BAFF levels in 173 UM patients (36 with metastases and 137 without), and 23 healthy controls were measured with a multiplexed sandwich ELISA system and then correlated with clinicopathological characteristics such as primary tumor size, tumor location, histological cell type, sex, cancer stage, cytogenetic alterations of chromosome 3, and the metastatic burden. Immunohistochemical staining of 50 UM tissue specimens was also performed to evaluate the expression of BAFF and its receptors BAFF-R and TACI. RESULTS: The metastatic patients were identified to have significantly higher serum BAFF levels (mean ± SD, 1520.8 ± 1182.1 pg/ml) than those without metastases (950.4 ± 494.6 pg/ml) and controls (810.3 ± 140.5 pg/ml). While no distinctions were detected with regard to tumor location, histological cell type, gender, and monosomy 3, the patients in cancer stages II, III, and IV displayed higher serum BAFF levels than those in stage I. The serum BAFF level was significantly correlated with the metastatic burden. The serum BAFF level of 1120 pg/ml was identified to have the best performance for distinguishing the metastatic patients from non-metastatic patients. In the kinetic study, we noticed that 20.8% of the analysed patients already demonstrated elevated serum BAFF concentrations before the clinical diagnosis of metastases. Positive BAFF staining was detected in the cytoplasm of single tumor cells (in 13 specimens), macrophages (in 12 specimens), and tumor-infiltrating lymphocytes (TILs) (in 13 specimens). The expressions of BAFF-R and TACI were also observed in 17 and 36 of the 50 tested UM specimens, respectively. CONCLUSIONS: Our study first suggests that BAFF might be a promising serum marker for the detection of UM metastases.

A Scalable Computational Approach for Simulating Complexes of Multiple Chromosomes.

Significant efforts have been recently made to obtain the three-dimensional (3D) structure of the genome with the goal of understanding how structures may affect gene regulation and expression. Chromosome conformational capture techniques such as Hi-C, have been key in uncovering the quantitative information needed to determine chromatin organization. Complementing these experimental tools, co-polymers theoretical methods are necessary to determine the ensemble of three-dimensional structures associated to the experimental data provided by Hi-C maps. Going beyond just structural information, these theoretical advances also start to provide an understanding of the underlying mechanisms governing genome assembly and function. Recent theoretical work, however, has been focused on single chromosome structures, missing the fact that, in the full nucleus, interactions between chromosomes play a central role in their organization. To overcome this limitation, MiChroM (Minimal Chromatin Model) has been modified to become capable of performing these multi-chromosome simulations. It has been upgraded into a fast and scalable software version, which is able to perform chromosome simulations using GPUs via OpenMM Python API, called Open-MiChroM. To validate the efficiency of this new version, analyses for GM12878 individual autosomes were performed and compared to earlier studies. This validation was followed by multi-chain simulations including the four largest human chromosomes (C1-C4). These simulations demonstrated the full power of this new approach. Comparison to Hi-C data shows that these multiple chromosome interactions are essential for a more accurate agreement with experimental results. Without any changes to the original MiChroM potential, it is now possible to predict experimentally observed inter-chromosome contacts. This scalability of Open-MiChroM allow for more audacious investigations, looking at interactions of multiple chains as well as moving towards higher resolution chromosomes models.

Characterization of inv(3) cell line OCI-AML-20 with stroma-dependent CD34 expression.

Acute myeloid leukemia (AML) is a complex, heterogeneous disease with variable outcomes following curative intent chemotherapy. AML with inv(3) is a genetic subgroup characterized by a very low response rate to current induction type chemotherapy and thus has among the worst long-term survivorship of the AMLs. Here, we describe OCI-AML-20, a new AML cell line with inv(3) and deletion of chromosome 7; the latter is a common co-occurrence in inv(3) AML. In OCI-AML-20, CD34 expression is maintained and required for repopulation in vitro and in vivo. CD34 expression in OCI-AML-20 shows dependence on the co-culture with stromal cells. Transcriptome analysis indicates that the OCI-AML-20 clusters with other AML patient data sets that have poor prognosis, as well as other AML cell lines, including another inv(3) line, MUTZ-3. OCI-AML-20 is a new cell line resource for studying the biology of inv(3) AML that can be used to identify potential therapies for this poor outcome disease.

The 3p14.2 tumour suppressor ADAMTS9 is inactivated by promoter CpG methylation and inhibits tumour cell growth in breast cancer.

Chromosome region 3p12-14 is an important tumour suppressor gene (TSG) locus for multiple cancers. ADAMTS9, a member of the metalloprotease large family, has been identified as a candidate 3p14.2 TSG inactivated by aberrant promoter CpG methylation in several carcinomas, but little known about its expression and function in breast cancer. In this report, ADAMTS9 expression and methylation was analysed in breast cancer cell lines and tissue samples. ADAMTS9 RNA was significantly down-regulated in breast cancer cell lines (6/8). After treating the cells with demethylation agent Aza and TSA, ADAMTS9 expression was dramatically increased. Bisulphite genomic sequencing and methylation-specific PCR detected promoter methylation, which was associated with decreased ADAMTS9 expression. Hypermethylation was also detected in 130/219 (59.4%) of primary tumours but only in 4.5% (2/44) of paired surgical margin tissues. Ectopic expression of ADAMTS9 in tumor cells induced significant growth suppression, cell cycle arrest at the G0/G1 phase, enhanced apoptosis and reduced cell migration and invasion. Conditioned culture medium from ADAMTS9-transfected BT549 cells markedly disrupted tube formation ability of human umbilical vein endothelial cell (HUVEC) in Matrigel. Furthermore, ADAMTS9 inhibited AKT signaling and its downstream targets (MDM2, p53, p21, p27, E-cadherin, VIM, SNAIL, VEGFA, NFκB-p65 and MMP2). In addition, we demonstrated, for the first time, that ADAMTS9 inhibits AKT signaling, through suppressing its upstream activators EGFR and TGFß1/TßR(I/II) in breast cancer cells. Our results suggest that ADAMTS9 is a TSG epigenetically inactivated in breast cancer, which functions through blocking EGFR- and TGFß1/TßR(I/II)-activated AKT signaling.

LncRNA SOX2-OT is a novel prognostic biomarker for osteosarcoma patients and regulates osteosarcoma cells proliferation and motility through modulating SOX2.

Long noncoding RNA (LncRNA) SOX2 overlapping transcript (SOX2-OT) has been shown to serve an oncogenic role in human lung cancer, hepatocellular carcinoma, and gastric cancer. However, the clinical significance and biological function of lncRNA SOX2-OT in osteosarcoma are still unclear. LncRNA SOX2-OT expression was measured in osteosarcoma tissues and cell lines. Loss-of-function and gain-of-function studies were performed to observe the effects of lncRNA SOX2-OT on osteosarcoma cells proliferation, migration, invasion, and expressions of cancer stem cell biomarker. The relationship between lncRNA SOX2-OT and SOX2 was analyzed in osteosarcoma tissues and cells. Rescued-function studies were conducted to confirm the role of SOX2 in the regulation of lncRNA SOX2-OT in osteosarcoma cells migration, invasion, and expression of cancer stem cell biomarkers. In our results, lncRNA SOX2-OT expression was increased in osteosarcoma tissues and cell lines, and associated with malignant status and overall survival in osteosarcoma patients. LncRNA SOX2-OT regulated osteosarcoma cells proliferation, migration, invasion, and expression of cancer stem cell biomarkers. LncRNA SOX2-OT positively regulated SOX2 expression in osteosarcoma cells and positively associated with SOX2 expression in osteosarcoma tissues. The rescued-function studies suggested that SOX2 is necessary for lncRNA SOX2-OT induced osteosarcoma cells migration, invasion, and expression of cancer stem cell biomarkers. In conclusion, lncRNA SOX2-OT is a prognostic biomarker for osteosarcoma patients and serves an oncogenic role to regulate osteosarcoma cells migration, invasion, and expression of cancer stem cell biomarkers. © 2017 IUBMB Life, 69(11):867-876, 2017.

An Update on P2Y13 Receptor Signalling and Function.

The distribution of nucleotide P2Y receptors across different tissues suggests that they fulfil key roles in a number of physiological and pathological conditions. P2Y13 is one of the latest P2Y receptors identified, a novel member of the Gi-coupled P2Y receptor subfamily that responds to ADP, together with P2Y12 and P2Y14. Pharmacological studies drew attention to this new ADP receptor, with a pharmacology that overlaps that of P2Y12 receptors but with unique features and roles. The P2RY12-14 genes all reside on human chromosome 3 at 3q25.1 and their strong sequence homology supports their evolutionary origin through gene duplication. Polymorphisms of P2Y13 receptors have been reported in different human populations, yet their consequences remain unknown. The P2Y13 receptor is versatile in its signalling, extending beyond the canonical signalling of a Gi-coupled receptor. Not only can it couple to different G proteins (Gs/Gq) but the P2Y13 receptor can also trigger several intracellular pathways related to the activation of MAPKs (mitogen-activated protein kinases) and the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3 axis. Moreover, the availability of P2Y13 receptor knockout mice has highlighted the specific functions in which it is involved, mainly in the regulation of cholesterol and glucose metabolism, bone homeostasis and aspects of central nervous system function like pain transmission and neuroprotection. This review summarizes our current understanding of this elusive receptor, not only at the pharmacological and molecular level but also, in terms of its signalling properties and specific functions, helping to clarify the involvement of P2Y13 receptors in pathological situations.

Effect of 3q oncogenes SEC62 and SOX2 on lymphatic metastasis and clinical outcome of head and neck squamous cell carcinomas.

Chromosome 3q26 amplification represents a frequent alteration in head and neck squamous cell carcinomas (HNSCCs). Overexpression of 3q26 encoded genes SEC62 and SOX2 was detected in various cancers, including HNSCCs, indicating their potential function as oncogenes. In our study, we elucidated the function of SEC62 and SOX2 in HNSCC patients, with a main focus on their effect on lymphatic metastasis and patient survival. We analyzed SEC62 and SOX2 expression in tissue specimens from 65 HNSCC patients and 29 patients with cervical cancer of unknown primary (CUP); a higher SEC62 and lower SOX2 expression was observed in the lymph node metastases from HNSCC patients compared with the respective primary tumor. Lymph node metastases from CUP patients showed higher SEC62 and lower SOX2 expression compared with lymph node metastases from HNSCC patients. When proceeding from the N1 to the N3 stage, SEC62 expression in the lymph node metastases showed an increase and SOX2 expression showed a decrease. Moreover, both genes showed a highly significant relevance as prognostic biomarkers, with the worst prognosis for patients with high SEC62 and low SOX2 expression levels. In functional analyses, knockdown of SEC62 resulted in an inhibition of HNSCC cell migration while, conversely, SEC62 and SOX2 overexpression stimulated cell migration. Taken together, our study showed that the expression of the 3q oncogenes SEC62 and SOX2 affects lymphatic metastasis and cell migration in HNSCC and CUP patients and has a high prognostic relevance in these diseases.

The chromosome 3q26 OncCassette: A multigenic driver of human cancer.

Recurrent copy number variations (CNVs) are genetic alterations commonly observed in human tumors. One of the most frequent CNVs in human tumors involves copy number gains (CNGs) at chromosome 3q26, which is estimated to occur in >20% of human tumors. The high prevalence and frequent occurrence of 3q26 CNG suggest that it drives the biology of tumors harboring this genetic alteration. The chromosomal region subject to CNG (the 3q26 amplicon) spans from chromosome 3q26 to q29, a region containing ∼200 protein-encoding genes. The large number of genes within the amplicon makes it difficult to identify relevant oncogenic target(s). Whereas a number of genes in this region have been linked to the transformed phenotype, recent studies indicate a high level of cooperativity among a subset of frequently amplified 3q26 genes. Here we use a novel bioinformatics approach to identify potential driver genes within the recurrent 3q26 amplicon in lung squamous cell carcinoma (LSCC). Our analysis reveals a set of 35 3q26 amplicon genes that are coordinately amplified and overexpressed in human LSCC tumors, and that also map to a major LSCC susceptibility locus identified on mouse chromosome 3 that is syntenic with human chromosome 3q26. Pathway analysis reveals that 21 of these genes exist within a single predicted network module. Four 3q26 genes, SOX2, ECT2, PRKCI and PI3KCA occupy the hub of this network module and serve as nodal genes around which the network is organized. Integration of available genetic, genomic, biochemical and functional data demonstrates that SOX2, ECT2, PRKCI and PIK3CA are cooperating oncogenes that function within an integrated cell signaling network that drives a highly aggressive, stem-like phenotype in LSCC tumors harboring 3q26 amplification. Based on the high level of genomic, genetic, biochemical and functional integration amongst these 4 3q26 nodal genes, we propose that they are the key oncogenic targets of the 3q26 amplicon and together define a "3q26 OncCassette" that mediates 3q26 CNG-driven tumorigenesis. Genomic analysis indicates that the 3q26 OncCassette also operates in other major tumor types that exhibit frequent 3q26 CNGs, including head and neck squamous cell carcinoma (HNSCC), ovarian serous cancer and cervical cancer. Finally, we discuss how the 3q26 OncCassette represents a tractable target for development of novel therapeutic intervention strategies that hold promise for improving treatment of 3q26-driven cancers.

Interaction of human minichromosome maintenance protein-binding protein with minichromosome maintenance 2-7.

It has been reported that minichromosome maintenance protein-binding protein (MCM-BP) functions in the formation of the pre-replication complex, unloading of minichromosome maintenance (MCM)2-7 from chromatin in late S phase, and formation of the cohesion complex by interacting with MCM3-7 proteins, suggesting that MCM-BP functions in several different reactions during the cell cycle. Here, we examined the interaction of human MCM-BP with MCM2-7 and structural maintenance of chromosome 3 in synchronized HeLa cells by immunoprecipitation. The results show that MCM-BP mainly interacts with MCM7 in the Triton-soluble fraction from S phase and G(2) phase cells, and it also interacts with structural maintenance of chromosome 3 in the fraction from G(2) phase cells. In vitro studies show that MCM-BP disassembles MCM2-7 bound to DNA with a fork-like structure by interacting with MCM3, MCM5, and MCM7. These results suggest that MCM-BP functions in disassembling MCM2-7 on chromatin during S phase and G2 phase by interacting with MCM3, MCM5, and MCM7.

Genome-wide scan on total serum IgE levels identifies no common variants in a healthy Chinese male population.

Immunoglobulin E (IgE) provides important information on the humoral immune status, and the IgE level is routinely detected in clinical practice. There are many diseases associated with IgE, such as atopic disease, autoimmune diseases, and so on. IgE is a genetically complex trait, but comprehensive genetic assessment of the variability in serum IgE levels is lacking. Previous genome-wide association studies (GWAS) on total serum IgE levels have identified FCER1A as the susceptibility locus; however, the candidate gene association study in southern Chinese patients reported no association. Given the genetic difference in different populations, we firstly conducted this two-stage GWAS in a Chinese population of 3,495 men, including 1,999 unrelated subjects in the first stage and 1,496 independent individuals replicated in the second stage. In the first stage, we totally identified three single nucleotide polymorphisms (SNPs) which reached a P value of 1.0 × 10⁻5. Rs17090302 on chromosome 3 and Rs28708846 on chromosome 13 are intergenic. Rs432085 from chromosome 3p28 is located in the gene CCDC50. When the two-stage data was combined, none of the SNPs reached the genome-wide significant level. Collectively, we did not identify novel loci associated with the serum IgE level in Chinese males, but we hypothesized that CCDC50 was a candidate gene in regulation on IgE level.

Diagnosis of blue nevus-like metastatic uveal melanoma confirmed by fluorescence in situ hybridization (FISH) for monosomy 3.

Metastatic melanoma can on rare occasion simulate the appearance of a blue nevus clinically and/or histopathologically, which may lead to diagnostic confusion and delay in treatment. Given the known difficulty in recognizing a small dermal blue nevus-like melanoma metastasis by light microscopic findings alone, recent discoveries of unique cytogenetic aberrations in various types of melanomas have led pathologists to explore cytogenetic techniques as an ancillary diagnostic tool. Herein, we report a case of a 58-year-old man with a history of uveal melanoma, in which fluorescence in situ hybridization (FISH) analysis for monosomy 3 helped confirm a diagnosis of blue nevus-like uveal melanoma metastasis. The patient had presented clinically with a new small 1-mm dark blue-gray macule on the forehead. Histopathologically, a small dermal nodule of pigmented epithelioid melanocytes and melanophages was found with a rare mitotic figure. The pathologist's suspicion of a blue nevus-like melanoma metastasis was confirmed by FISH analysis: both the tumor cells of the patient's prior uveal melanoma and the melanocytes of the new dermal blue nevus-like nodule carried only one copy of chromosome 3. Furthermore, deletion of 1p36 and amplifications of 8q32 were also identified.

Genetic and epigenetic analysis of non-small cell lung cancer with NotI-microarrays.

This study aimed to clarify genetic and epigenetic alterations that occur during lung carcinogenesis and to design perspective sets of newly identified biomarkers. The original method includes chromosome 3 specific NotI-microarrays containing 180 NotI clones associated with genes for hybridization with 40 paired normal/tumor DNA samples of primary lung tumors: 28 squamous cell carcinomas (SCC) and 12 adenocarcinomas (ADC). The NotI-microarray data were confirmed by qPCR and bisulfite sequencing analyses. Forty-four genes showed methylation and/or deletions in more than 15% of non-small cell lung cancer (NSCLC) samples. In general, SCC samples were more frequently methylated/deleted than ADC. Moreover, the SCC alterations were observed already at stage I of tumor development, whereas in ADC many genes showed tumor progression specific methylation/deletions. Among genes frequently methylated/deleted in NSCLC, only a few were already known tumor suppressor genes: RBSP3 (CTDSPL), VHL and THRB. The RPL32, LOC285205, FGD5 and other genes were previously not shown to be involved in lung carcinogenesis. Ten methylated genes, i.e., IQSEC1, RBSP3, ITGA 9, FOXP1, LRRN1, GNAI2, VHL, FGD5, ALDH1L1 and BCL6 were tested for expression by qPCR and were found downregulated in the majority of cases. Three genes (RBSP3, FBLN2 and ITGA9) demonstrated strong cell growth inhibition activity. A comprehensive statistical analysis suggested the set of 19 gene markers, ANKRD28, BHLHE40, CGGBP1, RBSP3, EPHB1, FGD5, FOXP1, GORASP1/TTC21, IQSEC1, ITGA9, LOC285375, LRRC3B, LRRN1, MITF, NKIRAS1/RPL15, TRH, UBE2E2, VHL, WNT7A, to allow early detection, tumor progression, metastases and to discriminate between SCC and ADC with sensitivity and specificity of 80-100%.