Integrated Genomic Analysis of Sézary Syndrome

Sézary syndrome (SS) is a rare variant of primary cutaneous T-cell lymphoma. Little is known about the underlying pathogenesis of S. To address this issue, we used Affymetrix 10K SNP microarray to analyse 13 DNA samples isolated from 8 SS patients and qPCR with ABI TaqMan SNP genotyping assays for the validation of the SNP microarray results. In addition, we tested the impact of SNP loss of heterozygosity (LOH) identified in SS cases on the gene expression profiles of SS cases detected with Affymetrix GeneChip U133A. The results showed: (1) frequent SNP copy number change and LOH involving 1, 2p, 3, 4q, 5q, 6, 7p, 8, 9, 10, 11, 12q, 13, 14, 16q, 17, and 20, (2) reduced SNP copy number at FAT gene (4q35) in 75% of SS cases, and (3) the separation of all SS cases from normal control samples by SNP LOH gene clusters at chromosome regions of 9q31q34, 10p11q26, and 13q11q12. These findings provide some intriguing information for our current understanding of the molecular pathogenesis of this tumour and suggest the possibility of presence of functional SNP LOH in SS tumour cells

Upregulation of FOXM1 induces genomic instability in human epidermal keratinocytes

<p>Abstract</p> <p>Background</p> <p>The human cell cycle transcription factor FOXM1 is known to play a key role in regulating timely mitotic progression and accurate chromosomal segregation during cell division. Deregulation of FOXM1 has been linked to a majority of human cancers. We previously showed that FOXM1 was upregulated in basal cell carcinoma and recently reported that upregulation of FOXM1 precedes malignancy in a number of solid human cancer types including oral, oesophagus, lung, breast, kidney, bladder and uterus. This indicates that upregulation of FOXM1 may be an early molecular signal required for aberrant cell cycle and cancer initiation.</p> <p>Results</p> <p>The present study investigated the putative early mechanism of UVB and FOXM1 in skin cancer initiation. We have demonstrated that UVB dose-dependently increased FOXM1 protein levels through protein stabilisation and accumulation rather than de novo mRNA expression in human epidermal keratinocytes. FOXM1 upregulation in primary human keratinocytes triggered pro-apoptotic/DNA-damage checkpoint response genes such as p21, p38 MAPK, p53 and PARP, however, without causing significant cell cycle arrest or cell death. Using a high-resolution Affymetrix genome-wide single nucleotide polymorphism (SNP) mapping technique, we provided the evidence that FOXM1 upregulation in epidermal keratinocytes is sufficient to induce genomic instability, in the form of loss of heterozygosity (LOH) and copy number variations (CNV). FOXM1-induced genomic instability was significantly enhanced and accumulated with increasing cell passage and this instability was increased even further upon exposure to UVB resulting in whole chromosomal gain (7p21.3-7q36.3) and segmental LOH (6q25.1-6q25.3).</p> <p>Conclusion</p> <p>We hypothesise that prolonged and repeated UVB exposure selects for skin cells bearing stable FOXM1 protein causes aberrant cell cycle checkpoint thereby allowing ectopic cell cycle entry and subsequent genomic instability. The aberrant upregulation of FOXM1 serves as a 'first hit' where cells acquire genomic instability which in turn predisposes cells to a 'second hit' whereby DNA-damage checkpoint response (eg. p53 or p16) is abolished to allow damaged cells to proliferate and accumulate genetic aberrations/mutations required for cancer initiation.</p

The diagnosis of inherited metabolic diseases by microarray gene expression profiling

<p>Abstract</p> <p>Background</p> <p>Inherited metabolic diseases (IMDs) comprise a diverse group of generally progressive genetic metabolic disorders of variable clinical presentations and severity. We have undertaken a study using microarray gene expression profiling of cultured fibroblasts to investigate 68 patients with a broad range of suspected metabolic disorders, including defects of lysosomal, mitochondrial, peroxisomal, fatty acid, carbohydrate, amino acid, molybdenum cofactor, and purine and pyrimidine metabolism. We aimed to define gene expression signatures characteristic of defective metabolic pathways.</p> <p>Methods</p> <p>Total mRNA extracted from cultured fibroblast cell lines was hybridized to Affymetrix U133 Plus 2.0 arrays. Expression data was analyzed for the presence of a gene expression signature characteristic of an inherited metabolic disorder and for genes expressing significantly decreased levels of mRNA.</p> <p>Results</p> <p>No characteristic signatures were found. However, in 16% of cases, disease-associated nonsense and frameshift mutations generating premature termination codons resulted in significantly decreased mRNA expression of the defective gene. The microarray assay detected these changes with high sensitivity and specificity.</p> <p>Conclusion</p> <p>In patients with a suspected familial metabolic disorder where initial screening tests have proven uninformative, microarray gene expression profiling may contribute significantly to the identification of the genetic defect, shortcutting the diagnostic cascade.</p

Identification of FBXL4 as a Metastasis Associated Gene in Prostate Cancer

Prostate cancer is the most common cancer among western men, with a significant mortality and morbidity reported for advanced metastatic disease. Current understanding of metastatic disease is limited due to difficulty of sampling as prostate cancer mainly metastasizes to bone. By analysing prostate cancer bone metastases using high density microarrays, we found a common genomic copy number loss at 6q16.1–16.2, containing the FBXL4 gene, which was confirmed in larger series of bone metastases by fluorescence in situ hybridisation (FISH). Loss of FBXL4 was also detected in primary tumours and it was highly associated with prognostic factors including high Gleason score, clinical stage, prostate-specific antigen (PSA) and extent of disease, as well as poor patient survival, suggesting that FBXL4 loss contributes to prostate cancer progression. We also demonstrated that FBXL4 deletion is detectable in circulating tumour cells (CTCs), making it a potential prognostic biomarker by ‘liquid biopsy’. In vitro analysis showed that FBXL4 plays a role in regulating the migration and invasion of prostate cancer cells. FBXL4 potentially controls cancer metastasis through regulation of ERLEC1 levels. Therefore, FBXL4 could be a potential novel prostate cancer suppressor gene, which may prevent cancer progression and metastasis through controlling cell invasion

Distinctive Patterns of MicroRNA Expression Associated with Karyotype in Acute Myeloid Leukaemia

Acute myeloid leukaemia (AML) is the most common acute leukaemia in adults; however, the genetic aetiology of the disease is not yet fully understood. A quantitative expression profile analysis of 157 mature miRNAs was performed on 100 AML patients representing the spectrum of known karyotypes common in AML. The principle observation reported here is that AMLs bearing a t(15;17) translocation had a distinctive signature throughout the whole set of genes, including the up regulation of a subset of miRNAs located in the human 14q32 imprinted domain. The set included miR-127, miR-154, miR-154*, miR-299, miR-323, miR-368, and miR-370. Furthermore, specific subsets of miRNAs were identified that provided molecular signatures characteristic of the major translocation-mediated gene fusion events in AML. Analysis of variance showed the significant deregulation of 33 miRNAs across the leukaemic set with respect to bone marrow from healthy donors. Fluorescent in situ hybridisation analysis using miRNA-specific locked nucleic acid (LNA) probes on cryopreserved patient cells confirmed the results obtained by real-time PCR. This study, conducted on about a fifth of the miRNAs currently reported in the Sanger database (microrna.sanger.ac.uk), demonstrates the potential for using miRNA expression to sub-classify cancer and suggests a role in the aetiology of leukaemia

Identification of ZDHHC14 as a novel human tumour suppressor gene

Genomic changes affecting tumour suppressor genes are fundamental to cancer. We applied SNP array analysis to a panel of testicular germ cell tumours to search for novel tumour suppressor genes and identified a frequent small deletion on 6q25.3 affecting just one gene, ZDHHC14. The expression of ZDHHC14, a putative protein palmitoyltransferase with unknown cellular function, was decreased at both RNA and protein levels in testicular germ cell tumours. ZDHHC14 expression was also significantly decreased in a panel of prostate cancer samples and cell lines. In addition to our findings of genetic and protein expression changes in clinical samples, inducible overexpression of ZDHHC14 led to reduced cell viability and increased apoptosis through the classic caspase-dependent apoptotic pathway and heterozygous knockout of ZDHHC14 decreased cell colony formation ability. Finally, we confirmed our in vitro findings of the tumour suppressor role of ZDHHC14 in a mouse xenograft model, showing that overexpression of ZDHHC14 inhibits tumourigenesis. Thus, we have identified a novel tumour suppressor gene that is commonly down-regulated in testicular germ cell tumours and prostate cancer, as well as given insight into the cellular functional role of ZDHHC14, a potential protein palmitoyltransferase that may play a key protective role in cancer

Gothic Revival Architecture Before Horace Walpole's Strawberry Hill

The Gothic Revival is generally considered to have begun in eighteenth-century Britain with the construction of Horace Walpole’s villa, Strawberry Hill, Twickenham, in the late 1740s. As this chapter demonstrates, however, Strawberry Hill is in no way the first building, domestic or otherwise, to have recreated, even superficially, some aspect of the form and ornamental style of medieval architecture. Earlier architects who, albeit often combining it with Classicism, worked in the Gothic style include Sir Christopher Wren, Nicholas Hawksmoor, William Kent and Batty Langley, aspects of whose works are explored here. While not an exhaustive survey of pre-1750 Gothic Revival design, the examples considered in this chapter reveal how seventeenth- and eighteenth-century Gothic emerged and evolved over the course of different architects’ careers, and how, by the time that Walpole came to create his own Gothic ‘castle’, there was already in existence in Britain a sustained Gothic Revivalist tradition

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival