Comparison of actionable events detected in cancer genomes by whole-genome sequencing, in silico whole-exome and mutation panels.

BACKGROUND: Next-generation sequencing is used in cancer research to identify somatic and germline mutations, which can predict sensitivity or resistance to therapies, and may be a useful tool to reveal drug repurposing opportunities between tumour types. Multigene panels are used in clinical practice for detecting targetable mutations. However, the value of clinical whole-exome sequencing (WES) and whole-genome sequencing (WGS) for cancer care is less defined, specifically as the majority of variants found using these technologies are of uncertain significance. PATIENTS AND METHODS: We used the Cancer Genome Interpreter and WGS in 726 tumours spanning 10 cancer types to identify drug repurposing opportunities. We compare the ability of WGS to detect actionable variants, tumour mutation burden (TMB) and microsatellite instability (MSI) by using in silico down-sampled data to mimic WES, a comprehensive sequencing panel and a hotspot mutation panel. RESULTS: We reveal drug repurposing opportunities as numerous biomarkers are shared across many solid tumour types. Comprehensive panels identify the majority of approved actionable mutations, with WGS detecting more candidate actionable mutations for biomarkers currently in clinical trials. Moreover, estimated values for TMB and MSI vary when calculated from WGS, WES and panel data, and are dependent on whether all mutations or only non-synonymous mutations were used. Our results suggest that TMB and MSI thresholds should not only be tumour-dependent, but also be sequencing platform-dependent. CONCLUSIONS: There is a large opportunity to repurpose cancer drugs, and these data suggest that comprehensive sequencing is an invaluable source of information to guide clinical decisions by facilitating precision medicine and may provide a wealth of information for future studies. Furthermore, the sequencing and analysis approach used to estimate TMB may have clinical implications if a hard threshold is used to indicate which patients may respond to immunotherapy.

MutSα and MutLα immunoexpression analysis in diagnostic grading of oral epithelial dysplasia and squamous cell carcinoma.

OBJECTIVES: This study explored the expression of DNA mismatch repair (MMR) proteins in a range of oral biopsies. We further evaluated the significance of MMR protein expression combined with basic demographic data in differentiating grades of oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC). STUDY DESIGN: Immunohistochemical expression of MutSα (hMLH1 and hPMS2) and MutLα (hMSH2 and hMSH6) were compared in 98 formalin-fixed paraffin embedded oral biopsies: 21 normal, 24 mild-dysplasia (MD), 8 moderate-to-severe-dysplasia (SD), and 45 OSCC. RESULTS: Expression of hMLH1, hPMS2, and hMSH2 was reduced in MD, SD, and OSCC compared with the normal. Reduced hMSH2 immunoreactivity discriminated poorly differentiated OSCC from well-differentiated OSCC. The diagnostic model correctly classified 71.4% of cases and revealed that hPMS2-negative biopsies were more likely to be cancerous (odds ratio [OR], 0.11; 95% confidence interval [CI], 0.000-0.813; P = .040). CONCLUSION: The results suggested a diagnostic role for MMR proteins in OED and OSCC.

Epithelial to mesenchymal transition (EMT) biomarkers--E-cadherin, beta-catenin, APC and Vimentin--in oral squamous cell carcinogenesis and transformation.

OBJECTIVES: To investigate immunohistochemical (IHC) analysis of E-cadherin, ß-catenin, APC and Vimentin for prediction of oral malignant transformation. MATERIALS AND METHODS: Immunoreactivity for E-cadherin, ß-catenin, APC and Vimentin were determined for 100 oral biopsies classified as normal, mild dysplasia, moderate-severe dysplasia or OSCC, using the IHC scoring or label index scoring systems. Co-expression of biomarkers and correlation with histopathological grading was analysed. Vimentin and E-cadherin results were confirmed by RT-PCR and further investigated in vitro using a novel organotypic cell invasion model based on human dermis. RESULTS: A trend for decreased E-cadherin expression but increased Vimentin expression that correlated with increased disease severity was observed. Epithelial ß-catenin localisation shifted from being membranous to cytoplasmic/nuclear with increased histopathological grade severity. Relative to normal, APC expression was decreased for mild dysplasia but increased for OSCC. Co-expression of ß-catenin, APC and Vimentin (Spearman rank correlation) suggests interdependence of these molecules and involvement of the Wnt pathway in oral malignant transformation. Relative mRNA expression of E-cadherin for dysplasia and OSCC were less than 1% of normal tissue values, and mRNA expression of Vimentin was 3.7 times higher for OSCC than normal. After 63 days of organotypic culture neoplastic oral keratinocytes (PE/CA-PJ15) lost expression of E-cadherin and gained expression of Vimentin relative to their non-invasive counterparts in the epithelium. CONCLUSIONS: Trends in the expression of EMT markers - E-cadherin, ß-catenin, APC and Vimentin - suggest their involvement in oral carcinogenesis via Wnt pathway dysregulation. Aberrant expression of ß-catenin, APC and Vimentin are potential markers of malignant transformation.

Keratinocyte-driven contraction of reconstructed human skin.

We have previously reported that reconstructed human skin, using deepidermized acellular sterilized dermis and allogeneic keratinocytes and fibroblasts, significantly contracts in vitro. Contracture of split skin grafts in burns injuries remains a serious problem and this in vitro model provides an opportunity to study keratinocyte/mesenchymal cell interactions and cell interactions with extracted normal human dermis. The aim of this study was to investigate the nature of this in vitro contraction and explore several approaches to prevent or reduce contraction. Three different methodologies for sterilization of the dermal matrix were examined: glycerol, ethylene oxide and a combination of glycerol and ethylene oxide. While the nature of the sterilization technique influenced the extent of contraction and thinner dermal matrices contracted proportionately more than thicker matrices, in all cases contraction was driven by the keratinocytes with relatively little influence from the fibroblasts. The contraction of the underlying dermis did not represent any change in tissue mass but rather a reorganization of the dermis which was rapidly reversed (within minutes) when the epidermal layer was removed. Pharmacological approaches to block contraction showed forskolin and mannose-6-phosphate to be ineffective and ascorbic acid-2-phosphate to exacerbate contraction. However, Galardin, a matrix metalloproteinase inhibitor and keratinocyte conditioned media, both inhibited contraction.

The requirement for basement membrane antigens in the production of human epidermal/dermal composites in vitro.

The importance of a dermal element when providing permanent wound cover for skin loss has become evident as the shortcomings of pure epidermal grafts are recognized. We are developing a skin composite formed from sterilized human de-epidermized acellular dermis, keratinocytes and fibroblasts with the ultimate aim of using this composite to cover full-thickness excised burn wounds. These composites can be prepared with or without basement membrane (BM) antigens initially present on the dermis. This study investigates the importance of retaining BM antigens on the dermis to the production and appearance of these composites in vitro. Skin composites prepared from dermis with BM antigens either present or absent initially were studied throughout 3 weeks. Composites with BM antigens present initially were significantly better than those initially lacking BM antigens in: (i) the degree of epithelial cell attachment to the underlying dermis (hemidesmosomes were seen only in the former); (ii) the morphology of the epithelial layer; (iii) the consistent presence of collagen IV and laminin and the increasing expression of tenascin; and (iv) the amount of soluble collagen IV and fibronectin detected in the conditioned media. We conclude that an initial BM antigen template is vital in this skin composite model for the attachment and differentiation of the epithelial layer and for the subsequent remodelling of the BM in vitro.

Urinary and serum type III collagen: markers of renal fibrosis.

BACKGROUND: The progression of chronic renal failure is characterized by the progressive fibrosis of the kidneys. Such fibrosis reflects the increased deposition of collagens (I, III, and IV) as well as fibronectin within scarred kidneys. In this study, we determined whether changes in renal extracellular matrix (ECM) components are reflected by parallel changes in their circulating or urinary levels. PATIENTS AND METHODS: We studied 40 patients with a range of subacute and chronic nephropathies who underwent a renal biopsy. At the time of the biopsy, their serum and urinary levels of collagens III (amino terminal peptide of procollagen III; PIIINP) and IV, as well as fibronectin were measured. Clinical, biochemical and histological parameters were correlated. Multiple regression analysis was applied to determine the predictive value of circulating and urinary ECM components for the severity of renal fibrosis. RESULTS: We noted an increase in circulating and urinary levels of collagens III and IV but not fibronectin in patients with nephropathies compared to healthy volunteers. Increased immunoreactivity for these ECM components was also detected in kidney biopsies when compared to normal kidneys. A strong positive correlation was detected between circulating and urinary procollagen III (PIIINP) and the severity of renal interstitial fibrosis (serum PIIINP: r = 0.49, P < 0.01; urine PIIINP: r = 0.51, P < 0.01). CONCLUSION: We conclude that the measurements of urinary collagen III (PIIINP), and to a lesser extent serum collagen III (PIIINP), are useful indicators of the extent of renal fibrosis. This may have diagnostic implications and may prove useful for the monitoring of disease progression.

Keratinocytes contract human dermal extracellular matrix and reduce soluble fibronectin production by fibroblasts in a skin composite model.

Composites of human de-epidermised acellular dermis and normal adult human keratinocytes and fibroblasts were examined for the ability of cells to contract these composites. Image analysis of the outline of the composites showed that, in this model, keratinocytes alone or in the presence of fibroblasts caused highly significant contraction (of the order of 25% by day 12). There was no significant contraction of the dermis with fibroblasts alone or in the absence of cells. The presence or absence of basement membrane antigens did not influence the effect of keratinocytes on dermal contraction. Analysis of the conditioned media from these composites showed that the greatest fibronectin production was seen with fibroblasts alone in the presence of basement membrane. Keratinocytes alone produced little fibronectin irrespective of the presence of the basement membrane. If keratinocytes were present with fibroblasts, however, then fibronectin production was significantly reduced both in the presence and absence of the basement membrane, indicating that keratinocytes modify dermal fibroblast extracellular matrix production. This study shows that while keratinocytes and fibroblasts are clearly influencing each other's activity in this human skin composite model, under the circumstances we describe it is the keratinocyte and not the fibroblast which causes contraction of the human de-epidermised acellular dermis.