Cancer biomarker development from basic science to clinical practice

The amount of published literature on biomarkers has exponentially increased over the last two decades. Cancer biomarkers are molecules that are either part of tumour cells or secreted by tumour cells. Biomarkers can be used for diagnosing cancer (tumour versus normal and differentiation of subtypes), prognosticating patients (progression free survival and overall survival) and predicting response to therapy. However, very few biomarkers are currently used in clinical practice compared to the unprecedented discovery rate. Some of the examples are: carcino-embryonic antigen (CEA) for colon cancer; prostate specific antigen (PSA) for prostate; and estrogen receptor (ER), progesterone receptor (PR) and HER2 for breast cancer. Cancer biomarkers passes through a series of phases before they are used in clinical practice. First phase in biomarker development is identification of biomarkers which involve discovery, demonstration and qualification. This is followed by validation phase, which includes verification, prioritisation and initial validation. More large-scale and outcome-oriented validation studies expedite the clinical translation of biomarkers by providing a strong ‘evidence base’. The final phase in biomarker development is the routine clinical use of biomarker. In summary, careful identification of biomarkers and then validation in well-designed retrospective and prospective studies is a systematic strategy for developing clinically useful biomarkers

Profiling, Comparison and Validation of Gene Expression in Gastric Carcinoma and Normal Stomach

Gastric carcinoma is the second most common cause of cancer death world-wide but its molecular biology is not well understood. My aims were to catalogue the genes expressed in gastric carcinoma and normal stomach and to identify differentially expressed and gastric-specific transcripts. Serial analysis of gene expression (SAGE) produces comprehensive, quantitative and reproducible expression profiles. The method of SAGE was established in this laboratory then used to study normal gastric antral mucosa and two gastric adenocarcinomas of distal, intestinal type. The libraries were compared on-line with other glandular epithelial tissues. Selected genes were validated in a panel of 19 normal and tumour gastro-intestinal tissues and cell lines by Northern blotting and immunohistochemistry. 29,480 transcripts, derived from 10,866 genes, were identified. The validation studies corroborated the SAGE profiles although tumour heterogeneity was noted. 1% of genes were differentially expressed (by over five-fold and with a p-value below 0.01) between the pooled gastric carcinomas and normal stomach. The most abundant transcripts included ribosomal and mitochondrial proteins, of which most were up- regulated in the tumours, as were other widely expressed genes including transcription factors (Id1), signalling molecules (fibroblast growth factor receptor and serine/threonine protein kinases), coatomer and proteasome components, thymosin beta 10 and collagenase I. In contrast, cytoskeletal proteins (alpha actinin and profilin) were down-regulated in the tumours. Many genes which were more highly expressed in normal stomach are important in normal gastric function, including gastrin, immunoglobulin alpha, lysozyme, mucin (MUC5), trefoil peptides (pS2 and spasmolytic polypeptide) and pepsinogens, which were amongst 55 gastric-specific transcripts. Some transcripts had previously been characterised only minimally (prostate stem cell antigen) or not at all (aquaporin 5) in the stomach. Some genes (intestinal trefoil factor) which were up-regulated in gastric carcinoma reflect the intestinal-type histology. Some genes abundant in normal gastric antrum had previously been regarded as markers of pancreatic carcinoma. Many differentially expressed species, some tumour-associated, were novel and await investigation. One new gene which was identified was highly expressed in normal stomach but absent from gastric carcinomas. This new gene was selected for further investigation. The SAGE expression profile was confirmed by Northern blotting and in situ hybridisation by which the mRNA was located in the superficial/foveolar (pit) epithelium of the gastric mucosa, so the gene was termed foveolin. The transcript was expressed outwith the stomach only in metaplastic gastric epithelium, in Barrett's oesophagus or the ulcer-associated cell lineage in the gut, and outwith the gut only in ovarian mucinous tumours. The mRNA was present in the stomach of mouse, rat and cow in the same location as in humans. The 5' and 3' ends of the mRNA were characterised by Rapid Amplification of cDNA Ends (RACE). Homologous mouse and cow mRNAs were identified, characterised and compared. The full-length genomic sequences for the human and mouse were obtained using on-line databases, characterised and compared. A partial human genomic clone was obtained from a PAC library, and used to map the gene by fluorescent in situ hybridisation (FISH) to human chromosome 2. The predicted protein product, like the mRNA and DNA sequences, is highly conserved between the human, mouse and cow species. The protein shows no homology to any known protein sequences or motifs, but bears an initial signal peptide and is therefore predicted to have an extracellular location, being either retained on the outer cell surface or secreted into the gastric lumen, much like gastric mucin (MUC5) and the trefoil peptide pS2 (TFF1), with which foveolin shares a similar location in the superficial and foveolar gastric epithelium. These are the first global profiles of gene expression in the stomach. The molecular anatomy correlated with the morphology. The gastric carcinoma profiles resembled other tumours, which supports the existence of common cancer-associated molecular targets. The normal gastric profile differed from other normal glandular tissues but agreed with existing literature. Many new transcripts were identified, of which one has been further characterised here in its first detailed description. These data increase our knowledge about the genes involved in normal gastric function and in malignant change in the stomach, and provide a catalogue of candidates from which to develop markers for better diagnosis and therapy of gastric carcinoma

A33 shows similar sensitivity to but is more specific than CDX2 as an immunomarker of colorectal carcinoma

Aims: CDX2 is widely used as a sensitive and specific immunomarker for colorectal carcinoma (CRC) but neither this sensitivity nor specificity is absolute. This study is the first known comparison of CDX1 and A33 against CDX2 as immunomarkers for CRC. Methods and Results: As a pilot study, whole sections of 51 cases of liver metastatic carcinoma of different origins - colorectum (n=32), breast (n=3), oesophagogastric tract (n=4), lung (n=3), pancreas (n=8), and prostate (n=1) - were immunostained with CDX1, CDX2 and A33. Compared with CDX1, A33 showed higher sensitivity as a CRC immunomarker, greater interobserver reproducibility for assessment of expression, and less background cross-reactivity. Therefore, only A33 was compared with CDX2 for a tissue microarray-based study of primary adenocarcinomas of different origin: CRC (n=55), liver deposits of metastatic CRC (n=60), breast (n=101), lung (n=40), oesophagogastric tract (n=134), ovary (n= 67), pancreas (n= 77), and prostate (n= 56). Combining the whole section and TMA cases of CRC, A33 had a sensitivity of 95.9% and CDX2 a sensitivity of 97.2%. Combining all the whole section and TMA cases of non-colorectal carcinomas, A33 showed 85.4% specificity as a marker of CRC compared to CDX2 which showed a specificity of 64.3%. The higher specificity of A33 as a colorectal carcinoma immunomarker compared with CDX2 was particularly seen amongst pancreatic and ovarian carcinomas. Further, unlike with CDX2, none of the prostatic and lung carcinomas studied showed A33 positivity. Conclusions: A33 shows similar sensitivity to but is more specific than CDX2 as an immunomarker of CRC

Prevalent low-grade dysplasia: the strongest predictor of malignant progression in Barrett's columnar-lined oesophagus

No abstract

Time for change: a new training programme for morpho-molecular pathologists?

The evolution of cellular pathology as a specialty has always been driven by technological developments and the clinical relevance of incorporating novel investigations into diagnostic practice. In recent years, the molecular characterisation of cancer has become of crucial relevance in patient treatment both for predictive testing and subclassification of certain tumours. Much of this has become possible due to the availability of next-generation sequencing technologies and the whole-genome sequencing of tumours is now being rolled out into clinical practice in England via the 100 000 Genome Project. The effective integration of cellular pathology reporting and genomic characterisation is crucial to ensure the morphological and genomic data are interpreted in the relevant context, though despite this, in many UK centres molecular testing is entirely detached from cellular pathology departments. The CM-Path initiative recognises there is a genomics knowledge and skills gap within cellular pathology that needs to be bridged through an upskilling of the current workforce and a redesign of pathology training. Bridging this gap will allow the development of an integrated 'morphomolecular pathology' specialty, which can maintain the relevance of cellular pathology at the centre of cancer patient management and allow the pathology community to continue to be a major influence in cancer discovery as well as playing a driving role in the delivery of precision medicine approaches. Here, several alternative models of pathology training, designed to address this challenge, are presented and appraised

Digital pathology access and usage in the UK: results from a national survey on behalf of the National Cancer Research Institute's CM-Path initiative.

Aim To canvass the UK pathology community to ascertain current levels of digital pathology usage in clinical and academic histopathology departments, and prevalent attitudes to digital pathology. Methods A 15-item survey was circulated to National Health Service and academic pathology departments across the UK using the SurveyMonkey online survey tool. Responses were sought at a departmental or institutional level. Where possible, departmental heads were approached and asked to complete the survey, or forward it to the most relevant individual in their department. Data were collected over a 6-month period from February to July 2017. Results 41 institutes from across the UK responded to the survey. 60% (23/39) of institutions had access to a digital pathology scanner, and 60% (24/40) had access to a digital pathology workstation. The most popular applications of digital pathology in current use were undergraduate and postgraduate teaching, research and quality assurance. Investigating the deployment of digital pathology in their department was identified as a high or highest priority by 58.5% of institutions, with improvements in efficiency, turnaround times, reporting times and collaboration in their institution anticipated by the respondents. Access to funding for initial hardware, software and staff outlay, pathologist training and guidance from the Royal College of Pathologists were identified as factors that could enable respondent institutions to increase their digital pathology usage. Conclusion Interest in digital pathology adoption in the UK is high, with usage likely to increase in the coming years. In light of this, pathologists are seeking more guidance on safe usage

Pathology and regulation for research in the UK: An overview [version 2; peer review: 3 approved]

The input of pathologists is essential for the conduct of many forms of research, including clinical trials. As the custodians of patient samples, pathology departments have a duty to ensure compliance with the relevant regulations, standards and guidelines to ensure the ethical and effective use for their intended investigational analysis, including when patients are participating in a research study. The results of research studies have impacts beyond the research study itself as they may inform changes in policy and practice or support the licensing of medicines and devices. Compliance with regulations and standards provides public assurance that the rights, safety and wellbeing of research participants are protected, that the data have been collected and processed to ensure their integrity and that the research will achieve its purpose. The requirements of the regulatory environment should not be seen as a barrier to research and should not significantly impact on the work of the laboratory once established and integrated into practice. This paper highlights important regulations, policy, standards and available guidance documents that apply to research involving NHS pathology departments and academic laboratories that are contributing to research involving human subjects

Pathology and regulation for research in the UK: An overview [version 2; peer review: 3 approved]

The input of pathologists is essential for the conduct of many forms of research, including clinical trials. As the custodians of patient samples, pathology departments have a duty to ensure compliance with the relevant regulations, standards and guidelines to ensure the ethical and effective use for their intended investigational analysis, including when patients are participating in a research study. The results of research studies have impacts beyond the research study itself as they may inform changes in policy and practice or support the licensing of medicines and devices. Compliance with regulations and standards provides public assurance that the rights, safety and wellbeing of research participants are protected, that the data have been collected and processed to ensure their integrity and that the research will achieve its purpose. The requirements of the regulatory environment should not be seen as a barrier to research and should not significantly impact on the work of the laboratory once established and integrated into practice. This paper highlights important regulations, policy, standards and available guidance documents that apply to research involving NHS pathology departments and academic laboratories that are contributing to research involving human subjects

Expression of KOC, S100P, mesothelin and MUC1 in pancreatico-biliary adenocarcinomas: development and utility of a potential diagnostic immunohistochemistry panel

<b>Background</b> Pancreatico-biliary adenocarcinomas (PBA) have a poor prognosis. Diagnosis is usually achieved by imaging and/or endoscopy with confirmatory cytology. Cytological interpretation can be difficult especially in the setting of chronic pancreatitis/cholangitis. Immunohistochemistry (IHC) biomarkers could act as an adjunct to cytology to improve the diagnosis. Thus, we performed a meta-analysis and selected KOC, S100P, mesothelin and MUC1 for further validation in PBA resection specimens.<p></p> <b>Methods</b> Tissue microarrays containing tumour and normal cores in a ratio of 3:2, from 99 surgically resected PBA patients, were used for IHC. IHC was performed on an automated platform using antibodies against KOC, S100P, mesothelin and MUC1. Tissue cores were scored for staining intensity and proportion of tissue stained using a Histoscore method (range, 0–300). Sensitivity and specificity for individual biomarkers, as well as biomarker panels, were determined with different cut-offs for positivity and compared by summary receiver operating characteristic (ROC) curve.<p></p> <b>Results</b> The expression of all four biomarkers was high in PBA versus normal ducts, with a mean Histoscore of 150 vs. 0.4 for KOC, 165 vs. 0.3 for S100P, 115 vs. 0.5 for mesothelin and 200 vs. 14 for MUC1 (p < .0001 for all comparisons). Five cut-offs were carefully chosen for sensitivity/specificity analysis. Four of these cut-offs, namely 5%, 10% or 20% positive cells and Histoscore 20 were identified using ROC curve analysis and the fifth cut-off was moderate-strong staining intensity. Using 20% positive cells as a cut-off achieved higher sensitivity/specificity values: KOC 84%/100%; S100P 83%/100%; mesothelin 88%/92%; and MUC1 89%/63%. Analysis of a panel of KOC, S100P and mesothelin achieved 100% sensitivity and 99% specificity if at least 2 biomarkers were positive for 10% cut-off; and 100% sensitivity and specificity for 20% cut-off.<p></p> <b>Conclusion</b> A biomarker panel of KOC, S100P and mesothelin with at least 2 biomarkers positive was found to be an optimum panel with both 10% and 20% cut-offs in resection specimens from patients with PBA.<p></p&gt

Tumour inflammatory infiltrate predicts survival following curative resection for node-negative colorectal cancer

<b>Background</b>: A pronounced tumour inflammatory infiltrate is known to confer a good outcome in colorectal cancer. Klintrup and colleagues reported a structured assessment of the inflammatory reaction at the invasive margin scoring low grade or high grade. The aim of the present study was to examine the prognostic value of tumour inflammatory infiltrate in node-negative colorectal cancer. <b>Methods</b>: Two hundred patients had undergone surgery for node-negative colorectal cancer between 1997 and 2004. Specimens were scored with Jass’ and Klintrup’s criteria for peritumoural infiltrate. Pathological data were taken from the reports at that time. <b>Results</b>: Low-grade inflammatory infiltrate assessed using Klintrup’s criteria was an independent prognostic factor in node-negative disease. In patients with a low-risk Petersen Index (n = 179), low-grade infiltrate carried a threefold increased risk of cancer death. Low-grade infiltrate was related to increasing T stage and an infiltrating margin. <b>Conclusion</b>: Assessment of inflammatory infiltrate using Klintrup’s criteria provides independent prognostic information on node-negative colorectal cancer. A high-grade local inflammatory response may represent effective host immune responses impeding tumour growth