Epidermal growth factor receptor immunohistochemistry: new opportunities in metastatic colorectal cancer

The treatment of cancer is becoming more precise, targeting specific oncogenic drivers with targeted molecular therapies. The epidermal growth factor receptor has been found to be over-expressed in a multitude of solid tumours. Immunohistochemistry is widely used in the fields of diagnostic and personalised medicine to localise and visualise disease specific proteins. To date the clinical utility of epidermal growth factor receptor immunohistochemistry in determining monoclonal antibody efficacy has remained somewhat inconclusive. The lack of an agreed reproducible scoring criteria for epidermal growth factor receptor immunohistochemistry has, in various clinical trials yielded conflicting results as to the use of epidermal growth factor receptor immunohistochemistry assay as a companion diagnostic. This has resulted in this test being removed from the licence for the drug panitumumab and not performed in clinical practice for cetuximab. In this review we explore the reasons behind this with a particular emphasis on colorectal cancer, and to suggest a way of resolving the situation through improving the precision of epidermal growth factor receptor immunohistochemistry with quantitative image analysis of digitised images complemented with companion molecular morphological techniques such as in situ hybridisation and section based gene mutation analysis.

The prognostic and therapeutic value of EpHA2 in early colorectal cancer (CRC).

Background: EpHA2 is a 130 kD transmembrane glycoprotein belonging to ephrin receptor subfamily and involved in angiogenesis/tumour neovascularisation. High EpHA2 mRNA level has recently been implicated in cetuximab resistance. Previously, we found high EpHA2 levels in a panel of invasive colorectal cancer (CRC) cells, which was associated with high levels of stem-cell marker CD44. Our aim was to investigate the prognostic value of EpHA2 and subsequently correlate expression levels to known clinico-pathological variables in early stage CRC. Methods: Tissue samples from 509 CRC patients were analysed. EpHA2 expression was measured using IHC. Kaplan-Meier graphs were used. Univariate and multivariate analyses employed Cox Proportional Hazards Ratio (HR) method. A backward selection method (Akaike’s information criterion) was used to determine a refined multivariate model. Results: EpHA2 was highly expressed in CRC adenocarcinoma compared to matched normal colon tissue. In support of our preclinical invasive models, strong correlation was found between EpHA2 expression and CD44 and Lgr5 staining (p<0.001). In addition, high EpHA2 expression significantly correlated with vascular invasion (p=0.03).HR for OS for stage II/III patients with high EpHA2 expression was 1.69 (95%CI: 1.164-2.439; p=0.003). When stage II/III was broken down into individual stages, there was significant correlation between high EpHA2 expression and poor 5-years OS in stage II patients (HR: 2.18; 95%CI: 1.28-3.71; p=0.005).HR in the stage III group showed a trend to statistical significance (HR: 1.48; 95%CI=0.87-2.51; p=0.05). In both univariate and multivariate analyses of stage II patients, high EpHA2 expression was the only significant factor and was retained in the final multivariate model. Higher levels of EpHA2 were noted in our RAS and BRAF mutant CRC cells, and silencing EpHA2 resulted in significant decreases in migration/invasion in parental and invasive CRC sublines. Correlation between KRAS/NRAS/BRAFmutational status and EpHA2 expression in clinical samples is ongoing. Conclusions: Taken together, our study is the first to indicate that EpHA2 expression is a predictor of poor clinical outcome and a potential novel target in early stage CRC.

Quantification of HER2 heterogeneity in breast cancer-implications for identification of sub-dominant clones for personalised treatment

Breast cancer is a heterogeneous disease, at both an inter- and intra-tumoural level. Appreciating heterogeneity through the application of biomarkers and molecular signatures adds complexity to tumour taxonomy but is key to personalising diagnosis, treatment and prognosis. The extent to which heterogeneity exists, and its interpretation remains a challenge to pathologists. Using HER2 as an exemplar, we have developed a simple reproducible heterogeneity index. Cell-to-cell HER2 heterogeneity was extensive in a proportion of both reported 'amplified' and 'non-amplified' cases. The highest levels of heterogeneity objectively identified occurred in borderline categories and higher ratio non-amplified cases. A case with particularly striking heterogeneity was analysed further with an array of biomarkers in order to assign a molecular diagnosis. Broad biological complexity was evident. In essence, interpretation, depending on the area of tumour sampled, could have been one of three distinct phenotypes, each of which would infer different therapeutic interventions. Therefore, we recommend that heterogeneity is assessed and taken into account when determining treatment options.

Stratified analysis reveals chemokine-like factor (CKLF) as a potential prognostic marker in the MSI-immune consensus molecular subtype CMS1 of colorectal cancer

The Colorectal Cancer (CRC) Subtyping Consortium (CRCSC) recently published four consensus molecular subtypes (CMS’s) representing the underlying biology in CRC. The Microsatellite Instable (MSI) immune group, CMS1, has a favorable prognosis in early stage disease, but paradoxically has the worst prognosis following relapse, suggesting the presence of factors enabling neoplastic cells to circumvent this immune response. To identify the genes influencing subsequent poor prognosis in CMS1, we analyzed this subtype, centered on risk of relapse.
In a cohort of early stage colon cancer (n=460), we examined, in silico, changes in gene expression within the CMS1 subtype and demonstrated for the first time the favorable prognostic value of chemokine-like factor (CKLF) gene expression in the adjuvant disease setting [HR=0.18, CI=0.04-0.89]. In addition, using transcription profiles originating from cell sorted CRC tumors, we delineated the source of CKLF transcription within the colorectal tumor microenvironment to the leukocyte component of these tumors. Further to this, we confirmed that CKLF gene expression is confined to distinct immune subsets in whole blood samples and primary cell lines, highlighting CKLF as a potential immune cell-derived factor promoting tumor immune-surveillance of nascent neoplastic cells, particularly in CMS1 tumors. Building on the recently reported CRCSC data, we provide compelling evidence that leukocyte-infiltrate derived CKLF expression is a candidate biomarker of favorable prognosis, specifically in MSI-immune stage II/III disease.

QUADrATiC: scalable gene expression connectivity mapping for repurposing FDA-approved therapeutics

Background: Gene expression connectivity mapping has proven to be a powerful and flexible tool for research. Its application has been shown in a broad range of research topics, most commonly as a means of identifying potential small molecule compounds, which may be further investigated as candidates for repurposing to treat diseases. The public release of voluminous data from the Library of Integrated Cellular Signatures (LINCS) programme further enhanced the utilities and potentials of gene expression connectivity mapping in biomedicine. Results: We describe QUADrATiC (http://go.qub.ac.uk/QUADrATiC), a user-friendly tool for the exploration of gene expression connectivity on the subset of the LINCS data set corresponding to FDA-approved small molecule compounds. It enables the identification of compounds for repurposing therapeutic potentials. The software is designed to cope with the increased volume of data over existing tools, by taking advantage of multicore computing architectures to provide a scalable solution, which may be installed and operated on a range of computers, from laptops to servers. This scalability is provided by the use of the modern concurrent programming paradigm provided by the Akka framework. The QUADrATiC Graphical User Interface (GUI) has been developed using advanced Javascript frameworks, providing novel visualization capabilities for further analysis of connections. There is also a web services interface, allowing integration with other programs or scripts.Conclusions: QUADrATiC has been shown to provide an improvement over existing connectivity map software, in terms of scope (based on the LINCS data set), applicability (using FDA-approved compounds), usability and speed. It offers potential to biological researchers to analyze transcriptional data and generate potential therapeutics for focussed study in the lab. QUADrATiC represents a step change in the process of investigating gene expression connectivity and provides more biologically-relevant results than previous alternative solutions.

Embracing an integromic approach to tissue biomarker research in cancer: Perspectives and lessons learned

Modern approaches to biomedical research and diagnostics targeted towards precision medicine are generating ‘big data’ across a range of high-throughput experimental and analytical platforms. Integrative analysis of this rich clinical, pathological, molecular and imaging data represents one of the greatest bottlenecks in biomarker discovery research in cancer and other diseases. Following on from the publication of our successful framework for multimodal data amalgamation and integrative analysis, Pathology Integromics in Cancer (PICan), this article will explore the essential elements of assembling an integromics framework from a more detailed perspective. PICan, built around a relational database storing curated multimodal data, is the research tool sitting at the heart of our interdisciplinary efforts to streamline biomarker discovery and validation. While recognizing that every institution has a unique set of priorities and challenges, we will use our experiences with PICan as a case study and starting point, rationalizing the design choices we made within the context of our local infrastructure and specific needs, but also highlighting alternative approaches that may better suit other programmes of research and discovery. Along the way, we stress that integromics is not just a set of tools, but rather a cohesive paradigm for how modern bioinformatics can be enhanced. Successful implementation of an integromics framework is a collaborative team effort that is built with an eye to the future and greatly accelerates the processes of biomarker discovery, validation and translation into clinical practice.

Challenging the cancer molecular stratification dogma: Intratumoral heterogeneity undermines consensus molecular subtypes and potential diagnostic value in colorectal cancer

Purpose:
A number of independent gene expression profiling studies have identified transcriptional subtypes in colorectal cancer (CRC) with potential diagnostic utility, culminating in publication of a CRC Consensus Molecular Subtype classification. The worst prognostic subtype has been defined by genes associated with stem-like biology. Recently, it has been shown that the majority of genes associated with this poor prognostic group are stromal-derived. We investigated the potential for tumor misclassification into multiple diagnostic subgroups based on tumoral region sampled.

Experimental Design:
We performed multi-region tissue RNA extraction/transcriptomic analysis using Colorectal Specific Arrays on invasive front, central tumor and lymph node regions selected from tissue samples from 25 CRC patients.

Results:
We identified a consensus 30 gene list which represents the intratumoral heterogeneity within a cohort of primary CRC tumors. Using a series of online datasets, we showed that this gene list displays prognostic potential (HR=2.914 (CI 0.9286-9.162) in stage II/III CRC patients, but in addition we demonstrated that these genes are stromal derived, challenging the assumption that poor prognosis tumors with stem-like biology have undergone a widespread Epithelial Mesenchymal Transition (EMT). Most importantly, we showed that patients can be simultaneously classified into multiple diagnostically relevant subgroups based purely on the tumoral region analysed.

Conclusions:
Gene expression profiles derived from the non-malignant stromal region can influence assignment of CRC transcriptional subtypes, questioning the current molecular classification dogma and highlighting the need to consider pathology sampling region and degree of stromal infiltration when employing transcription-based classifiers to underpin clinical decision-making in CRC.

Immune-derived PD-L1 gene expression defines a subgroup of stage II/III colorectal cancer patients with favorable prognosis that may be harmed by adjuvant chemotherapy

A recent phase 2 study of metastatic colorectal carcinoma (CRC) patients showed that mismatch repair gene status was predictive of clinical response to PD-1-targeting immune checkpoint blockade. Further examination revealed strong correlation between PD-L1 protein expression and microsatellite instability (MSI) in stage IV CRC, suggesting that the amount of PD-L1 protein expression could identify late stage patients who may benefit from immunotherapy. To assess whether the clinical associations between PD-L1 gene expression and MSI identified in metastatic CRC are also present in stage II/III CRC, we used in silico analysis to elucidate the cell types expressing the PD-L1 gene. We found a significant association of PD-L1 gene expression with MSI in early stage CRC (P < 0.001) and show that unlike in non-CRC tumors, PD-L1 is derived predominantly from the immune infiltrate. We demonstrate that PD-L1 gene expression has positive prognostic value in the adjuvant disease setting (PD-L1low v PD-L1high HR = 9.09; CI, 2.11-39.10). PD-L1 gene expression had predictive value, as patients with high PD-L1 expression appear to be harmed by standard-of-care treatment (HR = 4.95; CI,1.10-22.35). Building on the promising results from the metastatic CRC PD-1-targeting trial, we provide compelling evidence that PD-L1high/MSI/immunehigh stage II/III CRC patients should not receive standard chemotherapy. This conclusion supports the rationale to clinically evaluate this patient subgroup for PD-1 blockade treatment.

Vetus Testamentum cum glossa ordinaria, pars.

Avec arguments. Cant. canticorum V,11-VIII,14 (1) ; Osee I,3-XIV,10 (9) ; Joel I,6-III,21 (38) ; Amos I,1-IX,15 (48) ; Abdias I,7-21 (68) ; Jonas I,1-IV,11 (70v) ; Mich. I,6-VII,20 (77) ; Nahum I,1-III,19 (89v) ; Habacuc I,2-III,19 (95) ; Sophonias I,1-III,20 (102v) ; Agg. I,2-II,24 (110) ; Zachar. I,3-XIV,21 (115) ; Malach. I,4-IV,6 (139).

Morell (André). Correspondance numismatique d'André Morell, d'Arnstatt, pendant les années 1702 et 1703.

Copie du temps comprenant à la fois les lettres de Morell et celles de ses correspondants, savoir : André Fountaine ; — Al. Ferd. comes Marsilii ; — le prince seneschal de Ligne ; — Francesco de Ficoroni ; — Jo. Mich. Dilherrus ; — Jo. Lupt. Marinele Armengol. ; — Doctor J. C. Bon Cretensis ; — Jac. Perizonius ; — Gisb. Cuperus.

A Souvenir of the Niagara Falls, showing Summer and Winter views of Niagara Falls and their surroundings. --

Cover title: Niagara Falls illustrated, Summer and Winter.

Niagara and vicinity : views of the world's greatest wonder and its nearby cities. --

Cover title: Beauty spots of Niagara & vicinity.

Summer resorts reached by the Grand Trunk Railway and its connections : Including Niagara Falls, The Thousand Islands, Rapids of the St. Lawrence River, The White Mountains, Montreal, Quebec, The Saquenay River, and the seashore. --

Compliments of Passenger Department.