Neil Gray

Vascular Endothelial Growth Factor Receptors VEGFR-2 and VEGFR-3 Are Localized Primarily to the Vasculature in Human Primary Solid Cancers

Purpose: Vascular endothelial growth factor (VEGF) signaling is key to tumor angiogenesis and is an important target in the development of anticancer drugs. However, VEGF receptor (VEGFR) expression in human cancers, particularly the relative expression of VEGFR-2 and VEGFR-3 in tumor vasculature versus tumor cells, is poorly defined. Experimental Design: VEGFR-2– and VEGFR-3–specific antibodies were identified and used in the immunohistochemical analysis of human primary cancers and normal tissue. The relative vascular localization of both receptors in colorectal and breast cancers was determined by coimmunofluorescence with vascular markers. Results: VEGFR-2 and VEGFR-3 were expressed on vascular endothelium but not on malignant cells in 13 common human solid tumor types (n > 400, bladder, breast, colorectal, head and neck, liver, lung, skin, ovarian, pancreatic, prostate, renal, stomach, and thyroid). The signal intensity of both receptors was significantly greater in vessels associated with malignant colorectal, lung, and breast than adjacent nontumor tissue. In colorectal cancers, VEGFR-2 was expressed on both intratumoral blood and lymphatic vessels, whereas VEGFR-3 was found predominantly on lymphatic vessels. In breast cancers, both receptors were localized to and upregulated on blood vessels. Conclusions: VEGFR-2 and VEGFR-3 are primarily localized to, and significantly upregulated on, tumor vasculature (blood and/or lymphatic) supporting the majority of solid cancers. The primary clinical mechanism of action of VEGF signaling inhibitors is likely to be through the targeting of tumor vessels rather than tumor cells. The upregulation of VEGFR-3 on tumor blood vessels indicates a potential additional antiangiogenic effect for dual VEGFR-2/VEGFR-3–targeted therapy. Clin Cancer Res; 16(14); 3548–61. ©2010 AACR.

Antibody validation of immunohistochemistry for biomarker discovery: Recommendations of a consortium of academic and pharmaceutical based histopathology researchers

As biomarker discovery takes centre-stage, the role of immunohistochemistry within that process is increasing. At the same time, the number of antibodies being produced for “research use” continues to rise and it is important that antibodies to be used as biomarkers are validated for specificity and sensitivity before use. This guideline seeks to provide a stepwise approach for the validation of an antibody for immunohistochemical assays, reflecting the views of a consortium of academic and pharmaceutical based histopathology researchers. We propose that antibodies are placed into a tier system, level 1–3, based on evidence of their usage in immunohistochemistry, and that the degree of validation required is proportionate to their place on that tier.

Benefits of mTOR kinase targeting in oncology: pre-clinical evidence with AZD8055

AZD8055 is a small-molecule inhibitor of mTOR (mammalian target of rapamycin) kinase activity. The present review highlights molecular and phenotypic differences between AZD8055 and allosteric inhibitors of mTOR such as rapamycin. Biomarkers, some of which are applicable to clinical studies, as well as biological effects such as autophagy, growth inhibition and cell death are compared between AZD8055 and rapamycin. Potential ways to develop rational combinations with mTOR kinase inhibitors are also discussed. Overall, AZD8055 may provide a better therapeutic strategy than rapamycin and analogues.

The establishment of a network of European human research tissue banks.

This is a report of a workshop held on the establishment of human research tissue banking which was held in Levi, Finland 21–24 March 2002.There were 21 participants from 7 European countries. This meeting was attended by representatives from academia, research tissue banks and from the Biotech and Pharmaceutical Industries. The principal aim of the workshop was to find a way to progress the recommendations from ECVAM workshop 44 (ATLA 29, 125–134,2001) and ECVAM workshop 32 (ATLA 26, 763–777, 1998). The workshop represented the first unofficial meeting of the European Network of Research Tissue Banks (ENRTB) steering group. It is expected that in the period preceding the next workshop the ENRTB steering group will co-ordinate the ethical,legislative and organisational aspects of research tissue banking. Key issues dealt with by the Levi workshop included the practical aspects of sharing expertise and experiences across the different European members. Such collaboration between research tissue banks and end users of such material seeks to ultimately enable shared access to human tissue for medical and pharmaco-toxicological research while maintaining strict adherence to differences in legal and ethical aspects related to the use of human tissue in individual countries.

A retrospective analysis of RET translocation, gene copy number gain and expression in NSCLC patients treated with vandetanib in four randomized Phase III studies

BackgroundTo determine the prevalence of RET rearrangement genes, RET copy number gains and expression in tumor samples from four Phase III non-small-cell lung cancer (NSCLC) trials of vandetanib, a selective inhibitor of VEGFR, RET and EGFR signaling, and to determine any association with outcome to vandetanib treatment.MethodsArchival tumor samples from the ZODIAC (NCT00312377, vandetanib ± docetaxel), ZEAL (NCT00418886, vandetanib ± pemetrexed), ZEPHYR (NCT00404924, vandetanib vs placebo) and ZEST (NCT00364351, vandetanib vs erlotinib) studies were evaluated by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in 944 and 1102 patients.ResultsThe prevalence of RET rearrangements by FISH was 0.7% (95% CI 0.3–1.5%) among patients with a known result. Seven tumor samples were positive for RET rearrangements (vandetanib, n = 3; comparator, n = 4). 2.8% (n = 26) of samples had RET amplification (innumerable RET clusters, or ≥7 copies in > 10% of tumor cells), 8.1% (n = 76) had low RET gene copy number gain (4–6 copies in ≥40% of tumor cells) and 8.3% (n = 92) were RET expression positive (signal intensity ++ or +++ in >10% of tumor cells). Of RET-rearrangement-positive patients, none had an objective response in the vandetanib arm and one patient responded in the comparator arm. Radiologic evidence of tumor shrinkage was observed in two patients treated with vandetanib and one treated with comparator drug. The objective response rate was similar in the vandetanib and comparator arms for patients positive for RET copy number gains or RET protein expression.ConclusionsWe have identified prevalence for three RET biomarkers in a population predominated by non-Asians and smokers. RET rearrangement prevalence was lower than previously reported. We found no evidence of a differential benefit for efficacy by IHC and RET gene copy number gains. The low prevalence of RET rearrangements (0.7%) prevents firm conclusions regarding association of vandetanib treatment with efficacy in the RET rearrangement NSCLC subpopulation.Trial registrationRandomized Phase III clinical trials (NCT00312377, ZODIAC; NCT00418886, ZEAL; NCT00364351, ZEST; NCT00404924, ZEPHYR).

Validation of the BRCA1 antibody MS110 and the utility of BRCA1 as a patient selection biomarker in immunohistochemical analysis of breast and ovarian tumours

BRCA1 protein measurement has previously been evaluated as a potential diagnostic marker without reaching a conclusive recommendation. In this study, we applied current best practice in antibody validation to further characterize MS110, a widely used antibody targeting BRCA1. Antibody specificity was investigated using different biochemical validation techniques. We found that BRCA1 could not be reliably detected using immunoprecipitation and Western blot in endogenously expressing cells. We used immunohistochemistry on formalin-fixed paraffin-embedded cell pellets to establish compatibility with formalin-fixed paraffin-embedded samples. We demonstrated that in transfected cells and cell lines with known genetic BRCA1 status, MS110 successfully detected BRCA1 giving the expected level of staining in immunohistochemistry. Following this, we investigated the use of BRCA1 protein measurement by immunohistochemistry in a cohort of triple negative breast and serous ovarian tumour samples to explore the use of BRCA1 protein measurement by immunohistochemistry for patient stratification. Using MS110 in repeated standardized experiments, on serial sections from a panel of patient samples, results demonstrated considerable run-to-run variability. We concluded that in formalin-fixed tissue samples, MS110 does detect BRCA1; however, using standard methodologies, BRCA1 expression levels in tissue samples is incompatible with the use of this protein as a statistically robust patient selection marker in immunohistochemistry. These results demonstrate the need for further development to deliver BRCA1 protein quantification by immunohistochemistry as a patient stratification marker.

Identification of a Subset of Human Non–Small Cell Lung Cancer Patients with High PI3Kβ and Low PTEN Expression, More Prevalent in Squamous Cell Carcinoma

Purpose: The phosphoinositide 3-kinase (PI3K) pathway is a major oncogenic signaling pathway and an attractive target for therapeutic intervention. Signaling through the PI3K pathway is moderated by the tumor suppressor PTEN, which is deficient or mutated in many human cancers. Molecular characterization of the PI3K signaling network has not been well defined in lung cancer; in particular, the role of PI3Kβ and its relation to PTEN in non–small cell lung cancer NSCLC remain unclear. Experimental Design: Antibodies directed against PI3Kβ and PTEN were validated and used to examine, by immunohistochemistry, expression in 240 NSCLC resection tissues [tissue microarray (TMA) set 1]. Preliminary observations were extended to an independent set of tissues (TMA set 2) comprising 820 NSCLC patient samples analyzed in a separate laboratory applying the same validated antibodies and staining protocols. The staining intensities for PI3Kβ and PTEN were explored and colocalization of these markers in individual tumor cores were correlated. Results: PI3Kβ expression was elevated significantly in squamous cell carcinomas (SCC) compared with adenocarcinomas. In contrast, PTEN loss was greater in SCC than in adenocarcinoma. Detailed correlative analyses of individual patient samples revealed a significantly greater proportion of SCC in TMA set 1 with higher PI3Kβ and lower PTEN expression when compared with adenocarcinoma. These findings were reinforced following independent analyses of TMA set 2. Conclusions: We identify for the first time a subset of NSCLC more prevalent in SCC, with elevated expression of PI3Kβ accompanied by a reduction/loss of PTEN, for whom selective PI3Kβ inhibitors may be predicted to achieve greater clinical benefit. Clin Cancer Res; 20(3); 595–603. ©2013 AACR.

Banking human tissue for research: vision to reality

Advances in scientific understanding of disease together with introduction of new high throughput technologies have led to increased demand for human tissue in research. In general, patients are willing to donate for research, particularly samples that are surplus to diagnostic or therapeutic requirements. New tissue-specific regulations in the UK are intended to facilitate the use of human tissue in research. Despite this positive environment there are challenges to researcher access. Coordinated, systematic collection and storage, via a biobank can provide easier access. However translating a vision for a biobank into reality whether in the public or private sector, has never been simple. But it can be done.

Providing Human Tissue for Research: 1996-2006

This article details the authors’ experience establishing infrastructure for tissue collection, storage and distribution for biomedical research, firstly within a public healthcare service and latterly in the pharmaceutical industry. Access to human tissue in the context of public-private collaboration in research and development is essential to the provision of high-quality medicines and healthcare and is now supported by a new legal framework in England and Wales. Through collaborations there are opportunities for mutual benefit for patients and professionals alike. Attention to the wishes of tissue donors through informed consent at the outset ensured confidence and continued activity when so-called ‘organ retention scandals’ emerged midway through this period. The overwhelming majority of potential donors support the use of their tissues in biomedical research irrespective of where the research is carried out.

Cadaveric Tissue Supply to the Commercial Sector For Research: Collaboration between NHS Pathology and NBS Tissue Services in the U.K., Extending the Options for Donors.

The Peterborough Hospital Human Tissue Bank (PHHTB) and National Blood Service Tissue Services (London and South East Zone) (NBSTS) operate within the U.K. National Health Service (NHS) and have a system in place to retrieve cadaveric tissues for commercial sector research. The collaboration meets the aims of PHHTB and NBSTS and is legal, ethical and safe. This paper presents the results of the first 20 successful retrievals referred from NBSTS to PHHTB. Cadaveric retrieval of tissue for research extends the options for donors and their relatives. The research option is particularly welcomed in cases where clinical retrieval for tissue transplantation is contraindicated. We believe the system is applicable to other centres.