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Identification and Capture of Enriched Tumor Zones with Preservation of Labile Biomarkers from Ultra-Cold Biopses

Personalized medicine approaches allow the treatment of patient tumors with drugs tailored to their tumors, which increase the probability of a beneficial response.  In the last decade, a number of pharmacodynamic (PD) markers have been identified that help the physician know that the drug is hitting the target or target pathway in the patient?s tumor.  Recently approved cancer treatments target either the cell surface receptors at the head of these signaling pathways or the intermediate phosphoproteins and kinases in the pathway signaling cascades. Understanding the phosphoprotein activation state of key signaling molecules in tumor cells can yield critical information on the type, stage and status of those cells, aiding in the diagnosis, prognosis and treatment of an individual?s disease.  Unfortunately, target analyte lability, especially with phosphoproteins, requires adoption of rapid and highly controlled tissue collection and handling before widespread clinical use of the assays in predicting drug response.  Also, in some cases, the magnitude of drug modulation of biomarker may be small but still significant in correlating to overall tumor response, which mandates the use of quantitative assays in enriched samples of tumor.  NCI/DCTD has developed analytic multiplexed ELISA type immunoassays to cancer drug targets/pathways that quantitate actual analyte levels in tumor lysates.
However, there is an inherent problem in the use of tissue lysates in that a tissue is comprised not only of tumor cells but also of normal parenchyma, stromal cells, inflammatory cells, vessels, and often significant necrosis, so when a specimen sample is homogenized the macromolecules extracted is a sum of  all cellular/matrix components, proportional to each representative element. Thus, the presence of non-target cells and necrosis can significantly affect the quantification of protein levels in ?viable? tumor cells.  Even the most sophisticated testing methods are of limited value when the input DNA, RNA, or protein is contaminated or diluted by non-target cells and necrotic/acellular matrix. The requirement for relatively pure cell populations has led to various technical solutions, most notable Laser Capture Microdissection (LCM). Microdissection techniques permit analysis of various molecular signatures within a specific cell population of a tissue and reduce the interference from non-target cell populations and acellular matrix such as fibrosis/necrosis. Although there are numerous reports of genetic and gene expression analyses of microdissected tumor populations, and of proteomic assessment using Western blot, 2D-PAGE, mass spectrometry, and peptide sequencing, studies using ELISA/immunoassay quantitation of key drug targets are limited. Tumor enrichment techniques that allow the rapid capture of tumor rich zones from core needle biopsies (not sections) have the potential to provide sufficient tumor amounts for analyte quantitation in tumor cells residing within a tissue with the sensitivity and specificity of ELISA. This technology will have significant clinical value. 
The purpose/goal of this SBIR topic is the development and commercialization of a visualization/microdissection system that is capable of identifying and capture of ?viable? tumor rich zones in frozen solid tumor biopsies under conditions that preserve labile pharmacodynamics (PD) biomarkers for antibody mediated quantitation. 
Microdissection technologies are powerful tools for the isolating enriched populations of tumor cells from cellular heterogeneous tissues. It has been shown that the harvested cells can be used for many molecular investigations including DNA, RNA, protein, microRNA, and protein analyses (See Figure 1 below). The goal of this SBIR topic is to adapt / improve existing visualization strategies for frozen solid tumor needle biopsies allowing microdissection of large tumor rich zones with 50% tumor region enrichment while preserving labile biomarkers. The desire is to include quantitative ELISA/immunoassay in the list of molecular analyses (see chart below) that can be performed reliably on microdissected cell populations by developing a system for the rapid identification and dissection of enriched tumor cell zones from frozen biopsies. These tumor rich zones should provide sufficient material for multiplex ELISA quantitation of biomarkers. This system will be much more time-effective and productive than the labor intensive microdissection of multiple tissue sections from a biopsy. The ability to quantify biomarkers via immunoassay of isolated biopsy zones enriched for ?viable? tumor cells is especially important when a biomarker is only modulated to a small extent requiring very sensitive and specific analytic assays. Of note, the development of this technology has the potential to improve the ?specificity? of any molecular test that involves homogenization of the tissue and macromolecular isolation.
 
Applied Immunohistochemistry & Molecular Morphology. 21(1):31-47, January 2013
References
1.      Wikipedia: https://en.wikipedia.org/wiki/Laser_capture_microdissection
2.      Applied Immunohistochemistry & Molecular Morphology: January 2013 – Volume 21 – Issue 1 – p 31?47, Cheng, Liang et al.
3.      Veterinary Pathology Onlinevet.sagepub.com
4.      Veterinary PathologyJanuary 2014vol. 51 no. 1 257-269.
5.      Laser Capture Microdissection for the Investigative Pathologist, H. Liu et al
6.      Published online before print November 13, 2013, doi: 10.1177/0300985813510533
7.      Espina V, Heiby M, Pierobon M, Liotta LA (2007). “Laser capture micro-dissection technology”. Expert Rev. Mol. Diagn. 7 (5): 647?57. doi:10.1586/14737159.7.5.647. PMID 17892370.
8.      Orba Y, Tanaka S, Nishihara H, Kawamura N, Itoh T, Shimizu M, Sawa H, Nagashima K (2003). “Application of laser capture microdissection to cytologic specimens for the detection of immunoglobulin heavy chain gene rearrangement in patients with malignant lymphoma”. Cancer 99 (4): 198?204. doi:10.1002/cncr.11331. PMID 12925980.
9.      Laser Microdissection & Pressure Catapulting”. University of Gothenburg.
10.   LCM User Community: https://abcommunity.lifetechnologies.com/community/laser_capture_microdissection_%28lcm%29
11.   Am J Pathol. 1999 Jan;154(1):61-6.Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis.Fend F1, Emmert-Buck MR, Chuaqui R, Cole K, Lee J, Liotta LA, Raffeld M.
12.   Molecular & Cellular Proteomics 9:2529?2544, 2010.In Situ Proteomic Analysis of Human Breast Cancer Epithelial Cells Using Laser Capture Microdissection: Annotation by Protein Set Enrichment Analysis and Gene Ontology*_SSangwon Cha?, Marcin B. Imielinski?, Tomas Rejtar?, Elizabeth A. Richardson?,Dipak Thakur?, Dennis C. Sgroi??**, and Barry L. Karger?**
Project Goals
ELISA/Immunoassays have the capacity to quantify the levels of a specific analytes in a specimen.  There are two important considerations in the reliability of immunoassays to accurately measure the levels of PD biomarkers and the use of the information in clinical care:
1.      Many of the drug biomarkers are very labile and require immediate processing after collection which usually involves rapid freezing or lysis in buffers with specific inhibitors (e.g., phosphatase inhibitors).
2.      Tumors are not of a homogeneous cell type and often contain significant amounts of necrotic and normal cell zones which will impinge on the accurate measurement of an analyte in the tumor population if the entire tissue sample is homogenized. 
It is believed that the development of microdissection techniques for identification and collection of viable tumor zones with at least 50% tumor enrichment from frozen solid tumor biopsies under conditions that preserve the stability of labile biomarkers can lead to detailed quantitative assessment of key protein targets modulated by drugs in tumor cells in their natural microenvironment and increase the PD utility of selected biomarkers.
The use of an appropriate visualization technologies and microdissection method such laser capture microdissection (LCM) is proposed.  The LCM process has the advantage of working with tissue in various physical states, particular frozen (a state that would preserve labile phosphoprotein biomarkers), and does not alter the morphology or biochemistry of the sample collected. LCM has demonstrated success in collecting selected cells for molecular analyses, particular when frozen sections are used. Limitations of LCM include difficulties of microdissection due to decreased optical resolution of tissue sections.  Possible options to improve cell resolution include the use of specialized optics to identify tumor zones via architectural/morphometric/light refraction/density parameters or the use of special stains or immunohistochemistry/immunofluorescence. This topic goal is to develop ?identification? or visualization and capture methods for frozen solid tumor needle biopsies or thick sections.  These techniques must not impinge on the stability of the labile biomarkers (i.e., frozen conditions must be maintained). 
The goalof this SBIR topic is to develop a reliable visualization approach to identify and capture zones of ?viable? tumor cells from frozen solid tumor biopsies that lead to at least 50% enrichment of tumor zones.  The technology should focus on distinguishing tumor regions from non-tumor zones and acellular matrix, such as necrosis, upon scanning frozen solid tumor biopsies or thick sections. This technology will result in the collection of relatively large areas of biopsy material that is enriched in tumor cells with preserved labile biomarkers and of sufficient amounts to be amenable to quantitation by ELISA/Immunoassay. 
The first objective should be to develop reliable methods to distinguish enriched ?viable? tumor zones from necrotic zones or viable tumor zones from normal cell types in frozen solid tumor biopsies or thick sections. Malignant transformation is associated with structural, genotypic/phenotypic cellular modifications, and biochemical changes, which as a consequence, alter the spectroscopic, metabolic and microscopic properties. These and other alterations may be exploited to develop a means to scan frozen solid tumor biopsies /thick sections and identify zones that correlate to cellular/acellular areas in the biopsy. The optimal scanning system should be able to extract information about the morphological/ architecture/ spectroscopic properties in the frozen biopsy that may be used to distinguish normal and malignant areas; such as, the measurement of color, overall density or light reflectance which may correlate to cellular density and/or vascularity which in turn may reflect tumor zones.
The second objective is to microdissect and capture the enriched tumor zones from frozen solid tumor biopsies/thick sections in a manner that preserves the label PD biomarkers, while maintaining the tumor zone as frozen throughout the process. Such a product will enable the reliable identification and capture of relatively large amounts of pure populations of tumor cells from frozen solid tumor biopsies which are amenable for quantification of label biomarkers via immunoassays, thus increasing the sensitivity and specificity of the assays for the target in tumor cells and their PD utility.   
Phase I Activities and Deliverables
The essential characteristics of a tumor enrichment microdissection system of frozen solid tumor biopsies should include all or some of the following features:
 
1)     a biopsy/thick section scanning/microdissection method that is adaptable for use with most common microscopic/microdissection systems with the capability to maintain the specimens under freezing temperatures;
2)     able to generate an easily interpretable signal indicative of parameters that can distinguish between necrotic and viable tumor, and if possible, also between tumor and non-tumor tissue;
3)     be capable of microdissection of enriched tumor zones without inducing significant cellular damage or change in labile PD biomarkers (i.e. maintain frozen state); and
4)     able to perform as designed and intended in fit-for-purpose studies in relevant clinical veterinary models (i.e. the method  has to produce tumor tissue of sufficient quantity and quality for ELISA based  immunoassay).
To accomplish the goal of this SBIR topic to develop microdissection techniques that reliably identifies and captures tumor-rich ?viable? zones in frozen, unfixed tumor biopsy cores or thick sections in a manner that preserves labile drug target/pathway PD biomarkers for quantitative ELISA/Immunoassay analyses, the Phase I deliverables are:
?       Develop a microscopic visualization/microdissection method to identify and capture ?viable/cellular? tumor zones from frozen tumor biopsies/thick sections from at least two solid tumor types while maintaining the frozen state of the specimen.  The use of the entire needle tumor biopsy is preferred. Any gauge needle biopsy can be used for Phase I development, but 18 gauge is desired for Phase II. The readout from the visualization/scan of the frozen biopsy should be easily interpretable (e.g. cellular rich or acellular rich) and is associated with the level of necrosis and ?viable? cell-rich tumor zones, and if possible, between tumor and non-tumor ?normal? zones.  Xenograft tumors may be used for developing this technology.  NCI can recommend specific xenografts that have sufficient levels of specific labile PD biomarkers and are known for developing necrosis. (See Table 1 below).
?       The purity and cellularity of the captured zones can be assessed by H&E staining and imaging.  Initially the histology of the entire biopsy will need to be analyzed to associate the visualization measurement with the histology (i.e. the cellular quantity and cellular types present throughout the biopsy).  One option is that during the development phase, the biopsy can be cut longitudinally into 2 halves with one side being subjected to frozen sectioning and H&E staining for histological evaluation and the 2nd half to being scanned via the visualization method. This will allow the visualization measures to be related to the histology of the biopsy. As to the quantitative needs, it will depend on the assay; for example, for the DCTD apoptosis multiplex immunoassay, 20 ug of protein is required per panel, preferably with 50% tumor cell content.
?       Demonstrate that the visualization/capture technology preserves in the enriched tumor cells at least one of labile protein biomarkers of interest to NCI (see Table 1 below).  Maintaining the biopsy specimen under freezing conditions is mandatory and it is preferred that the enriched tumor zones be kept frozen. If specimen loss after capture is an issue, then the use of special lysis buffer may be approved by NCI. NCI may provide assistance in the analysis of the biomarker levels in isolated tumor zones. 
?       The device and methodology need to be independently tested  at a different laboratory.  NCI may be willing to perform the independent validation/field testing of the breadboard prototype device and associated methodology.
Table 1: Suggested Phospho-protein PD biomarkers and associated xenograft models

Priority

Name

Phospho-Site(s)

Xenograft Models

1

MET Receptor

pY1234/1235, pY1356

MKN45, GTL16

2

Akt1  Kinase

pT308, pS473

Calu-1, H-23

3

ERK1/ERK2 Kinases
(* Internal control for one and two)

ERK1: pY202, pT204
ERK2: pY185, pT187

 
Calu-1, H-23

 
Phase II Activities and Deliverables
The Phase II activities should be focused on the design criteria/specification and fabrication of the alpha prototype biopsy visualization/microdissection system. The size of the needle biopsy is recommended to be 18 gauge for Phase II activities.  The activities/deliverables are:
?       Processes/instrumentation should be optimized to reproducibly identify and allow microdissection of ?viable? cell rich tumor zones from frozen tumor biopsies/thick sections of at least 4 different tumor types, evaluating a minimum of 6 specimens from each type (e.g., xenograft models).  Reproducibility should be demonstrated by 2 users on 3 different days. Images of H&E stained slides of the isolated tumor zones should be provided to demonstrate reliability of the technique.
?       Demonstrate preservation of 2 labile PD biomarkers via ELISA/immunoassay. NCI may provide assistance in this task.
?       The alpha prototype device and associated methodology should undergo independent validation/field testing at a separate laboratory. NCI may be willing to do this.  
 
?       Provide the program and contract officers with a letter of commercial interest. 

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