Circulating cells in cancer detection.

The purpose of this PA is to develop novel technologies for capturing, enriching, and preserving exfoliated abnormal cells and macromolecules in body fluids or effusions and to develop methods for concentrating the enriched cells for biomarker studies. In the context of this PA, we have extended the definition of exfoliation to include not only the cellular materials, but also subcellular materials, such as DNA and proteins. In body fluids, such as sputum, the number of exfoliated tumor cells is often small compared to the number of non-neoplastic cells. Therefore, the detection of exfoliated abnormal cells by routine cytopathology is often limited because few atypical cells may be present in the specimen. There may be difficulty in separating dysplastic cells from non-specific reactive changes and degenerating cells or variation in diagnostic criteria. Furthermore, exfoliated cells are frequently contaminated with normal cells, bacteria, and other cellular debris, which makes molecular analysis difficult without physical separation of the neoplastic cells. Thus, the development of enrichment methods becomes prerequisite for the routine detection of small numbers of exfoliated cells and small amounts of subcellular materials in biological fluids for molecular analysis. Similarly, subcellular materials are in amounts that may not be detectable by available technologies and therefore the enrichment of such materials is of paramount importance. Enrichment will allow exfoliated cells and subcellular molecules, for example from urine, to be used for genomic, proteomic, and epigenomic analyses that may lead to improvements in the detection of bladder cancer through measurements of alterations in expressed genes, peptide profiles, and epigenetic markers.

The most common human tumors arise from epithelial surfaces (e.g. colon, lung, prostate, oral cavity, esophagus, stomach, uterine cervix, bladder). Their development often becomes apparent when tumor cells exfoliate spontaneously into sputum, urine, or even into various effusions. The molecular and genetic abnormalities within these exfoliated cells could be used to detect and identify precancerous lesions or very early stage cancer if highly sensitive technologies were clinically available to identify the few abnormal cells among millions of normal cells. For example, detection of widespread microsatellite instability (MSI), as demonstrated by expansion or deletion of repeat elements of DNA, may be adapted for exfoliated cells in general. With the advent of PCR-based detection of DNA from rare neoplastic cells in body fluids, mutations have been detected in ras genes from the stools of patients with colorectal cancer, in p53 from the urine of patients with bladder cancer, and in p53 genes in the sputum of patients with lung cancer. As these assays are complex and technically challenging, they depend on the development of novel technologies for isolating and enriching cells or subcellular materials of interest. Abnormal exfoliated cells can be routinely identified by cytologic examination of brushings and fluids, for instance, from bronchi, pancreatic ducts, voided urine, and effusions. Currently, fluids are usually processed by centrifugation or membrane filtration. However, the detection of abnormal exfoliated cells, for instance, cancer cells by routine cytopathological examination may be limited because the number of abnormal cells may be very small compared to the number of normal cells, is difficult. Alternatively, the cellular and nuclear changes in abnormal cells may be minimal compared to normal cells. This is particularly true of cytological examinations of urine cytology, where many low-grade papillary lesions are often missed. New PCR-based technologies may substantially enhance the sensitivity, but current technologies for isolating and analyzing exfoliated cells are too cumbersome to be of practical utility. The cellular and molecular changes that ensue during tumor progression do so over a number of years and in an apparently stochastic manner. This progressive accumulation of genetic and epigenetic changes in precancerous cell populations eventually confers the malignant phenotype on emerging clonal subpopulations. In human and animal clinical and experimental models, the progression of precancer to cancer is known to be lengthy. For example, it takes an average of estimated 15 to 20 years for a small adenomatous polyp to become malignant. Prior to the appearance of a morphologically identified precancerous lesion, numerous genetic and molecular alterations would have already occurred. During histological progression into a morphologically identifiable lesion, the stochastic process of molecular events in different cells confers genetic heterogeneity.

Finding molecular and genetic biomarkers of malignancy is particularly important in detecting the emergence of precancerous cell populations and is what the NCI considers to be an "Extraordinary Opportunity." In these earliest stages of neoplasia, lesions should be amenable to complete eradication. This principle has been well-demonstrated in cervical neoplasia, where screening for dysplastic exfoliated cells can result in a 70% or greater reduction in the cervical cancer mortality. During the early stages of cancer development, there is a window of opportunity to detect precancerous cells with genetic or molecular biomarkers that identify and characterize their progression towards cancer. Detection of genetic abnormalities in preneo-plastic lesions poses challenges because of the small size of lesions, the heterogeneity of precancerous cells, and their dilution by normal cellular constituents. Therefore, assays should be tailored to detect a small number of abnormal cells or molecules among a large number of normal cells or molecules in biological fluids, such as in colonic washes of the gastrointestinal tract, in sputa, and in bronchial biopsies.

In order to detect and analyze precancerous and cancerous cells in biologic fluids, there are a variety of approaches. The most appropriate approach depends upon 1) the type of biological fluid (sputum, bronchial washing, cervical brushing, voided urine, etc.), and 2) the form of analysis to be performed (e.g., cytopathological analysis, morphometric analysis, molecular biomarkers for specific receptors or genetic changes, FISH-or-PCR based analyses). All of these approaches require an enrichment of atypical epithelial cells through selective processing to concentrate the assay target of interest. The enrichment methods currently used can be grouped into the following two broad categories: 1) mechanical (centrifugation, cytospin, sucrose gradients, etc.) and 2) antibody-based selection with mechanical separation (FACS--flow assisted cell sorting, MACS--magnetic assisted cell sorting, etc.). While one type of enrichment process can be sequentially added to another to improve the yield, all of these methods have good but not adequate sensitivity or specificity required for detecting precancerous cells in body fluids. Given that the concentration of these cells or molecules can be very low compared to other commonly present cell types or molecules, one needs enrichment factors of 1 to 10,000 or 1 to million.

More than 80 percent of human tumors originate from epithelial cells, often at a mucosal surface, and are clonal in origin. Precancerous exfoliated cells can be routinely identified in pathology departments by cytologic examination of washings or brushings from bronchi, oral cavity, esophagus, stomach, bile and pancreatic ducts, sputum and urine; however, the detection of exfoliated cancer cells by routine cytopathological examination is limited because of the presence of few atypical cells in specimens, the difficulty of distinguishing low grade dysplasias from non-specific reactive or inflammatory changes, and the low sensitivity and specificity of the available diagnostic methodology. These limitations are particularly true of urine cytology, where most low-grade papillary lesions are missed on cytologic examination of urine. New PCR-based technologies may substantially enhance sensitivity, but current technologies for isolating exfoliated cells are too cumbersome to be of practical utility. For example, exfoliated cells are frequently contaminated with normal cells, bacteria, and other cellular debris, making molecular analysis difficult without further physical separation of neoplastic cells. Therefore, the development of novel, high-throughput, sensitive technologies for sample preparation is a prerequisite for the successful detection of small numbers of exfoliated cells or small amounts of subcellular materials, such as DNA and proteins.

There are occasions in which the only biologic materials available from patients are stored plasma or serum samples. The amount of DNA in these samples are generally very low when they are obtained from normal (healthy) individuals, but increased amounts of circulating DNA have been found in cancer. The circulating DNA in plasma/serum of cancer patients has been shown to reflect the characteristics of the tumor DNA including molecular changes, such as methylation, point mutations, and microsatellite instability. Fragmented nucleosomal DNA in plasma resulting from apoptotic death of the tumor cells may also provide an indication for tumor DNA. There is a need to develop high-yield technologies to isolate circulating DNA that can be used for early detection of cancer and the follow-up of the disease.

The primary purpose of this initiative is to encourage the development of high-throughput technologies to facilitate the isolation and enrichment of exfoliated cells and subcellular materials. In pursuit of these goals, the NCI invites applications that address the following areas: 1) Development of high-throughput technologies for identifying abnormal exfoliated ceils and subcellular materials in body fluids; 2) Development of sampling technologies for capturing and preserving exfoliated tumor cells and subcellular materials in body fluids; 3) Development of enrichment methods for the isolation of tumor cells and subcellular materials; 4) Development of sensitive, high-throughput molecular, cytomorphometric, immunologic, and other relevant technologies to isolate tumor cells or subcellular materials in malignant effusions to help detect low tumor burden and distinguish reactive cells from tumor cells. The long-term goal, to which this initiative will eventually lead, is the development of panels of well-characterized biomarkers derived from exfoliated cells that can be sampled in the clinical setting. These methodologies will be tested and validated in future population-based clinical trials, and integrated into a comprehensive information system that will be developed under the Early Detection Research Network.

This PA will use the NIH exploratory/ developmental (R21) award mechanism. As an applicant, you will be solely responsible for planning, directing, and executing the proposed project. The applicant may request a project period of up to two years with a combined budget for direct costs of up $275,000 for the two year period. For example, the applicant may request $100,000 in the first year and $175,000 in the second year. The request should be tailored to the needs of the project. Normally, no more than $200,000 may be requested in any single year. These grants are non-renewable and continuation of projects developed under this PA will be through the traditional unsolicited investigator initiated grant program. This PA uses just-in-time concepts. It also uses the modular budgeting format. (see http://grants.nih.gov/grants/funding/ modular/modular.htm). Specifically, if you are submitting an application with direct costs in each year of $250,000 or less, use the modular format. This program does not require cost sharing as defined in the current NIH Grants Policy Statement at http://grants.nih.gov/ grants/policy/nihgps_2001/part_i_1.htm.

Applications must be prepared using the PHS 398 research grant application instructions and forms (rev. 5/2001). Applications must have a Dun and Bradstreet (D&B) Data Universal Numbering System (DUNS) number as the Universal Identifier when applying for Federal grants or cooperative agreements. The DUNS number can be obtained by calling [phone] or through the web site at http://www.dunandbradstreet.com/. The DUNS number should be entered on line 11 of the face page of the PHS 398 form. The PHS 398 document is available at http://grants.nih.gov/grants/funding/phs398/ phs398.html in an interactive format. For further assistance contact GrantsInfo, 301-435-0714, e-mail: [e-mail].

The title and number of the PA must be typed on line 2 of the face page of the application form and the YES box must be checked.

Supplementary Instructions: All instructions for the PHS 398 (rev. 5/2001) must be followed, with these exceptions: Research Plan: Items a--d of the Research Plan (Specific Aims, Background and Significance, Preliminary Studies, and Research Design and Methods) may not exceed a total of 15 pages. No preliminary data is required but may be included if it is available. Please note that a Progress Report is not needed; competing continuation applications for an exploratory/developmental grant will not be accepted. Appendix: Use the instructions for the appendix detailed in the PHS 398 except that no more than 5 manuscripts, previously accepted for publication, may be included. For the NIH Exploratory/Developmental Grant (R21), applicants may request direct costs in $25,000 modules, up to a total direct cost of $275,000 for the combined two year award period.

Applications must be received by or mailed on or before the receipt dates described at http://grants.nih.gov/grants/ funding/submissionschedule.htm.

The CSR will nor accept any application in response to this PA that is essentially the same as one currently pending initial review unless the applicant withdraws the pending application. The CSR will not accept any application that is essentially the same as one already reviewed under this PA. This does not preclude the submission of a substantial revision of an unfunded version of an application already reviewed, but such application must include an introduction addressing the previous critique. Unfunded applications previously reviewed as investigator-initiated applications under a different research grant mechanism may be resubmitted as a new application under this PA (see http:// grants.nih.gov/grants/guide/notice-files/NOT-OD-03-019.html).

Contact: Mukesh Verma, Division of Cancer Prevention, NCI, Executive Plaza North, EPN 3144, Bethesda, MD 20892-0001 USA, Rockville, MD 20852 (for express/courier service), 301-496-3893, fax: [phone], e-mail: [e-mail]; Sudhir Srivastava, Division of Cancer Prevention, NCI, Executive Plaza North, EPN 3142, Bethesda, MD 20892-0001 USA, Rockville, MD 20852 (for express/ courier service), 301-496-3983, fax: [phone], e-mail: [e-mail]