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SBIR/STTR: circulating cells and DNA in cancer detection.

Notice: This Request for Application (RFA) must be read in conjunction with the current Omnibus Solicitation of the National Institutes of Health (NIH), Centers for Disease Control and Prevention (CDC), and Food and Drug Administration (FDA) for Small Business Innovation Research (SI31R) Small Business Technology Trandfer (STFR) Grant Applications. The solicitation (see sbirsttr1/index.pdf or contains information about the SBIR and STTR programs, regulations governing the programs, and instructional information for submission. All of the instructions within the SBIR/STTR Omnibus Solicitation apply with the exception of the following: special receipt dates, and initial review convened by the National Cancer Institute (NC1) Division of Extramural Activities.

The Division of Cancer Prevention of the NCI invites small business applications for research projects to develop novel technologies for capturing, enriching, and preserving exfoliated abnormal cells and circulating DNA from body fluids or effusions and to develop methods to concentrate these cells and DNA for cancer biomarker detection.

In body fluids, such as sputum, the number of exfoliated tumor cells is often low compared to the number of normal cells, making it difficult to detect these abnormal cells by routine cytopathology. Separation of dysplastic cells from degenerating cells and cells undergoing non-specific reactive changes is problematic. Moreover, exfoliated cells are frequently contaminated with normal cells, bacteria, and cellular debris. Therefore, enrichment methods are needed to allow for routine detection and molecular analysis of small numbers of exfoliated cells.

Circulating extracelular DNA was first reported in 1948. It has been shown that the circulating DNA in the blood of cancer patients has genetic characteristics identical to those of the primary tumors. Thus, circulating DNA is an important material that may be useful for cancer detection. Currently available methods for isolating undegraded circulating DNA are limited, and there is a need to develop novel methods which improve the yield of undegraded DNA and to adapt detection assays so that this DNA can be used to detect mutations, microsatellite instabilities, loss of heterozygosity, epigenetic changes, and other molecular genetic changes.

This RFA will utilize the SBIR and STTR mechanisms, but will be run in parallel with a program announcement of identical scientific scope (PA-04035) that will utilize the exploratory/developmental (R21) grant mechanism.

Cellular and molecular changes that ensue during tumor progression occur over a number of years and to an apparently stochastic manner. For example, it takes an average of 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 have occurred. 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 cancel. Finding molecular and genetic biomarkers of malignancy is an extraordinary opportunity for the NCI and is particularly important in detecting the emergence of precancerous cell populations. In these earliest stages of neoplasia, lesions are more likely to be amenable to eradication. This principle has been well-demonstrated in cervical neoplasia, where screening for dysplastic exfoliated cells can result in a 70 percent or greater reduction in mortality due to cervical cancer. Detection of genetic abnormalities in preneoplastic lesions poses challenges because of the small size of lesions, the heterogeneity of precancerous cells, and the relatively low number of abnormal cells compared to normal cells.

More than 80 percent of human tumors (e.g. colon, lung, prostate, oral cavity, esophagus, stomach, uterine cervix, and bladder) originate from epithelial cells, often at a mucosal surface, and are clonal in origin. Cells from these rumors exfoliate spontaneously into blood, sputum, urine, and various effusions. Abnormalities within these exfoliated cells could be used to detect and identify' precancerous lesions or very early stage cancers if highly sensitive technologies were available to identify the presence of a few abnormal cells among millions of normal cells. For example, PCP, has been used to detect mutant DNAs in neoplastic exfoliated cells; mutations have been detected in ras genes present in stool samples obtained from patients with colorectal cancel, and in p53 from the urine of patients with bladder cancer and in the sputa of patients with lung cancer. Assays to detect genetic mutations, microsatellite instability, or hypermethylation may be adapted for use with exfoliated cells. As these assays are complex and technically challenging, their general use will require the development of novel technologies for isolating and enriching abnormal exfoliated cells.

Studies performed in the early 1970s showed that increased quantities of DNA are found in the plasma of patients suffering from different malignancies, but it was not until the 1990s that this circulating DNA was shown to exhibit tumor-related alterations. Mutant DNA has been found in the plasma of patients with colorectal, pancreatic, biliary tree, skin, head-and-neck, lung, breast, kidney, ovarian, nasopharyngeal, liver, bladder, gastric, prostate, and cervical cancers as well as in haematologic malignancies. Allelic imbalance (AI), which involves the loss or gain of chromosomal regions, is found in many cancers. AI can be detected in genomic tumor DNA released into the blood after cellular necrosis or apoptosis. These observations indicate that plasma/serum may be a suitable specimen source for noninvasive diagnostic, prognostic, and follow-up tests for cancer.

Precancerous exfoliated cells can be identified by cytologic examination of washings or brushings from bronchi, oral cavity, esophagus, stomach, bile and pancreatic ducts, as well as of sputum and urine specimens. However, the detection of these exfoliated cancer cells by routine cytopathological examination is very difficult because the number of abnormal cells in the specimens is usually very low compared to the number of normal cells. It is also difficult to distinguish low grade dysplasia from non-specific reactive or inflammatory changes due to the low sensitivity and specificity of current diagnostic methodologies. This is particularly true of urine cytology, where most low-grade papillary lesions are missed by cytologic examination. Even with new PCR-based technologies with enhanced sensitivity, current technologies for isolating exfoliated cells are too inefficient to be of practical utility. Therefore, the development of novel, high-throughput, sensitive technologies for sample preparations is a prerequisite for the successful detection of the small number of exfoliated cells or of the small amounts of DNA, RNA and proteins in these cells.

There are a variety of approaches to detect and analyze precancerous and cancerous cells in body fluids [e.g., cytopathological analysis, morphometric analysis, molecular biomarkers for specific receptors or genetic changes, Fluorescence in Situ Hybridization (FISH) analysis, or PCR-based analysis]. The selection of approach, in many instances, depends on the type of biological specimens (sputum, bronchial washing, cervical brushing, voided urine, etc.). Given that the concentration of the atypical epithelial cells can be very low compared to that of normal cells, all of these approaches require between 1 to 10,000 and 1 million enrichments of the atypical cells. Currently, there are two broad categories of enrichment methods: mechanical (centrifugation, cytospin, sucrose gradients, etc.) and antibody-based selection with mechanical separation (FACS--flow-assisted cell sorting, MACS--magnetic assisted cell sorting, etc.). While these two types of enrichment processes can be used in series to improve the yield, none of the currently available methods achieve sufficient enrichment of atypical cells to allow them to be routinely used for cancer detection.

The single largest barrier to using circulating DNA for cancer detection is the amount of circulating undegraded DNA that can be isolated is low, making it unsuitable for currently available assay technologies. Several factors affect the yield and purity of circulating DNA. Intracellular nuclease activity in both apoptotic and necrotic cells in a particular organ affect the degree of DNA degradation found in body fluids. Also, the degree to which a particular tissue is represented in the total circulating DNA is dependent on the mechanism and efficiency by which apoptotic cells are eliminated front the tissue.

As with any other diagnostic technique, practical application of circulating DNA technology is dependent on concurrent increase in the sensitivity and reproducibility of molecular based-assays. The potential use of circulating DNA for cancer detection could be greatly enhanced by developing isolation methods that result in less degradation and by adapting assay methods to use the low amounts that can be isolated. Because of the limitations of "conventional" markers, there has been a search for additional sources of specificity so as to expand the target pool of cancer-associated molecules. Circulating cells and DNA offer such opportunity for detection molecular aberrations in plasma/serum, or other body fluids, that accurately reflect the situation in primary tumor. This will, however, require the development of methodological consistencies so as to allow valid comparisons between various assays based on circulating cells or DNA.

The primary purpose of this initiative is to encourage the development of technologies for isolating and characterizing exfoliated cells, circulating cells, and plasma/serum DNA. A secondary purpose is the analytical validation of existing and/or newly developed technologies for their usefulness in cancer detection. Analytical validation refers to the measurement of sensitivity and reproducibility of the proposed assay/technology. The long-term goal of the technology development is to identify a panel of well-characterized biomarkers derived from exfoliated cells and/or circulating DNA that can be sampled in a 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 ( In pursuit of these goals, the NCI invites applications which address the following areas: 1) Development of high-throughput, high-yield technologies for isolating exfoliated cells, circulating cells and DNA in body fluids; 2) Development of methods for enrichment and preservation of exfoliated cells, circulating cells and DNA isolated front body fluids; 3) Development of sensitive, high-throughput molecular, cytomorphometric, immunologic, and other relevant technologies to isolate and characterize tumor cells in malignant effusions for detection of low tumor burden, to help distinguish reactive cells from rumor cells, and to perform accurate assays on circulating DNA; 4) Validation of the sensitivity and reproducibility of current technologies for isolating and characterizing exfoliated cells, circulating cells and DNA isolated from body fluids.

This RFA uses the SBIR and STTR mechanisms, which are set-aside programs. As an applicant, you will be solely responsible for planning, directing, and executing the proposed project. Future unsolicited, competing-continuation applications based on this project will compete with all SBIR/STTR applications and will be reviewed according to the customary peer review procedures. The anticipated award date is approximately 9-11 months from the respective receipt date. Applications that are not funded in the competition described in this RFA may be resubmitted as new SBIR/STTR applications using the standard receipt dates for new applications described in the current SBIR/STTR Omnibus Solicitation. As there are multiple receipt dates, it is possible that an unfunded application can be resubmitted under this RFA as a revised application.

This RFA uses just-in-time concepts. It also uses the modular budgeting as well as the non-modular budgeting formats. Specifically, if you are submitting an application budget of $100,000 total costs (direct, F&A and fee) or less, use the modular budget format. For applications requesting more than $100,000, rise the non-modular budget format. Instructions for both are described in the current SBIR/STTR Omnibus Solicitation. This program does not require cost sharing as defined in the current NIH Grants Policy Statement at

Except as otherwise stated in this RFA, awards will be administered under NIH grants policy as stated in the NIH Grants Policy Statement, December 2003, available at grants/policy/nihgps_2003/.

Applications may be submitted for support as Phase I STTR (R41) or Phase I SBIR (R43) grants; Phase II SBIR (R42) or Phase II SBIR (R44) grants; or the SBIR/STTR FAST-TRACK option as described in the SBIR/STTR Omnibus Solicitation. Phase II applications in response to this RFA will only be accepted as competing continuations of previously funded NIH Phase I SBIR/STTR awards. A Phase II application must be a logical extension of the Phase I research but not necessarily a Phase I project supported in response to this RFA. Fast Track applications will benefit from expedited evaluation of progress following the Phase I feasibility study for transition to Phase II funding for expanded developmental work.

Prospective applicants are asked to submit a letter of intent that includes the following information: descriptive title of the proposed research; name, address, and telephone number of the Principal Investigator; names of other key personnel; participating institutions, number and title of this RFA. Although a letter of intent is not required, is not binding, and does not enter into the review of a subsequent application, the information that it contains allows IC staff to estimate the potential review workload and plan the review.

The PHS 398 research grant application must be used for all SBIR/STTR Phase I, Phase II, and FastTrack applications (new and revised). Effective 1 October 2003, 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 866-705-5711 or through the website at http://www. The DUNS number should be entered on line 11 of the Face page of the PHS 398 form. The PHS 398 is available at http://grants.nih. gov/grants/funding/phs398/phs398.html. Prepare your application in accordance with the SBIR/STTR Omnibus Solicitation and the PHS 398. Helpful information for advice and preparation of the application can be obtained at The NIH will return applications that are not submitted on the 5/2001 version of the PHS 398. For further assistance, contact Grantslnfo 301-435-0714; e-mail:

Applications hand delivered by individuals to the NCI will no longer be accepted. This policy does not apply, to courier deliveries (i.e., FEDEX, UPS, DHL, etc.) (see This policy is similar to and consistent with the policy for applications addressed to Centers for Scientific Review as published in the NIH Guide Notice at

The Center for Scientific Research (CSR) will not accept any application in response to this RFA 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. However, when a previously unfunded application, originally submitted as an investigator-initiated application, is to be submitted in response to an RFA, it is to be prepared as a new application. That is, the application for the RFA must not include an introduction describing the changes and improvements made, and the text must not be marked to indicate the changes from the previous unfunded version of the application.

Letters of Intent are due 17 January 2005, 16 May 2005, and 14 September 2005. Applications are due 14 February 2005, 13 June 2005, and 12 October 2005. The earliest anticipated start dates are January 2006, April 2006, and July 2006.

Contact: Sudhir Srivastava, Division of Cancer Prevention, NCI, 6130 Executive Blvd, EPN Rm 3144, Bethesda, MD 20892-0001 USA, Rockville, MD 20852 (for express/courier service), 301-496-3983, fax: 301-402-8990, e-mail:; (for peer review issues) Referral Officer, NCI, Division of Extramural Activities, 6116 Executive Blvd, Rm 8041 MSC 8329, Bethesda, MD 20892-8329 USA, Rockville, MD 20852 (for express/courier service), 301-496-3428, fax: 301-402-0275, e-mail:

Reference: RFA No. RFA-CA-06-001
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Title Annotation:Announcements / Fellowships, Grants, & Awards
Publication:Environmental Health Perspectives
Date:Aug 15, 2004
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