DEVELOPMENT OF HIGH-YIELD TECHNOLOGIES FOR ISOLATING EXFOLIATED CELLS IN BODY 
FLUIDS
 
Release Date:  November 15, 2000
 
PA NUMBER: PAR-01-019 (see replacement PA-02-086)
 
National Cancer Institute

Letter of Intent Date:  March 6, 2001, October 11, 2001 
Application Receipt Date: April 10, 2001, November 15, 2001

THIS PA USES THE "MODULAR GRANT" AND "JUST-IN-TIME" CONCEPTS. IT INCLUDES 
DETAILED MODIFICATIONS TO STANDARD APPLICATION INSTRUCTIONS THAT MUST BE USED 
WHEN PREPARING APPLICATIONS IN RESPONSE TO THIS PA.

PURPOSE

The purpose of this Program Announcement (PA) is to develop novel technologies 
for capturing, enriching, and preserving exfoliated abnormal cells in body 
fluids or effusions and to develop methods for concentrating the enriched 
cells for biomarker studies. 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. Furthermore, 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 is a 
prerequisite for the routine detection of small numbers of exfoliated cells 
and small amounts of subcellular materials in biological fluids for molecular 
analysis.

Small businesses are encouraged to respond to a parallel RFA CA-01-016 (see 
http://grants.nih.gov/grants/guide/rfa-files/RFA-CA-01-016.html) of identical 
scientific scope that uses the SBIR and STTR mechanisms.

RESEARCH OBJECTIVES 

Background

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 stool of patients 
with colorectal cancer, and in p53 from the urine of patients with bladder 
cancer and 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 exfoliated cells.  

Abnormal exfoliated cells can be routinely identified by cytologic examination 
of brushings and fluids, for instance, from bronchi, pancreatic ducts, voided 
urine and tapping of 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. Alternatively, the cellular and 
nuclear changes in abnormal cells may be minimal compared to normal cells. 
This is particularly true of urine cytology, where many low-grade papillary 
lesions are often missed on cytological examination.  New PCR-based 
technologies may substantially enhance the  sensitivity, but current 
technologies for isolating exfoliated cells are too cumbersome to be of 
practical utility. 

Finding molecular and genetic biomarkers of early cancer represents an 
extraordinary opportunity for the National Cancer Institute (NCI) and is 
particularly important in detecting the emergence of precancerous cell 
populations.  In these earliest stages of neoplastic development, lesions 
should be amenable to complete eradication.  This has been well demonstrated 
in cervical neoplasia, where screening for brushed exfoliated cells has 
resulted in a 70% or greater reduction in cervical cancer mortality. Brushing 
of the esophagus or stomach is commonly performed during endoscopic 
examination.

Goals and Scope

In pursuit of these goals, the NCI invites applications to address the 
following areas: 
o  development of novel technologies for identifying abnormal exfoliated cells 
in body fluids; 
o  development of novel technologies for capturing, enriching, and preserving 
abnormal exfoliated cells in body fluids;
o  development of enrichment methods for the isolation of tumor cells; 
o  development of sensitive, high-throughput molecular, cytomorphometric, 
immunologic, and other relevant technologies to isolate tumor cells in 
malignant effusions for detection of low tumor burden and to help distinguish 
reactive cells from tumor cells. 

Applicants are encouraged to address the technology of enriching and isolating 
exfoliated cells, 
but they should also address their viability and usefulness for cytologic and 
molecular studies.
 
MECHANISM OF SUPPORT 

This PA will use the National Institutes of Health (NIH) 
exploratory/developmental (R21) grant mechanism.  Responsibility for the 
planning, direction, and execution of the proposed project will be solely that 
of the applicant.  The total project period for an application submitted in 
response to this PA may not exceed TWO years. Though the size of award may 
vary with the scope of research proposed, it is expected that applications 
will stay within the budgetary guidelines for an exploratory/developmental 
project; direct costs are limited to $100,000 (four budget modules) per year 
unless the application includes consortium costs, in which case the limit is 
$125,000 direct costs (five budget modules) per year. These grants are non-
renewable and continuation of projects developed under this PA will be through 
the traditional unsolicited investigator initiated grant program. 

Specific application instructions have been modified to reflect "MODULAR 
GRANT" and "JUST-IN-TIME" streamlining efforts being examined by the NIH. 
Complete and detailed instructions and information on Modular Grant 
applications can be found at 
http://grants.nih.gov/grants/funding/modular/modular.htm

ELIGIBILITY REQUIREMENTS
 
Applications may be submitted by foreign and domestic, for-profit and not-
for-profit organizations, public and private, such as universities, colleges, 
hospitals, laboratories, units of State and local governments, and eligible 
agencies of the Federal government.  Racial/ethnic minority individuals, 
women, and persons with disabilities are encouraged to apply as principal 
investigators. 

INQUIRIES
  
Inquiries are encouraged.  The opportunity to clarify any issues or questions 
from potential applicants is welcome.
 
Direct inquiries regarding programmatic issues to:
 
Sudhir Srivastava, Ph.D., M.P.H.
Division of Cancer Prevention
National Cancer Institute
EPN, Room 330
Rockville, MD  20852
Telephone:  (301) 435-1594
FAX:  (301) 402-0816
Email:  ss1a@nih.gov
 
Direct inquiries regarding review matters to:

Ms. Toby Friedberg
Referral Officer
Division of Extramural Activities
National Cancer Institute
6116 Executive Boulevard, Room 8109, MSC 8329
Bethesda, MD 20892-8329
Rockville, MD 20852 (for express/courier service)
Telephone: (301) 496 -3428
FAX: (301) 402-0275 
Email: tf12w@nih.gov

Direct inquiries regarding fiscal matters to: 
 
Ms. Sara Stone 
Grants Administration Branch
National Cancer Institute 
6120 Executive Boulevard, Room 243, MSC 7150 
Bethesda, MD  20892-7150 
Rockville, MD 20852 (express courier)
Telephone:  (301) 496-9927 
Fax:  (301) 496-8601
Email:  stones@gab.nci.nih.gov

LETTER OF INTENT 

Prospective applicants are asked to submit, by the dated listed on the first 
page of this PA, a letter of intent that includes a descriptive title of the 
proposed research, the name, address, and telephone number of the Principal 
Investigator, the identities of other key personnel and participating 
institutions, and the number and title of the PA in response to which the 
application may be submitted.  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 letter of intent is to be sent to Dr. Sudhir Srivastava by the letter of 
intent receipt date listed.

Schedule

Letter of Intent Receipt:           March 6, 2001, October 11, 2001
Application Receipt Date:           April 10, 2001, November 15, 2001
Peer Review Date:                   June 2001, Feb 2002
Review by NCAB Advisory Board:      September 2001, May 2002
Earliest Anticipated Start Date:    December 2001, July 2002

APPLICATION PROCEDURES
 
The modular grant concept establishes specific modules in which direct costs 
may be requested as well as a maximum level for requested budgets. Only 
limited budgetary information is required under this approach. The just-in-
time concept allows applicants to submit certain information only when there 
is a possibility for an award. It is anticipated that these changes will 
reduce the administrative burden for the applicants, reviewers and Institute 
staff. The research grant application form PHS 398 (rev. 4/98) is to be used 
in applying for these grants, with the modifications noted below.  
Applications kits are available at most institutional offices of sponsored 
research and may be obtained from the Division of Extramural Outreach and 
Information Resources, National Institutes of Health, 6701 Rockledge Drive, 
MSC 7910, Bethesda, MD 20892-7910, telephone 301/435-0714, email: 
grantsinfo@nih.gov.   The title and number of the program announcement must be 
typed on line 2 of the face page of the application form and the YES box must 
be marked.  For those applicants with internet access, the 398 kit may be 
found at: http://grants.nih.gov/grants/forms.htm.

Applicants are strongly encouraged to call the program contacts listed in 
INQUIRIES below with any questions regarding the adherence to the guidelines 
of their proposed project to the goals of this PA.
 
SPECIFIC INSTRUCTIONS FOR MODULAR GRANT APPLICATIONS

BUDGET INSTRUCTIONS

Modular Grant applications will request direct costs in $25,000 modules, up to 
a total direct cost request of $100,000 per year ($125,000 if there are 
consortium/contractual costs).The total direct costs must be requested in 
accordance with the program guidelines and the modifications made to the 
standard PHS 398 application instructions described below:

o  FACE PAGE:  Items 7a and 7b should be completed, indicating Direct Costs 
(in $25,000 increments) and Total Costs [Modular Total Direct plus Facilities 
and Administrative (F&A) costs] for the initial budget period.  Items 8a and 
8b should be completed indicating the Direct and Total Costs for the entire 
proposed period of support.

o  DETAILED BUDGET FOR THE INITIAL BUDGET PERIOD - Do not complete Form Page 4 
of the PHS 398.  It is not required and will not be accepted with the 
application.

o  BUDGET FOR THE ENTIRE PROPOSED PERIOD OF SUPPORT - Do not complete the
categorical budget table on Form Page 5 of the PHS 398.  It is not required 
and will not be accepted with the application.

o  NARRATIVE BUDGET JUSTIFICATION -  Prepare a Modular Grant Budget Narrative 
page (see http://grants.nih.gov/grants/funding/modular/modular.htm for sample 
pages). At the top of the page, enter the total direct costs requested for 
each year.  This is not a form page.  Provide an additional narrative budget 
justification for any variation in the number of modules requested.

o  Under Personnel, list all project personnel, including their names, percent 
of effort, and roles on the project. No individual salary information should 
be provided. However, the applicant should use the NIH appropriation language 
salary cap and the NIH policy for graduate student compensation in developing 
the budget request.

For Consortium/Contractual costs, provide an estimate of total costs (direct 
plus facilities and administrative) for each year, each rounded to the nearest 
$1,000. List the individuals/organizations with whom consortium or contractual 
arrangements have been made, the percent effort of all personnel, and the role 
on the project. Indicate whether the collaborating institution is domestic or 
foreign.  The total cost for a consortium/contractual arrangement is included 
in the overall requested modular direct cost amount. Include the Letter of 
Intent to establish a consortium

o  BIOGRAPHICAL SKETCH - The Biographical Sketch provides information used by 
reviewers in the assessment of each individual's qualifications for a specific 
role in the proposed project, as well as to evaluate the overall 
qualifications of the research team.  A biographical sketch is required for 
all key personnel, following the instructions below.  No more than three pages 
may be used for each person.  A sample biographical sketch may be viewed at: 
http://grants.nih.gov/grants/funding/modular/modular.htm

- Complete the educational block at the top of the form page;
- List position(s) and any honors;
- Provide information, including overall goals and responsibilities, on 
research projects ongoing or completed during the last three years;
- List selected peer-reviewed publications, with full citations.

o  CHECKLIST -  This page should be completed and submitted with the 
application. If the F&A rate agreement has been established, indicate the type 
of agreement and the date. All appropriate exclusions must be applied in the 
calculation of the F&A costs for the initial budget period and all future 
budget years.

The applicant should provide the name and phone number of the individual to 
contact concerning fiscal and administrative issues if additional information 
is necessary following the initial review.

Applications not conforming to these guidelines will be considered 
unresponsive to this PAR and will be returned without further review.

Submit a signed, typewritten original of the application, including the 
checklist, and three signed, exact, single-sided photocopies, in one package 
to:

Center for Scientific Review
National Institutes of Health
6701 Rockledge Drive, Room 1040 - MSC 7710
Bethesda, MD  20892-7710
Bethesda, MD  20817 (for express/courier service)

To expedite the review process, at the time of submission, send two additional
copies of the application to:  

Ms. Toby Friedberg  
Referral Officer
National Cancer Institute
6116 Executive Boulevard, Room 8109, MSC 8329
Bethesda, MD 20892-8329
Rockville, MD 20852 (for overnight/courier service)
Telephone:  (301) 496-3428
FAX:  (301) 402-0275

Applications must be received by the receipt dates listed at the beginning of 
this PA. 

REVIEW CONSIDERATIONS

Applications will be reviewed for completeness by the Center for Scientific 
Review and for adherence to the guidelines to this PA by the National Cancer 
Institute.  Incomplete applications will be returned to the applicant without 
further consideration.  Applications that are complete and adhere to the 
guidelines of this PA will be evaluated for scientific and technical merit by 
an appropriate scientific review group convened by the Division of Extramural 
Activities of the NCI in accordance with the standard NIH peer review 
procedures.  As part of the initial merit review, all applications will 
receive a written critique, and may undergo a process in which only those 
applications deemed to have the highest scientific merit, generally the top 
half of applications under review, will be discussed, assigned a priority 
score, and receive a second level review by the National Cancer Advisory 
Board. 

Review Criteria
 
The five criteria to be used in the evaluation of grant applications are 
listed below.
 
The goals of NIH-supported research are to advance our understanding of 
biological systems, improve the control of disease, and enhance health.  The 
reviewers will comment on the following aspects of the application in their 
written critiques in order to judge the likelihood that the proposed research 
will have a substantial impact on the pursuit of these goals.  Each of these 
criteria will be addressed and considered by the reviewers in assigning the 
overall score weighting them as appropriate for each application.  Note that 
the application does not need to be strong in all categories to be judged 
likely to have a major scientific impact and thus deserve a high priority 
score.  For example, an investigator may propose to carry out important work 
that by its nature is not innovative but is essential to move a field forward.
 
1.  Significance. Does this study address the objectives of the PA? If the 
aims of  the application are achieved, how will this project enable the 
technological application of exfoliated cells?  What will be the effect of 
these studies on the concepts or methods that drive the non-invasive 
approaches to cancer detection?
 
2.  Approach.  Are the conceptual framework, design, methods, and analyses 
adequately developed, well-integrated, and appropriate to the aims of the 
project?  Does the applicant acknowledge potential problem areas and consider 
alternative tactics?
 
3.  Innovation.  Does the project employ novel concepts, approaches or method?  
Are the aims original and innovative? Does the project challenge existing 
paradigms or develop new methodologies or technologies?
 
4.  Investigator.  Is the investigator appropriately trained and well suited 
to carry out this work?  Is the work proposed appropriate to the experience 
level of the principal investigator and other researchers (if any)?
 
5.  Environment.  Does the scientific environment in which the work will be 
done contribute to the probability of success?  Do the proposed experiments 
take advantage of unique features of the scientific environment or employ 
useful collaborative arrangements? Is there evidence of institutional support?

The initial review group will also examine: the appropriateness of proposed 
project budget and duration; the adequacy of plans to include both genders and 
minorities and their subgroups, and children as appropriate for the scientific 
goals of the research and plans for the recruitment and retention of subjects;  
the provisions for the protection of human and animal subjects; and the safety 
of the research environment.

AWARD CRITERIA
  
Applications will compete for available funds with all other recommended 
applications. The following will be considered in making funding decisions:  
Quality of the proposed project as determined by peer review, availability of 
funds, and program priority.

INCLUSION OF WOMEN AND MINORITIES IN RESEARCH INVOLVING HUMAN SUBJECTS
 
It is the policy of the NIH that women and members of minority groups and 
their subpopulations must be included in all NIH supported biomedical and 
behavioral research projects involving human subjects, unless a clear and 
compelling rationale and justification is provided that inclusion is 
inappropriate with respect to the health of the subjects or the purpose of the 
research.  This policy results from the NIH Revitalization Act of 1993 
(Section 492B of Public Law 103-43).
 
All investigators proposing research involving human subjects should read the 
UPDATED "NIH Guidelines for Inclusion of Women and Minorities as Subjects in 
Clinical Research," published in the NIH Guide for Grants and Contracts on 
August 2, 2000  (http://grants.nih.gov/grants/guide/notice-files/NOT-OD-00-
048.html); a complete copy of the updated Guidelines are available at 
http://grants.nih.gov/grants/funding/women_min/guidelines_update.htm: The 
revisions relate to NIH defined Phase III clinical trials and require: a) all 
applications or proposals and/or protocols to provide a description of plans 
to conduct analyses, as appropriate, to address differences by sex/gender 
and/or racial/ethnic groups, including subgroups if applicable; and b) all 
investigators to report accrual, and to conduct and report analyses, as 
appropriate, by sex/gender and/or racial/ethnic group differences.

INCLUSION OF CHILDREN AS PARTICIPANTS IN RESEARCH INVOLVING HUMAN SUBJECTS.

It is the policy of NIH that children (i.e., individuals under the age of 21) 
must be included in all human subjects research, conducted or supported by the 
NIH, unless there are clear and compelling scientific and ethical reasons not 
to include them. This policy applies to all initial (Type 1) applications 
submitted for receipt dates after October 1, 1998.

All investigators proposing research involving human subjects should read the 
“NIH Policy and Guidelines on the Inclusion of Children as Participants in 
Research Involving Human Subjects” that was published in the NIH Guide for 
Grants and Contracts, March 6, 1998, and is available at the following URL 
address:  http://grants.nih.gov/grants/guide/notice-files/not98-024.html

Investigators also may obtain copies of the policy from the program staff 
listed under INQUIRIES.  Program staff may also provide additional relevant 
information concerning the policy.

URLS IN NIH GRANT APPLICATIONS OR APPENDICES

All applications and proposals for NIH funding must be self-contained within 
specified page limitations.  Unless otherwise specified in an NIH 
solicitation, internet addresses (URLs) should not be used to provide 
information necessary to the review because reviewers are under no obligation 
to view the Internet sites.  Reviewers are cautioned that their anonymity may 
be compromised when they directly access an Internet site.

HEALTHY PEOPLE 2010

The Public Health Service (PHS) is committed to achieving the health promotion 
and disease prevention objectives of "Healthy People 2010," a PHS led national 
activity for setting priority areas. This PA, Development of High-Yield 
Technologies for Isolating exfoliated Cells in Body Fluids., is related to 
priority area of Cancer Research.  Potential applicants may obtain a copy of 
"Healthy People 2010" at http://www.health.gov/healthypeople/.
 
AUTHORITY AND REGULATIONS

This program is described in the Catalog of Federal Domestic Assistance No. 
93.393 Cancer Cause and Prevention Research. Awards are made under 
authorization of Sections 301 and 405 of the Public Health Service Act as 
amended (42 USC 241 and 284) and administered under NIH grants policies and 
Federal Regulations 42 CFR 52 and 45 CFR Parts 74 and 92. This program is not 
subject to the intergovernmental review requirements of Executive Order 12372 
or Health Systems Agency review.

The PHS strongly encourages all grant recipients to provide a smoke-free 
workplace and promote the non-use of all tobacco products. In addition, Public 
Law 103-227, the Pro-Children Act of 1994, prohibits smoking in certain 
facilities (or in some cases, any portion of a facility) in which regular or 
routine education, library, day care, health care, or early childhood 
development services are provided to children. This is consistent with the PHS 
mission to protect and advance the physical and mental health of the American 
people.


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