Dr. Melvyn S. Tockman
Johns Hopkins School of Hygiene and Public Health
615 North Wolfe Street
Baltimore, MD 21205-2179
Phone: 410-955-4587
Fax: 410-955-1811
Email: mtockman@phnet.sph.jhu.edu
Projects are approved by an IRB located at: Johns Hopkins School of Hygiene and Public Health.
The approving IRB operates under a Multiple Project Assurance (MPA) recognized by DOE or by the Department of Health and Human Services (HHS).
MPA number of the IRB: M-1090
Number of Human Subjects Projects reported: 1
Project Identifier: JHSHPH-91-DEFG0293ER61572
Project Title:
Early Detection of Lung Cancer
Principle Investigator:
Dr. Melvyn S. Tockman
Project started in: 1991
Project Funding Information:
Project received funding in Fiscal Year 1995.
Project did not use human subjects in Fiscal Year 1995.
Explanation:
Specimen collection was completed by June 1, 1994.
Funding Sources:
Project does not involve use of multiple protocols/subprojects.
IRB Review:
Type of Review: Full Board
Most Recent Approval: July 06, 1994
IRB Approval Number: H.18.91.06.19.A
Number of Human Subjects in the Last Reporting Period for this Project: 120
(Reporting periods vary.)
Type of Human Subjects Involvement:
A. Objectives:
The focus of the present study has been to optimize the in-vitro uptake and
quantitation of H2TCPP, and then evaluate the potential of 5,10,15,20 tetrakis
(4-carboxyphenyl) porphine (H2TCPP) as a biomarker of early (pre-malignant) lung
cancer.
B. Methods:
Two archives of sputum specimens collected at progressive degrees of
morphologic dysplasia along with corresponding histologically confirmed lung
tumors are available to confirm the link between a potential biomarker (i.e.,
H2TCPP) and standard, pathologically confirmed lung cancer. These archives were
collected during an 8-year National Institutes of Health/National Cancer
Institute (NIH/NCI) early lung cancer detection trial at the Johns Hopkins Lung
Project (JHLP) and a pilot lung cancer screening study among Chinese Yunnan Tin
Corp (YTC) miners, respectively.
1. Using video-enhanced transmitted light and fluorescent microscopy, we acquired shading-corrected images of cells, stained and unstained with H2TCPP, from freshly harvested human tissue culture cell lines of epidermoid American Type Culture Collection (ATCC) HTB58, adenocarcinoma (ATCC Calu-3) and undifferentiated small cell (ATCC OH3, H345) lung cancer, plus archived human specimens. We established the optimal conditions under which neoplastic and non-neoplastic cells take up H2TCPP by varying reagent concentration, temperature, incubation time, and pH (acidity).
2. Using optimal technique, we compared three biomarkers for their ability to distinguish three groups of cells: normal sputum cells only, ATCC lung cancer cells only and 1:1 mixture of tumor and sputum cells.
3. Fluorescent staining methods:
a. 7-amino Actinomycin-D (7-AAD)
7-AAD is a membrane-impermeant fluorescent DNA intercalator which
binds particularly well to Guanine Cytosine (GC)-rich regions of DNA. Upon
binding, 7-AAD undergoes a spectral shift resulting in an emission maximum at
655 nm after excitation at 555 nm. Following a phosphate buffered saline (PBS)
wash, cells are resuspended in cold PBS and fixed by addition of freshly
prepared 2% phosphate-buffered paraformaldehyde to a final concentration of
0.25%. Fixation at 4 degrees Celsius (C) for 1 hour is followed by
permeabilization with 0.2% between-20 in PBS for 15 minutes. After fixation and
permeabilization, DNA was stained with 7-AAD by 30 minute incubation of cells in
PBS containing 25 ug/ml of 7-AAD.
b. 5,10,15,20-tetrakis (4-carboxyphenyl) porphine (H2TCPP)
Cell spreads and cytospin preparations are fixed in 95% ethanol
for 10 minutes, then rinsed two times with PBS. Under reduced ambient light,
H2TCPP is applied in concentrations from 50-200 ug/ml to slides maintained in a
dark humid chamber for 4 to 24 hours, at temperatures from 37 to 4 degrees C.
At the conclusion of incubation, excess porphyrin is removed by 2 quick PBS
washes. Slides are then dehydrated, mounted in glycerol, cover slipped and
sealed with colorless nail enamel.
c. Fluorescein-labeled monoclonal antibody (703D4) to alpha-2,
beta-1 hnRNP, p31.
Monoclonal antibody 703D4, which binds to a tumor-associated
hnRNP, p31, was applied to cells which have been paraformaldehyde fixed and
permeabilized as described above. Cells are suspended in a 1:50 dilution of Mab
703D4 and incubated overnight at 4 degrees C. After washing with PBS two times,
the cells are resuspended in 1:50 dilution of biotinylated horse anti-mouse
Immunoglobulin G (IgG), incubated for 30 minutes at room temperature and again
washed with PBS. Cell pellets are then resuspended in a 1:50 dilution of
Fluorescein avidin D, incubated for 30 minutes at 4 deg C in the dark and washed
in PBS prior to measurement of FITC fluorescence.
C. There is no human subject contact with chemicals, radioactive substances or ionizing radiation in the present study.
D. Involvement of Human Subjects
1. Procedures involving human subjects.
Human specimens used in the present study were collected by the JHLP, conducted from 4/71 through 11/87, and by the YTC, from 3/91 through 9/93. In both studies, an annual sputum induction was performed consisting of a 15-minute inhalation of hypertonic saline performed before a laminar-flow hood. Fresh sputum was smeared on glass slides for Papanicolaou staining and interpretation. The remaining sputum was homogenized, concentrated and placed in Saccomanno's preservative solution using standard techniques. A total of 4,545 sputum specimens from 953 participants are presently stored in 30 cc screw-top glass bottles, each containing the specimen in a 2% carbowax/50% alcohol (Saccomanno's) preservative solution, arranged in boxes of 50. Box and bottle location, specimen cytological interpretation, participant identification and outcome are available on a computerized data base.
2. Risks to human subjects.
It is very unlikely that research subjects will experience side effects from performing the sputum induction procedure. Occasionally, individuals may experience transient shortness of breath or wheezing as a result of induction/deep coughing.