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CANCER DRUG(s) SCREENING TECHNICAL PROPOSAL
Background:
Hartech's PC based Bactobridge is a diagnostic tool that can be used to determine the sensitivity of human tumors to various anticancer drugs in order to select specific cancer drug regimens. The needs and potential benefits patients will derive from the availability of non-invasive approaches to the evaluation of cancers (linked to minimally debilitating treatments that are tailored to target the precise molecular alterations in the individual tumor) are numerous. "The development of the Bactobridge has created a processing and analytical methodology that can accurately determine quantum dose responses from human tumors to anticancer drugs". The electrical impedance characteristics of each sample cell suspension can be determined at different concentrations and under two conditions, namely viable and killed.As an incubation instrument with associated computers and conductivity measuring test tubes the Bactobridge can be used to track (monitor) the activity associated with the metabolism of both bacterial and malignant cells (i.e., human cancer cells). Analysis over time, utilizing a closed system and the principles of conductance/impedance measurement can be achieved presumably, prior to the onset of tumor cell mutation. Reference tests for the instrument involve detecting dose responses to anticancer drugs and the presence or absence of pathogenic microorganisms within minutes. The fact that most bacteria multiply every twenty minutes they provide a convenient tool to correlate with cancer cell metabolism in controlled clinical trials.
Purpose:
This project is designed to use an electrical impedance measuring device i.e., a Bactobridge for in vitro screening and evaluation of selected chemopreventive agents that may inhibit tumor cell transformation. The ability to monitor the electrical/physical characteristics of human tumors [in real time] has created a rapid and efficient way to qualify these agents for further evaluation for the prevention of cancer in humans. Reference tests for the Bactobridge will involve detecting the quantal dose (drug) response of specific chemotherapeutic agents (compounds) against biopsied human tumors and cultured tumor cell lines provided by the National Cancer Institutes (NCI). Instrument verification and accuracy will be confirmed by determining the presence or absence of pathogenic microorganisms in commercial food sources within hours of their arrival at our testing facility, which will impact on industrial microbiological analyses, a secondary application of the instrument.
Results to be achieved
: By using the Bactobridge we expect to develop a processing and analysis methodology to rapidly determine the chemosensitivity of various tumor types from individual patients and/or cultured tumor cell lines, within minutes. It will be shown that near real time analysis utilizing a closed system and the principles of conductance/impedance measurement can be achieved presumably, prior to the onset of tumor cell mutation.
Technical Approach and Scope of Work:
[Details are provided for real-time evaluation and analysis of biopsied human tumors as no current comparable data exists].4.1. Neoplastic Tissue
Collection sites: Mercy Medical Center, 301 St. Paul Place, Department of Pathology - 2nd Fl. Baltimore, Maryland, North Anne Arundel Hospital and the U.S. Veterans Hospital, Baltimore.
At the pathology gross bench, a Pathologist will assess specimen adequacy. If after all tissue requirements for routine pathologic evaluation have been fulfilled and excess tumor is present, 1-2 grams of tumor tissue will be sampled from an area identified by the Pathologist as representative of the tumor.
As a control, sampling from the same site will be submitted for permanent section/frozen section to ensure proper representation of the tumor.
Sampling will be done under sterile conditions with tumor immediately placed in culture/transport media for immediate transport or temporary storage, the latter not to exceed 16-20 hours.
4.2. Cell lines will be obtained from the NCI (specific cell lines are not known at this time).
4.3. Preparation of Cell Suspension
(Developing methodology).Upon receipt of the specimen at the DMRT Laboratory Research Facility it will be assigned a unique identifier with all clinical information logged into the database.
Processing will proceed under sterile conditions in producing a single cell suspension [for Procedure, see Appendix G].
Cell viability will be established by the trypan-blue exclusion (or other approved) methods.
Cell suspensions with known cell counts will be washed in non-nutritive media i.e., Hank’s balanced salt solution without phenol red and sodium bicarbonate-to ensure serum-free preparations eliminating possible spurious impedance changes.
5.1. Two types of preparations from each sample will be run on the Bactobridge to evaluate performance characteristics of the cells in (1) viable cell suspension; and, (2) killed cell suspension with mitomycin-C.
5.2. Following optimization of the procedure for sample type, exposure of the cell suspensions to pre-determined anti-cancer drugs utilizing different concentrations will be performed.
Note: Specific anti-cancer drug(s) will be determined by tumor type. Samples will be run as six matched pairs (see Table in 5.3).
5.3. Electrical impedance changes between each matched pair will be measured (see Table below).
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Pair 1 |
Pair 2 |
Pair 3 |
Pair 4 |
Pair 5 |
Pair 6 |
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Control Cell ( C Cell ) Run 1 Viable cells Run 2 Killed cells |
Run2 |
Run 2 |
Run 2 |
Run 2 |
Run 2 |
Run 2 |
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Reaction Cell ( R Cell ) |
Viable Cells |
Conc 1 |
Conc 2 |
Conc 3 |
Conc 4 |
Conc 5 |
5.4. Expected Results
Run 1 in which
C cell contains a viable cell suspension, changes in electrical impedance will indicate tumor cell chemosensitivity.Run 2 in which
C cell contains a killed cell suspension, a lack of change in electrical impedance will indicate tumor cell chemosensitivity.6. Comparative/Reference Methodology
[ PHASE I]Various tumor types will be analyzed by the above methodology and procedures optimized per specific tumor type.
The length of this phase will vary depending upon tumor amount, resultant viable cell count and the time it takes to determine appropriate suspension media. This phase will also determine the appropriate panel of chemotherapeutic agents to be tested.Tissue collection sites to include: University of Maryland Baltimore Tissue Bank (UMH-NBW), Mercy Hospital, North Anne Arundel Hospital and the U.S. Veteran's Hospital in Baltimore.
Following the optimization phase of study, all tumor tissue submitted for Bactobridge testing will also be simultaneously submitted for testing for cell culture drug resistance testing using a reference laboratory.
7. Clinical Trial Testing Phase
[PHASE II]Following optimization per specific tumor type and with appropriate chemotherapeutic panels established prospective trials will be initiated.
Collection sites to expand to multiple institutions and to major laboratories.
Prospective clinical data for each patient will be followed during chemotherapy 6-12 months after to monitor for persistence/recurrence. Data for specific cell lines will be made available to the NCI.
ANTICIPATED RESULTS
Tumor cell evaluation will show that isolating the glycolytic pathway of metabolizing tumors within the closed system of the reaction cell can provide a convenient indicator of alterations in the cellular milieu (following exposure to chemotherapeutic agents); and, that the Bactobridge utilizing bridge technologies and principles of conductance/impedance will display resultant characteristic patterns correlating with chemosensitivity, in near real time.
We expect that each tumor type will produce subtle but specific conductance curves. We also expect that some tumor types may not be viable candidates for evaluation by the Bactobridge.
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PROJECT SUMMARY
The potential chemopreventive agents which can be examined by the Bactobridge range from all of the retinoid compounds, antioxidants, growth factor analogs, inhibitors of promotion, antibodies to promoters, to synthetic viral polypeptide vaccines.
Cancer chemoprevention, a priority research area at NCI is aimed attesting the concept that certain natural or synthetic agents may lower cancer incidence. Potential chemopreventive agents include naturally occurring and synthetic substances, approved drugs, and others still in the realm of the laboratory such as phenolic antioxidants, protease and prostaglandin synthesis inhibitors, secondary plant constituents, and miscellaneous chemicals (e.g., oltipraz, ellagic acid, etc.). Retinoids, that class of compounds structurally similar to retinol (Vitamin A), have been actively investigated for their anti-carcinogenic potential. Human trials in cancer prevention are underway with three such retinoids, 4-hydroxyphenylretinamide and the all-trans and 13-cis isomers of retinoic acid.
Current research data, collected largely from laboratory studies, suggest that chemopreventive agents have the capacity to inhibit the neoplastic effects of chemical carcinogens and may prevent, inhibit, retard or reverse one or more of the stages of carcinogenesis. For example, laboratory studies indicate that chemopreventive agents (e.g. retinoids) can inhibit the expression of the transformed cell state in a number of in vitro cell culture systems as well as prevent the development of cancers (including skin, bladder, colon, trachea and mammary gland) in a number of in vivo animal systems.
The use of the in vitro screening system for preventive agents shall serve to: (1) improve the criteria for the selection of chemicals which shall be tested later for efficacy and toxicology in whole animal systems and for assigning priorities to chemicals for further studies, (2) improve the breadth of data on the inhibiting potential of the chemical, (3) evaluate the effect on actual target sites in one or more in vitro systems, (4) decrease later toxicology testing costs by reducing the number of inappropriate compounds reaching that stage in the screening sequence and (5) accelerate the rate that compounds are evaluated for use in man
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OTHER PERTINENT INFORMATION
Duration of Phase I
Tumor material for processing is expected to be limited. A preliminary period of two months will be needed to optimize the Bactobridge. The scope of testing for each individual sample will be defined by the viable cell count following processing. "Optimization" requires the identification of the requirements of each specific tumor type for media, incubator parameters, and sufficient data to have statistical predictability power. These factors are dependent on tumor biology specific for each tumor type.
Duration of Phase II
Phase II is designed to test all tumor types including solid and hematopoetic submitted for analyses from various sources (biopsied and cultured) for both comparative and for prospective (in-vivo) analysis. After conclusion of this project patient follow up testing (for up to twelve months) is recommended.
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The Department of Medical and Research Technology, |
