Claims for Patent: 4,582,787

Summary for Patent: 4,582,787

Title:	Method of testing a patient for a predisposition to lung cancer, certain other cancers, neurofibromatosis and certain other hereditary disorders
Abstract:	A method is disclosed for testing a patient to determine a predisposition to lung cancer and neurofibromatosis. The method comprises the steps of carrying out a biopsy on each patient of a first group of patients known to have a cancer or a cancer with a known hereditary component such as neurofibromatosis, the biopsy including the removal of a skin sample from each patient. The removed skin samples are prepared for in vitro testing thereof, initially to prove contamination is not present. In vitro testing is carried out to determine whether viral transformation of cultures established from the skin samples exists. Portions of Kirsten murine sarcoma virus are prepared and added to the uncontaminated cultured skin cells. The skin cells treated with the Kirsten murine sarcoma virus are incubated and a viral assay is performed to determine which skin cultures have been transformed due to the treatment with Kirsten murine sarcoma virus. The foregoing steps are performed relative to a second group of cancer and neurofibromatosis-free patients. An index is composed from the results of the assays performed on the first and second groups of patients. The foregoing steps are repeated relative to a subsequent patient, the predisposition to lung cancer and/or neurofibromatosis of which is unknown and a comparison of the results obtained from the assay relative to the subsequent patient is compared with the index to determine whether a predisposition to lung cancer and neurofibromatosis exists relative to the subsequent patient.
Inventor(s):	Frankel; Jack W. (Treasure Island, FL)
Assignee:
Application Number:	06/454,880
Patent Claims:	1. A method of diagnostically testing a patient for degree of risk for the particular presence of lung cancer, skin cancer, liver cancer, leukemia, neurofibromatosis or emphysema which comprises the steps of: carrying out a biopsy on each patient of a first group of patients known to have lung cancer, skin cancer, liver cancer, neurofibromatosis or emphysema, the biopsy including the removal of a skin sample from each of the patients of the first group; preparing the skin samples removed from the first group of the patients for in vitro testing thereof; performing in vitro testing of the skin samples of the first group of patients to determine the existence of bacterial and mycoplasma contamination; preparing portions of Kirsten murine sarcoma virus; treating the uncontaminated prepared skin cells of the first group of patients with the prepared portions of Kirsten murine sarcoma virus; incubating the virus treated skin cells of the first group of patients; carrying out a viral assay upon the incubated treated skin cells to determine which skin samples have been transformed due to the treatment with Kirsten murine sarcoma virus and to determine the greatest dilution of Kirsten murine sarcoma virus that is capable of transforming the cells characterized by a cytological change relative to one or more surface areas of the culture cells; repeating the foregoing steps carried out relative to the skin cell cultures taken from the first group of patients of skin cells obtained by carrying out a biopsy on each patient of a second group of cancer and hereditary disease-free patients; composing an index from a comparison of the results obtained from the assay relative to the first group of patients with the results obtained from the assay relative to the second group of patients; repeating the foregoing steps relative to the first group of patients on a test patient utilizing either a skin sample or an amniotic fluid sample to obtain cells for in vitro testing where the predisposition of the subsequent patient to lung cancer, skin cancer, liver cancer, leukemia, neurofibromatosis or emphysema is not known; and utilizing the index to determine whether a predisposition to lung cancer, skin cancer, liver cancer, leukemia or emphysema exists relative to the test patient by a comparison of the results obtaned by the assay to determine the greatest dilution of Kirsten murine sarcoma virus that is capable of transforming the cells characterized by a cytological change relative to one or more surface areas of the culture cells carried out relative to the test patient with the index. 2. The method as set forth in claim 1 wherein skin cells are removed from the patient by punch biopsy. 3. The method as set forth in claim 1 wherein the skin sample is removed from the patient and aseptically transferred to a self-buffering medium to provide a stable pH value for the skin sample. 4. The method as set forth in claim 3 wherein the self-buffering medium is cold relative to the ambient temperature. 5. The method as set forth in claim 4 wherein the self-buffering medium is Leibovitz L-15 medium including fetal bovine serum, gentomycin and fungizone. 6. The method as set forth in claim 5 wherein the Leibovitz medium includes fetal bovine serum within the rage 1-10%, gentamycin within the range 50-200 micrograms per milliliter, and fungizone within the range 10-100 micrograms per milliliter. 7. The method as set forth in claim 6 wherein the fetal bovine serum is heated. 8. The method as set forth in claim 3 wherein the skin sample is finely minced or treated overnight at 4.degree. C. directly with EDTA-trypsin or collagenase. 9. The method as set forth in claim 8 wherein a growth medium is added to a portion of the minced skin sample or to the portion treated with EDTA-trypsin or collagenase. 10. The method as set forth in claim 9 wherein the growth medium comprises minimal essential medium, Earle's balanced salt solution, the salt solution being supplemented by glutamine, fetal bovine serum, penicillin, streptomycin and fungizone. 11. The method as set forth in claim 10 wherein the growth medium has a volume of 3 milliliters, the glutamine is within the range 1-5 mM, the fetal bovine serum is within the range 5-50% by volume, the penicillin is within the range 50-150 units, the streptomycin is within the range 50-150 micrograms and the fungizone is within the range 5-50 micrograms. 12. The method as set forth in claim 10 wherein for a growth medium having a volume of 3 milliliters, the glutamine is 2 mM, the fetal bovine serum is 20% by volume, the penicillin is 100 units, the streptomycin is 100 micrograms and the Fungizone is 25 micrograms. 13. The method as set forth in claim 10 wherein for a unit volume of salt solution in milliliters, the glutamine is 0.66 mM, the fetal bovine srum is 20% by volume, the penicillin is 33 units, the streptomycin is 33 micrograms and the Fungizone is 8.33 micrograms. 14. The method as set forth in claim 8 wherein the skin sample is subjected to incubation. 15. The method as set forth in claim 14 wherein the skin sample is subjected to a temperature within the range 30.degree.-40.degree. C. for a period of between one-half to two hours. 16. The method as set forth in claim 15 wherein the skin sample is subjected to a temperaure of approximately 37.degree. C. for a period of approximately one hour, or overnight at 4.degree. C. with EDTA-trypsin or collagenase. 17. The method as set forth in claim 9 wherein the skin sample or the EDTA-trypsin or collagenase-treated cells with the growth medium are subjected to incubation. 18. The method as set forth in claim 17 wherein the skin sample or the EDTA-trypsin or collagenase-treated skin samples within the growth medium are subjected to a temperature of 37.degree. C. for a period of one to twenty-eight days within an atmosphere comprising air to which is added an additional 5% by volume of carbon dioxide. 19. The method as set forth in claim 17 wherein the monolayer of incubated cells of the skin sample are removed by means of a EDTA-trypsin mixture, the removed cells are washed and transferred to a growth medium. 20. The method as set forth in claim 19 wherein the cells are subjected to a temperature of approximately 37.degree. C. for a further perid within the range of seven to twenty-one days, within an atmosphere comprising air to which is added an additional 5% by volume of carbon dioxide, the atmosphere being changed approximately every three days. 21. The method as set forth in claim 20 wherein a portion of the prepared skin cells are monitored to establish the absence of bacterial contamination and mycoplasma and the monitored sample is stored in liquid nitrogen. 22. The method as set forth in claim 21 wherein the Kirsten murine sarcoma virus is produced by the rat kidney cell line in vitro and the Kirsten murine sarcoma virus is purified and a special clone prepared by passage through human skin fibroblasts. 23. The method as set forth in claim 1 wherein the uncontaminated prepared cultured skin cells from the patient are inoculated into a plurality of containers; a portion of DEAE-dextran is added to each container, and the resultant culture mixture is incubated for approximately one hour at approximately 37.degree. C. 24. The method as set forth in clam 23 wherein a portion of standard frozen Kirsten murine sarcoma virus stock is added to the culture mixture within each container. 25. The method as set forth in claim 23 wherein the dextran has a molecular weight greater than 2.times.10.sup.6 daltons and a concentration of approximately 25 micrograms per milliliter where the total incubation volume within each container is approximately 4 milliliters. 26. The method as set forth in claim 24 wherein each portion of the Kirsten murine sarcoma virus is approximately 0.25 milliliters by volume. 27. The method as set forth in claim 1 wherein the viral assay includes observing the highest dilution of Kirsten murine sarcoma virus that induces transformation of the uncontaminated prepared cultured skin cells after removing from the patient within a unit period of time. 28. The method as set forth in claim 27 wherein the induced transformation includes a cytological change in at least one area. 29. The method as set forth in claim 28 wherein the unit period of time is approximately 14 days. 30. The method as set forth in claim 1 wherein analyzing of the results of the viral assay includes comparing the transformation results relative to skin samples taken from patients known to have lung cancer or neurofibromatosis with the transformation results relative to skin samples taken from normal patients which have been age matched to the patients known to have lung cancer or neurofibromatosis. 31. The method of claim 1 for diagnostically testing a patient for a degree of risk for the presence of lung cancer. 32. The method of claim 1 for diagnostically testing a patient for a degree of risk for the presence of neurofibromatosis.

Details for Patent 4,582,787

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2039-03-29