Stain-directed molecular analysis for cancer prognosis and diagnosis

1. A prognostic/diagnostic method for detecting and diagnosing cancerous and precancerous tissue, the method comprising, in combination and in sequence, the steps of:

(a) topically applying to epithelial tissue a dye that selectively stains cancerous and precancerous tissue to locate suspect tissue, wherein the dye comprises:

(1) conformational isomers of toluidine blue O, the compounds having the structures

(2) N-demethylation derivatives of the isomers, the compounds having the structures

 a ratio of combined areas of 254 nm HPLC peaks representing the isomers to combined areas of 254 nm HPLC peaks representing the N-demethylation derivatives being at least about 6:1;

(b) separating cells from the suspect tissue; and

(c) subjecting the cells to molecular analysis to determine whether the separated cells exhibit characteristics associated with cell differentiation or cancer.

2. The method of claim 1 wherein step (a) is preceded by saliva test cancer screening to determine whether cancerous or precancerous tissue exists in head and neck tissues and step (b) is then performed on the head and neck tissues.

3. A prognostic/diagnostic method for detecting and diagnosing cancerous and precancerous tissue in a patient's mouth, the method comprising, in combination and in sequence, the steps of:

(a) conducting a visual examination of the patient's mouth to determine the condition and appearance of soft tissue and teeth in the patient's mouth;

(b) rinsing the patient's mouth with a solution comprising a staining compound, wherein the staining compound comprises:

(1) conformational isomers of toluidine blue O, the compounds having the structures

(2) N-demethylation derivatives of the isomers, the compounds having the structures

 a ratio of combined areas of 254 nm HPLC peaks representing the isomers to combined areas of the peaks representing the N-demethylation derivatives being at least about 6:1;

(c) rinsing the patient's mouth with water;

(d) observing and recording the characteristics of suspect lesions, including their location, size, morphology, color and surface;

(e) removing cells from the suspect lesions;

(f) subjecting the cells to microsatellite analysis to determine loss of heterozygosity (LOH) at critical loci, wherein LOH is indicative of cancer or precancerous cells.

4. The method of claim 3 wherein the visual examination is conducted without using any instruments which might cause nicks or cuts to the soft tissue.

5. The method of claim 3 , wherein the critical loci chosen for testing are located in chromosomal regions selected from the group consisting of 9p21 and 3p21.

6. The method of claim 5 , wherein the critical loci are selected from any one or a combination of loci selected from the group consisting of D9S171, D9S736, and D3S1067.

7. A prognostic/diagnostic method for detecting and diagnosing cancerous and precancerous tissue, the method comprising, in combination and in sequence, the steps of:

(a) topically applying to epithelial tissue a dye that selectively stains cancerous and precancerous tissue to locate suspect tissue, wherein the dye comprises:

(1) conformational isomers of toluidine blue O, the compounds having the structures

 in which the isomer of subparagraph (i) comprises at least 58% of a total organic dye content of the composition,

(b) separating cells from the suspect tissue; and

(c) subjecting the cells to molecular analysis to determine whether the separated cells exhibit characteristics associated with cell differentiation or cancer.

8. The method of claim 7 wherein step (a) is preceded by saliva test cancer screening to determine whether cancerous or precancerous tissue exists in head and neck tissues and step (b) is then performed on the head and neck tissues.

9. The method of claim 7 wherein the visual examination is conducted without using any instruments which might cause nicks or cuts to the soft tissue.

10. A prognostic/diagnostic method for detecting and diagnosing cancerous and precancerous tissue, the method comprising, in combination and in sequence, the steps of:

(a) topically applying to epithelial tissue a dye that selectively stains cancerous and precancerous tissue to locate suspect tissue, wherein the dye is made using a process for manufacturing toluidine blue O, which comprises the steps of

(1) oxidizing N,N-dimethyl-p-phenylene diarnine in a first reaction mixture, to form a first intermediate, 2-amino-5-dimethylaminophenyl thiosulfonic acid,

(2) oxidizing the first intermediate and condensing the oxidizate in a second reaction mixture with o-toluidine, forming a second intermediate, indamine thio-sulfonic acid,

(3) oxidizing the second intermediate to close the indamine ring thereof, forming a TBO-containing reaction product dissolved in a third reaction mixture,

(4) introducing a complexing reagent into the third reaction mixture, to form a TBO-complex product dissolved in the third reaction mixture,

(5) precipitating the TBO-complex product from the third reaction mixture, and

(6) separating the TBO-complex product, containing conformational isomers of TBO;

wherein the improved process comprises introducing the complexing reagent to a reaction mixture before formation of the third reaction mixture, the complexing reagent being a compound that forms with the N,N-dimethyl-p-phenylenediamine, the first intermediate and/or the second intermediate, a complex that provides steric hinderence to demethylation thereof;

(b) separating cells from the suspect tissue; and

(c) subjecting the cells to molecular analysis to determine whether the separated cells exhibit characteristics associated with cell differentiation or cancer.

11. The method of claim 10 wherein step (a) is preceded by saliva test cancer screening to determine whether cancerous or precancerous tissue exists in head and neck tissues and step (b) is then performed on said head and neck tissues.