Dynamic genomic deletion expansion and formulation of molecular marker panels for integrated molecular pathology diagnosis and characterization of tissue, cellular fluid, and pure fluid specimens
Provided are materials, methods, platforms, and kits for diagnosing, prognosing, and/or determining the biological aggressiveness of a tumor (malignant and non-malignant) based in part on the temporal profile of genomic deletion expansion of the tumor. Also provided are methods of determining marker panels for different diseases and/or tissues and markers identified by these methods.
1 . A method of determining tumor aggressiveness in a patient comprising the steps of:
(a) amplifying DNA from a microdissection section of a biological sample from the patient;
(b) analyzing two or more genes for the presence of a nucleotide deletion wherein a deletion is the acquisition of genetic mutation;
(c) analyzing each gene with an array of markers to determine the extent of nucleotide deletion;
(d) determining the order of acquisition of nucleotide deletions in the patient; and
(e) collating the data from steps (a) to (d) to determine tumor aggressiveness.
2 . The method of claim 1 , wherein the biological sample is pretreated with a proteinase in lysis buffer, which contains a nonionic detergent.
3 . The method of claim 2 , wherein the proteinase is selected from the group consisting of proteinase K, pronase, subtilisin, thermolysin, papain, or a combination of proteinases.
4 . The method of claim 3 , wherein the proteinase or combination of proteinases is present in the amount of about 0.5% to about 2.0% final volume.
5 . The method of claim 4 , wherein the proteinase is present in the amount of about 1.0% and is proteinase K.
6 . The method of claim 2 , wherein the nonionic detergent is Nonidet P40, Tween, Triton X, or Nikkol.
7 . The method of claim 6 , wherein the nonionic detergent is present in the amount of about 0.5% to about 2.0%.
8 . The method of claim 1 , wherein the amplifying step is performed in the presence of about 5 to about 10 mM magnesium chloride, and about 5 to about 20 g/100 mL sucrose.
9 . The method of claim 8 , wherein magnesium chloride is present in the amount of about 8 mM, and sucrose is present in the amount of about 12 g/100 mL.
10 . The method of claim 1 , wherein the biological sample is from a tissue sample from two different organ sites in said patient, wherein a first organ site is the first diagnosed cancer and a second organ site is a putative metastatic lesion or a second primary tumor.
11 . The method of claim 1 , wherein the biological sample is a cell-free fluid sample, a blood sample, a cytology sample, resected tissue, or a combination thereof.
12 . The method of claim 11 , wherein the cell-free fluid sample contains non-nuclear DNA.
13 . The method of claim 11 , wherein the resected tissue is frozen, fresh, stained, fixative-treated, or stained and fixative-treated.
14 . The method of claim 1 , wherein step (c) is repeated to obtain replicate data.
15 . The method of claim 1 , further comprising the step of identifying a treatment plan to treat the tumor of said patient which best treats the tumor based on the determination of tumor aggression.
16 . The method of claim 1 , wherein the biological sample is microdissected tissue, and the steps of (b) and/or (c) are performed on DNA obtained from two or more microdissected sections of the tissue sample.
17 . The method of claim 16 , wherein step (c) is repeated to obtain replicate data.
18 . The method of claim 1 , further comprising determining whether genetic mutation acquisition is due to familial inheritance, an environmental factor, or a spontaneous mutation based on the order of the mutation acquisition.
19 . The method of claim 16 , wherein a patient fluid sample undergoes analysis comprising:
(a) amplifying DNA from the fluid sample of the patient;
(b) analyzing two or more genes for the presence of nucleotide deletion from the amplified DNA, wherein a deletion is the acquisition of genetic mutation;
(c) analyzing each nucleotide deletion with an array of markers to determine the extent of nucleotide deletion;
(d) determining the order of acquisition of nucleotide deletion in the patient; and
(e) validating the collated data of claim 16 with the data obtained from the fluid sample.
20 . A kit for determining tumor aggressiveness comprising:
(i) a device for amplifying DNA and analyzing the presence of a nucleotide deletion and order of acquisition of said nucleotide deletion; and
(ii) sets of cancer specific markers for assessing nucleotide deletion and extent of nucleotide deletion.
21 . The kit of claim 20 , wherein the device also has a data storage component for storing patient information regarding sex, age, weight, medical history, family medical history, prior cancer history and genetic analysis on a prior cancer, and genomic deletion acquisition data in a relational database.
22 . The kit of claim 20 , wherein the markers are markers for environmentally-induced mutations, germ line mutations, and spontaneous mutations.
23 . The kit of claim 22 , wherein the markers for environmentally-induced mutations are trichloroethylene markers.
24 . The kit of claim 20 , wherein the kit further comprises reagents for amplifying DNA, wherein the reagents when admixed contain about 5 to about 10 mM magnesium chloride; about 5 to about 20 g/100 mL sucrose; about 0.5% to about 2.0% nonionic detergent; and about 0.5% to about 2.0% proteinase or a combination of proteinases.
25 . The kit of claim 24 , wherein the magnesium chloride when admixed is present in the amount of about 8 mM; the sucrose is present in the amount of 12 g/100 mL; the nonionic detergent is nonidet P-40 and is present in the amount of about 1.0%; and the proteinase is proteinase K and is present in the amount of 2 mg/mL.
26 . A method of creating a panel of molecular markers for detecting a condition in a patient comprising the steps of:
(a) determining gene targets for detection of a mutation to include in a molecular marker analysis for a marker panel;
(b) delineating genomic regions for each gene target;
(c) identifying a 4 to 1500 nucleotide repeat microsatellite and/or a minisatellite in the genomic region that will constitute the marker panel;
(d) identifying at least two 4 to 1500 nucleotide microsatellites and/or minisatellites positioned a certain distance from each other and performing genomic deletional expansion;
(e) determining the amount of DNA in the biological sample;
(f) determining the quality of DNA in the biological sample by quantitative PCR;
(g) determining the quality of DNA in the biological sample by means on competitive template PCR (CT-PCR);
(h) determining the amplifiability of DNA for each 4 to 1500 repeat microsatellite and/or minisatellite;
(i) defining a normal range of allele variation thereby defining allelic imbalance comprising:
(i) defining different normal ranges for each allele for two or more quantities of DNA; and
(ii) defining different normal ranges for each allele with two or more qualities of DNA; and
(j) defining minimum thresholds for significant allelic imbalance thereby obtaining an indication of mutation change in a significant percentage of evaluated cells comprising:
(i) defining minimum thresholds for significant allelic imbalance for different amounts of DNA; and
(ii) defining minimum thresholds for significant allelic imbalance for different qualities of DNA; and
(k) calculating a percentage of mutated cells based upon ratios of a tested sample using a calculated normal for each 4 to 1500 microsatellite and/or minisatellite, thereby creating a panel of molecular markers for detecting a condition in a patient.
27 . The method of claim 26 , wherein the microsatellite is 4 to 20 nucleotides.
28 . The method of claim 27 , wherein the microsatellite is 4-nucleotide microsatellite.
29 . The method of claim 26 , wherein the condition is a neoplasia, a hyperplasia, or a benign growth.
30 . The method of claim 26 , wherein the condition arises from a germ line mutation, an environmental factor, or a spontaneous mutation, or a combination thereof.
31 . A marker or panel of markers identified by the method of claim 26 .
32 . A method of determining tumor aggressiveness in a patient comprising the steps of:
(a) analyzing two or more genes from DNA amplified from a microdissection section of a patient biological sample for the presence of a nucleotide deletion wherein a deletion is the acquisition of genetic mutation;
(b) analyzing each gene with an array of markers to determine the extent of nucleotide deletion;
(c) determining the order of acquisition of nucleotide deletion in the patient; and
(d) collating the data from steps (a) to (d) to determine tumor aggressiveness.