Methods for treating Barrett's metaplasia and esophageal adenocarcinoma
Disclosed herein are methods for treating Barrett's metaplasia and esophageal adenocarcinoma and methods for determining mutational load as a predictor of the risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
1. A method, the method comprising:
amplifying DNA sequences from a biological specimen from a subject having Barrett's metaplasia;
detecting the presence or absence of a mutation in microsatellite regions of 1q22, 1p34.2, 1p22.3, 1p36.21, 1p36.21, B Catenin E3, 3p25, 3p12.3, 3p22.3, 3p24.3, 3p26.3, 3p11.2, 3p24.2, C-KIT E11, C-KIT E17, 5q23.1, 5q23.2, BRAF E15, EGFR E 19, EGFR E 21, 7p12.3, 7p12.1, 9p21.1, 9p23, 10q23.32, 10q23.33, HRAS1 E1, Kras2.E1, Kras2.E2, 17q22, 17q21.2, 17q11.2, 17p13.1 17p13, 17p13.1, 18q21.33, 19q13.2, 19q13.32, 21q21.2, 22q13.2, and Xp22.2 present in the amplified DNA sequences;
categorizing clonality of each mutation;
calculating a mutational load based on the sum of low and high clonality mutations;
comparing the mutational load with a series of pre-determined mutational load cut-offs defining risk categories;
assigning the subject to a risk category corresponding to the subject's mutational load, wherein each risk category is indicative of the risk of disease progression;
determining if the subject is in a high risk category for disease progression from Barrett's metaplasia to esophageal adenocarcinoma; and
administering to the subject identified in the high risk category at least one treatment modality selected from endoscopic mucosal resection, endoscopic submucosal dissection, a therapeutically effective amount of radiofrequency ablation, a therapeutically effective amount of cryoablation, a therapeutically effective amount of photodynamic therapy and combinations thereof.
2. The method of claim 1 , wherein the pre-determined mutational load cut-offs defining risk categories are derived from a pre-determined patient population distribution with known mutational loads corresponding to a known disease state diagnosis.
3. The method of claim 2 , wherein the known disease state diagnosis is selected from normal squamous, columnar epithelium without Barrett's metaplasia, Barrett's metaplasia, Barrett's metaplasia intermediate for dysplasia, low grade dysplasia and high grade dysplasia.
4. The method of claim 2 , wherein the risk categories are selected from no mutational load, low mutational load, and high mutational load.
5. The method of claim 4 , wherein the subject is assigned to the no mutational load risk category when the subject has mutational load of 0.0.
6. The method of claim 4 , wherein the wherein no mutational load is indicative of no risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
7. The method of claim 4 , wherein no mutational load is indicative of the absence of actionable disease.
8. The method of claim 7 , wherein the absence of actionable disease is categorized as Barrett's metaplasia with a lower risk of progression than the baseline risk for Barrett's metaplasia, wherein surveillance of the patient can be safely discontinued.
9. The method of claim 4 , wherein the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than or equal to 2.0.
10. The method of claim 4 , wherein the wherein a low mutational load is indicative of a low risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
11. The method of claim 4 , wherein a low mutational load is indicative of suitability of the subject for monitoring.
12. The method of claim 4 , wherein the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than 2.0.
13. The method of claim 4 , wherein the wherein a high mutational load is indicative of high risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
14. The method of claim 1 , wherein the subject is a human.
15. The method of claim 1 , wherein the subject is a human diagnosed with Barrett's esophagus.
16. The method of claim 1 , wherein the biological specimen is a mucosal lining of the esophagus.
17. The method of claim 1 , wherein the biological specimen is representative of a disease region.
18. The method of claim 1 , wherein amplifying DNA sequences comprises:
selecting a primer pair corresponding to a specific microsatellite region;
adding the primer pair to the DNA sequences;
and performing quantitative polymerase chain reaction on the DNA sequences with the primer.
19. The method of claim 1 , wherein detecting mutations comprises determining the sequence of the amplified DNA and comparing the amplified DNA to a known wild type control sequence for the specific microsatellite region and identifying differences between the sequence of the amplified DNA and the known wild type control sequence.
20. The method of claim 1 , wherein categorizing clonality of each mutation comprises assigning one of three categories selected from the group consisting of no clonality, low clonality and high clonality.
21. The method of claim 20 , wherein high clonality is assigned where loss of heterozygosity is present in greater than about 75% of DNA analyzed.
22. The method of claim 20 , wherein low clonality is assigned where loss of heterozygosity is present in about 50% to about 75% of DNA analyzed.
23. The method of claim 20 , wherein no clonality is assigned where loss of heterozygosity is present in less than about 50% of DNA analyzed.
24. The method of claim 1 , wherein calculating the mutational load comprises assigning a score to each mutation based on a categorization of low or high clonality of each mutation, wherein the score for low clonality is 0.5x, wherein x is the number of low clonality mutations and the score for high clonality is y, wherein y is the number of high clonality mutations; and wherein the overall mutational load is y+0.5x.
25. The method of claim 1 , wherein calculating the mutational load further comprises assigning a score to each mutation based on detection of DNA microsatellite instability at a particular locus, wherein DNA microsatellite instability at a single locus is defined as 0.75z, wherein z is the number of loci displaying DNA microsatellite instability; wherein the score for low clonality is 0.5x, wherein x is the number of low clonality mutations and the score for high clonality is y, wherein y is the number of high clonality mutations; and wherein the overall mutational load is y+0.5x+0.75z.
26. The method of claim 24 , wherein DNA microsatellite instability is determined by the presence of at least one of the shortening and lengthening of a DNA microsatellite region.
27. The method of claim 1 , wherein calculating the mutational load further comprises assigning a score to each mutation based on detection of DNA microsatellite instability at multiple loci, wherein DNA microsatellite instability at multiple loci is defined as 2z, wherein z is the number of loci displaying DNA microsatellite instability; wherein the score for low clonality is 0.5x, wherein x is the number of low clonality mutations and the score for high clonality is y, wherein y is the number of high clonality mutations; and wherein the overall mutational load is y+0.5x+2z.
28. The method of claim 26 , wherein DNA microsatellite instability is determined by the presence of at least one of the shortening and lengthening of a DNA microsatellite region.
29. The method of claim 1 , wherein calculating a mutational load further comprises summing the clonality weighting for each specific microsatellite region showing a mutation or DNA microsatellite instability.
30. The method of claim 1 , wherein determining mutational load as a predictor of disease progression is independent of a histological standard.