Methods for detecting braf in cancer
AssigneeBeth Israel Deaconess Medical Center, Inc.
InventorDavid J. Panka
Life of this patent
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Abstract
The invention features compositions and methods for detecting and quantifying BRAFV600E in a sample of a subject. In particular embodiments, the invention provides a highly sensitive and precise quantitative method for diagnosis, post-operative surveillance, and monitoring evidence of disease over time in the adjuvant setting (i.e. treatment with BRAF-inhibitors).
Claims24
A method for detecting a BRAFV600E mutation in a biological sample, identifying a subject, or quantitatively detecting thyroid cancer or melanoma comprising a BRAFV600E mutation in a subject, the method comprising
- (a) isolating RNA from a biological sample of a subject;
- (b) using reverse transcriptase to obtain a cDNA;
- (c) amplifying said cDNA to obtain a BRAF amplicon;
- and
- (d) digesting said amplicon with TspR1 to distinguish wild-type BRAF from BRAFV600E and measuring the level of BRAFV600E in the sample, wherein a level of BRAFV600E greater than about 5 pg quantitatively detects the presence of thyroid cancer or melanoma comprising a BRAFV600E mutation in the subject.
2-3. (canceled)
A method for detecting resistance and/or relapse or detecting the clinical responsiveness of a thyroid cancer or melanoma comprising a BRAFV600E mutation, the method comprising
- (a) isolating RNA from two or more biological samples of a subject, wherein the samples are obtained prior to and following initiation of anti-cancer therapy;
- (b) using reverse transcriptase to obtain a cDNA;
- (c) amplifying said cDNA to obtain a BRAF amplicon;
- and
- (d) digesting said amplicon with TspR1 to distinguish wild-type BRAF from BRAFV600E and measuring the level of BRAFV600E in the sample relative to the level present in the sample obtained prior to treatment, wherein a decrease in the level of BRAFV600E following treatment indicates the clinical responsiveness of the thyroid cancer or melanoma to treatment and an increase in the level of BRAFV600E is indicative that the thyroid cancer or melanoma has developed resistance and/or has relapsed.
9-14. (canceled)
16-18. (canceled)
A method of treating thyroid cancer or melanoma in a pre-selected subject, the method comprising administering an effective amount of a BRAF inhibitor to the subject, and wherein the preselection comprises:
- (a) isolating RNA from a biological sample of a subject;
- (b) using reverse transcriptase to obtain a cDNA;
- (c) amplifying said cDNA to obtain a BRAF amplicon;
- and
- (d) digesting said amplicon with TspR1 to distinguish wild-type BRAF from BRAFV600E and detecting a level of a BRAFV600E mutation greater than about 5 or 10 pg in the sample.
(canceled)
A method for detecting thyroid cancer in a subject comprising a BRAFV600E mutation in a subject, the method comprising
- (a) isolating RNA from a biological sample of a subject;
- (b) using reverse transcriptase to obtain a cDNA;
- (c) amplifying said cDNA to obtain a BRAF amplicon;
- and
- (d) digesting said amplicon with TspR1 to distinguish wild-type BRAF from BRAFV600E and measuring the level of BRAFV600E in the sample, thereby detecting thyroid cancer in the subject.
A kit for quantifying thyroid cancer or melanoma in a blood sample obtained from a subject, the kit comprising at least one primer used to obtain a BRAF amplicon, a TspR1 restriction enzyme, and an oligonucleotide control comprising a portion of a BRAF polynucleotide encoding a valine or glutamate residue at position 600 of the BRAF protein.
Dependencies are inferred from the claim text.