Method to determine responsiveness of cancer to epidermal growth factor receptor targeting treatments
Disclosed herein are methods and reagents for determining the responsiveness of cancer to an epidermal growth factor receptor (EGFR) targeting treatment. The detection of these mutations will allow for the administration of gefitinib, erlotinib and other tyrosine kinase inhibitors to those patients most likely to respond to the drug.
1. An assay comprising:
(a) adding primers specific for at least one of the following nucleotide variances in an epidermal growth factor receptor (EGFR) gene, where the nucleotide variance is selected from:
i. a mutation in exon 18 that results in a substitution of cysteine for glycine at position 719 (G719C) or in a substitution of serine for glycine at position 719 (G719S) or in a substitution of an alanine for glycine at position 719 (G719A) of SEQ ID NO: 512;
ii. an in-frame deletion in exon 19 that results in a deletion of at least amino acids leucine, arginine, glutamic acid and alanine at codons 747, 748, 749, and 750 of SEQ ID NO: 512;
iii. a mutation in exon 20 that results in an insertion of amino acids asparagine, proline and glycine between position 770 and 771 (D770_N771insNPG), or in an insertion of amino acids serine, valine and aspartic acid between position 770 and 771 (D770_N771insSVD), or in an insertion of amino acid valine between position 772 and 773 (P772 H773insV), or in a substitution at position 790 of SEQ ID NO: 512; and
iv. a mutation in exon 21 that results in an amino acid substitution of arginine for leucine at position 858 (L858R) or of glutamine for leucine at position 861 (L861Q) of SEQ ID NO: 512;
to a biological sample obtained from the blood of a human patient afflicted with non-small cell lung cancer;
(b) performing an amplification step by polymerase chain reaction (PCR) wherein the PCR is allele-specific amplification for at least one of the nucleotide variances; and
(c) detecting whether at least one of the above-described variances is present.
2. The assay of claim 1 , wherein the allele-specific amplification is performed using at least one primer pair designed to anneal to an EGFR nucleic acid, wherein one primer of the pair comprises a sequence that selectively hybridizes to the nucleotide variance under high stringency conditions and amplifies the nucleotide variance sequence but does not amplify a corresponding wild type EGFR sequence.
3. The assay of claim 1 or 2 , wherein the blood is further processed to produce plasma.
4. The assay of claim 1 or 2 , wherein the nucleotide variance is mutation in exon 18 that results in a substitution of cysteine for glycine at position 719 (G719C).
5. The assay of claim 1 or 2 , wherein the nucleotide variance is a mutation in exon 18 that results in a substitution of serine for glycine at position 719 (G719S) of SEQ ID NO: 512.
6. The assay of claim 1 or 2 , wherein the nucleotide variance is a mutation in exon 18 that results in a substitution of alanine for glycine at position 719 (G719A) of SEQ ID NO: 512.
7. The assay of claim 1 or 2 , wherein the nucleotide variance is an in-frame deletion in exon 19 that results in a deletion of at least amino acids leucine, arginine, glutamic acid and alanine at codons 747, 748, 749, and 750 of SEQ ID NO: 512.
8. The assay of claim 1 or 2 , wherein the nucleotide variance is a substitution in exon 20 that results in an amino acid change at position 790 of SEQ ID NO: 512.
9. The assay of claim 1 or 2 , wherein the nucleotide variance is a mutation in exon 20 that results in an insertion of amino acids asparagine, proline and glycine between position 770 and 771 (D770_N771insNPG) of SEQ ID NO: 512.
10. The assay of claim 1 or 2 , wherein the nucleotide variance is mutation in exon 20 that results in an insertion of amino acids serine, valine and aspartic acid between position 770 and 771 (D770_N771insSVD) of SEQ ID NO: 512.
11. The assay of claim 1 or 2 , wherein the nucleotide variance is a mutation in exon 20 that results in an insertion of amino acid valine between position 772 and 773 (P772_H773insV) of SEQ ID NO: 512.
12. The assay of claim 1 or 2 , wherein the nucleotide variance is a mutation in exon 21 that results in an amino acid substitution of arginine for leucine at position 858 (L858R).
13. The assay of claim 1 or 2 , wherein the nucleotide variance is a mutation in exon 21 that results in an amino acid substitution of glutamine for leucine at position 861 (L861Q) of SEQ ID NO: 512.
14. An assay comprising:
(a) adding primers specific for at least one of the following nucleotide variances in an epidermal growth factor receptor (EGFR) gene, where the nucleotide variance is selected from:
i) a mutation in exon 18 that results in a substitution of cysteine for glycine at position 719 (G719C) or in a substitution of serine for glycine at position 719 (G719S) or a substitution of an alanine for glycine at position 719 (G719A) of SEQ ID NO: 512;
ii) an in-frame deletion in exon 19 that results in a deletion of at least amino acids leucine, arginine, glutamic acid and alanine at codons 747, 748, 749, and 750 of SEQ ID NO: 512;
iii) a mutation in exon 20 that results in an insertion of amino acids asparagine, proline and glycine between position 770 and 771 (D770_N771insNPG), or in an insertion of amino acids serine, valine and aspartic acid between position 770 and 771 (D770 N771insSVD), or in an insertion of amino acid valine between position 772 and 773 (P772 H773insV), or in a substitution at position 790 of SEQ ID NO: 512; and
iv) a mutation in exon 21 that results in an amino acid substitution of arginine for leucine at position 858 (L858R) or of glutamine for leucine at position 861 (L861Q) of SEQ ID NO: 512;
to a biological sample obtained from the blood of a human patient afflicted with non-small cell lung cancer;
(b) performing an amplification step by polymerase chain reaction (PCR) to amplify part of exon 18, 19, 20, or 21 of the EGFR gene; and
(c) detecting whether at least one of the above-described nucleotide variances is present by hybridizing at least one allele-specific nucleic acid probe specific for the nucleotide variance to the EGFR gene.
15. The assay of claim 14 , wherein the nucleic acid probe comprises a label.
16. An assay comprising:
(a) adding primers specific for at least one of the following nucleotide variances in an epidermal growth factor receptor (EGFR) gene, where the nucleotide variance is selected from:
i) a mutation in exon 18 that results in a substitution of cysteine for glycine at position 719 (G719C) or in a substitution of serine for glycine at position 719 (G719S) or a substitution of an alanine for glycine at position 719 (G719A) of SEQ ID NO: 512;
ii) an in-frame deletion in exon 19 that results in a deletion of at least amino acids leucine, arginine, glutamic acid and alanine at codons 747, 748, 749, and 750 of SEQ ID NO: 512;
iii) a mutation in exon 20 that results in an insertion of amino acids asparagine, proline and glycine between position 770 and 771 (D770_N771insNPG), or in an insertion of amino acids serine, valine and aspartic acid between position 770 and 771 (D770_N771insSVD), or in an insertion of amino acid valine between position 772 and 773 (P772 H773insV), or in a substitution at position 790 of SEQ ID NO: 512; and
iv) a mutation in exon 21 that results in an amino acid substitution of arginine for leucine at position 858 (L858R) or of glutamine for leucine at position 861 (L861Q) of SEQ ID NO: 512;
to a biological sample obtained from the blood of a human patient afflicted with non-small cell lung cancer;
(b) performing an amplification step by polymerase chain reaction (PCR) wherein the PCR is allele-specific amplification for at least one of the nucleotide variances, to thereby generate an amplification product; and
(c) detecting whether at least one of the above-described nucleotide variances is present by sequencing the region where the nucleotide variance is found within the amplification product of step (b).