# US 12,006,555 B2 · Method to determine responsiveness of cancer to epidermal growth factor receptor targeting treatments

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- Number: US 12,006,555 B2
- Document ID: US12006555B2
- Status: Granted
- Kind: B2
- Application number: 16/854,654
- Filed: 2020-04-21
- Published: 2021-01-21
- Granted: 2024-06-11
- Earliest claimed: 2004-03-31
- Assignees: The General Hospital Corporation; Dana-Farber Cancer Institute, Inc.
- Applicants: The General Hospital Corporation; Dana-Farber Cancer Institute, Inc.
- Inventors: Daphne Winifred Bell; Daniel A. Haber; Pasi Antero Janne; Bruce E. Johnson; Thomas J. Lynch; Matthew Meyerson; Juan Guillermo Paez; William R. Sellers; Jeffrey E. Settleman; Raffaella Sordella
- Web page: https://patentgrep.com/patent/US12006555B2

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- A61P 35/00: Antineoplastic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12Q 1/485: {involving kinase}
- G01N 33/575: for cancer
- G01N 33/74: involving hormones {or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors}
- C12Q 2600/106: Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
- C12Q 2600/118: Prognosis of disease development
- C12Q 2600/136: Screening for pharmacological compounds
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/158: Expression markers
- C12Q 2600/16: Primer sets for multiplex assays
- G01N 2333/485: Epidermal growth factor [EGF] (urogastrone)
- G01N 2800/52: Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1, 2)

The assay of claim 1 or 2, wherein the blood is further processed to produce plasma.

### Claim 4 (depends on 1, 2)

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).

### Claim 5 (depends on 1, 2)

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.

### Claim 6 (depends on 1, 2)

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.

### Claim 7 (depends on 1, 2)

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.

### Claim 8 (depends on 1, 2)

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.

### Claim 9 (depends on 1, 2)

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.

### Claim 10 (depends on 1, 2)

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.

### Claim 11 (depends on 1, 2)

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.

### Claim 12 (depends on 1, 2)

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).

### Claim 13 (depends on 1, 2)

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.

### Claim 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.

### Claim 15 (depends on 14)

The assay of claim 14, wherein the nucleic acid probe comprises a label.

### Claim 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).