Methods for lung cancer detection
The invention provides methods for detecting single nucleotide variants in lung cancer, especially stage 3a lung adenocarcinoma and lung squamous cell carcinoma. Additional methods and compositions, such as reaction mixtures and solid supports comprising clonal populations of nucleic acids, are provided.
1. A method for treatment of an individual known to have lung squamous cell carcinoma, the method comprising:
(a) extracting cell-free DNA (cfDNA) from a plasma sample of blood obtained from the individual known to have lung squamous cell carcinoma;
(b) preparing a fraction of the DNA useful for tracking single nucleotide variants present in the individual known to have lung squamous cell carcinoma by:
generating a set of amplicons, wherein the set of amplicons are generated by performing a multiplex PCR amplification reaction on nucleic acids of the DNA extracted in (a) and the multiplex PCR amplification reaction comprises forming an amplification reaction mixture by combining a polymerase, nucleotide triphosphates, nucleic acid fragments from a nucleic acid library generated from the plasma sample, and a set of forward and reverse primers that each binds an effective distance from a single nucleotide variant loci within 25 base pairs of the single nucleotide variant loci, or a set of primer pairs that each span an effective region comprising the single nucleotide variant loci within 25 base pairs of the single nucleotide variant loci, an annealing time of 15 minutes, 20 mM etramethyl ammonium chloride (TMAC), and a primer concentration of 10 nM, wherein the single nucleotide variant loci are known to be associated with lung squamous cell carcinoma and each amplicon of the set of amplicons spans at least one single nucleotide variant loci of a set of 25 to 1000 single nucleotide variant loci known to be associated with lung squamous cell carcinoma;
(c) determining presence of at least one single nucleotide variant associated with the lung squamous cell carcinoma in the plasma sample by next generation sequencing at a depth of read of greater than 1,000,000 and determining a sequence of at least a segment of each amplicon in the set of amplicons, wherein determining the presence of the at least one single nucleotide variant further comprises:
1) estimating an efficiency of the multiplex PCR amplification reaction and per cycle error rate using normal control samples, wherein the efficiency of the multiplex PCR amplification reaction indicates a probability that each molecule in the plasma sample is replicated in the multiplex PCR amplification reaction;
2) computing a starting number of molecules in the plasma sample using the efficiency obtained in 1);
3) for a range of potential real mutant fraction values between 0 and 0.15, estimating a mean and variance for a total number of molecules, background error molecules, and real mutation molecules using parameters obtained in 1) and 2);
4) computing a likelihood L(θ) for each potential real mutant fraction;
5) selecting a value of θ that maximizes the likelihood L(θ) and denoting the maximized likelihood by {circumflex over (θ)} MLE ,
6) computing a confidence score as:
L
(
θ
^
MLE
)
L
(
0
)
+
L
(
θ
^
MLE
)
;
and
7) determining presence of a single nucleotide variant if the confidence score is ≥0.95% for transitions and ≥0.98% for transversions; wherein the segment comprises one or more single nucleotide variant loci; and
administering a compound to the individual, wherein the compound is known to be specifically effective in treating the lung squamous cell carcinoma having the one or more single nucleotide variants.
2. The method according to claim 1 , wherein the lung squamous cell carcinoma is a stage 1a, 1b, or 2a squamous cell carcinoma.
3. The method according to claim 1 , wherein the lung squamous cell carcinoma is a stage 1a or 1b squamous cell carcinoma.
4. The method according to claim 1 , wherein the individual is not subjected to surgery.
5. The method according to claim 1 , wherein the individual is not subjected to a biopsy.
6. The method according to claim 1 , wherein the method further comprises determining the variant allele frequency for each of the single nucleotide variants from the sequence determination.
7. The method according to claim 6 , wherein a lung squamous cell carcinoma treatment plan is identified based on the variant allele frequency determinations.
8. The method according to claim 6 , where the compound is known to be specifically effective in treating the lung squamous cell carcinoma having one of the single nucleotide variants with a variable allele frequency greater than at least one half of the other single nucleotide variants that were determined.
9. The method according to claim 1 , wherein the nucleic acids are isolated from a tumor of the individual and single nucleotide variants are identified in the tumor for the set of single nucleotide variant loci before determining the sequence of the at least a segment of each amplicon of the set of amplicons for the sample of blood.
10. The method of claim 1 , wherein the set of single nucleotide variant loci comprises clonal single nucleotide variant loci.
11. The method according to claim 10 , wherein the compound targets the clonal single nucleotide variants.
12. The method according to claim 10 , wherein a variant allele frequency of greater than 1.0% is indicative a clonal single nucleotide variant.