Patent Application
20220073993
The Sequence Listing for this application is labeled “Seq-List.txt” which was created on Dec. 21, 2020 and is 39 KB. The entire content of the sequence listing is incorporated herein by reference in its entirety.
The present invention relates to methods, materials and kits for detecting mutations in the EGFR gene. The invention also relates to methods for detecting or diagnosing cancer in a subject, as well as for determining the clinical stage, and clinical progression of a cancer.
The epidermal growth factor receptor (EGFR) is a trans-membrane tyrosine-kinase protein whose deregulated activation can lead to cellular proliferation, inhibition of apoptosis, angiogenesis and to phenotypic changes towards cell adhesion, migration and invasion. Activating EGFR mutations are clustered within exons 18-21 which encode for a portion of the EGFR kinase domain. EGFR mutations are very frequent in lung adenocarcinoma and are estimated to affect approximately 15% of US patients and more than 30% patients of Asian descent. In this respect, small-molecule EGFR tyrosine kinase inhibitors (TKIs) have been designed and developed, which launched the era of targeted, personalized and precise medicine for lung cancer. EGFR tyrosine kinase inhibitors are associated with high response rate and progression-free survival for patients with non-small cell lung cancer with EGFR-mutant genotype. Iressa® (gefitinib) and Tarceva® (erlotinib) constitute the first generation of EGFR tyrosine kinase inhibitors. However, most patients develop acquired drug resistance mostly driven by the T790M mutation occurring within the exon 20 of the EGFR gene. Table 1 below presents a list of EGFR mutations conferring either sensitivity or resistance to the first-generation TKIs.
Although the second-generation EGFR TKIs, such as Giotrif® (afatinib), dacomitinib and neratinib, demonstrated promising activity against T790M in preclinical models, they have failed to overcome resistance in patients due to dose-limiting toxicity.
Recently, the third-generation EGFR TKIs Tagrisso® (osimertinib) have shown to be effective against cell lines and murine models harbouring T790M mutations whilst sparing EGFR wild-type cells, which represented a promising breakthrough approach in overcoming T790M-mediated resistance in NSCLC patients.
On Mar. 30, 2017, following the positive results obtained by the AURA study (ClinicalTrials.gov identifier: NCT01802632) the U.S. Food and Drug Administration granted regular approval to Tagrisso® for the treatment of patients with metastatic EGFR T790M mutation-positive NSCLC, whose disease has progressed on or after EGFR TKI therapy. Positive EGFR T790M mutation status needs to be determined by an FDA-approved test, either performed on tissue samples or from circulating tumour DNA obtained from plasma samples.
Accurate and sensitive detection systems for prompt genotyping of sensitizing mutations to EGFR TKIs treatments and for the identification of pre-existing or acquired resistance to first-generation TKIs drugs are hence pivotal for providing lung cancer patients with the best and most effective personalized treatment, based on their genetic profiles.
The invention provides novel methods, kits and materials allowing fast and reliable detection of mutations in the EGFR gene. The invention more specifically provides methods and kits for optimized detection of mutations in EGFR sequence from cell-free nucleic acids (“cfNA”). The invention is suitable to detect any type of mutation, such as nucleotide substitution(s), deletion(s) and/or insertion(s) in the EGFR gene sequence.
In a first aspect, the invention more particularly relates to a method for determining the presence of a mutation in the EGFR gene in a sample from a subject, the method comprising amplifying, in a sample of a biological fluid from the subject containing cell free nucleic acids, at least a first target sequence from the EGFR gene, the first target sequence containing a site of a mutation on said gene, wherein the first amplified target sequence is 50-95 nt in length and amplification of the first target sequence is conducted with a first set of reagents comprising (i) a mutation-specific primer of 15-25 nt in length, (ii) a paired primer of 15-25 nt in length and (iii) an oligoblocker, wherein the oligoblocker hybridizes specifically to the first target sequence in non-mutated form and blocks polymerase elongation.
In a preferred embodiment, the method further comprises amplifying a second non-mutated target sequence on the EGFR gene, said second target sequence being located between 100 and 400 bp from the first target sequence on said gene, the first and second amplified sequences each being 50-95 nt in length and of a similar length.
In a further aspect, the invention relates to a kit for determining the presence of a mutation in the EGFR gene in a sample of a biological fluid containing cell free nucleic acids from a subject, the kit comprising at least a first set of reagents, wherein the first set of reagents amplifies a first target sequence from the EGFR gene, said first target sequence containing a site of a mutation in the EGFR gene, said first set comprising (i) a mutation-specific primer of 15-25 nt in length, (ii) a paired primer of 15-25 nt in length positioned such that the amplified first target sequence is 50-95 nt in length, and (iii) an oligoblocker, wherein the oligoblocker hybridizes specifically to the first target sequence in non-mutated form and blocks polymerase elongation.
Preferably, the kit comprises a second set of reagents (b) which amplifies a second non-mutated target sequence from the EGFR gene, said second target sequence being located between 100 and 400 bp from the first target sequence on said gene, and wherein said amplified second target sequence is 50-95 nt in length and of a length similar to that of the first amplified target sequence.
The invention may be used preferably to detect a mutation selected from G719A, G719C, G719S, S768I, T790M, L858R, L861Q, exon19DEL and exon20INS, or a combination thereof.
A further object of the invention relates to a nucleic acid molecule consisting of a nucleotide sequence selected from any one of SEQ ID NOs: 1 to 55.
The invention also relates to the use of a nucleic acid molecule as defined above for in vitro amplification of a target sequence in the EGFR gene.
The invention also relates to a method for diagnosing a patient, comprising detecting a mutation in the EGFR gene in a sample from the patient using a method or kit or nucleic acid as defined above.
The invention also relates to a method for treating a patient, comprising detecting a mutation in the EGFR gene in a sample from the patient using a method or kit or nucleic acid as defined above, and treating subjects having cancer.
The present invention relates to improved methods and kits for detecting mutations in EGFR gene sequence, as well as the uses thereof. EGFR mutations are correlated to cancers, particularly lung cancer, and the invention may be used e.g., in the diagnosis, drug development, or clinical monitoring of subjects with cancer, as well as to adjust/define optimized treatment.
The invention is suitable for detecting any mutation in EGFR. The term “mutation” designates any alteration in the nucleotide sequence encoding EGFR, such as nucleotide substitution(s), deletion(s), or insertion(s). In a particular embodiment, the mutation is a single nucleotide substitution in a coding (exon) region of the gene. In another particular embodiment, the mutation is a deletion or insertion of one or several (typically from 1 to 15) contiguous nucleotides, typically in an exon of the gene sequence.
The invention utilizes particular sets of allele-specific primers and oligoblockers allowing specific and sensitive detection of mutations in the gene sequence from circulating cell-free nucleic acids. The inventors have selected optimized target sequences within the EGFR gene and optimized primer design, which allow controlled and specific amplification from cell free nucleic acids. The invention can be implemented from any fluid sample containing cfNAs, e.g., cell free DNA (cfDNA), cell free RNA, cell free siRNA and/or cell free miRNA. Cell free nucleic acids are typically circulating nucleic acids contained in biological fluids, so that the method is advantageous as compared to an analysis from tissue biopsy. The invention may be implemented with samples from any subject, including any human patient having or suspected to have cancer.
The method of the invention typically comprises amplifying, in a sample of a biological fluid from the subject containing cell free nucleic acids, at least a first target sequence from the EGFR gene, the first target sequence containing a site of a mutation on said gene, wherein the first amplified target sequence is 50-95 nt in length and amplification of the first target sequence is conducted with a first set of reagents comprising (i) a mutation-specific primer of 15-25 nt in length, (ii) a paired primer of 15-25 nt in length and (iii) an oligoblocker which hybridizes specifically to the first target sequence in non-mutated form resulting in a blockade of polymerase elongation.
The invention may be conducted with any fluid sample containing cfNA, such as blood, plasma, serum, pleural fluid, saliva, urine, or the like. The sample may be pretreated prior to being used in the invention (e.g., diluted, concentrated, separated, partially purified, frozen, etc.). The method is typically performed on a sample of, or derived from, blood, serum or plasma, such as a pretreated blood sample.
The invention uses a first set of primers which is designed to amplify a target sequence of 50-95 nt in length within the EGFR gene, said sequence carrying a site of a targeted mutation in the gene.
The term “primer” designates, within the context of the present invention, nucleic acid molecules that can hybridize specifically to a target genetic sequence and serve to initiate amplification. Primers of the invention are typically single-stranded nucleic acids, with a length advantageously comprised between 10 and 30 bases, preferably between 15 and 30 bases, even more preferably between 15 and 25 bases. Primers should perfectly match with the targeted sequence in the EGFR gene, allowing specific hybridization thereto and essentially no hybridization to another region.
Preferred primers of the invention shall have a sequence with a GC content between 40 and 60%. The GC content is the number of G's and C's in the primer as a percentage of the total bases. Such a level of GC content provides the optimized combination of specific amplification and efficacy. Also, in a set of reagents of the invention, all of the primers shall have a similar Tm. More particularly, the Tm difference between the mutation-specific primer and its paired WT primer should preferably not be more than 5° C. Indeed, with such low difference, the difference of hybridization of each partner primer is low and does not affect specific mutant amplification. More preferably, the Tm of the primers should be between 40-60° C. and should not differ between mutation-specific and paired by more than 5° C. Furthermore, in a preferred embodiment, the primers of the invention should not be self-annealing. Indeed, self-annealing may decrease the quantity of primer available for the amplification and reduce efficacy. Moreover, it contributes to non-specific amplification. The risk of self-annealing and self-hairpin loops of primers may be determined in silico using available software such as the software Oligoanalyzer®, which analyzes the change in Gibbs free energy required for the breaking of secondary structures (ΔG). Typically, if this energy is negative, it should not be below −4 kcal/mol, otherwise risk of self-annealing or self-hairpin loops could occur. In a particular embodiment, primers of the invention shall thus have a ΔG superior or equal to −4 kcal/mol as determined with Oligoanalyzer®. When using online tools as IDT OligoAnalyzer, which rely on different algorithms than Oligoanalyzer® for analyzing the change in Gibbs free energy required for the breaking of secondary structures, the ΔG should not be below −7 kcal/mol otherwise risk of self-annealing or self-hairpin loops could occur. Accordingly, in another particular embodiment, primers of the invention shall have a ΔG superior or equal to −7 kcal/mol as determined with IDT OligoAnalyzer.
As indicated, the invention uses a first set of reagents comprising (i) a mutation-specific primer, (ii) a paired primer and (iii) an oligoblocker.
The mutation-specific primer is designed to hybridize to the mutated sequence, if and when present. In this regard, when the mutation is a nucleotide substitution, the mutation-specific primer preferably contains the mutated position at one terminal nucleotide thereof. Indeed, the invention shows such conformation allows increased specificity of the method. In a preferred embodiment, the mutated position is at the 3′-terminal nucleotide of the mutation-specific primer, or at the 5′-terminal nucleotide of the mutation-specific primer.
In another embodiment, when the mutation is a deletion, the mutation-specific primer overlaps with the junction region in the gene created by said deletion. The primer sequence is preferably centered on the targeted junction.
In still another embodiment, when the mutation is an insertion, the mutation-specific primer overlaps with at least one junction region in the gene created by said insertion. The primer sequence is preferably centered on the targeted junction.
The second primer, the “paired” primer, hybridizes to a sequence in the EGFR gene which is non-mutated and amplifies, with its paired mutation-specific primer, a target sequence of 50-95 nt in length.
The first set of reagent contains an oligoblocker, which increases specificity of the method. The oligoblocker is an oligonucleotide which hybridizes specifically to the first target sequence in non-mutated form, such hybridization resulting in a blockade of amplification. When the targeted mutation is present, the oligoblocker does not hybridize and amplification of the mutated sequence occurs. In contrast, when the mutation is not present, the oligoblocker hybridizes to the target sequence and prevents non-specific amplification. The oligoblocker hybridizes “specifically” to the first target sequence in non-mutated form, which means that its sequence is exactly complementary to the non-mutated sequence. As a result, the oligoblocker essentially cannot hybridizes to the gene when the mutation is present. The oligoblocker preferably has a length similar to that of the two primers. It is thus preferably between 15-25 nucleotides in length. Furthermore, in order to increase specificity, in case of a single nucleotide mutation, the site of the non-mutated nucleotide targeted by the oligoblocker shall be preferably centered on the oligoblocker sequence, more preferably located within 80% central nucleotides, even more preferably within 60% central nucleotides, even further preferably within 40% central nucleotides.
Furthermore, in all embodiments, the oligoblocker is modified, preferably terminally, to prevent amplification upon hybridization, e.g. to prevent polymerase elongation. As a result, when the mutation is not present, the oligoblocker hybridizes to the target sequence and prevents any non-specific amplification. In a preferred embodiment, the oligoblocker is phosphorylated in 3′, resulting in a blockade of polymerase elongation.
Amplification using the above set of reagents from a sample containing cfNA allows specific production of a 50-95 nt long amplicon sequence containing a mutation when the targeted mutation is present, and no production of such an amplicon when the mutation is not present. The results contained in the present application show that such design provides high specificity and high sensitivity with any sample containing cell-free nucleic acids. Detection of an amplification product thus indicates the presence of the mutation in the sample.
Amplification may be performed using any technique known per se in the art such as with a polymerase, e.g., by PCR, Q-PCR, droplet-digital PCR and crystal-digital PCR.
In a preferred embodiment, the method uses two sets of reagents to amplify two distinct target sequences in the EGFR gene: The first set, as described above, targets a sequence containing a (site of a) mutation; and the second set targets a wild-type (“WT”) sequence. By combining the two sets of reagents, the method of the invention allows measuring not only the presence or amount of a mutation, but also the level of total cfNA, thereby providing with high reliability a mutant allele frequency in circulating nucleic acids. The second targeted sequence should be located at a distance of about 100-400 bp of the mutant sequence. Furthermore, the second amplified sequence should have a length comprised between 50-95 nt and similar to that of the first amplified sequence.
In a preferred embodiment, the method thus comprises a step of amplifying a second non-mutated target sequence on the EGFR gene, said second target sequence being located at a distance of 100 to 400 bp from the first target sequence on said gene, the first and second amplified sequences each being 50-95 nt in length and of a similar length.
The term “similar length” indicates that the two amplified sequences should not differ in length by more than 15%, even more preferably by not more than 10%, further preferably by not more than 5%. In other words, if one set amplifies a sequence of 80 nt, the other shall amplify a sequence of 68-92 nt, preferably of 72-88 nt, more preferably of 76-84 nt.
The distance between the two targeted sequence shall be comprised between 100-400 pb. Such a distance designates typically the number of nucleotides contained between the first amplified nucleotide of the first target sequence and the first amplified nucleotide of the second target sequence. Preferably, the distance is comprised between 200-400, more preferably between 250 and 350. Further preferably, at a distance of 300 nucleotides±10%.
The second set of reagents contains a pair of amplification primers, but no oligoblocker. Indeed, the second amplified sequence is non-mutated. The primers in the second set shall have a length between 15-25 nt, and shall preferably have the characteristics as defined above for the first set in terms of Tm, GC content and self-annealing.
Both amplifications are preferably conducted in parallel, by separately incubating aliquots of a test sample with the two sets of reagents and suitable enzymes and nucleotides to perform amplification. The reactions thus produce:
one mutation-specific amplicon of 50-95 nt produced by the first set of reagents (if the mutation is present); and
one WT amplicon of 50-95 nt, produced by the second set of reagents.
Detection of the mutation-specific amplicon indicates the presence of the mutation in the sample. Detection of the other amplicon allows determination of a frequency of mutation in circulating NAs of the subject, e.g., by determining a ratio mt/WT. As illustrated in the experimental section, the method is highly selective and very sensitive.
Alternatively, the two amplifications may be conducted simultaneously, by incubating an aliquot of a test sample with the two sets of reagents and suitable enzymes and nucleotides to perform amplification. Such a reaction produces the following amplicons:
one mutation-specific amplicon of 50-95 nt produced by the first set of reagents (if the mutation is present);
one WT amplicon of 50-95 nt, produced by the second set of reagents; and
one amplicon of 200-400 nt, produced by the combination of primers of the two sets of reagents.
Furthermore, the method may be used to detect several different mutations in the EGFR gene from a subject sample, either sequentially, or in parallel. In such a case, the method uses one set of mutation-specific reagents as defined above for each targeted mutation. Typically, the method also uses at least one set of reagents that amplify a WT target sequence located 100-400 bp from one of the targeted mutated sequence.
Most preferred mutations that are detected with the present invention are described in the following table:
These mutations are correlated to cancer, particularly lung cancer, such as NSCLC. The detection of these mutations, alone or in combinations, allows reliable diagnosis or prognosis of cancer in a subject, and also allows staging of the cancer.
In a particular embodiment, the invention is for detecting T790M mutation. EGFR T790M is a substitution mutation with a high frequency rate, especially in patients having acquired resistance to cancer treatment with tyrosine kinase inhibitors. Detecting the presence of this mutation allows an adaptation of the treatment. The T790M mutation results in an amino acid substitution at position 790, from a threonine (T) to a methionine (M). This mutation occurs within exon 20, which encodes part of the kinase domain. Nomenclature of EGFR T790M point mutation is c: 2369 C>T. It means that the 2369th nucleotide on the cDNA reference sequence is a Cytosine that is substituted by a Thymine.
In a preferred embodiment, the mutation is EGFR T790M and the first set of reagents comprises a mutation-specific primer selected from SEQ ID NO: 1 or 2 (T790M 16; T790M 17), a paired primer selected from SEQ ID NO: 3 or 4 (T790 P19; T790 P20), and an oligoblocker selected from SEQ ID NO: 5 (T790 OB19). Most preferably, the mutation-specific primer is SEQ ID NO: 1 (T790M 16), the paired primer is SEQ ID NO: 3 (T790 P19) and the oligoblocker is SEQ ID NO: 5 (T790 OB19). In a preferred embodiment, the method amplifies a second WT target sequence, located preferably 200-400 bp from the first mutated target sequence, using a second set of reagents. In a more preferred embodiment, the second set of reagents comprises a first primer SEQ ID NO: 6 (T790 WT L19) and a second primer SEQ ID NO: 7 (T790 WT R19).
In another embodiment, the mutation is EGFR L858R and the first set of reagents for detecting the EGFR L858R mutation comprises a mutation-specific primer selected from any one of SEQ ID NOs: 8-11 (L858R 16, L858R 17, L858R 18, L858R 19), a paired primer selected from SEQ ID NO: 12 (L858 P20), and an oligoblocker selected from any one of SEQ ID NOs: 13-14 (L858 OB19/3, L858 OB20/3). Most preferably, the mutation-specific primer is SEQ ID NO: 11 (L858R 19), the paired primer is SEQ ID NO: 12 (L858 P20) and the oligoblocker is SEQ ID NO: 13 (L858 OB19/3). In a preferred embodiment, the method amplifies a second WT target sequence, located preferably 200-400 bp from the first mutated target sequence, using a second set of reagents. In a more preferred embodiment, the second set of reagents comprises a first primer SEQ ID NO: 15 (L858 WT L) and a second primer SEQ ID NO: 16 (L858 WT R).
In another embodiment, the mutation is EGFR L861Q and the first set of reagents for detecting the EGFR L861Q mutation comprises a mutation-specific primer selected from any one of SEQ ID NOs: 17-18 (L861Q i17, L861Q i18), a paired primer selected from SEQ ID NO: 19 (L861 iP20), and an oligoblocker selected from any one of SEQ ID NOs: 20-21 (L861 iOB19/2, L861 iOB20/2). Most preferably, the mutation-specific primer is SEQ ID NO: 18 (L861Q i18), the paired primer is SEQ ID NO: 19 (L861 iP20), and the oligoblocker is SEQ ID NO: 21 (L861 iOB20/2). In a preferred embodiment, the method amplifies a second WT target sequence, located preferably 200-400 bp from the first mutated target sequence, using a second set of reagents. In a more preferred embodiment, the second set of reagents comprises a first primer SEQ ID NO: 22 (L861 WT L) and a second primer SEQ ID NO: 23 (L861 WT R).
In another embodiment, the mutation is EGFR G719S and the first set of reagents for detecting EGFR G719S mutation comprises a mutation-specific primer selected from any one of SEQ ID NOs: 24-25 (G719S 19, G719S 20), a paired primer selected from SEQ ID NO: 26 (G719 P19), and an oligoblocker selected from any one of SEQ ID NOs: 27-28 (G719 OB18/2, G719 OB19/2). Most preferably, the mutation-specific primer is SEQ ID NO: 25 (G719S 20), the paired primer is SEQ ID NO:26 (G719 P19), and the oligoblocker is SEQ ID NO: 28 (G719 OB19/2). In a preferred embodiment, the method amplifies a second WT target sequence, located preferably 200-400 bp from the first mutated target sequence, using a second set of reagents. In a more preferred embodiment, the second set of reagents comprises a first primer SEQ ID NO:29 (G719 WT L) and a second primer SEQ ID NO: 30 (G719 WT R).
In another embodiment, the mutation is EGFR G719A and the first set of reagents for detecting the EGFR G719A mutation comprises a mutation-specific primer selected from any one of SEQ ID NOs: 31-33 (G719A 18, G719A 19, G719A 20), a paired primer selected from SEQ ID NO: 26 (G719 P19), and an oligoblocker selected from any one of SEQ ID NOs: 27-28 (G719 OB18/2, G719 OB19/2). Most preferably, the mutation-specific primer is SEQ ID NO: 31 (G719A 18), the paired primer is SEQ ID NO: 26 (G719 P19) and the oligoblocker is SEQ ID NO: 27 (G719 OB18/2). In a preferred embodiment, the method amplifies a second WT target sequence, located preferably 200-400 bp from the first mutated target sequence, using a second set of reagents. In a more preferred embodiment, the second set of reagents comprises a first primer SEQ ID NO: 29 (G719 WT L) and a second primer SEQ ID NO: 30 (G719 WT R).
In another embodiment, the mutation is EGFR S768I and the first set of reagents for detecting the EGFR S768I mutation comprises a mutation-specific primer selected from any one of SEQ ID NOs: 34-35 (S768I i16, S768I i17), a paired primer selected from SEQ ID NO: 36 (S768 iP18), and an oligoblocker selected from SEQ ID NO: 37 (S768 iOB19). Most preferably, the mutation-specific primer is SEQ ID NO: 35 (S768I i17), the paired primer is SEQ ID NO: 36 (S768 iP18) and the oligoblocker is SEQ ID NO: 37 (S768 iOB19). In a preferred embodiment, the method amplifies a second WT target sequence, located preferably 200-400 bp from the first mutated target sequence, using a second set of reagents. In a more preferred embodiment, the second set of reagents comprises a first primer SEQ ID NO:38 (S768 WT L) and a second primer SEQ ID NO: 39 (S768 WT R).
In another embodiment, the mutation is EGFR exon19 Δ(E746-A750) and wherein the mutation-specific primer is SEQ ID NO: 40 (DEL19 17N), the paired primer is SEQ ID NO: 41 (DEL19 P19), and the oligoblocker is SEQ ID NO: 42 (DEL19 OB21/3). In a preferred embodiment, the method amplifies a second WT target sequence, located preferably 200-400 bp from the first mutated target sequence, using a second set of reagents. In a more preferred embodiment, the second set of reagents comprises a first primer SEQ ID NO: 43 (DEL19 WT L) and a second primer SEQ ID NO: 44 (DEL19 WT R).
In another embodiment, the mutation is EGFR V769-D770 insASV (Ins20) and the first set of reagents for detecting the EGFR Ins20 mutation comprises a mutation-specific primer selected from any one of SEQ ID NOs: 45-49 (INS20 AVS i16, INS20 AVS i16/2, INS20 AVS i16/3, INS20 AVS i16/4, INS20 AVS i16/5), a paired primer selected from any one of SEQ ID NOs: 50-52 (Ins20 iP18, Ins20 iP17, Ins20 iP17/2), and an oligoblocker selected from SEQ ID NO: 53 (Ins20AVS iOB18). Most preferably, the mutation-specific primer is SEQ ID NO: 45 (INS20 AVS i16), the paired primer is SEQ ID NO: 51 (Ins20 iP17) and the oligoblocker is SEQ ID NO: 53 (Ins20AVS iOB18). In a preferred embodiment, the method amplifies a second WT target sequence, located preferably 200-400 bp from the first mutated target sequence, using a second set of reagents. In a more preferred embodiment, the second set of reagents comprises a first primer SEQ ID NO:54 (Ins20 WT L) and a second primer SEQ ID NO: 55 (Ins20 WT R).
In another particular embodiment, the invention is for detecting a combination of two or more mutations in the EGFR gene.
In this regard, in a particular embodiment, the method is for detecting at least two nucleotide substitutions, preferably at least T790M and L858R.
In another embodiment, the method is for detecting at least one nucleotide substitution and one deletion.
In a particular embodiment, the method is for detecting T790M, L858R and DelExon19 (such as exon19 Δ(E746-A750)).
Such combination of mutations is particularly indicative of a subject condition. In particular, the detection of L858R and of deletions of Exon 19 covers approximately 91% of EGFR positive cases where targeted therapy with first-generation TKIs is predicted to work effectively. Moreover, the addition of T790M enables to identify the 5% of treatment-naïve patients T790M-positive that would be resistant to first-generation TKIs, for whom third-generation TKIs should be considered as favored therapeutic option.
In a further aspect, the invention relates to kits suitable for detecting EGFR mutations in a sample of a biological fluid containing cell free nucleic acids from a subject. Kits generally comprise reagents, containers and, optionally, instructions to perform the method. The reagents may be in a same container, in discrete parts thereof, of in separate containers. The user may then use any portion of the reagents to perform the reaction, typically following the instructions.
In a more particular embodiment, the kits of the invention comprise at least a first set of reagents (a), wherein the first set of reagents amplifies a first target sequence from the EGFR gene, said first target sequence containing a site of a mutation in the EGFR gene, said first set of reagents comprising (i) a mutation-specific primer of 15-25 nt in length, (ii) a paired primer of 15-25 nt in length positioned such that the amplified first target sequence is 50-95 nt in length, and (iii) an oligoblocker, wherein the oligoblocker hybridizes specifically with the first target sequence in non-mutated form and blocks amplification.
In a further particular embodiment, the kits of the invention further comprise a second set of reagents (b) which amplifies a second non-mutated target sequence from the EGFR gene, said second target sequence being located between 100 and 400 bp from the first target sequence on said gene, and wherein said amplified second target sequence is 50-95 nt in length and of a length similar to that of the first amplified target sequence.
In a further particular embodiment, the kits of the invention comprise at least a third set of reagents (c), wherein the third set of reagents amplifies a third target sequence from the EGFR gene, said third target sequence containing a site of a mutation in the EGFR gene distinct from the first mutation targeted by set of reagent (a), said third set of reagents comprising (i) a mutation-specific primer of 15-25 nt in length, (ii) a paired primer of 15-25 nt in length positioned such that the amplified third target sequence is 50-95 nt in length, and (iii) an oligoblocker, wherein the oligoblocker hybridizes specifically with the third target sequence in non-mutated form and blocks amplification.
Sets of reagents (a), (b) and (c) are preferably in separate containers of the kit, allowing amplification in parallel on different aliquots of a test sample.
In a particular embodiment, the kit further comprises other reagents to perform an amplification reaction, such as an enzyme (e.g., a polymerase such as Taq), nucleotides, and/or a buffer. Such reagents may be in separate containers, to be mixed by the user.
The kit is preferably suitable to detect a mutation in EGFR gene selected from G719A, G719C, G719S, S768I, T790M, L858R, L861Q, exon19DEL and exon20INS.
In a preferred embodiment, the kit of the invention is suitable for detecting the mutation EGFR T790M and the first set of reagents comprises a mutation-specific primer selected from any one of SEQ ID NOs: 1-2 (T790M 16; T790M 17), a paired primer selected from SEQ ID NO: 3 or 4 (T790 P19; T790 P20), and an oligoblocker selected from SEQ ID NO:5 (T790 OB19). Most preferably, the mutation-specific primer is SEQ ID NO: 1 (T790M 16), the paired primer is SEQ ID NO: 3 (T790 P19) and the oligoblocker is SEQ ID NO: 5 (T790 OB19). In a preferred embodiment, the method amplifies a second WT target sequence, located preferably 200-400 bp from the first mutated target sequence, using a second set of reagents. In a more preferred embodiment, the second set of reagents comprises a first primer SEQ ID NO: 6 (T790 WT L19) and a second primer SEQ ID NO: 7 (T790 WT R19).
In another embodiment, the kit of the invention is suitable for detecting the mutation EGFR L858R and the first set of reagents for detecting the EGFR L858R mutation comprises a mutation-specific primer selected from any one of SEQ ID NOs: 8-11 (L858R 16, L858R 17, L858R 18, L858R 19), a paired primer selected from SEQ ID NO: 12 (L858 P20), and an oligoblocker selected from any one of SEQ ID NOs: 13-14 (L858 OB19/3, L858 OB20/3). Most preferably, the mutation-specific primer is SEQ ID NO: 11 (L858R 19), the paired primer is SEQ ID NO: 12 (L858 P20) and the oligoblocker is SEQ ID NO: 13 (L858 OB19/3). In a preferred embodiment, the method amplifies a second WT target sequence, located preferably 200-400 bp from the first mutated target sequence, using a second set of reagents. In a more preferred embodiment, the second set of reagents comprises a first primer SEQ ID NO: 15 (L858 WT L) and a second primer SEQ ID NO: 16 (L858 WT R).
In another embodiment, the kit of the invention is suitable for detecting the mutation EGFR L861Q and the first set of reagents for detecting the EGFR L861Q mutation comprises a mutation-specific primer selected from any one of SEQ ID NOs: 17-18 (L861Q i17, L861Q i18), a paired primer selected from SEQ ID NO: 19 (L861 iP20), and an oligoblocker selected from any one of SEQ ID NOs: 20-21 (L861 iOB19/2, L861 iOB20/2). Most preferably, the mutation-specific primer is SEQ ID NO: 18 (L861Q i18), the paired primer is SEQ ID NO: 19 (L861 iP20), and the oligoblocker is SEQ ID NO: 21 (L861 iOB20/2). In a preferred embodiment, the method amplifies a second WT target sequence, located preferably 200-400 bp from the first mutated target sequence, using a second set of reagents. In a more preferred embodiment, the second set of reagents comprises a first primer SEQ ID NO: 22 (L861 WT L) and a second primer SEQ ID NO: 23 (L861 WT R).
In another embodiment, the kit of the invention is suitable for detecting the mutation EGFR G719S and the first set of reagents for detecting EGFR G719S mutation comprises a mutation-specific primer selected from any one of SEQ ID NOs: 24-25 (G719S 19, G719S 20), a paired primer from SEQ ID NO: 26 (G719 P19), and an oligoblocker selected from any one of SEQ ID NOs: 27-28 (G719 OB18/2, G719 OB19/2). Most preferably, the mutation-specific primer is SEQ ID NO: 25 (G719S 20), the paired primer is SEQ ID NO: 26 (G719 P19), and the oligoblocker is SEQ ID NO: 28 (G719 OB19/2). In a preferred embodiment, the method amplifies a second WT target sequence, located preferably 200-400 bp from the first mutated target sequence, using a second set of reagents. In a more preferred embodiment, the second set of reagents comprises a first primer SEQ ID NO: 29 (G719 WT L) and a second primer SEQ ID NO: 30 (G719 WT R).
In another embodiment, the kit of the invention is suitable for detecting the mutation EGFR G719A and the first set of reagents for detecting the EGFR G719A mutation comprises a mutation-specific primer selected from any one of SEQ ID NOs: 31-33 (G719A 18, G719A 19, G719A 20), a paired primer from SEQ ID NO: 26 (G719 P19), and an oligoblocker selected from any one of SEQ ID NOs: 27-28 (G719 OB18/2, G719 OB19/2). Most preferably, the mutation-specific primer is SEQ ID NO: 31 (G719A 18), the paired primer is SEQ ID NO: 26 (G719 P19) and the oligoblocker is SEQ ID NO: 27 (G719 OB18/2). In a preferred embodiment, the method amplifies a second WT target sequence, located preferably 200-400 bp from the first mutated target sequence, using a second set of reagents. In a more preferred embodiment, the second set of reagents comprises a first primer SEQ ID NO: 29 (G719 WT L) and a second primer SEQ ID NO: 30 (G719 WT R).
In another embodiment, the kit of the invention is suitable for detecting the mutation EGFR S768I and the first set of reagents for detecting the EGFR S768I mutation comprises a mutation-specific primer selected from any one of SEQ ID NOs: 34-35 (S768I i16, S768I i17), a paired primer from SEQ ID NO: 36 (S768 iP18), and an oligoblocker from SEQ ID NO: 37 (S768 iOB19). Most preferably, the mutation-specific primer is SEQ ID NO: 35 (S768I i17), the paired primer is SEQ ID NO: 36 (S768 iP18) and the oligoblocker is SEQ ID NO: 37 (S768 iOB19). In a preferred embodiment, the method amplifies a second WT target sequence, located preferably 200-400 bp from the first mutated target sequence, using a second set of reagents. In a more preferred embodiment, the second set of reagents comprises a first primer SEQ ID NO: 38 (S768 WT L) and a second primer SEQ ID NO: 39 (S768 WT R).
In another embodiment, the kit of the invention is suitable for detecting the mutation EGFR exon19 Δ(E746-A750) and wherein the mutation-specific primer is SEQ ID NO: 40 (DEL19 17N), the paired primer is SEQ ID NO: 41 (DEL19 P19), and the oligoblocker is SEQ ID NO: 42 (DEL19 OB21/3). In a preferred embodiment, the method amplifies a second WT target sequence, located preferably 200-400 bp from the first mutated target sequence, using a second set of reagents. In a more preferred embodiment, the second set of reagents comprises a first primer SEQ ID NO: 43 (DEL19 WT L) and a second primer SEQ ID NO: 44 (DEL19 WT R).
In another embodiment, the kit of the invention is suitable for detecting the mutation EGFR V769-D770 insASV (Ins20) and the first set of reagents comprises a mutation-specific primer selected from any one of SEQ ID NOs: 45-49 (INS20 AVS i16, INS20 AVS i16/2, INS20 AVS i16/3, INS20 AVS i16/4, INS20 AVS i16/5), a paired primer selected from any one of SEQ ID NO:50-52 (Ins20 iP18, Ins20 iP17, Ins20 iP17/2), and an oligoblocker selected from SEQ ID NO: 53 (Ins20AVS iOB18). Most preferably, the mutation-specific primer is SEQ ID NO: 45 (INS20 AVS i16), the paired primer is SEQ ID NO: 51 (Ins20 iP17) and the oligoblocker is SEQ ID NO: 53 (Ins20AVS iOB18). In a preferred embodiment, the method amplifies a second WT target sequence, located preferably 200-400 bp from the first mutated target sequence, using a second set of reagents. In a more preferred embodiment, the second set of reagents comprises a first primer SEQ ID NO: 54 (Ins20 WT L) and a second primer SEQ ID NO: 55 (Ins20 WT R).
The kits of the invention may comprise reagents for detecting one mutation in the EGFR gene.
Alternatively, kits of the invention may comprise reagents suitable for detecting several distinct mutations in the EGFR gene. In this regard, particular preferred kits of the invention comprise reagents as described above for detecting at least T790M and L858R. A particular kit of the invention comprises reagents as described above for detecting T790M and L858R and Exon19del (in particular exon19 Δ(E746-A750)). Such reagents are preferably in separate containers of the kit, allowing amplification in parallel on different aliquots of a test sample.
In this regard, a particular kit of the invention comprises:
a first set of reagents (a) comprising a mutation-specific primer selected from any one of SEQ ID NOs: 1-2 (T790M 16; T790M 17), a paired primer selected from any one of SEQ ID NOs: 3 or 4 (T790 P19; T790 P20), and an oligoblocker selected from SEQ ID NO:5 (T790 OB 19). Most preferably, the mutation-specific primer is SEQ ID NO: 1 (T790M 16), the paired primer is SEQ ID NO: 3 (T790 P19) and the oligoblocker is SEQ ID NO: 5 (T790 OB19);
a second set of reagents (b) for detecting a WT target sequence, located preferably 200-400 bp from the mutated sequence detected by the first set of reagents. In a more preferred embodiment, the second set of reagents comprises a first primer SEQ ID NO: 6 (T790 WT L19) and a second primer SEQ ID NO: 7(T790 WT R19);
a third set of reagents (c) for detecting the EGFR L858R mutation comprising a mutation-specific primer selected from any one of SEQ ID NOs: 8-11 (L858R 16, L858R 17, L858R 18, L858R 19), a paired primer from SEQ ID NO: 12 (L858 P20), and an oligoblocker selected from any one of SEQ ID NOs: 13-14 (L858 OB19/3, L858 OB20/3). Most preferably, the mutation-specific primer is SEQ ID NO: 11 (L858R 19), the paired primer is SEQ ID NO: 12 (L858 P20) and the oligoblocker is SEQ ID NO: 13 (L858 OB 1 9/3).
In a particular embodiment, the kit further comprises a fourth set of reagents (d) for detecting the mutation EGFR exon19 Δ(E746-A750) and wherein the mutation-specific primer is SEQ ID NO: 40 (DEL19 17N), the paired primer is SEQ ID NO: 41 (DEL19 P19), and the oligoblocker is SEQ ID NO: 42 (DEL19 OB21/3).
The invention also relates to the use of kits as described above for detecting EGFR mutations in a sample from a subject, preferably a human subject.
The invention also relates to a nucleic acid molecule consisting of a nucleotide sequence selected from any one of SEQ ID NOs: 1 to 55. Preferred nucleic acid molecules are selected from 1-5, 8-14, 17-21, 24-28, 31-33, 34-37, 40-42 and 45-53. Particular nucleic acid molecules of the invention are mutation-specific primers consisting of a nucleotide sequence selected from any one of SEQ ID Nos: 1,2,8,11,17,18,24,25,31-33,34,35,40 and 45-49.
The invention also relates to the use of a nucleic acid molecule as defined above for in vitro amplification of a target sequence in an EGFR gene.
The invention also relates to a method for diagnosing a patient, comprising detecting a mutation in the EGFR gene in a sample from the patient using a method or kit or nucleic acid as defined above.
The invention also relates to a method for treating a patient, comprising detecting a mutation in the EGFR gene in a sample from the patient using a method or kit or nucleic acid as defined above, and treated subjects having cancer.
Further aspects and advantages of the invention will be disclosed in the following experimental section.
This section presents a summary of optimal criteria followed to ensure optimal efficiency and sensitivity for all mutation detection systems herein described, in combination with absence of any non-specificity.
I1) Design of the Allele-Specific Primer Targeting the Mutation of Interest
To optimize specificity and sensitivity of the allele-specific primer targeting the single nucleotide mutation of interest, the mutant base was generally positioned as 3′ end last nucleotide. The 3′ end of an allele-specific primer is mismatched for one allele (the WT allele) and is a perfect match for the other allele (the mutated allele). The 3′ positioning of the mismatch has the effect of partially blocking the amplification of the WT allele. Indeed, the Taq polymerase enzyme typically used for DNA amplification preferably extends DNA in presence of a perfect match at the 3′ primer end with a free hydroxyl group present. Furthermore, any residual non-specific amplification from the WT allele is further avoided using an oligoblocker, as discussed in section I3.
The positioning of the targeted mutation as the 3′ end of the allele-specific primers generates two possible options for assay design: one in “conventional configuration”, with the primer sequence matching the reference sequence on the positive strand (hence with the primer annealing to the negative strand) with the mutation in 3′; one in “inverse configuration”, with the primer sequence matching the negative strand (hence with the primer annealing to the positive strand) with the mutation in 3′.
Both options were considered in the experimental section, and the thermodynamic characteristics of both options were generally compared, to ensure optimal Tm and GC % with absence of primer-dimers, blocker-primer dimers, self-dimerization, and hairpins.
In addition, different combinations of primers at different lengths were tested, to provide optimal results.
In the selection of the primers targeting the mutation of interest the following recommendations were followed to prevent the risk of non-specific amplification and to improve the method's sensitivity:
I2) Paired Wild-Type Primer to the Allele-Specific Primer
In the selection of the paired wild-type primer the following criteria were followed to prevent the risk of non-specific amplification and to improve the method's sensitivity:
I3) Oligoblocker Design
The oligoblocker is an oligonucleotide complementary to the WT allele with respect to the mutation of interest. It is modified (e.g., phosphorylated in 3′) to block the polymerase elongation. The role of the oligoblocker is to avoid non-specific amplification of the WT sequence at the mutation locus. When the oligoblocker is hybridized to the WT sequence it avoids non-specific hybridization of the primer targeting the mutation to the WT sequence, as it partially overlaps the primer target sequence.
The criteria for an optimal oligoblocker design are herein described, to prevent the risk of non-specific amplification and to improve the detection of low-frequency mutations.
I4) Combinatorial Hetero-Dimer Analysis
The absence of hetero-annealing among the oligonucleotides (allele-specific primer, paired WT primer and oligoblocker) should preferably be verified. This strategy prevents the risk of forming primer-dimers and non-specific amplification, improving the efficiency of the method. The software Oligoanalyzer® can be used for this purpose and, based on its algorithms for the calculations of Gibbs free energy, just values above −4 Kcal/mol can be accepted. Oligonucleotide combinations with ΔG<−4 kcal/mol need to be discarded and re-designed.
I5) Design of the Wild-Type Primer Pair
This primer set should target a nucleic acid sequence located 100-350 bp, typically 300±10 bases pair, from the nucleic acid sequence targeted by the allele-specific primer set. Moreover, it should preferably target a nucleic acid sequence of the same size than the one targeted by the allele-specific primer set±10%. Preferred characteristics of the primers that ensure optimal performance are listed below:
I6) In Vitro Validation for Mutated Region
EGFR T790M is a substitution mutation. The T790M mutation results in an amino acid substitution at position 790, from a threonine (T) to a methionine (M). This mutation occurs within EGFR exon 20, which encodes part of the kinase domain. Nomenclature of EGFR T790M point mutation is c: 2369 C>T. It means that the 2369th nucleotide on the cDNA reference sequence is a Cytosine that is substituted by a Thymine.
The inventors have designed specific sets of primers allowing improved detection of the presence (or absence, or frequency) of such a mutation from a sample containing cfNA.
II1) Design of the Allele-Specific T790M Primer
Various allele-specific primers were considered in the design of this invention and their thermodynamic characteristics were analyzed, as shown in Table 2:
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II2) Paired Wild-Type Primer
Results Obtained with Primer 3
All allele-specific primers that met our criteria (see Table 2) were used on Primer3 to find a compatible paired primer. Results are summarized in Table 3.
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From the results presented in Table 2 it was not possible to select a definite candidate as paired WT primer for the T790M allele-specific primer. The candidates with the best thermodynamic characteristics produced amplicons considered borderline for low frequency detection of mutations from tumour-derived cfDNA fragments. On the other hand, the primer T790 P20/66 bp in combination with the allele-specific primer T790M 16 produced an amplicon of suitable size but contains a region causing high self-dimerization.
In view of the unsuccessful generation of a paired primer using conventional tools, we designed such primers manually. All primers are listed in Table 4. Their characteristics were analyzed.
The following combinations were thus found, that meet the thermodynamic criteria required:
Blat alignments (Human GRCh38/hg38) for all the PCR amplicons generated by the combinations of primers meeting the thermodynamic criteria were performed. All primer pairs produced just one single amplicon at the expected chromosomal position. No other homologies were reported.
We further designed paired primers in inverse configuration, as listed in Table 5.
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As can be seen from Table 5, no suitable combinations could be identified by designing the T790M assay in inverse configuration.
II3) Oligoblocker for the Allele-Specific T790M Assay
We generated several oligoblocker that target the wild-type sequence for T790, and tested their characteristics in combination with the primers.
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The best candidate was T790 OB 19, despite the possibility of hairpins with borderline values (−3.01 Kcal/mol). The hairpin was predicted to open at a temperature of 43.7° C. Given that the oligoblocker annealing takes place at a temperature of 60° C., the hairpin should not cause any efficiency problem. All further thermodynamic analyses were thus conducted considering T790 OB 19 as best oligoblocker candidate.
II4) Thermodynamic Analysis on All Candidate Primers and Oligoblockers
The following heterodimers combinations were considered for all the primers/oligoblocker candidates that met the thermodynamic criteria described in the previous sections:
The results show all oligonucleotide candidates meet the thermodynamic criteria required to ensure absence of non-specific amplification and to improve the amplification efficiency.
II5) Design of the T790M Wild-Type Primer Set
To produce optimal T790M Wild-Type primer sets, we chose to target the DNA region shown in
The tested primer candidates are disclosed in Table 8.
Our results confirm the candidate primers exhibit the required characteristics. The pair was thus validated for further analysis.
The Blat local alignment highlighted the presence of a secondary homology region. Nevertheless, by analyzing the structure and extension of the homology region reported on chr14, we concluded that it would at most produce just a partial annealing region for the T790 WT L primer but, as no homology is present in the target region of the T790 WT R primer, no secondary product would be expected to be produced. This primer combination could thus be validated.
The oligonucleotide candidates of the T790M WT set met the thermodynamic criteria required to ensure absence of non-specific amplification and to improve the amplification efficiency.
II6) In Vitro Validation of the T790M Primer Pairs
The specificity of the combination was tested. They showed one peak only, indicating the specificity criteria are met.
A substantial non-specificity was observed when using this oligonucleotide combination. This combination is thus not optimal.
The specificity of the combination was tested. The results show one peak only, indicating the specificity criteria are met.
Melting curves of genomic DNA WT for EGFR T790M from the Difi cell line was compared to the melting curves of commercial DNA mutant for EGFR T790M using this combination of oligonucleotides. A substantial non-specificity was observed when using this oligonucleotide combination. This combination is thus not optimal.
The specificity of the combination was tested. Melting curves of a commercial DNA mutant for EGFR T790M was determined using this oligonucleotide system. Just one peak was observed, as presented in
Melting curves of genomic DNA WT for T790M from the Difi cell line was compared to melting curves of commercial DNA mutant for EGFR T790M using this combination of oligonucleotides (see
The efficiency analysis for the mutation-specific set comprised of T790M 16+T790 P19+T790 OB19 was performed in conjunction with the proposed combination for the T790 WT primer set. The results are presented in Table 9.
The observed mutant allele frequency of the T790M mutation-specific system on the T790 WT system is 62%, close to expected 50% reported by the supplier of the positive control used for the experiment.
The efficiency is thus appropriate and this oligonucleotide combination was further evaluated for sensitivity.
The results are presented in Table 10.
The system could detect the T790M mutation down to a frequency of 0.1 pg/μl. The sensitivity is thus very high.
The results here presented confirmed that the method comprised of the mutation-specific set T790M 16+T790 P19+T790 OB19 and of the wild-type set T790 WL L+T790 WT R exhibits high specificity efficiency and sensitivity.
II7) Comparison with Prior Art Method
We compared the efficiency of our method to the technique described in Zhao et al., Oncology Letters 11:2573:2579, 2016. This system uses a mismatch at the third base of 3′ ends to decrease the non-specific combination of the allele-specific primer with the WT allele.
We employed the Zhao system and compared its sensitivity to our system on mutant genomic DNA and on circulating DNA from clinical samples. Zhao primer system targets a 106 bp mutant sequence whilst the invention targets a mutant sequence of 73 bp.
The result obtained show that Zhao system is less efficient than our method, as in same cases it does not detect mutations present at 11% in frequency when analyzing clinical samples from lung cancer patients (see
Such results highlight the strong efficacy and sensitivity of our method compared with other mutation detection systems
II8) Conclusions
Our method is specific, sensitive and more efficient than prior art techniques for detecting T790M. Among tested primer combinations, the best system to detect the EGFR T790M mutation is comprised by the mutation specific oligonucleotides: T790M 16+T790 P19+T790 OB19, in combination with the wild-type set T790 WT L+T790 WT R.
Other oligonucleotide combinations that allow efficient detection include:
EGFR L858R is a substitution mutation. The L858R mutation results in an amino acid substitution at position 858, from a Leucine (L) to an Arginine (R). This mutation occurs within exon 21, which encodes part of the kinase domain. Nomenclature of EGFR L858R point mutation is c: 2573 T>G. It means that the 2573rd nucleotide on the cDNA reference sequence is a Thymine that is substituted by a Guanine. After positioning the variant position on the cDNA sequence, it is positioned on the genomic DNA sequence.
The inventors have designed specific sets of primers allowing improved detection of the presence (or absence, or frequency) of such a mutation from a sample containing cfNA.
III1) Design for the Allele-Specific L858R Primer
Various allele-specific primers were considered in the design of this invention and their thermodynamic characteristics were analyzed, as shown in Table 12:
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This preliminary analysis suggested that primer design in inverse sense is thermodynamically less favorable.
III2) Paired Wild-Type Primer
Results Obtained with Primer 3
All allele-specific primers that met our criteria (see Table 12) were used on Primer3 to find a compatible paired primer. Results are summarized in Table 13.
The only candidate identified via Primer3 has acceptable thermodynamic characteristics but it produces a PCR amplicon considered borderline for low frequency detection of mutations from tumour-derived cfDNA fragments. It was thus not possible to find appropriate primer using such approach.Manually generated primer pair candidates in conventional configuration.
In view of the unsuccessful generation of a paired primer using conventional tools, we designed such primers manually. All designed primers are listed in Table 14. Their characteristics were analyzed.
The following combinations were found, that meet the thermodynamic criteria required:
Blat alignments (Human GRCh38/hg38) for all the PCR amplicons generated by the combinations of primers meeting the thermodynamic criteria were performed. All primer pairs produced just one single amplicon at the expected chromosomal position. No other homologies are reported.
III3) Oligoblocker Design for the EGFR L858R Position
The presence of a region of self-annealing prevents the design of the L858 oligoblocker with the WT base towards the middle of the oligonucleotide sequence. The oligoblocker was designed with the variant position towards its 3′ end. The oligoblockers and their characteristics are described in Table 15.
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Oligoblockers L858 OB20/3 and L858 OB19/3 present the best thermodynamic characteristics.
From the Tm analysis of Table 16, all the combinations of allele-specific primers and oligoblocker seem to fit the optimal criteria.
III4) Thermodynamic Analysis on All Candidate Primers and Oligoblockers
The following heterodimers combinations were considered for all the primers/oligoblockers that met the thermodynamic criteria:
The results are presented in Table 17:
The results show all oligonucleotide candidates meet the thermodynamic criteria required to ensure absence of non-specific amplification and to improve the amplification efficiency.
III5) Design of the L858 Wild-Type Primer Set
To produce optimal L858R wild-type primer sets, we chose to target the DNA region presented in
The tested primer candidates are disclosed in Table 18.
Our results confirm the primers satisfy the required optimal characteristics.
Blat alignments (Human GRCh38/hg38) for all the PCR amplicons generated by the combinations of primers were performed.
The oligonucleotide candidates of the L858 WT set meet the thermodynamic criteria required to ensure absence of non-specific amplification and to improve the amplification efficiency.
III6) In Vitro Validation of the L858R IntPlex® System
The specificity of the combination was tested. The results are presented in
Melting curves of genomic DNA WT for the L858R mutation were compared to melting curves of commercial DNA mutant for L858R using this oligonucleotide combination (see
The efficiency analysis for the mutation-specific set comprised of L858R 19+L858 P20+L858 OB19 was performed in conjunction with the proposed combination for the L858WT primer set. The results are presented in Table 19.
The observed mutant allele frequency of the L858R mutation-specific system on the L858WT system is 76%, close to expected 50% reported by the supplier of the positive control used for the experiment.
The efficiency is thus appropriate and this oligonucleotide combination was further evaluated for sensitivity.
The results are presented in Table 20.
The system can detect the L858R mutation down to a frequency of 0.1 pg/μl. The sensitivity is thus very high.
The results here presented confirm that the method comprised of the mutation-specific set L858R 19+L858 P20+L858 OB19/3 and of the wild-type set L858 WT L+L858 WT R exhibits high specificity, efficiency and sensitivity.
III7) Conclusion
Our method allows detection of L858R with high specificity, sensitivity and efficiency. Among tested primers, the optimal combination is L858R 19+L858 P20/64 bp+L858 OB19/3. Other suitable primer combinations include:
EGFR L861Q is a substitution mutation. The L861Q mutation results in an amino acid substitution at position 861, from a Leucine (L) to a Glutamine (Q). This mutation occurs within exon 21, which encodes part of the kinase domain. Nomenclature of EGFR L861Q point mutation is c: 2582 T>A. It means that the 2582th nucleotide on the cDNA reference sequence is a Thymine that is substituted by an Adenine.
The inventors have designed specific sets of primers allowing improved detection of the presence (or absence, or frequency) of such a mutation from a sample containing cfNA.
IV1) Design of the Allele-Specific EGFR L861Q Primer
Various allele-specific primers were considered in the design of this invention and their thermodynamic characteristics were analyzed, as shown in Table 22.
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IV2) Paired Wild-Type Primer
Results Obtained with Primer 3
All allele-specific primers that met our IntPlex® criteria (see Table 22) were used on Primer3 to find a compatible paired primer. Unfortunately, no suitable primers were identifiable through this approach.
New potential paired-wild type primer candidates will be manually designed to overcome this issue.
In view of the unsuccessful generation of a paired primer using conventional tools, we designed such primers manually. All primers are listed in Table 23. There characteristics were analyzed.
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The following combinations were thus found, that meet the thermodynamic criteria required:
Blat alignments (Human GRCh38/hg38) for all the PCR amplicons generated by the combinations of primers meeting the thermodynamic criteria were performed.
The oligonucleotide candidates of the L861Q set met the thermodynamic criteria required to ensure absence of non-specific amplification and to improve the amplification efficiency.
IV3) Oligoblocker for the Allele-Specific EGFR L861Q Assay
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IV4) Thermodynamic Analysis on All Candidate Primers and Oligoblockers
The following heterodimers combinations to consider for all the primers/oligoblocker candidates that met the thermodynamic criteria described in the previous sections:
The results show all oligonucleotide candidates meet the thermodynamic criteria required to ensure absence of non-specific amplification and to improve the amplification efficiency.
IV5) Design of the EGFR L861Q Wild-Type Set
To produce optimal L861Q Wild-Type primer sets, we chose to target the DNA region shown in
The tested primer candidates are disclosed in Table 26.
Our results confirm the candidate primers exhibit the required characteristics. The pair was thus validated for further analysis.
Blat alignments (Human GRCh38/hg38) for the PCR amplicons generated by the combinations of primers meeting the thermodynamic criteria were performed.
The Blat local alignment highlighted the presence of secondary homology regions. Nevertheless, none of the secondary homology region overlapped with the primer sequences. Hence no secondary product was produced. This primer combination could hence be validated. The oligonucleotide candidates of the L861 WT set met the thermodynamic criteria required to ensure absence of non-specific amplification and to improve the amplification efficiency.
IV6) In Vitro Validation of the L861Q Primer Pairs
L861Q i18+L861 iP20+L861 iOB20/2
The specificity of the combination was tested. The results are presented in
Melting curves of genomic DNA WT for the L861Q mutation were compared to melting curves of commercial DNA mutant for L861Q using this oligonucleotide combination (see
The efficiency analysis for the mutation-specific set comprised of L861Q i18+L861 iP20+L861 iOB20 was performed in conjunction with the proposed combination for the L861 WT primer set. The results are presented in Table 27.
The observed mutant allele frequency of the L861Q mutation-specific system on the L861Q WT system is 62%, close to expected 50% reported by the supplier of the positive control used for the experiment.
The efficiency is thus appropriate and this oligonucleotide combination was further evaluated for sensitivity.
The results are presented in Table 28.
IV7) Conclusions
Our method exhibits high specificity, efficiency and sensitivity. The results here presented confirm that among all tested primer combinations the method comprised of the mutation-specific set L861Q i18+L861 iP20+L861 iOB20/2 and of the wild-type set L861 WT L+L861 WT R is the best system to detect the EGFR L861Q.
Other systems that allow efficient detection include:
EGFR S768I is a substitution mutation. The S768I mutation results in an amino acid substitution at position 768, from a Serine (S) to an Isoleucine (I). This mutation occurs within exon 20, which encodes part of the kinase domain. Nomenclature of EGFR S768I point mutation is c: 2303 G>T. It means that the 2303th nucleotide on the cDNA reference sequence is a Guanine that is substituted by a Thymine.
The inventors have designed specific sets of primers allowing improved detection of the presence (or absence, or frequency) of such a mutation from a sample containing cfNA.
V1) Design for the Allele-Specific EGFR S768I Primer
Various allele-specific primers considered in the design of this invention and their thermodynamic characteristics were analyzed, as shown in Table 29.
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V2) Paired Wild-Type Primer
Results Obtained with Primer 3
All allele-specific primers that met our criteria (see Table 29) were used on Primer3 to find a compatible paired primer. Results are summarized in Table 30.
The paired wt primer candidates identified by Primer3 has suitable thermodynamic characteristics but produces amplicons that are considered borderline for low frequency detection of mutations from tumour-derived cfDNA fragments.
The paired-wild type primer was thus manually designed following the thermodynamic criteria described in the previous sections to try and reduce the PCR amplicon size.
Paired wild-type primers were manually generated to reduce the PCR amplicon size. All primers are listed in Table 31. Their characteristics were analyzed.
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The following combinations were thus found, that met the thermodynamic criteria required:
Blat alignments were produced (Human GRCh38/hg38) for the PCR amplicons generated by the combination of primers meeting the thermodynamic criteria.
The primer pair in analysis produced just one single amplicon at the expected chromosomal position. No other homologies are reported.
V3) Oligoblocker for the EGFR S768 Position
We generated several oligoblockers that target the wild-type sequence for S768 and tested their characteristics in combination with the primers.
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The best candidate is the S768 i OB19, despite slightly high GC % and Tm, as all other combination presented excessive too high Tm. All further thermodynamic analyses were thus conducted considering S768 i OB19 as best oligoblocker candidate.
V4) Thermodynamic Analysis on All Candidate Primers and Oligoblockers
The following heterodimers combinations were considered for all the primers/oligoblocker candidates that met the thermodynamic criteria described in the previous sections:
The results are presented in Table 33.
The results show all oligonucleotide candidates meet the thermodynamic criteria required to ensure absence of non-specific amplification and to improve the amplification efficiency.
V5) Design of the S768 Wild-Type Set
To produce optimal S768 wild-type sets, we chose to target the DNA region shown in
The tested primer candidates are disclosed in Table 34.
Our results confirm the candidate primers exhibit the required characteristics. The pair was thus validated for further analysis.
Blat alignments were produced (Human GRCh38/hg38) for the PCR amplicons generated by the combination of primers meeting the thermodynamic criteria.
The Blat local alignment highlighted the presence of some secondary homology region. Nevertheless, by analyzing the structure and extension of the homology region reported on chrX we concluded that it would at most produce a partial annealing region for the S768 WT L primer but, as no homology is present in the target region of the S768 WT R primer, no secondary product would be produced. None of the other homology regions spans the primer target sequence. This primer combination could hence be validated.
The oligonucleotide candidates of the S768 WT set met the thermodynamic criteria required to ensure absence of non-specific amplification and to improve the amplification efficiency.
V6) In Vitro Validation of the S768I Primer Pairs
S768I i16+S768 iP18+S768 iOB19
The specificity of the combination was tested. The results showed one peak-only, indicating the specificity QC criteria are met.
A slight amplification was observed in the negative control and in the NTC, with a 4° C. Tm difference from the expected amplification peak of the positive control. Moreover, the concentration of the non-specific amplification is <0.5 pg/μl, in compliance with IntPlex® acceptance QC criteria.
As the Tm difference between the tested mutation-specific primer S768I i16 and the paired WT primer S768 iP18 is 6° C., the mutation-specific primer S768I i17 will be also tested.
S768I i17+S768 iP18+S768 iOB19
The specificity of the combination was tested. The results are presented in
Melting curves of genomic DNA WT for the S768I mutation were compared to melting curves of commercial DNA mutant for S768I using this oligonucleotide combination (see
The efficiency analysis for the mutation-specific set comprised of S768 i17+S768 iP18+S768 iOB19 was performed in conjunction with the proposed combination for the S768 WT primer set. The results are presented in Table 35.
The observed mutant allele frequency of the S768I mutation-specific system on the S768 WT system is 28.33%, close to expected 50% reported by the supplier of the positive control used for the experiment.
The efficiency is thus appropriate and this oligonucleotide combination was further evaluated for sensitivity.
The results are presented in Table 36.
The system can detect the S768I mutation down to a frequency of 0.04 pg/μl. The sensitivity is thus very high.
V7) Conclusions
Our method allows detection of the S768I mutation with high specificity, efficiency and sensitivity.
Among the tested primers, the optimal combination is comprised of the mutation-specific set S768I i17+S768 iP18+S768 iOB19 and of the wild-type set S768 WT L+S768 WT R.
Other suitable primer combinations include:
The G719S mutation results in an amino acid substitution at position 719, from a glycine (G) to a serine (S). This mutation occurs within exon 18, which encodes part of the kinase domain. Nomenclature of EGFR G719S point mutation is c: 2155 G>A. It means that the 2155th nucleotide on the cDNA reference sequence is a Guanine that is substituted by an Adenine.
The inventors have designed specific sets of primers allowing improved detection of the presence (or absence, or frequency) of such a mutation from a sample containing cfNA.
VI1) Design of the Allele-Specific EGFR G719S Primer
Various allele-specific primers were considered in the design of this invention and their thermodynamic characteristics were analyzed, as shown in Table 37.
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This preliminary analysis suggested that primer design in inverse configuration is thermodynamically less stable.
VI2) Paired Wild-Type Primer
Results Obtained with Primer 3
All allele-specific primers that met our criteria (see Table 37) were used on Primer3 to find a compatible paired primer. Results are summarized in Table 38.
The only suitable paired primer obtained using the Primer3 software has a very borderline Tm difference with all mutation-specific primer candidates. A manual design of the paired WT primer was thus favored to reduce the Tm difference and ensure optimal amplification efficiency.
In view of the unsuccessful generation of a paired primer using conventional tools, we designed such primers manually. All designed primers are listed in Table 39. Their characteristics were analyzed.
The following combinations were found, that met the thermodynamic criteria required:
Blat alignments (Human GRCh38/hg38) for the PCR amplicon generated by the combinations of primers meeting the thermodynamic criteria were performed.
All primer pairs produced just one single amplicon at the expected chromosomal position. No other homologies are reported.
VI3) Oligoblocker Design for the EGFR G719 Position
We generated several oligoblockers that target the wild-type sequence for G719 and tested their characteristics in combination with the primers.
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When the variant position is kept at the middle of the potential G719 oligoblocker, the self-dimerization values are too high. By shifting the variant position towards the 3′ end of the oligoblocker more favorable thermodynamic values were obtained.
The best oligoblockers were G719 BO19/2 and G719 BO18/2 that were thus considered for all further thermodynamic analyses.
VI4) Thermodynamic Analysis on All Candidate Primers and Oligoblockers
The following heterodimers combinations were considered for all the primers/oligoblocker candidates that met the thermodynamic criteria described in the previous sections:
The results show all oligonucleotide candidates meet the thermodynamic criteria required to ensure absence of non-specific amplification and to improve the amplification efficiency.
VI5) Design of the EGFR G719S Wild-Type Set
To produce optimal G719S Wild-Type primer sets we chose to target the DNA region shown in
The tested primer candidates are disclosed in Table 42.
Our results confirm that the candidate primers exhibit the required characteristics. The pair was thus validated for further analysis.
Blat alignments (Human GRCh38/hg38) were performed for the PCR amplicons generated by the combinations of primers meeting the thermodynamic criteria.
The oligonucleotide candidates of the G719 WT set produced one single amplicon, thus meeting the thermodynamic criteria required to ensure absence of non-specific amplification and to improve the amplification efficiency.
VI6) In Vitro Validation of the EGFR G719S Primer Pairs
The specificity of the combination was tested. The results are presented in
Melting curves of genomic DNA WT for the G719S mutation were compared to melting curves of commercial DNA mutant for G719S using this oligonucleotide combination (see
The efficiency analysis for the mutation-specific set comprised of G719S 20+G719 P19+G719 OB19 was performed in conjunction with the proposed combination for the G719 WT primer set. The results are presented in Table 43.
The target G719 WT corresponds to the combination G719 WL L+G719 WT R.
The observed mutant allele frequency of the G719S mutation-specific system on the G719 WT system is 44.97%, close to expected 50% reported by the supplier of the positive control used for the experiment. The efficiency was thus appropriate and this oligonucleotide combination was further evaluated for sensitivity.
Sensitivity
The results are presented in Table 44.
The system can detect the G719S mutation down to a frequency of 0.1 pg/μl. The sensitivity is thus very high.
VI7) Conclusions
Our method has high specificity, efficiency and sensitivity.
The results here presented confirm that the best system to detect the EGFR G719S mutation is comprised of the mutation-specific set G719S 20+G719 P19+G719 OB19 and of the wild-type set G719 WT L+G719 WT R.
Other possible oligonucleotide systems include:
EGFR G719A is a substitution mutation. The G719A mutation results in an amino acid substitution at position 719, from a glycine (G) to an alanine (A). This mutation occurs within exon 18 which encodes part of the kinase domain. Nomenclature of the EGFR G719A point mutation is c: 2156 G>C. This means that the 2156th nucleotide on the cDNA reference sequence is a Guanine that is substituted by a Cytosine.The inventors have designed specific sets of primers allowing improved detection of the presence (or absence, or frequency) of such a mutation from a sample containing cfNA.
VII1) Design of the Allele-Specific EGFR G719A Primer
Various allele-specific primers were considered in the design of this invention and their thermodynamic characteristics were analyzed, as shown in Table 45.
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This preliminary analysis suggested that primer design in inverse configuration is thermodynamically less favorable.
VII2) Paired Wild-Type Primer
As the EGFR G719A assay is designed to target the same codon of the validated EGFR G719S assay (see chapter VI), it was extremely likely that the validated paired wild-type primer G719 P19 would work fine in combination with the proposed G719A mutation-specific primer candidates proposed in section VIII 1. A thermodynamic analysis was performed to exclude the presence of hetero-dimers with the mutation-specific primer candidates.
The following combinations were found, that met the thermodynamic criteria required:
Blat alignments (Human GRCh38/hg38) for all the PCR amplicons generated by the combinations of primers meeting the thermodynamic criteria were performed.
All primer pairs produced just one single amplicon at the expected chromosomal position. No other homologies are reported.
VII3) Oligoblocker Design for the EGFR G719 Position
As the EGFR G719A assay is designed to target the same codon of the validated EGFR G719A assay (see chapter VI), it was extremely likely that the validated oligoblocker G719 OB19/2 or the other thermodynamically validated oligoblocker candidate G719 OB18/2 would work fine in combination with the primer candidates proposed in section VII.1/VII.2. A thermodynamic analysis was performed to exclude the presence of hetero-dimers with all primer candidates.
VII4) Thermodynamic Analysis on All Candidate Primers and Oligoblockers
The following heterodimers combinations were considered for all the primers/oligoblocker candidates that met the thermodynamic criteria described in the previous sections:
The results are presented in Table 47.
The results show all oligonucleotide candidates meet the thermodynamic criteria required to ensure absence of non-specific amplification and to improve the amplification efficiency.
VII5) Design of the EGFR G719A Wild-Type Set
As the EGFR G719A assay is designed to target the same codon of the validated EGFR G719A assay (see chapter VI), the validated wild-type EGFR G719 Wild-type assay had to respect all constraints in terms of distance from the mutation-specific primer set to be validated. The WT primer combination G719 WT L+G719 WT R was thus used for the in-vitro validation of the EGFR G719A IntPlex® assay.
VII6) In Vitro Validation of the EGFR G719A Primer Pairs
The specificity of the combination was tested. The results showed one peak-only, indicating the specificity criteria are met.
A substantial non-specificity was observed when using this oligonucleotide combination. The QC criteria for non-specificity are not met. This combination is thus not optimal.
The specificity of this combination was tested. The results are presented in
Melting curves of genomic DNA WT for the G719A mutation were compared to melting curves of commercial DNA mutant for G719A using this oligonucleotide combination (see
The efficiency analysis for the mutation-specific set comprised of G719A 18+G719 P19+G719 OB18/2 was performed in conjunction with the proposed combination for the G719 WT primer set. The results are presented in Table 48.
The observed mutant allele frequency of the G719A mutation-specific system on the G719 WT system is 60.66%, close to expected 50% reported by the supplier of the positive control used for the experiment. The efficiency is thus appropriate and this oligonucleotide combination was further evaluated for sensitivity.
The results are presented in Table 49.
The system can detect the G719A mutation down to a frequency of 0.5 pg/μl. The sensitivity is thus very high.
VII7) Conclusions
Our method presents high specificity, efficiency and sensitivity. Among the tested primer combinations, the best system to detect the EGFR G719A mutation is comprised of the mutation-specific set G719A 18+G719 P19+G719 OB18/2 and of the wild-type set G719 WT L+G719 WT R.
Other oligonucleotide combinations that allow efficient detection include:
EGFR exon 19 deletions are in-frame deletions occurring within exon 19, which encodes part of the kinase domain. The most frequent deletion in EGFR exon 19 is Δ(E746-A750) resulting from a 15 nucleotide deletion event. The nucleotidic nomenclature of EGFR E746-A750 deletion is c:2235-2249. The 15 nucleotides from 2235 to 2249 on the coding DNA reference sequence are deleted. The inventors have designed specific sets of primers allowing improved detection of the presence (or absence, or frequency) of such a mutation from a sample containing cfNA.
VIII1) Strategy for Detecting Deletion Mutations
Strategy for the qualitative detection of the deletions is described in
As previously described, the addition of an oligoblocker allows avoiding non-specific amplification of WT DNA sequences. The strategy for optimal positioning of the oligoblocker is described in
The system presented here allows highly specific and sensitive qualitative detection of the deletion. For quantitative detection, the total DNA concentration can be determined using a B1/B2 WT primer set and by measuring the difference between the total DNA concentration determined with B1/B2 primer set to the WT DNA concentration determined using A1/A2 primer set (
VIII2) Design of the Allele-Specific EGFR DEL19 Primer
The allele-specific primers generated and considered in the design of this invention and their thermodynamic characteristics are listed in Table 50.
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ATTCCTT (SEQ
VIII3) Paired Wild-Type Primer
Results Obtained with Primer 3
All allele-specific primers that met our criteria (see Table 50) were used on Primer3 to try to find a compatible paired primer. No valid output results were generated applying the suggested parameters.
In view of the unsuccessful generation of a paired primer using conventional tools, we designed such primers manually. All designed primers are listed below, with their characteristics.
Blat alignments (Human GRCh38/hg38) were performed. They showed the selected optimal primer pairs produced just one single amplicon at the expected chromosomal position.
VIII4) Oligoblocker Design for the EGFR DEL19 Position
We generated several oligoblockers and verified their characteristics, as shown in Table 52.
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The best oligoblocker appeared to be DEL 19 OB21/3 and it was thus selected for all further thermodynamic analyses.
VIII5) Thermodynamic Analysis on All Candidate Primers and Oligoblockers
The following heterodimers combinations were considered for all the primers/oligoblocker candidates that met the thermodynamic criteria:
The results are presented in Table 53.
The results show all oligonucleotide candidates meet the thermodynamic criteria required to ensure absence of non-specific amplification and to improve the amplification efficiency.
VIII6) Design of the DEL19 Wild-Type Primer Set
The optimal positioning of the DEL19 wild-type primer candidates on the genomic nucleic acid sequence is shown in
The tested primers are disclosed in Table 54.
The primers exhibited the required characteristics.
Blat alignments (Human GRCh38/hg38) were performed for the PCR amplicons generated by the combinations of primers.
The Blat local alignment highlighted the presence of a secondary homology region. Nevertheless, by analyzing the structure and extension of the homology region reported on chr3, we concluded that it would produce just a partial annealing region for the DEL19 WT L primer but, as no homology is present in the target region of the DEL19 WT R primer, no secondary product would be produced.
The oligonucleotide candidates of the DEL19 WT set thus met the thermodynamic criteria required to ensure absence of non-specific amplification and to improve the amplification efficiency.
VIII7) In Vitro Validation of the EGFR DEL19 Primers
A substantial non-specificity was observed when using this oligonucleotide combination.
This oligonucleotide combination is highly specific.
The efficiency analysis for the mutation-specific set comprised of DEL19 17N+DEL19 P19+DEL19 OB21/3 was performed in conjunction with the proposed system for the DEL19 WT primer set.
The observed mutant allele frequency of the EGFR DEL19-specific system on the DEL19 WT system is 47%, close to expected 50% reported by the supplier of the positive control used for the experiment. The method thus provides high efficiency.
The method can detect the EGFR deletion Δ(E746-A750) down to a frequency of 0.1 pg/μl. The sensitivity is thus very high.
VIII8) Conclusions
The results here presented confirm that the method allows detection of DEL19 with high specificity, efficiency and sensitivity. Optimal primers include the mutation-specific set DEL19 17N+DEL19 P19 with and without oligoblocker DEL19 OB21/3 and of the wild-type set DEL19 WT L+DEL19 WT R.
IX) EGFR Exon 20 Insertions (Ins20) V769-D770 insASV
EGFR exon 20 insertions are in-frame insertions occurring within exon 20. Most EGFR exon 20 insertions occur in between amino acids 767 to 774 of exon 20, within the loop that follows the C-helix of the kinase domain of EGFR (Yasuda, Kobayashi, and Costa 2012). In terms of coding DNA reference sequence refers to c: 2302-2320. Insertions can vary from 3 to 12 nucleotides, with the most common being EGFR V769-D770 insASV (c.2308_2309insGCCAGCGTG).
The inventors have designed specific sets of primers allowing improved detection of the presence (or absence, or frequency) of such a mutation from a sample containing cfNA.
IX1) Design of the Allele-Specific EGFR Ins20 Primer
The allele-specific primers generated and considered in the design of this invention and their thermodynamic characteristics are listed in Table 57.
The design in conventional configuration was strongly unflavored: the presence of a region of self-dimerization impaired any potential primer design in conventional sense spanning the insertion junction from the gene 5′. Only allele-specific primer candidates in inverse configuration were thus considered for all thermodynamic analyses.
TGGC (SEQ ID
CTGG (SEQ ID
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GGGGGTTGTCCA
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ACGC (SEQ ID
Because of the very high GC % of the area, longer allele-specific primers were not considered as the Tm would have been too close to 60° C. (Optimal annealing temperature for the oligoblocker).
All primer pairs do have the same high GC % but there were no alternative options. According to Oligoanalyzer all primers possessed similar thermodynamic characteristics. Nevertheless, as general precaution when designing primers, it is recommended to avoid G quadraplexes. This rule was applied when analyzing allele-specific primer with similar thermodynamic characteristics.
IX2) Paired Wild-Type Primer
Results Obtained with Primer 3
All allele-specific primers that met our Icriteria (see Table 57) were used on Primer3 to try to find a compatible paired primer. Results are summarized in Table 58.
From the results presented in Table 58 it was not possible to select a suitable candidate as the only output presented too high melting temperature.
By manually reducing the size of the primer candidate proposed by Primer3 a better fit could be reached. The paired-wild type primer was manually designed.
In view of the unsuccessful generation of a paired primer using conventional tools, we designed such primers manually. All designed primers are listed in Table 59, with their characteristics.
All WT primer candidates represented suitable choices for the assay in inverse configuration. The GC % was quite high but as it is region-dependent it could not be lowered further.
Blat alignments (Human GRCh38/hg38) for all the PCR amplicons generated all combinations of primers were performed. The primer combinations provided one principal hit on the EGFR gene. The percentage of identity is <100% as the presence of the insertion would add 9 nucleotides not present in the reference sequence. All secondary hits did not span any primer landing site.
All primer pair combinations produced just one single amplicon at the expected chromosomal position.
IX3) Oligoblocker Design for the Allele-Specific EGFR Ins20 AVS Position
We generated several oligoblockers and verified their characteristics, as shown in Table 60.
CCACGCTGG
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The best oligoblocker appeared to be Ins20AVS iOB 18 that was thus selected for all further thermodynamic analyses.
IX4) Thermodynamic Analysis on All Candidate Primers and Oligoblockers
The following heterodimers combinations were considered for all the primers/oligoblocker candidates that met the thermodynamic criteria:
The results are presented in Table 61.
The results show all oligonucleotide candidates meet the thermodynamic criteria required to ensure absence of non-specific amplification and to improve the amplification efficiency.
IX5) Design of the Ins20 Wild-Type Primer Set
The optimal positioning of Ins20 wild-type primer candidates on the genomic nucleic acid sequence is presented in
The primers exhibit the required characteristics.
Blat alignments (Human GRCh38/hg38) were performed for the PCR amplicons generated by all combinations of primers. All primer pairs produced one single amplicon only at the expected chromosomal position. No other homologies were reported.
The oligonucleotide candidates of the EGFR Ins20 WT set met the thermodynamic criteria required to ensure absence of non-specific amplification and to improve the amplification efficiency.
IX6) Conclusions
The results here presented confirmed that the method allows detection of EGFR Ins20 AVS insertion with high specificity, efficiency and sensitivity. Optimal primers include the following combinations:
Other suitable combinations include:
The following combination of primers can be used as paired wild-type assay for the EGFR Ins20 AVS insertion: Ins20 WT L+Ins20 WT R.
| Number | Date | Country | Kind |
|---|---|---|---|
| 18305788.4 | Jun 2018 | EP | regional |
This application is the U.S. national stage application of International Patent Application No. PCT/EP2019/066371, filed Jun. 20, 2019.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/066371 | 6/20/2019 | WO | 00 |
