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WO2008151031A1 - Détection de séquences de gènes mutés par séquençage avec enrichissement en mutant - Google Patents

Détection de séquences de gènes mutés par séquençage avec enrichissement en mutant Download PDF

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WO2008151031A1
WO2008151031A1 PCT/US2008/065343 US2008065343W WO2008151031A1 WO 2008151031 A1 WO2008151031 A1 WO 2008151031A1 US 2008065343 W US2008065343 W US 2008065343W WO 2008151031 A1 WO2008151031 A1 WO 2008151031A1
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pik3ca
wild
mutant
gene sequences
cancer
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PCT/US2008/065343
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Gloria Huei-Ting Su
Wanglong Qui
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The Trustees Of Columbia University In The City Of New York
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • HNSCC head and neck tumors
  • HNSCCs are squamous cell carcinomas
  • HNSCCs are defined as cancer that originates from the cuboidal cells along the basement membrane of the mucosa. The disease is characterized by local tumor aggressiveness, early recurrence, and a high frequency of a second primary tumor. Patients with HNSCC often face a poor prognosis secondary to a late presentation and ineffective therapies.
  • the 5-year survival rate is 40%.
  • the annual incidence of HNSCC is estimated at 41,000 cases and 12,300 associated deaths in the United States (16), and an estimated 270,500 deaths annually worldwide (17).
  • overall survival has hardly improved for the last 25 years.
  • HNSCC The genetic profile of HNSCC is a work in progress (Fig 1).
  • Fig 1 The genetic profile of HNSCC is a work in progress (Fig 1).
  • Fig 1 The genetic profile of HNSCC is a work in progress (Fig 1).
  • Fig 1 The genetic profile of HNSCC is a work in progress (Fig 1).
  • p53 is inactivated in -50%
  • Cyclin Dl is amplified in -30-60% of HNSCC (1, 18).
  • some candidate genes have been named for the frequent LOH or amplification observed in HNSCC, such as Rb (Chr 13q LOH), p21, TNF (Chr 6p amplification), MIN (Chr 4q LOH) 1 and PTEN (Chr 1Oq LOH)
  • Rb Chor 13q LOH
  • p21 TNF
  • MIN Chir 4q LOH
  • PTEN Chor 1Oq LOH
  • PBK phosphatidylinositol 3-kinase
  • PIKSCA is located on chromosome 3q26.32 and encodes for the catalytic subunit pi 10a of class IA PI3 -kinase. It has been implicated to function as an oncogene in human cancer both because increased kinase levels and genomic amplifications have been appreciated in tumor samples (4-6, 27-29). Recently high frequencies of somatic mutations in the PIK3CA gene have been reported in several human cancer types, including colon, brain, stomach, breast, and ovary (3, 9-11, 30, 31). More than 80% of these mutations are clustered in the helical (exon 9) and kinase domains (exon 20) of the gene (3) (Fig 3).
  • PIK3CA The three most frequently reported mutation hot spots in PIK3CA, named E542K, E545K and Hl 047R, have been shown to elevate the lipid kinase activity of PIK3CA and lead to the activation of its downstream Akt signaling pathway (3, 7). Theses hot-spot mutations account for 78.6% of all reported PIK3CA mutations and are completely oncogenic in vivo (2, 14). Interestingly, PIK3CA mutations and PTEN loss are nearly mutually exclusive, suggesting that the homeostasis of phosphatidylinositoI-3,4,5- triphosphate is regulated by both PIK3CA and PTEN, both of which are critical to carcinogenesis (32). This conclusion further supports the importance of the PI3K pathway in the tumorigenesis of many cancer types. Of note, no mutation has ever been detected in other members of the PI3K gene family (3).
  • the present invention provides a method of identifying mutated gene sequences, comprising the steps of: obtaining a sample comprising mutated and wild-type gene sequences; conducting a first round of PCR; treating the sample with a restriction enzyme that would digest the wild-type gene sequences but not the mutated gene sequences; conducting a second round of PCR to amplify the mutated gene sequences but not the wild-type gene sequences; and performing sequencing to identify the mutated gene sequences.
  • the present invention also provides a method of identifying mutated gene sequences in the PIK3CA gene encoding catalytic subunit pi 10a of phosphatidylinositol 3-kinase, comprising the steps of: obtaining a sample comprising mutated and wild-type gene sequences; conducting a first round of PCR; treating the sample with a restriction enzyme that would digest the wild-type gene sequences but not the mutated gene sequences; conducting a second round of PCR to amplify the mutated gene sequences but not the wild-type gene sequences; and performing sequencing to identify the mutated gene sequences.
  • the present invention also provides a method of identifying mutated gene sequence in the PIKiCA gene encoding catalytic subunit pi 10a of phosphatidylinositol 3-kinase, comprising the steps of: obtaining a sample comprising mutated and wild-type gene sequences; conducting a first round of PCR, wherein primers used in the PCR introduce one or more unique restriction enzyme site into the wild-type gene sequence; treating the sample with a restriction enzyme that would digest the wild-type gene sequence but not the mutated gene sequence; conducting a second round of PCR to amplify the mutated gene sequence but not the wild-type gene sequence; and performing sequencing to identify the mutated gene sequence.
  • Figure 1 shows the genetic profile of HNSCC tumorigenesis (1).
  • Figure 2 shows the signaling pathway of PI3K (2).
  • PI3K signaling involves PTEN, Akt, and mTOR.
  • Figure 3 shows E545K and H1047R are the two most frequent hot-spot mutations of PlKiCA.
  • Figure 3A shows more than 80% of the PIK3CA mutations have been detected in its exons 9 and 20 (3).
  • Figure 3B shows the three hot-spot mutations, E542K, E545K and H1047R, account for 78.6% of all PIK3CA mutations reported (2).
  • Figure 4 shows target therapies in development for PIK3CA and its signaling pathway. Many target therapies are in clinical trials that target EGFR, Akt, or mTOR. These are potential therapeutic drugs, whose treatment outcome can be impacted by the activities and genetic status of PlKiCA. Evidence suggests that it would be beneficial for a patients' tumor to be tested for PlKiCA and PTEN mutations, before enrolling the patient in a target therapy for EGFR, Akt, or mTOR.
  • Figure 5 shows the schematic of the mutant-enriched sequencing methods for detecting
  • Figure 5A shows the protocol for detecting PIK3CA mutation H1047R.
  • Enzyme BsaBI (GATNNNNATC) specifically cuts the wild-type sequences of the exon 20, but not the mutant copies with A3140G nucleotide alteration. After digesting the first PCR product with the enzyme BsaBI, the second PCR selectively amplifies the mutant copies.
  • Figure 5B shows a unique restriction enzyme site Hpy 1881 (TCNGA) was introduced by mismatch PCR for detecting PIKiCA mutation E545K.
  • the mismatch primer (PIK-E9MF: TCTACACGAGATCCTCTCTCTGTAATCTC) has two A ⁇ T nucleotide substitutions in the forward primer to create a unique enzyme site Hpy 1881 (TCNGA) in the wild-type sequences of the PIKiCA exon 9, but not the mutant sequences.
  • Figure 5C shows the mismatch primer PIK-2E9MR was designed to create a unique restriction enzyme site EcoRI for enriching PIK3CA mutation E542K (G1624A) and E542G (A1625G) with the similar strategy for the other hot-spot mutation E545K.
  • Figure 6 shows high-risk patients will be consented to contribute saliva and sputum specimens at the time of clinical examinations.
  • the results of the PIKiCA mutation analyses will not affect patient care decisions. However, we will investigate whether there is a correlation between PIK3CA mutation status and the needs for CT scan and/or biopsy.
  • Figure 7 shows the detection of PIKiCA hot-spot mutations by mutant-enriched sequencing.
  • Figure 7al-a2 show the sensitivity of the mutant-enriched sequencing protocol for the exon 20 H1047R mutation was investigated in head and neck cell line Detroit 562, whose genome had been reported to harbor a H1047R mutation.
  • Figure 7al shows both wild-type A and mutant G peaks were detected at 1 : 1 ratio as expected by conventional genomic sequencing of the cell line Detroit 562 DNA (nondiluted).
  • Figure 7a2 shows when the ratio of mutant and wild-type DNA reached 1:360, the mutant G peak was still the only peak detected in the cell line Detroit 562 DNA by mutant-enriched sequencing.
  • Figure 7bl-b2 show a patient sample with a known E545K mutation was used to test the mutant-enriched sequencing protocol for the exon 9 E545K mutation.
  • Figure 7bl shows antisense sequencing of the mutated site by the conventional genomic sequencing (marked by the black arrow) detected both the wild-type and the mutant alleles.
  • Figure 7b2 shows the peak representing the wild-type allele in the same sample disappeared when the mutant-enriched sequencing method was applied (indicated by the black arrow).
  • the red arrow marks the nucleotide Al 63 OT change that was introduced by mismatch primer (PIK-E9MF) to generate the restriction enzyme Hpyl88I (TCNGA) site.
  • Figure 7cl-c4 show the mutant-enriched sequencing method identified an undetected PIK3CA hotspot mutation E542K (Gl 624A) by the conventional sequencing.
  • Figure 7cl shows forward sequencing of a clinical sample with a known E542K mutation by the conventional genomic sequencing method (marked by the black arrow) displayed a dominant presence of the wild-type allele over the mutant allele.
  • Figure 7c2 shows the wild-type allele in the same sample disappeared when the mutant-enriched sequencing method was applied (indicated by the black arrow).
  • the red arrow marks the nucleotide A1627T change that was introduced by mismatch primer (PIK-2E9MF) to generate a unique restriction enzyme EcoRI (GAATTC) site.
  • PIK-2E9MF mismatch primer
  • GATTC unique restriction enzyme EcoRI
  • the present invention provides a method of identifying mutated gene sequences, comprising the steps of: obtaining a sample comprising mutated and wild-type gene sequences; conducting a first round of PCR; treating the sample with a restriction enzyme that would digest the wild-type gene sequences but not the mutated gene sequences; conducting a second round of PCR to amplify the mutated gene sequences but not the wild-type gene sequences; and performing sequencing to identify the mutated gene sequences.
  • samples comprising mutated and wild-type gene sequences can be, but are not limited to, tissue swab sample, saliva sample, biopsy sample, pancreatic duct juice, blood, bodily fluid, and surgically resected samples.
  • the samples can be obtained from a subject having head and neck cancer, breast cancer, liver cancer, lung cancer, colorectal cancer, brain cancer, gastric cancer, or ovarian cancer.
  • the samples are obtained from a subject having a disease or abnormality comprising mutated sequences in the PIKSCA gene.
  • the first and second rounds of PCR in the above method are performed with one or more primers comprising DNA sequences that anneal to a desired gene locus with an annealing temperature between 45-85 0 C and aim to amplify exonic sequences of the desired gene.
  • the primers that bind to coding or non-coding sequences inside the desired locus are from about 10-bp long to about 100-bp long, and can amplify regions from about 20-bp to 20-kb inside the desired locus.
  • the first round of PCR is performed with primers that introduce one or more unique restriction enzyme site into the wild-type gene sequences so that only the wild-type gene sequences will be digested by the one or more restriction enzymes.
  • This method can detect at least 1 mutated gene sequence among 360 copies of wild-type gene sequence.
  • the sensitivity of this method can be enhanced and amplified by optimizing the restriction enzyme digestion and PCR amplification conditions. For example, the above method can detect 1 mutated gene sequence among 200 copies of wild-type gene sequence, or 1 in 500, or 1 in 750, or 1 in 1000, or 1 in 2000, or 1 in 3000 copies of wild-type gene sequence.
  • the present invention also provides a method of identifying mutated gene sequences in the PIK3CA gene encoding catalytic subunit pi 10a of phosphatidylinositol 3 -kinase, comprising the steps of: obtaining a sample comprising mutated and wild-type gene sequences; conducting a first round of PCR; treating the sample with a restriction enzyme that would digest the wild-type gene sequences but not the mutated gene sequences; conducting a second round of PCR to amplify the mutated gene sequences but not the wild-type gene sequences; and performing sequencing to identify the mutated gene sequences.
  • samples comprising mutated and wild-type gene sequences can be, but are not limited to, tissue swab sample, saliva sample, biopsy sample, pancreatic duct juice, blood, bodily fluid, and surgically resected samples.
  • the samples can be obtained from a subject having head and neck cancer, breast cancer, liver cancer, lung cancer, colorectal cancer, brain cancer, gastric cancer, or ovarian cancer.
  • the samples are obtained from a subject having a disease or abnormality comprising mutated sequences in the PIK3CA gene.
  • primers that bind to coding or non-coding sequences inside the PIK3CA locus are from about 10-bp long to about 100-bp long, and can amplify regions from about 20-bp to
  • the first and second rounds of PCR are performed with one or more primers comprising DNA sequences that anneal to the PIK3CA locus with an annealing temperature between 45-85 0 C and aim to amplify sequence in exon 20 of the PIK3CA gene.
  • the first round of PCR is performed with primers GACATTTGAGCAAAGACCTGAA and ATCAAACCCTGTTTGCGTTT, whereas the second round of PCR is performed with primers CATTTGCTCCAAACTGACCA and
  • the restriction enzyme employed in the above method recognizes wild-type but not mutated sequences of exon 20 of the PIK3CA gene.
  • the restriction enzyme is BsaBI or any enzyme that is specific for the A3140 and does not digest the
  • the mutated gene sequence detected by the above method is hot- spot mutation A3140G (H 1047R).
  • This method can detect at least 1 mutated gene sequence among 360 copies of wild-type gene sequence.
  • the sensitivity of this method can be enhanced and amplified by optimizing the restriction enzyme digestion and PCR amplification conditions. For example, the above method can detect 1 mutated gene sequence among 200 copies of wild-type gene sequence, or 1 in 500, or 1 in 750, or 1 in 1000, or 1 in 2000, or 1 in 3000 copies of wild-type gene sequence.
  • the present invention also provides a method of identifying mutated gene sequence in the PIK3CA gene encoding catalytic subunit pi 10a of phosphatidylinositol 3-kinase, comprising the steps of: obtaining a sample comprising mutated and wild-type gene sequences; conducting a first round of PCR, wherein primers used in the PCR introduce one or more unique restriction enzyme site into the wild-type gene sequence; treating the sample with a restriction enzyme that would digest the wild-type gene sequence but not the mutated gene sequence; conducting a second round of PCR to amplify the mutated gene sequence but not the wild-type gene sequence; and performing sequencing to identify the mutated gene sequence.
  • Samples comprising mutated and wild-type gene sequences have been described above.
  • the first and second rounds of PCR are performed with one or more primers comprising DNA sequences that anneal to the PIK3CA locus with an annealing temperature between 45-85 0 C and aim to amplify sequence in exon 9 of the PIK3CA gene.
  • the mutated gene sequence detected by this method is hot-spot mutation G1633A (E545K) or G1624A (E542K).
  • a non hot-spot mutation, E542G (A1625G) can also be detected by this method.
  • the first round of PCR for detecting mutation E545K is performed with primers TCTACACGAGATCCTCTCTCTGTAATCTC and GCATTTAATGTGCCAACTACCA, wherein the first round of PCR for detecting mutation E542K is performed with primers GATTGGTTCTTTCCTGTCTCTG and CATAGAAAATCTTTCTCCTGCTCAGTGAAT.
  • the second round of PCR for detecting mutation E545K is performed with primers TCTACACGAGATCCTCTCTCTGTAATCTC and CTGAGATCAGCCAAATTCAGT TATTTTTTC, wherein the second round of PCR for detecting mutation E542K is performed with primers TTGCTTTTTCTGTAAATCATCTGTG and CATAGAAAAT CTTTCTCCTGCTCAGTGAAT.
  • the restriction enzyme employed in the above method recognizes wild-type but not mutated sequences of exon 9 of the PIK3CA gene.
  • the restriction enzyme is Hpy 1881, EcoRl, or any enzymes that can distinguish between wild-type and mutant sequences at E542K and E545K. The sensitivity of this method is the same as that described above.
  • restriction enzymes and primers described above can be used together so that mutated sequences in both exons 9 and 20 of the PIK3CA gene can be detected.
  • the present invention also provides uses of the methods described above for the detection or diagnosis of cancers, diseases or abnormalities comprising mutated sequences in the PIK3CA gene. Cancers or diseases comprising mutated PIK3CA gene have been described herein.
  • kits comprising reagents for the practice of the methods described above.
  • the kits comprise the primers and restriction enzymes described above for detecting mutated gene sequences in the PIK3CA gene.
  • PIK3CA phosphatidylinositol 3- kinase, catalytic subunit pllO ⁇
  • HNSCC head and neck squamous cell carcinoma
  • mutant-enriched sequencing protocols that specifically detect the three hot-spot changes in PIK3CA. These protocols are unique in that they enhance the sensitivity with which hot-spot changes are detected (15).
  • the sensitivity of sequencing protocols is especially relevant because traditional mutation analyses require that tissue samples contain a predominance of tumor cells with only a minor contribution from normal tissues. Unfortunately, in the clinical setting, these traditional analyses often prove inadequate because patient specimens often contain a predominance of normal cells with only a minor contribution from tumor tissues. Our novel methods should mitigate this problem by increasing the sensitivity with which frequent hot-spot changes are detected. In this proposal we attempt to investigate the clinical applicability of these new mutant-enriched sequencing protocols.
  • HNSCC status This will include patients who come to clinic a) because of a suspicious clinical symptoms (often primary symptom is a neck mass), with a diagnosis that has not been confirmed by biopsy, b) for follow-up after tumor resection, and c) for follow-up after remission.
  • This high-risk population is chosen, rather than a general healthy population (for whom our early detection assay is ultimately meant for), so we can evaluate the sensitivity and specificity of our methods in a better controlled, short-term experiment.
  • These patients will be asked to contribute a salivary specimen and a sputum specimen during a routine clinical examination. We will correlate the mutant-enrich mutation results from the saliva and sputum with whether a patient has undergone a CT scan or a biopsy. This experiment will inform us as to the utility with which our assay predicts whether a patient will require a CT scan or a biopsy.
  • the present invention provides novel assays that allow for enhanced sensitivity in detecting the most frequent PIK3CA hot-spot mutations (E542K, E545K and H1047R) (15).
  • the three hot-spot mutations account for 78.6% of all reported cancer-causing PIK3CA mutations (2).
  • the assay we have developed for H1047R allows detection at a sensitivity of 1 mutant DNA copy per 360 wild-type DNA copies in a mixed population of wild-type and mutant DNA.
  • PIK3CA mutation can impact cancer patients in several potential aspects: 1) patients diagnosed with PIK3CA mutation can undergo pathway-specific or target therapy 2) PIK3CA mutation can be utilized as a prognosis and recurrent marker 3) PIK3CA mutation can be used as an early detection marker.
  • PIK3CA Potential Therapy Targeting PIK3CA
  • PI3K can be activated by EGFR 5 Her-2, and Ras, while Akt and mTOR are downstream mediators of PI3-K.
  • PTEN can reverse the action of PI3K by removing the 3 '-phosphate from the inositol ring of PI3,4P2 and PIP3, thus preventing the activation of downstream molecules like Akt.
  • Emerging evidence demonstrates, that targeted cancer therapies against EGFR (such as Gefitinib/Iressa by AstraZeneca, Erlotinib/Tarceva by OSI Pharmaceuticals, Cetuximab/Erbitux by Imclone, and Lapatinib/Tykerb by GlaxoSmithKline) or Her-2 (CM 033 by Pfizer and Lapatinib/Tykerb by GlaxoSmithKline ) may have had mixed results possibly due to the upregulation or activation of the PI3K pathway. If we can better determine the mutational status of PIK3 CA in a patient's tumor, we can make immediate impacts in the EGFR and Her-2 target therapies by excluding patients with PIK3CA mutations.
  • EGFR such as Gefitinib/Iressa by AstraZeneca, Erlotinib/Tarceva by OSI Pharmaceuticals, Cetuximab/Erbitux by Imclone, and Lapatinib/Tykerb by Gla
  • Inhibitors of Akt and mTOR are also available; some are in clinical use, while the others are at various phases of clinical trials (33). Rapamycin (an anti-proliferative inhibitor of mTOR) has been shown to be effective treatment for glioma tumors that are EGFR-dependent and PTEN-deficient (35, 36). Similarly, in clinical trials we can expect to treat patients with PIKiCA mutations with an Akt inhibitor (Perifosine by Keryx Biopharmaceuticals) or an mTOR inhibitor (Rapamycin, RADOO 1/Everolimus by Novartis, CCI-779/Temsirolimus by Wyeth, and AP23573 by Ariad).
  • PIK3CA has tremendous impacts on the success of target therapies for EGFR, Her-2, mTOR, and Akt. Furthermore, it is imperative that we determine the activity of the PI3K pathway (by the combination of mutational analysis of PIK3CA and immunohistochemistry analysis of PTEN) in order for affected patients to reap the full benefits of these targeted therapies.
  • PIK3CA as a tumor, prognosis, or recurrent marker
  • Amplification of chromosome 3q26 is observed in up to 40% of HNSCC and is linked to tumor progression and negatively correlated with clinical outcome (37-39).
  • Gene amplification and overexpression of PJK3CA may be a critical early event of HNSCC tumorigenesis because they are observed in low to moderate dysplasic cases, and their increased frequencies are associated with transition to invasive cancer (40, 41).
  • Mutations of PIK3CA have been reported in early lesions of breast cancer carcinoma, hepatocellular carcinoma, and gastric carcinoma, also supporting the notion that PIKSCA mutation is an early event in tumorigenesis (32, 42). Given the involvement of PIK3CA in many aspects of tumorigenesis, our ability to detect PIKSCA mutation with enhanced sensitivity will impact patient care in many cancer fronts.
  • HNSCC Amplification of chromosome 3q26 is frequently observed in HNSCC and is linked to tumor progression and negatively correlated with clinical outcome (37-39). Gene amplification and overexpression of PIKSCA are observed in low to moderate dysplasic cases, but their increased frequencies are associated with transition to invasive cancer (40, 41).
  • PIK3CA activating mutation is a common mechanism involved in the tumorigenesis of HNSCC.
  • missense mutations of the PIK3CA gene were identified in the 38 HNSCC specimens (4/38, 11%). Two of the mutations were in the exon 9 (E545K, E542K), one was in the exon 20 (H1047Y) and one was in the exon 4 (Y343C). None of these mutations were detected in the corresponding normal tissues except for the H 1047 Y mutation, which was identified in HNSCC cell line Detroit 562. Three of the four PIK3CA missense mutations are hot-spot mutations (3). The mutation in the exon 4 nucleotide 1028 A ⁇ G, which leads to alteration at codon 343 TAC (Y) - ⁇ TGC(C), has not been described before.
  • pharynx (Table 1). Cancer of the pharynx is the 9 th most common cancer worldwide (46). It is characterized as the following subsites: posterior pharynx, hypopharynx and lateral pharyngeal walls. A total of six pharyngeal squamous cell carcinoma cases were examined in this study, suggesting that as high as 50% (3/6) of pharyngeal tumor samples may harbor PIK3CA mutations.
  • Mutant-enriched sequencing methods generally involve a first-round PCR, restriction enzyme digest, and a second-round PCR.
  • mutant- enriched sequencing methods selectively amplifies the mutant copy of a targeting gene by reducing the wild-type copy via a restriction enzyme digestion that is specific for the wild-type DNA after the first round PCR.
  • the second PCR amplification only the mutant strands but not the wild-type would be further amplified.
  • mutant-enriched DNA sequencing is particularly helpful to screen gene mutation in samples with high concentrations of normal cells.
  • This mutant-enriched sequencing method was tested in a previously reported patient sample with a known PIK3CA E542K mutation (13). We showed that only the mutant peak remained while the corresponding wild-type peak completely vanished when the mutant- enriched sequencing was applied (data not shown). Interestingly, this method can detect not only the E542K (Gl 624A) mutation, but also a previously described non-hot-spot E542G (A1625G) mutation (3)(data not shown). We screened the 24 biopsy specimens of pharyngeal cancer and an additional case of PlKiCA E542K (G1624A) mutation was identified (data not shown).
  • HNSCC Patients with newly diagnosed HNSCC or unresectable HNSCC will be asked to enroll. Consented patients will be asked to contribute a salivary specimen, a sputum specimen, and for a cotton swab of the tumor mucosa at the time of routine examination. In addition, permission to access previously taken biopsy and/or surgical specimens will also be procured from the patients. Patients will be admitted and attended to by the medical residents of the Department of Otolaryngology and Head and Neck Surgery (OTO/HNS) under the supervision of Drs. Lanny Close and Spiros Manolidis. Specimens can be collected at all OTO/HNS clinical locations, including HP 7 th , VC 10 th , East 60 th St.
  • OTO/HNS Department of Otolaryngology and Head and Neck Surgery
  • the participants will be asked not to eat, drink, smoke, or brush their teeth for at least 60 minutes prior to sample collection.
  • the subjects will be swabbed first (a scope is only necessary for cancers outside the oral cavity, oropharynx, hypopharyx, and pharynx.
  • a laryngoscope, nasopharyngoscope, or esophagoscope will be used), then asked to swish and gargle with 25 ml of sterile NaCI solution (0.9%) for 3 minutes for saliva collection (49).
  • Swab samples and Saliva samples will be stored at 4°C before transferring to the research lab (unprocessed samples can be stored up to 2 weeks at 4°C).
  • Genomic DNA from exfoliated cells will be digested with proteinase K and extracted with ChargeSwitch gDNA Buccal Cell Kit (Invitrogen) according to the manufacturer's direction. Up to 6 ⁇ g of genomic DNA can be extracted per sample. The participants will then be asked to provide an induced sputum sample using a variation of the ultrasonic nebulization technique described by Saccomanno et al.(50, 51).
  • Subjects will use water or saline to brush tongue, buccal surfaces, teeth, and gingiva gently to remove superficial epithelial cells and bacteria, followed by gargling and rinsing with tap water. Participants then inhale a nebulized 3% saline solution from an ultrasonic nebulizer for 20 to 30 minutes. Sputum will be collected in a sterile specimen cup and an equal volume of Saccomanno solution was added immediately. Sputum samples will be defined as adequate by the presence of deep lung macrophages or Curschmann's spiral (50) and, irrespective of adequacy, processed for DNA extraction by extensive vortex mixing, washing once with Saccomanno solution, and storage at room temperature until analysis.
  • Genomic DNA was isolated from sputum as by digestion with Pronase in SDS (1%), followed by standard phenol-chloroform extraction and ethanol precipitation (52).
  • Six slides (10 ⁇ m thick each) of biopsy or resected tumors will be microdissected and processed into genomic DNA using QIAmp DNA Mini Kit (Qiagen) according to manufacturer's instruction for paraffin-embedded tissues.
  • QIAmp DNA Mini Kit Qiagen
  • saliva, sputum, tumor swab, biopsy, and/or resected tumors will be collected from the participants and processed into genomic DNA for mutation analyses. All the samples will be subject to mutant-enriched sequencing for H1047R and E545K as described previously. Assuming 10% of the 400 cases harbor a PIK3CA mutation, and 67.8% (two hot-spots) of the mutations is a hot-spot mutation, we estimate that we will detect a PIK3CA hot-spot mutation in the biopsy and/or resected tumor specimens of ⁇ 27 cases. The number of cases may be higher if pharyngeal cancer predominates the study cohort.
  • Recruitment will include patients who come to clinic a) because of suspicious clinical symptoms (whose primary symptom is neck mass), but not yet confirmed by biopsy, b) for follow-up after tumor resection, and c) for follow-up after remission.
  • a high-risk patient comes to the clinic, he/ she will receive a clinical examination and possibly a CT scan. A biopsy is subsequently taken only if the CT scan is positive (larger than lcm). All high-risk patients will be asked to contribute a salivary specimen and a sputum specimen regardless whether a CT scan or a biopsy is ordered at the time of clinical examination. This will be a double-blind experiment where the clinicians will not know the results of the mutation analyses of the specimens while providing patient care.
  • ROC Receiver Operating Characteristics
  • mutant-enriched sequencing methods to selectively amplify and sequence three most frequent mutations (H1047R, E545K, and E542K) of PIK3CA in specimens with low tumor DNA contribution, such as biopsy samples.
  • Multiplex PCR with three primer sets will be performed in the first-round PCR, restriction enzyme digest involving three enzymes, and multiplex PCR with three primer sets will be performed in the second-round PCR.
  • the PCR product from the second-round PCR will be divided and submitted for sequencing with three sequencing primers in three separate reactions. Optimization will involve primer designs, enzyme selection, digestion conditions (e.g.
  • DNA amount, enzyme amount, buffer, temperature, and duration), and PCR protocols e.g. annealing temperature, number of cycles, and buffer.
  • the sensitivity will be determined again by testing on a series of dilutions with various mutant to wild-type DNA.
  • the combination assay for high-throughput analysis is a novel proposal, because it has not been reported on other mutant-enriched sequencing methods.
  • the optimization can also be designed to complement high-throughput sequencers available on the market. For example, primers can be re-designed to include "fusion primers" necessary for amplicon sequencing on the Roche's 454 sequencing (Genome Sequencing FLX System). Or the primers can be re-designed to allow the annealing of "adaptors" required for the Illumina's sequencing technology.
  • mutant-enriched assay identifies mutations undetectable by the conventional method in the resected tumors, we will then suspect that multiple tumor clones co-exist in those resected tumors and our assay is sensitive enough to detect presences of tumor subclones.
  • the DNA of those suspected tumor samples will be PCR-amplified, subcloned into bacterial vectors, and transfected into bacteria to generate bacterial clones. Approximately 100 bacterial clones will be analyzed per tumor sample to confirm the presence of the subclones and determine the percentage of mutant PlKSCA DNA copies.
  • HNSCC head and neck cancer
  • PIK3CA Gene is Mutated with High Frequency in Human Breast Cancers. Cancer Biol Ther, 3: 772-775, 2004. 31. Wang, Y., Helland, A., Holm, R., Kristensen, G. B., and Borresen-Dale, A. L. PIK3CA mutations in advanced ovarian carcinomas. Hum Mutat, 25: 322, 2005.
  • Pharyngeal Cancer Frequent somatic mutation of PIK3CA has been identified in many human cancer types. We previously reported four PIK3CA mutations in 38 head and neck cancer samples; three of which were identified in six pharyngeal cancer samples. To determine the mutation frequency of PIK3CA in pharyngeal cancer, we studied 24 additional cases of pharyngeal squamous cell carcinoma in this study. Using both direct genomic DNA sequencing and novel mutant- enriched sequencing methods developed specifically for the three hot-spot mutations (H1047R, E545K and E452K) of PIK3CA, we detected five mutations of PIK3CA in the 24 pharyngeal cancers (20.8%).
  • mutant-enriched sequencing methods for the H1047R hot-spot mutation can identify the mutation in a mixed population with wild-type DNA sequences at minimum sensitivity of 0.0028 (1 mutant: 360 wild-type DNA copies).
  • novel mutant-enriched sequencing methods allow the detection of the PlKiCA hot-spot mutations in clinical specimens which often contain limited tumor tissues (i.e. biopsy specimens).
  • the mutant-enriched sequencing methods also allow the detection of the hot-spot mutations existing in tumor subclones that usually go undetected by the conventional sequencing method because of their minor cellular populations.
  • the data further support that oncogenic PIK3CA may play a critical role in pharyngeal carcinogenesis, and the mutant-enriched sequencing methods for PlKiCA are sensitive and reliable ways to detect PIKiCA mutations in clinical samples.
  • PIKiCA and its pathway are potential targets for chemotherapy and radiation therapy, and frequent somatic mutation of PIKiCA has been identified in many human cancer types (e.g. breast cancer, colorectal cancer), the abilities to detect PIKiCA mutations with enhanced sensitivities have great potential impacts on target therapies for many cancer types.
  • HIPAA Health Organization
  • the 24 specimens came from five female and nineteen male patients, with ages ranging from 38 to 78 years-old (average 57.9 ⁇ 12.2 years-old). Of the 24 patients, eleven were heavy smokers (more than 40 packs per year), two of the 11 were also with heavy alcohol consumption, and one of the heavy smokers abused cocaine; two were moderate smokers, two had no smoking and had only occasional alcohol use, and the remaining nine patients' history was not available.
  • One patient was a HTV carrier. All patients were diagnosed as squamous cell carcinomas of the pharynx. The grade of cancer in these patients was four well-, 18 moderately-, and two poorly-differentiated.
  • DNA isolations from paraffin embedded tissue The cases were reviewed by two pathologists and the diagnosis confirmed. The paraffin embedded blocks containing tumor tissues were selected and five 10 ⁇ m thickness sections were cut for each case. The 24 cases studied included five surgical resection specimens and nineteen cases of small biopsy specimens. Genomic DNA were extracted from the tumor tissues using QIAmp DNA Kit
  • the primers for the PIK3CA exons 9 and 20 are PIK-E9F: CCAGAGGGGAAAAATATGACA; PIK-E9R: CATTTTAGC ACTTACCTGTGAC; PIK- E20F: CATTTGCTCCAAACTGACCA; PIK-E20R: TGAGCTTTCATT
  • PCR products were purified using the Geneclean Turbo Nucleic Acid purification Kit (Qbiogene, Irvine, CA). Finally, purified DNA fragments were sequenced using the corresponding forward PCR primers. Samples found to have a genetic alteration in the target gene were subsequently sequenced in the reverse direction to confirm the mutation using the reverse PCR primers. The mutation was then further verified by sequencing of a second PCR product derived independently from the original template. All sequencings were performed with ABI' s 3100 capillary automated sequencers at the DNA facility of Columbia University Medical Center in New York (Qiu et al., Clin. Cancer Res. 12:1441-1446 (2006)).
  • each sample 40ng of genomic DNA was first amplified using outer primers PIK-E20OF (GACATTTGAGCAAAGACCTGAA) and PIK-E20OR (ATCAAACCCTGTTTGCGTTT) for 30 cycles.
  • PIK-E20OF GACATTTGAGCAAAGACCTGAA
  • PIK-E20OR ATCAAACCCTGTTTGCGTTT
  • the primers for the second PCR are PIK-E20IF (CATTTGCTCCAAACTGACCA) and PIK-E20IR (TGAGCTTTCATTTTCTCAGTTATCTTTTC).
  • PIK-E20IF or PIK-E20IR TGAGCTTTCATTTTCTCAGTTATCTTTTC.
  • Fig. 5A PIK-E20IF or PIK-E20IR
  • a mismatch primer PIK-E9MF (TCTACACGAGATCCTCTCTCTGTAATCTC) was used as the forward primer for both rounds of PCR.
  • the reverse primers for the first and second PCR were respectively PIK-E9OR (GCATTTAATGTGCCAACTACCA) and PIK-E9IR
  • the PCR strategy of mutant- enriched sequencing is the same as the one described above for the hot-spot mutation G 1633 A (E545K), in which a mismatch primer is designed to create a unique restriction enzyme site EcoRI in the PIK3CA exon 9 region.
  • the mismatch primer PIK-2E9MR CATAGAAAATCTTTCTCCTGCTCAGTGAAT
  • the forward primers for the first and second PCR were respectively PIK- 2E9OF (GATTGGTTCTTTCCTGTCTCTG) and PIK-2E9IF
  • TTGCTTTTTCTGTAAATCATCTGTG The restriction enzyme EcoRI digestion was performed at 37 0 C overnight.
  • the forward PCR primer PIK-2E9IF was also used as the DNA sequencing primer (Fig. 5C).
  • the PCR condition for all the PCR reactions is 94°C, 2 minutes; (94 ⁇ C, 30 seconds; 60 0 C, 30 seconds; 72°C, 30 seconds) x 40 cycles; 72°C, 5 minutes.
  • PIK3CA hot-spot mutation G1633A E545K
  • GAG codon 545 glutamic acid
  • AAG lysine
  • the other mutation was a missense mutation in exon 20 nucleotide 3127 A ⁇ G, led to a codon 1043 ATG (Met) ⁇ GTG (VaI) substitution.
  • This missense mutation of PIK3CA has been reported previously. Both mutations were not detected in the surrounding normal tissues, thus both were somatic mutations.
  • Mutant-enriched sequencing identified PIK3CA hotspot mutation H1047R in samples screened negative by conventional DNA sequencing approach.
  • Conventional DNA sequencing method only recognizes the mutant DNA if it is present in more than 10 percent of a mutant/wild-type mixed population in primary tumor tissue (Levi et al., Cancer Res. 51:3497 (1991); Nakahori et al., J. Gastroenterol. Hepatol. 10:419 (1995)). Therefore we hypothesized that the low tumor to normal cell ratio in our biopsy specimens may have caused some false negative results and contributed to the lower frequency of PIKiCA mutation observed in the current study (2/24, 8.3%) than our previous report (3/6, 50%) (Qiu et al., Clin. Cancer Res.
  • the unexpectedly low mutation frequency of PIK3CA in the biopsy samples might also have been caused by the overall low or poor DNA contents available in these tissues.
  • Mutant-enriched sequencing methods generally involve a first-round PCR, restriction enzyme digest, and a second-round PCR.
  • mutant- enriched sequencing methods selectively amplify the mutant copy of a targeted gene by reducing the wild-type copy number via a restriction enzyme digestion that is specific for the wild-type DNA after the first round PCR.
  • the second PCR amplification only the mutant strands but not the wild-type ones would be further amplified.
  • mutant-enriched DNA sequencing is particularly valuable when the ratio of mutant DNA is expected to be low.
  • Mutant-enrich sequencing for PIK3CA exon 9 hotspot mutation E545K For mutant- enriched sequencing of PIK3 CA exon 9 hotspot mutation, G1633A (E545K), mismatch primer (PIK-E9MF) was designed to introduce 2 A ⁇ T nucleotide mismatches in the forward primer to create a unique restriction enzyme site Hpyl88I (TCNGA), because there is no natural unique restriction enzyme site specific for the wild-type but not the mutant DNA sequences at this hot- spot.
  • TCNGA unique restriction enzyme site
  • mutant-enriched sequencing can identify the H1047R mutant DNA in a mixed population with wild-type DNA at sensitivity of 0.0028 (1 mutant: 360 wild-type DNA copies).
  • mutant-enriched sequencing protocol for hotspot mutation H542K G 1624A
  • the primers for the mutant-enriched sequencing analyses of the PIK3CA exon 9 hotspot mutations were designed to avoid interference from a homologous pseudogene located on chromosome 22ql l.2 cat eye syndrome region. At least one of the 2 primers for each PCR amplification contains mismatched base pairs to the pseudogene. Those mismatched nucleotides in either the forward and/or reverse primers seemed sufficient to prevent PCR amplification of the pseudogene in our mutant-enriched sequencing analyses. This was supported by the fact that we never observed the A1634C change in our samples.
  • mutant-enriched sequencing method is more sensitive and specific because of the following (i) there is the possibility of the nonspecific digestion by the restriction enzyme when confirming the mutation by PCR-RFLP; (ii) small amount of mutant DNA after digestion may not be enough to be visualized on an agarose gel in the PCR-RFLP analysis.
  • the mutant-enriched sequencing method directly displays the exact nucleotide sequence; (iii) the mutant-enrich sequencing method is not limited by available restriction enzyme sites.
  • a unique enzyme site could be introduced by mismatch PCR, as we have done for the detection of PlKSCA exon 9 hot-spot mutations E545K and E542K.
  • the mutant-enriched sequencing method is more superior to both the PCR-RFLP analysis and the conventional genomic sequencing assay for detecting hot-spot mutations. This method is particularly valuable in clinical applications where tumor samples are often mixed with a large population of normal cells.

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Abstract

La présente invention concerne de nouveaux procédés de séquençage avec enrichissement en mutant spécifiquement mis au point pour les trois mutations de points chauds (H 1047R, E545K et E452K) de PIK3CA qui peuvent être utilisés pour détecter des mutations non détectées par le procédé de séquençage conventionnel. Les nouveaux procédés de séquençage avec enrichissement en mutant permettent la détection des mutations de points chauds de PIK3CA dans des échantillons cliniques qui contiennent souvent des quantités limitées de tissus tumoraux. Les données confirment en outre que PIK3CA oncogène pourrait jouer un rôle critique dans la carcinogenèse du pharynx, et la capacité à détecter des mutations de PIK3CA avec de meilleures sensibilités a des impacts potentiels importants sur les thérapies ciblées pour de nombreux types de cancers (par exemple, cancer du sein, cancer colorectal).
PCT/US2008/065343 2007-05-30 2008-05-30 Détection de séquences de gènes mutés par séquençage avec enrichissement en mutant WO2008151031A1 (fr)

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CN104593515A (zh) * 2015-02-06 2015-05-06 中国医科大学 检测pik3ca基因h点突变的pcr-rflp方法
CN105238799A (zh) * 2015-10-19 2016-01-13 昆明理工大学 一种乳腺癌pik3ca突变基因及其应用
EP3246332A1 (fr) * 2010-01-12 2017-11-22 Siemens Healthcare Diagnostics Inc. Oligonucléotides et procédés et détection de mutations de pik3ca
CN105238799B (zh) * 2015-10-19 2018-08-31 昆明理工大学 一种乳腺癌pik3ca突变基因及其应用
WO2019178606A1 (fr) 2018-03-16 2019-09-19 Gopath Laboratories Llc Procédés pour la détection personnalisée de la récidive d'un cancer ou d'une métastase et/ou l'évaluation d'une réponse à un traitement
CN112410329A (zh) * 2020-10-16 2021-02-26 深圳乐土生物科技有限公司 引物组合、试剂盒及其在卵巢癌早期筛查中的应用
CN113470745A (zh) * 2021-08-25 2021-10-01 南京立顶医疗科技有限公司 SARS-CoV2潜在突变位点的筛选方法及其应用
CN114480642A (zh) * 2022-01-05 2022-05-13 合肥艾迪康医学检验实验室有限公司 检测smad4基因全外显子突变的引物、方法及试剂盒

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EP2377933A1 (fr) * 2008-12-23 2011-10-19 Guangzhou Surexam Bio-Tech Co., Ltd. Sondes, puces en phase liquide et procédés de détection de mutations du gène pik3ca
EP2377933A4 (fr) * 2008-12-23 2012-10-31 Guangzhou Surexam Bio Tech Co Sondes, puces en phase liquide et procédés de détection de mutations du gène pik3ca
WO2010072118A1 (fr) * 2008-12-23 2010-07-01 广州益善生物技术有限公司 Sondes, puces en phase liquide et procédés de détection de mutations du gène pik3ca
US9938584B2 (en) 2010-01-12 2018-04-10 Siemens Healthcare Diagnostics Inc. Oligonucleotides and methods for detecting KRAS and PIK3CA mutations
US10968487B2 (en) 2010-01-12 2021-04-06 Siemens Healthcare Diagnostics Inc. Oligonucleotides and methods for detecting KRAS and PIK3CA mutations
EP3246332A1 (fr) * 2010-01-12 2017-11-22 Siemens Healthcare Diagnostics Inc. Oligonucléotides et procédés et détection de mutations de pik3ca
CN104593515A (zh) * 2015-02-06 2015-05-06 中国医科大学 检测pik3ca基因h点突变的pcr-rflp方法
CN105238799B (zh) * 2015-10-19 2018-08-31 昆明理工大学 一种乳腺癌pik3ca突变基因及其应用
CN105238799A (zh) * 2015-10-19 2016-01-13 昆明理工大学 一种乳腺癌pik3ca突变基因及其应用
WO2019178606A1 (fr) 2018-03-16 2019-09-19 Gopath Laboratories Llc Procédés pour la détection personnalisée de la récidive d'un cancer ou d'une métastase et/ou l'évaluation d'une réponse à un traitement
EP3752604A4 (fr) * 2018-03-16 2021-04-21 Gopath Laboratories LLC Procédés pour la détection personnalisée de la récidive d'un cancer ou d'une métastase et/ou l'évaluation d'une réponse à un traitement
CN112410329A (zh) * 2020-10-16 2021-02-26 深圳乐土生物科技有限公司 引物组合、试剂盒及其在卵巢癌早期筛查中的应用
CN113470745A (zh) * 2021-08-25 2021-10-01 南京立顶医疗科技有限公司 SARS-CoV2潜在突变位点的筛选方法及其应用
CN113470745B (zh) * 2021-08-25 2023-09-08 南京立顶医疗科技有限公司 SARS-CoV-2潜在突变位点的筛选方法及其应用
CN114480642A (zh) * 2022-01-05 2022-05-13 合肥艾迪康医学检验实验室有限公司 检测smad4基因全外显子突变的引物、方法及试剂盒

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