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WO2008005559A2 - Stratégie servant à détecter des mutations de faible abondance - Google Patents

Stratégie servant à détecter des mutations de faible abondance Download PDF

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Publication number
WO2008005559A2
WO2008005559A2 PCT/US2007/015631 US2007015631W WO2008005559A2 WO 2008005559 A2 WO2008005559 A2 WO 2008005559A2 US 2007015631 W US2007015631 W US 2007015631W WO 2008005559 A2 WO2008005559 A2 WO 2008005559A2
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mutations
dna
pcr
sample
pancreatic
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PCT/US2007/015631
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WO2008005559A3 (fr
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Michael Goggins
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Johns Hopkins University
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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • This invention relates, e.g. , to a method for detecting the presence of one or more mutations, particularly low abundance mutations, in one or more genes of interest.
  • the method comprises the use of limit dilution PCR (LD-PCR), coupled with a method that can detect a plurality of mutations, e.g. at undefined sites in the DNA, such as temperature gradient capillary electrophoresis (TGCE) or cycle sequencing.
  • LD-PCR limit dilution PCR
  • TGCE temperature gradient capillary electrophoresis
  • pancreatic cancer Of particular interest is the detection/diagnosis of pancreatic cancer.
  • Pancreatic ductal adenocarcinoma is the fourth leading cause of cancer death in the USA and has the lowest survival rate for any solid cancer (about 2%). This poor survival occurs in part because only about 15% of patients are diagnosed with pancreatic cancer while they have surgically resectable disease. Pancreatic cancer survival is better for patients with the smallest tumors. Such a poor survival is particularly of concern to patients with inherited susceptibility to the disease.
  • Screening asymptomatic individuals with a significant risk of developing pancreatic cancer has demonstrated that pre-invasive pancreatic neoplasms can be detected by endoscopic ultrasound in some individuals. These results suggest that some form of clinical screening for high-risk individuals will eventually become an important part of their management.
  • pancreatic neoplasia • more accurate markers of pancreatic neoplasia, which could help differentiate pancreatic cancers from chronic pancreatitis and help identify pre-invasive pancreatic neoplasms such as intraductal papillary mucinous neoplasms (EPMNs) and pancreatic intraepithelial neoplasia (PanIN).
  • EPMNs intraductal papillary mucinous neoplasms
  • PanIN pancreatic intraepithelial neoplasia
  • Pancreatic neoplasms evolve with many genetic and epigenetic alterations, but few such alterations are useful diagnostic markers. Genetic alterations of pancreatic neoplasia include oncogene (K-ras, BRAF) and suppressor gene mutations (p!6, p53, SMAD4, BRCA2, STKlJ, hMLHl, hCDC4, MKK4, and FancC). Mitochondrial mutations and microsatellite instability also occur in pancreatic cancers as do many gene expression changes. DNA methylation markers are currently undergoing evaluation as markers of pancreatic neoplasia.
  • oncogene K-ras, BRAF
  • suppressor gene mutations p!6, p53, SMAD4, BRCA2, STKlJ, hMLHl, hCDC4, MKK4, and FancC.
  • Mitochondrial mutations and microsatellite instability also occur in pancreatic cancers as do many gene expression changes. DNA methylation markers are currently
  • the secondary fluid, pancreatic juice, is readily obtainable during endoscopic investigation.
  • mutant K-ras is most readily detectable in such secondary fluids.
  • ⁇ K-ra? mutations are not specific for invasive pancreatic cancer; they are also found in the pancreatic juice and in the stool of patients with chronic pancreatitis, individuals who smoke, and in
  • PanINs Quantifying mutant K-ras levels in pancreatic juice helps to distinguish patients with pancreatic cancer from those with pancreatitis, but is not sufficient for pancreatic cancer diagnosis.
  • the p53 gene is mutated in ⁇ 75% of pancreatic cancers. Mutations in the p53 gene are located throughout the gene (e.g. at any of several hundred nucleotides, within exons 5, 6, 7 and/or 8). Some current strategies can detect only specific mutations, or mutations at a specified nucleotide site in a DNA. Therefore, such methods are not suitable for identifying one of the myriad mutations that might be expected in patients with pancreatic cancer. Furthermore, among patients with pancreatic cancer, mutations seen in pancreatic juice are generally present at low concentration (about 1-10%). Although some methods currently exist which can detect a wider range of mutations, these methods can only detect mutant DNA when present at concentrations of about 5-10% or higher of the total DNA in a sample, so many low abundance mutations are missed using such methods of analysis.
  • pancreatic ductal adenocarcinomas harbor point mutations in exons 1 and 2 of pi 6.
  • pancreatic cancers harbor homozygous deletions, and in most remaining cancers, pi 6 is inactivated by promoter methylation.
  • pi 6 mutations are located through these two mentioned exons and are present in low abundance in secondary fluids, such mutations are not readily detected in secondary fluids, such as pancreatic duct juice, using currently available methods.
  • Figure 1 shows a schematic of LD-PCR. PCR on diluted pancreatic DNA samples followed by heteroduplex analysis using TGCE discriminates between wild-type and mutant alleles.
  • Figures 2A-2D show examples of heteroduplex analysis of pl6 exon 2.
  • Fig.2 A top left shows a heteroduplex pattern indicative of a mutation in exon 2 of pi 6 in the pancreatic juice DNA of a patient with pancreatic cancer (PJ-8 from patient No.8).
  • Fig.2B bottom left shows a homoduplex pattern indicative of normal pl6 in pancreatic juice from a subject without a detectable pl6 exon 2 mutation in their cancer
  • Fig. 2C top right shows a mutation in exon 2 of pi 6 in the patient's corresponding primary pancreatic cancer (patient No. 8).
  • Fig.2D shows a normal pi 6 heteroduplex pattern from the pancreatic cancer DNA of a patient without a detectable pi 6 mutation.
  • This invention relates, e.g., to a method for detecting mutations that are at low abundance in a DNA sample.
  • the method does not rely on the detection of specific mutations, or of mutations at specific nucleotide sites within a DNA molecule.
  • the assay strategy involves PCR (polymerase chain reaction) amplification of DNA at limiting dilution (LD-PCR), followed by screening the PCR products for mutations, using a method that can identify an undefined mutation (rather than a specific mutation, or a mutation at a specific site).
  • Suitable procedures for analyzing the amplified DNA (amplicons) for mutations include temperature gradient capillary electrophoresis (TGCE) or DNA sequence analysis (e.g. cycle sequencing).
  • a limiting dilution of DNA can be used to identify rare species of mutant PCR amplicons admixed with wild type DNA.
  • the limiting dilution PCR strategy involves analyzing DNA samples for mutations by performing many PCR reactions on the sample, with each PCR amplifying only a few DNA templates. That is, DNA from a sample is diluted and distributed in a large number of aliquots, such that only a few DNA templates are present in each aliquot; and the DNA in each aliquot is amplified by PCR with primer sets that are specific for one or more segments of the DNA of interest (segments that are expected to contain mutants).
  • mutant DNA In samples with low concentrations of mutant molecules, mutant DNA will be present in only a few of the amplicons, but in these amplicons, the mutant DNA will be at sufficient concentration to be detected by a detection method of the invention (e.g., TGCE or cycle sequencing). As a result, the majority wild-type DNA in the sample will no longer obscure the few mutant DNA molecules.
  • a detection method of the invention e.g., TGCE or cycle sequencing
  • temperature gradient capillary electrophoresis is used to analyze PCR products of limit dilution.
  • This method is shown diagrammatically in Figure 1.
  • TGCE temperature gradient capillary electrophoresis
  • TGCE detects heteroduplexes created from mixes of mutant to wild-type DNA and can detect the majority of mutations in a PCR fragment as long as there is sufficient concentration of mutant DNA in a given sample (usually 10-50% of total DNA).
  • a DNA sample obtained from pancreatic juice by this method, one quantifies the DNA accurately, using a conventional procedure, then performs 48 PCR reactions with each PCR containing 6 molecules of DNA. Each PCR is then subjected to TGCE. As a result, 48 x 6 (288) molecules of DNA are screened for mutations.
  • Example IV illustrates the detection ofp53 and pi 6 mutations in the pancreatic juice of patients with pancreatic cancer, using limiting dilution TCGE.
  • mutations in the limit dilution PCR products are detected by cycle sequencing rather than by heteroduplex detection.
  • cycle sequencing such a procedure is accomplished by PCR amplifying DNA molecules at about 10 picogram amounts (comparable to about 3 molecules of human genomic DNA, measured using a conventional procedure, such as real time PCR).
  • Most of the PCR wells (aliquots) contain 2-4 such DNA molecules, and when one of the molecules contains mutant DNA, the ratio of wild-type to mutant molecules enables the mutation to be detected by cycle sequencing in most cases.
  • performing 93 PCRs that contain about 3 molecules per well about 279 DNA molecules are screened for mutations.
  • a method of the invention can detect mutations which are in low abundance (e.g., somatic mutations that are present in only about 1 -10% of the cells in a population, or that are only present in about 1-10% of the total DNA in a sample, such as in a clinical sample from a cancer patient); can detect unknown mutations (e.g., multiple mutations) at unspecified sites within a defined segment of DNA (or RNA that has been reverse transcribed) in a gene, e.g.
  • a method of the invention can be used for a wide variety of applications, including diagnostic methods (e.g. to detect cancer, such as pancreatic cancer); research purposes (e.g. to study factors involved in ageing, the pathogenesis of neoplasia or metastasis, etc.); or to identify mutations that are correlated with a disease or condition of interest, such as a cancer.
  • diagnostic methods e.g. to detect cancer, such as pancreatic cancer
  • research purposes e.g. to study factors involved in ageing, the pathogenesis of neoplasia or metastasis, etc.
  • One aspect of the invention is a method of screening for mutations (variants) in a DNA sample, comprising a) diluting the sample and distributing the diluted sample to obtain a plurality of aliquots which, on the average, contain between about 2-10 genome equivalents of the DNA (e.g., of a mammalian DNA); b) PCR amplifying the DNA in a sufficient number of aliquots, with at least one set of PCR primers, so as to generate amplicons such that, if mutations are present at a frequency of about 1-
  • a "genome equivalent,” as used herein, is an amount of DNA that includes one copy of each allele in the genome.
  • each of the aliquots may contain between about 3 and 8 genome equivalents of DNA (e.g., between about 6 and 7 genome equivalents). All ranges used herein include the end points of the range.
  • the PCR step is carried out by selecting sets of PCR primers which flank segments of interest in a gene (e.g., exons which are thought or known to contain a plurality of mutations in a cancer of interest), hi one embodiment, in which it is desirable to identify previously unknown mutations present in a gene of interest, a series of adjacent regions of the gene of interest can be independently amplified by suitable PCR primers.
  • mRNA is converted to DNA by RT-PCR (reverse transcriptase PCR), and PCR primers are selected to flank the splice site of an alternatively spliced variant. In this manner, different sized splice variants will be observed following heteroduplex analysis.
  • RT-PCR reverse transcriptase PCR
  • PCR primers are selected which will amplify a segment of a nucleic acid of a desired size.
  • Typical amplicons range in size from between about 100 and 1,000 bases pairs (bp), e.g. between about 200 and 800 bp.
  • bp bases pairs
  • the optimal length of an amplicon to be analyzed is between about 10 and 800 bp. Improvements in gel chemistry in the future will likely allow larger PCR products to be screened for heteroduplexes.
  • the length limitation is also determined by the ratio of deoxy to dideoxy nucleotides in the sequencing chemistry: too high a didoxy to deoxy nucleotide ratio results in predominantly short sequencing products, too low a ratio in mostly long sequencing products. Because of this sequencing chemistry, the accuracy of sequencing of the first about 25-50 base pairs of any PCR product is also typically reduced and the sequencing of a PCR product utilizes a sequencing primer at one end of the amplicon. For this reason, PCR products of at least 100 base pairs in length are generally sequenced.
  • the number of aliquots for PCR amplification can be determined empirically, depending on variety of factors, including the frequency of mutations, the number of genome equivalent templates in each aliquot, the number of sets of PCR primers, etc.
  • the number of aliquots that are subjected to PCR analysis is determined by how low a concentration of templates containing mutant molecules one is trying to detect. A typical range would be about 40 to 200 aliquots for each PCR primer set, with each aliquot containing about 2 to 8 molecules.
  • a skilled worker can readily determine the optimal limiting dilution PCR strategy for mutation detection.
  • Increasing numbers of aliquots may be used as increasing numbers of PCR primer pairs are used in each PCR reaction. For example, one can amplify simultaneously several regions within a gene of interest (e.g., the four exons of p53 in which mutations are most often detected); regions of several different genes, such as p53 and pi 6; or other variations that will be evident to a skilled worker. For example, at least about 2, 4, 6, 8, 10, or more, sets (pairs) of primers can be used. Many aliquots (e.g., 1,000 or more) can be used in a method of the invention.
  • Methods for designing PCR primers and for carrying out PCR reactions including reaction conditions, such as the presence of salts, buffers, ATP, dNTPs, etc. and the times and temperature of incubation, are conventional and can be optimized readily by one of skill in the art. See, e.g., Innis et ⁇ l., editors, PCR Protocols (Academic Press, New York, 1990); McPherson et ⁇ l., editors, PCR: A Practical Approach, Volumes 1 and 2 (IRL Press, Oxford, 1991, 1995); Barany( 1991) PCRMethods and Applications JL, 5-16; Diffenbach et al., editors, PCR Primers, A Laboratory Manual (Cold Spring Harbor Press); etc.
  • a high fidelity DNA polymerase can be used. See, e.g., the discussion in Example VI.
  • Any of a variety of reaction chambers e.g., containers, wells of a plate, etc.
  • reaction chambers e.g., containers, wells of a plate, etc.
  • Containers can be closed to form a leak-proof seal, in order to reduce or prevent cross-contamination of samples.
  • Suitable formats for performing PCR reactions include computer-controlled thermal cyclers.
  • TCGE temperature gradient capillary electrophoresis
  • Variations of this method include the performance of multiple different sized PCRs during the same TGCE run, which can reduce assay cost.
  • the sensitivity of TGCE for detecting mutations can also be improved by using GC clamps as part of the PCR products, and by using fluorescently labeled primers.
  • amplicons that have resulted in the formation of a heteroduplex are sequenced, using a conventional procedure, to characterize more precisely the nature of the mutation that gave rise to the heteroduplex.
  • the detection of mutations in the amplified DNAs is performed by cycle sequencing.
  • One sequencing method is dye-terminator sequencing, in which a sequencing chemistry reaction that includes DNA polymerases and nucleotides also includes four dideoxynucleotide chain terminators, one for each nucleotide, which are labeled with different fluorescent dyes. Once the dideoxy labeled nucleotide is incorporated into a template sequence, additional nucleotides can no longer be added and the sequence can be detected by automated high-throughput DNA sequence analyzers that detect the fluorescently labeled template.
  • templates are resuspended in buffer after a clean up step, typically precipitation, and loaded onto a capillary sequencer.
  • High throughput capillary sequencers now have as many as 384 capillaries and can run multiple reactions (4-8 or more depending on the length of the PCR products being sequenced) each day. Thus, thousands of PCR products can be sequenced in one day at current costs of less than $1 per sequence.
  • Chromatograms of sequencing data are managed by software packages that can compare the sequencing data to identify sequence variants.
  • dHPLC denaturing high performance liquid chromatography
  • SSCP single- strand conformation polymorphism
  • SSCP detection can also be applied to capillary electrophoresis and it is possible to combine SSCP with heteroduplex analysis to improve the detection of mutations (Kozlowski et al. (2005) Electrophoresis 26, 71-81; Kozlowski etal. (2001) Nucleic Acids Res 29, E71).
  • Samples for analysis can be obtained from any suitable source. Suitable subjects from which cells, tissues or fluids can be isolated include eukaryotes, such as plants or invertebrate or vertebrate animals, e.g. mammals (including pets, farm animals, research animals, and primates, including humans).
  • the samples maybe, e.g., tumor samples, biopsy samples or other tissues.
  • the samples may be secondary fluids, e.g. fluids which may contain DNA products of cancer cells, ⁇ e.g., fluids into which DNA from cancer cells has leaked).
  • the analysis of such secondary fluids is advantageous because it offers a non-invasive sampling method;,and is especially useful for the detection of a cancer in an inaccessible tissue.
  • Secondary fluids include, e.
  • urine/plasma blood or blood fractions
  • urine, seminal fluid can be tested for the presence of mutations associated with cancers of the bladder, colorectum, and lung, respectively.
  • Mutant sequences from the DNA of neoplastic cells have been found in the blood of cancer patients, so the detection of residual disease in lymph nodes or surgical margins may also be useful in predicting which patients might benefit most from further therapy.
  • pancreatic duct juice (sometimes referred to herein as “pancreatic juice” or “juice") obtained during endoscopy, fine needle aspirates of tumor masses, brushings of the pancreatic duct, bile duct or aspirates of cyst fluid.
  • samples can be taken from small primary tumors, allowing a diagnosis at a stage when the primary tumors are still curable and the patients asymptomatic.
  • Samples for analysis can also be obtained from cultured cells (e.g., primary cells or cell lines of interest).
  • the sample is a cell-free lysate.
  • a method of the invention can be used to find a tumor mutation in a population of cells which is not purely tumor cells.
  • RNA can be converted to cDNA and amplified, using conventional procedures, such as RT-PCR (reverse transcriptase PCR), and the resulting DNA assayed as described herein.
  • the method can detect mutations is RNAs such as, e.g., mRNA (transcribed mutations in coding sequences, splice variants, etc.), tRNA, rRNA, microRNAs, etc.
  • Methods of RT-PCR are conventional and well- known in the art.
  • a variety of types of mutations can be identified by a method of the invention. Although much of the discussion herein is directed to "mutations," it is to be understood that any type of variant DNA, including naturally occurring variants, e.g. allelic differences, SNPs, etc. can be detected by a method of the invention.
  • a DNA mutation may differ from the wild type by a single base (point mutations, including transversions, transitions, base substitutions, etc.), two or more non-contiguous bases (including mutations that result in frame shifts), small or large deletions or insertions (e.g. deletions or insertions of between about 1-50, 1-25,1-10 bp, etc.), inversions, truncations, combinations thereof, etc.
  • Chromosomal translocations e.g., which are characteristic of leukemias or lymphomas
  • gene amplifications can also be detected.
  • the mutations can be in any type of nucleic acid, including, e.g., genomic cellular DNA, mitochondrial DNA (mtDNA), messenger RNA (mRNA), viral DNA or RNA genomes, etc.
  • a method of the invention may be used for a variety of applications.
  • One aspect of the invention is a method for testing a subject for (diagnosing) pancreatic cancer, comprising screening a sample from the subject for mutations ⁇ np53 and/or pi 6 genes, using a method of the invention.
  • the baseline value may be, e.g, a reference standard, or the number of such mutations in a subject known not to have pancreatic cancer (such as a "normal" control or a subject having chronic pancreatitis).
  • an empirical cut-off value may be used, as described, e.g., in Example rv.
  • Such a method can be used in conjunction with other methods for diagnosing pancreatic cancer. For example, since not every pancreatic neoplasm has mutations in p53 or pi 6, the detection of these mutations can be combined with quantification of mutant K-r ⁇ s, aberrantly methylated DNA, telomerase activity, or the detection of LOH (loss of heterozygosity) in microdissected samples to facilitate pancreatic cancer diagnosis. Such other methods can be carried out before atest of the invention, as part of a preliminary screen.
  • Another aspect of the invention is a method for diagnosing cancers other than pancreatic cancer for which somatic mutations are diagnostic.
  • a method of the invention to detect pi 6 and p53 mutations in lung or biliary tract cancers, or mutations in other genes, particularly when mutations are not limited to a few single nucleotide hotspots, such as mutations of the EGFR gene found in non-small cell lung carcinomas, or mutations in the cluster region pfAPC (codons 1254-1631) found in colorectal cancer.
  • Another aspect of the invention is a method to identify previously unknown somatic mutations that are associated with a cancer of interest.
  • DNA-containing samples from the cancer can be analyzed by a method of the invention, and amplicons in which heteroduplexes are observed can be further sequenced to characterize the mutation. Once such mutations are identified, and shown to be correlated with the presence of the cancer or, in some cases, to be causal, the newly identified mutations can serve as the basis for diagnosis of the cancer.
  • Another aspect of the invention is a method to determine the future risk of cancer in a patient having one of certain chronic inflammatory diseases (e.g., ulcerative colitis, primary sclerosing cholangitis or chronic pancreatitis) that are associated with an increased risk of cancer, typically 10- 20 or more years after the onset of the chronic inflammatory disease.
  • chronic inflammatory conditions are characterized by the accumulation of somatic mutations (e.g., in exons 1 and 2 of pi 6 in chronic pancreatitis and primary sclerosing cholangitis, and in the mutation cluster region of APC (codons 1254—1631) in colon neoplasms, polyps and colorectal cancer).
  • one aspect of the invention is to periodically (e.g., about once every year or two years) screen subjects having one of these conditions, starting, e.g., about 10 years after the incidence of the inflammatory condition, in order to detect cancer or advanced precancerous lesions (e.g., to determine if a subject is likely to develop cancer).
  • Suitable secondary fluids for testing will be evident to a skilled worker and include, e.g., plasma, colonic washings (ulcerative colitis), bile (primary sclerosing cholangitis) or pancreatic fluids (chronic pancreatitis). Markers for neoplasia, such as, e.g.,p53 mutations, can be detected even if present at low concentrations (such as about 1%). Preferable genes for use in this method are those in which mutations are concentrated in particular regions of the gene.
  • Another aspect of the invention is a method to screen a sample from the circulation of a subject (e.g. a non-symptomatic subject) for the presence of cancer in the subject, using a method of the invention.
  • a subject e.g. a non-symptomatic subject
  • the detection of, e.g., p53, pl6 and/or APC mutations in the circulation would indicate a high probability of cancer and necessitate a search for cancer.
  • Another aspect of the invention is a method for predicting the response of a subject to a chemotherapy procedure, comprising testing a secondary fluid (e.g. from the circulation) from the subject for the presence of somatic mutations by a method of the invention.
  • somatic mutations of the EGFR gene in lung cancers predict response to EGFR inhibitors. It is often not possible to access cancer tissue without thoracotomy if the lung cancer is beyond the reach of a needle biopsy. Detecting mutations in the plasma or serum or bronchoalveolar lavage require a strategy that can detect such mutations when they are at low concentrations.
  • Many other targets of novel therapies are being identified, such as c-kit and other tyrosine kinases, and could be detected in a similar fashion.
  • Methods of the invention can be readily adapted to a high throughput format, using automated (e.g. robotic) systems, which allow many measurements to be carried out simultaneously. Furthermore, the methods can be miniaturized (e.g. , carried out in reaction buffers of about 25 ⁇ l, 1 ⁇ l, 0.1 ⁇ l, or less).
  • any combination of the materials useful in the disclosed methods can be packaged together as a kit for performing any of the disclosed methods.
  • reagents for performing PCR and for heteroduplexing could be packaged along with suitable PCR primers.
  • Components for performing cycle sequencing may also be included.
  • the reagents are packaged in single use form, suitable for carrying one set of analyses.
  • Kits may supply reagents in pre-measured amounts so as to simplify the performance of the subject methods.
  • kits of the invention comprise instructions for performing the method.
  • Other optional elements of a kit of the invention include suitable buffers, packaging materials, etc.
  • the kits of the invention may further comprise additional reagents that are necessary for performing the subject methods.
  • the reagents of the kit may be in containers in which they are stable, e.g., in lyophilized form or as stabilized liquids.
  • Lymphocytes were obtained from 7 healthy controls.
  • the pancreatic juice and pancreatic •-cancer tissues of 20 patients with Stage 1 or 2 disease undergoing Whipple resection between 2000 and 2003 were included.
  • Pancreatic duct juice was also obtained during endoscopy from 8 patients with pancreatitis and 8 patients with a normal pancreas by clinical evaluation undergoing ERCP (endoscopic retrograde cholangiopancreatography) as part of their diagnostic workup. These patients were studied as part of a protocol approved by the Johns Hopkins Joint Committee for Clinical Investigation.
  • the pancreatic cancers of 7 patients were grown in athymic nude mice to enable human pancreatic cancer cells to grow along with mouse stroma.
  • pancreatic cancer DNA Genetic characterization of the pancreatic cancer DNA (such as identifying homozygous deletions) is facilitated because the pancreatic cancer is not admixed with human stroma. Mutation analysis of the remaining 13 patients was performed in microdissected fresh frozen pancreatic cancer tissues. Pancreatic juice was collected from these 20 individuals during their pancreatic surgery by aspirating the pancreatic juice present just after the pancreatic duct is cut.
  • B. Cell Lines The cancer cell lines used in this study include 9 pancreatic cell lines (AsPcI, BxPc3,
  • DNA was extracted from tissue samples using the QIAGEN DNeasy Tissue Kit (QIAGEN Inc, Valencia, CA) C.
  • 10 ⁇ l PCRs were performed in 96-well PCR plates (2OmM Tris-HCl (pH 8.4), 3.OmM MgCl 2 , 0.2mM of each dNTPs, 0.5 ⁇ M of each primer F (forward) and R (reverse), 0.2 units/ ⁇ l Platinum Tag polymerase, Invitrogen, Carisbad, CA). 10% DMSO (vol/vol) was added to amplify pl6. Input DNA is quantified using a real-time Q-PCR assay (Quantifier, Applied Biosystems, Foster City, CA).
  • PCR was carried out in a ThermoHybaid Thermal cycler (Middlesex, U.K.) at 94°C for 2 min; 60 cycles of 94°C, 30s; 60 0 C (pl6 exon I) 5 59°C (pl ⁇ exon 2 and p53 exon 5-6), or 56°C (p53 exon 7-8), 45s; 72°C for 1 min; and 72°C for 10 minutes.
  • PCR products were visualized on 2% agarose gels prior to heteroduplex analysis.
  • C. Heteroduplex Analysis PCR products were diluted in a 1 : 1 ratio with IX AmpliTaq PCR buffer containing 1.5mM
  • heteroduplexes MgCl 2 ) in 96-well PCR plates and overlaid with mineral oil.
  • samples were thermocycled: typically 95°C for 3 min, 95°C-80°C for 3°C/min, 80°C-50°C for l°C/min, 50 0 C for 20min, 50°C-45°C for l°C/min, 45°C-25°C for 2°C/min, 4°C hold.
  • Samples were then subjected to temperature gradient capillary electrophoresis (TGCE) using a SCE9610 automated sequencer (SpectruMedix Corporation, State College, PA). Heteroduplex analysis of PCR products were performed twice, subjecting PCR products to two different temperature protocols during capillary electrophoresis.
  • TGCE temperature gradient capillary electrophoresis
  • the temperature protocol used varied with the size of the PCR product (e.g.400bp PCR products underwent a ramping between 50-60 0 C over 30 minutes).
  • a typical analysis of 96 PCR products screened for heteroduplexes, only 2 PCRs had discordant results between the first and second temperature protocol runs.
  • Heteroduplexes were identified using Revelation Mutational Discovery Software, version 2.4 (SpectruMedix Corporation, State College. PA). The software is programmed to score each chromatogram and to automatically call abnormal chromatograms suggestive of heteroduplexes that are above a set mutation score of 200.
  • PCR products were purified using QIAquick PCR Purification Kit (QIAGEN Inc, Valencia, CA). Sequencing was performed using BigDye terminator mix vl .1 (Applied Biosystems, Foster City, CA). Sequence analysis was performed with ABI PRISM 377XL DNA sequencer (The Perkin- Elmer Corporation, Wellesley, MA).
  • Example II Sensitivity and Specificity of Heteroduplex assay by limiting dilution PCR
  • DNAs from pancreatic and breast cancer cell lines were analyzed including 4 base substitutions and 2 insertion/deletions. Characteristics of all these sequence variants are shown in Table 2. Table 2. Characteristics of sequence variants of cancer cell lines used in the study
  • the number of DNA molecules per PCR and the ratio of mutant and wild type DNA was adjusted from 1 : 1 to 1 :20 and screened for heteroduplex changes using TGCE.
  • TGCE could detect heteroduplexes as long as the mutant to wild-type ratio of input DNA was 1 :6 ratio or more (1:4 etc), but at higher ratios such as 1:8 or 1:10 some mutations were not detected, and no mutations were detected at lower ratios using our protocol (1 :20).
  • PCRs in which 1 of 7 DNA molecules (21 pg of DNA) would reliably identify mutations we examined 8 additional DNA samples from breast cancer cell lines with known mutation in p53. Mutant and wild type DNA was mixed at a ratio of 1:6.
  • Heteroduplex changes were found in all PCR products from these mutant/wild type DNA mixtures. We therefore chose to amplify DNA samples and screen the PCR products for heteroduplexes by including about 7 DNA molecules for each PCR. This way PCRs that amplified a mutant DNA molecule would have a ratio of mutant to wild-type DNA of about 1 :6, a concentration sufficient to allow the mutation to be detected by TGCE.
  • heteroduplex changes identified in limiting dilution PCR products by TGCE.
  • seven lymphocyte DNA samples from healthy controls were analyzed.
  • multiple PCRs were performed with 12-24 pg of input DNA (equivalent to 4-8 DNA molecules per PCR) and screened for heteroduplexes.
  • Three heteroduplex changes (one inpl ⁇ exon 1 and two inp53 exon 5-6) were found in 967 PCRs analyzed yielding an assay specificity of 99.7%.
  • the 3 PCRs with false positive heteroduplexes likely arose either from sequence alterations generated during PCR 3 from incorrect heteroduplex formation, or calls during the TGCE step.
  • DNA samples generating PCRs with heter ⁇ duplexes above healthy control levels are likely to contain sequence alterations.
  • Example IV Heteroduplex assay by LD-PCR on DNA from pancreatic juice
  • pancreatic juice samples were subjected to 45 PCRs with 7 DNA molecules per PCR (21 pg of DNA per PCR) for a total of ⁇ 315 DNA molecules screened.
  • Three additional control wells were analyzed including a positive • control (a DNA sample with a 1 :6 mixture of mutant cancer cell line DNA and wild type DNA) 3 one PCR of wild type DNA 5 and a negative control PCR (water instead of DNA).
  • pancreatic juice samples from patients with pancreatic cancer had increased amounts of mutant j ⁇ i ⁇ 5 DNA and 6 of 20 (30%) had increased mutant p53. In contrast only 1 of 8 (12%) juice samples from patients with chronic pancreatitis had increased mutant p53 and none had increased mutant pi 6.
  • pancreatic cancer DNA from 20 patients that underwent pancreaticoduodenectomy for mutations in p53 and pi 6. Alterations of pi 6 were found in 55% (11/20) of the pancreatic cancers including five point mutations, 5 homozygous deletions (confirmed by multiplex PCR using larger primer sets), and one 12-base pair deletion (Table 4).
  • pancreatic cancer xenografts Four of the five homozygous deletions were in pancreatic cancer xenografts. p53 mutations were detected in 80% (16/20) of the pancreatic cancers.
  • pancreatic juice DNA samples that were not detected in the corresponding pancreatic cancer.
  • the LD-PCR assay also detected mutations arising elsewhere in the pancreas (such as from PanlN) or from other regions of the cancer reflecting intra-tumoral heterogeneity. It is also possible that despite careful microdissection and molecular analysis, one or more of pancreatic cancer mutations were not detected. Previous studies comparing pancreatic cancer and matching pancreatic juice samples have found that mutations in pancreatic juice that are not found in the patient' s primary pancreatic cancer.
  • Example VI Discussion In this study, we demonstrated that limiting dilution PCR followed by TGCE is more sensitive at detecting low-abundance mutations in pancreatic juice samples than conventional TGCE.
  • PanINs are small neoplasms, 5mm or less, and are thought to be the commonest precursor to invasive pancreatic ductal adenocarcinoma. PanINs have been shown to harbor mutations in p53 and pi 6. PanINs are too small to be detected directly by imaging. Using molecular assays to predict whether or not a pancreas harbors advanced PanIN can help predict future pancreatic cancer risk among high-risk individuals undergoing pancreas screening. Since not all pancreatic cancers harbor mutations in p53 or pi 6, it is not surprising that our assay did not identify evidence of mutations in the pancreatic juice of all patients with pancreatic (cancer.
  • pancreatic cancer such as quantifying levels of aberrantly methylated DNA or K-r ⁇ s mutations in pancreatic juice.
  • the specificity of these markers for cancer increases by quantifying their levels in pancreatic juice, as it is uncommon for individuals without cancer to have mutant K-r ⁇ s or aberrantly methylated DNA above a certain threshold concentration (-1%).
  • DNA molecules are PCR amplified at about 10 picogram concentrations (about 3 molecules of DNA, measured using real time PCR). By adding 10 picograms of template DNA per
  • PCR most PCR aliquots will contain 2-4 DNA molecules. When one of the molecules in a PCR contains mutant DNA, the ratio of mutant to wild-type molecules (1 :1 to 1 :3) will enable the mutation to be detected by cycle sequencing of the resulting PCR product. In a typical scenario, 93 PCRs are performed where each aliquot contains about 3 molecules per well, and about 279 DNA molecules are screened for mutations. Therefore, if one analyzes a clinical sample such as pancreatic juice for mutations ⁇ np53 (exons 5-8) sax ⁇ pl ⁇ (exons 1 and 2), one uses 4 sets of PCR primers (p53 exon 5-6, p53 exon 7-8, pl6 exon 1, pl ⁇ exon 2).
  • One 96 well plate is used for each primer, 93 wells containing about lOpg of pancreatic juice DNA, 1 well containing a water lane, one well containing a wild type DNA control and one well containing a mutant DNA control.
  • 93 wells containing about lOpg of pancreatic juice DNA
  • 1 well containing a water lane one well containing a wild type DNA control
  • one well containing a mutant DNA control By sampling about 279 DNA molecules, there is about a 95% probability of sampling at least one mutant DNA molecule when the mutant to wild-type ratio of DNA is 1/100.
  • Each PCR is sequenced and the ability to detect the mutation by sequencing approaches the accuracy of sequencing to detect such a mutation. There is a small chance that a mutation could by chance be mixed in an aliquot that contains 5 or more templates and the other 4 templates are wild type. If so, the cycle sequencing reaction may not be able to detect the mutation.
  • this ratio of wild-type to mutant molecules is still generally detectable by heteroduplex analysis.
  • the number of wells containing 5 or more templates will depend on the accuracy of pipetting. Also, some wells will not contain any DNA but the average number of DNA templates screened in a 96 well plate is still the same, hi many clinical samples the concentration of mutant molecules is in the 2-10% range, and so this limiting dilution strategy for sequencing is useful.
  • the availability of low-cost high-throughput sequencing makes this assay approach feasible to use in the clinical setting. Direct comparison of limiting dilution sequencing and limiting dilution heteroduplex detection can be used to determine which method has better sensitivity and specificity for mutation detection. Since this assay strategy is designed to detect mutations at low concentrations, the false positive rate could be reduced by combining sequencing and heteroduplex analysis to each sample.

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Abstract

La présente invention concerne, par exemple, un procédé servant à rechercher par criblage des mutations dans un échantillon d'ADN, ledit procédé consistant à (a) diluer et diviser l'échantillon pour obtenir une pluralité de petites quantités d'échantillon qui, en moyenne, contiennent entre environ 2 et 10 équivalents génome de l'ADN ; (b) amplifier par ACP l'ADN dans un nombre suffisant de petites quantités d'échantillon, avec au moins un ensemble d'amorces pour ACP, de façon à générer des amplicons tels que, si des mutations sont présentes à une fréquence d'environ 1-10 % dans un gène donné présent dans l'échantillon, la probabilité qu'il y ait une mutation dans ce gène dans au moins l'un des amplicons soit d'au moins 95 % ; et (c) cribler les amplicons en ce qui concerne la présence de mutations, en utilisant un procédé qui peut détecter des mutations non spécifiées au niveau de sites non spécifiés à l'intérieur d'un amplicon (par exemple par électrophorèse capillaire à gradient de température (TGCE) ou séquençage cyclique de l'ADN).
PCT/US2007/015631 2006-07-07 2007-07-09 Stratégie servant à détecter des mutations de faible abondance WO2008005559A2 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109517899A (zh) * 2018-12-05 2019-03-26 山东普瑞斯分子诊断技术有限责任公司 用于检测egfr基因突变的试剂盒及检测方法
CN113628683A (zh) * 2021-08-24 2021-11-09 慧算医疗科技（上海）有限公司 一种高通量测序突变检测方法、设备、装置及可读存储介质

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Title
DANI ET AL GENETICS AND MOLECULAR RESEARCH vol. 3, no. 3, September 2004, pages 395 - 409 *
MARGRAF ET AL JOURNAL OF CLINICAL MICROBIOLOGY vol. 42, no. 10, October 2004, pages 4545 - 4551 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109517899A (zh) * 2018-12-05 2019-03-26 山东普瑞斯分子诊断技术有限责任公司 用于检测egfr基因突变的试剂盒及检测方法
CN109517899B (zh) * 2018-12-05 2021-07-20 山东普瑞斯分子诊断技术有限责任公司 用于检测egfr基因突变的试剂盒及检测方法
CN113628683A (zh) * 2021-08-24 2021-11-09 慧算医疗科技（上海）有限公司 一种高通量测序突变检测方法、设备、装置及可读存储介质
CN113628683B (zh) * 2021-08-24 2024-04-09 慧算医疗科技（上海）有限公司 一种高通量测序突变检测方法、设备、装置及可读存储介质

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