WO2010014920A1 - Circulating mutant dna to assess tumor dynamics - Google Patents

Circulating mutant dna to assess tumor dynamics Download PDF

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Publication number
WO2010014920A1
WO2010014920A1 PCT/US2009/052436 US2009052436W WO2010014920A1 WO 2010014920 A1 WO2010014920 A1 WO 2010014920A1 US 2009052436 W US2009052436 W US 2009052436W WO 2010014920 A1 WO2010014920 A1 WO 2010014920A1
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Prior art keywords
dna
mutation
tumor
gene
patient
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PCT/US2009/052436
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English (en)
French (fr)
Inventor
Frank Diehl
Luis Diaz
Kenneth W. Kinzler
Bert Vogelstein
Kerstin Schmidt
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Johns Hopkins University
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Johns Hopkins University
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Application filed by Johns Hopkins University filed Critical Johns Hopkins University
Priority to CA2732623A priority Critical patent/CA2732623C/en
Priority to JP2011521359A priority patent/JP2011529691A/ja
Priority to EP09803662.7A priority patent/EP2315849B1/en
Publication of WO2010014920A1 publication Critical patent/WO2010014920A1/en
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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
    • C12Q1/6876—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
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/197—Modifications characterised by incorporating a spacer/coupling moiety
    • 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
    • C12Q2535/00—Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides
    • C12Q2535/131—Allele specific probes
    • 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
    • C12Q2549/00—Reactions characterised by the features used to influence the efficiency or specificity
    • C12Q2549/10—Reactions characterised by the features used to influence the efficiency or specificity the purpose being that of reducing false positive or false negative signals
    • C12Q2549/119—Reactions characterised by the features used to influence the efficiency or specificity the purpose being that of reducing false positive or false negative signals using nested primers
    • 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
    • C12Q2600/112—Disease subtyping, staging or classification
    • 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
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
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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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • Figs. 4A-4C show comparison of ctDNA, CEA and imaging dynamics in individual study subjects.
  • the top, middle, and bottom graphs represent ctDNA level, tumor volume as assessed by imaging, and CEA level.
  • the horizontal lines represent the upper bound of the normal levels: one mutant DNA fragment per sample for ctDNA levels, 0.0 cm for tumor diameter, and 5.0 ng ml "1 for CEA abundance.
  • Fig. 4C Patient 8 had a sigmoid adenocarcinoma and solitary metastases in both hepatic lobes.
  • the subject underwent a sigmoidectomy and left lateral hepatic sectorectomy (Surgery 1).
  • Fig. 15 shows total DNA fragments in plasma prior to and after surgery.
  • the Wisker box plot shows the total number of DNA fragments in 2 ml plasma, estimated by real-time PCR at baseline (day 0), post-surgery (day 1), day of discharge (days 2-5), and at the 1 st follow-up (days 13-56).
  • ctDNA levels reflect the total systemic tumor burden, in that ctDNA levels decreased upon complete surgery and generally increased as new lesions became apparent upon radiological examination.
  • Radiographs are inaccurate, because lesions that are observed upon imaging are composed of live neoplastic cells, dead neoplastic cells and varying amounts of nonneoplastic cells (stromal fibroblasts, inflammatory cells, vasculature, and the like) 11 .
  • the proportion of these cell types in any lesion is unknown.
  • micrometastatic lesions that are smaller than a few millimeters, which in aggregate may make a large contribution to the total tumor burden, are not detectable by positron emission tomography, computed tomography or magnetic resonance imaging scans.
  • Plasma samples were drawn in BD Vacutainer tubes with EDTA (Becton Dickinson, Franklin Lakes, NJ USA) from 16 of the 25 patients.
  • Plasma was prepared by centrifugation of blood at 1380 g for 30 min. The supernatant was transferred to a fresh tube and re-centrifuged. After centrifugation, the plasma was transferred to a Millipore Ultrafree-MC 0.45 micron filter device (Millipore, Billerica, MA, USA) to remove remaining cellular debris. The filter device was subjected to centrifugation at 1380 g for 15 min. The cleared plasma was transferred to a new tube and stored at -20° C until processed.
  • Millipore Ultrafree-MC 0.45 micron filter device Millipore Ultrafree-MC 0.45 micron filter device
  • Each reaction consisted of 5* Phusion high fidelity buffer, 1.5 U of Hotstart Phusion polymerase (both NEB), 0.2 ⁇ M of each primer, 0.25 mM of each dNTP, and 0.5 mM MgCl 2 .
  • Nested PCR reactions were performed for selected target regions; for the second amplification, 2 ⁇ l of the first PCR was added to a 20- ⁇ l PCR reaction of the same makeup as described above except that different primers were used.
  • Primer sequences and cycling conditions are listed in Fig. 9.
  • PCR products were pooled, diluted, and quantified using the PicoGreen dsDNA assay (Invitrogen). The BEAMing procedure has been described previously 10 and modifications used in the current study are described here.
  • the first PCR was performed in a 10 ⁇ l reaction volume containing 50-100 genome equivalents (GEs) of template DNA (1 GE equals 3.3 pg of human genomic DNA), 0.5 U of Platinum Taq DNA Polymerase (Invitrogen), Ix PCR buffer (67 mM of Tris-HCl, pH 8.8, 67 mM of MgCl 2 , 16.6 mM of (NH 4 ) 2 SO 4 , and 10 mM of 2-mercaptoethanol), 2 mM ATP, 6% (v/v) DMSO, 1 mM of each dNTP, and 0.2 ⁇ M of each primer.
  • GEs genome equivalents
  • the second (nested) PCR reaction was temperature cycled using the following conditions: 2 min at 94°C; 15 cycles of 94 0 C for 15 s, 58°C for 30 s, 70°C for 15 s.
  • the PCR products were purified using the AMpure system (Agencourt, Beverly, MA) and sequenced from both directions using BigDye Terminator v3.1 (Applied Biosystems).
  • Patient 7 has a prior history of a resected T3N2M1 rectosigmoid adenocarcinoma.
  • the patient underwent surgical excision of two recurrent liver lesions, and an additional 4 liver lesions were treated with radiofrequency ablation (Surgery).
  • Post-operative imaging revealed no evidence of disease, however, imaging three months later revealed new liver disease and new lung metastases.
  • the patient was started on irinotecan, cetuximab, and bevacizumab (Chemotherapy). Despite chemotherapy, on follow-up imaging the patient was noted to have persistent and progressing disease.

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  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
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  • Molecular Biology (AREA)
  • Hospice & Palliative Care (AREA)
  • Biophysics (AREA)
  • Oncology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
PCT/US2009/052436 2008-07-31 2009-07-31 Circulating mutant dna to assess tumor dynamics Ceased WO2010014920A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CA2732623A CA2732623C (en) 2008-07-31 2009-07-31 Circulating mutant dna to assess tumor dynamics
JP2011521359A JP2011529691A (ja) 2008-07-31 2009-07-31 腫瘍動態を評価するための循環変異型ｄｎａ
EP09803662.7A EP2315849B1 (en) 2008-07-31 2009-07-31 Circulating mutant dna to assess tumor dynamics

Applications Claiming Priority (4)

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US8517508P 2008-07-31 2008-07-31
US61/085,175 2008-07-31
US12/512,585 2009-07-30
US12/512,585 US20100041048A1 (en) 2008-07-31 2009-07-30 Circulating Mutant DNA to Assess Tumor Dynamics

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CA (1) CA2732623C (https=)
WO (1) WO2010014920A1 (https=)

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