Research ArticleCancer Genetics

Development of Personalized Tumor Biomarkers Using Massively Parallel Sequencing

See allHide authors and affiliations

Science Translational Medicine  24 Feb 2010:
Vol. 2, Issue 20, pp. 20ra14
DOI: 10.1126/scitranslmed.3000702
  • Fig. 1.

    Schematic of PARE approach. The method is based on next-generation mate-paired analysis of resected tumor DNA to identify individualized tumor-specific rearrangements. Such alterations are used to develop PCR-based quantitative analyses for personalized tumor monitoring of plasma samples or other bodily fluids.

  • Fig. 2.

    Detection of tumor-specific rearrangements in breast and colorectal cancers. Two representative rearrangements are shown for each tumor sample. (A) PCR amplification across breakpoint regions. MW, molecular weight; T, tumor; N, normal. (B) Genomic coordinates for a representative mate pair of each rearrangement.

  • Fig. 3.

    Detection of tumor-specific rearrangements in mixtures of tumor and normal DNA. Decreasing amounts of tumor DNA were mixed with increasing amounts of normal tissue DNA (300 ng total) and were used as template molecules for PCR using chromosome 4:8 translocation-specific primers or chromosome 3 control primers (see Materials and Methods for additional information).

  • Fig. 4.

    Detection of tumor-specific rearrangements in plasma of cancer patients. (A) The identified chromosome 4:8 and 16 rearrangements were used to design PCR primers spanning breakpoints and to amplify rearranged DNA from tumor tissue and plasma from patients Hx402 and Hx403, respectively. A plasma sample from an unrelated healthy individual was used as a control for both rearrangements. (B) Plasma samples from patient Hx402 were analyzed at different time points using digital PCR to determine the fraction of genomic equivalents of plasma DNA containing the chromosome 4:8 rearrangement. The fraction of rearranged DNA at day 137 was 0.3%, consistent with residual metastatic lesions present in the remaining lobe of the liver.

  • Table 1.

    Summary of mate-paired tag libraries.

    SamplesSingle tag analysesMate-paired tag analyses
    Number of
    beads*
    Number
    of tags
    matching human
    genome
    Total
    bases
    sequenced
    (bp)
    Expected
    coverage
    per
    3-kb bin
    Number of
    mate-paired
    tags matching
    human genome
    Distance
    between
    mate-paired
    tags (bp)
    Total
    physical
    coverage by
    mate-paired
    tags (bp)
    Expected
    genome
    coverage
    Colon cancer
    Co108
    tumor
    526,209,780121,527,7073,038,192,67512221,899,809137130,024,693,71410.0
    Co108
    normal
    328,599,03386,032,2532,150,806,3258611,694,361125414,665,530,8044.9
    Co84
    tumor
    677,137,128256,065,4376,401,635,92525658,678,410148887,292,060,00629.1
    Co84
    normal
    486,663,520218,280,1465,457,003,65021859,019,031138481,690,396,37927.2
    Hx402
    tumor
    523,745,015198,342,7494,958,568,72519843,457,431162970,789,547,65323.6
    Hx403
    tumor
    475,658,760164,061,9384,101,548,45016437,123,395170563,295,388,47521.1
    Breast cancer
    B7
    tumor
    840,979,999281,027,2747,025,681,85028127,548,989122033,604,662,40411.2
    B7
    normal
    705,704,265253,482,2626,337,056,55025357,878,644140481,271,654,77027.1
    B5
    tumor
    444,249,217147,612,9413,690,323,52514829,961,045119335,730,144,65111.9
    B5
    normal
    549,237,156220,669,7955,516,744,87522153,611,974120564,591,276,02521.5

    *Number of beads corresponds to the number of magnetic beads containing clonally amplified DNA fragments and represents the maximal number of raw sequence reads for each run.

  • Table 2.

    Summary of rearrangements identified in tumor samples.

    SampleRearrangement typeTotal
    rearrangements
    Tested
    rearrangements
    Confirmed somatic
    rearrangements
    IntrachromosomalInterchromosomal
    Tumor and normal libraries
    B5741175 (71%)
    B7174211615 (94%)
    Co8407764 (67%)
    Co108612181311 (85%)
    Tumor libraries
    Hx40272994 (44%)
    Hx40317017127 (58%)

Additional Files

  • Supplementary Material for:

    Development of Personalized Tumor Biomarkers Using Massively
    Parallel Sequencing

    Rebecca J. Leary, Isaac Kinde, Frank Diehl, Kerstin Schmidt, Chris Clouser, Cisilya Duncan, Alena Antipova, Clarence Lee, Kevin McKernan, Francisco M. De La Vega, Kenneth W. Kinzler, Bert Vogelstein, Luis A. Diaz Jr., Victor E. Velculescu*

    *To whom correspondence should be addressed. E-mail: velculescu{at}jhmi.edu

    Published 24 February 2010, Sci. Transl. Med. 2, 20ra14 (2010)
    DOI: 10.1126/scitranslmed.3000702

    This PDF file includes:

    • Table S1. Comparison of SOLiD sequencing, Illumina SNP arrays, and Digital Karyotyping for analysis of copy number alterations.
    • Table S2. Putative copy number alterations identified by SOLiD sequencing in Co84 that were not identified by Illumina SNP arrays or Digital Karyotyping.
    • Table S3. Confirmed somatic rearrangements in breast and colorectal cancer samples.
    • Fig. S1. Flow chart of approach used to identify rearranged sequences.
    • Fig. S2. Comparison of Digital Karyotyping, Illumina SNP array, and SOLiD sequencing results on chromosome 8.

    [Download PDF]

Navigate This Article