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      Whole exome sequencing of circulating tumor cells provides a window into metastatic prostate cancer

      research-article
      Author(s): 1 , 2 , 3 , 1 , 4 , 1 , 1 , 2 , 3 , 1 , 1 , 1 , 2 , 1 , 2 , 5 , 1 , 1 , 1 , 4 , 4 ,   1 , 1 , 1 , 6 , 1 , 1 , 1 , 2 , 3 , 2 , 3 , 2 , 2 , 2 , 2 , 7 , 2 , 3 , 2 , 3 , 6 , 1 , 2 , 3 , 6 , 1 , 8 , 9 , 1 , 2 , 3 , 1 , 2 , 3 , 6 , 2 , 3 , 1 , 2 , 3 , 9 , 1 , 7 , ** , 1 , ** , 1 , 4 , 10 , **
      Publication date (Electronic):
      Journal: Nature biotechnology

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          Abstract

          Comprehensive analyses of cancer genomes promise to inform prognoses and precise cancer treatments. A major barrier, however, is inaccessibility of metastatic tissue. A potential solution is to characterize circulating tumor cells (CTCs), but this requires overcoming the challenges of isolating rare cells and sequencing low-input material. Here we report an integrated process to isolate, qualify and sequence whole exomes of CTCs with high fidelity, using a census-based sequencing strategy. Power calculations suggest that mapping of >99.995% of the standard exome is possible in CTCs. We validated our process in two prostate cancer patients including one for whom we sequenced CTCs, a lymph node metastasis and nine cores of the primary tumor. Fifty-one of 73 CTC mutations (70%) were observed in matched tissue. Moreover, we identified 10 early-trunk and 56 metastatic-trunk mutations in the non-CTC tumor samples and found 90% and 73% of these, respectively, in CTC exomes. This study establishes a foundation for CTC genomics in the clinic.

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          Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases.

          W Jeffrey Allard, Jeri Matera, M. Craig Miller … (2004)
          The purpose of this study was to determine the accuracy, precision, and linearity of the CellSearch system and evaluate the number of circulating tumor cells (CTCs) per 7.5 mL of blood in healthy subjects, patients with nonmalignant diseases, and patients with a variety of metastatic carcinomas. The CellSearch system was used to enumerate CTCs in 7.5 mL of blood. Blood samples spiked with cells from tumor cell lines were used to establish analytical accuracy, reproducibility, and linearity. Prevalence of CTCs was determined in blood from 199 patients with nonmalignant diseases, 964 patients with metastatic carcinomas, and 145 healthy donors. Enumeration of spiked tumor cells was linear over the range of 5 to 1,142 cells, with an average recovery of >/=85% at each spike level. Only 1 of the 344 (0.3%) healthy and nonmalignant disease subjects had >/=2 CTCs per 7.5 mL of blood. In 2,183 blood samples from 964 metastatic carcinoma patients, CTCs ranged from 0 to 23,618 CTCs per 7.5 mL (mean, 60 +/- 693 CTCs per 7.5 mL), and 36% (781 of 2,183) of the specimens had >/=2 CTCs. Detection of >/=2 CTCs occurred at the following rates: 57% (107 of 188) of prostate cancers, 37% (489 of 1,316) of breast cancers, 37% (20 of 53) of ovarian cancers, 30% (99 of 333) of colorectal cancers, 20% (34 of 168) of lung cancers, and 26% (32 of 125) of other cancers. The CellSearch system can be standardized across multiple laboratories and may be used to determine the clinical utility of CTCs. CTCs are extremely rare in healthy subjects and patients with nonmalignant diseases but present in various metastatic carcinomas with a wide range of frequencies.
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            Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells

            Alex Shalek, Rahul Satija, Xian Adiconis … (2013)
            Recent molecular studies have revealed that, even when derived from a seemingly homogenous population, individual cells can exhibit substantial differences in gene expression, protein levels, and phenotypic output 1–5 , with important functional consequences 4,5 . Existing studies of cellular heterogeneity, however, have typically measured only a few pre-selected RNAs 1,2 or proteins 5,6 simultaneously because genomic profiling methods 3 could not be applied to single cells until very recently 7–10 . Here, we use single-cell RNA-Seq to investigate heterogeneity in the response of bone marrow derived dendritic cells (BMDCs) to lipopolysaccharide (LPS). We find extensive, and previously unobserved, bimodal variation in mRNA abundance and splicing patterns, which we validate by RNA-fluorescence in situ hybridization (RNA-FISH) for select transcripts. In particular, hundreds of key immune genes are bimodally expressed across cells, surprisingly even for genes that are very highly expressed at the population average. Moreover, splicing patterns demonstrate previously unobserved levels of heterogeneity between cells. Some of the observed bimodality can be attributed to closely related, yet distinct, known maturity states of BMDCs; other portions reflect differences in the usage of key regulatory circuits. For example, we identify a module of 137 highly variable, yet co-regulated, antiviral response genes. Using cells from knockout mice, we show that variability in this module may be propagated through an interferon feedback circuit involving the transcriptional regulators Stat2 and Irf7. Our study demonstrates the power and promise of single-cell genomics in uncovering functional diversity between cells and in deciphering cell states and circuits.
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              Detection of mutations in EGFR in circulating lung-cancer cells.

              Shyamala Maheswaran, Lecia V. Sequist, Sunitha Nagrath … (2008)
              The use of tyrosine kinase inhibitors to target the epidermal growth factor receptor gene (EGFR) in patients with non-small-cell lung cancer is effective but limited by the emergence of drug-resistance mutations. Molecular characterization of circulating tumor cells may provide a strategy for noninvasive serial monitoring of tumor genotypes during treatment. We captured highly purified circulating tumor cells from the blood of patients with non-small-cell lung cancer using a microfluidic device containing microposts coated with antibodies against epithelial cells. We performed EGFR mutational analysis on DNA recovered from circulating tumor cells using allele-specific polymerase-chain-reaction amplification and compared the results with those from concurrently isolated free plasma DNA and from the original tumor-biopsy specimens. We isolated circulating tumor cells from 27 patients with metastatic non-small-cell lung cancer (median number, 74 cells per milliliter). We identified the expected EGFR activating mutation in circulating tumor cells from 11 of 12 patients (92%) and in matched free plasma DNA from 4 of 12 patients (33%) (P=0.009). We detected the T790M mutation, which confers drug resistance, in circulating tumor cells collected from patients with EGFR mutations who had received tyrosine kinase inhibitors. When T790M was detectable in pretreatment tumor-biopsy specimens, the presence of the mutation correlated with reduced progression-free survival (7.7 months vs. 16.5 months, P<0.001). Serial analysis of circulating tumor cells showed that a reduction in the number of captured cells was associated with a radiographic tumor response; an increase in the number of cells was associated with tumor progression, with the emergence of additional EGFR mutations in some cases. Molecular analysis of circulating tumor cells from the blood of patients with lung cancer offers the possibility of monitoring changes in epithelial tumor genotypes during the course of treatment. 2008 Massachusetts Medical Society
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                Author and article information

                Journal
                Journal ID (nlm-journal-id): 9604648
                Journal ID (pubmed-jr-id): 20305
                Journal ID (nlm-ta): Nat Biotechnol
                Journal ID (iso-abbrev): Nat. Biotechnol.
                Title: Nature biotechnology
                ISSN (Print): 1087-0156
                ISSN (Electronic): 1546-1696
                Publication date Nihms-submitted: 9 May 2014
                Publication date (Electronic): 20 April 2014
                Publication date (Print): May 2014
                Publication date PMC-release: 01 November 2014
                Volume: 32
                Issue: 5
                Pages: 479-484
                Affiliations
                [1 ]The Eli and Edythe Broad Institute, Cambridge, Massachusetts 02412, USA
                [2 ]Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA
                [3 ]Harvard Medical School, Boston, Massachusetts 02115, USA
                [4 ]Koch Institute for Integrative Cancer Research at MIT, Massachusetts Institute of Technology, 77 Massachusetts Ave., Bldg. 76-231, Cambridge, Massachusetts 02139, USA
                [5 ]Department of Chemistry and Chemical Biology and Department of Physics, Harvard University, Cambridge, Massachusetts 02138, USA
                [6 ]Brigham and Women’s Hospital, Boston, Massachusetts 02115, USA
                [7 ]Massachusetts General Hospital, Boston, Massachusetts 02114, USA
                [8 ]Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
                [9 ]Howard Hughes Medical Institute, Chevy Chase, Maryland 20815, USA
                [10 ]Ragon Institute of MGH, MIT, and Harvard, Cambridge, Massachusetts 02139, USA
                Author notes
                [** ] Corresponding authors. J. Christopher Love, 77 Massachusetts Ave., Bldg. 76-253, Cambridge, MA 02139, Phone: 617-324-2300, clove@ 123456mit.edu , Jesse S. Boehm, 7 Cambridge Center, 4021, Cambridge, MA 02142, Phone: 617-714-7494, boehm@ 123456broadinstitute.org , Gad Getz, 301 Binney Street, Cambridge, MA 02142, Phone: 617-714-7621, Fax: 617-714-8931, gadgetz@ 123456broadinstitute.org
                [11*]

                These authors contributed equally to this work.

                Article
                Manuscript ID: NIHMS581049
                DOI: 10.1038/nbt.2892
                PMC ID: 4034575
                PubMed ID: 24752078
                SO-VID: 680b5f46-4de2-4185-a33f-d63d65587ec3
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                ScienceOpen disciplines: Biotechnology

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