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Microscopic lymph node tumor burden quantified by macroscopic dual-tracer molecular imaging

Nature Medicine volume 20, pages 1348–1353 (2014)Cite this article

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Abstract

Lymph node biopsy is employed in many cancer surgeries to identify metastatic disease and to determine cancer stage, yet morbidity and diagnostic delays associated with lymph node biopsy could be avoided if noninvasive imaging of nodal involvement were reliable. Molecular imaging has potential in this regard; however, variable delivery and nonspecific uptake of imaging tracers have made conventional approaches ineffective clinically. Here we present a method of correcting for nonspecific uptake with injection of a second untargeted tracer that allows for quantification of tumor burden in lymph nodes. We confirmed the approach in an athymic mouse model of metastatic human breast cancer by targeting epidermal growth factor receptor, a cell surface receptor overexpressed by many cancers. We observed a significant correlation between in vivo (dual-tracer) and ex vivo measures of tumor burden (r = 0.97, P < 0.01), with an ultimate sensitivity of approximately 200 cells (potentially more sensitive than conventional lymph node biopsy).

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Figure 1: Animal model.
Figure 2: LN-MCI.
Figure 3: Dual-tracer compared to single tracer imaging.
Figure 4: Estimation of tumor burden.
Figure 5: Modeling and simulations.

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References

  1. Chen, S.L., Iddings, D.M., Scheri, R.P. & Bilchik, A.J. Lymphatic mapping and sentinel node analysis: current concepts and applications. CA Cancer J. Clin. 56, 292–309; quiz 316–297 (2006).

    PubMed  Google Scholar 

  2. Tobler, N.E. & Detmar, M. Tumor and lymph node lymphangiogenesis—impact on cancer metastasis. J. Leukoc. Biol. 80, 691–696 (2006).

    CAS  PubMed  Google Scholar 

  3. Schrenk, P., Rieger, R., Shamiyeh, A. & Wayand, W. Morbidity following sentinel lymph node biopsy versus axillary lymph node dissection for patients with breast carcinoma. Cancer 88, 608–614 (2000).

    CAS  PubMed  Google Scholar 

  4. Sampath, L., Wang, W. & Sevick-Muraca, E.M. Near infrared fluorescent optical imaging for nodal staging. J. Biomed. Opt. 13, 041312 (2008).

    PubMed  PubMed Central  Google Scholar 

  5. Reilly, R.M. et al. Problems of delivery of monoclonal antibodies. Pharmaceutical and pharmacokinetic solutions. Clin. Pharmacokinet. 28, 126–142 (1995).

    CAS  PubMed  Google Scholar 

  6. Liu, J.T. et al. Quantifying cell-surface biomarker expression in thick tissues with ratiometric three-dimensional microscopy. Biophys. J. 96, 2405–2414 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Pogue, B.W. et al. Imaging targeted-agent binding in vivo with two probes. J. Biomed. Opt. 15, 030513 (2010).

    PubMed  PubMed Central  Google Scholar 

  8. Baeten, J., Haller, J., Shih, H. & Ntziachristos, V. In vivo investigation of breast cancer progression by use of an internal control. Neoplasia 11, 220–227 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Tichauer, K.M. et al. In vivo quantification of tumor receptor binding potential with dual-reporter molecular imaging. Mol. Imaging Biol. 14, 584–592 (2012).

    PubMed  PubMed Central  Google Scholar 

  10. Davis, S.C. et al. Dynamic dual-tracer MRI-guided fluorescence tomography to quantify receptor density in vivo. Proc. Natl. Acad. Sci. USA 110, 9025–9030 (2013).

    CAS  PubMed  Google Scholar 

  11. Saltz, L.B. et al. Phase II trial of cetuximab in patients with refractory colorectal cancer that expresses the epidermal growth factor receptor. J. Clin. Oncol. 22, 1201–1208 (2004).

    CAS  PubMed  Google Scholar 

  12. Nicholson, R.I., Gee, J.M. & Harper, M.E. EGFR and cancer prognosis. Eur. J. Cancer 37 (suppl. 4), S9–S15 (2001).

    CAS  PubMed  Google Scholar 

  13. van Agthoven, T., Timmermans, M., Dorssers, L.C. & Henzen-Logmans, S.C. Expression of estrogen, progesterone and epidermal growth factor receptors in primary and metastatic breast cancer. Int. J. Cancer 63, 790–793 (1995).

    CAS  PubMed  Google Scholar 

  14. Wei, Q. et al. EGFR, HER2, and HER3 expression in laryngeal primary tumors and corresponding metastases. Ann. Surg. Oncol. 15, 1193–1201 (2008).

    PubMed  Google Scholar 

  15. Wei, Q. et al. EGFR, HER2 and HER3 expression in esophageal primary tumours and corresponding metastases. Int. J. Oncol. 31, 493–499 (2007).

    PubMed  Google Scholar 

  16. Shen, L. et al. EGFR and HER2 expression in primary cervical cancers and corresponding lymph node metastases: implications for targeted radiotherapy. BMC Cancer 8, 232 (2008).

    PubMed  PubMed Central  Google Scholar 

  17. Carlsson, J., Shen, L., Xiang, J., Xu, J. & Wei, Q. Tendencies for higher co-expression of EGFR and HER2 and downregulation of HER3 in prostate cancer lymph node metastases compared with corresponding primary tumors. Oncology Lett. 5, 208–214 (2013).

    CAS  Google Scholar 

  18. Bue, P. et al. Expression of epidermal growth factor receptor in urinary bladder cancer metastases. Int. J. Cancer 76, 189–193 (1998).

    CAS  PubMed  Google Scholar 

  19. Italiano, A. et al. Epidermal growth factor receptor (EGFR) status in primary colorectal tumors correlates with EGFR expression in related metastatic sites: biological and clinical implications. Ann. Oncol. 16, 1503–1507 (2005).

    CAS  PubMed  Google Scholar 

  20. Real, F.X. et al. Expression of epidermal growth factor receptor in human cultured cells and tissues: relationship to cell lineage and stage of differentiation. Cancer Res. 46, 4726–4731 (1986).

    CAS  PubMed  Google Scholar 

  21. Kern, K.A. Sentinel lymph node mapping in breast cancer using subareolar injection of blue dye. J. Am. Coll. Surg. 189, 539–545 (1999).

    CAS  PubMed  Google Scholar 

  22. Brader, P. et al. Imaging of lymph node micrometastases using an oncolytic herpes virus and [F]FEAU PET. PLoS ONE 4, e4789 (2009).

    PubMed  PubMed Central  Google Scholar 

  23. Tafreshi, N.K. et al. Noninvasive detection of breast cancer lymph node metastasis using carbonic anhydrases IX and XII targeted imaging probes. Clin. Cancer Res. 18, 207–219 (2012).

    CAS  PubMed  Google Scholar 

  24. Tafreshi, N.K. et al. A mammaglobin-A targeting agent for noninvasive detection of breast cancer metastasis in lymph nodes. Cancer Res. 71, 1050–1059 (2011).

    CAS  PubMed  Google Scholar 

  25. Savariar, E.N. et al. Real-time in vivo molecular detection of primary tumors and metastases with ratiometric activatable cell-penetrating peptides. Cancer Res. 73, 855–864 (2013).

    CAS  PubMed  Google Scholar 

  26. Weaver, D.L. Pathology evaluation of sentinel lymph nodes in breast cancer: protocol recommendations and rationale. Mod. Pathol. 23 (suppl. 2), S26–S32 (2010).

    PubMed  Google Scholar 

  27. Sexton, K. et al. Pulsed-light imaging for fluorescence guided surgery under normal room lighting. Opt. Lett. 38, 3249–3252 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Porter, C.J. & Charman, S.A. Lymphatic transport of proteins after subcutaneous administration. J. Pharm. Sci. 89, 297–310 (2000).

    CAS  PubMed  Google Scholar 

  29. Gerlinger, M. et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N. Engl. J. Med. 366, 883–892 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Hettiarachchi, K., Kim, H. & Faris, G.W. Optical manipulation and control of real-time PCR in cell encapsulating microdroplets by IR laser. Microfluid. Nanofluid. 13, 967–975 (2012).

    CAS  Google Scholar 

  31. Liebert, A. et al. Time-resolved multidistance near-infrared spectroscopy of the adult head: intracerebral and extracerebral absorption changes from moments of distribution of times of flight of photons. Appl. Opt. 43, 3037–3047 (2004).

    PubMed  Google Scholar 

  32. Zhu, Q. et al. Benign versus malignant breast masses: optical differentiation with US-guided optical imaging reconstruction. Radiology 237, 57–66 (2005).

    PubMed  PubMed Central  Google Scholar 

  33. Flynn, B.P., Dsouza, A.V., Kanick, S.C., Davis, S.C. & Pogue, B.W. White light-informed optical properties improve ultrasound-guided fluorescence tomography of photoactive protoporphyrin IX. J. Biomed. Opt. 18, 046008 (2013).

    PubMed  PubMed Central  Google Scholar 

  34. Koral, K.F. et al. SPECT dual-energy-window Compton correction: scatter multiplier required for quantification. J. Nucl. Med. 31, 90–98 (1990).

    CAS  PubMed  Google Scholar 

  35. Jenkins, D.E., Hornig, Y.S., Oei, Y., Dusich, J. & Purchio, T. Bioluminescent human breast cancer cell lines that permit rapid and sensitive in vivo detection of mammary tumors and multiple metastases in immune deficient mice. Breast Cancer Res. 7, R444–R454 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Contag, C.H., Jenkins, D., Contag, P.R. & Negrin, R.S. Use of reporter genes for optical measurements of neoplastic disease in vivo. Neoplasia 2, 41–52 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Reilly, R.M. et al. A comparison of EGF and MAb 528 labeled with 111In for imaging human breast cancer. J. Nucl. Med. 41, 903–911 (2000).

    CAS  PubMed  Google Scholar 

  38. Peng, X. et al. Phthalocyanine dye as an extremely photostable and highly fluorescent near-infrared labeling agent. Proc. SPIE 6097, 19 (2006).

    Google Scholar 

  39. Wu, F. et al. Fluorescence imaging of the lymph node uptake of proteins in mice after subcutaneous injection: molecular weight dependence. Pharm. Res. 29, 1843–1853 (2012).

    CAS  PubMed  Google Scholar 

  40. Alcoser, S.Y. et al. Real-time PCR-based assay to quantify the relative amount of human and mouse tissue present in tumor xenografts. BMC Biotechnol. 11, 124 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Becker, M. et al. Sensitive PCR method for the detection and real-time quantification of human cells in xenotransplantation systems. Br. J. Cancer 87, 1328–1335 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Hamzei, N. et al. Comparison of kinetic models for dual-tracer receptor concentration imaging in tumors. Austin J. Biomed. Eng. 1, 9 (2014).

    Google Scholar 

  43. Samkoe, K.S. et al. High vascular delivery of EGF, but low receptor binding rate is observed in AsPC-1 tumors as compared to normal pancreas. Mol. Imaging Biol. 14, 472–479 (2012).

    PubMed  PubMed Central  Google Scholar 

  44. Tichauer, K.M. et al. Tumor endothelial marker imaging in melanomas using dual-tracer fluorescence molecular imaging. Mol. Imaging Biol. 16, 372–382 (2014).

    PubMed  Google Scholar 

  45. Tichauer, K.M. et al. Accounting for pharmacokinetic differences in dual-tracer receptor density imaging. Phys. Med. Biol. 59, 2341–2351 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Tichauer, K.M., Samkoe, K.S., Klubben, W.S., Hasan, T. & Pogue, B.W. Advantages of a dual-tracer model over reference tissue models for binding potential measurement in tumors. Phys. Med. Biol. 57, 6647–6659 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Tichauer, K.M. et al. Improved tumor contrast achieved by single time point dual-reporter fluorescence imaging. J. Biomed. Opt. 17, 066001 (2012).

    PubMed  PubMed Central  Google Scholar 

  48. Lammertsma, A.A. & Hume, S.P. Simplified reference tissue model for PET receptor studies. Neuroimage 4, 153–158 (1996).

    CAS  PubMed  Google Scholar 

  49. Innis, R.B. et al. Consensus nomenclature for in vivo imaging of reversibly binding radioligands. J. Cereb. Blood Flow Metab. 27, 1533–1539 (2007).

    CAS  PubMed  Google Scholar 

  50. Jacques, S.L. Optical properties of biological tissues: a review. Phys. Med. Biol. 58, R37–R61 (2013).

    Google Scholar 

  51. Ichise, M. et al. Linearized reference tissue parametric imaging methods: application to [11C]DASB positron emission tomography studies of the serotonin transporter in human brain. J. Cereb. Blood Flow Metab. 23, 1096–1112 (2003).

    PubMed  Google Scholar 

  52. Lammertsma, A.A. et al. Comparison of methods for analysis of clinical [11C]raclopride studies. J. Cereb. Blood Flow Metab. 16, 42–52 (1996).

    CAS  PubMed  Google Scholar 

  53. Kety, S.S. The theory and applications of the exchange of inert gas at the lungs and tissues. Pharmacol. Rev. 3, 1–41 (1951).

    CAS  PubMed  Google Scholar 

  54. Patel, D. et al. Monoclonal antibody cetuximab binds to and down-regulates constitutively activated epidermal growth factor receptor vIII on the cell surface. Anticancer Res. 27, 3355–3366 (2007).

    CAS  PubMed  Google Scholar 

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Acknowledgements

This work was sponsored by US National Institutes of Health research grants R01 CA109558, R01 CA156177, and U54 CA151662 as well as a the Canadian Institutes of Health Research postdoctoral fellowship for K.M.T.

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Authors and Affiliations

  1. Department of Biomedical Engineering, Illinois Institute of Technology, Chicago, Illinois, USA

    Kenneth M Tichauer

  2. Department of Surgery, Geisel School of Medicine, Hanover, New Hampshire, USA

    Kimberley S Samkoe, P Jack Hoopes, Richard J Barth & Brian W Pogue

  3. Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire, USA

    Jason R Gunn, Stephen C Kanick, P Jack Hoopes & Brian W Pogue

  4. Department of Medicine, Geisel School of Medicine, Lebanon, New Hampshire, USA

    Peter A Kaufman

  5. Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, USA

    Tayyaba Hasan & Brian W Pogue

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Contributions

K.M.T. designed the experiments, developed the kinetic modeling methodology, carried out the experiments, analyzed all imaging data and wrote the paper. K.S.S. helped design the experiments and validation procedure and helped write the paper. J.R.G. carried out much of the animal imaging and carried out all qPCR and bioluminescence imaging. S.C.K. carried out photon propagation simulations. P.J.H. analyzed all H&E stains. R.J.B. and P.A.K. provided clinical support for design and direction of the study. T.H. helped supervise the project. B.W.P. provided full support for the project and gave substantial feedback on all aspects of the project.

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Correspondence to Kenneth M Tichauer or Brian W Pogue.

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Tichauer, K., Samkoe, K., Gunn, J. et al. Microscopic lymph node tumor burden quantified by macroscopic dual-tracer molecular imaging. Nat Med 20, 1348–1353 (2014). https://doi.org/10.1038/nm.3732

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