Molecular Diagnostics

Detection and prognostic role of heterogeneous populations of melanoma circulating tumour cells

Abstract

Background

Circulating tumour cells (CTCs) can be assessed through a minimally invasive blood sample with potential utility as a predictive, prognostic and pharmacodynamic biomarker. The large heterogeneity of melanoma CTCs has hindered their detection and clinical application.

Methods

Here we compared two microfluidic devices for the recovery of circulating melanoma cells. The presence of CTCs in 43 blood samples from patients with metastatic melanoma was evaluated using a combination of immunocytochemistry and transcript analyses of five genes by RT-PCR and 19 genes by droplet digital PCR (ddPCR), whereby a CTC score was calculated. Circulating tumour DNA (ctDNA) from the same patient blood sample, was assessed by ddPCR targeting tumour-specific mutations.

Results

Our analysis revealed an extraordinary heterogeneity amongst melanoma CTCs, with multiple non-overlapping subpopulations. CTC detection using our multimarker approach was associated with shorter overall and progression-free survival. Finally, we found that CTC scores correlated with plasma ctDNA concentrations and had similar pharmacodynamic changes upon treatment initiation.

Conclusions

Despite the high phenotypic and molecular heterogeneity of melanoma CTCs, multimarker derived CTC scores could serve as viable tools for prognostication and treatment response monitoring in patients with metastatic melanoma.

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Fig. 1: CTC isolation platforms comparison.
Fig. 2: Phenotypic and morphological heterogeneity of melanoma CTCs after microfluidic enrichment.
Fig. 3: Comparison of gene expression profiles of CTC fractions using the five melanoma-specific RT-PCR and the 19 genes ddPCR.
Fig. 4: Association between CTC detection and clinical outcomes.
Fig. 5: CTC scores and ctDNA comparison.

References

  1. 1.

    Cancer Genome Atlas N. Genomic classification of cutaneous melanoma. Cell 161, 1681–1696 (2015).

  2. 2.

    Tirosh, I., Izar, B., Prakadan, S. M., Wadsworth, M. H. 2nd, Treacy, D., Trombetta, J. J. et al. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science 352, 189–196 (2016).

  3. 3.

    Krepler, C., Sproesser, K., Brafford, P., Beqiri, M., Garman, B., Xiao, M. et al. A comprehensive patient-derived xenograft collection representing the heterogeneity of melanoma. Cell Rep. 21, 1953–1967 (2017).

  4. 4.

    Pogrebniak, K. L. & Curtis, C. Harnessing tumor evolution to circumvent resistance. Trends Genet. 34, 639–651 (2018).

  5. 5.

    Dive, C. & Brady, G. SnapShot: circulating tumor cells. Cell 168, 742–e1 (2017).

  6. 6.

    Marsavela, G., Aya-Bonilla, C. A., Warkiani, M. E., Gray, E. S. & Ziman, M. Melanoma circulating tumor cells: benefits and challenges required for clinical application. Cancer Lett. 424, 1–8 (2018).

  7. 7.

    Freeman, J. B., Gray, E. S., Millward, M., Pearce, R. & Ziman, M. Evaluation of a multi-marker immunomagnetic enrichment assay for the quantification of circulating melanoma cells. J. Transl. Med. 10, 192 (2012).

  8. 8.

    Khoja, L., Shenjere, P., Hodgson, C., Hodgetts, J., Clack, G., Hughes, A. et al. Prevalence and heterogeneity of circulating tumour cells in metastatic cutaneous melanoma. Melanoma Res. 24, 40–46 (2014).

  9. 9.

    Gray, E. S., Reid, A. L., Bowyer, S., Calapre, L., Siew, K., Pearce, R. et al. Circulating melanoma cell subpopulations: their heterogeneity and differential responses to treatment. J. Invest. Dermatol. 135, 2040–2048 (2015).

  10. 10.

    Aya-Bonilla, C. A., Marsavela, G., Freeman, J. B., Lomma, C., Frank, M. H., Khattak, M. A. et al. Isolation and detection of circulating tumour cells from metastatic melanoma patients using a slanted spiral microfluidic device. Oncotarget 8, 67355–67368 (2017).

  11. 11.

    Chudziak, J., Burt, D. J., Mohan, S., Rothwell, D. G., Mesquita, B., Antonello, J. et al. Clinical evaluation of a novel microfluidic device for epitope-independent enrichment of circulating tumour cells in patients with small cell lung cancer. Analyst 141, 669–678 (2016).

  12. 12.

    Ozkumur, E., Shah, A. M., Ciciliano, J. C., Emmink, B. L., Miyamoto, D. T., Brachtel, E. et al. Inertial focusing for tumor antigen-dependent and -independent sorting of rare circulating tumor cells. Sci. Transl. Med. 5, 179ra47 (2013).

  13. 13.

    Hong, X., Sullivan, R. J., Kalinich, M., Kwan, T. T., Giobbie-Hurder, A., Pan, S. et al. Molecular signatures of circulating melanoma cells for monitoring early response to immune checkpoint therapy. Proc. Natl Acad. Sci. USA 115, 2467–2472 (2018).

  14. 14.

    Yanagita, M., Luke, J. J., Hodi, F. S., Janne, P. A. & Paweletz, C. P. Isolation and characterization of circulating melanoma cells by size filtration and fluorescent in-situ hybridization. Melanoma Res. 28, 89–95 (2018).

  15. 15.

    Aya-Bonilla, C., Gray, E. S., Manikandan, J., Freeman, J. B., Zaenker, P., Reid, A. L. et al. Immunomagnetic-enriched subpopulations of melanoma circulating tumour cells (CTCs) exhibit distinct transcriptome profiles. Cancers (Basel). 11, E157 (2019).

  16. 16.

    Reid, A. L., Freeman, J. B., Millward, M., Ziman, M. & Gray, E. S. Detection of BRAF-V600E and V600K in melanoma circulating tumour cells by droplet digital PCR. Clin. Biochem. 48, 999–1002 (2015).

  17. 17.

    Calapre, L., Giardina, T., Robinson, C., Reid, A. L., Al-Ogaili, Z., Pereira, M. R. et al. Locus-specific concordance of genomic alterations between tissue and plasma circulating tumor DNA in metastatic melanoma. Mol. Oncol. 13, 171–184 (2019).

  18. 18.

    McEvoy, A. C., Calapre, L., Pereira, M. R., Giardina, T., Robinson, C., Khattak, M. A. et al. Sensitive droplet digital PCR method for detection of TERT promoter mutations in cell free DNA from patients with metastatic melanoma. Oncotarget 8, 78890–78900 (2017).

  19. 19.

    Gray, E. S., Rizos, H., Reid, A. L., Boyd, S. C., Pereira, M. R., Lo, J. et al. Circulating tumor DNA to monitor treatment response and detect acquired resistance in patients with metastatic melanoma. Oncotarget 6, 42008–42018 (2015).

  20. 20.

    Girotti, M. R., Lopes, F., Preece, N., Niculescu-Duvaz, D., Zambon, A., Davies, L. et al. Paradox-breaking RAF inhibitors that also target SRC are effective in drug-resistant BRAF mutant melanoma. Cancer Cell. 27, 85–96 (2015).

  21. 21.

    Wong, S. Q., Raleigh, J., Callahan, J., Vergara, I. A., Ftoumi, S., Hatzimihalis, A., et al. Circulating tumor DNA analysis and functional imaging provide complementary approaches for comprehensive disease monitoring in metastatic melanoma. J. Clin. Oncol. Precis. Oncolog. 1, 1–14 (2017).

  22. 22.

    Lee, J. H., Long, G. V., Boyd, S., Lo, S., Menzies, A. M., Tembe, V. et al. Circulating tumour DNA predicts response to anti-PD1 antibodies in metastatic melanoma. Ann. Oncol. 28, 1130–1136 (2017).

  23. 23.

    Ramskold, D., Luo, S., Wang, Y. C., Li, R., Deng, Q., Faridani, O. R. et al. Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nat. Biotechnol. 30, 777–782 (2012).

  24. 24.

    Khoja, L., Lorigan, P., Zhou, C., Lancashire, M., Booth, J., Cummings, J. et al. Biomarker utility of circulating tumor cells in metastatic cutaneous melanoma. J. investigative Dermatol. 133, 1582–1590 (2013).

  25. 25.

    Luo, X., Mitra, D., Sullivan, R. J., Wittner, B. S., Kimura, A. M., Pan, S. et al. Isolation and molecular characterization of circulating melanoma cells. Cell Rep. 7, 645–653 (2014).

  26. 26.

    Vetrini, F., Auricchio, A., Du, J., Angeletti, B., Fisher, D. E., Ballabio, A. et al. The microphthalmia transcription factor (Mitf) controls expression of the ocular albinism type 1 gene: link between melanin synthesis and melanosome biogenesis. Mol. Cell Biol. 24, 6550–6559 (2004).

  27. 27.

    Rambow, F., Rogiers, A., Marin-Bejar, O., Aibar, S., Femel, J., Dewaele, M. et al. Toward minimal residual disease-directed therapy in melanoma. Cell 174, 843–855 e19 (2018).

  28. 28.

    Grahovac, J., Becker, D. & Wells, A. Melanoma cell invasiveness is promoted at least in part by the epidermal growth factor-like repeats of tenascin-C. J. investigative Dermatol. 133, 210–220 (2013).

  29. 29.

    Blake, J. A. & Ziman, M. R. Pax3 transcripts in melanoblast development. Dev. Growth Differ. 47, 627–635 (2005).

  30. 30.

    Lang, D., Lu, M. M., Huang, L., Engleka, K. A., Zhang, M. Z., Chu, E. Y. et al. Pax3 functions at a nodal point in melanocyte stem cell differentiation. Nature 433, 884–887 (2005).

  31. 31.

    Medic, S. & Ziman, M. PAX3 expression in normal skin melanocytes and melanocytic lesions (naevi and melanomas). PLoS ONE 5, e9977 (2010).

  32. 32.

    Potterf, S. B., Furumura, M., Dunn, K. J., Arnheiter, H. & Pavan, W. J. Transcription factor hierarchy in Waardenburg syndrome: regulation of MITF expression by SOX10 and PAX3. Hum. Genet. 107, 1–6 (2000).

  33. 33.

    Kupas, V., Weishaupt, C., Siepmann, D., Kaserer, M. L., Eickelmann, M., Metze, D. et al. RANK is expressed in metastatic melanoma and highly upregulated on melanoma-initiating cells. J. Invest. Dermatol. 131, 944–955 (2011).

  34. 34.

    Schatton, T., Murphy, G. F., Frank, N. Y., Yamaura, K., Waaga-Gasser, A. M., Gasser, M. et al. Identification of cells initiating human melanomas. Nature 451, 345–349 (2008).

  35. 35.

    Wilson, B. J., Saab, K. R., Ma, J., Schatton, T., Putz, P., Zhan, Q. et al. ABCB5 maintains melanoma-initiating cells through a proinflammatory cytokine signaling circuit. Cancer Res. 74, 4196–4207 (2014).

  36. 36.

    Klinac, D., Gray, E. S., Freeman, J. B., Reid, A., Bowyer, S., Millward, M. et al. Monitoring changes in circulating tumour cells as a prognostic indicator of overall survival and treatment response in patients with metastatic melanoma. BMC Cancer 14, 423 (2014).

  37. 37.

    Khattak A., Gray E., Reid A., Pereira M., McEvoy A. C., Aya-Bonilla C. A. et al. PD-L1 expression on pre-treatment circulating tumour cells, but not serum VEGF, is predictive of response to pembrolizumab in melanoma. Ann. Oncolog. 2019–0557 (2019).

  38. 38.

    McEvoy, A. C., Warburton, L., Al-Ogaili, Z., Celliers, L., Calapre, L., Pereira, M. R. et al. Correlation between circulating tumour DNA and metabolic tumour burden in metastatic melanoma patients. BMC Cancer 18, 726 (2018).

  39. 39.

    Jerby-Arnon, L., Shah, P., Cuoco, M. S., Rodman, C., Su, M. J., Melms, J. C. et al. A cancer cell program promotes T cell exclusion and resistance to checkpoint blockade. Cell 175, 984–997. e24 (2018).

  40. 40.

    Sade-Feldman, M., Yizhak, K., Bjorgaard, S. L., Ray, J. P., de Boer, C. G., Jenkins, R. W. et al. Defining T cell states associated with response to checkpoint immunotherapy in melanoma. Cell 175, 998–1013. e20 (2018).

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Acknowledgements

We thank patients and healthy volunteers for contributing samples to this study. We also acknowledge the clinical, nursing and phlebotomy staff of participating hospitals in the acquisition of blood samples. We thank Pauline Zaenker, Daniele Bartlett, Aaron Beasley, Gabriela Marsavela for their help with blood collection from healthy controls. In particular, we thank Paula Van Miert for helping with sample processing and Michelle Pereira for carrying out ctDNA analysis. We thank Peggy Robinson and Christopher Wagner from Angle plc, for their generous scientific and technical support and deploying the Parsortix platform. We thank Julie Lang (University of Southern California-USC, Los Angeles US) for conceptual advice on this project, and Daniel Haber and Shyamala Maheswaran (Harvard University, Boston US) for their comments on the results and interpretation.

Author information

C.A.B., M.Z., E.S.G., R.J.S. designed the study. C.A.B., M.Mo., J.F., L.C. performed experimental work. X.H. provided protocol details. C.A.B., M.Mo., X.H., L.C. and E.S.G. analysed data and performed statistical analysis. A.C.M., M.A.D., T.M. and M.Mi. enrolled patients into the study, collected and curated clinical data. C.A.B., M.Mo. and E.S.G. wrote the paper. All authors reviewed and approved the final version of the paper.

Correspondence to Elin Solomonovna Gray.

Ethics declarations

Ethics approval and consent to participate

Healthy volunteers and melanoma patients signed consent forms approved by the Human Research Ethics Committees at Edith Cowan University (No. 11543) and Sir Charles Gairdner Hospital (No. 2013–246). The study was performed in accordance with the Declaration of Helsinki.

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N/A.

Data availability

Raw data is available upon request.

Competing interests

The authors declare no competing interests.

Funding infromation

This study was funded by a National Health and Medical Research Council (NHMRC) grant 1013349 to M.Z., a Cancer Council WA (CCWA) grant to E.S.G., M.Z., M.Mi. and C.A.B., a CCWA Suzanne Cavanagh Early Career Investigator (2016) to C.A.B. as well as an Edith Cowan University Early Career Research grant (2016) to C.A.B. E.S.G. was supported by fellowships from the Cancer Research Trust and CCWA. A.C.M. was supported by a Western Australian Health Translation Early Career Fellowship.

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Aya-Bonilla, C.A., Morici, M., Hong, X. et al. Detection and prognostic role of heterogeneous populations of melanoma circulating tumour cells. Br J Cancer (2020). https://doi.org/10.1038/s41416-020-0750-9

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