Characterization of spatial distribution of tumor-infiltrating CD8+ T cells refines their prognostic utility for pancreatic cancer survival

Abstract

The abundance of cytotoxic T-cell infiltrates has important implications for patient outcome and therapeutic design for pancreatic ductal adenocarcinoma. However, intratumoral heterogeneity remains a challenge to understanding the complex immune microenvironment. We hypothesized that characterizing CD8+ cell distribution within pancreatic adenocarcinoma tissues might refine the prognostic value of tumor-infiltrating CD8+ lymphocytes. Using multiplex immunohistochemistry-based image analysis on whole-tissue sections of 214 pancreatic ductal adenocarcinomas, we measured CD8+ cell densities in the tumor center, the tumor margin, and the whole tumor, along with the proximity of CD8+ cells to carcinoma cells. Multivariable Cox regression analysis was performed to assess the associations of CD8+ cell densities with pancreatic cancer-specific survival, adjusting for clinicopathologic and immune-related features, including tumor expressions of TP53, SMAD4, and the programmed cell death 1 ligand 1 (CD274, PD-L1) and the extent of tertiary lymphoid structures. There was substantial heterogeneity in CD8+ cell density, with the mean density in the tumor center less than half that in the tumor margin. Tumor CD274 expression and extensive tertiary lymphoid structures were appeared to be associated with higher CD8+ cell density in the tumor margin (P = 0.037 and P = 0.005, respectively), but not with that in the tumor center (P > 0.50). The association of higher CD8+ cell density with prolonged survival was significant for the whole tumor (Ptrend = 0.009); however, the association was stronger for the tumor center (Ptrend = 0.002) and insignificant for the tumor margin (Ptrend = 0.07). Tumor cell–CD8+ cell distance correlated strongly with CD8+ cell density, whereas the density of CD8+ cells proximate to cancer cells exhibited no prognostic association. In conclusion, spatial computational analysis on pancreatic ductal adenocarcinoma reveals the prognostic validity of CD8+ cell density in the tumor center, where CD8+ cell infiltration is ununiformly restricted, likely suggesting pro-tumorigenic roles of the immunosuppressive tumor microenvironment of pancreatic cancer.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. 1.

    Chen Daniel S, Mellman I. Oncology meets immunology: the cancer-immunity cycle. Immunity. 2013;39:1–10.

  2. 2.

    Seo YD, Pillarisetty VG. T-cell programming in pancreatic adenocarcinoma: a review. Cancer Gene Ther. 2017;24:106–13.

  3. 3.

    Vonderheide RH. The immune revolution: a case for priming, not checkpoint. Cancer Cell. 2018;33:563–9.

  4. 4.

    Foucher ED, Ghigo C, Chouaib S, Galon J, Iovanna J, Olive D. Pancreatic ductal adenocarcinoma: a strong imbalance of good and bad immunological cops in the tumor microenvironment. Front Immunol. 2018;9:1044.

  5. 5.

    Hiraoka N, Ino Y, Yamazaki-Itoh R, Kanai Y, Kosuge T, Shimada K. Intratumoral tertiary lymphoid organ is a favourable prognosticator in patients with pancreatic cancer. Br J Cancer. 2015;112:1782–90.

  6. 6.

    Sautes-Fridman C, Lawand M, Giraldo NA, Kaplon H, Germain C, Fridman WH, et al. Tertiary lymphoid structures in cancers: prognostic value. Front Immunol. 2016;7:407.

  7. 7.

    Lutz ER, Wu AA, Bigelow E, Sharma R, Mo G, Soares K, et al. Immunotherapy converts nonimmunogenic pancreatic tumors into immunogenic foci of immune regulation. Cancer Immunol Res. 2014;2:616–31.

  8. 8.

    Karakhanova S, Ryschich E, Mosl B, Harig S, Jäger D, Schmidt J, et al. Prognostic and predictive value of immunological parameters for chemoradioimmunotherapy in patients with pancreatic adenocarcinoma. Br J Cancer. 2015;112:1027.

  9. 9.

    Vennin C, Murphy KJ, Morton JP, Cox TR, Pajic M, Timpson P. Reshaping the tumor stroma for treatment of pancreatic cancer. Gastroenterology. 2018;154:820–38.

  10. 10.

    Neesse A, Bauer CA, Ohlund D, Lauth M, Buchholz M, Michl P, et al. Stromal biology and therapy in pancreatic cancer: ready for clinical translation? Gut. 2019;68:159–71.

  11. 11.

    Johnson BA 3rd, Yarchoan M, Lee V, Laheru DA, Jaffee EM. Strategies for increasing pancreatic tumor immunogenicity. Clin Cancer Res. 2017;23:1656–69.

  12. 12.

    Bailey P, Chang DK, Nones K, Johns AL, Patch A-M, Gingras M-C, et al. Genomic analyses identify molecular subtypes of pancreatic cancer. Nature. 2016;531:47.

  13. 13.

    Moffitt RA, Marayati R, Flate EL, Volmar KE, Loeza SGH, Hoadley KA, et al. Virtual microdissection identifies distinct tumor- and stroma-specific subtypes of pancreatic ductal adenocarcinoma. Nat Genet. 2015;47:1168.

  14. 14.

    Collisson EA, Sadanandam A, Olson P, Gibb WJ, Truitt M, Gu S, et al. Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy. Nat Med. 2011;17:500–3.

  15. 15.

    Li J, Byrne KT, Yan F, Yamazoe T, Chen Z, Baslan T, et al. Tumor cell-intrinsic factors underlie heterogeneity of immune cell infiltration and response to immunotherapy. Immunity. 2018;49:178–93 e7.

  16. 16.

    Lafaro KJ, Melstrom LG. The paradoxical web of pancreatic cancer tumor microenvironment. Am J Pathol. 2019;189:44–57.

  17. 17.

    Birnbaum DJ, Bertucci F, Finetti P, Birnbaum D, Mamessier E. Molecular classification as prognostic factor and guide for treatment decision of pancreatic cancer. Biochim Biophys Acta. 2018;1869:248–55.

  18. 18.

    Notta F, Hahn SA, Real FX. A genetic roadmap of pancreatic cancer: still evolving. Gut. 2017;66:2170–8.

  19. 19.

    Knudsen ES, Vail P, Balaji U, Ngo H, Botros IW, Makarov V, et al. Stratification of pancreatic ductal adenocarcinoma: combinatorial genetic, stromal, and immunologic markers. Cancer Res. 2017;23:4429–40.

  20. 20.

    Yachida S, Jones S, Bozic I, Antal T, Leary R, Fu B, et al. Distant metastasis occurs late during the genetic evolution of pancreatic cancer. Nature. 2010;467:1114–7.

  21. 21.

    Carstens JL, Correa de Sampaio P, Yang D, Barua S, Wang H, Rao A, et al. Spatial computation of intratumoral T cells correlates with survival of patients with pancreatic cancer. Nat Commun. 2017;8:15095.

  22. 22.

    Pagès F, Mlecnik B, Marliot F, Bindea G, Ou F-S, Bifulco C, et al. International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study. Lancet. 2018;391:2128–39.

  23. 23.

    Galon J, Mlecnik B, Bindea G, Angell HK, Berger A, Lagorce C, et al. Towards the introduction of the ‘Immunoscore’ in the classification of malignant tumours. J Pathol. 2014;232:199–209.

  24. 24.

    Masugi Y, Nishihara R, Hamada T, Song M, da Silva A, Kosumi K, et al. Tumor PDCD1LG2 (PD-L2) expression and the lymphocytic reaction to colorectal cancer. Cancer Immunol Res. 2017;5:1046–55.

  25. 25.

    Ogino S, Nowak JA, Hamada T, Phipps AI, Peters U, Milner DA, et al. Integrative analysis of exogenous, endogenous, tumour and immune factors for precision medicine. Gut. 2018;67:1168–80.

  26. 26.

    Kloppel G, Lingenthal G, von Bulow M, Kern HF. Histological and fine structural features of pancreatic ductal adenocarcinomas in relation to growth and prognosis: studies in xenografted tumours and clinico-histopathological correlation in a series of 75 cases. Histopathology. 1985;9:841–56.

  27. 27.

    Japan Pancreas Society. Classification of pancreatic carcinoma, Fourth English Edition 4th English ed. Kanehara: Tokyo, 2017. 70–9 pp.

  28. 28.

    Brierley JD, Gospodarowics MK, Wittekind C. TNM Classification of Malignant Tumours, 8th edn. John Wiley & Sons, Ltd: Oxford, 2017.

  29. 29.

    Rimm DL, Han G, Taube JM, Yi ES, Bridge JA, Flieder DB, et al. A prospective, multi-institutional, pathologist-based assessment of 4 immunohistochemistry assays for PD-L1 expression in non-small cell lung cancer. JAMA Oncol. 2017;3:1051–8.

  30. 30.

    Qian ZR, Rubinson DA, Nowak JA, Morales-Oyarvide V, Dunne RF, Kozak MM, et al. Association of alterations in main driver genes with outcomes of patients with resected pancreatic ductal adenocarcinoma. JAMA Oncol. 2018;4:e173420.

  31. 31.

    Yamazaki K, Masugi Y, Effendi K, Tsujikawa H, Hiraoka N, Kitago M, et al. Upregulated SMAD3 promotes epithelial-mesenchymal transition and predicts poor prognosis in pancreatic ductal adenocarcinoma. Lab Investig. 2014;94:683–91.

  32. 32.

    Towards generalized nuclear segmentation in histological images. Proceedings of the 13th IEEE International Conference on BioInformatics and BioEngineering; 10–13 Nov 2013.

  33. 33.

    Otsu N. A threshold selection method from gray-level histograms. IEEE Trans Syst Man Cyber. 1979;9:62–6.

  34. 34.

    Kanda Y. Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics. Bone Marrow Transplant. 2013;48:452–8.

  35. 35.

    Topalian SL, Drake CG, Pardoll DM. Immune checkpoint blockade: a common denominator approach to cancer therapy. Cancer Cell. 2015;27:450–61.

  36. 36.

    Balachandran VP, Luksza M, Zhao JN, Makarov V, Moral JA, Remark R, et al. Identification of unique neoantigen qualities in long-term survivors of pancreatic cancer. Nature. 2017;551:512–6.

  37. 37.

    Bailey P, Chang DK, Forget MA, Lucas FA, Alvarez HA, Haymaker C, et al. Exploiting the neoantigen landscape for immunotherapy of pancreatic ductal adenocarcinoma. Sci Rep. 2016;6:35848.

  38. 38.

    Poschke I, Faryna M, Bergmann F, Flossdorf M, Lauenstein C, Hermes J, et al. Identification of a tumor-reactive T-cell repertoire in the immune infiltrate of patients with resectable pancreatic ductal adenocarcinoma. Oncoimmunology. 2016;5:e1240859.

  39. 39.

    Ene-Obong A, Clear AJ, Watt J, Wang J, Fatah R, Riches JC, et al. Activated pancreatic stellate cells sequester CD8+ T cells to reduce their infiltration of the juxtatumoral compartment of pancreatic ductal adenocarcinoma. Gastroenterology. 2013;145:1121–32.

  40. 40.

    Stromnes IM, Hulbert A, Pierce RH, Greenberg PD, Hingorani SR. T-cell localization, activation, and clonal expansion in human pancreatic ductal adenocarcinoma. Cancer Immunol Res. 2017;5:978–91.

  41. 41.

    Mirlekar B, Michaud D, Searcy R, Greene K, Pylayeva-Gupta Y. IL35 hinders endogenous antitumor T-cell immunity and responsiveness to immunotherapy in pancreatic. Cancer Cancer Immunol Res. 2018;6:1014–24.

  42. 42.

    Nywening TM, Belt BA, Cullinan DR, Panni RZ, Han BJ, Sanford DE, et al. Targeting both tumour-associated CXCR2(+) neutrophils and CCR2(+) macrophages disrupts myeloid recruitment and improves chemotherapeutic responses in pancreatic ductal adenocarcinoma. Gut. 2018;67:1112–23.

  43. 43.

    Feig C, Jones JO, Kraman M, Wells RJ, Deonarine A, Chan DS, et al. Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer. Proc Natl Acad Sci USA. 2013;110:20212–7.

  44. 44.

    Jiang H, Hegde S, Knolhoff BL, Zhu Y, Herndon JM, Meyer MA, et al. Targeting focal adhesion kinase renders pancreatic cancers responsive to checkpoint immunotherapy. Nat Med. 2016;22:851–60.

  45. 45.

    Ozdemir BC, Pentcheva-Hoang T, Carstens JL, Zheng X, Wu CC, Simpson TR, et al. Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival. Cancer Cell. 2014;25:719–34.

  46. 46.

    Balachandran VP, Beatty GL, Dougan SK. Broadening the impact of immunotherapy to pancreatic cancer: challenges and opportunities. Gastroenterology. 2019;156:2056–72.

  47. 47.

    Cannon A, Thompson C, Hall BR, Jain M, Kumar S, Batra SK. Desmoplasia in pancreatic ductal adenocarcinoma: insight into pathological function and therapeutic potential. Genes Cancer. 2018;9:78–86.

  48. 48.

    Jiang H, Hegde S, DeNardo DG. Tumor-associated fibrosis as a regulator of tumor immunity and response to immunotherapy. Cancer Immunol Immunother. 2017;66:1037–48.

  49. 49.

    Biffi G, Oni TE, Spielman B, Hao Y, Elyada E, Park Y, et al. IL-1-induced JAK/STAT signaling is antagonized by TGF-beta to shape CAF heterogeneity in pancreatic ductal adenocarcinoma. Cancer Discov. 2018;9:282–301.

  50. 50.

    Mahajan UM, Langhoff E, Goni E, Costello E, Greenhalf W, Halloran C, et al. Immune cell and stromal signature associated with progression-free survival of patients with resected pancreatic ductal adenocarcinoma. Gastroenterology. 2018;155:1625–39 e2.

  51. 51.

    Lianyuan T, Dianrong X, Chunhui Y, Zhaolai M, Bin J. The predictive value and role of stromal tumor-infiltrating lymphocytes in pancreatic ductal adenocarcinoma (PDAC). Cancer Biol Ther. 2018;19:296–305.

  52. 52.

    Lohneis P, Sinn M, Bischoff S, Juhling A, Pelzer U, Wislocka L., et al. Cytotoxic tumour-infiltrating T lymphocytes influence outcome in resected pancreatic ductal adenocarcinoma. Eur J of Cancer. 2017;83:290–301.

  53. 53.

    Ino Y, Yamazaki-Itoh R, Shimada K, Iwasaki M, Kosuge T, Kanai Y, et al. Immune cell infiltration as an indicator of the immune microenvironment of pancreatic cancer. Br J cancer. 2013;108:914–23.

  54. 54.

    Ogino S, Giannakis M. Immunoscore for (colorectal) cancer precision medicine. Lancet. 2018;391:2084–6.

  55. 55.

    Donnem T, Kilvaer TK, Andersen S, Richardsen E, Paulsen EE, Hald SM, et al. Strategies for clinical implementation of TNM-Immunoscore in resected nonsmall-cell lung cancer. Ann Oncol. 2016;27:225–32.

  56. 56.

    Hruban RH, Gaida MM, Thompson E, Hong SM, Noe M, Brosens LAA, et al. Why is pancreatic cancer so deadly? The Pathologist’s View. J Pathol. 2019;248:131–41.

  57. 57.

    Masugi Y, Yamazaki K, Hibi T, Aiura K, Kitagawa Y, Sakamoto M. Solitary cell infiltration is a novel indicator of poor prognosis and epithelial-mesenchymal transition in pancreatic cancer. Hum Pathol. 2010;41:1061–8.

  58. 58.

    Feng Z, Bethmann D, Kappler M, Ballesteros-Merino C, Eckert A, Bell RB, et al. Multiparametric immune profiling in HPV- oral squamous cell cancer. JCI insight. 2017;2:93652.

  59. 59.

    Angelo M, Bendall SC, Finck R, Hale MB, Hitzman C, Borowsky AD, et al. Multiplexed ion beam imaging of human breast tumors. Nat Med. 2014;20:436–42.

  60. 60.

    Levenson RM, Borowsky AD, Angelo M. Immunohistochemistry and mass spectrometry for highly multiplexed cellular molecular imaging. Lab Invest. 2015;95:397.

Download references

Acknowledgements

We are grateful to the Fourth Laboratory of the Department of Pathology in Keio University School of Medicine for assistance with tissue processing and staining.

Financial support

This work was supported by KAKENHI (Grant number 18K15094 to YM) from the Japan Society for the Promotion of Science, and Keio University Academic Development Funds for Individual Research from Keio University (to YM).

Author information

Correspondence to Michiie Sakamoto.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Table 1

Supplementary Figure 1

Supplementary Figure 2

Supplementary Figure 3

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark