Clinicopathological factors associated with tumor-infiltrating lymphocyte reactivity in breast cancer

Abstract

Background

The clinical significance of adoptive tumor-infiltrating lymphocyte (TIL) therapy has been demonstrated in many clinical trials. We analyzed the in vitro reactivity of cultured TILs against autologous breast cancer cells.

Methods

TILs and cancer cells were cultured from 31 breast tumor tissues. Reactivity of TILs against cancer cells was determined by measuring secreted interferon-gamma. Expression levels of epithelial markers, major histocompatibility complex molecules, and programmed death-ligand 1 (PD-L1) in cancer cells, and T cell markers (memory, T cell activation and exhaustion, and regulatory T cell markers) in expanded TILs were analyzed and compared between the reactive and non-reactive groups.

Results

In seven cases, TILs showed reactivity to autologous cancer cells. Six of these cases were associated with triple-negative breast cancer (TNBC). All reactive TNBCs were derived from surgical specimens after neoadjuvant chemotherapy (NAC). Higher expression of Ki67 in tumor tissues and lower expression of PD-L1 in cultured cancer cells were associated with reactivity. Proliferation of reactive TILs was high. High proportions of T cells and PD-1+CD4+ and PD1+CD8+ T cells were associated with reactivity in TNBC cases, while other activation or exhaustion markers were not.

Conclusion

TILs from approximately half the TNBC cases with NAC showed reactivity against autologous cancer cells. The proportion of PD-1+ T cells was higher in the reactive group. Adoptive TIL therapy combined with PD-1 inhibitors might be promising for TNBC patients with residual tumors after NAC.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

ACT:

Adoptive cell transfer

CK:

Cytokeratin

EpCAM:

Epithelial cell adhesion molecule

ER:

Estrogen receptor

HER2+ :

Human epidermal growth factor receptor 2-positive

HR:

Hormone receptor

IFN-γ:

Interferon-gamma

MDSCs:

Myeloid-derived suppressor cells

MHC:

Major histocompatibility complex

NAC:

Neoadjuvant chemotherapy

NK:

Natural killer

NKT:

Natural killer T

PBMC:

Peripheral blood mononuclear cell

PDX:

Patient-derived xenograft

PR:

Progesterone receptor

REP:

Rapid expansion

TCR:

T cell receptor

TIL:

Tumor-infiltrating lymphocyte

TLS:

Tertiary lymphoid structure

TNBC:

Triple-negative breast cancer

Treg:

Regulatory T cell

References

  1. 1.

    Waks AG, Winer EP (2019) Breast cancer treatment: a review. JAMA 321(3):288–300

    CAS  PubMed  Article  Google Scholar 

  2. 2.

    Bernhard H, Neudorfer J, Gebhard K, Conrad H, Hermann C, Nahrig J, Fend F, Weber W, Busch DH, Peschel C (2008) Adoptive transfer of autologous, HER2-specific, cytotoxic T lymphocytes for the treatment of HER2-overexpressing breast cancer. Cancer Immunol Immunother 57(2):271–280

    PubMed  Article  Google Scholar 

  3. 3.

    Sparano JA, Fisher RI, Weiss GR, Margolin K, Aronson FR, Hawkins MJ, Atkins MB, Dutcher JP, Gaynor ER, Boldt DH et al (1994) Phase II trials of high-dose interleukin-2 and lymphokine-activated killer cells in advanced breast carcinoma and carcinoma of the lung, ovary, and pancreas and other tumors. J Immunother Emphas Tumor Immunol 16(3):216–223

    CAS  Article  Google Scholar 

  4. 4.

    Tchou J, Zhao Y, Levine BL, Zhang PJ, Davis MM, Melenhorst JJ, Kulikovskaya I, Brennan AL, Liu X, Lacey SF et al (2017) Safety and efficacy of intratumoral injections of chimeric antigen receptor (CAR) T cells in metastatic breast cancer. Cancer Immunol Res 5(12):1152–1161

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. 5.

    Martinez M, Moon EK (2019) CAR T cells for solid tumors: new strategies for finding, infiltrating, and surviving in the tumor microenvironment. Front Immunol 10:128

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 6.

    Zacharakis N, Chinnasamy H, Black M, Xu H, Lu YC, Zheng Z, Pasetto A, Langhan M, Shelton T, Prickett T et al (2018) Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer. Nat Med 24(6):724–730

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. 7.

    Egelston CA, Avalos C, Tu TY, Simons DL, Jimenez G, Jung JY, Melstrom L, Margolin K, Yim JH, Kruper L et al (2018) Human breast tumor-infiltrating CD8(+) T cells retain polyfunctionality despite PD-1 expression. Nat Commun 9(1):4297

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  8. 8.

    Stanton SE, Adams S, Disis ML (2016) Variation in the incidence and magnitude of tumor-infiltrating lymphocytes in breast cancer subtypes: a systematic review. JAMA Oncol 2(10):1354–1360

    PubMed  Article  Google Scholar 

  9. 9.

    Rosenberg SA, Yang JC, Sherry RM, Kammula US, Hughes MS, Phan GQ, Citrin DE, Restifo NP, Robbins PF, Wunderlich JR et al (2011) Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy. Clin Cancer Res 17(13):4550–4557

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. 10.

    Creelan B, Teer J, Toloza E, Mullinax J, Landin A, Gray J, Tanvetyanon T, Taddeo M, Noyes D, Kelley L et al (2018) OA05.03 safety and clinical activity of adoptive cell transfer using tumor infiltrating lymphocytes (TIL) combined with nivolumab in NSCLC. J Thorac Oncol 13(10):S330–S331

    Article  Google Scholar 

  11. 11.

    Jazaeri AA, Zsiros E, Amaria RN, Artz AS, Edwards RP, Wenham RM, Slomovitz BM, Walther A, Thomas SS, Chesney JA et al (2019) Safety and efficacy of adoptive cell transfer using autologous tumor infiltrating lymphocytes (LN-145) for treatment of recurrent, metastatic, or persistent cervical carcinoma. J Clin Oncol 37(15_suppl):2538–2538

    Article  Google Scholar 

  12. 12.

    Fujita K, Ikarashi H, Takakuwa K, Kodama S, Tokunaga A, Takahashi T, Tanaka K (1995) Prolonged disease-free period in patients with advanced epithelial ovarian cancer after adoptive transfer of tumor-infiltrating lymphocytes. Clin Cancer Res 1(5):501–507

    CAS  PubMed  Google Scholar 

  13. 13.

    Tran E, Robbins PF, Lu YC, Prickett TD, Gartner JJ, Jia L, Pasetto A, Zheng Z, Ray S, Groh EM et al (2016) T-cell transfer therapy targeting mutant KRAS in cancer. N Engl J Med 375(23):2255–2262

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14.

    Lee HJ, Kim YA, Sim CK, Heo SH, Song IH, Park HS, Park SY, Bang WS, Park IA, Lee M et al (2017) Expansion of tumor-infiltrating lymphocytes and their potential for application as adoptive cell transfer therapy in human breast cancer. Oncotarget 8(69):113345–113359

    PubMed  PubMed Central  Article  Google Scholar 

  15. 15.

    June CH, O'Connor RS, Kawalekar OU, Ghassemi S, Milone MC (2018) CAR T cell immunotherapy for human cancer. Science 359(6382):1361–1365

    CAS  PubMed  Article  Google Scholar 

  16. 16.

    Salgado R, Denkert C, Demaria S, Sirtaine N, Klauschen F, Pruneri G, Wienert S, Van den Eynden G, Baehner FL, Penault-Llorca F et al (2015) The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014. Ann Oncol 26(2):259–271

    CAS  PubMed  Article  Google Scholar 

  17. 17.

    Lee HJ, Park IA, Song IH, Shin SJ, Kim JY, Yu JH, Gong G (2016) Tertiary lymphoid structures: prognostic significance and relationship with tumour-infiltrating lymphocytes in triple-negative breast cancer. J Clin Pathol 69(5):422–430

    PubMed  Article  Google Scholar 

  18. 18.

    Van Acker HH, Capsomidis A, Smits EL, Van Tendeloo VF (2017) CD56 in the immune system: More than a marker for cytotoxicity? Front Immunol 8:892

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  19. 19.

    Ohtani H (2007) Focus on TILs: prognostic significance of tumor infiltrating lymphocytes in human colorectal cancer. Cancer Immun 7(1):4

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    Liakou CI, Narayanan S, Ng Tang D, Logothetis CJ, Sharma P (2007) Focus on TILs: prognostic significance of tumor infiltrating lymphocytes in human bladder cancer. Cancer Immun 7(1):10

    PubMed  PubMed Central  Google Scholar 

  21. 21.

    Dunn GP, Dunn IF, Curry WT (2007) Focus on TILs: prognostic significance of tumor infiltrating lymphocytes in human glioma. Cancer Immun 7(1):12

    PubMed  PubMed Central  Google Scholar 

  22. 22.

    Uppaluri R, Dunn GP, Lewis JS (2008) Focus on TILs: prognostic significance of tumor infiltrating lymphocytes in head and neck cancers. Cancer Immun 8(1):16

    PubMed  PubMed Central  Google Scholar 

  23. 23.

    Oble DA, Loewe R, Yu P, Mihm MC (2009) Focus on TILs: prognostic significance of tumor infiltrating lymphocytes in human melanoma. Cancer Immun 9(1):3

    PubMed  PubMed Central  Google Scholar 

  24. 24.

    Denkert C, von Minckwitz G, Darb-Esfahani S, Lederer B, Heppner BI, Weber KE, Budczies J, Huober J, Klauschen F, Furlanetto J et al (2018) Tumour-infiltrating lymphocytes and prognosis in different subtypes of breast cancer: a pooled analysis of 3771 patients treated with neoadjuvant therapy. Lancet Oncol 19(1):40–50

    PubMed  Article  Google Scholar 

  25. 25.

    Shang B, Liu Y, Jiang S-J, Liu Y (2015) Prognostic value of tumor-infiltrating FoxP3+ regulatory T cells in cancers: a systematic review and meta-analysis. Sci Rep 5:15179

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. 26.

    Shen T, Zhou L, Shen H, Shi C, Jia S, Ding GP, Cao L (2017) Prognostic value of programmed cell death protein 1 expression on CD8+ T lymphocytes in pancreatic cancer. Sci Rep 7(1):7848

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  27. 27.

    Zhou C, Tang J, Sun H, Zheng X, Li Z, Sun T, Li J, Wang S, Zhou X, Sun H et al (2017) PD-L1 expression as poor prognostic factor in patients with non-squamous non-small cell lung cancer. Oncotarget 8(35):58457–58468

    PubMed  PubMed Central  Article  Google Scholar 

  28. 28.

    Jung HI, Jeong D, Ji S, Ahn TS, Bae SH, Chin S, Chung JC, Kim HC, Lee MS, Baek MJ (2017) Overexpression of PD-L1 and PD-L2 is associated with poor prognosis in patients with hepatocellular carcinoma. Cancer Res Treat 49(1):246–254

    CAS  PubMed  Article  Google Scholar 

  29. 29.

    Koganemaru S, Inoshita N, Miura Y, Miyama Y, Fukui Y, Ozaki Y, Tomizawa K, Hanaoka Y, Toda S, Suyama K et al (2017) Prognostic value of programmed death-ligand 1 expression in patients with stage III colorectal cancer. Cancer Sci 108(5):853–858

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30.

    Nair S, Dhodapkar MV (2017) Natural killer T cells in cancer immunotherapy. Front Immunol 8:1178

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  31. 31.

    Shi B, Li Q, Ma X, Gao Q, Li L, Chu J (2018) High expression of programmed cell death protein 1 on peripheral blood T-cell subsets is associated with poor prognosis in metastatic gastric cancer. Oncol Lett 16(4):4448–4454

    PubMed  PubMed Central  Google Scholar 

  32. 32.

    Badoual C, Hans S, Merillon N, Van Ryswick C, Ravel P, Benhamouda N, Levionnois E, Nizard M, Si-Mohamed A, Besnier N et al (2013) PD-1-expressing tumor-infiltrating T cells are a favorable prognostic biomarker in HPV-associated head and neck cancer. Cancer Res 73(1):128–138

    CAS  PubMed  Article  Google Scholar 

  33. 33.

    Carreras J, Lopez-Guillermo A, Roncador G, Villamor N, Colomo L, Martinez A, Hamoudi R, Howat WJ, Montserrat E, Campo E (2009) High numbers of tumor-infiltrating programmed cell death 1-positive regulatory lymphocytes are associated with improved overall survival in follicular lymphoma. J Clin Oncol 27(9):1470–1476

    PubMed  Article  PubMed Central  Google Scholar 

  34. 34.

    Li Y, Liang L, Dai W, Cai G, Xu Y, Li X, Li Q, Cai S (2016) Prognostic impact of programed cell death-1 (PD-1) and PD-ligand 1 (PD-L1) expression in cancer cells and tumor infiltrating lymphocytes in colorectal cancer. Mol Cancer 15(1):55

    PubMed  PubMed Central  Article  Google Scholar 

  35. 35.

    Brockhoff G, Seitz S, Weber F, Zeman F, Klinkhammer-Schalke M, Ortmann O, Wege AK (2018) The presence of PD-1 positive tumor infiltrating lymphocytes in triple negative breast cancers is associated with a favorable outcome of disease. Oncotarget 9(5):6201–6212

    PubMed  Article  Google Scholar 

  36. 36.

    Simon S, Labarriere N (2017) PD-1 expression on tumor-specific T cells: Friend or foe for immunotherapy? Oncoimmunology 7(1):e1364828

    PubMed  PubMed Central  Article  Google Scholar 

  37. 37.

    Josefowicz SZ, Lu LF, Rudensky AY (2012) Regulatory T cells: mechanisms of differentiation and function. Annu Rev Immunol 30:531–564

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38.

    Niakan A, Faghih Z, Talei AR, Ghaderi A (2019) Cytokine profile of CD4(+)CD25(-)FoxP3(+) T cells in tumor-draining lymph nodes from patients with breast cancer. Mol Immunol 116:90–97

    CAS  PubMed  Article  Google Scholar 

  39. 39.

    Ferreira RC, Simons HZ, Thompson WS, Rainbow DB, Yang X, Cutler AJ, Oliveira J, Castro Dopico X, Smyth DJ, Savinykh N et al (2017) Cells with Treg-specific FOXP3 demethylation but low CD25 are prevalent in autoimmunity. J Autoimmun 84:75–86

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. 40.

    Schuster M, Plaza-Sirvent C, Visekruna A, Huehn J, Schmitz I (2019) Generation of Foxp3(+)CD25(-) regulatory T-cell precursors requires c-Rel and IkappaBNS. Front Immunol 10:1583

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. 41.

    Thornton AM, Lu J, Korty PE, Kim YC, Martens C, Sun PD, Shevach EM (2019) Helios(+) and Helios(-) treg subpopulations are phenotypically and functionally distinct and express dissimilar TCR repertoires. Eur J Immunol 49(3):398–412

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. 42.

    Han S, Toker A, Liu ZQ, Ohashi PS (2019) Turning the tide against regulatory T cells. Front Oncol 9:279

    PubMed  PubMed Central  Article  Google Scholar 

  43. 43.

    Barroso-Sousa R, Jain E, Cohen O, Kim D, Buendia-Buendia J, Winer E, Lin N, Tolaney SM, Wagle N (2020) Prevalence and mutational determinants of high tumor mutation burden in breast cancer. Ann Oncol 31(3):387–394

    CAS  PubMed  Article  Google Scholar 

  44. 44.

    Lee HJ, Song IH, Park IA, Heo SH, Kim YA, Ahn JH, Gong G (2016) Differential expression of major histocompatibility complex class I in subtypes of breast cancer is associated with estrogen receptor and interferon signaling. Oncotarget 7(21):30119–30132

    PubMed  PubMed Central  Article  Google Scholar 

Download references

Funding

This study was supported by the Korean Health Technology R&D Project, Ministry of Health & Welfare (HI15C0708 and HI17C0337) and the Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea (2018IL0169, 2019IL0169, and 2019IL0839).

Author information

Affiliations

Authors

Contributions

HjL interpreted the data associated with tumor-infiltrating lymphocyte reactivity in breast cancer and was a major contributor in writing the manuscript. YAK and HSP participated in sample preparation and analysis. YK and JHS analyzed the TCR repertoire of the TILs. HL, GG, and HJL contributed as pathologists and revised the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Gyungyub Gong or Hee Jin Lee.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Ethics approval and consents to participate

Tumor tissues were obtained from patients along with their consent to the use of the specimen for research purposes. This study was approved by the Institutional Review Board of Asan Medical Center (IRB#2015-0438).

Consent for publication

Not applicable.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file 1 (PPTX 557 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Lee, H., Kim, Y., Kim, Y. et al. Clinicopathological factors associated with tumor-infiltrating lymphocyte reactivity in breast cancer. Cancer Immunol Immunother (2020). https://doi.org/10.1007/s00262-020-02633-5

Download citation

Keywords

  • Tumor-infiltrating lymphocytes
  • Breast cancer
  • Adoptive cell therapy
  • Reactivity