Breast Cancer Research and Treatment

, Volume 151, Issue 3, pp 619–627 | Cite as

Prognostic and predictive value of NanoString-based immune-related gene signatures in a neoadjuvant setting of triple-negative breast cancer: relationship to tumor-infiltrating lymphocytes

  • Hee Jin Lee
  • Jeong-Ju Lee
  • In Hye Song
  • In Ah Park
  • Jun Kang
  • Jong Han Yu
  • Jin-Hee Ahn
  • Gyungyub GongEmail author
Clinical Trial


The prognostic significance of tumor-infiltrating lymphocytes and immune signals has been described previously in triple-negative breast cancer (TNBC). Furthermore, recent studies have shown that immunologic parameters are relevant for the response to neoadjuvant chemotherapy (NAC) in breast cancer as well as for outcomes after adjuvant chemotherapy. However, immune signals are variable, and which signals are important is largely unknown. We, therefore, evaluated the expression of immune-related genes in TNBC treated with NAC. We retrospectively evaluated biopsy tissue from 55 patients with primary TNBC treated with NAC (anthracycline, cyclophosphamide, and docetaxel) against the NanoString nCounter GX Human Immunology Panel (579 immune-related genes). Higher expression of cytotoxic molecules, T cell receptor signaling pathway components, cytokines related to T helper cell type 1 (Th1), and B cell markers was associated with a pathologic complete response (pCR). Higher expression of NFKB1, MAPK1, TRAF1, CXCL13, GZMK, and IL7R was significantly associated with pCR, higher Miller-Payne grade, and lower residual cancer burden class. Expression of NFKB1, TRAF1, and CXCL13genes, in particular, was significantly correlated with a longer disease-free survival rate. Conversely, patients those who failed to achieve a pCR showed increased expression of genes related to neutrophils. Higher expression of cytotoxic molecules, T cell receptor signaling pathway components, Th1-related cytokines, and B cell markers is correlated with pCR and survival in TNBC patients treated with NAC. Our results suggest that the activation status of neutrophils may provide additional predictive information for TNBC patients treated with NAC.


Triple-negative breast cancer Prognosis Tumor-infiltrating lymphocytes NanoString 



This study was supported by a grant (2013-0866) from the Asan Institute for Life Sciences, Seoul, Korea. We thank professor Chan Sik Park, Asan Medical Center for comments that greatly improved the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10549_2015_3438_MOESM1_ESM.pdf (62 kb)
Supplementary material 1 (PDF 61 kb)
10549_2015_3438_MOESM2_ESM.pdf (123 kb)
Supplementary material 2 (PDF 123 kb)
10549_2015_3438_MOESM3_ESM.pdf (32 kb)
Supplementary material 3 (PDF 32 kb)
10549_2015_3438_MOESM4_ESM.pdf (91 kb)
Supplementary material 4 (PDF 91 kb)


  1. 1.
    Penault-Llorca F, Viale G (2012) Pathological and molecular diagnosis of triple-negative breast cancer: a clinical perspective. Ann Oncol 23(Suppl 6):vi19–vi22CrossRefPubMedGoogle Scholar
  2. 2.
    Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA, Lickley LA, Rawlinson E, Sun P, Narod SA (2007) Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res 13:4429–4434CrossRefPubMedGoogle Scholar
  3. 3.
    Dawson SJ, Provenzano E, Caldas C (2009) Triple negative breast cancers: clinical and prognostic implications. Eur J Cancer 45(Suppl 1):27–40CrossRefPubMedGoogle Scholar
  4. 4.
    Stagg J, Allard B (2013) Immunotherapeutic approaches in triple-negative breast cancer: latest research and clinical prospects. Ther Adv Med Oncol 5:169–181CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Fisher CS, Ma CX, Gillanders WE, Aft RL, Eberlein TJ, Gao F, Margenthaler JA (2012) Neoadjuvant chemotherapy is associated with improved survival compared with adjuvant chemotherapy in patients with triple-negative breast cancer only after complete pathologic response. Ann Surg Oncol 19:253–258CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Kong X, Moran MS, Zhang N, Haffty B, Yang Q (2011) Meta-analysis confirms achieving pathological complete response after neoadjuvant chemotherapy predicts favourable prognosis for breast cancer patients. Eur J Cancer 47:2084–2090CrossRefPubMedGoogle Scholar
  7. 7.
    Osako T, Horii R, Matsuura M, Domoto K, Ide Y, Miyagi Y, Takahashi S, Ito Y, Iwase T, Akiyama F (2010) High-grade breast cancers include both highly sensitive and highly resistant subsets to cytotoxic chemotherapy. J Cancer Res Clin Oncol 136:1431–1438CrossRefPubMedGoogle Scholar
  8. 8.
    Darb-Esfahani S, Loibl S, Muller BM, Roller M, Denkert C, Komor M, Schluns K, Blohmer JU, Budczies J, Gerber B, Noske A, du Bois A, Weichert W, Jackisch C, Dietel M, Richter K, Kaufmann M, von Minckwitz G (2009) Identification of biology-based breast cancer types with distinct predictive and prognostic features: role of steroid hormone and HER2 receptor expression in patients treated with neoadjuvant anthracycline/taxane-based chemotherapy. Breast Cancer Res 11:R69CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Pages F, Berger A, Camus M, Sanchez-Cabo F, Costes A, Molidor R, Mlecnik B, Kirilovsky A, Nilsson M, Damotte D, Meatchi T, Bruneval P, Cugnenc PH, Trajanoski Z, Fridman WH, Galon J (2005) Effector memory T cells, early metastasis, and survival in colorectal cancer. N Engl J Med 353:2654–2666CrossRefPubMedGoogle Scholar
  10. 10.
    Zhang L, Conejo-Garcia JR, Katsaros D, Gimotty PA, Massobrio M, Regnani G, Makrigiannakis A, Gray H, Schlienger K, Liebman MN, Rubin SC, Coukos G (2003) Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N Engl J Med 348:203–213CrossRefPubMedGoogle Scholar
  11. 11.
    Zitvogel L, Kroemer G (2008) The immune response against dying tumor cells: avoid disaster, achieve cure. Cell Death Differ 15:1–2CrossRefPubMedGoogle Scholar
  12. 12.
    Apetoh L, Ghiringhelli F, Tesniere A, Criollo A, Ortiz C, Lidereau R, Mariette C, Chaput N, Mira JP, Delaloge S, Andre F, Tursz T, Kroemer G, Zitvogel L (2007) The interaction between HMGB1 and TLR4 dictates the outcome of anticancer chemotherapy and radiotherapy. Immunol Rev 220:47–59CrossRefPubMedGoogle Scholar
  13. 13.
    Denkert C, Loibl S, Noske A, Roller M, Muller BM, Komor M, Budczies J, Darb-Esfahani S, Kronenwett R, Hanusch C, von Torne C, Weichert W, Engels K, Solbach C, Schrader I, Dietel M, von Minckwitz G (2010) Tumor-associated lymphocytes as an independent predictor of response to neoadjuvant chemotherapy in breast cancer. J Clin Oncol 28:105–113CrossRefPubMedGoogle Scholar
  14. 14.
    Issa-Nummer Y, Darb-Esfahani S, Loibl S, Kunz G, Nekljudova V, Schrader I, Sinn BV, Ulmer HU, Kronenwett R, Just M, Kuhn T, Diebold K, Untch M, Holms F, Blohmer JU, Habeck JO, Dietel M, Overkamp F, Krabisch P, von Minckwitz G, Denkert C (2013) Prospective validation of immunological infiltrate for prediction of response to neoadjuvant chemotherapy in HER2-negative breast cancer–a substudy of the neoadjuvant GeparQuinto trial. PLoS One 8:e79775CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    West NR, Milne K, Truong PT, Macpherson N, Nelson BH, Watson PH (2011) Tumor-infiltrating lymphocytes predict response to anthracycline-based chemotherapy in estrogen receptor-negative breast cancer. Breast Cancer Res 13:R126CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Yamaguchi R, Tanaka M, Yano A, Tse GM, Yamaguchi M, Koura K, Kanomata N, Kawaguchi A, Akiba J, Naito Y, Ohshima K, Yano H (2012) Tumor-infiltrating lymphocytes are important pathologic predictors for neoadjuvant chemotherapy in patients with breast cancer. Hum Pathol 43:1688–1694CrossRefPubMedGoogle Scholar
  17. 17.
    Schmidt M, Hellwig B, Hammad S, Othman A, Lohr M, Chen Z, Boehm D et al (2012) A comprehensive analysis of human gene expression profiles identifies stromal immunoglobulin kappa C as a compatible prognostic marker in human solid tumors. Clin Cancer Res 18:2695–2703CrossRefPubMedGoogle Scholar
  18. 18.
    Loi S, Sirtaine N, Piette F, Salgado R, Viale G, Van Eenoo F, Rouas G, Francis P, Crown JP, Hitre E, de Azambuja E, Quinaux E, Di Leo A, Michiels S, Piccart MJ, Sotiriou C (2013) Prognostic and predictive value of tumor-infiltrating lymphocytes in a phase III randomized adjuvant breast cancer trial in node-positive breast cancer comparing the addition of docetaxel to doxorubicin with doxorubicin-based chemotherapy: BIG 02-98. J Clin Oncol 31:860–867CrossRefPubMedGoogle Scholar
  19. 19.
    Gu-Trantien C, Loi S, Garaud S, Equeter C, Libin M, de Wind A, Ravoet M, Le Buanec H, Sibille C, Manfouo-Foutsop G, Veys I, Haibe-Kains B, Singhal SK, Michiels S, Rothe F, Salgado R, Duvillier H, Ignatiadis M, Desmedt C, Bron D, Larsimont D, Piccart M, Sotiriou C, Willard-Gallo K (2013) CD4(+) follicular helper T cell infiltration predicts breast cancer survival. J Clin Invest 123:2873–2892CrossRefPubMedCentralPubMedGoogle Scholar
  20. 20.
    Ignatiadis M, Singhal SK, Desmedt C, Haibe-Kains B, Criscitiello C, Andre F, Loi S, Piccart M, Michiels S, Sotiriou C (2012) Gene modules and response to neoadjuvant chemotherapy in breast cancer subtypes: a pooled analysis. J Clin Oncol 30:1996–2004CrossRefPubMedGoogle Scholar
  21. 21.
    Bianchini G, Gianni L (2014) The immune system and response to HER2-targeted treatment in breast cancer. Lancet Oncol 15:e58–e68CrossRefPubMedGoogle Scholar
  22. 22.
    Spitale A, Mazzola P, Soldini D, Mazzucchelli L, Bordoni A (2009) Breast cancer classification according to immunohistochemical markers: clinicopathologic features and short-term survival analysis in a population-based study from the South of Switzerland. Ann Oncol 20:628–635CrossRefPubMedGoogle Scholar
  23. 23.
    Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM, Allison KH, Allred DC, Bartlett JM, Bilous M, Fitzgibbons P, Hanna W, Jenkins RB, Mangu PB, Paik S, Perez EA, Press MF, Spears PA, Vance GH, Viale G, Hayes DF (2014) Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. Arch Pathol Lab Med 138:241–256Google Scholar
  24. 24.
    Salgado R, Denkert C, Demaria S, Sirtaine N, Klauschen F, Pruneri G, Wienert S et al (2014) The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group. Ann Oncol 26:259–271CrossRefPubMedGoogle Scholar
  25. 25.
    Lakhani SREI, Schnitt SJ, Tan PH, van de Vijver MJ (eds) (2012) WHO classification of tumours of the breast. International Agency for Research on Cancer, LyonGoogle Scholar
  26. 26.
    Symmans WF, Peintinger F, Hatzis C, Rajan R, Kuerer H, Valero V, Assad L, Poniecka A, Hennessy B, Green M, Buzdar AU, Singletary SE, Hortobagyi GN, Pusztai L (2007) Measurement of residual breast cancer burden to predict survival after neoadjuvant chemotherapy. J Clin Oncol 25:4414–4422CrossRefPubMedGoogle Scholar
  27. 27.
    Geiss GK, Bumgarner RE, Birditt B, Dahl T, Dowidar N, Dunaway DL, Fell HP, Ferree S, George RD, Grogan T, James JJ, Maysuria M, Mitton JD, Oliveri P, Osborn JL, Peng T, Ratcliffe AL, Webster PJ, Davidson EH, Hood L, Dimitrov K (2008) Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat Biotechnol 26:317–325CrossRefPubMedGoogle Scholar
  28. 28.
    Reich M, Liefeld T, Gould J, Lerner J, Tamayo P, Mesirov JP (2006) GenePattern 2.0. Nat Genet 38:500–501CrossRefPubMedGoogle Scholar
  29. 29.
    Salgado R, Denkert C, Demaria S, Sirtaine N, Klauschen F, Pruneri G, Wienert S et al (2015) The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014. Ann Oncol 26:259–271CrossRefPubMedGoogle Scholar
  30. 30.
    Ladoire S, Arnould L, Apetoh L, Coudert B, Martin F, Chauffert B, Fumoleau P, Ghiringhelli F (2008) Pathologic complete response to neoadjuvant chemotherapy of breast carcinoma is associated with the disappearance of tumor-infiltrating foxp3+ regulatory T cells. Clin Cancer Res 14:2413–2420CrossRefPubMedGoogle Scholar
  31. 31.
    Ruffell B, Au A, Rugo HS, Esserman LJ, Hwang ES, Coussens LM (2012) Leukocyte composition of human breast cancer. Proc Natl Acad Sci USA 109:2796–2801CrossRefPubMedCentralPubMedGoogle Scholar
  32. 32.
    Gobert M, Treilleux I, Bendriss-Vermare N, Bachelot T, Goddard-Leon S, Arfi V, Biota C, Doffin AC, Durand I, Olive D, Perez S, Pasqual N, Faure C, Ray-Coquard I, Puisieux A, Caux C, Blay JY, Menetrier-Caux C (2009) Regulatory T cells recruited through CCL22/CCR4 are selectively activated in lymphoid infiltrates surrounding primary breast tumors and lead to an adverse clinical outcome. Cancer Res 69:2000–2009CrossRefPubMedGoogle Scholar
  33. 33.
    Cimino-Mathews A, Ye X, Meeker A, Argani P, Emens LA (2013) Metastatic triple-negative breast cancers at first relapse have fewer tumor-infiltrating lymphocytes than their matched primary breast tumors: a pilot study. Hum Pathol 44:2055–2063CrossRefPubMedCentralPubMedGoogle Scholar
  34. 34.
    Alistar A, Chou JW, Nagalla S, Black MA, D’Agostino R Jr, Miller LD (2014) Dual roles for immune metagenes in breast cancer prognosis and therapy prediction. Genome Med 6:80CrossRefPubMedCentralPubMedGoogle Scholar
  35. 35.
    Bindea G, Mlecnik B, Tosolini M, Kirilovsky A, Waldner M, Obenauf AC, Angell H, Fredriksen T, Lafontaine L, Berger A, Bruneval P, Fridman WH, Becker C, Pages F, Speicher MR, Trajanoski Z, Galon J (2013) Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer. Immunity 39:782–795CrossRefPubMedGoogle Scholar
  36. 36.
    King C, Tangye SG, Mackay CR (2008) T follicular helper (TFH) cells in normal and dysregulated immune responses. Annu Rev Immunol 26:741–766CrossRefPubMedGoogle Scholar
  37. 37.
    Denkert C, von Minckwitz G, Brase JC, Sinn BV, Gade S, Kronenwett R, Pfitzner BM, Salat C, Loi S, Schmitt WD, Schem C, Fisch K, Darb-Esfahani S, Mehta K, Sotiriou C, Wienert S, Klare P, André F, Klauschen F, Blohmer JU, Krappmann K, Schmidt M, Tesch H, Kümmel S, Sinn P, Jackisch C, Dietel M, Reimer T, Untch M, Loibl S (2015) Tumor-infiltrating lymphocytes and response to neoadjuvant chemotherapy with or without carboplatin in human epidermal growth factor receptor 2-positive and triple-negative primary breast cancers. J Clin Oncol 33:983–991Google Scholar
  38. 38.
    West NR, Kost SE, Martin SD, Milne K, Deleeuw RJ, Nelson BH, Watson PH (2013) Tumour-infiltrating FOXP3(+) lymphocytes are associated with cytotoxic immune responses and good clinical outcome in oestrogen receptor-negative breast cancer. Br J Cancer 108:155–162CrossRefPubMedCentralPubMedGoogle Scholar
  39. 39.
    Salama P, Phillips M, Grieu F, Morris M, Zeps N, Joseph D, Platell C, Iacopetta B (2009) Tumor-infiltrating FOXP3+ T regulatory cells show strong prognostic significance in colorectal cancer. J Clin Oncol 27:186–192CrossRefPubMedGoogle Scholar
  40. 40.
    Jacquemier J, Bertucci F, Finetti P, Esterni B, Charafe-Jauffret E, Thibult ML, Houvenaeghel G, Van den Eynde B, Birnbaum D, Olive D, Xerri L (2012) High expression of indoleamine 2,3-dioxygenase in the tumour is associated with medullary features and favourable outcome in basal-like breast carcinoma. Int J Cancer 130:96–104CrossRefPubMedGoogle Scholar
  41. 41.
    Loi S, Michiels S, Salgado R, Sirtaine N, Jose V, Fumagalli D, Kellokumpu-Lehtinen PL, Bono P, Kataja V, Desmedt C, Piccart MJ, Loibl S, Denkert C, Smyth MJ, Joensuu H, Sotiriou C (2014) Tumor infiltrating lymphocytes are prognostic in triple negative breast cancer and predictive for trastuzumab benefit in early breast cancer: results from the FinHER trial. Ann Oncol 25:1544–1550CrossRefPubMedGoogle Scholar
  42. 42.
    Callahan MK, Postow MA, Wolchok JD (2014) CTLA-4 and PD-1 pathway blockade: combinations in the clinic. Front Oncol 4:385PubMedCentralPubMedGoogle Scholar
  43. 43.
    Terlizzi M, Casolaro V, Pinto A, Sorrentino R (2014) Inflammasome: cancer’s friend or foe? Pharmacol Ther 143:24–33CrossRefPubMedGoogle Scholar
  44. 44.
    Bhatelia K, Singh K, Singh R (2014) TLRs: linking inflammation and breast cancer. Cell Signal 26:2350–2357CrossRefPubMedGoogle Scholar
  45. 45.
    Azab B, Bhatt VR, Phookan J, Murukutla S, Kohn N, Terjanian T, Widmann WD (2012) Usefulness of the neutrophil-to-lymphocyte ratio in predicting short- and long-term mortality in breast cancer patients. Ann Surg Oncol 19:217–224CrossRefPubMedGoogle Scholar
  46. 46.
    Forget P, Machiels JP, Coulie PG, Berliere M, Poncelet AJ, Tombal B, Stainier A, Legrand C, Canon JL, Kremer Y, De Kock M (2013) Neutrophil:lymphocyte ratio and intraoperative use of ketorolac or diclofenac are prognostic factors in different cohorts of patients undergoing breast, lung, and kidney cancer surgery. Ann Surg Oncol 20(Suppl 3):S650–S660CrossRefPubMedGoogle Scholar
  47. 47.
    Noh H, Eomm M, Han A (2013) Usefulness of pretreatment neutrophil to lymphocyte ratio in predicting disease-specific survival in breast cancer patients. J Breast Cancer 16:55–59CrossRefPubMedCentralPubMedGoogle Scholar
  48. 48.
    Dirican A, Kucukzeybek BB, Alacacioglu A, Kucukzeybek Y, Erten C, Varol U, Somali I, Demir L, Bayoglu IV, Yildiz Y, Akyol M, Koyuncu B, Coban E, Ulger E, Unay FC, Tarhan MO (2015) Do the derived neutrophil to lymphocyte ratio and the neutrophil to lymphocyte ratio predict prognosis in breast cancer? Int J Clin Oncol 20:70–81Google Scholar
  49. 49.
    Sato H, Tsubosa Y, Kawano T (2012) Correlation between the pretherapeutic neutrophil to lymphocyte ratio and the pathologic response to neoadjuvant chemotherapy in patients with advanced esophageal cancer. World J Surg 36:617–622CrossRefPubMedGoogle Scholar
  50. 50.
    Jin H, Zhang G, Liu X, Chen C, Yu H, Huang X, Zhang Q, Yu J (2013) Blood neutrophil-lymphocyte ratio predicts survival for stages III–IV gastric cancer treated with neoadjuvant chemotherapy. World J Surg Oncol 11:112CrossRefPubMedCentralPubMedGoogle Scholar
  51. 51.
    Koh YW, Lee HJ, Ahn JH, Lee JW, Gong G (2014) Prognostic significance of the ratio of absolute neutrophil to lymphocyte counts for breast cancer patients with ER/PR-positivity and HER2-negativity in neoadjuvant setting. Tumour Biol 35:9823–9830CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Hee Jin Lee
    • 1
  • Jeong-Ju Lee
    • 2
  • In Hye Song
    • 1
  • In Ah Park
    • 1
  • Jun Kang
    • 3
  • Jong Han Yu
    • 4
  • Jin-Hee Ahn
    • 5
  • Gyungyub Gong
    • 1
    Email author
  1. 1.Department of Pathology, Asan Medical CenterUniversity of Ulsan College of MedicineSeoulKorea
  2. 2.Department of PathologyMyongji HospitalGoyangKorea
  3. 3.Department of Pathology, Incheon St. Mary’s Hospital, College of MedicineThe Catholic University of KoreaIncheonKorea
  4. 4.Department of Surgery, Asan Medical CenterUniversity of Ulsan College of MedicineSeoulKorea
  5. 5.Department of OncologyUniversity of Ulsan College of Medicine, Asan Medical CenterSeoulKorea

Personalised recommendations