Diffuse distribution of tumor-infiltrating lymphocytes is a marker for better prognosis and chemotherapeutic effect in triple-negative breast cancer
High-density tumor-infiltrating lymphocytes (TILs) are a prognostic marker for triple-negative breast cancer (TNBC). However, lymphocytic infiltration is heterogeneous in its pattern. We aimed to explore the utility of TIL distribution patterns against TIL density for predicting TNBC prognosis and chemotherapeutic effects.
Primary invasive TNBC cases were retrieved from a single institutional cohort, and archived samples were reviewed by two board-certificated pathologists. We used 154 consecutive surgical specimens from patients with standard adjuvant therapy, and 80 biopsies taken before primary systemic chemotherapy. The average density of stromal TILs was scored at 10% intervals, while the distribution pattern of TILs was evaluated as diffuse or non-diffuse. The association between TILs and prognosis or pathological complete response (pCR) was statistically analyzed.
A diffuse pattern of TILs at primary surgery correlated with better prognosis (relapse-free survival [RFS], hazard ratio [HR] 3.71, 95% confidence interval [CI] 1.60–8.57; overall survival [OS], HR 3.87, 95% CI 1.46–10.27), as well as high TIL density (≥ 50%; RFS, HR 4.51, 95% CI 2.06–9.90; OS, HR 3.28, 95% CI 1.32–8.14). Diffuse TIL pattern and nodal status were independent prognostic factors in multivariate analysis. Diffuse TIL pattern upon biopsy was associated with higher pCR rate (diffuse, 46%; non-diffuse, 21%; P = 0.032). All high TIL cases had diffuse patterns and the best outcome. Interobserver concordance was moderate (k = 0.53–0.55; distribution pattern) to good (weighted k = 0.67–0.69; density), and it was faster to assess the distribution pattern than to assess the density of TIL.
Showing similar clinical impacts to the TIL density, diffuse TILs could be a predictive marker for better prognosis and higher pCR. The assessment of TIL distribution pattern is simple, faster, and practical. Heterogeneous tumor immunity may contribute to further stratification of TNBC treatment.
KeywordsTumor-infiltrating lymphocytes Triple-negative breast cancer Heterogeneous tumor immunity Pathological complete response Interobserver concordance
Triple-negative breast cancer
Human epithelial growth factor receptor 2
Pathological complete response
Primary systemic chemotherapy
Lymphocytic-predominant breast cancer
The authors would like to thank S. Haraguchi at the research center of Sagara Hospital for extracting the necessary information from the database, and the technicians at the Department of Pathology at Sagara Hospital for their assistance in dealing with archived samples. We thank H. Nikki March, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.
AH, KA, and YO planned the study. AH and TW conducted pathological assessments. Yasuaki S., Yoshiaki S., and MK performed biopsies and/or surgeries, and provided oncological treatment. KA supervised the whole study. The manuscript was mainly written by AH, and amended by YO and AT. All authors read and approved the final manuscript.
This study was funded by the Division of Clinical Research Promotion at the National Hospital Organization Shikoku Cancer Center.
Compliance with ethical standards
Conflict of interest
AH received personal fees as honoraria from Chugai Pharmaceutical, Taiho Pharmaceutical, and Novartis Pharma. YS received personal fees as honoraria from AstraZeneca, Chugai Pharmaceutical, Pfizer, Eisai, Novartis Pharma, Taiho Pharmaceutical, and Takeda Pharmaceutical. MK received personal fees as honoraria from Chugai Pharmaceutical, Eisai, AstraZeneca, Pfizer, Taiho Pharmaceutical, Novartis Pharma, Takeda Pharmaceutical, Daiichi Sankyo, Kyowa Hakko Kirin, Shionogi, and Asashi Kasei. KA received personal fees as honoraria from Chugai Pharmaceutical, Eisai, AstraZeneca, Taiho Pharmaceutical, Novartis Pharma, Daiichi Sankyo, Mochida Pharmaceutical, Ono Pharmaceutical, and Eli Lilly Japan, and his institution received research funds from Chugai Pharmaceutical, Eisai and Sanofi. The other authors have no competing interests to declare.
All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional research committee (No. 14-06 & 17-33) and with the 1964 Helsinki declaration and its later amendments.
Formal consent was not required for this type of study.
- 1.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(7):860–867. https://doi.org/10.1200/JCO.2011.41.0902 Google Scholar
- 2.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, Andre F, Klauschen F, Blohmer JU, Krappmann K, Schmidt M, Tesch H, Kummel 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(9):983–991. https://doi.org/10.1200/JCO.2014.58.1967 Google Scholar
- 3.Adams S, Gray RJ, Demaria S, Goldstein L, Perez EA, Shulman LN, Martino S, Wang M, Jones VE, Saphner TJ, Wolff AC, Wood WC, Davidson NE, Sledge GW, Sparano JA, Badve SS (2014) Prognostic value of tumor-infiltrating lymphocytes in triple-negative breast cancers from two phase III randomized adjuvant breast cancer trials: ECOG 2197 and ECOG 1199. J Clin Oncol 32(27):2959–2966. https://doi.org/10.1200/JCO.2013.55.0491 Google Scholar
- 5.Loi S, Drubay D, Adams S, Pruneri G, Francis PA, Lacroix-Triki M, Joensuu H, Dieci MV, Badve S, Demaria S, Gray R, Munzone E, Lemonnier J, Sotiriou C, Piccart MJ, Kellokumpu-Lehtinen PL, Vingiani A, Gray K, Andre F, Denkert C, Salgado R, Michiels S (2019) Tumor-infiltrating lymphocytes and prognosis: a pooled individual patient analysis of early-stage triple-negative breast cancers. J Clin Oncol 37(7):559–569. https://doi.org/10.1200/jco.18.01010 Google Scholar
- 7.Morris GJ, Naidu S, Topham AK, Guiles F, Xu Y, McCue P, Schwartz GF, Park PK, Rosenberg AL, Brill K, Mitchell EP (2007) Differences in breast carcinoma characteristics in newly diagnosed African-American and Caucasian patients: a single-institution compilation compared with the National Cancer Institute’s surveillance, epidemiology, and end results database. Cancer 110(4):876–884. https://doi.org/10.1002/cncr.22836 Google Scholar
- 11.Cimino-Mathews A, Thompson E, Taube JM, Ye X, Lu Y, Meeker A, Xu H, Sharma R, Lecksell K, Cornish TC, Cuka N, Argani P, Emens LA (2016) PD-L1 (B7-H1) expression and the immune tumor microenvironment in primary and metastatic breast carcinomas. Hum Pathol 47(1):52–63. https://doi.org/10.1016/j.humpath.2015.09.003 Google Scholar
- 12.Zacharakis N, Chinnasamy H, Black M, Xu H, Lu YC, Zheng Z, Pasetto A, Langhan M, Shelton T, Prickett T, Gartner J, Jia L, Trebska-McGowan K, Somerville RP, Robbins PF, Rosenberg SA, Goff SL, Feldman SA (2018) Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer. Nat Med 24(6):724–730. https://doi.org/10.1038/s41591-018-0040-8 Google Scholar
- 13.Schmid P, Adams S, Rugo HS, Schneeweiss A, Barrios CH, Iwata H, Dieras V, Hegg R, Im SA, Shaw Wright G, Henschel V, Molinero L, Chui SY, Funke R, Husain A, Winer EP, Loi S, Emens LA (2018) Atezolizumab and nab-paclitaxel in advanced triple-negative breast cancer. N Engl J Med 379(22):2108–2121. https://doi.org/10.1056/NEJMoa1809615 Google Scholar
- 15.Denkert C, von Minckwitz G, Darb-Esfahani S, Lederer B, Heppner BI, Weber KE, Budczies J, Huober J, Klauschen F, Furlanetto J, Schmitt WD, Blohmer JU, Karn T, Pfitzner BM, Kummel S, Engels K, Schneeweiss A, Hartmann A, Noske A, Fasching PA, Jackisch C, van Mackelenbergh M, Sinn P, Schem C, Hanusch C, Untch M, Loibl S (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. https://doi.org/10.1016/s1470-2045(17)30904-x Google Scholar
- 16.Salgado R, Denkert C, Demaria S, Sirtaine N, Klauschen F, Pruneri G, Wienert S, VandenEynden G, Baehner FL, Penault-Llorca F, Perez EA, Thompson EA, Symmans WF, Richardson AL, Brock J, Criscitiello C, Bailey H, Ignatiadis M, Floris G, Sparano J, Kos Z, Nielsen T, Rimm DL, Allison KH, Reis-Filho JS, Loibl S, Sotiriou C, Viale G, Badve S, Adams S, Willard-Gallo K, Loi S, International TWG (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. https://doi.org/10.1093/annonc/mdu450 Google Scholar
- 19.Greer LT, Rosman M, Mylander WC, Hooke J, Kovatich A, Sawyer K, Buras RR, Shriver CD, Tafra L (2013) Does breast tumor heterogeneity necessitate further immunohistochemical staining on surgical specimens? J Am Coll Surg 216(2):239–251. https://doi.org/10.1016/j.jamcollsurg.2012.09.007 Google Scholar
- 20.Hou Y, Nitta H, Wei L, Banks PM, Portier B, Parwani AV, Li Z (2017) HER2 intratumoral heterogeneity is independently associated with incomplete response to anti-HER2 neoadjuvant chemotherapy in HER2-positive breast carcinoma. Breast Cancer Res Treat 166(2):447–457. https://doi.org/10.1007/s10549-017-4453-8 Google Scholar
- 24.Hida AI, Sagara Y, Yotsumoto D, Kanemitsu S, Kawano J, Baba S, Rai Y, Oshiro Y, Aogi K, Sagara Y, Ohi Y (2016) Prognostic and predictive impacts of tumor-infiltrating lymphocytes differ between Triple-negative and HER2-positive breast cancers treated with standard systemic therapies. Breast Cancer Res Treat 158(1):1–9. https://doi.org/10.1007/s10549-016-3848-2 Google Scholar
- 25.Watanabe T, Hida AI, Inoue N, Imamura M, Fujimoto Y, Akazawa K, Hirota S, Miyoshi Y (2018) Abundant tumor infiltrating lymphocytes after primary systemic chemotherapy predicts poor prognosis in estrogen receptor-positive/HER2-negative breast cancers. Breast Cancer Res Treat 168(1):135–145. https://doi.org/10.1007/s10549-017-4575-z Google Scholar
- 26.Hida AI, Mizuno Y, Ueda N, Oshiro Y, Sugita A, Matsukage S, Maeda T, Kito K, Ohtsuki Y, Aogi K (2017) Simple assessment reveals the importance of distribution and density of tumor-infiltrating lymphocytes in triple-negative breast cancer. Breast 32:S33–S34. https://doi.org/10.1016/S0960-9776(17)30134-0 Google Scholar
- 27.Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, Dowsett M, Fitzgibbons PL, Hanna WM, Langer A, McShane LM, Paik S, Pegram MD, Perez EA, Press MF, Rhodes A, Sturgeon C, Taube SE, Tubbs R, Vance GH, van de Vijver M, Wheeler TM, Hayes DF, American Society of Clinical O, College of American P (2007) American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol 25(1):118–145. https://doi.org/10.1200/jco.2006.09.2775 Google Scholar
- 28.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, American Society of Clinical O, College of American P (2013) 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. J Clin Oncol 31(31):3997–4013. https://doi.org/10.1200/jco.2013.50.9984 Google Scholar
- 30.Dieci MV, Criscitiello C, Goubar A, Viale G, Conte P, Guarneri V, Ficarra G, Mathieu MC, Delaloge S, Curigliano G, Andre F (2014) Prognostic value of tumor-infiltrating lymphocytes on residual disease after primary chemotherapy for triple-negative breast cancer: a retrospective multicenter study. Ann Oncol 25(3):611–618. https://doi.org/10.1093/annonc/mdt556 Google Scholar
- 31.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(1):105–113. https://doi.org/10.1200/JCO.2009.23.7370 Google Scholar
- 32.Pruneri G, Gray KP, Vingiani A, Viale G, Curigliano G, Criscitiello C, Lang I, Ruhstaller T, Gianni L, Goldhirsch A, Kammler R, Price KN, Cancello G, Munzone E, Gelber RD, Regan MM, Colleoni M (2016) Tumor-infiltrating lymphocytes (TILs) are a powerful prognostic marker in patients with triple-negative breast cancer enrolled in the IBCSG phase III randomized clinical trial 22-00. Breast Cancer Res Treat 158(2):323–331. https://doi.org/10.1007/s10549-016-3863-3 Google Scholar
- 33.Miyashita M, Sasano H, Tamaki K, Hirakawa H, Takahashi Y, Nakagawa S, Watanabe G, Tada H, Suzuki A, Ohuchi N, Ishida T (2015) Prognostic significance of tumor-infiltrating CD8+ and FOXP3+ lymphocytes in residual tumors and alterations in these parameters after neoadjuvant chemotherapy in triple-negative breast cancer: a retrospective multicenter study. Breast Cancer Res 17:124. https://doi.org/10.1186/s13058-015-0632-x Google Scholar
- 34.Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pages C, Tosolini M, Camus M, Berger A, Wind P, Zinzindohoue F, Bruneval P, Cugnenc PH, Trajanoski Z, Fridman WH, Pages F (2006) Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 313(5795):1960–1964. https://doi.org/10.1126/science.1129139 Google Scholar
- 35.Zaretsky JM, Garcia-Diaz A, Shin DS, Escuin-Ordinas H, Hugo W, Hu-Lieskovan S, Torrejon DY, Abril-Rodriguez G, Sandoval S, Barthly L, Saco J, Homet Moreno B, Mezzadra R, Chmielowski B, Ruchalski K, Shintaku IP, Sanchez PJ, Puig-Saus C, Cherry G, Seja E, Kong X, Pang J, Berent-Maoz B, Comin-Anduix B, Graeber TG, Tumeh PC, Schumacher TN, Lo RS, Ribas A (2016) Mutations Associated with acquired resistance to PD-1 Blockade in Melanoma. N Engl J Med 375(9):819–829. https://doi.org/10.1056/NEJMoa1604958 Google Scholar
- 36.Konig L, Mairinger FD, Hoffmann O, Bittner AK, Schmid KW, Kimmig R, Kasimir-Bauer S, Bankfalvi A (2019) Dissimilar patterns of tumor-infiltrating immune cells at the invasive tumor front and tumor center are associated with response to neoadjuvant chemotherapy in primary breast cancer. BMC Cancer 19(1):120. https://doi.org/10.1186/s12885-019-5320-2 Google Scholar
- 37.Masuda N, Lee SJ, Ohtani S, Im YH, Lee ES, Yokota I, Kuroi K, Im SA, Park BW, Kim SB, Yanagita Y, Ohno S, Takao S, Aogi K, Iwata H, Jeong J, Kim A, Park KH, Sasano H, Ohashi Y, Toi M (2017) Adjuvant capecitabine for breast cancer after preoperative chemotherapy. N Engl J Med 376(22):2147–2159. https://doi.org/10.1056/NEJMoa1612645 Google Scholar
- 38.Swisher SK, Wu Y, Castaneda CA, Lyons GR, Yang F, Tapia C, Wang X, Casavilca SA, Bassett R, Castillo M, Sahin A, Mittendorf EA (2016) Interobserver agreement between pathologists assessing tumor-infiltrating lymphocytes (TILs) in breast cancer using methodology proposed by the International TILs Working Group. Ann Surg Oncol 23(7):2242–2248. https://doi.org/10.1245/s10434-016-5173-8 Google Scholar
- 40.Steele KE, Tan TH, Korn R, Dacosta K, Brown C, Kuziora M, Zimmermann J, Laffin B, Widmaier M, Rognoni L, Cardenes R, Schneider K, Boutrin A, Martin P, Zha J, Wiestler T (2018) Measuring multiple parameters of CD8+ tumor-infiltrating lymphocytes in human cancers by image analysis. J Immunother Cancer 6(1):20. https://doi.org/10.1186/s40425-018-0326-x Google Scholar
- 41.Klauschen F, Muller KR, Binder A, Bockmayr M, Hagele M, Seegerer P, Wienert S, Pruneri G, de Maria S, Badve S, Michiels S, Nielsen TO, Adams S, Savas P, Symmans F, Willis S, Gruosso T, Park M, Haibe-Kains B, Gallas B, Thompson AM, Cree I, Sotiriou C, Solinas C, Preusser M, Hewitt SM, Rimm D, Viale G, Loi S, Loibl S, Salgado R, Denkert C (2018) Scoring of tumor-infiltrating lymphocytes: From visual estimation to machine learning. Semin Cancer Biol 52(Pt 2):151–157. https://doi.org/10.1016/j.semcancer.2018.07.001 Google Scholar