Pitfalls in machine learning-based assessment of tumor-infiltrating lymphocytes in breast cancer: A report of the International Immuno-Oncology Biomarker Working Group on Breast Cancer.
The clinical significance of the tumor-immune interaction in breast cancer is now established, and tumor-infiltrating lymphocytes (TILs) have emerged as predictive and prognostic biomarkers for patients with triple-negative (estrogen receptor, progesterone receptor, and HER2-negative) breast cancer and HER2-positive breast cancer. How computational assessments of TILs might complement manual TIL assessment in trial and daily practices is currently debated. Recent efforts to use machine learning (ML) to automatically evaluate TILs have shown promising results. We review state-of-the-art approaches and identify pitfalls and challenges of automated TIL evaluation by studying the root cause of ML discordances in comparison to manual TIL quantification. We categorize our findings into four main topics: (1) technical slide issues, (2) ML and image analysis aspects, (3) data challenges, and (4) validation issues. The main reason for discordant assessments is the inclusion of false-positive areas or cells identified by performance on certain tissue patterns or design choices in the computational implementation. To aid the adoption of ML for TIL assessment, we provide an in-depth discussion of ML and image analysis, including validation issues that need to be considered before reliable computational reporting of TILs can be incorporated into the trial and routine clinical management of patients with triple-negative breast cancer. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Thagaard J
，Broeckx G
，Page DB
，Jahangir CA
，Verbandt S
，Kos Z
，Gupta R
，Khiroya R
，Abduljabbar K
，Acosta Haab G
，Acs B
，Akturk G
，Almeida JS
，Alvarado-Cabrero I
，Amgad M
，Azmoudeh-Ardalan F
，Badve S
，Baharun NB
，Balslev E
，Bellolio ER
，Bheemaraju V
，Blenman KR
，Botinelly Mendonça Fujimoto L
，Bouchmaa N
，Burgues O
，Chardas A
，Chon U Cheang M
，Ciompi F
，Cooper LA
，Coosemans A
，Corredor G
，Dahl AB
，Dantas Portela FL
，Deman F
，Demaria S
，Doré Hansen J
，Dudgeon SN
，Ebstrup T
，Elghazawy M
，Fernandez-Martín C
，Fox SB
，Gallagher WM
，Giltnane JM
，Gnjatic S
，Gonzalez-Ericsson PI
，Grigoriadis A
，Halama N
，Hanna MG
，Harbhajanka A
，Hart SN
，Hartman J
，Hauberg S
，Hewitt S
，Hida AI
，Horlings HM
，Husain Z
，Hytopoulos E
，Irshad S
，Janssen EA
，Kahila M
，Kataoka TR
，Kawaguchi K
，Kharidehal D
，Khramtsov AI
，Kiraz U
，Kirtani P
，Kodach LL
，Korski K
，Kovács A
，Laenkholm AV
，Lang-Schwarz C
，Larsimont D
，Lennerz JK
，Lerousseau M
，Li X
，Ly A
，Madabhushi A
，Maley SK
，Manur Narasimhamurthy V
，Marks DK
，McDonald ES
，Mehrotra R
，Michiels S
，Minhas FUAA
，Mittal S
，Moore DA
，Mushtaq S
，Nighat H
，Papathomas T
，Penault-Llorca F
，Perera RD
，Pinard CJ
，Pinto-Cardenas JC
，Pruneri G
，Pusztai L
，Rahman A
，Rajpoot NM
，Rapoport BL
，Rau TT
，Reis-Filho JS
，Ribeiro JM
，Rimm D
，Roslind A
，Vincent-Salomon A
，Salto-Tellez M
，Saltz J
，Sayed S
，Scott E
，Siziopikou KP
，Sotiriou C
，Stenzinger A
，Sughayer MA
，Sur D
，Fineberg S
，Symmans F
，Tanaka S
，Taxter T
，Tejpar S
，Teuwen J
，Thompson EA
，Tramm T
，Tran WT
，van der Laak J
，van Diest PJ
，Verghese GE
，Viale G
，Vieth M
，Wahab N
，Walter T
，Waumans Y
，Wen HY
，Yang W
，Yuan Y
，Zin RM
，Adams S
，Bartlett J
，Loibl S
，Denkert C
，Savas P
，Loi S
，Salgado R
，Specht Stovgaard E
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The path to a better biomarker: application of a risk management framework for the implementation of PD-L1 and TILs as immuno-oncology biomarkers in breast cancer clinical trials and daily practice.
Immune checkpoint inhibitor therapies targeting PD-1/PD-L1 are now the standard of care in oncology across several hematologic and solid tumor types, including triple negative breast cancer (TNBC). Patients with metastatic or locally advanced TNBC with PD-L1 expression on immune cells occupying ≥1% of tumor area demonstrated survival benefit with the addition of atezolizumab to nab-paclitaxel. However, concerns regarding variability between immunohistochemical PD-L1 assay performance and inter-reader reproducibility have been raised. High tumor-infiltrating lymphocytes (TILs) have also been associated with response to PD-1/PD-L1 inhibitors in patients with breast cancer (BC). TILs can be easily assessed on hematoxylin and eosin-stained slides and have shown reliable inter-reader reproducibility. As an established prognostic factor in early stage TNBC, TILs are soon anticipated to be reported in daily practice in many pathology laboratories worldwide. Because TILs and PD-L1 are parts of an immunological spectrum in BC, we propose the systematic implementation of combined PD-L1 and TIL analyses as a more comprehensive immuno-oncological biomarker for patient selection for PD-1/PD-L1 inhibition-based therapy in patients with BC. Although practical and regulatory considerations differ by jurisdiction, the pathology community has the responsibility to patients to implement assays that lead to optimal patient selection. We propose herewith a risk-management framework that may help mitigate the risks of suboptimal patient selection for immuno-therapeutic approaches in clinical trials and daily practice based on combined TILs/PD-L1 assessment in BC. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Gonzalez-Ericsson PI
，Stovgaard ES
，Sua LF
，Reisenbichler E
，Kos Z
，Carter JM
，Michiels S
，Le Quesne J
，Nielsen TO
，Laenkholm AV
，Fox SB
，Adam J
，Bartlett JM
，Rimm DL
，Quinn C
，Peeters D
，Dieci MV
，Vincent-Salomon A
，Cree I
，Hida AI
，Balko JM
，Haynes HR
，Frahm I
，Acosta-Haab G
，Balancin M
，Bellolio E
，Yang W
，Kirtani P
，Sugie T
，Ehinger A
，Castaneda CA
，Kok M
，McArthur H
，Siziopikou K
，Badve S
，Fineberg S
，Gown A
，Viale G
，Schnitt SJ
，Pruneri G
，Penault-Llorca F
，Hewitt S
，Thompson EA
，Allison KH
，Symmans WF
，Bellizzi AM
，Brogi E
，Moore DA
，Larsimont D
，Dillon DA
，Lazar A
，Lien H
，Goetz MP
，Broeckx G
，El Bairi K
，Harbeck N
，Cimino-Mathews A
，Sotiriou C
，Adams S
，Liu SW
，Loibl S
，Chen IC
，Lakhani SR
，Juco JW
，Denkert C
，Blackley EF
，Demaria S
，Leon-Ferre R
，Gluz O
，Zardavas D
，Emancipator K
，Ely S
，Loi S
，Salgado R
，Sanders M
，International Immuno-Oncology Biomarker Working Group
... -
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The journey of tumor-infiltrating lymphocytes as a biomarker in breast cancer: clinical utility in an era of checkpoint inhibition.
In 2014, we described a method to quantify percentage of tumor-infiltrating lymphocytes (TILs) on hematoxylin and eosin-stained slides of breast cancer samples using light microscopy that could be performed easily by pathologists with no extra stains. The aim of detailing the method was to facilitate independent research groups replicating our prognostic findings using TIL quantity in early-stage breast cancers. A global working group of breast pathologists was convened to standardize, test reproducibility, and refine the method. A website was also established which allowed free training (www.tilsinbreastcancer.org). As a result of this work, TIL data have been collected in over 20 000 primary breast cancer samples worldwide and the robust associations with better prognoses in triple-negative breast cancer (TNBC) and HER2+ BC have been confirmed. This has resulted in the inclusion of the TIL biomarker in several international breast cancer guidelines as well as in national criteria for routine pathology reporting. TIL therefore represents the first biological prognostic biomarker for early-stage TNBCs, and here its prognostic effect is linear, with values of 30%-50% being suggested as suitable for use in potential chemotherapy de-escalation studies. The efficacy of immune checkpoint-targeted agents in breast cancer now provides direct evidence that host immune responses can modify tumor growth in some patients. With the recent granting of accelerated approvals for the first PD-1/PD-L1 targeting agents in early and advanced TNBC, our focus has now moved to investigating the clinical utility of TIL in the setting of immune checkpoint agents, with or without PD-L1 protein assessment. Emerging data suggest that TIL quantity can help clinicians identify patients with breast cancer who benefit most from PD-1/PD-L1 inhibition. In patients with advanced TNBC and HER2+ disease a TIL cut-off of 5% or 10%, with PD-L1 expression can define 'immune-enriched' tumors and currently seems to have the most clinical relevance in this context.
Loi S
，Michiels S
，Adams S
，Loibl S
，Budczies J
，Denkert C
，Salgado R
... -
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Tumour-infiltrating lymphocytes and prognosis in different subtypes of breast cancer: a pooled analysis of 3771 patients treated with neoadjuvant therapy.
Tumour-infiltrating lymphocytes (TILs) are predictive for response to neoadjuvant chemotherapy in triple-negative breast cancer (TNBC) and HER2-positive breast cancer, but their role in luminal breast cancer and the effect of TILs on prognosis in all subtypes is less clear. Here, we assessed the relevance of TILs for chemotherapy response and prognosis in patients with TNBC, HER2-positive breast cancer, and luminal-HER2-negative breast cancer.
Patients with primary breast cancer who were treated with neoadjuvant combination chemotherapy were included from six randomised trials done by the German Breast Cancer Group. Pretherapeutic core biopsies from 3771 patients included in these studies were assessed for the number of stromal TILs by standardised methods according to the guidelines of the International TIL working group. TILs were analysed both as a continuous parameter and in three predefined groups of low (0-10% immune cells in stromal tissue within the tumour), intermediate (11-59%), and high TILs (≥60%). We used these data in univariable and multivariable statistical models to assess the association between TIL concentration and pathological complete response in all patients, and between the amount of TILs and disease-free survival and overall survival in 2560 patients from five of the six clinical trial cohorts.
In the luminal-HER2-negative breast cancer subtype, a pathological complete response (pCR) was achieved in 45 (6%) of 759 patients with low TILs, 48 (11%) of 435 with intermediate TILs, and 49 (28%) of 172 with high TILs. In the HER2-positive subtype, pCR was observed in 194 (32%) of 605 patients with low TILs, 198 (39%) of 512 with intermediate TILs, and 127 (48%) of 262 with high TILs. Finally, in the TNBC subtype, pCR was achieved in 80 (31%) of 260 patients with low TILs, 117 (31%) of 373 with intermediate TILs, and 136 (50%) of 273 with high TILs (p<0·0001 for each subtype, χ2 test for trend). In the univariable analysis, a 10% increase in TILs was associated with longer disease-free survival in TNBC (hazard ratio [HR] 0·93 [95% CI 0·87-0·98], p=0·011) and HER2-positive breast cancer (0·94 [0·89-0·99], p=0·017), but not in luminal-HER2-negative tumours (1·02 [0·96-1·09], p=0·46). The increase in TILs was also associated with longer overall survival in TNBC (0·92 [0·86-0·99], p=0·032), but had no association in HER2-positive breast cancer (0·94 [0·86-1·02], p=0·11), and was associated with shorter overall survival in luminal-HER2-negative tumours (1·10 [1·02-1·19], p=0·011).
Increased TIL concentration predicted response to neoadjuvant chemotherapy in all molecular subtypes assessed, and was also associated with a survival benefit in HER2-positive breast cancer and TNBC. By contrast, increased TILs were an adverse prognostic factor for survival in luminal-HER2-negative breast cancer, suggesting a different biology of the immunological infiltrate in this subtype. Our data support the hypothesis that breast cancer is immunogenic and might be targetable by immune-modulating therapies. In light of the results in luminal breast cancer, further research investigating the interaction of the immune system with different types of endocrine therapy is warranted.
Deutsche Krebshilfe and European Commission.
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
，Kümmel 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
... -
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