# The tale of TILs in breast cancer: A report from The International Immuno-Oncology Biomarker Working Group

Khalid El Bairi, Harry Haynes, Elizabeth Blackley, Susan Fineberg, Jeffrey Shear, Sophia Turner, Juliana Ribeiro de Freitas, Daniel Sur, Luis Claudio Amendola, Masoumeh Gharib, et al.

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## To cite this version:

Khalid El Bairi, Harry Haynes, Elizabeth Blackley, Susan Fineberg, Jeffrey Shear, et al.. The tale of TILs in breast cancer: A report from The International Immuno-Oncology Biomarker Working Group. npj Breast Cancer, 2021, 7 (1), pp.150. 10.1038/s41523-021-00346-1. hal-03467047

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Submitted on 19 Dec 2023

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# The tale of TILs in breast cancer: A report from The International Immuno-Oncology Biomarker Working Group

Khalid El Bairi<sup>1,3</sup>, Harry R. Haynes<sup>2,3</sup>, Elizabeth Blackley<sup>4</sup>, Susan Fineberg<sup>5</sup>, Jeffrey Shear<sup>6</sup>, Sophia Turner<sup>7</sup>, Juliana Ribeiro de Freitas<sup>8</sup>, Daniel Sur<sup>9</sup>, Luis Claudio Amendola<sup>10</sup>, Masoumeh Gharib<sup>11</sup>, Amine Kallala<sup>12</sup>, Indu Arun<sup>13</sup>, Farid Azmoudeh-Ardalan<sup>14</sup>, Luciana Fujimoto<sup>15</sup>, Luz F. Sua<sup>16</sup>, Shi-Wei Liu<sup>17</sup>, Huang-Chun Lien<sup>18</sup>, Pawan Kirtani<sup>19</sup>, Marcelo Balancin<sup>20</sup>, Hicham El Attar<sup>21</sup>, Prerna Guleria<sup>22</sup>, Wenxian Yang<sup>23</sup>, Emad Shash<sup>24</sup>, I-Chun Chen<sup>25,26</sup>, Veronica Bautista<sup>27</sup>, Jose Fernando Do Prado Moura<sup>28</sup>, Bernardo L. Rapoport<sup>29,30</sup>, Carlos Castaneda<sup>31,32</sup>, Eunice Spengler<sup>33</sup>, Gabriela Acosta-Haab<sup>34</sup>, Isabel Frahm<sup>35</sup>, Joselyn Sanchez<sup>36</sup>, Miluska Castillo<sup>36</sup>, Najat Bouchmaa<sup>37</sup>, Reena R. Md Zin<sup>38</sup>, Ruohong Shui<sup>39</sup>, Timothy Onyuma<sup>40</sup>, Wentao Yang<sup>39</sup>, Zaheed Husain<sup>41</sup>, Karen Willard-Gallo<sup>42</sup>, An Coosemans<sup>43</sup>, Edith A. Perez<sup>44</sup>, Elena Provenzano<sup>45</sup>, Paula Gonzalez Ericsson<sup>46</sup>, Eduardo Richardet<sup>47</sup>, Ravi Mehrotra<sup>48</sup>, Sandra Sarancone<sup>49</sup>, Anna Ehinger<sup>50</sup>, David L. Rimm<sup>51</sup>, John M. S. Bartlett<sup>52,53,54</sup>, Giuseppe Viale<sup>55</sup>, Carsten Denkert<sup>56</sup>, Akira I. Hida<sup>57</sup>, Christos Sotiriou<sup>58</sup>, Sibylle Loibl<sup>59</sup>, Stephen M. Hewitt<sup>60</sup>, Sunil Badve<sup>61</sup>, William Fraser Symmans<sup>62</sup>, Rim S. Kim<sup>63</sup>, Giancarlo Pruneri<sup>64</sup>, Shom Goel<sup>4,65</sup>, Prudence A. Francis<sup>65,66</sup>, Gloria Inurrigarro<sup>67</sup>, Rin Yamaguchi<sup>68</sup>, Hernan Garcia-Rivello<sup>69</sup>, Hugo Horlings<sup>70</sup>, Said Afqir<sup>1</sup>, Roberto Salgado<sup>4,71</sup>, Sylvia Adams<sup>72</sup>, Marleen Kok<sup>73</sup>, Maria Vittoria Dieci<sup>74,75</sup>, Stefan Michiels<sup>76</sup>, Sandra Demaria<sup>77</sup>, Sherene Loi<sup>4,65</sup> and The International Immuno-Oncology Biomarker Working Group*

The advent of immune-checkpoint inhibitors (ICI) in modern oncology has significantly improved survival in several cancer settings. A subgroup of women with breast cancer (BC) has immunogenic infiltration of lymphocytes with expression of programmed death-ligand 1 (PD-L1). These patients may potentially benefit from ICI targeting the programmed death 1 (PD-1)/PD-L1 signaling axis. The use of tumor-infiltrating lymphocytes (TILs) as predictive and prognostic biomarkers has been under intense examination. Emerging data suggest that TILs are associated with response to both cytotoxic treatments and immunotherapy, particularly for patients with triple-negative BC. In this review from *The International Immuno-Oncology Biomarker Working Group*, we discuss (a) the biological understanding of TILs, (b) their analytical and clinical validity and efforts toward the clinical utility in BC, and (c) the current status of PD-L1 and TIL testing across different continents, including experiences from low-to-middle-income countries, incorporating also the view of a patient advocate. This information will help set the stage for future approaches to optimize the understanding and clinical utilization of TIL analysis in patients with BC.

npj Breast Cancer (2021)7:150; https://doi.org/10.1038/s41523-021-00346-1

### INTRODUCTION

The use of immune-checkpoint blockade (ICI) in clinical oncology has revolutionized patient care and improved survival outcomes in many patients with malignancies¹. This therapeutic strategy has significantly expanded in the setting of advanced and early-stage breast cancer (BC), but much more work is needed to optimize patient selection based on tumor-based biomarkers. The presence of tumor-infiltrating lymphocytes (TILs) is believed to be predictive of response to immunotherapy, chemotherapy, and other targeted therapies²,³ in addition to their role as a prognostic biomarker⁴,⁵. Moreover, TILs in the tumor and the surrounding microenvironment are thought to reflect ongoing anti-tumor host immune response. Three main categories of the tumor microenvironment (TME) have been defined across different tumor types: immune-desert ("cold" tumors largely devoid of lymphocytes), immune-excluded (lymphocytes are present in the peritumoral stroma only), and immune-infiltrated/inflamed ("hot" tumors)⁶,⁷. Conceptually, each of these TME categories reflects a specific interaction between the tumor genotype/phenotype and the host immune system, which can impact the response to both conventional anticancer therapies and ICI⁸. However, there is considerable heterogeneity within each TME category, adding uncertainty to the reproducibility of

the current classification of "cold" vs. "hot" vs. "intermediate" immune-subtypes. Also, there are no validated criteria to define these subtypes, either using morphology, immunostaining, transcriptomics, or their combination, limiting the impact of these descriptors in clinical trials and daily practice.

In BC, the molecular subtype of the tumor has a major influence on its interaction with the immune system. Triple-negative BC (TNBC) and HER2-positive BC are more frequently infiltrated by higher numbers of TILs than hormone receptor (HR)-positive tumors⁹,¹⁰. However, all BC subtypes include cases with TIL-infiltration. The degree of TIL infiltration has been hypothesized to reflect the tumor mutational burden (TMB), which is lower in HR-positive BC¹¹,¹². Higher TMB is linked to the expression of more neoantigens and has been shown to predict survival after ICI therapy in several cancer types, and recent evidence indicates this may also be the case for TNBC¹³⁻¹⁵. However, the correlation between mutational burden and immune composition is complex, with the degree and nature of clonality of the mutations playing a key role in determining whether they favor or hinder immune-mediated tumor control¹⁶. In TNBC, higher TMB and greater genomic heterogeneity have been associated with lower TILs¹⁷. Conceptually, this can be explained by immunoediting, which is a result of a selection of cancer cell clones with decreased
immunogenicity despite the presence of many mutations¹⁸. This escape from immune surveillance is associated with a reduced TIL component and increased tumor clonal heterogeneity, explaining the negative association between TMB and TILs¹⁹⁻²¹.

In TNBC, scoring TILs in the stromal compartment (sTILs) is demonstrably reproducible and generally well-correlated with intra-epithelial TILs, with higher stromal TILs (sTILs) predicting longer survival⁹,²²⁻²⁴. Generally, intra-epithelial TIL density tends to be lower than stromal TIL density²³, raising the question of whether a tumor nest-stromal barrier precludes more robust T cell infiltration²⁵, and/or whether the intra-epithelial TILs have always been present (in an inactive state), as tissue-resident T-cells. CD8+ tissue-resident memory T (TRM) cells were shown to mediate BC immunosurveillance²⁶. Notably, high BC infiltration by TILs contained CD8+ T lymphocytes with TRM patterns which highly express immune-checkpoint molecules²⁶.

Tertiary lymphoid structures (TLS) are lymph node-like structures that arise in tissues at sites of chronic inflammation²⁷. TLS has been detected in the stroma of up to 60% of BC, with the highest frequencies in HER2+ and TNBC⁷,²⁸,²⁹. These findings support a critical role for the stroma in shaping the TME of BC. For example, fibroblastic reticular cells, concentrated in the T cell zone of TLS, promote, maintain or suppress T cell activities via their cytokine and chemokine secretion³⁰. TLS architecture is distinguished by a T cell zone adjacent to a B cell follicle, similar to secondary lymphoid organs²⁷. Immune responses generated in tumor-associated TLS would thus produce immunological memory to multiple BC neoantigens and could potentially control the growth of disseminated tumor cells³¹⁻³⁴. However, there are significant concerns that TLS cannot be assessed in a reproducible manner by analysis of HE-stained slides, and that B- and T-cell immunostains are needed³⁴. In addition, TLS is frequently found at the tumor perimeter, often contain high endothelial venules, and when present in the tumor area are generally considered as an aggregate when quantifying TILs in BC. Their role in BC still remains to be addressed.

The importance of expanding our current understanding of the complex TME in breast and other cancers has driven the development of diverse techniques, including molecular biomics profiling³⁵ coupled with computational deconvolution of immune cell populations³⁶,³⁷, global and single-cell transcriptomics³⁸,³⁹, and multiplex imaging⁴⁰, to quantify TIL distribution, functional orientation and relative frequencies in individual tumor types. However, these cutting-edge investigational tools are often limited by reproducibility across studies, particularly when attempting to resolve specific immune cell subtypes. In addition, tumors with low TILs seem to pose a significant challenge to any of these techniques and platforms⁴¹. Machine learning techniques are in development to evaluate TIL distribution patterns and integrate the spatial information with sTILs and with molecular profiling data⁴²,⁴³. A study examining clonal heterogeneity, TMB, copy number variations, somatic mutations, and germline polymorphisms, as well as the neoantigen load for their association with immune metagene expression in the BC subtypes, did not find any distinct recurrent single gene or pathway level mutations associated with immune infiltration²¹. However, lower clonal heterogeneity was observed in TNBC and HER2+ BC associated with higher immune gene expression, which is consistent with immunoediting²¹. There is also evidence for immune escape during the in situ to invasive BC transition, with a decrease in immune activation measurable using a combination of global profiling and single-cell transcriptomics⁴⁴. It may be hypothesized that similar immune evasion mechanisms also occur during the transition from stage I TNBC to more advanced stages of the disease. This paradigm was also recently reported in lung cancer in which immune-evasion seems to be triggered by neoantigen editing during tumor evolution⁴⁵. In the remainder of this review, the analytical and clinical impact of sTILs in BC

management will be discussed based on recent updates from human studies, particularly clinical trials.

# UPDATES ON THE ANALYTICAL AND CLINICAL VALIDITY OF TILS

## Challenges in establishing the analytical validity of TILs

Analytical validity is defined as how accurately a test predicts the presence or absence of a biological variable. In other words, can the “test” correctly distinguish between TILs and other immune cells? Previous TIL inter-pathologists-reproducibility studies (RING-studies) have shown that pathologists (using an H&E slide) can reliably assess TILs with very high concordance between many pathologists on a powered number of cases and at different cut-offs²⁴. There is overwhelming evidence for the clinical validity of this evaluation⁴⁶,⁴⁷. However, analytical validity cannot be formally demonstrated due to the lack of a “gold standard” against which to assess the proposed method. This may not be that important for the clinical implementation of sTILs. However, for machine learning approaches this question is crucial. Comparing two methods, for example, a pathologist vs. an automated image-analysis system provides evidence on concordance, but not accuracy⁴³. All the previous TIL-RING studies have been performed with the assumption of histological accuracy⁴⁶,⁴⁷.

In order to define accuracy in the context of assessing TILs using an H&E-stained slide, it is necessary to define a “gold standard” against which to assess the proposed method, i.e., an orthogonal-type method which is used as the “gold standard”. For example, a pan-lymphocyte marker can establish the accuracy of lymphocyte identification, answering “how often is what a pathologist calls a TIL actually a TIL?”. This is crucial when applying machine learning methods to identify TILs⁴², since the unequivocal differentiation of a myofibroblast, an invasive lobular carcinoma (ILC) cell, and lymphocyte is not always possible on an H&E slide. Moreover, the concordance between TIL assessment by machine learning-based methods and pathologist-TIL scores is unlikely to be 100%⁴⁸. Currently, a TIL is defined as being a lymphocyte or a plasma cell, and both are scored and defined according to classic morphological definitions. Then, we need to define a “stromal TIL” (sTIL), i.e., what is the maximal distance between a tumor cell and a TIL to define it as an sTIL. Furthermore, uncertainty amongst pathologists exists about the inclusion of stroma abutting the tumor, or all of the stroma within the total tumoral area to include intervening stroma with low/very low sTILs. Indeed, accumulating evidence suggests that sTILs touching the tumor cells may have a different molecular phenotype than sTILs distant to the main tumor bulk²⁵. Kos et al.²⁴ have recently reported further factors that may impact the assessment of TILs including pre- and post-analytical histology factors, particularly in the setting of retrospective analysis of trial material, and the recognition of common artifacts. In an effort to improve concordance, the International Immuno-Oncology Biomarker Working Group, also called the TILs Working Group (https://www.tilsinbreastcancer.org/pitfalls/) recently provided reference images and digital slides as well as accessible guidance regarding the analysis of heterogeneous immune cell infiltrates.

## The importance to assess the clinical validity of TILs in the context of clinical utility

Clinical validity refers to the presence of sufficient evidence, usually level 1 evidence of the effect of a test (biomarker) to demonstrate its validity in a clinical setting. That is, there is robust statistically validated evidence that the test (biomarker) relates to a clinical outcome (prognostic or predictive) or a specific phenotype (TILs), etc. However clinical validity alone, whilst required for changes in clinical practice, does not drive a change in practice. For this to occur, the test must have clinical utility. Clinical utility essentially requires that the test in question
addresses a direct clinical need (prediction of response to therapy, prognosis, or diagnostic classification of subtypes) and *will when implemented, impact patient management*. Simply put, a test only has clinical utility when it impacts physician and patient choice on treatment or management options. The level 1 requirement for clinical validity clearly differs according to the setting⁴⁹. However, it is clearly not optimal to assess the clinical validity of TILs without first framing the question of clinical utility correctly. Once the correct clinical question is framed, appropriate studies must be designed to generate evidence to support the clinical validity of TILs in the setting of clinical utility. In this respect, we agree with Simon et al. that prospective trials not originally designed to address tissue biomarker studies can be used to "accommodate biomarker utility" using archived samples⁴⁹. Since only simple tools, like a microscope, are needed for the assessment of TILs, this provides tremendous opportunities to test the "clinical utility" of TILs in various settings.

## The clinical validity and utility of TILs

TILs in TNBC. Level 1 evidence for a biomarker⁴⁹ can either be reached by incorporating a biomarker into a properly powered prospective clinical trial (level 1A) or by achieving reproducible results in archived tissues from independent randomized trials, designed, conducted, and analyzed as per REMARK criteria (level 1B)⁵⁰. Using these widely accepted criteria, level 1B evidence for clinical validity of TILs as a prognostic biomarker in early-stage TNBC is well established⁹,²². Two pooled analyses of TILs, in the adjuvant setting for TNBC²², and in the neoadjuvant setting across BC-subtypes⁹, included studies that have evaluated TILs on archived tissue samples based on our published guidelines²³.

In a pooled analysis (n = 2148), the clinical value of TILs in predicting prognosis of early-stage TNBC, including adjuvant trials of anthracycline-based chemotherapy alone or in combination with taxanes was investigated²². The average age of enrolled patients was 50 years, and 33% of them were lymph node-negative. The quantification of TILs showed that their average was 23%, and 77% of patients had at least 1% sTILs. Notably, sTILs were found to be significantly reduced with advanced age, larger tumor size, more positive lymph nodes, and lower histological grade. In the multivariable Cox regression model, sTILs were an independent prognostic predictor for all endpoints; each 10% increment in sTILs corresponding to an invasive disease-free survival (DFS) hazard ratio (HR) of 0.87 (95% CI: 0.83–0.91), a distant DFS HR of 0.83 (95% CI: 0.79–0.88) and an overall survival (OS) HR of 0.84 (95% CI: 0.79–0.89) (p < 10⁻⁶)²². Histological grade was not a prognostic factor in this study. The second pooled analysis included women with primary BC treated with neoadjuvant chemotherapy (NACT) in six randomized trials conducted by the German Breast Cancer Group⁹. It assessed the predictive value of sTILs for chemotherapy response and prognostic estimation in patients with TNBC, HER2-positive, and luminal A/B–HER2-negative BC. sTILs quantified in diagnostic core biopsies from 3771 patients were associated with pathologic complete response (pCR) after NACT across all BC subgroups. For instance, based on three predefined groups of low (0–10% immune cells in peritumoral stromal tissue), intermediate (11–59%), and high TILs (≥60%), pCR was achieved in 31% (80/260) of TNBC patients with low TILs, 31% (117/373) of TNBC patients with intermediate TILs, and 50% (136/273) of TNBC patients with high TILs (p < 0.0001). OS was analyzed in 2560 patients across all BC subtypes from five of the six neoadjuvant clinical trial cohorts. However, increased sTILs were associated with longer OS only in TNBC (HR: 0.92; CI: 0.86–0.99, p = 0.032).

Recently, Park et al.⁵¹ investigated the prognostic impact of sTILs in early-stage TNBC based on four multicenter cohorts (476 patients) who were not treated with (neo)adjuvant chemotherapy. The presence of sTILs at baseline was correlated with several

clinical endpoints including OS. Multivariate analyses demonstrated that sTILs are an independent prognostic biomarker of OS (p = 0.015), invasive DFS and distant DFS for TNBC (p < 0.001 for both)⁵¹. A 10% increase in sTILs also positively correlated with OS (HR: 0.88; 95% CI: 0.79–0.98), invasive DFS (HR: 0.90; 95% CI: 0.82–0.97) and distant DFS (HR: 0.86; 95% CI: 0.77–0.95). In a subgroup of TNBC patients with stage I tumors and sTILs ≥ 30%, excellent 5-year survival outcomes were reached including 98% 5-year OS⁵¹. Indeed, the expert opinion at the 16th St. Gallen International Breast Cancer Conference has endorsed the routine reporting of sTILs in TNBC as a prognostic factor²² although guidelines have not yet recommended de-escalation of standard systemic therapy according to TILs. *The WHO Classification of Tumors: Breast Tumors, 5th Edition* has also endorsed histopathological TIL quantification in TNBC and HER2-positive BCs, expressed as a mean percentage of lymphoplasmacytic infiltration of the tumor stroma⁵³.

The 5th Edition of the WHO classification also re-classifies medullary carcinoma, in which prominent TILs have long been recognized, into *invasive breast carcinoma of no special type with medullary pattern*⁵³. These carcinomas have been characterized as showing high immune-related gene⁵⁴ expression and it is likely that the high TILs component of these tumors contributes significantly to their favorable clinical outcomes⁵⁵,⁵⁶. The role of TILs in the histological spectrum of TNBC is yet to be fully elucidated although early data on metaplastic carcinoma suggest that TILs may have prognostic relevance⁵⁷,⁵⁸. The role of TILs in TNBC subtypes recognized as low grade and with good prognosis histological features, such as adenoid cystic carcinoma of the breast⁵⁹, is at present unknown. The importance of histological grade in TNBC is likely to be of utility in identifying the so-called low-grade TNBC, including the rarer subtypes (for example adenoid cystic carcinoma), that have low TILs but an excellent outcome compared to the lack of clinical utility and prognostic significance of histological grading alone in TNBC NOS.

TILs and PD-L1 in triple-negative BC. The phase 2 double-blind placebo-controlled GeparNuevo study (NCT02685059), randomized 174 early stage TNBC patients to receive durvalumab (a PD-L1 inhibitor) combined with standard NACT and used sTILs as a biomarker for patient stratification during randomization⁶⁰. This proof-of-concept study showed that patients with higher sTILs in both arms of the trial had significantly improved pCR rates (p < 0.01), although the sTILs were not specifically related to durvalumab-response. A recent translational analysis of this trial demonstrated that both continuous TMB and TILs were independent predictors of pCR⁶¹. Patients with high TMB had excellent pCR rates of 82% (95% CI: 60–95%) highlighting the potential of this emerging biomarker in tailoring therapy in this setting⁶¹. The German phase II trial (NCT03289819) compares neoadjuvant pembrolizumab combined with nab-paclitaxel vs. pembrolizumab with epirubicin and cyclophosphamide in patients with early TNBC will also investigate sTILs at baseline, in addition to other potential biomarkers such as mutational load and microbiota. In this setting, the recently released findings of IMpassion031⁶², KEYNOTE-522⁶³, and I-SPY²⁴ studies suggest that response to ICIs is independent of PD-L1 status. Thus, other biomarkers of response are needed to predict outcomes in TNBC patients treated with NACT and immune-checkpoint blockade.

In metastatic or locoregionally advanced TNBC, several immunotherapy trials have demonstrated the value of immune cell infiltrate in estimating survival outcomes including phase I (NCT01375842)⁶⁵, phase II KEYNOTE-086 (NCT02447003)⁶⁶,⁶⁷, and phase III IMpassion130 (NCT02425891)⁶⁸ clinical trials. TILs were used to assess the activity of atezolizumab as monotherapy in a phase Ia expansion cohort in metastatic TNBC⁶⁹. Patients' stratification based on TILs at baseline indicated that median OS was improved in those with >10% of sTILs cut-off (12.6 vs.
6.6 months, *p* = 0.0028)⁶⁹. In the phase III study, Schmid et al. showed that the addition of atezolizumab to nab-paclitaxel improved progression-free survival (PFS) in TNBC as compared with the addition of placebo to nab-paclitaxel, particularly in those with PD-L1 positive tumors. Median OS in this subgroup was consistently improved in both interim analyses⁶⁸,⁷⁰, but no formal statistical testing of OS in the PD-L1 positive subgroup could be performed due to the absence of OS benefit for the entire study population⁷⁰. Hence, the FDA approval for this particular drug and assay was based on PFS, not on OS. The available tumor samples from this study were also re-assessed for the analytical concordance of various assays for PD-L1 immunohistochemistry (IHC) and the clinical utility of these assays⁷¹. This post-hoc analysis demonstrated that all subgroup results based on different PD-L1-based assays were suggestive of some clinical benefit (with SP142 immunopositivity apparently indicating most clinical benefit), with different HR and low concordance between the immunohistochemical assays used (SP142 vs. 22C3 vs. SP263)⁷¹,⁷². This analysis also raises confusion as to whether HR or under-powered subgroup-analyses combining different antibodies, should be used to make claims on the performance of assays or not; taking into consideration that only a biomarker-treatment interaction analysis, in the biomarker positive vs biomarker negative population and only in powered subgroup-analysis, can inform the performance of assays. It was also shown that when >20% of sTILs were present, nearly all patients had a positive PD-L1 essay, irrespective of the assay used. This indicates that sTILs are important drivers of response and that sTILs could help mitigate the well-known assay- and reproducibility issues associated with PD-L1 assessment⁷¹,⁷². In fact, numerically higher counts of sTILs were noted in TNBC patients with a positive FDA-approved SP142 assay⁷¹. Furthermore, in the recent biomarker-analysis of Impassion130⁷³, it was shown that TILs predict benefit to atezolizumab for any PD-L1-expression.

KEYNOTE-086 enrolled 170 patients with advanced TNBC who were treated with at least one prior line of therapy and showed a 5% response rate (RR) and 20% stable disease of the subgroup with PD-L1 positive tumors (based on the 22C3 pharmDx assay) when treated with pembrolizumab monotherapy⁶⁶. Similarly, the cohort of the trial treated with first-line pembrolizumab displayed a durable response in patients with positive-PD-L1 (21.4%; 95% CI: 13.9–31.4)⁶⁷. These findings confirm the previously noted improved outcomes in this setting with higher TIL levels⁷⁴. In fact, higher sTILs were associated with significantly increased ORR (OR: 1.26, 95% CI: 1.03–1.55, *p* = 0.01) and disease control rates (OR: 1.22, 95% CI: 1.02–1.46; *p* = 0.01). PD-L1 expression was also significantly correlated with the levels of sTILs (*p* < 0.001) in metastatic TNBC treated with pembrolizumab⁷⁴.

A recent advance resulted from the KEYNOTE-119 phase III trial (NCT02555657) in which 622 previously treated metastatic TNBC patients were randomized to receive pembrolizumab as monotherapy or cytotoxic chemotherapy¹³. This study did not demonstrate significantly prolonged OS in the overall cohort nor in the PD-L1-positive subgroup (combined positive score -CPS- ≥1 or ≥10)¹³. Exploratory analysis revealed that a marked increase in the efficacy of pembrolizumab was seen when the cut-off for a combined positive score was increased to ≥20. In this study, TILs were then evaluated according to our established guidelines²³. In the pembrolizumab arm, high distribution of TILs was observed in responders with better survival outcomes, an effect not observed in the chemotherapy arm⁷⁵. Since sTILs were not prespecified in the trial protocol, sTILs were not considered "regulatory-proof", despite the OS benefit demonstrated in this phase 3 setting. TILs and combined positive scores were moderately correlated and were independent predictors of outcome in patients treated with pembrolizumab. More recently, KEYNOTE-355 (NCT02819518) randomized 847 women with metastatic TNBC to receive pembrolizumab in combination with chemotherapy vs.

chemotherapy plus placebo⁷⁶. This demonstrated that patients with CPS ≥10 treated in the immunotherapy arm had improved median PFS as compared to the placebo group (9.7 vs. 5.6 months, HR: 0.65, 95% CI: 0.49–0.86; *p* = 0.0012) but not in those with CPS of 1 or more (7.6 vs. 5.6 months (HR: 0.74, 95% 0.61–0.90; one-sided *p* value not significant)⁷⁶.

Nevertheless, biomarker analyses of TILs in such studies are all exploratory. These findings should, therefore, ideally be validated in independent prospective studies that use suitably powered phase III randomized and controlled trial design to accelerate the clinical validation of TIL-guided therapy and to provide Level 1A evidence. We strongly propose pre-specifying the use of TILs as an integral biomarker in future trial protocols. However, the FDA has recently approved pembrolizumab for adults and children with TMB-H solid tumors. Treatment efficacy was studied in a prospectively planned retrospective analysis of 10 cohorts of patients with solid malignancies with unresectable or metastatic TMB-H tumors. These patients were enrolled in a multicenter, non-randomized, open-label clinical trial (KEYNOTE-158; NCT02628067). This approval raises interesting questions, for example: is the old paradigm for obtaining level 1A evidence for biomarkers becoming obsolete or is it simply underused? Are prospective-retrospective analyses for biomarkers that are predictive for immune therapy and indicate OS, with the level of evidence IB, and with proven analytical and clinical validity (like TILs) sufficient for regulatory approval as a predictive marker for selection of patients for immune checkpoint inhibition? Will level 1B evidence drive clinical change? Regulatory agencies approve assays and drugs, but the scientific community still must apply that knowledge in a clinical context based on a synthesis of all evidence. Is it the role of regulatory agencies to approve clinical practice changes or should this be the role of the scientific community, in a partnership with industry and the regulatory agencies?

TILs in ER-positive BC. Although a high percentage of sTILs in primary TNBC and HER2-positive BCs suggests a favorable prognosis, the significance in estrogen receptor (ER)-positive, HER2-negative tumors remains uncertain. This is partly because there has been less focus on ER-positive carcinomas, which are traditionally considered to demonstrate lower immunogenicity⁹,⁷⁷,⁷⁸. For example, the mean TILs count is significantly lower than in TNBC and HER2-positive BC⁹,⁷⁹,⁸⁰ The average TMB, which frequently correlates with neantigen load, is also lower in these tumors⁸¹. Small studies of single-agent immune checkpoint blockade have yielded low responses in patients with pretreated metastatic ER-positive cancer⁸²,⁸³. Nevertheless, there is marked heterogeneity for TILs infiltration and mutational burden seen in ER-positive BCs, with a considerable proportion above the mean observed in triple-negative tumors⁷⁷. The importance of TILs in ER +/HER2-ve BC can be most easily demonstrated by combining two aspects of BC; firstly, recent population-based surveys of over 350,000 BCs in the UK and USA suggest that 73% of newly diagnosed BCs are ER+/HER2-⁸⁴,⁸⁵. Given that TILs have been detected in the stroma of up to 60% of BC⁷,²⁸,²⁹ the majority (over half) of all BCs with TILs must therefore be in the ER+/HER2-subgroup. Put simply, the population of ER+/HER2- BCs with TILs exceeds the combined population of HER2+/TNBCs with this feature. This demonstrates the large unmet potential for exploiting immune targeted agents in ER+/HER2- patients.

Several studies have examined the impact of TIL levels on patient prognosis in primary ER-positive BC, and five of the largest studies are summarized in Table 1,9,78,86–88. These studies retrospectively examined histological slides either from randomized controlled trials or annotated clinical cohorts. However, the material varied considerably (tissue microarray vs. core biopsy vs full face section) as did the TILs analysis methodology (H&E staining vs. IHC) and the TILs quantification statistical methods
<table><caption>**Table 1.** Summary of previous studies on the prognostic value of TILs in ER+ breast cancer.</caption><thead><tr><th>Study</th><th>Patient population</th><th>Treatment</th><th>Method of lymphocyte assessment</th><th>Impact of sTILs in prognosis</th><th>Impact of iTILs on prognosis</th></tr></thead><tbody><tr><td>Loi et al.<sup>78</sup></td><td>BIG 02-98 trial (n = 1078)</td><td>Adjuvant anthracycline chemotherapy ± taxane</td><td>Full face H&amp;E sections—TILs assessed as a continuous variable</td><td>Each 10% increment in sTILs associated with worse OS (HR: 1.1, p = 0.04044) on univariate analysis</td><td>No association with DFS or OS.</td></tr><tr><td>Ali et al.<sup>86</sup></td><td>Mixed RCT samples and clinical cohorts (n = 5961)</td><td>Variable (adjuvant)</td><td>IHC for CD8+ cells on tissue microarrays (TILs assessed as a categorical variable, present vs. not present)</td><td>No association with BCSS.</td><td>The presence of any iTILs is associated with worse BCSS (HR: 1.16; 95% CI: 1.02–1.32).<br/>Association lost on multivariate analysis.</td></tr><tr><td>Dieci et al.<sup>87</sup></td><td>Two RCT cohorts (n = 463)</td><td>Adjuvant chemotherapy vs. no chemotherapy</td><td>Full face H&amp;E sections. TILs assessed as both continuous and categorical variables.</td><td>No association with DFS or OS.</td><td>No association with DFS or OS.</td></tr><tr><td>Denkert al.<sup>9</sup></td><td>Meta-analysis of 6 neoadjuvant trials (n = 832)</td><td>Various neoadjuvant chemotherapy regimens</td><td>Core biopsies. TILs were classified as low (0–10%), intermediate (11–59%), or high (60–100%).</td><td>Intermediate TILs were associated with worse DFS (HR: 1.50, 95% CI: 1.09–2.06) and OS (HR: 2.45, 95% CI: 1.61–3.70) vs. low TILs.<br/>High TILs associated with worse OS (HR: 1.79, 95% CI: 1.02–3.15) vs. low TILs. No association with DFS.<br/>CD4 and FOXP3 lymphocytes were not significantly associated with prognosis. Patients with high CD8 cells had an increased risk of recurrence (HR = 1.98; 95% CI: 1.14–3.41)</td><td>N/A</td></tr><tr><td>Sobral-Leite et al.<sup>88</sup></td><td>Retrospective analysis of a multicenter trial (n = 563)</td><td>Tamoxifen or no adjuvant therapy</td><td>IHC for CD4, CD8, and FOXP3 cells</td><td></td><td>N/A</td></tr></tbody></table>

BCSS breast cancer-specific survival, DFS disease-free survival, IHC immunohistochemistry, iTILs intratumoural TILs, OS overall survival, RCT randomized controlled trial, sTILs stromal TILs.

(continuous vs categorical variable). With these caveats in mind, the most notable finding from these analyses is that no study demonstrated a favorable impact of TILs on disease-free, BC-specific, or OS in ER-positive BC. In some analyses, TILs were associated with an unfavorable prognosis. For example, in a recent meta-analysis of six NACT trials, higher TILs levels were associated with a significant reduction in OS⁹. Furthermore, two studies analyzing mixed trial and/or clinical cohorts (of almost 6000 and 1400 ER-positive tumors, respectively) reported that higher levels of intratumoral CD8⁺ TILs were associated with a significant reduction in BC-specific survival⁸⁰,⁸⁶.

These observations must be interpreted with caution given that the molecular subtype of ER⁺ BC is a major confounder in all these analyses. With the introduction of PAM-50 assays in the last decade, ER-positive cancers have been divided into luminal A and luminal B tumors—the latter characterized by higher proliferation and reduced dependence on endocrine signaling⁸⁹. Luminal A tumors have both lower TIL infiltration and TMB than luminal B tumors⁷⁹ and luminal B cancers are associated with worse clinical outcomes⁹⁰. In keeping with this, the significant associations between CD8⁺ TIL infiltration and worse prognosis in the studies described above were lost after adjusting for tumor grade. Similarly, the significant reduction in OS observed in the ER⁺/HER2⁻ cancers in the BIG 02-98 analysis was not upheld in a multivariate analysis adjusting for other prognostic features⁷⁸.

ILC is the second most common histological subtype of BC and is frequently ER+/HER2-. Compared to IDC, fewer data are currently available on the immune microenvironment in ILC⁹¹. Nevertheless, ILC has been shown to harbor lower TIL levels when compared to IDC⁹²,⁹³, although gene expression profiling has indicated that the transcriptomic immune response signatures may in fact be upregulated in ILC⁹⁴. High TIL ILC has been associated with poor prognostic factors and, to date, emergent data suggest that high TILs are suggestive of less favorable clinical outcomes in ILC⁹⁵,⁹⁶. Indeed, it has been suggested that high TILs may drive aromatase inhibitor resistance which may in part account for some of the ILC-TIL signal identified⁹⁷,⁹⁸. However, the role of TILs in ILC is still unsolved, and more and larger, preferably pooled studies are needed to define the importance of TILs in this specific subtype.

The data on TIL levels and benefits from anti-estrogen endocrine therapy is even sparser. Sobral-Leite and colleagues evaluated CD8 expression in over 500 ER-positive BC patients who were randomized between no adjuvant endocrine treatments or one or three years of tamoxifen⁸⁸. Patients with high CD8 infiltration in the tumor had a significant unfavorable prognosis; however, this effect was seen regardless of tamoxifen treatment. In addition, a study of 79 patients demonstrated a smaller reduction in tumor cell proliferation after two weeks of neoadjuvant aromatase inhibitor treatment in tumors with a significant lymphocytic infiltrate⁹¹. Another analysis from a prospective study of neoadjuvant letrozole ± lapatinib (n = 73) showed that significant Ki-67 reduction was observed in both patients with high and low baseline TILs, with high TILs tumors achieving more frequently a relative Ki-67 suppression >50% (55% vs. 35% of patients with low TILs)⁹⁹. The long-term clinical significance of these observations remains unclear. There is also very limited data regarding the impact of targeted systemic therapy on TILs infiltration in ER-positive BC. Recently, CDK4/6 inhibitors have been reported to enhance antitumor immune responses associated with the upregulation of interferon-driven gene expression signatures¹⁰⁰. However, this has not been associated with an increased TILs level in tumors¹⁰¹.

At present, there is no role for routine assessment of TILs in primary ER-positive BC, and their presence cannot be used to guide prognosis nor as a predictive biomarker. We propose that new studies should take a uniform approach to the assessment of TILs, such as those outlined in recent guidelines²³,¹⁰² and should
report data for luminal A and B tumors separately. In addition, a more refined understanding of the ER-positive BC immune environment, including the significance of immunosuppressive regulatory T cells, the spatial distribution of lymphocytes, and the role of estrogenic signaling will be required to define meaningful immune biomarkers in ER-positive disease<sup>103-105</sup>. Notably, a recent retrospective analysis (n = 563) of the multicenter IKA trial that randomized stage 1-3 ER-positive BC patients to receive tamoxifen or no adjuvant therapy showed that CD8-positive sTILs were significantly higher in patients with *PIK3CA*-mutated tumors (OR: 1.65; 95% CI: 1.03-2.68)<sup>88</sup>. In addition, this population of BC patients had an increased risk of recurrence (HR: 1.98; 95% CI: 1.14-3.41) on multivariate analysis as compared to those with CD4 and FOXP3 sTILs that were not statistically associated with prognostic outcomes<sup>88</sup>. This enrichment of TILs in tumors with mutated *PIK3CA*and their association with outcomes provided additional evidence on the crosstalk between mutational status and TME.

TILs in HER2-positive BC. Patients with HER2-positive early BC have a higher infiltration of TILs and improved pCR with NACT and trastuzumab<sup>106,107</sup>. Moreover, improved event-free survival in primary disease treated with trastuzumab and lapatinib was also noticed<sup>107</sup>. The predictive and prognostic value of TILs as a continuous variable in HER2-positive BC in the neoadjuvant setting was demonstrated in another analysis (n = 498) that evaluated TILs from two trials (GeparQuattro and GeparQuinto)<sup>108</sup>. In the group of patients with lymphocyte-predominant (≥60% sTILs) phenotype, a marked increase of pCR rates was noted<sup>108</sup>. Moreover, an increment of TILs by 10% was found to be an independent predictor of pCR in multivariate analysis (p = 0.014)<sup>108</sup>. Notably, this effect was more relevant in patients with ER+, PR+, and HER2+ tumors<sup>108</sup>. In the N9831 phase III trial (NCT00005970), tumor samples from patients with early HER2-positive BC were collected at baseline for TILs quantification in deciles with a prespecified categorical cutoff of ≥60%<sup>109</sup>. TILs were associated with recurrence-free survival in the cohort treated in the control arm with chemotherapy alone. Unexpectedly, higher TILs predicted a lack of response to trastuzumab in HER2 positive BC patients<sup>109</sup>. However, an immune gene-expression analysis on the same dataset did show a strong association with benefit<sup>110</sup>. A recent meta-analysis of 5 randomized and controlled trials that pooled data from 1256 patients provided additional evidence of the prognostic impact of TILs in the neoadjuvant setting<sup>111</sup>. Higher TILs at baseline were predictive of pCR irrespective of the neoadjuvant treatments used in HER2 positive BC<sup>111</sup>.

Recently, supporting evidence of the association of sTILs with distant DFS in this setting has emerged from the Short-HER phase III randomized study that compared different adjuvant regimens combined with trastuzumab (9 weeks vs. 12 months) (NCT00629278). On multivariate analysis, TILs predicted improved distant DFS for each 10% increment (HR: 0.73, 95% CI: 0.59-0.89, *p* = 0.006)<sup>112</sup>. At five years of follow-up, distant DFS rates were significantly higher in patients with TILs ≥ 20% (*p* = 0.025)<sup>112</sup>. Importantly, the 10% TIL increments were significantly associated with distant DFS only in patients randomized to receive nine weeks trastuzumab-based regimen (HR: 0.60, 95% CI: 0.41-0.88; *p* = 0.009)<sup>112</sup>.

In the National Surgical Adjuvant Breast and Bowel Project B-31 stromal TILs were also assessed<sup>47</sup>. Not only was the concordance between 6 different pathologists 90.8% when evaluated on a set of 100 representative cases from this trial, but sTILs were associated also with improved DFS. Higher sTILs were found associated with a higher response to trastuzumab-based on the 8-gene prediction model (*p* < 0.001)<sup>47</sup>. The association between TILs and prognosis in the advanced HER2-positive setting is less well established. A recent analysis of the pre-treatment samples from the CLEOPATRA study population revealed that higher

quantities of TILs predicted an improved survival benefit in first-line treatment with taxane-based chemotherapy, trastuzumab, and pertuzumab<sup>113</sup>. Conversely, analysis of the samples from MA.31, a randomized clinical trial of lapatinib or trastuzumab in combination with taxane-based chemotherapy, reported that 35% of the population were categorized as high TIL but that this did not show significant prognostic or predictive effects<sup>114</sup>. It is worthwhile noting that the cutoff used for high TILs was 5%, much lower than the 20% cutoff used in the binary component of the CLEOPATRA analysis. MA.31 assessed TILs also on the primary tumor samples and found a mean of 9.2%; median, 5%; IQR, 1-10%. Provision of tumor tissue from a metastatic site pre and post-study treatment was an optional subsidy with low uptake— hence metastatic samples were not included in the TIL analysis. Conversely, the CLEOPATRA TILs analysis used samples of either the primary (93%) or metastatic (7%) sites and found the mean stromal TILs value was 21.07% and the median stromal TILs value was 10% (IQR 5-30). Although only a small number of metastatic samples were analyzed, the TILs values of samples from lung and lymph nodes were significantly higher than those from primary tumor, liver, bone, or skin. Furthermore, in the PANACEA-trial<sup>115</sup>, combined TILs and PD-L1 predicted benefit for pembrolizumab combined with trastuzumab in trastuzumab-resistant advanced HER2 positive BC. Whilst the early TNBC studies used a similar cutoff for high TIL groups, more recent analyses using TIL as a continuous variable has shown a linear association with survival outcomes. The ideal threshold for high TILs remains unclear in advanced HER2-positive disease, but variation in cut-offs may account for the conflicting results seen thus far<sup>114</sup>.

The incorporation of TILs into routine clinical practice and clinical trial design. It should be emphasized that formally speaking, the clinical utility of sTILs is not proven since no prospectively designed phase 3 TILs-driven trials have yet been performed, and evidence is lacking that treatment decisions based on sTILs favorably affect patient outcomes. Therefore, the *TILs Working Group* does not currently recommend the use of sTILs in isolation to guide treatment decisions in clinical practice. However, sTILs could be used to help identify patients with an excellent outcome to study *de-escalation* approaches and thereby reduce toxicity. In addition, based on the data of Luen et al., *escalation* of treatment regimens may be clinically beneficial for TNBC patients with a high post-neoadjuvant residual disease burden and low sTILs due to the poor prognosis<sup>116</sup>. Along those lines, the retrospective evaluation of sTILs in trials that have evaluated adjuvant treatments for those patients who did not reach a pCR, such as the CREATE-X trial—is planned<sup>117</sup>. In practice, some clinicians are using TILs *in conjunction* with other clinical variables to decide on the type and duration of cytotoxic chemotherapy. It may be emphasized that for most of the prognostic factors currently used for decades in BC daily practices, there is no level of 1A-evidence. To achieve clinical utility in TNBC, TILs must be included as pre-specified biomarkers in clinical trials and/or prospectively/retrospectively evaluated in randomized trials.

To successfully integrate TILs as a stratification factor in clinical trials, it is necessary to develop digital pathology tools to ensure accurate and rapid sTILs scoring by independent pathologists. Web-based platforms that allow easy scanning, viewing, and scoring of H&E slides should be optimized. A risk-based framework, aiming to reduce variability in TIL assessment within a clinical trial was used in the first stage of an adaptive non-comparative phase II trial (TONIC-trial)<sup>118</sup>. A total of 67 patients with metastatic TNBC were randomized to nivolumab (anti-PD-1) without induction or to one of four induction treatments, consisting of irradiation of one metastatic lesion (3 × 8 Gy) or a 2-week low-dose regimen of cyclophosphamide, cisplatin, or doxorubicin, all followed by nivolumab (NCT02499367)<sup>118</sup>. Concordance values between four pathologists were >90% in this trial,
showing the applicability of this concept in real-world trial-
settings¹¹⁹. The added value of web based-tools is that the
biomarker scores of local pathologists can be integrated into the
flow of a clinical trial, thereby enhancing local pathologists'
compliance and experience within a trial, bridging trial- with daily
pathology practices. This is in contrast to the current situation,
where the tissue may be sent to a central laboratory with limited
to no involvement of local pathologists. Is the current trial-
paradigm of "single-reference laboratory-approach" therefore in
need of revision?

PD-L1 TESTING AND TILS IN WORLDWIDE SETTINGS FOR BC

Access to novel therapeutic agents, including ICIs in low- and
middle-income countries needs global support and importantly,
standardization of testing. The "interchangeability of PD-L1
antibodies", with a recent ESMO presentation showing all PD-L1
antibodies predict some benefit in TNBC for immunotherapy⁷¹ is
still debated, illustrating the complexity and confusion that exists
on these assays in the scientific community. In addition, the TILs
Working Group, in a partnership with the College of American
Pathologists, the European Society of Pathology, the Latin
American Society of Pathology, and the European Working Group
of Breast Screening Pathology argued how the current assay-
approval policies, exemplified by the current PD-L1-assay situa-
tion, are leading to (unintended) imprecision medicine, which is
not in the best interest of our patients. Hence, this pathology
partnership proposed concrete solutions to improve the current
assay approval pathway¹²⁰. A framework for better evaluation of
risks associated with suboptimal patient selection and stratifica-
tion for ICI-based treatments in future clinical trials and real-world
settings based on TILs/PD-L1 is proposed by the *TILs Working
Group*⁷². The use of TILs as a predictive biomarker of response to
ICI's may considerably support cancer care in countries that have
difficulty with PD-L1 implementation, mainly related to costs.
Many ongoing clinical trials will report data that are expected to
support TILs as a valid biomarker for response (see Table 2).

In Europe

In the United Kingdom (UK), the National Institute for Health and Care Excellence (NICE) reported that atezolizumab combined with nab-paclitaxel (Abraxane®) does not have plausible potential to be cost-effective in advanced TNBC. Nevertheless, PD-L1 testing and atezolizumab can be offered to patients with advanced TNBC. In Sweden, TILs have been incorporated in the Swedish Breast Cancer Guidelines as of June 2020¹²¹. Based on the results of Impassion 131, the New Therapies Council (NT-Rådet) does not recommend the use of atezolizumab in combination with paclitaxel or nab-paclitaxel for the treatment of advanced TNBC. The implementation of PD-L1 testing (SP142 assay) in Denmark has been hampered by the lack of analytical validity and reproducibility of immunostaining. Consequently, PD-L1 IHC is not in widespread use. In addition, the process concerning approval of atezolizumab for metastatic TNBC is ongoing in the Danish Medicines Council. TILs are included in the national Danish Breast Cancer Cooperative Group (DBCG) pathology guidelines. In Romania, PD-L1 testing is not routinely used. The testing of patients is made primarily by private pathology laboratories on request. National Health Insurance does not reimburse the bill for testing, so patients can enroll in a clinical trial or get free testing from the Pharmaceutical Industry accessing special testing programs. Atezolizumab is currently not approved for BC in Romania. Atezolizumab is approved and reimbursed in Italy for the treatment of patients with locally advanced or metastatic TNBC, and the SP142 assay has been identified as a companion diagnostic.

In Africa

In Morocco, many breast pathologists practicing at university hospitals have benefited from the Roche® training for the implementation of SP142 PD-L1 IHC. However, this assay has a high cost, and most of the national efforts to support cancer patients including health insurance do not cover this testing. Alternatively, several private pathology laboratories offer this assay on-demand with some oncological institutions providing access to ICI. Data from Kenya show HER-2 type BC prevalence of 17.6% and TNBC prevalence of 20.2%. Most patients with BC are still unable to meet the costs of expensive immunohistochemical PD-L1-testing and subsequent immunotherapy. In South Africa, PD-L1 testing is available on Dako and Ventana platforms but the cost is very high and not routinely performed in the public health sectors, which covers 75% of the population. In the private sector, PD-L1 testing is performed upon clinician request. The cost of ICIs drugs remains prohibitively high for use in the public sector. ICIs in BC are available only in the clinical trial setting, as these drugs are not licensed for use in TNBC. Assessment of TILs in BC care is currently not routinely done in either sector.

In North and South America

In the United States, the PD-L1 SP142 assay has been approved as a companion assay for the selection of patients with TNBC to offer atezolizumab and taxane chemotherapy (http://www.svfp.se/ foreningar/uploads/L15178/kvast/brostpatologi/Brostjuni2020.pdf). In Brazil, some central pathology laboratories perform PD-L1 testing based on principal investigator-sponsored agreements. PD-L1 IHC is tested mostly in private institutions with some programs offering pro bono testing to assess eligibility for approved ICI. The Brazilian Pathology Society follows WHO recommendations; hence the inclusion of TILs in daily practice can be envisaged in the near future. In Chile, PD-L1 testing is available on several platforms but their cost is high and this is not covered by public health insurance which covers 75% of the population. Anti-PD-L1 drugs remain at a high cost and are usually only funded by additional insurance or in clinical trial settings. In Argentina, PD-L1 testing is not covered by public funds. A few central private pathology laboratories perform PD-L1 testing on-demand funded by industry. Some pathologists have started to incorporate TILs into pathology reports for TNBC; however, there are no current national guidelines. In Perú, TILs evaluation has been included in TNBC protocols by pathologists in public and private centers. SP142 IHC is available in a few private labs while Dako 22C3 is being processed for non-breast malignancies in private and public centers. There are a few BC patients who have received atezolizumab funded via private insurance or clinical trials. It is not expected that the public system will support anti-PD-1 drugs in the near future.

In Asia. In India, PD-L1 and TIL assessments are not being
performed routinely with significant discrepancies of results due
to the different clones available to date. PD-L1 reporting is
assessed on the platform as recommended by the standard
reporting guidelines. TILs reporting is performed simultaneously
with PD-L1 testing. Insurance companies do not offer reimburse-
ment for PD-L1 testing. In April 2020, atezolizumab in combination
with nab-paclitaxel received Drug Controller General of India
(DCGI) approval as a first-line treatment for metastatic TNBC
patients. This may pave the way for its use in selected patients
if affordable (https://www.expresspharma.in/amp/latest-updates/
roches-atezolizumab-receives-dcgi-approval-for-treatment-of-
metastatic-triple-negative-breast-cancer-in-india/). OptiView PD-L1
(SP142) on the Ventana platform was approved in Japan as the
companion diagnostic for metastatic TNBC treatment with
atezolizumab. Other antibodies including 22C3, 28-8, and SP236
or OptiView PD-L1 (SP-142) on other platforms are un-funded.
<table><caption>**Table 2.** Ongoing phase II/III clinical trials investigating TILs as a biomarker in invasive breast cancer.</caption><thead><tr><th>Trial identifier and phase</th><th>Sample size</th><th>Setting</th><th>Purpose</th><th>Estimated primary completion date</th><th>Sponsor</th></tr></thead><tbody><tr><td>NCT04188119 (IMpALA) Phase II</td><td>42</td><td>Triple-negative breast cancer (TNBC)</td><td>Assessment of post treatment tumor-infiltrating lymphocytes (TILs) by immunohistochemistry (as secondary outcome measure)</td><td>June 2021</td><td>The Christie NHS Foundation Trust</td></tr><tr><td>NCT03863301 (ABLATIVE-2) Phase II</td><td>70</td><td>Non-lobular invasive BC</td><td>Assessment TILs at baseline on biopsies and after surgery and their correlation with the pathological response (as a secondary outcome measure)</td><td>November 2022</td><td>UMC Utrecht, Netherland</td></tr><tr><td>NCT03820141 (DTP Trial) Phase II</td><td>39</td><td>HER2-amplified BC</td><td>Correlation of pathological complete response rate in BC patients with TILs (as secondary outcome measure)</td><td>March 2023</td><td>AstraZeneca</td></tr><tr><td>NCT03359954 Phase II</td><td>20</td><td>HR+/ HER2- BC</td><td>Evaluation of changes in TILs as a continuous variable before and after preoperative radiotherapy (RT) (as primary objective)</td><td>October 2019</td><td>M.D. Anderson Cancer Center</td></tr><tr><td>NCT03971045 (PERICLES) Phase II</td><td>46</td><td>Locally advanced 'hest wall" BC</td><td>Positive PD-L1 (≥1%) and/or TILs (≥1%) as biomarkers for patient selection in locally advanced BC previously treated with chemotherapy or radiation therapy</td><td>July 2021</td><td>European Institute of Oncology</td></tr><tr><td>NCT02883062 Phase II</td><td>72</td><td>Newly diagnosed, stage II-III TNBC</td><td>Comparison of TILs percentage, PD-L1 and necantigen load at baseline before and after therapy in patients receiving neoadjuvant chemotherapy (NACT) alone and those treated with NACT and atezolizumab in a randomized fashion</td><td>July 2020</td><td>National Cancer Institute (NCI)</td></tr><tr><td>NCT03366844 Phase/III</td><td>60</td><td>Invasive BC</td><td>TILs score changes (by Salgado et al. criteria)</td><td>January 2021</td><td>Cedars-Sinai Medical Center</td></tr><tr><td>NCT02435680 Phase II</td><td>50</td><td>Advanced TNBC with high tumor-associated macrophages (TAMs)</td><td>Assessment of TILs and TAMs content in pre- and post-dose tumor biopsies (as secondary outcome measure)</td><td>December 2019</td><td>Novartis Pharmaceuticals</td></tr><tr><td>NCT03894007 (PREDIX II HER2) Phase II</td><td>190</td><td>HER2-amplified early BC</td><td>TILs and gut microbiota as predictors of treatment response in patients receiving atezolizumab in a randomized fashion</td><td>December 2024</td><td>Karolinska University Hospital, Sweden</td></tr><tr><td>NCT03004183 (STOMP) Phase II</td><td>57</td><td>Metastatic TNBC</td><td>Measurement of the changes of TILs in tumor biopsy tissues as a biomarker of response to ADV/HSV-tk + valacyclovir therapy in combination with stereotactic body radiation therapy followed by immune-checkpoint blockade</td><td>May 2022</td><td>Merck Sharp & Dohme Corp.</td></tr><tr><td>NCT03979508 (BEAUTY Study) Phase II</td><td>100</td><td>Surgically resectable and chemotherapy-resistant TNBC</td><td>Quantification of TILs in TNBC patients treated with abemaciclib</td><td>July 2022</td><td>Mayo Clinic in collaboration with NCI</td></tr><tr><td>NCT02003209 Phase III</td><td>312</td><td>Locally advanced BC</td><td>Quantification of TILs in BC patients treated with neoadjuvant chemotherapy, trastuzumab, and pertuzumab with or without estrogen deprivation</td><td>August 2021</td><td>NCI</td></tr><tr><td>NCT02990845 (PEER) Phase I/II</td><td>25</td><td>Premenopausal HR+/ HER2- locally advanced or metastatic BC</td><td>Study of potentially predictive biomarkers of the efficacy of the combination of pembrolizumab and exemestane/euprolide including TILs, PD-L1, circulating tumor cells, and mutational load</td><td>December 2019</td><td>National Taiwan University Hospital in collaboration with Merck Sharp & Dohme Corp.</td></tr><tr><td>NCT03125928 Phase II</td><td>50</td><td>Metastatic HER2-positive BC</td><td>Investigation of predictive biomarkers of response (PD-L1 and TILs) to immune-checkpoint blockade (as secondary outcome measure)</td><td>December 2020</td><td>Fox Chase Cancer Center in collaboration with Genentech, Inc.</td></tr><tr><td>NCT02971761 Phase II</td><td>29</td><td>Androgen receptor-positive metastatic TNBC</td><td>Evaluation of pre-treatment PD-L1 and TILs as predictive biomarkers of pembrolizumab and enobosarm</td><td>November 2020</td><td>City of Hope Medical Center in collaboration with NCI</td></tr><tr><td>NCT02849496 Phase II</td><td>72</td><td>Locally advanced or metastatic non-HER2-positive BC</td><td>Measurement of TILs level changes in patients treated with olaapib with or without atezolizumab</td><td>August 2020</td><td>NCI</td></tr><tr><td>NCT02536794 Phase II</td><td>30</td><td>Metastatic HER2-negative BC</td><td>Evaluation of tissue-based immunohistochemical expression TILs, PD-L1, and other immune-related candidate biomarkers as predictors of response to MEDI4736 in combination with tremelimumab (as secondary outcome measure)</td><td>September 2019</td><td>Northwestern University in collaboration with MedImmune LLC</td></tr><tr><td>NCT03289819 Phase II</td><td>50</td><td>Early TNBC</td><td>Assessment of stromal and intratumoral TILs collected at baseline, 12 weeks after treatment initiation, and at the surgery</td><td>November 2019</td><td>Avon Breast Cancer Foundation and NCI</td></tr><tr><td>NCT03740893 (PHOENIX) Phase II</td><td>81</td><td>NACT resistant and residual TNBC</td><td>Study of frequency and function of subsets of TILs in patients treated with DNA damage response inhibition and/or immune-checkpoint blockade (as secondary outcome measure)</td><td>May 2021</td><td>Institute for Women's Health in collaboration with Merck Sharp & Dohme Corp. And Celgene Corporation</td></tr></tbody></table>

ADV/HSV-tk adenovirus-mediated herpes simplex virus thymidine kinase, BC breast cancer, DNA deoxyribonucleic acid, HER2 human epidermal growth factor receptor, HR hormone receptor, NACT neoadjuvant chemotherapy, PD-L1 programmed death-ligand 1, RT radiotherapy, TAMs tumor-associated macrophages, TILs tumor-infiltrating lymphocytes, TNBC triple-negative breast cancer. Data from ClinicalTrials.gov (accessed 15 December 2019).
Pathology laboratories can use any PD-L1 assay for clinical research but only PD-L1-testing using SP142 on the Ventana platform companion diagnostic is permitted for funded treatment with atezolizumab in clinical practice. Many Japanese pathologists have limited clinical experience interpreting PD-L1 staining with no current national guidelines. The Japanese Society of Pathology does not provide oversight for quality control, which leaves this mostly to the pharmaceutical industry and individual laboratories. In Taiwan, PD-L1 assays are obligatory when considering immune checkpoint inhibition for BC. The fee for a PD-L1 assay is very high and the assay is not covered by the national health insurance and has to be paid at the patients' own expense. The PD-L1 essay is only available in medical centers and some large hospitals.

In Malaysia, some ministry/government-based pathologists do not deem the PD-L1 interpretation as practical given the fact that immunotherapy is not generally provided to patients due to high costs and the lack of subsidized initiatives from the government. On the other hand, similar to that seen in Morocco, assays are being actively performed in the private setting with private health insurance covering the costs of the tests and the ICI. PD-L1 testing is not standardized in Iran, and this assay is not routinely requested in the setting of BC by oncologists. Additionally, sTILs are not routinely reported by pathologists in Iran, and those who do, use the method developed by the International TILS working group.

Cost issues are an extremely relevant topic for low-to-middle-income countries (LMIC) pertaining to PD-L1 testing. There is a considerable difference between the costing of the standard PD-L1-antibodies which are for most patients unaffordable. The high cost of PD-L1 assays in LMIC can be mitigated by selecting patients using the sTILs on H&E-slides to include cases for further analysis. Furthermore, as argued in detail before<sup>72</sup>, TILs and PD-L1 assays for immune cells are complementary as both are part of the same immune spectrum in cancer. However, the landscape of PD-L1 testing is clearly complex for both oncologists and pathologists. The guidelines for interpretation of immunohistochemical results as well as the selection of the appropriate companion diagnostic antibody vary with both tumor type and specific ICI under consideration. The Canadian evidence-based recommendations are a good example of the efforts provided to guide pathologists in establishing fit-for-purpose PD-L1 biomarker assays to select patients to benefit from ICIs in any tumor type<sup>122</sup>.

The International Immuno-Oncology Biomarker Working Group supports efforts to advance knowledge and best practices for immune-based therapies in all solid tumors. In this regard, the TIL Working Group has created an online tool (www.tilsinbreastcancer.org, "NEW PD-L1 Help Desk) to help oncologists and pathologists select and interpret appropriate anti-PD-1/PD-L1 therapies and associated companion diagnostics. This will be updated regularly to reflect the most current standards for eligibility for ICI for all solid tumors.

## A PERSONAL PATIENT ADVOCATE PERSPECTIVE ON TILS

"As a 5-year TNBC survivor who supports other BC patients to navigate and cope with their treatment, I know there's no standard set of information that a patient uses to make their informed choice on how to treat their tumor. Some use Ki-67, some use PET scans; some are shown online risk tools like Predict, most use receptor status. Patients want as much information as possible to make the informed decisions forced upon them. We understand that nothing is "absolute", that one hospital's benchmark of a "clear margin" is different to another's. We know that the thresholds defining hormone receptor subtypes differ between hospitals and that Ki-67 scoring can vary as much as three times between different pathologists. We

understand the variability of response to drugs in different patients. We accept that pathology is an art but struggle to accept that cancer treatment plans arise from a random clutch of data-points and estimates of risk. Patients would benefit from owning their pathology reports. We live longer, we move between hospitals, and even when IT systems are compatible (which is rare) much of our data exist in unstructured reports that future clinicians do not access, either because they do not know it exists or they can't find it amidst the hundreds of files that we generate. If patients had a checklist of markers that were important to their disease, we could help ensure evidence-based treatment plans. Patients suffer unnecessarily; it is stressful enough to face your own mortality but to trawl social media comparing your treatment to that of strangers creates more stress. Patient-ownership of pathology reports would help remove fears of age-bias and variation in care, and furthermore could help generate complete datasets for audits and real-world evidence studies.

We know that cancer is an ecosystem of cancer cells and the stromal cells between them. We know the well-defined markers for the cancer cells that direct chemotherapy and hormone therapy. What markers do we have for the intra-tumor stroma? PD-L1 SP142 is an expensive assay and notoriously difficult to achieve consensus on, but nevertheless if you ever once had PD-L1 recorded for any one of your TNBC primary tumors or metastases, you are eligible for one of the latest drugs, atezolizumab. Would it be wise therefore for all TNBC patients to request PD-L1 testing of their primaries for the event that their metastases can't be biopsied? Shouldn't we be seeking an easier and cheaper marker (e.g., TILs) to understand the stroma and direct treatment? I still feel very lucky that my TILs were scored in my pathology report. They have given me a more positive outlook and this alone is a reason for scoring them. I'm also very aware (through atezolizumab's licensing) that details on my pathology report today might be my gateway to a life-prolonging drug tomorrow."

## CONCLUSION

TILs are an inexpensive, robust prognostic biomarker that represents a surrogate for anti-tumor T cell-mediated immunity. Incorporating TILs into standard clinical practice should be strongly considered in both early and advanced TNBC and HER2-positive BC. TILs assessment at the time of diagnosis may enable a clinician to assess prognosis and in future inform therapeutic decision-making more accurately, is informative for predictive purposes, can help to interpret PD-L1 assays, and, certainly in LMIC, may be considered as a screening tool before embarking on expensive immune-assays, being PD-L1 or others. Further information on the clinical utility of TILs in HR positive BC is needed to identify their role in this subtype of the disease.

Education and standardization of testing across the pathology community by providing centralized training and educational tools are required to up-skill clinicians to utilize this new biomarker. The TIL-WG host's pathologists from academic hospitals, community hospitals, and industry, supported by expert clinicians and statisticians, incorporating the view of patients also. We encourage transparent and efficient communication with the regulatory authorities. This collaboration of experts and patients is imperative to guide the development of this new biomarker and optimize its role across academia, industry, and the clinical setting. Looking to the future, a collaboration between the oncology and pathology communities across countries and continents is integral
to define how best TILs can be integrated into a multivariate prognostic model with standard variables such as age, tumor size, and nodal status to optimize outcomes of patients with BC. In addition, trials being developed using baseline, on-treatment, and post-treatment TILs may improve our understanding of the complex interaction between host immunity and the TME and will improve our approach to fit as best as possible our patient's needs.

Received: 3 November 2020; Accepted: 28 September 2021;
Published online: 01 December 2021

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## ACKNOWLEDGEMENTS

Shom Goel' work is supported by the National Health and Medical Research Council of Australia (Investigator Grant GNT1177357 to S.G.); Susan G. Komen for the Cure (CCR18547966 to S.G.); the Royal Australasian College of Physicians (Research Establishment Fellowship to S.G.); and the NIH/NCI (P50 CA168504 to S.G.). Sherene Loi is supported by the National Breast Cancer Foundation of Australia Endowed Chair and the Breast Cancer Research Foundation, New York. Khalid El Bairi would like to report that the content of this review reflects the authors' perspectives and not of his institution and/or affiliation. Roberto Salgado is supported by the Breast Cancer Research Foundation (BCRF, grant N° 17-194).

## AUTHOR CONTRIBUTIONS

All authors made a substantial contribution to the conception or design of the work. All authors participated in either drafting the work or revising it critically for important intellectual content. All authors have approved the final completed version of this paper and assume accountability for all aspects of the work.

## COMPETING INTERESTS

S. Loi has received research funding to her institution from Novartis, Bristol Meyers Squibb, Merck, Roche-Genentech, Puma Biotechnology, Pfizer, Eli Lilly, Nektar Therapeutics AstraZeneca, and Seattle Genetics. S. Loi has acted as a consultant (not compensated) to Seattle Genetics, Pfizer, Novartis, BMS, Merck, AstraZeneca, and Roche-Genentech. S. Loi has acted as a consultant (paid to her institution) to Aduro Biotech, Novartis, GlaxoSmithKline, Roche-Genentech, Puma Biotechnology, AstraZeneca, and G1 Therapeutics. S. Loi is a Scientific Advisory Board Member of Akamara Therapeutics. S.D. has received research funding from institutions from Lytix Biopharma and has acted as an ad-hoc consultant (compensated) to Ono Pharma USA Inc, EMD Serono, and Mersana Therapeutics. S.G. is the recipient of lab research funding from Eli Lilly and Co and G1 Therapeutics. Shom Goel has served as a paid advisory board member for Eli Lilly and Co, G1 Therapeutics, Pfizer, and Novartis. S.G. also conducts clinical research sponsored by Eli Lilly and Co. S. Loibl reports grants and others from Abbvie, grants and other from Amgen, grants and other from Celgene, grants and other from Novartis, grants and other from Roche, other from Seattle Genetics, other from PriME/Medscape, personal fees from Chugai, grants and other from Daiichi-Sankyo, other from Lilly, other from Samsung, other from BMS, other from Puma, other from MSD, grants from Immunomedics, grants and other from AstraZeneca, grants and other from Pfizer, other from Pierre Fabre and other from Merck outside the submitted work; In addition, Dr. Loibl has a patent EP14153692.0 pending. J.M.S. B. BSc, PhD, FRCPath has consultancies for Insight Genetics, Inc. BioNTech AG Biotheranostics, Inc. Pfizer RNA Diagnostics Inc. oncoXchange/MedcomXchange Communications Inc. Herbert Smith French Solicitors OncoCyte Corporation. SCIENTIFIC ADVISORY BOARD MedcomXchange Communications Inc. HONORARIA NanoString Technologies, Inc. Oncology Education Biotheranostics, Inc. MedcomX-change Communications Inc. RESEARCH FUNDING Thermo Fisher Scientific Genop-tixAgenciaNanoString Technologies, Inc. Stratifier GmbH Biotheranostics, Inc. TRAVEL, ACCOMMODATIONS, EXPENSES Biotheranostics, Inc. NanoString Technolo-gies, Inc. Breast Cancer Society of Canada PATENTS—APPLIED Jan 2017: Methods and Devices for Predicting Anthracycline Treatment Efficacy, US utility—15/325,472; EPO—15822898.1; Canada—not yet assigned Jan 2017: Systems, Devices and Methods for Constructing and Using a Biomarker, US utility—15/328,108; EPO—15824751.0; Canada—not yet assigned Oct 2016: Histone gene module predicts anthracycline benefit, PCT/CA2016/000247 Dec 2016: 95-Gene Signature of Residual Risk Following Endocrine Treatment, PCT/CA2016/000304 Dec 2016: Immune Gene Signature Predicts Anthracycline Benefit, PCT/CA2016/000305 June 2020: Use of Molecular Classifiers to Diagnose, Treat and Prognose Prostate Cancer, US Provisional 63/040.692 INVENTION DISCLOSURE Disclosure Name: A Molecular Classifier for Personalized Risk Stratification for Patients with Prostate Cancer, Date: 21/08/2019. P.A.F. has received Honoraria from AstraZeneca and Novartis and Traveled to give overseas lectures from Ipsen. B.L.R. reports non-financial support from HalioDx, in the form of a collaborative association on a non-remunerative basis, during the conduct of the study. D.R. reports grants and personal fees from Amgen and AstraZeneca, Cepheid, Konica Minolta InVicro, NextCure, Ultivue, and Eli Lilly; personal fees from Bristol Myers Squibb, Cell Signaling Technology, Daiichi Sankyo, Danaher, GSK, Merck, Nanostring Technologies, Odonate, Paige, Al, Roche, Sanofi, and Ventana; grants from Navigate Biopharma; and personal royalties from RareCyte related to a patent on circulating cancer cells outside of the submitted work. A.E. is a Roche advisory board and Lecturer paid by Roche, Amgen, and Novartis. M.V.D. has personal fees for
advisory/consultancy roles from Eli Lilly, Celgene, Novartis, and Genomic Health. R.Y. has received support from Chugai pharmaceutical company. S.M. has conflicts of interest not related to this study including statistical advice for IDDI and Janssen Cilag and is an Independent Data Monitoring Committee member for Hexal, Steba, IQVIA, Roche, Sensorion, Biophytis, Servier, and Yuan. M.K. Institution receives funding from BMS, Roche, AZ/Medimmune. M.K. has an advisory role for BMS, Roche, MSD, and Daiichi. R.S. reports non-financial support from Merck and Bristol Myers Squibb; research support from Merck, Puma Biotechnology, and Roche; and personal fees from Roche for an advisory board related to a trial-research project. The remaining authors declare no competing interests.

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© The Author(s) 2021

¹Department of Medical Oncology, Mohammed VI University Hospital, Faculty of Medicine and Pharmacy, Mohammed Ist University, Oujda, Morocco. ²Department of Cellular Pathology, Great Western Hospital, Swindon, UK. ³Translational Health Sciences, University of Bristol, Bristol, UK. ⁴Division of Research, Peter MacCallum Cancer Centre, Melbourne, Australia. ⁵Department of Pathology, Montefiore Medical Center and the Albert Einstein College of Medicine, Bronx, NY, USA. ⁶Chief Information Officer, WISS & Company, LLP and President J. Shear Consulting, LLC-Arsleys, Ardsley, NY, USA. ⁷ICPV, Independent Cancer Patient Voice, London, UK. ⁸Department of Pathology and Legal Medicine, Medical School of the Federal University of Bahia, Salvador, Brazil. ⁹Department of Medical Oncology, University of Medicine “I. Hatieganu”, Cluj Napoca, Romania. ¹⁰Brazilian Society of Oncology, Salvador, Brazil. ¹¹Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. ¹²Hospital Charles Nicolle, Tunis, Tunisia. ¹³Department of Histopathology, Tata Medical Center, Kolkata, India. ¹⁴Department of Pathology, Imam Khomeini Hospital Complex, Tehran University of Medical Sciences, Tehran, Iran. ¹⁵Pathology and Legal Medicine, Amazon Federal University, Belém, Brazil. ¹⁶Department of Pathology and Laboratory Medicine, Fundacion Valle del Lili, and Faculty of Health Sciences, Universidad ICESI, Cali, Colombia. ¹⁷Sichuan Cancer Hospital, Chengdu, China. ¹⁸Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan. ¹⁹Department of Histopathology, Manipal Hospitals Dwarka, New Delhi, India. ²⁰Department of Pathology, Faculty of Medicine, University of São Paulo, São Paulo, Brazil. ²¹Apc labs - Annasr Pathology Center, El Jadida, Morocco. ²²Army Hospital Research and Referral, Delhi Cantt, New Delhi, India. ²³Aginome Scientific Pte Ltd, Xiamen, China. ²⁴Breast Cancer Comprehensive Center, National Cancer Institute, Cairo University, Cairo, Egypt. ²⁵Department of Oncology, National Taiwan University Cancer Center, Taipei, Taiwan. ²⁶Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan. ²⁷Department of Pathology, Breast Cancer Center FUCAM, Mexico City, Mexico. ²⁸Centro de Pesquisas Clínicas do IMIP, Recife, Brazil. ²⁹The Medical Oncology Centre of Rosebank, Johannesburg, South Africa. ³⁰Department of Immunology, Faculty of Health Sciences, University of Pretoria, corner Doctor Savage Road and Bophelo Road, Pretoria 0002, South Africa. ³¹Department of Medical Oncology, Instituto Nacional de Enfermedades Neoplásicas, Lima 15038, Peru. ³²Faculty of Health Sciences, Universidad Científica del Sur, Lima, Peru. ³³Departmento de Patologia, Hospital Universitario Austral, Pilar, Argentina. ³⁴Department of Pathology, Hospital de Oncologia Maria Curie, Buenos Aires, Argentina. ³⁵Department of Pathology, Sanatorio Mater Dei, Buenos Aires, Argentina. ³⁶Department of Research, Instituto Nacional de Enfermedades Neoplásicas, Lima 15038, Peru. ³⁷Institute of Biological Sciences, Mohammed VI Polytechnic University (UM6P), 43 150 Ben-Guerir, Morocco. ³⁸Department of Pathology, Faculty of Medicine, UKM Medical Centre, Kuala Lumpur, Malaysia. ³⁹Department of Pathology, Fudan University Cancer Center, Shanghai, China. ⁴⁰Kenyatta National Hospital, Nairobi, Kenya. ⁴¹Praava Health, Dhaka, Bangladesh. ⁴²Molecular Immunology Unit, Institut Jules Bordet, Université Libre de Bruxelles, Brussels, Belgium. ⁴³Laboratory of Tumour Immunology and Immunotherapy, Department of Oncology, KU Leuven, Leuven, Belgium. ⁴⁴Department of Hematology/Oncology, Mayo Clinic, Jacksonville, FL, USA. ⁴⁵Department of Histopathology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK. ⁴⁶Breast Cancer Program, Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, USA. ⁴⁷Clinical Oncology Unit, Instituto Oncológico Córdoba, Córdoba, Argentina. ⁴⁸India Cancer Research Consortium-ICMR, Department of Health Research, New Delhi, India. ⁴⁹Department of Pathology, Laboratorio QUANTUM, Rosario, Argentina. ⁵⁰Department of Clinical Genetics and Pathology, Skåne University Hospital, Lund University, Lund, Sweden. ⁵¹Department of Pathology, Yale School of Medicine, New Haven, CT, USA. ⁵²Diagnostic Development, Ontario Institute for Cancer Research, Toronto, Canada. ⁵³Cancer Research UK Edinburgh Centre, Institute of Genetics and Cancer, The University of Edinburgh, Edinburgh, UK. ⁵⁴Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada. ⁵⁵Department of Pathology, Istituto Europeo di Oncologia IRCCS, and University of Milan, Milan, Italy. ⁵⁶Institute of Pathology, Universitätsklinikum Gießen und Marburg GmbH, Standort Marburg and Philipps-Universität Marburg, Marburg, Germany. ⁵⁷Department of Pathology, Matsuyama Shimin Hospital, Matsuyama, Japan. ⁵⁸Department of Medical Oncology, Institut Jules Bordet, Université Libre de Bruxelles, Brussels, Belgium. ⁵⁹German Breast Group, Neu-Isenburg, Germany. ⁶⁰Laboratory of Pathology, National Cancer Institute, NIH, Bethesda, MD, USA. ⁶¹Department of Pathology and Laboratory Medicine, Indiana University, Indianapolis, USA. ⁶²Department of Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA. ⁶³National Surgical Adjuvant Breast and Bowel Project (NSABP)/NRG Oncology, Pittsburgh, PA, USA. ⁶⁴Department of Pathology, RCCS Fondazione Istituto Nazionale Tumori and University of Milan, School of Medicine, Milan, Italy. ⁶⁵Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, VIC, Australia. ⁶⁶Medical Oncology Department, Peter MacCallum Cancer Centre, Melbourne, Australia. ⁶⁷Department of pathology Sanatorio Mater Dei, Buenos Aires, Argentina. ⁶⁸Department of Pathology and Laboratory Medicine, Kurume University Medical Center, Kurume, Fukuoka, Japan. ⁶⁹Servicio de Anatomía Patológica, Hospital Italiano de Buenos Aires, Buenos Aires, Argentina. ⁷⁰Division of Pathology, The Netherlands Cancer Institute, Amsterdam, The Netherlands. ⁷¹Department of Pathology, GZA-ZNA Hospitals, Antwerp, Belgium. ⁷²Perlmutter Cancer Center, New York University Medical School, New York, NY, USA. ⁷³Divisions of Medical Oncology, Molecular Oncology & Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands. ⁷⁴Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy. ⁷⁵Medical Oncology 2, Istituto Oncologico Veneto IOV-IRCCS, Padova, Italy. ⁷⁶Service de Biostatistique et d'Epidémiologie, Gustave Roussy, Oncostat U1018, Inserm, University Paris-Saclay, labeled Ligue Contre le Cancer, Villejuif, France. ⁷⁷Department of Radiation Oncology, Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA. *A list of authors and their affiliations appears at the end of the paper. ⁸⁵email: k.elbairi@ump.ac.ma

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Khalid El Bairi¹,², Harry R. Haynes²,³, Elizabeth Blackley⁴, Susan Fineberg⁵, Jeffrey Shear⁶, Sophia Turner⁷,
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Watson303, Sam Leung304, Torsten Nielsen305, Leming Shi306, Eva Balslev307, Jeppe Thagaard308, Alhadi Almangush309, Antti Makitie310, Heikki Joensuu311, Johan Lundin312, Damien Drubay313,314, Elvire Roblin315,316, Fabrice Andre317, Frederique Penault-Llorca318, Jerome Lemonnier319, Julien Adam320, Magali Lacroix-Triki321, Nils Ternes322, Nina Radosevic-Robin323, Frederick Klaushen324, Karsten Weber59, Nadia Harbeck325, Oleg Gluz326, Stephan Wienert327,328, Gabor Cserni329,330, Andrea Vingiani331, Carmen Criscitiello332, Cinzia Solinas333, Giuseppe Curigliano334, Eiichi Konishi335, Eiji Suzuki336, Katsuhiro Yoshikawa337, Kosuke Kawaguchi338, Masahiro Takada336, Masakazu Toi338, Mitsuaki Ishida339, Nobuhiro Shibata340, Shigehira Saji341, Takahiro Kogawa342, Takashi Sakatani343, Takeru Okamoto344, Takuya Moriya345, Tatsuki Kataoka346, Tatsunori Shimoi347, Tomohagu Sugie348, Tomoharu Sugie349, Toru Mukohara347, Yazaki Shu347, Yuichiro Kikawa340, Yuji Kozuka349, Shahin Sayed350, Reena Rahayu351, Reena Ramsaroop352, Elżbieta Senkus-Konefka353, Ewa Chmielik354, Fatima Cardoso355, Joana Ribeiro356, Jack Chan357, Rebecca Dent358, Miguel Martin359, Carlos Hagen360, Angel Guerrero361, Federico Rojo362,363, Laura Comerma364, Paolo Nuciforo365, Victor Vivo Serrano366, Vincente Peg Cамаеа367, Tessa Steenbruggen368, Francesco Ciompi369, Iris Nederlof370, Jan Hudecek371, Jeroen van der Laak369, Jose van den Berg372, Leonie Voorwerk373, Mark van de Vijver374, Michiel de Maaker370, Sabine Linn375, Hayley McKenzie376, Navita Somaiah377, Andrew Tutt378, Charles Swanton379, Crispin Hiley379, David A. Moore380,381, Jacqueline A. Hall382, John Le Quesne383, Khalid Abdul Jabbar384, Maise al Bakir379, Robert Hills385, Sheeba Irshad386,387, Yinyin Yuan384, Zaibo Li388, Minetta Liu389, Jonathan Klein390, Oluwole Fadare391, Alastair Thompson392, Alexander J. Lazar393, Allen Gown394, Amy Lo261, Ana C. Garrido Castro395, Anant Madabhushi396, Andre Moreira397, Andrea Richardson398, Andrew H. Beck399, Andrew M. Bellizzi400, Antonio Wolff401, Aparna Harbhajanka402, Ashish Sharma403, Ashley Cimino-Mathews404, Ashok Srinivasan405, Baljit Singh406, Chakra S. Chennubhotla407, Cynthia Chauhan408, Deborah A. Dillon409,410, Dimitrios Zardavas411, Douglas B. Johnson412, Aubrey E. Thompson413, Edi Brogi414,415, Emily Reisenbichler51, Erich Huang416, Fred R. Hirsch417, Heather McArthur418, James Ziai261,
Jane Brock⁴⁰⁹,⁴¹⁰, Jennifer Kerner⁴¹¹, Jiping Zha⁴²⁰, Jochen K. Lennerz⁴²¹, Jodi M. Carter⁴²², Jorge Reis-Filho⁴¹⁴, Joseph Sparano⁴²³, Justin M. Balko⁴²⁴, Katherine Pogue-Geile⁴²⁵, Keith E. Steele⁴²⁰, Kim R. M. Blenman⁴²⁶, Kimberly H. Allison⁴²⁷, Lajos Puszta⁴²⁸, Lee Cooper⁴²⁹, Valeria M. Estrada⁴³⁰, Margaret Flowers⁴³¹, Mark Robson⁴³², Marlon C. Rebelatto⁴²⁰, Matthew G. Hanna⁴¹⁴,⁴¹⁵, Matthew P. Goetz⁴³³, Mehrnoush Khojasteh⁴³⁴, Melinda E. Sanders⁴³⁵, Meredith M. Regan⁴¹⁴,⁴³⁶,⁴³⁷, Michael Misialek⁴³⁸, Mohamed Amgad⁴³⁹, Nadine Tung⁴⁴⁰, Rajendra Singh⁴⁴¹, Richard Huang⁴⁴², Robert H. Pierce⁴⁴³, Roberto Leon-Ferre⁴³³, Sandra Swain⁴⁴⁴, Scott Ely⁴⁴⁵, Seong-Rim Kim⁴⁵⁰, Shahinaz Bedri⁴⁴⁶, Soonmyung Paik⁴⁵⁰, Stuart Schnitt⁴⁰⁹,⁴¹⁰, Timothy d'Alfons⁴¹⁴,⁴¹⁵, Udy Kurkure⁴³⁴, Veerle Bossuyt⁴²¹, Weida Tong⁴⁴⁷, Yihong Wang⁴⁴⁸, Carlos Henrique Dos Anjos⁴⁴⁹, Fabien Gaire⁴⁵⁰ and Paul J. Van Diest⁴⁵¹

⁷⁸Department of Pathology, AZ Sint-Maarten, Mechelen, Belgium. ⁷⁹Department of Pathology, VCU, Richmond, VA, USA. ⁸⁰Pathomorphology, Buzoo KOD, Omsk, Russia.

⁸¹Anatomical Pathology, HUFA, Alcorcón, Spain. ⁸²Pathology, Hospital Clinic Barcelona, Barcelona, Spain. ⁸³Pathology, The Catholic University of Korea, Seoul, South Korea.

⁸⁴Breast Surgery, the First Hospital of Jiaxing, Jiaxing, China. ⁸⁵Oncology, General Teaching hospital Prague, Prague, Czech Republic. ⁸⁶Medical Oncology, Centro Oncologico De Galicia, A Coruña, Spain. ⁸⁷Pathology, Istanbul University-Cerrahpasa, Medical School, Istanbul, Turkey. ⁸⁸Pathology, Tata Memorial Hospital, ACTREC, Mumbai, India. ⁸⁹Pathology, Hospital israelita Albert Einstein, São Paulo, Brazil. ⁹⁰Histopathology, Sullivan Nicolaides, Darwin City, NT, Australia. ⁹¹Anatomical Pathology, Singapore General Hospital, Singapore, Singapore. ⁹²Dentistry, Universidade Estadual de Montes Claros (UNIMONTES), Montes Claros, Brazil. ⁹³Anatomical Pathology, Faculdade de Medicina do ABC, Santo André, Brazil. ⁹⁴Institut für Pathologie, Klinikum Bayreuth, Bayreuth, Germany. ⁹⁵Department of Pathology, Mater Misericordiae University Hospital, Dublin, Ireland.

⁹⁶Department of Pathology, ULSS 6 Euganea, Padua, Italy. ⁹⁷Department of Oncology/Pathology, Karolinska Institutet, Solna, Sweden. ⁹⁸Breast surgery, Memorial Sloan Kettering Cancer Center, Boston, MA, USA. ⁹⁹Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Ulsan, Korea. ¹⁰⁰Pathology, Complejo hospitalario universitario insular materno infantile, Las Palmas de Gran Canaria, Las Palmas, Spain. ¹⁰¹Medical Oncology, Kyungpook National University Hospital, Daegu, South Korea.

¹⁰²Department of Pathology, Mexican Oncology Hospital, IMSS, Mexico, Mexico. ¹⁰³Medical Oncology, Clínica AMO, Salvador, Brazil. ¹⁰⁴Pathology, Complex oncology center Plovdiv, Plovdiv, Bulgaria. ¹⁰⁵Anatomical Pathology, The Oncology Institute Cluj-Napoca, Cluj-Napoca, Romania. ¹⁰⁶Pathology, Imagepat, Porto Alegre, Brazil. ¹⁰⁷Medical Oncology, Azienda Ospedaliera Regionale San Carlo, San Carlo, Italy. ¹⁰⁸Pathology, Virginia Commonwealth University Health, Virginia, VA, USA. ¹⁰⁹Pathology, AllMS, Bhopal, India.

¹¹⁰Pathology, Katedra Patomorfologii UJ CM, Kraków, Poland. ¹¹⁰Pathology, Unipath, Jaipur, India. ¹¹²Department of Pathology, Medical Faculty of Porto University, Porto, Portugal. ¹¹³Pathology, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil. ¹¹⁴Department of Pathology, Keio University School of Medicine, Tokyo, Japan. ¹¹⁵Pathology, Unidad de Patología y Reumatología, Mexico, Mexico. ¹¹⁶Pathology, Hospital Universitario de Canarias, Tenerife, Spain. ¹¹⁷Pathology, IRCCS sacrocuore don calabria, Verona, Italy. ¹¹⁸U.O. Anatomia Patologica, ASST Lariana-Ospedale "Sant'Anna di Como", Vicenza, Italy. ¹¹⁹Laboratory Medicine, De La Salle University Medical Center, De La Salle, Philippines. ¹²⁰Pathology, Instituto Nacional de Cancerologia, Colombia, USA. ¹²¹Pathology, Liverpool Clinical Laboratories, Liverpool University Hospitals NHS Trust, Liverpool, UK. ¹²²Pathology, NSW Health, Gosford, NSW, Australia. ¹²³Life Sciences, Ahmedabad University, Gujarat, India. ¹²⁴Pathology, Tata Memorial Centre, Mumbai, India. ¹²⁵Faculté de Médecine, Université Mouloud MAMMERI, Tizi Ouzou, Algeria. ¹²⁶Pathology, Hung Vuong Hospital, Ho Chin Minh, Hanoi, Vietnam. ¹²⁷Driving, Imperial Driving School, Kisumu City, Kenya. ¹²⁸lovance Biotherapeutics, San Carlos, CA, USA. ¹²⁹Breast Surgery, Nakagami Hospital, Okinawa, Japan. ¹³⁰Pathology, Q2 Solutions, Bathgate, UK. ¹³¹NSW Health Pathology, St George Hospital, Sydney, NSW, Australia. ¹³²Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA, USA. ¹³³Histogenex, Beijing, China. ¹³⁴Department of Pathology, NSW Health Pathology, Sydney, NSW, Australia. ¹³⁵ASL Napoli 1 Centro, Ospedale Pellegrini, Napoli, Italy.

¹³⁶Anatomic Pathology for Clinical Trials, Quest Diagnostics, California, USA. ¹³⁷Department of Pathology, Argos - Fortaleza-CE, Fortaleza, Brazil. ¹³⁸Anatomic Pathology, Q2 Solutions, California, USA. ¹³⁹Pathology, Q2 lab solutions and Henry Mayo Newhall Hospital, California, USA. ¹⁴⁰UOC Anatomia Patologica, ULSS5 Polesana Rovigo, Rovigo, Italy.

¹⁴¹Pathology, DPMG, Sacramento, USA. ¹⁴²Anatomic Pathology, Q2 Lab Solutions, San Juan Capistrano, California, USA. ¹⁴³Anatomic Pathology, Q2 Lab Solutions, Bathgate, UK.

¹⁴⁴Department of Oncology, IMIP, Northeast, Brazil. ¹⁴⁵Surgical Pathology, San Cecilio Hospital, Granada, Spain. ¹⁴⁶Anatomical Pathology, NSW Health, Sydney, Australia.

¹⁴⁷Histopathology Department, Betsi Cadwaladr University Health Board, North Wales, UK. ¹⁴⁸Pathology, Federal University of Pernambuco, Recife, Brazil. ¹⁴⁹School of Control Science and Engineering, Shandong University, Jinan, China. ¹⁵⁰Pathology, Gustave Roussy Institut, Villejuif, France. ¹⁵¹Anatomical Pathology, Royal North Shore Hospital, St Leonards, NSW, Australia. ¹⁵²Department of Pathology, Stavanger University Hospital, Stavanger, Norway. ¹⁵³Q2 Solutions, Atlanta, GA, USA. ¹⁵⁴Pathology, Krasnoyarsk State Medical University, Krasnoyarsk, Russia. ¹⁵⁵Pathological Anatomy, Padova University Hospital, Padua, Italy. ¹⁵⁶Division of Imaging, Diagnostics, and Software Reliability, US Food and Drug Administration, Silver Spring, MD, USA. ¹⁵⁷School of Medicine, Medical Science and Nutrition, University of Aberdeen, Aberdeen, Scotland. ¹⁵⁸Multidisciplinary Oncologic Centre Antwerp (MOCA), Antwerp University Hospital, Edegem, Belgium. ¹⁵⁹Public Health and Primary Care, Katholieke Universiteit Leuven, Leuven, Belgium.

¹⁶⁰Pathology, Washington University at St. Louis, St. Louis, MO, USA. ¹⁶¹Optimisation and Pattern Recognition Group, University of Melbourne, Melbourne, Australia. ¹⁶²Anatomic Pathology, ASST Bergamo Ovest, Treviglio, Italy. ¹⁶³Pathology Department, Hospital Quiron Salud, Madrid, Spain. ¹⁶⁴Department of Pathology, University of Pennsylvania, Philadelphia, USA. ¹⁶⁵Department of Pathology, Nakagami Hospital, Okinawa, Japan. ¹⁶⁶Department of Pathology, Erasmus MC, Cancer Institute, Rotterdam, The Netherlands. ¹⁶⁷Medical Oncology, San Raffaele Scientific Institute, Milan, Italy. ¹⁶⁸Anatomic Pathology, Istanbul Florence Nightingale Hospital, İstanbul, Turkey. ¹⁶⁹Cancer

Biology and Therapeutics Laboratory, School of Biomolecular and Biomedical Science, UCD Conway Institute, Dublin, Ireland. ¹⁷⁰Precision Oncology Ireland, Conway Institute, UCD, Dublin, Ireland. ¹⁷¹Ohio State University Comprehensive Cancer Center, Columbus, OH, USA. ¹⁷²Medical Oncology, Centro Hospitalar Universitario do Algarve, Faro, Portugal. ¹⁷³Medical Biophysics, University of Toronto, Toronto, Canada. ¹⁷⁴Histogenex, Antwerpen, Belgium. ¹⁷⁵Institute of Pathology Heidelberg (IPH), University Hospital Heidelberg, Heidelberg, Germany. ¹⁷⁶Head breast Cancer Program, Oncology Department, IOB institute of Oncology, Quiron Group, Barcelona, Spain. ¹⁷⁷Department of Pathology, University Hospital, Sofia, Bulgaria. ¹⁷⁸Public Health and Primary Care, KU Leuven, Leuven, Belgium. ¹⁷⁹Department of Oncology, Hospital de Mataro, Barcelona, Spain.

¹⁸⁰Department of Pathology, University of Kansas Medical Center, KansasCity, KS, USA. ¹⁸¹Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

¹⁸²Adelson School of Medicine, Ariel University, Ramat Gan, Israel. ¹⁸³Institute of Cancer Research, London, UK. ¹⁸⁴Memorial Sloan Kettering Cancer Centre, Department of Pathology, New York, NY, USA. ¹⁸⁵Department of Pathology, Léon Bérard Cancer Center, Lyon, France. ¹⁸⁶Department of Pathology, Saint-Petersburg clinical scientific and practical center for specialised types of medical care (oncological), St.Petersburg, Russia. ¹⁸⁷Fundacion Instituto Valenciano de Oncologia, Valencia, Spain. ¹⁸⁸Histopathology, Aster Labs, Shoreview, St Paul, MN, USA. ¹⁸⁹St. Vincent's Hospital, The Catholic University of Korea, Seoul, South Korea. ¹⁹⁰Department of Pathology, University of Marburg, Marburg, Germany. ¹⁹¹Department of Oral and Maxillofacial Diseases, Helsinki, Finland. ¹⁹²Faculty of Medicine and Health Sciences, Macquarie University, Sydney, Australia. ¹⁹³KU Leuven- University of Leuven, Department of Imaging and Pathology, Laboratory of Translational Cell & Tissue Research and KU Leuven- University Hospitals Leuven, Department of Pathology, Leuven, Belgium. ¹⁹⁴Institut für Pathologie, UK Hamburg, Hamburg, Germany. ¹⁹⁵Department of Surgical Pathology, JR Kyushu Hospital, Kitakyushu, Fukuoka, Japan. ¹⁹⁶Biomedical Quality Assurance Research Unit, KU Leuven, Leuven, Belgium. ¹⁹⁷Cancer Bioinformatics Lab, Cancer Centre at Guy's Hospital, London, UK. ¹⁹⁸School of Life Sciences and Medicine, King's College London, London, UK. ¹⁹⁹Department of Veterinary Medicine, University of Perugia, Perugia, Italy. ²⁰⁰Department of Pathology, Karolinska Institute, Solna, Sweden. ²⁰¹Consulting, PixelMed, Bangor, PA, USA. ²⁰²Department of Pathology and Laboratory Medicine, UNC School of Medicine, Chapel Hill, NC, USA. ²⁰³Department of Laboratory Medicine and Molecular Diagnostics, Sunnybrook Health Sciences Centre, Toronto, Canada. ²⁰⁴Roche Diagnostics, Brussel, Belgium.

²⁰⁵Department of Radiation Oncology, Sunnybrook Health Sciences Centre, Toronto, ON, Canada. ²⁰⁶Department of Radiation Oncology, University of Toronto, Toronto, Canada.

²⁰⁷Breast Cancer Now Research Centre, The Institute of Cancer Research, London, UK. ²⁰⁸Merck & Co., Inc, Kenilworth, NJ, USA. ²⁰⁹Oncology Merck & Co., Kenilworth, NJ, USA.

²¹⁰Pathology Department, Hospital General Universitario Gregorio Marañón, Madrid, Spain. ²¹¹Catalan Institute of Oncology, Hospital Germans Trias i Pujol, Badalona, Spain.

²¹²Pathology and Molecular Diagnostics department, MD Anderson Cancer Center Madrid, Madrid, Spain. ²¹³Roche, Tucson, AZ, USA. ²¹⁴Department of Biomedical Informatics and Department of Pathology, Stony Brook School of Medicine, Stony Brook, NY, USA. ²¹⁵Comprehensive Cancer Center, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Università Cattolica del Sacro Cuore, Roma, Italy. ²¹⁶Medical Oncology Unit, Agostino Gemelli IRCCS Polyclinic Foundation, Rome, Italy. ²¹⁷Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden. ²¹⁸Departments of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. ²¹⁹Department of Clinical Pathology, Akademiska University Hospital, Uppsala, Sweden. ²²⁰Department of pathology, Maastricht University Medical Centre, Maastricht, The Netherlands. ²²¹GHI Le Raincy-Montfermeil, Chelles, Île-de-France, Montfermeil, France. ²²²Department of Medical Biology and Pathology, Gustave Roussy Cancer Campus, Villejuif, France. ²²³Laboratory of Molecular and Tumor Immunology, Earle A. Chiles Research Institute/ Providence Cancer Center, Portland, Oregon, USA. ²²⁴Department
of Pathology, Breast Pathology Section, Northwestern University, Chicago, IL, USA.²²⁵Harry Perkins Institute of Medical Research, University of Western Australia, Crawley, WA, Australia.²²⁶Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, USA.²²⁷Department de Anatomia Patologica, Hospital de Terrassa, Barcelona, Spain.²²⁸Department of Pathology, Netherlands Cancer Institute, Amsterdam, The Netherlands.²²⁹Department of Anatomic Pathology, Faculty of Medicine, Universidad De La Frontera, Temuco, Chile.²³⁰Department of Pathology, Universidad de La Frontera, Temuco, Chile.²³¹Department of Pathology and Laboratory Medicine, Tufts Medical Center, Boston, USA.²³²Institute of Cancer Research Clinical Trials and Statistics Unit, The Institute of Cancer Research, Surrey, UK.²³³Department of Medical Sciences, University of Turin, Turin, Italy.²³⁴The Fingerland Department of Pathology, University Hospital Hradec Kralove, Kralove, Czech Republic.²³⁵Department of Surgical Pathology Zealand University Hospital, Zealand, Denmark.²³⁶Department of Pathology, Semmelweis University, Budapest, Hungary.²³⁷Department of Pathology, St Vincent's University Hospital and University College Dublin, Dublin, Ireland.²³⁸University of Turin / Candiolo Cancer Institute - FPO, IRCCS, Candiolo, Italy.²³⁹Servicio de Anatomia Patologica, Centro Hospitalar Universitario de Sao Joao and Ipatimup, Porto, Portugal.²⁴⁰University of Turin at Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Italy.²⁴¹Nuffield Department of Population Health, University of Oxford, Oxford, UK.²⁴²Department of Medical Oncology, University Hospitals Bristol NHS Foundation Trust, Bristol, UK.²⁴³Institut Curie, Department of Pathology, Inserm U934,Paris Sciences Lettres University, Paris, France.²⁴⁴Pharmaceutical Research and Early Development (pRED), Roche Innovation Center Munich, Penzberg, Germany.²⁴⁵Department of Surgical Pathology, gSaint Savvash Regional Anticancer Hospital, Athens, Greece.²⁴⁶Department of Pathology, Herlev and Gentofte University Hospital, Herlev, Denmark.²⁴⁷Dipartimento di Scienze della Salute (DSS), Firenze, Italy.²⁴⁸Department of Pathology, Medical University of Vienna, Vienna, Austria.²⁴⁹Department of Physics and Astronomy, University of California, Irvine, Irvine, CA, USA.²⁵⁰Diagnostic and Research Institute of Pathology, Medical University of Graz, Graz, Austria.²⁵¹The Gillies McIndoe Research Institute, Wellington, New Zealand.²⁵²Department of Pathology, BC Cancer Agency Vancouver Centre, Department of Pathology, Vancouver, Canada.²⁵³Department of Pathology, University Hospital, Geneva, Switzerland.²⁵⁴Division of Imaging, Diagnostics, and Software Reliability, Office of Science and Engineering Laboratories, Center for Devices and Radiological Health, US Food and Drug Administration, Silver Spring, MD, USA.²⁵⁵University Hospital Halle (Saale), Institute of Pathology, Halle (Saale), Germany.²⁵⁶Oncology Department, Cancer Institute, University College London, London, UK.²⁵⁷Department of Pathology, University Hospital of Bellvitge, Oncobell, IDIBELL, LfHospitalet del Llobregat, Barcelona 08906 Barcelona, Spain.²⁵⁸Department of molecular pathology, Netherlands Cancer Institute, Amsterdam, The Netherlands.²⁵⁹Translational Research, Forbus, Montreal, Canada.²⁶⁰Vall dfHebron Hospital Research Institute, Autoimmune Diseases department, Barcelona, Spain.²⁶¹Research Pathology, Genentech Inc., South San Francisco, CA, USA.²⁶²Department of Pathology, GZA-ZNA Ziekenhuizen, Wilrijk, Belgium.²⁶³Translational Gerontology Branch, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.²⁶⁴Experimental Gerontology Section and Translational Gerontology Branch, NIA, NIH, Baltimore, USA.²⁶⁵Masaryk Memorial Cancer Institute, Brno, Czech Republic.²⁶⁶Department of Pathology and Cell Biology, Faculty of Medicine, Montreal University, Montreal, Canada.²⁶⁷Scientific Director, Image Analysis, AstraZeneca Computational Pathology, Munich Area, Germany.²⁶⁸Pathology, CHU UCL, Namur, Belgium.²⁶⁹Anatomical Pathology, Hospital Italiano de Buenos Aires, Buenos Aires, Argentina.²⁷⁰Anatomical Pathology, Austin Health, Heidelberg, Australia.²⁷¹University of Sydney, Sydney, Australia.²⁷²Department of Medical Oncology, Peter MacCallum Cancer Centre, Melbourne, Australia.²⁷³Department of Medicine, University of Melbourne, Parkville, Australia.²⁷⁴Directorate of Surgical Pathology, SA Pathology, Adelaide, Australia.²⁷⁶Department of Anatomical Pathology, Royal Melbourne Hospital, Parkville, Australia.²⁷⁷The Cancer Research Program, Garvan Institute of Medical Research, Darlinghurst, Australian Clinical Labs, Darlinghurst, Australia.²⁷⁸Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Australia.²⁷⁹Division of Research and Cancer Medicine, Peter MacCallum Cancer Centre, University of Melbourne, Victoria, Australia.²⁸⁰The University of Queensland Centre for Clinical Research and Pathology Queensland, Brisbane, Australia.²⁸¹NSW Health Pathology, Sydney, Australia.²⁸²Department of Medical Oncology, Austin Health, Heidelberg, Australia.²⁸³Institute of Pathology, Medical University of Graz, Graz, Austria.²⁸⁴Department of Medicine, Clinical Division of Oncology, Comprehensive Cancer Centre Vienna, Medical University of Vienna, Vienna, Austria.²⁸⁵Department of Pathology, Hospital Graz II, Academic Teaching Hospital of the Medical University Graz, Graz, Austria.²⁸⁶Department of Radiation Oncology, Iridium Kankernetwerk, Wilrijk- Antwerp, Belgium.²⁸⁷Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk-Antwerp, 2610 Antwerpen, Belgium.²⁸⁸Department of Radiology, Jules Bordet Institute, Bruxelles, Belgium.²⁸⁹Department of Pathology, AZ Turnhout, Turnhout, Belgium.²⁹⁰Department of Pathology, Jules Bordet Institute, Brussels, Belgium.²⁹¹HistoGeneX NV, Antwerp, Belgium and AZ Sint-Maarten Hospital, Mechelen, Belgium.²⁹²Department of Pathology, University Hospital Antwerp, Edegem, Belgium.²⁹³Department of Pathology, University Hospital Ghent, Gent, Belgium.²⁹⁴Medical Oncology, GZA, Antwerp, Belgium.²⁹⁵Pathology department of ZNA and GZA hospitals in Antwerp, Antwerp, Belgium.²⁹⁶Institut Jules Bordet, Universite Libre de Bruxelles, Brussels, Belgium.²⁹⁷Department of Pathology, Cliniques Universitaires Saint-Luc, Brussels, Belgium.²⁹⁸Department of Pathology, Institut Jules Bordet, Universite Libre de Bruxelles, Brussels, Belgium.²⁹⁹Department of Pathology, Institut Jules Bordet, Brussels, Belgium.³⁰⁰Department of Development and Regeneration, Laboratory of Experimental Urology, KU Leuven, Leuven, Belgium.³⁰¹Bioinformatics and Computational Genomics Laboratory, Princess Margaret Cancer Center, Toronto, Canada.³⁰²Trev & Joyce Deeley Research Centre, British Columbia Cancer Agency, Victoria, Canada.³⁰³Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada.³⁰⁴University of British Columbia, Vancouver, British Columbia, Canada.³⁰⁵Genetic Pathology Evaluation Centre, Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada.³⁰⁶Center for Pharmacogenomics and Fudan-Zhangjiang, Center for Clinical Genomics School of Life Sciences and Shanghai Cancer Center, Fudan University, Shanghai, China.³⁰⁷Department of Pathology, Herlev and Gentofte Hospital, Herlev, Denmark.³⁰⁸DTU Compute, Department of Applied Mathematics, Technical University of Denmark, Visiopharm A/S, Horsholm, Denmark.³⁰⁹Department of Pathology, University of Helsinki and University of Turku, Turku, Finland.³¹⁰Department of Otorhinolaryngology - Head and Neck Surgery, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.³¹¹Helsinki University Central Hospital, Helsinki, Finland.³¹²Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, Finland.³¹³Gustave Roussy, Universite Paris-Saclay, Villejuif, France.³¹⁴Université Paris-Sud, Institut National de la Santé et de la Recherche Médicale, Villejuif, France.³¹⁵Université Paris-Saclay, Univ. Paris-Sud, Villejuif, France.³¹⁶Service de biostatistique et d'epidemiologie, Gustave Roussy, Villejuif, France.³¹⁷Department of Medical Oncology, Gustave Roussy, Villejuif, France.³¹⁸Centre de Lutte Contre le cancer - Centre Jean Perrin, Clermont-Ferrand, France.³¹⁹R&D UniCancer, Paris, France.³²⁰Department of Pathology, Gustave Roussy, Grand Paris, France.³²¹Department of Pathology, Gustave Roussy, Villejuif, France.³²²Service de Biostatistique et d'Epidémiologie, Gustave Roussy, CESP, Université-Paris Sud, Université Paris-Saclay, Villejuif, France.³²³Department of Surgical Pathology and Biopathology, Jean Perrin Comprehensive Cancer Centre, Clermont-Ferrand, France.³²⁴Institute of Pathology, Charité Universitätsmedizin Berlin, Berlin, Germany.³²⁵Breast Center, Dept. OB&GYN and CCC (LMU), University of Munich, Munich, Germany.³²⁶Johanniter GmbH - Evangelisches Krankenhaus Bethesda Mönchengladbach, West German Study Group, Monchengladbach, Germany.³²⁷Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany.³²⁸Berlin Institute of Health, Institute of Pathology, Charitéplatz 1, 10117 Berlin, Germany.³²⁹Department of Pathology, Bäcs-Kiskun County Teaching Hospital, Kecskemét, Hungary.³³⁰Department of Pathology, University of Szeged, Szeged, Hungary.³³¹Department of Pathology, Istituto Europeo di Oncologia, University of Milan, Milan, Italy.³³²Department of Medical Oncology, Istituto Europeo di Oncologia, Milan, Italy.³³³Azienda AUSL, Regional Hospital of Aosta, Aosta, Italy.³³⁴University of Milano, Istituto Europeo di Oncologia, IRCCS, Milano, Italy.³³⁵Departments of Surgical Pathology, Kyoto Prefectural University of Medicine, Kyoto, Japan.³³⁶Kyoto University, Department of Breast Surgery, Kyoto, Japan.³³⁷Kansai Medical University Medical Center, Osaka, Japan.³³⁸Department of Breast Surgery, Kyoto University, Kyoto, Japan.³³⁹Department of Pathology and Laboratory Medicine, Kansai Medical University, Osaka, Japan.³⁴⁰Breast Surgery, Kansai Medical University Hospital, Osaka, Japan.³⁴¹Medical Oncology, Fukushima Medical University Hospital, Fukushima, Japan.³⁴²Japanese Foundation for Cancer Research, Tokyo, Japan.³⁴³Department of Diagnostic Pathology, Nippon Medical School Hospital, Tokyo, Japan.³⁴⁴Chugai Pharmaceutical Co., Ltd, Tokyo, Japan.³⁴⁵Department of Pathology, Kawasaki Medical School, Okayama, Japan.³⁴⁶Department of Diagnostic Pathology, Kyoto University, Kyoto, Japan.³⁴⁷National Cancer Center Hospital, Tokyo, Japan.³⁴⁸Department of Surgery, Kansai Medical School, Hirakata, Japan.³⁴⁹Department of Pathology, Mie University School of Medicine, Mie, Japan.³⁵⁰Pathology, Aga Khan University Hospital, Nairobi, Kenya.³⁵¹Department of Pathology and Diagnostic Laboratory Services, UKM Medical Centre, Kuala Lumpur, Malaysia.³⁵²Surgical Pathologist, North Shore Hospital, WDHB, Auckland, New Zealand.³⁵³Department of Oncology & Radiotherapy, Medical University of Gdaś, Gdansk, Poland.³⁵⁴Tumor Pathology Department, Maria Sklodowska-Curie Memorial Cancer Center, Gliwice, Poland.³⁵⁵Breast Unit, Champalimaud Clinical Center/Champalimaud Foundation, Lisbon, Portugal.³⁵⁶Breast Unit, Champalimaud Clinical Centre, Lisbon, Portugal.³⁵⁷Department of Oncology, National Cancer Centre, Singapore, Singapore.³⁵⁸Division of Medical Oncology, National Cancer Centre Singapore, Singapore, Singapore.³⁵⁹Medical Oncology Service, Hospital General Universitario Gregorio Maranon, Universidad Complutense, Madrid, Spain.³⁶⁰Palex Medical SA - Exact Sciences Corp, Madrid, Spain.³⁶¹Department of Oncology, IVO Valencia, Valencia, Spain.³⁶²Pathology Department, Instituto de Investigación Sanitaria Fundación Jimenez Diaz (IIS-FJD), Madrid, Spain.³⁶³GEICAM-Spanish Breast Cancer Research Group, Madrid, Spain.³⁶⁴Pathology Department, Hospital del Mar, Parc de Salut Mar, Barcelona, Spain.³⁶⁵Molecular Oncology Group, Vall dfHebron Institute of Oncology, Barcelona, Spain.³⁶⁶Department of Pathology, Hospital General Universitario de Castelló, Castello, Spain.³⁶⁷Pathology Department, H.U. Vall dfHebron, Barcelona, Spain.³⁶⁸Medical Oncology, The Netherland Cancer Institute, Amsterdam, The Netherlands.³⁶⁹Computational Pathology Group, Department of Pathology, Radboud University Medical Center, Nijmegen, The Netherlands.³⁷⁰Division of Molecular Pathology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.³⁷¹Department of Research IT, The Netherlands Cancer Institute, Amsterdam, The Netherlands.³⁷²Department of Pathology, The
Netherlands Cancer Institute, Amsterdam, The Netherlands. ³⁷³Division of Molecular Oncology & Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

³⁷⁴Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands. ³⁷⁵Medical Oncology, Netherlands Cancer Institute, Amsterdam, The Netherlands.

³⁷⁶Medical Oncology, University Hospital Southampton NHS Foundation Trust, Southampton, UK. ³⁷⁷Translational Breast Radiobiology, Institute of Cancer Research, Honorary Consultant Clinical Oncologist (Breast), The Royal Marsden, London, UK. ³⁷⁸Breast Cancer Now Toby Robins Research Centre, The Institute of Cancer Research, London, UK.

³⁷⁹Cancer Research UK Lung Cancer Centre of Excellence, University College London Cancer Institute, University College London, London, UK. ³⁸⁰Department of Pathology, UCL Cancer Institute, UCL, London, UK. ³⁸¹University College Hospitals NHS Trust, London, UK. ³⁸²Vivactiv Ltd, Bellingdon, Bucks, UK. ³⁸³Leicester Cancer Research Centre, University of Leicester, Leicester, and MRC Toxicology Unit, University of Cambridge, Cambridge, UK. ³⁸⁴Centre for Evolution and Cancer; Division of Molecular Pathology, The Institute of Cancer Research, London, UK. ³⁸⁵Clinical Trial Service Unit & Epidemiological Studies Unit, University of Oxford, Oxford, UK. ³⁸⁶Guy's Hospital, London, UK. ³⁸⁷King's College London, London, UK. ³⁸⁸Department of Pathology, Wexner Medical Center at the Ohio State University, Columbus, OH, USA. ³⁸⁹Mayo Clinic, Rochester, Minnesota Joint Appointment, Department of laboratory Medicine and Pathology, Research Chair, Division of Medical Oncology, Robert Mutter Radiation Oncology, Mayo Clinic, Rochester, MN, USA. ³⁹⁰Albert Einstein College of Medicine in New York, New York, NY, USA. ³⁹¹Division of Anatomic Pathology, University of California San Diego Health System, San Diego, CA, USA. ³⁹²Surgical Oncology, Baylor College of Medicine, Houston, TX, USA. ³⁹³Departments of Pathology, Genomic Medicine, Dermatology, and Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. ³⁹⁴PhenoPath Laboratories, Seattle, WA, USA. ³⁹⁵Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. ³⁹⁶Case Western Reserve University; Louis Stokes Cleveland Veterans Health Administration Medical Center, Cleveland, OH, USA. ³⁹⁷Pulmonary Pathology, New York University Center for Biospecimen Research and Development, New York University, New York, NY, USA. ³⁹⁸Department of Pathology, Johns Hopkins Hospital, Baltimore, MD, USA. ³⁹⁹PathAI, Inc., Cambridge, MA, USA. ⁴⁰⁰Department of Pathology, University of Iowa Hospitals and Clinics, Iowa City, IA, USA. ⁴⁰¹Breast Cancer Trials, Women's Malignancies Disease Group, The Johns Hopkins Kimmel Cancer Center, Baltimore, MD, USA. ⁴⁰²Department of Pathology, University Hospitals Cleveland Medical Center affiliated with Case Western University, Cleveland, OH, USA. ⁴⁰³Department of Biomedical Informatics, Emory University, Atlanta, GA, USA. ⁴⁰⁴Departments of Pathology and Oncology, The Johns Hopkins Hospital, Baltimore, MD, USA. ⁴⁰⁵National Surgical Adjuvant Breast and Bowel Project Operations Center/NRG Oncology, Pittsburgh, PA, USA.

⁴⁰⁶Department of Pathology, New York University Langone Medical Centre, New York, NY, USA. ⁴⁰⁷Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA, USA. ⁴⁰⁸Mayo Clinic Breast SPORE, Mayo Clinic, Rochester, MN, USA. ⁴⁰⁹Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA.

⁴¹⁰Dana-Farber Cancer Institute, Boston, MA, USA. ⁴¹¹Oncology Clinical Development, Bristol-Myers Squibb, Princeton, NJ, USA. ⁴¹²Department of Medicine, Vanderbilt University Medical Centre, Nashville, TN, USA. ⁴¹³Department of Cancer Biology, Mayo Clinic, Jacksonville, FL, USA. ⁴¹⁴Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA. ⁴¹⁵Department of Pathology, Dana Farber Cancer Institute, Boston, MA, USA. ⁴¹⁶National Cancer Institute, Bethesda, MD, USA.

⁴¹⁷Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA. ⁴¹⁸Medical Oncology, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA. ⁴¹⁹PathAI Inc., Cambridge, MA, USA. ⁴²⁰Translational Sciences, MedImmune, Gaithersberg, MD, USA. ⁴²¹Department of Pathology, Massachusetts General Hospital, Boston, MA, USA. ⁴²²Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, NY, USA. ⁴²³Department of Medicine, Department of Obstetrics & Gynecology and Women's Health, Albert Einstein Medical Center, Bronx, NY, USA. ⁴²⁴Departments of Medicine and Cancer Biology, Vanderbilt University Medical Centre, Nashville, TN, USA. ⁴²⁵NSABP/NRG Oncology, Pittsburgh, PA, USA. ⁴²⁶Yale Cancer Center Genetics, Genomics and Epigenetics Program, Yale School of Medicine, New Haven, CT, USA. ⁴²⁷Pathology Department, Stanford University Medical Centre, Stanford, CA, USA. ⁴²⁸Department of Medical Oncology, Yale University School of Medicine, New Haven, CN, USA. ⁴²⁹Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA. ⁴³⁰Biorepository and Tissue Technology Shared Resources, University of California San Diego, San Diego, CA, USA. ⁴³¹Breast Cancer Research Foundation, New York, NY, USA. ⁴³²Memorial Sloan Kettering Cancer Center, New York, NY, USA. ⁴³³Department of Oncology, Mayo Clinic, Rochester, NY, USA. ⁴³⁴Roche Tissue Diagnostics, Digital Pathology, Santa Clara, CA, USA. ⁴³⁵Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Centre, Nashville, TN, USA. ⁴³⁶Division of Biostatistics, Dana-Farber Cancer Institute, Boston, MA, USA.

⁴³⁷Harvard Medical School, Boston, MA, USA. ⁴³⁸Vernon Cancer Center, Newton-Wellesley Hospital, Newton, MA, USA. ⁴³⁹Department of Biomedical Informatics, Emory University School of Medicine, Atlanta, GA, USA. ⁴⁴⁰Division of Hematology-Oncology, Beth Israel Deaconess Medical Center, Boston, MA, USA. ⁴⁴¹Icahn School of Medicine at Mt. Sinai, New York, NY, USA. ⁴⁴²Department of Pathology, Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA. ⁴⁴³Cancer Immunotherapy Trials Network, Central Laboratory and Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. ⁴⁴⁴Department of Pathology, Weill-Cornell Medicine, New York, NY, USA.

⁴⁴⁵Translational Medicine, Bristol-Myers Squibb, Princeton, NJ, USA. ⁴⁴⁶Anatomic Pathology, Boston, MA, USA. ⁴⁴⁷Division of Bioinformatics and Biostatistics, U.S. Food and Drug Administration, Silver Spring, MD, USA. ⁴⁴⁸Department of Pathology and Laboratory Medicine, Rhode Island Hospital and Lifespan Medical Center, Providence, RI, USA.

⁴⁴⁹Oncology Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA. ⁴⁵⁰Pathology and Tissue Analytics, Roche, Basel, Switzerland.

⁴⁵¹Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.