Assessment Of Tumor-infiltrating Lymphocytes Research Articles

Evaluating the prognostic significance of tumor-infiltrating lymphocytes in solid tumor: practice of a standardized method from the International Immuno-Oncology Biomarkers Working Group.

PurposeTumor-infiltrating lymphocytes (TILs) become increasingly relevant to tumor progression. This study aims to evaluate (a) methods of TILs assessment and (b) their prognostic significance in gastric cancer (GC).MethodsThe percentage of stromal TILs (psTIL) was reported semi-quantitatively by H&E evaluation. Herein, we screened two independent cohorts of breast cancer (n=240) and GC (n=481) for psTIL characterization. Correlations between psTIL and clinic-pathological features, as well as overall survival (OS) were further explored. Additionally, the prediction role of psTIL in GC was evaluated by receiver operating characteristic curve (ROC) analysis.ResultsTILs could be demonstrably distinguished from other stromal areas and surrounding tumor nests according to the assessment method. More importantly, it is reproducible, easily to determine, and quickly performed. In GC, a two-grade scale for psTIL was appropriate to be divided into low and high subgroups by using the median value of 10% as the threshold. High psTIL was correlated with no serosa invasion, earlier TNM stage and better survival state (P<0.05 for all), and identified as a favorable prognostic factor both by univariate (HR: 0.734, P=0.047) and multivariate analyses (HR: 0.722, P=0.030). A beneficial OS of high psTIL was found in a linear manner with increasing TILs infiltrates associated with improved survival by Kaplan–Meier survival curve (P=0.030) and ROC analysis (AUC: 0.432, P=0.012).ConclusionTILs provide a reproducible method for assessment that can potentially be used to guide management. The parameter psTIL could be served as an independent, favorable prognostic factor of GC.

Abstract P4-03-10: Identifying germline APOBEC3B deletion using hereditary cancer panel in Korean patients with operable breast cancer

Abstract Background: APOBEC3B is a cytosine deaminase implicated in host immune defense to virus and mutagenesis in cancer. Germline APOBEC3B deletion is known as risk factors for breast cancer with hypermutation and immune activation from previous database-based studies. This study was aimed to evaluate the incidence of germline APOBEC3B deletion in Korean patients with operable breast cancer. Method: The copy number variants of germline APOBEC3B deletion was analyzed from leukocyte DNA of 103 breast cancer patients whose bloods were collected in 2009 for pharmacogenomic study at Seoul National University Bundang Hospital. Hybrid-capture based next-generation sequencing panel targeting 53 hereditary cancer genes were used. We also measured tumor infiltrating lymphocytes (TILs) and programmed cell death-ligand 1 (PD-L1) expression in tumor or immune cell with a rabbit monoclonal antibody (E1L3N). Results: Median age of breast cancer diagnosis was 46 (25-72). In APOBEC3B deletion analysis, 10 (9.7%), 36 (35.0%), and 57 (55.3%) patients were identified as two-copy deletion (A3Bdel/del), one-one copy deletion (A3Bdel/wt) and no deletion (A3Bwt/wt), respectively. In non-APOBEC3B analysis, 9 (8.7%) patients were identified as pathogenic variant: RAD51D(n=1), GJB2(n=1), BRCA1(n=1), BRCA2 (n=2), ATM(n=1), USH2A(n=1), RET(n=1), BARD1(n=1). We observed no significant association between germline APOBEC3B deletion with any clinicopathologic features of breast cancer such as age, family history of cancer, and bilateral breast cancer. Triple-negative subtype was associated with A3Bwt/wt Tumors (35.1% in A3Bwt/wt vs. 5.6% in A3Bdel/wt vs20% in A3Bdel/del; P=0.018). After a median follow-up time of 92.8 months, APOBEC3B deletion was not predictive of recurrence or survival. In patients with sufficient tumor samples for the assessment of TIL (n=63) and PD-1 (n=71), A3Bdel/del tumor was associated with higher TILs (&amp;gt;10%) than other tumor types (6/7 patients in A3Bdel/del vs. 13/24 in A3Bdel/wt vs. 15/32 in A3Bwt/wt: Fisher's exact test in A3Bdel/del, P=0.029). However, PD-L1 expression was not associated with APOBEC3B deletion status (1/7 patients &amp;gt;1% PD-L1 in A3Bdel/del vs. 4/26 in A3Bdel/wt vs. 8/38 in A3Bwt/wt: P=0.901). Germline APOBEC3B deletion and TILs (n=63) TIL (0-10%)TIL (&amp;gt;10%)TotalA3B(wt/wt)17 (53.1%)15 (46.9%)32A3B(del/wt)11 (45.8%)13 (54.2%)24A3B(del/del)1 (14.3%)6 (85.7%)7 Conclusion: We identified germline APOBEC3B deletion in 9.7% of Korean patients with operable breast cancer. The relationship between A3Bdel/del tumor and high TILs suggests that these tumors might be potential candidates for future immunotherapy. Citation Format: Kim SH, Koung Jin S, Kim YJ, Ahn S, Park SY, Chae SM, Kang E, Kim E-K, Kim IA, Kim JH. Identifying germline APOBEC3B deletion using hereditary cancer panel in Korean patients with operable breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-03-10.

Assessment of TILs, IDO-1, and PD-L1 in resected non-small cell lung cancer: an immunohistochemical study with clinicopathological and prognostic implications.

Several cancers, especially non-small cell lung cancer (NSCLC), are able to escape the immunosurveillance of tumor-infiltrating lymphocytes (TILs); among the molecules involved, the indoleamine 2,3-dioxygenase 1 (IDO-1) and the programmed cell death ligand-1 (PD-L1) play a crucial role. These aspects are of great interest in the current immunotherapeutic era, therefore the current study analyses the TILs, IDO-1, and PD-L1 interactions and their correlations with clinicopathological parameters and prognosis in NSCLC. One hundred ninety-three NSCLC surgical specimens, formalin-fixed, and paraffin-embedded were assessed for TILs density, TILs localization, IDO-1 (clone 4.16H1), and PD-L1 (clone E1L3N) immunohistochemical expressions. This data was correlated with clinicopathological parameters, disease free, and overall survivals. IDO-1 and PD-L1 high expressions were related to the solid pattern of adenocarcinomas (respectively p = 0.036 and p = 0.026); high PD-L1 expression was correlated with squamous histotype (p = 0.048). IDO-1 overexpression correlated with former smokers (p = 0.041), higher adenocarcinoma stages (p = 0.039), and with both higher TILs density and PD-L1 expression (respectively p = 0.025 and p = 0.0003). A better prognosis was associated with TILs intratumoral or mixed localizations (p = 0.029). TILs localization affects NSCLC prognosis; the higher expression of IDO-1 and PD-L1 in poorly differentiated and more aggressive lung adenocarcinomas, as well as the correlation between high PD-L1 expression and squamous cell histotype, confirm the more efficient immunoescaping of these NSCLC subgroups.

The prognostic and predictive value of tumor-infiltrating lymphocytes and hematologic parameters in patients with breast cancer

BackgroundCarcinogenesis and tumor growth are associated with chronic inflammation and the host immune system. Here, we investigated the clinical significance and relationship between tumor-infiltrating lymphocytes (TILs) and hematologic parameters in patients with breast cancer.MethodsInvasive ductal breast cancer patients (N = 145) who underwent surgery were retrospectively evaluated. Samples were obtained using a core needle biopsy for CD8+, FOXP3+ TIL assessment. Blood lymphocytes, neutrophils, monocytes, and platelets were obtained by peripheral venous punctures.ResultsCD8 + TILs were significantly associated with absolute lymphocyte count (ALC) and the absolute monocyte count (AMC). Low LMR (ALC/AMC) (cut-off - 5.3, range = 0.73–12.31) was associated with poor overall survival (OS) (p = 0.010), disease-free survival (DFS) (p = 0.005). However, in subgroup analysis, LMR did not have any value as a prognostic factor in HER2-positive breast cancers. TILs had different prognostic impacts across breast cancer subtypes, although they were not statistically significant. The treatment response after NAC tended to improve in breast cancer patients with high FOXP3+ TILs, low NLR (neutrophil count/ALC) (FOXP3 p for trend = 0.006, NLR p for trend = 0.063).ConclusionsA relevance between TILs and hematologic parameters in breast cancer was demonstrated. The influence of the immune system on breast cancer progression may differ by subtype.

Prognostic significance of tumor-infiltrating lymphocytes in ductal carcinoma in situ of the breast

Tumor-infiltrating lymphocytes (TILs) provide prognostic value in invasive breast cancer and guidelines for their assessment have been published. This study aims to evaluate: (a) methods of TILs assessment, and (b) their prognostic significance in breast ductal carcinoma in situ (DCIS). Hematoxylin and eosin sections from two clinically annotated DCIS cohorts; a training set (n = 150 pure DCIS) and a validation set (n = 666 comprising 534 pure DCIS and 132 cases wherein DCIS and invasive breast carcinoma were co-existent) were assessed. Seven different scoring methods were applied to the training set to identify the most optimal reproducible method associated with strongest prognostic value. Among different methods, TILs touching ducts' basement membrane or away from it by one lymphocyte cell thickness provided the strongest significant association with outcome and highest concordance rate [inter-cluster correlation coefficient = 0.95]. Assessment of periductal TILs at increasing distances from DCIS (0.2 , 0.5 , and 1 mm) as well as percent of stromal TILs were practically challenging and showed lower concordance rates than touching TILs. TILs hotspots and lymphoid follicles did not show prognostic significance. Within the pure DCIS validation set, dense TILs were associated with younger age, symptomatic presentation, larger size, higher nuclear grade, comedo necrosis and estrogen receptor negativity as well as shorter recurrence-free interval (p = 0.002). In multivariate survival analysis, dense TILs were independent predictor of shorter recurrence-free interval (p = 0.002) in patients treated with breast conservation. DCIS associated with invasive carcinoma showed denser TILs than pure DCIS (p = 9.0 × 10−13). Dense TILs is an independent prognostic variable in DCIS. Touching TILs provides a reproducible method for their assessment that can potentially be used to guide management

Abstract CT101: D-BEYOND: A window of opportunity trial evaluating denosumab, a RANK-ligand (RANKL) inhibitor and its biological effects in young pre-menopausal women diagnosed with early breast cancer

Abstract Background Breast cancer (BC) in young women has unique biology and poor prognosis. Previous reports suggest that they often express RANKL, which was also shown to play a role in mammary tumorigenesis and the etiology of BRCA1/2 related BC. Here, we present the primary results of D-BEYOND, a window of opportunity study investigating the biological activity of the RANKL inhibitor; denosumab in pre-menopausal BC patients. Methods D-BEYOND is a prospective, phase IIa, single-arm, multicenter study assessing the effect of denosumab on BC biology in premenopausal women with early BC (NCT01864798). Patients received two subcutaneous injections of denosumab (120mg), one week apart, followed by breast surgery. Blood, tumor and normal adjacent breast tissue were collected at baseline and at surgery. The primary endpoint was geometric mean change in tumor Ki67 assessed by immunohistochemistry (IHC) from baseline to surgery. Absolute Ki67 responders were defined as having &amp;lt;2.7% IHC staining in the post-treatment tumor. Serum levels of soluble RANKL (sRANKL), OPG and C-terminal telopeptide (CTX) were assessed by ELISA. Ki67, RANK and RANKL expression were assessed by IHC. The percentage of tumor infiltrating lymphocytes (TILs) were also evaluated. Pre- and post-treatment values were compared using a paired t-test. Results A total of 27 patients were enrolled in the study between October 2013 and July 2016. The median age was 45 years (range 35-51 years). Tumors of 21 patients were hormone receptor positive (77.8%), 4 were HER2 positive (14.8%) and 2 were triple negative (7.4%). No serious adverse events were reported, the most frequent non-serious adverse event being arthralgia (14.8%). After treatment, serum levels of CTX and sRANKL decreased in all patients (P &amp;lt; 0.001) whereas serum levels of OPG increased in 76.9% of patients (P = 0.009, 95% CI 0.56-0.91). There was no significant reduction of Ki67 values from baseline (geometric mean change after treatment; 0.98, 95% CI 0.76-1.26; P = 0.90) and there were no absolute Ki67 responders. Twenty-four pre- and post-treatment tumor pairs were available for RANK/L staining and TILs assessment. There was no significant difference in RANKL and RANK H-score in tumors after treatment (P = 0.842, P = 0.142, respectively) but we observed a decrease of RANKL H-score in 3 tumors (12.5%) and an increase of RANK H-score in 5 tumors (20%). Interestingly, there was a significant increase in the percentage of stromal TILs after treatment (geometric mean change of 2.51, 95% CI 1.58-3.97; P = 0.004). There were 10/24 patients (41.7%) with a change in TILs of at least 10%, all of them having an increase in TILs presence (P = 0.002). Conclusion Short course of denosumab did not reduce tumor proliferation rate. However, it induced a significant increase in TILs. These findings suggest that denosumab may potentiate immunotherapy efficacy, at least in young BC patients. Additional results including immune cell profiling by multiplex IHC and RNA-sequencing of tumor and normal tissues will be presented at the meeting. Citation Format: Bastien Nguyen, Marion Maetens, Roberto Salgado, David Venet, Peter Vuylsteke, Laura Polastro, Hans Wieldiers, Philippe Simon, Geoff Lindeman, Denis Larsimont, Gert Van den Eynden, Chloe Velghe, Francoise Rothe, Soizic Garaud, Stefan Michiels, Karen Willard-gallo, Hatem A. Azim, Sherene Loi, Martine Piccart, Christos Sotiriou. D-BEYOND: A window of opportunity trial evaluating denosumab, a RANK-ligand (RANKL) inhibitor and its biological effects in young pre-menopausal women diagnosed with early breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT101.

Abstract 4069: Evaluation of inflammatory infiltration (tumor infiltrating lymphocytes - TIL) in malignant melanoma

Abstract Assessment of tumor infiltrating lymphocytes (TIL) in malignant melanoma (MM) should be a standard part of biopsy examination. This parameter is of interest regarding its prognostic, but potentially also predictive significance. However, the usefulness of this marker in routine practice is limited due to unequal methodological approach and equivocal results of previous studies. The aim of our study is to compare 5 different methods of TIL assessment with respect to their prognostic significance. TIL were double-blindly evaluated by two pathologists in 221 cases of primary cutaneous malignant melanoma. The sample set comprised of pT1 - 48 cases, pT2 - 61 cases, pT3 - 69 cases, and pT4 - 43 cases. Five different scoring methods were used, including: i) Clark scoring; ii) Melanoma Institute Australia (MIA) system; iii) scoring system used in the study of Saldanha et al.; iv) scoring system used in the TCGA study and modified by Park et al.; v) the system recently proposed by “International Immuno-Oncology Biomarker Working Group“ for TIL scoring in all solid tumors. Cox's Proportional Hazard Method was used to test 10-year survival with three main outcomes - disease-specific survival (DFS), local recurrence-free survival (LFS), and distant metastasis-free survival (MFS). The evaluation of TIL with respect to its prognostic value was statistically significant for all 3 outcomes when using Clark scoring (score 0, 1 vs. score 2; DFS p=0.027, LFS p=0.019, and MFS p=0.010); MIA system (groups 0, 1 vs. groups 2, 3; DFS p=0.060, LFS p=0.035, and MFS p=0.012) and the system proposed by “International Immuno-Oncology Biomarker Working Group“. In this system, however, only the assessment of stromal TIL (not intratumoral or in the invasive margin) showed prognostic significance (DFS p=0.034, LFS p=0.033, and MFS p=0.033). Our results did not prove prognostic significance of the scoring systems used in the study published by Saldanha et al. and Park et al. In conclusion, the results of our study showed that the evaluation of TIL in MM is a prognostic marker, but its significance is dependent on the chosen scoring method. From a practical point of view, application of a unified methodology is desirable. This would allow for a comparison across studies, and, even more interestingly, also for the assessment of the predictive value of TIL evaluation, especially with respect to immunotherapy. However, out of the five different methods used in our study, only 3 showed statistically significant results and none of the 3 systems seem to be superior to others. This work was supported by Ministry of Health, Czech Republic (Conceptual development of research organization 64165, General University Hospital in Prague, and by project AZV 16-30954A). Citation Format: Kristyna Nemejcova, Ivana Ticha, Ondrej Kodet, Miroslav Dura, Michaela Bartu, Pavel Dundr. Evaluation of inflammatory infiltration (tumor infiltrating lymphocytes - TIL) in malignant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4069.

Abstract 1624: Reliability of immune biomarker assessment in breast cancer: A report on interobserver variability from studies at a single institution

Abstract Reproducible evaluation of immune infiltration parameters is needed for the development of reliable biomarkers in the era of cancer immunotherapy. This study investigated inter-observer reproducibility when evaluating tumor-infiltrating lymphocytes (TIL), tertiary lymphoid structures (TLS), PD-1 and PD-L1 positivity on immunohistochemically-stained (IHC) breast cancer (BC) tissues, including detailed analysis of spatial positioning in the tumor microenvironment and expression on individual positive cells. Retrospectively collected archival BC samples (N=441) were dual IHC-stained for CD3 (pan T cells) plus CD20 (pan B cells) for TIL and TLS assessment with a consecutive tissue section dual-IHC stained for PD-1 and PD-L1. The 882 tissue sections were independently read by two immuno-pathologists blinded to the clinical data for specific studies between 2014 and 2017. Five independent study series were used for the current analysis: 1) samples of BC relapses, 2) residual disease in breast after neodjuvant treatment, and 3) three independent series of untreated primary tumors. Intraclass correlation coefficients for evaluations are shown in the table below: RelapsesResidual diseaseUntreated primary tumorsSeries 1: N=88Series 2: N=20Series 3: N=85Series 4: N=113Series 5: N=135201420142014-201520162017ICCICCICCICCICC%Stromal TIL0.85 [0.76-0.90]0.71 [0.27-0.89]0.79 [0.68-0.87]0.74 [0.62-0.82]0.77 [0.68-0.83]%Intratumoral TIL0.40 [0.09-0.61]0.63 [0.08-0.86]0.73 [0.58-0.82]0.87 [0.81-0.91]0.64 [0.51-0.74]%Global TIL0.87 [0.80-0.92]0.79 [0.47-0.92]0.83 [0.74-0.89]0.80 [0.72-0.86]0.81 [0.73-0.86]%CD30.83 [0.74-0.89]0.71 [0.28-0.89]0.81 [0.70-0.88]0.72 [0.60-0.80]0.73 [0.63-0.81]%CD200.74 [0.62-0.81]0.81 [0.52-0.92]0.76 [0.63-0.84]0.78 [0.68-0.84]0.78 [0.70-0.84]TLS0.80 [0.70-0.87]0.78 [0.45-0.91]0.83 [0.74-0.89]0.95 [0.93-0.97]0.97 [0.96-0.98]%PD-L1 tumor cells0.78 [0.66-0.86]0.56 [-0.12-0.82]0.82 [0.73-0.89]0.62 [0.45-0.74]0.45 [0.25-0.61]%PD-L1 stromal cells0.61 [0.40-0.74]-1.56 [-1.53-0.60]0.43 [0.12-0.63]0.61 [0.44-0.73]0.31 [0.05-0.5]%PD-L1 immune cells0.53 [0.29-0.70]0.94 [0.84-0.97]0.29 [-0.09-0.54]0.38 [0.10-0.57]0.10 [-0.25-0.35]%PD-1 TLS-0.00 [-0.54-0.35]NA0.72 [0.57-0.82]0.57 [0.35-0.72]0.76 [0.62-0.84] Cohen's κ coefficients reveal poor agreement (κ&amp;lt;0.4) for assessment of the categorical global PD-1 and PD-L1 variables (cut-off &amp;gt;1%). Sporadically, fair agreement (κ&amp;gt;0.4) was observed for PD-L1 expressed by immune and tumor cells. Globally, the inter-observer agreement remained constant overtime. Poor reproducibility for categorical global PD-1 and global PD-L1 assessment was observed. On the contrary, pathologists have excellent reproducibility for assessing global TIL and TLS, good/excellent reproducibility for PD-1 in TLS (primary tumors) and for PD-L1 expression on tumor cells which make these of great interest for clinical use. Citation Format: Cinzia Solinas, Gert Van den Eynden, Alexandre De Wind, François Richard, Anaïs Boisson, Soizic Garaud, Karen Willard-Gallo. Reliability of immune biomarker assessment in breast cancer: A report on interobserver variability from studies at a single institution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1624.

Evaluation of tumor-infiltrating lymphocytes using routine H&E slides predicts patient survival in resected non–small cell lung cancer

The presence of tumor-infiltrating lymphocytes (TILs) positively impacts the outcome of non-small cell lung cancer (NSCLC) patients. Most previous studies have assessed TILs using different immunohistochemical assays. The purpose of this study was to develop and validate a histopathological scoring model for the assessment of TILs in whole-tissue hematoxylin and eosin (H&E)-stained section slides of NSCLC patients and to evaluate the model in an immunoscore setting. Therefore, TIL was evaluated manually on H&E slides from 537 surgical specimens of primary resected stage I-III NSCLC patients. Using stromal TIL score as a stepwise discrete variable, increasing survival was seen with rising TIL level: disease-specific survival (DSS; P = .008), overall survival (P = .036) and disease-free survival (P = .006). Subgroup analysis revealed that high stromal TILs level was associated with superior DSS (P = .047) in patients with squamous cell carcinoma, but not in patients with adenocarcinoma. Multivariable analysis confirmed that high TIL levels independently predict improved prognosis for all endpoints in the overall cohort. In conclusion, high stromal TIL level is an independent favorable prognostic factor in stage I-III NSCLC patients. The comprehensive histological evaluation conducted in this study may be helpful in streamlining TIL quantification for routine clinical use in a future NSCLC immunoscore setting.

Abstract P2-09-29: Potential recurrence markers of locally advanced triple negative breast cancer treated by combined neoadjuvant EGFR targeting and chemotherapy, revealed by genomic analyses and assessment of tumor-infiltrating lymphocytes

Abstract Background: Epidermal growth factor receptor (EGFR) is expressed in ˜50% of triple negative breast cancer (TNBC) and has been proposed as a therapeutic target in this disease. However, trials testing EGFR blockade in TNBC failed to show significant clinical benefit. Probable reasons for such results were patient selection based on EGFR expression and the enrollment of heavily pretreated metastatic patients. Our team has conducted two neoadjuvant trials testing the activity of the anti-EGFR antibodies panitumumab (PTB) and cetuximab (CTX) combined with chemotherapy in locally advanced TNBC. Biomarkers predictive of pathological complete response (pCR) were the level of tumor cell EGFR protein expression and tumor-infiltrating lymphocytes' (TILs) profile (PMIDs 24827135, 26649807). The PTB-treated pts had a higher pCR rate (47%) than the CTX-treated pts (24%), but also a twice higher relapse rate, after 5 years of follow-up. Here we report results of genomic and TILs studies, performed in order to reveal possible determinants of recurrences in those trials. Methods: Tumor tissues sampled before and after neoadjuvant therapy (NAT) have been analyzed by next generation sequencing (NGS) using a targeted exome panel (MSK-IMPACT) of 410 cancer-related genes. Gene expression was evaluated by Affymetrix arrays. TIL density was assessed on pre-NAT samples according to Salgado et al, 2014 (PMID 25214542). The correlation between response, recurrences, genomic and TIL findings was analyzed in a case-by-case fashion. Results: Sixteen tumors that achieved pCR (PTB: 11, CTX: 5) and 23 non-pCR tumors (PTB: 11, CTX: 12) have been analyzed. For 14 non-pCR tumors (PTB: 6, CTX: 8) data have been obtained both from the pre-NAT and the post-NAT sample. Among those tumors, 6 recurred within 2 years after surgery (PTMB: 3, CTX: 3) and assays are on-going on several others that relapsed. Several genomic aberrations that potentially played a causative role in opposing to therapy were identified. We observed multiple mutations in the PI3K pathway in several non-pCR or relapsing pts. Interestingly, in a residual tumor (RT) of a non-pCR patient we found 3 different activating mutations in PIK3CA and one in PTEN. Another example of genomic selection induced by pharmacological pressure is the emergence of a HRAS G12S mutation in a RT after CTX. Additional novel findings include in-frame mutations and deletions in ARID1B and PARP1 amplification in non-pCR pts. Most of the tumors which recurred had ≤10% TILs (9/13) and only 4/13 had ≥30%. Among the tumors with a post-NAT RT but without recurrence, 17/33 had ≤10% TILs and 16/33 ≥30%. No particular link between TIL density and mutation pattern was observed. Conclusions: This is an example of a case-by-case approach where we captured the intrinsic inter-tumor heterogeneity, which is likely responsible for the different responses to EGFR-targeting in TNBC. Genes/pathways candidate of resistance to therapy are currently being validated. Citation Format: Radosevic-Robin N, Cocco E, Privat M, Abrial C, Penault-Llorca F, Scaltriti M. Potential recurrence markers of locally advanced triple negative breast cancer treated by combined neoadjuvant EGFR targeting and chemotherapy, revealed by genomic analyses and assessment of tumor-infiltrating lymphocytes [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-29.

PD-L1 and Emerging Biomarkers in Immune Checkpoint Blockade Therapy.

PD-L1 checkpoint blockade is revolutionizing cancer therapy, and biomarkers capable of predicting which patients are most likely to respond are highly desired. The detection of PD-L1 protein expression by immunohistochemistry can enrich for response to anti-PD-(L)1 blockade in a variety of tumor types, but is not absolute. Limitations of current commercial PD-L1 immunohistochemical (IHC) assays and improvements anticipated in next-generation PD-L1 testing are reviewed. Assessment of tumor-infiltrating lymphocytes in conjunction with PD-L1 testing could improve specificity by distinguishing adaptive (interferon γ driven and cytotoxic T-lymphocyte associated) from constitutive (non-immune mediated) expression. The presence of a high tumor mutational burden also enriches for response to therapy, and early data indicate that this may provide additive predictive value beyond PD-L1 IHC alone. As candidate biomarkers continue to emerge, the pathologist's assessment of the tumor microenvironment on hematoxylin-eosin stain combined with PD-L1 IHC remains a rapid and robust way to evaluate the tumor-immune dynamic.

Interobserver reproducibility of tumor-infiltrating lymphocyte evaluations in breast cancer.

Tumor-infiltrating lymphocytes (TILs) have a prognostic value in breast cancer (BC); however, because of the lack of standard evaluation methods, we aimed to assess the interobserver agreement of stromal TILs (sTILs) and intratumoral TILs (iTILs) as well as the effect of hot spot areas and molecular subtyping on the overall agreement. The study consisted of 121 haematoxylin and eosin (H and E)-stained slides of invasive BC samples obtained from the pathology archives. The TIL assessment was based on the International TIL Working Group recommendations for the percentage of sTILs and was conducted by four pathologists. The percentage of iTILs, the number of lymphocytes in hot spot areas (iTILs-HS), and the overall interobserver agreement for the molecular subtypes were evaluated. The interclass correlation coefficient (ICC) was used to assess interobserver agreement among the four pathologists. The ICC score among the observers for the sTIL percentages was 0.74, and the individual ICC values for each molecular subtype were 0.55, 0.88, and 0.79 for luminal, HER2-positive, and triple-negative tumors, respectively. The compliance value for the iTILs was 0.29 (95% confidence interval (CI) = 0.06-0.48], whereas the compliance value for the iTILs-HS was 0.63 (95% CI = 0.49-0.71). The compliance values for the iTILs-HS subtypes were 0.72, 0.43, and 0.55 for luminal, HER2-positive, and triple-negative tumors, respectively. The IWTILG recommendations are reproducible and reliable. The interobserver agreement of the sTIL percentages was considerably higher for the triple-negative and HER2-positive cases than the luminal cases, whereas the interobserver agreement for the assessment of iTILs-HS in tumors was higher for the luminal subtype.

Standardized assessment of tumor-infiltrating lymphocytes in breast cancer: an evaluation of inter-observer agreement between pathologists

Introduction: In breast cancer, there is a growing body of evidence that tumor-infiltrating lymphocytes (TILs) may have clinical utility and may be able to direct clinical decisions for subgroups of patients. Clinical utility is, however, not sufficient for warranting the implementation of a new biomarker in the routine practice, and evaluation of the analytical validity is needed, including testing the reproducibility of decentralized assessment of TILs. The aim of this study was to evaluate the inter-observer agreement of TILs assessment using a standardized method, as proposed by the International TILs Working Group 2014, applied to a cohort of breast cancers reflecting an average breast cancer population.Material and methods: Stromal TILs were assessed using full slide sections from 124 breast cancers with varying histology, malignancy grade and ER- and HER2 status. TILs were estimated by nine dedicated breast pathologists using scanned hematoxylin–eosin stainings. TILs results were categorized using various cutoffs, and the inter-observer agreement was evaluated using the intraclass coefficient (ICC), Kappa statistics as well as individual overall agreements with the median value of TILs.Results: Evaluation of TILs led to an ICC of 0.71 (95% CI: 0.65–0.77) corresponding to an acceptable agreement. Kappa values were in the range of 0.38–0.46 corresponding to a fair to moderate agreement. The individual agreements increased, when using only two categories (‘high’ vs. ‘low’ TILs) and a cutoff of 50–60%.Discussion: The results of the present study are in accordance with previous studies, and shows that the proposed methodology for standardized evaluation of TILs renders an acceptable inter-observer agreement. The findings, however, indicate that assessment of TILs needs further refinement, and is in support of the latest St. Gallen Consensus, that routine reporting of TILs for early breast cancer is not ready for implementation in a clinical setting.

Assessing Tumor-Infiltrating Lymphocytes in Solid Tumors: A Practical Review for Pathologists and Proposal for a Standardized Method from the International Immuno-Oncology Biomarkers Working Group: Part 2: TILs in Melanoma, Gastrointestinal Tract Carcinomas, Non-Small Cell Lung Carcinoma and Mesothelioma, Endometrial and Ovarian Carcinomas, Squamous Cell Carcinoma of the Head and Neck, Genitourinary Carcinomas, and Primary Brain Tumors.

Assessment of the immune response to tumors is growing in importance as the prognostic implications of this response are increasingly recognized, and as immunotherapies are evaluated and implemented in different tumor types. However, many different approaches can be used to assess and describe the immune response, which limits efforts at implementation as a routine clinical biomarker. In part 1 of this review, we have proposed a standardized methodology to assess tumor-infiltrating lymphocytes (TILs) in solid tumors, based on the International Immuno-Oncology Biomarkers Working Group guidelines for invasive breast carcinoma. In part 2 of this review, we discuss the available evidence for the prognostic and predictive value of TILs in common solid tumors, including carcinomas of the lung, gastrointestinal tract, genitourinary system, gynecologic system, and head and neck, as well as primary brain tumors, mesothelioma and melanoma. The particularities and different emphases in TIL assessment in different tumor types are discussed. The standardized methodology we propose can be adapted to different tumor types and may be used as a standard against which other approaches can be compared. Standardization of TIL assessment will help clinicians, researchers and pathologists to conclusively evaluate the utility of this simple biomarker in the current era of immunotherapy.

Update on tumor-infiltrating lymphocytes (TILs) in breast cancer, including recommendations to assess TILs in residual disease after neoadjuvant therapy and in carcinoma in situ: A report of the International Immuno-Oncology Biomarker Working Group on Breast Cancer.

Morphological evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer is gaining momentum as evidence strengthens the clinical relevance of this immunological biomarker. TILs in the post-neoadjuvant residual disease setting are acquiring increasing importance as a stratifying marker in clinical trials, considering the raising interest on immunotherapeutic strategies after neoadjuvant chemotherapy. TILs in ductal carcinoma in situ, with or without invasive carcinoma, represent an emerging area of clinical breast cancer research. The aim of this report is to update pathologists, clinicians and researchers on TIL assessment in both the post-neoadjuvant residual disease and the ductal carcinoma in situ settings. The International Immuno-Oncology Working Group proposes a method for assessing TILs in these settings, based on the previously published International Guidelines on TIL Assessment in Breast Cancer. In this regard, these recommendations represent a consensus guidance for pathologists, aimed to achieve the highest possible consistency among future studies.

Assessing Tumor-infiltrating Lymphocytes in Solid Tumors: A Practical Review for Pathologists and Proposal for a Standardized Method From the International Immunooncology Biomarkers Working Group: Part 1: Assessing the Host Immune Response, TILs in Invasive Breast Carcinoma and Ductal Carcinoma In Situ, Metastatic Tumor Deposits and Areas for Further Research.

Assessment of tumor-infiltrating lymphocytes (TILs) in histopathologic specimens can provide important prognostic information in diverse solid tumor types, and may also be of value in predicting response to treatments. However, implementation as a routine clinical biomarker has not yet been achieved. As successful use of immune checkpoint inhibitors and other forms of immunotherapy become a clinical reality, the need for widely applicable, accessible, and reliable immunooncology biomarkers is clear. In part 1 of this review we briefly discuss the host immune response to tumors and different approaches to TIL assessment. We propose a standardized methodology to assess TILs in solid tumors on hematoxylin and eosin sections, in both primary and metastatic settings, based on the International Immuno-Oncology Biomarker Working Group guidelines for TIL assessment in invasive breast carcinoma. A review of the literature regarding the value of TIL assessment in different solid tumor types follows in part 2. The method we propose is reproducible, affordable, easily applied, and has demonstrated prognostic and predictive significance in invasive breast carcinoma. This standardized methodology may be used as a reference against which other methods are compared, and should be evaluated for clinical validity and utility. Standardization of TIL assessment will help to improve consistency and reproducibility in this field, enrich both the quality and quantity of comparable evidence, and help to thoroughly evaluate the utility of TILs assessment in this era of immunotherapy.

Clinical Validity and Utility of Tumor-Infiltrating Lymphocytes in Routine Clinical Practice for Breast Cancer Patients: Current and Future Directions.

The interest in tumor-infiltrating lymphocytes (TILs) as a prognostic biomarker in breast cancer has grown in recent years. Biomarkers must undergo comprehensive evaluation in terms of analytical validity, clinical validity and clinical utility before they can be accepted as part of clinical practice. The International Immuno-Oncology Biomarker Working Group has developed a practice guideline on scoring TILs in breast cancer in order to standardize TIL assessment. The prognostic value of TILs as a biomarker in early-stage breast cancer has been established by assessing tumor samples in thousands of patients from large prospective clinical trials of adjuvant therapy. There is a strong linear relationship between increase in TILs and improved disease-free survival for triple-negative and HER2-positive disease. Higher levels of TILs have also been associated with increased rates of pathological complete response to neoadjuvant therapy. TILs have potential clinical utility in breast cancer in a number of areas. These include prediction of responders to immune checkpoint blockade, identification of primary HER2-positive and triple-negative patients who have excellent prognoses and may thus be appropriate for treatment de-escalation, and potentially incorporation into a neoadjuvant endpoint which may be a better surrogate maker for drug development.

DNA methylation-based immune response signature improves patient diagnosis in multiple cancers.

The tumor immune response is increasingly associated with better clinical outcomes in breast and other cancers. However, the evaluation of tumor-infiltrating lymphocytes (TILs) relies on histopathological measurements with limited accuracy and reproducibility. Here, we profiled DNA methylation markers to identify a methylation of TIL (MeTIL) signature that recapitulates TIL evaluations and their prognostic value for long-term outcomes in breast cancer (BC). MeTIL signature scores were correlated with clinical endpoints reflecting overall or disease-free survival and a pathologic complete response to preoperative anthracycline therapy in 3 BC cohorts from the Jules Bordet Institute in Brussels and in other cancer types from The Cancer Genome Atlas. The MeTIL signature measured TIL distributions in a sensitive manner and predicted survival and response to chemotherapy in BC better than did histopathological assessment of TILs or gene expression-based immune markers, respectively. The MeTIL signature also improved the prediction of survival in other malignancies, including melanoma and lung cancer. Furthermore, the MeTIL signature predicted differences in survival for malignancies in which TILs were not known to have a prognostic value. Finally, we showed that MeTIL markers can be determined by bisulfite pyrosequencing of small amounts of DNA from formalin-fixed, paraffin-embedded tumor tissue, supporting clinical applications for this methodology. This study highlights the power of DNA methylation to evaluate tumor immune responses and the potential of this approach to improve the diagnosis and treatment of breast and other cancers. This work was funded by the Fonds National de la Recherche Scientifique (FNRS) and Télévie, the INNOVIRIS Brussels Region BRUBREAST Project, the IUAP P7/03 program, the Belgian "Foundation against Cancer," the Breast Cancer Research Foundation (BCRF), and the Fonds Gaston Ithier.

Abstract P1-09-08: Predictive effect of cytotoxic tumor infiltrating lymphocytes in HER2-positive metastatic breast cancer: A correlative study with CCTG MA.31

Abstract Background and Objectives: The presence of tumor infiltrating lymphocytes (TILs), particularly CD8+ cytotoxic T-cells, has been associated with improved prognosis in patients with HER2+ breast cancer. Increasing levels of TILs also appear to predict response to adjuvant trastuzumab in early breast cancer, although they did not predict benefit of combined trastuzumab-lapatinib neoadjuvant dual therapy over monotherapy in NeoALLTO. CCTG MA.31 randomized 652 women with HER2+ metastatic breast cancer to treatment with trastuzumab (T) vs. lapatinib (L), in combination with taxane (Tax) chemotherapy for 24 weeks, followed by the same HER2-targeted monotherapy. Final results from MA.31 found trastuzumab was superior to lapatinib for the primary endpoint of progression free survival (PFS): the hazard ratio (HR) for lapatinib to trastuzumab was 1.37 (95% CI, 1.13-1.65). Although both agents block HER2 signaling, trastuzumab has additional mechanisms of action via the immune system. We hypothesized that TIL levels may predict response to HER2-targeted therapy (trastuzumab vs. lapatinib). Methods: MA.31 included HER2+ metastatic breast cancer patients, median age 55 years, and median follow-up 21.5 months. Overall TILs were counted per published guidelines on the original H&amp;E stained sections used for pathology review at study entry. Immunohistochemistry (IHC) was performed on unstained sections from tissue microarrays or individual formalin-fixed paraffin-embedded blocks to test expression of lymphocyte biomarkers CD8, FOXP3, CD56 and PD-1 on stromal and intra-tumoral TILs (sTILs, iTILs). Statistical analysis was conducted by CCTG for a total of 9 prespecified biomarker tests. Associations of TILs with PFS were evaluated by univariate stratified log-rank test with graphical Kaplan-Meier curves, and by stratified multivariate Cox proportional hazards regression analysis. Predictive effect was examined with a test of interaction between treatment allocation and biomarker classification (high vs. low, using pre-established cutpoints). Results: Of the 652 cases, 614 had slides for overall TIL assessment and 427 for IHC biomarker assessments. In this correlative study set, superiority of trastuzumab over lapatinib for PFS was confirmed in multivariate analysis (LTax/T vs. TTax/L: HR = 2.55, 95% CI = 1.43-4.55, p = 0.001). TIL counts by H&amp;E were neither prognostic nor predictive in this set of metastatic HER2+ breast cancers. Lymphocyte IHC markers were not prognostic. However, prespecified stratified univariate analysis detected a significantly higher risk for lapatinib over trastuzumab (HR = 2.94, 95% CI = 1.40-6.17, p = 0.003) in patients with low CD8+ sTIL (&amp;lt; 3) than was observed among those with high CD8+ sTIL (HR = 1.36, 95% CI = 1.05-1.75, p = 0.019). This differential effect was confirmed in multivariate analysis (interaction test p = 0.042). The other tested biomarkers did not demonstrate significant predictive effects. Conclusions: In this correlative study of metastatic HER2+ breast cancer, a low level of pre-existing stromal cytotoxic T cell infiltration predicts women who benefit most from trastuzumab over lapatinib. Overall TIL counts were neither prognostic nor predictive. Citation Format: Liu S, Chen B, Burugu S, Leung S, Gao D, Virk S, Kos Z, Parulekar WR, Shepherd L, Gelmon K, Nielsen TO. Predictive effect of cytotoxic tumor infiltrating lymphocytes in HER2-positive metastatic breast cancer: A correlative study with CCTG MA.31 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-09-08.

Abstract P2-04-07: Immune profiling of post neoadjuvant high metastatic risk (RCB-II/III) residual disease in patients with early triple negative breast cancers

Abstract Background: Poor prognosis in TNBC can be predicted in the significant fraction of patients with large volume residual cancer burden (RCB-II/III) after neoadjuvant chemotherapy (NACT). Whilst residual disease has been characterised to identify “driver” mutations and copy number variations, the contribution of the immune response within its tumour microenvironment remains unclear. Here we aimed to: 1) assess the potential spatial heterogeneity of immune transcript related gene expression between areas of tumour approximately 1cm apart as might still occur with a radiologically guided biopsy through the residual disease; and 2) assess the immune stroma composition of the TNBC high metastatic risk RCB II/III disease. Method: 12 TNBC post NACT RCB II/III residual cases were identified from the KHP biobank. H&amp;E sections were reviewed and areas of tumor 1cm apart within a residual resection specimen marked as area A and area B. HistoQuest analysis software was used to quantify the proportion of tumor infiltrating lymphocytes (per total cell count) within both areas. RNA was extracted from both areas and immune gene expression profiling performed using a Nanostring nCounter® on all 24 samples. The immune PanCancer panel consisted of 770 genes combining markers for different immune cell populations. Differential genes between paired samples were compared and unsupervised hierarchical clustering using 770 genes and immune cell types performed. Results:Quantitative comparison of the tumour infiltrating lymphocytes (TILS) between area A and B revealed that 73% (8/11) of the cases had a &amp;lt;2-fold difference in the percentage of TILS within a residual specimen; and 27% (3/11) displayed a &amp;gt;2-fold (range 2.03-3.16) difference in the TILS. When comparing the 770 gene expression profiles between sampling areas in the same tumour, we found little spatial heterogeneity with areas A/B clustering together in 10 out of 12 cases. Interestingly, the two cases that revealed spatial heterogeneity within the paired samples displayed little immune cell heterogeneity histologically (i.e. &amp;lt;1-fold change in the TILS percentage score between area A and B). Comparing patient samples by immune gene expression profiling divided the patients into two groups: i) those with immunologically enriched tumors in whom gene signatures for majority of the immune cell types (DC, Macrophages, CD8+T-cells, T-helper cells (Th17, Th2, Th1), Tregs, NK, B-cells, Neutrophils, Mast cells) were highly expressed and ii) those with immunologically inert tumors in whom the immune cell signatures were not highly expressed. Within our patient cohort, patients with immunologically enriched gene expression profiles were also seen to display higher TILS score (ranging between 28.17% to 40.66%) as compared to patients with immunologically inert gene expression with scores ranging from 11.82% to 16.80%. Conclusion: The findings that high metastatic risk residual disease can be further characterized as either “immunologically inert” or “immunologically enriched” at the level of extensive immunological transcript gene expression and by histological assessment of TILS requires further investigation; and is being validated in a larger sample set. Citation Format: Irshad S, Cheang M, Gazinka P, Naidoo K, Buus R, Pinder S, Dowsett M, Tutt A. Immune profiling of post neoadjuvant high metastatic risk (RCB-II/III) residual disease in patients with early triple negative breast cancers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-07.