Tumor-infiltrating Lymphocytes Working Group Research Articles

The Value of Tumor Infiltrating Lymphocytes (TIL) for Predicting the Response to Neoadjuvant Chemotherapy (NAC) in Breast Cancer according to the Molecular Subtypes.

The antitumor host immune response is an important factor in breast cancer, but its role is not fully established. The role of tumor infiltrating lymphocytes (TIL) as an immunological biomarker in breast cancer has been significantly explored in recent years. The number of patients treated with neoadjuvant chemotherapy (NAC) has increased and the identification of a biomarker to predict the probability of pCR (pathological complete response) is a high priority. We evaluated 334 cases of BC treated with NAC followed by surgical resection from 2020-2022 at the Ist Clinic of Oncological Surgery, Oncological Institute "Prof Dr I Chiricuta" Cluj Napoca. Of the above, 122 cases were available for histological evaluation both in pre-NAC biopsy and post-NAC resection tissue. Evaluation of biopsy fragments and resection parts were performed using hematoxylin eosin (H&E). The TIL evaluation took place according to the recommendations of the International TIL Working Group (ITILWG). There was a strong association between elevated levels of pre-NAC TIL. At the same time, there is a statistically significant correlation between stromal TIL and tumor grade, the number of lymph node metastases, the molecular subtype and the number of mitoses (p < 0.005). Intratumoral TIL showed a significant correlation with tumor size, distant metastasis, molecular subtype, number of mitosis, stage and lymph node metastasis (p < 0.005). We also demonstrated that high pre-NAC STIL represents a strong predictive marker for pCR. This study reveals the role of TIL as a predictive biomarker in breast cancer not only for the well-established TNBC (triple negative breast cancer) and HER2+ (Her2 overexpressed) subtypes but also in Luminal A and B molecular subtypes. In this scenario, the evaluation of sTIL as a novel predictive and therapy-predicting factor should become a routinely performed analysis that could guide clinicians when choosing the most appropriate therapy.

THE PROGNOSTIC INDICATOR OF TUMOR INFILTRATING LYMPHOCYTES IN BREAST CARCINOMA

Introduction: Breast cancer is one of the common malignant tumors in women. The use of biomarkers ensures breast cancer patients receive optimal treatment. Tumor-infiltrating lymphocytes (TILs) are emerging as one of the key prognostic markers of breast cancer. Our study aims to evaluate percentage of TILs in Objectives And Methodology: tumor tissue, grading TILs based on the recommendations by an International TIL Working Group 2014 &amp; compare with ER &amp; PR status and in relation to concentration of TILs we assess prognosis of breast carcinoma for a period of 6 months. Results: Among 30 study subjects, TILs were of intermediate, high &amp; lower grade in 53.3%, 40% and 6.7% cases respectively. TILs expression was statistically significant in both ER and PR, positive &amp; negative groups (p&lt;0.05). TILs were statistically highly significant as a prognostic marker in hormone negative breast cancers (p&lt;0.05). Conclusion: Our study concludes that high TILs are correlated with favourable survival and response to treatment in breast cancer patients with ER, PR negative molecular subtype

Stromal Tumor Infiltrating Lymphocytes (TIL) as a potential prognostic biomarker for recurrence in Locally Advanced Breast Cancer (LABC) patients

Background: Tumor-infiltrating lymphocytes (TIL) serves as the host adaptive immune response in breast cancer. TIL has the potential to be an independent prognostic factor in breast cancer patients. In this study, we aimed to examine the association between stromal TIL and recurrence in locally advanced breast cancer patients. Methods: A cohort retrospective study was conducted using medical records of female breast cancer patients with locally advanced breast cancer. We collected patients' data, including demographic data from the medical record. Stromal TIL was examined following the recommendations of the International TIL Working Group 2014. Results: 75 samples were included with an average age of 49.5±8.4. Ductal carcinoma was the most common type histologically (88.0%). Luminal B Her2-negative was the predominant breast cancer subtype (32.0%). There was a significant association between the breast cancer subtype and disease-free survival (P – 0.014). The optimal cut-off to determine the recurrence of breast cancer was 15%. The sensitivity, specificity, PPV, NPV, and accuracy of TIL to predict the 24-month disease-free survival (DFS) were 80.5%, 82.4%, 84.6%, 77.8%, and 81.3%, respectively. Every 10% increase in TIL percentage could raise the DFS by 5.45 months (p=0.001). Patients with high TIL values had higher survival than those with low TIL values. Conclusion: There was a significant correlation between the stromal TIL and the recurrence rate. The pre-therapy stromal TIL percentage can be employed as a potential biomarker to predict breast cancer recurrence, particularly in the first two years.

Combined assessment of metabolic response and tumor infiltrating lymphocytes as a predictor of outcomes following neoadjuvant therapy for HER2-positive breast cancer: Results from the randomized PREDIX HER2 trial.

593 Background: Abundance of tumor infiltrating lymphocytes (TIL) is prognostic in early HER2-positive breast cancer (BC). Response to neoadjuvant therapy (NAT) according to positron emission tomography combined with computed tomography (PET-CT) has been shown to predict pathologic complete response (pCR). There is paucity of data regarding long-term prognostication using PET-CT and the potential value of the combined assessment of both these biomarkers. Methods: PREDIX HER2 (NCT02568839) is a prospective randomized phase 2 trial that compared standard NAT (docetaxel, trastuzumab, pertuzumab) with trastuzumab emtansine, in patients with HER2-positive BC. Overall, 202 patients were included (197 evaluable) and the primary efficacy analysis showed no difference in pCR or event-free survival (EFS) between the two groups (Hatschek, JAMA Oncology 2021). Assessment with fluorine 18–labeled fluorodeoxyglucose PET-CT was performed at baseline and after 2 and 6 treatment cycles, and SUVmax was evaluated as a continuous variable. TILs were assessed at baseline biopsies according to guidelines from the International TIL Working Group (J.H.). The aim of this secondary analysis was to investigate the combined assessment of TIL and PET-CT as an early predictor of response to NAT. Results: Overall, 112 patients underwent baseline PET-CT and 109 after C2, whereas 173 had baseline TIL. In multivariable analysis, baseline SUVmax did not predict pCR (ORadj= 1.04, 95% CI 0.97-1.12, p = 0.259) or EFS (HRadj= 1.07, 95% CI 0.98-1.17, p = 0.117). In contrast, higher SUVmax at C2 predicted lower pCR (ORadj= 0.65, 95% CI 0.48-0.87, p = 0.005) and worse EFS (HRadj= 1.18, 95% CI 1.04-1.34, p = 0.01). Baseline TIL &gt; 10% (median cut-off) provided additional prognostic information to clinical parameters (stage and hormone receptor expression) and C2 SUVmax (LR-Δχ2 = 7.19, p = 0.007; ORadj= 3.52, 95% CI 1.37 – 9.06, p = 0.009). 75% of patients with high TIL and C2 SUVmax &lt; 2.49 achieved pCR, compared with 13.8% of those with low TIL and high C2 SUVmax and 39.1%-41.3% for the intermediate groups (p = 0.001). Conclusions: SUVmax after two cycles of NAT for HER2-positive BC is an independent predictor of both short- and long-term outcomes. A combined assessment with TIL may facilitate early selection of good responders for de-escalation and poor responders for alternative treatment strategies. Clinical trial information: NCT02568839.

Abstract P5-06-14: A novel combination of a 2 gene score &amp; TIL as a predictive Biomarker for responders to novel therapies in Indian TNBC - A population with greater proportion of TNBCs

Abstract Introduction: Triple-Negative Breast Cancer (TNBC) comprises approximately 30% of all breast cancers in Indian women. Given their aggressive nature, TNBCs have high rates of systemic metastasis and mortality with only chemotherapy available for treatment. Compelling evidence has demonstrated the prognostic value of tumor-infiltrating lymphocytes (TILs), in many cancers especially in Breast Cancer and play a critical role in tumour progression, response to therapeutics and prognosis. They are typically measured by H&amp;E staining and immunohistochemistry for research purpose and degree of infiltrate is also known to differ among subtypes. Nonetheless, the literature regarding the types of immune cells characterizing TILs and their prognostic utility in TNBC has been conflicting for lack of accurate “functional” TIL assessments. Herein we have used a combination signature of TILs and a 2 gene immune function expression based signature to develop an unique classifier, to identify a subgroup within TNBC that has better clinical outcome, such as survival and response to treatment. Our findings also suggest a possible use of the score as a predictive biomarker for response to immune checkpoint therapy Patients and Methods: Surgically excised TNBC tumor specimens from 44 women from a single treating hospital in Bangalore, India were accessed under IERB approved protocols and assayed on nCounter® PanCancer Immune Profiling Panel which comprised of 740 genes and characterises 14 different immune cell types. Analysis using Non-negative Matrix factorization (NMF) based unsupervised clustering was done to arrive at stable subtypes characterized by different tumor microenvironments within TNBC. SSGSEA analysis was done to identify immune cell types. TILs were characterised by a pathologist on H&amp;E sections according to guidelines from TIL working group. The Immune classifier was validated on Breast Cancer data sets from TCGA. Results: NMF followed by SAM and PAM analysis yielded 2 stable subtypes (ST1 and ST2) of proportions 59% and 41% respectively. TIL groups also were divided into Dense and Mild groups which were 52% and 48% respectively. Despite similar distributions of the groups, ST1 and ST2 had distinct survival with ST2 having poorer outcome (p=0.023) while the dense and mild TIL groups did not separate as distinct groups. On a closer examination using SSGSEA analysis with Rooney et al signature, ST1 was enriched with T-cells, Dendritic cells, MHC-class 1 and very significantly high cytolytic score (CYT), which was defined by Granzyme A and Perforin expressions, proteins secreted by cytotoxic T cells (p=0.0074). The CYT Score ranged from -35 to 170 and cutoff was based on 75th percentile. We next hypothesized that a combined score of TIL and CYT would represent a functional TIL group with high immune activity. We arrived at a CYT+TIL score based on 75th percentile cut off of CYT and 2 groups of TIL. Of the resultant four groups, TIL High-CYT high that constituted about 20% of all TNBCs indicated an elevated immune response because of their microenvironment constitution and this can be identified using our simple immune classifier. On the other hand, in the TIL-high and CYT-low group, despite lymphocyte migration, outcome was not favorable. The classifier was applied to TNBC from TCGA (n=96) and similar results seen, with ST1 and ST2 which had distinctly separate survival and had similar patterns on CYT high groups. Conclusion: We developed a simple immune classifier with 2 gene signature c and H&amp;E slide TIL assessment for identifying a group with TNBC that can be better targeted with therapies. Our signature is predictive for selecting TNBCs which are potential responders to Immune therapies and has the potential for quick clinical adoption though it requires validation on a larger set. Citation Format: Aruna Korlimarla, Hari PS, Jyothi S Prabhu, Chantirika Ragulan, Ravi B Diwakar, Sandhya Apachu, Maggie Cheang, Rekha V Kumar, BS Srinath, TS Sridhar, Savitha Rajarajan, Annie Alexander, Anguraj Sadanandam. A novel combination of a 2 gene score &amp; TIL as a predictive Biomarker for responders to novel therapies in Indian TNBC - A population with greater proportion of TNBCs [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-14.

Association of pathological complete response rates and TILs in triple-negative breast cancer patients.

e12596 Background: Triple negative breast cancers (TNBC), characterized by the lack of expression of estrogen receptors and progesterone receptors as well as human epidermal growth factor receptor 2, are associated with high distant recurrence rate and death. As a result, the majority of patients with TNBC are treated with perioperative chemotherapy with the goal of eradicating micrometastases and preventing distrant relapse. The preoperative systemic therapy offers the advantages of permitting an assessment of chemo-sensitivity, increased rates of breast conserving surgery and the ability to adapt postoperative therapies depending on the response. Recently, neoadjuvant chemotherapy has been used at an increasing frequency. The response to neoadjuvant chemotherapy, as measured by the residual cancer burden index, for example, is correlated with the long-term prognosis of TNBC and HER2 expressing breast cancers. Previous studies suggest that tumor-infiltrating lymphocytes (TILs) may correlate with pathological complete response (pCR) rates in TNBC patients treated with neoadjuvant chemotherapy. The pathologic evaluation of TILs in TNBC has been recommended by the International TILs working group since 2014. In this study we sought to analyze the association of TILs with pCR in a cohort of TNBC patients treated with neoadjuvant chemotherapy. Methods: An IRB-approved single-institution retrospective analysis was performed on 127 patients diagnosed with TNBC who received neoadjuvant anthracycline and taxane based chemotherapy at the Ohio State University Comprehensive Cancer Center between January 1st, 2012 and November 31st, 2018. We analyzed TILs as a continuous variable as a part of a secondary analysis of this data. Whole tissue sections from archived H&amp;E stained glass slides were scanned using Philips UltraFast Scanner at ×40 magnification with a single-focus layer. TIL scoring was performed according to guideline recommendations from the International TILs Working Group (2014). Results: A total of 127 female patients with TNBC were identified. The median age at diagnosis was 52.0 years (range 32.0, 74.0) and patients were predominately white (103, 81%), post-menopausal (68, 53.5%) and presented with invasive ductal cancer (113, 89%), stage II (88, 69%), and high grade (108, 85%). Of those patients, 56 had TILs measurement available. pCR was associated with statistically higher level of TILs in core biopsies taken prior to chemotherapy had (Wilcoxon rank-sum test, p = 0.04). Conclusions: The long-term prognosis of patients with TNBC is predicted by the response to neoadjuvant chemotherapy. Consistent with other studies, our study revealed that TILs are associated with a higher probability of pCR. Our future goals are to identify which TIL subsets correlate best with pCR and to identify the mechanism for the increased chemotherapy responsiveness of lymphocyte-infiltrated tumors.

Abstract P3-08-12: An open source, automated tumor infiltrating lymphocyte algorithm for prognosis in triple-negative breast cancer

Abstract Background: The presence of high TILs (tumor infiltrating lymphocytes) have been shown to be predictive of response to chemotherapy and is also a prognostic factor associated with better outcome in breast cancer, especially in early stage triple-negative (TNBC) and HER2-positive breast cancers. TIL assessment, while now more standardized due to the efforts of Salgado and the International TIL Working Group (https://www.tilsinbreastcancer.org/), are still a subjective test with variability in evaluation that has prevented broad adoption. Given the advances in application of artificial intelligence to pathology images, we believe the next step for TILs is to make them automated and objective and to identify a standardized and meaning TIL cut-point. The aim of this study is to build an open source, H&amp;E image-based automated TIL assessment algorithm for breast cancer that allows global standardization of TILs for prognostic value. Materials and methods: Using QuPath open source software, we first built a neural network classifier for image-based, automated assessment of TILs. It distinguishes tumor cells, lymphocytes, stromal cells and other cells on hematoxylin-eosin (H&amp;E) stained sections. We then defined “eTILs%” calculated as follows for the percentage of machine defined TILs: (TILs/TILs+Tumor cells) *100. A retrospective collection of 63 TNBC cases was used as the training set (Set A) and then tested for cell classifier accuracy and the optimal “eTILs%” cut point. The validation sets were a retrospective collection of 354 TNBC patients comprised of three independent validation subsets (Set B; N=87, Set C; N=183, and Set D; N=83) in both tissue microarray (TMA) and whole tissue section (WTS) format. Results: Using an optimal cut point (30%) derived from TNBC cohort training set A, patients with high eTILs% displayed an overall survival benefit (HR 0.4, p=0.0150). This algorithm was then applied in other three TNBC validation sets (Set B: HR=0.42, p=0.0033; Set C: HR=0.42, p=0.0127; Set D in TMA format: HR=0.39, p=0.0089). For Set D, we also tested WTS format which showed HR=0.23, (p=0.0155). The validation sets were combined to assess independence from clinical status in a multi-variable analysis where eTILs% was independently associated with improved overall survival (HR=0.35, p&amp;lt;0.0001). Conclusion: We have constructed a single institutional algorithm built from open source QuPath software that is a robust and independent prognostic factor. With further validation in tissues from other institutions and larger cohorts, this algorithm could be potentially useful as a broadly applicable, standardized and globally available method for assessment of TILs in triple negative breast cancer patients. Citation Format: Yalai Bai, Balazs Acs, Jon Zugazagoitia, Sandra Martinez-Morilla, Fahad Shabbir Ahmed, David L. Rimm. An open source, automated tumor infiltrating lymphocyte algorithm for prognosis in triple-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-12.

Interaction between Molecular Subtypes and Stromal Immune Infiltration before and after Treatment in Breast Cancer Patients Treated with Neoadjuvant Chemotherapy.

High levels of tumor-infiltrating lymphocytes (TIL) before neoadjuvant chemotherapy (NAC) are associated with higher pathologic complete response (pCR) rates and better survival in triple-negative breast cancer (TNBC) and HER2-positive breast cancer. We investigated the value of TIL levels by evaluating lymphocyte infiltration before and after NAC. We assessed stromal TIL levels in 716 pre- and posttreatment matched paired specimens, according to the guidelines of the International TIL Working Group. Pre-NAC TIL levels were higher in tumors for which pCR was achieved than in cases with residual disease (33.9% vs. 20.3%, P = 0.001). This was observed in luminal tumors and TNBCs, but not in HER2-positive breast cancers (P Interaction = 0.001). The association between pre-NAC TIL levels and pCR was nonlinear in TNBCs (P = 0.005). Mean TIL levels decreased after chemotherapy completion (pre-NAC TILs: 24.1% vs. post-NAC TILs: 13.0%, P < 0.001). This decrease was strongly associated with high pCR rates, and the variation of TIL levels was strongly inversely correlated with pre-NAC TIL levels (r = -0.80, P < 0.001). Pre-NAC TILs and disease-free survival (DFS) were associated in a nonlinear manner (P < 0.001). High post-NAC TIL levels were associated with aggressive tumor characteristics and with impaired DFS in HER2-positive breast cancers (HR, 1.04; confidence interval, 1.02-1.06; P = 0.001), but not in luminal tumors or TNBCs (P Interaction = 0.04). The associations of pre- and post-NAC TIL levels with response to treatment and DFS differ between breast cancer subtypes. The characterization of immune subpopulations may improve our understanding of the complex interactions between pre- or post-NAC setting, breast cancer subtype, response to treatment, and prognosis.

291TiP - Correlation between the density of tumour-infiltrating lymphocytes, immune cell subsets in tumour stroma and response to systemic therapy in breast cancer

BackgroundThe density of tumor-infiltrating lymphocytes (TIL) is a predictive factor for response to neoadjuvant systemic therapy (NAST) in breast cancer. High TIL density correlates with higher complete pathologic remission (pCR) in triple negative and HER2 positive breast cancer. Additionally, not only TIL density but also subsets of immune cells in tumor stroma seem to play an important role in cancer and immune cell interaction and response to chemotherapy. Our aim is to determine TIL density and expression of CD 20, CD3, CD8, PD-L1, FOXP3 and PGM1 on core biopsy specimen and find a correlation with pCR. Trial designWe will conduct a prospective study on early breast cancer patients (n=180) treated with NAST. Patients with breast tumor >2cm will be suitable. All patients will underwent mammography, MR of breast, core biopsy of breast tumor, implantation of radiopaque marker into tumor, axillary ultrasound and fine needle biopsy of suspect nodes, CT scan of thorax and abdomen and bone scintigraphy. On core biopsy specimen histologic type, grade, estrogen and progesterone receptors, HER 2 status and MIB-1 will be determined. Additionally, TIL density will be determined on stromal part of tumor on H&E slides in accordance with International TIL Working Group. TIL density will be defined as the percentage of lymphocytes in tumor stroma: as a continuous variable and as two categories: low (0-59%) or high (60-100%). Immunohistochemical detection of CD 20, CD3, CD8, PD-L1, FOXP3 and PGM1 will be performed and each subpopulation of immune cells defined as percentage of all immune cells in tumor stroma. Patients with metastatic tumours and luminal A-like characteristics will be excluded. All other patients will undergo NAST with anthracyclines and taxanes and anti-HER2 therapy if indicated, followed by surgical treatment. The breast and axilla specimen will undergo pathologic examination. pCR will be defined as absence of invasive and carcinoma in situ in breast and axillary nodes. The primary objective will be correlation of CD20, CD3, CD8, PD-L1, FOXP3 and PGM1 with TIL density. The secondary objective will be correlation of subsets of immune cells in tumor stroma with pCR. Clinical trial identificationEudraCT: 2018-000 547-11. Legal entity responsible for the studyInstitute of Oncology Ljubljana. FundingSlovenian Research Agency P3-0321. DisclosureAll authors have declared no conflicts of interest.

72P - Clinico-pathological relationship between androgen receptor (AR) and tumor infiltrating lymphocytes (TILs) in triple negative breast cancer (TNBC)

Background: Triple negative breast cancer (TNBC) is an aggressive subtype of with ill-defined therapeutic targets. Androgen receptor (AR) expression ranged from 32-53% in different reports presenting emerging biomarker in TNBC. Tumor-infiltrating lymphocytes (TIL) play an important prognostic and predictive value in TNBC. The relationship between AR, TIL and clinical behavior is still not fully understood. Methods: Thirty-six TNBC patients were retrospectively selected from a cohort of 800 patients diagnosed in 2012. Clinico-demographic data were collected. Formalin-fixed, paraffin embedded tissue specimens were evaluated for AR (+ ve if ≥ 1% expression), CD3, CD20, CD4, CD8 with IHC. Stromal TIL were quantified following the TIL Working Group recommendations. Lymphocyte-predominant breast cancer (LPBC) are defined as stromal TILs of ≥ 50%. Results: The mean age was 52.5 years and 27.8% were ≥60 years. The majority of patients (58.3%) were invasive duct carcinoma (IDC), while IDC with medullary features and invasive lobular carcinoma were 22% and 14% respectively. Grade III tumors were 52.8%. Stage III, IV were 54.3%, 8.6% respectively and N+ve were 77%. Seven patients of the total (19.4%) were AR+ve. TIL were assessed in 31 patients where LPBC was 32.3%. Median TIL was 15% and 33% (p = 0.07) and CD20 was 7% and 18% (p = 0.01) in AR+ve and AR-ve respectively. Mean CD3 was 93.3% and 80.6% (p = 0.007) and CD8 was 80.8% and 75% (p = 0.41) in AR+ve and AR-ve respectively. Majority of patients ≥60 years (66.7%) were lymphocyte deficient (TIL˂50%). CD8 was significantly lower in stage III/IV vs I/II (p = 0.04). LPBC type was significantly lower in N+ve vs N-ve patients (p = 0.03). Significant positive correlation presented between CD8 and CD3 (p˂0.001) while significant negative correlation presented between CD20 and CD3 (p = 0.03) and CD4 and CD8 (p = 0.04). Non statistically significant increased median OS in AR+ve vs AR-ve (44 vs 40 months, p = 0.84). Conclusions: The present study show that T cell marker (CD3) was more predominant in AR+ve TNBC. Older patients and N+ve were lymphocyte deficient (TIL˂50%). More studies are needed to focus on the clinical impact of the relation between AR and TIL in TNBC. Legal entity responsible for the study: Hagar Elghazawy. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.

Standardized determination of tumor-infiltrating lymphocytes in breast cancer : A prognostic marker for histological diagnosis

Tumor-infiltrating lymphocytes (TILs) have been identified as prognostic parameter in breast cancer. The aim of this review article is to provide an overview on the clinical and analytical validation of TILs in breast cancer. Summary of international guidelines of the TIL working group as well as clinical and translational studies. Breast carcinomas with ahigh TIL level have an improved response to neoadjuvant chemotherapy. Triple-negative and HER2-positive carcinomas with increased TIL levels have improved survival. TILs are anew prognostic biomarker for routine histopathological diagnosis.

Interobserver Variability in Scoring Percentages of Tumor-Infiltrating Lymphocytes in Breast Core Needle Biopsies

Recent research has shown that tumor-infiltrating lymphocytes (TILs) are correlated with improved survival in a continuous fashion in primary breast cancers. Additionally, the TIL score on breast core biopsies has been shown to correlate with higher rates of pathological complete response after neoadjuvant chemotherapy. In 2014, an International TIL Working Group created a standardized evaluation method for scoring TILs. We sought to determine the interobserver variability in scoring TILs after training among three pathologists with different levels of experience. Fifty-five cases of diagnostic core biopsies from 2010 to 2012 of primary breast carcinoma were selected for the cohort. Prior to scoring the breast biopsies, the evaluators reviewed the standardized scoring method from the 2014 TIL Working Group. The biopsies were assessed for the average percent of stromal lymphocytes in increments of 5%. Each of the cases was scored by the three pathologists who were blinded to the other’s results. The scores were then tabulated and Kendall’s correlation coefficient was calculated using SPSS software from IBM. Values of Kendall’s concordance less than 0.4 can be considered poor agreement, values between 0.4 and 0.59 are fair, between 0.6 and 0.74 are good, and above 0.75 is excellent agreement. The stromal TIL scores ranged from 1% to 90%. The calculated Kendall’s correlation coefficients varied from 0.704 to 0.762 between the three evaluators. Minor interobserver disagreements were attributed to heterogeneity within the tumor, with alternating areas of relatively scant and dense stromal lymphocytic infiltration. The data show that with adequate training, a high degree of correlation is possible between observers at varying levels of experience on core needle biopsies. Scoring TILs on core needle biopsy may be used as an additional biomarker to estimate a patient’s response to neoadjuvant therapy.

Abstract P3-05-03: Metaplastic breast cancers: Genomic profile, mutational burden and TILs

Abstract Background: Metaplastic breast cancers (MPBC) are rare, typically triple negative aggressive tumors composed of both, adenocarcinoma and metaplastic elements. Recent evidence that TNBC and MPBC can respond vigorously to immune checkpoint inhibitor therapy (Adams et al, ASCO 2017 and npj Breast Cancer 2017) has prompted the following comprehensive genomic profiling (CGP) and histopathologic assessment of tumor infiltrating lymphocytes (TIL) designed to uncover potential biomarkers of immunotherapy response for MPBC, including mutational burden, Microsatellite Instability (MSI) status and gene amplification of 9p21.4 (or CD274, which includes the PD-L1 locus). Methods: 12,214 locally aggressive, relapsed and metastatic breast malignancies (mBM) were subjected to CGP using DNA extracted from 40 µm of FFPE sections and adaptor ligation-based libraries to a mean coverage depth &amp;gt;650X for up to 315 cancer-related genes. The results were analyzed for all classes of genomic alterations (GA) including base substitutions, insertions and deletions, select rearrangements, and copy number changes. Tumor mutational burden (TMB) was determined on 1.1 Mbp of sequenced DNA. MSI status was determined by an algorithm based on the sequencing results. TIL were assessed on archived H&amp;E tumor sections and enumerated per guidelines established by the TIL Working Group (Salgado, Ann Oncol 2015) in a subset of MPBC with the highest TMB and compared with low TMBC cases. Results: 165 of mBM cases were MPBC (1.4%) and are included in this study. All patients were female with a median age of 60 (range 24-86). 165 of the MPBC cases (100%) harbored a wide variety of GA involving more than 100 individual genes. The most common GA were identified in TP53 (65%), followed by PIK3CA (37%). No cases of MSI hi status (0/103) and only one case with amplification of 9p21.4 (1/165, 0.6%) were observed. Most MPBC had a low mutational burden, with a median TMB of 2.7 mutations/Mb (range 0-39.6). Only 11/165 tumors (6.7%) were found to have a TMB over 10 mutations/Mb, including 3 cases (1.8%) with TMB &amp;gt;20. Tumor sections were available for TIL review from 9/11 cases with highest TMB, as well as 11 control cases with lowest TMB. TIL were more frequently observed in high versus low TMB MPBC, with median TIL percentage of 40 and 20 (range 10-80 and range 10-60), respectively, although this difference was not statistically significant (Wilcoxon rank-sum test, p=0.15). Conclusions: Genomic profiling in the largest cohort of MPBC reported to date confirms that MPBC is enriched for TP53 and PIK3CA mutations and many tumors harbor targetable GA. The frequently observed tumoral PD-L1 expression in MPBC is not based on gene amplification as amplification of 9p21.4 is rare. Most tumors had a low mutation burden, and no significant association of TIL with TMB was observed, suggesting additional processes underlying MPBC immunogenicity. Citation Format: Taff J, Suh J, Singh B, Denkert C, Troxel AB, Ross JS, Adams S. Metaplastic breast cancers: Genomic profile, mutational burden and TILs [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 P3-05-03.

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.

Abstract ES4-3: Tumor-infiltrating lymphocytes (TILs) and genomic signatures of immune cells

Abstract Increased levels of tumor-infiltrating lymphocytes have been described in subsets of breast cancer, in particular triple-negative (TNBC) and HER2-positive (HER2+) tumors. Neoadjuvant therapy approaches in breast cancer have been used as a strategy to characterize subgroups of tumors with increased tumor-infiltrating lymphocytes (TILs). We have described tumor-infiltrating lymphocytes (TILs) as predictors of pathological complete response to neoadjuvant chemotherapy in breast cancer trials conducted by the German Breast Group. It has been shown that increased TILs are linked to an increased response rate to neoadjuvant therapy and improved prognosis after adjuvant therapy, in particular in triple-negative and HER2 positive breast cancer. In addition to tumor-infiltrating lymphocytes, immune mRNA markers have been studied in breast cancer and are associated with increased chemotherapy response and improved prognosis. These results suggest that some subtypes of breast cancer are immunogenic and that there is an ongoing low-level immune response present even in clinically progressing tumors. Therapeutic approaches to modulate tumor-associated inflammation by immune checkpoint inhibitors have shown clinical responses in a subset of triple-negative breast cancers, and some of these responses were durable. These targeted therapies have stimulated interest in the interaction between immune cells and tumor cells. Based on the concept that conventional chemotherapy might be immunogenic, some studies are evaluating the combination of an immune checkpoint inhibitor and a chemotherapy. Considering the fact that responses to immune checkpoint inhibitors are observed in only a subset of tumors, biomarkers that will be able to identify those tumors are needed. Possible biomarkers include the therapeutic targets, in particular PD-L1 and PD1, tumor-infiltrating lymphocytes, predictive mRNA signatures, immunogenic neoepitope signatures that are generated by mutations as well as other factors. Due to the low costs of a simple H&amp;E based test, tumor infiltrating lymphocytes might become a promising biomarker as part of the pathology report. In preparation for this, the international immune-oncology working group (TIL working group) has published standardized guidelines for TIL evaluation in breast cancer, and has conducted a first international TIL ring trial. This ring trial suggests that decentral TIL evaluation is feasible. In some situations, in particular in tumors with intermediate and heterogeneous TILs, additional tests, including gene expression signatures might be necessary. It should be emphasized that the clinical utility of TILs for current medical decisions is very limited at the present time, and the ultimate usefulness of these biomarker concepts will be obtained in the ongoing and future clinical immunotherapy trials. The presentation will provide an overview on recent results on immune markers for prediction of response to neoadjuvant chemotherapy and prognosis with a focus in standardized histomorphological evaluation of tumor-infiltrating lymphocytes as well as clinical trials concepts and biomarker strategies for future immune checkpoint inhibitor therapies in breast cancer. Citation Format: Denkert C. Tumor-infiltrating lymphocytes (TILs) and genomic signatures of immune cells [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 ES4-3.

Interobserver Agreement Between Pathologists Assessing Tumor-Infiltrating Lymphocytes (TILs) in Breast Cancer Using Methodology Proposed by the International TILs Working Group.

The presence of tumor-infiltrating lymphocytes (TILs) in breast tumors is prognostic and predictive, suggesting that TILs may be an important biomarker. Recently, an international TILs working group formulated consensus recommendations for TIL evaluation. The current study was performed to determine interobserver agreement using that methodology. Tumor-infiltrating lymphocytes were assessed on a single hematoxylin and eosin (H&E)-stained slide obtained from the core biopsy of 75 triple-negative breast cancers. Four pathologists independently reviewed each slide and evaluated stromal TILs (sTILs) and intratumoral TIL (iTILs). The kappa statistic was used to estimate interobserver agreement for identification of sTILs, and the intraclass correlation coefficient (ICC) was used to estimate the agreement among observers for iTILs. Cases with poor agreement were reviewed to identify pathologic factors that may contribute to the lack of agreement. The kappa statistic for sTIL evaluation was 0.57 (standard error, 0.04). For iTILs, the ICC calculated to determine internal consistency within raters was 0.65 (95% confidence interval [CI] 0.56-0.74; p<0.0001), and the ICC calculated to determine agreement among raters was 0.62 (95% CI 0.50-0.72; p<0.0001). In 10 cases (13%), there was not agreement between three of four pathologists. The pathologic features contributing to difficulty in TIL enumeration included marked individual tumor cell necrosis or apoptosis, the presence of reactive plasma cells mimicking tumor cells, plasmatoid tumor cells, and accurate quantification of TILs in specimens with focal areas of heavy immune infiltrate. Acceptable agreement in TIL enumeration was observed, suggesting that the proposed methodology can be used to facilitate the use of TILs as a biomarker in research and clinical trial settings.

Clinical validity of tumor-infiltrating lymphocytes analysis in patients with triple-negative breast cancer

Although tumor-infiltrating lymphocytes (TILs) have been associated with a favorable prognosis in triple-negative breast cancer (TNBC) patients, this marker is not currently considered robust enough for entering the clinical practice. In the present study, we assessed the clinical validity of the guidelines recently issued by the International TIL Working Group in a large retrospective series of well-annotated TNBC patients. TILs were evaluated in all the full-face H&E sections from 897 consecutive TNBC (i.e. tumors with <1% of ER and PgR immunoreactivity and absence of HER2 overexpression or amplification) patients diagnosed and treated at the European Institute of Oncology between 1995 and 2010 (median follow-up 8.2 years, range 6 months to 18 years). All mononuclear cells were evaluated in the stromal area within the borders of the invasive tumor, reported as a percentage value and treated as a continuous variable in survival analysis. The median percentage of TILs was 20%, and 21.9% of the cases had ≥50% (lymphocyte predominant breast cancer, LPBC) TILs. At univariable survival analysis, TILs were a significant predictor of better disease-free survival (DFS), distant disease-free survival (DDFS) and overall survival (OS) (P < 0.0001). Multivariable analysis confirmed that each 10% increase in TILs strongly predicted better survival, independent of patients' age, lymph node status, tumor size, histological grade, peritumoral vascular invasion and Ki-67 labeling index. Patients with LPBC had a 10-year survival rate of 71%, 84% and 96% for DFS, DDFS and OS, respectively. Stratified analysis revealed a positive correlation between TILs and OS across all the subgroups analyzed. Our data support the analytical validity of the recently issued TILs evaluation guidelines in the clinical practice.