High Levels Of Tumor-infiltrating Lymphocytes Research Articles

High level of TILs is an independent predictor of negative sentinel lymph node in women but not in men.

Factors that are most associated with positive lymph node status in melanoma are Breslow thickness and ulceration. However, there are other factors that have been little explored and could help in the identification of "at risk patients" harbouring occult metastasis. The objective of this study was to determine whether intensity of tumour-infiltrating lymphocytes (TILs) in a cohort study (N = 4133) is an independent predictor of sentinel lymph node (SLN) status in patients with primary cutaneous melanoma. Of the patients with cutaneous melanoma who resulted negative for nodal metastasis, 50.7% had moderate/marked TILs versus 27.7% among those patients who resulted positive for nodal metastasis. In the multivariate analysis, controlling for sex, age, mitotic rate, ulceration and Breslow, high levels of TILs in primary invasive melanoma was associated with a lower risk of developing SLN metastasis (OR 0.46; 95% CI 0.23-0.95, p = 0.037). When the analysis was stratified by sex, the protective effect of moderate/marked TIL remained only for women (OR 0.30; 95% CI 0.10-0.93, p = 0.037) but not for men (OR 0.51; 95% CI 0.19-1.34, p = 0.172). Other independent predictors of negative lymph node were low Breslow thickness (≤ 2.0mm) and low mitotic rate. Besides predicting a negative lymph node response, TILs were also associated with a decreased risk of 10-year mortality among females with positive lymph node. Our findings suggest that high level of TILs is an independent predictor of negative SLN status among women. Further research is warranted to confirm our findings.

Prognostic and Clinicopathological Value of PD-L1 in Melanoma: A Meta-Analysis

BackgroundThere is a growing interest in using programmed death ligand-1 (PD-L1) as a prognostic marker for melanoma. We conducted this meta-analysis to explore the prognostic and clinicopathological value of PD-L1 in melanoma. Materials and MethodsThe electronic databases PubMed, Web of Science and the Cochrane Library were searched for relevant studies. The major investigated parameters were PD-L1 expression levels in relation to patient gender, tumor-infiltrating lymphocytes (TILs), tumor stage, lymph node (LN) metastasis, histological type, progression-free survival (PFS) and overall survival (OS). Odds ratios (ORs) and hazard ratios (HRs) were computed using the fixed-effect or random-effects model according to data heterogeneity. ResultsPositive PD-L1 expression was significantly associated with high levels of TILs (OR = 7.56, 95% CI 2.04-28.02), metastatic melanoma (OR = 0.45, 95% CI 0.30-0.67) and LN-positive melanoma (OR = 2.56, 95% CI 1.31-4.99) but not gender or histological type. In addition, the pooled HRs showed no relation between PD-L1 expression and PFS (HR = 1.18, 95% CI 0.83-1.69) or OS (HR = 0.77, 95% CI 0.47-1.25). When restricted to metastatic melanoma, positive PD-L1 expression was significantly related to prolonged OS (HR = 0.57, 95% CI 0.46-0.70). ConclusionsPositive PD-L1 expression may be an important prognostic factor for longer OS in patients with metastatic melanoma.

APOBEC3B expression and its prognostic potential in breast cancer.

Apolipoprotein B mRNA editing enzyme catalytic subunit 3B (APOBEC3B) mRNA expression is associated with the poor prognosis of estrogen receptor positive (ER+) breast cancer. However, the clinical relevance of APOBEC3B protein expression in patients with breast cancer remains unclear. The present study evaluated the association of APOBEC3B protein expression with clinicopathological features, as well as survival outcomes of patients with breast cancer. Furthermore, the association between APOBEC3B protein expression and tumor infiltrating lymphocytes (TILs) was investigated. APOBEC3B protein expression in 120 patients with breast cancer was evaluated via immunohistochemistry, using a constructed tumor microarray, and TILs were analyzed by hematoxylin and eosin staining. The relevance of APOBEC3B mRNA expression in breast cancer was assessed using a Kaplan-Meier Plotter online tool, as well as the Tumor Immune Estimation Response and The Cancer Genome Atlas databases. The present study assessed APOBEC3B expression in 116 patients with breast cancer and demonstrated that protein expression was significantly associated with ER and progesterone receptor expression, as well as different subtypes of breast cancer. Notably, APOEBC3B protein expression was significantly associated with TILs. Overall, high expression levels of APOBEC3B protein and high levels of TILs were indicative of longer disease-free survival rate. High APOBEC3B mRNA expression was associated with poor relapse-free survival rate, overall survival rate and distant metastasis-free survival rate in patients with breast cancer, particularly for the Luminal A subtype. APOBEC3B mRNA expression was also indicated to be associated with the immune status of patients with breast cancer. Overall, the results of the present study demonstrated that APOBEC3B mRNA and protein expression levels presented different prognostic values in the survival of patients with breast cancer. However, both APOBEC3B mRNA and protein expression levels were associated with TILs in breast cancer. Therefore, APOBEC3B may be a prognostic biomarker for breast cancer.

Nab-paclitaxel and atezolizumab for the treatment of PD-L1-positive, metastatic triple-negative breast cancer: review and future directions

ABSTRACTIntroduction: Breast cancer is the most common malignancy in women in the United States and triple-negative breast cancer (TNBC) accounts for 15–20%. The standard of care for metastatic TNBC has been limited to cytotoxic chemotherapy with modest efficacy. TNBC is associated with high levels of tumor-infiltrating lymphocytes and PD-L1 expression, supporting the investigation of immune checkpoint inhibitors in this breast cancer subtype.Areas covered: This review summarizes the clinical data supporting the use of atezolizumab and nab-paclitaxel in the treatment of metastatic PD-L1-positive TNBC. It examines the pharmacology and toxicity profile of the combination in patients with metastatic TNBC.Expert opinion: The addition of atezolizumab to nab-paclitaxel prolonged progression-free survival in both the intention-to-treat and PD-L1-positive subgroups in the first-line setting in patients with metastatic TNBC. The IMpassion 130 trial led to FDA-approval of this combination in patients with PD-L1-positive, metastatic TNBC and represents the first approval of immunotherapy for TNBC. This work supports ongoing investigations of other immunotherapy combinations in TNBC, predictive biomarker development, and immunotherapy in patients with early-stage TNBC. Immunotherapy combinations in TNBC have the potential to lead to improved survival in this group of patients with high-risk disease.

Abstract PD5-04: Tumor-infiltrating lymphocytes evaluation using machine learning image analysis based on core needle biopsy in HER2-positive breast cancer

Abstract Purpose: To evaluate tumor-infiltrating lymphocytes (TILs) quantitatively in breast cancer and to verify the utility of machine learning image analysis in TIL assessment. Methods: Stromal TILs in 105 Human epidermal growth factor receptor 2 (HER2)-positive invasive breast cancers, diagnosed from patients treated with neoadjuvant anti-HER2 therapy, were evaluated by image analysis of hematoxylin and eosin-stained slides from core needle biopsies. TIL level was determined as the number of TILs per square millimeter of stromal tissue. Associations of TIL level with clinicopathological parameters, pathological response, and clinical outcomes were assessed. Results: Median TIL level were 1287/mm2 (range, 123 - 8101/mm2). Higher TIL level was associated with higher histological grade (P = 0.02), estrogen receptor (ER) negativity (P = 0.036), and pathological complete response (pCR) (P< 0.0001). According to analysis of the receiver operating characteristic curve, a threshold TIL level of 2420/mm2 stroma best discriminated pCR from non-pCR. In multivariate analysis, high TIL levels (>2420/mm2) were significantly associated with pCR (P< 0.0001). On Kaplan-Meier analysis, prognosis was not associated with high or low TIL levels. However, high TIL levels tended to be associated with better prognosis in the HER2+/ER+ subgroup (P = 0.131), but not in the HER2+/ER- subgroup. Conclusions: A machine learning image analysis algorithm could assess TIL level as the number of TILs per square millimeter, contrary to semiquantitative evaluation. TIL level derived from this method could offer an independent predictor of pCR. Citation Format: Norie Abe, Hirofumi Matsumoto, Reika Takamatsu, Kentaro Tamaki, Naoko Takigami, Kanou Uehara, Yoshihiko Kamada, Nobumitsu Tamaki, Tokiwa Motonari, Norihiro Nakada, Hisamitsu Zaha, Naoki Yoshimi. Tumor-infiltrating lymphocytes evaluation using machine learning image analysis based on core needle biopsy in HER2-positive 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 PD5-04.

Abstract P5-04-26: Expression of cytotoxic T lymphocyte antigen-4 (CTLA-4) is associated with tumor-infiltrating lymphocytes (TILs) levelin HER2-positive breast cancers

Abstract Though CTLA4 expression was reported in various tumor types, expression of CTLA4 is not well known in breast cancers, especially HER2-positive breast cancers. TILs level has significant role in treatment response and prognosis in HER2-positive breast cancers. Total 167 patients with HER2-positive breast cancers were included. All patients were pre-operatively chemotherapy and radiotherapy naïve. All received adjuvant standard treatment. Histologic TILs evaluation was performed with full face hematoxylin and eosin sections. Immunohistochemistry for CTLA4 was performed with tissue microarray sections. CTLA4 expression was evaluated separately for tumor cells and TILs. Cytoplasmic and/or membranous stains were assessed and human appendix was used as positive control. The intensity and percentage of positivity were evaluated collectively. Immunostaining score was calculated by multiplying percentage of positive cells by staining intensity. Total immunostaining score was obtained by adding tumor cells score and TILs score. Expression level was divided into two categories (low and high) according to mean value. Only 11 patients (6.6%), 47 patients (28.1), and 10 patients (6.0) showed total negative staining for CTLA4 on tumor cells, TILs and both on tumor cells and TILs, respectively. According to mean score on tumor cells, 97 (58.1%) patients showed low level of CTLA4 expression and 70 (41.9%) showed high level of CTLA4 expression. One hundred and two (61.1%) patients had low expression and 65 (38.9%) patients had high expression of CTLA4 on TILs based on the mean value of score of TILs. According to mean value of total score both on tumor cells and TILs, 70 patients (41.9%) were categorized as high expression and 97 patients (58.1%) were categorized as low CTLA4 expression. TILs level, CTLA4 immunostaining score on tumor cells, CTLA4 immunostaining score on TILs, and total CTLA4 immunostaining score on tumor cells and TILs were significantly correlated each other. High expression level of CTLA4 on tumor cells was significantly associated with negative expression of hormone receptor and high TILs level. Also, high CTLA4 expression on TILs significantly correlated with high histologic grade, negative expression of hormone receptor and high TILs level. High level of total CTLA4 expression both on tumor cells and TILs was significantly correlated with negative expression of hormone receptor and high TILs level. However, expression level of CTLA4 was not associated with overall survival and disease-free survival. In conclusions, CTLA4 expression was common in HER2-positive breast cancers and the level of CTLA4 expression was associated with hormone receptor negativity and TILs level in HER2-positive breast cancers. Table 1. Correlation of TILs level, CTLA4 immunostaining score on tumor cells, TILs, and total immunostaining scoreimmunostaining score on tumor cellsimmunostaining score on TILsTotal immunostaining score on tumor cells and TILsTILs level0.254 (<0.001)0.394 (<0.001)0.312 (<0.001)Immunostaining score of CTLA-4 in tumor cells0.565 (<0.001)0.815 (<0.001)Immunostaining score of CTLA-4 in TILs0.783 (<0.001) Table 2. Correlation between CTLA-4 expression and clinicopathologic factorsLowHighLowHighLowHighHistologic grade1.0000.0390.754Low (grade 1, 2)51 (52.6)36 (51.4)60 (58.8)27 (41.5)52 (53.6)35 (50.0)High (grade 3)46 (47.4)34 (48.6)42 (41.2)38 (58.5)45 (46.4)35 (50.0)Necrosis0.3251.0000.325Absent30 (30.9)27 (38.6)35 (34.3)22 (33.8)30 (30.9)27 (38.6)Present67 (69.1)43 (61.4)67 (65.7)43 (66.2)67 (69.1)43 (61.4)Lymphovascular invasion0.7540.2650.754Absent51 (52.6)39 (55.7)51 (50.0)39 (60.0)51 (52.6)39 (55.7)Present46 (47.4)31 (44.3)51 (50.0)26 (40.0)46 (47.4)31 (44.3)Hormone receptor expression0.0120.0400.005Negative37 (38.1)41 (58.6)41 (40.2)37 (56.9)36 (37.1)42 (60.0)Positive60 (61.9)29 (41.4)61 (59.8)28 (43.1)61 (62.9)28 (40.0)TILs level0.001<0.001<0.001≤10%42 (43.3)19 (27.1)50 (49.0)11 (16.9)44 (45.4)17 (24.3)20-60%45 (46.4)29 (41.4)43 (42.2)31 (47.7)43 (44.3)31 (44.3)>60%10 (10.3)22 (31.4)9 (8.8)23 (35.4)10 (10.3)22 (31.4) Citation Format: Ahrong Kim, Jee Yeon Kim, So Jeong Lee. Expression of cytotoxic T lymphocyte antigen-4 (CTLA-4) is associated with tumor-infiltrating lymphocytes (TILs) levelin HER2-positive breast cancers [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 P5-04-26.

Abstract P4-10-16: Reactive stroma and trastuzumab resistance in HER2-positive early breast cancer

Abstract Purpose We investigated the value of reactive stroma as a predictor for trastuzumab resistance in patients with early HER2-positive breast cancer receiving adjuvant therapy. Experimental Design The pathological reactive stroma and the mRNA gene signatures that reflect reactive stroma in 209 HER2-positive breast cancer samples from the FinHer adjuvant trial were evaluated. Levels of stromal gene signatures were determined as a continuous parameter, and pathological reactive stromal findings were defined as stromal predominant breast cancer (SPBC; ≥50% stromal) and correlated with distant disease-free survival (DDFS). Results Gene signatures associated with reactive stroma in HER2-positive early breast cancer (N=209), were significantly associated with trastuzumab resistance in estrogen receptor (ER)-negative tumors (HR=1.27 p-interaction=0.014 [DCN], HR=1.58, p-interaction=0.027 [PLAU], HR=1.71, p-interaction=0.019 [HER2STROMA, novel HER2 stromal signature]), but not in ER-positive tumors (HR=0.73 p-interaction=0.47 [DCN], HR=0.71, p-interaction=0.73 [PLAU], HR=0.84; p-interaction=0.36 [HER2STROMA]). Pathological evaluation of HER2-positive/ER-negative tumors suggested an association between SPBC and trastuzumab resistance. Reactive stroma did not correlate with tumor-infiltrating lymphocytes (TILs), and the expected benefit from trastuzumab in patients with high levels of TILs was pronounced only in tumors with low stromal reactivity (SPBC <50%). Conclusions Reactive stroma in HER2-positive/ER-negative early breast cancer tumors may predict resistance to adjuvant trastuzumab therapy. Citation Format: Amir Sonnenblick, Mali Salmon-Divon, Roberto Salgado, Noam Pondé, Sibylle Loibl, Carsten Denkert, Heikki Joensuu, Pirkko-Liisa Kellokumpu-Lehtinen, Amos Azaria, Sherene Loi, Stefan Michiels, Christos Sotiriou. Reactive stroma and trastuzumab resistance in HER2-positive early 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 P4-10-16.

Abstract PD5-10: Impact of immune markers on response to neoadjuvant de-escalated T-DM1 or trastuzumab with/or without endocrine therapy in HR+/HER2+ early breast cancer: A translational subproject of the WSG-ADAPT-HER2+/HR+ trial

Abstract Background: Among patients with highly heterogeneous HER2+ breast cancer (BC), pathological complete response (pCR) (ypT0/is/ypN0) is associated with improved prognosis. The prospective, phase-II neoadjuvant 3-arm WSG HER2+/HR+ trial (ADAPT-TP) showed pCR rates of about 41.0% after 4xT-DM1+/-ET and 15% after trastuzumab+ET. Pre-planned translational analysis of ADAPT-TP has revealed several biomarkers with impact on pCR, such as PIK3CA mutation status, HER2-enriched subtype (by PAM50), Bcl-2 and most notably immune biomarkers (e.g.CD8/tumor center). In particular, higher CD8 protein expression values either at baseline and/or after one cycle (3w) of neoadjuvant therapy were favorable for pCR. Moreover, CD8 and PDL1 dynamics were indicators of early therapy response, and dynamical response was itself favorable for pCR. The impact of immune biomarkers suggests that tumor infiltrating lymphocytes (TILs) could serve as predictive markers for de-escalated anti-HER2 therapy, in particular T-DM1. Methods: Semi-quantitative, triplicate TIL measurements were performed in tissue obtained at baseline (TIL-0) and at 3 weeks (TIL-3). At each timepoint, the median of the three measurements was used for analysis. CD8 and PD-L1 expression measurements were obtained by mRNA at baseline at both time points by immunohistochemistry (IHC). Associations of semiquantitative TIL-0/TIL-3 levels with pCR and with other clinical/pathological measurements were analyzed using logistic regression, rank correlations, t and chi-square statistics. Results: TIL-0 and TIL-3 were available in 355/375 and 301/375 randomized patients, respectively. Associations of TIL-0, TIL-3 and TIL-dynamics (increase from TIL-0 to TIL-3) with CD8 and PD-L1 (including dynamics) and with clinical factors (tumor size, nodal status, ER and PR status, grade, patient age, Ki67 and menopausal status) revealed several prominent relationships: Both TIL-0 and TIL-3 were at least moderately correlated with IHC measurements of CD8 (at center, not at invasive margin) and of PD-L1 at both timepoints. At cycle 2, the correlation of TILs and CD8 was strong, particularly in the T-DM1 arms (r=.7, p<.001). CD8 by mRNA was moderately correlated with TIL-0. Correlations between clinical factors and TILs at both timepoints were weak. These associations with known factors influencing pCR suggest a possible direct association of TILs with pCR; logistic regression using standardized TIL-0 revealed a modest (standardized) odds ratio of about 1.4 for pCR for TIL-0 in the T-DM1 arms, though not in the whole cohort. In particular, patients receiving T-DM1 with TIL0≥40% (about 8%) had pCR of 70% vs. 40% pCR for others (p=.016). We observed a dynamic increase of TILs during T-DM1 treatment of about 10 percentage points. However, neither these dynamics nor TIL-3 were significantly associated with pCR (in contrast to CD8 protein expression at cycle 2). Interpretation: To the best of our best knowledge, these are the first prospective data regarding the predictive value of TILs and TIL dynamics in HR+/HER2+ BC treated with de-escalated anti-HER2 therapy. Very high TIL levels (>40%) were associated with an excellent pCR rate of 70% in TDM1treated patients. However, CD8 expression and dynamics of CD8 were more strongly predictive for efficacy of de-escalated treatment than TILs – despite promising data on baseline and/or on-treatment TILs as markers for improved prognosis and higher sensitivity to (dual) anti-HER2 blockade (with or without chemotherapy) in unselected HER2+ BC. Further research on static and dynamic immune markers (according to HR+ and HR-/HER2+ subtypes) is needed prior to final conclusions regarding the clinical impact of TILs in early HER2+ BC. Citation Format: Claudia Biehl, Cornelia Kolberg-Liedtke, Oleg Gluz, Friedrich Feuerhake, Matthias Christgen, Ronald Kates, Eva-Maria Grischke, Helmut Forstbauer, Michael Braun, Mathias Warm, John Hackmann, Christoph Uleer, Bahriye Aktas, Claudia Schumacher, Sherko Kuemmel, Sanne L de Haas, Regula Deurloo, Rachel Wuerstlein, Ulrike Nitz, Hans H. Kreipe, Nadia Harbeck, West German Study Group. Impact of immune markers on response to neoadjuvant de-escalated T-DM1 or trastuzumab with/or without endocrine therapy in HR+/HER2+ early breast cancer: A translational subproject of the WSG-ADAPT-HER2+/HR+ trial [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 PD5-10.

Abstract OT1-01-02: Neoadjuvant pembrolizumab + decitabine followed by standard neoadjuvant chemotherapy for locally advanced HER2-breast cancer

Abstract Background: Higher levels of tumor-infiltrating lymphocytes (TIL) in breast cancers are associated with increased likelihood of pathologic complete response (pCR) to chemotherapy and improved outcomes. DNA methyltransferase inhibitors (DNMTi) can augment immune responses to cancers by upregulating tumor antigen expression, increasing MHC Class I expression and antigen presentation, decreasing numbers and activity of myeloid derived suppressor cells (MDSC) and increasing responsiveness of T lymphocytes. We have shown that decitabine augments the effectiveness of immunotherapy against murine triple negative breast cancer (TNBC). Trial design: Patients with HER-2-negative breast cancer who are candidates for neoadjuvant chemotherapy will receive decitabine (15 mg/kg x 4 doses over 5 days) followed by 2 doses of pembrolizumab (200 mg, 2 weeks apart) prior to starting neoadjuvant chemotherapy. Two research biopsies are obtained - 1 prior to the window immunotherapy and 1 afterwards, prior to starting chemotherapy. Chemotherapy regimens: TNBC - ddAC X 4 followed by weekly Taxol + Carboplatin X 12; HR+ - ddAC X 4 followed by weekly Taxol X 12. After the safely lead-in, the sequence of regimens can be reversed, per treating physician preference. Safety Lead-In: During immunotherapy and dose-dense AC in the first 11 patients enrolled and who have received at least 3 doses of decitabine and at least 1 dose of pembrolizumab, clinically significant immune related adverse events (irAEs) will be reported in an expedited manner. Accrual was halted until the safety lead-in phase was completed. Brief Eligibility criteria: Primary breast cancer, candidates for neoadjuvant chemotherapy Cohort A: TNBC (ER<10%, PR<10%, Her-2 negative) cT2, N1 or N2 cT2, 3-5 cm, N0 Any T3 or T4 Cohort B: ER+ or PR+, Her-2 negative cT2, N1 or N2 Any T3 or T4ECOG status 0-1 No Active autoimmune diseases Absence of metastatic disease too extensive to consider surgery for the primary Specific aims: Primary endpoint: Changes in Lymphocyte infiltration - by 10-15% Tumor area Stromal area Secondary endpoints: Gene expression (Nanostring) Multiplex Cell Markers (DAPI/CD4/CD8/FOXP3/CK/PDL1/PDL2, and MDSCs) pCR Percentage of patients with lymphocyte-predominant breast cancer Correlation of PD-L1 expression with pCR MDSC in blood samples pre- and post- window portion Sample Size and Statistical methods: Cohort A (TNBC) is the primary target group in this trial. When Cohort A has achieved its required sample size of 28 evaluable patients, Cohort B (HR+) will be closed to accrual irrespective of accrual to that cohort. However, in the unlikely event that accrual to Cohort B occurs at a faster rate than in Cohort A, Cohort B will be capped at 18 patients. Therefore, the projected sample size for this study is 32-50 patients. A one-sided paired t-test will be used to compare baseline and post-treatment percentage of tumor and stromal areas with infiltrating lymphocytes. The paired t-test will be applied separately to Cohorts A and B. Present accrual and target accrual: 12 subjects have been accrued; 1 withdrew prior to any protocol therapy. The 11 patient safety lead-in has been completed. As described above, the projected total sample size is 32-50 patients. Citation Format: Harry Douglas Bear, Michael O. Idowu, Andrew Poklepovic, Adam Sima, Maciej Kmieciak. Neoadjuvant pembrolizumab + decitabine followed by standard neoadjuvant chemotherapy for locally advanced HER2-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 OT1-01-02.

Abstract P1-10-20: Serial TILs: Evaluating the role of mid-treatment tumor infiltrating lymphocytes (TIL) in predicting pathologic complete response (pCR) in early-stage triple negative breast cancer (TNBC)

Abstract Introduction High levels of TIL at baseline are associated with higher pCR rates and better overall survival in TNBC. Recent studies have also indicated that higher TIL in post-NACT residual disease in TNBC are an important independent predictor of improved survival. We evaluated the role of mid-treatment (post-AC; Adriamycin/Cyclophosphamide) TIL in predicting pCR rates in early-stage TNBC. Methods Of 242 patients with stage I-III TNBC enrolled in the ARTEMIS trial (NCT02276443), 156 patients had pre-AC TIL and pCR status available for this analysis. Both pre-and post-AC TIL counts were available in 29 patients. Post-AC TIL counts for the remaining patients were imputed using linear regression with age, race, stage III, vimentin >50% and post-AC tumor volume reduction. Using these imputed TIL counts we evaluated the association of post-AC TIL with pCR. We also evaluated the change in TIL before and after treatment with AC. Results At baseline the median TIL count was 10% (n=156). In the post-AC samples, the median TIL count was 5%. Using imputed TIL counts, we did not conclude that post-AC TIL was associated with pCR (p= 0.28). Using a cut-point of 15% TIL, our analysis showed that baseline TIL is more strongly correlated with pCR than post-AC TIL (Table 1). In our univariable logistic regression, both baseline TIL and the difference in TIL pre-and post- treatment were significantly associated with pCR (p= 0.0015 and p=0.0068, respectively), however in the multivariable analysis only baseline TIL was significant. Our analysis did show that a decrease in TIL from pre- to post-treatment was significantly associated with pCR (p=0.022). However, this measure was not significant in our logistic regression model when pre-TIL was also included. Conclusion Higher pre-treatment TIL correlated more strongly with pCR rate when compared to post-AC TIL. Pre-treatment high TIL was associated with pCR regardless of changes in TIL pre and post treatment. Table 1. Changes in TIL before and after treatmentBaseline TILPost-AC TILN#pCR (%)LowLow6217 (27%)LowHigh4012 (30%)HighLow2513 (52%)HighHigh2916 (55%)TIL; Low: <15, High >15 Citation Format: Nour Abuhadra, Kenneth Hess, Jennifer Litton, Gaiane Rauch, Alastair Thompson, Bora Lim, Beatriz Adrada, Elizabeth Mittendorf, Senthil Damodaran, Rosalind Candelaria, Banu Arun, Wei Tse Yang, Naoto Ueno, Lumarie Santiago, Rashmi Murthy, Nuhad Ibrahim, Sahin Aysegul, William Symmans, Lei Huo, Stacy Moulder. Serial TILs: Evaluating the role of mid-treatment tumor infiltrating lymphocytes (TIL) in predicting pathologic complete response (pCR) in early-stage triple negative breast cancer (TNBC) [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 P1-10-20.

Abstract P4-10-23: Expression of PD-L1 is independent of PIK3CA/AKT1/PTEN alterations in triple-negative breast cancer (TNBC) and is not associated with response to ipatasertib (IPAT) plus paclitaxel (PAC)

Abstract Background: In the FAIRLANE trial (NCT02301988) in early TNBC, adding the oral AKT inhibitor IPAT to neoadjuvant PAC led to numerical increases in rates of pathologic complete response (pCR; primary endpoint) and complete response by MRI in unselected patients, with a numerically greater treatment effect in patients with PIK3CA/AKT1/PTEN-altered tumors [Oliveira et al., Ann Oncol 2019]. The IMpassion130 trial established the efficacy of first-line atezolizumab + nab-PAC in patients with PD-L1-positive advanced TNBC [Schmid et al., NEJM 2018]. Preliminary phase 1b results showed promising activity with a triplet combination of IPAT, atezolizumab, and taxane as first-line therapy for advanced TNBC across PIK3CA/AKT1/PTEN and PD-L1 biomarker subgroups [Schmid et al., AACR 2019]. Using samples from the FAIRLANE trial, PD-L1 expression was characterized to evaluate relationships between IPAT treatment effect and expression of PD-L1 and biomarkers relevant to IPAT. Methods: Pretreatment tumor samples were evaluated for genomic alterations using the FoundationOne® assay (Foundation Medicine). Pretreatment PD-L1 status was determined using the SP142 immunohistochemistry assay (VENTANA Medical Systems), with PD-L1 positivity defined as PD-L1 expression on ≥1% of tumor-infiltrating immune cells. Tumor-infiltrating lymphocytes (TILs) were quantified using the Salgado method [Salgado et al., Ann Oncol 2015]. Gene expression was measured by RNA sequencing. Results: Samples were evaluable for genomic alterations in 144 patients, TILs in 135 patients, gene expression in 111 patients, and PD-L1 expression in 99 patients. The prevalence of PD-L1 positivity was 39%, consistent with the reported prevalence in IMpassion130. Compared with PD-L1-negative tumor samples, PD-L1-positive tumor samples showed significantly higher levels of TILs (mean 37% vs 24%; p=0.004) and significantly higher expression of the genes encoding for PD-L1 (CD274: geometric mean 10.5 vs 3.9 counts per million; p<0.0001) and PD-1 (PDCD1:geometric mean 2.3 vs 0.9 counts per million; p=0.006). The prevalence of PD-L1 positivity was similar in PIK3CA/AKT1/PTEN-altered and -nonaltered tumor samples (36% vs 40%, respectively). For patients receiving IPAT + PAC, no difference was observed according to PD-L1 status for either pCR (16% in PD-L1-positive samples vs 15% in PD-L1-negative samples; p=1) or complete/partial response by MRI (75% vs 64%, respectively; p=0.5). Conclusions: PIK3CA/AKT1/PTEN alterations and PD-L1 expression are independent biomarkers in TNBC. The prevalence of PD-L1 positivity in FAIRLANE (early TNBC) is similar to that in IMpassion130 (advanced TNBC). Pretreatment PD-L1 expression is not associated with pCR or response by MRI to IPAT + PAC. Citation Format: Matthew J Wongchenko, Mafalda Oliveira, Cristina Saura, Paolo Nuciforo, Isabel Calvo, Jay Andersen, José L Passos Coelho, Miguel Gil Gil, Begoña Bermejo, Debra A Patt, Eva Ciruelos, Zhen Shi, Na Xu, Patricia Villagrasa, Aruna Mani, Steven J Isakoff. Expression of PD-L1 is independent of PIK3CA/AKT1/PTEN alterations in triple-negative breast cancer (TNBC) and is not associated with response to ipatasertib (IPAT) plus paclitaxel (PAC) [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 P4-10-23.

Abstract P5-02-10: Overexpression of Indoleamine 2,3-dioxygenase 1 in HER2 positive breast cancer correlates with increase of tumor-infiltrating lymphocytes level and worse overall survival

Abstract Introduction: HER2+ breast cancer (BC) shows a large amount of mutation burden and high lymphocyte infiltration in the tumor. Tumor-infiltrating lymphocytes (TILs) reflect the host immune reaction against tumor cells, and TILs levels have been studied as prognostic and predictive factors in breast cancers, particularly in the triple-negative and HER2+ subtype. Conversely, tumor cells develop immunosuppressive tumor microenvironment by change the gene expression. This study evaluated the differences in expression of immune-related genes particularly involving immune evasion mechanism according to TILs level and confirmed protein expression in the tumor tissue. Materials and Methods: This study included 167 patients with primary HER2+ BC who underwent surgery. All hematoxylin and eosin slides of the tumors were reviewed and their TILs level was recorded. All tumors were divided into high and low TILs groups with a cutoff value of 60%. RNA was extracted from formalin-fixed paraffin embedded (FFPE) block of 12 cases to determine their immune-related gene RNA expression levels using the NanoString nCounter® PanCancer Immune Profiling Panel. Among the expressed genes with significance, indoelamine-2,3-dioxygenase 1 (IDO1)expression was further evaluated by immunohistochemistry (IHC) in tissue microarray. The IDO1 expression on tumor cells and TILs was categorized as low or high. Results: The IDO1 gene expression level was 7.1-fold higher in high-TILs group compared to low-TILs group (p=0.024). High IDO1 expression in the tumor cells and TILs by IHC analysis were related to high-TILs group (Table 1). Tumors with high IDO1 expression showed a high nuclear grade and related to worse overall survival (OS), particularly in hormone receptor-/HER2+ patients (all, p < 0.05). Multivariate analysis indicated that high IDO1 expression in tumor cells was an independent poor prognostic factor for OS (p=0.045, Table 2) Conclusion: This study demonstrated that HER2+ BC with high TILs level overexpressed IDO1 in tumor tissue. High IDO1 expression in the tumor cell is related to higher nuclear grade and poor OS in patients with HER2+ BC. In conclusion, this study showed that HER2-positive breast cancer has high immunogenicity and is related to the IDO1 immunosuppressive mechanism and related with poor OS. This result indicates that IDO1 targeted therapy may be effective in patients with HER2-positive breast cancer patients. Table 1. Clinicopathologic features according to IDO1 expression level in HER2+ BCFactorsIDO1TC expressionIDO1TIL expressionLowHighP-value†LowHighP-value‡Age (mean, yr)51.6753.680.20254.0751.780.163Size (median, cm)3.103.350.3763.213.210.997Histologic grade (n, %)0.0540.740Low40 (44.0)22 (28.9)23 (39.0)39 (36.1)High51 (56.0)54 (71.1)36 (61.0)69 (63.9)Nuclear grade (n, %)0.040*0.523Low21 (23.1)8 (10.5)12 (20.3)17 (15.7)High70 (76.9)68 (89.5)47 (79.7)91 (84.3)Lymphovascular invasion(n, %)0.7580.257Absent48 (52.7)42 (55.3)28 (47.5)62 (57.4)Present43 (47.3)34 (44.7)31 (52.5)46 (42.6)Node metastasis(n, %)0.3500.630Absent46 (50.5)32 (42.1)26 (44.1)52 (48.1)Present45 (49.5)44 (57.9)33 (55.9)56 (51.9)AJCC 7th Stage (n, %)0.4710.853Early71 (78.0)55 (72.4)44 (74.6)82 (75.9)Advanced20 (22.0)21 (27.6)15 (25.4)26 (24.1)HR expression (n, %)0.2810.517Positive52 (57.1)37 (48.7)29 (49.2)60 (55.6)Negative39 (42.9)39 (51.3)30 (50.8)48 (44.4)TILs level (n, %)0.047*0.002*Low (≤60%)79 (86.8)56 (73.7)55 (93.2)80 (74.1)High (>60%)12 (13.2)20 (26.3)4 (6.8)28 (25.9)TLSs in DCIS (n, %)0.5240.067Absent58 (63.7)44 (57.9)42 (71.2)60 (55.6)Present33 (36.3)32 (42.1)17 (28.8)48 (44.4)TLSs in invasive component (n, %)0.7440.308Absent58 (63.7)51 (67.1)42 (71.2)67 (62.0)Present33 (36.3)25 (32.9)17 (28.8)41 (38.0)Abb: IDO1TC, IDO1 in tumor cells; IDO1TIL, IDO1 in TILs; TLS, tertiary lymphoid structure Table 2. Univariate and multivariate analysis of IDO expression for OS in HER2+ BCUnivariateMultivariateFactorsHR95% CIp-valueHR95% CIp-valueIDO1TC expression0.022*0.045*Low(ref)(ref)(ref)(ref)High3.7411.206-11.6023.2161.028-10.060IDO1TIL expression0.771Low(ref)(ref)High0.8610.313-2.368TLSs in DCIS0.036*0.079Absent(ref)(ref)(ref)(ref)Present0.2060.047-0.9050.2620.059-1.169TLSs in invasive0.364Absent(ref)(ref)Present0.5920.191-1.836Abb: OS, overall survival; HR, hazard ratio; CI, confidence interval; IDO1TC, IDO1 in tumor cells; IDO1TIL, IDO1 in TILs; TLS, tertiary lymphoid structure Citation Format: Jeeyeon Kim, Ahrong Kim, So Jeong Lee. Overexpression of Indoleamine 2,3-dioxygenase 1 in HER2 positive breast cancer correlates with increase of tumor-infiltrating lymphocytes level and worse overall survival [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 P5-02-10.

Reactive stroma and trastuzumab resistance in HER2-positive early breast cancer.

We investigated the value of reactive stroma as a predictor for trastuzumab resistance in patients with early HER2-positive breast cancer receiving adjuvant therapy. The pathological reactive stroma and the mRNA gene signatures that reflect reactive stroma in 209 HER2-positive breast cancer samples from the FinHer adjuvant trial were evaluated. Levels of stromal gene signatures were determined as a continuous parameter, and pathological reactive stromal findings were defined as stromal predominant breast cancer (SPBC; ≥50% stromal) and correlated with distant disease-free survival. Gene signatures associated with reactive stroma in HER2-positive early breast cancer (N = 209) were significantly associated with trastuzumab resistance in estrogen receptor (ER)-negative tumors (hazard ratio [HR] = 1.27 p interaction = 0.014 [DCN], HR = 1.58, p interaction = 0.027 [PLAU], HR = 1.71, p interaction = 0.019 [HER2STROMA, novel HER2 stromal signature]), but not in ER-positive tumors (HR = 0.73 p interaction = 0.47 [DCN], HR = 0.71, p interaction = 0.73 [PLAU], HR = 0.84; p interaction = 0.36 [HER2STROMA]). Pathological evaluation of HER2-positive/ER-negative tumors suggested an association between SPBC and trastuzumab resistance. Reactive stroma did not correlate with tumor-infiltrating lymphocytes (TILs), and the expected benefit from trastuzumab in patients with high levels of TILs was pronounced only in tumors with low stromal reactivity (SPBC <50%). In conclusion, reactive stroma in HER2-positive/ER-negative early breast cancer tumors may predict resistance to adjuvant trastuzumab therapy.

Prognostic and clinicopathological significance of PD-L1 and tumor infiltrating lymphocytes in hypopharyngeal squamous cell carcinoma.

Limited information is available regarding programmed death-ligand 1 (PD-L1) and tumor-infiltrating lymphocytes (TILs) in hypopharyngeal squamous cell carcinoma (HPSCC). Therefore, we investigated the expression of PD-L1 and the volume of TILs in HPSCC to determine whether these biomarkers were associated with patient clinicopathologic characteristics and prognosis. Furthermore, we explored p16 status and analyzed its possible correlations with clinical outcomes. Tissues of 111 HPSCC patients were immunohistochemically analyzed for PD-L1, CD8, CD4, and Foxp3 expression to assess the microenvironment. We also assessed the p16 status. The expression of PD-L1 and TILs were analyzed with respect to patient clinicopathologic variables and prognosis. Twenty-four (21.6%) patients had PD-L1 expression in ≥1% of tumor cells. PD-L1 expression was significantly correlated with a high level of TILs (P<0.05). Kaplan-Meier analysis showed that higher CD8+ and FoxP3+ TIL infiltration was strongly associated with superior overall survival (OS, P=0.005 and P=0.008) and disease-free survival (DFS, P=0.015 and P=0.048). Univariate and multivariate analyses confirmed that CD8+ TIL exhibited strong prognostic significance. The combination of PD-L1+ with CD8high expression was a prognostic factor and was associated with better OS (P=0.025). Moreover, p16 positivity was detected in five patients (4.5%) and was only occasionally involved in HPSCC. Our findings indicate that high CD8 and FoxP3 expression in HPSCC contributes to longer patient survival. Although PD-L1 expression was not associated with outcome, PD-L1 positivity in combination with CD8high expression may have greater predictive potential.

Sarcomatoid Dedifferentiation in Renal Cell Carcinoma: From Novel Molecular Insights to New Clinical Opportunities.

Sarcomatoid features in renal cell carcinoma (RCC) have long been associated with dismal prognosis and poor response to therapy, while biological mechanisms underpinning sarcomatoid dedifferentiation remained obscure. Several efforts have been conducted to break down the molecular profile of sarcomatoid RCC and investigate different targeted therapeutic approaches. Mutations enriched for in sarcomatoid RCC involve, notably, TP53, BAP1, cell cycle, and chromatin-remodeling genes. The immunological landscape of these tumors is also gradually being uncovered, showing frequent expression of programmed cell death ligand-1 (PD-L1) and high levels of tumor-infiltrating lymphocytes. These features may be major determinants for the activity of immune checkpoint inhibitors in this population, which has been confirmed by retrospective studies and subgroup analyses of large randomized phase 3 trials. Combinations based on PD-1/PD-L1 inhibition have demonstrated response rates and complete responses in >50% and >10% of patients in the first-line metastatic setting, respectively, with median overall survival exceeding two years. This remarkable improvement in outcomes effectively establishes immune checkpoint inhibitor combinations as a new standard of care in patients with sarcomatoid RCC. New research fields, including epigenetic regulations and tumor–microenvironment interactions, may further sharpen understanding of sarcomatoid RCC and advance therapeutic developments.

The prognostic value of tumour-infiltrating lymphocytes (TILs) in pancreatic cancer: A systematic review and meta-analysis

Abstract Background Pancreatic cancer (PC) contributes to over 7 % of all cancer related deaths worldwide with a relative 5-year survival of less than 8 %. High levels of tumor-infiltrating lymphocytes (TILs) are associated with improved survival in many cancer types. Examining the impact of TILs on survival in PC could provide better prognostication and help clinicians tailor therapy for patients. Methods A systematic search based on the PICO-process was conducted on PubMed, Embase, The Cochrane Library and Web of Science. Outcome of interest was overall survival (OS). Studies examining high vs. low levels of TILs in pancreatic tumor tissue and its impact on OS was included. Following data extraction a random-effects model meta-analysis was conducted on the reported outcome with corresponding lymphocyte subset. The Newcastle-Ottowa Scale was used for study quality assessment. Results In total, 43 studies were included in the systematic review and 40 were eligible for meta-analysis. A time-to-event meta-analysis on the different lymphocyte subtypes revealed, that high vs. low infiltration of CD3+ T and CD8+ T cells was significantly associated with improved OS (HR = 0.59, 95 % CI: 0.51 - 0.68, I2: 0 % and HR = 0.61, 95 % CI: 0.57 – 0.67, I2: 0 %, respectively). High infiltration of FoxP3+ T cells was associated with decreased OS (HR = 1.41, 95 % CI: 1.15 – 1.73, I2: 85 %). The prevalence of CD4+ and CD20+ lymphocytes in pancreatic tumor tissue was not significantly associated with increased OS (HR = 0.87, 95 % CI: 0.65 - 1.17, I2: 80 % and HR = 0.86, 95 % CI: 0.54 – 1.35, I2: 66 %, correspondingly). Conclusion High infiltration of CD3+ and CD8+ lymphocytes is associated with improved OS among patients with resected PC, whereas high infiltration of FoxP3+ T cells is associated with decreased OS. CD4+ and CD20+ lymphocytes did not have a significant impact on OS. Based on these findings, staining for TILs, especially CD3+, CD8+ and FoxP3+ T cells, might be an important tool for future assessment of patient survival and prognosis, as well as for tailored oncological therapy. Clinical trial identification CRD42019134744. Legal entity responsible for the study The authors. Funding Center for Surgical Science. Disclosure All authors have declared no conflicts of interest.

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.

Correlation between PD-L1 expression and clinicopathological characteristics of non-small cell lung cancer: A real-world study of a large Chinese cohort.

Programmed death ligand-1 (PD-L1) is a predictive marker of anti-programmed death protein 1 (PD-1)/PD-L1 therapies for non-small cell lung cancer (NSCLC). However, little is known between PD-L1 expression and the clinicopathological characteristics of NSCLC in the Chinese population in a real-world setting. We analyzed PD-L1 expression by immunohistochemistry (IHC) in NSCLC patients using the 22C3 clone on the Dako Autostainer Link 48 platform. We then examined the associations of PD-L1 expression with clinicopathological characteristics, stromal tumor-infiltrating lymphocytes (TILs) and major molecular features. A total of 1,156 recently NSCLC specimens including 827 sequentially resected specimens and 293 biopsy specimens were enrolled in our study. PD-L1 high expression was observed in 9.7% of 827 NSCLC patients, including 6.5% with adenocarcinoma (ADC, n=690), and 27.4% with squamous cell carcinoma (SqCC, n=117). These results showed higher expression rates than those in archived samples (>5 years old, n=329), that were previously reported by our group (4.9%, 0.5%, and 13.9% in NSCLC, ADC, and SqCC, respectively). The prevalence of PD-L1 expression was lower in surgical resection samples than in small biopsy samples. PD-L1 high expression in the lung biopsy was less likely present in the primary cancer than in metastases, and was also associated with a high level of stromal TILs (P=0.029) and PD-L1-positive immune cells (IC) (P<0.001). Both PD-L1 high and low expressions were more frequent in EGFR-wild type than in mutant type (P<0.001). This study demonstrates that expression of PD-L1 is linked to the type of tumor specimens, resection versus biopsy specimens, and biopsies of primary versus metastatic cancers. These findings have substantial implications for clinical practice.

Investigating the role of chromosomal instability in immune cell activation

Abstract Background Patients with high numbers of neoepitopes responds better to immunotherapy, but the correlation to response is imperfect, indicating that multiple layers of modulation and/or suppression plays a role in triggering an ongoing immune response. The STING/cGAS pathway senses cytosolic DNA and interact with P53 to trigger the innate immune system. Cytosolic DNA may be generated through chromosomal instability (CIN), a hallmark of cancer associated with poor prognosis. Here we investigate the interplay between tumour infiltrating lymphocytes (TILs) and CIN in a bladder cancer cohort from the The Cancer Genome Atlas. Methods Measures of CIN (Marquard AM, 2015, Biomark Res, Burrell RA, Nature 2013) were determined based on ASCAT segmented SNP6 data. TILs were estimated from RNAseq data as described (Danaher P, Journal for ImmunoTherapy of Cancer, 2017). CIN and TIL scores were subjected to hierarchical clustering to determine groups of high and low CIN and TIL. Results We performed hierarchical clustering separately on measures of CIN and measures of TILs, revealing for each analysis a low, intermediate and high group. To improve signal, we discarded samples in the intermediate group. This resulted in samples classified into four groups, as high/low CIN and high/low TILs. Biological characterization revealed the high CIN-low TIL group as an outlier, with fewer mutations overall, relatively higher expression of STING, and low proliferation. Kaplan-Meier analysis shows how overall survival associates with both CIN and TILs, with high-CIN associating with poor survival in high-TIL samples, but with improved survival in low-TIL samples. Conclusions Mobilization of the immune system to fight cancer is currently revolutionizing patient treatment. The adaptive immune system recognizes cancer neoepitopes and may drive an anti-cancer response. Stratifying tumours into high and low levels of CIN and TILs shows subgroup-specific biological variation potentially indicating how a dysfunctional STING/cGAS pathway may result in a reduced activation of the innate immune response with an association to overall survival. These observations need further confirmation in larger cohorts, and the implications of CIN must be explored in the context of immuno-oncology agents. Legal entity responsible for the study The authors. Funding Aarhus University Research Foundation. Disclosure All authors have declared no conflicts of interest.

16P - Functional analysis of tumour infiltrating lymphocytes in triple negative breast cancer focusing on granzyme B

BackgroundTriple-negative breast cancer (TNBC) is one of the most aggressive subtypes in breast cancer and has a worse prognosis than others. Tumor infiltrating lymphocytes (TIL) are considered to be a prognostic factor in TNBC. Cytotoxic T cells (CTL) produce cytokines and cytotoxic substances such as perforin and granzyme B, and attack target cells. We have previously shown that combination of PD-L1 expression and TIL is useful as a functional indicator of tumor immune activation. Granzyme B released from CTL induces apoptosis by passing through pores formed by perforin on the cell membrane of target cells. We focused on the cytotoxic substance granzyme B and explored functional of TIL. MethodsThis study included 228 patients with primary TNBC who underwent resection without neoadjuvant chemotherapy at Kyushu University Hospital (Japan), between January 2004 and December 2014. We retrospectively analyzed granzyme B, CD8, PD-L1 expression assessed by immunohistochemistry and TIL in 228 TNBCs. Granzyme B was evaluated in TIL, ≥ 3% was defined as high expression, and < 3% as low. We also explored the correlation between immunologic features on tumors and immune cells and the clinicopathological characteristics of the tumors, their response to chemotherapy and clinical outcome. ResultsAmong the 228 tumors, granzyme B expression was classified as high in 106 (46.5%), low in 122 (53.5%). Granzyme B-high is correlated with high levels of TIL (p=0.004), CD8 expression of T cell (p=0.016) and PD-L1 of tumor cells (p<.0001). In the TIL-high and granzyme B-high group, it is considered that the anti-tumor immune system is activated, and it tend to be the most favorable prognosis. On the other hand, in the TIL-high and granzyme B-low group, despite lymphocyte migration, it is presumed that they do not recognize the antigen, that is, they are in a state of immune tolerance and thus have a poor prognosis. In the group with granzyme B-high, the patients treated with anthracycline as adjuvant chemotherapy significantly improved their prognosis (p=0.043). The high expression of granzyme B may be a predictor of postoperative chemotherapy. ConclusionsGranzyme B is an important substance of cytotoxic pathway and has been possible to be an indicator of TIL activation. Legal entity responsible for the studyMasafumi Nakamura. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.