High Levels Of Tumor-infiltrating Lymphocytes Research Articles

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.

Imaging and Clinicopathologic Features Associated With Pathologic Complete Response in HER2-positive Breast Cancer Receiving Neoadjuvant Chemotherapy With Dual HER2 Blockade

BackgroundIn human epidermal growth factor receptor 2-positive (HER2+) breast cancer, the incorporation of a dual HER2 blockade into neoadjuvant chemotherapy (NAC) has been shown to induce a higher rate of pathologic complete response (pCR). The purpose of this study was to investigate whether pretreatment imaging and clinicopathologic features show any association with pCR in HER2+ breast cancer receiving NAC plus dual blockade. Materials and MethodsThis retrospective study evaluated 94 consecutive patients (mean age, 49.8 ± 9.9 years) with HER2+ breast cancer who underwent NAC plus dual blockade with trastuzumab and pertuzumab between April 2016 and June 2018. All patients underwent mammography, ultrasound, and magnetic resonance imaging prior to NAC. Clinicopathologic and imaging features acquired before NAC were evaluated for their ability to predict the pathologic response after surgery. Multivariate analysis was used to identify independent predictors of pCR. ResultsFifty patients (53.2%) showed pCR and 44 (46.8%) did not. According to a univariate analysis, fine pleomorphic/fine linear or linear-branching calcification morphology on mammography, parallel orientation on ultrasound, intratumoral high signal intensity on T2-weighted magnetic resonance imaging, progesterone receptor negativity, and high levels of tumor-infiltrating lymphocytes were associated with pCR. On multivariate analysis, fine pleomorphic/fine linear or linear-branching calcification morphology on mammography (odds ratio [OR], 7.23), progesterone receptor negativity (OR, 6.76), and a high tumor-infiltrating lymphocyte level (OR, 5.92) remained significant independent factors associated with pCR. ConclusionSeveral pretreatment imaging and clinicopathologic features were shown to be independent variables predicting pCR in patients with HER2+ breast cancer receiving NAC with dual blockade.

Abstract 5252: High expression of PLK1, polo-like kinase 1, is significantly associated with DNA repair deficiency, inactivated TP53, and worse prognosis in breast cancer

Abstract Backgrounds: Polo-like kinase 1 (PLK1), the representative member of the PLK family, plays a pivotal role both in mitosis, especially in G2/M phase, and in the regulation of DNA damage repair. It is suggested that overexpression of PLK1 in cancer cells may disturb the behavior of G2-M DNA damage checkpoint for cancer cell survival. Therefore it was of interest whether PLK1 expression has any clinical significance in breast cancer (BC), which is known to have less DNA damage and mutations compared from other cancers. Methods: A total of 1025 women with BC from The Cancer Genome Atlas (TCGA) and a total of 237 women with BC from neoadjuvant (NAC) cohort are enrolled. We developed computer-based analysis to verify the clinical significance of PLK1 mRNA expression in these large BC cohorts. Results: In TCGA cohort, PLK1 high expressing tumors were statistically significantly associated with more aggressive clinical factor. Gene Set Enrichment Analysis shows that PLK1 high expressing tumors were significantly associated with cell cycle related gene sets. In addition, PLK1 high expressing tumors were significantly associated with deficiency of homologous recombination (HR), which is one of the important mechanisms of DNA repair, and high expression of TP53, which is an intermediary to the HR of PLK1. All these results suggest that PLK1 high expressing tumors associate with worse cancer biology. Recently tumor immune microenvironment (TIM) attracted many researchers’ interest for its association with better response to NAC and better survival. In analysis of TIM, we found that PLK1 high expressing tumors were significantly correlated with anti-cancer related tumor infiltrating lymphocytes (TILs) and higher immune cytolytic activity CYT. Thus, PLK1 high expressing tumors associate with TIM that is against cancer. Since both higher HR deficiency (HRD) and anti-cancer related TILs are predictors of high sensitivity to chemotherapy, response of PLK1 high expressing tumors to NAC were analyzed, which did not show significant association. On the other hand, survival analysis demonstreated that PLK1 high expressing tumors were statistically significantly associated with poor prognosis in the whole and the hormone receptor-positive/human epidermal growth factor receptor 2-negative group. Our result suggest that although PLK1 high expressing tumors attract TILs and assoaciate with HRD, those anti-tumor effects are overweighed by its association with aggressive cancer biology that translate to worse survival particularly in the subtype known to have less mutations. Conclusions: We have demonstrated the clinical significance of PLK1 mRNA expression in BC. High PLK1 expression was associated with more aggressive clinical factors, cell cycle related genes, higher HRD, inactivated TP53, high levels of TILs, and worse prognosis in BC. Citation Format: Takashi Takeshita, Mariko Asaoka, Eriko katsuta, Yan Li, Kazuaki Takabe. High expression of PLK1, polo-like kinase 1, is significantly associated with DNA repair deficiency, inactivated TP53, and worse prognosis in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5252.

Abstract 1175: A pro-tumorigenic mechanism of M2 tumor-associated macrophages in triple-negative breast cancer

Abstract Introduction: Triple-negative breast cancers (TNBCs) are defined as tumors that are negative for both estrogen, progesterone and HER-2 receptors. It is a heterogeneous subtype of breast cancer with a high propensity for systemic metastases and poor survival with only chemotherapy available for treatment. Compared to other hormone-positive breast cancer subtypes, TNBC features a unique tumor microenvironment (TME) characterized by a large number of tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) (1), and the increase of immune infiltrate with high levels of TILs predicts response to neoadjuvant chemotherapy as well as improved survival. TAMs can be described as classically activated M1 types with pro-inflammatory antitumor functions, versus alternatively activated M2 types with immunosuppressive pro-tumor functions. It has been reported that the presence of M2 TAMs positively correlates with TNBC, hormone receptor negativity, as well as higher tumor proliferation (2-3). However, the mechanisms by which TAMs interact with TNBC cells and their phenotypes in TNBC versus estrogen receptor positive (ER+) patients is not well understood. Experimental Procedures: Using MultiOmyx, a proprietary, immunofluorescence (IF) multiplexing assay that enables visualization and characterization of up to 60 biomarkers on a single FFPE section (4), we have characterized TIL phenotypes, tumor proliferation and TAM activation in 15 FFPE tumors from TNBC and 5 tumors from ER+ patients. Results: Using a multiplex panel of 9 markers we found a 10-fold higher number of TILs and a 5-fold higher number of proliferating tumor cells in TNBC versus ER+ tumors. Furthermore, the proportion of T helper cells (CD3+CD4+) that were Tregs (CD3+CD4+FoxP3+) was increased from 26% to 43% in TNBC tumors. Interestingly, when analyzing TAMs we found that while ER+ tumors had a significantly higher proportion of M1 TAMs (CD68+HLA-DR+) than M2 TAMs (CD68+CD163+), this expression pattern was reversed in TNBC. When analyzing a possible correlation between the activation state of TAMs and the proliferation of tumor cells, we found a positive significant correlation between the presence of M2 TAMs and proliferating tumor cells (Pearson's correlation p&amp;lt;0.05), only in TNBC and not in ER+ tumors. Conversely, the presence of M1 tumors showed a weak negative correlation to tumor cell proliferation, in both ER+ and TNBC tumors. Conclusion: These data are suggestive of a possible pathway in which an increase in alternatively activated immunosuppressive M2 TAMs in hormone receptor negative breast cancer tumors, are responsible for providing a suitable environment for tumor proliferation.

Differences in immune contextures among different molecular subtypes of gastric cancer and their prognostic impact.

Gastric cancers have been recently classified in accordance with their molecular characteristics, thus demonstrating the complex nature of cancers and an association with the immune contexture within the tumor microenvironment. This study aimed to investigate the correlation between the molecular subtype and immune contexture of gastric cancers. The immune contexture, including the type, density, and location of tumor-infiltrating lymphocytes (TILs), of gastric cancer patients was examined and immune subtypes were classified based on it. In particular, PD-L1 expression on tumor cells and TILs and Foxp3+ TILs was assessed in accordance with molecular subtypes. High levels of visual TIL estimates and Foxp3+ TILs were markedly associated with increased overall survival (P = 0.001, P < 0.001, separately). Immune subtypes were associated with tumor size, gross type, depth of invasion, lymph node metastatic status, lymphovascular invasion, perineural invasion, and microsatellite instability status. EBV-positive (C1) and MSI (C2) gastric cancers, considered subtypes with better prognosis, were significantly associated with high TIL levels (P < 0.05). In contrast, epithelial-mesenchymal transition (EMT, C3) gastric cancers with poor overall survival displayed low levels of Foxp3+ TILs. Type II tumors (low level of TILs/low PD-L1 expression) displayed a significant correlation with poor overall survival (P = 0.004) and accounted for the highest proportion in the aberrant p53-expressing (C4) gastric cancers. The molecular subtype of gastric cancers is correlated with the immune subtype, including immune contexture and PD-L1 expression, within the tumor microenvironment.

Correlation between magnetic resonance imaging and the level of tumor-infiltrating lymphocytes in patients with estrogen receptor-negative HER2-positive breast cancer.

High levels of tumor-infiltrating lymphocytes (TILs) are associated with improved prognosis and response to therapy in patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer. This study investigated the associations between TIL levels and magnetic resonance imaging (MRI) findings in patients with estrogen receptor (ER)-negative HER-2 positive breast cancer. This study included 110 consecutive patients with surgically confirmed ER-negative HER2-positive breast cancers who underwent preoperative MRI from January to December 2015. Images of all lesions were reviewed in accordance with the BI-RADS lexicon by radiologists blinded to clinicopathologic findings. Tumor kinetic features were acquired by computer-aided diagnosis (CAD). Patients were divided into three TIL groups: low (<10%); intermediate (10-50%); and high (>50%). Associations between TIL levels and clinicopathologic and imaging features were evaluated; independent predictors of high and low TIL were identified by multiple logistic regression analysis. The 110 patients included 29 (26.4%) with low, 45 (40.9%) with intermediate, and 36 (32.7%) with high TIL levels. Multiple logistic regression analysis showed that older age (odds ratio [OR] = 1.08; P = 0.017), high peak enhancement (OR = 1.01; P = 0.019), positive CK5/6 (OR = 4.36; P = 0.024), and low Ki-67 (OR = 14.29; P = 0.037) were significantly associated with low TILs; low peak enhancement (OR = 1.01; P = 0.020) was significantly associated with high TILs. MRI features may predict TIL levels in patients with ER-negative HER-2 positive breast cancer, enhancing the ability to diagnose and treat these patients.

Immune biomarker expression in the tumor microenvironment in Chinese patients with esophageal squamous cell carcinoma was explored.

e15542 Background: Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies in China, of which the standard treatment of advanced esophageal cancer is chemotherapy followed by surgery. Recently, immunotherapy has shown great potential in ESCC treatment. The aim of this study was to explore the landscape of immune-related gene expression in the tumor microenvironment (TIME) and further explore the relationship between immune biomarkers and therapeutic effect. Methods: We retrospectively analyzed PDL1(clone sp142)/PD1 expression in a cohort of Chinese patients (male = 92, female = 18; median age = 64, range from 36 to 84) with ESCC. Patient samples were detected in Genecast. Co. from February 2017 to November 2018. Another 6 patients (male = 4, female = 2) who accepted anti-PD1/PD-L1 antibody treatment were analyzed. PD-L1/PD-1 expression and the incidence of CD8, CD57, CD68, CD163 TILs were immunohistochemically. Results: In tumor cells, more than 32% (36/110) samples of ESCC patients was stained with PDL1 (≥1%), while52.8% (21/36) of these patients highly expressed PDL1 (≥10%, 10%-98%). In tumor infiltrating lymphocytes (TILs), 31.82% (35/110) patients expressed PDL1 (≥1%) and 1.82% (2/110) highly expressed PDL1 (≥10%, 10%-50%). For PD1 expression in TILs, 17.27% (19/110) samples were positively stained with PD1 antibody (≥1%). In a subset of ESCC patients who received immune checkpoint blockade therapy (including 2 responders and 4 non-responders), the median percentage of CD163, CD68, PDL1, CD8, PD1 and CD57 were 1.49% (0.44%-9.98%), 0.40% (0.16%-19.08%), 3.74% (0%-45%), 9.52% (4.20%-13.79%), 4.51% (1.37%-33.43%) and 0.68% (0.08%-1.74%), respectively in tumor area and 4.14% (1.85%-15.99%), 1.62% (0.21%-11.47%), 4.87% (0.33%-18.97%), 7.74% (5.88%-14.35%), 3.93% (1.92%-20.54%) and 0.92% (0.27%-2.18%), respectively in stroma area. When compared with non-responders, samples from the responders showed higher CD8 expression in tumor area. Conclusions: Percentage of PDL1/PD1 expression is relatively high in Chinese patients with ESCC. High levels of CD8 positive TILs might be beneficial to patients with ESCC. Clinical investigations with large sample sizes are warranted to prove our findings.

Clinical relevance and significance of programmed death-ligand 1 expression, tumor-infiltrating lymphocytes, and p16 status in sinonasal squamous cell carcinoma.

Purpose: Immunotherapy may be a potential alternative for patients with sinonasal squamous cell carcinoma (SNSCC). Data regarding potential immunotherapy targets, such as programmed death-ligand 1 (PD-L1) and tumor-infiltrating lymphocytes (TILs), in SNSCC are limited. In this study, we assessed the prevalence and prognostic value of PD-L1 expression and TILs in p16-negative and p16-positive SNSCC.Patients and methods: Tissues from 96 patients with SNSCC were stained using immunohistochemistry against PD-L1, CD8, and Foxp3 to assess the immune environment. The correlations between PD-L1 expression, TILs, and p16 status were analyzed. Additionally, PD-L1, CD8, and Foxp3 expressions, as well as p16 status, were analyzed in relation to patient clinicopathological variables and prognosis.Results: Twenty-nine (30.2%) patients with SNSCC showed PD-L1 expression in >5% of tumor cells. PD-L1 expression was significantly correlated with poor differentiation and a high level of TILs. PD-L1 expression and the CD8+ and Foxp3+ T-cell infiltrates in p16-negative patients (n=78, 81.2%) and p16-positive patients (n=18, 18.8%) were not significantly different. PD-L1 expression and p16 status were not associated with overall survival (OS) and disease-free survival (DFS). Patients with high CD8+ or Foxp3+ cell infiltration had better clinical outcomes. A multivariate analysis confirmed that CD8 TILs were a significant independent and favorable prognostic factor for OS (p=0.023) and DFS (p=0.008).Conclusion: TILs can play a prognostic role in SNSCC. We did not find differences in immune marker expression between p16-positive and p16-negative SNSCC tissues. The high correlation between PD-L1 expression and TILs indicates that the PD-1/PD-L1 pathway is a promising immunotherapeutic target for SNSCC.

Expression of PD-L1 Attenuates the Positive Impacts of High-level Tumor-infiltrating Lymphocytes on Prognosis of Triple-negative Breast Cancer

ABSTRACT Background: Among all breast cancer subtypes, triple-negative breast cancer (TNBC) has aggressive clinical manifestations including more frequent relapses and metastases. The roles of PD-L1 expression and tumor-infiltrating lymphocytes (TILs) in TNBC clinicopathological behaviors and patients’ survival outcomes remain unclear. Methods: TNBC (108 cases) patients with at least 5-year follow-up were analyzed for PD-L1 expression and TILs by immunohistochemistry. We also analyzed the relationships between PD-L1 expression, TILs and clinicopathological characteristics. Furthermore, we explored the effect of PD-L1 expression and TILs on prognosis as illustrated by disease-free survival (DFS). Results: The expression of PD-L1 was related to more aggressive clinicopathological behaviors in TNBC patients including a larger tumor size, higher incidence of PL-1-ALN, more frequent distant metastasis, and a reduced disease-free survival. In contrast, patients with high-level TILs showed less aggressive disease progression hence a better prognosis compared to patients with low-level TILs. Among patients with high-level TILs, PD-L1 expression was correlated with adverse prognosis. Conclusions: Expression of PD-L1 and low-level TILs in TNBC patients were associated with adverse clinical outcomes. However, the positive impact of high-level TILs was attenuated by PD-L1 expression. Our results suggest potential biomarkers for a selection of indicated cases in the TNBC patients for anti-PD-L1/anti-PD1 immunotherapy.

Association between p53 Expression and Amount of Tumor-Infiltrating Lymphocytes in Triple-Negative Breast Cancer.

BackgroundMost triple-negative breast cancers (TNBCs) have a high histologic grade, are associated with high endoplasmic stress, and possess a high frequency of TP53 mutations. TP53 missense mutations lead to the production of mutant p53 protein and usually show high levels of p53 protein expression. Tumor-infiltrating lymphocytes (TILs) accumulate as part of the anti-tumor immune response and have a strong prognostic and predictive significance in TNBC. We aimed to elucidate the association between p53 expression and the amount of TILs in TNBC. MethodsIn 678 TNBC patients, we evaluated TIL levels and expression of endoplasmic stress molecules. Immunohistochemical examination of p53 protein expression was categorized into three groups: no, low, and high expression. ResultsNo, low, and high p53 expression was identified in 44.1% (n = 299), 20.1% (n = 136), and 35.8% (n = 243) of patients, respectively. Patients with high p53 expression showed high histologic grade (p < .001), high TIL levels (p = .009), and high expression of endoplasmic reticulum stress-associated molecules (p-eIF2a, p = .013; XBP1, p = .007), compared to patients with low p53 expression. There was no significant difference in disease-free (p = .406) or overall survival rates (p = .444) among the three p53 expression groups. ConclusionsHigh p53 expression is associated with increased expression of endoplasmic reticulum stress molecules and TIL influx.

Abstract P2-08-53: Tumor elasticity and clinicopathologic factors affecting neoadjuvant chemotherapy response in breast cancer patients

Abstract Background: Neoadjuvant chemotherapy for breast cancer has been increased. Many studies have reported on clinicopathologic factors to predict neoadjuvant chemotherapy response. Elastography, which is usually used to differentiate benign and malignant tumors, can be performed to evaluate tissue elasticity during conventional ultrasonography. The purpose of this study was to determine the clinicopathologic factors, including tumor elasticity, that affect neoadjuvant chemotherapy response in stage II or III breast cancer patients. Methods: From April 2014 to March 2017, 95 patients received neoadjuvant chemotherapy for clinical stage IIa-IIIc primary breast cancer. To evaluate tumor elasticity, strain elastography was performed in 74 patients before neoadjuvant chemotherapy. Patients were divided into two groups by the Tsukuba elasticity scoring system (soft group ≤3 vs. hard group ≥4). Histologic type, nuclear grade, tumor infiltrating lymphocytes (TILs), tumor cellularity, characteristics of stroma, and hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) status were evaluated using core needle biopsy specimens obtained before neoadjuvant chemotherapy. Pathologic complete response (pCR) was defined as the absence of invasive carcinoma in breast (ypT0 and ypTis) and axillary lymph node (ypN0). Residual cancer burden (RCB) was also calculated in 79 cases and the cases were categorized into 2 groups; favorable RCB group (RCB-0 and I) and unfavorable RCB group (RCB-II and III). Results: The mean age of patients was 46.43±8.62 years (range, 27-71 years) and the mean initial tumor size was 3.63±1.95cm (range, 2.1-12.8cm). Twenty-four patients (32.4%) were categorized into the soft group and 50 patients (67.6%) into the hard group. The mean tumor cellularity on core needle biopsy specimens and characteristics of stroma were not significantly different between the two groups (p=0.35 and p=0.79, respectively). Twenty-two patients achieved pCR (23.2%). The patients with pCR were more likely to have estrogen receptor (ER) or progesterone receptor (PR) negative breast cancer (p=0.04 and p=0.03). The rate of nuclear grade 3 was higher in patients with pCR than those without (p=0.03). Tumor elasticity was not correlated with pCR (p=0.28). Thirty patients (38.0%) achieved favorable RCB and forty-nine patients (62.0%) had unfavorable RCB. Not only the rates of ER negativity (p=0.05), PR negativity (p=0.03), nuclear grade 3 (p=0.01), and high TILs level (≥ 10%) (p=0.04) but also the mean TILs level (p=0.05) were significantly higher in the favorable RCB group compared withthe unfavorable RCB group. No significant difference in tumor elasticity was observed between the two groups (p=0.30). In univariate analyses, nuclear grade 3 (p=0.03), and high TILs level (≥10%) (p=0.04) were significantly correlated with favorable RCB. HR negativity was an independent predictor of favorable RCB in multivariate analysis (odds ratio, 2.93; 95% confidence interval, 1.04-8.28; p=0.04). Conclusion: Tumor elasticity was not associated with pCR or RCB. HR negativity was an independent predictor for favorable RCB.Nuclear grade and TILs were also potential predictive factors for neoadjuvant chemotherapy response. Citation Format: Park JY, Choi JE, Bae YK, Lee SJ. Tumor elasticity and clinicopathologic factors affecting neoadjuvant chemotherapy response in breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-53.

Abstract P2-08-46: Prognostic value of tumor-infiltrating lymphocytes (TILs) and clinical values for prediction of breast cancer recurrence in HER2 positive early breast cancer after surgery in King Chulalongkorn Memorial Hospital

Abstract Background:Tumor-infiltrating lymphocytes (TILs), one of immunological biomarkers have been investigated in breast cancer and other cancers. High levels of TILs or lymphocyte-predominant breast cancer subgroup (LPBC) were associated with better prognosis in HER2 positive and triple negative breast cancers from various studies. Recently, TILs is also the predictive biomarker for response to neoadjuvant chemotherapy. Clinical utility of TILs has been recommended in routine clinical practice for breast cancer patients. However, limitation of TILs in HER2 positive women in Thai ethnicity was reported. So, the aim of this study was to evaluate TILs in combination with clinical values as the prognostic value for predicting the recurrent breast cancer in Thai population. Methods: Four hundred and eighty-six patients with early stage HER-2 positive breast cancer who were diagnosed and treated at King Chulalongkorn Memorial Hospital from January 2005 to December 2016 were reviewed retrospectively. Clinico-pathological features, stromal TILs classified as low, intermediate and high, and survival outcomes were analyzed. Results: The median age was 52 years (26-85). Of the 486 HER2 positive women, 56% had postmenopause, 54.5% had T2 tumor, 47.7% had node negative, 66.8% had stage I-II, 44.6% had lymphovascular invasion, and 47.5% had positive hormonal receptor. For the primary treatment, 67.3% underwent modified radical mastectomy, 96.5% received neoadjuvant/adjuvant chemotherapy, 69.7% received adjuvant trastuzumab, 46.7% received adjuvant hormonal therapy. In 92 recurrent patients (18.9%), distant metastasis was identified in 67.4%. In 100 available tissues for evaluating stromal TILs, only 14 (14%) had high stromal TILs (at least 50%) and 46 patients (46%) had recurrent disease. Twenty three of 39 (59%)in low stromal TILs group had disease recurrence while only 4 out of 14 (28.6%) in high stromal TILs had recurrent disease. Median percentage of stromal TILs in recurrent and non-recurrent group was 17.5% and 27.5%, respectively. From multivariate analysis, high stromal TILs (HR 0.52 [95%CI 0.32-0.85]; p = 0.01) and trastuzumab used (HR 0.39 [95%CI 0.25-0.61]; p &amp;lt;0.001) were associated with decreased risk of recurrences. After median follow up of 4.1 years, 5-years disease free survival (DFS) and 5-years overall survival (OS) were 80.9%, 86.8%, 69% and 92.6%, 91.5%, 94.5% in overall populations, trastuzumab and non-trastuzumab used group, respectively. Additionally, 5-year DFS and 5-year OS were 34.3%, 60.8%, 75.7% and 86.2%, 82.5%, 83.1% in low, intermediate and high stromal TILs subgroup, respectively. Conclusion: High stromal TILs and trastuzumab used were statistically significant prognostic values for predicting disease recurrence in HER-2 positive early breast cancer. Stromal TILs together with clinical values established clinical utility in Thai HER2 positive breast cancer women. Level of Stromal TILs in Overall PatientsStromal TILsTotal (n = 100)No recurrence (n = 54)Recurrence (n = 46)P valueLow (0-15%)39(39%)16(41%)23(59%)0.03Intermediate (20-40%)47(47%)28(59.6%)19(40.4%) LPBC (50-90%)14(14%)10(71.4%)4(28.6%) Citation Format: Prapatsornvichit S, Atikankul T, Sriuranpong V, Poovorawan N, Parinyanitikul N. Prognostic value of tumor-infiltrating lymphocytes (TILs) and clinical values for prediction of breast cancer recurrence in HER2 positive early breast cancer after surgery in King Chulalongkorn Memorial Hospital [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-46.

Abstract PD3-08: Assessment of the tumor immune environment in inflammatory breast cancer treated with neoadjuvant dual-HER2 blockade

Abstract Background: Inflammatory breast cancer (IBC) is an aggressive form of breast cancer that remains relatively understudied. We examined the efficacy of neoadjuvant dual-HER2 blockade (trastuzumab (H) and pertuzumab (P)) combined with paclitaxel (T) in HER2+ IBC, including a planned analysis to elucidate associations between the tumor immune microenvironment profile and response to therapy. Methods: An IRB-approved, single-arm phase II trial for patients (pts) with newly diagnosed HER2+ IBC was conducted. Pts had a pre-treatment biopsy of the affected breast (D1) followed by a loading dose of HP. A second biopsy was performed 1 week (wk) later (D8), when T (80mg/m2/wk x 16 wks) was added to HP. Responding pts underwent modified radical mastectomy (MRM) where residual disease was collected. The primary objective was to determine the rate of pathologic complete response (pCR) defined as ypT0/isN0. Residual Cancer Burden (RCB) was also determined. Tumor specimens from D1, D8 and MRM were assessed for disease cellularity and scored for percentage of tumor infiltrating lymphocytes (TILs): low=0-10%, intermediate=11-59%, high&amp;gt;60%. RNA-sequencing was performed on tumor tissue from D1 and D8 to explore the impact of short-term HP treatment on the tumor transcriptomic profile and to identify potential predictors of pCR. Results: 23 pts with HER2+ IBC were enrolled between 8/2013-6/2017. Mean age was 48 years (range 32-74); 11 pts (48%) had estrogen and progesterone receptor (ER/PR) negative disease. Matched tumor biopsies (D1, D8) were obtained in all 23 pts; 21 underwent MRM; 1 was lost to follow-up and 1 had disease progression. In the intent to treat analysis, 10/23 (43%) pts achieved a pCR and 7 (30%) had RCB-1. Ten of the 22 evaluable pts achieved a pCR (45.5%). TILs were evaluable in 20/23 (87%) matched tumor biopsies (D1, D8). Among the D1 biopsy specimens: 19 (95%) had low levels, 2 (10%) had intermediate levels, and none had high levels. When D1 TIL levels were compared with D8 levels, 3(15%) had an increase in TILs, 16(80%) had no change in TIL levels, and 1(5%) had a decrease in the level of TILs. Both samples with intermediate levels and 2 of 3 samples with high levels of TILs on D1 and D8 were seen in ER/PR negative disease. An evaluation of biopsy specimens associated with subsequent pCR using GO enrichment analysis from the RNA-Seq data showed significant upregulation of several immune-process related gene expression signatures both at D1 and D8 (e.g. antigen processing and presentation, TCR signaling, NK cell cytotoxicity, p-value: 2.99E-48 to 1.39E-16) when compared with those associated with residual disease at the time of MRM. Across the entire cohort, D8 biopsies showed evidence of upregulated anti-tumor immunity compared to D1 biopsies (p-value: 9.57E-06 to 0.012). Notably, this change from D1 to D8 was largely restricted to tumors that achieved a pCR. Conclusion: THP for 16 weeks was a highly effective treatment for HER2+ IBC. Immune activation as determined by gene expression signatures predicted pCR, and moreover upregulation of anti-tumor immunity after 1 wk of HP might further predict a complete pathologic response to therapy. ClinicalTrials.gov identifier: NCT01796197 Citation Format: Pernas S, Goel S, Harrison BT, Hu J, Johnson N, Regan M, Chichester LA, Nakhlis F, Schlosnagle EJ, Winship G, Guerriero JL, Parsons H, Mittendorf EA, Overmoyer B. Assessment of the tumor immune environment in inflammatory breast cancer treated with neoadjuvant dual-HER2 blockade [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD3-08.

Abstract P3-11-01: Interaction between molecular subtype and stromal immune infiltration dynamics in breast cancer patients treated with neoadjuvant chemotherapy

Abstract Purpose: High levels of tumor-infiltrating lymphocytes (TILs) before neoadjuvant chemotherapy (NAC) are associated with higher pathological complete response (pCR) rates, and better survival in TNBC and HER2-positive breast cancers (BCs). We investigated the value of changes in TIL levels and final TIL levels after treatment, by evaluating lymphocyte infiltration before and after NAC in a real-life BC cohort. Patients and methods: We assessed stromal TIL levels in 716 pre- and post-treatment matched paired specimens, according to the guidelines of the international TIL working group. Results: Pre-NAC TIL levels were higher in tumors for which pCR was achieved than in cases of residual disease (33.9% versus 20.3%, p=0.001), in luminal tumors and TNBCs, but not in HER2-positive BCs, (pInteraction =0.001). The association between pre-NAC TIL levels and pCR was non-linear in TNBCs (p=0.005). Mean TIL levels decreased during NAC (pre-NAC TILs: 24.1% versus post-NAC TILs: 13.0%, p&amp;lt;0.001). This decrease was strongly associated with high pCR rates, and TIL level variation was strongly inversely correlated with pre-NAC TIL levels (r=-0.80, p&amp;lt;0.001). Pre-NAC TILs and disease-free survival (DFS) were associated in a non-linear manner (p&amp;lt;0.001). High post-NAC TIL levels were associated with aggressive tumor characteristics and with impaired DFS in HER2-positive BCs (HR=1.04, CI [1.02-1.06], p=0.001), but not in luminal tumors or TNBCs (pInteraction =0.04). Conclusion: The associations of pre, post-NAC TIL levels with response to treatment and DFS differ between BC subtypes and may deviate from linearity. The characterization of immune subpopulations may improve our understanding of the complex interactions between pre- or post-NAC setting, BC subtype, response to treatment and prognosis. Citation Format: Hamy A-S, Bonsang-Kitzis H, De Croze D, Laas E, Darrigues L, Topciu L, Menet E, Vincent-Salomon A, Lerebours F, Pierga J-Y, Brain E, Feron J-G, Benchimol G, Lam G-T, Laé M, Reyal F. Interaction between molecular subtype and stromal immune infiltration dynamics in breast cancer patients treated with neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-11-01.

Abstract PD5-10: Immune characterization of matched primary and multiple metastatic samples issued from an institutional autopsy cohort

Abstract Introduction While immune infiltrates have already been extensively characterized in primary tumors (P), data on breast cancer metastases (M) remains limited. To this end we quantified and qualified the immune cells in a unique cohort of multiple matched P and M samples selected from an institutional breast cancer autopsy cohort. Patients and methods Twenty-three patients were selected from an institutional autopsy program (Semmelweis University, Budapest, Hungary) based on matched P and M sample availability (124 samples). All samples were centrally characterized for estrogen (ER), progesterone (PR) and HER2 receptors. The primary molecular subtypes were as follows: 9 ER+/PR+/HER2-, 8 triple negative and 6 HER2+. Ten patients relapsed ≤1 year after diagnosis and were further referred to as “early relapsers”, as opposed to the remaining qualified as “late relapsers”. Immunohistochemistry (IHC) was carried out against CD3/CD20 and CD4/CD8 in 21 patients (119 samples). Tumor infiltrating lymphocytes (TILs) were assessed on hematoxylin and eosin (H&amp;E) and CD3-stained slides. Gene expression data were generated using the NanoString nCounter assay (PanCancer Immune Profiling Panel) for 11 patients (35 samples) and analyzedusing the R package NanoStringQCPro. The scores from published immune gene signatures were calculated as a weighted sum of the expressions of their genes. All samples were analyzed for 22 immune cell subtypes relative abundance using CIBERSORT. Results TILs assessed on H&amp;E and CD3-stained slides were weakly correlated (Rho= 0.38, p&amp;lt;.001). TIL levels as well as the number of tertiary lymphoid structures (TLS) were significantly lower in Ms as compared to Ps (p&amp;lt;.001). Among the different metastatic sites, the lung was more infiltrated when considering CD3+ and CD4+ cells (p=.01 and .02, respectively). We further observed significantly higher levels of TILs, CD3+, CD4+ and CD8+ cells in the Ms but not in the Ps from late relapsers as opposed to those from early relapsers. Gene expression analyses further confirmed these observations as several immune gene signatures displayed significantly higher scores in the Ms from late compared to early relapsers. An unsupervised analysis identified 13 genes significantly differentially expressed between Ps and Ms: CSF1R, CXCL14, CYBB, IL21R, IL2RB, TNF and TNFSF15 were upregulated in Ps while BCL2L1, C7, HSD11B1, and PSMB7 were upregulated in Ms. The matched P/M CIBERSORT analyses revealed a distinct composition of immune cell types between P and M of a same patient. Apart from a potential increase in M0 macrophages, no common trait was observed in immune cell composition between the Ms from the different patients. Conclusion This is to the best of our knowledge the first study characterizing the immune infiltration in patients with multiple matched P and M samples. The results suggest that Ms have not only a globally lower immune infiltration as compared to Ps, but also a different immune composition. Additionally, Ms from late relapsers are more infiltrated as compared to early relapsers. The present data also uncovers not only important inter-patient but also intra-patient immune heterogeneity, which should be taken into consideration for optimal treatment decision. Citation Format: Szekely B, Bareche Y, Van den Eynden G, Salgado R, Buisseret L, Garaud S, Willard-Gallo K, Hatzis C, Szasz M, Kulka J, Larsimont D, Sotiriou C, Pusztai L, Desmedt C. Immune characterization of matched primary and multiple metastatic samples issued from an institutional autopsy cohort [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD5-10.

Scoring System for Tumor-Infiltrating Lymphocytes and Its Prognostic Value for Gastric Cancer.

The tumor microenvironment (TME) is the internal environment of malignant tumor progression, and the host antitumor immune response and normal tissue destruction occur in the TME. Tumor-infiltrating lymphocytes (TIL) is a crucial component of the TME and reflect the host antitumor immune response. The purpose of this study was to discuss the methodology for TIL evaluation and assess the prognostic value of TIL in gastric cancer. In total, we reviewed 1,033 gastrectomy cases between 2002 and 2008 at the Third Affiliated Hospital of Soochow University. To understand the prognostic value of TIL in gastric cancer (GC), TIL were assessed by optical microscopy, and verified by immunohistochemistry. There is no current consensus on TIL scoring in GC. In this study, we discussed a TIL evaluation system that includes an analysis of the amount and percentage of TIL in a tumor. Ultimately, 439 (52.7%) cases showed high levels of TIL and 394 (47.3%) cases had low levels. There was a statistically significant relationship among TIL, tumor size, histological grade, LN metastasis, nerve invasion, tumor thrombus, pTN stage, and WHO subtypes (p < 0.001, respectively). TILhi was a positive significant predictor of overall survival (OS) in Kaplan–Meier survival analysis (P < 0.001) and multivariate Cox regression analysis (HR = 0.431, 95% CI: 0.347–0.534, P < 0.001). After surgery, patients with malignant tumors underwent chemoradiotherapy according to standard therapeutic guidelines based on TNM stage. The TNM scoring system cannot reflect the full information of TME; therefore, TIL can be used as a diagnostic supplement. We constructed a nomogram model that showed more predictive accuracy for OS than pTN stage. In summary, this study proves that high levels of TIL are associated with a positive prognosis and that TIL reflect the protective host antitumor immune response.

PD-L1 and CD8 are associated with deficient mismatch repair status in triple-negative and HER2-positive breast cancers

Triple-negative and HER2-positive breast cancers (BCs) are more aggressive than hormone receptor-positive/HER2-negative BCs and show higher levels of tumor-infiltrating lymphocytes (TILs) and PD-L1 expression. Recently, US Food and Drug Administration approved anti-PD-L1 immunotherapy for solid tumors with deficient mismatch repair (MMR). In this study, we aimed to examine the prevalence of deficient MMR and its association with checkpoint immune markers in BCs. Immunohistochemistries (IHCs) with anti-MMR proteins (MLH1, PMS2, MSH2 and MSH6) and multiplex IHCs with anti-PD-L1, anti-CD8 or anti-CD163 were performed on tissue microarrays (TMAs) with 101 triple-negative BCs (TNBC) and 197 HER2-positive BCs. Additional IHCs for MMR proteins were also performed on whole-tissue sections from selected cases. Thirteen cases (4.4%) showed complete loss of MMR protein on TMAs, including 7 TNBCs (6.9%) and 6 HER2-positive BCs. On whole-tissue sections, only one of 13 cases showed complete loss of MMR proteins, while the other 12 cases showed partial loss. PD-L1 expression was identified in 37% of cases and was significantly higher in TNBCs than in HER2-positive BCs (71% versus 19%). Furthermore, BCs with complete/partial loss of MMR demonstrated significantly more PD-L1 and CD8 expressions than BCs with preserved MMR proteins. Although complete loss of MMR proteins exists in an extremely low frequency, partial loss is not uncommon in BCs. The association of partial loss of MMR proteins with increased PD-L1 and CD8 expression suggests a potential use of MMR testing as a screening method for anti-PD-L1 immunotherapy in BCs.

A nomogram to predict pathologic complete response (pCR) and the value of tumor-infiltrating lymphocytes (TILs) for prediction of response to neoadjuvant chemotherapy (NAC) in breast cancer patients.

The value of tumor-infiltrating lymphocytes (TILs) for prediction of pathologic complete response (pCR) in breast cancer (BC) patients treated with neoadjuvant chemotherapy (NAC) has received increasing attention. In human epidermal growth factor receptor 2 (HER2)-positive BC, advances in HER2-targeted therapy have not yet clarified the clinical implications of pre-NAC TILs. Likewise, the prognostic role of TILs for long-term survival is not well established. Pre- and post-NAC TIL levels were evaluated in 248 pair-matched pre-NAC biopsy and post-NAC resection samples, and analyzed for predictive and prognostic significance with other clinicopathologic parameters. Additional 60 pre-NAC biopsy samples of HER2-positive BC treated with a TCHP regimen (docetaxel, carboplatin, and a combination of trastuzumab and pertuzumab) were also assessed. High pre-NAC TILs, clinical nodal stage 0-1 (cN0-1), and negative ER expression were shown to be strong predictive markers for pCR. A nomogram based on these significant clinicopathologic predictors was developed, providing integrated probability of achieving pCR after NAC. The association between high pre-NAC TIL levels and significantly increased pCR rate was also confirmed in HER2-positive BC patients treated with a TCHP regimen. After chemotherapy, increased quantity of post-NAC TILs was shown to have extended BC-specific survival and disease-free survival in univariable and multivariable analyses. High pre-NAC TIL levels were significantly predictive of pCR in BC, and can act as a surrogate marker for predicting therapeutic effects of a TCHP regimen for HER2-positive BC. Post-NAC TILs in residual disease were a new prognostic marker of risk stratification for long-term survival.

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

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