Assessment Of Tumor-infiltrating Lymphocytes Research Articles

Abstract P5-02-04: Upfront dichotomous histopathological assessment of ductal carcinoma in situ of the breast to reduce inter-observer variability: The DCISion study

Abstract Background. Ductal carcinoma in situ (DCIS) of the breast is considered to be a non-obligatory precursor of invasive breast cancer. Its histopathological assessment is characterized by considerable inter-observer discordance. In a previous study, we performed post hoc dichotomization of multi-categorical variables to determine the ‘ideal’ cut-offs for dichotomous histopathological assessment. In the present multicenter study, inter-observer variability is evaluated among 39 pathologists who performed upfront dichotomous evaluation of a consecutive series of 149 DCIS. Methods. All participants were board-certified pathologists with a special interest in breast disease. The participants assessed at least 50 primary oncologic breast cancer resection specimens per year, in accordance with the EUSOMA criteria for dedicated breast pathologists. No training set was used. Instead, a written guideline with associated DCISion poster was provided, which contained all definitions of the histopathological features of interest. Representative digital slides of 149 DCIS were accessible via an online platform. All pathologists independently assessed the following histopathological features: nuclear atypia, necrosis, solid DCIS architecture, calcifications, stromal architecture and lobular cancerization. Stromal inflammation was assessed semi-quantitatively. Stromal tumor-infiltrating lymphocytes (TILs) were quantified as percentages and were also dichotomously assessed with a cut-off at 50%. Krippendorff’s alpha (KA), Cohen’s kappa (K) and intraclass correlation coefficient (ICC) were calculated for the appropriate variables. Results. Intraductal calcifications (KA 0,676) and solid DCIS architecture (KA 0,602) were characterized by the highest inter-observer concordance. Stromal inflammation (KA 0,564), dichotomously assessed TILs (KA 0,520) and comedonecrosis (KA 0,539) showed slightly higher inter-observer agreement. Lobular cancerization (KA 0,396), nuclear atypia (KA 0,422) and stromal architecture (KA 0,450) showed the lowest inter-observer concordance. Assessment of TILs as a percentage showed good overall agreement with a mean ICC of 0,821 (range 0,566 - 0,933). Semi-quantitative assessment of stromal inflammation (KA 0,564) resulted in somewhat lower inter-observer variability than upfront dichotomous TILs assessment (KA 0,520). High stromal inflammation corresponded best with dichotomously assessed TILs when the TILs cut-off was set at 10% (K 0,881). Nevertheless, a post hoc TILs cut-off set at 20% resulted in the highest inter-observer agreement (KA 0,669). Experience and time dedicated to breast pathology did not influence the degree of concordance. Conclusion. The DCSion study shows that, despite upfront dichotomous evaluation, the inter-observer variability remains considerable and is at most acceptable. Nevertheless, the discordance rate varies among the different histopathological features. Future studies should investigate its impact on DCIS risk stratification. Differences in prognostic value among the different methods to quantify TILs are of particular interest, since inter-observer variability may partly explain different outcomes among different studies. Artificial intelligence might be able to tackle this diagnostic challenge. Development of deep learning algorithms could result in more objective histopathological assessment. Although machine learning might represent the next “pathologist’s best friend”, we should be careful not to introduce inter-observer variability into these deep learning algorithms. The DCISion study therefore provides an excellent setting to investigate the value of such algorithms in rendering the final diagnosis more robust. Citation Format: Mieke Rosalie Van Bockstal, Hélène Dano, Serdar Altinay, Laurent Arnould, Noella Bletard, Cecile Colpaert, Franceska Dedeurwaerdere, Benjamin Dessauvagie, Valérie Duwel, Giuseppe Floris, Stephen Fox, Clara Gerosa, Shabnam Jaffer, Eline Kurpershoek, Magali Lacroix-Triki, Andoni Laka, Kathleen Lambein, Gaëtan Marie MacGrogan, Caterina Marchió, Dolores Martin Martinez, Sharon Nofech-Mozes, Dieter Peeters, Alberto Ravarino, Emily Reisenbichler, Erika Resetkova, Souzan Sanati, Anne-Marie Schelfhout, Vera Schelfhout, Abeer M Shaaban, Renata Sinke, Claudia Maria Stanciu-Pop, Claudia Stobbe, Carolien HM van Deurzen, Koen Van de Vijver, Anne-Sophie Van Rompuy, Stephanie Verschuere, Anne Vincent-Salomon, Hannah Wen, Caroline Bouzin, Christine Galant. Upfront dichotomous histopathological assessment of ductal carcinoma in situ of the breast to reduce inter-observer variability: The DCISion study [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 P5-02-04.

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 P6-06-13: Racial/ethnic differences in tumor infiltrating lymphocytes

Abstract Background/Rationale: There have been increasing studies regarding the morphologic evaluation of tumor-infiltrating lymphocytes (TILs) and the tumor microenvironment. In breast cancer, TILs are more abundant in stroma (sTILs) compared to intratumoral (iTILs) areas and iTILs have been an unreliable marker due to difficulty in standardizing their evaluation. The International Immuno-Oncology Working Group published recommendations to guide the accurate assessment of TILs. TILs have relevance in the setting of neoadjuvant therapy (NAT), primarily in triple negative and HER2 positive breast cancers. Studies report that sTIL infiltration is predictive of response to NAT in these two breast cancer subtypes, but not in the estrogen positive breast cancer subtype. The presence of TILs is a good prognostic indicator and correlates with axillary lymph node negativity, lower histological grade and improved recurrence-free survival. Therefore, evaluating TILs prior to NAT can provide important predictive and prognostic information for clinicians. There are insufficient data exploring differences in sTILs among different racial/ethnic groups. Previous data showed Asians had more sTILs compared to White and African American patients, consistent with data from Asia that found substantial sTILs in their respective patient populations. Our diverse population has known racial/ethnic disparities, where Pacific Islanders have a higher mortality compared to Asian and White patients. Evaluating sTILs within this population may reveal tumor biology differences that could contribute to health disparities. Research objectives: To determine the stromal tumor infiltrating lymphocytes on core biopsy specimens and correlate them with clinical characteristics: ethnicity, age, BMI and stage To determine differences in pathologic complete response (pCR) with ethnicity, age, BMI, neoadjuvant therapy and stage Table 1. % Stromal tumor infiltrating lymphocytes (sTILs) compared to age, BMI, ethnicity, stage and pCR adjusted for breast cancer subtypeVariableValueNMeanSDPAge30838.615.140927.123.0.30501834.626.6.67601431.121.3.4570+826.822.8.30BMI< 252825.918.825-301638.521.9.07> 301337.228.0.12EthnicityAsian1636.724.2.01Pacific Islander2038.021.3.004White1818.515.9Other348.130.6.02Stage1A818.48.01B933.425.5.192A1631.923.0.172B938.620.6.113A630.823.2.313B937.225.8.10pCRno3233.019.8yes2530.825.6.72 Table 2. Pathologic complete tesponse (pCR) compared to age, BMI, neoadjuvant therapy, ethnicity and stage adjusted for breast cancer subtypeVariableValueNMeanSDPAge3080.5500.5354090.4060.527.5650180.4450.511.6360140.3030.469.2770+80.5860.518.89BMI< 25280.5260.50825-30160.2730.447.11> 30130.4530.519.65Neoadjuvant TherapyACT140.2680.469.88TC110.2370.505TCHP160.7480.479.02THP120.4280.515.41Letrozole40.3850.500.62EthnicityAsian160.4720.512.77Pacific Islander200.3890.503.85White180.4200.511other30.7060.577.36Stage1A80.2410.3541B90.4270.527.412A160.7240.479.022B90.0320.441.403A60.8220.516.023B90.2680.500.90 Results: We evaluated 57 neoadjuvant breast cancer cases for sTILs according to the International Immuno-Oncology Working Group recommendations and reviewed their clinical characteristics. We found significant % sTILs differences in ethnicity, specifically Asian (35%), Pacific Islander (38.5%) and Other (46%) category compared to White (19%) Table 1. There were no differences found in sTILs according to age, BMI or stage. We also found no significant pCR differences according to age, BMI or ethnicity, however for certain stages and neoadjuvant therapy, there is an increased rate of pCR, although these are a small number of cases, no conclusions can be made, Table 2. Future studies to evaluate the significance of sTILs in different ethnicities may be informative to delineate possible mechanisms contributing to known disparities they experience. Citation Format: Jami Fukui, Alana Taniguchi, Madison Meister, Ian Pagano, Jeffrey Killeen. Racial/ethnic differences in tumor infiltrating lymphocytes [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 P6-06-13.

Prediction of Response to Neoadjuvant Chemotherapy in Egyptian Patients with Locally Advanced Breast Cancer: The Evolving Role of Tumour Infiltrating Lymphocytes (TILs)

Background & objectives: Presence of TILs in breast cancer indicates better therapeutic responses to neoadjuvant chemotherapy (NAC), increased pCR and improved outcome. We aim at evaluation of TILs in breast cancer biopsies in correlation with pathological response after NAC in locally advanced breast cancer Methods. This study was conducted at Ain Shams university hospital and Maadi Military hospital in Egypt. 45 Female patients with locally advanced breast cancer were treated with NAC; pathological response was assessed. Tumours were categorized into: luminal A, luminal B, Her2 enriched and TNBC. Assessment of TILs (CD4 and CD8 lymphocytes) was based on Immuno-Oncology Biomarker Working Group guidelines. Results: Tumours were classified into high and low TIL groups using the interquartile range cutoff (29%). High CD4 group showed increased pCR (p = 0.003) and smaller residual tumours (p = 0.04). High CD8 group showed a significant association with smaller residual tumours (p = 0.003). At follow-up of 24-months, CD4 and CD8 high groups showed significantly higher 2-years DFS. The difference between CD4 high and low groups was significant in regards to estrogen receptor status, showing higher levels in hormone-negative tumors (p = 0.029). Patients with Her2 subtype showed higher CD4 (p = 0.007) and CD8 expression (p = 0.018). In CD4 & CD8 low groups, more patients developed local recurrence and distant metastasis (p = 0.025). Conclusion: We concluded that TILs may predict response to NAC and overall prognosis of breast cancer. The evaluation of TILs in correlation with morphological or genomics-based parameters helps to stratify patients of breast cancer.

Quantitative assessment of tumor-infiltrating lymphocytes in mismatch repair proficient colon cancer

ABSTRACT Tumor infiltrating lymphocytes (TIL), which represent host adaptive response to the tumor, were first identified at scanning magnification to select areas with the highest counts on hematoxylin and eosin slides, quantitated per high-power field (HPF), and analyzed for association with recurrence-free survival (RFS) in 848 patients. Highest TIL in a single HPF was analyzed as a continuous and categorical variable, and optimal cutoff analysis was performed to predict RFS. Highest TIL count in a single HPF ranged from 0 to 45, and the optimal cutoff for TIL high vs TIL low was determined to be ≥ 3 vs < 3 with a concordance probability estimate of 0.74. In the entire cohort, 5-year RFS was 90.2% (95% CI = 83.7–94.2) in TIL high compared to 78.9% (95% CI = 74.1–82.9) in TIL low (log rank P < .0001). TIL remained significant in the mismatch repair-proficient (pMMR) cohort where 5-year RFS was 94.6% (95% CI = 88.3–97.5) in TIL high compared to 77.9% (95% CI = 69.2–84.4) in TIL low (P = .008). On multivariable analysis, TIL and AJCC Stage were independently associated with RFS in the pMMR cohort. Qualitatively in the pMMR cohort, RFS in Stage II TIL high patients was similar to that in Stage I patients and RFS in Stage III TIL high was similar to that in Stage II TIL low patients. Assessment of TIL in a single HPF using standard H&E slides provides important prognostic information independent of MMR status and AJCC stage.

Usefulness of imaging findings in predicting tumor-infiltrating lymphocytes in patients with breast cancer.

Tumor-infiltrating lymphocytes (TILs) have been determined as a new prognostic indicator of immunotherapy response in breast cancer (BC). The aim of this study is to investigate the effectiveness of imaging features in predicting the TIL levels in invasive BC patients. A total of 158 patients with invasive BC were included in our study. All lesions were evaluated based on the BIRADS lexicon. US was performed for all the patients and 89 of them underwent MRI. The histologic stromal TIL (sTIL) levels were assessed and associations between the sTIL levels and imaging features were evaluated. Tumors with high sTIL levels had more circumscribed margins, round shape, heterogeneous echogenicity, and larger size on ultrasonography (p < 0.005). There was a statistically significant positive correlation between the sTIL levels and ADC value (p < 0.001). Tumors with high sTIL levels had significantly more homogeneous enhancement than the tumors with low sTIL levels (p = 0.001). Logistic regression analysis showed that the ADC was the most statistically significant parameter in predicting the sTIL levels (the odds ratio was 90.952; p = 0.002). The optimal cutoff value for ADC in predicting low and high sTIL levels was found to be 0.87 × 10-3 mm2 s-1 (AUC = 0.726, 73% specificity, and 60% sensitivity). Imaging findings, especially the ADC, may play an important role as an adjunct tool in cases of uncertain situations and may improve the accuracy of biopsy results. The prediction of sTIL levels using imaging findings may give an opportunity to predict prognosis. • Preoperative assessment of TILs is an important biomarker of prognosis and treatment efficacy. • ADC value can be a useful tool in distinguishing high and low sTIL levels as a non-invasive method. • The prediction of sTIL levels using imaging findings may give an opportunity to predict prognosis and an optimal treatment for the BC patients.

Prognostic significance of tumour-infiltrating lymphocytes on survival outcomes of patients with resected pancreatic ductal adenocarcinoma: A systematic review and meta-analysis

Abstract Background Pancreatic cancer remains a leading cause of cancer-related mortality worldwide. Tumor-infiltrating lymphocytes (TILs) play an important role in mediating tumor progression and treatment resistance in pancreatic cancer. However, the prognostic value of TILs in pancreatic cancer remains uncertain. This meta-analysis evaluated the prognostic significance of TIL subsets (CD3+, CD4+, CD8+, FoxP3+ T cells) on overall survival (OS) and disease-free survival (DFS) of patients with resected pancreatic ductal adenocarcinoma. Methods Pertinent studies were gathered through systematic search of PubMed, Google Scholar and Cochrane Library databases up to August 1, 2019. Using Review Manager version 5.3, pooled hazard ratios and 95% confidence intervals were calculated using random or fixed effects models, depending on the heterogeneity of studies. Results A total of 11 studies comprising 1760 patients were included. Pooled analysis revealed that high levels of CD8+ TILs were associated with improved OS (HR = 0.59, 95% CI = 0.51-0.69, p Conclusion TILs are a promising prognostic biomarker in pancreatic cancer. High levels of CD8+ TILs correlated with favorable OS and DFS, while high levels of FoxP3+ TILs associated with poor OS. Nonetheless, results of this meta-analysis should be approached with caution due to the lack of established standards in assessment of TILs and the small number of available studies. Prospective studies that assess TILs in a more comprehensive and standardized manner are needed. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Overall assessment of tumour-infiltrating lymphocytes in early-stage nasopharyngeal carcinoma

Abstract Background Nasopharyngeal carcinoma (NPC) has distinct histopathology and associated with high mortality rate. Identification of NPC cases that have aggressive behavior at an early-stage can aid in improvement of the survival. Assessment of tumor-infiltrating lymphocytes (TILs) has shown a promising prognostic value in many epithelial cancers. Methods This study included a multicenter cohort of 44 cases treated for early-stage (I-II) NPC at the five Finnish university hospitals (Helsinki, Turku, Tampere, Kuopio and Oulu). Hematoxylin and eosin-stained cancer sections were used to evaluate TILs. We followed the guidelines that recently introduced by International Immuno-Oncology Biomarkers Working Group for assessment of TILs in different tumors including head and neck cancer. Results The score of intra-tumoral TILs showed a promising prognostic value for predicting survival in early NPC. A hazard ratio of 2.45 and 95% confidence interval of 1.07 to 5.62 (P = 0.03) was reported, indicates that tumors with low TILs have a higher risk of mortality. Conclusion In early-stage of NPC, assessment of TILs has a significant prognostic value that can be useful to identify cases with aggressive behavior. Further studies are necessary to validate the importance of TILs in larger cohorts of early NPC. Legal entity responsible for the study Alhadi Almangush. Funding Has not received any funding. Disclosure The author has declared no conflicts of interest.

Abstract LB-B06: A novel ex vivo 3D tumor organoid model of fresh patient tumors (3D-ACT) to assess efficacy of cellular therapy in immuno-oncology

Abstract Introduction: The spatial organization and dynamic interplay of the complex cell-to-cell interactions in patient tumors play an important role in cellular phenotypes that can result in permanent alterations in cellular functions and response to immuno-oncology (IO) treatments. Therefore, to assess the therapeutic efficacy of IO treatments, including cellular therapeutics, it is imperative to develop models that preserve the stromal-stoichiometry of the tumor microenvironment. Here we describe a patient-derived high content confocal imaging approach using an unpropagated tumor organoid model with intact tumor stroma for quantitative assessment of infiltration and target tumor cell killing activity of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) in non-small cell lung cancer. Materials and Methods: All human tumor samples were obtained with patient consent and relevant IRB approval. For the ex vivo assays 3D tumoroids measuring 150 micron in size were prepared and cryopreserved during the process of ex vivo propagation of autologous TILs. Ex vivo propagated TILs were characterized using multiparameter flow analysis, fluorescently labeled and exposed to fresh tumor organoids. Computational confocal image analysis was used to quantify TILs infiltration into the tumor organoids and target tumor cell killing. Multiplex cytokine assays and flow cytometry analysis were performed to assess activation of TILs upon exposure to tumor organoids. Results: We successfully prepared matched autologous TILs and unpropagated 3D tumor organoids from 75% of NSCLC patient tumors. The characteristics of tumor immune microenvironment and tumor cell viability was evaluated in previously cryopreserved organoids using a custom image analysis algorithm that was developed for the collection of data in a structurally relevant environment on quantification of marker-specific cell number, cell viability and apoptosis in addition to structural and functional analysis of cells in intact 3D tumor organoids. High content confocal imaging analysis demonstrated that CD3/CD28 pre-activated TILs with increased activation phenotypes and enhanced pro-inflammatory cytokine release had marked infiltration into the 3D organoids compared to untreated TILs and PBMCs. The data was correlated with quantitative tumor cell killing assessment for each tumor organoid. Conclusion: These results demonstrate that our 3D-ACT model using ex vivo expanded TILs and 3D tumor organoids is an effective tool for the therapeutic assessment of autologous TILs and indicate that it can also be used to assess efficacy of other cellular therapy applications in Immuno-oncology. Furthermore, implementation of this platform in the clinical studies may also allow determining the most effective combinatorial cellular therapy strategies for individual patients. Citation Format: Vijayendra Agrawal, Mibel Pabon, Melba Marie Page, Jenny Kreahling, Soner Altiok. A novel ex vivo 3D tumor organoid model of fresh patient tumors (3D-ACT) to assess efficacy of cellular therapy in immuno-oncology [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-B06. doi:10.1158/1535-7163.TARG-19-LB-B06

An open source automated tumor infiltrating lymphocyte algorithm for prognosis in melanoma

Assessment of tumor infiltrating lymphocytes (TILs) as a prognostic variable in melanoma has not seen broad adoption due to lack of standardization. Automation could represent a solution. Here, using open source software, we build an algorithm for image-based automated assessment of TILs on hematoxylin-eosin stained sections in melanoma. Using a retrospective collection of 641 melanoma patients comprising four independent cohorts; one training set (N = 227) and three validation cohorts (N = 137, N = 201, N = 76) from 2 institutions, we show that the automated TIL scoring algorithm separates patients into favorable and poor prognosis cohorts, where higher TILs scores were associated with favorable prognosis. In multivariable analyses, automated TIL scores show an independent association with disease-specific overall survival. Therefore, the open source, automated TIL scoring is an independent prognostic marker in melanoma. With further study, we believe that this algorithm could be useful to define a subset of patients that could potentially be spared immunotherapy.

Assessment of Tumor-infiltrating Lymphocytes Using International TILs Working Group (ITWG) System Is a Strong Predictor of Overall Survival in Colorectal Carcinoma: A Study of 1034 Patients.

The presence of increased tumor-infiltrating lymphocytes (TILs) is established as a positive prognostic factor in many malignancies including colorectal carcinoma (CRC). However, multiple different approaches have been used to assess TILs. In 2014, the International TILs Working Group (ITWG) proposed a standardized methodology for evaluating TILs, initially in the context of breast cancer, but subsequently expanded to other malignancies. To date, the efficacy of the ITWG system has not been investigated in a large cohort of all-stage CRC. We, therefore, sought to validate this system in CRC. We used the ITWG system to assess the density of stromal TILs in an unselected cohort of 1034 CRC patients undergoing primary tumor resection at our institution. The percentage TILs' score was categorized into 3 groups: low (0% to 10%), intermediate (15% to 50%), and high (55% to 100%). The mean survival was 53, 67, and 75 months, respectively (P=0.0001). This survival benefit remained statistically significant in multivariate analyses (P=0.0001) and subgroup analyses of mismatch repair-proficient CRCs (P=0.0001), mismatch repair-deficient CRCs (P=0.031), BRAFV600E-mutant CRCs (P=0.0001), and BRAF wild-type CRCs (P=0.001). The predictive value of TILs assessed using the ITWG system was superior to the assessment of intraepithelial lymphocyte performed prospectively using a standard system requiring ≥5 lymphocytes per high-powered field in direct contact with tumor cells or between tumor clusters. We conclude that the ITWG system for assessing TILs is a powerful predictor of all-cause survival in CRC independent of many prognostic factors and superior to the assessment of intraepithelial lymphocytes using a traditional system.

Changes in Tumor Immune Micro-Environment in Diffuse Large B-Cell Lymphoma (DLBCL): A Comparative Study of Relapsed Versus Diagnostic DLBCL

Introduction The tumor immune microenvironment (TiME) of DLBCL at diagnosis bears many biomarkers known to predict sensitivity to checkpoint blockade therapy (CBT). Approximately 25% of DLBCL, harboring PDL1 gene alteration, exhibit an immune inflamed phenotype. However, CBT studies in relapsed DLBCL show very low response rates. The immune landscape of DLBCL in the setting of relapse has not yet been characterized. We hypothesized that the TiME can be influenced by the therapy, explaining the CBT lack of efficacy. We explored the TiME evolution by comparing the tumor infiltrating lymphocytes (TIL) and the immune evasion signature by protein and gene expression (GE) in paired biopsies of DLBCL at diagnosis and at relapse. Method This study included patients with DLBCL treated with first-line R-CHOP-like regimen, in which we had access to nodal biopsies obtained pre-treatment and at relapse. Assessment of TIL and PDL1+ cell content was carried out by CD3, CD4, CD8 immunostaining and with PDL1 (Ventana, SP263) and PAX5 double staining. These were performed on formalin-fixed, paraffin-embedded (FFPE) tissue sections cut at 4µm. The proportion of each cell-type was expressed in relation to total nucleated cells. PDL1+ cell proportion was expressed for both total cells and tumor cells. The paired non-parametric Wilcoxon test, double-sided with p=0.05, was used for comparison. GE using a panel of 760 cancer immune genes was performed with the digital hybridization NanoString platform on paired FFPE specimens. NSolver software was used for normalization on housekeeping genes, differential expression (DE) and cell-type profiling. The correlation between IHC and GE variation were computed using the Spearman test. Analysis of enriched pathways on combined patient data were performed with GSEA software, using two gene sets for T-cell activation and immune evasion validated in B-cell lymphoma, and Ingenuity Pathway Analysis (IPA) for the 339 highest changed genes. The pathway enrichment analysis was reported for false discovery rate (FDR) &lt;0.05. Results Twenty DLBCL patients with paired diagnostic and relapse biopsies were studied. IHC was performed on 19 patients (necrotic tissue for one patient) and GE by NanoString on 6 patients. The median number of treatments received prior to obtaining the relapsed biopsy was 3 (1-6). Three patients had prior exposure to CBT, none from the patients in whom GE was performed. Our population was composed of 2/3 germinal center B-cell (GCB) DLBCL. Six patients had transformed DLBCL. At relapse, CD4 and CD8 cells density was significantly reduced compared to diagnosis. The median percentages of CD3, CD4 and CD8 were 15%, 7.5% and 5% at diagnosis and 7.5%, 2.5% and 2.5% at relapse, respectively (Figure 1A). The median proportion of PDL1+ cells (n=15) did not differ at relapse compared to pre-treatment (5.25% and 5%, respectively) (Figure 1B). At diagnosis, 87% of biopsies were found to be positive for PDL1 (≥1% of positive cells) and 73% at relapse. The PDL1 positive cells were mostly immune cells; only 3 samples had tumor cells expressing PDL1 at pre-treatment and 4 at relapse with &lt;1% of positive tumor cells in all cases. The GE profile detected 63 genes that were significantly different in relapse vs pre-treatment samples (p&lt;0.005), including 8 up-regulated and 55 down-regulated. The correlation for T-cell abundance variation between IHC and GE was high (r=0.94, p=0.017). For the subset of 6 patients in whom GE was performed, the T-cells were not significantly reduced as measured by both IHC and GE. However, the GSEA for immune escape genes and T-cell activation signatures were both down-regulated (FDR=0.033 and 0.010, respectively) (Figure 2). IPA predicted decreased activation of leukocytes (z-score=-6.317) and Th2 activation pathway (z-score=-3.182). Conclusion At relapse, a proportion of DLBCL have a significant reduction in T cell infiltration. As well, they acquire a non-T cell-inflamed TiME. The T-cell inhibition seems to be independent of local T-cell immunoregulatory mechanisms as the immune escape signature is also reduced. This non immunogenic, "cold" condition could explain the lack of efficacy of CBT in the relapsed setting. Prior exposure to steroid containing therapy might be a trigger for intra-tumor T-cell desertion and downregulation. This observation must be confirmed on a larger population. Further analyses will be presented. Disclosures Johnson: BD Biosciences: Other: Provided a significant proportion of the antibodies used in this project free of cost.; Lundbeck: Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; Seattle Genetics: Honoraria; Merck: Consultancy, Honoraria; Roche: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; BMS: Consultancy, Honoraria; Abbvie: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Assouline:Janssen: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Speakers Bureau; Abbvie: Consultancy, Honoraria.

14P - Validation of the optimum timing of assessment of tumour infiltrating lymphocytes during preoperative chemotherapy for breast cancer

BackgroundHost tumor microenvironment (TME) assessment is considered to play an important role in the prognostic and therapeutic predictions of breast cancer treatment. No consensus has been reached regarding evaluation methods despite reports of the utilization of tumor-infiltrating lymphocytes (TILs) for tumor immune microenvironment (TIME) monitoring. While the International Working Group recommends a tumor stromal evaluation, the timing of TIME assessment has not yet been established. Here, we focused on the assessment time and verified clinical predictions of neoadjuvant chemotherapy (NAC) effects using TILs. MethodsTwo hundred thirty-nine patients were treated with neoadjuvant chemotherapy (NAC). During the period from diagnostic needle biopsy to NAC initiation for breast cancer, the optimal evaluation timing was examined using a receiver operating characteristic (ROC) curve analysis. ResultsThe prognostic analysis revealed that both disease-free survival (DFS) and overall survival (OS) were significantly prolonged in the short-term group (118 patients) relative to the long-term group (121 patients) (p = 0.020, p = 0.010, log-rank). A significant correlation between TILs and pathological complete response (pCR) was only observed in the short-term group (p = 0.033). Prognostic analysis revealed that in the short-term group, the high TIL group had a significantly better survival prognosis relative to the low TIL group (DFS: p = 0.001, OS: p = 0.021, log-rank). TILs were also found to be a significant factor affecting DFS in the univariate analysis (p = 0.004, hazard ratio = 0.115), as well as an independent factor affecting the DFS in a multivariate analysis (p = 0.008, hazard ratio = 0.130). ConclusionsThe timing of TIL assessment during NAC for breast cancer may be more sensitive index in the short term (≤35 days). Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Tumor-infiltrating lymphocytes in primary tumors of colorectal cancer and their metastases.

The presence of tumor cells in the large intestine stimulates hypoxia and local inflammatory mediators that activate numerous inflammatory cells, including a diverse lymphoid tumor cell population. The aim of the present study was to evaluate tumor-infiltrating lymphocytes (TILs) located in the invasive primary tumor, surrounding deposits of tumor cells and those present in distal metastatic cells in the liver of patients with colorectal cancer. Furthermore, the correlation of TILs with anatomical parameters was assessed. The study group included 123 patients with primary tumor colorectal cancer without distant metastasis, 25 cases with deposits of colorectal cancer cells and 15 cases of colorectal cancer liver metastasis. TILs were assessed in tissues stained with hematoxylin-eosin using light microscopy and evaluated by two independent pathologists blinded to the clinical information. Infiltration of TILs in the invasive front of primary tumor was stronger compared with those surrounding deposits of cancer cells and liver metastases (P<0.001). TILs in the invasive front of primary tumor masses were associated with various variables linked with tumor progression and inflammatory cell infiltrate. TILs distributed around the deposits of cancer cells were associated with postoperative treatment; however, those localized in the invasive front of liver metastases were correlated with preoperative therapy. In conclusion, TILs assessment in primary tumors of colorectal cancer, surrounding deposits of tumor cells and in the metastatic cells in the liver may be helpful in understanding the role of these cells in the organization of immune response.

Metastatic breast cancers have reduced immune cell recruitment but harbor increased macrophages relative to their matched primary tumors

The interplay between the immune system and tumor progression is well recognized. However, current human breast cancer immunophenotyping studies are mostly focused on primary tumors with metastatic breast cancer lesions remaining largely understudied. To address this gap, we examined exome-capture RNA sequencing data from 50 primary breast tumors (PBTs) and their patient-matched metastatic tumors (METs) in brain, ovary, bone and gastrointestinal tract. We used gene expression signatures as surrogates for tumor infiltrating lymphocytes (TILs) and compared TIL patterns in PBTs and METs. Enrichment analysis and deconvolution methods both revealed that METs had a significantly lower abundance of total immune cells, including CD8+ T cells, regulatory T cells and dendritic cells. An exception was M2-like macrophages, which were significantly higher in METs across the organ sites examined. Multiplex immunohistochemistry results were consistent with data from the in-silico analysis and showed increased macrophages in METs. We confirmed the finding of a significant reduction in immune cells in brain METs (BRMs) by pathologic assessment of TILs in a set of 49 patient-matched pairs of PBT/BRMs. These findings indicate that METs have an overall lower infiltration of immune cells relative to their matched PBTs, possibly due to immune escape. RNAseq analysis suggests that the relative levels of M2-like macrophages are increased in METs, and their potential role in promoting breast cancer metastasis warrants further study.

Assessment of Tumor-infiltrating Lymphocytes Predicts the Behavior of Early-stage Oral Tongue Cancer.

Tumor-infiltrating lymphocytes (TILs) have shown a promising prognostic value in many epithelial cancers. We sought to assess the prognostic value of TILs in a multicenter cohort of early oral tongue squamous cell carcinoma (OTSCC). The percentage of TILs was assessed on the surgical resection slides stained with hematoxylin and eosin. The assessment of TILs was performed in the stromal compartment and in the intraepithelial compartment (at the invasive front and at the center of the tumor). We followed the method that was described recently by the International Immuno-Oncology Biomarker Working Group for the assessment of TILs. A total of 308 cases from the 5 Finnish university hospitals and from A.C. Camargo Cancer Center, São Paulo, Brazil, were included. We found a promising prognostic value for stromal TILs at the invasive front in the multivariable analysis with a hazard ratio of 2.61 (95% confidence interval [CI], 1.77-3.83; P<0.001) for overall survival, 1.99 (95% CI, 1.07-3.69; P=0.040) for disease-specific survival, and 1.94 (95% CI, 1.14-3.29; P=0.020) for disease-free survival. In conclusion, evaluation of TILs is simple and can aid in identifying the high-risk cases of early OTSCC. The method introduced by the International Immuno-Oncology Biomarker Working Group can be used for standardized determination of TILs in early OTSCC.

120P - Early on-treatment vs pre-treatment tumour transcriptomes as predictors of response to neoadjuvant therapy for HER2-positive inflammatory breast cancer

BackgroundInflammatory breast cancer (IBC) is an understudied form of breast cancer (BC). The incidence of HER2-positive disease in IBC is 2-fold greater than that in non-IBC. Several studies evaluating dual-HER2 blockade in HER2-positive BC have demonstrated an association of specific gene expression signatures with response. Those analyses were based on pretreatment tumor characteristics and did not focus specifically on IBC. We used RNA-Seq to elucidate the impact of short term neoadjuvant dual-HER2 blockade on IBC transcriptomic profile and to identify early predictors of response. MethodsWe analyzed fresh frozen tumor samples prospectively obtained from 23 patients (pts) with HER2-positive IBC, treated at a single institution in a phase 2 trial (NCT01796197) with neoadjuvant trastuzumab, pertuzumab (HP) and paclitaxel for 16 weeks (wk). Breast biopsies were performed at baseline (D1) and 1 wk later (D8) after a single dose of dual blockade with HP before adding paclitaxel. Primary endpoint was pathologic complete response (pCR) defined as no evidence of invasive disease in breast or lymph nodes. Tumors from D1 and D8 were used for RNA-Seq analysis and assessment of tumor-infiltrating lymphocytes (TILs) and CelTIL score. ResultsPaired breast tumor biopsies (D1, D8) were obtained in all pts; 2 pts did not have surgery. In the intent-to-treat analysis, 10/23 (43%) pts achieved a pCR. Across all metrics, D8 biopsies were significantly better predictors of response than D1 (p-value: 1.0X10-15). Upregulation of immune signatures by RNA-Seq was observed at D1 and D8. D8 biopsies showed a greater upregulation of anti-tumor immunity and changes in multiple signalling pathways. Neither TILs nor CelTIL were associated with pCR. ConclusionsWe identified an accurate predictor of response based on transcriptomic profiling by RNAseq, following a single dose of neoadjuvant dual-HER2 blockade in HER2-positive IBC. It outperforms a similar predictor constructed on a pretreatment profile in the same cohort. Assessing early-changes in gene expression level by RNA-seq following one dose of treatment may provide insights for the molecular mechanisms underlying response or resistance to anti-HER2 therapy. Clinical trial identificationNCT01796197. Legal entity responsible for the studyDana-Farber Cancer Institute. FundingGenentech, Inc and the Inflammatory Breast Cancer (IBC) Network. DisclosureS. Pernas: Travel / Accommodation / Expenses: Roche; Advisory / Consultancy: Polyphor. S. Goel: Advisory / Consultancy, Research grant / Funding (institution): Eli Lilly; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy: G1 Therapeutics; Research grant / Funding (institution): Merck. J.L. Guerriero: Advisory / Consultancy, Research grant / Funding (institution): Glaxo-Smith Kline; Research grant / Funding (self): Eli Lilly. E.A. Mittendorf: Advisory / Consultancy: Amgen; Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Advisory / Consultancy, Research grant / Funding (institution): Genentech; Advisory / Consultancy: Genomic Health; Advisory / Consultancy: Merck; Advisory / Consultancy: Peregrine Pharmaceuticals; Advisory / Consultancy, Research grant / Funding (institution): Sellas Lifesciences; Advisory / Consultancy: Tapimmune. B. Overmoyer: Research grant / Funding (institution): Genentech; Research grant / Funding (institution): Incyte; Research grant / Funding (institution): Eisai. All other authors have declared no conflicts of interest.

128P - Multiplex chromogenic Immunohistochemistry (IHC) for spatial analysis of checkpoint-positive tumour infiltrating lymphocytes (TILs)

BackgroundSpatial localization of TILs has been shown to correlate with immunotherapy response. Chromogenic IHC (cIHC) enables assessment of lineage (eg, CD8) or immune checkpoint expression (eg, T-cell immunoglobulin and mucin-domain–containing-3 [TIM-3]). Codetection of lineage and checkpoint molecules may help to better inform responders to checkpoint inhibitors, but colocalization is challenging by cIHC. Fluorescence (fIHC) allows colocalization but not spatial assessment and is not an approved CDx platform. Consequently, CDxs have been limited to single-plex cIHC assays. We developed a multiplex cIHC platform that enables image analysis colocalization and spatial assessment of TILs. MethodsImage analysis of cIHC via HALO software was performed to detect colocalized CD8 and TIM-3. We compared % TIM-3+/CD8+ by cIHC to TIL multiparameter flow cytometry (MFC) on 10 procured renal cell carcinoma (RCC) samples. Pathologist annotation and a trained random forest machine-learning tissue classifier were used to determine spatial localization (tumor vs peritumoral vs nontumor) of CD8+ and TIM-3+/CD8+ TIL subsets. We also performed a validated fIHC assay, including CD8 and TIM-3, on 5 additional procured RCC samples. ResultsHigh concordance (r=0.92) was observed between % TIM3+/CD8+ TILs measured by cIHC and MFC. Image analysis showed that within the CD8+ population, median % TIM-3+/CD8+ TILs were primarily intratumoral (18.0%), with fewer in peritumoral (9.4%) and nontumoral (6.4%) regions (intratumoral vs peritumoral, and intratumoral vs nontumor, both P=0.015). TIM-3/CD8 coexpression detection appeared similar between cIHC and fIHC. ConclusionsWe developed a multiplex cIHC method that enables simultaneous quantitation of TIL subpopulations, checkpoint expression, and spatial analysis. cIHC, fIHC and MFC performed similarly in coexpression detection, but only multiplex cIHC enabled spatial localization. Specifications of this assay show promise for development as a CDx. Additional data will demonstrate the capability to substitute TIM-3 with other markers, suggesting an assay template for other lineage/target combinations. Editorial acknowledgementAmrita Dervan, MSc, and Jay Rathi, MA, of Spark Medica Inc, funded by Bristol-Myers Squibb. Legal entity responsible for the studyBristol-Myers Squibb. FundingBristol-Myers Squibb. DisclosureS. Ely: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. G. Lee: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. L. Menard: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. J. Yan: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. P. Fischer: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. B. Kakrecha: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. D. Locke: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. P. Patah: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. K. Urbanska: Full / Part-time employment: Bristol-Myers Squibb.

Prognostic and clinicopathological value of PD-L1 expression in primary breast cancer: a meta-analysis.

To evaluate the association between PD-L1 expression (PD-L1+) and clinicopathological characteristics and effect on prognosis in primary breast cancer (PBC). A systematic search of the PubMed, Web of Science, and Embase databases was conducted in November 2018. Studies detecting PD-L1 using immunohistochemistry, and concerning its prognostic or clinicopathological significance in PBC were included. The HR with 95% CI for survival, and the events for clinicopathological features were pooled. Forty-seven studies were included, with a total of 14,367 PBC patients. PD-L1+ tumor cells (TCs) were associated with ductal carcinomas, large tumor size, histological Grade 3 tumors, high Ki-67, ER and PR negative, and triple-negative breast cancer; and also, related to high tumor-infiltrating lymphocytes (TILs) and PD-1 expression. PD-L1+ TCs were significantly associated with shorter disease-free survival (DFS, HR = 1.43, 95% CI 1.21-1.70, P < 0.0001) and overall survival (OS, HR = 1.58, 95% CI 1.14-2.20, P = 0.006). And the HRs of PD-L1+ TCs on DFS and OS were higher (1.48 and 1.70, respectively) and homogeneous when using whole tissue section, compared with tumor microarrays. However, PD-L1+ TILs related to better DFS (HR = 0.45, 95% CI 0.28-0.73, P = 0.001) and OS (HR = 0.41, 95% CI 0.27-0.63, P < 0.0001). PD-L1 expression on TCs associates with high-risk clinicopathological parameters and poor prognosis in PBC patients, while PD-L1+ TILs may relate to a better survival. Comprehensive assessment of TCs and TILs is required when evaluating the clinical relevance of PD-L1 expression in future studies.

Programmed death-1 receptor (PD-1) and PD-ligand-1 (PD-L1) expression in non-small cell lung cancer and the immune-suppressive effect of anaerobic glycolysis.

The microenvironment of a tumor may regulate the anti-tumor immune response. Intratumoral acidosis and hypoxia may suppress lymphocyte proliferation and migration, and this may have important implications in modern immunotherapy. The expression of PD-L1 by cancer cells and of PD-1 by tumor infiltrating lymphocytes (TILs) was assessed in tissue specimens from 98 operable NSCLC patients. Their prognostic role and their association with makers of glycolysis and anaerobic metabolism were assessed. Strong cytoplasmic/membrane PD-L1 expression was noted in 45/98 cases. Intense presence of TILs was noted in 42/98 cases (high TIL-score), and intense presence of PD-1 expressing TILs (high PIL-score) in 17/98 cases. PD-L1 expression was directly correlated with high PIL-score (p = 0.005). A significant inverse relationship was found between lactate dehydrogenase LDH5 expression and PIL-score (p = 0.008). Similarly, low PIL-score was significantly linked with high-hexokinase HXKII and monocarboxylate transporter MCT2 expression (p < 0.04). Cases with both intense TIL-score and PIL-score had significantly better survival (p < 0.05). For patients with high TIL-score or high PIL-score, PD-L1 overexpression defined significantly poorer survival (p = 0.01 and p = 0.03, respectively). In multivariate analysis, stage (p = 0.002, HR 3.33, 95%CI 1.4-4.5) and TIL-score (p = 0.02, HR 2.12, 95%CI 1.1-4.0) were independent predictive variables of death events. Given the low specificity of PD-L1 as a biomarker for anti-PD-1/PD-L1 immunotherapy, a combined assessment of TIL, PD-L1, PD-1, and LDH5 provides a tool for an immunological/metabolic classification of NSCLC tumors, with a different prognosis and different expected response to anti-PD-1/PD-L1 immunotherapy, which should be considered in relevant clinical trials.