Intraepithelial Tumor-infiltrating Lymphocytes Research Articles

The Relationship of Microsatellite Instability with BRAF and p53 Mutations and Histopathological Parameters in Colorectal Adenocarcinoma.

This study aims to elucidate the associations between microsatellite instability (MSI) status, BRAF mutation, and p53 reactions with pathological parameters and survival outcomes in colorectal carcinoma. MutL homologous 1 (MLH1), Postmeiotic segregation increased 2 (PMS2), MutS homologous 2 (MSH2), MutS homologous 6 (MSH6), BRAF, and p53 antibodies were performed on 130 adenocarcinoma samples, including 65 from the right colon and 65 from the left colon. The relationships of MSI status with BRAF mutation, p53 reaction, clinical and pathological parameters, and survival times were statistically analyzed. A statistically significant relationship was found between MSI and right colon localization, tumor size, histological grade, intraepithelial tumor-infiltrating lymphocytes, Crohn-like lymphocytic reaction, expansive growth pattern, and BRAF mutation (p < 0.05). No significant correlation was found between MSI status and the disease-free or overall survival times (p > 0.05). In colorectal adenocarcinoma, MSI and BRAF mutation are associated with parameters, indicating the host immune response and prognostic histopathological parameters, including tumor size and histological grade. The evaluation of MSI status and BRAF mutation can be particularly informative for predicting the prognosis and guiding the treatment management in poorly differentiated colorectal adenocarcinoma. Understanding the mechanisms of molecular carcinogenesis in colorectal carcinoma and organizing treatment algorithms based on molecular foundations will increase the success of the treatment.

A Deep Learning–Based Assessment Pipeline for Intraepithelial and Stromal Tumor-Infiltrating Lymphocytes in High-Grade Serous Ovarian Carcinoma

Tumor-infiltrating lymphocytes (TILs) are associated with improved survival in patients with epithelial ovarian cancer. However, the evaluation of TILs has not been applied to routine clinical practice due to reproducibility issues. We developed two convolutional neural network models to detect TILs and to determine their spatial location in whole-slide images, and established a spatial assessment pipeline to objectively quantify intraepithelial and stromal TILs in patients with high-grade serous ovarian carcinoma. The predictions of the established models showed a significant positive correlation with the number of CD8+ T cells and immune gene expressions. We demonstrated that patients with a higher density of intraepithelial TILs had a significantly prolonged overall survival (OS) and progression-free survival (PFS) in multiple cohorts. Based on the density of intraepithelial and stromal TILs, we classified patients into three immunophenotypes: immune-inflamed, excluded, and desert. The immune-desert subgroup showed the worst prognosis. Gene expression analysis showed that the immune-desert subgroup had lower immune cytolytic activity (CYT) and T-cell-inflamed gene-expression profile (GEP) scores, whereas immune-excluded subgroup had higher expression of interferon-γ and programmed death 1 receptor (PD-1) signaling pathway. Our established evaluation method provides detailed and comprehensive quantification of intraepithelial and stromal TILs throughout hematoxylin and eosin (H&E)-stained slides, and has potential for clinical application for personalized treatment of patients with ovarian cancer.

Intraepithelial tumor-infiltrating lymphocytes shape loco-regional PET/CT spread of locally advanced cervical cancer

BackgroundData suggest an association between positron emission tomography/CT (PET/CT) metabolic metrics and tumor microenvironment in several malignancies, and a potential role of PET/CT to monitor response to immunotherapy.ObjectiveTo evaluate the...

Machine Learning Quantification of Intraepithelial Tumor-Infiltrating Lymphocytes as a Significant Prognostic Factor in High-Grade Serous Ovarian Carcinomas.

The prognostic and predictive role of tumor-infiltrating lymphocytes (TILs) has been demonstrated in various neoplasms. The few publications that have addressed this topic in high-grade serous ovarian carcinoma (HGSOC) have approached TIL quantification from a semiquantitative standpoint. Clinical correlation studies, therefore, need to be conducted based on more accurate TIL quantification. We created a machine learning system based on H&E-stained sections using 76 molecularly and clinically well-characterized advanced HGSOC. This system enabled immune cell classification. These immune parameters were subsequently correlated with overall survival (OS) and progression-free survival (PFI). An intense colonization of the tumor cords by TILs was associated with a better prognosis. Moreover, the multivariate analysis showed that the intraephitelial (ie) TILs concentration was an independent and favorable prognostic factor both for OS (p = 0.02) and PFI (p = 0.001). A synergistic effect between complete surgical cytoreduction and high levels of ieTILs was evidenced, both in terms of OS (p = 0.0005) and PFI (p = 0.0008). We consider that digital analysis with machine learning provided a more accurate TIL quantification in HGSOC. It has been demonstrated that ieTILs quantification in H&E-stained slides is an independent prognostic parameter. It is possible that intraepithelial TIL quantification could help identify candidate patients for immunotherapy.

Prognostic and Predictive Role of Tumor-Infiltrating Lymphocytes (TILs) in Ovarian Cancer

Simple SummaryIn this review, we present current knowledge about the prognostic and predictive role of tumor-infiltrating lymphocytes (TILs) in ovarian cancer since this tumor shows a potential immunogenicity which makes it suitable for immunotherapy treatment; although, to date, the immunotherapy has not yielded the expected results. Understanding the factors which drive infiltration could be the key to unravel the clinical outcome heterogeneity in this cancer. Furthermore, understanding molecular mechanisms underlying the crosstalk between cancer and immune cells within the tumor microenvironment (TME) could help to identify and select subsets of OC patients who may benefit from immunotherapy.In the last decade, tumor-infiltrating lymphocytes (TILs) have been recognized as clinically relevant prognostic markers for improved survival, providing the immunological basis for the development of new therapeutic strategies and showing a significant prognostic and predictive role in several malignancies, including ovarian cancer (OC). In fact, many OCs show TILs whose typology and degree of infiltration have been shown to be strongly correlated with prognosis and survival. The OC histological subtype with the higher presence of TILs is the high-grade serous carcinoma (HGSC) followed by the endometrioid subtype, whereas mucinous and clear cell OCs seem to contain a lower percentage of TILs. The abundant presence of TILs in OC suggests an immunogenic potential for this tumor. Despite the high immunogenic potential, OC has been described as a highly immunosuppressive tumor with a high expression of PD1 by TILs. Although further studies are needed to better define their role in prognostic stratification and the therapeutic implication, intraepithelial TILs represent a relevant prognostic factor to take into account in OC. In this review, we will discuss the promising role of TILs as markers which are able to reflect the anticancer immune response, describing their potential capability to predict prognosis and therapy response in OC.

Stromal or intraepithelial tumor-infiltrating lymphocytes: which one has more prognostic significance in cervical cancer?

To investigate the effect of tumor-infiltrating lymphocytes (TILs) and tumor associated macrophages (TAMs) on treatment results in patients with cervical squamous cell carcinoma who underwent definitive or adjuvant radiotherapy (RT) or chemoradiotherapy (CRT). Pathological specimens were evaluated from 96 cervical cancer patients who were treated with definitive or adjuvant RT/CRT between April 2001 and January 2020. The percentage of intraepithelial TILs (iTILs) and stromal TILs (sTILs) were calculated, and immunohistochemistry was used for identifying lymphocyte lineage with CD4, CD8, and CD20 antibodies and macrophages with CD68 antibody. Prognostic values of TILs/TAMs on oncological outcomes were evaluated. Thirty patients had early-stage disease and 66 patients had advanced-stage disease. Sixty-three and 33 patients received adjuvant RT and definitive CRT, respectively. Low number of sCD20 positive cells was associated with large tumor size and parametrial invasion. In multivariate analysis, low percentage of sTILs and advanced-stage disease were independent poor prognostic factors for overall survival, disease-free survival (DFS), and distant metastasis-free survival; low number of sCD4 positive cells was also an independent poor prognostic factor for DFS. Low percentage of sTILs and low number of sCD8 positive cells was correlated with high rates of distant metastasis (p = 0.038 and p = 0.025, respectively). sTILs have superior predictive value than iTILs in terms of prognosis. Stromal compartment should be investigated as a routine practice in TIL studies in cervical cancer. Intensifying the treatment in cervical cancer patients with low number of sTILs should be studied in further investigations.

Abstract P1-08-26: Morphologic characterization of tumor-infiltrating lymphocytes and its relation with pathological response in a series of breast cancer patients treated with primary chemotherapy

Abstract Introduction: Tumor-Infiltrating Lymphocytes (TILS) is a well-known predictor of response to primary chemotherapy1,2 and a prognostic factor for improved survival in different breast cancer subtypes3. We present data on the association of the level of TILs and morphologic characteristics of such infiltrate with pCR. Material and methods: A series of 477 breast cancer patients (479 tumors) treated with primary chemotherapy at Catalan Institut of Oncolgy - H.U. Bellvitge between January 2009 and December 2016. Chemotherapy consisted on anthracyclines and taxanes (plus trastuzumab if Her-2 positive disease). Levels of percentage of TILs using hematoxylin-eosin-stained sections of diagnostic core-needle biopsy were evaluated according to international guidelines in a prospectively defined retrospective analysis. Characterization of TILS consisted on identification of plasma cells, intraepithelial infiltrate vs stromal infiltrate and homogeneous vs heterogeneous infiltrate. A sub-classification using levels of TILS and heterogeneity was done for statistical purposes. Levels of TILS and their morphological characteristics were examined for their associations with pCR adjusted for predictive clinic-pathological factors, by univariate and multivariate logistic regression, statistical significance set at 0.05. A ROC curve was performed to look for a cut-point of TILs to predict PCR. Results: The mean value of TILs was 23.79% (SD, 24%). TILs were significantly higher in ductal carcinomas (39.1% vs 0%, p=0.007), grade 3 (55.2 vs 17.7%p&amp;lt;0.001), ki 67 &amp;gt;30 (48.8% vs 24.5%, p&amp;lt;0.001), HER2 (56.6%) and triple negative tumors (TNBC)(56.6%) vs luminal (20%), p&amp;lt;0.001). The level of TILS was set in 20% to predict pCR by a ROC curve (S=62.3% E=71%). Characterization of TILs found plasma cells in 41.1% of the samples, intraepithelial infiltrate in 11.2% and homogeneous infiltrate in 60.1%. Homogeneously high infiltrate was found in 20% of the samples. In the univariate analysis pCR was higher in samples with TILs &amp;gt; 20% (15.2% vs 41.5%, OR: 3.96 [95%CI, 2.57-6.10]; P &amp;lt; 0.001); plasma cells (OR 6.61 [95%CI, 1.51-28.8]; P=0.01), intraepithelial TILS (OR: 10.34 [95%CI, 2.22-48.01]; P =0.003), homogeneous high infiltrate (OR: 13.6 [95%CI, 3.04-60.77]; P =0.001). In luminal tumors, TILs over 20% predicted pCR (OR 12.3 [95%CI, 4.0-37.7]; P &amp;lt; 0.001) as well as in TNBC (OR 4.32 [95%CI, 1.77-10.53]; P=0 .001) but not in those cases with HER2 positive tumors (luminalB HER2 + HER2) (OR 1.65 [95%CI, 0.88-3.07]; P=0.118). In the multivariate analyses, levels of TILs &amp;gt; 20% were associated with higher pCR rates (adjusted odds ratio, 2.44 [95%CI, 1.48-4.01]; P &amp;lt; .001). Conclusions: The presence of TILs over 20% at diagnosis is an independent, positive, predictive marker of pCR in early breast cancer treated with neoadjuvant chemotherapy. Interestingly, the predictive information added by TILs &amp;gt;20% was higher in luminal and triple negative tumors compared to HER2 positive cases. Careful morphological characterization of TILS may add valuable predictive information and can be done in current pathologic laboratories with a well-trained breast cancer pathologist.

Combinatory statuses of tumor stromal percentage and tumor infiltrating lymphocytes as prognostic factors in stage III colorectal cancers.

Tumor stroma and tumor-infiltrating lymphocytes (TILs) are major constituents of the tumor microenvironment, although they have different effects on the prognosis of patients with colorectal cancer (CRC). Combinatory statuses of tumor-stromal percentage (TSP) and TILs are expected to provide more powerful prognostic information but have never been studied in CRCs. Stage III CRCs from patients (n=487) treated with adjuvant chemotherapy were assessed for their TSP and CD3-TIL or CD8-TIL densities using computer-aided methodology. With cut-off values set at median values for intraepithelial TIL (iTIL) and stromal TIL (sTIL) densities, CRCs were sorted into low and high iTIL or sTIL groups. CRCs were classified into five quintile (Q1-Q5) groups according to their TSP and divided into high TSP (Q5) and low TSP (Q1-4) groups. The combination of CD8 iTIL density and TSP was found to be an independent prognostic parameter in multivariate survival analysis in terms of cancer-specific survival and recurrence-free survival. CRCs with low CD8 iTIL density and high TSP showed the worst survival. The combinatory status showed more prognostic power than CD8 iTIL density or TSP alone. Multivariate survival analysis in an independent cohort of stage III CRC validated the prognostic power of the combinatory statuses. The findings suggest that the combinatory status might serve as a prognostic parameter in stage III CRCs. Further research in a large-scale cohort of patients with stage III CRC is needed to validate the prognostic power of the combinatory status.

Tumor infiltrating lymphocytes (TILs) in endometrioid and clear cell ovarian carcinoma: Characterization, association with mismatch repair system deficiency and endometriosis, and prognostic implications.

e17549 Background: In contrast to high-grade serous ovarian cancer, relationship between tumor infiltrating lymphocytes (TILs) with endometrioid (EC) and clear cell ovarian carcinomas (CC) are less known. However, these tumors are thought to arise from a transformed endometriosis, a disease with a marked inflammatory response, and are also more related to Lynch syndrome. The characterization of TILs in these tumors could lead to stratify patients according to their prognosis and to identify candidates for immumotherapy in case of relapse. Methods: We conducted a retrospective observational study of 121 patients with EC and CC, diagnosed and treated in La Paz University Hospital, from November 1992 until December 2013. An expert pathologist reviewed the histopathological diagnosis, using the immunohistochemical algorithm with WT1, p53, Napsin A and progesterone receptor, and the presence of endometriosis. Clinicopathological data, genetic testing and personal and familial background were collected. Tissue-microarrays (TMAs) were used to analyze CD8+, CD4+ and CD3+ intra-epithelial TILs, according to the score designed by the Ovarian Tumor Tissue Analysis Consortium, peritumoral and endometriosis lymphocytes, ARID1A, MLH1, PMS2, MSH2 and MSH6. Software SPSS18 version was used for descriptive and statistical analysis. Results: Mean age was 54.3 years, and the median follow-up 10.02 years. 48.8% were CC and 51.2% EC, of which, 55.9% were well-differentiated, 32.2% moderately and 11.9% poorly. 58.8% of patients were diagnosed in early stages and 41.2% in advanced ones. 9.1% had family history of ovarian, colorectal or endometrial cancer and 16.5% of breast cancer. 37.2% of patients had a relapse (median progression-free survival of 31 months) and 41.6% died (32.6% related to the tumor). 55.4% of the patients had endometriosis (53.2% were EC and 57.6% CC). 8.2% had lost of MLH1, PMS2, MSH2 or MSH6 and 42% lost ARID1A expression. Moderate or high levels of CD3+, CD8+ and CD4+ TILs were seen in the 57.9%, 53.7% and 1.1%, respectively. There was no relationship between TILs and the lost of MLH1, PMS2, MSH2 and MSH6. Moderate or higher levels of CD3+ TILs were related with better overall survival (OS) only in early stages (p = 0.045). Increased CD8+ TILs in EC patients were associated with a trend towards an improved OS in comparison with those patients with lower or negative CD8+ TILs. Moderate o higher levels of CD8+ TILs were also associated, without statistical significance, with the presence of endometriosis and ARID1A. Conclusions: In our study CD3+ TILs correlated to a better OS, but only in early stages of CC and EC. CD8+ TILs were also associated with a tendency to better OS in EC. We did not find statistically significant association between TILs and Mismatch Repair System deficiency, ARID1A or endometriosis.

Adenoma-like adenocarcinoma: clinicopathologic characterization of a newly recognized subtype of colorectal carcinoma

The 5th edition of the World Health Organization (WHO) Classification of Tumours (Digestive System) recognizes a new subtype of colorectal adenocarcinoma, called adenoma-like adenocarcinoma. In this study, we sought to determine its clinicopathologic associations and how it is comparable with adenocarcinoma, of no special type (NOS). We retrospectively reviewed all available archival slides of stage I-III colonic adenocarcinoma resection specimens at our institution from 2013 to 2016.Ninety-one cases were classified as adenoma-like adenocarcinoma, and 251 cases were classified as adenocarcinoma, NOS. Of the adenoma-like adenocarcinoma cases, a majority (65 cases, 71%) were composed exclusively of adenoma-like features, designated as pure adenoma-like adenocarcinoma, whereas in the rest, the component of adenoma-like morphology was more than 50% but less than 100%, designated as mixed adenoma-like adenocarcinoma. Compared with adenocarcinoma, NOS, adenoma-like adenocarcinoma cases were significantly associated with the absence of tumor budding (P<0.001), the absence of an immature/myxoid desmoplastic reaction (P<0.001), the presence of intraepithelial tumor-infiltrating lymphocytes (P=0.006), involvement of fewer lymph nodes ( P<0.001), fewer tumor deposits (P=0.042), lower pT stage (P=0.047), lower pN stage (P<0.001), and consequently the pTNM prognostic group (P<0.001), as well as better recurrence-free survival (RFS), as per univariate analysis than adenocarcinoma, NOS cases (P=0.026) but not as per multivariate analysis. However, mixed adenoma-like adenocarcinoma had a worse RFS than pure adenoma-like adenocarcinoma (hazard ratio = 1.639, 95% confidence interval = 0.494-5.437). Our findings not only support the importance of distinguishing this new subtype of colorectal adenocarcinoma but also raisethe question whether mixed adenoma-like adenocarcinoma cases should be included in this category, and if so, whether 50% is an appropriate cutoff, as currently defined by the WHO.

Neoadjuvant chemotherapy alters the balance of effector to suppressor immune cells in advanced ovarian cancer

BackgroundAt diagnosis, tumor-infiltrating lymphocytes (TILs) are prognostic in epithelial ovarian cancer (EOC). We recently demonstrated that neoadjuvant chemotherapy (NACT) significantly increased stromal TILs. Here, we investigated the impact of NACT on immune subpopulations with a particular focus on the balance of immune-reactive to tolerant subpopulations.Materials and methodsTissue microarrays of EOC (145 pre-NACT, 139 post-NACT) were analyzed for CD3+, CD8+, FOXP3+, CD68+, and CD163+ by immunohistochemistry and CD4+ cells from deduction. Stromal TILs scored as percentage of stromal area, while intra-epithelial TILs scored as number of TILs in contact with tumor cells/HPF. Differences were evaluated by Wilcoxon or Chi square tests, Wilcoxon signed-rank for paired analyses, and cox model for PFS and OS.ResultsNACT significantly increased stromal CD3+ (p = 0.003) and CD8+ (p = 0.001) and intra-epithelial CD8+ (p = 0.022) and CD68+ (p = 0.0003) infiltration in unmatched samples and among paired samples for stromal CD3+ and CD8+. Neither CD3+, CD8+, CD4+, and CD68+ nor CD163+ expression correlated with outcome at diagnosis or post NACT. Using median value as a cut-off, high stromal CD8+/FOXP3+ ratio (HR = 0.59; p = 0.017) and high stromal CD3+/FOXP3+ ratio post NACT were associated with prolonged PFS (p = 0.0226). The more the balance shifted in favor of effector versus regulatory TILs, the better the survival. Similarly, high CD68+/CD163+ ratio post NACT improved PFS (p = 0.0445).ConclusionNACT has a significant impact on the balance of immune-reactive to immune-tolerant subpopulations and a high ratio of CD8+/FOXP3+, CD3+/FOXP3+, and CD68+/CD163+ post NACT was significantly associated with improved outcomes. Whether this could select patients for immunotherapy in the post-operative setting should be investigated.

Tumor-infiltrating lymphocytes and tumor budding refine prognostication in patients with low- and high-risk stage III colon cancers (NCCTG N0147)[Alliance

4065 Background: Tumor infiltrating lymphocytes (TILs) and tumor budding (linked to epithelial mesenchymal transition) may influence metastatic potential and patient prognosis. We analyzed these features and their relative contribution to survival among low (T1-3 N1) and high (T4 and/or N2) risk groups, defined by the IDEA study, used to inform the duration of adjuvant chemotherapy in stage III colon cancer. Methods: Among 1,532 patients (low risk n=804; high risk n=728) treated in a phase III adjuvant trial of FOLFOX + cetuximab (x 6 months), intraepithelial TIL densities and tumor budding were quantified at microscopy in routine histologic sections. Optimal cutpoints were determined in association with 5-yr disease-free survival (DFS). Relative contribution of variables to DFS was calculated using χ2 from Harrell’s rms R package based on multivariable Cox regression models. Results: In the overall cohort, the tumor budding/TILs combined variable was more robust for predition of DFS than either alone. Budding/TILs was significantly associated with DFS in both low (HRadj, 1.59; 95% CI, 1.02-2.48; p=.0273) and high (HRadj, 2.82; 95% CI, 1.72-4.63; p&lt;.0001) risk patients. We then determined its relative contribution (%) to DFS (Table). Among low risk, budding/TILs ranked second (24.4%) behind KRAS status (45.5%) and ahead of treatment arm (7.2%) and mismatch repair (MMR) status (6.1%). Among high risk, budding/TILs contributed the most to DFS (45.4%) followed by primary tumor sidedness (13.0%), performance status (12.0%), and MMR (10.4%). Conclusions: Tumor budding/TILs provides robust prognostic stratification by risk group to improve anatomic tumor staging. The relative contribution of budding/TILs to DFS was second only to KRAS status in low risk patients, and was the most important predictor of DFS in high risk patients. Evaluation in patients treated with 3 vs 6 mos of adjuvant chemotherapy is warranted. [Table: see text]

Abstract P3-08-13: Prognostic significance of intraepithelial CD103-positive tumor-infiltrating lymphocytes in breast cancer

Abstract Cluster of differentiation 103 (CD103), also known as αEβ7 integrin, is expressed on subsets of CD8+ T lymphocytes and a biomarker for tissue resident memory CD8+ T cells (TRM). CD103 expression is upregulated upon antigen recognition concurrent with transforming growth factor β (TGFβ) stimulation. CD103 is primarily expressed in the intra-epithelial tumor-infiltrating lymphocytes (iTILs) compared with that in stromal TILs (sTILs). Here, we investigated the prognostic significance of CD103+ iTILs in invasive breast cancer (IBC). Immunohistochemistry was performed for CD103, TGFβ isoforms (1, 2, and 3), and TGFβ receptors on tissue microarrays of 1173 IBC samples. CD103 expression was analyzed using Aperio ScanScope slide scanner. An area with the highest density of CD103+ iTILs was captured at a magnification of × 200, and CD103+ iTILs per unit area (mm2) were enumerated. The threshold for CD103+ iTILs (high vs low) was determined based on the mean number. Immunohistochemical staining for TGFβ isoforms (1, 2, and 3) and their receptors was quantitated using H-score and calculated as: staining intensity (0-3) × the percentage of positive tumor cells (0-100%). For statistical analyses, we categorized cases as positive or negative based on the mean value of H-score. The number of CD103+ iTILs were compared with clinicopathological characteristics, and tumor cell expression of TGFβ and its receptors. Further, the relationship between CD103+ iTILs density and prognosis was investigated according to the molecular subtypes. CD103+ iTILs were observed in 713 (60.8%) cases with a mean ± standard deviation of 34.2 ± 94.6. Increased abundance of CD103+ iTILs was found to be significantly associated with histological grade 3, high Ki-67 labelling index, non-luminal subtypes, high sTIL density, and positive expression of TGFβ3 and its receptor (all, P&amp;lt; 0.001). Furthermore, high CD103+ iTIL density was significantly associated with prolonged overall survival (OS) (P = 0.02) and disease-free survival (DFS) (P = 0.001). Subgroup analysis according to molecular subtypes showed that CD103+ iTILs was prognostic only for triple-negative breast cancer (OS, P = 0.003; DFS, P = 0.005). Multivariate analysis of CD103+ iTILs with clinicopathological prognostic factors revealed that CD103+ iTILs was an independent prognostic factor for OS (P = 0.005) and DFS (P = 0.006) for the triple-negative subtype. Our study revealed that breast cancer cell expression of TGFβ3 may be involved in recruitment of CD103+ iTILs. In breast cancer, CD103+ iTILs indicates good prognosis which is an independent favorable prognostic factor in patients with triple negative subtype and more reliable than sTIL density. Citation Format: Young Kyung Bae, Min Hui Park, Jung Eun Choi, Su Hwan Kang, Gyungyub Gong. Prognostic significance of intraepithelial CD103-positive tumor-infiltrating lymphocytes in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-13.

Whole-Slide Image Analysis Reveals Quantitative Landscape of Tumor-Immune Microenvironment in Colorectal Cancers.

Despite the well-known prognostic value of the tumor-immune microenvironment (TIME) in colorectal cancers, objective and readily applicable methods for quantifying tumor-infiltrating lymphocytes (TIL) and the tumor-stroma ratio (TSR) are not yet available. We established an open-source software-based analytic pipeline for quantifying TILs and the TSR from whole-slide images obtained after CD3 and CD8 IHC staining. Using a random forest classifier, the method separately quantified intraepithelial TILs (iTIL) and stromal TILs (sTIL). We applied this method to discovery and validation cohorts of 578 and 283 stage III or high-risk stage II colorectal cancers patients, respectively, who were subjected to curative surgical resection and oxlaliplatin-based adjuvant chemotherapy. Automatic quantification of iTILs and sTILs showed a moderate concordance with that obtained after visual inspection by a pathologist. The K-means-based consensus clustering of 197 TIME parameters that showed robustness against interobserver variations caused colorectal cancers to be grouped into five distinctive subgroups, reminiscent of those for consensus molecular subtypes (CMS1-4 and mixed/intermediate group). In accordance with the original CMS report, the CMS4-like subgroup (cluster 4) was significantly associated with a worse 5-year relapse-free survival and proved to be an independent prognostic factor. The clinicopathologic and prognostic features of the TIME subgroups have been validated in an independent validation cohort. Machine-learning-based image analysis can be useful for extracting quantitative information about the TIME, using whole-slide histopathologic images. This information can classify colorectal cancers into clinicopathologically relevant subgroups without performing a molecular analysis of the tumors.

Analysis of tumor microenvironmental features to refine prognosis by T, N risk group in patients with stage III colon cancer (NCCTG N0147) (Alliance)

Tumor-infiltrating lymphocytes (TILs), tumor budding, and micropapillary architecture may influence tumor growth and metastatic potential, thereby enhancing prognostic stratification. We analyzed these features and their relative contribution to overall outcome and in low (T1-3 N1) and high (T4 and/or N2) risk groups that are used to inform the duration of adjuvant chemotherapy in patients with resected stage III colon cancers. Among 1532 patients treated in a phase III adjuvant trial of FOLFOX-based therapy, intraepithelial TIL densities, tumor budding, and micropapillary features were analyzed and quantified in routine histopathological sections with light microscopy. Optimal cut-points were determined in association with disease-free survival (DFS) in training and validation sets. Associations or relative contributions of individual features or combined variables with DFS were determined using multivariable Cox regression models. TILs, tumor budding, and micropapillary features were shown to differ significantly by T, N risk groups and by mismatch repair (MMR) status. Low TILs, high budding, and their combined variable [hazard ratio= 2.07 (95% CI, 1.50% to 2.88%); Padj < 0.0001], but not micropapillary features, were each significantly associated with poorer DFS in a training data set and confirmed in a validation set. TILs were prognostic in proficient mismatch repair (pMMR) and deficient mismatch repair (dMMR) tumors; budding was prognostic only in pMMR tumors. The percentage relative contribution of budding/TILs to DFS was second only to nodal status overall, was second (24.4%) after KRAS in low-risk patients, and was the most important contributor (45.4%) in high-risk patients. TIL density and tumor budding were each validated as significant prognostic variables and their combined variable provided robust prognostic stratification by T, N risk groups, being the strongest predictor of DFS among high-risk stage III patients. CLINICALTRIALS. NCT00079274.

Prognostic Significance of Stromal and Intraepithelial Tumor-Infiltrating Lymphocytes in Small Intestinal Adenocarcinoma.

Assessment of tumor-infiltrating lymphocytes (TILs) may predict the prognosis and therapeutic benefit of immunotherapy in small intestinal adenocarcinoma (SIAC) patients. TILs were evaluated in 231 surgically resected SIACs and compared with microsatellite instability (MSI) and clinicopathologic variables. The average number of intraepithelial TILs (iTILs) and the average density of stromal TILs (sTILs) were calculated separately. High iTIL count (≥2 per high-power field) was associated with MSI-high, whereas high sTIL density (≥20% on ×200 magnification) was not. High iTIL count and high sTIL density were related to distal tumor location, medullary carcinoma, high Crohn-like lymphoid reaction counts, and fewer pancreatic invasions. SIAC patients with high iTIL count or high sTIL density had better survival than those with low values. On multivariate analysis, MSI, high sTIL density, proximal locations, lower N category, and absence of lymphovascular invasions and retroperitoneal seeding were the best independent prognostic predictors. High sTIL density can be used as a prognostic indicator and high iTIL count may provide a basis for the clinical use of targeted immunotherapy in SIAC patients.

The inhibitory receptor CD94/NKG2A on CD8+ tumor-infiltrating lymphocytes in colorectal cancer: a promising new druggable immune checkpoint in the context of HLAE/β2m overexpression.

We previously demonstrated that HLA-E/β2m overexpression by tumor cells in colorectal cancers is associated with an unfavorable prognosis. However, the expression of its specific receptor CD94/NKG2 by intraepithelial tumor-infiltrating lymphocytes, their exact phenotype and function, as well as the relation with the molecular status of colorectal cancer and prognosis remain unknown. Based on a retrospective cohort of 234 colorectal cancer patients, we assessed the expression of HLA-E, β2m, CD94, CD8, and NKp46 by immunohistochemistry on tissue microarray. The expression profile of HLA-E/β2m on tumor cells and the density of tumor-infiltrating lymphocytes were correlated to the clinicopathological and molecular features (Microsatellite status, BRAF and RAS mutations). Then, from the primary tumors of 27 prospective colorectal cancers, we characterized by multiparameter flow cytometry the nature (T and/or NK cells) and the co-expression of the inhibitory NKG2A or activating NKG2C chain of ex vivo isolated CD94+ tumor-infiltrating lymphocytes. Their biological function was determined using an in vitro redirected cytolytic activity assay. Our results showed that HLA-E/β2m was preferentially overexpressed in microsatellite instable tumors compared with microsatellite stable ones (45% vs. 19%, respectively, p = 0.0001), irrespective of the RAS or BRAF mutational status. However, HLA-E/β2m+ colorectal cancers were significantly enriched in CD94+ intraepithelial tumor-infiltrating lymphocytes in microsatellite instable as well as in microsatellite stable tumors. Those CD94+ tumor-infiltrating lymphocytes mostly corresponded to CD8+ αβ T cells, and to a lesser extent to NK cells, and mainly co-expressed a functional inhibitory NKG2A chain. Finally, a high number of CD94+ intraepithelial tumor-infiltrating lymphocytes in close contact with tumor cells was independently associated with a worse overall survival. In conclusion, these findings strongly suggest that HLA-E/β2m-CD94/NKG2A represents a new druggable inhibitory immune checkpoint, preferentially expressed in microsatellite instable tumors, but also in a subgroup of microsatellite stable tumors, leading to a new opportunity in colorectal cancer immunotherapies.

PD-L1 expression on stromal tumor-infiltrating lymphocytes is a favorable prognostic factor in ovarian serous carcinoma

BackgroundPD-L1 expression levels determined by immunostaining are known to be related to the survival rate and prognosis of patients with various types of cancers, as well as to the therapeutic response to immune checkpoint inhibitors. Recently, the U.S. Food and Drug Administration approved an immune checkpoint inhibitor for the treatment of non-small cell lung cancer along with the clones used for PD-L1 immunostaining to predict the resulting response. In this study, we performed PD-L1 immunostaining of tissue microarrays from ovarian epithelial cancer using SP263, an approved clone, and examined the effect of PD-L1 expression on survival rate and prognosis.MethodsTissue microarrays were constructed from ovarian epithelial cancer tissues of 248 patients and PD-L1 immunostaining was performed using the SP263 clone. PD-L1 expression levels in tumor cells, intraepithelial tumor-infiltrating lymphocytes, and stromal tumor-infiltrating lymphocytes were evaluated, and the effect of PD-L1 expression on survival and prognosis was analyzed.ResultsPD-L1 was detected in tumor cells as well as intraepithelial tumor-infiltrating lymphocytes and stromal tumor-infiltrating lymphocytes. It was most frequently expressed in stromal tumor-infiltrating lymphocytes. The Kaplan-Meier curve analysis and log rank test showed that only high stromal PD-L1 expression was associated with increased overall survival in ovarian serous carcinoma. Multivariate and univariate Cox regression analyses revealed that stromal PD-L1 expression was an independent prognostic factor, especially in ovarian serous carcinoma.ConclusionsPD-L1 immunostaining using SP263 was observed in tumor cells as well as intraepithelial and stromal tumor-infiltrating lymphocytes. PD-L1-expressing stromal tumor-infiltrating lymphocytes were associated with an increased overall survival rate and may serve as a favorable prognostic factor in ovarian cancer, particularly serous carcinoma.

Local environment in biopsy better predict the pathological response to neoadjuvant chemoradiotherapy in rectal cancer

Neoadjuvant chemoradiotherapy (nCRT) followed by surgery is the standard treatment for locally advanced rectal cancer. Here, we analyzed the impact of local and systemic environments on the tumor response to preoperative chemoradiotherapy in rectal cancer. We recruited 141 patients with rectal cancer treated with nCRT. We evaluated the local tumor environment, including tumor-infiltrating lymphocytes (TILs), intratumor budding (ITB), and the systemic inflammatory environment, including the neutrophil-to-lymphocyte ratio (NLR) and C-reactive protein (CRP) level. Our finding revealed that tumor regression was significantly associated with the density of CD8+ TILs in the intraepithelial, the presence of ITB, the combination of NLR and CRP (NLR-CRP) value, and the combination of CD8+ intraepithelial TIL (iTIL) density and ITB presence. Moreover, multivariate analysis showed that only the combination of CD8+ iTILs and ITB was an independent predictive factor for the pathological response to nCRT in rectal cancer. Our finding demonstrate that the local tumor environment was a better predictor of the tumor response than the systemic environment and thus provided new insight into screening for patients who are more likely to benefit from cancer treatment.

Abstract PD5-12: Spatial mapping of the immune microenvironment in primary triple-negative breast cancer (TNBC) and association with neoadjuvant therapy response

Abstract Background: Emerging data suggest that some patients with TNBC could benefit from the addition of immune-based therapy. This observation is in part due to the significant association between distinct tumor-infiltrating lymphocytes (TILs) and prognosis in TNBC. Efforts to comprehensively characterize the immune microenvironment of TNBCs are critically important to gain a better understanding of the immune landscape and how it influences response to both standard chemotherapy and immunotherapy. We previously assessed both stromal TILs (sTILs) and intraepithelial TILs (iTILs) from pre-treatment tumor samples from patients enrolled on a phase II study of neoadjuvant gemcitabine, carboplatin and iniparib (PrECOG 0105; NCT00813956). We found that both iTILS and sTILS significantly associated with pathologic response. Furthermore, we assessed a novel 'in silico flow cytometry' gene expression-based method, CIBERSORT, designed to assess overall immune content and deconvolute the relative levels of distinct leukocyte subsets in tumors. Specific leukocyte subsets significantly associated with pCR included activated memory CD4+ T cells, CD8+ T cells and M1 macrophages (all p&amp;lt;0.05). To validate these findings and further explore spatial mapping of these immune cell subsets we undertook this analysis. Methods: We performed SPARTA - Spatial Perception And Regional Tumor Analysis - a multiplexed immunohistochemistry-based technology with data visualization and analysis modules that is designed to capture clinically relevant information about the tumor microenvironment in 72 pre-treatment FFPE tumor sections from patients enrolled on PrECOG 0105. SPARTA was used to test and analyze the coordinated expression of PD-L1, PD-1, CD45RO, CD4, CD8 and HLA-DRA in the study cohort from whole slide scanned images. Pathologic response at the time of surgery was evaluated using the residual cancer burden index. Germline BRCA1 and BRCA2 status was known for all patients. Results: Of 72 samples, 67 were evaluable for SPARTA analysis. Within a subset of tumors separately profiled by both SPARTA (FFPE/IHC microscopy) and CIBERSORT (Frozen/RNA GEP), we found significant correlation for clinically relevant TIL subpopulations, including for CD8+ T cells (r=0.83, p&amp;lt;0.0001), and activated memory CD4+ T cells (r=0.78, p&amp;lt;0.0001). On average, the SPARTA frequency of CD8, PD-L1 and CD4 expressing cell types were higher in samples from the pCR group when compared to the no pCR group. Subpopulation analysis revealed that, on average, the frequency of CD4+HLA-DRA+ cells were higher in samples from the pCR group when compared to the no pCR group. Samples from the pCR group were associated with a higher local spatial density of CD45RO+HLA-DRA+CD4+ cells when compared to the no pCR group. Location- and proximity-based analyses are ongoing and will be presented. Analyses based on germline BRCA1/2 mutant versus wild-type status, as well as associations between tumor mutation burden and neoantigen burden will also be presented. Conclusions: Spatial mapping of the immune microenvironment in primary TNBC reveals distinct immune cell populations associated with response to neoadjuvant platinum-based therapy. Citation Format: Telli ML, Vinayak S, Khododoust MS, Gruber JJ, Ford JM, Sanchez P, Banayan N, Azimi S, Tumeh PC, Newman AM, Alizadeh AA. Spatial mapping of the immune microenvironment in primary triple-negative breast cancer (TNBC) and association with neoadjuvant therapy response [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 PD5-12.