Density Of Tumor-infiltrating Lymphocytes Research Articles

Theragnostic significance of tumor-infiltrating lymphocytes and Ki67 in BRAFV600-mutant metastatic melanoma (BRIM-3 trial)

We conducted a retrospective tumor tissue analysis as part of the BRIM3 trial to evaluate the theragnostic significance of tumor-infiltrating lymphocytes (TILs) and melanoma cell proliferation. Using manual semi-quantitative analyses, we assessed the density of TILs by pathology review of tissue sections stained with hematoxylin and eosin (H&E TIL score) and by immunohistochemistry (IHC) with an anti–CD8 antibody (CD8 TIL score); also, the melanoma cell proliferation by IHC with an anti–Ki67 antibody. Three hundred and fifty-three, 280, and 172 patients’ tumor tissue samples were available for H&E, CD8, and Ki67 IHC analysis, respectively. There was no association between high (2+, 3+) peritumoral and intratumoral H&E and/or TIL CD8 score or high Ki67 proliferation index (>15%) with serum LDH level and stage IV melanoma. Neither high Ki67 proliferation, nor high peritumoral and/or intratumoral TIL score was significantly associated with objective antitumor response in any treatment arm. High intratumoral and high peritumoral CD8 TIL score was significantly associated with progression-free survival (PFS) only in DTIC-treated patients (P = 0.002 and 0.037, respectively); in vemurafenib-treated patients, high intratumoral and/or peritumoral CD8 TIL score was not significant (log-rank P = 0.053 and 0.062, respectively). Nevertheless, a high peritumoral CD8 TIL score was a significant predictor of PFS and overall survival after adjustment for age, sex, serum LDH, ECOG performance status, and treatment arm in a Cox regression model. Vemurafenib does not only benefit patients bearing brisk TILs; even vemurafenib-treated patients with absent and/or non–brisk TILs tend to have longer PFS compared to DTIC-treated patients with brisk TILs. High peritumoral CD8 TIL score is a favorable prognostic factor independent of well-established AJCC staging factors.

UV-induced local immunosuppression in the tumour microenvironment of eccrine porocarcinoma and poroma

Eccrine porocarcinoma (EPC) is a rare malignant adnexal tumour of the skin. Part of EPCs develop from their benign counterpart, poroma (EP), with chronic light exposure and immunosuppression hypothesized to play a role in the malignant transformation. However, the impact of chronic light exposure on the microenvironment of EPCs and EPs has not been investigated yet. Although the clinical relevance of tumour infiltrating lymphocytes (TILs) and tertiary lymphoid structures (TLSs) has been established in various tumours, their distribution and significance in EPCs and EPs is still poorly understood. We characterized the distribution of TILs and TLSs using CD3, CD4, CD8, CD20 immunohistochemistry in a cohort of 10 EPCs and 49 EPs. We then classified our samples using solar-elastosis grading, analyzing the influence of ultraviolet (UV) damage on TIL density. A negative correlation between UV damage and TIL density was observed (CD4 r = −0.286, p = 0.04. CD8 r = −0.305, p = 0.033). No significant difference in TIL density was found between EPCs and EPs. TLS was scarse with the presence rate 10% in EPCs and 8.3% in EPs. The results suggest that UV has an immunosuppressive effect on the microenvironment of EPCs and EPs.

Up-to-Date Pathologic Classification and Molecular Characteristics of Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) is an aggressive primary liver malignancy with an increasing incidence worldwide. Recently, histopathologic classification of small duct type and large duct type iCCA has been introduced. Both these types of tumors exhibit differences in clinicopathological features, mutational profiles, and prognosis. Small duct type iCCA is composed of non-mucin-producing cuboidal cells, whereas large duct type iCCA is composed of mucin-producing columnar cells, reflecting different cells of origin. Large duct type iCCA shows more invasive growth and poorer prognosis than small duct type iCCA. The background liver of small duct type iCCA often shows chronic liver disease related to hepatitis B or C viral infection, or alcoholic or non-alcoholic fatty liver disease/steatohepatitis, in contrast to large duct type iCCA that is often related to hepatolithiasis and liver fluke infection. Cholangiolocarcinoma is a variant of small duct type iCCA composed of naïve-looking cuboidal cells forming cords or ductule-like structures, and shows better prognosis than the conventional small duct type. Fibrous tumor stroma, one of the characteristic features of iCCA, contains activated fibroblasts intermixed with innate and adaptive immune cells. The types of stroma (mature versus immature) are related to tumor behavior and prognosis. Low tumor-infiltrating lymphocyte density, KRAS alteration, and chromosomal instability are related to immune-suppressive tumor microenvironments with resistance to programmed death 1/ programmed death ligand 1 blockade. Data from recent large-scale exome analyses have revealed the heterogeneity in the molecular profiles of iCCA, showing that small duct type iCCA exhibit frequent BAP1, IDH1/2 hotspot mutations and FGFR2 fusion, in contrast to frequent mutations in KRAS, TP53, and SMAD4 observed in large duct type iCCA. Multi-omics analyses have proposed several molecular classifications of iCCA, including inflammation class and proliferation class. The inflammation class is enriched in inflammatory signaling pathways and expression of cytokines, while the proliferation class has activated oncogenic growth signaling pathways. Diverse pathologic features of iCCA and its associated multi-omics characteristics are currently under active investigation, thereby providing insights into precision therapeutics for patients with iCCA. This review provides the latest knowledge on the histopathologic classification of iCCA and its associated molecular features, ranging from tumor microenvironment to genomic and transcriptomic research.

HLA class II immunogenic mutation burden predicts response to immune checkpoint blockade

Whereas human leukocyte antigen (HLA) class I mutation-associated neoantigen burden has been linked with response to immune checkpoint blockade (ICB), the role of HLA class II-restricted neoantigens in clinical responses to ICB is less studied. We used computational approaches to assess HLA class II immunogenic mutation (IMM) burden in patients with melanoma and lung cancer treated with ICB. We analyzed whole-exome sequence data from four cohorts of ICB-treated patients with melanoma (n= 110) and non-small-cell lung cancer (NSCLC) (n= 123). MHCnuggets, a neural network-based model, was applied to estimate HLA class II IMM burdens and cellular fractions of IMMs were calculated to assess mutation clonality. We evaluated the combined impact of HLA class II germline genetic variation and class II IMM burden on clinical outcomes. Correlations between HLA class II IMM burden and density of tumor-infiltrating lymphocytes were computed from expression data. Responding tumors harbored a significantly higher HLA class II IMM burden for both melanoma and NSCLC (P ≤ 9.6e-3). HLA class II IMM burden was correlated with longer survival, particularly in the NSCLC cohort and in the context of low intratumoral IMM heterogeneity (P < 0.001). HLA class I and II IMM landscapes were largely distinct suggesting a complementary role for class II IMMs in tumor rejection. A higher HLA class II IMM burden was associated with CD4+ T-cell infiltration and programmed death-ligand 1 expression. Transcriptomic analyses revealed an inflamed tumor microenvironment for tumors harboring a high HLA class II IMM burden. HLA class II IMM burden identified patients with NSCLC and melanoma that attained longer survival after ICB treatment. Our findings suggest that HLA class II IMMs may impact responses to ICB in a manner that is distinct and complementary to HLA class I-mediated responses.

Expression of PD-L1 in Early-Stage Invasive Breast Carcinoma and Its Relation to Tumor-Infiltrating Lymphocytes.

Objectives:Immunotherapeutic targets became one of the promising approaches in breast cancer (BC), especially in advanced stage triple-negative subtype (TNBC). However, the role of programmed cell death ligand 1 (PD-L1) targeting in other BC subtypes, especially in early-stage carcinoma is less explored. We aimed in this study to investigate the prevalence of PD-L1 in early-stage invasive BC of different molecular subtypes and to elucidate its relation to tumor-infiltrating lymphocytes (TILS) density (cytotoxic and regulatory T-cells), established clinicopathological factors and patients’ outcome. Material and Methods:One hundred and nine cases of early-stage BC were enrolled in our study. Cases were classified into five molecular subtypes according to the Immunohistochemical data. PD-L1, FOXP3 and CD8 immunostaining were analyzed for all studied cases. PD-L1 expression was correlated with CD8+ cytotoxic T-cells, FOXP3+ regulatory T-cells, histopathologic parameters, BC molecular subtypes, 7-years disease-free survival (DFS) and overall survival (OS). Results:PD-L1 was expressed in 11% of the studied early-stage BC cases. It showed a significant correlation with high tumor grade (p= <0.001), development of metastasis (p=0.037), high FOXP3+ T-cell density (p= <0.001) and low CD8+ T-cells density (p= <0.001). PD-L1 expression was higher in TNBC (16.1%), followed by HER2/neu-enriched group (14.3%). All luminal A cases showed negative PD-L1 expression. PD-L1 was found to be an independent prognostic factor for patients’ survival (DFS; p=0.031 and OS: p=0.04). Conclusion:Although the impact of PD-L1 on early-stage BC outcomes had not been clearly established, our results indicated that PD-L1 is a negative prognostic marker in early settings. PD-L1 can serve as a new therapeutic target for patients with high-grade early-stage breast carcinoma.

Correlation between Lymphocyte-to-Monocyte Ratio (LMR), Neutrophil-to-Lymphocyte Ratio (NLR), Platelet-to-Lymphocyte Ratio (PLR) and Tumor-Infiltrating Lymphocytes (TILs) in Left-Sided Colorectal Cancer Patients.

Simple SummaryColorectal cancer (CRC) is one of the most common cancers worldwide. Novel markers have been investigated in order to better predict the course of disease and adjust the treatment. Markers associated with cancer-related inflammation (CRI), both in the bloodstream and the tumor tissue, have been in the spotlight for years. In this study, we investigate whether blood-based markers: lymphocyte-to-monocyte ratio, neutrophil-to-lymphocyte ratio or platelet-to-lymphocyte ratio, correlate with tissue-based markers, such as tumor-infiltrating lymphocytes. We retrospectively analyzed 87 patients with locally advanced left-sided CRC treated with radical surgery. Fifty patients were found suitable for the study. We compared the results of their blood tests from the time of the surgical intervention and the density of lymphocytes in the resected tumors. We found no correlation between local and peripheral markers of CRI. Further prospective studies are needed to confirm the results.Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality worldwide. Novel markers are required in order to select high-risk patients and better adjust the treatment. Both peripheral and local markers of cancer-related inflammation (CRI) such as lymphocyte-to-monocyte ratio (LMR), neutrophil-to-lymphocyte ratio (NLR) or platelet-to-lymphocyte ratio (PLR) and tumor-infiltrating lymphocytes (TILs) have been thoroughly investigated in recent years and deemed to be highly prognostic. We hypothesized that there is an association between local and peripheral CRI indices and that blood-based biomarkers may serve as a surrogate of TILs. We retrospectively analyzed 87 patients with locally advanced left-sided CRC treated with radical-intent surgery in the Maria Skłodowska-Curie National Research Institute of Oncology in Warsaw, Poland, between January 2014 and December 2015. Fifty patients were found eligible for the study. The patients were divided in terms of pre-treatment values of systemic inflammatory response (SIR) markers into LMR/NLR/PLR-high and low groups. We evaluated the resected specimens by immunohistochemistry in order to assess the densities of CD3+ and CD8+ lymphocytes in the center of the tumor and in the invasive margin. We found that the level of CD3+ lymphocytes in the center of the tumor was statistically significantly higher in patients with low pre-treatment NLR (p = 0.044); however, no correlation between any of the SIR markers and CD3+ or CD8+ TILs was observed. Five-year overall survival (OS) was longer in patients with high LMR (p < 0.001), low NLR (p = 0.001) and low PLR (p = 0.095). No correlation between the density of TILs and OS was demonstrated. In conclusion, based on our study, peripheral blood-based markers and CD3+ and CD8+ TILs are not interrelated.

Immunoarchitectural patterns as potential prognostic factors for invasive ductal breast cancer

Currently, tumor-infiltrating lymphocytes (TILs) in invasive breast cancers are assessed solely on the basis of their number, whereas their spatial distribution is rarely investigated. Therefore, we evaluated TILs in 579 patients with invasive breast cancer of no special type (IBC-NST) with a focus on their spatial distributions in tumor center (TC) and invasive margin (IM). We also assessed a new factor, namely para-tumor infiltrating lymphocytes (PILs) in the para-tumor lobular area (Para). Five immunoarchitectural patterns (IPs) were observed, which were significantly associated with clinicopathological features, especially molecular subtypes, histological grades, clinical stages, and programmed death-ligand 1 (PD-L1) expression. High-TIL density (IP1/2) correlated with favorable disease-free survival (DFS) in TNBC patients (p = 0.04), but opposite results were observed for luminal B subtype patients (both the lowest TIL and PIL densities (IP5) correlated with good DFS, p = 0.013). Luminal B patients with high TILs in the IM and low TILs in the TC (IP3) exhibited the worst DFS, whereas those with low TILs (similar to IP5) and high PILs (IP4) exhibited poor DFS. We also identified TIL subpopulations with significantly different IPs. Our findings suggest that IP can be a potential prognostic factor for tumor immunity in IBC.

Clinical characteristics and patient outcomes of molecular subtypes of small cell lung cancer (SCLC)

BackgroundRecent studies have shown that according to the expression levels of achaete-scute homolog 1 (ASCL1), neurogenic differentiation factor 1 (NEUROD1), and POU class 2 homeobox 3 (POU2F3), small cell lung cancer (SCLC) can be divided into four subtypes: SCLC-A (ASCL1-dominant), SCLC-N (NEUROD1-dominant), SCLC-P (POU2F3-dominant), and SCLC-I (triple negative or SCLC-inflamed). However, there are limited data on the clinical characteristics and prognosis of molecular subtypes of SCLC.MethodsImmunohistochemistry (IHC) was used to detect the expression levels of ASCL1, NEUROD1, and POU2F3 in 53 patient samples of resectable SCLC. The subtype was defined by the differential expression of the transcription factors for ASCL1, NEUROD1, and POU2F3 or the low expression of all three factors with an inflamed gene signature (SCLC-A, SCLC-N, SCLC-P, and SCLC-I, respectively). The clinicopathological characteristics, immunological features (programmed death ligand 1 [PD-L1] expression and CD8+ tumor infiltrating lymphocyte [TIL] density), and patient outcomes of the four subtypes of SCLC were analyzed.ResultsPositive ASCL1, NEUROD1, and POU2F3 staining was detected in 43 (79.2%), 27 (51.0%), and 17 (32.1%) SCLC specimens by IHC. According to the results of IHC analysis, SCLC was divided into four subtypes: SCLC-A (39.6%), SCLC-N (28.3%), SCLC-P (17.0%), and SCLC-I (15.1%). The 5-year overall survival (OS) rates of these four subtypes were 61.9%, 69.3%, 41.7%, and 85.7%, respectively (P=0.251). There were significant differences in smoking status among different subtypes of SCLC (P= 0.031). However, we did not confirm the correlation between subtypes of SCLC and other clinicopathological factors or immune profiles. Cox multivariate analysis showed that N stage (P=0.025), CD8+ TILs (P=0.024), Ki-67 level (P=0.040), and SCLC-P (P=0.023) were independent prognostic factors for resectable SCLC.ConclusionsOur IHC-based study validated the proposed classification of SCLC using the expression patterns of key transcriptional regulatory factors. We found that SCLC-P was associated with smokers and was one of the poor prognostic factors of limited-stage SCLC. In addition, no correlation was found between PD-L1 expression or CD8+ TIL density and SCLC subtypes.

Differences in tumor-infiltrating lymphocyte density and prognostic factors for breast cancer by patient age

BackgroundLymphocytes that surround cancer participate in tumor-related immune responses and are called tumor-infiltrating lymphocytes (TILs). Several recent reports suggest TILs index the tumor microenvironment and predict the therapeutic effect of chemotherapy. However, only few studies have studied the relationship between age and TILs. Aging reduces host immunity, and we predict that it may also affect TILs. Thus, we hypothesized that older breast cancer (BC) patients may have low TIL density than younger BC patients. Here, we retrospectively analyzed the differences in TILs by age and the therapeutic effects of preoperative chemotherapy (POC) in BC patients who were aged either less than 45 years or more than 60 years.MethodsWe retrospectively examined the data of 356 breast cancer patients who underwent POC, including 75 patients aged ≤ 45 years and 116 patients aged > 60 years. Using pre-treatment needle biopsy specimens, TIL density was compared for each age group by Student’s t-test. After analyzing different factors that affect TIL density, prognostic factors were also examined.ResultsOlder patients with triple-negative BC had significantly lower TIL density than younger patients, while in human epidermal growth factor receptor 2 (HER2)-enriched BC, TIL density was significantly higher in the younger age group than that in the older age group. In addition, younger patients with HER2-rich breast cancer showed significantly higher complete pathological response rates than older patients with HER2-rich BC. In addition, significant differences in overall survival were observed among these patients with triple-negative BC.ConclusionsOur study suggests that younger BC patients possess significantly higher TIL density than older patients. These differences may influence the therapeutic efficacy in highly immunogenic subtypes.

Association of L-type amino acid transporter 1 (LAT1) with the immune system and prognosis in invasive breast cancer

L-type amino acid transporter 1 (LAT1), also referred to as SLC7A5, is believed to regulate tumor metabolism and be associated with tumor proliferation. In invasive breast cancer, we clinicopathologically investigated the utility of LAT1 expression. LAT1 expression was evaluated via immunohistochemistry analyses in 250 breast cancer patients undergoing long-term follow-up. We assessed the relationships between LAT1 expression and patient outcomes and clinicopathological factors. Breast cancer-specific survival stratified by LAT1 expression was assessed. Human epidermal growth factor receptor 2 (HER2)-positive patients with metastasis received trastuzumab therapy. The density of tumor-infiltrating lymphocytes (TILs) was evaluated according to the International Working Group guidelines. In the current study, high LAT1 expression was significantly correlated with estrogen receptor (ER) negativity, progesterone receptor negativity, high histological grade, increased TILs, and programmed death ligand 1 positivity. Among the ER-positive and HER2-negative patients, high LAT1 was an independent indicator of poor outcomes (hazard ratio (HR) = 2.97; 95% confidence interval (CI), 1.16–7.62; p = 0.023). Moreover, high LAT1 expression was an independent poor prognostic factor in luminal B-like breast cancer with aggressive features (HR = 3.39; 95% CI 1.35–8.52; p = 0.0094). In conclusion, high LAT1 expression could be used to identify a subgroup of invasive breast cancer characterized by aggressive behavior and high tumor immunoreaction. Our findings suggest that LAT1 might be a candidate therapeutic target for breast cancer patients, particularly those with luminal B-like type breast cancer.

Abstract P5-13-32: Mucin 4 expression in high risk breast cancer: Predicting and overcoming resistance to immunotherapy

Abstract Background HER2-positive (+) and triple negative breast cancer (TNBC) have the worst survival among BC. BC patients are treated with chemotherapy (CT) and/or radiotherapy (RT), and HER2+ BC patients also receive targeted therapies, such as trastuzumab (Tz). The abundance of tumor infiltrating lymphocytes (TILs), in both HER2+ and TNBC, has a major good prognostic value. Thus, indicating that immunological evasion mechanisms are present in the tumor microenvironment (TME) hampering the efficacy of the treatments. We previously showed that soluble tumor necrosis factor α (sTNF) induces upregulation of mucin 4 (MUC4), which shields Tz epitope on HER2 impairing Tz binding and its effects. In preclinical models of de no5vo Tz-resistant tumors, administration of the sTNF blocking agent INB03 (DN) together with Tz inhibited tumor growth. We proved that MUC4 expression is an independent predictor of poor DFS in patients treated with adjuvant Tz. Our goal is to study whether MUC4 plays a role in tumor immune evasion in HER2+ and TNBC. Methods Untreated primary BC samples were assessed for TILs density (H&amp;E) and MUC4 expression by immunohistochemistry. Tumors with TILs ≥30% and &amp;gt;50%, for TNBC and HER2+ BC respectively, and MUC4 scores 2 and 3 (0-3) were deemed positive. A cohort of 56 TNBC and 90 HER2+BC, stage I-III were retrospectively retrieved from Hospital Fernández and Instituto Henry Moore from 2013-2017, and clinicopathological and treatment characteristics were obtained from electronic records. TNBC were treated with adjuvant (41) or neoadjuvant CT +/- RT (15). HER2+BC patients received adjuvant Tz + CT. The association between MUC4 and OS was assessed by Kaplan Meier and log rank test and between MUC4 and TILs using Chi2. JIMT-1 HER2+ BC, de novo resistant tumors to Tz, containing a doxycycline (Dox)-inducible shRNA MUC4 plasmid (JIMT-1shMUC4) growing in nude mice were treated with IgG, Tz, DN or Tz + DN. Tumor growth was measured and macrophages and NK cells were determined in the TME by flow cytometry. Anti-asialo GM1 and clodronate-encapsulated liposomes were used to deplete NK cells and macrophages, respectively. Results We found an inverse relationship between TILs and MUC4 expression in HER2+ and TNBC (P=0.02 and P= 5 x10-5, respectively). Patients with MUC4+ TNBC have a shorter OS (P=0.03) and MUC4 was an independent predictor of OS [P=0.01; HR 4.9 (95%CI 1.4-17.0)]. To study MUC4 involvement in macrophage and NK cells recruitment in a Tz resistant model, nude mice bearing JIMT-1-shMUC4 tumors were treated or not with Dox to abolish MUC4 expression. Both groups received IgG, Tz, DN or DN + Tz. In control groups (without Dox), only Tz + DN administration was able to inhibit tumor growth (75% inhibition, P&amp;lt;0.0001 vs. IgG), in line with our previous results, and DN treatment reduced MUC4 expression. Knockdown of MUC4 expression by Dox, showed that Tz alone was effective in inhibiting JIMT-shMUC4 tumor growth at similar levels than Tz + DN group. Tumor growth inhibition was accompanied by an increase in NK cells activation and degranulation, and a rise in M1/M2 macrophage ratio. Depletion of macrophages or NK cells totally blunted antitumor effect of Tz + DN in control tumors. In MUC4-silenced tumors only macrophage depletion was able to abolish Tz antitumor effect. Conclusion Our results suggest that i) MUC4 expression is associated with immunologically “cold” HER2+ and TNBC, inducing an immunosuppressive TME that reflects in poor DFS/OS, and it confers resistance to Tz in HER2+ BC; ii) elimination of MUC4 expression reverses resistance to Tz; iii) tumor infiltrating macrophages are critical to the anti-tumor response in HER2+ BC. Patients with MUC4+ HER2+ or MUC4+ TNBC should benefit from sTNF blockade treatment leading to MUC4 downregulation and higher TILs, which would result in a better response to Tz and probably to immune checkpoint inhibitors. Citation Format: Roxana Schillaci, Sofia Bruni, Florencia Mauro, María F Mercogliano, Agustina Roldan-Deamicis, Cecilia J Proietti, Rosalía Cordo-Russo, Gloria Inurrigarro, Agustina Dupont, Carla Adami, Daniel Lopez Della Vecchia, Sabrina Barchuck, Silvina Figurelli, Ernesto Gil Deza, Sandra Ares, Felipe G Gercovich, Patricia V Elizalde. Mucin 4 expression in high risk breast cancer: Predicting and overcoming resistance to immunotherapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-32.

Abstract P4-07-22: Evaluation of human epididymis protein 4 serum and tissue expression in ductal carcinoma in situ of the breast

Abstract Background: Human epididymis protein 4 (HE4), also known as WFDC2, is a member of whey acidic protein (WAP) family with a presumptive role in natural immunity. HE4 expression has been increased in many cancer types, particularly in gynecologic and pulmonary cancers. HE4 has been identified as an important serum biomarker in the diagnosis and monitoring of epithelial ovarian cancer. Recent researches have demonstrated that HE4 levels in serum and tissues are elevated in patients with breast cancer and HE4 is thought be involved in breast cancer progression. In contrast, expression of HE4 in ductal carcinoma in situ (DCIS) has not been well characterized yet. The present work was undertaken to evaluate serum and tissue levels of HE4 in DCIS and their correlations with clinicopathological features of DCIS. Materials and methods: Preoperative serum HE4 levels in 59 DCIS patients was analyzed using the ARCHITECT assay. Tissue microarray containing DCIS and adjacent normal tissues from the same cohort were tested for HE4 mRNA and protein by RNAscope in situ hybridization (ISH) and immunohistochemistry analysis, respectively. Tissue microarray containing only DCS tissues from 41 patients were also tested for HE4 mRNA and protein. To validate prognostic potential of HE4 in breast cancer patients, the BreastMark database was used. Results: HE4 levels in 59 DCIS patients ranged from 23.5 to 86.3 pmol/L (mean ± SD: 39.4 ± 11.9). Based on the cutoff level, there were no abnormal cases for HE4. Serum HE4 levels did not differ according to clinicopathological parameters except for menopause status. RNAscope ISH and immunohistochemistry confirmed an increase in HE4 in DCIS tissues compared with their corresponding normal tissues. Spearman’s correlation analyses revealed no significant correlation between serum and tissue levels of HE4. Among 100 DCIS tissues, there was a positive correlation between HE4 mRNA ISH scores and HE4 immunohistochemical staining scores (r = 0.771, P &amp;lt; 0.001). High mRNA and protein expression of HE4 were observed in 25 of 99 (25.3%) and 34 of 99 (34.3%) cases, respectively. High mRNA HE4 expression was significantly associated with low stromal tumor infiltrating lymphocyte (TIL) density scores, ER positivity, HER2 negativity, and HR+/HER2- subtype. High protein HE4 expression was also significantly associated with absence of comedo-type necrosis, low stromal TIL density scores, ER positivity, HER2 negativity, and HR+/HER2- subtype. HE4 mRNA and protein expression did not correlate with recurrence of DCIS. In breast cancer patients, high HE4 expression was significantly associated with good survival in the overall group (HR = 0.838, P = 0.003, n = 2,652) and lymph node negative group (HR = 0.791, P = 0.029, n = 1,183). Conclusions: Our study revealed that serum HE4 is not elevated in patients with DCIS. High expression of HE4 mRNA and protein in DCIS tissues are associated with good clinicopathological characteristics, which warrant. further investigations. Citation Format: Ji Shin Leee, Nah Ihm Kim, Min Ho Park. Evaluation of human epididymis protein 4 serum and tissue expression in ductal carcinoma in situ of the breast [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-07-22.

Computational image features of immune architecture is associated with clinical benefit and survival in gynecological cancers across treatment modalities

BackgroundWe present a computational approach (ArcTIL) for quantitative characterization of the architecture of tumor-infiltrating lymphocytes (TILs) and their interplay with cancer cells from digitized H&E-stained histology whole slide images and...

Tumor-Infiltrating Lymphocytes as Biomarkers of Treatment Response and Long-Term Survival in Patients with Rectal Cancer: A Systematic Review and Meta-Analysis.

Simple SummaryThis study investigated tumor-infiltrating lymphocytes (TILs) in pretherapeutic biopsies as biomarkers of treatment response and long-term prognosis in patients with locally advanced rectal cancer undergoing neoadjuvant chemoradiotherapy. A systematic review and meta-analysis was performed in accordance with the PRISMA guidelines. The results indicate that it is possible to identify a sub-group of patients with improved treatment response and long-term prognosis by assessing the density of CD8+ TILs at the time of diagnosis.Neoadjuvant chemoradiotherapy (NCRT) is indicated in locally advanced rectal cancer (LARC) to downstage tumors before surgery. Watchful waiting may be a treatment option to avoid surgery in patients, obtaining a complete clinical response. However, biomarkers predictive of treatment response and long-term prognosis are lacking. Here we investigated tumor-infiltrating lymphocytes (TILs) in pretherapeutic biopsies as predictive and prognostic biomarkers. A systematic review and meta-analysis was performed in accordance with the PRISMA guidelines. In total, 429 articles were identified, of which 19 studies were included in the systematic review and 14 studies in the meta-analysis. Patients with high pretherapeutic CD8+ TILs density had an increased likelihood of achieving a pathological complete response (RR = 2.71; 95% CI: 1.58–4.66) or a complete or near-complete pathological treatment response (RR = 1.86; 95% CI: 1.50–2.29). Furthermore, high CD8+ TILs density was a favorable prognostic factor for disease-free survival (HR = 0.57; 95% CI: 0.38–0.86) and overall survival (HR = 0.43; 95% CI: 0.27–0.69). CD3+, CD4+, and FOXP3+ TILs were not identified as predictive or prognostic biomarkers. Thus, assessing pretherapeutic CD8+ TILs density may assist in identifying patients with increased sensitivity to NCRT and favorable long-term prognosis.

Coexpression of HHLA2 and PD-L1 on Tumor Cells Independently Predicts the Survival of Spinal Chordoma Patients.

BackgroundImmunotherapy only achieves efficacy in some cancer patients, and less is known about other immune checkpoint molecules in chordoma. Here, we aimed to determine the expression of PD-L1, HHLA2, B7H3, IDO-1 and Galectin-9 in spinal chordoma and evaluated their association with tumor infiltrating lymphocytes (TILs), clinicopathological characteristics and survival of patients.MethodsUsing multiplexed quantitative immunofluorescence (QIF), we simultaneously measured the levels of five different immune checkpoint molecules and major TIL subsets in 92 human spinal chordoma samples.ResultsTumor HHLA2 and PD-L1 were positive in 80.0% and 86.0% of cases, respectively. However, B7H3, IDO-1 and Galectin-9 positivity on tumor cells were only seen in 21.0% of cases, despite all showing predominantly stromal expression. Coexpression of these QIF markers in the tumor compartment was scarcely detected except for PD-L1 and HHLA2, which was observed in 69.6% of cases. While tumoral HHLA2 and stromal B7H3 expressions were associated with an aggressive tumor phenotype, suppressive immune response (specifically including elevated PD-1+ TILs level and decreased CD8+ TIL density) and poor prognosis, stromal levels of PD-L1 and Galectin-9 predicted the opposite outcomes. Importantly, HHLA2 and PD-L1 coexpression on tumor cells independently predicted both worse local recurrence-free survival and overall survival.ConclusionThese data provide a better understanding of the immunosuppressive mechanism in chordoma and may be useful for the development of combination or novel immunotherapy approaches aiming to improve therapeutic efficacy and survival.

CD70 in Thymic Squamous Cell Carcinoma: Potential Diagnostic Markers and Immunotherapeutic Targets.

CD70 – a ligand protein of CD27 on lymphocytes – is expressed in a large spectrum of malignancies. It is an attractive target for antibody-based therapy and several clinical trials are currently being conducted. However, there is no evidence regarding the expression of CD70 and its relationship with expression of programmed death ligand-1 (PD-L1) and CD27+ tumor-infiltrating lymphocytes (TIL) in formalin-fixed paraffin-embedded (FFPE) tissues of thymic tumors. FFPE tissues of thymic squamous cell carcinoma (TSCC) (operative specimens, n = 31; biopsy specimens, n = 11), thymoma (n = 60), thymic carcinoid (n = 3), and lung squamous cell carcinoma (LSCC) (n = 30) were analyzed immunohistochemically. Immunoreactivity for CD70 was semi-quantitatively scored according to the proportion of positive tumor cells. Moreover, the densities of CD27-positive intratumoral TIL (iTIL) and stromal TIL of TSCC were assessed and survival was compared. Most TSCC cases (87%; 27/31) were CD70-positive. In contrast, all thymoma and thymic carcinoid cases were CD70-negative. In LSCC cases, CD70-positivity was significantly lower than TSCC cases (20%; 6/30). Biopsy and resected specimens obtained from the same patients demonstrated a consistent staining pattern (6/6 patients). The proportion of CD70-positive TSCC was comparable with those of CD5 (87%) and CD117 (90%). Correlation between CD70 and PD-L1 expression score was observed. There was no significant difference in survival between the CD70-high and CD70-low expression groups. Meanwhile, patients with CD27-positive iTIL-high tumors exhibited better survival than those with iTIL-low tumors. This tendency was weaker in the CD70-high subset. CD70 immunohistochemistry is useful in diagnosing TSCC. CD70 may prevent anti-tumor immunity via CD27. Immunotherapy targeting the CD70–CD27 axis may be a promising option for the treatment of TSCC.

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.

Tumour infiltrating lymphocyte density differs by meningioma type and is associated with prognosis in atypical meningioma

Tumour infiltrating lymphocyte (TIL) density is prognostically significant in various tumours, but few studies have investigated its significance in meningioma. This study aimed to investigate how TIL density differs by meningioma histology and whether it is a predictor of meningioma recurrence. We studied CD3, CD8, CD4, FOXP3 and PD-1 positive (+) TIL density in a continuous cohort of 476 meningiomas resected at Auckland Hospital between 2002 and 2011 using tissue microarrays and computer assisted image analysis. TILs were identified in all meningiomas except one (median CD3+ TIL density across entire cohort 53.0 cells/mm2). Most TILs were CD8+ (median 33.6 cells/mm2) with smaller numbers of CD4+ TILs (median 2.9 cells/mm2). PD-1+ (median 0.32 cells/mm2) and FOXP3+ (median 0.0 cells/mm2) TILs were scarce. Reduced CD3+ (p=0.0066), CD8+ (p=0.0029) and PD-1+ (p=0.0375) TIL density was seen in WHO grade II/III meningioma compared with WHO grade I. Pairwise comparison confirmed statistically significant differences in TIL density existed between meningioma types (CD3, CD8, CD4, p<0.0001; FOXP3, p=0.0096; PD-1, p=0.0090) with chordoid meningioma having the lowest overall CD3+ TIL density (median 12.5 cells/mm2). Despite its low TIL density, chordoid meningioma had a higher FOXP3:CD8 ratio than several meningioma types. Atypical meningioma had a higher FOXP3:CD8 ratio than transitional meningioma (p=0.0045). No association between TIL density and recurrence was seen across the entire cohort or by WHO grade. However, CD3+ and CD8+ TIL density was associated with recurrence in atypical meningioma on multivariable analysis (CD3, p=0.0012; CD8, p=0.0071). A higher CD3+ and CD8+ TIL density was associated with improved recurrence free survival. Our findings suggest CD3+ and CD8+ TIL density is prognostically significant in atypical meningioma. Further investigation of this observation and its biological basis is warranted. The differences in TIL density by meningioma histology may be of relevance in studies of therapeutic immune checkpoint inhibition.

Methylation statuses of NCOR2, PARK2, and ZSCAN12 signify densities of tumor-infiltrating lymphocytes in gastric carcinoma

Individual cell types of human tissues have their own CpG site methylation profiles, which might be utilized for the development of methylation markers to denote tumor-infiltrating lymphocytes (TILs). We aimed to develop DNA methylation markers that recapitulate the densities of TILs in gastric carcinoma (GC). Through genome-wide methylation profiling, NCOR2, PARK2, and ZSCAN12 were found to be highly methylated in CD3-positive and CD8-positive cells and rarely methylated in tumor cells. Scores of the three methylation markers were analyzed for their relationship with the overall survival and recurrence-free survival of patients with advanced GC (n = 471). The scores of three methylation markers were closely associated with densities of CD3-positive or CD8-positive cells at the tumor center or invasive front of GCs and found to be a significant prognostic factor in univariate analysis of overall survival and recurrence-free survival. In multivariate analysis, the highest score showed hazard ratios of 0.513 (CI 0.306–0.857) and 0.434 (CI 0.261–0.720) for overall survival and recurrence-free survival, respectively. The findings suggest that methylation markers signifying TILs might be utilized for the recapitulation of TIL density in GCs and serve as biomarkers for predicting prognosis in patients with GC.

Lymphocytes and sinus histiocytosis in tumor and matched lymph nodes as predictors of survival in non-small-cell lung cancer.

Aim: To analyzeimmune cell populations in non-small-cell lung cancer (NSCLC) tumors and matched tumor-bearingand non-tumor-bearing lymph nodes (ntbLNs) to predict prognosis. Patients & methods: 71 patients with long-term disease-free survival and 80 patients with relapse within 3 years were included in this study. We used Cox regression to identify factors associated with overall survival (OS) and progression-free survival (PFS). Results: Sinus histiocytosis and tumor-infiltrating lymphocyte density in the tumor were positively associated with PFS and OS. CD4 expression in node 1 (hazard ratio =0.72; p=0.02) and node 2 (hazard ratio=0.91; p=0.04) ntbLNs were positively correlated with OS and PFS, respectively. Discussion: Immunological markers in ntbLNs could be used to predict survival in NSCLC.