Immunochemistry-based quantification of tumor-infiltrating lymphocytes and immunoscore as prognostic biomarkers in bladder cancer.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) and the derived immunoscore (IS) have gained considerable attention over the last decade as prognostic markers in many solid cancers. However, in bladder cancer (BC), their prognostic value is not clearly established. METHODS: The present study aimed to quantify the TILs rates in BC, assess the derived immunoscore, and investigate their prognostic value. An immunochemistry-based quantification of the different subtypes of TILS was performed on paraffin-embedded blocks from patients with invasive urothelial carcinoma of the bladder. We have assessed the rates of TILs, respectively, on peri-tumoral (PT) and intra-tumoral (IT) areas and calculated for each case the corresponding IS which is the index: CD8+/CD3+ TILs. The IS was then classified as low (I0, I1) or high (I2, I3, I4). We included 30 cases in the analysis. RESULTS: The median age of patients was 65 years with a sex ratio of 9. TILs densities and distribution were significantly variable between IT and PT areas CD3+ (p = 0.03) and CD8+ (p = 0.004) with the highest rates on the PT areas. In univariate analysis, a low density of CD8+ TILs was significantly associated with an advanced age (p = 0.05), with the presence of lympho-vascular invasion (p = 0.02) and with the absence of specific histological subtype (p = 0.05). A low immunoscore was significantly associated with the presence of lympho-vascular invasion (p = 0.004). No significant association was found between TILs subpopulations, the IS, and the other clinicopathological and survival data. The overall survival (OS) and disease-free survival (DFS) medians were slightly superior in highly T (CD3+/CD8+)-cell infiltrated tumors as well as tumors with a high IS densities. However, the univariate analysis showed that TILs and immunoscore did not impact overall survival (OS) and disease-free survival (DFS). CONCLUSION: TILs and immunoscore might be effective prognostic tools in BC. However, standardized quantification methods and further investigation on larger samples are highly recommended to definitively attest the prognostic value of TILs and IS in BC.

Treatment Response, Tumor Infiltrating Lymphocytes and Clinical Outcomes in Inflammatory Breast Cancer-Treated with Neoadjuvant Systemic Therapy.

Inflammatory breast cancer (IBC) is a rare (1%-5%), aggressive form of breast cancer, accounting for approximately 10% of breast cancer mortality. In the localized setting, standard of care is neoadjuvant chemotherapy (NACT) ± anti-HER2 therapy, followed by surgery. Here we investigated associations between clinicopathologic variables, stromal tumor-infiltrating lymphocytes (sTIL), and pathologic complete response (pCR), and the prognostic value of pCR. We included 494 localized patients with IBC treated with NACT from October 1996 to October 2021 in eight European hospitals. Standard clinicopathologic variables were collected and central pathologic review was performed, including sTIL. Associations were assessed using Firth logistic regression models. Cox regressions were used to evaluate the role of pCR and residual cancer burden (RCB) on disease-free survival (DFS), distant recurrence-free survival (DRFS), and overall survival (OS). Distribution according to receptor status was as follows: 26.4% estrogen receptor negative (ER-)/HER2-; 22.0% ER-/HER2+; 37.4% ER+/HER2-, and 14.1% ER+/HER2+. Overall pCR rate was 26.3%, being highest in the HER2+ groups (45.9% for ER-/HER2+ and 42.9% for ER+/HER2+). sTILs were low (median: 5.3%), being highest in the ER-/HER2- group (median: 10%). High tumor grade, ER negativity, HER2 positivity, higher sTILs, and taxane-based NACT were significantly associated with pCR. pCR was associated with improved DFS, DRFS, and OS in multivariable analyses. RCB score in patients not achieving pCR was independently associated with survival. In conclusion, sTILs were low in IBC, but were predictive of pCR. Both pCR and RCB have an independent prognostic role in IBC treated with NACT. SIGNIFICANCE: IBC is a rare, but very aggressive type of breast cancer. The prognostic role of pCR after systemic therapy and the predictive value of sTILs for pCR are well established in the general breast cancer population; however, only limited information is available in IBC. We assembled the largest retrospective IBC series so far and demonstrated that sTIL is predictive of pCR. We emphasize that reaching pCR remains of utmost importance in IBC.

Association between CD8+ tumor-infiltrating lymphocytes and prognosis of non-small cell lung cancer patients treated with PD-1/PD-L1 inhibitors: a systematic review and meta-analysis.

BACKGROUND: A meta-analysis method was used to investigate the prognostic value of CD8+ tumor-infiltrating lymphocytes (TILs) in non-small cell lung cancer (NSCLC) patients treated with PD-1/PD-L1 inhibitors. METHODS: A database search of PubMed, Embase, Web of Science and Cochrane Library up until 7 February, 2023. A clinical study on the relationship between CD8+ TILs and PD-1/PD-L1 inhibitors in the therapeutics of NSCLC. RevMan 5.3 and StataMP 17.0 software were used for meta-analysis. The outcome indicators incorporated overall survival (OS), progression-free survival (PFS) and objective response rate (ORR). RESULTS: Nineteen articles with 1488 patients were included. The analysis results showed that high CD8+ TILs were associated with better OS (HR = 0.60, 95% CI: 0.46-0.77; P < 0.0001), PFS (HR = 0.68, 95% CI: 0.53-0.88; P = 0.003) and ORR (OR = 2.26, 95% CI: 1.52-3.36; P < 0.0001) in NSCLC patients treated with PD-1/PD-L1 inhibitors. Subgroup analysis indicated that patients with high CD8+ TILs had good clinical prognostic benefits whether the location of CD8+ TILs was intratumoral or stromal, and compared with East Asian, high CD8+ TILs in Caucasians showed a better prognosis. High CD8+ TILs in peripheral blood did not improve OS (HR = 0.83, 95% CI: 0.69-1.01; P = 0.06) and PFS (HR = 0.93, 95% CI: 0.61-1.14; P = 0.76) in NSCLC patients receiving PD-1/PD-L1 inhibitors. CONCLUSION: In spite of the location of CD8+ TILs, high densities of CD8+ TILs were predictive of treatment outcomes in NSCLC patients treated with PD-1/PD-L1 inhibitors. However, high CD8+ TILs in peripheral blood had no predictive effect.

Predictive and Prognostic Role of Tumor-Infiltrating Lymphocytes in Patients with Advanced Breast Cancer Treated with Primary Systemic Therapy.

INTRODUCTION: Tumor-infiltrating lymphocytes (TILs) are gaining recognition as an important immunological biomarker with therapeutic potential in breast cancer. In this cohort study conducted on patients with advanced breast cancer treated with primary systemic therapy (PST), the TILs concentration was correlated with response to PST and survival outcomes. METHODS: Patients with primary breast cancer treated with PST between 2016 and 2020 were included in this study, approved by IEC, and registered on ClinicalTrials.gov (NCT05250336). Tumor core biopsies obtained prior to starting treatment from 489 patients were assessed for the proportion of stromal TILs by standardized method and categorized into low (0-10% immune cells), intermediate (11-59%), and high (≥ 60%) TILs. TIL concentration and complete pathological response (pCR), disease-free survival (DFS), and overall survival (OS) were correlated. RESULTS: Of the 489 patients, 372 matched the eligibility criteria for assessment of TILs and made the final study cohort. Among these, 135 were luminal, 129 HER2-enriched, and 108 triple-negative breast cancers (TNBC). Proportions of patients with high TILs were greater in TNBC (15.7%) and HER2-enriched (9.3%), compared to luminal cancers (4.4%). High TIL concentration was correlated with higher pCR in all subtypes. A pCR was achieved in 33.3, 50, and 52.9% of high TIL patients in luminal, HER2-enriched, and TNBC subtypes, respectively (p < 0.05). High TILs were linked to longer DFS and OS in TNBC and HER2-enriched breast cancers. CONCLUSION: In this first study of its kind from a low- and middle-income country, high TILs concentration was found to be a predictor of response to PST across all breast cancer subtypes. TILs concentration was found predictive of better DFS and OS in TNBC and HER2-enriched cancers. Prognostic role of TILs in luminal cancers was not so apparent.

Prognostic impact of tumor-infiltrating lymphocytes in non-small cell lung carcinomas.

INTRODUCTION: Tumor-infiltrating lymphocytes represent a pivotal component of the host anti-tumor response. Thus, they considerably influence the evolution of cancers including non-small cell lung carcinomas. Even if, this important role is consensual, many discordant results are published in the literature about the prognostic role of the different populations of tumor-infiltrating lymphocytes. The aim of our work was to evaluate the prognostic impact of CD8+, CD4+, and forkhead box protein P3+ lymphocytes in the tumor microenvironment of non-small cell lung carcinomas. METHODS: We conducted a retrospective descriptive study, which included non-small cell lung carcinomas diagnosed in the department of pathology and followed in the medical oncology department of the same hospital between 2011 and 2015. Tumor-infiltrating lymphocytes were analyzed by the immunohistochemical method for forkhead box protein P3, CD4, and CD8. Intratumoral and stromal-labeled lymphocytes were quantified by manual counting at high magnification (×400). Forkhead box protein P3+/CD8+, forkhead box protein P3+/CD4+, and CD8+/CD4+ ratios were subsequently calculated. The prognostic value of tumor-infiltrating lymphocytes was assessed in respect of overall survival, recurrence-free survival, and relapse-free survival. RESULTS: Thirty-nine patients were included. The mean age of patients was 59.6 years. A complete surgical resection (p = 0.009), and a CD8/CD4 ratio (p = 0.008) were prognostic factors for overall survival. Complete surgical resection (p = 0.003), the forkhead box protein P3/CD8 (p = 0.005), and forkhead box protein P3/CD4 (p = 0.037) ratios were prognostic factors for recurrence-free survival. The CD8+ tumor-infiltrating lymphocytes rate (p = 0.037) was a prognostic factor for relapse-free survival with a threshold of 67.8/high power field. Microscopic subtype (p = 0.037) was a prognostic factor for relapse-free survival when only adenocarcinoma and squamous cell carcinoma were considered. In multivariate analysis, age (p = 0.004) and a CD8/CD4 ratio (p = 0.016) were independent predictors of overall survival. CONCLUSION: Despite the limitations of our study, our results confirm the prognostic value of tumor-infiltrating lymphocytes in non-small cell lung carcinomas and the importance of the combined quantification of their different subpopulations.

CD11c+CD163+ Cells and Signal Transducer and Activator of Transcription 3 (STAT3) Expression Are Common in Melanoma Leptomeningeal Disease.

Leptomeningeal disease (LMD) in melanoma patients is associated with significant neurological sequela and has a dismal outcome, with survival measured typically in weeks. Despite the therapeutic benefit of targeted therapies and immunotherapies for Stage IV melanoma, patients with LMD do not typically benefit. A deeper understanding of the tumor microenvironment (TME) of LMD may provide more appropriate therapeutic selection. A retrospective analysis of subjects who underwent surgical resection with LMD (n=8) were profiled with seven color multiplex staining to evaluate the expression of the global immune suppressive hub - the signal transducer and activator of transcription 3 (STAT3) and for the presence of CD3+ T cells, CD68+ monocyte-derived cells, CD163+ immune suppressive macrophages, and CD11c+ cells [potential dendritic cells (DCs)] in association with the melanoma tumor marker S100B and DAPI for cellular nuclear identification. High-resolution cellular imaging and quantification was conducted using the Akoya Vectra Polaris. CD11c+ cells predominate in the TME (10% of total cells), along with immunosuppressive macrophages (2%). Another potential subset of DCs co-expressing CD11c+ and the CD163+ immunosuppressive marker is frequently present (8/8 of specimens, 8%). Occasional CD3+ T cells are identified, especially in the stroma of the tumor (p=0.039). pSTAT3 nuclear expression is heterogeneous in the various immune cell populations. Occasional immune cluster interactions can be seen in the stroma and on the edge. In conclusion, the TME of LMD is largely devoid of CD3+ T cells but is enriched in immune suppression and innate immunity.

Rapid Identification of the Tumor-Specific Reactive TIL Repertoire via Combined Detection of CD137, TNF, and IFNγ, Following Recognition of Autologous Tumor-Antigens.

Detecting the entire repertoire of tumor-specific reactive tumor-infiltrating lymphocytes (TILs) is essential for investigating their immunological functions in the tumor microenvironment. Current in vitro assays identifying tumor-specific functional activation measure the upregulation of surface molecules, de novo production of antitumor cytokines, or mobilization of cytotoxic granules following recognition of tumor-antigens, yet there is no widely adopted standard method. Here we established an enhanced, yet simple, method for identifying simultaneously CD8+ and CD4+ tumor-specific reactive TILs in vitro, using a combination of widely known and available flow cytometry assays. By combining the detection of intracellular CD137 and de novo production of TNF and IFNγ after recognition of naturally-presented tumor antigens, we demonstrate that a larger fraction of tumor-specific and reactive CD8+ TILs can be detected in vitro compared to commonly used assays. This assay revealed multiple polyfunctionality-based clusters of both CD4+ and CD8+ tumor-specific reactive TILs. In situ, the combined detection of TNFRSF9, TNF, and IFNG identified most of the tumor-specific reactive TIL repertoire. In conclusion, we describe a straightforward method for efficient identification of the tumor-specific reactive TIL repertoire in vitro, which can be rapidly adopted in most cancer immunology laboratories.

CTLA-4 Synergizes With PD1/PD-L1 in the Inhibitory Tumor Microenvironment of Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is highly invasive and carries high mortality due to limited therapeutic strategies. In other solid tumors, immune checkpoint inhibitors (ICIs) target cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD1), and the PD1 ligand PD-L1 has revolutionized treatment and improved outcomes. However, the relationship and clinical significance of CTLA-4 and PD-L1 expression in ICC remains to be addressed. Deciphering CTLA-4 and PD-L1 interactions in ICC enable targeted therapy for this disease. In this study, immunohistochemistry (IHC) was used to detect and quantify CTLA-4, forkhead box protein P3 (FOXP3), and PD-L1 in samples from 290 patients with ICC. The prognostic capabilities of CTLA-4, FOXP3, and PD-L1 expression in ICC were investigated with the Kaplan-Meier method. Independent risk factors related to ICC survival and recurrence were assessed by the Cox proportional hazards models. Here, we identified that CTLA-4+ lymphocyte density was elevated in ICC tumors compared with peritumoral hepatic tissues (P <.001), and patients with a high density of CTLA-4+ tumor-infiltrating lymphocytes (TILsCTLA-4 High) showed a reduced overall survival (OS) rate and increased cumulative recurrence rate compared with patients with TILsCTLA-4 Low (P <.001 and P = .024, respectively). Similarly, patients with high FOXP3+ TILs (TILsFOXP3 High) had poorer prognoses than patients with low FOXP3+ TILs (P = .021, P = .034, respectively), and the density of CTLA-4+ TILs was positively correlated with FOXP3+ TILs (Pearson r = .31, P <.001). Furthermore, patients with high PD-L1 expression in tumors (TumorPD-L1 High) and/or TILsCTLA-4 High presented worse OS and a higher recurrence rate than patients with TILsCTLA-4 LowTumorPD-L1 Low. Moreover, multiple tumors, lymph node metastasis, and high TumorPD-L1/TILsCTLA-4 were independent risk factors of cumulative recurrence and OS for patients after ICC tumor resection. Furthermore, among ICC patients, those with hepatolithiasis had a higher expression of CTLA-4 and worse OS compared with patients with HBV infection or undefined risk factors (P = .018). In conclusion, CTLA-4 is increased in TILs in ICC and has an expression profile distinct from PD1/PD-L1. TumorPD-L1/TILsCTLA-4 is a predictive factor of OS and ICC recurrence, suggesting that combined therapy targeting PD1/PD-L1 and CTLA-4 may be useful in treating patients with ICC.

Programmed death ligand-1 and CD8 tumor-infiltrating lymphocytes (TILs) as prognostic predictors in ovarian high-grade serous carcinoma (HGSC).

BACKGROUND: P D-L1 is expressed in tumor cells and plays a crucial role in tumor immune escape. Tumor-infiltrating lymphocytes (TILs) as CD8 T cells contribute to reduced tumor growth. Few studies investigated the prognostic effect of PD-L1 and CD8 TILs in ovarian high-grade serous carcinoma (HGSC). In the present study, we analyzed the expression of PD-L1 and CD8 TILs in HGSC by immunohistochemistry, and results were correlated to prognosis. It was carried on 54 cases of ovarian HGSC who attended the Oncology Centre, Mansoura University, Egypt, from 2012 till 2019. RESULTS: Nearly 60% of cases showed positive PD-L1 expression in tumor cells. Regarding the clinicopathological characteristics, higher PD-L1 expression was found among patients with residual tumor (82.4%) compared to patients with no residual tumor (54.5%), with marginal statistical significance (p 0.07). PD-L1 was significantly associated with CD8 TILs expression. Higher PD-L1 expression was found among tumors with low expression of CD8 TILs with statistically significant difference (p≤0.001). Disease-free survival (DFS) was significantly lower among the group with positive expression of PD-L1 compared to the group with negative expression of PD-L1 (p 0.01), while overall survival (OS) was not associated with PD-L1 expression. On the other hand, the overall survival (OS) in patients with high CD8 expression was significantly higher than patients with low CD8 expression (p 0.043), while DFS was not significantly different among both CD8 TILS groups. CONCLUSIONS: PD-L1 and CD8 TILs may become a promising therapeutic target for patients with ovarian HGSC. More studies are needed to further validate their prognostic effect. Precise identification of patients who will benefit from PD-L1 checkpoint blockade and TILs adaptive immunotherapy is mandatory.

The relation of CD3, CD4, CD8 and PD-1 expression with tumor type and prognosis in epithelial ovarian cancers.

OBJECTIVES: Ovarian cancer is a heterogeneous disease, where chronic inflammation plays a key role in carcinogenesis. In this study, it is aimed to analyze the relationship with prognosis and chemotherapy response to clinicopathologicalnvariables in epithelial ovarian cancers such as proliferation of PD-1 +, CD8 +, CD4 +, CD3 + T-lymphocytes infiltrating the tumor and tumor stroma. MATERIAL AND METHODS: Seventy-six cases diagnosed with primary epithelial ovarian tumor from biopsy or surgical resection materials were included in the study. Immunreactivity of CD3, CD4, CD8, PD1 was evaluated immunohistochemically in lymphocytes in tumor infiltrating lymphocytes and stromal lymphocytes. RESULTS: Seventeen (22.4%) of the cases were Type I, 59 (77.6%) of them were Type II ovarian carcinoma. PD-1 positivity was observed in stromal and intraepithelial lymphocytes in 22 (28.9%) of 76 cases. In the presence of PD-1 + T-lymphocytes that infiltrate tumor and stroma, disease-free survival are shorter (p = 0.037). The presence of stromal CD4 + and CD8 + T-lymphocytes was more common in late stage patients (p = 0.012, p = 0.036; respectively). The disease-free and overall survival rate was statistically significantly shorter in the presence of CD8 + T lymphocytes (p = 0.009, p = 0.003; respectively). CONCLUSIONS: CD3, CD4 and CD8 may contribute to PD-1 mediated tumor control. Anti PD-1 therapy may be an alternative to chemotherapy in PD-1 positive patients. Identifying patients who do not respond to chemotherapy through PD-1 expression prior to immunotherapy will help develop potential personalized immunotherapy.

The prognostic significance of TILs as a biomarker in triple-negative breast cancer: what is the role of TILs in TME of TNBC?

Triple-negative breast cancer (TNBC) accounts for approximately 15-20% of all breast cancers, and its poor response to treatment has been a major problem in the field of breast cancer. In recent years, the application of immune checkpoint inhibitors has introduced a new era of treatment. The IMpassion 130 trial used PD-L1 as a mature biomarker for immunotherapy in metastatic TNBC, but the population screened, which was only patients with positive PD-L1 expression, was too narrow. Otherwise, it could not be determined whether the PD-L1-positive group benefited from immunotherapy in early TNBC, but this was confirmed in the KEYNOTE-522 and IMpassion 031 studies, in which there was no significant difference in the benefit, whether patients were PD-L1-positive or not. Therefore, how to screen more suitable biomarkers for accurate immunotherapy has become a burning and persistent problem to be solved. In fact, immune infiltration has always been our focus in the process of exploring immunotherapy in TNBC, and tumor infiltrating lymphocytes (TILs) have been well-known for decades as a prognostic factor in early TNBC. Furthermore, TILs are positively correlated with both patient survival and pathological complete response (pCR) after neoadjuvant chemotherapy. Recently, increasingly more foundational experiments and clinical trial verifications suggest that TILs could contain more biomarkers. Thus, in this review, we will assess the composition and heterogeneity of TILs, their evaluation standards, their relationship with TNBC prognosis and the prediction ability of different treatment options in TNBC, and their correlation with other biomarkers in the clinical application. We also summarize new studies that show the future potential of TILs.

T Cells Expanded from PD-1+ Peripheral Blood Lymphocytes Share More Clones with Paired Tumor-Infiltrating Lymphocytes.

Both tumor-infiltrating lymphocytes (TIL) and PD-1+ peripheral blood lymphocytes (PBL) are enriched for tumor-reactive clones recognizing known and unknown tumor antigens. However, the relationship between the T-cell receptor-ß (TCRß) repertoires of the TILs and T cells expanded from paired PD-1+ PBLs, and whether T cells expanded from PD-1+ PBLs can be used to treat patients with cancer as TIL substitutes remain unclear. Here, we established a highly efficient protocol to prepare polyclonal T cells from PD-1+ PBLs. A functional T-cell assay and tetramer staining revealed that cells from PD-1+ PBLs were relatively enriched for tumor-reactive T cells. Furthermore, deep TCRß sequencing data revealed that an average of 11.29% (1.32%-29.06%; P = 0.015; n = 8) tumor-resident clonotypes were found in T cells expanded from paired PD-1+ PBLs, and the mean accumulated frequency of TIL clones found in T cells expanded from PD-1+ PBLs was 35.11% (7.23%-78.02%; P = 0.017; n = 8). Moreover, treatment of four patients, who failed multiline therapy and developed acquired resistance to anti-PD-1, with autologous T cells expanded from PD-1+ PBLs combined with anti-PD-1 antibody elicited objective responses from three of them. These results indicate that T cells expanded from PD-1+ PBLs share more clones with paired TILs and could be used to treat patients with cancer as TIL substitutes. SIGNIFICANCE: This study harnesses the tumor reactivity of PD-1+ PBLs, developing a method to expand T cells from these clones as a potential therapeutic strategy and TIL substitute in patients with cancer.See related commentary by Ladle, p. 1940.

PET Imaging of TIGIT Expression on Tumor-Infiltrating Lymphocytes.

PURPOSE: Therapeutic checkpoint inhibitors on tumor-infiltrating lymphocytes (TIL) are being increasingly utilized in the clinic. The T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory receptor expressed on T and natural killer cells. The TIGIT signaling pathway is an alternative target for checkpoint blockade to current PD-1/CTLA-4 strategies. Elevated TIGIT expression in the tumor microenvironment correlates with better therapeutic responses to anti-TIGIT therapies in preclinical models. Therefore, quantifying TIGIT expression in tumors is necessary for determining whether a patient may respond to anti-TIGIT therapy. PET imaging of TIGIT expression on TILs can therefore aid diagnosis and in monitoring therapeutic responses. EXPERIMENTAL DESIGN: Antibody-based TIGIT imaging radiotracers were developed with the PET radionuclides copper-64 (64Cu) and zirconium-89 (89Zr). In vitro characterization of the imaging probes was followed by in vivo evaluation in both xenografts and syngeneic tumor models in mouse. RESULTS: Two anti-TIGIT probes were developed and exhibited immunoreactivity of >72%, serum stability of >95%, and specificity for TIGIT with both mouse TIGIT-expressing HeLa cells and ex vivo-activated primary splenocytes. In vivo, the 89Zr-labeled probe demonstrated superior contrast than the 64Cu probe due to 89Zr's longer half-life matching the TIGIT antibody's pharmacokinetics. The 89Zr probe was used to quantify TIGIT expression on TILs in B16 melanoma in immunocompetent mice and confirmed by ex vivo flow cytometry. CONCLUSIONS: This study develops and validates novel TIGIT-specific 64Cu and 89Zr PET probes for quantifying TIGIT expression on TILs for diagnosis of patient selection for anti-TIGIT therapies.

Atypical BCL6/GATA3+ Primary Cutaneous Acral CD8-Positive T-Cell Lymphoma: A Diagnostic Challenge.

ABSTRACT: Primary cutaneous acral CD8-positive T-cell lymphoma consists of slow-growing nodules in acral sites with a histopathology, suggesting high-grade lymphoma despite the indolent clinical course. It has been recently included in WHO-EORTC classification for primary cutaneous lymphomas as a provisional entity. A correct diagnosis of this entity is important because its differential diagnosis include more aggressive cutaneous lymphomas. We present a 53-year-old woman with an indolent solitary nodule on her right leg, which histopathologically showed features of CD8-positive T-cell lymphoma, although with some peculiarities, including epidermotropism, absence of CD68 expression, and positivity for GATA3 and Bcl6 in neoplastic cells. This case could contribute to better define the spectrum of this rare cutaneous lymphoma.

Prognostic impact of CD73 expression and its relationship to PD-L1 in patients with radically treated pancreatic cancer.

Immune suppressing molecule CD73 is overexpressed in various cancers and associated with poor survival. Little is so far known about the predictive value of CD73 in pancreatic ductal adenocarcinoma (PDAC). The purpose of this study was to investigate the prognostic significance of CD73 in PDAC. The study material consisted of 110 radically treated patients for PDAC. Tissue microarray blocks were constructed and stained immunohistochemically using CD73 antibody. Staining intensity and numbers of stained tumour cells, inflammatory cells, stroma, and blood vessels were assessed. High-level CD73 expression in tumour cells was positively associated with PD-L1 expression, perineural invasion, and histopathological grade. CD73 positivity in tumour-infiltrating lymphocytes was significantly associated with lymph node metastasis. Lymphocytic CD73 positivity was also associated with staining positivity in both stroma and vascular structures. In addition, CD73 positivity in vascular structures and stroma were associated with each other. There were no significant associations between CD73 positive tumour cells and CD73 positivity in any other cell types. PD-L1 expression was associated with CD73 staining positivity in stroma (p = 0.007) and also with histopathological grade (p = 0.033) and T class (p = 0.016) of the primary tumour. CD73 positivity in tumour cells was significantly associated with poor disease-specific (p = 0.021) and overall survival (p = 0.016). In multivariate analysis, CD73 positivity in tumour cells was an independent negative prognostic factor together with histopathological grade, TNM stage, and low immune cell score. In conclusion, high CD73 expression in tumour cells is associated with poor survival in PDAC independently of the number of tumour-infiltrating lymphocytes or TNM stage.

Prognostic impact of CD8-positive tumour-infiltrating lymphocytes and PD-L1 expression in salivary gland cancer.

OBJECTIVES: Aim of the study was to evaluate the prognostic impact of CD8-positive (CD8+) tumour-infiltrating lymphocytes (TILs) and PD-L1 expression on the outcome of patients with malignant salivary gland neoplasms. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded tissue samples and clinicopathological data from patients treated for salivary gland carcinoma in a head and neck cancer centre were retrospectively retrieved. Immunohistochemical staining was applied on sections of 84 specimens of 12 different histological subtypes. Both CD8 and PD-L1 expression were rated by semi-automated cell counts by a digital image analysis programme. Survival analyses were performed by the log-rank test on the univariate level, and the Cox model was applied on the multivariate level. Associations between immunological markers and clinicopathological variables were estimated by the Pearson chi-squared test. Additionally, PD-1 was estimated as an exhaustion marker of CD8+ TILs. RESULTS: Patients exceeding a tumour proportion score ≥5% regarding PD-L1 expression demonstrated a significantly decreased survival, as did individuals with an overall high CD8+ cell density. Particularly, high CD8+ cell counts in the invasive front of the respective tumour tissue significantly coincided with a poor outcome. Also, high numbers of CD8+ TILs significantly matched with a high quantity of PD-1+ TILs. CONCLUSION: CD8+ TILs abundance in the peritumoural microenvironment correlates with impaired outcome of patients with salivary gland carcinoma. The simultaneous negative prognostic impact of PD-L1 expression and presence of PD-1+ TILs advocates an immune checkpoint-controlled mechanism of CD8+ TILs exhaustion for these tumours and paves the way for future treatment strategies.

Assessment of lipid load in tumor-infiltrating Tregs by flow cytometry.

Regulatory T cells (Tregs), expressing the transcription factor Foxp3, are defined as immunosuppressive cells able to modulate a variety of immune cells in order to avoid unwanted and excessive immune responses; however, in the tumor context, Treg function contribute to inhibit immune surveillance, thus promoting cancer progression. In tumor microenvironment, where the availability of metabolic resources is strongly limited, Tregs are expanded and may exploit different metabolic routes to achieve a metabolic advantage, prevailing over effector cells. In this context an important role of lipid metabolism has been described thanks to the possibility to evaluate the intracellular lipid content selectively in tumor-infiltrating Tregs (TUM-Tregs). Taking into account the heterogeneous and complex build of tumor mass, we set-up a combined protocol that optimizes tumor-infiltrating lymphocytes (TIL) extraction from the tissue through a Percoll density gradient, and assesses ex vivo the lipid load in whole TUM-Treg population, evaluating by flow cytometry the incorporation of an intensely fluorescent lipophilic fluorophore able to specifically stain neutral lipids. This method provides an important advantage compared to the traditional technique based on microscopy, whose lipid level evaluation is limited to a tissue section, and hence may not be representative of the entire population.

Does a polarization state exist for mast cells in cancer?

The data of literature are discordant about the role of mast cells in different types of neoplasms. In this paper the authors propose the hypothesis that tumor-associated mast cells may switch to different polarization states, conditioning the immunogenic capacities of the different neoplasms. Anti-inflammatory polarized mast cells should express cytokines such as interleukin-10 (IL-10) and then mast cells number should be inversely related to the intensity of inflammatory infiltrate. On the contrary, when mast cells do not express anti-inflammatory cytokines their number should be directly related to the intensity of the inflammatory infiltrate. In this paper we briefly argue around feasible approaches, based on the retrospective studies of tumor tissue samples from neoplasms considered "immunologically hot" and neoplasms considered "immunologically cold", through immunohistochemistry and immunofluorescence techniques (confocal microscopy). The establishment of the actual existence of a polarization interchange of mast cells, could lead to a new vision in prognostic terms, useful to contrive new approaches in immunotherapy of tumors.

Prognostic impact of p16 and PD-L1 expression in patients with oropharyngeal squamous cell carcinoma receiving a definitive treatment.

AIMS: Limited information is available regarding the precise differences in the tumour immune microenvironment (TIM) of patients with human papilloma virus (HPV)-associated and non-HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). Here, we retrospectively reviewed 137 patients with OPSCC treated with a definitive treatment to identify molecular relationships in the TIM. MATERIALS AND METHODS: We used immunohistochemical analysis to assess p16 status, programmed death ligand 1 (PD-L1) level, and/or CD8+ tumour-infiltrating lymphocyte (TIL) density, followed by prognostic evaluation of these immune-related parameters. RESULTS: Multivariate analyses demonstrated that PD-L1 level on immune cells but not on tumour cells or CD8+ TIL density was a significant predictive factor of disease-free survival (DFS) and overall survival (OS). Additionally, subgroup analyses demonstrated that patients positive for p16 and PD-L1 expression on immune cells had favourable DFS and OS, whereas patients negative for p16 and PD-L1 expression on immune cells showed worse DFS and OS. CONCLUSIONS: We demonstrated that PD-L1 expression on immune cells but not tumour cells might represent a useful prognostic biomarker in patients with OPSCC receiving a definitive treatment. We propose that a co-assessment of p16 and PD-L1 expression on immune cells would have greater prognostic potential compared with evaluation of each factor alone in patients with OPSCC.

Clinical implications of heterogeneity in PD-L1 immunohistochemical detection in hepatocellular carcinoma: the Blueprint-HCC study.

Programmed cell death ligand-1 immunohistochemical detection (PD-L1 IHC) is a putative predictor of response to PD-1/PD-L1-targeted checkpoint inhibitors. However, there is no gold standard assay in hepatocellular carcinoma (HCC). We evaluated 5 PD-L1 IHC assay platforms (E1LN3, 28-8, 22c3, SP263 and SP142) in 100 HCCs reporting PD-L1 expression in malignant (M) and tumour-infiltrating immune cells (TICs) and non-tumorous cirrhotic tissues (NTICs). We found substantial inter-assay heterogeneity in detecting PD-L1 expression in M (R2 = 0.080-0.921), TICs (Cohen's κ = 0.175-0.396) and NTICs (κ = 0.004-0.505). Such diversity may impact on the reliability and reproducibility of PD-L1 IHC assays as a predictor of response to immune checkpoint inhibitors.