Study of Tumor-Infiltrating Lymphocytes in Breast Carcinoma and Their Association With Pathological and Prognostic Factors and Pathological Tumor-Node-Metastasis (pTNM) Staging.

OBJECTIVES: Breast carcinoma is the second most frequent type of cancer globally, with an estimated 2.08 million new carcinoma cases identified in 2018. Breast cancer prognosis is influenced by a number of variables, including the patient's age, morphological variant, stromal inflammatory reaction, elastotic, fibrotic focus, lymphovascular emboli, recurrence of tumor, etc. Recently, the morphological evaluation and extent of tumor-infiltrating lymphocytes (TIL) have also been studied in breast cancer. An attempt is being made to understand the role of TIL in determining the prognostication of carcinoma breast. Thus, the goal of the current academic study is to assess TIL in breast carcinoma. MATERIALS AND METHOD: The study was performed at a medical institution's pathology department, which covered newly diagnosed cases of infiltrating ductal carcinoma of the breast on histopathology during the January to December 2019 time frame. The gross and hematoxyline-eosin-stained paraffin sections were studied for histopathological examination. RESULTS: The study included 50 cases of infiltrating ductal carcinoma of the breast with a female-to-male ratio of 24:1. Stromal TIL was negative (0-10%) in 12 cases, while was positive (11-100 %) in 38 cases. The results of the receiver operating characteristic (ROC) curve study indicated that the specificity was 70.7% and the sensitivity was 85.3% when the cutoff of stromal TIL <11% was used to predict the live status of patients. CONCLUSION: Stromal TIL is an important parameter that must be reported in breast carcinoma cases. Positive stromal TIL shows a statistically significant difference with pathological tumor-node-metastasis (pTNM) staging, tumor laterality, size of the tumor, and involvement of nipple and areola.

Triple negative breast cancer: Immunogenicity, tumor microenvironment, and immunotherapy.

Triple negative breast cancer (TNBC) is a biologically diverse subtype of breast cancer characterized by genomic and transcriptional heterogeneity and exhibiting aggressive clinical behaviour and poor prognosis. In recent years, emphasis has been placed on the identification of mechanisms underlying the complex genomic and biological profile of TNBC, aiming to tailor treatment strategies. High immunogenicity, specific immune activation signatures, higher expression of immunosuppressive genes and higher levels of stromal Tumor Infiltrating Lymphocytes, constitute some of the key elements of the immune driven landscape associated with TNBC. The unprecedented response of TNBC to immunotherapy has undoubtedly changed the standard of care in this disease both in the early and the metastatic setting. However, the extent of interplay between immune infiltration and mutational signatures in TNBC is yet to be fully unravelled. In the present review, we present clinical evidence on the immunogenicity and tumour microenvironment influence on TNBC progression and the current treatment paradigms in TNBC based on immunotherapy.

Insulin-like growth factor 2 receptor is a key immune-related gene that is correlated with a poor prognosis in patients with triple-negative breast cancer: A bioinformatics analysis.

Background: Immunotherapy plays an important role in the treatment of triple-negative breast cancer (TNBC). This study aimed to identify immune-related genes that are associated with the prognosis of patients with TNBC as possible targets of immunotherapy, alongside their related tumor-infiltrating lymphocytes (TILs). Methods: The clinical data and gene expression profiles of patients with breast cancer were extracted from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and divided into training (n = 1,053) and verification (n = 508) groups. CIBERSORT was used to predict the differences in immune cell infiltration in patient subsets that were stratified according to risk. Gene Ontology (GO) enrichment analysis was used to identify pathways associated with immune-related genes in patient subsets that were stratified according to risk. The clinical data and insulin-like growth factor 2 receptor (IGF2R) expression profiles of patients with breast cancer were extracted from METABRIC. The expression of IGF2R and TILs were evaluated in a cohort containing 282 untreated patients with TNBC. The correlations of IGF2R expression, TILs, and clinicopathological parameters with patient prognosis were analyzed in the whole cohort. Results: The prognostic model, which was composed of 26 immune-related gene pairs, significantly distinguished between high- and low-risk patients. Univariate and multivariate analyses indicated that the model was an independent prognostic factor for breast cancer. Among the identified genes, the expression of IGF2R significantly distinguished between high- and low-risk patients in TCGA (P = 0.008) and in METABRIC patients (P < 0.001). The expression of IGF2R was significantly associated with clinical risk factors such as TNBC, estrogen receptor (ER)-negative expression, human epidermal growth factor receptor 2 (HER2)-positive expression, and age ≤60 years old in METABRIC patients. In addition, the patients with IGF2R-positive expression had lower disease-free survival (DFS) rates than those with IGF2R-negative expression in the TNBC cohort (67.8% vs. 78.5%, P = 0.023). IGF2R expression also was significantly negatively correlated with TILs, particularly with CD8+ TILs and CD19+ TILs in the cohort of patients with TNBC. Conclusion: IGF2R can be used as an indicator of a poor prognosis in patients with TNBC and as a potential target and research direction for TNBC immunotherapy in the future.

Transcriptome Analysis of Tumor-Infiltrating Lymphocytes Identifies NK Cell Gene Signatures Associated With Lymphocyte Infiltration and Survival in Soft Tissue Sarcomas.

Purpose: Clinical successes using current T-cell based immunotherapies have been limited in soft tissue sarcomas (STS), while pre-clinical studies have shown evidence of natural killer (NK) cell activity. Since tumor immune infiltration, especially tumor-infiltrating lymphocytes, is associated with improved survival in most solid tumors, we sought to evaluate the gene expression profile of tumor and blood NK and T cells, as well as tumor cells, with the goal of identifying potential novel immune targets in STS. Experimental Design: Using fluorescence-activated cell sorting, we isolated blood and tumor-infiltrating CD3-CD56+ NK and CD3+ T cells and CD45- viable tumor cells from STS patients undergoing surgery. We then evaluated differential gene expression (DGE) of these purified populations with RNA sequencing analysis. To evaluate survival differences and validate primary DGE results, we also queried The Cancer Genome Atlas (TCGA) database to compare outcomes stratified by bulk gene expression. Results: Sorted intra-tumoral CD3+ T cells showed significant upregulation of established activating (CD137) and inhibitory genes (TIM-3) compared to circulating T cells. In contrast, intra-tumoral NK cells did not exhibit upregulation of canonical cytotoxic genes (IFNG, GZMB), but rather significant DGE in mitogen signaling (DUSP4) and metabolic function (SMPD3, SLC7A5). Tumors with higher NK and T cell infiltration exhibited significantly increased expression of the pro-inflammatory receptor TLR4 in sorted CD45- tumor cells. TCGA analysis revealed that tumors with high TLR4 expression (P = 0.03) and low expression of STMN1 involved in microtubule polymerization (P < 0.001) were associated with significantly improved survival. Conclusions: Unlike T cells, which demonstrate significant DGE consistent with upregulation of both activating and inhibiting receptors in tumor-infiltrating subsets, NK cells appear to have more stable gene expression between blood and tumor subsets, with alterations restricted primarily to metabolic pathways. Increased immune cell infiltration and improved survival were positively correlated with TLR4 expression and inversely correlated with STMN1 expression within tumors, suggesting possible novel therapeutic targets for immunotherapy in STS.

Development and therapeutic manipulation of the head and neck cancer tumor environment to improve clinical outcomes.

The clinical response to cancer therapies involves the complex interplay between the systemic, tumoral, and stromal immune response as well as the direct impact of treatments on cancer cells. Each individual's immunological and cancer histories are different, and their carcinogen exposures may differ. This means that even though two patients with oral tumors may carry an identical mutation in TP53, they are likely to have different pre-existing immune responses to their tumors. These differences may arise due to their distinct accessory mutations, genetic backgrounds, and may relate to clinical factors including previous chemotherapy exposure and concurrent medical comorbidities. In isolation, their cancer cells may respond similarly to cancer therapy, but due to their baseline variability in pre-existing immune responses, patients can have different responses to identical therapies. In this review we discuss how the immune environment of tumors develops, the critical immune cell populations in advanced cancers, and how immune interventions can manipulate the immune environment of patients with pre-malignancies or advanced cancers to improve therapeutic outcomes.

Metabolites Profiling of Melanoma Interstitial Fluids Reveals Uridine Diphosphate as Potent Immune Modulator Capable of Limiting Tumor Growth.

Tumor interstitial fluid (TIF) surrounds and perfuses tumors and collects ions, metabolites, proteins, and extracellular vesicles secreted by tumor and stromal cells. Specific metabolites, accumulated within the TIF, could induce metabolic alterations of immune cells and shape the tumor microenvironment. We deployed a metabolomic approach to analyze the composition of melanoma TIF and compared it to the plasma of C57BL6 mice, engrafted or not with B16-melanoma cells. Among the classes of metabolites analyzed, monophosphate and diphosphate nucleotides resulted enriched in TIF compared to plasma samples. The analysis of the effects exerted by guanosine diphosphate (GDP) and uridine diphosphate (UDP) on immune response revealed that GDP and UDP increased the percentage of CD4+CD25+FoxP3- and, on isolated CD4+ T-cells, induced the phosphorylation of ERK, STAT1, and STAT3; increased the activity of NF-κB subunits p65, p50, RelB, and p52; increased the expression of Th1/Th17 markers including IFNγ, IL17, T-bet, and RORγt; and reduced the expression of IL13, a Th2 marker. Finally, we observed that local administrations of UDP in B16-engrafted C57BL6 mice reduced tumor growth and necrotic areas. In addition, UDP-treated tumors showed a higher presence of MHCIIhi tumor-associated macrophage (TAM) and of CD3+CD8+ and CD3+CD4+ tumor-infiltrating T-lymphocytes (TILs), both markers of anti-tumor immune response. Consistent with this, intra-tumoral gene expression analysis revealed in UDP-treated tumors an increase in the expression of genes functionally linked to anti-tumor immune response. Our analysis revealed an important metabolite acting as mediator of immune response, which could potentially represent an additional tool to be used as an adjuvant in cancer immunotherapy.

Tumor Infiltrating Lymphocytes Target HLA-I Phosphopeptides Derived From Cancer Signaling in Colorectal Cancer.

There is a pressing need for novel immunotherapeutic targets in colorectal cancer (CRC). Cytotoxic T cell infiltration is well established as a key prognostic indicator in CRC, and it is known that these tumor infiltrating lymphocytes (TILs) target and kill tumor cells. However, the specific antigens that drive these CD8+ T cell responses have not been well characterized. Recently, phosphopeptides have emerged as strong candidates for tumor-specific antigens, as dysregulated signaling in cancer leads to increased and aberrant protein phosphorylation. Here, we identify 120 HLA-I phosphopeptides from primary CRC tumors, CRC liver metastases and CRC cell lines using mass spectrometry and assess the tumor-resident immunity against these posttranslationally modified tumor antigens. Several CRC tumor-specific phosphopeptides were presented by multiple patients' tumors in our cohort (21% to 40%), and many have previously been identified on other malignancies (58% of HLA-A*02 CRC phosphopeptides). These shared antigens derived from mitogenic signaling pathways, including p53, Wnt and MAPK, and are therefore markers of malignancy. The identification of public tumor antigens will allow for the development of broadly applicable targeted therapeutics. Through analysis of TIL cytokine responses to these phosphopeptides, we have established that they are already playing a key role in tumor-resident immunity. Multifunctional CD8+ TILs from primary and metastatic tumors recognized the HLA-I phosphopeptides presented by their originating tumor. Furthermore, TILs taken from other CRC patients' tumors targeted two of these phosphopeptides. In another cohort of CRC patients, the same HLA-I phosphopeptides induced higher peripheral T cell responses than they did in healthy donors, suggesting that these immune responses are specifically activated in CRC patients. Collectively, these results establish HLA-I phosphopeptides as targets of the tumor-resident immunity in CRC, and highlight their potential as candidates for future immunotherapeutic strategies.

Strategies to Overcome Failures in T-Cell Immunotherapies by Targeting PI3K-δ and -γ.

PI3K-δ and PI3K-γ are critical regulators of T-cell differentiation, senescence, and metabolism. PI3K-δ and PI3K-γ signaling can contribute to T-cell inhibition via intrinsic mechanisms and regulation of suppressor cell populations, including regulatory T-cells and myeloid derived suppressor cells in the tumor. We examine an exciting new role for using selective inhibitors of the PI3K δ- and γ-isoforms as modulators of T-cell phenotype and function in immunotherapy. Herein we review the current literature on the implications of PI3K-δ and -γ inhibition in T-cell biology, discuss existing challenges in adoptive T-cell therapies and checkpoint blockade inhibitors, and highlight ongoing efforts and future directions to incorporate PI3K-δ and PI3K-γ as synergistic T-cell modulators in immunotherapy.

Epigenetic strategies synergize with PD-L1/PD-1 targeted cancer immunotherapies to enhance antitumor responses.

Immunotherapy strategies targeting the programmed cell death ligand 1 (PD-L1)/programmed cell death 1 (PD-1) pathway in clinical treatments have achieved remarkable success in treating multiple types of cancer. However, owing to the heterogeneity of tumors and individual immune systems, PD-L1/PD-1 blockade still shows slow response rates in controlling malignancies in many patients. Accumulating evidence has shown that an effective response to anti-PD-L1/anti-PD-1 therapy requires establishing an integrated immune cycle. Damage in any step of the immune cycle is one of the most important causes of immunotherapy failure. Impairments in the immune cycle can be restored by epigenetic modification, including reprogramming the environment of tumor-associated immunity, eliciting an immune response by increasing the presentation of tumor antigens, and by regulating T cell trafficking and reactivation. Thus, a rational combination of PD-L1/PD-1 blockade and epigenetic agents may offer great potential to retrain the immune system and to improve clinical outcomes of checkpoint blockade therapy.

Immune signature of tumor infiltrating immune cells in renal cancer.

Tumor-associated immune cells have been discussed as an essential factor for the prediction of the outcome of tumor patients. Lymphocyte-specific genes are associated with a favorable prognosis in colorectal cancer but with poor survival in renal cell carcinoma (RCC). Flow cytometric analyses combined with immunohistochemistry were performed to study the phenotypic profiles of tumor infiltrating lymphocytes (TIL) and the frequency of T cells and macrophages in RCC lesions. Data were correlated with clinicopathological parameters and survival of patients. Comparing oncocytoma and clear cell (cc)RCC, T cell numbers as well as activation-associated T cell markers were higher in ccRCC, whereas the frequency of NK cells was higher in oncocytoma. An intratumoral increase of T cell numbers was found with higher tumor grades (G1:G2:G3/4 = 1:3:4). Tumor-associated macrophages slightly increased with dedifferentiation, although the macrophage-to-T cell ratio was highest in G1 tumor lesions. A high expression of CD57 was found in T cells of early tumor grades, whereas T cells in dedifferentiated RCC lesions expressed higher levels of CD69 and CTLA4. TIL composition did not differ between older (>70 y) and younger (<58 y) patients. Enhanced patients' survival was associated with a higher percentage of tumor infiltrating NK cells and Th1 markers, e.g. HLA-DR+ and CXCR3+ T cells, whereas a high number of T cells, especially with high CD69 expression correlated with a worse prognosis of patients. Our results suggest that immunomonitoring of RCC patients might represent a useful tool for the prediction of the outcome of RCC patients.

IL-12 secreting tumor-targeted chimeric antigen receptor T cells eradicate ovarian tumors in vivo.

A novel approach for the treatment of ovarian cancer includes immunotherapy with genetically engineered T cells targeted to ovarian cancer cell antigens. Using retroviral transduction, T cells can be created that express an artificial T cell receptor (TCR) termed a chimeric antigen receptor (CAR). We have generated a CAR, 4H11-28z, specific to MUC-16ecto antigen, which is the over-expressed on a majority of ovarian tumor cells and is the retained portion of MUC-16 after cleavage of CA-125. We previously demonstrated that T cells modified to express the 4H11-28z CAR eradicate orthotopic human ovarian cancer xenografts in SCID-Beige mice. However, despite the ability of CAR T cells to localize to tumors, their activation in the clinical setting can be inhibited by the tumor microenvironment, as is commonly seen for endogenous antitumor immune response. To potentially overcome this limitation, we have recently developed a construct that co-expresses both MUC16ecto CAR and IL-12 (4H11-28z/IL-12). In vitro, 4H11-28z/IL-12 CAR T cells show enhanced proliferation and robust IFNγ secretion compared to 4H11-28z CAR T cells. In SCID-Beige mice with human ovarian cancer xenografts, IL-12 secreting CAR T cells exhibit enhanced antitumor efficacy as determined by increased survival, prolonged persistence of T cells, and higher systemic IFNγ. Furthermore, in anticipation of translating these results into a phase I clinical trial which will be the first to study IL-12 secreting CAR T cells in ovarian cancer, an elimination gene has been included to allow for deletion of CAR T cells in the context of unforeseen or off-tumor on-target toxicity.

Are BiTEs the "missing link" in cancer therapy?

Conventional treatment for cancer routinely includes surgical resection and some combination of chemotherapy and radiation. These approaches are frequently accompanied by unintended and highly toxic collateral damage to healthy tissues, which are offset by only marginal prognostic improvements in patients with advanced cancers. This unfortunate balance has driven the development of novel therapies that aim to target tumors both safely and efficiently. Over the past decade, mounting evidence has supported the therapeutic utility of T-cell-centered cancer immunotherapy, which, in its various iterations, has been shown capable of eliciting highly precise and robust antitumor responses both in animal models and human trials. The identification of tumor-specific targets has further fueled a growing interest in T-cell therapies given their potential to circumvent the non-specific nature of traditional treatments. Of the several strategies geared toward achieving T-cell recognition of tumor, bispecific antibodies (bsAbs) represent a novel class of biologics that have garnered enthusiasm in recent years due to their versatility, specificity, safety, cost, and ease of production. Bispecific T-cell Engagers (BiTEs) are a subclass of bsAbs that are specific for CD3 on one arm and a tumor antigen on the second. As such, BiTEs function by recruiting and activating polyclonal populations of T-cells at tumor sites, and do so without the need for co-stimulation or conventional MHC recognition. Blinatumomab, a well-characterized BiTE, has emerged as a promising recombinant bscCD19×CD3 construct that has demonstrated remarkable antitumor activity in patients with B-cell malignancies. This clinical success has resulted in the rapid extension of BiTE technology against a greater repertoire of tumor antigens and the recent US Food and Drug Administration's (FDA) accelerated approval of blinatumomab for the treatment of a rare form of acute lymphoblastic leukemia (ALL). In this review, we dissect the role of T-cell therapeutics in the new era of cancer immunotherapy, appraise the value of CAR T-cells in the context of solid tumors, and discuss why the BiTE platform may rescue several of the apparent deficits and shortcomings of competing immunotherapies to support its widespread clinical application.

Unleashing the immune system: PD-1 and PD-Ls in the pre-treatment tumor microenvironment and correlation with response to PD-1/PD-L1 blockade.

Focal tumor cell PD-L1 expression adjacent to TIL can be used as a surrogate marker of an ongoing antitumor host response, which may be unleashed by PD-1 blockade. Tumor cell PD-L1 expression is superior to TIL PD-1 expression and the presence of TIL alone, when predicting response to anti-PD-1 therapy.

Interleukin-6 receptor and its ligand interleukin-6 are opposite markers for survival and infiltration with mature myeloid cells in ovarian cancer.

An increased level of interleukin-6 (IL-6) in epithelial ovarian cancer (EOC) is correlated with a worse prognosis. IL-6 stimulates tumor-growth and inflammation. We investigated the intricate interaction between the IL-6 signaling pathway and tumor-infiltrating myeloid cells (TIMs) to determine their prognostic impact in EOC. 160 EOC samples were analyzed for the expression of IL-6, its receptor (IL-6R) and downstream signaling via pSTAT3 by immunohistochemistry. Triple color immunofluorescence confocal microscopy was used to identify myeloid cell populations by CD14, CD33, and CD163. The relationship between these markers, tumor-infiltrating immune cells, clinical-pathological characteristics and survival was investigated. EOC displayed a dense infiltration with myeloid cells, in particular of the CD163+ type. The distribution pattern of all myeloid subtypes was comparable among the different histological subtypes. Analysis of the tumor cells revealed a high expression of IL-6R in 15% and of IL-6 in 23% of patients. Interestingly, tumors expressing IL-6 or IL-6R formed two different groups. Tumors with a high expression of IL-6R displayed low mature myeloid cell infiltration and a longer disease-specific survival (DSS), especially in late stage tumors. High expression of IL-6R was an independent prognostic factor for survival by multivariate analyses (hazard ratio = 0.474, p = 0.011). In contrast, tumors with high epithelial IL-6 expression displayed a dense infiltration of mature myeloid cells and were correlated with a shorter DSS. Furthermore, in densely CD8+ T-cell infiltrated tumors, the ratio between these lymphoid cells and CD163+ myeloid cells was predictive for survival. Thus, IL-6 and IL-6R are opposite markers for myeloid cell infiltration and survival.