Signaling via a CD28/CD40 chimeric costimulatory antigen receptor (CoStAR™), targeting folate receptor alpha, enhances T cell activity and augments tumor reactivity of tumor infiltrating lymphocytes.

Transfer of autologous tumor infiltrating lymphocytes (TIL) to patients with refractory melanoma has shown clinical efficacy in a number of trials. However, extending the clinical benefit to patients with other cancers poses a challenge. Inefficient costimulation in the tumor microenvironment can lead to T cell anergy and exhaustion resulting in poor anti-tumor activity. Here, we describe a chimeric costimulatory antigen receptor (CoStAR) comprised of FRα-specific scFv linked to CD28 and CD40 intracellular signaling domains. CoStAR signaling alone does not activate T cells, while the combination of TCR and CoStAR signaling enhances T cell activity resulting in less differentiated T cells, and augmentation of T cell effector functions, including cytokine secretion and cytotoxicity. CoStAR activity resulted in superior T cell proliferation, even in the absence of exogenous IL-2. Using an in vivo transplantable tumor model, CoStAR was shown to improve T cell survival after transfer, enhanced control of tumor growth, and improved host survival. CoStAR could be reliably engineered into TIL from multiple tumor indications and augmented TIL activity against autologous tumor targets both in vitro and in vivo. CoStAR thus represents a general approach to improving TIL therapy with synthetic costimulation.

Epithelial ovarian cancer is infiltrated by activated effector T cells co-expressing CD39, PD-1, TIM-3, CD137 and interacting with cancer cells and myeloid cells.

Introduction: Despite predicted efficacy, immunotherapy in epithelial ovarian cancer (EOC) has limited clinical benefit and the prognosis of patients remains poor. There is thus a strong need for better identifying local immune dynamics and immune-suppressive pathways limiting T-cell mediated anti-tumor immunity. Methods: In this observational study we analyzed by immunohistochemistry, gene expression profiling and flow cytometry the antigenic landscape and immune composition of 48 EOC specimens, with a focus on tumor-infiltrating lymphocytes (TILs). Results: Activated T cells showing features of partial exhaustion with a CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ surface profile were exclusively present in EOC specimens but not in corresponding peripheral blood or ascitic fluid, indicating that the tumor microenvironment might sustain this peculiar phenotype. Interestingly, while neoplastic cells expressed several tumor-associated antigens possibly able to stimulate tumor-specific TILs, macrophages provided both co-stimulatory and inhibitory signals and were more abundant in TILs-enriched specimens harboring the CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ signature. Conclusion: These data demonstrate that EOC is enriched in CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ T lymphocytes, a phenotype possibly modulated by antigen recognition on neoplastic cells and by a combination of inhibitory and co-stimulatory signals largely provided by infiltrating myeloid cells. Furthermore, we have identified immunosuppressive pathways potentially hampering local immunity which might be targeted by immunotherapeutic approaches.

Targeting myeloid-derived suppressor cells with gemcitabine to enhance efficacy of adoptive cell therapy in bladder cancer.

Background: New therapeutics in development for bladder cancer need to address the recalcitrant nature of the disease. Intravesical adoptive cell therapy (ACT) with tumor infiltrating lymphocytes (TIL) can potentially induce durable responses in bladder cancer while maximizing T cells at the tumor site. T cells infused into the bladder directly encounter immunosuppressive populations, such as myeloid derived suppressor cells (MDSCs), that can attenuate T cell responses. Intravesical instillation of gemcitabine can be used as a lymphodepleting agent to precondition the bladder microenvironment for infused T cell products. Methods: Urine samples from bladder cancer patients and healthy donors were analyzed by flow cytometry and cytometric bead array for immune profiling and cytokine quantification. MDSCs were isolated from the urine and cocultured with stimulated T cells to assess effects on proliferation. An orthotopic murine model of bladder cancer was established using the MB49-OVA cell line and immune profiling was performed. MDSCs from tumor-bearing mice were cocultured with OT-I splenocytes to assess T cell proliferation. Mice received intravesical instillation of gemcitabine and depletion of immune cells was measured via flow cytometry. Bladder tumor growth of mice treated with intravesical gemcitabine, OT-I transgenic T cells, or combination was monitored via ultrasound measurement. Results: In comparison to healthy donors, urine specimen from bladder cancer patients show high levels of MDSCs and cytokines associated with myeloid chemotaxis, T cell chemotaxis, and inflammation. T cells isolated from healthy donors were less proliferative when cocultured with MDSCs from the urine. Orthotopic murine bladder tumors also presented with high levels of MDSCs along with enrichment of cytokines found in the patient urine samples. MDSCs isolated from spleens of tumor-bearing mice exerted suppressive effects on the proliferation of OT-I T cells. Intravesical instillation of gemcitabine reduced overall immune cells, MDSCs, and T cells in orthotopic bladder tumors. Combination treatment with gemcitabine and OT-I T cells resulted in sustained anti-tumor responses in comparison to monotherapy treatments. Conclusion: MDSCs are enriched within the microenvironment of bladder tumors and are suppressive to T cells. Gemcitabine can be used to lymphodeplete bladder tumors and precondition the microenvironment for intravesical ACT.

Dual-Armed Oncolytic Myxoma Virus Encoding IFN-γ and CD47 Promotes Lymphocyte Infiltration and Tumor Suppression of Syngeneic Murine Melanoma.

Myxoma virus (MyxV) is a rabbit-specific poxvirus. However, its ability to selectively target tumor cells has established it as a safe and effective anticancer therapy. To strengthen its preclinical efficacy, transgenes that can prolong cancer cell infection and enhance anti-tumor effector functions are currently being investigated. We engineered MyxV armed with CD47, to turn on a 'do not eat me' signal within infected cells with actively replicating viruses, and with IFN-γ to further activate host immune anticancer responses. Tumor suppressive activities were significantly enhanced by the dual-armed MyxV_CD47/IFN-γ compared to parental MyxV or single-armed MyxV_CD47 or MyxV_IFN-γ. In addition, significant increases in IFN-γ+ CD8+T-cells and CD4+ T-cells populations within tumor-infiltrating lymphocytes (TIL) were observed after MyxV_CD47/IFN-γ treatment. Notably, all groups treated with MyxV showed a marked reduction in Foxp3+ CD4+ regulatory T-cells (Tregs) within TIL. We also show that MyxV infection induces PD-L1 up-regulation in cancer cells, and combinational treatment of MyxV with anti-mouse PD-L1 antibodies (αPD-L1) further controlled tumor burden and increased survival in the syngeneic melanoma model B16F10. Our data demonstrate that a CD47 and IFNγ dual-armed MyxV is an effective oncolytic viral immunotherapeutic. These findings strongly support further preclinical investigations to develop next-generation MyxV-based immunotherapy approaches.

Personalized Immunotherapy of Patients: Defining by Single-cell RNA-seq with Artificial Intelligence.

Immunotherapy, including immune cell therapy and targeted therapy, is gradually developed through the ongoing discovery of molecular compounds or immune cells. Choosing the best one or the best combination of target compounds and immune-cell therapy is a challenge for clinical scientists and clinicians. We have found variable efficacy individually after tumor-infiltrating lymphocyte (TIL) therapy, and now TILs have been discovered in a group of heterogeneous immune cells. To select the best immunotherapy for each patient, we started to study TIL genomics, including single-cell mRNA differential display from TIL published in 2007 and single-cell RNA-seq from TIL published in 2013, set up TIL quantitative network in 2015, researched machine-learning model for immune therapy in 2022. These manual reports single-cell RNA-seq data combined with machine learning to evaluate the optimal compounds and immune cells for individual patients. The machine-learning model, one of artificial intelligence, can estimate targeting genomic variance from single-cell RNA-seq so that they can cover thirteen kinds of immune cell therapies and ongoing FDA-approved targeted therapies such as PD1 inhibitors, PDL1 inhibitors, and CTLA4 inhibitors, as well as other different treatments such as HDACI or DNMT1 inhibitors, FDA-approved drugs. Moreover, also cover Phase-1, Phase-2, Phase-3, and Phase-4 of clinical trials, such as TIL, CAR T-cells, TCR T-cells. Single-cell RNA-seq with an Artificial intelligence estimation system is much better than our published models from microarrays or just cell therapy. The medical goal is to address three issues in clinical immunotherapy: the increase of efficacy; the decrease of adverse effects and the decrease of the cost in clinical applications.

Association of Molecular Profiles and Mutational Status With Distinct Histological Lung Adenocarcinoma Subtypes. An Analysis of the LACE-Bio Data.

BACKGROUND: Adjuvant chemotherapy (AC) is indicated for stage II and stage III lung adenocarcinomas (ADC). Using the LACE Bio II database, we analyzed the distribution of various mutations across the subtypes of ADCs and studied the prognostic and predictive roles of PD-L1, TMB, and Tumor Infiltrating Lymphocytes (TILs). MATERIALS AND METHODS: Clinical and genomic data from the LACE Bio II data were extracted. Patients were divided into ADC subtypes, in which the grouping was done based on their known clinical behavior (Lepidic [LEP], Acinar/Papillary [ACI or PAP], Micropapillary/Solid [MIP or SOL], Mucinous [MUC] and Others). Kaplan-Meier (KM) and log-rank test were used to compare survival based on PD-L1, TMB, TILs and combinations of TMB with PD-L1 and TILs. Adjusted Hazard Ratios (HR) were analyzed with Overall Survival (OS), Disease-Free Survival (DFS) and Lung Cancer-Specific Survival (LCSS) as endpoints. RESULTS: A total of 375 ADC patients were identified. MIP/SOL was the subtype most commonly positive for various biomarkers. PD-L1 Negative/high TMB was associated with better outcomes in terms of OS (HR = 0.46 [0.23-0.89], P = .021) and DFS (HR = 0.52 [0.30-0.90], P = .02), relative to PD-L1 Negative/low TMB. High TMB predicted worse outcome with AC use in terms of OS (ratio of hazard ratio rHR = 2.75 [1.07-7.04], P = .035). Marked TILs had better outcome with AC for DFS (rHR = 0.22 [0.06-0.87], P = .031 and LCSS (rHR = 0.08 [0.01-0.66], P = .019) respectively. There was also a beneficial effect of AC among patients with Marked TILs/low TMB in terms of DFS (rHR = 0.06 [0.01-0.53], P = .011). CONCLUSION: High TMB has a prognostic role in resectable lung ADC. The high TMB group had a poor outcome with AC, suggesting that this group may be better served with immune checkpoint therapy.

Identifying immune infiltration by deep learning to assess the prognosis of patients with hepatocellular carcinoma.

BACKGROUND: The treatment situation for hepatocellular carcinoma remains critical. The use of deep learning algorithms to assess immune infiltration is a promising new diagnostic tool. METHODS: Patient data and whole slide images (WSIs) were obtained for the Xijing Hospital (XJH) cohort and TCGA cohort. We wrote programs using Visual studio 2022 with C# language to segment the WSI into tiles. Pathologists classified the tiles and later trained deep learning models using the ResNet 101V2 network via ML.NET with the TensorFlow framework. Model performance was evaluated using AccuracyMicro versus AccuracyMacro. Model performance was examined using ROC curves versus PR curves. The percentage of immune infiltration was calculated using the R package survminer to calculate the intergroup cutoff, and the Kaplan‒Meier method was used to plot the overall survival curve of patients. Cox regression was used to determine whether the percentage of immune infiltration was an independent risk factor for prognosis. A nomogram was constructed, and its accuracy was verified using time-dependent ROC curves with calibration curves. The CIBERSORT algorithm was used to assess immune infiltration between groups. Gene Ontology was used to explore the pathways of differentially expressed genes. RESULTS: There were 100 WSIs and 165,293 tiles in the training set. The final deep learning models had an AccuracyMicro of 97.46% and an AccuracyMacro of 82.28%. The AUCs of the ROC curves on both the training and validation sets exceeded 0.95. The areas under the classification PR curves exceeded 0.85, except that of the TLS on the validation set, which might have had poor results (0.713) due to too few samples. There was a significant difference in OS between the TIL classification groups (p < 0.001), while there was no significant difference in OS between the TLS groups (p = 0.294). Cox regression showed that TIL percentage was an independent risk factor for prognosis in HCC patients (p = 0.015). The AUCs according to the nomogram were 0.714, 0.690, and 0.676 for the 1-year, 2-year, and 5-year AUCs in the TCGA cohort and 0.756, 0.797, and 0.883 in the XJH cohort, respectively. There were significant differences in the levels of infiltration of seven immune cell types between the two groups of samples, and gene ontology showed that the differentially expressed genes between the groups were immune related. Their expression levels of PD-1 and CTLA4 were also significantly different. CONCLUSION: We constructed and tested a deep learning model that evaluates the immune infiltration of liver cancer tissue in HCC patients. Our findings demonstrate the value of the model in assessing patient prognosis, immune infiltration and immune checkpoint expression levels.

Modulating the proliferative and cytotoxic properties of patient-derived TIL by a synthetic immune niche of immobilized CCL21 and ICAM1.

A major challenge in developing an effective adoptive cancer immunotherapy is the ex-vivo generation of tumor-reactive cells in sufficient numbers and with enhanced cytotoxic potential. It was recently demonstrated that culturing of activated murine CD8+ T-cells on a "Synthetic Immune Niche" (SIN), consisting of immobilized CCL21 and ICAM-1, enhances T-cell expansion, increases their cytotoxicity against cultured cancer cells and suppresses tumor growth in vivo. In the study reported here, we have tested the effect of the CCL21+ICAM1 SIN, on the expansion and cytotoxic phenotype of Tumor Infiltrating Lymphocytes (TIL) from melanoma patients, following activation with immobilized anti-CD3/CD28 stimulation, or commercial activation beads. The majority of TIL tested, displayed higher expansion when cultured on the coated SIN compared to cells incubated on uncoated substrate and a lower frequency of TIM-3+CD8+ cells after stimulation with anti-CD3/CD28 beads. Comparable enhancement of TIL proliferation was obtained by the CCL21+ICAM1 SIN, in a clinical setting that included a 14-day rapid expansion procedure (REP). Co-incubation of post-REP TIL with matching target cancerous cells demonstrated increased IFNγ secretion beyond baseline in most of the TIL cultures, as well as a significant increase in granzyme B levels following activation on SIN. The SIN did not significantly alter the relative frequency of CD8/CD4 populations, as well as the expression of CD28, CD25, several exhaustion markers and the differentiation status of the expanded cells. These results demonstrate the potential capacity of the CCL21+ICAM1 SIN to reinforce TIL-based immunotherapy for cancer patients.

Tumor Infiltrating Lymphocyte (TIL) Therapy for Solid Tumor Treatment: Progressions and Challenges.

Over the past decade, immunotherapy, especially cell-based immunotherapy, has provided new strategies for cancer therapy. Recent clinical studies demonstrated that adopting cell transfer of tumor-infiltrating lymphocytes (TILs) for advanced solid tumors showed good efficacy. TIL therapy is a type of cell-based immunotherapy using the patient's own immune cells from the microenvironment of the solid tumor to kill tumor cells. In this review, we provide a comprehensive summary of the current strategies and challenges in TIL isolation and generation. Moreover, the current clinical experience of TIL therapy is summarized and discussed, with an emphasis on lymphodepletion regimen, the use of interleukin-2, and related toxicity. Furthermore, we highlight the clinical trials where TIL therapy is used independently and in combination with other types of therapy for solid cancers. Finally, the limitations, future potential, and directions of TIL therapy for solid tumor treatment are also discussed.

Clinicopathological Significance, Related Molecular Changes and Tumor Immune Response Analysis of the Abnormal SWI/SNF Complex Subunit PBRM1 in Gastric Adenocarcinoma.

Background: PBRM1 gene abnormalities were recently found to play a role in tumor development and tumor immune activity. This article will explore the clinicopathological and molecular changes and tumor immune activity of the abnormal SWI/SNF complex subunit PBRM1 in gastric adenocarcinoma (GAC) and its significance. Methods: The cBioPortal, LinkedOmics and TISIDB datasets were used to analyze the abnormality of the PBRM1 gene in GAC and its relationship with prognosis, related molecular changes and tumor-infiltrating lymphocytes (TILs). In addition, 198 GAC cases were collected to further study the relationship between the loss/attenuation of PBRM1 expression and clinicopathology, prognosis, microsatellite stability, PD-L1 expression and TIL in GAC. DNA whole-exome sequencing was performed on 7 cases of gastric cancer with loss of PBRM1 expression. Results: The cBioPortal data showed that PBRM1 deletion/mutation accounted for 7.32% of GAC and was significantly associated with several molecular changes, such as molecular subtypes of GAC. The LinkedOmics dataset showed that PBRM1 mutation and its promoter DNA methylation showed lower PBRM1 mRNA expression, and PBRM1 mutation cases showed significantly higher mRNA expression of PD-L1 (CD274). TISIDB data showed that PBRM1 abnormalities were significantly positively associated with multiple TILs. In our group of 198 cases, the loss/attenuation of PBRM1 expression was significantly positively correlated with intra-tumoral tumor infiltrating lymphocytes (iTILs) and deficient MMR and PD-L1 expression. Kaplan-Meier survival analysis showed that the overall survival of GAC patients with loss/attenuation of PBRM1 expression was significantly better (p = 0.023). iTIL was an independent prognostic factor of GAC. Loss of PBRM1 expression often co-occurs with mutations in other SWI/SNF complex subunit genes, and there are some repetitive KEGG signaling changes. Conclusion: Abnormality of the PBRM1 gene may be related to the occurrence of some GACs and can affect tumor immune activity, thereby affecting clinicopathology and prognosis. It may be a potentially effective predictive marker for immunotherapy and a novel therapeutic approach associated with synthetic lethality.

A Paradoxical Role for Regulatory T Cells in the Tumor Microenvironment of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is considered to be a poorly immunogenic cancer type that combines a low mutation burden with a strong immunosuppressive tumor microenvironment. Regulatory T cells (Tregs) are major drivers of immune suppression but their prognostic role, particularly in gastrointestinal malignancies, remains controversial. Lymphocytic infiltration in 122 PDAC samples was assessed by multispectral immunofluorescence with anti-Keratin, -CD3, -CD8, -FOXP3 and -CD163 antibodies. Differential infiltration by Tregs was analyzed in the context of transcriptomic profiles that were available for 65 tumors. High infiltration of CD3+CD8- (mainly CD4+) T cells and, especially, of the subset expressing FOXP3 (Tregs) was associated with improved patient survival, whilst cytotoxic CD3+CD8+ T cell infiltration did not have an impact on overall survival. Transcriptomic analysis revealed three signatures in PDAC tumors comprising of epithelial-mesenchymal transition (EMT)/stromal, metabolic, and secretory/pancreatic signature. However, none of these signatures explained differences in Treg infiltration. We show that Tregs associate with improved overall survival in PDAC patients. This effect was independent of cytotoxic T cell infiltration and the transcriptomic profiles of their respective tumors. These findings provide a new layer of complexity in the study of PDAC tumor microenvironment that must be considered when developing immunotherapeutic interventions for this disease.

Tertiary Lymphatic Structures in Primary Hepatic Carcinoma: Controversy Cannot Overshadow Hope.

Tertiary lymphoid structures (TLSs) are organized aggregates of immune cells found in the tumor microenvironment. TLS can influence primary hepatic carcinoma (PHC) occurrence and have an active role in cancer. TLS can promote or inhibit the growth of PHC depending on their location, and although available findings are controversial, they suggest that TLS have a protective role in PHC tissues and a non-protective role in paracancerous tissues. In addition, the cellular composition of TLS can also influence the outcome of PHC. As an immunity marker, TLS can act as a marker of immunotherapy to predict its effect and help to identify patients who will respond well to immunotherapy. Modulation of TLS formation through the use of chemokines/cytokines, immunotherapy, or induction of high endothelial vein to interfere with tumor growth has been studied extensively in PHC and other cancers. In addition, new tools such as genetic interventions, cellular crosstalk, preoperative radiotherapy, and advances in materials science have been shown to influence the prognosis of malignant tumors by modulating TLS production. These can also be used to develop PHC treatment.

The role of tumor-infiltrating lymphocytes in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is the second most common primary liver cancer and associated with a dismal prognosis due to the lack of an efficient systemic therapy. In contrast to other cancers, new immunotherapies have demonstrated unsatisfactory results in clinical trials, underlining the importance of a deeper understanding of the special tumor microenvironment of CCA and the role of immune cells interacting with the tumor. Tumor-infiltrating lymphocytes (TILs) are an important component of the adaptive immune system and the foundation of current immunotherapy. Therefore, the aim of this systemic review is to summarize the current literature focusing on the proportions and distribution, molecular pathogenesis, prognostic significance of TILs and their role in immunotherapy for CCA patients.In CCA, CD8+ and CD4+ T lymphocytes represent the majority of TILs and are mostly sequestered around the cancer cells. CD20+ B lymphocytes and Natural Killer (NK) cells are less frequent. In contrast, Foxp3+ cells (regulatory T cells, Tregs) are observed to infiltrate into the tumor. In the immune microenvironment of CCA, cancer cells and stromal cells such as TAMs, TANs, MSDCs and CAFs inhibit the immune protection function of TILs by secreting factors like IL-10 and TGF-ß. With respect to molecular pathogenesis, the Wnt/-catenin, TGF-signaling routes, aPKC-i/P-Sp1/Snail Signaling, B7-H1/PD-1Pathway and Fas/FasL signaling pathways are connected to the malignant potential and contributed to tumor immune evasion by increasing TIL apoptosis. Distinct subtypes of TILs show different prognostic implications for the long-term outcome in CCA. Although there are occasionally conflicting results, CD8+ and CD4+ T cells, and CD20+ B cells are positively correlated with the oncological prognosis of CCA, while a high number of Tregs is very likely associated with worse overall survival. TILs also play a major role in immunotherapy for CCA.In summary, the presence of TILs may represent an important marker for the prognosis and a potential target for novel therapy, but more clinical and translationaldata is needed to fully unravel the importance of TILs in the treatment of CCA.

PD-L1 tumor expression is associated with poor prognosis and systemic immunosuppression in glioblastoma.

PURPOSE: Glioblastoma is the most common primary malignant brain tumor in the adult, whose grim prognosis largely relates to the absence of effective treatment targets. Given its success in other cancers, immunotherapy has been trialed in glioblastoma and failed to demonstrate the expected benefit. Importantly, these disappointing results highlight the importance of understanding the unique and transforming biology of glioblastoma and its microenvironment. Our goal was to evaluate and characterize the expression of PD-L1 through immunohistochemistry in a large glioblastoma cohort. We further studied PD-L1 expression-associated prognosis and its correlation to systemic and neuropathological parameters. METHODS: A series of 352 glioblastoma specimens (313 initial resection, 39 matched recurrences) was collected, with a detailed characterization of tumor neuropathological characteristics, including the presence, density and location of tumor infiltrating lymphocytes (TIL). Two hematological markers, absolute lymphocyte count and neutrophil-lymphocyte ratio (NLR), were used to analyze and correlate with systemic inflammation and immunosuppression. Immunohistochemistry was performed to evaluate PD-L1 expression. RESULTS: Membranous PD-L1 expression was identified in 31% (98/313) of newly diagnosed and 46% (18/39) of matched recurrent tumors. TIL were found in 26% (82/313) of primary tumors and both density and location were found to be significantly associated with PD-L1 expression (p < 0.001). Interestingly, PD-L1 expressing tumors had more frequently areas with sarcomatous differentiation (p < 0.001) and were significantly associated with lower lymphocyte count (p = 0.018) and higher NLR ratio (p = 0.004) upon diagnosis. Importantly, PD-L1 expression was an independent poor prognostic marker in our cohort. CONCLUSION: Taken together, our data points to a putative role for PD-L1 expression in glioblastoma biology, which correlates to poor patient overall survival, as well as with a general systemic inflammatory status and immunosuppression.

Is There a Relationship Between TILs and Regression in Melanoma?

BACKGROUND: The relationship between tumor-infiltrating lymphocytes (TILs) and regression in melanoma is unknown. This report describes a large multicenter study assessing the association between TILs and regression. METHODS: The Sentinel Lymph Node Working Group database was queried from 1993 to 2018 for cases with TILs and regression data. Clinicopathologic factors were correlated with regression and TIL status, sentinel lymph node (SLN) status, and overall survival (OS). RESULTS: The study enrolled 2450 patients. In 1811 cases, TILs (73.9%) were present, with regression present in 328 of these 1811 (18.1%) cases and in 49 (7.7%) of 639 cases without TILs. The presence of TILs was significantly associated with regression (p < 0.0001) as well as a negative SLN (p < 0.05). However, when TILs were stratified by regression status, only absence or presence of both TILs and regression were significantly associated with SLN metastases (p = 0.038). Although the presence of TILs was associated with OS (p < 0.05), regression status by itself was not (p = 0.2058 and 0.252, respectively). Furthermore, when TILs were stratified by regression status, only the presence of TILs with or without regression was significantly associated with improved OS (p = 0.0081 and 0.0137, respectively) versus the absence of both TILs and regression, with regression status not significantly affecting OS for patients with or without TILs (p = 0.2314 and 0.65, respectively). CONCLUSIONS: Regression is highly correlated with TILs, but only TILs are significantly associated with SLN metastasis and OS in melanoma patients, whereas regression is not. The impact of regression on outcomes ultimately appears dependent upon the absence or presence of TILs.

Isolation and Maintenance of Tumor-Infiltrating Lymphocytes for Translational and Clinical Applications: Established Methods and New Developments.

Adoptive cell transfer (ACT) of in vitro expanded tumor-infiltrating lymphocytes (TIL) for the treatment of patients with advanced stages of metastatic melanoma remains one of the most beneficial therapies eliciting long-lasting responses. Methods and protocols used to expand TIL have evolved over time, utilizing different culture devices and other tools, to streamline and maximize the end product in both numbers and quality. Summarized in this chapter are the latest protocols used in the TIL program at MDACC.

Improving Breast Cancer Responses to Immunotherapy-a Search for the Achilles Heel of the Tumor Microenvironment.

PURPOSE OF REVIEW: To explore the role of the tumor microenvironment (TME) in breast cancer, identify the changes that occur in the TME during breast cancer progression, and explore the possibility of modifying the TME to improve immune checkpoint inhibitor responses. RECENT FINDINGS: Emerging evidence shows the TME may be shaped by internal and external factors. Preclinical data suggests it may be possible to shift the TME to allow for better immune infiltration. In this review, we summarize emerging evidence of changes in the TME and how it can affect prognosis and responses to therapy. We also examine pre-clinical and clinical research aiming at modulating TME to increase proportion of patients who benefit from immune checkpoint inhibitors. The composition of the TME in breast cancer is likely dynamic and may be altered. These changes may lead to more or less responses to immunotherapy.

Prognostic value of tumor infiltrating lymphocytes combined with PD-L1 expression for patients with solitary colorectal cancer liver metastasis.

BACKGROUND: The aim of this study was to assess the prognostic value of CD8+ tumor infiltrating lymphocytes (TIL) combined with programmed cell death-ligand 1 (PD-L1) expression for patients with solitary colorectal cancer liver metastasis (SCLM) undergoing R0 resection. METHODS: Patients undergoing curative hepatectomy for SCLM were reviewed. Immunohistochemical multiplex technique was used for quantifying CD8+ TIL, and immunohistochemical staining was used for assessing PD-L1 expression. The tumor immune microenvironment (TIME) was classified as strong for high CD8+ TIL and low PD-L1, weak for low CD8+ TIL and high PD-L1, and mild for the rest. Recurrence-free survival (RFS) and overall survival (OS) was compared between these groups. RESULTS: Among the 94 patients included, a high CD8+ TIL and high PD-L1 expression was observed in 51 (54.3%) and 47 (50.0%) patients, respectively. Strong, mild, and weak TIME was observed in 24 (25.5%), 42 (44.7%), and 28 (29.8%) patients, respectively. Patients with a high CD8+ TIL had a significant longer RFS than patients with a low CD8+ TIL (3-year RFS rate, 71.6% vs. 55.3%, P=0.018). The 3-year RFS rate in the strong TIME group was significantly higher than that in the mild and weak TIME groups (89.5% vs. 71.7% and 28.8%, P<0.001), as was the 3-year rate of OS (93.8% vs. 81.8% and 61.6%, P<0.001). CD8+ TIL combined with PD-L1 expression showed better predicting accuracy for RFS than CD8+ TIL alone. CONCLUSIONS: The density of CD8+ TIL combined with PD-L1 expression in liver metastasis was a predictor of RFS for patients with SCLM undergoing R0 resection, and therefore can be used for guiding the postoperative treatment of these patients.

Effect of preoperative chemoradiotherapy on the immunological status of rectal cancer patients.

The aim of the study was to investigate the effect of chemo-radiation on the genetic and immunological status of rectal cancer patients who were treated with preoperative chemoradiotherapy (CRT). The expression of immune response-associated genes was compared between rectal cancer patients treated (n = 9) and not-treated (n = 10) with preoperative CRT using volcano plot analysis. Apoptosis and epithelial-to-mesenchymal transition (EMT) marker genes were analysed by quantitative PCR (qPCR). Other markers associated with the tumor microenvironment (TME), such as tumor-infiltrating lymphocytes (TIL) and immune checkpoint molecules, were investigated using immunohistochemistry (IHC). The clinical responses of preoperative CRT for 9 rectal cancer patients were all rated as stable disease, while the pathological tumor regression score (TRG) revealed 6 cases of grade2 and 3 cases of grade1. According to the genetic signature of colon cancers, treated tumors belonged to consensus molecular subtype (CMS)4, while not-treated tumors had signatures of CMS2 or 3. CRT-treated tumors showed significant upregulation of EMT-associated genes, such as CDH2, TGF-beta and FGF, and cancer stem cell-associated genes. Additionally, qPCR and IHC demonstrated a suppressive immunological status derived from the upregulation of inflammatory cytokines (IL-6, IL-10 and TGF-beta) and immune checkpoint genes (B7-H3 and B7-H5) and from M2-type macrophage accumulation in the tumor. The induction of EMT and immune-suppressive status in the tumor after strong CRT treatment urges the development of a novel combined therapy that restores immune-suppression and inhibits EMT, ultimately leading to distant metastasis control.

The Relationship Between Tumor-Infiltrating Lymphocytes, PD-L1 Expression, Driver Mutations and Clinical Outcome Parameters in Non-Small Cell Lung Cancer Adenocarcinoma in Patients with a Limited to no Smoking History.

Tumor infiltrating lymphocytes (TIL), programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) expression are important prognostic markers. This study aimed to investigate these markers in lung adenocarcinoma (ADC) biopsies from patients with stage IIIB or IV ADC with little or no smoking history, to investigate their prognostic value and to correlate these results with the presence of driver mutations in the tumors. TIL were retrospectively evaluated on hematoxylin and eosin stained slides from 152 tumor samples. PD-1/PD-L1 expression was retrospectively evaluated with PD-1/PD-L1 immunohistochemistry (IHC) double staining on 74 tumor samples with sufficient residual tissue. PD-L1 expression was analysed on stromal cells of the tumor compartment, the tumor cells and TIL and PD-1 on TIL. Median overall survival (OS) was longer in patients with high stromal TIL infiltration compared to patients with low stromal TIL infiltration (68 weeks vs. 35 weeks respectively; p = 0.003). This was observed most prominently in KRAS mutant tumors (95 weeks vs. 12 weeks; p = 0.003). Only PD-L1 expression on tumor stromal cells influenced OS and indicated a worse prognosis (77 weeks vs 25 weeks; p = 0.013). Stromal TIL counts nor PD-1/PD-L1 expression levels were associated with the presence of driver mutations. The results of the current study reinforce the prognostic role of TIL in lung ADC, which is most prominent in KRAS mutant cancers. The results of the PD-1/PD-L1 analysis suggest that stromal cells can effectively suppress the anti-tumor immune response via the PD-L1 pathway.