Ratio Of Tumor-infiltrating Lymphocytes Research Articles

Tumor-Intrinsic Enhancer of Zeste Homolog 2 Controls Immune Cell Infiltration, Tumor Growth, and Lung Metastasis in a Triple-Negative Breast Cancer Model.

Triple-negative breast cancer (TNBC) is an aggressive and highly metastatic type of tumor. TNBC is often enriched in tumor-infiltrating neutrophils (TINs), which support cancer growth in part by counteracting tumor-infiltrating lymphocytes (TILs). Prior studies identified the enhancer of zeste homolog 2 (EZH2) as a pro-tumor methyltransferase in primary and metastatic TNBCs. We hypothesized that EZH2 inhibition in TNBC cells per se would exert antitumor activity by altering the tumor immune microenvironment. To test this hypothesis, we used CRISPR to generate EZH2 gene knockout (KO) and overexpressing (OE) lines from parent (wild-type-WT) 4T1 cells, an established murine TNBC model, resulting in EZH2 protein KO and OE, respectively. In vitro, EZH2 KO and OE cells showed early, transient changes in replicative capacity and invasiveness, and marked changes in surface marker profile and cytokine/chemokine secretion compared to WT cells. In vivo, EZH2 KO cells showed significantly reduced primary tumor growth and a 10-fold decrease in lung metastasis compared to WT cells, while EZH2 OE cells were unchanged. Compared to WT tumors, TIN:TIL ratios were greatly reduced in EZH2 KO tumors but unchanged in EZH2 OE tumors. Thus, EZH2 is key to 4T1 aggressiveness as its tumor-intrinsic knockout alters their in vitro secretome and in vivo primary tumor growth, TIN/TIL poise, and metastasis.

Clinical impact of heterogeneously distributed tumor-infiltrating lymphocytes on the prognosis of colorectal cancer.

Tumor-infiltrating lymphocytes (TILs) exist in various malignancies, and have been viewed as a promising biomarker to predict the efficacy and outcome of treatment. However, the marked inter- and intra-tumor heterogeneity of TILs has resulted in some confusion regarding their impact on the prognosis of colorectal cancer (CRC). In this study, 78 CRC patients were enrolled and the CD3+ and CD8+ TILs densities at the tumor center (TC), the invasive margin (IM) and the tumor stroma (TS) were assessed by immunohistochemical staining. Their associations with clinicopathological features and progression free survival (PFS) were analyzed to evaluate the predictive and prognostic values of TILs. TILs were mainly distributed along the invasive margin. High density of TILs in tumor center and invasive margin was associated with smaller tumor size (CD3+TILsIM), reduced tumor invasion (CD3+TILsIM), absence of lymph node metastasis (CD3+TILsIM and CD8+TILsTC), earlier stage (CD3+TILsIM and CD8+TILsIM), and lower tumor grade (CD3+TILsIM and CD8+TILsTC). However, stromal TILs were not associated with any clinicopathological features. Kaplan-Meier survival analysis revealed that high densities of TILs always correlated with prolonged patient survival. The pathological N stage, CD3+ TILsIM and CD8+ TILsTC were found to be independent prognostic indicators. Additionally, early-stage CRC patients who developed recurrence after surgery, showed a higher CD3+/CD8+ TILs ratio in invasive margin. In the present study, it was clarified that CD3+ and CD8+ TILs were heterogeneously distributed in tumor tissues of CRC. The increase in intratumoral and peritumoral TILs had been shown to be strongly predictive of improved clinical outcome. More importantly, the immune signatures enabled to stratify early-stage CRC patients with high risk of recurrence, highlighting the prognostic power of TILs.

Metformin Alters Tumor Immune Microenvironment, Improving the Outcomes of Breast Cancer Patients With Type 2 Diabetes Mellitus.

This study investigated the clinical effect of metformin on breast cancer patients with preexisting type 2 diabetes mellitus (T2DM). We analyzed 177 patients with T2DM who underwent breast cancer surgery and assessed tumor-associated macrophages (TAMs) and tumor-infiltrating lymphocytes (TILs) in patients who underwent tumor resection with or without metformin treatment using multiplex immunohistochemistry (IHC). Patients who received metformin either pre- or postoperatively exhibited reduced distant organ recurrence and improved postoperative recurrence-free survival compared to those of patients who did not. Additionally, in a subgroup of 40 patients receiving preoperative systemic therapy, metformin treatment was associated with increased rates of pathological complete response. IHC analysis revealed significantly lower levels of cluster of differentiation (CD) 68(+) CD163(+) M2-type TAMs (p < 0.01) but higher CD3(+) and CD8(+) TIL densities in the metformin-treated group compared with the same parameters in those without metformin treatment, with a significant difference in the CD8(+)/CD3(+) TIL ratio (p < 0.01). Despite the constraints posed by our small sample size, our findings suggest a potential role for metformin in modulating the immunological microenvironment, which may contribute to improved outcomes in diabetes patients with breast cancer.

Prognostic and Predictive Significance of Tumor Immune Microenvironment in Breast Ductal Carcinoma In Situ

This study aims to identify the role of different subtypes of tumor infiltrating lymphocytes (TILs) in ductal carcinoma in situ (DCIS) in predicting risk of recurrence and benefit of whole breast irradiation (WBI). Immunohistochemical stain for CD3, CD4, CD8, FOXP3 and CD20 were carried in a well characterized DCIS cohort who received breast-conserving surgery (BCS) from Jan 2009 to Dec 2018. All the TILs subtypes were evaluated by the average numbers of touching-TILs which defined as TILs touching or within one lymphocyte cell thickness from the malignant ducts' basement membrane. The optimal cut-off values of TILs subtypes were selected by the X-tile. In total, 167 patients were enrolled in this analysis with 114 patients received WBI. After a median follow-up of 67 months, 15 IBTR events occurred with 6 invasive-IBTRs. Nine out of 15 IBTRs occurred outside of the original quadrant (elsewhere failure event, EFE). CD3+ lymphocytes were the predominant cell subtype while Treg showed the lowest levels. High abundance of TILs subtypes was associated with high tumor grade, presence of microinvasion, high Ki67 index, ER negativity and HER2 positivity. For various TILs subtypes, the multivariate analyses showed that dense CD4+ TILs (HR = 9.84, 95% CI 2.43-39.91, p<0.01) and dense Treg (HR = 4.22, 95% CI 1.24-14.36, p = 0.02) were independent prognostic factors for higher IBTR. As the infiltration of TIL subsets was correlated with one another, we also analyzed the relationship between IBTR and the ratios of different TILs subtypes. By adjusted by clinicopathological parameters, high ratios of CD4+/CD8+, Treg/CD4+ and Treg/CD8+ were found to be independent prognostic factors for higher IBTR (HR = 11.31, 95% CI 3.14-40.76, p<0.01; HR = 3.09, 95% CI 1.05-9.11, p = 0.04; HR = 7.14, 95% CI 1.98-25.73, p<0.01). Consistent with the results of IBTR, the 5-y rate of invasive-IBTR and EFE was both significantly associated with the high CD4+/CD8+, Treg/CD4+ and Treg/CD8+ TILs ratios (all p<0.01). WBI reduced the rate of 5y-IBTR risk from 8.4% to 1.3% (p = 0.02) in the low Treg/CD8+ group, but there was no benefit of WBI in the high group. With respect to EFE, WBI significantly reduced the rate from 2.8% to 0.0% (p = 0.03) in the low Treg/CD8+ group while not in the high group. The benefits of WBI in reducing IBTR and EFE were not significant difference between different CD4+/CD8+ and Treg/CD4+ groups. Assessment of overall TILs provides a tool for comprehensive evaluation of the DCIS immune microenvironment. Patients with pro-tumoral immune infiltrate (high Treg, high ratios of CD4+/CD8+, Treg/CD4+ and Treg/CD8+) in tumor microenvironment show an increased risk of IBTR and less benefit from breast radiotherapy.

The prognostic value of expressions of STAT3, PD-L1, and PD-L2 in Ta/T1 urothelial carcinoma before and after BCG treatment

IntroductionNonmuscle invasive bladder cancers (NMIBC) are common tumors diagnosed in older individuals and men (median age: 69 years). Immunotherapy is a treatment option in cases resistant to Bacillus-Calmette-Guerin (BCG) therapy. We aimed to evaluate the prognostic role of programmed-cell-death ligand (PD-L)-1 (PD-L1), PD-L2, and signal transducer and activator of transcription 3 (STAT3) expressions, which are closely related to immune mechanisms, in the response to BCG treatment of NMIBC. MethodsThe data of patients at the Ta and T1 stages of the cancer without muscularis propria invasion, who were treated with intravesical BCG therapy between 2017 and 2022 were retrospectively analyzed. Immunohistochemical staining for PD-L1, PD-L2, and STAT3 was performed on transurethral resection materials. ResultsThe mean age of 59 patients was 66.5 ±7.7 and 83.9% were male. The percentage of patients with complete response to BCG treatment was 66.1% and that of BCG refractory patients was 33.9%. Demographic and clinical data did not differ significantly according to BCG treatment response (P> 0.05). The proportion of patients with tumor-infiltrating lymphocytes (TILs) ≥20% were 9.7% among those with Ta-stage tumors and 46.4% among those with T1-stage tumors (P = 0.0014). The percentages of tumor cell scoring (TCS), immune cell scoring (ICS), combined scoring (CS), and expression levels of PD-L1, PD-L2, and H-score of STAT3 did not differ significantly according to tumor stage and treatment response (P > 0.05). However, the median ICS and CS for PD-L1 and median H-score for STAT3 were significantly higher among patients in T1 stage compared to those in Ta stage (P = 0.0487, 0.0462, 0.0112, respectively). Among BCG refractory patients, median STAT3 of patients in T1 stage was significantly higher than those at stage Ta (P = 0.0356) and the rate of TILs was ≥20% in only 3 patients in T1 stage (P = 0.031). Among all patients, significant positive correlation was found between TCS for PD-L1 and H-score for STAT3 (P = 0.0302); and between ICS for PD-L1 and TCS for PD-L2 (P = 0.0053) and TILs ratio (P < 0.0001). Among BCG-refractory cases, pretreatment and post-treatment TCS for PD-L2 and H-scores for STAT3 were significantly correlated (P = 0.0361 and 0.0021, respectively). ConclusionsThe success of BCG treatment in NMIBC was not related to PD-L1, PD-L2, and STAT3 expression status, but PD-L1 expression was correlated with both PD-L2 and STAT3 as well as TILs rate, but this correlation was lost after BCG treatment.

The Peritumoral CD8+/FOXP3+ Cell Ratio Has Prognostic Value in Triple-negative Breast Cancer.

Compelling data has demonstrated the prognostic significance of tumor-infiltrating lymphocytes (TILs) in triple-negative breast cancer (TNBC), a subtype generally associated with a poor clinical outcome but highly heterogeneous in nature. There have been limited studies investigating the importance of subsets of T cells in TILs. Further, the significance of intratumoral versus peritumoral TILs remains controversial. We examined the prognostic value of tumor-associated CD8+ cytotoxic T cells and FOXP3+ regulatory T cells in 35 chemotherapy-naive TNBC cases with a tumor-host interface in the tissue sections. The CD8+ and FOXP3+ cell count was expressed by immunoreactive cells per high-power field in an average of 10 high-power fields. There was a wide range of CD8+ and FOXP3+ T cells within the peritumoral and intratumoral stroma. Both CD8+ and FOXP3+ TILs were significantly higher at the former location as compared with the latter (P<0.0001 and 0.003, respectively). The numbers of CD8+ and FOXP3+ T cells, either within peritumoral or intratumoral stroma, were not significantly associated with distant relapse-free or disease-specific survival. However, the peritumoral CD8+/FOXP3+ ratio of TILs was significantly associated with prolonged relapse-free survival (P=0.04) and disease-specific survival (P=0.02). This association was not observed with the CD8+/FOXP3+ ratio of intratumoral TILs. These observations suggest that the immunologic balance in the tumor microenvironment might determine antitumor immunity. Further, the peritumoral TILs appear to play a more important role in the progression of TNBC when compared with the intratumoral TILs, thus reaffirming the necessity of revisiting the method for the assessment of TILs.

Pentoxifylline changes the balance of immune cell population in breast tumor-infiltrating lymphocytes

Immunotherapy utilizing tumor-infiltrating lymphocytes (TILs) is a promising approach for cancer treatment. Pentoxifylline (PTXF), a xanthine derivative, exhibits antitumor properties. This study aimed to investigate the impact of PTXF on the phenotype and function of TILs and splenocytes in a triple-negative breast cancer (TNBC) mouse model. TNBC was subcutaneously induced in BALB/c mice, followed by nine intraperitoneal injections of 100 mg/kg PTXF. TILs were then isolated by enzymatic digestion of tumors and cocultured with 4T1 cells. The proportion of regulatory T cells (Tregs) and cytotoxic T cells in TILs and splenocytes was assessed using flow cytometry. Transforming growth factor (TGF)-β and interferon (IFN)-γ production in TILs and splenocytes cultures was measured by ELISA. Relative expression of t-bet, foxp3, gata-3, and ror-γt in TILs and splenocytes was evaluated using real-time PCR. Tumor growth in PTXF-treated mice was significantly lower than that in the controls (P < 0.01). The frequency of regulatory and cytotoxic TILs in PTXF-treated mice was approximately half (P < 0.01) and twice (P < 0.05) that of the control group, respectively. The level of TGF-β and IFN-γ in the supernatant of PTXF-treated TILs was decreased and increased, respectively (P < 0.05). The relative expression of t-bet and foxp3 in the PTXF-treated mice compared to controls was increased and decreased, respectively (P < 0.05). Changes in the immune cell balance were less significant in the spleen compared to the TILs. PTXF treatment could limit the tumor growth and modify the regulatory-to-cytotoxic TILs ratio, as well as cytokine balance of TILs, in favor of antitumor responses.

Tumor-Infiltrating CD45RO+ Memory Cells Are Associated with Favorable Prognosis in Oral Squamous Cell Carcinoma Patients

Tumor-infiltrating lymphocytes (TILs) have been used to predict the prognosis of solid tumors. In this study, we investigated which molecules in TILs play a role in the prognosis of patients with oral squamous cell carcinoma (OSCC). In a retrospective case-control study, we immunohistochemically evaluated the expression of CD3, CD8, CD45RO, Granzyme B, and the major histocompatibility complex class I chain-related molecule A (MICA) of the histocompatibility complex as predictors of prognosis in 33 patients with OSCC. The patients were classified as TILsHigh or TILsLow according to the number of TILs for each molecule in the central tumor (CT) and invasive margin (IM). Furthermore, MICA expression scores were determined based on the intensity of the staining. CD45RO+/TIL in the nonrecurrent group were significantly higher than those in the recurrent group in the CT and IM areas (p < 0.05). The disease-free survival/overall survival rate of the CD45RO+/TILsLow group in the CT and IM areas and the Granzyme B+/TILsLow group in the IM area was significantly lower than that of the CD45RO+/TILsHigh group and the Granzyme B+/TILsHigh group, respectively (p < 0.05). Furthermore, the MICA expression score of tumors around the CD45RO+/TILsHigh group was significantly higher than that of the CD45RO+/TILsLow group (p < 0.05). A high ratio of CD45RO-expressing TILs was associated with a disease-free/overall survival improvement in OSCC patients. Furthermore, the number of TILs that express CD45RO was associated with the expression of MICA in tumors. These results suggest that CD45RO-expressing TILs are useful biomarkers for OSCC.

A Fraction of CD8+ T Cells from Colorectal Liver Metastases Preferentially Repopulate Autologous Patient-Derived Xenograft Tumors as Tissue-Resident Memory T Cells.

Simple SummaryTreatment options for colorectal cancer (CRC) patients with liver metastases are often limited to liver surgery with or without chemotherapy. However, not all patients present operable colorectal liver metastases (CRLMs). Thus, alternative therapies that exploit the anti-tumor potential of tumor-infiltrating lymphocytes (TILs) are being evaluated. The establishment of markers connecting the phenotype to the function of tumor-reactive CD8+ TILs could aid diagnostic and therapeutic advances. In this regard, tissue-resident memory T cells (TRM cells) could be a potential candidate for therapies targeting TILs. Putative tumor-reactive TRM cells among CD8+ TILs likely co-express CD103 and CD39, since these markers indicate stable tumor residency and repeated response to antigens from the tumor environment, respectively. Our phenotypic and functional analyses of TILs in CRLM, with a specific focus on CD103+CD8+ TRM cells, may guide the improvement of TIL-mediated CRC treatments.The diversity of T cells in the human liver may reflect the composition of TILs in CRLM. Our ex vivo characterization of CRLM vs. adjacent liver tissue detected CD103+CD39+CD8+ TRM cells predominantly in CRLM, which prompted further assessments. These TRM cells responded to cognate antigens in vitro. As functional activities of autologous TILs are central to the implementation of personalized cancer treatments, we applied a patient-derived xenograft (PDX) model to monitor TILs’ capacity to control CRLM-derived tumors in vivo. We established PDX mice with CRLMs from two patients, and in vitro expansion of their respective TILs resulted in opposing CD4+ vs. CD8+ TIL ratios. These CRLMs also displayed mutated KRAS, which enabled trametinib-mediated inhibition of MEK. Regardless of the TIL subset ratio, persistent or transient control of CRLM-derived tumors of limited size by the transferred TILs was observed only after trametinib treatment. Of note, a portion of transferred TILs was observed as CD103+CD8+ TRM cells that strictly accumulated within the autologous CRLM-derived tumor rather than in the spleen or blood. Thus, the predominance of CD103+CD39+CD8+ TRM cells in CRLM relative to the adjacent liver and the propensity of CD103+CD8+ TRM cells to repopulate the autologous tumor may identify these TILs as strategic targets for therapies against advanced CRC.

MIF improves immune microenvironment of Lewis lung cancer brain metastases after radiotherapy via reducing M2 macrophages.

e21026 Background: Our previous studies showed that inhibition of MIF/CD74 signaling pathway can promote the transformation of intracranial macrophages from M2 to M1 type after radiotherapy in brain metastases(BMs). It is reported that M2 macrophages are the main components of tumor immunosuppression microenvironment, and the surface marker, ARG-1, can decompose the Arginine which is essential in the growth and development of T cells. Therefore, this paper will focus on the mechanism of MIF regulating tumor infiltrating lymphocytes (TILs) by inducing macrophage phenotype transformation. Methods: MIF-Knockdown in Lewis cells were constructed by lentiviral shRNA interference technique. The expression levels of MIF in Lewis and iNOS (M1 type marker), Arg-1 (M2 type marker) in BV2 cells were detected by WB. The RNA expression levels of M1 and M2 tags were detected by qPCR. The expression of iNOS, Arg-1 and Iba-1 in lung cancer BMs model mice was detected by IF. The content of arginine in tumor tissue and clinical patient specimens were detected by Arginine kit. The proportion of TILs in vivo was measured by flow cytometry. BMs tumor growth in mice was observed by Small Animals Imaging Technology. Results: In animal experiments, WB、PCR and IF results showed that MIF knockdown (sh-MIF) combined with whole brain irradiation(IR) can raise the expression of iNOS, while the Arg-1 lessend, suggesting that downregulation of MIF can promote M1 phenotype and reduce M2 phenotype. Flow cytometry results showed that sh-MIF combined with IR increased the proportion of CD8+T/CD4+T cells . After clearance of macrophage, Flow cytometry showed no difference between the sh-MIF group and the control group (NC). Small Animals Imaging results also showed that the inhibition of tumor growth by sh-MIF was less significant than macrophages presence. It is indicated that down-regulation of MIF expression combined with irradiation promote T cell infiltration and inhibit tumor may be mediated by macrophages. Arginine kit results showed that the content of arginine increased after IR or sh-MIF, while it increased overall after macrophage clearance, but the difference between each groups was not significant, suggesting that IR or sh-MIF could increase the content of arginine by reducing M2-type macrophages. Flow cytometry and animals imaging results showed that the CD8+/CD4+TILs ratio was increased and tumor growth was inhibited after the addition of exogenous arginine. In clinical samples, the content of arginine in CSF decreased after radiotherapy, which suggests that arginine supplementation has the potential to improve the effect of radiotherapy. Conclusions: Inhibitory MIF expression can reduce the expression of Arg-1 from M2 type macrophages in BMs, thus raise the content of arginine, promote the infiltration of lymphocytes, improve the immune microenvironment, achieve radiotherapy sensitization finally.

Tumor-infiltrating lymphocytes-based subtypes and genomic characteristics of EBV-associated lymphoepithelioma-like carcinoma.

Tumor-infiltrating lymphocytes (TILs) offer a key for morphological diagnosis of lymphoepithelioma-like carcinoma (LELC) and are the foundation of oncoimmunology. To date, no reports have found a specific risk stratification value of TILs and related it to genomic variation in LELC. Based on the stromal TILs (str-TILs) ratio, we classified 105 Epstein-Barr virus (EBV)-associated LELC cases into two subtypes: patients with ≥60% str-TILs area ratio in tumor were classified as subtype I, and otherwise as subtype II. Subtype I patients had significantly better progression-free survival (PFS) and overall survival (OS). We also explored the genomic characteristics of EBV-associated LELC within different involved organs. We performed whole-exome sequencing for 51 patients with enough tissue and analyzed the genomic characteristics of EBV-associated LELC. Overall, EBV-associated LELCs were characterized by a low somatic mutation rate and copy number variations; the enriched genetic lesions affected RTK-RAS, PI3K, and cell cycle pathways. Moreover, EBV-associated LELCs from different organs were more similar to each other genetically as compared with other traditional carcinomas of the same sites-as evidenced by unsupervised clustering based on the quantitative data from both mutation signature and chromosomal aneuploidies. Notably, EBV-associated LELC patients with oncogenic driver alterations showed a worse prognosis compared with patients without such alterations. © 2022 The Pathological Society of Great Britain and Ireland.

Prognostic Relevance of CD4+, CD8+ and FOXP3+ TILs in Oral Squamous Cell Carcinoma and Correlations with PD-L1 and Cancer Stem Cell Markers

This study investigates the relevance of tumor-infiltrating lymphocytes (TILs) in oral squamous cell carcinoma (OSCC). Immunohistochemical analysis of stromal/tumoral CD4+, CD8+ and FOXP3+ TILs is performed in 125 OSCC patients. Potential relationships with the expression of tumoral PD-L1 and cancer stem cell (CSC) markers (NANOG, SOX2, OCT4, Nestin and Podoplanin (PDPN)) are assessed. CD4+ and CD8+ TILs are significantly associated with smoking and alcohol habits. CD4+ and CD8+ TILs show an inverse relationship with NANOG and SOX2 expression, and FOXP3+ TILs is significantly correlated with Nestin and PDPN expression. High infiltration of CD4+ and CD8+ TILs and a high tumoral CD8+/FOXP3+ ratio are significantly associated with tumors harboring positive PD-L1 expression. Infiltration of stromal/tumoral FOXP3+ TILs and a low stromal CD8+/FOXP3+ ratio are significantly associated with better disease-specific survival. Multivariate analysis reveals that the stromal CD8+/FOXP3+ TILs ratio is a significant independent prognostic factor. Regarding OSCC patient survival, the CD8+/FOXP3+ TILs ratio is an independent prognostic factor. TILs may act as biomarkers and potential therapeutic targets for OSCC.

Prognostic Impact of APOBEC3B Expression in Metastatic Urothelial Carcinoma and Its Association with Tumor-Infiltrating Cytotoxic T Cells.

APOBEC3B enzymes are endogenous carcinogenic mutagens. Metastatic urothelial carcinomas often harbor APOBEC3B-mediated mutations in which tCw to T or G substitution occurs. Here, we evaluated patient survival and CD8+ T-cell density according to APOBEC3B expression in patients with metastatic urothelial carcinoma who underwent cytotoxic chemotherapy. We performed a retrospective study on 94 patients with urothelial carcinoma who were treated with first line palliative chemotherapy. APOBEC3B expression and CD8+/CD3+ ratio of tumor-infiltrating lymphocytes were evaluated using immunohistochemistry. Kaplan–Meier survival curves were generated and the log-rank test was employed. The association between APOBEC3B expression and tumor-infiltrating lymphocytes was analyzed using Pearson’s chi-squared test. High APOBEC3B expression was detected in 71 of the 94 patients (75.5%). The median overall survival was longer in patients with high APOBEC3B expression (15 months) than in those with low expression (p = 0.045). The hazard ratio obtained based on the Cox regression analysis was 0.292 (95% confidence interval 0.118–0.723, p = 0.008). APOBEC3B expression was associated with the CD8+/CD3+ ratio (2.914, 95% confidence interval 1.030–8.249, p = 0.039). Collectively, APOBEC3B expression was an independent prognostic factor in patients with metastatic urothelial carcinoma treated with platinum-based chemotherapy. Tumor-infiltrating cytotoxic T cells were associated with APOBEC3B expression.

HHLA2 in intrahepatic cholangiocarcinoma: an immune checkpoint with prognostic significance and wider expression compared with PD-L1

BackgroundIntrahepatic cholangiocarcinoma (ICC) is a highly mortal malignancy with limited therapeutic options. Immunotherapies targeting PD-1/PD-L1 pathway represent a promising treatment for ICC. However, PD-L1 expression and microsatellite instability are not common in ICC. This study aimed to investigate whether HHLA2, a newly identified B7 family immune checkpoint for T cells, could be a therapeutic target next to PD-L1 in ICC.MethodsExpression levels of PD-L1 and HHLA2 as well as infiltrations of CD3+, CD8+, CD4 + Foxp3+, CD68+, CD163+ and CD20+ cells were evaluated by immunohistochemistry in 153 resected ICC samples. Comprehensive comparisons were made between PD-L1 and HHLA2 in terms of the expression rates, clinicopathological features and infiltrations of different immune cells. The expression level and prognostic significance of HHLA2 were further validated in an independent cohort.ResultsExpression of HHLA2 is more frequent than PD-L1 in ICC (49.0% vs 28.1%). Co-expression of both immune checkpoints was infrequent (13.1%) and 50% PD-L1 negative cases were with elevated HHLA2. HHLA2 overexpression was associated with sparser CD3+ tumor infiltrating lymphocytes (TILs), CD8+ TILs and a higher CD4 + Foxp3+/CD8+ TIL ratio, whereas PD-L1 expression was associated with prominent T cells and CD163+ tumor associated macrophages infiltrations. PD-L1 failed to stratify overall survival (OS) but HHLA2 was identified as an independent prognostic indicator for OS in two independent cohorts.ConclusionsCompared with PD-L1, HHLA2 is more prevalent and possesses more explicit prognostic significance, which confer the rationale for HHLA2 as a potential immunotherapeutic target next to PD-L1 for ICC patients.

Immune Cell Infiltration in Liver Oligometastasis Microenvironment from Colorectal Cancer: Intratumoral CD8/CD3 Ratio Represents a Valuable Prognostic Index for Patients Undergoing Liver Metastasectomy

Background: The overview of the tumor microenvironment on liver oligometastasis from colorectal cancer remains largely unclear. A comprehensive investigation on the immune cell infiltration and its constituent ratio might provide a more accurate and reliable prognostic information for the patients with colorectal liver oligometastasis (CLO) after liver metastasectomy. Methods: A simultaneous detection of immune constituents CD3+, CD8+, Foxp3+ T cells, and α-SMA fibroblast on the liver oligometastasis of 133 patients were conducted by using a five-color immunohistochemical multiplex technique. The immune cells were quantified and tumor-infiltrating lymphocytes (TILs) ratios were subsequently calculated. Correlation analysis was performed using Pearson's correlation. Recurrence-free survival (RFS) and overall survival (OS) for TIL ratios were analyzed using the Kaplan-Meier method and Cox regression models. Results: The number of CD3+, CD8+, and Foxp3+ T cells were significantly fewer in the intratumoral subsection by compared to the peritumoral subsection of metastases. CD3+, CD8+, Foxp3+ T cells, and α-SMA+ fibroblast showed a significantly positive correlation between each other both in the intratumoral and peritumoral subsection of liver metastases. As to the correlation of TIL ratios, only the CD8/CD3 ratio demonstrated a positive correlation in intratumoral and peritumoral metastases (r=0.541, P<0.001). Patients with high intratumoral CD8/CD3 ratio had a significantly higher 3-year RFS (59.0% vs. 47.4%, P = 0.035) and 3-year OS rate (83.3% vs. 65.8%, P = 0.007) than those with low intratumoral CD8/CD3 ratio. Multivariate analyses revealed that intratumoral CD8/CD3 ratio was independently associated with RFS (HR 0.572; 95% CI 0.346-0.948; P = 0.030). Conclusions The current study showed a novel insight into the tumor microenvironment on CLO. The intratumoral CD8/CD3 ratio was found to be a valuable prognostic index for CLO undergoing liver metastasectomy. Funding: This work was supported by grants from the National Natural Science Foundation of China (grant No.81772595), Sun Yat-sen University Clinical Research 5010 Program (grant No. 2015024), Sun Yat-sen University Clinical Research 5010 Program (grant No. 2013013), and Guangzhou Science and Technology Plan Projects (Health Medical Collaborative Innovation Program of Guangzhou) (grant No. 201803040019). Declaration of Interest: The authors declare that they have no conflict of interest. Ethical Approval: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. This study was approved by the Institutional Research Ethics Committee of Sun Yat-sen University Cancer Center (Approval number: GZR2017-006). Informed consents for using tissue samples before the initial treatment were obtained from the patients.

Effect of CTLA-4 overexpression on response to ipilimumab in melanoma.

190 Background: CD8 positive tumor infiltrating lymphocytes (TILS) are highly associated with immune response and prognosis, and are also under investigation as a marker of response to checkpoint inhibitors. Given lack of predictive biomarkers for ipilimumab, growing number of trials for new indications for combination ipilimumab + nivolumab, and evidence to support therapeutic target overexpression as markers of response, we examined the role of CTLA-4 expression and TILS in response to ipilimumab and combination ipilimumab + nivolumab in melanoma. Methods: Formalin-fixed paraffin embedded melanoma samples taken prior to treatment by ipilimumab (n = 36) or combination ipilimumab + nivolumab (n = 10) were evaluated by a comprehensive immune gene expression profile to establish the relationship between CTLA-4 and CD8 and therapeutic response (RECIST v1.1). Results: Increased CTLA-4 expression was moderately associated with increased TILS (r2= .41, p = .004). This was observed in the monotherapy group (r2= .38, p = .02), and was higher in the smaller combination therapy group, though not statistically significant (r2= .59, p = .06). Higher levels of TILS were observed in responders who achieved clinical benefit from either regimen within 6 months (n = 20). No significant difference was observed between responders (M = 57.1, SD = 30.2) and nonresponders (M = 48.6, SD = 32.9); t(44) = -.895, p = .376. Lower levels of CTLA-4 were observed in responders who achieved clinical benefit from either regimen within 6 months. No significant difference was observed between responders (M = 54, SD = 35) and nonresponders (M = 38.7, SD = 26.8); t(44) = 1.70, p = .09. The ratio of TILS to CTLA-4 expression was higher in responders who achieved clinical benefit within 6 months (n = 20).No significant difference was observed between responders (M = 5.2, SD = 14.0) and nonresponders (M = 1.4, SD = 2.7); t(41) = -1.2, p = .212. Conclusions: While not statistically significant, CTLA-4 expression in melanoma patients treated with either ipilimumab or combination ipilimumab + nivolumab was lower in responders compared to nonresponders. This analysis does not support the concept that over-expression of CTLA-4 is a biomarker of response to anti-CTLA-4 therapy.

The role of tumor-infiltrating lymphocytes (TILs) as a predictive biomarker of response to anti-PD1 therapy in patients with metastatic non-small cell lung cancer or metastatic melanoma.

Immunotherapy plays an important role in cancer treatment. Biomarkers that can predict response, including tumor-infiltrating lymphocytes (TILs), are in the spotlight of many studies. This cohort study was designed to evaluate the role of CD4+ and CD8+ TILs as predictive factors for response to anti PD-1 treatment in patients with metastatic non-small cell lung cancer (NSCLC) or metastatic melanoma. We evaluated the expression of CD4+ and CD8+ TILs in tissue samples of 56 patients with metastatic NSCLC or melanoma treated with anti-PD1 immunotherapy. The study included 30 patients with melanoma and 26 with NSCLC. An association was found between CD8+/CD4+ TILs ratio and response to anti-PD1 treatment in both cancers. Regarding melanoma patients, ratios of CD8+/CD4+ lower than 2 predicted lack of response to treatment (0%) (p=0.006), while CD8+/CD4+ ratios higher than 2.7 had an 81.3% response rate (p=0.0001). In addition, we found that the presence of more than 1900/mm2 of CD8+ lymphocytes in the melanoma tumor predicted a 90% response to therapy. In the metastatic NSCLC group, tumors with CD8+ lymphocyte count under 886/mm2 showed low response rates (16.7%, p=0.046). When the CD8+ lymphocyte count was in the range of 886-1899/mm2, the response rate was high (60%, p=0.017). In CD8+/CD4+ ratios lower than 2, the response rate was low (13.3%), and in ratios higher than 2, response rates ranged between 43 and 50% (p=0.035). The use of CD8+/CD4+ TILs ratios in tumor biopsies may predict response to anti-PD1 treatment in metastatic melanoma and NSCLC.

Combined Effect of IL-12Rβ2 and IL-23R Expression on Prognosis of Patients with Laryngeal Cancer

Background/Aims: This study aimed to pathologically elucidate the roles of interleukin-12 receptor (IL-12R) β2 and interleukin-23 receptor (IL-23R) expression in tumor cells and tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment and to determine their combined effect on prognosis of laryngeal cancer (LC). Methods: The tumor-cell expression scores and TIL positivity ratiosof IL-12Rβ2 and IL-23R in matched LC and normal laryngeal tissue samples from 61 LC patients were measured via immunohistochemistry (IHC). We adopted a linear regression model to analyze the correlation between IL-12Rβ2 and IL-23R expression in tumor cells and TIL ratios. TheKaplan-Meier log-rank test and Cox regression hazard ratios were used to analyze survival. Results: LC tumor cells had a higher IL-12Rβ2 expression and TIL ratio than IL-23R expression and TIL ratio. The significant correlations between IL-12Rβ2 and IL-23R expression and TIL ratios were identified in LC tissues, particularly in well-differentiated LC. Furthermore, either high tumor cell IL-12Rβ2 or low IL-23R expression had better survival than its corresponding low or high expression, respectively. Similar results did for IL-12Rβ2 ratio and IL-23R ratio. Finally, patients with both high IL-12Rβ2 and low IL-23R had the best prognosis among any other combined groups with both gene expression (HR, 0.1; 95% CI, 0.0-0.8). Likewise, patients with positive ratios of high IL-12Rβ2 and low IL-23R TILs had the best survival (HR, 0.1; 95% CI, 0.0-0.4). Conclusion: IL-12Rβ2 and IL-23R create a homeostasis within the tumor cells and TILs, and this homeostasis affects prognosis. While the intrinsic mechanisms of epigenetic immunoediting for IL-12Rβ2 and IL-23R remain unknown, additional larger and functional studies are warranted for validation.

A high ratio of IL-12Rβ2-positive tumor-infiltrating lymphocytes indicates favorable prognosis in laryngeal cancer

BackgroundThe purpose of this study was to elucidate IL-12Rβ2’s roles as a tumor-associated immunological molecule, delineate the complex roles of IL-12Rβ2+ tumor-infiltrating lymphocytes (TILs) and tumor cell IL-12Rβ2 expression in the tumor microenvironment, and determine the correlation of IL-12Rβ2+ TILs and tumor cell IL-12Rβ2 expression with clinical prognosis. MethodsWe assessed mRNA and protein levels in matched laryngeal cancer tissues (LTs) and adjacent normal mucous membrane tissues (ANMMTs) from 3 laryngeal cancer (LC) patients and ratios of IL-12Rβ2+ TILs in matched LTs and ANMMTs from 61 LC patients. We used the Kaplan-Meier log-rank test and Cox regression hazard ratios to analyze survival. ResultsComparative proteomic and transcriptomic assays revealed that matched LTs and ANMMTs from the 3 patients had significantly different IL-12Rβ2 and IFN-γ expression; the ratio of IL-12Rβ2+ TILs decreased with lower degrees of tumor differentiation. Among all 61 LC patients, the IL-12Rβ2+ TIL ratio in ANMMTs (38.5% ± 22.8%) was significantly higher than that in LTs (29.7% ± 19%; p<.001). Kaplan-Meier analysis revealed that patients with an IL-12Rβ2+ TIL ratio ≥35% had significantly better survival than those with an IL-12Rβ2+ TIL ratio <35% (log rank p=0.041). Multivariable analysis showed a significant association between a high IL-12Rβ2+ TIL ratio and overall survival (hazard ratio, 0.14; 95% confidence interval, 0.03–0.77). ConclusionTumor cell differentiation is associated with TILs’ expression of IL-12Rβ2, and an IL-12Rβ2+ TIL ratio ≥35%) indicates favorable prognosis in LC.

The CD4/CD8 ratio of tumor-infiltrating lymphocytes at the tumor-host interface has prognostic value in triple-negative breast cancer

Compelling evidence has demonstrated the prognostic value of tumor-infiltrating lymphocytes (TILs), especially in triple-negative breast cancer (TNBC). However, only a limited number of studies to investigate the importance of the subsets of T cells in TILs have been carried out, less so the significance of the location of these TILs. In this study, we explored in a cohort of 42 consecutive TNBC cases the prognostic significance of TIL subsets at the tumor-host interface (within 1 high-power field [0.5 mm] of the invasive front) and compared them with TILs within the intratumoral stroma. Given the reported importance of TILs in HER2-overexpressing breast cancer, a subset of such tumors was also included for comparison. The range was wide in both locations; nevertheless, the mean CD4+ and CD8+ T cell count was significantly higher at the tumor-host interface than that found within the intratumoral stroma (both P<.0001). The number of CD4+ or CD8+ T cells at either location was not significantly associated with distant relapse-free or overall survival. However, the CD4/CD8 ratio at the tumor-host interface was significantly associated with both relapse-free survival (hazard ratio 0.2, P=.002) and overall survival (hazard ratio 0.13, P=.002), whereas this association was not seen for the CD4/CD8 ratio within the intratumoral stroma. As expected, both tumor size and nodal status were significantly associated with survival outcomes. The findings further support the contention that TILs, as markers of regional immune escape, are of prognostic importance in TNBC progression and that the CD4/CD8 ratio of TILs at the tumor-host interface plays a distinctive role, thus appearing to be of clinical relevance.