CMTM6 Expression Research Articles

P0548 - CMTM6 expression as a potential biomarker for immunotherapy in metastatic renal cell carcinoma

P0548 - CMTM6 expression as a potential biomarker for immunotherapy in metastatic renal cell carcinoma

CMTM6 expression in M2 macrophages is a potential predictor of PD-1/PD-L1 inhibitor response in colorectal cancer

BackgroundCMTM6 is a novel key regulator of PD-L1. High expression of both CMTM6 and PD-L1 may predict the benefit of PD-1 axis blockade in lung cancer. We aimed to investigate the expression pattern of CMTM6 between mismatch repair-defective (dMMR) and mismatch repair-proficient (pMMR) colorectal cancer (CRC) tissues and assess its correlation with the response to PD-1/PD-L1 pathway blockade.MethodsImmunohistochemistry (IHC) was used to analyze CMTM6 and PD-L1 expression and immune cell density in dMMR/pMMR CRC. Quantitative multiplex immunofluorescence (IF) was performed to detect CMTM6, PD-L1, CD4, CD8, CD68 and CD163 expression in CRC patients treated with PD-1/PD-L1 inhibitors.ResultIHC analysis showed that CMTM6 and PD-L1 were both expressed in tumor cells (TCs) and invasion front immune cells (ICs). CMTM6 and PD-L1 expression and CD4+, CD8+, CD68+ or CD163+ cell density were significantly higher in dMMR CRC patients than in pMMR CRC patients. CMTM6 expression was positively correlated with PD-L1 expression and CD163+ M2 macrophage density in dMMR CRC. IF analysis showed that the coexpression rate of CMTM6/PD-L1 and the expression rate of CMTM6 in CD8+ T cells and CD163+ M2 macrophages were significantly increased in the group that exhibited clinical benefit. CMTM6 expression in M2 macrophages was identified as the best biomarker for predicting the responsiveness to PD-1/PD-L1 inhibitors.ConclusionsCMTM6 expression in M2 macrophages may predict the PD-1/PD-L1 inhibitor response rate in CRC patients more accurately than dMMR/microsatellite instability-high (MSI-H) status. It can also identify pMMR CRC patients who could benefit from PD-1/PD-L1 inhibitors.

CMTM6 promotes migration, invasion, and EMT by interacting with and stabilizing vimentin in hepatocellular carcinoma cells

BackgroundCKLF like MARVEL transmembrane domain containing 6 (CMTM6) has been associated with the development in many kinds of cancers. However, the roles of CMTM6 in hepatocellular carcinoma (HCC) are largely unknown. Thus, the present study aimed to investigate the function of CMTM6 in HCC.MethodsWe analysed CMTM6 levels and functions using human HCC cell lines, paired HCC and adjacent non-tumorous tissues, and a tissue microarray. CMTM6 expression was silenced using short hairpin RNAs and its was overexpressed from a lentivirus vector. CMTM6 mRNA and protein levels were determined using quantitative real-time reverse transcription PCR and western blotting, respectively. Proliferation, colony formation, migration, and invasion were assessed using a Cell counting kit-8, colony formation, wound-healing, and Matrigel invasion assays, respectively. Immunohistochemistry was used to score the expression of CMTM6 in tissue samples. The localization and binding partners of CMTM6 were investigated using immunofluorescence and coimmunoprecipitation experiments, respectively. A mouse xenograft model was used for in vivo studies.ResultsCompared with that in adjacent, non-cancerous tissue, Here, CMTM6 levels were increased in HCC tissue samples. Silencing of CMTM6 suppressed the proliferation, migration, and invasion of HCC cells. Conversely, CMTM6 overexpression enhanced HCC cell invasion, migration, and proliferation. Mechanistically, CMTM6 physically interacts with and stabilizes vimentin, thus inducing epithelial–mesenchymal transition (EMT), which promotes proliferation, migration and invasion. Importantly, in HCC tissues, CMTM6 expression correlated positively with vimentin levels. Poor prognosis of HCC was associated significantly with higher CMTM6 expression.ConclusionsCMTM6 has an important function in HCC proliferation, migration, and invasion, via its interaction with and stabilization of vimentin. CMTM6 might represent a potential biomarker and therapeutic target to treat HCC.

P14.18 Expression and Clinical Significance of CMTM6 in Non-Small Cell Lung Cancer

Lung cancer is one of the most serious malignancies, which bearing high estimated new cancer cases and deaths. Although death rate of lung cancer have reduced over the past decade in USA, it still caused more deaths than breast, prostate, colorectal and brain cancers combined. Similarly, the lung cancer situation in China is not optimistic. Nowadays, many drugs against NSCLC have been approved and applicated in clinical, such as PD-1 inhibitors (nivolumab and pembrolizumab) and PD-L1 inhibitors (atezolizumab). However, there are only 15%-20% of patients can respond to this therapy. For a better understanding and treatment of NSCLC, it is important to explore more and better diagnostic and prognostic biomarkers. Chemokine-like factor (CKLF)-like MARVEL transmembrane domain containing protein 6 (CMTM6) is a widely expressed protein. In 2017, two independent researches published on Nature reported the biological function of CMTM6 in tumor immunity. As a critical regulator of PD-L1 protein in cancer cells, CMTM6 can bind to PD-L1 and prevent PD-L1 from being targeted for lysosome-mediated degradation, resulting in enhanced inhibitory effect on immune system and successful escape of immune surveillance.All of research mentioned above have been done at the cellular and genetic levels, and it is not entirely clear the expression of CMTM6 in various cancers, such as non-small cell lung cancer. In view of the relationship between CMTM6 and PD-L1 and the critical role of PD-L1 in the immunotherapy of NSCLC, we evaluated the expression and clinical significance of CMTM6 in NSCLC in this study. Firstly, the 109 NSCLC patients’ tissue samples were collected from affiliated hospital of zunyi medical university. Then the expression of CMTM6 in NSCLC patients’ tissue samples and its correlation with prognosis were explored by immunohistochemistry (IHC). Finally, the relationship between CMTM6 expression and clinical characteristics of NSCLC patients were evaluated as well as the survival of NSCLC patients. 109 patients who were diagnosed with NSCLC and underwent surgical resection[何1] were included in this retrospective study. The expression of CMTM6 in NSCLC patients’ tissue samples were measured by immunohistochemistry and the staining intensity were assessed by the multiplication of staining intensity and staining proportion: score ≤ 5 was considered low CMTM6 expression and score > 5 belong to high CMTM6 expression. Sixty out 109 cases (55.05%) had high CMTM6 expression in this study. The Chi-square test showed that the expression of CMTM6 in NSCLC was significantly related with smoking (p = 0.017) and differentiation (p = 0.029). The Kaplan-Meier analysis suggested that the high[何1] expression of CMTM6 was associated with better prognosis of NSCLC patients. The univariate analysis revealed that high CMTM6 expression in NSCLC patients’ tumor tissues was correlated with pathological differentiation (p = 0.001), metastasis (p = 0.009), LNM (p = 0.007) and T stage (p = 0.042). In addition, multivariate analysis indicated that high CMTM6 expression was an independent prognostic factor for NSCLC patients (p = 0.002). The tumor tissue CMTM6 expression may have the potential to be a biomarker assisting in disease monitoring and prognosis in NSCLC.

HuR up-regulates cell surface PD-L1 via stabilizing CMTM6 transcript in cancer

Despite the well-established role of CMTM6 in the stabilization of cell surface PD-L1 in cancer cells, the mechanisms underlying CMTM6 expression and regulation are still largely unknown. Here we unexpectedly find a strikingly positive correlation between CMTM6 and Hu-Antigen R (HuR) expression in most types of cancer. Mechanistically, we elucidate HuR stabilizes CMTM6 mRNA via direct association with AU-rich elements (AREs) in its 3′UTR and predominantly up-regulates CMTM6, which is readily abolished by HuR-specific inhibitor, MS-444. Phenotypically, we notice abundant cell surface PD-L1 in HuR-high cancer cells, which significantly inhibits immune activation of co-cultured T cells as indicated by IL-2 production. Treatment with MS-444 completely relieves immune suppression imposed by HuR-overexpression and further stimulates immune responses. Ectopic HuR accelerates allograft tumor progression in vivo, which is greatly compromised by simultaneous administration with MS-444. Our study uncovers a novel mechanism in control of CMTM6 and therefore PD-L1 expression, and suggests the potential of combining HuR inhibitor with PD-1/PD-L1 antibodies for cancer immunotherapy.

CMTM6 expression as a potential biomarker for immunotherapy in metastatic renal cell carcinoma.

The introduction of immune checkpoint inhibitors (ICIs) has revolutionized the treatment of metastatic renal cell carcinoma (mRCC) [1]. The expression of programmed death-ligand 1 (PD-L1) as assessed by immunohistochemistry (IHC) has been extensively studied as predictive biomarker for patients receiving ICIs. However, results in clinical RCC trials concerning the predictive value of PD-L1 expression were highly controversial [2]. In the Checkmate 214 trial [3], patients with PD-L1 positivity were found to benefit most from ICI combination. On the contrary, PD-L1 expression was not found to be predictive for nivolumab in the 2nd line setting [4]. Thus, all ICIs (combination and monotherapy) were approved in mRCC, irrespective of PD-L1 status.

Co-expression of CMTM6 and PD-L1: a novel prognostic indicator of gastric cancer

BackgroundCKLF Like MARVEL Transmembrane Domain Containing 6 (CMTM6) is involved in the epigenetic regulation of genes and tumorigenesis. Programmed cell death ligand 1 (PD-L1) is closely related to the prognosis of some human cancers. CMTM6 is a key regulator of PD-L1 in many cancers. The purpose of this study was to investigate the expressions of these proteins in gastric cancer and the correlations with clinicopathological features and survival.MethodsThe expression levels of CMTM6 and PD-L1 were examined in 185 gastric cancer specimens using immunohistochemistry, quantitative real-time PCR and Western blot. Immunofluorescence was used to examine the localizations of CMTM6 and PD-L1. Chi-square test was used to analyze the relationship between CMTM6 and PD-L1 expressions and clinicopathological characteristics. Kaplan–Meier method and log-rank test were used to analyze the survival data of patients.ResultsThe positive expression rates of CMTM6 and PD-L1 in gastric cancers were 78.38% (145/185) and 75.68% (140/185), respectively. CMTM6 and PD-L1 were both mainly expressed in the cell membrane and nucleus of gastric cancer tumor cells. High expression of CMTM6 and PD-L1 was correlated with Borrmann type (P < 0.001), N stage (P = 0.002), peritoneal metastasis (P = 0.007) and TNM stage (P = 0.038). CMTM6 and PD-L1 expression in gastric cancer tissues showed a positive correlation (Pearson’s coefficient test, r = 0.260; P < 0.001). CMTM6 may positively regulate PD-L1 expression. High expression of CMTM6 was correlated with poor prognosis of gastric cancer patients (HR = 1.668; 95% CI = 1.032–2.695; P = 0.037). High expression of both CMTM6 and PD-L1 may be an independent factor for overall survival (HR = 1.554; 95% CI = 1.011–2.389; P = 0.044).ConclusionThe combined detection of CMTM6 and PD-L1 may be used as an indicator for judging the prognosis of gastric cancer patients.

CMTM6 drives cisplatin resistance by regulating Wnt signaling through the ENO-1/AKT/GSK3β axis.

Rewiring tumor cells to undergo drug-induced apoptosis is a promising way to overcome chemoresistance. Therefore, identifying causative factors for chemoresistance is of high importance. Unbiased global proteome profiling of sensitive, early, and late cisplatin-resistant oral squamous cell carcinoma (OSCC) lines identified CMTM6 as a top-ranked upregulated protein. Analyses of OSCC patient tumor samples demonstrated significantly higher CMTM6 expression in chemotherapy (CT) nonresponders as compared with CT responders. In addition, a significant association between higher CMTM6 expression and poorer relapse-free survival in esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, and lung squamous cell carcinoma was observed from Kaplan-Meier plot analysis. Stable knockdown (KD) of CMTM6 restored cisplatin-mediated cell death in chemoresistant OSCC lines. Upon CMTM6 overexpression in CMTM6-KD lines, the cisplatin-resistant phenotype was rescued. The patient-derived cell xenograft model of chemoresistant OSCC displaying CMTM6 depletion restored the cisplatin-induced cell death and tumor burden substantially. The transcriptome analysis of CMTM6-KD and control chemoresistant cells depicted enrichment of the Wnt signaling pathway. We demonstrated that CMTM6 interaction with membrane-bound Enolase-1 stabilized its expression, leading to activation of Wnt signaling mediated by AKT–glycogen synthase kinase-3β. CMTM6 has been identified as a stabilizer of programmed cell death ligand 1. Therefore, as CMTM6 facilitates tumor cells for immune evasion and mediates cisplatin resistance, it could be a promising therapeutic target for treating therapy-resistant OSCC.

The association of CMTM6 expression with prognosis and PD-L1 expression in triple-negative breast cancer.

BackgroundImmune checkpoint inhibitors play a vital role in triple-negative breast cancer (TNBC) immunotherapy. A recent study showed that chemokine-like factor (CKLF)-like MARVEL transmembrane domain containing 6 (CMTM6) has a crucial role in programmed death-ligand 1 (PD-L1) stability. The aim of this study was to investigate the relationship between CMTM6 and PD-L1 in TNBC and the association with clinical characteristics.MethodsA total of 143 patients, including 75 with human epidermal growth factor receptor 2 (HER2)-driven breast cancer and 68 with TNBC, were included in this study. In 83 paired primary breast cancers (PBCs) and metastatic breast cancers (MBC) comprising 45 HER2-driven breast cancers and 38 TNBC, CMTM6 and PD-L1 were detected based on immunohistochemistry (IHC) with FFPE tissues. Another 60 PBCs comprising 30 HER2-driven breast cancers and 30 TNBC in order to detect CMTM6 and PD-L1 mRNA expressions based on real-time polymerase chain reaction (RT-PCR) using frozen tissues. Furthermore, 153 patients comprising 30 TNBC and 123 HER2-driven breast cancer based on The Cancer Genome Atlas (TCGA) database were used to confirm the difference mRNA expression.ResultsThe expression of CMTM6 in patients with TNBC was significantly higher than in those with HER2-driven PBC (IHC, P=0.036, mRNA, P=0.036, TCGA dataset, P=0.039). CMTM6 was correlated with PD-L1 based on IHC in triple-negative MBC (P=0.004); the same result was found based on mRNA data in triple- negative PBC (P=0.021). Moreover, a high expression of CMTM6 in TNBC was associated with poor progression-free survival (PFS) (P=0.030, 95% CI: 1.08–4.57, HR =2.22). After multiple Cox regression analysis, CMTM6 in TNBC emerged as an independent risk factor for PFS (P=0.027, 95% CI: 1.11–5.20, HR =2.40). The expression of PD-L1 was negatively correlated with lymph node metastasis (P=0.026) and was not associated with PFS.ConclusionsThe expression of CMTM6 was higher in TNBC than in HER2-driven breast cancer. In TNBC, CMTM6 was correlated with PD-L1 expression, and potentially could be used as an independent risk factor for predicting PFS.

Quantitative analysis of CMTM6 expression in tumor microenvironment in metastatic melanoma and association with outcome on immunotherapy

ABSTRACT Chemokine-like factor (CKLF)-like MARVEL transmembrane domain containing 6 (CMTM6) modulates degradation of a number of proteins, including programmed death ligand-1 (PD-L1) by protecting it from ubiquitin-mediated degradation. In this role, it could modulate the effectiveness of immunotherapy. Here, for the first time, we characterize CMTM6 expression in melanoma and evaluate its association with response to immune checkpoint inhibitors (ICI). We evaluated the expression of CMTM6, PD-L1 and other immune-related proteins in 60 pretreatment biopsies from metastatic melanoma patients who received immunotherapy, in a tissue microarray (TMA) using quantitative immunofluorescence (QIF). Expression of mRNA from control patients obtained from The Cancer Genome Atlas (TCGA) database was also compared. CMTM6 expression was positively correlated with PD-L1, CD3, CD20, and CD68 markers, at protein (Pearson’s r = 0.53–0.81, all P < .0001) and mRNA (Spearman’s r = 0.15–0.44, all P < .002, except for CD68 where P = .26) levels. CMTM6 protein was associated with longer survival after immunotherapy when measured in the stromal (P = .007) and all the immune compartments tested (T cells, B cells, and macrophages). Multivariable analyses also revealed significant CMTM6 survival associations when measured in stromal (Hazard Ratio (HR) = 0.12, P = .001) and CD68-positive (HR = 0.30, P = .043) compartments. Additionally, PD-L1 but not CMTM6 showed prognostic value in control patients. Finally, high CMTM6 and PD-L1 co-expression in the stromal compartment was significantly associated with longer survival in treated patients (P = .028). Consequently, CMTM6 expression shows potential as a predictive factor for ICI treatments.

Expression and Clinical Significance of CMTM6 in Nonsmall Cell Lung Cancer.

Chemokine-like factor (CKLF)-like MARVEL transmembrane domain containing protein 6 (CMTM6) is a ubiquitously expressed protein, which plays a critical role in the stability of programmed death-ligand 1. However, the expression of CMTM6 in a variety of cancer pathological tissues is not clear. Therefore, 109 patients who were diagnosed with nonsmall cell lung cancer (NSCLC) and underwent surgical resection were included in this retrospective study. The expression of CMTM6 in NSCLC patients' tissue samples were measured by immunohistochemistry and the results showed that 60 cases (55.05%) had high CMTM6 expression. The chi-square test showed that the expression of CMTM6 in NSCLC was significantly related to smoking (p = 0.017) and differentiation (p = 0.029). The Kaplan-Meier survival analysis suggested that the high expression of CMTM6 was associated with better prognosis of NSCLC patients. The univariate analysis revealed that the prognosis of NSCLC patients was correlated with T stage (p = 0.042), lymph node metastasis (p = 0.007), metastasis (p = 0.009), pathological differentiation (p = 0.001), and CMTM6 expression level (p < 0.001). In addition, multivariate analysis indicated that CMTM6 was an independent prognostic factor for NSCLC patients (p = 0.002). CMTM6 expression may have the potential to be a biomarker assisting in disease monitoring and prognosis in NSCLC.

CMTM6 is positively correlated with PD-L1 expression and immune cells infiltration in lung squamous carcinoma

BackgroundThe aim of this study was to clarify the association between CMTM6 and PD-L1 expression as well as microenvironment in lung squamous carcinoma (LUSC). Material and methodsUsing Spearman's correlation and Tumor Immune Estimation Resource (TIMER), we analyzed the relationship between CMTM6 and PD-L1 mRNA in LUSC. Immunohistochemistry (IHC) assay was applied to validate the correlation between CMTM6 and PD-L1 protein level in 80 LUSC samples originated from Shandong Provincial Hospital. Then, using The Cancer Genome Atlas (TCGA) database and fisher test, we analyzed the differential mutation genes in high and low CMTM6 expression group. TISIDB was used to explore the distribution of CMTM6 across immune- and molecular-subtypes. TCGA database and Gene Set variation analysis (GSVA) were used to analyze the relationship between CMTM6 and immune genes, immune related pathways. ResultPositive correlation between CMTM6 and PD-L1 in mRNA and protein level was found in LUSC patients. More gene mutations were found in CMTM6 high expression group compared with low expression group. Meanwhile, we also found the correlation between CMTM6 expression and molecular subtypes, immune genes, immune related pathways. Furthermore, our result revealed that B cells memory, T cells memory testing, T cells folicular helper, macrophages M0, macrophages M1 and neutrophils varied significantly between patients with CMTM6 high and low expression group. Finally, we found that CMTM6 expression was positively related to CD8 + T cell, macrophage, neutrophil and dendtritic cell (all, P < 0.05) and negatively related to CD4 + T cell (P = 0.018). ConclusionCMTM6 is positively associated with PD-L1 expression and correlates with infiltration of immune cells in microenvironment of lung squamous carcinoma.

Molecular and immune characteristics for lung adenocarcinoma patients with CMTM6 overexpression

BackgroundCMTM6 was identified as an important regulator of the PD-L1 protein. The role of CMTM6 in lung adenocarcinoma (LUAD) has so far remained unclear. We aimed at investigating the role of CMTM6 in LUAD at transcriptome and genomic levels and its relationship with tumor-infiltrating immune cells (TIICs). MethodsWe downloaded the data sets of LUAD from TCGA. The genomic profiles containing somatic mutations were analyzed and the transcriptome level of CMTM6 was also obtained. Gene set variation analysis (GSVA) was used to predict the pathway change. In addition, we explored the association between CMTM6 and LUAD immune infiltrates by means of CIBERSORT. The association between CMTM6 and PD-L1 mRNA was analyzed using an integrated repository portal for tumor-immune system interactions (TISIDB) and was further validated in 80 LUAD patients. Kaplan-Meier survival curve and the log-rank test was used to analyze the survival significance of CMTM6. ResultsWe found that CMTM6 was downregulated in LUAD. Patients with low CMTM6 expression were more likely to be frequent with somatic mutations. Moreover, GSVA analysis exhibited that CMTM6 was associated with immune responses and inflammatory activities. Specifically, a positive correlation between increased CMTM6 expression and immune infiltrating level of Dendritic cells resting, Eosinophils, Macrophages M1, Macrophages M2, Neutrophils, T cells CD4 memory activated and T cells CD4 memory resting was established. The CMTM6 expression was positively correlated with PD-L1 in both mRNA and protein level. Clinically, patients with high expression of CMTM6 tended to have a better survival. ConclusionCMTM6 expression likely had an important effect on TIICs composition and prognosis in LUAD patients. The CMTM6 expression was positively correlated with PD-L1 in LUAD. These findings establish CMTM6 as a promising target for immunotherapeutic prospects.

Targeting CMTM6 Suppresses Stem Cell-Like Properties and Enhances Antitumor Immunity in Head and Neck Squamous Cell Carcinoma.

CMTM6, a regulator of PD-L1 expression, also modulates tumor immunity. Little is known about the function of CMTM6 and its mechanism of action in head and neck squamous cell carcinoma (HNSCC). In this study, we found by IHC analysis that CMTM6 overexpression predicted a poor prognosis for patients with HNSCC. We discovered that CMTM6 expression was correlated with increased activity through the Wnt/β-catenin signaling pathway, which is essential for tumorigenesis, maintenance of cancer stem cells (CSC), and the epithelial-to-mesenchymal transition (EMT) characteristic of multiple cancers. We used short hairpin RNA to eliminate expression of CMTM6, which led, in HNSCC cells, to reduced expression of nuclear β-catenin as well as inhibition of stem cell-like properties, TGFβ-induced EMT, and cell proliferation. Consistent with these results, we identified a significant positive correlation between expression of CMTM6 and EMT- and CSC-related genes in The Cancer Genome Atlas (TCGA). We found positive correlations for both RNA and protein between expression of CMTM6 and immune checkpoint components. CMTM6 silencing-induced PD-L1 downregulation delayed SCC7 tumor growth and increased CD8+ and CD4+ T-cell infiltration. The proportions of PD-1+, TIM-3+, VISTA+, LAG-3+, and B7-H3+ exhausted T cells were decreased significantly in the CMTM6 knockdown group. CMTM6 thus regulates stemness, EMT, and T-cell dysfunction and may be a promising therapeutic target in the treatment of HNSCC.

Quantitative Assessment of CMTM6 in the Tumor Microenvironment and Association with Response to PD-1 Pathway Blockade in Advanced-Stage Non–Small Cell Lung Cancer

IntroductionCKLF like MARVEL transmembrane domain containing 6 (CMTM6) has been described as a programmed death ligand 1 (PD-L1) regulator at the protein level by modulating stability through ubiquitination. In this study, we describe the patterns of CMTM6 expression and assess its association with response to programmed cell death 1 pathway blockade in NSCLC. MethodsWe used multiplexed quantitative immunofluorescence to determine the expression of CMTM6 and PD-L1 in 438 NSCLCs represented in tissue microarrays, including in two independent retrospective cohorts of immunotherapy-treated (n = 69) and non–immunotherapy-treated (n = 258) patients and a third collection of EGFR- and KRAS-genotyped tumors (n = 111). ResultsTumor and stromal CMTM6 expression was detected in approximately 70% of NSCLCs. CMTM6 expression was not associated with clinical features or EGFR/KRAS mutational status and showed a modest correlation with T-cell infiltration (R2 < 0.40). We found a significant correlation between CMTM6 and PD-L1, which was higher in the stroma (R2 = 0.51) than in tumor cells (R2 = 0.35). In our retrospective NSCLC cohort, neither CMTM6 nor PD-L1 expression alone significantly predicted immunotherapy outcomes. However, high CMTM6 and PD-L1 coexpression in the stromal and CD68 compartments (adjusted hazard ratio = 0.38, p = 0.03), but not in tumor cells (p = 0.15), was significantly associated with longer overall survival in treated patients but was not observed in the absence of immunotherapy. ConclusionThis study supports the mechanistic role for CMTM6 in stabilization of PD-L1 in patient tumors and suggests that high coexpression of CMTM6 and PD-L1, particularly in stromal immune cells (macrophages), might identify the greatest benefit from programmed cell death 1 axis blockade in NSCLC.

Abstract 4105: CD8 positive tumor infiltrated lymphocytes as prognostic factor in sinonasal squamous cell carcinoma

Abstract Introduction Sinonasal squamous cell carcinoma (SNSCC) patients have a poor survival rate and treatment options are limited. We evaluated CD8-positive tumor-infiltrated lymphocytes (TILs) in 57 SNSCC and the relation to PD-L1 expression. Furthermore, we analyzed PD-L1 mutations and CMTM6 expression in relation to enhanced ability to inhibit TILs. Our aim was to identify patients that may benefit from therapy with immune checkpoint inhibitors. Experimental Procedures Four tissue microarray blocks were constructed including three 1 mm cores from different areas of 57 SNSCC. Each block also contained normal mucosa samples as controls. The presence of CD8+ TILs, tumoral PD-L1 and CMTM6 expression was evaluated by immunohistochemistry. The stainings were evaluated by 3 independent observers and results were correlated to clinico-pathological characteristics and follow-up data. Co-localization of CD8+ TILs and PD-L1+ cells was performed in 2-μm-thick FFPE sections by immunofluorescence. Exon 6 of the PD-L1 gene, which encodes the DTSSK motif, was amplified by PCR and products were sequenced by Sanger sequencing. Results High level presence of intratumoral CD8+ TILs occurred in 11/57 (19%), low level in 39/57 (69%) and absence in 7/57 (12%) cases. Five-year disease-specific survival (DSS) was 34% of cases with intratumoral CD8+ versus 71% in cases CD8- (P=0.03). Five-year DSS was 0%, 24% and 43% in high level, low level and absence of intratumoral CD8+ TILs, respectively (p=0.006). PD-L1 expression was observed in 26/57 (46%) of cases and did not correlate with clinico-pathological parameters or DSS. All patients with high intratumoral CD8+ expression were PD-L1 positive and 75% of patients with strong PD-L1 staining showed high intratumoral CD8+ expression. All intratumoral CD8+/PD-L1+/- patients died of disease before 5 years compared to 24% CD8-/PD-L1- and 48% CD8-/PD-L1+ patients who survived up to 5 years (P=0.003). Tumors CD8+/PD-L1+/- had a significant worse DSS than CD8-/PD-L1+/- cases (p=0.015). No mutations were found in the DTSSK domain of PD-L1 and CMTM6 showed expression in 43/57 (75%) of patients while 17 of those 43 (40%) were also PD-L1+. Conclusion Our data showed that SNSCC are immunogenic tumors with up to 88% of cases harboring some level of CD8+ TILs. The presence of intratumoral CD8+ TILs was correlated to worse survival, as has been observed previously in other tumor types. We also found 46% of tumors to express PD-L1. Together these results indicate that SNSCC patients could benefit from immunotherapy releasing the PD-1/PD-L1 immune checkpoint and reactivating the already present cytotoxic CD8+ TILs to exercise their antitumor activity. Pembrolizumab and nivolumab have already received FDA approval for clinical application in recurrent or metastatic HNSCC and may also be considered for treatment of recurrent SNSCC. Citation Format: ROCIO GARCIA-MARIN, CRISTINA RIOBELLO, VIRGINIA N. CABAL, LAURA SUAREZ-FERNANDEZ, SARA REDA, FERNANDO LOPEZ, JOSE L LLORENTE, MARIO HERMSEN. CD8 positive tumor infiltrated lymphocytes as prognostic factor in sinonasal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4105.

Therapeutic Co-targeting of WEE1 and ATM Downregulates PD-L1 Expression in Pancreatic Cancer.

PurposePancreatic cancer (PC) is one of the most lethal cancers worldwide, but there are currently no effective treatments. The DNA damage response (DDR) is under investigation for the development of novel anti-cancer drugs. Since DNA repair pathway alterations have been found frequently in PC, the purpose of this study was to test the DDR-targeting strategy in PC using WEE1 and ATM inhibitors.Materials and MethodsWe performed in vitro experiments using a total of ten human PC cell lines to evaluate antitumor effect of AZD1775 (WEE1 inhibitor) alone or combination with AZD0156 (ATM inhibitor). We established Capan-1–mouse model for in vivo experiments to confirm our findings.ResultsIn our research, we found that WEE1 inhibitor (AZD1775) as single agent showed anti-tumor effects in PC cells, however, targeting WEE1 upregulated p-ATM level. Here, we observed that co-targeting of WEE1 and ATM acted synergistically to reduce cell proliferation and migration, and to induce DNA damage in vitro. Notably, inhibition of WEE1 or WEE1/ATM downregulated programmed cell death ligand 1 expression by blocking glycogen synthase kinase-3β serine 9 phosphorylation and decrease of CMTM6 expression. In Capan-1 mouse xenograft model, AZD1775 plus AZD0156 (ATM inhibitor) treatment reduced tumor growth and downregulated tumor expression of programmed cell death ligand 1, CMTM6, CD163, and CXCR2, all of which contribute to tumor immune evasion.ConclusionDual blockade of WEE1 and ATM might be a potential therapeutic strategy for PC. Taken toget

Increased CMTM6 can predict the clinical response to PD-1 inhibitors in non-small cell lung cancer patients

ABSTRACTCKLF-like MARVEL transmembrane domain containing 6 (CMTM6) plays a crucial role in the stability of the programmed death-ligand 1 (PD-L1). However, there has been no previous study of CMTM6 in non-small cell lung cancer (NSCLC) and its association with PD-L1 has not been confirmed. The aim of this study was to investigate the expression of CMTM6 and PD-L1 and to confirm their predictive roles for anti-PD-1 therapy in non-small cell lung cancer. CMTM6 and PD-L1 immunohistochemical expressions were evaluated in 35 advanced, treatment-refractory NSCLC patients who received PD-1 inhibitor therapy. The correlation between CMTM6 and PD-L1 expression was also determined based on immunohistochemistry and RNA-sequencing data obtained from The Cancer Genome Atlas (TCGA) database. CMTM6 expression was positively correlated with PD-L1 expression in immunohistochemical data (Pearson’s r = 0.342 and p = .044). A positive correlation was also identified in the mRNA expression data. Using receiver operating characteristic curves, the levels of CMTM6 and PD-L1 expression which provided the best distinguishing point between responder versus non-responder to PD-1 inhibitors were 70 and 75 H-scores, respectively. The patients in the PD-1 inhibitor responder group had higher CMTM6 expressions in univariate logistic regression analysis (odds ratio (OR) = 5.333, p = .037). However, PD-L1 expression was not associated with response to PD-1 inhibitor (p = .288). In multivariate analysis, CMTM6 was also found to be an independent predictor of the response to PD-1 inhibitors (OR = 6.226, p = .032). CMTM6 expression can be a promising predictor useful for therapeutic decision-making regarding PD-1 inhibitors.

Expression and Clinical Significance of CMTM6 in Hepatocellular Carcinoma.

This study aimed to examine the expression level and clinical significance of chemokine-like factor-like MARVEL transmembrane domain-containing family member 6 (CMTM6) in paired hepatocellular carcinoma (HCC) and adjacent nontumor tissues. The expression of CMTM6 was detected in 75 paired HCC and adjacent nontumor tissues by immunohistochemistry. Chi-square test was used to compare the difference of CMTM6 expression between HCC tissues and adjacent nontumor tissues. The clinic-pathological features and prognosis of HCC patients were collected to analyze the relationship with CMTM6 expression. The positive expression of CMTM6 in HCC tissues was significantly lower than that of adjacent nontumor tissues. The difference of CMTM6 expression between HCC tissues and paired adjacent nontumor tissues was statistically significant (p < 0.05). Furthermore, CMTM6 expression was correlated with HCC metastasis and alpha-fetoprotein (AFP) (p < 0.05). Multivariate logistic regression analysis showed tumor staging, metastasis, and AFP had a significant relationship with CMTM6 expression. In addition, the survival time of HCC patients was different between CMTM6 positive group and CMTM6 negative group by Kaplan-Meier survival analysis (p < 0.05). Downregulation of CMTM6 is related to HCC metastasis and the prognosis of HCC patients.

CMTM6 overexpression is associated with molecular and clinical characteristics of malignancy and predicts poor prognosis in gliomas.

CMTM6, a previously uncharacterized protein, was identified as a critical regulator of PD-L1, which is reported as an immune checkpoint inhibitor, to modulate the T cell activities both in vitro and in vivo of other tumors. However, the role of CMTM6 has so far remained unclear in glioma. To investigate the role of CMTM6 in gliomas, we analyzed the transcriptome level, genomic profiles and its relationship with clinical practice. 1862 glioma samples with transcriptome data were enrolled in this study, including CGGA RNA-seq, TCGA RNA-seq, CGGA-microarray, GSE16011 and IVY GBM databases. Clinical information and genomic profiles containing somatic mutations and DNA copy numbers were also obtained. We found that CMTM6 expression was highly correlated with major clinical and molecular characteristics. Cases with high CMTM6 expression were more likely to be predicted as malignant entities and frequent with genomic aberrations of driver oncogenes. Moreover, gene ontology analysis based on significantly correlated genes of CMTM6 expression exhibited that CMTM6 was associated with immune responses and inflammatory activities. CMTM6 was synthetic with other immune checkpoint inhibitors. Additionally, CMTM6 was involved in immune functions via modulating T-lymphocyte-mediated anti-tumor immunity. Finally, high CMTM6 expression was associated with reduced survival time and may serve as a strong indicator of poor prognosis in gliomas. In brief, High level of CMTM6 expression is closely related to high malignant gliomas. Meanwhile, CMTM6 plays an important role in regulating T cell activation and antitumor responses. Therefore, CMTM6 is a promising target for developing immunotherapy of gliomas.