PD-L1 Protein Expression Research Articles

PD-L1 IHC 22C3 pharmDx Demonstrates Precision and Reproducibility in Triple-negative Breast Cancer

In the United States, the PD-L1 IHC 22C3 pharmDx indications for use have expanded to include the assay’s use as an aid in identifying triple-negative breast cancer (TNBC) patients for treatment with pembrolizumab (KEYTRUDA®). Analytical validation for TNBC was conducted by Agilent Technologies in support of the device performance and FDA-approval. Analytical validation studies included device precision (inter-instrument, inter-operator, inter-day, inter-lot, and intra-run) and external reproducibility (inter- and intra-site, inter- and intra-observer) using the Combined Positive Score (CPS) ≥ 10 cutoff. All precision and external reproducibility studies achieved confidence interval lower bound values of greater than 85% for positive, negative, and overall percent agreement. These analytical validation studies demonstrate that PD-L1 IHC 22C3 pharmDx is precise and reproducible in evaluating PD-L1 protein expression at a CPS ≥ 10 cutoff when used for testing TNBC tissues.

PD-L1 (CD274) promoter hypomethylation predicts immunotherapy response in metastatic urothelial carcinoma

ABSTRACT PD-L1 status assessed by immunohistochemistry (IHC) has failed to reliably predict outcomes for patients with metastatic urothelial carcinoma (mUC) on immune checkpoint blockade (ICB). PD-L1 promoter methylation is an epigenetic mechanism that has been shown to regulate PD-L1 mRNA expression in various malignancies. The aim of our present study was to evaluate the predictive potential of PD-L1 promoter methylation status (mPD-L1) in ICB-treated mUC compared to conventional IHC-based PD-L1 assessment. We quantified mPD-L1 in formalin-fixed and paraffin-embedded tissue sections using an established quantitative methylation-specific PCR assay (qMSP) in a well-characterized multicenter ICB-treated cohort comprising N = 107 patients with mUC. Additionally, PD-L1 protein expression in tumor tissues was assessed using regulatory approved IHC protocols. The effect of pharmacological hypomethylation by the DNA methyltransferase inhibitor decitabine in combination with interferon-γ stimulation in urothelial carcinoma cell lines was investigated by IHC and FACS. mPD-L1 hypomethylation predicted objective response rate at the first staging on ICB. Patients with tumors categorized as PD-L1 hypomethylated (lower quartile) showed significantly prolonged progression-free (PFS) and overall survival (OS) after ICB initiation. In contrast, PD-L1 protein expression status neither correlated with response nor survival. In multivariable Cox regression analyses, PD-L1 promoter hypermethylation remained an independent predictor of unfavorable PFS and OS. In urothelial carcinoma cell lines, pharmacological demethylation led to an upregulation of membranous PD-L1 expression and an enhanced inducibility of PD-L1 expression by interferon γ. Hypomethylation of the PD-L1 promoter is a promising predictive biomarker for response to ICB in patients with mUC.

LncTUG1 promotes hepatocellular carcinoma immune evasion via upregulating PD-L1 expression

HCC is one of the most common malignant tumors worldwide. Although traditional treatment methods have been improved in recent years, the survival rate of HCC patients has not been significantly improved. Immunotherapy has shown extremely high clinical value in a variety of tumors. In this study, we found that TUG1 could regulate the expression of PD-L1 through JAK2/STAT3 to mediate immunosuppression. Here, The expression of TUG1 and PD-L1 in HCC tissues was evaluated through analysis of databases and verified in HCC tissue and HCC cancer cells by qRT-PCR. The effect of TUG1 on tumor immune escape was detected by coculture, and cell viability was detected with a CCK8 assay. The results demonstrated that TUG1 was closely associated with anticancer immunity. TUG1 and PD-L1 were highly expressed in HCC tissues and HCC cancer cells, and high expression of TUG1 and PD-L1 was related to the poor prognosis of HCC patients. In addition, knocking down TUG1 expression could reduce PD-L1 expression and enhance the cancer cell-killing capability of T cells. Downregulating TUG1 expression could also decrease the mRNA and protein expression of JAK2 and STAT3. To sum up, TUG1 and PD-L1 are overexpressed in patients with liver cancer and are related to the poor prognosis of these patients. Silencing TUG1 expression reduced the mRNA and protein expression of PD-L1 by affecting the JAK2/STAT3 pathway.

CMTM6 shapes antitumor Tcell response through modulating protein expression of CD58 and PD-L1.

The dysregulated expression of immune checkpoint molecules enables cancer cells to evade immune destruction. While blockade of inhibitory immune checkpoints like PD-L1 forms the basis of current cancer immunotherapies, a deficiency in costimulatory signals can render these therapies futile. CD58, a costimulatory ligand, plays a crucial role in antitumor immune responses, but the mechanisms controlling its expression remain unclear. Using two systematic approaches, we reveal that CMTM6 positively regulates CD58 expression. Notably, CMTM6 interacts with both CD58 and PD-L1, maintaining the expression of these two immune checkpoint ligands with opposing functions. Functionally, the presence of CMTM6 and CD58 on tumor cells significantly affects Tcell-tumor interactions and response to PD-L1-PD-1 blockade. Collectively, these findings provide fundamental insights into CD58 regulation, uncover a shared regulator of stimulatory and inhibitory immune checkpoints, and highlight the importance of tumor-intrinsic CMTM6 and CD58 expression in antitumor immune responses.

300P Correlation of PD-L1 protein and mRNA expression and their prognostic impact in triple-negative breast cancer

300P Correlation of PD-L1 protein and mRNA expression and their prognostic impact in triple-negative breast cancer

PD-L1 mRNA Detection in Immunohistochemically Negative Patients: A Complementary Method for a Better Treatment Selection?

PD-L1 inhibitors have been approved for cisplatin-ineligible urothelial cancer patients relapsing after radical cystectomy. A prerequisite for therapy is a positive PD-L1 expression in the tumor tissue, whereas no options are available for patients with negative PD-L1 status. However, studies revealed that many PD-L1-negative patients also responded to PD-L1 therapy. This study investigated the feasibility of PD-L1 mRNA complementary RNA in situ hybridization (RNAish) analysis to detect PD-L1-responders independent of PD-L1 protein status. Immunohistochemistry and RNA in situ hybridization were used to assess PD-L1 protein and mRNA in radical cystectomy tissue from patients with advanced and metastasized urothelial cancer. In this study, PD-L1 protein and mRNA were detected in ≥90% of the examined tissue. Positive PD-L1 mRNA expression (≥1%) on TC and IC could be evaluated in 77% and 31% of the cases, respectively. Moreover, scatterplot analysis revealed a PD-L1 mRNA positive and PD-L1 protein negative subpopulation. According to the CPS score, positive PD-L1 protein expression could be evaluated in 88% and positive PD-L1 mRNA expression in 71% of the cases. Scatterplot analysis of the CPS scores revealed a CPS protein negative but CPS mRNA positive small subpopulation. The feasibility of RNAish on formalin-fixed tissue could be proven. Moreover, complementary PD-L1 RNAish identified a sub-population of PD-L1 protein-negative and PD-L1 mRNA-positive patients, which may benefit from PD-L1 therapy.

PD-L1 protein expression by Combined Positive Score (CPS) in patients with muscle invasive or advanced urothelial carcinoma: a single institution experience

IntroductionImmune checkpoint inhibitors have revolutionized the treatment of patients with advanced urothelial carcinoma (UC) in the frontline and relapsed settings. Lebanon has one of the highest incidence of UC worldwide, yet no data exists regarding the expression of PD-L1 by Combined Positive Score (CPS) in advanced disease.MethodsWe reviewed all patients treated at our institution for high grade UC, stage pT2 and above, between January 2017 and March 2021. We assessed the expression of PD-L1 by immunohistochemistry using 22C3 clone, and analyzed the association between PD-L1 expression and clinicopathological characteristics. PD-L1 positivity was defined as CPS score ≥ 10.ResultsA total of 101 patients with advanced UC were included, with a median age of 71 years (range, 38 to 96 years); 78% were ever-smokers. Ninety-three of 101 patients (92%) had conventional UC and 43 patients (43%) had positive PD-L1 expression, with 12 patients having CPS of 100. The analysis by molecular subtype showed that patients with maximal CPS of 100 were enriched in “basal” molecular subtype. However, no association was found between PD-L1 expression (positive versus negative) and clinicopathological characteristics.ConclusionThe positivity of PD-L1 expression as assessed by CPS using the 22C3 clone in our population was almost comparable to the results reported in the occidental literature. Therefore, PD-L1 expression, as a potential predictor of response to immunotherapy, concerns the same percentage of the Lebanese UC patients.

Silencing of Perilipin 3 Inhibits Lung Adenocarcinoma Cell Immune Resistance by Regulating the Transcription of PD-L1 Through c-Myc

ABSTRACT Background Perilipin 3 (PLIN3), a lipid droplet-associated protein, is found to be highly expressed in human cancers. This study aimed to investigate the biological functions and underlying mechanism of PLIN3 in lung adenocarcinoma (LUAD). Methods To analyse PLIN3 expression in normal and cancerous tissues, relevance between PLIN3 expression and survival prognosis, and to predict the pathways related to PLIN3, bioinformatic analysis was performed. In A549 and H1299 cells, qRT-PCR or western blotting was used to determine mRNA/protein expression of PLIN3, PD-L1, and c-Myc. In A549 and H1299 cells, CCK-8 assay, EdU, and flow cytometry were used to assess cell viability, proliferation, and apoptosis. Chip and luciferase reporter assays were performed to verify the binding of PD-L1 with c-Myc. The functions of PLIN3 were examined in vivo in a xenograft tumor model. Results In LUAD tissues and cells, PLIN3 expression was downregulated. A shorter survival time was observed in patients with high PLIN3 expression than in patients with low PLIN3 expression. Silencing of PLIN3 inhibited cell proliferation, PD-L1 expression, and Myc pathway, as well as induced apoptosis in LUAD cells. c-Myc acts as a transcription factor of PD-L1. Moreover, the inhibitory actions of PLIN3 silencing on c-Myc and PD-L1 expression as well as cell proliferation and stimulatory action of PLIN3 silencing on cell apoptosis were reversed by c-Myc overexpression. In vivo, PLIN3 silencing inhibited the growth of xenograft tumour and reduced PLIN3, PD-L1, and c-Myc protein expression. Conclusion Silencing of PLIN3 inhibited tumour growth by regulating the Myc/PD-L1 pathway.

Low co-expression of PD-L1 and oncogenic receptor tyrosine kinases HER2 and cMET in urothelial carcinoma is associated with discordant expression between primary and metastatic sites

Novel regimens targeting immune checkpoints and the cMET or HER2 pathways are under investigation in metastatic urothelial carcinoma (mUC) though co-expression of these molecular targets has not been defined. We sought to characterize the protein co-expression rates of PD-L1, cMET and HER2 in primary and metastatic mUC lesions and agreement rates in paired biopsies.We assessed PD-L1, cMET and HER2 protein expression by immunohistochemistry (IHC) in archival mUC samples identified from an institutional database (n = 143). Correlation of expression between primary and metastatic biopsies was performed in patients with available paired biopsies (n = 79). Protein expression levels by predefined thresholds were measured, and Cohen's kappa statistics (κ) were utilized to assess the agreement in expression between paired primary and metastatic samples.In primary tumors (n = 85), high expression of PD-L1, cMET, and HER2 was observed in 14.1%, 34.1%, and 12.9%, respectively. In metastatic samples (n = 143), high expression of PD-L1, cMET and HER2 was detected in 9.8%, 41.3%, and 9.8%, respectively. Expression agreement rates between paired specimens (n = 79) were PD-L1: 79.7% (κ = 0.09), cMET: 69.6% (κ = 0.35), HER2: 84.8% (κ = 0.17). High PD-L1/cMET co-expression was observed in only 5.1% (n = 4) of primary and 4.9% (n = 7) of metastatic specimens. High co-expression of PD-L1/HER2 occurred in 3.8% (n = 3) of primary samples and no metastatic samples. The overall co-expression agreement between paired samples was 55.7% (κ = 0.22) for PD-L1/cMET and 67.1% (κ = 0.06) for PD-L1/HER2, but agreement for high co-expression between paired samples was very low (2.5% for PD-L1/cMET and 0% for PD-L1/HER2).Tumor co-expression of high cMET or HER2 and PD-L1 is low in this cohort. Agreement of high co-expression between primary and metastatic sites is rare. Biomarker-based strategies used in selection of patients for contemporary trials testing combinations of immune checkpoint inhibitors with either cMET or HER2-targeted agents should account for discordant biomarker expression between primary and metastatic sites.

NUP62CL as an Immunological and Prognostic Biomarker of Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common head and neck malignancy with a high mortality rate and poor prognosis. The exploration and understanding of biomarkers will help to further improve the diagnosis and treatment of OSCC. Tumor tissue samples from 319 OSCC patients were retrospectively collected, along with their clinical information. In combination with bioinformatics tools and multiplex immunohistochemistry (mIHC) analyses, we evaluated NUP62CL protein expression and its relationship to tumor-infiltrating immune cells (TIICs) and immune checkpoints in the tumor microenvironment (TME), as well as its association with clinical features and prognosis of OSCC. We identified high-NUP62CL expression in OSCC tissues, and high-NUP62CL protein expression was associated with large tumor size, advanced clinical stage and poor prognosis. In addition, NUP62CL protein expression was positively associated with the abundance of CD3+CD4+ T cells (P<0.01), CD3+CD8+ T cells (P<0.01), CD56+ NK cells (P<0.05), CD68+CD86+ macrophages (P<0.01) and CD68+CD163+ macrophages (P<0.01), as well as the immune checkpoints, including PD-1 (P<0.001), PD-L1 (P<0.001), and CTLA-4 (P<0.001) protein expression. In conclusion, NUP62CL could be an effective prognostic and immunological biomarker for OSCC patients.

Si Jun Zi decoction inhibits the growth of lung cancer by reducing the expression of PD-L1 through TLR4/MyD88/NF-κB pathway

Ethnopharmacological relevanceSi Jun Zi decoction (SJZT) is a traditional Chinese medicine (TCM) formula with the effect of invigorating the spleen qi and replenishing qi. TCM believes that a strong spleen qi helps to strengthen lung qi. Lung cancer is often caused by a deficiency of lung qi. Based on this theory, TCM often applies SJZT to the treatment of lung cancer and has achieved remarkable results. However, the mechanism of SJZT in the treatment of lung cancer remains unclear and requires further study. Aim of the studyThe main purpose of this study is to explore the mechanism of SJZT against lung cancer. Materials and methodsIn this study, the chemical constituents in SJZT were analyzed by UPLC-Q-Exactive-MS/MS. MTT and cell scratch test were used to determine the cell viability and inhibition of migration in vitro. The effect of SJZT on the expression of PD-L1 protein in A549 cells was detected by Western Blotting (WB). Apoptosis was detected by crystal violet staining. The mouse model of Lewis lung cancer was established in vivo, and the levels of serum TNF-α and IL-2 were detected by enzyme linked immunosorbent assay (ELISA). The protein levels of TLR4, MyD88, NF-κB and PD-L1 in tumor tissues of mice were detected by WB. Quantitative real-time PCR (qRT-PCR) was used to detect the levels of TLR4, MyD88, NF-κB and PD-L1 mRNA. Finally, hematoxylin and eosin (H&E) staining were used to detect the pathological status of tumor tissues in mice. ResultsA total of 16 active chemical constituents were identified in SJZT. In vitro experiments showed that SJZT could inhibit the growth of A549, induce apoptosis and reduce the expression of PD-L1. In vivo experiments showed that SJZT regulated TLR4/MyD88/NF-κB signaling pathway, decreased the expression of PD-L1, and inhibited tumor growth. ConclusionsSJZT inhibits the growth of lung cancer by regulating TLR4/MyD88/NF-κB signal pathway and reducing the expression of PD-L1.

Tumor-TME Bipartite Landscape of PD-1/PD-L1 in Endometrial Cancers.

The bipartite landscape of tumor cells and stromal cells determines a tumor's response to treatment during disease management. In endometrial cancers (ECs), the mechanistic contribution of PD-L1/L2 and PD-1 signaling of the host's tumor microenvironment (TME) (CAF and immune cells) in the context of the tumor cells is elusive. To understand the tumor-stroma-immune crosstalk, we studied the compartmental pattern of PD-L1/L2 and PD-1 expression in EC tissues and their matched CAFs. Over 116 surgically resected tumors (T) and the tumor-adjacent normal tissues (N) were obtained from consented unselected consecutive patients. IHC was performed in T, N-epi-thelium, and the stromal mesenchymal environment (SME; mesenchyme) in the T and N tissues. The staining intensity and distribution patterns of PD-L1/L2 and PD-1 in the FFPE sections of T and N were evaluated by a pathologist using a standard scoring system of TPS and CPS. We tested the PD-L1/L2 and PD-1 immune landscape of tumor-TME pair and normal epithelial-stromal mesenchyme pairs from patients with different grades of disease vis-à-vis their CAF PD-L1 levels. We used qRT-PCR to determine the expressions of mRNAs, while the flow cytometry and ICC determined the level of expression of proteins. We observed higher levels of PD-L1 mRNA and protein expression in primary CAFs from the resected tumor tissue compared to the tumor-adjacent normal tissues. We also determined the expression of patients' soluble PD-L1/L2 as peripheral readouts of PD-L1/L2 and PD-1. As we evaluated the results in the context of their pathological parameters, such as grades, stages, lymphovascular invasion, percentage of myometrial invasion, and dMMR in patients, the dominance of PD-L1 expression in TME was positively correlated to the higher pathological grades of tumors, and its relationship with the dMMR. Since the neutralization of CD8-positive cytotoxic T-cells is PD-L1-dependent, our data indicate that irrespective of the PD-L1 positivity of tumor cells, the PD-L1-positive CAFs can play a critical role in bringing out an additional load of PD-L1 for an effective engagement of PD-1 within a tumor mass.

PPARγ agonist inhibits c-Myc-mediated colorectal cancer tumor immune escape.

As a master transcription factor, c-Myc plays an important role in promoting tumor immune escape. In addition, PPARγ (peroxisome proliferator-activated receptor γ) regulates cell metabolism, inflammation, and tumor progression, while the effect of PPARγ on c-Myc-mediated tumor immune escape is still unclear. Here we found that cells treated with PPARγ agonist pioglitazone (PIOG) reduced c-Myc protein expression in a PPARγ-dependent manner. qPCR analysis showed that PIOG had no significant effect on c-Myc gene levels. Further analysis showed that PIOG decreased c-Myc protein half-life. Moreover, PIOG increased the binding of c-Myc to PPARγ, and induced c-Myc ubiquitination and degradation. Importantly, c-Myc increased PD-L1 and CD47 immune checkpoint protein expression and promoted tumor immune escape, while PIOG inhibited this event. These findings suggest that PPARγ agonist inhibited c-Myc-mediated tumor immune escape by inducing its ubiquitination and degradation.

Update on lung cancer

Lung cancer is the leading cause of cancer deaths in Canada and worldwide. Major scientific advances in the last decade have dramatically changed the way we detect and treat lung cancers. British Columbia, Ontario and Quebec have recently launched lung cancer screening programs for high-risk individuals using evidence-based risk prediction models, and many other provinces are in the active planning phase. This review focuses on non-small cell lung cancer (NSCLC), which comprises 80-85% of lung cancers. For patients diagnosed with advanced or metastatic NSCLC, we now have systemic therapy options—targeted therapies, chemotherapies and immune checkpoint inhibitors (ICIs)—that are tailored to the molecular characterization of individual tumor samples for DNA/RNA alterations and PD-L1 protein expression. Molecular characterization is also becoming increasingly crucial for patients diagnosed with earlier and potentially curable NSCLC, as targeted therapies and ICIs make their way into the adjuvant and neoadjuvant therapy space, partnering with radiation and surgery to improve outcomes for patients receiving multimodality curative-intent treatment.

Immunohistochemical scoring of LAG-3 in conjunction with CD8 in the tumor microenvironment predicts response to immunotherapy in hepatocellular carcinoma.

Immune checkpoint blockade (ICB) is a systemic therapeutic option for advanced hepatocellular carcinoma (HCC). However, low patient response rates necessitate the development of robust predictive biomarkers that identify individuals who will benefit from ICB. A 4-gene inflammatory signature, comprising CD8, PD-L1, LAG-3, and STAT1, was recently shown to be associated with a better overall response to ICB in various cancer types. Here, we examined whether tissue protein expression of CD8, PD-L1, LAG-3, and STAT1 predicts response to ICB in HCC. HCC samples from 191 Asian patients, comprising resection specimens from 124 patients (ICB-naïve) and pre-treatment specimens from 67 advanced HCC patients treated with ICB (ICB-treated), were analyzed for CD8, PD-L1, LAG-3, and STAT1 tissue expression using multiplex immunohistochemistry followed by statistical and survival analyses. Immunohistochemical and survival analyses of ICB-naïve samples showed that high LAG-3 expression was associated with shorter median progression-free survival (mPFS) and overall survival (mOS). Analysis of ICB-treated samples revealed that high proportions of LAG-3+ and LAG-3+CD8+ cells pre-treatment were most closely associated with longer mPFS and mOS. Using a log-likelihood model, adding the total LAG-3+ cell proportion to the total CD8+ cell proportion significantly increased the predictive values for mPFS and mOS, compared with the total CD8+ cell proportion alone. Moreover, levels of CD8 and STAT1, but not PD-L1, were significantly correlated with better responses to ICB. After analyzing viral-related and non-viral HCC samples separately, only the LAG3+CD8+ cell proportion was significantly associated with responses to ICB regardless of viral status. Immunohistochemical scoring of pre-treatment levels of LAG-3 and CD8 in the tumor microenvironment may help predict ICB benefits in HCC patients. Furthermore, immunohistochemistry-based techniques offer the advantage of being readily translatable in the clinical setting.

Salvia mitiorrhiza Bunge aqueous extract attenuates infiltration of tumor-associated macrophages and potentiates anti-PD-L1 immunotherapy in colorectal cancer through modulating Cox2/PGE2 cascade

Ethnopharmacological relevanceBased on the notion of traditional Chinese medicine, the theory of invigorating the circulation of blood is a prominent treatment for cancer in clinic. Therefore, Salvia miltiorrhiza Bunge, as a representative of Chinese medicine of invigorating the circulation of blood, has been proved to be an effective medicinal herb for treating cancer. Aim of the studyTo clarify the anti-cancer effect of Salvia miltiorrhiza Bunge aqueous extract (SMAE) on colorectal cancer (CRC) and investigate whether the therapeutic effect of SMAE was mediated by attenuating the infiltration of tumor-associated macrophages (TAMs) into the tumor microenvironment (TME). Materials and methodsHigh-performance liquid chromatography (HPLC) was used for determined the main compounds of SMAE. MC38 cells were subcutaneously injected into the mice to establish the mouse model of CRC. Tumor growth curve was detected by tumor volume measurement. The model group received distilled water irrigation once a day. SMAE-treated group received 5 g/kg or 10 g/kg SMAE once a day. Anti-PD-L1 treated group received 5 mg/kg anti-PD-L1 once every three days. Protein expression of Cox2 and PD-L1 was determined by Western blot assay. The secretion levels of PGE2, IL-1β, IL-6, MCP-1, and GM-CSF were evaluated through ELISA. The mRNA expression of CSF1, CCL2, CXCL1, CXCL2, and CXCL3 was measured by using RT-qPCR. Staining of Ki67, TUNEL and Caspase3 was used to investigate cell proliferation and apoptosis. Immunohistochemical staining was used to determine CD8+ T cell distribution. H&E staining was used to confirm histopathological changes. The expressions of F4/80 and CD68 were measured by flow cytometry to identify macrophages in tumors and lymph nodes. The number of CD8+ T cells and the expression of PD-1, IFN-γ, and Granzyme B (GZMB) were determined by flow cytometry. ResultsSMAE significantly retarded the growth of MC38 mouse colorectal cancer. SMAE strikingly inhibited the expression of Cox2 and impaired the secretion of PGE2 in tumors, contributing to the attenuated intra-tumoral infiltration of TAMs via Cox2/PGE2 cascade. Meanwhile, SMAE augmented anti-tumor immunity by the elevated proportion of IFN-γ+ CD8+ T cells and GZMB+ CD8+ T cells, which decreased the tumor load. Furthermore, the combination of SMAE and anti-PD-L1 showed a higher therapeutic efficacy than either monotherapy in controlling tumor growth in MC38 xenograft model. ConclusionsSMAE attenuated the infiltration of TAMs into tumors and synergized with anti-PD-L1 to treat CRC via modulating Cox2/PGE2 cascade.

Comprehensive profiling of clock genes expression in hepatocellular carcinoma (HCC).

4108 Background: The circadian clock mechanism controls the physiological homeostasis of the liver and plays a key role in hepatocarcinogenesis. Recent evidence unveiled core clock proteins as novel therapeutic targets in cancer. Our group showed that clock regulators BMAL1 and CLOCK can promote proliferation of liver cancer cells by modulating the cell cycle checkpoint kinase Wee1. Here we further evaluated the molecular landscape of clock pathway alterations in HCC leveraging multi-platform profiling of patient tumor samples. Methods: 780 HCC tested at Caris Life Sciences (Phoenix, AZ) with WTS (Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES) were analyzed. Clock gene Score (CS) was determined using expression of core clock genes Z scores (positives of CLOCK, ARNTL, RORA/B/C and negatives of repressors CRY1/2, PER1/2/3, REVERBA/B) stratified by quartiles. xCell was used to quantify cell infiltration in the tumor microenvironment (TME). Significance was determined as P-values and adjusted for multiple testing ( q) of &lt; .05. Gene expression profiles were analyzed for transcriptional signatures predictive of response to immunotherapy including the T cell inflamed score (TIS) and IFG score. Real world survival was obtained from insurance claims data and Kaplan-Meier estimates were calculated for comparison. Results: CS was higher in metastatic sites than primary tumors (median transcripts per million [TPM]: 0.81 vs 0.37, P &lt; .05). No significant differences in patient age and sex were observed between CS Q1 (lowest) and Q4 (highest) cohorts, although a trend towards a higher frequency of males was observed in Q4 (76% vs 68%, Q4 vs Q1, P = .07). CS was positively associated with telomerase subunit TERT mutations (64% vs 52%, Q4 vs Q1, P = .04) and negatively correlated with FGF3 copy number amplification (2% vs 6%, P = .04) and WEE1 gene expression (median TPM: 15 vs 28, q &lt; .05). No dMMR/MSI-H tumors were observed in our series and there were no significant associations with tumor mutational burden and PD-L1 protein expression. Expression of immune related genes was lower in tumors with high CS, including IDO1, CD80, PD-L1, LAG3, CD86, TIM3, PD-1 and PD-L2 (fold change: 0.57-0.67 q &lt; .05). NK cell infiltration in the TME and the TIS score were also significantly lower in CS-high HCC ( q &lt; .05). Individually, lower CLOCK and CRY1 tumor mRNA expression were associated with longer OS (Q1 vs Q4: CLOCK HR 0.71, 95%CI [0.51-0.98], P = .04 and CRY1 HR 0.70 [0.51-0.95], P = .02, respectively). Conclusions: This is the most extensive profiling study to investigate the expression of clock genes in HCC. Our data show that clock genes expression impacts patient survival and is associated with alterations in immune-related gene expression and TIS score which suggest a role in the modulation of anti-tumor immunity. These results support the clock pathway role as a oncogenic driver and its potential as a therapeutic target in HCC.

Metastatic breast cancer (MBC) with ultra-high tumor mutational burden (UHTMB): A comprehensive genomic profiling (CGP) study.

1036 Background: Pts with MBC whose tumors feature high TMB (≥ 10 mutations/Mb) are eligible for on label immune checkpoint inhibitor (ICI) treatment. This study evaluated the genomic landscape of MBC with “Ultra high” TMB, defined at &gt; 20 mutations/Mb. Methods: 2049 MBC underwent hybrid capture-based CGP to evaluate all classes of genomic alterations (GA), TMB, microsatellite instability (MSI) and trinucleotide mutational signatures. HER2 IHC results were available in a subset of pts. PD-L1 expression on immunocytes was determined by IHC (Ventana SP142). Results: 45/2049 (2.2%) of MBC were UHTMB. 45 (100%) pts had metastatic disease. 38 (84%) had documented Stage IV disease and 7 documented axillary LN metastases at the time of sequencing. Local breast tumor was used for CGP in 19 (42.2%) MBC and metastatic site biopsy was used in 26 (57.8%). When compared with 2004 non-UHTMB pts with UHTMB were older (mean 64.6 yrs vs 58.2 yrs p &lt; .0001), more often had lobular histology (40.0% vs 14.5% p &lt; .0001) and ER+ disease (86.6% vs 70.0%). Of the 35 UHTMB cases with HER2 IHC data available, 11 (31.4%%) were HER2 IHC negative (0+), 21 (60.0%) were HER2-low status (9 1+ and 12 2+/ISH negative) and 3 (8.6%) were HER2 IHC positive (3+). 1/3 HER2 IHC2+ cases and 2/45 (4.4%) of all UHTMB cases were positive for HER2 copy number gain on CGP. UHTMB cases had more driver GA/tumor (mean 9.8 vs 5.7 p &lt; .0001) and were less often TNBC (13.3% vs 27.0% p = .041) compared to non-UHTMB high cancers. Mutation signature analysis revealed APOBEC was predominant in UHTMB samples (82.5%); MMR signature was also observed in 10% of cases. MSI high status was significantly more frequent in UHTMB high cases (11.6% vs 0.4% p &lt; .0001). GA more frequently identified in UHTMB cases included CDH1 (45.5% vs 14.3% p &lt; .0001), PIK3CA (81.8% vs 37.9% p &lt; .0001), CDKN2A (11.4% vs 3.2% p = .017), ARID1A (25.0% vs 5.0% p &lt; .0001) and NF1 (20.5% vs 5.9% p = .0014). PD-L1 ( CD274) gene amplification (2.3% vs 1.3%) or protein expression by the Ventana SP142 assay (57.14% vs 51.10%) were not significantly different among groups. Conclusions: UHTMB MBC is a rare, yet clinically important subset of clinically advanced breast cancer driven by APOBEC mutagenesis, with high incidence of ER+ lobular histology and frequent alterations in CDH1 and PIK3CA. In addition to potential benefit from ICI based treatment, UHTMB MBC present with a high frequency of HER2-low status which may impact therapy decisions for this rare disease. [Table: see text]

Association of long noncoding RNA MALAT1 with the radiosensitivity of lung adenocarcinoma cells via the miR-140/PD-L1 axis

ObjectiveTo investigate the effect of MALAT1 on the modulating the radiosensitivity of lung adenocarcinoma, through regulation of the expression of the miR-140/PD-L1 axis. MethodsThe online databases UALCAN and dbDEMC were searched for the MALAT1 and miR-140 expressions in patients with lung adenocarcinoma (LUAD), respectively. Then analyze their relationship with overall survival rates separately in the UALCAN and ONCOMIR databases. A functional analysis was performed for A549 cells by transfecting small-interfering RNAs or corresponding plasmids after radiotherapy. Xenograft models of LUAD exposed to radiation were established to further observe the effects of MALAT1 on the radiosensitivity of LUAD. The luciferase assay and reverse transcription–polymerase chain reaction were performed to assess the interaction between miR-140 and MALAT1 or PD-L1. ResultsMALAT1 were overexpressed in human LUAD tumor tissues and cell lines, while miR-140 were inhibited. MALAT1 knockdown or miR-140 increase suppressed cell proliferation and promoted cell apoptosis in LUAD after irradiation. LUAD xenograft tumor growth was also inhibited by MALAT1 knockdown combined with irradiation. miR-140 could directly bind with MALAT1 or PD-L1. Furthermore, MALAT1 knockdown inhibited PD-L1 mRNA and protein expressions by upregulating miR-140 in LUAD cells. ConclusionMALAT1 may function as a sponge for miR-140a-3p to enhance the PD-L1 expression and decrease the radiosensitivity of LUAD. Our results suggest that MALAT1 might be a promising therapeutic target for the radiotherapy sensitization of LUAD.

PD-L1 expression in Chinese patients with solid tumor and its correlation with gene alternations.

e14640 Background: Immune checkpoint blockade are recommended in more and more solid tumors for first-line or posterior treatment, and PD-L1 expression is a crucial predictive biomarker for immunotherapy. However, the expression can be influenced by many factors. Methods: A total of 1743 Chinese patients with solid tumor was retrospectively identified. The Dako PD-L1 IHC 22C3 pharmDx assay was used to detect PD-L1 protein expression. And Tumor Proportion Score (TPS) ≥ 1% was defined as positive expression, TPS ≥ 50% was defined as high expression. Genomic profiling was performed on tumor tissue and matched peripheral blood samples. Only the tumor subtypes with sufficient sample size were used for correlation analysis. Results: PD-L1 positive expression occurred in 45.6% patients and high expression occurred in 8.1% patients. The highest expression incidences occurred in cervical cancer as of 78.8%, followed by lung cancer (53.8%), esophageal cancer (53.7%) and gastric cancer (42.7%). The PD-L1 expression assessed by TPS was consistent with that assessed by Combined Positive Score (CPS) in multiple tumors. However, it was significantly different in gastrointestinal tumors, especially in colorectal cancer (TPS&gt;1%: 30.4% vs. CPS&gt;1: 80.6%). In addition, gene alternations including TP53, CDKN2A, LRP1B, KRAS and PIK3CA apparently occurred in patients with high PD-L1 expression. Moreover, high tumor mutation burden (TMB-H) was associated with PD-L1 positive expression in these Chinese patients. Besides, no correlation was found between microsatellite instability (MSI) and PD-L1 expression. Conclusions: Our results illustrated genomic correlates of PD-L1 expression in Chinese solid tumor patients. These findings may broaden the application of immune checkpoint blockade to patients harboring specific molecular characteristics.