Tumor Expression Research Articles

Unveiling the RKIP and EGFR Inverse Relationship in Solid Tumors: A Case Study in Cervical Cancer.

Raf Kinase Inhibitor Protein (RKIP) is recognized as a bona fide tumor suppressor gene, and its diminished expression or loss is associated with the progression and poor prognosis of various solid tumors. It exerts multifaceted roles in carcinogenesis by modulating diverse intracellular signaling pathways, including those governed by HER receptors such as MAPK. Given the significance of HER receptor overexpression in numerous tumor types, we investigated the potential oncogenic relationship between RKIP and HER receptors in solid tumors. Through a comprehensive in silico analysis of 30 TCGA PanCancer Atlas studies encompassing solid tumors (10,719 samples), we uncovered compelling evidence of an inverse correlation between RKIP and EGFR expression in solid tumors observed in 25 out of 30 studies. Conversely, a predominantly positive association was noted for the other HER receptors (ERBB2, ERBB3, and ERBB4). In particular, cervical cancer (CC) emerged as a tumor type exhibiting a robust inverse association between RKIP and EGFR expression, a finding that was further validated in a cohort of 202 patient samples. Subsequent in vitro experiments involving pharmacological and genetic modulation of EGFR and RKIP showed that RKIP depletion led to significant upregulation of EGFR mRNA levels and induction of EGFR phosphorylation. Conversely, EGFR overactivation decreased RKIP expression in CC cell lines. Additionally, we identified a common molecular signature among patients depicting low RKIP and high EGFR expression and demonstrated the prognostic value of this inverse correlation in CC patients. In conclusion, our findings reveal an inverse association between RKIP and EGFR expression across various solid tumors, shedding new light on the underlying molecular mechanisms contributing to the aggressive phenotype associated with RKIP and EGFR in cervical cancer.

Comprehensive characterization of B7 family members in breast cancer: B7-H5 switch reverses breast cancer from “immuno-cold” into “immuno-hot” status

The members of the classic B7 family regulate the immune microenvironment of several malignant tumors. However, the potential relationship between the B7 family and the breast cancer (BrCa) tumor immune microenvironment has remained elusive. In the present study, we provide a comprehensive explanation of the expression, clinical significance, mutation, and immune cell infiltration of B7 family molecules in BrCa. First, we recruited 10 patients with BrCa surgery from the Wuxi Maternal and Child Health Hospital and performed single-cell RNA sequencing (scRNA-seq) analysis to investigate the distribution of B7 family members in multiple immune cell subsets. We focused on B7-2, B7-H3, and B7-H5 molecules of the B7 family and constructed tumor microarrays by self-recruiting patients to perform multiple immunohistochemical (mIHC) analyses and study tumor expression of B7-2, B7-H3, B7-H5 and CD8+ immune cell infiltration. B7-H5 displayed a strong correlation with CD8+ immune cell infiltration. In summary, B7-H5 provides a new perspective for the identification of immunothermal subtypes of BrCa and could function as a switch to reverse BrCa from an “immunologically cold” state to an “immunologically hot” state.Graphical abstract

MiR-26a and miR-191 are upregulated while PLAG1 and HIF2 are downregulated in pleomorphic adenomas of the salivary glands compared to Warthin tumors.

Salivary gland tumors (SGTs) are a heterogenous group of pathologies, which still represents a challenge regarding differential diagnosis and therapy. Although histological findings govern SGTs management, detection of molecular alterations is emerging as an effective additional tool. The aim of this study was to analyze the relative expression levels of three micro RNAs (miR-26a, miR-26b, and miR-191), and three pro-oncogenic molecular markers (PLAG1, MTDH, and HIF2) in SGTs and normal salivary gland (NSG) tissues and evaluate them as potential differential diagnosis markers. This cross-sectional study included 58 patients with SGTs (23 pleomorphic adenomas, 27 Warthin tumors, and 8 malignant SGTs) and 10 controls (normal salivary gland tissues). Relative gene expression levels of all investigated molecules were determined by reverse transcriptase-real-time polymerase chain reaction. All three micro RNAs exhibited highest expression levels in benign SGTs, whereas miR-26a And miR-191 were significantly more expressed in PAs compared to WTs (p = 0.045 and p = 0.029, respectively). PLAG1 And HIF2 were both overexpressed in WTs compared to PAs (p = 0.048 and p = 0.053, respectively). Bioinformatic analysis suggested that all investigated micro RNAs function as negative regulators of MTDH. The results of this study suggest that all three micro RNAs have a considerable negative impact on MTDH oncogene expression in malignant tumors, while the differences between levels of miR-26a, miR-191, PLAG1, and HIF2 in PA and WT represent possible differential diagnosis markers.

Calprotectin and β-Catenin Expression in Canine Hepatoid Gland Tumors and Correlation with Macrophage Infiltration

β-catenin is deregulated in cancer malignancies and drives the epithelial-to-mesenchymal transition (EMT). Calprotectin plays antioxidant activities, modulates inflammation and immune responses, and influences cell migration and invasion. Calprotectin can contribute to the progression of various types of cancer. Macrophages expressing calprotectin (MAC387) have been related to M1 polarization and promote EMT. In this study, β-catenin and calprotectin expression in canine hepatoid gland tumors and its relationship with MAC387-positive macrophages is reported. Β-catenin was membranous and strong in hyperplasia and adenomas, moderate or weak in well-differentiated carcinomas, and absent in less-well-differentiated carcinomas. In cells with squamous differentiation, β-catenin was weak or absent. In benign and malignant lesions, MAC/387 positivity was found in both macrophages and clusters of cells with squamous differentiation arranged in whorls centered on ductal-like spaces. These clusters were more voluminous in carcinomas, sometimes with a center of lamellar keratin (horny pearls) and were surrounded by neoplastic hepatoid cells variably positive to calprotectin. The number of calprotectin-positive macrophages progressively increased in the stroma of carcinomas. These findings suggest that hepatoid glands are a useful model for studying the different roles of β-catenin and calprotectin in the tumor milieu and their involvement in tumor differentiation and EMT.

Caspase-4 promotes metastasis and interferon-γ-induced pyroptosis in lung adenocarcinoma

Caspase-4 (CASP4) is a member of the inflammatory caspase subfamily and promotes inflammation. Here, we report that CASP4 in lung adenocarcinoma cells contributes to both tumor progression via angiogenesis and tumor hyperkinesis and tumor cell killing in response to high interferon (IFN)-γ levels. We observe that elevated CASP4 expression in the primary tumor is associated with cancer progression in patients with lung adenocarcinoma. Further, CASP4 knockout attenuates tumor angiogenesis and metastasis in subcutaneous tumor mouse models. CASP4 enhances the expression of genes associated with angiogenesis and cell migration in lung adenocarcinoma cell lines through nuclear factor kappa-light chain-enhancer of activated B cell signaling without stimulation by lipopolysaccharide or tumor necrosis factor. CASP4 is induced by endoplasmic reticulum stress or IFN-γ via signal transducer and activator of transcription 1. Most notably, lung adenocarcinoma cells with high CASP4 expression are more prone to IFN-γ-induced pyroptosis than those with low CASP4 expression. Our findings indicate that the CASP4 level in primary lung adenocarcinoma can predict metastasis and responsiveness to high-dose IFN-γ therapy due to cancer cell pyroptosis.

Integrin αVβ1-activated PYK2 promotes the progression of non-small-cell lung cancer via the STAT3-VGF axis

BackgroundNon-small-cell lung cancer (NSCLC) accounts for 80–85% of all lung cancer and is the leading cause of cancer-related deaths globally. Although various treatment strategies have been introduced, the 5-year survival rate of patients with NSCLC is only 20–30%. Thus, it remains necessary to study the pathogenesis of NSCLC and develop new therapeutic drugs. Notably, PYK2 has been implicated in the progression of many tumors, including NSCLC, but its detailed mechanism remains unclear. In this study, we aimed to elucidate the mechanisms through which PYK2 promotes NSCLC progression.MethodsThe mRNA and protein levels of various molecules were measured using qRT-PCR, western blot (WB), and immunohistochemistry (IHC), respectively. We established stable PYK2 knockdown and overexpression cell lines, and CCK-8, EdU, and clonogenic assays; wound healing, transwell migration, and Matrigel invasion assays; and flow cytometry were employed to assess the phenotypes of tumor cells. Protein interactions were evaluated with co-immunoprecipitation (co-IP), immunofluorescence (IF)-based colocalization, and nucleocytoplasmic separation assays. RNA sequencing was performed to explore the transcriptional regulation mediated by PYK2. Secreted VGF levels were examined using ELISA. Dual-luciferase reporter system was used to detect transcriptional regulation site. PF4618433 (PYK2 inhibitor) and Stattic (STAT3 inhibitor) were used for rescue experiments. A public database was mined to analyze the effect of these molecules on NSCLC prognosis. To investigate the role of PYK2 in vivo, mouse xenograft models of lung carcinoma were established and examined.ResultsThe protein level of PYK2 was higher in human NSCLC tumors than in the adjacent normal tissue, and higher PYK2 expression was associated with poorer prognosis. PYK2 knockdown inhibited the proliferation and motility of tumor cells and caused G1-S arrest and cyclinD1 downregulation in A549 and H460 cells. Meanwhile, PYK2 overexpression had the opposite effect in H1299 cells. The siRNA-induced inhibition of integrins alpha V and beta 1 led to the downregulation of p-PYK2(Tyr402). Activated PYK2 could bind to STAT3 and enhance its phosphorylation at Tyr705, regulating the nuclear accumulation of p-STAT3(Tyr705). This further promoted the expression of VGF, as confirmed by RNA sequencing in a PYK2-overexpressing H1299 cell line and validated by rescue experiments. Two sites in promoter region of VGF gene were confirmed as binding sites of STAT3 by Dual-luciferase assay. Data from the TGCA database showed that VGF was related to the poor prognosis of NSCLC. IHC revealed higher p-PYK2(Tyr402) and VGF expression in lung tumors than in adjacent normal tissues. Moreover, both proteins showed higher levels in advanced TNM stages than earlier ones. A positive linear correlation existed between the IHC score of p-PYK2(Tyr402) and VGF. Knockdown of VGF inhibited tumor progression and reversed the tumor promoting effect of PYK2 overexpression in NSCLC cells. Finally, the mouse model exhibited enhanced tumor growth when PYK2 was overexpressed, while the inhibitors PF4618433 and Stattic could attenuate this effect.ConclusionsThe Integrin αVβ1-PYK2-STAT3-VGF axis promotes NSCLC development, and the PYK2 inhibitor PF4618433 and STAT3 inhibitor Stattic can reverse the pro-tumorigenic effect of high PYK2 expression in mouse models. Our findings provide insights into NSCLC progression and could guide potential therapeutic strategies against NSCLC with high PYK2 expression levels.

The Comprehensive Characterization of B7-H3 Expression in the Tumor Microenvironment of Lung Squamous Cell Carcinoma: A Retrospective Study.

Lung squamous cell carcinoma (LSCC) is refractory to various therapies for non-small cell cancer; therefore, new therapeutic approaches are required to improve the prognosis of LSCC. Although immunotherapies targeting B7 family molecules were explored as treatments for several cancer types, the expression and significance of B7-H3 in the tumor microenvironment (TME) and its relationship with other immune checkpoint molecules have not yet been investigated in detail. We used high-throughput quantitative multiplex immunohistochemistry to examine B7-H3 expression in the TME. We investigated the relationship between B7-H3 expression and prognosis as well as changes in the TME with B7-H3 expression using 110 surgically resected pathological specimens retrospectively. We examined the correlation between B7-H3 and programmed cell death-ligand 1 (PD-L1) expression in single cells. High B7-H3 expression in tumor cells was associated with a better prognosis and a significant increase in the number of CD163+PD-L1+ macrophages. Quantitative analysis revealed that there is a positive correlation between B7-H3 and PD-L1 expression in tumor and stromal cells, as well as in intratumoral tumor-infiltrating lymphocytes and tumor-associated macrophages in the same cells. CD68+, CD163+, and CK+ cells with PD-L1+ phenotypes had higher B7-H3 expression compared to PD-L1- cells. Our findings demonstrate a correlation between B7-H3 and PD-L1 expression in the same cells, indicating that therapies targeting B7-H3 could provide additional efficacy in patients refractory to PD-L1-targeting therapies.

Development of novel MUC1-C targeting antibody-drug conjugate for pancreatic cancer treatment.

e15025 Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive forms of cancers, which is usually diagnosed in advanced stages and presents an abysmal 5-year survival rate of 3-10%. With late detection of metastasized/advanced disease, curative surgery is not an option for most patients. Accordingly, there is an urgent unmet need for development of therapeutics targeting the pancreatic cancer cells. MUC1 is one such cell surface antigen that is aberrantly overexpressed in most solid tumors including pancreatic cancer. In this study we report preclinical efficacy of pharmacological inhibition of MUC1 signaling by GO-203 and targeted delivery of toxin conjugated to novel MUC1-C antibodies (7B8 and 3D1) binding to the extra cellular domain (ECD) to the PDAC xenografts. Methods: Subjects from TCGA-PAAD were stratified into high (75th percentile) and low (25th percentile) MUC1-expressing cohorts. Differential expression was performed using DESeq2, followed by GSEA, and signaling network analysis using partial correlation networks, ARACNE, and BC3NET. Anti-MUC1 antibody-drug conjugates (ADCs) were generated by coupling monomethyl auristatin E (3D1-MMAE, 7B8-MMAE) via V-C-PAB linker and characterized for anti-tumor activity against pancreatic cancer cells including MIA PaCa-1, CAPAN2 and HAPF-II. Paired-end RNA-seq (50bp) was aligned and quantified to the Human Ensembl GRC38 reference using STAR aligner. Subcutaneous xenografts were obtained in nude mice by injecting 5X106 HPAF-II tumor cells. Results: Differential expression, pathway, and network analyses revealed consistent dysregulation of cancer survival-related processes, and P53-related pathways in the cohort with increased MUC1 expression. The ADCs (DAR of ~3.5) exhibited efficient internalization in cancer cells and associated apoptotic cell death (Kds = 2 - 5nM). Pharmacological inhibition of MUC1 signaling by GO-203 demonstrated reduction in canonical pathways including P53 and EMT. Treatment with 3D1-MMAE compared to both vehicle and GO-203 showed dysregulation of cellular metabolism pathways (cholesterol homeostasis, glycolysis) and exhibited several strong indicators of cellular stress including activation of unfolded protein response processes and mTorc1 signaling. Excellent tumor-suppressive effects of ADCs were observed in HPAF-II xenografts in nude mice. The 7.5 mg/kg (i.v., q7dx3), treatment showed complete tumor regression from 14th day (end of treatment) till the date of termination (Day 50). Doses up to 40 mg/kg were well tolerated by the mice and this anti-tumor efficacy was observed without any appreciable loss in body weight or adverse effects on tissues. Conclusions: Targeted delivery of MMAE toxin to the pancreatic cancer cells presents an exciting opportunity for a novel therapeutic intervention for the treatment of pancreatic cancer patients with MUC1 over expression in tumors.

The correlation between the MYBL2/CDCA8 signaling pathway of malignant melanoma

ObjectiveInvestigating the effects of MYB proto-oncogene like 2 (MYBL2)-mediated regulation of Cell division cycle associated 8 (CDCA8) expression on the biological activity of cutaneous malignant melanoma cells. MethodsA375 cells with MYBL2 and CDCA8 overexpression and knockdown were evaluated using migration, invasion, and proliferation assays. Besides, cell apoptosis was quantified by flow cytometry. To investigate the tumorigenic effects of MYBL2 knockdown in vivo, A375 cells with MYBL2 knockdown were injected in BALB/C nude mice. ResultsThe levels of MYBL2 and CDCA8 gene expression were notably elevated in A375 cells in comparison to HaCat cells (P < 0.05). Downregulation of MYBL2 led to a notable reduction in the migratory and invasive capability of A375 cells in vitro (P < 0.001). On the contrary, overexpression of MYBL2 enhanced migration and invasion ability (P < 0.001). There existed a positive correlation between CDCA8 and MYBL2 gene and protein expression levels after overexpression or knockdown of MYBL2 (P < 0.001). In the in vivo tumorigenic study, the MYBL2 knockdown group displayed a substantial decrease in tumor volume (P < 0.01) and exhibited decreased CDCA8 expression in tumors in comparison to the control group. ConclusionWe arrived at such a conclusion that MYBL2 promoted the migration, invasion and proliferation ability of cutaneous malignant melanoma cells by targeted regulation of CDCA8 expression in this study.

Safety, efficacy, and biomarker results from an open-label, multicenter, phase 1 study of RP2 alone or combined with nivolumab in a cohort of patients with uveal melanoma.

9511 Background: Uveal melanoma is the most common primary intraocular tumor in adults and has a high risk of liver metastasis. Effective treatments for metastatic uveal melanoma (MUM) are limited, as MUM responds poorly to immune checkpoint inhibition. RP2 is an enhanced-potency oncolytic HSV-1–expressing human GM-CSF, a fusogenic glycoprotein (GALV-GP-R−), and an anti–CTLA-4 antibody-like molecule. We present safety, efficacy, and biomarker data for RP2 monotherapy and RP2 +nivolumab (nivo; anti–PD-1) in patients (pts) with MUM (NCT04336241). Methods: Pts ≥18 years old with advanced or metastatic non-neurologic solid tumors (including MUM) who progressed on, or could not tolerate, standard therapy were included in the clinical trial; pts had ≥1 measurable and injectable tumor (≥1 cm). After determination of the recommended phase 2 dose of intratumoral RP2 (1×106 PFU/mL once, then ≤7 doses at 1×107 PFU/mL; ≤10 mL total/treatment day), pts received RP2 Q2W as monotherapy or in addition to nivo (240 mg Q2W/480 mg Q4W). Responses were assessed per modified RECIST v1.1.Tumor biopsies and peripheral blood mononuclear cells were collected pre-treatment and at day 43 and were analyzed by immunohistochemistry (IHC) and/or sequencing of the CDR3β region of the T-cell receptor (TCR) by immunoSEQ. Results: A total of 17 pts with MUM were enrolled (RP2 monotherapy, n = 3; RP2 + nivo, n = 14). Most pts had received both anti–PD-1 and anti–CTLA-4 therapy (12/17 [70.6%]), and 3/17 (17.6%) had received ≥3 prior lines of therapy. The ORR was 29.4% (5/17; all PRs; RP2 monotherapy, 1/3; RP2 + nivo, 4/14). The DCR (CR + PR + SD) was 58.8% (10/17). The median (range) duration of response at the data cutoff was 11.5 (2.8–21.2) months, with some responses and disease stabilizations ongoing. The most common overall grade 1–2 treatment-related AEs (TRAEs; ≥20% overall) were pyrexia, chills, fatigue, hypotension, and pruritus. The only grade 3 TRAE in &gt; 1 pt was hypotension (2 pts receiving RP2 + nivo); no grade 4/5 TRAEs occurred. Evaluable baseline and post-treatment biopsies were used for IHC (n = 8) and TCR sequencing analyses. Paired biopsies from pts with clinical benefit (n = 5; 2 PR, 3 SD) showed an increase in tumor PD-L1 expression by IHC analysis, and 4 of these pts (2 PR, 2 SD) exhibited an increase in CD8+ T-cell infiltration into tumors. TCR sequencing following treatment with RP2 + nivo revealed expansion of pre-existing TCRs and generation of new T-cell clones. Conclusions: RP2 alone or combined with nivo demonstrated a favorable safety profile and meaningful antitumor activity in pts with previously treated MUM. Biomarker data indicated immune cell infiltration and increased PD-L1 expression in tumors and changes in the peripheral T-cell repertoire following RP2 ± nivo therapy. Based on these results, a randomized, controlled clinical development plan is being planned. Clinical trial information: NCT04336241 .

Association of N-adhesin and focal adhesion kinase with frequency of tumor spread through the airspace (STAS) in stage I lung carcinoma and correlation with recurrence.

e20020 Background: Spread through air spaces (STAS) is one of the many modes of lung cancer dissemination. Aberrant activation of epithelial-mesenchymal transition (EMT) has been associated with enhanced tumor cell migration and invasiveness, a process that is accompanied by a decrease in adhesion molecules that maintain contact between epithelial cells, leading to cell separation and increased cell motility. Our objectives were to investigate the effect of adhesion molecule expression levels on STAS occurrence and postoperative recurrence in patients undergoing radical resection for stage I lung carcinoma. Methods: We examined the association of adhesion molecules with STAS as the primary endpoint and the Disease-Free Survival (DFS) as a secondary endpoint. E-cadherin, P-cadherin, N-cadherin, focal adhesion kinase (FAK), epithelial cell adhesion molecule (EpCAM) neural cell adhesion molecule 1 (NCAM1), vascular cell adhesion molecule 1 (VCAM1), intercellular cell adhesion molecule-1 (ICAM-1) were analyzed by immunohistochemistry in patients undergoing radical resection for stage I lung carcinoma. Associations between those adhesion molecules expression levels and STAS were determined by using the chi-square test and logistic regression model. DFS was analyzed by using the log-rank test and Cox proportional risk model. Results: As of January 1, 2024, 12 of 60 patients undergoing radical resection for stage I lung carcinoma had a disease recurrence. All of 60 patients’ tissue specimens were retrospectively analyzed, and there were no significant differences between patients with STAS-positive(n=30) and STAS-negative(n=30) in baseline clinicopathologic features, except for histological growth patterns, and lymphovascular invasion.Higher incidence of STAS was significantly associated with low expression of E-cadherin (P=0.002), high expression of N-cadherin (P=0.018) and FAK (P=0.027), and males (P=0.026), independent of tumor size, age, and clinical stage. Multivariate analysis showed that STAS (P=0.025), low E-cadherin/high N-cadherin (P=0.038), low E-cadherin/high FAK (P=0.025), and high N-cadherin/FAK (P=0.003) were significantly correlated with a higher risk of recurrence. In addition, females (P=0.034), and high N-cadherin/FAK (P=0.032) were associated with a high risk of recurrence in patients with STAS. Conclusions: Low expression of e-cadherin, high expression of N-cadherin and FAK were independent predictors of higher incidence of STAS. Tumors with low E-cadherin/high N-cadherin, low E-cadherin/high FAK, and high N-cadherin/FAK expression were important predictors of recurrence in patients with stage I lung carcinoma. Additionally, high N-cadherin/FAK expression in tumors provides information about a higher risk of recurrence in STAS-positive stage I lung carcinoma.

Phase 1b/2a study evaluating the combination of MN-166 (ibudilast) and temozolomide in patients with newly diagnosed and recurrent glioblastoma (GBM).

2016 Background: Ibudilast is a selective inhibitor of macrophage inhibitory factor (MIF) activity, which is upregulated in GBM and promotes stem cell growth. It also disrupts the interaction between MIF and CD74. When combined with temozolomide (TMZ) in preclinical studies, it attenuates the immunosuppressive properties of myeloid-derived suppressor cells and enhances tumor regression. This is a phase 1b/2a single-center, open-label, dose escalation study evaluating the safety, tolerability, and efficacy of the combination of Ibudilast and TMZ in newly diagnosed (nGBM) and recurrent (rGBM) GBM was initiated (NCT03782415). Methods: We included patients with nGBM who had completed concurrent chemoradiation, and patients with first GBM relapse who had a measurable enhancing disease. Patients were treated with monthly cycles of temozolomide (5 days of a 28-day cycle) and daily ibudilast, with a starting dose of 30 mg twice daily (BID), and escalated to a maximal dose of 50 mg BID. The primary objectives were to evaluate the safety and tolerability of the combination and determine the phase 2 recommended dose (R2PD) for phase 1b, and to evaluate the efficacy of the combination, determined by the 6-month progression-free survival rate (PFS-6) for phase 2a. A standard 3+3 design was used for phase 1b. For the rGBM cohort, a sample size of 30 provided 80% power to discriminate between historical 15% and 35% PFS-6 rates for rGBM patients. The trial would be considered successful if at least 8 patients were progression free at 6 months. Secondary objectives included overall survival (OS). Immunohistochemistry (IHC) studies were performed on archival tumor samples to evaluate factors of the tumor immune microenvironment. Results: 36 patients with nGBM and 26 patients with rGBM were included; 61% were males, with median age of 59 years. Ibudilast 50 mg BID was deemed to be the R2PD. PFS-6 was 44% (16 patients) in nGBM, and 31% (8 patients) in rGBM. The median OS was 21.0 months (17.7, 23.1) for nGBM and 8.6 months (7.8, 10.5) for rGBM. IHC evaluation on the original tumor tissue for patients with rGBM revealed a significantly higher CD3 positive cells infiltration, and elevated CD74 expression in tumors of patients who progressed at 5 months compared to those who did not. Conclusions: This study met its primary endpoint for rGBM, as the combination of ibudilast and TMZ was associated with a higher PFS-6 rate than historical controls. CD3 expression was a good predictor for tumor progression at 5 months in patients with rGBM. Encouraging preclinical studies suggest enhanced efficacy of ibudilast in GBM when combined to immune checkpoint blockade agents. Clinical trial information: NCT03782415 .

Impact of the tumor proportion score (TPS) and the combined positive score (CPS) on the use of immunotherapy in patients with metastatic non-small cell lung cancer.

e14611 Background: Expression of programmed cell death ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC) has often been measured by the Tumor Proportion Score (TPS) (1). Recently, the Combined Positive Score (CPS), which evaluates PD-L1 expression in tumor and inflammatory cells and applies to other types of cancer including gastric and head and neck cancers, was found to be equally predictive of the response to immunotherapy in NSCLC (1). In this study, we aimed to conduct a single center retrospective analysis to evaluate the clinical use and impact of using TPS and CPS data in patients with metastatic NSCLC. Methods: Patients with NSCLC who had TPS and CPS scores and received PD-1/PD-L1 inhibitors between 2021-2022 as a monotherapy or in combination with chemotherapy were included. Patients’ data was retrieved from the electronic medical record data and analysis included histopathological data, lung cancer surgical history, presence of EGFR mutation, ALK mutation, Ki67, use of adjuvant chemotherapy, type of immunotherapy used, and development of immune checkpoint inhibitor- related toxicity and treatment as applicable. TPS and CPS positivity was defined as &gt;1 % and &gt;1 respectively. Statistical methods employed included descriptive approaches such as calculated means as well as the Kaplan-Meier estimate. Results: 40 patients with NSCLC were included. Patients’ age ranged from 19 to 82 years old with an average age of 64.3 years old. Most patients had adenocarcinoma (65 %) and were at stage IV (72.5 %). TPS positivity was identified in 29 patients (72.5 %) and CPS positivity was identified in 35 patients (87.5 %). Patients received immunotherapy mostly as first or second line of treatment (90 %). Immune checkpoint inhibitors received by patients were pembrolizumab (50 %), nivolumab (35 %), atezolizumab (12.5 %), and durvalumab (2.5 %). The median overall survival was 16, 12, and 17 months in the &lt;1 %, 1-49 % and &gt; 49 % TPS groups respectively (p= 0.354); while the median overall survival was 11, 16 and 17 months in the &lt;1, 1-49, and &gt; 49 CPS groups respectively (p= 0.417). It is critical to highlight that there were 7 patients (17.5 %) with TPS of zero who had CPS positivity (score range: 1-10). Most of these patients had adenocarcinoma (71.4 %), received pembrolizumab or atezolizumab (85.7 %) and had a median survival of 16 months. Conclusions: The Combined Positive Score (CPS) has proven to be an effective method in evaluating the level of expression of PD-L1 in NSCLC (1,2). Similar to TPS, CPS provides critical information that can be applied to guide the use of immunotherapy, and which can greatly impact patients with NSCLC. 1. De Marchi et al., 2021. 2. Ulas et al., 2023.

EVICTION study: ICT01, an anti-Butyrophilin 3A monoclonal antibody activating γ9δ2 T cells in combination with pembrolizumab in checkpoint inhibitor refractory melanoma.

9534 Background: γ9δ2 T-cell tumor infiltration is associated with a favorable prognosis making these cells a new potential target for immunotherapy. The anti-BTN3A mAb ICT01 selectively activates γ9δ2 T cells and is being studied in the phase1/2a EVICTION Trial (NCT04243499). In preclinical studies, ICT01 induces upregulation of PD-1 on γ9δ2 T-cells and combination with pembrolizumab leads to enhanced cancer cell killing, providing scientific rationale to evaluate this combination. Here we present interim results from EVICTION of ICT01 in combination with pembrolizumab in patients with checkpoint inhibitor (CPI) refractory melanoma. Methods: EVICTION evaluated ICT01 (20 µg to 200 mg, Q3W) plus pembrolizumab (200mg IV Q3W) in a dose escalation solid tumor basket design that resulted in an ongoing expansion cohort of patients with CPI refractory melanoma (2 dose levels, 7 and 200 mg ICT01). Patients are selected based on higher baseline γ9δ2 T cells. Efficacy evaluations by i/RECIST 1.1 are conducted every 8 weeks. Disease Control Rate (DCR) as primary efficacy endpoint is defined as the sum of complete response (CR), partial response (PR) and stable disease (SD, minimum week 16). Baseline and on-treatment blood and biopsy specimens are collected for correlative translational work. Results: ICT01 plus pembrolizumab has a favorable safety profile with first-dose Grade 1/2 infusion related reactions (IRR) and cytokine release syndrome (CRS) as most common adverse events (in 38% and 19% of patients respectively, 5%and 2% Grade 3) among all doses and indications. To date, 35 CPI refractory melanoma patients have been enrolled, of which 65% have received 2+ prior lines of CPI. Currently 21 patients are evaluable at week 16 with a DCR of 42% (including 3 PR) and a 6-month progression free survival of 38%. In the circulation, full BTN3A receptor occupancy on immune cells was achieved at 7 mg dose, with down-modulation of BTN3A observed at doses greater than 20 mg. Clinical response was related to baseline tumoral BTN3A expression, sustained elevation of IFNg levels, and tumor microenvironment remodeling, including increased PD-L1 expression and CD8 T cells proliferation and activation. Conclusions: ICT01 in combination with pembrolizumab has a favorable safety profile and promising efficacy data. Patient selection based on BTN3A tumor expression will be further evaluated as an enrichment strategy. Clinical trial information: NCT04243499 .

Association between GPRC5A expression and carboplatin efficacy in triple-negative breast cancer patients.

e13142 Background: Triple-negative breast cancer (TNBC) patients often face a grim prognosis, primarily due to their resistance to chemotherapy. Carboplatin is a common treatment for TNBC, particularly effective in BRCA1/2 mutations, but its efficacy varies in BRCA1/2 wild-type patients. A previous study identified GPRC5A expression as a biomarker for carboplatin chemotherapy in ovarian cancer, yet its association with carboplatin efficacy in TNBC remains unknown. This study aims to investigate whether GPRC5A can serve as a biomarker for predicting carboplatin efficacy in TNBC and to elucidate the underlying mechanisms. Methods: Analyzing clinical data from the past 5 years in Division of Breast &amp; Thyroid Surgery in Jiangsu Province Hospital, we identified 199 TNBC patients treated with carboplatin. GPRC5A expression in TNBC tumors was assessed via immunohistochemistry (IHC) and evaluated by three individual pathologists. Expression levels were categorized as low (low), low (mild), high (moderate), and high (high). The Chi-square test was employed to assess the correlation between GPRC5A expression and carboplatin efficacy. TNBC-derived organoids were used to validate the relationship between GPRC5A expression and carboplatin sensitivity. Results: Among the 199 TNBC patients, 31 (17.2%) had BRCA1/2 mutations. The pathologic complete response (pCR) rate in BRCA1/2 mutation patients receiving carboplatin was 83.9%, compared to 61.3% in BRCA1/2 wild-type patients (p=0.016). Analyzing the relationship between GPRC5A expression and BRCA1/2 gene mutations in 145 patient samples revealed no significant association (p=0.37). Irrespective of BRCA1/2 mutation status, patients with low/mild GPRC5A expression had a higher pCR rate (74.1%) compared to those with moderate/high GPRC5A expression (45%, p&lt;0.001). TNBC-derived organoids treated with carboplatin showed a cell survival rate of 31.25% (±8.5%) in low GPRC5A expression organoids and 56.25% (±12.6%) in high GPRC5A expression organoids (p=0.017). Conclusions: Our study demonstrates a correlation between GPRC5A expression and BRCA1/2 mutation status in TNBC. Furthermore, TNBC patients with low GPRC5A expression exhibited a more favorable response to carboplatin treatment, suggesting GPRC5A could be a potential biomarker for predicting carboplatin efficacy in TNBC patients.

Spatial transcriptomic and proteomic insight into Trop-2, HER2, and AR expression: A pathway to tailored therapies in triple-negative breast cancer.

e12576 Background: Triple-negative breast cancer’s (TNBC) dismal prognosis demands more effective therapy selection. Previously described molecular subtypes reveal its heterogeneity but lack spatial insight and are not currently used in clinical practice. Here, we aim to evaluate by immunohistochemistry (IHC) the expression of key biomarkers in TNBC and its correlation with spatial gene expression obtained by spatial transcriptomics (ST). Methods: We studied a retrospective cohort of 92 patients (pts) treated at the Institut Jules Bordet in Belgium with primary surgery for early TNBC. ST (Visium Spatial Gene Expression, 10X Genomics) was previously performed on fresh frozen surgical samples. Using serial sections of these samples, we performed duplex IHC staining for Trop-2/androgen receptor (AR) and HER2/Ki-67. IHC staining was scored using H-score (Trop-2), Allred score (AR), and current guidelines (Ki67/HER2), adding the ultra-low and null HER2 categories. Statistical analyses included descriptive statistics, Spearman correlation, Mann-Whitney test, and Kaplan-Meier with log-rank tests for estimating outcome distribution. Results: Of 92 pts included, 4 (4.3%), 25 (27.2%), 28 (30.4%), and 35 (38%) pts had HER2 2+, 1+, ultra-low, and null, respectively, tumors. Importantly, we observed a significant difference in tumor cell-derived ERBB2 gene expression of HER2 ultra-low versus HER2 null tumors ( p&lt;0.0001), while there was no difference in ERBB2 expression between HER2 ultra-low and HER2 1+ tumors ( p=0.49). HER2 expression was not correlated with prognosis. Trop-2 expression by IHC was high, medium, and low in 67%, 23%, and 10% of pts, respectively. IHC expression correlated well with tumor-derived gene expression (r=0.54), with intrasample heterogeneity observed both in IHC and spatial gene expression. Trop-2 expression was prognostic, pts with high Trop-2 H-score tumors having significantly worse outcome ( p=0.021). In this cohort, 25%, 40.2%, and 34.8% of pts had AR Allred scores of 0-2, 3-5, and 6-8, respectively. Tumor AR gene expression was well correlated with IHC AR expression (67.7%). IHC AR expression was associated with the presence of adipose tissue in the stroma (r=0.23, p=0.025) but not with prognosis. Of note, we report for the first time, to our knowledge, for TNBC, AR expression both in tumor and stroma cells, both at the IHC and gene expression levels. Importantly, we could construct a classifier that identifies luminal androgen receptor tumors, using AR Allred score &gt; 5 and Ki67 ≤ 40%, with a PPV of 93% and an NPV of 96%. Conclusions: ST data has the potential to improve easily accessible IHC classifiers, with the aim of refining patient selection for ADC and anti-AR therapies in TNBC.

Evaluation of Paxillin Expression in Odontogenic Cysts and Tumors.

Paxillin (PXN) is one of the proteins involved in cell adhesion. PXN and integrins constitute a key site for the focal adhesion between the cell and extracellular matrix. Several studies have shown that PXN is a factor in tumor formation, progression, invasion, and metastasis. This study evaluated PXN expression in four types of odontogenic lesions with different aggressive behaviors. In this retrospective cross-sectional study, PXN expression was immunohistochemically assessed in 68 paraffin-embedded tissue samples from patients with the confirmed diagnosis of four types of odontogenic lesions, including 14 dentigerous cysts (DC), 20 odontogenic keratocyst (OKC), 16 unicystic ameloblastoma, and 18 solid ameloblastoma. The PXN expression in these samples were scored based on the percentage and intensity of immunoreactivity, and compared among the groups by Chi-square test. The PXN marker was detected in the cytoplasm of tumor cells (unicystic and solid ameloblastoma) and the epithelial layer of cysts (DC and OKC). The intensively stained marker of PXN was observed in 9 cases (64.3%) of the DC, 14 cases (70%) of OKC, 12 cases (75%) of unicystic ameloblastoma, and 13 cases (72.2%) of solid ameloblastoma. However, there was not statistical difference of PXN protein expression between DC and OKC (p Value = 0.51) and unicystic and solid ameloblastoma (p = 0.58), also the same was true for cysts and tumors (p = 0.37). The expression of PXN is not related to the biological behaviors of odontogenic lesions.

The role of stress in modulating AIPB expression and estradiol synthesis in triple-negative breast tumors.

e13146 Background: In the United States, invasive breast cancer affects 1 in 8 women during their lifetime, with over 3.8 million individuals living as survivors. Breast cancers vary, with some being hormone-responsive and categorized on the presence or absence of estrogen (ER), progesterone (PR), and HER2 receptors. While targeted therapies exist for Estrogen Receptor positive (Er+) and Progesterone Receptor positive (PR+) cancers, triple-negative breast cancer (TNBC), which lacks all three receptors, affects about 15% of patients and is unresponsive to effective molecular treatments. Only a fraction of the cases respond to chemotherapy which emphasizes a need for new prognostic markers and treatments. Aromatase is the key enzyme in converting testosterone to estradiol which plays a crucial role in tumor growth, especially after menopause. Our research lab has identified the “Aromatase Interacting Partner in Breast or AIPB, whose overexpression inhibits aromatase, leading to a reduction in estradiol levels. However, in triple-negative breast cancer, there is an increase in AIPB expression when compared to ER+/PR+/Her2- breast tumors or unaffected women. This devastating prognosis and low survival rates can contribute to a considerable amount of stress in the diagnosed patients, in turn leading to an increase in cortisol production. Our hypothesis suggests that this stress or increase in cortisol could alter AIPB production in the tumorigenic cells which could potentially lead to a decrease in estradiol synthesis. Methods: Stress stimulation was achieved in a lab setting by subjecting engineered triple-negative breast cancer cells, capable of conditional AIPB overexpression upon tetracycline addition, to a 42 °C heat shock for various durations (0,15, 30, 45 minutes). Results: Following the 45 minutes of stress, MDA-MMD-231 cells exhibited a significant reduction in estradiol levels, decreasing from 160ng/ml to 100ng/ml. MDA-MD-231 cells engineered to overexpress AIPB produced only 62ng/ml of estradiol, marking an 18.75% decrease. The introduction of doxycycline led to a further reduction in estradiol levels to 47ng/ml, which is approximately a 29.4% drop. Western blot analysis using a CT antibody revealed heightened AIPB expression in MDA-MD-231-AIPB cells when stress and doxycycline treatments were combined. Conclusions: Given the absence of aromatase in triple-negative breast cancer, our findings suggest that AIPB’s role in estradiol production is independent of aromatase, highlighting its significance in these cancer cells. The data supports the hypothesis that enhancing AIPB expression could potentially improve patient outcomes by reducing estradiol synthesis.

CRPA1A2: A novel TCR-based T-cell engager targeting MAGEA1-positive solid tumors.

e14589 Background: MAGEA1, identified as a cancer/testis antigen, is highly expression in various solid tumors, including hepatocellular carcinoma, non-small cell lung cancer, but absent in normal tissues except male germ cells and placenta, both of which lacking HLA class molecules. This selective expression of HLA-MAGEA1 epitope makes it a promising target for cancer therapy. However, its intracellular localization poses a challenge for conventional antibodies designed to target surface-expressed antigens. TCR-based T-cell engagers, such as Tebentafusp, overcome this hurdle by targeting intracellular protein-derived peptides presented on cancer cell surfaces via HLA, enabling precise cytotoxic T cell attacks. Notably, Tebentafusp has shown significant clinical benefits by improving survival rates in metastatic uveal melanoma, underscoring the efficacy of TCR-based T-cell engagers in treating MAGEA1-positive tumors. Methods: We have developed an advanced structural format, CR62, consisting of a anti-CD3 single chain fragment, a soluble modified TCR, and an Fc region. Additionally, the use of CorreGene's SMART-TCR system has facilitated the acquisition of TCRs with high affinities in the pico-molar range. By leveraging CR62 and a high-affinity TCR, we have conceptualized the TCR-based T-cell engager, CRPA1A2. Comprehensive preclinical assessments have been conducted to evaluate CRPA1A2's safety and efficacy. Results: CRPA1A2 is designed to specifically target the HLA-A*02:01-restricted MAGEA1 epitope, effectively inhibiting tumor growth. CRPA1A2 is characterized by its exceptional soluble expression in CHO cells, minimal homodimer miss-assembly and simplifying purification. It is strategically engineered with a high-affinity TCR and a low-affinity anti-CD3 scFv to optimize the efficacy and safety. In vitro studies, CRPA1A2 showed strong ability to eliminate tumor cells and induce IFN-γ release, effective even at low MAGEA1 epitope densities, with a therapeutic window that spans over three orders of magnitude, suggesting a 1000-fold greater activity against antigen-positive cancer cells compared to antigen-negative cells. In vivo, using a tumor-bearing mouse model, CRPA1A2 has been shown to recruit T cells into subcutaneous solid tumors, expand CD8+ T cell populations, and significantly reduce tumor mass. Notably, CRPA1A2 has an extended half-life of 120 hours and demonstrates preferential accumulation in tumor tissues, thereby enhancing its therapeutic potential. Moreover, the safety assessments for CRPA1A2 highlight its exceptional specificity, with in vitro studies showing no evidence of off-target toxicity, on-target off-tumor toxicity, or alloreactivity and favorable tolerance in mice during in vivo studies. Conclusions: CRPA1A2 has emerged as a superior TCR-based T-cell engager with exceptional antitumor efficacy and promising safety profile.

9P Synergistic potential of vitamin D receptor and cancer stem cells markers expression in ovarian tumors

9P Synergistic potential of vitamin D receptor and cancer stem cells markers expression in ovarian tumors