Mesothelin Levels Research Articles

Mesothelin expression correlates with elevated inhibitory immune activity in patients with colorectal cancer

The expression of the protein Mesothelin (MSLN) is highly variable in several malignancies, including colorectal cancer (CRC), and high levels are associated with aggressive clinicopathological features and worse patient survival. Colorectal cancer is both a common and deadly cancer; being the third most common in incidence and second most common cause of cancer-related death. While systemic therapy remains the primary therapeutic option for most patients with stage IV (metastatic; m) CRC, their disease eventually becomes treatment refractory, and 85% succumb within 5 years. Microsatellite-stable (MSS) CRC tumors, which constitute more than 90% of patients with mCRC, are generally refractory to immunotherapeutic interventions. In our current work, we characterize MSLN levels in CRC, specifically correlating expression with clinical outcomes in relevant CRC subtypes, and explore how MSLN expression impacts the status of immune activation and suppression in the peritumoral microenvironment. Higher MSLN expression is prevalent in CMS1 and CMS4 CRC subtypes and correlates with higher gene mutation rates across the patient cohorts. Further, MSLN-high patients exhibit increased M1/M2 macrophage infiltration, PD-L1 staining, immune-inhibitory gene expression, enrichment in inflammatory, TGF-β, IL6/JAK/STAT3, IL2/STAT5 signaling pathways, and mutation in KRAS and FBXW7. Together, these results suggest that MSLN protein is a potential target for antigen-specific therapy and supports investigation into its tumorigenic effects to identify possible therapeutic interventions for patients with high MSLN expressing MSS CRC.

Abstract LB069: Repurposing anti-mesothelin CAR-NK immunotherapy against colorectal cancer

Abstract Colorectal cancer (CRC) is the third most common cancer worldwide, with highly variable prognosis and response to treatment. A large subset of patients does not respond to standard treatments or develops resistance. As an alternative, adoptive immunotherapy based on chimeric antigen receptor (CAR)-transduced immune cells has been proposed, however with significant adverse events. We therefore evaluated alternative CAR targets already tested in other tumour types, and employed the natural killer cell line NK-92 for CAR transduction because of its more favourable toxicity profile. As an alternative antigen, we considered mesothelin (MSLN), the most represented in CAR-based clinical studies for solid tumours. MSLN RNA expression was analysed in large series of CRC patient-derived xenografts (PDXs, n=500), cell lines (n=150) and tumours (n=640), to evaluate its distribution and to identify MSLN-overexpressing models. NK-92 cells were transduced with anti-MSLN CAR, and subsequently sorted and cloned. Activity of CAR-NK-92 cells against target-expressing ovarian and CRC cells was assessed in vitro and in vivo. Statistical significance of efficacy was evaluated by t-test. The large-scale expression analysis highlighted that about 10% of CRCs overexpress MSLN at levels comparable to those of ovarian cancer, a typical target of MSLN-CAR-based therapy. Intriguingly, MSLN overexpression is more frequent in the CRIS-B poor prognosis subtype and KRAS/BRAF-mutant CRC. Lentiviral transduction of NK-92 cells with the MSLN-CAR, followed by sorting and cloning, led to the identification of one clone, NK-92cl45, stably expressing the CAR and retaining the NK phenotype. As expected, the clone demonstrated significant in vitro and in vivo activity against ovarian cancer cells. When repurposed against models of CRC expressing high MSLN levels, it displayed comparable efficacy, both in vitro and in vivo. Specificity of NK-92cl45 was confirmed by the absence of activity on control models with low or absent MSLN. Our results provide preclinical evidence that a subset of colorectal cancers expressing high mesothelin levels can be effectively targeted by MSLN-CAR-based immunotherapy. The potential therapeutic impact of these findings is enhanced by the fact that frequently MSLN-overexpressing CRCs display worse prognosis and resistance to standard care. Citation Format: Erica Torchiaro, Marco Cortese, Alice D'Andrea, Consalvo Petti, Enzo Medico. Repurposing anti-mesothelin CAR-NK immunotherapy against colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB069.

DETERMINATION OF IL-1β mRNA GENE EXPRESSION AND HMGB-1 mRNA GENE EXPRESSION IN WOMEN WITH GENITAL ENDOMETRIOSIS ASSOCIATED WITH INFERTILITY AT THE STAGE OF PREPARATION FOR ASSISTED REPRODUCTIVE TECHNOLOGIES

Despite the large number of studies on the identification and search for noninvasive early diagnostic markers of genital endometriosis, this problem remains relevant today. It is important to introduce early diagnosis of genital endometriosis for the purpose of timely prevention and treatment.Objective: to study the expression levels of IL-1β and HMGB-1 mRNA genes in women with genital endometriosis associated with infertility, and to evaluate the effect of our proposed preparation for assisted reproductive technologies on the level of IL-1β and HMGB-1 mRNA gene expression.Material and methods. To analyze gene expression IL-1β and HMGB-1 and determine relative normalized mRNA expression IL-1β and HMGB-1, the polymerase chain reaction method was used with real-time reverse transcription(RT-PCR. The object of molecular genetic studies using the RT-PCR method was the fraction of mononuclear cells isolated from the whole blood of patients with endometriosis. In this study, we performed a retrospective analysis of the medical records of 30 women with infertility undergoing an assisted reproductive technology program. The main group consisted of 20 women diagnosed with external genital endometriosis who underwent assisted reproductive technologies. Women in the main group, in addition to standard training for assisted reproductive technologies, received a probiotic containing Lactobacilli 1010 manufactured by Unic Biotech Ltd, India. They took one pill twice a day for one month as part of a general treatment before undergoing assisted reproductive technologies. We determined the expression level of IL-1β and HMGB-1 before and after this preparation stage. The control group consisted of 10 women who had tubal infertility due to a previous inflammatory disease, but according to the results of a comprehensive clinical and laboratory examination, they were compared to healthy women. These women, aged 21 to 42 years, with a mean age of 29,75 years, did not receive our proposed preparation with the inclusion of a probiotic. This study was conducted at the Bukovyna State Medical University and the clinic "Yuzko medical center".Results. In the main group, the IL-1β mRNA gene expression level before training was (26,7877±0,01), which was significantly higher than the level after training (0,1610±0,01*). Consequently, the use of the probiotic led to a dramatic reduction in elevated mesothelin levels, underscoring its efficacy and potential application in assisted reproductive technology training programs. Also, a high level of expression of mRNA genes can be noted HMGB-1in whole blood of women of the main group before preparation for assisted reproductive technologies (table 2). Expression level of mRNA genes HMGB-1in whole blood of women of the main group before preparation for assisted reproductive technologies was (11,9198±0,01). After our proposed preparation for assisted reproductive technologies with the inclusion of a probiotic, the mRNA gene expression level HMGB-1in the whole blood of women of the main group was (0,2821±0,01*). Therefore, our proposed preparation for assisted reproductive technologies with the inclusion of a probiotic is quite effective, since the expression levels of IL-1β mRNA genes and HMGB-1sharply decreased.Conclusions. Extremely high expression of mRNA genes IL-1β and HMGB-1 indicates a close connection between the pathogenesis of endometriosis and inflammation. The inclusion of probiotics in the complex scheme of preparation for assisted reproductive technologies leads to a noticeable improvement in the well-being of the patient and a significant decrease in the expression of IL-1β and HMGB-1 mRNA genes. Therefore, we recommend our proposed preparation for assisted reproductive technologies with the inclusion of a probiotic.

Novel combinatorial therapy of oncolytic adenovirus AdV5/3-D24-ICOSL-CD40L with anti PD-1 exhibits enhanced anti-cancer efficacy through promotion of intratumoral T-cell infiltration and modulation of tumour microenvironment in mesothelioma mouse model.

Malignant mesothelioma is a rare and aggressive form of cancer. Despite improvements in cancer treatment, there are still no curative treatment modalities for advanced stage of the malignancy. The aim of this study was to evaluate the anti-tumor efficacy of a novel combinatorial therapy combining AdV5/3-D24-ICOSL-CD40L, an oncolytic vector, with an anti-PD-1 monoclonal antibody. The efficacy of the vector was confirmed in vitro in three mesothelioma cell lines - H226, Mero-82, and MSTO-211H, and subsequently the antineoplastic properties in combination with anti-PD-1 was evaluated in xenograft H226 mesothelioma BALB/c and humanized NSG mouse models. Anticancer efficacy was attributed to reduced tumour volume and increased infiltration of tumour infiltrating lymphocytes, including activated cytotoxic T-cells (GrB+CD8+). Additionally, a correlation between tumour volume and activated CD8+ tumour infiltrating lymphocytes was observed. These findings were confirmed by transcriptomic analysis carried out on resected human tumour tissue, which also revealed upregulation of CD83 and CRTAM, as well as several chemokines (CXCL3, CXCL9, CXCL11) in the tumour microenvironment. Furthermore, according to observations, the combinatorial therapy had the strongest effect on reducing mesothelin and MUC16 levels. Gene set enrichment analysis suggested that the combinatorial therapy induced changes to the expression of genes belonging to the "adaptive immune response" gene ontology category. Combinatorial therapy with oncolytic adenovirus with checkpoint inhibitors may improve anticancer efficacy and survival by targeted cancer cell destruction and triggering of immunogenic cell death. Obtained results support further assessment of the AdV5/3-D24-ICOSL-CD40L in combination with checkpoint inhibitors as a novel therapeutic perspective for mesothelioma treatment.

Mesothelin expression remodeled the immune-matrix tumor microenvironment predicting the risk of death in patients with malignant pleural mesothelioma.

The combination of immunobiological agents with immune checkpoint proteins is a promising treatment for malignant pleural mesothelioma (MPM). Mesothelin and anti-PD-L1 antibody-drug conjugates specifically target malignant neoplastic cells, inhibit the migration and invasion of neoplastic cells, and restore the immune landscape. In this study, we confirmed the importance of mesothelin and examined the relationship between mesothelin and the immune landscape of the tumor microenvironment (TME) in two MPM cohorts. The discovery cohort included 82 MPM cases. Tissue microarray slides were generated, and samples were processed for hematoxylin & eosin staining, immunohistochemistry, and immunofluorescence assays. The relationship between mesothelin, biomarkers of histogenesis, histological aggressiveness, PD-L1, immune cells (CD4, CD8, CD20, CD68), and collagen type I and type V fibers was evaluated by quantitative digital analyses. The outcome was the survival time until death from disease recurrence. The exploratory cohort included 87 malignant mesothelioma (MESO) patients from The Cancer Genome Atlas database. Most patients were male (70.7%) with a history of asbestos exposure (53.7%) and with the epithelioid subtype (89%). Surgical resection was performed in 85.4% of patients, and 14.6% received chemotherapy; 59.8% of patients died from disease extension to the mediastinum. Low tumor mesothelin expression was associated with tumor necrosis and nuclear grade 1, whereas high mesothelin expression was significantly associated with the epithelioid histotype and high density of T cells CD8+, macrophages CD68+, and collagen type I fibers. Cox multivariate analysis showed a high risk of death for non-operated patients [hazard ratio (HR), 3.42 (1.15-10.16)] with low tumor mesothelin levels [HR, 2.58 (1.09-6.10)] and high PD-L1 and low infiltration of T cells CD4+ [HR, 3.81 (1.58-9.18)]. In the exploratory cohort, low mesothelin and high COL1A1 and COL5A1 expression were associated with poor overall survival. Tumor mesothelin expression associated with the TME immune landscape predicts the risk of death for patients with MPM and could be a new target for immunotherapy in MPM.

Longitudinal monitoring of response to chemotherapy in patients with malignant pleural mesothelioma by biomarkers.

The aim of the study was to longitudinally investigate the serum levels of mesothelin, sestrin1, hyaluronan synthase 2 (HAS2), midkine, and high mobility group box 1 (HMGB1) before and after chemotherapy and at the time of relapse in malignant pleural mesothelioma (MPM) patients treated with chemotherapy and to compare the changes in biomarker levels with radiological treatment outcome. A total of 64 MPM patients treated with chemotherapy were enrolled in the study and longitudinally followed for changes in biomarker levels in response to treatment. Biomarkers evels were measured in serum using a human ELISA kit. Relative and absolute changes in biomarker levels were compared with the best radiological overall response at each time point. Median survival was 20.0 ± 2.4 (15.3-24.7) months in patients with partial and complete response, 17.0 ± 1.0 (15.0-19.0) months in patients with stable disease, and 9.0 ± 1.0 (7.0-11.0) months in patients with progressive disease. A significant decrease in serum levels of mesothelin, midkine, and HMGB1 was observed in patients with radiologically partial and complete responses to chemotherapy (p< 0.001, p= 0.016, and p= 0.039, respectively). In these patients, mesothelin levels decreased by 15%, midkine levels by 7%, and HMGB1 levels by 15%. In addition, HMGB1 serum levels were found to significantly increase by 15% in patients with radiologically progressive responses to chemotherapy compared to pretreatment serum levels (p= 0.035). In patients with partial and complete response to chemotherapy, mesothelin levels increased by 15%, midkine by 12%, and sestrin1 by 8% when the disease recurred (p= 0.004, p= 0.004 and p= 0.044, respectively). Biomarkers may be useful in the longitudinal monitoring of response to treatment in MPM. However, the results of our study should be validated in larger groups with sufficient case numbers from multicenter institutions.

High expression of mesothelin in plasma and tissue is associated with poor prognosis and promotes invasion and metastasis in gastric cancer

Mesothelin (MSLN), a tumor-associated antigen, is upregulated in various malignancies, including gastric cancer (GC). In addition, MSLN is found in the blood-stream of affected individuals, where it is referred to as soluble MSLN-related protein (SMRP). This study aims to investigate the role of MSLN in GC and evaluate its potential as a plasma biomarker for diagnosis and prognosis. Toward that end, GC tissues were obtained, upon signed consent, from affected individuals undergoing surgery or endoscopy (n = 82). Quantitative RT-PCR and immunohistochemistry were performed to determine MSLN expression. Simultaneously, The Cancer Genome Atlas (TCGA) database was mined to evaluate global status of MSLN gene expression in gastric cancer. Next, in vitro cell-culture studies were conducted to evaluate MSLN-driven proliferation properties. Using ELISA, sera from 55 GC-affected individuals were tested for MSLN level. Additionally, plasma mesothelin levels were compared in 6 cases before and after surgery. Upregulated MSLN expression was found in GC tissues, compared to adjacent normal tissues (p < 0.001). Cell culture studies with a MSLN-overexpressing stable GC line showed increased cell proliferation and invasion with ectopic MSLN. Additionally, gene-set-enrichment-analysis (GSEA) revealed an association of MSLN with the genes involved in the epithelial-mesenchymal transition and G2/M checkpoint. GC-affected cases showed higher serum MSLN levels, compared to healthy controls, with rapid decrease post-surgery. We found that MSLN upregulation correlates with poor clinical outcome and promotes growth advantage to GC cells in vitro. With further experimental evidences, we propose that MSLN could potentially be used as a plasma biomarker for diagnosis of GC.

INFLUENCE OF PROBIOTICS ON THE MESOTHELIN LEVEL IN WOMEN WITH ENDOMETRIOSIS ASSOCIATED WITH INFERTILITY IN COMPLEX PREPARATION FOR ASSISTED REPRODUCTIVE TECHNOLOGIES.

The aim: To study the determination of Mesothelin level in women with endometriosis associated with infertility and estimate influence of probiotic on endometriosis according of Mesothelin level in complex preparation before assisted reproductive technologies. Materials and methods: In this study, we conducted a retrospective analysis of the medical records of 40 infertile women who underwent assisted reproductive technologies while also using the probiotic "Femina Probiz." We divided the participants into two groups. The control group comprised 11 women who had tubal infertility due to a previous inflammatory condition but were otherwise found to be in good health through comprehensive clinical and laboratory assessments. These women, aged between 21 and 42 with an average age of 29.75 years, did not use the probiotic "Femina Probiz." The main group consisted of 29 women diagnosed with external genital endometriosis who were undergoing assisted reproductive technologies. Women in the main group received the probiotic "Femina Probiz" from Unic Biotech Ltd, India. They took one tablet twice a day for one month as part of their overall treatment before undergoing assisted reproductive technologies. We measured the Mesothelin levels before and after this preparation phase. This study was conducted at Bukovinian State Medical University and Centre of Reproductive Medicine. It's worth noting that the primary infertility incidence was significantly higher in the main group of patients. Results: In the main group, we observed that the Mesothelin level was 0.73±0.01, which was significantly higher than the post-preparation level (0.59±0.01). In contrast, the control group had a Mesothelin level of 0.49±0.01. Interestingly, we noted that the Mesothelin level in patients increased approximately twofold before preparation compared to those who had undergone preparation. This suggests that the use of the probiotic led to a sharp reduction in the elevated Mesothelin levels. Consequently, the significant decrease in Mesothelin levels after using the probiotic indicates its effectiveness and potential utility in the preparation phase of assisted reproductive technologies programs. Conclusions: The elevated Mesothelin levels indicate a strong association between the pathogenesis of endometriosis and inflammation, as well as damage to the peritoneum. The incorporation of a probiotic as part of a comprehensive preparation regimen prior to assisted reproductive technologies notably enhances the overall health of patients and leads to a reduction in Mesothelin levels. Based on our findings, we highly recommend the inclusion of this probiotic preparation in clinical practice.

Mesothelin Gene Variants Affect Soluble Mesothelin-Related Protein Levels in the Plasma of Asbestos-Exposed Males and Mesothelioma Patients from Germany.

Malignant mesothelioma (MM) is a severe disease mostly caused by asbestos exposure. Today, one of the best available biomarkers is the soluble mesothelin-related protein (SMRP), also known as mesothelin. Recent studies have shown that mesothelin levels are influenced by individual genetic variability. This study aimed to investigate the influence of three mesothelin (MSLN) gene variants (SNPs) in the 5'-untranslated promoter region (5'-UTR), MSLN rs2235503 C &gt; A, rs3764246 A &gt; G, rs3764247 A &gt; C, and one (rs1057147 G &gt; A) in the 3'-untranslated region (3'-UTR) of the MSLN gene on plasma concentrations of mesothelin in 410 asbestos-exposed males without cancer and 43 males with prediagnostic MM (i.e., with MM diagnosed later on) from the prospective MoMar study, as well as 59 males with manifest MM from Germany. The mesothelin concentration differed significantly between the different groups (p &lt; 0.0001), but not between the prediagnostic and manifest MM groups (p = 0.502). Five to eight mutations of the four SNP variants studied were associated with increased mesothelin concentrations (p = 0.001). The highest mesothelin concentrations were observed for homozygous variants of the three promotor SNPs in the 5'-UTR (p &lt; 0.001), and the highest odds ratio for an elevated mesothelin concentration was observed for MSLN rs2235503 C &gt; A. The four studied SNPs had a clear influence on the mesothelin concentration in plasma. Hence, the analysis of these SNPs may help to elucidate the diagnostic background of patients displaying increased mesothelin levels and might help to reduce false-positive results when using mesothelin for MM screening in high-risk groups.

Assessment of miR-103a-3p in leukocytes-No diagnostic benefit in combination with the blood-based biomarkers mesothelin and calretinin for malignant pleural mesothelioma diagnosis.

Malignant pleural mesothelioma (MPM) is a cancer associated with asbestos exposure and its diagnosis is challenging due to the moderate sensitivities of the available methods. In this regard, miR-103a-3p was considered to increase the sensitivity of established biomarkers to detect MPM. Its behavior and diagnostic value in the Mexican population has not been previously evaluated. In 108 confirmed MPM cases and 218 controls, almost all formerly exposed to asbestos, we quantified miR-103-3a-3p levels in leukocytes using quantitative Real-Time PCR, together with mesothelin and calretinin measured in plasma by ELISA. Sensitivity and specificity of miR-103-3a-3p alone and in combination with mesothelin and calretinin were determined. Bivariate analysis was performed using Mann-Whitney U test and Spearman correlation. Non-conditional logistic regression models were used to calculate the area under curve (AUC), sensitivity, and specificity for the combination of biomarkers. Mesothelin and calretinin levels were higher among cases, remaining as well among males and participants ≤60 years old (only mesothelin). Significant differences for miR-103a-3p were observed between male cases and controls, whereas significant differences between cases and controls for mesothelin and calretinin were observed in men and women. At 95.5% specificity the individual sensitivity of miR-103a-3p was 4.4% in men, whereas the sensitivity of mesothelin and calretinin was 72.2% and 80.9%, respectively. Positive correlations for miR-103a-3p were observed with age, environmental asbestos exposure, years with diabetes mellitus, and glucose levels, while negative correlations were observed with years of occupational asbestos exposure, creatinine, erythrocytes, direct bilirubin, and leukocytes. The addition of miR-103a-3p to mesothelin and calretinin did not increase the diagnostic performance for MPM diagnosis. However, miR-103a-3p levels were correlated with several characteristics in the Mexican population.

LBA71 First-line nivolumab (NIVO) plus ipilimumab (IPI) vs chemotherapy (chemo) in patients (pts) with unresectable malignant pleural mesothelioma (uMPM): 4-year update from CheckMate 743

In the randomized, phase 3 CheckMate 743 study (NCT02899299), NIVO + IPI significantly prolonged OS vs chemo in pts with uMPM. Identifying pts with uMPM who could benefit from immunotherapy is an unmet need. Here, we report clinical results and exploratory biomarker analyses from CheckMate 743 at 4y minimum follow-up (min f/u). Pts with previously untreated uMPM, stratified by histology and sex, were randomized 1:1 to receive NIVO (3 mg/kg Q2W) + IPI (1 mg/kg Q6W) for up to 2 y, or chemo (Q3W) for ≤ 6 cycles. Primary endpoint (EP) was OS; secondary EPs included PFS and ORR; exploratory EPs were safety and biomarker analyses. OS was assessed by baseline (BL) soluble mesothelin (sMESO) levels (high, mid, low by ELISA-based assay) and MPM-specific tumor suppressor mutations (TP53, BAP1, SETD2, NF2, LATS2 using whole-exome sequencing). At 47.5-month min f/u (database lock [DBL] 6 May 2022), NIVO + IPI continued to show OS benefit vs chemo with 4y OS rates of 16.8% vs 10.7%; 4y PFS rates were 9.0% vs 0%, respectively (Table). High (vs low or mid) BL sMESO level was associated with shorter OS in both arms; NIVO + IPI showed a trend of improved OS vs chemo across BL sMESO levels (HRs [95% CI], high: 0.72 [0.53–0.98], mid: 0.77 [0.56–1.06], low: 0.77 [0.56–1.05], respectively). OS favored NIVO + IPI vs chemo for both wild-type (HRs = 0.67–0.72) and MPM-specific tumor suppressor mutation subgroups (HRs = 0.41–0.55), except SETD2 mutation (HR = 1.37). Consistent with the prior DBL, the most common grade 3/4 immune-mediated adverse events with NIVO + IPI were hepatitis (5%), diarrhea/colitis (4%), and rash (3%).Table: LBA71Efficacy outcomes with NIVO + IPI vs chemoNIVO + IPI (n = 303)Chemo (n = 302)OSMedian (95% CI), moHR (95% CI) vs chemo4y OS rate (95% CI), %18.1 (16.8–21.0)0.73 (0.61–0.87)16.8 (12.7–21.5)14.1 (12.4–16.3)-10.7 (7.5–14.7)4y PFSa rate (95% CI), %9.0 (5.2–14.1)– cORRa (95% CI), %39.3 (33.7–45.0)44.4 (38.7–50.2)Median DORa,b (95% CI), mo11.6 (8.2–16.8)6.8 (5.6–7.1)aPer blinded independent central review;bCalculated in patients with a response (NIVO + IPI, n = 119; chemo, n = 134);cAll pts either had disease progression or were censored by month 39. DOR, duration of response; ORR, objective response rate; OS, overall survival; PFS, progression-free survival. Open table in a new tab aPer blinded independent central review; bCalculated in patients with a response (NIVO + IPI, n = 119; chemo, n = 134); cAll pts either had disease progression or were censored by month 39. DOR, duration of response; ORR, objective response rate; OS, overall survival; PFS, progression-free survival. With 4y min f/u, NIVO + IPI continued to provide long-term, durable OS benefit vs chemo in pts with uMPM. No new safety signals were seen. High BL sMESO levels were prognostic for poor OS. OS benefit with NIVO + IPI vs chemo was generally observed regardless of MPM-specific tumor suppressor mutations.

Assessment of the value of mesothelin in differentiating between different pleural malignancies by different interventional modalities

Abstract Context Malignant mesothelioma is an insidious neoplasm, with malignant pleural mesothelioma as the most common type, which is characterized by poor prognosis in most patients owing to late presentation. The use of noninvasive markers for early diagnosis of malignant pleural mesothelioma is of great importance in decreasing the incidence of associated morbidity and mortality. Aims To evaluate the value of soluble mesothelin level in serum and pleural fluid as a diagnostic marker in patients with pleural malignancies and in differentiation of different causes of pleural effusion. Patients and methods We included 48 patients who were classified into three groups: group A (mesothelioma), group B (metastatic malignancy), and group C (transudative effusion). History taking, clinical examination, and laboratory assessment were performed in all groups, in addition to measuring mesothelin level in serum and pleural fluid. Histopathological examination of pleural biopsies was performed in groups A and B. Results Mesothelin levels in serum and pleural fluid demonstrated a statistically significant increase in group A as compared with the cases in groups B and C (P&lt;0.001). Moreover, mesothelin levels in serum and pleural fluid demonstrated a statistically significant increase in the samples taken from the cases in group B as compared with the cases in group C (P&lt;0.001). Conclusion Soluble mesothelin-related peptides (SMRP) could be used as a valuable marker in diagnosis of different causes of pleural effusion. The diagnostic accuracy of pleural fluid SMRP was higher as compared with the serum SMRP.

Defining a prognostic risk score in ovarian cancer patients by integrated analysis of serum mesothelin, HE4, CA125 and surgical outcome.

e17611 Background: In ovarian cancer, serum CA125 is currently the only biomarker used in routine clinical practice and has several limitations, e.g. it is not entirely specific for ovarian malignancy. Alternative serum biomarkers have been proposed for ovarian cancer, such as mesothelin or human epididymis protein 4 (HE4). Although it is known that single marker analysis is often insufficient for diagnostic purposes or relapse prediction, no comprehensive studies on the combination of these markers have been conducted, yet. Using a multi-marker prediction model, we herein investigated the clinical usefulness of mesothelin and HE4, detected in a sequence of pre- and post-operative serum samples, in complementing the standard marker CA125. Methods: Serum mesothelin, HE4 and CA125 were quantified by Lumipulse G chemiluminescent enzyme immunoassay (Fujirebio) in a total of 432 serum samples from 90 healthy controls and 192 ovarian cancer patients (thereof 81.8% with FIGO III/IV), including 151 paired pre- and post-operative samples. Results: Pre-operative serum mesothelin and HE4 were significantly elevated compared to healthy controls with a median ratio (IQR) to controls of 2.6 and 9.6, respectively (p &lt; 0.0001). High pre-operative levels of mesothelin or HE4 alone were significantly correlated with advanced FIGO-stage (p &lt; 0.0001) and predicted surgical outcome (p &lt; 0.0001). We retrospectively compiled mesothelin and HE4 levels with CA125, BRCA mutational status and surgical outcome, in order to define a multi-marker setting. Both, mesothelin and HE4 levels were independent from BRCA mutational status, however they positively correlated with CA125. Detection of pre-operative HE4 &gt; 498pM (OR = 4.1, p &lt; 0.0001) combined with pre-operative CA125 &gt; 516U/mL (OR = 2.2, p = 0.001) was the best combination to predict surgical outcome (AUC = 0.815). Finally, we defined a prognostic signature, including pre-operative CA125, post-operative mesothelin, HE4 and surgical outcome, which was superior to the individual prognostic capacity of all included parameters. Our model allowed the calculation of a weighted risk score, stratifying patient at “lower risk” (score 0-4) or at “higher risk” (score 5-10). Patients with higher risk had significantly shorter PFS (19.0 months (95%CI = 16.1-21.8) vs 64.1 months (95%CI = 56.8-71.3) p &lt; 0.0001) and OS (34.0 months (95%CI = 28.6-39.3) vs 71.2 months (95%CI = 64.6-77.7) p &lt; 0.0001). Conclusions: This is the first study, proposing an integrated prognostic risk score for ovarian cancer patients, which could easily be implemented into routine diagnostics and may guide personalized treatment decisions. Patients at “higher risk”, as identified by this model, could potentially benefit from an intensified therapy regime, whereas patients at “lower risk” might be suitable for treatment de-escalation.

Variation rs2235503 C > A Within the Promoter of MSLN Affects Transcriptional Rate of Mesothelin and Plasmatic Levels of the Soluble Mesothelin-Related Peptide.

Soluble mesothelin-related peptide (SMRP) is a promising biomarker for malignant pleural mesothelioma (MPM), but several confounding factors can reduce SMRP-based test’s accuracy. The identification of these confounders could improve the diagnostic performance of SMRP. In this study, we evaluated the sequence of 1,000 base pairs encompassing the minimal promoter region of the MSLN gene to identify expression quantitative trait loci (eQTL) that can affect SMRP. We assessed the association between four MSLN promoter variants and SMRP levels in a cohort of 72 MPM and 677 non-MPM subjects, and we carried out in vitro assays to investigate their functional role. Our results show that rs2235503 is an eQTL for MSLN associated with increased levels of SMRP in non-MPM subjects. Furthermore, we show that this polymorphic site affects the accuracy of SMRP, highlighting the importance of evaluating the individual’s genetic background and giving novel insights to refine SMRP specificity as a diagnostic biomarker.

Multiplex Soluble Biomarker Analysis from Pleural Effusion.

Malignant pleural mesothelioma (MPM) is a highly aggressive and therapy resistant pleural malignancy that is caused by asbestos exposure. MPM is associated with poor prognosis and a short patient survival. The survival time is strongly influenced by the subtype of the tumor. Dyspnea and accumulation of pleural effusion in the pleural cavity are common symptoms of MPM. The diagnostic distinction from other malignancies and reactive conditions is done using histopathology or cytopathology, always supported by immunohistochemistry, and sometimes also by analyses of soluble biomarkers in effusion supernatant. We evaluated the soluble angiogenesis related molecules as possible prognostic and diagnostic biomarkers for MPM by Luminex multiplex assay. Pleural effusion from 42 patients with malignant pleural mesothelioma (MPM), 36 patients with adenocarcinoma (AD) and 40 benign (BE) effusions were analyzed for 10 different analytes that, in previous studies, were associated with angiogenesis, consisting of Angiopoietin-1, HGF, MMP-7, Osteopontin, TIMP-1, Galectin, Mesothelin, NRG1-b1, Syndecan-1 (SDC-1) and VEGF by a Human Premixed Multi-Analyte Luminex kit. We found that shed SDC-1 and MMP-7 levels were significantly lower, whereas Mesothelin and Galectin-1 levels were significantly higher in malignant mesothelioma effusions, compared to adenocarcinoma. Galectin-1, HGF, Mesothelin, MMP-7, Osteopontin, shed SDC-1, NRG1-β1, VEGF and TIMP-1 were significantly higher in malignant pleural mesothelioma effusions compared to benign samples. Moreover, there is a negative correlation between Mesothelin and shed SDC-1 and positive correlation between VEGF, Angiopoietin-1 and shed SDC-1 level in the pleural effusion from malignant cases. Shed SDC-1 and VEGF have a prognostic value in malignant mesothelioma patients. Collectively, our data suggest that MMP-7, shed SDC-1, Mesothelin and Galectin-1 can be diagnostic and VEGF and SDC-1 prognostic markers in MPM patients. Additionally, Galectin-1, HGF, Mesothelin, MMP-7, Osteopontin, shed SDC-1 and TIMP-1 can be diagnostic for malignant cases.

Microfluidic Immunoassay System for Rapid Detection and Semi-Quantitative Determination of a Potential Serum Biomarker Mesothelin.

Mesothelin (MSLN) is considered as a potential serological tumor biomarker for early diagnosis of pancreatic cancer. Nevertheless, low sensibility, high reagent consumption, and time costs of traditional detection methods limit their utility in clinical disease diagnoses. Here, we combined the immunoassay technique with microfluidic chips to develop a microfluidic immunoassay system (MIAS) that can be used for rapid semi-quantitative detection of serum MSLN levels. The MIAS was composed of 12 uniform structures, including 12 inlets, 12 reaction chambers, and one outlet allowing measurement of four samples with three repeats, simultaneously. A unique microarray cylinder was located at the end of each reaction chamber where immunoassay was performed to trap microspheres. A feasible interception efficiency (∼80%) was attained, with microspheres filling the reaction column. It has been demonstrated that fluorescence intensity is proportional to the MSLN concentration on the MIAS (R2 = 0.95). Subsequently, 16 clinical serum samples collected from Changhai Hospital, Shanghai were selected from eight patients with pancreatic cancers, four with pancreatitis, and four with other digestive system diseases (2 gastric cancer, 1 bile duct stricture, and 1 bile duct stones). MSLN levels for these samples were detected via MIAS. The results showed a significant correlation between MIAS and traditional enzyme-linked immunosorbent assay (ELISA), with the correlation coefficient, 0.93. The detection limit of MSLN fluorescence intensity and concentration was ∼6 a.u. and ∼20 pg/mL, respectively. The entire duration of analysis by MIAS decreased to ∼40 min compared to 2 h by ELISA. Statistical analysis of MIAS data revealed that MSLN was overexpressed in pancreatic cancer than in the others (P value = 0.0014). Moreover, the diagnostic accuracies of MSLN detected by MIAS and CA19-9 detected by ELISA in hospitals were 87.5 and 81.3%, respectively. MSLN is helpful for the early diagnosis of pancreatic cancer and other diseases, and it had a significant ability to discriminate between pancreatic and nonpancreatic cancers (P value = 0.0159). The results from this study show that MIAS has the potential to become a new serological tumor marker detection platform for rapid detection and semi-quantitative determination of MSLN and would have broad applications in early clinical diagnosis.

P2.06-19 A Meta-Analysis of Mesothelin, Osteopontin, and Fibulin-3 as Biomarkers of Malignant Pleural Mesothelioma

Malignant Pleural Mesothelioma (MPM) is a rare and aggressive cancer; the use of mesothelin, fibulin-3, and osteopontin as biomarkers has been proposed as a screening tool. The following meta-analysis examines average levels of these biomarkers in blood and pleura in comparison to several control groups. A PubMed search was conducted for studies that measured the levels of mesothelin, osteopontin, and fibulin-3 in patients with MPM compared to several control groups (other malignancies, benign lung disease, or healthy participants). Thirty two studies with 28 distinct datasets were identified as reporting mesothelin levels. Statistically significant mean differences (MD) were observed between mesothelin in the blood of MPM patients and each of the control groups. The overall MD (95% confidence interval) between MPM and non-MPM controls was 3.88 (2.73-5.05) nM/L (nMPM=455). Statistically significant MDs in pleural levels of mesothelin were also seen between MPM patients and non-MPM cancers and benign lung diseases. The overall MD (95%CI) was 31.45 (23.63-39.27) nM/L (nMPM=571). Twelve articles with 10 distinct datasets reported the mean level of osteopontin in MPM cases and controls. Statistically significant MDs in blood levels of osteopontin were seen between MPM patients and benign lung diseases or healthy controls (overall MD (95% CI) 103.75 (54.77-152.72) ng/mL (nMPM=504)), but not with other cancers. Studies on pleural levels of osteopontin were too scarce to conduct a meta-analysis. Nine articles measured fibulin-3 in blood or pleura of MPM patients compared to controls. There was a statistically significant MD in blood levels of fibulin-3 between MPM and each of the control groups. The overall MD (95%CI) was 46.52 (34.04-59.00) ng/mL (nMPM=330). There was also a statistically significant MD in the pleural levels of fibulin-3 between MPM and the cancer and benign lung disease groups. The overall MD (95% CI) was 385.35 (287.98-482.73)ng/mL (nMPM=137). This meta-analysis supports the concept that mesothelin and fibulin-3 in control groups are significantly lower than in MPM, and thus they might be useful as screening biomarkers. Further studies are necessary to better assess the value of these promising biomarkers.

Determination of Mesothelin Levels in Pleural Effusion Does Not Help Predict Survival of Patients With Malignant Pleural Mesothelioma.

This study evaluated the prognostic value of soluble mesothelin-related protein (SMRP) levels in pleural effusions (PE) from patients with pleural mesothelioma (MPM). SMRP level in PE was tested using an enzyme-linked immunosorbent assay (ELISA) in 109 patients with MPM at diagnosis before any treatment. The Kaplan-Meier method and the Cox regression were applied to compare overall survival probabilities across tertile categories of SMRP level. No significant differences in Kaplan-Meier overall survival probabilities among the SMRP categories were found. A statistically non-significant trend for increased death rate ratio (RR) was computed (p=0.327) when the higher (>46.5 nM, RR=1.38) and intermediate (8.5-46.5 nM, RR=1.18) SMRP categories were compared to the lower category (<8.5 nM, RR=1.00). Cox regression modelling including a restricted cubic spline showed a moderately rising non-linear trend in death rate. The SMRP level in PE does not appear to have prognostic significance and its detection is not recommended in routine clinical management of patients with MPM.

Biomarkers for malignant pleural mesothelioma: a meta-analysis.

Malignant pleural mesothelioma (MPM) is a rare but aggressive cancer, and early detection is associated with better survival. Mesothelin, fibulin-3 and osteopontin have been suggested as screening biomarkers. The study conducted a meta-analysis of the mean differences of mesothelin, osteopontin and fibulin-3 in blood and pleural samples. PubMed searches were conducted for studies that measured levels of mesothelin, osteopontin and fibulin-3 in participants with MPM compared with malignancy, benign lung disease or healthy participants. Thirty-two studies with mesothelin levels, 12 studies with osteopontin levels and 9 studies with fibulin-3 levels were included in the meta-analysis. Statistically significant mean differences were seen between MPM patients and all other comparison groups for mesothelin blood and pleural levels. Statistically significant differences in blood osteopontin levels were seen between participants with benign lung disease and healthy participants compared with participants with MPM, but not when comparing participants with cancer with MPM participants. There were not enough studies that reported osteopontin levels in pleural fluid to complete a meta-analysis. Statistically significant differences were seen in both blood and pleural levels of fibulin-3 in MPM patients compared with all other groups. On the basis of these results, mesothelin and fibulin-3 levels appear to be significantly lower in all control groups compared with those with MPM, making them good candidates for screening biomarkers. Osteopontin may be a useful biomarker for screening healthy individuals or those with benign lung disease but would not be useful for screening patients with malignancies.

Immunotherapeutic Targeting of Mesothelin in Acute Myeloid Leukemia in Vitro with Anetumab Ravtansine and a Novel Antibody-Drug Conjugate

Acute myeloid leukemia (AML) is a high-risk malignancy with overall poor outcomes across the age spectrum. Attempts to intensify cytotoxic chemotherapeutic regimens in recent decades have been limited by toxic acute and late effects, establishing the necessity of targeted therapy development. Immunotherapy is a highly promising treatment strategy in AML; however, its development has been limited as agents with potent anti-leukemic activity often confer significant hematopoietic toxicities. We previously reported the identification of an ideal potential immunotherapeutic target, mesothelin (MSLN), a cell surface protein expressed in 37% of childhood and young adult AML as well as older adult AML that, importantly, is absent in normal hematopoietic cells. Mesothelin is overexpressed in a number of solid tumors and has been studied as a target in solid tumors with various immunotherapeutic agents, including the antibody-drug conjugate (ADC) anetumab ravtansine (AR), comprised of an anti-MSLN antibody conjugated to the maytansinoid tubulin inhibitor DM4. We evaluated AR as well as a novel MSLN-targeted ADC for their ability to induce MSLN-directed cytotoxicity in several cell lines engineered to express MSLN.As we have previously reported, the leukemia cell line K562 transduced to overexpress MSLN (K562+MSLN) was highly sensitive to AR in vitro with an IC50 of 1.4 nM (Fig. 1a). Furthermore, AR was highly active in vivo in K562+MSLN xenograft-bearing mice. We sought to extend the study of AR's cytotoxicity in AML, to illustrate its potential as a therapeutic agent, in 3 additional AML cell lines, MV4;11, Kasumi-1, and Me-1, lentivirally-transduced to express MSLN, with cell-surface MSLN levels confirmed by flow cytometry. In vitro cytotoxicity assays demonstrated potent MSLN-dependent cytotoxicity of AR in MV4;11+MSLN cells with IC50 0.009 nM, compared to 1.9-3.8 nM in control experiments with parental cells or isotype control (IC) treatment (Fig. 1b). AR had limited MSLN-dependent activity in Kasumi-1+MSLN, with an IC50 of 28 nM, compared to 76-87 nM in controls (Fig. 1c) and no MSLN-dependent cytotoxicity in Me-1+MSLN, compared to controls (Fig. 1d). Previous reports suggest that DM4 is a substrate of the drug resistance protein P-glycoprotein (ABCB1). We evaluated drug resistance protein expression, including ABCB1, MRP1-6, LRP1, and BRCP, using RNASeq in representative AR-sensitive and AR-resistant cell lines, MV4;11 and Kasumi-1, respectively, and examined published RNASeq data for K562 and Me-1, and found that P-glycoprotein expression correlated with resistance to AR, measured by the IC50 (R2 = 0.995, p = 0.002).To optimize ADC targeting of MSLN in AML, we created a novel MSLN-targeted ADC using an alternative MSLN antibody with an alternative chemotherapeutic payload, pyrrolobenzodiazepine (PBD) dimer, and molecular linker (anti-MSLN-PBD) as well as 3 control compounds: an anti-MSLN ADC with a DM4 payload (anti-MSLN-DM4) and isotype controls comprised of anti-Tetanus toxin antibody conjugated to PBD (IC-PBD) or DM4 (IC-DM4). In vitro cytotoxicity assays in the 4 aforementioned MSLN+ leukemia cell lines revealed potent MSLN-dependent cell kill with the novel anti-MSLN-PBD compared to controls in all but Me-1+MSLN, with IC50 of 1.6 pM-8.3 nM (Fig. 1e-h). Notably, unlike AR, anti-MSLN-PBD was able to confer MSLN-dependent cytotoxicity in Kasumi-1+MSLN. Sensitivity to anti-MSLN-PBD was independent of P-glycoprotein expression.Mesothelin is a cell-surface protein previously recognized as an attractive immunotherapeutic target in solid tumors that is aberrantly expressed in a sizeable subset of AML patients of all ages, and herein we report successful targeting of MSLN in leukemia cells in vitro with antibody-drug conjugates. AR, an ADC well-tolerated in phase I testing for solid tumors, showed dramatic MSLN-dependent cytotoxicity in 2 cell lines at clinically-achievable doses. To optimize MSLN targeting in AML, we created a novel MSLN-targeted ADC with a PBD dimer payload, which conferred a high degree of MSLN-dependent cytotoxicity in vitro in 3 of 4 MSLN+ leukemia cell lines tested. Mesothelin-directed ADCs hold great promise for the treatment of MSLN+ AML across the age spectrum and warrant additional pre-clinical and clinical evaluation as potential novel immunotherapeutics for AML. DisclosuresNo relevant conflicts of interest to declare.