Monoclonal antibodies to the exon 18 encoded moiety of NCAM

Aim: Exon 18 expression of NCAM has been recognized as a biomarker for small cell lung cancer (SCLC). To use this finding for an improved diagnosis of SCLC and personalized treatment of patients, techniques to identify and quantitate E18, the exon 18 encoded protein moiety of NCAM, are needed. We developed three monoclonal antibodies for this purpose.Methods: The his-tagged E18 antigen was expressed in E. coli and, after purification, used to immunize mice. Hybridoma’s were isolated by standard procedures and tested for their reaction with E18.Results: Three monoclonal antibodies, MUM-1, MUM-4 and MUM-6 were obtained. They reacted with E18 in western blots, with SCLC cell line NCI-H82, but not with unrelated his-tagged proteins. Only permeabilized NCI-H82 cells stained with the antibodies, confirming the intracellular position of E18. Next an enzyme-linked immunosorbent assay was developed using the earlier isolated monoclonal antibody MUMi-21B2, coated on the surface of microtiter wells as capture antibody and biotinylated MUM-6 as second antibody. Using streptavidin conjugated to horse radish peroxidase a linear dose response curve to his-tagged E18 antigen was obtained between 0 and 5 µg/mL with a sensitivity of at least 0.5 µg/mL or 50 ng/well.Conclusion: Four monoclonal antibodies are available to be used in assays for the identification and quantification of SCLC biomarker E18. This will enable the development of liquid biopsies to follow the tumor load in patients

The 180 splice variant of NCAM-containing exon 18-is specifically expressed in small cell lung cancer cells

Background: The Neural Cell Adhesion Molecule (NCAM) is a glycoprotein expressed as 120, 140 and/or 180 kDa isoforms, all derived through alternative splicing of a single gene. NCAM 120 contains no intracellular domain, whereas NCAM 140 and 180 have different intracellular domains determined by alternative splicing of exon 18. NCAM has been described as a biomarker to discriminate small cell lung cancer (SCLC) from non-SCLC (NSCLC). However, peripheral blood mononuclear cells (PBMC) also express NCAM. We studied the expression of NCAM splice variants in cell lines, tumor tissues and control cells. Methods: Using reverse transcriptase-PCR we evaluated the expression of NCAM exon 18 splice variants in lung cancers cell lines, control cell lines, PBMC of healthy controls and SCLC tissue. In addition we studied the expression of the NCAM exon 18 encoded protein (E18) in SCLC by immunocytochemistry and flow cytometry using an E18-specific monoclonal antibody obtained by hybridoma fusion of E18-immunized mouse spleen cells. Finally we looked at immune responses to E18 in mice. Results: We found expression of RNA encoding the NCAM 180 variant in all SCLC cell lines. NCAM exon 18 was not expressed in 23/28 (82%) of the other tumor and leukemia cell lines tested and PBMC. Next, we also evaluated the expression of NCAM exon 18 in human SCLC tissue. Expression of NCAM exon 18 in 8 of the 10 (80%) SCLC biopsy samples was found. The newly raised E18-specific antibodies stained NCAM at the adherent junctions between adjacent cells in SCLC cell lines. The data demonstrate the intracellular location of E18 in SCLC. Furthermore, a specific cytotoxic T cell (CTL) response and significant antibody titers were found in mice upon immunization with recombinant E18 and its encoding DNA. Conclusions: The results of this study can be applied in the diagnosis and immunotherapy of SCLC. A larger study investigating E18 as a marker for SCLC is indicated

Decentralization of Next-Generation RNA Sequencing-Based MammaPrint® and BluePrint® Kit at University Hospitals Leuven and Curie Institute Paris.

A previously developed and centrally validated MammaPrint® (MP) and BluePrint® (BP) targeted RNA next-generation sequencing (NGS) kit was implemented and validated in two large academic European hospitals. Additionally, breast cancer molecular subtypes by MP and BP RNA sequencing were compared with immunohistochemistry (IHC). Patients with early breast cancer diagnosed at University Hospitals Leuven and Curie Institute Paris were prospectively included between September 2017 and January 2018. Formalin-fixed paraffin-embedded tissue sections were analyzed with MP and BP NGS technology at the beta sites and with both NGS and microarray technology at Agendia. Raw NGS data generated on Illumina MiSeq instruments at the beta sites were interpreted and compared with NGS and microarray data at Agendia. MP and BP NGS molecular subtypes were compared to surrogate IHC breast cancer subtypes. Equivalence of MP and BP indices was determined by Pearson's correlation coefficient. Acceptable limits were defined a priori, based on microarray data generated at Agendia between 2012 and 2016. The concordance, the Negative Percent Agreement and the Positive Percent Agreement were calculated based on the contingency tables and had to be equal to or higher than 90%. Out of 124 included samples, 48% were MP Low and 52% High Risk with microarray. Molecular subtypes were BP luminal, HER2 or basal in 82%, 8% and 10% respectively. Concordance between MP microarray at Agendia and MP NGS at the beta sites was 91.1%. Concordance of MP High and Low Risk classification between NGS at the beta sites and NGS at Agendia was 93.9%. Concordance of MP and BP molecular subtyping using NGS at the beta sites and microarray at Agendia was 89.5%. Concordance between MP and BP NGS subtyping, and IHC was 71.8% and 76.6%, for two IHC surrogate models. The MP/BP NGS kit was successfully validated in a decentralized setting.status: Published onlin

Identification of Proteomic Differences between Squamous Cell Carcinoma of the Lung and Bronchial Epithelium*S⃞

Proteins that exhibit different expression levels in normal and malignant lung cells are good candidate biomarkers to improve early diagnosis and intervention. We used a quantitative approach and compared the proteome of microdissected cells from normal human bronchial epithelium and squamous cell carcinoma tumors of histopathological grades G2 and G3. DIGE analysis and subsequent MS-based protein identification revealed that 32 non-redundant proteins were differentially regulated between the respective tissue types. These proteins are mainly involved in energy pathways, cell growth or maintenance mechanisms, protein metabolism, and the regulation of DNA and RNA metabolism. The expression of some of these proteins was analyzed by immunohistochemistry using tissue microarrays containing tissue specimen of 55 patients, including normal bronchial epithelium, squamous cell carcinomas, adenocarcinomas, and large cell carcinomas. The results of the immunohistochemical studies correlated with the proteome study data and revealed that particularly HSP47 and a group of cytokeratins (i.e. cytokeratins 6a, 16, and 17) are significantly co-regulated in squamous cell carcinoma. Furthermore cytokeratin 17 showed significantly higher abundance in G2 grade compared with G3 grade squamous cell carcinomas in both the gel-based and the immunohistochemical analysis. Therefore this protein might be used as a marker for stratification between different tumor grades