Karen Zwaenepoel

CD70: An emerging target in cancer immunotherapy

Over the last decades, advances in the knowledge of immunology have led to the identification of immune checkpoints, reinvigorating cancer immunotherapy. Although normally restricted to activated T and B cells, constitutive expression of CD70 in tumor cells has been described. Moreover, CD70 is implicated in tumor cell and regulatory T cell survival through interaction with its ligand, CD27. In this review, we summarize the targetable expression patterns of CD70 in a wide range of malignancies and the promising mechanism of anti-CD70 therapy in stimulating the anti-tumor immune response. In addition, we will discuss clinical data and future combination strategies.

Circulating cell-free nucleic acids and platelets as a liquid biopsy in the provision of personalized therapy for lung cancer patients.

Lung cancer is the predominant cause of cancer-related mortality in the world. The majority of patients present with locally advanced or metastatic non-small-cell lung cancer (NSCLC). Treatment for NSCLC is evolving from the use of cytotoxic chemotherapy to personalized treatment based on molecular alterations. Unfortunately, the quality of the available tumor biopsy and/or cytology material is not always adequate to perform the necessary molecular testing, which has prompted the search for alternatives. This review examines the use of circulating cell-free nucleic acids (cfNA), consisting of both circulating cell-free (tumoral) DNA (cfDNA-ctDNA) and RNA (cfRNA), as a liquid biopsy in lung cancer. The development of sensitive and accurate techniques such as Next-Generation Sequencing (NGS); Beads, Emulsion, Amplification, and Magnetics (BEAMing); and Digital PCR (dPCR), have made it possible to detect the specific genetic alterations (e.g. EGFR mutations, MET amplifications, and ALK and ROS1 translocations) for which targeted therapies are already available. Moreover, the ability to detect and quantify these tumor mutations has enabled the follow-up of tumor dynamics in real time. Liquid biopsy offers opportunities to detect resistance mechanisms, such as the EGFR T790M mutation in the case of EGFR TKI use, at an early stage. Several studies have already established the predictive and prognostic value of measuring ctNA concentration in the blood. To conclude, using ctNA analysis as a liquid biopsy has many advantages and allows for a variety of clinical and investigational applications.

Detection of ALK expression in non‐small‐cell lung cancer with ALK gene rearrangements – comparison of multiple immunohistochemical methods

Testing for ALK rearrangements in advanced, non‐squamous non‐small‐cell lung cancers that are wild‐type for activating EGFR mutation has become standard care. Fluorescence in‐situ hybridization is considered the gold standard for this evaluation. Pre‐screening with immunohistochemistry has been suggested, to reduce testing costs and to make testing more widely available. By analysing the sensitivity and specificity of different ALK immunohistochemical assays, we aimed to identify the most reliable assay to detect ALK rearrangement.

A Comparison of Cell-Free DNA Isolation Kits: Isolation and Quantification of Cell-Free DNA in Plasma

The analysis of cell-free DNA (cfDNA) as a sensitive biomarker for cancer diagnosis and monitoring has resulted in a need for efficient and standardized cfDNA isolation. In this study, we compared the isolation efficiency of the QIAamp circulating nucleic acid kit (QIA) with four other cfDNA isolation kits: the PME free-circulating DNA Extraction Kit (PME), the Maxwell RSC ccfDNA Plasma Kit (RSC), the EpiQuick Circulating Cell-Free DNA Isolation Kit (EQ), and two consecutive versions of the NEXTprep-Mag cfDNA Isolation Kit (NpMV1/2). cfDNA was isolated from 10 plasma samples, of which five contained KRAS mutated cell-free tumor DNA (ctDNA). Digital droplet PCR was used to quantify the total cfDNA concentration as well as the KRAS mutated ctDNA fraction. cfDNA integrity was assessed with real-time quantitative PCR. The QIA and the RSC kits displayed similar isolation efficiencies of both KRAS mutated ctDNA and nonmutated cfDNA, whereas the yield generated by the PME and NpMV2 kits was significantly lower. Real-time quantitative PCR indicated the presence of digital droplet PCR inhibiting agents in the eluates of the NpMV1 and EQ kits. To conclude, this study presents two highly efficient isolation kits for cfDNA isolation, of which the RSC kit has the advantage of a fully automated protocol over the labor-intensive QIA kit.

The relevance of external quality assessment for molecular testing for ALK positive non-small cell lung cancer: results from two pilot rounds show room for optimization.

Background and Purpose Molecular profiling should be performed on all advanced non-small cell lung cancer with non-squamous histology to allow treatment selection. Currently, this should include EGFR mutation testing and testing for ALK rearrangements. ROS1 is another emerging target. ALK rearrangement status is a critical biomarker to predict response to tyrosine kinase inhibitors such as crizotinib. To promote high quality testing in non-small cell lung cancer, the European Society of Pathology has introduced an external quality assessment scheme. This article summarizes the results of the first two pilot rounds organized in 2012–2013. Materials and Methods Tissue microarray slides consisting of cell-lines and resection specimens were distributed with the request for routine ALK testing using IHC or FISH. Participation in ALK FISH testing included the interpretation of four digital FISH images. Results Data from 173 different laboratories was obtained. Results demonstrate decreased error rates in the second round for both ALK FISH and ALK IHC, although the error rates were still high and the need for external quality assessment in laboratories performing ALK testing is evident. Error rates obtained by FISH were lower than by IHC. The lowest error rates were observed for the interpretation of digital FISH images. Conclusion There was a large variety in FISH enumeration practices. Based on the results from this study, recommendations for the methodology, analysis, interpretation and result reporting were issued. External quality assessment is a crucial element to improve the quality of molecular testing.

Expression analysis on archival material revisited : isolation and quantification of RNA extracted from FFPE samples

Background:Formalin-fixed paraffin-embedded (FFPE) tissue is the most readily available source of RNA for the gene expression studies. The main disadvantage is the poor quality of isolated RNA. Our group recently compared 5 commercially available RNA isolation kits and concluded that the RNeasy FFPE kit from Qiagen was the most appropriate one. However, this kit has been discontinued and replaced by a new version. In this study both kits were compared, and spectrophotometric and fluorometric analyses for quantification of RNA samples extracted from FFPE tissue. Methods:Both RNeasy FFPE kits were compared for the total RNA and DNA yields, purity, and raw cycle threshold. Quantity and quality of the isolated RNA was measured using the NanoDrop ND-1000 spectrophotometer and Qubit 2.0 fluorometer. Results:The average concentration of RNA extracted from FFPE tissue measured using the NanoDrop was 32.0%±9.5% higher than the concentration measured using the Qubit. When measuring an RNA sample extracted from a cell line, the concentration measured using both methods was similar. When comparing both RNeasy FFPE kits, marginal differences were observed for total RNA yield, purity, and raw cycle threshold. However, the residual DNA in the samples isolated using the old kit was higher than in the samples isolated using the new kit. Conclusions:A fluorometric analysis is more suitable for quantification of RNA samples extracted from FFPE tissue compared with spectrophotometric analysis. For RNA isolation from FFPE tissue, both old and new RNeasy FFPE kits were adequate. The new kit resulted in more efficient DNA removal.

Prognostic and predictive aspects of the tumor immune microenvironment and immune checkpoints in malignant pleural mesothelioma

ABSTRACT Malignant pleural mesothelioma (MPM) is an aggressive cancer with a poor prognosis and an increasing incidence, for which novel therapeutic strategies are urgently required. Since the immune system has been described to play a presumed role in the protection against MPM, characterization of its tumor immune microenvironment (TME) and immune checkpoints can identify new immunotherapeutic targets and their predictive and/or prognostic value. To characterize the TME and the immune checkpoint expression profile, we performed immunohistochemistry (IHC) on formalin-fixed paraffin embedded (FFPE) tissue sections from 54 MPM patients (40 at time of diagnosis; 14 treated with chemotherapy). We stained for PD-1, PD-L1, TIM-3, LAG-3, CD4, CD8, CD45RO, granzyme B, FoxP3 and CD68. Furthermore, we analyzed the relationship between the immunological parameters and survival, as well as response to chemotherapy. We found that TIM-3, PD-1 and PD-L1 were expressed on both immune and tumor cells. Strikingly, PD-1 and PD-L1 expression on tumor cells was only seen in unpretreated samples. No LAG-3 expression was observed. CD45RO expression in the stroma was an independent negative predictive factor for response on chemotherapy, while CD4 and TIM-3 expression in lymphoid aggregates were independent prognostic factors for better outcome. Our data propose TIM-3 as a promising new target in mesothelioma. Chemotherapy influences the expression of immune checkpoints and therefore further research on the best combination treatment schedule is required.

CD70 Expression and Its Correlation with Clinicopathological Variables in Squamous Cell Carcinoma of the Head and Neck.

Objective: Over the last decade, efforts have been made to get a better understanding of the tumor microenvironment and the role of the immune system in it. New insights into the CD27/CD70 signaling pathway point towards a role in tumor immunology, making CD70 an attractive target for immunotherapy. Here, we evaluate CD70 expression in squamous cell carcinoma of the head and neck (SCCHN). Methods: CD70 immunohistochemistry was retrospectively performed on 95 tumor samples. Tumoral CD70 expression was scored and correlated with clinicopathological variables and overall survival (OS). Results: CD70 expression in tumor cells was observed in 66 samples (69%) and was strongly associated with tumor differentiation grade (p < 0.001). CD70 expression was also observed in tumor-associated fibroblasts and endothelial cells. Additionally, the density of tumor-infiltrating lymphocytes correlated with OS (p = 0.042). Conclusion: This study describes the tumoral expression of CD70 in SCCHN. Results highlight the role of CD70 in tumor biology and identify CD70 as a novel therapeutic target. Further research is warranted.

CD70 and PD-L1 in anaplastic thyroid cancer : promising targets for immunotherapy

During recent years, immune checkpoint inhibition has proved to be effective in several solid malignancies. The aim of this study was to identify novel targets for immunotherapy in anaplastic thyroid cancer by analysis of the expression of tumour antigens for which therapeutic agents are available.

Clinical performance evaluation of a sensitive, rapid low-throughput test for KRAS mutation analysis using formalin-fixed, paraffin-embedded tissue samples

BackgroundTesting for KRAS mutations in metastatic colorectal cancer (mCRC) on formalin-fixed, paraffin embedded (FFPE) tumor tissue has become standard of care. Different molecular methods exist to determine hotspot KRAS mutations in exon 2, 3 and 4, but testing is often limited by the sensitivity and the speed of analysis. The aim of this retrospective study was to establish the clinical performance of the Idylla™ KRAS Mutation Test on FFPE tumor samples of patients with mCRC.MethodsKRAS mutation analysis was performed using the therascreen KRAS on the RotorGene Q platform (CE-IVD; Qiagen) and results were subsequently compared to the Idylla™ KRAS Mutation Test. Discordant result testing was performed with massive parallel sequencing or alternative routine approaches.ResultsData from 182 samples were used to show that the overall agreement between the two methods for mutation characterization was 96.7% [95%CI: 93.0%-98.5%]. Six out of 182 samples (3.3%) showed true discordant results.ConclusionThe Idylla™ KRAS Mutation Test allows for a fast and reliable analysis of FFPE samples with a turnaround-time of two hours without the need of molecular infrastructure or expertise in order to guide the personalized treatment of colorectal cancer patients.