Daan van den Broek

Case Report of a Fatal Serious Adverse Event Upon Administration of T Cells Transduced With a MART-1-specific T-cell Receptor

Here, we describe a fatal serious adverse event observed in a patient infused with autologous T-cell receptor (TCR) transduced T cells. This TCR, originally obtained from a melanoma patient, recognizes the well-described HLA-A*0201 restricted 26-35 epitope of MART-1, and was not affinity enhanced. Patient 1 with metastatic melanoma experienced a cerebral hemorrhage, epileptic seizures, and a witnessed cardiac arrest 6 days after cell infusion. Three days later, the patient died from multiple organ failure and irreversible neurologic damage. After T-cell infusion, levels of IL-6, IFN-γ, C-reactive protein (CRP), and procalcitonin increased to extreme levels, indicative of a cytokine release syndrome or T-cell-mediated inflammatory response. Infused T cells could be recovered from blood, broncho-alveolar lavage, ascites, and after autopsy from tumor sites and heart tissue. High levels of NT-proBNP indicate semi-acute heart failure. No cross reactivity of the modified T cells toward a beating cardiomyocyte culture was observed. Together, these observations suggest that high levels of inflammatory cytokines alone or in combination with semi-acute heart failure and epileptic seizure may have contributed substantially to the occurrence of the acute and lethal event. Protocol modifications to limit the risk of T-cell activation-induced toxicity are discussed.

Swarm Intelligence-Enhanced Detection of Non-Small-Cell Lung Cancer Using Tumor-Educated Platelets

Summary Blood-based liquid biopsies, including tumor-educated blood platelets (TEPs), have emerged as promising biomarker sources for non-invasive detection of cancer. Here we demonstrate that particle-swarm optimization (PSO)-enhanced algorithms enable efficient selection of RNA biomarker panels from platelet RNA-sequencing libraries (n = 779). This resulted in accurate TEP-based detection of early- and late-stage non-small-cell lung cancer (n = 518 late-stage validation cohort, accuracy, 88%; AUC, 0.94; 95% CI, 0.92–0.96; p < 0.001; n = 106 early-stage validation cohort, accuracy, 81%; AUC, 0.89; 95% CI, 0.83–0.95; p < 0.001), independent of age of the individuals, smoking habits, whole-blood storage time, and various inflammatory conditions. PSO enabled selection of gene panels to diagnose cancer from TEPs, suggesting that swarm intelligence may also benefit the optimization of diagnostics readout of other liquid biopsy biosources.

Multicenter evaluation of a new progastrin-releasing peptide (ProGRP) immunoassay across Europe and China

BACKGROUND We performed a multicenter evaluation of the Elecsys® progastrin-releasing peptide (ProGRP) immunoassay in Europe and China. METHODS The assay was evaluated at three European and two Chinese sites by imprecision, stability, method comparison and differentiation potential in lung cancer. RESULTS Intermediate imprecision across five analyte concentrations ranged from 2.2% to 6.0% coefficient of variation. Good stability for plasma and serum samples was shown for various storage conditions. There was excellent correlation between the Elecsys® and ARCHITECT assays in plasma (slope 1.02, intercept -2.72pg/mL). The Elecsys® assay also showed good correlation between serum and plasma samples (slope 0.93, intercept 2.35pg/mL; correlation coefficient 0.97). ProGRP differentiated small-cell and non-small-cell lung cancer (NSCLC; area under the curve 0.90, 95% CI 0.87-0.93; 78.3% sensitivity, 95% specificity; at 84pg/mL), with no relevant effects of ethnicity, age, gender or smoking. Median ProGRP concentrations were low in benign diseases (38pg/mL), other malignancies (40pg/mL) or NSCLC (39pg/mL), except chronic kidney disease above stage 3 (>100pg/mL). CONCLUSIONS Increased stability of the Elecsys® ProGRP assay in serum and plasma offers clear benefits over existing assays. This first evaluation of a ProGRP assay in China demonstrated comparable differentiation potential among different ethnicities.

Preanalytical blood sample workup for cell-free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics

In current molecular cancer diagnostics, using blood samples of cancer patients for the detection of genetic alterations in plasma (cell‐free) circulating tumor DNA (ctDNA) is an emerging practice. Since ctDNA levels in blood are low, highly sensitive Droplet Digital PCR (ddPCR) can be used for detecting rare mutational targets. In order to perform ddPCR on blood samples, a standardized procedure for processing and analyzing blood samples is necessary to facilitate implementation into clinical practice. Therefore, we assessed the technical sample workup procedure for ddPCR on blood plasma samples. Blood samples from healthy individuals, as well as lung cancer patients were analyzed. We compared different methods and protocols for sample collection, storage, centrifugation, isolation, and quantification. Cell‐free DNA (cfDNA) concentrations of several wild‐type targets and BRAF and EGFR‐mutant ctDNA concentrations quantified by ddPCR were primary outcome measurements. Highest cfDNA concentrations were measured in blood collected in serum tubes. No significant differences in cfDNA concentrations were detected between various time points of up to 24 h until centrifugation. Highest cfDNA concentrations were detected after DNA isolation with the Quick cfDNA Serum & Plasma Kit, while plasma isolation using the QIAamp Circulating Nucleic Acid Kit yielded the most consistent results. DdPCR results on cfDNA are highly dependent on multiple factors during preanalytical sample workup, which need to be addressed during the development of this diagnostic tool for cancer diagnostics in the future.

Crizotinib-induced fatal fulminant liver failure

Herein we describe a case of a 62-year-old female in good clinical condition with non-small-cell lung cancer who was treated with crizotinib. After 24 days of crizotinib therapy she presented with acute liver failure. Serum aspartate aminotransferase and alanine aminotransferase levels had increased from normal prior to crizotinib start to 2053 IU/L and 6194 IU/L, respectively. Total bilirubin and prothrombin time (PT-INR) increased up to 443 IU/L and 5.33, respectively, and symptoms of hepatic encephalopathy and hepatorenal syndrome emerged. Despite crizotinib discontinuation and intensive supportive therapy, the patient died 40 days after treatment with crizotinib was initiated due to acute liver failure with massive liver cell necrosis.

A serum and platelet-rich plasma serotonin assay using liquid chromatography tandem mass spectrometry for monitoring of neuroendocrine tumor patients

BACKGROUND Serotonin is used for the diagnosis and follow-up of neuroendocrine tumors (NET). We describe the analytical and clinical validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) based serotonin assay for serum and platelet-rich plasma (PRP). METHODS An LC-MS/MS based method for serum and PRP serotonin was validated by determination of assay imprecision, carry-over, linearity, interference, recovery, sample stability and a matrix/method comparison of serum and PRP serotonin was made with whole blood serotonin. Furthermore, upper limits of normal were determined and serotonin concentrations of healthy individuals, 14 NET patients without evidence of disease and 51 NET patients with evidence of disease were compared. RESULTS For serum and PRP fractions, total assay imprecision was <5%. All correlation coefficients were 0.98 and the serum and platelet-rich serotonin upper limit of normal were 5.5nmol/109 platelet and 5.1nmol/109 platelet, respectively. NET patients with confirmed evidence of disease had significantly higher serum and PRP serotonin levels when compared to NET patients without evidence of disease and healthy volunteers. CONCLUSIONS LC-MS/MS based serum and PRP serotonin assays were developed with suitable analytical characteristics. Furthermore, serum and PRP serotonin was found to be useful for monitoring NET patients.

A proteomics-based approach identifies secreted protein acidic and rich in cysteine as a prognostic biomarker in malignant pleural mesothelioma.

Background:We aimed to identify prognostic blood biomarkers using proteomics-based approaches in malignant pleural mesothelioma (MPM).Methods:Plasma samples from 12 MPM patients were used for exploratory mass spectrometry and ELISA analyses. The significance of secreted protein acidic and rich in cysteine (SPARC) was examined in sera from a Dutch series (n=97). To determine the source of the circulating SPARC, we investigated SPARC expression in MPM tumours and healthy controls, as well as the expression and secretion from cell lines and xenografts.Results:Secreted protein acidic and rich in cysteine was identified as a putative prognostic marker in plasma. Validation in the Dutch series showed that the median survival was higher in patients with low SPARC compared with those with high SPARC (19.0 vs 8.8 months; P=0.01). In multivariate analyses, serum SPARC remained as an independent predictor (HR 1.55; P=0.05). In MPM tumour samples, SPARC was present in the tumour cells and stromal fibroblasts. Cellular SPARC expression was higher in 5 out of 7 cell lines compared with two immortalized mesothelial lines. Neither cell lines nor xenograft tumours secreted detectable SPARC.Conclusions:Low circulating SPARC was associated with favourable prognosis. Secreted protein acidic and rich in cysteine was present in both tumour cells and stromal fibroblasts; and our in vitro and in vivo experiments suggest that stromal fibroblasts are a potential source of circulating SPARC.

Food‐effect study on uracil and dihydrouracil plasma levels as marker for dihydropyrimidine dehydrogenase activity in human volunteers

This study aimed to determine the effect of food intake on uracil and dihydrouracil plasma levels. These levels are a promising marker for dihydropyrimidine dehydrogenase activity and for individualizing fluoropyrimidine anticancer therapy.

CMET-12. TUMOR CELL DETECTION BY IMMUNOFLOW CYTOMETRY AND BRAF MUTATION ANALYSIS IN CEREBROSPINAL FLUID OF MELANOMA PATIENTS WITH SUSPECTED LEPTOMENINGEAL METASTASES

INTRODUCTION Approximately five percent of melanoma patients develop leptomeningeal metastases (LM). Diagnosis of LM can be made on clinical symptoms and typical contrast enhancement of the leptomeninges on MRI brain or spine and/or CSF cytology. However, MRI has a sensitivity of 76% and a specificity of 77%. Sensitivity of CSF cytology is also low: 55% at first analysis, increasing to 80% upon second sampling.

Abstract LB-248: RNA-sequencing of tumor-educated platelets enables nivolumab immunotherapy response prediction

I: Studies have shown the activity of anti-PD(L)-1 therapies in patients with advanced non-small-cell lung cancer (NSCLC), however response rates are rather low (~20%). Therefore, there is an urgent need to identify biomarkers that predict patient outcome to immunotherapy (IT). Previously we have shown that RNA signatures of tumor-educated platelets (TEPs) may have predictive value for tumor type-specific diagnostics. Platelets are intimately involved in immune responses. We hypothesized that TEP RNA profiles have predictive value for IT response. M: We collected baseline platelet pellets, isolated from whole blood by differential centrifugation, from 389 stage IV NSCLC patients treated with Nivolumab (table 1). Tumor response was evaluated at 3 and 6 months and reported according to RECIST 1.1. Platelet pellets were subjected to total RNA isolation, SMARTer cDNA amplification, and libraries were sequenced on the HiSeq platform. Raw data (~20 M reads per sample) was mapped to the human genome, intron-spanning spliced RNA reads were selected for analysis. Gene panels were calculated by ANOVA statistics. R: Until now 64 samples were sequenced, 30 of patients with clinical benefit (PR or SD at six months; CB) and 34 of patients with no clinical benefit (PD; no CB). 40 randomly selected samples (20 CB, 20 no CB) were used for training of the support vector machine (SVM)-based therapy response classification algorithm. 24 samples were used for independent evaluation of the classifier. Hierarchical clustering of genes with p Citation Format: Mirte Muller, Myron Best, Nik Sol, Anna-Larissa N. Niemeijer, Adrienne Vancura, Robert D. Schouten, Jeroen J.N. Hiltermann, Sjors G.J.G. In 9t Veld, Daan van den Broek, Vincent van der Noort, Adrianus J. de Langen, Ed M.D. Schuuring, Thomas Wurdinger, Michel M. van den Heuvel. RNA-sequencing of tumor-educated platelets enables nivolumab immunotherapy response prediction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-248. doi:10.1158/1538-7445.AM2017-LB-248