The Monoclonal Anti-CD157 Antibody Clone SY11B5, Used for High Sensitivity Detection of PNH Clones on WBCs, Fails to Detect a Common Polymorphic Variant Encoded by BST-1.

BACKGROUND: CD157, encoded by BST-1, has been described as a useful flow cytometric marker for the analysis of paroxysmal nocturnal hemoglobinuria (PNH) as it is a glycosylphosphatidylinositol (GPI)-linked molecule highly expressed on normal monocytes and neutrophils. We and others observed isolated CD157 signal dropouts during intended PNH analysis. We hypothesize that these negative populations occur due to an antibody failure. To investigate the reason for this finding, we compared two different anti-CD157 antibody clones for PNH analysis. METHODS: We sequenced BST-1 of CD157-negative probands that are not suffering from PNH and expressed wild type and a discovered variant form of CD157 in HEK293 cells. We compared the binding patterns of two different anti-CD157 antibody clones (SY11B5 and RF3) by flow cytometry and western blot analysis. RESULTS: When sequencing two CD157-negative probands we detected a common SNP (p.Arg145Gln) in exon 3 of BST-1. We found that only anti-CD157 antibody clone RF3 but not the more widely used clone SY11B5 was able to detect both, the wild type and the variant form of CD157 in flow cytometric experiments. CONCLUSION: The failure of anti-CD157 antibody clone SY11B5 to detect a common SNP can explain some CD157-negative cytometric data. This provides crucial knowledge for laboratories performing PNH analyses as such results can potentially lead to false-positive PNH interpretation. Our results confirm the importance of published PNH guidelines. © 2018 International Clinical Cytometry Society.

Metronomic topotecan impedes tumor growth of MYCN-amplified neuroblastoma cells in vitro and in vivo by therapy induced senescence.

Poor prognosis and frequent relapses are major challenges for patients with high-risk neuroblastoma (NB), especially when tumors show MYCN amplification. High-dose chemotherapy triggers apoptosis, necrosis and senescence, a cellular stress response leading to permanent proliferative arrest and a typical senescence-associated secretome (SASP). SASP components reinforce growth-arrest and act immune-stimulatory, while others are tumor-promoting. We evaluated whether metronomic, i.e. long-term, repetitive low-dose, drug treatment induces senescence in vitro and in vivo. And importantly, by using the secretome as a discriminator for beneficial versus adverse effects of senescence, drugs with a tumor-inhibiting SASP were identified.We demonstrate that metronomic application of chemotherapeutic drugs induces therapy-induced senescence, characterized by cell cycle arrest, p21(WAF/CIP1) up-regulation and DNA double-strand breaks selectively in MYCN-amplified NB. Low-dose topotecan (TPT) was identified as an inducer of a favorable SASP while lacking NFKB1/p50 activation. In contrast, Bromo-deoxy-uridine induced senescent NB-cells secret a tumor-promoting SASP in a NFKB1/p50-dependent manner. Importantly, TPT-treated senescent tumor cells act growth-inhibitory in a dose-dependent manner on non-senescent tumor cells and MYCN expression is significantly reduced in vitro and in vivo. Furthermore, in a mouse xenotransplant-model for MYCN-amplified NB metronomic TPT leads to senescence selectively in tumor cells, complete or partial remission, prolonged survival and a favorable SASP.This new mode-of-action of metronomic TPT treatment, i.e. promoting a tumor-inhibiting type of senescence in MYCN-amplified tumors, is clinically relevant as metronomic regimens are increasingly implemented in therapy protocols of various cancer entities and are considered as a feasible maintenance treatment option with moderate adverse event profiles.