Introduction
Genomic instability is the basic prerequisite for a Darwinian-type evolution of neoplasia and as such represents a fundamental hallmark of cancer. Centrosomal aberrations have been identified as potent drivers of genomic instability (Cosenza et al., Cell Reports 2017; Krämer et al., Leukemia 2003). The current standard to investigate centrosomal aberrations in cancer patients is immunofluorescence (IF) staining. Although this method is fast and easily scalable, its diagnostic significance is controversially discussed. Moreover, ultrastructural analysis of centrosomes in cancer patients is required to gain a mechanistical understanding of the relationship between genomic instability and centrosomal aberrations. To address this, we combined semi-automated analysis of immunofluorescence (IF) images with high-throughput electron tomography (ET) of different cell lines and subentities of primary plasma cell neoplasia, which serve as surrogate for clonal evolution.
Methods
CD138+ plasma cells were isolated from bone marrow aspirates of consenting patients with plasma cell neoplasia. Each sample was split to be subsequently processed for IF and ET. The IF workflow included (1) chemical fixation, (2) staining for nuclei, cells, centrin and pericentrin, (3) semi-automated acquisition of >1000 cells, (4) semi-automated analysis of IF data using the software Konstanz Information Miner (KNIME) (Berthold et al., GfKL 2007). The ET workflow included (1) chemical fixation (2) agarose embedding, (3) dehydration and epoxy resin embedding, (4) serial sectioning at 200 nm, (5) semi-automated screening for centrioles with transmission electron microscopy (TEM) (Schorb et al., Nature Methods 2019), (6) semi-automated acquisition of previously identified centriole regions with serial section ET.
Results
So far, four patients with relapsed refractory myeloma as well as two cell lines (U2OS-PLK4, RPMI.8226) have been screened with TEM. No centrosomal amplification was apparent by IF in any of these patients. Within 5598 cells, 205 centrosomes have been detected. A total of 659 electron tomograms were performed on 141 regions of interest that were distributed on average over five sections. One patient with highly refractory multiple myeloma (resistance to eight prior therapies) showed over-elongated and partially fragmented centrioles (Figure), similar to recently reported findings in tumor cell lines (Marteil et al., Nature Communications 2018). Six out of 10 mother centrioles in this patient were longer than 500 nm, which is supposed to be the physiological length. The dimensions (mean [range]) of mother (decorated with appendages) and daughter centrioles in this patient were: length 919 nm [406 nm - 2620 nm] and 422 nm [367 nm - 476 nm]; diameter 221 nm [99 nm - 470 nm] and 236 nm [178 nm - 450 nm]. Moreover, the mother centrioles showed multiple sets of appendages (mean [range]: 5.9 [2 - 13]), while one set of appendages would be physiological. This is an ongoing study and additional results are expected by the date of presentation.
Conclusions
We present a semi-automated methodological setup that combines high-throughput IF and cutting-edge ET to study centrosomal aberrations. To our knowledge, this is the first study that systematically analyzes the centrosomal phenotype of cancer patients at the ultrastructural level. Our preliminary IF results suggest that supernumerary centrosomes in plasma cell neoplasia might be less common than previously reported. Moreover, we for the first time describe and characterize over-elongated centrioles in myeloma patients, reminiscent of previous findings in tumor cell lines. With increasing numbers of patients, we will be also able to correlate results from IF and ET to address the current uncertainty with respect to IF screens for centrosomal aberrations.
Better insight into centrosomal aberrations will likely increase our understanding on karyotype evolution in plasma cell neoplasia and possibly facilitate the development of novel targeted therapies.
Figure
Disclosures
Goldschmidt: John-Hopkins University: Research Funding; John-Hopkins University: Research Funding; MSD: Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dietmar-Hopp-Stiftung: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Research Funding; Molecular Partners: Research Funding; Janssen: Consultancy, Research Funding; Mundipharma: Research Funding; Chugai: Honoraria, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Müller-Tidow:MSD: Membership on an entity's Board of Directors or advisory committees. Schönland:Medac: Other: Travel Grant; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Krämer:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Daiichi-Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding.
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