Introduction
The current standard to investigate centrosome aberrations on a single cell level is labeling of known centrosomal as well as core centriolar proteins by immunofluorescence (IF) staining ...and subsequent manual quantification of centrosomes and centrioles. This approach is, however, very time-consuming and prone to inter-observer variations. In order to systematically evaluate centrosomal aberrations as potential predictors of malignancy, reliable high throughput analyses of IF images are required. To address this unmet need, we developed a semi-automated workflow using the highly versatile data analysis platform Konstanz Information Miner (KNIME) (Berthold et al., 2008).
Input data
U2OS-PLK4 cells (Konotop et al., Cancer Res. 2016) were induced for centrosomal amplification and immediately processed including IF staining against pericentrin and centrin and image acquisition using a Zeiss Cell Observer and 40× 1.3 NA Plan Apochromat objective. Per condition, 300 cells were observed and allocated manually to the phenotype classes - cells with normal centrosomes, cells with clustered amplified centrosomes and cells with declustered amplified centrosomes. The workflow settings were trained with 20% of the entire data.
KNIME workflow and neural network for centrosome analysis
Our KNIME workflow for centrosome analysis is composed of three main functional parts: (1) Input node groups, where image data is loaded and user-specific settings are pre-defined, (2) the Image Analysis metanode which carries out the central workflow functions and is outlined in the Figure and (3) the output node groups where all data is organized into results data tables and verification views are generated. Briefly, the Image Analysis metanode identifies nuclei, cells and centrosome areas (single or clustered centrosomes) based on thresholding and extracts various features of all objects. The centrosome detection is supposed to be highly sensitive to ensure a low number of false-negative detections. False-positive detections are filtered out or tagged as “uncertain” by a pre-defined set of rules, however, the thresholds used in these rules are adapted automatically based on features of “reference spots”. These are identified centrosome candidates with a high likelihood to be true centrosomes. The workflow structure allows easy adjustment to changing parameters for a broad spectrum of user applications with a similar readout.
To discriminate between cells with normal and amplified centrosomes we used a feed-forward neural network classifier that assigns the cells into these classes by evaluating relevant extracted feature parameters. The network is a multilayer perceptron (layer widths 70, 10, 10, 10, 2) with nonlinear sigmoid activation functions; the output layer carries a softmax activation and yields a probability distribution over the two classes “normal” and “clustered”. The training was performed by maximizing the cross-entropy loss on a dataset of 554 manually-labelled samples, 154 of which were retained for validation.
Comparison of manual and automated quantification
As expected, centrosomal amplification increased upon TET-induction according to manual quantification (clustered 71% vs. 21%, declustered 7.4% vs. 0.3%).
The KNIME workflow was used for feature extraction and to assign the cells into the phenotype class declustered in case of >2 spots per cell. It tagged 43/615 cells (7%) as uncertain and almost all of the remaining cells were labeled in agreement with the manual count (one false-positive and one false-negative).
Subsequently, using the features exported from the KNIME workflow, a neural network was trained to discriminate between normal and clustered amplified centrosomes. The training loss converged to 95%, i.e. 381 of 400 training samples were correctly classified, and on the hitherto unseen validation samples the network correctly classified 88%, i.e. 135 of 154 were labeled in agreement with the manual method.
The combined detection of centrosome amplification by the KNIME workflow and neural network was (TET-induced vs. control): clustered 79% vs. 21%, declustered 5.6% vs. 0.3%.
Conclusions
We present a reliable semi-automated workflow for high throughput analysis of IF images. This tool will be particularly useful for screens of centrosomal aberrations, but can also be easily adjusted for different experimental and infrastructural setups.
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Goldschmidt:Mundipharma: Research Funding; MSD: Research Funding; Amgen: Consultancy, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Dietmar-Hopp-Stiftung: Research Funding; John-Hopkins University: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; John-Hopkins University: Research Funding; Takeda: 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; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Honoraria, Research Funding; Molecular Partners: Research Funding; Janssen: Consultancy, Research Funding. Schönland:Janssen: 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; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Medac: Other: Travel Grant. Krämer:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; BMS: Research Funding.
Plasma cell disorders are clonal outgrowths of pre-malignant or malignant plasma cells, characterized by extensive chromosomal aberrations. Centrosome abnormalities are a major driver of chromosomal ...instability in cancer but their origin, incidence, and composition in primary tumor cells is poorly understood. Using cutting-edge, semi-automated high-throughput electron tomography, we characterized at nanoscale 1386 centrioles in CD138
plasma cells from eight healthy donors and 21 patients with plasma cell disorders, and 722 centrioles from different control populations. In plasma cells from healthy individuals, over-elongated centrioles accumulated with age. In plasma cell disorders, centriole over-elongation was notably frequent in early, pre-malignant disease stages, became less pronounced in overt multiple myeloma, and almost entirely disappeared in aggressive plasma cell leukemia. Centrioles in other types of patient-derived B cell neoplasms showed no over-elongation. In contrast to current belief, centriole length appears to be highly variable in long-lived, healthy plasma cells, and over-elongation and structural aberrations are common in this cell type. Our data suggest that structural centrosome aberrations accumulate with age in healthy CD138
plasma cells and may thus play an important role in early aneuploidization as an oncogenic driver in plasma cell disorders.
Background
Systemic light-chain amyloidosis (AL) is a life-threatening disease caused by the misfolding and deposition of clonal free light-chains (FLC). The current standard of therapy is targeting ...the malignant plasma cells with chemotherapy to reduce the circulating FLC burden.
Daratumumab is a human monoclonal IgG kappa antibody targeting CD38 which is mainly expressed on plasma cells. It has been approved as a monotherapy in the European Union in May 2016 for treatment in patients with multiple myeloma (MM) exposed to proteasome inhibitors (PI) and immunomodulatory drugs (IMID). Sher et al. (Blood 2016) first published a case report with an FLC response in a patient with cardiac AL and a hematologic complete remission (CR) in a patient with renal AL. Kaufman et al. (Blood 2017) reported a hematologic response (>PR) in 19 of 25 pretreated patients with systemic AL.
Patients and Methods
This is a retrospective analysis of 32 consecutive patients treated with daratumumab monotherapy starting from October 2016 for symptomatic AL at our institution. Only patients with an underlying MM (smoldering or CRAB+) were initiated on daratumumab. Hematologic remission and NT-ProBNP response were assessed according to Palladini et al., JCO 2012, renal response according to Palladini et al., Blood 2014. Daratumumab 16 mg/kg of total body weight was applied intravenously after premedication with dexamethasone (commonly 20 mg), intravenous paracetamol and antihistamine. Infusions were initiated weekly in 17 patients and every other week in 15 patients due to reduced performance status. For evaluation of hematologic progression-free survival (PFS) the addition of a second therapeutic agent was also considered as an event. Hematologic PFS was calculated using SPSS v.24.0 statistical software (IBM, Armonk, New York, USA)
Patient cohort: For patient characteristics please refer to the tables .
Results
Hematologic response: Twenty-three of 32 patients achieved a hematologic remission after a median duration of 49 days (range: 5-287). Patients received a median of 8 (range: 2-17) infusions of daratumumab. CR was achieved in 3 patients, a very good partial remission (VGPR) in 14 and a partial remission (PR) in 6 patients (see figure).
The 3 month overall response rate among 27 evaluable patients was 63% with CR in 3 patients, VGPR in 10 patients and PR in 4 patients.
Progression and survival: After a median follow-up time for patients alive of 134 days from the first daratumumab infusion median PFS is not reached (figure). Currently, 21 patients are still on and four patients are off therapy without hematologic progress. One patient died from a cardiac event after discontinuing daratumumab for no response. Another patient with a VGPR died after infectious complications.
Organ response at 3 months: For 14 of 25 patients with cardiac AL NT-ProBNP analysis was performed. Two patients had an NT-ProBNP response and six patients had an NT-ProBNP increase. Renal response assessment was performed in 9 of 15 patients with renal involvement not on dialysis and showed a response in 3 patients and a progression in 3 patients.
Complications: One patient with cardiac AL and terminal renal failure on peritoneal dialysis died after developing peritonitis after 90 days of first daratumumab infusion. Another four patients with cardiac AL were admitted to hospital for lower respiratory tract infections (3) or cardiac decompensation (1). Mild upper respiratory tract infections were reported in six patients. One patient newly developed atrial fibrillation. Two patients with symptomatic MM experienced dyspnoea during the first infusion. One patient with symptomatic MM required a blood transfusion.
Summary
Daratumumab monotherapy can induce fast hematologic responses in heavily pre-treated AL patients who cannot tolerate or are refractory to proteasome inhibitors or immunomodulatory drugs. NT-ProBNP and renal responses can be achieved, although NT-ProBNP progression under therapy was common in our cohort. As expected, patients with advanced cardiac AL are at a higher risk of severe complications.
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Schönland:Sanofi: Research Funding; Janssen: Honoraria, 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, Speakers Bureau; Takeda: Speakers Bureau. Hegenbart:Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Prothena: Membership on an entity's Board of Directors or advisory committees.
Summary
Lenalidomide and dexamethasone (RD) is a standard treatment in relapsed/refractory immunoglobulin light chain (AL) amyloidosis (RRAL). We retrospectively investigated toxicity, efficacy and ...prognostic markers in 260 patients with RRAL. Patients received a median of two prior treatment lines (68% had been bortezomib‐refractory; 33% had received high‐dose melphalan). The median treatment duration was four cycles. The 3‐month haematological response rate was 31% very good haematological response (VGHR) in 18%. The median follow‐up was 56·5 months and the median overall survival (OS) and haematological event‐free survival (haemEFS) were 32 and 9 months. The 2‐year dialysis rate was 15%. VGHR resulted in better OS (62 vs. 26 months, P < 0·001). Cardiac progression predicted worse survival (22 vs. 40 months, P = 0·027), although N‐terminal prohormone of brain natriuretic peptide (NT‐proBNP) increase was frequently observed. Multivariable analysis identified these prognostic factors: NT‐proBNP for OS hazard ratio (HR) 1·71; P < 0·001; gain 1q21 for haemEFS (HR 1·68, P = 0·014), with a trend for OS (HR 1·47, P = 0·084); difference between involved and uninvolved free light chains (dFLC) and light chain isotype for OS (HR 2·22, P < 0·001; HR 1·62, P = 0·016) and haemEFS (HR 1·88, P < 0·001; HR 1·59, P = 0·008). Estimated glomerular filtration rate (HR 0·71, P = 0·004) and 24‐h proteinuria (HR 1·10, P = 0·004) were prognostic for renal survival. In conclusion, clonal and organ biomarkers at baseline identify patients with favourable outcome, while VGHR and cardiac progression define prognosis during RD treatment.
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.
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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.
We investigated whether breast tumor cells can modulate the function of mesenchymal stromal cells (MSCs) with a special emphasis on their chemoattractive activity towards hematopoietic stem and ...progenitor cells (HSPCs). Primary MSCs as well as a MSC line (SCP‐1) were cocultured with primary breast cancer cells, MCF‐7, MDA‐MB231 breast carcinoma or MCF‐10A non‐malignant breast epithelial cells or their conditioned medium. In addition, the frequency of circulating clonogenic hematopoietic progenitors was determined in 78 patients with breast cancer and compared with healthy controls. Gene expression analysis of SCP‐1 cells cultured with MCF‐7 medium revealed CXCL12 (SDF‐1) as one of the most significantly downregulated genes. Supernatant from both MCF‐7 and MDA‐MB231 reduced the CXCL12 promoter activity in SCP‐1 cells to 77% and 47%, respectively. Moreover, the CXCL12 mRNA and protein levels were significantly reduced. As functional consequence of lower CXCL12 levels, we detected a decreased trans‐well migration of HSPCs towards MSC/tumor cell cocultures or conditioned medium. The specificity of this effect was confirmed by blocking studies with the CXCR4 antagonist AMD3100. Downregulation of SP1 and increased miR‐23a levels in MSCs after contact with tumor cell medium as well as enhanced TGFβ1 expression were identified as potential molecular regulators of CXCL12 activity in MSCs. Moreover, we observed a significantly higher frequency of circulating colony‐forming hematopoietic progenitors in patients with breast cancer compared with healthy controls. Our in vitro results propose a potential new mechanism by which disseminated tumor cells in the bone marrow may interfere with hematopoiesis by modulating CXCL12 in protected niches.
What's new?
Disseminated breast tumor cells tend to metastasize to the bones, possibly using similar pathways as hematopoietic stem and progenitor cells (HSPCs) to colonize bone marrow—such as the CXCL12/CXCR4 chemotactic pathway. This study reports a novel mechanism by which breast cancer cells interfere with CXCL12 production by bone marrow‐derived mesenchymal stromal cells in vitro. Such effect reduces the stromal support for CD34+ HSPCs and involves regulation by SP1 transcription factors, miR‐23a, and TGFβ1. The authors also found a higher frequency of circulating colony‐forming hematopoietic progenitors in patients with breast cancer, compared to healthy controls, that may result from this interference.
High-dose melphalan with autologous stem cell transplantation (ASCT) can induce durable haematological and organ responses in systemic AL amyloidosis (AL). Stringent selection criteria have improved ...safety of ASCT in AL but most patients are transplant-ineligible. We report our experience of deferred ASCT in AL patients who were transplant-ineligible at presentation but had improvements in organ function after induction chemotherapy, enabling them to undergo ASCT. Twenty-two AL patients underwent deferred ASCT from 2011 to 2017. All had serial organ function and clonal response assessment. Organ involvement and responses were defined by amyloidosis consensus criteria. All patients were transplant-ineligible at presentation, predominantly due to advanced cardiac involvement. All received bortezomib-based therapy, with 100% haematologic response (86% complete response (CR)/very good partial response (VGPR)), enabling reversal of ASCT exclusion criteria. Patients underwent deferred ASCT for haematologic progression (45%) or consolidation (55%). There was no transplant-related mortality. Haematologic responses post-ASCT: CR 50%, VGPR 27%, PR 18%, non-response 5%. In all, 85.7% achieved cardiac responses. Median overall survival (OS) was not reached. Median progression-free survival (PFS) was 54 months. This selected cohort achieved excellent haematologic responses, organ responses, PFS and OS with deferred ASCT. If larger studies confirm these findings, this may widen the applicability of ASCT in AL.
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