Introduction
Immune-checkpoint blockade has emerged as an effective therapeutic strategy in solid tumor and in hematologic malignancies, including classical Hodgkin Lymphoma (cHL).
cHL represents ...about 11% of all malignant lymphoma and it is generally highly curable with standard frontline therapies, although about 20% of the patients will relapse or become refractory after initial treatment.
The hallmark of cHL is the presence of malignant Hodgkin and Reed-Sternberg Cells (HRS) that represent only a small fraction (about 1%) of the surrounding heterogeneous immune infiltrate. Despite this extensive inflammatory microenvironment, HRS are able to escape immune surveillance using several mechanisms, including the overexpression of PD-1 ligands (PD-Ls) that bind PD-1 on reactive T-cells, inhibiting their activity and proliferation and causing ultimately T-cell exhaustion. The PD-Ls expression is upregulated in a dose-dependent manner by copy number alterations of chromosome 9p24.1, a locus encoding for PD-L1/PD-L2 as well as JAK2, which further enhances PD-Ls expression through JAK2/STAT pathway.
Here we present a method for the isolation and the genetic characterization of single purified HRS, which overcomes the limitations posed by the low tumor cellularity of cHL biopsies and gives an estimation of inter-tumor and intra-tumor heterogeneity which may be useful to guide immune treatment selection.
Methods
FFPE tissue sections from 4 cHL patients were dissociated down to single-cell suspension and stained using anti-CD30 and anti-PD-L1 antibodies. Since CD30 is not expressed exclusively by malignant cells, beyond the positivity to CD30 and PD-L1 HRS were selected according to morphological criteria, such as cell size and the presence of nuclei with ploidy higher than the surrounding lymphocytes.
DEPArray™ NxT system (Menarini Silicon Biosystems) was used to isolate single target cells. After recovery, single cells were whole genome amplified (Ampli1™ WGA, Menarini Silicon Biosystems), and genome-wide copy-number alterations (CNAs) profiles were obtained using Ampli1™ LowPass kits (Menarini Silicon Biosystems) on Illumina® and Ion Torrent™ platforms.
Results
For each patient, at least 8 HRS cells and infiltrating lymphocytes were identified and isolated from lymphoid tissue using DEPArray™ NxT system.
Copy-number analyses of recovered cells allowed us to precisely discriminate HRS, characterized by extensive gains and losses, from non-tumor cells, showing flat profiles as expected (Fig.1). Ploidy of HRS was automatically determined, based on best-fitting of profiles with underlying copy number levels.
Hierarchical clustering showed that some alterations are highly conserved among patients, e.g. the region containing PD-L1/PD-L2/JAK2 has several copy gains in the majority of malignant cells. Interestingly, these alterations show high variable copy-number levels between different HRS even in the same patient, ranging from few copy-gains to amplifications, suggesting some level of heterogeneity.
Different CNAs are also detected in regions containing genes belonging to pathways already known to be altered in cHL, like REL/NFKB and JAK/STAT pathways, which may be involved in the constitutive activation of proliferative and antiapoptotic phenotype of HRS.
Conclusion
Single HRS sorting combined with low-pass whole genome sequencing offer a valuable tool to uncover genetic alterations hidden by the massive cHL immune infiltrate and to estimate inter-tumor and intra-tumor heterogeneity in cHL patients. Considering that PD-Ls locus amplifications are associated with advanced stages of the disease and with a shorter progression free survival, the analysis of purified HRS could be helpful for patient stratification for the adoption of immune therapy.
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Mangano:Menarini Silicon Biosystems: Employment. Edoardo:Menarini Silicon Biosystems: Employment. Garonzi:Menarini Silicon Biosystems: Employment. Lanzellotto:Menarini Silicon Biosystems: Employment. Papadopulos:Menarini Silicon Biosystems: Employment. Bolognesi:Menarini Silicon Biosystems: Employment. Buson:Menarini Silicon Biosystems: Employment. Ferrarini:Menarini Silicon Biosystems: Employment. Forcato:Menarini Silicon Biosystems: Employment. Fontana:Menarini Silicon Biosystems: Employment. Ceccolini:Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Fabbri:Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Fici:Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Gallerani:Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Simonelli:Menarini Silicon Biosystems: Employment. Medoro:Menarini Silicon Biosystems: Employment. Manaresi:Menarini Silicon Biosystems: Employment.
Introduction: Multiple myeloma (MM) is a malignancy of terminally differentiated plasma cells. The high heterogeneity of MM cells is one of the major cause of disease relapse. Detection of ...circulating MM cells (CMMC) from peripheral blood is a useful procedure to investigate tumor heterogeneity and provides a painless alternative to the classic bone marrow biopsy to monitor disease progression. Here we demonstrate that the synergy between CellSearch® (CS) and DEPArray™ (DA) technologies can be used to identify, isolate and characterize at the genetic level single and pure CMMCs .
Methods: 4.0 ml of peripheral blood samples were obtained from 3 patients with MM. Putative CMMCs were enriched with CS using anti-CD138 or anti-CD138/CD38 as positive selection marker and subsequently stained with CD38-PE, CD19/CD45-APC immunofluorescent probes. Cells detection and enumeration was performed based on the co-localization of nuclei DAPI staining and CD38-PE. Single CMMCs (CD38+/CD19- and CD45-/DAPI+) and White Blood Cells (WBCs: CD38-/CD19+ or CD45+/DAPI+) were then isolated using the DA NxT system. Single cells genomic DNA was amplified using Ampli1™ Whole Genome Amplification (WGA) kit and Illumina®-compatible libraries were obtained using Ampli1™ LowPass kit and a high-throughput, customized automated protocol using Hamilton STARLet Liquid handler. Highly-multiplexed, genome-wide single-cell Low-Pass Copy Number Alteration (LPCNA) analysis was performed using HiSeq 2500 Illumina® platform.
Results: CS and DA workflow* enabled the isolation of 215 single CMMC, selected for LPCNA analysis. 42 single WBCs were also included as normal controls. Copy-number profiles of single CMMCs showed relevant gains and losses of chromosomal segments, as result of a high-level genomic instability. Notably, intra-patient CMMCs revealed overall conserved CNA patterns with subclonal alterations, suggesting a certain level of branched tumor evolution. Conversely, a higher degree of heterogeneity in CMMCs CNA profiles was observed among different patients. Interestingly, CNAs detected in all patients are located in regions containing genes involved in cell cycle regulation (MAPK, NOTCH pathways) and cell signaling (IL6R), which might be involved in proliferative processes and immuno-surveillance escape.
Conclusion: The combination of CS and DA workflow* with a streamlined automated protocol allowed to obtain hundreds of genomic libraries from pure single CMMCs. The presented workflow constitutes a non-invasive, rapid and high-throughput approach for characterizing MM tumor heterogeneity and progression, suggesting a possible future implementation in clinical applications.
*For Research Use Only. Not for use in diagnostic procedures.
Raspadori:Menarini Silicon Biosystems: Employment. Forcato:Menarini Silicon Biosystems: Employment. Edoardo:Menarini Silicon Biosystems: Employment. Papadopulos:Menarini Silicon Biosystems: Employment. Ferrarini:Menarini Silicon Biosystems: Employment. Del Monaco:Menarini Silicon Biosystems: Employment. Terracciano:Menarini Silicon Biosystems: Employment. Morano:Menarini Silicon Biosystems: Employment. Gross:Menarini Silicon Biosystems: Employment. Bolognesi:Menarini Silicon Biosystems: Employment. Buson:Menarini Silicon Biosystems: Employment. Fontana:Menarini Silicon Biosystems: Employment. Connelly:Menarini Silicon Biosystems, Inc.: Employment, Other: Chief R&D Officer, USA. Simonelli:Menarini Silicon Biosystems: Employment. Medoro:Menarini Silicon Biosystems: Employment. Manaresi:Menarini Silicon Biosystems: Employment.
Critical lower limb ischemia is a severe disease. A common approach is infrainguinal bypass. Synthetic vascular prosthesis, are good conduits in high-flow low-resistance conditions but have ...difficulty in their performance as small diameter vessel grafts. A new approach is the use of native decellularized vascular tissues. Cell-free vessels are expected to have improved biocompatibility when compared to synthetic and are optimal natural 3D matrix templates for driving stem cell growth and tissue assembly in vivo. Decellularization of tissues represent a promising field for regenerative medicine, with the aim to develop a methodology to obtain small-diameter allografts to be used as a natural scaffold suited for in vivo cell growth and pseudo-tissue assembly, eliminating failure caused from immune response activation. Material and methods. Umbilical cord-derived mesenchymal cells isolated from human umbilical cord tissue were expanded in advanced DMEM. Immunofluorescence and molecular characterization revealed a stem cell profile. A non-enzymatic protocol, that associate hypotonic shock and low-concentration ionic detergent, was used to decellularize vessel segments. Cells were seeded cell-free scaffolds using a compound of fibrin and thrombin and incubated in DMEM, after 4 days of static culture they were placed for 2 weeks in a flow-bioreactor, mimicking the cardiovascular pulsatile flow. After dynamic culture, samples were processed for histological, biochemical and ultrastructural analysis. Discussion. Histology showed that the dynamic culture cells initiate to penetrate the extracellular matrix scaffold and to produce components of the ECM, as collagen fibres. Sirius Red staining showed layers of immature collagen type III and ultrastructural analysis revealed 30 nm thick collagen fibres, presumably corresponding to the immature collagen. These data confirm the ability of cord-derived cells to adhere and penetrate a natural decellularized tissue and to start to assembly into new tissue. This achievement makes natural 3D matrix templates prospectively valuable candidates for clinical bypass procedures
L’ischemia critica degli arti inferiori è una condizione morbosa che riduce gravemente il flusso sanguigno e mette a repentaglio la conservabilità dell’arto colpito. Il trattamento standard è rappresentato dal bypass femoro-popliteo. Per questo tipo di rivascolarizzazione ci si avvale di protesi sintetiche o di graft in vena. Un approccio alternativo è l'uso di tessuti vascolari nativi decellularizzati, dotati di maggiore biocompatibilità rispetto agli altri e presumibilmente ottimi modelli 3D per guidare la crescita di cellule staminali. Lo scopo di questa ricerca è sviluppare una metodologia per ottenere allograft di piccolo calibro cell-free da utilizzare come scaffold naturali compatibili con la crescita cellulare in vivo e il loro assemblaggio in pseudo-tessuti, eliminando fallimenti dovuti all’attivazione della risposta immunitaria. Materiali e metodi: Abbiamo isolato dal cordone ombelicale umano cellule mesenchimali. La caratterizzazione con tecniche di immunofluorescenza e molecolari ha rivelato un profilo staminale. La decellularizzazione di segmenti vascolari è stata effettuata utilizzando un protocollo, che associa shock ipotonico e l’utilizzo di detergenti ionici. Le cellule sono state seminate su cell-free scaffold utilizzando come carrier fibrina e trombina, dopo 4 giorni di coltura statica in DMEM sono stati posti per 2 settimane in un bireattore a flusso che mima il fisiologico flusso pulsatile cardiovascolare. Al termine della coltura dinamica, i campioni sono stati processati per analisi istologiche, biochimiche e ultrastrutturali. Risultati e discussione: L’analisi istologica ha mostrato una propensione delle cellule a aderire e penetrare nel tessuto. Inoltre la colorazione Sirius Red ha rivelato la neoproduzione fibre di collagene III dato confermato dall'analisi ultrastrutturale dalla quale è emersa la presenza di fibre di collagene di 30 nm, presumibilmente corrispondente al collagene immaturo. Questi dati confermano la capacità delle cellule ombelicali di ricellularizzare tessuti nativi cell-free. Questo risultato rende gli scaffold naturali decellularizzati validi candidati per interventi di bypass.
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