Proteomic Exploration of Plasma Exosomes and Other Small Extracellular Vesicles in Pediatric Hodgkin Lymphoma: A Potential Source of Biomarkers for Relapse Occurrence

Exosomes and other small extracellular vesicles (EVs) are potential sources of cancer biomarkers. Plasma-derived EVs have not yet been studied in pediatric Hodgkin lymphoma (HL), for which predictive biomarkers of relapse are greatly needed. In this two-part proteomic study, we used two-dimensional difference gel electrophoresis (2D-DIGE) followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) to analyze EV proteins of plasma collected at diagnosis from children with nodular sclerosis HL, relapsed or not. EVs isolated using membrane affinity had radii ranging from 20 to 130 nm and contained the programmed cell death 6-interacting (ALIX) and the tumor susceptibility gene 101 (TSG101) proteins, whereas calnexin (CANX) was not detected. 2D-DIGE identified 16 spots as differentially abundant between non-relapsed and relapsed HL (|fold change| ≥ 1.5, p < 0.05). LC–MS/MS identified these spots as 11 unique proteins, including five more abundant in non-relapsed HL (e.g., complement C4b, C4B; fibrinogen γ chain, FGG) and six more abundant in relapsed HL (e.g., transthyretin, TTR). Shotgun LC–MS/MS on pooled EV proteins from non-relapsed HL identified 161 proteins, including 127 already identified in human exosomes (ExoCarta data). This EV cargo included 89 proteins not yet identified in exosomes from healthy plasma. Functional interrogation by the Database for Annotation, Visualization and Integrated Discovery (DAVID) revealed that the EV proteins participate in platelet degranulation and serine-type endopeptidase activity as the most significant Gene Ontology (GO) biological process and molecular function (p < 0.01).

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Proteomic Profiles and Biological Processes of Relapsed vs. Non-Relapsed Pediatric Hodgkin Lymphoma

International Journal of Molecular Sciences ◽

10.3390/ijms21062185 ◽

2020 ◽

Vol 21 (6) ◽

pp. 2185

Author(s):

Ombretta Repetto ◽

Valli De Re ◽

Lara Mussolin ◽

Massimo Tedeschi ◽

Caterina Elia ◽

...

Keyword(s):

Hodgkin Lymphoma ◽

Gel Electrophoresis ◽

Predictive Biomarkers ◽

Biological Processes ◽

Label Free ◽

Pediatric Hodgkin Lymphoma ◽

Difference Gel Electrophoresis ◽

Thrombospondin 1 ◽

Serum Paraoxonase ◽

Differentially Abundant Proteins

The identification of circulating proteins associated with relapse in pediatric Hodgkin lymphoma (HL) may help develop predictive biomarkers. We previously identified a set of predictive biomarkers by difference gel electrophoresis. Here we used label-free quantitative liquid chromatography-mass spectrometry (LC-MS/MS) on plasma collected at diagnosis from 12 children (age 12–16 years) with nodular sclerosis HL, including six in whom the disease relapsed within 5 years of treatment in the LH2004 trial. Plasma proteins were pooled in groups of three, separately for non-relapsing and relapsing HL, and differentially abundant proteins between the two disease states were identified by LC-MS/MS in an explorative and validation design. Proteins with a fold change in abundance >1.2 or ≤0.8 were considered “differentially abundant”. LC-MS/MS identified 60 and 32 proteins that were more abundant in non-relapsing and relapsing HL plasma, respectively, in the explorative phase; these numbers were 39 and 34 in the validation phase. In both analyses, 11 proteins were more abundant in non-relapsing HL (e.g., angiotensinogen, serum paraoxonase/arylesterase 1, transthyretin), including two previously identified by difference gel electrophoresis (antithrombin III and α-1-antitrypsin); seven proteins were more abundant in relapsing HL (e.g., fibronectin and thrombospondin-1), including two previously identified proteins (fibrinogen β and γ chains). The differentially abundant proteins participated in numerous biological processes, which were manually grouped into 10 biological classes and 11 biological regulatory subclasses. The biological class Lipid metabolism, and its regulatory subclass, included angiotensinogen and serum paraoxonase/arylesterase 1 (more abundant in non-relapsing HL). The biological classes Immune system and Cell and extracellular matrix architecture included fibronectin and thrombospondin-1 (more abundant in relapsing HL). These findings deepen our understanding of the molecular scenario underlying responses to therapy and provide new evidence about these proteins as possible biomarkers of relapse in pediatric HL.

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The Evolution of Pediatric Hodgkin Lymphoma Therapy: Contemporary Protocols Significantly Reduce Radiation Exposure of Normal Tissues

Klinische Pädiatrie ◽

10.1055/s-0034-1371160 ◽

2014 ◽

Vol 226 (02) ◽

Author(s):

R Zhou ◽

A Ng ◽

R Weathers ◽

A McDonald ◽

W Leisenring ◽

...

Keyword(s):

Radiation Exposure ◽

Hodgkin Lymphoma ◽

Reduce Radiation Exposure ◽

Normal Tissues ◽

Pediatric Hodgkin Lymphoma ◽

Lymphoma Therapy

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Immunological Status of the Host in Aspect of Epstein-Barr Virus Latency in Pediatric Hodgkin Lymphoma

Klinische Pädiatrie ◽

10.1055/s-0034-1371105 ◽

2014 ◽

Vol 226 (02) ◽

Author(s):

T Klekawka ◽

W Balwierz

Keyword(s):

Hodgkin Lymphoma ◽

Epstein Barr Virus ◽

Barr Virus ◽

Immunological Status ◽

Virus Latency ◽

Pediatric Hodgkin Lymphoma ◽

Epstein Barr

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The GATLA (Argentinian Collaborative Group) experience through international cooperation in Pediatric Hodgkin Lymphoma (HL)

10.1055/s-0040-1701830 ◽

2020 ◽

Author(s):

D Veron ◽

P Streitenberger ◽

D Freigeiro ◽

M Castellanos ◽

M Metzger

Keyword(s):

Hodgkin Lymphoma ◽

International Cooperation ◽

Collaborative Group ◽

Pediatric Hodgkin Lymphoma ◽

Group Experience

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Special Drug Use Surveillance for Brentuximab Vedotin Intravenous Infusion "Relapsed or Refractory CD30-positive Peripheral T Cell Lymphoma or Pediatric Hodgkin Lymphoma"

Case Medical Research ◽

10.31525/ct1-nct04213209 ◽

2019 ◽

Author(s):

Keyword(s):

T Cell ◽

Drug Use ◽

Hodgkin Lymphoma ◽

Intravenous Infusion ◽

Cell Lymphoma ◽

Brentuximab Vedotin ◽

T Cell Lymphoma ◽

Peripheral T Cell Lymphoma ◽

Pediatric Hodgkin Lymphoma

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Automated quantification of baseline imaging PET metrics on FDG PET/CT images of pediatric Hodgkin lymphoma patients

EJNMMI Physics ◽

10.1186/s40658-020-00346-3 ◽

2020 ◽

Vol 7 (1) ◽

Author(s):

Amy J. Weisman ◽

Jihyun Kim ◽

Inki Lee ◽

Kathleen M. McCarten ◽

Sandy Kessel ◽

...

Keyword(s):

Hodgkin Lymphoma ◽

Correlation Coefficient ◽

Pet Imaging ◽

Fdg Pet ◽

Relative Difference ◽

Ct Images ◽

Imaging Features ◽

Pediatric Hodgkin Lymphoma ◽

Pet Ct ◽

Fdg Pet Ct

Abstract Purpose For pediatric lymphoma, quantitative FDG PET/CT imaging features such as metabolic tumor volume (MTV) are important for prognosis and risk stratification strategies. However, feature extraction is difficult and time-consuming in cases of high disease burden. The purpose of this study was to fully automate the measurement of PET imaging features in PET/CT images of pediatric lymphoma. Methods 18F-FDG PET/CT baseline images of 100 pediatric Hodgkin lymphoma patients were retrospectively analyzed. Two nuclear medicine physicians identified and segmented FDG avid disease using PET thresholding methods. Both PET and CT images were used as inputs to a three-dimensional patch-based, multi-resolution pathway convolutional neural network architecture, DeepMedic. The model was trained to replicate physician segmentations using an ensemble of three networks trained with 5-fold cross-validation. The maximum SUV (SUVmax), MTV, total lesion glycolysis (TLG), surface-area-to-volume ratio (SA/MTV), and a measure of disease spread (Dmaxpatient) were extracted from the model output. Pearson’s correlation coefficient and relative percent differences were calculated between automated and physician-extracted features. Results Median Dice similarity coefficient of patient contours between automated and physician contours was 0.86 (IQR 0.78–0.91). Automated SUVmax values matched exactly the physician determined values in 81/100 cases, with Pearson’s correlation coefficient (R) of 0.95. Automated MTV was strongly correlated with physician MTV (R = 0.88), though it was slightly underestimated with a median (IQR) relative difference of − 4.3% (− 10.0–5.7%). Agreement of TLG was excellent (R = 0.94), with median (IQR) relative difference of − 0.4% (− 5.2–7.0%). Median relative percent differences were 6.8% (R = 0.91; IQR 1.6–4.3%) for SA/MTV, and 4.5% (R = 0.51; IQR − 7.5–40.9%) for Dmaxpatient, which was the most difficult feature to quantify automatically. Conclusions An automated method using an ensemble of multi-resolution pathway 3D CNNs was able to quantify PET imaging features of lymphoma on baseline FDG PET/CT images with excellent agreement to reference physician PET segmentation. Automated methods with faster throughput for PET quantitation, such as MTV and TLG, show promise in more accessible clinical and research applications.

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