Erratum: Cuceu, C., et al. Independent Mechanisms Lead to Genomic Instability in Hodgkin Lymphoma: Microsatellite or Chromosomal Instability. Cancers 2018, 10, 233.

The authors wish to make the following corrections to this paper [...].

Establishment and Characterization of a Reliable Xenograft Model of Hodgkin Lymphoma Suitable for the Study of Tumor Origin and the Design of New Therapies.

To identify the cells responsible for the initiation and maintenance of Hodgkin lymphoma (HL) cells, we have characterized a subpopulation of HL cells grown in vitro and in vivo with the aim of establishing a reliable and robust animal model for HL. To validate our model, we challenged the tumor cells in vivo by injecting the alkylating histone-deacetylase inhibitor, EDO-S101, a salvage regimen for HL patients, into xenografted mice. Methodology: Blood lymphocytes from 50 HL patients and seven HL cell lines were used. Immunohistochemistry, flow cytometry, and cytogenetics analyses were performed. The in vitro and in vivo effects of EDO-S101 were assessed. Results: We have successfully determined conditions for in vitro amplification and characterization of the HL L428-c subline, containing a higher proportion of CD30-/CD15- cells than the parental L428 cell line. This subline displayed excellent clonogenic potential and reliable reproducibility upon xenografting into immunodeficient NOD-SCID-gamma (-/-)(NSG) mice. Using cell sorting, we demonstrate that CD30-/CD15- subpopulations can gain the phenotype of the L428-c cell line in vitro. Moreover, the human cells recovered from the seventh week after injection of L428-c cells into NSG mice were small cells characterized by a high frequency of CD30-/CD15- cells. Cytogenetic analysis demonstrated that they were diploid and showed high telomere instability and telomerase activity. Accordingly, chromosomal instability emerged, as shown by the formation of dicentric chromosomes, ring chromosomes, and breakage/fusion/bridge cycles. Similarly, high telomerase activity and telomere instability were detected in circulating lymphocytes from HL patients. The beneficial effect of the histone-deacetylase inhibitor EDO-S101 as an anti-tumor drug validated our animal model. Conclusion: Our HL animal model requires only 10³ cells and is characterized by a high survival/toxicity ratio and high reproducibility. Moreover, the cells that engraft in mice are characterized by a high frequency of small CD30-/CD15- cells exhibiting high telomerase activity and telomere dysfunction.

Independent Mechanisms Lead to Genomic Instability in Hodgkin Lymphoma: Microsatellite or Chromosomal Instability †.

Background: Microsatellite and chromosomal instability have been investigated in Hodgkin lymphoma (HL). Materials and Methods: We studied seven HL cell lines (five Nodular Sclerosis (NS) and two Mixed Cellularity (MC)) and patient peripheral blood lymphocytes (100 NS-HL and 23 MC-HL). Microsatellite instability (MSI) was assessed by PCR. Chromosomal instability and telomere dysfunction were investigated by FISH. DNA repair mechanisms were studied by transcriptomic and molecular approaches. Results: In the cell lines, we observed high MSI in L428 (4/5), KMH2, and HDLM2 (3/5), low MSI in L540, L591, and SUP-HD1, and none in L1236. NS-HL cell lines showed telomere shortening, associated with alterations of nuclear shape. Small cells were characterized by telomere loss and deletion, leading to chromosomal fusion, large nucleoplasmic bridges, and breakage/fusion/bridge (B/F/B) cycles, leading to chromosomal instability. The MC-HL cell lines showed substantial heterogeneity of telomere length. Intrachromosmal double strand breaks induced dicentric chromosome formation, high levels of micronucleus formation, and small nucleoplasmic bridges. B/F/B cycles induced complex chromosomal rearrangements. We observed a similar pattern in circulating lymphocytes of NS-HL and MC-HL patients. Transcriptome analysis confirmed the differences in the DNA repair pathways between the NS and MC cell lines. In addition, the NS-HL cell lines were radiosensitive and the MC-cell lines resistant to apoptosis after radiation exposure. Conclusions: In mononuclear NS-HL cells, loss of telomere integrity may present the first step in the ongoing process of chromosomal instability. Here, we identified, MSI as an additional mechanism for genomic instability in HL.

The Transition between Telomerase and ALT Mechanisms in Hodgkin Lymphoma and Its Predictive Value in Clinical Outcomes.

Background: We analyzed telomere maintenance mechanisms (TMMs) in lymph node samples from HL patients treated with standard therapy. The TMMs correlated with clinical outcomes of patients. Materials and Methods: Lymph node biopsies obtained from 38 HL patients and 24 patients with lymphadenitis were included in this study. Seven HL cell lines were used as in vitro models. Telomerase activity (TA) was assessed by TRAP assay and verified through hTERT immunofluorescence expression; alternative telomere lengthening (ALT) was also assessed, along with EBV status. Results: Both TA and ALT mechanisms were present in HL lymph nodes. Our findings were reproduced in HL cell lines. The highest levels of TA were expressed in CD30-/CD15- cells. Small cells were identified with ALT and TA. Hodgkin and Reed Sternberg cells contained high levels of PML bodies, but had very low hTERT expression. There was a significant correlation between overall survival (p < 10-3), event-free survival (p < 10-4), and freedom from progression (p < 10-3) and the presence of an ALT profile in lymph nodes of EBV+ patients. Conclusion: The presence of both types of TMMs in HL lymph nodes and in HL cell lines has not previously been reported. TMMs correlate with the treatment outcome of EBV+ HL patients.

Retrospective cohort study and biobanking of patients treated for hemangioma in childhood - telomeres as biomarker of aging and radiation exposure.

PURPOSE: Cohorts allowing joint epidemiological and biological analyses are essential for radiation risk assessment. The French Hemangioma Cohort (FHC), studied within the European project EpiRadBio, is one of the rare cohorts suitable for studying the effect of low dose radiation exposure (<100 mGy at organs), with a long-term follow-up. This highly homogeneous cohort consists of healthy individuals belonging to a normal population, except for the presence of skin hemangioma (age at exposure: between 6 months and 3 years of age). Published epidemiological studies have demonstrated that the risk of developing cancer is three times higher in the exposed individuals than in the general population. Here, we present the biobanking of samples (nucleated blood cells, cytogenetic slides of T and B lymphocytes) from the FHC and a primary feasibility study of biomarker analysis focusing on mean telomere length (MTL). Telomeres act as an internal clock, regulating the lifetime of the cell by their shortening during cell division. MTL is thus a biomarker of age. Many in vitro studies have linked MTL and radiosensitivity. The FHC will make it possible to discriminate between the effects of aging and radiation on this biomarker. CONCLUSION: The establishment of a biobank of essentially healthy individuals (369 in total), exposed 40-70 years before, during their early childhood, is a logistical challenge. Even among those who previously participated to a self-questionnaire based study, the response rate was only 30%. The first biomarker to be studied was the MTL to discriminate age effects from those of radiation exposure. MTL showed significant variation within age groups (4-11 kb) in both the exposed and non-exposed groups. MTL within the limited age window (i.e. 40-73 year) examined, showed age-dependent changes of 46 bp/year, consistent with the age-dependent decline of 41 bp/year previously reported. We observed no significant changes in MTL according to the average active bone marrow dose. However, we were able to demonstrate that exposure to radiation causes the loss of cells with, on average, shorter telomeres, by applying a model in which both the heterogeneity of the individual dose received at the bone marrow and the heterogeneity of the intercellular distribution of MTL were taken into account.

Proteome alterations in rat mitochondria caused by aging.

Analysis of the protein profile of mitochondria and its age-dependent variation is a promising approach to unravel mechanisms involved in aging and age-related diseases. Our studies focus on the mammalian mitochondrial membrane proteome, especially of the inner mitochondrial membrane with the respiratory chain complexes and other proteins possibly involved in life-span control and aging. Variations of the mitochondrial proteome during aging, with the emphasis on the abundance, composition, structure, and activity of membrane proteins, are examined in various rat tissues by native polyacrylamide gel electrophoresis techniques in combination with MALDI-TOF mass spectrometry. In rat brain, age-modulated differences in the abundance of various mitochondrial and nonmitochondrial proteins, such as Na,K-ATPase, HSP60, mitochondrial aconitase-2, V-type ATPase, MF(o)F(1) ATP synthase, and the OXPHOS complexes I-IV are detected. During aging, a decrease in the amount of intact MF(o)F(1) ATP synthase occurs in the cortex. As analytical technique, native PAGE separates not only individual proteins but also multi-subunit (membrane) proteins, (membrane) protein supercomplexes as well as interacting proteins in their native state. It reveals the occurrence and architecture of supramolecular assemblies of proteins. The age-related alterations in the oligomerization of the MF(o)F(1) ATP synthase observed by us in rat cortex might be one clue for understanding the link between respiration and longevity. Also, the abundance of OXPHOS supercomplexes, that is, the natural assemblies of the respiratory complexes I, III, and IV into supramolecular stoichiometric entities, such as I(1)III(2)IV(0-4), can differ between young and aged cortex tissue. Age-related changes in the supramolecular architecture of OXPHOS complexes might explain alterations in ROS production during aging.