Proteomic profile of melanoma cell-derived small extracellular vesicles in patients' plasma: a potential correlate of melanoma progression.

Molecular profiling of small extracellular vesicles (sEV) isolated from plasma of cancer patients emerges as promising strategy for biomarkers discovery. We investigated the proteomic profiles of sEV immunoselected using anti-CSPG4 antibodies from 15 melanoma patients' plasma. The proteomes of sEV separated into melanoma cell-derived (MTEX) and non-malignant cell-derived (NMTEX) were compared using high-resolution mass spectrometry. Paired analysis identified the MTEX-associated profile of 16 proteins that discriminated MTEX from NMETEX. We also identified the MTEX profile that discriminated between seven patients with no evidence of melanoma (NED) after therapy and eight with progressive disease (PD). Among 75 MTEX proteins overexpressed in PD patients, PDCD6IP (ALIX) had the highest discriminating value, while CNTN1 (contactin-1) was upregulated only in MTEX of NED patients. This is the first report documenting that proteomes of tumour-derived sEV in patients' plasma discriminate cancer from non-cancer and identify proteins with potential to serve as prognostic biomarkers in melanoma.

The mutation profile of differentiated thyroid cancer coexisting with undifferentiated anaplastic cancer resembles that of anaplastic thyroid cancer but not that of archetypal differentiated thyroid cancer.

Differentiated thyroid cancer (DTC) has one of the lowest cancer mutational burdens, while anaplastic thyroid cancer (ATC) has a much higher mutation frequency. A fraction of ATC has an associated differentiated component, which suggests the coevolution of both cancers. Here, we aimed to compare mutation frequency in coexisting ATC and DTC diagnosed concurrently in the same thyroid gland (3 cases) as well as in archetypal DTC and ATC alone (5 cases each). Single-nucleotide variations (SNV) and copy number variations (CNV) were analyzed in each case based on the next-generation sequencing data. We found a similar extent of mutational events, both SNV and CNV, in undifferentiated and differentiated components of thyroid cancers coexisting in one patient. The magnitude of these mutations was comparable to the level of mutations observed in ATC alone; yet, it was much higher than in archetypal DTC. This suggested that, despite histopathological features of differentiated tumors, molecular characteristics of such cancers coexisting with ATC and archetypal DTC could be significantly different. Pairwise comparison of mutational profiles of coexisting cancers enabled assumption on the possible evolution of both components, which appeared distinct in 3 analyzed cases. This included independent development of ATC and DTC diagnosed concurrently in two lobes of the same thyroid, as well as the development of anaplastic and differentiated cancer from the common ancestor that putatively gained a key driver mutation (BRAFV600E or KRASQ61R), which was followed either by early or late molecular separation of both cancers.

MicroRNA Profile of Exosomes and Parental Cells is Differently Affected by Ionizing Radiation.

Exosomes are key mediators of cell-to-cell communication involved in different aspects of the response to ionizing radiation. The functional role of exosomes depends on their molecular cargo, including protein and miRNA content. In this work, we compared the miRNA profile of cells exposed to a high-dose of radiation and the exosomes released by those cells. FaDu cells (derived from human head and neck cancer) were exposed to 2 and 8 Gy doses, exosomes were purified from culture media at 36 h postirradiation using a combination of differential centrifugation, ultrafiltration and precipitation, then microRNA was analyzed using the RNA-seq approach. There were 439 miRNA species quantified, and significant differences in their relative abundance were observed between the cells and exosomes; several low-abundance miRNAs were over-represented while high-abundance miRNA were under-represented in exosomes. There were a few miRNA species markedly affected in irradiated cells and in exosomes released by these cells. However, markedly different radiation-induced effects were observed in both miRNA sets, which could be exemplified by miR-3168 significantly downregulated in cells and upregulated in exosomes. On the other hand, both 2 and 8 Gy radiation doses induced similar effects. Radiation-affected miRNA species present in exosomes are linked to genes involved in the DNA damage and cytokine-mediated response, which may suggest their hypothetical role in the exosome-mediated radiation-induced bystander effect reported elsewhere.

Germline DNA Retention in Murine and Human Rearranged T Cell Receptor Gene Coding Joints: Alternative Recombination Signal Sequences and V(D)J Recombinase Errors.

The genes coding for the antigenic T cell receptor (TR) subunits are assembled in thymocytes from discrete V, D, and J genes by a site-specific recombination process. A tight control of this activity is required to prevent potentially detrimental recombination events. V, D, and J genes are flanked by semi-conserved nucleotide motives called recombination signal sequences (RSSs). V(D)J recombination is initiated by the precise introduction of a DNA double-strand break exactly at the border of the genes and their RSSs by the RAG recombinase. RSSs are therefore physically separated from the coding region of the genes before assembly of a rearranged TR gene. During a high throughput profiling of TRB genes in mice, we identified rearranged TRB genes in which part or all of a flanking RSS was retained in V-D or D-J coding joints. In some instances, this retention of germline DNA resulted from the use of an upstream alternative RSS. However, we also identified TRB sequences where retention of germline DNA occurred in the absence of alternative RSS, suggesting that RAG activity was mis-targeted during recombination. Similar events were also identified in human rearranged TRB and TRG genes. The use of alternative RSSs during V(D)J recombination illustrates the complexity of RAG-RSSs interactions during V(D)J recombination. While the frequency of errors resulting from mis-targeted RAG activity is very low, we believe that these RAG errors may be at the origin of oncogenic translocations and are a threat for genetic stability in developing lymphocytes.

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 [...].

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.

Low-dose radiation accelerates aging of the T-cell receptor repertoire in CBA/Ca mice.

While the biological effects of high-dose-ionizing radiation on human health are well characterized, the consequences of low-dose radiation exposure remain poorly defined, even though they are of major importance for radiological protection. Lymphocytes are very radiosensitive, and radiation-induced health effects may result from immune cell loss and/or immune system impairment. To decipher the mechanisms of effects of low doses, we analyzed the modulation of the T-cell receptor gene repertoire in mice exposed to a single low (0.1 Gy) or high (1 Gy) dose of radiation. High-throughput T-cell receptor gene profiling was used to visualize T-lymphocyte dynamics over time in control and irradiated mice. Radiation exposure induces "aging-like" effects on the T-cell receptor gene repertoire, detectable as early as 1 month post-exposure and for at least 6 months. Surprisingly, these effects are more pronounced in animals exposed to 0.1 Gy than to 1 Gy, where partial correction occurs over time. Importantly, we found that low-dose radiation effects are partially due to the hematopoietic stem cell impairment. Collectively, our findings show that acute low-dose radiation exposure specifically results in long-term alterations of the T-lymphocyte repertoire.