Low-Coverage Whole Genome Sequencing of Cell-Free DNA From Immunosuppressed Cancer Patients Enables Tumor Fraction Determination and Reveals Relevant Copy Number Alterations.

Cell-free DNA (cfDNA) analysis is a minimally invasive method that can be used to detect genomic abnormalities by directly testing a blood sample. This method is particularly useful for immunosuppressed patients, who are at high risk of complications from tissue biopsy. The cfDNA tumor fraction (TF) varies greatly across cancer type and between patients. Thus, the detection of molecular alterations is highly dependent on the circulating TF. In our study, we aimed to calculate the TF and characterize the copy number aberration (CNA) profile of cfDNA from patients with rare malignancies occurring in immunosuppressed environments or immune-privileged sites. To accomplish this, we recruited 36 patients: 19 patients with non-Hodgkin lymphoma (NHL) who were either human immunodeficiency virus (HIV)-positive or organ transplant recipients, 5 HIV-positive lung cancer patients, and 12 patients with glioma. cfDNA was extracted from the patients' plasma and sequenced using low-coverage whole genome sequencing (LC-WGS). The cfDNA TF was then calculated using the ichorCNA bioinformatic algorithm, based on the CNA profile. In parallel, we performed whole exome sequencing of patient tumor tissue and cfDNA samples with detectable TFs. We detected a cfDNA TF in 29% of immune-suppressed patients (one patient with lung cancer and six with systemic NHL), with a TF range from 8 to 70%. In these patients, the events detected in the CNA profile of cfDNA are well-known events associated with NHL and lung cancer. Moreover, cfDNA CNA profile correlated with the CNA profile of matched tumor tissue. No tumor-derived cfDNA was detected in the glioma patients. Our study shows that tumor genetic content is detectable in cfDNA from immunosuppressed patients with advanced NHL or lung cancer. LC-WGS is a time- and cost-effective method that can help select an appropriate strategy for performing extensive molecular analysis of cfDNA. This technique also enables characterization of CNAs in cfDNA when sufficient tumor content is available. Hence, this approach can be used to collect useful molecular information that is relevant to patient care.

TspanC8 tetraspanins differentially regulate ADAM10 endocytosis and half-life.

ADAM10 is a transmembrane metalloprotease that is essential for development and tissue homeostasis. It cleaves the ectodomain of many proteins, including amyloid precursor protein, and plays an essential role in Notch signaling. ADAM10 associates with six members of the tetraspanin superfamily referred to as TspanC8 (Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33), which regulate its exit from the endoplasmic reticulum and its substrate selectivity. We now show that ADAM10, Tspan5, and Tspan15 influence each other's expression level. Notably, ADAM10 undergoes faster endocytosis in the presence of Tspan5 than in the presence of Tspan15, and Tspan15 stabilizes ADAM10 at the cell surface yielding high expression levels. Reciprocally, ADAM10 stabilizes Tspan15 at the cell surface, indicating that it is the Tspan15/ADAM10 complex that is retained at the plasma membrane. Chimeric molecules indicate that the cytoplasmic domains of these tetraspanins contribute to their opposite action on ADAM10 trafficking and Notch signaling. In contrast, an unusual palmitoylation site at the end of Tspan15 C-terminus is dispensable. Together, these findings uncover a new level of ADAM10 regulation by TspanC8 tetraspanins.

Interactions between tumor-associated macrophages and tumor cells in glioblastoma: unraveling promising targeted therapies.

INTRODUCTION: Glioblastoma (GBM) is the deadliest primary malignant central nervous system (CNS) tumor with a median overall survival of 15 months despite a very intensive therapeutic regimen including maximal safe surgery, radiotherapy, and chemotherapy. Therefore, GBM treatment still raises major biological and therapeutic challenges. Areas covered: One of the hallmarks of the GBM is its tumor microenvironment including tumor-associated macrophages (TAM). TAM, accounting for approximately 30% of the GBM bulk cell population, may explain, at least in part, the immunosuppressive features of GBMs. The TAM are active and highly plastic immune cells and include two major ontogenetically different cell populations: (i) microglia and, (ii) monocytes-derived macrophages (MDM). TAM recruited to the tumor bulk can be reprogramed by GBM cells resulting in an ineffective anti-tumor response. Interestingly, interactions between TAM and GBM cells promote tumor oncogenesis (i.e. tumor cells proliferation and migration/invasion). This review aims to explore TAM targeting in GBM as a promising therapeutic option in the near future. Expert Commentary: A better understanding of TAM-GBM interactions and dynamics will certainly uncover new anti-GBM therapeutic avenues.

TspanC8 tetraspanins differentially regulate the cleavage of ADAM10 substrates, Notch activation and ADAM10 membrane compartmentalization.

The metalloprotease ADAM10 mediates the shedding of the ectodomain of various cell membrane proteins, including APP, the precursor of the amyloid peptide Aß, and Notch receptors following ligand binding. ADAM10 associates with the members of an evolutionary conserved subgroup of tetraspanins, referred to as TspanC8, which regulate its exit from the endoplasmic reticulum. Here we show that 4 of these TspanC8 (Tspan5, Tspan14, Tspan15 and Tspan33) which positively regulate ADAM10 surface expression levels differentially impact ADAM10-dependent Notch activation and the cleavage of several ADAM10 substrates, including APP, N-cadherin and CD44. Sucrose gradient fractionation, single molecule tracking and quantitative mass-spectrometry analysis of the repertoire of molecules co-immunoprecipitated with Tspan5, Tspan15 and ADAM10 show that these two tetraspanins differentially regulate ADAM10 membrane compartmentalization. These data represent a unique example where several tetraspanins differentially regulate the function of a common partner protein through a distinct membrane compartmentalization.

Tetraspanins at a glance.

Tetraspanins are a family of proteins with four transmembrane domains that play a role in many aspects of cell biology and physiology; they are also used by several pathogens for infection and regulate cancer progression. Many tetraspanins associate specifically and directly with a limited number of proteins, and also with other tetraspanins, thereby generating a hierarchical network of interactions. Through these interactions, tetraspanins are believed to have a role in cell and membrane compartmentalization. In this Cell Science at a Glance article and the accompanying poster, we describe the basic principles underlying tetraspanin-based assemblies and highlight examples of how tetraspanins regulate the trafficking and function of their partner proteins that are required for the normal development and function of several organs, including, in humans, the eye, the kidney and the immune system.

Breakpoint features of genomic rearrangements in neuroblastoma with unbalanced translocations and chromothripsis.

Neuroblastoma is a pediatric cancer of the peripheral nervous system in which structural chromosome aberrations are emblematic of aggressive tumors. In this study, we performed an in-depth analysis of somatic rearrangements in two neuroblastoma cell lines and two primary tumors using paired-end sequencing of mate-pair libraries and RNA-seq. The cell lines presented with typical genetic alterations of neuroblastoma and the two tumors belong to the group of neuroblastoma exhibiting a profile of chromothripsis. Inter and intra-chromosomal rearrangements were identified in the four samples, allowing in particular characterization of unbalanced translocations at high resolution. Using complementary experiments, we further characterized 51 rearrangements at the base pair resolution that revealed 59 DNA junctions. In a subset of cases, complex rearrangements were observed with templated insertion of fragments of nearby sequences. Although we did not identify known particular motifs in the local environment of the breakpoints, we documented frequent microhomologies at the junctions in both chromothripsis and non-chromothripsis associated breakpoints. RNA-seq experiments confirmed expression of several predicted chimeric genes and genes with disrupted exon structure including ALK, NBAS, FHIT, PTPRD and ODZ4. Our study therefore indicates that both non-homologous end joining-mediated repair and replicative processes may account for genomic rearrangements in neuroblastoma. RNA-seq analysis allows the identification of the subset of abnormal transcripts expressed from genomic rearrangements that may be involved in neuroblastoma oncogenesis.

Characterization of rearrangements involving the ALK gene reveals a novel truncated form associated with tumor aggressiveness in neuroblastoma.

Activating mutations of the ALK gene have been identified in sporadic and familial cases of neuroblastoma (NB), a cancer of the peripheral nervous system, and are thought to be the primary mechanism of oncogenic activation of this receptor in this pediatric neoplasm. To address the possibility that ALK activation may occur through genomic rearrangements as detected in other cancers, we first took advantage of high-resolution array-comparative genomic hybridization to search for ALK rearrangements in NB samples. Using complementary experiments by capture/paired-end sequencing and FISH experiments, various types of rearrangements were fully characterized, including partial gains or amplifications, in several NB cell lines and primary tumors. In the CLB-Bar cell line, we described a genomic rearrangement associated with an amplification of the ALK locus, leading to the expression of a 170 kDa protein lacking part of the extracellular domain encoded by exons 4 to 11, named ALK(Δ4-11). Analysis of genomic DNA from the tumor at diagnosis and relapse revealed that the ALK gene was amplified at diagnosis but that the rearranged ALK allele was observed at the relapse stage only, suggesting that it may be implicated in tumor aggressiveness. Consistently, oncogenic and tumorigenic properties of the ALK(Δ4-11) variant were shown after stable expression in NIH3T3 cells. Moreover, we documented an increased constitutive kinase activity of this variant, as well as an impaired maturation and retention into intracellular compartments. These results indicate that genomic rearrangements constitute an alternative mechanism to ALK point mutations resulting in receptor activation.