Robust detection of translocations in lymphoma FFPE samples using targeted locus capture-based sequencing.

In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens.

TEG001 Insert Integrity from Vector Producer Cells until Medicinal Product.

Despite extensive usage of gene therapy medicinal products (GTMPs) in clinical studies and recent approval of chimeric antigen receptor (CAR) T cell therapy, little information has been made available on the precise molecular characterization and possible variations in terms of insert integrity and vector copy numbers of different GTMPs during the complete production chain. Within this context, we characterize αßT cells engineered to express a defined γδT cell engineered to express a defined γδT receptor (TEG) currently used in a first-in-human clinical study (NTR6541). Utilizing targeted locus amplification in combination with next generation sequencing for the vector producer clone and TEG001 products, we report on five single-nucleotide variants and nine intact vector copies integrated in the producer clone. The vector copy number in TEG001 cells was on average a factor 0.72 (SD 0.11) below that of the producer cell clone. All nucleotide variants were transferred to TEG001 without having an effect on cellular proliferation during extensive in vitro culture. Based on an environmental risk assessment of the five nucleotide variants present in the non-coding viral region of the TEG001 insert, there was no altered environmental impact of TEG001 cells. We conclude that TEG001 cells do not have an increased risk for malignant transformation in vivo.

Lack of major genome instability in tumors of p53 null rats.

Tumorigenesis is often associated with loss of tumor suppressor genes (such as TP53), genomic instability and telomere lengthening. Previously, we generated and characterized a rat p53 knockout model in which the homozygous rats predominantly develop hemangiosarcomas whereas the heterozygous rats mainly develop osteosarcomas. Using genome-wide analyses, we find that the tumors that arise in the heterozygous and homozygous Tp53C273X mutant animals are also different in their genomic instability profiles. While p53 was fully inactivated in both heterozygous and homozygous knockout rats, tumors from homozygous animals show very limited aneuploidy and low degrees of somatic copy number variation as compared to the tumors from heterozygous animals. In addition, complex structural rearrangements such as chromothripsis and breakage-fusion-bridge cycles were never found in tumors from homozygous animals, while these were readily detectable in tumors from heterozygous animals. Finally, we measured telomere length and telomere lengthening pathway activity and found that tumors of homozygous animals have longer telomeres but do not show clear telomerase or alternative lengthening of telomeres (ALT) activity differences as compared to the tumors from heterozygous animals. Taken together, our results demonstrate that host p53 status in this rat p53 knockout model has a large effect on both tumor type and genomic instability characteristics, where full loss of functional p53 is not the main driver of large-scale structural variations. Our results also suggest that chromothripsis primarily occurs under p53 heterozygous rather than p53 null conditions.

Genomic and functional overlap between somatic and germline chromosomal rearrangements.

Genomic rearrangements are a common cause of human congenital abnormalities. However, their origin and consequences are poorly understood. We performed molecular analysis of two patients with congenital disease who carried de novo genomic rearrangements. We found that the rearrangements in both patients hit genes that are recurrently rearranged in cancer (ETV1, FOXP1, and microRNA cluster C19MC) and drive formation of fusion genes similar to those described in cancer. Subsequent analysis of a large set of 552 de novo germline genomic rearrangements underlying congenital disorders revealed enrichment for genes rearranged in cancer and overlap with somatic cancer breakpoints. Breakpoints of common (inherited) germline structural variations also overlap with cancer breakpoints but are depleted for cancer genes. We propose that the same genomic positions are prone to genomic rearrangements in germline and soma but that timing and context of breakage determines whether developmental defects or cancer are promoted.

Extensive localization of long noncoding RNAs to the cytosol and mono- and polyribosomal complexes.

BACKGROUND: Long noncoding RNAs (lncRNAs) form an abundant class of transcripts, but the function of the majority of them remains elusive. While it has been shown that some lncRNAs are bound by ribosomes, it has also been convincingly demonstrated that these transcripts do not code for proteins. To obtain a comprehensive understanding of the extent to which lncRNAs bind ribosomes, we performed systematic RNA sequencing on ribosome-associated RNA pools obtained through ribosomal fractionation and compared the RNA content with nuclear and (non-ribosome bound) cytosolic RNA pools. RESULTS: The RNA composition of the subcellular fractions differs significantly from each other, but lncRNAs are found in all locations. A subset of specific lncRNAs is enriched in the nucleus but surprisingly the majority is enriched in the cytosol and in ribosomal fractions. The ribosomal enriched lncRNAs include H19 and TUG1. CONCLUSIONS: Most studies on lncRNAs have focused on the regulatory function of these transcripts in the nucleus. We demonstrate that only a minority of all lncRNAs are nuclear enriched. Our findings suggest that many lncRNAs may have a function in cytoplasmic processes, and in particular in ribosome complexes.

High-resolution identification of balanced and complex chromosomal rearrangements by 4C technology.

Balanced chromosomal rearrangements can cause disease, but techniques for their rapid and accurate identification are missing. Here we demonstrate that chromatin conformation capture on chip (4C) technology can be used to screen large genomic regions for balanced and complex inversions and translocations at high resolution. The 4C technique can be used to detect breakpoints also in repetitive DNA sequences as it uniquely relies on capturing genomic fragments across the breakpoint. Using 4C, we uncovered LMO3 as a potentially leukemogenic translocation partner of TRB@. We developed multiplex 4C to simultaneously screen for translocation partners of multiple selected loci. We identified unsuspected translocations and complex rearrangements. Furthermore, using 4C we detected translocations even in small subpopulations of cells. This strategy opens avenues for the rapid fine-mapping of cytogenetically identified translocations and inversions, and the efficient screening for balanced rearrangements near candidate loci, even when rearrangements exist only in subpopulations of cells.