Circulating cell-free DNA fragmentation is a stepwise and conserved process linked to apoptosis.

BACKGROUND: Circulating cell-free DNA (cfDNA) is a pool of short DNA fragments mainly released from apoptotic hematopoietic cells. Nevertheless, the precise physiological process governing the DNA fragmentation and molecular profile of cfDNA remains obscure. To dissect the DNA fragmentation process, we use a human leukemia cell line HL60 undergoing apoptosis to analyze the size distribution of DNA fragments by shallow whole-genome sequencing (sWGS). Meanwhile, we also scrutinize the size profile of plasma cfDNA in 901 healthy human subjects and 38 dogs, as well as 438 patients with six common cancer types by sWGS. RESULTS: Distinct size distribution profiles were observed in the HL60 cell pellet and supernatant, suggesting fragmentation is a stepwise process. Meanwhile, C-end preference was seen in both intracellular and extracellular cfDNA fragments. Moreover, the cfDNA profiles are characteristic and conserved across mammals. Compared with healthy subjects, distinct cfDNA profiles with a higher proportion of short fragments and lower C-end preference were found in cancer patients. CONCLUSIONS: Our study provides new insight into fragmentomics of circulating cfDNA processing, which will be useful for early diagnosis of cancer and surveillance during cancer progression.

Shallow whole-genome sequencing of bone marrow aspirates in myelodysplastic neoplasms: A retrospective comparison with cytogenetics.

Copy number alterations (CNA) are powerful prognostic markers in myelodysplastic neoplasms (MDS) and are routinely analyzed by conventional cytogenetic analysis (CCA) on bone marrow (BM). Although CCA is still the gold standard, it requires extensive hands-on time and highly trained staff for the analysis, making it a laborious technique. To reduce turn-around-time per case, shallow whole genome sequencing (sWGS) technologies offer new perspectives for the diagnostic work-up of this disorder. We compared sWGS with CCA for the detection of CNAs in 33 retrospective BM samples of patients with MDS. Using sWGS, CNAs were detected in all cases and additionally allowed the analysis of three cases for which CCA failed. The prognostic stratification (IPSS-R score) of 27 out of 30 patients was the same with both techniques. In the remaining cases, discrepancies were caused by the presence of balanced translocations escaping sWGS detection in two cases, a subclonal aberration reported with CCA that could not be confirmed by FISH or sWGS, and the presence of an isodicentric chromosome idic(17)(p11) missed by CCA. Since sWGS can almost entirely be automated, our findings indicate that sWGS is valuable in a routine setting validating it as a cost-efficient tool.

Biomarkers of outcome to weekly paclitaxel in epithelial ovarian cancer.

OBJECTIVE: We sought to evaluate the role of intrinsic chromosomal aberrations in determining favorable outcome to weekly paclitaxel (WP) in patients with epithelial ovarian cancer (EOC). METHODS: We evaluated the common genomic aberrations of two patients with EOC and exceptional WP response in the GENIUS study (NCT03740503). We then searched for potential markers of unusual outcomes to WP in a validation cohort. We performed shallow whole genome sequencing (sWGS) in the tumor tissue of women with EOC considered as short-responders (SR; progression with ≤3 cycles) and long-responders (LR; response at ≥8 cycles) to WP monotherapy. RESULTS: We identified two women with exceptional response to WP, lasting over four years, who shared chromosome 8 gain as a common genomic aberration. In order to validate our findings, we reviewed 188 patients with EOC treated with WP and selected 61 women (39 SR, 22 LR) with unusual responses. By sWGS, there was no differential alterations in the copy number changes in chromosome 8, or in genes related to angiogenesis, tubulin superfamily, cell-cycle, apoptosis and paclitaxel metabolism or transportation pathways. Amongst the LR group, we identified six exceptionally long responders (ExLR), with responses lasting over a year. In an exploratory analysis, there was increased amplification of angiogenesis (VEGFB, MMP9), tubulin superfamily (TSC2) and apoptosis related genes (BCL2L1, BAD) in ExLR compared to SR. We identified one patient with a complete response to WP for over 7 years. Molecular profiling identified unique amplifications in interleukin related genes (CXCR1, CXCR2, IL1A, IL1B), not detected in other patients. CONCLUSION: Intrinsic tumor pathways may impact outcome with weekly paclitaxel monotherapy and further investigations are required.

Sequencing Structural Variants in Cancer for Precision Therapeutics.

The identification of mutations that guide therapy selection for patients with cancer is now routine in many clinical centres. The majority of assays used for solid tumour profiling use DNA sequencing to interrogate somatic point mutations because they are relatively easy to identify and interpret. Many cancers, however, including high-grade serous ovarian, oesophageal, and small-cell lung cancer, are driven by somatic structural variants that are not measured by these assays. Therefore, there is currently an unmet need for clinical assays that can cheaply and rapidly profile structural variants in solid tumours. In this review we survey the landscape of 'actionable' structural variants in cancer and identify promising detection strategies based on massively-parallel sequencing.

Shallow whole genome sequencing for robust copy number profiling of formalin-fixed paraffin-embedded breast cancers.

Pathology archives with linked clinical data are an invaluable resource for translational research, with the limitation that most cancer samples are formalin-fixed paraffin-embedded (FFPE) tissues. Therefore, FFPE tissues are an important resource for genomic profiling studies but are under-utilised due to the low amount and quality of extracted nucleic acids. We profiled the copy number landscape of 356 breast cancer patients using DNA extracted FFPE tissues by shallow whole genome sequencing. We generated a total of 491 sequencing libraries from 2 kits and obtained data from 98.4% of libraries with 86.4% being of good quality. We generated libraries from as low as 3.8 ng of input DNA and found that the success was independent of input DNA amount and quality, processing site and age of the fixed tissues. Since copy number alterations (CNA) play a major role in breast cancer, it is imperative that we are able to use FFPE archives and we have shown in this study that sWGS is a robust method to do such profiling.

Dynamic changes in circulating tumor DNA assessed by shallow whole-genome sequencing associate with clinical efficacy of checkpoint inhibitors in NSCLC.

Immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis are the main therapeutic option for patients with advanced non-small cell lung cancer (NSCLC) without a druggable oncogenic alteration. Nevertheless, only a portion of patients benefit from this type of treatment. Here, we assessed the value of shallow whole-genome sequencing (sWGS) on plasma samples to monitor ICI benefit. We applied sWGS on cell-free DNA (cfDNA) extracted from plasma samples of 45 patients with metastatic NSCLC treated with ICIs. Over 150 samples were obtained before ICI treatment initiation and at several time points throughout treatment. From sWGS data, we computed the tumor fraction (TFx) and somatic copy number alteration (SCNA) burden and associated them with ICI benefit and clinical features. TFx at baseline correlated with metastatic lesions at the bone and the liver, and high TFx (≥ 10%) associated with ICI benefit. Moreover, its assessment in on-treatment samples was able to better predict clinical efficacy, regardless of the TFx levels at baseline. Finally, for a subset of patients for whom SCNA burden could be computed, increased burden correlated with diminished benefit following ICI treatment. Thus, our data indicate that the analysis of cfDNA by sWGS enables the monitoring of two potential biomarkers-TFx and SCNA burden-of ICI benefit in a cost-effective manner, facilitating multiple serial-sample analyses. Larger cohorts will be needed to establish its clinical potential.

Shallow Whole-Genome Sequencing of Cell-Free DNA (cfDNA) Detects Epithelial Ovarian Cancer and Predicts Patient Prognosis.

Despite the progress in diagnostics and therapeutics, epithelial ovarian cancer (EOC) remains a fatal disease. Using shallow whole-genome sequencing of plasma cell-free DNA (cfDNA), we investigated biomarkers that could detect EOC and predict survival. Plasma cfDNA from 40 EOC patients and 20 healthy subjects were analyzed by shallow whole-genome sequencing (WGS) to identify copy number variations (CNVs) and determine the Z-scores of genes. In addition, we also calculated the genome-wide scores (Gi scores) to quantify chromosomal instability. We found that the Gi scores could distinguish EOC patients from healthy subjects and identify various EOC histological subtypes (e.g., high-grade serous carcinoma). In addition, we characterized EOC CNVs and demonstrated a relationship between RAB25 amplification (alone or with CA125), and disease-free survival and overall survival. This study identified RAB25 amplification as a predictor of EOC patient survival. Moreover, we showed that Gi scores could detect EOC. These data demonstrated that cfDNA, detected by shallow WGS, represented a potential tool for diagnosing EOC and predicting its prognosis.

Erratum: Aegilops crassa Boiss. repeatome characterized using low-coverage NGS as a source of new FISH markers: application in phylogenetic studies of the Triticeae.

[This corrects the article DOI: 10.3389/fpls.2022.980764.].

Comparative Characterization of Pseudoroegneria libanotica and Pseudoroegneria tauri Based on Their Repeatome Peculiarities.

Pseudoroegneria species play an important role among Triticeae grasses, as they are the putative donors of the St genome in many polyploid species. Satellite repeats are widely used as a reliable tool for tracking evolutionary changes because they are distributed throughout the genomes of plants. The aim of our work is to perform a comparative characterization of the repeatomes of the closely related species Ps. libanotica and Ps. tauri, and Ps. spicata was also included in the analysis. The overall repeatome structures of Ps. libanotica, Ps. tauri, and Ps. spicata were similar, with some individual peculiarities observed in the abundance of the SIRE (Ty1/Copia) retrotransposons, Mutator and Harbinger transposons, and satellites. Nine new satellite repeats that have been identified from the whole-genome sequences of Ps. spicata and Ps. tauri, as well as the CL244 repeat that was previously found in Aegilops crassa, were localized to the chromosomes of Ps. libanotica and Ps. tauri. Four satellite repeats (CL69, CL101, CL119, CL244) demonstrated terminal and/or distal localization, while six repeats (CL82, CL89, CL168, CL185, CL192, CL207) were pericentromeric. Based on the obtained results, it can be assumed that Ps. libanotica and Ps. tauri are closely related species, although they have individual peculiarities in their repeatome structures and patterns of satellite repeat localization on chromosomes. The evolutionary fate of the identified satellite repeats and their related sequences, as well as their distribution on the chromosomes of Triticeae species, are discussed. The newly developed St genome chromosome markers developed in the present research can be useful in population studies of Ps. libanotica and Ps. tauri; auto- and allopolyploids that contain the St genome, such as Thinopyrum, Elymus, Kengyilia, and Roegneria; and wide hybrids between wheat and related wild species.

The feasibility of using liquid biopsies as a complementary assay for copy number aberration profiling in routinely collected paediatric cancer patient samples.

BACKGROUND: Paediatric tumours are often characterised by the presence of recurrent DNA copy number alterations (CNAs). These DNA copy number profiles, obtained from a tissue biopsy, can aid in the correct prognostic classification and therapeutic stratification of several paediatric cancer entities (e.g. MYCN amplification in neuroblastoma) and are part of the routine diagnostic practice. Liquid biopsies (LQBs) offer a potentially safer alternative for such invasive tumour tissue biopsies and can provide deeper insight into tumour heterogeneity. PROCEDURE: The robustness and reliability of LQB CNA analyses was evaluated. We performed retrospective CNA profiling using shallow whole-genome sequencing (sWGS) on paired plasma circulating cell-free DNA (cfDNA) and tissue DNA samples from routinely collected samples from paediatric patients (n = 128) representing different tumour entities, including osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, Wilms tumour, brain tumours and neuroblastoma. RESULTS: Overall, we observed a good concordance between CNAs in tissue DNA and cfDNA. The main cause of CNA discordance was found to be low cfDNA sample quality (i.e. the ratio of cfDNA (<700 bp) and high molecular weight DNA (>700 bp)). Furthermore, CNAs were observed that were present in cfDNA and not in tissue DNA, or vice-versa. In neuroblastoma samples, no false-positives or false-negatives were identified for the detection of the prognostic marker MYCN amplification. CONCLUSION: In future prospective studies, CNA analysis on LQBs that are of sufficient quality can serve as a complementary assay for CNA analysis on tissue biopsies, as either cfDNA or tissue DNA can contain CNAs that cannot be identified in the other biomaterial.

Aegilops crassa Boiss. repeatome characterized using low-coverage NGS as a source of new FISH markers: Application in phylogenetic studies of the Triticeae.

Aegilops crassa Boiss. is polyploid grass species that grows in the eastern part of the Fertile Crescent, Afghanistan, and Middle Asia. It consists of tetraploid (4x) and hexaploid (6x) cytotypes (2n = 4x = 28, D1D (Abdolmalaki et al., 2019) XcrXcr and 2n = 6x = 42, D1D (Abdolmalaki et al., 2019) XcrXcrD2D (Adams and Wendel, 2005), respectively) that are similar morphologically. Although many Aegilops species were used in wheat breeding, the genetic potential of Ae. crassa has not yet been exploited due to its uncertain origin and significant genome modifications. Tetraploid Ae. crassa is thought to be the oldest polyploid Aegilops species, the subgenomes of which still retain some features of its ancient diploid progenitors. The D1 and D2 subgenomes of Ae. crassa were contributed by Aegilops tauschii (2n = 2x = 14, DD), while the Xcr subgenome donor is still unknown. Owing to its ancient origin, Ae. crassa can serve as model for studying genome evolution. Despite this, Ae. crassa is poorly studied genetically and no genome sequences were available for this species. We performed low-coverage genome sequencing of 4x and 6x cytotypes of Ae. crassa, and four Ae. tauschii accessions belonging to different subspecies; diploid wheatgrass Thinopyrum bessarabicum (Jb genome), which is phylogenetically close to D (sub)genome species, was taken as an outgroup. Subsequent data analysis using the pipeline RepeatExplorer2 allowed us to characterize the repeatomes of these species and identify several satellite sequences. Some of these sequences are novel, while others are found to be homologous to already known satellite sequences of Triticeae species. The copy number of satellite repeats in genomes of different species and their subgenome (D1 or Xcr) affinity in Ae. crassa were assessed by means of comparative bioinformatic analysis combined with quantitative PCR (qPCR). Fluorescence in situ hybridization (FISH) was performed to map newly identified satellite repeats on chromosomes of common wheat, Triticum aestivum, 4x and 6x Ae. crassa, Ae. tauschii, and Th. bessarabicum. The new FISH markers can be used in phylogenetic analyses of the Triticeae for chromosome identification and the assessment of their subgenome affinities and for evaluation of genome/chromosome constitution of wide hybrids or polyploid species.

Shallow whole-genome sequencing: a useful, easy to apply molecular technique for CNA detection on FFPE tumor tissue-a glioma-driven study.

Copy number alterations (CNAs) have increasingly become part of the diagnostic algorithm of glial tumors. Alterations such as homozygous deletion of CDKN2A/B, 7 +/ 10 - chromosome copy number changes or EGFR amplification are predictive of a poor prognosis. The codeletion of chromosome arms 1p and 19q, typically associated with oligodendroglioma, implies a more favorable prognosis. Detection of this codeletion by the current diagnostic standard, being fluorescence in situ hybridization (FISH), is sometimes however subject to technical and interpretation problems. In this study, we evaluated CNA detection by shallow whole-genome sequencing (sWGS) as an inexpensive, complementary molecular technique. A cohort of 36 glioma tissue samples, enriched with "difficult" and "ambiguous" cases, was analyzed by sWGS. sWGS results were compared with FISH assays of chromosomes 1p and 19q. In addition, CNAs relevant to glioblastoma diagnosis were explored. In 4/36 samples, EGFR (7p11.2) amplifications and homozygous loss of CDKN2A/B were identified by sWGS. Six out of 8 IDH-wild-type glioblastomas demonstrated a prognostic chromosome 7/chromosome 10 signature. In 11/36 samples, local interstitial and terminal 1p/19q alterations were detected by sWGS, implying that FISH's targeted nature might promote false arm-level extrapolations. In this cohort, differences in overall survival between patients with and without codeletion were better pronounced by the sequencing-based distinction (likelihood ratio of 7.48) in comparison to FISH groupings (likelihood ratio of 0.97 at diagnosis and 1.79 ± 0.62 at reobservation), suggesting sWGS is more accurate than FISH. We recognized adverse effects of tissue block age on FISH signals. In addition, we show how sWGS reveals relevant aberrations beyond the 1p/19q state, such as EGFR amplification, combined gain of chromosome 7 and loss of chromosome 10, and homozygous loss of CDKN2A/B. The findings presented by this study might stimulate implementation of sWGS as a complementary, easy to apply technique for copy number detection.

Shallow whole genome sequencing of adenoid cystic carcinomas of the salivary glands identifies specific chromosomal aberrations related to tumor progression.

BACKGROUND AND PURPOSE: Adenoid cystic carcinomas (ACC) are characterized by high rate of local recurrence and late distant metastasis. Chromosomal changes in the evolution from primary tumors to metastatic disease of ACC have not been appointed. Here we investigated the chromosomal alterations of 53 primary tumors from ACC patients with different progressive states by shallow whole genome sequencing to identify potential new markers for metastatic spread. METHODS: Illumina paired-end libraries were generated using DNA from the primary tumor of 53 ACC patients. Fragmented DNA was end-repaired, A-tailed and multiplex sequencing adapters were ligated. Sequence data were mapped to HG19 and a copy-number analysis was conducted using the QDNAseq R package (version 1.10.0). Outliers were removed and data was smoothed by applying the circular binary segmentation algorithm implemented in the R package copynumber version 1.22.0. A modified chromosomal instability (CNI) score was used to analyze deletions and amplifications. RESULTS: Cluster analysis of the whole genome sequencing revealed that the frequency of chromosomal aberrations were increased in ACC with local recurrence and distant metastases in comparison to ACC patients with no metastatic spread. Specifically, chromosome 6 and 12 and exclusively the entire chromosome 4 showed an increased frequency of chromosomal alterations with tumor progression. CONCLUSION: Our data show a molecular evolution from primary tumors to local recurrences and distant metastases and pinpoint the critical chromosomal regions involved in this process. These regions should be in the focus of the search for therapeutic targets of progressive ACC.

Shallow whole-genome sequencing of plasma cell-free DNA accurately differentiates small from non-small cell lung carcinoma.

BACKGROUND: Accurate lung cancer classification is crucial to guide therapeutic decisions. However, histological subtyping by pathologists requires tumor tissue-a necessity that is often intrinsically associated with procedural difficulties. The analysis of circulating tumor DNA present in minimal-invasive blood samples, referred to as liquid biopsies, could therefore emerge as an attractive alternative. METHODS: Concerning adenocarcinoma, squamous cell carcinoma, and small cell carcinoma, our proof of concept study investigates the potential of liquid biopsy-derived copy number alterations, derived from single-end shallow whole-genome sequencing (coverage 0.1-0.5×), across 51 advanced stage lung cancer patients. RESULTS: Genomic abnormality testing reveals anomalies in 86.3% of the liquid biopsies (16/20 for adenocarcinoma, 13/16 for squamous cell, and 15/15 for small cell carcinoma). We demonstrate that copy number profiles from formalin-fixed paraffin-embedded tumor biopsies are well represented by their liquid equivalent. This is especially valid within the small cell carcinoma group, where paired profiles have an average Pearson correlation of 0.86 (95% CI 0.79-0.93). A predictive model trained with public data, derived from 843 tissue biopsies, shows that liquid biopsies exhibit multiple deviations that reflect histological classification. Most notably, distinguishing small from non-small cell lung cancer is characterized by an area under the curve of 0.98 during receiver operating characteristic analysis. Additionally, we investigated how deeper paired-end sequencing, which will eventually become feasible for routine diagnosis, empowers tumor read enrichment by insert size filtering: for all of the 29 resequenced liquid biopsies, the tumor fraction could be increased in silico, thereby "rescuing" three out of five cases with previously undetectable alterations. CONCLUSIONS: Copy number profiling of cell-free DNA enables histological classification. Since shallow whole-genome sequencing is inexpensive and often fully operational at routine molecular laboratories, this finding has current diagnostic potential, especially for patients with lesions that are difficult to reach.

Characterization of Somatically-Acquired Copy Number Alterations in Chronic Lymphocytic Leukaemia Using Shallow Whole Genome Sequencing.

Shallow whole genome sequencing (sWGS) is a simple, robust, and cost-effective technique recently optimized for the identification of copy number aberrations (CNAs) in tumor samples. This multiplexed methodology sequences 50 bp from one end of the DNA molecule, generating ˜0.1× coverage, and utilizes the observed sequence depth across the genome to infer copy number. It is amenable to low quantities of input DNA, sequencing costs are modest, processing is compatible with low-output instruments, and downstream analysis is simplified by the use of freely available bioinformatics tools and a data analysis package written especially for the analysis of sWGS data. It is the aim of this chapter to introduce the fundamental concepts of sWGS and to provide an overview of the steps involved in a successful sWGS experiment.

Genome-Wide Copy Number Alteration Detection in Preimplantation Genetic Diagnosis.

Shallow whole genome sequencing has recently been introduced for genome-wide detection of chromosomal copy number alterations (CNAs) in preimplantation genetic diagnosis (PGD), using only 4-7 trophectoderm cells biopsied from day-5 embryos. This chapter describes the complete method, starting from whole genome amplification (WGA) on isolated blastomere(s), up to data analysis for CNA detection. The process is described generically and can also be used to perform CNA analysis on a limited number of cells (down to a single cell) in other applications. This unique description also includes some tips and tricks to increase the chance of success.

Cell-Free DNA From Metastatic Pancreatic Neuroendocrine Tumor Patients Contains Tumor-Specific Mutations and Copy Number Variations.

Background: Detection of tumor-specific alterations in cell-free DNA (cfDNA) has proven valuable as a liquid biopsy for several types of cancer. So far, use of cfDNA remains unexplored for pancreatic neuroendocrine tumor (PNET) patients. Methods: From 10 PNET patients, fresh frozen tumor tissue, buffy coat and plasma samples were collected. Whole-exome sequencing of primary tumor and germline DNA was performed to identify tumor-specific variants and copy number variations (CNVs). Subsequently, tumor-specific variants were quantified in plasma cfDNA with droplet digital PCR. In addition, CNV analysis of cfDNA was performed using shallow whole-genome sequencing. Results: Tumor-specific variants were detected in perioperative plasma samples of two PNET patients, at variant allele fractions (VAFs) of respectively 19 and 21%. Both patients had metastatic disease at time of surgery, while the other patients presented with localized disease. In the metastatic patients, CNV profiles of tumor tissue and cfDNA were significantly correlated. A follow-up plasma sample of a metastatic patient demonstrated an increased VAF (57%) and an increased chromosomal instability, in parallel with an increase in tumor burden. Conclusions: We are the first to report the presence of tumor-specific genetic alterations in cfDNA of metastatic PNET patients and their evolution during disease progression. Additionally, CNV analysis in cfDNA shows potential as a liquid biopsy.