Shallow Whole Genome Sequencing Research Articles

Detecting cell-of-origin and cancer-specific methylation features of cell-free DNA from Nanopore sequencing

The Oxford Nanopore (ONT) platform provides portable and rapid genome sequencing, and its ability to natively profile DNA methylation without complex sample processing is attractive for point-of-care real-time sequencing. We recently demonstrated ONT shallow whole-genome sequencing to detect copy number alterations (CNAs) from the circulating tumor DNA (ctDNA) of cancer patients. Here, we show that cell type and cancer-specific methylation changes can also be detected, as well as cancer-associated fragmentation signatures. This feasibility study suggests that ONT shallow WGS could be a powerful tool for liquid biopsy.Graphical

Pan-Cancer Detection and Typing by Mining Patterns in Large Genome-Wide Cell-Free DNA Sequencing Datasets.

Cell-free DNA (cfDNA) analysis holds great promise for non-invasive cancer screening, diagnosis, and monitoring. We hypothesized that mining the patterns of cfDNA shallow whole-genome sequencing datasets from patients with cancer could improve cancer detection. By applying unsupervised clustering and supervised machine learning on large cfDNA shallow whole-genome sequencing datasets from healthy individuals (n = 367) and patients with different hematological (n = 238) and solid malignancies (n = 320), we identified cfDNA signatures that enabled cancer detection and typing. Unsupervised clustering revealed cancer type-specific sub-grouping. Classification using a supervised machine learning model yielded accuracies of 96% and 65% in discriminating hematological and solid malignancies from healthy controls, respectively. The accuracy of disease type prediction was 85% and 70% for the hematological and solid cancers, respectively. The potential utility of managing a specific cancer was demonstrated by classifying benign from invasive and borderline adnexal masses with an area under the curve of 0.87 and 0.74, respectively. This approach provides a generic analytical strategy for non-invasive pan-cancer detection and cancer type prediction.

Abstract 3625: Clonal architecture and evolution of treatment-resistant prostate cancer via deep whole-genome ctDNA sequencing

Abstract Background: Circulating tumor DNA (ctDNA) in blood plasma is an emerging tool for clinical cancer genotyping and longitudinal disease monitoring. However, integration of ctDNA tests into clinical management is critically impeded by the poor understanding of the distinct somatic populations comprising bulk ctDNA—including their relationship to synchronous metastatic tissue, and their temporal dynamics during standard-of-care treatment. Prior approaches relying on targeted and/or low-resolution techniques (e.g. targeted exon sequencing, low-pass (shallow) whole-genome sequencing; WGS) do not permit comprehensive dissection of clonal architecture and unbiased analysis of putative resistance mechanisms. Methods: We performed deep WGS on serial plasma ctDNA (median depth: 185×) and synchronous metastatic tissue biopsies with high tumor purity from 35 patients with metastatic castration-resistant prostate cancer. We developed a subclonal reconstruction algorithm optimized for our data enabling resolution of per-patient evolutionary histories and ctDNA clonal composition. ctDNA nucleosome footprinting was used to infer mRNA abundance in synchronously biopsied metastases and androgen receptor (AR) transcription factor activity at 3224 AR binding sites (ARBS). Results: We comprehensively assess all classes of genomic alterations and demonstrate that ctDNA harbors greater populational heterogeneity than metastatic tissue (p<0.001). The evolutionary histories of ctDNA populations indicate frequent whole-genome doubling and attenuation of C>T aging-associated mutation signature during subclonal differentiation. Although driver alterations were largely concordant between tissue and ctDNA, each individual metastasis contributed only a minor share of total ctDNA (average ctDNA contribution: 17%). By comparing serial ctDNA before and after clinical progression on potent AR pathway inhibitors, we reveal population restructuring converging solely on AR copy augmentation as the dominant genomic driver of acquired treatment-resistance. Nucleosome depletion at transcription start-sites is highly correlated with same-patient metastatic tissue mRNA abundance, indicating that ctDNA fragmentomics can recapitulate transcriptomic patterns in metastatic lesions. Most ctDNA samples exhibited strong ARBS nucleosome depletion which correlated with AR gene copy number (R=0.36, p=0.003). Finally, serial ctDNA nucleosome profiling at ARBS revealed adaptive transcriptomic resistance to AR pathway inhibitors, including lineage switch to a neuroendocrine-like (AR-low) state. Conclusions: We show that the populations comprising ctDNA are typically complex and more heterogeneous than those found in bulk WGS of a synchronous metastasis. Our work advocates for liquid biopsy as a comprehensive multi-omic discovery tool for cancers with high ctDNA fractions. Citation Format: Cameron Herberts, Matti Annala, Joonatan Sipola, Sarah W. Ng, Xinyi E. Chen, Anssi Nurminen, Olga Korhonen, Aslı D. Munzur, Kevin Beja, Elena Schönlau, Cecily Q. Bernales, Elie Ritch, Jack V. Bacon, Nathan A. Lack, Matti Nykter, Rahul Aggarwal, Eric J. Small, Martin E. Gleave, David A. Quigley, Felix Y. Feng, Kim N. Chi, Alexander W. Wyatt. Clonal architecture and evolution of treatment-resistant prostate cancer via deep whole-genome ctDNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3625.

Abstract 1967: Potential of aqueous humor as a liquid biopsy for uveal melanoma

Abstract Introduction: Uveal melanoma (UM) is the most common primary intraocular tumor in adults and arises from the uvea (iris, ciliary body and choroid). Although there are prognostic biomarkers available, identifying them requires intraocular tumor biopsy. Blood biopsy has emerged as a significantly less invasive alternative that also offers the ability to longitudinally track disease. However, blood biopsy has been unsuccessful for clinical use in identifying prognostic biomarkers in UM due to low detection rates. Thus, eye-specific aqueous humor (AH) biopsy may be a better alternative. Methods: This case series includes 20 primary UM patients with 13 (65%) choroid and 7 (35%) ciliary body (CB) tumors. AH samples were collected prior to brachytherapy plaque placement (n = 17), after brachytherapy (n = 19) and at enucleation (n = 1). A tumor wash sample (n = 1) was collected after routine fine needle aspiration biopsy. Nucleic acids ([NA], dsDNA, RNA, ssDNA and miRNA) were quantitated by Qubit Assay Kits. All AH samples and cells from tumor washing underwent DNA isolation and was followed by shallow whole-genome sequencing for copy-number profiling. Results: Comparison of pre- and post-radiation samples revealed significantly higher concentration of ssDNA (n = 10 pairs, P = 0.039) and miRNA (n = 10 pairs, P = 0.008) in choroid post- samples and significantly higher concentration of dsDNA (n = 7 pairs, P = 0.031) and miRNA (n = 7 pairs, P = 0.016) in CB post- samples. Between choroid (n = 12) and CB (n = 7) post- samples, there was a significantly higher concentration of dsDNA (P = 0.022) and RNA (P = 0.009) in post- CB samples, suggesting NA content is released from the disturbance of tumor cells by radiation and due to the proximity of the tumor to the AH in CB. Highly recurrent UM somatic copy number alterations (SCNAs) of monosomy 3, 6p gain, 6q loss and 8q gain were identified in post- AH samples. There was a significant difference between altered SCNA profiles in CB and choroid (P= 0.038). Altered SCNA profiles were found in 1/12 (8.3%) and 4/7 (57.1%) post-radiation AH samples of choroid and CB, respectively. No SCNAs were found in the same eyes’ pre-radiation sample. A high concordance of DNA profiles between a tumor wash and post- AH sample was identified (r = 0.978) and UM mutations GNAQ and BAP1 were detected in 3/6 (50%) post- AH with 2/5 (40%) matching tumor samples, demonstrating AH dsDNA’s tumor-origin. Conclusions: The AH is a source of cfDNA, with a higher yield of NAs after radiation in UM. Given the significant increase in NA concentration after radiation and in CB compared to choroid tumors, we hypothesize that all detected NAs post-radiation are tumor-derived due to apoptosis of tumor cells and subsequent release of cfDNA and due to the proximity of the CB tumor to AH. These results represent the first time that UM SCNAs and mutations were identified in cfDNA isolated from the AH, and this suggests that the AH can serve as a liquid biopsy for UM, especially in CB tumors. Citation Format: Deborah H. Im, Chen-Ching Peng, Liya Xu, Mary E. Kim, Dejerianne Ostrow, Venkata Yellapantula, Moiz Bootwalla, Jaclyn A. Biegel, Xiaowu Gai, Peter Kuhn, James Hicks, Jesse L. Berry. Potential of aqueous humor as a liquid biopsy for uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1967.

Abstract 5263: Whole-genome sequencing as an alternative to analyze copy number abnormalities in acute myeloid leukemia and myelodysplastic syndrome

Abstract Genomic alternations especially copy number aberrations (CNAs) are particularly imperative for diagnostic classification and risk stratification in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). Although conventional cytogenetic analysis (CCA) has been mandatory in clinical routine to detect recurrent genomic abnormalities in AML/MDS, it presents a few obvious shortcomings, such as time-consuming in vitro cell-culturing procedure, low resolution (no smaller than 10 Mbp), and subjective bias introduced from the visual interpretation by cytogeneticists. With the technological advances of next generation sequencing (NGS), the current CCA test and the molecular tests are likely to be challenged by whole-genome sequencing (WGS), which is a more unbiased method to detect all types of genomic aberrations not only those well-characterized clinically actionable mutations but also cryptic alternations yet to be defined and annotated. In this study a shallow whole-genome sequencing (sWGS) based assay, LeukoPrint, was utilized to depict genomic CNA profiles from the bone marrow cells of 107 newly diagnosed patients with AML (n=63, 58.9%) or MDS (n=44, 41.1%). Bone marrow was collected and mononuclear cells were separated to harvest genomic DNA which was sequenced without capture at approximately 1x coverage depth. Genome-wide CNAs was analyzed for each sample using an automated pipeline. European Leukemia Net (ELN) or Revised International Prognostic Scoring System (IPSS-R)-defining CNAs were selected and used to assign patients to a genetic risk group through the same classification systems that are used for CCA. It demonstrated 98.1% concordance of CNA profiles at individual patient level with cytogenetics and/or FISH. It is advantageous in detecting CNAs of short segments (1 Mb) and from samples with low leukemic cell content, more accurate for describing complex karyotypes and less confounded by subjective bias. LeukoPrint improved the overall diagnostic yield by redefining the risk categories for 16 patients (15%) by presenting new information. Nearly identical CNA profiles from the paired plasma and bone marrow suggest that peripheral blood may serve as a substitute to spare bone marrow aspiration. In summary, LeukoPrint provided an automated, convenient and cost-effective approach to describe genomic CNA profiles in AML/MDS. It brought greater diagnostic yield and risk stratification information by incorporating into the routine cytogenetics based on the standard ELN/IPSS-R guidelines. Citation Format: Xiaodong Lyu, Tao Li, Dandan Zhu, Mengna Zhang, Yuexin Cheng, Yan Chen, Zhenling Li, Shiyong Li, Wei Wu, Shuaipeng Geng, Jingshuai Li, Xiangxiang He, Yangwei Li, Yinyin Chang, Zunmin Zhu, Mao Mao, Yongping Song. Whole-genome sequencing as an alternative to analyze copy number abnormalities in acute myeloid leukemia and myelodysplastic syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5263.

Longitudinal monitoring of disease burden and response using ctDNA from dried blood spots in xenograft models.

Whole‐genome sequencing (WGS) of circulating tumour DNA (ctDNA) is now a clinically important biomarker for predicting therapy response, disease burden and disease progression. However, the translation of ctDNA monitoring into vital preclinical PDX models has not been possible owing to low circulating blood volumes in small rodents. Here, we describe the longitudinal detection and monitoring of ctDNA from minute volumes of blood in PDX mice. We developed a xenograft Tumour Fraction (xTF) metric using shallow WGS of dried blood spots (DBS), and demonstrate its application to quantify disease burden, monitor treatment response and predict disease outcome in a preclinical study of PDX mice. Further, we show how our DBS‐based ctDNA assay can be used to detect gene‐specific copy number changes and examine the copy number landscape over time. Use of sequential DBS ctDNA assays could transform future trial designs in both mice and patients by enabling increased sampling and molecular monitoring.

Potential of Aqueous Humor as a Liquid Biopsy for Uveal Melanoma.

Tumor biopsy can identify prognostic biomarkers for metastatic uveal melanoma (UM), however aqueous humor (AH) liquid biopsy may serve as an adjunct. This study investigated whether the AH of UM eyes has sufficient circulating tumor DNA (ctDNA) to perform genetic analysis. This is a case series of 37 AH samples, taken before or after radiation, and one tumor wash sample, from 12 choroidal and 8 ciliary body (CB) melanoma eyes. AH was analyzed for nucleic acid concentrations. AH DNA and one tumor wash sample underwent shallow whole-genome sequencing followed by Illumina sequencing to detect somatic copy number alterations (SCNAs). Four post-radiation AH underwent targeted sequencing of BAP1 and GNAQ genes. Post-radiation AH had significantly higher DNA and miRNA concentrations than paired pre-radiation samples. Highly recurrent UM SCNAs were identified in 0/11 post-radiation choroidal and 6/8 post-radiation CB AH. SCNAs were highly concordant in a CB post-radiation AH with its matched tumor (r = 0.978). BAP1 or GNAQ variants were detected in 3/4 post-radiation AH samples. AH is a source of ctDNA in UM eyes, particularly in post-radiation CB eyes. For the first time, UM SCNAs and mutations were identified in AH-derived ctDNA. Suggesting that AH can serve as a liquid biopsy for UM.

On-treatment plasma ctDNA fraction and treatment outcomes in metastatic castration-resistant prostate cancer.

5051 Background: Androgen receptor pathway inhibitors (ARPI) are standard of care for treatment-naive metastatic castration-resistant prostate cancer (mCRPC), but primary or rapidly acquired resistance is common. Early identification of resistant disease is critical for improving management strategies. We investigated whether on-treatment changes in plasma ctDNA fraction (ctDNA%; the proportion of cell-free DNA that is tumor-derived) associate with ARPI treatment outcomes. Methods: We collected serial plasma cell-free DNA from 81 patients with mCRPC: at baseline and following 4 weeks of treatment during two prospective multi-center observational studies (NCT02426333; NCT02471469). CtDNA% was calculated by integrating deep targeted and shallow whole-genome sequencing. Samples were dichotomized at above and below 1%, and designated as high or low ctDNA%, respectively. Outcome measurements were radiographic and/or clinical progression-free survival (rcPFS) and overall survival (OS). Non-durable responses were defined as rcPFS < 6 months, excluding patients with PSA progression alone (n = 3) or treatment cessation for toxicity (n = 4). Results: Median follow-up was 27.4 months (IQR 17.7-34.9). CtDNA% was high in 47/81 (58%) patients at baseline and 29/81 (36%) patients at 4 weeks. The median ctDNA% for patients with high ctDNA was 15.0% (IQR 4.9-43.8%) at baseline and 5.0% (IQR 2.0-20.6%) at 4 weeks. High baseline ctDNA% was prognostic for rcPFS and OS (Table). RcPFS and OS was shortest for patients that retained high ctDNA% at 4 weeks. However, patients converting from high to low ctDNA% at 4 weeks did not experience different outcomes to patients with low ctDNA at both timepoints. ctDNA% associations with rcPFS and OS were independent of established clinical prognostic factors. 23/27 (85%) patients experiencing non-durable responses had high ctDNA% at baseline and 4 weeks. Only 3/47 patients (6%) experiencing durable responses had high ctDNA% at both timepoints. Sensitivity and specificity for predicting non-durable response was 85% and 94%, respectively. Conclusions: Early changes in ctDNA% are strongly linked to duration of first-line ARPI treatment benefit in mCRPC and may have utility for informing clinical trials testing early therapy switches in patients unlikely to experience durable disease responses. Clinical trial information: NCT02426333; NCT02471469. [Table: see text]

Whole-genome sequencing as an alternative to analyze copy number abnormalities in AML, MDS and MM.

e19023 Background: Genomic alternations especially copy number aberrations (CNAs) are particularly imperative for diagnostic classification and risk stratification in patients with acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and multiple myeloma (MM). With the technological advances of next generation sequencing (NGS), the current CCA test and the molecular tests are likely to be challenged by whole-genome sequencing (WGS), which is a more unbiased method to detect all types of genomic aberrations. Methods: 178 patients who met the clinical manifestations of blood malignancies were included in the study, including 89 patients with AML, 48 with MDS and 41 with MM. Bone marrow was collected and mononuclear cells were separated to harvest genomic DNA which was sequenced without capture at approximately 1x coverage depth (shallow whole-genome sequencing, sWGS). Genome-wide CNAs greater than 5 Mbp was analyzed for each sample. European Leukemia Net (ELN), Revised International Prognostic Scoring System (IPSS-R) or Revised International Staging System (R-ISS)-defining CNAs were selected and used to assign patients to a genetic risk group through the same classification systems that are used for CCA. Results: In this study, CNA events were identified in 109 (73.6%) patients via sWGS. By calculating frequencies of recurrent gains and losses at chromosome arm level, some recurrent events were detected like gains of chr 8 (observed in 12 AML and 9 MDS patients), chr 1 (observed in 18 MM patients), losses of chr 7 (observed in 9 AML and 3 MDS patients) and chr 13 (observed in 16 MM patients), which were consistent with previously reported cytogenetic events. Besides, amplifications of odd-numbered chromosomes were detected in 15(36.6%) of patients with multiple myeloma, including chromosomes 3, 5, 7, 9, 11, 15, 19 and/or 21. And the trisomy of chr 5, 7 or 9 are mainly observed with a frequency of 66.7%. Among 145 patients with CCA results, a high concordance of 97.7% (98.5%, 97.6% and 97.1% in AML, MDS and MM respectively) was achieved in CNA profiles at individual patient level with cytogenetics and/or FISH. Noteworthily, CNA events were detected in 20 of 28 (71.4%) MM patients with negative karyotyping results. Moreover, sWGS improved the overall diagnostic yield by reassign 18 patients (9 with AML and 9 with MDS) to risk categories with poorer prognosis. Nearly identical CNA profiles from the paired plasma and bone marrow suggest that peripheral blood may serve as a substitute to spare bone marrow aspiration. Conclusions: In summary, a streamlined sWGS provides an accurate, convenient and cost-effective approach to describe genomic profiles of CNAs in patients with AML, MDS and MM. Clinical trial information: ChiCTR2100052276.

Non-invasive detection of lymphoma with circulating tumor DNA features and plasma protein marker.

e19574 Background: Lymphoma is common cancer worldwide, a large group of lymphoid hematopoietic malignancies including Non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL) two major type. Lymphoma includes more than 30 unique subtypes, originating from B, T and NK cells. Currently, diagnosis and classification of lymphoma is based on excisional lymph node biopsy through immunohistochemistry and in situ hybridization. However, invasive biopsies have significant limitations and carry procedural risks to patients, and it cannot account for spatial inter- and intra-tumor heterogeneity due to sampling from only one location in a single tumor lesion. In the past few years, an emerging class of methods named Liquid biopsy can potentially improve on these limitations. Methods: 135 untreated stage I-IV lymphoma patients and 399 healthy individuals were enrolled in this study. 8ml peripheral blood was collected from each participant after enrollment. Cell-free DNA (cfDNA) was extracted and subjected to shallow whole-genome sequencing (sWGS), and partly plasma was used to quantify the expression of 7 tumor serum protein markers. We developed a newly blood-based assay, which integrated the newly next-generation technology of sWGS of cfDNA and protein marker (CA125), with artificial intelligence (AI) technology. Though our multi-dimensional assay, the cancer risk score (CRS) of each sample were calculated, and was used for lymphoma early diagnosis. Results: After compared each protein marker between lymphoma and healthy groups, only CA125 could be used to screen the lymphoma with a sensitivity of 25.9% at extremely high specificity 98.0% and an area under the curve (AUC) of 69.5%. As for the genomic feature of cfDNA, the characteristics of copy number aberrations (CNA) and fragment size (FS) were significantly different between lymphoma and healthy groups (p value < 0.0001 by student’s t-test). Both FS and CNA demonstrated improved sensitivity (36.3% and 67.4% respectively) compared to CA125. When the three signatures were incorporated into the CRS model, it achieved the best performance allowing 95 lymphoma cases to be identified with a sensitivity of 70.4% at 98.0% specificity, and an AUC of 88.0%. In stage I/II and III/IV lymphoma cases, the sensitivity was 40.0% and 85.7% respectively. For each lymphoma subtype, the lymphoma which from B-cell had higher sensitivity than that from NK/T cell (71.0% vs 64.3%), especially in HL (91.7%), all originated from B cell. Conclusions: We newly find the traditional protein marker CA125 can be used for lymphoma screening, which was integrated with genomic feature CNA and FS of cfDNA as a multidimensional assay for early detection of lymphoma with sufficient accuracy. The performance of our efficient and non-invasive assay for detecting lymphoma is 70.4% sensitivity at extremely high specificity 98.0%, especially for 40.0% sensitivity for early patients (stage I/II) screening.

High levels of chromosomal aberrations negatively associate with benefit to checkpoint inhibition in NSCLC

BackgroundImmune checkpoint inhibitors (ICIs) targeting the programmed cell death 1/programmed death-ligand 1 axis have transformed the management of advanced non-small cell lung cancer (NSCLC). However, many patients do not benefit...

The Effect of Preanalytical and Physiological Variables on Cell-Free DNA Fragmentation.

Assays that account for the biological properties and fragmentation of cell-free DNA (cfDNA) can improve the performance of liquid biopsy. However, preanalytic and physiological differences between individuals on fragmentomic analysis are poorly defined. We analyzed the impact of collection tube, plasma processing time, and physiology on the size distribution of cfDNA, their genome-wide representation, and sequence diversity at the cfDNA fragment ends using shallow whole-genome sequencing. Neither different stabilizing collection tubes nor processing times affected the cfDNA fragment sizes, but could impact the genome-wide fragmentation patterns and fragment-end sequences of cfDNA. In addition, beyond differences depending on the gender, the physiological conditions tested between 63 individuals (age, body mass index, use of medication, and chronic conditions) minimally influenced the outcome of fragmentomic methods. Fragmentomic approaches have potential for implementation in the clinic, pending clear traceability of analytical and physiological factors.

BIOME-Preserve: A novel storage and transport medium for preserving anaerobic microbiota samples for culture recovery.

Sequencing-based protocols for studying the human microbiome have unearthed a wealth of information about the relationship between the microbiome and human health. But these microbes cannot be leveraged as therapeutic targets without culture-based studies to phenotype species of interest and to establish culture collections for use in animal models. Traditional sample collection protocols are focused on preserving nucleic acids and metabolites and are largely inappropriate for preserving sensitive anaerobic bacteria for later culture recovery. Here we introduce a novel microbiome preservation kit (BIOME-Preserve) that facilitates recovery of anaerobic bacteria from human stool. Using a combination of culture recovery and shallow whole-genome shotgun sequencing, we characterized the anaerobes cultured from fresh human stool and from human stool held at room temperature in BIOME-Preserve for up to 120 hours. We recovered several species of interest to microbiome researchers, including Bifidobacterium spp., Bacteroides spp., Blautia spp., Eubacterium halii (now Anaerobutyricum hallii), Akkermansia muciniphila, and Faecalibacterium prausnitzii. We also demonstrated that freezing at -80°C did not adversely affect our ability to culture organisms from BIOME-Preserve, suggesting that it is appropriate both as a transport medium and as a medium for longer-term ultra-cold storage. Together, our results suggest BIOME-Preserve is practical for the collection, transport, and culture of anaerobic bacteria from human samples and can help enable researchers to better understand the link between the microbiome and human health and how to leverage that link through novel microbiome-based therapeutics.

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.

A phase Ib/II study of regorafenib and paclitaxel in patients with beyond first-line advanced esophagogastric carcinoma (REPEAT).

Purpose:Regorafenib monotherapy, a multikinase inhibitor of angiogenesis, tumor microenvironment, and tumorigenesis, showed promising results in gastric cancer. We aimed to assess the tolerability of regorafenib and paclitaxel in patients with advanced esophagogastric cancer (EGC) refractory to first-line treatment, and explore potential biomarkers.Methods:Patients received paclitaxel (80 mg/m2) on days 1, 8, and 15 of a 28-day cycle and regorafenib (80/120/160 mg) on days 1–21 in the dose-escalation cohort, and the maximum-tolerated dose (MTD) in the dose-expansion cohort. Exploratory, overall survival (OS) and progression-free survival (PFS) were compared to a propensity-score matched cohort receiving standard second-/third-line systemic treatment. Paclitaxel pharmacokinetics were assessed using samples from day 1 (D1) and day 15 (D15). We performed enzyme-linked immunosorbent assay measurements of galectin-1, RNA sequencing, and shallow whole-genome sequencing of metastatic tumor biopsies for biomarker analyses.Results:In the dose-escalation cohort (n = 14), the MTD of regorafenib was 120 mg. In all, 34 patients were enrolled in the dose-expansion cohort. Most common toxicities (all grades; grade ⩾ 3) were fatigue (79%; 4%) and sensory neuropathy (63%; 4%). Best responses achieved were partial response (28%) and stable disease (54%). Median OS and PFS were 7.8 and 4.2 months, respectively (median follow-up: 7.8 months). OS (p = 0.08) and PFS (p = 0.81) were not significantly improved compared to the matched cohort. Paclitaxel concentrations were significantly increased with regorafenib (D15) compared with paclitaxel only (D1; p < 0.05); no associations were observed with toxicity or efficacy. An increase in circulating galectin-1 compared to baseline was associated with shorter OS (p < 0.01). Enrichment of angiogenesis-related gene expression was observed in short survivors measured by RNA sequencing. Chromosome 19q13.12-q13.2 amplification was associated with shorter OS (p = 0.02) and PFS (p = 0.02).Conclusion:Treatment with regorafenib and paclitaxel is tolerable and shows promising efficacy in advanced EGC refractory to first-line treatment. Galectin-1 and chromosome 19q13.12-q13.2 amplification could serve as negative predictive biomarkers for treatment response.Registration:Clinicaltrials.gov, NCT02406170, https://clinicaltrials.gov/ct2/show/NCT02406170

Usefulness of combined NGS and QF-PCR analysis for product of conception karyotyping.

PurposeSince chromosomal abnormalities can be detected in more than half of miscarriages, cytogenetic testing of the product of conception (POC) can provide important information when preparing for a subsequent pregnancy. Conventional karyotyping is the common diagnostic method for a POC but can be problematic due to the need for cell culture.MethodsWe here conducted shallow whole‐genome sequencing (sWGS) using next‐generation sequencing (NGS) for alternative POC cytogenomic analysis. Since female euploidy samples can include 69,XXX triploidy, additional QF‐PCR was performed in these cases.ResultsWe here analyzed POC samples from miscarriages in 300 assisted reproductive technology (ART) pregnancies and detected chromosomal abnormalities in 201 instances (67.0%). Autosomal aneuploidy (151 cases, 50.3%) was the most frequent abnormality, consistent with prior conventional karyotyping data. Mosaic aneuploidy was detected in seven cases (2.0%). Notably, the frequency of triploidy was 2.3%, 10‐fold lower than the reported frequency in non‐ART pregnancies. Structural rearrangements were identified in nine samples (3%), but there was no case of segmental mosaicism.ConclusionsThese data suggest that NGS‐based sWGS, with the aid of QF‐PCR, is a viable alternative karyotyping procedure that does not require cell culture. This method could also assist with genetic counseling for couples who undergoes embryo selection based on PGT‐A data.

Early identification of disease progression in ALK-rearranged lung cancer using circulating tumor DNA analysis

Targeted kinase inhibitors improve the prognosis of lung cancer patients with ALK alterations (ALK+). However, due to the emergence of acquired resistance and varied clinical trajectories, early detection of disease progression is warranted to guide patient management and therapy decisions. We utilized 343 longitudinal plasma DNA samples from 43 ALK+ NSCLC patients receiving ALK-directed therapies to determine molecular progression based on matched panel-based targeted next-generation sequencing (tNGS), and shallow whole-genome sequencing (sWGS). ALK-related alterations were detected in 22 out of 43 (51%) patients. Among 343 longitudinal plasma samples analyzed, 174 (51%) were ctDNA-positive. ALK variant and fusion kinetics generally reflected the disease course. Evidence for early molecular progression was observed in 19 patients (44%). Detection of ctDNA at therapy baseline indicated shorter times to progression compared to cases without mutations at baseline. In patients who succumbed to the disease, ctDNA levels were highly elevated towards the end of life. Our results demonstrate the potential utility of these NGS assays in the clinical management of ALK+ NSCLC.

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

BackgroundPaediatric 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. ProcedureThe 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. ResultsOverall, 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. ConclusionIn 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.

Longitudinal Copy-Number Alteration Analysis in Plasma Cell-Free DNA of Neuroendocrine Neoplasms is a Novel Specific Biomarker for Diagnosis, Prognosis, and Follow-up.

As noninvasive biomarkers are an important unmet need for neuroendocrine neoplasms (NEN), biomarker potential of genome-wide molecular profiling of plasma cell-free DNA (cfDNA) was prospectively studied in patients with NEN. Longitudinal plasma samples were collected from patients with well-differentiated, metastatic gastroenteropancreatic and lung NEN. cfDNA was subjected to shallow whole-genome sequencing to detect genome-wide copy-number alterations (CNA) and estimate circulating tumor DNA (ctDNA) fraction, and correlated to clinicopathologic and survival data. To differentiate pancreatic NENs (PNEN) from pancreatic adenocarcinomas (PAAD) using liquid biopsies, a classification model was trained using tissue-based CNAs and validated in cfDNA. One hundred and ninety-five cfDNA samples from 43 patients with NEN were compared with healthy control cfDNA (N = 100). Plasma samples from patients with PNEN (N = 21) were used for comparison with publicly available PNEN tissue (N = 98), PAAD tissue (N = 109), and PAAD cfDNA (N = 96). Thirty percent of the NEN cfDNA samples contained ctDNA and 44% of the patients had at least one ctDNA-positive (ctDNA+) sample. CNAs detected in cfDNA were highly specific for NENs and the classification model could distinguish PAAD and PNEN cfDNA samples with a sensitivity, specificity, and AUC of 62%, 86%, and 79%, respectively. ctDNA-positivity was associated with higher World Health Organization (WHO) grade, primary tumor location, and higher chromogranin A and neuron-specific enolase values. Overall survival was significantly worse for ctDNA+ patients and increased ctDNA fractions were associated with poorer progression-free survival. Sequential genome-wide profiling of plasma cfDNA is a novel, noninvasive biomarker with high specificity for diagnosis, prognosis, and follow-up in metastatic NENs.

Karyosequencing: Integrating Genome-Wide and Targeted Sequencing for Comprehensive Diagnosis of Lymphoproliferative Disorders

Introduction: Molecular diagnostic testing for lymphoproliferative disorders (LPDs) includes detection of clonal immunoglobulin (IG) and/or T cell receptor (TCR) rearrangements, translocations, copy number alterations (CNA) and somatic mutations. To date, laboratories still rely on subjective and labour-intensive technologies, e.g. karyotyping/FISH to detect complex genomic alterations. Whole Genome Sequencing (WGS) can detect all genomic alterations, but factors such as cost, computation/storage, DNA requirements and poor detection of clinically relevant subclonal mutations limits the routine implementation of WGS in clinical diagnostics. We have developed “KaryoSequencing” (KS), a novel approach that combines targeted deep-sequencing, using a targeted hybridisation capture NGS panel, for rare mutation and translocation detection with shallow WGS (sWGS) for genome wide copy number analysis, in a single test.Methods: KS was validated using 138 clinical samples from patients with acute lymphoblastic leukaemia (ALL) (n=46), chronic lymphocytic leukaemia (CLL) (n=46) and plasma cell myeloma (PCM) (n=46) samples from 3 UK laboratories. Samples underwent library preparation and hybridisation using the EuroClonality-NDC assay. A KS library for each sample was prepared by combining the pre-capture and post-capture libraries at an optimised ratio to enable high coverage (>500 x) at regions covered by the targeted panel while providing 0.5-1x coverage genome-wide. Forty-six KS libraries were pooled and sequenced per NovaSeq 6000 run, using a 200-cycle SP flow cell and a 100bp paired-end strategy. For analysis of targeted regions, somatic mutation calling was performed using VarDict and structural variants (rearrangements and translocations) were detected using ARResT/Interrogate. For analysis of large chromosomal copy number variation using sWGS, a modified version of QDNASeq/ACE with a window size of 50kb was performed. For sWGS analysis, bioinformatic exclusion of all target capture regions and panel-specific off-target regions was performed using a panel of 48 DNA samples from healthy individuals ran on the same KS protocol.Results:Analysis of genome-wide copy number by sWGS was concordant in 477 of 503 (95%) evaluable FISH tests, including precise detection of hyper and hypo-diploidy and other complex karyotypes. The performance of the targeted deep-sequencing component of the KS approach was assessed to ensure comparable performance to previously validated results for the EuroClonality-NDC. The EuroClonality-NDC detected a clonal IG rearrangement in 46/46 (100%) CLL cases and in 42/46 (91.3%) PCM cases. Clonality was detected in 43/46 (93.5%) and 44/46 (95.7%) cases at an IG and TCR locus respectively in ALL, a disease entity known to exhibit cross-lineage rearrangements. Overall, 44 translocations were detected in the 138 samples by the EuroClonality-NDC. FISH results were available for 32 of the 44 translocations detected by NGS and were concordant in 29/32 (91%). In two PCM samples NGS and FISH reported a different translocation; in the ALL sample, the FISH pattern showed an additional copy but not a split signal. Across the entire cohort of 138 samples, KaryoSequencing detected 190 mutations with a variant allele frequency ranging from 4.0 - 97.6%. Of the detected mutations, 69/190 (36.3%) had a VAF <20% which can be difficult to detect reliably with typical coverage depths observed with conventional WGS. The most frequently mutated genes were KRAS [30.4%], NRAS [13.0%], KMT2D [10.9%] in ALL; SF3B1 [23.9%], NOTCH1 [19.6%], ATM [15.2%] and TP53[13.0%] in CLL and KRAS [19.6%], NRAS [15.2%] and TP53 [10.9%] in PCM, consistent with expected frequencies in each entity.Conclusions:KaryoSequencing demonstrated >95% sensitivity and specificity for detection of gross genome-wide copy number alterations while retaining the analytical performance for detection of rearrangements, translocations, and mutations for the lymphoid-specific targeted regions of the EuroClonality-NDC assay. KS is a cost-effective and high-throughput integrated alternative to current diagnostic strategies in haematological malignancies and can be implemented in routine clinical practice. DisclosuresJenner: Janssen: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy; Takeda: Consultancy; BMS/Celgene: Consultancy, Honoraria, Speakers Bureau. Gonzalez: Univ8 Genomics Ltd: Current holder of individual stocks in a privately-held company.