Current and future perspectives of liquid biopsies in genomics-driven oncology.

Precision oncology seeks to leverage molecular information about cancer to improve patient outcomes. Tissue biopsy samples are widely used to characterize tumours but are limited by constraints on sampling frequency and their incomplete representation of the entire tumour bulk. Now, attention is turning to minimally invasive liquid biopsies, which enable analysis of tumour components (including circulating tumour cells and circulating tumour DNA) in bodily fluids such as blood. The potential of liquid biopsies is highlighted by studies that show they can track the evolutionary dynamics and heterogeneity of tumours and can detect very early emergence of therapy resistance, residual disease and recurrence. However, the analytical validity and clinical utility of liquid biopsies must be rigorously demonstrated before this potential can be realized.

Correction: The population genetics of human disease: The case of recessive, lethal mutations.

[This corrects the article DOI: 10.1371/journal.pgen.1006915.].

Genetic ancestry analysis on >93,000 individuals undergoing expanded carrier screening reveals limitations of ethnicity-based medical guidelines.

PURPOSE: Carrier status associates strongly with genetic ancestry, yet current carrier screening guidelines recommend testing for a limited set of conditions based on a patient's self-reported ethnicity. Ethnicity, which can reflect both genetic ancestry and cultural factors (e.g., religion), may be imperfectly known or communicated by patients. We sought to quantitatively assess the efficacy and equity with which ethnicity-based carrier screening captures recessive disease risk. METHODS: For 93,419 individuals undergoing a 96-gene expanded carrier screen (ECS), correspondence was assessed among carrier status, self-reported ethnicity, and a dual-component genetic ancestry (e.g., 75% African/25% European) calculated from sequencing data. RESULTS: Self-reported ethnicity was an imperfect indicator of genetic ancestry, with 9% of individuals having >50% genetic ancestry from a lineage inconsistent with self-reported ethnicity. Limitations of self-reported ethnicity led to missed carriers in at-risk populations: for 10 ECS conditions, patients with intermediate genetic ancestry backgrounds-who did not self-report the associated ethnicity-had significantly elevated carrier risk. Finally, for 7 of the 16 conditions included in current screening guidelines, most carriers were not from the population the guideline aimed to serve. CONCLUSION: Substantial and disproportionate risk for recessive disease is not detected when carrier screening is based on ethnicity, leading to inequitable reproductive care.

The population genetics of human disease: The case of recessive, lethal mutations.

Do the frequencies of disease mutations in human populations reflect a simple balance between mutation and purifying selection? What other factors shape the prevalence of disease mutations? To begin to answer these questions, we focused on one of the simplest cases: recessive mutations that alone cause lethal diseases or complete sterility. To this end, we generated a hand-curated set of 417 Mendelian mutations in 32 genes reported to cause a recessive, lethal Mendelian disease. We then considered analytic models of mutation-selection balance in infinite and finite populations of constant sizes and simulations of purifying selection in a more realistic demographic setting, and tested how well these models fit allele frequencies estimated from 33,370 individuals of European ancestry. In doing so, we distinguished between CpG transitions, which occur at a substantially elevated rate, and three other mutation types. Intriguingly, the observed frequency for CpG transitions is slightly higher than expectation but close, whereas the frequencies observed for the three other mutation types are an order of magnitude higher than expected, with a bigger deviation from expectation seen for less mutable types. This discrepancy is even larger when subtle fitness effects in heterozygotes or lethal compound heterozygotes are taken into account. In principle, higher than expected frequencies of disease mutations could be due to widespread errors in reporting causal variants, compensation by other mutations, or balancing selection. It is unclear why these factors would have a greater impact on disease mutations that occur at lower rates, however. We argue instead that the unexpectedly high frequency of disease mutations and the relationship to the mutation rate likely reflect an ascertainment bias: of all the mutations that cause recessive lethal diseases, those that by chance have reached higher frequencies are more likely to have been identified and thus to have been included in this study. Beyond the specific application, this study highlights the parameters likely to be important in shaping the frequencies of Mendelian disease alleles.

Machine learning enables detection of early-stage colorectal cancer by whole-genome sequencing of plasma cell-free DNA.

BACKGROUND: Blood-based methods using cell-free DNA (cfDNA) are under development as an alternative to existing screening tests. However, early-stage detection of cancer using tumor-derived cfDNA has proven challenging because of the small proportion of cfDNA derived from tumor tissue in early-stage disease. A machine learning approach to discover signatures in cfDNA, potentially reflective of both tumor and non-tumor contributions, may represent a promising direction for the early detection of cancer. METHODS: Whole-genome sequencing was performed on cfDNA extracted from plasma samples (N = 546 colorectal cancer and 271 non-cancer controls). Reads aligning to protein-coding gene bodies were extracted, and read counts were normalized. cfDNA tumor fraction was estimated using IchorCNA. Machine learning models were trained using k-fold cross-validation and confounder-based cross-validations to assess generalization performance. RESULTS: In a colorectal cancer cohort heavily weighted towards early-stage cancer (80% stage I/II), we achieved a mean AUC of 0.92 (95% CI 0.91-0.93) with a mean sensitivity of 85% (95% CI 83-86%) at 85% specificity. Sensitivity generally increased with tumor stage and increasing tumor fraction. Stratification by age, sequencing batch, and institution demonstrated the impact of these confounders and provided a more accurate assessment of generalization performance. CONCLUSIONS: A machine learning approach using cfDNA achieved high sensitivity and specificity in a large, predominantly early-stage, colorectal cancer cohort. The possibility of systematic technical and institution-specific biases warrants similar confounder analyses in other studies. Prospective validation of this machine learning method and evaluation of a multi-analyte approach are underway.

Systematic design and comparison of expanded carrier screening panels.

PurposeThe recent growth in pan-ethnic expanded carrier screening (ECS) has raised questions about how such panels might be designed and evaluated systematically. Design principles for ECS panels might improve clinical detection of at-risk couples and facilitate objective discussions of panel choice.MethodsGuided by medical-society statements, we propose a method for the design of ECS panels that aims to maximize the aggregate and per-disease sensitivity and specificity across a range of Mendelian disorders considered serious by a systematic classification scheme. We evaluated this method retrospectively using results from 474,644 de-identified carrier screens. We then constructed several idealized panels to highlight strengths and limitations of different ECS methodologies.ResultsBased on modeled fetal risks for "severe" and "profound" diseases, a commercially available ECS panel (Counsyl) is expected to detect 183 affected conceptuses per 100,000 US births. A screen's sensitivity is greatly impacted by two factors: (i) the methodology used (e.g., full-exon sequencing finds more affected conceptuses than targeted genotyping) and (ii) the detection rate of the screen for diseases with high prevalence and complex molecular genetics (e.g., fragile X syndrome).ConclusionThe described approaches enable principled, quantitative evaluation of which diseases and methodologies are appropriate for pan-ethnic expanded carrier screening.

Validation of an Expanded Carrier Screen that Optimizes Sensitivity via Full-Exon Sequencing and Panel-wide Copy Number Variant Identification.

BACKGROUND: By identifying pathogenic variants across hundreds of genes, expanded carrier screening (ECS) enables prospective parents to assess the risk of transmitting an autosomal recessive or X-linked condition. Detection of at-risk couples depends on the number of conditions tested, the prevalence of the respective diseases, and the screen's analytical sensitivity for identifying disease-causing variants. Disease-level analytical sensitivity is often <100% in ECS tests because copy number variants (CNVs) are typically not interrogated because of their technical complexity. METHODS: We present an analytical validation and preliminary clinical characterization of a 235-gene sequencing-based ECS with full coverage across coding regions, targeted assessment of pathogenic noncoding variants, panel-wide CNV calling, and specialized assays for technically challenging genes. Next-generation sequencing, customized bioinformatics, and expert manual call review were used to identify single-nucleotide variants, short insertions and deletions, and CNVs for all genes except FMR1 and those whose low disease incidence or high technical complexity precluded novel variant identification or interpretation. RESULTS: Screening of 36859 patients' blood or saliva samples revealed the substantial impact on fetal disease-risk detection attributable to novel CNVs (9.19% of risk) and technically challenging conditions (20.2% of risk), such as congenital adrenal hyperplasia. Of the 7498 couples screened, 335 were identified as at risk for an affected pregnancy, underscoring the clinical importance of the test. Validation of our ECS demonstrated >99% analytical sensitivity and >99% analytical specificity. CONCLUSIONS: Validated high-fidelity identification of different variant types-especially for diseases with complicated molecular genetics-maximizes at-risk couple detection.

Clinical Utility of Expanded Carrier Screening: Reproductive Behaviors of At-Risk Couples.

Expanded carrier screening (ECS) analyzes dozens or hundreds of recessive genes to determine reproductive risk. Data on the clinical utility of screening conditions beyond professional guidelines are scarce. Individuals underwent ECS for up to 110 genes. Five-hundred thirty-seven at-risk couples (ARC), those in which both partners carry the same recessive disease, were invited to participate in a retrospective IRB-approved survey of their reproductive decision making after receiving ECS results. Sixty-four eligible ARC completed the survey. Of 45 respondents screened preconceptionally, 62% (n = 28) planned IVF with PGD or prenatal diagnosis (PNDx) in future pregnancies. Twenty-nine percent (n = 13) were not planning to alter reproductive decisions. The remaining 9% (n = 4) of responses were unclear. Of 19 pregnant respondents, 42% (n = 8) elected PNDx, 11% (n = 2) planned amniocentesis but miscarried, and 47% (n = 9) considered the condition insufficiently severe to warrant invasive testing. Of the 8 pregnancies that underwent PNDx, 5 were unaffected and 3 were affected. Two of 3 affected pregnancies were terminated. Disease severity was found to have significant association (p = 0.000145) with changes in decision making, whereas guideline status of diseases, controlled for severity, was not (p = 0.284). Most ARC altered reproductive planning, demonstrating the clinical utility of ECS. Severity of conditions factored into decision making.

Smith-Lemli-Opitz syndrome carrier frequency and estimates of in utero mortality rates.

OBJECTIVE: To tabulate individual allele frequencies and total carrier frequency for Smith-Lemli-Opitz syndrome (SLOS) and compare expected versus observed birth incidences. METHODS: A total of 262 399 individuals with no known indication or increased probability of SLOS carrier status, primarily US based, were screened for SLOS mutations as part of an expanded carrier screening panel. Results were retrospectively analyzed to estimate carrier frequencies in multiple ethnic groups. SLOS birth incidences obtained from existing literature were then compared with these data to estimate the effect of SLOS on fetal survival. RESULTS: Smith-Lemli-Opitz syndrome carrier frequency is highest in Ashkenazi Jews (1 in 43) and Northern Europeans (1 in 54). Comparing predicted birth incidence with that observed in published literature suggests that approximately 42% to 88% of affected conceptuses experience prenatal demise. CONCLUSION: Smith-Lemli-Opitz syndrome is relatively frequent in certain populations and, because of its impact on prenatal and postnatal morbidity and mortality, merits consideration for routine screening. © 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Noninvasive prenatal screening at low fetal fraction: comparing whole-genome sequencing and single-nucleotide polymorphism methods.

OBJECTIVE: Performance of noninvasive prenatal screening (NIPS) methodologies when applied to low fetal fraction samples is not well established. The single-nucleotide polymorphism (SNP) method fails samples below a predetermined fetal fraction threshold, whereas some laboratories employing the whole-genome sequencing (WGS) method report aneuploidy calls for all samples. Here, the performance of the two methods was compared to determine which approach actually detects more fetal aneuploidies. METHODS: Computational models were parameterized with up-to-date published data and used to compare the performance of the two methods at calling common fetal trisomies (T21, T18, T13) at low fetal fractions. Furthermore, clinical experience data were reviewed to determine aneuploidy detection rates based on compliance with recent invasive screening recommendations. RESULTS: The SNP method's performance is dependent on the origin of the trisomy, and is lowest for the most common trisomies (maternal M1 nondisjunction). Consequently, the SNP method cannot maintain acceptable performance at fetal fractions below ~3%. In contrast, the WGS method maintains high specificity independent of fetal fraction and has >80% sensitivity for trisomies in low fetal fraction samples. CONCLUSION: The WGS method will detect more aneuploidies below the fetal fraction threshold at which many labs issue a no-call result, avoiding unnecessary invasive procedures. © 2017 Counsyl Inc. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Group Testing Approach for Trinucleotide Repeat Expansion Disorder Screening.

BACKGROUND: Fragile X syndrome (FXS, OMIM #300624) is an X-linked condition caused by trinucleotide repeat expansions in the 5' UTR (untranslated region) of the fragile X mental retardation 1 (FMR1) gene. FXS testing is commonly performed in expanded carrier screening and has been proposed for inclusion in newborn screening. However, because pathogenic alleles are long and have low complexity (>200 CGG repeats), FXS is currently tested by a single-plex electrophoresis-resolved PCR assay rather than multiplexed approaches like next-generation sequencing or mass spectrometry. In this work, we sought an experimental design based on nonadaptive group testing that could accurately and reliably identify the size of abnormally expanded FMR1 alleles of males and females. METHODS: We developed a new group testing scheme named StairCase (SC) that was designed to the constraints of the FXS testing problem, and compared its performance to existing group testing schemes by simulation. We experimentally evaluated SC's performance on 210 samples from the Coriell Institute biorepositories using pooled PCR followed by capillary electrophoresis on 3 replicates of each of 3 pooling layouts differing by the mapping of samples to pools. RESULTS: The SC pooled PCR approach demonstrated perfect classification of samples by clinical category (normal, intermediate, premutation, or full mutation) for 90 positives and 1800 negatives, with a batch of 210 samples requiring only 21 assays. CONCLUSIONS: Group testing based on SC is an implementable approach to trinucleotide repeat expansion disorder testing that offers ≥10-fold reduction in assay costs over current single-plex methods.

Modeled Fetal Risk of Genetic Diseases Identified by Expanded Carrier Screening.

IMPORTANCE: Screening for carrier status of a limited number of single-gene conditions is the current standard of prenatal care. Methods have become available allowing rapid expanded carrier screening for a substantial number of conditions. OBJECTIVES: To quantify the modeled risk of recessive conditions identifiable by an expanded carrier screening panel in individuals of diverse racial and ethnic backgrounds and to compare the results with those from current screening recommendations. DESIGN, SETTING, AND PARTICIPANTS: Retrospective modeling analysis of results between January 1, 2012, and July 15, 2015, from expanded carrier screening in reproductive-aged individuals without known indication for specific genetic testing, primarily from the United States. Tests were offered by clinicians providing reproductive care. EXPOSURES: Individuals were tested for carrier status for up to 94 severe or profound conditions. MAIN OUTCOMES AND MEASURES: Risk was defined as the probability that a hypothetical fetus created from a random pairing of individuals (within or across 15 self-reported racial/ethnic categories; there were 11 categories with >5000 samples) would be homozygous or compound heterozygous for 2 mutations presumed to cause severe or profound disease. Severe conditions were defined as those that if left untreated cause intellectual disability or a substantially shortened lifespan; profound conditions were those causing both. RESULTS: The study included 346,790 individuals. Among major US racial/ethnic categories, the calculated frequency of fetuses potentially affected by a profound or severe condition ranged from 94.5 per 100,000 (95% CI, 82.4-108.3 per 100,000) for Hispanic couples to 392.2 per 100,000 (95% CI, 366.3-420.2 per 100,000) for Ashkenazi Jewish couples. In most racial/ethnic categories, expanded carrier screening modeled more hypothetical fetuses at risk for severe or profound conditions than did screening based on current professional guidelines (Mann-Whitney P < .001). For Northern European couples, the 2 professional guidelines-based screening panels modeled 55.2 hypothetical fetuses affected per 100,000 (95% CI, 51.3-59.3 per 100,000) and the expanded carrier screening modeled 159.2 fetuses per 100,000 (95% CI, 150.4-168.6 per 100,000). Overall, relative to expanded carrier screening, guideline-based screening ranged from identification of 6% (95% CI, 4%-8%) of hypothetical fetuses affected for East Asian couples to 87% (95% CI, 84%-90%) for African or African American couples. CONCLUSIONS AND RELEVANCE: In a population of diverse races and ethnicities, expanded carrier screening may increase the detection of carrier status for a variety of potentially serious genetic conditions compared with current recommendations from professional societies. Prospective studies comparing current standard-of-care carrier screening with expanded carrier screening in at-risk populations are warranted before expanded screening is adopted.

SCISSORS: practical considerations.

Molecular similarity has been effectively applied to many problems in cheminformatics and computational drug discovery, but modern methods can be prohibitively expensive for large-scale applications. The SCISSORS method rapidly approximates measures of pairwise molecular similarity such as ROCS and LINGO Tanimotos, acting as a filter to quickly reduce the size of a problem. We report an in-depth analysis of SCISSORS performance, including a mapping of the SCISSORS error distribution, benchmarking, and investigation of several algorithmic modifications. We show that SCISSORS can accurately predict multiconformer similarity and suggest a method for estimating optimal SCISSORS parameters in a data set-specific manner. These results are a useful resource for researchers seeking to incorporate SCISSORS into molecular similarity applications.

High-resolution genome-wide mapping of chromosome-arm-scale truncations induced by CRISPR-Cas9 editing.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is a powerful tool for introducing targeted mutations in DNA, but recent studies have shown that it can have unintended effects such as structural changes. However, these studies have not yet looked genome wide or across data types. Here we performed a phenotypic CRISPR-Cas9 scan targeting 17,065 genes in primary human cells, revealing a 'proximity bias' in which CRISPR knockouts show unexpected similarities to unrelated genes on the same chromosome arm. This bias was found to be consistent across cell types, laboratories, Cas9 delivery methods and assay modalities, and the data suggest that it is caused by telomeric truncations of chromosome arms, with cell cycle and apoptotic pathways playing a mediating role. Additionally, a simple correction is demonstrated to mitigate this pervasive bias while preserving biological relationships. This previously uncharacterized effect has implications for functional genomic studies using CRISPR-Cas9, with applications in discovery biology, drug-target identification, cell therapies and genetic therapeutics.

An empirical estimate of carrier frequencies for 400+ causal Mendelian variants: results from an ethnically diverse clinical sample of 23,453 individuals.

PURPOSE: Recent developments in genomics have led to expanded carrier screening panels capable of assessing hundreds of causal mutations for genetic disease. This new technology enables simultaneous measurement of carrier frequencies for many diseases. As the resultant rank-ordering of carrier frequencies impacts the design and prioritization of screening programs, the accuracy of this ranking is a public health concern. METHODS: A total of 23,453 individuals from many obstetric, genetics, and infertility clinics were referred for routine recessive disease carrier screening. Multiplex carrier screening was performed and results were aggregated for this study. RESULTS: Twenty-four percent of individuals were identified as carriers for at least one of 108 disorders, and 5.2% were carriers for multiple disorders. We report tabulations of carrier frequency by self-identified ethnicity and disease. CONCLUSION: To our knowledge, this study of a large, ethnically diverse clinical sample provides the most accurate measurements to date of carrier frequencies for hundreds of recessive alleles. The study also yields information on the clinical considerations associated with routine use of expanded panels and provides support for a pan-ethnic screening paradigm that minimizes the use of "racial" categories by the physician, as recommended by recent guidelines.

Error bounds on the SCISSORS approximation method.

The SCISSORS method for approximating chemical similarities has shown excellent empirical performance on a number of real-world chemical data sets but lacks theoretically proven bounds on its worst-case error performance. This paper first proves reductions showing SCISSORS to be equivalent to two previous kernel methods: kernel principal components analysis and the rank-k Nyström approximation of a Gram matrix. These reductions allow the use of generalization bounds on these techniques to show that the expected error in SCISSORS approximations of molecular similarity kernels is bounded in expected pairwise inner product error, in matrix 2-norm and Frobenius norm for full kernel matrix approximations and in root-mean-square deviation for approximated matrices. Finally, we show that the actual performance of SCISSORS is significantly better than these worst-case bounds, indicating that chemical space is well-structured for chemical sampling algorithms.

Anatomy of high-performance 2D similarity calculations.

Similarity measures based on the comparison of dense bit vectors of two-dimensional chemical features are a dominant method in chemical informatics. For large-scale problems, including compound selection and machine learning, computing the intersection between two dense bit vectors is the overwhelming bottleneck. We describe efficient implementations of this primitive as well as example applications using features of modern CPUs that allow 20-40× performance increases relative to typical code. Specifically, we describe fast methods for population count on modern x86 processors and cache-efficient matrix traversal and leader clustering algorithms that alleviate memory bandwidth bottlenecks in similarity matrix construction and clustering. The speed of our 2D comparison primitives is within a small factor of that obtained on GPUs and does not require specialized hardware.

PAPER--accelerating parallel evaluations of ROCS.

Modern graphics processing units (GPUs) are flexibly programmable and have peak computational throughput significantly faster than conventional CPUs. Herein, we describe the design and implementation of PAPER, an open-source implementation of Gaussian molecular shape overlay for NVIDIA GPUs. We demonstrate one to two order-of-magnitude speedups on high-end commodity GPU hardware relative to a reference CPU implementation of the shape overlay algorithm and speedups of over one order of magnitude relative to the commercial OpenEye ROCS package. In addition, we describe errors incurred by approximations used in common implementations of the algorithm.

SCISSORS: a linear-algebraical technique to rapidly approximate chemical similarities.

Algorithms for several emerging large-scale problems in cheminformatics have as their rate-limiting step the evaluation of relatively slow chemical similarity measures, such as structural similarity or three-dimensional (3-D) shape comparison. In this article we present SCISSORS, a linear-algebraical technique (related to multidimensional scaling and kernel principal components analysis) to rapidly estimate chemical similarities for several popular measures. We demonstrate that SCISSORS faithfully reflects its source similarity measures for both Tanimoto calculation and rank ordering. After an efficient precalculation step on a database, SCISSORS affords several orders of magnitude of speedup in database screening. SCISSORS furthermore provides an asymptotic speedup for large similarity matrix construction problems, reducing the number of conventional slow similarity evaluations required from quadratic to linear scaling.