ClonEvol: clonal ordering and visualization in cancer sequencing.

BACKGROUND: Reconstruction of clonal evolution is critical for understanding tumor progression and implementing personalized therapies. This is often done by clustering somatic variants based on their cellular prevalence estimated via bulk tumor sequencing of multiple samples. The clusters, consisting of the clonal marker variants, are then ordered based on their estimated cellular prevalence to reconstruct clonal evolution trees, a process referred to as 'clonal ordering'. However, cellular prevalence estimate is confounded by statistical variability and errors in sequencing/data analysis, and therefore inhibits accurate reconstruction of the clonal evolution. This problem is further complicated by intra- and inter-tumor heterogeneity. Furthermore, the field lacks a comprehensive visualization tool to facilitate the interpretation of complex clonal relationships. To address these challenges we developed ClonEvol, a unified software tool for clonal ordering, visualization, and interpretation. MATERIALS AND METHODS: ClonEvol uses a bootstrap resampling technique to estimate the cellular fraction of the clones and probabilistically models the clonal ordering constraints to account for statistical variability. The bootstrapping allows identification of the sample founding- and sub-clones, thus enabling interpretation of clonal seeding. ClonEvol automates the generation of multiple widely used visualizations for reconstructing and interpreting clonal evolution. RESULTS: ClonEvol outperformed three of the state of the art tools (LICHeE, Canopy and PhyloWGS) for clonal evolution inference, showing more robust error tolerance and producing more accurate trees in a simulation. Building upon multiple recent publications that utilized ClonEvol to study metastasis and drug resistance in solid cancers, here we show that ClonEvol rediscovered relapsed subclones in two published acute myeloid leukemia patients. Furthermore, we demonstrated that through noninvasive monitoring ClonEvol recapitulated the emerging subclones throughout metastatic progression observed in the tumors of a published breast cancer patient. CONCLUSIONS: ClonEvol has broad applicability for longitudinal monitoring of clonal populations in tumor biopsies, or noninvasively, to guide precision medicine. AVAILABILITY: ClonEvol is written in R and is available at https://github.com/ChrisMaherLab/ClonEvol.

U2AF1 mutations alter sequence specificity of pre-mRNA binding and splicing.

We previously identified missense mutations in the U2AF1 splicing factor affecting codons S34 (S34F and S34Y) or Q157 (Q157R and Q157P) in 11% of the patients with de novo myelodysplastic syndrome (MDS). Although the role of U2AF1 as an accessory factor in the U2 snRNP is well established, it is not yet clear how these mutations affect splicing or contribute to MDS pathophysiology. We analyzed splice junctions in RNA-seq data generated from transfected CD34+ hematopoietic cells and found significant differences in the abundance of known and novel junctions in samples expressing mutant U2AF1 (S34F). For selected transcripts, splicing alterations detected by RNA-seq were confirmed by analysis of primary de novo MDS patient samples. These effects were not due to impaired U2AF1 (S34F) localization as it co-localized normally with U2AF2 within nuclear speckles. We further found evidence in the RNA-seq data for decreased affinity of U2AF1 (S34F) for uridine (relative to cytidine) at the e-3 position immediately upstream of the splice acceptor site and corroborated this finding using affinity-binding assays. These data suggest that the S34F mutation alters U2AF1 function in the context of specific RNA sequences, leading to aberrant alternative splicing of target genes, some of which may be relevant for MDS pathogenesis.

Clonal diversity of recurrently mutated genes in myelodysplastic syndromes.

Recent studies suggest that most cases of myelodysplastic syndrome (MDS) are clonally heterogeneous, with a founding clone and multiple subclones. It is not known whether specific gene mutations typically occur in founding clones or subclones. We screened a panel of 94 candidate genes in a cohort of 157 patients with MDS or secondary acute myeloid leukemia (sAML). This included 150 cases with samples obtained at MDS diagnosis and 15 cases with samples obtained at sAML transformation (8 were also analyzed at the MDS stage). We performed whole-genome sequencing (WGS) to define the clonal architecture in eight sAML genomes and identified the range of variant allele frequencies (VAFs) for founding clone mutations. At least one mutation or cytogenetic abnormality was detected in 83% of the 150 MDS patients and 17 genes were significantly mutated (false discovery rate ≤0.05). Individual genes and patient samples displayed a wide range of VAFs for recurrently mutated genes, indicating that no single gene is exclusively mutated in the founding clone. The VAFs of recurrently mutated genes did not fully recapitulate the clonal architecture defined by WGS, suggesting that comprehensive sequencing may be required to accurately assess the clonal status of recurrently mutated genes in MDS.

A 3.5 kb deletion in the glycophorin C gene accounts for the Gerbich-negative blood group in Melanesians.

The Gerbich-negative blood group types are rare in most populations, but reach appreciable frequencies in certain Melanesian groups in Papua New Guinea. The recent cloning of the human glycophorin C (GPC) gene, that encodes Gerbich (Ge) blood group antigens, has facilitated study of its genetic variants. We have obtained partial genomic clones of a normal GPC gene, for molecular analysis of Ge: -1, -2, -3 types in Melanesians, and have shown that a 3.5 kb deletion in the GPC gene that removes all of exon 3 accounts for at least one Gerbich-negative phenotype in Melanesians. Population distributions of GPC RFLP have shown that the deletion-type GPC is not confined to mainland Papua New Guinea as previously thought, but occurs sporadically in Melanesians from Fiji as well as in Micronesians.

Powerlessness and the pulmonary alveolar edema patient.

Everyone wants to maintain control over events in their life. The need for personal control does not end when the patient is hospitalized; instead the patient's need for personal control usually intensifies in critical care situations. The nursing diagnosis of powerlessness is common for most critical care patients, and especially so for the patient experiencing respiratory difficulties such as Pulmonary Alveolar Edema. These authors describe a model of powerlessness which suggests strategies for increasing the patient's control over his or her situation.

Effect of random abnormal cell proportion on specimen classifier performance.

A series of papers had analyzed a simplified model of an automated cytology prescreening configuration consisting of a two-class cell classifier followed by a two-class specimen classifier. This has shown, among other things, that the proportion (p) of abnormal cells on an abnormal specimen dictates the number (N) of cells that must be classified before the specimen can be classified with specified accuracy (Anal Quant Cytol, 2:117-122, 1980). It has also shown that if a system designed assuming one fixed value, po, encounters a specimen with a different fixed value, p, then the specimen classifier false negative rate will deviate significantly from the design value, increasing for p < po and vice versa (Cytometry, 2: 155-158, 1981). Using a Gaussian approximation, Timmers and Gelsema (Cytometry, 6:22-25, 1985) extended this to the case where p is a Beta-distributed random variable. They showed that N increases dramatically with the width (coefficient of variation) of the distribution of p. They also concluded that the randomness of p imposes a fundamental lower limit on the specimen false negative rate below which it is impossible to go, even with an error-free cell classifier. In this paper we also extend the basic model to cover the case of random p, but by using an asymptotic expansion (rather than the Gaussian approximation), to develop an expression for N. We show that the limit cited by Timmers and Gelsema is not real, but is actually an artifact of the breakdown of the Gaussian approximation.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary alveolar edema: preventing complications.

The critical care nurse uses a wide range of interventions to prevent or reduce complications for the PAE patient. The goal of the interventions are to enhance cardiac performance, improve oxygenation, and decrease myocardial workload.

Nursing management of high permeability pulmonary oedema.

In summary, the goal in managing HPPE is to recognise its occurrence and initiate appropriate treatment. While there may be a wide range of possible nursing diagnoses that have application to the HPPE patient, eight essential diagnoses were discussed and outcomes identified: impaired gas exchange; ineffective breathing pattern; ineffective airway clearance; cardiac output, alteration in; fluid volume deficit; infection, potential for; coping, ineffective individual: depression; and powerlessness.

Powerlessness and personal control model applied to the myocardial infarction patient.

The MI patient can experience powerlessness through loss of personal control. The feeling of powerlessness can limit the patient's ability to understand the diagnosis of MI, care or decisions necessary to restore health. The MI patient can react by experiencing a sense of physiological, cognitive, environmental and decisional loss of control. Regardless of the specific component of the powerless model, the coronary care nurse diagnoses powerlessness according to defining characteristics. Nursing interventions are organized to facilitate physiological, cognitive, environmental and decisional powerfulness. Research is needed to clarify the MI patient's perception of control or lack of control within each component of the presented model, and to evaluate the effectiveness of nursing interventions created to foster personal control or uncontrol. Research will enable the nurse to scientifically determine strategies and outcomes that correlate with the patient's need for control in the illness situation.

High permeability pulmonary oedema: pathophysiology and mechanisms of injury.

Many critically ill patients are at risk for developing HPPE. Since 60% of patients develop HPPE within 24 hours of the pulmonary insult with 11% developing respiratory failure within 72 hours, it is imperative that the critical care nurse understand the pathophysiological responses (Bernard & Bradley, 1986). While the pathophysiological responses are specific, injury to the alveolar-capillary membrane, the mechanisms of injury are diffuse. Knowing the mechanisms can alert health care providers to those patients who are at risk for developing HPPE and more quickly mobilize interventions to alleviate or lessen its occurrence.

HLA-DR and -DQ DNA polymorphisms: new linkage relationships established by RFLP genomic typing in Polynesians and Melanesians.

Class II restriction fragment length polymorphisms (RFLPs) of DR beta, DQ beta, and DQ alpha loci were examined in Polynesians of the southwest Pacific and in non-Austronesian-speaking Melanesians from the Papua New Guinean Highlands. Polynesians, previously considered to have a restricted set of HLA-DR antigens, showed class II gene heterogeneity associated with DR2, DR5, DRw6, and DRw8 RFLPs. Furthermore, Melanesians and Polynesians share certain antigens such as DRw6 and DRw8, but the DR beta 2 genes associated with DRw6 and the DQ genes associated with DRw8 are population-specific and show little or no overlap. This study has shown that genetic analysis of closely linked polymorphic genes is a powerful anthropological tool and supports the view that Polynesians represent an independent colonizing group in the Pacific, rather than a group evolved from within Melanesia.

The role of prostacyclin and thromboxane in rat burn and freeze injuries.

Deep dermal injuries elicit discrete reaction patterns dependent on the type of injury sustained. Full thickness burn injuries produce an avascular focus of dead and dying tissue surrounded by a peripheral zone of secondary vascular dilation. In contrast, equivalent freeze injuries demonstrate vascular patency both centrally and peripherally. The basis for these differences are unknown. Because of their potent vasoactive and hematologic properties, the presence of two endogenously generated eicosanoids, thromboxane A2 (Tx A2) and prostacyclin (PGI2), were examined in this process. Implanted stainless steel mesh chambers served as an in vivo interstitial collecting reservoir permitting repeated sampling of the wound fluid without tissue disruption. Standard burn and freeze injuries were administered to the skin covering the implanted chambers. The major metabolites of these eicosanoids: 6-keto PGF1 alpha and TxB2 were measured in wound fluid during the first 24 hr following injury. Although both TxB2 and 6-keto PGF1 alpha increased significantly following either injury, treatment with indomethacin did not alter the vascular sequelae despite evident cyclooxygenase inhibition. Latex infusion of whole rats confirmed the considerable difference between these two types of injury, with or without indomethacin. Thus, little evidence was found to support the importance of either TxA2 or PGI2 in the vascular alterations which follow burn or freeze injury.

An expanded cervical cell classification system validated by automated measurements.

The commonly used nomenclature for classifying cervical cells was expanded to include more classes, reflecting finer gradations of disease-related morphologic changes. Using a set of subjective visual criteria developed for this purpose, we visually classified 7,000 randomly selected cells and then subjected them to morphologic measurement by digital image analysis. Several of the measurements showed statistically significant differences among all the cell classes, indicating that it is possible to distinguish the finer morphologic gradations incorporated in the new system of cell classes. These same measurements showed a continuous trend of change from class to class along the scale from borderline dysplasia to carcinoma. This is consistent with the notion of a continuous progression of disease development. Finally, we found that those measurements that reflect the visual criteria used in the manual classification were significantly different between classes, indicating that the computer system can successfully quantify many of the important visual criteria.

Endocervical columnar cell atypia coincident with cervical neoplasia characterized by digital image analysis.

In the course of classifying uterine cervical epithelial cells for digital image analysis, certain changes in endocervical cells were observed. These changes coincided with the process ongoing in the squamous or metaplasia epithelium. Specifically, in severe dysplasias or carcinomas in situ (CIS), the endocervical nuclei reflected some of the same cytologic changes observed in the dysplastic or CIS squamous cells and yet definitely retained their columnar shape and cytoplasmic quality. This paper deals with almost unexplored area of the endocervical columnar cell in the face of significant cervical neoplasia. Correlation is made between the cytologic criteria observed by the human eye with the light microscope and significant parameters measured by digital image analysis. These measurements suggest that endocervical columnar cells may participate in the dysplastic progression toward CIS.

The effect of abnormal cell proportion on specimen classifier performance.

In two previous papers we developed formulas relating the performance (error rates) of a two-class specimen classifier to the performance of a preceding two-class classifier and the number of cells examined (K. R. Castleman and B. S. White, Analytical and Quantitative Cytology 2:117, 1980 and K. R. Castleman and B. S. White, Cytometry 1:156, 1980.). This analysis assumed a certain proportion (p) of abnormal cells on an abnormal specimen. In this paper we examine what happens when a system designed assuming one value of p is presented with a positive specimen having a different abnormal cell proportion. We show that the specimen false negative error rate increases dramatically as p decreases below the design value, and conversely. This suggests that the specimen classification performance of a particular system should be quoted only with reference to the abnormal cell proportion of the specimens used for testing.

The tradeoff of cell classifier error rates.

In gynecologic cytodiagnosis it is generally agreed that specimen false negative errors are more serious than false positives. When classifying individual cells, however, it is not obvious how one should adjust the parameters that control the two cell error rates. In the case where a specimen classifier follows a cell classifier, one can calculate the sample size required to achieve specified overall performance. This analysis shows that for the small abnormal cell proportions encountered in cervical cytology, cell false positives should be kept so low that a substantial portion of the abnormal cells are missed.

Optimizing cervical cell classifiers.

In an automated prescreening system where a cell classifier and a specimen classifier operate in cascade, the false-positive and false-negative error rates of each classifier can be traded off to obtain the best overall performance. It is usually desirable to keep the specimen false-negative rate below the false-positive rate. An analysis of the classifier cascade shows that, in contrast, the cell classifier should have its false-positive rate much lower than its false-negative rate. A procedure is presented for selecting the best operating point on the ROC curve of the cell classifier. This minimizes the sample size required to achieve prescribed specimen error rates.