Clinical characterization of the mutational landscape of 24,639 real-world samples from patients with myeloid malignancies.

Myeloid neoplasms represent a broad spectrum of hematological disorders for which somatic mutation status in key driver genes is important for diagnosis, prognosis and treatment. Here we summarize the findings of a targeted, next generation sequencing laboratory developed test in 24,639 clinical myeloid samples. Data were analyzed comprehensively and as part of individual cohorts specific to acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN). Overall, 48,015 variants were detected, and variants were found in all 50 genes in the panel. The mean number of mutations per patient was 1.95. Mutation number increased with age (Spearman's rank correlation coefficient, ρ = 0.29, P < 0.0001) and was higher in patients with AML than MDS or MPN (Student's t-test, P < 0.0001). TET2 was the most common mutation detected (19.1% of samples; 4,695/24,639) including 7.7% (1,908/24,639) with multi-hit TET2 mutations. Mutation frequency was correlated between patients with cytopenias and MDS (Spearman's, ρ = 0.97, P < 2.2×10-16) with the MDS diagnostic gene SF3B1 being the only notable outlier. This large retrospective study shows the utility of NGS testing to inform clinical decisions during routine clinical care and highlights the mutational landscape of a broad population of myeloid patients.

Assessing CAR T-Cell Therapy Response Using Genome-Wide Sequencing of Cell-Free DNA in Patients With B-Cell Lymphomas.

Methods that enable monitoring of therapeutic efficacy of autologous chimeric antigen receptor (CAR) T-cell therapy will be clinically useful. The aim of this study is to demonstrate the feasibility of blood-derived cell-free DNA (cfDNA) to predict CAR T-cell therapy response in patients with refractory B-cell lymphomas. Whole blood was collected before and throughout CAR T-cell therapy until day 154. Low-coverage (∼0.4×), genome-wide cfDNA sequencing, similar to that established for noninvasive prenatal testing, was performed. The genomic instability number (GIN) was used to quantify plasma copy number alteration level. Twelve patients were enrolled. Seven (58%) patients achieved a complete response (CR); 2 (25%), a partial response. Median progression-free survival was 99 days; median overall survival was not reached (median follow-up, 247 days). Altogether, 127 blood samples were analyzed (median, 10 samples/patient [range 8-13]). All 5 patients who remained in CR at the time of last measurement had GIN <170 (threshold). Two patients who attained CR, but later relapsed, and all but one patient who had best response other than CR had last GIN measurement of >170. In 5 of 6 patients with relapsed or progressive disease, increasing GIN was observed before the diagnosis by imaging. The abundance of CAR T-cell construct (absolute number of construct copies relative to the number of human genome equivalents) also showed a trend to correlate with outcome (day 10, P = .052). These data describe a proof-of-concept for the use of multiple liquid biopsy technologies to monitor therapeutic response in B-cell lymphoma patients receiving CAR T-cell therapy.

Genome-wide Sequencing of Cell-free DNA Enables Detection of Copy-number Alterations in Patients with Cancer Where Tissue Biopsy is Not Feasible.

When tissue biopsy is not medically prudent or tissue is insufficient for molecular testing, alternative methods are needed. Because cell-free DNA (cfDNA) has been shown to provide a representative surrogate for tumor tissue, we sought to evaluate its utility in this clinical scenario. cfDNA was isolated from the plasma of patients and assayed with low-coverage (∼0.3×), genome-wide sequencing. Copy-number alterations (CNA) were identified and characterized using analytic methods originally developed for noninvasive prenatal testing (NIPT) and quantified using the genomic instability number (GIN), a metric that reflects the quantity and magnitude of CNAs across the genome. The technical variability of the GIN was first evaluated in an independent cohort comprising genome-wide sequencing results from 27,754 women who consented to have their samples used for research and whose NIPT results yielded no detected CNAs to establish a detection threshold. Subsequently, cfDNA sequencing data from 96 patients with known cancers but for whom a tissue biopsy could not be obtained are presented. An elevated GIN was detected in 35% of patients and detection rates varied by tumor origin. Collectively, CNAs covered 96.6% of all autosomes. Survival was significantly reduced in patients with an elevated GIN relative to those without. Overall, these data provide a proof of concept for the use of low-coverage, genome-wide sequencing of cfDNA from patients with cancer to obtain relevant molecular information in instances where tissue is difficult to access. These data may ultimately serve as an informative complement to other molecular tests.

Correction: Blood RNA analysis can increase clinical diagnostic rate and resolve variants of uncertain significance.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Blood RNA analysis can increase clinical diagnostic rate and resolve variants of uncertain significance.

PURPOSE: Diagnosis of genetic disorders is hampered by large numbers of variants of uncertain significance (VUSs) identified through next-generation sequencing. Many such variants may disrupt normal RNA splicing. We examined effects on splicing of a large cohort of clinically identified variants and compared performance of bioinformatic splicing prediction tools commonly used in diagnostic laboratories. METHODS: Two hundred fifty-seven variants (coding and noncoding) were referred for analysis across three laboratories. Blood RNA samples underwent targeted reverse transcription polymerase chain reaction (RT-PCR) analysis with Sanger sequencing of PCR products and agarose gel electrophoresis. Seventeen samples also underwent transcriptome-wide RNA sequencing with targeted splicing analysis based on Sashimi plot visualization. Bioinformatic splicing predictions were obtained using Alamut, HSF 3.1, and SpliceAI software. RESULTS: Eighty-five variants (33%) were associated with abnormal splicing. The most frequent abnormality was upstream exon skipping (39/85 variants), which was most often associated with splice donor region variants. SpliceAI had greatest accuracy in predicting splicing abnormalities (0.91) and outperformed other tools in sensitivity and specificity. CONCLUSION: Splicing analysis of blood RNA identifies diagnostically important splicing abnormalities and clarifies functional effects of a significant proportion of VUSs. Bioinformatic predictions are improving but still make significant errors. RNA analysis should therefore be routinely considered in genetic disease diagnostics.

Maternal Obesity during Pregnancy Alters Daily Activity and Feeding Cycles, and Hypothalamic Clock Gene Expression in Adult Male Mouse Offspring.

An obesogenic diet adversely affects the endogenous mammalian circadian clock, altering daily activity and metabolism, and resulting in obesity. We investigated whether an obese pregnancy can alter the molecular clock in the offspring hypothalamus, resulting in changes to their activity and feeding rhythms. Female mice were fed a control (C, 7% kcal fat) or high fat diet (HF, 45% kcal fat) before mating and throughout pregnancy. Male offspring were fed the C or HF diet postweaning, resulting in four offspring groups: C/C, C/HF, HF/C, and HF/HF. Daily activity and food intake were monitored, and at 15 weeks of age were killed at six time-points over 24 h. The clock genes Clock, Bmal1, Per2, and Cry2 in the suprachiasmatic nucleus (SCN) and appetite genes Npy and Pomc in the arcuate nucleus (ARC) were measured. Daily activity and feeding cycles in the HF/C, C/HF, and HF/HF offspring were altered, with increased feeding bouts and activity during the day and increased food intake but reduced activity at night. Gene expression patterns and levels of Clock, Bmal1, Per2, and Cry2 in the SCN and Npy and Pomc in the ARC were altered in HF diet-exposed offspring. The altered expression of hypothalamic molecular clock components and appetite genes, together with changes in activity and feeding rhythms, could be contributing to offspring obesity.

High-Risk Human Papillomavirus Detection in Urine Samples From a Referral Population With Cervical Biopsy-Proven High-Grade Lesions.

The aim of the study was to evaluate the performance of the HPV-HR test to detect high-risk human papillomavirus (HPV) in urine samples in comparison with a commercial molecular HPV test. MATERIALS AND METHODS: This is a prospective study, in which 350 patients diagnosed previously with cervical intraepithelial neoplasia (CIN) grade 2 or higher were enrolled. Urine and cervical specimens were collected. Urine was tested with the HPV-HR test and cervical specimens were tested with the Cobas. RESULTS: Of the 336 evaluable patients, there were 271 cases of CIN 2+, of which 202 were CIN 3+ and the remaining 65 patients were less than CIN 2. Positivity was 77.1% (95% confidence interval [CI] = 72.5-81.5) for the urine samples and 83.6% (95% CI = 79.6-87.6) for the cervical samples. Agreement between cervical and urine samples for HPV detection was 79.8% (κ = 0.363; 95% CI = 0.243-0.484). Sensitivity for CIN 2+ was 83.4% (95% CI = 78.4-87.6) for urine and 90.8% (95% CI = 86.7-92.9) for cervical samples. The sensitivity for CIN 3+ was 85.6% (95% CI = 80.0-90.2) for urine and 92.6% (95% CI = 88.0-95.8) for cervical samples. Specificity for worse than CIN 2 was 50.8% (95% CI = 33.7-59.0) and 46.2% (95% CI = 33.7-59.0) for urine and cervical samples, respectively. CONCLUSIONS: Although these results demonstrated slightly higher detection rates for HR-HPV and clinical sensitivity in cervical samples than in urine, when compared with histological diagnoses, urine sampling is a viable alternative to access women who do not participate in routine screening programs.

Genomic profiling of ER+ breast cancers after short-term estrogen suppression reveals alterations associated with endocrine resistance.

Inhibition of proliferation in estrogen receptor-positive (ER+) breast cancers after short-term antiestrogen therapy correlates with long-term patient outcome. We profiled 155 ER+/human epidermal growth factor receptor 2-negative (HER2-) early breast cancers from 143 patients treated with the aromatase inhibitor letrozole for 10 to 21 days before surgery. Twenty-one percent of tumors remained highly proliferative, suggesting that these tumors harbor alterations associated with intrinsic endocrine therapy resistance. Whole-exome sequencing revealed a correlation between 8p11-12 and 11q13 gene amplifications, including FGFR1 and CCND1, respectively, and high Ki67. We corroborated these findings in a separate cohort of serial pretreatment, postneoadjuvant chemotherapy, and recurrent ER+ tumors. Combined inhibition of FGFR1 and CDK4/6 reversed antiestrogen resistance in ER+FGFR1/CCND1 coamplified CAMA1 breast cancer cells. RNA sequencing of letrozole-treated tumors revealed the existence of intrachromosomal ESR1 fusion transcripts and increased expression of gene signatures indicative of enhanced E2F-mediated transcription and cell cycle processes in cancers with high Ki67. These data suggest that short-term preoperative estrogen deprivation followed by genomic profiling can be used to identify druggable alterations that may cause intrinsic endocrine therapy resistance.

A novel Bcr-Abl-mTOR-eIF4A axis regulates IRES-mediated translation of LEF-1.

Internal ribosome entry sites (IRESs) in cellular mRNAs direct expression of growth-promoting factors through an alternative translation mechanism that has yet to be fully defined. Lymphoid enhancer factor-1 (LEF-1), a Wnt-mediating transcription factor important for cell survival and metastasis in cancer, is produced via IRES-directed translation, and its mRNA is frequently upregulated in malignancies, including chronic myeloid leukaemia (CML). In this study, we determined that LEF1 expression is regulated by Bcr-Abl, the oncogenic protein that drives haematopoietic cell transformation to CML. We have previously shown that the LEF1 5' untranslated region recruits a complex of proteins to its IRES, including the translation initiation factor eIF4A. In this report, we use two small molecule inhibitors, PP242 (dual mTOR (mammalian target of rapamycin) kinase inhibitor) and hippuristanol (eIF4A inhibitor), to define IRES regulation via a Bcr-Abl-mTOR-eIF4A axis in CML cell lines and primary patient leukaemias. We found that LEF1 and other IRESs are uniquely sensitive to the activities of Bcr-Abl/mTOR. Most notably, we discovered that eIF4A, an RNA helicase, elicits potent non-canonical effects on the LEF1 IRES. Hippuristanol inhibition of eIF4A stalls translation of IRES mRNA and triggers dissociation from polyribosomes. We propose that a combination drug strategy which targets mTOR and IRES-driven translation disrupts key factors that contribute to growth and proliferation in CML.

Poliovirus infection induces the co-localization of cellular protein SRp20 with TIA-1, a cytoplasmic stress granule protein.

Different types of environmental stress cause mammalian cells to form cytoplasmic foci, termed stress granules, which contain mRNPs that are translationally silenced. These foci are transient and dynamic, and contain components of the cellular translation machinery as well as certain mRNAs and RNA binding proteins. Stress granules are known to be induced by conditions such as hypoxia, nutrient deprivation, and oxidative stress, and a number of cellular factors have been identified that are commonly associated with these foci. More recently it was discovered that poliovirus infection also induces the formation of stress granules, although these cytoplasmic foci appear to be somewhat compositionally unique. Work described here examined the punctate pattern of SRp20 (a host cell mRNA splicing protein) localization in the cytoplasm of poliovirus-infected cells, demonstrating the partial co-localization of SRp20 with the stress granule marker protein TIA-1. We determined that SRp20 does not co-localize with TIA-1, however, under conditions of oxidative stress, indicating that the close association of these two proteins during poliovirus infection is not representative of a general response to cellular stress. We confirmed that the expression of a dominant negative version of TIA-1 (TIA-1-PRD) results in the dissociation of stress granules. Finally, we demonstrated that expression of wild type TIA-1 or dominant negative TIA-1-PRD in cells during poliovirus infection does not dramatically affect viral translation. Taken together, these studies provide a new example of the unique cytoplasmic foci that form during poliovirus infection.

Viral proteinase requirements for the nucleocytoplasmic relocalization of cellular splicing factor SRp20 during picornavirus infections.

Infection of mammalian cells by picornaviruses results in the nucleocytoplasmic redistribution of certain host cell proteins. These viruses interfere with import-export pathways, allowing for the cytoplasmic accumulation of nuclear proteins that are then available to function in viral processes. We recently described the cytoplasmic relocalization of cellular splicing factor SRp20 during poliovirus infection. SRp20 is an important internal ribosome entry site (IRES) trans-acting factor (ITAF) for poliovirus IRES-mediated translation; however, it is not known whether other picornaviruses utilize SRp20 as an ITAF and direct its cytoplasmic relocalization. Also, the mechanism by which poliovirus directs the accumulation of SRp20 in the cytoplasm of the infected cell is currently unknown. Work described in this report demonstrated that infection by another picornavirus (coxsackievirus B3) causes SRp20 to relocalize from the nucleus to the cytoplasm of HeLa cells, similar to poliovirus infection; however, SRp20 is relocalized to a somewhat lesser extent in the cytoplasm of HeLa cells during infection by yet another picornavirus (human rhinovirus 16). We show that expression of poliovirus 2A proteinase is sufficient to cause the nucleocytoplasmic redistribution of SRp20. Following expression of poliovirus 2A proteinase in HeLa cells, we detect cleavage of specific nuclear pore proteins known to be cleaved during poliovirus infection. We also find that expression of human rhinovirus 16 2A proteinase alone can cause efficient cytoplasmic relocalization of SRp20, despite the lower levels of SRp20 relocalization observed during rhinovirus infection compared to poliovirus. Taken together, these results further define the mechanism of SRp20 cellular redistribution during picornavirus infections, and they provide additional insight into some of the differences observed between human rhinovirus and other enterovirus infections.

An RNA virus hijacks an incognito function of a DNA repair enzyme.

A previously described mammalian cell activity, called VPg unlinkase, specifically cleaves a unique protein-RNA covalent linkage generated during the viral genomic RNA replication steps of a picornavirus infection. For over three decades, the identity of this cellular activity and its normal role in the uninfected cell had remained elusive. Here we report the purification and identification of VPg unlinkase as the DNA repair enzyme, 5'-tyrosyl-DNA phosphodiesterase-2 (TDP2). Our data show that VPg unlinkase activity in different mammalian cell lines correlates with their differential expression of TDP2. Furthermore, we show that recombinant TDP2 can cleave the protein-RNA linkage generated by different picornaviruses without impairing the integrity of viral RNA. Our results reveal a unique RNA repair-like function for TDP2 and suggest an unusual role in host-pathogen interactions for this cellular enzyme. On the basis of the identification of TDP2 as a potential antiviral target, our findings may lead to the development of universal therapeutics to treat the millions of individuals afflicted annually with diseases caused by picornaviruses, including myocarditis, aseptic meningitis, encephalitis, hepatitis, and the common cold.

Re-localization of cellular protein SRp20 during poliovirus infection: bridging a viral IRES to the host cell translation apparatus.

Poliovirus IRES-mediated translation requires the functions of certain canonical as well as non-canonical factors for the recruitment of ribosomes to the viral RNA. The interaction of cellular proteins PCBP2 and SRp20 in extracts from poliovirus-infected cells has been previously described, and these two proteins were shown to function synergistically in viral translation. To further define the mechanism of ribosome recruitment for the initiation of poliovirus IRES-dependent translation, we focused on the role of the interaction between cellular proteins PCBP2 and SRp20. Work described here demonstrates that SRp20 dramatically re-localizes from the nucleus to the cytoplasm of poliovirus-infected neuroblastoma cells during the course of infection. Importantly, SRp20 partially co-localizes with PCBP2 in the cytoplasm of infected cells, corroborating our previous in vitro interaction data. In addition, the data presented implicate the presence of these two proteins in viral translation initiation complexes. We show that in extracts from poliovirus-infected cells, SRp20 is associated with PCBP2 bound to poliovirus RNA, indicating that this interaction occurs on the viral RNA. Finally, we generated a mutated version of SRp20 lacking the RNA recognition motif (SRp20ΔRRM) and found that this protein is localized similar to the full length SRp20, and also partially co-localizes with PCBP2 during poliovirus infection. Expression of this mutated version of SRp20 results in a ∼100 fold decrease in virus yield for poliovirus when compared to expression of wild type SRp20, possibly via a dominant negative effect. Taken together, these results are consistent with a model in which SRp20 interacts with PCBP2 bound to the viral RNA, and this interaction functions to recruit ribosomes to the viral RNA in a direct or indirect manner, with the participation of additional protein-protein or protein-RNA interactions.

Stress-inducible alternative translation initiation of human cytomegalovirus latency protein pUL138.

We have previously characterized a 21-kDa protein encoded by UL138 (pUL138) as a viral factor inherent to low-passage strains of human cytomegalovirus (HCMV) that is required for latent infection in vitro. pUL138 is encoded on 3.6-, 2.7-, and 1.4-kb 3' coterminal transcripts that are produced during productive and latent infections. pUL138 is encoded at the 3' end of each transcript and is preceded by an extensive 5' sequence (approximately 0.5 to 2.5 kb) containing several putative open reading frames (ORFs). We determined that three putative ORFs upstream of UL138 (UL133, UL135, and UL136) encode proteins. The UL138 transcripts are polycistronic, such that each transcript expresses pUL138 in addition to the most-5' ORF. The upstream coding sequences (CDS) present a significant challenge for the translation of pUL138 in mammalian cells. We hypothesized that sequences 5' of UL138 mediate translation initiation of pUL138 by alternative strategies. Accordingly, a 663-nucloetide (nt) sequence overlapping the UL136 CDS supported expression of a downstream cistron in a bicistronic reporter system. We did not detect cryptic promoter activity or RNA splicing events that could account for downstream cistron expression. These data are consistent with the sequence element functioning as an internal ribosome entry site (IRES). Interestingly, pUL138 expression from the 3.6- and 2.7-kb transcripts was induced by serum stress, which concomitantly inhibited normal cap-dependent translation. Our work suggests that an alternative and stress-inducible strategy of translation initiation ensures expression of pUL138 under a variety of cellular contexts. The UL138 polycistronic transcripts serve to coordinate the expression of multiple proteins, including a viral determinant of HCMV latency.

Bridging IRES elements in mRNAs to the eukaryotic translation apparatus.

IRES elements are highly structured RNA sequences that function to recruit ribosomes for the initiation of translation. In contrast to the canonical cap-binding, ribosome-scanning model, the mechanism of IRES-mediated translation initiation is not well understood. IRES elements, first discovered in viral RNA genomes, were subsequently found in a subset of cellular RNAs as well. Interestingly, these cellular IRES-containing mRNAs appear to play important roles during conditions of cellular stress, development, and disease (e.g., cancer). It has been shown for viral IRESes that some require specific IRES trans-acting factors (ITAFs), while others require few if any additional proteins and can bind ribosomes directly. Current studies are aimed at elucidating the mechanism of IRES-mediated translation initiation and features that may be common or differ greatly among cellular and viral IRESes. This review will explore IRES elements as important RNA structures that function in both cellular and viral RNA translation and the significance of these structures in providing an alternative mechanism of eukaryotic translation initiation.

Embryonic even skipped-dependent muscle and heart cell fates are required for normal adult activity, heart function, and lifespan.

The Drosophila pair-rule gene even skipped (eve) is required for embryonic segmentation and later in specific cell lineages in both the nervous system and the mesoderm. We previously generated eve mesoderm-specific mutants by combining an eve null mutant with a rescuing transgene that includes the entire locus, but with the mesodermal enhancer removed. This allowed us to analyze in detail the defects that result from a precisely targeted elimination of mesodermal eve expression in the context of an otherwise normal embryo. Absence of mesodermal eve causes a highly selective loss of the entire eve-expressing lineage in this germ layer, including those progeny that do not continue to express eve, suggesting that mesodermal eve precursor specification is not implemented. Despite the resulting absence of a subset of muscles and pericardial cells, mesoderm-specific eve mutants survive to fertile adulthood, providing an opportunity to examine the effects of these developmental abnormalities on adult fitness and heart function. We find that in these mutants, flying ability, myocardial performance under normal and stressed conditions, and lifespan are severely reduced. These data imply a nonautonomous role of the affected pericardial cells and body wall muscles in developing and/or maintaining cardiac performance and possibly other functions contributing to normal lifespan. Given the similarities of molecular-genetic control between Drosophila and vertebrates, these findings suggest that peri/epicardial influences may well be important for proper myocardial function.