PKC signaling contributes to chromatin decondensation and is required for competence to respond to IL-2 during T cell activation.

The clonal proliferation of antigen-specific T cells during an immune response critically depends on the differential response to growth factors, such as IL-2. While activated T cells proliferate robustly in response to IL-2 stimulation, naïve (quiescent) T cells are able to ignore the potent effects of growth factors because they possess chromatin that is tightly condensed such that transcription factors, such as STAT5, cannot access DNA. Activation via the T cell receptor (TCR) induces a rapid decondensation of chromatin, permitting STAT5-DNA engagement and ultimately promoting proliferation of only antigen-specific T cells. Previous work demonstrated that the mobilization of intracellular calcium following TCR stimulation is a key event in the decondensation of chromatin. Here we examine PKC-dependent signaling mechanisms to determine their role in activation-induced chromatin decondensation and the subsequent acquisition of competence to respond to IL-2 stimulation. We found that a calcium-dependent PKC contributes to activation-induced chromatin decondensation and that the p38 MAPK and NFκB pathways downstream of PKC each contribute to regulating the proper decondensation of chromatin. Importantly, we found that p44/42 MAPK activity is required for peripheral T cells to gain competence to properly respond to IL-2 stimulation. Our findings shed light on the mechanisms that control the clonal proliferation of antigen-specific peripheral T cells during an immune response.

A (fire)cloud-based DNA methylation data preprocessing and quality control platform.

BACKGROUND: Bisulfite sequencing allows base-pair resolution profiling of DNA methylation and has recently been adapted for use in single-cells. Analyzing these data, including making comparisons with existing data, remains challenging due to the scale of the data and differences in preprocessing methods between published datasets. RESULTS: We present a set of preprocessing pipelines for bisulfite sequencing DNA methylation data that include a new R/Bioconductor package, scmeth, for a series of efficient QC analyses of large datasets. The pipelines go from raw data to CpG-level methylation estimates and can be run, with identical results, either on a single computer, in an HPC cluster or on Google Cloud Compute resources. These pipelines are designed to allow users to 1) ensure reproducibility of analyses, 2) achieve scalability to large whole genome datasets with 100 GB+ of raw data per sample and to single-cell datasets with thousands of cells, 3) enable integration and comparison between user-provided data and publicly available data, as all samples can be processed through the same pipeline, and 4) access to best-practice analysis pipelines. Pipelines are provided for whole genome bisulfite sequencing (WGBS), reduced representation bisulfite sequencing (RRBS) and hybrid selection (capture) bisulfite sequencing (HSBS). CONCLUSIONS: The workflows produce data quality metrics, visualization tracks, and aggregated output for further downstream analysis. Optional use of cloud computing resources facilitates analysis of large datasets, and integration with existing methylome profiles. The workflow design principles are applicable to other genomic data types.

The rs387907171 SNP in TYRP1 is not associated with blond hair color on the Island of Bougainville.

OBJECTIVES: The aims of this study are to characterize the frequency of the derived allele at rs387907171 in populations from the islands of New Britain and Bougainville in Northern Island Melanesia, to confirm its association with lighter hair color, and to refine hypotheses regarding its evolutionary history. METHODS: rs387907171 was genotyped in 93 individuals from New Britain and 101 from Bougainville for whom quantitative assessments of skin and hair pigmentation were available. Combining these with existing data from other Melanesian islands we tested for differences in allele frequencies between islands and for associations with skin and hair pigmentation using ANOVA, including sex, age, and island affiliations as covariates. RESULTS: The derived allele at rs387907171 was observed in a single copy in the New Britain and Bougainville populations genotyped here. Its frequency differs significantly among islands in the region (χ(2) = 206.5, df = 3, P < 0.001). rs387907171 remains significantly, although weakly, associated with lighter hair pigmentation (F = 10.28, R(2) = 0.0125, P = 0.0014). This association increases when sex and age (F = 20.68, R(2) = 0.074, P < 7.92 × 10(-13) ) are included as covariates. CONCLUSIONS: The rs387907171 SNP exhibits strong allele frequency differences among islands in Northern Island Melanesia. Its absence from Bougainville, as well as the weak association with decreased hair color, indicates that additional alleles contribute to the blondism phenotype. Its geographic distribution suggests that a Lapita-mediated model for the dispersal of the derived allele at rs387907171 remains a viable evolutionary scenario. Am. J. Hum. Biol. 28:431-435, 2016. © 2015 Wiley Periodicals, Inc.

Loss of ATRX, genome instability, and an altered DNA damage response are hallmarks of the alternative lengthening of telomeres pathway.

The Alternative Lengthening of Telomeres (ALT) pathway is a telomerase-independent pathway for telomere maintenance that is active in a significant subset of human cancers and in vitro immortalized cell lines. ALT is thought to involve templated extension of telomeres through homologous recombination, but the genetic or epigenetic changes that unleash ALT are not known. Recently, mutations in the ATRX/DAXX chromatin remodeling complex and histone H3.3 were found to correlate with features of ALT in pancreatic neuroendocrine cancers, pediatric glioblastomas, and other tumors of the central nervous system, suggesting that these mutations might contribute to the activation of the ALT pathway in these cancers. We have taken a comprehensive approach to deciphering ALT by applying genomic, molecular biological, and cell biological approaches to a panel of 22 ALT cell lines, including cell lines derived in vitro. Here we show that loss of ATRX protein and mutations in the ATRX gene are hallmarks of ALT-immortalized cell lines. In addition, ALT is associated with extensive genome rearrangements, marked micronucleation, defects in the G2/M checkpoint, and altered double-strand break (DSB) repair. These attributes will facilitate the diagnosis and treatment of ALT positive human cancers.

Activating mutations in ALK provide a therapeutic target in neuroblastoma.

Neuroblastoma, an embryonal tumour of the peripheral sympathetic nervous system, accounts for approximately 15% of all deaths due to childhood cancer. High-risk neuroblastomas are rapidly progressive; even with intensive myeloablative chemotherapy, relapse is common and almost uniformly fatal. Here we report the detection of previously unknown mutations in the ALK gene, which encodes a receptor tyrosine kinase, in 8% of primary neuroblastomas. Five non-synonymous sequence variations were identified in the kinase domain of ALK, of which three were somatic and two were germ line. The most frequent mutation, F1174L, was also identified in three different neuroblastoma cell lines. ALK complementary DNAs encoding the F1174L and R1275Q variants, but not the wild-type ALK cDNA, transformed interleukin-3-dependent murine haematopoietic Ba/F3 cells to cytokine-independent growth. Ba/F3 cells expressing these mutations were sensitive to the small-molecule inhibitor of ALK, TAE684 (ref. 4). Furthermore, two human neuroblastoma cell lines harbouring the F1174L mutation were also sensitive to the inhibitor. Cytotoxicity was associated with increased amounts of apoptosis as measured by TdT-mediated dUTP nick end labelling (TUNEL). Short hairpin RNA (shRNA)-mediated knockdown of ALK expression in neuroblastoma cell lines with the F1174L mutation also resulted in apoptosis and impaired cell proliferation. Thus, activating alleles of the ALK receptor tyrosine kinase are present in primary neuroblastoma tumours and in established neuroblastoma cell lines, and confer sensitivity to ALK inhibition with small molecules, providing a molecular rationale for targeted therapy of this disease.

A Rare Presentation of Lyme Disease in an Immunocompromised Patient.

Lyme disease is a progressive infectious disease caused by the Borrelia species that affects multiple organ systems, including the brain, heart, skin, and musculoskeletal systems. The cardiac manifestations of Lyme disease typically present with atrioventricular nodal conduction abnormalities and, more rarely, myocarditis. We report a case of an immunocompromised 57-year-old woman who presented with acute onset shortness of breath, hypervolemia, injective conjunctiva, and global vision loss of the left eye in the setting of a recent tick bite. Serologic testing confirmed borreliosis, and cardiac testing demonstrated acute isolated systolic heart failure without any cardiac conduction system abnormalities on the electrocardiogram. The diagnosis of Lyme carditis was made, and the patient was started on doxycycline with complete recovery of cardiac systolic function. This case demonstrates atypical cardiac manifestations of Lyme disease and highlights the difficulty in workup and understanding of Lyme carditis particularly in immunocompromised patients.

Characterizing the cancer genome in lung adenocarcinoma.

Somatic alterations in cellular DNA underlie almost all human cancers. The prospect of targeted therapies and the development of high-resolution, genome-wide approaches are now spurring systematic efforts to characterize cancer genomes. Here we report a large-scale project to characterize copy-number alterations in primary lung adenocarcinomas. By analysis of a large collection of tumours (n = 371) using dense single nucleotide polymorphism arrays, we identify a total of 57 significantly recurrent events. We find that 26 of 39 autosomal chromosome arms show consistent large-scale copy-number gain or loss, of which only a handful have been linked to a specific gene. We also identify 31 recurrent focal events, including 24 amplifications and 7 homozygous deletions. Only six of these focal events are currently associated with known mutations in lung carcinomas. The most common event, amplification of chromosome 14q13.3, is found in approximately 12% of samples. On the basis of genomic and functional analyses, we identify NKX2-1 (NK2 homeobox 1, also called TITF1), which lies in the minimal 14q13.3 amplification interval and encodes a lineage-specific transcription factor, as a novel candidate proto-oncogene involved in a significant fraction of lung adenocarcinomas. More generally, our results indicate that many of the genes that are involved in lung adenocarcinoma remain to be discovered.

An Unexpected Turn of Events in a Patient With Mitral Valve Endocarditis.

A variety of gram-positive infections can be treated with daptomycin. Daptomycin-induced acute eosinophilic pneumonia (DEP) is a rare adverse drug reaction with nonspecific clinical findings of dyspnea, dry cough, and fever. Although diagnostic criteria exist, prompt recognition is important to prevent rapid progression and respiratory failure. In this case, a 69-year-old female was initially admitted due to a prosthetic joint infection; however, her case was complicated by DEP.

Hemorrhagic Pericardial Effusion as the Presenting Symptom of Newly Diagnosed Rheumatoid Arthritis.

Hemorrhagic pericardial effusion is a rare presenting sign of undiagnosed rheumatoid arthritis (RA). We present a case of a 58-year-old female with a history of mucinous cystadenoma with subsequent omental caking status-post small bowel resection, chronic intermittent bilateral knee pain, carpal tunnel syndrome of the left hand, and drainage of a peritoneal inclusion cyst two days prior to admission. The patient had pleuritic chest pain and acute-onset shortness of breath but was hemodynamically stable on presentation. Transthoracic echocardiogram and CT scan demonstrated a large pericardial effusion measuring 1.5 cm anteriorly, 2.21 cm posteriorly, and 2.5 cm laterally. Diagnostic pericardiocentesis revealed a hemorrhagic pericardial fluid with a glucose level of 133 mg/dL, pH of 7.34, albumin of 2.6 g/dL, red blood cell count of 401,000 cells per cubic millimeters (CUMM), white blood cell count of 1,400 CUMM, lactate dehydrogenase (LDH) of 930 U/L, and protein of 5 g/dL. Infectious and malignancy workups were negative. Rheumatologic workup was positive for elevated rheumatoid factor and anti-cyclic citrullinated peptide. The patient was diagnosed with RA; she was started on methotrexate with folic acid, and a pericardial drain was kept in place for three days. We present a brief review of the workup, etiologies, and therapeutic approach for patients who present with hemorrhagic pericardial effusion secondary to undiagnosed RA.

Drug-sensitive FGFR2 mutations in endometrial carcinoma.

Oncogenic activation of tyrosine kinases is a common mechanism of carcinogenesis and, given the druggable nature of these enzymes, an attractive target for anticancer therapy. Here, we show that somatic mutations of the fibroblast growth factor receptor 2 (FGFR2) tyrosine kinase gene, FGFR2, are present in 12% of endometrial carcinomas, with additional instances found in lung squamous cell carcinoma and cervical carcinoma. These FGFR2 mutations, many of which are identical to mutations associated with congenital craniofacial developmental disorders, are constitutively activated and oncogenic when ectopically expressed in NIH 3T3 cells. Inhibition of FGFR2 kinase activity in endometrial carcinoma cell lines bearing such FGFR2 mutations inhibits transformation and survival, implicating FGFR2 as a novel therapeutic target in endometrial carcinoma.

The perils of recreational marijuana use: relationships with mental health among emergency department patients.

INTRODUCTION: Marijuana is a commonly used drug in the United States. Many states have legalized the recreational use of marijuana. The effects of marijuana on mental health are unknown. METHODS: In this prospective survey study, eligible participants included ED patients age 18 and older, who had ever used recreational marijuana. A survey instrument was developed, piloted, and revised. Data collected included reasons for marijuana use, marijuana's perceived effectiveness, and history of mental health conditions, including depression, anxiety, and suicidal thoughts. RESULTS: Among 303 participants (86% response rate), the median age of first marijuana use was 16 ([IQR 14, 19], range 6-65). The most commonly cited reasons for marijuana use included recreational use (70%; n = 211), to treat anxiety (30%; n = 89), to treat pain (25%; n = 74), and to treat depression (17%; n = 51). Mental health issues were common in the study population. A majority of patients reported anxiety in the last 30 days (59%; n = 176), and a significant minority of patients reported serious depression in the last 30 days (46%; n = 137). Some patients reported suicidal thoughts in the last 30 days (9%; n = 29). Participants who used marijuana more frequently reported more days of anxiety (median 15.5, compared to 1; P = 0.001). Among participants with mental health conditions, most began using marijuana before the onset of the mental health conditions (77%, n = 167). Earlier age of starting to use marijuana was correlated with higher number of years of anxiety or tension in lifetime (r = -0.11, P = 0.05, n = 301). Perceived effects of marijuana use on mental health were variable. Most participants stated that marijuana improved their mental health (62%; n = 163), and some reported that marijuana did not improve their mental health (37%; n = 98). CONCLUSIONS: Many ED patients have used marijuana, either currently or in the past. Mental health conditions are also common, including anxiety, depression, and suicidal thoughts. Most participants reported marijuana use starting at an age under 18. Marijuana use preceded the onset of mental health conditions in the majority of participants.

Author Correction: Liquid versus tissue biopsy for detecting acquired resistance and tumor heterogeneity in gastrointestinal cancers.

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

Liquid versus tissue biopsy for detecting acquired resistance and tumor heterogeneity in gastrointestinal cancers.

During cancer therapy, tumor heterogeneity can drive the evolution of multiple tumor subclones harboring unique resistance mechanisms in an individual patient1-3. Previous case reports and small case series have suggested that liquid biopsy (specifically, cell-free DNA (cfDNA)) may better capture the heterogeneity of acquired resistance4-8. However, the effectiveness of cfDNA versus standard single-lesion tumor biopsies has not been directly compared in larger-scale prospective cohorts of patients following progression on targeted therapy. Here, in a prospective cohort of 42 patients with molecularly defined gastrointestinal cancers and acquired resistance to targeted therapy, direct comparison of postprogression cfDNA versus tumor biopsy revealed that cfDNA more frequently identified clinically relevant resistance alterations and multiple resistance mechanisms, detecting resistance alterations not found in the matched tumor biopsy in 78% of cases. Whole-exome sequencing of serial cfDNA, tumor biopsies and rapid autopsy specimens elucidated substantial geographic and evolutionary differences across lesions. Our data suggest that acquired resistance is frequently characterized by profound tumor heterogeneity, and that the emergence of multiple resistance alterations in an individual patient may represent the 'rule' rather than the 'exception'. These findings have profound therapeutic implications and highlight the potential advantages of cfDNA over tissue biopsy in the setting of acquired resistance.

Comparison of Prevalence and Types of Mutations in Lung Cancers Among Black and White Populations.

IMPORTANCE: Lung cancer is the leading cause of cancer death in the United States in all ethnic and racial groups. The overall death rate from lung cancer is higher in black patients than in white patients. OBJECTIVE: To compare the prevalence and types of somatic alterations between lung cancers from black patients and white patients. Differences in mutational frequencies could illuminate differences in prognosis and lead to the reduction of outcome disparities by more precisely targeting patients' treatment. DESIGN, SETTING, AND PARTICIPANTS: Tumor specimens were collected from Baptist Cancer Center (Memphis, Tennessee) over the course of 9 years (January 2004-December 2012). Genomic analysis by massively parallel sequencing of 504 cancer genes was performed at Dana-Farber Cancer Institute (Boston, Massachusetts). Overall, 509 lung cancer tumors specimens (319 adenocarcinomas; 142 squamous cell carcinomas) were profiled from 245 black patients and 264 white patients. MAIN OUTCOMES AND MEASURES: The frequencies of genomic alterations were compared between tumors from black and white populations. RESULTS: Overall, 509 lung cancers were collected and analyzed (273 women [129 black patients; 144 white patients] and 236 men [116 black patients; 120 white patients]). Using 313 adenocarcinomas and 138 squamous cell carcinomas with genetically supported ancestry, overall mutational frequencies and copy number changes were not significantly different between black and white populations in either tumor type after correcting for multiple hypothesis testing. Furthermore, specific activating alterations in members of the receptor tyrosine kinase/Ras/Raf pathway including EGFR and KRAS were not significantly different between populations in lung adenocarcinoma. CONCLUSIONS AND RELEVANCE: These results demonstrate that lung cancers from black patients are similar to cancers from white patients with respect to clinically actionable genomic alterations and suggest that clinical trials of targeted therapies could significantly benefit patients in both groups.

Colorectal cancers from distinct ancestral populations show variations in BRAF mutation frequency.

It has been demonstrated for some cancers that the frequency of somatic oncogenic mutations may vary in ancestral populations. To determine whether key driver alterations might occur at different frequencies in colorectal cancer, we applied a high-throughput genotyping platform (OncoMap) to query 385 mutations across 33 known cancer genes in colorectal cancer DNA from 83 Asian, 149 Black and 195 White patients. We found that Asian patients had fewer canonical oncogenic mutations in the genes tested (60% vs Black 79% (P = 0.011) and White 77% (P = 0.015)), and that BRAF mutations occurred at a higher frequency in White patients (17% vs Asian 4% (P = 0.004) and Black 7% (P = 0.014)). These results suggest that the use of genomic approaches to elucidate the different ancestral determinants harbored by patient populations may help to more precisely and effectively treat colorectal cancer.

Structural, biochemical, and clinical characterization of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in lung cancer.

Epidermal growth factor receptor (EGFR) gene mutations (G719X, exon 19 deletions/insertions, L858R, and L861Q) predict favorable responses to EGFR tyrosine kinase inhibitors (TKIs) in advanced non-small cell lung cancer (NSCLC). However, EGFR exon 20 insertion mutations (~10% of all EGFR mutations) are generally associated with insensitivity to available TKIs (gefitinib, erlotinib, and afatinib). The basis of this primary resistance is poorly understood. We studied a broad subset of exon 20 insertion mutations, comparing in vitro TKI sensitivity with responses to gefitinib and erlotinib in NSCLC patients, and found that most are resistant to EGFR TKIs. The crystal structure of a representative TKI-insensitive mutant (D770_N771insNPG) reveals an unaltered adenosine triphosphate-binding pocket, and the inserted residues form a wedge at the end of the C helix that promotes the active kinase conformation. Unlike EGFR-L858R, D770_N771insNPG activates EGFR without increasing its affinity for EGFR TKIs. Unexpectedly, we find that EGFR-A763_Y764insFQEA is highly sensitive to EGFR TKIs in vitro, and patients whose NSCLCs harbor this mutation respond to erlotinib. Analysis of the A763_Y764insFQEA mutant indicates that the inserted residues shift the register of the C helix in the N-terminal direction, altering the structure in the region that is also affected by the TKI-sensitive EGFR-L858R. Our studies reveal intricate differences between EGFR mutations, their biology, and their response to EGFR TKIs.

The genetic landscape of high-risk neuroblastoma.

Neuroblastoma is a malignancy of the developing sympathetic nervous system that often presents with widespread metastatic disease, resulting in survival rates of less than 50%. To determine the spectrum of somatic mutation in high-risk neuroblastoma, we studied 240 affected individuals (cases) using a combination of whole-exome, genome and transcriptome sequencing as part of the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. Here we report a low median exonic mutation frequency of 0.60 per Mb (0.48 nonsilent) and notably few recurrently mutated genes in these tumors. Genes with significant somatic mutation frequencies included ALK (9.2% of cases), PTPN11 (2.9%), ATRX (2.5%, and an additional 7.1% had focal deletions), MYCN (1.7%, causing a recurrent p.Pro44Leu alteration) and NRAS (0.83%). Rare, potentially pathogenic germline variants were significantly enriched in ALK, CHEK2, PINK1 and BARD1. The relative paucity of recurrent somatic mutations in neuroblastoma challenges current therapeutic strategies that rely on frequently altered oncogenic drivers.

Integrative genomic analysis implicates gain of PIK3CA at 3q26 and MYC at 8q24 in chronic lymphocytic leukemia.

PURPOSE: The disease course of chronic lymphocytic leukemia (CLL) varies significantly within cytogenetic groups. We hypothesized that high-resolution genomic analysis of CLL would identify additional recurrent abnormalities associated with short time-to-first therapy (TTFT). EXPERIMENTAL DESIGN: We undertook high-resolution genomic analysis of 161 prospectively enrolled CLLs using Affymetrix 6.0 SNP arrays, and integrated analysis of this data set with gene expression profiles. RESULTS: Copy number analysis (CNA) of nonprogressive CLL reveals a stable genotype, with a median of only 1 somatic CNA per sample. Progressive CLL with 13q deletion was associated with additional somatic CNAs, and a greater number of CNAs was predictive of TTFT. We identified other recurrent CNAs associated with short TTFT: 8q24 amplification focused on the cancer susceptibility locus near MYC in 3.7%; 3q26 amplifications focused on PIK3CA in 5.6%; and 8p deletions in 5% of patients. Sequencing of MYC further identified somatic mutations in two CLLs. We determined which catalytic subunits of phosphoinositide 3-kinase (PI3K) were in active complex with the p85 regulatory subunit and showed enrichment for the α subunit in three CLLs carrying PIK3CA amplification. CONCLUSIONS: Our findings implicate amplifications of 3q26 focused on PIK3CA and 8q24 focused on MYC in CLL.