Tissue architecture delineates field cancerization in BRAFV600E-induced tumor development.

Cancer cells hijack developmental growth mechanisms but whether tissue morphogenesis and architecture modify tumorigenesis is unknown. Here, we characterized a new mouse model of sporadic thyroid carcinogenesis based on inducible expression of BRAF carrying a Val600 Glu (V600E) point mutation (BRAFV600E) from the thyroglobulin promoter (TgCreERT2). Spontaneous activation of this Braf-mutant allele due to leaky activity of the Cre recombinase revealed that intrinsic properties of thyroid follicles determined BRAF-mutant cell fate. Papillary thyroid carcinomas developed multicentrically within a normal microenvironment. Each tumor originated from a single follicle that provided a confined space for growth of a distinct tumor phenotype. Lineage tracing revealed oligoclonal tumor development in infancy and early selection of BRAFV600E kinase inhibitor-resistant clones. Somatic mutations were few, non-recurrent and limited to advanced tumors. Female mice developed larger tumors than males, reproducing the gender difference of human thyroid cancer. These data indicate that BRAFV600E-induced tumorigenesis is spatiotemporally regulated depending on the maturity and heterogeneity of follicles. Moreover, thyroid tissue organization seems to determine whether a BRAF-mutant lineage becomes a cancerized lineage. The TgCreERT2;BrafCA/+ sporadic thyroid cancer mouse model provides a new tool to evaluate drug therapy at different stages of tumor evolution.

Marinacci Anastomosis: A Case Report.

Marinacci anastomosis or the reverse Martin-Gruber anastomosis is a rare anomalous intercommunication from the ulnar nerve to the median nerve in the forearm. A young lady was involved in a road traffic accident that resulted in the fracture of both forearm bones for which she was treated surgically. A nerve conduction study was performed later on, which incidentally demonstrated findings of this communication. Knowledge of this anastomosis can help prevent misdiagnosis of clinical findings and misinterpretation of electrophysiological studies and help mitigate iatrogenic injury during surgical procedures.

Effect of head down tilt on heart rate variability.

CONTEXT: Sirshasan is supposed to increase blood flow to the brain and considered to be beneficial for intellectual function, however mastering these techniques may be difficult. AIMS: To see the effect of headstand using a tilt table on heart rate variability (HRV). SETTINGS AND DESIGN: A cross-sectional study that was done on 26 healthy volunteers. METHODS AND MATERIAL: HRV was assessed in the supine position and 30° head tilt position for 5 min. HRV recording was done on the power lab (AD Instruments P Ltd, Castle Hill Australia). The tilt table used was Medica Podium, New Delhi, HLT-200. STATISTICAL ANALYSIS USED: Paired t-test. RESULTS: All the HRV parameters showed non-significant change except low-frequency parameters which showed significant change during head tilt. CONCLUSIONS: Headstand to a 30° using tilt table cause a decrease in the autonomic activity which is mainly because of decrease in sympathetic activity.

Electrodiagnostic description of a rare variant of Berrettini anastomois: A case report.

INTRODUCTION: Berrettini Anastomosis is a common purely sensory anastomosis between ulnar and median nerves in palm. Here, via a communicating branch, ulnar nerve can provide sensory supply to digits 3 and 2.There have been electrodiagnostic (EDX) descriptions of the former. However, till date, to the best of our knowledge, there have been no EDX descriptions of the latter. Here, in our case report we would like to describe first instance of the same. PRESENTATION OF CASE: During an assault with a knife a 25-year-old male sustained laceration injury of right median nerve and flexor tendons which were repaired surgically. During rehabilitation, a nerve conduction study (NCS) was performed which incidentally revealed that ulnar nerve was responsible for sensory innervation of digit 2. DISCUSSION: Until recently, it was generally believed that EDX of BA was not possible. However, recent studies on EDX features in BA, have recorded small sensory nerve action potentials (SNAP) from digit 3 on distal ulnar nerve stimulation. But there are no published reports where SNAP from digit 2 on ulnar nerve stimulation were studied, even though anatomical evidence of the same exists. In our patient, we incidentally recorded the same. CONCLUSION: Although our patient had a complete laceration of median nerve, he did not have major sensory disturbances. NCS findings suggested sensory supply of digits 5, 4, 3 and 2 were by ulnar nerve. Without adequate knowledge of communicating branch crossovers in palm, there is a possibility that clinical findings can be misdiagnosed and NCS features can be misinterpreted. For surgeons, awareness of these communicating branches can prevent iatrogenic injuries during surgical interventions.

Global RNA Expression and DNA Methylation Patterns in Primary Anaplastic Thyroid Cancer.

Anaplastic thyroid cancer (ATC) is one of the most malignant tumors, with a median survival of only a few months. The tumorigenic processes of this disease have not yet been completely unraveled. Here, we report an mRNA expression and DNA methylation analysis of fourteen primary ATCs. ATCs clustered separately from normal thyroid tissue in unsupervised analyses, both by RNA expression and by DNA methylation. In expression analysis, enrichment of cell-cycle-related genes as well as downregulation of genes related to thyroid function were seen. Furthermore, ATC displayed a global hypomethylation of the genome but with hypermethylation of CpG islands. Notably, several cancer-related genes displayed a correlation between RNA expression and DNA methylation status, including MTOR, NOTCH1, and MAGI1. Furthermore, TSHR and SLC26A7, encoding the thyroid-stimulating hormone receptor and an iodine receptor highly expressed in normal thyroid, respectively, displayed low expression as well as aberrant gene body DNA methylation. This study is the largest investigation of global DNA methylation in ATC to date. It shows that aberrant DNA methylation is common in ATC and likely contributes to tumorigenesis in this disease. Future explorations of novel treatments should take this into consideration.

Correction: A urinary extracellular vesicle microRNA biomarker discovery pipeline; from automated extracellular vesicle enrichment by acoustic trapping to microRNA sequencing.

[This corrects the article DOI: 10.1371/journal.pone.0217507.].

A urinary extracellular vesicle microRNA biomarker discovery pipeline; from automated extracellular vesicle enrichment by acoustic trapping to microRNA sequencing.

Development of a robust automated platform for enrichment of extracellular vesicles from low sample volume that matches the needs for next-generation sequencing could remove major hurdles for genomic biomarker discovery. Here, we document a protocol for urinary EVs enrichment by utilizing an automated microfluidic system, termed acoustic trap, followed by next-generation sequencing of microRNAs (miRNAs) for biomarker discovery. Specifically, we compared the sequencing output from two small RNA library preparations, NEXTFlex and CATS, using only 130 pg of input total RNA. The samples prepared using NEXTflex was found to contain larger number of unique miRNAs that was the predominant RNA species whereas rRNA was the dominant RNA species in CATS prepared samples. A strong correlation was found between the miRNA expressions of the acoustic trap technical replicate in the NEXTFlex prepared samples, as well as between the acoustic trap and ultracentrifugation enrichment methods. Together, these results demonstrate a robust and automated strategy for biomarker discovery from small volumes of urine.

Proteogenomics and Hi-C reveal transcriptional dysregulation in high hyperdiploid childhood acute lymphoblastic leukemia.

Hyperdiploidy, i.e. gain of whole chromosomes, is one of the most common genetic features of childhood acute lymphoblastic leukemia (ALL), but its pathogenetic impact is poorly understood. Here, we report a proteogenomic analysis on matched datasets from genomic profiling, RNA-sequencing, and mass spectrometry-based analysis of >8,000 genes and proteins as well as Hi-C of primary patient samples from hyperdiploid and ETV6/RUNX1-positive pediatric ALL. We show that CTCF and cohesin, which are master regulators of chromatin architecture, display low expression in hyperdiploid ALL. In line with this, a general genome-wide dysregulation of gene expression in relation to topologically associating domain (TAD) borders were seen in the hyperdiploid group. Furthermore, Hi-C of a limited number of hyperdiploid childhood ALL cases revealed that 2/4 cases displayed a clear loss of TAD boundary strength and 3/4 showed reduced insulation at TAD borders, with putative leukemogenic effects.

Identification of Targetable Lesions in Anaplastic Thyroid Cancer by Genome Profiling.

Anaplastic thyroid cancer (ATC) is a rare and extremely malignant tumor with no available cure. The genetic landscape of this malignancy has not yet been fully explored. In this study, we performed whole exome sequencing and the RNA-sequencing of fourteen cases of ATC to delineate copy number changes, fusion gene events, and somatic mutations. A high frequency of genomic amplifications was seen, including 29% of cases having amplification of CCNE1 and 9% of CDK6; these events may be targetable by cyclin dependent kinase (CDK) inhibition. Furthermore, 9% harbored amplification of TWIST1, which is also a potentially targetable lesion. A total of 21 fusion genes in five cases were seen, none of which were recurrent. Frequent mutations included TP53 (55%), the TERT promoter (36%), and ATM (27%). Analyses of mutational signatures showed an involvement of processes that are associated with normal aging, defective DNA mismatch repair, activation induced cytidine deaminase (AID)/apolipoprotein B editing complex (APOBEC) activity, failure of DNA double-strand break repair, and tobacco exposure. Taken together, our results shed new light on the tumorigenesis of ATC and show that a relatively large proportion (36%) of ATCs harbor genetic events that make them candidates for novel therapeutic approaches. When considering that ATC today has a mortality rate of close to 100%, this is highly relevant from a clinical perspective.

A novel human in vitro papillomavirus type 16 positive tonsil cancer cell line with high sensitivity to radiation and cisplatin.

BACKGROUND: Human papillomavirus (HPV) is an established risk factor for oropharyngeal squamous cell carcinoma (OSCC). The aim was to establish cell lines from HPV-positive tonsil carcinomas to be used for treatment development. METHODS: Fresh samples from 23 HPV-positive tonsil carcinomas were cultivated in vitro. The established cell line was analyzed for viral characteristics, cell karyotype, TP53 status, and growth capabilities in nude mice. In vitro studies of sensitivities to radiation, cisplatin and cetuximab were performed. RESULTS: After 19 months (eight passages), one cell line, LU-HNSCC-26, was established in vitro and also grew as xenografts. The tumor was from a 48 year old non-smoking man with non-keratinizing, p16 positive tonsil OSCC, stage T2N0M0 with HPV16. It contained 19.5 (CV% 3.7) HPV16 copies/cell (passage 8). The complete HPV16 genome sequence was obtained. Episomal HPV16 was present with an E2/E7 ratio of 1.1 (CV% 2.6). In addition, HPV16 mRNA specific for the intact E2 gene was detected. The viral expression manifested 1.0 (CV% 0.1) E7 mRNA copies per HPV16 genome. The karyotype was determined and the cell line demonstrated wild type TP53. The ID50 for radiation was 0.90 Gy and the IC50 for cisplatin was 0.99 µmol/L. The cell line was inhibited to a maximum of 18% by cetuximab. CONCLUSIONS: We established an in vitro tonsil carcinoma cell line containing episomal HPV16. This is an important step towards efficient treatment development.

Genomic complexity and targeted genes in anaplastic thyroid cancer cell lines.

Anaplastic thyroid cancer (ATC) is a highly malignant disease with a very short median survival time. Few studies have addressed the underlying somatic mutations, and the genomic landscape of ATC thus remains largely unknown. In the present study, we have ascertained copy number aberrations, gene fusions, gene expression patterns, and mutations in early-passage cells from ten newly established ATC cell lines using single nucleotide polymorphism (SNP) array analysis, RNA sequencing and whole exome sequencing. The ATC cell line genomes were highly complex and displayed signs of replicative stress and genomic instability, including massive aneuploidy and frequent breakpoints in the centromeric regions and in fragile sites. Loss of heterozygosity involving whole chromosomes was common, but there were no signs of previous near-haploidisation events or chromothripsis. A total of 21 fusion genes were detected, including six predicted in-frame fusions; none were recurrent. Global gene expression analysis showed 661 genes to be differentially expressed between ATC and papillary thyroid cancer cell lines, with pathway enrichment analyses showing downregulation of TP53 signalling as well as cell adhesion molecules in ATC. Besides previously known driver events, such as mutations in BRAF, NRAS, TP53 and the TERT promoter, we identified PTPRD and NEGR1 as putative novel target genes in ATC, based on deletions in six and four cell lines, respectively; the latter gene also carried a somatic mutation in one cell line. Taken together, our data provide novel insights into the tumourigenesis of ATC and may be used to identify new therapeutic targets.