Bizarre giant cells in human angiosarcoma exhibit chemoresistance and contribute to poor survival outcomes.

Giant cells (GC) are a poorly understood subset of tumor cells that have been increasingly recognized as a potential contributor to tumor heterogeneity and treatment resistance. We aimed to characterize the biological and clinical significance of GC in angiosarcoma, an aggressive rare cancer of endothelial origin. Archival angiosarcoma samples were examined for the presence of GC and compared with clinicopathological as well as NanoString gene expression data. GC were examined in angiosarcoma cell lines MOLAS and ISOHAS using conventional and electron microscopy, single cell whole genome profiling, and other assays. In the cell lines, GC represented a rare population of mitotically active, non-senescent CD31+ cells, and shared similar genomic profiles with regular-sized cells, consistent with a malignant endothelial phenotype. GC remained viable and persisted in culture following exposure to paclitaxel and doxorubicin. In patient samples, GC were present in 24 of 58 (41.4%) cases. GC was correlated with poorer responses to chemotherapy (25.0% vs 73.3%, P = 0.0213) and independently contributed to worse overall survival outcomes (hazard ratio 2.20, 95% confidence interval 1.17-4.15, P = 0.0142). NanoString profiling revealed overexpression of genes, including COL11A1, STC1, and ERO1A, accompanied by upregulation of immune-related metabolic stress and metastasis/matrix remodeling pathways in GC-containing tumors. In conclusion, GC may contribute to chemoresistance and poor prognosis in angiosarcoma.

Genetic differences between benign phyllodes tumors and fibroadenomas revealed through targeted next generation sequencing.

Breast fibroepithelial lesions are biphasic tumors which comprise the common benign fibroadenomas (FAs) and the rarer phyllodes tumors (PTs). This study analyzed 262 (42%) conventional FAs, 45 (7%) cellular FAs, and 321 (51%) benign PTs contributed by the International Fibroepithelial Consortium, using a previously curated 16 gene panel. Benign PTs were found to possess a higher number of mutations, and higher rates of cancer driver gene alterations than both groups of FAs, in particular MED12, TERT promoter, RARA, FLNA, SETD2, RB1, and EGFR. Cases with MED12 mutations were also more likely to have TERT promoter, RARA, SETD2, and EGFR. There were no significant differences detected between conventional FAs and cellular FAs, except for PIK3CA and MAP3K1. TERT promoter alterations were most optimal in discriminating between FAs and benign PTs. Our study affirms the role of sequencing and key mutations that may assist in refining diagnoses of these lesions.

Morphologic and genetic heterogeneity in breast fibroepithelial lesions-a comprehensive mapping study.

Breast fibroepithelial lesions (FELs) encompass the common fibroadenoma (FA) and relatively rare phyllodes tumour (PT); the latter entity is usually classified as benign, borderline or malignant. Intratumoural heterogeneity is frequently present in these tumours, making accurate histologic evaluation challenging. Despite their rarity, PTs are an important clinical problem due to their propensity for recurrence and, in the case of malignant PT, metastasis. Surgical excision is the mainstay of management. Recent work has uncovered myriad genetic alterations in breast FELs. In this study, exome sequencing was performed on seven cases of morphologically heterogeneous breast FELs, including FAs, PTs of all grades, and a case of metaplastic spindle cell carcinoma arising in PT, in order to elucidate their intratumoural genetic repertoire. Gene mutations identified encompassed cell signalling, tumour suppressor, DNA repair and cell cycle regulating pathways. Mutations common to multiple tumour regions generally showed higher variant allele frequency. Frequent mutations included MED12, TP53, RARA and PIK3CA. Histological observations of increased cellular density and pleomorphism correlated with mutational burden. Phylogenetic analyses revealed disparate pathways of possible tumour progression. In summary, histological heterogeneity correlated with genetic changes in breast FELs.

Genomic characterisation of breast fibroepithelial lesions in an international cohort.

Fibroepithelial lesions (FELs) are a heterogeneous group of tumours comprising fibroadenomas (FAs) and phyllodes tumours (PTs). Here we used a 16-gene panel that was previously discovered to be implicated in pathogenesis and progression, to characterise a large international cohort of FELs via targeted sequencing. The study comprised 303 (38%) FAs and 493 (62%) PTs which were contributed by the International Fibroepithelial Consortium. There were 659 (83%) Asian and 109 (14%) non-Asian FELs, while the ethnicity of the rest was unknown. Genetic aberrations were significantly associated with increasing grade of PTs, and were detected more in PTs than FAs for MED12, TERT promoter, RARA, FLNA, SETD2, TP53, RB1, EGFR, and IGF1R. Most borderline and malignant PTs possessed ≥ 2 mutations, while there were more cases of FAs with ≤ 1 mutation compared to PTs. FELs with MED12 mutations had significantly higher rates of TERT promoter, RARA, SETD2, EGFR, ERBB4, MAP3K1, and IGF1R aberrations. However, FELs with wild-type MED12 were more likely to express TP53 and PIK3CA mutations. There were no significant differences observed between the mutational profiles of recurrent FAs, FAs with a history of subsequent ipsilateral recurrence or contralateral occurrence, and FAs without a history of subsequent events. We identified recurrent mutations which were more frequent in PTs than FAs, with borderline and malignant PTs harbouring cancer driver gene and multiple mutations. This study affirms the role of a set of genes in FELs, including its potential utility in classification based on mutational profiles. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Genomic profile of breast sarcomas: a comparison with malignant phyllodes tumours.

PURPOSE: We aimed to investigate the genomic profile of breast sarcomas (BS) and compare with that of malignant phyllodes tumours (MPT). METHODS: DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) specimens from 17 cases of BS diagnosed at Singapore General Hospital from January 1991 to December 2014. Targeted deep sequencing and copy number variation (CNV) analysis on 16 genes, which included recurrently mutated genes in phyllodes tumours and genes associated with breast cancer, were performed on these samples. Genetic alterations (GA) observed were summarised and analysed. RESULTS: Nine cases met the quality control requirements for both targeted deep sequencing and CNV analysis. Three (33.33%) were angiosarcomas and 6 (66.67%) were non-angiosarcomas. In the non-angiosarcoma group, 83.33% (n = 5) of the patients had GA in the TERT gene. The other commonly mutated genes in this group of tumours were MED12 (n = 4, 66.67%), BCOR (n = 4, 66.67%), KMT2D (n = 3, 50%), FLNA (n = 3, 50%) and NF1 (n = 3, 50%). In contrast, none of the angiosarcomas had mutations or copy number alterations in TERT, MED12, BCOR, FLNA or NF1. Eighty percent of patients with GA in TERT (n = 5) had concurrent mutations in MED12. Sixty percent (n = 3) of these cases also demonstrated GA in NF1, PIK3CA or EGFR which are known cancer driver genes. CONCLUSIONS: The non-angiosarcoma group of BS was found to share similar GA as those described for MPT, which may suggest a common origin and support their consideration as a similar group of tumours with regard to management and prognostication.

Molecular insights into paediatric breast fibroepithelial tumours.

AIMS: This study aims to examine the molecular genetics of paediatric breast fibroepithelial tumours through the targeted sequencing of 50 genes. METHODS AND RESULTS: Formalin-fixed paraffin-embedded tissues of fibroepithelial tumours diagnosed in a cohort of patients aged 18 years and below were subjected to next generation sequencing using the Haloplex Target Enrichment System. Twenty-five conventional and 17 juvenile fibroadenomas were studied, with MED12 mutations found in 53.8 and 35% of the tumours, respectively. There was also one benign fibroepithelial neoplasm with hybrid features of juvenile papillomatosis and infarcted benign phyllodes tumour-like areas. Most tumours did not have mutations in well-known cancer driver genes, none harboured TERT promoter mutations, while 25.6% (11 of 43) showed no mutations. Metachronous and synchronous tumours were found to have mutational heterogeneity with some containing mutations in MED12; other genes or no mutations were detected at all. Four of eight giant fibroadenomas (size 5 cm or larger) had no mutations detected, suggesting that there are other molecular mechanisms driving their growth. Tumours with MED12 mutations incidentally had a significantly higher stromal mitotic count compared with those without. CONCLUSION: While paediatric fibroepithelial lesions can have cellular stroma potentially raising concern for phyllodes tumour, their lack of TERT promoter and cancer driver mutations is reassuring. The absence of mutations in a significant proportion of tumours, especially the giant fibroadenomas, warrants investigation of pathogenetic mechanisms beyond those involving the 50 genes.

Clear cell sarcomas of the kidney are characterised by BCOR gene abnormalities, including exon 15 internal tandem duplications and BCOR-CCNB3 gene fusion.

AIMS: Clear cell sarcoma of the kidney (CCSK) is a rare paediatric renal malignant tumour. The majority of CCSKs have internal tandem duplications (ITDs) of the BCOR gene, whereas a minority have the YWHAE-NUTM2 gene fusion. A third 'double-negative' (DN) category comprises CCSKs with neither BCOR ITDs nor YWHAE-NUTM2 fusion. The aim of this study was to characterise 11 histologically diagnosed CCSKs immunohistochemically (with CCND1, BCOR and CCNB3 stains) and genetically. METHODS AND RESULTS: By next-generation sequencing, 10 cases (90.9%) had BCOR exon 15 ITDs, with positive BCOR immunoreactivity being found in four (36%) or eight (72%) cases, depending on the antibody clone. By reverse transcription polymerase chain reaction, none had the YWHAE-NUTM2 fusion. The DN case had a BCOR-CCNB3 fusion and strong nuclear CCNB3 and BCOR immunoreactivity. Quantitative polymerase chain reaction showed markedly elevated BCOR expression in this case, whereas BCOR ITD cases had lower levels of elevated BCOR expression. CONCLUSIONS: The majority of the CCSKs in our cohort had BCOR ITDs, and none had the YWHAE-NUTM2 fusion. We verified the strong, diffuse cyclin D1 (CCND1) immunoreactivity in CCSKs described in recent reports. BCOR immunoreactivity was not consistently positive in all CCSKs with BCOR ITDs, and therefore cannot be used as a diagnostic immunohistochemical stain to identify BCOR ITD cases. The DN case was a BCOR-CCNB3 fusion sarcoma. BCOR-CCNB3 sarcoma is typically a primary bone sarcoma affecting male adolescents, and this is the first report of it presenting in a kidney of a young child as a CCSK. The full spectrum of DN CCSKs awaits more comprehensive characterisation.

SETD2 histone modifier loss in aggressive GI stromal tumours.

BACKGROUND: GI stromal tumours (GISTs) are clinically heterogenous exhibiting varying degrees of disease aggressiveness in individual patients. OBJECTIVES: We sought to identify genetic alterations associated with high-risk GIST, explore their molecular consequences, and test their utility as prognostic markers. DESIGNS: Exome sequencing of 18 GISTs was performed (9 patients with high-risk/metastatic and 5 patients with low/intermediate-risk), corresponding to 11 primary and 7 metastatic tumours. Candidate alterations were validated by prevalence screening in an independent patient cohort (n=120). Functional consequences of SETD2 mutations were investigated in primary tissues and cell lines. Transcriptomic profiles for 8 GISTs (4 SETD2 mutated, 4 SETD2 wild type) and DNA methylation profiles for 22 GISTs (10 SETD2 mutated, 12 SETD2 wild type) were analysed. Statistical associations between molecular, clinicopathological factors, and relapse-free survival were determined. RESULTS: High-risk GISTs harboured increased numbers of somatic mutations compared with low-risk GISTs (25.2 mutations/high-risk cases vs 6.8 mutations/low-risk cases; two sample t test p=3.1×10-5). Somatic alterations in the SETD2 histone modifier gene occurred in 3 out of 9 high-risk/metastatic cases but no low/intermediate-risk cases. Prevalence screening identified additional SETD2 mutations in 7 out of 80 high-risk/metastatic cases but no low/intermediate-risk cases (n=29). Combined, the frequency of SETD2 mutations was 11.2% (10/89) and 0% (0/34) in high-risk and low-risk GISTs respectively. SETD2 mutant GISTs exhibited decreased H3K36me3 expression while SETD2 silencing promoted DNA damage in GIST-T1 cells. In gastric GISTs, SETD2 mutations were associated with overexpression of HOXC cluster genes and a DNA methylation signature of hypomethylated heterochromatin. Gastric GISTs with SETD2 mutations, or GISTs with hypomethylated heterochromatin, showed significantly shorter relapse-free survival on univariate analysis (log rank p=4.1×10-5). CONCLUSIONS: Our data suggest that SETD2 is a novel GIST tumour suppressor gene associated with disease progression. Assessing SETD2 genetic status and SETD2-associated epigenomic phenotypes may guide risk stratification and provide insights into mechanisms of GIST clinical aggressiveness.

Transcriptomic convergence despite genomic divergence drive field cancerization in synchronous squamous tumors.

Introduction: Field cancerization is suggested to arise from imbalanced differentiation in individual basal progenitor cells leading to clonal expansion of mutant cells that eventually replace the epithelium, although without evidence. Methods: We performed deep sequencing analyses to characterize the genomic and transcriptomic landscapes of field change in two patients with synchronous aerodigestive tract tumors. Results: Our data support the emergence of numerous genetic alterations in cancer-associated genes but refutes the hypothesis that founder mutation(s) underpin this phenomenon. Mutational signature analysis identified defective homologous recombination as a common underlying mutational process unique to synchronous tumors. Discussion: Our analyses suggest a common etiologic factor defined by mutational signatures and/or transcriptomic convergence, which could provide a therapeutic opportunity.

Comprehensive molecular characterization of collecting duct carcinoma for therapeutic vulnerability.

Collecting duct carcinoma (CDC) is an aggressive rare subtype of kidney cancer with unmet clinical needs. Little is known about its underlying molecular alterations and etiology, primarily due to its rarity, and lack of preclinical models. This study aims to comprehensively characterize molecular alterations in CDC and identify its therapeutic vulnerabilities. Through whole-exome and transcriptome sequencing, we identified KRAS hotspot mutations (G12A/D/V) in 3/13 (23%) of the patients, in addition to known TP53, NF2 mutations. 3/13 (23%) patients carried a mutational signature (SBS22) caused by aristolochic acid (AA) exposures, known to be more prevalent in Asia, highlighting a geologically specific disease etiology. We further discovered that cell cycle-related pathways were the most predominantly dysregulated pathways. Our drug screening with our newly established CDC preclinical models identified a CDK9 inhibitor LDC000067 that specifically inhibited CDC tumor growth and prolonged survival. Our study not only improved our understanding of oncogenic molecular alterations of Asian CDC, but also identified cell-cycle machinery as a therapeutic vulnerability, laying the foundation for clinical trials to treat patients with such aggressive cancer.

PCAF interacts with XBP-1S and mediates XBP-1S-dependent transcription.

X-box binding protein 1 (XBP-1) is a key regulator required for cellular unfolded protein response (UPR) and plasma cell differentiation. In addition, involvement of XBP-1 in host cell-virus interaction and transcriptional regulation of viruses, such as human T-lymphotropic virus type 1 (HTLV-1), has been revealed recently. Two XBP-1 isoforms, XBP-1U and XBP-1S, which share an identical N-terminal domain, are present in cells. XBP-1S is a transcription activator while XBP-1U is the inactive isoform. Although the transactivation domain of XBP-1S has been identified within the XBP-1S-specific C-terminus, molecular mechanism of the transcriptional activation by XBP-1S still remains unknown. Here we report the interaction between p300/CBP-associated factor (PCAF) and XBP-1S through the C-terminal domain of XBP-1S. No binding between XBP-1U and PCAF is detected. In a cell-based reporter assay, overexpression of PCAF further stimulates the XBP-1S-mediated cellular and HTLV-1 transcription while knockdown of PCAF exhibits the opposite effect. Expression of endogenous XBP-1S cellular target genes, such as BiP and CHOP, is significantly inhibited when PCAF is knocked down. Furthermore, PCAF is recruited to the promoters of XBP-1S target genes in vivo, in a XBP-1S-dependent manner. Collectively, our results demonstrate that PCAF mediates the XBP-1S-dependent transcription through the interaction with XBP-1S.

A comprehensive next generation sequencing tissue assay for Asian-prevalent cancers-Analytical validation and performance evaluation with clinical samples.

Introduction: A well-validated diagnostic assay with curated biomarkers complements clinicopathological factors to facilitate early diagnosis and ensure timely treatment delivery. This study focuses on an Asian-centric cancer diagnostic assay designed and thoroughly validated against commercially available standard references and a cohort of over 200 clinical specimens spanning 12 diverse Asian-centric cancer types. Methods: The assay uses hybrid-capture probes capable of profiling DNA aberrations from 572 cancer-related genes and 91 RNA fusion partners. The panel can detect clinically-tractable biomarkers such as microsatellite instability (MSI) and tumor mutation burden (TMB). Results: Analytical evaluation demonstrated 100% specificity and 99.9% sensitivity within a ≥5% VAF limit of detection (LoD) for SNV/Indels. RNA-based fusion features an LoD of ≥5 copies per nanogram input when evaluated against commercial references. Excellent linearity and concordance were observed when benchmarking against orthogonal methods in identifying MSI status, TMB scores and RNA fusions. Actionable genetic alterations were identified in 65% of the clinical samples. Conclusion: These results demonstrate a molecular diagnostic assay that accurately detects genomic alterations and complex biomarkers. The data also supports an excellent performance of this assay for making critical diagnoses and well-informed therapeutic decisions in Asian prevalent cancers.

NEAR trial: A single-arm phase II trial of neoadjuvant apalutamide monotherapy and radical prostatectomy in intermediate- and high-risk prostate cancer.

OBJECTIVE: Treatment efficacy of androgen deprivation therapy with radical prostatectomy for intermediate- to high-risk prostate cancer is less well-studied. The NEAR trial is a single-arm, phase II investigation of neoadjuvant apalutamide monotherapy and radical prostatectomy (RP) in the treatment of D'Amico intermediate- and high-risk prostate cancer (NCT03124433). MATERIALS AND METHODS: Patients with histologically-proven, D'Amico intermediate- to high-risk prostate adenocarcinoma received apalutamide 240 mg once-daily for 12 weeks followed by RP + /-lymphadenectomy. Primary outcome was pathological complete response (pCR) rate. Secondary outcomes included rate of biochemical response (defined by PSA < 0.03 ng/mL at week 24 from starting apalutamide without subsequent PSA relapse), treatment-related adverse events, and RP complication rates. Correlative biomarker analyses were performed to examine for molecular predictors of treatment responses. RESULTS: From 2017 to 2019, 30 patients were recruited, of which 20 and 10 were high and intermediate risk, respectively; 25 completed treatment as per-protocol. We did not observe any pCR on trial; median reduction of cancer burden was 41.7% (IQR: 33.3%-60.0%). 18 out of 25 patients were classified as having a biochemical response (4 did not achieve PSA of <0.03 ng/mL at week 24 and 3 developed PSA relapse subsequently). Dry skin (N = 16; 53.3%), fatigue (N = 10; 33.3%) and skin rash (N = 9; 30.0%) were the most common adverse events, and there was no major peri-operative complication. We observed an association between tumours of low androgen receptor activity and PAM50 basal status with biochemical non-responders, albeit these molecular phenotypes were not associated with pathological response. CONCLUSIONS: A 12-week course of neoadjuvant apalutamide prior to RP did not meet the primary endpoint of pCR in this trial. Tumours with low androgen receptor activity or of the PAM50 basal subtype may have a reduced response to apalutamide.

Elephant shark sequence reveals unique insights into the evolutionary history of vertebrate genes: A comparative analysis of the protocadherin cluster.

Cartilaginous fishes are the oldest living phylogenetic group of jawed vertebrates. Here, we demonstrate the value of cartilaginous fish sequences in reconstructing the evolutionary history of vertebrate genomes by sequencing the protocadherin cluster in the relatively small genome (910 Mb) of the elephant shark (Callorhinchus milii). Human and coelacanth contain a single protocadherin cluster with 53 and 49 genes, respectively, that are organized in three subclusters, Pcdhalpha, Pcdhbeta, and Pcdhgamma, whereas the duplicated protocadherin clusters in fugu and zebrafish contain >77 and 107 genes, respectively, that are organized in Pcdhalpha and Pcdhgamma subclusters. By contrast, the elephant shark contains a single protocadherin cluster with 47 genes organized in four subclusters (Pcdhdelta, Pcdhepsilon, Pcdhmu, and Pcdhnu). By comparison with elephant shark sequences, we discovered a Pcdhdelta subcluster in teleost fishes, coelacanth, Xenopus, and chicken. Our results suggest that the protocadherin cluster in the ancestral jawed vertebrate contained more subclusters than modern vertebrates, and the evolution of the protocadherin cluster is characterized by lineage-specific differential loss of entire subclusters of genes. In contrast to teleost fish and mammalian protocadherin genes that have undergone gene conversion events, elephant shark protocadherin genes have experienced very little gene conversion. The syntenic block of genes in the elephant shark protocadherin locus is well conserved in human but disrupted in fugu. Thus, the elephant shark genome appears to be less prone to rearrangements compared with teleost fish genomes. The small and "stable" genome of the elephant shark is a valuable reference for understanding the evolution of vertebrate genomes.

Lack of Targetable FGFR2 Fusions in Endemic Fluke-Associated Cholangiocarcinoma.

PURPOSE: Cholangiocarcinoma (CCA) remains a disease with poor prognosis and limited therapeutic options. Identification of driver genetic alterations may lead to the discovery of more effective targeted therapies. CCAs harboring FGFR2 fusions have recently demonstrated promising responses to FGFR inhibitors, highlighting their potential relevance as predictive biomarkers. CCA incidence is high in the northeast of Thailand and its neighboring countries because of chronic infection with the liver fluke Opisthorchis viverrini (Ov). However, there are currently no available data on the prevalence of FGFR alterations in fluke-associated CCA in endemic countries. MATERIALS AND METHODS: In this study, we performed anchored multiplex polymerase chain reaction target enrichment RNA sequencing of FGFR1-3, validated by fluorescence in situ hybridization and Sanger sequencing, in 121 Ov-associated and 95 non-Ov-associated CCA tumors. RESULTS: Compared with non-fluke-associated CCA (11/95; 11.6%), FGFR2 fusions were significantly less common in fluke-associated CCA (1/121; 0.8%; P = .0006). All FGFR fusions were detected exclusively in intrahepatic CCAs and were mutually exclusive with KRAS/ERBB2/BRAF/FGFR mutations, pointing to their potential roles as oncogenic drivers. CONCLUSION: FGFR2 fusions are rare in fluke-associated CCA, underscoring how distinct etiologies may affect molecular landscapes in tumors and highlighting the need to discover other actionable genomic alterations in endemic fluke-associated CCA.

An Optimised Protocol Harnessing Laser Capture Microdissection for Transcriptomic Analysis on Matched Primary and Metastatic Colorectal Tumours.

Generation of large amounts of genomic data is now feasible and cost-effective with improvements in next generation sequencing (NGS) technology. Ribonucleic acid sequencing (RNA-Seq) is becoming the preferred method for comprehensively characterising global transcriptome activity. Unique to cytoreductive surgery (CRS), multiple spatially discrete tumour specimens could be systematically harvested for genomic analysis. To facilitate such downstream analyses, laser capture microdissection (LCM) could be utilized to obtain pure cell populations. The aim of this protocol study was to develop a methodology to obtain high-quality expression data from matched primary tumours and metastases by utilizing LCM to isolate pure cellular populations. We demonstrate an optimized LCM protocol which reproducibly delivered intact RNA used for RNA sequencing and quantitative polymerase chain reaction (qPCR). After pathologic annotation of normal epithelial, tumour and stromal components, LCM coupled with cDNA library generation provided for successful RNA sequencing. To illustrate our framework's potential to identify targets that would otherwise be missed with conventional bulk tumour sequencing, we performed qPCR and immunohistochemical technical validation to show that the genes identified were truly expressed only in certain sub-components. This study suggests that the combination of matched tissue specimens with tissue microdissection and NGS provides a viable platform to unmask hidden biomarkers and provides insight into tumour biology at a higher resolution.

Amplification and overexpression of PPFIA1, a putative 11q13 invasion suppressor gene, in head and neck squamous cell carcinoma.

Chromosomal amplifications of the 11q13 genomic region are frequent in head and neck squamous cell carcinoma (HNSCC). To identify novel 11q13 amplification targets, we integrated high-resolution array-based comparative genomic hybridization and Affymetrix gene-expression profiling of eight HNSCC cell lines. We found that PPFIA1 was the highest upregulated gene in the 11q13 amplicon of HNSCC cell lines when compared with HNSCC lines without 11q13 amplification and confirmed the upregulation of PPFIA1 in primary HNSCCs by real-time PCR. Using siRNA knockdown, we investigated PPFIA1 function in three HNSCC lines using both in vitro invasion assays and wound-healing assays. Surprisingly, we found that cancer cells become more invasive when the PPFIA1 protein levels were reduced, suggesting that PPFIA1 may act as an invasion inhibitor in HNSCC. This unexpected result suggests that the 11q13 amplicon may comprise both positive and negative regulators involved in HNSCC. Our study is the first to evaluate the role of PPFIA1 in head and neck carcinogenesis and suggests a potential link between PPFIA1 activity and cell-extracellular matrix interactions. This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

A novel genomic panel as an adjunctive diagnostic tool for the characterization and profiling of breast Fibroepithelial lesions.

BACKGROUND: Known collectively as breast fibroepithelial lesions (FELs), the common fibroadenomas (FAs) and the rarer phyllodes tumors (PTs) are a heterogenous group of biphasic neoplasms. Owing to limited tissue availability, inter-observer variability, overlapping histological features and heterogeneity of these lesions, diagnosing them accurately on core biopsies is challenging. As the choice management option depends on the histological diagnosis; a novel 16-gene panel assay was developed to improve the accuracy of preoperative diagnosis on core biopsy specimens. METHODS: Using this 16-gene panel, targeted amplicon-based sequencing was performed on 275 formalin-fixed, paraffin-embedded (FFPE) breast FEL specimens, archived at the Singapore General Hospital, from 2008 to 2012. RESULTS: In total, 167 FAs, 24 benign, 14 borderline and 6 malignant PTs, were profiled. Compared to FAs, PTs had significantly higher mutation rates in the TERT promoter (p <  0.001), RARA (p <  0.001), FLNA, RB1 and TP53 (p = 0.002, 0.020 and 0.018, respectively). In addition to a higher mutational count (p <  0.001), TERT promoter (p <  0.001), frameshift, nonsense and splice site (p = 0.001, < 0.001 and 0.043, respectively) mutations were also frequently observed in PTs. A multivariate logistic regression model was built using these as variables and a predictive scoring system was developed. It classifies a FEL at low or high risk (score <  1 and ≥ 1, respectively) of being a PT. This scoring system has good discrimination (ROC area = 0.773, 95% CI: 0.70 to 0.85), calibration (p = 0.945) and is significant in predicting PTs (p <  0.001). CONCLUSION: This novel study demonstrates the ability to extract DNA of sufficient quality and quantity for targeted sequencing from FFPE breast core biopsy specimens, along with their successful characterization and profiling using our customized 16-gene panel. Prospective work includes validating the utility of this promising 16-gene panel assay as an adjunctive diagnostic tool in clinical practice.

Sequencing and comparative analysis of fugu protocadherin clusters reveal diversity of protocadherin genes among teleosts.

BACKGROUND: The synaptic cell adhesion molecules, protocadherins, are a vertebrate innovation that accompanied the emergence of the neural tube and the elaborate central nervous system. In mammals, the protocadherins are encoded by three closely-linked clusters (alpha, beta and gamma) of tandem genes and are hypothesized to provide a molecular code for specifying the remarkably-diverse neural connections in the central nervous system. Like mammals, the coelacanth, a lobe-finned fish, contains a single protocadherin locus, also arranged into alpha, beta and gamma clusters. Zebrafish, however, possesses two protocadherin loci that contain more than twice the number of genes as the coelacanth, but arranged only into alpha and gamma clusters. To gain further insight into the evolutionary history of protocadherin clusters, we have sequenced and analyzed protocadherin clusters from the compact genome of the pufferfish, Fugu rubripes. RESULTS: Fugu contains two unlinked protocadherin loci, Pcdh1 and Pcdh2, that collectively consist of at least 77 genes. The fugu Pcdh1 locus has been subject to extensive degeneration, resulting in the complete loss of Pcdh1gamma cluster. The fugu Pcdh genes have undergone lineage-specific regional gene conversion processes that have resulted in a remarkable regional sequence homogenization among paralogs in the same subcluster. Phylogenetic analyses show that most protocadherin genes are orthologous between fugu and zebrafish either individually or as paralog groups. Based on the inferred phylogenetic relationships of fugu and zebrafish genes, we have reconstructed the evolutionary history of protocadherin clusters in the teleost fish lineage. CONCLUSION: Our results demonstrate the exceptional evolutionary dynamism of protocadherin genes in vertebrates in general, and in teleost fishes in particular. Besides the 'fish-specific' whole genome duplication, the evolution of protocadherin genes in teleost fishes is influenced by lineage-specific gene losses, tandem gene duplications and regional sequence homogenization. The dynamic protocadherin clusters might have led to the diversification of neural circuitry among teleosts, and contributed to the behavioral and physiological diversity of teleosts.

The draft genome of tropical fruit durian (Durio zibethinus).

Durian (Durio zibethinus) is a Southeast Asian tropical plant known for its hefty, spine-covered fruit and sulfury and onion-like odor. Here we present a draft genome assembly of D. zibethinus, representing the third plant genus in the Malvales order and first in the Helicteroideae subfamily to be sequenced. Single-molecule sequencing and chromosome contact maps enabled assembly of the highly heterozygous durian genome at chromosome-scale resolution. Transcriptomic analysis showed upregulation of sulfur-, ethylene-, and lipid-related pathways in durian fruits. We observed paleopolyploidization events shared by durian and cotton and durian-specific gene expansions in MGL (methionine γ-lyase), associated with production of volatile sulfur compounds (VSCs). MGL and the ethylene-related gene ACS (aminocyclopropane-1-carboxylic acid synthase) were upregulated in fruits concomitantly with their downstream metabolites (VSCs and ethylene), suggesting a potential association between ethylene biosynthesis and methionine regeneration via the Yang cycle. The durian genome provides a resource for tropical fruit biology and agronomy.