Suzanne Manning

Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.

Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.

Oesophageal adenocarcinoma (EAC) incidence is rapidly increasing in Western countries. A better understanding of EAC underpins efforts to improve early detection and treatment outcomes. While large EAC exome sequencing efforts to date have found recurrent loss-of-function mutations, oncogenic driving events have been underrepresented. Here we use a combination of whole-genome sequencing (WGS) and single-nucleotide polymorphism-array profiling to show that genomic catastrophes are frequent in EAC, with almost a third (32%, n=40/123) undergoing chromothriptic events. WGS of 22 EAC cases show that catastrophes may lead to oncogene amplification through chromothripsis-derived double-minute chromosome formation (MYC and MDM2) or breakage-fusion-bridge (KRAS, MDM2 and RFC3). Telomere shortening is more prominent in EACs bearing localized complex rearrangements. Mutational signature analysis also confirms that extreme genomic instability in EAC can be driven by somatic BRCA2 mutations. These findings suggest that genomic catastrophes have a significant role in the malignant transformation of EAC.

The importance of epigenetic modifications such as DNA methylation in tumorigenesis is increasingly being appreciated. To define the genome‐wide pattern of DNA methylation in pancreatic ductal adenocarcinomas (PDAC), we captured the methylation profiles of 167 untreated resected PDACs and compared them to a panel of 29 adjacent nontransformed pancreata using high‐density arrays. A total of 11,634 CpG sites associated with 3,522 genes were significantly differentially methylated (DM) in PDAC and were capable of segregating PDAC from non‐malignant pancreas, regardless of tumor cellularity. As expected, PDAC hypermethylation was most prevalent in the 5′ region of genes (including the proximal promoter, 5′UTR and CpG islands). Approximately 33% DM genes showed significant inverse correlation with mRNA expression levels. Pathway analysis revealed an enrichment of aberrantly methylated genes involved in key molecular mechanisms important to PDAC: TGF‐β, WNT, integrin signaling, cell adhesion, stellate cell activation and axon guidance. Given the recent discovery that SLIT‐ROBO mutations play a clinically important role in PDAC, the role of epigenetic perturbation of axon guidance was pursued in more detail. Bisulfite amplicon deep sequencing and qRT‐PCR expression analyses confirmed recurrent perturbation of axon guidance pathway genes SLIT2, SLIT3, ROBO1, ROBO3, ITGA2 and MET and suggests epigenetic suppression of SLIT‐ROBO signaling and up‐regulation of MET and ITGA2 expression. Hypomethylation of MET and ITGA2 correlated with high gene expression, which was associated with poor survival. These data suggest that aberrant methylation plays an important role in pancreatic carcinogenesis affecting core signaling pathways with potential implications for the disease pathophysiology and therapy.

Pancreatic cancer is molecularly diverse, with few effective therapies. Increased mutation burden and defective DNA repair are associated with response to immune checkpoint inhibitors in several other cancer types. We interrogated 385 pancreatic cancer genomes to define hypermutation and its causes. Mutational signatures inferring defects in DNA repair were enriched in those with the highest mutation burdens. Mismatch repair deficiency was identified in 1% of tumors harboring different mechanisms of somatic inactivation of MLH1 and MSH2. Defining mutation load in individual pancreatic cancers and the optimal assay for patient selection may inform clinical trial design for immunotherapy in pancreatic cancer. Pancreatic cancer is molecularly diverse, with few effective therapies. Increased mutation burden and defective DNA repair are associated with response to immune checkpoint inhibitors in several other cancer types. We interrogated 385 pancreatic cancer genomes to define hypermutation and its causes. Mutational signatures inferring defects in DNA repair were enriched in those with the highest mutation burdens. Mismatch repair deficiency was identified in 1% of tumors harboring different mechanisms of somatic inactivation of MLH1 and MSH2. Defining mutation load in individual pancreatic cancers and the optimal assay for patient selection may inform clinical trial design for immunotherapy in pancreatic cancer. Pancreatic ductal adenocarcinoma has a 5-year survival of <5%, with therapies offering only incremental benefit,1Vogelzang N.J. et al.J Clin Oncol. 2012; 30: 88-109Crossref PubMed Scopus (85) Google Scholar potentially due to the diversity of its genomic landscape.2Bailey P. et al.Nature. 2016; 531: 47-52Crossref PubMed Scopus (1973) Google Scholar, 3Biankin A.V. et al.Nature. 2012; 491: 399-405Crossref PubMed Scopus (1379) Google Scholar, 4Waddell N. et al.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1466) Google Scholar Recent reports link high mutation burden with response to immune checkpoint inhibitors in several cancer types.5Le D.T. et al.N Engl J Med. 2015; 372: 2509-2520Crossref PubMed Scopus (6099) Google Scholar Defining tumors that are hypermutated with an increased mutation burden and understanding the underlying mechanisms in pancreatic cancer has the potential to advance therapeutic development, particularly for immunotherapeutic strategies. Whole genome sequencing (WGS, n = 180) and whole exome sequencing (n = 205) of 385 unselected predominantly sporadic pancreatic ductal adenocarcinoma (Supplementary Table 1) defined a mean mutation load of 1.8 and 1.1 mutation per megabase (Mb), respectively (Supplementary Table 2). Outlier analysis identified 20 tumors with the highest mutation burden (5.2%, 15 WGS and 5 exome) (Table 1 and Supplementary Figure 1A), 5 of which were considered extreme outliers and classified as hypermutated as they contained ≥12 somatic mutations/Mb, the defined threshold for hypermutation in colorectal cancer.6Cancer Genome Atlas NetworkNature. 2012; 487: 330-337Crossref PubMed Scopus (5894) Google Scholar Immunohistochemistry for mismatch repair (MMR) proteins (MSH2, MSH6, MLH1, and PMS2) identified 4 MMR-deficient tumors, all of which were hypermutated (n = 180, Figure 1).Table 1Clinical and Histologic Features and Proposed Etiology for Highly Mutated Pancreatic Ductal Adenocarcinoma Tumors (n = 20)Sample IDPersonal and family history of malignancyHistologyMutation load, mutations/MbIHC resultMSIsensor scoreKRAS mutationPredominant mutation signature (mutations/Mb)SV subtype (no. of events)Proposed etiologyHypermutation (extreme outliers) ICGC_0076aSample sequenced by WGS, other samples by exome sequencing.NoneMixed signet ring, mucinous and papillary adenocarcinoma38.55Absent MLH1 and PMS228.3p.G12VMMR (18.3)Scattered (131)MMR deficiency: >280 kb somatic homozygous deletion over MSH2. ICGC_0297aSample sequenced by WGS, other samples by exome sequencing.NoneUndifferentiated adenocarcinoma60.62Absent MSH2 and MSH627.33WTMMR (33.4)Scattered (75)MMR deficiency: Somatic MLH1 promoter hypermethylation. ICGC_0548aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, moderately differentiated30.13Absent MSH2 and MSH617.47WTMMR (16.6)Stable (49)MMR deficiency: >27 kb somatic inversion rearrangement disrupting MSH2. ICGC_0328aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma16.63Normal3.2p.G12DUnknown (11.9)Scattered (110)Cell line with signature: etiology unknown. ICGC_00901 FDR, father CRCDuctal adenocarcinoma, moderately differentiated12.9Absent MSH2 and MSH60.21p.G12CNANAMMR deficiency: somatic MSH2 splice site c.2006G>A.Highly mutated tumors ICGC_0054aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated6.52Normal0.01p.G12VHR deficiency (1.3)Unstable (310)HR deficiency: no germline or somatic cause found. ICGC_0290aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated6.54Not available0.07p.G12VHR deficiency (3.1)Unstable (558)HR deficiency: Germline BRCA2 mutation c.7180A>T, p.A2394*. Somatic CN-LOH. ICGC_0215aSample sequenced by WGS, other samples by exome sequencing.2 FDR lung cancer, 2 FDR prostate cancer. Previous CRC and melanomaDuctal adenocarcinoma, moderately differentiated6.27Normal0.01p.G12VHR deficiency (1.9)Scattered (111)HR deficiency: Germline ATM mutation c.7539_7540delAT, p.Y2514*. Somatic CN-LOH. ICGC_0324NoneDuctal adenocarcinoma, moderately differentiated6.24Normal0p.G12DNANAUndefined ICGC_0034aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated6.09Normal4.02p.G12DHR deficiency (3.4)Unstable (366)HR deficiency: Germline BRCA2 mutation c.5237_5238insT, p.N1747*. Somatic CN-LOH. ICGC_0131aSample sequenced by WGS, other samples by exome sequencing.Lung cancer after PCDuctal adenocarcinoma, moderately differentiated5.63Normal0p.G12DT>G at TT sites (3.0)Focal (147)T>G at TT sites signature: etiology potentially associated with DNA oxidation ICGC_0006aSample sequenced by WGS, other samples by exome sequencing.1 FDR, father lung cancerAdenocarcinoma arising from IPMN, moderately differentiated5.29Normal0.01p.G12DHR deficiency (1.2)Unstable (211)HR deficiency: Somatic BRCA2 c.5351dupA, p.N1784KfsTer3. Somatic CN-LOH. ICGC_0321aSample sequenced by WGS, other samples by exome sequencing.2 FDR, mother and cousin breast cancerDuctal adenocarcinoma, poorly differentiated4.79Not available0p.G12DHR deficiency (2.1)Unstable (286)HR deficiency: Germline BRCA2 c.6699delT, p.F2234LfsTer7. Somatic CN loss- 1 copy. ICGC_0309aSample sequenced by WGS, other samples by exome sequencing.NoneAdenocarcinoma arising from IPMN, moderately differentiated4.74Normal0.03p.G12VT>G at TT sites (3.1)Unstable (232)T>G at TT sites signature: etiology potentially associated with DNA oxidation ICGC_0005aSample sequenced by WGS, other samples by exome sequencing.1 FDR, mother CRCDuctal adenocarcinoma, poorly differentiated4.72Not available1p.G12VHR deficiency (1.1)Focal (95)HR deficiency: No germline or somatic cause found. ICGC_0016aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated4.61Normal3.03p.G12VHR deficiency (1.7)Unstable (447)HR deficiency: potentially linked to Somatic RPA1 c.273G>T, p.R91S ICGC_00461 FDR, brother PCDuctal adenocarcinoma, poorly differentiated4.3Normal0p.Q61HNANAUndefined GARV_0668aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated4.3Not available2.19p.G12VHR deficiency (1.6)Unstable (464)HR deficiency: Germline BRCA2 c.7068_7069delTC, p.L2357VfsTer2. Somatic CN loss - 1 copy. ICGC_0291NoneDuctal adenocarcinoma, well differentiated3.84Not available0.03p.G12RNANAHR deficiency: Somatic BRCA2 c.7283T>A, p.L2428*. ICGC_0256NoneDuctal adenocarcinoma, poorly differentiated3.72Not available0.06p.G12DNANAUndefinedCRC, colorectal cancer; FDR, first-degree relative; IHC, immunohistochemistry; IPMN, intraductal papillary mucinous neoplasm; CN-LOH, copy neutral loss of heterozygosity; CN, copy number; PC, pancreatic cancer; NA, not applicable to exome data.a Sample sequenced by WGS, other samples by exome sequencing. Open table in a new tab CRC, colorectal cancer; FDR, first-degree relative; IHC, immunohistochemistry; IPMN, intraductal papillary mucinous neoplasm; CN-LOH, copy neutral loss of heterozygosity; CN, copy number; PC, pancreatic cancer; NA, not applicable to exome data. KRAS mutation status and histopathologic characteristics have been associated with MMR-deficient pancreatic tumors.7Goggins M. et al.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar Of the 4 MMR-deficient tumors in our cohort, 2 were KRAS wild-type; 3 had undifferentiated to moderately differentiated histology and one had a signet-ring component. These features were not predictive of MMR deficiency in our cohort, as 11 additional non−MMR-deficient tumors had a signet-ring cell component or colloid morphology, and 131 of 347 assessable tumors had poorly or undifferentiated histology. Mutational signature analysis can detect MMR deficiency indirectly based on the pattern of somatic mutations.8Alexandrov L.B. et al.Nature. 2013; 500: 415-421Crossref PubMed Scopus (6213) Google Scholar An MMR-deficient signature dominated the MMR-deficient tumors (with WGS), and was minimal in MMR intact tumors (Supplementary Figure 1). In addition, microsatellite instability (MSI), a hallmark of MMR deficiency in colorectal cancer, was detected in all three MMR deficient tumors with WGS using MSIsensor9Niu B. Ye K. et al.Bioinformatics. 2014; 30: 1015-1016Crossref PubMed Scopus (294) Google Scholar (Supplementary Table 2). MSI was not identified for the fourth MMR deficient sample potentially due to the reduced number of microsatellite loci in exome data. The underlying causes of MMR deficiency in the 4 cases were private somatic events. For 2 cases, MSH2 was disrupted by different structural rearrangements, 1 case contained a missense MSH2 mutation and the last, methylation of the MLH1 promoter (Figure 1). The missense mutation caused an MSH2 splice acceptor site mutation that alters the same nucleotide results in a pathogenic skipping of exon 13 in germline studies.10Thompson B.A. et al.Nat Genet. 2014; 46: 107-115Crossref PubMed Scopus (346) Google Scholar Hypermethylation of the MLH1 promoter is the predominant mechanism of MSI in sporadic colon cancer.11Boland C.R. et al.Gastroenterology. 2010; 138: 2073-2087 e3Abstract Full Text Full Text PDF PubMed Scopus (1359) Google Scholar The remaining hypermutated tumor contained an intact MMR pathway, and was a cell line (ATCC, CRL-2551) with an unidentified mutational signature, therefore the high mutation burden in this sample may be the result of long-term cell culture. The 15 samples (11 WGS and 4 exome) identified in the outlier analysis with high mutation burden, but not hypermutated (∼4 to 12 mutations/Mb) contained no evidence of MMR deficiency. Mutational signature analysis of the WGS samples indicated homologous recombination (HR) repair deficiency as the most substantial (range, 1.0–3.4 mutations/Mb) contributor to the mutation burden for 8 WGS mutation load outlier tumors. In support of a HR defect4Waddell N. et al.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1466) Google Scholar; 7 of these tumors contained high levels of genomic instability with >200 structural variants and mutations in genes involved in HR were present for 6 of 8 cases (Supplementary Table 2). In addition, 1 case that had undergone exome sequencing had a somatic BRCA2 nonsense mutation that likely contributed to HR deficiency in this case. A mutational signature associated with T>G mutations at TT sites previously described in other cancers, including esophageal cancer12Nones K. Waddell N. Wayte N. et al.Nat Commun. 2014; : 5Google Scholar was the major contributor (>3 mutations/Mb) in 2 samples. For these 2 and the remaining 4 cases, no potential causative event could be identified. Although germline defects in MMR genes are well reported in pancreatic cancer13Grant R.C. Selander I. et al.Gastroenterology. 2015; 148: 556-564Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar in our cohort, they did not contribute to MMR deficiency even in those with familial pancreatic cancer or a personal or family history of Lynch-related tumors. A germline truncating variant was detected in PMS2 in 1 case, but did not have loss of the second allele, had normal immunohistochemistry staining and did not display a MMR mutational signature (Supplementary Table 2). MMR deficiency is important in the evolution in a small, but meaningful proportion of pancreatic cancers with a prevalence of 1% (4 of 385) in our cohort. This is consistent with recent studies using the Bethesda polymerase chain reaction panel,14Laghi L. et al.PLoS One. 2012; 7: e46002Crossref PubMed Scopus (55) Google Scholar and with previous estimates of MSI prevalence of 2%−3%.15Nakata B. et al.Clin Cancer Res. 2002; 8: 2536-2540PubMed Google Scholar However, in tumors with low epithelial content that underwent exome sequencing, the sensitivity of somatic mutation detection is reduced, which will affect mutation burden and signature analysis. While cognizant of small numbers, immunohistochemistry was the most accurate in defining MMR due to multiple genomic mechanisms of MMR gene inactivation. Multiple methods to define MMR deficiency may be required for clinical trials that aim to recruit MMR-deficient participants to assess the potential efficacy of checkpoint inhibitors or other therapies in pancreatic cancer. Homologous recombination-deficient tumors, and those with a novel signature seen in esophageal cancer had an increased mutation burden, and need further evaluation as potential patient selection markers for clinical trials of checkpoint inhibitor and other therapies that target tumors with a high mutation burden. The authors would like to thank Cathy Axford, Deborah Gwynne, Mary-Anne Brancato, Clare Watson, Michelle Thomas, Gerard Hammond, and Doug Stetner for central coordination of the Australian Pancreatic Cancer Genome Initiative, data management, and quality control; Mona Martyn-Smith, Lisa Braatvedt, Henry Tang, Virginia Papangelis, and Maria Beilin for biospecimen acquisition; and Sonia Grimaldi and Giada Bonizzato of the ARC-Net Biobank for biospecimen acquisition. For a full list of contributors see Australian Pancreatic Cancer Genome Initiative: http://www.pancreaticcancer.net.au/apgi/collaborators. The cohort consisted of 385 patients with histologically verified pancreatic exocrine carcinoma, prospectively recruited between 2006 and 2013 through the Australian Pancreatic Cancer Genome Initiative (www.pancreaticcancer.net.au) as part of the International Cancer Genome Consortium.1Hudson T.J. et al.Nature. 2010; 464: 993-998Crossref PubMed Scopus (1689) Google Scholar Ethical approval was granted at all treating institutions and individual patients provided informed consent upon entry to the study. The clinicopathologic information for the cohort is described in (Supplementary Table 1), and the global mutation profile has previously been reported for some of these tumors (Supplementary Table 2). Tumor and normal DNA were extracted after histologic review from fresh frozen tissue samples collected at the time of surgical resection or biopsy, as described previously.2Biankin A.V. et al.Nature. 2012; 491: 399-405Crossref PubMed Scopus (1513) Google Scholar Tumor cellularity was determined from single-nucleotide polymorphism array data using qpure.3Song S. et al.PLoS One. 2012; 7: e45835Crossref PubMed Scopus (85) Google Scholar Tumors with epithelial content ≥40% underwent WGS lower cellularity tumors underwent whole exome sequencing. DNA from patient-derived pancreas cell lines and matched normal was also extracted. Exome and WGS were performed using paired 100-bp reads on the Illumina HiSeq 2000, as described previously.2Biankin A.V. et al.Nature. 2012; 491: 399-405Crossref PubMed Scopus (1513) Google Scholar, 4Waddell N. et al.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1686) Google Scholar Regions of germline and somatic copy number change were detected using Illumina SNP BeadChips with GAP.5Popova T. et al.Genome Biol. 2009; 10 (R128−R128)Crossref PubMed Scopus (151) Google Scholar Somatic structural variants were identified from WGS reads using the qSV tool.4Waddell N. et al.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1686) Google Scholar, 6Patch A.M. et al.Nature. 2015; 521: 489-494Crossref PubMed Scopus (930) Google Scholar Single nucleotide variants were called using 2 variant callers: qSNP7Kassahn K.S. et al.PLoS One. 2013; 8: e74380Crossref PubMed Scopus (52) Google Scholar and GATK.8McKenna A. et al.Genome Res. 2010; 20: 1297-1303Crossref PubMed Scopus (14755) Google Scholar Mutations identified by both callers or, those that were unique to a caller but verified by an orthogonal sequencing approach, were considered high confidence and used in all subsequent analyses. Small indels (<200 bp) were identified using Pindel9Ye K. et al.Bioinformatics. 2009; 25: 2865-2871Crossref PubMed Scopus (1391) Google Scholar and each indel was visually inspected in the Integrative Genome Browser. The distribution of the total number of small somatic mutations (coding and noncoding single nucleotide and indel variants) identified per megabase for exome and WGS sequence data were analyzed separately. The group of samples with high mutation load, at the top of each distribution, were defined as the upper distribution outliers for mutations per megabase, that is, ≥75th centile + (1.5× interquartile range). The threshold for detecting outliers in the exome and WGS groups was 3.4 and 4.2 mutations/Mb, respectively. From within the highly mutated set of tumors, hypermutated samples were identified as those with a mutation rate exceeding the thresholds for extreme distribution outliers (≥75th centile + [5× interquartile range]) of 7.4 and 8.1 mutations/Mb for exome and WGS sequencing, respectively. MSIsensor was used to detect microsatellite instability by directly comparing microsatellite repeat lengths between paired normal and tumor sequencing data.10Niu B. et al.Bioinformatics. 2014; 30: 1015-1016Crossref PubMed Scopus (378) Google Scholar A MSIsensor score of >3.5% of somatic microsatellites with repeat length shifts was the detection threshold used to indicate microsatellite instability as published for endometrial cancer.10Niu B. et al.Bioinformatics. 2014; 30: 1015-1016Crossref PubMed Scopus (378) Google Scholar This correlated well with the 5 and 7 microsatellite panels recommended in the Bethesda guidelines.10Niu B. et al.Bioinformatics. 2014; 30: 1015-1016Crossref PubMed Scopus (378) Google Scholar, 11Umar A. et al.J Natl Cancer Inst. 2004; 96: 261-268Crossref PubMed Scopus (2461) Google Scholar Tissue microarrays were constructed using at least three 1-mm formalin-fixed, paraffin-embedded tumor cores. Immunohistochemistry for MSH6 and PMS2 proteins was performed on tissue microarray sections as a screen for MMR deficiency due to MMR proteins forming heterodimers with concordant mismatch repair loss (ie, loss of MLH1 and PMS2 or loss of MSH2 and MSH6).12Hall G. et al.Pathology. 2010; 42: 409-413Abstract Full Text PDF PubMed Scopus (98) Google Scholar Immunohistochemistry on full tumor sections for MSH2, MLH1, MSH6, and PMS2 was performed in those with abnormal staining in core sections. The immunohistochemistry was performed as described previously12Hall G. et al.Pathology. 2010; 42: 409-413Abstract Full Text PDF PubMed Scopus (98) Google Scholar and scored by a senior pathologist. Somatic mutational signatures were extracted from the whole genome sequenced samples using the framework described previously.13Alexandrov L.B. et al.Cell Rep. 2013; 3: 246-259Abstract Full Text Full Text PDF PubMed Scopus (734) Google Scholar High confidence somatic substitutions were classified by the substitution change and sequence context, that is, the type of immediately neighboring bases to the variant. The framework processes the counts of somatic mutations at each context within each sample using non-negative factorization to produce the different signature profiles that are present in the data. The profiles identified were matched against reported signatures from the Cancer of Somatic Mutations in Cancer (http://cancer.sanger.ac.uk/cosmic/signatures). The major contributory signatures, defined as the mutational signature with the highest number of contributing somatic substitution variants, is reported for highly mutated whole genome samples. Bisulfite-converted whole-genome amplified DNA was hybridized to Infinium Human Methylation 450K Beadchips according to the manufacturers protocol (Illumina). Methylation arrays were performed on DNA from 174 pancreatic ductal adenocarcinoma samples, which were compared to DNA from 29 adjacent nonmalignant pancreata. A subset of the methylation data has been published previously.14Nones K. et al.Int J Cancer. 2014; 135: 1110-1118Crossref PubMed Scopus (156) Google Scholar We examined the data for evidence of tumor-specific hypermethylation of the promoter region of MLH1 and MSH2 genes. The methylation array data have been deposited into the International Cancer Genome Consortium data portal (dcc.icgc.org, project PACA-AU). Download .xlsx (.08 MB) Help with xlsx files Supplementary Tables 1 and 2

Abstract Treatment options for patients with brain metastases ( BMs ) have limited efficacy and the mortality rate is virtually 100%. Targeted therapy is critically under‐utilized, and our understanding of mechanisms underpinning metastatic outgrowth in the brain is limited. To address these deficiencies, we investigated the genomic and transcriptomic landscapes of 36 BMs from breast, lung, melanoma and oesophageal cancers, using DNA copy‐number analysis and exome‐ and RNA ‐sequencing. The key findings were as follows. (a) Identification of novel candidates with possible roles in BM development, including the significantly mutated genes DSC2 , ST7 , PIK3R1 and SMC5 , and the DNA repair, ERBB – HER signalling, axon guidance and protein kinase‐A signalling pathways. (b) Mutational signature analysis was applied to successfully identify the primary cancer type for two BMs with unknown origins. (c) Actionable genomic alterations were identified in 31/36 BMs (86%); in one case we retrospectively identified ERBB2 amplification representing apparent HER2 status conversion, then confirmed progressive enrichment for HER2 ‐positivity across four consecutive metastatic deposits by IHC and SISH , resulting in the deployment of HER2 ‐targeted therapy for the patient. (d) In the ERBB / HER pathway, ERBB2 expression correlated with ERBB3 ( r 2 = 0.496; p &lt; 0.0001) and HER3 and HER4 were frequently activated in an independent cohort of 167 archival BM from seven primary cancer types: 57.6% and 52.6% of cases were phospho‐ HER3 Y1222 or phospho‐ HER4 Y1162 membrane‐positive, respectively. The HER3 ligands NRG1 / 2 were barely detectable by RNAseq , with NRG1 (8p12) genomic loss in 63.6% breast cancer‐ BMs , suggesting a microenvironmental source of ligand. In summary, this is the first study to characterize the genomic landscapes of BM . The data revealed novel candidates, potential clinical applications for genomic profiling of resectable BMs , and highlighted the possibility of therapeutically targeting HER3 , which is broadly over‐expressed and activated in BMs , independent of primary site and systemic therapy. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley &amp; Sons, Ltd.

We show that 1 of the type II bone morphogenetic protein (BMP) receptor ligands, BMP4, is widely expressed in the adult mouse lung and is upregulated in hypoxia-induced pulmonary hypertension (PH). Furthermore, heterozygous null Bmp4(lacZ/+) mice are protected from the development of hypoxia-induced PH, vascular smooth muscle cell proliferation, and vascular remodeling. This is associated with a reduction in hypoxia-induced Smad1/5/8 phosphorylation and Id1 expression in the pulmonary vasculature. In addition, pulmonary microvascular endothelial cells secrete BMP4 in response to hypoxia and promote proliferation and migration of vascular smooth muscle cells in a BMP4-dependent fashion. These findings indicate that BMP4 plays a dominant role in regulating BMP signaling in the hypoxic pulmonary vasculature and suggest that endothelium-derived BMP4 plays a direct, paracrine role in promoting smooth muscle proliferation and remodeling in hypoxic PH.

Somatic mutation calling from next-generation sequencing data remains a challenge due to the difficulties of distinguishing true somatic events from artifacts arising from PCR, sequencing errors or mis-mapping. Tumor cellularity or purity, sub-clonality and copy number changes also confound the identification of true somatic events against a background of germline variants. We have developed a heuristic strategy and software (http://www.qcmg.org/bioinformatics/qsnp/) for somatic mutation calling in samples with low tumor content and we show the superior sensitivity and precision of our approach using a previously sequenced cell line, a series of tumor/normal admixtures, and 3,253 putative somatic SNVs verified on an orthogonal platform.

The incidence of esophageal adenocarcinoma (EAC) has risen significantly over recent decades. Although survival has improved, cure rates remain poor, with <20% of patients surviving 5 years. This is the first study to explore methylome, transcriptome and ENCODE data to characterize the role of methylation in EAC. We investigate the genome-wide methylation profile of 250 samples including 125 EAC, 19 Barrett's esophagus (BE), 85 squamous esophagus and 21 normal stomach. Transcriptome data of 70 samples (48 EAC, 4 BE and 18 squamous esophagus) were used to identify changes in methylation associated with gene expression. BE and EAC showed similar methylation profiles, which differed from squamous tissue. Hypermethylated sites in EAC and BE were mainly located in CpG-rich promoters. A total of 18575 CpG sites associated with 5538 genes were differentially methylated, 63% of these genes showed significant correlation between methylation and mRNA expression levels. Pathways involved in tumorigenesis including cell adhesion, TGF and WNT signaling showed enrichment for genes aberrantly methylated. Genes involved in chromosomal segregation and spindle formation were aberrantly methylated. Given the recent evidence that chromothripsis may be a driver mechanism in EAC, the role of epigenetic perturbation of these pathways should be further investigated. The methylation profiles revealed two EAC subtypes, one associated with widespread CpG island hypermethylation overlapping H3K27me3 marks and binding sites of the Polycomb proteins. These subtypes were supported by an independent set of 89 esophageal cancer samples. The most hypermethylated tumors showed worse patient survival.

Recent studies in prostate tissues and especially cell lines have suggested roles for arachidonic acid (AA) metabolizing enzymes in prostate adenocarcinoma (Pca) development or progression. The goal of this study was to more fully characterize lipoxygenase (LOX) and cyclooxygenase-2 (COX-2) gene expression and AA metabolism in benign and malignant prostate using snap-frozen tissues obtained intraoperatively and mRNA analyses and enzyme assays. Formation of 15-hydroxyeicosatetraenoic acid (15-HETE) was detected in 23/29 benign samples and 15-LOX-2 mRNA was detected in 21/25 benign samples. In pairs of pure benign and Pca from the same patients, 15-HETE production and 15-LOX-2 mRNA were reduced in Pca versus benign in 9/14 (P=.04) and 14/17 (P=.002), respectively. Under the same conditions, neither 5-HETE nor 12-HETE formation was detectable in 29 benign and 24 tumor samples; with a more sensitive assay, traces were detected in some samples, but there was no clear association with tumor tissue. COX-2 mRNA was detected by nuclease protection assay in 7/16 benign samples and 5/16 tumors. In benign and tumor pairs from 10 patients, COX-2 was higher in tumor versus benign in only 2, with similar results by in situ hybridization. Paraffin immunoperoxidase for COX-2 was performed in whole mount sections from 87 additional radical prostatectomy specimens, with strong expression in ejaculatory duct as a positive control and corroboration with in situ hybridization. No immunostaining was detected in benign prostate or tumor in 45% of cases. Greater immunostaining in tumor versus benign was present in only 17% of cases, and correlated with high tumor grade (Gleason score 8 and 9 vs. 5 to 7). In conclusion, reduced 15-LOX-2 expression and 15-HETE formation is the most characteristic alteration of AA metabolism in Pca. Increased 12-HETE and 5-HETE formation in Pca were not discernible. Increased COX-2 expression is not a typical abnormality in Pca in general, but occurs in high-grade tumors.

15-Lipoxygenase (15-LOX)-2 is expressed in benign prostate secretory cells and benign prostate produces 15S-hydroxyeicosatetraenoic acid (15S-HETE) from exogenous arachidonic acid (AA). In contrast, 15S-LOX-2 and 15S-HETE formation are reduced in prostate carcinoma (Pca). The mechanisms whereby reduced 15-LOX-2 may contribute to Pca development or progression are not known. We investigated the expression of peroxisome proliferator-activated receptor (PPAR) gamma in benign and malignant prostate tissues and the ability of 15S-HETE to activate PPARgamma-dependent transcription and modulate proliferation of the Pca cell line PC3. In contrast to benign prostate and similar to most Pca tissues, 15-LOX-2 mRNA was not detected in PC3 cells, and they did not produce detectable 15-HETE from [14C]AA. By reverse transcription-PCR, PPARgamma mRNA was present in 18 of 18 benign and 9 of 9 tumor specimens. The PPARgamma ligand BRL 49653 and 15S-HETE caused a dose-dependent inhibition of PC3 proliferation in a 14-day soft agar colony-forming assay (IC50 of 3 and 30 microM, respectively). 15S-HETE (10 microM) caused greater inhibition than 10 microM 15R-HETE. At 3 days, BRL 49653 and 15S-HETE caused a slight increase in cells in G0-G1 and a corresponding decrease in cells in S phase. In PC3 cells transiently transfected with a luciferase reporter linked to a PPAR response element, 1 microM BRL 49653 and 10 microM 15S-HETE caused approximately threefold and greater than twofold induction of PPAR-dependent transcription, respectively. By quantitative real-time reverse transcription-PCR and Northern analysis, 3-day treatment with BRL 49653 and 15S-HETE caused a reduction of PPARgamma expression but a marked up-regulation of the PPAR response element containing adipocyte type fatty acid binding protein. These results support the hypothesis that 15-LOX-2-derived 15S-HETE may constitute an endogenous ligand for PPARgamma in the prostate and that loss of this pathway by reduced expression of 15-LOX-2 may contribute to increased proliferation and reduced differentiation in prostate carcinoma.

Arachidonic acid (AA) metabolites are implicated in the oncogenesis of several tumors, including prostate cancer. 15-Lipoxygenase-2 (15-LOX-2) is a novel AA-metabolizing enzyme with a limited tissue distribution, which includes prostate, lung, skin, and cornea. Previous studies have shown that 15-LOX-2 is present in benign prostate secretory cells and reduced in prostate adenocarcinoma and that production of the 15-LOX-2 metabolite 15S-hydroxyeicosatetraenoic acid is reduced in malignant compared with benign prostate. The objective of this study was to determine the frequency with which 15-LOX-2 immunostaining is reduced in prostate carcinoma and to correlate reduced expression with tumor differentiation (grade) and other pathologic parameters in radical prostatectomy specimens. Paraffin immunoperoxidase with a polyclonal antibody specific for 15-LOX-2 was performed on tumors and benign portions from 70 cases, and the percentage of tumor immunostaining for 15-LOX-2 was assessed. Whereas uniform 15-LOX-2 immunostaining was observed in secretory cells of benign glands, it was markedly reduced or absent in most adenocarcinomas: 23 of 70 tumors showed completely absent 15-LOX-2 immunostaining, and 45 of 70 cases showed negative immunostaining in more than 50% of the tumor. The extent of reduced 15-LOX-2 immunostaining correlated with tumor differentiation, with retained expression particularly in Gleason score 5 tumors versus a significant reduction of 15-LOX-2 in higher-grade tumors (mean +/- SD tumor 15-LOX-2 positive: Gleason score 5 = 67%+/-30%, Gleason score 6 = 16%+/-30%, Gleason score 7 = 23%+/-28%, Gleason score > or =8 = 41%+/-46%). In 16 cases with multifocal tumors or different foci of the same tumor with different grades, the higher-grade foci had significantly reduced 15-LOX-2 expression compared with the lower-grade foci. In peripheral zone tumors without complete loss of 15-LOX-2 expression, there was a significant inverse relationship between 15-LOX-2 immunostaining and tumor volume. There was not a significant correlation between 15-LOX-2 immunostaining and serum PSA or pathologic stage. In a subset of 27 cases, 15-LOX-2 expression in high-grade prostatic intraepithelial neoplasia (HGPIN) glands was significantly reduced compared with benign glands. These data show that in contrast to the uniform expression of 15-LOX-2 in differentiated secretory cells of benign prostate, reduced 15-LOX-2 is a common alteration in prostate carcinoma, and this correlates with tumor cell differentiation. That reduced expression is seen in HGPIN suggests that this may be an early alteration in carcinoma development.

We have examined the expression and structure of several genes belonging to two classes of vegetative specific genes of the simple eukaryote, Dictyostelium discoideum. In amebae grown on bacteria, deactivation of all vegetative specific genes occurred at the onset of development and very little mRNA exists by 8 to 10 hours. In contrast, when cells were grown in axenic broth, the mRNA levels remained constant until a dramatic drop occurred around 10 to 12 hours. Thus, regulation of both classes of genes during the first several hours of development is dependent upon the prior growth conditions. Analysis of genomic clones has resulted in the identification of two V genes, V1 and V18, as ribosomal protein genes. Several other V genes were not found to be ribosomal protein genes, suggesting that in Dictyostelium non-ribosomal protein genes may be coordinately regulated with the ribosomal protein genes. Finally, using deletion analysis we show that the promoters of two of the V genes are composed of a constitutive positive element(s) located upstream of sequences involved in the regulated expression of these genes and within the first 545 upstream bp for V18 and 850 bp for V14. The regions involved in regulated expression were localized between -7 and -222 for V18 and -70 and -368 for V14. The sequences conferring protein synthesis sensitivity were shown to reside between -502 and -61 of the H4 promoter.

Nature 521, 489–494 (2015); doi: 10.1038/nature14410 In this Article, the affiliations of authors Michael Quinn and Orla McNally should read “22Department of Obstetrics and Gynaecology, The University of Melbourne, and The Royal Women’s Hospital, Parkville, Victoria 3052, Australia”. Their affiliations have been corrected in the HTML and PDF versions online.

The rate of parvovirus B19 (PV) infection in cases of "idiopathic" nonimmune hydrops fetalis (NIHF) is reported to be approximately 16% with polymerase chain reaction (PCR)-based methods. Antibodies for use in paraffin-embedded tissue have not been systematically compared with PCR or with the presence of inclusions at varying gestational ages. All autopsy cases of NIHF and those with effusions of multiple serous membranes examined between 1991 and 1996 (n = 29) were evaluated for the presence of PV DNA by PCR analysis of paraffin-embedded liver tissue. PCR-positive cases and "idiopathic" cases were examined for the presence of inclusions in routine histological sections and for PV protein using a monoclonal antibody (NovoCastra R92F6). Among the four clinically idiopathic cases, one (25%) was positive for PV using PCR. The three negative idiopathic cases had no inclusions and were negative for PV by PCR and immunohistochemistry (IHC); all were third-trimester gestations (28, 31, and 32 weeks). Identifiable risk factors for NIHF other than PV in the remaining 25 cases included cystic hygroma, seven (three 45,X; two 46,XX; two no growth); complex cardiac anomaly, six; infection, three (two CMV, one chlamydia); twin-twin transfusion, two; lymphangiectasia, two; diaphragmatic hernia, tracheal atresia, trisomy 21, congenital cystic adenomatoid malformation, one each. One of these nonidiopathic cases, a fetus with cystic hygroma and a 45,X karyotype, was positive for PV DNA only on the blot, consistent with a low titer; no inclusions were present, and IHC was negative in multiple organs in this instance. One of four (25%) cases of idiopathic NIHF cases contained PV DNA by PCR analysis; there were abundant inclusions in multiple organs, and IHC was strongly positive as well. Of 25 cases of nonidiopathic NIHF, one (4%) was also positive for PV DNA by PCR. PV protein was detected by IHC only in the presence of inclusions; IHC thus may be useful for highlighting sparse inclusions. No second-trimester case of NIHF was unexplained. Late (third-trimester) cases of "idiopathic" NIHF are likely to be negative by all methods, either because they are not attributable to PV infection or because PV protein and DNA are below detectable levels or are no longer present. Maternal serology for PV and TORCH agents may be the best method for investigating third-trimester losses to otherwise unexplained NIHF.

Changes in expression of arachidonic acid (AA) metabolizing enzymes are implicated in the development and progression of human prostate carcinoma (Pca). Transgenic mouse models of Pca that progress from high-grade prostatic intraepithelial neoplasia (HGPIN) to invasive and metastatic carcinoma could facilitate study of the regulation and function of these genes in Pca progression. Herein we characterize the AA-metabolizing enzymes in transgenic mice established with a prostate epithelial-specific long probasin promoter and the SV40 large T antigen (LPB-Tag mice) that develop extensive HGPIN and invasive and metastatic carcinoma with neuroendocrine (NE) differentiation. Murine 8-lipoxygenase (8-LOX), homologue of the 15-LOX-2 enzyme that is expressed in benign human prostatic epithelium and reduced in Pca, was not detected in wild-type or LPB-Tag prostates as determined by enzyme assay, reverse transcription-PCR, and immunohistochemistry. The most prominent AA metabolite in mouse prostate was 12-HETE. Wild-type prostate (dorsolateral lobe) converted 1.6 +/- 0.5% [(14)C]AA to 12-HETE (n = 7), and this increased to 8.0 +/- 4.4% conversion in LPB-Tag mice with HGPIN (n = 13). Quantitative real-time reverse transcription-PCR and immunostaining correlated the increased 12-HETE synthesis with increased neoplastic epithelial expression of 12/15-LOX, the leukocyte-type (L) of 12-LOX and the murine homologue of human 15-LOX-1. Immunostaining showed increased L12-LOX in invasive carcinoma and approximately one-half of metastatic foci. COX-2 mRNA was detectable in neoplastic prostates with HGPIN but not in wild-type prostate. By immunostaining, COX-2 was increased in the neoplastic epithelium of HGPIN but was absent in foci of invasion and metastases. We conclude that (a) AA metabolism in wild-type mouse prostate differs from humans in the basal expression of LOXs (15-LOX-2 in human, absence of its 8-LOX homologue in mouse prostate); (b) increased expression of 12/15-LOX in HGPIN and invasive carcinoma of the LPB-Tag model is similar to the increased 15-LOX-1 in high-grade human Pca; and (c) the LPB-Tag model shows increased COX-2 in HGPIN, and therefore, it may allow additional definition of the role of this enzyme in the subset of human HGPINs or other precursor lesions that are COX-2 positive, as well as investigation of its contribution to neoplastic cell proliferation and tumor angiogenesis in Pca.

In this study, we evaluated the impact of hemoglobin (Hb) variants on the performance of the Abbott Alinity c and Bio-Rad Variant II Turbo 2.0 HPLC Hb A1c assays.The analytical performance of the Abbott Alinity c Hb A1c (enzymatic) assay was compared to the Bio-Rad Variant II Turbo 2.0 HPLC method using leftover whole blood EDTA samples with and without the presence of a hemoglobin variant. Assay precision was determined from an analysis of controls. Bias was estimated from analysis of a set of 40 samples analyzed by a Tosoh G8 HPLC instrument at the University of Missouri Diabetes Diagnostic Laboratory, an NGSP Secondary Reference Laboratory.Between-day precision was excellent for both methods (<3%). Bias met NGSP criteria of ±5% to target value. Correlation between the Alinity and Bio-Rad methods was good for patient samples without a hemoglobinopathy (y = 1.028x - 0.38, standard error of the estimate (SEE) = 0.16, n = 36, mean bias = -0.22). A total of 700 hemoglobin variant samples were evaluated on the 2 methods. Of the hemoglobin variants, 640/700 gave results on both methods: hemoglobin (Hb) S trait (n = 452), C trait (n = 131), D trait (n = 23), E trait (n = 26), and a mixture of other hemoglobinopathies (n = 8) including beta thalassemia, high hemoglobin F, transfused Hb SC, transfused Hb SD, and transfused Hb SS, or unknown variant. There was good agreement for the 640 Hb variants between the methods with a range of mean differences of -0.10 to +0.06 depending on the variant, but more variability (SEE 0.25 to 0.39). Sixty samples did not have paired results.To our knowledge, this study was the largest investigation of the effect of hemoglobinopathies on the Abbott Alinity c Hb A1c assay. Analytical performance varied depending on the specific hemoglobin variant trait when compared to the Bio-Rad Variant II Turbo 2.0 HPLC method.

Several genes which are deactivated on the initiation of development of Dictyostelium discoideum were identified by differential screening of various cDNA libraries. These genes have in common a decrease in the steady-state levels of their corresponding mRNAs on the onset of development and as development proceeds. When development was carried out in the absence of protein synthesis by inhibition with cycloheximide, the decrease in mRNA levels for most genes (V genes) was normal or slightly accelerated. For about 5% of the genes (H genes), however, cycloheximide caused an apparent induction of expression, as revealed by a slight or dramatic increase in mRNA levels, instead of the normal decrease. This effect was due to inhibition of protein synthesis and not to cycloheximide per se. The induction was found to be due to an enhancement of the transcription rate; normal rates of transcription for the H genes were dependent on continued protein synthesis during vegetative growth and development. Thus, two general regulatory classes exist for deactivation of gene expression on initiation of development, one of which is dependent on and one of which is independent of protein synthesis. Analysis of expression of these genes in mutant strains which are aggregation deficient allowed the classes to be subdivided further. Taken together, these characterizations allow several distinct regulatory mechanisms to be identified that are involved in the deactivation of gene expression on the onset of development in D. discoideum.

We have identified and characterized three genes, the I genes (I for induced), which are induced during the preaggregative phase of the developmental program of Dictyostelium discoideum. None of these genes are expressed in cells growing vegetatively on bacteria or in axenic broth, and their induction during early development is due to transcriptional activation. Developmental expression of I6, I8, and I11 occurs even in the absence of protein synthesis. Their induction is very rapid and occurs essentially at the onset of development. The expression is transient, peaking between 2 and 4 hr followed by a rapid loss of expression. These characteristics suggest that the induction of I6, I8, and I11 is a primary result of the initiation of development, and thus they represent the first such genes isolated. Although their expression behavior shares these characteristics, examination of their expression under various conditions of development and in a variety of aggregation-deficient mutant strains reveals that the details of the regulation and developmental control of these three genes are distinct.

We have examined the expression and structure of vegetative specific genes belonging to the V and H gene classes. Both classes of genes are deactivated at the onset of development by a reduction in the rate of transcription. Thus, the genes must be reactivated when the terminally differentiated spores germinate and the resulting amebae return to the vegetative state. During germination, activation of expression of most members of the V gene class was found to parallel the emergence of amoebae from the spore coats. The activation of the V genes did not occur when protein synthesis was inhibited. The timing of activation of the H genes was more heterogeneous and did not parallel emergence. H gene activation occurred even when protein synthesis was inhibited. V4 was found to be the only vegetative specific gene that was responsive to the presence of bacteria. V4 expression was induced by 25-100 fold via transcriptional activation when bacteria were added to amebae growing axenically. Isolation and sequence analysis of the corresponding genomic clones revealed that two V genes, V18 and V1, encode ribosomal proteins. Promoter analysis has delineated the sequences necessary for expression and regulation for several of the V and H genes. In all cases, expression was determined by sequences within the first several hundred base pairs of the transcription start site. For V18 and V14, a positive constitutive element was identified in addition to the sequences involved in regulation. Finally, all of the characterizations and findings are discussed in terms of postulated models for V and H gene expression and regulation.

We examined gene and surface expression and activity of the endothelin (ET)-1 receptors (ETA and ETB) in subendothelial (L1) and inner medial (L2) cells from the main pulmonary artery of sheep with continuous air embolization (CAE)-induced chronic pulmonary hypertension (CPH). According to quantitative real-time RT-PCR, basal gene expression of both receptors was significantly higher in L2 than L1 cells, and hypertensive L2 cells showed significantly higher gene expression of ETB than controls. Expression of both genes in hypertensive L1 cells was similar to controls. Fluorescence-activated cell sorter analysis confirmed the increased distribution of ET(B) in hypertensive L2 cells. Although only the ETA receptors in control L2 cells showed significant binding of [125I]-labeled ET-1 at 1 h, both receptors bound ET-1 to hypertensive cells. Exposure to exogenous ET-1 for 18 h revealed that only the L2 cells internalized ET-1, and internalization by hypertensive L2 cells was significantly reduced when compared with controls. Treatment with ETA (BQ-610) and ETB (BQ-788) receptor antagonists demonstrated that both receptors contributed to internalization of ET-1 in control L2 cells, whereas in hypertensive cells only when both receptor antagonists were used in combination was significant suppression of ET-1 internalization found. We conclude that in sheep receiving CAE, alterations in ETB receptors in cells of the L2 layer may contribute to the maintenance of CPH via alterations in their expression, distribution, and activity.

Loss of heterozygosity and microsatellite instability have been often reported in breast cancer and seldom in proliferative breast disease (PBD). DNA samples from microdissected PBD lesions, including papillomas (25 lesions), from 8 women were analyzed by polymerase chain reaction for loss of heterozygosity and microsatellite instability at 10 loci including INT-2 oncogene locus, D17S796 (the p53 gene region), and D17S579 (in the region of the BRCA-1 gene). In a patient, five loci with microsatellite instability and two loci with loss of heterozygosity were identified in one papilloma with florid hyperplasia and atypia, and 10 other PBD lesions were negative for genetic alteration (GA) and atypia. Three loci with microsatellite instability were identified in another PBD lesion without atypia, whereas another lesion from this second patient had minimal atypia without GAs. These two patients have been well for more than 20 years. No other patient, including a woman developing cancer, had GAs. We detected GAs in PBD (25% of women, 8% of lesions). Incomplete correlation between GAs and anatomic atypia was suggested. It seems evident that several GAs in PBD lesions may not indicate clinically meaningful premalignancy for remaining breast.

To the Editor: Cells that have altered or absent DNA repair mechanisms may be more susceptible to genetic alteration and, thus, neoplastic transformation. Alteration in the integrity of the DNA mismatch repair mechanism can be measured by documenting microsatellite instability (MSI). Microsatellite

Abstract Several genes that are deactivated upon the initiation of development of Dictyostelium discoideum have been identified by differential screening of various cDNA libraries. These genes have in common a decrease in the steady‐state levels of their corresponding mRNAs as development proceeds. When development was carried out in the absence of protein synthesis by inhibition with cycloheximide, the decrease in mRNA levels for most genes (V genes) was normal or slightly accelerated. However, for about 5% of the genes (H genes), cycloheximide caused an apparent induction of expression, as revealed by a slight or dramatic increase in mRNA levels instead of the normal decrease. This effect was due to inhibition of protein synthesis and not to cycloheximide per se. The induction was found to be due to an enhancement of the trascription rate; normal rates of transcription for the H genes were dependent upon continued protein synthesis during vegetative growth and during development. Thus, two general regulatory classes exist for deactivation of gene expression upon initiation of development, one dependent and one independent of protein synthesis. Models concerning the control of expression of these two classes of genes are discussed here. Analysis of expression of these genes in mutant strains that are aggregation‐deficient has also been performed, and the results lead to subdivisions of the classes.

Abstract During tumorigenesis, a heterotypic interface exists between cancer and stromal cells that can both support and repress tumor growth. In the breast, studies have demonstrated a pro-tumorigenic role for adipocytes. However, the molecular mechanisms by which breast cancer cells coopt adipocytes remain elusive. Studying breast tumors and normal adjacent tissue (NAT) from several patient cohorts, patient-derived xenografts and mouse models, we show that lipolysis and lipolytic signaling are activated in NAT. We investigated the tumor-adipocyte interface and find that functional gap junctions form between breast cancer cells and adipocytes. As a result, cAMP, a critical lipolysis-inducing signaling molecule, is transferred from breast cancer cells to adipocytes and activates lipolysis in a gap junction-dependent manner. We found that gap junction formation depends upon connexin 31 (Cx31), and that Cx31 is essential for breast tumor growth and activation of lipolysis in vivo . Thus, direct tumor cell-adipocyte interaction is critical for tumorigenesis, and may serve as a new therapeutic target in breast cancer. One sentence summary Gap junctions between breast cancer cells and adipocytes transfer cAMP and activate lipolysis in the breast tumor microenvironment.

Abstract Background Mass spectrometry (MS) remains the gold standard, however immunoassays can provide a preliminary test result while the patient is still in the emergency room or clinic. Methods The ARK Fentanyl II assay (ARK Diagnostics Inc, Fremont, CA) was compared to the Immunalysis SEFRIA Fentanyl (Immunalysis Corporation, Pomona, CA) assay on the Abbott Alinity c analyzer (Abbott Park, IL) using leftover deidentified urine samples. MS analysis was performed at ARUP Laboratories (Salt Lake City, UT) to quantitate the amount of fentanyl or norfentanyl metabolite in each sample. Medications prescribed at the time of sampling were reviewed to assess for possible cross reactivity with the assays. Results A total of 142 urine samples were analyzed with 70 MS positive and 72 MS negative samples. Positive and negative assay specific quality control precision (N = 10) was 3.0% and 9.6% CV for the ARK Fentanyl II assay and 10.2% and 15.9% CV for the Immunalysis SEFRIA assays respectively. ARK Fentanyl II Sensitivity = 95.7%, Specificity = 94.4%, PPV = 94.4%, NPV = 95.8% and Efficiency = 95.1% compared to the Immunalysis SEFRIA Fentanyl Sensitivity = 95.7%, Specificity = 81.9%, PPV = 83.8%, NPV = 95.2% and Efficiency = 88.7%. Upon repeat analysis, 4 of 4 ARK and 6 of 13 Immunalysis false positives reported as negative, indicating some variability around the cutoff concentration. Possible carryover from high samples has also been reported (ARK Technical Note 20-001-August 2020) and high samples were analyzed prior to some of the false positive samples. Six MS positive samples were reported as negative (false negatives), 3 samples by each of the assays. However, 4/6 of those samples had combined fentanyl and norfentanyl concentrations &amp;lt;3 ng/mL by MS. Conclusion The ARK Fentanyl II and Immunalysis SEFRIA assays demonstrated acceptable performance in the detection of fentanyl in our patient population.

Medical Journal of AustraliaVolume 181, Issue 3 p. 171-172 Letter Temporary protection visas and child refugees Christine B Phillips, Corresponding Author Christine B Phillips Senior Lecturer christine.phillips@calvary-act.com.au Academic Unit of General Practice and Community Health, Australian National University Medical School, PO Box 254, Jamison Centre, Jamison, ACT 2614Correspondence: christine.phillips@calvary-act.com.auSearch for more papers by this authorSuzanne Manning, Suzanne Manning Intern Department of Psychology, Australian National University, Acton, ACT.Search for more papers by this author Christine B Phillips, Corresponding Author Christine B Phillips Senior Lecturer christine.phillips@calvary-act.com.au Academic Unit of General Practice and Community Health, Australian National University Medical School, PO Box 254, Jamison Centre, Jamison, ACT 2614Correspondence: christine.phillips@calvary-act.com.auSearch for more papers by this authorSuzanne Manning, Suzanne Manning Intern Department of Psychology, Australian National University, Acton, ACT.Search for more papers by this author First published: 02 August 2004 https://doi.org/10.5694/j.1326-5377.2004.tb06213.xCitations: 1Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume181, Issue3August 2004Pages 171-172 RelatedInformation

Abstract Proteomic analysis of cancer tissues by mass spectrometry (MS) is complicated by the fact that histological context is lost in the analysis. Knowledge on the abundance of key proteins for the different histological compartments may allow an assessment of the contribution of each of these components to the proteomic profile obtained. We studied 18 colorectal carcinoma specimens and isolated carcinoma, necrosis, normal epithelium, stroma, smooth muscle, and lymphoid infiltrate compartments using laser capture microdissection (LCM). Proteins extracted from 60 LCM caps were subjected to short-stack, gel electrophoresis, in-gel trypsin digestion and analyzed on a Thermo-Fisher QExactive Orbitrap MS instrument. Data were searched using myrimatch and MS-GF+ on a RefSeq protein database and identified proteins were assembled using IDpicker 3. The analysis identified about 2,000 non-redundant proteins at a protein FDR&amp;lt;5%. Unsupervised cluster analysis of the most abundant proteins clustered each of the compartments together with few exceptions. Surprisingly, the necrotic compartment was almost identical from the carcinoma compartment, indicating the stability of the small peptides used for MS sequence identifications. Comparisons of spectral count data between each of the compartments and the remainder of the dataset by protein enrichment analyses was performed using the online tool www.webgestalt.org. The carcinoma compartment was enriched for proteins involved in nucleic acid and general metabolism processes. Smooth muscle was enriched for proteins involved in muscle contraction and cell adhesion. The lymphoid infiltrates were enriched for chromatin and nucleosome proteins, possibly indicating the relative contribution of nuclear proteins from these small cells. The stromal component was enriched for extracellular matrix proteins and proteins involved in glycosylation processes. Using data from ProteinAtlas.org, we confirmed histological associations of the most prominent protein markers with their respective histological component. For instance, COL12A1 was highly expressed in the stromal compartment, while SMTN was highly specific for smooth muscle. Expression levels characterizing each of the histological components can thus be described in protein signatures that can serve as a measure of the relative contribution of each of the components in the complex composition of a colorectal carcinoma sample. Supported by NIH/NCI grant U24CA159988. Citation Format: Robbert JC Slebos, Lisa J. Zimmerman, Suzanne Manning, Melinda E. Sanders, Chanjuan Shi, M Kay Washington, Daniel C. Liebler. Proteomic features of histological compartments in colorectal carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2000. doi:10.1158/1538-7445.AM2015-2000

Abstract HER2 (ERBB2) is overexpressed in about 25% of breast cancers and predicts clinical benefit from trastuzumab, as well as response to anthracycline-based chemotherapy. Fluorescence in situ hybridization (FISH) to detect HER2 gene copy number and immunohistochemistry (IHC) to detect HER2 protein levels are approved by the FDA to identify HER2−positive (H2) tumors. However, the 2007 ASCO/CAP report concluded that approximately 20% of HER2 testing may be inaccurate. Further, the available data did not clearly demonstrate clear superiority of either IHC or FISH as a predictor of benefit from anti-HER2 therapy. Discordance between these methods is as high as 5%. Thus, novel complementary quantitative methods for interrogating HER2 expression in tumors are needed. Targeted protein analysis by multiple reaction monitoring mass spectrometry (MRM-MS) offers a powerful approach to configure assays for specific proteins without using antibodies. Our studies using this platform have demonstrated applicability to formalin-fixed, paraffin-embedded (FFPE) specimens. In the current studies, we used this approach to measure signals from two tryptic peptides specific to HER2, one each from the extracellular and intracellular domains, selected from among 28 candidates based on their signal intensity and sharpness of their chromatographic profiles. Preliminary studies with a HER2−overexpressing BT474 xenograft in mice demonstrated quantitation and detected previously reported HER2 ectodomain shedding. Subsequent analysis of FFPE tissue from five H2 and five triple-negative (TN) tumors yielded measurement of at least 1 femptomole of receptor for H2 tumors and less than 0.2 femptomole of receptor for TN tumors per microgram of digest analyzed. If we assume 200 picograms of protein per cell, the results suggest 110,00 to 468,000 receptors per cell in the H2 tumors and only 2,000 to 14,000 receptors per cell in the TN tumors. Despite significant biological variability in receptor levels measured among the specimens of each type, a clear separation of the H2 and TN tumors was achieved based on the peptide quantitation. This preliminary study demonstrates the potential of MRM-MS in FFPE tissue to provide an alternate approach to IHC-based protein analysis. MRM-MS offers the potential for more, accurate and robust HER2 quantification in clinical breast cancer tissues. The next phase of this work will encompass a larger sample set, including tumors with equivocal and negative FISH and/or IHC test results. Correlation with response to anti-Her2 therapy will be performed in samples with available follow-up data. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-19.

Somatic rearrangements, which are commonly found in human cancer genomes, contribute to the progression and maintenance of cancers. Conventionally, the verification of somatic rearrangements comprises many manual steps and Sanger sequencing. This is labor intensive when verifying a large number of rearrangements in a large cohort. To increase the verification throughput, we devised a high-throughput workflow that utilizes benchtop next-generation sequencing and in-house bioinformatics tools to link the laboratory processes. In the proposed workflow, primers are automatically designed. PCR and an optional gel electrophoresis step to confirm the somatic nature of the rearrangements are performed. PCR products of somatic events are pooled for Ion Torrent PGM and/or Illumina MiSeq sequencing, the resulting sequence reads are assembled into consensus contigs by a consensus assembler, and an automated BLAT is used to resolve the breakpoints to base level. We compared sequences and breakpoints of verified somatic rearrangements between the conventional and high-throughput workflow. The results showed that next-generation sequencing methods are comparable to conventional Sanger sequencing. The identified breakpoints obtained from next-generation sequencing methods were highly accurate and reproducible. Furthermore, the proposed workflow allows hundreds of events to be processed in a shorter time frame compared with the conventional workflow.

A significant point has recently been made by Miller and Nelson in the ongoing debate about how and when to obtain pediatric assent.1Miller V.A. Nelson R.M. A developmental approach to child assent for nontherapeutic research.J Pediatr. 2006; 149: S25-S30Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar They appropriately note that assent, based on the framework of informed consent, holds children to the unreasonable ethical standard of autonomy rather than upon respect for persons. There is another compelling reason to reject the reliance on informed consent as the model for pediatric assent. Apart from providing the wrong ethical framework, informed consent provides the wrong legal foundation as well. One only has to consider the legal evolution of informed consent to recognize its limitations. The law of informed consent developed through a series of fragmented retrospective decisions resulting in the propagation of a series of complicated and poorly executed legal formalities. Where once medical judgment dictated the degree of medical disclosure, now legal requirements developed in the courts have been imposed on the doctor-patient relationship. The notion of pediatric assent is currently developing as a malleable concept. Federal regulations require assent but fall short of either defining it or providing guidance as to how to obtain it.2Department of Health and Human ServicesProtection of Human Subjects. US Government Printing Office, Washington, DC1991Google Scholar As we have seen from the example of informed consent, where there is a void left by standards and guidelines, courts will step in to fill the gap. The potential exists for a resulting patchwork of laws placing practitioners, researchers, and institutions in legal jeopardy while ultimately failing to uphold the underlying ethical principles. Miller and Nelson rightly caution against the tendency to conflate assent with informed consent. Pediatric assent must not follow the same trajectory. Rather than waiting for the courts to fill the gaps left by federal regulations, a proactive approach is needed to define and shape pediatric assent in a way that fully embodies the underlying ethical values. A developmental approach to child assent for nontherapeutic researchThe Journal of PediatricsVol. 149Issue 1PreviewThe question of when and how to obtain child assent for nontherapeutic research has received increasing attention in recent years. Although child assent and parent permission are grounded in the principle of respect for persons, assent is often understood from the more narrow principle of respect for autonomy. When viewed in this way, “assent” is frequently conflated with “consent,” and children are held to a higher standard than what might be sufficient for meaningful involvement in decision-making about research participation. Full-Text PDF

Abstract During mammary tumorigenesis, a cell-cell interface exists between adipocytes and cancer cells. Several studies have demonstrated that breast tumor cells can secrete cytokines that induce lipolysis in adjacent adipocytes. However, evidence of tumor-adjacent lipolysis in clinical samples has been lacking. We therefore assayed for lipolysis in normal tissue adjacent to breast tumors (NAT) using (1) the three-component breast composition measure, a radiographic imaging method derived from dual-energy mammography that allows lipid content of a tissue to be quantified, on breast tumors and NAT from 46 patients, (2) a publically available dataset of gene expression in primary breast tumors and NAT from 9 patients, (3) laser capture microdissection and proteomics on primary breast tumors, stroma and NAT from 75 patients, and (4) immunoblot analysis of NAT from several patient-derived and transgenic mouse models of breast cancer. We found strong evidence in all cases that lipolysis is activated in breast cancer-adjacent adipose tissue. We next set out to model the breast cancer-adipocyte interface and determine the contribution of cell-cell contact to induced lipolysis. Gap junctions are cell-cell junctions formed by proteins called connexins, which are known to transport a variety of small molecules (&amp;lt;1kD) including cAMP, a critical pro-lipolytic signaling molecule. Using established dye transfer assays, we determined that gap junctions form between breast cancer cells, and between breast cancer cells and adipocytes. Using biochemical assays, we demonstrated that cAMP is a substrate of breast cancer cell gap junctions, that transfer of cAMP from breast cancer cells to adipocytes occurs, and that breast cancer cells activate lipolytic signaling, all in a gap junction-dependent manner. Finally, we found that gap junction communication in this context is dependent upon connexin 31 (Cx31), and we establish the importance of Cx31 for breast tumor growth and activation of lipolysis in tumor-adjacent adipose tissue in vivo. Citation Format: Roman Camarda, Jeremy Williams, Serghei Malkov, Lisa J. Zimmerman, Suzanne Manning, Dvir Aran, Andrew Beardsley, Daniel Van de Mark, Jeffrey van Haren, Yong Chen, Charles Berdan, Sharon Louie, Celine Mahieu, Juliane Winkler, Elizabeth Willey, John D. Gagnon, Kosaku Shinoda, K. Mark Ansel, Zena Werb, Daniel C. Nomura, Shingo Kajimura, Torsten Wittmann, Atul J. Butte, Melinda E. Sanders, Daniel C. Liebler, Gregor Krings, John A. Shepherd, Andrei Goga. Tumor cell-adipocyte gap junctions activate lipolysis in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2398.

&nbsp;Metaphors are powerful devices for guiding the practice of Learning Advisors, but are often used implicitly. This can&nbsp;result in such personal metaphors&nbsp;mis-guiding practice, or constraining the thinking about practice through a&nbsp;monocultural&nbsp;perspective. This paper is a response to the paper of Golding et al. (2015) in the first issue of the ATLAANZ journal, which evaluated a variety of different metaphors for Learning Advisors. The metaphor suggested in this response is based on the process of helping someone bake for a ‘please bring a plate’ function. It is an attempt to develop a metaphor that has resonance for Learning Advisors of Palagi/Western cultural backgrounds, but which is based on the values embedded in Pacific cultures. This metaphor is offered as a contribution to the philosophy of learning advising discussion: it is my ‘plate’ that I am bringing to help feed our academic community.&nbsp

In the course of a study on the impacts of changing early childhood policy in Aotearoa New Zealand since 1989, the illustrations accompanying three major government reports and policies stood out as encapsulating the changes in underlying discourses. This enabled the illustrations from these three policy reports to be used for a historical analysis of shifting discourses, which highlighted the trends towards human capital theory and an increasing educationalisation of early childhood as the basis for policy. It is noted that the latter two reports feature cover pictures with block play. The particular historic positioning of block play at the juncture between acceptable early childhood and school pedagogy is explored, suggesting a compromise between discourses of early childhood care and education for human capital development purposes, and for the holistic development of the child.

Several genes which are deactivated on the initiation of development of Dictyostelium discoideum were identified by differential screening of various cDNA libraries. These genes have in common a decrease in the steady-state levels of their corresponding mRNAs on the onset of development and as development proceeds. When development was carried out in the absence of protein synthesis by inhibition with cycloheximide, the decrease in mRNA levels for most genes (V genes) was normal or slightly accelerated. For about 5% of the genes (H genes), however, cycloheximide caused an apparent induction of expression, as revealed by a slight or dramatic increase in mRNA levels, instead of the normal decrease. This effect was due to inhibition of protein synthesis and not to cycloheximide per se. The induction was found to be due to an enhancement of the transcription rate; normal rates of transcription for the H genes were dependent on continued protein synthesis during vegetative growth and development. Thus, two general regulatory classes exist for deactivation of gene expression on initiation of development, one of which is dependent on and one of which is independent of protein synthesis. Analysis of expression of these genes in mutant strains which are aggregation deficient allowed the classes to be subdivided further. Taken together, these characterizations allow several distinct regulatory mechanisms to be identified that are involved in the deactivation of gene expression on the onset of development in D. discoideum.