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The hallmark of Hodgkin’s Lymphoma (HL) is the presence of large Hodgkin’s and Reed/Sternberg (HRS) cells that comprise only ~1–3% of the cellular infiltrate. Given the paucity of genomic analyses of these cells, we sought to characterize their DNA copy number alterations (CNA) by array comparative genomic hybridization (aCGH) using DNA isolated from laser capture microdissected (LCM) CD30+ HRS cells. Primary paraffin-embedded diagnostic samples were obtained from 27 patients (pts), including 15 pts with chemotherapy responsive and 12 pts with primary refractory HL. From each sample, 150 HRS cells were isolated by LCM from 5-μm thick tissue sections, amplified using whole genome amplification (WGA), and hybridized to a minimal tiling 19K whole genome bacterial artificial chromosome (BAC) array (BAC center to center distance of 165 kb). To define thresholds for calling gains and loss and to control for WGA noise, 1.05 ng DNA (DNA equivalent of 150 cells) was obtained from six normal (3 males and 3 females) individuals, amplified and analyzed by aCGH. Triplicate samples from a single HL pt confirmed mean replicate correlation as approximately Pearson r = 0.90. DNA gains and losses observed in >35% of the HL samples were localized to 22 and12 chromosome regions, respectively. Region-specific FISH analyses confirmed the presence or absence of aCGH-defined CNAs in CD30+ HRS for 10 of these regions (3–5 pt samples/FISH probe). CNA gains in >60% of HL samples included genes associated with growth and proliferation (AHR, BAI1, BOP1, COMMD5, LY6E, PTP4A3, SLURP1, CBFA2T3, SLC7A5, NOTCH1, FOXF1, TRAF2, IRF8, S100B, MYH14), cell cycle (AKT1, CDK10, SUMO3), drug metabolism (CYP11B1, CYP11B2, SLC19A1), angiogenesis and cell adhesion (COL18A1, CDH4, ITGB2), apoptosis regulation (FOXC2, FOXF1, GPR132), immune and lymphatic development (CBFA2T3, IL17C, IRF8, CLEC11A, RXRA, SPIB, ICOSLG) and invasion, metastasis or cancer-relatedness (VAV2, PSCA PTP4A3, GINS2, FUT7, TUBB2C, KLK, POLD1, TFF2). Losses observed in >40% of HL samples included SPRY1, NELL1, SLC1A3, GDNF, IL7R, SKP2, GRIA1, ID4, PPARGC1A, and TXNIP. Different CNA patterns between sensitive and refractory HL were identified. Genomic differences observed either specifically or in >35% of the HL chemosensitive pts included ~25 CNA gains including genes known to regulate T-cell trafficking or NFkB activation (CCL22, CX3CL1, CCL17, DOK4 and IL10). The refractory signature showed a higher frequency of CNA gains for three genes involved in the H4 ubiquitin ligase complex that play a role in the cellular response to DNA damage (HDAC4, CUL4A, and DDB2), and gains of ILKAP, GAS6, MADD, SPI1, PVR, MTCH2 CCND3, GPCI, MAPK11 with CNA losses of ELAC2, IL2, GRIA1, SLC17A6, IL21, and MAP2K4 genes. Of interest, CCL22 and CX3CL1 have been associated with a more favorable outcome and a lower risk of recurrence in other cancers, whereas gains or overexpression of SKP2, MTCH2 and CCND3 have been associated with a poor prognosis and a more aggressive cancer phenotype. Moreover, genomewide discrimination analyses on CNAs revealed two distinct clusters that correlated closely with the favorable and unfavorable IPS scores for the 27 HL pts evaluated. Overall, our proof-of-principle, exploratory genomic analyses of HD show that genomic profiles of small numbers of HRS cells is possible, HL samples shared many CNAs but differences may reflect disease course, and highlights the potential to build a genetic CNA map for HL to potentially guide prognosis, therapy decisions and drug discovery.

Background: Leukemia is the most common pediatric malignancy, accounting for nearly 40% of all new childhood cancer. The cure rates for pediatric ALL have increased to more than 80% and the cure rates for pediatric AML now approach 50%. Much of this progress can be attributed to cytogenetic and molecular risk stratification with subsequent randomized control trials. Genomic instability events may also serve as relevant prognostic biomarkers. A novel high-throughput genomic technology called Molecular Inversion Probes (MIPs) quantifies genomic instability, gene copy number and allelic imbalances at the highest genomic resolution. MIPs can analyze genetic target sequences in parallel with high specificity and sensitivity. Further classifying leukemic blasts using more precise molecular techniques could prove useful in further risk stratifying and developing new treatment strategies. Objective: To adapt MIPs to characterize and define unique molecular subtypes of childhood leukemia. Methods: DNA was extracted from AML and ALL clinical samples obtained at diagnosis. 400 ng of leukemic DNA was mixed with a previously synthesized MIP cancer probe set (667 probes representing all human chromosomes from exon sequences of 298 cancer genes); each probe contains two unique recognition sequences to targeted genomic DNA and a unique barcode (tag) sequence. A thermostable polymerase and ligase were added, and the mixture was denatured. Probe homology sequences were then annealed to complementary genome sites. Standard PCR reagants were added including one fluorescent-labeled primer and unlabeled primer. The reaction was thermocycled and probes circularized in the nucleotide specific “gap fill” reaction were amplified. The PCR mixture was hybridized to a barcode microarray overnight. The chip was stained and washed, and then interrogated by an Affymetrix scanner via argon laser excitation at 488-nm. ‘Cluster along Chromosomes’ analysis was performed to detect significant amplifications or deletions. Results: Childhood ALL and AML samples had unique patterns of multiple gene deletions. Additionally, overlap was found in several of these deleted genes. Significantly deleted genes in common between ALL and AML samples included: RAP1GA1, HYAL1, HSPA9B, CSF1R, TNF, NOTCH4, FANCE, CCND3, BAG2, PLAGL1, MAD1L1, POLM, POLD2, IGFBP3, PSD, SUFU, CYP17A1, MXI1, HRAS, IGF2, RRM1, BRCA2, TP53, ERBB2, ERCC2, and POLD1. Conclusions: MIPs represent a novel technology for highly sensitive and specific gene copy analysis of childhood leukemia. Unique and distinguishing signatures of allelic imbalance can be determined between ALL and AML clinical samples using MIP technology. The unexpected overlap of deleted genes in both ALL and AML may represent a common molecular mechanism that requires further investigation. We currently are adapting MIPs for an expanded cancer probe set and are exploring new techniques to determine loss of heterozygosity (LOH) in pediatric leukemia.

A small minority of colorectal cancers (CRCs) (≤5%) are caused by a single, inherited faulty gene. These diseases, the Mendelian colorectal cancer (CRC) syndromes, have been central to our understanding of colorectal carcinogenesis in general. Most of the approximately 13 high-penetrance genes that predispose to CRC primarily predispose to colorectal polyps, and each gene is associated with a specific type of polyp, whether conventional adenomas ( APC, MUTYH, POLE, POLD1, NTHL1), juvenile polyps ( SMAD4, BMPR1A), Peutz-Jeghers hamartomas ( LKB1/STK11) and mixed polyps of serrated and juvenile types ( GREM1). Lynch syndrome ( MSH2, MLH1, MSH6, PMS2), by contrast, is associated primarily with cancer risk. Major functional pathways are consistently inactivated in the Mendelian CRC syndromes: certain types of DNA repair (proofreading of DNA replication errors, mismatch repair and base excision repair) and signalling (bone morphogenetic protein (BMP), Wnt signalling and mTOR). The inheritance of the CRC syndromes also varies: most are dominant but some of the DNA repair deficiencies are recessive. Some of the Mendelian CRC genes are especially important because they play a role through somatic inactivation in sporadic CRC ( APC, MLH1, SMAD4, POLE). Additional Mendelian CRC genes may remain to be discovered and searches for these genes are ongoing, especially in patients with multiple adenomas and hyperplastic polyps.

Inhibition of the kinase ATR, a central regulator of the DNA damage response, eliminates subsets of cancer cells in certain tumors. As previously shown, this is at least partly attributable to synthetic lethal interactions between ATR and POLD1, the catalytic subunit of the polymerase δ. Various POLD1 variants have been found in colorectal cancer, but their significance as therapeutic targets for ATR pathway inhibition remains unknown. Using CRISPR/Cas9 in the colorectal cancer cell line DLD-1, which harbors four POLD1 variants, we established heterozygous POLD1-knockout clones with exclusive expression of distinct variants to determine the functional relevance of these variants individually by assessing their impact on ATR pathway activation, DNA replication, and cellular sensitivity to inhibition of ATR or its effector kinase CHK1. Of the four variants analyzed, only POLD1R689W affected POLD1 function, as demonstrated by compensatory ATR pathway activation and impaired DNA replication. Upon treatment with ATR or CHK1 inhibitors, POLD1R689W strongly decreased cell survival in vitro, which was attributable at least partly to S phase impairment and apoptosis. Similarly, treatment with the ATR inhibitor AZD6738 inhibited growth of murine xenograft tumors, harboring the POLD1R689W variant, in vivo. Our POLD1-knockout model thus complements algorithm-based models to predict the pathogenicity of tumor-specific variants of unknown significance and illustrates a novel and potentially clinically relevant therapeutic approach using ATR/CHK1 inhibitors in POLD1-deficient tumors.

Werner Syndrome (WS) is an adult-onset segmental progeroid syndrome. Bisulfite pyrosequencing of repetitive DNA families revealed comparable blood DNA methylation levels between classical (18 WRN-mutant) or atypical WS (3 LMNA-mutant and 3 POLD1-mutant) patients and age- and sex-matched controls. WS was not associated with either age-related accelerated global losses of ALU, LINE1, and α-satellite DNA methylations or gains of rDNA methylation. Single CpG methylation was analyzed with Infinium MethylationEPIC arrays. In a correspondence analysis, atypical WS samples clustered together with the controls and were clearly separated from classical WS, consistent with distinct epigenetic pathologies. In classical WS, we identified 659 differentially methylated regions (DMRs) comprising 3,656 CpG sites and 613 RefSeq genes. The top DMR was located in the HOXA4 promoter. Additional DMR genes included LMNA, POLD1, and 132 genes which have been reported to be differentially expressed in WRN-mutant/depleted cells. DMRs were enriched in genes with molecular functions linked to transcription factor activity and sequence-specific DNA binding to promoters transcribed by RNA polymerase II. We propose that transcriptional misregulation of downstream genes by the absence of WRN protein contributes to the variable premature aging phenotypes of WS. There were no CpG sites showing significant differences in DNA methylation changes with age between WS patients and controls. Genes with both WS- and age-related methylation changes exhibited a constant offset of methylation between WRN-mutant patients and controls across the entire analyzed age range. WS-specific epigenetic signatures occur early in life and do not simply reflect an acceleration of normal epigenetic aging processes.

Polymerases ϵ and δ are the main enzymes that replicate eukaryotic DNA. Accurate replication occurs through Watson–Crick base pairing and also through the action of the polymerases' exonuclease (proofreading) domains. We have recently shown that germline exonuclease domain mutations (EDMs) of POLE and POLD1 confer a high risk of multiple colorectal adenomas and carcinoma (CRC). POLD1 mutations also predispose to endometrial cancer (EC). These mutations are associated with high penetrance and dominant inheritance, although the phenotype can be variable. We have named the condition polymerase proofreading-associated polyposis (PPAP). Somatic POLE EDMs have also been found in sporadic CRCs and ECs, although very few somatic POLD1 EDMs have been detected. Both the germline and the somatic DNA polymerase EDMs cause an ‘ultramutated’, apparently microsatellite-stable, type of cancer, sometimes leading to over a million base substitutions per tumour. Here, we present the evidence for POLE and POLD1 as important contributors to the pathogenesis of CRC and EC, and highlight some of the key questions in this emerging field. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

Three DNA polymerases — Pol α, Pol δ and Pol ɛ — are essential for DNA replication. After initiation of DNA synthesis by Pol α, Pol δ or Pol ɛ take over on the lagging and leading strand respectively. Pol δ and Pol ɛ perform the bulk of replication with very high fidelity, which is ensured by Watson–Crick base pairing and 3′exonuclease (proofreading) activity. Yeast models have shown that mutations in the exonuclease domain of Pol δ and Pol ɛ homologues can cause a mutator phenotype. Recently, we identified germline exonuclease domain mutations (EDMs) in human POLD1 and POLE that predispose to ‘polymerase proofreading associated polyposis’ (PPAP), a disease characterised by multiple colorectal adenomas and carcinoma, with high penetrance and dominant inheritance. Moreover, somatic EDMs in POLE have also been found in sporadic colorectal and endometrial cancers. Tumors with EDMs are microsatellite stable and show an ‘ultramutator’ phenotype, with a dramatic increase in base substitutions.

Background: Ovarian cancer is a cancerous growth arising from the ovary. Objective: This study was aimed to explore the molecular mechanism of the development and progression of the ovarian cancer. Methods: We first identified the differentially expressed genes (DEGs) between the ovarian cancer samples and the healthy controls by analyzing the GSE14407 affymetrix microarray data, and then the functional enrichments of the DEGs were investigated. Furthermore, we constructed the protein-protein interaction network of the DEGs using the STRING online tools to find the genes which might play important roles in the progression of ovarian cancer. In addition, we performed the enrichment analysis to the PPI network. Results: Our study screened 659 DEGs, including 77 up- and 582 down-regulated genes. These DEGs were enriched in pathways such as Cell cycle, p53 signaling pathway, Pathways in cancer and Drug metabolism. CCNE1, CCNB2 and CYP3A5 were the significant genes identified from these pathways. Protein-protein interaction (PPI) network was constructed and network Module A was found closely associated with ovarian cancer. Hub nodes such as VEGFA, CALM1, BIRC5 and POLD1 were found in the PPI network. Module A was related to biological processes such as mitotic cell cycle, cell cycle, nuclear division, and pathways namely Cell cycle, Oocyte meiosis and p53 signaling pathway. Conclusions: It indicated that ovarian cancer was closely associated to the dysregulation of p53 signaling pathway, drug metabolism, tyrosine metabolism and cell cycle. Besides, we also predicted genes such as CCNE1, CCNB2, CYP3A5 and VEGFA might be target genes for diagnosing the ovarian cancer.