DNA Copy Number Abnormalities Research Articles

Mitochondrial DNA copy number and risk of cardiovascular disease and all-cause mortality: a systematic review and meta-analysis of observational studies.

Increasing studies have explored the correlation of mitochondrial DNA copy number (mtDNA-CN) abnormalities with cardiovascular disease (CVD) and all-cause mortality; however, their findings are contradictory. This systematic review and meta-analysis sought to quantitatively summarize current studies to elucidate the impact of mtDNA-CN on CVD outcomes and all-cause mortality. Relevant studies were searched for in PubMed, Embase, and Web of Science databases, up to October 23, 2023. Summary relative risks (RRs) and 95% confidence intervals (CIs) were calculated with the random-effects model. In total, 22 articles were included in the systematic review, 13 of which were included in the meta-analysis of CVD outcomes and 8 in all-cause mortality. Compared to the highest mtDNA-CN level, the summary RR (95% CI) for the lowest mtDNA-CN level was 2.09 (95% CI 1.59-2.75) for CVD, 1.70 (95% CI 1.29-2.24) for coronary heart disease (CHD), 1.43 (95% CI 1.15-1.79) for heart failure (HF), 1.88 (95% CI 1.08-3.28) for stroke, and 1.33 (95% CI 1.21-1.47) for all-cause mortality. Lower mtDNA-CN may increase the risk of CVD, including CHD, HF, and stroke, as well as all-cause mortality. MtDNA-CN is a potential predictor of CVD and all-cause mortality.

High level of aneuploidy and recurrent loss of chromosome 11 as relevant features of somatotroph pituitary tumors

BackgroundSomatotroph neuroendocrine pituitary tumors (sPitNET) are a subtype of pituitary tumors that commonly cause acromegaly. Our study aimed to determine the spectrum of DNA copy number abnormalities (CNAs) in sPitNETs and their relevance.MethodsA landscape of CNAs in sPitNETs was determined using combined whole-genome approaches involving low-pass whole genome sequencing and SNP microarrays. Fluorescent in situ hybridization (FISH) was used for microscopic validation of CNAs. The tumors were also subjected to transcriptome and DNA methylation analyses with RNAseq and microarrays, respectively.ResultsWe observed a wide spectrum of cytogenetic changes ranging from multiple deletions, recurrent chromosome 11 loss, stable genomes, to duplication of the majority of the chromosomes. The identified CNAs were confirmed with FISH. sPitNETs with multiple duplications were characterized by intratumoral heterogeneity in chromosome number variation in individual tumor cells, as determined with FISH. These tumors were separate CNA-related sPitNET subtype in clustering analyses with CNA signature specific for whole genome doubling-related etiology. This subtype encompassed GNAS-wild type, mostly densely granulated tumors with favorable expression level of known prognosis-related genes, notably enriched with POUF1/NR5A1-double positive PitNETs. Chromosomal deletions in sPitNETs are functionally relevant. They occurred in gene-dense DNA regions and were related to genes downregulation and increased DNA methylation in the CpG island and promoter regions in the affected regions. Recurrent loss of chromosome 11 was reflected by lowered MEN1 and AIP. No such unequivocal relevance was found for chromosomal gains. Comparisons of transcriptomes of selected most cytogenetically stable sPitNETs with tumors with recurrent loss of chromosome 11 showed upregulation of processes related to gene dosage compensation mechanism in tumors with deletion. Comparison of stable tumors with those with multiple duplications showed upregulation of processes related to mitotic spindle, DNA repair, and chromatin organization. Both comparisons showed upregulation of the processes related to immune infiltration in cytogenetically stable tumors and deconvolution of DNA methylation data indicated a higher content of specified immune cells and lower tumor purity in these tumors.ConclusionssPitNETs fall into three relevant cytogenetic groups: highly aneuploid tumors characterized by known prognostically favorable features and low aneuploidy tumors including specific subtype with chromosome 11 loss.

Analysis of the value of chromosomal instability test for evaluating early efficacy assessment in advanced gastric cancer.

e16060 Background: Gastric Cancer is featured by frequent somatic copy number alterations (SCNA) and high chromosomal instability (CIN). Circulating tumor DNA (ctDNA) is a minimally invasive approach, effective for the real-time follow-up of cancer. However, the clinical significance in gastric cancer is not yet determined.We performed this study to to evaluate whether ctDNA-based CIN analysis can predict treatment efficacy in advanced gastric cancer (AGC). Methods: The patients with surgery or cytopathology confirmed AGC and who received chemotherapy were enrolled. The blood sample (10ml) for ctDNA sequencing were obtained at baseline (BL) (before first-line chemotherapy) and at the time of efficacy assessment, respectively.The ctDNA of gastric cancer patients was isolated from plasma using QIAseq ctDNA extraction kit (Qiagen), and then repaired and amplified. The whole genome data were analyzed for DNA copy number abnormality, which was expressed as TFx value, and TFx ≥ 0.06% was taken as the positive value of chromosomal instability.Clinical responses (by RECIST 1.1 criteria) were estimated at the time points after 2 cycles of treatment. Results: A total of 18 patients were registered.There were 12 males and 6 females with a median age of 64 years (range 43-72 years).According to the RECIST1.1 criteria, there were 7cases of PR, 9 cases of SD, and 1 case of PD after treatment,1 case was under observation. Patients with CR+PR+SD were defined as the treatment-effective group, and with PD were defined as the treatment-ineffective group.The treatment-effective group mean value of CIN TFx value before treatment was 3.47±7.69, after treatment was 0.59±1.46(P<0.05),14 (82.35%) of these patients experienced a decrease in TFx values after treatment.Patients in the treatment-naïve group showed elevated TFx values.We divided the patients into group A and group B,group A was defined as the TFx value did not fall into the normal range after treatment, and group B was defined as the TFx value fell into the normal range and was called the rapid clearance group.Comparing the efficacy to the CIN group, 71.4% of patients with PR showed rapid clearance of ctDNA, while no clearance of ctDNA was observed in patients with PD,50.0% of patients with SD showed rapid clearance of ctDNA. Rapid clearance of ctDNA was also associated with longer survival, with a median OS of 7.2 months in group A and 17.85 months in group B, with HR of 0.55 (95% CI: 0.081-3.775 P > 0.05) However, there was no significant difference, probably due to too little data.In terms of PFS, Group B did not demonstrate benefit, with a median PFS of 5.9 months in Group A compared to only 3.9 months in Group B (P = 0.647). Conclusions: Dynamic ctDNA-based CIN assay can be used as a biomarker for efficacy assessment in metastatic gastric cancer. Rapid clearance of ctDNA is associated with longer survival, but this still needs to be validated with extensive data.

Differential microRNAs Expression during Cancer Development, and Chemoprevention by Natural Compounds: A Comprehensive Review.

MicroRNAs are short non-coding RNAs that inhibit gene expression at the post-transcriptional level. Abnormal microRNA expression has been associated with different human diseases, including cancer. Epigenetic changes, mutation, transcriptional deregulation, DNA copy number abnormalities, and defects in the biogenesis machinery play an important role in abnormal microRNA expression. Modulation of microRNAs by natural agents has emerged to enhance the efficacy of conventional chemotherapy through combinatorial therapeutic approach. This review summarizes the current understanding of abnormal microRNA expression in cancer, the different cellular mechanisms of microRNA, and their prevention by natural compounds. Understanding microRNA expression patterns during cancer development may help to identify stage-specific molecular markers. Natural compounds that exert regulatory effects by modulating microRNAs can be used in better cancer chemopreventive strategies by directly targeting microRNAs or as a way to increase sensitivity to existing chemotherapy regimens.

Abstract 426: Ongoing TCRβ VDJ rearrangement in pre-T LBL cell lines

Abstract The prognosis of recurrent precursor T cell lymphoblastic leukemia/lymphoma (pre-T LBL) is poor, mainly due to chemotherapy resistance. The specific mechanism(s) leading to chemoresistance at relapse is still poorly understood. We previously characterized a mouse strain that contains a CreERT2 cassette “knocked into” the 3′ UTR of Mcm2. Mice with 2 copies of this knock-in allele, designated Mcm2cre/cre show decreases Mcm2 protein expression. They die of pre-T LBL, due to acquired DNA copy number abnormalities (CNA), involving important tumor suppressor or oncogenes. We hypothesize this unique mutator phenotype may reveal specific genetic drivers leading to chemoresistance. We selected six drugs with different mechanisms that are known to be active against pre-T LBL, including cytarabine, etoposide, melphalan, methotrexate, prednisone, and vincristine. We used 4 mcm2 Pre-T LBL cell lines (#2883, 2696, 2641 and 2869). After 1 year of treatment, we obtained resistant cell lines that can tolerate drug concentrations which are up to 10,000X that of parental cell lines. These samples will be characterized by Sparse WGS (for CNA), RNA-Seq and WES. Before sequencing, we wanted to verify that cell lines had not become cross-contaminated during drug selection. During thymocyte differentiation, noncontiguous genomic V, D, and J gene segments become juxtaposed, and form an exon which encodes the variable region of a T cell receptor protein. The TCRβ VDJ rearrangement provides a unique identifier for pre-T LBL cell lines. For the 2869 cell line, we found that the VDJ sequences of cytarabine, vincristine, melphalan, and prednisone resistant 2869 were identical to the parental 2869 cells. However, analysis of 2883 cell lines were more complex. Although there were multiple VDJ rearrangements in parental 2883, they had an identical junction of D2J2S4, suggesting that the variable VDJ rearrangements were derived from a single clone with a unique DJ rearrangement, and subsequent different V-DJ rearrangements. Moreover, following drug selection, the VDJ rearrangement of chemo-resistant 2883 and parental 2883 were not identical. However, the chemo-resistant 2883 cells retain the fingerprint of parental 2883, namely the unique junction of D2J2S4. The VDJ rearrangement of etoposide resistant and vincristine resistant 2883 (2883ER and 2883VR, respectively) clones were identical, and could be identified as a subclone of parental 2883. Interestingly, the2883ER and 2883VR clones had nearly identical CNA of a number of genes, including Abcb1a (also known as Multi-Drug Resistance gene 1a), suggesting selection of a pre-existing resistant clone. Although the VDJ rearrangement of cytarabine 2883 was not found in the parental 2883 subclone, however, the unique D2J2S4 was present. These findings indicate that pre-T LBL cell lines do not invariably have stable TCRβ VDJ rearrangements, and can undergo V-DJ rearrangement event after several years of continuous cell culture. Citation Format: Dengchao Cao, Mianmian Yin, Toshihiro Matsukawa, Taimour Baslan, Peter Aplan. Ongoing TCRβ VDJ rearrangement in pre-T LBL cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 426.

The association between mitochondrial DNA copy number, telomere length, and tubal pregnancy

Growing evidence has demonstrated association between the occurrence of tubal ectopic pregnancy (TP) and oxidative stress (OS) status, in which mitochondria and telomeres play important roles. However, little is known about the underlying correlation between TP and the mitochondrial DNA copy number (mtDNAcn) or telomere length (TL) abnormalities. In this study, we found OS level was elevated in TP patients. We hierarchically detected the relative mtDNAcn and TL of villi from normal pregnancy (NP) and TP samples according to different gestational age, fetal sex, maternal age, and BMI. The results revealed that the relative mtDNAcn was significantly lower in the villi in the TP group compared with the NP cohort, which was negatively correlated with OS status. In the NP group, the mtDNAcn in the female subgroup was apparently lower than that in the male subgroup, while no statistical difference was found in the mtDNAcn in the TP group between the female and male subgroups. Moreover, the relative TL in the TP group was at a similar level to the NP group, and no statistical correlation was observed between relative TL and OS level. In summary, our findings indicate that the abnormal level of mtDNAcn rather than TL is correlated with TP, which provides new insights into the mechanism of TP.

Integrative analysis of genomic and epigenetic regulation of endometrial cancer.

Endometrial carcinomas (EC) are characterized by high DNA copy numbers and DNA methylation aberrations. In this study, we sought to comprehensively explore the effect of these two factors on development and progression of EC by analyzing integrated genomic and epigenetic analysis to. We found high DNA copy number and DNA methylation abnormalities in EC, with 6308 copy-number variation genes (CNV-G) and 4376 methylation genes (MET-G). We used these CNV-G and MET-G to subcategorize the samples for prognostic analysis, and identified three molecular subtypes (iC1, iC2, iC3). Moreover, the subtypes exhibited different tumor immune microenvironment characteristics. A further analysis of their molecular characteristics revealed three potential prognostic markers (KIAA1324, nonexpresser of pathogenesis-related genes1 (NPR1) and idiopathic hypogonadotropic hypogonadism (IHH)). Notably, all three markers showed distinct CNV, DNA methylation, and gene expression profiles. Analysis of mutations among the three subtypes revealed that iC2 had fewer mutations than the other subtypes. Conversely, iC2 showed significantly higher CNV levels than other subtypes. This comprehensive analysis of genomic and epigenetic profiles identified three prognostic markers, therefore, provides new insights into the multi-layered pathology of EC. These can be utilized for accurate treatment of EC patients.

Bithalamic gliomas may be molecularly distinct from their unilateral high‐grade counterparts

Bithalamic gliomas are rare cancers diagnosed based on poorly defined radiologic criteria. Infiltrative astrocytomas account for most cases. While some previous studies reported dismal outcomes for patients with bithalamic gliomas irrespective of therapy and histologic grade, others described better prognoses even without anticancer therapy. Little is known about their molecular characteristics. We reviewed clinical, radiologic, and histologic features of patients with bithalamic gliomas treated at our institution over 15 years. Targeted sequencing of mutational hotspots in H3F3A, HIST1H3B, IDH1/2, and BRAF, and genome-wide analysis of DNA methylation and copy number abnormalities was performed in available tumors. Eleven patients with bithalamic gliomas were identified. Their median age at diagnosis was 4.8 years (range: 1-15.7). Additional involvement of the brainstem, basal ganglia, and cerebral lobes occurred in 11, 9, and 3 cases, respectively. All patients presented with hydrocephalus. Two-thirds of the patients had a histologic diagnosis of anaplastic astrocytoma. Despite aggressive therapy, our youngest patient, the only one diagnosed before 1 year of age, is the sole long-term survivor. DNA methylation could be performed in seven tumors, all of which clustered with the RTK I 'PDGFRA' subgroup by unsupervised hierarchical analysis of methylation array against a previously published cohort of 59 pediatric high-grade gliomas. Sequencing of hotspots mutations could be done in 10 tumors, none of which harbored H3F3A p.K27 and/or the respective DNA methylation signature, and any other hotspot mutations. Amplification of MDM4 (n = 2), PDGFRA (n = 2), and ID2 combined with MYCN (n = 1) were observed in 7 tumors available for analysis. In comparison with the previously published experience with unilateral high-grade thalamic astrocytomas where H3F3A p.K27 was present in two-thirds of cases, the absence of this molecular subgroup in bithalamic gliomas was striking. This finding suggests that unilateral and bithalamic high-grade gliomas may represent two distinct molecular entities.

Chromothripsis Is a Recurrent Genomic Abnormality in High-Risk Myelodysplastic Syndromes.

To explore novel genetic abnormalities occurring in myelodysplastic syndromes (MDS) through an integrative study combining array-based comparative genomic hybridization (aCGH) and next-generation sequencing (NGS) in a series of MDS and MDS/myeloproliferative neoplasms (MPN) patients. 301 patients diagnosed with MDS (n = 240) or MDS/MPN (n = 61) were studied at the time of diagnosis. A genome-wide analysis of DNA copy number abnormalities was performed. In addition, a mutational analysis of DNMT3A, TET2, RUNX1, TP53 and BCOR genes was performed by NGS in selected cases. 285 abnormalities were identified in 71 patients (23.6%). Three high-risk MDS cases (1.2%) displayed chromothripsis involving exclusively chromosome 13 and affecting some cancer genes: FLT3, BRCA2 and RB1. All three cases carried TP53 mutations as revealed by NGS. Moreover, in the whole series, the integrative analysis of aCGH and NGS enabled the identification of cryptic recurrent deletions in 2p23.3 (DNMT3A; n = 2.8%), 4q24 (TET2; n = 10%) 17p13 (TP53; n = 8.5%), 21q22 (RUNX1; n = 7%), and Xp11.4 (BCOR; n = 2.8%), while mutations in the non-deleted allele where found only in DNMT3A (n = 1), TET2 (n = 3), and TP53 (n = 4). These cryptic abnormalities were detected mainly in patients with normal (45%) or non-informative (15%) karyotype by conventional cytogenetics, except for those with TP53 deletion and mutation (15%), which had a complex karyotype. In addition to well-known copy number defects, the presence of chromothripsis involving chromosome 13 was a novel recurrent change in high-risk MDS patients. Array CGH analysis revealed the presence of cryptic abnormalities in genomic regions where MDS-related genes, such as TET2, DNMT3A, RUNX1 and BCOR, are located.

Genome-Wide Uniparental Disomy and Copy Number Variations in Renal Cell Carcinomas Associated with Birt-Hogg-Dubé Syndrome

Birt-Hogg-Dubé syndrome is an inherited disorder caused by germline mutations of the folliculin gene (FLCN). The affected patients are prone to developing renal cell carcinomas (RCCs). Most mutant FLCN-associated RCCs (mFLCN-RCCs) are histologically chromophobe RCCs and hybrid oncocytic/chromophobe tumors. It is incompletely understood whether mFLCN-RCCs have different chromosomal abnormalities compared with their sporadic histological counterparts. Herein, we describe somatic mutations of FLCNand DNA-copy number abnormalities using a high-density, whole-genome, single-nucleotide polymorphism array. The histological types included chromophobe RCC (n = 12), hybrid oncocytic/chromophobe tumor (n = 5), and clear-cell RCC (n = 2). Of 19 tumors, 8 had pathological somatic mutations of FLCN. Among 11 mFLCN-RCCs investigated by single-nucleotide polymorphism array, 8showed balanced genomic profiles, 2 had gains in chromosome 3q, and 1 had gains in chromosomes 1q and 7. All had copious numbers of loss of heterozygosity in a wide range of chromosomes. The common loss-of-heterozygosity regions were chromosomes 3p24, 8q11, 16q11, Xp22-21, Xp11, Xq11, Xq13, and Xq23. Most of the loss of heterozygosity was because of uniparental disomy. Common uniparental disomy patterns in chromophobe RCCs and hybrid oncocytic/chromophobe tumors indicated that these types were relatively similar in cytogenetic events. Two clear-cell RCCs also shared several uniparental disomy regions with chromophobe RCCs and hybrid oncocytic/chromophobe tumors. mFLCN-RCCs may have common therapeutic targets among different histological types.

Prognostic Relevance of CD200/Btla Deletions in Pediatric Precursor-B Cell Acute Lymphoblastic Leukemia Treated According to the EORTC-CLG 58951 Protocol

Introduction: Despite the use of current risk classification in children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), a substantial proportion of so-called standard risk patients will experience a hematological relapse. Detection of DNA copy number abnormalities in BCP-ALLs has revealed additional genetic alterations, some of which are associated with outcome and may be included in future stratification strategies.Materials and methods: Using array-comparative genomic hybridization in a selected cohort of 70 intermediate risk pediatric BCP-ALLs, we characterized a recurrent RAG-mediated deletion of the CD200 and BTLA genes in 10% of patients. A breakpoint-specific PCR assay was designed and used to screen an independent non-selected cohort of 1154 genetically well-characterized BCP-ALLs uniformly treated according to the BFM-based EORTC-58951 protocol.Results: CD200/BTLA deletions were identified in 56 patients of the non-selected cohort (4.8%). Survival analysis revealed that CD200/BTLA deletions are associated with an inferior 8-year event free survival (EFS) of 70.2% ± 1.2% for patients with the deletions versus 83.5% ± 6.4% for non-deleted cases (HR 2.02; 95% CI 1.23-3.32; p=0.005) in the complete cohort of 1154 BCP-ALL patients. We observed a strong association between CD200/BTLA deletions and ETV6-RUNX1 positive leukemias (Table 1). The presence of ETV6-RUNX1 is a good prognostic marker in BCP-ALL and CD200/BTLA deletions did not affect prognosis within this genetic subtype. However and most notably, CD200/BTLA deletions were also identified in patients without any known genetic lesion, who are classified as having an intermediate outcome and belong to the intermediate-risk genetic group (defined in Table 1). Within this genetic group an inferior 8-year EFS rate of 33.3% (95% CI 7.8%-62.3%) was observed for patients with the deletions versus 76.2% (95% CI 71.0%-80.6%) for non-deleted cases (HR 4.00; 99% CI 1.34-11.93; p <0.001). Similarly, CD200/BTLA deleted cases were characterized by an inferior 8-year overall survival rate of 48.6% (95% CI 12.8-77.6%) versus 86.9% (95% CI 82.6-90.2%) for non-deleted cases (HR 4.43; 99% CI 1.15-17.03; p=0.002) (event distribution in Table 2). Multivariate analysis confirmed the independent prognostic importance of CD200/BTLA deletions, after adjusting for the presence of IKZF1 deletions, gender, response to prephase treatment and CNS involvement.Discussion and conclusion: Altogether, these findings suggest that the prognostic value of CD200/BTLA deletions is restricted to intermediate risk BCP-ALL cases that lack any of the currently known prognostic indicators and underscore the rationale for implementing additional genetic markers in future risk stratification strategies.Table 1:Frequency of CD200/BTLA deletions according to BCP-ALL genetic subtypes and genetic risk groupsNot deletedDeletedp-valueNo (%)No (%)All patients109856Genetic subtypes<0.0001ERGdel36 (3.3)1 (1.8)ETV6-RUNX1248 (22.6)34 (60.7)High hyperdiploidy382 (34.8)4 (7.1)BCR-ABL124 (2.2)3 (5.4)Low hypo/near-haploidy9 (0.8)1 (1.8)MLL translocation17 (1.5)0 (0.0)iAMP2121 (1.9)4 (7.1)TCF3-PBX147 (4.3)0 (0.0)B-other314 (28.6)9 (16.1)IKZF1del172 (15.7)10 (17.9)0.7Genetic groups *0.004Good-risk666 (60.7)39 (69.6)Intermediate-risk361 (32.9)9 (16.1)Poor-risk71 (6.5)8 (14.3)*Genetic risk groups were defined as follows: good-risk group include all patients with high hyperdiploidy, ETV6-RUNX1 or ERGdel; intermediate-risk group includes patients with TCF3-PBX1 and B-other patients; poor-risk group includes all patients with MLL translocation, low hypodiploidy/near-haploidy or iAMP21.Table 2:Event types in CD200/BTLA deleted versus non-deleted patientsNot deletedDeletedNo (%)No (%)Intermediate-risk genetic group3619EFS statusNoCR7 (1.9)2 (22.2)CCR283 (78.4)3 (33.3)Relapse67 (18.6)4 (44.4)TRM4 (1.1)0 (0.0)Abbreviations: CR, complete remission; CCR, continuous complete remission; TRM, treatment related mortality. DisclosuresNo relevant conflicts of interest to declare.

TigaR: integrative significance analysis of temporal differential gene expression induced by genomic abnormalities.

BackgroundTo determine which changes in the host cell genome are crucial for cervical carcinogenesis, a longitudinal in vitro model system of HPV-transformed keratinocytes was profiled in a genome-wide manner. Four cell lines affected with either HPV16 or HPV18 were assayed at 8 sequential time points for gene expression (mRNA) and gene copy number (DNA) using high-resolution microarrays. Available methods for temporal differential expression analysis are not designed for integrative genomic studies.ResultsHere, we present a method that allows for the identification of differential gene expression associated with DNA copy number changes over time. The temporal variation in gene expression is described by a generalized linear mixed model employing low-rank thin-plate splines. Model parameters are estimated with an empirical Bayes procedure, which exploits integrated nested Laplace approximation for fast computation. Iteratively, posteriors of hyperparameters and model parameters are estimated. The empirical Bayes procedure shrinks multiple dispersion-related parameters. Shrinkage leads to more stable estimates of the model parameters, better control of false positives and improvement of reproducibility. In addition, to make estimates of the DNA copy number more stable, model parameters are also estimated in a multivariate way using triplets of features, imposing a spatial prior for the copy number effect.ConclusionWith the proposed method for analysis of time-course multilevel molecular data, more profound insight may be gained through the identification of temporal differential expression induced by DNA copy number abnormalities. In particular, in the analysis of an integrative oncogenomics study with a time-course set-up our method finds genes previously reported to be involved in cervical carcinogenesis. Furthermore, the proposed method yields improvements in sensitivity, specificity and reproducibility compared to existing methods. Finally, the proposed method is able to handle count (RNAseq) data from time course experiments as is shown on a real data set.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2105-15-327) contains supplementary material, which is available to authorized users.

Abstract 998: Rise and fall of subclones from diagnosis to relapse in pediatric B-acute lymphoblastic leukemia (B-ALL): A report from the children's oncology group (COG) - Target - St. Jude Pediatric Cancer Genome Project

Abstract Tumor clonal heterogeneity has been demonstrated in several cancers; however, the rise and fall of subclones under therapy have not been well characterized. To gain insight into the subclonal population architecture of pediatric B-ALL, we analyzed somatic lesions including sequence mutations, structural alterations and DNA copy number abnormalities derived from high coverage (200X) exome sequencing, whole-genome sequencing and SNP arrays of diagnosis (Dx)-remission-relapse DNA from 20 patients (median 7 yrs, range 2 to 19) treated on recent COG B-ALL trials. Cases were selected for analysis based upon occurrence of an early bone marrow relapse (median 19.2 months, range 3.8 to 35.7), which is associated with poor survival. We identified a high frequency of mutations in B-cell development (80%, e.g. PAX5, IKZF1, TCF3, BTG1, TOX, and EBF1 ), RAS signaling (65%, e.g. NRAS, KRAS, PTPN11 and FLT3), TP53 (60%, e.g. CDKN2A, TP53 and RB1), kinase signaling (25%, e.g. JAK2, CRLF2 and SH2B3) and chromatin remodeling (60%, e.g. WHSC1, MLL2, CREBBP, ARID2 and SETD2) pathways. Somatic lesions in these pathways were mostly retained (68%) between Dx and relapse. In contrast, mutations in the purine metabolism genes NT5C2 (45%) and NT5CB1 (15%) were relapse-specific. We constructed clonal lineage of somatic lesions for 15 cases based on subclonal population fractions estimated by a binomial mixture model that adjusts for sequence coverage of mutant alleles. The subclone number (median 3, range 1-5) at Dx was comparable to that at relapse (median=3, range 2-4). Notably, 6 Dx samples were observed with multiple subclones harboring distinct driver mutations in the same oncogene (e.g. NRAS, KRAS and JAK2). In almost all cases only one subclone from Dx arose to be the predominant clone present at relapse. Moreover, the mutation burden in an emergent subclone was comparable to those eradicated by therapy (P=0.43, Wilcoxon rank sum). In 80% of cases, the predominant clone at relapse originated from the smallest Dx subclone; in 45% of those cases, mutant allele present in relapse was detectable in remission DNA (∼0.1%) obtained at the end of the 1st month of therapy. Clonal lineage shows that the earliest acquired mutations at relapse are NT5C2 (n=5), NRAS (n=3), USH2A (n=3), WHSC1 (n=2), TP53 (n=2), IKZF1 (n=1) and CREBBP (n=1). Our study is the first that analyzes the genetic composition and population architecture of subclones that rise or fall after B-ALL therapy. The majority of the “rising” subclones are oligoclonal at Dx, which may explain acquisition of mutations such as those found in NT5C2 to confer growth advantage in relapse. Additional studies of patients who were cured of B-ALL are needed to determine if the presence of mutant clone at end induction might help to predict risk and tempo of relapse. Citation Format: Xiaotu Ma, Mignon L. Loh, Michael Rusch, Michael Edmonson, Richard C. Harvey, David A. Wheeler, Oliver A. Hampton, John Easton, Donald Yergeau, Bhavin Vadodaria, Gang Wu, William L. Carroll, I-Ming Chen, Daniela S. Gerhard, Julie M. Gastier-Foster, Mary V. Relling, Malcolm A. Smith, Meenakshi Devidas, Jaime M. Guidry Auvil, James R. Downing, Cheryl L. Willman, Charles G. Mullighan, Stephen P. Hunger, Jinghui Zhang. Rise and fall of subclones from diagnosis to relapse in pediatric B-acute lymphoblastic leukemia (B-ALL): A report from the children's oncology group (COG) - Target - St. Jude Pediatric Cancer Genome Project. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 998. doi:10.1158/1538-7445.AM2014-998

Comparison of Cancer-Associated Genetic Abnormalities in Columnar-Lined Esophagus Tissues With and Without Goblet Cells

To determine and compare the frequency of cancer-associated genetic abnormalities in esophageal metaplasia biopsies with and without goblet cells. Barrett's esophagus is associated with increased risk of esophageal adenocarcinoma (EAC), but the appropriate histologic definition of Barrett's esophagus is debated. Intestinal metaplasia (IM) is defined by the presence of goblet cells whereas nongoblet cell metaplasia (NGM) lacks goblet cells. Both have been implicated in EAC risk but this is controversial. Although IM is known to harbor genetic changes associated with EAC, little is known about NGM. We hypothesized that if NGM and IM infer similar EAC risk, then they would harbor similar genetic aberrations in genes associated with EAC. Ninety frozen NGM, IM, and normal tissues from 45 subjects were studied. DNA copy number abnormalities were identified using microarrays and fluorescence in situ hybridization. Targeted sequencing of all exons from 20 EAC-associated genes was performed on metaplasia biopsies using Ion AmpliSeq DNA sequencing. Frequent copy number abnormalities targeting cancer-associated genes were found in IM whereas no such changes were observed in NGM. In 1 subject, fluorescence in situ hybridization confirmed loss of CDKN2A and amplification of chromosome 8 in IM but not in a nearby NGM biopsy. Targeted sequencing revealed 11 nonsynonymous mutations in 16 IM samples and 2 mutations in 19 NGM samples. This study reports the largest and most comprehensive comparison of DNA aberrations in IM and NGM genomes. Our results show that IM has a much higher frequency of cancer-associated mutations than NGM.

Abstract PD4-6: Combined analysis of gene expression, DNA copy number and mutation profiling data to display biological process anomalies in individual cancers

Abstract Background: The goal of this analysis was to develop a computational tool that integrates gene expression, DNA copy number and sequence abnormalities in individual cancers in the framework of biological processes. Methods and Findings: We used the hierarchical structure of the Gene Ontology (GO) database to create a reference network and projected mRNA expression, DNA copy number and mutation anomalies detected in single samples into this space. We applied our method to 59 breast cancers where all three types of molecular data were available. Each cancer had a large number of disturbed biological processes. Locomotion, multicellular organismal process and signal transduction pathways were the most commonly affected GO terms but the individual molecular events were different from case-to-case. Estrogen receptor-positive and -negative cancers had different repertoire of anomalies. We tested the functional impact of 27 mRNAs that had overexpression in cancer with variable frequency (&amp;lt;2-42%) using an siRNA screen. Each of these genes inhibited cell growth in at least some of 18 breast cancer cell lines. Conclusions We developed a free, on-line software tool (http://netgoplot.org) to display the complex genomic abnormalities in individual cancers in the biological framework of the GO biological processes. We show that each cancer harbors a large number and unique combination of pathway anomalies, the individual molecular events that underlie the pathway level disturbances vary from case-to-case. Our in vitro experiments indicate that even rare case-specific molecular abnormalities can play a functional role and driver events may vary from case to case depending on the constellation of other molecular anomalies. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD4-6.

Prognostic Value of Fibroblast Growth Factor Receptor 1 Gene Locus Amplification in Resected Lung Squamous Cell Carcinoma

Fibroblast growth factor receptor 1 (FGFR1) gene amplification was recently reported as a recurrent abnormality in 10% to 20% of primary lung squamous cell carcinomas (SqCCs), and has attracted significant interest as a potential therapeutic target. Limited data are available for its prognostic impact in early-stage SqCC. Tissue microarrays containing 135 primary lung SqCCs and 58 matching lymph node metastases were tested by interphase fluorescence in situ hybridization for DNA copy number (CN) abnormalities at the 8p12 region including FGFR1. FGFR1amplification was found in 18.2% (22 of 121 evaluable) of primary SqCC, using a definition of average copies of FGFR1 per cell of 5.0 or more. Concordance rate between primaries and matching lymph node metastases was 97.7% (43 of 44; 7 amplified and 37 nonamplified), with the only discordant case showing CN at approximately the dichotomous cutoff. Similarly, concordance between two separate lymph node metastases in each of 10 patients was 100% (1 amplified and 9 nonamplified). Using various CN cutoffs, we found no statistically significant association between FGFR1 CN abnormalities and patient age, sex, tumor grade, stage, smoking status, disease-free survival, cause-specific survival, or overall survival. FGFR1 amplification is not prognostic in resected lung squamous cell carcinoma patients.

Multinodular and Vacuolating Neuronal Tumors of the Cerebrum: 10 Cases of a Distinctive Seizure‐Associated Lesion

We report 10 cases of a non-neurocytic, purely neuronal tumor affecting adults. Situated in the cerebral hemispheres, with 7 of 10 confined to the temporal lobes, most presented with seizures as their principal clinical manifestations. On magnetic resosnance imaging (MRI), the tumors generally appeared solid and non-contrast enhancing with minimal diffuse infiltration, edema, or mass effect. Six examples demonstrated internal nodularity. Microscopically, the tumor cells were largely distributed into discrete and coalescent nodules exhibiting varying degrees of matrix vacuolization, principally within the deep cortical ribbon and superficial subcortical white matter. Populating elements ranged from morphologically ambiguous to recognizably neuronal, with only two cases manifesting overt ganglion cell cytology. In all cases, tumor cells exhibited widespread nuclear immunolabeling for the HuC/HuD neuronal antigens, although expression of other neuronal markers, including synaptophysin, neurofilament and chromogranin was variable to absent. Tumor cells also failed to express GFAP, p53, IDH1 R132H, or CD34, although CD34-labeling ramified neural elements were present in the adjoining cortex of seven cases. Molecular analysis in a subset of cases failed to reveal DNA copy number abnormalities or BRAF V600E mutation. Follow-up data indicate that this unusual neuronal lesion behaves in benign, World Health Organization (WHO) grade I fashion and is amenable to surgical control.

Integrative Genomics in Combination with RNA Interference Identifies Prognostic and Functionally Relevant Gene Targets for Oral Squamous Cell Carcinoma

In oral squamous cell carcinoma (OSCC), metastasis to lymph nodes is associated with a 50% reduction in 5-year survival. To identify a metastatic gene set based on DNA copy number abnormalities (CNAs) of differentially expressed genes, we compared DNA and RNA of OSCC cells laser-microdissected from non-metastatic primary tumors (n = 17) with those from lymph node metastases (n = 20), using Affymetrix 250K Nsp single-nucleotide polymorphism (SNP) arrays and U133 Plus 2.0 arrays, respectively. With a false discovery rate (FDR)<5%, 1988 transcripts were found to be differentially expressed between primary and metastatic OSCC. Of these, 114 were found to have a significant correlation between DNA copy number and gene expression (FDR<0.01). Among these 114 correlated transcripts, the corresponding genomic regions of each of 95 transcripts had CNAs differences between primary and metastatic OSCC (FDR<0.01). Using an independent dataset of 133 patients, multivariable analysis showed that the OSCC–specific and overall mortality hazards ratio (HR) for patients carrying the 95-transcript signature were 4.75 (95% CI: 2.03–11.11) and 3.45 (95% CI: 1.84–6.50), respectively. To determine the degree by which these genes impact cell survival, we compared the growth of five OSCC cell lines before and after knockdown of over-amplified transcripts via a high-throughput siRNA–mediated screen. The expression-knockdown of 18 of the 26 genes tested showed a growth suppression ≥30% in at least one cell line (P<0.01). In particular, cell lines derived from late-stage OSCC were more sensitive to the knockdown of G3BP1 than cell lines derived from early-stage OSCC, and the growth suppression was likely caused by increase in apoptosis. Further investigation is warranted to examine the biological role of these genes in OSCC progression and their therapeutic potentials.

Somatic copy-neutral loss of heterozygosity and copy number abnormalities in Malaysian sporadic colorectal carcinoma patients

Colorectal cancer is one of the most common cancers in many countries, including Malaysia. The accumulation of genomic alterations is an important feature of colorectal carcinogenesis. A better understanding of the molecular events underlying the stages of colorectal carcinogenesis might be helpful in the detection and management of the disease. We used a commercially available single-nucleotide polymorphism genotyping array to detect both copy number abnormalities (CNAs) and copy-neutral loss of heterozygosity (LOH) in sporadic colorectal carcinomas. Matched tumor and normal tissues of 13 colorectal carcinomas (Dukes' stages A-D) were analyzed using a 250K single nucleotide polymorphism array. An additional assay was performed to determine the microsatellite instability status by using the National Cancer Institute-recommended BAT-26 panel. In general, copy number gain (92.3%) was most common, followed by copy number loss (53.8%) and copy-neutral LOH (46.2%). Frequent CNAs of gains and losses were observed on chromosomes 7p, 8, 13q, 17p, 18q, and 20q, and copy-neutral LOH was observed on chromosomes 2, 6, 12, 13q, 14q, 17, 20p, 19q, and 22q. Even though genomic alterations are associated with colorectal cancer progression, our results showed that DNA CNAs and copy-neutral LOH do not reflect disease progression in at least 50% tumors. Copy-neutral LOH was observed in both early and advanced tumors, which favors the involvement of these genomic alterations in the early stages of tumor development.

Microarray comparative genomic hybridization in prenatal diagnosis: a review

G-band chromosomal karyotyping of fetal cells obtained by invasive prenatal testing has been used since the 1960s to identify structural chromosomal anomalies. Prenatal testing is usually performed in response to parental request, increased risk of fetal chromosomal abnormality associated with advanced maternal age, a high-risk screening test and/or the presence of a congenital malformation identified by ultrasonography. The results of karyotyping may inform the long-term prognosis (e.g. aneuploidy being associated with a poor outcome or microscopic chromosomal anomalies predicting global neurodevelopmental morbidity). Relatively recent advances in microarray technology are now enabling high-resolution genome-wide evaluation for DNA copy number abnormalities (e.g. deletions or duplications). While such technological advances promise increased sensitivity and specificity they can also pose difficult challenges of interpretation and clinical management. This review aims to give interested clinicians without an extensive prior knowledge of microarray technology, an overview of its use in prenatal diagnosis, the literature to date, advantages, potential pitfalls and experience from our own tertiary center.