High-resolution Comparative Genomic Hybridization Research Articles

Comprehensive Study of Chromosomal Copy Number Variations and Genomic Variations Predicting Overall Survival in Myelodysplastic Syndromes

Introduction: Myelodysplastic syndrome (MDS) is a heterogeneous disease characterized by cytopenia, marrow dysplasia and has a propensity to develop into acute myeloid leukemia. The disease progression is majorly affected by genetic defects. However, about 40–50% of patients with MDS present with a normal karyotype and develop different courses of disease. Hence, there remains a room to advance the biological understanding and find molecular prognostic markers for cytogenetically normal MDS. Methods: We performed a high-resolution CGH + SNP array along with next-generation sequencing (NGS) of 77 primary diagnosed MDS patients, and also they were clinically followed up. Results: Our study revealed 82 clinically significant genomic lesions (losses/gains) in 49% of MDS patients. CGH + SNP array reduced the proportion of normal karyotype by 30%. SNP array in combination with NGS confirmed the biallelic loss of function of the TP53 gene (2/6), which is a clinically relevant biomarker and new genetic-based MDS entity, i.e., MDS-biTP53, as per the new WHO classification 2022. Genomic region 2p22.3 presented with frequent lesions and also with a more hazard ratio (2.7, 95% CI: 0.37–21) when analyzed by Kaplan-Meier survival analysis. Conclusion: CGH + SNP array changed the cytogenetic and IPSS-R risk group in 18% and 13% of patients, respectively, with an improved prediction of prognosis. This study emphasizes the cytogenetic heterogeneity of MDS and highlights that abnormality with chromosome 2 may have a diagnostic and prognostic impact.

Array CGH + SNP in ALL Enhances Detection of Complex Cytogenetic Profiles and Suggests That PRSS1 Loss May Play a Role in Leukemogenesis

Introduction Cytogenetic alterations are essential prognostic biomarkers in patients (pts) with acute lymphoblastic leukemia/lymphoma (ALL), but are limited by the propensity of leukemic blasts to undergo apoptosis in culture, poor-quality chromosomes, and overgrowth by normal cells. Array CGH + SNP has 10,000-fold higher resolution than conventional cytogenetics and can identify submicroscopic gains, losses and copy number neutral loss of heterozygosity (cnLOH) irrespective of mitotic rate. Few studies outside of clinical trials have investigated microarray findings in adults with ALL or correlated microarray findings with clinical outcomes. Methods We retrospectively reviewed pts with ALL and mixed phenotype acute leukemia (MPAL) aged ≥18 years at diagnosis at our institution between 2017 and 2022. Array analysis was performed on diagnostic bone marrow or peripheral blood samples using high resolution CGH + SNP Genetisure cancer array platform (Agilent). Statistical analysis was done using R software (R core team, 2022). Results Baseline characteristics of included pts (n = 60) are shown in Table 1. CGH + SNP analysis identified 93.3% (56/60) of pts with genomic changes with a mean of 7 (range 0 - 28) aberrations per pt. Among pts with a normal karyotype, 81.8% (9/11) had new genomic abnormalities revealed by CGH + SNP, but significantly fewer aberrations were detected per pt compared to those with abnormal karyotypes (mean 3.09 v. 7.67, p = .008). Pts with Ph-like B-ALL/MPAL had significantly more aberrations on array than pts without Ph-like phenotype (mean 12.0 v. 5.85, p.= .002). Among the 78.3% of pts without complex cytogenetics on chromosome analysis, 44.7% (21/47) were found to have a complex genomic profile (≥5 aberrations) with the addition of CGH + SNP. However, there was no difference in CR rates or OS in pts with <5 or ≥5 microarray aberrations (p = .515 and p = .970, respectively). Quantity of copy number alterations (CNAs) as detected on array was not predictive of achieving end of induction CR/CRi/CRh (mean 6.08 in pts who achieved CR/CRi/CRh v. 7.02 in pts with primary refractory disease, p = .310). Hispanic pts had the same number of CNAs as non-Hispanic pts (mean 6.69 v. 6.94, p = .435). On linear regression analysis, there was no correlation between number of CNAs and age (r 2 = -.01, F = 0.1941, p = .661) or BMI (r 2 = 5.21 x10 -5, F = 0.0029, p = .957). The most frequent focal aberrations were detected in CDKN2A/B (n = 19), IKZF1 (n = 17), PRSS1 (n = 11), PAX5 (n = 8), BTG1 (n = 8) and RB1 (n = 8) (Figure 1). RB1 loss was enriched in pts with T-ALL (45.5% v. 6%, p = .003),and occurred at significantly higher rates than previously reported in T-ALL (Schraders, 2009). Loss of PRSS1 was detected in 18.3% (11/60) of the cohort. Homozygous PRSS1 deletions were identified in 3/11 (27.3%) pts. We identified the minimal overlapping deleted region on 7q34 in all 11 pts, which included the PRSS1 gene within the 124 kb region. Five of 6 pts who had repeat arrays performed at the end of induction had resolution of this aberration. The one pt who retained PRSS1 loss after induction had primary refractory disease, but after further treatment and attainment of CR1, this pt cleared the PRSS1 loss. Asparaginase associated pancreatitis, which has been linked to PRSS1 variations, occurred in only 1 pt with PRSS1 loss. Pts harboring PRSS1 loss were significantly less likely to achieve MRD negativity after induction (50% vs. 82%, P = .041) and had numerically more relapses (37.5% vs. 19%, p = .300), but no OS difference was observed (p = .290). Conclusions In our institutional cohort, the addition of CGH + SNP array reclassified almost half (44.7%) of pts without complex cytogenetics to harbor complex genomic changes (≥5 aberrations). Pts with Ph-like ALL had significantly more aberrations on array than those with Ph-positive or -negative B-ALL. Focal PRSS1 loss was associated with reduced rates of MRD clearance at the end of induction. This aberration resolved upon disease remission, raising the possibility that both homozygous and/or heterozygous deletions of 124 kb from the 7q34 chromosomal region of the PRSS1 gene may potentially have a role in ALL pathogenesis. Further studies with larger cohorts utilizing CGH + SNP analyses at additional time points during treatment are needed to explore the potential integration of microarray data for refined ALL risk stratification as well as to elucidate the functional role of PRSS1 in leukemogenesis.

Characterization of copy-number variants in a large cohort of patients with von Willebrand disease reveals a relationship between disrupted regions and disease type

BackgroundGenetic analysis for von Willebrand disease (VWD) commonly utilizes DNA sequencing to identify variants in the von Willebrand factor (VWF) gene; however, this technique cannot always detect copy-number variants (CNVs). Additional mapping of CNVs in patients with VWD is needed. ObjectivesThis study aimed to characterize CNVs in a large sample of VWF mutation-negative VWD patients. MethodsTo determine the role of CNVs in VWD, a VWF high-resolution comparative genomic hybridization array was custom-designed to avoid multiple sequence variations, repeated sequences, and the VWF pseudogene. This was performed on 204 mutation-negative subjects for whom clinical variables were also available. ResultsAmong the 204 patients, 7 unique CNVs were found, with a total of 24 CNVs (12%). Of the 7 unique CNVs, 1 was novel, 1 was found in a VWF database, and 5 were previously reported. All patients with type 1C VWD and a CNV had the same exon 33 and 34 in-frame deletion. Certain clinical variables were also significantly different between those with and without CNVs. ConclusionThe in-frame deletion in patients with type 1C VWD exactly matches the D4N module of the D4 domain, a region where mutations and deletions are known to affect clearance. We observed significantly higher VWF–to–ristocetin cofactor levels in patients with type 1C VWD and a CNV than in patients without a CNV, suggesting a relationship between CNVs and the increased clearance observed in patients with type 1C VWD. Glycoprotein IbM activity was significantly lower in patients with type 1 VWD and a CNV than in patients without a CNV, suggesting that platelet binding is more affected by CNVs than single base pair mutations. This work elucidates some of the underlying genetic mechanisms of CNVs in these patients.

Genomic amplification of chromosome 20q13.33 is the early biomarker for the development of sporadic colorectal carcinoma

BackgroundColorectal carcinoma (CRC) is the third most common cancer in the world and also the third leading cause of cancer-related mortality in Taiwan. CRC tumorigenesis is a multistep process, starting from mutations causing loss of function of tumor suppressor genes, canonically demonstrated in adenomatous polyposis coli pathogenesis. Although many genes or chromosomal alterations have been shown to be involved in this process, there are still unrecognized molecular events within CRC tumorigenesis. Elucidating these mechanisms may help improve the management and treatment.MethodsIn this study, we aimed to identify copy number alteration of the smallest chromosomal regions that is significantly associated with sporadic CRC tumorigenesis using high-resolution array-based Comparative Genomic Hybridization (aCGH) and quantitative Polymerase chain reaction (qPCR). In addition, microsatellite instability assay and sequencing-based mutation assay were performed to illustrate the initiation event of CRC tumorigenesis.ResultsA total of 571 CRC patients were recruited and 377 paired CRC tissues from sporadic CRC cases were used to define the smallest regions with chromosome copy number changes. In addition, 198 colorectal polyps from 160 patients were also used to study the role of 20q13.33 gain in CRC tumorigenesis. We found that gain in 20q13.33 is the main chromosomal abnormalities in this patient population and counts 50.9 and 62.8% in CRC and colon polyps, respectively. Furthermore, APC and KRAS gene mutations were profiled simultaneously and co-analyzed with microsatellite instability and 20q13.33 gain in CRC patients. Our study showed that the frequency of 20q13.33 copy number gain was highest among all reported CRC mutations.ConclusionAs APC or KRAS mutations are currently identified as the most important targets for CRC therapy, this study proposes that 20q13.33 copy number gain and the associated chromosomal genes function as promising biomarkers for both early stage detection and targeted therapy of sporadic CRCs in the future.

Exon-focused targeted oligonucleotide microarray design increases detection of clinically relevant variants across multiple NHS genomic centres

In recent years, chromosomal microarrays have been widely adopted by clinical diagnostic laboratories for postnatal constitutional genome analysis and have been recommended as the first-line test for patients with intellectual disability, developmental delay, autism and/or congenital abnormalities. Traditionally, array platforms have been designed with probes evenly spaced throughout the genome and increased probe density in regions associated with specific disorders with a resolution at the level of whole genes or multiple exons. However, this level of resolution often cannot detect pathogenic intragenic deletions or duplications, which represent a significant disease-causing mechanism. Therefore, new high-resolution oligonucleotide comparative genomic hybridisation arrays (oligo-array CGH) have been developed with probes targeting single exons of disease relevant genes. Here we present a retrospective study on 27,756 patient samples from a consortium of state-funded diagnostic UK genomic centres assayed by either oligo-array CGH of a traditional design (Cytosure ISCA v2) or by an oligo-array CGH with enhanced exon-level coverage of genes associated with developmental disorders (CytoSure Constitutional v3). The new targeted design used in Cytosure v3 array has been designed to capture intragenic aberrations that would have been missed on the v2 array. To assess the relative performance of the two array designs, data on a subset of samples (n = 19,675), generated only by laboratories using both array designs, were compared. Our results demonstrate that the new high-density exon-focused targeted array design that uses updated information from large scale genomic studies is a powerful tool for detection of intragenic deletions and duplications that leads to a significant improvement in diagnostic yield.

Integrated analysis revealing genome-wide chromosomal copy number variation in supraglottic laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (LSCC) is a genetically complex tumor type and one of the leading causes of cancer-associated disability and mortality. Genetic instability, such as chromosomal instability, is associated with the tumorigenesis of LSCC. Copy number variations (CNVs) have been demonstrated to contribute to the genetic diversity of tumor pathogenesis. Comparative genomic hybridization (CGH) has emerged as a high-throughput genomic technology that facilitates the aggregation of high-resolution data of cancer-associated genomic imbalances. In the present study, a total of 38 primary supraglottic LSCC cases were analyzed by high-resolution array-based CGH (aCGH) to improve the understanding of the genetic alterations in LSCC. Additionally, integration with bioinformatic analysis of microarray expression profiling data from the Gene Expression Omnibus (GEO) database provided a fundamental method for the identification of putative target genes. Genomic CNVs were detected in all cases. The size of net genomic imbalances per case ranged between a loss of 682.3 Mb (~24% of the genome) and a gain of 1,958.6 Mb (~69% of the genome). Recurrent gains included 2pter-q22.1, 3q26.1-qter, 5pter-p12, 7p22.3p14.1, 8p12p11.22, 8q24.13q24.3, 11q13.2q13.4, 12pter-p12.2, 18pter-p11.31 and 20p13p12.1, whereas recurrent losses included 3pter-p21.32, 4q28.1-q35.2, 5q13.2-qter, 9pter-p21.3 and monosomy 13. Gains of 3q26.1-qter were associated with tumor stage, poor differentiation and smoking history. Additionally, through integration with bioinformatic analysis of data from the GEO database, putative target oncogenes, including sex-determining region Y-box 2, eukaryotic translation initiation factor 4 gamma 1, fragile X-related gene 1, disheveled segment polarity protein 3, defective n cullin neddylation 1 domain containing 1, insulin like growth factor 2 mRNA binding protein 2 and CCDC26 long non-coding RNA, and tumor suppressor genes, such as CUB and sushi multiple domains 1, cyclin dependent kinase inhibitor 2A, protocadherin 20, serine peptidase inhibitor Kazal type 5 and Nei like DNA glycosylase 3, were identified in supraglottic LSCC. Supraglottic LSCC is a genetically complex tumor type and aCGH was demonstrated to be effective in the determination of molecular profiles with higher resolution. The present results enable the identification of putative target oncogenes and tumor suppressor gene mapping in supraglottic LSCC.

Clinical impact of chromothriptic complex chromosomal rearrangements in newly diagnosed multiple myeloma

Complex Chromosomal Rearrangements (CCRs) are increasingly being reported as genetic risk factors of clinical significance in cancer owing to their identification using high resolution whole genome profiling technologies. This study employed high resolution CGH + SNP microarrays for whole genome copy number variations (CNV) profiling and identified CCRs in 11/107(10%) newly diagnosed Multiple Myeloma (MM) patients. Six patients exhibited Chromothripsis (CTH) among seven chromosomes that were confirmed with automated CTLPscanner web tool and; five cases displayed chromoplexy (CPL) which involved multiple chromosomes. Presence of chromothripsis in chromosome 17 in three out of six patients indicate a link between TP53 aberrations and incidence of CTH. Multivariable Cox regression model demonstrated a significant association of CTH with poor PFS (HR = 3.09, p = 0.010) and OS (HR = 3.31, p = 0.024) which suggests that CTH is an additional independent prognostic marker in multiple myeloma. Addition of CTH in risk stratification models in clinical setting in multiple myeloma may help in upfront identification of high risk patients for suitable customized therapy.

Rare Copy Number Variants in Array-Based Comparative Genomic Hybridization in Early-Onset Skeletal Fragility.

Early-onset osteoporosis is characterized by low bone mineral density (BMD) and fractures since childhood or young adulthood. Several monogenic forms have been identified but the contributing genes remain inadequately characterized. In search for novel variants and novel candidate loci, we screened a cohort of 70 young subjects with mild to severe skeletal fragility for rare copy-number variants (CNVs). Our study cohort included 15 subjects with primary osteoporosis before age 30 years and 55 subjects with a pathological fracture history and low or normal BMD before age 16 years. A custom-made high-resolution comparative genomic hybridization array with enriched probe density in >1,150 genes important for bone metabolism and ciliary function was used to search for CNVs. We identified altogether 14 rare CNVs. Seven intronic aberrations were classified as likely benign. Five CNVs of unknown clinical significance affected coding regions of genes not previously associated with skeletal fragility (ETV1-DGKB, AGBL2, ATM, RPS6KL1-PGF, and SCN4A). Finally, two CNVs were pathogenic and likely pathogenic, respectively: a 4 kb deletion involving exons 1–4 of COL1A2 (NM_000089.3) and a 12.5 kb duplication of exon 3 in PLS3 (NM_005032.6). Although both genes have been linked to monogenic forms of osteoporosis, COL1A2 deletions are rare and PLS3 duplications have not been described previously. Both CNVs were identified in subjects with significant osteoporosis and segregated with osteoporosis within the families. Our study expands the number of pathogenic CNVs in monogenic skeletal fragility and shows the validity of targeted CNV screening to potentially pinpoint novel candidate loci in early-onset osteoporosis.

Genetic fine-mapping of the Iowan SNCA gene triplication in a patient with Parkinson\u2019s disease

The “Iowa kindred,” a large Iowan family with autosomal-dominant Parkinson’s disease, has been followed clinically since the 1920s at the Mayo Clinic. In 2003, the genetic cause was determined to be a 1.7 Mb triplication of the alpha-synuclein genomic locus. Affected individuals present with an early-onset, severe parkinsonism-dementia syndrome. Here, we present a descendant of the Iowa kindred with novel, disease-associated non-motor findings of reduced heart rate variability, complete anosmia, and a rare skin condition called colloid milium. At autopsy, key neuropathological findings were compatible with diffuse Lewy body disease. Using high-resolution comparative genomic hybridization (CGH) array analysis to fine-map the genomic breakpoints, we observed two independent recombination events of the SNCA locus that resulted in a genomic triplication of twelve genes, including SNCA, and the disruption of two genes, HERC6 and CCSER1, at the genomic breakpoints. In conclusion, we provide further evidence that the mere two-fold overexpression of alpha-synuclein leads to a fulminant alpha-synucleinopathy with rapid progression and severe clinical and neuropathological features.

Molecular characterization of a series of 990 index patients with albinism.

Albinism is a clinically and genetically heterogeneous disease characterized by variable degrees of hypopigmentation and by nystagmus, foveal hypoplasia, and chiasmatic misrouting of the optic nerves. The wide phenotypic heterogeneity impedes the establishment of phenotype-genotype correlations. To obtain a precise diagnosis, we screened the 19 known albinism genes in 990 index patients using targeted next-generation sequencing (NGS) and high-resolution comparative genomic hybridization. A molecular diagnosis was obtained in 72.32% of patients. A total of 243 new pathogenic variants were identified. Intragenic rearrangements represented 10.8% of all pathogenic alleles. NGS panel analysis allowed establishing a diagnosis for the rarest forms of the disease, which could not be diagnosed otherwise. Because of the clinical overlap between the different forms of the disease, diagnosis nowadays clearly relies on molecular grounds.

Genomic imbalances are involved in miR-30c and let-7a deregulation in ovarian tumors: implications for HMGA2 expression.

The High-mobility group AT-hook 2 protein (HMGA2) is involved in different processes during tumorigenesis. High expression levels of HMGA2 are found in various types of cancer, with recent studies highlighting the important role of miRNAs in the regulation of HMGA2 expression. We report a study of 155 ovarian tumors (30 sex-cord stromal tumors, 22 borderline tumors, and 103 carcinomas) analyzed for HMGA2 expression as well as the expression of two miRNAs targeting this gene, let-7a and miR-30c. We also evaluated the expression of the fragile histidine triad (FHIT) and lin28 homologues (LIN28A/B) genes which are known to be an enhancer of miR-30c expression and a repressor of let-7a, respectively. HMGA2 was found expressed at high levels in most samples analyzed, with clear cell carcinomas as the only exception. let-7a and miR-30c were highly deregulated in all tumor types. LIN28A and FHIT were found overexpressed in all examined tumor types. The chromosomal imbalances that might lead to loss of the genes expressing let-7a and miR-30c could be evaluated on the basis of previously generated karyotypic and high resolution comparative genomic hybridization (CGH) data on 103 tumors. 76% of the samples with an imbalanced genome had at least one chromosomal aberration leading to a deletion of a miRNA cluster for let-7a and miR-30c. FISH using locus specific probes for these clusters validate the aberrations at the gene level. Our study shows that genomic imbalances are involved in miR-30c and let-7a deregulation. One can reasonably assume that dysregulation of these miRNAs is a cause leading to HMGA2 upregulation in ovarian tumors.

Abstract 3178: Metastatic urothelial cancer 10 years after transplanting a filial kidney: genomically investigating the perpetrator

Abstract BACKGROUND Polyomavirus (BKV) infection has been associated with development of urothelial carcinoma (UC). We report on a patient with conventional UC of the bladder (Ta, G3) 8 years after receiving a kidney graft from a 4 year old male donor. After a recurrence (T1, G3) 9 years after transplantation (NTx), he was diagnosed with a SV-40 positive metastatic micropapillary and muscle-invasive UC 10 years after NTx with involvement of the graft kidney pelvis (pT3), the bladder (pT3) and a pelvic lymphnode metastasis. The aim of this study was to investigate the genealogy and clonal relationship between these cancer locations. METHODS Cancer cell nuclei derived from formalin fixed tumor specimens were processed and sorted based on DNA content with a BD Influx FACS. DNA from sorted aneuploid populations was analyzed by whole genome high resolution CGH microarrays (aCGH). In addition, short tandem repeat (STR) analyses were done with the PowerPlex® ESI Kit commercial testing kit. Each run was performed on an Applied Biosystems Genetic Analyzer 3500 and analyzed with the GeneMapper ID-X Software. Integration of PV-genom was investigated by RT-PCR. The IonTorrent platform by LifeTech (PGM, IonAmpliseq Cancer Hotspot Panel v2) was used for next-generation-sequencing (NGS). RESULTS Array CGH clarified that the primary two non-muscle invasive bladder tumors differed completely from each other and from the subsequent cancer tissues. The tumor manifestations 10 years after NTx shared the same genomic profile, except for a private amplification at 6p12.3 and a private deletion at Xp22.33 - Xp22.11, both in the bladder tumor. STR identified all three tumors diagnosed 10 years after NTx to originate from the allograft donor. NGS identified two shared mutations of unknown relevance (KDR / InDel chr4:55962545; Akt1 / c.138C>A) in the 10 year after NTx UC sites. No known somatic mutation was found. RT-PCR identified the PV to be integrated in the human cancer genome. Further studies are currently ongoing to define the exact integration sites of BKV and the potentially influenced human genes. CONCLUSIONS This case is an impressive example of a, donor-derived, metastatic micropapillary UC developed under immuno-suppression and subsequent BKV-reactivation in a presumably healthy transplanted kidney of a four years old donor. Citation Format: David C. Müller, Maarit Raemoe, Christian Wetterauer, Valeria Perrina, Luca Quagliata, Tatjana Vlajnic, Christian Ruiz, Beate Balitzki, Hans H. Hirsch, Rainer Grobholz, Lukas Bubendorf, Cyrill A. Rentsch. Metastatic urothelial cancer 10 years after transplanting a filial kidney: genomically investigating the perpetrator. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3178.

Epilepsy and cataplexy in Angelman syndrome. Genotype-phenotype correlations

BackgroundAngelman syndrome (AS) is a neurogenetic disorder characterized by intellectual disability, epilepsy, and low threshold for laughter. AimsWe investigated the occurrence and severity of epilepsy and laughter-induced loss of postural muscle tone determined by the different genetic subtypes. MethodsThis study included 39 children with AS. Deletion breakpoints were determined by high resolution CGH microarray (1×1M Agilent). Clinical data were based on a parent interview and medical record review. ResultsAll patients with AS based on a deletion had epilepsy. Epilepsy was present in 3/4 children with UBE3A mutation, and 4/5 with pUPD. Onset of epilepsy occurred earlier in deletion cases compared to pUPD or UBE3A mutations cases. Laughter-induced postural muscle tone loss occurred only among deletion cases. We found no differences in severity of epilepsy between children with a larger Class I or a smaller Class II deletions, or between the total group with a deletion compared to children with pUPD or a UBE3A mutation. The drugs most frequently prescribed were benzodiazepines in monotherapy, or a combination of benzodiazepines and valproic acid. ConclusionEpilepsy is very common in patients with AS, especially in patients with a deletion. Postural muscle tone loss and collapsing during outbursts of laughter were seen in patients with a deletion only.

An investigation of the potential for epigenetic inactivation by transcription read-through in a sporadic colorectal cancer

Aberrant transcription read-through of a gene promoter as a result of genetic structural rearrangements can cause the epigenetic inactivation of a neighbouring gene. All reported cases have involved copy number alterations that remove the 3′ poly(A) transcription terminator sequence of a gene leading to transcription read-through (TRT) and methylation of the gene promoter of a downstream gene. We aimed to determine whether deletion of poly (A) transcription terminator sequences was associated with the methylation of neighbouring genes in a CRC with extensive copy number alterations. We performed a high resolution CGH array and methylation analysis on a CRC specimen to identify such alterations. Analysis of the CRC using high-resolution CGH identified 6 genes with deletions in the 3′ part of the gene that encompassed the poly(A) transcription terminator sequence. Bisulphite sequencing of the promoter region of neighbouring (affected) genes at these six regions showed all candidate genes were unmethylated. Considering the fact that six TRT affected genes in a CRC with multiple deletions show no signs of hypermethylated promoters, it would be fairly appropriate to suggest that epigenetic inactivation by TRT might be a rare phenomenon in sporadic CRCs.

Association between chromosomal aberration of COX8C and tethered spinal cord syndrome: array-based comparative genomic hybridization analysis.

Copy number variations have been found in patients with neural tube abnormalities. In this study, we performed genome-wide screening using high-resolution array-based comparative genomic hybridization in three children with tethered spinal cord syndrome and two healthy parents. Of eight copy number variations, four were non-polymorphic. These non-polymorphic copy number variations were associated with Angelman and Prader-Willi syndromes, and microcephaly. Gene function enrichment analysis revealed that COX8C, a gene associated with metabolic disorders of the nervous system, was located in the copy number variation region of Patient 1. Our results indicate that array-based comparative genomic hybridization can be used to diagnose tethered spinal cord syndrome. Our results may help determine the pathogenesis of tethered spinal cord syndrome and prevent occurrence of this disease.

P35 Genomic profiling of intestinal-type sinonasal adenocarcinoma reveals a subgroup of patients with favourable clinical outcome

Intestinal-type sinonasal adenocarcinomas (ITAC) are rare tumors, related to occupational exposure to wood and leather dust. Patients with ITAC carry a poor prognosis and recurrent disease is the main cause of mortality. The purpose of this study was to analyse genome-wide copy number alterations (CNAs) in a large series of cases, aiming to identify genetic events involved in ITAC development and progression. DNA was isolated from 61 frozen ITAC tissue samples including all histological subtypes and analysed by high-resolution microarray CGH. Results were correlated to previously obtained genetic and immunohistochemical data and to clinico-pathological characteristics and follow-up data. All but three cases showed CNAs. Most frequent gains were found at chromosomal loci 5p, 7p, 8q, 12p and 20, whereas recurrent deletions were observed at 4q, 5q, 8p, 10q, 17p and 18q. High level amplifications included 7p12, 8q24, 11q13 and 19q11. Cluster analysis revealed a subgroup of cases characterized by carrying few CNAs (mean 8 versus 45). In addition, these cases showed absence of TP53 mutation and more frequent overexpression of COX2 and NFkB compared to the other cases. Overall survival was significantly better in this subgroup (p = 0.012). Genomic profiling revealed a subset of patients with ITAC with favourable clinical outcome, independent from established prognostic clinico-pathological parameters such as tumor stage and histological subtype. It may be speculated that this subset of tumors arises through a process of tumorigenesis that does not involved gross chromosomal instability as in the majority of epithelial tumors.

Fusion genes with ALK as recurrent partner in ependymoma-like gliomas: a new brain tumor entity?

We have previously characterized 19 ependymal tumors using Giemsa banding and high-resolution comparative genomic hybridization. The aim of this study was to analyze these tumors searching for fusion genes. RNA sequencing was performed in 12 samples. Potential fusion transcripts were assessed by seed count and structural chromosomal aberrations. Transcripts of interest were validated using fluorescence in situ hybridization and PCR followed by direct sequencing. RNA sequencing identified rearrangements of the anaplastic lymphoma kinase gene (ALK) in 2 samples. Both tumors harbored structural aberrations involving the ALK locus 2p23. Tumor 1 had an unbalanced t(2;14)(p23;q22) translocation which led to the fusion gene KTN1-ALK. Tumor 2 had an interstitial del(2)(p16p23) deletion causing the fusion of CCDC88A and ALK. In both samples, the breakpoint of ALK was located between exons 19 and 20. Both patients were infants and both tumors were supratentorial. The tumors were well demarcated from surrounding tissue and had both ependymal and astrocytic features but were diagnosed and treated as ependymomas. By combining karyotyping and RNA sequencing, we identified the 2 first ever reported ALK rearrangements in CNS tumors. Such rearrangements may represent the hallmark of a new entity of pediatric glioma characterized by both ependymal and astrocytic features. Our findings are of particular importance because crizotinib, a selective ALK inhibitor, has demonstrated effect in patients with lung cancer harboring ALK rearrangements. Thus, ALK emerges as an interesting therapeutic target in patients with ependymal tumors carrying ALK fusions.

Oncogenic mutations and chromosomal aberrations in primary extranodal diffuse large b-cell lymphomas of the thyroid – A study of 21 cases

Context: Primary extranodal diffuse large B-cell lymphomas of the thyroid (ptDLBCL) constitute a rare entity, which until now was not fully explored. Objective: Due to recently published data genetically linking ptDLBCL to a subset of thyroid carcinoma, we assessed the occurrence of oncogenic mutations and copy number alterations. Design: A high-resolution array-based comparative genomic hybridization approach was applied to quantify genomic aberrations in a study population of 21 ptDLBCL patients. In addition, we investigated the frequency of mutations involving the BRAF, NRAS, and MYD88 genes in correlation with immunohistochemical data. Results: Chromosomal gains were recurrently detected at 6p21.33-p21.31, 6p22.2, 12p13.31, 14q31.1, 14q32.33, 19p13.3, and 22q11.22; numeric losses were most frequently observed at 6p21.3-p21.31, 10q26.3, 19p13.3, 20q13.33, and 21q11.2. Aberrations affecting 6p22.2 and 14q32.33 as well as 22q11.22 differed slightly between germinal center B-cell (GCB) and non-GCB groups....

Distinct Patterns of Genomic Alterations in Adult T-Cell Leukemia-Lymphoma Endemic in the Western World

Introduction: Acute T-cell leukemia/lymphoma (ATLL) is a highly aggressive malignancy caused by HTLV-I, which is endemic in Japan, the Caribbean, and South America. ATLL carries a dismal prognosis and is generally incurable with conventional chemotherapy. ATLL is challenging to study at the molecular level, in part due to its complex genetic alterations likely resulting from years of HTLV-I driven T-cell proliferation and accumulation of genetic damage prior to malignant transformation. While no specific chromosome or genetic abnormalities have been proven to contribute to the pathogenesis of ATLL, older comparative genomic hybridization (CGH) studies performed in Japanese patients have demonstrated frequent genetic lesions (gains and losses) involving specific chromosomal regions, thus limited information exists about the chromosomal abnormalities occurring in the African ATLL variant commonly seen in the Western World.Methods: In this study, we used a high-density oligo array 244K platform CGH platform (Agilent Technologies) with an average resolution of 8.9 Kb, to perform a comprehensive genomic analysis of 47 ATLL patient tumor specimens obtained from African-descendants in the United States, Caribbean and Brazil. Patients were sub-classified as acute-type (A) ATLL (n=31), lymphomatous (L) (n=8), chronic (n=7, six whom had unfavorable features), and one with smouldering type according to Shimoyama criteria. DNA samples were extracted from peripheral blood mononuclear cells or tumor samples from these patients and checked for quality.Results: ATLL tumors exhibited complex genomic abnormalities and high copy number changes (CNCs). The average of copy number (CN) aberrations per sample was 238 in the L-group vs. 114 in acute/unfavorable chronic (A/UC) group. However, many chromosomal alterations were observed in this cohort, which had not been previously reported by other studies. The common CNCs were gains at 1q21-q44, 3, 3p, 7q22-q36, 8, 18, 19p13.1-p13.3, 21q21.1-q22.3, 22q12-q13 and losses at 5q13.2-q32, 6q11-q15, 9q13-q21. Gains in the 14 q32 (IGH) regions and losses in the 7p14.1 (TCRG), 7q34 (TCRB) and 14q11.2 (TCRA) regions involving small DNA segments were frequently observed. Genomic losses involving at least one or more known or candidate tumor suppressor genes were found in nearly all tumors, including some genes not previously implicated in ATLL. Some of the most significant gene or locus specific losses occurring in at least 20 % of the tumors in aggressive ATLL subtypes (A/UC and L) are summarized in Table 1. Losses of CDKN2A and CDKN2B tumor suppressor genes have been previously implicated in ATLL and other cancers. Several other genes found by this analysis have been implicated in apoptosis or cancer (i.e. CBLB, ANKRD11, IKZF1, and EPC1). IMMP2L deletion was associated with shorter survival time (2.3 weeks) compared with those cases without this gene deletion (29 weeks) in the A/UC group (p=0.005). ANKRD11 homo- or heterozygous deletions were seen in 37% of L-type and 19% of acute-type cases, and were associated with a shorter survival (13 vs. 43 weeks, p=<0.05) in the A group. CPN2/LRRC15 locus gains in 32% of A-type were linked to poorer survival (16 vs. 42 weeks, p=0.05).Table 1GENEFUNCTIONLOSSES (n) GAINS (n) NRXN3Membrane receptor, cell adhesion23 A/UC 3 LNSIMMP2LMitochondrial inner membrane peptidase16 A/UC* 5 L4 A/UCCDKN2A/ CDKN2B (P16INK4/p15INK4b)Cyclin-dependent kinase inhibitors, tumor suppressors16 A/UC 2 L1 A/UC 1 LCBLBE3 ubiquitin protein ligase12 A/UC 1 LNSANKRD11Transcriptional inhibitor, co-activator of p536 A/UC* 3 LNSCPN2CarboxypeptidaseNS10 A/UC* 2 LIKZF1Zinc-finger DNA binding proteins , lymphocyte differentiationNS6 A/UC 3 LINSIG1Endoplasmic reticulum membrane protein, intracellular lipid metabolismNS10 A/UC 1 LEPC1Member of the polycomb group family, transcriptional activator and repressor10 A/UC 2 LNS* are genomic imbalances associated with a statistically significant reduction in survival. NS: non-significant.Conclusion: In sum, using a high resolution CGH array we observed distinct patterns ofgenetic aberrations in ATLL endemic in the Western World. We have successfully narrowed the genomic regions containing potential candidate genes that could be relevant to the pathogenesis of this fatal disease. Functional studies are required to determine the role of some of these genes in the pathogenesis of ATLL. DisclosuresO’Hara:BioDiscovery, Inc.: Employment.

Abstract A9: Uridine diphospho glucuronosyl-transferase 2B17 genotype and a risk of acute lymphoblastic leukemia and lymphoblastic lymphoma among Japanese children

Abstract Background: Using clinical samples and tumor cell lines, chromosomal aberrations and somatic mutations have been well explored in the field of pediatric oncology. However, genes susceptible to pediatric acute lymphoblastic leukemia (ALL) as well as lymphoblastic lymphoma (LBL) have not been delineated yet. Therefore, we examined copy number variations (CNVs) of genomic DNA by means of high-resolution comparative genomic hybridization (CGH) microarray to find significant susceptible genes to ALL and LBL. Methods: We investigated CNVs by array CGH in genomic DNA derived from a total of 58 children with ALL and LBL as case, and compared with control I (n=67): acute myeloid leukemia (AML)(n=10), other type of leukemia (n=4), non-LBL (n=7) and solid tumors such as neuroblastoma (n=46) as well as control II (N=301): adults patients with diabetic nephropathy and receiving dialysis (n=86), with neurological diseases (n=76) and with esophageal, gastric and colon cancers (n=72) plus control I (n=67). Normalization was performed using the Log2 signal ratio (sample DNA/control DNA) for individual 180K probes distributed throughout the whole genome. Control DNA was obtained from a single Japanese person. To compare data between two groups (e.g., ALL+LBL vs. control I or control II), an unpaired t test was used and subsequently corrected by the Benjamini-Hochberg false-discovery rate using GeneSpringGX v.11.5.1. Probes with a corrected p-value (=q-value) less than 0.05 and fold change greater than 1.8 or less than -1.8 were considered statistically significant. Significant gene was confirmed using real time polymerase chain reaction (PCR). Results: CNV of uridine diphospho glucuronosyltansferase 2B17 (UGT2B17) gene was statistically less deleted in children with ALL+LBL, compared with control I (q-value=5.51 x 10-12) and with control II (q-value=6.58 x 10-6). Presence or absence of UGT2B17 gene was reconfirmed by real time PCR. CNVs of other genes were similar in cases and controls. UGT2B17 glucuronidates several endogenous and exogenous compounds in particular steroid hormones as well as several pharmaceutical drugs such as anthraquinones and histone deacetylase inhibitor. As previously reported in some paper of adult solid tumors, CNV of UGT2B17 gene showed significant correlation to drug response. The frequency of CNV shows exceptional differences between populations from Africa, Europe, and Asia. Interindividual variability in UGT2B17 allele-frequency is accompanied by pronounced differences in gene expression characterized by more than 29 times higher mRNA levels in Caucasian compared with Japanese. Conclusion: These results suggest that homozygous or heterozygous gains of the UGT2B17 gene may be associated with a significant risk for pediatric ALL and LBL. Citation Format: Sae Ishimaru, Yuya Saito, Yuichi Yokokawa, Yuki Yuza, Takashi Kaneko, Mitsuyoshi Urashima. Uridine diphospho glucuronosyl-transferase 2B17 genotype and a risk of acute lymphoblastic leukemia and lymphoblastic lymphoma among Japanese children. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A9.