Genomic Hybridization Array Research Articles

Right ventricle hypertrophy in fetuses diagnosed with Noonan syndrome: Can ductus venosus agenesis represent a contributing cause?

Noonan syndrome (NS) is the most common genetic syndrome in fetuses with increased nuchal translucency (NT) and normal karyotype. NS patients present typical patterns of cardiac defects, such as pulmonary stenosis, polyvalvular dysplasia and hypertrophic cardiomyopathy. This study aims to evaluate onset and trend of cardiac disease in fetuses with molecular diagnosis of NS and to search for other cardiovascular sonographic markers of NS. We performed a fetal echocardiographic follow-up of pregnancies with increased NT in the first trimester. All the fetuses with normal Karyotype and Comparative Genomic Hybridizationarray underwent a targeted next-generation sequencing panel for RASopathies. Increased NT was the only finding up to the second trimester in half of the fetuses, while in the others ductus venosus agenesis was also observed. In all of them, during the late second trimester, a severe cardiopathy arouse.

Contribution of amniocentesis in fetuses small for gestational age without other sonographic signs.

Our study evaluated the contribution of amniocentesis in the diagnostic approach of small-for-gestational-age fetuses (SGA) without morphological abnormality identified on ultrasound by studying FISH (fluorescence in situ hybridization) for chromosomes 13, 18 and 21, CMV PCR (cytomegalovirus polymerase chain reaction), karyotype and CGH (genomic hybridization array) METHODS: Our single-center retrospective cohort study included pregnant women referred for prenatal diagnosis for a SGA fetus in whom amniocentesis was performed between 2016 and 2019. A SGA fetus was defined as a fetus with an estimated fetal weight (EFW) below the 10th percentile according to referral growth curves in use. We evaluated the number of amniocenteses with an abnormal result and identified factors that may be associated with this outcome. Among the 79 amniocenteses performed, there were 5 (6.3%) abnormalities: karyotype (1.3%) and CGH (5.1%). No complications were described. We did not find any statistically significant factors associated with abnormal amniocentesis findings even if some elements seemed reassuring such as late discovery (p=0.31), moderate SGA (p=0.18), normal head, abdomen and femur measurements (p=0.57), but without statistically significant difference. Our study found 6.3% pathological analysis of amniocenteses, of which several would have been missed by conventional karyotyping. Patients must be informed about the risk of detecting abnormalities of low severity, with low penetrance or with unknown fetal consequences that could be source of anxiety.

Ptpn20 deletion in H-Tx rats enhances phosphorylation of the NKCC1 cotransporter in the choroid plexus: an evidence of genetic risk for hydrocephalus in an experimental study

BackgroundCongenital hydrocephalus occurs with some inheritable characteristics, but the mechanisms of its development remain poorly understood. Animal models provide the opportunity to identify potential genetic causes in this condition. The Hydrocephalus-Texas (H-Tx) rat strain is one of the most studied animal models for investigating the causative genetic alterations and analyzing downstream pathogenetic mechanisms of congenital hydrocephalus.MethodsComparative genomic hybridization (CGH) array on non-hydrocephalic and hydrocephalic H-Tx rats was used to identify causative genes of hydrocephalus. Targeted gene knockout mice were generated by CRISPR/Cas9 to study the role of this gene in hydrocephalus.ResultsCGH array revealed a copy number loss in chromosome 16p16 region in hydrocephalic H-Tx rats at 18 days gestation, encompassing the protein tyrosine phosphatase non-receptor type 20 (Ptpn20), a non-receptor tyrosine phosphatase, without change in most non-hydrocephalic H-Tx rats. Ptpn20-knockout (Ptpn20−/−) mice were generated and found to develop ventriculomegaly at 8 weeks. Furthermore, high expression of phosphorylated Na-K-Cl cotransporter 1 (pNKCC1) was identified in the choroid plexus (CP) epithelium of mice lacking Ptpn20 from 8 weeks until 72 weeks.ConclusionsThis study determined the chromosomal location of the hydrocephalus-associated Ptpn20 gene in hydrocephalic H-Tx rats. The high level of pNKCC1 mediated by Ptpn20 deletion in CP epithelium may cause overproduction of cerebrospinal fluid and contribute to the formation of hydrocephalus in Ptpn20−/− mice. Ptpn20 may be a potential therapeutic target in the treatment of hydrocephalus.

SMAD4 haploinsufficiency in small intestinal neuroendocrine tumors

BackgroundPatients with small intestinal neuroendocrine tumors (SINETs) frequently present with lymph node and liver metastases at the time of diagnosis, but the molecular changes that lead to the progression of these tumors are largely unknown. Sequencing studies have only identified recurrent point mutations at low frequencies with CDKN1B being the most common harboring heterozygous mutations in less than 10% of all tumors. Although SINETs are genetically stable tumors with a low frequency of point mutations and indels, they often harbor recurrent hemizygous copy number alterations (CNAs) yet the functional implications of these CNA are unclear.MethodsUtilizing comparative genomic hybridization (CGH) arrays we analyzed the CNA profile of 131 SINETs from 117 patients. Two tumor suppressor genes and corresponding proteins i.e. SMAD4, and CDKN1B, were further characterized using a tissue microarray (TMA) with 846 SINETs. Immunohistochemistry (IHC) was used to quantify protein expression in TMA samples and this was correlated with chromosome number evaluated with fluorescent in-situ hybridization (FISH). Intestinal tissue from a Smad4+/− mouse model was used to detect entero-endocrine cell hyperplasia with IHC.ResultsAnalyzing the CGH arrays we found loss of chromosome 18q and SMAD4 in 71% of SINETs and that focal loss of chromosome 12 affecting the CDKN1B was present in 9.4% of SINETs. No homozygous loss of chromosome 18 was detected. Hemizygous loss of SMAD4, but not CDKN1B, significantly correlated with reduced protein levels but hemizygous loss of SMAD4 did not induce entero-endocrine cell hyperplasia in the Smad4+/− mouse model. In addition, patients with low SMAD4 protein expression in primary tumors more often presented with metastatic disease.ConclusionsHemizygous loss of chromosome 18q and the SMAD4 gene is the most common genetic event in SINETs and our results suggests that this could influence SMAD4 protein expression and spread of metastases. Although SMAD4 haploinsufficiency alone did not induce tumor initiation, loss of chromosome 18 could represent an evolutionary advantage in SINETs explaining the high prevalence of this aberration. Functional consequences of reduced SMAD4 protein levels could hypothetically be a potential mechanism as to why loss of chromosome 18 appears to be clonally selected in SINETs.

Case report of 49, XXXXY syndrome: first case in Oman

Background: The incidence of sex chromosome aneuploidies is 1:400. Klinefelter syndrome is considered to be the most common type of sex chromosome aneuploidy, manifested as variants, such as 48,XXXY, 48,XXYY and 49,XXXXY. Nondisjunction of the X chromosome during meiosis I and II is considered as the cause of this type of aneuploidy. The classic clinical presentation of Klinefelter Syndrome is a triad of hypogonadism, mental retardation, and musculoskeletal anomalies. Case Presentation: In this case report, we describe a 2-year-old male child identified postnatally to have low birth weight, congenital heart defect, inguinal hernia, facial dysmorphism, and genital organs anomalies. Chromosomal analysis revealed a karyotype of 49,XXXXY and the comparative genomic hybridization (CGH) array analysis revealed a 155,065 kilo base pair duplication on chromosome Xp22.33q28 (168,546, −155,233,731) X4. Conclusion: 49,XXXXY karyotype is considered as the rarest sex chromosome aneuploidy syndrome. To our knowledge, this study is the first report of a patient with 49,XXXXY syndrome from Oman.

Do the risks of preimplantation genetic testing for aneuploidy outbalance the benefits?

Much has been achieved in the field of preimplantation genetic testing for aneuploidy (PGT-A; previously preimplantation genetic screening, PGS) in recent years, particularly with the novel and more precise methods for analyzing biopsied cells such as comparative genomic hybridization (CGH) and single-nucleotide polymorphism (SNP) arrays. Although these methods have been used in individual studies and shown an improvement in pregnancy and birth rates in some cases, the final results from prospective randomized studies are still awaited. Until these are available, the promising results from individual studies remain experimental. We also owe it to our patients to apply promising novel methods outside of studies, if a positive effect has been demonstrated. The same applies to the polar body diagnosis (PBD) practiced in Germany for aneuploidy screening. Thus, one can conclude that neither PGT-A by means of analyzing blastomeres nor polar body screening to indirectly investigate oocytes currently have a positive effect on pregnancy and birth rates. The large specialist societies, the American Society for Reproductive Medicine (ASRM) and the European Society of Human Reproduction and Embryology (ESHRE), also advise against routine use of these methods.

Abstract 1552: SATB1 is a novel prognostic factor in esophageal cancer patients and promotes migration and invasion of esophageal cancer cells

Abstract Introduction: Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy with extremely poor overall survival. There is no available molecular marker in clinical application until now. SATB1(special AT-rich binding protein 1) tether genomic DNA in nucleolus and hold three-dimensional structure of genomic DNA. We evaluated SATB1 expression using clinical samples from patients of ESCC and analyzed the association with clinicopathological factors and prognosis to identify novel prognostic marker in ESCC. In addition, we investigated the function of SATB1 using ESCC cells. We focused on matrix metalloproteinase protein (MMP3), which has a SATB1 binding site in its upstream. Materials and Methods: We accumulated 71 samples of ESCC patients who underwent primary tumor resection. The relative expression levels of SATB1 were obtained by normalizing the amount of SATB1 mRNA with respect to GAPDH mRNA as an endogenous control for each sample. We divided 71 ESCC samples into 2 groups, which are SATB1 low expression group (n=35) and SATB1 high expression group (n=36) according to the median of SATB1 expression level, and performed the comparison between two groups. In order to elucidate biological significance, we generated SATB1 overexpression stable transfectants in KYSE150, and performed migration and invasion assays. Next, we knockdowned SATB1 expression of KYSE410, which are ESCC lines, using SATB1 siRNA and evaluated the capability of migration and invasion. Moreover, to validate whether SATB1 altered MMP3 genomic locus, we analyzed comparative genomic hybridization (CGH) array obtained from 54 esophageal cancer samples. We examined MMP3 expression in SATB1 overexpression cells using qRT-PCR. Result: The SATB1 high expression group showed greater tumor depth than the low expression group (P=0.003). In the overall survival curve, patients in the SATB1 high expression group had a significant poorer prognosis than those in the SATB1 low expression group (P=0.013). Multivariate analysis showed that the level of SATB1 expression was an independent prognostic predictor (OR; 1.56, 95%CI; 1.04 - 2.44, P = 0.043). SATB1 transfectants significantly increased the capability of migration and invasion compared to mock (P<0.05). KYSE410 cells transfected with SATB1 siRNA significantly decreased the capability of migration and invasion compared to control cells (P<0.05). Genomic alterations in SATB1 locus were strongly correlated with those in MMP3 locus (P<0.0001, ρ=0.5156). Overexpression of SATB1 showed the elevated MMP3 level compared to mock (P<0.05). Conclusion: We indicated that SATB1 is novel prognostic factor in ESCC and that SATB1 promote migration and invasion of ESCC cells. SATB1 may be a promising new candidate for targeted therapies for ESCC. Citation Format: Yusuke Tsuruda, Ryunosuke Kogo, Shuhei Ito, Akihiro Kitagawa, Atsushi Fujii, Shotaro Kuramitsu, Dai Simizu, Yosuke Kuroda, Hidetoshi Eguchi, Tkaaki Masuda, Shoji Natsugoe, Masaki Mori, Koshi Mimori. SATB1 is a novel prognostic factor in esophageal cancer patients and promotes migration and invasion of esophageal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1552.

Genome profiling is an efficient tool to avoid the STUMP classification of uterine smooth muscle lesions: a comprehensive array-genomic hybridization analysis of 77 tumors

AbstractThe diagnosis of a uterine smooth muscle lesion is, in the majority of cases, straightforward. However, in a small number of cases, the morphological criteria used in such lesions cannot differentiate with certainty a benign from a malignant lesion and a diagnosis of smooth muscle tumor with uncertain malignant potential (STUMP) is made. Uterine leiomyosarcomas are often easy to diagnose but it is difficult or even impossible to identify a prognostic factor at the moment of the diagnosis with the exception of the stage. We hypothesize, for uterine smooth muscle lesions, that there is a gradient of genomic complexity that correlates to outcome. We first tested this hypothesis on STUMP lesions in a previous study and demonstrated that this 'gray category' could be split according to genomic index into two groups. A benign group, with a low to moderate alteration rate without recurrence and a malignant group, with a highly rearranged profile akin to uterine leiomyosarcomas. Here, we analyzed a large series of 77 uterine smooth muscle lesions (from 76 patients) morphologically classified as 19 leiomyomas, 14 STUMP and 44 leiomyosarcomas with clinicopathological and genomic correlations. We confirmed that genomic index with a cut-off=10 is a predictor of recurrence (P<0.0001) and with a cut-off=35 is a marker for poor overall survival (P=0.035). For the tumors confined to the uterus, stage as a prognostic factor was not useful in survival prediction. At stage I, among the tumors reclassified as molecular leiomyosarcomas (ie, genomic index ≥10), the poor prognostic markers were: 5p gain (overall survival P=0.0008), genomic index at cut-off=35 (overall survival P=0.0193), 13p loss including RB1 (overall survival P=0.0096) and 17p gain including MYOCD gain (overall survival P=0.0425). Based on these findings (and the feasibility of genomic profiling by array-comparative genomic hybridization), genomic index, 5p and 17p gains prognostic value could be evaluated in future prospective chemotherapy trials.

Distal deletion at 22q11.2 in a patient with Oculo-auriculo-vertebral Spectrum

Oculo-auriculo-vertebral Spectrum (OAVS), also known as Craniofacial Microsomia or Goldenhar Syndrome, presents wide phenotypic and etiological heterogeneity. It affects mainly the structures originated from the first and second pharyngeal arches. In addition, other major anomalies also may be observed, including congenital heart diseases, which are the main cause of OAVS deaths. Some recent studies have been investigated genomic imbalances in patients with this condition and deletions or duplications in the 22q11.2 region represent the most frequent alterations found in OAVS. The chromosome 22 has eight low copy repeats (LCRs) named from A to H, being the proximal deletion of ~3Mb (LCR22A-LCR22D) associated with 22q11.2 deletion Syndrome. There are five cases of OAVS with distal 22q11.2 deletion described in the literature. In this study, we report a patient with OAVS and a distal deletion in the 22q11.2 regions between LCRs D and E. The proband is a girl, first child of healthy, young and non-consanguineous parents. She was referred for genetic evaluation because she presented cleft lip and palate, intestinal atresia, annular pancreas, hydronephrosis and congenital heart disease. The dysmorphologic evaluation also revealed bilateral pre-auricular tags, left auditory canal stenosis, right nasal cleft and zygomatic hypoplasia with facial asymmetry. The genomic imbalances investigation was performed by MLPA technique using P250-B2 DiGeorge (MRC-Holland MLPA®) kit, and array genomic hybridization (aGH) using the CytoScan 750K Array chip (Affymetrix®, Santa Clara, CA, USA), which revealed a distal deletion of 1.498 kb at 22q11.2 encompassing the region from LCR22-D to LCR22-E. This is the sixth case of OAVS associated of distal 22q11.2 deletions and the second encompassing the region between LCR22-D to LCR22-E (type I deletion). All share some phenotypic signs, such as pre-auricular tags, facial asymmetry, cleft lip and palate, and congenital heart diseases. Some candidate genes in this region have been studied by having an important role in pharyngeal arches development and in congenital heart diseases, such as YPEL1, HIC2 e MAPK1/ERK2. This case contributes to genotype-phenotype correlation and reinforces that candidate genes for OAVS, in the 22q11.2 region, may be located between LCRs D and E.Financial Support: FAEPEX-UNICAMP, FAPESP, CAPES e CNPq

Application of Molecular Karyotyping in Acute Myeloid Leukemia: A Review

Acute myeloid leukemia (AML) is an aggressive disease characterized by the overproduction of immature myeloid cells that accumulate in blood and bone marrow. Integration of genetic findings and clinicopathological information is crucial in establishing the diagnosis, prognosis and determining the therapeutic approach in the management of AML patients. In recent years, the AML classification has evolved from morphology to cytogenetics/molecular genetics-based findings, which is essential in the detection of chromosomal abnormalities and has provided the framework for the diagnosis and risk-stratification in AML. Moreover, with advances in molecular karyotyping such as comparative genomic hybridization (CGH) and single nucleotide polymorphism (SNP) arrays, various limitations of conventional diagnostic approaches have been overcome. Hence, this review focuses on the insights into molecular karyotyping using CGH and SNP arrays which enable the identification of copy number variations (CNVs) at a higher resolution and facilitate the detection of copy neutral loss of heterozygosity (CN-LOH) otherwise undetectable by conventional cytogenetics. Technical hindrances of these methods (e.g. regions of losses, gains, or “undulating waves”) are also discussed in the context of AML.

Severe Mental Retardation Patient With a De Novo Chromosomal Deletion 5q14-22 Can Be a Carrier of a Rectal And Duodenal Cancer Associated With Over 200 Colorectal Polyps: A Case Report

Introduction Familial adenomatous polyposis (FAP) caused by a de novo 5q chromosomal deletion is rare, and precise cytogenetic analysis using comparative genomic hybridization (CGH) arrays have been performed in a few cases. Case presentation We herein present the case of a 38-year-old man with advanced rectal and duodenal cancer, FAP, and severe mental retardation caused by a de novo chromosomal deletion at 5q14–22. He was referred to our outpatient clinic because of a positive fecal occult blood test result. He was found to have advanced rectal cancer, with over 200 colorectal polyps, and duodenal cancer. He underwent laparoscopic total proctocolectomy with definitive ileostomy followed by partial resection of the duodenum. Comparative genomic hybridization to characterize deleted genes identified a large 5q deletion expanding approximately 24.9 Mbp, including the APC gene. Conclusion Patients who harbor a chromosomal deletion involving 5q21 are at risk of developing rectal cancer and polyposis. Therefore, such patients need early cancer screening following the availability of genetic information.

Abstract 2553: High-density array-CGH with targeted NGS unmask multiple non-contiguous minute deletions on chromosome 3p21 in mesothelioma

Abstract We used a custom-made comparative genomic hybridization-array (aCGH; average probe interval 254 bp) to screen 33 malignant mesothelioma (MM) biopsies for somatic copy number loss throughout the 3p21 region (10.7 Mb) that harbors 251 genes, including BRCA1-associated protein 1 (BAP1), the most commonly mutated gene in MM. We identified frequent minute biallelic deletions (&amp;lt;3 Kb) in 46/251 genes: four were cancer associated genes: SETD2 (7/33), BAP1 (8/33), PBRM1 (3/33) and SMARCC1 (2/33). These four genes were further investigated by targeted Next Generation Sequencing (tNGS), which revealed sequence-level mutations causing biallelic inactivation. Combined high-density aCGH and tNGS revealed biallelic gene inactivation in SETD2 (9/33, 27%), BAP1 (16/33, 48%), PBRM1 (5/33, 15%) and SMARCC1 (2/33, 6%). The incidence of genetic alterations detected is much higher than reported in the literature because minute deletions are not detected by NGS or commercial aCGH. Many of these minute deletions were not contiguous but rather they alternated with segments showing oscillating copy number changes along the 3p21 region. Functional assays revealed that SETD2, PBRM1 and SMARCC1 act as tumor suppressor in MM. In summary, we found that in MM: 1) multiple minute simultaneous biallelic deletions are frequent in chromosome 3p21, where they occur as distinct events involving multiple genes; 2) in addition to BAP1, mutations of SETD2, PBRM1 and SMARCC1 are frequent in MM; 3) our results suggest that high-density aCGH combined with tNGS provide a more precise estimate of the frequency and types of genes inactivated in human cancer, than approaches based exclusively on NGS strategy. Citation Format: Michele Carbone, Yoshie Yoshikawa, Mitsuru Emi, Tomoko Hashimoto-Tamaoki, Masaki Ohmuraya, Ayuko Sato, Tohru Tsujimura, Seiki Hasegawa, Takashi Nakano, Masaki Nasu, Sandra Pastorino, Agata Szymiczek, Angela Bononi, Harvey I. Pass, Haining Yang. High-density array-CGH with targeted NGS unmask multiple non-contiguous minute deletions on chromosome 3p21 in mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2553. doi:10.1158/1538-7445.AM2017-2553

Genomic Investigation of Balanced Chromosomal Rearrangements in Patients with Abnormal Phenotypes

Balanced chromosomal rearrangements (BCR) are associated with abnormal phenotypes in approximately 6% of balanced translocations and 9.4% of balanced inversions. Abnormal phenotypes can be caused by disruption of genes at the breakpoints, deletions, or positional effects. Conventional cytogenetic techniques have a limited resolution and do not enable a thorough genetic investigation. Molecular techniques applied to BCR carriers can contribute to the characterization of this type of chromosomal rearrangement and to the phenotype-genotype correlation. Fifteen individuals among 35 with abnormal phenotypes and BCR were selected for further investigation by molecular techniques. Chromosomal rearrangements involved 11 reciprocal translocations, 3 inversions, and 1 balanced insertion. Array genomic hybridization (AGH) was performed and genomic imbalances were detected in 20% of the cases, 1 at a rearrangement breakpoint and 2 further breakpoints in other chromosomes. Alterations were further confirmed by FISH and associated with the phenotype of the carriers. In the analyzed cases not showing genomic imbalances by AGH, next-generation sequencing (NGS), using whole genome libraries, prepared following the Illumina TruSeq DNA PCR-Free protocol (Illumina®) and then sequenced on an Illumina HiSEQ 2000 as 150-bp paired-end reads, was done. The NGS results suggested breakpoints in 7 cases that were similar or near those estimated by karyotyping. The genes overlapping 6 breakpoint regions were analyzed. Follow-up of BCR carriers would improve the knowledge about these chromosomal rearrangements and their consequences.

Abstract P2-03-11: Genomic pattern of breast carcinomas carrying mutations of non-BRCA homologous recombination genes

Abstract Background BRCA1 and BRCA2 are involved in the homologous recombination (HR) double-strand DNA break repair and genomic patterns of breast tumors with defective BRCA are characterized by increased genomic instability. The pre-clinical and clinical studies show that tumors with defective BRCA or other HR genes can response to platinum and PARP inhibitors. However, the genomic pattern of tumors carrying mutations of non-BRCA HR genes are not investigated. Methods Genomic patterns of breast carcinomas were performed by comparative genomic hybridization (CGH) array containing 60000 probes covering the whole genome with an average spacing of 40kb. The frequency of gains and losses for each regions detected by probes was calculated by ratio thresholds of 0.25 and -0.25, respectively. Large-scale genomic structural aberration was defined as the region of gains and losses of at least 10Mb. We analyzed the difference of large-scale aberration, including numbers, length and specific regions, between tumors with BRCA mutation (mtBRCA), non-BRCA HR mutation (mtHR) and wild type. Results We examined 41 breast carcinomas, including 15 cases with BRCA mutations, 14 with non-BRCA HR gene mutations and 12 without mutations (control). The 14 non-BRCA HR gene were 1 ATM, 1 BRIP1, 1 BARD1, 1 FANCA, 2 FANCB, 1 FANCI, 1 PALB2, 2 RAD50, 2 RAD51C and 2 RAD51D. The number and length of large-scale genomic structural aberration of mtBRCA tumors were significantly higher than wild type tumors (number p=0.005; length p=0.005), indicating CGH can distinguish the mtBRCA from control tumors. We then checked the mtHR tumors, which also revealed significantly increased number and longer length of structural aberrations compared to wild type tumors (number p=0.035; length p=0.022), but were not different from mtBRCA tumors (number p=0.204; length p=0.425). Among the specific regions on chromosomes, mtBRCA and mtHR tumors contained similar genomic aberration regions but different from wild type tumors. The most frequent aberration regions of mtBRCA and mtHR tumors are chromosome 6p22.1-p25, 6q21-q27, 8q11.1-q24, 11p11.2-p14.1, 11q, 12p and 19p, which are less revealed in the wild type (all p value &amp;lt;0.05). Conclusions Our study demonstrated a direct evidence that increased genomic instability were the common characteristics of mtBRCA and non-BRCA mtHR tumors. In addition, we identify the specific genomic patterns of mtBRCA and mtHR tumors, which can be a biomarker indicating HR deficiency and response to platinum and PARP inhibitors. Citation Format: Lin P-H, Kuo W-H, Wang M-Y, Lo C, Lin C-H, Lu Y-S, Chiu C-F, Huang C-S. Genomic pattern of breast carcinomas carrying mutations of non-BRCA homologous recombination genes [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-03-11.

Testing the Complex Child: CGH Array, WES, Clinical Exome, WGS

Purpose of Review The purpose of this review was to compare existing strategies for evaluation of complex paediatric patients with newer techniques. Comparative genomic hybridization (CGH) array is the currently accepted first tier genetic test in the evaluation of a pediatric patient with complex physical and developmental anomalies. CGH provides an answer in only 15–20 % cases, and further genetic testing is required in the majority of cases. This has previously involved sequential single-gene tests, with low yield, significant costs and delay in diagnosis.

Abstract 3696: Biological significance of the wild-type p53-induced phosphatase 1(Wip1) expression in invasive breast cancer

Abstract Backgrounds The wild-type p53-induced phosphatase 1(Wip1) is a member of the serine/threonine protein phosphatases, and plays an important role in the nucleus as one of the key components in the DNA damage response (DDR) network. Wip1 is encoded by the protein phosphatase magnesium dependent 1 delta (PPM1D), sited on locus 17q23. PPM1D amplification has been reported in breast cancer. Aims We evaluated the expression of Wip1 mRNA, Wip1 protein and PPM1D DNA copy number to clarify the relationship between Wip1 expression and the clinicopathological features and prognosis to determine the biological significance of Wip1. Materials and Methods Breast cancer cells (MCF7, T47D, MDA-MB231, HCC1937, HS578T, BT20 and SKBr3) were used for Wip1 expression analysis and copy number analysis. Primary invasive ductal carcinoma specimens were obtained from Japanese patients who underwent surgery without neoadjuvant chemotherapy or endocrine therapy. Wip1 mRNA expression was evaluated in 140 cases by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Wip1 protein expression was evaluated in 192 cases by immunohistochemistry (IHC). The PPM1D DNA copy number was analyzed by genomic PCR in 33 breast cancer cases and by the single-nucleotide polymorphism-Comparative Genomic Hybridization (SNP-CGH) array in 12 cases. The effects on the cell growth of the Wip1 inhibitor (GSK2830371) was analyzed by the viability assay in MCF7. Results Wip1 mRNA expression was significantly higher in MCF7, luminal type cell line. Wip1 mRNA expression was divided into four groups, very high, high, low and very low. The very high Wip1 mRNA expression was significantly associated with positive estrogen receptor (ER) expression (p = 0.02). There was no significant correlation between Wip1 mRNA expression and the prognosis. Wip1 protein nuclear expression was positive in 21 cases (10.9%). There was no significant association between the Wip1 protein expression levels and the clinicopathological factors and the prognosis. PPM1D DNA copy number significantly correlated with Wip1 protein expression (p = 0.0035). Amplification at 17q23 was detected in 6 cases by SNP-CGH array, and all of these six cases showed positive nuclear Wip1 expression. PPM1D amplification was not observed in Wip1 negative cases. In the cell viability assay, the suppression of the MCF7 cell growth was observed by Wip1 inhibitor. Conclusions Wip1 nuclear protein expression may be regulated by PPM1D amplification, and the Wip1 inhibitor may have the therapeutic effects for the breast cancer with PPM1D amplification or high Wip1 protein expression. Citation Format: Yuka Inoue, Nami Yamashita, Eriko Tokunaga, Hiroyuki Kitao, Kimihiro Tanaka, Hiroki Ueo, Hiroshi Saeki, Eiji Oki, Yoshihiko Maehara. Biological significance of the wild-type p53-induced phosphatase 1(Wip1) expression in invasive breast cancer. [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 3696.

Abstract 3660: ATF4 is a putative tumor suppressor gene in medullary thyroid cancer

Abstract Medullary thyroid carcinoma (MTC) is derived from the calcitonin-producing C-cells of the thyroid gland. Oncogenic mutations of the RET proto-oncogene are found in all hereditary MTC and ∼45% of the sporadic cases. However, it is clear that additional genetic lesions contribute to the development of both RET-dependent and RET independent MTC. We analyzed 30 primary medullary thyroid cancer (MTC) tumors by comparative genomic hybridization (CGH) arrays (Agilent 244K) and a subset of those tumors by ultra-high-resolution SNP arrays (Affymetrix SNP6.0). A consistent finding with both arrays was a loss localized to 22q13.1 in 40% of tumors. This region contains the ATF4 gene and loss of this gene was validated by real time PCR. ATF4 is a central mediator of integrated stress response pathway and activates expression of a cohort of downstream target genes known to be involved in cell survival, apoptosis, autophagy and senescence. We hypothesize that the loss of ATF4 expression contributes to MTC progression through mechanisms involving aberrant proliferation and escape from apoptosis. We present several findings to support this. First, ATF4 protein levels were found downregulated in 50% MTC tumor tissues (n = 43) and low ATF4 expression was associated with poor overall survival in MTC patients (HR = 5.015, 95% CI: 1.38-10.64), P = 0.013). A negative correlation was observed between RET and ATF4 protein expression in MTC tumors (r = -0.89, R square = 0.799, p&amp;lt;0.0001). Second, we showed that targeted heterozygote and homozygous deletion of Atf4 in mice causes C cell hyperplasia, a precancerous lesion preceding MTC. Third, ATF4 overexpression inhibits proliferation and survival of MTC derived cell lines. ATF4 decreases RET protein levels by inducing ubiquitination and degradation of activated RET. In contrast, ATF4 depletion upregulates RET protein levels as well as RET autophosphorylation. Finally, combination treatment of tyrosine kinase inhibitor (sunitinib) and eeyarestatin that stimulates ATF4 transcription, induces apoptosis of the MTC cells in a synergistic manner through activation of ATF4 and its target genes NOXA and PUMA as well. These results suggest ATF4 as a potential tumor suppressor gene in MTC. We postulate that inactivation of ATF4 is a central impediment to induction of apoptosis in MTC tumors. Therefore, strategies to upregulate ATF4 during TKI therapy would render tumor cells exquisitely sensitive to stress induced apoptosis, offering a novel synergistic treatment. Citation Format: Rozita Bagheri-Yarmand, Gilbert J. Cote, Elizabeth G. Grubbs, Michelle D. Williams, Steven I. Sherman, Robert F. Gagel. ATF4 is a putative tumor suppressor gene in medullary thyroid cancer. [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 3660.

Abstract 3179: Germline deletion of FH in hereditary leiomyomatosis and renal cell carcinoma (HLRCC)

Abstract Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a familial cancer syndrome characterized by germline mutations in the fumarate hydratase (FH) gene that encodes the enzyme responsible for conversion of fumarate to malate in the Kreb's Cycle. HLRCC is associated with the development of cutaneous and uterine leiomyomas, and a highly aggressive form of type 2 papillary kidney cancer. HLRCC renal tumors can be extremely aggressive with patients demonstrating locally advanced or disseminated disease even when associated with small tumors 1-2 cm in size. Therefore, accurate identification of the “at risk” individuals is very important. In our experience, FH point mutations are identified in 90% of individuals that are clinically diagnosed with HLRCC, while in the remainder, FH may be inactivated by deletion or other causes. Several studies have reported complete deletions of the FH gene and a few have estimated the extent of these deletions and identified the additional genes that would be lost in conjunction with FH. Interestingly, families with complete FH deletion have demonstrated cutaneous and uterine leiomyomas but no families have yet been reported with evidence of kidney cancer. A single family has been identified that demonstrates a partial deletion resulting in the loss of exon 1, and in this case kidney cancer has identified within the family. In this study, patients with suspected HLRCC hereditary kidney cancer but no germline point mutation were enrolled on an NCI-IRB approved protocol and an Agilent custom high-definition comparative genomic hybridization (CGH) array was used to identify FH gene deletions. The assay identified complete germline deletion of a single copy of FH within seven suspected HLRCC patients deletion ranging from 249kb to 4.74Mb that in all cases resulted in a minimum addition loss of the KMO, OPN3, CHML, and WDR6 genes. All results were confirmed by CLIA approved tests and a further five new patients were identified by direct CLIA testing, including a novel partial deletion. In contrast to previous reports, seven out of twelve (58.3%) patients/families in our cohort demonstrated a personal or family history of HLRCC associated Type 2 papillary kidney cancer. In conclusion, complete or partial deletion of the FH gene is an infrequent but important cause of HLRCC and has no protective effect on the incidence of HLRCC associated type 2 papillary kidney cancer. Citation Format: Cathy D. Vocke, Christopher J. Ricketts, Lindsay A. Middelton, Youfeng Yang, W. Marston Linehan. Germline deletion of FH in hereditary leiomyomatosis and renal cell carcinoma (HLRCC). [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 3179.

Abstract 4949: 18q copy number variation in primary prostate tumors as a predictor of long-term outcome

Abstract The goal of this study is to use primary prostate tumors with long-term outcome to identify regions of copy number variation (CNV) on 18q along with other known loci to determine which regions correlate with poor prognosis. Most prostate cancers (PCa) grow slowly and remain indolent, yet some become aggressive and metastasize. Clinical decision-making requires prognostic markers that can be utilized at the time of diagnosis to determine which tumors will become aggressive. Previous studies have shown a correlation between alterations on the long arm of chromosome 18 (18q) and metastatic PCa. Prostatectomy specimens were collected and banked in the University of Texas Health Science Center San Antonio Genitourinary tissue biorepository. Subjects were categorized as No Evidence of Disease recurrence (NED) or those who developed Metastases (MET) with a minimum of five years follow-up. Tumor foci were marked after H&amp;E staining. Using Agilent's SureDesign software, a custom comparative genomic hybridization (CGH) array was created containing high-density tiling of 18q, as well as 15 genes located elsewhere in the genome that have been implicated in PCa prognosis. Tumor DNA was isolated and analyzed for CNV using standard CGH methodology and the Agilent Cytogenomics software. Chromosome-wide (18q) and genome-wide statistical significance of the relative CNV anomaly prevalence (NED vs. MET) was determined with permutation testing. Thirty-five primary prostate tumors were analyzed; 17 were classified as NED and 18 as MET. Two significant regions of copy number gains were found on 18q. One gain at 18q21.31 was found only in MET samples while another gain at 18q11.2 was found in NED samples. Additionally, confirmatory gains and losses in the regions containing 15 PCa related genes were found, only three of which were statistically different between the two sets. Our arrayCGH results implicate DNA gain of copy at 18q21.31 as being predictive of poor outcome. MicroRNA-122, which lies within this region, has been shown to have increased expression in prostate tumors with higher Gleason score and has been implicated in breast cancer metastasis. Our data suggests it may be an important prognostic marker for aggressive PCa. The 18q11.2 region found to be amplified in NED samples warrants further study to identify genes in this region that may predict good outcome. Of the 15 gene regions included, MYC and Cyclin D1 regions both exhibit gain of copy number in primary tumors with poor outcome, in line with other reports that these gains that these are early events in PCa progression. Citation Format: Keith A. Ashcraft, Teresa L. Johnson-Pais, Jonathan A.L Gelfond, Javier Hernandez, Ian M. Thompson, Robin J. Leach. 18q copy number variation in primary prostate tumors as a predictor of long-term outcome. [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 4949.

Tetrasomy 3q26.32-q29 due to a supernumerary marker chromosome in a child with pigmentary mosaicism of Ito.

Pigmentary mosaicism of Ito (PMI) is a skin abnormality often characterized by hypopigmentation of skin, following, in most cases, the Blaschko lines, usually associated with extracutaneous abnormalities, especially abnormalities of the central nervous system (CNS). It is suggested that this pattern arises from the presence and migration of two cell lineages in the ectoderm layer during the embryonic period and embryonic cell migration, with different gene expression profiles associated with pigmentation. Several types of chromosomal aberrations, with or without mosaicism, have been associated with this disorder. This study comprised clinical description and cytogenetic analysis of a child with PMI. The G-banded karyotype analysis revealed a supernumerary marker chromosome in 76% of the analyzed metaphases from peripheral blood lymphocytes. Array genomic hybridization analysis showed a copy number gain between 3q26.32-3q29, of approximately 20.5 Mb. Karyotype was defined as 47,XX,+mar[38]/46,XX[12].arr 3q26.32-3q29(177,682,859- 198,043,720)x4 dn. Genes mapped in the overlapping region among this patient and three other cases described prior to this study were listed and their possible involvement on PMI pathogenesis is discussed.