Array Comparative Genomic Hybridization Studies Research Articles

Molecular Cytogenetic Characterization of the Murine Melanoma Cell Lines S91 Clone M3 and B16-F1 with Variant B16-4A5

Melanoma is considered to be one of the most aggressive human tumors. Thus, early molecular diagnosis with risk factor stratification could be an efficacious strategy to increase the survival rates in affected patients. Murine cell lines B16-F1, B16-4A5, and S91 clone M3 are the ones most commonly applied in melanoma research. However, genetic peculiarities of these 3 cell lines have not been studied in detail before. Here, we closed this gap by molecular cytogenetic and array-comparative genomic hybridization studies and the translation of the characterized imbalances into the human genome. This study revealed severely rearranged karyotypes with in parts similar imbalances for all 3 cell lines. Interestingly, they involve genes known to play major roles in human melanoma. These are specifically the oncogenes and tumor suppressor genes, being associated with aggressive forms of melanoma. B16-F1, B16-4A5, and S91 clone M3 revealed aberrations which were similarly observed in human eye and skin but not in human uveal melanoma. Thus, they can be considered as model systems for advanced eye and skin melanoma.

RUNX1 associated Familial Platelet Disorder with Myeloid Malignancy (FPD-MM) in Children: A Novel New Phenotype with Juvenile and Chronic Myelomonocytic Leukemia (JMML/CMML) Characteristics

Introduction:RUNX1 (aka AML1; 21q22.12) is indispensable in the establishment of definitive hematopoiesis in humans. Activating RUNX1 mutations are associated with both Acute Myeloid and Lymphoblastic Leukemias (AML, ALL). On the other hand, hypofunctioning RUNX1 mutations cause dominantly inherited Familial Platelet Disorder (FPD). RUNX1 FPD has a high risk for progression to pancytopenia, myeloproliferative disorders (MPD) or AML, hence the new WHO category FPD with myeloid malignancy (FPD-MM). Those with MM carry mutations in other genes seen in AML, MDS. It is a relatively rare disorder with ~75 affected kindreds reported worldwide (Sood, et al. Blood 2017). Detailed reviews of pediatric cases are few.Case Histories: We encountered two children with RUNX1 associated thrombocytopenia; the mutations are novel. The first family is that of 14 yr old AAF, presenting with fainting- blood counts are shown in Table 1; fetal hemoglobin (HbF) was elevated; bone marrow was hypercellular with 6% type 1 blasts, extreme paucity of megakaryocytes, erythroid hyperplasia and large numbers of sea blue histiocytes. The high HbF suggested JMML while the monocyte CD16;14 profile (95.6% CD14+ cells) was similar to that seen in the adult type Chronic Myelomonocytic Leukemia (CMML). Her mother has pancytopenia without excess blasts in the marrow. The second case presented with neonatal thrombocytopenia; father has history of excessive bruising.Results: Blood counts and values for HbF are listed in Table 1.Molecular testing:Case1: A Myeloid gene panel showed RUNX1 - NM_001754.4:c.501delT, p.Ser167Argfs*9; PHF6 - NM_032458.2:c.902dupA, p.Tyr301*; CUX1- NM_001202543.1:c.2378delC, p.Pro793Argfs*26. No mutations were noted in PTPN11, CBL or RAS genes, the latter confirmed by JMML panel done at University of California, San Francisco. UCSF panel identified a mutation in SH2B3, a gene linked to erythrocytosis not caused by JAK2 mutations. Her mother has the same RUNX1 mutation, thus identifying a germline mutation of RUNX1 in her and her child but not the PHF6, CUX1 or the SH2B3 mutations seen in her daughter. A half sibling is unaffected and is a potential transplant donor for the mother.Case2: No coding sequence mutations were detected in genes associated with familial thrombocytopenia including ETV6, GATA1 and RUNX1. Array Comparative Genomic Hybridization studies (Prevention Genetics) identified a heterozygous deletion of the entire exon 5 of RUNX1.To understand the complex findings in family 1 additional studies were done- DRAQ5, CD71, Fetal Hb staining showed that NRBC in Case 1 contained predominantly high HBF cells. LIN28B was markedly elevated in the proband but not the mother (HbF- normal); LIN28B expression was normal in Case 2.Treatment/Outcome: In Case 1, low dose decitabine therapy resulted in the control of MPD features with good Hb recovery and normalization of the monocyte CD16;14 profiles. There was no platelet response to decitabine nor to a course of valproic acid. The child died of fulminant acute graft vs host disease affecting the liver following a 4/6 cord mismatch transplantation. Mother continues to show moderately severe pancytopenia requiring frequent transfusion support. The second child is symptom free with mild thrombocytopenia.Discussion: The hybrid JMML/CMML features in the index child are likely caused by the concurrent CUX1/PHF6/SH2B3 mutations. We are unable to establish if these are true de novo mutations as the father was not available for study; she had no full siblings. Neither high HbF nor high LIN28B are known feature of FPD by itself nor CMML or Polycythemia Vera (p Vera). Recently, the high HbF in JMML has been linked to high expression of LIN28B. SH2B3 mutation may have contributed to the high erythroid proliferation observed in our case. Induced CUX1 haploinsufficiency in mice causes MPD akin to CMML and megakaryocytic (Meg) proliferation (An N, et al. Blood 2018). The virtual absence of Megs in our case indicates that the CUX1 mutation was unable to overcome the Meg ploidization defect caused by the RUNX1 mutation. PHF6 mutations occur in T-ALL and AML but have not been linked to high HbF.Conclusions: Normal HbF and normal LIN28B expression in the mother of Case1 and in Case2 indicate that increased LIN28B is linked to the high HbF in Case 1 and that high LIN28B itself is a consequence of the malignant transformation caused by the concurrent CUX1/PHF6/SH2B3 mutations. DisclosuresChitlur:Baxter, Bayer, Biogen Idec, and Pfizer: Honoraria; Novo Nordisk Inc: Consultancy. Ravindranath:AGIOS: Other: Site Investigator for Pyruvate Kinase Deficiency.

Familial Chiari Type 1: A Molecular Karyotyping Study in a Turkish Family and Review of the Literature

The etiology of Chiari I malformation (CMI) has not been fully elucidated. Therefore, we performed a genetic study of a Turkish family in which 3 sisters had a diagnosis of CMI with or without syringomyelia. In a family with 7 children, 4 daughters complained of occipital headaches. In 2 of these daughters, CMI had been diagnosed during their 30s, and CMI plus syrinx had been diagnosed in the other daughter in her 40s. Cranial magnetic resonance imaging of the fourth daughter who had developed headaches during her 30s showed normal findings. Because the other siblings in the family were asymptomatic, radiological examinations were not performed. The family had a history of distant consanguineous marriage between parents. Additionally, the father had died, and the mother was asymptomatic, with radiologically normal findings. Array comparative genome hybridization studies were performed for 12 persons from 3 generations of this family. None of the 12 cases examined harbored copy number variations. This family with 3 sisters having CMI suggested a possible autosomal recessive single-gene etiology. Cases of familial CMI are unusual but important to study because they could reveal the specific genes involved in posterior fossa/foramen magnum structure and function and provide insights into the cause of sporadic cases.

Whole-exome sequencing demonstrates recurrent somatic copy number alterations and sporadic mutations in specialized stromal tumors of the prostate

In a previous array comparative genomic hybridization study, we detected common deletions of chromosomes 13 and 14 in prostatic stromal sarcoma and stromal tumor of uncertain malignant potential (STUMP). In this study, we performed whole-exome sequencing (WES) and fluorescence in situ hybridization to explore somatic mutations in 1 low-grade stromal sarcoma, 1 high-grade stromal sarcoma, and 12 STUMPs including 5 cases of degenerative atypia type, 1 myxoid type, 1 phyllodes type, and 5 cases of recently described round cell type. WES was successful on 13 cases that revealed frequent somatic copy number alterations including losses of chromosomes 13 (11 cases), 14 (11 cases), and 1p (9 cases), and partial or complete loss of chromosome 10 (7 cases). Fluorescence in situ hybridization was done on 9 cases and showed compatible chromosome 13 copy numbers with the WES results. STUMPs and the low-grade stromal sarcoma carried moderate tumor mutation burdens that ranged from 1.23 to 7.24 mutations per megabase, while the high-grade stromal sarcoma harbored a significantly higher mutation burden (11.55 mutations per megabase). Sporadic somatic mutations were observed, but no recurrent driver mutations could be discerned. In conjunction with prior array comparative genomic hybridization, we have demonstrated the consistent gene dosage profiles that support the clonal nature and the concept of specialized stromal tumors of the prostate as a distinctive tumor entity.

Unexpected frequency of genomic alterations in histologically normal colonic tissue from colon cancer patients.

As shown by genomic studies, colorectal cancer (CRC) is a highly heterogeneous disease, where copy number alterations (CNAs) may greatly vary among different patients. To explore whether CNAs may be present also in histologically normal tissues from patients affected by CRC, we performed CGH + SNP Microarray on 15 paired tumoral and normal samples. Here, we report for the first time the occurrence of CNAs as a common feature of the histologically normal tissue from CRC patients, particularly CNAs affecting different oncogenes and tumor-suppressor genes, including some not previously reported in CRC and others known as being involved in tumor progression. Moreover, from the comparison of normal vs paired tumoral tissue, we were able to identify three groups: samples with an increased number of CNAs in tumoral vs normal tissue, samples with a similar number of CNAs in both tissues, and samples with a decrease of CNAs in tumoral vs normal tissue, which may be likely due to a selection of the cell population within the tumor. In conclusion, our approach allowed us to uncover for the first time an unexpected frequency of genetic alteration in normal tissue, suggesting that tumorigenic genetic lesions are already present in histologically normal colonic tissue and that the use in array comparative genomic hybridization (CGH) studies of normal samples as reference for the paired tumors can lead to misrepresented genomic data, which may be incomplete or limited, especially if used for the research of target molecules for personalized therapy and for the possible correlation with clinical outcome.Electronic supplementary materialThe online version of this article (doi:10.1007/s13277-016-5181-0) contains supplementary material, which is available to authorized users.

Abstract 1534: Amplification of PITPNC1 affects breast cancer progression

Abstract Introduction Identification of driver mutations at single gene level and determination of their respective contribution to the enhanced invasive potential of cancer cells, via expression profiling and functional assays, are indispensable steps toward elucidating breast cancer pathobiology. In our previous array comparative genomic hybridization studies, gain in a region of chromosome 17, 17q23.3-24.3, was frequently observed in invasive duct carcinoma (IDC) patients with lymph node metastasis. Particularly, phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1), found in 17q24.2, was associated with higher histological grade, larger tumor size, positive HER2 staining, and poorer prognosis from the analysis of a NKI dataset. PITPNC1 is involved in signal transduction and intracellular lipid transport, and may be an essential part of the epidermal growth factor signaling pathway. Additionally, amplification of PITPNC1 via the loss of microRNA-126 suppression has been implicated in metastatic angiogenesis and colonization. In this study, we aim to assess the role of PITPNC1 in the development of aggressive breast cancer by evaluating its effects on breast cancer progression and invasion. Method A total of 21 samples - 13 pure duct carcinoma in situ (DCIS), 8 IDC with or without lymph node metastasis - were used for the preliminary round of this study. Formalin-fixed and paraffin-embedded tissue blocks were microdissected and whole genome amplified using ligation-mediated PCR. Amplified DNA was subjected to quantitative real-time PCR. Copy number alterations of PITPNC1 were calculated with Livak method, using B2M as the reference gene. Lipofectamine transfection overexpressing PITPNC1 in non-invasive MCF-10A and invasive MCF7, MD-MB-231 cell lines was prepared for subsequent in vitro assays. Proliferation and migration capabilities associated with PITPNC1 overexpression is being evaluated using MTT assay and transwell migration assay. Result From preliminary qPCR data, PITPNC1 was shown to be amplified in DCIS samples (p = 0.0025) and IDC samples (p = 0.0093). The 95% confidence intervals of fold difference against normal breast tissue control are [7.1, 23.4] and [5.7, 24.4] respectively. PITPNC1 overexpressing MCF-10A cells exhibit a higher proliferation rate in culture compared to controls. Conclusion Consistent amplification of PITPNC1 is seen in both DCIS and IDC cases. However, it is still unclear if PITPNC1 amplification is a result of a single mutational event occurring early in cancer progression or a cumulative effect occurring over time. Additional qPCR on paired samples against a pooled control would be necessary to determine its contribution to invasion. The regularity of copy number gain in the PITPNC1 locus and its association with HER2 expression highlight its predictive potential as a novel biomarker for tumorigenesis or invasion. Citation Format: Peiqi Wang, Ranju Nair, Nisha Kanwar, Grace Cheung, Susan J. Done. Amplification of PITPNC1 affects breast cancer progression. [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 1534.

De novo deletions and duplications of 17q25.3 cause susceptibility to cardiovascular malformations.

BackgroundGenomic disorders resulting from deletion or duplication of genomic segments are known to be an important cause of cardiovascular malformations (CVMs). In our previous study, we identified a unique individual with a de novo 17q25.3 deletion from a study of 714 individuals with CVM.MethodsTo understand the contribution of this locus to cardiac malformations, we reviewed the data on 60,000 samples submitted for array comparative genomic hybridization (CGH) studies to Medical Genetics Laboratories at Baylor College of Medicine, and ascertained seven individuals with segmental aneusomy of 17q25. We validated our findings by studying another individual with a de novo submicroscopic deletion of this region from Cytogenetics Laboratory at Cincinnati Children’s Hospital. Using bioinformatic analyses including protein-protein interaction network, human tissue expression patterns, haploinsufficiency scores, and other annotation systems, including a training set of 251 genes known to be linked to human cardiac disease, we constructed a pathogenicity score for cardiac phenotype for each of the 57 genes within the terminal 2.0 Mb of 17q25.3.ResultsWe found relatively high penetrance of cardiovascular defects (~60 %) with five deletions and three duplications, observed in eight unrelated individuals. Distinct cardiac phenotypes were present in four of these subjects with non-recurrent de novo deletions (range 0.08 Mb–1.4 Mb) in the subtelomeric region of 17q25.3. These included coarctation of the aorta (CoA), total anomalous pulmonary venous return (TAPVR), ventricular septal defect (VSD) and atrial septal defect (ASD). Amongst the three individuals with variable size duplications of this region, one had patent ductus arteriosus (PDA) at 8 months of age.ConclusionThe distinct cardiac lesions observed in the affected patients and the bioinformatics analyses suggest that multiple genes may be plausible drivers of the cardiac phenotype within this gene-rich critical interval of 17q25.3.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-015-0291-0) contains supplementary material, which is available to authorized users.

Abstract P3-06-28: Alterations in HER2 status and outcome following neoadjuvant chemotherapy in HER2-positive breast cancer (BC)

Abstract Background: Emerging data suggest that chemotherapy and anti-HER2 agents can change HER2 status in HER2+ BC and that this may have prognostic implications. The findings suggest a role for post-treatment (tx) retesting of HER2 status in surgical specimens. However, few studies have specifically examined this phenomenon, and direct comparisons of post-tx HER2 testing modalities are lacking. Furthermore, the biologic basis for HER2 alterations remains poorly understood. The aims of this study were to systematically evaluate the effects of neoadjuvant chemotherapy (NAT) including anti-HER2 agents on HER2 protein expression (P-EXP) and gene amplification (G-AMP), and to examine the potential prognostic impact of HER2 alterations on outcomes in HER2+ BC. Design: We retrospectively identified 129 patients with HER2+ BC who received NAT at our institution. HR (ER and PR) and HER2 P-EXP were evaluated by immunohistochemistry (IHC) and HER2 G-AMP by fluorescence in situ hybridization (FISH) in pre- and post-tx samples. Pathologic tumor responses were categorized as no residual disease (RD; no invasive or in situ cancer), residual ductal carcinoma in situ (DCIS) only, and residual invasive carcinoma. Pathologic complete response (pCR) was defined as DCIS only or no RD. Association between groups was determined by Fisher exact test. For survival analysis, Kaplan Meier curves were constructed and significance in curve separation was assessed by log rank test. Results: Mean follow-up was 52.6 months (range 6-162). Eighty-four (65%) cases had residual invasive cancer, 21 (16%) residual DCIS only and 24 (19%) no RD. Post-tx HER2 status was available in 70 cases with residual invasive cancer, and 27 (39%) of these showed reduced HER2 P-EXP (staining intensity 0, 1+, 2+) by IHC. Twenty-one (78%) of 27 cases with reduced HER2 P-EXP retained HER2 G-AMP (IHC-/FISH+), and 6 (22%) were negative for both HER2 P-EXP and G-AMP (IHC-/FISH-). The subset of IHC-/FISH- patients showed 100% 5-year recurrence free survival (RFS), whereas IHC-/FISH+ and IHC+ (intensity 3+) cases demonstrated similarly decreased 5-year RFS of 67% and 71%, respectively. There was a trend towards reduced HER2 expression in HR+ versus HR- cases (46% vs 26%, p=0.19). HR-HER2+ tumors were more likely to achieve pCR than HR+HER2+ cases (48% vs 26%; p=0.014, OR=0.375). In the HR- subset, RFS was significantly better in patients with no RD compared to those with residual invasive cancer (p=0.047); this relationship was not observed in the HR+ group (p=0.693). Conclusion: A significant fraction of pre-tx HER2+ BC demonstrate reduced HER2 P-EXP following NAT. In a subset of these, post-tx decreased HER2 P-EXP is associated with retained HER2 G-AMP, suggestive of HER2 protein downregulation. In another subset, decreased HER2 P-EXP with associated lack of HER2 G-AMP indicates selection for HER2 non-amplified clones in HER2 heterogeneous tumors. Array comparative genomic hybridization studies are in progress to further elucidate these mechanisms. Lastly, our results suggest that post-tx evaluation of HER2 status by FISH in addition to IHC may have prognostic and/or predictive value in HER2+ BC. Further studies in larger prospective study populations are needed to confirm our findings. Citation Format: Dianna Ng, Gregor Krings, Christina Yau, Kristie White, Jie Hou, Anthony Chua, James P Grenert, Yunn-Yi Chen. Alterations in HER2 status and outcome following neoadjuvant chemotherapy in HER2-positive breast cancer (BC) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-28.

A case of Sotos syndrome presented with end-stage renal disease due to the posterior urethral valve

Sotos syndrome (SS, OMIM 117550) is characterized by prenatal and postnatal overgrowth with multiple congenital anomalies. However, there have been few cases of growth retardation caused by renal failure from infancy. We report a case of dysplasia of the bilateral kidneys with renal failure and poor postnatal growth. A 2-month-old boy visited the emergency room owing to poor oral intake and abdominal distension. He was born at the gestational age of 38 weeks with a birth weight of 4,180 g. After birth, he had feeding difficulty and abdominal distension. Upon physical examination, his height and weight were in less than the 3rd percentile, while his head circumference was in the 50th percentile on the growth curve. He also showed a broad and protruding forehead and high hairline. Blood laboratory tests showed severe azotemia; emergent hemodialysis was needed. Abdominal ultrasonography revealed bilateral renal dysplasia with multiple cysts and diffuse bladder wall thickening. A posterior urethral valve was suggested based on vesicoureterography and abdominal magnetic resonance findings. Results of a colon study to rule out congenital megacolon did not reveal any specific findings. The conventional karyotype of the patient was 46, XY. Array comparative genomic hybridization study revealed a chromosome 5q35 microdeletion including the NSD1 gene, based on which SS was diagnosed. We describe a case of SS presenting with end stage renal disease due to posterior urethral valve. The typical somatic overgrowth of SS in the postnatal period was not observed due to chronic renal failure that started in the neonatal period.

Chromophobe renal cell carcinoma with neuroendocrine differentiation/morphology: A clinicopathological and genetic study of three cases

Chromophobe renal cell carcinoma (ChRCC) with neuroendocrine differentiation/morphology (NED/NEM) is exceedingly rare. We present three cases of ChRCC with NED/NEM, two of which showed positivity for neuroendocrine markers on immunohistochemical analysis. Patients ranged in age from 49 to 79 years (mean: 64.3 years). One of the three patients died of metastatic disease to multiple organs. Of the remaining two patients, one is currently alive without disease and the other is alive with disease. Histologically, all three tumors were composed of conventional ChRCC and NEM showed glandular and rosette formation. Immunohistochemically, tumor cells were positive for CK7, KAI1, E-cadherin, and c-kit in both ChRCC and neuroendocrine areas in three cases. CD56 and synaptophysin immunoreactivity were detected in two cases; in only the neuroendocrine area in one case and in both components in the other. Neuroendocrine granules were ultrastructurally observed at both neuroendocrine and conventional areas of ChRCC. Array comparative genomic hybridization (CGH) study indicated losses of chromosomes 1, 2, 6, 10, 17, 21, and Y in both conventional ChRCC and NED in one case. In addition, losses of chromosomes 1, 2, 4, 6, 9, 10, 13, 16p, 17, and 21 were observed in both components of the remaining one tumor. Furthermore, loss of chromosome 5 was identified only in the neuroendocrine area in this case. We concluded that the neuroendocrine area may reflect dedifferentiation within ChRCC. It is possible that losses of chromosomes 4, 5, and 16p may be involved in the neuroendocrine differentiation or progression of ChRCC.

Recurrent chromosomal aberrations in intravenous leiomyomatosis of the uterus: high-resolution array comparative genomic hybridization study.

Uterine intravenous leiomyomatosis (IVL) is a distinct smooth muscle neoplasm with a potential of clinical aggressiveness due to its ability to extend into intrauterine and extrauterine vasculature. In this study, chromosomal alterations analyzed by oligonucleotide array comparative genomic hybridization were performed in 9 cases of IVL. The analysis was informative in all cases with multiple copy number losses and/or gains observed in each tumor. The most frequent recurrent loss of 22q12.3-q13.1 was observed in 6 tumors (66.7%), followed by losses of 22q11.23-q13.31, 1p36.13-p33, 2p25.3-p23.3, and 2q24.2-q32.2 and gains of 6p22.2, 2q37.3 and 10q22.2-q22.3, in decreasing order of frequency. Copy number variants were identified at 14q11.2, 15q11.1-q11.2, and 15q26.2. Genes mapping to the regions of loss include CHEK2, EWS, NF2, PDGFB, and MAP3K7IP1 on chromosome 22q, HEI10 on chromosome 14q, and succinate dehydrogenase subunit B, E2F2, ARID1A KPNA6, EIF3S2 , PTCH2, and PIK3R3 on chromosome 1p. Regional losses on chromosomes 22q and 1p and gains on chromosomes 12q showed overlaps with those previously observed in uterine leiomyosarcomas. In addition, presence of multiple chromosomal aberrations implies a higher level of genetic instability. Follow-up polymerase chain reaction (PCR) sequencing analysis of MED12 gene revealed absence of G> A transition at nucleotides c.130 or c.131 in all 9 cases, a frequent mutation found in uterine leiomyoma and its variants. In conclusion, this is the first report of high-resolution, genome-wide investigation of IVL by oligonucleotide array comparative genomic hybridization. The presence of high frequencies of recurrent regional loss involving several chromosomes is an important finding and likely related to the pathogenesis of the disease.

Intracystic papillary carcinoma of breast: interrelationship with in situ and invasive carcinoma and a proposal of pathogenesis: array comparative genomic hybridization study of 14 cases

Classifying intracystic papillary carcinoma under invasive or in situ ductal carcinoma is still a matter of debate. The purpose of this study was to explore the genomic relationship of this tumor to its concurrent invasive ductal carcinoma and ductal carcinoma in situ using array comparative genomic hybridization. Intracystic papillary carcinoma cases were classified into three categories: pure, with concurrent ductal carcinoma in situ or with concurrent invasive ductal carcinoma. Each component was dissected using laser capture microdissection. DNA was extracted and array comparative genomic hybridization was performed. The test of difference in copy number changes among the three tumors was carried out using CGHMultiArray. Intracystic papillary carcinoma clustered with four of five concurrent ductal carcinoma in situ cases and with two of two invasive ductal carcinoma cases. Intracystic papillary carcinoma showed the highest proportions of genome copy number aberration, followed by ductal carcinoma in situ, and then by invasive ductal carcinoma (P=0.06). Comparing intracystic papillary carcinoma with invasive ductal carcinoma vs without invasive ductal carcinoma, the former had 11q22.1–23.3 loss (P=0.031) and chr5 gain (P=0.085), and was enriched with matrix metalloproteinase genes. Comparing intracystic papillary carcinoma with ductal carcinoma in situ vs without ductal carcinoma in situ, the former had gain in 5q35.3 (P=0.041), 8q24.3 (P=0.041) and 21q13.2 to 21q13.31 (P=0.011). Comparing intracystic papillary carcinoma with ductal carcinoma in situ, the latter acquired a group of genes involved in cell adhesion and motility, whereas intracystic papillary carcinoma differentially expressed genes that are involved in papillary carcinomas of other organs (thyroid and kidney). We conclude that the overall molecular change in intracystic papillary carcinoma is closer to ductal carcinoma in situ than to invasive ductal carcinoma, which may explain the indolent behavior of this tumor. We offer herein a proposal of intracystic papillary carcinoma pathogenesis through its relation to invasive ductal carcinoma and ductal carcinoma in situ.

Recurrent structural malformations identified among Mowat–Wilson syndrome fetuses

What's already known about this topic? Mowat–Wilson syndrome (MWS) (OMIM#235730) is a genetic disease caused by mutations or deletions of the ZEB2 gene and characterized by typical facial dysmorphism. It is rarely diagnosed prenatally.What does this study add? We report and review the ultrasound features of MWS fetuses identified by array comparative genomic hybridization study. The MWS should be considered in fetuses with increased nuchal translucency, ACC, hypospadias, and congenital heart defect. The prevalence of MWS in Hong Kong is reported.

Abstract P3-04-07: Functional BRCA-pathway defects in sporadic breast cancers rarely show a significant reduction in BRCA-pathway proteins

Abstract Background: Array comparative genomic hybridization (aCGH) studies have revealed that a subgroup of sporadic breast cancers have a similar genetic landscape as BRCA-mutation carriers, characterized by widespread large-size deletions across the genome. We have previously shown that a functional assay of homologous recombination, using radiation-induced RAD51 foci, correlates closely with the aCGH findings. The prevalence of functional defects in the BRCA-pathway is estimated to be ∼25%, and present in all subtypes of breast cancer regardless of estrogen receptor or HER2 status. Here we sought to investigate the candidate genes and proteins that may drive this BRCA-like phenotype in sporadic breast cancers. Methods: Fresh human breast cancer samples were obtained to query the BRCA-pathway, under an IRB-approved study. As previously, the ability to assemble RAD51 foci was the defining functional assay. A 4-day growth assay assessed whether BRCA-pathway defective tumors were sensitive to crosslinking agents. Nanostring-based gene expression analysis was performed to interrogate the transcript level of key players in the BRCA-pathway. Massively parallel whole exome sequencing analysis is in progress, since tumor and normal germline DNA was obtained for all these samples. Results: Previously we found the incidence of BRCA-pathway defective tumors was 18/71 (25.3%). The current analysis relates to samples obtained in the last 3 years, including 42 patients in the initial series and 18 additional samples, for which DNA, RNA and immunohistochemistry analyses were successfully performed. Known mutation carriers were excluded from the study. RAD51 focus induction revealed a bimodal distribution: no induction (0.75-1.25) or an induction by > 75%. 17 tumors were repair-defective and 43 were proficient, consistent with our previous analysis. 16 of the 17 repair-defective tumors had the lowest IC50 to mitomycin C, further supporting the functional phenotype classification. Since many of the BRCA-pathway proteins are cell cycle regulated, we determined that cell proliferation markers (e.g. Ki67, PCNA) were not different between the repair defective and proficient tumors. Nanostring gene expression analysis revealed no difference in the expression levels of BRCA1, BRCA2 and RAD51. Since BRCA1 protein recruitment was defective in the majority of repair-defective tumors, we also analysed RNF8, RNF168, RAP80, ABRAXAS, MERIT40 or BRCC36 and found no abnormality. The methylation status of the BRCA1 promoter is being tested for all cases, however our results suggest that reduced mRNA and protein levels of BRCA1, BRCA2 or RAD51 are not a major cause of BRCA-pathway defects. Conclusions: The cause of BRCA-pathway defects in sporadic breast cancer cannot be explained by a reduction in mRNA or protein for members of the BRCA-pathway in the vast majority of cases. Methylation of BRCA1 to produce a reduction in mRNA sufficient to cause a repair defective phenotype is likely to be relatively uncommon. Whole exome analysis may reveal a common explanation for this biologically and clinically relevant phenotype. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-04-07.

Abstract 2998: Identification of AURKA- and PTPRJ-interacting human colorectal cancer susceptibility alleles.

Abstract Colorectal cancer (CRC) causes nearly 50,000 deaths in the United States each year and therefore represents the third leading cause of cancer-related deaths. The identification of susceptibility variants that interact to modulate risk for sporadic CRC would have immense value as a tool to identify individuals with increased genetic predisposition to disease. Polymorphisms in Aurora Kinase A (AURKA) and Protein Tyrosine Phosphatase Receptor Type J (PTPRJ) have been shown to increase CRC risk in human case-control studies. Crosses between mice resistant and susceptible to chemically induced skin cancer have led to the identification of two loci, Skts1 and Skts5, each of which statistically interacts with the Aurka locus to promote skin cancer in a murine model. Using a similar approach, the mouse Ptprj locus has been shown to interact synergistically with the loci Scc5 and Scc13 to increase colon cancer susceptibility. Array comparative genomic hybridization studies in human colorectal tumor DNAs have shown high frequencies of genomic copy number gains or losses at the human orthologs of Skts1, Skts5, Scc5, and Scc13. Our goal is to identify sporadic CRC risk variants and to characterize genetic interactions at the AURKA- and PTPRJ-interacting loci. We hypothesize that the human orthologs of these mouse CRC susceptibility loci will interact genetically with AURKA and PTPRJ to influence CRC risk. By generating normal colon transcriptome data from CRC-susceptible and CRC–resistant mouse strains using RNA-Seq, we prioritized candidate genes on the basis of differential expression, differential splicing or potentially functional polymorphisms. Tagging single nucleotide polymorphisms (tagSNPs) for the human orthologs of these genes were tested for allele-specific imbalance by Sequenom MassARRAY quantitative genotyping of 194 paired human colorectal tumor/non-tumor DNA samples. TagSNPs exhibiting point-wise p-values < 0.1 were further tested in a replication sample set of 296 paired colorectal tumor/non-tumor DNA samples. Among ∼900 tagSNPs tested, 75 polymorphisms reproducibly showed evidence of allele-specific imbalance (point-wise p-values < 0.05). Our data indicate that allele-specific imbalance occurs in candidate genes (e.g. KLF13, HDAC9, FBN2, SNX10 and PRDM5) whose murine orthologs map to cancer susceptibility loci. Our next step is to explore expression patterns of these candidate genes in human colorectal tumor and non-tumor tissues and to investigate the effects of gene silencing or overexpression on critical cancer phenotypes such as proliferation, apoptosis, and migration in mouse and human colon cancer cell lines. Additionally, an ongoing human case-control association study of 6000 colorectal cancer cases and 6000 controls is expected to yield results shortly, which will be presented at the 2013 AACR meeting. Citation Format: Madelyn M. Gerber, Nathan P. Schulz, Mehmet Deveci, Heather L. Hampel, Umit V. Catalyurek, Albert de la Chapelle, Amanda Ewart Toland. Identification of AURKA- and PTPRJ-interacting human colorectal cancer susceptibility alleles. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2998. doi:10.1158/1538-7445.AM2013-2998

Functional genomic analysis of chromosomal aberrations in a compendium of 8000 cancer genomes

A large database of copy number profiles from cancer genomes can facilitate the identification of recurrent chromosomal alterations that often contain key cancer-related genes. It can also be used to explore low-prevalence genomic events such as chromothripsis. In this study, we report an analysis of 8227 human cancer copy number profiles obtained from 107 array comparative genomic hybridization (CGH) studies. Our analysis reveals similarity of chromosomal arm-level alterations among developmentally related tumor types as well as a number of co-occurring pairs of arm-level alterations. Recurrent (“pan-lineage”) focal alterations identified across diverse tumor types show an enrichment of known cancer-related genes and genes with relevant functions in cancer-associated phenotypes (e.g., kinase and cell cycle). Tumor type-specific (“lineage-restricted”) alterations and their enriched functional categories were also identified. Furthermore, we developed an algorithm for detecting regions in which the copy number oscillates rapidly between fixed levels, indicative of chromothripsis. We observed these massive genomic rearrangements in 1%–2% of the samples with variable tumor type-specific incidence rates. Taken together, our comprehensive view of copy number alterations provides a framework for understanding the functional significance of various genomic alterations in cancer genomes.

Homozygous deletions of cadherin genes in chondrosarcoma—an array comparative genomic hybridization study

Chondrosarcoma is a malignant bone tumor that is often resistant to chemotherapy and radiotherapy. We applied high resolution oligonucleotide array comparative genomic hybridization to 46 tumor specimens from 44 patients with chondrosarcoma and identified several genes with potential importance for the development of chondrosarcoma. Several homozygous deletions were detected. The tumor suppressor genes CDKN2A and MTAP were each homozygously deleted in four of the cases, and the RB1 gene was homozygously deleted in one. Two homozygous deletions of MTAP did not affect CDKN2A. Deletions were also found to affect genes of the cadherin family, including CDH4 and CDH7, each of which had a targeted homozygous loss in one case, and CDH19, which had a targeted homozygous loss in two cases. Loss of the EXT1 and EXT2 genes was uncommon; EXT1 was homozygously deleted in none and EXT2 in two of the cases, and large heterozygous losses including EXT1 and/or EXT2 were seen in three cases. Targeted gains and amplifications affected the MYC, E2F3, CDK6, PDGFRA, KIT, and PDGFD genes in one case each. The data indicate that chondrosarcomas develop through a combination of genomic imbalances that often affect the RB1 signaling pathway. The inactivation of cadherin genes may also be critical in the pathogenesis of the tumor.

Overexpression of PAFAH1B1 is associated with tumor metastasis and poor survival in non-small cell lung cancer

Our previous array-comparative genomic hybridization study showed that PAFAH1B1 gene locus was amplified in lung cancer patients, suggesting that PAFAH1B1 is a potential oncogene in lung cancer. Here, we investigate the oncogenic mechanisms of PAFAH1B1 in lung cancer. PAFAH1B1 was characterized in cell and animal models of lung cancer by in vitro migration and invasion assays and in vivo metastasis studies. The mRNA and protein expression levels of PAFAH1B1 were further determined and the prognostic effects of PAFAH1B1 overexpression in lung cancer patients were analyzed. Overexpression of PAFAH1B1 enhanced migration and invasion in lung cancer cells, whereas knockdown of PAFAH1B1 decreased cell migration and invasion, and disrupted cell microtubule organization and pericellular poly-fibronectin assemblies. In vivo tumor metastasis assay confirmed that PAFAH1B1 knockdown in lung cancer cells markedly reduced their metastasis capabilities in animals. The frequencies of overexpressed PAFAH1B1 mRNA and protein were 62.4% (63/101) and 57.4% (58/101) in lung cancer patients, respectively. The clinical correlation results showed that overexpression of PAFAH1B1 was significantly associated with late stage (mRNA: P=0.008, protein: P=0.008) and poor survival in lung adenocarcinoma (P=0.020) and male patients (P=0.049). Our results provide the first evidence that PAFAH1B1 overexpression contributes to lung tumorigenesis and poor prognosis. These effects are partly mediated through disruption of microtubule network and pericellular poly-fibronectin assembly to promote migration and invasiveness of lung cancer cells.

Advances in Whole-Genome Genetic Testing: From Chromosomes to Microarrays

Whole-genome genetic diagnostics has changed the clinical landscape of pediatric and adolescent medicine. In this article, we review the history of clinical cytogenetics as the field has progressed from studying chromosomes prepared from cells squashed between 2 slides to the high-resolution, whole-genome technology in use today, which has allowed for the identification of numerous previously unrecognized microdeletion and microduplication syndromes. Types of arrays and the data they collect are addressed, as are the types of results that array comparative genomic hybridization studies may generate. Throughout the review, we present case stories to illustrate the familiar (Down syndrome) and the new (a never-before reported microdeletion on the long arm of chromosome 12).

Molecular karyotyping by array CGH in a Russian cohort of children with intellectual disability, autism, epilepsy and congenital anomalies

BackgroundArray comparative genomic hybridization (CGH) has been repeatedly shown to be a successful tool for the identification of genomic variations in a clinical population. During the last decade, the implementation of array CGH has resulted in the identification of new causative submicroscopic chromosome imbalances and copy number variations (CNVs) in neuropsychiatric (neurobehavioral) diseases. Currently, array-CGH-based technologies have become an integral part of molecular diagnosis and research in individuals with neuropsychiatric disorders and children with intellectual disability (mental retardation) and congenital anomalies. Here, we introduce the Russian cohort of children with intellectual disability, autism, epilepsy and congenital anomalies analyzed by BAC array CGH and a novel bioinformatic strategy.ResultsAmong 54 individuals highly selected according to clinical criteria and molecular and cytogenetic data (from 2426 patients evaluated cytogenetically and molecularly between November 2007 and May 2012), chromosomal imbalances were detected in 26 individuals (48%). In two patients (4%), a previously undescribed condition was observed. The latter has been designated as meiotic (constitutional) genomic instability resulted in multiple submicroscopic rearrangements (including CNVs). Using bioinformatic strategy, we were able to identify clinically relevant CNVs in 15 individuals (28%). Selected cases were confirmed by molecular cytogenetic and molecular genetic methods. Eight out of 26 chromosomal imbalances (31%) have not been previously reported. Among them, three cases were co-occurrence of subtle chromosome 9 and 21 deletions.ConclusionsWe conducted an array CGH study of Russian patients suffering from intellectual disability, autism, epilepsy and congenital anomalies. In total, phenotypic manifestations of clinically relevant genomic variations were found to result from genomic rearrangements affecting 1247 disease-causing and pathway-involved genes. Obviously, a significantly lesser part of them are true candidates for intellectual disability, autism or epilepsy. The success of our preliminary array CGH and bioinformatic study allows us to expand the cohort. According to the available literature, this is the first comprehensive array CGH evaluation of a Russian cohort of children with neuropsychiatric disorders and congenital anomalies.