Xp22.33p22.13 Duplication in a Male Patient Carrying a Recombinant X Chromosome Derived from an Inherited Intrachromosomal Insertion.

Intrachromosomal insertions are complex structural rearrangements that are challenging to interpret using classical cytogenetic methods. We report a male patient carrying a recombinant X chromosome derived from a maternally inherited intrachromosomal insertion. The patient exhibited developmental delay, intellectual disability, behavioral disorder, and dysmorphic facial features. To accurately identify the rearrangements in the abnormal X chromosome, additional cytogenetic studies were conducted, including fluorescence in situ hybridization (FISH), multicolor-banding FISH, and array comparative genomic hybridization. The results showed a recombinant X chromosome, resulting in a 13.05 Mb interstitial duplication of segment Xp22.33-Xp22.13, which was inserted at cytoband Xq26.1. The duplicated region encompasses 99 genes, some of which are associated with the patient's clinical manifestations. We propose that the combined effects of the Xp-duplicated genes may contribute to the patient's phenotype.

Correction to: Use of multicolor fluorescence in situ hybridization to detect deletions in clinical tissue sections.

Figure 2 is incorrect in the original version of this article. The correct figure 2 is provided below.

Use of multicolor fluorescence in situ hybridization to detect deletions in clinical tissue sections.

A variety of laboratory methods are available for the detection of deletions of tumor suppressor genes and losses of their proteins. The clinical utility of fluorescence in situ hybridization (FISH) for the identification of deletions of tumor suppressor genes has previously been limited by difficulties in the interpretation of FISH signal patterns. The first deletion FISH assays using formalin-fixed paraffin-embedded tissue sections had to deal with a significant background level of signal losses affecting nuclei that are truncated by the cutting process of slide preparation. Recently, more efficient probe designs, incorporating probes adjacent to the tumor suppressor gene of interest, have increased the accuracy of FISH deletion assays so that true chromosomal deletions can be readily distinguished from the false signal losses caused by sectioning artifacts. This mini-review discusses the importance of recurrent tumor suppressor gene deletions in human cancer and reviews the common FISH methods being used to detect the genomic losses encountered in clinical specimens. The use of new probe designs to recognize truncation artifacts is illustrated with a four-color PTEN FISH set optimized for prostate cancer tissue sections. Data are presented to show that when section thickness is reduced, the frequency of signal truncation losses is increased. We also provide some general guidelines that will help pathologists and cytogeneticists run routine deletion FISH assays and recognize sectioning artifacts. Finally, we summarize how recently developed sequence-based approaches are being used to identify recurrent deletions using small DNA samples from tumors.

Assessing the cost of implementing the 2011 Society of Obstetricians and Gynecologists of Canada and Canadian College of Medical Genetics practice guidelines on the detection of fetal aneuploidies.

BACKGROUND: The Society of Obstetricians and Gynecologists of Canada and the Canadian College of Medical Genetics published guidelines, in 2011, recommending replacement of karyotype with quantitative fluorescent polymerase chain reaction when prenatal testing is performed because of an increased risk of a common aneuploidy. STUDY OBJECTIVE: This study's objective is to perform a cost analysis following the implementation of quantitative fluorescent polymerase chain reaction as a stand-alone test. RESULTS: A total of 658 samples were received between 1 April 2014 and 31 August 2015: 576 amniocentesis samples and 82 chorionic villi sampling. A chromosome abnormality was identified in 14% (93/658) of the prenatal samples tested. The implementation of the 2011 Society of Obstetricians and Gynecologists of Canada and the Canadian College of Medical Genetics guidelines in Edmonton and Northern Alberta resulted in a cost savings of $46 295.80. The replacement of karyotype with chromosomal microarray for some indications would be associated with additional costs. CONCLUSION: The implementation of new test methods may provide cost savings or added costs. Cost analysis is important to consider during the implementation of new guidelines or technologies. © 2017 John Wiley & Sons, Ltd.

PTEN losses exhibit heterogeneity in multifocal prostatic adenocarcinoma and are associated with higher Gleason grade.

Prostatic adenocarcinoma is an epithelial malignancy characterized by marked histological heterogeneity. It most often has a multifocal distribution within the gland, and different Gleason grades may be present within different foci. Data from our group and others have shown that the genomic deletion of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor gene and the disruption of the ETS gene family have a central role in prostate cancer and are likely to be associated with Gleason grade. In this study, prostate cancer samples were systematically analyzed to determine whether there was concordance between PTEN losses and TMPRSS2-ERG fusion rearrangements, within or between foci in multifocal disease, using well-annotated tissue microarrays (TMAs) consisting of 724 cores derived from 142 radical prostatectomy specimens. Three-color fluorescence in situ hybridization analysis of both the PTEN deletion and the TMPRSS2-ERG fusion was used to precisely map genetic heterogeneity, both within and between tumor foci represented on the TMA. PTEN deletion was observed in 56 of 134 (42%) patients (hemizygous=42 and homozygous=14). TMPRSS2-ERG fusion was observed in 63 of 139 (45%) patients. When analyzed by Gleason pattern for a given TMA core, PTEN deletions were significantly associated with Gleason grades 4 or 5 over grade 3 (P<0.001). Although TMPRSS2-ERG fusions showed a strong relationship with PTEN deletions (P=0.007), TMPRSS2-ERG fusions did not show correlation with Gleason grade. The pattern of genetic heterogeneity of PTEN deletion was more diverse than that observed for TMPRSS2-ERG fusions in multifocal disease. However, the marked interfocal discordance for both TMPRSS2-ERG fusions and PTEN deletions was consistent with the concept that multiple foci of prostate cancer arise independently within the same prostate, and that individual tumor foci can have distinct patterns of genetic rearrangements.

PTEN genomic deletions that characterize aggressive prostate cancer originate close to segmental duplications.

Deletion of PTEN at 10q23.3 occurs in ∼40% of human prostate cancers and is associated with aggressive metastatic potential, poor prognosis, and androgen-independence. This high frequency of recurrent PTEN deletions in prostate cancer suggests there may be unusual genomic features close to this locus that facilitate DNA alteration at 10q23.3. To explore possible mechanisms for deletions in the PTEN region, a meta-analysis of 311 published human genome array datasets was conducted and determined that the minimal prostate cancer-associated deletion at 10q23.3 corresponds to ∼2.06 MB region flanked by BMPR1A and FAS. On a separate cohort comprising an additional 330 tumors, four-color fluorescence in situ hybridization analysis using probes for BMPR1A, FAS, cen(10), and PTEN showed that 132 of 330 (40%) tumors had PTEN loss, 50 (15%) of which were homozygous losses (comprising in total 100 deletion events). Breakpoints between PTEN and BMPR1A or FAS were subsequently mapped in 100 homozygous and 82 hemizygous PTEN losses, revealing that 125/182 PTEN microdeletions occurred within the 940 kB interval between BMPR1A and PTEN. Furthermore, this breakpoint interval coincides with a repeat-rich region of 414 kB containing the SD17 and SD18 segmental duplications, which contain at least 13 homologous inverted repeat sequences. Together, these data suggest that a strong selective growth advantage for loss of PTEN and upregulation of PI3K/AKT, combined with the close proximity of PTEN to a large unstable segment of repeated DNA comprising SD17 and SD18, can lead to recurrent microdeletions of the PTEN gene in prostate cancer. © 2011 Wiley Periodicals, Inc.

Copy number alterations of c-MYC and PTEN are prognostic factors for relapse after prostate cancer radiotherapy.

Despite the use of PSA, Gleason score, and T-category as prognosticators in intermediate-risk prostate cancer, 20-40% of patients will fail local therapy. In order to optimize treatment approaches for intermediate-risk patients, additional genetic prognosticators are needed. Previous reports using array comparative genomic hybridization (aCGH) in radical prostatectomy cohorts suggested a combination of allelic loss of the PTEN gene on 10q and allelic gain of the c-MYC gene on 8q were associated with metastatic disease. We tested whether copy number alterations (CNAs) in PTEN (allelic loss) and c-MYC (allelic gain) were associated with biochemical relapse following modern-era, image-guided radiotherapy (mean dose 76.4 Gy). We used aCGH analyses validated by fluorescence in-situ hybridization (FISH) of DNA was derived from frozen, pre-treatment biopsies in 126 intermediate-risk prostate cancer patients. Patients whose tumors had CNAs in both PTEN and c-MYC had significantly increased genetic instability (percent genome alteration; PGA) compared to tumors with normal PTEN and c-MYC status (p < 0.0001). We demonstrate that c-MYC gain alone, or combined c-MYC gain and PTEN loss, were increasingly prognostic for relapse on multivariable analyses (hazard ratios (HR) of 2.58/p = 0.005 and 3.21/p = 0.0004; respectively). Triaging patients by the use of CNAs within pre-treatment biopsies may allow for better use of systemic therapies to target sub-clinical metastases or locally recurrent disease and improve clinical outcomes.

Interactions and relationships of PTEN, ERG, SPINK1 and AR in castration-resistant prostate cancer.

AIMS: Recently, ETS-related gene (ERG) gene rearrangements, phosphatase tensin homologue (PTEN) deletions and serine protease inhibitor Kazal type 1 (SPINK1) overexpression were investigated as potential markers for molecularly subtyping prostate cancer (PCA). However, their incidence and co-association in castration-resistant PCA (CRPC) has not been characterized fully. METHODS AND RESULTS: A cohort of 59 CRPC patients was investigated for ERG rearrangements, PTEN deletions and androgen receptor (AR) amplification by fluorescence in-situ hybridization. SPINK1 overexpression was assessed by immunohistochemistry. ERG rearrangements and PTEN deletions were detected in 22 of 53 (41.5%) and 35 of 55 (63.6%) of cases, with 15 of 22 (68.1%) of ERG rearrangements occurring through deletions. SPINK1 overexpression occurred in three of 51 (5.8%) of cases exclusively in non-ERG rearranged and AR amplification was detected in 12 of 49 (24.4%) of cases. Only PTEN deletions showed intrafocal heterogeneity occurring in nine of 35 (25.7%) of cases. PTEN deletions were significantly associated with each of ERG rearrangements occurring by deletions only (P = 0.001), AR amplification (P = 0.002) and SPINK1 overexpression (P = 0.002). None of the SPINK1 overexpressing tumours showed AR amplification (P = 0.005) and all occurred in PTEN deleted foci (P = 0.002). CONCLUSION: Te study supports the heterogeneous nature of CRPC and confirms a significant association between PTEN, ERG, AR and SPINK1. Characterizing combined markers will aid in defining PCA subgroups relevant to prognosis contributing to the design of improved therapeutic approaches for CRPC.

PTEN deletion and heme oxygenase-1 overexpression cooperate in prostate cancer progression and are associated with adverse clinical outcome.

Overexpression of the pro-survival protein heme oxygenase-1 (HO-1) and loss of the pro-apoptotic tumour suppressor PTEN are common events in prostate cancer (PCA). We assessed the occurrence of both HO-1 expression and PTEN deletion in two cohorts of men with localized and castration-resistant prostate cancer (CRPC). The phenotypic cooperation of these markers was examined in preclinical and clinical models. Overall, there was a statistically significant difference in HO-1 epithelial expression between benign, high-grade prostatic intraepithelial neoplasia (HGPIN), localized PCA, and CRPC (p < 0.0001). The highest epithelial HO-1 expression was noted in CRPC (2.00 ± 0.89), followed by benign prostate tissue (1.49 ± 1.03) (p = 0.0003), localized PCA (1.20 ± 0.95), and HGPIN (1.07 ± 0.87) (p < 0.0001). However, the difference between HGPIN and PCA was not statistically significant (p = 0.21). PTEN deletions were observed in 35/55 (63.6%) versus 68/183 (37.1%) cases of CRPC and localized PCA, respectively. Although neither HO-1 overexpression nor PTEN deletions alone in localized PCA showed a statistically significant association with PSA relapse, the combined status of both markers correlated with disease progression (log-rank test, p = 0.01). In a preclinical model, inhibition of HO-1 by shRNA in PTEN-deficient PC3M cell line and their matched cells where PTEN is restored strongly reduced cell growth and invasion in vitro and inhibited tumour growth and lung metastasis formation in mice compared to cells where only HO-1 is inhibited or PTEN is restored. In summary, we provide clinical and experimental evidence for cooperation between epithelial HO-1 expression and PTEN deletions in relation to the PCA patient's outcome. These findings could potentially lead to the discovery of novel therapeutic modalities for advanced PCA.

Identification of interactive networks of gene expression associated with osteosarcoma oncogenesis by integrated molecular profiling.

Altered gene expression in tumors can be caused by copy number alterations to DNA or mutation affecting coding or regulatory regions of genes. However, epigenetic events may also influence gene expression. Malignant cells can show major disruptions in DNA methylation profiles, which are manifested as aberrant hypermethylation or as hypomethylation of gene promoters, as well as global genomic hypomethylation. In this study we performed integrative whole-genome analysis of DNA copy number, promoter methylation and gene expression using 10 osteosarcomas. We identified significant changes including: hypomethylation, gain, and overexpression of histone cluster 2 genes at chromosome 1q21.1-q21.3; loss of chromosome 8p21.2-p21.3 and underexpression of DOCK5 and TNFRSF10A/D genes; and amplification-related overexpression of RUNX2 at chromosome 6p12.3-p21.1. Amplification and overexpression of RUNX2 could disrupt G2/M cell cycle checkpoints, and downstream osteosarcoma-specific changes, such as failure of bone differentiation and genomic polyploidization. Failure of DOCK5-signaling, together with p53 and TNFRSF10A/D-related cell cycle and death pathways, may play a critical role in abrogating apoptosis. Our analyses show that the RUNX2 interactome may be constitutively activated in osteosarcoma, and that the downstream intracellular pathways are strongly associated with the regulation of osteoblast differentiation and control of cell cycle and apoptosis in osteosarcoma.

PTEN genomic deletion is an early event associated with ERG gene rearrangements in prostate cancer.

OBJECTIVE: To investigate the interaction between, and significance of, ERG gene rearrangements and PTEN genomic deletions in relation to the development and progression of prostate cancer (PCA). PATIENTS AND METHODS: We interrogated an initial cohort of 220 men with localized PCA using fluorescence in situ hybridization for ERG rearrangements and PTEN genomic deletions. RESULTS: The incidences of ERG rearrangements and PTEN deletions in PCA were significantly higher than in high-grade prostatic intra-epithelial neoplasia (HGPIN) and benign prostate tissue (P < 0.001). ERG rearrangements and PTEN deletions were detected in 41.9 and 42.6% of patients' tumours, respectively. ERG rearrangements were never detected in benign prostate tissue, while PTEN aberrations were present at a basal level of 4.6%. PTEN hemizygous deletions showed higher frequency than homozygous deletions within each diagnostic category from benign prostate tissue to HGPIN and PCA (P ≤ 0.001). Furthermore, in 29 patients where all three tissues were available, PTEN genomic aberrations in PCA were significantly different from those in benign tissue (P = 0.005) and HGPIN (P = 0.02), reflecting the accumulation of genomic aberrations in the early stages of disease progression. Within this cohort, 71.4% of homozygous and 44.2% of hemizygous PTEN deletions occurred simultaneously with ERG rearrangements (P ≈ 0). Stratified according to Gleason score (GS), hemizygous PTEN deletions across various GS groups were observed at a higher frequency than homozygous deletions. However, PTEN homozygous deletions showed positive trends with higher GS, increasing in poorly differentiated PCA (GS 8-10) in comparison to moderately and well differentiated tumours (GS 6 and 7). CONCLUSION: We show significant association between ERG gene rearrangements and PTEN genomic aberrations in subset of PCA. Our analysis also provides further support for the observation that homozygous PTEN deletions can occur within the subset of HGPIN lesions, and shows accumulating genetic aberrations with disease progression, evidenced by higher detection in PCA than in HGPIN and more PTEN homozygous deletions in GS 8-10 than in 6-7.

Development of metastatic and non-metastatic tumor lines from a patient's prostate cancer specimen-identification of a small subpopulation with metastatic potential in the primary tumor.

BACKGROUND: Metastasis is the major cause of prostate cancer deaths. Tumor heterogeneity in both primary and metastatic prostate cancers is a major hurdle in elucidating the mechanisms of metastasis in this disease. To circumvent this obstacle and improve our understanding of prostate cancer metastasis, we developed multiple tumor tissue lines from one patient's primary prostate cancer specimen to examine differences in metastatic ability. METHODS: Pieces of tissue from different foci of a patient's primary prostate tumor were grafted into subrenal capsules of NOD-SCID mice and transplantable tumor sublines were established by serial passage. The metastatic ability of each subline was tested via orthotopic grafting into mice. Chromosomal alterations exclusively presented in a metastatic subline were examined by SKY and investigated for their presence in parental tissues by fluorescence in situ hybridization. RESULTS: Three transplantable sublines were developed, resembling the primary tumor histologically, exhibiting poor differentiation, and different growth rates. Importantly, the LTL-220N and LTL-221N sublines were non-metastatic, whereas the LTL-220M subline was spontaneously metastatic in vivo. SKY analysis showed limited but unique chromosomal alterations in each subline. Some chromosomal alterations, exclusively present in the metastatic LTL-220M subline, were also observed in a small portion of the parental cancer tissues. CONCLUSION: The results indicate that in primary prostate tumors metastatic potential can be confined to a minority of cancer cells. Subrenal capsule xenograft methodology can be used to dissect heterogeneous cancer cells in a patient's primary tumor and sublines derived from such cells provide valuable tools for investigating mechanisms underlying prostate cancer metastasis.

A rare case of trisomy 15pter-q21.2 due to a de novo marker chromosome.

Supernumerary marker chromosomes (sSMC) may or may not be associated with an abnormal phenotype, depending on the presence of euchromatin, on their chromosomal origin and whether they are inherited. Over 80% of sSMCs are derived from acrocentric chromosomes and half of them include the short arm of chromosome 15. Generally, they appear as bisatellited isodicentric marker chromosomes, most of them are symmetric. These chromosomes are normally originated de novo and are associated with mild to severe intellectual disability but not with physical abnormalities. We report on a patient with an SMC studied using classical and molecular cytogenetic procedures (G and C banding, NOR staining, painting and centromeric fluorescent in situ hybridization (FISH), BAC-FISH, and SKY). The MLPA technique and DNA polymorphic markers were used in order to identify its parental origin. The marker chromosome, monosatellited and monocentric, was found to be derived from a maternal chromosome 15 and was defined as 15pter-q21.2. This is the report of the largest de novo monosatellited 15q marker chromosome ever published presenting detailed cytogenetic and clinical data. It was associated with a phenotype including cardiac defect, absence of septum pellucidum, and dysplasia of the corpus callosum.

PTEN genomic deletion is associated with p-Akt and AR signalling in poorer outcome, hormone refractory prostate cancer.

PTEN haploinsufficiency is common in hormone-sensitive prostate cancer, though the incidence of genomic deletion and its downstream effects have not been elucidated in clinical samples of hormone refractory prostate cancer (HRPC). Progression to androgen independence is pivotal in prostate cancer and mediated largely by the androgen receptor (AR). Since this process is distinct from metastatic progression, we examined alterations of the PTEN gene in locally advanced recurrent, non-metastatic human HRPC tissues. Retrospective analyses of PTEN deletion status were correlated with activated downstream phospho-Akt (p-Akt) pathway proteins and with the androgen receptor. The prevalence of PTEN genomic deletions in transurethral resection samples of 59 HRPC patients with known clinical outcome was assessed by four-colour FISH analyses. FISH was performed using six BAC clones spanning both flanking PTEN genomic regions and the PTEN gene locus, and a chromosome 10 centromeric probe. PTEN copy number was also evaluated in a subset of cases using single nucleotide polymorphism (SNP) arrays. In addition, the samples were immunostained with antibodies against p-Akt, p-mTOR, p-70S6, and AR. The PTEN gene was deleted in 77% of cases, with 25% showing homozygous deletions, 18% homozygous and hemizygous deletions, and 34% hemizygous deletions only. In a subset of the study group, SNP array analysis confirmed the FISH findings. PTEN genomic deletion was significantly correlated to the expression of downstream p-Akt (p < 0.0001), AR (p = 0.025), and to cancer-specific mortality (p = 0.039). PTEN deletion is common in HRPC, with bi-allelic loss correlating to disease-specific mortality and associated with Akt and AR deregulation.

High-resolution array CGH identifies novel regions of genomic alteration in intermediate-risk prostate cancer.

Approximately one-third of prostate cancer patients present with intermediate risk disease. Interestingly, while this risk group is clinically well defined, it demonstrates the most significant heterogeneity in PSA-based biochemical outcome. Further, the majority of candidate genes associated with prostate cancer progression have been identified using cell lines, xenograft models, and high-risk androgen-independent or metastatic patient samples. We used a global high-resolution array comparative genomic hybridization (CGH) assay to characterize copy number alterations (CNAs) in intermediate risk prostate cancer. Herein, we show this risk group contains a number of alterations previously associated with high-risk disease: (1) deletions at 21q22.2 (TMPRSS2:ERG), 16q22-24 (containing CDH1), 13q14.2 (RB1), 10q23.31 (PTEN), 8p21 (NKX3.1); and, (2) amplification at 8q21.3-24.3 (containing c-MYC). In addition, we identified six novel microdeletions at high frequency: 1q42.12-q42.3 (33.3%), 5q12.3-13.3 (21%), 20q13.32-13.33 (29.2%), 22q11.21 (25%), 22q12.1 (29.2%), and 22q13.31 (33.3%). Further, we show there is little concordance between CNAs from these clinical samples and those found in commonly used prostate cancer cell models. These unexpected findings suggest that the intermediate-risk category is a crucial cohort warranting further study to determine if a unique molecular fingerprint can predict aggressive versus indolent phenotypes.

Absence of TMPRSS2:ERG fusions and PTEN losses in prostate cancer is associated with a favorable outcome.

TMPRSS2:ERG gene fusions and PTEN deletions are the most common genomic aberrations in prostate cancer. Recent work has suggested that the TMPRSS2:ERG fusion is associated with a more aggressive phenotype. Similarly, PTEN deletion has been associated with biochemical recurrence and lymph node metastasis. To date, there has been no systematic analysis of the combined influence of genomic PTEN deletion with TMPRSS2:ERG gene fusions on clinical parameters of prostate cancer progression. We carried out a retrospective analysis of 125 prostate cancers with known clinical outcome using interphase fluorescence in situ hybridization to detect the relative prevalence of TMPRSS2:ERG rearrangements and/or PTEN genomic deletions. TMPRSS2:ERG rearrangement was found in 60 of 125 (48%) prostate cancers. Duplication of TMPRSS2:ERG fusion was observed in seven (6%) tumors. Gleason grade (P=0.0002)/score (P=0.001), median tumor volume (P=0.0024), preoperative PSA (P=0.001) and perineural invasion (P=0.0304) were significantly associated with biochemical recurrence by univariate analysis with TMPRSS2:ERG approaching significance (P=0.0523). By multivariate analysis, relevant factors associated with recurrence were Gleason scores 7 (P=0.001) and 8-10 (P=0.015), PTEN homozygous deletion (P=0.013) and concurrent TMPRSS2:ERG fusion and PTEN deletion (P=0.036). Kaplan-Meier analysis indicated that the presence of TMPRSS2:ERG fusion was marginally less favorable in comparison to no fusion. Duplication of fusion gene showed worse prognosis. It was possible to determine the relative frequencies of PTEN deletion and/or TMPRSS2:ERG fusions in 82 of 125 prostate cancers. With biochemical recurrence as an endpoint, the genomic biomarkers identified three patient groups: (1) 'poor genomic grade' characterized by both PTEN deletion and TMPRSS2:ERG fusions (23/82, 28%); (2) 'intermediate genomic grade' with either PTEN deletion or TMPRSS2:ERG fusion (35/82, 43%) and (3) 'favorable genomic grade' in which neither rearrangement was present (24/82, 29%). Kaplan-Meier and multivariate analysis indicate that TMPRSS2:ERG fusion and PTEN loss together are a predictor of earlier biochemical recurrence of disease.

Characterization of trisomy 8 in pediatric undifferentiated sarcomas using advanced molecular cytogenetic techniques.

Pediatric undifferentiated soft tissue sarcomas (USTS) are a rare group of neoplasms that are unclassifiable despite the application of immunohistochemical, cytogenetic, and molecular techniques. To date, there is a dearth of studies looking at the cytogenetic and molecular genetic alterations in such tumors. Trisomy 8, a frequent molecular alteration in neoplasia, is seen in several soft tissue sarcomas, including Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET), synovial sarcoma, and leiomyosarcoma. Because USTS share several clinicobiological features with the aforementioned tumors, the occurrence of alterations in chromosome 8 was studied in 11 pediatric USTS using a combination of interphase fluorescence in situ hybridization (FISH), spectral karyotyping (SKY), and genomic profiling with oligonucleotide array comparative genomic hybridization (aCGH). The copy number status of MYC was also assessed on the same tumors using dual-color FISH, with the aim of delineating the degree and intratumoral distribution of MYC amplification in this tumor. A near-uniform presence of an increase in MYC copy number was observed, along with an increase in chromosome 8 copy number in all the tumors. SKY and aCGH analysis of tumors exhibiting trisomy 8 confirmed the numerical imbalances. The occurrence of trisomy 8 in a subset of pediatric USTS confirms a shared genomic alteration with several other soft tissue sarcomas. Further studies are required to determine the clinical implications of such a finding.

Novel 6p rearrangements and recurrent translocation breakpoints in retinoblastoma cell lines identified by spectral karyotyping and mBAND analyses.

Gain of the short arm of chromosome 6, usually through isochromosome 6p formation, is present in approximately 50% of retinoblastoma tumors. The minimal region of gain maps to chromosome band 6p22. Two genes, DEK and E2F3, are implicated as candidate oncogenes. However, chromosomal translocations have been overlooked as a potential mechanism of activation of oncogenes at 6p22 in retinoblastoma. Here, we report combined spectral karyotyping), 4',6-diamidino-2-phenylindole banding, mBAND, and locus-specific fluorescence in situ hybridization analyses of four retinoblastoma cell lines, RB1021, RB247c, RB383, and Y79. In RB1021 and RB247c, 6p undergoes structural rearrangements involving a common translocation breakpoint at 6p22. These data imply that 6p translocations may represent another mechanism of activation of 6p oncogene(s) in a subset of retinoblastomas, besides the copy number increase. In addition to 6p22, other recurrent translocation breakpoints identified in this study are 4p16, 11p15, 17q21.3, and 20q13. Common regions of gain map to chromosomal arms 1q, 2p, 6p, 17q, and 21q.

Identification of cryptic microaberrations in osteosarcoma by high-definition oligonucleotide array comparative genomic hybridization.

Osteosarcoma (OS) is an aggressive bone tumor characterized by complex abnormal karyotypes and a high level of genomic instability. Using high-resolution array comparative genomic hybridization (aCGH), a novel class of localized copy number variations called microaberrations has been detected. These genomic anomalies typically involve DNA imbalances affecting 700 kb to 1 Mb DNA, and are often associated with some type of genetic syndromes. Because the origin of instability in OS is poorly understood, we used aCGH to determine whether microaberrations were a characteristic of four OS cell lines: U-2 OS, HOS, MG-63, and SAOS-2. TP53 is mutated in SAOS-2, a line in which 17 microaberrations were found. In contrast, U-2 OS, which has a wild-type TP53, had only six such anomalies, the lowest incidence. A 500-kb microaberration within a region of gain at 5p15.33 in SAOS-2 was confirmed by fluorescence in situ hybridization. Significantly, this genomic location is close to the TERT gene, a region of gain in all four cell lines. To our knowledge, this is the first systematic analysis of the incidence of microaberrations in OS. The high levels of these anomalies detected suggest that the instability processes in OS that lead to a highly abnormal karyotypes may also be associated with acquisition of genomic microaberrations.

Expression of major vault protein gene in osteosarcoma patients.

Osteosarcoma (OS) is a primary malignant tumor of bone. Despite the successful use of multiple chemotherapeutic agents in the treatment of OS, more than 30% of OS tumors remain resistant to treatment. Elucidation of cellular resistance mechanisms may lead to better treatments for cancer patients. In this study, we used the low-density expression cDNA array, GEArray Q Series Human Cancer Drug Resistance and Metabolism Gene Array to screen genes related to drug resistance in 15 OS tumors. Expression patterns of the MPV gene were validated by real time PCR on 45 OS patient tumor samples and correlated with clinical and pathological data. Major vault protein (MVP) expression was present in 24 (53%) tumor samples and absent in 21 (47%). Samples from surgery showed correlation between the expression of MVP, metastatic disease at diagnosis and event free survival (EFS). The MVP gene expression correlates with metastatic disease at diagnosis after neoadjuvant chemotherapy (p=0.048), and is also associated with worse EFS (p=0.036). These findings suggest that MVP expression is involved in one of the mechanisms of drug resistance in OS and is induced by chemotherapy.