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.

Placental Molar Disease: What are the Benefits and Barriers to the Adoption of a Comprehensive Diagnostic Service?

The molecular cytogenetic analysis of specimens (genotyping) suspicious for hydatidiform mole (HM) significantly improves diagnostic accuracy over histopathology and immunohistochemical analysis alone, particularly in the classification of partial mole. However, the implementation of this advance in diagnostics has been slow. This study sought to identify the major benefit and potential barriers to the adoption of genotyping. A pilot Placental Molar Diagnostic (PMD) Service was established combining histopathology, p57 immunohistochemistry, and molecular genotyping analysis for both in-house and referred-in cases suspicious for HM or with a preliminary diagnosis of HM. A retrospective analysis of 117 cases received in the first 16 mo was conducted to identify the utility of the PMD Service and factors or barriers which precluded optimal results. A final diagnosis of HM was made in 73 cases (37 complete HMs and 36 partial HMs). The remaining 44 cases were hydropic abortuses. Three potential barriers were identified that could lead to less than optimal results from a PMD Service: prevalence of noninformative genotyping, lack of any available or appropriate paraffin blocks, and inappropriate deferral of genotyping. The major utility of this pilot PMD Service was to increase the specificity of a diagnosis of HM, and avoid unnecessary clinical follow-up in 37% of cases with an initial suspicion or diagnosis of HM. Measures can be undertaken to address potential barriers to the implementation of a comprehensive placental diagnostic platform. Underutilization of molecular genotyping in the diagnosis of HM likely leads to inappropriate management and "downstream" costs in a significant proportion of patients suspected of having HM.

PGD for a carrier of an intrachromosomal insertion using aCGH.

Intrachromosomal insertions are rare and difficult to diagnose. However, making the correct diagnosis is critical for genetic risk assessment, and prenatal and preimplantation genetic diagnosis outcomes. We present a case of preimplantation genetic diagnosis (PGD) using array comparative genomic hybridization (aCGH) following trophectoderm biopsy of embryos created after in vitro fertilization for a carrier of an intrachromosomal insertion on chromosome 1 [46,XX, ins(1)(q44q23q32.1)]. The PGD analysis of 6 blastocysts demonstrated 67% unbalanced embryos. No pregnancy was achieved after the transfer of 2 euploid embryos. To the best of our knowledge, this is the first reported case of PGD using aCGH following trophectoderm biopsy for a carrier of an intrachromosomal insertion.

QF-PCR rapid aneuploidy screen and aCGH analysis of cell free fetal (cff) DNA in supernatant of compromised amniotic fluids (AF).

OBJECTIVE: The aim of this study was to determine whether cell free fetal (cff) DNA in residual amniotic fluid (AF) supernatant obtained from bloody, low-volume and late gestation samples can be used for prenatal diagnosis by quantitative fluorescence polymerase chain reaction (QF-PCR) and array comparative genomic hybridization (aCGH). METHOD: A total of 49 compromised AFs were analyzed in this case-control, double-blinded study. The samples were processed through: a conventional cytogenetic approach utilizing Fluorescence in situ Hybridization and/or karyotype (Approach I); QF-PCR analysis to establish the presence of maternal cell contamination (MCC) (Approach II) and a newly proposed approach using AF supernatant cff DNA (Approach III). Data on clinical impact and turn-around-time was collected. RESULTS: Evidence of MCC was not detected in any of the cff DNA samples, and informative results were provided for all cases, including nine aneuploidies. In contrast, the conventional approach (I) failed to provide results either due to MCC or culture failure in a significant proportion of cases. An adequate amount of quality cff DNA was obtained for successful aCGH testing. CONCLUSION: We have shown that it is feasible to isolate pure cff DNA from routinely discarded AF supernatant to perform QF-PCR and microarray analyses, providing timely and informative results even for problematic grossly bloody and otherwise compromised AF samples or culture failures.

RNF168 ubiquitylates 53BP1 and controls its response to DNA double-strand breaks.

Defective signaling or repair of DNA double-strand breaks has been associated with developmental defects and human diseases. The E3 ligase RING finger 168 (RNF168), mutated in the human radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties syndrome, was shown to ubiquitylate H2A-type histones, and this ubiquitylation was proposed to facilitate the recruitment of p53-binding protein 1 (53BP1) to the sites of DNA double-strand breaks. In contrast to more upstream proteins signaling DNA double-strand breaks (e.g., RNF8), deficiency of RNF168 fully prevents both the initial recruitment to and retention of 53BP1 at sites of DNA damage; however, the mechanism for this difference has remained unclear. Here, we identify mechanisms that regulate 53BP1 recruitment to the sites of DNA double-strand breaks and provide evidence that RNF168 plays a central role in the regulation of 53BP1 functions. RNF168 mediates K63-linked ubiquitylation of 53BP1 which is required for the initial recruitment of 53BP1 to sites of DNA double-strand breaks and for its function in DNA damage repair, checkpoint activation, and genomic integrity. Our findings highlight the multistep roles of RNF168 in signaling DNA damage.

A newly characterized human well-differentiated liposarcoma cell line contains amplifications of the 12q12-21 and 10p11-14 regions.

While surgery is the usual treatment for localized well-differentiated and dedifferentiated liposarcomas (WDLPS/DDLPS), the therapeutic options for patients with advanced disease are limited. The classical antimitotic treatments are most often inefficient. The establishment of genetically characterized cell lines is therefore crucial for providing in vitro models for novel targeted therapies. We have used spectral karyotyping, fluorescence in situ hybridization with whole chromosome painting and locus-specific probes, and array-comparative genomic hybridization to identify the chromosomal and molecular alterations of a novel cell line established from a recurring sclerosing WDLPS. The karyotype was hypertriploid and showed multiple structural anomalies. All cells retained the presence of a giant marker chromosome that had been previously identified in the primary cell cultures. This giant chromosome contained high-level amplification of chromosomal regions 12q13-21 and lacked the alpha-satellite centromeric sequences associated with WDLPS/DDLPS. The 12q amplicon was large, containing 370 amplified genes. The DNA copy number ranged from 3 to 57. The highest levels of amplification were observed at 12q14.3 for GNS, WIF1, and HMGA2. We analyzed the mRNA expression status by real-time reverse transcription polymerase chain reaction for six genes from this amplicon: MDM2, HMGA2, CDK4, TSPAN31, WIF1, and YEATS4. mRNA overexpression was correlated with genomic amplification. A second amplicon originating from 10p11-14 was also present in the giant marker chromosome. The 10p amplicon contained 62 genes, including oncogenes such as MLLT10, previously described in chimeric fusion with MLL in leukemias, NEBL, and BMI1.

A clinical algorithm for efficient, high-resolution cytogenomic analysis of uncultured perinatal tissue samples.

PURPOSE: Cytogenetic analysis of solid tissue is indispensable in perinatal care, reproductive planning, and detection of gestational trophoblastic disease. Unfortunately, methods in common use suffer from drawbacks including culture artifact, low resolution, and high cost. We propose a new diagnostic algorithm based on direct genetic analysis of tissues (without cell culture) using QF-PCR and array CGH. METHODS: Study samples consisted of specimens submitted to the cytogenetics laboratory between January and June of 2011 that were split and analyzed in parallel by our traditional algorithm (culture and G-banding, plus an interphase FISH aneuploidy panel for culture failures) and the proposed "no-culture" algorithm (first line QF-PCR, plus array CGH on normal QF-PCRs). Data on clinical impact, cost, and turnaround time were collected. RESULTS: Forty specimens were included. The algorithms produced results that were fully concordant in 22 cases, partially concordant in 9 cases, and discordant in 9 cases. The no-culture algorithm detected new, clinically-significant abnormalities in 8 of 40 cases (20%), corrected the sex chromosome assortment in 1 case, reduced the analysis failure rate from 10% to 0%, and provided at least one of these three important benefits in 12 of 40 cases (30%). The algorithm also demonstrated a reduced cost per-specimen and per diagnosis, as well as improved turnaround time, with virtually all cases reported per guidelines. CONCLUSION: These striking results favor the "no-culture" algorithm, which may have the potential to replace standard cytogenetic methods in the clinical laboratory.

Caspase-8 is essential for maintaining chromosomal stability and suppressing B-cell lymphomagenesis.

In addition to its proapoptotic function, caspase-8 is also important for several other processes, including suppressing necroptosis, cell migration, and immune cell survival. In the present study, we report that the loss of caspase-8 in B lymphocytes leads to B-cell malignancies and that the risk for these tumors is further enhanced in the absence of p53. We also report that deficiency of caspase-8 results in impaired cytokinesis and that casp8(-/-) lymphomas display remarkably elevated levels of chromosomal aberrations. Our data support an important role for caspase-8 in the maintenance of genomic integrity and highlight its tumor-suppressive function.

Analysis of miRNA-gene expression-genomic profiles reveals complex mechanisms of microRNA deregulation in osteosarcoma.

Osteosarcoma is an aggressive sarcoma of the bone characterized by a high level of genetic instability and recurrent DNA deletions and amplifications. This study assesses whether deregulation of microRNA (miRNA) expression is a post-transcriptional mechanism leading to gene expression changes in osteosarcoma. miRNA expression profiling was performed for 723 human miRNAs in 7 osteosarcoma tumors, and 38 miRNAs differentially expressed ≥10-fold (28 under- and 10 overexpressed) were identified. In most cases, observed changes in miRNA expression were DNA copy number-correlated. However, various mechanisms of alteration, including positional and/or epigenetic modifications, may have contributed to the expression change of 23 closely linked miRNAs in cytoband 14q32. To develop a comprehensive molecular genetic map of osteosarcoma, the miRNA profiles were integrated with previously published array comparative genomic hybridization DNA imbalance and mRNA gene expression profiles from a set of partially overlapping osteosarcoma tumor samples. Many of the predicted gene targets of differentially expressed miRNA are involved in intracellular signaling pathways important in osteosarcoma, including Notch, RAS/p21, MAPK, Wnt, and the Jun/FOS pathways. By integrating data on copy number variation with mRNA and miRNA expression profiles, we identified osteosarcoma-associated gene expression changes that are DNA copy number-correlated, DNA copy number-independent, mRNA-driven, and/or modulated by miRNA expression. These data collectively suggest that miRNAs provide a novel post-transcriptional mechanism for fine-tuning the expression of specific genes and pathways relevant to osteosarcoma. Thus, the miRNA identified in this manner may provide a starting point for experimentally modulating therapeutically relevant pathways in this tumor.

Recurrent RECQL4 imbalance and increased gene expression levels are associated with structural chromosomal instability in sporadic osteosarcoma.

Osteosarcoma (OS) is an aggressive bone tumor with complex abnormal karyotypes and a highly unstable genome, exhibiting both numerical- and structural-chromosomal instability (N- and S-CIN). Chromosomal rearrangements and genomic imbalances affecting 8q24 are frequent in OS. RECQL4 gene maps to this cytoband and encodes a putative helicase involved in the fidelity of DNA replication and repair. This protective genomic function of the protein is relevant because often patients with Rothmund-Thomson syndrome have constitutional mutations of RECQL4 and carry a very high risk of developing OS. To determine the relative level of expression of RECQL4 in OS, 18 sporadic tumors were studied by reverse transcription-polymerase chain reaction. All tumors overexpressed RECQL4 in comparison to control osteoblasts, and fluorescence in situ hybridization analysis of tumor DNA showed that expression levels were strongly copy number-dependent. Relative N- and S-CIN levels were determined by classifying copy number transitions within array comparative genomic hybridization profiles and by enumerating the frequency of break-apart fluorescence in situ hybridization within 8q24 using region-specific and control probes. Although there was no evidence that disruption of 8q24 in OS led to an elevated expression of RECQL4, there was a marked association between increased overall levels of S-CIN, determined by copy number transition frequency and higher levels of RECQL4.

Variability of origin for the neocentromeric sequences in analphoid supernumerary marker chromosomes of well-differentiated liposarcomas.

Well-differentiated liposarcomas (WDLPS) and dedifferentiated liposarcomas are cytogenetically characterized by the presence of supernumerary ring or giant chromosomes containing amplified material from the 12q14-15 region. These chromosomes contain neocentromeres, which are able to bind the kinetochore proteins and to ensure a stable mitotic transmission although they do not show detectable alpha-satellite sequences. WDLPS is the sole solid tumor for which the presence of a neocentromere is a consistent and specific feature. By immunostaining with anti-centromere antibodies in combination with FISH analysis (immunoFISH) in four cases of WDLPS, we have shown that sequences from the region 12q14-21 region were not located at the neocentromere site. In addition, we have microdissected the neocentromeric region from a giant supernumerary chromosome in the 94T778 WDLPS cell line. By using immunoFISH and positional cloning we have shown that the neocentromere of this cell line originated from a region at 4p16.1, rich in AT sequences and in long interspersed nucleotide element (LINE)1, that was co-amplified with 12q14-15. We have observed that this 4p sequence was not involved in the neocentromere of the supernumerary giant chromosome present in the 93T449 WDLPS cell line derived from a metachronous recurrence of the same primary WDLPS than 94T778. Altogether, these results indicate that the neocentromeres in WDLPS originate from amplified chromosomal regions other than 12q14-15 and do not involve a specific and recurrent DNA sequence. These sequences might be activated for centromeric function by epigenetic mechanisms.

In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma.

Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks.

Complex t(5;8) involving the CSPG2 and PTK2B genes in a case of dermatofibrosarcoma protuberans without the COL1A1-PDGFB fusion.

Dermatofibrosarcoma protuberans (DFSP) is a rare, dermal neoplasm of intermediate malignancy. It is made of spindle-shaped tumor cells in a storiform pattern positive for CD34. Cytogenetically, DFSP cells are characterized by either supernumerary ring chromosomes composed of sequences derived from chromosomes 17 and 22 or more rarely of translocations t(17;22). These chromosomal rearrangements lead to the formation of a specific chimeric gene fusing COL1A1 to PDGFB. So far, the COL1A1-PDGFB fusion gene remains the sole fusion gene identified in DFSP. However, some observations suggest that genes, other than COL1A1 and PDGFB, might be involved in some DFSP cases. We report in this paper a DFSP case presenting as a unique chromosomal abnormality a complex translocation between chromosomes 5 and 8. This is the first report of a DFSP case where the lack of chromosomes 17 and 22 rearrangement and the absence of COL1A1-PDGFB fusion gene have been demonstrated. Using fluorescence in situ hybridization analysis, we showed that the CSPG2 gene at 5q14.3 and the PTK2B gene at 8p21.2 were disrupted by this rearrangement. Although rare, the existence of cases of DFSP negative for the COL1A1-PDGFB fusion has to be taken in consideration when performing molecular diagnosis for a tumor suspected to be a DFSP.

Complex rearrangement of chromosomes 19, 21, and 22 in Ewing sarcoma involving a novel reciprocal inversion-insertion mechanism of EWS-ERG fusion gene formation: a case analysis and literature review.

EWS-ERG Ewing sarcoma (ES) gene fusions often result from complex chromosomal rearrangements. We report an unusually aggressive case of ES with an EWS-ERG fusion gene that appeared to be a result of a simple balanced and reciprocal translocation, t(19;22)(q13.2;q12.2). Subsequent molecular investigation of the primary tumor, the metastasis, and a cell line generated from this ES permitted reconstruction of each genomic step in the evolution of this complex EWS-ERG fusion. We elucidated a new mechanism of reciprocal insertion inversion between chromosome 21 and 22, involving cryptic alterations to both the ERG and EWS genes. Molecular cytogenetic investigation, using systematic analysis with locus-specific probes, identified the cognate genomic breakpoints within chromosome 21 and 22, mandatory for the excision and exchange of both 3'ERG and 3'EWS, resulting in the formation of the EWS-ERG fusion gene present on the der(22). Array comparative genomic hybridization and fluorescence in situ hybridization studies of the ES cell line derived from this tumor identified additional acquired chromosomal and genomic abnormalities, likely associated with establishment and adaptation to in vitro growth. Notably, the cell line had lost one copy of the RB1 gene within the 13q13.1 approximately q14.2 region, and also had a near-tetraploid karyotype. The significance of these findings and their relationship to other reports of variant and complex ES translocations involving the ERG gene are reviewed.

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.

Detection of MDM2-CDK4 amplification by fluorescence in situ hybridization in 200 paraffin-embedded tumor samples: utility in diagnosing adipocytic lesions and comparison with immunohistochemistry and real-time PCR.

Atypical lipomatous tumor/well-differentiated liposarcomas and dedifferentiated liposarcomas are characterized by the amplification of MDM2 and CDK4 genes. To evaluate the accuracy of fluorescence in situ hybridization (FISH) analysis in the differential diagnosis of adipose tissue tumors, we investigated MDM2-CDK4 status by FISH, real-time polymerase chain reaction (PCR) [quantitative PCR (Q-PCR)] and immunohistochemistry (IHC) in a series of 200 adipose tumors. First, we evaluated MDM2-CDK4 amplification and expression in a series of 94 well-defined adipose tissue tumors. Results showed that FISH was interpretable in 45 of 50 cases (90%), and was more specific and sensitive than Q-PCR and IHC. We then used the same techniques as complementary diagnostic tools in a series of 106 adipose and soft tissue tumors of unclear diagnosis to distinguish between (i) lipomas and atypical lipomatous tumor/well-differentiated liposarcomas, (ii) malignant undifferentiated tumors and dedifferentiated liposarcomas, and (iii) a variety of benign tumors and liposarcomas. Our results indicate that although helpful, IHC alone is often insufficient to solve diagnostic problems. FISH and Q-PCR methods gave concordant results and were equally informative in most cases. However, the proportion of noninterpretable cases was slightly higher with FISH than with Q-PCR. When tumor cells represented a minor component of the tumor tissue, such as with inflammatory tumors, FISH was more powerful than Q-PCR by allowing visualization of individual cells. In conclusion, we recommend that the evaluation of MDM2-CDK4 amplification using FISH or Q-PCR be used to supplement IHC analysis when diagnosis of adipose tissue tumors is not possible based on clinical and histologic information alone.

[From cytogenetics to cytogenomics of dermatofibrosarcoma protuberans family of tumors]. / De la cytogénétique à la cytogénomique du dermatofibrosarcome de Darier-Ferrand (dermatofibrosarcoma protuberans) et des tumeurs apparentées.

Dermatofibrosarcoma protuberans (DP) is a rare, slow growing dermal neoplasm of intermediate malignancy. It is made of spindle-shaped tumor cells in a storiform pattern often positive for CD34. The preferred treatment for DP is a surgical wide excision with pathologically sane margins of 3 cm. At the cytogenetic level, DP cells are characterized by either supernumerary ring chromosomes composed of sequences derived from chromosomes 17 and 22 or more rarely of translocations t(17;22). Rings have been mainly observed in adults whereas translocations have been reported in all pediatric cases. These chromosomal rearrangements lead to the formation of a specific fusion gene : COL1A1-PDGFB detected in rings as well as in translocations. DP is therefore a unique example of tumor in which the same molecular event occurs either on rings or linear translocation derivatives and the chromosomal abnormalities display an age-related pattern. So far, the COL1A1-PDGFB fusion gene remains the only fusion gene identified in this tumor. It is also present in variant forms of DP such as giant cell fibroblastoma, Bednar tumor, adult superficial fibrosarcoma and the granular cell variant of DP demonstrating that these tumors are not distinct entities but morphological variants of DP. The breakpoint localization in PDGFB was found to be remarkably constant, placing exon 2 of PDGFB under the control of the COL1A1 promoter. In contrast, the COL1A1 breakpoint was found to be variably located within the exons of the alpha-helical coding region (exons 7-47). No correlation between the breakpoint location in COL1A1 and the age of the patient or any clinical or histological particularity has been established. Moreover, no preferential breakpoint appears to be more particularly linked to one or another variant of DP. The COL1A1-PDGFB fusion gene is detectable either by multiplex RT-PCR with a combination of forward primers designed from a variety of COL1A1 exons and one reverse primer for PDGFB exon 2, or by in situ fluorescence hybridization (FISH) on interphase nuclei from frozen or fixed paraffin-embbeded sections. The COL1A1-PDGFB fusion gene is not found in approximately 8% of DP cases, suggesting that genes other than COL1A1 or PDGFB might be involved in a small subset of cases. It has been proposed that PDGFB acts as a mitogen in DP cells by autocrine stimulation of the PDGF receptor. The PDGF receptor tyrosine kinase antagonist imatinib mesylate has recently been used in clinical trials; its efficiency has been demonstrated in several cases, which allows it to be considered as a novel treatment strategy for metastatic or locally advanced DP.

A clinical, histologic, and molecular study of 9 cases of congenital dermatofibrosarcoma protuberans.

BACKGROUND: The diagnosis of dermatofibrosarcoma protuberans (DFSP) in childhood is often difficult because of the deceptive appearance of the lesions. Little is known about congenital DFSP, the frequency of which is probably underestimated because the initial lesion may pass unnoticed. OBSERVATIONS: We studied 9 DFSP congenital cases (8 plaques and 1 nodule) initially suspected to be benign lesions. The first biopsies or excisions were performed after a delay of 5(1/2) months to 15 years. All cases were CD34+. Histologic patterns were similar to the DFSP adult classic pattern in 4 cases. One case was a Bednar tumor. The histologic diagnosis of the 4 remaining cases was difficult. The collagen, type I, alpha 1-platelet-derived growth factor beta fusion gene (COL1A1-PDGFB) was detected by means of reverse transcriptase-polymerase chain reaction or fluorescence in situ hybridization. CONCLUSIONS: All cases of congenital DFSP were difficult to identify clinically. The diagnosis was suspected by means of histologic and immunohistochemical evaluation and was confirmed using molecular analyses. This study illustrates the difficulties and pitfalls of the recognition of congenital DFSP and emphasizes the value of immunohistochemical study with anti-CD34 and complementary molecular analysis for all cutaneous spindle cell tumors and plaques in neonates and infants.

Global analysis of chromosome X gene expression in primary cultures of normal ovarian surface epithelial cells and epithelial ovarian cancer cell lines.

The interpretation of loss of heterozygosity (LOH) in cancers is complicated as genes that map to LOH regions may be transcriptionally active (Xa) or inactive (Xi) due to X chromosome inactivation (XCI). We have analyzed the chromosome X transcriptome in four epithelial ovarian cancer (EOC) cell lines (TOV21G, TOV81D, TOV112D, and OV90) and 12 primary cultures of normal ovarian surface epithelial (NOSE) cells in relation to chromosome X integrity. Two-way comparative analysis using HuGeneFL Affymetrix GeneChips of TOV21G, TOV81D and OV90 relative to the NOSE samples was highly correlated (> 89%) in contrast to that of TOV112D (56-69%). TOV112D, followed by TOV21G, exhibited the largest number of up-regulated genes. XIST expression by RT-PCR was not detectable in TOV112D or TOV21G. Allele-specific transcription by cDNA sequence analysis of genes known to be subjected to XCI revealed maintenance of XCI in TOV81D and OV90, but not TOV21G. Biallelic expression could not be assessed in TOV112D due to reduction to hemizygosity of chromosome X. Chromosome X rearrangements were observed in FISH analysis of TOV112D and TOV21G, and both of these EOC cell lines were negative for Barr body analysis. The differentially expressed genes did not appear to map to any particular region of the X chromosome in any EOC cell line. The absence of XIST expression is consistent with Barr body loss in TOV112D and TOV21G. The combined evidence is consistent with two proposed mechanisms to account for absence of Xi in female cancers: Xi loss followed by Xa duplication (exemplified by TOV112D) and transcriptional reactivation of Xi (exemplified by TOV21G). Despite an alteration in XIST expression and differences in allelic content in the EOC cell lines, the chromosome X transcriptome was modified modestly when compared with that of NOSE samples.