Teresa Maxwell

Her-2/neu expression in osteosarcoma increases risk of lung metastasis and can be associated with gene amplification.

The purpose of this study was to investigate whether Her-2/ neu expression at diagnosis of osteosarcoma could provide biologic and prognostic information that predicts the risk of pulmonary metastases and outcome. Human epidermal growth factor (Her-2/ neu) expression in 25 initial pretreatment osteosarcoma biopsies and 12 posttreatment pulmonary metastatic osteosarcoma resection specimens was assessed by standard immunohistochemical techniques on formalin-fixed paraffin-embedded tissue. As a screening analysis to determine if gene amplification may be a mechanism for increased Her-2/ neu expression, FISH analysis was conducted on seven Her-2/ neu immunostain-positive samples and five Her-2/ neu immunostain-negative samples. Cytoplasmic Her-2/ neu reactivity was identified in 11/25 (44%) of primary tumors and in 7/12 (58%) resection specimens from pulmonary metastases. Cytoplasmic Her-2/ neu expression was associated with shorter overall metastasis-free survival. Her-2/ neu gene amplification was identified by FISH analysis in six of the seven immunostain-positive samples but was also identified in two of the five immunostain-negative samples. Her-2/ neu expression in patients with osteosarcoma is associated with an increased risk of metastasis and may define a subset of patients with a more aggressive tumor phenotype. Her-2/ neu gene amplification may provide a mechanism for Her-2/ neu overexpression in certain cases of osteosarcoma. Whether Her-2/ neu expression influences outcome needs to be examined further in a prospective fashion. The hope is that Her-2/ neu expression will identify patients who may benefit from the addition of directed biologic therapy.

Chromosomal clues to the development of prostate tumors.

Cytogenetic, molecular cytogenetic, and molecular studies of prostate cancer have revealed an enormous amount of data regarding chromosomal loci that are aberrant in prostate tumors.

Array Comparative Genomic Hybridization for Genetic Evaluation of Fetal Loss Between 10 and 20 Weeks of Gestation

OBJECTIVE: To estimate genomic copy number changes in fetal loss between 10 and 20 weeks of gestation using array comparative genomic hybridization. METHODS: This was a prospective series of 35 women who experienced pregnancy loss between 10–20 weeks of gestation with either normal karyotype (n=9) or no conventional cytogenetic testing (n=26). DNA was isolated from fetal tissue and parental blood. Array comparative genomic hybridization was performed on DNA from fetal tissue using a whole genome BAC array chip. Copy number changes in fetal tissue were then compared against databases of benign copy number changes. Parental DNA was analyzed using the same BAC array in cases that contained suspected pathogenic copy number changes. In cases where de novo copy number changes were detected in fetal DNA, further characterization was performed using a 244K oligonucleotide microarray. RESULTS: DNA was successfully isolated in 30 of 35 (86%) of cases. Array comparative genomic hybridization was performed in all of these. De novo copy number changes were detected in six (20%) cases using the Spectral chip and confirmed in four (13%) cases using the Agilent chip. These ranged in size from 93–289 Kb and mapped on 5p, 13q and Xq22. In the cases with de novo copy number changes, the higher-density Agilent array detected additional changes (20–1,310 Kb). CONCLUSION: Array comparative genomic hybridization detected de novo copy number changes in 13% of cases where routine cytogenetic testing was normal or not performed. These involved large regions of DNA and may provide novel explanations for some cases of otherwise unexplained pregnancy loss. LEVEL OF EVIDENCE: III

Chromosome 7 Abnormalities in Prostate Cancer Detected by Dual-Color Fluorescence In Situ Hybridization

Aneusomy of chromosome 7 and loss at 7q (especially 7q31.1) have been reported in prostate cancer. To further investigate abnormalities of 7q and the relationship with whole chromosome 7 changes, we have conducted a dual-color fluorescence in situ hybridization (FISH) analysis on isolated nuclei from 28 primary prostate cancers. A pericentromeric probe for chromosome 7, five newly isolated sequence-specific bacterial artificial chromosome (BAC) probes from 7q31.1, and one BAC for the epidermal growth factor receptor (EGFR) gene at 7p12 were used in dual color hybridizations. Pericentromeric probes for chromosomes X and 4 were also used as controls. Sixteen (57.1%) of the 28 tumors showed clonal aberrations. Nine of them were trisomy 7 and four were hypertetrasomy for chromosome 7. Deletions at 7q31.1 were found in two of the high grade tumors. With the exception of these two cases, all other cases showed concordant results using all probes. These findings confirm previous studies that aneusomy of 7 is associated with prostate cancer progression, and there may be a tumor suppressor gene (TSG) at 7q31.1 which is associated with tumor progression. In addition, our study indicates: (1) the deletion pattern of individual nuclei infers that deletions at 7q31.1 precede reduplications of chromosome 7; and (2) the amplification of EGFR was not detected at the DNA level, suggesting that activation of this oncogene may play a minor role in prostate cancer.

Comparison of targeted and whole genome analysis of postnatal specimens using a commercially available array based comparative genomic hybridisation (aCGH) microarray platform

Purpose: The University of Utah Comparative Genomic Hybridization Microarray Laboratory was one of the first US laboratories to offer comparative genomic hybridisation (CGH) microarray testing using a commercial platform in a clinical setting. Results for 1076 patients (1598 chips) are presented. Methods: The Spectral Genomics/PerkinElmer Constitutional ChipTM (targeted array), SpectralChip 2600TM (whole genome array) and a “Combo” chip (both arrays run simultaneously) were the tests offered. Abnormal results were confirmed by an alternative method, most often fluorescence in situ hybridisation. Results: In 669 cases with known normal cytogenetics, an abnormal detection rate of 10.8% was observed, (5.3%, 12.2% and 14.1% for the Constitutional ChipTM, SpectralChip 2600TM and Combo assay, respectively). Known copy number variants and single clone abnormalities are not included in these rates. Single clone abnormalities are reported separately. For 1076 total cases, we report an average abnormal rate of 16.9% (8.7%, 23.7% and 18.6% for the three assays). This rate includes characterisation of some abnormalities previously identified by cytogenetics. Conclusions: CGH microarray provides a likely aetiology for the clinical phenotype in many cytogenetically normal cases, and a whole genome array generally identifies copy number changes more effectively than a targeted chip alone.

Co-occurrence of 4p16.3 deletions with both paternal and maternal duplications of 11p15: modification of the Wolf-Hirschhorn syndrome phenotype by genetic alterations predicted to result in either a Beckwith-Wiedemann or Russell-Silver phenotype.

Paternal duplications of chromosome region 11p15 can result in Beckwith–Weidemann syndrome (BWS), whereas maternal duplications of the same region on 11p15 can result in Russell–Silver syndrome (RSS). These two syndromes have numerous opposing phenotypes, especially with regards to growth parameters. The differences in the phenotype are proposed to be due to altered dosage of imprinted genes that control growth within this region of 11p15. Wolf–Hirschhorn syndrome (WHS) is due to deletions of a region in 4p16.3 and there is no known parent‐of‐origin effect for deletions of the WHS critical region, and no genes are known to be imprinted in this region. We report on three individuals with very similar unbalanced translocations resulting in a derivative chromosome 4 with both a deletion of 4p16.3 and a duplication of 11p15. Two of these individuals are family members with one inheriting the derivative 4 from her balanced mother and the other inheriting the derivative 4 from his balanced father. The third individual is unrelated and inherited his derivative 4 from his balanced father. While the findings of these individuals included some features of WHS and RSS or BWS, the phenotypes as an aggregate are distinct from these syndromes. The genomic and phenotypic characterization of these three individuals demonstrates how unbalanced translocations can result in the modification of chromosome duplication and deletion syndromes and identifies genomic architecture that may be responsible for mediating a recurrent translocation between 4p and 11p.

A new genomic mechanism leading to cri-du-chat syndrome†

Using standard banding techniques, a within‐arm intrachromosomal insertion can be mistakenly interpreted as a paracentric inversion. The need to correctly distinguish between these two types of chromosome rearrangements is emphasized by their different reproductive risks. For carriers of an intrachromosomal insertion, the empiric risk of having a liveborn child with a recombinant chromosome leading to a genetic imbalance is at least 15%, whereas the risk for a carrier of a paracentric inversion having a liveborn child with a recombinant chromosome leading to a genetic imbalance is thought to be practically negligible. We report a unique observation in which a paracentric inversion in the short arm of chromosome 5, 46,XX,inv(5)(p13.3p15.3), was identified in a women who had a daughter with an apparently terminal deletion in the distal short arm of chromosome 5, 46,XX,del(5)(p14.3), and the clinical diagnosis of cri‐du‐chat syndrome. We further characterized the rearrangement, and fluorescence in situ hybridization (FISH) and microsatellite analyses confirmed the paracentric inversion in the mother and showed the deletion in the daughter was maternal in origin. Therefore, this represents a case in which a confirmed paracentric inversion likely resulted in a viable terminal deletion. We propose a mechanism involving dicentric chromosome formation with subsequent breakage and telomere healing during meiosis. This illustrates a new genomic mechanism of chromosome rearrangement leading to cri‐du‐chat syndrome and should provide significant information for the medical management of patients with other terminal deletion syndromes.

Expansion in size of a terminal deletion: a paradigm shift for parental follow-up studies

Background: Parental studies are often necessary subsequent to the identification of a chromosome abnormality. The recommended studies are based on assumptions about how chromosome rearrangements occur. One such assumption is that deletion size is stable through generations. Results: We have identified a family where a small subtelomeric deletion in a phenotypically and cytogenetically normal mother expanded nearly 10-fold into a clinically consequential and cytogenetically visible deletion in her affected daughter. Conclusion: Traditional parental follow-up studies would have not identified this expansion, but would have rather classified the deletion in the daughter as either de novo or benign. Only by sizing the deletion by array comparative genomic hybridisation in both the mother and the daughter was the expansion recognised. Previous assumptions about chromosome behaviour suggest that this phenomenon may have been easily missed in other cases of chromosomal deletions. Therefore, this case illustrates the need for more comprehensive analyses of parental chromosome structure when characterising an abnormality in a child.

Detection of a de novo interstitial 2q microdeletion by CGH microarray analysis in a patient with limb malformations, microcephaly and mental retardation

We describe the cytogenetic diagnosis using BAC‐ and oligonucleotide microarrays of a 16‐year‐old Laotian‐American female, who first presented at 2½ years of age with microcephaly, developmental retardation, and skeletal abnormalities of the upper limb including mild syndactyly of the second and third and the third and fourth fingers, short middle phalanges and clinodactyly of the fifth digit at the distal interphalangel joint on both hands, and symphalangism of the metacarpal‐phalangeal joints of the second and fifth digits bilaterally. Her lower limbs displayed symphalangism of the metatarsal‐phalangeal joint of the second, third, and fourth digits on both feet, with fusion of the middle and distal phalanges of the second and fifth digits and hallux valgus bilaterally. G‐banded chromosomal study at age 4 was normal. However, comparative genomic hybridization at age 15 with the Spectral Genomics 1 Mb Hu BAC array platform indicated a microdeletion involving two BAC clones, RP11‐451F14 → RP11‐12N7 at 2q31.1. The maximal deletion on initial analysis comprised the HOXD cluster, which is implicated in limb development. Florescence in situ hybridization (FISH) using the RP11‐451F14 probe confirmed the deletion. Both parents were negative for the deletion. Additional FISH using BAC RP11‐387A1, covering the HOXD cluster, limited the maximal deletion to approximately 2.518 Mb, and excluded involvement of the HOXD cluster. The Agilent 44K and 244K platforms demonstrated a deletion of approximately 2,011,000 bp, which did not include the HOXD cluster. The malformations in our patient may be caused by deletion of a regulatory element far upstream of the HOXD cluster.

Mandibulofacial Dysostosis in a Patient with a de novo 2;17 Translocation that Disrupts the HOXD Gene Cluster

Treacher Collins syndrome (TCS) is the prototypical mandibulofacial dysostosis syndrome, but other mandibulofacial dysostosis syndromes have been described. We report an infant with mandibulofacial dysostosis and an apparently balanced de novo 2;17 translocation. She presented with severe lower eyelid colobomas requiring skin grafting, malar and mandibular hypoplasia, bilateral microtia with external auditory canal atreasia, dysplastic ossicles, hearing loss, bilateral choanal stenosis, cleft palate without cleft lip, several oral frenula of the upper lip/gum, and micrognathia requiring tracheostomy. Her limbs were normal. Chromosome analysis at the 600‐band level showed a 46,XX,t(2;17)(q24.3;q23) karyotype. Sequencing of the entire TCOF1 coding region did not show evidence of a sequence variation. High‐resolution genomic microarray analysis did not identify a cryptic imbalance. FISH mapping refined the breakpoints to 2q31.1 and 17q24.3‐25.1 and showed the 2q31.1 breakpoint likely affects the HOXD gene cluster. Several atypical findings and lack of an identifiable TCOF1 mutation suggest that this child has a provisionally unique mandibulofacial dysostosis syndrome. The apparently balanced de novo translocation provides candidate loci for atypical and TCOF1 mutation negative cases of TCS. Based on the agreement of our findings with one previous case of mandibulofacial dysostosis with a 2q31.1 transocation, we hypothesize that misexpression of genes in the HOXD gene cluster produced the described phenotype in this patient.