Gregg Magrane

End-sequence profiling: Sequence-based analysis of aberrant genomes

Genome rearrangements are important in evolution, cancer, and other diseases. Precise mapping of the rearrangements is essential for identification of the involved genes, and many techniques have been developed for this purpose. We show here that end-sequence profiling (ESP) is particularly well suited to this purpose. ESP is accomplished by constructing a bacterial artificial chromosome (BAC) library from a test genome, measuring BAC end sequences, and mapping end-sequence pairs onto the normal genome sequence. Plots of BAC end-sequences density identify copy number abnormalities at high resolution. BACs spanning structural aberrations have end pairs that map abnormally far apart on the normal genome sequence. These pairs can then be sequenced to determine the involved genes and breakpoint sequences. ESP analysis of the breast cancer cell line MCF-7 demonstrated its utility for analysis of complex genomes. End sequencing of ≈8,000 clones (0.37-fold haploid genome clonal coverage) produced a comprehensive genome copy number map of the MCF-7 genome at better than 300-kb resolution and identified 381 genome breakpoints, a subset of which was verified by fluorescence in situ hybridization mapping and sequencing.

HER2 Protein Expression and Gene Amplification in Androgen-Independent Prostate Cancer

The role of the HER2 receptor remains uncertain in the pathogenesis and progression of human prostate cancer. Previous studies have reported widely divergent rates for HER2 expression in primary prostate tumors, probably owing to significant methodologic differences in the studies. Few data exist about the frequency of HER2 protein overexpression and gene amplification in androgen-independent prostate cancer (AIPC), although recent xenograft models suggest HER2 expression may be up-regulated in the transition from androgen-dependent to androgen-independent disease. We studied the role of HER2 protein in AIPC by immunohistochemical and fluorescence in situ hybridization (FISH) analyses on AIPC specimens using well-characterized and validated reagents. Fourteen (36%) of 39 specimens expressed HER2; however, only 2 (5%) had moderate (2+) expression, and 2 (5%) had high-level (3+) expression. Two (6%) of 36 specimens had gene amplification by FISH. These data suggest that HER2 protein overexpression and gene amplification are relatively uncommon in AIPC.

Selective Inactivation of p53 Facilitates Mouse Epithelial Tumor Progression without Chromosomal Instability

ABSTRACT We examined the selective pressure for, and the impact of, p53 inactivation during epithelial tumor evolution in a transgenic brain tumor model. In TgT121 mice, cell-specific inactivation of the pRb pathway in brain choroid plexus epithelium initiates tumorigenesis and induces p53-dependent apoptosis. We previously showed that p53 deficiency accelerates tumor growth due to diminished apoptosis. Here we show that in a p53+/− background, slow-growing dysplastic tissue undergoes clonal progression to solid angiogenic tumors in all animals. p53 is inactivated in all progressed tumors, with loss of the wild-type allele occurring in 90% of tumors. Moreover, similar progression occurs in 38% of TgT121p53+/+ mice, also with loss of at least one p53 allele and inactivation of p53. Thus, the selective pressure for p53 inactivation, likely based on its apoptotic function, is high. Yet, in all cases, p53 inactivation correlates with progression beyond apoptosis reduction, from dysplasia to solid vascularized tumors. Hence, p53 suppresses tumor progression in this tissue by multiple mechanisms. Previous studies of fibroblasts and hematopoietic cells show that p53 deficiency can be associated with chromosomal instability, a mechanism that may drive tumor progression. To determine whether genomic gains or losses are present in tumors that progress in the absence of p53, we performed comparative genomic hybridization analysis. Surprisingly, the only detectable chromosomal imbalance was partial or complete loss of chromosome 11, which harbors the p53 gene and is thus the selected event. Flow cytometry confirmed that the majority of tumor cells were diploid. These studies indicate that loss of p53 function is frequent under natural selective pressures and furthermore that p53 loss can facilitate epithelial tumor progression by a mechanism in addition to apoptosis reduction and distinct from chromosomal instability.

Objective aneuploidy detection for fetal and neonatal screening using comparative genomic hybridization (CGH)

Comparative genomic hybridization (CGH) allows entire genomes to be scanned for whole and segmental aneuploidy and thus may be an appropriate tool for the detection of clinically important abnormalities during fetal and neonatal screening. Criteria to distinguish between significant aberrations and experimental artifacts are essential for these applications. This report describes the use of a t-statistic to detect changes in CGH profiles that differ significantly from variations that occur in CGH profiles of normal samples. Eleven cell lines derived from fetal or neonatal patients were analyzed in this study. Aneuploidies in these lines included trisomies for chromosomes 13, 16, 18, and 21 and monosomy for distal 5p and tetrasomy 18p. Aneuploidy was detected in all samples by using the t-statistic, although the extent of the aneuploid region was not correctly estimated in some cases. A detailed description of the t-statistic fused for making these CGH comparisons is described in a companion paper (Moore et al., Cytometry 28:183-190, 1997.

Anaplastic Thyroid Cancer: Cytogenetic Patterns by Comparative Genomic Hybridization

We studied chromosomal abnormalities by comparative genomic hybridization (CGH) and flow cytometry in anaplastic thyroid cancer (ATC), and when present in coexisting or previous differentiated thyroid cancer (DTC). Overall 10 frozen tissues from patients with ATC and 5 cell lines (1 ATC and 4 DTCs) were analyzed. We found chromosomal abnormalities in 5 of 10 ATC tissues, with 24 abnormalities (22 gains and 2 losses). Among 8 ATCs that were associated with prior or concurrent DTC, more chromosomal abnormalities were found in ATC associated with follicular thyroid cancer (FTC) than those associated with PTC (median numbers 9.5 and 0.5, respectively, p = 0.046) or no associated differentiated thyroid cancer. Gain of 1q was relatively common in ATCs (30%). By flow cytometry, we found aneuploidy in 6 of 10 ATC tissues and diploidy in 4. There was concordance between DNA aneuploidy and the presence of chromosomal abnormalities by CGH in 4 of the 5 ATCs (p = 0.048). We also found 26 chromosomal abnormalities in an ATC cell line, 14.3 in 3 FTC cell line, and 3 in a PTC cell line. In conclusion, chromosomal abnormalities are frequent in ATCs associated with FTC, but uncommon in those associated with PTC and in ATCs with no associated differentiated thyroid cancer. These findings support the concept that PTC and FTC have different genetic backgrounds and, even after the transformation to ATC, they may retain some of their cytogenetic characteristics.

Chromosomal aberrations by comparative genomic hybridization in thyroid tumors in patients with familial nonmedullary thyroid cancer.

PURPOSE Nonmedullary thyroid cancer is the most common form of thyroid cancer and its familial form (FNMTC) is increasingly recognized as a distinct clinical entity. However, the genetic background of FNMTC is still poorly understood and the causative gene(s) have not yet been identified. METHODS Because comparative genomic hybridization allows for screening of the entire tumor genome simultaneously for chromosomal gains and/or losses without prior knowledge of potential aberrations, we used this technique in thyroid normal and neoplastic samples from FNMTC patients (1) to analyze whether chromosomal aberrations would correlate with inheritance pattern, and/or clinicopathologic features and (2) to compare comparative genomic hybridization (CGH) findings in familial tumors with those already known in sporadic differentiated thyroid cancers. RESULTS No common germline or somatic chromosomal aberrations were observed in patients with FNMTC because the frequencies and most locations of chromosomal aberrations in familial tumors were also common in sporadic tumors. However, some somatic aberrations were only found in familial tumors (gains in 2q, 3q, 18p, and 19p). Common aberrations in familial tumors corresponded to several locations of candidate genes already reported for sporadic thyroid tumorigenesis. CONCLUSIONS Our findings suggest that chromosomal aberrations in thyroid tumors in patients with FNMTC are not related to inheritance pattern but rather to tumorigenesis.

Fluorescence in situ hybridization of ductal lavage samples identifies malignant phenotypes from cytologically normal cells in women with breast cancer

Ductal lavage (DL) does not routinely identify cytologically malignant cells. For this study, the authors asked whether molecular analyses of DL specimens from women with cancer would identify abnormal cells, even if they appeared cytologically normal.

Morphologically similar epithelial and stromal cells in primary bilateral breast tumors display different genetic profiles: implications for treatment.

The morphologic features of primary bilateral breast carcinoma have been well elucidated, but it is not known whether tumors at two sides share a common genetic profile and undergo the same clinical course. To address this issue, morphologically comparable epithelial and stromal cells in 18 paired primary bilateral breast tumors were microdissected and subjected to comparisons for the frequency and pattern of loss of heterozygosity (LOH) and microsatellite instability (MI), as well as the profiles of comparative genomic hybridization. Of 18 paired bilateral epithelial samples assessed with 10 DNA markers at five chromosomes, 78 altered loci were found; of these, 23 (29.5%) displayed concurrent and 55 (70.5%) showed independent LOH, MI, or both. Of 18 paired bilateral stromal samples assessed with the same markers, 70 altered loci were seen; of these, 9 (12.9%) displayed concurrent and 61 (87.1%) showed independent LOH, MI, or both. Collectively, all the markers and 30 (83.3%) of 36 paired bilateral epithelial and stromal cells displayed significantly more (P < 0.01) independent than concurrent LOH, MI, or both. In contrast, the epithelial cells of a pulmonary small cell carcinoma metastasized to both breasts displayed concurrent LOH at each of the four altered loci. Of seven selected cases for comparative genomic hybridization, six (86%) displayed chromosomal changes, but none showed an identical pattern and frequency of changes in both breasts. The significantly higher rate of independent genetic alterations in morphologically comparable cells of paired bilateral primary breast tumors supports the notion that the development and clinical course of tumors in two sides differ substantially; consequently, different interventions might be needed for the optimal management of bilateral breast tumors.

Duplication of C7orf58, WNT16 and FAM3C in an Obese Female with a t(7;22)(q32.1;q11.2) Chromosomal Translocation and Clinical Features Resembling Coffin-Siris Syndrome

We characterized the t(7;22)(q32;q11.2) chromosomal translocation in an obese female with coarse features, short stature, developmental delay and a hypoplastic fifth digit. While these clinical features suggest Coffin-Siris Syndrome (CSS), we excluded a CSS diagnosis by exome sequencing based on the absence of deleterious mutations in six chromatin-remodeling genes recently shown to cause CSS. Thus, molecular characterization of her translocation could delineate genes that underlie other syndromes resembling CSS. Comparative genomic hybridization microarrays revealed on chromosome 7 the duplication of a 434,682 bp region that included the tail end of an uncharacterized gene termed C7orf58 (also called CPED1) and spanned the entire WNT16 and FAM3C genes. Because the translocation breakpoint on chromosome 22 did not disrupt any apparent gene, her disorder was deemed to result from the rearrangement on chromosome 7. Mapping of yeast and bacterial artificial chromosome clones by fluorescent in situ hybridization on chromosome spreads from this patient showed that the duplicated region and all three genes within it were located on both derivative chromosomes 7 and 22. Furthermore, DNA sequencing of exons and splice junctional regions from C7orf58, WNT16 and FAM3C revealed the presence of potential splice site and promoter mutations, thereby augmenting the detrimental effect of the duplicated genes. Hence, dysregulation and/or disruptions of C7orf58, WNT16 and FAM3C underlie the phenotype of this patient, serve as candidate genes for other individuals with similar clinical features and could provide insights into the physiological role of the novel gene C7orf58.

Comparative Genomic Hybridization

Comparative genomic hybridization (CGH) allows the entire genome of an organism to be scanned for relative changes in DNA copy number (copy number aberrations) (1–3) in a single experiment. Standard CGH can detect aneuploidies, deletions, and unbalanced translocations at a resolution of 5–10 megabases. This is particularly useful in cases where DNA is unstable and where changes in copy number occur. Thus, tumors are often analyzed by CGH, but other applications could include diagnosis of other chromosome based illnesses.