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[Cancer Research 61, 4951-4955, July 1, 2001]
© 2001 American Association for Cancer Research

Advances in Brief

Structural Analysis of the 17q22–23 Amplicon Identifies Several Independent Targets of Amplification in Breast Cancer Cell Lines and Tumors1

Guo-jun Wu2, Colleen Sinclair2, Shannon Hinson, James N. Ingle, Patrick C. Roche and Fergus J. Couch3

Departments of Laboratory Medicine and Pathology [G-j. W., C. S., S. H., P. C. R., F. J. C.], Oncology [J. N. I.], and Biochemistry and Molecular Biology [F. J. C.], Mayo Clinic, Rochester, Minnesota 55905


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
A novel region of amplification in breast tumors has recently been identified on chromosome 17q22–23. In an effort to identify the oncogenes in the region that are targeted by the amplification process, we determined the structure of the amplicon in breast cancer cell lines and tumors. Physical and transcription maps of the ~3.5-Mb region were established and used as the basis for copy number analysis within the region by Southern blot and fluorescence in situ hybridization. Seven specific and independent amplification maxima were identified in breast cancer cell lines and breast tumors. We present correlative amplification and overexpression studies for the FLJ21316 and Hs.6649 genes suggesting a role for these candidates as amplification-dependent oncogenes.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Gene amplification is a mechanism allowing for increased expression of critical genes involved in initiation and progression of cancer and response to therapy. A number of regions of recurrent amplification containing dominant oncogenes have been identified in breast cancer. These include ERBB2 at 17q12, PRAD1/CCND1 at 11q13, and CMYC at 8q24, which have been associated with poor patient survival, increased metastatic potential, and decreased disease-free survival (1, 2, 3) . More recently, comparative genomic hybridization of breast tumors and cancer cell lines has identified about 20 regions of amplification including 17q22–23 (4 , 5) .

Initial comparative genomic hybridization studies detected amplification of the 17q22–23 region in 18% of primary breast tumors (4) . Subsequent studies detected amplification in 18–31% of primary tumors (5 , 6) , 41% of breast metastases (6) , 50% of tumors containing BRCA1 mutations, 87% of breast tumors containing BRCA2 mutations (5) , and aneuploid tumors (7) . Amplification of the region has also been observed in meningiomas (8) . More detailed analysis of the region in breast cancer cell lines identified two independent peaks of amplification (9) and suggested that one or more genes located on 17q22–23 are preferentially selected for amplification and overexpression during tumor progression.

Several candidate oncogenes from the region have been identified. The TBX2 protein enhances cell proliferation by inhibiting senescence after amplification and overexpression of the gene (10) , whereas amplification of the RPSK6B1 gene, which encodes the p70-S6-kinase regulator of early response gene translation, correlates with poor patient survival (11 , 12) . Both genes are located at amplification maxima in the amplicon in MCF7 cells, which suggests that each peak of amplification harbors an oncogene. Here we describe in detail the complex structure of the amplicon in breast cancer cell lines and tumors. We identify seven independent amplification maxima and 20 genes that are amplified and overexpressed in cell lines and tumors and that are strong candidates as oncogenes.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Cell Lines and Tumors.
The MDA-MB157, MDA-MB361, MDA-MB468, MCF7, BT474, UACC812, UACC893 breast cancer cell lines were obtained from American Type Culture Collection. The 93 primary infiltrating ductal adenocarcinomas of the breast were described previously (13) . Corresponding paraffin-embedded tumor specimens were obtained from the Tissue Registry at the Mayo Clinic (Rochester, MN). Normal mammary epithelial cells were microdissected from reduction mammoplasty specimens.

Physical Map.
A total of 74 ESTs4 and STSs from the 17q22–23 region were identified from the GeneMap 1999 database.5 PAC and BAC clones from the region were identified by screening GenBank, the NCBI MapViewer database,6 the Whitehead Institute for Genomic Research contig database,7 and the Washington University Restriction Fingerprinting Map.8 BAC and PAC clones were obtained from Research Genetics Inc. (Huntsville, AL) or from BACPAC Resources Inc. (Oakland, CA). All of the clones in the minimal BAC and PAC contig were mapped to normal metaphase cells using FISH to confirm cytogenetic location.

Copy Number Analysis by Southern Blotting.
Genomic DNA from seven breast cancer cell lines and 93 primary breast tumors was used to generate Southern blots as described previously (11 , 13) . Southern hybridization with PCR-based probes from each of 50 ESTs and STSs was as described previously (13) . GAPDH was used as a control probe to assess loading differences on the blots. Signals were measured using a Molecular Dynamics PhosphorImager, and amplification levels were quantified by calculating a gene specific to GAPDH signal ratio for each sample, and by normalizing this ratio relative to the ratio in a normal breast epithelium sample on the same blot.

Copy Number Analysis by FISH.
Interphase dual-color FISH analysis of the MCF7 and BT474 cell lines and of paraffin-embedded breast tumor specimens was performed using BAC probes from the minimal contig and a CEP17 chromosome 17 centromeric probe (Vysis Inc.). Sixty intact nuclei were scored for each probe. Probe signals and CEP17 signals were counted in each nucleus and a ratio of mean probe signal to mean CEP17 signal was calculated. Ratios of >2.5 correspond to high-level amplification.

Transcription Map.
Analysis of the UCSC Human Genome Project,9 MapViewer, and GeneMap 1999 databases identified 36 candidate genes from the region covered by the physical map. Transcript size of known and novel genes was estimated by Northern blot analysis of breast cancer cell line RNA (13) . GAPDH was used as a control probe to assess loading differences. Levels of overexpression of each gene in the cell lines relative to a normal breast epithelium sample were calculated as described for Southern blotting.

Quantitative RT-PCR.
High quality total RNA was prepared from 59 of the 93 frozen breast tumors, primary mammary epithelial cells (HMEC; Clonetics) and microdissected normal mammary epithelial cells as described previously (13) . RT-PCR was performed as described (13) with primers as follows. For Hs.6649: forward, 5'-GGGACCATCATCACCAAACGA-3', and reverse, 5'-GGATGTTCCGAAGATGCAG-3'; for FLJ21316: forward, 5'-TGAGGCAAAGGGAAATGA-3', and reverse, 5'-AGGCTGAGGCAGGAGAAT-3'; and for GAPDH: forward, 5'-CAACTACATGGTTTACATGTTC-3', and reverse, 5'-GCCAGTGGACTCCACGAC-3'. The level of expression of the genes in tumors relative to normal mammary epithelial cells was calculated using the ratios described for Southern blotting.

Gene Assembly.
Unigene clusters containing ESTs from the region were assembled into extended cDNA sequences, and cDNAs were extended by 5' RACE, XGRAIL (Oak Ridge National Laboratory), and GeneScan (NCBI) analysis of BAC and PAC sequences from the region. cDNA sequences were verified by PCR of human testis cDNA.

Immunofluorescence.
293T cells were transiently transfected with a FLAG-tagged FLJ21316-pCR3.1 expression plasmid, fixed, and hybridized with a monoclonal anti-FLAG antibody (Sigma Chemical Co.) at 1:1000 dilution. Cells were washed and incubated with goat antimouse secondary antibody conjugated to Texas Red (Molecular Probes) at 1:800 and were incubated with 1 µg/ml HOECHST 33342. Images were obtained using a Zeiss confocal microscope.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Assembly of a 3.5-Mb Physical Map on 17q22–23.
We previously defined a region of amplification on chromosome 17q22–23 between positions 372 and 385 cR on the GeneMap 1999 radiation hybrid database (13) . In an effort to define the structure of this amplicon, we first established a physical map of the region. ESTs from chromosome 17q22–23 on the GeneMap 1999 database, were used to identify BACs and PACs by BLASTN homology searching of GenBank, and by screening the CITB BAC library. A partial contig was assembled by sequence analysis and by mapping of the BAC and PAC clones with 66 STSs. Twelve novel STSs, identified by end-sequencing, were also used to screen the BAC library. Analysis of the chromosome 17 restriction fingerprinted map verified the order and overlap of the clones and identified three more BAC clones that completed the contig. The result is a complete 3.5-Mb physical map of the region between Hs.97140 gene and the TRAP240 gene, containing a minimal contig of 33 BAC and PAC clones. Full-length or partial DNA sequence is available for 29 of the 33 BACs and PACs. A representation of the minimal contig, displaying BACs, PACs, ESTs, and STSs, is shown in Fig. 1Citation .10



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  		Fig. 1. Physical map of 17q22–23 between Hs.97140 and TRAP240. The contig is drawn as two overlapping sections. BAC and PAC clones are drawn as horizontal lines. The lengths of lines are proportional to the size of the clones. Mapped STSs and ESTs are shown as vertical lines that intersect BAC and PAC clones. All identified genes in the region are drawn above the physical map. The TRE-17 pseudogene (TRE17-like) is shaded in gray.

 
Candidate genes from the amplicon were originally identified as ESTs and known genes on the GeneMap 1999 radiation hybrid map and the UCSC Genome Database. In total, 36 candidate genes were identified (Fig. 1)Citation , including 13 known genes, 9 genes predicted from genomic sequences, and 14 genes based on ESTs or that do not have a known function. In addition, a pseudogene with 91% homology to the TRE2 oncogene from the 17q11.2 region is located 69 kb distal to the RPS6KB1 gene.

Amplification Analysis in Cell Lines.
We have previously reported that the amplicon contains more than one peak of amplification (11 , 13) . To better define the actual number of amplification targets in the region, seven breast cancer cell lines were screened for amplification (more than five copies) by Southern blotting with 52 probes representing all of the unique ESTs, STSs, and cDNAs from the region around the RPS6KB1 gene. By plotting copy number against physical map location for each probe, the structure of the amplicon in each breast cancer cell line was defined (Fig. 2A)Citation .



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  		Fig. 2. A, Southern blot analysis of copy number for 45 markers in the MCF7 and BT474 breast cancer cell lines relative to normal cellular DNA. Y axis, the fold increase in copy number relative to normal tissue; X axis, the markers used for Southern blotting. Markers are positioned according to their relative location in the physical map. Solid lines connect the measurements for each cell line. B, FISH analysis of amplification in the MCF7 and BT474 cell lines and two informative tumors using BAC probes from the physical map. Y axis, the amplification ratio of BAC-probe-signal-number:chromosome-17-centromere-signal-number. Ratios >2.5 reflect amplification. The 16 probes used in the analysis are shown on the X axis. BAC probes are positioned on the X axis to reflect their position in the physical map. Measurements for each sample are connected. C, Alu repeat frequency in each BAC and PAC clone in the physical map. Y axis, the percentage of Alu repeat sequence in each clone. X axis, each clone in its relative position in the physical map. D, frequency of marker amplification in breast tumors. The frequency of amplification of each marker in 93 breast tumors, as measured by Southern blot, is shown. Markers are positioned on the X axis relative to their position on the physical map.

 
Three independent amplification peaks were detected between Hs.97104 at 372 cR and TRAP240 at 385 cR on the GeneMap 1999 radiation hybrid map in MCF7 cells (Fig. 2A)Citation . The proximal peak covers about 400 kb and maximizes at 32 copies. The second peak covers ~1.5 Mb, extends from the FLJ21316 gene to the Hs.6649 gene, has a consistently high copy number of between 15 and 30, and maximizes at 32 copies. This peak contains the RPS6KB1 gene. The third peak extends from Hs.6649 to Hs.97515, covers 500 kb and maximizes at 50 copies. In BT474 cells, we detected a single 500-kb peak of between 15 and 29 copies, that maximizes at EST 24133 (Fig. 2A)Citation . This peak was not detected in MCF7 cells. Analysis of the other five breast cancer cell lines detected smaller regions of amplification that aligned well with the four peaks described. In total, 22 ESTs had more than 20 copies in at least one of the cell lines.

Amplification in the MCF7 and BT474 cell lines was verified by FISH analysis with 16 of the 33 BACs and PACs from the physical map (Fig. 2B)Citation . In MCF7 cells, all of the probes showed a signal to CEN17 ratio of greater than 3.0, indicating that the entire region is amplified. Three peaks of amplification corresponding to the peaks detected by Southern blot were observed. In BT474 cells, high-level amplification was observed across the proximal 2.5-Mb of the region. An amplification maximum of 25 copies between BACs 329E11 and 1073F15 corresponded to the Southern blot results (Fig. 2B)Citation .

Next, we investigated the density of Alu repeats in each BAC from the physical map because Alu repeat density corresponds to amplification maxima in several known amplicons.11 Five independent peaks of Alu repeat frequency corresponded to regions of high- level amplification in the cell lines, suggesting an association between amplification and Alu repeat density in this amplicon, and further strengthening the hypothesis that the amplicon contains at least five regions of independent amplification in cell lines and tumors.

Amplification Analysis in Breast Tumors.
Genomic DNA from 93 breast tumors was analyzed for copy number increases by Southern blot using probes from 36 known or predicted genes in the region. Whereas amplification levels were lower than detected in breast cancer cell lines, high-level amplification (>10 copies) of at least two probes was detected in 22 tumors. By setting the threshold for high-level amplification at seven gene copies because of underestimation of copy number by Southern blotting relative to FISH in cell lines, 40 (43%) tumors were found to display moderate- to high-level amplification of at least one part of the amplicon. A total of eight ESTs or STSs were amplified in at least 15% of the breast tumors as shown in Fig. 2DCitation . While the tumor studies verified the four amplification peaks in the cell lines, three additional peaks maximizing at CGI-147, PAT1, and 47342, were detected.

Amplification in tumors was also quantified by interphase FISH analysis of paraffin-embedded tumor samples with 13 BAC probes. Two representative samples are shown in Fig. 2BCitation . Tumor 1 is amplified at low levels across most of the region and has an amplification maximum in the region containing the WIP1 gene, which corresponds to one of the peaks suggested by Southern blotting. The peak of amplification in tumor 2 overlaps with the major peak identified in BT474 cells. These results indicate a complex pattern of amplification in this region and suggest that several independent candidate oncogenes may be present.

Expression of Amplified Genes.
To determine whether the amplified genes in the region were overexpressed in association with amplification, Northern blot analysis of breast cancer cell lines was performed. Of the genes tested, 20 were overexpressed in MCF7 and/or BT474 (Table 1)Citation . The FLJ21316 and Hs.6649 genes were the most highly expressed. In addition, the TBX2 and Hs.97868 genes were highly overexpressed in MCF7 cells, but the level of overexpression could not be calculated because of the complete absence of expression in other cell lines and in normal breast tissue.


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  		Table 1 Expression level of amplified genes in breast cancer cell lines

 
Characterization of Candidate Oncogenes.
We selected the FLJ21316 and Hs.6649 genes for further study because of their high levels of expression in cell lines and amplification in cell lines and breast tumors. The full length 2530-bp cDNA sequence of FLJ21316 was assembled by Unigene database analysis and by 5' RACE studies. The gene is composed of 12 exons including 11 coding exons. Southern blot analysis detected 15 gene copies in MCF7 cells and nine copies in BT474 cells (Fig. 3A)Citation , and amplification in 13% of breast tumors (Fig. 3B)Citation . Amplification corresponded well with overexpression in cell lines (Fig. 3C)Citation . The gene was expressed in all of the tissues, with elevated levels in ovarian and prostate tissue (Fig. 3D)Citation . qRT-PCR analysis of 59 tumors detected overexpression in four of seven amplified tumors and in six nonamplified tumors. Examples are shown in Fig. 3ECitation . The FLJ21316 protein is predicted to contain seven transmembrane domains and a single N-glycosylation site at amino acids 23–26 (Fig. 3F)Citation . Membrane localization of the protein was verified by immunofluorescence studies with FLAG-tagged FLJ21316 in 293T cells (Fig. 3G)Citation .



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  		Fig. 3. Characterization of the FLJ21316 gene. A, Southern blot analysis of FLJ21316 copy number in seven breast cancer cell lines. B, Southern blot analysis of the FLJ21316 in a subset of the breast tumors. N, a normal DNA sample. *, amplified samples. C, Northern blot expression analysis of FLJ21316 in the seven breast cancer cell lines. A GAPDH loading control is shown for each Southern and Northern blot. D, multiple tissue Northern blot analysis of the FLJ21316 gene. E, qRT-PCR analysis of FLJ21316 gene expression in the same breast tumors and in the same order as shown in B. GAPDH was coamplified as an internal normalizing control. F, predicted amino acid sequence of FLJ21316. Underlined, seven putative transmembrane domains; bold, a putative N-glycosylation site; arrowheads, exon boundaries determined by genomic sequence alignment. G, immunofluorescence analysis of FLAG-tagged FLJ21316 protein in 293T cells using an anti-FLAG antibody (red) and HOECHST 33342 DNA dye (blue). Left, FLAG-FLJ21316 with HOECHST 33342 staining; center, FLAG-FLJ21316 alone; right, HOECHST 33342 alone.

 
We also characterized the Hs.6649 gene. This gene is amplified 15-fold in MCF7 cells (Fig. 4A)Citation , and in 8% of breast tumors (Fig. 4B)Citation . Overexpression of multiple isoforms, especially a 1.5-kb isoform was detected in MCF7 cells by Northern blot (Fig. 4C)Citation , in three of the five amplified tumors tested by qRT-PCR, and in 10 other nonamplified tumors. Examples are shown in Fig. 4DCitation . The cDNA sequences of several Hs.6649 isoforms are available in GenBank, but the overexpressed 1.5-kb isoform has not yet been identified.



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  		Fig. 4. Characterization of the Hs.6649 gene; a GAPDH loading control is shown for each analysis. A, Southern blot analysis of Hs.6649 copy number with a 3' probe in seven breast cancer cell lines. B, Southern blot analysis of Hs.6649 with a 3' probe in a subset of the breast tumors. N, normal DNA sample. C, Northern blot expression analysis of Hs.6649 in the seven breast cancer cell lines. D, qRT-PCR analysis of Hs.6649 gene expression in the same breast tumors and in the same order as in B; *, amplified samples. GAPDH was coamplified as an internal normalizing control.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
In this study, we have extended our understanding of the 17q22–23 amplicon by accurately mapping its structure in breast cancer cell lines and in breast tumors. We have shown that the RPS6KB1 and TBX2 candidate oncogenes are among the most highly amplified genes from the region in MCF7 cells, but that these genes are infrequently amplified in breast tumors. We have determined that the amplicon has a highly complex structure, with at least seven independent amplification maxima in cell lines and breast tumors. In addition, we have detected different combinations of amplified genes in many of the 43% of breast tumors displaying amplification of 17q22–23. These data suggest that the amplicon may contain as many as seven different oncogenes, one from each peak of amplification, that are initially coamplified but are subsequently independently selected and retained during tumor progression.

In an effort to identify these oncogenes, we mapped the positions of the 36 candidate genes from the region onto the structural map of the amplicon. All of them are amplified in breast cancer cell lines and/or breast tumors, albeit at different levels and frequencies, whereas 22 of the genes are highly amplified and map to amplicon peaks. This number of candidate genes is likely to increase as other as-yet-unknown genes are identified through genome sequence analysis. In an attempt to reduce the number of candidate oncogenes, amplification of each gene in the amplicon was correlated with overexpression in cell lines. The MTMR4, NACA, PNUTL2, and CGI147 genes are all amplified, but are not overexpressed, and have been excluded as candidate oncogenes (Table 1)Citation . In contrast, we have verified that 20 of the genes from the amplicon peaks are overexpressed. Although the genes located at amplification maxima may be considered the best candidate oncogenes, it is important to consider that amplified genes that are not positioned at amplification maxima, such as the BTAK gene on 20q13.2, can function as oncogenes (14) .

In the course of our studies, we identified the FLJ21316 and Hs.6649 genes as attractive candidate oncogenes. The FLJ21316 gene is centrally located in the core of the amplicon at the peak of Alu repeat density, and encodes a seven-transmembrane-domain protein that is localized to the plasma membrane. This suggests that the protein is a member of the superfamily of seven-transmembrane-domain hormone, cytokine, growth-factor, and G-protein receptors. Because many of these proteins regulate cell growth and proliferation, it is possible that amplification and overexpression of FLJ21316 contributes to tumor progression. FLJ21316 is amplified in 13% of tumors and is overexpressed in all of the highly amplified tumors but not in several tumors with low-level amplification. Future functional studies will elucidate the relevance of the FLJ21316 amplification and expression to tumor progression.

Our analysis of the Hs.6649 gene has yielded some interesting findings. First, we have identified a highly overexpressed isoform that is not accounted for by the known cDNA sequence. Perhaps the expression of this isoform is regulated by an alternative, and as yet unknown, promoter in the region. Second, our initial copy number analysis using a probe in the 3' end of the gene detected high levels of amplification (30 copies) in the MCF7 cell line. After the completion of the physical map, we found that Hs.6649 is spread over a 300-kb region. Subsequent copy number analyses using STSs located at the center (WI-22728) and 5' end (264B14X) of the gene detected low levels of amplification (<10 copies) in MCF7 cells. This suggests that the high level of recombination within the amplicon results in breakage within the gene and amplification of the 3' end independently of the promoter and 5' end. Incomplete amplification of the gene is reflected in the 5-fold increase in expression level detected by Northern blotting when excluding the 1.5-kb overexpressed isoform. Thus, hyperrecombination and incomplete amplification within the amplicon may also explain the poor correlation between amplification and overexpression for other genes, although this may be restricted to the larger genes in the region.


   ACKNOWLEDGMENTS
 
We thank Kari Anderl, Mark Law, and Dr. Robert Jenkins for assistance with FISH analysis, and Christene Hettinga for assistance with the preparation of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by the Medical Research and Materiel Command of the United States Army (DAMD17-99-9282), by the Breast Cancer Research Foundation, and by NIH Training Grant CA 75926 (to C. S.). Back

2 G-j. W. and C. S. contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 First Street, S.W., Rochester, MN 55905. Phone: (507) 266-0878; Fax: (507) 266-0824; E-mail: couch.fergus{at}mayo.edu Back

4 The abbreviations used are: EST, expressed sequence tag; FISH, fluorescence in situ hybridization; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; STS, sequence tagged site; RT-PCR, reverse transcription-PCR; qRT-PCR, quantitative RT-PCR; BAC, bacterial artificial chromosome; PAC, P1 artificial chromosome; NCBI, National Center for Biotechnology Information; cR, centiRay. Back

5 Internet address: http://www.ncbi.nlm.nih.gov/genemap/. Back

6 Internet address: http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/hum_srch. Back

7 Internet address: http://www-genome.wi.mit.edu/. Back

8 Internet address: http://genome.wustl.edu/gsc/human/human_database.shtml. Back

9 Internet address: http://genome.ucsc.edu. Back

10 The sequences reported in this manuscript have been reported to GenBank (accession no. AF214006 and AF260269). Back

11 Colin Collins, personal communication. Back

Received 2/22/01. Accepted 5/16/01.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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L. Melchor, S. Alvarez, E. Honrado, J. Palacios, A. Barroso, O. Diez, A. Osorio, and J. Benitez
The Accumulation of Specific Amplifications Characterizes Two Different Genomic Pathways of Evolution of Familial Breast Tumors
Clin. Cancer Res., December 15, 2005; 11(24): 8577 - 8584.
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M. Nessling, K. Richter, C. Schwaenen, P. Roerig, G. Wrobel, S. Wessendorf, B. Fritz, M. Bentz, H.-P. Sinn, B. Radlwimmer, et al.
Candidate Genes in Breast Cancer Revealed by Microarray-Based Comparative Genomic Hybridization of Archived Tissue
Cancer Res., January 15, 2005; 65(2): 439 - 447.
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A. Hirasawa, F. Saito-Ohara, J. Inoue, D. Aoki, N. Susumu, T. Yokoyama, S. Nozawa, J. Inazawa, and I. Imoto
Association of 17q21-q24 Gain in Ovarian Clear Cell Adenocarcinomas with Poor Prognosis and Identification of PPM1D and APPBP2 as Likely Amplification Targets
Clin. Cancer Res., June 1, 2003; 9(6): 1995 - 2004.
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F. Saito-Ohara, I. Imoto, J. Inoue, H. Hosoi, A. Nakagawara, T. Sugimoto, and J. Inazawa
PPM1D Is a Potential Target for 17q Gain in Neuroblastoma
Cancer Res., April 15, 2003; 63(8): 1876 - 1883.
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X. Huang, S. M. Gollin, S. Raja, and T. E. Godfrey
High-resolution mapping of the 11q13 amplicon and identification of a gene, TAOS1, that is amplified and overexpressed in oral cancer cells
PNAS, August 20, 2002; 99(17): 11369 - 11374.
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C. S. Sinclair, C. Adem, A. Naderi, C. L. Soderberg, M. Johnson, L. Wadum, V. L. Couch, T. A. Sellers, D. Schaid, J. Slezak, et al.
TBX2 Is Preferentially Amplified in BRCA1- and BRCA2-related Breast Tumors
Cancer Res., July 1, 2002; 62(13): 3587 - 3591.
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 Neuro Oncol DukeHome page
G. D. Eley, J. L. Reiter, A. Pandita, S. Park, R. B. Jenkins, N. J. Maihle, and C. D. James
A chromosomal region 7p11.2 transcript map: Its development and application to the study of EGFR amplicons in glioblastoma
Neuro-oncol, April 1, 2002; 4(2): 86 - 94.
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