SEARCH

SEARCH BY CITATION

Keywords:

  • lobular breast cancer;
  • CDH1 mutation;
  • array CGH;
  • SCID mouse xenograft model;
  • chemoresistance

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Supporting information
  9. REFERENCES
  10. Supporting Information

Infiltrating lobular breast cancer (ILBC) is a clinically and biologically distinct tumour entity defined by a characteristic linear cord invasion pattern and inactivation of the CDH1 tumour suppressor gene encoding for E-cadherin. ILBCs also lack β-catenin expression and show aberrant cytoplasmic localization of the E-cadherin binding protein p120-catenin. The lack of a well-characterized ILBC cell line has hampered the functional characterization of ILBC cells in vitro. We report the establishment of a permanent ILBC cell line, named IPH-926, which was derived from a patient with metastatic ILBC. The DNA fingerprint of IPH-926 verified genetic identity with the patient and had no match among the human cell line collections of several international biological resource banks. IPH-926 expressed various epithelial cell markers but lacked expression of E-cadherin due to a previously unreported, homozygous CDH1 241ins4 frameshift mutation. Detection of the same CDH1 241ins4 mutation in archival tumour tissue of the corresponding primary ILBC proved the clonal origin of IPH-926 from this particular tumour. IPH-926 also lacked β-catenin expression and showed aberrant cytoplasmic localization of p120-catenin. Array-CGH analysis of IPH-926 revealed a profile of genomic imbalances that included many distinct alterations previously observed in primary ILBCs. Spectral karyotyping of IPH-926 showed a hyperdiploid chromosome complement and numerous clonal, structural aberrations. IPH-926 cells were anti-cancer drug-resistant, clonogenic in soft agar, and tumourigenic in SCID mice. In xenograft tumours, IPH-926 cells recapitulated the linear cord invasion pattern that defines ILBCs. In summary, IPH-926 significantly extends the biological spectrum of the established breast cancer cell lines and will facilitate functional analyses of genuine human ILBC cells in vitro and in vivo. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Supporting information
  9. REFERENCES
  10. Supporting Information

Infiltrating lobular breast carcinoma (ILBC) accounts for approximately 15% of all breast cancer cases 1. ILBCs consist of small, uniform tumour cells arranged in characteristic single-file linear cords that invade the stroma. This peculiar histoarchitecture distinguishes ILBCs from the more common infiltrating ductal breast cancers (IDBCs). ILBCs also display distinctive genetic, molecular, and clinical features. They lack expression of E-cadherin 2 and this serves as a supportive parameter in their clinical diagnosis 1. Loss of E-cadherin expression is always accompanied by a loss of β-catenin expression and is frequently associated with aberrant cytoplasmic localization of the E-cadherin binding protein p120-catenin in ILBCs 3, 4.

The diverse mechanisms of CDH1 inactivation in ILBCs comprise truncating mutations and allelic loss as well as epigenetic silencing 5–7. Germline CDH1 mutation is the genetic defect underlying some rare cases of hereditary ILBCs 3, 8. Profiling approaches focusing on genomic imbalances established that nearly all ILBCs harbour a loss of 16q22, the locus of CDH19. This alteration is frequently accompanied by gain of 1q, loss of 8p23–p22, concomitant gains of 8p12–p11 and 11q12–q13, and loss of 11q14-qter 9, 10. In the clinics, ILBCs frequently metastasize to the peritoneum and abdominal organs 11. ILBCs are mostly oestrogen receptor (ER)-positive and adjuvant endocrine therapy represents the treatment of choice 12. Several studies have demonstrated the chemoresistance of ILBCs towards conventional anti-cancer agents 12–14, perhaps related to the slow proliferation of ILBC cells 13.

Investigation of ILBC at a cell biological level has been hampered by the absence of ILBC-like lesions in most genetically engineered mouse models and the lack of well-characterized human ILBC cell lines 15, 16. Derksen et al developed a mouse tumour model with an ILBC-like histoarchitecture 17. Of human breast cancer cell lines, only two, HCC-2185 and MDA-MB-330, have been proposed to derive from classical ILBCs 15, 18, 19 but the corresponding primary tumours were not documented 18–20 and their mutational status of CDH1 was undefined. Furthermore, HCC-2185 and MDA-MB-330 were not tumourigenic in vivo18, 19. Recently, it was proposed that the cell line MDA-MB-134 might be derived from an ILBC 10. However, MDA-MB-134 was originally reported to be derived from a ductal mammary carcinoma 21 is widely perceived as a ductal breast cancer cell line [e.g., 15], usually non-tumourigenic in vivo15, 18, 21–23 and lacking E-cadherin expression due to a homozygous mutation of the CDH1 gene 24. However, identical allozyme phenotype patterns in the MDA-MB-134 and the MDA-MB-309 cell lines raised concerns about a potential cross-contamination or mis-identification of both cell lines early after their establishment 25.

Therefore the establishment and accurate characterization of a tumourigenic ILBC cell line with a defined CDH1 mutation have not yet been achieved. This may reflect a particular difficulty in the in vitro propagation of tumour cells from this slow-growing tumour entity 1, 9, 13. Here, we report the establishment and detailed molecular, genetic, and functional characterization of a novel human ILBC cell line named IPH-926.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Supporting information
  9. REFERENCES
  10. Supporting Information

Cell lines

The human breast carcinoma cell lines MDA-MB-134, MCF-7, and UACC-893 were obtained from ATCC (Manassas, USA). The human ductal breast carcinoma cell line KPL-1 and the HL-60, IM-9, and RAJI cell lines were obtained from DSMZ (Braunschweig, Germany). Additional cell lines and their culture conditions have been described previously 26. For functional analyses, cells were incubated in medium containing the FGFR1 inhibitor SU5402 (Calbiochem, Darmstadt, Germany) 27.

Culture conditions of IPH-926

With the approval of the local ethics committee, IPH-926 carcinoma cells were derived from malignant ascites of a 72-year-old female with peritoneal metastases from ILBC. In 1990, at the age of 56, this patient had first been diagnosed with ER-positive ILBC (66 fmol/mg protein) and underwent breast-conserving surgery, axillary dissection, and radiotherapy. The patient received tamoxifen (20 mg/day) for 5 years but presented with a locally recurrent ILBC in 1996 and underwent mastectomy. In 1998, the patient experienced an axillary relapse (dermal metastasis) and was subsequently diagnosed with liver and peritoneal metastases. The patient was treated with various regimes of chemotherapy including epirubicin, cyclophosphamide, paclitaxel, and capecitabine. In 2005–2006, the peritoneal cavity was repeatedly punctured to drain malignant ascites for palliation. Ascites-derived cells were sieved through a 100 µm cell strainer; resuspended in 150 ml of serum-free DMEM/F12 containing 10 mM HEPES, 1 mM sodium pyruvate, 10 µg/ml bovine insulin, and 0.1 mM non-essential amino acids; transferred to 6 × 140 mm (diameter) cell culture dishes; and subjected to a short-term culture of 30 days with regular medium changes starting at day 5. Clonal isolation was then performed as described previously 28. Larger aggregates of cells of epitheloid appearance, which adhered to the bottom of the culture flask, were identified under the microscope and picked and placed into separate vessels on a 24-well plate. Three out of 12 clones continued to grow and one was chosen for further cultivation. Cells were then expanded in RPMI-1640 containing 10 mM HEPES, 2 mM glutamine, 1 mM sodium pyruvate, 2.5 g/l glucose, 10 µg/ml bovine insulin, and 20% FCS in a water-saturated atmosphere containing 5% CO2 at 37 °C.

DNA typing

Genomic DNA was isolated from cell lines and formalin-fixed, paraffin-embedded (FFPE) tissue using proteinase K digestion and organic extractions according to standard procedures. Highly polymorphic short tandem repeat (STR) DNA markers (SE33, VWA31A, FGA) were amplified in standard PCR reactions and analysed on an ABI Prism 310 system. PCR primers are given in the Supporting information, Supplementary Methods. An additional DNA fingerprinting analysis, using an extended panel of STR markers according to the international reference standards for authentication of human cell lines, was independently performed by the DSMZ 29.

mRNA expression analyses

Total RNA was extracted from cells as described previously 26. The cDNA synthesis was performed using Superscript II reverse transcriptase (Invitrogen, Karlsruhe, Germany) and 1 µg of DNAse I-treated total RNA as template in a 20 µl reaction volume. Standard end-point PCR was performed with 1 µl of cDNA as template in a 25 µl reaction volume using Platinum Taq DNA polymerase (Invitrogen) according to the manufacturer's recommendations. Quantitative assessment of gene expression normalized to the housekeeping gene β-GUS was performed with Sybr Green I (Invitrogen) on an ABI Prism 7700 system as described previously 26. PCR primers are given in the Supporting information, Supplementary Methods.

Western blot

For the detection of protein expression, cell lines were lysed in RIPA buffer and 30 µg of total cellular proteins was separated by 12% SDS-PAGE and transferred to nitrocellulose membranes 26. Membranes were probed with antibodies against the following proteins: KRT18, KRT19, ErbB2, ER, E-cadherin, β-catenin, p120-catenin, FGFR1, PARP, and β-actin. Antibody clones and dilutions may be found in the Supporting information, Supplementary Methods.

Flow cytometry

Flow cytometric detection of TACSTD1/Ep-CAM was performed with the Ber-EP4 antibody (Dako, Hamburg, Germany) using a MoFlo cell sorter as described in the Supporting information, Supplementary Methods.

Immunohistochemistry

Immunohistochemical staining of FFPE tissue and cell lines was performed with the same antibodies as those used for western blots and the tyramine amplification technique as previously described 26.

Confocal microscopy

Indirect immunofluorescent staining was performed on LAB-TEK-II chamber slides with an anti-p120-catenin antibody (clone #98, BD Transduction Laboratories) and observed with a Leica DM IRB confocal laser scanning microscope equipped with a TCS SP2 AOBS scan head.

Laser microdissection and loss of heterozygosity (LOH) analysis

Laser microdissection and DNA extraction of tumour cells from FFPE tissue were performed with a PALM Laser-MicroBeam System and proteinase K digestion as described previously 30. Allelic loss at the CDH1 locus was assessed with the microsatellite marker D16S752, which maps to 16q22 close to the CDH1 gene 3.

Analysis of CDH1 gene alterations

For mutational analysis of the CDH1 gene, all 16 coding exons and flanking regulatory regions of CDH1 were amplified 5. Purified PCR products were subjected to direct sequencing. Oligonucleotide primers designed to amplify the CDH1 mutation site in IPH-926 cells were forward, 5′-CTCGACACCCGATTCAAAGT-3′; reverse, 5′-CCG- TAGAGGCCTTTTGACTG-3′. Methylation-specific PCR was done with bisulphite-treated genomic DNA 31 and CDH1 methylation-sensitive PCR primers described elsewhere 32.

Array CGH

DNA chips containing around 8000 individual BAC/ PAC (DKFZ, Heidelberg, Germany) clones spotted in triplicate were used. They allow a genome-wide resolution of 1 Mb and an even higher resolution of up to 100 kb for chromosome regions recurrently involved in human tumours. Isolation and spotting of DNA probes were performed as described previously 33. Probe processing and image analysis were carried out as published earlier 34. Data normalization and analysis were done using the software packages marray and a-CGH from R software3 (http://www.r-project.org). Raw fluorescence intensity values were normalized applying the print-tip LOESS normalization function. Spot quality criteria were set as foreground to background greater than 3.0 and standard deviation of triplicates less than 0.2. For breakpoint calling, GLAD software was used 35. Thresholds for copy number loss or gain were set at − 0.2 and + 0.2, respectively. CNAs concerning one single BAC/PAC clone only were assessed as outliers and not further analysed.

Spectral karyotyping

Spectral karyotyping (SKY) analysis was performed with SKYPaint reagents (ASI Ltd, Migdal Ha'Emek, Israel) as described previously 36. Details are given in the Supporting information, Supplementary Methods.

Fluorescence in situ hybridization (FISH)

FISH analyses on cells fixed in absolute ethanol were performed as described previously 37. Probe generation and hybridization conditions are described in the Supporting information, Supplementary Methods.

Quantitative gene dosage PCR

Quantitative gene dosage PCR was performed essentially as described before 30. Details of the reaction conditions, PCR primers and data evaluation are given in the Supporting information, Supplementary Methods.

Soft agar assay

To test the ability of cells to form colonies under anchorage-independent conditions in a semi-solid medium, a soft agar assay was carried out as described previously 38.

Cell death and proliferation assays

Detection of cell death was performed with the cytotoxicity detection kit plus (Roche, Mannheim, Germany) according to the manufacturer's recommendations. Relative cell proliferation was determined with the WST-1 proliferation reagent plus (Roche) according to the manufacturer's recommendations. Detection of PARP cleavage, indicative of apoptosis, was performed as described before 39.

Tumour xenografts

Animal experiments were in accordance with international guidelines and approved by the local ethics committee. Female NOD/SCID mice (n = 5), 6 weeks of age, were obtained from the Institute of Laboratory Animal Science (Hannover, Germany) and inoculated subcutaneously with 3 × 106 tumour cells. After 18 weeks, animals were sacrificed and tumours were harvested.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Supporting information
  9. REFERENCES
  10. Supporting Information

Epithelial origin and lack of E-cadherin expression in IPH-926

IPH-926 was derived from the malignant ascites of a 72-year-old female with peritoneal metastases from ILBC. Figure 1A shows the histomorphology of the corresponding primary tumour, a lobular mammary carcinoma (Figure 1A). IPH-926 cells have been continuously cultivated for more than 34 months (approximately 72 doubling times and 32 passages). DNA typing at passages 4, 7, and 15 demonstrated genetic identity of IPH-926 with primary tissue of the patient (Figure 1B). An additional DNA fingerprinting analysis was performed by the DSMZ (German Collection of Micro-organisms and Cell Cultures) (Supporting information, Supplementary Figure 1). The IPH-926 DNA fingerprint does not match any other known human cell line at the DSMZ (Germany), the ATCC (USA) or the JCRB (Japan) cell line collections. The epithelial origin of IPH-926 was verified by expression analyses at passages 3–7 (Figures 1C–1F). IPH-926 expressed the epithelial cell markers TACSTD1/Ep-CAM, KRT18, and KRT19 at the mRNA as well as at the protein level (Figures 1C–1F). In contrast, IPH-926 entirely lacked mRNA expression of the mesothelial cell marker CALB2 and of the B-lymphoblastoid cell marker BLAST140, 41 (Figure 1C). Moreover, IPH-926 cells completely lacked mRNA expression of ESR1, encoding for ER; CDH1, encoding for E-cadherin; and CTNNB1, encoding for β-catenin (Figures 1C and 1D). Consistently, IPH-926 also lacked protein expression of ER, E-cadherin, and β-catenin (Figure 1E, lower panel). ERBB2 mRNA was expressed at a very low level in IPH-926 (Figure 1D). Similar to the ErbB2-negative cell line MCF-7, which also shows a very low level of ERBB2 mRNA, IPH-926 cells completely lacked ErbB2 protein expression (Figure 1E, lower panel). IPH-926 also entirely lacked ErbB2 immunoreactivity (Figure 1G). Hence, IPH-926 was classified as ErbB2-negative. The ILBC of the patient also lacked ErbB2, E-cadherin, and β-catenin expression (Figure 1G and Table 1). A detailed comparison of the immunoreactivity profiles and molecular and genetic properties of IPH-926 and the corresponding primary and recurrent ILBC of the patient is given in Table 1.

thumbnail image

Figure 1. Characterization of IPH-926 cells by DNA typing and expression analyses. (A) H&E-stained paraffin section of the primary ILBC and IPH-926 cells in vitro. Arrows indicate a single-file linear cord of tumour cells and an intracytoplasmic lumen. (B) DNA typing demonstrating genetic identity of IPH-926 passage 4 and the patient. Identical results were obtained at passages 7 and 15. (C) Analysis of mRNA expression by standard end-point reverse transcription (RT)-PCR. Primary mesothelioma tissue (MST-156) served as a positive control for CALB2, a mesothelial cell marker. The B-lymphoblastoid cell line IM-9 served as a positive control for BLAST1, a B-lymphoblastoid cell marker. The breast cancer cell line MCF-7, its subclone KPL-1, and the breast cancer cell line UACC-893 served as controls for TACSTD1, encoding for the epithelial cell-specific adhesion molecule Ep-CAM; the epithelial keratins KRT18 and KRT19, ERBB2, and ESR1, encoding for oestrogen receptor (ER); CDH1, encoding for E-cadherin; and CTNNB1, encoding for β-catenin. Amplification of the housekeeping gene β-GUS served as a control for the cDNA preparations. (D) Analysis of mRNA expression by quantitative RT-PCR. Cell lines and target genes correspond to the end-point RT-PCR shown in C. Note that ERBB2 mRNA is detected at a very low level in IPH-926. (E) Protein expression analysis by western blot. HL-60 and KPL-1 served as negative and positive controls for the KRT18 and KRT19. UACC-893 served as a positive control for ErbB2. MCF-7 and KPL-1 served as positive controls for ER. MCF-7, KPL-1, and UACC-893 served as controls for E-cadherin and β-catenin. Detection of β-actin verified equal loading. (F) Ep-CAM protein expression analysis by flow cytometry using the Ber-EP4 antibody. HL-60 and KPL-1 served as negative and positive controls. (G) Lack of immunoreactivity for ErbB2, ER, E-cadherin, and β-catenin in IPH-926 cells. UACC-893 cells served as a positive control

Download figure to PowerPoint

Table 1. Comparison of the features of the IPH-926 cell line and the ILBC of the corresponding patient
 Primary ILBCLocally recurrent ILBCAxillary relapseIPH-926
  1. Disease stage was determined according the UICC–TNM Classification 42. Grading was determined according to the Bloom–Scarf–Richardson score. LIN = lobular intraepithelial neoplasia; ILBC = infiltrating lobular breast cancer; Pos. = positive; Pos. (cyt.) = positive with strong cytoplasmatic reactivity; Neg. = negative; n.d. = not determined; LI = labelling index.

Year of sampling1990199619982006
Patient age, years566264
HistologyLIN + ILBCILBCILBC metastasis
 TpT1crpT1bcT0
 NpN0pN0cN0
 MpM0pM0pM1
 GradingG1G2G3
Immunoreactivity
 E-cadherinNeg.Neg.Neg.Neg.
 ERPos.Neg.Neg.Neg.
 PRNeg.Neg.Neg.Neg.
 ErbB2Neg.Neg.Neg.Neg.
 KRT18Pos.Pos.Pos.Pos.
 FGFR1n.d.Pos.Pos.Pos.
 β-cateninNeg.Neg.Neg.Neg.
 p120-cateninn.d.Pos. (cyt.)Pos. (cyt.)Pos. (cyt.)
 Ki67 LI< 101515< 10
CDH1 status
 LOHn.d.n.d.Pos.Pos.
 Mutation241ins4241ins4241ins4241ins4

A unique CDH1 mutation in IPH-926 and its primary ILBC

Truncating CDH1 gene mutations and allelic loss or epigenetic silencing account for the absence of E-cadherin expression in human ILBCs 5–7. IPH-926 passage 4 and 7 cells showed a loss of heterozygosity (LOH) at the CDH1 locus at chromosome 16q22, indicating that one 16q22 allele was deleted (Figure 2B, lane 3). Deletion of the same 16q22 allele in the corresponding ILBC of the patient was demonstrated by PCR analyses of laser-microdissected tumour cells from archival FFPE tissue (purity > 95% tumour cells) (Figure 2B, lane 5). IPH-926 did not exhibit aberrant methylation of the CDH1 promoter, suggesting that the remaining CDH1 allele is inactivated by mutation in these cells (Figure 2A). Mutational analysis of CDH1 in IPH-926 cells at passage 4 identified a previously unreported homozygous 4 base pair insertion in exon 3, which corresponds to a 241ins4 nucleotide change (Figure 2C). This mutation causes a frameshift and a premature stop at codon 93. The resulting truncated protein lacks all functionally important domains (Figure 2E). The 241ins4 mutation was also detectable by PCR using flanking primers (Figure 2D). IPH-926 cells at various passages as well as archival tumour tissue showed a size shift of the amplification product that indicated the 241ins4 mutation. This was not detectable in adjacent normal mammary tissue or other established breast cancer cell lines (Figure 2D). Analysis of laser-microdissected ILBC cells from archival tumour tissue verified that the 241ins4 mutation was homozygous in the ILBC of the patient (Figure 2D, lanes 6 and 7). Detection of this 241ins4 mutation in the primary ILBC and its subsequent relapses represents a unique genetic marker that firmly establishes the clonal relationship between the different tumour samples (Table 1) and the cell line IPH-926.

thumbnail image

Figure 2. CDH1 gene alterations in IPH-926. (A) Analysis of CDH1 promoter methylation in IPH-926 passage 4 by methylation-specific PCR using primers specific for the methylated (M) or unmethylated (U) CDH1 promoter region. RAJI cells served as a positive control. (B) CDH1 LOH analysis using the D16S752 microsatellite marker in IPH-926 passage 7 cells. Identical results were obtained with IPH-926 at passage 4. Note the loss of the same allele in laser-microdissected (md.) tumour cells from archival FFPE tissue of the axillary relapse of the corresponding patient (purity > 95% tumour cells). (C) Sequencing of CDH1 in IPH-926 cells at passage 4. The rectangle in the top panel accentuates a previously unreported CDH1 mutation corresponding to a 241ins4 nucleotide change according to the common nomenclature 6. The bottom panel shows the genomic organization of the 16 exons of CDH1. (D) PCR amplification of the region spanning the insertion site. A 60 base-pair product indicates wild type (WT); a 64 base-pair product indicates the 241ins4 mutation. The presence of both the WT allele and the 241ins4 mutation allele reflects the mixture of cancerous and non-cancerous tissue components in the clinical tumour specimens. Note that the WT allele is virtually undetectable in laser-microdissected (md.) tumour cells from archival FFPE tissue of the patient. (E) Schematic representation of human E-cadherin protein. N = N-terminus; C = C-terminus; SIG = signal peptide; PRE = precursor sequence; ECD = extracellular domains; TMD = transmembrane domain; CPD = cytoplasmic domain. The 241ins4 mutation results in a frameshift and a premature stop at codon 93. The corresponding truncated E-cadherin protein is shown at the bottom

Download figure to PowerPoint

Genomic imbalances in IPH-926 are characteristic of ILBC

Genomic imbalances recurrently observed in primary ILBCs include gain of 1q, gain of 5p15, gain of 9q33–q34, loss of 8p23–p22, concomitant gains of 8p12–p11 and 11q12–q13, loss of 11q14-qter, gain of 12q23–q24, loss of 15q21, loss of 16q22, gain of 19p13, gain of 20q11–q13, and several other distinct alterations 9, 10. The total number of chromosomal arms with genomic imbalances in primary ILBC ranges from 3 to 15 10. The total number of chromosomal arms with genomic imbalances in IPH-926 passage 4 cells was 31. Interestingly, IPH-926 harboured most of the above-mentioned distinct genomic imbalances (Figure 3A, Figure 6 and Supporting information, Supplementary Data Table 1). IPH-926 lacked a loss of 16q22, the locus of CDH1, as determined by array CGH (Figure 3A). As CDH1 mutational analyses identified a homozygous CDH1 mutation and LOH analyses demonstrated that one 16q22 allele was deleted, these results indicate that the mutated CDH1 allele was duplicated. This was also supported by spectral karyotyping of IPH-926 passage 14 cells showing two copies of chromosome 16 and an additional, aberrant chromosome involving material of the chromosomes 16 and 5 (Figures 3B and 3C). In line with the complex genomic imbalances detected by array CGH, spectral karyotyping revealed a plethora of further structural aberrations, which were all clonal and thus verified the clonality of the IPH-926 cell line.

thumbnail image

Figure 3. Genomic imbalances in IPH-926. (A) Array-based CGH plot of IPH-926 passage 4 cells. Log2 ratio is plotted on the Y-axis against each clone according to the genomic location on the X-axis. Vertical dotted lines, centromeres; horizontal dotted lines, log2 ratios of − 1, − 0.5, 0.5, and 1; red triangles, losses characteristic of ILBC; green triangles, gains characteristic of ILBC; grey triangle, CDH1 locus at 16q. (B) Spectral karyotyping of IPH-926 passage 14 cells. A representative metaphase spread is shown as an inverted DAPI-banded image (left) and in SKY classification colours (right). (C) SKY karyotype after chromosomal classification. Note the two copies of chromosome 16 and the additional aberrant chromosome involving material from chromosomes 16 and 5

Download figure to PowerPoint

Functional characterization of IPH-926

IPH-926 ILBC cells grow extraordinarily slowly in vitro (Figure 4A). However, IPH-926 passage 15 cells were clonogenic in soft agar (Figure 4B) and tumourigenic in SCID mice (Figure 4C). Subcutaneous inoculation of 3 × 106 IPH-926 ILBC cells resulted in macroscopically visible tumour growth within 18 weeks (Figure 4C). Xenograft tumours displayed a high tumour cell density in the central areas (Figure 4D). Tumour cells were comparatively small, uniform, and arranged in strands. At the invasion front, tumour cells infiltrated the surrounding tissue in characteristic single-file linear cords, which are typical for human ILBCs (Figure 4E, left panel). Some IPH-926 cells were individually dispersed through the tissue (Figure 4E, right panel). Furthermore, IPH-926 xenograft tumour cells displayed the occasional occurrence of intracytoplasmic lumina with central mucoid inclusions, another highly characteristic feature of human ILBCs (Figure 4E, right panel) 1. Hence, the histomorphology of IPH-926 xenograft tumours was well in line with the origin of IPH-926 cells from a human ILBC. Several clinical studies have demonstrated the poor responsiveness of ILBCs to chemotherapy 12–14. IPH-926 passage 16–18 cells were highly resistant to doxorubicin, a commonly used anti-cancer agent (Figure 4F). Continuous exposure to doxorubicin for 2 days induced the cell death of MCF-7 and UACC-893 cells, but not of IPH-926 cells (Figure 4F). Doxorubicin failed to induce apoptosis, as determined by PARP cleavage, in IPH-926 ILBC cells (Figure 4G).

thumbnail image

Figure 4. Functional characterization of IPH-926. (A) In vitro growth kinetics of IPH-926 assessed at passage 5 (rectangles) and 10 (circles). Doubling time was about 14 days. (B) Clonogenic growth of IPH-926 passage 15 cells in soft agar. Photographs were taken 18 weeks after plating at 40-fold magnification. (C) Macroscopic appearance of IPH-926 passage 15 xenograft tumour arising 18 weeks after subcutaneous inoculation in SCID mice. Arrows indicate the tumour. (D) Two representative micrographs of the central areas of IPH-926 xenograft tumours. Central areas displayed a high tumour cell density. Note the strand-like formation of the comparatively small, uniform tumour cells. (E) Two representative H&E-stained paraffin sections of IPH-926 xenograft tumours showing the histomorphological characteristics at the invasion front. Arrows indicate single-file linear cords and an intracytoplasmic lumen. (F) Resistance of IPH-926 cells to doxorubicin. Human breast cancer cells were exposed to various concentrations of doxorubicin for 2 days and cell survival was assessed. Error bars represent SEM. Experiments were performed at passages 16–18. Representative results of one of three experiments are shown. (G) PARP cleavage, indicative of cell death, in human breast cancer cells treated with 50 µ M doxorubicin (dox.) for 2 days

Download figure to PowerPoint

Comparison of IPH-926 and MDA-MB-134

Having established IPH-926 as the first cancer cell line with a proven origin from human ILBC, a systematic comparison with the MDA-MB-134 cell line, whose potential origin from a misdiagnosed or occult ILBC has recently been suggested 10, was performed. Both cell lines are characterized by grape-like clusters of small, round cells and the occasional appearance of larger, flattened cells (Figure 1A) 21, 22. In comparison to IPH-926 cells (doubling time of about 2 weeks), MDA-MB-134 cells proliferate more rapidly (reported doubling times of 22–60 h) 18, 22, 43. Contrary to MDA-MB-134 cells, which are loosely adherent in regular tissue culture flasks 21, IPH-926 cells grow in an adherent manner in vitro. Both cell lines lack expression of E-cadherin, albeit due to different CDH1 mutations (241ins4 in IPH-926 and exon 6 deletion in MDA-MB-134) (Figures 5A and 5B) 24. Loss of E-cadherin expression is accompanied by a loss of β-catenin expression and a diffuse scattering of the E-cadherin binding protein p120-catenin over the whole cytoplasm in both cell lines (Figure 5C). Aberrant cytoplasmic localization of p120-catenin, which is virtually never observed in ductal mammary carcinomas 4, was also clearly evident in IPH-926 tumours grown in SCID mice and the ILBC of the corresponding patient (Figure 5D). Both cell lines are also ErbB2-negative (Figure 5A). Array-CGH analyses identified an almost identical pattern of losses and gains at chromosomes 8p and 11q in primary ILBCs, in both the IPH-926 ILBC cell line and the MDA-MB-134 cell line (Figure 6 and Supporting information, Supplementary Data Tables 1 and 2). It has previously been reported that FGFR1, which maps to 8p11, functions as an oncogene in MDA-MB-134 cells 10. Interestingly, IPH-926 and MDA-MB-134 both harbour a loss at 8p23 and a gain at 8p12–p11 (Figure 7A). This was confirmed by quantitative gene dosage PCR for DLGAP2 at 8p23 and FGFR1 at 8p11 30. Based on quantitative gene dosage PCR, the FGFR1 gene copy number was clearly elevated in both IPH-926 and MDA-MB-134 (Figure 7B). In line with these results, FISH analyses of MDA-MB-134 cells revealed one centromere 8 signal associated with one FGFR1 signal, and two additional clusters of FGFR1 signals, in 72/100 cells analysed (Figure 7C, lower panel). In the remaining 28/100 MDA-MB-134 cells analysed, two centromere 8 signals, each associated with one FGFR1 signal, and three additional clusters of FGFR1 signals were detected. The heterogeneity of centromere signals and clusters of FGFR1 signals is most likely related to aberrant cell division processes. FISH analyses of IPH-926 passage 9 cells revealed three centromere 8 signals, each associated with one FGFR1 signal, and an additional centromere 8 signal associated with a cluster of four to eight individual FGFR1 signals, in nearly all of the 100 cells analysed (Figure 7C, upper panel). A few IPH-926 cells showed two centromere 8 signals, each associated with one FGFR1 signal, and the additional centromere 8 signal associated with the above-mentioned cluster of FGFR1 signals. This indicates that at least two out of five aberrant chromosomes shown to involve chromosome 8 material by SKY analysis of IPH-926 do not contain a chromosome 8 centromere. Although both cell lines showed a gain at 8p12–p11 (Figure 7A) and an increased FGFR1 gene copy number (Figure 7B), the FGFR1 mRNA expression level was approximately 100-fold higher in MDA-MB-134 than in IPH-926 (Figure 7D). Yet in comparison to MDA-MB-134, IPH-926 displayed a relatively low-level amplification of FGFR1, which may well explain the much lower FGFR1 mRNA expression (Figure 7D). However, in comparison to other established breast cancer cell lines, IPH-926 cells did not display significant overexpression of FGFR1 mRNA (Figure 7D). In fact, FGFR1 protein expression was barely detectable in IPH-926 cells by western blotting of 30 µg of total cellular proteins separated by SDS-PAGE using the well-established monoclonal anti-FGFR1 antibody M19B2 (Figure 7E) 44. This was additionally confirmed with another anti-FGFR1 antibody, 10646, whose specificity has also previously been demonstrated (Figure 7F) 10. In line with the very weak expression of FGFR1 protein, the FGFR1 inhibitor SU5402 had no apparent impact on IPH-926 cells (Figures 7G and 7H). Continuous exposure of IPH-926 cells for 3 days to various concentrations of SU5402 in normal growth medium failed to induce cell death or to modulate cell proliferation (Figures 7G and 7H).

thumbnail image

Figure 5. Comparison of IPH-926 and MDA-MB-134. (A) Protein expression analysis of IPH-926 passage 6 and MDA-MB-134 by western blot. UACC-893 served as a positive control for E-cadherin, β-catenin, and ErbB2. Detection of β-actin verified equal loading. (B) PCR-based detection of CDH1 mutations in IPH-926 passage 4 and MDA-MB-134. The upper panel shows results of the PCR reactions performed with oligonucleotide primers flanking CDH1 exon 6 as described by Berx et al5. The middle panel shows results of the PCR reactions performed with oligonucleotide primers flanking the insertion site of the CDH1 241ins4 mutation. Amplification of ErbB3 exon 3 is shown at the bottom and served as a control for the DNA preparations. (C) Cytoplasmic localization of p120-catenin in IPH-926 passage 25 and MDA-MB-134 in vitro (confocal laser scanning microscopy). E-cadherin-positive UACC-893 cells showing strictly membranous immunoreactivity served as a control. (D) Cytoplasmic localization of p120-catenin in IPH-926 passage 15 cells grown in vivo as SCID mouse xenografts (conventional immunohistochemistry). UACC-893 cells served as a control. Note also the cytoplasmic p120-catenin immunoreactivity in the locally recurrent ILBC of the corresponding patient

Download figure to PowerPoint

thumbnail image

Figure 6. Similar genomic imbalances on chromosomes 8p and 11q in IPH-926, MDA-MB-134, and primary ILBCs. Losses and gains in the array-CGH profiles of IPH-926 passage 4 (n = 52) and MDA-MB-134 (n = 15) were matched with identical or overlapping gains and losses from a compilation of genomic imbalances recurrently observed in primary ILBCs (n = 68) 10. For details see Supporting information, Supplementary Data Tables 1 and 2

Download figure to PowerPoint

thumbnail image

Figure 7. Genomic imbalances on chromosome 8 in IPH-926 and MDA-MB-134. (A) Array-based CGH plots for chromosome 8 of IPH-926 passage 4 (left) and MDA-MB-134 (right). Log2 ratios are plotted on the Y-axis against each clone for chromosome 8 from pter to qter on the X-axis. Horizontal dotted lines indicate log2 ratios of − 0.2 and 0.2. Vertical lines indicate the gain of 8p12–p11. (B) Quantitative gene dosage PCR for FGFR1 at 8p11 and DLGAP2 at 8p23. Data were normalized to a value of 2 for normal control DNA. Bars represent means of six individual PCR reactions. Error bars represent SEM. Note the elevated gene dosage for FGFR1 in IPH-926 passage 4 and the axillary relapse of the corresponding patient. Also note that contrary to IPH-926, the axillary relapse had no loss of DLGAP2. (C) Representative dual-colour fluorescence in situ hybridization of IPH-926 passage 9 and MDA-MB-134 for FGFR1 (red) and centromere 8 (green). (D) FGFR1 mRNA expression in IPH-926 passage 6 cells, MDA-MB-134 cells, and in a panel of established human ductal breast cancer cell lines. Note that the FGFR1 mRNA level of MDA-MB-134 is 100-fold higher than the FGFR1 mRNA level of IPH-926. (E) FGFR1 protein expression determined by western blot with the anti-FGFR1 antibody M19B2. Detection of β-actin verified equal loading. (F) FGFR1 protein expression determined by western blot with the anti-FGFR1 antibody 10646. Detection of β-actin verified equal loading. (G) IPH-926 cells were continuously exposed to various concentrations of the FGFR1 inhibitor SU5402 and relative cell proliferation was determined. Similar results were obtained with various passages of IPH-926. (H) IPH-926 cells were continuously exposed to various concentrations of the FGFR1 inhibitor SU5402 for 3 days and cell death was determined. Similar results were obtained with various passages of IPH-926

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Supporting information
  9. REFERENCES
  10. Supporting Information

There is compelling evidence that ILBCs are clinically and biologically distinct from other human breast tumours 1, 12. The investigation of ILBC has been hampered by the lack of appropriate functional models. We derived a novel cancer cell line, named IPH-926, from a patient with peritoneal metastases from ILBC. A previously unreported, homozygous CDH1 mutation, 241ins4, was identified in IPH-926. Detection of this unique mutation in the primary ILBC of the patient proved the clonal origin of IPH-926 from this particular ILBC. The presence of this unique CDH1 241ins4 mutation in the subsequent tumour relapses of the patient also confirmed the clonal relationship of the primary ILBC and its recurrences in the patient (Table 1). The CDH1 241ins4 mutation will serve as a genetic marker to verify the correct identity of this novel cell line in future studies. Comprehensive molecular, genetic, and functional analyses indicate that IPH-926 recapitulates key properties of ILBC. These include absence of E-cadherin expression, lack of ErbB2 and β-catenin expression, aberrant cytoplasmic localization of p120-catenin, slow proliferation, anti-cancer drug resistance, and numerous ILBC-typical genomic imbalances, such as gain of 5p15, gain of 9q33–q34, loss of 8p23–p22, gain of 8p12–p11, gain of 11q12–q13, loss of 11q14-qter, gain of 12q23–q24, loss of 15q21, gain of 19p13, and gain of 20q11–q13. Importantly, the novel IPH-926 cell line also recapitulates histomorphological features of human ILBCs in xenograft tumours in vivo.

Typical properties of ILBC that are not reflected by IPH-926 include expression of ER and gain of 1q. Concerning the lack of ER expression in IPH-926, it is important to note that the corresponding primary ILBC from 1990 was ER-positive. After the patient had been treated with the selective ER modulator tamoxifen for 5 years, the recurrent ILBC from 1996 and the ILBC metastasis from 1998 were ER-negative (Table 1), yet the ER-negative recurrences had evolved from the same tumour cell clone with the CDH1 241ins4 mutation. This is the first demonstration that an ILBC lost ER expression during tumour progression and tamoxifen treatment.

We have shown that MDA-MB-134, a cell line of uncertain origin, has many similarities with genuine human ILBC cells. MDA-MB-134 cells may indeed have derived from an occult or misdiagnosed ILBC and may represent a less advanced state of ILBC progression, as they are still ER-positive, show fewer genomic imbalances, and are still sensitive to anti-cancer drug treatment (data not shown). Interestingly, IPH-926 and MDA-MB-134 both harbour a gain at chromosome 8p12–p11 encompassing the FGFR1 gene, which functions as an oncogene in MBA-MB-134 cells 10. This is not the case in the IPH-926 cells, since they lack overexpression of FGFR1 mRNA and FGFR1 protein, and were not sensitive to the FGFR1 inhibitor SU5402. Genes such BAG4, C8orf4, and UNC5D might be the target of the genomic alterations on chromosome 8 45, 46.

In contrast to IPH-926 cells, MDA-MB-134 cells proliferate more rapidly 18, 22, 43. This appears to be quite untypical for ILBC cells 1, 9, 13, yet some cell lines derived from highly proliferative basal-like ductal breast cancers also showed a surprisingly slow growth in vitro43, 47. This underscores that proliferation properties of tumours in vivo are not necessarily predictive of the proliferation properties of the corresponding cell lines in vitro. Nonetheless, in view of the fact that no other cell line except IPH-926 has so far been proven to originate from a human ILBC, it might be advantageous to employ IPH-926 and MDA-MB-134 side by side in future studies. The establishment of a broad panel of human ILBC cell lines is clearly warranted but may take many years.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Supporting information
  9. REFERENCES
  10. Supporting Information

We would like to thank Dr Wilhelm Dirks from the DSMZ (German Collection of Micro-organisms and Cell Cultures) for authentication of the novel cell line IPH-926 and for cross-checking the IPH-926 STR profile with the DSMZ, ATCC, and JCRB STR profile databases.

Supporting information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Supporting information
  9. REFERENCES
  10. Supporting Information

Supporting information may be found in the online version of this article.

REFERENCES

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Supporting information
  9. REFERENCES
  10. Supporting Information

Note: References 48–56 are cited in the Supporting information to this article.

  • 1
    Tavassoli F, Devilee P. World Health Organzation Classification of Tumours. Pathology and Genetics of Tumours of the Breast and Female Genital Organs. IARC Press: Lyon, 2003.
  • 2
    Moll R, Mitze M, Frixen UH, Birchmeier W. Differential loss of E-cadherin expression in infiltrating ductal and lobular breast carcinomas. Am J Pathol 1993; 143: 17311742.
  • 3
    Sarrio D, Moreno-Bueno G, Hardisson D, Sanchez-Estevez C, Guo M, Herman JG, et al. Epigenetic and genetic alterations of APC and CDH1 genes in lobular breast cancer: relationships with abnormal E-cadherin and catenin expression and microsatellite instability. Int J Cancer 2003; 106: 208215.
  • 4
    Sarrio D, Perez-Mies B, Hardisson D, Moreno-Bueno G, Suarez A, Cano A, et al. Cytoplasmic localization of p120ctn and E-cadherin loss characterize lobular breast carcinoma from preinvasive to metastatic lesions. Oncogene 2004; 23: 32723283.
  • 5
    Berx G, Cleton-Jansen AM, Nollet F, de Leeuw WJ, van de Vijver M, Cornelisse C, et al. E-cadherin is a tumour/invasion suppressor gene mutated in human lobular breast cancers. EMBO J 1995; 14: 61076115.
  • 6
    Berx G, Cleton-Jansen AM, Strumane K, de Leeuw WJ, Nollet F, van Roy F, et al. E-cadherin is inactivated in a majority of invasive human lobular breast cancers by truncation mutations throughout its extracellular domain. Oncogene 1996; 13: 19191925.
  • 7
    Droufakou S, Deshmane V, Roylance R, Hanby A, Tomlinson I, Hart IR. Multiple ways of silencing E-cadherin gene expression in lobular carcinoma of the breast. Int J Cancer 2001; 92: 404408.
  • 8
    Masciari S, Larsson N, Senz J, Boyd N, Kaurah P, Kandel MJ, et al. Germline E-cadherin mutations in familial lobular breast cancer. J Med Genet 2007; 44: 726731.
  • 9
    Nishizaki T, Chew K, Chu L, Isola J, Kallioniemi A, Weidner N, et al. Genetic alterations in lobular breast cancer by comparative genomic hybridization. Int J Cancer 1997; 74: 513517.
  • 10
    Reis-Filho JS, Simpson PT, Turner NC, Lambros MB, Jones C, Mackay A, et al. FGFR1 emerges as a potential therapeutic target for lobular breast carcinomas. Clin Cancer Res 2006; 12: 66526662.
  • 11
    Borst MJ, Ingold JA. Metastatic patterns of invasive lobular versus invasive ductal carcinoma of the breast. Surgery 1993; 114: 637641; discussion 641–642.
  • 12
    Biglia N, Mariani L, Sgro L, Mininanni P, Moggio G, Sismondi P. Increased incidence of lobular breast cancer in women treated with hormone replacement therapy: implications for diagnosis, surgical and medical treatment. Endocr Relat Cancer 2007; 14: 549567.
  • 13
    Mathieu MC, Rouzier R, Llombart-Cussac A, Sideris L, Koscielny S, Travagli JP, et al. The poor responsiveness of infiltrating lobular breast carcinomas to neoadjuvant chemotherapy can be explained by their biological profile. Eur J Cancer 2004; 40: 342351.
  • 14
    Cristofanilli M, Gonzalez-Angulo A, Sneige N, Kau SW, Broglio K, Theriault RL, et al. Invasive lobular carcinoma classic type: response to primary chemotherapy and survival outcomes. J Clin Oncol 2005; 23: 4148.
  • 15
    Lacroix M, Leclercq G. Relevance of breast cancer cell lines as models for breast tumours: an update. Breast Cancer Res Treat 2004; 83: 249289.
  • 16
    Holland EC. Mouse Models of Human Cancer. Wiley-Liss: Hoboken, NJ, 2004.
  • 17
    Derksen PW, Liu X, Saridin F, van der Gulden H, Zevenhoven J, Evers B, et al. Somatic inactivation of E-cadherin and p53 in mice leads to metastatic lobular mammary carcinoma through induction of anoikis resistance and angiogenesis. Cancer Cell 2006; 10: 437449.
  • 18
    Engel LW, Young NA. Human breast carcinoma cells in continuous culture: a review. Cancer Res 1978; 38: 43274339.
  • 19
    Gazdar AF, Kurvari V, Virmani A, Gollahon L, Sakaguchi M, Westerfield M, et al. Characterization of paired tumor and non-tumor cell lines established from patients with breast cancer. Int J Cancer 1998; 78: 766774.
  • 20
    Fogh J, Wright WC, Loveless JD. Absence of HeLa cell contamination in 169 cell lines derived from human tumors. J Natl Cancer Inst 1977; 58: 209214.
  • 21
    Cailleau R, Young R, Olive M, Reeves WJ Jr. Breast tumor cell lines from pleural effusions. J Natl Cancer Inst 1974; 53: 661674.
  • 22
    Brinkley BR, Beall PT, Wible LJ, Mace ML, Turner DS, Cailleau RM. Variations in cell form and cytoskeleton in human breast carcinoma cells in vitro. Cancer Res 1980; 40: 31183129.
  • 23
    Fogh J, Fogh JM, Orfeo T. One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. J Natl Cancer Inst 1977; 59: 221226.
  • 24
    Hiraguri S, Godfrey T, Nakamura H, Graff J, Collins C, Shayesteh L, et al. Mechanisms of inactivation of E-cadherin in breast cancer cell lines. Cancer Res 1998; 58: 19721977.
  • 25
    Siciliano MJ, Barker PE, Cailleau R. Mutually exclusive genetic signatures of human breast tumor cell lines with a common chromosomal marker. Cancer Res 1979; 39: 919922.
  • 26
    Christgen M, Ballmaier M, Bruchhardt H, von Wasielewski R, Kreipe H, Lehmann U. Identification of a distinct side population of cancer cells in the Cal-51 human breast carcinoma cell line. Mol Cell Biochem 2007; 306: 201212.
  • 27
    Mohammadi M, McMahon G, Sun L, Tang C, Hirth P, Yeh BK, et al. Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors. Science 1997; 276: 955960.
  • 28
    Band V, Zajchowski D, Swisshelm K, Trask D, Kulesa V, Cohen C, et al. Tumor progression in four mammary epithelial cell lines derived from the same patient. Cancer Res 1990; 50: 73517357.
  • 29
    Dirks WG, Faehnrich S, Estella IA, Drexler HG. Short tandem repeat DNA typing provides an international reference standard for authentication of human cell lines. Altex 2005; 22: 103109.
  • 30
    Lehmann U, Glockner S, Kleeberger W, von Wasielewski HF, Kreipe H. Detection of gene amplification in archival breast cancer specimens by laser-assisted microdissection and quantitative real-time polymerase chain reaction. Am J Pathol 2000; 156: 18551864.
  • 31
    Lehmann U, Hasemeier B, Christgen M, Muller M, Romermann D, Langer F, et al. Epigenetic inactivation of microRNA gene hsa-mir-9-1 in human breast cancer. J Pathol 2008; 214: 1724.
  • 32
    Bae YK, Brown A, Garrett E, Bornman D, Fackler MJ, Sukumar S, et al. Hypermethylation in histologically distinct classes of breast cancer. Clin Cancer Res 2004; 10: 59986005.
  • 33
    Zielinski B, Gratias S, Toedt G, Mendrzyk F, Stange DE, Radlwimmer B, et al. Detection of chromosomal imbalances in retinoblastoma by matrix-based comparative genomic hybridization. Genes Chromosomes Cancer 2005; 43: 294301.
  • 34
    Steinemann D, Skawran B, Becker T, Tauscher M, Weigmann A, Wingen L, et al. Assessment of differentiation and progression of hepatic tumors using array-based comparative genomic hybridization. Clin Gastroenterol Hepatol 2006; 4: 12831291.
  • 35
    Willenbrock H, Fridlyand J. A comparison study: applying segmentation to array CGH data for downstream analyses. Bioinformatics 2005; 21: 40844091.
  • 36
    Rudolph C, Liehr T, Steinemann D, Emura M, Daibata M, Matsuo Y, et al. Different breakage-prone regions on chromosome 1 detected in t(11;14)-positive mantle cell lymphoma cell lines and multiple myeloma cell lines are associated with different tumor progression-related mechanisms. Cytogenet Genome Res 2006; 112: 213221.
  • 37
    Schlegelberger B, Metzke S, Harder S, Zuhlke-Jenisch R, Zhang Y, Siebert R. Classical and molecular cytogenetics of tumor cells. In: Diagnostic Cytogenetics, WegenerRD (ed.). Springer: Berlin-Heidelberg, 1999; 151185.
  • 38
    Lasfargues EY, Coutinho WG, Redfield ES. Isolation of two human tumor epithelial cell lines from solid breast carcinomas. J Natl Cancer Inst 1978; 61: 967978.
  • 39
    Christgen M, Schniewind B, Jueschke A, Ungefroren H, Kalthoff H. Gemcitabine-mediated apoptosis is associated with increased CD95 surface expression but is not inhibited by DN-FADD in Colo357 pancreatic cancer cells. Cancer Lett 2005; 227: 193200.
  • 40
    Fetsch PA, Abati A. Immunocytochemistry in effusion cytology: a contemporary review. Cancer 2001; 93: 293308.
  • 41
    Thorley-Lawson DA, Schooley RT, Bhan AK, Nadler LM. Epstein–Barr virus superinduces a new human B cell differentiation antigen (B-LAST 1) expressed on transformed lymphoblasts. Cell 1982; 30: 415425.
  • 42
    Sobin L, Wittekind C. TNM Classification of Malignant Tumours. John Wiley & Sons Inc: New York, 1997.
  • 43
    Reddel RR, Murphy LC, Hall RE, Sutherland RL. Differential sensitivity of human breast cancer cell lines to the growth-inhibitory effects of tamoxifen. Cancer Res 1985; 45: 15251531.
  • 44
    Aziz KA, Till KJ, Chen H, Slupsky JR, Campbell F, Cawley JC, et al. The role of autocrine FGF-2 in the distinctive bone marrow fibrosis of hairy-cell leukemia (HCL). Blood 2003; 102: 10511056.
  • 45
    Yang ZQ, Streicher KL, Ray ME, Abrams J, Ethier SP. Multiple interacting oncogenes on the 8p11–p12 amplicon in human breast cancer. Cancer Res 2006; 66: 1163211643.
  • 46
    Pole JC, Courtay-Cahen C, Garcia MJ, Blood KA, Cooke SL, Alsop AE, et al. High-resolution analysis of chromosome rearrangements on 8p in breast, colon and pancreatic cancer reveals a complex pattern of loss, gain and translocation. Oncogene 2006; 25: 56935706.
  • 47
    Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, et al. A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 2006; 10: 515527.
  • 48
    Courjal F, Cuny M, Simony-Lafontaine J, Louason G, Speiser P, Zeillinger R, et al. Mapping of DNA amplifications at 15 chromosomal localizations in 1875 breast tumors: definition of phenotypic groups. Cancer Res 1997; 57: 43604367.
  • 49
    Rodriguez C, Hughes-Davies L, Valles H, Orsetti B, Cuny M, Ursule L, et al. Amplification of the BRCA2 pathway gene EMSY in sporadic breast cancer is related to negative outcome. Clin Cancer Res 2004; 10: 57855791.
  • 50
    Janssen JW, Cuny M, Orsetti B, Rodriguez C, Valles H, Bartram CR, et al. MYEOV: a candidate gene for DNA amplification events occurring centromeric to CCND1 in breast cancer. Int J Cancer 2002; 102: 608614.
  • 51
    Courjal F, Louason G, Speiser P, Katsaros D, Zeillinger R, Theillet C. Cyclin gene amplification and overexpression in breast and ovarian cancers: evidence for the selection of cyclin D1 in breast and cyclin E in ovarian tumors. Int J Cancer 1996; 69: 247253.
  • 52
    Baumgartner A, Weier JF, Weier HU. Chromosome-specific DNA repeat probes. J Histochem Cytochem 2006; 54: 13631370.
  • 53
    Fiegler H, Carr P, Douglas EJ, Burford DC, Hunt S, Scott CE, et al. DNA microarrays for comparative genomic hybridization based on DOP-PCR amplification of BAC and PAC clones. Genes Chromosomes Cancer 2003; 36: 361374.
  • 54
    Letessier A, Sircoulomb F, Ginestier C, Cervera N, Monville F, Gelsi-Boyer V, et al. Frequency, prognostic impact, and subtype association of 8p12, 8q24, 11q13, 12p13, 17q12, and 20q13 amplifications in breast cancers. BMC Cancer 2006; 6: 245.
  • 55
    Lambros MB, Natrajan R, Reis-Filho JS. Chromogenic and fluorescent in situ hybridization in breast cancer. Hum Pathol 2007; 38: 11051122.
  • 56
    Freier K, Schwaenen C, Sticht C, Flechtenmacher C, Muhling J, Hofele C, et al. Recurrent FGFR1 amplification and high FGFR1 protein expression in oral squamous cell carcinoma (OSCC). Oral Oncol 2007; 43: 6066.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Supporting information
  9. REFERENCES
  10. Supporting Information
FilenameFormatSizeDescription
PATH2495_suppinfo_methods.doc87KSupporting Information: Supplementary Methods
path_2495_sm_supportinginformationsT1.doc131KSupporting Information; Table S1
path_2495_sm_supportinginformationsT2.doc143KSupporting Information: Table S2

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.