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Short Communication |
From the Departments of Pathology,*Surgery,¶ Oncology,|| andMedicine,** The Johns Hopkins MedicalInstitutions, Baltimore, Maryland; Gene LogicInc., Gaithersburg, Maryland; the Departmentof Pathology, University of TexasSouthwestern Medical Center, Dallas, Texas; and The EppleyInstitute, University of Nebraska,Omaha, Nebraska
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Abstract |
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The utility of RNA-based global gene expression profiling biotechniques in identifying new markers of cancer is established.3,4 For example, we have identified two new potential markers of pancreatic carcinoma, mesothelin and prostate stem cell antigen, using serial analysis of gene expression (SAGE).5,6 Both markers are expressed specifically by the neoplastic epithelium of infiltrating carcinomas of the pancreas as compared to normal duct epithelium, and both offer new possibilities for the development of screening markers and therapeutic targets.
In an effort to identify additional potential markers of pancreatic carcinoma, we used the Gene Logic Inc. BioExpress platform and Affymetrix GeneChip arrays to identify genes differentially expressed in a large series of pancreatic cancers. Biocomputational tools were used to determine those genes most highly expressed within pancreatic cancer samples compared to normal pancreatic tissue. Genes found to be significantly expressed in SAGE libraries of normal pancreatic ductal cells were excluded, and the expression of selected genes was confirmed by immunohistochemical labeling, in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR). Here we report 97 genes differentially overexpressed in pancreatic cancer, 69 of which are novel.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Samples (0.5 g) of normal pancreas (n = 11); normal duodenal, jejunal, or colonic mucosa (n = 22); or infiltrating pancreatic adenocarcinoma (n = 14) were collected from surgical specimens from patients at The Johns Hopkins Hospital. In each case, the specimens were harvested within 10 minutes of resection from the patient and snap-frozen in liquid nitrogen before storage at -80°C. The resected cancers were not microdissected because we were interested in not only identifying the genes expressed by neoplastic epithelial calls, but also the genes expressed as a result of the neoplastic cell-stroma interaction. Hematoxylin and eosin-stained sections of adjacent sections of the tissue were prepared before snap-freezing to confirm the diagnosis. The neoplastic cellularity of these tissue samples ranged from 5 to 55%. Normal gastrointestinal mucosa was included in the analyses to facilitate the identification of markers of pancreatic cancer that would be useful in screening secondary sources, such as in duodenal fluid samples.
Cell Lines
Human pancreatic cancer cell lines AsPc1, BxPc3, CAPAN1, CAPAN2, CFPAC1, COLO357, Hs766T, MiaPaCa2, Panc-1, and Su86.86, and human pancreatic normal duct epithelial line HPDE6, were obtained from the American Type Culture Collection, Rockville, MD. PL cell lines (PL1-6, PL8-14) were low-passage pancreatic carcinoma cell lines kindly provided by Dr. Elizabeth Jaffee from the Department of Oncology, The Johns Hopkins Hospital, Baltimore, MD.7 Cell lines were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). CAPAN1 and CAPAN2 cell lines were cultured in RPMI 1640 medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin), respectively. Use of different media minimized the variance in growth rates that would otherwise be exaggerated with a single medium. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 in air.
mRNA Extractions and Affymetrix GeneChip Hybridization
Sample preparation and processing procedure was performed as described in the Affymetrix GeneChip Expression Analysis Manual (Santa Clara, CA). Briefly, each frozen tissue was crushed to powder by using the Spex Certiprep 6800 Freezer Mill (Metuchen, NJ). Total RNA was then extracted from the crushed normal and neoplastic tissues or cell pellets (BxPC3, COLO357, Hs766T, MiaPaCa2, Panc1, PL3, PL4, PL8) using TRIzol (Life Technologies, Inc., Rockville, MD) and cleaned using RNeasy columns according to the manufacturer’s protocol (Qiagen, Valencia, CA). Using 5 to 40 µg of total RNA, double-stranded cDNA was synthesized following SuperScript Choice system (Life Technologies, Inc., Rockville, MD). T7-(dT24) oligomer was used for priming the first-strand cDNA synthesis. The resultant cDNA was purified using Phase Lock Gel, phenol/chloroform extraction, and precipitated with ethanol. The cDNA pellet was collected and dissolved in appropriate volume. Using cDNA as template, cRNA was synthesized using a T7 MegaScript In Vitro Transcription (IVT) Kit (Ambion, Austin, TX). Biotinylate-11-CTP and 16-UTP ribonucleotides (Enzo Diagnostics Inc., Farmingdale, NY) were added to the reaction as labeling reagents. IVT reactions were performed at 37°C for 6 hours and, the labeled cRNA obtained was purified using RNeasy columns (Qiagen, Valencia, CA). The cRNA was fragmented in fragmentation buffer (40 mmol/L Tris-Acetate, pH 8.1, 100 mmol/L KOAc, 30 mmol/L MgOAc) for 35 minutes at 94°C. Fragmented cRNA prepared from each sample (10 to 11 µg/probe array) was hybridized to the human GeneChip set (HG_U95 A, B, C, D, and E) noncompetitively at 45°C for 24 hours in a hybridization oven with constant rotation (60 rpm). Fragmented cRNAs are hybridized to the GeneChip set by way of multiple 20 to 25 oligonucleotide probes specific for each gene, with each probe corresponding to a different region of the mRNA of interest. The probes specific for each mRNA are scattered across the surface of each GeneChip to control for technical issues that occur with each hybridization. The chips were washed and stained using Affymetrix fluidics stations. Staining was performed using streptavidin-phycoerythrin conjugate (SAPE; Molecular Probes, Eugene, OR), followed by the addition of biotinylated antibody to streptavidin (Vector Laboratories, Burlingame, CA), and finally with streptavidin-phycoerythrin conjugate. Probe arrays was scanned using fluorometric scanners (Hewlett Packard Gene Array Scanner; Hewlett Packard Corporation, Palo Alto, CA).
The scanned images were inspected and analyzed using established quality control measures, with the hybridization intensities reflecting in a linear manner the mRNA expression in the tissues or cells being assayed. Hybridization was controlled for each probe by the use of a mismatch control that has a single base mismatch. This mismatch control is analyzed using the GeneLogic informatics filter that compares the hybridization intensity of mismatched to perfect matched probes (to eliminate those that are nonspecific over a specified threshold) as well as different probes to the same gene.
Statistical Data Analysis
The GeneExpress Software System Fold Change Analysis tool was used to identify genes expressed at least fivefold greater in the pancreatic cancers compared to normal tissues. For each gene fragment, the ratio of the geometric means of the expression intensities in the normal control tissues and the pancreas cancer samples was calculated, and the fold change then calculated on a per fragment basis. Confidence limits were calculated using a two-sided Welch modified t-test on the difference of the means of the logs of the intensities.
SAGE
Short-term cultures of nonneoplastic pancreatic ductal epithelial cells (HX and H126) were prepared as described and validated as having the characteristics of ductal epithelium.8 SAGE libraries were previously constructed as described by Ryu and colleagues,9,10 and sequencing was performed by the CGAP SAGE consortium at the Lawrence Livermore National Laboratories and Washington University Human Genome Center (St. Louis, MO). SAGE library data from the short-term cultures of nonneoplastic pancreatic duct epithelial cells have been posted on the CGAP web site as part of the SAGEmap database (http://www.ncbi.nlm.nih.gov/SAGE).
In Situ Hybridization
Preparation of digoxigenin-labeled sense and antisense riboprobes and in situ hybridization were performed as previously described in detail.11
RT-PCR
Total RNA was isolated from cultured cells by using TRIzol reagent (Life Technologies, Inc.). Cell lines used for RT-PCR were PL1-6, PL8-14, CAPAN1, CFPAC, AsPc1, BxPC3, Hs766T, MiaPaCa2, Panc1, and HPDE6. An aliquot of 1 µg of total RNA from each sample was reverse-transcribed to cDNA using the SuperScript II kit (Life Technologies, Inc.) according to the manufacturer’s instructions, with oligo(dT)12-18 primer. PCR primers were designed to amplify cDNA fragments with various sizes using standard PCR conditions. The PCR reaction products were resolved by electrophoresis in a 3% agarose gel and stained with ethidium bromide. Loading was controlled by the simultaneous PCR of glyceraldehyde-3-phosphate dehydrogenase cDNA.
Immunohistochemistry
Sections of infiltrating primary ductal adenocarcinoma of the
pancreas were formalin-fixed and paraffin-embedded, and unstained
4-µm sections were then cut from the paraffin blocks. For detection
of heat shock protein 47 (hsp47), sections were deparaffinized by
routine techniques before placing in 200 ml of Target Retrieval
Solution, pH 6.0 (Envision Plus Detection kit, DAKO, Carpinteria, CA)
for 20 minutes at 100°C. After cooling for 20 minutes, slides were
quenched with 3% H2O2 for
5 minutes, before incubating with a 1:800 dilution of monoclonal
antibody (colligin m16.10A1) against heat shock protein 47
(Stressgen Biotechnologies, Victoria, BC, Canada) for 30
minutes using the DAKO Autostainer. Labeling was detected with the DAKO
Envision system following the manufacturer’s protocol. For detection
of topoisomerase II
and fascin, slides were steamed for 20 minutes
in sodium citrate buffer (diluted to 1x from 10x heat-induced epitope
retrieval buffer; Ventana-Bio Tek Solutions, Tucson, AZ). After cooling
for 5 minutes, slides were labeled with a 1:3200 dilution of mouse
monoclonal antibody against topoisomerase II (clone TG100; Neomarkers,
Freemont,CA) or a 1:500 dilution of mouse monoclonal antibody against
fascin (DAKO) using the Bio Tek 1000 automated stainer (Ventana).
Labeling was detected by adding biotinylated secondary antibodies,
avidin-biotin complex, and 3,3'-diaminobenzidine. All sections were
counterstained with hematoxylin, and staining was evaluated by three of
the authors (CID, AM, and RHH) with agreement in all cases evaluated.
Staining was considered positive if at least 10% of the cells showed
immunolabeling.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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RNA samples were hybridized to the complete Affymetrix Human Genome U95 GeneChip set (arrays U95 A, B, C, D, and E) for simultaneous analysis of 60,000 fragments, with 12,000 fragments covering full-length genes and 48,000 fragments covering ESTs. Affymetrix GeneChips were analyzed for all genes with a fivefold or greater increase in expression in the pancreatic adenocarcinoma tumor tissues or cell lines compared to all normal tissues, using a 95% confidence limit. We identified 180 fragments expressed at least fivefold greater in pancreatic cancer samples as compared to normal tissues, 12 of which were expressed greater than 10-fold. The level of significance for each gene fragment ranged from less than P = 0.00001 to P = 0.01 (modified Welch t-test).
Identification of Highly Expressed Genes in Pancreatic Cancer
Characterization of the 180 fragments identified revealed that 56
fragments corresponded to ESTs, and 124 fragments corresponded to known
genes. Among these 124 fragments, 10 genes were represented by two or
more fragments, resulting in 107 known genes identified as expressed at
least fivefold or greater in pancreatic cancers as compared to normal
(Table 1)
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. The first pattern (the A or
cancer-specific pattern) demonstrated elevated expression of the
fragment in both pancreas cancer cell lines and in resected pancreatic
cancer tissues compared to normal tissues. Ninety-five fragments showed
this pattern. The second pattern (the B or invasion-specific pattern)
showed elevated expression of the fragment in the resected pancreatic
cancer tissues only, but not in the cancer cell lines or normal
tissues. This B pattern was observed for 29 fragments. Genes that were
represented by more than one fragment showed the same e-Northern
pattern for each fragment analyzed.
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. Thus, based on the initial
results of e-Northern analysis and SAGE filtering, 97 candidate genes
were identified as differentially overexpressed in pancreatic cancer. Literature Search of Genes Highly Expressed in Pancreatic Cancer
For each of the 97 genes identified, a search was performed using
the online NCBI database PubMed using the known gene name together with
the terms "pancreas" or "pancreas cancer." Of the 97 genes
analyzed, 28 genes were previously reported to be associated with
pancreatic cancer, whereas 69 genes were not (Table 1)
. Of these 69
genes not identified in this PubMed search as having been reported in
pancreatic cancer, 21 have been reported before in association with
tumor types other than pancreatic cancer, whereas 48 genes have not
been reported in association with any neoplasm.
These 97 candidate tumor markers of pancreatic cancer represented a
variety of cellular functions. Genes identified included those involved
in cell membrane junctions (claudin 1, connexin 26),12,13
signal transduction (tumor-associated calcium signal transducer 2, ras
GTPase-activating protein-like),14,15
calcium homeostasis
(S100 calcium-binding protein P),16
cytoskeletal assembly
(fascin, keratin 7, rabkinesin6 and pleckstrin),17-20
cell surface adhesion and recognition (integrin ß-like
1),21
DNA transcription (topoisomerase II,
transcription factor BMAL2, and AML1),22-24
DNA repair
(ATDC),25
or extracellular matrix remodeling and function
(collagens 11, 12, and X11, heat shock protein 47, MMP14, and
MMP7).11,26,27
The cellular localization of the
corresponding gene products was also determined using the online
database OMIM available through the NCBI web site
(http://www.ncbi.nlm.nih.gov/entrez/query). Genes
were found to encode membrane-bound proteins (prostate stem cell
antigen, OB-cadherin), cytoplasmic proteins (fascin, ATDC), nuclear
proteins (topoisomerase II, paraneoplastic antigen MA1), as well as
extracellular proteins, such as those involved in extracellular matrix
homeostasis (hsp47, thrombospondin 2) or secreted protein products
(osteopontin).
Verification of Selected Candidate Tumor Markers
Candidate genes were selected for verification of expression in
samples of pancreatic cancer tissues or cell lines (Figures 2 and 3)
. Four genes were selected for
analysis by immunohistochemical or in situ
hybridization techniques: fascin,
topoisomerase II, hsp47, and pleckstrin.
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Topoisomerase II showed strong positive nuclear immunolabeling
within eight of eight pancreatic duct adenocarcinomas studied (100%).
Normal duct epithelium and the surrounding desmoplastic
stroma did not express detectable
levels of topoisomerase II (Figure 2B)
.
In contrast to fascin and topoisomerase II, hsp47 showed a B pattern
of expression on e-Northern, indicating elevated expression of hsp47 in
the resected neoplastic tissues only, but not in the cancer cell lines
or normal tissues. (Figure 2C)
. In concordance with this pattern,
immunolabeling for hsp47 showed strong labeling of the desmoplastic
stroma within the invasive cancer in eight of eight pancreatic duct
adenocarcinomas studied (100%). In one of the eight cases, the
neoplastic epithelium also labeled. No expression of hsp47 was detected
within normal pancreatic duct epithelium or within the intralobular
stroma of normal pancreas tissue within the same paraffin-embedded
tissue sections.
Pleckstrin was also identified as differentially expressed in
pancreatic cancer and displayed an A pattern of expression by
e-Northern. No commercially available antibody for pleckstrin was
available. Therefore, a digoxigenin-labeled probe was generated to
match the coding region of the pleckstrin gene for use in in
situ hybridization. In situ hybridization using the
anti-sense probe showed expression within the neoplastic epithelium in
all eight cases (100%), seen as variably sized granules throughout the
cytoplasm of the neoplastic epithelium, in contrast to normal duct
epithelium or the surrounding desmoplastic stroma, which did not
express this gene (Figure 3D)
.
Eight additional genes were selected for validation by an RT-PCR study
of 20 pancreas cancer cell lines and the immortal human pancreatic
ductal epithelial cell line (HPDE6) (Figure 3)
. Genes selected for
validation using RT-PCR were claudin 1, S100 calcium-binding protein P
(S100P), interferon-induced transmembrane protein 1 (IFITM1), lamin B2,
DKFZP564G013 protein, KIAA0470 gene product, KIAA1265 protein, and
KIAA1363 protein. Expression of these eight genes were detected in 19
of the 20 cell lines analyzed, in support of their initial
identification as differentially expressed genes by Affymetrix
GeneChip.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The finding of 97 genes significantly overexpressed in infiltrating
pancreatic duct carcinomas has immediate diagnostic potential.
Overexpression of these novel tumor markers of pancreatic cancer can be
used to differentiate infiltrating pancreatic duct adenocarcinoma from
chronic pancreatitis, particularly in small tissue samples or
cytological material. Our initial studies to validate these markers
support this possibility. Immunohistochemical and in situ
labeling for these differentially expressed genes, including fascin,
topoisomerase II, and pleckstrin, specifically label the neoplastic
epithelium of infiltrating pancreatic duct adenocarcinomas, but not by
normal duct epithelium included in the same tissue sections.
These 97 differentially expressed markers of pancreatic cancer also have potential for the development of new screening tests for pancreatic cancer. For example, the development of tagged antibodies to one or more of these genes may be useful in the diagnostic radiological imaging of small primary pancreatic cancers or metastases before they become clinically apparent. Several of these genes were found to be membranous or secreted proteins, suggesting they may be shed into the blood or pancreatic secretions. If so, these proteins may also serve as diagnostic markers in such specimens, not only for identification of primary pancreatic cancers at an earlier stage, but also for the identification of recurrent disease at an earlier phase when it may be more responsive to adjuvant therapies. In addition, whereas use of any one marker individually may have a limited sensitivity or specificity in detecting pancreatic cancer, the development of a panel of markers may significantly increase the specificity of detecting clinically inapparent pancreatic cancers without decreasing the sensitivity.28
The identification of these differentially expressed genes in pancreatic cancer also has important therapeutic applications for pancreatic cancer. For example, Jaffee and colleagues29 have recently shown that cell-mediated immunotherapy can be both safe and effective in patients with pancreatic cancer, and each of the differentially expressed genes represents a potential target for the development of a cell-mediated vaccine. Similarly, as a number of the genes identified were found to encode for cell surface proteins (ie, OB-cadherin, CD83, claudin 1, prostate stem cell antigen, and retinoic acid-induced 3), these proteins hold promise for the development of antibody-based immunotherapy against pancreatic carcinoma.7,30 In addition, signal transduction pathways in which these differentially expressed genes may function are potential targets for molecular therapeutics.
Overexpression of several of the genes found in pancreatic duct
adenocarcinomas, such as ataxia-telangiectasia group D-associated
protein (ATDC), topoisomerase II (TOP2A), and transglutaminase II
(TGM2), may offer new insights into the biology of pancreatic cancer.
ATDC protein has been shown to be induced by ionizing radiation and to
suppress the radiosensitivity of ataxia telangiectasia (A-T) fibroblast
cell lines.31
The overexpression of ATDC in pancreatic
cancers may therefore contribute to the radioresistance often observed
for this tumor type.32
Chemotherapeutic resistance in
pancreatic cancers may also, in part, be contributed to by genes such
as TOP2A or TGM2.22
TOP2A is a target for several
chemotherapeutic agents, including doxorubicin, that have been used for
treatment of advanced pancreatic cancer.33
The high levels
of TOP2A expression in some pancreatic cancers might indicate
amplification of this gene, an occurrence that contributes to the
ineffectiveness of this chemotherapeutic agent in other tumor
types.22,33
Similarly, the overexpression of TGM2 has also
been associated with drug resistance.34
Other highly expressed genes in pancreatic cancer, such as interleukin-8 (IL-8) or the AML1 oncogene, may contribute to the aggressiveness of this tumor by alternative mechanisms. IL-8 overexpression in pancreatic cancers is thought to result from low oxygen tension and hypoxia of the tumor microenvironment. Consequently, IL-8 overexpression is thought to contribute to the aggressiveness of pancreatic cancer by inducing angiogenesis and promoting tumor metastasis.35 The AML1 oncogene is a transcription factor that is commonly overexpressed by translocation in acute myeloid leukemias.24 The overexpression of AML1 in pancreatic cancer suggests that this gene may also play a role in the pathogenesis of this tumor type. Thus, our finding of differentially expressed genes related to the aggressiveness of pancreatic cancers may be used to develop more effective therapeutic protocols for this tumor type.
Invasive pancreatic cancers represent an aggregate of diverse cell
types, such as invasive neoplastic epithelial cells, fibroblasts,
inflammatory cells, smooth muscle cells, endothelial cells, and cells
of residual nonneoplastic pancreatic parenchyma.9
Thus,
comparative studies of gene expression in pancreatic cancer tissues and
cell cultures provide valuable information of gene expression by the
different cellular compartments of the neoplasm. By e-Northern
analysis, we found that 29 genes were overexpressed in pancreas cancer
tumor tissues only, as compared to cancer cell lines or normal tissues.
Studies suggest that most of these genes are likely to be expressed by
the nonneoplastic host stromal response to the neoplasm.11
Genes with this pattern of expression (B pattern on e-Northern), which
included hsp47; apolipoprotein C-1; collagens type 11, 12, and
XI1; osteopontin, and thrombospondin 2, highlight the prominent host
stromal response characteristic of infiltrating pancreatic duct
adenocarcinomas. In some instances, however, the gene expression
identified in association with pancreas cancer tumor tissues does not
always indicate stromal gene expression, but instead may reflect the
gene expression of epithelial cells only when such cells are within a
tumor in vivo (as opposed to the environment of cell
culture).11
Prostate stem cell antigen exemplifies this
observation. It had a B pattern of expression by e-Northern analysis
(Table 1)
, and a striking epithelial-specific pattern of expression by
immunohistochemical labeling in a majority of resected infiltrating
pancreatic cancers.6
In summary, we have identified 97 differentially expressed genes in infiltrating pancreatic cancer, all with immediate potential utility for the development of screening tools, radiological imaging techniques, or therapies for pancreatic cancer. Approximately one-third of these 97 known genes have previously been reported in association with pancreatic cancer, and an additional 12 genes were confirmed by immunohistochemical labeling, in situ hybridization, or RT-PCR, thus validating our approach in identifying these new markers. These genes not only provide insights into the complex cellular biology of pancreatic duct adenocarcinoma, but also represent novel clinical targets for this tumor type.
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Footnotes |
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Supported by the National Institutes of Health Specialized Programs of Research Excellence in Gastrointestinal Cancer (grant CA62924), the Michael Rolfe Fund for Pancreatic Cancer Research, and a grant from GeneLogic Inc.
Presented at the 70th meeting of the United States and Canadian Academy of Pathology, February 23 to March 1, 2002.
Accepted for publication January 8, 2002.
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J. Koopmann, P. Buckhaults, D. A. Brown, M. L. Zahurak, N. Sato, N. Fukushima, L. J. Sokoll, D. W. Chan, C. J. Yeo, R. H. Hruban, et al. Serum Macrophage Inhibitory Cytokine 1 as a Marker of Pancreatic and Other Periampullary Cancers Clin. Cancer Res., April 1, 2004; 10(7): 2386 - 2392. [Abstract] [Full Text] [PDF]
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J. Koopmann, N. S. Fedarko, A. Jain, A. Maitra, C. Iacobuzio-Donahue, A. Rahman, R. H. Hruban, C. J. Yeo, and M. Goggins Evaluation of Osteopontin as Biomarker for Pancreatic Adenocarcinoma Cancer Epidemiol. Biomarkers Prev., March 1, 2004; 13(3): 487 - 491. [Abstract] [Full Text]
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N. Sato, N. Fukushima, A. Maitra, C. A. Iacobuzio-Donahue, N. T. van Heek, J. L. Cameron, C. J. Yeo, R. H. Hruban, and M. Goggins Gene Expression Profiling Identifies Genes Associated with Invasive Intraductal Papillary Mucinous Neoplasms of the Pancreas Am. J. Pathol., March 1, 2004; 164(3): 903 - 914. [Abstract] [Full Text] [PDF]
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 Y. Y. Ng, B. van Kessel, H. M. Lokhorst, M. R. M. Baert, C. M. M. van den Burg, A. C. Bloem, and F. J. T. Staal Gene-expression profiling of CD34+ cells from various hematopoietic stem-cell sources reveals functional differences in stem-cell activity J. Leukoc. Biol., February 1, 2004; 75(2): 314 - 323. [Abstract] [Full Text] [PDF]
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J. Koopmann, Z. Zhang, N. White, J. Rosenzweig, N. Fedarko, S. Jagannath, M. I. Canto, C. J. Yeo, D. W. Chan, and M. Goggins Serum Diagnosis of Pancreatic Adenocarcinoma Using Surface-Enhanced Laser Desorption and Ionization Mass Spectrometry Clin. Cancer Res., February 1, 2004; 10(3): 860 - 868. [Abstract] [Full Text] [PDF]
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C. A. Iacobuzio-Donahue, R. Ashfaq, A. Maitra, N. V. Adsay, G. L. Shen-Ong, K. Berg, M. A. Hollingsworth, J. L. Cameron, C. J. Yeo, S. E. Kern, et al. Highly Expressed Genes in Pancreatic Ductal Adenocarcinomas: A Comprehensive Characterization and Comparison of the Transcription Profiles Obtained from Three Major Technologies Cancer Res., December 15, 2003; 63(24): 8614 - 8622. [Abstract] [Full Text] [PDF]
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J. Walter-Yohrling, X. Cao, M. Callahan, W. Weber, S. Morgenbesser, S. L. Madden, C. Wang, and B. A. Teicher Identification of Genes Expressed in Malignant Cells That Promote Invasion Cancer Res., December 15, 2003; 63(24): 8939 - 8947. [Abstract] [Full Text] [PDF]
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A. Maitra, D. E. Hansel, P. Argani, R. Ashfaq, A. Rahman, A. Naji, S. Deng, J. Geradts, L. Hawthorne, M. G. House, et al. Global Expression Analysis of Well-Differentiated Pancreatic Endocrine Neoplasms Using Oligonucleotide Microarrays Clin. Cancer Res., December 1, 2003; 9(16): 5988 - 5995. [Abstract] [Full Text] [PDF]
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 M. Ruel, C. Bianchi, T. A. Khan, S. Xu, J. R. Liddicoat, P. Voisine, E. Araujo, H. Lyon, I. S. Kohane, T. A. Libermann, et al. Gene expression profile after cardiopulmonary bypass and cardioplegic arrest J. Thorac. Cardiovasc. Surg., November 1, 2003; 126(5): 1521 - 1530. [Abstract] [Full Text] [PDF]
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K. G. Kohlgraf, A. J. Gawron, M. Higashi, J. L. Meza, M. D. Burdick, S. Kitajima, D. L. Kelly, T. C. Caffrey, and M. A. Hollingsworth Contribution of the MUC1 Tandem Repeat and Cytoplasmic Tail to Invasive and Metastatic Properties of a Pancreatic Cancer Cell Line Cancer Res., August 15, 2003; 63(16): 5011 - 5020. [Abstract] [Full Text] [PDF]
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N. Sato, A. Maitra, N. Fukushima, N. T. van Heek, H. Matsubayashi, C. A. Iacobuzio-Donahue, C. Rosty, and M. Goggins Frequent Hypomethylation of Multiple Genes Overexpressed in Pancreatic Ductal Adenocarcinoma Cancer Res., July 15, 2003; 63(14): 4158 - 4166. [Abstract] [Full Text] [PDF]
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D. E. Hansel, A. Rahman, M. Hidalgo, P. J. Thuluvath, K. D. Lillemoe, R. Shulick, J.-L. Ku, J.-G. Park, K. Miyazaki, R. Ashfaq, et al. Identification of Novel Cellular Targets in Biliary Tract Cancers Using Global Gene Expression Technology Am. J. Pathol., July 1, 2003; 163(1): 217 - 229. [Abstract] [Full Text] [PDF]
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C. D. Logsdon, D. M. Simeone, C. Binkley, T. Arumugam, J. K. Greenson, T. J. Giordano, D. E. Misek, and S. Hanash Molecular Profiling of Pancreatic Adenocarcinoma and Chronic Pancreatitis Identifies Multiple Genes Differentially Regulated in Pancreatic Cancer Cancer Res., May 15, 2003; 63(10): 2649 - 2657. [Abstract] [Full Text] [PDF]
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C. A. Iacobuzio-Donahue, A. Maitra, M. Olsen, A. W. Lowe, N. T. Van Heek, C. Rosty, K. Walter, N. Sato, A. Parker, R. Ashfaq, et al. Exploration of Global Gene Expression Patterns in Pancreatic Adenocarcinoma Using cDNA Microarrays Am. J. Pathol., April 1, 2003; 162(4): 1151 - 1162. [Abstract] [Full Text] [PDF]
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 A. Spector, D. Li, W. Ma, F. Sun, and P. Pavlidis Differential Amplification of Gene Expression in Lens Cell Lines Conditioned to Survive Peroxide Stress Invest. Ophthalmol. Vis. Sci., October 1, 2002; 43(10): 3251 - 3264. [Abstract] [Full Text] [PDF]
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