Karin D. Berg

Discovery of Novel Tumor Markers of Pancreatic Cancer using Global Gene Expression Technology

Despite several advances in our basic understanding and in the clinical management of pancreatic cancer, virtually all patients who will be diagnosed with pancreatic cancer will die from this disease. The high mortality of pancreatic cancer is predominantly because of diagnosis at an advanced stage of disease and a lack of effective treatments. We used the Gene Logic Inc. BioExpress platform and Affymetrix GeneChip arrays to identify genes differentially expressed in pancreatic cancer. cDNA was prepared from samples of normal pancreas (n = 11), normal gastrointestinal mucosa (n = 22), resected pancreas cancer tissues (n = 14), and pancreas cancer cell lines (n = 8), and was hybridized to the complete Affymetrix Human Genome U95 GeneChip set (arrays U95 A, B, C, D, and E) for simultaneous analysis of 60,000 cDNA fragments, with 12,000 fragments covering full-length genes and 48,000 fragments covering expressed sequence tags (ESTs). Genes expressed at levels at least fivefold greater in the pancreatic cancers ascompared to normal tissues were identified. Serial analysis of gene expression (SAGE) libraries (http://www.ncbi.nlm.nih.gov/SAGE/) of two normal pancreatic ductal cell cultures (HX and H126) were used to exclude genes expressed in the normal ducts (more than five tags per library). Differential expression of selected candidate genes was validated by immunohistochemical analysis (n = 3), by in situ hybridization (n = 1), and by reverse transcriptase-polymerase chain reaction (n = 8). One hundred eighty fragments were identified as having fivefold or greater expression levels in pancreas cancer specimens as compared to normal tissue, of which 124 corresponded to known genes and 56 to ESTs. Of these 124 fragments, 10 genes were represented by two or more fragments, resulting in 107 known genes identified as differentially expressed in pancreatic cancer. An additional 10 genes were expressed in the SAGE libraries of normal pancreatic duct epithelium, and were excluded from further analysis. A literature search indicated that 28 of the remaining 97 genes have been reported in association with pancreatic cancer, validating this approach. The remaining 69 genes have not been implicated in pancreatic cancer before, and have immediate potential as novel therapeutic targets and tumor markers of pancreatic cancer.

Conversion of diploidy to haploidy.

Individuals susceptible to multigene disorders may now be spotted more easily.

Met Proto-Oncogene and Insulin-Like Growth Factor Binding Protein 3 Overexpression Correlates with Metastatic Ability in Well-Differentiated Pancreatic Endocrine Neoplasms

Pancreatic endocrine neoplasms are neoplastic proliferations of islet cells or islet cell precursors and are capable of secreting a variety of synthetic products, including insulin, glucagon, gastrin, and vasoactive intestinal peptide. The biological behavior of pancreatic endocrine neoplasms is often unpredictable, and there are few reliable histopathologic criteria reliably correlating with metastatic ability. We have used the Affymetrix U133 GeneChip set (HG_U133 A and B; Affymetrix; Santa Clara, CA) representing ∼33,000 characterized transcripts to examine global gene expression profiles from well-differentiated nonmetastatic (n = 5) and metastatic (n = 7) pancreatic endocrine neoplasms to determine molecular markers that predict disease progression. Microarray hybridization data were normalized using the GeneLogic GeneExpress Software System to identify differentially up- and down-regulated genes in metastatic versus nonmetastatic pancreatic endocrine neoplasms. Using a 3-fold change in gene expression as a threshold, we have identified 65 overexpressed and 57 underexpressed genes in metastatic pancreatic endocrine neoplasms as compared with nonmetastatic pancreatic endocrine neoplasms. Several classes of genes, including growth factors and growth factor-related molecules (IGFBP1, IGFBP3, and MET), developmental factors (TBX3 and MEIS2), cytoskeletal factors (β 1 tubulin and ACTN2), cholesterol homeostasis mediators (LRP5, SLC27A2, and RXRG), intracellular signaling pathway mediators (DYRK1A, PKIB, and AK2), methyltransferases (MGMT and GAMT), and DNA repair and regulatory molecules (CHEK1 and ZNF198), were identified as differentially over- or underexpressed via this method. Immunohistochemical validation of microarray data were performed for two overexpressed genes, namely, the met proto-oncogene (MET) and insulin-like growth factor binding protein 3 (IGFBP3) with tissue microarrays of nonmetastatic (n = 24) and metastatic (n = 15) pancreatic endocrine neoplasms. Increased expression of IGFBP3 was confirmed in metastatic versus nonmetastatic pancreatic endocrine neoplasms (12 of 15, 80% versus 10 of 24, 42%), as well as in lymph node (6 of 7, 86%) and liver (9 of 9, 100%) metastases. Similarly, overexpression of MET was confirmed in metastatic versus nonmetastatic pancreatic endocrine neoplasms (5 of 15, 33% versus 4 of 24, 17%), as well as in lymph node metastases (4 of 7, 57%) and liver metastases (5 of 9, 56%). The majority of genes that demonstrated altered expression has not been previously identified as differentially expressed in metastatic pancreatic endocrine neoplasm lesions and may therefore represent newly identified molecules in the progression of these lesions.

Detection of Microsatellite Instability by Fluorescence Multiplex Polymerase Chain Reaction

We have created a clinical molecular diagnostic assay to test for microsatellite instability (MSI) at multiple loci simultaneously in paraffin-embedded surgical pathology colon resection specimens. This fluorescent multiplex polymerase chain reaction (PCR) assay analyzes the five primary microsatellite loci recommended at the 1997 National Cancer Institute-sponsored conference on MSI for the identification of MSI or replication errors in colorectal cancer: Bat-25, Bat-26, D2S123, D5S346, and D17S250. Amplicon detection is accomplished by capillary electrophoresis using the ABI 310 Genetic Analyzer. Assay validation compared 18 specimens previously assessed by radioactive PCR and polyacrylamide gel electrophoresis detection to results generated by the reported assay. Germline and tumor DNA samples were amplified in separate multiplex PCR reactions, sized in separate capillary electrophoresis runs, and compared directly to identify novel length alleles in tumor tissue. A concordance of 100% between the two modalities was achieved. The multiplex assay routinely detected a subpopulation of 10% tumor alleles in the presence of 90% normal alleles. A novel statistical model was generated that corroborates the validity of using results generated by analysis of five independent microsatellites to achieve a single overall MSI diagnosis. The assay presented is superior to standard radioactive monoplex PCR, polyacrylamide gel electrophoretic analysis, primarily due to the multiplex PCR format.

Molecular Diagnosis of Oligodendroglioma in Paraffin Sections

Distinction of oligodendrogliomas from other gliomas is clinically important, but the histologic diagnosis of oligodendroglioma has been a difficult and notoriously subjective task. Testing for loss of heterozygosity (LOH) on chromosomal arms 1p and 19q, the genetic signature of oligodendroglioma, has emerged as a useful, objective adjunct to the traditional histologic evaluation. However, LOH testing of glioma specimens has not yet been widely implemented, presumably because of a lack of a practical LOH assay. We describe a 1p and 19q LOH assay suitable for routine diagnostics. In contrast to traditional microsatellite-based LOH analysis, we show that detection of LOH is usually possible even without normal tissue or blood from the same patient. A small area of tumor on a single paraffin section is sufficient for the assay. The assay protocol consists of a one-step DNA extraction, multiplex PCR for microsatellites on 1p and 19q, and capillary electrophoresis of the PCR products. LOH is detected by analysis of the allelic patterns and by integration of data from multiple highly polymorphic microsatellites. In a validation study on 19 gliomas, the results were concordant with results obtained by established methods and correlated well with histologic diagnoses. Because only a paraffin section is required, the pathologist can perform both the traditional histopathologic evaluation and this supporting molecular assay from the material at hand.

Little or no residual prostate cancer at radical prostatectomy: Vanishing cancer or switched specimen? A microsatellite analysis of specimen identity

With more vigilant screening for prostate cancer, there has been an associated increase in patients with little or no residual cancer at radical prostatectomy after an initial diagnosis of minute cancer on needle biopsy. This raises a critical question as to whether the biopsy and subsequent radical prostatectomy in these patients are from the same patient. We used PCR-based microsatellite marker analysis to perform identity test in 46 men (35 with minute cancer and 11 with no residual cancer). Of them, 41 were interpretable, including 31 with minute cancer and 10 with no residual cancer. All 31 interpretable cases with minute cancer showed match between the initial biopsy and radical prostatectomy specimens. Nine of the 10 interpretable cases with no residual cancer showed match and 1 showed mismatch. The remaining 5 cases (4 with minute cancer and 1 with no residual cancer) were considered uninterpretable due to technical problems. The initial biopsy of the mismatched case had high-grade cancer (Gleason score 4 + 4 = 8) measuring 9.6 mm in length with perineural invasion. Our results confirm that, in most cases of “vanishing cancer” in radical prostatectomy specimens, it reflects a chance sampling of a minute cancer and not a switch in specimens. However, specimen switch can rarely occur, and if there is high grade or a lot of cancer on the biopsy with no or very minimal cancer in the radical prostatectomy specimen, one should evaluate for patient identity.

FLT3 mutations in myeloid sarcoma

Myeloid sarcoma is an extramedullary tumour that typically occurs in the setting of acute myeloid leukaemia (AML), or myeloproliferative disorders. In AML, two types of mutations in Fms‐like tyrosine kinase 3 (FLT3) have been described; internal tandem duplications (ITD) and point mutations at aspartic acid residue 835 (D835). We analysed 24 myeloid sarcoma specimens from 20 patients for FLT3 ITD and D835 mutations. FLT3 ITD mutations were identified in three of 20 cases (15%); no D835 mutations were identified. The ITD inserts ranged in size from 33 to 198u2003base pairs (bp) and represented approximately 20–40% of the FLT3 alleles. Two cases showed discordance in FLT3 ITD mutational status. In one case, the leukaemia specimen was positive for a FLT3 ITD mutation and the myeloid sarcoma specimen was negative. In the second case, the myeloid sarcoma was positive for a FLT3 ITD mutation at diagnosis, but negative in subsequent relapse samples. Our findings suggest that small molecule inhibitors of FLT3 may be useful therapeutic agents for treatment of myeloid sarcomas‐containing FLT3 mutations, however, the potential for discordance between the leukaemia and myeloid sarcoma, necessitates that the myeloid sarcoma tumour itself be analysed for FLT3 mutations.

Gene expression profiling identifies markers of ampullary adenocarcinoma

Ampullary adenocarcinoma is an aggressive cancer with a poor prognosis. Without surgical resection, ampullary adenocarcinomas can be difficult to distinguish from ampullary adenomas. The aim of this study was to identify differentially expressed genes in ampullary adenocarcinoma in order to identify candidate markers of the disease. The Affymetrix Human Genome U133 GeneChip set (HG-U133A and HG-U133B) was used to obtain gene expression profiles of 5 ampullary adenocarcinomas and 10 normal duodenal samples. Using fold change analysis we identified 235 fragments expressed at least fivefold higher in ampullary cancers than in normal duodenum. The expression profiles of eight candidate overexpressed genes (osteopontin, mesothelin, tissue inhibitor of metalloproteinases 1, mucin-1, mucin-5, fascin, heat shock protein 47, fibronectin 1) were confirmed by immunohistochemistry or in situ hybridization on tissue microarrays (TMA) containing 54 ampullary adenocarcinomas. One of these genes, osteopontin, was expressed 27-fold higher levels in ampullary adenocarcinomas compared to normal duodenum by genechip analysis. We therefore determined serum osteopontin levels in patients with ampullary neoplasms, patients with other periampullary diseases and in normal controls. Mean pre-operative serum osteopontin levels as measured by competitive ELISA were 906 ± 268 ng/ml in patients with ampullary cancer, 867 ± 160 ng/ml in patients with an ampullary adenoma, 327.1±195.6 ng/ml in patients with non-malignant periampullary diseases and 204 ± 65 ng/ml in age-matched healthy controls (P

Microsatellite Instability Is Infrequent in Medullary Breast Cancer

Microsatellite instability (MSI), characterized by contraction or expansion in microsatellite length or short tandem repeats compared with germline lengths, is found in 85% to 90% of colon cancer arising in hereditary nonpolyposis colorectal cancer families. These cancers commonly have characteristic histologic appearances, including medullary features with intense lymphoid infiltrates. In pancreatic cancer, a rare medullary histologic subtype more often demonstrates MSI than the more common adenocarcinoma subtype. We hypothesized that the medullary histologic pattern might correlate with MSI in additional tumor types and analyzed 8 cases of typical and atypical medullary carcinoma of the breast. Tumor and normal DNA was extracted from paraffinized tissue blocks of tumor and histologically uninvolved axillary lymph nodes, respectively. We analyzed the tumors for instability in 5 primary (BAT25, BAT26, D17S250, D5S346, D2S123) and 3 alternative (BAT40, D18S55, D18S58) microsatellites recommended at the National Cancer Institute--sponsored conference for diagnosis of MSI in colorectal cancer. All 8 tumors were microsatellite stable at the 8 loci, suggesting that MSI is not commonly associated with medullary or atypical medullary breast carcinoma, in contrast with the reported association with medullary tumors of the colon and pancreas.

Functional Genomics, Gene Arrays, and the Future of Pathology

The human genome project has attracted a great deal of attention in recent years among the general public as well as the scientific community. Although it is likely to be a number of years before many of the expected benefits of the genomics revolution are realized, the impact of these scientific breakthroughs on diagnostic pathology is likely to become apparent relatively quickly. In particular, gene array technology, which allows gene expression measurements of thousands of genes in parallel, provides a powerful tool for pathologists seeking new markers for diagnosis. Several recent studies demonstrate how the gene array approach can not only recognize markers for known categories of neoplasia but also lead to recognition of different categories not previously appreciated. Although this approach shows great potential, the successful application of gene arrays to diagnostic problems will require thoughtful interpretation, just as immunochemical technologies require careful planning and analysis.