Scott D. Jewell

Effect of Fixatives and Tissue Processing on the Content and Integrity of Nucleic Acids

Clinical and molecular medicines are undergoing a revolution based on the accelerated advances in biotechnology such as DNA microarrays and proteomics. Answers to fundamental questions such as how does the DNA sequence differ between individuals and what makes one individual more prone for a certain disease are eagerly being sought in this postgenomic era. Several government and nonprofit organizations provide the researchers access to human tissues for molecular studies. The tissues procured by the different organizations may differ with respect to fixation and processing parameters that may affect significantly the molecular profile of the tissues. It is imperative that a prospective investigator be aware of the potential contributing factors before designing a project. The purpose of this review is to provide an overview of the methods of human tissue acquisition, fixation, and preservation. In addition, the parameters of procurement and fixation that affect the quality of the tissues at the molecular level are discussed.

Microsatellite Instability Predicts Improved Response to Adjuvant Therapy With Irinotecan, Fluorouracil, and Leucovorin in Stage III Colon Cancer: Cancer and Leukemia Group B Protocol 89803

PURPOSE Colon cancers exhibiting DNA mismatch repair (MMR) defects demonstrate distinct clinical and pathologic features, including better prognosis and reduced response to fluorouracil (FU) -based chemotherapy. This prospective study investigated adjuvant chemotherapy containing FU and irinotecan in patients with MMR deficient (MMR-D) colon cancers. PATIENTS AND METHODS Cancer and Leukemia Group B 89803 randomly assigned 1,264 patients with stage III colon cancer to postoperative weekly bolus FU/leucovorin (LV) or weekly bolus irinotecan, FU, and LV (IFL). The primary end point was overall survival; disease-free survival (DFS) was a secondary end point. Tumor expression of the MMR proteins, MLH1 and MSH2, was determined by immunohistochemistry (IHC). DNA microsatellite instability was also assessed using a panel of mono- and dinucleotide markers. Tumors with MMR defects were those demonstrating loss of MMR protein expression (MMR-D) and/or microsatellite instability high (MSI-H) genotype. RESULTS Of 723 tumor cases examined by genotyping and IHC, 96 (13.3%) were MMR-D/MSI-H. Genotyping results were consistent with IHC in 702 cases (97.1%). IFL-treated patients with MMR-D/MSI-H tumors showed improved 5-year DFS as compared with those with mismatch repair intact tumors (0.76; 95% CI, 0.64 to 0.88 v 0.59; 95% CI, 0.53 to 0.64; P = .03). This relationship was not observed among patients treated with FU/LV. A trend toward longer DFS was observed in IFL-treated patients with MMR-D/MSI-H tumors as compared with those receiving FU/LV (0.57; 95% CI, 0.42 to 0.71 v 0.76; 95% CI, 0.64 to 0.88; P = .07; hazard ratio interaction between tumor status and treatment, 0.51; likelihood ratio P = .117). CONCLUSION Loss of tumor MMR function may predict improved outcome in patients treated with the IFL regimen as compared with those receiving FU/LV.

Norepinephrine Up-regulates the Expression of Vascular Endothelial Growth Factor, Matrix Metalloproteinase (MMP)-2, and MMP-9 in Nasopharyngeal Carcinoma Tumor Cells

Recent studies using ovarian cancer cells have shown that the catecholamine hormones norepinephrine (norepi) and epinephrine (epi) may influence cancer progression by modulating the expression of matrix metalloproteinases (MMP) and vascular endothelial growth factor (VEGF). The purpose of this study is to determine if the stress hormone norepi can influence the expression of MMP-2, MMP-9, and VEGF in nasopharyngeal carcinoma (NPC) tumors by using three NPC tumor cell lines. The NPC cell lines HONE-1, HNE-1, and CNE-1 were treated with norepi. The effects of norepi on MMP-2, MMP-9, and VEGF synthesis were measured by ELISA; functional MMP activity was measured by the invasive potential of the cells using a membrane invasion culture system whereas functional activity of VEGF was analyzed using a human umbilical vein endothelial cell tube formation assay. Norepi treatment increased MMP-2, MMP-9, and VEGF levels in culture supernatants of HONE-1 cells, which could be inhibited by the beta-blocker propranolol. Norepi induced the invasiveness of all NPC cell lines in a dose-dependent manner, which was blocked by CMT-3, an MMP inhibitor, and propranolol. Norepi stimulated the release of functional angiogenic VEGF by HONE-1 cells as well. Finally, HONE-1 cells were shown to express beta-adrenergic receptors as did seven of seven NPC biopsies examined. The data suggest that catecholamine hormones produced by the sympathetic-adrenal medullary axis may affect NPC tumor progression, in part, through modulation of key angiogenic cytokines.

Eicosanoid Modulation in Advanced Lung Cancer: Cyclooxygenase-2 Expression Is a Positive Predictive Factor for Celecoxib + Chemotherapy—Cancer and Leukemia Group B Trial 30203

PURPOSE Increased expression of eicosanoids in cancer has been associated with adverse prognosis. We performed a randomized phase II trial to test the hypothesis that inhibitors of two eicosanoid pathways (cyclooxygenase-2 [COX-2], celecoxib and 5-lipoxygenase [5-LOX], zileuton) added to chemotherapy would improve outcome in advanced non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS Patients with advanced NSCLC, a performance status of 0 to 2, and no prior therapy were eligible. All patients received carboplatin area under the curve (AUC) 5.5 mg/mL x min day 1 + gemcitabine (1,000 mg/m(2)) days 1 and 8. Patients were randomly assigned to: (a) zileuton 600 mg PO qid, (b) celecoxib 400 mg PO bid, or (c) celecoxib and zileuton at the same doses. Immunohistochemical staining for COX-2 and 5-LOX was performed without knowledge of outcomes. RESULTS One hundred forty patients were entered and 134 were eligible and treated. There was no survival difference between the arms. COX-2 expression was a negative prognostic marker for overall survival (OS; hazard ratio [HR] = 2.51, P = .019 for index >or= 4; HR = 4.16, P = .005 for index = 9) for patients not receiving celecoxib. Patients with increased COX-2 expression (index >or= 4), receiving celecoxib had better survival than did COX-2-expressing patients not receiving drug (HR = .342, P = .005 for OS; HR = .294, P = .002 for failure-free survival). Multivariate analysis confirmed the interaction of COX-2 and celecoxib on survival. 5-LOX expression was neither prognostic nor predictive. CONCLUSION This study failed to demonstrate the value of dual eicosanoid inhibition or benefit from either agent alone in addition to chemotherapy. However, a prospectively defined subset analysis suggests an advantage for celecoxib and chemotherapy for patients with moderate to high COX-2 expression.

Analysis of the Molecular Quality of Human Tissues An Experience From the Cooperative Human Tissue Network

The scientific usefulness of the data obtained from tissue analysis is related to specimen quality, which may be affected by conditions that may contribute to the degradation of the specimen before processing and analysis. We determined the usability of nucleic acids extracted from banked human tissues for further molecular analyses. We assayed 151 tissue specimens, storedfor various times at 4 divisions of the Cooperative Human Tissue Network, National Cancer Institute, Bethesda, MD, for DNA and RNA degradation. Simple electrophoresis, polymerase chain reaction (PCR), reverse-transcriptase (RT)-PCR, and Northern blot analysis were compared to determine the optimal quality control procedure. In addition, a time course degradation procedure was performed on human lung tissue. Gel electrophoresis was as informative as PCR, RT-PCR, and Northern blot analysis in determining the molecular usefulness of the human tissues. Overall, 80% of the stored human tissues had good-quality DNA, and 60% had good-quality RNA. Electrophoresis procedures for DNA and RNA offer a quick and valuable measure of the molecular quality of stored human tissues. The DNA and RNA degradation of one tissue type (lung) was stable for both nucleic acids for up to 5 hours after excision.

Accurate Molecular Characterization of Formalin-Fixed, Paraffin-Embedded Tissues by microRNA Expression Profiling

Formalin-fixed, paraffin-embedded tissues are an invaluable tool for biomarker discovery and validation. As these archived specimens are not always compatible with modern genomic techniques such as gene expression arrays, we assessed the use of microRNA (miRNA) as an alternative means for the reliable molecular characterization of formalin-fixed, paraffin-embedded tissues. Expression profiling using two different microarray platforms and multiple mouse and human formalin-fixed, paraffin-embedded tissue types resulted in the correlation ratios of miRNA expression levels between frozen and fixed tissue pairs ranging from 0.82 to 0.99, depending on the cellular heterogeneity of the tissue type. The same miRNAs were identified as differentially expressed between tissues using both fixed and frozen specimens. While formalin fixation time had only marginal effects on microarray performance, extended storage times for tissue blocks (up to 11 years) resulted in a gradual loss of detection of miRNAs expressed at low levels. Method reproducibility and accuracy were also evaluated in two different tissues stored for different lengths of time. The technical variation between full process replicates, including independent RNA isolation methods, was approximately 5%, and the correlation of expression levels between microarray and real-time quantitative reverse transcriptase polymerase chain reaction was 0.98. Together, these data demonstrate that miRNA expression profiling is an accurate and robust method for the molecular analysis of archived clinical specimens, potentially extending the use of miRNAs as new diagnostic, prognostic, and treatment response biomarkers.

Analysis of Micrometastatic Disease in Sentinel Lymph Nodes From Resectable Colon Cancer: Results of Cancer and Leukemia Group B Trial 80001

PURPOSE To determine whether sentinel lymph node (LN) sampling (SLNS) could reduce the number of nodes required to characterize micrometastatic disease (MMD) in patients with potentially curable colon cancer. PATIENTS AND METHODS Cancer and Leukemia Group B 80001 was a study to determine whether SLNS could identify a subset of LNs that predicted the status of the nodal basin for resectable colon cancer and, therefore, could be extensively evaluated for the presence of micrometastases. Patients enrolled onto this study underwent SLNS after injection of 1% isosulfan blue, and both sentinel nodes (SNs) and non-SNs obtained during primary tumor resection were sectioned at multiple levels and stained using anti-carcinoembryonic antigen and anticytokeratin antibodies. RESULTS Using standard histopathology, SNs failed to predict the presence of nodal disease in 13 (54%) of 24 node-positive patients. Immunostains were performed for patients whose LNs were negative by standard histopathology. Depending on the immunohistochemical criteria used to assign LN positivity, SN examination resulted in either an unacceptably high false-positive rate (20%) or a low sensitivity for detection of MMD (40%). CONCLUSION By examining both SNs and non-SNs, this multi-institutional study showed that SNs did not accurately predict the presence of either conventionally defined nodal metastases or MMD. As a result, SLNS is not a useful technique for the study of MMD in patients with colon cancer.

Recommendations for Collection and Handling of Specimens From Group Breast Cancer Clinical Trials

Recommendations for specimen collection and handling have been developed for adoption across breast cancer clinical trials conducted by the Breast International Group (BIG)-sponsored Groups and the National Cancer Institute (NCI)-sponsored North American Cooperative Groups. These recommendations are meant to promote identifiable standards for specimen collection and handling within and across breast cancer trials, such that the variability in collection/handling practices that currently exists is minimized and specimen condition and quality are enhanced, thereby maximizing results from specimen-based diagnostic testing and research. Three working groups were formed from the Cooperative Group Banking Committee, BIG groups, and North American breast cancer cooperative groups to identify standards for collection and handling of (1) formalin-fixed, paraffin-embedded (FFPE) tissue; (2) blood and its components; and (3) fresh/frozen tissue from breast cancer trials. The working groups collected standard operating procedures from multiple group specimen banks, administered a survey on banking practices to those banks, and engaged in a series of discussions from 2005 to 2007. Their contributions were synthesized into this document, which focuses primarily on collection and handling of specimens to the point of shipment to the central bank, although also offers some guidance to central banks. Major recommendations include submission of an FFPE block, whole blood, and serial serum or plasma from breast cancer clinical trials, and use of one fixative and buffer type (10% neutral phosphate-buffered formalin, pH 7) for FFPE tissue across trials. Recommendations for proper handling and shipping were developed for blood, serum, plasma, FFPE, and fresh/frozen tissue.

Evaluation of TGF-α and EGFR Expression in Oral Leukoplakia and Oral Submucous Fibrosis by Quantitative Immunohistochemistry

Objective: Oral leukoplakia (OL) and oral submucous fibrosis (OSMF) are clinically distinct preneoplastic states that precede the development of oral squamous cell carcinoma. Recently, attempts are being made to identify specific molecular event(s) as prognostic markers to identify oral precancerous lesions with higher malignant potential. The goal of the present study was to evaluate the expression of EGFR and its ligand TGF-α in OL with dysplasia and OSMF as intermediate markers of malignancy by quantitative immunohistochemistry. Methods: Oral tissues were stained with monoclonal antibodies to TGF-α and EGFR. The results were analyzed with Photoquant image analysis software. Results: The expression of TGF-α and EGFR was upregulated in OL, OSMF and oral squamous cell carcinomas relative to normal oral mucosa. The area and intensity of staining of TGF-α in the proliferative layers (stratum germinativum) increased linearly in OL with mild, moderate and severe dysplasia as compared to control mucosa (p < 0.05). EGFR levels increased linearly in the stratum spinosum in OL with increasing degrees of dysplasia (p < 0.05). In general, the expression of both proteins in OSMF was similar to that in OL with moderate dysplasia. Conclusions: EGFR and TGF-α represent early markers of malignancy in OL with dysplasia. Quantitative measurement of these proteins may provide intermediate endpoints in prospective chemopreventive trials.

Suppression of Experimental Autoimmune Encephalomyelitis Using Peptide Mimics of CD28

The B7:CD28/CTLA-4 costimulatory pathway plays a critical role in regulating the immune response and thus provides an ideal target for therapeutic manipulation of autoimmune disease. Previous studies have shown that blockade of CD28 signaling by mAbs can both prevent and exacerbate experimental autoimmune encephalomyelitis (EAE). In this study, we have designed two CD28 peptide mimics that selectively block B7:CD28 interactions. By surface plasmon resonance, both the end group-blocked CD28 peptide (EL-CD28) and its retro-inverso isomer (RI-CD28) compete effectively with the extracellular domain of CD28 for binding to B7-1. Both the CD28 peptide mimics inhibited expansion of encephalitogenic T cells in vitro. A single administration of EL-CD28 or RI-CD28 peptide significantly reduced disease severity in EAE. Importantly, we show that either CD28 peptide mimic administered during acute disease dramatically improved clinical signs of EAE, suppressing ongoing disease. The ratio of CD80:CD86 expression was significantly lower on CD4+ and F4/80+ spleen cells in CD28 peptide-treated mice. Peripheral deletion of Ag-specific CD4+ T cells occurs following in vivo blockade of CD28 with synthetic CD28 peptides.