Mary Lowery Nordberg

Potential roles of cell-derived microparticles in ischemic brain disease

Abstract Purpose: The objective of this study is to review the role of cell-derived microparticles in ischemic cerebrovascular diseases. Materials and methods: An extensive PubMed search of literature pertaining to this study was performed in April 2009 using specific keyword search terms related to cell-derived microparticles and ischemic stroke. Some references are not cited here as it is not possible to be all inclusive or due to space limitation. Discussion: Cell-derived microparticles are small membranous vesicles released from the plasma membranes of platelets, leukocytes, red cells and endothelial cells in response to diverse biochemical agents or mechanical stresses. They are the main carriers of circulating tissue factor, the principal initiator of intravascular thrombosis, and are implicated in a variety of thrombotic and inflammatory disorders. This review outlines evidence suggesting that cell-derived microparticles are involved predominantly with microvascular, as opposed to macrovascular, thrombosis. More specifically, cell-derived microparticles may substantially contribute to ischemic brain disease in several settings, as well as to neuroinflammatory conditions. Conclusion: If further work confirms this hypothesis, novel therapeutic strategies for minimizing cell-derived microparticles-mediated ischemia are available or can be developed, as discussed.

EGFR expression as an ancillary tool for diagnosing lung cancer in cytology specimens.

Lung cancer evolves in a multistep process, and its early detection portends a better prognosis. Bronchial washings/brushings and fine-needle aspirations are often used as early screening and cytological diagnosis of lung cancer. In some cases, it is difficult to differentiate morphologically malignant from reactive cells. Epidermal growth factor receptor (EGFR) is a transmembrane receptor overexpressed in high percentage lung cancers, and contributes to tumor growth. Assessing EGFR expression levels by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) may provide critical information of tumor marker abnormalities, assist in the cytological diagnosis, and stratify patients for EGFR inhibitor therapy. Fifty patients with bronchial washings/brushings or fine-needle aspiration specimens, and corresponding histologically confirmed lung biopsies, were studied for EGFR expression with FISH and IHC. Copy numbers of the EGFR gene locus were analyzed with those of chromosome 7 by FISH. EGFR and FISH results were compared to our FISH data with combined EGFR, c-myc, 5p15.2, and chromosome 6 probes in selected cases. Cell blocks, if available, and tissue biopsy sections were used for EGFR IHC. The intensity of IHC was scored, and quantified. Only balanced aneuploidy of EGFR was identified by FISH. Gene amplification was not detected. The chromosomal abnormalities of EGFR were often accompanied by other chromosomal aneuploidies demonstrated in c-myc (8q24), 5p15.2 or 6p, indicating a general genomic instability. About half of the specimens with confirmed malignancy showed EGFR balanced aneuploidy by FISH, and gene copy number was not coupled with protein expression in many cases. The benign or reactive cytology specimens confirmed by biopsies had high specificity by FISH (96%) and IHC (88%). FISH and IHC analysis of EGFR, possibly along with other tumor markers, may be a useful ancillary tool to classify difficult cytology cases and inform clinicians arranging targeted chemotherapy.

Comparison of FISH and Quantitative RT-PCR for the Diagnosis and Follow-Up of BCR-ABL-Positive Leukemias

AbstractBackground: For Philadelphia chromosome positive (Ph+) leukemias (chronic myelogenous leukemia [CML], acute lymphoblastic leukemia [ALL], and rare other leukemias), both allogeneic transplantation and treatment with tyrosine kinase inhibitors offer chances of molecular remission (the molecular marker being consistently undetectable). Molecular remission is defined as a reduction in the quantification of BCR-ABL transcripts to an undetectable level by molecular diagnostic methods, and is considered as a surrogate marker for cure or long-term disease control. The molecular diagnostic methods including fluorescence in situ hybridization (FISH) and quantitative reverse transcription-polymerase chain reaction (QRT-PCR) are more sensitive than classical cytogenetic analysis for the detection of BCR-ABL positive cells. QRT-PCR, due to its superior sensitivity, is considered the gold standard for the follow-up of Ph+ leukemias treated with imatinib. Aim: The objective of our study was to compare the diagnostic and clinical usefulness of FISH and QRT-PCR at different timepoints for Ph+ leukemias. Patients and methods: We investigated 23 unselected patients with Ph+ CML (n = 21) or Ph+ ALL (n = 2) at 77 different timepoints in a comparative study with both FISH and QRT-PCR using commercially available reagents in a routine laboratory. Results: Our study demonstrated a good correlation of QRT-PCR with FISH in detecting the BCR-ABL fusion gene among patients with CML or ALL (coefficient of correlation = 0.77493, p < 0.0001, using Spearman’s correlation procedure). All newly diagnosed or untreated cases were positive with both methods. Lower coefficients of correlation were found when FISH and QRT-PCR were correlated with the white blood cell count (WBC). An overall concordance of FISH and QRT-PCR (being either negative or positive in both tests) was found in 65 cases (84.4%) and a discrepancy identified in 12 cases (15.6%). Conclusions: We confirm that QRT-PCR allows precise measurement of low levels of BCR-ABL transcripts and can serve as a sensitive indicator for minimal residual disease. In addition, we demonstrate in most cases a good correlation of QRT-PCR with FISH in detecting the BCR-ABL fusion gene among patients with CML or Ph+ ALL. FISH is not suitable for monitoring minimal residual disease.

Gastrointestinal Stromal Tumors: Current Concepts and Controversies

A large number of mesenchymal lesions can occur in the gastrointestinal tract. Stromal tumors represent the most important category of these lesions and have been a source of controversy in regards to classification, histogenesis/ differentiation and perhaps most important, determination of biologic potential. Recently with the advent of new therapeutic interventions, proper characterization and conceptualization of these tumors has acquired a new meaning, and emphasis is being placed on the role that various diagnostic techniques play in the evaluation of these neoplasms. The information that has become available is still in the process of being collated and expert opinions vary according to their own biases. There is no consensus of opinion in some very important and fundamental issues. While most of the issues pertaining to stromal gastrointestinal tumors may eventually be clarified with well-designed studies addressing the controversies, it may take quite some time before this happens and proper clinico-pathologic testing of the conclusions is completed. In the meantime, these neoplasms need to be carefully examined pathologically in a way that clinically relevant information is obtained to be conveyed to the clinicians. The challenge today remains to decide how these stromal tumors should be worked up by surgical pathologists and reported to the clinicians to manage their patients appropriately. This article will address some of the important issues that remain unsettled. In this article, two representative cases will be illustrated to highlight a practical approach to address issues pertaining to gastrointestinal stromal tumors.

Fluorescence In Situ Hybridization as an Ancillary Tool in Urine Cytology to Diagnose Urothelial Carcinoma

Urothelial carcinoma represents a diagnostic challenge for cytopathologists, especially for diagnosing low-grade tumors. The poor sensitivity of urine cytology makes it difficult for the pathologist to identify these tumors. In the past few years, researchers have sought more sensitive and specific methods, and the focus has been on identifying chromosomal abnormalities in tumor cells. A multicolor, multitarget, interphase fluorescence in situ hybridization (FISH) probe set containing probes to the centromeres of chromosomes 3, 7, 17, and 9p21 band has been shown to be more sensitive and specific for detecting urothelial carcinoma in urine specimens when compared with urine cytology. This is helpful in cases in which the urine cytology is equivocal. Early diagnosis can significantly reduce the morbidity and mortality of urothelial carcinoma. FISH can also be used for monitoring patients with noninvasive urothelial carcinoma for tumor recurrence.

Abstract 1573: MLPA and REMBRANDT data predict potential modulation of vitamin D via increased CYP27B1 in aggressive primary brain tumors

Glioblastomas are lethal, aggressive primary brain tumors that resist therapies. We hypothesized that their analysis using multiplex ligation probe amplification (MLPA) kits (MRC Holland, The Netherlands) with probes for 81 genes could aid in selection of individualized treatments for patients. Genes tested in P171, P172, P173 MLPA kits are known to have increased copy numbers in various tumors. DNA was purified from 4 glioblastomas, 1 meningioma, & 1 metastatic carcinoma (lung primary). No gene amplifications were shown in the meningioma or brain metastasis. Probes detected gene amplifications in the glioblastomas: all 4 had amplified EGFR and two (50%) had additional amplifications of CDK4 and CYP27B1. One of these also had amplification of PDGFRA and MDM2. Amplification of CYP27B1 at 12q13.3 was related to CDK4 at 12q14 as shown by simultaneous occurrence in 2 of the 4 glioblastomas. Products of the amplified genes are related to cell proliferation with CYP27B19s effects indirectly mediated through its metabolism of vitamin D (not shown here). Gene amplifications in tumor samples were quantified by calculating the fold increase versus the same gene in commercial normal brain DNA tested concurrently. At least 6 other genes (no apparent changes between normal and tumor) in each MLPA kit were used for normalization between the two samples in each case. Genes amplified in at least 2 tumors, included EGFR, 6.8 − 31.5 fold, CDK4, 6.7 − 17.8 fold and CYP27B1, 3.3 − 9.7 fold increased. All were reported previously as being amplified in some glioblastomas, with a lower percent noted for CYP27B1 than here. Although survival data for copy numbers of these genes in the NIH9s REMBRANDT database are limited to EGFR, survival data for their gene expressions (median reporters) are available for 297 gliomas with poorer survival indicated for increased EGFR (3-fold, p = 0.0017), CYP27B1 (2-fold, p = 0.0284) and MDM2 (2-fold, p = 0.0397) in 112, 102, and 100 patients, respectively, but not for increased expression of CDK4 or PDGFRA. Each group with poorer survival contained approximately 80% high grade gliomas, grades III-IV/IV. Chi-square analysis of REMBRANDT data confirmed the relationship between CYP27B1 and CDK4 with p = 9.04 × 10 −5 for co-expression of both when increased 2-fold and p = 3.97 × 10 −13 for 2-fold increased CYP27B1 with 3-fold increased CDK4. Analysis of CYP27B1 should be included in genomic surveys of brain tumors performed for designing individualized treatments in that vitamin D levels are potentially modulated due to amplification of the gene encoding one of its metabolic enzymes. We acknowledge Louisiana State University Health Sciences Center - Shreveport9s Fall 2008 Grant-In-Aid funding, Dwain D9Souza, LSUHSC-S, for technical support and use of the Repository of Molecular Brain Neoplasia Database (REMBRANDT) at NCI & NINDS at NIH, http://rembrandt.nci.nih.gov. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1573.

Molecular Methods for Detection of Tumor Markers in Glioblastomas

Glioblastoma (grade IV/IV astrocytoma), the most malignant type of brain tumor, occurs in all age groups with the highest incidence in older patients. Novel therapeutic strategies and molecular assays to follow treatments of suppressible targets are urgently needed. Surveys of large numbers of genes in the initial phase of tumor marker identification are currently being performed in multi-center studies. Queries of the results in national databases, such as the Respository of Molecular Brain Neoplasia Database (REMBRANDT) can yield candidate tumor markers, such as those involved with upregulated glycolysis. We found that ENO1, encoding enolase 1, emerged as a tumor marker in glioblastomas. Although treatments directed at glycolytic enzymes, such as enolase, are not technically feasible, ENO1 can be used to identify associated genes that are feasible targets. Real-time quantitative polymerase chain reaction (RQ-PCR) provides the sensitivity required to detect ENO1-associated tumor markers that can serve as suppressible treatment targets. The sensitivity of RQ-PCR is also ideal for monitoring treatment response to successful cancer drugs that are developed for molecular targets. This approach to tumor marker discovery in glioblastomas is described.

Genetics Primer: What Is Behind a Molecular Test?

Molecular pathology is a broad but distinct, new dimension added to the traditional approach of laboratory medicine in the investigation of human disease. This new division incorporates basic molecular biology themes for use in the clinical arena to investigate and characterize nucleic acids. We now know that many clinical entities have a genetic component central to their etiology. It becomes the role of the pathologist to recognize and provide support for clinicians by applying these new molecular methods in the diagnosis, classification, and prognosis of human disease. This affects anatomic and clinical pathology because new standards for disease diagnoses are being altered to reflect molecular pathology where appropriate. DNA tests may aid in the diagnosis and subsequent treatment; however, they frequently cannot change the course of the disease in affected individuals. Nonetheless, early diagnosis of specific mutations associated with morbidity and/or mortality assists in the diagnosis of human disease by providing additional diagnostic data not obtainable by conventional methodologies. This review introduces basic molecular foundations used in genetic approaches to define and characterize human disease. It is intended to serve as an introduction and encourage interested readers to increase their fund of knowledge by perusing many of the excellent text and reference books in this specialty.

HER-2/neu Status: Comparison of Quantitative Immunohistochemistry by Automated Cellular Imaging and Fluorescence In Situ Hybridization

Amplification of the HER-2/neu gene is generally accompanied by overexpression of its protein product in invasive breast carcinoma. Accurate determination of HER2 status is imperative, particularly when considering therapeutic intervention with the anti-HER2 monoclonal antibody-based therapy, Herceptin. Correlation of gene and protein status is at a maximum at both ends of the protein expression spectrum (3 and negative by IHC). While image analyses have improved the accuracy of interpretation, the relationship of HER-2/neu gene amplification and protein expression at intermediate levels of immunostaining (2 ) remains an issue of controversy. To compare the efficacy and accuracy of FISH and quantitative IHC in determining HER-2/neu status in human breast cancer, levels of immunostaining for HER-2/neu (HercepTest), were scored using image analysis (ChromaVision ACIS). Reflex testing for HER-2/neu FISH (PathVysion) was performed on a subset of cases with intermediate IHC values. Of the cases evaluated, some cases remained negative for HER-2/neu gene amplification by FISH yet were considered to be positive for HER2 overexpression by IHC. Despite the improvements in IHC interpretation, it is evident that FISH testing may provide a strategic advantage for patients to most benefit from Herceptin therapy.