Gregory J. Tsongalis

Correlation of polymorphisms to coagulation and biochemical risk factors for cardiovascular diseases

Currently, the established risk factors for cardiovascular disease (CVD) are largely environmental in nature. Conflicting studies have suggested that mutations in specific coagulation genes may also provide a genetic basis for CVD risk. We reviewed clinical studies that examined the role of single nucleotide polymorphisms in coagulation and platelet factors, and a biochemical factor to determine if specific genotypes are correlated with patients with a history of arterial thrombotic diseases (acute coronary syndromes or stroke). A meta-analysis was performed on studies for factors II (G20210A variant), V Leiden (G1691A), VII (R353Q), glycoprotein (GP) IIIa receptor (PI(A1/A2)), and methylenetetrahydrofolate reductase (MTHFR, C677T). There was no correlation for factor II or factor V polymorphisms to coronary artery disease (CAD) in 5,607 and 5,431 patients studied, respectively. There was also no correlation for factor II variants and stroke in 3,451 patients studied. For factor V, statistical significance was achieved for the G1691A variant on 3,399 patients with stroke (odds ratio [OR] 1.43, 95% confidence intervals [CI] 1.03 to 1.97). The GP IIIa PI(A1/A2) genotype was associated with increased risk for CAD in 7,920 patients (OR 1.12, 95% CI 1.01 to 1.24), but not for 1,855 patients who had a stroke (OR 0.80, 95% CI 0.62 to 1.04). The combined RQ and RR genotypes of factor VII R353Q were correlated to a reduced risk for CVD in 2,574 patients (OR 0.78, 95% CI 0.65 to 0.93), whereas the QQ genotype had offered more protection (OR 0.53, 95% CI 0.27 to 1.03). The TT homozygous variant of MTHFR was associated with CAD risk in 5,644 patients studied (OR 1.30, 95% CI 1.11 to 1.52) but not for 3,075 patients with stroke. This study shows that for some genes, further studies are unnecessary, whereas for others, no more enrollments are needed. The impact of certain genotypes must be examined in relation to other established risk factors and potentially new therapeutic strategies.

Pharmacogenomics: will it change the field of medicine?

Tailoring preventive medicine to an individual’s predisposition for specific diseases, predicting a patient’s response to therapeutic agents, designing new therapeutic agents to optimize their effectiveness, and predicting an individual’s response to exercise based on their DNA are only a few concepts that were once thought unlikely but have become reality. It has always been known that an individual’s genetic makeup plays a major role in disease and drug effectiveness, but the exact details have not been known. The near completion of the Human Genome Project and the advanced biotechnology, however, are allowing this mystery to become unraveled through a new applied discipline, pharmacogenomics. The aim of pharmacogenomics is to decrease adverse responses to therapy through determining new therapeutic targets and genetic polymorphisms that effect drug specificity and toxicity. A gene sequence is considered polymorphic if one or more genetic variations occur in at least 1% of the popula-

Search for evidence of recurring or persistent viruses in Crohn's disease

New‐onset Crohns disease and acute flares are often associated with viral infections. The aim of this study was to search for evidence of persistent or recurrent viruses in patients. Tissue blocks were obtained from surgical specimens from patients and a control population. 111 samples were tested by PCR or RT‐PCR, for EBV, CMV, HSV 1, HSV 2, HHV 8, pestiviruses, and enteroviruses. Additionally, seven sets of serum samples, including pre‐operative and post‐operative samples, from CD patients were analyzed serologically for antibodies to EBV. The tests revealed evidence of EBV nucleic acid in tissues of 11 patients from a total of 70 tested (15.7%) and in tissues of 3 of 41 control subjects (7.3%). Evidence of pestivirus was found in one CD patient, while one patient and one control were positive for CMV. No HSV 1 or 2, HHV 8 or enteroviruses were found. The serologic tests revealed that five of seven CD patients had antibodies against the early protein, the capsid protein and the EBV nuclear antigen (EBNA). The titers were not significantly altered post‐surgically. None of the patients had antibodies of the IgM isotype. Our findings vary from those of Ruther et al. who demonstrated evidence of EBV in tissues from 7 of 11 (64%) German CD patients. Antibodies to early EBV viral antigen and to nuclear antigen in five of seven Belgian patients suggest persistent active viral infection.

p185HER2 Overexpression in Human Breast Cancer Using Molecular and Immunohistochemical Methods

Abstract With the successful clinical trials of the engineered antibody Herceptin(tm) (in advanced-stage breast cancer) and adriamycin-based chemotherapy regimens (in the adjuvant setting), the need to detect pi85HER2 overexpression or associated amplification of the coding gene HER2 in breast cancer patients is escalating. Twenty to 30% of breast carcinomas have overexpression of p185HEK2. This condition correlates with poor patient prognosis and predicts response to chemotherapy in lymph node-positive patients. In this study we compare quantisation of p185HER2 in breast cancer at the gene and protein levels using differential polymerase chain reaction (PCR) and immunohistochemistiy, respectively. To assign HER2 gene copy numbers, a calibration curve was constructed using normal breast epithelia and breast carcinoma cell lines having known dosages of amplified HER2. We found corresponding molecular and immunohistochemical results in 85% of the 13 paraffin-embedded breast carcinoma cases examined. Two cases were found to have minimum gene amplification but marked p185HER2 overexpression, suggesting an alternative mechanism to overexpression such as transcriptional activation. although the differential PCR assay exhibits saturation approaching 20 HER2 gene copies, this may not be clinically significant because the immunohistochemical assay also appears to saturate in this gene copy number range.

Correlation between Morphologic and Nonmorphologic Prognostic Markers of Neuroblastoma

Morphologic (Shimada classification--SC, original and modified histologic grades--OHG and MHG) and nonmorphologic (serum LDH, 1 p del, DNA index, N-myc copy number, telomerase activity, and expression of MRP, MDR1, and TRK) prognostic markers for NB have been reviewed. The functional role of these nonmorphologic markers in the development and progression of this disease include abnormal cell proliferation, resistance to chemotherapeutic agents, and induction of apoptosis. A statistically significant association between high OHG/MHG (grade 3), DNA index of 1 (diploidy), > 1 copy of N-myc per haploid genome and serum LDH of > or = 1500 IU/1 (p < 0.001 for each) has been described. In SC, undifferentiated histology and high MKI are associated with N-myc amplification. However, a lack of correlation between morphology and N-myc amplification has been found in localized NB. Confirmation of these observations must now be obtained on larger numbers of prospectively studied cases. Data on correlation for various prognostic markers could provide guidelines for identification of subsets of NB having strongly significant, readily determinable, reproducible, and relatively inexpensive prognostic markers that could ultimately be used to design an algorithm for risk-specific therapy.

Rapid Detection of HSV from Cytologic Specimens Collected into ThinPrep Fixative

OBJECTIVE Herpes simplex virus (HSV) infection is associated with substantial morbidity and mortality in neonates. A diagnosis of HSV on cervical cytologic studies could lead to a cesarean section, with an increase in the risk of maternal morbidity. The identification of viral lesions in sexually active women has medical and social implications. There have been reports of false positive diagnoses of HSV in patients with altered endocervical cells and with cervical intraepithelial neoplasia 3. We evaluated a polymerase chain reaction (PCR)-based assay to detect HSV-1 and HSV-2 in routinely collected cervical cytology specimens in ThinPrep fixative (Cytyc Corp., Marlborough, Massachussets, U.S.A.). STUDY DESIGN DNA was extracted from five cases that demonstrated cytologic changes suggestive of an HSV infection. PCR amplification with subsequent gel electrophoresis was performed to detect the presence of HSV. RESULTS HSV DNA was detected in three of five cases that were cytologically diagnosed as suspicious or strongly suspicious for HSV infection. CONCLUSION The combination of the ThinPrep liquid-based method for cervical cytology with PCR allows prompt confirmation of the diagnosis of HSV without sacrificing the diagnostic morphology on the slide.

The effect of pathologic substances and adulterants on the DNA typing of urine.

Human urine has not been adequately investigated as a potential source of DNA for forensic identity testing. The advent of polymerase chain reaction technology has made possible the analysis of previously undetectable levels of nucleic acids from human urine and other body fluids lacking nucleated cells. In this study, we evaluated the ability to genotype DNA extracted from adulterated urine specimens using the AmpliType PM + DQA1 PCR amplification and typing system. Fresh, first-void male urine specimens were contaminated with household bleach, E. coli, human serum albumin, glucose and saponin (a strong detergent). All of the adulterated samples were typed without difficulty. Frozen male urine specimens were split into equal volumes; one aliquot was adulterated with either E. coli or saponin, and the other was left free of contaminants. Seventy-one percent of all frozen urine specimens tested (adulterated and unadulterated) were successfully typed using this amplification and typing system. Our data, therefore, suggest that the AmpliType PM + DQA1 PCR amplification and typing system described is suitable for genotype analysis of adulterated fresh and frozen urine specimens.

The Role of Genomic Instability in the Development of Human Cancer

Cancer development is a multi-step process through which cells acquire increasingly abnormal proliferative and invasive behaviors. Furthermore, cancer represents a unique form of genetic disease, characterized by the accumulation of multiple somatic mutations in a population of cells undergoing neoplastic transformation (1–5). Several forms of molecular alteration have been described in human cancers, including gene amplifications, deletions, insertions, rearrangements, and point mutations (5, 6). In many cases specific genetic lesions have been identified that are associated with the process of neoplastic transformation and/or tumor progression in a particular tissue or cell type (4). Statistical analyses of age-specific mortality rates for different forms of human cancer predict that multiple (three to eight) mutations in specific target genes are required for the genesis and outgrowth of most clinically diagnosable tumors (7). In accordance with this prediction, it has been suggested that tumors grow through a process of clonal expansion driven by mutation (1,2,8–10). In this model, the first mutation leads to limited expansion of progeny of a single cell, and each subsequent mutation gives rise to a new clonal outgrowth with greater proliferative potential. The idea that carcinogenesis is a multi-step process is supported by morphologic observations of the transitions between premalignant (benign) cell growths and malignant tumors. In some tumor systems (such as colon), the transition from benign to malignant can be easily documented and occurs in discernible stages, including benign adenoma, carcinoma in situ, invasive carcinoma, and eventually local and distant metastasis (11,12). Moreover, specific genetic alterations have been shown to correlate with each of these well-defined histopathologic stages of tumor development and progression (13,14). However, it is important to recognize that it is the accumulation of multiple genetic alterations in affected cells, and not necessarily the order in which these changes accumulate, that determines tumor formation and progression. These observations suggest strongly that the molecular alterations observed in human cancers represent integral (necessary) components of the process of neoplastic transformation and tumor progression.

Identification of Urine Specimen Donors by the PM+DQA1 Amplification and Typing Kit

We evaluated the ability to genotype DNA extracted from urine samples, which were previously submitted for toxicological analysis, by either the AmpliType HLA DQ alpha or the combined PM+DQA1 amplification and typing systems. Initial experiments were conducted on fresh urine, which was either processed fresh or frozen for one week at -20 degrees C, from male and female volunteers. Although male urine is noted for containing minimal numbers of nucleated cells when compared with female urine, we were able to type these samples without difficulty. Male urine specimens that were stored frozen for one year in the Toxicology Laboratory provided sources of low concentration, poor quality genomic DNA with respect to degradation of nucleic acid. These samples, however, were also easily typed using the amplification typing kits. Our data, therefore, suggest that the PM+DQA1 amplification and typing systems described here are suitable for typing analysis of donor urine specimens.

Certification in Molecular Pathology in the United States (Training and Education Committee, the Association for Molecular Pathology)

Training in molecular pathology in the United States is undergoing development toward a more structured format involving accreditation of training programs and the availability of recognized professional credentials. The traditional apprenticeship for molecular pathology, composed of experience in a molecular biology laboratory with a strong research focus is being replaced by formal training in residency, fellowship, and other postdoctoral training programs that have a clinical focus. This is a reflection of increasing importance of this field to clinical practice, and to a growing desire of persons interested in molecular pathology to undertake formal training programs. These developments are bringing molecular pathology in line with other clinical laboratory specialties for which structured training and certification have long been the norm. An important measure of educational achievement is success in professional examinations leading to recognized credentials. These credentials should attest to the holder’s professional competence as a practitioner in the field and are frequently used for this purpose by licensing and other regulatory agencies. In the past decade, and especially in the last 5 years, a number of routes for certification in molecular pathology by examination have been offered by nationally recognized credentialing agencies. This paper provides an update on these certification routes reflective of the growing interest in molecular pathology education. The principal Boards that offer certification in the United States are the American Board of Pathology, the American Board of Medical Genetics, the American Board for Clinical Chemistry, the National Credentialing Agency for Laboratory Personnel, and the American Board of Bioanalysis. The American Board of Medical Genetics offered a combined examination in Clinical Molecular Genetics and Clinical Biochemical Genetics in 1990 and has offered an examination in Clinical Molecular Genetics alone since 1993. Because of the overlap between molecular genetics and molecular pathology, we have included this examination in this listing. The information provided here is intended to summarize the requirements for these exams. Complete requirements are available from the boards listed here.