Quantification of HER2/neu gene amplification by competitive pcr using fluorescent melting curve analysis.

BACKGROUND: Molecular detection methods for HER2/neu gene amplification include fluorescence in situ hybridization (FISH) and competitive PCR. We designed a quantitative PCR system utilizing fluorescent hybridization probes and a competitor that differed from the HER2/neu sequence by a single base change. METHODS: Increasing twofold concentrations of competitor were coamplified with DNA from cell lines with various HER2/neu copy numbers at the HER2/neu locus. Competitor DNA was distinguished from the HER2/neu sequence by a fluorescent hybridization probe and melting curve analysis on a fluorescence-monitoring thermal cycler. The percentages of competitor to target peak areas on derivative fluorescence vs temperature curves were used to calculate copy number. RESULTS: Real-time monitoring of the PCR reaction showed comparable relative areas throughout the log phase and during the PCR plateau, indicating that only end-point detection is necessary. The dynamic range was over two logs (2000-250 000 competitor copies) with CVs < 20%. Three cell lines (MRC-5, T-47D, and SK-BR-3) were determined to have gene doses of 1, 3, and 11, respectively. Gene amplification was detected in 3 of 13 tumor samples and was correlated with conventional real-time PCR and FISH analysis. CONCLUSION: Use of relative peak areas allows gene copy numbers to be quantified against an internal competitive control in < 1 h.

Immunoelectron microscopy in the age of molecular pathology.

The introduction of molecular biology-based diagnostic procedures in pathology has created substantial expectations in regard to screening, characterization, monitoring, and detection of predisposition to a variety of diseases, most notably malignant neoplasms. It should be emphasized, however, that molecular studies are only one component of the diagnostic process and that more traditional methods are still required in the evaluation of tumors and management of patients. The data obtained from the molecular biology-based studies must be always interpreted in conjunction with the clinical history, immunomorphologic findings, and other pertinent ancillary data. Routine evaluation of tissues using traditional light microscopy remains the backbone of pathologic evaluation. The additive role of molecular diagnostics often depends on how accurate the initial evaluation has been. Ancillary techniques such as immunohistochemistry and electron microscopy remain essential in properly characterizing diseased tissues and in speciation of tumors. Ultrastructural immunolabeling capitalizes on combining these two techniques and providing exquisite immunomorphologic evaluation. The extra time and effort required are more than compensated by the degree of sophistication that can be achieved when this diagnostic technique is utilized and the added expense is rather reasonable. The value of molecular biology-based diagnostics is potentially questionable if the tissue samples are not initially accurately characterized. The question that molecular diagnostics may be trying to answer may be the wrong one or the answer obtained may be interpreted incorrectly if the context of the clinicopathologic situation has not been clearly defined using traditional diagnostic techniques.

Fluorescence in situ hybridization (FISH) for detection of HER-2/neu amplification in breast cancer: a multicenter portability study.

Amplification and/or overexpression of HER-2/neu has been shown to be both a prognostic and predictive marker in breast cancer. Recent studies have also confirmed the efficacy of Herceptin (trastuzumab) as adjuvant therapy for patients with overexpression of HER-2/neu. Therefore, it is critical that precise and reproducible assays be used in the clinical laboratory setting for determination of the HER-2/neu status in patients with breast cancer. The objective of this study was to determine the portability (reproducibility between different institutions) of the PathVysion HER-2 fluorescence in situ hybridization (FISH) assay used for detection of amplification of the HER-2/neu gene in formalin-fixed, paraffin-embedded tissue sections of invasive ductal carcinoma of the breast. Study specimens consisted of one breast tumor with a normal HER-2/neu copy number, two tumors with a low level, and one tumor with a high level of HER-2/neu amplification. The PathVysion HER-2 assay was shown to be highly reproducible on different assay days (n = 3) and between different institutions (n = 5) in the detection of amplification of the HER-2/neu gene in routinely processed clinical specimens of breast carcinoma. In addition, this study examined the feasibility of enumerating FISH signals in 20 nuclei in contrast to 60 nuclei per specimen. Although a modest increase in variation was observed when analyzing 20 compared to 60 nuclei, the mean ratios were similar. Therefore, analysis of as few as 20 nuclei with this FISH HER-2/neu assay may be sufficient for determining the amplification level of the HER-2/neu gene.

Strong correlation of elastin deletions, detected by FISH, with Williams syndrome: evaluation of 235 patients.

Williams syndrome (WS) is generally characterized by mental deficiency, gregarious personality, dysmorphic facies, supravalvular aortic stenosis, and idiopathic infantile hypercalcemia. Patients with WS show allelic loss of elastin (ELN), exhibiting a submicroscopic deletion, at 7q11.23, detectable by FISH. Hemizygosity is likely the cause of vascular abnormalities in WS patients. A series of 235 patients was studied, and molecular cytogenetic deletions were seen in 96% of patients with classic WS. Patients included 195 solicited through the Williams Syndrome Association (WSA), plus 40 clinical cytogenetics cases referred by primary-care physicians. Photographs and medical records of most WSA subjects were reviewed, and patients were identified as "classic" (n = 114) or "uncertain" (n = 39). An additional 42 WSA patients were evaluated without clinical information. FISH was performed with biotinylated ELN cosmids on metaphase cells from immortalized lymphoblastoid lines from WSA patients and after high-resolution banding analysis on clinical referral patients. An alpha-satellite probe for chromosome 7 was included in hybridizations, as an internal control. Ninety-six percent of the patients with classic WS showed a deletion in one ELN allele; four of these did not show a deletion. Of the uncertain WS patients, only 3 of 39 showed a deletion. Of the 42 who were not classified phenotypically, because of lack of clinical information, 25 patients (60%) showed a deletion. Thirty-eight percent (15/40) of clinical cytogenetics cases showed an ELN deletion and no cytogenetic deletion by banded analysis. These results support the usefulness of FISH for the detection of elastin deletions as an initial diagnostic assay for WS.

Identification of hyperdiploidy in fixed cells from pediatric acute lymphoblastic leukemia cases using flow cytometry and cytogenetic analysis.

Cytogenetic analysis is of value in predicting clinical outcome of pediatric cases of acute lymphoblastic leukemia (ALL). Hyperdiploidy in these patients has significant impact on therapeutic outcome. Because of the technologic limitations of cytogenetic analysis in determining hyperdiploidy in some of these cases, we devised a method to analyze cytogenetically fixed material for cellular DNA content by standard flow cytometric methods. This technique greatly enhances out ability to interpret the observance of single cell anomalies and assess whether these cells are predictive of clonal stemlines or are a result of random events.

Physician peer review surveys: a management tool for improving quality of patient care.

An increasing number of patients are presenting multiple medical problems requiring the collaboration of two or more physician specialists or subspecialists for effective treatment. The quality of care delivered to multiple-problem patients depends greatly on how well the physician specialists interact with one another. The Cleveland Clinic Foundation (CCF) has developed and implemented a physician peer review survey that enables physicians to receive anonymous feedback on the service they provide to their colleagues. The survey has been implemented in both medical and surgical departments. Colleagues have identified areas for improvement to increase collaboration and enhance effectiveness in treating multiproblem patients. The data have led to a variety of specific service-related improvements and changes in physician behavior. Though originally conceived as a quality improvement technique, the physician review survey has become an internal marketing and management tool for physician managers.

Male-mediated behavioral abnormalities.

The assessment of behavioral development in the progeny of males exposed to known mutagenic chemicals is a potentially sensitive endpoint for detecting transmissible abnormalities. A genetic component is demonstrated as these behavioral abnormalities can also be passed on to the F2 generation. In this review, experimental studies exploring transmission of behavioral deficits from paternal exposure to drugs, chemicals and radiation are addressed. Additionally included is a brief synopsis of recent work performed in our laboratory investigating such abnormalities in offspring from male rats exposed to ionizing radiations. The implications of these behavioral endpoints to humans is also discussed.