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Gene expression–based assays have been introduced into the clinical arena to assist in the diagnosis of poorly differentiated or undifferentiated tumors. The US Food and Drug Administration has cleared the microarray-based Pathwork Tissue of Origin (TOO) Test (Pathwork Diagnostics, Sunnyvale, CA) for the molecular characterization of such challenging specimens. We aimed at verifying the analytic and clinical performance of this test on 43 poorly differentiated and undifferentiated tumor samples, including 6 off-panel cases and 7 cancers of unknown primary (CUP). Our results showed 97% (95% confidence interval, 80.4%-99.8%) agreement between the Pathwork TOO Test result and the complete diagnosis, which included clinical correlations and immunohistochemical staining, after the original diagnosis. We concluded that for off-panel and CUP samples, the tissue type and the cell type may be confounded by the Pathwork TOO Test and that careful clinicopathologic assessment is needed when interpreting results from this helpful ancillary tool for pathologists.

BACKGROUND: The application of ancillary molecular testing is becoming more important for the diagnosis and classification of disease. The use of fine-needle aspiration (FNA) biopsy as the means of sampling tumors in conjunction with molecular testing could be a powerful combination. FNA is minimally invasive, cost effective, and usually demonstrates accuracy comparable to diagnoses based on excisional biopsies. Quality control (QC) and test validation requirements for development of molecular tests impose a need for access to pre-existing clinical samples. Tissue banking of excisional biopsy specimens is frequently performed at large research institutions, but few have developed protocols for preservation of cytologic specimens. This study aimed to evaluate cryopreservation of FNA specimens as a method of maintaining cellular morphology and ribonucleic acid (RNA) integrity in banked tissues. METHODS: FNA specimens were obtained from fresh tumor resections, processed by using a cryopreservation protocol, and stored for up to 27 weeks. Upon retrieval, samples were made into slides for morphological evaluation, and RNA was extracted and assessed for integrity by using the Agilent Bioanalyzer (Agilent Technologies, Santa Clara, Calif). RESULTS: Cryopreserved specimens showed good cell morphology and, in many cases, yielded intact RNA. Cases showing moderate or severe RNA degradation could generally be associated with prolonged specimen handling or sampling of necrotic areas. CONCLUSIONS: FNA specimens can be stored in a manner that maintains cellular morphology and RNA integrity necessary for studies of gene expression. In addition to addressing quality control (QC) and test validation needs, cytology banks will be an invaluable resource for future molecular morphologic and diagnostic research studies. Cancer (Cancer Cytopathol) 2011;000:000–000. V C 2011 American Cancer Society.

Previous cross-platform reproducibility studies have compared consistency of intensities as well as consistency of fold changes across different platforms using Pearson's correlation coefficient. In this study, we propose the use of measurement error models for estimating gene-specific correlations. Additionally, gene-specific reliability estimates are shown to be useful in prioritizing clones for sequence verification rather than selecting clones using a simple random sample. The proposed 'disattenuated' correlation may prove useful in a wide variety of studies when both X and Y are measured with error, such as in confirmation studies of microarray gene expression values, wherein more reliable laboratory assays such as real-time polymerase chain reaction are used.

This chapter attempts to summarize what is known regarding the organization and functional role of DNA and RNA in normal cell growth and differentiation that may be relevant to cancer cells. Information accruing over the last 15 years and particularly the last 5–10 years is emphasized but early discoveries important to the development of our current understanding are referenced. New technologies such as next generation DNA sequencing and its derivatives including ChIP-seq, RNA-seq, and chromosome conformation capture techniques along with huge collaborative projects including sequencing of the human genome, ENCODE, the Roadmap Epigenomics Program, and most recently the 4D Nucleome project are extending the ability of medical scientists to probe the organization of chromatin and through this determine the regulatory networks of specific cell types. While this chapter can only touch briefly on this broad range of topics it is hoped that it can provide a starting point for expanding understanding of the many opportunities as well as some of the potential pitfalls that are certain to arise in the growing field of cancer molecular testing.

A pretest-posttest, repeated-measures design was used to evaluate the effects of two stress management interventions on a battery of outcomes derived from a psychoneuroimmunological (PNI) framework. The effects of cognitive-behavioral relaxation training groups (CBSM) and social support groups (SSG) were compared with a WAIT-listed control group on the outcomes of psychosocial functioning, quality of life, neuroendocrine mediation, and somatic health. Participants were 148 individuals (119 men, 29 women), diagnosed with HIV disease; 112 (76%) completing the study groups. Using analysis of covariance, the CBSM group was found to have significantly higher postintervention emotional well-being and total quality-of-life scores than did either the SSG or WAIT groups. SSG participants had significantly lower social/family well-being scores immediately postintervention and lower social support scores after 6 months. The findings point to a pressing need for further, well-controlled research with these common intervention modalities.

From the Editor: Our world is changing, and our field is changing with it. Each new advance in technology, each new application, each step forward in automation and reduction in time and work required to perform molecular testing expands the potential of molecular diagnostics to impact the clinical management of patients and their families. The completion of the Human Genome Project will propel our field forward rapidly, and has already had a major impact in raising the visibility of our field in the public eye. Concomitantly, we have become more visible to government and regulatory agencies as well. Many of you have carefully followed the discussions of the Food and Drug Administration (FDA) over the years, and more recently the deliberations of the Secretaries Advisory Committee on Genetic Testing (SACGT) and the Clinical Laboratory Improvement Advisory Committee (CLIAC). Both groups have been at work these past two years, discussing the means by which genetic testing should be ordered, performed, reported, and overseen in the United States. Obviously, the results of their discussions would have enormous implications for those involved in molecular diagnostics. The primary concern leading to the establishment of SACGT approximately two years ago was protection of the public from harm due to inappropriately used or performed genetic testing. SACGT is a federally mandated group acting under the auspices of the Secretary of Health and Human Services. They hold that increased oversight of genetic testing is necessary, particularly since much of genetic testing is currently done via methods developed by individual laboratories and not by FDA-approved kits, and have recommended that the FDA be the agency to provide this oversight. In their deliberations, the definition of genetic test is very broad and would include both somatic and germline alterations. They have worked to develop a mechanism by which tests could be stratified based on their intended use and potential impact on patient care. In theory, this would allow triaging of “high-risk” tests such as those for Huntington’s disease or BRCA1 into a high scrutiny category necessitating high level oversight, whereas less critical tests such as Factor V Leiden, for example, would require a lower intensity scrutiny. Such algorithms have thus far proven unwieldy and ineffective for risk stratification, however, in part due to the multiple intended uses for many of these assays (disease confirmation, prenatal assessment, carrier detection, population screening, etc) and thus are still under discussion. SACGT holds firmly that some sort of FDA approval mechanism is needed before these tests can be applied to clinical decision-making. Numerous professional groups have responded to SACGT and have worked with the FDA through the FDA-Professional Organization Roundtable discussions in an effort to help develop a practical oversight plan. Many of these groups, including the Association for Molecular Pathology (AMP), hold that existing mechanisms for laboratory accreditation and oversight are best suited to be the nidus for any new oversight programs. Laboratories performing clinical testing must currently be certified by the College of American Pathologists (CAP) and Clinical Laboratory Improvement Act (CLIA). Inspection and accreditation of laboratories by CAP provides a very detailed assessment of laboratory and quality control practices in molecular diagnostics laboratories. For example, laboratories are required to keep on file details and data of validation studies for each assay performed. CLIA requires additional quality-oriented practices. At the FDA Roundtable discussions, a genetic “template” was developed that could be used to help gather data on the use, performance, interpretation, and reporting of a genetic test. The template was very well received by SACGT, and discussion is underway regarding utilization of this template. The template would certainly help to gather and organize information on genetic testing and detailed laboratory practices, but numerous questions remain unanswered regarding further benefits of implementing use of the template. Would this move truly improve the quality of genetic testing in the United States, and in doing so, protect the public? Or would it duplicate existing regulatory mechanisms such as CAP accreditation, add to the administrative burden and cost of the labs, and thus limit public access to genetic testing? The views of AMP were presented at the most recent SACGT hearing in May 2001 by Dr. Debra Leonard, Past-President of AMP; her comments follow this introduction. Another federal group active in this arena is CLIAC. CLIAC has focused on defining genetic testing and addressing pre- and postanalytic issues in genetic testing, such as informed consent and reporting of results. Of note is that the definition of a genetic test put forth by CLIAC is extremely broad, to include not only nucleic acids but also proteins and metabolites. The recommendations of CLIAC are discussed in more detail by Dr. Andrea Gonzalez, Chair of the AMP Policy Committee, in the update to follow. During the coming months, much will become clear with these various regulatory issues. I anticipate that we will be satisfied with some aspects of the increased oversight, and very dissatisfied with others. Regardless of the outcomes, we will have to find ways to live with the recommendations of these committees. In the meantime, we must remain actively involved, commenting and working with federal agencies when possible to effect workable solutions. In the end, better patient care is the goal for all of us.

Although most solid tumors are treated surgically, determining the genetic changes present in the tumor of an individual patient is becoming increasingly important for managing the oncology patient. Our knowledge of the genetic alterations that characterize and predispose to solid tumors continues to expand. Concurrently, the advent of newer technologies such as DNA chips has the potential to enable a more rapid and comprehensive assessment of these changes. The ultimate goal of this new information and technology is to provide sensitive and specific tests that reduce unnecessary procedures and optimize therapy. This review addresses the utility of molecular testing in evaluating cancer. A review of the current technology and hereditary cancer syndromes is also presented. Semin. Surg. Oncol. 18:358–370, 2000. © 2000 Wiley-Liss, Inc.

We present a child with mild to moderate global developmental delay including severe speech impairment, inappropriate happy demeanor, wide-based gait, frequent ear infections with mild hearing loss, deep-set eyes, a wide mouth, widely-spaced teeth, normal head circumference, and no seizures. Results of peripheral blood lymphocyte chromosomal analysis with GTG banding were normal. However, fluorescence in situ hybridization (FISH) studies showed mosaicism for a deletion of probes (D15S10 and SNRPN) from the Angelman syndrome (AS) critical region with approximately 40% of peripheral lymphocytes having the deletion. The deleted chromosome 15 also showed centromeric duplication, which was detected with a D15Z1 probe [46,XX, dic(15)(pter-->q11.1::p11.2-->q11. 1::q13-->qter)]. The same duplication pattern was observed in 30% of the nuclei obtained from a buccal smear. Methylation studies using polymerase chain reaction with sodium bisulfite-treated DNA demonstrated a normal biparental methylation pattern. To the best of our knowledge, this is the first case with AS and a FISH detectable deletion in a mosaic pattern. We recommend FISH studies for the detection of mosaicism in the patients with AS clinical findings even if results of the methylation studies are normal.

Background. Accurate and rapid diagnosis of human cytomegalovirus (HCMV) disease in solid organ transplant patients remains a challenge. We evaluated the clinical utility of a quantitative polymerase chain reaction (QPCR) method to diagnose transplant patients with HCMV disease. Methods. A total of 429 plasma samples from 121 solid organ transplant patients were prospectively collected and evaluated for HCMV using a QPCR assay. To enhance the sensitivity of the QPCR assay, plasma samples were centrifuged in a manner designed to concentrate the virions before nucleic acid extraction. Quantitation was achieved by co-amplifying an internal quantitative standard (IS) that contained the same primer sequences as for HCMV. Polymerase chain reaction products were detected by hybridization to 96-well microtiter plates coated with either a HCMV- or an IS-specific probe. Results. A total of 103 patients had all samples negative by QPCR. None of the 103 patients developed HCMV disease during the study. In contrast, 18 patients showed at least 1 sample positive by the QPCR assay, but only 8 of these developed HCMV disease. The mean viral load value for patients without HCMV disease was 93 viral copies (vc) per ml of plasma (range: 35‐325 vc/ml plasma) and for the 8 patients with HCMV disease was 67,686 vc/ml plasma (range: 167‐1,325,000 vc/ml plasma) (P

Background. Cytomegalovirus (CMV) is the most prevalent viral disease in solid organ transplantation. Detection of CMV DNA in peripheral blood mononuclear cells (PBMC) by polymerase chain reaction (PCR) frequently occurs in renal allograft recipients, yielding false positive results in seropositive patients free of CMV disease. We evaluated the clinical utility of a quantitative PCR-enzyme-linked immunosorbent assay (ELISA) for identifying patients with CMV disease. Methods. Three hundred and fifty samples from 65 consecutive renal transplant recipients were studied. DNA was extracted from PBMC weekly up to the day of discharge and after any further admission. Samples were tested by a qualitative PCR method, and all positive samples were further studied by a quantitative PCR-ELISA method. The quantitative PCR-ELISA method used an internal standard (IS) that contained the primer sequences used in the qualitative CMV PCR. Detection and quantification was performed in 96-well plates coated with IS or CMV specific probes. Results. Forty-one of 65 patients (63.1%) showed positive results by the qualitative PCR, but only 8 of these patients were diagnosed with CMV disease. Positive samples were re-analyzed by the quantitative assay. The 8 patients with CMV disease had a mean CMV viral load of 1,438±687 viral copies (VC)/10 6 PBMC, and the 33 without CMV disease had a mean value of 219.6±117.2 VC/10 6 PBMC (P

The p21 protein inhibits cyclin-dependent kinases and mediates cell-cycle arrest and cell differentiation. It is induced by wild-type p53, but not by mutant p53. This study of 75 patients with endometrial carcinoma investigates the relationship between p21 expression and the functional status of p53, and the usefulness of p21 as a prognostic marker. Correlations were determined between p21 immunoreactivity, p53 overexpression as examined by immunohistochemistry, p53 DNA mutations as examined by polymerase chain reaction-single-stranded conformation polymorphism (PCR-SSCP) analysis, and clinicopathological features, including the clinical outcome. Immunoreactivity for p21 and p53 mutations were detected in 47 (62.7 per cent), 37 (49 per cent), and 23 (31 per cent) patients, respectively. There were no significant correlations between the presence or absence of p21 immunoreactivity and p53 overexpression and DNA mutations. Survival curves revealed that patients with p53 overexpression tended to have a poorer prognosis than those without p53 overexpression (P = 0.104), that patients with p53 mutations had a significantly worse prognosis than those without mutations (P = 0.035), and that patients with p21 expression tended to have a better prognosis than those without p21 expression (P = 0.074). Immunohistochemical analysis of p21 was not useful for evaluating the functional status of p53 in patients with endometrial carcinoma. Both p21 expression and p53 abnormalities were considered as prognostic indicators in patients with endometrioid endometrial carcinoma.

Background: Detection of hepatitis C virus (HCV) RNA in serum or plasma is currently the best means of identifying active HCV infection. In this study, we assessed the clinical utility of the HCV Amplicor Monitor (Roche Molecular Diagnostics, Branchburg, NJ) quantitative assay for monitoring viral burden and its implications for identifying responders among alpha-interferon-treated patients with chronic hepatitis C. Methods and Results: Precision and linearity were determined on aliquots of a pooled control serum. Error of the mean was normally distributed. The coefficient of variation of log10-transformed titers was 2%-6% over a range of 1.5 x 104-1.5 x 105 copies/mL. Linearity over this range was high (R=.98-.99). Accuracy, as evaluated by comparison of split samples, showed that the Amplicor assay provided an unbiased estimate of the values from a reference laboratory. In a sample of 36 patients treated with alpha-interferon for chronic hepatitis C disease, mean viral titer declined with improvement of disease. The assay demonstrated heterogeneity among clinical responders with regard to their ability to actually clear their viral burden. Conclusions: Decreased viral burden as measured by the HCV Amplicor assay is potentially useful for monitoring individuals with HCV infection. (Mol Diagn 1996 Jun;1(2):109-120)

Diseases are increasingly being defined in terms of genetic alterations. A body of information termed “molecular pathogenesis” is evolving which provides a framework for integrating the rapidly accumulating genetic information with the related disease process. Molecular methods with their increased sensitivity and specificity are required to gather this information. A major challenge now lies in the transfer of this molecular technology from a research environment to the clinical testing arena. Technical issues, patented technology, special facilities, personnel, and regulatories issues imposed by CLIA'88, require those desiring to perform molecular tests to pay special attention to laboratory design, personnel training, and test menu development. Although establishing a successful molecular diagnostics laboratory is a complex and difficult task, the added value of these tests can have a tremendous impact in disease diagnosis and patient management.

Colorectal and breast cancers account for a significant number of deaths due to malignant neoplasia. Laboratory medicine plays an important role in the diagnosis and management of these tumors through the application of histopathology, immunohistochemis- try, and serologic identification of tumor markers. Approximately 5% to 10% of colorectal and breast cancers result from an inherited predisposition. The genes responsible for most genetically transmitted cancers have been identified, and the application of findings from molecular pathology are being evaluated. This article reviews the genetic changes that occur as a result of somatic mutation and inherited or germline mutations.

Background. Adenocarcinoma of the fallopian tube is a rare tumor with a poor prognosis. Whether these carcinomas possess any genetic changes that contribute to their malignant behavior is unknown and to date few studies regarding the molecular pathogenesis of these tumors have been reported. In adenocarcinoma of the endo-metrium, mutations in the first exon of K-ras, although relatively infrequent, were observed to be an independent risk factor for poor clinical outcome. Methods. Eight patients with adenocarcinoma of the fallopian tube were examined for mutations in the 12th codon of K-ras. DNA was obtained from single sections of paraffin embedded tumor tissue and the first exon of K-ras was amplified by the polymerase chain reaction. Point mutations were assayed using a nonradioactive restriction fragment length polymorphism technique. Results. The eight patients in this study varied in clinical stage from I-IV and were all treated with surgery and chemotherapy. Six of eight of the patients died and one of the surviving patients had metastases in the vertebrae. K-ras point mutations were detected at codon 12 in seven of the eight tumors (87.5%). Conclusions. K-ras mutations occurred with high frequency in this series of eight patients with fallopian tube carcinoma, suggesting that mutations of this protooncogene could play an important role in the molecular pathogenesis of this lesion.

The development of a reliable polymerase chain reaction (PCR) technique for the routine detection of clonal immunoglobulin heavy chain (IgH) gene rearrangements would represent an attractive alternative to Southern hybridization analysis because of the relative simplicity of PCR protocols, and because the requirements for both quality and quantity of DNA would be considerably less stringent. To assess the utility of PCR for the routine detection of clonal IgH gene rearrangements, samples from 123 adult patients were evaluated and analysis by PCR amplification using IgH Framework 1 or Framework 3 variable region consensus primers was compared with analysis by restriction endonuclease digestion and Southern hybridization with genomic, IgH probes. The authors found that 90% of IgH genes found to be rearranged by Southern hybridization are detected by the PCR technique. An additional 9 patient samples had clonal IgH gene rearrangements that were detectable by PCR alone. Eight of these nine patients had a history of a clonal hematopoietic process at either the morphologic or molecular level, and six had a history of a B-cell malignancy. It is likely that these specimens contained clonal lymphoid populations undetected by the Southern hybridization technique. Thus, the diagnostic sensitivity and specificity of the PCR method for the detection of B-cell tumors were 91% and 95%, respectively. The combination of improved analytical sensitivity and specimen flexibility of the IgH PCR assay could make it the method of choice for the routine detection of clonal IgH gene rearrangements, if minor improvements in the diagnostic sensitivity of the assay can be achieved.

Gene expression-based assays have been introduced into the clinical arena to assist in the diagnosis of poorly differentiated or undifferentiated tumors. The US Food and Drug Administration has cleared the microarray-based Pathwork Tissue of Origin (TOO) Test (Pathwork Diagnostics, Sunnyvale, CA) for the molecular characterization of such challenging specimens. We aimed at verifying the analytic and clinical performance of this test on 43 poorly differentiated and undifferentiated tumor samples, including 6 off-panel cases and 7 cancers of unknown primary (CUP). Our results showed 97% (95% confidence interval, 80.4%-99.8%) agreement between the Pathwork TOO Test result and the complete diagnosis, which included clinical correlations and immunohistochemical staining, after the original diagnosis. We concluded that for off-panel and CUP samples, the tissue type and the cell type may be confounded by the Pathwork TOO Test and that careful clinicopathologic assessment is needed when interpreting results from this helpful ancillary tool for pathologists.

The current model predicts that MDM2 is primarily overexpressed in cancers with wild-type (WT) p53 and contributes to oncogenesis by degrading p53. Following a correlated expression of MDM2 and NF-κB2 transcripts in human lung tumors, we have identified a novel transactivation function of MDM2. Here, we report that in human lung tumors, overexpression of MDM2 was found in approximately 30% of cases irrespective of their p53 status, and expression of MDM2 and NF-κB2 transcripts showed a highly significant statistical correlation in tumors with WT p53. We investigated the significance of this correlated expression in terms of mechanism and biological function. Increase in MDM2 expression from its own promoter in transgenic mice remarkably enhanced expression of NF-κB2 compared with its non-transgenic littermates. Knockdown or elimination of endogenous MDM2 expression in cultured non-transformed or lung tumor cells drastically reduced expression of NF-κB2 transcripts, suggesting a normal physiological role of MDM2 in regulating NF-κB2 transcription. MDM2 could up-regulate expression of NF-κB2 transcripts when its p53-interaction domain was blocked with Nutlin-3, indicating that the MDM2-p53 interaction is dispensable for up-regulation of NF-κB2 expression. Consistently, analysis of functional domains of MDM2 indicated that although the p53-interaction domain of MDM2 contributes to the up-regulation of the NFκB2 promoter, MDM2 does not require direct interactions with p53 for this function. Accordingly, MDM2 overexpression in non-transformed or lung cancer cells devoid of p53 also generated a significant increase in the expression of NF-κB2 transcript and its targets CXCL-1 and CXCL-10, whereas elimination of MDM2 expression had the opposite effects. MDM2-mediated increase in p100/NF-κB2 expression reduced cell death mediated by paclitaxel. Furthermore, knockdown of NF-κB2 expression retarded cell proliferation. Based on these data, we propose that MDM2-mediated NF-κB2 up-regulation is a combined effect of p53-dependent and independent mechanisms and that it confers a survival advantage to lung cancer cells.

HIV-associated immune-mediated renal disease. Although focal glomerulosclerosis is the most common renal disease, other proliferative glomerulonephritides are encountered in HIV-infected patients. We studied four HIV-infected patients with renal insufficiency, proteinuria, and proliferative glomerulonephritis, consistent with immune-mediated disease, to investigate the role of the virus and immune complexes in the pathogenesis of the nephropathy. Circulating immune complexes (CICs) and HIV-reactive antibodies were measured and characterized in each patient. Renal biopsy tissue was acid eluted, and the eluate analyzed. DNA extracted from biopsies was subjected to the polymerase chain reaction (PCR) to detect HIV genome. CICs were detected in each patient: an IgA-p24 HIV antigen complex and an IgG antibody-gp120 HIV antigen complex in two patients; two patients had an IgG-p24 HIV antigen complex. Identical complexes were eluted from renal tissue in the first three patients; p24 HIV antigen, and complement from the fourth. The eluted antibodies reacted with the HIV antigens from the isolated CICs. Direct immunofluorescence for viral antigen in the eluted glomerular tissue revealed HIV antigens; PCR confirmed the presence of gag genome in all four biopsies. We conclude both circulating and in-situ HIV antigen-specific immune complexes may be associated with glomerulonephritis in HIV infected patients. Viral incorporation into renal tissue may be important in the pathogenesis of HIV-associated renal disease.

SEVERAL renal syndromes have been described in patients with human immunodeficiency virus (HIV) infection. The most common renal abnormality is focal segmental glomerulosclerosis,1 2 3 but some patients have evidence of immune-complex nephropathy,1 2 3 including membranoproliferative and diffuse proliferative glomerulonephritis,4 membranous nephropathy,5 and IgA nephropathy.6 , 7 HIV-related antigen–antibody circulating immune complexes have been demonstrated in patients with HIV infection,8 9 10 11 12 13 but not in renal tissue from patients with both the infection and renal disease; therefore, the importance of the circulating complexes is unknown. Although the pathogenesis of IgA nephropathy in patients with HIV infection6 , 7 is unknown, genetic factors or immune-complex mechanisms may be involved.14 , 15 Idiotypes, . . .

Objectives: This is a pilot study designed to identify gene expression profiles able to stratify head and neck squamous cell carcinoma (HNSCC) tumors that may or may not respond to chemoradiation or radiation therapy. Study Design: We prospectively evaluated 14 HNSCC specimens, arising from patients undergoing chemoradiotherapy or radiotherapy alone with curative intent. A complete response was assessed by clinical evaluation with no evidence of gross tumor after a 2-year follow-up period. Methods: Residual biopsy samples from eight complete responders (CR) and six nonresponders (NR) were evaluated by genome-wide gene expression profiling using HG-U133A 2.0 arrays. Univariate parametric t-tests with proportion of false discoveries controlled by multivariate permutation tests were used to identify genes with significantly different gene expression levels between CR and NR cases. Six different prediction algorithms were used to build gene predictor lists. Three representative genes showing 100% crossvalidation support after leave-one-out crossvalidation (LOOCV) were further validated using real-time QRT-PCR. Results: We identified 167 significant probe sets that discriminate between the two classes, which were used to build gene predictor lists. Thus, 142 probe sets showed an accuracy of prediction ranging from 93% to 100% across all six prediction algorithms. The genes represented by these 142 probe sets were further classified into different functional networks that included cellular development, cellular movement, and cancer. Conclusions: The results presented herein offer encouraging preliminary data that may provide a basis for a more precise prognosis of HNSCC, as well as a molecular-based therapy decision for the management of these cancers. Laryngoscope, 119:91–101, 2009

Two hundred fifteen patients infected with human immunodeficiency viras (HIV) participated in a prospective longitudinal study of HIV-related heart disease. Evaluation included signal-averaged etectrocardiography and echocardiography. Fifteen patients underwent endomyocardial biopsy, 5 had cardiovascular symptoms and 10 did not. Cardiac myocytes or dendritic cells were prepared by individual cell microdissection to sort them from other cell types such as interstitial cells or circulating blood elements. HIV proviral sequences were amplified in samples of 15 to 20 cells of each type by multiplex, nested, polymerase chain reaction and hybridized to 32 P-labeled probes specific for regions within the gag and pol genes of HIV-1. The results showed the presence of HIV sequences in myocytes of 2 of 5 patients with cardiac symptoms and in 6 of 10 without. Thus, symptomatic HIV cardiomyopathy did not appear to be a direct consequence of the viras on myocardial cells. In dendritic cells, HIV sequences were detected in 5 of 5 patients with cardiac symptoms and in 8 of 10 with apparently normal ventricular function. Furthermore, dendritic cells were somewhat more numerous in the myocardium of symptomatic than asymptomatic patients. Our studies are the first to directly detect the HIV genome in purified cardiac myocytes from patients with and without cardiac dysfunction. Our findings do not support a direct role of the viras in myocardial dysfunction. However, the results do suggest that the interstitial dendritic cells may be involved in some manner in the development of cardiac dysfunction observed in HIV-infected patients.

A patient presenting with marked inflammatory lymphadenitis and Jaccoud's arthritis was found to have a rearranged gene for the beta-chain of the T-cell receptor (TCR-beta) antigen in the lymph node DNA digests and normal germ line DNA in the peripheral blood lymphocytes. Four months later, the patient was diagnosed to have poorly differentiated adenocarcinoma of the lung with small foci of metastatic tumor in lymph nodes that contained the same extensive lymphocytic and inflammatory cell infiltrates noted earlier. Rearranged TCR-beta chain genes were detected in both lymph node and peripheral blood lymphocyte DNA at this time. The most likely explanation for the florid lymph node reaction and the unusual arthropathy appears to be a paraneoplastic immune response. The rearranged TCR-beta genes indicate a clonal T-cell expansion that most likely resulted from the aberrant immunologic response to the lung cancer.

The utilization of genome-wide gene expression microarray technology in tumor stratification has proven a powerful tool to identify gene expression signatures associated with cancer prognosis and is currently under evaluation in clinical laboratories. Standardized protocols, including tumor tissue postoperatively handling guidelines are yet to be defined. We aimed at assessing a systematic effect of devitalization in ovarian tumors' gene expression profiling, using high-density oligonucleotide microarrays, under a standardized protocol following strict quality control criteria. Residual tissue from the surgical pathology specimen was divided into 5 samples. Half of each was immediately snap frozen in liquid nitrogen. The remaining halves were kept at room temperature for 0, 15, 30, 60, and 120 minutes, at which time the tissue was snap frozen in liquid nitrogen, and stored at -80 degrees C until RNA extraction. The entire process from RNA extraction through feature intensity distribution was rigorously monitored for quality. Identification of altered gene expression among each pair of snap frozen and devitalized samples per ovarian tumor specimen was assessed by using the Significance score (S-score) method. We identified only 4 probe sets that seemed to correlate with devitalization time in one of the ovarian tumor specimens, suggesting that they are not likely to have an impact on gene expression profiling tumor stratification. Our study suggests that with proper sample handling and rigorous quality control procedures for RNA extraction and microarray analysis, tumor classification based on global gene expression data will not be adversely affected if devitalization times are kept within a 120-minute window.

The altered expression of the human retinoblastoma (RB) gene has been demonstrated to play an important role in the pathogenesis of RB and other tumors. To determine whether the RB gene might be involved in the pathogenesis of human ovarian and endometrial cancer, DNA from 24 human ovarian tumors, 3 normal ovaries, 3 endometrial carcinomas, and 1 endometrial hyperplasia was examined with an RB complementary DNA probe. Evidence for homozygous deletion of the RB gene was observed in only one specimen. Interestingly, the specimen was an endometrioid tumor of the ovary of low malignant potential (LMP). This patient experienced rapid progression of the tumor and died 8 months after diagnosis. Abnormalities of the RB gene may be involved in the aggressive biologic behavior of certain forms of ovarian carcinoma, particularly those of LMP.

DNA from 24 ovarian tumors, including 16 carcinomas, was examined for amplification of the proto-oncogenes c-myc, int-2, and rc-erbB-2. All cases of carcinoma were also examined by flow cytometry for DNA ploidy and cell cycle analysis, and eight cases of carcinoma were examined for estrogen and progesterone receptors. Protooncogene amplification was not detected in the DNA of benign ovarian neoplasms, or of ovarian carcinomas with low malignant potential. Amplification of c-myc was detected in six of 12 cases of invasive carcinoma, int-2 amplification was present in one case, and c-erbB-2 amplification was not detected in any case. Among the seven cases evidencing protooncogene amplification, three cases showed aneuploidy in tumor DNA, while four showed diploidy. Two cases which showed aneuploidy in tumor DNA did not demonstrate any degree of protooncogene amplification. Protooncogene amplification was frequently associated with morphologic nuclear anaplasia and high mitotic count. Six of the seven cases demonstrating c-myc or int-2 were of the serous type or showed some degree of serous differentiation, while none of the four cases of purely mucinous carcinoma had evidence of amplification. While the total number of cases in the study was limited, it would appear from the trend demonstrated by the data that protooncogene amplification (particularly c-myc) may be involved in the pathogenesis of aggressive common epithelial tumors of the ovary.

The polymerase chain reaction (PCR) technique offers a promising alternative to tissue culture for the rapid and sensitive detection of cytomegalovirus (CMV) infection. However, high levels of background amplification detected in samples containing water but no DNA make interpretation of borderline positive samples extremely difficult and reduce the sensitivity of the assay. The signal from amplification of water or positive samples can be eliminated by DNase treatment, but not by filtration through anisotropic membrane, autoclaving, or ultraviolet irradiation. A lag time of 10 to 12 cycles is observed before the reactions with water will show product formation by liquid hybridization detection. The use of nested PCR eliminates the background and, in serial dilutions of a positive sample, shows a 500- to 1000-fold increase in sensitivity by liquid hybridization detection. We suggest that the background signal is arising from small fragments of DNA, which may be produced by autoclaving viral culture material. Such fragments would escape filtration, and overlapping fragments of DNA can prime one another to form complete mosaic sequences that will then amplify. Nested PCR, appropriately controlled for the number of cycles at each step, should successfully overcome such false positives caused by fragmented DNA, no matter if the contamination occurs at the collection site, in processing, or at the facility performing the test.

Background Recent research examining cross-platform correlation of gene expression intensities has yielded mixed results. In this study, we demonstrate use of a correction factor for estimating cross-platform correlations. Results In this paper, three technical replicate microarrays were hybridized to each of three platforms. The three platforms were then analyzed to assess both intra- and cross-platform reproducibility. We present various methods for examining intra-platform reproducibility. We also examine cross-platform reproducibility using Pearson's correlation. Additionally, we previously developed a correction factor for Pearson's correlation which is applicable when X and Y are measured with error. Herein we demonstrate that correcting for measurement error by estimating the "disattenuated" correlation substantially improves cross-platform correlations. Conclusion When estimating cross-platform correlation, it is essential to thoroughly evaluate intra-platform reproducibility as a first step. In addition, since measurement error is present in microarray gene expression data, methods to correct for attenuation are useful in decreasing the bias in cross-platform correlation estimates.

Background: Quantitative real-time PCR has become the predominant molecular technique to monitor BCRABL levels in response to treatment in Ph+ leukemia patients. However, without some form of standardized methodology between laboratories, the correlation of results is difficult. Methods: Using TaqMan®-based assays, parallel quantitative real-time PCR analysis was performed on 70 clinical specimens at Vanderbilt University Medical Center and Virginia Commonwealth University. While the same positive control cell line (K562) and quality control gene (BCR) were used, the RNA isolation technique, cDNA synthesis, BCR control cell line, and PCR primer and probe sequences were different. Results: The detection of BCRABL-positive results spanned a dynamic range from 100 to 105/100 000 cells. Forty-three samples were negative at both facilities. A Spearman rank correlation analysis was performed for the 22 BCRABL-positive paired results. The correlation coefficient, rs, was 0.9435 (p < 0.00001), suggesting a strong correlation of the results. One discordant result was obtained for consecutive samples from one patient with a low BCRABL copy number as a result of a minimal RNA yield at one laboratory. Conclusions: These results suggest that quantitative real-time PCR assays for BCRABL detection can be comparable between laboratories despite significant differences in methodologies if the same positive control cell line and quality control gene are used. It is imperative that some level of assay standardization be adopted between laboratories, not only for patients who are monitored at different facilities, but also for larger investigative studies in which hematologic, cytogenetic and molecular responses are to be compared.

One of the fastest growing areas in clinical laboratory testing is molecular diagnostics. This growth has been in part the result of the tremendous amount of knowledge gained in the last decade from the Human Genome Project regarding organization, regulation, and expression of genomic information in humans and microbes. This knowledge has allowed us to better understand the pathogenesis of many diseases at the molecular level. As a consequence, diseases are being increasingly defined in terms of their molecular pathogenesis. This has lead to the development of new clinical molecular assays for diagnosis, prognosis, selection of therapeutic modalities, and monitoring of diseases. Until a few years ago, practical laboratory methods for detecting differences in nucleic acid sequences were not sufficiently simple or robust for the clinical laboratory. Recent advances in new technology, instrumentation, and efforts in standardization have overcome many of these limitations. The development and introduction of new technologies that allow automation of the testing process, as well as higher-throughput testing, have improved the diagnosis of diseases and patient care. In addition to the increasing number of kits and reagents used for molecular diagnostics approved by the Food and Drug Administration (FDA), a large number of molecular tests are still manufactured in-house by different clinical laboratories. In its role supporting US science and industry, the National Institute of Standards and Technology (NIST), a nonregulatory agency of the US Department of Commerce, provides physical and chemical standards in support of national commerce, manufacturing, and science (3). These materials are available internationally as Standard Reference Materials (SRMs) for use by industry developing assays and/or technology platforms for diagnostic use, by regulatory agencies ensuring the quality and efficacy of these assays, and by clinical laboratories providing diagnostic tests for patients. Traditionally, NIST responds to standard needs as defined by these communities. Consensus is developed through NIST workshops attended by representatives of these communities as well as direct request by other governmental agencies. Specific examples of ongoing programs within the Biotechnology Division at NIST are described in this chapter.

Clinical repositories containing large amounts of biological, clinical, and administrative data are increasingly becoming available as health care systems integrate patient information for research and utilization objectives. To investigate the potential value of searching these databases for novel insights, we applied a new data mining approach, HealthMiner ® , to a large cohort of 667,000 inpatient and outpatient digital records from an academic medical system. HealthMiner ® approaches knowledge discovery using three unsupervised methods: CliniMiner ® , Predictive Analysis, and Pattern Discovery. The initial results from this study suggest that these approaches have the potential to expand research capabilities through identification of potentially novel clinical disease associations.

Background: Development of quality-control criteria to ensure reproducibility of microarray results for potential clinical application is still in its infancy. Methods: In the present studies we developed quality-control criteria and evaluated their effect in microarray data analysis using total RNA from cell lines, frozen tumors, and a commercially available reference RNA. Quality-control criteria such as A260/A280 ratios, percentage of rRNA, and median size of cDNA and cRNA synthesis products were evaluated for robustness in microarray analysis. Furthermore, precision studies using a reference material were performed on the Affymetrix® HG-U133A high-density oligonucleotide microarrays. The same reference RNA sample was examined in 16 different chips run on 2 different days in the four different modules of the Affymetrix fluidics workstation. Fresh and frozen fragmented cRNAs were also compared. An ANOVA model was fit to identify the main sources of variation. Results: Good-quality samples showed >30% rRNA in the electropherograms and cDNA and cRNA synthesis products with median sizes of 2.0 and 3.0 kb, respectively. Precision studies showed that the main source of variation was the day-to-day variability, minimally affecting hybridization exogenous control genes. Altogether, the results showed that the Affymetrix Genechip® system is highly reproducible when RNA that meet the quality-control criteria are used (overall P >0.01). Conclusions: These results confirm the need to establish defined quality-control criteria for sample quality to distinguish between analytical and biological variability.

High-density oligonucleotide microarray analysis has proven to be an excellent approach for gene expression profiling in human cancers. This technique assesses the expression of thousands of genes simultaneously, from at least 5 μg of total RNA per sample per experiment. This total RNA requirement poses a challenge when studying small, unique clinical samples, like biopsies. Recently, a new standardized protocol for small samples was released by Affymetrix, which includes a linear amplification step. To evaluate the impact of such amplification in the gene expression profiling of human ovarian cancer, we compared results obtained from 5 μg and 100 ng of total RNA from the same tumor sample, using the standard Affymetrix protocol and the new linear RNA amplification protocol, respectively. We identified a small bias in gene expression data caused by linear amplification, potentially due to shorter elongation products leading to misclassification of probe sets directed to the middle-5′ region of the transcripts. Interestingly, the magnitude of the bias varied when different normalization and expression summary algorithms were used. However, this bias does not affect tumor gene expression profiling. Consequently, linear amplification may be of utility in cases of extremely low RNA recovery from critical and unique samples, such as small biopsies.

Hepatic artery thrombosis (HAT) after liver transplantation is associated with a high incidence of graft failure. The incidence ranges between 2% and 25%, with an overall incidence of approximately 7%. Different risk factors have been associated, but the participation of genetic factors in the cause of HAT is less well studied. A single-base change (G to A) at position 20210 in the 3' untranslated region of the prothrombin gene is associated with increased plasma levels of prothrombin and might therefore increase the risk for thrombosis. We reviewed our HAT experience in 11 years at Medical College of Virginia hospitals of 491 patients undergoing 533 liver transplantations. There were 14 liver grafts with documented HAT (2.62%) in 13 patients. Prothrombin G20210A polymorphism was found in the DNA obtained from 2 of 14 liver allograft tissues (14.2%) but not in the DNA from leukocytes obtained from the peripheral blood of recipients with HAT.

The present study was performed to evaluate the impact of the international unit standard for measuring HCV RNA in the management of patients with chronic hepatitis C virus (HCV) infection.The three assays used were Amplicor Monitor PCR, the National Genetics Institute PCR assay, and branched chain DNA. HCV RNA was measured at four time points (baseline, 3 months after the start of therapy, at the end of treatment, and 6 months after discontinuation of therapy) in 106 consecutive patients who received interferon and ribavirin for chronic HCV.The mean age of the patients was 44 yr. Of the patients, 62% were male, 24% were African American, 38% had bridging fibrosis or cirrhosis, and 75% were HCV genotype 1. Of the 424 samples analyzed, 82-89% of values were within 1 log unit and 85-92% were within 2 log units by the various assays. This variability was not dependent upon HCV genotype. HCV RNA was undetectable in 1.4-6.8% of samples when virus was detected by another assay. The mean HCV RNA in these discordant samples was 1.47-6.33 log IU/ml (30-2,100,000 IU/ml).These data demonstrate that approximately 90% of serum values for HCV RNA were within 1 log unit by the international unit standard regardless of which virological assay was used. However, false positive and false negative results as well as variations in the HCV RNA level of more than 1 to 2 log units can occur with any of the assays, and these results may have an impact upon the management of patients receiving interferon therapy. It is therefore unwise in clinical practice to base important treatment decisions upon a single HCV RNA determination.

Loss of heterozygosity (LOH) of chromosomal regions is crucial in tumor progression. In this study we assessed the potential of the Affymetrix GeneChip HuSNP mapping assay for detecting genome-wide LOH in prostate tumors. We analyzed two human prostate cell lines, P69SV40Tag (P69) and its tumorigenic subline, M12, and 11 prostate cancer cases. The M12 cells showed LOH in chromosomes 3p12.1-p22.1, 11q22.1-q24.2, 19p13.12, and 19q13.42. All of the prostate cases with informative single-nucleotide polymorphism (SNP) markers showed LOH in 1p31.2, 10q11.21, 12p13.1, 16q23.1-q23.2, 17p13.3, 17q21.31, and 21q21.2. Additionally, a high percentage of cases showed LOH at 6p25.1-p25.3 (75%), 8p22-p23.2, and 10q22.1 (70%). Several tumor suppressor genes (TSGs) have been mapped in these loci. These results demonstrate that the HuSNP mapping assay can serve as an alternative to comparative genomic hybridization for assessing genome-wide LOH and can identify chromosomal regions harboring candidate TSGs implicated in prostate cancer.

Transcript variants of the same gene may play distinct functions in the tissue where they are expressed. Absolute quantitation of different transcript variants in malignant and normal tissues can address the specific role of each particular isoform in cancer development and progression. We have recently demonstrated differential expression of the wild-type Wilms’ tumor transcript (wtWT1) and a novel truncated WT1 transcript (trWT1) which lacks the first five exons of wtWT1, among human prostate cancer, leukemia, and breast cancer cell lines. Here we report the analytical validation of a real-time RT-PCR assay for the absolute quantitation of these two different WT1 transcripts with specific primers and probes that ensure specificity for each WT1 variant. By cloning each WT1 transcript in a T3 promoter-containing plasmid, we obtained two WT1 transcript-specific in vitro-generated RNA calibrators for absolute quantitation. Serial dilution of each RNA calibrator demonstrated a 5 log linear dynamic range (5×10 1 to 5×10 6 copies/reaction, R 2 =0.9963 for wtWT1 and R 2 =0.9993 for trWT1). Dilution of the calibrators in total RNA from 1×10 3 non-WT1-expressing cells showed a decreased sensitivity without affecting the linear dynamic range. Precision studies for values within the linear dynamic range showed a coefficient of variation of less than 4% for both transcripts. The described method provides a sensitive and reliable technique for quantitating different WT1 mRNA transcripts.

Telomerase activity has been associated with almost 90% of malignant human cancers from a variety of tissue sources, making it one of the most prominent molecular cancer markers known to date. As such, telomerase has become a very attractive diagnostic and therapeutic target. The advent of the telomeric repeat amplification protocol (TRAP) has allowed for the semiquantitative detection of telomerase from limiting sample amounts. Both the standard TRAP assay and a real-time assay using Amplifluor technology with primers designed specifically for telomerase activity amplification were used to quantitatively assess telomerase activity in primary tumors and tumor-derived cell lines. We have adapted the recently developed TRAPeze XL telomerase detection kit (Intergen, Gaithersburg, MD) for use with real-time polymerase chain reaction for more accurate quantification and high-throughput capabilities. In doing so, the reliability, assay time, and accuracy of quantitation have all been dramatically improved. A comparison of the quantitative analysis for the standard TRAP assay versus the real-time assay using 19 breast tumors revealed telomerase quantitation and standardization using the real-time assay was superior to the standard assay. Our data suggest that this assay will be useful for clinical and research studies involving detection of telomerase activity as it relates to cancer diagnosis.

Many HIV patients develop a progressive syndrome of abnormal body fat distribution accompanied by hypertriglyceridemia. Antiretroviral agents are thought to be etiologic in the syndrome, often termed “highly active antiretroviral therapy (HAART)‐associated lipodystrophy.” In the course of clinical HIV genotype testing, we observed that our HIV patients with hypertriglyceridemia had viral genotypes that were more highly mutated than those of our therapy‐matched control patients. Hypertriglyceridemia was statistically associated with predicted resistance for three nucleoside reverse transcriptase inhibitors: zidovudine, abacavir, and stavudine. Statistical analysis of 51 patients in retrospect revealed a strong association of mutations at reverse transcriptase codons M41 and T215 with hypertriglyceridemia (chi‐square (χ(2)) = 8.375, P=.0038; and χ(2)=7.445, P=.0064, respectively). This was in contrast to silent mutations, which occurred at equivalent rates in retroviral genotypes of patients with and without hypertriglyceridemia. The findings imply that the HIV genotype itself may be a significant etiologic factor in antiretroviral‐associated lipodystrophy. © 2002 Wiley‐Liss, Inc.

Background: The Wilms’ tumor 1 (WT1) gene encodes a transcription factor critical in urogenital development. Using a new model of prostate cancer progression that permits comparison of the cellular and molecular properties of increasingly aggressive sublines of simian virus 40 large T-antigen—immortalized human prostate epithelial cells within the same lineage, the role of WT1 in tumorigenesis was investigated. Method and Results: Using RT-PCR and northern blotting, we identified a novel truncated WT1 transcript in these prostate cancer cell lines. This 2.1-kb transcript consisted of the coding region of the zinc-finger domain of WT1, together with a portion of intron 5 at the 5′ end of the transcript. Furthermore, two peptides were detected by western blotting using antibodies to epitopes of the COOH terminus of WT1. Using RT-PCR, the 2.1-kb transcript was also detected in leukemia cell line K562, breast cancer cell line MCF7, and blood samples from patients with acute leukemia. Conclusion: These novel findings in both cell lines and patient-derived specimens suggest this new WT1 gene alteration has a potential role in the development of new diagnostic assays for some human malignancies.