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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.

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.

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)

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.

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.

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.

Clinical workup of metastatic malignancies of unknown origin is often arduous and expensive and is reported to be unsuccessful in 30 to 60% of cases. Accurate classification of uncertain primary cancers may improve with microarray-based gene expression testing. We evaluated the analytical performance characteristics of the Pathwork tissue of origin test, which uses expression signals from 1668 probe sets in a gene expression microarray, to quantify the similarity of tumor specimens to 15 known tissues of origin. Sixty archived tissue specimens from poorly and undifferentiated tumors (metastatic and primary) were analyzed at four laboratories representing a wide range of preanalytical conditions (eg, personnel, reagents, instrumentation, and protocols). Cross-laboratory comparisons showed highly reproducible results between laboratories, with correlation coefficients between 0.95 to 0.97 for measurements of similarity scores, and an average 93.8% overall concordance between laboratories in terms of final tissue calls. Bland-Altman plots (mean coefficients of reproducibility of 32.48+/-3.97) and kappa statistics (kappa0.86) also indicated a high level of agreement between laboratories. We conclude that the Pathwork tissue of origin test is a robust assay that produces consistent results in diverse laboratory conditions reflecting the preanalytical variations found in the everyday clinical practice of molecular diagnostics laboratories.

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: 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.

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.

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.

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.

Accurate and rapid diagnosis of CMV disease in immunocompromised individuals remains a challenge. Quantitative polymerase chain reaction (QPCR) methods for detection of CMV in peripheral blood mononuclear cells (PBMC) have improved the positive and negative predictive value of PCR for diagnosis of CMV disease. However, detection of CMV in plasma has demonstrated a lower negative predictive value for plasma as compared with PBMC. To enhance the sensitivity of the QPCR assay for plasma specimens, plasma samples were centrifuged before nucleic‐acid extraction and the extracted DNA resolubilized in reduced volume. Optimization of the nucleic‐acid extraction focused on decreasing or eliminating the presence of inhibitors in the pelleted plasma. Quantitation was achieved by co‐amplifying an internal quantitative standard (IS) with the same primer sequences as CMV. PCR products were detected by hybridization in a 96‐well microtiter plate coated with a CMV or IS specific probe. The precision of the QPCR assay for samples prepared from untreated and from pelleted plasma was then assessed. The coefficient of variation for both types of samples was almost identical and the magnitude of the coefficient of variations was reduced by a factor of ten if the data were log transformed. Linearity of the QPCR assay extended over a 3.3‐log range for both types of samples but the range of linearity for pelleted plasma was 20 to 40,000 viral copies/ml (vc/ml) in contrast to 300 to 400,000 vc/ml for plasma. Thus, centrifugation of plasma before nucleic‐acid extraction and resuspension of extracted CMV DNA in reduced volume enhanced the analytical sensitivity approximately tenfold over the dynamic range of the assay. J. Clin. Lab. Anal. 14:32–37, 2000. © 2000 Wiley‐Liss, Inc.

Hereditary predisposition to thrombosis due to activated protein C resistance (APCR) has been attributed to a missense mutation in the factor V gene at nucleotide 1691 (G to A), causing replacement of arginine at codon 506 with glutamine. Using an RFLP‐PCR assay to detect this mutation, we measured a prevalence of 3.3% in healthy Caucasians and 1.25% in healthy African‐Americans. In addition, we evaluated a total of 90 consecutive specimens submitted to the coagulation laboratory at the Medical College of Virginia for the presence of this mutation. We compared our results for 78 of these specimens with the values measured by a modified partial thromboplastin assay, the COATEST. Twelve of the 90 samples could not be tested using the COATEST because the patients were undergoing anticoagulant therapy. One of the latter 12 specimens was positive by the RFLP‐PCR test. Using the genetic test as the definitive assay and the cutoff value established for distinguishing between normal and abnormal results by the COATEST, the COATEST had a sensitivity of 50% and specificity of 93% for the detection of factor V mutation. Analysis of the 90 samples stratified by ethnic groups revealed a frequency of mutation of 13.3% for Caucasians and 6.88% for African‐Americans, although with the present sample size, the difference was not statistically significant. Although the COATEST is technically simpler to perform than the genetic test for diagnosing the presence of the factor V mutation, its use for this purpose is limited due to low sensitivity. Thus where this disorder is clinically suspected, submission of the specimen directly for genetic testing by RFLP‐PCR or equivalent assay should be considered. J. Clin. Lab. Anal. 11:328–335, 1997. © 1997 Wiley‐Liss, Inc.

The present study was conducted to evaluate the relationship between biochemical, virological, and histological response during the course of interferon therapy. Ninety consecutive patients with well-documented chronic hepatitis C virus (HCV) were treated with 5 MU of interferon alfa-2b three times weekly for 6 months. Liver biopsy was performed, and serum HCV RNA titer was measured before and at the completion of interferon treatment. Normalization of serum alanine transaminase (ALT) concentration (biochemical response) was observed in 50% of patients. In these patients, Knodell score declined significantly from 9.6 +/- 0.5 to 5.0 +/- 0.5 (P < .01), and 75% became HCV RNA negative. The remaining patients (50%) were biochemical nonresponders; mean Knodell score declined from 9.6 +/- 0.5 to 7.7 +/- 0.5 (P < .01), and 11% became HCV RNA negative. For both biochemical responders and nonresponders, the decline in Knodell score was confined to the components of hepatic inflammation (piecemeal necrosis + lobular + portal inflammation); no change in fibrosis was observed. Hepatic inflammation declined by 5 points or more in 69% of biochemical responders and 48% of biochemical nonresponders, and by at least 50% from pretreatment values in 74% and 38% of biochemical responders and biochemical nonresponders, respectively. For all patients (both biochemical responders and nonresponders) who remained viremic at the conclusion of interferon therapy, the reduction in hepatic inflammation was a linear function of the decline in HCV RNA titer. We conclude that more than one third of patients who had no biochemical response after 6 months of interferon therapy achieved a similar improvement in hepatic histology as was observed in patients with biochemical response. This improvement in hepatic histology appeared to correlate with a reduction in HCV RNA titer, especially in patients who remained viremic.

Clinical Application of Genetic, Oncogenic, and Differentiation Markers of Cancer. ras Proto-Oncogene Activation in Human Malignancy. C-myc as a Tumor Marker for Primary Human Cancers. p53 in Human Cancer. Oncogenes and Tumor Suppressor Genes in Gynecological Malignancies. Genetic Alterations in Colon Cancer. Genetic Changes in Breast Cancer. Genetic Alterations in Lung Cancer. Wilms Tumor-Susceptibility Loci. Non-Hodgkin"s Lymphomas. Phenotype Expression of Hodgkin"s Disease: Biologic, Immunologic, and Functional Properties. Melanoma Markers. Detection of Minimal Residual Disease (MRD) in Leukemia and Lymphoma. P-Glycoproteins in Tumors. Genetically Engineered Antitumor Monoclonal Antibodies.Targeting Antisense Oligonucleotide Chemotherapy to the Type I Regulatory Subunit of c-AMP Dependent Protein Kinase. Summary and Perspective: Assessing Test EffectivenessThe Identification of Good Tumor Markers. Index

Human colon cancer has proven to be an excellent model system for studying the development of malignant disease in humans. Based on clinical and pathological findings, colon cancer is observed to develop through a variety of stages involving premalignant adenomatous polyps followed by localized invasive malignant carcinoma and subsequently disseminated malignant tumor (Muto et al., 1975; O’Brien et al., 1992). Premalignant lesions vary from small relatively well-differentiated hyperplastic lesions to larger less-well-differentiated tubular adenomas and subsequently to lesions that demonstrate still greater size, and reduced cytologic differentiation called villus adenomas (Muto et al., 1975; Jass et al., 1986; O’Brien et al., 1992). This process occurs over a period of years and is believed to involve the occurrence of four to six separate genetic changes (Fearon and Vogelstein, 1990).

PCR is 100 times more sensitive than traditional tube culture for detecting cytomegalovirus (CMV) but may require up to 12 reactions per specimen (Sandin et al., 1991). In order to make the assay practical for use in a clinical laboratory the procedure used to detect CMV must be simplified. In this study, the effect of reducing the number of reactions per specimen on sensitivity and specificity of the PCR assay was evaluated. 53 residual samples from specimens processed for CMV by shell vial assay/routine tube tissue culture (SVA/TTC) were analyzed by PCR. The residual samples were separated into a supernatant and pellet fractions, then tested for CMV with primers to the immediate early (IEP) and late protein (LP) genes using a nested procedure. To exclude false negatives due to the presence of inhibitors in the sample fractions, all fractions were tested for the presence of the human myosin heavy chain gene also using a nested procedure. SVA/TTC had a sensitivity and specificity of 52/96% in comparison to PCR when data from all 12 PCR reactions was considered. However, high sensitivity and specificity were retained when only the data of the IEP primers with two samples were considered. The results from examining only the 1:10 dilution of pellet and the undiluted supernatant by PCR provided a 60% increase in sensitivity over SVA/TTC, high specificity and a clinically feasible assay.

Although traditional tube culture (TTC) is still considered by many as the 'gold standard' for the laboratory diagnosis of human cytomegalovirus (HCMV), the shell vial assay (SVA) offers greater speed of detection. This technique utilizes immunofluorescence (IF) to detect early or immediate early nuclear antigens (IEA). The detection capabilities of these two tests were compared with the polymerase chain reaction (PCR), a technique that amplifies enzymatically selected DNA target sequences. Serial dilutions of crude culture harvests from 2 HCMV strains, Towne and a clinical urine isolate, were made up to 1:1 000,000. Ten-microliters aliquots of the original sample and each dilution were tested by PCR, TTC and SVA. For PCR, the nested-primer approach was used. Outer primers delimited a 721-bp sequence contained within the 2nd to 4th exons of the immediate-early protein. Inner nest primers delimited a 167-bp sequence in the third exon, detected by a 32P-labelled probe. The results show that: (1) control samples which contained all PCR reagents but no DNA were uniformly negative; (2) radiolabelled-probe detection (RPD) of PCR products is, on average, 100 x more sensitive than detection by ethidium bromide; (3) PCR is, on average, 100 x more sensitive than evaluation of cytopathic effect (CPE) in the TTC; (4) the predictive value of a negative SVA result is low compared to PCR.

Colposcopically directed cervical biopsies, smears, and swabs obtained from 210 women with a previous abnormal cervical cytology were evaluated for the presence of human papilloma virus (HPV) using morphology and dot-blot hybridization. The diagnosis of HPV infection in biopsies and smears examined morphologically was rendered using established criteria for condyloma/cervical intraepithelial neoplasia (CIN). In hybridization studies, DNA was isolated from cells obtained from cervical swabs and annealed with probes that detected HPV types 6/11, 16/18, and 31/33/35 using a dot-blot procedure. Ninety-five cases demonstrated morphologic evidence of condyloma/CIN; 51 of these (54%) were positive for HPV DNA (five cases 6/11, 21 cases 6/18, 20 cases 31/33/35, and five cases two different probes). HPV DNA was also detected in 6 of the 115 cases (5.2%) that were morphologically negative (three cases 16/18, three cases 31/33/35). The results demonstrated that morphology was more sensitive than dot-blot hybridization for detection of HPV-related lesions. The dot-blot hybridization did detect HPV DNA in a small percentage of the cases that showed no morphologic abnormality and was useful for typing of the HPV. At this juncture, however, the clinical significance of the latter findings is unclear.

Cryopreservation of patient bone marrow specimens allows for future purification of tumor cells for research or diagnostic purposes. It has not been determined whether this practice causes significant changes in gene expression. In order to evaluate this, we performed microarray analysis (Affymetrix U133A and U133A 2.0 gene chips) on mononuclear cells from 5 acute myelogenous leukemia (AML) patient samples (>70% blast count) that had been either a) cryopreserved, b) snap frozen, or c) resuspended in TRIzol and stored at −80°C. Samples remained frozen for a period of 1–4 weeks. While gene expression changes between groups were minor, there were some differences. Thirteen probe sets between TRIzol and cryopreserved samples, and 60 probe sets between TRIzol and snap frozen samples, varied significantly (>2-fold difference and p

int-2 is a proto-oncogene that is partially homologous to angiogenesis-inducing fibroblast growth factor and is believed to play a role in mouse mammary carcinogenesis. Recent evidence has suggested that this proto-oncogene may also play a role in human breast cancer. In the present study, we used Southern hybridization analysis to examine DNA from 79 primary and 11 recurrent human breast cancers for evidence of activation of int-2 through either gene rearrangement or amplification. A similar analysis was performed for two other proto-oncogenes, c-erbB-2 and c-myc, also suspected of playing a role in the development of human breast cancer. Proto-oncogene status was correlated with estrogen (ER) and progesterone (PR) receptor status, patient age, and lymph node (LN) status at the time of surgery. Gene rearrangement was not a frequent occurrence with any of the proto-oncogenes. However, amplification of int-2 occurred at a significantly higher frequency in recurrent breast cancers than in primary cancers and in patients with primary cancers who were less than or equal to 50 years of age versus greater than 50 years of age at surgery. Although amplification of all three proto-oncogenes occurred at a greater frequency in primary tumors from patients with lymph node metastases than from those without lymph node metastases, a significant difference was noted only in the case of c-myc amplification. These findings confirm and extend earlier results of studies of int-2, c-erbB-2 and c-myc amplification in human breast cancers and point to a role for int-2 activation in certain cases of recurrent breast malignant neoplasia.

The ability of di(2-ethylhexyl) phthalate (DEHP), a widely used plasticizer and environmental contaminant, to suppress development of putative preneoplastic lesions in rat liver was evaluated. gamma-Glutamyl transpeptidase-positive (GGT+) foci were initiated in the livers of Sprague-Dawley male rats with a single dose of diethylnitrosamine (DEN) following partial hepatectomy. Promotion of foci was commenced by feeding a choline-deficient diet (CD). A group of control rats was fed a choline-supplemented diet (CS). The ability of DEHP to suppress the emergence of GGT+ foci was evaluated by feeding additional groups of rats the CD diet containing either 0.1%, 0.5%, 1.0% or 2.0% DEHP. The CD diet promoted the number of GGT+ foci above levels in control livers. Inclusion of the plasticizer to the levels of 0.5%, 1.0% and 2.0% in the CD diet effectively inhibited the appearance of the foci. However, DEHP was unable to inhibit the promoting effect of the CD diet at a concentration of 0.1%. DEHP's ability to block development of GGT+ foci correlated with its ability to increase liver weight and to induce carnitine acetyltransferase (EC 2.3.1.7), a marker of peroxisome proliferation.

DNase I and α-amylase are commercial products obtained from mammalian sources and may be separated from contaminating RNase by treatment with bentonite. The completeness of RNase removal is demonstrated by using a highly sensitive technique employing E. coli ribosomal RNA as substrate.

The possible role that informosomal mRNA may play in the stimulation of hepatic protein synthesis induced by the acute administration of tryptophan was investigated. L-tryptophan (30 mg/100 g body weight) tube-fed to rats induced an increase (20%) in the amount of polyribosome-associated poly(A+)mRNA in the liver within 1 hour. On the other hand, the amount of the informosomal poly(A+)- mRNA revealed no significant increase or decrease. The size of the increase in the polyribosome-associated poly(A+)mRNA pool was equal to the entire amount of informosomal poly(A+)mRNA present in the hepatic cells. Therefore it was concluded that the failure to detect a significant decrease in the size of the informosomal mRNA pool indicated that the increase in the polyribosome-associated poly(A+)mRNA must be due to a different mechanism, such as enhanced nuclear-to-cytoplasmic translocation of nuclear poly(A+)mRNA, which had been reported earlier. To determine whether any qualitative or quantitative changes occurred in the RNA sequences along with the increase in poly(A+)mRNA following tryptophan administration, DNA/RNA hybridization studies were conducted by using hepatic polyribosome-associated poly(A+)- mRNA from tryptophan-treated and control rats. Although qualitatively no new species of mRNA were detected in the mRNA from tryptophan-treated rats, kinetic analysis of the hybridization curves indicated that there was a shift or accumulation of hepatic poly(A+)mRNA belonging to the intermediate and possibly the high frequency classes of polyribosome-associated poly(A+)mRNA in the livers of the tryptophan-treated rats.

5-Fluoroorotic acid treatment lowered the (Guanine + Cytosine)/(Adenine + Uracil) base ratio of 32P-labeled microsomal RNA from a control value of 1.36 to 1.00. Low doses of actinomycin D, which are effective in inhibiting ribosomal RNA synthesis without significantly affecting messenger RNA synthesis, caused a similar decrease in the base ratio. Microsomal RNA labeled by [3H]orotate in the presence of 5-fluoroorotic acid had approximately 1 2 the specific radioactivity but twice the hybridization efficiency of RNA labeled in its absence. Evidence is presented that this RNA (1) has a different structure from that of ribosomal RNA, (2) hybridizes to DNA with an efficiency consistent with that of other published studies of polysome-associated messenger RNA, and (3) possesses sequences which are present in other samples of liver microsomal RNA but not in kidney microsomal RNA. These properties differ from those known to be exhibited by 18 S and 28 S ribosomal RNA. Electrophoretic analysis of this [3H]orotate-labeled microsomal RNA indicated that the analogue greatly inhibited precursor incorporation into ribosomal RNA but had little or no effect on incorporation into messenger RNA. Ribosomal RNA and polyadenylate-rich nonribosomal RNA were prepared from total polyribosomes by phenol extraction at pH 7.6 and pH 9.0, respectively. 5-Fluoroorotic acid inhibited [3H]orotate or 32Pi incorporation into the pH 7.6 fraction much more effectively than incorporation into the pH 9.0 fraction. A subfraction of the pH 9.0 RNA which was retained by a polythymidylate-cellulose column had a greatly increased adenylate content.