A disattenuated correlation estimate when variables are measured with error: illustration estimating cross-platform correlations.

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.

Polymorphisms in cytokines and growth factor genes and their association with acute rejection and recurrence of hepatitis C virus disease in liver transplantation.

Acute rejection (AR) and recurrence of hepatitis C virus (HCV) infection are complications after liver transplantation (LTx). Genetic factors play a role in cytokine production as a consequence of polymorphisms within cytokine genes. Our goal was to identify genetic factors that might be associated with AR and recurrence of HCV in liver transplant recipients (LTxRs). We studied 77 Caucasian LTxRs and 100 Caucasian healthy individuals. We studied single-nucleotide polymorphisms (SNPs) in tumor necrosis factor-alpha[TNF-alpha, interleukin-6 (IL-6), IL-10, transforming growth factor-beta1, and angiotensin-converting enzyme genes by SNaPSHOT trade mark Multiplex assay. SNPs were classified as high producers (HP), intermediate producers (IP), or low producers (LP), and their association with AR and recurrence of HCV were studied. The frequency of TNF-alpha IP and HP genotypes was significantly higher in LTxRs with AR in comparison to patients without AR (TNF-alpha HP -238: 63 vs 20%, p < 0.001; TNF-alpha HP -308: 47.4 vs 20%, p = 0.02). The frequency of IL-6 IP and HP genotypes was higher in patients with AR episodes, but the difference was not statistically significant (p = 0.14). However, when we analyzed the simultaneous presence of pro-inflammatory genotypes in the same patient, we found a significant difference between patients with and without AR, respectively (42.1 vs 14.6%, p = 0.012). Moreover, the frequency of the IL-10 LP genotype was higher in LTx patients with AR (p = 0.001) compared to patients without AR. There was an association between pro-inflammatory genotypes and HCV recurrence. Our data suggest that cytokine gene polymorphisms might play a role in AR and HCV recurrence in LTxRs.

Prospective multicenter clinical evaluation of AMPLICOR and COBAS AMPLICOR hepatitis C virus tests.

We conducted a multicenter clinical evaluation of the second versions of the manual AMPLICOR and the semiautomated COBAS AMPLICOR tests for hepatitis C virus (HCV) RNA (Roche Molecular Systems, Inc., Pleasanton, Calif.). The performance characteristics of these HCV RNA tests for diagnosis of active viral infection were determined by comparison to anti-HCV serological test results, alanine aminotransferase levels, and liver biopsy histology results. A total of 878 patients with clinical or biochemical evidence of liver disease were enrolled at four hepatology clinics. A total of 1,089 specimens (901 serum and 188 plasma) were tested with the AMPLICOR test. Sensitivity compared to serology was 93.1% for serum and 90.6% for plasma. The specificity was 97% for serum and 93.1% for plasma. A total of 1,084 specimens (896 serum and 188 plasma) were tested with the COBAS test. Sensitivities for serum and plasma were the same as with the AMPLICOR test. The specificity was 97.8% for serum and 96.6% for plasma. Of the 69 specimens with false-positive and false-negative AMPLICOR test results relative to those of serology, alternative primer set (APS) reverse transcription (RT)-PCR analysis showed that the AMPLICOR test provided the correct result relative to the specimens containing HCV RNA in 64 (92.7%) specimens. Similarly, 66 of 67 (98.5%) false-positive and false-negative COBAS test results were determined to be correct by APS RT-PCR analysis. There were no substantive differences in clinical performances between study sites, patient groups, specimen types, storage conditions (-20 to -80 degrees C versus 2 to 8 degrees C), or anticoagulants (EDTA versus acid citrate dextrose) for either test. Both tests showed >99% reproducibility within runs, within sites, and overall. We conclude that these tests can reliably detect the presence of HCV RNA, as evidence of active infection, in patients with clinical or biochemical evidence of liver disease.

Validation of a multiplex methylation-sensitive PCR assay for the diagnosis of Prader-Willi and Angelman's syndromes.

BACKGROUND: Prader-Willi (PWS) and Angelman's (AS) syndromes are two distinct clinical entities caused by alterations in an identical but differentially methylated region of DNA on chromosome 15q. Highly complex laboratory tests are required for diagnosis because the disorders are caused by several genetic mechanisms. Methylation-specific PCR (MSPCR) is a relatively simple alternative method to detect the methylation status of the PWS/AS region. METHODS AND RESULTS: DNA was treated with sodium bisulfite, with alterations to the published method in which a neutralization step after the alkali treatment of the modified DNA enabled the use of the modified product directly in the PCR, eliminating the need for ethanol precipitation. Multiplex MSPCR using primers to methylated and unmethylated DNA was optimized to yield equal amplification efficiency for both products. Complete concordance was observed during the clinical validation of 40 previously characterized samples, except for one patient with mosaic AS detected by fluorescence in situ hybridization. CONCLUSION: We have developed and validated a multiplex MSPCR assay with alterations of the original published protocol that is technically robust and reproducible and can be used as a screening assay to detect PWS and AS.

Detection of a novel truncated WT1 transcript in human neoplasia.

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. METHODS 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.

Fluorescence in situ hybridization detectable mosaicism for Angelman syndrome with biparental methylation.

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.

Genetic changes in solid tumors.

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.

Can polymerase chain reaction help distinguish benign from malignant lymphoid aggregates in bone marrow aspirates?

OBJECTIVE: Although morphologic and immunologic clues are helpful in distinguishing benign from malignant lymphoid aggregates in bone marrow biopsies, there remain some cases in which it is not possible to arrive at a definitive diagnosis. Since the malignant aggregates are monoclonal B-cell proliferations, we sought to determine whether performing polymerase chain reaction for the immunoglobulin heavy-chain locus would be helpful in distinguishing these 2 entities. METHODS AND RESULTS: Scrapings from unstained bone marrow aspirate smears or touch preparations of bone marrow biopsies from 15 patients with benign bone marrow lymphoid aggregates and 18 patients with malignant lymphoid infiltrates were analyzed for rearrangements of the FR3 region of the immunoglobulin heavy-chain gene locus by a heminested polymerase chain reaction procedure. All specimens had amplifiable DNA, as shown by amplification of the ras proto-oncogene. None of the 15 cases of benign bone marrow lymphoid aggregates demonstrated clonality upon amplification of the immunoglobulin heavy-chain gene locus. In contrast, 8 of the 18 malignant samples were positive (P =.01 by chi(2) test; sensitivity, 44%; specificity, 100%; positive predictive value, 100%; negative predictive value, 60%). There was a tendency for there to be more lymphocytes in stained bone marrow aspirate smears from the cases of malignant lymphoid aggregates with a positive polymerase chain reaction result than in those without demonstrable clonality (36.0 +/- 35.4% vs 9.8 +/- 8.0%, P =.13). CONCLUSIONS: Polymerase chain reaction for the immunoglobulin heavy-chain gene locus may help distinguish benign from malignant bone marrow lymphoid aggregates. Although the presence of false-negative samples may be related to the relative lack of lymphocytes in the bone marrow aspirates, other factors, such as the lack of amplification of the FR3 region of the immunoglobulin heavy-chain gene locus in particular tumors, cannot be ruled out with certainty.

Enhanced analytical sensitivity of a quantitative PCR for CMV using a modified nucleic-acid extraction procedure.

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.

Clinical utility of a quantitative polymerase chain reaction for diagnosis of cytomegalovirus disease in solid organ transplant patients.

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<0.001). Using a cut-off value of 100 vc/ml plasma and clinical diagnosis of HCMV disease, the QPCR assay showed a sensitivity of 100% and specificity of 99.1%. CONCLUSION: HCMV viral load may be useful in the diagnosis of HCMV disease in solid organ transplant patients.

Utility of cytomegalovirus viral load in renal transplant patients in Argentina.

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<0.01). None of the 33 patients without CMV disease had viral loads higher than 500 VC/10(6) PBMC. Using 500 VC/10(6) PBMC as a cut-off value for CMV disease, the quantitative PCR showed a sensitivity and specificity of 100% compare to clinical diagnosis. CONCLUSION: CMV viral load may be useful in the diagnosis of CMV disease in renal transplant patients.

Relationship between biochemical, virological, and histological response during interferon treatment of chronic hepatitis C.

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.

Detection of a common mutation in factor V gene responsible for resistance to activate protein C causing predisposition to thrombosis.

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.

K-ras point mutations in endometrial carcinoma: effect on outcome is dependent on age of patient.

Mutations involving the K-ras proto-oncogene are believed to play an important role in the mechanism of tumorigenesis for many human cancers and occur in 10-30% of endometrial carcinomas. In the present study 221 cases of endometrioid endometrial carcinoma obtained from Japanese patients with average follow-up of 41 months were examined for point mutations in codon 12 of K-ras through use of the polymerase chain reaction. In 103 cases lymph node dissection had been performed. K-ras mutations were significantly associated with the presence of lymph node metastases (P < 0.04). Since endometrial carcinoma in premenopausal women generally behaves less aggressively than tumors of similar histologic grade arising in postmenopausal patients, we evaluated the effect of K-ras mutation on outcome in patients stratified into three different age categories (<53 years, premenopausal; 54-59 years, perimenopausal; >60 years, postmenopausal). In the postmenopausal age group (>60 years), the presence of K-ras mutations was statistically significantly associated with patients who died or experienced recurrence (41.2% vs 13.0%; P < 0.03). This was related to a dramatic (greater than eightfold; P = 0.011) increase in the likelihood of adverse outcome between the premenopausal and postmenopausal states for patients whose tumors contained mutant K-ras. These findings point to a possible role for K-ras activation in the mechanism(s) responsible for more aggressive clinical behavior of endometrioid endometrial cancer that is observed in postmenopausal patients.

Pitfalls in establishing a molecular diagnostic laboratory.

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.

Genetics of colorectal and breast cancer.

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, immunohistochemistry, 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.

High incidence of point mutation in K-ras codon 12 in carcinoma of the fallopian tube.

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 endometrium, 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.