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For more than 50 years, clinicians have relied primarily on the same visual and mechanical assessment methods to diagnose and classify periodontal disease. Clinical signs are simply a measurement of the past damage of a disease process. While clinical presentation and probing depths are indicators that the disease exists, these alone cannot determine the types and quantities of the responsible pathogens. Likewise, clinical signs alone cannot determine if therapy has achieved the goal of suppression of the etiological agent(s). Genetic presymptomatic testing complements bacterial DNA testing by providing insight into the patient's genetic predisposition to periodontal disease before symptoms appear, or when disease is already present. DNA-PCR testing of saliva can help the clinician provide an earlier and more specific diagnosis of disease based on causation. Treatment planning is also enhanced, as therapy can be appropriately modified based on both clinical and biological inflammatory factors. Finally, patient communication and case acceptance can be more readily achieved because the test reports elicit a persuasive "seeing is believing" attitude when reviewing test results with patients. Through the use of accurate periodontal charting, medical and dental risk assessments, and other diagnostic screening tests such as OralNA's MyPerioPath and MyPerioID PST tests, highly personalized periodontal therapy can be developed and carried out by the general practitioner. There has never been a better time to become more aware, and keep tighter control, of the periodontal status of one's patient base. Many patients are asking dentists about the connection between periodontal health and general health, While it currently would not be appropriate to suggest a causative relationship, there is abundant ongoing research that suggests a correlative relationship between periodontal disease and other whole-body ailments. Many patients have refused periodontal care or denied the importance of maintaining their periodontal health. The use of tools such as the OralDNA test report can assist in achieving patient acceptance of needed treatment and cooperation with the clinician to improve their periodontal health. The contents of the report and the visual presentation demonstrate that many patients have an active infection that can be stabilized if treated. Patients also provide more information about other factors that may contribute to their periodontal condition. Further, the OralDNA report enhances dentist-physician communication and a team approach to patient care.

Objective: The purpose of this study was to report type-specific prevalence and persistence of human papillomavirus (HPV) in women who underwent cytologic screening., Study Design: We examined HPV prevalence in 73,371 women who had type-specific HPV testing in 1 of 23 clinical laboratories in the United States. Persistence was evaluated in 963 women who were tested within 8-16 months of their index test., Results: HPV was detected in 31% of the women, and high-risk HPV was detected in 23% of the women. HPV-16, -53, -52, and -31 were the most prevalent types. Of the 953 women with 2 tests, 39% of the women had persistent HPV infection. High-risk HPV persistence was detected in 34% of the women who were positive initially for high-risk HPV., Conclusion: Approximately one-third of our sample had HPV; of those women who were retested within 8-16 months, more than one-third had persistent infection. Among women with high-risk HPV infections, the likelihood of persistence was highest with HPV genotypes that were phylogenetically similar to HPV-16.

Background: Detection of serum monoclonal proteins is a common laboratory analysis used in the evaluation of patients with B-cell disorders. Since many individuals with elevated immunoglobulin have no symptoms, it is important to have simple methods for initial screening of patients with suspected B-cell disorders., Methods: Samples of serum from healthy donors and from patients with elevated immunoglobulin levels were tested using a technology named Droplet MicroChromatography (DMC). DMC was developed at Artann Laboratories (West Trenton, New Jersey, USA) for the rapid assessment of changes in the composition of serum. DMC is based on the dynamics of the sediment pattern formation during drying of a fluid microdroplet., Results: Results of this pilot study confirm the hypothesis that the pattern formation created by drying droplets of serum would differ between normal samples and those containing monoclonal proteins. Reproducible differences in the patterns formed by the two types of specimens are shown. Strong correlation between abnormally elevated levels of immunoglobulins in the serum of myeloma patients and the patterns formed by drying droplets of serum indicates that the DMC technique may be suitable for semi-quantitative analysis of serum samples. We also demonstrate that computer identification of the drying droplet structure and dynamics is a tractable issue., Conclusions: DMC has significant diagnostic potential and can serve as a basis for development of a simple, rapid, and inexpensive method for initial screening of patients suspected of having multiple myeloma and other pathologies of lymphoid origin that are associated with the overproduction of monoclonal immunoglobulins. The DMC test requires only approximately 1 microL of serum and could therefore be performed in any facility where it is safe to work with serum.

The conventional Papanicolaou smear (CPS) is not considered accurate for the diagnosis of Trichomonas vaginalis (T. vaginalis), and women noted to carry the organism on CPS are recommended to undergo confirmatory testing. Liquid-based preparations have been shown to facilitate the diagnosis of squamous lesions and may also facilitate the diagnosis of T. vaginalis. We used polymerase chain reaction (PCR) to investigate the accuracy of the diagnosis of T. vaginalis by the liquid-based Pap test (LBP). LBP with the diagnosis of T. vaginalis from a 12-mo period were identified. Residual samples from these cases were subjected to PCR for T. vaginalis as were the residual samples of a control group of 195 LBP (including 103 inflammatory LBP and 69 cases of atypical squamous cells) in which T. vaginalis was not diagnosed cytologically. PCR confirmed the presence of T. vaginalis in 50 of 51 (98%) LBP and identified 2 additional cases. Morphologic identification of T. vaginalis on LBP is highly accurate and should not require confirmatory testing.

We have developed a low-density DNA array for the detection and typing of human papillomavirus (HPV) DNA. The gene chemistry strategy involves using a combination of the polymerase chain reaction (PCR) with the consensus oligonucleotide primers MY09/MY11 followed by a ligase detection reaction (LDR). Fluorochrome-labeled HPV-specific primers are joined to a common primer modified with a unique anchoring sequence called a zip code on its 3' end. The result is a series of 60-70 base pair and single-stranded ligation products that are then hybridized to their respective zip code complements affixed to glass slide based arrays. Nine separate zip codes were assigned, one for each HPV type (6,11,16,18, 31, 33, 35, and 53) and one for a beta-globin internal control marker. Two additional zip-codes were reserved for a pair of consensus HPV LDR products: the cLDR1 and cLDR2 primers hybridize to a conserved sequence within the HPV L1 open reading frame internal to the MY09/MY11 fragment. These consensus primers were shown to detect over 40 different HPV types. The purpose of this study was to evaluate the analytic performance of this low-density microarray based assay for HPV, as well as to introduce our simplified read-out instrumentation, shown here to be a low cost and highly efficient way to detect and genotype HPV for clinical testing.

Objective: To make recommendations regarding the appropriate evaluation for the prothrombin G20210A mutation, as reflected by published evidence and the consensus opinion of recognized experts in the field. DAT SOURCES: Review of the medical literature, primarily since 1996., Data Extraction and Synthesis: After an initial assessment of the literature, key points defining the condition, and review of the clinical study design, a draft manuscript was prepared and circulated to every participant in the College of American Pathologists Conference on Diagnostic Issues in Thrombophilia before the meeting. Each of the key points and associated recommendations were then presented for discussion at the conference. Recommendations were accepted if a consensus of 70% of experts attending the conference was reached. The results of the discussion were used to revise the manuscript into its final form., Conclusions: Consensus was reached on several recommendations concerning the criteria for testing for the prothrombin G20210A mutation and for the method of testing. First, a major point of consensus was that the prothrombin G20210A mutation is a significant risk factor for venous thromboembolism (VTE) and that testing should be considered in the initial evaluation of suspected inherited thrombophilia. Second, although several analytic methods are commonly used for genetic testing for the prothrombin mutation, all are generally robust and reliable. The recommendations for testing for the prothrombin mutation parallel those for the factor V Leiden mutation and include patients with a history of recurrent VTE, a first episode of VTE before the age of 50 years, a history of an unprovoked VTE at any age, thromboses in unusual anatomic sites, or an affected first-degree relative with VTE. A history of VTE related to pregnancy or estrogen use and unexplained pregnancy loss during the second or third trimesters were also considered to be indications for testing. Other scenarios remain controversial or not recommended, including general population screening.

Objective: To review the role of an elevated total plasma homocysteine level (hyperhomocyst[e]inemia) in patients with venous or arterial thrombosis, as reflected by the medical literature and the consensus opinion of recognized experts in the field., Data Sources: Review of the medical literature, primarily from the last 10 years., Data Extraction and Synthesis: The literature was reviewed to identify key points defining the condition, and the clinical study design of each article was examined. A draft manuscript was prepared and circulated prior to the conference to every participant in the College of American Pathologists Conference XXXVI: Diagnostic Issues in Thrombophilia. Each of the key points and associated recommendations was then presented for discussion at the conference. Recommendations were accepted if a consensus of the 70% of the experts attending the conference was reached. The results of the discussion were used to revise the manuscript into its final form., Conclusions: Consensus was reached on 9 recommendations concerning the criteria for diagnosis, the method of testing, and the approach for clinical management. A major point of consensus was that no causal role of hyperhomocyst(e)inemia in venous or arterial thrombosis is yet established. Testing methods used to measure homocysteine directly are sensitive and reliable, and provide more information than does genotyping for markers linked to abnormal plasma homocysteine.

Structural rearrangements involving the MLL gene at 11q23 are common recurring abnormalities in de novo and therapy-related hematologic disorders. MLL rearrangement most often results from translocation or partial tandem duplication, although recent published reports suggest a different mechanism by which MLL might participate in leukemogenesis: MLL amplification. We report two patients with myeloid disorders who showed amplification of MLL at diagnosis and who, like the majority of reported cases, had an older age at onset and on aggressive clinical course. Additionally, we summarize the salient clinical, cytogenetic and molecular findings of the 29 other cases of MLL amplification that have thus far been reported.

We describe a retrospective study of 4 cases of sporadic fatal infectious mononucleosis (IM), 1 case of fatal IM, and 1 case of sporadic severe IM. Patients were 26 months to 17 years old; 3 were male. Five died of complications of IM. All 5 of these patients had the Epstein-Barr virus (EBV) present in examined tissue specimens; EBV was monoclonal in 3 patients and biclonal in 1. EBV clonality studies were not performed in the remaining patient. All 5 patients also had monoclonal gene rearrangements. The sixth patient survived despite a life-threatening clinical course; EBV was oligoclonal, and gene rearrangements were not detected. EBV clonality and gene rearrangement studies may be usefulfor predicting which patients with clinically aggressive IM are at highest risk for fatal outcome. Patients in whom IM has a fatal outcome are more likely to have monoclonal or biclonal EBV and immunoglobulin heavy chain or T-cell receptor gene rearrangements. In contrast, patients with nonfatal IM may lack monoclonal EBV and monoclonal rearrangements of the aforementioned genes. The reasons EBV induces a monoclonal proliferation only in some patients remain to be elucidated.

Background: Inherited thrombophilia is caused by mutations in genes central to the clotting cascade. Analysis of the factor V Leiden (FVL) and prothrombin G20210A mutations are the most prevalent in thrombophilia., Methods and Results: We have optimized an allele-specific PCR assay for the simultaneous detection of both wild-type and mutant alleles. This method is adapted for clinical use with the FVL and prothrombin G20210A assays and is significant in its intentional use of nucleotide mismatches at the 3' end of allele-specific primers. Two internal allele-specific primers are designed to amplify in opposite directions on opposite strands that reduce differential amplification. Our results show concordance with methods involving PCR with restriction endonuclease digestion, yet are simpler to perform., Conclusion: The simultaneous allele-specific amplification method allows simultaneous detection of wild-type and mutant alleles by PCR using four distinct primers. Nucleotide mismatches in the primers reduce competitive amplification.

Background: Molecular diagnostics devices are becoming smaller. With the advancement of miniaturization technologies, microchip-based systems will soon be available for genetic testing. The purpose of this review is to highlight the underlying principles in miniaturization, the strategies being developed for bioanalysis, and the potential impact on the practice of this rapidly growing medical discipline., Approach: The author discusses DNA microchips and their practical importation into the clinical laboratory, based on his background in medical device and microchip design and development. His discussion is supported by a body of literature covering both biomedical and electrical engineering and more recent publications in the field of molecular genetics and pathology., Content: This review is descriptive and intended to outline the technologic and methodologic approaches to the creation of an integrated genetic analysis instrument based on miniature components. The review draws on published scientific evaluations of these devices without regard to the companies involved in their development., Summary: The intent of this review is that the reader will better understand the variety of technical approaches toward the miniaturization of molecular genetic testing for the clinical laboratory. With insight into the principles underlying the operation of these chips and the integrated systems, the end user can better evaluate the value to the field in terms of making molecular genetics testing simpler, faster, and less expensive.

Bone marrow transplantation is used as a primary treatment for many diseases, including leukemia, lymphoma, and inborn errors of metabolism. The procedure involves ablation of the recipient's bone marrow by chemotherapy and/or radiation therapy, followed by transplantation of harvested bone marrow. In autologous bone marrow transplantation (BMT), the patient's own marrow is harvested and treated to remove malignant cells before it is replaced into the patient. In allogeneic BMT, bone marrow is obtained from a donor who is a close antigenic match to the patient. In either case, the goal of BMT is full, permanent replacement of the recipient's original bone marrow by donor hematopoietic elements.

The process of lymphocyte differentiation involves structural alterations of specific genes including those for the immunoglobulin (Ig) and T-cell receptor (TCR) antigen genes. This process occurs very early in the differentiation of B- and T-lymphocytes and involves an ordered program for splicing and rearranging segments of these genes, depending on cell lineage and level of differentiation. Specific DNA cutting and splicing enzymes result in the removal of a number of constant, joining, and variable segments of the Ig and TCR genes. Rearrangement of the VDJ and C segments occurs randomly during the process of B- and T-cell development; hence, the resultant gene rearrangement varies from cell to cell. This results in a unique rearrangement of these genes that encode for a specific Ig or TCR protein. A clonal population of lymphocytes, however, will have a specific molecular structure of rearrangements. Identification of this clonal population is central to the diagnosis of lymphomas and lymphocytic leukemias, because virtually all forms of lymphoid malignancies contain rearrangements of one or more antigen receptor genes. Furthermore, as a clonal expansion, an individual neoplasm will contain the identical rearranged gene throughout the population, serving as a unique clonal marker (1). However, it is important to be aware that lymphocyte clonality is not equivalent to malignancy (2). Benign and reactive conditions may show monoclonal rearrangements. Correlation with histology and immunophenotypic studies is important in order to establish a definitive diagnosis of malignancy. Similarly, the absence of clonal gene rearrangement may be seen in cases that appear malignant by histologic and immunophenotypic criteria. In these instances, it is important to be aware of technical limitations of the assays and sampling errors, which may result in a false-negative result.

HPVs have evolved to accomplish the task of controlling host cell proliferation and differentiation to the end of producing more infectious virions. Coincident with the viral life cycle, however, is the risk that the viral genome will be disrupted and its DNA integrated into the host cell chromosomes. Integration of the viral genome is potentiated by host factors and extracellular effectors that alone may increase genetic instability. But it is consequent to viral integration that most HPV-associated malignancies develop. Investigations of the potential for HPV to immortalize primary cells or transform immortalized cells in vitro demonstrate two distinct classes of genital viral types: (1) oncogenic, exemplified by HPV 16 and 18; and (2) the nononcogenic types 6 and 11. Subsequently, localization of the HPV oncogene implicated that E6 and E7 act by uncoupling the checkpoint controls of the cell cycle principally by inhibiting the normal functioning of p53 and pRb, respectively. By in large, the nononcogenic viruses do not effect irreversible growth properties through these same viral genes and the same cellular counterparts.

Background: Clinical descriptions of the dominantly inherited ataxic motor syndromes in a 7-generation family of German origin were first reported in 1951., Objective: To provide follow-up clinical, pathological, and genetic data for 9 patients in this family., Design: Clinical histories and neurologic findings, gross and microscopic pathological features, and DNA analysis., Results: Clinical presentations in this closely followed up portion of the family include fairly uniform ataxic and upper motor neuron symptoms. Nystagmus was a conspicuous and early sign, but generational anticipation was not evident. Although often present, amyotrophy was not a major source of disability. Major pathological degeneration was noted in the pons, spinal cord, and upper brainstem, where ubiquitin-immunoreactive intranuclear inclusion bodies were demonstrated. The diagnosis of Machado-Joseph disease (SCA3 [spinocerebellar ataxia type 3] genotype) was established from autopsy tissue in 1 patient and from blood specimens in 6 others., Conclusions: Clinical variation within this family and between this family and families with the SCA1 and SCA3 genotypes is so broad as to make the genetic diagnosis from clinical criteria alone practically impossible. The pathological definition of Machado-Joseph disease is more reliable, but some findings do overlap those of other genotypes. To our knowledge, the basis for the phenotypic variations in Machado-Joseph disease, genetic or otherwise, has not been established.

Several methods have been used to evaluate engraftment after allogeneic bone marrow transplantation (BMT). We assessed the usefulness of a multiple short tandem repeat (STR) amplification kit combined with a capillary electrophoresis unit for DNA identity analysis in the evaluation of engraftment after BMT. For 17 of 18 patients, at least 1 locus showed unique alleles for the donor and the recipient. In all cases, at least 1 locus was informative for the presence of small amounts of recipient DNA. The results from STR analysis were the same as Southern blot analysis in 14 of 17 cases. Differences included mixed chimerism detected only with STR analysis, informative loci present only with STR analysis, and informative loci present only with Southern blot analysis (1 case each). By using mock mixed chimeras, minor populations of 5% were detected routinely in all loci using the kit manufacturer's default protocol. By increasing the amount of amplified DNA, minor populations of 1% were detected in all cases but not in all loci. This single reaction technique provides for faster results, reduced workforce needs, and greater sensitivity than traditional Southern blot.

Objectives: To determine the underlying prevalence of cervical intraepithelial neoplasia (CIN) in women with benign cellular changes on a Papanicolaou smear, and to evaluate follow-up strategies to identify women at high risk for serious underlying pathology., Methods: Nonpregnant women aged 18 to 75 years with benign cellular changes on a Papanicolaou smear were recruited from primary care clinics of an urban teaching hospital. The subjects (N = 132) were tested at baseline for the presence of human papillomavirus using the polymerase chain reaction technique, and underwent repeated cervicovaginal smears at 3, 6, and 9 months. At 12 months colposcopy was performed. The main study outcome was the proportion of subjects with CIN as determined by colposcopic biopsy specimens. We determined the sensitivity, specificity, and predictive values of historical risk factor information, human papillomavirus testing, and repeated cervicovaginal smears for the detection of CIN., Results: Cervical intraepithelial neoplasia was found in 30 of 132 women, of whom 27 (20%) had low-grade CIN (CIN I) and 3 (2%) had high-grade CIN (CIN II). Underlying CIN was significantly more common in women younger than 35 years or who had a history of Trichomonas infection or venereal warts, a positive human papillomavirus test result, or abnormal follow-up smears. However, no follow-up strategy combined high sensitivity with a low referral rate for colposcopy., Conclusions: The prevalence of underlying high-grade CIN in women with benign cellular changes is low. However, further prospective studies in other settings are needed before routine follow-up can unequivocally be recommended.

A patient with juvenile Huntington's disease (HD) of probable maternal inheritance is reported. The expanded IT-15 allele was only detected with the use of modified PCR and Southern transfer techniques, which showed a CAG trinucleotide repeat expansion of approximately 250 repeats-the largest CAG expansion reported within the huntingtin gene. This case emphasizes the need for communication between the diagnostic laboratory and the clinician to define the molecular genetics of unusual cases.

We evaluated 118 cases of mantle cell lymphoma by polymerase chain reaction (PCR) for the major translocation cluster (MTC) region and another breakpoint corresponding to probe p94PS, located 24 kb telomeric to the MTC locus on chromosome 11. The specimens included 64 frozen, 19 formalin-fixed, and 9 B-5-fixed lymph nodes and 26 B-5-fixed bone marrow biopsy specimens. We also analyzed DNA from the 64 frozen lymph nodes by Southern transfer analysis (SB) using three separate bcl-1 breakpoint probes. Gene rearrangements were identified in 17 (PCR) and 18 (SB) of 64 frozen lymph nodes and by PCR in 6 of 19 formalin-fixed lymph nodes, 3 of 9 B-5-fixed lymph nodes, and 12 of 26 B-5-fixed bone marrow cores with MTC locus primers and probe. Only one case showed rearrangement with the p11EH probe that corresponds to breakpoints situated 63 kb telomeric to the MTC locus. No rearrangements were detected by PCR or SB for the breakpoint site corresponding to the p94PS probe, but we identified a polymorphic restriction site with HinD III digest in approximately 25% of the cases. In agreement with other studies, these results confirmed that breakpoints in the MTC region of the bcl-1 locus are tightly clustered and associated with 30 to 40% of mantle cell lymphomas. Other breakpoints in the bcl-1 locus seem to be heterogeneous and cannot be detected by PCR or SB with use of existing probes or primer sequences. The most important finding of our study is optimization of the methodology for the detection of immunoglobulin heavy chain gene rearrangement and MTC region breakpoints by PCR from the DNA isolated from B-5-fixed, paraffin-embedded lymph nodes and bone marrow biopsy specimens. The results obtained from these tissues are comparable to those obtained from frozen or formalin-fixed tissue.

Background: Competitive reverse transcription polymerase chain reaction (RT-PCR) has been used increasingly to quantitate messenger RNA (mRNA) levels; however, conventional competitive RT-PCR methods require four or five reactions per sample of RNA, employing serial dilutions of an internal competitor sequence, making analysis of multiple samples of tedious process. A modified method is described by which multiple samples and multiple RNA transcripts can be analyzed easily by an automated process. Methods and Results: Transforming growth factor beta-1 (TGF-beta-1) mRNA was assayed in total RNA extracted from cultured human skin fibroblasts. A standard solution of total RNA was first prepared by pooling RNA from several cell lines and stored in aliquots. A 270-bp competitor RNA molecule (RNA mimic) was prepared by in vitro transcription and was added to each reaction. PCR was performed with a fluorescent dye (Hex; Applied Biosystems, Foster City, CA)-labeled sense primer to amplify a 161-bp-long c DNA product of target TGF-beta-1 mRNA sequence and the RNA mimic. The PCR products were analyzed with an automated laser-scanned gel electrophoresis system and the area under the curve (AUC) was used for quantitation. The concentration TGF-beta-1 mRNA in standard RNA was determined by conventional competitive RT-PCR. Subsequently, equal amounts of RNA mimic were mixed with four serial dilutions of standard RNA and 0.1 µg of sample total RNA for RT-PCR. A standard curve was generated using the known dilutions of a standard target RNA solution and ratio of AUC for target to that for mimic for each dilution. The unknown sample was then quantitated by interpolation of its area under the curve ratio on the standard curve. This method had inter- and intra-assay coefficients of variation of less than 10%. Conclusions: This modification is highly reproducible for quantitation of mRNA and significantly reduces the number of PCR reactions required for each assay. It can be used to assay several RNA molecules in a given sample by designing RNA mimics and PCR primers to generate PCR products of different lengths so that they can be analyzed by the laser scanning of a single lane of electrophoretic gel.

The Coagulation and Molecular Diagnostic laboratories at the University of Minnesota Medical School (Minneapolis) have collaborated to develop a diagnostic algorithm to identify all factor VLeiden mutation carriers without performing unnecessary and expensive genetic testing. The algorithm uses a coagulation assay for activated protein C resistance (APCR) to determine the need for genetic testing. We report the results of our experience validating this program. We compared the sensitivity, specificity, and positive and negative predictive values of two measures of APCR, the APCR ratio and the normalized ratio. We found that the normalized ratio was the more sensitive but less specific parameter to determine the need for genetic testing. By using the normalized ratio as the standard by which to refer patients to the Molecular Diagnostics Laboratory, all mutation carriers were identified. We found a large overlap in both measures of APCR between symptomatic patients with normal genotype and mutation carriers. Furthermore, we demonstrated that increased factor VIII levels with a normal genotype are associated with apparent APCR. In this article we also review other correlates of apparent APCR.

Bone marrow biopsy is the conventional staging and posttherapy evaluation method for assessing marrow involvement by lymphoma. Polymerase chain reactions (PCR) for antigen receptor rearrangements have the potential to increase the detection of minimal degrees of marrow involvement. The present study is a concurrent morphologic and PCR evaluation of 225 staging or posttherapy marrow biopsies from 127 patients with B-lineage non-Hodgkin's lymphoma. The biopsies were morphologically categorized into four groups: group 1 (positive for lymphoma), 60 biopsies (27%); group 2 (suspicious for lymphoma), 20 biopsies (9%); group 3 (lymphocytic lesions of indeterminate biology), 22 biopsies (10%); and group 4 (negative for lymphoma), 123 biopsies (54%). Molecular studies were performed on concurrently obtained aspirates and used consensus immunoglobulin-heavy-chain (IgH) and IgH/bcl-2 gene PCR primers. A molecular clone was detected in 53 of the 225 aspirates (24%): group 1, 34 aspirates (57%); group 2, five aspirates (25%); group 3, one aspirate (5%); and group 4, 13 aspirates (11%). A PCR-positive aspirate was present in 47% of follicular lymphomas, 58% of diffuse large cell lymphomas, and 72% of the other lymphomas in the group I specimens. Morphology or PCR was positive in 79 of the 225 cases (35%). The molecular detection of clonality in the aspirate DNA from cases with positive morphologic findings was lower than anticipated. The discordance between morphology and PCR results may be related to sample variation between the trephine biopsy and aspirate, a failure to aspirate sufficient lymphoma cells, or insufficient primer homology for amplification. DNA extracted from trephine sections may provide results more concordant with morphology, because PCR detected a clone in 10 of 11 DNA specimens extracted from trephine biopsies with positive morphologic findings and PCR negative aspirates.

T-lineage non-Hodgkin's lymphomas are a diverse group of malignancies for which routine histology and immunophenotyping alone may not be diagnostic. Molecular genetic studies can be used to detect unique T-cell receptor gene rearrangements and establish clonality in these cases. The methods for evaluating T-cell receptor clonality are less well studied and produce more varied results than those for the immunoglobulin genes. Whereas clonality is a hallmark of malignancy, its presence or absence in T-lineage disorders does not always correlate with subsequent biologic behavior. Since the molecular testing of T-lineage lymphoproliferative disorders is increasing, this review summarizes the polymerase chain reaction and Southern blotting methods for T-cell receptor gene rearrangements. Interpretation of the results of these methods should always be done in the context of complete clinical and histologic data.

We examined the effect of two rhesus papillomavirus 1 (RhPV) oncogenes on cytokine-induced signal transduction pathways leading to the possible activation of Ras protein (p21ras) and phosphatidylinositol kinase. p21ras in both the activated (GTP-bound) and inactivated (GDP-bound) states were quantitated. NIH 3T3 cell lines expressing the RhPV 1 E5 gene or epidermal growth factor receptor cDNA had about a sixfold higher ratio of p21ras-bound GTP to p21ras-bound GDP as compared with parental NIH 3T3 cells or a cell line expressing the RhPV 1 E7 gene under normal culture conditions, yet expressed similar levels of p21ras. Quiescent cells had dramatically reduced levels of activated p21ras, except those containing RhPV 1 E7. Levels were restored by stimulation with epidermal growth factor or platelet-derived growth factor. Both epidermal growth factor and platelet-derived growth factor receptor of RhPV 1 E5- and E7-containing cells responded to cytokine stimulation. Endogenous phosphatidylinositol-3'-kinase was up-regulated in NIH 3T3 cells transformed with the E5 genes of RhPV 1 and bovine papillomavirus 1. These results suggest that E5 genes of papillomaviruses play a major role in the regulation of transduction pathways.

T-cell non-Hodgkin's lymphomas are an uncommon occurrence after solid-organ transplantation. We describe a morphologically and immunophenotypically distinct group of T-cell lymphoproliferative disorders that occurred late in the course of six patients with solid-organ transplants. The patients ranged in age from 31 to 56 years (median, 43). Three were male; all were splenectomized. The interval from transplant to the diagnosis of lymphoma ranged from 4 to 26 years (median, 15). Symptoms at presentation were related to sites of involvement. Pulmonary, marrow, and CNS involvement were present in five, four, and one case, respectively. No patient had lymphadenopathy. Five patients had an elevated lactate dehydrogenase level (range, 226 to 4,880 IU/L; median, 1,220 IU/L). Five of six patients had a leukoerythroblastic reaction. All cases had large-cell histology and frequently contained cytoplasmic granules. Those cases tested expressed CD2, CD3, and CD8 and were negative for B-cell antigens. T-cell receptor beta- and gamma-chain genes were clonally rearranged in three of three and one of three cases, respectively. All T-cell posttransplant lymphoproliferative disorders (T-PTLDs) studied were negative for Epstein-Barr virus (EBV), human T-cell leukemia/lymphoma virus type 1 (HTLV-1), human T-cell leukemia/lymphoma virus type 2 (HTLV-2), and human herpes virus type 8 (HHV-8) genomes. Treatment with acyclovir (three patients) or chemotherapy (three patients) resulted in two responses. All patients had an aggressive course, with a median survival duration of 5 weeks. In conclusion, a clinically aggressive T-PTLD may be a late complication of solid-organ transplantation and does not appear to be related to EBV, HTLV-1, HTLV-2, or HHV-8 infection.

Routine clinical molecular testing of diseases associated with unstable or dynamic trinucleotide repeat syndromes poses unique technical, medical, and ethical challenges to the laboratory. Although the pathophysiology of these disorders is to date still largely undefined, the uniformity of their genetics has led to the development of highly informative diagnostic tests. In general, amplification techniques, such as the polymerase chain reaction (PCR), are used to determine the size of alleles within the genes linked to these disorders. Technically, these assays require empirical optimization so that the PCR reactions are both robust and reproducible, and occasionally other methods must be used to confirm diagnoses. Beyond execution of the test, however, the molecular diagnostics laboratory needs also to be fundamentally involved in the process of interpreting these tests in the correct clinical context and in setting policy as to how these data are presented to patients.

Although sebaceous glands are prominent on the vulva, sebaceous carcinomas of the vulva rarely occur. In fact, there have been only two cases of sebaceous carcinomas of the vulva reported in the literature. Eighty percent of vulvar cancers are squamous in origin with human papillomavirus (HPV) DNA detected in approximately 60% of these cancers. We present a third patient with sebaceous carcinoma of the vulva and the first to our knowledge that has been analyzed for HPV DNA. The case report and a review of the literature are presented.

We have previously demonstrated the presence of rhesus monkey papillomavirus type 1 (RhPV-1), from molecular and pathological evidence, in a mating group within a single institution. We have now also obtained a number of fresh or archival tissues of rhesus monkeys from other geographically distinct institutions. Using PCR amplification, we observed two animals from one of these institutions and five animals from another which demonstrated RhPV-1 DNA sequences. In addition we molecularly cloned the E7, E2, E4, L2 and L1 genes of RhPV-1 into bacterial expression vectors. The fusion gene products were used to test for serological response to RhPV-1 antigens by Western blot analysis. Responses were observed in up to 52% of the animals tested. While some serologically positive animals were also RhPV-1 DNA-positive, most were not.

The polymerase chain reaction (PCR) clonality assay based on the principle of random X chromosome methylation in females provides a potentially important tool in both cancer research and diagnostics. This assay, however, has not been compared to the standard Southern blot assay and is limited by the rate of heterozygosity of the X-linked phosphoglycerate kinase (PGK) and androgen receptor genes, the only two genes yet described with which this technique may be used. Using 46 marrow and blood specimens from females with and without hematological malignancies, the PCR and Southern blot methods of clonality were compared. In addition, a new technique based on the highly polymorphic fragile X (FMR1) locus was examined. The rate of heterozygosity was 25% for the PGK gene and 45% for the FMR1 gene. In the PCR assay, 7 of 8 and 11 of 14 normal control specimens showed a polyclonal methylation pattern in the PGK and FMR1 genes, respectively. Of the malignant specimens, 17 of 17 and 17 of 18 showed a monoclonal methylation pattern in the PGK and FMR1 genes, respectively. The Southern blot and PCR assay gave similar results with regards to the PGK gene. It is concluded that the PCR and Southern blot clonality assays are comparable with regards to the PGK gene and that both the PGK and FMR1 genes may be reliably used in the determination of clonality. The methods, however, are limited by the skewed methylation patterns seen in hematological specimens in a significant number of normal females.

The transforming potential of the E6 open reading frame (ORF) of the human papillomavirus type 16 was investigated with transformation assays in cotransfections with an activated ras gene. The E6 ORF driven by the heterologous CMV promoter could fully transform baby rat kidney cells (BRK) in cooperation with ras. The transformed cells grew in soft agar and induced tumors in athymic nude mice. The E6 ORF with mutations at the splicing donor site, which only encodes the full length E6 but not E6*s, could also fully transform the BRK cells at a similar efficiency as the wild-type E6 ORF, indicating that the full-length E6 was sufficient for the transformed phenotype.

Quantitative analysis of DNA products derived from polymerase chain reaction (PCR)-based assays depends on the careful optimization of each of the reaction parameters to achieve highly efficient amplification of target sequences. In practice, however, measurement of the accumulated PCR product is reliable only when analyses are performed at points in the exponential phase of the PCR amplification curve and before the onset of the plateau phase. The recent development of more sensitive DNA product detection systems has permitted the analysis of PCR assays after fewer amplification cycles, where the accumulation of product approaches linearity, while at the same time maintaining superior assay specificity. These methods include the use of high performance liquid chromatography, automated fluorescence detection, electrochemiluminescence, and the ligase chain reaction. Clinical applications of these methods are numerous and include diagnostic testing as well as therapeutic monitoring for neoplastic, infectious, and inherited genetic disease.

CTC-96, a cobalt containing complex, was tested as a putative topical therapeutic agent for the treatment of papillomavirus-induced tumors in our cottontail rabbit papillomavirus (CRPV)-rabbit model system. Following experimental infection of domestic rabbits with CRPV, CTC-96 was applied to infection sites twice daily, 5 days a week for a total of 8 weeks. Two levels of concentrations of aqueous CTC-96 were compared to placebo control-treated animals. With increasing dose of CTC-96 we observed tumors earlier, larger, and more often across eight infected sites on each animal.

The antiviral drug ribavirin was used as an adjunct to laser surgery for the treatment of patients with laryngeal papillomatosis (LP). An uncontrolled clinical trial for four patients with ribavirin treatment at a daily dose of 23 mg/kg was performed. Three adults received drug prior to laser surgery and continuing orally for 6 months. One infant was treated for 3 months. Two adults achieved complete remissions for at least 2 consecutive months, and both patients developed only minimal recurrent disease in 4 months of follow-up. The other adult and the child sustained a partial response and an increased interval between the required surgeries. Ribavirin caused only a mild, reversible reduction in hemoglobin and reticulocytosis. This preliminary trial shows that ribavirin may be an effective therapy in combination with surgery for LP in a larger controlled clinical trial.

Rhesus papillomavirus (RhPV) type 1 was recently shown to cooperate with the activated ras oncogene to transform primary rodent epithelial cells at a level comparable to HPV 16. In similar cotransfection studies, subgenomic portions of RhPV 1 driven by either their natural or a strong heterologous promoter were used in primary baby rat kidney cells to demonstrate that transforming properties of RhPV 1 could be localized individually to the E5, E6, and E7 open reading frames. Fully transformed cells were observed when either E5 or E7 were downstream of a strong heterologous promoter. Similarly, either E6 or E6 and E7 downstream of the native promoter fully transformed these cells as determined by immortalization, anchorage independent growth and tumorigenicity studies.

Infection with human papillomavirus type 11 (HPV 11) is associated with benign epithelial proliferations and rarely with malignant and metastasizing tumors. Because of the biological diversity displayed in tissues infected with HPV 11, we have examined the capacity of various isolates of HPV 11 to transform cultured cells and compared their molecular differences by DNA sequence analysis. Five isolates of HPV 11 were examined for their ability to transform primary neonatal rat kidney epithelial cells and NIH 3T3 mouse fibroblasts in DNA transfection experiments using calcium phosphate precipitation. Included in these studies are the prototype isolate from a laryngeal papilloma (HPV 11P); HPV 11VC from a verrucous carcinoma of the penis; HPV 11Epi from the viral episomes of a primary squamous cell carcinoma; and two integrated genomes (HPV 11Int 1 and HPV 11Int 2) of the metastases. Only HPV 11VC cotransfected with the oncogene Ha-ras transformed neonatal rat kidney epithelial cells with an efficiency comparable to that of HPV 16 DNA. HPV 11VC DNA alone transformed NIH 3T3 cells. Analysis of the DNA sequence of HPV 11P and 11VC revealed 16 single nucleotide changes in the upstream regulatory region and open reading frames E1, E2, E4, and E5, five resulting in amino acid substitutions. This is the first demonstration of cellular transformation by a natural isolate HPV 11 DNA in vitro and illustrates that minimal changes in the DNA sequence of certain viruses confer oncogenicity to what are normally nontransforming viruses.

The challenge to develop antiviral agents effective against DNA viruses such as human papillomavirus (HPV) has been dependent on finding an animal model which mimics the human forms of the disease. We have used an existing model system for the purpose of measuring the effect of antiviral drugs on the inhibition of growth of these lesions. This was based upon domestic rabbits which efficiently grow cutaneous papillomas (warts) when infected with cottontail rabbit papillomavirus (CRPV). One agent which had shown significant success in achieving these goals was ribavirin. Ribavirin was administered intradermally shortly prior to infection at multiple sites with CRPV. Following daily injections of this drug for eight weeks, we have shown a dose-dependent response which had markedly reduced the number of warts, the time of first appearance of warts and reduced the tumor mass as compared to placebo-treated control animals. At the highest dose of ribavirin tested, 30 mg/kg/day, compared to controls, the average reduction in the number of warts was 52%, the average time of first appearance of warts was 49% longer, and the average mass of the warts was reduced by 98%. No detectable antibodies to CRPV were observed in any of the animals. The only side effects which were observed was focal alopecia, and a decrease in body growth upon prolonged treatment, both of which were completely reversible. Pharmacokinetic studies established the metabolism of ribavirin over a 24-h period of time. Ribavirin administered beginning 12 or 30 days post-infection, while not reducing the number of warts, slightly retarded the growth of warts as determined by date of first appearance of warts and mass of warts.

Human papillomavirus infection of the uterine cervix is associated with a spectrum of benign, premalignant, and malignant epithelial lesions, a process that appears to require the coordinated effects of secondary cellular and environmental events. We have used flow cytometry and immunohistochemistry to examine the expression of the cellular markers for proliferation (interleukin-1, epidermal growth factor receptor, and transferrin receptor) and the markers of cellular differentiation (filaggrin and low-molecular-weight cytokeratin) in normal and human papillomavirus--infected human cervical tissues representing the natural range of human papillomavirus--induced disease. The results were correlated with the histologic grade of disease, human papillomavirus type, cellular deoxyribonucleic acid content, and cell cycle status. Interleukin-1 and transferrin receptor were slightly increased in high-grade dysplasias and in squamous cell carcinomas. Filaggrin expression was found to be inversely related and cytokeratin and epidermal growth factor receptor expression directly related to the degree of neoplasia. These findings indicate that cytokeratin and epidermal growth factor receptor are useful markers of cell proliferation in human papillomavirus--infected tissues and that their expression may directly increase as a result of infection.

Rhesus Papillomavirus type 1 (RhPV-1) was recently cloned from a rhesus monkey lymph node metastasis of a penile squamous cell carcinoma. In this paper, we demonstrate that RhPV-1 cooperates with the activated ras oncogene to transform primary cells at a level comparable to human papillomavirus type 16. The viral DNAs were cloned such that their expression was under the control of their natural promoter elements. Unlike human papillomavirus type 16, RhPV-1 DNA cooperated with ras independently of the hormone dexamethasone. However, dexamethasone did have a positive influence on the ability of some RhPV-1 cotransformed cells to grow in soft-agar assays. The transformed cells are highly tumorigenic in vivo in nude mice.

Recently we molecularly cloned and characterized a papillomavirus from a lymph node metastasis of a primary penile carcinoma found in a rhesus monkey; this virus species, rhesus papillomavirus type 1 (RhPV-1), is similar to oncogenic human papillomaviruses (HPVs), such as HPV-16 or HPV-18, in that the RhPV-1 DNA was found to be integrated in the tumor cell DNA. To compare the sexual transmission and oncogenic nature of RhPV-1 with these HPVs, we undertook an extensive retrospective study of a group of rhesus monkeys whose sexual mating and offspring histories were known. These animals had mated directly with the index male mentioned above or were secondarily exposed to this virus through intermediate sexual partners. This study combines cytological, histopathological, and several complementary hybridization and DNA amplification techniques on multiple tissue samples to demonstrate the sexually transmitted nature of RhPV-1. The oncogenic potential of RhPV-1 is suggested in several of the infected animals by the presence of various degrees of neoplasia including squamous cell cancer of the cervix.

Pulmonary artery sarcomas are rare tumors that arise in the region of the bulbus cordis, the embryologic structure that gives rise to the pulmonary trunk. Nearly 100 cases have been reported in the literature, yet considerable debate exists regarding the histogenesis and biologic properties of these neoplasms. We report four additional cases in which ultrastructural and immunohistochemical studies demonstrated that these tumors contain cellular constituents with features of myofibroblastic, cartilaginous, and osteogenic differentiation. Polyphenotypic expression of several mesenchymal lineages suggests that the progenitor cell has pluripotential properties. Our findings and a review of the literature appear to confirm that pulmonary artery sarcomas are histopathologically heterogeneous, possibly reflecting the indeterminate character of the mesenchymal cell(s) of origin. The poor prognosis is attributable to the critical anatomic location of the neoplasm rather than its metastatic potential, which is low.