Diltiazem inhibits transferrin receptor expression and causes G1 arrest in normal and neoplastic T cells.

Transferrin receptor expression is essential for the proliferation of both normal and malignant T cells. While transferrin receptor expression in normal T cells is tightly coupled to interleukin-2 receptor expression, transferrin receptor expression in malignant cells is usually constitutive and is released from this constraint. Temporally, the appearance of these membrane receptors is preceded by changes in the expression of the proto-oncogenes c-myc and c-myb. In addition, although an increase in the level of intracellular free calcium occurs early in the sequence of T-cell activation, the activation events dependent on this calcium flux have not been resolved. In the present study we report that diltiazem, an ion channel-blocking agent that inhibits calcium influx, arrested the growth in vitro of both normal and malignant human T cells in the G1 phase of the cell cycle. However, diltiazem did not inhibit the expression of c-myc or interleukin-2 receptor mRNA and protein in normal mitogen-activated T cells or the constitutive expression of c-myc and c-myb mRNA in malignant T cells (T acute lymphoblastic leukemia cells). In contrast, diltiazem prevented the induction of transferrin receptor (mRNA and protein) in normal T cells and caused a progressive loss of transferrin receptor (mRNA and protein) in malignant T cells. These data demonstrate that diltiazem can dissociate several growth-related processes normally occurring in G1 and thereby disrupt the biochemical cascade leading to cell proliferation.

Clonal determination by the fragile X (FMR1) and phosphoglycerate kinase (PGK) genes in hematological malignancies.

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.

Characterization of integrated human papillomavirus type 11 DNA in primary and metastatic tumors from a renal transplant recipient.

A primary perianal squamous cell carcinoma and two metastatic tumors from a renal transplant recipient with a previous history of condyloma acuminatum were analyzed by filter hybridization for the presence of human papillomavirus (HPV) DNA. Each of the DNA extracts from these three tissues was found to contain HPV DNA. Stringent hybridization and restriction endonuclease analysis identified this viral DNA as HPV 11 related, which largely comigrated with cellular DNA, suggesting the presence of integrated viral DNA. Each DNA extract was analyzed by two-dimensional gel electrophoresis, which separates circular and linear forms of DNA and can demonstrate linear viral DNA, which comigrated with high molecular weight linear cellular DNA, thus implying viral integration. In all three cases the vast majority of viral DNA was found to comigrate with linear DNA; in addition, a significant portion comigrated with high molecular weight cellular DNA, suggesting the presence of integrated viral DNA in these tumors. Restriction endonuclease analysis of high molecular weight cellular DNA from each of these tumors revealed identical banding patterns, indicating that the integration site in each tissue is identical and, therefore, that all three tumors most likely originated from a single clonal event. These molecular results are presented in light of the clinical history of this patient with a histologically "low grade," but biologically aggressive, squamous cell carcinoma and suggest that HPV 11 may be associated with the initiation of malignant epithelial neoplasms.

Cellular transformation by a unique isolate of human papillomavirus type 11.

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.

Hereditary spastic paraplegia and hereditary ataxia, Part 2: A family demonstrating various phenotypic manifestations with the SCA3 genotype.

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.

Analysis of a very large trinucleotide repeat in a patient with juvenile Huntington's disease.

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.

Correlation of PCR-detected clonal gene rearrangements with bone marrow morphology in patients with B-lineage lymphomas.

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.

B-cell gene rearrangement in benign and malignant lymphoid proliferations of mucosa-associated lymphoid tissue and lymph nodes.

The polymerase chain reaction (PCR) with polyacrylamide gel electrophoresis was used to study patterns of immunoglobulin heavy chain (IgH) gene rearrangement (GR) in formalin-fixed, paraffin-embedded specimens of lymphomas and reactive conditions of mucosa-associated lymphoid tissue (MALT) and lymph node. DNA amplification was performed directly on sections obtained from paraffin blocks. Five patterns of PCR products were observed: a single band, two or more discrete bands, smearing, a single band overlying a smear, and two or more bands over a smear. A pure polyclonal pattern (smear) was observed in all of the reactive lymph nodes but in only 15% of cases of Helicobacter pylori (HP) gastritis with lymphoid hyperplasia, 25% of cases of HP gastritis without lymphoid hyperplasia, and 37% of colonic specimens of various types. Patterns consisting of multiple bands with or without background smearing were common in gastritis, colitis, and gastric lymphomas. Single bands or dominant bands were present in all lymph node and salivary gland lymphomas, 12 of 14 cases of gastric lymphoma, and 17 of 20 cases of HP gastritis with lymphoid hyperplasia. These bands were reproducible in deeper sections from the same paraffin block or similar areas sampled in different blocks in all of the lymph node and salivary gland lymphomas, 11 of 12 gastric lymphomas, but only 1 of 17 cases of HP gastritis with lymphoid hyperplasia. Bands were also found in 3 of 20 cases of HP gastritis without lymphoid hyperplasia and 17 of 38 colonic specimens, but these were not reproducible. The complexity of patterns of IgH GR in acquired MALT compared with lymph nodes may be the result of a relative paucity of B-cell clones or preferential proliferation of B-cell clones with a limited area of distribution.

Quantitative analysis in molecular diagnostics.

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.

Topical CTC-96 accelerates wart growth in rabbits infected with cottontail rabbit papillomavirus.

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.

Ribavirin mitigates wart growth in rabbits at early stages of infection with cottontail rabbit papillomavirus.

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.

Graft-versus-leukemia is sufficient to induce remission in juvenile myelomonocytic leukemia.

It has been unclear whether a graft-versus-leukemia (GVL) effect assists in the control of juvenile myelomonocytic leukemia (JMML) following allogeneic bone marrow transplant. We describe a patient with JMML who relapsed early after an unrelated donor transplant, and following withdrawal of immunosuppression developed graft-versus-host disease (GVHD). Associated with GVHD the proportion of donor cells measured by variable nucleotide tandem repeat (VNTR) analysis increased, and peripheral blasts and cutaneous disease were eliminated. These findings strongly suggest that GVL has a role in the control of JMML.

Non-leukemic autologous reconstitution after allogeneic bone marrow transplantation for Ph-positive chronic myelogenous leukemia: extended remission preceding eventual relapse.

Autologous reconstitution is the recovery of autologous hematopoietic function after failure of an allogeneic graft to establish sustained hematopoiesis either with or without preceding donor engraftment. We reviewed 9 years experience of the University of Minnesota and identified 10 of 291 patients who underwent allogeneic BMT for Ph-positive CML and developed non-leukemic autologous reconstitution. All patients received the same preparative regimen with cyclophosphamide and total body irradiation. Eight patients had a 6/6-antigen matched donor. Eight patients received their graft from an unrelated donor. In five cases the graft was T cell-depleted. Non-malignant autologous reconstitution initially manifested as mixed chimerism in nine of 10 patients and lasted for a median of 11 (3-41) months. Eight patients have relapsed and four are still alive. The two relapse-free patients have died 24 and 48 months post transplant. Of the four surviving patients, two are in interferon-induced cytogenetic remission at 53+ and 101+ months of follow-up. Autologous non-leukemic reconstitution is uncommon, but appears to be a distinct clinical syndrome, perhaps occurring more frequently after unrelated donor BMT. Although usually followed by relapse, relapse-free survival may be prolonged.

Leukemia in donor cells after allogeneic hematopoietic stem cell transplant.

The development of leukemia in donor cells after allogeneic hematopoietic stem cell transplant is an extremely rare event. We report here the case of a patient who developed myelodysplastic syndrome/acute myeloid leukemia, in cells of donor origin 3.5 years after related donor HSCT for refractory chronic lymphocytic leukemia and therapy-induced myelodysplastic syndrome. The origin of the leukemia was determined by analysis of minisatillite polymorphism tested on CD34(+) cells.

Detection of the factor VLeiden mutation. Development of a testing algorithm combining a coagulation assay and molecular diagnosis.

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.

Evaluation of an automated technique for assessment of marrow engraftment after allogeneic bone marrow transplantation using a commercially available kit.

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.

Simultaneous allele-specific amplification: a strategy using modified primer-template mismatches for SNP detection--application to prothrombin 20210A (factor II) and factor V Leiden (1691A) gene mutations.

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.

Human papillomavirus oncogenesis.

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.

Molecular diagnosis of cystic fibrosis.

The clinical and pathologic features of patients with cystic fibrosis are summarized, and generalized genotype or phenotype correlations are discussed in this article. Incorporation of modern molecular biologic techniques into a rapid, cost-efficient, and specific diagnostic laboratory test is outlined. The protocol for the multiplex polymerase chain reaction detection of five common cystic fibrosis transmembrane conductance regulator (CFTR) mutations by ASO hybridization is detailed. Neonatal screening and issues involved in the genetic counseling of families at risk for cystic fibrosis are presented. Recommendations for molecular diagnostic testing in cystic fibrosis are made.

Methods to detect clonal gene rearrangements in lymphomas and leukemias.

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.