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.

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.

Monitoring of bone marrow transplant engraftment.

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.

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.

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.

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.

Are benign cellular changes on a Papanicolaou smear really benign? A prospective cohort study.

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.

bcl-1 gene rearrangements in mantle cell lymphoma: a comprehensive analysis of 118 cases, including B-5-fixed tissue, by polymerase chain reaction and Southern transfer analysis.

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.

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.

Posttransplant T-cell lymphoproliferative disorders--an aggressive, late complication of solid-organ transplantation.

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.

The E5 gene product of rhesus papillomavirus is an activator of endogenous Ras and phosphatidylinositol-3'-kinase in NIH 3T3 cells.

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.

Sebaceous carcinoma of the vulva: a case report and review of the literature.

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.

Serological and molecular evidence of rhesus papillomavirus type 1 infections in tissues from geographically distinct institutions.

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.

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.

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.

The E6 gene of human papillomavirus type 16 is sufficient for transformation of baby rat kidney cells in cotransfection with activated Ha-ras.

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.

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.

Pilot trial of ribavirin for the treatment of laryngeal papillomatosis.

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.

The products of the E5, E6, or E7 open reading frames of RhPV 1 can individually transform NIH 3T3 cells or in cotransfections with activated ras can transform primary rodent epithelial cells.

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.

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.