Naive T-cells in myelodysplastic syndrome display intrinsic human telomerase reverse transcriptase (hTERT) deficiency.

Telomeres are specialized structures providing chromosome integrity during cellular division along with protection against premature senescence and apoptosis. Accelerated telomere attrition in patients with myelodysplastic syndrome (MDS) occurs by an undefined mechanism. Although the MDS clone originates within the myeloid compartment, T-lymphocytes display repertoire contraction and loss of naive T-cells. The replicative lifespan of T-cells is stringently regulated by telomerase activity. In MDS cases, we show that purified CD3+ T-cells have significantly shorter telomere length and reduced proliferative capacity upon stimulation compared with controls. To understand the mechanism, telomerase enzymatic activity and telomerase reverse transcriptase (hTERT), gene expression were compared in MDS cases (n=35) and healthy controls (n=42) within different T-cell compartments. Telomerase activity is greatest in naive T-cells illustrating the importance of telomere repair in homeostatic repertoire regulation. Compared with healthy controls, MDS cases had lower telomerase induction (P<0.0001) that correlated with significantly lower hTERT mRNA (P<0.0001), independent of age and disease stratification. hTERT mRNA deficiency affected naive but not memory T-cells, and telomere erosion in MDS occurred without evidence of an hTERT-promoter mutation, copy number variation or deletion. Telomerase insufficiency may undermine homeostatic control within the hematopoietic compartment and promote a change in the T-cell repertoire in MDS.

Interferon-alpha induces dendritic cell differentiation of CML mononuclear cells in vitro and in vivo.

The ability of interferon-alpha (IFN-alpha) to induce dendritic cell (DC) differentiation in chronic myeloid leukemia (CML) was evaluated. Peripheral blood mononuclear cells from CML patients cultured with IFN-alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF) developed a dendritic morphology. Fluorescence in situ hybridization demonstrated that the DCs harbored the bcr/abl translocation. The DCs prepared with IFN-alpha/GM-CSF expressed significantly higher levels of class I and II HLA than those grown in interleukin-4 (IL-4) and GM-CSF. The DCs prepared from newly diagnosed CML patients using IFN-alpha/GM-CSF expressed immunoregulatory proteins at levels comparable to normal DCs. In contrast, DCs cultured from CML patients who did not achieve a cytogenetic response to IFN-alpha expressed significantly lower levels of class I HLA, CD40, CD54, CD80 and CD86 than normal DCs. The expression of CD86 by CML DCs was enhanced when they were cultured with IFN-alpha/IL-4/GM-CSF, or when IFN-alpha/GM-CSF-treated cells were induced to mature by CD40 ligand. The DCs from IFN-alpha failures were less stimulatory than normal DCs in the allogeneic mixed leukocyte reaction. CML patients who had a cytogenetic response to IFN-alpha initially had low numbers of bone marrow DCs that increased significantly with treatment, while nonresponders had more prevalent DCs at baseline that showed no consistent change with treatment. Therefore, IFN-alpha can induce DC differentiation from CML progenitor cells both in vitro and in vivo. The therapeutic activity of IFN-alpha in CML may be due to its ability to stimulate the generation of DCs that can present CML-specific antigens. Resistance to IFN-alpha may result when DC differentiation becomes impaired.

Ex vivo expanded unselected peripheral blood: progenitor cells reduce posttransplantation neutropenia, thrombocytopenia, and anemia in patients with breast cancer.

The safety and efficacy of administering ex vivo expanded peripheral blood progenitor cells (PBPC) to patients with breast cancer who undergo high-dose chemotherapy and PBPC transplantation was investigated. Unselected PBPC were cultured in gas-permeable bags containing 1-L serum-free media, granulocyte colony-stimulating factor, stem cell factor, and pegylated megakaryocyte growth and development factor for 9 days. Cell dose cohorts were assigned to have between 2 and 24 x 10(9) PBPC cultured at 1, 2, or 3 x 10(6) cells/mL. Twenty-four patients received high-dose chemotherapy followed by infusion of the cultured PBPC and at least 5 x 10(6) CD34(+) uncultured cryopreserved PBPC per kilogram. No toxicities resulted from infusions of the ex vivo expanded PBPC. The study patients had shorter times to neutrophil (P =.0001) and platelet (P =.01) recovery and fewer red cell transfusions (P =.02) than 48 historical controls who received the same conditioning regimen and posttransplantation care and at least 5 x 10(6) CD34(+) PBPC per kilogram. Improvements in all these endpoints were significantly correlated with the expanded cell dose. Nine of 24 (38%) patients recovered neutrophil counts above 500/microL by day 5 or 6 after transplantation, whereas none of the controls had neutrophil recovery before the eighth day. Seven (29%) patients had neutropenia for 3 or fewer days, and 9 (38%) patients did not experience neutropenic fevers or require broad-spectrum antibiotics. Therefore, ex vivo expanded PBPC are capable of ameliorating posttransplantation neutropenia, thrombocytopenia, and anemia in patients receiving high-dose chemotherapy.

Interferon-alpha and granulocyte-macrophage colony-stimulating factor differentiate peripheral blood monocytes into potent antigen-presenting cells.

The diverse roles of interferon-alpha (IFN-alpha) in regulating the immune response to infectious agents suggested that it might affect dendritic cell (DC) development. Peripheral blood mononuclear cells cultured with IFN-alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF) developed a dendritic morphology and expressed high levels of the class I and II human leukocyte antigens (HLA), B7 co-stimulatory molecules, adhesion proteins, and CD40. Elevated DC expression of B7-2 and HLA-DR was observed with increasing IFN-alpha concentrations up to 5000 U/mL. The effects of IFN-alpha on DC immunophenotype were not reversed by adding neutralizing antibodies against interleukin-4 (IL-4) or tumor necrosis factor alpha to the cell cultures or by eliminating lymphocytes from the cultures. The addition of IFN-alpha to cultures containing optimal concentrations of IL-4 and GM-CSF significantly increased the B7-2 and HLA-DR levels above those present on DCs grown in two cytokines. The DCs generated with IFN-alpha and GM-CSF were potent antigen-presenting cells in allogeneic mixed leukocyte reactions. They also were capable of taking up, processing, and presenting tetanus toxin to autologous T lymphocytes. These results demonstrate an important role for IFN-alpha in the generation of DCs with potent antigen-presenting capabilities from peripheral blood monocytes.

Thrombotic microangiopathy following allogeneic bone marrow transplantation is associated with intensive graft-versus-host disease prophylaxis.

Thrombotic microangiopathy (TM), manifesting clinically as thrombotic thrombocytopenic purpura or hemolytic uremic syndrome, is an uncommon complication after bone marrow transplantation (BMT). A retrospective analysis of potential risk factors for TM following allogeneic BMT was performed. Clinical data were analyzed from seven patients diagnosed with severe TM and 409 patients who underwent BMT during the same time period and who survived for at least 100 days afterwards. Six of the seven patients with TM received intensive GVHD prophylaxis consisting of cyclosporine, methotrexate and glucocorticoids, whereas only 66 of the 409 patients without TM received this regimen (P < 0.001, Fisher's exact test). This regimen was administered to patients older than 40 years, or recipients of a mismatched or unrelated allograft. Univariate analysis also revealed an increased risk of TM associated with the use of an unrelated bone marrow donor (P = 0.02), but no significant association with patient age or gender, diagnosis, amount of prior chemotherapy, transplant conditioning regimen or severity of GVHD. A multivariate exact logistic regression analysis revealed that only the type of GVHD prophylaxis had a significant impact on the risk for TM. The combined use of cyclosporine, methotrexate and glucocorticoids as GVHD prophylaxis may predispose to the development of TM following BMT.

Hepatitis C virus infection in acquired aplastic anemia.

Hepatitis-associated aplastic anemia (HAAA) is an uncommon disorder that usually is not due to hepatitis A or B virus infection. Hepatitis C virus (HCV) seropositivity is infrequently observed in aplastic anemia (AA) patients who have not been extensively transfused. However, HCV seropositivity may not be detected until several weeks or months after viral infection and AA patients may exhibit defective humoral immunity. Therefore, we evaluated sera from AA patients for the presence of HCV viremia using a reverse transcriptase polymerase chain reaction (RT-PCR) based assay and several serologic assays for HCV antibodies. Serum samples from 90 AA patients who presented to the UCLA Medical Center between March 1984 and February 1990 were analyzed. Overall, 17 patients were found to have HCV viremia by RT-PCR assay, of whom 14 had a positive second-generation HCV enzyme immunoassay (EIA-2) and only 6 were EIA-1 reactive. The frequency of HCV viremia increased with the duration of time between diagnosis and sample procurement, and the number of blood products transfused prior to sampling (P = 0.026). No patient who received fewer than 20 U of blood products or who was sampled less than 20 days after diagnosis had a positive HCV RT-PCR result. Of four patients with hepatitis-associated AA (HAAA), one who was sampled 23 days after diagnosis had hepatitis C viremia and a reactive EIA-2 assay. Therefore, the high frequency of HCV viremia in this patient population is most likely due to transfusion with contaminated blood products prior to the introduction of routine blood donor screening for HCV.

Ex vivo expansion and differentiation of unselected peripheral blood progenitor cells in serum-free media.

The ability to expand and differentiate unselected PBPC was investigated. Cells were grown in serum-free media containing stem cell factor, GCSF and megakaryocyte growth and development factor (pegylated PEG-rHuMGDF) with or without supplemental serum. Optimal proliferation occurred when PBPC were cultured without prior Ficoll-Paque separation in serum-free media. Cell yields after 17 days of culture were proportional to the percentage of CD34+ cells in the starting population and were 1170+/-302-fold higher than the starting numbers of CD34+ cells. Granulocyte-macrophage colony-forming units increased over 12 days of culture, whereas the numbers of erythroid colony-forming cells peaked between 4 and 7 days. Elimination of PEG-rHuMGDF from cell cultures resulted in significantly lower yields of myeloid and erythroid colony-forming cells and total cell numbers. Cell differentiation into neutrophils was indicated by progressive increases in CD11b, CD15, and CD66b expression. Expanded neutrophils phagocytosed and killed bacteria as efficiently as neutrophils from normal donors. Large-scale expansion studies yielded similar proliferation and differentiation results as parallel small-scale cultures. Therefore, unselected PBPC can be efficiently expanded and differentiated into large numbers of functional mature neutrophils.

Clinical characteristics predict response to antithymocyte globulin in paroxysmal nocturnal haemoglobinuria.

Seven patients with paroxysmal nocturnal haemoglobinuria (PNH) were treated with antithymocyte globulin (ATG). Each patient received ATG (20 mg/kg/d) for 8 d and prednisone to prevent or control serum sickness. Three patients experienced a sustained improvement in at least one peripheral blood cytopenia, including one patient who had a complete trilineage response. Several pretreatment clinical features appeared to be associated with response to ATG. All responding patients had hypoproliferative features including depressed platelet counts (< 30 x 10(9)/l), and a minor degree of chronic haemolysis as indicated by relatively low reticulocyte counts (< 100 x 10(9)/l), lactate dehydrogenase (< 1000 U/l) and total bilirubin (< 17 mumol/l) levels. Responding patients continued to have chronic low-grade haemolysis after their response to immunosuppression that was similar to that observed prior to treatment. The non-responding patients had a classic haemolytic form of PNH characterized by elevated reticulocyte counts (> 100 x 10(9)/l), lactate dehydrogenase (> 2000 U/l) and total bilirubin (> 17 mumol/l) levels. The impaired haemopoiesis that occurs in hypoproliferative PNH may respond to ATG treatment, but the haemolytic component of the disease, and hence the PNH clone, is not altered by immunosuppressive therapy.

Analysis of c-kit gene integrity in aplastic anemia.

Mice harboring the "white spotting" (W) locus have abnormalities in hematopoiesis due to one of various mutations of the c-kit proto-oncogene, which encodes the stem cell factor (SCF) receptor. The c-kit mutations identified in W mice cause diminished, absent or dominant negative receptor function. This study explores the hypothesis that acquired mutations of c-kit in the hematopoietic stem cell participate in the pathogenesis of aplastic anemia (AA). Genomic DNA was prepared from granulocytes and monocytes of 11 patients with acquired AA and one patient with a non-Fanconi's form of inherited AA. DNA was subjected to polymerase chain reaction (PCR) amplification and single-stranded conformation polymorphism (SSCP) analysis for all 21 exons of the c-kit gene. Two patients were heterozygous for a previously described polymorphism involving exon 17. Two other patient samples had an extra band on SSCP analysis of exon 10. DNA extracted from epithelial cells of one patient revealed the same SSCP pattern as that from the blood cells, suggesting that the alteration was in the germ-line. PCR-SSCP analysis of leukocyte DNA from 12 normal individuals revealed that 2 samples also had an extra band in exon 10. DNA sequencing of PCR-amplified and cloned DNA from the patients and the normal individuals with the aberrant SSCP results showed them all to be heterozygous for an ATG to CTG transition in codon 541, resulting in substitution of methionine by leucine in the transmembrane region of the protein. The same two patients and two controls were heterozygous for a silent change in codon 862 (exon 18). Matching serum samples from 4 of 6 AA patients tested had SCF levels more than two standard deviations above the normal mean value. These results suggest that neither c-kit mutations nor decreased soluble SCF levels are commonly involved in the pathogenesis of acquired AA.

An interferon-gamma activation sequence mediates the transcriptional regulation of the IgG Fc receptor type IC gene by interferon-gamma.

Expression of the IgG Fc receptor type I (Fc gamma RI) on myeloid cells is dramatically increased by treatment with interferon-gamma (IFN-gamma). We observed that Fc gamma RI transcript levels in monoblast-like U937 cells were elevated within 3 hr and peaked 12 hr after exposure to IFN-gamma. Treatment of U937 with IFN-gamma for 9 hr in the presence of cycloheximide led to super-induction of Fc gamma RI expression. Nuclear run-on analysis revealed that the rate of Fc gamma RI transcription was increased by IFN-gamma. Genomic sequence upstream of the Fc gamma RIC gene was cloned and subjected to primer extension analysis, which demonstrated a single transcription initiation site without a TATA box. Transient transfections of CAT reporter gene constructs containing various Fc gamma RIC promoter sequences into U937 cells revealed that a 20-bp region surrounding the transcription start site (-7 to +13) was capable of mediating transcription initiation and that an IFN-gamma responsive element (GIRE) was present within 74 bp upstream of the transcription initiation site. A 17-bp sequence between positions -51 and -35 conferred IFN-gamma responsiveness on a heterologous promoter. Double-stranded GIRE sequence, but not a scrambled sequence, was specifically bound by nuclear proteins from IFN-gamma treated U937 cells. Gel shift experiments further showed that the STAT1 alpha protein bound to the Fc gamma RIC GIRE in response to IFN-gamma treatment of U937 cells. The Fc gamma RIC GIRE is homologous to the IFN-gamma activation sequence (GAS) of the guanylate binding protein and to X box elements of class II MHC genes. Our results demonstrate that transcriptional regulation of the Fc gamma RIC gene by IFN-gamma involves the binding of STAT1 alpha to a 17-bp GAS homology in the proximal promoter.

Irradiation increases manganese superoxide dismutase mRNA levels in human fibroblasts. Possible mechanisms for its accumulation.

Irradiation induces the production of superoxide radicals (O2.-), which play an important causative role in radiation damage. Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme involved in scavenging O2..-. This study examined MnSOD gene regulation by irradiation in WI38 human fibroblasts. Unstimulated fibroblasts constitutively expressed MnSOD activity and mRNA; irradiation markedly increased MnSOD activity and mRNA levels. The increase in MnSOD transcripts by irradiation was both time- and dose-dependent. WI38 fibroblasts constitutively produce low levels of interleukin-1 (IL-1). The induction of MnSOD mRNA by irradiation was partially blocked by anti-IL-1 antibodies, and treatment of cells with IL-1 also increased MnSOD mRNA levels. Inhibition of the cyclo-oxygenase pathway with indomethacin augmented the induction MnSOD mRNA by irradiation and prostaglandin E2 inhibited the accumulation of MnSOD mRNA by irradiation. Transcriptional run-on analysis showed that irradiation increased the rate of MnSOD transcription 2-fold. Stability studies of MnSOD mRNA in these cells showed that the half-life increased from < 1.5 h in unirradiated cells to > 4 h in irradiated cells. These results suggest that induction of the MnSOD gene after irradiation is regulated, at least in part, by IL-1 production and that increased levels of MnSOD transcripts also occur through a pathway of endogenous prostaglandin E2 production. Our data indicate that the increase in MnSOD mRNA observed after irradiation occurs through both transcriptional and post-transcriptional mechanisms.

A phase I trial of recombinant human interleukin-6 in patients with myelodysplastic syndromes and thrombocytopenia.

To evaluate the hematologic effects of recombinant human interleukin-6 (rhIL-6, Escherichia coli, SDZ ILS 969, IL-6), and determine its toxicity profile, we performed a phase I trial of IL-6 in 22 patients with various myelodysplastic syndromes (MDS), platelet counts < 100,000/microL, and < 5% bone marrow (BM) blasts. Patients received one of four doses of IL-6 (1.0, 2.5, 3.75, and 5.0 micrograms/kg/d) as a subcutaneous injection on day 1, followed by a 7-day wash-out period, and then 28 days of IL-6 therapy. Dose-limiting toxicities of fatigue, fever, and elevated alkaline phosphatase were seen at 5.0 micrograms/kg/d; the maximum tolerated dose was 3.75 micrograms/kg/d. All patients experienced at least grade II fever and all had an increase in acute phase proteins. Eight patients (36%) experienced at least a transient improvement in platelet counts; three fulfilled the criteria for response, whereas five others had clinically significant increases that failed to meet response criteria. Various IL-6-related toxicities prevented more than three patients from receiving maintenance therapy. Two of the three patients who received maintenance IL-6 therapy had a persistent increase in platelet counts, during 3 and 12 months of IL-6 therapy, respectively. Laboratory studies indicated that IL-6 increased the frequency of higher ploidy megakaryocytes but did not significantly increase the number of assayable megakaryocytic progenitor cells, suggesting that IL-6 acts as a maturational agent rather than a megakaryocyte colony-stimulating factor. Although IL-6 therapy can promote thrombopoiesis in some MDS patients, its limited activity and significant therapy-related toxicity preclude its use as a single agent in this patient population. Further studies, combining low doses of IL-6 with other hematopoietic growth factors, are underway.

Long-term outcome of aplastic anemia in adults treated with antithymocyte globulin: comparison with bone marrow transplantation.

The outcome of 155 adult aplastic anemia (AA) patients treated with antithymocyte globulin (ATG, Upjohn, Kalamazoo, MI) at University of California, Los Angeles from 1977 to 1988 was evaluated. The median survival of the 146 patients who did not undergo bone marrow transplantation was 5.6 years, with 49% +/- 4% surviving more than 6 years. The most important predictor of survival was positive response to ATG (P < 0.001), which was observed in 48% of patients. Among pretreatment variables, disease severity was the best predictor of survival. Patients with moderate AA (MAA) had significantly better survival than those with severe (SAA) or very severe (VSAA) disease (P = 0.04). The 6-year actuarial survival rates of the three groups were 71% +/- 9%, 48% +/- 7% and 38% +/- 7%, respectively. Cox regression analysis found disease severity to be the only pretreatment variable significantly associated with survival (P = .02). Patient age, sex, disease etiology, concurrent treatment with androgens, or duration of ATG therapy were not associated with differences in survival or response to ATG. Late clonal hematologic complications (ie, myelodysplasia, acute myelogenous leukemia) were observed in 5 of the 77 patients followed for more than 2 years after ATG treatment. In addition, one case of non-Hodgkin's lymphoma and three solid tumors occurred in the ATG-treated patients. The survival of 56 ATG-treated patients with SAA or VSAA between the ages of 16 and 43 did not differ significantly from that of 55 adult AA patients who underwent bone marrow transplant (BMT) during the same time period (P = 0.6). However, 6-year survival rates improved from 43% for patients transplanted before 1984, to 72% for those who underwent BMT between 1984 and 1989. In contrast, there was no difference in the survival rates of patients treated with ATG during these two time periods (46% v 45%, respectively). The results suggest a superior long-term outcome for adult patients with SAA treated with BMT rather than with ATG alone, using current protocols.

A phase I/II study of interleukin-3 in patients with aplastic anemia and myelodysplasia.

We performed a phase I/II study of recombinant human interleukin-3 (rhIL-3) in 21 patients with aplastic anemia (AA) or myelodysplasia (MDS). Patients received 21-day cycles of IL-3 (0.5, 1.25, 2.5, 5.0, or 10 micrograms/kg/d) by subcutaneous injection followed by a 10- to 14-day washout period. Nineteen patients completed at least one 21-day cycle of IL-3. Frequent toxicities of IL-3 included headache, low-grade fever, and erythema at the injection site; at higher doses, weight gain and peripheral edema was seen. Eleven patients developed eosinophilia. Of the 20 evaluable patients, eight had increases in absolute neutrophil counts (seven with MDS, one with AA) including six of the nine patients receiving > or = 5.0 micrograms/kg/d. One AA patient became transfusion-independent for 8 months, while another AA patient had decreased transfusion requirements. Three patients with MDS had at least a doubling of their platelet count, and another patient experienced a 1.9-fold increase. One patient with RAEB progressed to aleukemic AML by the end of one treatment cycle. IL-3 was well-tolerated, but multilineage effects were seen in only 25% of patients with primary bone marrow failure states (five of 20 evaluable) and more commonly in patients with myelodysplastic syndromes. Its optimal use may be as part of combination hematopoietic growth factor therapy.

Autoimmune myelofibrosis. A steroid-responsive cause of bone marrow fibrosis associated with systemic lupus erythematosus.

Autoimmune myelofibrosis is an uncommon disorder in which patients present with anemia and thrombocytopenia in conjunction with limited clinical manifestations of autoimmune disease or an exacerbation of previously established SLE. The presence of leukoerythroblastosis in a patient with SLE may suggest the presence of myelofibrosis. Conversely, the absence of splenomegaly in a patient with presumed idiopathic myelofibrosis may suggest an autoimmune etiology. Patients with autoimmune myelofibrosis universally have a positive ANA test and frequently have either elevated anti-DNA titers or a positive LE cell preparation. Because physical manifestations of autoimmune disease may not be evident at presentation, all patients found to have myelofibrosis should have an ANA test. Peripheral blood cytopenias in autoimmune myelofibrosis frequently respond to glucocorticoids but regression of bone marrow fibrosis may be incomplete. Hematologic response to treatment parallels that of the associated autoimmune disease.

p53 mutations in HPV-negative cervical carcinoma.

Human papillomavirus (HPV) infection has been strongly linked to the development of cervical carcinoma. Two viral oncoproteins, E6 and E7, produced by HPV, have been shown to immortalize primary human genital epithelial cells by interacting with the protein products of cellular tumor suppressor genes p53 and Rb, respectively. E6 binds to the cellular p53 protein promoting p53 degradation and inactivity. This mechanism has been suggested to contribute to the oncogenesis of HPV-positive anogenital cancers. In HPV-negative cervical carcinoma, p53 mutation is thought to be the possible mechanism of oncogenesis. We have studied 257 cervical carcinoma specimens for HPV infection by Southern blot analysis and polymerase chain reaction (PCR). Of 257 samples, 39 were HPV-negative. We have further studied 21 HPV-negative specimens for p53 mutations utilizing PCR amplification of genomic DNA followed by single-stranded conformation polymorphism (SSCP) analysis and DNA sequencing. We found only two missense point mutations of p53 gene. In summary, although inactivation of p53 mediated either by E6 or by mutations may be an important key step in the development of cervical carcinoma, our study suggests that other mechanisms may also be involved in development of cervical cancer.

Mutations of p53 and human papillomavirus infection in cervical carcinoma.

BACKGROUND: Oncogenic human papillomavirus (HPV) infection has been implicated in the pathogenesis of cervical carcinoma. The HPV oncoproteins E6 and E7 are though to play a crucial role in this process by their interactions with the p53 protein and the retinoblastoma susceptibility gene product, respectively. The E6 protein binds to and stimulates the degradation of the p53 protein. Mutations involving evolutionary conserved regions of the p53 gene also can alter p53 function. Point mutations of p53 frequently have been identified in a wide variety of human tumors. METHODS: Forty-five cervical carcinoma samples were evaluated for the presence of mutations involving exons 5-8 of the p53 gene with polymerase chain reaction (PCR) amplification of genomic DNA, followed by single-stranded conformation polymorphism analysis and/or direct sequencing. The status of oncogenic HPV infection in the tumor tissues was analyzed by Southern blot and PCR. RESULTS: Forty-two of 45 cervical carcinomas showed oncogenic HPV DNA: Of three HPV-negative samples, one harbored a missense point mutation of the p53 gene. An additional p53 point mutations was identified in a tumor with HPV 18 infection. CONCLUSIONS: Oncogenic HPV DNA can be identified in most cervical carcinomas. Mutations involving conserved regions of p53, although infrequent in cervical cancer, occur preferentially in tumors without HPV infection. Inactivation of p53 function is important in the pathogenesis of cervical carcinoma.

N-ras mutations are associated with poor prognosis and increased risk of leukemia in myelodysplastic syndrome.

To evaluate the clinical significance of N-ras mutations in the myelodysplastic syndrome (MDS) archival bone marrow samples from 252 patients were studied for the presence of N-ras exon I mutations using polymerase chain reaction amplification and differential oligonucleotide hybridization. Subsequently, clinical information about these patients was obtained and analyzed. Of 220 evaluable patients, 20 (9%) had point mutation of N-ras involving codon 12. Individuals with N-ras mutation had a significantly shorter survival period than those who were N-ras negative (P = .02). An increased risk of acute myelogenous leukemia (AML) was also found in patients with N-ras mutations (P = .005). N-ras mutations were not associated with any French-American-British (FAB) subtype, with the presence of increased myeloblasts, or with chromosomal aberrations in the bone marrow. However, the presence of increased bone marrow blasts was strongly associated with poor survival rate and risk of AML (P < .001 for each). After stratifying for the percentage of blasts, N-ras mutations remained significantly associated with shorter survival period (P = .04) and increased risk of AML (P = .02). Bone marrow cytogenetic abnormalities, particularly when multiple abnormalities were present, were significantly associated with a poor prognosis (P < .001). In conclusion, N-ras mutation, although relatively infrequent in MDS, is associated with short survival period and increased probability of developing AML.

Differentiation therapy.

A variety of compounds are able to induce myeloid leukemia cells to differentiate into morphologically and functionally mature cells. Vitamin D derivatives, retinoids, interferons, and polar-planar compounds are agents that demonstrate such activity in vitro and have been employed as therapy for the myelodysplastic syndromes.

Oncogenes in multiple myeloma: point mutation of N-ras.

Alterations of ras, c-myc and bcl-1 have been described in hematologic malignancies of lymphoid origin. We investigated the structure of these genes and evaluated the frequency of point mutations involving H-, K- or N-ras in bone marrow samples from patients with multiple myeloma. No abnormalities were detected in the c-myc and bcl-1 genes, but two of 17 patients were found to have N-ras mutations by differential oligonucleotide hybridization and dideoxynucleotide sequencing following amplification by polymerase chain reaction. Bone marrow DNA from both patients had identical missense mutations of N-ras codon 61 changing CAA to AAA, resulting in a substitution of lysine for glutamine in the encoded protein. Multiple myeloma is the first mature B cell neoplasm found to harbor ras mutations.