Fludarabine triphosphate inhibits nucleotide excision repair of cisplatin-induced DNA adducts in vitro.

Fludarabine (9-beta-arabinofuranosyl-2-fluoroadenine-5'-monophosphate) is clinically active against chronic lymphocytic leukemia and low-grade lymphomas. We reported previously that fludarabine nucleoside synergistically enhanced cisplatin (CDDP)-induced cytotoxicity in vitro, and that the synergism was concomitant with inhibition of removal of cellular CDDP-induced DNA interstrand cross-links, which are presumably repaired by homologous recombinational repair. To extend our work, we investigated whether fludarabine inhibits nucleotide excision repair (NER) of CDDP-induced DNA intrastrand adducts. The effect of fludarabine on NER was determined using a cell-free system in which a plasmid containing the DNA adducts served as the substrate for repair enzymes in whole-cell extracts from repair-competent cells. To prevent the cell-bound high mobility group box-containing proteins from interfering with repair, cell extracts were depleted with high mobility group box proteins by immunoprecipitation prior to the assay. Repair synthesis, measured by the incorporation of [(32)P]dATP or [(32)P]dCTP, was inhibited by 50% at 26 or 43 microM fludarabine triphosphate, respectively; the effect was dose dependent and may have resulted from the termination of repair-patch elongation. These results were consistent with those from pulse-chase experiments demonstrating the conversion of nicked circular plasmid to the closed circular form by cell extracts filling the repair gaps. When proliferating cell nuclear antigen-depleted cell extracts were used and aphidicolin was added in the repair assay to arrest NER at the incision/excision stage, 100 microM fludarabine triphosphate inhibited about 55% of the conversion of nicked plasmids from the closed circular damaged plasmid substrate; the inhibition was dose dependent. We conclude that fludarabine triphosphate inhibited NER at the steps of incision and repair synthesis. These results suggest that fludarabine may serve as a potential repair modulator to improve the antitumor efficacies of combination regimens containing agents that induce NER.

Homozygous deletions at 8p22 and 8p21 in prostate cancer implicate these regions as the sites for candidate tumor suppressor genes.

Frequent loss of an allele at specific chromosomal regions implicates these regions as sites of tumor suppressor genes (TSG) that become inactivated during tumor progression. We have studied chromosome 8p allele losses in 32 primary human prostate carcinomas with 16 polymorphic microsatellite sequences. Overall, 22 of 32 (69%) informative specimens showed loss of allele in at least one locus. The most frequent losses of heterozygosity (LOH) occurred at the LPL locus (46%) on chromosome 8p22 and at the D8S360 (45%) and NEFL (43%) loci on chromosome 8p21. Homozygous deletions were detected at the LPL and NEFL loci at 8p22 and 8p21, respectively. The minimal region with frequent LOH and homozygous deletion, around the LPL locus, was restricted between the MSR locus and the D8S258 marker, separated by less than 9 cM. The second region was restricted between markers D8S1128 and D8S131 separated by 12 cM. The results suggest the existence of two chromosome 8p sites for candidate TSGs in prostate cancer.

Early detection of relapse by hypermetaphase fluorescence in situ hybridization after allogeneic bone marrow transplantation for chronic myeloid leukemia.

PURPOSE: Standard G-band cytogenetic analysis (CG) provides information on approximately 25 metaphases for monitoring the presence of Philadelphia chromosome positive (Ph+) cells in chronic myelogenous leukemia (CML) patients, making the detection of a low frequency of Ph+ cells problematic. The purpose of this study was to improve the detection of a low frequency of Ph+ cells. PATIENTS AND METHODS: We combined fluorescence in situ hybridization (FISH) with long-term colcemid exposure, capturing several hundred metaphases in bone marrow cultures (hypermetaphase FISH [HMF]). Using probes that identify Ph+ cells, HMF was compared with CG analysis in the follow-up evaluations of 51 patients with CML at various time points after allogeneic bone marrow transplant (BMT). RESULTS: Thirty-five patients never showed the presence of Ph+ cells by either method. In four patients, high frequencies of Ph+ cells were detected by both methods. In the remaining 12 patients, Ph+ cells were detected by HMF at time points after BMT when they were not detected by CG. In seven of the 12 patients, low but statistically significant frequencies of Ph+ cells (0.37% to 5.20%) were detected 3 months or later after BMT, and when no intervention was initiated, all seven patients later relapsed. Based on those data, an eighth patient with mixed chimerism and a similar HMF-detected Ph+ frequency (1.8% at 27 months after BMT) was reinfused with donor lymphocytes and achieved remission with 0% Ph+ cells studied by HMF (up to 50 months after BMT). Ph+ cells detected by HMF but not by CG less than 3 months after BMT disappeared on later examination in two of four patients. After detection of Ph+ cells by HMF only, the median time to cytogenetic progression (detection of Ph+ cells by CG) was 101 days. CONCLUSION: The results demonstrate the ability of HMF to detect low but clinically relevant levels of leukemic cells not detected by CG in transplant patients. The data indicate that HMF can detect low levels of Ph+ cells before standard cytogenetics at a time that may be useful in monitoring disease status and planning clinical interventions.

Prognostic factors for survival of patients treated systemically for disseminated melanoma.

PURPOSE: The current American Joint Commission on Cancer (AJCC) staging system distinguishes between soft tissue and visceral metastases in advanced (stage IV) melanoma. We sought to verify these staging criteria and to identify prognostic variables that could be used to evaluate the impact of systemic therapy on long-term survival during the prior decade. PATIENTS AND METHODS: We conducted a retrospective study of patients with advanced cutaneous melanoma enrolled in clinical trials between 1979 and 1989 at The University of Texas M.D. Anderson Cancer Center. Pretreatment age, sex, number of organs with metastases, serum levels of lactate dehydrogenase (LDH) and albumin, and period of enrollment were analyzed using a Cox proportional hazards model of survival. RESULTS: In univariate and multivariate analyses that involved 318 stage IV patients, normal serum levels of LDH and albumin, soft tissue and/or single visceral organ metastases (especially lung), female sex, and enrollment late in the decade were independent positive predictors for survival. In multivariate analyses, the current AJCC criteria did not significantly predict outcome. Systemic treatment response did not bias these results, and only 4% of patients had a complete response. Patients who lived more than 2 years (11%) had a mix of favorable prognostic characteristics and a high frequency of systemic or surgically induced complete response. CONCLUSION: This study supports the use of stratification parameters that reflect the favorable prognostic impact of soft tissue or single visceral organ metastases and normal serum levels of LDH and albumin at time of enrollment in advanced melanoma trials. Improved survival over the prior decade probably reflects advances in diagnostic and palliative interventions.

Detection of chromosome 11q13 breakpoints by interphase fluorescence in situ hybridization. A useful ancillary method for the diagnosis of mantle cell lymphoma.

We assessed cytologic specimens from 11 mantle cell lymphomas (MCLs) and 32 other B-cell non-Hodgkin lymphomas (NHLs) for 11q13 breakpoints using a 2-color fluorescence in situ hybridization (FISH) assay that uses an 11q13 probe centered on the CCND1 gene and a centromeric chromosome 11 probe (CEP11). The number of nuclei in 200 cells were counted, and results were expressed as an 11q13/CEP11 ratio. All MCLs showed a high percentage of interphase nuclei with 3 or more 11q13 signals (mean, 74.8%; range 57%-90%). In contrast, in other B-cell NHLs the mean percentage of cells with 3 or more 11q13 signals was 9.2%. All MCLs had an elevated 11q13/CEP11 ratio (mean, 1.38). The mean ratio for other B-cell NHLs was 0.99. Two non-MCL cases, 1 large B-cell and 1 B-cell unclassified NHL, had high 11q13/CEP11 ratios of 1.15 and 1.30, respectively. Conventional cytogenetic analysis performed on the former case revealed a t(5;11)(q31;q13). Interphase FISH analysis using 11q13 and CEP11 probes is a convenient ancillary method for assisting in the diagnosis of MCL. This commercially available assay is simple to use on cytology or imprint specimens, and results can be obtained within 24 hours.

Adoptive transfer of vaccine-induced resistance to Leishmania donovani.

BALB/c mice were immunized with three subcutaneous injections combining killed parasites and glucan, or were untreated. Spleen cells were transferred to syngeneic recipients. Mice which received 5 X 10(8) spleen cells from vaccinated donors demonstrated significant protection against Leishmania donovani challenge as compared to untreated mice receiving immune sera, or mice which received untreated spleen cells.

Leishmania donovani: immunization against infection as a function of parasite growth phase.

C57BL/6 mice were immunized against Leishmania donovani infection with a subcutaneous vaccination protocol. Groups received 3 injections at 4-day intervals combining glucan and killed promastigotes harvested from either logarithmic or stationary phase cultures. Controls were immunized with glucan alone, stationary or log phase promastigotes alone, or were untreated. All groups were challenged intravenously with stationary phase promastigotes at day 45 post-immunization. Results revealed that animals immunized with the glucan-killed parasite vaccine, utilizing promastigotes derived from either log (GPL) or stationary phase cultures (GPS), demonstrated significant resistance against infection as compared to controls or untreated mice. Additionally, the reduction in hepatic amastigote proliferation in mice immunized with GPS was significantly greater than in mice immunized with GPL.

der(1;15)(q10;10): a nonrandom chromosomal abnormality of myeloid neoplasia.

The occurrence of an unusual karyotypic abnormality der(1;15)(q10;q10) is reported in three patients, one with acute megakaryoblastic leukemia and two with myelodysplastic syndrome. A literature review shows that this cytogenetic abnormality is a rare but nonrandom change in myeloid neoplasia/neoplasia.

The relationship between age and karyotypic abnormalities in myelodysplastic syndromes.

We performed a retrospective analysis of 143 consecutive patients with myelodysplastic syndrome to study the possible relationship between patient age and cytogenetic findings in this disorder. There were 96 men and 47 women, with a mean age of 63 years. Eighty-six patients were 63 years old and above, and 57 patients were younger than 63 years of age. The distributions of the five FAB subtypes were comparable in both groups of patients, except for a higher percentage of refractory anemia with ringed sideroblasts in older patients. The incidences of cytogenetic abnormalities were similar in the two groups. However, the younger patients tended to have a higher frequency of involvement of chromosomes 5 or 7 than the elderly patients (p < 0.05). The implications of our findings in relation to the biology of myelodysplastic syndrome are discussed.

Translocation (X;10)(p10;p10): A rare but nonrandom chromosomal abnormality in acute leukemia of myeloid differentiation.

Structural abnormality of chromosome X is uncommonly seen in patients with acute leukemia, and translocation between chromosome X and 10 is an exceedingly rare event. In this report, we describe the occurrence of t(X;10)(p10;p10) in two patients with acute leukemia, one with acute monocytic leukemia and the other with myeloblastic relapse arising from bilineage leukemia. To our knowledge, similar chromosomal abnormality has been reported only twice in the literature.

The biologic function of PML and its role in acute promyelocytic leukemia.

Patients with acute promyelocytic leukemia (APL) are characterized by the presence of a t(15;17) chromosomal translocation. The fusion protein PML-RAR alpha encoded from the breakpoint can form a heterodimer and acts as a dominant negative inhibitor against the normal function of PML. Recently we demonstrated that PML is a growth suppressor and transcription suppressor expressed in all cell lines tested. We also found that PML suppresses the clonogenicity and tumorigenicity of APL-derived NB4 cells, as well as the transformation of rat embryo fibroblasts by cooperative oncogenes and NIH/3T3 by neu. Overexpression of PML in human tumor cell lines induces a remarkable reduction in growth rate in vitro and in vivo. More recently, we have shown that PML is a phosphoprotein associated with the nuclear matrix and that its expression is cell cycle related. PML expression is altered during human oncogenesis, implying that PML may be an anti-oncogene involved not only in APL but also in other oncogenic events. Mutation analysis of the functional domains of PML demonstrated that its ability to form PML nuclear bodies or PODs (PML oncogenic domains) is essential for suppressing growth and transformation. In light of the above studies it appears that disruption of the normal function of PML plays a critical role in the pathogenesis of APL.

Antibodies to A19 and Bw35 complexes of human leukocyte antigens are present in infertile subjects with sperm antibodies.

Sera and secretions from 100 couples with unexplained infertility were tested for sperm antibodies by cytotoxicity and passive hemagglutination and also for antibodies to human leukocyte antigen (HLA) by cytotoxicity assays. Lymphocytes of the study subjects were typed for 61 HLA-A and B alleles. Thirteen of 30 (43%) men with sperm autoimmunity also had HLA antibodies in their serum and/or seminal plasma samples, in contrast to 2 of 35 (6%) nonautoimmune males (P = 0.0003). Twenty-five of 35 (71%) sperm antibody-positive infertile women had HLA antibodies in their sera and/or secretions, while only 7 of 31 (23%) women without sperm antibodies were positive (P = 0.00007). Antibodies to HLA-A19 (A26, A29, AW30, AW31, AW32, AW33, and AW34) and Bw35 (B5, B15, B17, and B18) complexes were present in 19 of 22 (86%) infertile men and 44 of 48 (92%) infertile women positive for HLA antibodies (P less than 0.01). The presence of antibodies to HLA-A19 and/or Bw35 in the infertile subjects did not correlate with the presence of HLA-A19 and/or Bw35 in the husbands. The presence of antibodies to HLA-A19 and/or Bw35 in the cervical mucus of the infertile women correlated with their presence in the seminal plasma of their husbands. It is suggested that antibodies to sperm antigens cross-reactive with HLA-A19 and/or Bw35 may be relevant to infertility.

Apheresis, exchange, adsorption and filtration of plasma: four approaches to the removal of undesirable circulating substances.

Plasmapheresis and plasma exchange have been widely used in the treatment of a variety of conditions, not always with a clear rationale. The most favorable results have been observed in the hyperviscosity syndrome and in a group of antibody-mediated diseases which includes post-infectious cold agglutinin disease, red cell aplasia, post-transfusion purpura, Goodpasture's syndrome, hemophilic patients with anti-factor VIII antibodies, and also in thrombotic thrombocytopenic purpura. In contrast, disappointing or conflicting results have been obtained in immune complex diseases, myasthenia gravis, cryoglobulinemia, etc. It seems likely that at least some of the difficulties may arise from the non-specific removal of a variety of substances as well as for our lack of understanding of immunological feed-back mechanisms which are disturbed by plasmapheresis and related techniques. Future developments are likely to be centered in the development of more specific approaches as based on filtration, affinity chromatography and adsorption to antibodies or other substrates. These approaches appear promising for the removal of "blocking factors" in patients with cancer, lipoproteins in patients with hypercholesterolemia, immune complexes, and toxic compounds. A cautiously optimistic approach to such new developments and their testing in animal models and in carefully controlled patient trials are essential. The principles on which these therapeutic approaches lie are valid, and the skepticism that surrounds them may be underserved, since it was largely the result of an indiscriminate use of non-selective procedures.

Cytogenetics. An evolving role in the diagnosis and treatment of cancer.

Conventional cytogenetics has been used for many years in the diagnosis and follow-up of myeloproliferative disorders. Molecular techniques including FISH and gene rearrangements are complementary in the evaluation of myeloproliferative and myelodysplastic disorders. Lymphomas and lymphocytic leukemias have nonrandom cytogenetic patterns that are useful in the clinical diagnosis, prognosis, and therapeutic follow-up. Solid tumors have complex karyotypic and genetic abnormalities, and clinical utilization of conventional cytogenetics and molecular techniques is in the developmental stages of applicability. The benefits of karyotype analysis in myeloproliferative and lymphoproliferative diseases include guidance for diagnostic and therapeutic approaches as well as assessment of minimal residual disease. Conventional cancer cytogenetics and commercially available FISH reagents enhance these applications. Molecular diagnostic techniques are analytically sensitive and specific. The complexities of the function and role of genetic abnormalities in solid tumors present challenges in the choice, interpretation, and application of conventional cytogenetic and molecular data. These challenges offer exciting potential for future advances.

Chromosomal abnormalities in acute leukemias.

Conventional cytogenetic techniques are the standard for the diagnosis and follow-up of patients with AML and ALL. Some characteristic translocations associated with various groups of AML diagnoses, such as t(8;21), t(15;17), and inv(16) for M2, M3, and M4eo, respectively, have been recognized for years. The most common cytogenetic abnormality found in childhood ALL and hyperdiploid adult ALL is t(9;22). Future directions include increased use of FISH and molecular diagnostic techniques. The clinical cytogenetics laboratory plays a major role in the diagnosis and management of AML and ALL.

DNA and protein synthesis in normal and transformed lymphocytes exposed to abrin.

The dose dependent effects of abrin, a toxic D-galactose binding plant lectin, on 3H-TdR and 14C-leucine uptake are studied in normal and Epstein Barr Virus (EBV) transformed lymphocyte cultures. Results show that while abrin is highly toxic to both the DNA and protein synthesis in EBV lymphocytes, some toxicity to the normal cells is also seen. It is postulated that lymphocyte DNA synthesis is affected by ribosomal shutdown induced by the abrin.

Platelet abnormalities in hepatobiliary diseases.

Platelet abnormalities associated with hepatobiliary diseases include increased (thrombocytosis) and decreased (thrombocytopenia) numbers of platelets as well as abnormalities in function (thrombocytopathy or thrombasthenia). Hepatic diseases that are accompanied by platelet abnormalities include hepatitis, cirrhosis, portal hypertension, and neoplastic disorders both benign and malignant. The objective of this work is to examine the platelet abnormalities that occur with a variety of hepatobiliary disorders. Thrombocytosis is seen as a reactive entity following splenectomy. Thrombocytopenia is associated with hypersplenism, dysproteinemias and liver disease related disseminated intravascular coagulation (DIC). Qualitative platelet abnormalities are found in hepatic failure, liver diseases associated with high or low levels of lipid, and with medications given for a variety of hepatocellular diseases. Clinically common and significant platelet abnormalities associated with liver disease are thrombocytopenia secondary to portal hypertension and the thrombasthenias following metabolic changes and/or therapeutic interventions of liver disease.

Hepatobiliary imaging: imaging modalities.

Imaging modalities are a major part of the diagnosis and follow-up to therapy of hepatobiliary disease. Changes of the liver, spleen, gallbladder and pancreas can be assessed by a variety of highly technical and evolving methods. The purpose of this paper is to review briefly some diseases of the hepatobiliary tree and pancreas and how they may be diagnosed through imaging modalities. Hepatic diseases may include those that affect the parenchyma, biliary tree, or gallbladder. The spleen is often altered in size secondary to liver disease. Pancreatic diseases present particular difficulties in diagnosis as they relate to clinical chemistry tests, tumor markers or imaging. Imaging modalities for hepatobiliary disease include x-ray studies with or without contrast, and with or without computerized tomographic enhancements. Some of the x-ray procedures are done in association with percutaneous or endoscopic retrograde approaches. Ultrasound is an excellent technique for evaluation of cholelithiasis and gives useful information for hepatic and splenic size. Radionuclide studies can be either anatomically or physiologically based. Magnetic resonance imaging is evolving as a very useful, specific and sensitive method of evaluating the liver, gallbladder, spleen and pancreas. There is a diverse armamentarium of imaging modalities available for the evaluation of hepatobiliary disease. Ultrasound is the first choice for cholelithiasis. Endoscopic retrograde cholecystography (ERCP) is a definitive method for evaluating the biliary tree. Radionuclide studies with the iminodiacetic acid (IDA) derivatives are very useful for functional disorders of the gallbladder. Computer assisted tomography and magnetic resonance imaging (MRI) provide optimal anatomic resolution for tumors, abscesses, and other metastatic lesions involving the liver and spleen.

Thrombocytopenia: proposed mechanisms and treatment in human immunodeficiency virus infection.

Thrombocytopenia (TC) is noted in three to nine percent of patients who are seropositive for human immunodeficiency virus (HIV). Causes of this may be immune, infectious, platelet destruction, or underproduction. Thrombocytopenia associated with HIV will be seen with increasing frequency. Corticosteroids and splenectomy are the usual therapeutic approaches. Intravenous gammaglobulin and antiviral agents may be indicated and useful in some patients.

Human immunodeficiency virus type II (HIV-2) testing: a perspective.

Testing of blood for antibodies to the human immunodeficiency virus type 2 (HIV2) has begun. A major benefit of this testing will be the elimination of the exclusion of blood donors on the basis of geographic origin. Will this test be helpful? Present anti-HIV1 testing detects between 60 to 90 percent of those samples containing the HIV2 antibody. A Food and Drug Administration (FDA) licensed enzyme immunoassay for HIV1/HIV2 antibodies became available for clinical use in March of 1992. (There is an FDA unlicensed HIV-2 Western Blot (WB) Test). The specificity and sensitivity of either the HIV2 EIA or HIV2 WB are not well defined. In a survey of 24 million U.S. donors, no antibodies to HIV2 were detected. With the incidence of HIV2 infection being so small, it is probable that the majority of positive tests will be false-positives causing needles concern for the blood donor who must be counseled. The benefits to the blood supply remain to be evaluated.