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J S Kovach

Publications and source records attributed to J S Kovach.

At least 19 recordsLinked to original sources
PubMed1992

Current status of adjuvant chemotherapy for colorectal cancer. Can molecular markers play a role in predicting prognosis?

BACKGROUND: Recent clinical trials establish a beneficial effect for adjuvant chemotherapy after surgical resection of the primary tumor (1) as single treatment for patients with colonic cancer and (2) combined with radiation therapy for patients with rectal cancer. Because adjuvant chemotherapy is not universally effective and is associated with toxicity and some degree of risk, it would be desirable to supplement standard pathologic staging criteria to define more precisely the subset of patients at high risk for tumor recurrence who would benefit most from adjuvant therapy. Tumor cell DNA content and cell proliferation measured by flow cytometry were identified as important and independent prognostic factors for patients undergoing curative resection of colorectal cancer. Basic laboratory investigations show a series of more specific molecular and genetic abnormalities that might provide better prognostic discrimination. Recent molecular studies suggest that the process of tumorigenesis in colorectal cancer proceeds through a series of genetic alterations that include both dominant and recessive protooncogenes. Characterization of these molecular genetic abnormalities may provide valuable prognostic information for use in patient management. METHODS: Allelic loss was studied for chromosomes 5, 17, and 18, and immunohistochemical analysis was done of the p53 protein product in tumors from 91 patients with colorectal cancer. RESULTS: Preliminary analysis of disease-free survival after surgical resection in 60 patients with Dukes' B or C tumors suggests a poorer prognosis associated with allelic loss on chromosome 18q (P = 0.08). CONCLUSIONS: Additional studies involving a much larger population of patients with Dukes' B and C colorectal cancer are needed to define the true prognostic significance of these molecular markers.

Aged↗
PubMed1992

Levamisole potentiation of fluorouracil antiproliferative activity mimicked by orthovanadate, an inhibitor of tyrosine phosphatase.

BACKGROUND: Levamisole is an effective antihelminthic drug with immunomodulatory and anticancer activities in model systems. Combined with fluorouracil (5-FU) as adjuvant treatment following resection of Dukes' stage C colon carcinomas, levamisole significantly reduces mortality. However, neither 5-FU nor levamisole alone has a significant effect on survival in this patient group. Previously, we noted that in vitro levamisole potentiated the antiproliferative activity of 5-FU. PURPOSE: Because levamisole is known to inhibit alkaline phosphatases and has been reported to inhibit dephosphorylation of some membrane phosphoproteins, we studied the effects of levamisole analogues and of chemically unrelated inhibitors of phosphatases for their ability to potentiate 5-FU inhibition of tumor cell line proliferation in vitro. METHODS: Human cancer cell lines were exposed to drugs alone or in combination with 5-FU. Antiproliferative activity was measured by determining the extent of reduction of colony formation by the cell lines in test plates compared with control plates. RESULTS: We found that potentiation of 5-FU cytotoxicity by levamisole and by p-hydroxytetramisole, a metabolite of levamisole, is mimicked by orthovanadate, an inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor of serine and threonine phosphatases, Furthermore, l-p-bromotetramisole, a synthetic analogue of levamisole that is 10-fold more potent in inhibition of alkaline phosphatase than levamisole, potentiates the antiproliferative activity of 5-FU to a greater extent than d-p-bromotetramisole, a stereoisomer of l-p-bromotetramisole with little antiphosphatase activity. CONCLUSION: Inhibition of tyrosine phosphatases may be responsible for the potentiation by levamisole of the inhibitory activity of 5-FU in vitro. IMPLICATIONS: Inhibition of dephosphorylation of regulatory phosphoproteins may be related to the therapeutic efficacy of the combination of levamisole and 5-FU in the adjuvant treatment of colon carcinoma and may underlie at least some of the multiple effects of levamisole on immune parameters.

Breast Neoplasms↗
PubMed1992

Expression of p53 and 17p allelic loss in colorectal carcinoma.

Mutations in the p53 gene are the most common genetic changes in cancer thus far. Many p53 mutations result in a protein product having a prolonged half-life compared to wild-type p53. The mutant protein is frequently detectable immunohistochemically, whereas the wild-type p53 present in normal cells is not. We examined 90 colorectal carcinomas for increased expression of p53 using 3 p53 specific monoclonal antibodies, PAb1801, PAb421, and PAb240. Overall, 70% of the colorectal carcinomas stained for p53. Each tumor's DNA was also assessed for loss of heterozygosity on chromosome 17p, the location of the p53 gene. Of those tumors that reacted with the anti-p53 antibodies, 76% showed loss on chromosome 17p. Tumors with loss of heterozygosity on 17p generally stained with all 3 antibodies, whereas those without loss tended to stain with just one antibody, typically PAb240. Fifteen tumors were examined for the presence of specific p53 mutations. A total of 10 mutations were found, 6 were missense and 2 were deletions, and all but one of the tumors with missense mutations stained for p53.

Alleles↗
PubMed1992

Pattern of p53 gene mutations in breast cancers of women of the midwestern United States.

BACKGROUND: Mutation in the p53 gene is the most common genetic lesion in human cancers. The pattern of mutation in the p53 gene differs among cancers and may be a useful epidemiological tool for identification of factors contributing to carcinogenesis. PURPOSE: Our purpose was to determine if the pattern of p53 mutation in breast carcinomas in our population of women residing in the midwestern region of the United States is similar to the pattern of p53 mutation in breast cancers in patients from other regions of the United States and Europe and in other epithelial tumors. METHODS: With a technique we recently developed for the analysis of p53 mutations in genomic DNA from tumor cell clusters in touch preparations of solid tumors, we sequenced exons 5-9 and adjacent splice junctions of the gene in 44 breast cancers. Cells from each tumor were also stained with three monoclonal antibodies which recognize different epitopes of the p53 protein. RESULTS: We detected p53 mutations in 14 (32.6%) of 44 breast carcinomas. Only half of the mutations were missense changes. The other half included five microdeletions (three producing frame-shifts), one single-base substitution generating a stop codon, and one single-base substitution generating a splice junction abnormality. Nuclear expression of p53 antigen was present in eight of 44 cancers, including six with hemizygous missense mutations in the p53 gene. CONCLUSIONS: The pattern of p53 mutations in our breast cancer population differs from that reported in breast cancer populations by other investigators in which most p53 mutations were missense. Among 14 mutations in our population, at least five drastically altered the structure of p53, suggesting that a recessive mechanism of inactivation of the p53 gene may be more common than in other populations. IMPLICATIONS: Differences in the pattern of p53 mutation in breast cancers in Midwestern women and in breast cancers in other populations may reflect selection bias or small sample sizes currently available. However, our data are compatible with the possibility that an endogenous or exogenous factor influences p53 carcinogenesis in some women with breast cancer in the Midwest to a greater extent than in other regions of the United States and Europe.

Adult↗
PubMed1992

Distribution of transforming growth factor-beta 1 in human astrocytomas.

We used immunohistochemical techniques to study the distribution of transforming growth factor-beta 1 (TGF-beta 1) and infiltrating lymphocytes and macrophages in human astrocytomas. Thirteen of 15 grade 4 astrocytomas (glioblastomas) showed staining with anti-TGF-beta 1 antibody, predominantly in proliferating endothelial complexes and surrounding small and medium-sized blood vessels. Brain tissue microscopically free of tumor cells (n = 8) and more differentiated astrocytomas of varying grade (1 to 3; n = 6) devoid of endothelial proliferation did not stain with anti-TGF-beta 1. Normal brain contained only rare lymphoreticular cells. The majority of astrocytomas studied, however, contained T lymphocytes and macrophages with smaller numbers of B lymphocytes. The lymphoreticular infiltrates were concentrated primarily in close proximity to blood vessels. Within an individual tumor perivascular regions staining for TGF-beta 1 never contained more than occasional T lymphocytes. Perivascular regions not staining for TGF-beta 1 frequently contained low to high numbers of T lymphocytes. The inverse relationship in the distribution of TGF-beta 1 and lymphocyte infiltrates is compatible with a functional relationship between this cytokine and an immune effector cell response to glioblastomas.

Astrocytoma↗
PubMed1991

Administration of interleukin-2 (IL-2) results in increased plasma concentrations of IL-5 and eosinophilia in patients with cancer.

Peripheral eosinophilia is almost invariably observed during the course of interleukin-2 (IL-2) therapy and is frequently accompanied by the development of a capillary leak syndrome characterized by edema, weight gain, and oliguria. We studied five patients with advanced malignancy treated with IL-2. Eosinophilia was not present initially but developed in all patients late in the course of therapy, with counts ranging from 2,328/mm3 to 15,958/mm3. In all patients, there was a temporal relationship between the infusion of IL-2 and the appearance of elevated plasma concentrations of IL-5, a growth factor for eosinophils. Granulocyte-macrophage colony-stimulating factor was not detectable in plasma. IL-4 and gamma-interferon plasma levels were variably elevated. Plasma concentrations of major basic protein, a toxic eosinophil granule protein, began increasing before eosinophil counts increased. By the time of the third IL-2 infusion, high concentrations of major basic protein were present in all five patients (up to 5,600 ng/mL) and skin biopsies showed major basic protein deposition in the dermis. Four patients developed significant capillary leak syndrome and all of these patients showed markedly elevated major basic protein levels. The lowest peak plasma concentration of major basic protein (1,751 ng/mL) was observed in the one patient who did not develop edema and weight gain. These results suggest that IL-2 induces IL-5 leading to marked peripheral eosinophilia and extravascular eosinophil degranulation. The release of toxic eosinophil products at extravascular sites and in the circulation may contribute to the pathogenesis of the capillary leak syndrome complicating IL-2 therapy.

Aged↗
PubMed1991

Direct sequencing from touch preparations of human carcinomas: analysis of p53 mutations in breast carcinomas.

A new technique for characterizing somatic mutations in very small samples of cellularly heterogeneous human cancer tissue was developed and tested using mutations in the p53 gene in breast carcinomas as a model system. The technique combines touch preparation of specimens to obtain homogeneous clusters of carcinoma cells free of normal cells with a nested pair of polymerase chain reaction (PCR) amplifications of DNA to increase the amount of target gene sequence sufficiently to permit direct sequencing of the p53 gene. Touch preparations of fresh or previously frozen tissue from human adenocarcinomas derived from several organs were stained, and clusters of 10-50 malignant cells were transferred by pipette into microfuge tubes for PCR amplification. Exons 5-9 of the p53 gene, which contain the major mutational hot spots associated with most human cancers, were sequenced by the following steps: 1) two rounds of PCR amplification using DNA Taq polymerase and two sets of oligonucleotide primers, the second set being nested within the segment amplified by the first set and having attached T7 and SP6 phage promoter sequences, 2) transcription of the amplified DNA sequences with T7 and SP6 RNA polymerases, and 3) dideoxy sequencing of single-stranded RNA transcripts with reverse transcriptase and with additional oligonucleotide primers to achieve specificity for this unique region of the genome. The utility of this approach is illustrated by our success in detecting and analyzing point mutations in cell clusters from four of 11 primary adenocarcinomas of the human breast.

Adenocarcinoma↗
PubMed1991

Phase I-II study of pibenzimol hydrochloride (NSC 322921) in advanced pancreatic carcinoma.

Pibenzimol is a fluorescent molecule known to bind to double stranded DNA. It also induces prolongation of the G2 phase of the cell cycle, inhibition of DNA replication and cessation of the growth of some cells in late S phase after DNA content has been doubled. It has been shown to increase the life span of mice bearing intraperitoneally implanted L1210 and P388 leukemia. These factors coupled with the affinity of pibenzimol for pancreatic tissue led us to conduct a phase I-II trial of pibenzimol hydrochloride in patients with advanced pancreatic cancer. Twenty-six patients were treated with a five day continuous infusion of pibenzimol at a dose ranging from 6-28 mg/m2/d. There were no treatment related deaths. Major toxicity was hyperglycemia which was self-limited. No objective responses were noted.

Adult↗
PubMed1991

Transforming growth factor-alpha, epidermal growth factor receptor, and proliferating potential in benign and malignant gliomas.

Surgical specimens from six benign and 16 malignant human gliomas were investigated immunohistochemically to correlate the degree of malignancy, the distribution of transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF) receptor, and the potential for cell proliferation using monoclonal antibodies to TGF-alpha, EGF receptor, and Ki-67. Fourteen (88%) of the malignant gliomas and one (17%) of the benign gliomas were found to be positive for TGF-alpha, and 14 (88%) of the malignant gliomas and two (33%) of the benign gliomas expressed EGF receptor. The proliferation index with Ki-67 was 18.8% +/- 8.1% (mean +/- standard deviation) in malignant gliomas and 1.9% +/- 1.8% in benign gliomas. In general, cells positive for EGF receptor and Ki-67 were randomly distributed throughout the tumor tissue, and cells positive for TGF-alpha tended to be clustered without obvious relationship to areas of necrosis or blood vessels. In some tumors, cells positive for TGF-alpha, EGF receptor, and Ki-67 were associated in a focal distribution. The more frequent expression of TGF-alpha and EGF receptor in the highly proliferative malignant gliomas is compatible with a role for TGF-alpha and EGF receptor in the induction or stimulation of malignant gliomas.

Astrocytoma↗
PubMed1990

Phase I and clinical pharmacological evaluation of a parenteral hexamethylmelamine formulation.

Hexamethylmelamine has been evaluated in single agent and combination regimen studies for many years, but only following p.o. administration. Pharmacological studies in animals and humans have shown that systematic availability of parent drug following p.o. administration is relatively low and variable due to extensive first-pass metabolism rather than due to poor absorption. Two Phase I clinical trials, with accompanying pharmacokinetic studies, have been conducted by using a parenteral formulation in which hexamethylmelamine was prepared by Intralipid 10%. The parenteral formulation was well tolerated by all patients receiving hexamethylmelamine by 1-day and by daily for 5-days schedules. Nausea and vomiting were the dose-limiting toxicities. Maximally tolerated doses on the 1-day and daily for 5-days schedules were approximately 850 mg/m2 and 630 mg/m2/day, respectively. No responses were observed in either study. Following i.v. administration of 540 mg/m2 hexamethylmelamine, plasma elimination was best described by a three-compartment open model with terminal half-life, total body clearance, and steady-state volume of distribution values of 10.4 h, 0.75 liter/min/m2 and 460 liters/m2, respectively. Twenty-four h urinary recoveries of parent drug were less than 1% for all patients. Accumulation of hexamethylmelamine during the 5-day treatment at 945 mg/m2 suggested possible saturation of parent drug elimination at that dose. Phase II studies are currently under way with the parenteral formulation of hexamethylmelamine.

Altretamine↗
PubMed1990

Effector function of lymphokine-activated killer cells and cytotoxic T lymphocytes in ovarian epithelial carcinoma.

From 20 patients with ovarian carcinoma (4 with stage I and 16 with stage III disease), we obtained peripheral blood mononuclear cells (MNC) from each, tumor-associated MNC from 9, and ascitic MNC from 6. These cells were stimulated with interleukin 2 (IL-2), and radioactive chromium-release cytotoxicity assays were used to evaluate the lymphokine-activated killer (LAK) activity against autologous tumor cells and two natural killer-resistant cell lines. The LAK response was consistently better with control peripheral blood MNC than with patient-derived MNC against all targets. However, within the ascitic MNC population, LAK activity against two cell lines (Daudi and OVCAR) was not statistically different from that of the control MNC. Phenotypic analysis of the ascitic MNC with monoclonal antibodies and flow cytometry revealed an activated population (IL-2 receptor-positive) characterized by predominantly monocytes/macrophages and T cells. In addition, the peripheral blood LAK activity for patients with stage I disease was statistically better against each target than that from patients with stage III disease. These results suggest an immunosuppressive effect directly related to tumor burden. Except perhaps for the ascitic MNC, autologous MNC do not appear to be effective LAK sources. Follow-up of the stage III patients revealed no difference in 2-year survival associated with in vitro LAK response.

Ascitic Fluid↗
PubMed1989

LAK cell generation in normal subjects: Ficoll-Hypaque vs. light spin purification.

Research in adoptive immunotherapy such as lymphokine-activated killer cell (LAK) generation requires a large number of mononuclear cells (MNCs). The original procedure for generation of LAK cells used a Ficoll-Hypaque (FH) purification of the MNCs collected by a blood separator to remove granulocytes, platelets, and red blood cells. Since the Fenwal CS 3000 blood separator produces MNCs with reduced granulocyte contamination, it is possible that FH purification could be replaced by a light centrifuge spin (LS) purification. We report on a study comparing the MNC recovery, platelet and red cell removal, purification time, in vitro LAK cytotoxicity, and phenotypic characterization of the lymphocytes, collected by FH purification vs. LS purification by using the CS 3000. Six donors were leukapheresed with the CS 3000 using a shortened LAK Cell procedure. Following an LS (3 minutes at 1,000 rpm), the MNC collection was divided into two equal aliquots, one aliquot purified by FH and the other aliquot purified by LS. Both aliquots were then washed, cultured for 5 days in IL-2, and harvested with the CS 3000. Our findings suggest the LS purification resulted in improved 1) postpurification MNC recovery (89% vs. 60%), 2) purification time (54 minutes vs. 129 minutes), and 3) LAK cell generation by phenotype. LS purification was similar to FH purification in 1) platelet removal and 2) in vitro LAK cytotoxicity. We found the LS superior to FH when considering efficiency, economics, simplicity, and LAK cell generation and comparable in platelet removal and in vitro LAK cytotoxicity.

Centrifugation↗
PubMed1989

Cellular pharmacology of fluorinated pyrimidines in vivo in man.

Fluorinated pyrimidines, particularly 5-fluorouracil (FUra), have been the subject of intense and almost continuous basic and clinical study since development in the late 1950's by Dr. Charles Heidelberger. Despite this intensive effort, the most important mechanisms by which FUra influences tumor growth in individual cancer patients and the therapeutically optimum method of administration of the drug alone and in combination with other drugs or ionizing radiation continue to be questions of interest. This article reviews aspects of the study of FUra pertinent to the thesis that for this drug as for other agents used to treat human cancers, data on intracellular concentrations of drug and metabolites as a function of dose, schedule of administration and time are needed for correlation with effects on tumor proliferation if a rational basis for individualization of therapy is to be achieved. A preliminary description of ongoing studies of the tissue concentrations of FUra and metabolites in human colorectal carcinoma and in adjacent normal bowel after rapid injection and after 24-hour infusion of radiolabelled pharmacologically active doses of FUra is included as one approach to learning more about the cellular pharmacology of fluorinated pyrimidines.

Antineoplastic Agents↗
PubMed1989

Phenotypic and functional characteristics of mononuclear cells in ovarian carcinoma tumors.

By using monoclonal antibodies and flow cytometry, the phenotype and activation state [based on presence of interleukin 2 (IL-2) receptors] were determined on the mononuclear cell (MNC) fraction of ovarian carcinoma tissue from 12 patients. Thymidine incorporation response to IL-2 and phytohemagglutinin stimulation was measured in five cases. By FACS analysis, tumor MNCs contained 79% T cells, 11% monocytes/macrophages, 5% B cells, and 5% natural killer cells. Similar findings were noted in the patients' peripheral blood MNC population. The respective T-helper:cytotoxic/suppressor ratios in tumor and blood MNC populations were less than 1 and approximately 2. The percentage of IL-2 receptor-positive cells was low in both populations. The proliferative response of tumor MNCs was lower than that of blood MNCs from patients and from normal volunteers. These results suggest an intratumor immune suppression, perhaps secondary to the absence of a specific responder population or to the presence of suppressor cells or a soluble factor secreted by the tumor that directly suppresses or affects MNC transport into and out of the tumor.

B-Lymphocytes↗
PubMed1989

Cancer immunotherapy: current status of treatment with interleukin 2 and lymphokine-activated killer cells.

In recent years, the medical community has witnessed a growing interest in the use of adoptive immunotherapy in patients with malignant lesions refractory to standard treatments. Systemic administration of interleukin 2, in combination with the adoptive transfer of a patient's own activated immune cells, has resulted in objective regression of several types of advanced cancers. Pronounced regression of tumor has also been observed with use of systemic interleukin 2 alone. This ability to augment the immune defense system of the host against cancer has stimulated intense clinical and laboratory investigations.

Animals↗
PubMed1989

Recycling of tumor necrosis factor-alpha receptor in MCF-7 cells.

Kinetics of regulation of membrane receptors for tumor necrosis factor-alpha (TNF) in human breast adenocarcinoma MCF-7 cells was investigated. When MCF-7 cells were incubated with radioiodinated human recombinant TNF, they bound TNF specifically and accumulated it intracellularly. Preincubation of cells with native TNF up to 1 x 10(-9) M for 12 h stimulated specific binding of TNF, indicating that concentrations of membrane receptors for TNF were regulated by the ligand. Accumulation of radioactivity in cells incubated with [125I]TNF proceeded at a constant rate for up to 24 h. Kinetics of binding and internalization of TNF were similar in the presence and absence of protein synthesis for at least 1 h, suggesting that the TNF receptor was either replenished from an intracellular pool of receptors or was recycled (reutilized) during the course of TNF internalization. Data were analyzed kinetically by fitting equations of compartmental models of ligand-cell interactions with and without the term for receptor recycling. Fits were obtained only for the model with receptor recycling; absence of the term for receptor recycling resulted in physically impossible best-fit parameter values. Analysis of the best-fit parameters indicated that both internalization and recycling of the receptor were stimulated by the ligand.

Adenocarcinoma↗