Biomedical subjects
Publications and source records attributed to Z Estrov.
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Mycosis fungoides (MF) and Sezary syndrome (SS) are the two main clinical entities of cutaneous T-cell lymphoma (CTCL). As the disease progresses from MF to SS, a switch from a type 1 (interleukin [IL]-2 and gamma interferon [IFN-gamma]) to a type 2 (IL-4) cytokine production profile occurs. Although roles for type 1 and type 2 cytokines in the pathogenesis of CTCL have been proposed, the cellular origins of these cytokines are unclear. Using flow cytometry to identify individual T-cell subsets, we studied cytokine synthesis by the T cells of 13 patients with SS and 12 with MF and 9 hematologically healthy donors. Upon activation with phorbol 12-myristate 13-acetate (PMA), the numbers of T cells synthesizing IL-2 were similar for all study groups. Whereas the predominant T-cell producing IL-2 in healthy donors and in those with MF was CD7(+), in patients with SS, it was CD7(-). Although the number of IL-4(+) CD4(+) T cells was low for all study groups, there was a significantly higher number of IL-4(+) CD8(+) T cells in patients with MF than in those with SS or healthy donors. There was a decline in the number of IFN-gamma-producing T cells in CTCL donors compared to that in healthy donors. More importantly, there was a significant decrease in the number of IFN-gamma-producing T cells with disease progression from MF to SS. The inability of these T cells to synthesize IFN-gamma may have prognostic value in CTCL, since it may be responsible for the progression of the disease from MF to SS.
Several techniques developed in recent years provide us with the capability to detect sub-microscopic leukemia during remission. Quantitative polymerase chain reaction (PCR) is thus far the most sensitive assay that is applicable in most patients with acute lymphoblastic leukemia (ALL) of childhood. However, false-positive and false-negative results may provide the clinician with misleading data and therefore PCR analysis should be accompanied by another assay and changes in the level of residual disease should be confirmed at different time points following treatment. Furthermore, several studies did not determine a threshold of residual disease level above which relapse is likely to occur, and more recent data show that long-term remission may be sustained in the presence of residual disease. Thus, additional studies of the biology of residual disease in childhood ALL should be performed before sensitive assays of residual disease detection and quantitation can be clinically utilized.
In almost no other area of medical oncology has the introduction of new drugs, combinations of chemotherapeutic agents, and novel biologic treatments caused such dramatic responses as it has in the treatment of malignant hematologic disorders. However, despite some therapeutic success, many patients relapse and die from recurrence of their disease. The implications of minimal residual disease (MRD), a term referring to disease that is undetectable by conventional morphologic methods, have therefore attracted increasing attention in recent years. New and powerful laboratory tools such as polymerase chain reaction assays have extraordinary sensitivity and provide exciting new insights into the detection, nature, quantification, and kinetics of MRD. This article summarizes methods used in the identification of MRD and its importance as exemplified in the case of acute leukemias and chronic myelogenous leukemia.
BCL2 protein exerts an antiapoptotic effect on cells and decreases chemosensitivity. To determine whether BCL2 expression is prognostic of patient outcome in acute myelogenous leukemia (AML), we measured its level in 198 newly diagnosed, untreated AML patients by Western blotting using whole-cell lysates from low-density peripheral blood cells. BCL2 expression was not associated with the percentage of blasts (R2 = 0.10), French-American-British classification type, or cytogenetic abnormality. Smoothed martingale residual plots indicated that high BCL2 protein level was an adverse prognostic factor for patients with either favorable or intermediate prognosis cytogenetics [FIPC; inv(16), t(8:21), t(15:17), or diploid or insufficient metaphases] but was a favorable prognostic factor for patients with unfavorable prognosis cytogenetics (UC; -5, -7, +8, 11q23, Ph1, or miscellaneous changes). Patients with FIPC and high BCL2 (highest quartile) had a significantly shorter median survival (78 weeks versus not reached; P = 0.009) than did those with lower (lower three quartiles) levels of BCL2. Among those with UC, as BCL2 level decreased from the fourth quartile to the third or the combined first and second quartiles, the median survival decreased (from 94 to 45 or 17 weeks, respectively; P = 0.003). Lower expression of BCL2 in UC was associated with shorter remission duration (P = 0.05). In multivariate analyses performed using either overall or event-free survival as the end point, for either all patients or within either cytogenetic subgroup, BCL2 level was an independent prognostic factor. Similar analysis revealed that BCL2 level was an independent predictor of remission duration for UC patients as well. These data suggest that BCL2 is involved differently in different types (favorable versus unfavorable) of AML and that therapeutic strategies aimed at modulating BCL2 function may be more likely to work in patients with favorable cytogenetic abnormalities.
Cytogenetic/molecular abnormalities significantly influence the prognosis of patients with acute leukemia. Recently, two genes, p16INK4a and p15INK4b, encoding two cyclin-dependent kinase inhibitor proteins of the INK4 family of Mr 15,000 and 16,000, respectively, have been localized to 9p21. Remarkably, the p16INK4a locus has been found to encode a second protein, p14ARF, known as p19ARF in mice, with a distinct reading frame. Like p16INK4a, p14ARF is involved in cell cycle regulation, blocking cells at the G1 restriction point through the activity of MDM-2 and p53. We studied bone marrow samples of 42 newly diagnosed and untreated patients with acute lymphoblastic leukemia for the incidence of deletions of p16INK4a/p14ARF and p15INK4b using Southern blot analysis and determined the clinical outcome with regard to complete remission (CR) duration, event-free survival, and overall survival. We found deletions of p16INK4a/p14ARF in 17 of 42 patients (40%), with homozygous deletions in 11 of 42 patients (26%) and hemizygous deletions in 6 of 42 patients (14%). The gene for p15INK4b was codeleted in most, but not all, cases and was never deleted without deletion of p16INK4a/ p14ARF. No correlation was observed between molecular studies and karyotype abnormalities as determined by conventional cytogenetics. Furthermore, no difference was found in the CR rate, CR duration, event-free survival, and overall survival in patients with homozygous gene deletions compared to patients with no deletions or loss of only one allele.
This review of the molecules and pathways involved in programmed cell death (apoptosis) discriminates triggers of apoptosis (e.g. chemotherapy, radiation, Fas ligation), modulators of apoptosis (e.g. Bcl-2 family members, Bcl-2 interacting proteins, Apafs, IAPs, and Fas/FasL modulators including FLICE and FLIPs), effectors (caspases 1-13) and cleavage substrates (e.g. PARP). Special consideration is given to the structure-function relationship of Bcl-2 family members and to their post-transcriptional modification. Brief references are made to the role of apoptotic pathway in leukemias and lymphomas and to strategies of modulating apoptotic pathways.
To evaluate the safety, toxicity, and maximum tolerated dose (MTD) of IFN beta-1a (Rebif, Serono Laboratories, Inc.) in patients with malignant diseases unresponsive to standard therapies and to assess the pharmacodynamics and pharmacokinetics associated with IFN beta-1a administration, an open-label, single-center phase I study was designed. Thirty-four patients were enrolled and treated with IFN beta-1a. All had measurable solid neoplasms or evaluable hematological malignancies. All patients received a single i.v. bolus dose of IFN-beta-1a on day 1, followed 7 days later by daily s.c. injections for 28 consecutive days. Successive groups of three patients received increasingly higher doses (in geometric progression from 1.5 million international units (MIU)/m2 to 24 MIU/m2) until dose-limiting toxicities were noted. Pharmacokinetic and biological studies, including measurement of the activity of 2',5'-oligoadenylate synthetase (2',5'-OAS) in peripheral blood mononuclear cells and serum levels of soluble Tac (CD 25) and beta-2 microglobulin, were performed on patients who agreed to participate. i.v. and s.c. doses of IFN beta-1a up to 24 MIU/m2 were administered. The most frequent adverse events (AEs) were constitutional symptoms. Grade III AEs during i.v. dosing included fever, elevation of bilirubin, and infection unrelated to therapy. No grade IV events were seen. AEs noted during continuous s.c. therapy included fever, liver transaminase increase, albuminuria, fatigue, nausea, myalgia, and rigors. Dose-limiting toxicities were encountered during s.c. dosing at the 24-MIU/m2 and 18-MIU/m2 dose levels and included gastrointestinal toxicity, elevations of aspartate aminotransferase and alanine aminotransferase, and albuminuria. The s.c. MTD was determined to be 12 MIU/m2, although there was great variability in the individual patient's ability to tolerate IFN beta-1a. 2',5'-OAS activity, thought to be indicative of IFN activity, increased within hours after i.v. and s.c. dosing, with the level remaining persistently elevated during the s.c. daily injections. The highest peak level was attained in the 6-MIU/m2 group. There was no evidence that the increase in 2',5'-OAS activity decayed with repetitive dosing, nor was there evidence of accumulation in this pharmacodynamic marker. Serum beta-2-microglobulin levels showed a modest time- and dose-dependent increase after s.c. administration of IFN beta-1a, with the largest increase seen at the 24-MIU/m2 dose level. There were no clear dose-dependent responses noted in soluble Tac serum levels. IFN beta-1a was well-tolerated when administered by a single i.v. bolus injection at doses up to and including 24 MIU/m2. Daily s.c. injections for at least 28 days were well-tolerated at doses up to and including 12 MIU/m2, with some patients tolerating doses twice as high as this. The MTD for the i.v. route could not be clearly determined according to the guidelines of the protocol. However, i.v. bolus doses up to 24 MIU/m2 were relatively well-tolerated. For the s.c. route, the MTD was determined to be 12 MIU/m2, but there was great interpatient variability, with some patients able to tolerate higher doses.
Dysregulation of apoptosis is an important mechanism in leukemogenesis. Caspases are cysteine proteases that play a major role in the activation of apoptotic pathways and chemotherapy-induced cell death. High levels of inactive, uncleaved caspase 2 and caspase 3 have recently been associated with poor survival in patients with acute myelogenous leukemia. We hypothesized a similarly significant role for caspase 2 and caspase 3 in patients with acute lymphoblastic leukemia. We determined levels of uncleaved caspase 2 and caspase 3 by quantitative Western blot analysis in peripheral blood samples of 45 adults with newly diagnosed ALL. We evaluated patient prognostic variables and caspase levels using multivariate logistic and Cox regression models to determine their impact on complete remission rate and overall survival probability. Levels of caspase 2 and, to a lesser degree, caspase 3 were highly associated with cytogenetic abnormalities, with lower levels in the diploid group (P = 0.016 and P = 0.10, respectively). No association between either caspase level and the percentage of bone marrow blasts was found. A high level of caspase 3 (>0.37 as determined graphically) was significantly associated with achieving complete remission (CR; P = 0.006). A multivariate logistic regression analysis including age, WBC count, percentage of peripheral and marrow blasts, hemoglobin, albumin, lactate dehydrogenase, bilirubin, and creatinine determined that a high level of caspase 3 was the most significant predictor of CR (P = 0.025, adjusted), with albumin the only other significant variable (P = 0.031). Caspase 2 levels were not associated with probability of CR. In a multivariate Cox model for survival, however, levels of caspase 2 above 0.37 were associated with a lower survival probability than were levels below that threshold (P = 0.064). High levels of caspase 3 may have a significant effect on achieving CR. Because of the limited power (n = 45) of our study, the significance of caspase 2 and caspase 3 on overall survival remains to be validated by further investigations.
Cytogenetic changes are of pivotal prognostic significance in patients with de novo acute lymphoblastic leukemia (ALL). However, in some cases leukemic blasts can harbor gene lesions on a submicroscopic level without evidence of a corresponding abnormality by conventional cytogenetic studies. This can result in failure to recognize chromosomal abnormalities and inappropriate evaluation with respect to therapy assignments. To study the discrepancy in the detection of deletions of the short arm of chromosome 9 and deletions of tumor suppressor genes p15/p16/p14 on chromosome 9p21, we analyzed bone marrow samples from 92 patients with ALL both by cytogenetic analysis and by Southern blot. In 41 patients (45%), we found deletions of p15/p16/p14, which were homozygous in 27 and hemizygous in 14. Cytogenetic analysis demonstrated abnormalities of the short arm of chromosome 9 in the form of 9p- or del(9p21-22) in only 5 of the 41 patients (12%). Only 2 of 51 patients without gene deletions as detected by Southern blot revealed a 9p- abnormality, which was found only in a subpopulation of the cells. We demonstrate that deletions of the p15/p16/p14 genes on chromosome 9p21 are more frequent than indicated by cytogenetic analysis. Molecular techniques in addition to cytogenetic studies are necessary to detect otherwise-unrecognized genetic lesions of the short arm of chromosome 9.
Because caspase activation is an essential step in programmed cell death (apoptosis) and cytotoxic drug-induced apoptosis is mediated by caspase 2 and caspase 3, we hypothesized that caspase 2 and 3 levels predict clinical outcome in acute myelogenous leukemia (AML). Using quantitative Western blot analysis, we studied the levels of nonactivated (uncleaved) caspase 2 and 3 in peripheral blood low-density cells from 185 patients with newly diagnosed AML. We also measured the level of activated (cleaved) caspase 3 in 41 randomly selected samples from the 185 patients. Finally, we analyzed the effect of caspase 2 and 3 levels and other prognostic variables on patient survival using a multivariate Cox model. We found that median levels of nonactivated caspase 2 and 3 were higher in AML than in normal peripheral blood cells (P < .001 and P <.02, respectively). There was no association between caspase level and either the percentage of peripheral blasts or any specific type of leukemia cell cytogenetic abnormalities. When the effect of each uncleaved caspase was considered individually, a high level of uncleaved caspase 3 (P = .04), but not of caspase 2 (P = .16), was associated with decreased survival. Conversely, a high level of cleaved caspase 3 denoted improved survival and correlated with the inactivation of the DNA-repair enzyme poly(ADP-ribose) polymerase. Thus, cleaved caspase 3 could stimulate the apoptotic cascade further, and lack of its activation likely caused an accumulation of the uncleaved caspase. Although uncleaved caspase 2 level per se had no prognostic significance, the interactive effect of high levels of both uncleaved caspase 2 and 3 denoted very poor survival (P < .001) and had the largest effect of all prognostic variables (P < .001; estimated relative risk, 2.49; 95% confidence interval, 1.59 to 3. 90). Taken together, caspase 2 and caspase 3 protein levels obtained at diagnosis may constitute a reliable prognostic factor in AML.
CD38 is expressed during early stages of differentiation in normal and leukemic myeloid cells. Recently, CD38 has been shown to participate in intracellular signal transduction pathways following its ligation with CD38-specific mAbs. In this study we report that ligation of CD38 by one such agonistic mAb (IB4) induced proliferation of cultured leukemic cells in vitro. In HL-60, KG-1A, NB4, and OCI-AML-3 myeloid leukemia cell lines, IB4 mAb induced an increase in the proliferating cell fraction as determined by cell number, clonogenic assay, and flow cytometric analysis. The presence of Ab caused a dose-dependent increase in the number of CFU and an increase in cell divisions. HL-60-Dox cells (a HL-60-doxorubicin-resistant cell line), which have no detectable CD38 expression, failed to respond to IB4 mAb. The effect of CD38 ligation on cell growth was also evaluated in freshly isolated leukemic cells from patients with acute myelogenous leukemia (AML). A significant increase in the proliferating cell fraction (S+G2M) was observed in 50% of the patients incubated with IB4 mAb. In five of the six AML patients, anti-CD38 mAb stimulated the proliferation of AML colony-forming cells. These results suggest that ligation of CD38 can induce the proliferation of leukemic cells and may play a role in the propagation of leukemic cell clones in certain cohorts of AML patients.
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Oncostatin M (OSM) is a cytokine produced by activated T lymphocytes and macrophages. OSM is structurally and functionally related to leukaemia inhibitory factor (LIF), another cytokine in the interleukin 6 (IL-6) family. The biological activities of OSM are mediated through two types of receptor complexes, the LIF/OSM shared receptor (type I) and OSM-specific receptor (OSM-R, type II), which is composed of gp130 as a binding subunit and a newly identified affinity conversion subunit, OSM-R beta. Previous research conducted in the authors' laboratory has shown that OSM inhibits the growth of several breast cancer cell lines. To investigate whether OSM has a similar effect in primary normal human mammary epithelial (HME) cells, the activity of OSM in HME cells derived from four donors was examined. OSM produced a dose-dependent inhibition of DNA synethesis in these cells. In order to determine the receptor subtypes mediating OSM activity in HME and breast cancer cells, flow cytometry analysis using anti-gp130mAb and anti-OSM-R beta mAb was performed. In these studies, the authors were able to examine expressions of gp130 and OSM-R beta. In addition, quantitative RT-PCR assays were conducted to measure expressions of the mRNAs of the subunits for type I and type II OSM receptor. The results show that HME cells and most breast cancer cell lines express both the type I and the type II OSM receptors. However, type II, OSM-specific receptors are expressed at a higher levels than type I, OSM/LIF shared receptors. Accordingly, we compared the growth regulatory activities of OSM with LIF in HME cells and in breast cancer cells. In contrast to the inhibitory activity of OSM, LIF stimulated the growth of breast cancer cells, whereas it had no effect on normal mammary epithelial cell growth. Together, these data suggest that OSM plays an inhibitory role in normal and malignant mammary epithelial cell growth in vitro. OSM activity is mediated by the OSM-specific receptor (type II), not by the OSM/LIF shared receptor.
The LYT-10 gene was initially cloned by virtue of its disruption by the translocation breakpoint in some t(10;14) lymphoid neoplasms. LYT-10 is now known to encode a component of the NF-kappaB family of transcriptional activators and has therefore also been designated NFkappaB2. Activation of NF-kappaB is generally associated with its transfer to the nucleus and is followed by a rapid increase in expression of its target genes, which include cytokines such as interleukin-6 (IL-6). IL-6 can also be induced by other transcription factors such as NF-IL6. We studied the interaction of IL-1 and these transcription factors in two renal cell carcinoma cell lines (ACHN and Caki-1). These lines produce high levels of IL-6, show endogenous chloramphenicol acetyltransferase activity for the IL-6 promoter, and have high basal levels of transcripts encoding the NF-kappaB components Lyt-10, p50, and p65 as well as the NF-IL6 transcription factor. IL-1alpha and IL-1beta markedly increased steady-state levels of LYT-10 (NFkappaB2) transcripts and nuclear Lyt-10 protein in both cell lines. Levels of the NFkappaB1 (p50-encoding), p65, and NF-IL6 transcripts also increased after IL-1 exposure. These changes were accompanied by a 20-fold or greater increase in levels of IL-6 messenger ribonucleic acid (mRNA) and protein. Our observations suggest that the mechanism by which IL-1alpha or IL-1beta induces IL-6 may be mediated through increases in LYT-10 mRNA and protein levels as well as increases in expression of other transcription factors (NFkappaB1, p65, and NF-IL6), in addition to the known ability of IL-1 to post-translationally activate NF-kappaB.
We describe a new mature B-cell acute lymphoblastic leukemia (ALL) cell line designated Z-138 that was derived from a patient with chronic lymphocytic leukemia (CLL) whose disease underwent transformation to a rare, aggressive form of mature B-cell ALL. This cell line has an L3 morphology, ultrastructural characteristics of lymphoblasts, B-lineage surface markers and an immunoglobulin heavy-chain gene rearrangement identical to the rearrangement observed in the patient's blasts from whom the cell line was derived. Z-138 cells produce granulocyte-macrophage colony-stimulating factor (GM-CSF) and high levels of granulocyte-CSF (G-CSF), but they do not exhibit a proliferative response to either cytokine. Both the patient's lymphoblasts and Z-138 cells exhibited cytogenetic abnormalities including t(8;14), t(14;18) and a chromosome 11 abnormality similar to the t(11;14) of the parental cells, resulting in marked overexpression of cyclin D1 (BCL-1 (PRAD1)) mRNA in Z-138 cells. Since these karyotypic anomalies have been associated with low grade (t(14;18)), intermediate grade (t(11;14)) and high grade (t(8;14)) lymphomas, their development may be involved in the unusual aggressive transformation of this patient's CLL.
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Leukemia inhibitory factor (LIF) is a pluripotent cytokine which has a diverse array of effects on hematopoietic and epithelial cells. Depending on the nature of the target cells, these effects can be growth-stimulatory or growth-inhibitory. Receptors for leukemia inhibitory factor (LIFR) have been identified on a variety of hematopoietic and epithelial cells. We have recently demonstrated in vitro growth stimulation of human breast cancer cells, both primary tumors and cultured cell lines, by LIF. To begin to understand the in vivo relevance of these observations, we investigated the expression of LIF and LIFR in human breast cancer specimens. Specimens from 50 cases were immunostained with mouse monoclonal antibodies D62.3 and M1 (to stain for LIF and LIFR, respectively). LIF expression was observed in 78% of the specimens and correlated with favorable biological features, i.e. low S-phase fraction (SPF) (P = 0.001) and diploidy (P = 0.08). LIFR expression was observed in 80% of the tumors and correlated with the presence of estrogen receptor (ER) (P = 0.04) and diploidy (P = 0.07). Coexpression of LIF and LIFR was associated with diploidy (P = 0.02) and low SPF (P = 0.05). LIF staining was primarily cytoplasmic whereas LIFR staining was cytoplasmic in the majority of cases and membranous in a minority of cases. The presence of LIFR in the primary tumor specimens correlated with the growth stimulation of tumor cells (derived from the same specimens) by exogenous LIF in methylcellulose colony assays. The findings support a widespread but probably complex role for LIF and LIFR in breast tumor growth regulation which should be investigated in greater detail in larger cohorts of tumors.