P-glycoprotein expression in human plasma cell myeloma: correlation with prior chemotherapy.

Multidrug-resistant (MDR) myeloma patients failing chemotherapy may express P-glycoprotein (PGP), which serves as an efflux pump protecting the neoplastic cells. Unknown is whether PGP expression might relate to prior cytotoxic drug exposure. To address this question, we studied 106 consecutive bone marrow samples from 104 myeloma patients with samples studied either before or after therapy and at the time of relapse. We performed an established immunocytochemical assay of PGP using an MDR-1-specific monoclonal antibody and correlated PGP status with prior chemotherapy dosage. Myeloma patients with no prior therapy had a low incidence of PGP expression (6%, 3/47), whereas those receiving chemotherapy had a significantly higher incidence (43%, 21/49) (P < .0001). A substantially higher incidence of PGP expression (50%, 83%, respectively) occurred when the total vincristine dose exceeded 20 mg and when doxorubicin exceeded 340 mg. In the 11 patients who received both high vincristine and doxorubicin dosages (> 20 mg, > 340 mg total dose) there was 100% incidence of PGP expression in the tumor cells. These data provided the basis for a predictive mathematical model from which dose-related PGP expression normograms were generated. Time with myeloma for PGP-negative patients (mean 33 months) had overlapping confidence limits with PGP-positive patients (mean 42 months), suggesting that disease duration was not a significant variable. PGP expression did not correlate with other clinical factors or immunophenotypic factors. Our findings indicate a strong correlation between PGP expression in myeloma and past chemotherapy in myeloma, in particular, related to prior exposure to the natural product agents vincristine and doxorubicin. Additionally, the proportion of PGP-positive plasma cells was significantly higher in the doxorubicin-treated patients than the nondoxorubicin-treated patients (87.7% v 65.17%; P = .013). Combined high vincristine and doxorubicin total dosage appear highly predictive of PGP expression.

Diffuse small cleaved-cell lymphoma: a heterogeneous disease with distinct immunobiologic subsets.

PURPOSE AND METHODS: Diffuse small cleaved-cell lymphoma (DSCL) is a relatively uncommon non-hodgkin's lymphoma (NHL) in the United States and has not been the subject of recent in-depth study of factors predictive of outcome. It is unique among the NHL of intermediate grade because there is no evidence of a curable subset of patients. To investigate whether any laboratory data might predict outcome, we studied 33 cases collected during a 12-year period and correlated morphology, immunohistochemistry, and serum lactate dehydrogenase (LDH) with clinical data and outcome. RESULTS: We found that proliferative rate (Ki-67), cell lineage (T v B cell), and serum LDH were associated with significant differences in survival. A Ki-67 value greater than or equal to 20% was associated with a median survival of 20 months compared with 80 months for lower values (P = .0002); patients with tumors of T-cell lineage had a median survival of 20 months compared with 40 months for those with B-cell neoplasms (P = .0143); and a serum LDH greater than 225 IU/L was associated with a median survival of 8 months compared with 40 months for lower LDH levels (P = .0004). Blastoid morphology was also linked to a trend toward poor outcome (P = .08). Neither a history of low-grade lymphoma nor the presence of residual immunologically detectable follicles influenced outcome (P = .93 and .97, respectively). CONCLUSION: We conclude that high Ki-67, high LDH, and T-cell lineage each identify DSCL patients with poor outcome.

Reduction of murine cutaneous UVB-induced tumor-infiltrating T lymphocytes by dietary canthaxanthin.

The effect of dietary canthaxanthin, retinyl palmitate, or their combination on the tumor-infiltrating T-lymphocyte response (T-TIL) in de novo murine ultraviolet type B irradiation-induced tumors was investigated to elucidate potential mechanisms of action of these compounds. We found that dietary canthaxanthin greatly reduced the number of tumor-infiltrating helper/inducer, suppressor/cytotoxic, and interleukin-2 receptor-positive T lymphocytes and also observed a concomitant statistically significant increase in tumour incidence in canthaxanthin-fed animals. The addition of retinyl palmitate to the canthaxanthin diet ameliorated this negative effect on TIL and the development of skin tumors. We conclude that dietary retinyl palmitate and canthaxanthin can modulate the host T-cell immune response within a growing tumor and may affect tumorigenicity.

Prediction of Outcome in Diffuse Large Cell Lymphoma by the Major Histocompatibility Complex Class II (HLA-DR, DP, DQ) and Class I (HLA-A, B, C) Phenotype.

Ninety-nine consecutive diffuse large cell lymphoma (DLCL) patients were studied by immunohistochemistry to determine whether clinical outcome was predicted by major histocompatibility complex (MHC) antigen phenotypic expression. Statistically significantly shorter disease free survival (p = 0.005), but not overall survival (p = 0.47), was observed when patient lymphomas failed to express class I MHC antigens. We also observed significantly reduced survival of class II MHC HLA-DP negative patients (p = 0.038). This extends our previous finding of poor outcome with absent class II MHC HLA-DR in DLCL(1) to other MHC antigens and demonstrates that the phenomenon of defective class II antigen expression comprises 16% of these DLCL patients. Known clinical parameters predictive of prognosis were equally distributed between phenotypic groups. These findings indicate that aberrancy of immune phenotype in DLCL is critical to patient outcome and we speculate that loss of MHC expression may confound host immunosurveillance and tumor containment.

Optimization of immunocytochemical P-glycoprotein assessment in multidrug-resistant plasma cell myeloma using three antibodies.

Seeking to optimize the immunocytochemical assay of P-glycoprotein, a 170-kilodalton (P-170) molecule associated with multidrug resistance, we experimented with a variety of antibodies (JSB-1, C219, and MRK-16), fixation conditions, and titers using both human myeloma cell lines and clinical myeloma specimens. Under optimized conditions, using all three antibodies and the cell lines as standards and controls, the ICC method proved sensitive, specific, reliable, rapid, and within the realm of everyday hospital laboratory expertise. The 3 anti-P-glycoprotein antibodies revealed different reactivities with P-170. Both C219 and JSB1 were optimized by fixation in cold acetone. With MRK-16 optimal results were obtained on unfixed or formalin fixed specimens. Under optimal fixation and titering conditions, low level (DOX 4) detection was possible. Given that the three antibodies differ in reactivity and recognize different P-170 epitopes, it follows that using the antibodies in a small panel is a useful strategy in increasing the likelihood of detecting true P-glycoprotein expression by the immunocytochemical method. In dilution experiments, the immunocytochemical method was as sensitive as RNase protection assay and more sensitive than Western blot detection. Immunocytochemistry coupled to computer-assisted single-cell densitometry, showed a strong correlation (R = 0.98) between cellular P-170 density and in vitro resistance to doxorubicin. Multidrug-resistant specific probes for RNA expression and Western blot assays confirmed the specificity of P-170 expression in both cell lines and clinical samples. Thus, a small panel of antibodies, under optimized immunocytochemical conditions, appears to have potential as a rapid, sensitive, clinically useful assay for multidrug resistance in myeloma.

Immunobiologic factors predictive of clinical outcome in diffuse large-cell lymphoma.

The prognostic importance of immunobiologic factors in diffuse large-cell lymphoma (DLCL) is studied in 105 consecutive DLCL patients. Multivariate results using the Cox proportional hazards model clearly indicate that the Ki-67 index (P = .002), a marker of cell proliferation activity, and the presence or absence of human leukocyte antigen-DR (HLA-DR) (P = .007) are strong predictors of survival even in the presence of established clinical factors of stage (P = .015) and symptoms (P = .050). Using these four variables, prognostic groups were formed identifying patient groups with varying degrees of risk. The group of patients with three or four risk factors present at the time of diagnosis had a median survival of 4 months compared with a median survival of 59 months for the group with no risk factors. Similarly, prognostic groups for disease-free survival (DFS) were constructed based on the proportional hazards model that involved B versus T phenotype (P = .035) and HLA-DR (P = .054). Median DFS for the patient group with one or two risk factors present was 11 months compared with 43 months with no risk factors present. This study suggests immunobiologic parameters are important predictors of clinical outcome in DLCL patients and are of value in identifying subgroups of patients who have not responded to currently available therapy. The practical significance of this study is to identify parameters that may suggest specific changes in therapy of patient subgroups.

Tumor-infiltrating T-lymphocytes in B-cell diffuse large cell lymphoma related to disease course.

Recent data from studies of experimental murine tumors and certain human tumors (primarily melanoma) suggest that tumor-infiltrating T-lymphocytes (T-cell TILs) represent a highly potent and specific host antitumor response. We conducted an immunohistochemical analysis of the T-cell TIL subpopulations in frozen tissue sections taken from 82 consecutive B-cell diffuse large cell lymphoma (DLCL) patients. Initially, we analyzed the relationship in these patients between relapse-free survival (RFS) and their T-cell TIL characteristics. Nineteen patients had a low percentage (less than 6% of Leu-2+ (suppressor/cytotoxic) T-cell TILs, and 63 patients had a high percentage (greater than 6%) of Leu-2+ TILs. We found that a low percentage of Leu-2+ TILs correlated with a reduction in RFS: at 20 mo follow-up, all 19 low Leu-2+ patients had relapsed, whereas 70% of the 63 high Leu-2+ patients remained relapse-free (P = 0.008). No significant correlations appeared between patients' T-cell TIL subsets and overall survival. The percentage of newly diagnosed tumors with low counts of Leu-4+ (pan-T) TILs was marginally greater among interleukin-2 (IL-2) receptor-positive tumors than among IL-2 receptor-negative tumors (50 versus 28%, P = 0.098), which suggests that specific phenotypic characteristics of B-cell DLCL may modulate the host T-cell TIL response. Our results indicate that the host's T-cell TIL response in B-cell DLCL can be quantitated from frozen tissue sections and that this response may be related to disease course. Further related TIL studies may lead to new immunorestorative therapeutic approaches for patients with deficient or aberrant cytotoxic T-lymphocyte host responses.

Ultrastructural morphometric analysis of papillary neoplasms: biological and diagnostic relevance.

Ten papillary adenocarcinomas of thyroid origin (P-Thy), ten papillary adenocarcinomas of ovarian origin (P-Ov), and eight papillary neoplasms of non-thyroid/non-ovarian origin (P-Other) were morphometrically compared using 19 distinct quantitative nuclear and nucleolar parameters as a database for diagnosis. The selected cases consisted of 16 primary and 12 metastatic neoplasms. It was determined that the P-Thy group had a significantly smaller nucleolar area (NuA) and nucleolar perimeter (NuP), and smaller SDs of nuclear area (NA), NuA, and NuP compared with the P-Ov and P-Other groups (P less than .05). The P-Ov group had a significantly smaller SD of NA compared with the P-Other group (P less than .05). The P-Ov group exhibited the greatest variability among the papillary neoplasms. Linear regression analysis indicated that in the P-Thy group alone there was a significant correlation between mean nuclear form factor (4 pi A/P2) and mean NuA (r = -.82; P less than .01), and mean NP and mean NuA (r = +.77; P less than .01). Linear regression analysis also indicated that in the P-Ov group alone, there was a significant correlation between mean NA and mean NuA (r = +.75; P less than .02). Morphometric domains were established using statistically significant sets of variables that distinguished between the groups. The application of three-dimensional computerized cluster analysis techniques indicated that the P-Thy group consistently had the smallest morphometric domains. It was concluded that ultrastructural morphometric analysis of papillary neoplasms has diagnostic potential and reveals interesting biological relationships among distinct nuclear features in the different groups of neoplasms.

Human sera and culture supernatants from human tumors and diploid fetal fibroblasts suppress tumor necrosis factor secretion in vitro.

Human sera and culture supernatants from human tumors and diploid fetal fibroblasts suppressed peripheral blood leukocyte secretion of tumor necrosis factor (TNF). The suppressive activities of all three fluids had similar characteristics: each was heat and acid stable, removed by adsorption on immobilized lectins, and abrogated the stimulatory effect of interferon-gamma. Inhibition of leukocyte TNF secretion was observed only when either serum or conditioned medium was added to leukocytes at the initiation of culture; delaying the addition by 2 h failed to suppress cytokine secretion. Suppression by all fluids was also found to be reversible by washing cells free of suppressive activity. Although serum, tumor, and fibroblast culture supernatants inhibited cytokine secretion, they failed to alter the cytotoxic activity of recombinant human TNF on murine L929 cells. This study suggests that factors which can inhibit TNF secretion are present in human blood and are secreted by both fibroblasts and tumor cells. These suppressive factors may play an important role in the regulation of TNF secretion and cytokine homeostasis.

Alpha-globulins suppress human leukocyte tumor necrosis factor secretion.

The secretion of tumor necrosis factor (TNF) by human peripheral blood mononuclear cells was suppressed by either whole human plasma alpha-globulins or purified alpha 1-acid-glycoprotein, alpha 1-antitrypsin and alpha 2-macroglobulin in a concentration-dependent manner. alpha 1-Antitrypsin was found to be the most suppressive of the purified proteins tested and completely blocked TNF release at concentrations above 1.25 mg/ml. Both alpha 1-acid glycoprotein and alpha 1-antitrypsin blocked TNF secretion by leukocytes which were simultaneously stimulated with either recombinant human interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS). IFN-gamma- and LPS-activated cells were also susceptible to suppression mediated by these two alpha-globulins and the inhibition produced by 5 mg/ml alpha 1-antitrypsin was greater than that caused by either 1 microM prostaglandin E2 or 10 ng/ml transforming growth factor-beta 1. The level of TNF mRNA in TNF-secreting and alpha-globulin-suppressed cells was examined and found to be equal in both groups. The suppressive effect of whole alpha-globulins was confined to the inhibition of TNF secretion and these plasma proteins had no effect on the cytolytic activity of the recombinant cytokine as measured on murine L-929 target cells. Thus the alpha-globulins, which are a major fraction of the circulating plasma proteins, may function in TNF homeostasis by controlling TNF secretion without inhibiting the biological activity of the released cytokine.

Beta-carotene stimulates human leukocytes to secrete a novel cytokine.

The effect of beta-carotene on cytokine production by human peripheral blood leukocytes was tested. Beta-carotene stimulated the secretion of a novel cytotoxic cytokine when peripheral blood cells were exposed to carotenoid concentrations between 10(-6) and 10(-10) M. Beta-carotene-treated supernatants caused the cytolysis of four out of the six human tumor cell lines tested. Low level toxicity was also observed when normal diploid fibroblast lines were exposed to beta-carotene-treated leukocyte supernatants. The cytotoxic activity elicited by beta-carotene was found to be distinct from characterized cytokines based on both antisera neutralization and target cell specificity studies. This study demonstrates that beta-carotene can induce human leukocytes to secrete one or more cytokines that can manifest cytotoxic activity against human tumor cells in vitro.

Immunohistochemical detection and quantitation of P-glycoprotein in multiple drug-resistant human myeloma cells: association with level of drug resistance and drug accumulation.

Using several multiple drug-resistant human myeloma cell lines as standards, we developed an immunohistochemical staining technique and means of quantitating P-glycoprotein in individual myeloma cells. The level of staining intensity for P-glycoprotein in individual myeloma cells was quantitated by measuring the average optical density of each cell with a microscopic computerized cell analysis system. Using this system, we observed that the level of P-glycoprotein for individual cells within a cell population of known drug sensitivity was very homogeneous (coefficient of variation less than or equal to 13%). Analysis of cell lines with gradually increasing levels of multidrug resistance (8226/S, 8226/Dox6 and 8226/Dox40) demonstrated a close association between the level of resistance to doxorubicin, defined by the mean lethal dose (D0) and the amount of P-glycoprotein on individual cells determined by the optical density (r = 0.82, P less than 0.0005). Intracellular doxorubicin (DOX) accumulation in the individual cell lines was inversely related to the level of drug resistance expressed as D0. P-glycoprotein was also detected in the marrow-derived myeloma cells of patients with drug refractory disease using immunohistochemical staining. The amount of P-glycoprotein in the cells of one patient was directly compared to the amount found in the simultaneously stained standard cell lines (8226/Dox6 and 8226/Dox40) by comparing the optical densities for individual cells. Using this immunohistochemical technique to detect and quantitate P-glycoprotein in patient myeloma cells and comparing it to standard multidrug resistant myeloma cell lines may be of value in determining the contribution of P-glycoprotein to clinical drug resistance in patients with multiple myeloma.

Effect of fibronectin on antigen-induced lymphoproliferation and antibody synthesis in rats.

Macrophages are important positive and negative regulators of both primary and secondary antibody responses, and their activity may, in turn, be controlled by soluble mediators secreted by other cells. Fibronectin is a 440,000 dalton normal constituent of plasma and extracellular membranes that acts through macrophages to inhibit mitogen- and alloantigen-stimulated lymphoproliferation. We examined the effect of Fn on the antigen-stimulated lymphoproliferative and antibody responses in cells from trinitrophenol-derivitized keyhole limpet hemocyanin (TNP-KHL) primed rats. Fn in concentrations equivalent to normal plasma levels inhibited TNP-KLH-stimulated lymphoproliferation by unseparated lymph node leukocytes. When the experiment was repeated using purified lymph node T cells and added thioglycollate-induced peritoneal exudate macrophages or splenic adherent macrophages, Fn alone and TNP-KLH alone stimulated lymphoproliferation, but in combination they were strongly inhibitory. The effect was not due to decreased lymphocyte viability in the presence of both TNP-KLH and Fn. Nor was it due to complexes between TNP-KLH and Fn or to a simple alteration in the kinetics of lymphoproliferation. Fn had to be present with the TNP-KLH within the 1st hour of incubation. If macrophages were coincubated with TNP-KLH and Fn for 24 h, washed, and added to enriched T cells, inhibition was equivalent to that seen with continuous coculture. Similarly, coculture of TNP-KLH and Fn inhibited both total immunoglobulin and TNP-KLH-specific antibody synthesis at optimal concentrations of splenic adherent cells. However, at suboptimal levels of splenic macrophages, the combination was synergistic, stimulating more total immunoglobulin synthesis than either TNP-KLH or Fn alone. These data suggest that the inhibitory effect was dependent upon the concentration and phenotype of macrophages present in culture.

The aberrancy of immunophenotype and immunoglobulin status as indicators of prognosis in B cell diffuse large cell lymphoma.

To assess the prognostic significance of the immunophenotype in diffuse large cell lymphoma (DLCL), 105 DLCL patients were studied between 1978 and 1987 using a panel of 40 monoclonal antibodies applied to frozen tissue. Eighty-three patients were found to have B cell phenotypes, and 20 patients had T cell phenotypes. Focusing on markers relevant to clinical outcome among B cell LCL showed that lack of expression of the pan B antigens Leu14 and Leu16 were correlated with decreased survival (Leu14, P = 0.01; Leu16, P = 0.06; log-rank). HLA-DR activity also showed that lack of expression of this antigen correlated with poor survival (P = 0.004, log-rank). Kappa light chain immunoglobulin lack of expression showed predictive value for decreased survival as well (P = 0.005, log-rank). Multivariate analyses of known clinically important variables and the immune phenotypes confirm that the loss of HLA-DR and B cell aberrancy are independent factors predicting a poor clinical outcome. Losing some B activation/kappa antigens appears to be a broad biologic phenomenon linking surface antigen lack of expression with decreased survival. This suggests that aberrancy of immunophenotype and immunoglobulin status are key predictors of survival in B-LCL.

Independent prognostic significance of a nuclear proliferation antigen in diffuse large cell lymphomas as determined by the monoclonal antibody Ki-67.

To assess the prognostic significance of the growth fraction in diffuse large cell lymphoma (DLCL), we studied 105 DLCL patients with the monoclonal antibody Ki-67 applied to frozen tissue sections. Ki-67 detects a nuclear antigen associated with cell proliferation not found in resting cells. Ki-67 findings and other clinical prognostic factors were correlated with outcome using univariate and multivariate analyses in the proportional hazards model. High proliferative activity, defined as nuclear Ki-67 expression in greater than 60% of malignant cells (Ki-67 greater than 60), was found to be a strong predictor of poor survival among these patients (P = .003, log-rank). The 19 patients with Ki-67 greater than 60% had a median survival of 8 months compared with a median survival of 39 months for the 86 patients with Ki-67 less than or equal to 60%. Examination of pretreatment clinical variables indicated the patient groups were similar with regard to age, sex, stage, B symptoms, tumor bulk, and lactate dehydrogenase (LDH). Both patient groups received comparable curative intent therapy and showed comparable complete response rate precluding treatment differences as modifying outcome. Multivariate analysis indicated Ki-67 is an independent predictor of survival (multivariate P = .006). Further statistical analysis using only B-cell DLCL patients treated with CHOP (63 patients) indicated that Ki-67 greater than 60 retained strong prediction of poor outcome (P = .002, log-rank) among this homogeneous group. We conclude that high proliferative activity (Ki-67 greater than 60) is an independent factor allowing laboratory prediction of probable poor outcome of DLCL.

Control of ornithine decarboxylase activity in Physarum by polyamines.

The addition of putrescine, spermidine, or spermine, to cultures of Physarum polycephalum rapidly reduced the activity of ornithine decarboxylase, with maximal inhibition, 80-90%, occurring after 90 min. This response was not due to a decrease in enzyme molecules, but rather to the rapid conversion of the active enzyme to a stable, catalytically less active form. This response to exogenous polyamines was not accompanied by the appearance of a macromolecular inhibitor (antizyme) either free, or bound to the enzyme. Physiological levels of the polyamines were also found to inhibit this enzyme in vitro both competitively and non-competitively, and to promote complete yet reversible inactivation of this enzyme in the absence of reducing agents. The data suggest that the control of this enzyme by endogenous polyamine levels may be distinct from its response to exogenous polyamines.