Peripheral blood B cell labeling indices are a measure of disease activity in patients with monoclonal gammopathies.

Labeling indices (LI) provide a rapid measure of the bone marrow (BM) plasma cell proliferation rate and are useful in the diagnosis and prognosis of monoclonal gammopathies. Because circulating B cells may be a part of the neoplastic clone, we examined peripheral blood B cells that were producing the same cytoplasmic light chain isotype as the patient's monoclonal; protein (M-protein) and determined the peripheral blood LI (PBLI) by a two-color immunofluorescence bromodeoxyuridine method. The 105 patients studied were divided into three disease activity groups by standard clinical criteria. Median PBLI was 0.2% for the 29 patients with inactive monoclonal gammopathies (monoclonal gammopathy of undetermined significance [MGUS] and smoldering multiple myeloma [SMM]), 0.8% for the 35 patients with new, untreated multiple myeloma (MM), and 1.7% for the 41 patients with relapsed MM. These differences between groups were statistically significant (P less than .001, Wilcoxon). Four patients had high PBLI but clinically inactive gammopathy at the time of study, and all developed active MM within 6 months that required treatment. In 92 patients a BMLI was performed simultaneously with the PBLI (rank correlation coefficient, 0.69). In patients with new, untreated MM, use of both tests identified 72% of patients (23 of 32) with high LI, rather than 56% (18 of 32) by BMLI alone or 63% (20 of 32) by PBLI alone. These results suggest that PB B cells bearing the same cytoplasmic light chain isotype as the monoclonal protein are part of the malignant clone and can be kinetically active. The LI of these cells can provide a measure of disease activity and may help to differentiate active from inactive disease.

DNA ploidy and percent S-phase as prognostic factors in node-positive breast cancer: results from patients enrolled in two prospective randomized trials.

PURPOSE AND METHODS: To help clarify the clinical utility of flow-cytometric parameters, we performed flow cytometry on archival paraffin-embedded primary breast cancers from 502 patients treated on two adjuvant chemotherapy protocols performed by the North Central Cancer Treatment Group (NCCTG) and Mayo Clinic. DNA ploidy and percent S-phase (%S) were examined in univariate and Cox model multivariate analyses along with tumor size, menopausal and estrogen receptor status, Quetelet's index (QI), number of positive nodes and nodes examined, and Fisher and nuclear grades. RESULTS: Ploidy analysis showed that 40% of tumors were DNA diploid and 60% were DNA nondiploid (12% tetraploid and 48% aneuploid). There was no difference in relapse-free survival (RFS) (P = .82) or overall survival (OS) (P = .78) between the ploidy groups. Tetraploid patients had the longest RFS and OS of any group, but this did not achieve statistical significance. The %S was computed in 98% of cases and the medians were 9.0% for all patients, 6.4% for diploid patients, and 11.7% for nondiploid patients (P < .0001). By use of a %S greater than 12.3 as a prognostic variable in a univariate analysis, there was a significant difference in the RFS (P = .02) and OS (P = .007) of patients with low- versus high-proliferative tumors. However, when the %S was adjusted for clinical characteristics in the multivariate analysis, it was not a significant factor for RFS (P = .23) or OS (P = .36). CONCLUSION: These results indicate that DNA content and %S measurements by flow cytometry are not clinically useful independent prognostic factors in women with resected node-positive breast cancer administered adjuvant chemotherapy.

Controlled clinical trial of interferon-gamma as postoperative surgical adjuvant therapy for colon cancer.

PURPOSE: The primary goal of this study was to assess the effectiveness of interferon gamma (IFN-gamma) to prevent tumor relapse following potentially curative surgery in patients with high-risk colon cancer. A secondary goal was to determine the effect of IFN-gamma on immune function and to correlate alterations in immune parameters with survival. PATIENTS AND METHODS: Three to 4 weeks after undergoing resection of all known malignant disease, 99 patients with stage II, III, or IV colon cancer were randomly assigned to receive IFN-gamma 0.2 mg total dose by subcutaneous injection daily for 6 months or observation. Serial assessment of human leukocyte antigen (HLA)-DR expression and Fc receptors on peripheral-blood monocytes was conducted in 24 patients who received IFN-gamma and 27 control patients. RESULTS: With a median follow-up duration of 59 months in patients still alive, there was evidence of a detrimental effect on time to relapse (P = .03) among patients who received IFN-gamma. There was no significant difference in patient survival (P = .12). This study has sufficient power to rule out a 25% reduction in death rate for patients who received IFN-gamma (P < .05). Significant enhancement of immune function was observed in patients treated with IFN-gamma as measured by HLA-DR expression (P < .01) and Fc receptors (P < .001) on peripheral-blood monocytes. CONCLUSION: This study effectively rules out any clinically meaningful benefit for IFN-gamma as surgical adjuvant treatment for patients with high-risk colon cancer. Although significant enhancement of nonspecific immune function was seen with this dosage administration schedule of IFN-gamma, this was not associated with any demonstrable antitumor effect.

Blood group antigen A and flow cytometric analysis in resected early-stage non-small cell lung cancer.

The loss of blood group antigen A on tumor tissue has been reported to be a strong adverse prognostic marker for patients with resected non-small cell lung cancer (NSCLC). Results have varied with respect to the prognostic significance of flow cytometric data. We sought to confirm the prognostic significance of blood group antigen A loss and flow cytometry in a large cohort of patients with early-stage NSCLC. Two hundred and sixty patients with surgically resected stage I (n = 193) and II (n = 67) NSCLC with at least a 5-year follow-up were identified. Using paraffin-embedded primary tumor, immunohistochemical stains for blood group antigen A were performed on 90 patients with blood type A or AB. The DNA index and percentage of cells in S phase were successfully obtained on 188 and 152 patients, respectively. The median survival time of the patients with primary tumors negative for blood group antigen A was 38 months (n = 36), compared with 98 months (n = 54) for those with antigen A-positive tumors (P < 0.01). The median disease-free survival times for antigen A-negative and -positive tumors were 26 months and 98 months, respectively (P < h 0.01). The median survival time of the patients with aneuploid tumors was 51 months (n = 131), compared with 50 months (n = 57) for those with diploid tumors (P = 0.42). The median survival time of the patients with S phase >8% was 44 months (n = 105), compared with 60 months (n = 47) for those with S phase

A flow cytofluorometric double staining technique for simultaneous determination of human mononuclear cell surface phenotype and cell cycle phase.

A double staining technique for the simultaneous determination by flow cytofluorometry of cell surface phenotype and cell cycle phase is described. Peripheral blood mononuclear cells were stained with fluorescein-conjugated monoclonal antibodies for cell surface phenotype, fixed serially with 2% paraformaldehyde and 71.25% ethanol, and stained with propidium iodide to label cellular DNA. The cells were then analyzed by flow cytofluorometry for both green and red fluorescence. A variety of cells, including T cells and their subsets, B cells, NK cells and monocyte/macrophages, can be identified by this technique with simultaneous determination of cell cycle phase.

T cell depletion using anti-CD2 coated magnetic beads simplifies the detection of peripheral blood plasma cells.

The detection of peripheral blood plasma cells is clinically important, but difficult to perform by use of routine smears. To simplify the detection process, the peripheral blood T cells from ten patients with known active multiple myeloma were depleted using anti-CD2 coated magnetic beads. In all cases, there was enrichment of the immunoglobulin (Ig) positive cells after T cell depletion (mean enrichment factor, 3.4; median, 3.2; range, 1.2-5.1) with a mean pre-bead %Ig+ cells of 7.3 compared to 20.4 for the post-bead sample (p = 0.005). The monoclonal plasma cells were similarly enriched and more easily enumerated on the microscope slides prepared from the T cell depleted sample.

S-phase detection with an antibody to bromodeoxyuridine. Role of DNase pretreatment.

We have previously described a monoclonal antibody (BU-1) to 5-bromo-2-deoxyuridine (BrdUrd) that is useful for measurement of cell cycle S-phase. BU-1 hybridoma supernatant reacted with incorporated BrdUrd after the cells had been ethanol fixed; without a requirement for acid or base denaturation. We have found that this reactivity is lost if purified antibody is used, if the culture supernatants are heated, or if a mycoplasma-free hybridoma line is isolated. The supernatant contained endogenous DNase activity that was a result of mycoplasma infection of the cell line. This DNase activity was required for staining the cells with BU-1 in the absence of other denaturation steps. The endogenous DNase could be substituted for by the addition of bovine pancreatic DNase I. The disruption of the double stranded DNA structure with an enzyme rather than with harsh chemical or heat treatments does not affect protein structure or cellular morphology and allows the detection of incorporated BrdUrd of morphologic or antigenic cell subsets. DNase pre-treatment may also be useful for detection of other 'hidden' DNA antigens.

Immunofluorescence labeling indices in myeloma and related monoclonal gammopathies.

We measured plasma cell labeling indices (LI) in 52 patients with monoclonal gammopathies. Cytoplasmic reactivity with polyspecific or kappa- and lambda-specific light chain anti-Ig reagents identified monoclonal plasma cells, plasmablasts, and lymphocytoid plasma cells. Among newly diagnosed untreated patients, a high immunofluorescence LI distinguished those with multiple myeloma (MM) from those with stable monoclonal gammopathies (P less than 0.002). Among treated patients with MM, those in the plateau phase of the disease had low LI, whereas patients in the relapse phase or early in treatment had high LI. The immunofluorescence LI correctly classified three more patients with newly diagnosed MM than did the tritiated thymidine LI technique. LI specific for the neoplastic plasma cells resulted in excellent discrimination of patients with active disease. Because results are easily and rapidly obtained, this technique is useful clinically.

Infusible platelet membranes retain partial functionality of the platelet GPIb/IX/V receptor complex.

Infusible platelet membranes (IPMs) prepared from fresh or outdated human platelets have been shown to correct prolonged bleeding times in thrombocytopenic rabbits. In previous trials, IPMs did not seem to be immunogenic and lacked dose-limiting toxicity. The present study was undertaken to explore whether the platelet glycoprotein (GP) Ib/IX/V complex might retain functionality in the IPM preparation. IPMs did not spontaneously bind von Willebrand factor (vWF), but saturable binding could be induced by ristocetin, with a dissociation constant (Kd) of 0.31 +/- 0.03 microgram/mL at 1.0 mg/mL of ristocetin. Of 4 anti-GPIb-alpha monoclonal antibodies tested, AN-51 inhibited vWF binding 67.8% +/- 5.8%, whereas AS-2, AS-7, and SZ-2 were ineffective. Maximal vWF binding induced by botrocetin was only 10% to 15% of that observed with ristocetin. Retention of partial functionality of the GPIb/IX/V receptor allowing vWF binding in a modulated manner seems to represent a critical mechanism by which IPMs may provide hemostatic efficacy.

Rapid immunotyping of B-cell non-Hodgkin's lymphomas by flow cytometry. A comparison with the standard frozen-section method.

The authors compared immunotyping (IT) results obtained by both standard frozen section (FS) and flow cytometry (FC) methods on 218 biopsies suggestive of lymphoma to learn the advantages of each method. The independent interpretations of the FS and FC IT results were concordant in 93% (202 of 218) of cases. The 16 cases with discordance were reviewed and seven causes for discrepancy found: methodologic problems, focal lymphomatous involvement, more sensitive light chain detection by FC, inadequate sample for FC, interpretation error, sample mislabeling for FC, and unexplained. Eleven of the concordant B-cell non-Hodgkin's lymphomas (NHLs) studied by FC did not have a kappa:lambda ratio of 3 or greater or 0.5 or less and were shown to express light chain restriction by a D-value of 15 or greater with the use of statistical analysis of the kappa and lambda histograms or by multiparameter analysis of large versus small cells. The authors found both methods to be effective for phenotyping lymphomas, however, each has distinct features, making them complementary in their applications.

Rapid S-phase determination of non-Hodgkin's lymphomas with the use of an immunofluorescence bromodeoxyuridine labeling index procedure.

Cell kinetic measurements are currently being investigated to determine if they are useful in the clinical management of patients with non-Hodgkin's lymphomas (NHLs). Although the tritiated thymidine labeling index (TLI) is the standard method of S-phase measurement, it is difficult to perform. The authors describe a slide-based immunofluorescence labeling index (LI) method that uses 5-bromo-2-deoxyuridine (BrdUrd) as the pulsing medium and a monoclonal antibody (BU-1) to BrdUrd. The BrdUrd LI was performed on 217 NHLs and compared with routine histologic results. The authors found a median BrdUrd LI of 0.9% for low-grade NHLs; 7.5% for intermediate-grade; 10.4% for high-grade; and 2.2% for T-cell NHLs. This method provides a rapid, reliable S-phase measurement that can be easily performed in the clinical laboratory. It should replace the TLI and allow wider application of S-phase measurements in the NHL.

Pleomorphic fibrohistiocytoma of the breast: a potential pitfall in breast biopsy interpretation.

We describe a benign mammary mesenchymal tumour with atypical stromal giant cells in the contralateral breast of a 66-year-old woman with infiltrating ductal carcinoma. The clinical, morphological and immunohistochemical features of this tumour suggest a pleomorphic variant of fibrous histiocytoma. This benign lesion represents a possible pitfall in breast pathology when interpreting a frozen section or fine needle aspiration biopsy.

Renal cell carcinoma in young and old patients. Comparison of prognostic pathologic variables (cell type, tumor grade and stage, and DNA ploidy pattern) and their impact on disease outcome.

A group of 41 young patients (age less than or equal to 40 years; mean, 35.7 years) and a group of 34 old patients (age greater than or equal to 80 years; mean, 82.4 years) who underwent operation for renal cell carcinoma between 1970 and 1986 were compared. Sex, grade, and DNA ploidy pattern distributions were similar between the groups. Granular cell and papillary cancers with lower stages at presentation were more common among the young. In patients with high-stage disease, 73 percent of the older group but none of the younger had DNA diploid tumors. Low-stage clear cell carcinoma caused cancer death only in the young. Stage I nondiploid clear cell carcinomas were associated with death (33%) only in the young. Overall, death rates seem similar for both groups but among the young most (63%) occurred with low-stage disease and a nondiploid pattern only; among the old, 88 percent occurred with high-stage disease and independent of DNA ploidy pattern.

CD8+ polylobated T-cell leukemia/lymphoma. A case report with immunophenotypic, ultrastructural, gene rearrangement, karyotypic, and DNA content analysis and autopsy description.

Polylobated lymphoma is a morphologic variant of malignant lymphoma characterized by large pleomorphic neoplastic cells with polylobated nuclei. We report an unusual case in a 57-year-old man with a 9-year history of an antecedent low-grade peripheral T-cell lymphoma with dermal involvement. The polylobated lymphoma expressed the CD2, 8, 45, and DR surface antigens and had a clonally rearranged T-cell receptor beta-chain gene. The DNA content analysis indicated that most of the neoplasm was DNA diploid and tetraploid, with a high S phase. Cytogenetic analysis demonstrated the presence of a clone with an abnormal karyotype 45, X, -Y, -1, -10, -10, -17, -19, +5 mar. Serology and polymerase chain reaction analysis showed no evidence of retroviral infection (human immunodeficiency virus types 1 and 2 and human T-cell lymphotropic virus types I and II). We review the literature on polylobated T-cell lymphoma.

Expression of pS2, c-erbB-2, and cathepsin D during the menstrual cycle in human breast cancers.

BACKGROUND: Many studies have addressed the effect of the timing of surgery for breast cancer relative to menstrual cycle phase, with conflicting results. Explanations for the possibility that survival could be altered by the appropriate timing of breast cancer surgery in humans remain speculative. METHODS: We examined the expression of three estrogen related proteins (c-erbB-2, cathepsin D, pS2) in the breast tumors from 69 premenopausal women sampled in different phases of the menstrual cycle. Data on S-phase fraction and hormone receptor expression were also analyzed. Immunohistochemical assays were used to measure the proteins of interest. S-phase fraction was determined by flow cytometry. Analyses were performed based on fraction of cells staining positive for the protein, density of stain, and a histoscore that combined both fraction of positive cells and density. RESULTS: We found no differences in c-erbB-2, cathepsin D, hormone receptor, or S-phase levels in tumors sampled in the follicular versus luteal phase, or perimenstrual versus periovulatory phase. The exception was pS2, which was expressed at greater levels during the luteal than during the follicular phase of the cycle (p < 0.01); but there was no difference in pS2 expression when the patients were classified as periovulatory versus perimenstrual. CONCLUSIONS: Our findings do not support a variation in c-erbB-2, cathepsin D, S-phase fraction, or receptor expression as an explanation for the differences in breast cancer prognosis when surgery is timed by menstrual cycle phase. The finding that pS2 (an indicator of hormone sensitivity, and possibly better prognosis) is expressed at higher levels in tumor samples during the luteal phase suggests that the biologic profile of breast tumors may vary with the menstrual cycle and that these variations deserve further study.

Molecular ordering in HIV-induced apoptosis. Oxidative stress, activation of caspases, and cell survival are regulated by transaldolase.

Dysregulated apoptosis may underlie the etiology of T cell depletion by human immunodeficiency virus type 1 (HIV-1). We show that HIV-induced apoptosis is preceded by an exponential increase in reactive oxygen intermediates (ROIs) produced in mitochondria. This leads to caspase-3 activation, phosphatidylserine (PS) externalization, and GSH depletion. Since mitochondrial ROI levels are regulated by the supply of NADPH from the pentose phosphate pathway (PPP), the effect of transaldolase (TAL), a key enzyme of PPP, was investigated. Jurkat and H9 human CD4+ T cells were transfected with TAL expression vectors oriented in the sense or antisense direction. TAL overexpression down-regulated glucose-6-phosphate dehydrogenase activities and GSH levels. Alternatively, decreased TAL expression up-regulated glucose-6-phosphate dehydrogenase activities and GSH levels. HIV-induced 1) mitochondrial ROI production, 2) caspase-3 activation, 3) proteolysis of poly(ADP-ribose) polymerase, and 4) PS externalization were accelerated in cells overexpressing TAL. In contrast, suppression of TAL abrogated these four activities. Thus, susceptibility to HIV-induced apoptosis can be regulated by TAL through controlling the balance between mitochondrial ROI production and the metabolic supply of reducing equivalents by the PPP. The dominant effect of TAL expression on oxidative stress, caspase activation, PS externalization, and cell death suggests that this balance plays a pivotal role in HIV-induced apoptosis.

Immunofluorescent plasma cell labeling indices (LI) using a monoclonal antibody (BU-1).

Tritiated thymidine labeling indices (LI), although useful in diagnosis and prognosis of multiple myeloma, have not found wide-spread application because autoradiographic analysis is difficult and time consuming. Using a monoclonal antibody (BU-1) reactive with 5-bromo-2-deoxyuridine (BrdUrd), we have developed an immunofluorescent procedure that allows DNA S-phase measurements to be determined in 4 hr. Plasma cells are easily identified by reactivity with a fluorescein isothiocyanate-conjugated antihuman immunoglobulin, and cells in DNA S phase are detected via BU-1 and a rhodamine-conjugated antimouse immunoglobulin. Results using this method on 12 patients with multiple myeloma compare favorably (correlation coefficient 0.84), with those obtained by tritiated thymidine. This immunofluorescent slide method will facilitate application of labeling indices as a clinical test to measure disease activity in patients with multiple myeloma and other hematologic neoplasms.

Interleukin 2 induces proliferation of normal "resting" human T cells in the absence of other known external stimulation.

Interleukin 2(IL-2) is known to stimulate the progression of activated T cells from G1 through the rest of the cell cycle. We have demonstrated that addition of purified recombinant human IL-2 (rIL-2) to fresh normal human peripheral blood mononuclear cells (PBM), which were IL-2 receptor (Tac) negative by FACS analysis, stimulated marked proliferation of the PBM. IL-2-induced proliferation was also observed with umbilical cord blood mononuclear cells. Monocyte depletion of PBM resulted in a marked reduction of rIL-2-induced proliferative response which could be restored by adding back autologous irradiated monocytes but not by interleukin 1. The T cells preincubated with rIL-2 showed a five to six times enhanced autologous mixed-lymphocyte reaction (AMLR) compared to controls. The rIL-2-induced proliferative response of PBM was inhibited in a concentration-dependent fashion by preincubation of PBM with an anti-HLA-DR framework monoclonal antibody. The proliferating cells were shown by two-color flow cytometric analysis to be primarily Leu-1+ and Leu-4+ T cells (both leu-3+ and Leu-2+ subsets); however, 6 to 19% of responding cells had surface markers for B cells or NK cells. The data demonstrate that rIL-2 can induce proliferation of "resting" human T cells. The phenomenon may be related to a monocyte-dependent AMLR which induces IL-2 receptors and IL-2 responsiveness in a subset of T cells.

Apoptotic index by Annexin V flow cytometry: adjunct to morphologic and cytogenetic diagnosis of myelodysplastic syndromes.

The myelodysplastic syndromes (MDS) are clonal hematologic malignancies characterized by pancytopenia, dysplastic hematopoiesis, and a propensity to leukemic transformation. Increased apoptosis has been noted in MDS as a possible explanation for ineffective hematopoiesis, with lower levels in progression to and in de novo acute leukemia. Apoptosis can be measured by binding of Annexin V to exposed membrane phosphatidylserine. We postulated that the apoptotic index would aid in the differential diagnosis of MDS versus other hematopoietic diseases. We examined 33 bone marrow aspirates suspected of hematopoietic malignancy for apoptotic index by Annexin V analysis using a Becton Dickinson FACStar+ flow cytometer. The apoptotic index was expressed as the percentage of Annexin V-positive cells divided by total mononuclear cells in the gate. By standard morphologic analysis, 16 cases were diagnosed as MDS (9 refractory anemia [RA], 2 refractory anemia with ringed sideroblasts [RARS], 1 refractory anemia with excess of blasts [RAEB], 3 chronic myelomonocytic leukemia [CMML], and 1 unclassified), 11 as acute leukemia (AL), 6 as myeloproliferative disorders (MPD). Eight cases (uninvolved marrow of five patients with lymphoproliferative disorders [LPD], one patient with multiple myeloma, and two patients with anemia of chronic disease) served as nonneoplastic controls. A higher degree of apoptosis was observed in MDS (mean = 44.7%; range = 29.5--60%) compared with MPD (mean = 8.2%; range = 2.3--15.4%), AL (mean = 16.1%; range = 5.1--29.4%), and control marrow samples (mean = 11.6%; range = 1.5--21%). Additionally, the apoptotic index was significantly higher in MDS compared with MPD (P < 0.0001). In conclusion, a high apoptotic index occurs in MDS, supporting previous reports and suggesting that Annexin V analysis can be used as an adjunct in the diagnosis of MDS versus MPD. This would be particularly useful for the often-difficult distinction between early MDS and early MPD cases with equivocal morphology.