Impact of flow cytometry on predicting recurrence and survival in breast cancer patients.
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Analysis of Variance↗
Biomedical subjects
B Barlogie
Publications and source records attributed to B Barlogie.
The synergistic lethal interaction of cis-diamminedichloroplatinum and natural nucleosides is related to increased DNA cross-links.
Human tumor cells were treated in vitro with combinations of cis- or trans-dichlodiammineplatinum (DDP) and natural nucleosides (thymidine, uridine, cytidine and adenosine). Effects were measured by inhibition of colony-formation (cell survival) and DNA alkaline elution (DNA cross-links). No increments in cell lethality or DNA cross-links were elicited by any combination of trans-DDP and nucleosides. In contrast, every combination of cis-DDP and nucleoside was eminently synergistic with 5- and 10-fold increases in cell lethality over the predicted sum of each agent alone. These increments in cell kill correlated linearly with increases in DNA crosslinks suggesting that the nucleosides interact with cis-DDP to enhance its cytotoxic crosslinking mode of action.
Adenosine↗
Pretreatment of cytokinetics in acute myelogenous leukemia. Age-related prognostic implications.
To determine the clinical and biologic relevance of cellular kinetics in leukemia, DNA flow cytometric analysis was performed on bone marrow biopsy specimens from 148 previously untreated adult patients with acute myelogenous leukemia. The proportion of cells in synthesis, second growth, and mitosis (S + G2M) ranged from 4% to 33% with a median of 14%. The overall incidence of complete remission was not affected by the pretreatment cell cycle distribution. As in earlier studies, there was a marked decline in remission rate with advancing age from 73% for patients age less than or equal to 50 yr to 50% for those greater than 50 (P less than 0.01). Although not affecting remission induction overall, an increasing proportion of cells in S + G2M phase was favorable in patients under the age of 50 yr, but was associated with a progressive decline in remission rate in older patients (P = 0.01). This age-related divergent effect of cell cycle kinetics on initial response to therapy was confined to the less favorable subgroup of patients with karyotypic abnormalities, whereas patients with normal diploid cytogenetics had a consistently higher response rate regardless of proliferative activity. A positive correlation was also observed between percent of S + G2M cells and the proportion of diploid metaphases in young patients, contrasting with a negative correlation in the older age group. Our observations strongly suggest that the well-recognized prognostic effect of age on remission induction is not entirely host-mediated, but is at least partly an expression of disease-intrinsic differences between young and older patients.
Adult↗
Cytoplasmic immunoglobulin content in multiple myeloma.
Bone marrow cells of 82 patients with multiple myeloma were subjected to flow cytometric analysis of DNA and cytoplasmic immunoglobulin (CIg) content using propidium iodide and direct immunofluorescence assays. Except for two patients with nonsecretory myeloma, there was conformity in the immunoglobulin type derived from immunoelectrophoresis and plasma cell CIg staining. One patient with nonsecretory myeloma exhibited monotypic CIg staining, while the second showed no reaction. In eight patients with IgG lambda myeloma, the same tumor cells contained both lambda and kappa light chains, suggesting the productive rearrangement of both light chain genes. 14 patients with previously unrecognized plasma cells of low RNA content, all of whom were resistant to chemotherapy, were identified by CIg staining. By revealing previously unrecognized plasma cells with low RNA content, CIg analysis identified more patients with treatment-refractory myeloma.
Bone Marrow Cells↗
Intensive combination chemotherapy (ROAP 10) and splenectomy in the management of chronic myelogenous leukemia.
To investigate the role of intensive chemotherapy in chronic myelogenous leukemia (CML), we treated 37 patients who had Philadelphia-positive benign-phase disease with rubidazone 300 mg/m2/d 1 (or daunorubicin 30 mg/m2/d X 4), cytosine arabinoside 80 mg/m2/d X 10, vincristine 2 mg/d 1, and prednisone 100 mg/d X 5 (ROAP 10), every four weeks for a median of three cycles. This treatment was followed by splenectomy and by subsequent maintenance therapy with 1 to 5 g hydroxyurea daily in intermittent courses. After a median follow-up of 42 months (range, 24 to 54 months), 20 patients (54%) remain in benign phase. The projected median survival is 52 months, and the three-year survival rate is 67%. Six patients (16%) developed blastic crisis, and eight died in the benign phase. A significant cytogenetic response, defined as a fall in the percentage of Philadelphia-positive cells to less than or equal to 30%, occurred in 18 (53%) of 34 patients who had serial cytogenetic studies. Six patients (18%) had reductions to 35% to 90%, whereas ten remained 100% positive. Cytogenetic response lasted for a median of six months from the time of maximal response (range, 1 to 18 months). Blastic crisis or accelerated disease developed in seven (44%) of the 16 patients who manifested minimal or no cytogenetic response, compared to only two of the 18 patients (11%) who achieved a significant cytogenetic response. Toxicity, which resulted in one death, was due to myelosuppression and consisted of febrile episodes during neutropenia (24% of courses), documented infections (8% of courses), and bleeding (8% of courses). ROAP 10 intensive therapy produces moderate survival improvement for CML patients compared to a matched historical control group of patients treated at our institution, but it has considerable myelosuppressive toxicity. The Philadelphia chromosome response is an important treatment-related prognostic factor.
Adolescent↗
DNA-RNA measurements in patients with acute leukemia undergoing remission induction therapy.
Prior to therapy, 111 newly diagnosed adult patients with acute leukemia had DNA content measured (cell-cycle distributions) and 91 had RNA content measured using flow cytometry of acridine orange-stained bone marrow biopsies. The RNA index (RI) (ratio of mean RNA in G0/G1 of the sample to the median RNA in G0/G1 of normal lymphocytes) distinguished acute myelogenous leukemia (AML) from acute lymphocytic leukemia (ALL). The mean RI in AML was 2.2 and in ALL 1.5. RI did not predict for achievement of complete remission (CR). In AML the mean S-phase percent was 11.2 in patients who achieved CR and 13.1 in those who failed to respond to therapy, whereas in ALL it was 14.5 in those responding and 8.0 in those not responding (P = .02). In the 77 patients with either AML or ALL who achieved CR, a low pretreatment S-phase percent and a high RI correlated with a long duration of CR. S-phase percent and RI were also measured during remission induction and during maintenance therapy. Between days 8 and 18, the RI of responders decreased. In both AML and ALL an increase in S-phase percent between days 18 and 22, prior to morphological CR, was observed in responders but not in nonresponders. The mean S-phase percent at the time of morphological remission was 17.3. A high S-phase percent at this time correlated with a longer duration of CR. Although neither pretreatment S-phase percent nor RI was found to predict for achievement of CR in AML, both predicted for length of CR. Increases in S-phase percent during therapy indicated recovery of normal hematopoiesis.
Acridine Orange↗
Prognostic implications of tumor cell DNA and RNA content in multiple myeloma.
Flow cytometric studies of bone marrow DNA and RNA content were conducted in 71 previously untreated patients with multiple myeloma. There was a progressive increase in response rate with rising plasma cell RNA content. The DNA-derived ploidy level also affected chemotherapy sensitivity: only one of 11 patients with either hypodiploidy or biclonal DNA stemlines responded. DNA-RNA-defined marrow plasmacytosis was the only tumor mass-related variable adversely affecting remission induction. Survival was longer in patients with low tumor burden and favorable DNA features. The availability of objective and quantitative pretreatment variables associated with both initial response and survival should permit a risk-based selection of patients for novel treatment approaches.
Bone Marrow↗
Heterogeneity of non-Hodgkin's lymphoma probed by nucleic acid cytometry.
Flow cytometric analyses of cellular DNA, RNA, and double-stranded RNA content were performed on lymph nodes and extranodal tissue from 177 patients with non-Hodgkin's lymphoma. With increasing histologic grade, a higher incidence of aneuploidy, higher proliferative activity, and higher total and double-stranded RNA content were found. Despite considerable cytometric heterogeneity within histologic grades and morphologic subdivisions, conformity between cytometric and morphologic classifications was observed in 85% of cases. Among intermediate-grade and high-grade lymphomas, increased proliferative activity and diploidy were associated with more frequent responses to treatment. Thus, nucleic acid-derived parameters relate to morphologic subtypes and permit an objective approach to lymphoma classification based on ploidy, proliferation, and RNA characteristics that also had prognostic implications.
Aneuploidy↗
Preferential expression of double-stranded ribonucleic acid in tumor versus normal cells: biological and clinical implications.
In an effort to develop a new tumor marker suitable for flow cytometric analysis, we examined the value of double-stranded ribonucleic acid (ds-RNA) measurements using propidium iodide after DN'ase treatment. Cellular ds-RNA content was evaluated both in experimental cell lines and in clinical specimens. Higher levels of ds-RNA were present in tumor cells as compared with normal cells. In tumor cells, fluorescence was intensely localized in the nucleolus and was more diffuse in the cytoplasm. Change of less than 10% in the ds-RNA levels was observed in cell lines as a function of cytokinetic determinants such as cycle phase, culture age, and cycle traverse rate. Tumor differentiation by dimethylsulfoxide resulted in a significant decrease in cellular ds-RNA content. For quantitative comparison of clinical material, a ds-RNA excess was defined in relationship to normal peripheral blood lymphocytes. ds-RNA excess greater than 30% was observed in only one of 34 normal tissues (3%) as compared with 124 of 201 neoplastic tissue samples (62%). This incidence was higher in patients with acute leukemia (76%), high-grade and intermediate-grade lymphoma (75%), and high tumor stage myeloma (83%), as compared with chronic leukemia (20%), low-grade lymphoma (25%), and intermediate or low tumor mass myeloma (43%). Prognostically, a high pretreatment ds-RNA excess in myeloma was associated with a lower remission rate. The persistence of ds-RNA excess in the bone marrow of patients with acute myelogenous leukemia in remission predicted for a shorter remission duration (seven v 22 months; P = .05). We conclude that ds-RNA excess, as readily measured objectively and quantitatively by flow cytometry, may have important diagnostic and prognostic implications for the management of patients with malignant disease.
Animals↗
Aggressive non-Hodgkin's lymphomas in immunocompromised homosexual males.
During the period from 1981 through 1984, 14 immunocompromised homosexual males with intermediate or high-grade non-Hodgkin's lymphoma were seen at University of Texas M.D. Anderson Hospital and Tumor Institute. Six patients had diffuse large-cell lymphoma, seven had diffuse undifferentiated lymphoma, and one had unclassifiable lymphoma that suggested large-cell lymphoma. Eight patients had the acquired immunodeficiency syndrome (AIDS) and five had the AIDS-related complex. Kaposi's sarcoma was initially present in four patients and developed later in two others. The patients with diffuse large-cell lymphoma were characterized by more severely altered immune parameters, multicentric brain mass lesions, pretherapy opportunistic infections, lower performance status, poor response to therapy, and death in all within six months. The undifferentiated lymphoma group had preceding generalized reactive lymphadenopathy, less severe immune dysfunction, and excellent response to combination chemotherapy, with survival time greater than 19 months in three patients. Twelve of the patients had extranodal sites of lymphoma at presentation. There is a definite trend for the development of aggressive non-Hodgkin's lymphomas with unusual sites of extranodal involvement in immunocompromised homosexual males, with the potential for good tolerance to combination chemotherapy and improved survival in the subgroup without severe concomitant opportunistic infections.
Abscess↗
Multiple myeloma: current management.
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Antineoplastic Combined Chemotherapy Protocols↗
The hematologic malignancies. Leukemia, lymphoma, and myeloma.
Many of the conceptual advances in the treatment of advanced cancer have resulted from studies of the hematologic malignancies: The signal importance of complete remission, the complete disappearance of evident disease, as the major contributor to significant palliation; the first studies of adjuvant therapy, that is chemotherapy given to patients free of disease, which demonstrated prolongation of disease-free periods; the first studies of intensification, including early, intermittent, and late; combination chemotherapy; and finally, the important observation that advanced metastatic malignancies can be cured were made in studies of these important diseases. Because of treatment advances that have occurred over the last 10 to 20 years, the majority of patients with adult hematologic malignancies that were once considered universally fatal can be either cured or have substantial palliation. Treatment for adult acute leukemia has advanced such that 15% to 20% of patients have prolonged disease and treatment-free survivorship; in Hodgkin's disease, over 70% of patients can be cured; and for the lymphomas, the majority or 50% to 60% of patients can be cured with available treatments. Major treatment advances in supportive treatment such as allogeneic transfusion and allogeneic and autologous bone marrow transplantation improve the perspective for control of the hematologic malignancies. In addition, the potential for biologic response modifiers or the biologic products of normal cells that are normally involved in the regulation of both proliferation and differentiation show enormous potential for the treatment of advanced disease. Studies of interferon have shown promising early results in chronic granulocytic leukemia and in hairy cell leukemia. A new class of drugs, the acridine analogs, of which AMSA (4'-[9-acridinylamino]methanesulfon-M-anisidide) is a member, has been introduced and has established activity against acute leukemia. VP-16 (etoposide) has just become commercially available and is an important drug both in leukemia and lymphoma. Finally, the discovery of new knowledge about the biochemical pharmacology of drugs such as arabinosyl cytosine has offered a major advance in salvage treatment and the potential for substantial further improvement in the frontline management of these diseases. The rapid advances in both palliative and curative treatment for the hematologic malignancies have generally found broad application to the management of advanced cancer arising from other organ systems.
Antineoplastic Combined Chemotherapy Protocols↗
ARH-77, an established human IgG-producing myeloma cell line. II. Growth kinetics, clonogenic capacity, chalone production, xenogeneic transplantations, and response to melphalan.
The growth curve of monolayer cultures of ARH-77 cells, a human myeloma cell line propagated in vitro, is represented by an everbending curve on a semilogarithmic plot; however, the curve can be fitted by a straight line on a linear-linear plot. This unusual growth pattern suggests that, instead of a fixed proportion of the population, a fixed number of ARH-77 cells divide per unit time. The following are cell cycle transit time parameters calculated from percent labeled mitosis experiments: TG1, 10.0 +/- 3.5 hours; Ts, 14.3 +/- 2.3 hours; TG2, 4.3 hours; TM, 1.4 +/- 1.3 hours; and Tc, 30.0 +/- 6.1 hours. For cells exposed continuously to 3H-thymidine the values are: growth fraction, 67%; TG1, 6.5 hours; Ts, 13.0 hours; and TG2 + M, 3.0 hours. The average doubling time is 4.6 days (range, 3.8-4.7 days); after about 10 to 15 days in culture, the growth rate of freshly passaged cells declines markedly, as reflected by a growth curve with a much shallower slope. The changes are accompanied by a marked decline in the labeling index from 41.3% (range, 28.9%-53.7%) during the first 3 days of culture to less than 5% measured on day 21. Flow cytometry for DNA content-dependent cell cycle compartment distribution demonstrates an obvious decline in the proportion of S-phase cells and a marked accumulation of G2 phase cells as the cultures age. When the supernatant medium of ARH-77 cells grown for 10 days is replaced by fresh medium, a new burst of vigorous cellular growth is observed with a curve slope similar to that observed during the first 5 days of culture. If the 10-day-old supernatant medium is used to set up cultures with freshly harvested ARH-77 cells, their growth curve resembles that of 10-day-old cultures. However, this supernatant medium induces no decrease in the growth rate of other human tumor cells, suggesting that inhibition of cellular growth does not result from exhaustion of nutrients, but that ARH-77 cells produce a molecular mediator that specifically inhibits the growth of these cells. ARH-77 cells could be synchronized with a single treatment of 3 or 5 mM thymidine; (dThd) and cloning efficiency was 2% to 4% in a double-layer soft agar assay. Treatment for 1 hour with increasing concentrations of melphalan produced a threshold exponential survival curve (Dq = 0.45 microgram/ml and D0 = 0.35 microgram/ml, 1 hour).(ABSTRACT TRUNCATED AT 400 WORDS)
Adult↗
Estrogen receptor analysis by flow cytometry.
A fluorescently labeled estradiol, N'-fluoresceino-N'-(17 beta-estradiol hemisuccinamide) thiourea (FE) was used for measuring estrogen receptor content per cell in tumor cells. The cellular content of FE was measured quantitatively by flow cytometry. Binding of FE occurs in the nanomolar concentration range, an indication of the high affinity of the labeled estradiol. Competition of FE for binding sites is observed with estrogens, but not with progestins, androgens, or glucocorticosteroids, indicating the specificity of FE binding. In contrast to other estrogen receptor assays, this new technique requires a small sample size (about 5000 cells) and permits the assessment of heterogeneity in estrogen receptor expression among tumor cells.
Binding, Competitive↗
Effective treatment of advanced multiple myeloma refractory to alkylating agents.
Twenty-nine patients with advanced refractory multiple myeloma were treated with intermittent high-dose dexamethasone in combination with four-day infusions of vincristine and doxorubicin. Rapid and marked tumor-mass reduction (greater than 75 per cent) was noted in 14 of 20 patients whose disease was resistant to alkylating agents and in 3 of 9 patients with additional resistance to doxorubicin--a result far superior to those in previous trials with similar patients. In responsive patients, remissions were of excellent quality, and survival was significantly longer than in unresponsive patients.
Alkylating Agents↗
Evolution of oligoleukemia.
Chronic lithium administration to 22 patients with oligoleukemia did not alleviate cytopenia or stimulate bone marrow proliferative activity. The authors identified, however, pretreatment characteristics discriminating two evolutionary endpoints of oligoleukemia (marrow failure, 10 patients; overt acute leukemia, 12 patients): higher marrow leukemic infiltrate, normal myeloid precursor proportion, platelet count, and female sex all favored eventual transition to overt leukemia which, in comparison with marrow failure, was associated with a significantly longer survival duration from symptoms. For patients developing overt leukemia, survival from diagnosis was inversely correlated with the degree of marrow leukemic infiltrate. The lack of lithium responsiveness in oligoleukemia is consistent with the concept of differentiated leukemia with abnormalities either at the level of a lithium-responsive adherent cell elaborating colony stimulating activity (CSA) or at the level of CSA-responsive CFUs.
Acute Disease↗
Flow-cytometric analysis of DNA content in postmortem tissue.
In order to determine the heterogeneity of ploidy in disseminated cancer, we assessed the suitability of postmortem tissue for flow-cytometric analysis of cellular DNA content. Forty tissue samples from various tumor-bearing sites in 12 patients with widely metastatic cancer were analyzed. In eight cases studied there was a single unimodal ploidy level in all disease sites studied. The remaining four cases revealed bimodal tumor populations present in different proportions in the various sites studied. No difference in ploidy could be detected between irradiated and nonirradiated sites in patients who had received radiation therapy. Samples taken from normal tissues all revealed diploid DNA content, suggesting that DNA fluorescence is not altered in the immediate postmortem period. Systematic analysis of cellular DNA content in autopsy material is feasible and may contribute to our understanding of patterns of metastatic spread in patients with cancer.
Autopsy↗
Convention on nomenclature for DNA cytometry. Committee on Nomenclature, Society for Analytical Cytology.
Analysis of cellular DNA content by cytometry is important in clinical and biological research. Measurements are used widely to assess the relative DNA content of tumor stemlines and to assist in the detection and evaluation of malignant diseases. A review of the literature on DNA measurements in solid tumor and leukemias reveals a confusing variety of terms applied for the description of similar results. In order to facilitate the understanding of data and to standardize the terminology for DNA analyses, a questionnaire was distributed to more than 500 investigators. Subsequently, a workshop on terminology was held at the Combined Conference on Analytical Cytology and Cytometry IX and VIth International Symposium on Flow Cytometry, Schloss Elmau, West Germany, 18-23 October, 1982. The workshop nominated a nine-member committee to develop guidelines for nomenclature to be used in reporting results from analyses by DNA cytometry. The committee was charged by the Council of the Society for Analytical Cytology to complete this task and to publish its recommendations in Cytometry and in Cancer Genetics and Cytogenetics. The following guidelines are based on the questionnaires returned and the discussion at the workshop; they represent the unanimous recommendations of the committee. The five guidelines given herein apply to measurements of relative DNA content of cells that have been stained appropriately and analyzed by cytometry.
Animals↗