Barthel Barlogie

Cellular DNA content as a marker of neoplasia in man

Abstract Cellular DNA content was determined by means of flow cytometry with the use of DNA specific fluorochromes (ethidium bromide and mithramycin) in 516 human tissue samples from 440 subjects. Compared to human granulocytes as diploid reference standard, there was a 91 percent incidence of DNA content abnormality difference in DNA content of tumor G10 cells indicating aneuploidy in 118 patients with neoplastic disease (including nine patients who lacked histopathologic evidence of malignancy at the time of study). Ninety-four percent of aneuploid tumors were hyperdiploid. Except for six solid tumors with biclonal abnormalities in DNA content, the remainder of neoplasms were characterized by uniform DNA content with little dispersion (small coefficient of variation of tumor G10 populations). For the entire group of patients with malignant disease, three modal values of DNA content were recognized at low-degree hyperdiploidy, near triploidy and tetraploidy. Except for the prevalence of high-degree hyperdiploidy in melanomas and low-degree hyper- and hypodiploid abnormalities in malignant lymphomas, significant disease-specific patterns of abnormal DNA content were not apparent. The magnitude of ploidy abnormality was further influenced by patient age and proliferative activity of the tumor. Female patients displayed a preponderance of small-degree hyperdiploid and tetraploid tumors, whereas near-triploid abnormalities prevailed among male patients, who also harbored five of six biclonal tumors. Tumor cell ploidy did not vary among different sites of disease and upon sequential long-term follow-up examination. All 121 benign tumors had a diploid DNA content. Among the group of 209 patients with normal histology or reactive changes were seven patients with a previously established diagnosis of cancer with ploidy abnormality. This discrepancy indicates that monodispersal of the entire tissue aliquot for DNA flow cytometry is superior to histologic examination of focal neoplasia. There were two patients, one with recurrent benign pleural effusions and one with reactive lymphadenopathy, with ploidy abnormality by DNA content in whom malignant lymphoma developed. We conclude that flow cytometry of cellular DNA content is a rapid, objective, quantitative and sensitive method to determine a highly specific and stable tumor cell marker.

Results of the vincristine, doxorubicin, and dexamethasone regimen in adults with standard- and high-risk acute lymphocytic leukemia.

One hundred five untreated adult patients with acute lymphocytic leukemia (ALL) were entered on the vincristine, Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH), and Decadron (dexamethasone; Merck Sharp and Dohme, West Point, PA) (VAD) regimen. Induction therapy with VAD and VAD plus cyclophosphamide (CVAD) was followed by a 2-year rotating maintenance program with multiple antileukemic combinations, and included early intensifications with Adriamycin and high-dose cytarabine (ara-C) and a late intensification with cyclophosphamide, carmustine (BCNU), and etoposide (VP-16) (CBV) followed by autologous bone marrow transplantation (BMT). Duration of therapy was 24 to 30 months. Eight-eight patients (84%) achieved complete remission (CR) with VAD-CVAD, and 94 (90%) ultimately had CR with continuation of the maintenance as planned. Induction mortality was 3%; only half of the patients required prolonged hospitalization of 1 week or longer, or intravenous antibiotics. Maintenance therapy was given to 79 patients, while nine with histocompatibility locus antigen (HLA)-matched related donors underwent allogeneic BMT. The median remission duration was 22 months, and the median survival was 19 months. Factors associated with significantly worse CR rates were older age, the presence of hypoalbuminemia or hyperbilirubinemia, L2 or L3 morphology, and myeloid markers on leukemic cells. Those associated with significantly worse remission durations were the presence of elevated leukocyte or absolute peripheral blast counts, Philadelphia chromosome (Ph)-positive or B-cell ALL, L2 morphology, and more than one course to achieve CR. Patients could be divided into standard-risk ALL (28% of patients) and high-risk ALL (72% of patients) with long-term remission rates of 70% versus less than 30%. The 26 patients who underwent CBV autologous BMT had similar long-term outcome compared with 21 patients who did not (older age, medical contraindications, or socioeconomic problems). The presence or absence of myeloid markers on leukemic cells did not affect long-term prognosis. We conclude that VAD therapy is a well-tolerated effective induction regimen. High-risk ALL patients require alternative maintenance investigational approaches.

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.

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.

High-dose cytosine arabinoside in non-Hodgkin's lymphoma

Thirty-two patients with refractory non-Hodgkins lymphoma were treated with high-dose cytosine arabinoside (ara-C) given at 2 g/m2 IV over three hours every 12 hours for 4-8 g/m2/course repeated at three to four week intervals. There were eight partial responses (29%) and two minor responses among 28 evaluable patients. The median response duration was 10 weeks (range, 6-33 weeks). The median survival was significantly prolonged in responders compared to nonresponders (28 versus 15 weeks; p = 0.03). Two additional patients treated with 12 g/m2/course died of sepsis and myelosuppression. The dose-limiting toxicity was myelosuppression, which was more pronounced in patients with prior extensive radiation therapy and bone marrow involvement. In vivo measurements of intracellular concentrations of ara-CTP, the active metabolite of ara-C, showed significantly higher values in bone marrows with lymphomatous involvement compared to normal bone marrows (210 versus 95 microM; p = 0.05), probably indicating a preferential formation and retention of ara-CTP in malignant cells compared to normal hemopoietic cells. In addition, higher ara-CTP levels were found in bone marrows that had higher percentages of cells in S phase.

Fusarium. A newly recognized fungal pathogen in immunosuppressed patients

Two cases of disseminated fungal infection due to Fusarium species, that occurred during a 4‐month period, are reported. Both patients had a myeloproliferative disorder for which they had received intensive cytotoxic and immunosuppressive therapy, and both died despite treatment with amphotericin B. This report and review of the recent literature suggest that Fusarium is emerging as a newly recognized fungal pathogen in immunosuppressed patients.

Etoposide, dexamethasone, cytarabine, and cisplatin in vincristine, doxorubicin, and dexamethasone-refractory myeloma.

Based on remarkable activity in refractory lymphomas, a combination of etoposide, cisplatin (both administered by 4-day continuous infusions), cytarabine (Ara-C), and dexamethasone (EDAP) was evaluated in 20 patients with advanced myeloma refractory to standard melphalan and prednisone (MP) and/or vincristine, Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH), and dexamethasone (VAD) and even to high doses of melphalan (HDM) (seven patients). Forty percent of patients responded regardless of previously recognized risk factors (eg, duration of drug resistance, tumor mass, and serum lactic dehydrogenase [LDH] level). While the median survival was only 4.5 months, patients with good performance (Zubrod less than 2) and low or intermediate tumor stage survived more than 14 months compared with only 2 months for the remaining group. EDAP could be readily administered in the outpatient clinic, but neutropenic fever prompted hospital admission in 80% of patients, half of whom developed penumonia and sepsis, a fatal outcome in four patients. Severe myelosuppression was of short duration, so that subsequent cycles could be administered every 3 to 4 weeks. No serious extramedullary toxicity, including renal toxicity, was encountered. Marrow toxicity and hence infectious complications may be reduced by elimination of Ara-C without compromising treatment efficacy. We conclude that the lack of cross-resistance with VAD and even HDM makes EDAP or a similar combination an attractive regiment to be formally explored in an alternating sequence with VAD in high-risk myeloma.

Ph-positive chronic myeloid leukemia with near-haploid conversion in vivo and establishment of a continuously growing cell line with similar cytogenetic pattern.

Blast cells from a 39-year-old man in the blastic phase of chronic myeloid leukemia, with a benign phase of 15 years duration, as well as a cell line arising from this cell population, were studied. Cellular morphology, cytochemical staining pattern, and absence of terminal deoxynucleotidyl transferase showed the blast cells to be of myeloid character. Cytogenetic studies revealed the presence of two near-haploid cell populations with +8 and +8, +15, respectively, both of them containing the translocation t(9;22) in the original tumor cell sample. The cell line derived from this patients leukemic cell sample contained both near-haploid and hyperdiploid clones, the hyperdiploid clones being multiples of the near-haploid clone(s). All of the clones carried the t(9;22) in the form of a Philadelphia chromosome.

Marrow cytometry and prognosis in myeloma.

We have previously shown that flow cytometric analysis of acridine orange-stained bone marrow cells is useful for the objective enumeration and characterization of plasma cells from patients with myeloma, frequently exhibiting an abnormal DNA and an elevated RNA content. In this report on 77 previously untreated patients, we have investigated the biologic and prognostic implications of these quantitative tumor cell parameters. The degree of marrow involvement by tumor, both by microscopic and cytometric analysis, correlated with the clinically derived tumor mass stage. Examination of the product of relative tumor cell RNA content and marrow tumor infiltrate (as a measure of metabolic capacity for immunoglobulin production) in relationship to the myeloma protein concentration in the serum revealed differences in the efficiency of immunoglobulin production and/or catabolism. There was an inverse relationship between the degree of marrow tumor involvement and RNA index, suggesting a more aggressive behavior of myeloma in patients with a low tumor cell RNA content. Prognostically, high tumor cell RNA content identified patients with a high likelihood of response to both initial treatment (32 patients, P = 0.004) and salvage therapy (29 patients, P = 0.01). Favorable factors for survival were low clinical tumor mass stage (P = 0.07) and low marrow tumor infiltrate as determined morphologically (P = 0.04) and cytometrically (P = 0.004). Thus, the direct examination of marrow cellular DNA and RNA content permitted assessment of tumor burden and was useful in the prediction of response and survival.

Accumulation of S-phase cells in the bone marrow of patients with acute leukemia by cytosine arabinoside

11 adult patients with acute myeloblastic or myelomonocytic leukemia received a continuous infusion of cytosine arabinoside (6 mg/kg bw) for 48 hrs. Before and after application bone marrow punctures were made. The ceils were fluorochromed with ethidiumbromide [6] and measured in the pulse cytophotometer [5] (Phywe AG G6ttingen) within 1 hr. In 8 of these 11 cases and in altogether 13 courses DNAhistograms exhibited a 1.3-4.0 fold (mean 2.4; s. d. 1.0) increase of cells in the Sphase range (Fig.), whereas the cellular components in the smear had not changed significantly. Pulse cytophotometry has been found to combine simple cell preparation and staining, ultrarapid and accurate measurement by through flow technique and automatic registration of the DNA-histogram of 50 000-100 000 cells. RNAse treatment of cells did not remove the cytokinetic effects reported, so that fuorescence of double strain RNA by staining with ethidiumbromide [6] is negligable. An accumulation of peripheral blood cells in the early or the entire S-phase range under cytosine arabinoside therapy has been found in preceding studies [3]. In clinical tests good remission inducing effects of cytosine arabinoside resulted especially from combination with other cytostatics [1,2,4]. Our findings suggest that besides the cytocide effect of cytosine arabinoside there is an essential cytokinetic action causing accumulation of S-phase cells either by retardation of DNA synthesis or by triggering resting (Go) cells into the proliferation cycle. Cytokinetic studies in the clinic will be necessary to evaluate an optimal timing of therapy combination. We would propose a therapy regime which combines the cytocide and cytokinetic effects of cytosine arabinoside with a S-phase specific cytocide substance, controlled by pulse cytophotometry. In our cases with a marked cytokinetic effect we selected Ifosfamid ~ as second cytostatic drug by a 4 hrs. infusion of 3-3.5 g per patient.