Immunologically mediated aplastic anemia in mice: evidence of hematopoietic stromal injury and injury to hematopoietic stem cells.

Immunologically mediated aplastic anemia (AA) results when lymph node cells (LNC) from C3H/He mice are injected intravenously (i.v.) into H-2 identical CBA/J mice previously given 600 cGy sublethal total-body gamma irradiation (TBI). Previously, we showed that T lymphocytes injure pluripotent hematopoietic stem cells and cause severe pancytopenia and death in 80 to 100% of mice within 3 to 4 weeks, with changes in the bone marrow suggesting stromal injury. The following models were used to study the stroma: (1) Transplantation of femurs from AA mice into normal syngeneic CBA/J mice. After 6 weeks, colony-forming unit-spleen (CFU-S) levels in the femur implants were measured in both AA and control mice (600 cGy TBI only). (2) Development of Dexter long-term bone marrow cultures from AA and control mice, which were used to support hematopoietic bone marrow cells (colony-forming units-granulocyte/macrophage [CFU-GM]) from normal mice. (3) Cellulose ester membranes (CEM) were coated with hematopoietic stroma from AA and control mice and then implanted intraperitoneally (i.p.) into syngeneic CBA/J mice. Six months later, the CEM were removed and analyzed for the presence of trilineal hematopoiesis and bone. Injury to the hematopoietic stroma was documented by the following: (1) Femurs from AA mice had a decreased number of CFU-S compared to controls; (2) Dexter cultures from AA mice formed abnormal stromal layers with a decreased capacity to support CFU-GM from normal donor mice; and (3) CEM coated with stromal cells from AA mice had a decreased capacity to support trilineal hematopoiesis and bone compared to CEM coated with marrow stroma from control mice.

Normal and aberrant expression of cytokines in neoplastic cells from chronic lymphocytic leukemias.

Molecular expression of cytokines and cytokine receptors associated with B-cell growth and differentiation was examined in cells from B-cell chronic lymphocytic leukemia patients using the PCR. These studies were undertaken in order to determine whether a particular cytokine could be associated with leukemic transformation in this disease. The precursor-lymphoid and pro-B, pre-B-cell growth factor, interleukin-7, was found to be expressed in 30 of 30 patients, whereas, it was not expressed in normal donor peripheral blood lymphocytes (0 of 8) or in purified B-cell subsets from normal individuals. IL-1 beta and IL-2 receptors, on the other hand, were expressed by B cells from both normal and B-CLL patients. Other cytokine and cytokine receptors examined were not consistently expressed by all donors. Thus IL-7 was found to be the only cytokine tested that was expressed in CLL cells and not in normal cells.

Hematopoiesis on cellulose ester membranes. XIII. A combination of cloned stromal cells is needed to establish a hematopoietic microenvironment supportive of trilineal hematopoiesis.

A mixture of stromal cells from murine bone marrow placed upon cellulose ester membranes (CEM) and then implanted intraperitoneally (i.p.) in mice results in a regenerated hematopoietic microenvironment which supports trilineal hematopoiesis. We used this model to study the capacity of 5 cloned murine stromal cell lines of marrow origin to support hematopoiesis in vivo: MBA-1 (fibroblast); MBA-2 (endothelial); MBA-13 (fibroendothelial); 14F1.1 (endothelial-adipose); and 14M1.4 (macrophage).10(7) stromal cells of a single cell line were applied to 1.5 cm2 CEM, which were folded into tubes and implanted i.p. into mice. Similarly, combinations of 4, 3 and 2 stromal cell lines were applied to CEM and implanted i.p. Single lines were implanted into syngeneic hosts of the same murine strain from which the clone was derived and into nude mice. Combinations of stromal cells were implanted only in nude mice to avoid allogeneic incompatibility. CEM implants were removed after intervals of 5 to 36 weeks and examined histologically. 1) Stromal cells of a single phenotype did not develop hematopoiesis. 2) A combination of 4 stromal phenotypes (MBA-1, MBA-2, MBA-13 and 14F1.1) formed a hematopoietic microenvironment supportive of trilineal hematopoiesis and bone. 3) The combination of 14F1.1 (endothelial adipose) + a second stromal phenotype--MBA-1 (fibroblast) or MBA-2 (endothelial) or MBA-13 (fibroendothelial) also supported trilineal hematopoiesis and bone. 4) CEM coated with MBA-13 or MBA-1 developed bone but no hematopoiesis. The endothelial-adipose phenotype appears to be essential to support hematopoiesis but requires other types of stromal cells--fibroblast, fibroendothelial or endothelial phenotype.

Comparison of chlorambucil and prednisone versus cyclophosphamide, vincristine, and prednisone as initial treatment for chronic lymphocytic leukemia: long-term follow-up of an Eastern Cooperative Oncology Group randomized clinical trial.

The Eastern Cooperative Oncology Group (ECOG) conducted a study in which patients with advanced chronic lymphocytic leukemia (CLL) were randomized between a regimen consisting of chlorambucil (30 mg/m2 orally day 1) and prednisone (80 mg orally days 1 to 5) (C + P) administered every 2 weeks and a more intensive regimen of cyclosphosphamide (300 mg/m2 orally days 1 to 5), vincristine (1.4 mg/m2 intravenously [IV] day 1), and prednisone (100 mg/m2 orally days 1 to 5) (CVP) given every 3 weeks. Treatment was continued for up to 18 months to maximal response. Of the 122 eligible patients, 60 received C + P, while 62 received CVP. With a median follow-up of 7 years, there were no significant differences in survival (4.8 v 3.9 years, P = .12), complete remission (CR) rate (25% v 23%; P = .83), or duration of response (2.0 v 1.9 years; P = .78) between C + P and CVP. Toxicity was modest despite the prolonged treatment. The long median survival of 4.1 years for stage III and IV patients is superior to that usually reported. This could stem from continuing treatment to maximal response rather than an increase in intensity of therapy. These results are comparable to those reported with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) therapy by other investigators. The data suggest that intermittent C + P administered to maximal response continues to be the standard treatment approach for advanced CLL.

High-dose dexamethasone for refractory or relapsing multiple myeloma.

In order to assess the efficacy and toxicity of dexamethasone as a single agent without the concomitant infusion of Adriamycin and vincristine (VAD), an ECOG pilot study was initiated using 40 mg by mouth daily for 4 days every week for 8 weeks. Patients who responded were then maintained on the same treatment, but at 2 week intervals. Of the 32 patients evaluable for response, three were completely refractory to all prior therapy. All patients had advanced disease and 26 had received multiple prior treatments. There were 13/32 (40%) objective responses by ECOG criteria. Of the 28 patients evaluable for subjective response, i.e., significant decrease in performance status and/or bone pain, eight (28.5%) responded. Of the 34 patients evaluable for toxicity, 19 patients (55%) had moderate to severe side effects, including nine who had central nervous system effects, three who had gastrointestinal bleeding, two who had pulmonary emboli, one with psychosis, and four who had serious infections with one death. Median survival for the entire group was 19 weeks, with 31 weeks in the responders and 9 weeks in the non-responders. Although high-dose dexamethasone is capable of producing a significant number of partial responses (40%), it is associated with excessive toxicity. Less frequent administration of the dexamethasone at 2 week intervals was well tolerated in the maintenance of partial response, but has not been studied for efficacy in induction of remission.

Hematopoiesis on cellulose ester membranes (CEM). XII. Effect of membrane enrichment by purified matrix proteins.

Cellulose ester membranes (CEM) were enriched with the following purified matrix proteins: collagen I, II, IV, proteoglycan and laminin. Fifteen milligrams of each were placed on CEM which were then folded into open-ended tubes implanted i.p. and s.c. CEM were removed after 3, 6 and 12 months and examined histologically. There was no evidence of hematopoiesis or new bone formation on the implanted, enriched CEM at any of the intervals examined. Collagen I and proteoglycan-enriched CEM showed evidence of increased sinusoid-like vascular structures.

Hematopoiesis on cellulose ester membranes. XI. Induction of new bone and a hematopoietic microenvironment by matrix factors secreted by marrow stromal cells.

Cellulose ester membranes (CEM) were coated with stromal cells from bone marrow (BM) or bone and implanted intraperitoneally (IP) in CAF1 mice for intervals of 1 to 6 months. Previous studies indicated that matrix factors [glycoproteins (GPs), proteoglycans (PGs), and glycosaminoglycans (GAGs)] were secreted by the regenerating stromal cells and adsorbed by the CEM. After 1 to 6 months, the CEMs were removed, scraped free of adherent cells, and irradiated in vitro with 40 Gy. The scraped and irradiated CEMs were then reimplanted IP or subcutaneously (SC) for periods of 1 to 6 months in secondary syngeneic murine hosts. They were then removed for histologic study. CEMs reimplanted in SC sites developed bone and hematopoiesis as early as 1 month after implantation. Maximum hematopoiesis and bone formation was observed after 3 months. CEMs coated during the initial implantation with bone-derived stromal cells contained more bone and hematopoietic cells than did CEMs coated with marrow-derived stromal cells after SC implementation. Neither the CEMs coated with bone stromal cells nor those coated with marrow stromal cells developed new bone or trilineal hematopoiesis after being implanted IP. A few CEMs contained small foci of granulopoiesis only. We conclude that noncellular matrix substances deposited on CEMs by bone, and to a lesser degree by marrow cells, can induce prestromal cells in the SC tissues to produce a microenvironment suitable for trilineal hematopoiesis.

Attenuated high-dose cytosine arabinoside in the treatment of the elderly patient with acute nonlymphocytic leukemia. An Eastern Cooperative Oncology Group Pilot Study.

Seventeen elderly patients with acute nonlymphocytic leukemia (EP-ANLL) were treated with cytarabine, either 1.5 g/m2 or 2.0 g/m2 q 12 h for 4 days [attenuated high-dose ARA-C (HDARAC)]. One complete and one partial response was seen in 15 evaluable cases. Toxicity, evaluated in all 17 patients, was severe, with 47% showing a variety of Eastern Cooperative Oncology Group grade 3, 4, or 5 toxicities. Forty-one percent of all patients died within 33 days of initiating treatment. We conclude that attenuated HDARAC is ineffective in inducing remission and is very toxic in the EP-ANLL.

Inhibitor of granulocyte-macrophage colony formation in plasma of mice rendered aplastic by allogeneic lymph node cells.

Sublethally irradiated CBA/J mice injected with lymph node cells (LNC) of C3H/He mice exhibit aplastic anemia within 3 weeks. Aplastic anemia plasma (AAP) from these mice was found to inhibit granulocyte-macrophage colony (GM-CFU) formation. This inhibitory action was not strain specific and was not generated in donor:host combination involving other strains. AAP also inhibited the formation of colonies derived from leukemic cell lines. Though this activity inhibited GM-CFU, it did not affect erythroid colony formation. Two experiments were performed to examine the mechanism of inhibition. Superoptimal concentrations of recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) did not reverse AAP-induced inhibition of colony formation. Bone marrow cells preincubated with AAP for 24 h and washed were unchanged in their ability to form GM-CFU colonies. Thus, the inhibitory activity acted neither as a competitive nor a cytotoxic agent. Interferons and certain prostaglandins, known to inhibit colony formation, were not found in active concentrations in AAP. The inhibitory activity of AAP was heat stable, nondialyzable, inextractable with chloroform, precipitable with 50% ammonium sulfate, and had a molecular weight of 100,000 daltons. In contrast, control plasma from mice given only sublethal irradiation and injected with saline had significantly less inhibitory activity, which was not heat stable and was extractable with chloroform. Thus, LNC in certain host mouse strains generate a plasma activity that can inhibit the formation of normal and leukemic GM-CFU colonies.

The ultrastructure of hematopoietic stroma on cellulose ester membranes implanted intraperitoneally into Sl/Sld and Sl+/Sl+ mice.

The structural features of hematopoietic stromal elements forming on cellulose ester membranes (CEM) implanted intraperitoneally into hematopoietically impaired, anemic Sl/Sld mice and their normal Sl+/Sl+ littermates were compared by combined light and electron microscopy. The generally thicker, multilayered stroma lining the Sl+/Sl+ CEM implants developed from a bed--a syncytium--of large, highly pleomorphic macrophage-type lining cells whose filopodial extensions exhibited extensive interactions (i.e., nurse cell interactions) with both stromal and hematopoietic elements. In contrast, the thinner stromal layers lining the CEM of Sl/Sld mice formed from a base of dysplastic lining elements. These CEM-lining macrophage-type cells had much reduced cytoplasmic volumes, less extensive interactive surface projections, and an absence of select types of cytoplasmic organelles (e.g., membrane-bound crystalline inclusions). These observations suggest that the reduction of cell layering and, in turn, hematopoietic support activity, is due to an impaired interactive capacity of these elemental lining cells, i.e., pleomorphic macrophagic cell types, in the hematopoietically impaired strain of Sl/Sld mice.

Hematopoiesis on cellulose ester membranes (CEM). IX. Enrichment of membranes with adherent layers from long-term marrow culture from normal or drug-treated mice (hematopoietic microenvironment study II).

Cellulose ester membranes (Millipore) or polytetrafluoroethylene (Mitex) membranes were coated with adherent layers taken from Dexter-type long-term cultures, 4-5, 8 or 12 weeks after initiation of culture. The cultures were established with marrow taken from untreated mice or, in some cases, from mice treated with a single lethal dose (LD10) of carmustine (BCNU) or cyclophosphamide. In the studies using untreated mice, the cultures went for 8 or 12 weeks and in the drug studies, for 4-5 weeks. The 8 and 12 week cultures were reseeded at 4 weeks. The membranes were implanted into the peritoneal cavities of mice for 3-12 months after which they were removed, fixed, sectioned and stained for histologic study. After 6 months of implantation, about 40% of the membranes coated with cells from non-drug-treated mice and 60% of the membranes coated with cells from drug-treated mice contained hematopoietic elements; often there were foci of trilineal hematopoiesis. Hematopoiesis never occurred without bone formation, but the reverse was not true. Membranes coated with adherent layers established from marrow of mice treated with cyclophosphamide or BCNU showed two main characteristics: 1) they supported hematopoiesis normally, and 2) the regeneration of stroma and hematopoiesis occurred earlier than in membranes coated with stroma derived from normal mice, perhaps because the cells from the drug-treated mice spent a shorter time in culture. In vitro culture may damage cells required to condition the membrane for hematopoiesis.

Acute leukemia following treatment of polycythemia vera and essential thrombocythemia with uracil mustard.

Transformation to acute leukemia (AL) is known to occur in polycythemia vera (PV) and essential thrombocythemia (ET). Myelosuppressive therapy with agents such as 32P and alkylating agents increase this risk in both disorders. The alkylating agent, uracil mustard (UM), which is an effective agent for controlling thrombocytosis, has not been reported to be leukemogenic. We have treated 29 patients with UM (9 treated continuously and 20 treated intermittently): II with PV, 16 with ET, and 2 with myelofibrosis (MF). Three patients developed AL, two after continuous therapy. These two patients with PV had received the fourth highest and highest total dose of UM, and their duration of treatment was the third and fourth longest among the nine patients treated continuously, respectively. One out of 20 patients treated intermittently with UM developed AL. This patient (3) with ET had received the highest total dose of UM, and her duration of treatment was the longest among the 20 patients treated intermittently.

Possible cytogenetic marker associated with myelofibrosis in chronic granulocytic leukemia and its prognostic significance.

An association between myelofibrosis (MF) and chronic granulocytic leukemia (CGL) has been recognized. MF is usually a sign of a poor prognosis but its relation to other important parameters of CGL is not known. We observed a 54-year-old, white male patient who was well until May 1983 when he began developing gradually increasing right hip and left shoulder pain. Clinical evaluation 3 months later revealed splenomegaly and a white blood count of 126,000 with 29 segmented neutrophils, 22 bands, 7 metamyelocytes, 11 myelocytes, 6 promyelocytes, 5 blasts, 2 eosinophils, 5 basophils, and 3 lymphocytes. Cytogenetic analysis by G-banding technique showed a male karyotype with all 20 bone marrow cells examined positive for the Philadelphia chromosome. The patient was placed on busulfan therapy with good symptomatic improvement, but later suffered severe thrombocytopenia. At the end of October 1983, he was admitted with blast crisis and thrombocytopenia and was initiated on vincristine and cytosine arabinoside therapy. His bone marrow was repeatedly inaspirable and the biopsy was characterized by diffuse fibrosis. Chromosome analysis of 16 spontaneously dividing cells in the blood at this time revealed that 86% of cells had a karyotype of 46,XY,t(9;22)(q34;q11),t(1;3)(p32;p21) with the rest of the cells having only the Ph chromosome. The patient died 4 months later of intracranial hemorrhage. Chromosome #3 involvement has been reported in acute MF and essential thrombocytosis, but no specific cytogenetic abnormalities have been found in MF associated with CGL. It is unclear whether t(1;3) in this case represents a cytogenetic marker of MF or blast transformation, but it is certainly associated with poor prognosis and short survival.

Immunologically mediated aplastic anemia in mice: effects of varying the source and composition of donor cells.

Experimental aplastic anemia (EAA) can be induced in CBA/J mice when they are sublethally irradiated and injected with lymph node cells (LNC) from C3H/He mice. Mice injected with LNC die of severe pancytopenia and marrow aplasia. In the present study, cells from other anatomical locations and subsets of LNC were examined for their ability to induce and to modulate EAA. Of peritoneal, splenic, and thymic cells, only cells from the thymus had EAA activity. C3H/He bone marrow cells did not induce any adverse effects in sublethally or lethally irradiated CBA/J mice. LNC, when depleted of B or phagocytic cells, retained EAA activity. In contrast, LNC depleted of T cells had significantly less EAA activity. Furthermore, when T cells of LNC were separated into peanut agglutinin (PNA)-receptor-positive and negative fractions, only the PNA- cells were able to induce EAA. EAA activity was lost when LNC were irradiated (1000 rad). The inability of splenic or bone marrow cells to induce EAA could have been due to the presence of cells that suppressed EAA activity. When splenic or bone marrow cells were coinjected with LNC, EAA was not induced. Coinjected irradiated splenic cells, but not bone marrow cells, were still able to inhibit EAA activity. Bone marrow cells seemed to inhibit EAA by replacing stem cells that were lost during the EAA process. On the other hand, splenic cells appeared to suppress EAA activity of LNC. Thus, radiosensitive PNA- T cells of lymph nodes or thymus were capable of inducing EAA, and their activity could be modulated by radioresistant splenic cells.

Prolonged unmaintained remission after intensive consolidation therapy in adult acute nonlymphocytic leukemia.

Thirty-five adults with acute nonlymphocytic leukemia who were in complete remission after initial induction therapy received a single course of high-dose cytarabine and amsacrine as consolidation therapy. No further therapy was administered. Despite substantial toxicity, the median duration of disease-free survival was 12 months, and 30% of patients are projected to be alive in continuous complete remission at 3 years. A single course of intensive postremission chemotherapy provides long-term disease-free survival in the absence of any further treatment.

Amsacrine [4'-(9-acridinylamino)-methanesulfon-m-anisidide] (m-AMSA) and 5-azacytidine (AZA) for remission reinduction in patients with relapsed adult acute nonlymphocytic leukemia. An ECOG pilot study. Eastern Cooperative Oncology Group.

Twenty-two evaluable adult patients with relapsed, acute nonlymphocytic leukemia (ANLL) were treated with the combination of amsacrine (m-AMSA) and 5-azacytidine (AZA) as part of an Eastern Cooperative Oncology Group (ECOG) pilot study to evaluate efficacy and toxicity. Each drug was given in a dosage of 150 mg/m2 i.v. daily for 5 consecutive days. A complete response (CR) was obtained in 8 of 22 patients (36%) and a partial response was seen in two others, yielding an overall response rate of 45%. Median survival for all 22 patients was 2.5 months, but medium survival was 7.2 months (range 4.3-13 months) for those with a CR. Twelve of 22 died during the first 3 months, seven of these during the period of drug-induced aplasia. Moderate to severe toxicity included serious infection (16 of 22); nausea, vomiting, and diarrhea (19 of 22); and mucositis (10 of 22). There were four instances each of cardiac abnormalities and hepatic abnormalities but all reversed spontaneously. We conclude that this combination therapy cannot be recommended for further investigation in relapsed patients with ANLL since there was no notable increase in long-term survival and since there were 10 treatment-related deaths out of 22 patients.

Hematopoiesis on cellulose ester membranes (CEM). X. Effects of in vitro irradiation of stromal cells prior to application on CEM.

Cellulose ester membranes (CEM) were coated with stromal cells from murine bone or bone marrow irradiated in vitro with 1000, 2000, or 4000 rad and then implanted i.p. in CAF1 mice for periods of six and 12 months. CEM coated with stromal cells from bone showed excellent regeneration of bone and hematopoiesis after 1000 rad in vitro irradiation. After 2000 rad, hematopoietic and bone regeneration was reduced by about 50%, and after 4000 rad it was completely absent in CEM coated with stromal cells from bone. CEM coated with stromal cells from bone marrow showed no regeneration of hematopoiesis or bone after 1000, 2000, and 4000 rad in vitro irradiation and residence i.p. for six and 12 months. These results indicate that regeneration of the hematopoietic microenvironment is dependent upon living stromal cells. A difference in radiation sensitivity is demonstrated between stromal cells from bone and from bone marrow.

Hematologic emergencies.

Hematologic emergencies require thorough but prompt evaluation of the patient, availability of relatively routine laboratory tests, and immediate decisions. This article discusses the emergencies associated with red blood cell disorders, white blood cell disorders, hemostatic disorders, and transfusion reactions.

Treatment of chronic lymphocytic leukemia using chlorambucil and prednisone with or without cycle-active consolidation chemotherapy. A Southeastern Cancer Study Group Trial.

Patients with untreated chronic lymphocytic leukemia (CLL) received protocol treatment with 6 months of chlorambucil (CB) (30 mg/M2) and prednisone (P) (80 mg/d X 5) every 2 weeks. Complete and partial responders (CR, PR) were then randomized to consolidation with six more courses of CB and P or to four courses of cytosine arabinoside (25 mg/M2 every 12 hours X 8, subcutaneously) and cyclophosphamide (25 mg/M2 every 12 hours X 8, orally) every three weeks. Of the 178 eligible patients entered, 138 (78%) were evaluable for induction therapy which produced a 22% hematologic CR and an overall response rate (CR + PR) of 74%. Eighty-two patients received adequate consolidation, at the end of which 43 were in CR. No difference was seen in response or survival between the two consolidation treatments. Responders had longer survival than nonresponders (P = 0.0001) even when a 6-month "guarantee time" was excluded, but there was no survival difference between CR and PR. Thus, intermittent CB and P is a well-tolerated, useful therapy for CLL but the addition of cyclophosphamide and cytosine arabinoside does not improve results.