Implications of the anemia of chronic disorders in patients anticipating radiotherapy.

The authors assess prevalence of anemia in a population of patients anticipating radiation therapy. They also characterize the anemia and determine its relationship to stage, inflammation, and mortality. Blood counts were recorded for 81 of 103 patients surveyed during August 1988. When available, iron studies were used to characterize anemia. Stage, treatment, and 3-year mortality were obtained from tumor registry data. Many patients had anemia of chronic disorders. Therefore, 16 adults with solid tumors anticipating radiation therapy were evaluated with complete blood counts, iron studies, chemistries, erythropoietin, and measures of inflammation. Of 81 patients, more than half were anemic. Thirteen of 17 patients with anemia and with iron studies had anemia of chronic disorders. Two-thirds of patients with anemia had advanced cancer, versus one third of patients without anemia. Twice as many patients with anemia compared with those without anemia died within 3 years. Of 16 patients studied intensively, 4 had anemia, advanced cancer, and died within 6 months, whereas all 12 patients without anemia lived longer. The 4 patients with anemia had an elevated iron index (7.95), erythrocyte sedimentation rate (86 mm per hour), and C-reactive protein (4.48 mg/dL) versus those of 12 patients without anemia and 10 volunteers. Erythropoietin levels were not different significantly among groups. Anemia indicated a poor prognosis during and before radiotherapy, and anemia of chronic disorders was associated with advanced cancer and a short survival rate.

Paraneoplastic erythrocytosis in a young adult with an erythropoietin-producing Wilms' tumor.

Paraneoplastic erythrocytosis in patients with Wilms' tumors is exceedingly rare, with only three reported cases in the literature. We report a case of a young man with Wilms' tumor with a significant erythrocytosis but a normal serum erythropoietin level and a tumor that elaborated erythropoietin.

Suppressive effects of pentoxifylline on natural killer cell activity.

The methylxanthine derivative pentoxifylline, widely used as a hemorrheologic agent in the treatment of peripheral vascular disease, is now being evaluated for potential applications in patients with cancer. Recent studies have shown that pentoxifylline can modulate a number of neutrophil functions at in vitro concentrations of at least 50 micrograms/ml. Using a standard51chromium-release assay, we studied the suppressive effects of pentoxifylline on natural killer (NK) cell activity and found that pentoxifylline, at concentrations of 50 and 100 micrograms/ml, suppressed the in vitro NK cell activity of healthy volunteers by 25% and 75%, respectively. Postreaction supernatants from chromium-release studies were then assayed for prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNF-alpha) by using enzyme-linked immunosorbent assay. Concentrations of both PGE2 and TNF-alpha were increased by more than five times in those assay wells containing pentoxifylline. Moreover, the addition of 1 microgram/ml indomethacin to the NK assay system containing pentoxifylline, completely inhibited PGE2 production and abrogated the pentoxifylline-induced NK suppression. The addition of PGE2 (1 x 10(-6) mol/L) to the assay system suppressed NK activity, whereas addition of 1 or 10 ng/ml TNF-alpha did not. Theophylline, another methylxanthine, failed to suppress NK activity like pentoxifylline at equimolar concentrations. Our studies provide the first evidence that concentrations of pentoxifylline of at least 50 micrograms/ml suppressed NK cell function by inducing PGE2 synthesis from effector peripheral-blood mononuclear cells.

Effect of recombinant human erythropoietin on renal function in humans.

To assess the effect of recombinant human erythropoietin (r-HuEPO) treatment on renal function, the slopes of the regression lines of the reciprocal of serum creatinine versus time were compared in 26 patients with renal insufficiency (serum creatinine ranged from 2.3 to 11.7 mg/dl) followed for a period of 2.7 to 24 months. Ten patients received r-HuEPO and the anemia was corrected (Group I). Sixteen patients did not receive r-HuEPO. Ten of them were anemic (Group II) and six had normal hematocrits (Group III). All study groups were matched for age, diagnosis and degree of renal insufficiency. All cohorts were followed prospectively (Period B, from the first day of the study to the time of data analysis or dialysis and transplantation); renal function was also examined retrospectively (Period A, from the first day of the study to the time of first renal function measurement). Hematocrit was lowest in Group II control patients, 27%, highest in the Group III control subjects, 43%, and intermediate in Group I EPO-treated patients, 36%. Serum creatinine uniformly increased in all three groups of patients. The rate of progression, as measured by the slopes of the reciprocal of serum creatinine versus time, however, was similar in all three groups of subjects and during both periods. The mean slopes for Group I patients before and after r-HuEPO were, respectively, -0.0058 and -0.0054, that of the control cohorts with low and normal hematocrit during period B were -0.0063 and -0.0010, respectively. Thus, it appeared that neither r-HuEPO administration nor a normal hematocrit accelerated the deterioration of renal function in these patients with renal insufficiency.

The safety and the efficacy of maintenance therapy of recombinant human erythropoietin in patients with renal insufficiency.

Ten anemic predialysis renal patients participated in a study to examine the long-term effects of recombinant human erythropoietin (r-HuEPO) treatment. The drug was initially given intravenously three times a week for 1 to 5 months, then by subcutaneous injections three times each week for 4 to 8 months, and finally by subcutaneous injection once weekly for 3 to 18 months. The duration of follow-up ranged from 11 to 29 months. Anemia was ameliorated in all participants. Mean hematocrit increased from a basal value of 26.8% to 35.1% during the intravenous phase and to 36.7% and 34.6% during the two subcutaneous periods. Mean weekly doses of erythropoietin (EPO) were 276 units/kg during intravenous therapy and 134 and 108 units/kg in the two subcutaneous periods. The differences in the doses were significant only between the intravenous and the two subcutaneous periods. Mean erythrocyte mass increased from a baseline value of 13.6 mL/kg to 20.4 mL/kg 8 months after initiation of treatment. Mean erythrocyte survival half-time was increased from 23 days before to 26 days, 8 months after r-HuEPO treatment, P less than 0.002. Mean blood pressure (mm Hg) was 105 before and 95 after treatment. Mean serum creatinine was 513 mumol/L (5.8 mg/dL) at the beginning of the study. At the time of this writing (11 to 29 months after treatment), seven patients have required dialysis treatment. There were three episodes of transient refractoriness to r-HuEPO documented during periods of infection and surgical procedures. All subjects tolerated the medication well, and no serious side effects attributable to the medication were noted. Furthermore, circulating antibodies against r-HuEPO were consistently negative.

Recombinant human erythropoietin treatment in pre-dialysis patients. A double-blind placebo-controlled trial.

STUDY OBJECTIVE: To determine the efficacy and safety of recombinant human erythropoietin (r-HuEPO) in predialysis renal patients. DESIGN: Randomized, double-blind, placebo-controlled trial for 8 weeks. SETTING: Inpatient and outpatient facility in the Clinical Research Center of a university-based hospital. PATIENTS: Fourteen adult subjects with renal insufficiency (mean serum creatinine, 473 mumol/L +/- 61 [6.2 +/- 0.8 mg/dL]) and anemia (mean hematocrit, 0.27 +/- 0.01). INTERVENTIONS: Recombinant human erythropoietin, 50, 100, or 150 IU/kg body weight or placebo given intravenously three times per week. MEASUREMENTS AND MAIN RESULTS: Subjects who received active r-HuEPO showed a dose-dependent rise in hematocrit; mean hematocrit increased 41% from 0.27 +/- 0.01 to 0.38 +/- 0.01. At the same time, erythrocyte mass rose 43% from 13.7 +/- 0.6 mL/kg in the baseline state to 19.6 +/- 1.0 mL/kg after treatment. Maximal oxygen consumption during exercise increased 9% from 16.0 mL/min.kg +/- 1.8 to 17.5 mL/min.kg +/- 1.9. CONCLUSIONS: Recombinant human erythropoietin is effective and safe in ameliorating the anemia of pre-dialysis patients.

Differential elaboration of prostaglandin E2 by cells of the hemopoietic microenvironment in response to endotoxin.

Eight daily intraperitoneal injections of endotoxin (LPS) induced hematologic abnormalities in mice like those previously observed with chronic inflammation, sterile abscess, and tumor bearing. By the ninth day, anemia, leukocytosis, hypocellularity of the bone marrow, and compensatory hemopoietic hyperplasia of the spleen had occurred. The suppressed hemopoietic recovery and impaired survival of mice with these abnormalities, after receiving an ordinarily sublethal dose of total body irradiation (600 cGy T.B.), confirmed their importance to the intact mouse and suggested that splenic hyperplasia was insufficient to compensate for a total body deficit of functional hemopoietic stem cells. Atrophy of hemopoietic tissue in the marrow with hyperplasia in the spleen implicated changes in the hemopoietic microenvironment to account for the different responses to endotoxin. Prostaglandin E2 (PGE2) serves as an important mediator of the inflammatory response and profoundly affects hemopoiesis. Previous studies had shown that low concentrations of PGE2 enhanced, and high concentrations suppressed erythropoiesis in vitro; therefore, we wondered whether stromal cells from the marrow's microenvironment produced more PGE2 in response to LPS than splenic stromal cells to explain the suppression of hemopoiesis in the marrow and its enhancement in the spleen. Indeed, synthesis of PGE2 in primary short-term cultures of adherent marrow stromal cells in response to LPS proved much greater than that observed in cultures of splenic stromal cells. Extending adherence times from 3 to 24 to 48 hours did not change the relationship. We believe that the results of our studies point to a role of PGE2 in the microenvironmental modulation of hemopoiesis in mice with activation of the inflammatory response.

Chronic inflammation impairs hematopoiesis and survival after irradiation.

Our studies show that the induction of a chronic inflammatory lesion in the left hind legs of mice by administration of 5000 rad produced distinct abnormalities of the hematopoietic system. A peripheral neutrophilia accompanied reduced numbers of total nucleated cells, stem cells, stromal cells, erythroblasts, and lymphocytes in the unirradiated femoral marrow, and the spleen was enlarged. Mice with these hematopoietic abnormalities promptly succumbed with bone marrow failure to a sublethal dose of total body irradiation (600 rad TB). Acute inflammation associated with a sterile abscess also impaired survival after 600 rad TB. Hematopoietic abnormalities resembling those in mice with inflammation had been reported in mice bearing a solid extramedullary tumor of sarcoma-180. Concomitant studies showed that bone marrow failure and impaired survival after 600 rad TB administered to mice bearing sarcoma-180 occurred at the same time as that in mice with chronic inflammation. We concluded that chronic inflammation or tumor produced similar abnormalities in the bone marrow and spleen that led to markedly impaired survival and death from bone marrow failure after a sublethal dose of total body irradiation. Although the extramedullary hematopoiesis in the enlarged spleen indicated that its microenvironment supported hematopoiesis, whereas that in marrow was reduced, it was insufficient to compensate for a total body deficit of functional stem cells.

Tumor bearing impairs hemopoietic recovery and survival after irradiation.

These investigations were undertaken to determine whether the hemopoietic abnormalities induced by bearing a solid sarcoma-180 (S-180) impaired recovery of hemopoiesis and shortened survival of mice after sublethal total body irradiation. Mice, intramuscularly injected with 4 x 10(6) S-180 cells before or after 600 or 700 rad total body (TB) irradiation were followed for over 1 month postirradiation compared to irradiated control mice or to tumor bearing mice receiving no irradiation. Whereas more than half of the control mice survived doses of 600 to 700 rad TB, none of the tumor bearing mice survived the same dose for over 3 weeks. Irradiated tumor bearing mice all died with smaller tumors before unirradiated tumor bearing mice began to die. Because bearing tumor profoundly suppressed medullary hemopoietic stem cells (CFUS), it was not surprising to observe impaired recovery of hemopoiesis after irradiation. Except for myeloid hyperplasia induced by tumor bearing, hemopoietic repopulation in locally irradiated femoral bone marrow (1000 rad LI) by endogenous cells was greatly diminished for at least 3 weeks. The results of these studies led us to conclude that bearing a small solid tumor of S-180 dramatically shortened survival after hematosuppressive treatment with sublethal doses of total body irradiation. Since the tumor suppressed medullary stem cells (CFUS) and inhibited endogenous repopulation of locally irradiated bone marrow, we postulate that the tumor impaired recovery of hemopoiesis after irradiation leading to bone marrow failure and premature death.

Residual injury to the hemopoietic microenvironment following sequential radiation and busulfan.

Our earlier studies in mice showed that sequential radiation and cyclophosphamide suppressed marrow stromal cells (MSC) and prevented local hemopoietic repopulation for several months. Because others have shown that busulfan administration caused marrow aplasia, we studied its ability, combined with radiation, to produce a persistent microenvironmental defect in mice. Intraperitoneal administration of four weekly doses of 20 mg/kg busulfan, starting one week after 1500 rad leg irradiation, produced a severe microenvironmental lesion for 6 months reflected by lack of repopulation in femoral marrow to greater than 50% of normal by MSC, hemopoietic stem cells (CFU-S), and granulocyte-macrophage precursors. Differential marrow cell counts revealed that precursors of hemopoietic cells were more profoundly affected than their progeny. Hemopoietic stem cells and MSC failed to recover in busulfan-treated mice at 6 months to the same extent as those treated with cyclophosphamide. In addition, the busulfan-treated mice had an excessive number of myeloid blast cells and a severe erythroid depletion suggesting that these animals were preleukemic. We conclude that: 1) sequential radiation and busulfan administration caused long-term microenvironmental damage reflected by incomplete repopulation of the femoral marrow and suppression of MSC, and 2) multiple courses of busulfan prevented hemopoietic repopulation longer than a similar regimen of cyclophosphamide.

Tumor-induced suppression of marrow stromal colonies.

The studies reported in this paper evaluated one of the mechanisms by which extramedullary tumor caused anemia, neutrophilia, medullary erythroblastopenia, and suppression of marrow stromal cells (MSC) in tumor bearing mice. Since MSC have been shown to support hemopoiesis, we asked whether tumor released a suppressor which directly inhibited MSC colonies, and if it did, whether or not it was prostaglandin-E (PGE). We found that co-culture of Ehrlich ascites carcinoma (EAC) and Sarcoma (S-180) cells with normal mouse bone marrow cells profoundly suppressed formation of MSC colonies. At concentrations exceeding 15% of the medium, tumor-conditioned media not only suppressed MSC colonies but also enhanced the growth of granulocyte-macrophage colonies (CFUC) in vitro like Colony Stimulating Factor (CSF) from other sources. It did not suppress CFUE, nor did it inhibit growth or kill cells other than MSC in bone marrow cultures. Fibroblasts grew luxuriantly in 20% conditioned media from Ehrlich ascites carcinoma cells. Concentrations of PGE2 required to suppress MSC colonies greatly exceeded those detected in media conditioned by S-180. Production of PGE by S-180 cells was inhibited by growing the tumor cells in the presence of indomethacin, but the supernatant media, devoid of PGE, still markedly suppressed the growth of MSC from normal marrow. Because tumor produced CSF, we tested the suppressive effect of CSF in post-endotoxin serum to find that concentrations of 10 to 15% inhibited MSC colony growth. The results show that these tumors produced a substance, possibly CSF, that selectively inhibited MSC colony growth in vitro. It is conceivable that suppression of the supportive tissue (MSC) for erythropoiesis in the bone marrow by tumor led to diminished erythroblasts in that site.

Prostaglandin E and the erythropoietic and stromal insufficiency induced by extramedullary tumor.

This paper reports the results of our studies concerning the specificity and mechanism of anemia in tumor-bearing mice. Three different types of transplanted extramedullary tumors, including a carcinoma (EAC), a sarcoma (S-180), and a leukemia (L-1210) produced anemia, neutrophilia, and medullary erythroblastopenia. Because the most striking effects were observed with S-180, it was selected for detailed study. Although erythroblasts were greatly decreased in the bone marrow to about 1% in the differential count, CFU-E and BFU-E were not, suggesting inhibited maturation of erythroid progenitors. Suppression of MSC to 1/3 of normal occurred at 21 days of tumor bearing, and qualitatively abnormal MSC at 35 days failed to enhance CFU-E and BFU-E in split-phase culture. We found that these MSC from tumor-bearing mice produced suppressive levels of PGE. PGE production and erythroid colony enhancement of MSC from either normal or tumor-bearing mice was abrogated by including 5 micrograms/ml indomethacin in the split-phase culture. Medium conditioned by S-180 that was capable of suppressing the growth of MSC colonies had no direct effect on erythroid colony formation. Our results support a hypothesis that extramedullary tumors are capable of producing a lesion in the supportive tissue of the bone marrow, leading to anemia and medullary erythroblastopenia. We believe that early, the tumor suppresses the number of MSC required for maturation of erythroid precursors and later induces the normal numbers of MSC to produce suppressive levels of PGE.

Prostaglandin-mediated enhancement of erythroid colonies by marrow stromal cells (MSC).

Marrow stromal cells (MSC) released a diffusible substance in split-phase culture that enhanced the response of erythroid colonies to erythropoietin when MSC were present in small numbers and suppressed when they were present in large numbers. Incremental concentrations of indomethacin inhibited the enhancement, but it did not suppress growth of MSC colonies. Radioimmunoassay of conditioned media demonstrated that MSC produced E-type prostaglandin (PGE) that was inhibited by non-lethal concentrations of indomethacin sufficient to suppress MSC enhancement of erythroid colonies. Finally, when increasing concentrations of PGE2 were added to cultures, erythroid colonies were enhanced at levels similar to those produced by small numbers of MSC which enhanced and suppressed at higher levels, producing a biphasic curve similar to that noted with increasing numbers of MSC in the underlayer. These data support our hypothesis that E-type prostaglandins are released by marrow stromal cells (MSC) to mediate the enhancement and suppression of erythroid colonies, in vitro.

Characterization of marrow stromal (fibroblastoid) cells and their association with erythropoiesis.

Our previous studies suggested that a defect in the hemopoietic microenvironment of bone marrow occurred in the anemia of chronic inflammatory disease. We examined the in vivo hemopoietic and marrow stromal cell (MSC) response of shielded marrow to 5000 rad leg-irradiation (5000 rad LI), the in vitro growth of BFUE, CFUE, and CFUC in co-culture with MSC, and the in vitro characteristics of MSC. One month after 5000 rad LI, erythroblasts and MSC fell to 18% and 26% respectively. BFUE and CFUE in shielded marrow increased 1.5 to 2 times. Total CFUS and CFUC were normal. In co-culture, small numbers of MSC enhanced BFUE and CFUE, but suppressed CFUC. MSC in colonies were polygonal cells with a plating efficiency of 10 per 10(6) nucleated marrow cells and 1 per 10(6) spleen or nucleated blood cells. MSC were not phagocytic and did not stain with nonspecific esterase. We conclude that in mice with an inflammatory response to 5000 rad LI: 1) Erythroblasts and MSC decreased while erythroid progenitors (BFUE and CFUE) increased in the shielded marrow, 2) MSC stimulated BFUE and CFUE in co-culture, 3) MSC in colonies were adherent, large polygonal cells that may enhance erythroid maturation in the bone marrow.

Remission of giant lymph node hyperplasia with anemia after radiotherapy.

We have described a 51-year-old patient with unresectable mesenteric giant lymph node hyperplasia of the plasma cell type, severe systemic manifestations, and profound anemia. Supression of erythropoiesis may have been related to the presence of a circulating erythropoietic inhibitor produced by the lymphoid tumor. Markedly elevated titers to Epstein-Barr virus capsid antigen suggest that this virus may be important in the etiology of the abnormal lymphoid proliferation. The marked clinical response and decrease in the size of the tumor following irradiation suggests that radiation therapy may be an alternative form of treatment for similar patients with unresectable lesions.