The role of clonal selection in the pathogenesis of an autoreactive human B cell lymphoma.

To study the association of autoimmunity and human B cell neoplasia, we have established a model of a B cell lymphoma which expresses a pathogenic autoantibody of defined specificity. The Ig VH gene expressed in this neoplasm was analyzed longitudinally using clinical specimens taken from the splenic lymphoma (S) at diagnosis and from lymph node relapses 3 and 4 yr later (N3 and N4). Southern analysis and oligonucleotide hybridization experiments demonstrated that clonally related predominant and minor tumor cell populations were present in S at diagnosis, and that the minor population became the predominant population in the relapse specimens, N3 and N4. Although the Ig specificity and idiotype were the same at diagnosis and at both relapses, analysis of the expressed VH gene sequences showed 14 base changes between S and N3, and 2 further changes at N4. Little sequence heterogeneity was observed at each sampling time, indicating that the ongoing mutation frequency was low. The relevant germline precursor VH gene was determined from autologous germline DNA and compared to the expressed genes. Based on the pattern of shared and unshared mutations, we were able to establish the genealogic relationship of the germline VH gene and the expressed clonotypes of S, N3 and N4. Taken together, the findings from Southern blotting, oligonucleotide hybridization, and sequence analysis permit us to describe a molecular aspect of tumor progression, "clonotypic shift", wherein subpopulations of the malignant clone, marked by different V gene clonotypes, emerge and predominate at different time points in the evolution of the lymphoma. Furthermore, the sequential and nonrandom pattern of the VH mutations, correlated with the observed conservation of autospecificity and idiotype, implies that clonal selection may have influenced the pathogenesis of the lymphoma.

Effective erythropoiesis induced by 5beta-pregnane-3beta-hydroxy-20-one in squirrel monkeys.

The erythropoietic effect of 5beta-pregnane-3beta-hydroxy-20-one, a naturally occurring steroid metabolite of progesterone, was evaluated in the squirrel monkey by ferrokinetic studies. red cell survival, and blood volume measurements. The intramuscular administration of this steroid in pharmacologic doses shortened the (59)Fe plasma clearance and increased the plasma iron turnover, thereby indicating an increase in erythropoiesis. A normal (59)Fe red cell uptake was observed, and the bone marrow maturation time was not altered. Red cell survival was the same in the treated and control groups. After five weekly injections of the steroid, the monkeys increased their red cell mass by 57%. A significant increase in white blood cells and a slight elevation of platelet counts in the treated monkeys also suggest a possible direct stimulation of hemopoietic stem cells by the steroid metabolite. These observations indicate that some steroid metabolites can stimulate an early increase in iron turnover (within 48 h) that is not secondary to hemolysis. The increased red cell mass indicates effective erythropoiesis in primates.

Survey of exposure to genotoxic agents in primary myelodysplastic syndrome: correlation with chromosome patterns and data on patients without hematological disease.

Exposure to genotoxic agents such as insecticides, pesticides, and solvents correlated with abnormal karyotypes and development of acute nonlymphocytic leukemia (ANLL) similar to, but to a lesser degree compared to, patients exposed to irradiation and alkylating drugs in several reports. Because of the natural progression of myelodysplastic syndrome (MDS) to ANLL, we investigated the relationship of exposure to these carcinogens in patients with primary MDS by having 52 such patients diagnosed and referred to our center answer an occupational/environmental questionnaire. We excluded all secondary MDS patients with exposure to previous chemotherapy and radiation for a previous malignancy. In addition, we prospectively gave the same questionnaire to a similar number of age- and sex-matched comparable control patients from the same socioeconomic group based on their residence, health insurance coverage, and occupation. We found a 46% exposure rate to implicated genotoxic agents in our patients with MDS. Patients with MDS who were exposed had 75% incidence of a poor prognosis French-American-British classification compared to 57% in the nonexposed group but the difference was not significant (P = 0.089). However, the karyotypic abnormalities that were associated with exposure in ANLL were found equally in both exposed (55%) and nonexposed groups (50%) of our MDS patients and our control group had a similarly high exposure rate at 40% to genotoxins. Implicating a relationship between exposure to pesticides and solvents in ANLL and MDS is difficult. All the previous studies indicating such a relationship did not use a control group of patients. Our findings indicate the pitfalls of historical data without investigating the bias of obtaining an exposure history. However, our findings that the majority of our MDS patients came from the middle socioeconomic group which has a high exposure rate as shown by our control group indicate a relationship and that prospective follow-up of the exposed cohort of control patients should be done to determine if ANLL and MDS will increase after a latent period compared to the nonexposed controls.

Demonstration of two distinct antibodies in autoimmune hemolytic anemia with reticulocytopenia and red cell aplasia.

To determine the mechanism of the aplastic crisis in a patient with autoimmune hemolytic anemia (AIHA) and reticulocytopenia who developed red cell aplasia simultaneously, serum- and IgG-separated fractions were examined for the presence of erythroid progenitor cell inhibitors. The patient's red cell autoantibody was a complement-independent IgG that reacted with the little-e antigen of the Rh complex. A complement-dependent serum IgG inhibitor directed against erythroid colony- and burst-forming units but not granulocyte-macrophage units was detected in samples before treatment with extracorporeal staphylococcal protein-A immunoadsorption and corticosteroids. The erythroid progenitor cell inhibitor persisted in samples multiply adsorbed against type-ee red cells and was not detected in heat eluates prepared from these red cells. A reticulocytosis occurred when serum IgG levels were reduced to 27% of pretreatment values. At this point, the erythroid progenitor cell inhibitor was not detectable in vitro. These findings suggest that the development of the aplastic crisis in some patients with AIHA may be associated with the presence of two distinct IgG antibodies, one directed at the mature red cell and the other at the erythroid progenitor cells.

Clinical heterogeneity reflects biologic diversity in chronic lymphocytic leukemia.

As the incidence of B cell chronic lymphocytic leukemia increases in an aging population, it becomes more important to re-evaluate our understanding of the disease process and current therapy. Previous treatment strategies have been, for the most part, unsuccessful in prolonging survival and thus new approaches are needed. More intense cellular and molecular research on the biologic diversity of this neoplasm will further our understanding of the causes of clinical heterogeneity and refine our ability to predict progression. New approaches, based on alterations of neoplastic cell growth by cytokines or chemotherapeutic agents, may enable clinicians to 'customize' individual treatments based on the stages of CLL B cell differentiation and our understanding of factors involved in the regulation of apoptosis and proliferation at those stages. Taken together, these efforts should ultimately yield much new information that will lead to reduced morbidity and mortality in B-CLL, the most common form of human leukemia.

Rapid transient reversal of anemia and long-term effects of maintenance intravenous immunoglobulin for autoimmune hemolytic anemia in patients with lymphoproliferative disorders.

Seven patients with autoimmune hemolytic anemia (AIHA) associated with an underlying lymphoproliferative disorder were treated with intravenous immunoglobulin. Five patients with chronic lymphocytic leukemia, a patient with Hodgkin's lymphoma with severe AIHA associated with a "warm" IgG antibody, and a patient with non-Hodgkin's lymphoma with an IgM "cold" antibody were treated with intravenous immunoglobulin G (0.4 g/kg) daily for five doses followed by maintenance therapy every 21 to 28 days if evidence of recurrence was noted. Two additional patients with refractory chronic lymphocytic leukemia and hypogammaglobulinemia were given maintenance therapy with intravenous immunoglobulin G every 21 days for previously recurrent AIHA and infections. Hematocrit levels of patients with AIHA stabilized followed by a gradual improvement at 21 days after intravenous immunoglobulin G infusion without steroids. Treatment with steroids and intravenous immunoglobulin G resulted in faster and higher increments in hematocrit levels in these patients. Other patients who had partial responses to steroids showed further improvement in their hematocrit levels by the addition of intravenous immunoglobulin G. Another patient with a cold agglutinin disease was refractory to intravenous immunoglobulin G therapy. Five patients with chronic lymphocytic leukemia and acute AIHA and two patients with previous recurrences of AIHA required maintenance intravenous immunoglobulin G every 21 days. All seven patients except one did not have any episodes of AIHA from six months to as long as four years while receiving the three-week intravenous immunoglobulin G therapy. These observations indicate a role for intravenous immunoglobulin G in the management of IgG-mediated but not IgM-associated autoimmune hemolysis in immunocompromised patients with lymphoproliferative diseases.

Use of intravenous immunoglobulin in chronic lymphocytic leukemia.

Five patients with clinical stage III and IV chronic lymphocytic leukemia were treated with a five-day infusion of high-dose intravenous immunoglobulin for either autoimmune hemolytic anemia or immune thrombocytopenic purpura or by a dose of intravenous immunoglobulin G every three to four weeks for prevention and control of infections associated with hypogammaglobulinemia. The hematocrit level stabilized with a decrease in red blood cell destruction by intravenous immunoglobulin G in chronic lymphocytic leukemia patients with AIHA without altering red blood cell autoantibody titers, but severe hemolysis recurred after 21 days. A long-term remission was observed when intravenous immunoglobulin G was combined with steroids, chemotherapy, plasmapheresis, and splenectomy. The platelet count increased and platelet transfusion requirement was eliminated in a chronic lymphocytic leukemia patient with immune thrombocytopenic purpura by the intravenous immunoglobulin G, and chemotherapy was administered and a complete remission resulted. An interesting observation was the lymphocytopenic effects observed in the five patients during a daily infusion of intravenous immunoglobulin for five days. Furthermore, the maintenance intravenous immunoglobulin G given every three weeks controlled both infections and lymphocyte counts with the chemotherapy. Changing the schedule to every four weeks resulted in poor control of the lymphocyte counts with chemotherapy. These observations indicate a direct macrophage blocking effect in autoimmune hemolytic anemia and immune thrombocytopenic purpura, but the lymphocytopenic effect suggests immunomodulating effects of intravenous immunoglobulin G, which may alter the response of chronic lymphocytic leukemia cells to chemotherapy.

Treatment with 13-cis-retinoic acid in transfusion-dependent patients with myelodysplastic syndrome and decreased toxicity with addition of alpha-tocopherol.

PURPOSE: The purpose of this study was to determine the response and tolerance to long-term treatment using 13-cis-retinoic acid (13-CRA) in transfusion-dependent patients with the myelodysplastic syndrome (MDS) and to determine the effects of therapy on the natural history of the disease. PATIENTS AND METHODS: Sixty-six consecutive patients with transfusion-dependent MDS seen in a medical school hospital and outpatient clinic from 1981 to 1988 were studied. The first 21 patients were treated with 13-CRA alone and the next 45 patients with 13-CRA plus alpha-tocopherol (AT). We compared responses to and toxicities of therapy, rates of transformation, and survival from onset of therapy in 20 evaluable patients treated with 13-CRA alone and 43 patients treated with 13-CRA plus AT. RESULTS: Four patients responded (20%) at 4 to 8 months to 13-CRA alone, but this response was associated with considerable toxicity and resulted in cessation of therapy. Among the responders, only one continued therapy and is currently in remission, whereas three discontinued therapy because of toxicity and have had a relapse and died. In the 13-CRA plus AT group, we observed one prolonged complete remission and 10 partial remissions (26%), with a decrease in skin and constitutional toxicities by the addition of AT, which enabled the continuation of 13-CRA indefinitely. Although the response rates were similar in both groups, fewer patients (28% versus 60%) experienced progression to acute leukemia in the 13-CRA plus AT group than in the group receiving 13-CRA alone, who terminated treatment (p = 0.018). A twofold increase in median survival of the RA/RARS and RAEB/CMML patient groups was observed with 13-CRA plus AT but was not significant (p greater than 0.5). CONCLUSION: This study shows a 20% to 26% response rate to 13-CRA and suggests that 13-CRA, if given continuously, decreases the rate of progression or transformation to acute leukemia in patients with MDS. The addition of AT ameliorates the toxicity of 13-CRA and allows for long-term treatment with 13-CRA. Since the standard treatment for MDS is currently unsatisfactory, these findings indicate that longer treatment with a non-marrow-suppressive agent such as 13-CRA is important, and further trials to determine the role of 13-CRA plus AT in combination with new recombinant growth factors in the therapy for transfusion-dependent MDS should offer a new approach to a disease common in the elderly population.

Specific immunoadsorption of IgG antibody in a patient with chronic lymphocytic leukemia and autoimmune hemolytic anemia. A new form of therapy for the acute critical stage.

Progressive and severe autoimmune hemolytic anemia developed in a patient with chronic lymphocytic leukemia (CLL) despite treatment with chlorambucil, high doses of corticosteroids and attempts to transfuse packed red blood cells. Splenectomy was not performed because of severe coronary artery disease. Direct antiglobulin tests revealed a warm red blood cell autoantibody of IgG-type with anti-e specificity. The patient was treated by extracorporeal immunoadsorption of plasma IgG using a cell separator and protein A as the immunoadsorbent. The patient responded by an increase in the hemoglobin levels and platelet counts after two treatments. Specificity of the procedure was shown by a decrease in the serum IgG and by the demonstration of the same reactivity to ficin-treated reagent red blood cell panel of the eluate from the protein A. Antibody titers of the patient's red blood cell eluate decreased from 1:128 to 1:64 and eventually the anti-e specificity was lost. This is a report of a novel approach to treatment of the acute phase of an autoimmune hemolytic anemia.

Thrombospondin levels in patients with malignancy.

Thrombospondin (TSP), a large glycoprotein present in platelets, and various normal and tumor tissues, has recently been shown to promote cell adhesion and platelet aggregation. Most importantly because TSP has been shown to promote metastasis of melanoma tumor cells to the lung in a murine model (1) and since thromboembolic events commonly occur in patients afflicted with metastatic tumors, we explored the role of TSP in human cancer by measuring TSP blood levels in patients with various malignant neoplasms. Blood TSP levels were measured by indirect enzyme-linked immunoadsorbent assay (ELISA) from 20 control subjects, 22 patients with gastrointestinal (GI) cancer, 18 patients with breast cancer, and 17 patients with lung cancer. Control subjects consisted both of healthy subjects and acutely ill patients with no malignancies. TSP levels of both healthy and acutely ill controls were found to range between 245-440 ng/ml with a mean of 365 ng/ml. In contrast, elevated levels of TSP greater than the mean value of 400 ng/ml for controls ranging between 590-3,650 ng/ml were found in 20/22 (91%) patients with GI malignancies, 13/18 (72%) patients with breast cancer, and 15/17 (88%) with lung cancer. Mean TSP levels of GI, breast, and lung cancer patients were 3, 2, and 3 fold greater than controls, respectively. Increased blood TSP levels in patients were not due to increased levels of platelets since both control and patient groups had platelet counts within the normal range. These results suggest that TSP may play a role in tumor cell metastasis in man and could serve as a blood marker for metastasis.

A pilot trial of 13-cis-retinoic acid and alpha-tocopherol with recombinant human erythropoietin in myelodysplastic syndrome patients with progressive or transfusion-dependent anemias. The Central Pennsylvania Oncology Group.

A pilot study was performed to determine the efficacy of 13-cis-retinoic acid (CRA) and alpha-tocopherol (AT) with increasing doses of recombinant human erythropoietin (rHuEPO) in anemic patients with primary myelodysplastic syndrome (MDS), to determine response rate and to determine the dose requirement and long-term effects of rHuEPO therapy on the transition to acute non-lymphocytic anemia and survival of these patients. Twenty-four consecutive MDS patients were entered into the study. Patients were stratified according to their FAB classification at study entry. Therapy consisted of a 6 month trial of CRA (100 mg m(-2) day(-1) and AT (800 mg day(-1)) with rHuEPO (150 units kg(-1) body weight subcutaneously three times a week). The rHuEPO dose was escalated to daily doses at 2 months, and 300 U kg(-1) body weight given three times a week for another 2 months and continuing therapy after 6 months in responsive patients. Response was measured by elimination of transfusions requirement (partial response, PR) and normal hemoglobin level and complete blood counts (complete response, CR). Observed responses for the 23 evaluable patients were 2 CR and 6 PR (34.8%). Odds ratio analysis showed that patients with anemia alone were 14 times more likely to respond than patients with pancytopenia (p = 0.06). In our study, two patients (8%) transformed to acute leukemia in CRA + AT + rHuEPO-treated patients. Median survival of 34 months with a median follow-up of 17 months (range 3-70 months) was observed. The response rates with the addition of rHuEPO to CRA and AT was similar but occurs earlier at 2 months compared to 6-10 months with CRA and AT alone and did not alter survival. There was no increase in the risk for leukemia in the group treated with rHuEPO. Response to either therapy appeared to be limited to patients in the early stages of MDS.

Richter's syndrome associated with loss of response to transforming growth factor-beta.

A patient with chronic lymphocytic leukemia developed a large cell lymphoma apparently derived from the same neoplastic B-cell clone (Richter's syndrome). At the same time, mitogen-stimulated proliferation of the patient's circulating leukemic B-cells was no longer inhibited by the regulatory cytokine transforming growth factor-beta (TGF-beta), suggesting that such loss of inhibition might be contributing to the clinical and biological progression of the disease.

Prognostic significance of single chromosome abnormalities in preleukemic states.

Clinical outcome was evaluated in 43 patients with a myelodysplastic syndrome or myeloproliferative disorder and a bone marrow clone containing a single chromosome abnormality: monosomy 7/del(7q), trisomy 8, i(17q), del(5q), del(20q), or a t(2;11). Those with one of the first three abnormalities (22 patients) had shorter survival, more frequent progression to leukemia, and less response to treatment with 13-Cis-retinoic acid than did those in the latter three groups (21 patients). Additional data on these subgroups of preleukemic patients may confirm the prognostic value of such karyotypic information.

Karyotypic stability in chronic B-cell leukemia.

Twenty-one patients with B-cell chronic lymphocytic leukemia (B-CLL) have been followed for more than 2 years with serial cytogenetic studies, including 11 cases for more than 5 years and three others for more than 10 years. A chromosomally abnormal clone was present at the time of initial study in 10 of these patients, and neither these nor the 11 individuals with a normal karyotype had any cytogenetic evolution during the follow-up period, although clinical progression, requiring therapy, was observed in 13 cases. In an additional 12 B-CLL patients who had repeat chromosome studies but were followed for less than 2 years, two patients with advanced disease and multiple cytogenetic abnormalities developed minor additional karyotypic changes and died within 18 months, and two patients with a normal karyotype developed rapidly progressive disease associated with an emerging chromosomally abnormal clone and survived only 1 year. These results demonstrate that karyotypic evolution is rare in B-CLL. Its occurrence indicates a poor prognosis, but its rarity suggests that clinical progression in this disease is usually more dependent on other factors.

Myelodysplastic syndromes (refractory anemia). A perspective of the biologic, clinical, and therapeutic issues.

The myelodysplastic syndromes (MDS) are a diverse group of hematologic disorders that have an abnormality in differentiation of the hematopoietic stem cell resulting in varying degrees of peripheral blood cytopenias. The author reviews the hematologic and clinical aspects of MDS, summarizes the recent pertinent aspects of the advances in our understanding of the disease, and explores the current approaches in the new treatment programs.

The onset of a refractory response to androgens in the anemia of bone marrow failure.

Patients with bone marrow failure may have a hematopoietic response to an androgen preparation after they have no response from the use of a previous testosterone preparation. The failure to try a variety of hormone preparations may lead to improper conclusions as to a potential marrow proliferation for a clinical response. In some instances the use of a 5beta steroid metabolite may initiate stem cell proliferation in patients previously refractory to testosterone or nontestosterone esters. These obsevations suggest that some patients refractory to testosterone may be unable to form adequate 5beta metabolities by alterations in 5alpha reductase or enhanced metabolic transformation of the androgen. All patients should be evaluated with several androgen esters before concluding that their marrow cannot achieve a hematopoietic response.

Approach to mild anemia in the elderly.

There is evidence that indicates a physiologic decrease in bone marrow function as a part of aging, perhaps as an adjustment to a decrease in metabolic demand and tissue requirement for oxygen. In general, aging is associated with a decline in activity and an increase in chronic inflammatory as well as cardiovascular disorders. These alter not only red blood cell production but also plasma volume, leading to an increased frequency of a lower hemoglobin concentration and hematocrit level. However, there is resistance to using new geriatric norms for these hematologic parameters to define anemia, since the majority of patients without disease fall within the usual established ranges. Yet surveys have shown that there is no increase in iron deficiency with age, and the majority of elderly patients with mild anemia show no demonstrable cause and are unresponsive to therapy. The statement that anemia is often a presenting sign of a serious underlying illness and that patients with anemia must undergo a complete work-up should be reconsidered in the elderly, and the criteria for anemia should be redefined to prevent unnecessary studies for individuals who fall in the lower limits of normalcy because of age. This statement is supported by the results of long-term follow-up of patients in a geriatric care institution. A group of patients with mild anemia, without any apparent cause, who were often not examined thoroughly because of the relative mildness of symptoms and lack of progression, lived a satisfactory life and seem to be well adjusted to low hemoglobin levels. Asymptomatic elderly patients with mild anemia may not be anemic if we adjust the expected levels for the elderly. We can save 95 per cent of these patients from undergoing unnecessary testing.

Intravenous immunoglobulin. A promising new therapy for idiopathic thrombocytopenic purpura.

Intravenous administration of human immunoglobulin has been shown to have clinical efficacy in the treatment of idiopathic thrombocytopenic purpura. Physicians who use this remarkable clinical tool face the dilemma of balancing the safety and efficacy of therapy against the cost, which is higher than that of the more toxic forms of therapy. Cost-effectiveness studies of specific clinical situations indicate that costs for children are decreased by a shortened hospital stay, the need for less antibiotic therapy, and avoidance of splenectomy. Therapy for adults must be individualized and varies depending on whether the goal is a rapid platelet response (eg, before emergency surgery) or avoidance of the risks of corticosteroid therapy and splenectomy. The availability of a safe immunomodulating agent makes intravenous immunoglobulin therapy a potentially useful tool for a wide variety of diseases.