[Clinical use of hematopoietic growth factors].
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Antineoplastic Agents↗
Biomedical subjects
S Siena
Publications and source records attributed to S Siena.
Single high doses of cyclophosphamide enable the collection of high numbers of hemopoietic stem cells from the peripheral blood.
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Cell Division↗
Total body irradiation and high-dose melphalan with bone marrow transplantation at Istituto Nazionale Tumori, Milan, Italy.
The technique of total body irradiation (TBI) developed at Istituto Nazionale Tumori, Milan, Italy, is described. This technique consists of i) administration of 12.5 Gy and 14.85 Gy TBI for autologous and allogeneic bone marrow transplantation respectively; ii) in all cases in vivo dosimetry of absorbed TBI dose; and iii) radiation doses to lungs higher than previously described. As of June 1988, seventeen patients with Hodgkin's disease and four with lymphoblastic lymphoma received TBI and 120-180 mg/m2 melphalan. Respiratory function was prospectively evaluated demonstrating moderate and transient reduction of pulmonary function.
Adolescent↗
Large-scale collection of circulating haematopoietic progenitors in cancer patients treated with high-dose cyclophosphamide and recombinant human GM-CSF.
Circulating haematopoietic progenitors from 36 cancer patients were collected by continuous-flow leukapheresis during the phase of rapid haematopoietic recovery after pancytopenia induced by high-dose cyclophosphamide and then cryopreserved for autologous transplantation. 20 of the patients also received intravenous infusion of recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) for 7, 10 or 14 days after cyclophosphamide. 106 leukapheresis procedures were done for 2-5 consecutive days. Leukapheresis was started significantly earlier in patients receiving rhGM-CSF. In these patients, yields of peripheral blood elements (leucocytes, mononuclear cells, haematopoietic progenitors and platelets) were significantly higher than in controls treated with cyclophosphamide only. In particular, the mean number of granulocyte-monocyte colony-forming cells was 43.88 X 10(4) vs. 6.16 X 10(4) per kg patient body weight per leukapheresis. Side-effects of leukapheresis were limited to central venous catheter occlusion and fever in 4% and 2% of all procedures, respectively.
Adult↗
Prospective evaluation of pulmonary function in cancer patients treated with total body irradiation, high-dose melphalan, and autologous hematopoietic stem cell transplantation.
Pulmonary function tests (standard vital capacity, SVC; total lung capacity, TLC; forced expiratory volume in 1 second-forced vital capacity ratio, FEV1/FVC; carbon monoxide transfer factor, DLCO) were prospectively evaluated in patients (median age 25 years, 13-52 years; median follow-up 20 months, 6-51 months) with Hodgkin's disease (15 patients), non-Hodgkin's lymphoma (9 patients), and inflammatory breast cancer (3 patients) treated with sequential high-dose therapy comprising the following phases over approximately 2 months: a) cyclophosphamide (7 g/m2); b) vincristine (1.4 mg/m2), methotrexate (8 g/m2), and cisplatinum (120 mg/m2) or etoposide (2 g/m2); c) total body irradiation (TBI; 12.5 gy, 5 fractions over 48 hours), intravenous melphalan (120-180 mg/m2), and transplantation of autologous peripheral blood and/or bone marrow hematopoietic stem cells. Within 2 months after transplantation, 12 patients also received 25 Gy radiotherapy boost to mediastinum and clavicular regions. In vivo dosimetry evaluations of fractionated TBI treatments showed that mean radiation dose absorbed by lungs was 12.18 Gy (97.4% of TBI dose). Despite such a high radiation dose, we observed only transient and subclinical decrease of SVC, TLC, and DLCO. The decrease of SVC, TLC, and DLCO was more evident and prolonged in patients receiving radiotherapy boost. All parameters progressively recovered to normal values within 2 years after transplantation. In contrast, FEV1/FVC remained within normal limits in all patients, thus demonstrating the absence of obstructive ventilatory changes. In addition, no interstitial pneumonia was observed.
Adolescent↗
Recombinant human granulocyte-macrophage colony-stimulating factor reduces hematologic toxicity and widens clinical applicability of high-dose cyclophosphamide treatment in breast cancer and non-Hodgkin's lymphoma.
High-dose administration of anticancer agents is attractive both on theoretic and clinical grounds. Yet, high-dose regimens are usually used as salvage treatments, mainly as a consequence of their considerable hematologic toxicity. One pertinent example is represented by cyclophosphamide, an alkylating agent with a wide spectrum of marked antitumor activity. When used at doses up to 7 g/m2 (190 to 200 mg/kg) this drug does not cause myeloablation, but induces a severe, albeit transient, myelosuppression, which requires platelet transfusions in approximately 50% of treated patients, and is frequently complicated by infectious episodes, occasionally lethal. To accelerate hematopoietic recovery, we continuously infused for 14 consecutive days 5.5 micrograms/kg/d of the glycosylated human recombinant granulocyte-macrophage colony-stimulating factor (rhGM-CSF) into 15 patients with breast cancer and non-Hodgkin's lymphoma treated with 7 g/m2 cyclophosphamide. This schedule was chosen having obtained the fastest hematopoietic recovery among four different options during an initial schedule-finding phase on 12 overall patients. Twenty-one comparable subjects with solid tumors served as controls. We report here that this relatively low, well-tolerated dose of rhGM-CSF reduces from 20 to 14 (median) and from 24 to 14, the number of days required to recover circulating granulocyte counts over 1,000 and 2,500/microL, respectively. The stimulatory effect was associated with a remarkable clinical benefit. In fact, treated patients experienced less infectious complications (7% v 24%) were eligible to receive chemotherapy earlier (median, by day +14 v day +20 for controls), and fewer required prophylactic platelet transfusions (13% v 43%). Our results show that even very high doses of cyclophosphamide can be administered with improved hematologic toxicity, tolerable morbidity, and reduced supportive care requirements. The increase in the therapeutic index made possible by rhGM-CSF infusion prompts the use of high-dose cyclophosphamide, and possibly of other agents with similar myelotoxic activity, early in the clinical course of chemotherapy-sensitive tumors.
Adolescent↗
Durable and complete hematopoietic reconstitution after autografting of rhGM-CSF exposed peripheral blood progenitor cells.
Two patients with poor prognosis stage III multiple myeloma have been treated with myeloablative chemoradiotherapy, i.e. 10 Gy fractionated total body irradiation plus 120 mg/m2 intravenous melphalan, and then transplanted with autologous peripheral blood cells harvested by four leukaphereses during the phase of rapid hematopoietic recovery following induction therapy with high-dose (2 g/m2) etoposide and recombinant human glycosylated granulocyte macrophage-colony stimulating factor (rhGM-CSF). Following myeloablative therapy and autologous peripheral blood cell transplantation, both patients experienced brief pancytopenia followed by rapid hematopoietic recovery of leukocytes (time to greater than 500 x 10(6)/l = 12 days) and platelets (time to greater than 100 x 10(9)/l = 14 days). In particular, single donor platelet transfusion requirements were limited to one and two transfusions per patient, respectively. Reconstitution has so far been maintained throughout the follow-up period for the two patients (9 and 6 months, respectively). These two cases show that rhGM-CSF-exposed peripheral blood cells are capable of producing prompt and sustained hematopoietic reconstitution in patients treated with myeloablative chemoradiotherapy.
Adult↗
Circulation of CD34+ hematopoietic stem cells in the peripheral blood of high-dose cyclophosphamide-treated patients: enhancement by intravenous recombinant human granulocyte-macrophage colony-stimulating factor.
We report that hematopoietic progenitor cells expressing the CD34 antigen (CD34+ cells) transiently circulate in the peripheral blood (PB) of cancer patients treated with 7 g/m2 cyclophosphamide (HD-CTX) with or without recombinant human granulocyte macrophage-colony stimulating factor (rHuGM-CSF). In adult humans, CD34+ cells represent a minor fraction (1% to 4%) of bone marrow (BM) cells, comprising virtually all hematopoietic colony-forming progenitors in vitro and probably also stem cells capable of restoring hematopoiesis of lethally irradiated hosts. We show that CD34+ cell circulation is fivefold enhanced by rHuGM-CSF 5.5 protein micrograms/kg/day by continuous intravenous infusion for 14 days after HD-CTX. During the third week after HD-CTX (ie, when CD34+ cells peak in the circulation), large-scale collection of PB leukocytes by three to four continuous-flow leukaphereses allows the yield of 2.19 to 2.73 x 10(9) or 0.45 to 0.56 x 10(9) CD34+ cells depending on whether or not patients receive rHuGM-CSF. The number of CD34+ cells retrieved from the circulation by leukaphereses exceeds the number that can be harvested by multiple BM aspirations under general anesthesia. Thus, after therapy with HD-CTX and rHuGM-CSF, PB represents a rich source of hematopoietic progenitors possibly usable for restoring hematopoiesis after myeloablative chemoradiotherapy. To determine whether CD34+ cells found in the PB are equivalent to their marrow counterpart, we evaluated their in vitro growth characteristics and immunological phenotype by colony assays and dual-color immunofluorescence, respectively. We show that PB CD34+ cells possess qualitatively normal hematopoietic colony growth and high cloning efficiency comparable to that observed with BM CD34+ cells. In addition, PB CD34+ cells display heterogeneous surface membrane differentiation antigens analogous to BM CD34+ cells. The availability of large quantities of CD34+ cells by leukapheresis is relevant to the field of stem cell transplantation and possibly to genetic manipulations of the hematopoietic system in humans.
Antigens, CD34↗
Granulocyte-macrophage colony-stimulating factor to harvest circulating haemopoietic stem cells for autotransplantation.
Granulocyte-macrophage colony-stimulating factor (GM-CSF), given to accelerate recovery from cytopenia induced by high-dose (7 g/m2) cyclophosphamide, reproducibly brought about a dramatic increase (up to 1000-fold) in the number of peripheral blood granulocyte-macrophage colony-forming units (CFU-GM). These circulating progenitors were harvested by leucapheresis and reinfused, together with autologous bone marrow cells, in seven patients with cancer after total body irradiation and melphalan. Complete haemopoietic recovery occurred in all seven transplanted patients in a very short time: mean (SD) 9.1 (0.9) days (range 8-11) to achieve more than 0.5 x 10(9)/l neutrophils, 9.9 (1.7) days (range 8-13) to over 1 x 10(9)/l neutrophils, 10.7 (2.6) days (range 9-16) to over 0.5 x 10(11)/l platelets, and 13.6 (4.2) days (range 13-21) to over 1.0 x 10(11)/l platelets. A reduction in the severity of mucositis was also observed. The rapid haematological recovery made possible by this approach promises to increase the therapeutic index of high-dose chemoradiotherapy regimens and to widen their role as treatment for chemoradiosensitive tumours.
Adult↗
Immunotoxin-mediated inhibition of chronic lymphocytic leukemia cell proliferation in humans.
We evaluated the cytotoxicity of the immunotoxin OKT1-SAP on fresh B-chronic lymphocytic leukemia (B-CLL) cells from 31 consecutive patients. OKT1-SAP comprised the OKT1 (CD5) monoclonal antibody disulfide linked to saporin-6 (SAP) ribosome-inactivating protein from the plant Saponaria officinalis. The effect of OKT1-SAP on target CD5-positive B-CLL cells was estimated using an in vitro proliferation inhibition assay in which control or OKT1-SAP-treated B-CLL cells were induced to proliferate by sequential stimulation with insolubilized anti-C3b receptor CB04 (CD35) antibody and low molecular weight B-cell growth factor. In 90% of patients, OKT1-SAP specifically suppressed B-CLL cell proliferation in a dose-related manner (50% inhibitory concentration, 4.0-6.8 nM). Taken together the findings reported in this article provide information relevant to the clinical development of immunotoxins because: (a) the in vitro conditions under which B-CLL cell proliferation is inhibited by OKT1-SAP are achievable in vivo without nonspecific toxicity according to our previous toxicology and pharmacokinetics studies in primates; and (b) the B-CLL cell proliferation inhibition assay described here provides a basis for future comparative studies.
Aged↗
In vivo effects in mice of an anti-T cell immunotoxin.
We have evaluated both the proliferative response as well as the Thy-1 Ag expression of lymphocytes from mice treated in vivo with an anti-Thy-1 immunotoxin (IT). The IT was a rat IgG2c mAb recognizing the Thy-1 Ag, disulfide-linked to a ribosome-inactivating protein isolated from the seeds of the plant Saponaria officinalis (soapwort). Toxicity studies showed that a single i.v. injection of doses up to 20 micrograms IT/mouse was well tolerated and allowed indefinite survival. The Con A-induced proliferative response of spleen cells from mice killed 1 day after treatment with sublethal doses of IT was inhibited in a dose-dependent manner, with complete inhibition observed at doses of greater than or equal to 5 micrograms IT/mouse. Control experiments showed that the inhibition was due to the IT and not to its single components. Moreover, the IT effect was abolished by a large (100-fold) excess of anti-Thy-1 mAb alone given concurrently, but not by an unrelated, isotype-matched rat mAb. At all IT doses, the proliferative response to a B cell mitogen (LPS) was normal. Kinetic studies showed a time- and dose-dependent reconstitution of Con A responsiveness. In limiting dilution cultures of spleen cells from mice treated with 5 micrograms IT 1 or 4 days before death, a 97% depletion of T lymphocytes capable of proliferation was observed. Limiting dilution cultures showed that also the thymus of IT-treated mice was depleted by more than 90% of growth-competent T lymphocytes. Cytofluorographic studies of Thy-1+ cells from the spleens of IT-treated mice gave results which did not correlate with those obtained in functional assays. Thus, a dose-dependent reduction, followed by a time-dependent reconstitution of Thy-1+ cells was observed in this case too, but the depletion occurred at later time points and was less complete than that observed in functional assays. Moreover, the mean fluorescence intensity of the residual Thy-1+ cells decreased below normal levels.
Animals↗
B-cell restricted saporin immunotoxins: activity against B-cell lines and chronic lymphocytic leukemia cells.
B cell-restricted immunotoxins were constructed by conjugating anti-B monoclonal antibodies to saporin, the major ribosome inactivating protein from the seeds of the plant Saponaria officinalis. HD37-SAP is directed against CD19, the broadest B cell-specific determinant. HD39-SAP and HD6-SAP recognize two different epitopes on the CD22 molecule, an antigen present on the cell surface of B cells at late stages of differentiation. All three immunotoxins inhibited DNA synthesis and protein synthesis in target B lymphoma cells with a dose-related effect, in short incubation times and in the absence of potentiators. A clonogenic assay demonstrated that all immunotoxins could eliminate more than two logs of clonogenic malignant B cells with a two-hour incubation at concentrations not toxic to cells not bearing target antigens. The immunotoxin activity was evaluated by DNA synthesis inhibition in fresh B-chronic lymphocytic leukemia cells (B-CLL) stimulated to proliferate by incubation with an antibody specific for the receptor of C3b complement component (CR1) plus B cell growth factor. B-CLL cell DNA synthesis was actively inhibited by treatment at low immunotoxin concentration without need of potentiators. Immunotoxins exerted their effect also in whole blood of CLL patients under conditions achievable in vivo. We conclude that B cell-restricted immunotoxins HD37-SAP, HD39-SAP, and HD6-SAP are good candidates for in vivo therapy of B-cell malignancies.
Antibodies, Monoclonal↗
Minimal residual disease in acute lymphoblastic leukemia detected by immune selection and gene rearrangement analysis.
We have developed an assay for the detection of malignant residual cells in the bone marrow from patients with B- or T-lineage acute lymphoblastic leukemia (ALL) in clinical remission. This assay involves an immune selection step followed by immunoglobulin or T-cell receptor gene rearrangement analysis and allows the detection of one contaminating tumor cell out of 1,000 normal bone marrow cells. We have examined the bone marrow of 11 patients with adult ALL in remission over a 24-month period. Five patients relapsed in the bone marrow and one in the CNS. The assay allowed the detection of minimal residual disease in four of five patients that subsequently relapsed in the bone marrow, 1.5 to 9 months before the relapse became morphologically and clinically manifest. Residual disease was not found in the bone marrow from patients in continuous remission and from the single patient who relapsed in the CNS. We conclude that the ability of the assay described here to detect minimal residual disease with high specificity can provide information for further understanding of the biology of ALL and hopefully for the clinical management of patients with this disease.
Adolescent↗
[Use of recombinant hematopoietic growth factors after chemotherapy and high-dose chemo-radiotherapy].
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Anemia, Aplastic↗
Activity of a monoclonal antibody-saporin-6 conjugate against B-lymphoma cells.
A monoclonal antibody reactive with the immunoglobulin heavy chain (TEC IgM) has been conjugated to saporin-6 (SAP), which is the major ribosome-inactivating protein from the seeds of the plant Saponaria officinalis. Studies with Burkitt's lymphoma cell line Bjab 113 demonstrate that this immunotoxin is capable of killing 3 logs (99.9%) of clonogenic lymphoma cells after a 2-hour incubation. The presence of human bone marrow inhibits the activity of the conjugate. However, full potency of TEC IgM-SAP immunotoxin is restored by adding 1 mM amantadine to the incubation medium. The reaction is highly specific and is inhibited by the presence of excess anti-mu-antibody or human serum. Clonal growth of other Burkitt's lymphoma cell lines is inhibited to a lesser extent by the immunotoxin. The presence of surface IgM on the different cell lines is directly correlated to target cell killing by TEC IgM-SAP. Isolation of Bjab 113 clones surviving treatment demonstrates that only a minority are truly resistant and that the others randomly escape the treatment. The highly potent and specific activity of this conjugate in the presence of bone marrow buffy coat and its exceptionally rapid onset of action make this conjugate a good candidate for the ex vivo elimination of neoplastic cells from the bone marrow of non-Hodgkin's lymphoma patients.
Antibodies, Monoclonal↗
Evaluation of antihuman T lymphocyte saporin immunotoxins potentially useful in human transplantation.
We have synthesized 3 immunotoxins (ITs) by covalently coupling the saporin-6 hemitoxin (SAP) to OKT11, SOT3, and SOT1a murine monoclonal antibodies that recognize human T lymphocyte CD2, CD3, and CD5 surface antigens, respectively. The resulting ITs, referred to as OKT11-SAP, SOT3-SAP, and SOT1a-SAP, are equally effective in inhibiting eukaryotic protein synthesis in a cell-free system, and all 3 ITs bind to human T lymphocytes in an almost comparable manner. However, these reagents differ markedly in their ability to kill target T lymphocytes as assessed by measuring the inhibition of DNA synthesis and growth of clonable T lymphocytes in response to mitogenic and allogeneic stimuli. Whereas the anti-CD2 IT, OKT11-SAP, shows moderate cytotoxicity against T lymphocytes, the anti-CD3 IT, SOT3-SAP, and the anti-CD5 IT, SOT1a-SAP, are highly effective in eliminating the same target cells. The concentrations inhibiting 50% (IC50) of T lymphocyte DNA synthesis are 60 nM, 4.5 nM, and 1.4 nM for OKT11-SAP, SOT3-SAP, and SOT1a-SAP, respectively. Among 3 tested lysosomotropic amines, i.e., ammonium chloride, chloroquine, and amantadine, the latter only moderately potentiates the cytotoxicity of SOT1a-SAP (IC50 0.36 nM). We show that the conditions under which T lymphocyte killing is accomplished require less than 10 min exposure of T lymphocytes to the ITs, in the absence of adjuvant molecules artificially added to the incubation medium and at physiologic culture pH. These experimental characteristics of unprecedented closeness to a physiologic in-vivo model are likely to reflect the biophysical properties of the SAP moiety of the ITs. We conclude that clinical studies are warranted to define the advantage of using SAP ITs over previously described immunoconjugates.
Amines↗
Synthesis and characterization of an antihuman T-lymphocyte saporin immunotoxin (OKT1-SAP) with in vivo stability into nonhuman primates.
The authors conjugated, by a disulphide bond, the antihuman T-lymphocyte (CD5) monoclonal antibody (MoAb) OKT1 to the saporin-6 (SAP) ribosome-inactivating protein of the plant Saponaria officinalis. The resulting OKT1-SAP immunotoxin bound to CD5-expressing target cells and under standard culture conditions specifically suppressed mitogen-induced-T-lymphocyte DNA and protein synthesis in a dose-related manner. T-lymphocyte killing was achieved by five-minute exposure of the target cells to OKT1-SAP. The concentration inhibiting 50% (IC50) of T-lymphocyte DNA synthesis was 0.32 nmol/L. The potency of OKT1-SAP was moderately enhanced by amantadine (IC50 0.08 nmol/L) but not by ammonium chloride or chloroquine. Whole blood components did not interfere with the efficacy of OKT1-SAP, as in vitro treatment of fresh whole blood resulted in effective elimination of clonable peripheral blood T-lymphocytes assessed by a limiting dilution assay. Because these characteristics of T-lymphocyte killing by OKT1-SAP (ie, rapidity of action, potency also without potentiators) and lack of inhibition by whole blood components may be relevant for the use of an immunotoxin as a therapeutic agent in humans, the authors evaluated the stability in vivo and the circulatory clearance of OKT1-SAP in cynomolgus monkeys. Following a single intravenous (IV) injection of nontoxic dosages (0.16 to 1.3 mg/kg), an initial rapid decline (t1/2 alpha = 1.0 to 4.1 hours) was followed by a long-lasting slower decrease (t1/2 beta = 11.6 to 20.6 hours) of OKT1-SAP plasma concentrations as detected by double-antibody solid phase enzyme-linked immunosorbent assay (ELISA) assay. Not only did OKT1-SAP remain intact immunologically but it also retained its biological activity, as measured by the ability of plasma samples from monkeys given immunotoxin to inhibit DNA synthesis in human T-lymphocytes. Taken together the findings presented in this article indicate the feasibility of using OKT1-SAP as a therapeutic tool and provide information that will facilitate the rational use of immunotoxins as a treatment modality in humans.
Animals↗
High sensitivity and specificity assay for detection of leukemia/lymphoma cells in human bone marrow.
An assay of high sensitivity and specificity for detection of residual malignant cells in remission bone marrow from patients with acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL) has been developed. The assay combines an immunoselection step with immunoglobulin gene rearrangement analysis by high specific-activity DNA probes. In experimental conditions, less than one contaminating tumor cell out of 1000 bone marrow cells was detected. A bone marrow contamination was detected in morphologically negative bone marrow specimens from two intermediate-grade non-Hodgkin's lymphoma patients. This method can be of value in determining a true remission status in high-risk ALL and NHL patients.
Antibodies, Monoclonal↗