I. C. M. Slaper-Cortenbach

Subsets of CD34+ Cells and Rapid Hematopoietic Recovery After Peripheral-Blood Stem-Cell Transplantation

PURPOSE To study whether there is a relationship between transplanted cell dose and rate of hematopoietic recovery after peripheral-blood stem-cell (PBSC) transplantation, and to obtain an indication whether specific subsets of CD34+ cell populations contribute to rapid recovery of neutrophils or platelets. PATIENTS AND METHODS Based on data from 59 patients, we calculated for each day after PBSC transplantation the dose of CD34+ cells that resulted in rapid recovery of either neutrophils or platelets in the majority (> 70%) of patients. Using dual-color flow cytometry, subsets of peripheral-blood CD34+ cells were quantified and the numbers of CD34+ cells belonging to each of the reinfused subsets correlated with hematopoietic recovery following high-dose chemotherapy. RESULTS The calculated threshold values with a high probability of engraftment showed a steep dose-effect relationship between CD34+ cell dose and time to recovery of both neutrophils or platelets. Predominantly CD34+ cells with the phenotype of myeloid precursors were mobilized. A minority of CD34+ cells expressed the erythroid and megakaryocytic lineage-associated antigens and a low but distinct population of CD34+ cells expressed antigens associated with multipotent stem cells. Analysis showed that the number of CD34+CD33- cells (r = -.74, P < .05), as well as the number of CD34+CD41+ cells (r = -.81, P < .005), correlated significantly better with time to neutrophil and platelet recovery, respectively, than with the total number of CD34+ cells (r = -.55 and r = -.56, respectively). CONCLUSION The numbers of CD34+CD33- cells and CD34+CD41+ cells may help to predict short-term repopulation capacity of PBSCs, especially when relatively low numbers of CD34+ cells per kilogram are reinfused.

Feasibility of multiple courses of high-dose cyclophosphamide, thiotepa, and carboplatin for breast cancer or germ cell cancer.

PURPOSE To determine the feasibility and safety of multiple, closely timed courses of high-dose cyclophosphamide, thiotepa, and carboplatin (CTC) with peripheral-blood progenitor-cell transplantation (PBPCT). PATIENTS AND METHODS Forty-eight patients with advanced cancer were scheduled to undergo either two or three courses of CTC with PBPCT. All PBPCs were harvested before high-dose therapy began. Full-dose CTC courses incorporated cyclophosphamide (6,000 mg/m2), thiotepa (480 mg/m2), and carboplatin (1,600 mg/m2) divided over days -6, -5, -4, and -3. Tiny CTC courses (tCTC) contained 67% of the doses of each of these agents. Second or third courses of CTC or tCTC began on day 28. RESULTS A sufficient number of PBPC could be harvested from all but two patients. Thirty-five first full-dose courses of CTC were given, 28 second courses, and 10 third courses. Second courses could be given on time and at full dose in 80% of the patients, but there was one toxic death from venoocclusive disease (VOD). Only four of 12 patients scheduled to receive three courses of full-dose CTC could be treated at the time and dose planned. There were three toxic deaths: one of VOD, one of sepsis, and one of hemolytic uremic syndrome (HUS). Eight patients were scheduled to receive three courses of tCTC. Eight first, seven second, and six third courses were given. One of the third courses had to be delayed and one had to be reduced in dose. CONCLUSION A sufficient number of PBPCs for two or three transplantations can be harvested from most patients without much difficulty before high-dose therapy. Two full-dose CTC courses or three tCTC courses can be given safely and with acceptable toxicity at 5-week intervals. Organ toxicity rather than bone marrow toxicity has become dose-limiting for alkylating agents.

Preclinical studies with radiolabeled monoclonal antibodies for treatment of patients with b-cell malignancies

Background. Studies on radiolabeled monoclonal antibodies (MoAb) have dealt mainly with single antibodies. However, major differences may exist among different radiolabeled MoAb that bind to the same antigen and between switch variants of the same antibody. This study evaluates and compares a series of radiolabeled MoAb of different specificities, subclasses, and isotypes applicable in treatment of patients with B cell malignancies.

High-dose carboplatin, thiotepa and cyclophosphamide (CTC) with peripheral blood stem cell support in the adjuvant therapy of high-risk breast cancer: a practical approach.

In 29 chemotherapy-naive patients with stage II-III breast cancer, peripheral blood stem cells (PBSCs) were mobilised following fluorouracil 500 mg m-2, epirubicin 90-120 mg m-2 and cyclophosphamide 500 mg m-2 (FEC) and granulocyte colony-stimulating factor (G-CSF; Filgrastim) 300 microgram s.c. daily. In all but one patient, mobilisation was successful, requiring three or fewer leucocytopheresis sessions in 26 patients; 28 patients subsequently underwent high-dose chemotherapy consisting of carboplatin 1600 mg m-2, thiotepa 480 mg m-2 and cyclophosphamide 6 g m-2 (CTC) followed by PBSC transplantation. Haemopoietic engraftment was rapid with a median time to neutrophils of 500 x 10(6) l(-1) of 9 days (range 8-10) in patients who received G-CSF after PBSC-transplantation; platelet transfusion independence was reached within a median of 10 days (range 7-16). Neutropenic fever occurred in 96% of patients. Gastrointestinal toxicity was substantial but reversible. Renal, neural or ototoxicity was not observed. Complications related to the central venous catheter were encountered in 64% of patients, with major vein thrombosis occurring in 18%. High-dose CTC-chemotherapy with PBSC-transplantation, harvested after mobilisation with FEC and G-CSF, is reasonably well tolerated without life-threatening toxicity and is a suitable high-dose strategy for the adjuvant treatment of breast cancer.

PCR-positivity in harvested bone marrow predicts relapse after transplantation with autologous purged bone marrow in children in second remission of precursor B-cell acute leukaemia.

Purging of autologous bone marrow (BM) grafts of children in second remission after a relapse of precursor B acute lymphoblastic leukaemia (ALL) in the BM has been carried out in our laboratory since 1987, initially by complement mediated cell lysis. This protocol was extended by performing an immunorosette depletion before lysis with complement. The aim of the present study was to assess by polymerase chain reaction the presence of residual leukaemic cells in the BM grafts before and after purging. The results were then correlated to clinical outcome. In 24/28 patients a PCR product was obtained by amplification of IgH and/or TcR junctional regions. BM before purging was available for analysis in 13 patients. We found that leukaemic cells could be detected in 8/13 (62%) of these grafts before purging . All these eight patients experienced a relapse, regardless of whether the purging procedure had been successful (defined as achievement of PCR‐negativity) or not. In contrast, none of the five patients with PCR‐negative grafts before purging relapsed (P = 0.0008). One patient died due to transplant‐related toxicity. Of the remaining 23 patients, nine patients received a PCR‐positive BM graft after purging. All these nine patients experienced a relapse as compared to 6/14 whose BM was PCR‐negative after purging (P = 0.0072). Two of eight PCR‐positive BM grafts could be purged to PCR‐negativity. Thus, improvements both in treatment of leukaemia and in purging efficacy are still needed.

99mTc-CD19 monoclonal antibody is not useful for imaging of B cell non-Hodgkin’s lymphoma

Abstract In this study we investigated the applicability of 99mTc-labeled CD19 monoclonal antibody (mAb) for tumor imaging in patients with B cell non-Hodgkin’s lymphoma. A 1-mg sample of murine CD19 mAb was labeled with approximately 550 MBq [99mTc]pertechnetate. The labeled mAb was administered i. v. to seven patients, four without and three with pretreatment with 10 mg unlabeled CD19 mAb. The number of circulating B cells was decreased by 44±5% 1 h after injection of the radiolabeled mAb. Peripheral B cells were coated with CD19, resulting in partial modulation of CD19, most pronounced in the three pretreated patients. Whole-body images were obtained with a gamma camera and compared with results obtained by conventional imaging techniques. Initially, blood-pool activity dominated, whereas 24 h after injection the radioactivity was mainly located in the spleen, kidneys and liver. In two patients, a lesion in the spleen appeared as an unlabeled spot. In one patient, a lesion in the femur, which was detected by computed tomography (CT) and gallium-67 scans, was also seen on the CD19 scan from 1 h after administration of the radioimmunoconjugate onwards. Good imaging of bone marrow infiltration was observed in one of three patients. Lymph node involvement was not observed in any of the patients in whom affected lymph nodes were detected by CT or gallium-67 scan. In conclusion, in the present study radioimmunodetection with 99mTc-labeled CD19 mAb was found to be inferior to CT and gallium-67 scanning in the diagnosis of patients with B cell non-Hodgkin’s lymphoma.

Stroma‐supported progenitor production as a prognostic tool for graft failure following autologous stem cell transplantation

To analyse the involvement of a possible numerical or qualitative stem cell defect in the development of sustained graft failure after autologous transplantation, we have determined the graft content of CD34+ nucleated cells, colony‐forming cells and cobblestone area‐forming cell subsets, as well as transplant ability to produce progenitors using the long‐term culture colony‐forming cell (LTC‐CFC) assay. We evaluated material from the graft reference ampoules of 13 graft failure patients after bone marrow transplantation (BMT), four graft failure patients and four isolated thrombocytopenia patients after peripheral blood stem cell transplantation (PBSCT). We compared these data with those from six successfully engrafted BMT patients and 20 engrafted PBSCT patients respectively. In the BMT setting, the LTC‐CFC 6‐week assay represented a highly significant graft failure predictor. In the PBSCT setting, the total number of 2‐week and 6‐week LTC‐CFCs transplanted per kg bodyweight (BW) showed the highest significant difference between the engrafted and the graft failure patients, as well as between the engrafted patients and the patients suffering from isolated thrombocytopenia after transplantation. These data show that the ability of a graft to generate progenitors in vitro rather than the number of primitive progenitors transplanted can have prognostic value for post‐transplant haematological reconstitution.

Triple immunofluorescence staining for prediction of relapse in childhood precursor B acute lymphoblastic leukaemia.

In this study we describe a fast and sensitive method using three‐colour immunofluorescence for the detection of cells with phenotypes that are rare in normal bone marrow (BM) but occur frequently in children with precursor B acute lymphoblastic leukaemia. We show that, in the first year after initiation of therapy, in 17/18 patients (10 patients were analysed after first diagnosis and nine patients after first BM relapse; one patient was analysed on both occasions) the percentage of CD10+CD19+ cells and CD20−CD22+ cells in the CD34+ cell population indicated the likelihood of relapse. A suppression of cells expressing these phenotypes after initiation of therapy was followed by an outgrowth of normal precursor B cells after 12 months. Therefore this early test for impending relapse (which occurred 10–28 months after starting chemotherapy) was only applicable in the first year after beginning the treatment. However, despite this predictive value, comparison of fluorescence data with PCR results obtained from the same BM samples indicated that only a subpopulation of the CD34+CD10+CD19+ and CD34+CD20−CD22+ cells above the determined threshold value represented malignant cells. A large prospective study to confirm the predictive value of this three‐colour immunofluorescence assay is warranted.

Degradation of radioiodinated B cell monoclonal antibodies : inhibition via a FCγ-receptor-II-mediated mechanism and by drugs

Abstract Our aim is to treat patients with B cell malignancies with radioimmunotherapy using monoclonal antibodies (mAb) such as CD19, CD20 and CD22. In this study we investigated the rate of internalization and catabolism of these mAb. After 24 h at 37°C, 20% – 25% of initially cell-bound 125I-CD19 mAb and 125I-CD22 mAb was degraded in B cells, whereas almost no degradation occurred after binding of 125I-CD20 mAb. For B cells expressing Fcγ receptor II (FcγRII), isotype-dependent degradation was noted as the CD19 IgG1 mAb showed an enhanced degradation rate compared to the switch variant IgG2a. The effect of various pharmaceutical agents that delay the internalization or subsequent degradation of mAb was evaluated. The degradation was inhibited most effectively by a combination of etoposide and vinblastine, resulting in accumulation of radioactivity in the target cell. Also the simultaneous application of CD20 or CD22 with 125I-CD19 mAb or of CD20 with 125I-CD22 mAb proved to be a potent inhibitor of the rapid degradation of these mAb, by inhibiting internalization via an FcγRII-mediated mechanism. Both methods of reducing the degradation of radioiodinated mAb are expected to prolong irradiation of malignant B cells and consequently result in an enhanced therapeutic effect in vivo.

Separation of G‐CSF‐mobilized PBSC transplants by counterflow centrifugal elutriation: modest enrichment of CD34+ cells but no loss of primitive haemopoietic progenitors

The suitability of counterflow centrifugal elutriation (CCE) for reduction of the number of non‐stem cells in autologous G‐CSF‐mobilized peripheral blood stem cell (PBSC) transplants was investigated. By cell size‐monitored CCE, small cells could be rapidly separated from the haemopoietic progenitor cells present in leukapheresis product (LP) samples. The large cell fraction contained an average 86 ± 25% of the CD34+ cells and 76 ± 20% of the granulocyte‐macrophage progenitors (CFU‐GM) loaded into the separation chamber, and was depleted of 75 ± 18% of the lymphocytes, 89 ± 7% of the erythrocytes and 98 ± 2% of the platelets (n = 21). Due to the presence of high numbers of large immature myeloid cells, which co‐elutriated with progenitor cells, enrichment of CD34+ cells in the large cell fraction was only modest (average 1.8 times). No indication of preferential co‐elutriation of primitive stem cells with the small cells was obtained. There was no difference in expression of CD38 or Thy‐1 on CD34+ cells between the two elutriation fractions. Frequencies of cobblestone‐area‐forming cells (CAFC) week 6, which are considered to represent cells with long‐term repopulating ability, were reduced in the small cell fractions as compared to those in the unseparated samples and the large cell fractions. On average, 100% of CAFC week 6 were recovered in the large cell fractions (n = 5). In conclusion erythrocytes, platelets and 40–50% of leucocytes can be depleted from G‐CSF‐mobilized PBSC samples by CCE with an almost complete recovery of both clonogenic and primitive stem cells.