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Biomedical subjects

W Krüger

Publications and source records attributed to W Krüger.

At least 55 records · Page 3Linked to original sources
PubMed1998

Successful mobilization of peripheral blood stem cells in heavily pretreated myeloma patients with G-CSF alone.

We investigated the feasibility of mobilizing peripheral blood stem cells (PBSC) with G-CSF alone in 24 patients with multiple myeloma. The median age was 53 years (range 33-62). All patients had stage II/III disease and responded to standard first-line (n = 6) or salvage chemotherapy (n = 18). The median number of previous chemotherapy cycles was 7 (4-18) and the median number of prior melphalan-cycles was 6 (0-14). Nine (35%) patients had experienced prior radiation therapy. The patients received either 10 microg/kg G-CSF (n = 18) or 24 microg/kg G-CSF (n = 7, including one patient with previous 10 microg/kg G-CSF stimulation) daily s.c. for 5 or more consecutive days until completion of harvesting, starting apheresis on the fifth day. G-CSF treatment was well tolerated, with only slight bone pain in half of the patients (51%). After a median of three (range 1-7) apheresis procedures, medians of 3.8 (0.3-17) x 10(6) CD34+ cells/kg, 8.5 (4.5-24) x 10(8) MNC/kg, 2.9 (0.6-39.4) x 10(4) CFU-GM/kg, and 5.6 (0.9-49) x 10(4) BFU-E/kg were harvested. Three patients (12%) with extensive melphalan pretreatment failed the target collection of at least 2.0 x 10(6) CD34+ cell/kg. Pretreatment with six or more cycles of melphalan yielded a smaller number of CD34+ cells than pretreatment with fewer than six cycles (2.5 vs 5.3 x 10(6)/kg; p = 0.001). Nineteen patients underwent high-dose chemotherapy consisting of either total marrow irradiation (9 Gy)/busulfan (12 mg/kg) and cyclophosphamide (120 mg/kg) (n = 10), or busulfan (14 mg/kg)/cyclophosphamide (120 mg/kg) (n = 5), or tandem melphalan (200 mg/m2). The median time for granulocyte (> 1.0/nl) and platelet (> 50/nl) recovery was 10 and 14 days (ranges 7-12 and 8-40), respectively. G-CSF alone is a safe, alternative approach to mobilizing sufficient PBSC in patients with multiple myeloma and allows an exact prediction of harvest time. G-CSF-mobilized PBSCs ensure rapid engraftment after myeloablative therapy. Melphalan treatment should be avoided in patients who are candidates for high-dose chemotherapy.

Adult↗
PubMed1998

Specificity of reverse transcriptase polymerase chain reaction assays designed for the detection of circulating cancer cells is influenced by cytokines in vivo and in vitro.

Several reverse transcriptase polymerase chain reaction (RT-PCR) assays have been described for the detection of circulating tumour cells in blood and bone marrow. Target mRNA sequences for this purpose are the cytokeratins (CK) 19 and 20, the carcinoembryonic antigen (CEA), and the prostate-specific antigen messages. In this study, we investigated biological factors influencing the specificity of the CK19 and CEA RT-PCR assays. Bone marrow, granulocyte colony-stimulating factor (G-CSF)-mobilized blood stem cells and peripheral blood samples obtained from healthy volunteers (n = 15; CEA n = 7), from patients with epithelial (n = 29) and haematological (n = 23) cancer and from patients with chronic inflammatory diseases (n = 16) were examined. Neither CEA nor cytokeratin 19 messages could be amplified from bone marrow samples from healthy subjects and from patients with haematological malignancies. In contrast, specimens from patients with inflammatory diseases scored positive up to 60%. To investigate the influence of inflammation on target mRNA expression, haemopoietic cells were cultured with and without cytokine stimulation in vitro. CK19 messages could be easily detected in cultured marrow cells without further stimulation, CEA messages only after gamma-interferon (gamma-INF) stimulation. In contrast, G-CSF-mobilized peripheral blood stem cells were positive for CK19 messages only after stem cell factor (SCF) or interleukin stimulation. We conclude that transcription of so-called tissue-specific genes is inductible in haemopoietic tissues under certain conditions. These factors have to be considered in future applications of RT-PCR for the detection of minimal residual disease.

Abdominal Neoplasms↗
PubMed1998

Long-term disease-free survival of patients with advanced mantle-cell lymphoma following high-dose chemotherapy.

In advanced stage mantle cell lymphoma, conventional chemotherapy yields a complete remission rate below 40%, and the median survival rate is only about 3 years. Between 1991 and 1996 we treated nine such patients (six male; three female) with high-dose chemotherapy (six of these also with 12 Gy fractionated total body irradiation (TBI)) and peripheral stem cell support (n = 8) or allogeneic bone marrow transplantation (n = 1). The median age was 47 years (range, 28-61). At the time of high-dose chemotherapy, five patients were in first complete remission (CR), two in first partial remission (PR) and two in second remission (CR = 1; PR = 1). High-dose chemotherapy included TBI (12 Gy), etoposide and cyclophosphamide (patients 1-5), TBI and cyclophosphamide (patient 7), busulfan, etoposide and cyclophosphamide (patients 6 and 9), cyclophosphamide and busulfan (patient 8). The patterns of toxicity according to the Bearman score were usually mild (mucositis grade 2, n = 7; renal grade I, n = 2) with no therapy-related fatality. Only one patient developed hepatic toxicity grade III (veno-occlusive disease) but recovered completely. The median time to neutrophil engraftment was 10 days (range, 8-15). After high-dose chemotherapy all patients achieved complete remission. After a median follow-up of 22 months (range, 9.4-64) all patients remain in continuous complete remission. These encouraging results suggest that high-dose chemotherapy can be applied safely and leads to long-term disease-free survival in otherwise incurable disease.

Adult↗
PubMed1998

Purging and haemopoietic progenitor cell selection by CD34+ cell separation.

Tumour cell contamination of autologous peripheral blood stem cell samples (PBSC) and bone marrow (BM) is frequent. Enrichment of CD34+ stem cells is a promising approach to purging tumour cells from autografts without damaging progenitor cells. Breast cancer cells were seeded (10(-3)-10(-7)) into mononuclear cells from G-CSF-mobilised PBSC and BM harvests from patients without breast cancer. CD34+ cells were enriched from mixtures either by immunomagnetic separation (Isolex-50, and MiniMACS) or by biotin-streptavidin immunoaffinity columns (Ceprate-LC). CD34+ cell fractions were determined by FACS, cancer cells were detected immunocytochemically with an anti-pancytokeratin antibody. The CD34+ cells were enriched with a median purity of 92.2% (43.5-96.1) (n = 17) (Isolex-50), 96.5% (66.6-99.2) (n = 17) (MiniMACS) and 77.9% (31.4-93.6) (n = 15) (Ceprate-LC) from PBSC and BM harvests. The percentages of median recovery of CD34+ cells were 30.8% (18.6-71.8) (Isolex-50), 69.9% (39.1-100) (MiniMACS) and 42.9% (23.7-100) (Ceprate-LC). The median tumour cell reductions in log steps were 3.7 (2.9-4.3) (n = 13) (Isolex-50), 3.5 (2.6-4.3) (n = 13) (MiniMACS) and 1.5 (0.9-2.9) (n = 17) (Ceprate-LC). Results were compared statistically by univariate analysis. Purity was significantly (P < 0.05) better after MiniMACS selection. Recovery rates were significantly different between all devices tested. Tumour cell purging was superior after immunomagnetic separation (P < 0.001). Tumour cell purging is a main objective of CD34+ selection in the autologous setting. Our in vitro data clearly indicate that immunomagnetic separation is more efficient in the prevention of accidental reinfusion of contaminating tumour cells compared to immunoaffinity. However, it is not yet known if the same results can be obtained with fresh contaminating tumour cells.

Antigens, CD34↗
PubMed1998

Busulfan, cyclophosphamide and etoposide as high-dose conditioning therapy in patients with malignant lymphoma and prior dose-limiting radiation therapy.

Relapse after transplant for malignant lymphomas remains the main cause of treatment failure. Most conditioning regimens contain total body irradiation (TBI). We investigated the toxicity and efficacy of an intensified chemotherapy conditioning regimen without TBI in patients with relapsed or high-risk malignant lymphoma who had received prior radiation therapy and were therefore not eligible for TBI. Twenty patients with a median age of 38 (18-56) and relapsed or high-risk malignant non-Hodgkin's lymphoma (NHL, n = 16) or Hodgkin's disease (HD, n = 4) underwent high-dose chemotherapy consisting of busulfan (16 mg/kg), cyclophosphamide (120 mg/kg) and etoposide 30 mg/kg (n = 8) or 45 mg/kg (n = 12) followed by peripheral stem cell support (n = 14), autologous bone marrow (n = 3), allogeneic (n = 2) or syngeneic (n = 1) transplantation. All but two had chemosensitive disease before high-dose chemotherapy. The main toxicity -- according to the Bearman score -- was mucositis II in 18 (90%) patients; five patients (25%) suffered a grade I hepatic toxicity. GI toxicity I occurred in three (15%) and renal toxicity I in two patients (10%). Sixty percent of the patients developed transient dermatitis with erythema and three of them (15%) had skin desquamation; one patient experienced asymptomatic pancreatitis. Toxicity was slightly higher in patients treated with 45 mg/kg etoposide. One patient (5%) died of treatment-related venoocclusive disease. After a median follow-up of 50 months (24-84) the disease-free and overall survival were 50% and 55%. One of the nine relapsing patients developed secondary AML 18 months after transplant. High-dose busulfan, cyclophosphamide and etoposide is an effective regimen resulting in long-term disease-free survival in 50% of patients with relapsed malignant lymphoma and prior radiation therapy. The toxicity is moderate with a low treatment-related mortality (5%).

Adolescent↗
PubMed1998

Intensified conditioning regimen in bone marrow transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia.

We investigated an intensified conditioning regimen including fractionated total body irradiation (12 Gy), etoposide (30-45 mg/kg) and cyclophosphamide (120 mg/kg), followed by autologous (n = 5), allo-related (n = 13) or allo-unrelated (n = 6) bone marrow (n = 22) or peripheral stem cell (n = 2) transplantation in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia. One patient received busulfan (16 mg/kg) instead of TBI. Nineteen patients were transplanted in 1CR, two in 2CR, one in 1PR and two in relapse. Major toxicity was mucositis grade II according to the Bearman scale in all patients. The treatment-related mortality was 25%, mainly due to infection or GVHD after allogeneic transplantation. After a median follow-up of 45 months (range 2-93), nine patients (37.5%) remain alive in CR. Nine patients (37.5%) relapsed and eight (33.3%) of these subsequently died. After autologous transplantation, four of five patients (80%) relapsed and died. Late relapse was seen after allogeneic, as well as autologous transplantation, at 33 and 59 months, respectively. The Kaplan-Meier estimate of leukemia-free survival for all patients is 38% at 3 years (95% CI: 18-58%) and 35% at 5 years (95% CI: 15-55%). For allogeneic transplants in first CR (n = 15) the estimate of disease-free survival was 46% at 3 years (95% CI: 19-73%) and 34% at 5 years (95% CI: 17-51%). Patients aged below 30 years had a better estimated overall survival at 3 years (61% vs 11%, P < 0.001). The bcr-abl fusion transcript (p210 vs p190 vs p210/190) did not affect disease-free or overall survival. In our experience, an intensified conditioning regimen seems to improve the results of bone marrow transplantation in patients with Ph+ acute lymphoblastic leukemia. However, the high relapse rate warrants novel approaches to enhance anti-leukemic efficacy.

Adolescent↗
PubMed1998

Mobilizing peripheral blood stem cells with high-dose G-CSF alone is as effective as with Dexa-BEAM plus G-CSF in lymphoma patients.

We compared retrospectively the efficacy of granulocyte colony stimulating factor (G-CSF) alone with chemotherapy plus G-CSF in mobilizing CD34-positive cells in patients with malignant lymphoma. 35 patients underwent peripheral blood stem cell (PBSC) collection following mobilization either with 24 microg/kg G-CSF for 4 consecutive days (n = 18) or Dexa-BEAM chemotherapy plus 5 microg/kg G-CSF (n = 17). High-dose G-CSF was well tolerated with only slight bone pain and/or myalgia. The Dexa-BEAM therapy required hospitalization with a median duration of 21 d. The median number of apheresis procedures in both groups was two (range two to four), resulting in a median of 5.3 and 5.1 x 10(6) CD34+ cells/kg. No patients in the G-CSF group, but one in the Dexa-BEAM group, failed to reach the target of collecting >2.0 x 10(6) CD34+ cells/kg. The number of CFU-GM (10.4 v 6.0 x 10(5)/kg) and of BFU-E (10.6 v 4.5 x 10(5)/kg; P = 0.04) was higher in the G-CSF group than in the Dexa-BEAM group. A subset analysis of CD34+ cells was performed in 16 patients showing a higher mean of Thy-1 (CD90w) coexpression in the G-CSF than in the Dexa-BEAM group (4.8 v 1.8%, P = 0.12). Additionally the percentage of CD34+/CD38- cells was higher in the G-CSF group (10.66% v 8.8%). However, these differences were not statistically significant. The median time to leucocyte and platelet engraftment after high-dose chemotherapy was slightly shorter in the G-CSF than in the Dexa-BEAM group (9 v 10 and 12 v 13.5 d, respectively). These results demonstrate that high-dose G-CSF is as effective as Dexa-BEAM plus G-CSF in mobilizing peripheral blood stem cells and produces prompt engraftment. The major advantages of G-CSF mobilization were the safe outpatient self-application and the fixed-day apheresis.

Adolescent↗
PubMed1997

Factors influencing haematological recovery after allogeneic bone marrow transplantation in leukaemia patients treated with methotrexate-containing GVHD prophylaxis. A single-centre experience.

In the present single institution study of 66 leukaemia patients (28 AML, 23 ALL, 15 CML), the factors influencing haematological recovery after allogeneic bone marrow transplantation (alloBMT) were analysed retrospectively to identify the optimal conditions required for rapid haematological recovery after alloBMT. All patients received GVHD prophylaxis with cyclosporine A plus methotrexate. The mean number of days required to achieve a neutrophil count > or = 0.5 x 10(9)/l after alloBMT was 17 (range 9-27), 19 patients (28.8%) had rapid neutrophil recovery within 15 days after alloBMT. Haematological recovery was more rapid in the 38 patients without GVHD or with only grade I GVHD. Also, 50% and 40% of patients receiving 10 (n = 18) or 5 (n = 20) micrograms/kg G-CSF per day, respectively, had rapid neutrophil recovery within 15 days after alloBMT, as against only 7.1% of patients not receiving G-CSF after the transplant (n = 28); P < 0.001. The neutrophil recovery was similar in patients receiving either fresh or cryopreserved allografts and either a TBI-containing or a busulfan-containing conditioning regimen. A significant correlation was found between the neutrophil recovery and either the MNCs or CFU-GM contents of the allografts. The mean number of days required for neutrophil recovery was only 16 (range 9-24) in patients receiving allografts containing > 1 x 10(5) CFU-GM/kg (n = 28), as against 19 (range 13-27) in patients receiving allografts containing < or = 1 x 10(5) CFU-GM/kg (n = 35). Three patients receiving allografts containing < 0.5 x 10(5) CFU-GM/kg had primary neutrophil engraftment failure. The mean number of days required to achieve a platelet count > or = 20 x 10(9)/l was 21 (range 11-50), and 30 patients (46.9%) had platelet recovery within 20 days after alloBMT. The platelet recovery after alloBMT was not affected by the type of leukaemia, conditioning regimen, or G-CSF administration. The mean number of days required for platelet recovery after alloBMT was 20 in patients receiving allografts containing > 1.0 x 10(5) BFU-E/kg (n = 35), as against 23 days in patients receiving allografts containing < or = 1.0 x 10(5) BFU-E/kg (n = 24). Seven patients receiving allografts containing < 0.5 x 10(5) BFU-E/kg had primary platelet engraftment failure. The present study has identified the high number of progenitor cells in the allografts infused and the daily administration of G-CSF posttransplant as the optimal combination for rapid neutrophil recovery after alloBMT. More significantly, the number of BFU-E in allografts was the most significant determining factor in platelet recovery after alloBMT. The development of GVHD of grade II or more during the first weeks after alloBMT was associated with slower haematological recovery, a longer period of fever during neutropenia and longer hospitalization.

Adolescent↗
PubMed1997

Can dentists recognize manipulated digital radiographs?

OBJECTIVES: To determine the ability of dentists to recognize digitally manipulated radiographs. METHODS: A poster was presented at the Annual Meeting of the German Society for Periodontology displaying the intra-oral radiographs of 12 different patients. Half of the radiographs were subjected to digital manipulation to add or remove specific features. Dentists were asked to identify these radiographs by means of a questionnaire. RESULTS: Thirty-nine dentists submitted usable questionnaires. Statistical evaluation revealed a distribution of hits similar to the random distribution. None of the dentists detected all the six manipulated radiographs; three dentists had five correct, but there were five with only one. An authentic radiograph scored highest as a manipulation. CONCLUSIONS: Image manipulations which alter the diagnostic content of a radiograph are unlikely to be detected by dentists. Digital radiography requires additional measures for data protection.

Clinical Competence↗
PubMed1997

Long-term follow-up of leukaemia patients after related cryopreserved allogeneic bone marrow transplantation.

We have previously shown that allogeneic bone marrow transplantation (BMT) with cryopreserved donor marrow cells can be used without prolonging the engraftment time or interfering with the reconstitution of haemopoiesis. In this report we extend our initial observations of the first 40 patients who underwent allogeneic bone marrow transplantation from related donors with cryopreserved donor bone marrow for haematological malignancies, including the long-term follow-up data of the previously reported patients. The outcome of these patients was compared with that of 40 related BMT recipients receiving fresh donor bone marrow (historic control group). Time until engraftment of all patients receiving cryopreserved bone marrow was not different from the control group (ANC > 0.5 x 10(9)/l 17d (range 11-24 d) versus 17.5 d (range 10-28 d): platelets > 20 x 10(9)/l 21 d (range 11-85 d) versus 22 d (range 13-69 d), respectively). There was the same incidence of acute and chronic GvHD in patients receiving either cryopreserved bone marrow or fresh bone marrow (acute GvHD > or = II 61% v 60% and chronic GvHD 56% v 52%, respectively). Chimaerism studies showed no difference between the patient groups. Furthermore, the two groups did not differ in day 100 survival (82% v 72%). With a median follow-up of 520 d (range 47-1365 d) and 1289 d (range 48-1849 d), 60% of the patients receiving cryopreserved and 53% of the patients receiving fresh allogeneic donor bone marrow, respectively, are alive. We conclude that cryopreservation of allogeneic related donor bone marrow does not adversely affect engraftment, does not decrease the incidence of severe acute GvHD, and does not seem to affect the day 100 survival or long-term haemopoiesis.

Adolescent↗
PubMed1997

Allogeneic transplantation with CD34+-selected cells.

We report the outcome of eight patients with different hematological malignancies who were transplanted with allogeneic CD34-selected mononuclear cells following myeloablative therapy. Four patients received G-CSF mobilized CD34-enriched peripheral blood progenitor cells (PBPC) together with CD34-enriched bone marrow (BM), two patients were transplanted with allogeneic G-CSF mobilized CD34-enriched PBPC alone, and two patients received only allogeneic CD34-enriched BM cells. On average, patients received 2.66 x 10(6) CD34+-cells/kg BW (range: 0.53-8.40 x 10(6) CD34+-cells/kg body weight) and 0.57 x 10(6) CD3+-cells/kg BW (range: 0.20-1.10 x 10(6) CD3+-cells/kg BW), respectively. Seven of the eight patients engrafted (ANC > 0.5 x 10(9)/L median: day +19 (range: 16-23 days); platelets > 20 x 10(9)/L median: day +34 (range: 21-47 days); one patient died on day +16 after transplantation and was not evaluable for engraftment. Three of seven patients evaluable for acute graft-versus-host disease (GvHD) developed acute GvHD grade II which resolved upon steroid treatment. Five of the eight patients are still alive and in remission with a median follow-up of 215 days (range: 80-420 days). Causes of death included fungal infection, cerebral bleeding and sepsis. These preliminary data suggest that CD34-enriched cells can be successfully given during for allogenic transplantation following myeloablative therapy in hematological malignancies. The impact of T-cell depletion by enrichment for CD34+-cells in an attempt to reducing the incidence and/or severity of acute and/or chronic GvHD still remains to be determined.

Adult↗
PubMed1997

Antimycotic therapy with liposomal amphotericin-B for patients undergoing bone marrow or peripheral blood stem cell transplantation.

Suspected deep or systemic mycosis in patients undergoing high-dose therapy and autologous or allogeneic bone marrow transplantation (BMT) requires an immediate systemic antimycotic therapy. Intravenous therapy with the standard drug conventional amphotericin-B is associated with severe adverse effects like nephrotoxicity and chills. Furthermore, BMT patients often receive other potential nephrotoxic drugs such as CsA or virustatics. In this study, we report 74 BMT-patients treated with liposomal amphotericin-B for culture-documented aspergillosis (n = 5) or candidiasis (n = 6), or for serologically (n = 35) or clinically suspected mycosis or as prophylaxis (n = 2). Therapy was initiated with a median dose of 2.8 (0.64-5.09) mg/kg body-weight and continued for 13 (1-55) days. The drug was excellently tolerated and only in one was therapy stopped due to severe chills and fever. Severe organ impairment was not observed under therapy with liposomal amphotericin-B. Creatinine decreased in five patients after an increase under preceding therapy with the conventional formulation. Influence of liposomal amphotericin-B on bilirubin and transaminases was difficult to evaluate due to therapy-related toxicity, veno-occlusive disease (VOD), and graft-versus-host disease (GvHD). 10/11 culture-positive patients died from aspergillosis (5/5) or candidiasis (5/6), but in 9/11 of these subjects the immunity was additionally compromised by GvHD, steroid therapy, and VOD. Liposomal amphotericin-B was effective in preventing relapse of systemic mycosis in 10/12 patients with a history of aspergillosis (n = 11) or candidiasis (n = 1). We conclude, that favourable toxicity of liposomal amphotericin-B should encourage dose escalation studies of liposomal amphotericin-B randomised against the conventional formulation and that the comparison of patients undergoing BMT with patients under standard chemotherapy might be difficult because of additional risk factors of the BMT-patients.

Adolescent↗
PubMed1997

High dose chemotherapy with busulfan, cyclophosphamide, and etoposide as conditioning regimen for allogeneic bone marrow transplantation for patients with acute myeloid leukemia in first complete remission.

We explored the combination of busulfan/cyclophosphamide/etoposide as conditioning regimen prior to bone marrow transplantation in 31 patients with acute myeloid leukemia (AML) in first complete remission. The preparative regimen consisted of 16 mg/kg busulfan, 30-60 mg/kg VP-16, and 120 mg/kg cyclophosphamide. With a median follow-up of 30.5 months (range, 5-60 months), 25 patients are alive in continuous complete remission. Estimated disease-free survival at 5 years is 80.5%. Death was due to transplant-related toxicity (graft-versus-host disease and cytomegalovirus infection, graft-versus-host disease and pneumonia, sepsis and mucositis, respectively). None of the patients have relapsed. As demonstrated by the results of this analysis, the conditioning regimen busulfan/cyclophosphamide/etoposide is effective and well tolerated in patients with AML in first complete remission. Main nonhematological toxicities were mucositis and hepatotoxicity. The low mortality and relapse rate appears to justify allogeneic bone marrow transplantation for patients with AML in first complete remission who have an HLA-identical donor. Whether this regimen offers a substantial improvement in disease-free and overall survival over presently used regimens warrants further investigation.

Acute Disease↗
PubMed1996

Comparison between bone marrow and G-CSF-mobilized peripheral blood allografts undergoing clinical scale CD34+ cell selection.

Allogeneic transplantation of selected CD34+ cells, rather than conventional transplantation of bone marrow (BM) harvest or peripheral blood (PB) leukapheresis products, has the advantage of reducing volume, facilitating storage and decreasing the amount of dimethylsulfoxide (DMSO) and cell lysis products, as well as reducing the number of T-lymphocytes responsible for graft-versus-host disease (GVHD). Using biotinavidin immunoaffinity columns (Ceprate SC system, CellPro; Bothell, WA), CD34+ cells were selected from each of 20 allografts (12 G-CSF-mobilized PB and 8 BM) collected from 14 HLA-identical normal healthy donors for transplantation. After the clinical-scale selection, the median concentration of CD34+ cells was 44.6% (range, 13% to 91%) in BM and 50.4% (range, 15% to 77%) in PB. Whereas 75% of the PB allografts had a CD34+ cell yield of more than 65%, only 37.5% of the BM allografts achieved such a yield, p < 0.01. The number of T-lymphocytes in the selected CD34+ cell allografts was reduced by two to three logs from a median of 4.2 x 10(9) to 7.8 x 10(5) CD3+ cells. The enrichment in CD34+ cells was 240-fold (range, 24- to 382-fold) in PB versus only 34-fold (range, 14- to 108-fold) in BM. Also, the enrichment in clonogenic cells was significantly more in PB (median value of 38.6-fold) than in BM (median value of 19.2-fold) and more in allografts from younger (< 50 years old) rather than older (> or = 50 years old) adult donors. A correlation was found between the percentage of CD34 or CD3+ cells before and after selection (r = 0.58 or r = 0.60, respectively, p < 0.05). Selective enrichment of the colony forming units-granulocyte-macrophage (CFU-GM) was found in all 20 allografts. The progenitor cell recovery after freezing and thawing was similar in BM and PB allografts, with a mean of about 60% for the CFU-GM and BFU-E. In the same six donors, the CD34+ cell yield was significantly more in the PB after mobilization (median 78.5%, range 50% to 90%) than in the BM before mobilization (median 41.5%, range 25% to 87%), p < 0.01. Ten patients with hematologic malignancies have been allotransplanted with 14 of the 20 selected CD34+ cells either combined BM + PB (n = 4) or single (n = 6) grafts. Seven patients did not develop acute GVHD, and only two patients developed > or = grade II GVHD, one of whom developed only grade II GVHD that resolved after brief treatment with corticosteriods. Only one patient showed chronic GVHD (skin and liver). The low incidence and severity of GVHD seen in the present study (only 30%) could be due to the two- to three-log reduction of T-lymphocytes in the selected CD34+ cell allotransplants. All 10 patients had stable hematological recovery, and seven had full donor hematopoiesis. In conclusion, G-CSF-mobilized PB leukapheresis products undergoing selection of CD34+ cells have a greater yield and enrichment of progenitor cells than BM harvests collected from HLA-identical normal healthy donors for allogeneic transplantation. The low incidence and severity of both acute and chronic GVHD (30%) seen in the present study are very encouraging.

Adult↗
PubMed1996

Reverse transcriptase/polymerase chain reaction detection of cytokeratin-19 mRNA in bone marrow and blood of breast cancer patients.

A two-step reverse-transcriptase-based polymerase chain reaction (PCR) with nested primer pairs was developed to amplify sensitive and specific cytokeratin-19 (CK-19) mRNA sequences from human breast cancer cells. No CK-19 pseudogene interference was seen. The larger DNA-derived amplification products could be clearly discriminated from mRNA-derived products. The CK-19 message was not amplified from bone marrow or blood of healthy volunteers and patients with haematological malignancies nor from myeloid and lymphoid cell lines. Breast cancer cells were diluted in buffy coat cells up to 10(-6) and CK-19 mRNA sought by PCR. The CK-19 message was detected in 14 of 26 blood samples and 14 of 24 marrow samples but in neither of two peripheral blood stem cell samples taken from 35 breast cancer patients. By sequence-analysis control of two of these samples and two cell lines, the amplified DNA fragments were confirmed to be homologous with the CK-19 sequence. The CK-19 message was further sought in matched blood/marrow samples taken from 13 untreated women in the same cohort at the time of diagnosis. In 3 of these, CK-19 RNA was detected in blood and marrow and, in 3 others, only in blood, but never in marrow alone. The results show that CK-19 assay by reverse transcriptase/PCR is a sensitive and specific technique for the detection of cancer cells in bone marrow and blood. It could be helpful in diagnosis and monitoring of metastatic breast cancer and detection of micrometastases. This should be evaluated on larger numbers of patients, with different clinical samples and epithelial malignancies.

Base Sequence↗
PubMed1996

Comparison of the content and subpopulations of CD3 and CD34 positive cells in bone marrow harvests and G-CSF-mobilized peripheral blood leukapheresis products from healthy adult donors.

Recombinant human granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem/progenitor cells (PBPC) have replaced bone marrow (BM) harvests for autologous transplantation after myeloablative therapy in cancer patients. G-CSF-mobilized PBPC from healthy donors contain one log excess of T lymphocytes representing a potential risk for graft-versus-host disease (GVHD). However, recent pilot clinical studies of G-CSF-mobilized allogeneic PBPC transplantation have shown rapid haematological recovery and no severe acute GVHD except in a very few cases. Therefore, the risk of inducing severe acute GVHD is not as high as was expected during the pioneering period of allogeneic PBPC transplantation. The present study was performed to address the possible reasons for the rapid haematological recovery and the absence of severe acute GVHD observed after allogeneic PBPC transplantation by comparing the contents and subsets of CD3+ and CD34+ G-CSF-mobilized PBPC (n = 31) with those of BM (n = 26) allografts from healthy adult donors. The present results revealed that the phenotypic profiles of CD3+ and CD34+ cells differ between PBPC and BM allografts. The single PBPC leukapheresis product contained 10 times more mononuclear cells, 1.5 times more CD34+ cells, 5.5 times more CD3+ T lymphocytes, 3 times more CD19+ B lymphocytes and 3.8 times more CD14+ monocytes than the single BM harvest. Both CD34+CD33+ myeloid progenitor cells and CD34+HLA-DR-long-term reconstituting haemopoietic stem cells were significantly increased in the CD34+ G-CSF-mobilized PBPC compared with the CD34+ BM cells; median 73.1% and 30.4% vs 60.6% and 5.0%, respectively, P < 0.01. The percentage of CD3+ cells coexpressing CD4 (T helper/inducer) was similar in both PBPC and BM allografts, 47.2% and 45.6%, respectively, whereas the percentage of CD3+ cells coexpressing CD8 (T suppressor/cytotoxic) was significantly decreased in PBPC compared with BM; 37.0% vs 55.9%, p < 0.01. The rapid haematological recovery after allogeneic PBPC transplantation could be due to the increased content of CD34+CD33+ myeloid committed cells and the CD34+HLA-DR-long-term reconstituting haemopoietic stem cells in the PBPC allografts. Also, the absence of an increased risk of severe acute GVHD after allogeneic PBPC transplantation could be due to the increased T lymphocyte ratio of CD4+/CD8+ in the PBPC allografts. In conclusion, rapid haematological recovery without an increased risk of severe acute GVHD can be achieved using G-CSF-mobilized PBPC rather than BM for allogeneic transplantation.

Adult↗