ALBERT

Description: Graft-versus-host disease (GVHD) represents the major nonrelapse complication of allogeneic hematopoietic cell transplantation. Although rare, the CNS and the eye can be affected. In this study, manifestation in the retina as part of the CNS and T-cell epitopes recognized by the allogeneic T cells were evaluated. In 2 of 6 patients with posttransplantation retina diseases and 6 of 22 patients without ocular symptoms, antigen-specific T-cell responses against retina-specific epitopes were observed. No genetic differences between donor and recipient could be identified indicating T-cell activation against self-antigens (graft versus self). Transplantation of a preexisting immunity and cross-reactivity with ubiquitous epitopes was excluded in family donors and healthy individuals. In summary, an immunological reaction against retina cells represents a mechanism of graft-versus-host interaction following hematopoietic cell transplantation.

Description: Background The selection pressure exercised by antibiotic drugs is an important consideration for the wise stewardship of antimicrobial treatment programs. Treatment decisions are currently based on crude assumptions, and there is an urgent need to develop a more quantitative knowledge base that can enable predictions of the impact of individual antibiotics on the human gut microbiome and resistome. Results Using shotgun metagenomics, we quantified changes in the gut microbiome in two cohorts of hematological patients receiving prophylactic antibiotics; one cohort was treated with ciprofloxacin in a hospital in Tübingen and the other with cotrimoxazole in a hospital in Cologne. Analyzing this rich longitudinal dataset, we found that gut microbiome diversity was reduced in both treatment cohorts to a similar extent, while effects on the gut resistome differed. We observed a sharp increase in the relative abundance of sulfonamide antibiotic resistance genes (ARGs) by 148.1% per cumulative defined daily dose of cotrimoxazole in the Cologne cohort, but not in the Tübingen cohort treated with ciprofloxacin. Through multivariate modeling, we found that factors such as individual baseline microbiome, resistome, and plasmid diversity; liver/kidney function; and concurrent medication, especially virostatic agents, influence resistome alterations. Strikingly, we observed different effects on the plasmidome in the two treatment groups. There was a substantial increase in the abundance of ARG-carrying plasmids in the cohort treated with cotrimoxazole, but not in the cohort treated with ciprofloxacin, indicating that cotrimoxazole might contribute more efficiently to the spread of resistance. Conclusions Our study represents a step forward in developing the capability to predict the effect of individual antimicrobials on the human microbiome and resistome. Our results indicate that to achieve this, integration of the individual baseline microbiome, resistome, and mobilome status as well as additional individual patient factors will be required. Such personalized predictions may in the future increase patient safety and reduce the spread of resistance. Trial registration ClinicalTrials.gov, NCT02058888. Registered February 10 2014

Description: Abstract 3540 Historically, allogeneic hematopoietic cell transplantation (HCT) has been offered only to patients with good performance status and below the age of 60. However, the peak incidence of most hematologic malignancies is above 60 years of age. The introduction of reduced intensity conditioning (RIC) regimens enabled successful allogeneic HCT in patients with considerable comorbidities and older than 60 years. The impact of age on outcome of allogeneic HCT in patients ≥60 years has not been evaluated extensively. We retrospectively analyzed 109 consecutive patients (f=43, m=66) aged≥60 who received allogeneic HCT 2000–2010 at our institution. Median age of the patients was 65 years (range, 60–76). Patients were grouped in two cohorts depending on age: group 1 aged 60–65 years (n=60, median age=63) and group 2 aged 66–76 years (n=49, median age=68). Diagnoses were acute leukemia (AML n=65, ALL n=1), myelodysplastic syndrome (n=14), osteomyelofibrosis (n=7), non-Hodgkin lymphoma (n=9), multiple myeloma (n=8), aplastic anemia (n=1), chronic myeloid leukemia (n=2) and chronic lymphatic leukemia (n=2). At time of HCT, 41 of the patients were in complete remission (CR), 68 in partial remission (PR) (group 1: CR 21, PR 39; group 2: CR 20, PR 29) and 18 patients had a preceding HCT, 14 in group 1. Conditioning regimens were grouped in high (TBI/Bu+Cy, n=5, all group 1), intermediate (FLAMSA, Flu/Mel/BCNU, n=28, group 1=11, group 2=17), low (FLU+alkylans, n=48, group 1=32, group 2=16) and minimal (2GyTBI/Flu, n=28, group 1=12, group 2=16) intensity. Intermediate intensity conditioning was particularly used for high risk patients in PR (25/28). 22 patients were transplanted from matched related (MRD), 46 from matched unrelated (MUD) and 41 from mismatched unrelated donors (MMUD). Kaplan-Meier-estimated 3-year overall survival (OS) was 45% for all patients, 32% for group 1 and 62%, for group 2, respectively (p=0.02), with more patients with high risk constellation in group 1. 3-year OS for patients transplanted with MUD was 57%, with MMUD 46% vs. with MRD 0% (p=0.01). Non-relapse-mortality was 28% for all patients, 40% in group 1 and 12% in group 2, probably due to the higher intensity in conditioning in group 1. The outcomes with intermediate, low and minimal intensity conditioning were comparable, while all patients after high intensity conditioning died. Table 1 describes Kaplan-Meier estimated 3-year-OS and statistical univariate analysis by log-rank test in the different subgroups. Table 1. 3-year OS (in%) All Group 1 Age 60–65 Group 2 Age 66–76 Remission CR 52 p=0.25 31 p=0.76 77 p=0.15 PR 40 32 50 Conditioning high 0 p=0.5 0 p=0.08 – p=0.38 intermediate 52 50 53 low 48 43 57 minimal 45 17 67 Donor MRD 0 p=0.01 0 p=0.06 73 p=0.45 MUD 57 53 65 MMUD 46 40 33 GVHD acute no 18 p=0.003 13 p=0.008 33 p=0.27 ≥II 43 53 58 chronic no 39 p=0.25 36 p=0.70 52 p=0.08 limited 52 30 100 extensive 50 30 67 In group 1 the outcome of minimal conditioning was inferior compared to intermediate and low conditioning while patients in group 2 had a better outcome with minimal vs. low and intermediate conditioning. Incidences of acute GVHD ≥II, limited and extensive chronic GVHD (cGVHD) were 10%, 28% and 13%, respectively. In group 1, acute GVHD ≥II occurred in 13% and cGVHD in 35%, in group 2 in 5% and 41% of the patients, respectively. Acute GVHD ≥II was associated with inferior outcome (3-year OS of 18% vs. 43%, p=0.003) while cGVHD had a positive impact on OS. In group 2 patients with limited cGVHD showed better 3-year OS than patients without cGVHD (67% vs. 52%, p=0.12). Age alone had no major impact on outcome of allogeneic HCT. Patients aged ≥60 seemed to benefit from the use of MUD rather than an older MRD. Chronic GVHD had a positive influence on survival. Our data indicate that the regimen used should be tailored to disease risk and patient performance status. Disclosures: No relevant conflicts of interest to declare.

Description: NK-cells have been shown to play a pivotal role in haploidentical hematopoietic cell transplantation (HHCT) for engraftment, GvL effects and to combat infectious complications. Different strategies have been employed to hasten NK-cell recovery after HHCT. Here we compare the immune recovery of 17 patients after CD34 selected HHCT receiving additional adoptive CD3-depleted CD56-enriched NK cells 2 days after HHCT (adoptive NK-cells), with 18 patients receiving CD3/CD19 depleted grafts (CD3/CD19) for HHCT. Transplantations were performed at two different institutions with a median follow-up of 〉1 year. Conditioning consisted of 12 Gy TBI, thiotepa (10mg/kg), fludarabine (150 mg/m2) and OKT3 (day −4 to +2) in the group receiving CD34 selected grafts and adoptive NK-cell transfusions. All patients in the CD3/CD19 group received conditioning with fludarabine (150–200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2) and OKT-3 (day −5 to +14). No postgrafting immunosuppression was used in both groups. Seven out of the 17 patients in the adoptive NK-cell group received IL-2 activated NK cells. Median age was 37 years in the adoptive NK-cell group compared to 40 years in the CD3/CD19 group. Diagnoses in the adoptive NK-cell group included AML (n=10), ALL (n=3), CML (n=2), and Hodgkin’s disease (n=1) and MDS (n=1). Diagnoses in the CD3/CD19 group were AML (n=10), ALL (n=5), NHL (n=1), CML (n=1) and multiple myeloma (n=1). The grafts contained a median of 12.5x10E6 CD34+ cells/kg and 1.1×10E4 CD3+ cells/kg in the CD34 selected group versus 9.2×10E6 CD34+cells/kg and 2.3×10E4 CD3+cells/kg in the CD3/CD19 group. The number of transferred CD56+ cells was 8.3×10E6/kg in the adoptive NK-cell group and 7.2×10E7/kg cells in the CD3/CD19 group. Hematopoietic recovery was similar in both groups. Among the patients receiving adoptive NK-cells we observed a striking difference in immune recovery between the patients receiving IL2-activated and those treated with non-activated NK cells: patients receiving activated NK cells showed significantly lower numbers of NK- and T cells during the first months post transplant (p=

Description: Allogeneic hematopoietic cell transplantation (HCT) using reduced intensity conditioning (RIC) regimens offers a potential curative therapy to patients with advanced NHL. RIC HCT induces potent graft-versus-lymphoma effects with best results in patients with low tumor burden at time of HCT. Combined use of radioimmunotherapy (RIT) with RIC may increase anti-lymphoma activity of RIC while HCT provides rescue from hematologic toxicity of RIT. This may allow dose escalation of RIT. A multicenter phase I/II study of allogeneic HCT combining RIT using yttrium-90-ibritumomab tiuxetan (Y90-CD20) with two RIC regimens for treatment of patients with NHL has been initiated. Patients with indolent NHL (Arm A) receive RIT with Y90-CD20 (0.4 mCi/kg) on day −14 combined with RIC using fludarabine (30 mg/m^2 day −4 to−2) and 2 Gy TBI (day 0). Patients with aggressive NHL (Arm B) receive an escalated dose of Y90-CD20 (0,6–0,8 mCi/kg) on day −14 combined with RIC using fludarabine (30 mg/m^2 day −8 to−4), melphalan (140 mg/m^2 day −3) and campath (20–30 mg day −3 to−2). For postgrafting immunosuppression either CSA/MMF (Arm A) or CSA alone (Arm B) is used. To date, 31 patients have been enrolled (Arm A=23, Arm B=8). Diagnoses in Arm A were FL (n=12), MCL (n=6), CLL (n=4) and Immunocytoma (n=1). Diagnoses in Arm B were DLBCL (n=6), blastoid MCL (n=1) and transformed CLL (n=1). Median age was 55 (range, 34–67) years. PBSC grafts were either from matched related (n=10) or matched unrelated donors (n=21). All patients were high risk with refractory disease or relapse after preceding HCT. Disease status after salvage therapy at time of HCT was in Arm A: CR=1, PR=18, SD=4 and in Arm B: PR=8. No additional toxicity due to RIT was observed. Engraftment was rapid and sustained with no graft rejections. In Arm A median time to 〉500 granulocytes/μL was 13 (range, 0–69) days and to 〉20000 platelets/μL 3 (range, 0–69) days (in 11 patients platelets never dropped 500 granulocytes/μL was 17 (range, 9–23) days and to 〉20000 platelets/μL 11 (range, 8–29) days. TRM in the first 100 days was 3%, overall 19%. Incidence of grade II-IV GVHD in Arm A was 35% (II=3, III=4, IV=1) and in Arm B 25% (II=2). Best disease response observed was in Arm A: CR=18, PR=5 and in Arm B: CR=3, PR=5. To date, 16/23 patients in Arm A and 6/8 patients in Arm B are alive with a median follow-up of 271 (range, 20–390) days, resulting in a Kaplan-Meier 1 year survival estimate of 65% in Arm A and 62% in Arm B. Causes of death were infection=5, GVHD=1, relapse=1 in Arm A and relapse=2 in Arm B. A combination of RIT with RIC is feasible with no additional toxicity due to RIT and stable engraftment in all patients. Preliminary response data suggest that this strategy may improve early post-transplant disease control, but long-term disease-free survival remains to be determined.

Description: Haploidentical hematopoietic cell transplantation (HHCT) after high dose conditioning with a megadose of CD34-selected stem cells has been complicated by regimen related toxicities, slow engraftment and delayed immune reconstitution leading to high treatment related mortality (TRM). A new regimen using reduced intensity conditioning (RIC) and immunomagnetic CD3/CD19 graft depletion may allow HHCT with lower toxicity and faster engraftment. CD3/CD19 depleted grafts not only contain CD34+ stem cells but also graft-facilitating cells, CD34- progenitors, dendritic and natural killer cells which may allow stable engraftment even without a megadose of CD34+ cells. A multicenter phase I/II study of HHCT using RIC with fludarabine (150–200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2), OKT-3 (5 mg/day, day -5 to +14) and CD3/CD19 graft depletion has been initiated. No post grafting immunosuppression was applied if the graft contained 500 granulocytes/μL of 12 days (range, 9–50) and to 〉20000 platelets/μL of 11 days (range, 7–38). Full donor chimerism was reached after 2–4 weeks in all but four patients (median of 14 days (range, 7–215)). Four patients experienced rejection/non-engraftment, two were rescued by a second CD3/CD19 depleted graft from another haploidentical donor. Incidence of grade II-IV acute GVHD was 51% with grade II=16, III=6 and IV=4. So far there are six cases of limited chronic GVHD and one case of extensive chronic GVHD. TRM in the first 100 days was 11/51 (22%) and overall 20/51 (39%). Overall survival is 17/51 patients (33%) with deaths due to relapse (n=14), infection (n=15), PML (n=2), GVHD (n=2) and cardiac failure (n=1), with a median follow-up of alive patients of 397 days (range, 62–1180). This results in a Kaplan-Meier estimate 1-year survival of 37%. So far, we did not observe a statistical significant survival advantage for patient transplanted from a KIR-mismatched donor (28/51 patients). This regimen with low toxicity as well as fast and sustained engraftment is promising in high risk patients lacking a suitable donor. To investigate the treatment protocol earlier in course of disease a new study for high-risk patients with acute leukemia in first complete remission is in preparation.

Description: No standard treatment is available for relapse of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) after allogeneic stem cell transplantation (alloSCT). Efficacy of donor lymphocyte transfusion (DLT) was limited, at least in part, by the rapid pace of the disease, overwhelming any allogeneic immune response. However, induction chemotherapy before DLT was complicated by severe toxicity. Based on a pilot trial conducted in Munich, a prospective, multicenter phase II trial for relapsed AML and MDS after alloSCT was initiated in 2000 by the German transplant cooperative group. The study evaluated the sequence of low-dose(ld)AraC for leukemic control (intensive chemotherapy for progressive disease only), transfusion of donor PBSC without immunosuppression as adoptive immunotherapy, and systemic application of GM-CSF. GM-CSF was included, since in combination with other cytokines, it has been able to improve the antigene presentation capacity of myeloid blasts in vitro. Between 2000 and 2006, 41 patients with hematological relapse of AML or MDS 〉3 months after alloSCT were included. Median age was 47y, 50% had an unrelated donor. Median remission after SCT was 223d (93–1614), median percentage of BM blasts at relapse was 40%. Median follow up was 28 months. Control of leukemic proliferation by ldAraC was achieved in 61%, allowing outpatient care in the majority of these patients. 39% required intensive chemotherapy. Three patients died from infections before donor cell transfusion (DCT), one patient was not transfused due to progressive leukemia. Median time from relapse to DCT was 52 days. 25/34 evaluable patients were found to be free of blasts in BM at d35 after PBSC and were considered initial responders. In an intent-to-treat analysis, overall survival (OS) of the entire cohort at 1 and 2 years from relapse was 41% and 32%. Among initial responders, 1y- and 2y-OS was 68% and 49%. A remission 〉6months after alloSCT, and control of leukemia by ldAraC prior to DCT were associated with better outcome. At last follow up, 8 patiens were in continous CR, 2 were alive with second relapse, 18 had died from leukemia, and 13 had died in remission or aplasia. In conclusion, ldAraC seems to be effective for initial control of leukemic proliferation in relapsed AML and MDS after alloSCT. A longer duration of post-transplant remission and response to ldAraC may identify patients who will benefit from adoptive immunotherapy. On an intend-to-treat-basis, the overall results of our trial compare favorably to other published strategies; nevertheless, outcome is still unsatisfying and warrants further investigation.

Description: Abstract 3539 The introduction of reduced intensity conditioning (RIC) regimens in allogeneic hematopoietic cell transplantation (HCT) allows the use of this treatment option even in older, comorbid or heavily pretreated patients. The impact of donor source and match on outcome of HCT after RIC remains to be evaluated. We retrospectively analyzed 254 consecutive adult patients (f=104, m=150) who received allogeneic HCT using RIC treated at our institution between 2000–2010. Median age of patients was 58 years (range, 18–76). Diagnoses were acute leukemia (AML, n=127, ALL, n=9), Non-Hodgkin lymphoma (n=30), multiple myeloma (n=29), myelodysplastic syndrome (n=27), osteomyelofibrosis (n=19), chronic myeloid leukemia (n=5), chronic lymphatic leukemia (n=5) and Hodgkin lymphoma (n=3). 91 patients were transplanted in complete remission (CR), 163 in partial remission (PR). 49 patients were transplanted from a matched related donor (MRD), 93 from a matched unrelated donor (MUD), 68 from a mismatched unrelated donor (MMUD, up to 2 allel/antigen mismatches), 3 from a mismatched related donor and 41 from a haploidentical donor (haplo). Remission status at time of HCT in patients transplanted from haplo donors (21 CR, 20 PR) was balanced, whereas patients transplanted from MRD (10 CR, 39 PR), MUD (36 CR, 57 PR) or MMUD (24 CR, 47 PR) were predominantly in PR. 82 of the patients were high-risk because of preceding autologous or allogeneic HCT (MRD=19, MUD=27, MMUD=11, haplo=25). Kaplan-Meier estimated 3-year overall survival (OS) for all patients was 45%, 47% with MRD, 46% with MUD, 53% with MMUD and 29% with haplo (p=0.07). Kaplan Meier estimated 3-year-OS and statistical analysis by univariate log-rank test in different subgroups are described in table 1. Table 1 3-year OS (in %) MRD p MUD p MMUD p Haplo p Remission CR 39 49 62 29 0.30 PR 49 45 49 28 0.19 NRM 22 34 28 39 0.24 aGVHD 3II 100 0.49 13 0.001 53 0.91 25 0.91 no aGVHD 3II 40 51 50 23 cGVHD no 48 0.97 40 0.07 56 0.93 16 0.03 limited 75 39 50 50 extensive 35 66 57 53 Donor source and match had no significant impact on OS in the different subgroups. Non-relapse mortality was 31% without a difference between different donors. 3-year OS in the biggest diagnostic group AML was 47% with MUD, 51% with MMUD, 30% with MRD and 31% with haplo, respectively (p=0.38). Incidence of acute GVHD (aGVHD) ≥II was 14% (4% with MRD, 15% with MUD, 15% with MMUD and 17% with haplo) with a lower incidence of acute GVHD with MRD compared to haplo (p=0.02). Incidence of chronic GVHD (cGVHD) was 37% (33% with MRD, 45% with MUD, 33% with MMUD and 34% with haplo). Presence of aGVHD ≥II was associated with an inferior outcome (3-year OS 30 vs. 45%, p=0.02) while presence of limited or extensive cGVHD was associated with an improved OS (52% and 50% vs. 39% without cGVHD, p= 0.25). cGVHD had a positive impact on OS especially after HCT with MUD (p=0.07) or haplo (p=0.03) Thus, RIC for allogeneic HCT in elderly or heavily pretreated patients shows promising results irrespective of donor source. The lack of a MRD should not preclude MUD or MMUD allogeneic HCT using RIC in elderly patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1938 Introduction: Patients with relapsed or primary refractory AML have a dismal prognosis. Even salvage therapy with allogeneic hematopoietic cell transplantation (HCT) could not improve outcome due to high incidence of relapse and high non-relapse mortality (NRM). Recently, promising results in patients with unfavorable karyotype or treatment refractory AML have been reported using a sequential treatment with aplasia inducing chemotherapy consisting of Fludarabine, Ara-C and Amsacrine (FLAMSA) followed within 3 days by reduced intensity conditioning (RIC) for allogeneic HCT (Schmid et al., Blood 2006 Aug 1;108(3):1092–9). Methods: We report a retrospective analysis of our single center experience with FLAMSA-RIC in primary refractory or relapsed AML patients. We searched our database for patients receiving FLAMSA-RIC in the past 10 years. Details on characteristics and clinical course of the patients were confirmed by retrospective chart review. Results: We retrospectively identified and analyzed 51 consecutive patients (f=22, m=29) transplanted after FLAMSA-RIC at our institution from 2006–2011. At time of HCT patients were refractory after chemotherapy (n=22) or had an untreated relapse (n=29). Data on molecular and cytogenetic markers were available in 36 and 44 patients, respectively. 34 were initially high-risk because of unfavorable karyotype (n=25) or molecular genetic alterations (n=9). Median age of patients was 56 years (range, 20–72) and diagnosis of all patients was acute myeloid leukemia (de-novo AML, n=27, secondary AML, n=24). FLAMSA (Fludarabine 30 mg/m2 day −12 to −9, AraC 2000 mg/m2 day −12 to −11 and Amsacrine 100 mg/m2 day −12 to −9) was used as salvage therapy followed by RIC (Fludarabine 30 mg/m2 day −5 to −4/Busulfan 0.8 mg/kg day −6 to −4, n=10; TBI 4Gy on day −5/Cyclophosphamide 60 mg/kg on day −4 to −3, n=28; Busulfan 0.8 mg/kg day −6 to −4/Cyclophosphamide 60 mg/kg for matched and mismatched unrelated donors (MUD/MMUD) or 40 mg/kg for matched related donors (MRD) on day −3 to −2, n=13). As GVHD prophylaxis calcineurin inhibitor combined with mycophenolate mofetil and anti-thymocyte globuline (ATG-Fresenius®, 10 mg/kg for MRD and 20 mg/kg for MUD/MMUD) was used. 10 patients were transplanted from MRD, 16 from MUD, 21 from a MMUD and 4 from a MMRD. 14 patients received DLI (2 × 106 - 1 × 108 /kg after a median of 186 days, range 72–922) in absence of GVHD in case of mixed chimerism or relapse after HCT. Current overall survival (OS) was 18/51 patients with a median follow-up of 410 days (range, 179–1557) of patients alive resulting in a Kaplan-Meier estimated 2-year OS and event-free-survival (EFS) of 34% and 29%, respectively. There was no significant difference between the different RIC regimens with 50% Fludarabine / Busulfan vs. 26% TBI 4Gy / Cyclophosphamide and 40% Fludarabine / Busulfan (p=0.37). Causes of death were relapse (n=19), infections (n=5), GVHD (n=2), multi-organ-failure (n=5), cerebral hemorrhage (n=1) and progressive multifocal leukencephalopathy (n=1). Cumulative incidence of relapse at 2 years with death due to NRM as competing risk was 40% and cumulative incidence at 2 years of NRM with death due to relapse as competing risk was 27%. 2-year OS was inferior in patients with secondary AML compared to patients with de-novo AML (28% vs. 38% p=0.79). The outcome in the elderly subgroup defined by age ≥60 years (median age 67, n=22) was similar to the group of younger patients (median age 46, n=29) with 2-year OS of 31% vs. 37% (p=0.87). Patients with a blast count 〈 10% in the bone marrow at time of HCT had a better outcome with 64% vs. 25% OS (p=0.09). 2-year-OS was inferior in patients being refractory after chemotherapy (25% vs 38%, p=0.78). Incidence of acute GVHD (aGVHD) ≥II and chronic GVHD (cGVHD, limited, n=11, extensive, n=3) was 22% and 27%, respectively. Presence of aGVHD did not influence survival while presence of cGVHD was associated with an improved overall survival after HCT (58% vs 24%, p=0.009). Conclusion: FLAMSA-RIC followed by allogeneic HCT enables long-term disease free survival, even in primary refractory or relapsed AML patients. The sequential approach of this regimen seems to overcome the dismal prognosis of these patients. Its moderate toxicity allows the application of this curative salvage therapy option even in an elderly patient population. Disclosures: Off Label Use: The use of some agents in the conditioning is off-label.

Description: We have previously described a novel culture protocol to grow MSC from bone marrow (BM) and non-amniotic placenta (PL) with an immature phenotype and multi-lineage differentiation capacity (1). Using the low affinity nerve growth factor receptor (CD271) (2) as a key marker for isolation of MSC derived from femur shaft bone marrow cells (BM-MSC) of patients undergoing a total hip replacement, we could identify two CD271+ distinct populations: CD271dull and CD271bright cells. Two-color flow cytometer analysis revealed that only the CD271bright population coexpressed the mesenchymal markers CD10, CD13, CD73, and CD105, but was negative for CD45. CD271dull cells were positive for CD45 and HLA-DR but negative for the other markers. To analyze the mesenchymal stem cell potential, colony-forming-unit fibroblast (CFU-F) assays were performed. Not surprisingly, the CFU-F were exclusively detected in the CD271bright but not in the CD271dull fraction. By screening a battery of antibodies against known and unknown antigens, we identified several reagents that selectively detected the CD271brightCD45- population but no other bone marrow cells. These markers included the PDGF-RB (CD140b), the embryonic stem cell marker TRA-1-49, the clustered markers HER-2/erbB2 (CD340), the recently described W8B2 antigen (3), as well as the cell surface antigens defined by the antibodies W1C3, W3D5, W4A5, W5C4, W5C5, W7C6, 9A3, 58B1, F9-3C2F1, and HEK-3D6. In conclusion we identified several novel markers for the prospective isolation and characterization of BM-MSC.