Human-Leukocyte-Histocompatibility Antigens Predict Response to Rituximab and Donor Lymphocyte Infusion (DLI) After Non-Myeloablative Allogeneic Stem Transplantation (NST) for Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2548-2548
Author(s):  
Issa F. Khouri ◽  
Roland Bassett ◽  
Pedro Cano ◽  
Susan O'Brien ◽  
Yvonne Hsu ◽  
...  
Keyword(s):  
T Cell ◽  
Conflicts Of Interest ◽  
Chronic Gvhd ◽  
Hla Class I ◽  
Median Number ◽  
Class I ◽  
Hematopoietic Stem ◽  
Hla Alleles ◽  
Cell Counts ◽  
Significant Difference

Abstract Abstract 2548 Background: The effectiveness of allogeneic NST in CLL has been attributed to a graft-versus-leukemia effect due to elimination of tumor cells by alloimmune effector lymphocytes. In a previous study (Khouri et al, Exp Hematol 2004), we found that when patients with CLL develop relapse after NST, immunomanipulation (IMM) via withdrawal of immunosuppression and DLI with rituximab can induce sustained remissions in some patients. The underlying mechanism of this GVL effect is unknown. The simultaneous presence of the killer-immunoglobulin-like receptor (KIR) 3DL1 and its corresponding human leucocyte antigen (HLA) class I ligands bearing the Bw4 epitope has been described in CLL (Verheyden S, Leukemia 2006). Purpose: Considering that the HLA class I molecules may act as restriction elements for GVL targets after NST in CLL, we investigated the potential association of certain common class I HLA alleles and response to IMM. Methods: We studied all 43 CLL pts who required IMM after NST in sequential phase II protocols at the U.T. MD Anderson Cancer Center from February 1996 to August 2007 because of persistent disease or because of progression. In our analysis, we examined the most common alleles and serotypes expressed in the patients studied. This included HLA-A1, A2, A3, A24, B7, B8, B35, B44, B60, B62, BW4, BW6, CW7. These allele groups are known to be common in most world populations. Rituximab was given at a dose of 375 mg/m2 intravenously followed by 3 weekly doses of 1000 mg/m2. A DLI of 1 × 107 CD3-positive T cells/kg was given after the first two doses of rituximab if no GVHD occurred. An escalated DLI dose was given at 6-week intervals if there was persistent active disease and no GVHD. Results: Median age (range) was 55 years (39-73) and the median Hematopoietic Stem Cell Comorbidity Index was 3 (range, 0–8). The median number of prior chemotherapies was 3. At their transplant, 48% of pts had refractory disease, 90% had Binet stage B/C, and 60% had a beta-2 microglobulin of =/> 3. P53 deletion was detected in 11 of 35 pts (31%) tested. Mixed T-cell chimerism was observed in 60 % of pts at day 90. The median number of DLI infused was 2 (range, 1–6); their median maximal dose was 43.6 (range, 1–200) × 106 CD3+/Kg. In these 43 pts, 20 (47%) experienced complete remission (CR), by CT scans, marrow and flow analysis. Pts characteristics at time of study entry (described above) for NST, and at the time of initiation of IMM (this included white blood cell counts, LDH, % lymphocyte in marrow, % CD5-CD19, lymph node size by computed tomography, maximum dose DLI, number of DLIs, GVHD prior IMM and grade, T-cell chimerism), as well as HLA subtypes were assessed. The major determinants to achieve CR following IMM included receipt of a PBSC graft and achievement of a good (> 90%) donor T-cell chimerism at day 90 (p = 0.035), and having a combination of HLA-A1-positive, HLA-A2-negative, and HLA-B44-negative (p= 0.0009). The rate of CR to IMM was 9%, 36%, 50%, 91% respectively in patients who had none of the HLA factors described, I, 2, and all 3 respectively. There was no statistically significant difference in pts and disease characteristics between HLA-A1-positive or negative, HLA-A2 positive or negative nor between HLA-B44-positive or negative pts. In addition the risks of acute II-IV [Cumulative incidence (CI)=23%) and chronic GVHD (CI=69%) were not different between the respective subtypes. With a median follow-up in surviving pts of 37.2 months (range, 11.4–131.1), the progression-free survival rates at 5-year for patients with HLA-A1+/A2-/B44- vs those who had none of those HLA types were 68% vs 15%, respectively, (p<0.0001). Conclusions: Our results represent the first report showing certain HLA alleles might be predictive for response to GVL and achieving long-term remission in CLL. Verification of our findings in a larger cohort of pts is highly warranted for better selecting pts for IMM for the treatment of recurrent malignancy in CLL. Disclosures: No relevant conflicts of interest to declare.

Impact of Missing KIR Ligands in HLA-Matched Unrelated Donor HSCT.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1185-1185
Author(s):  
Aining Sun ◽  
Weiyang Li ◽  
Wu Depei ◽  
Jun He ◽  
Xiaojing Bao ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Chronic Gvhd ◽  
Hla Class I ◽  
Unrelated Donor ◽  
Prognostic Impact ◽  
Matched Unrelated Donor ◽  
Hematopoietic Stem ◽  
Immunoglobulin Receptor ◽  
Significant Difference ◽  
Hla Genotype

Abstract Abstract 1185 Poster Board I-207 Objective: To analyze the prognostic impact of missing ligands for inhibitory killer-immunoglobulin receptor(KIR) in HLA-matched hematopoietic stem cell transplantation(HSCT) using unrelated donor. Methods: HLA genotype of 51 patients (ALL 22 cases, AML 13 cases, CML 14 cases, MDS 1 cases and HAL 1 cases) and their matched unrelated donors was determined by polymerase chain reaction sequence oligonucleotide probes(PCR-SSOP) and sequence specific primers (PCR-SSP). The KIR genotype was determined by PCR-SSP. Results: Patients were divided into those with(n=37) and those without(n=14) missing 1 or more HLA class I ligands for donor inhibitory KIR. The period of platelet reconstruction was shorter in patients [ 13 d(10d∼27d)] with missing KIR ligands than those[14d(12d∼21d)] without missing KIR ligands(P=0.046). There was no significant difference in neutrophil recovery, ≥II° acute GVHD(13.5% vs 35.7%, P>0.05) and extensive chronic GVHD (16.2% vs 28.6%, P>0.05) between the two groups. The 3-year continuous complete remission(CCR) rate for patients with and without missing KIR ligands was 73.6% and 37.4%, respectively(P=0.183). The 3-year overall survival(OS) rate for the two groups was 77.5% and 52.4%, respectively(P=0.533). Conclusions: In HLA-matched unrelated donor HSCT, missing KIR ligands may be associated with enhanced engraftment, decreased severe GVHD, improved CCR and OS. Disclosures: No relevant conflicts of interest to declare.

Haploidentical Stem Cell Transplantation After Negative Depletion Of T Cells Expressing The αβ Chain Of The T-Cell Receptor (TCR) For Adults With Hematological Malignancies

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4609-4609 ◽  
Author(s):  
Lucia Prezioso ◽  
Sabrina Bonomini ◽  
Chiara Lambertini ◽  
Chiara Schifano ◽  
Elena Rossetti ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Conflicts Of Interest ◽  
Chronic Gvhd ◽  
Cell Depletion ◽  
T Cell Depletion ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Αβ T Cells

Introduction For many years, T cell depletion (TCD) of hematopoietic stem cells (HSCs) has been based on either positive or negative selection of mobilised peripheral blood cells (PBPCs). After CD34+ cell selection, the T cell repertoire is very narrow since the number of T lymphocytes in the graft has to be particularly low to prevent GvHD and ATG in the conditioning exerts an additional in vivo T cell depletion. Thus the immune recovery is slow and patients tend to remain susceptible to opportunistic infections for several months after HSCT. To hasten and improve post-transplant immune reconstitution broad repertoire various strategies of adoptive donor T cell immunotherapy (e.g. engineering with a suicide gene; depleting alloreactivity by means of photodynamic purging or through the use of freshly purified regulatory T cells) have been investigated over the past years. More recently, selective elimination of αβ+ T cells has been performed to achieve a 4,5–5 log TCD and to retain in the graft NK, dendritic cells, monocytes and γδT lymphocytes. Under this approach, a rapid immunological reconstitution and very promising outcome have been reported in pediatric patients. With the aims of confirming these results even in adults, we have recently launched this programme and here we report our preliminar clinical data. Methods Thirteen patients, median age 40 years (range 19-65), with AML (n=9), ALL (n=2), HL (n=1) or Rhabdomyosarcoma (n=1) entered the study. All but two patients, who were in first remission, were in advanced-stage disease at transplant with five patients in chemoresistant relapse. Conditioning consisted of ATG 1,5 mg/kg from day -13 to day -10, Treosulfan 12gr/sqm from -9 to –7, Fludarabine 30mg/sqm from -6 to -2 and Thiotepa 5mg/Kg on days -5 and -4. Ten μg/kg G-CSF was used to mobilize PBPCs from one-haplotype mismatched donors (4 mothers, 4 brothers, 2 sisters, 1 son, 1 daughter and 1 cousin). Mobilized mononuclear cells were incubated with a biotinylated anti-TcRαβ antibody and subsequently with an antibiotin antibody conjugated to magnetic microbeads (Miltenyi Biotec, Germany). Under a strong magnetic field, TcRαβ T lymphocytes were retained, whereas all nonmagnetized cells were recovered. Short sirolimus (1mg/day x3 weeks) was used as additional GVHD prophylaxis in 3 cases whose grafts contained more than 2x105/kg αβ+Tcells. Results Grafts contained a median of 12,3x106/kg CD34+ cells(range7-19), 6 x106 CD3+Tcells/kg (range 2,3-13)with 10,4x104/kg αβ+T cells (range 1,38-62) and 5,8x106 γδ+Tcells/kg (range2,1-12,6), 6x104B cells/kg (range 0,2–32) and 34x108 CD56+NKcells/kg (range10-91). All but one patient, who required a second graft from the same donor to boost hematopoietic reconstitution, achieved a full donor sustained engraftment. Median time to reach 500 neutrophils and 50,000 platelets was 13 (range 9-18) and 11 days (range 9-13), respectively. Four patients had skin grade I/II aGVHD. No patients has so far developed chronic GvHD. Median CD4+ cell counts at 30, 60, 90 and 120 days since the transplant were 33, 122, 190 and 251 n/mL, respectively. CMV reactivation occurred in only 2 cases (in one, CMV serology was unfavourable: CMV-negative donor/CMV-positive recipient). Overall, 3 patients have so far died (2 non-hematologic causes and 1 early relapse). Ten survive disease-free at a median follow-up of 104 days (range 30-178). Conclusions The infusion of αβ/CD19-depleted grafts was safe and effective also in adult setting, resulting into rapid donor hematopoietic engraftment and early expansion of donor-derived γδT lymphocytes, without life-threatening infectious complications. Disclosures: No relevant conflicts of interest to declare.

Impact of HLA Class I Typing-Resolution and Mismatches on Survival and Graft-Versus-Host Disease in Patients after Hematopoietic Stem Cell Transplantations from Unrelated Donors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2060-2060
Author(s):  
Miroslaw Markiewicz ◽  
Jerzy Wojnar ◽  
Malgorzata Krawczyk-Kulis ◽  
Iwona Wylezol ◽  
Sebastian Giebel ◽  
...  
Keyword(s):  
High Resolution ◽  
Relapse Rate ◽  
Graft Failure ◽  
Statistical Significance ◽  
Chronic Gvhd ◽  
Hla Class I ◽  
Class I ◽  
Hematopoietic Stem ◽  
Unrelated Donors ◽  
Grade 3

Abstract We analyzed 150 consecutive patients (pts) transplanted from unrelated donors (URD) at single institution- Silesian Medical Academy in Katowice- with use of the same standard operating procedure from February 1997 until December 2004 for CML (74 pts), AML (28), ALL (27), SAA (9), MDS (7), MM (1), NHL (1), PNH (1), OMF (1), bi-phenotypic AL (1). 92 pts were transplanted from matched donors, including 59 with complete 10 alleles (HLA-A,B,C,DRB1,DQB1) high resolution (HR) DNA-typing; and 33 with first class match established on base of low-resolution (LR) (13), serological (7) or un-complete typing without HLA-C (13) and second class HR typing. 58 pts were transplanted from mismatched donors (first class typing was HR in 43, LR in 14 and serological in 1 pt): 22 with single allelic mismatch (2 HLA-A, 7-B, 6-C, 7-DQB1), 33 with single HLA-C antigen mismatch and 3 with double mismatches (2 B+C, 1 C+C). Survival advantage at 4 years, although without statistical significance (p=0.16), was observed in the group of pts transplanted from 10/10 alleles matched donors (36+/− 11%) over those with mismatched donors (24+/− 11%). Oppositely, pts transplanted from matched donors who were not completely typed in HR did not achieve better survival (23+/− 17%). Poorest survival (13+/− 12%, p=0.007) was observed in patients transplanted from mismatched homozygous donors (n=10). The risk of acute GVHD grade 3–4 was increased (p=0.007) in pts with mismatched donors (31+/− 6%) when compared to matched completely typed in HR (10+/− 4%). Also the rate of graft failure tended to be lower in pts with matched than mismatched donors (5.1% versus 10.2%, p=0.25). In contrast, relapse rate was lower in mismatched (23+/− 10%) than in HR matched pts (34+/− 12%, p=0.55) what may reflect better GVL effect in mismatched transplant recipients. Unexpectedly, the rate of chronic GVHD was similar in pts with 10/10 alleles matched and mismatched donors (40+/− 10% versus 42+/− 9%, p=0.75). These results indicate that complete high resolution HLA class I typing is necessary for adequate selection of unrelated donors. Class I HLA-B and -C mismatches influence both survival and serious a-GVHD incidence. 10/10 alleles matched mismatched p survival at 4 years 36+/− 11% 24+/− 11% 0.16 aGVHD grade 3–4 10+/− 4% 31+/− 6% 0.007 graft failure rate 5.1% 10.2% 0.25 relapse rate 34+/− 12% 23+/− 10% 0.55 cGVHD 40+/− 10% 42+/− 9% 0.75 10/10 alleles matched mismatched homozygous donors p survival at 4 years 36+/− 11% 13+/− 12% 0.007

Outcome of Patients Developing GVHD after DLI for CML Relapse from HLA-Identical Sibling or VUD HSCT.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1817-1817
Author(s):  
Yves Chalandon ◽  
Christoph Schmid ◽  
Kimmo Porkka ◽  
Alvaro Urbano-Ispizua ◽  
Bernd Hertenstein ◽  
...  
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
Chronic Gvhd ◽  
Median Number ◽  
Unrelated Donor ◽  
Hematopoietic Stem ◽  
Initial Cell ◽  
Cell Dose ◽  
Stem Cell Source ◽  
Related Mortality

Abstract Using data submitted to the EBMT registry, we analyzed outcome on 344 patients (pts) who had received donor lymphocyte infusions (DLI) for relapse after allogeneic hematopoietic stem cell transplantation (HSCT) for chronic myeloid leukemia (CML) in 31 centers. 113/344 pts (33%) developed acute graft-versus-host disease (aGVHD) a median of 50 days post DLI (max grade: I=42, II=30, III=31, IV=6)(60% grade II–IV). Organs involved (%): skin (88), liver (42), gut (30). Median age was 38 (4–59), 58% pts were male, 62 transplants were HLA-identical sibling and 51 unrelated. 74 were T-cell depleted, 92 transplanted in CP1, 21 beyond CP1. Relapse was molecular in 19 pts, cytogenetic in 31, hematological in 49, accelerated or blastic in 12. Median initial cell dose was 107CD3+ cells/kg (0.01–32), median number of DLI was 1 (1–10). aGvHD was treated with prednisone in 92% of pts, CSA in 52 %, ATG and monoclonal antibodies in 2% and other in 19%. aGVHD resolved in 53% of the pts within a median of 63 d (7–546). 82/344 pts (24%) had chronic GVHD (cGVHD)(30 limited, 50 extensive, 2 not specified), of those 46 (56%) following aGVHD post DLI. Organs involved (%): skin (75), liver (35), lungs (13), mouth (43), eyes (22) and gut (5). Median age was 35 (6–58), 51% were male, stem cell source was PB in 15% and marrow in 85%, 43 underwent HLA-identical sibling HSCT and 39 unrelated donor HSCT. Forty-three were T-cell depleted, 66 transplanted in CP1, 16 beyond CP1. Relapse was molecular in 21 pts, cytogenetic in 29, hematological in 22, accelerated or blastic in 7. Median initial cell dose was 107 CD3+ cells/kg (0.05–40), median number of DLI was 1 (1–7). 61 pts are alive with a median follow-up of 50 mth. Treatment was with steroids in 83% of pts, CSA in 58 %, MMF in 20%, thalidomide in 15%, photopheresis in 15%, PUVA in 10% and other in 17%. cGVHD resolved in 39% of the pts within a median of 354 d (44–1588). The estimated 5-y OS post-DLI was significantly lower in pts who developed aGVHD post-DLI, 61 ± 10% vs 74 ± 7% in the one that did not, p=0.007 and also a tendency to have a lower 5-y EFS, 58 ± 10% vs 65 ± 7%, p=0.19. Median duration of response to DLI in aGVHD pts was 4 y. aGVHD post-DLI did not influence the relapse rate (5 ± 5% vs 6 ± 5% in the absence of aGVHD). 5-y DLI related mortality was significantly higher in aGVHD pts, 31 ± 8% vs 4 ± 4%, p<0.00001. On the other hand, pts that developed cGVHD post-DLI had a tendency to have a better 5-y OS and EFS, 74 ± 11% and 71 ± 11% respectively vs 69 ± 6% and 62 ± 7% in those that did not, p=0.32 and 0.09. This was related to a tendency to lower incidence of relapse, 2 ± 3% in pts with cGVHD vs 9 ± 6% without, p=0.2. DLI related mortality was not different, 11 ± 8% vs 10 ± 5%, p=0.77. aGVHD post-DLI for CML relapse is mainly of advanced stage and negatively influence OS and EFS with a higher DLI related mortality. cGVHD post-DLI is mainly extensive, but pts with cGHVD tend to have better outcome with better 5-y OS, EFS and less relapse than those without, although this was not statistically significant.

Anti-Leukemia Activity of Alloreactive NK Cells in Haploidentical HSCT in Pediatric Patients: Re-Defining the Role of Activating and Inhibitory KIR

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3002-3002 ◽  
Author(s):  
Daniela Pende ◽  
Stefania Marcenaro ◽  
Michela Falco ◽  
Stefania Martini ◽  
Maria Ester Bernardo ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
Cell Subset ◽  
Hla Class I ◽  
Class I ◽  
Leukemia Cells ◽  
Hla Alleles ◽  
Time Points ◽  
Selection Of

Abstract T-cell depleted hematopoietic stem cell transplantation from haploidentical donors (haplo-HSCT) has been reported to benefit from the graft-versus-leukemia effect mediated by natural killer (NK) cells when donor displays NK alloreactivity versus the recipient. NK alloreactivity is mediated by NK receptors, namely Killer Ig-like receptors (KIR) which are specific for allotypic determinants that are shared by different HLA-class I alleles (referred to as KIR ligands). It is known that KIR2DL1 recognizes HLA-C alleles characterized by Lys at position 80 (C2 group), KIR2DL2/3 recognize HLA-C alleles characterized by Asn at position 80 (C1 group), KIR3DL1 recognizes HLA-B alleles sharing the Bw4 supertypic specificity (Bw4 group) and KIR3DL2 recognizes HLA-A3 and –A11 alleles. KIR2D/3DL are inhibitory receptors that, upon engagement with the cognate ligand, inhibit lysis. Activating KIRs, highly homologous in the extracellular domain to the inhibitory counterparts, are KIR2DS1, KIR2DS2 and KIR3DS1, but only KIR2DS1 has been shown to specifically recognize C2 group of alleles expressed on B-EBV cells. We analyzed 21 children with leukemia receiving haplo-HSCT from a relative after a myeloablative conditioning regimen; in all pairs, the expression of a given KIR ligand (HLA class I allele) of the donor was missing in the patient (i.e. KIR ligand-mismatched haplo-HSCT). T-cell depletion was performed through positive selection of CD34+ cells; no pharmacological immune suppression was employed after HSCT. KIR genotype of all donors was evaluated to detect the presence of the various inhibitory and activating KIR genes. Phenotypic analyses were performed on NK cells derived from the donor and the patient at different time points after HSCT. Thanks to the availability of new mAbs able to discriminate between the inhibitory and the activating forms of a certain KIR, we could identify the alloreactive NK cell subset at the population level. These alloreactive NK cells express the KIR specific for the KIR ligand-mismatch (permissive inhibitory KIR) and the activating KIR (if present), while they do not express all inhibitory KIR specific for the patient HLA alleles and NKG2A. Thus, in most instances, we could precisely identify the size of the alloreactive NK cell subset in the donor and in the reconstituted repertoire of the recipient. Functional assays were performed to assess alloreactivity, using appropriate B-EBV cell lines and, if available, patient’s leukemia blasts. In some cases, also NK cell clones were extensively studied, for phenotype and receptor involvement in killing activity. We found that, in most transplanted patients, variable proportions of donor-derived alloreactive NK cells displaying anti-leukemia activity were generated and maintained even at late time-points after transplantation. Donor-derived KIR2DL1+ NK cells isolated from the recipient displayed the expected capability of selectively killing C1/C1 target cells, including patient leukemia blasts. Differently, KIR2DL2/3+ NK cells displayed poor alloreactivity against leukemia cells carrying HLA alleles belonging to the C2 specificity. Unexpectedly, this was due to recognition of C2 by KIR2DL2/3, as revealed by receptor blocking experiments and by binding assays of soluble KIR to HLA-C transfectants. Remarkably, however, C2/C2 leukemia blasts were killed by KIR2DL2/3+ (or by NKG2A+) NK cells that co-expressed KIR2DS1. This could be explained by the ability of KIR2DS1 to directly recognize C2 on leukemia cells. A role for the KIR2DS2 activating receptor in leukemia cell lysis could not be established. Taken together, these findings provide new information on NK alloreactivity in haplo-HSCT that may greatly impact on the selection of the optimal donor.

DynaChip Anti-HLA Antibody Analysis In Sera of Patients Following Allo-HSCT From HLA-Mismatched Unrelated Donors.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4575-4575
Author(s):  
Miroslaw Markiewicz ◽  
Urszula Siekiera ◽  
Tomasz Kruzel ◽  
Monika Dzierzak-Mietla ◽  
Patrycja Zielinska ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Hla Class I ◽  
Class I ◽  
Hla Antibodies ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Study Results ◽  
Reactive Groups ◽  
Unrelated Donors ◽  
Class Ii Antigens

Abstract Abstract 4575 Introduction: Anti-HLA antibodies constitute potentially important factor that may influence outcomes of HLA-mismatched allogeneic hematopoietic stem cell transplantation (allo-HSCT). The rationale of this study was to detect presence of anti-HLA antibodies in recipients of allo-HSCT from HLA-mismatched unrelated donors. Patients and Methods: Anti-HLA-A,B,C,DR,DQ,DP antibodies were identified in sera collected from 46 recipients of allo-HSCT from HLA-mismatched unrelated donors. Sera were collected between 1 month and 5.5 years after allo-HSCT, and additionally before allo-HSCT in 17 pts. We have used microchips spotted with purified HLA class I and HLA class II antigens to allow binding of anti-HLA antibodies present in tested sera to the surface of the microchip, pre-optimised reagents and DynaChip Processor (Dynal Invitrogen Corporation) for assay processing, data acquisition and analysis. Results: Antibodies against HLA class I, II or I and II were detected in 15%, 11% and 35% of pts whereas no antibodies were detected in 39% of patients. Antibodies were directed against HLA-A, B, C, DR and DQ in 37%, 46%, 35%, 48% and 35% of pts, respectively. Pre-transplant anti-HLA antibodies have been detected in 7 pts (41%) out of 17 tested before allo-HSCT. In this group percent of Panel Reactive Antibodies (% PRA) increased following allo-HSCT in 3 pts and decreased in 4. In 5 out of 10 remaining pts without pre-transplant antibodies, %PRA increased post-transplant. DynaChip software allowed to define specificities of HLA-A,B,C,DR and DQ antibodies on low and high resolution levels. The specificity of antigens that masked results of antibody identification has been also defined in 2 pts. At this stage we did not define exactly whether detected anti-HLA antibodies were donor-specific. Cross-reactive groups (CREG's) analysis has been also used to compare antibodies’ reactivity. Anti-HLA-DP antibodies were not detected in the examined group of transplanted patients. Conclusions: Presented preliminary study results indicate, that anti-HLA antibodies can appear post-transplant in mismatched allo-HSCT recipients. Further analysis aiming to evaluate their influence on transplant outcomes is ongoing. We intend to extend the search for anti-HLA antibodies with use of Luminex LabScreen method. Disclosures: No relevant conflicts of interest to declare.

“RUNX1-IRES-GFP Knock-in” Mice Lack Expression of Runx1a: A Versatile Tool for Hematopoietic Stem Cell Analysis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2329-2329
Author(s):  
Yukiko Komeno ◽  
Ming Yan ◽  
Shinobu Matsuura ◽  
Miao-Chia Lo ◽  
James R. Downing ◽  
...  
Keyword(s):  
Body Weight ◽  
Conflicts Of Interest ◽  
Lymphoid Cells ◽  
Hematopoietic Stem ◽  
Blood Indices ◽  
Cell Counts ◽  
Significant Difference ◽  
Versatile Tool ◽  
Exon 4 ◽  
Runx1 Expression

Abstract Abstract 2329 Previously reported “RUNX1-IRES-GFP knock-in mice” (Blood 2004;103:2522) (KI mice) were generated by replacing exon 4 of runx1 gene with cDNA of Runx1b/c from exon 4 to exon 8 followed by IRES-GFP, aiming to evaluate Runx1 expression in specific lineages and developmental stages during adult hematopoiesis. They are phenotypically normal, fertile, and blood indices are normal. GFP intensity correlates with Runx1 expression level, and shows lineage-specific changes during maturation in myeloid, erythroid, and lymphoid cells. However, the behavior in the hematopoietic stem cells (HSCs) had not been carefully examined. Interestingly, we discovered that this knock-in strategy eliminated Runx1a expression. Since Runx1a expression is relatively higher in HSCs than in differentiated cells, we analyzed HSCs in these mice to evaluate its roles in stable and stress hematopoiesis. We found that LSK fraction in bone marrow (BM) was significantly decreased in KI mice compared to wild type (WT) mice (0.043% vs 0.085%, p = 0.001). Among subpopulations in LSK, short-term HSC and multipotent progenitor fractions were significantly decreased (0.024% vs 0.046%, p = 0.003, 0.0021% vs 0.0026%, p = 0.001, respectively). SLAM marker staining using CD150 and CD48 showed similar results. Competitive repopulation assay showed less functional HSCs in KI mice. However, there was no significant difference in recovery of cell counts after single-dose 5-FU intraperitoneal injection (150 mg/kg body weight) or sublethal irradiation (5 Gy), or survival after weekly 5-FU injection. After G-CSF subcutaneous injection (125 μg/kg body weight, twice daily for 5 days), mobilized WBC or neutrophil in PB showed no difference. However, LSK and long-term HSC in PB were significantly less in KI mice (0.078% vs 0.135%, p = 0.010, 0.043% vs 0.092%, p = 0.029, respectively) while those in BM did not show significant difference (increased to 0.295% and 0.346% in KI and WT mice, respectively). In conclusion, Runx1a plays some non-redundant roles in stable hematopoiesis, while it is dispensable for tested stress hematopoiesis. RUNX1-GFP KI mice are a versatile tool to evaluate roles of Runx1a in normal hematopoiesis and leukemogenesis when combined with other genetic modifications. Disclosures: No relevant conflicts of interest to declare.

Comparative Analysis on Cellular Components of Bone Marrow Microenvironment in Normal and Aberrant Hematopoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4808-4808
Author(s):  
Young-Ho Lee ◽  
Young-hee Kwon ◽  
Kyoujung Hwang ◽  
Hyunju Jun ◽  
Byungbae Park ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Conflicts Of Interest ◽  
T Cell Subsets ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Stromal Cell Derived Factor ◽  
And Control ◽  
Cellular Components

Abstract Abstract 4808 Background: It is now evident that hematopoietic stem cells (HSCs) reside preferentially at the endosteal region within the bone marrow (BM) where bone-lining osteoblasts are a key cellular component of the HSC niche that directly regulates HSC fate. We investigated the microenvironmental differences including osteoblastic activities and HSC components in myeloproliferative (chronic myeloid leukemia, CML) and hypogenerative disease (aplastic anemia, AA) as well as normal control (NC). Methods: The immunohistochemistry for osteonectin, osteocalcin, stromal cell derived factor (SDF, CXCL12), T cell, T helper/inducer cell, T suppressor/cytotoxic cell, hematopoietic stem/progenitor (CD34, CD117) and megakaryocytes was performed on BM biopsy specimens from 10 AA patients, 10 CML patients and 10 NC (lymphoma without BM involvement). The positive cells for immunohistochemical stainings except osteocalcin on each slide were calculated on 10 high power fields (HPF, ×400), and then corrected by the cellularity. The positive cells for osteocalcin were counted on the peritrabecular line on each slide, and then corrected by the mean length measured. Results: The CD34+ cells (p=0.012) and megakaryocytes (p<0.0001) were significantly lower in AA than in NC, but CD117+ cells was comparable in AA, CML, and control samples. The osteonectin+ cells (p=0.0003) were lower in CML than in AA and NC, however the osteocalcin+ cells showed wide variation (0-903/2035um) and no significant difference. The SDF+ cells (p<0.0001) was significantly higher in AA and very lower in CML, compared with NC. The counts for T cell and T cell subsets were significantly lower in CML than in NC, and higher in AA than in NC (p<0.0001). Conclusions: Cellular components of BM microenvironment in 2 hematologic diseases representative of myeloproliferation (CML) and hyporegeneration (AA) respectively are quite different. Further studies would be required to explore the role of these components for hematopoiesis and the rationale for therapeutic application. Disclosures: No relevant conflicts of interest to declare.

Strong HLA Class I Expression Is Positively Correlated with the Number of PML Nuclear Bodies and Negatively with the Percentage of SATB1 Positive HRS Cells in EBV+ Classical Hodgkin Lymphoma (cHL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3633-3633
Author(s):  
Yuxuan Liu ◽  
Lydia Visser ◽  
Rianne Veenstra ◽  
Bea Rutgers ◽  
Anke Van Den Berg ◽  
...  
Keyword(s):  
Hodgkin Lymphoma ◽  
Conflicts Of Interest ◽  
Malignant Neoplasm ◽  
Hla Class I ◽  
Nuclear Bodies ◽  
Class I ◽  
Positive Staining ◽  
Classical Hodgkin Lymphoma ◽  
Pml Nuclear Bodies ◽  
Significant Difference

Abstract Abstract 3633 Introduction: Classical Hodgkin lymphoma (cHL) is a malignant neoplasm of the immune system, characterized by the presence of an abundant reactive infiltrate and a minority of Hodgkin Reed-Sternberg cells (HRS cells). HRS cells retain their professional antigen presenting phenotype in most cases. In EBV- cHL, membranous HLA class I expression is retained in HRS cells in approximately 30% of the cases, whereas in EBV+ cHL HLA class I expression is retained in HRS cells in 79% of the cases. Moreover, a proportion of the EBV+ cHL cases shows an enhanced HLA class I expression as compared to the surrounding infiltrating cells. The mechanism of the enhanced or lost HLA class I expression is unknown. Special AT-rich region binding protein 1 (SATB1) and promyelocytic leukemia protein (PML) are two proteins that have been shown to regulate HLA class I expression. Downregulation of SATB1 in Jurkat cells results in an enhanced expression of HLA-A, HLA-G, HLA-H and HCG4P6, whereas downregulation of PML results in a reduced HLA-A and HLA-G expression. PML is the main component of nuclear bodies (NBs) that organize the chromatin structure into loops by anchoring matrix attachment regions to the nuclear matrix. SATB1 has been shown to be associated with the PML-NBs in the HLA region. Aim: To investigate the possible role of SATB1 and PML-NBs in the regulation of HLA class I expression in EBV+ cHL. Methods: We analyzed 64 EBV+ cHL cases and as a control included 29 EBV- cHL cases. HLA class I membranous staining (HC10 antibody) by HRS cells was scored as positive or strongly positive, cases that lacked membrane staining were scored negative. ß2-microglobulin served as an additional marker for membranous HLA class I expression. For SATB1 (14/SATB1), we scored the percentage of HRS cells with nuclear SATB1 staining. For PML (PG-M3), we scored HRS cells based on the number of PML nuclear bodies in two categories; 10 or less NBs per cell or >10 NBs per cell (per 4 um tissue section). Results: In the EBV+ group 24 cases stained strongly positive, 22 positive and 18 negative for HLA class I. In the EBV- group, none of the cases showed a strong positive staining, 7 cases stained positive and 22 cases were negative for HLA class I. HLA class I staining results were consistent with the ß2-microglobulin staining results in all cases. The percentage of SATB1 positive HRS cells varied from 0–100% in both EBV+ and EBV- cHL. The number of PML-NBs was between 0–10 in 46 and >10 in 18 EBV+ cHL cases and between 0–10 in 23 and >10 in 6 EBV- cHL cases. We observed no correlation between HLA class I staining in the EBV- cHL cases and the percentage of SATB1 positive HRS cells or the number of PML-NBs in the HRS cells. In EBV+ cHL cases we observed significant differences in the percentages of SATB1 positive cells between the HLA class I negative, normal and strongly positive groups (p=0.0412). The cases with normal HLA class I staining had significantly higher percentages of SATB1 positive cells as compared to the cases with strong HLA class I staining pattern (p<0.05) (Figure 1). There was no significant difference between negative and normal or negative and strongly positive HLA class I groups with respect to the percentage of SATB1. The percentage of EBV+ cHL cases with >10 PML-NBs significantly increased from HLA class I negative (1 out of 18, i.e. 5%) to normal (4 out of 22, i.e. 18%) and strong (13 out of 24, i.e. 54%) positive cHL cases (p=0.0011). Conclusion: We found an inverse correlation between the percentages of SATB1 positive HRS cells in the HLA class I strong and normal EBV+ cHL groups. The number of cases with >10 PML-NBs were significantly increased in HLA class I strong as compared to the HLA class I normal and negative EBV+ cHL groups. Thus SATB1 and PML may play an important role in the regulation of HLA class I expression levels in EBV+ cHL. Disclosures: No relevant conflicts of interest to declare.

Hhex Is a Critical Gene In The Development Of Normal and Malignant Lymphoid Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3788-3788
Author(s):  
Charnise Goodings ◽  
Stephen B. Smith ◽  
Elizabeth Mathias ◽  
Elizabeth Smith ◽  
Rati Tripathi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Transgenic Mice ◽  
Conflicts Of Interest ◽  
Lymphoid Cells ◽  
Conditional Knockout ◽  
Hematopoietic Stem ◽  
Direct Transcriptional Target ◽  
Cell Counts ◽  
Transcriptional Target

Abstract Hematopoietically expressed homeobox (Hhex) is a T-cell oncogene. It is frequently deregulated in murine retroviral insertional mutagenesis screens and its enforced expression induces T-cell leukemia in bone marrow transduction and transplantation experiments. We discovered that HHEX is a direct transcriptional target of an LIM domain Only-2 (LMO2)-associated protein complex. HHEX clusters with LMO2-overexpressing T-ALLs and is especially overexpressed in Early T-cell Precursor (ETP) – ALL where it is a direct transcriptional target of LMO2. To further understand Hhex's function, we induced a conditional knockout in floxed Hhex mice with the Vav-iCre transgene. Mice were viable and showed normal blood cell counts with highly efficient deletion of Hhex in all hematopoietic tissues. Thymocytes from conditional knockouts showed a normal pattern of development. Most impressively, Hhex conditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2 transgenic mice (figure 1). Hhex conditional knockouts (Hhex cKOs) also had a significant decrease in mature B cells in the spleen and bone marrow. Interestingly, hematopoietic stem and progenitor cells plated on OP9-GFP or OP9-DL1 stromal cells showed proliferative defects and incomplete differentiation towards both B and T lineage. Also under stress conditions such as sublethal irradiation and competitive bone marrow transplants, Hhex conditional knockouts show a marked defect in both B and T lineages but an increase in early progenitor populations. Our experiments show that Hhex is a critical transcription factor in lymphoid development and in LMO2-induced T-ALL.Figure 1Hhex conditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2 transgenic miceFigure 1. Hhex conditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2 transgenic mice Disclosures: No relevant conflicts of interest to declare.

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