ALBERT

Description: Patients with hematologic malignancies that develop graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (HSCT) have a reduced risk of disease relapse compared to patients that do not develop GvHD, suggesting that GvHD and graft-versus-leukemia (GvL) responses are co-dependent. T cells are important in these processes, as T cell depletion from the graft reduces the risk of GvHD at the expense of disease relapse. Much less is known about the effect of B cell depletion on GvHD and GvL responses. Small patient series have demonstrated variable efficacy of Rituximab in the treatment of steroid-refractory chronic GvHD but the effect of depleting allo-reactive B cells on disease relapse remains to be determined. We here report on a patient with steroid-refractory GvHD whose AML relapsed after Rituximab treatment. This 39-year old male received an allogeneic HSCT for chemotherapy-related AML (AML-t) that however relapsed 8 weeks after the transplantation. Upon rapid cessation of immunosuppressive therapy (cyclosporine, mycophenolic acid) and without additional chemotherapy he obtained full remission, at the expense of severe GvHD of the skin, liver and intestine that was corticosteroid-refractory. B cell depletion with Rituximab was successful as second-line treatment for GvHD but eliminated the GvL response and the patient died of AML relapse several months after. To evaluate the B cell repertoire of this patient at the moment of maximal GvH and GvL responses, we isolated peripheral blood B lymphocytes that were transduced with Bcl-xL and Bcl-6 to create clonal B cell lines. These B cells were screened for binding to AML and host tissues. One clone was retrieved that specifically bound to AML cell lines and AML blasts freshly isolated from newly diagnosed AML patients. Antibodies from this clone induced antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity of AML cells in vitro. Several other clones were retrieved that were specific for host tissues such as liver (HepG2 and H69 cell lines), skin (primary fibroblasts) and/or colon (CaCo cell line). These data demonstrate the pivotal role of B lymphocytes in anti-leukemia and anti-host immune responses in an allogeneic HSCT recipient with relapsed AML-t. Disclosures No relevant conflicts of interest to declare.

Description: Key Points Chemotherapy and radiotherapy deplete ILCs from the blood; ILC reconstitution after allogeneic HSCT is slow. High frequencies of activated ILCs with tissue homing potential before allogeneic HSCT are associated with reduced risk for GVHD.

Description: Abstract 3026 Background Double umbilical cord blood transplantation (UCBT) results in higher engraftment rates as compared to single UCBT in adult patients. Sustained hematopoiesis is usually derived from a single cord blood unit (CBU) after double UCBT. So far, the mechanism of predominance of a particular CBU is unresolved. In a prospective single-arm phase II study (HOVON-106) we monitored early engraftment kinetics to determine whether graft predominance after double UCBT is driven by specific leukocyte subpopulations. Methods 36 consecutive patients (pts) from 5 Dutch centers with high-risk hematological diseases received a double UCBT, preceded by a reduced-intensity conditioning regimen (Cy 60 mg/kg/ Flu 160 mg/m2/ TBI 2×2 Gy). CBUs were selected by intermediate resolution typing for HLA-A and -B loci and by high-resolution typing for HLA-DRB1. The minimal required HLA-match grade was 4/6. Chimerism analysis (STR-PCR) of unseparated peripheral blood (PB) and bone marrow (BM) cells was performed as from day +32 onwards. In addition, chimerism analysis in PB leukocyte subpopulations by flowcytometry using lineage-specific (CD45, CD3, CD4, CD8, CD19, CD16/56, CD14 and CD33) monoclonal antibodies (mAbs) in combination with human HLA-antigen specific mAbs (HLA-mAbs) was performed at day +11, +18, +25 and +32 if discriminating HLA-mismatches between recipient and CBUs were present. Day +32 flowcytometry results were compared to day +32 PB STR-PCR results. Results The median number of prefreeze total nucleated cells (TNC) per CBU was 2.3×107/kg (range: 1.5–5.5). Median numbers of post-thaw viable CD34+ cells, T-, B- and NK cells were 0.32 (range: 0–1.7), 4.4 (range: 0.4–36), 9.7 (range: 0.7–79) and 7.2 (range: 0.24–45) x105/kg, respectively. One pt was non-evaluable for engraftment due to insufficient follow up after early relapse. The cumulative incidence of neutrophil recovery (≥0.5×109/l) was 91% with a median time to neutrophil recovery of 33 days (range: 15–82). Primary graft failure occurred in 1 pt. Chimerism analysis, performed at day +32 by STR-PCR revealed single CBU predominance in all pts whereas residual non-engrafting CBU and recipient cells were detectable in only 3 and 7 pts, respectively. Simultaneous 3-donor-origin detection of leukocyte subpopulations by flowcytometry based on HLA disparities was possible in 12 pts. Flowcytometry using HLA-mAbs demonstrated single CBU predominance in various leukocyte subsets as from day +11 onwards in the majority of pts. Moreover, ultimate engraftment of a particular CBU was reliably predicted for by chimerism within the CD4+ (in 90% of pts) and NK cell (in 90% of pts) subsets at this early time point. In contrast, predominance of the engrafting CBU in monocytic en myeloid subsets was observed in only 70% and 33% of pts, respectively, at day +11. The numbers of CD8+ and B-cells were too low for analysis in the majority of pts. Predominance of the ultimate engrafting CBU was established in all subpopulations at day +18. Furthermore, the contribution of the non-engrafting CBU to the different leukocyte subsets was negligible or even absent as from day +18 onwards. Recipient hematopoiesis did not contribute to PB cell recovery either. The results of day +32 flowcytometry (CD45+ population) were similar to results of day +32 PB STR-PCR. The number of prefreeze TNC did not predict for the ultimately engrafting CBU, nor did the number of post-thaw CD34+, T, B or NK cells. Engraftment was not associated with the degree of HLA mismatches or presence of KIR ligand mismatches among recipient and CBUs. Conclusions These results show that single donor chimerism is rapidly established after double UCBT, preceded by a 4 Gy TBI-based conditioning regimen without ATG. In addition, our flowcytometry data suggest the occurrence of CBU predominance within 2 weeks post transplant, in the course of which both CD4+ and NK cell predominance at day +11 are highly predictive for ultimate single donor chimerism. That early engraftment pattern of leukocyte subsets might suggest a key role for either CD4+ T cells or NK cells in CBU predominance. Disclosures: Janssen: Novartis: Consultancy.

Description: Abstract 4104 Introduction Patients undergoing hematopoietic stem cell transplantation (HSCT) are at increased risk of developing (severe) opportunistic infections, which can be attributed to the combination of a decimated immune system and disrupted epithelial barriers. Innate lymphoid cells (ILCs) represent a novel family of regulator and effector cells that share phenotypic and functional characteristics with both NK cells and T lymphocytes, but are non-cytotoxic and lack antigen specific receptors. ILCs serve important roles in the maintenance of epithelial integrity and - as part of the first line of defence - in protection against microbes. ILCs are mainly tissue-resident and can be identified by the absence of lineage markers (Lin-) and bright expression of the alpha chain of the IL-7 receptor (CD127). Extensive research has been carried out focussing on the relative contribution of different immune cells to immune deficiency and graft-versus-host disease (GvHD) following HSCT. However, little is known about ILCs in this context. We studied reconstitution dynamics of ILCs in order to explore their function in tissue repair and mucosal immunity following HSCT. Methods Blood samples of adult patients (n=15) receiving an allogeneic HSCT after reduced-intensity conditioning (RIC) for acute myeloid leukemia (AML) were obtained at sequential time points during induction, consolidation and conditioning chemotherapy, at the day of HSCT and up to 6 months following HSCT. Peripheral blood ILCs were analyzed using flow cytometry and defined as Lin-CD127hi. The ILC population was further divided into various subpopulations using the markers NKp44, CRTH2 and CXCR3. Results Induction and consolidation chemotherapy led to a significant decline in the number of circulating ILCs. Recovery of ILCs in between chemotherapy cycles was partial as ILC numbers improved without returning to healthy control values (n=10; reference values defined as upper and lower boundaries of 95% CI), in contrast to other innate cells such as neutrophils and NK cells. Following allogeneic HSCT, recovery dynamics of ILCs parallelled those of adaptive immune cells, rather than of innate cells: recovery was relatively slow, as normal numbers of circulating ILCs had not been reached 6 months after HSCT. In addition, we observed enrichment of a particular ILC subset, defined as Lin-CD127hiCD117+NKp44+ cells, in subgroups of patients after chemotherapy and HSCT. These RORγt-dependent ILCs have been found to comprise an important innate source of IL-22 (hence referred to as ILC22s), a cytokine crucial for epithelial homeostasis and induction of epithelial antimicrobial peptides. Discussion and conclusion Our observation of a relatively slow reconstitution of ILCs is notable since innate immune cells are generally known to recover within weeks to months after HSCT. In fact, ILC reconstitution following HSCT bears resemblance to post-transplantation adaptive lymphocyte recovery dynamics. Fitting with this notion, there is a certain degree of analogy between ILCs and T helper cells as ILCs with cytokine profiles resembling those of the various T helper subsets are found, i.e. ILCs producing mainly IL-13/5, IL-22, IL-17 or IFN-γ. Furthermore, proliferation and differentiation of the ILC subpopulations stands under the control of transcription factors known to be master regulators of the different T helper subsets (i.e. GATA3, Tbet, RORγt and AHR). Hence, our data show that the analogy between the ILC and T helper systems in terms of factors that control differentiation and function is matched by their similarities in reconstitution dynamics following chemotherapy and HSCT. Interestingly, following chemotherapy and allogeneic HSCT, ILC22s, known to be primarily dedicated to the maintenance of epithelial homeostasis, were found to be enriched in subgroups of patients. Damage to mucous membranes lining the gastrointestinal tract, due to chemotherapy-induced mucositis, conditioning radiotherapy or graft-versus-host disease, is a common problem that constitutes a significant limiting factor in HSCT. Therefore, improvements in the prevention or management of such epithelial damage are eagerly awaited. Our data suggest that ILCs may be involved in protection against and/or recovery of epithelial damage in patients with a hematological malignancy receiving chemotherapy and HSCT. Disclosures: No relevant conflicts of interest to declare.

Description: To improve the outcome of allogeneic stem cell transplantation (allo-SCT) in multiple myeloma as part of first-line treatment, we prospectively investigated the feasibility and efficacy of lenalidomide maintenance. Patients started maintenance 1 to 6 months after nonmyeloablative allo-SCT. Lenalidomide was dosed 10 mg on days 1 to 21 of a 28-day schedule for a total of 24 cycles. Peripheral blood samples were taken to evaluate immune modulating effects. Thirty-five eligible patients were enrolled, and 30 started with lenalidomide. After 2 cycles, 14 patients (47%) had to stop treatment, mainly because of the development of acute graft versus host disease (GVHD). In total, 13 patients (43%) stopped treatment because of development of GVHD, 5 patients (17%) because of other adverse events, and 5 patients (17%) because of progression. Responses improved in 37% of patients, and the estimated 1-year progression-free survival from start of maintenance was 69% (90% confidence interval, 53%-81%). Lenalidomide increased the frequency of human leukocyte antigen-DR+ T cells and regulatory T cells, without correlation with clinical parameters. In conclusion, lenalidomide maintenance 10 mg daily after nonmyeloablative allo-SCT with unmanipulated graft in multiple myeloma patients is not feasible, mainly because of the induction of acute GVHD. This trial was registered at www.trialregister.nl as #NTR1645.

Description: Introduction Acute myeloid leukemia (AML) is a high-risk malignancy with a poor prognosis. Allogeneic hematopoietic stem cell transplantation (HSCT) can be curative if it induces a potent graft versus leukemia (GvL) response. GvL responses and graft versus host disease (GvHD) are typically considered T cell mediated, because T cell depletion from hematopoietic stem cell grafts reduces the risk of GvHD at the cost of leukemia relapse. In addition, depletion of B-lymphocytes with rituximab has led to amelioration of GvHD in a number of studies suggesting that B cells are also important in the pathophysiology of GvHD and, in analogy, in GvL responses. However, the characteristics of the antibodies produced by these leukemic specific B cells have not yet been studied. Methods We selected three patients with high-risk myelomonocytic leukemia who remained disease free years after allogeneic HSCT, from whom we established clonal human B cell lines, using a unique and innovative technology that was developed in our laboratory (Kwakkenbos ea, Nat Med 2010). These B cell lines, that concomitantly express immunoglobulin on their membranes and secrete antibodies, were used to select antibodies specific for cell surface antigens on AML cell lines with similar morphologic and immunophenotypic characteristics as the patients’ leukemic blasts, using a FACS based assay. Results From each patient, several AML specific B cell clones were retrieved. Their antibodies recognized surface antigens on primary AML blasts derived from multiple patients and on AML cell lines, but not on healthy bone marrow, peripheral blood mononuclear cells or tissues such as liver, skin and colon. The majority of the antibodies were of the IgG3 isotype. Approximately 40% of the AML-specific antibodies induced direct death of cultured AML cell lines and of primary AML blasts. The cell death pathway induced by these cytotoxic antibodies was oncotic rather than apoptotic. Conclusion Our data demonstrate that high-risk AML patients with a potent GvL response mount robust antibody responses against surface antigens that are specifically expressed on tumor cells and binding of these antibodies induces direct cell death. The targets recognized by the recovered antibodies are also expressed on leukemic blast from other patients suggesting evidence for a common immune mechanism responsible for AML clearance. Our findings suggest that antibody responses are important in GvL and open up new ventures for specific antibody treatment of AML patients. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4322 Background Single nucleotide polymorphisms (SNPs) in the innate immunity receptor NOD2/CARD15 have been demonstrated to modulate the outcome of allogeneic haematopoietic stem cell transplantation. The effect of the NOD2/CARD15 polymorphism seems to be associated with donor source as well as type of conditioning regimen. Methods We reviewed NOD2/CARD15 mutations in all donor/recipient pairs of 192 consecutive patients who received non-myeloablative allogeneic stem cell transplantation(SCT) at our institution between 2002 and 2006. All patients were treated uniformly with fludarabine 30 mg/m2/day for 3 days followed by 200 cGy TBI (n=154) or TBI alone (n=38) and received grafts from HLA-matched related (n=132) or unrelated (n=60) donors. Results Mutated alleles were observed in 36 of 192 (19%) patients and in 35 of 192 (18%) donors. These SNPs, however, did not have a significant impact on clinical outcome data (P 〉 0.05, Kaplan Meier and Fine & Gray's test). Acute graft-versus-host disease (GVHD) occurred in 24 of 61 (39%) patients with the polymorphism and in 66 of 131 (50%) patients without the polymorphism. Chronic GVHD developed in 28 of 55 (51%) patients with SNP pairs and in 79 of 121 (65%) patients with the wild type. The incidence of transplant-related mortality was 21% in both groups, 13 of 61 patients in the group with the polymorphism and 27 of 131 without the polymorphism. Relapse was seen in 23 of 61 (38%) patients with the SNP pairs and in 48 of 131 (37%) wild type patients. Finally, overall survival was 43% (26/61) in patients with the polymorphism and 39% (51/131) in patients without the polymorphism. Conclusion These data indicate that mutations in the NOD2/CARD15 genes do not influence the clinical outcome of non-myeloablative allogeneic SCT directly. Since NOD2/CARD15 variants are not recognized as a single significant prognostic factor, screening for NOD2/CARD15 when selecting a donor does not seem to have additional value in patients undergoing non-myeloablative SCT. Disclosures: No relevant conflicts of interest to declare.

Description: Reduced intensity conditioning (RIC) protocols are increasingly used for allogeneic hematopoietic stem cell transplantation (HSCT) in elderly patients. We retrospectively analyzed the outcome of RIC HSCT in a homogeneous group of acute myeloid leukemia (AML) patients over the age of 40. Forty-three AML or high-risk myelodysplastic syndrome patients were treated with a fludarabine and low dose total body irradiation (TBI) based regimen, followed by a full peripheral stem cell graft. Antithymocyte globulin was given to matched unrelated recipients (34%) before infusion of fludarabine. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporin and mycophenolate mofetil. All but 2 AML patients were in complete remission at the time of transplantation. Seventy-six percent of patients had a poor risk profile. Hematologic recovery was fast and primary graft failure occurred in one patient. Two patients with active disease at the time of HSCT experienced ongoing relapse. Infections were diagnosed in 9 patients (21%) and 6 patients (14%) were treated for CMV reactivation. Sixty percent of patients developed acute GVHD, which was grade 2 in 40% and grade 3 in 12%. Chronic GVHD occurred in 33% of patients. The incidence and severity of both acute and chronic GVHD was similar in patients with related and unrelated donors (P = 0.84 and 0.74, respectively). Treatment-related mortality (TRM) was low (9%), total nonrelapse mortality was 19%. After a median follow-up of 571 days, 16 patients (37%) experienced relapse. One-year progression-free and overall survival were 61% and 67%, respectively. Median disease-free survival has not been reached yet (〉 58 months) and median overall survival was 31 months (Fig 1 and 2, respectively). Poor risk AML was significantly associated with disease-free survival in multivariate analysis (P = 0.02). Age 〉 60 years, gender, donor type, timing of RIC HSCT (upfront or after relapse) and acute GVHD were not identified as prognostic factors. In conclusion, fludarabine plus low-dose TBI-based RIC HSCT is effective in AML patients over the age of 40 without active disease at the time of transplant and is associated with low TRM. Figure Figure Figure Figure

Description: Lung injury limits the success of hematopoietic stem cell transplantation (HSCT). The overall incidence varies from 30–50% and noninfectious causes occur in one third to one half of these. We reviewed pulmonary complications in 369 patients who received either allo-BMT or allo-PBSCT at our institution between 1993 and 2003. Control subjects were selected from the same database and matched on sex, underlying diagnosis, age, type of transplantation and cytomegalovirus-serostatus. For all patients the conditioning myeloablative regimen consisted of cyclophosphamide (60 mg/kg/day for 2 days) followed by total body irradiation (total lung dose 850 cGy). The graft was partially T-cell depleted (1–2 x 105 T cells/kg). Sixty-one patients (16.5%) developed pulmonary complications, which were diagnosed at a median of 22 weeks after transplantation (range 2–263). Twenty-one patients (5.7%) developed infectious pneumonia. Non-infectious complications were further subclassified as BO (3.5%), BOOP (0.5%), DAH (0.8%), IPS (5.4%) or mixed etiology (0.5%). Acute GVHD ≥ grade II was significantly more common in patients with pulmonary complications than in the controls (36/61 versus 24/61 patients, P=0.02). There was no significant difference in the incidence of chronic GVHD (in 26/48 pulmonary patients versus 20/55 controls, P=0.1). Median survival was 41 weeks (range 4–583) for the pulmonary patients and 173 weeks (range 8–582) for the control subjects. These data illustrate that the incidence of pulmonary complications is low after T-cell depleted HSCT and demonstrate a clear association with acute GVHD. Improvement of the poor outcome of pulmonary complications is of utmost importance. Current studies at our institution are focused at the detection of early markers so that possible pre-emptive-like therapy can be initiated before symptomatic lung damage arises.