Carsten Grüllich

Evaluation of immunomodulatory treatment based on conventional and lineage-specific chimerism analysis in patients with myeloid malignancies after myeloablative allogeneic hematopoietic cell transplantation

Both conventional chimerism analysis (CCA) and lineage-specific chimerism analysis (LCA) have potential pitfalls as diagnostic means for the detection of minimal residual disease after allogeneic hematopoietic cell transplantation (aHCT). Therefore, the present study examines the results of both methods in order to determine how predictive consecutive evaluations were, with respect to the risk that the patient would relapse during post-transplant follow-up and with respect to responsiveness to immunomodulatory treatment. A total of 168 individuals with acute myeloid leukemia (AML) (n=137) and myelo dysplastic syndrome (n=31) were investigated with CCA and LCA at mean intervals of 24 days (range: 11–116). The median follow-up after myeloablative aHCT was 22 months (range: 4–49). Of 168 patients, 65 experienced a clinical relapse after aHCT. CCA and LCA were comparatively sensitive and specific for relapse at the intervals of chimerism testing employed in this study. Of 32 patients, 10 who were offered donor lymphocyte infusions (DLI) treatment for increasing (n=29) or stable (n=3) mixed chimerism (MC) achieved at least transitory CC. The observation that all patients with increasing MC relapsed despite DLI treatment (54%) or withdrawal of immune suppression (24%) indicates that novel strategies to deal with rapidly evolving relapse in AML patients, such as shortening of chimerism monitoring intervals, need to be evaluated.

A Novel GVHD-Prophylaxis with Low-Dose Alemtuzumab in Allogeneic Sibling or Unrelated Donor Hematopoetic Cell Transplantation: The Feasibility of Deescalation

Prophylaxis of acute graft-versus-host disease (aGVHD), while maintaining the graft-versus-leukemia (GVL)/lymphoma effect and preventing severe infectious diseases, remains the main challenge in allogeneic hematopoetic cell transplantation (allo-HCT). To evaluate this, we examined the feasibility of deescalating the dose of alemtuzumab (MabCampath) in combination with cyclosporine (CsA) as the sole GVHD-prophylaxis in patients after fludarabine (Flu)-based reduced-intensity conditioning (RIC) in an observational cohort study. We included 127 consecutive patients (median age 63 years) with an unrelated (UD; n=69) or related donor (SIB; n=58) after their first transplantation, mostly presenting with advanced disease. The first 30 patients received 20 mg/day on day -2 and -1 (40 mg), the following 48 patients 10 mg/day on day -2 and -1 (20 mg), and the last 49 patients 10 mg on day -1 (10 mg) alemtuzumab intravenous (i.v.) prior to transplant. We observed no statistical differences comparing the 40 mg, 20 mg, or 10 mg dose groups, in terms of cumulative incidences of aGVHD grade III-IV 7% (confidence interval [CI] 95%; 1-51), 12% (1-40), 6% (1-40), extensive chronic GVHD (cGVHD) 24.4% (3.3-55.8), 17% (2.5-42), and 14.2% (1.5-41.5) and of aGVHD grade II-IV 7 % (0-51.5), 29% (11.9-49.1), 21% (15.3-43.1), respectively. The difference between the 20-mg and 40-mg groups was significant for aGVHD grade II-IV(P < .05). In conclusion, we demonstrate the feasibility of reducing the dose of alemtuzumab as GVHD-prophylaxis to 10 mg absolute in combination with CsA only for UD transplantation in particular.

Rabbit anti T-lymphocyte globulin induces apoptosis in peripheral blood mononuclear cell compartments and leukemia cells, while hematopoetic stem cells are apoptosis resistant.

Polyclonal anti-T-lymphocyte globulins (ATG) are used in allogeneic stem cell transplantation (SCT) for the prophylaxis of graft versus host disease (GVHD) by in vivo T cell depletion. In this study we investigated the complement independent induction of apoptosis by rabbit ATG in peripheral blood mononuclear cell (PBMNC) compartments and hematopoetic stem cells (HSC). We also detected antileukemic activity of ATG by measuring apoptosis in myeloid and lymphatic leukemia cell lines and primary leukemia cells. We found ATG to induce apoptosis in T-lymphocytes (CD4(+), CD8+), B-lymphocytes (CD20+), natural killer (NK)-cells (CD56(+)), and monocytes (CD14(+)). HSC, in contrast, were apoptosis resistant and could be growth stimulated by low-dose ATG in the presence of bystander cells. The human leukemia cell lines Jurkat, Daudi, DG-75 (lymphoblastic), and K562, HL-60, KG1, and U937 (myeloblastic) underwent ATG-induced apoptosis, whereas the NK-cell line YT was resistant. Primary leukemia cells from 6 investigated patients with acute lymphoblastic leukemia, 9 of 10 patients with chronic lymphocytic leukemia, and 4 of 8 patients with acute myeloblastic leukemia underwent ATG-induced apoptosis. We conclude apoptosis induction in all PBMNC compartments contributes to GVHD prophylaxis. ATG might support engraftment. Finally, antileukemic activity of ATG could positively influence the transplantation outcome.

A fludarabine, thiotepa reduced toxicity conditioning regimen designed specifically for allogeneic second haematopoietic cell transplantation after failure of previous autologous or allogeneic transplantation.

We present a phase II study of fludarabine 5 × 30u2009mg/m2, thiotepa 3 × 5u2009mg/kg as preparative regimen specifically for allogeneic second haematopoietic stem cell transplantation (HCT) after failure of previous HCT. Forty-nine patients (median age 52 years, range 27–68) received an allogeneic second HCT after failed autologous (n=29) or allogeneic (n=20) HCT. Diagnoses were AML (n=18), ALL (n=3), multiple myeloma (n=11), lymphoma (n=16) and CML (n=1). GVHD prophylaxis consisted of CYA and mainly low dose alemtuzumab (40u2009mg). The median follow-up for patients alive is 528 days (range 217–1344). In 43 of 49 (88%) evaluable patients response rates were CR=19, PR=14 and SD=10 at one month. At one year, the probability (95% confidence interval) of relapse is 55.1 (38.2–72)% and the nonrelapse mortality (NRM) is 29 (14.2–44.4)%. Estimated survival at one year is 42.6 (28.7–56.6)% and event free survival is 38.1 (24.4–51.8)%. Survival was significantly better for patients experiencing relapse beyond one year, than for patients relapsing within one year from first transplantation (51.2 (33.5–68.9)% vs 27 (7–48.5)%; P=0.013). We conclude that this regimen is feasible and well tolerated for allogeneic second HCT.

Late cytomegalovirus polyradiculopathy following haploidentical CD34 + -selected hematopoietic stem cell transplantation

Summary:A 55-year-old man with acute myeloid leukemia in second relapse presented 4 months after haploidentical CD34+-selected hematopoietic stem cell transplantation (HSCT) with symmetric, progressive neurological deficits of the lower extremities. Although there was no molecular evidence for drug resistance in the cerebral-spinal fluid, antiviral combination therapy failed to control the rapidly progressing CMV polyradiculopathy (PRP) and encephalitis, which were confirmed by autopsy studies. Late CMV PRP as an unusual manifestation of CMV disease should be kept in mind in patients with suggestive neurological symptoms after HSCT.

Marrow versus Blood-Derived Stem Cell Grafts for Allogeneic Transplantation from Unrelated Donors in Patients with Active Myeloid Leukemia or Myelodysplasia

Peripheral blood stem cells (PBSCs) are increasingly used as the graft source in allogeneic hematopoietic cell transplantation. We compared long-term outcome after unrelated donor transplantation of 85 consecutive patients with acute myelogenous leukemia or myelodysplastic syndrome regarding disease status (early disease [CR1, refractory anemia); n = 25 and advanced/active disease [>CR1, >refractory anemia]; n = 60) who were treated with conventional conditioning regimens followed by bone marrow (BM) or PBSC grafts. Graft-versus-host disease prophylaxis consisted mainly of cyclosporine A, short-course methotrexate, and anti-T-lymphocyte globulin. After a median follow-up of 118xa0months (68-174), the 10-year event-free survival rate after peripheral blood stem cell transplantation (PBSCT) was 54.8% (95% confidence interval [CI], 39.7%-69.8%), and after bone marrow transplantation (BMT), it was 27.9% (14.5%-41.3%; P < .004). In the advanced/active disease group, the 10-year event-free survival rate after PBSCT was 50% (30.8%-69.2%), and after BMT, it was 23.5% (9.3%-37.8%; P < .007). Non relapse mortality was less after PBSCT than BMT (14.3% vs 30.2%), respectively. In multivariate Cox regression analysis, PBSCT showed a better overall survival (OS; hazard ratio [HR], 0.43; 95% CI, 0.23-0.79; P = .007) compared to BMT; unfavorable/unknown prognostic impact cytogenetic abnormalities were an adverse factor for all patients (HR, 2.202; 95% CI, 1.19-4.06; P = .011). In patients with advanced disease, the use of PBSCs showed a significant favorable outcome via multivariate analysis (HR, 0.49; 95% CI, 0.24-0.99; P = .046). Outcome of acute myelogenous leukemia/myelodysplastic syndrome after unrelated hematopoietic cell transplantation is adversely affected by cytogenetic abnormalities and state of remission at hematopoietic cell transplantation. PBSC as a graft source has a significant favorable influence on survival.

Ex vivo detection of primary leukemia cells resistant to granule cytotoxin-induced cell death: a rapid isolation method to study granzyme-B-mediated cell death

Cytotoxic T lymphocytes and natural killer cells (CTL/NK) induce cell death in leukemia cells by the granzyme B (grB)-dependent granule cytotoxin (GC) pathway. Resistance to GC may be involved in immune evasion of leukemia cells. The delivery of active grB into the cytoplasma is dependent on the presence of perforin (PFN) and grB complexes. We developed a rapid method for the isolation of GC to investigate GC-mediated cell death in primary leukemia cells. We isolated GC containing grB, grB complexes and PFN by detergent free hypotonic lysis of the human NK cell leukemia line YT. The GC induce grB-mediated, caspase-dependent apoptosis in live cells. The human leukemia cell lines KG-1, U937, K562 (myeloid leukemia), Jurkat, Daudi, and BV173 (lymphoblastic leukemia) treated with GC internalized grB and underwent cell death. In primary leukemia cells analyzed ex vivo, we found GC-resistant leukemia cells in three out of seven patients with acute myeloid leukemia and one out of six patients with acute lymphoblastic leukemia. We conclude that our method is fast (approximately 1xa0h) and yields active GC that induce grB-dependent cell death. Furthermore, resistance to GC can be observed in acute leukemias and may be an important mechanism contributing to leukemia cell immune evasion.

The death receptor pathway is not involved in alloreactive T-cell induced mitochondrial membrane permeability

Elimination of tumor cells by cytotoxic T lymphocytes (CTL) is mediated by two major pathways: the granule exocytosis and the death receptor pathway, transduced by Fas, TNF and TRAIL. The usage of these distinct pathways in the alloreactive setting across major and minor HLA barriers still remains controversial. We generated CTLs against allogeneic Epstein-Barr virus (EBV)-transformed cell lines (LCL) from HLA-unmatched healthy donors and assessed their cytotoxicity by flow cytometrically measuring mitochondrial membrane permeability (MMP) of target cells. Mitochondrial apoptosis induced by CTL was abrogated by selectively inhibiting the granule exocytosis-mediated pathway with Concanamycin A (CMA). Conversely, apoptosis was not decreased in the presence of the caspase 8 inhibitor IETD, which is specific for all death receptor pathways. In general, caspases were not involved in MMP as shown using the pan-caspase inhibitor zVAD. This effector mechanism was preserved when using purified CD4 + and CD8 + T-lymphocyte subsets to generate CTL. We further showed, that death receptor signalling was not used as a salvage mechanism when granule exocytosis was inhibited even at longer incubation times sufficient for slow kinetic death receptor caspase signalling. Our results clearly demonstrate that mitochondrial apoptosis induced by human alloreactive CTLs is mainly mediated by granule exocytosis but not by death receptor caspase dependent pathways. Furthermore, the granule exocytosis pathway does not require caspases to induce MMP.

Fas ligand is not constitutively expressed in low-grade B-cell lymphoma and B-lymphoblastoid cells.

The Fas ligand (FasL) pathway is one of the two major effector mechanisms of T‐cell‐mediated cell death. FasL expression by extralymphatic tissues is thought to maintain a status of immunity. Accordingly, it has been proposed that tumor cells express FasL as a mechanism of immunologic escape. However, data regarding FasL expression in normal or neoplastic tissues remain controversial. In the present study, we investigated the expression of FasL in normal peripheral blood B lymphocytes or malignant cells of the B‐lymphocyte lineage to elucidate a possible immunologic counterattack mechanism. FasL gene expression was analyzed by reverse transcriptase polymerase chain reaction in two non‐Hodgkins lymphoma (NHL) entities: the highly aggressive Burkitts lymphoma (BL) and indolent NHL chronic lymphocytic leukemia (CLL). FasL expression was found to be consistently negative in all BL cell lines and purified samples from patients with CLL. FasL is not constitutively expressed in normal peripheral blood or neoplastic B lymphocytes making the Fas counterattack, as described for gastrointestinal cancer, unlikely as a mechanism of immunologic escape of lymphoma cells.

Features of cell death, mitochondrial activation and caspase dependence of rabbit anti-T-lymphocyte globulin signaling in lymphoblastic Jurkat cells are distinct from classical apoptosis signaling of CD95

Rabbit anti-T-lymphocyte-globulin (ATG) is used for immunosuppression in organ and stem cell transplantation. The aim of this study was to investigate ATG-induced cell death compared to CD95-signaling of apoptosis. We measured features of cell death at the cell membrane, mitochondria, nuclei and caspase-3 cleavage. We used the following inhibitors: the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (O-Me)-fluoromethyl ketone (zVAD-fmk), the serine protease inhibitors 3,4 dichloroisocoumarin (DCI) and N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) and the reducing agent N-acetycysteine (NAC). ATG-induced cellular changes were rapid, included mitochondrial membrane permeability (MMP) induction and annexin V/propidium iodide (PI) positivity but little caspase-3 activation and nuclear morphology changes. MMP was not sensitive to caspase inhibition, serine protease inhibition with DCI moderately reduced MMP. These findings were in contrast to CD95-signaling. Interestingly, TLCK massively augmented CD95-induced MMP which could be abrogated by NAC. In conclusion, ATG-signaling differs in features and kinetics from CD95-induced apoptosis with caspase-independent mechanisms involved in MMP.