Dan Hauzenberger

Improved Survival after Allogeneic Hematopoietic Stem Cell Transplantation in Recent Years. A Single-Center Study

We analyzed the outcome of allogeneic hematopoietic stem cell transplantation (HSCT) over the past 2 decades. Between 1992 and 2009, 953 patients were treated with HSCT, mainly for a hematologic malignancy. They were divided according to 4 different time periods of treatment: 1992 to 1995, 1996 to 2000, 2001 to 2005, and 2006 to 2009. Over the years, many factors have changed considerably regarding patient age, diagnosis, disease stage, type of donor, stem cell source, genomic HLA typing, cell dose, type of conditioning, treatment of infections, use of granulocyte-colony stimulating factor (G-CSF), use of mesenchymal stem cells, use of cytotoxic T cells, and home care. When we compared the last period (2006-2009) with earlier periods, we found slower neutrophil engraftment, a higher incidence of acute graft-versus-host disease (aGVHD) of grades II-IV, and less chronic GVHD (cGHVD). The incidence of relapse was unchanged over the 4 periods (22%-25%). Overall survival (OS) and transplant-related mortality (TRM) improved significantly in the more recent periods, with the best results during the last period (2006-2009) and a 100-day TRM of 5.5%. This improvement was also apparent in a multivariate analysis. When correcting for differences between the 4 groups, the hazard ratio for mortality in the last period was 0.59 (95% confidence interval [CI]: 0.44-0.79; P < .001) and for TRM it was 0.63 (CI: 0.43-0.92; P = .02). This study shows that the combined efforts to improve outcome after HSCT have been very effective. Even though we now treat older patients with more advanced disease and use more alternative HLA nonidentical donors, OS and TRM have improved. The problem of relapse still has to be remedied. Thus, several different developments together have resulted in significantly lower TRM and improved survival after HSCT over the last few years.

A flow cytometric crossmatch test for simultaneous detection of antibodies against donor lymphocytes and endothelial precursor cells.

Complement-dependent cytotoxicity or flow cytometric lymphocyte crossmatch (LXM) tests may fail to detect clinically significant antibodies (Abs) against non-human leukocyte antigen (HLA). A flow cytometric endothelial precursor cell crossmatch (EPCXM) test (XM-ONE) is available for detection of Abs against donor endothelial precursor cells (EPCs). We showed that lymphocytes co-purified with EPCs can be used in LXM tests allowing simultaneous detection of Abs reactive with donor EPCs and lymphocytes. The lymphocyte population co-purified with EPCs on anti-Tie-2 Ab-coupled magnetic beads contained CD 8(+) and CD 4(+) T-cells, B-cells, and natural killer (NK)- and natural killer T (NKT)-cells. HLA class I antigen expression was slightly higher on CD 3(+) lymphocytes co-purified on Tie-2 Ab beads than on unseparated lymphocytes, whereas HLA class I and II antigen levels on CD 19(+) lymphocytes were not significantly different. Sera from 10 patients with panel-reactive Abs were tested on cells from nine donors using flow cytometric LXM and EPCXM tests. There was a very good correlation (R(2) = 0.94) between the channel shift values obtained on unseparated and Tie-2 Ab bead-isolated T-lymphocytes, whereas the correlation between the channel shift values obtained on the two B-lymphocyte populations was lower (R(2) = 0.71). T- and B-lymphocytes co-purified with EPCs can be used in LXM tests enabling simultaneous detection of donor lymphocyte- and EPC-reactive Abs in a single-tube XM-ONE assay.

Outcome of haematopoietic stem cell transplantation in patients transplanted with matched unrelated donors vs allele‐mismatched donors: a single centre study

We studied the importance of human leucocyte antigen (HLA)-A, -B and -DRB1 high-resolution matching on the outcome of haematopoietic stem cell transplantation (HSCT) with matched unrelated donors (MUDs) vs single allele-mismatched unrelated donors. Fifty consecutive HSCT patients receiving an HLA-A, -B or -DR allele-level-mismatched unrelated graft (mmUD) were compared with a matched cohort of 100 patients with an HLA-A, -B and -DR-MUD. Rejection occurred in seven patients (14%) in the mmUD group and in four patients (4%) in the MUD group (P = 0.04), but this was mainly an effect of HLA-C mismatch. The cumulative incidence of acute graft vs host disease (GVHD) grades II-IV were 61%, 26% and 33% in the class I mmUD, class II mmUD and MUD groups, respectively. In multivariate analysis, HLA class I mismatch was associated with an increased risk of acute GVHD grades II-IV (2.09, P = 0.007) and transplant-related mortality (TRM) (1.99, P = 0.06). The 5-year overall survival was 81% in patients with a class II allele-mismatched donor compared with 52% (P = 0.025) and 50% (P = 0.017) in patients with a class I mismatch and a MUD. In multivariate analysis, HLA class II allele mismatch was associated with improved survival (3.38, P = 0.019). Relapse-free survival were 53%, 37% and 42% in patients with a class II mmUD, class I mmUD and a MUD, respectively (not significant). An HLA-C or -DQ mismatch had no significant impact on survival, TRM and relapse. In conclusion, compared with MUD, HLA class I allele mmUD had an increased risk of acute GVHD and TRM.

Immune modulation to prevent antibody-mediated rejection after allogeneic hematopoietic stem cell transplantation

It has been shown that antibodies to donor CD34+/VEGFR-2+ stem cells or antibodies against mismatched HLA are associated with graft rejection after hematopoietic stem cell transplantation (HSCT). CD34/VEGFR-2 positive stem cells have been implicated to play a major role in engraftment after HSCT. In this study we treated four patients with an imminent risk of antibody-mediated rejection with immune modulation, i.e. plasma exchange, intravenous immunoglobulin (IVIG), and rituximab before HSCT. Three of the patients had been previously transplanted and rejected their initial grafts after 12 months, 1 month, and less than 1 month, respectively. The fourth patient was not transplanted previously but had HLA directed antibodies present against the graft. During the immune modulatory treatment we followed the pattern of antibodies in sera using FACS and microcytotoxicity assay. We could show that two patients had antibodies against donor CD34+/VEGFR-2+ cells while the other two had antibodies directed against HLA. All four patients tolerated the immune modulatory regimen without any side effects. In this preliminary study we show that immune modulatory treatment may be used to reduce antibody levels and to prevent rejection after HSCT. In two of the three patients which experienced previous rejections and had detectable anti-HLA or anti-CD34+/VEGFR-2+ antibodies, immune modulation resulted in engraftment. In the fourth patient with known anti-HLA-class I antibodies, the treatment also resulted in engraftment. Our results encourage further studies regarding this treatment regimen.

Evaluation of a new flow cytometry crossmatch procedure for simultaneous detection of cytotoxicity and antibody binding.

In this study we have evaluated an alternative 96-well format flow cytometry based (FCtox) method which enable simultaneous detection of cytotoxicity and human leukocyte antigen (HLA) antibody binding. Comparable results were obtained in side-by-side comparisons with conventional complement-dependent cytotoxicity (CDC) and flow cytometric crossmatch (FCXM) in terms of sensitivity and specificity. There was 91 and 93% agreement between results obtained by FCtox and CDC for T and B cells, respectively. In addition, comparable results were obtained with FCtox IgG and FCXM IgG for both T and B cells. Furthermore, compared with a recently developed and highly sensitive Luminex based C1q assay we obtained close to 90% method agreement with the FCtox assay. Our alternative cytotoxicity and IgG binding assay which exhibit low intra-and inter-assay variation will improve the workflow and speed up the pre-transplant testing and also allow continuous monitoring of assay performance and proper quality assurance.

No effect of HLA-C mismatch after allogeneic hematopoietic stem cell transplantation with unrelated donors and T-cell depletion in patients with hematological malignancies

HLA‐C mismatch in unrelated donors hematopoietic stem cell transplantation (HSCT) has been associated with poor patient outcome. However, the impact of HLA‐C mismatch in the context of HSCT combined with in vivo T‐cell depletion remains unclear. We therefore performed a single‐center, retrospective analysis of the clinical outcome on patients with hematological malignancies treated with allo‐HSCT, who underwent T‐cell depletion. The majority of the patients (n=276) received a HLA‐A, HLA‐B, HLA‐DRB1‐matched graft that were either also HLA‐C matched (n=260), or patients with the permissive HLA‐C*03:03/03:04 mismatch (n=16), while the remaining patients (n=95) received a HLA‐C‐mismatched graft (excluding HLA‐C*03:03/03:04 mismatches). We did not observe any significant differences between the HLA‐C‐matched patients (including the permissive HLA‐C*03:03/03:04 mismatch) and the HLA‐C‐mismatched patients regarding cumulative proportion surviving, graft failure, relapse‐free survival, relapse, or acute graft‐versus‐host disease. Our data suggest that in the context of high dose T lymphocyte‐depleting agents, HLA‐C matching is not essential for patients with hematological malignancies.

An unusual cause of fatal rapid-onset ataxia plus syndrome

BackgroundProgressive multifocal leukoencephalopathy (PML) is a demyelinating disorder of the central nervous system caused by reactivation of the JC-virus and is in most cases associated with underlying immunosuppression. Acquired immune deficiency syndrome (AIDS) and hematological malignancies are well-known predisposing factors for PML. However, in the past ten years, various pharmacological agents have been associated with increased risk of PML. Based on the phenomenology PML can be divided into the cerebral form and the rare cerebellar form.Case presentationHere we describe a man affected by polycythemia vera (PCV) that was treated with hydroxyurea (HU) and developed PML. The initially PML presentation included ataxia as one of the main features. Brain MRI displayed widespread supratentorial and infratentorial lesions. Immunological analysis revealed absence of reactivity to a wide range of antigens. The course of disease was rapidly progressive with fatal outcome - autopsy ruled out leukemic transformation.ConclusionThe occurrence of PML in PCV patients is very rare and has been reported only once. Movement disorders, such as ataxia, are also less frequent. In the present case the PML was likely multifactorial.

Extracellular Matrix and Mesothelioma: Some Clues to the Invasive Behavior of Mesothelioma

Malignant mesotheliomas are highly aggressive diffuse tumors arising from mesothelial-lined surfaces. Mesotheliomas spread along mesothelial-lined surfaces to involve the pericardium, contralateral hemithorax, and peritoneal cavity by invasion through the diaphragm. The resulting tumor often forms diffuse thickening of involved surfaces rather than solitary rounded lesions as seen in other neoplasms (1,2). Malignant mesotheliomas also invade the underlying basement membrane and produce metastases in up to 80% of patients (3–5). Invasion through needle biopsy tracts and incision in the thoracic wall is a common feature in malignant mesotheliomas (6,7). During this process mesothelioma cells must interact with extracellular matrix proteins, growth factors embedded in it, and stromal cells, which participate in synthesis and modifications of this microenvironment. Today, the central theme of research about tumor etiology, progression, and metastasis focuses more on the crosstalk between tumor cells, extracellular matrix, and a variety of host cells rather than the behavior of individual tumor cells taken out of their microenvironmental context (8). This chapter summarizes evidence describing various aspects of mesothelioma interactions with different components of the extracellular matrix, and the possible role of these interactions in the invasive behavior of this tumor.