Smart Conditioning with Venetoclax-Enhanced Sequential FLAMSA + RIC in Patients with High-Risk Myeloid Malignancies.

Up to 50% of patients with high-risk myeloid malignancies die of relapse after allogeneic stem cell transplantation. Current sequential conditioning regimens like the FLAMSA protocol combine intensive induction therapy with TBI or alkylators. Venetoclax has synergistic effects to chemotherapy. In a retrospective survey among German transplant centers, we identified 61 patients with myeloid malignancies that had received FLAMSA-based sequential conditioning with venetoclax between 2018 and 2022 as an individualized treatment approach. Sixty patients (98%) had active disease at transplant and 74% had genetic high-risk features. Patients received allografts from matched unrelated, matched related, or mismatched donors. Tumor lysis syndrome occurred in two patients but no significant non-hematologic toxicity related to venetoclax was observed. On day +30, 55 patients (90%) were in complete remission. Acute GvHD II°-IV° occurred in 17 (28%) and moderate/severe chronic GvHD in 7 patients (12%). Event-free survival and overall survival were 64% and 80% at 1 year as well as 57% and 75% at 2 years, respectively. The off-label combination of sequential FLAMSA-RIC with venetoclax appears to be safe and highly effective. To further validate these insights and enhance the idea of smart conditioning, a controlled prospective clinical trial was initiated in July 2023.

PTCY-Based Haploidentical Donor Transplantation versus HLA-Matched Related and Unrelated Donor Transplantations in Patients with Refractory or Relapsed Lymphoma-A Matched-Pair Analysis.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has demonstrated its potential as a curative option for patients with r/r lymphoma. With the introduction of post-transplant cyclophosphamide-based (PTCY) graft-versus-host disease (GvHD) prophylaxis, allo-HCT using haploidentical related donors (Haplo-HSCT) has emerged as a valuable alternative for patients without an available HLA-matched donor. In this study, we compared intermediate and long-term outcomes between Haplo-HSCT and HLA-matched related donor (MRD) and unrelated donor (URD) transplantations in 16 matched pairs using age, disease status, lymphoma classification and performance status as matching criteria. Of note, 88% of patients in each group presented with active disease at the time of conditioning. After a median follow-up of >10 years, 10-year overall and progression-free survival and non-relapse mortality incidence after Haplo-HSCT were 31%, 25% and 38%, respectively, and did not differ compared to the values observed in MRD-HSCT and URD-HSCT. A remarkable lower incidence of acute GvHD ≥ II and moderate and severe chronic GvHD was observed after Haplo-HSCT compared to MRD-HSCT (50%/50%, p = 0.03/0.03) and URD-HSCT (44%/38%, p = 0.04/0.08), resulting in slightly higher 10-year GvHD-free and relapse-free survival (25%) and chronic GvHD-free and relapse-free survival (25%) in the Haplo-HSCT group. In conclusion, Haplo-HSCT is an effective treatment in patients with non-remission NHL. Given its advantage of immediate availability, haploidentical donors should be preferably used in patients with progressive disease lacking an HLA-matched related donor.

Impact of extramedullary disease in AML patients undergoing sequential RIC for HLA-matched transplantation: occurrence, risk factors, relapse patterns, and outcome.

We sought to evaluate the role of extramedullary disease (EMD) in sequential RIC retrospectively analyzing data of 144 high-risk AML patients undergoing HLA-matched transplantation. Median long-term follow-up was 11.6 years. Eighteen percent of patients (n = 26/144) presented with extramedullary AML (EM AML) or a history of EMD at time of transplantation. Overall relapse rate was 25% (n = 36/144) with 15% (n = 21/144) of all patients developing isolated BM relapse and 10% (n = 15/144) developing EM AML relapse with or without concomitant BM relapse (EM ± BM). Manifestation of EM relapse after transplantation occurred frequently at multiple sites and presented mostly as solid tumor mass. Only 3/15 patients with EM ± BM relapse showed a prior EMD manifestation. EMD prior to allogeneic transplantation had no impact on post-transplant OS when compared to non-EMD (median post-transplant OS 3.8 years versus 4.8 years; ns). Risk factors (p = < 0.1) for EM ± BM relapse included younger age and a higher number of prior intensive chemotherapies, whereas the presence of chronic GVHD was a protective factor. Median post-transplant OS (15.5 months vs. 15.5 months), RFS (9.6 months vs 7.3 months), and post-relapse OS (6.7 months vs. 6.3 months) were not significantly different between patients with isolated BM vs. EM ± BM relapse. Taken together, occurrence of EMD prior to as well as of EM ± BM AML relapse after transplantation was moderate, presenting mostly as solid tumor mass after transplantation. However, diagnosis of those does not seem to influence outcomes after sequential RIC. A higher number of chemotherapy cycles prior to transplantation was identified as recent risk factor for EM ± BM relapse.

[Transplantation/cellular therapy in acute myeloid leukemia - Who, When, Why and How?] / Transplantation/Zelltherapie bei Akuter Myeloischer Leukämie.

To date allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the most effective immunotherapeutic approach for the treatment of acute myeloid leukemia (AML). It involves the transplantation of blood stem cells from a healthy donor into a patient, with the goal of using the donor's immune system to recognize and attack cancer cells (Graft-versus-leukemia effect). Thereby, allo-HSCT is more efficient than chemotherapy alone, as it combines high dose chemotherapy +/- irradiation with immunotherapy establishing a long-term control of leukemic cells while allowing reconstitution of a healthy donor hematopoiesis and a new immune system. However, the procedure carries significant risks, including the possibility of graft-versus-host disease (GvHD), and requires careful patient selection to ensure the best possible outcome. In AML patients with high-risk, relapsed or chemo-refractory disease allo-HSCT provides the only curative option.While allo-HSCT is currently the most established form of immunotherapy in the treatment of AML, other approaches that use the immune system to fight cancer are being researched. These may include immunomodulatory drugs or cell therapies such as CAR-T cells, which stimulate the immune system to attack cancer cells. Even if this does not yet play a role in current standard therapy, as our understanding of the immune system and its role in cancer grows, it is likely that targeted immunotherapies will become increasingly important in the treatment of AML The following article gives you an overview of allo-HSCT in AML patients and the current developments.

Impact of Cytogenetic Risk on Outcomes of Non-T-Cell-Depleted Haploidentical Hematopoietic Cell Transplantation in Patients with Relapsed or Refractory Acute Myeloid Leukemia.

Baseline cytogenetics and disease status are key factors predicting the outcomes of allogeneic hematopoietic cell transplantation (HCT) in patients with acute myeloid leukemia (AML). The importance of cytogenetic risk in patients with primary refractory or relapsed (R/R) AML undergoing haploidentical (Haplo) HCT is unknown. We studied the impact of cytogenetic risk in patients with R/R de novo AML with active disease who underwent non-T-cell-depleted Haplo-HCT with post-transplantation cyclophosphamide from 2010 to 2020. Four hundred forty patients with active disease at transplantation from the European Society for Blood and Marrow Transplantation database were analyzed (291 [66.1%] with intermediate-risk [AMLint] and 149 [44.1%] with adverse-risk cytogenetics [AMLadv]). Impact of baseline cytogenetic risk on various transplantation outcomes was evaluated. Pre-transplantation disease status was relapse in 48.1% and 26.8% and primary refractory in 51.9% and 73.2% of the patients with AMLint and AMLadv, respectively (P < .0001). Two-year leukemia-free survival (LFS, 35.5% versus 15.5%, P = .001) and overall survival (OS, 39.2% versus 20.1%, P = .001) were better in AMLint versus AMLadv. In multivariate analysis, the relapse rate was significantly higher (hazard ratio [HR] = 2.17 [95% confidence interval {CI} 1.57-3.0]) and LFS (HR = 1.71 [95% CI, 1.31-2.22]) and OS (HR = 1.69 [95% CI, 1.29-2.22]), significantly lower for patients with AMLadv compared to AMLint, conditioning intensity did not affect leukemia relapse rate. Non-relapse mortality (HR = 1.1 [95% CI, 0.7-1.74]) and graft-versus-host disease-free, relapse-free survival (HR = 1.37 [95% CI, 1.06-1.77]) did not differ significantly between the risk groups. Disease status before transplant (primary refractory versus relapsed) or conditioning intensity did not impact main transplant outcomes. Baseline cytogenetic risk remains a key prognostic factor for patients with R/R AML with persistent disease before non-T-cell-depleted Haplo-HCT.

Follow-up lung ultrasound to monitor lung failure in COVID-19 ICU patients.

OBJECTIVES: Point-of-care lung ultrasound (LU) is an established tool in the first assessment of patients with coronavirus disease (COVID-19). To assess the progression or regression of respiratory failure in critically ill patients with COVID-19 on Intensive Care Unit (ICU) by using LU. MATERIALS AND METHODS: We analyzed all patients admitted to Internal Intensive Care Unit, Ludwig-Maximilians-University (LMU) of Munich, from March 2020 to December 2020 suffering lung failure caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). LU was performed according to a standardized protocol at baseline and at follow up every other day for the first 15 days using a lung ultrasound score (LUSS). Ventilation data were collected simultaneously. RESULTS: Our study included 42 patients. At admission to ICU, 19 of them (45%) were mechanically ventilated. Of the non-invasive ventilated ones (n = 23, 55%), eleven patients required invasive ventilation over the course. While LUS did not differ at admission to ICU between the invasive ventilated ones (at baseline or during ICU stay) compared to the non-invasive ventilated ones (12±4 vs 11±2 points, p = 0.2497), LUS was significantly lower at d7 for those, who had no need for invasive ventilation over the course (13±5 vs 7±4 points, p = 0.0046). Median time of invasive ventilation counted 18 days; the 90-day mortality was 24% (n = 10) in our cohort. In case of increasing LUS between day 1 (d1) and day 7 (d7), 92% (n = 12/13) required invasive ventilation, while it was 57% (n = 10/17) in case of decreasing LUS. At d7 we found significant correlation between LU and FiO2 (Pearson 0.591; p = 0.033), p/F ratio (Pearson -0.723; p = 0.005), PEEP (Pearson 0.495; p = 0.043), pplat (Pearson 0.617; p = 0.008) and compliance (Pearson -0.572; p = 0.016). CONCLUSION: LUS can be a useful tool in monitoring of progression and regression of respiratory failure and in indicating intubation in patients with COVID-19 in the ICU.

The Feasibility of Percutaneous Dilatational Tracheostomy in Immunosuppressed ICU Patients with or without Thrombocytopenia.

Background: Percutaneous dilatational tracheostomy (PDT) has become the preferred method in several intensive care units (ICUs), but data on PDT performed in immunosuppressed and thrombocytopenic patients are scarce. This study aimed to analyze the feasibility of PDT in immunosuppressed and thrombocytopenic patients compared to conventional open surgical tracheostomy (OST). Methods: We retrospectively analyzed the charts of patients who underwent PDT or OST between May 2017 and November 2020. Our outcomes were stoma site infections and bleeding complications. Results: 63 patients underwent PDT, and 21 patients underwent OST. Distribution of gender ratio, age, SAPS II, time of ventilation before tracheostomy, and preexisting hematooncological diseases was comparable between the two groups. After allogeneic stem cell transplantation (alloSCT), patients were more likely to undergo PDT than OST (p=0.033). The PDT cohort suffered from mucositis more frequently (p=0.043). There were no significant differences in leucocyte or platelet count on the tracheostomy day. Patients with coagulation disorders and patients under immunosuppression were distributed equally among both groups. Stoma site infection was documented in five cases in PDT and eight cases in the OST group. Moderate infections were remarkably increased in the OST group. Smears were positive in six cases in the PDT group; none of these patients had local infection signs. In the OST group, smears were positive in four cases; all had signs of a stroma site infection. Postprocedural bleedings occurred in eight cases (9.5%) and were observed significantly more often in the OST group (p=0.001), leading to emergency surgery in one case of the OST group. Conclusion: PDT is a feasible and safe procedure in a predominantly immunosuppressed and thrombocytopenic patient cohort without an increased risk for stoma site infections or bleeding complications.

The feasibility of electromagnetic sensing aided post pyloric feeding tube placement (CORTRAK) in patients with thrombocytopenia with or without anticoagulation on the intensive care unit.

BACKGROUND: The successful initiation of enteral nutrition is frequently hampered by various complications occurring in patients treated in the intensive care unit (ICU). Successful placement of a nasojejunal tube by CORTRAK enteral access system (CEAS) has been reported to be a simple bedside tool for placing the postpyloric (PP) feeding tube. METHODS: We evaluated the efficacy and side effects using CEAS to establish EN in patients with critical illness, thrombocytopenia, and/or anticoagulation. RESULTS: Fifty-six mechanically ventilated patients were analyzed. Twenty-four of them underwent prior hematopoietic stem cell transplantation (SCT). Sixteen patients received extracorporeal membrane oxygenation treatment because of acute respiratory distress syndrome. The median platelet count at PP placement was 26 g/L (range, 4-106 g/L); 16 patients received therapeutic anticoagulation (activated partial thromboplastin time, 50-70 s). CEAS-assisted placement of a PP nasojejunal tube was performed successfully in all patients. The most frequent adverse event was epistaxis in 27 patients (48.2%), which was mostly mild (Common Terminology Criteria for Adverse Events grade 1, n = 21 [77.8%], and grade 2, n = 6). A significant association between a low platelet count and bleeding complications was observed (P < 0.001). CONCLUSION: Performed by an experienced operator, CEAS is a simple, rapidly available, and effective bedside tool for safely placing PP feeding tubes for EN in patients with thrombocytopenia, even when showing an otherwise-caused coagulopathy in the ICU. Higher-grade bleeding complications were not observed despite their obvious correlation to thrombocytopenia. A prospective study is in preparation.

Lung ultrasound predicts clinical course but not outcome in COVID-19 ICU patients: a retrospective single-center analysis.

BACKGROUND: Point-of-care lung ultrasound (LU) is an established tool in the first assessment of patients with coronavirus disease (COVID-19). Purpose of this study was to evaluate the value of lung ultrasound in COVID-19 intensive care unit (ICU) patients in predicting clinical course and outcome. METHODS: We analyzed lung ultrasound score (LUS) of all COVID-19 patients admitted from March 2020 to December 2020 to the Internal Intensive Care Unit, Ludwig-Maximilians-University (LMU) of Munich. LU was performed according to a standardized protocol at ICU admission and in case of clinical deterioration with the need for intubation. A normal lung scores 0 points, the worst LUS has 24 points. Patients were stratified in a low (0-12 points) and a high (13-24 points) lung ultrasound score group. RESULTS: The study included 42 patients, 69% of them male. The most common comorbidities were hypertension (81%) and obesity (57%). The values of pH (7.42 ± 0.09 vs 7.35 ± 0.1; p = 0.047) and paO2 (107 [80-130] vs 80 [66-93] mmHg; p = 0.034) were significantly reduced in patients of the high LUS group. Furthermore, the duration of ventilation (12.5 [8.3-25] vs 36.5 [9.8-70] days; p = 0.029) was significantly prolonged in this group. Patchy subpleural thickening (n = 38; 90.5%) and subpleural consolidations (n = 23; 54.8%) were present in most patients. Pleural effusion was rare (n = 4; 9.5%). The median total LUS was 11.9 ± 3.9 points. In case of clinical deterioration with the need for intubation, LUS worsened significantly compared to baseline LU. Twelve patients died during the ICU stay (29%). There was no difference in survival in both LUS groups (75% vs 66.7%, p = 0.559). CONCLUSIONS: LU can be a useful monitoring tool to predict clinical course but not outcome of COVID-19 ICU patients and can early recognize possible deteriorations.

Predictive and prognostic value of magnesium serum level in FOLFIRI plus cetuximab or bevacizumab treated patients with stage IV colorectal cancer: results from the FIRE-3 (AIO KRK-0306) study.

Magnesium wasting is a frequent side effect of epidermal growth factor receptor (EGFR)-antibody treatment as magnesium-absorption mechanisms are dependent on EGFR signaling. EGFR-inhibition results in decreased renal reabsorption. There is evidence that hypomagnesemia during cetuximab treatment correlates with response. The prognostic role of hypomagnesemia during bevacizumab treatment has not been studied yet. Here, we evaluate the prognostic value of hypomagnesemia in patients with metastatic colorectal cancer treated with FOLFIRI plus cetuximab or bevacizumab as first-line therapy. A total of 391 of 752 patients of the firstline irinotecan study population had magnesium levels measured at baseline and for the first three cycles (6 weeks) of treatment. Of those, 240 had Rat Sarkoma wildtype tumors. Overall hypomagnesemia was more common in the cetuximab compared to the bevacizumab arm (80 vs. 43%, P < 0.005). During therapy, magnesium showed a time-dependent decrease to 80% of baseline in the cetuximab and to 89% in the bevacizumab arm. Whereas magnesium continued to decrease over time in the cetuximab-treated patients, it remained stable in the bevacizumab-treated. Overall response rate (ORR) was associated with higher magnesium at week 6 (20.9 vs. 79.1%, P = 0.041). Bevacizumab-treated patients with magnesium levels below the median value at week 6 had a significantly longer progression-free survival (PFS; 11.7 vs. 9.9 months, P = 0.034; hazard ratio 0.73) and a trend towards longer overall survival (OS) (29.6 vs. 23.2 months, P = 0.089; hazard ratio 0.77). Hypomagnesemia at predefined time points and magnesium nadir had no significant effect on ORR, OS and PFS in the cetuximab arm. Our data show different magnesium kinetics in patients with metastatic colorectal cancer treated with cetuximab or bevacizumab. For patients treated with cetuximab, hypomagnesemia did not have an impact on response and survival. Hypomagnesemia might have a prognostic value in bevacizumab treatment.

Prognostic value of perioperative red blood cell transfusion and anemia on survival and recurrence in oral squamous cell carcinoma.

OBJECTIVE: To evaluate the prognostic effect of allogenic red blood cell transfusion (RBT) and preoperative anemia in patients with oral squamous cell carcinoma (OSCC) undergoing primary tumor resection. METHODS: We retrospectively analyzed a cohort of 621 patients, diagnosed with OSCC receiving tumor resection in curative intention. Preoperative anemia and perioperative RBT were evaluated according to WHO definition. Overall survival (OAS) as well as recurrence-free survival (RFS) was evaluated in transfused and non-transfused as well as in anemic and non-anemic patients. In addition, outcome parameters were calculated for distinct amounts of perioperatively administered RBTs. Data analysis was performed by uni- and multivariate statistics. Mean follow-up time was 7.3 years. RESULTS: Preoperative anemia was diagnosed in 29% of OSCC patients. Anemic patients displayed a significantly decreased five-year OAS (44%) in comparison to non-anemic equivalents (69%). 70% of non-transfused OSCC patients were alive after five years, whereas in case of RBT five-year OAS was 41%. These findings were substantiated by subgroup analysis in patients without preoperative anemia. For anemic patients however, no deleterious effect on survival in case of perioperative RBT was seen. Increasing numbers of received RBTs were shown to worsen outcome of OSCC patients in a dose-dependent manner. CONCLUSION: Preoperative anemia and RBT are significantly associated with impaired long-term outcome of patients suffering from OSCC. Future studies are needed to evaluate differentiated effects of RBTs in anemic and non-anemic OSCC patients and accordingly providing individual transfusion strategies to ameliorate outcome of patients suffering from OSCC.

Integrin-linked kinase controls retinal angiogenesis and is linked to Wnt signaling and exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is a human disease characterized by defective retinal angiogenesis and associated complications that can result in vision loss. Defective Wnt/ß-catenin signaling is an established cause of FEVR, whereas other molecular alterations contributing to the disease remain insufficiently understood. Here, we show that integrin-linked kinase (ILK), a mediator of cell-matrix interactions, is indispensable for retinal angiogenesis. Inactivation of the murine Ilk gene in postnatal endothelial cells results in sprouting defects, reduced endothelial proliferation and disruption of the blood-retina barrier, resembling phenotypes seen in established mouse models of FEVR. Retinal vascularization defects are phenocopied by inducible inactivation of the gene for α-parvin (Parva), an interactor of ILK. Screening genomic DNA samples from exudative vitreoretinopathy patients identifies three distinct mutations in human ILK, which compromise the function of the gene product in vitro. Together, our data suggest that defective cell-matrix interactions are linked to Wnt signaling and FEVR.

The role of checkpoint blockade after allogeneic stem cell transplantation in diseases other than Hodgkin's Lymphoma.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment option for many malignant high-risk hematological diseases. The Graft-vs.-Tumor (GvT) effect is the major hallmark of this treatment approach. However, disease relapse remains a major limitation. Boosting the GvT effect by checkpoint inhibitors (CI) is an attractive option in this desperate situation although potentially triggering Graft-vs.-Host Disease (GvHD). Early reports in patients with Hodgkin's lymphoma support the idea that CI therapy after HSCT is feasible and effective. We have retrospectively analyzed CI therapy for treatment of disease recurrence after allo-HSCT other than Hodgkin's lymphoma including 21 patients from eight German transplant centers. The median follow-up was 59 days. The overall response rate (ORR) was 43%. Patients receiving donor lymphocyte infusion (DLI) in combination with CI had superior response (ORR 80%). Severe acute GvHD grade III-IV and moderate to severe chronic GvHD were observed in 29% of all patients. Taken together, CI therapy in relapsed patients after HSCT, especially in combination with DLI, is effective but induces severe GvHD in a considerable proportion of patients. Thus, prospective trials or EBMT registry-based validation of different dosing and application schedules including immunosuppressive regimens in those patients are urgently needed.

Comparable outcome after haploidentical and HLA-matched allogeneic stem cell transplantation for high-risk acute myeloid leukemia following sequential conditioning-a matched pair analysis.

In acute myeloid leukemia (AML), primary refractory or relapsed disease, secondary AML, and leukemia with unfavorable genetics are considered high-risk AML (hrAML), with allogeneic stem cell transplantation (SCT) representing the standard treatment. Sequential conditioning has been successfully used for SCT in hrAML in HLA-matched transplants, and found its way into HLA-haploidentical SCT (haplo-SCT) later on. Hence, sequential conditioning had become standard for all patients with hrAML in our two centers, regardless of donor type. Thereby, HLA-matched family or unrelated transplants were first/second choice, post-transplant cyclophosphamide (PTCY)-based haplo-SCT was chosen in patients missing matched donors or requiring urgent transplantation. To compare the outcome after HLA-matched and haplo-SCT for hrAML following sequential conditioning, we performed a retrospective, matched-pair comparison, using disease stage, genetic subgroups and age as matching criteria. Thirty-four well-matched pairs were identified. At SCT, patients (median age 54 years) were untreated (9%), had remission (13%), or active disease (78%). Three-year overall and leukemia-free survival (OS/LFS) of the entire cohort was 56 ± 7%/49 ± 7%, without significant differences between donor types (OS after HLA-matched/haplo-SCT 62 ± 10%/52 ± 9% (p = 0.21), LFS 53 ± 10%/46 ± 9% (p = 0.26)). Similarly, the cumulative incidence of relapse, non-relapse-mortality and chronic GvHD, as well as GvHD-free, relapse-free survival (GRFS), and chronic GvHD-free, relapse-free survival (cGRFS), were comparable. However, a higher incidence of acute GvHD ≥ II° was observed after HLA-matched SCT (15 ± 1% versus 50 ± 2%, p = 0.001). In conclusion, sequential conditioning SCT achieved remarkable results in hrAML, independently from donor type. PTCY-based haplo-SCT produced results that were comparable to HLA-matched SCT and can be used as an alternative option.

Sequential HLA-haploidentical transplantation utilizing post-transplantation cyclophosphamide for GvHD prophylaxis in high-risk and relapsed/refractory AML/MDS.

This study evaluates the role of sequential therapy in HLA-haploidentical transplantation (haplo-HSCT) of high-risk, relapsed/refractory AML/MDS. We analyzed the course of 33 adults with active disease at time of transplantation (AML n = 30; MDS n = 3; median age 58 years, range: 32-71). Sequential therapy consisted of cytoreductive chemotherapy (FLAMSA n = 21; clofarabine n = 12) applied shortly prior to reduced intensity conditioning for T-cell-replete haplo-HSCT using post-transplantation cyclophosphamide as GvHD prophylaxis. No graft rejection was observed. Complete remission at day +30 was achieved in 97% of patients. CI of acute GvHD grade II-IV and chronic GvHD was 24% (no grade IV) and 23%, respectively. NRM at 1 and 3 years was 15%, each. Severe regimen-related toxicities (grade III-IV) were observed in 58%, predominantly involving the gastrointestinal tract (diarrhea 48%, mucositis 15%, transient elevation of transaminases 18%). Probability of relapse at 1 and 3 years was 28% and 35%. At a median follow-up of 36 months, the estimated 1- and 3-year overall survival was 56% and 48%. Disease-free survival was 49% and 40%, respectively. At 3 years, GvHD and relapse-free survival (GRFS) was 24% while chronic GvHD and relapse-free survival (CRFS) was 29%. Thus, our results indicate that sequential haplo-HSCT is an effective salvage treatment providing high anti-leukemic activity, favorable tolerance, and acceptable toxicity in patients suffering from advanced AML/MDS.

Cross-sectional analysis of CD8 T cell immunity to human herpesvirus 6B.

Human herpesvirus 6 (HHV-6) is prevalent in healthy persons, causes disease in immunosuppressed carriers, and may be involved in autoimmune disease. Cytotoxic CD8 T cells are probably important for effective control of infection. However, the HHV-6-specific CD8 T cell repertoire is largely uncharacterized. Therefore, we undertook a virus-wide analysis of CD8 T cell responses to HHV-6. We used a simple anchor motif-based algorithm (SAMBA) to identify 299 epitope candidates potentially presented by the HLA class I molecule B*08:01. Candidates were found in 77 of 98 unique HHV-6B proteins. From peptide-expanded T cell lines, we obtained CD8 T cell clones against 20 candidates. We tested whether T cell clones recognized HHV-6-infected cells. This was the case for 16 epitopes derived from 12 proteins from all phases of the viral replication cycle. Epitopes were enriched in certain amino acids flanking the peptide. Ex vivo analysis of eight healthy donors with HLA-peptide multimers showed that the strongest responses were directed against an epitope from IE-2, with a median frequency of 0.09% of CD8 T cells. Reconstitution of T cells specific for this and other HHV-6 epitopes was also observed after allogeneic hematopoietic stem cell transplantation. We conclude that HHV-6 induces CD8 T cell responses against multiple antigens of diverse functional classes. Most antigens against which CD8 T cells can be raised are presented by infected cells. Ex vivo multimer staining can directly identify HHV-6-specific T cells. These results will advance development of immune monitoring, adoptive T cell therapy, and vaccines.

Endothelial alpha-parvin controls integrity of developing vasculature and is required for maintenance of cell-cell junctions.

RATIONALE: Angiogenesis and vessel integrity depend on the adhesion of endothelial cells (ECs) to the extracellular matrix and to adjacent ECs. The focal adhesion protein α-parvin (α-pv) is essential for vascular development. However, the role of α-pv in ECs in vivo is not known. OBJECTIVE: To determine the function of α-pv in ECs during vascular development in vivo and the underlying mechanisms. METHODS AND RESULTS: We deleted the α-pv gene specifically in ECs of mice to study its role in angiogenesis and vascular development. Here, we show that endothelial-specific deletion of α-pv in mice results in late embryonic lethality associated with hemorrhages and reduced vascular density. Postnatal-induced EC-specific deletion of α-pv leads to retinal hypovascularization because of reduced vessel sprouting and excessive vessel regression. In the absence of α-pv, blood vessels display impaired VE-cadherin junction morphology. In vitro, α-pv-deficient ECs show reduced stable adherens junctions, decreased monolayer formation, and impaired motility, associated with reduced formation of integrin-mediated cell-extracellular matrix adhesion structures and an altered actin cytoskeleton. CONCLUSIONS: Endothelial α-pv is essential for vessel sprouting and for vessel stability.

Visualization of endothelial actin cytoskeleton in the mouse retina.

Angiogenesis requires coordinated changes in cell shape of endothelial cells (ECs), orchestrated by the actin cytoskeleton. The mechanisms that regulate this rearrangement in vivo are poorly understood - largely because of the difficulty to visualize filamentous actin (F-actin) structures with sufficient resolution. Here, we use transgenic mice expressing Lifeact-EGFP to visualize F-actin in ECs. We show that in the retina, Lifeact-EGFP expression is largely restricted to ECs allowing detailed visualization of F-actin in ECs in situ. Lifeact-EGFP labels actin associated with cell-cell junctions, apical and basal membranes and highlights actin-based structures such as filopodia and stress fiber-like cytoplasmic bundles. We also show that in the skin and the skeletal muscle, Lifeact-EGFP is highly expressed in vascular mural cells (vMCs), enabling vMC imaging. In summary, our results indicate that the Lifeact-EGFP transgenic mouse in combination with the postnatal retinal angiogenic model constitutes an excellent system for vascular cell biology research. Our approach is ideally suited to address structural and mechanistic details of angiogenic processes, such as endothelial tip cell migration and fusion, EC polarization or lumen formation.