90-yttrium-ibritumomab tiuxetan as first-line treatment for follicular lymphoma: updated efficacy and safety results at an extended median follow-up of 9.6 years.

Radioimmunotherapy with 90-yttrium-ibritumomab tiuxetan (90Y-IT) as first-line treatment in patients with follicular lymphoma (FL) demonstrated promising results with a complete remission (CR) rate of 56% and a median progression-free survival (PFS) of 26 months, when initially analyzed after a median follow-up of 30.6 months. The aim of this long-term follow-up was to investigate whether clinical benefits were maintained and new safety signals appeared. Fifty-nine patients, aged ≥ 50 years, with FL grade 1 to 3A in stages II to IV were treated with 90Y-IT as first-line therapy. If CR without evidence of minimal residual disease (MRD), partial response or stable disease was achieved 6 months after treatment, patients were observed without further treatment. Patients with CR but persisting MRD received consolidation therapy with rituximab. The primary endpoint was the clinical response rate. Secondary endpoints were time to progression, safety, and tolerability. After a median follow-up of 9.6 years, median PFS was 3.6 years, and 8-year PFS was 38.3%. Median overall survival (OS) was not reached during the extended follow-up, and 8-year OS amounted to 69.2%. Age 65 years and above or disease progression within 24 months of treatment were significantly associated with shorter OS. An important finding was the lack of new safety signals. In particular, no increase in secondary malignancies or transformation into aggressive lymphoma was observed compared to trials with a similar follow-up. In summary, 90Y-IT as first-line treatment demonstrates a favorable safety profile and long-term clinical activity in a substantial fraction of FL patients in need of therapy. ClinicalTrials.gov Identifier: NCT00772655.

Relapse of NPM1-Mutated AML with Extramedullary Manifestation 17 Years after Allogeneic Hematopoietic Stem Cell Transplantation.

The majority of patients with acute myeloid leukemia (AML) with the NPM1 mutation achieve remission with intensive chemotherapy. However, many patients subsequently relapse, which occurs frequently within the first 2-3 years after therapy, while late relapse after more than 10 years is rare and can also represent secondary/therapy-associated AML without the NPM1 mutation. Here, we present a case of NPM1-mutated AML that developed medullary and extramedullary relapse 17 years after allogeneic stem cell transplantation, maintaining the NPM1 mutation and all other genetic alterations detected at first diagnosis. This exceptionally long latency between diagnosis and relapse of a genetically highly related leukemic clone implies the existence of therapy-resistant, persisting dormant leukemic stem cells in NPM1 mutant AML.

Confocal endomicroscopy in diagnosis of intestinal chronic graft-versus-host disease.

Graft-versus-host disease (GvHD) is a major complication of allogeneic stem cell transplantation. High-resolution in vivo histology of the intestine by confocal endomicroscopy (CEM) detects acute GvHD (aGvHD) with high sensitivity. This pilot study aims to evaluate the diagnostic value of CEM for intestinal chronic GvHD (cGvHD). The study included 20 patients with gastrointestinal symptoms and confirmed cGvHD in other organs as well as 20 patients with clinically suspected acute GvHD for control. Confocal endomicroscopy was performed as gastroscopy followed by sigmoidoscopy after intravenous injection of fluorescein (10%) and topical application of acriflavine (0.05%). Histopathology from H&E-stained biopsy samples throughout the intestinal tract complemented the survey. All histological features of intestinal cGvHD were predominantly mild to moderate. Stroma fibrosis detected by standard histology (16/20 patients) was not seen by CEM. Apoptosis assessed by histology in 12/20 patients was concordant with CEM (8/12 patients). Confocal endomicroscopy revealed esophageal manifestation of cGvHD in 3 patients. For each biopsy site, CEM correlated with intestinal histology (r = 0.64). Classical histology from intestinal biopsy samples taken under CEM monitoring confirmed the final diagnosis of cGvHD. The sensitivity of CEM with 40% in cGvHD was significantly lower compared to 70% in patients with aGvHD. Confocal endomicroscopy detected acute features of cGvHD and contributed to the diagnosis of esophageal cGvHD but failed to display stroma fibrosis in vivo. Although CEM represents a useful noninvasive tool in routine diagnostic of intestinal aGvHD, the method is not sufficient to fully establish the diagnosis of cGvHD within the intestinal tract. Confocal endomicroscopy allowed acquisition of targeted biopsies in patients suspected of having cGvHD.

Physical and psychosocial aspects of adolescent and young adults after allogeneic hematopoietic stem-cell transplantation: results from a prospective multicenter trial.

PURPOSE: Allogeneic hematopoietic stem-cell transplantation (alloHSCT) is physically and psychosocially demanding. Among transplant recipients, adolescent and young adults (AYA) represent a special group, as disease occurs early in life, resulting in the prospect of long survival time and high burden of alloHSCT sequelae. However, data focusing on AYA undergoing alloHSCT are rare. METHODS: Data resulting from a prospective multicenter trial initially focusing on graft-versus-host disease (GvHD) after alloHSCT were reused to analyse the differences between AYA and elderly patients. In total, data of 205 alloHSCT recipients were evaluated. Patients completed the FACT-BMT, HAP, SF-36, 24-AM, LOT-R, BSSS, HADS, and GvHD questionnaires. RESULTS: Median age of AYA and non-AYA patients was 29 and 52 years. Using 24-AM-Test, evaluating personality traits, non-AYA reported to be more conscientious (p = 0.033). However, AYA described higher quality of life regarding physical role functioning (p = 0.001), physical functioning (p = 0.002), bodily pain (p = 0.023), and emotional role function (p = 0.027) in the SF-36. General health perception, vitality, social role functioning, and mental health were comparable among both groups. On HAP scale, AYA reported higher maximum (p = 0.003) and adjusted activity scores (p = 0.002), but showed similar restrictions regarding activity, self-supply, and self-determination. CONCLUSION: AYA represent a particular group characterized by higher physical well-being and activity scores, and significantly vary from non-AYA patients in psychosocial aspects. Studies covering distinctive features of AYA undergoing alloHSCT are warranted to improve awareness of the special needs of this group.

Rabbit antithymocyte globulin induces rapid expansion of effector memory CD8 T cells without accelerating acute graft versus host disease.

Rabbit antithymocyte globulin (Thymoglobulin(®)) is commonly used as graft-versus-host disease (GvHD) prophylaxis. Since we found similar total CD8 T cell numbers in patients with and without Thymoglobulin(®) therapy within the first six months after allogeneic hematopoietic stem cell transplantation, we have analyzed the reconstitution of the CD8 T cell compartment in detail. After T cell-depletion, higher and more sustained proliferative capacity of memory CD8 T cells resulted in their rapid expansion, whereas the fraction of naive CD8 T cells decreased. Importantly, this shift towards effector memory CD8 T cells did not accelerate the incidence of GvHD.

Rabbit antithymocyte globulin (thymoglobulin) impairs the thymic output of both conventional and regulatory CD4+ T cells after allogeneic hematopoietic stem cell transplantation in adult patients.

Rabbit antithymocyte globulin-Genzyme™ is used to prevent graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Common disadvantages of treatment are infectious complications. The effects of rabbit antithymocyte globulin-Genzyme™ on thymic function have not been well-studied. Multicolor flow cytometry was used to analyze the kinetics of conventional and regulatory T cells in adult patients treated (n=12) or not treated (n=8) with rabbit antithymocyte globulin-Genzyme™ during the first 6 months after allogeneic hematopoietic stem cell transplantation. Patients treated with rabbit antithymocyte globulin-Genzyme™ had almost undetectable levels of recent thymic emigrants (CD45RA(+)CD31(+)) of both conventional and regulatory CD4T cells throughout the 6 months after allogeneic hematopoietic stem cell transplantation whereas CD4(+)CD45RA-memory T cells were less affected, but their levels were also significantly lower than in patients not treated with rabbit antithymocyte globulin-Genzyme™. In vitro, rabbit antithymocyte globulin-Genzyme™ induced apoptosis and cytolysis of human thymocytes, and its cytotoxic effects were greater than those of rabbit antithymocyte globulin-Fresenius™. Rabbit antithymocyte globulin-Genzyme™ in combination with a conditioning regimen strongly impairs thymic recovery of both conventional and regulatory CD4(+) T cells. The sustained depletion of conventional and regulatory CD4(+)T cells carries a high risk of both infections and graft-versus-host disease. Our data indicate that patients treated with rabbit antithymocyte globulin-Genzyme™ could benefit from thymus-protective therapies and that trials comparing this product with other rabbit antithymocyte globulin preparations or lymphocyte-depleting compounds would be informative.

Evidence for the cure of HIV infection by CCR5Δ32/Δ32 stem cell transplantation.

HIV entry into CD4(+) cells requires interaction with a cellular receptor, generally either CCR5 or CXCR4. We have previously reported the case of an HIV-infected patient in whom viral replication remained absent despite discontinuation of antiretroviral therapy after transplantation with CCR5Δ32/Δ32 stem cells. However, it was expected that the long-lived viral reservoir would lead to HIV rebound and disease progression during the process of immune reconstitution. In the present study, we demonstrate successful reconstitution of CD4(+) T cells at the systemic level as well as in the gut mucosal immune system after CCR5Δ32/Δ32 stem cell transplantation, while the patient remains without any sign of HIV infection. This was observed although recovered CD4(+) T cells contain a high proportion of activated memory CD4(+) T cells, ie, the preferential targets of HIV, and are susceptible to productive infection with CXCR4-tropic HIV. Furthermore, during the process of immune reconstitution, we found evidence for the replacement of long-lived host tissue cells with donor-derived cells, indicating that the size of the viral reservoir has been reduced over time. In conclusion, our results strongly suggest that cure of HIV has been achieved in this patient.

Validation of the human activity profile questionnaire in patients after allogeneic hematopoietic stem cell transplantation.

Chronic graft-versus-host disease (cGVHD) associated morbidity and mortality remain major barriers for successful allogeneic hematopoietic stem cell transplantation (alloHSCT). Currently, no reliable measures are established to monitor cGVHD activity changes for use in clinical trials. The Human Activity Profile (HAP) patient self-report was proposed by the National Institutes of Health (NIH) cGVHD consensus project as an independent measure of patients' functional status that could also indirectly reflect improvement of cGVHD, but that has not been validated in an alloHSCT patient population. One hundred seventy-six patients (median age 44 years [range: 18-72 years] after alloHSCT were evaluated with a German translation of the HAP, the NIH criteria-based cGVHD activity assessment, the Lee cGVHD Symptom-Scale, FACT-BMT, SF36, Berlin Social Support Scale, 24-Item Adjective Measure (24-AM), Hospital Anxiety and Depression Scale, and the NCCN-Distress-Thermometer. Enrollment occurred a median of 286 (range: 85-4003) days after alloHSCT. Follow-up surveys were conducted at 1, 2, 3, 5, 8, and 12 months after the baseline survey. Although 117 patient had cGVHD at time of enrollment (mild n = 33, moderate n = 50, or severe n = 34), 59 patients were included into the study in the absence of cGVHD between days 85 and 395 after transplantation. The maximum activity score (MAS) and adjusted activity score (AAS) of the HAP correlated inversely with grading of cGVHD severity (mild, moderate, or severe) (r = -0.25 for MAS and -0.24 for AAS). Lung manifestations of cGVHD correlated with AAS (r = 0.17), but not with MAS. HAP scores correlated with subscales from other instruments measuring physical domains, especially the physical functioning scale of the SF36. Performance was improved by use of an HSCT-modified HAP scoring system that excluded activities prohibited within the first year after alloHSCT. No significant correlation of the HAP was found with personality, age, sex, symptom burden, or social functioning or social well-being. Moreover, the HAP displayed a higher sensitivity to change of cGVHD activity compared to the SF36 and the FACT-BMT. In addition, steroid myopathy correlated with both HAP scores, but not the SF36. The HAP is a simple and valid questionnaire for the evaluation of the physical activity in patients after alloHSCT, with the advantage of detecting changes in cGVHD status independently of other quality-of-life measures and with a superior sensitivity compared to the SF36.

The type of ATG matters -- natural killer cells are influenced differentially by Thymoglobulin, Lymphoglobulin and ATG-Fresenius.

Although ATG is frequently used in hematopoietic stem cell transplantation and solid organ transplantation, little is known on its effects on NK cells, which mediate important functions in post-transplantation immunology. We incubated peripheral blood lymphocytes of healthy donors with Thymoglobulin, Lymphoglobulin or ATG-Fresenius. Cell death and apoptosis of NK cells and T cells were determined by flow cytometry using propidium iodide and Annexin V. As expected, there were no significant differences between the different ATGs regarding their T cell toxicity. Surprisingly, we found profound differences between the different ATGs regarding their impact on NK cells: In clinically relevant concentrations Lymphoglobulin had less toxic effects on NK cells as compared to Thymoglobulin or ATG-Fresenius: the median percentages of apoptotic or necrotic NK cells in response to 1 mug/ml Lymphoglobulin, ATG-Fresenius and Thymoglobulin were 2%, 35% and 38%, respectively (p<0.001). This is the first report of differential effects of different ATGs on NK cells. Lymphoglobulin appears to be superior to Thymoglobulin or ATG Fresenius regarding the preservation of NK cell mediated immunity. Randomized trials addressing the impact of different ATGs on lymphocyte subpopulations in the clinical setting are urgently warranted.

Liver-specific increase of UTP and UDP-sugar concentrations in rats induced by dietary vitamin B6-deficiency and its relation to complex N-glycan structures of liver membrane-proteins.

This is the first known report on the influence of vitamin B6-deficiency on the concentration of UDP-sugars and other uracil nucleotides in rats. Animals aged 3 weeks or 2 months were fed a vitamin B6-free diet for periods varying from 3 days to 7 weeks. Nucleotides were quantified by enzymatic-photometry and by SAX-high precision liquid chromatography. In 3 week-old rats, vitamin B6-deficiency resulted in an up to 6.3-fold increase in the concentrations of UTP, UDP, UMP and UDP-sugars and less of CTP in rat liver, while no changes were observed in older rats. In young rats, the concentration of uracil nucleotides started to increase after 1 week diet, with a maximum after 2 weeks. After 5 weeks, the concentrations returned to normal values. In heart, lungs, kidney and brain, concentrations were measured after 2 weeks diet in young rats. In contrast to liver, the heart muscle uracil nucleotide concentrations were decreased by 40%. In kidney, the sum of UTP, UDP and UMP showed a decrease of 40%, whereas UDP-sugars were increased 1.4-fold. In the lungs, nucleotide concentrations were mostly unaffected by vitamin B6-deficiency, despite a 70% increase of UDP-GA. In brain, UDP-Glc, UDP-Gal and the sum of CTP and CDP showed an increase of 30-50%. We became surprised that the increased UDP-sugar concentrations did not influence the structure of liver plasma membrane-N-glycans. Despite the 4 to 6-fold increase of UTP and UDP-sugars, no changes in the complexity or sialylation of these N-glycans could be detected. This study demonstrates that, especially in liver, pyridoxal phosphate is closely involved in the control of uracil nucleotides during a defined period of development. In contrast to in vitro experiments, in vivo N-glycan biosynthesis in liver is regulated by a more complex or higher mechanism than substrate concentrations.

Mucosal FOXP3+ regulatory T cells are numerically deficient in acute and chronic GvHD.

CD4+CD25+ regulatory T cells (Tregs) control immune responses to self- and foreign antigens and play a pivotal role in autoimmune diseases, infectious and noninfectious inflammation, and graft rejection. Since recent experimental studies have indicated that Tregs were able to ameliorate graft-versus-host disease (GvHD), we analyzed the number of infiltrating Tregs in the intestinal mucosa as one site of GvH reactivity using immunoenzymatic labeling to enumerate FOXP3+ T cells in 95 intestinal biopsies from 49 allografted patients in comparison with healthy controls and patients with infectious inflammation. While patients with cytomegalovirus (CMV)-colitis or diverticulitis showed a concomitant increase of CD8+ effectors and Tregs, acute and chronic GvHD were characterized by the complete lack of a counter-regulation indicated by a FOXP3+/CD8+ T-cell ratio identical to healthy controls. In contrast, specimens without histologic signs of GvHD demonstrated increased numbers of FOXP3+ per CD8+ T cells, indicating that the potential for Treg expansion is principally maintained in allografted patients. Our findings provide evidence that GvHD is associated with an insufficient up-regulation of Tregs in intestinal GvHD lesions. The determination of FOXP3+/CD8+ ratio can be a helpful tool to discriminate GvHD from infectious inflammation after allogeneic stem cell transplantation.

Mesenchymal stem cells remain of host origin even a long time after allogeneic peripheral blood stem cell or bone marrow transplantation.

OBJECTIVE: Plasticity of hematopoietic stem cells (HSC) has gained major interest in stem cell research. In order to investigate whether HSC may differentiate into mesenchymal stem cells (MSC), we assessed chimerism in peripheral blood (PB), mononuclear cell fractions (MNC) of bone marrow, and MSC derived from bone marrow (BM) from 27 up to 4225 days after allogeneic transplantation. PATIENTS AND METHODS: We applied fluorescence in situ hybridization using X/Y gene probes in sex-mismatched and STR-PCR in sex-matched patients. MSC could have been generated in 27 of 55 bone marrow samples derived from 20 patients. Fifteen patients received peripheral blood stem cell transplants (PBSCT), including CD34-selected PBSCT in two. Five patients received bone marrow. RESULTS: While all patients had chimerism in PB and MNC of the BM, in all but one patient BM-derived MSC were of recipient origin. This single patient showed reproducibly MSC of donor origin in a frequency of 1% after having received a CD34-selected PBSCT. Looking at graft collections, MSCs were easily generated from BM specimens, while no MSC could be derived from PBSC samples. CONCLUSION: Even though HSC have been found to differentiate into a variety of nonhematological cell types, they usually do not differentiate into MSC after allogeneic transplantation.

Remission induction using alemtuzumab can permit chemotherapy-refractory chronic lymphocytic leukemia (CLL) patients to undergo allogeneic stem cell transplantation.

The outcome of allogeneic stem cell transplantation depends upon the disease status before transplantation. Patients with refractory disease are at high risk for relapse. To improve the curative potential of the transplant procedure, we treated 3 chemotherapy-refractory CLL patients with alemtuzumab before allogeneic stem cell transplantation. Prior to therapy, all patients suffered from B-symptoms, and had massive adenopathy, splenomegaly, thrombocytopenia, and anemia; two patients had hepatomegaly. Alemtuzumab greatly reduced tumor mass in blood and bone marrow, B-symptoms resolved, and organomegaly improved. Two patients became blood product independent. All patients proceeded to transplantation after conditioning with TBI 2 Gy (n=1) or Treosulfan (n=2) in combination with Fludarabine either from an HLA-matched sibling (n=2) or from an HLA-matched unrelated donor (n=1). All patients engrafted, and are alive and well. Two patients reached complete remission (CR); one patient attained stable partial remission (PR). These heavily pre-treated refractory patients gained substantial clinical benefit from alemtuzumab, and received successful allografts.

Progressive multifocal leukoencephalopathy with detection of JC virus in a patient with chronic lymphocytic leukemia parallel to onset of fludarabine therapy.

Along with the establishment of more intense chemotherapeutic regimens including fludarabine for the treatment of indolent lymphoproliferative diseases like chronic lymphocytic leukemia (CLL), an increasing amount of cases with progressive multifocal leukoencephalopathy (PML) due to JC virus have been observed. We report a patient with CLL who developed PML parallel to the onset of fludarabine therapy. Spinal fluid was tested positive for JC virus. Despite virostatic treatment with cidofovir, neurologic symptoms were progressive and the disease ultimately fatal. The present case suggests that immunosuppression caused by chronic lymphoproliferative malignancies alone may be a factor in the development of PML. Chemotherapy with fludarabine may act as an additional trigger. The question remains whether serologic screening for JC virus in patients with chronic lymphoproliferative disease undergoing intense chemotherapy might be valuable once sufficient antiviral treatment has been established.