Alloanatigenic Reactions after Hematopoietic Cell Transplantation Induce Genomic Alterations in Epithelial Cells as Shown in Human Studies and in an In Vitro Model.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3213-3213
Author(s):  
Alexandros Spyridonidis ◽  
Philipp Faber ◽  
Loizos Petrikkos ◽  
Jurgen Finke
Keyword(s):  
Cell Transplantation ◽  
Hematopoietic Cell Transplantation ◽  
Protein Translation ◽  
Hematopoietic Cell ◽  
Hygromycin B ◽  
Hygromycin Resistance ◽  
Hacat Cells ◽  
Genomic Alterations ◽  
Allogeneic Hct

Abstract Previous studies from our group demonstrated frequent genomic alterations measured by microsatellite instability (MSI) in non-neoplastic epithelial tissues of patients who underwent allogeneic hematopoietic cell transplantation (HCT) (Blood2006;107:3389–3396). These genomic alterations were found only after allogeneic but not after autologous HCT, and therefore we hypothesized that an “allogeneic” effect is substantially involved in the mutation process. We extended our previous analyses by examining 210 bucall swabs obtained from 70 patients between day (d+) 26 and d+3514 after allogeneic HCT for the presence of MSI. MSI analysis was performed by PCR and denaturing capillary electrophoresis at three tetranucleotide (THO-1, SEE33, D14S120) and three mononucleotide microsatellite (ZP3, BAT26, SRY) loci. MSI was found in the buccal smears of 38% allografted patients (median time of occurence 322 days). In a prospective trial, in which patients were followed from time before transplantation until d+365, 5 out of 14 (35%) patients exhibited MSI after transplantation although all of them showed stable microsatellites before transplantation. We are currently pefroming statistical analyses in order to identify which clinical factors influence the presence of MSI and we will present the data in the meeting. To test the hypothesis that an “alloantigenic” effect is responsible for th induction of MSI, we developed a model system in which keratinocyte (HaCaT) cells were transfected with a palsmid vector which carries a G418 (neo) selectable marker and a microsatellite repeat (CA) that places the sequence for Hygromycin Resistance (HygR) out of frame for protein translation. In this reporter system, DNA slippage mutations can restore the HygR reading frame and become detectable by hygromycin treatment as hygromycin resistant (HygR+) colonies. Pools of stably transfected (neo+) HaCaT cells were treated with supernatant (SN) of major histocompatibility complex nonmatched mixed lymphocyte cultures (MLC) and assayed for HygR+ colonies 48h later. Cells transfected with a control, in-frame hygromycin B gene construct (p12) were used as positive controls. Using this system, we found that HaCaT cells aquire hygromycin resistance after treatment with supernatatant from MLC. Treatment of cells with hydrogen hyperoxid which has been shown in a E. Coli system to induce MSI (PNAS1998, 95:12468–12473) generated HygR+ colonies at a >80% lower frequency than the SN-MLC treatment. Control p12 transfected cells were grown with high efficiency in the presence of hygromycin B. In summary, our in vivo data confirm our previous results and provide evidence of genomic alterations after allogeneic HCT and our in vitro data are compatible with the hypothesis that an “alloantigenic” factor is the driving force in producing detectable MSI in the allografted patients. Elucidating the ultimate mechanisms underlying the genomic instability following allogeneic HCT may prove to be of major therapeutic value.

Alloantigeneic Reactions after Human Hematopoietic Cell Transplantation Induce Genomic Alterations in Epithelium: A Confirmation in Vivo and in Vitro Study and Implications to Secondary Neoplasia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 452-452
Author(s):  
Maria Themeli ◽  
Loizos Petrikkos ◽  
Miguel Waterhouse ◽  
Hartmut Bertz ◽  
Eleni Lagadinou ◽  
...  
Keyword(s):  
Cell Transplantation ◽  
Hematopoietic Cell Transplantation ◽  
Hematopoietic Cell ◽  
Secondary Malignancy ◽  
Hacat Cells ◽  
Genomic Alterations ◽  
Allogeneic Hct ◽  
Dna Strand

Abstract We previously demonstrated frequent genomic alterations measured by microsatellite instability (MSI) in non-neoplastic epithelial tissues of pts who underwent allogeneic Hematopoietic Cell Transplantation (HCT) but not after autologous transplantation (Blood2006;107:3389–3396). Confirmation in a larger and independent patient cohort and demonstration in an in vitro system was needed. In total 176 buccal samples obtained from 71 unselected patients who underwent allogeneic HCT were analyzed for MSI with standard PCR for microsatellite markers (THO-1, SEE33, D14S120 and D1S80) and analysis by capillary electrophoresis. MSI was observed in 37 (52%) allo-transplanted pts but never in the 31 controls (16 healthy and 15 pts after auto-HCT, 47 samples). MSI+ pts were significantly older than MSI-pts (median age 60y vs 48y, p<0.05). Other clinical features were not significantly different between MSI+ and MSI− pts: gender, myeloid vs lymphoid disease, sibling vs unrelated HCT, myeloablative vs reduced intensity conditioning, sex mismatched transplantation, bone marrow vs mobilized peripheral blood graft, T-cell depletion of the graft, use of methotrexate for GvHD prophylaxis, number of CD3+ cells/kg b.w. infused, occurrence and grade of mucositis during neutropenia, CMV reactivation, median ferritin levels post-transplant and occurrence of GvHD vs no GvHD. When GVHD was analyzed according to its severity we found that there was a trend for increasing risk of MSI for pts who experienced severe GvHD (RR=1.8) as compared to pts with no GvHD (RR=1). Secondary malignancy (5 skin-, 1 adeno-Ca) was diagnosed in 5 (14%) of the MSI+ pts and only in 1 (3%) MSI− pts. In an in vitro model of mutation analysis we tested the hypothesis that an alloantigenic stimulus is substantially involved in the mutation process. Briefly, keratinocyte DNA-repair proficient HaCaT cells were transfected with a reporter plasmid and genomic instability (GI) was detected and measured as hygromycin resistant clones. Treatment of HaCaT cells with H2O2 resulted in a time and dose dependent GI induction (1.9–5.7 fold). Cytokines abundant in alloreactions such as IFN-γ, TNF-α, TGF-β didn’t result in any GI in various concentrations and incubation times. Also, treatment with supernatants from different MHC non-matched Mixed Lymphocyte Cultures (MLC-SN) or with MLC separated from HaCaT cells with a semipermeable membrane didn’t induce any significant GI. In contrast, exposure of HaCaT cells to the whole MLC which includes activated cellular components (measured as BrdU+) resulted in significant induction of GI in 6 out of 8 different cases (mean 2.4-fold). GI was also evaluated in terms of DNA strand break formation by using the neutral comet assay. Treatment of HaCaT cells with whole MLC resulted in significant increase of DNA strand breaks as compared to HaCaT cells exposed to MLC-SN (p=0.039) or untreated controls (p=0.048). Western blot analysis revealed no significant alterations in the levels of MMR proteins upon treatment with MLC or MLC-SN. Exposure of HaCaT cells to MLC or MLC-SN resulted in increase of intracellular ROS levels as compared to untreated cells. The MLC treatment caused significantly higher increase of ROS levels than MLC-SN. The results of the present study confirm that GI of epithelial cells is a frequent phenomenon in patients after allogeneic HCT and give in vivo and in vitro support that alloantigeneic processes can be the cause of such a phenomenon. Progress in understanding DNA damage and repair after allogeneic HCT can potentially provide molecular biomarkers and therapeutic targets.

Frequent genomic alterations in epithelium measured by microsatellite instability following allogeneic hematopoietic cell transplantation in humans

Blood ◽  
2006 ◽  
Vol 107 (8) ◽  
pp. 3389-3396 ◽  
Author(s):  
Philipp Faber ◽  
Paul Fisch ◽  
Miguel Waterhouse ◽  
Annette Schmitt-Gräff ◽  
Hartmut Bertz ◽  
...  
Keyword(s):  
Microsatellite Instability ◽  
Cell Transplantation ◽  
Hematopoietic Cell Transplantation ◽  
Hematopoietic Cell ◽  
Allogeneic Hematopoietic Cell Transplantation ◽  
Genomic Alterations ◽  
Graft Versus Host ◽  
Allogeneic Hct ◽  
Colonic Crypts ◽  
Autologous Hct

Abstract Although typically found in cancers, frameshift mutations in microsatellites have also been detected in chronically inflamed tissues. Allogeneic hematopoietic cell transplantation (HCT) may potentially produce chronic tissue stress through graft-versus-host reactions. We examined non-neoplastic epithelial tissues (colon, buccal) obtained 1 to 5061 days after human allogeneic HCT for the presence of genomic alterations at 3 tetranucleotide and 3 mononucleotide microsatellite loci. Novel bands indicative of microsatellite instability (MSI) at tetranucleotide repeats were detected in laser-microdissected colonic crypts and in buccal smears of 75% and 42% of patients who received an allograft, respectively. In contrast, no MSI was found in similar tissues from control subjects and from patients after intensive chemotherapy or in buccal cells from patients after autologous HCT. The MSI found in colon, which was often affected by graft-versus-host disease, was not due to loss of expression or nitrosylation of DNA repair proteins. MSI in clinically intact oral mucosa was more frequently found at later time points after HCT. MSI was also found in 3 posttransplant squamous cell cancers examined. Our data show that genomic alterations in epithelium regularly occur after allogeneic HCT and may be implicated in the evolution of posttransplantation diseases, including secondary cancer.

Frequent Genomic Alterations in Epithelium Measured by Microsatellite Instability Following Allogeneic Hematopoietic Cell Transplantation in Humans.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1117-1117
Author(s):  
Alexandros Spyridonidis ◽  
Philipp Faber ◽  
Paul Fisch ◽  
Miguel Waterhouse ◽  
Annette Schmitt-Graeff ◽  
...  
Keyword(s):  
Microsatellite Instability ◽  
Cell Transplantation ◽  
Hematopoietic Cell Transplantation ◽  
Hematopoietic Cell ◽  
Genomic Alterations ◽  
Graft Versus Host ◽  
Post Transplant ◽  
Allogeneic Hct ◽  
History Of ◽  
Oral Gvhd

Abstract While typically found in cancers, frameshift mutations in microsatellites have also been detected in chronically inflamed tissues. Allogeneic hematopoietic cell transplantation (HCT) may potentially produce chronic tissue stress through graft-versus-host reactions. Therefore, we examined non-neoplastic epithelial tissues (colon, buccal) obtained 1 to 5061 days after human allogeneic HCT for the presence of genomic alterations at three tetranucleotide (SEE33, THO-1, D14S120) and three mononucleotide (ZP3, BAT26, SRY) microsatellite loci. All except two colon biopsies examined had histological signs of GvHD. No signs of mucositis or oral GvHD were present at the time of buccal sampling. Novel bands indicative of microsatellite instability (MSI) were detected in laser-capture microdissected (LCM) colonic crypts in 12 out of 16 patients (75%) and in buccal smears in 10 out of 24 (42%) allografted patients. In contrast to the allografted patients, no MSI was found in the LCM crypts obtained from 4 patients after intensive chemotherapy or from 7 control subjects with no history of either colon malignancy or chemotherapy, and in the buccal smears obtained from 8 patients after autologous HCT or from 9 healthy controls. MSI was found only at tetranucleotide markers but not at mononucleotides. There was no statistical correlation between the presence of MSI in colon and the underlying disease, the previous history of multiple (>=3) intensive chemotherapies before transplantation, the type of preparative regimen for HCT, the overall GVHD grade any time before sampling or the GvHD stage in colon at sampling. The MSI found in colon, which was often affected by graft-versus-host disease, was not due to loss of expression or nitrosylation of DNA repair proteins (MLH1, MSH2, MSH3, APE-1, XPA). There was no significant association between MSI in the buccal mucosa and patient age, gender, disease, type of conditioning, history of oral GvHD or overall GvHD occured at any point before buccal sampling. Interestingly, we found a significant association between the time of buccal sampling after transplantation and the presence of MSI (p=0.008). MSI was also found in three post-transplant squamous cell cancers examined. The frameshift alterations found in post-transplant tumors often differed from those in the associated non-malignant tissues. Our data show that genomic alterations in epithelium regularly occur after allogeneic HCT. This previously unacknowledged genomic instability after allogeneic HCT may be implicated in the evolution of post-transplant diseases, including secondary cancer.

Abstract 135: Endothelial Dysfunction in Acute Graft-versus-host Disease After Allogeneic Hematopoietic Cell Transplantation. In vitro Evidence of the Protective Effect of Defibrotide

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Enrique Mir ◽  
Marta Palomo ◽  
Enric Carreras ◽  
Maribel Diaz-Ricart ◽  
Montse Rovira ◽  
...  
Keyword(s):  
Endothelial Dysfunction ◽  
Protective Effect ◽  
Cell Transplantation ◽  
Graft Versus Host Disease ◽  
Hematopoietic Cell Transplantation ◽  
Endothelial Damage ◽  
Hematopoietic Cell ◽  
Graft Versus Host ◽  
Acute Graft

Acute Graft-Versus-Host Disease (aGVHD) is the most common early complication after allogeneic Hematopoietic Cell Transplantation (allo-HCT). We demonstrated endothelial dysfunction (ED) in association with allo-HCT. According to this data, aGVHD has been linked to an inflammatory process that may affect the endothelium. To investigate the differential degree of endothelial damage in patients developing or not aGVHD, to identify potential biomarkers, and to explore the protective effect of defibrotide (DF) in this scenario. DF has orphan designation for GVHD prevention. Patients blood samples were collected before allo-HCT, at day 0, and every week till day 28 after HCT. Plasma proteins (sTNFR1, sVCAM-1, VWF and ADAMTS-13) were measured as biomarkers of ED in individual samples from patients developing (GVHD, n=24), or not (NoGVHD, n=13), aGVHD. In in vitro assays, endothelial cells (EC) in culture were exposed to media containing pooled sera from patients to evaluate changes in the: a) expression of VCAM-1 and ICAM-1 on cell surfaces; b) presence of VWF on the extracellular matrix (ECM) and c) reactivity of the ECM towards platelets, under flow. The effect of DF was explored in the in vitro experiments by previous exposure of the EC (for 24h) followed by continuous incubation (100 μg/ml, added every 24h). Levels of sTNFRI, sVCAM-1 and VWF in samples from group GVHD were significantly higher than in NoGVHD (increases of 100, 37 and 150% respectively, at diagnose, p<0.01). ADAMTS-13 activity and VWF levels were inversely related. In in vitro studies, cell surface expression of VCAM-1 and ICAM-1, presence of VWF and platelet adhesion on the ECM in response to GVHD samples were always superior (increases vs NoGVHD of 80, 40, 100 and 21%, respectively, at diagnose). In vitro exposure of EC to DF attenuated signs of endothelial injury reducing significantly (p<0.05) the expression of VCAM-1, ICAM-1 and VWF (reductions of 22, 30 and 30%, respectively) in the GVHD condition. Our results demonstrate endothelial damage in association with aGVHD, as evidenced by elevated plasma levels of several biomarkers. The in vitro approach showed a marked proinflammatory and prothrombotic phenotype in association with aGVHD, which could be significantly prevented by defibrotide.

High dose valacyclovir for cytomegalovirus prophylaxis following allogeneic hematopoietic cell transplantation

2021 ◽  
pp. 107815522110604
Author(s):  
Kelly G Hawks ◽  
Amanda Fegley ◽  
Roy T Sabo ◽  
Catherine H Roberts ◽  
Amir A Toor
Keyword(s):  
Cell Transplantation ◽  
Hematopoietic Cell Transplantation ◽  
Hematopoietic Cell ◽  
Allogeneic Hematopoietic Cell Transplantation ◽  
High Dose ◽  
Cmv Infection ◽  
Allogeneic Hct ◽  
Clinically Significant ◽  
Cmv Disease ◽  
Cmv Prophylaxis

Introduction Cytomegalovirus (CMV) is one of the most common and clinically significant viral infections following allogeneic hematopoietic cell transplantation (HCT). Currently available options for CMV prophylaxis and treatment present challenges related to side effects and cost. Methods In this retrospective medical record review, the incidence of clinically significant CMV infection (CMV disease or reactivation requiring preemptive treatment) following allogeneic HCT was compared in patients receiving valacyclovir 1 g three times daily versus acyclovir 400 mg every 12 h for viral prophylaxis. Results Forty-five patients who received valacyclovir were matched based on propensity scoring to 35 patients who received acyclovir. All patients received reduced-intensity conditioning regimens containing anti-thymocyte globulin. Clinically significant CMV infection by day + 180 was lower in the valacyclovir group compared to the acyclovir group (18% vs. 57%, p = 0.0004). Patients receiving valacyclovir prophylaxis also had less severe infection evidenced by a reduction in CMV disease, lower peak CMV titers, delayed CMV reactivation, and less secondary neutropenia. Conclusion Prospective evaluation of valacyclovir 1 g three times daily for viral prophylaxis following allogeneic HCT is warranted. Due to valacyclovir's favorable toxicity profile and affordable cost, it has the potential to benefit patients on a broad scale as an option for CMV prophylaxis.

Hematopoietic Cell Transplantation for Benign Hematological Disorders and Solid Tumors

Hematology ◽  
2003 ◽  
Vol 2003 (1) ◽  
pp. 372-397 ◽  
Author(s):  
Rainer F. Storb ◽  
Guido Lucarelli ◽  
Peter A. McSweeney ◽  
Richard W. Childs
Keyword(s):  
Autoimmune Diseases ◽  
Aplastic Anemia ◽  
Solid Tumors ◽  
Cell Transplantation ◽  
Hematopoietic Cell Transplantation ◽  
Family Members ◽  
Hematopoietic Cell ◽  
Allogeneic Hct ◽  
The Status ◽  
Autologous Hct

Abstract Allogeneic hematopoietic cell transplantation (HCT) has been successfully used as replacement therapy for patients with aplastic anemia and hemoglobinopathies. Both autologous and allogeneic HCT following high-dose chemotherapy can correct manifestations of autoimmune diseases. The impressive allogeneic graft-versus-tumor effects seen in patients given HCT for hematological malignancies have stimulated trials of allogeneic immunotherapy in patients with otherwise refractory metastatic solid tumors. This session will update the status of HCT in the treatment of benign hematological diseases and solid tumors. In Section I, Dr. Rainer Storb reviews the development of nonmyeloablative conditioning for patients with severe aplastic anemia who have HLA-matched family members. He also describes the results in patients with aplastic anemia given HCT from unrelated donors after failure of responding to immunosuppressive therapy. The importance of leuko-poor and in vitro irradiated blood product transfusions for avoiding graft rejection will be discussed. In Section II, Dr. Guido Lucarelli reviews the status of marrow transplantation for thalassemia major and updates results obtained in children with class I and class II severity of thalassemia. He also describes results of new protocols for class III patients and efforts to extend HCT to thalassemic patients without HLA-matched family members. In Section III, Dr. Peter McSweeney reviews the current status of HCT for severe autoimmune diseases. He summarizes the results of autologous HCT for systemic sclerosis, multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus, and reviews the status of planned Phase III studies for autologous HCT for these diseases in North America and Europe. He also discusses a possible role of allogeneic HCT in the treatment of these diseases. In Section IV, Dr. Richard Childs discusses the development and application of nonmyeloablative HCT as allogeneic immunotherapy for treatment-refractory solid tumors. He reviews the results of pilot clinical trials demonstrating graft-versus-solid tumor effects in a variety of metastatic cancers and describes efforts to characterize the immune cell populations mediating these effects, as well as newer methods to target the donor immune system to the tumor.

scholarly journals Erythropoietin therapy after allogeneic hematopoietic cell transplantation: a prospective, randomized trial

Blood ◽  
2014 ◽  
Vol 124 (1) ◽  
pp. 33-41 ◽  
Author(s):  
Aurélie Jaspers ◽  
Frédéric Baron ◽  
Évelyne Willems ◽  
Laurence Seidel ◽  
Kaoutar Hafraoui ◽  
...  
Keyword(s):  
Randomized Trial ◽  
Cell Transplantation ◽  
Hematopoietic Cell Transplantation ◽  
Hematopoietic Cell ◽  
Allogeneic Hematopoietic Cell Transplantation ◽  
Prospective Randomized Trial ◽  
Allogeneic Hct ◽  
Transfusion Requirements ◽  
Key Points

Key Points Erythropoietin therapy can be effective to hasten erythroid recovery and reduce transfusion requirements after allogeneic HCT.

The Effect of In Vivo T-Cell Depletion with Alemtuzumab on Reduced-Intensity Allogeneic Hematopoietic Cell Transplantation for Chronic Lymphocytic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3014-3014
Author(s):  
Julio Delgado ◽  
Srinivas Pillai ◽  
Reuben Benjamin ◽  
Dolores Caballero ◽  
Rodrigo Martino ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Cell Transplantation ◽  
Hematopoietic Cell Transplantation ◽  
Chronic Gvhd ◽  
Lymphocytic Leukemia ◽  
Hematopoietic Cell ◽  
Allogeneic Hematopoietic Cell Transplantation ◽  
Mixed Chimerism ◽  
Allogeneic Hct ◽  
Reduced Intensity

Abstract Reduced-intensity allogeneic hematopoietic cell transplantation (HCT) is increasingly considered as a therapeutic option for young patients with advanced chronic lymphocytic leukemia (CLL). We report 59 consecutive CLL patients who underwent allogeneic HCT following fludarabine and melphalan conditioning at four different institutions. For graft-versus-host disease (GVHD) prophylaxis, 38 patients (Cohort 1) received alemtuzumab (20–100 mg) and cyclosporine; and 21 patients (Cohort 2) received cyclosporine plus methotrexate or mycophenolate. Donors were 47 HLA-matched siblings and 12 unrelated volunteers, 6 of whom were mismatched. Median age at transplant was 53 (range, 34–64) years and median number of previous chemotherapy regimens was 3 (1–6), with 39% of patients being refractory to fludarabine. Nine patients had previously failed an autologous HCT. Fluorescent in-situ hybridization and IgVH mutation status data were available in 33 (56%) and 31 (53%) patients, respectively, being unfavorable (17p- or 11q-) in 22 (67%) and unmutated in 24 (77%) of them. All but 1 patient engrafted, and the median interval to neutrophil recovery (> 0.5 × 109/l) was 14 (range, 10–36) days. Twenty patients (34%), mostly from Cohort 1, received escalated donor lymphocyte infusions due to mixed chimerism or disease relapse. The overall complete response rate among 53 patients with measurable disease at the time of transplantation was 70%, whereas 21% had stable disease. Grade II-IV acute GVHD was observed in 14 (37%) and 12 (57%) patients from Cohorts 1 and 2, respectively (P = 0.17). Extensive chronic GVHD was observed in 3 (8%) and 10 (48%) patients from Cohorts 1 and 2, respectively (P < 0.01). The incidence of cytomegalovirus reactivation was not significantly different between cohorts (67% vs 47%, P = 0.23). With a median follow-up of 36 (range, 3–99) months for survivors, 18 (30%) patients have died, 3 of progressive disease and 15 of transplant-related complications. The 3-year overall survival (OS), progression-free survival (PFS) and non-relapse mortality were 66% (95% CI 48–84%), 38% (20–56%) and 21% (8–34%), respectively, for Cohort 1 and 65% (44–86%), 54% (32–76%) and 29% (10–48%) for Cohort 2 (P = 0.66; P = 0.33; and P = 0.53). Despite low patient numbers, alemtuzumab seemed particularly effective for unrelated donor recipients, with a 3-year OS and PFS of 54% and 40% for Cohort 1; and 33% and 0% for Cohort 2 (P = 0.02 and P = 0.07). In conclusion, results with reduced-intensity allogeneic HCT are promising for these poor-prognosis patients. Furthermore, the alemtuzumab-based regimen was effective in reducing the chronic GVHD rate with no negative effect on NRM, PFS or OS.

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