DEPARTMENT OF LABORATORY MEDICINE Karolinska Institutet, Stockholm, Sweden DIAGNOSTIC AND CLINICAL ASPECTS OF INVASIVE FUNGAL DISEASE AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION Ola Blennow Stockholm 2014 Cover picture: Thoracic CT showing a dense infiltrate with halo sign (top), and hyphae in a lung biopsy (bottom). All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by Åtta.45 Tryckeri AB © Ola Blennow, 2014 ISBN 978-91-7549-690-0 Institutionen för laboratoriemedicin Diagnostic and Clinical Aspects of Invasive Fungal Disease after Allogeneic Hematopoietic Stem Cell Transplantation AKADEMISK AVHANDLING som för avläggande av medicine doktorsexamen vid Karolinska Institutet offentligen försvaras i föreläsningssal R64, Karolinska Universitetsjukhuset, Huddinge Fredagen den 17 oktober 2014, kl 09.00 av Ola Blennow MD Huvudhandledare: Fakultetsopponent: Docent Jonas Mattsson Professor Kieren Marr Karolinska Institutet Johns Hopkins University School of Medicine Institutionen för onkologi och patologi Department of Infectious Diseases Centrum för allogen stamcellstransplantation (CAST) Betygsnämnd: Docent Stig Lenhoff Bihandledare: Lunds Universitet Professor Per Ljungman Avdelningen för hematologi och Karolinska Institutet transfusionsmedicin Institutionen för medicin, Huddinge Enheten för hematologi Professor Christine Wennerås Göteborgs Universitet Avdelningen för infektionssjukdomar Professor Bertil Christensson Lunds Universitet Stockholm 2014 Avdelningen för infektionsmedicin ABSTRACT Invasive fungal disease (IFD) is a major complication after allogeneic hematopoietic stem cell transplantation (HSCT). Effective prophylaxis has reduced the incidence of invasive candidiasis, but invasive mold infections (IMIs)—especially invasive aspergillosis (IA)— continue to be an important cause of non-relapse mortality. However, there are very few data regarding IFD and IMI after HSCT in the Nordic countries. The aim of this thesis work was to investigate epidemiological, diagnostic, and clinical aspects of IFD in HSCT recipients at Karolinska University Hospital, Huddinge. In paper I, 99 patients who received reduced-intensity conditioning (RIC) were followed with weekly fungal PCR during the first 100 days after HSCT. Patients with a positive fungal PCR result were randomized to either treatment with liposomal amphotericin B or no treatment. We found that a single positive PCR test was not associated with IFD, irrespective of treatment. The cumulative incidence rate of proven or probable IA during the first year after transplantation was 9%, and significant risk factors in a multivariate model were grades II–IV acute-graft-versus host disease (aGVHD), cytomegalovirus- (CMV-) seronegative recipient with CMV-seropositive donor, and conditioning with alemtuzumab. In paper II, a possible influence of the intensity of the conditioning on pneumonia-related death was investigated. We found no significant differences in the cumulative incidence of pneumonia- related death between patients receiving myeloablative conditioning (MAC) and those receiving RIC: early death (< 100 days after HSCT) 2.8% vs. 2.1%, and overall death 8.2% vs. 10.5%. Etiology could be established in 40 of 60 patients (67%) who died from pneumonia, with proven or probable IMI in 19 patients (48% of patients with established etiology, 32% of all patients with pneumonia-related death). In the multivariate analyses, grades II–IV aGVHD, CMV infection, and treatment with mesenchymal stromal cells (MSCs) were factors associated with overall pneumonia-related death. In paper III, posaconazole tissue concentrations were examined in vivo. Tissue concentrations of posaconazole were analyzed in biopsies taken at autopsy of seven patients who received posaconazole prophylaxis, and they were compared with plasma concentrations in samples taken before death. Accumulation of posaconazole was found in heart, lung, kidney, and liver tissue, while concentrations in brain were approximately equal to the concentrations in plasma. The apparent tissue accumulation in vivo is in agreement with earlier in vitro findings and may explain the low incidence of breakthrough infections seen in prophylaxis trials despite low serum concentrations. In paper IV, incidence and risk factors for IMI were retrospectively investigated in 843 patients. The cumulative incidences of proven and probable IMI were 2.2% at day 100, 5.2% after 1 year, and 6.3% after 2 years. Factors significantly associated with a new IMI were older age (risk hazard 4.26 for 41–60 years of age and 9.0 for > 60 years of age, with 0–20 years as reference), grades II–IV aGVHD, treatment with MSCs, and transplantation with female donor to male recipient. In patients with grade II aGVHD, no IMIs were seen after onset of GVHD in 113 HSCTs performed in patients < 40 years of age, compared to 14 IMIs in 106 HSCTs (13.2%) in patients > 40 years of age (p < 0.001). Twelve of these 14 patients had signs of poor immune reconstitution before onset of IMI. In patients with grade II aGVHD, few patients who are < 40 years of age appear to need mold-active prophylaxis, whereas in patients > 40 years of age prophylaxis is indicated if there are signs of poor immune reconstitution. LIST OF SCIENTIFIC PAPERS I. Blennow O, Remberger M, Klingspor L, Omazic B, Fransson K, Ljungman P, Mattsson J, Ringdén O. Randomized PCR-based therapy and risk factors for invasive fungal infection following reduced-intensity conditioning and hematopoietic SCT. Bone Marrow Transplantation 2010 Dec;45 (12):1710-8. II. Forslow U, Blennow O, LeBlanc K, Ringden O, Gustafsson B, Mattsson J, Remberger M. Treatment with mesenchymal stromal cells is a risk factor for pneumonia-related death after allogeneic hematopoietic stem cell transplantation. European Journal of Haematology 2012 Sep;89 (3):220-7. III. Blennow O, Eliasson E, Pettersson T, Pohanka A, Szakos A, El-Serafi I, Hassan M, Ringdén O, Mattsson J. Posaconazole concentrations in human tissues after allogeneic stem cell transplantation. Antimicrobial Agents and Chemotherapy 2014 Aug;58 (8):4941-3. IV. Blennow O, Remberger S, Törlén J, Ringdén O, Ljungman P, Mattsson J. Incidence and risk factors for invasive mold infections after allogeneic stem cell transplantation. Submitted manuscript. RELATED PUBLICATIONS NOT INCLUDED IN THE THESIS i. Blennow O, Mattsson J, Remberger M. Pre-engraftment blood stream infection is a risk factor for acute GVHD grades II-IV. Bone Marrow Transplantation 2013 Nov;48(12):1583-4. ii. Uhlin M, Wikell H, Sundin M, Blennow O, Maeurer M, Ringden O, Winiarski J, Ljungman P, Remberger M, Mattsson J. 2014. Risk factors for Epstein-Barr virus- related post-transplant lymphoproliferative disease after allogeneic hematopoietic stem cell transplantation. Haematologica 2014 Feb;99(2):346-52. iii. Blennow O, Ljungman P, Sparrelid E, Mattsson J, Remberger M. 2014. Incidence, risk factors, and outcome of bloodstream infections during the pre- engraftment phase in 521 allogeneic hematopoietic stem cell transplantations. Transplant Infectious Diseases 2014 Feb;16(1):106-14. iv. Blennow O, Fjaertoft G, Winiarski J, Ljungman P, Mattsson J, Remberger M. 2014. Varicella-Zoster Reactivation after Allogeneic Stem Cell Transplantation without Routine Prophylaxis-The Incidence Remains High. Biology of Blood and Marrow Transplantantation 2014 Jun 7. CONTENTS 1 Introduction ..................................................................................................................... 1 1.1 History ................................................................................................................... 1 1.2 Rationale and indications ...................................................................................... 1 1.3 Procedures .............................................................................................................. 2 1.4 Immune reconstitution ........................................................................................... 3 1.5 GVHD and immune reconstitution ....................................................................... 5 1.6 Infections after HSCT ........................................................................................... 6 1.6.1 Pre-engraftment phase ............................................................................... 6 1.6.2 Early post engraftment phase (< 100 days) .............................................. 8 1.6.3 Mid post engraftment phase (< 1 year) ..................................................... 8 1.6.4 Late post-engraftment phase (> 1 year) .................................................... 9 2 Invasive fungal disease after HSCT .............................................................................. 11 2.1 Definitions of invasive fungal disease ................................................................ 11 2.2 Epidemiology ....................................................................................................... 11 2.2.1 Candida .................................................................................................... 11 2.2.2 Aspergillus ............................................................................................... 13 2.2.3 Other molds ............................................................................................. 14 2.3 Antifungal agents for systemic use ..................................................................... 14 2.3.1 Amphotericin B derivates