Prevention of Cytomegalovirus Disease in Allogeneic Stem Cell Transplant: Are We Being Tricked to Treat?

Transcription

1 Prevention of Cytomegalovirus Disease in Allogeneic Stem Cell Transplant: Are We Being Tricked to Treat? Brittney Ramirez, PharmD PGY-1 Pharmacy Resident Methodist Hospital and Methodist Children s Hospital, San Antonio, TX Division of Pharmacotherapy, The University of Texas at Austin College of Pharmacy Pharmacotherapy Education and Research Center, UT Health San Antonio October 26 th, 2018 Objectives: 1. Understand the process of hematopoietic stem cell transplant and the risk of infections 2. Describe the epidemiology and classification of cytomegalovirus (CMV) 3. Review the methods for prevention of CMV 4. Formulate an evidence based recommendation for prevention of CMV disease 1 Ramirez

2 Hematopoietic Stem Cell Transplant (HSCT) I. Overview of HSCT a. HSCT involves the administration of autologous (recipient s own) or allogeneic (donor derived) stem cells into a recipient 1,2 i. Hematopoietic stem cells give rise to myeloid and lymphoid lineages of blood cells ii. HSCT is indicated for many oncologic and non-oncologic conditions b. U.S. Transplant Data from the Center for International Blood and Marrow Transplant Research state that approximately 21,766 transplants were performed in c. HSCT was first conceived as a rescue for the reconstitution of blood cells after the induction of life threatening irradiation 2 i. Adapting this theory of rescue, the first ever bone marrow transplant was performed in 1957 for acute leukemia patients after administration of supralethal chemotherapy 4 ii. Major complications developed including graft failure, graft versus host disease (GVHD), and/or death d. In 1958 the discovery of Human Leukocyte Antigens (HLA) occurred and numerous studies have helped elucidate the role of these antigens in HSCT 5 i. HLA class I (Region A,B,C) and class II antigens (Region DP, DQ, DR) can elicit an immune response by presentation to donor derived T-cells ii. HLA disparity between donor and recipient has been associated with graft failure, delayed immune reconstitution, GVHD, and mortality e. Transplant process overview HSCT Process Mobilization Hematopoietic Stem Cell Collection Conditioning Regimen Pre- Engraftment Engraftment Stem Cell Infusion Figure 1. Chart depicting HSCT stages Ramirez

3 i. Mobilization: involves the movement of stem cells from the bone marrow into the blood with the help of granulocyte-colony stimulating factors for allogeneic HSCT and plerixafor for autologous HSCT 6 ii. Hematopoietic stem cell collection: the process of collecting stem cells from the donor or recipient 7 a) Bone marrow harvesting: consists of aspirating bone marrow from the posterior iliac crest in a donor 7 b) Cord blood harvesting: following birth, an umbilical cord vein is collected, punctured and then drained into an anticoagulated sterile closed harvesting system 7 c) Peripheral blood stem cell collection: blood is run through an apheresis machine which separates stem cells from other blood parts which are then collected for storage for infusion into the recipient 7 iii. Conditioning regimen: process of eradication or suppression of the host s stem cells in order for donor stem cells to engraft adequately and eradicate underlying malignant disease (may include chemotherapy, monoclonal antibody therapy and radiation to the entire body) 8 iv. Pre-engraftment: period where patients are profoundly pancytopenic and are at highest risk for bleeding and infections 9 v. Engraftment phase: the process of transplanted stem cells traveling to the bone marrow of the recipient and beginning production of platelets, red blood cells and white blood cells 9 Table 1: Comparison of autologous versus allogeneic hematopoietic stem cell transplant 1 Autologous Graft Allogeneic Graft Stem cell source is self Moderate morbidity, low mortality No graft versus host disease Low infection risk Stem cell source is non-self (foreign) High morbidity, high mortality Improved outcomes in leukemia and diseases of the bone marrow Risk of GVHD Increased risk of infection due to prolonged immunosuppression II. Graft versus host disease a. A condition that may develop in allogeneic HSCT where the donated graft views the recipient s tissue as foreign and attacks the recipient s tissues 10 b. Patients may develop acute or chronic GVHD which can affect various systems of the body 10 c. About 35-50% of HSCT recipient will develop acute GVHD 10 d. Risk factors 11 i. Stem cell source ii. Donor and recipient mismatch iii. Age of the patient and donor iv. Conditioning regimen intensity v. GVHD prophylaxis e. Management 10 i. Classified based on grade and stage ii. Managed through immunosuppression 3 Ramirez

4 III. Infection Risk of HSCT iii. Degree of immunosuppression depends on the conditioning regimen and the level of HLA match iv. GVHD is typically managed with steroids, calcineurin inhibitors or mtor inhibitors 8,9, a. Depending on the intensity of the conditioning regimen, patients may experience a period of profound pancytopenia spanning days to weeks depending upon the donor source 8,9 Figure 2. Timing of Immune Reconstitution in HSCT 9 b. Neutrophil recovery varies but typically occurs: 9,12 i. Two weeks with G-CSF mobilized peripheral blood stem cell grafts ii. Two weeks with bone marrow grafts iii. Four weeks with umbilical cord blood grafts c. For several months patients experience a prolonged period of lymphocyte recovery with some patients not recovering fully for several years 9,12 i. Deficiency in lymphocytes is paramount to infection risk ii. Lymphocytes are vital for viral immunity d. T-cell recovery is prolonged with CD8+ cells recovering faster than CD4+ cells 13 i. CD4+ counts may provide the most readily available and predictive marker of the restoration of immune competence following HSCT ii. CD4+ recovery is associated with diminished infectious risk and improved transplant outcomes e. Risk Factors: 14,15,16 i. GVHD severity and degree of therapeutic immunosuppression ii. Age iii. Comorbidities iv. Pathogen exposure prior to transplant of recipient and donor v. Graft associated factors a) Peripheral blood stem cell grafts have a more rapid immune reconstitution b) Umbilical cord blood transplantation c) Transplantation of profoundly T cell-depleted grafts 4 Ramirez

5 Cytomegalovirus (CMV) d) Haploidentical grafts e) Stem cell dose is vital and levels of 3 X 10 6 or more are associated with an improved hematopoietic recovery Figure 3. Type of infection risk based on time post-transplant 9 Bacterial Viral Phase 1: Pre-engraftment Phase 2: Post-engraftment Phase 3: Late phase Neutropenia, barrier breakdown (mucositis, central venous access devices) Gram negative bacilli Gram positive organisms Gastrointestinal streptococci species Impaired cellular and humoral immunity; NK cells recover first, CD8 T cell numbers increasing, but restricted T cell repertoire Herpes Simplex Virus Cytomegalovirus Impaired cellular and humoral Immunity; B cell & CD4 T cell numbers recover slowly and repertoire diversifies Encapsulated bacteria Fungal Aspergillus Candida species Varicella/ Zoster virus Aspergillus Pneumocystis Day 0 Day Day 100 Day 365 and beyond I. Epidemiology 17,18 a. Transmission occurs via direct contact with infected secretions b. Infants may be infected in utero c. Cytomegaloviruses are among the most prevalent viral infections worldwide i % of the U.S. population is CMV seropositive ii. CMV seropositivity increases with age and correlates with socioeconomic level and race II. Microbiology 17 a. CMV is a icosapentahedral capsid member of the herpesviridae family b. Classification: Beta herpesvirus, it has a long replicative cycle and restricted host range c. Multiplication: transcription, genome replication and capsid assembly occur in the host cell nucleus III. Pathogenesis a. CMV carries mrnas in the virion particle into the host cell 17 5 Ramirez

6 b. Cytomegalovirus replicates mainly in the salivary glands and kidneys and is shed in saliva and urine 17 c. The cleaving of viral DNA and packaging of viral genes into preformed capsids is mediated by CMV-terminase complex (UL51, UL56, UL89) 18,19 d. UL97 is a viral kinase responsible for phosphorylation of natural viral and cellular protein substrates 20 e. UL54 is a DNA polymerase responsible for DNA replication 21 f. CMV grows in human epithelial, endothelial, smooth muscle, neurologic and fibroblast cells 22 g. CMV can establish latency in T-cells and macrophages 21 i. The latent viral genome may reactivate at any time, but typically occurs after immunosuppression, illness, or use of chemotherapeutic agents 22 ii. CMV is highly cell associated and spreads throughout the body within infected lymphocytes 21 IV. Clinical Manifestation 21 a. Cytomegalovirus clinical syndromes 21 i. Congenital CMV infection ii. CMV mononucleosis in healthy young adults iii. Life threatening disseminated disease in transplant recipients and human immunodeficiency virus (HIV) infected individuals b. Early CMV occurs 100 days post-transplant 9 c. Late CMV occurs >100 days post-transplant 9 Table 2. Type of CMV end-organ disease 22 CMV Pneumonia Signs/symptoms: fever, cough, hypoxemia and diffuse radiographic opacities Associated with high mortality CMV Gastrointestinal disease Can develop in the upper and lower GI tract Signs/symptoms of esophageal CMV: odynophagia, nausea, vomiting, weight loss, fever, diarrhea or abdominal pain Signs/symptoms of CMV colitis: abdominal pain, persistent small-volume diarrhea, and rectal bleeding CMV Hepatitis Signs/symptoms: acute right upper quadrant pain with cholestatic profile CMV CNS disease Signs/symptoms: diffuse encephalitis, ventriculoencephalitis, cerebral mass lesions CMV Retinitis Signs/symptoms: inflammation of the retina of the eye May lead to blindness V. Diagnostic Methods 23 a. Serology i. Detects CMV-specific antibodies (IgG and IgM) ii. Determines a patient s risk for CMV infection after transplantation iii. CANNOT be used for the diagnosis of CMV infection or disease b. Polymerase chain reaction (PCR) i. Most sensitive method for detecting CMV DNAemia ii. Relies on the amplification of quantitative measurement of CMV DNA iii. Maintains high specificity iv. Higher levels of DNA in blood are predictive of CMV disease in immunocompromised patients c. pp65 antigenemia 6 Ramirez

7 i. CMV pp65 antigenemia in peripheral blood leukocytes is a rapid and semi-quantitative method of diagnosing CMV infection ii. A positive pp65 assay is predictive for the development of invasive disease in transplant patients d. Histopathology is the gold standard for diagnosis of CMV disease e. Other testing options not routinely used: CMV RNA by nucleic acid, culture CMV Classifications CMV Viremia Detectable CMV DNA in blood CMV Infection Uncontrolled replication of CMV CMV Disease CMV organ invasion Figure 4. CMV classifications and definitions 22,23 VI. Diagnosis of CMV Disease 22 a. Negative DNA in blood does not rule out disease b. Positive DNA in blood does NOT equate to disease c. Histopathology of tissue biopsy is gold standard d. Risk Factors 24 i. Age ii. Sex match of donor/recipient iii. Type of conditioning regimen iv. Use of high dose corticosteroids v. GVHD severity vi. Use of alemtuzumab or antithymocyte globulin vii. Immune recovery after HSCT viii. Graft factors such as umbilical cord, haploidentical, T-cell depleted, mismatched or unrelated ix. Serostatus of the donor and recipient studies suggest is MOST IMPORTANT 24,25 Donor/Recipient Serostatus and Risk of CMV Recurrence D-/R- D+/R- D+/R+ D-/R+ 3.1% 12.9% 32.1% 35.8% Figure 5. Depicts the percentage risk of CMV recurrence based on donor/recipient serostatus VII. CMV Specific Cytotoxic T lymphocytes (CTLs) 24 7 Ramirez

8 a. Donor CMV seronegative: Does not have anti-cmv IgG there are NO CMV CTLs b. Donor CMV seropositive: Donor immune system has CMV in a latent phase after CMV primary infection. This donor would have CMV CTLs, but there is also the risk of CMV infection being transferred to a CMV seronegative recipient VIII. CMV Resistance a. Development of drug resistance requires prolonged exposure to the antiviral drug and persistent reactivation in the presence of drug, which will ultimately lead to the selection of resistant strains 30,31 b. CMV strains have amino acid deletions or substitutions of the UL97 or UL54 regions or point mutations in the DNA polymerase 30,32 c. UL97 mutations are resistant to ganciclovir, but susceptible to foscarnet and cidofovir 30,32 d. UL54 mutations can be resistant to ganciclovir, foscarnet and cidofovir 30,32 e. CMV strains with both mutations are highly resistant to ganciclovir 30,32 CMV Prevention Approaches I. Without any form of prevention, approximately 80% of CMV seropositive recipients are likely to reactivate CMV and 20%-35% will develop CMV disease after allogeneic HSCT 30 II. Primary Prophylaxis 30,31,33 a. Therapy used to prevent the development of CMV disease in a person who is at risk for, but has no prior history of the disease b. Usually started at engraftment and continued until day 100 after transplant c. Higher rates of late CMV disease have been seen with this strategy as recipients are unable to develop CMV-specific immune responses III. Preemptive therapy 30,31,33,34,35 a. Viral load is monitored at frequent intervals (usually weekly) with pp65 antigenemia or PCR detection to identify infection before the onset of symptoms or tissue invasion b. Supported by the observation that CMV can be detected 1-2 weeks prior to symptoms c. No well-defined threshold for the initiation of anti-cmv therapy d. Preemptive therapy does not aim to prevent viremia, but rather to prevent disease i. Green et al. conducted a single-center retrospective US study that showed any level of viremia is associated with an increased risk of mortality at one year after allogeneic transplant 34 ii. Teira et al. showed that early CMV reactivation is associated with increased transplant-related mortality 35 iii. Ljungman et al. state that some viral replication may be beneficial as it can stimulate immune responses and promote CMV-specific immune reconstitution leading to less CMV late disease (>3 months posttransplant) 30 8 Ramirez

9 Prevention Through the Years Acyclovir FDA Approved Ganciclovir FDA Approved Goodrich et al. showed ganciclovir prophylaxis significantly decreased disease. Failed to show a mortality benefit due to the large amount of neutropenia and bacterial associated infections Boeckh et al. pp65 antigenemia to guide initiation of ganciclovir, showed the noninferiority of ganciclovir prophylaxis vs preemptive therapy Valganciclovir FDA Approved Ayala et al. valganciclovir shown to be as efficacious as ganciclovir in the prevention of CMV Meyers et al. found that acyclovir significantly reduced the risk of both CMV infection and disease in seropositive patients Mond et al. applied the principals of preemptive therapy, showed that early treatment with ganciclovir in patients with positive surveillance cultures reduces the incidence of CMV disease and improves survival Valacyclovir FDA Approved Salzberger et al. reaffirmed the findings in previous ganciclovir prophylaxis study from Neutropenia determined an independent risk factor for mortality Vusirikala et al. valacyclovir prophylaxis showed no difference in CMV disease and survival compared to acyclovir 2017 Letermovir FDA Approved Figure 6. Depicts the milestones of CMV prevention through the years 36 9 Ramirez

10 a. Letermovir i. New agent approved in 2017 for prophylaxis of CMV infection and disease in adult CMV-seropositive recipients of an allogeneic HSCT ii. Novel mode of action that targets the UL56 subunit of the viral terminase complex specifically seen in CMV iii. In-vitro data has shown the development of CMV resistance to letermovir iv. Low to absent myelotoxic and nephrotoxic effects Clinical Question: Is preemptive therapy an appropriate approach to prevention with the introduction of a new, less myelosuppressive CMV prophylaxis option? Literature Review Boeckh M, Gooley TA, Myerson D, Cunningham T, Schoch G, Bowden RA. Cytomegalovirus pp65 antigenemia-guided early treatment with ganciclovir versus ganciclovir at engraftment after allogeneic marrow transplantation: a randomized double-blind study. Blood. 1996;88(10): Purpose This study compared CMV antigenemia-guided ganciclovir treatment to prophylaxis with ganciclovir by assessing CMV disease, neutropenia and bacterial, fungal, viral infections Study Design Patient Population Endpoints Randomized, double-blind, placebo controlled study conducted from stratified by presence of acute GVHD Randomized to either placebo or ganciclovir 5mg/kg BID for 5 days then daily for 6 days/week until day 100 after transplantation If high-grade CMV antigenemia or CMV viremia was detected the study drug was stopped and an open label ganciclovir treatment was started Inclusion CMV seropositive patients undergoing allogeneic marrow transplantation Primary Endpoint CMV disease and neutropenia Secondary Endpoints Monitored bacterial, fungal and viral infection rates Use of ganciclovir Hospitalization duration Exclusion Serum creatinine of 2.5mg/L CMV disease, viremia or antigenemia Received any antiviral drug the preceding 7 days Statistics Kaplan-meier method Log-rank test Cox proportional hazards models Results Baseline characteristics 226 patients randomized (antigenemia-ganciclovir n=114, ganciclovir n=112) No significant difference in age, sex, serostatus before transplant, underlying disease, disease stage, HLA donor matching, conditioning therapy, GVHD prophylaxis, GVHD grade 10 Ramirez

11 Authors Conclusion Critique Primary Endpoint Antigenemia-ganciclovir Ganciclovir P value (Preemptive) (Prophylaxis) CMV Disease Day 0-100, n (%) 16 (14.1) 3 (2.7) CMV Disease Day 0-400, n (%) 23 (20.2) 18 (16.1) 0.42 Incidence of neutropenia, n (%) 36 (32) 28 (25) 0.19 Endpoint Antigenemia-ganciclovir Ganciclovir P value (Preemptive) (Prophylaxis) Survival Day (87) 94 (84) 0.51 Survival Day (61) 66 (59) 0.80 Use of ganciclovir days < Hospitalization duration days Bacterial infections, n (%) 46 (40) 50 (45) 0.86 Fungal infections, n (%) 7 (6) 18 (16) 0.03 HSV infections, n (%) 38 (33) 14 (13) <0.001 Subset analysis showed GVHD grade 3-4 were significantly more likely to develop CMV disease More late CMV disease developed with 16% in the ganciclovir prophylaxis and only 8% in the preemptive ganciclovir group A trade-off exists for CMV-related morbidity versus antiviral drug toxicity More fatal fungal infections developed in the ganciclovir prophylaxis group Patients with higher grades of GVHD were more likely to progress to CMV disease Results showed no apparent difference in survival or CMV related mortality CMV antigenemia guided antiviral treatment prevents CMV disease Strengths Limitations Trial design Did not start antigenemia ganciclovir in Reported degree of GVHD patients with low grade antigenemia Reported anti-fungal prophylaxis Patients received acyclovir, famciclovir Reported detection with PCR based prophylaxis methods Single center Definitions for CMV disease Take away Preemptive therapy is the preferred method of CMV disease prevention as it showed similar survival rates when compared to primary prophylaxis Preemptive therapy should especially be considered in patients with lower CMV viral load and CMV disease risk Preemptive therapy reduces the amount of exposure of anti-cmv agents HSV= herpes simplex virus, ANC= absolute neutrophil count 11 Ramirez

12 Boeckh M, Nichols WG, Chemaly RF, et al. Valganciclovir for the prevention of complications of late cytomegalovirus infection after allogeneic hematopoietic cell transplantation: a randomized trial. Ann Intern Med. 2015;162(1):1-10. Purpose This study compared PCR guided valganciclovir therapy against valganciclovir prophylaxis Study Design Patient Population Endpoints by assessing: CMV disease, non-viral infectious events, toxicity, mortality Multicenter, double blind, placebo controlled, randomized clinical trial between , primary study period was until day 270 after HSCT Valganciclovir 900mg daily or matching placebo for 6 months Study drug was discontinued when CMV viral load was >1000 copies/ml or >5 times baseline value and preemptive treatment initiated Viral load was monitored weekly If ANC dropped < 1.0 X 10-9 /L study drug was held and growth factors were started Inclusion Allogeneic HSCT recipients 16 years who: If R+ and D-/D+, had one of the following: Previous CMV Infection GVHD that required immunosuppression Receipt of anti-cmv prophylaxis therapy If R-/D+, one of the following: CMV infection with appropriate treatment course pre-randomization Antiviral utilization prior to study entry permitted Exclusion Neutropenia within one week of study enrollment Renal insufficiency or inability to take oral medications CMV disease within 6 weeks prior to randomization Uncontrolled CMV load at time of evaluation Prophylactic use of high dose acyclovir or valacyclovir Imminent demise (<2 weeks) HIV infection Primary Endpoint Composite outcome consisting of CMV disease or invasive bacterial or fungal infection or death and assessment of neutropenic consequences Secondary Endpoint Primary endpoint at day 640 Neutropenia CMV lymphoproliferative response Days alive outside of hospital Treatment-emergent ganciclovir resistance Immunosuppression discontinuation at day 270 Statistics To demonstrate a 45% reduction of the primary endpoint, 92 patients per treatment arm were needed for 87% power Cox regression models Chi-square, Fisher s exact or Wilcoxan rank-sum tests as appropriate Interim analysis conducted after 50% of patients completed 90 days of study Results Baseline Characteristics N=184 (valganciclovir n=95, placebo n=89) 12 Ramirez

13 Authors Conclusion Critique Outcomes Valganciclovir, n (%) Placebo, n (%) P value Primary endpoint (composite)* 18 (20) 18 (21) 0.86 Secondary Endpoints CMV Disease* 2 (2) 2 (2) 0.88 CMV Infection* 10 (11) 31 (36) <0.001 Mortality* 6 (2) 2 (2) 0.89 Invasive bacterial and fungal 17 (23) 15 (28) 0.94 infections* Primary endpoint (composite) 32 (35) 34 (40) 0.45 day 640 HSV-VZV infection day (9) 3 (4) 0.22 ANC <1000mm 3 34 (43) 19 (29) ANC <500mm 3 5 (7) 6 (10) 0.57 ANC <200mm 3 2 (2) 3 (4) 0.62 Immunosuppression discontinuation* 45 (47) 43 (49) 0.76 No significant difference seen in gender, age, type of transplant, donor relationship, disease status, conditioning intensity, CMV donor and recipient serostatus, HSV and VZV status, CD34 selections, refractory GVHD prerandomization, neutropenia pre-randomization *Day 270 Valganciclovir prophylaxis did not improve the CMV disease-free and invasive infection free survival composite endpoint when compared to PCR-guided preemptive therapy Both strategies performed similarly with regard to most clinical outcomes Strengths Multi-center Primary endpoint included invasive bacterial and fungal infections No use of acyclovir Measured CMV lymphoproliferative response Limitations Some high risk patients not included Did not report use of other prophylactic antimicrobials Take Away No difference in CMV disease or mortality Preemptive therapy is a highly effective CMV prevention method HSV= herpes simplex virus, VZV= varicella zoster virus, ANC= absolute neutrophil count 13 Ramirez

14 Marty FM, Ljungman P, Chemaly RF, et al. Letermovir Prophylaxis for Cytomegalovirus in Hematopoietic-Cell Transplantation. N Engl J Med. 2017; Purpose Compared primary prophylaxis using the new anti-cmv agent, letermovir, placebo by assessing CMV infection, CMV disease, mortality Study June March 2016 phase 3, double-blind trial 2:1 (letermovir: placebo). Stratified by Design trial site and CMV disease risk Letermovir 480mg daily or placebo Patients were stratified into either high risk or low risk groups High Risk for CMV reactivation defined as: o Significant HLA mismatching o Haploidentical donor or umbilical cord graft o Ex-vivo T-cell depleted grafts o Moderate to severe GVHD If a certain threshold (PCR viral load of 1000 copies/ml for low risk or 500 copies/ml for high risk) was met, patients in either group were started on preemptive therapy Patient Population Endpoints with ganciclovir Inclusion Age 18 years undergoing allogeneic HSCT and were CMV seropositive Undetectable CMV before randomization Must enroll in trial by day 28 posttransplant Exclusion Severe hepatic impairment CrCl < 10mL/min Current or recent receipt of antiviral agents with anti-cmv activity Primary Endpoint Proportion of patients with clinically significant CMV infection through week 24 after transplantation o Patients who for any reason discontinued the trial before week 24 or who had missing data at week 24 were defined as having a primary-endpoint Secondary Endpoints Proportion of patients with clinically significant CMV infection through week 14 Time to clinically significant CMV infection Safety endpoints Cumulative all-cause mortality GVHD Rates of infection Statistics Mantel-Haenszel method Kaplan-Meier plots Results Baseline Characteristics Patients were well balanced at baseline 540 patients (letermovir n=325, placebo n=170) Baseline characteristics included: age, gender, race, CMV seropositive donor, reason for HSCT, HLA matching and donor type, haploidentical related donor, stem cell source, myeloablative conditioning regimen, antithymocyte globulin use, alemtuzumab use, ex vivo T-cell depletion, immunosuppressant use, acute GVHD of grade 2 at randomization and risk of CMV disease (high or low risk) 14 Ramirez

15 Authors Conclusion Critique Endpoint Letermovir, n (%) Placebo, n (%) P value Primary endpoint* 122 (37.5) 103 (60.6) <0.001 CMV disease* 5 (1.5) 3 (1.8) Preemptive therapy initiation 119 (36.6) 101 (59.4) < CMV infection week (7.7) 67 (39.4) <0.001 CMV disease week 14 1 (0.3) 2 (1.2) Mortality* 33 (10.2) 23 (15.9) 0.03 Mortality week (20.9) 43 (25.5) 0.12 Bacterial and fungal 78 (24) 37 (21.8) infections Absolute neutrophil count <500mm3 71 (19) 37(19) Serum Creatinine >2.5mg/dL 8 (2) 6 (3) *at week 24 Letermovir has a limited adverse effect profile it has not been associated with hematologic or renal toxicity One patient developed CMV resistance to letermovir with a UL56 mutation Letermovir prophylaxis resulted in lower risk of CMV infection Letermovir has an overall safety profile similar to placebo Strengths Limitations Stratified patients based on risk for CMV Primary endpoint was CMV infection infection rather than CMV disease Randomized controlled trial All patients received prophylaxis Developed thresholds for initiation of with acyclovir, valacyclovir or preemptive therapy in each risk category famciclovir with could provide anti- CMV activity Take Away A survival benefit was seen at week 24, but not seen at week 48 There was no difference seen in CMV disease incidence Letermovir is well-tolerated Studies comparing letermovir against preemptive ganciclovir with CMV disease as the primary endpoint are needed Table 3. Cost analysis of CMV therapies 44,49 Agent Dose Regimen Letermovir prophylaxis* $324 ~$28,000 Valganciclovir prophylaxis* $68 ~$5,800 Valganciclovir preemptive $68 ~$4,800 *assuming engraftment at day 14 average duration 35 days 15 Ramirez

16 Summary Parameters Preemptive Therapy Letermovir Prophylaxis Exposure to anti-cmv agents Drug toxicities + + Cost Antiviral resistance Viremia ++ + Development of CMV specific + - immunity Late CMV disease + ++ Conclusion I. No difference in mortality or CMV disease in preemptive and primary prophylaxis approaches II. Patients are at higher risk of bacterial and fungal infections with primary prophylaxis with ganciclovir III. Higher rates of CMV resistance are seen with prolonged exposure of anti-cmv agents IV. Letermovir for CMV a. Drastically more expensive than valganciclovir for primary prophylaxis b. A decrease in CMV infection existed, however, no benefit in CMV disease demonstrated c. Significant decrease in mortality at week 24 but none seen at week 48 Recommendations for Prevention of CMV High Risk Criteria Stem cells from a CMV seronegative donor Mismatched donor/recipient Haploidentical donor Umbilical cord graft T-cell depleted graft GVHD of grade 2 or greater requiring steroids Yes Does patient meet any high risk criteria? Assess Patients Risk for CMV Disease Recipient CMV seropositive? No Preemptive therapy No Yes Preemptive therapy Can consider primary prophylaxis with letermovir and continued viral monitoring 16 Ramirez

17 References: 1. Cutler C, Antin JH. An overview of hematopoietic stem cell transplantation. Clin Chest Med. 2005;26(4): Juric MK, Ghimire S, Ogonek J, et al. Milestones of hematopoietic stem cell transplantation - from first human studies to current developments. Front Immunol. 2016;7: Center for International Blood and Marrow Transplant, a contractor for the C.W. Bill Young Cell Transplantation Program operated through the U. S. Department of Health and Human Services, Health Resources and Services Administration, Healthcare Systems Bureau. U.S. Transplant Data by Center Report, CLL - Chronic lymphocytic leukemia, Number of Transplants Reported in U.S. Transplant Data by Center Report. Last Updated: May 17, Thomas ED, Lochte HL, Lu WC, et al. Intravenous infusion of bone marrow in patients receiving radiation and chemotherapy. N Engl J Med. 1957;257(11): Van Rood JJ, Eernisse JG, Van Leeuwen A. Leucocyte antibodies in sera from pregnant women. Nature. 1958;181(4625): Bensinger W, Dipersio JF, Mccarty JM. Improving stem cell mobilization strategies: future directions. Bone Marrow Transplant. 2009;43(3): Hequet O. Hematopoietic stem and progenitor cell harvesting: technical advances and clinical utility. J Blood Med. 2015;6: Bacigalupo A, Ballen K, Rizzo D, et al. Defining the intensity of conditioning regimens: working definitions. Biol Blood Marrow Transplant. 2009;15(12): Tomblyn M, Chiller T, Einsele H, et al. Guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective. Biol Blood Marrow Transplant. 2009;15(10): Jacobsohn DA, Vogelsang GB. Acute graft versus host disease. Orphanet J Rare Dis. 2007;2: Przepiorka D, Smith TL, Folloder J, et al. Risk factors for acute graft-versus-host disease after allogeneic blood stem cell transplantation. Blood. 1999;94(4): Storek J. Immunological reconstitution after hematopoietic cell transplantation - its relation to the contents of the graft. Expert Opin Biol Ther. 2008;8(5): Mackall CL, Fleisher TA, Brown MR, et al. Distinctions between CD8+ and CD4+ T-cell regenerative pathways result in prolonged T-cell subset imbalance after intensive chemotherapy. Blood. 1997;89(10): Hakim FT, Memon SA, Cepeda R, et al. Age-dependent incidence, time course, and consequences of thymic renewal in adults. J Clin Invest. 2005;115(4): Komanduri KV, St john LS, De lima M, et al. Delayed immune reconstitution after cord blood transplantation is characterized by impaired thymopoiesis and late memory T-cell skewing. Blood. 2007;110(13): Bittencourt H, Rocha V, Chevret S, et al. Association of CD34 cell dose with hematopoietic recovery, infections, and other outcomes after HLA-identical sibling bone marrow transplantation. Blood. 2002;99(8): Whitley RJ. Medical Microbiology. 4th edition. Galveston, TX: Univeristy of Texas Medical Branch at Galveston; Accessed October 10, Bogner E. Human cytomegalovirus terminase as a target for antiviral chemotherapy. Rev Med Virol. 2002;12(2): Borst EM, Kleine-Albers J, Gabaev I, et al. The human cytomegalovirus UL51 protein is essential for viral genome cleavage-packaging and interacts with the terminase subunits pul56 and pul89. J Virol. 2013;87(3): Prichard MN. Function of human cytomegalovirus UL97 kinase in viral infection and its inhibition by maribavir. Rev Med Virol. 2009;19(4): Ramirez

18 21. Varani S, Landini MP. Cytomegalovirus-induced immunopathology and its clinical consequences. Herpesviridae. 2011;2(1): Ljungman P, Griffiths P, Paya C. Definitions of cytomegalovirus infection and disease in transplant recipients. Clin Infect Dis. 2002;34(8): Ross SA, Novak Z, Pati S, et al. Overview of the diagnosis of cytomegalovirus infection. Infect Disord Drug Targets. 2011;11(5): Styczynski J. Who is the patient at risk of CMV recurrence: A review of the current scientific evidence with a focus on hematopoietic cell transplantation. Infect Dis Ther. 2018;7(1): Schmidt-Hieber M, Labopin M, Beelen D, et al. CMV serostatus still has an important prognostic impact in de novo acute leukemia patients after allogeneic stem cell transplantation: a report from the acute leukemia working party of EBMT. Blood. 2013;122(19): Teira P, Battiwalla M, Ramanathan M, et al. Early cytomegalovirus reactivation remains associated with increased transplant-related mortality in the current era: a CIBMTR analysis. Blood. 2016;127(20): Marty FM, Bryar J, Browne SK, et al. Sirolimus-based graft-versus-host disease prophylaxis protects against cytomegalovirus reactivation after allogeneic hematopoietic stem cell transplantation: a cohort analysis. Blood. 2007;110(2): Nichols WG, Corey L, Gooley T, et al. Rising pp65 antigenemia during preemptive anticytomegalovirus therapy after allogeneic hematopoietic stem cell transplantation: risk factors, correlation with DNA load, and outcomes. Blood. 2001;97(4): Zhou W, Longmate J, Lacey SF, et al. Impact of donor CMV status on viral infection and reconstitution of multifunction CMV-specific T cells in CMV-positive transplant recipients. Blood. 2009;113(25): Ljungman P, Hakki M, Boeckh M. Cytomegalovirus in hematopoietic stem cell transplant recipients. Hematol Oncol Clin North Am. 2011;25(1): Boeckh M, Ljungman P. How we treat cytomegalovirus in hematopoietic cell transplant recipients. Blood. 2009;113(23); Fillet AM, Auray L, Alain S, et al. Natural polymorphism of cytomegalovirus DNA polymerase lies in two nonconserved regions located between domains delta-c and II and between domains III and I. Antimicrob Agents Chemother. 2004;48(5): Boeckh M. Current antiviral strategies for controlling cytomegalovirus in hematopoietic stem cell transplant recipients: prevention and therapy. Transpl Infect Dis. 1999;1(3): Green ML, Leisenring W, Xie H, et al. Cytomegalovirus viral load and mortality after haemopoietic stem cell transplantation in the era of pre-emptive therapy: a retrospective cohort study. Lancet Haematol. 2016;3(3):e Teira P, Battiwalla M, Ramanathan M, et al. Early cytomegalovirus reactivation remains associated with increased transplant-related mortality in the current era: a CIBMTR analysis. Blood. 2016;127(20): Acyclovir. Lexi-Drugs. Lexicomp. Wolters Kluwer Heatlh, Inc. Riverwoods, IL. Available at: Accessed October 10, Meyers JD, Reed EC, Shepp DH, et al. Acyclovir for prevention of cytomegalovirus infection and disease after allogeneic marrow transplantation. N Engl J Med. 1988;318(2): Ganciclovir. Lexi-Drugs. Lexicomp. Wolters Kluwer Heatlh, Inc. Riverwoods, IL. Available at: Accessed October 10, Mond C, Cays M, Ebeling DF, et al. Early treatment with ganciclovir to prevent cytomegalovirus disease after allogeneic bone marrow transplantation. N Engl J Med. 1991;325(23): Goodrich JM, Bowden RA, Fisher L, et al. Ganciclovir prophylaxis to prevent cytomegalovirus disease after allogeneic marrow transplant. Ann Intern Med. 1993;118(3): Valacyclovir. Lexi-Drugs. Lexicomp. Wolters Kluwer Heatlh, Inc. Riverwoods, IL. Available at: Accessed October 10, Ramirez

19 42. Boeckh M, Gooley TA, Myerson D, et al. Cytomegalovirus pp65 antigenemia-guided early treatment with ganciclovir versus ganciclovir at engraftment after allogeneic marrow transplantation: a randomized double-blind study. Blood. 1996;88(10): r 43. Salzberger B, Bowden RA, Hackman RC, et al. Neutropenia in allogeneic marrow transplant recipients receiving ganciclovir for prevention of cytomegalovirus disease: risk factors and outcome. Blood. 1997;90(6): Valgaciclovir. Lexi-Drugs. Lexicomp. Wolters Kluwer Heatlh, Inc. Riverwoods, IL. Available at: Accessed October 10, Vusirikala M, Wolff SN, Stein RS, et al. Valacyclovir for the prevention of cytomegalovirus infection after allogeneic stem cell transplantation: a single institution retrospective cohort analysis. Bone Marrow Transplant. 2001;28(3): Ayala E, Greene J, Sandin R, et al. Valganciclovir is safe and effective as pre-emptive therapy for CMV infection in allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant. 2006;37(9): Marty FM, Ljungman P, Chemaly RF, et al. Letermovir prophylaxis for cytomegalovirus in hematopoieticcell transplantation. N Engl J Med. 2017;377(25): Chou S, Satterwhite LE, Ercolani RJ. New locus of drug resistance in the human cytomegalovirus UL56 gene revealed by exposure to letermovir and ganciclovir. Antimicrob Agents Chemother. 2018;62(9):AAC Letermovir. Lexi-Drugs. Lexicomp. Wolters Kluwer Heatlh, Inc. Riverwoods, IL. Available at: Accessed October 10, Boeckh M, Nichols WG, Chemaly RF, et al. Valganciclovir for the prevention of complications of late cytomegalovirus infection after allogeneic hematopoietic cell transplantation: a randomized trial. Ann Intern Med. 2015;162(1): Foscarnet. Lexi-Drugs. Lexicomp. Wolters Kluwer Heatlh, Inc. Riverwoods, IL. Available at: Accessed October 10, Mcintosh M, Hauschild B, Miller V. Human cytomegalovirus and transplantation: drug development and regulatory issues. J Virus Erad. 2016;2(3): *Black box warning 19 Ramirez

20 Appendix A Anti-CMV Options Drug Mechanism of Action Dosing Main Side Effects Acyclovir/Valacyclovir Not completely understood, thought to be phosphorylated by viral UL97 Prophylaxis dosing: IV acyclovir 500mg/m 2 TID then Oral: 800mg QID Valacyclovir 2gm PO TID- QID Begin at engraftment and continue to day 100 Malaise Renal toxicity (low risk) Ganciclovir/ Valganciclovir Foscarnet Phosphorylated by the UL97 viral kinase to ganciclovir monophosphate. Inhibits viral replication by competing with deoxyguanosine triphosphate as a substrate for viral polymerase UL54 Valganciclovir is the valine ester of ganciclovir. Hydrolyzed to ganciclovir after oral absorption Does not require phosphorylation and is virustatic through inhibition of viral DNA polymerase Ganciclovir preemptive dosing: 5mg/kg IV BID for 14 days with maintenance of 5mg/kg daily for a further 7-14 days Valganciclovir preemptive dosing: 900mg PO BID continue for a minimum of 2 weeks Ganciclovir prophylaxis: 5mg/kg/day from engraftment to day 100 post-transplant Valganciclovir prophylaxis: 900mg PO daily from engraftment to day 100 posttransplant Preemptive dosing: 60mg/kg BID for 7-14 days then 90mg/kg daily. Minimum total duration is 2 weeks Prophylaxis dosing: 60mg/kg BID for seven days followed by mg/kg daily until day 100 after transplant Pancytopenia Myelosuppression Increased rate of infection Electrolyte abnormalities* Nephrotoxicity* Seizures* Nausea/vomiting Cidofovir Nucleotide analogue that does not require phosphorylation by viral UL97 kinase Preemptive dosing: 5mg/kg per week for a medium duration of 3 weeks Nephrotoxicity* Neutropenia Carcinogenic* Teratogenic* Nausea/vomiting Marabavir Directly inhibits protein kinase enzyme UL97 Currently in phase 3 trials Taste disturbances Nausea/vomiting Brincidofovir Prodrug of cidofovir, designed to release cidofovir intracellularly allowing for higher intracellular and lower plasma concentrations of cidofovir Not on the market Diarrhea Gastrointestinal events Liver enzyme abnormalities Letermovir Targets the DNA terminase complex UL56 which is required for viral DNA processing and packaging Prophylaxis dosing: IV or PO 480mg once daily beginning between day 0-28 post HSCT for CMV seropositive recipients, continue through day 100 posttransplantation Dose adjustment with concomitant cyclosporine therapy 240mg daily Thrombocytopenia Peripheral edema Headache Nausea/vomiting 20 Ramirez