In Vivo Emergence of UL56 C325Y Cytomegalovirus Resistance to Letermovir in a Patient with Acute Myeloid Leukemia after Hematopoietic Cell Transplantation
Jochen J Frietsch1*, Detlef Michel2*, Thomas Stamminger2, Friederike Hunstig1, Sebastian Birndt1, Ulf Schnetzke1, Sebastian Scholl1, Andreas Hochhaus1 and Inken Hilgendorf1.
* Both authors contributed equally.
Received: September 21, 2018
Accepted: November 14, 2018
Mediterr J Hematol Infect Dis 2019, 11(1): e2019001 DOI 10.4084/MJHID.2019.001
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associated tissue-invasive disease is associated with a considerable
risk of morbidity and mortality after allogeneic hematopoietic stem
cell transplantation (HSCT). Recently, the terminase inhibitor
letermovir (LMV) has been approved for prophylaxis of CMV infection in
HSCT. We hereby report a 60-year-old female experiencing CMV
reactivation after HSCT in a CMV seronegative donor-constellation. Due
to ongoing elevated CMV viral load and drug-associated
myelosuppression, which prevented ganciclovir therapy, treatment was
replaced by foscarnet. Due to nephrotoxicity, foscarnet was switched to
LMV. The patient developed skin GvHD and prednisolone was started.
Subsequently, CMV viremia worsened despite LMV therapy. Genotyping
revealed the mutation C325Y of the CMV UL56 terminase being associated
with high-level resistance against LMV. Prolonged uncontrolled
low-level viremia due to prednisolone treatment may have favored the
selection of drug-resistant CMV. Despite the excellent toxicity profile
of LMV, physicians should be aware of risk factors for the emergence of
Loads of CMV were routinely monitored once a week by PCR technique using whole-blood (for detailed information about viral copy numbers see figure 1). CMV reactivation with 7 x 104 copies/ml was detected on day +43. Despite a change in antiviral medication to GCV at a dosage of 5 mg/kg bwt, bid, viral load kept on increasing up to 4.8 x 105 copies/ml after ten days of initiation of treatment. The viral load finally decreased underuse of FOS at a dosage of 90 mg/kg bwt bid and early but slow reduction of immunosuppressive therapy. Additionally, we administered 1 ml/kg bwt CMV Immunoglobulins (Cytotect® CP Biotest) on day +48. The CMV-treatment schedule is given in figure 1.
Due to delayed engraftment, several bone marrow aspirates were obtained, revealing increasing chimerism from 89% on day +30, 94% on day +54 up to 100% since day +82. As a result of minimal residual disease (MRD) of AML, we quickly reduced CsA. This resulted in decreasing leukemia associated molecular markers. However, CMV copy numbers raised from 1.5-3.5 x 103 copies/ml up to 3.9 x 105 copies/ml (please refer to figure 1, day 88 et seq.) and EBV reactivation (up to 0.83-1.65 x 105) despite sustained administration of FOS. Except for nephrotoxicity, no clinical side effects of FOS occurred. EBV reactivation was effectively treated with the monoclonal CD20 antibody rituximab throughout four weeks, at a dosage of 375 mg/m² per week (Figure 1).
At the same time, CMV copy number increased despite the continuation of treatment with FOS. Therefore, we attempted to exclude the existence of viral mutations by DNA sequencing following nested PCR amplification. With modifications, amplification and sequencing of UL56 were performed as described previously. The method allows identification of the UL56 coding region from amino acids 1 to 620. Albeit clinically expected, verifying the viral kinase UL97 and the viral polymerase UL54 as wild-type, no mutation conveying resistance was demonstrable. A retrospective analysis revealed no mutation in the viral terminase region UL56, too. As a consequence, administration of CsA was terminated at day +118.
Based on delayed engraftment, drug-associated myelotoxicity and nephrotoxicity, and prolonged hospitalization, we initiated LMV at a dose of 480 mg qd. The patient was discharged from stationary treatment, and LMV resulted in an increase first, and within a treatment period of five weeks in an impressive decrease of CMV copy numbers (from 90.000 at LMV initiation up to 1.000.000 to 8.200 per milliliter) as already reported for other cases. Due to the occurrence of herpes stomatitis, acyclovir was administered, adapted to renal function. Simultaneously, the patient developed a maculopapular rash on day +155 affecting the lower arms and the abdominal skin. Subsequently, a skin biopsy was performed, and suspected acute GvHD confirmed by histology.
Consequently, prednisolone 25 mg qd was started at day +165 in the absence of any signs of gastrointestinal or liver involvement of GvHD and tapered without recurrence of acute GvHD afterward. However, under the intensified immunosuppression the viral load increased up to 410.000 copies per milliliter despite the continuation of LMV treatment. Upon genotyping, mutation C325Y (cytosine at amino acid position 325 was substituted by tyrosine) was detected within UL56 which is supposed to confer high-level resistance to LMV.
Consequently, administration of LMV was stopped, and FOS application, adapted to renal function, 3.000 mg bid commenced once again in combination with administration of CMV-hyperimmune globulin. This change in antiviral treatment resulted in a decrease of viral loads. On day +292, the patient is alive in complete remission of AML without signs of GvHD or clinical signs of active CMV infection, still receiving FOS without any side effects. Of note, during the whole course of treatment, CMV was below 1 x 103 copies/ml only until day +42, between day +63 and +67 as well as day +75 and +84 after HSCT.
Discussion and Conclusion
Side effects like nephrotoxicity, electrolyte disturbances, and myelotoxicity sometimes restrict the treatment with distinct antiviral drugs. Concerning its safety profile, the newly approved drug LMV appears to be superior to other anti-cytomegaloviral substances. However, since LMV specifically interferes with cleavage and packaging of viral DNA, without affecting viral DNA replication, this may result in prolonged detection of CMV DNA after the initiation of LMV therapy. Furthermore, the case presented in this study underlines that LMV is highly specific for CMV without an inhibitory effect on related herpesviruses such as HSV or VZV. Consequently, concomitant prophylaxis with acyclovir is compulsory in order to prevent disease due to HSV/VZV reactivation as observed by the HSV-associated stomatitis in our patient.
Experimental in vitro data suggested an early selection of cytomegaloviruses with resistance-associated mutations in the presence of LMV. Thus, it was proposed that CMV may exhibit a low genetic barrier towards LMV resistance development necessitating continuous surveillance during treatment. So far, the UL56 V236M mutation has been selected in vivo during two clinical trials.[5,8] The authors of a subsequent phase III trial stated that the development of breakthrough CMV viremia with confirmed UL56 mutations had been observed.[4,13] DNA sequence analysis of the UL56 and UL89 coding regions was performed on samples obtained from 28 letermovir-treated patients who had received at least one dose of study drug and experienced prophylaxis failure. Two patients were identified as having a letermovir-resistance substitution, pUL56 V236M or C325W. These substitutions were identified from on-treatment samples (www.accessdata.fda.gov, Reference ID 4179078, ClinicalTrials.gov Identifier: NCT02137772). Here, we report for the first time in vivo cytomegalovirus carrying the UL56 mutation C325Y, which was detected by CMV genotyping upon rapidly increasing viral loads in a patient under LMV treatment. In vitro data indicate that this mutation is associated with high-grade LMV resistance increasing the 50% effective concentration of LMV >5.000-fold.[12,13] In line with the published risk factors by El Chaer et al., it is tempting to suggest, that prolonged uncontrolled low-level CMV viremia might have favored the emergence of letermovir resistance.
In the future, the combination of antiviral drugs with different mechanisms of action may be used synergistically to reduce the incidence of mutations and side effects. In addition, transfer of ex vivo-generated CMV-specific T-cells can suppress CMV-reactivation by re-establishing functional antiviral immune responses in immunocompromised hosts.
- Mohty B, Mohty M. Long-term complications and side
effects after allogeneic hematopoietic stem cell transplantation: an
update. Blood Cancer J. 2011 Apr;1(4):e16. https://doi.org/10.1038/bcj.2011.14
MK, Khanna R. Human cytomegalovirus: clinical aspects, immune
regulation, and emerging treatments. Lancet Infect Dis.
P, Leruez-Ville M. Maribavir, brincidofovir and letermovir: Efficacy
and safety of new antiviral drugs for treating cytomegalovirus
infections. Med Mal Infect. 2018. https://doi.org/10.1016/j.medmal.2018.03.006 PMid:29650261
FM, Ljungman P, Chemaly RF, Maertens J, Dadwal SS, Duarte RF, et al.
Letermovir Prophylaxis for Cytomegalovirus in Hematopoietic-Cell
Transplantation. N Engl J Med. 2017;377(25):2433-44. https://doi.org/10.1056/NEJMoa1706640 PMid:29211658
RR. Role of letermovir for prevention of cytomegalovirus infection
after allogeneic haematopoietic stem cell transplantation. Curr Opin
Infect Dis. 2018. https://doi.org/10.1097/QCO.0000000000000459 PMid:29746444
H, Estey E, Grimwade D, Amadori S, Appelbaum FR, Buchner T, et al.
Diagnosis and management of AML in adults: 2017 ELN recommendations
from an international expert panel. Blood. 2017;129(4):424-47. https://doi.org/10.1182/blood-2016-08-733196 PMid:27895058 PMCid:PMC5291965
J, van der Velden W, Fox CP, Engelhard D, de la Camara R, Cordonnier C,
et al. Management of Epstein-Barr Virus infections and post-transplant
lymphoproliferative disorders in patients after allogeneic
hematopoietic stem cell transplantation: Sixth European Conference on
Infections in Leukemia (ECIL-6) guidelines. Haematologica.
2016;101(7):803-11. https://doi.org/10.3324/haematol.2016.144428 PMid:27365460 PMCid:PMC5004459
P, Michel D, Zimmermann H. Characterization of Cytomegalovirus
Breakthrough Events in a Phase 2 Prophylaxis Trial of Letermovir
(AIC246, MK 8228). J Infect Dis. 2016;213(1):23-30. https://doi.org/10.1093/infdis/jiv352 PMid:26113373
S, Arns W, Renders L, Hummel J, Muhlfeld A, Stangl M, et al. Preemptive
treatment of Cytomegalovirus infection in kidney transplant recipients
with letermovir: results of a Phase 2a study. Transpl Int.
2014;27(1):77-86. https://doi.org/10.1111/tri.12225 PMid:24164420
G, Cazal R, Hantz S, Alain S. The human cytomegalovirus terminase
complex as an antiviral target: a close-up view. FEMS Microbiol Rev.
2018;42(2):137-45. https://doi.org/10.1093/femsre/fuy004 PMid:29361041 PMCid:PMC5972660
AH, Koldehoff M. Cytomegalovirus replication reduces the relapse
incidence in patients with acute myeloid leukemia. Blood.
2016;128(3):456-9. https://doi.org/10.1182/blood-2016-04-713644 PMid:27216219
S. Rapid In Vitro Evolution of Human Cytomegalovirus UL56 Mutations
That Confer Letermovir Resistance. Antimicrob Agents Chemother.
2015;59(10):6588-93. https://doi.org/10.1128/AAC.01623-15 PMid:26259791 PMCid:PMC4576131
T, Hempel C, Ruebsamen-Schaeff H, Zimmermann H, Lischka P. Geno- and
phenotypic characterization of human cytomegalovirus mutants selected
in vitro after letermovir (AIC246) exposure. Antimicrob Agents
Chemother. 2014;58(1):610-3. https://doi.org/10.1128/AAC.01794-13 PMid:24189264 PMCid:PMC3910730
(MK-8228) Versus Placebo in the Prevention of Clinically-Significant
Cytomegalovirus (CMV) Infection in Adult, CMV-Seropositive Allogeneic
Hematopoietic Stem Cell Transplant Recipients
(MK-8228-001) Available from: https://ClinicalTrials.gov/show/NCT02137772
Chaer F, Shah DP, Chemaly RF. How I treat resistant cytomegalovirus
infection in hematopoietic cell transplantation recipients. Blood.
R, Aigner M, Moi S, Schaffer S, Gottmann A, Maas S, et al.
Clinical-grade generation of peptide-stimulated CMV/EBV-specific T
cells from G-CSF mobilized stem cell grafts. J Transl Med.
2018;16(1):124. https://doi.org/10.1186/s12967-018-1498-3 PMid:29743075 PMCid:PMC5941463