VIROMARKERS GA n.101194735 - CMV and TTV Biomarkers Study Protocol

Human cytomegalovirus (CMV) infection represents a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT), mainly occurring as viral reactivation from latency in seropositive patients but also as de novo infection in HSCT recipients from seropositive donors. Indeed, CMV infection is a frequent event following HSCT and can cause severe end-organ disease (pneumonia, colitis, retinitis, hepatitis), as well as an increased risk of graft versus host disease (GVHD) and bacterial/fungal superinfections, overall responsible for a high transplant-related morbidity and mortality [Ljungman, 2025].

Recently, anti-CMV prophylaxis has substantially reduced the risk of CMV infection and disease, as well as its related mortality [Einsele, 2020]. To date, anti-CMV prophylaxis is mainly based on the usage of letermovir, an inhibitor of CMV terminase complex, that selectively blocks the cleavage of the concatemeric progeny CMV-DNA to single mature CMV genomes and is then associated with a limited drug toxicity [Ljungman, 2025]. Although letermovir prophylaxis has been demonstrated to be safe and effective at reducing CMV disease and mortality at early times post-HSCT, late CMV infection occurring after prophylaxis suspension (upon 100 days post-HSCT) continues to represent a major concern for patients undergoing HSCT [Ljungman, 2025].

Participant Selection

To be eligible for enrollment participants must be ≥ 18 years of age, have a signed informed consent, and fulfill all of the following criteria:

Having received or due to receive HSCT HSCT recipient is seropositive for CMV, or receiving HSCT from a CMV seropositive donor Eligible to receive antiviral prophylaxis with letermovir for preventing CMV infection for at least 100 days in Italy or 200 days in Germany after HSCT according to the current guidelines.

Study Plan Participants will be enrolled at participating clinical sites. At the time of receiving HSCT (enrollment T0), demographics, medical history, medications and treatments prescribed will be recorded.

Participants will be monitored post HCST by clinical visits as well as laboratory tests using stored plasma samples. The collection of samples will be based according to the duration of letermovir prophylaxis which varies by country: 100 days in Italy vs. 200 days in Germany. See Appendix B for the time schedule of plasma samples collection according to country of enrolment.

In all samples resulting positive to CMV-DNAemia, an additional aliquot of the same sample will be pre-treated with DNase prior to DNA extraction, to exclude DNA which is not protected by a viral capsid (e.g., concatemeric DNA) from Real Time-PCR amplification, thus inducing the loss of non-infectious viral DNA. The quantification of TTV-DNAemia will be carried at T0 and every month during and after letermovir prophylaxis up to the end of study period.

Proposed research utilizing stored clinical specimens collected per this protocol will be reviewed and approved by the Protocol Team, the VIROMARKERS Scientific Steering Committee, and EC Horizon Europe.

Background and Rationale Human cytomegalovirus (CMV) infection represents a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT), mainly occurring as viral reactivation from latency in seropositive patients but also as de novo infection in HSCT recipients from seropositive donors. Indeed, CMV infection is a frequent event following HSCT and can cause severe end-organ disease (pneumonia, colitis, retinitis, hepatitis), as well as an increased risk of graft versus host disease (GVHD) and bacterial/fungal superinfections, overall responsible for a high transplant-related morbidity and mortality.

In the setting of HSCT, CMV infection develops in >60% of CMV seropositive recipients (R+), and in approximately 10% of seronegative recipients (R-) transplanted from seropositive donors (D+) [George et al., 2010], in absence of any preventive strategy. Recently, anti-CMV prophylaxis has substantially reduced the risk of CMV infection and disease, as well as its related mortality [Einsele, 2020]. To date, anti-CMV prophylaxis is mainly based on the usage of letermovir, an inhibitor of CMV terminase complex, that selectively blocks the cleavage of the concatemeric progeny CMV-DNA to single mature CMV genomes and is then associated with a limited drug toxicity [Deleenheer, 2018]. Although letermovir prophylaxis has been demonstrated to be safe and effective at reducing CMV disease and mortality at early times post-HSCT, late CMV infection occurring after prophylaxis suspension (upon 100 days post-HSCT) continues to represent a major concern for patients undergoing HSCT.

Recently EMA and FDA approved letermovir prophylaxis for 200 days instead of 100, which further decreases the risk of clinically relevant CMV infection (PREVYMIS, INN-letermovir). Nevertheless, the availability of accurate virological markers to accurately monitor CMV activity in the early and late post-HSCT phases, remains a crucial issue to ensure a proper diagnosis of CMV infection and disease and to guide prophylactic and pre-emptive antiviral treatment.

With this regard, to date, the virological surveillance for CMV replication relies basically on the quantification of CMV-DNA in blood or plasma by using Real-Time PCR assays [Ljungman, 2025], and CMV-DNAemia is known to correlate with both CMV-related disease [Ljungman, 2025] and non-relapse mortality.

However, the detection of CMV-DNAemia is not always associated with an active CMV replication, particularly in patients exposed to letermovir as prophylaxis since this drug, acting as an inhibitor of viral terminase, does not inhibit viral DNA synthesis but only its further maturation into individual genomes. In particular, a recent study from Baldanti's group, demonstrated that CMV DNAemia during LMV prophylaxis can be positive even in absence of complete CMV replicative cycles, since non-infectious CMV-DNA are released from degrading abortively infected cells even in the absence of infectious virions. This issue is particularly relevant since the interpretation of CMV-DNAemia as active viral replication can lead to a potential overestimation of CMV infection during prophylaxis and to a consequent unjustified switch to high-toxicity antivirals as ganciclovir in a high proportion of patients.

Thus, the implementation of CMV diagnostics with the use of novel markers, capable of reflecting more accurately CMV replicative activity, represents a still unmet need that can significantly improve the management of CMV risk in patients undergoing HSCT.

In this setting, the quantification of CMV-RNA represents a potential candidate marker capable of better reflecting the presence of complete, infectious CMV virions.

In particular, the currently available assay for CMV-RNA quantification (EliTech) targets the UL21.5 spliced CMV mRNA, a late viral mRNA that has been demonstrated to be packaged within the circulating complete CMV virions [Nicastro, 2025]. Despite several data support a correlation of CMV UL21.5-mRNA with viral activity, studies investigating the kinetics of this viral mRNA among immune-suppressed patients at risk of CMV re-uptake are largely missing, even more in the setting of patients receiving antiviral prophylaxis with letermovir after HSCT.

Methodology Study Design This observational study will include at least 290 consecutive patients undergoing HSCT at risk of CMV infection. Clinical management of participants will be carried out according to international and local guidelines, without any modification due to the current study. Participants will be followed for the entire duration of prophylaxis treatment followed by a minimum of 3 months after suspension.

Endpoint definitions

Before suspension of prophylaxis with letermovir:

Clinically significant CMV infection requiring initiation of anti-CMV drugs: defined as CMV end-organ disease or any CMV-related clinical event, based on CMV-DNAemia (>1,000 IU/ml is the cut-off generally used by haematologists in Italy) and the clinical condition of the patient. These include the following clinical symptoms: neurological, gastrointestinal, pneumonia, retinitis.

After suspension of prophylaxis with letermovir (relapses):

CMV infection: defined as CMV-DNAemia >112IU/ml (lower limit of quantification [LLOQ] by the commercially available Real-Time EliTech CMV DNA ELITe MGB® Kit assay used at UTOV) in seropositive HSCT recipients and in seronegative Participants receiving HSCT from seropositive donors according to the criteria recommended by the CMV Drug Development Forum.

Clinically significant CMV infection: defined as CMV end-organ disease or any CMV-related clinical symptoms (neurological, gastrointestinal, pneumonia, retinitis, see above), based on documented DNAemia >112 IU/ml and the clinical condition of the patient.

a single value of CMV-DNAemia >1,000 IU/ml or with CMV-DNAemia increasing from 500 to 1,000 IU/ml without symptoms of CMV-related disease Participants with any CMV-DNAemia with symptoms of CMV-related disease will start a therapy based on gancyclovir, valganciclovir and other second line treatments (such as foscarnet, maribavir or letermovir-gancyclovir combination) for CMV-disease, according to the virological and clinical response. Dosage will be assessed based on renal function in terms of creatinine clearance. In cases of persistent or growing values of CMV viremia under ganciclovir treatment (suspected for clinical resistance) the switch to other antivirals (i.e. Foscarnet) will be evaluated, according to current guidelines.

Study Objectives Primary and secondary objectives are stated below. Primary In participants who received HSCT, to estimate the rate of initiation of anti-CMV therapy during letermovir-based prophylaxis and the rate of CMV reactivation (based on symptoms, signs of organ dysfunction and CMV-DNAemia) after suspension of letermovir.

To evaluate the kinetics of CMV-RNA, CMV-DNAemia and TTV-DNAemia and their correlation during prophylaxis with letermovir.

To establish whether early quantitative CMV-RNA level or the early kinetics of CMV-RNA and TTV-DNAemia during prophylaxis can predict initiation of anti-CMV therapy.

In participants not initiating anti-CMV therapy during prophylaxis, to establish whether quantitative CMV-RNA level at time of letermovir suspension or the kinetics of CMV-RNA and TTV-DNAemia during prophylaxis can predict CMV re-activation (based on symptoms signs of organ dysfunction and CMV-DNAemia) after suspension of letermovir.

Secondary To establish a cut-off for CMV-RNA levels and TTV DNAemia To maximize the accuracy of prediction of CMV re-activation after suspension of prophylaxis.

To explore the kinetics of CMV-RNAemia and TTV-DNAemia in participants treated with anti CMV drugs.

To explore the correlation between CMV-RNAemia and CMV-DNAemia after considering concatemeric DNA estimated from pre-treating the sample with DNase prior to DNA extraction Data collected to address these objectives will be rapidly analyzed and summarized so that more targeted protocols for treatment and monitoring of the response can be developed.