Frequency of Molecular Surveillance Testing Limited by Biopsy Protocol

First, it is important to note that all organ transplant recipients remain at risk for rejection indefinitely [1, 2]. Second, the prognosis of rejection worsens when it is recognized at a more advanced stage, and mild forms of rejection when treated have a favorable prognosis [3, 4]. Thus, rejection surveillance has always been and remains a critical component in the management of transplant patients [1]. Importantly, rejection surveillance also facilitates gradual reduction and individualization of immunosuppression for transplant patients, who are placed on very high doses of immunosuppression immediately after transplant [1]. For both purposes, surveillance strategies need to incorporate testing with optimal sensitivity and specificity to detect early forms of a disease such that timely interventions can occur, but, critically, they must do so without causing harm [5].

Historically, while only biopsies provided sufficient sensitivity to detect early rejection, it is now well established that biopsies, in fact, do cause harm; biopsies can cause mechanical complications, pain and anxiety, prolonged exposure to the health care system and consumption of health care resources [1, 6-9]. Surveillance biopsies are also impractical for the 39% of US heart transplant patients that live more than 50 miles from transplant centers, often requiring out-of-pocket patient expenditures for travel [10]. Moreover, in a contemporary surveillance population, the diagnostic yield of biopsies for acute rejection is low [1]. Finally, it has increasingly been recognized that biopsies are severely limited in their real-world sensitivity and specificity, due to both significant interobserver variability and sampling errors [4, 11, 12]. Due to all these limitations, as acknowledged by the 2023 ISHLT guidelines, transplant centers have reduced the length and frequency of protocoled biopsy surveillance but importantly, have not reduced the frequency or length of other forms of non-invasive surveillance, thus acknowledging the ongoing importance of rejection surveillance [1].

Surveillance molecular testing is not associated with pain, anxiety or mechanical complications and can be done at home, sparing exposure of patients to long and expensive travel, hospital pathogens and limiting hospital resource consumption [1, 6, 9]. Furthermore, molecular testing is also not subject to interobserver variability or sampling errors and as a consequence, may have superior sensitivity and specificity than biopsies [13]. Therefore, molecular testing is a suitable candidate to replace most surveillance biopsies and to allow surveillance testing when biopsies were not typically performed due to a more favorable risk benefit ratio. Specifically, extended surveillance with molecular testing is now reasonable both to detect late rejection but also to allow clinicians the freedom to continue to personalize immunosuppression throughout the transplant recipient’s lifetime. Specifically, molecular testing facilitates the precision medicine that has evaded cardiac transplant recipient management because of the difficulties and impracticalities of biopsy surveillance [14, 15]. This is particularly valuable for older patients who are at higher risk of toxicity from immunosuppressive therapy [16].

The optimal frequency and length of rejection surveillance for a heart transplant recipient likely varies by patient risk and need for optimizing immunosuppression, which ought to be determined by the treating physician. However, it ought not be limited by the timing and frequency of a historically performed biopsy, a highly flawed, invasive and impractical tool for surveillance.   

Multi-modality Molecular Testing

Multi-modality molecular surveillance testing using gene expression profiling and dd-cfDNA provides complementary and not redundant information about a transplant recipient. Gene expression profiling measures mRNA levels of peripheral blood mononuclear cells, informing on the immune status, while dd cfDNA measures levels of circulating DNA released by an injured graft [12]. In the context of rejection surveillance where the prevalence of disease is low, it is generally accepted that the most important characteristic of a surveillance test is its ability to predict which of the surveilled patients are most likely to have rejection [18, 19]. The characteristics of the test that closely aligns with this objective is the positive likelihood ratio. For patients undergoing surveillance for acute cellular rejection, the magnitude of the positive likelihood ratio of any one commercially available molecular test using recommended thresholds test is modest and at most 2.5 [11, 20, 21]. When transplant patient exceeds thresholds for both gene expression profiling and dd-cfDNA, the likelihood ratio is approximately 5, thus more accurately identifying patients at risk of acute cellular rejection [19]. Because of this synergy of using two molecular tests at the same time, clinicians have been able to safely and dramatically reduce their dependency on biopsies [22, 23].

There are also instances when patients present without overt signs/or symptoms of rejection but with an elevated pre-test probability of rejection, such as subtherapeutic drug levels, de novo DSA or non-specific symptoms. In these cases, testing is needed to rule out rejection and requires highly sensitive tests. Due to the relatively high levels of dd-cfDNA seen in the context of antibody mediated rejection (AMR), dd-cfDNA tests are highly sensitive for AMR, measuring 88% in two separate studies using two different commercially available assays [20, 21], and can be used to exclude it. Similarly, AlloMap can achieve high sensitivities, and can be used to rule out ACR [11, 24, 25]. Consequently, simultaneous dd-cfDNA and GEP testing can rule out ACR and AMR when the pre-test probability for both types of rejection is elevated.