**3.5. Genomic profiling of PTLD**

found to be associated with timing of development of PTLD and predicts survival in pediatric

Comprehensive phenotypic characterization of PTLD reveals potential reliance on EBV or NF-kappaB signaling instead of B-cell receptor signaling. Several signaling pathways, cells of origin of PTLD, and their relation to viruses were analyzed by immunohistochemistry and in situ hybridization. Most PTLDs are of activated B-cell origin. Two-thirds of cases show an Epstein-Barr virus (EBV) infection of the neoplastic cells. NF-kappaB signaling components are present in the majority of cases, except for EBV-infected cases with latency type III lacking CD19 and upstream B-cell signaling constituents. Proteins involved in B-cell receptor signaling like Bruton tyrosine kinase are seen only present in a minority of cases. Phosphoinositide 3-kinase (PI3K) is found to be expressed in 94% of cases and the druggable PI3K class 1 catalytic subunit p110 in 76%, while other signal transduction proteins are expressed only in occasional cases. Unsupervised cluster analysis has revealed three distinct subgroups: (I) related to EBV infection, mainly latency type III and lacking CD19, upstream B-cell signaling, and NF-kappa constituents; (ii) related to EBV infection with expression of the alternative NF-kappaB pathway compound including RelB, CD10, and FOXP1 or MUM1; and (iii) unrelated to virus infection with expression of the classic NF-kappaB pathway compound p65 [12]. EBV and NF-kappaB are important drivers in PTLD in contrast to B-cell receptor signaling. The main signal transduction pathway is related to PI3K. This links PTLD to other subgroups of EBV-related lymphomas, highlighting also new potential treatment approaches [4]. The diagnosis of PTLD relies on comprehensive morphologic examination, immunophenotyping, genetics, and EBV status. Most of PTLDs are of B-cell origin. EBV plays an important role in the pathogenesis of PTLD. The duration of disease onset is shorter in EBV-positive cases.

The majority of EBV infections that occur after transplantation, especially in adults, are clinically silent reactivations. This leads to a subsequent delay in the diagnosis of PTLD. A positive correlation between the degree of EBV DNAemia and the development of PTLD has significant implications for the importance of monitoring viral load after transplantation. In a study done by Holman et al., the risk of PTLD in viremic patients significantly increased with the peak quantity of EBV DNAemia [2, 13]. Since the occurrence of PTLD is significantly related to the viral load, constant monitoring and quantification of EBV-DNA load are utilized as prognostic markers for the development of PTLD.. In solid organ transplant (SOT) recipients, approximately 50% of patients develop detectable EBV DNAemia, but only a much smaller

The broad EBV latency profile (LMP1+/EBNA 2+) is found to be expressed in 59% of EBV (+) PT-DLBCL and is associated with a more elaborated inflammatory response than intermediate

PTLD in SOT [11].

**3.2. Other diagnostic markers**

398 Organ Donation and Transplantation - Current Status and Future Challenges

**3.3. EBV viremia and EBV detection by EBER**

subset develops PTLD.

**3.4. EBV antigens**

Clinical, pathological, and molecular genetic characteristics of PTLD show that EBV-positive and EBV-negative PTLDs have distinct gene expression profiling with clustering related to EBV status than immune status. Except for decreased T-cell signaling, EBV-negative PTLDs are inseparable from EBV-negative IC-DLBCL. In contrast, an EBV viral response signature is clearly shown to segregate EBV (+) PT-DLBCL from EBV (−) PT-DLBCL [14, 16].

#### **3.6. PTLD diagnostic algorithm**

Systematic morphological, immunophenotypic, and genetic analysis of each PTLD case should be performed. In DLBCL type, one may apply BCL-6, CD10, and MUM-1 immunostains in order to establish the cell of origin according to the Hans algorithm [17]; but the value of this assignment is not well established in this setting. Based on EBV protein expression, the latency type of EBV infection is defined as LMP1-/EBNA2- (type I, restricted), LMP1+/EBNA2- (type II, intermediate), and LMP1+/EBNA2+ (type III, broad). The stromal infiltrate can be estimated semiquantitatively based on the ratio of tumor cells and stromal cells in the entire tissue section. In situ hybridization: EBER (EBV-encoded RNA) in situ hybridization is considered the standard for diagnosis of EBV infection and should be performed in all PTLD cases. PTLD cases are defined as EBV (+) if EBER was expressed in all tumor cells in which RNA was preserved [14].
