Abstract

Pediatric liver transplantation represents a safe and long-lasting treatment option for various disease types, requiring the pathologist’s input. Indeed, an accurate and timely diagnosis is crucial in reporting and grading native liver diseases, evaluating donor liver eligibility and identifying signs of organ injury in the post-transplant follow-up. However, as the procedure is more frequently and widely performed, deceptive and unexplored histopathologic features have emerged with relevant consequences on patient management, particularly when dealing with long-term treatment and weaning of immunosuppression. In this complex and challenging scenario, this review aims to depict the most relevant histopathologic conditions which could be encountered in pediatric liver transplantation. We will tackle the conditions representing the main indications for transplantation in childhood as well as the complications burdening the post-transplant phases, either immunologically (i.e., rejection) or non-immunologically mediated. Lastly, we hope to provide concise, yet significant, suggestions related to innovative pathology techniques in pediatric liver transplantation.

Introduction

Pediatric liver transplantation (PLTx) has made crucial improvements since the first surgical interventions by Dr. Starzl in the late ’60 in Pittsburgh ^1,2, and now represents a relevant part of the annual liver transplant rate worldwide: in 2020, 5.2% of liver transplantations performed in Italy involved the pediatric population (0 to 17-year-old patients) and similar percentages are reported in Europe and North America ^3,4.

Thanks to improved technical procedures and patient management (i.e., donor-recipient matching strategies, surgical approaches and immunosuppressive protocols), PLTx now shows a 10-year and 20-year survival rate of more than 80% of transplanted children and young adults and represents an appropriate long-term therapeutic option for several end-stage/terminal hepatic conditions ^5-7. Similar to adult liver transplantation, the successful rate of PLTx is burdened by donor shortage, requiring (1) accurate recipient selection and stratification, (2) innovative surgical and organ preservation techniques, (3) early and specific recognition of graft disease, and (4) optimization of postoperative care. However, differently from the adult setting, PLTx presents peculiar features related to indications, immunosuppression and life-long postoperative follow-up ⁸. Indeed, acute complications requiring biopsy assessment are now uncommon but not negligible (and, subtle), while histologic diagnosis of long-term conditions is more frequently required and may be challenging.

To this end, a multidisciplinary approach to PLTx is essential, and pathologists can notably contribute in almost every step of the procedure ^9,10. In particular, pathologists are involved in diagnosing and grading native diseases, evaluating donor organ status and performance, identifying early signs of organ injury and differentiating transplant-related conditions [e.g., acute and chronic rejection, post-transplant lymphoproliferative disorder (PTLD)] from liver native disease recurrence and de novo diseases. Supporting our role in this challenging scenario, newly introduced next-generation pathology procedures (e.g., multiplex immunohistochemistry, tissue-tethered digital morphometrics, single-cell molecular analysis) have allowed us to extract innovative data from tissue biopsy and thoroughly advance our understanding of transplant-related conditions, particularly regarding immune activation and regulation ^11,12.

In this evolving and challenging panorama for pathologists approaching pediatric liver transplant pathology, this review will tackle the most significant aspects of PLTx, providing a pictural essay of the main histopathologic features, an Introduction. to the most innovative procedures of next-generation pathology, and, eventually, highlighting the role that pathologists should fulfill within the multidisciplinary management of pediatric transplantation.

Setting the stage of PLTx

At first sight, the pre-transplant setting could be considered somehow extraneous to the pathologist’s commitment: clinical- and laboratory-based organ allocation systems have been developed, namely the Pediatric End-Stage Liver Disease (PELD) and the Model of End-Stage Liver Disease (MELD), and are continuously updated ^13,14, whereas native liver disease diagnostic procedures tend to be as less invasive as possible, particularly in the neonatal setting ¹⁵. However, liver biopsy still maintains a relevant role in numerous pediatric hepatic conditions, particularly in diseases with atypical clinical pictures either to assess the diagnosis or to define the stage of the disease and potentially underestimated concurrent disease. Conversely, the histomorphologic evaluation of donor liver represents a crucial step of the whole procedure, especially considering the overall shortage of donor organs ¹⁵.

INDICATIONS AND CONTRAINDICATIONS OF PLTx

Pediatric conditions leading to PLTx are quite vast and heterogenous but classically distinguished in cholestatic diseases, metabolic and genetic disorders (either hepato-specific or systemic), acute liver failure scenarios (including drug induced liver injury) ¹⁶ and primary liver neoplasms (Tab. I, Fig. 1A) ^10,17-19.

Adult type conditions such as autoimmune hepatitis (AIH) ^20,21 may also impact the pediatric population, while a pathologic condition that is rapidly increasing and concerning pediatric hepatologists is fatty liver disease^22,23. Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are well-known conditions affecting adults in the Western world ²⁴, although this “fatty liver pandemic” is now moving from the adult to the pediatric population. Although NASH and NAFLD do not represent actually a leading indication of PLTx, we should expect an increasing rate in the next decades ^22,23.

Contraindications to PLTx are mainly represented by poor patient clinical conditions before transplantation and general contraindication to surgical procedure (e.g., septic status, overlapping multiorgan failure/life-threatening defects in other organs, irreversible and severe neurologic dysfunction) or presence of extrahepatic malignancy ^9,25,26. However, in this context the pathologist’s evaluation has a limited influence.

NON-NEOPLASTIC DISEASE AS PLTx INDICATION

An in-depth treatise on all causes of liver injury in the pediatric population is beyond the scope of this manuscript, although some important conditions will be briefly touched upon.

Cholestatic disorders

Although geographic and demographic variables could influence specific disease incidence, biliary atresia is globally reported as the leading cause of liver failure in the pediatric population, thus representing the primary indication for PLTx ²⁷. Apart from biliary atresia, other cholestatic diseases which may require PLTx include: Alagille syndrome, progressive familial intrahepatic cholestasis (PFIC), ductal plate abnormalities including Caroli syndrome and congenital hepatic fibrosis, autoimmune sclerosing cholangitis, bile acid synthesis defects and cystic fibrosis related disease. Some of these conditions are described in Table II.

Rare genetic and metabolic disorders may lead to PLTx for different reasons: 1) Disorders which affect the liver and PLTx is performed for end stage liver disease and complications such as tyrosinemia, alpha1 anti-trypsin deficiency, Wilson’s disease, glycogen storage disorders etc.; 2) Disorders in which enzymes are produced in the liver but manifestations are extra-hepatic (rare end stage liver damage) and PLTx is performed for extrahepatic organ involvement such as urea cycle deficits, primary hyperoxaluria, etc.; 3) Disorders in which enzymes are produced in the liver and in extrahepatic tissues for which PLTx only partially corrects enzyme deficiency and alleviates extrahepatic manifestations (but does not entirely cure them) such as methylmalonic acidemia, maple syrup urine disorder, etc.

NEOPLASTIC LESIONS AS PLTx INDICATION

Between 5 and 10% of PLTx are performed for neoplastic lesions, the most frequent being hepatoblastoma, which is also the most frequent primary liver cancer in children ²⁸.

Hepatoblastoma

The pathologic aspects of hepatoblastoma (HB) have been extensively described elsewhere in this issue. In the US, between 17 and 20% of surgically treated HB patients receive PLTx ²⁹ and 5-year overall survival is approximately 80%. In HB, PLTx is offered to children who cannot be safely and radically resected due to large tumor size or surgical/anatomic characteristics ³⁰ after pre-operative chemotherapy.

Hepatocellular carcinoma

Pediatric HCC is a rare malignancy more often seen in adolescents. Differences exist between adult and pediatric HCCs including ^31,32: 1) approximately 70% of pediatric HCC develop on normal liver background compared to 85% of adult HCCs developing in chronic liver disease; 2) pediatric underlying liver diseases include perinatal acquired hepatitis B virus, tyrosinemia, glycogen storage disease, Alagille syndrome, progressive familial intrahepatic cholestasis, and congenital portosystemic shunts; 3) differences at the molecular level between adult and pediatric HCCs have been identified; 4) pediatric HCCs are more often sensitive to chemotherapy compared to adult HCCs; 5) outcomes are better in pediatric HCC even at more advanced stages.

Approximately a quarter of pediatric HCCs (late childhood and adolescence) are of the fibrolamellar variant and show similar morphologic aspects as adult fibrolamellar HCC, together with the DNAJB1-PRKCA fusion transcript ³³. This variant occurs in normal liver background, expanding treatment options, nonetheless relatively recent studies show that it does not have better survival compared to conventional HCC ³⁴.

Pooled data have shown that outcomes of PLTx for HCC reach 70-80% 5-year survival rates ³⁵.

Sarcomas and Vascular Tumors

Embryonal Sarcoma has been discussed elsewhere and it represents a rare indication for PLTx as tumors are chemo-sensitive and can usually be cured with chemotherapy and surgery alone. Data on PLTx are scarce, but show excellent outcomes.

Malignant Rhabdoid Tumor (MRT) of the liver is a rare, aggressive malignancy of infancy characterized by round to polygonal cells with abundant dense eosinophilic cytoplasm with inclusions, large vesicular and eccentric nuclei, numerous nucleoli and loss of expression of INI1 in the nuclei of the tumor cells (but present in the nuclei of all normal cells) ³⁶. Few children have undergone PLTx with generally negative results.

The understanding of Vascular Lesions of the liver has expanded over the years through more accurate histologic description and use of immunohistochemistry, as well as molecular advancements. The International Society for the Study of Vascular Anomalies (ISSVA) in 2018, classified pediatric vascular tumors based on their behavior into benign (hepatic congenital hemangioma and hepatic infantile hemangioma), locally aggressive, and malignant neoplasms (hepatic hemangioendothelioma and hepatic angiosarcoma) The differential diagnosis requires immunostaining and (in case of EHE) molecular characterization ³⁷. PLTx for benign liver neoplasms, such as hepatic infantile hemangiomas, is rarely considered (and only in life threatening conditions as medical treatment is available) as most lesions are asymptomatic and, after rapid post-natal proliferation, tend to spontaneous involution. Malignant vascular tumors, such as epithelioid hemangioendothelioma and hepatic angiosarcoma, were historically not considered for transplantation, however few cases have been treated with PLTx with variable results ^38-40.

Assessment of transplantable liver

Pretransplant evaluation is often considered as the recipient assessment of disease severity and transplant urgency ¹⁷. However, we would like to shift the focus of the pretransplant evaluation from the recipient to the donor.

The shortage of donor organs is a well-known “side effect” of the successful therapeutic rate of transplantation and its resultant expansion of suitable indications (e.g., liver transplant oncology )⁴¹. As a consequence, the donor pool has been increased by implementing expanded criteria for donors [e.g., steatotic liver, cold ischemia time > 12 hours, partial allograft, and donation after circulatory death (DCD)] ⁴² and ex vivo machine perfusion techniques.

Among the expanded criteria, the most relevant for the pathologist approaching donor evaluation is the percentage of steatosis. Pre-transplant steatosis assessment is focused on reporting the percentage of large droplet macrovesicular steatosis that is defined as lipid vacuoles larger than a non steatotic hepatocyte and pushing the nucleus peripherally ⁴³. Lipid vacuoles not fulfilling these criteria should be considered as small droplet macrovesicular steatosis, whereas microvesicular steatosis represents a diffuse “foamy” cytoplasmic appearance of hepatocytes occurring in specific pathological subset (e.g., Reye syndrome) ⁴³. Recently, the recommendation of the Banff Working Group on Liver Allograft Pathology, introduced a detailed definition and diagnostic algorithm to specifically and accurately evaluate steatosis in the donor liver ⁴³. These consensus recommendations are helpful in standardizing the assessment of a variable with relevant consequences on organ management but burdened by relevant interobserver rates. Livers suitable for transplantation should present at most less than 60% (preferably less than 30%) of large droplet macrovesicular steatosis to prevent graft primary non-function and acute failure, but no consensus has been published regarding a specific acceptance cut-off.

In the adult setting, machine perfusion is demonstrating remarkable success, now introducing the opportunity to specifically and directly treat retrieved organs ^44,45. Nevertheless, limited evidence of machine perfusion implementation in the PLTx setting is available to date. The first report of a successful application of machine perfusion in the pediatric setting is dated 2019 and reported by Werner et al. ⁴⁶, followed by the experience reported by the Turin group⁴⁷. Indeed, PLTx requires a cautious approach, but once the benefits of machine perfusion will be ensured by multicenter studies, this technique would represent a valid support and eventually increase the PLTx donor pool.

Pathologic evaluation of the pediatric transplanted liver

Post-transplant liver biopsy evaluation represents the main core of the pathologist’s role in the PLTx setting. Indeed, features of acute injury and chronic evolution have to be promptly identified to guide clinical management, establish transplanted liver fitness, and predict functional decline and graft loss (Fig. 1B). Additionally, inflammatory and fibrotic findings have been identified in clinically-silent follow-up protocol biopsies, but the consequences of these features on patient management and long-term organ survival still need to be further explored.

ISCHEMIA/REPERFUSION INJURY AND OTHER NON-IMMUNOLOGICALLY MEDIATED ACUTE COMPLICATIONS

During organ procurement and transplantation phases, the liver is first exposed to metabolic stress due to oxygen deprivation and then to inflammatory and ROS effect, leading to so-called ischemia/reperfusion injury (IRI). Indeed, the abrupt vascularization interruption and subsequent replenishment characterizing organ transplant can cause endothelial cell swelling (particularly affecting the sinusoids) and terminal hepatic vein-based parenchymal injury (hepatocyte ballooning and apoptosis, neutrophil aggregates and parenchymal necrosis), and cholestatic features (Fig. 2) ⁴⁸. These signs of injury usually last for the first two-three weeks after transplantation, are relatively common and usually mild, but IRI may, rarely, evolve as a severe event leading to graft primary non-function and organ loss ⁴⁸.

Additional conditions related to inadequate vascularization/parenchymal perfusion are represented by subcapsular hemorrhage ^48,49 (Fig. 2) and small-for-size syndrome. The former is an effect caused by poorly vascularized peripheric parenchyma, particular evident in wedge biopsy sample where subcapsular parenchyma is more represented (Fig. 2) ^48,49. The latter is a condition of graft size mismatch in the context of elevated metabolic demands (i.e., elevated MELD score) and portal hypertension ⁵⁰, and is characterized by a portal-centered pattern of injury (portal/periportal hemorrhage, arterial occlusion and ischemic bile duct injury) due to portal hypoperfusion/hypertension and reflex arterial vasospasm ^50-52.

The biliary tree can also incur into early dysfunction, generally related to anastomotic complications and secondary bile duct obstruction. Histopathological changes are consistent with a biliary obstructive etiopathogenesis, showing features of cholangitis (peri- and intraductal neutrophills infiltration), ductular injury and proliferation, and bile leakage (portal edema) ⁴⁸.

Of note, all these conditions are not strictly related to immune system activation or rejection processes, and therefore it is mandatory to properly recognize their features to avoid diagnostic misinterpretation and overtreatment.

ALLOGRAFT REJECTION

Allograft rejection still represents a major threat to PLTx despite the liver’s tolerogenic immune environment and efficient immunosuppression protocols that dampen the graft directed immune response. Indeed, PLTx aims to represent a life-long treatment, thus requiring long-standing graft survival even though it is characterized by complicated immunosuppression management with a balance between the need to prevent rejection and to avoid infection and drug side effects ^9,53. To this end, the recognition of precocious signs of rejection is of pivotal interest to guide subsequent therapeutic management and prevent severe consequences (i.e., graft loss). Diagnostic criteria and grading schemes related to organ rejection pathology are continuously discussed and subsequently updated by The Banff Foundation for the Allograft Pathology ^54-56. Transplant rejection is classically differentiated into antibody and T-cell mediated rejection, although mechanistically different, considerable overlap exists between these two conditions and mixed episodes could occur.

Antibody-mediated rejection (AMR) is an immune-mediated condition triggered by donor sensitization and was especially described in cases of ABO incompatible transplantation. Differently from other transplanted organs (kidneys above all), AMR rarely occur in the liver due to its overall immunological resistance to AMR mechanism of injury ⁵⁷. Nevertheless, the 2016 Banff Working Group on Liver Allograft Pathology distinguishes two forms of AMR, namely acute and chronic AMR ^55,58.

Acute AMR usually occurs days to weeks after PLTx and presents histopathological features of an immune related micro/small vascular pathology (e.g., capillary and inlet venule hypertrophy, dilation and endotheliitis) affecting both portal/periportal small vessels and centrilobular veins associated with features of biliary compartment involvement (cholestasis, ductular reaction, portal edema). In addition to the histopathology picture of an acute injury, the complement fragment 4d (C4d) linear and granular vascular deposition (demonstrated through specific immunohistochemical or immunofluorescence stain) (Fig. 3) and positive serum Donor-Specific Antibodies (DSA) are required criteria to obtain a definitive diagnosis ^48,55,59,60

Chronic AMR still involves the vascular compartment, but presents also more non-specific inflammatory (portal inflammation, interface activity) and chronic (portal, sinusoidal, or perivenular moderate fibrosis) features, thus showing subtle morphologic features that could be easily misinterpreted and blurred by overlapping (and more frequent) conditions such as recurrent diseases ⁵⁵. In this regard, PLTx represents an ideal setting to evaluate putative chronic AMR features thanks to the low-rate of disease recurrence that could disguise diagnostic interpretation ⁵⁵. Similar to the acute form, a definitive diagnosis of chronic AMR requires C4d (portal microvascular) positivity and the correct clinical setting (DSA positivity) ⁵⁵.

Detailed acute and chronic AMR features are reported in Table III.

T-cell mediated rejection (TCMR) is the most frequent immune-mediated form of injury following transplantation. In its typical form, acute TCMR (a-TCMR) manifests as an inflammatory process mainly involving portal tracts, bile ducts, and venous endothelia usually occurring within the first trimester after transplantation and becomes less common as time pass. Accordingly, the Banff Working Group proposes diagnostic and grading criteria providing an overall evaluation and a more specific semi-quantitative index, namely the Rejection Activity Index (RAI), which specifically addresses the grades of involvement of the targeted structures. Typical a-TCMR presents portal tracts expansion by an inflammatory infiltrate mainly composed of lymphocytes, but other cell types (e.g., eosinophils, neutrophils, macrophages) are often involved, as well as immature and activated immune cells (e.g., lymphoblasts) (Fig. 4). Bile ducts are directly injured by the host’s immune activation, presenting various degrees of intraductal inflammatory infiltrates and morphologic signs of cell injury (e.g., apoptosis, nuclear overlapping/stratification) (Fig. 4). Similarly, vascular endothelia show lymphocyte infiltration and cell injury and detachment (i.e., endotheliitis) (Fig. 4). As a general rule, the more the inflammation and involvement of anatomical structures, the higher the RAI. Specific descriptors and grades are reported in Table IV. It is worth mentioning that RAI is not a diagnostic but grading index and it should be applied once the diagnosis of rejection has already established.

Chronic T-cell mediated rejection (c-TCMR) is described as an irreversible event occurring as a consequence of recurrent rejection episodes and patient inadequate immunosuppression compliance. Therefore, due to its temporal development and reiterated acute injury requirement, it is very unlikely that it could occur before the first six months after transplantation. A well-known and described caveat of c-TCMR diagnosis is related to the structures involved, particularly the larger hepatic arteries as an obliterative arteriopathy ⁴⁸. Indeed, these structures are usually not sampled with the (relatively) small biopsy performed, thus reducing the possibility of recognizing this event. Together with the larger arteries, c-TCMR also affects the bile ducts showing lesions ranging from initial cell injury (cytoplasmic eosinophilia and cell atrophy/nuclear loss) of early-phase disease to complete bile duct extinction and ductopenia characterizing the long-standing phase ⁴⁸. Diagnosing ductopenia can be challenging considering that portal tracts could physiologically lack bile ducts on biopsy (up to 7% of evaluable portal tracts, approximatively) ⁶¹. Additionally, liver biopsy can present an overall low number or partially sampled portal tracts. Thus, Banff guidelines recommend to perform the diagnosis of ductopenia (and therefore of c-TCMR) if the biopsy presents at least 10 portal tracts with at least > 50% showing clear ductopenia. A useful hint to the diagnosis is represented by the (almost) complete absence of ductular reaction that characterizes c-TCMR and differs it from other conditions affecting bile ducts (Fig. 4). Additionally, portal inflammation is usually mild/minimal, as well as endothelial injury. Similar to a-TCMR, the Banff Working Group on Liver Allograft Pathology published a grading system for c-TCMR, differentiating early-phase to late c-TCMR.

Furthermore, additional, non-canonical patterns of rejection have also been reported in the literature. They usually occur later after transplantation (starting from six months, approximatively) and prove to be particularly difficult to identify and relevant in PLTx.

1. Centrilobular rejection: refers to the isolated injury of the terminal hepatic vein (inflammation, endotheliitis and hepatocyte extinction) with no portal tract/bile duct involvement. It was described as an early sign of rejection in the PLTx setting ^48,62.
2. Plasma cell rich-rejection: previously reported as de novo AIH, the plasma cell rich mediated liver injury is now fully considered a form of rejection by the Banff guidelines ⁵⁵. Histopathologically, it does not differ greatly from usual AIH (plasma cell rich portal inflammation, interface hepatitis, lobular/bridging necrosis), but presents some additional peculiar features such as a more frequent severe bile ducts involvement (lymphocytic cholangitis) and a prevalent IgG4 positive plasma cell inflammation (Fig. 4) ^55,62. Typical features of both AMR (C4d positivity) and TCMR could also coexist. As a form of rejection, although atypical, it responds adequately to immunosuppression, but it occurs later (> 6 months) after transplantation ⁵⁵. Of note, morphological distinction between recurrent AIH and plasma cell rich-rejection represents, to date, a challenging scenario based on slight differences only.
3. Hepatitis-like rejection: a form of rejection presenting portal inflammation together with interface and lobular features mimicking chronic hepatitic processes ^62,63. It has emerged as a relatively common pattern of injury observed in clinically-silent protocol follow-up biopsy and currently represents a “hot-topic” of research studies trying to describe its temporal evolution (it is probably related to complicated therapeutic compliance from adolescent patients) and identify precocious signs of injury ^64-66.

DE NOVO AND RECURRENT DISEASES

Curiously, the diagnostic routine of a pathologist approaching PLTx biopsy can greatly vary depending on the follow-up protocols adopted by the specific transplant center. In particular, pathologists practicing in Institutions performing protocol liver follow-up biopsies will probably face a relatively high number of cases showing clinically silent (i.e., without associated clinical-serological signs and symptoms) signs of inflammation and fibrosis ^17,67-70.

On the other hand, if liver biopsies are performed following clinical indications only, the main features that we can encounter will be related to rejection processes, as described in the previous paragraph, or de novo and recurrent disease, both of them further addressed in this paragraph.

Recurrence disorders are particularly rare in the pediatric setting. Indeed, the main indications to PLTx are of metabolic and inherited nature (e.g., biliary atresia), thus harboring almost no recurrence potential. Additionally, disease with post-PLTx recurring capability (primitive sclerosing cholangitis, HBV and HCV hepatitis) do not present peculiar morphologic findings, and therefore we direct the readers to the specific literature addressing these conditions ^24,48,71.

On the other hand, de novo diseases (defined as the occurrence of a disease not experienced by the patient before transplantation) represent a relevant issue in long-term PLTx, particularly if considering infection-related conditions. Indeed, pediatric patients usually reach transplantation in an infection “naïve” condition and immunosuppression protocols to prevent rejection constitute a major risk factor for infection outbreak. In particular, infective agents can determine direct liver parenchymal injury (i.e., CMV, EBV and HSV hepatitis) or cause proliferative/neoplastic disorders (i.e., EBV and HHV8). The former group of infectious causes are mostly represented by hepatitic features (portal and lobular inflammation) with peculiar pathogen-specific characteristics. If suspected, ancillary techniques (immunohistochemical and RNA in situ hybridization) and laboratory tests are essential to perform a definitive diagnosis and prevent misinterpretation. Specific patterns of infective-related post-transplant relevant diseases are summarized in Table V.

Regarding infectious (viral) induced neoplastic disorders, two pathogens are particularly noteworthy: HHV8 and EBV. HHV8 leads to the development of Kaposi sarcoma, a malignant vascular tumor, fortunately only rarely reported in the PLTx setting ⁷². Similarly, EBV has been associated with induction of uncontrolled cell proliferation, particularly of B-cells, leading to the development of posttransplant lymphoproliferative disorder (PTLD) ⁷³. Indeed, PTLD is a group of multifaceted B-cell proliferative disorders affecting transplanted patients. Several patterns are described, grouped by the WHO in four categories, namely (1) non-destructive PTLD, (2) polymorphic PTLD, (3) monomorphic PTLD, and (4) classical Hodgkin’s lymphoma PTLD. Although PTLD rarely affects the graft liver of PLTx patients ^74-76, more in depth discussion is beyond the aim of this manuscript. Conversely, we would like to mention and briefly illustrate the features of another EBV-related condition, that is EBV related-smooth muscle tumor (EBV-SMT). EBV-SMT is a rare (and frequently unrecognized) neoplastic disorder affecting immunosuppressed patients (transplanted and immunodeficient patients) mainly developing in the liver (both native and transplanted). It is particularly frequent in the pediatric population presenting features of a benign-looking (no/minimal cytological atypia, no necrosis, low proliferative index/mitotic count) soft-tissue neoplasm with morphological (spindle cells arranged in fascicles) and immunohistochemical (smooth muscle actin and h-caldesmon diffuse expression, desmin focal expression) features of smooth muscle differentiation (Fig. 5) ^77-79. Neoplastic cell EBV expression is a required criterion to differentiate this entity from similar conditions (Fig. 5).

Additional neoplastic conditions that can newly develop with increased frequency in the PLTx population are represented by skin, lung, liver, kidney and anogenital cancer ⁸⁰.

Back to the future: next-generation pathology to revamp the benefit of the liver biopsy

Proclaiming a Liver Biopsy Manifesto is beyond the aim of this Review, but we believe that we are at the edge of a new era for liver histomorphologic evaluation, particularly in the PLTx setting. Indeed, liver biopsy is burdened by well-known and potentially severe complications, but next-generation pathology, namely the combination of multiplex immunohistochemistry and tissue-tethered digitally assisted analysis, harbor the potential to extract specific single-cell morphometric, phenotypic, and spatial characterization from the biopsy, thus obtaining a brand-new type of data enriched by the correlation with tissue architecture ^11,12,81,82. In particular, multiplex immunohistochemistry allows to contemporary evaluate multiple antigens within the same slide with cell detail and has proved particularly helpful in defining the specific phenotype of immune cells, whereas digital analysis sheds light on morphologic and spatial data that would have been otherwise remained unexplored. We believe that next-generation pathology will considerably renew liver biopsy indications in the next future, especially considering that it has already efficiently identified (pediatric) patients that would benefit from immunosuppression weaning, thus preserving them from long-term treatment side effects ^56,65,66,83.

Conclusions

PLTx is a life-saving treatment for several terminal conditions. Due to the life-long persistence of the transplanted organ, every step of PLTx needs to be thoroughly approached and analyzed, thus frequently requiring liver tissue evaluation. As pathologists, our role is to render the most specific and reliable yet early diagnosis. Pathologists are involved in every crucial step of PLTx including definition of liver graft function, immediate-post transplant injury recognition, rejection and long-term complications identification. Innovative next-generation pathology procedures are expanding our knowledge of graft pathology with unexplored data, allowing us to identify precocious signs of tissue injury, and renew the histological assessment of transplanted organs.

Figures and tables

Figure 1.Overall representation of the main indications to PLTx (A) and the main pathologies affecting the transplanted liver (B). (A) Main indications to PLTx are usually grouped in cholestatic disease [e.g., biliary atresia (hematoxylin and eosin stain showing bile duct injury in a portal tract)], metabolic disorder [e.g., alpha-1 antitrypsin deficiency (PAS stain after diastase digestion highlighting intracytoplasmic globules accumulation in periportal hepatocytes)], neoplasia [e.g., hepatoblastoma (hematoxylin and eosin stain of an embryonal subtype)], acute liver failure [e.g., Wilson disease (rhodamine stain highlighting granular coarse deposits in hepatocytes)], and “Other” categories [e.g., polycystic liver (hematoxylin and eosin stain showing a cluster of intrahepatic cysts)], the latter containing heterogeneous conditions that could not be otherwise classified. (B) Post-transplant liver diseases could be sorted in acute complications [ischemic-reperfusion injury (hematoxylin and eosin stain showing parenchymal necrosis and neutrophils aggregates)], allograft rejection [T-cell mediated rejection (hematoxylin and eosin stain showing a portal tract with severe inflammatory infiltration and injury)], recurrent disorders [primary biliary sclerosis (hematoxylin and eosin stain showing periductal concentric fibrosis)], and de novo disease [cytomegalovirus hepatitis (immunohistochemical staining for cytomegalovirus highlighting positive nuclei of infected hepatocytes)].

Figure 2.Non-immunological acute complications. (A) Terminal hepatic vein-centered pattern of injury in a case of ischemia-reperfusion injury (hematoxylin and eosin); (B) Several erythrocytes located underneath the rim of the liver capsule in a case of subcapsular hemorrhage.

Figure 3.Pathologic features of antibody-mediated rejection (AMR) 14 days post-transplant. (A) Portal tract showing inflammation and endotheliitis in small portal vessels. (B) Strong staining in the portal vein with C4d staining (used to detect complement deposition).

Figure 4.Pathologic features of rejection. (A) Mixed rejection infiltrate (lymphocytes, plasma cells, eosinophils) expanding a portal tract, presenting features of endotheliitis and bile duct injury. (B) Severe rejection infiltrate with conspicuous eosinophils, lymphoblasts, and plasma cells “obscuring” portal structures; notice how the infiltrate, although severe, is strictly confined to the portal tract, showing minimal parenchymal spillover only. (C) Severe centrilobular vein endotheliitis, showing endothelial cells swelling and detachment as well as immune cells aggressive behavior. (D) Bile duct loss in a portal tract of a liver affected by chronic rejection. (E) The absence of bile duct and ductular reaction are suggestive features of chronic rejection, whereas periportal hepatocyte ductular metaplasia is common and diffuse (cytokeratin 7 immunohistochemical staining). (F) Cytokeratin 19 further highlights the absence of bile duct and ductular reaction, similarly to cytokeratin 7, although it is not expressed by metaplastic hepatocytes (please, notice that tiles D, E, and F represent the same portal tract). (G) The plasma cell rich variant of rejection presents an inflammatory infiltrate with conspicuous plasma cells and interface hepatitis. (H) Centrilobular-based injury with hepatocyte apoptosis and necrosis and neutrophilic aggregates, is an additional characteristic feature of plasma cell rich rejection. (I) Immunohistochemical staining highlights plasma cells (here represented by MUM-1) and proves useful in evaluating the quantity of plasma cells and their location when dealing with plasma cell rich rejection,

Figure 5.Pathological features of EBV-SMT. (A) Spindle cells with none/minimal cytological atypia organized in fascicles, no/minimal necrosis, and low mitotic index are characteristic features of EBV-SMT (A; hematoxylin and eosin). (B) Diffuse expression of smooth-muscle actin helps confirm the nature of EBV-SMT. (B) EBV-SMT usually presents a focal/heterogeneous positivity to desmin. (C) Proof of diffuse positivity to EBV [here represented by Epstein-Barr encoding region (EBER) in situ hybridization] is a required criterion to perform the diagnosis of EBV-SMT.

References

1. Starzl TE, Groth CG, Brettschneider L. Extended survival in 3 cases of orthotopic homotransplantation of the human liver. Surgery. 1968; 63:549-563.
2. Starzl TE, Marchioro TL, Vonkaulla KN. Homotransplantation of the liver in Humans. Surg Gynecol Obstet. 1963; 117:659-676.
3. Kwong AJ, Kim WR, Lake JR. OPTN/SRTR 2019 Annual Data Report: Liver. Am J Transplant. 2021; 21:208-315. DOI
4. de Ville de Goyet J, Baumann U, Karam V, European Liver Intestine Transplant Association. European Liver Transplant Registry: donor and transplant surgery aspects of 16,641 liver transplantations in children. Hepatology. 2022; 75:634-645. DOI
5. Duffy JP, Kao K, Ko CY. Long-term patient outcome and quality of life after liver transplantation: analysis of 20-year survivors. Ann Surg. 2010; 252:652-661. DOI
6. Kerkar N, Lakhole A. Pediatric liver transplantation: a North American perspective. Expert Rev Gastroenterol Hepatol. 2016; 10:949-959. DOI
7. Ng VL, Alonso EM, Bucuvalas JC, Studies of Pediatric Liver Transplantation (SPLIT) Research Group. Health status of children alive 10 years after pediatric liver transplantation performed in the US and Canada: report of the studies of pediatric liver transplantation experience. J Pediatr. 2012; 160:820-6.e3. DOI
8. Hackl C, Schlitt HJ, Melter M. Current developments in pediatric liver transplantation. World J Hepatol. 2015; 7:1509-20. DOI
9. Pham YH, Miloh T. Liver transplantation in children. Clin Liver Dis. 2018; 22:807-821. DOI
10. Emre S, Umman V, Cimsit B. Current concepts in pediatric liver transplantation. Mt Sinai J Med. 2012; 79:199-213. DOI
11. Wood-Trageser MA, Lesniak AJ, Demetris AJ. Enhancing the Value of Histopathological Assessment of Allograft Biopsy Monitoring. Transplantation. 2019; 103:1306-1322. DOI
12. Wood-Trageser MA, Xu Q, Zeevi A. Precision transplant pathology. Curr Opin Organ Transplant. 2020; 25:412-419. DOI
13. Wiesner RH. Evidence-based evolution of the MELD/PELD liver allocation policy. Liver Transpl. 2005; 11:261-3. DOI
14. Freeman RB, Wiesner RH, Roberts JP. Improving liver allocation: MELD and PELD. Am J Transplant. 2004; 4:114-31. DOI
15. Ovchinsky N, Moreira RK, Lefkowitch JH. Liver biopsy in modern clinical practice: a pediatric point-of-view. Adv Anat Pathol. 2012; 19:250-262. DOI
16. Amin MD, Harpavat S, Leung DH. Drug-induced liver injury in children. Curr Opin Pediatr. 2015; 27:625-33. DOI
17. Kelly D, Wray J. The adolescent liver transplant patient. Clin Liver Dis. 2014; 18:613-632. DOI
18. Kriegermeier A, Green R. Pediatric cholestatic liver disease: review of bile acid metabolism and discussion of current and emerging therapies. Front Med (Lausanne). 2020; 7:149. DOI
19. Menon J, Vij M, Sachan D. Pediatric metabolic liver diseases: evolving role of liver transplantation. World J Transplant. 2021; 11:161-179. DOI
20. Mieli-Vergani G, Vergani D. Paediatric autoimmune liver disease. Arch Dis Child. 2013; 98:1012-7. DOI
21. Covelli C, Sacchi D, Sarcognato S. Pathology of autoimmune hepatitis. Pathologica. 2021; 113:185-193. DOI
22. Doycheva I, Watt KD, Alkhouri N. Nonalcoholic fatty liver disease in adolescents and young adults: The next frontier in the epidemic. Hepatology. 2017; 65:2100-2109. DOI
23. Mann JP, Valenti L, Scorletti E. Nonalcoholic fatty liver disease in children. Semin Liver Dis. 2018; 38:1-13. DOI
24. Cataldo I, Sarcognato S, Sacchi D. Pathology of non-alcoholic fatty liver disease. Pathologica. 2021; 113:194-202. DOI
25. Spada M, Riva S, Maggiore G. Pediatric liver transplantation. World J Gastroenterol. 2009; 15:648-74. DOI
26. Cuenca AG, Kim HB, Vakili K. Pediatric liver transplantation. Semin Pediatr Surg. 2017; 26:217-223. DOI
27. Sokol RJ, Shepherd RW, Superina R. Screening and outcomes in biliary atresia: summary of a National Institutes of Health workshop. Hepatology. 2007; 46:566-581. DOI
28. Sindhi R, Rohan V, Bukowinski A. Liver Transplantation for Pediatric Liver Cancer. Cancers (Basel). 2020; 12:720. DOI
29. Vinayak R, Cruz RJ, Ranganathan S. Pediatric liver transplantation for hepatocellular cancer and rare liver malignancies: US multicenter and single-center experience (1981-2015). Liver Transpl. 2017; 23:1577-1588. DOI
30. de Ville de Goyet J, Meyers RL, Tiao GM. Beyond the Milan criteria for liver transplantation in children with hepatic tumours. Lancet Gastroenterol Hepatol. 2017; 2:456-462. DOI
31. Khanna R, Verma SK. Pediatric hepatocellular carcinoma. World J Gastroenterol. 2018; 24:3980-3999. DOI
32. Renne SL, Sarcognato S, Sacchi D. Hepatocellular carcinoma: a clinical and pathological overview. Pathologica. 2021; 113:203-217. DOI
33. Honeyman JN, Simon EP, Robine N. Detection of a recurrent DNAJB1-PRKACA chimeric transcript in fibrolamellar hepatocellular carcinoma. Science. 2014; 343:1010-1014. DOI
34. Weeda VB, Murawski M, McCabe AJ. Fibrolamellar variant of hepatocellular carcinoma does not have a better survival than conventional hepatocellular carcinoma--results and treatment recommendations from the Childhood Liver Tumour Strategy Group (SIOPEL) experience. Eur J Cancer. 2013; 49:2698-2704. DOI
35. Romano F, Stroppa P, Bravi M. Favorable outcome of primary liver transplantation in children with cirrhosis and hepatocellular carcinoma. Pediatr Transplant. 2011; 15:573-579. DOI
36. Hoot AC, Russo P, Judkins AR, Perlman EJ, Biegel JA. Immunohistochemical analysis of hSNF5/INI1 distinguishes renal and extra-renal malignant rhabdoid tumors from other pediatric soft tissue tumors. Am J Surg Pathol. 2004; 28:1485-1491. DOI
37. Wassef M, Blei F, Adams D, ISSVA Board and Scientific Committee. Vascular Anomalies Classification: Recommendations From the International Society for the Study of Vascular Anomalies. Pediatrics. 2015; 136:e203-14. DOI
38. Otte JB, Zimmerman A. The role of liver transplantation for pediatric epithelioid hemangioendothelioma. Pediatr Transplant. 2010; 14:295-297. DOI
39. Guiteau JJ, Cotton RT, Karpen SJ. Pediatric liver transplantation for primary malignant liver tumors with a focus on hepatic epithelioid hemangioendothelioma: the UNOS experience. Pediatr Transplant. 2010; 14:326-331. DOI
40. Aldén J, Baecklund F, Psaros Einberg A. Is primary hepatic angiosarcoma in children an indication for liver transplantation?-A single-centre experience and review of the literature. Pediatr Transplant. 2021; 25:e14095. DOI
41. Panayotova G, Lunsford KE, Latt NL. Expanding indications for liver transplantation in the era of liver transplant oncology. World J Gastrointest Surg. 2021; 13:392-405. DOI
42. Alkofer B, Samstein B, Guarrera JV. Extended-donor criteria liver allografts. Semin Liver Dis. 2006; 26:221-233. DOI
43. Neil DAH, Minervini M, Smith ML. Banff consensus recommendations for steatosis assessment in donor livers. Hepatology. 2021. DOI
44. Boteon YL, Martins PN, Muiesan P. Machine perfusion of the liver: Putting the puzzle pieces together. World J Gastroenterol. 2021; 27:5727-5736. DOI
45. Tien C, Remulla D, Kwon Y. Contemporary strategies to assess and manage liver donor steatosis: a review. Curr Opin Organ Transplant. 2021; 26:474-481. DOI
46. Werner MJM, van Leeuwen OB, de Jong IEM. First report of successful transplantation of a pediatric donor liver graft after hypothermic machine perfusion. Pediatr Transplant. 2019; 23:e13362. DOI
47. Cussa D, Patrono D, Catalano G. Use of Dual Hypothermic Oxygenated Machine Perfusion to Recover Extended Criteria Pediatric Liver Grafts. Liver Transpl. 2020; 26:835-839. DOI
48. Husain AN, Chang A, Ranganathan S. Diagnosis in Pediatric Transplant Biopsies. Surg Pathol Clin. 2010; 3:797-866. DOI
49. Demetris AJ, Jaffe R, Starzl TE. A review of adult and pediatric post-transplant liver pathology. Pathol Annu. 1987; 22:347-386.
50. Hernandez-Alejandro R, Sharma H. Small-for-size syndrome in liver transplantation: New horizons to cover with a good launchpad. Liver Transpl. 2016; 22:33-36. DOI
51. Masuda Y, Yoshizawa K, Ohno Y. Small-for-size syndrome in liver transplantation: Definition, pathophysiology and management. Hepatobiliary Pancreat Dis Int. 2020; 19:334-341. DOI
52. Riddiough GE, Christophi C, Jones RM. A systematic review of small for size syndrome after major hepatectomy and liver transplantation. HPB (Oxford). 2020; 22:487-496. DOI
53. Demetris AJ, Bellamy CO, Gandhi CR. Functional Immune Anatomy of the Liver-As an Allograft. Am J Transplant. 2016; 16:1653-1680. DOI
54. Mengel M, Loupy A, Haas M. Banff 2019 Meeting Report: Molecular diagnostics in solid organ transplantation-Consensus for the Banff Human Organ Transplant (B-HOT) gene panel and open source multicenter validation. Am J Transplant. 2020; 20:2305-2317. DOI
55. Demetris AJ, Bellamy C, Hübscher SG. 2016 Comprehensive Update of the Banff Working Group on Liver Allograft Pathology: Introduction. of Antibody-Mediated Rejection. Am J Transplant. 2016; 16:2816-2835. DOI
56. Banff Working Group on Liver Allograft Pathology. Importance of liver biopsy findings in immunosuppression management: biopsy monitoring and working criteria for patients with operational tolerance. Liver Transpl. 2012; 18(10):1154-1170. DOI
57. Demetris AJ, Murase N, Nakamura K. Immunopathology of antibodies as effectors of orthotopic liver allograft rejection. Semin Liver Dis. 1992; 12:51-59. DOI
58. O’Leary JG, Michelle Shiller S, Bellamy C. Acute liver allograft antibody-mediated rejection: an inter-institutional study of significant histopathological features. Liver Transpl. 2014; 20:1244-1255. DOI
59. Zhou S, Mitsinikos T, Emamaullee J. Clinicopathologic Characteristics of Late Acute Antibody-mediated Rejection in Pediatric Liver Transplantation. Transplantation. 2021; 105:2045-2053. DOI
60. Wozniak LJ, Naini BV, Hickey MJ. Acute antibody-mediated rejection in ABO-compatible pediatric liver transplant recipients: case series and review of the literature. Pediatr Transplant.. 2017;21. DOI
61. Crawford AR, Lin XZ, Crawford JM. The normal adult human liver biopsy: a quantitative reference standard. Hepatology. 1998; 28:323-331. DOI
62. Oshima K, Voltaggio L. Survival Guide to Liver Biopsies. Innovative Science Press: Arlington VA (USA); 2021.
63. Neil DA, Hübscher SG. Current views on rejection pathology in liver transplantation. Transpl Int. 2010; 23:971-983. DOI
64. Feng S, Bucuvalas JC, Demetris AJ. Evidence of Chronic Allograft Injury in Liver Biopsies From Long-term Pediatric Recipients of Liver Transplants. Gastroenterology. 2018; 155:1838-1851.e7. DOI
65. Feng S, Demetris AJ, Spain KM. Five-year histological and serological follow-up of operationally tolerant pediatric liver transplant recipients enrolled in WISP-R. Hepatology. 2017; 65:647-660. DOI
66. Feng S, Bucuvalas JC, Mazariegos GV. Efficacy and Safety of Immunosuppression Withdrawal in Pediatric Liver Transplant Recipients: Moving Toward Personalized Management. Hepatology. 2021; 73:1985-2004. DOI
67. Hübscher S. What does the long-term liver allograft look like for the pediatric recipient?. Liver Transpl. 2009; 15:S19-24. DOI
68. Briem-Richter A, Ganschow R, Sornsakrin M. Liver allograft pathology in healthy pediatric liver transplant recipients. Pediatr Transplant. 2013; 17:543-549. DOI
69. Ekong UD, Melin-Aldana H, Seshadri R. Graft histology characteristics in long-term survivors of pediatric liver transplantation. Liver Transpl. 2008; 14:1582-1587. DOI
70. Kelly D, Verkade HJ, Rajanayagam J. Late graft hepatitis and fibrosis in pediatric liver allograft recipients: Current concepts and future developments. Liver Transpl. 2016; 22:1593-1602. DOI
71. Demetris AJ, Jaffe R, Sheahan DG. Recurrent hepatitis B in liver allograft recipients. Differentiation between viral hepatitis B and rejection. Am J Pathol. 1986; 125:161-172.
72. Ocwieja KE, Vargas SO, Elisofon SA. Pediatric post-transplant hepatic kaposi sarcoma due to donor-derived human herpesvirus 8. Pediatr Transplant. 2019; 23:e13384. DOI
73. Samant H, Vaitla P, Kothadia JP. Post Transplant Lymphoproliferative Disorders. StatPearls: Treasure Island (FL); 2021.
74. Liu Y, Sun LY, Zhu ZJ. Post-transplant lymphoproliferative disorder after paediatric liver transplantation. Int J Clin Pract. 2021; 75:e13843. DOI
75. Lo RC, Chan SC, Chan KL. Post-transplant lymphoproliferative disorders in liver transplant recipients: a clinicopathological study. J Clin Pathol. 2013; 66:392-398. DOI
76. Hsu CT, Chang MH, Ho MC. Post-transplantation lymphoproliferative disease in pediatric liver recipients in Taiwan. J Formos Med Assoc. 2019; 118:1537-1545. DOI
77. Hussein K, Rath B, Ludewig B. Clinico-pathological characteristics of different types of immunodeficiency-associated smooth muscle tumours. Eur J Cancer. 2014; 50:2417-2424. DOI
78. Deyrup AT, Lee VK, Hill CE. Epstein-Barr virus-associated smooth muscle tumors are distinctive mesenchymal tumors reflecting multiple infection events: a clinicopathologic and molecular analysis of 29 tumors from 19 patients. Am J Surg Pathol. 2006; 30:75-82. DOI
79. Jonigk D, Laenger F, Maegel L. Molecular and clinicopathological analysis of Epstein-Barr virus-associated posttransplant smooth muscle tumors. Am J Transplant. 2012; 12:1908-1917. DOI
80. Robinson CH, Coughlin CC, Chanchlani R. Post-transplant malignancies in pediatric organ transplant recipients. Pediatr Transplant. 2021; 25:e13884. DOI
81. De Smet F, Antoranz Martinez A, Bosisio FM. Next-Generation Pathology by Multiplexed Immunohistochemistry. Trends Biochem Sci. 2021; 46:80-82. DOI
82. Acs B, Hartman J. Next generation pathology: artificial intelligence enhances histopathology practice. J Pathol. 2020; 250:7-8. DOI
83. Shaked A, DesMarais MR, Kopetskie H. Outcomes of immunosuppression minimization and withdrawal early after liver transplantation. Am J Transplant. 2019; 19:1397-1409. DOI
84. Russo P, Magee JC, Anders RA, Childhood Liver Disease Research Network (ChiLDReN). Key histopathologic features of liver biopsies that distinguish biliary atresia from other causes of infantile cholestasis and their correlation with outcome: a multicenter study. Am J Surg Pathol. 2016; 40:1601-1615. DOI
85. Superina R. Biliary atresia and liver transplantation: results and thoughts for primary liver transplantation in select patients. Pediatr Surg Int. 2017; 33:1297-1304. DOI
86. Emerick KM, Rand EB, Goldmuntz E. Features of Alagille syndrome in 92 patients: frequency and relation to prognosis. Hepatology. 1999; 29:822-9. DOI
87. Kamath BM, Ye W, Goodrich NP, Childhood Liver Disease Research Network (ChiLDReN). Outcomes of childhood cholestasis in alagille syndrome: results of a multicenter observational study. Hepatol Commun. 2020; 4:387-398. DOI
88. Mehl A, Bohorquez H, Serrano MS. Liver transplantation and the management of progressive familial intrahepatic cholestasis in children. World J Transplant. 2016; 6:278-290. DOI
89. Herrmann U, Dockter G, Lammert F. Cystic fibrosis-associated liver disease. Best Pract Res Clin Gastroenterol. 2010; 24:585-92. DOI
90. Terziroli Beretta-Piccoli B, Vergani D, Mieli-Vergani G. Autoimmune sclerosing cholangitis: Evidence and open questions. J Autoimmun. 2018; 95:15-25. DOI
91. Sarcognato S, Sacchi D, Grillo F. Autoimmune biliary diseases: primary biliary cholangitis and primary sclerosing cholangitis. Pathologica. 2021; 113:170-184. DOI