Hepatoblastoma and Pediatric Hepatocellular Carcinoma: An Update

Abstract

Hepatoblastomas (HBs) and pediatric hepatocellular carcinomas (HCCs) together account for almost 80% of primary malignant liver tumors in children and adolescents/young adults. Children’s Hepatic International Collaboration (CHIC), Children’s Oncology Group (COG), SociétéInternationale d’Oncologie Pédiatrique (SIOP), and International Childhood Liver Tumors Strategy Group trials have contributed to define prognostic factors and risk stratification in these tumors. The recently proposed histologic International Consensus classification of HB and HCC in children based on retrospective analysis from CHIC cases represents the base to define entities with homogeneous clinicopathologic and molecular features. This review will provide a morphologic guide for the upcoming International Liver Tumor treatment trial (Pediatric Hepatic International Tumour Trial) to be conducted through several continents. There will be an emphasis on molecular features and immunohistochemical markers for the definition of the individual histologic subtypes of HB and to better characterize the group of liver tumors in the provisional category of hepatocellular neoplasm—not otherwise specified. A brief overview of HCC in children will also be provided.

Keywords

hepatoblastoma hepatocellular carcinoma hepatocellular neoplasm-NOS fibrolamellar HCC β-catenin fetal hepatoblastoma

Introduction

Primary malignant tumors of the liver are extremely rare accounting for only 1% of all malignancies in the pediatric age-group, with less than 1.5 cases per million children under 18 years.¹ Of these, more than two-thirds are hepatoblastomas (HBs), the remaining being hepatocellular carcinomas (HCCs) and the very rare embryonal sarcomas of liver. A recent increase in the incidence of HBs has been reported, probably in relation with the higher number of survivors of premature birth and the predisposition to develop HB in infants with birth weight lower than 1500 g.² HB are typical of the first 3 years of life and can be congenital.³ A subset of HB occurs in the context of familial syndromes, such as Beckwith–Weidemann syndrome, Simpson–Golabi–Behmel syndrome, Sotos syndrome, familial adenomatous polyposis coli, and constitutional trisomy 18.^4–6 Despite this syndromic association, the underlying liver is usually normal.

HCCs occur mostly in older children and adolescents either in a noncirrhotic liver or in association with a variety of fibrosing diseases or cirrhosis of different etiologies. Fibrolamellar HCC occurs in a normal liver and manifests in teenagers and young adults. Histology is crucial in planning the therapeutic strategies, since HB is highly chemosensitive while HCC is poorly responsive with need of an upfront surgical approach, including primary resection or early referral for liver transplantation. The histologic diagnosis is not always straightforward, and the morphologic overlap between HCC and HB may be challenging. Less than 2 decades ago, the identification of hybrid morphologic features in a set of HB, mostly pretreated, led to the introduction of the so-called transitional liver tumor, an histologically poorly defined epithelial liver tumor with HCC and HB-like features, typical of older children, clinically highly aggressive. The history of a previous treatment was a strong limitation of the study, being difficult to exclude the possibility that HCC-like features might represent posttherapy changes. Thus, the existence of this entity has been challenged, and the histologic criteria, poorly defined, have contributed to animate the scientific debate.^7,8

However, the recent molecular and cytogenetic studies shed new light on hybrid tumors, showing a genetic profile intermediate between HB and HCC. This evidence raised the need for the community of pathologists to better define the morphologic characteristics of these neoplasms. To highlight the characteristics of “work in progress” defining this group of hybrid tumors, the consensus conference in 2004 recognized a category of hepatocellular neoplasms—not otherwise specified (HCN-NOS).

This review will analyze the histologic criteria for the diagnosis of primary nonmesenchymal tumors of the liver in children with a special emphasis on HCN-NOS.⁹

Classifications

The term “hepatoblastoma” was introduced in 1962 by Willis based on the presence of hepatic epithelial parenchyma resembling fetal or embryonal liver.

Ishak and Glunz, in 1967, created the backbone for the current classification, dividing the tumors into an epithelial type, with a further break down into fetal and embryonal subtypes, and a mixed epithelial and mesenchymal type.

Over the years, the pediatric collaborative therapeutic protocols with systematic central histopathological review in the context of Children’s Oncology Group (COG), International Childhood Liver Tumors Strategy Group (SIOPEL), Gesellschaft fur Pädiatrische Onkologie undHämatologie (GPOH), and Japanese Study Group for Pediatric Liver Tumors (JPLT) contributed to redefine the clinicopathologic landscape of hepatocellular tumors in children and gave a strong impulse to the understanding of their biology.

New prognostically significant morphologic categories were identified: the favorable well-differentiated fetal (WDF) HB and the aggressive small cell undifferentiated (SCU) variant, reported by Finegold et al.^10–12; the “transitional liver tumor” and minor variants like cholangioblastic HB.^7,13 New immunohistochemical markers and molecular data have contributed to redefine the different groups, and the need to better standardize the nomenclature of pediatric liver tumors has emerged.

A retrospective review of all cases enrolled in various International study groups under the rubric of Children’s Hepatic International Collaboration (CHIC) led to the first International Pathology Symposium in 2011, with the elaboration of the Pediatric Liver Tumors Consensus Classification (PLTCC), currently used by many centers.^9,14,15 However, this classification was provisional and needed to be tested in large prospective studies with clinical correlation. PLTCC is the basis for the histologic diagnosis in the current Pediatric Hepatic International Tumor Trial (PHITT) clinical trial, just opened in the United States and Europe, and set to open in Japan. It is applied only on biopsies or primary resections. In fact, postchemotherapy specimens may show areas of HB indistinguishable from HCC or WDF morphology as a consequence of therapy-induced differentiation/maturation.¹⁶

The PHITT clinical trial has the goal to integrate clinicopathologic findings and biological parameters in a homogeneously treated population. The therapeutic stratification of HB is based on levels of alpha-fetoprotein (AFP) and PRETEXT stage (Figure 1).¹⁷ The pathologist plays an important role in the initial diagnosis in general, but he becomes crucial in addressing the further treatment in categorization of HCN-NOS or HCC and in primary resection of pure WDF HB (see below). For these histotypes, the PHITT (AHEP1531 in the United States) requires a rapid review by Central Pathology (Table 1).

Table 1.

Criteria for Rapid Review in Pediatric Hepatic International Tumour Trial.

Conditions Requiring Rapid Review	Rationale
Primary hepatic resection for low-stage HB	Need to capture pure well-differentiated fetal histology for risk stratification
AFP < 100 ng/dL	Exclude other liver malignancies (eg, malignant rhabdoid tumors)
Age at diagnosis of HB > 8 years	To capture HCN-NOS tumors
Provisional^a diagnosis of HCC and HCN-NOS	To correctly identify HCN-NOS for high-risk HB stratification

Abbreviations: AFP, alpha-fetoprotein; HB, hepatoblastoma; HCC, hepatocellular carcinomas; HCN-NOS, hepatocellular neoplasm—not otherwise specified.

Provisional because submitted to Children’s Oncology Group as an amendment pending approval.

Macroscopic Examination

Macroscopic examination is crucial to define the relationship between morphology and biology either in upfront surgery (primary resection) or surgery after chemotherapy.

The principal steps in macroscopic examination of the pediatric liver with a malignancy are reported in Table 2. Readers can also refer to the College of American Pathologists guidelines.¹⁸ It is important to map liver tumor resection specimens (similar to Wilms tumor or osteosarcoma) in order to facilitate the assessment of the response to chemotherapy, morphology of the residual tumor, and to study its correlation with prognosis (Figure 2).

Table 2.

Guide for Gross Evaluation of Resections/Explants for Tumors.

• Biopsy—triage for H&E and stains + frozen. Request 4 to 6 cores for all ancillary studies (more the better). If open biopsy then as needed. Normal tissue biopsy if possible (freeze and histology)

• Resection/explant:

- Ink margins, submit vascular margin first

- Serially cut to include right and left lobes in each

- Identify and document all tumor nodules

- Take photographs—use for mapping as shown

- Map the largest and second largest if many and submit the largest cross-section with designation of sections. Freeze tissue from parallel sections of tumor and normal liver (3–4 vials, sampling different areas and nodules)

- Fix tissue adequately and submit for paraffin sections

- Map allows opportunity to go back for more sections in worrisome/significant areas

- Attempt to give percent necrosis chemotherapy response for largest 2 nodules.

- Note vascular invasion, gross, and microscopic

Abbreviations: H&E, hematoxylin and eosin.

Hepatoblastoma

HB has an increased incidence in preterm infants of low birth weight. It is usually seen in children in the first 5 years of life but can occur in older children in the first decade and even in the second decade of life. The tumor usually presents as a mass with a protuberant abdomen. The liver enzymes are generally normal. Thrombocytosis with platelet counts 350 to 450 000 and higher is frequent. The AFP is elevated and may be in the millions.

HB reproduces the embryologic development of liver.^12,19,20 According to the cytoarchitectural characteristics of the tumor and the presence of only epithelial or also mesenchymal or undifferentiated components, several histologic variants with different components are recognized.

Epithelial HB

WDF HB (Figure 3) is composed of uniformly small, polygonal cells with central nucleus without nucleoli and an abundant eosinophilic to clear, vacuolated, cytoplasm. The mitoses are rare (≤2/10 hpf). There is no necrosis or pleomorphism. The tumor has classic light and dark areas based on cells with clear or eosinophilic cytoplasm that tend to show some degree of zonation. The cells are arranged in trabeculae 1 to 2 cells thick and rarely may be in an acinar arrangement. The diagnosis of pure WDF HB should be made only on primary resection specimens with 100% of tumor composed of fetal cells. Biopsies or postchemotherapy specimens cannot be diagnosed as pure WDF HB.

Immunostains show a strong positivity for glutamine synthetase (GS), and a weak but diffuse staining with glypican 3 (GPC3), usually in a fine stippled pericanalicular cytoplasmic staining pattern. A coarse, strong cytoplasmic staining with GPC3 should warrant reevaluation of the diagnosis. β-catenin (β-cat) is frequently membranous and even cytoplasmic, with rare nuclear staining. Strong diffuse nuclear staining for β-cat is rare.

The diagnosis of pure WDF in the PHITT protocol identifies the group of very low-risk patients (Group A), treated with upfront surgery with clear resection margins without subsequent chemotherapy.¹⁵

Crowded fetal (CF) HB (mitotically active fetal) is a fetal HB with closely packed cells and mitotic activity more than 2/10 hpf. Nuclear–cytoplasmic (N/C) ratio is higher, the nuclei are round, and the cytoplasm is more eosinophilic and dense (Figure 4). The light and dark areas seen with WDF histology are no longer evident. The increased N/C ratio gives this component a more closely packed (“crowded”) appearance. Nucleoli and extramedullary hematopoiesis (EMH) are frequent. Areas of WDF and embryonal HB are intermixed with focal transition from CF to embryonal without demarcation. Nuclear pleomorphism and atypical mitoses are absent. The presence of the latter 2 features would suggest a pleomorphic fetal HB. Areas of “crowded-like” histology without mitoses in fetal HB would be considered WDF component.

The tumor cells are strongly positive for GPC3 in a diffuse, coarse, cytoplasmic staining pattern and for GS. Many positive nuclei for β-cat and Cyclin D1 are seen. These tumors will require chemotherapy.

Embryonal HB: This morphology corresponds to the embryonic stage of liver development (Figure 5). The cells have high N/C ratio, scant cytoplasm with indistinct borders, and a large, angulated to oval (rather than round as seen in fetal subtypes) nucleus with a prominent nucleolus. The cell density is also increased. Mitoses are frequent; necrosis may be seen. Rosettes are easily seen and may resemble glandular or tubular structures. Serpentine and microcystic pattern can be seen. Myxoid change may be noted in the microcystic areas. Zonation, with embryonal cells in the center surrounded by CF cells, which are rimmed by varying proportions of WDF cells, is frequent. Immunohistochemistry shows uniform nuclear β-cat positivity. GPC3 ranges from absent to strong, coarse, diffuse cytoplasmic staining resembling that seen in CF areas. GS is also variable from patchy single cell staining to negative. The staining may not be uniform as embryonal HB recapitulates embryonic liver development, and the more immature tumors may be undifferentiated and negative for GPC3 and GS all markers that show up in later stages of development. The most undifferentiated areas may overlap phenotypically with the SCU areas (described below).

SCU HB: (Figure 6) This entity was in the past uniformly assigned a worse prognosis both in cases of diffuse small cell morphology and in tumors showing only a minimal small cell component.^11,19,21,22 SCU areas were represented as the “anaplastic variant of HB.”

The identification of INI1 loss of expression in tumors with a diffuse SCU morphology has contributed to clarify their histogenetic relationship with malignant rhabdoid tumors.^22,23 The cells show a variation from tightly packed cells in sheets with scarce cytoplasm to a frankly rhabdoid phenotype with eccentric nucleus and eosinophilic cytoplasmic globules and prominent nucleolus.

HBs with an SCU component show foci of small cells with high N/C ratio, pale vesicular to hyperchromatic nucleus, scant cytoplasm, and indistinct cell borders usually restricted to the centers of embryonal HB areas. The mitotic rate is variable from high to very low. INI1 is usually preserved, unlike in rhabdoid tumors.²⁴ Whether foci of SCU, usually less than 5% of tumor, with INI preserved are associated with an aggressive clinical behavior, remains to be determined.²⁴ Small series have reported a worse outcome and the review by Haas et al. suggested an overall poor outcome for this subtype, although it was based entirely on retrospective data.¹¹ While the previous COG study warranted the recognition of any small cell areas in the tumor for treatment on a higher risk stratum, the PHITT will collect data on this subgroup without necessarily assigning a higher risk. It is important to note that foci of extramedullary hematopoiesis are always INI1 lost and should be excluded in the evaluation of the stain.

Small cell component should be differentiated from the primitive blastemal areas of HB. These areas have a more ovoid to spindled morphology but can sometimes be composed of round to angulated cells (similar to the blastema seen in Wilms tumor) (Figure 7). Many blastemal areas are associated with mesenchymal elements within the tumor and, in some cases, may even blend with osteoid or they may be dispersed among all epithelial components and at the edge of fetal areas. The blastemal component may persist or may evolve into mixed HB in postchemotherapy specimens. By contrast, SCU areas are inconspicuous postchemotherapy and are more readily appreciated on untreated tumor resections or biopsies. Immunohistochemically, apart from retention of INI1, SCU and blastemal cells share a similar phenotype with strong and diffuse nuclear β-cat; express pancytokeratin, cytokeratin 19, and vimentin; and are negative for Hep Par 1, AFP, GS, and GPC3. SCU may occur in association with blastemal component, but their distinction may be difficult if not impossible; however, it does not impact the current therapeutic approach.

Macrotrabecular (MT) HB (Figure 8) is a provisional category, representing a morphologic pattern rather than a histotype. Its morphologic overlap with HCC and HCN-NOS may be a diagnostic challenge. It is characterized by trabeculae and cords greater than 5 cells in thickness, while the original descriptions proposed 20-cell thick trabeculae.^9,20 The cells can show fetal or embryonal morphology and are associated with other areas of typical HB but can rarely be the only pattern on a biopsy. In HCC, the trabeculae frequently are more than 10 to 20 cells thick and do not show associated areas of HB, although rare HB coexisting with HCC may be encountered as distinct nodules in the same liver. The immunohistochemical profile of the MT areas usually mimics the histologic component of CF or embryonal HB. They show strong nuclear β-cat expression in contrast with membranous β-cat staining in the thicker trabeculae of pediatric HCC.

Pleomorphic epithelial HB (Figure 8) shows bizarre, pleomorphic cells including giant cells. It is more frequent in postchemotherapy resection specimens.⁹ Rarely, it may be the predominant component within metastases. The cells resemble mitotically active fetal HB or embryonal HB but show pleomorphism and increased and abnormal mitoses. The presence of cells with large conspicuous nucleoli can simulate a HCC especially if seen as the only component in a pretreatment biopsy or may overlap with HCN-NOS.⁹ The pleomorphic cells frequently differ from HCC showing strong nuclear β-cat, strong GS, and variable GPC3 staining. Immunostains are less useful in the distinction from HCN-NOS.

Other epithelial components: Ductular differentiation may sometimes be seen at the periphery of other epithelial subtypes, especially fetal HB, and may be associated with a blastemal component. The ductular elements can be highlighted by a cytokeratin stain (CK19 and CK7) or MOC31.

Mixed epithelial HB is a category including HB showing an admixture of different epithelial components and should be distinguished from mixed HB (see below).

Cholangioblastic HB shows a prominent ductular differentiation. This variant needs to be differentiated from a ductular reaction noted at the periphery of the tumor, especially after chemotherapy, separating the treated tumor from normal liver. The cholangioblastic component of HB frequently shows nuclear β-cat stain, which is not seen in the reactive ductular population. These cells are negative for GS and GPC3, distinguishing them from acinar structures in areas of fetal HB.^13,25

Mixed HB

This morphologic designation comprises both epithelial and mesenchymal elements. The most common mesenchymal elements are osteoid and less often, cartilage (Figure 8). Muscle, especially skeletal muscle, fat, and primitive spindle cell mesenchyme may also be seen. The mesenchymal component represents an integral part of the tumor and is not a result of chemotherapy or a “metaplastic” change. It shows a similar staining as the epithelial component including nuclear β-cat in the primitive mesenchyme and in cells bordering bony elements (“osteoblasts”). The mesenchymal component is negative for GPC3.

Teratoid HB (HB with heterologous elements) is a mixed HB with neural/neuroectodermal differentiation represented by mature brain, primitive neuroepithelial components forming tubules and rosettes, as well as melanin and retinal pigment (Figure 8). Squamous and mucinous glands may also be present, but these can also be seen without neural elements in pure epithelial HB. Rarely glandular elements with subnuclear and supranuclear vacuoles can raise consideration for areas mimicking primary yolk sac tumor of the liver. Spindle cell mesenchyme with rhabdomyoblastic differentiation, cartilage, and bone may also occur. In general, teratoid HB shows larger areas of blastema when adequately sampled. Areas of neuroendocrine differentiation in epithelial components may be rarely encountered.

Hepatocellular Neoplasm-NOS

HCN-NOS are tumors with hybrid features, making their classification difficult. This “basket” has been created to include lesions previously defined as “transitional cell liver tumors” (TCLT), described as highly aggressive tumors with overlapping features of both HB and HCC.⁷ The existence of TCLT has been questioned since their original description. However, a subgroup of hepatocellular tumors not classifiable as HB or HCC for their hybrid morphology, with characteristic clinical features, has emerged from both SIOPEL and COG studies. These HCN-NOS occur in older children (although exceptions in younger children do exist), usually over the age of 8 years, and are associated with very high levels of AFP. Almost universally, HCN-NOS appear to occur in a background of normal liver with no predisposing liver disease; however, the findings are preliminary and need to be confirmed in large prospective series.

The change in nomenclature reflects the necessity to highlight the provisional nature of the category until molecular studies will better define the biology of these tumors. Interestingly, recent studies on animal models suggest that they might represent “derailed HB,” and in the future, it is possible that they might be renamed as HB with HCC features.²⁶ Histological features are heterogeneous, with cells varying from medium-sized or intermediate (between HCC and fetal HB), to large cells with clear to eosinophilic cytoplasm (Figures 9 and 10). Hepatobastoma type cells in a MT or nested pattern may be seen in association with intermediate cells. Pleomorphic or multinucleated cells may be present. In a subset of tumors, coexisting HCC and HB-like areas may be seen. β-cat nuclear stain supports the biologic relationship of these tumors with HB. HCN-NOS should be distinguished from fetal or embryonal HB containing small foci of pleomorphic or MT HB, which lack the intermediate cell component or the HCC component. Immunohistochemistry is useful to differentiate HCN-NOS from HCC. In fact, β-cat is usually negative or only focally positive (nuclear) in HCC compared to the heterogeneous pattern seen in HCN-NOS. HCN-NOS are treated as high-risk HB and not as HCC in the PHITT; thus their identification is critical for treatment.

Prognostic Parameters in HB

Tumor stage

In HB, the staging system is based on imaging using the PRETEXT system. While a low-stage disease amenable to surgery would be treated by upfront surgery, any higher stage tumor or unresectable tumor at diagnosis would undergo a biopsy confirmation of diagnosis prior to the start of therapy. A higher stage tumor, especially with distant metastasis, may preclude transplantation in most cases, unless chemotherapy leads to a resolution of the metastases.

AFP: Most HB are associated with elevated levels of AFP, and the level is not determined by the age, stage, or histotype of HB. AFP levels are usually elevated in the thousands or even in the millions. Significant subsets of patients have AFP levels between 100 and 1000 ng/mL. AFP levels below 100 ng/mL have been associated with a poor outcome, and these patients are stratified as high risk in the PHITT irrespective of histology; however, a rapid histologic review is required for these tumors to confirm their hepatoblastic nature. The CHIC database suggests that low AFP levels are distributed across all age groups and stages of PRETEXT.

Vascular invasion has been associated with higher stage tumors and recurrence/metastasis. Correlation with PRETEXT annotation factors prospectively in the PHITT will enable this assessment and, together with biology, may help identify prognostic biomarkers for this subgroup. Microscopic vascular invasion outside the tumor borders may indicate a worse outcome.

Margin of resection: A tumor-free resection margin is thought to be associated with a better outcome than a positive margin. This feature will be further assessed in the PHITT study.

Chemotherapy Response

The effect of chemotherapy is well recognized in HB; however, the lack of an adequate and standardized gross mapping of posttherapy specimens represents an important limitation in the evaluation of their prognostic significance. Most treated HBs show definitive shrinkage of the tumor radiologically. An isolated series²⁷ mentioned some relationship with percent necrosis in transplanted HB, but these data have not yet been validated across all HB stages. Saxena et al. reported osteoid in postchemotherapy specimens and speculated the role of maturation as a favorable prognostic feature.²⁸ In the study by Wang et al., tumor size and vascular invasion were significant predictors of metastatic disease at univariate analysis; moreover, pretreatment tumor size at imaging and vascular invasion were also significant independent predictors.¹⁶

Most frequent posttherapy changes reported include fibrosis, necrosis, vascular ectasia, peliosis-like appearance, and abundant hemosiderin deposition within tumor nodules.^16,29 Crowded fetal areas may be barely recognizable due to cellular differentiation/maturation and lack of mitoses. The embryonal components have a good response to therapy; however, small residual areas may be found. The blastemal component may persist or evolve into more mature mixed HB areas. Small foci of SCU tend to disappear and may not be recognizable after therapy. The teratoid component may show variable response. Pleomorphic areas or areas mimicking HCC have been reported in 20% of HB by Wang et al.;¹⁶ however, their incidence and the potential prognostic significance have to be systematically assessed. The PHITT study will analyze data to generate prognostic indicators in posttherapy specimens.

Hepatocellular Carcinoma

HCC represents 20% of all malignant liver tumors diagnosed in children and constitutes a clinically challenging group often presenting as large, unresectable lesions, typically in older children/adolescents. Pediatric HCC includes 2 groups. One is associated with underlying metabolic and/or genetic diseases either typical to the first decade of life or common to adults and children. The most common metabolic/genetic conditions associated with pediatric HCC include hemochromatosis, hereditary tyrosinemia, progressive familial cholestasis, especially Bile salt export pump disease, multidrug resistance protein 3 defect and tight junction protein 2 gene deficiency, alpha-1-antitrypsin deficiency (now rare due to treatment), glycogen storage diseases, and less often in biliary atresia and viral hepatitis. The second group arises in livers without underlying chronic disease. These tumors may demonstrate a wide morphologic spectrum^12,30 (Figure 11). The histologic classification of HCC is the same as described in the WHO Classification of Liver tumors, with grading into well-differentiated (WD), moderately differentiated (MD), and poorly differentiated (PD) tumors.³¹

WDHCC show uniform appearing hepatocytes arranged in trabeculae at least 3 cells thick with a prominent sinusoidal pattern, frequent bile production, and pseudoacinar arrangement. The nodule is usually separated from surrounding liver by a distinct fibrous pseudocapsule. No portal areas are seen within the tumor although a few entrapped portal areas may be seen at the infiltrating border. Unpaired arteries may be present. The individual tumor cells show vacuolated cytoplasm and round nuclei with mild nuclear pleomorphism and variable distinct nucleoli. Immunostaining shows variable GPC3 and GS staining, and β-cat is usually negative for nuclear staining.^32,33 Reticulin pattern is usually lost, unless the tumor is an early HCC.

MDHCC shows larger cells arranged in distinct trabecular arrangement with trabeculae 15 to 20 cells thick, lined by sinusoids. Bile production is evident, and a moderate degree of nuclear pleomorphism is evident. The cells show abundant cytoplasm, large nuclei with prominent large nucleoli and intranuclear inclusions, as well as eosinophilic cytoplasmic globules, possibly alpha-1-antitrypsin. The tumor nodules may be multinodular, and areas of necrosis may be seen. A pseudoacinar arrangement may be seen. GPC3 is positive along with GS in about half the tumors. β-cat is again variable, with only a subset showing weak and very focal nuclear staining. These tumors may be associated with a stronger GS expression.

PDHCC is composed of sheets or nests of small cells, not resembling hepatocytes. The N/C is high; there is a variable degree of nuclear pleomorphism. Prominent nucleoli and intranuclear inclusions may be seen. Numerous mitoses and necrosis are noted. Occasional cases may show pleomorphism and dense fibrosis, though these variants are rare in the pediatric age-group.

Other variants of HCC are unusual in the pediatric age-group. A few tumors may arise from an adenoma, especially those that are β-cat mutated.^34,35 The transformation from adenoma to WDHCC may be difficult to identify. Besides nuclear β-cat, GS expression may be strong and diffuse, and GPC3 may or may not be positive. A reticulin stain may, however, show a loss of normal pattern in the transformed nodule.³⁴

Fibrolamellar HCC has a hard consistency and may show a central scar. It is characterized by large, eosinophilic (oncocytic) hepatocytes with prominent nucleoli embedded within lamellar fibrotic tissue (Figure 11). Tumor cells express biliary, hepatocytic, and hepatic-progenitor markers and carry fewer genomic and epigenetic alterations than classic HCC. A recurrent-specific translocation PRKACA-DNAJB1 has been found recently.^36–38 Immunohistochemistry shows strong positivity for biliary markers such as CK7 and CK19 besides CD68. Hepatocellular markers such as Hep-par1 and Arginase 1 are more variable. β-cat, GS, and GPC3 are generally negative, although GPC3 stain shows a faint blush staining of cytoplasm. FISH for PRKACA break-apart probe is useful for diagnosis. The background liver is usually normal.

Combined hepatocellular-cholangiocarcinomas and HCCs with neuroendocrine component are extremely rare, and their incidence in pediatric age is not known. Their morphology overlaps with adult tumors.

Biology of Liver Tumors

HB: HBs are neoplasms with relatively stable genomes. Only a limited number of structural and numerical abnormalities are detected by cytogenetic or cytogenomic analysis in these tumors, including trisomies of chromosomes 2, 8, and 20, and rare structural abnormalities such as those involving chromosome 1q, including the t(1;4) unbalanced translocation.³⁹ Activation of the canonical Wnt-signaling pathway occurs in the vast majority of HBs through somatic mutations of CTNNB1, or rarely, other Wnt-signaling genes, but it can also be caused by germ line alterations, including APC mutations.^40,41 CTNNB1 mutations (encoding β-cat) can be found in over 80% of HBs tested, providing evidence of the important driver role of Wnt pathway activation in these tumors.^42–44 The second most commonly mutated gene in HB, found in only 5% to 10% of HB cases tested so far, and associated with poor prognosis, is NFE2L2 (also known as NRF2), which encodes a transcription factor involved in the antioxidant response pathway. Other reported features include aberrant reprogramming of imprinted genes such as IGF2, H19, DLK1, GTL2, PEG3, PEG10, and MEG3, abundantly expressed in the fetal liver.^45–47 Integrated genomic analysis demonstrated 3 distinct risk-stratifying molecular HB subtypes associated with low, intermediate, and high risk.^26,48 High-risk tumors are characterized by high NFE2L2 activity; high LIN28B, HMGA2, SALL4, and AFP expression; low let-7 expression; and HNF1A activity, as well as high coordinated expression of oncofetal proteins and stem cell markers.

Hepatocellular neoplasm-NOS: Molecular characterization demonstrates the presence of molecular and genetic characteristics of both HB and HCC. These changes include, almost invariably, CTNNB1 mutations, besides TERT promoter mutations (more than 60%) and/or other mutations involving pathways aberrantly activated in adult HCC.^26,49,50

HCC (pediatric): A number of alterations, including gain of chromosomes 1q, 8q, and 17q and loss of 4q, can be present in both HCC and HB. Gene expression profiling studies of adult HCCs has identified abnormalities in critical signaling pathways involved in hepatocarcinogenesis, particularly in hepatitis-associated HCC. Some of these pathways include the P53 pathway, mitogen-activated protein kinase, Wnt/β-cat, epidermal growth factor, and transforming growth factor-beta pathways.^{42,45,51–54}

Conclusions

The classification of pediatric epithelial liver tumors has evolved with advances in molecular characterization reflected in the addition of new immunohistochemical markers important for an adequate categorization of most of them. The expanded immunohistochemical panel available is useful in addressing the final diagnosis (Table 3). However, the challenge for the PHITT is to find an answer for many open issues: the prognostic significance of focal small cell areas in HB and the definition of HCN-NOS as an entity with a morphologic and molecular profile in between those of HB and HCC. The international cooperation and the pathologic and molecular correlation in the context of a clinical trial for tumors rare-like HB represents a unique opportunity to redefine the prognostic categories and disclose new perspectives in terms of tailored treatments.

Table 3.

Distinguishing Features of HB, Hepatocellular Neoplasm-NOS and HCC.

Characteristic	HB	Hepatocellular Neoplasm-NOS	HCC
Cytology	Mixed population of cells: Fetal, small polygonal with clear/eosinophilic cytoplasm and Embryonal—scant cytoplasm, hyperchromatic, angulated nuclei, frequent rosettes	Variable: Tumors with monotonous cytology showing high N/C ratio Tumors with coexisting HB-like cytology (no definitive embryonal/fetal features) and variable pleomorphic cells (HCC-like)	Usually pleomorphic (unless well-differentiated). Large nuclei with magenta nucleoli and intranuclear inclusions; cytoplasmic globules
Pattern	Trabecular, occasional macrotrabecular areas (up to 5 cells thick)	Sheets, macrotrabecular	Macrotrabeculae (15–20 cells thick). Sheets possible. Necrosis frequent
Background liver	Normal	Normal	Normal or cirrhotic
Age	0 to 8 years	Usually > 8 years	Usually second decade, rare in 2- to 10-year-old children
AFP	Elevated >100 ng/dL	Variable usually very high	Elevated or normal
Immunohistochemistry
β-catenin nuclear	Fetal HB: focal+; crowded fetal/embryonal HB 3+	1–2+, rarely negative	Usually negative; rare possible nuclei
GS—cytoplasmic	Fetal HB3+; embryonal HB 1-3+	2–3+ patchy	Variable; weak to positive
Glypican 3 cytoplasmic	1–3+	1–2+ patchy	1–2+ patchy
MOC31 membranous	1–3+	1–3+	1–2+
Prox 1 nuclear	2–3+	1-2+	–
SALL4 nuclear	3+	1-2+	Usually negative
Molecular	90% CTNNB1 mutation	CTNNB1 + TERT	Occasional CTNNB1; TERT+; p53+ others

Abbreviations: AFP, alpha-fetoprotein; GS, glutamine synthetase; HB, hepatoblastoma; HCC, hepatocellular carcinomas; NOS, not otherwise specified; N/C, nuclear–cytoplasmic; 1+, focal staining, intense up to 10% cells; 2+, staining in 10% to 50% cells, moderate intensity; 3+, staining in the majority of cells (>50%) intense staining.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Rita Alaggio has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 668596 (ChiLTERN).

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