Teratoid Hepatoblastoma—Our Experience

Simple Summary Hepatoblastomas (HB) are the most common pediatric liver tumor with a number of histologic patterns described. Teratoid HB is a rare subtype with only a few case reports documented in the current literature. The aim of this study is to analyze a large series of teratoid HB to better characterize the clinicopathologic features in order to aid in recognition and diagnosis, improve understanding of the biology of these tumors, and provide insights into clinical behavior that may inform future clinical trials. Abstract Hepatoblastomas (HB) are the most common pediatric liver tumor with several subgroups described, of which teratoid HB is the rarest. The aim of this study is to characterize the histologic and phenotypic spectrum of teratoid HB in order to better understand the biology and behavior of these tumors. A retrospective analysis of all teratoid HB diagnosed at a major pediatric hospital as well as the consultation files of one of the authors (SR) was performed with the available clinical data and surgical pathology material reviewed. A detailed immunohistochemical workup was also performed. A total of 28 cases were included from patients ranging from 5 to 84 months of age and a M:F ratio of 1.07:1. Four patients had syndromic associations. In 14/28 cases, the tumors contained primitive glandular elements with histologic and immunophenotypic overlap with the yolk sac tumor which in two cases became predominant in metastatic sites. One case had extensive primitive neural epithelium mimicking a primitive neuroectodermal tumor (PNET). Other unique elements included melanin, mature neuroglial tissue, rhabdomyoblastic differentiation, and neuroendocrine carcinoma-like areas (n = 2). In conclusion, this study provides the largest series of teratoid HB to date with clinical and outcome data, highlights previously undescribed or under-recognized histologic patterns in these tumors, and describes the immunohistochemical profile of these tumors to aid in diagnosis.


Introduction
Hepatoblastomas (HB) are the most common pediatric liver tumors with an annual incidence of 10.5 cases per million children less than 1 year of age (SEER database). The new consensus classification has described the histology of various subgroups of HB [1]. Teratoid HB is the least common subtype with no definite incidence known for this variant as only a small number of teratoid HB have been reported in the literature, mostly as isolated case reports [2]. By definition, the presence of primitive neuroepithelial elements or melanin is required to qualify for the diagnosis, but detailed clinical, pathologic, and immunohistochemical analyses of teratoid HB are lacking [1]. Recognition of the full spectrum of features that may be seen in these tumors is imperative to avoid overlooking a minor teratoid component which may become predominant in later specimens. In small biopsies with a significant teratoid component, failure to recognize the features as indicative of a hepatoblastoma could result in misdiagnosis of a germ cell tumor. In addition, the prognostic and biologic significance of the presence of teratoid elements is unknown and better characterization of teratoid HB is necessary to improve such knowledge. This study was conducted with the aim of characterizing the histologic and phenotypic spectrum of teratoid HB. Unusual variants and presentations not previously described are also illustrated.

Materials and Methods
A retrospective analysis of all hepatoblastomas seen at a large tertiary pediatric hospital, either as surgical or personal consults, was reviewed after IRB approval. Cases were excluded if surgical pathology material was not available or if the histologic features were insufficient to meet current criteria for the designation of teratoid hepatoblastoma as described in the most recent consensus classification. Detailed immunohistochemical workup of cases was reviewed with special emphasis on staining for beta-catenin, glypican-3, glutamine synthetase, MOC31, Prox1, and SALL4, with adequate controls. Details for these immunohistochemical stains are included in Table 1. Additional stains were performed where needed. Relevant clinical data were collected from the electronic medical record, including presenting symptoms, AFP levels, medical intervention and treatment history, and clinical follow-up when available.

Results
After retrospective review of previously diagnosed hepatoblastomas over a 42-year period, a total of 28 cases were confirmed as containing teratoid elements and included in the current series. Five cases were excluded from the series as they failed to meet the diagnostic criteria, with two cases only containing pigment equivocal for melanin, one case demonstrating only patchy expression of neuroendocrine markers by immunohistochemistry, and two cases failing to demonstrate any reproducible histologic teratoid features.

Clinical
The age range was 5 months to 84 months and the male to female ratio was 1.07:1 ( Table 2).
Alpha-fetoprotein (AFP) levels were documented as elevated at the time of presentation in 20 patients with available results. AFP levels ranged from 3183 to 1,260,000 ng/mL. Four patients had syndromic associations including Beckwith-Wiedemann syndrome (n = 3), trisomy 13 (n = 1), and Wolf-Hirschhorn syndrome (n = 1). Two patients had a documented history of prematurity. One 2-year-old male patient had an unusual presentation with precocious puberty and elevated AFP, testosterone, 17-OH progesterone, and beta-HCG (THB25). Six patients underwent liver transplants and nineteen patients had surgical resections. In the remaining patients, clinical data were limited as only biopsy specimens were received in consultation. Eight patients had lung, abdominal lymph node, peritoneal, or omental metastases. Since the early 2000s, most patients have been treated on standard Children's Oncology Group protocols for these tumors determined initially by their stage at presentation and ability to resect upfront and more recently on the latest protocol based on PRETEXT staging. Details of all treatments for individual patients are not available for the purposes of this study. Three patients have died of disease. One patient had a disease-free survival of 14 years but died due to renal failure at 18 years of age (THB3). Two patients had documented recurrence or progression during a follow-up period of 2 years (THB16, THB23). The other patients were well at varying follow-up periods (1 month-25 years).

Histology
The majority of tumors were mixed epithelial and mesenchymal hepatoblastomas (75%, 21/28). The epithelial components were most commonly composed of well-differentiated fetal (n = 14), crowded fetal (n = 21), embryonal (n = 24), and blastemal (n = 13) elements. A few cases also had cholangioblastic (n = 8), pleomorphic fetal (n = 6), and macrotrabecular patterns (n = 3). Mesenchymal components were typically composed of spindled cells or os-teoid/bone with a few tumors containing areas of skeletal muscle/rhabdomyoblastic (n = 3, Figure 1A,B) or smooth muscle differentiation (n = 1). The spectrum of teratoid elements are illustrated in Figure 1. Of the teratoid elements identified, one of the most common was melanin pigment (n = 10), often in association with squamous differentiation with or without keratin formation ( Figure 1C). These foci of melanin and squamous differentiation were sometimes located centrally within epithelial tumor islands with fetal morphology. In other cases, the melanin positive cells were intimately associated with osteoid (THB14, THB16, THB21). In 4/28 cases, melanin was the only teratoid component identified within the tumor and in some cases was very focal, only represented in the resection specimen and not present on initial biopsy (THB16, THB21). In a few of these cases significant post-therapy changes were present with abundant hemosiderin deposition in addition to melanin, and an iron stain was helpful to confirm the diagnosis (THB 21, Figure 1D). Other teratoid elements identified included neural elements with a range of maturation including mature neuroglial tissue, ganglion cells, and primitive neuroepithelium (n = 15, Figure 1E-H). One case was unique in that it had extensive primitive neuroepithelium with PNET-like areas which made the diagnosis of hepatoblastoma challenging (THB17, Figure 2). A significant number of teratoid tumors contained glandular components (n = 16). In a few cases (THB18, THB24) the glandular components demonstrated differentiation, recapitulating gastrointestinal or mature mucinous epithelium, but in many cases the glands appeared primitive, defying definitive classification. These primitive glandular components often had subnuclear and/or supranuclear vacuoles and a subset had significant morphologic overlap with yolk sac tumors. A few rare cases contained neuroendocrine-like elements (THB12, THB25, Figure 3).   A significant number of teratoid tumors contained glandular components (n = 16). In a few cases (THB18, THB24) the glandular components demonstrated differentiation, recapitulating gastrointestinal or mature mucinous epithelium, but in many cases the glands appeared primitive, defying definitive classification. These primitive glandular components often had subnuclear and/or supranuclear vacuoles and a subset had sig-   In those patients with metastasis, four had teratoid elements present at distant metastatic sites (THB12, THB15, THB20, THB23) consisting of ganglion cells, melanin, primitive glands or yolk-sac-tumor-like elements. In two cases, distant metastases were composed entirely of yolk-sac-tumor-like elements (THB12, THB23). One case initially had omental and extrahepatic tumors with mixed fetal, embryonal, and teratoid elements ( Figure 4) and subsequently developed peritoneal metastases exclusively composed of tubulopapillary yolk-sac-tumor-like areas ( Figure 5A,B, THB23). In those patients with metastasis, four had teratoid elements present at distant metastatic sites (THB12, THB15, THB20, THB23) consisting of ganglion cells, melanin, primitive glands or yolk-sac-tumor-like elements. In two cases, distant metastases were composed entirely of yolk-sac-tumor-like elements (THB12, THB23). One case initially had omental and extrahepatic tumors with mixed fetal, embryonal, and teratoid elements ( Figure 4) and subsequently developed peritoneal metastases exclusively composed of tubulopapillary yolk-sac-tumor-like areas ( Figure 5A,B, THB23).    The second case has been previously reported [3] and showed a mixed mesenchymal and epithelial hepatoblastoma at the time of resection with fetal and embryonal epithelial elements and osteoid as well as areas with insular to trabecular growth resembling a neuroendocrine tumor located between the osteoid. Subsequently, following The second case has been previously reported [3] and showed a mixed mesenchymal and epithelial hepatoblastoma at the time of resection with fetal and embryonal epithelial elements and osteoid as well as areas with insular to trabecular growth resembling a neuroendocrine tumor located between the osteoid. Subsequently, following salvage therapy the patient developed liver recurrence, retroperitoneal lymphadenopathy, and a perirenal mass which on biopsy were all composed of yolk-sac-tumor-like elements.

Immunohistochemistry
Primitive glandular and yolk-sac-tumor (YST)-like elements demonstrated nuclear positivity for nuclear SALL4 (10/11) and beta-catenin (9/11), although in two cases the nuclear beta-catenin was only focal (THB12, THB23) ( Figure 5C-H). The primitive glandular/YSTlike elements also frequently showed membranous positivity for MOC31 (7/10), although this was sometimes found in only a subset of the glandular elements (THB25, THB28). In 8/11 cases, the primitive glandular/YST-like elements demonstrated focal cytoplasmic glypican-3. These elements were largely negative for glutamine synthetase with a few cases (4/10) showing focal weak cytoplasmic expression (THB8, THB11, THB15, THB23). PROX-1 was also mostly negative (5/8) in these elements. In contrast, glandular elements with evidence of differentiation along gastrointestinal lineage did not show staining for SALL4 or demonstrate nuclear beta-catenin ( Figure 2E-H, THB18, THB24). Neural elements also showed predominantly cytoplasmic-to-membranous expression of beta-catenin with only rare nuclei staining (1/5), including in cases with more primitive elements. The staining pattern for SALL4 in neural elements largely reflected the degree of differentiation with mature elements being entirely negative and only a few cases (n = 3, Figure 2G) with very primitive elements showing some nuclear positivity. Neural elements were consistently negative for glutamine synthetase and glypican-3. MOC31 was variably positive in neural elements with two of four cases with available material for immunohistochemistry showing some expression and two of four cases being entirely negative. Synaptophysin highlighted neuroendocrine (Figure 3, THB12, THB25, THB27) and mature neuroglial areas with rare positive cells within glandular elements (THB14, THB15). Many cases additionally had small clusters of primitive embryonal cells with synaptophysin positivity (THB16, THB20, THB21, THB22, THB23).

Discussion
Teratoid hepatoblastomas (HB) are an unusual but enigmatic group of neoplasms that show differing patterns with only rare reported cases in the literature (Table 3).   Yr-years; mo-months; UK-unknown; F-fetal; E-embryonal; sq-squamous; YST-yolk sac tumor; DOD-died of disease; AWOD-alive without disease.
Teratoid HB was first described by Manivel et al. in reference to a tumor in an 8-month-old girl which demonstrated the unique presence of neuroepithelial-like embryonic tubules, melanocytes, and ganglion cells [6]. The authors of that paper postulated that the presence of these unusual elements was the result of neuroectodermal differentiation of multipotential neoplastic cells [6]. The authors noted that while other heterologous elements had been previously reported in hepatoblastoma, including squamous differentiation, primitive tubules or ducts recapitulating developing intestinal or respiratory epithelium, smooth muscle, cartilage, bone, and skeletal muscle, these were, in contrast, all endodermal-derived elements [6]. This is the largest reported series of teratoid HB in the current literature and the first study that highlights unique phenotypes and detailed IHC profile of these unusual tumors. Our series highlights that teratoid HB may not necessarily show neuroepithelial or melanin components in all cases and can have unusual clinical features.
While the more widely recognized teratoid elements of melanin (10/28) and variably differentiated neural elements (15/28) were present in many of our cases, a significant number (14/28) contained primitive glands with overlapping features with yolk sac tumors, indicating this is likely an under-recognized pattern that should be carefully evaluated for when reviewing an HB. In two of our cases, this pattern became predominant in subsequent metastases underscoring how recognition is imperative to avoid a misdiagnosis. It seems likely based on our study that previous reports in the historical literature of cases of "mixed HB and teratoma" [8,11,16] may actually represent additional examples of this unusual pattern of HB and add support to the idea that this pattern can cause diagnostic confusion. Histologically, these YST-like components can have a variety of appearances ranging from microcystic to glands with subnuclear vacuolization. In our study, we have demonstrated that IHC can be a useful adjunct in identifying YST-like elements and differentiating them from more differentiated glandular elements or neuroepithelium. These areas often demonstrate positivity for nuclear beta-catenin, glypican3, SALL4, and MOC31 in contrast to mature glands and neuroepithelium where positivity for these markers is absent in the case of mature glands or less consistently expressed, as in many neural elements in the current study. The underlying mechanism driving the evolution to a predominantly yolk-sac-tumor-like morphology and phenotype is unknown but the presence of nuclear beta-catenin supports a shared molecular pathogenesis with conventional HB. Considering the embryologic origin of the liver from the celomic cavity (yolk sac), it is not difficult to postulate that the presence of these primitive yolk sac elements may represent dedifferentiation of the stem cells that give rise to HB. This also reflects the totipotent capability of the HB stem cells. Further study is needed to identify whether these elements harbor additional molecular alterations that allow them to become dominant in the postchemotherapy setting. In at least two cases, the tumors were particularly aggressive, but whether presence of YST elements in a primary HB tumor portends a poorer outcome requires additional investigation.
Other unusual and unique histologic patterns identified in our series included one case with a predominance of very primitive PNET-like neural elements and two cases with neuroendocrine elements. One of the cases with neuroendocrine elements had a striking clinical presentation with precocious puberty and elevated levels of testosterone, 17-OHprogesterone, and beta-hCG in addition to elevated AFP. Prior to this case, there is only one previously reported case of HB presenting with endocrine manifestations, occurring in a 6-year-old female who had Cushing syndrome and whose liver tumor was found to be secreting ACTH, CRH, and PTHrP [17]. Unfortunately, this case report did not include a detailed histologic description so it is unknown if this case contained teratoid elements. A few studies have investigated the expression of neuroendocrine markers including synaptophysin, chromogranin, serotonin, and CD56 in HB by immunohistochemistry [18,24] describing clusters of positive cells in epithelial components with fetal and embryonal morphology as well as focal expression within glandular components. In our series, synaptophysin highlighted mature neural elements, showed focal and weak positivity in the glandular components of a few cases, and highlighted neuroendocrine elements. Notably, many of the cases in the current series without histologic evidence of neuroendocrine differentiation showed scattered small clusters of synaptophysin-positive cells which had morphology consistent with a primitive embryonal component. This finding illustrates that caution must be used when diagnosing a neuroendocrine component within an HB and the designation should be reserved for those cases in which both morphologic and immunophenotypic neuroendocrine features are present and not on focal expression of neuroendocrine markers alone. Interestingly, previous studies have demonstrated that subsets of hepatoblasts and the more primitive liver stem cells in the developing liver express neuroendocrine markers including NCAM and chromogranin [25]. We have also noted expression of synaptophysin within embryonal areas of HB. Synaptophysin and chromogranin are also expressed within the developing ductal plate, the site of the development of the intrahepatic bile ducts [25].
Whether teratoid elements originate from a population of multipotent or less committed stem cells remains an open question. In general, hepatoblastoma is hypothesized to arise either from primary hepatoblasts or a multipotent hepatic progenitor cell [26]. Embryologically, there is evidence of an early bipotential population of progenitor cells in the foregut endoderm which can go on to participate in either liver or pancreas development [27]. The liver primordium or liver bud which develops as hepatic cords grow into the mesenchyme of the transverse septum [27] contains multipotential cells that represent the most primitive stem cells of the liver. These stem cells undergo further differentiation to produce hepatoblasts which demonstrate expression of CK8, CK18, CK7, CK19, AFP, and glypican-3 [25] as well as MOC-31 and PROX-1 and exhibit bipotential to differentiate into both hepatocytes and cholangiocytes. The origin of hepatic stem cells is still debated, but evidence suggests that they may be derived from the bone marrow as studies in mouse models have demonstrated that purified hematopoietic stem cells have the capability to differentiate into mature hepatocytes [28]. Within the adult liver there are a subset of cells termed hepatic progenitor cells with overlapping molecular characteristics with primitive hepatoblasts which may be activated in severe injury to the liver [25]. More recently, a population of cells was identified within peribiliary glands in adults and infants with overlapping features to the stem cells identified in embryonic development and which were demonstrated to be capable of differentiating into intestinal and pancreatic epithelial cells, fat, bone, cartilage, and endothelial cells [25]. The presence of stem-like cells in hepatoblastoma, which might explain the presence of teratoid elements, has undergone much investigation. Ruck et al. identified a population of "small epithelial cells" with features by immunohistochemistry and ultrastructure which were intermediate between hepatocytes and cholangiocytes [10]. Zimmerman et al. also described a subset of socalled "immature-looking cells" which showed stronger beta-catenin expression, decreased Ki67 proliferative index and lower expression of hepatocyte antigens [27]. These descriptions likely correspond to those "small cell undifferentiated" elements now designated as "blastema" within hepatoblastoma. Some have proposed that these small epithelial cells resemble hepatic stem cells. Interestingly, larger areas of blastema are reportedly present in teratoid HB [2]. In our series, blastemal areas were present in 13/28 cases and in 9/16 cases with glandular components.
It has been suggested that teratoid elements may be less chemo-responsive given observations that in post-chemotherapy specimens, these elements may predominate as the more chemosensitive epithelial hepatoblastoma components regress [21]. Corroborating this idea is the fact that in our series the teratoid components were seen at metastatic sites in four cases, including two cases, one previously reported, where the metastases were composed entirely of yolk-sac-tumor-like elements [3]. Other reports have suggested that chemotherapy may induce maturation of teratoid elements [5,6,13,15]. This assertion is difficult to assess based on our series as the full spectrum of teratoid elements was often not represented on the small biopsies received prior to chemotherapy. As previously noted, more study is needed to further clarify the issue of prognostication in these rare tumors.

Conclusions
In conclusion, this study expands the current knowledge on the wide variety of patterns seen in teratoid HB, providing tools to the practicing pathologist to aid in recognition and to facilitate further investigation into the biology of these rare tumors. These are enigmatic tumors that almost always occur in the setting of classic mixed epithelial and mesenchymal hepatoblastoma but may rarely be noted in only the context of an epithelial hepatoblastoma. They highlight the multipotent capability of the HB stem cell, which is different than the stem cells seen in adult tumors which do not show this multidirectional dedifferentiation or lineages. This finding is similar to that seen in Wilms tumors and may suggest similar biology for these pediatric tumors despite their distinct molecular pathogenesis. It is interesting to note the association with BWS in these patients is similar to Wilms tumors.
The fact that nuclear beta-catenin is a feature in many of the primitive areas suggests the role of the Wnt/beta-catenin pathway in pathogenesis, more mature elements show less evidence of nuclear beta-catenin staining and may represent a more metaplastic differentiation in these tumors. Further molecular studies are in progress to better understand the pathogenesis of these tumors, which constitute less than 2% of all hepatoblastmas.
Author Contributions: Both authors contributed equally to all of the following components of this study: conceptualization; methodology; formal analysis; investigation; resources; data curation; writing-original draft preparation; writing-review and editing. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The study was approved by the Institutional Review Board of Cincinnati Children's Hospital Medical Center (IRB # 2020-0068).

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.