Protocol for the Examination of Specimens From Pediatric Patients With Hepatoblastoma

The College of American Pathologists offers these protocols to help pathologists provide clinically useful and relevant information when reporting results of examinations of surgical biopsy/resection specimens. The College regards the reporting elements in the “Surgical Pathology Cancer Case Summary (Checklist)” portion of the protocols as essential elements of the pathology report. However, the manner in which these elements are reported is at the discretion of each specific pathologist, taking into account clinician preferences, institutional policies, and individual practice.The College developed these protocols as an educational tool to assist pathologists in the useful reporting of relevant information. It did not issue the protocols for use in litigation, reimbursement, or other contexts. Nevertheless, the College recognizes that the protocols might be used by hospitals, attorneys, payers, and others. Indeed, effective January 1, 2004, the Commission on Cancer of the American College of Surgeons mandated the use of the checklist elements of the protocols as part of its Cancer Program Standards for Approved Cancer Programs. Therefore, it becomes even more important for pathologists to familiarize themselves with the document. At the same time, the College cautions that use of the protocols other than for their intended educational purpose may involve additional considerations that are beyond the scope of this document.This protocol applies to hepatoblastoma only. Other malignant primary hepatic tumors are excluded. There is no American Joint Committee on Cancer (AJCC)/International Union Against Cancer (UICC) TNM classification for hepatoblastoma. The Children's Oncology Group Staging System is recommended.First priority should be given to formalin-fixed tissues for morphologic evaluation. The second priority for tissue processing is snap-freezing up to 1 g (minimum of 100 mg) of tumor from grossly different regions, and nontumoral liver, for molecular studies, as well as submission of viable sterile tumor for cytogenetic studies (see Explanatory Note A). Samples from the same foci should be collected for histology and appropriately identified.For more information, contact The Children's Oncology Group Biopathology Center, Columbus, Ohio; telephone: 614-722-2890 or 800-347-2486.Intraoperative frozen sections should be avoided unless the operative procedure will be altered by the result. Biopsies of pediatric liver tumors present significant potential for diagnostic error, even on permanent sections. First priority should be given to formalin-fixed tissues for morphologic evaluation. For resection specimens, sections should be prepared from each major tumor nodule, with representative sampling of smaller nodules, if macroscopically different in appearance. The total number of sections taken should be equal to or greater than the greatest dimension of the tumor in centimeters, to better assure detection of areas of unfavorable (eg, small cell undifferentiated) histopathologic features. Sections from inked margins of resection and portal vein or hepatic vein–inferior vena cava involvement should also be submitted if this feature is seen grossly. Gross vascular invasion versus intravascular growth found only microscopically, and whether it is within the tumor mass or outside of it, should also be recorded.The second priority for tissue processing includes snap-freezing up to 1 g (minimum of 100 mg) of tumor from regions of different appearance and nontumoral liver for future molecular studies; viable sterile tumor should be submitted for cytogenetic studies whenever possible.Primary diagnosis by cytology (fine-needle aspiration) may be misleading because of difficulties in distinguishing well-differentiated hepatocellular malignancy from regenerative changes and benign proliferations, and because of the variability of histologic features in hepatoblastoma.Hepatoblastoma occurs in association with several well-described environmental factors and cancer genetic syndromes (see Table 1); however, these associations are not necessarily all of statistical significance. Environmental factors and prenatal exposure to different agents have been implicated in hepatoblastoma.1An increased incidence of hepatoblastoma—from 0.4 to 1.0 per million between 1971 and 1983—has been observed at a Children's Tumour Registry in Manchester, United Kingdom.2 The US National Cancer Institute's Surveillance, Epidemiology, and End Result (SEER) program includes approximately 14% of the population; it revealed an average 5.2% annual increase in the incidence of hepatoblastoma from 1973 to 1992.3 This change might be explained by the occurrence of hepatoblastoma in surviving premature infants. Hepatoblastomas in Japan accounted for 58% of all malignancies in children who weighed less than 1000 g at birth.4 Further analysis of the Japanese Children's Cancer Registry data revealed that 15 (5%) of 303 hepatoblastomas between 1985 and 1995 occurred in infants with history of prematurity and weight less than 1500 g at birth.5 This rate was greater than 10 times that for all live births. The relative risk for hepatoblastoma for children who weighed less than 1000 g at birth was 15.64 compared with 2.53 for those who weighed 1000 to 1499 g and 1.21 for those who weighed 2000 to 2499 g. Of 77 children with hepatoblastoma in the German registry,1 3 (4%) were premature infants who required parenteral nutrition, a treatment that has been lifesaving for many small premature infants but has been reported to lead to cirrhosis in many survivors. It has not previously been associated with hepatoblastoma. The histologic features of hepatoblastoma following prematurity are indistinguishable from those of other hepatoblastomas.The Children's Cancer Group has evaluated environmental or drug exposure. Seventy-five sets of parents of children with hepatoblastoma were compared with those of age-matched controls. Before and during pregnancy, there was a significant excess of maternal exposure to metals used in welding and soldering, lubricating oils, and protective greases.6 Paternal exposure to metals was also greater. At 23 weeks, a congenital hepatoblastoma was found in a stillborn fetus whose mother was an artist exposed to volatile hydrocarbons.7Karyotyping of hepatoblastomas has revealed a recurrent pattern of chromosomal abnormalities.89 The most common karyotypic changes are extra copies of entire chromosomes (trisomies), sometimes in conjunction with other complex structural changes and often in association with double-minute chromosomes. Trisomies of chromosomes 2 and 20 have each been reported most commonly,89 and each of these trisomies has been reported as a sole karyotypic event, suggesting that they may represent an early stage of tumor evolution. Trisomy of chromosome 20 and duplication of the long arm of chromosome 20 have been also observed in rhabdomyosarcoma, suggesting a link between these 2 embryonal tumors, both of which are also associated with losses at the Beckwith-Wiedemann syndrome locus.10 Trisomy of chromosome 8 is also common; other trisomies are seen with lesser frequency. Occasional losses of entire chromosomes are seen, and these, too, are not random. The clinical significance of trisomies is at present unknown, although a recent study using comparative genomic hybridization has suggested that chromosomal gains at chromosome 8 and 20 may be associated with an adverse prognosis.11 A unique translocation has been reported in undifferentiated small cell hepatoblastoma,12 a variant associated with a poor prognosis, although this cytogenetic variant has not been reported in other cases.Numerous recent studies have documented molecular genetic abnormalities in hepatoblastomas (see Table 2) and other hepatic tumors. Several genetic changes are shared with other embryonal tumors, such as loss of heterozygosity at chromosome 11p15, also described in rhabdomyosarcomas and Wilms tumors. Acquired mutations of the APC gene and the β-catenin gene, both members of the Wnt signaling pathway, have also been reported in hepatoblastoma.1314 The high frequency of β-catenin mutations in hepatoblastomas and the increased incidence of hepatoblastomas in familial adenomatous polyposis families suggests the important role of an overactivation of the wingless/Wnt pathway in the pathogenesis of hepatoblastoma. Collection of fresh or frozen hepatoblastoma tumor material as well as nontumoral liver tissue from these patients will be of great importance to the further investigation of the clinical relevance of these and other molecular genetic abnormalities in predicting the prognosis and clinical behavior of these tumors.Serum α-fetoprotein (αFP) is the most useful indicator of hepatocellular neoplasia. Levels of serum αFP are markedly elevated in 80% to 90% of hepatoblastomas and 60% to 70% of hepatocellular carcinomas.9 Lesser degrees of elevation in infants can be due to variations in the rate of decline after birth or secretion from regenerating hepatocytes adjacent to hemangioendotheliomas or mesenchymal hamartomas. Therefore, it is unacceptable practice to institute chemotherapy for mass lesions of the liver solely on the basis of imaging studies and serum αFP levels. α-Fetoprotein also can be elevated in yolk sac tumors, which may occur as primary tumors in the liver or together with hepatoblastoma. On the contrary, αFP levels will not be increased when hepatoblastomas are primarily composed of the small cell undifferentiated type or in most fibrolamellar carcinomas, but even some typical fetal hepatoblastomas have failed to produce detectable increases in serum αFP levels. Following the αFP level in patients with unresectable hepatoblastoma after chemotherapy had prognostic value in a retrospective analysis of 31 patients in a Children's Cancer Group series from 1986 to 1989.1There are many other proposed blood assays for the detection of hepatic malignancies. Other than αFP and human chorionic gonadotropin (HCG), none is used widely thus far because of relatively low specificity and predictive value. Occasionally, hypercholesterolemia is found in patients with hepatoblastoma, especially infants with fetal histology, and all those with high levels died.1516 Precocious puberty secondary to HCG or testosterone secretion has been observed in 6% of boys with hepatoblastoma.17 Thrombocytosis has been present in 25% to 65% of patients with hepatoblastoma.18The presenting symptom of almost all liver tumors in children is abdominal swelling secondary to hepatomegaly. When confronted with this symptom, it is useful to consider the age at which liver tumors tend to occur (see Table 3). Exceptions are frequent, but age can serve as a guide when the presenting symptoms lack specificity. In the Pediatric Oncology Group series from 1986 to 2002,1 66% of hepatoblastomas were manifest by the second year, and 11% before 6 months of age. Approximately 50% of those in infants were congenital, given their size when discovered by 2 to 3 months of age; 6% of hepatoblastomas occurred after age 5 years. Hepatocellular carcinomas have been observed as early as 6 months. Seven examples of mixed hepatoblastomas and hepatocellular carcinomas have been observed at a mean age of 8.5 years; perinatally acquired hepatitis B virus was responsible in 3 instances. Yolk sac tumors are more common in early childhood, but they also occur rarely in older adults. Systemic malignancies and metastatic disease must be considered at all ages because hepatomegaly due to megakaryoblastic leukemia, Langerhans cell histiocytosis, and neuroblastoma are important sources of confusion with hepatoblastoma in infancy, as are intra-abdominal desmoplastic small round cell tumors later in childhood.Primary malignant tumors of the liver account for approximately 1% of all childhood cancer. The most common type is hepatoblastoma, which has an annual incidence of 0.9 per million children. Not only are they rare, but their diversity significantly limits the experience of any one center and pathologist. A classification scheme for hepatoblastoma1 that divides the more frequently or prognostically influential features from infrequent or inconsequential (minor) components is presented in Table 4, based on study of tumor resection specimens.There is no relationship between the age of the child and predominant cell type in hepatoblastoma.1 Of all cases at all ages, 85% to 90% contain both fetal and embryonal derivatives in variable proportions; 20% have stromal derivatives. Because these histologic types tend to be randomly intermingled, both fine-needle aspiration and biopsies may capture a nonrepresentative sample of tumor.Distinguishing well-differentiated (mitotically inactive) fetal hepatoblastoma tumor cells from normal liver in an infant can be difficult. The fetal tumor cells are larger than normal fetal hepatocytes and have a higher nuclear cytoplasmic ratio. The nuclei are regular and round with little discernible mitotic activity (≤2 mitoses per 10 high-power [×40 objective] fields) in the well-differentiated variety. Fetal tumor cells grow in cords, as in normal liver, or nests or nodules. Clusters of normoblasts (extramedullary hematopoiesis) are commonly seen, as in fetal liver. The cytoplasm of the fetal tumor cells varies from eosinophilic to clear, depending on the amount of glycogen content. Fetal tumor cells may also contain abundant lipid, producing vacuolization. In well-differentiated fetal tumors, bile secretion may be observed.The embryonal cellular component of hepatoblastoma is less well differentiated than its fetal hepatocytic counterpart, with cells that are small and have a high nuclear cytoplasmic ratio with ovoid nuclei and that may assume a tubular or rosette-like configuration. Purely embryonal tumors are almost never encountered, and invariably show some fetal areas.A macrotrabecular pattern of hepatoblastoma growth is one in which fetal or embryonal cells numbered 20 or more within a cord or cluster, as opposed to the usual 2- to 6-cell-thick cords or plates.Some histopathologic subtypes of hepatoblastoma have prognostic and therapeutic importance. Well-differentiated (mitotically inactive) fetal histology was superior to embryonal differentiation in long-term survival; therefore, the current Children's Oncology Group study is treating stage I well-differentiated fetal hepatoblastoma (with low mitotic rate) with surgery alone.119 An urgent research need is to identify more effective medical therapy for both small cell undifferentiated hepatoblastoma and rhabdoid hepatoblastoma, the most aggressive forms of this malignancy.1When first distinguished from embryonal epithelium, small undifferentiated cells in hepatoblastoma were noted to resemble neuroblastoma, to have a low mitotic rate, and were called anaplastic, consistent with the dictionary definition, characterized by imperfect development. Since “anaplastic” was redefined by Faria et al20 for Wilms tumor as nuclear enlargement to 3 times those of typical tumor cells, hyperchromasia, and atypical mitoses, the small cell undifferentiated component no longer is designated as anaplastic. Beckwith-type anaplasia does occur rarely in hepatoblastoma, and its significance is unknown. The small cells have been considered a putative hepatic progenitor cells on the basis of immunohistochemical and electron microscopic studies. When present in a significant fraction of the hepatoblastoma (75%), or as the sole cell type, the small cell type is typically found in infants younger than 1 year; they have a poor prognosis, with poor response to current therapy. The prognostic significance of smaller proportions of the small cell undifferentiated type is still undetermined. Rhabdoid tumor cells have the characteristic eccentric pink cytoplasmic inclusions (periodic acid–Schiff/diastase positive, vimentin or cytokeratin positive) with vesicular nuclei and fibrillar inclusion bodies by electron microscopy. They may be associated with the small cell component in otherwise typical hepatoblastomas or as the exclusive cell type, in which case they occur in infancy and are associated with a poor prognosis.Often, mixed hepatoblastomas contain epithelial membrane antigen (EMA)–positive nests of squamous epithelium. The osteoid component of mixed hepatoblastomas is found to be a matrix of collagen surrounding cells expressing EMA and having ultrastructural features of epithelium rather than osteoblasts. Hepatoblastomas may contain other stromal derivatives, including cartilage and rhabdomyoblasts. There is no prognostic significance to the presence of mixed histologic features.Several other variant (stromal) histologic patterns in hepatoblastoma are placed into a “minor category” based on infrequency of the cell type or the lack of measurable prognostic consequence. Multinucleated tumor giant cells are found in rare hepatoblastomas, sometimes associated with HCG production and clinical virilization. Teratoid hepatoblastoma was initially depicted as having intestinal, neural, and melanocytic elements. These are distinguished from true teratomas, which can also occur in the livers of children, on the basis of organoid differentiation and even greater diversity of tissue elements in the teratomas.Postchemotherapy resection specimens often show eradication of the embryonal cells and more prevalent osteoid-like foci. Heifetz et al21 reported that vascular invasion, amount of mesenchyme, persistence of embryonal epithelium, extent of tumor necrosis, and mitotic activity of the epithelial component have predictive value in this type of specimen. This has yet to be confirmed, but the items should be documented, as should the presence of any small undifferentiated cells, which are known to negatively affect prognosis but may have been missed in the initial biopsies of stage III and IV lesions.Typical Hepatoblastoma Histologic Types1Epithelial, fetal, well differentiated (with minimal mitotic rate of ≤2 mitoses per 10, ×40 objective fields) (7%)Epithelial, fetal, mitotically active pattern (>2 mitoses per 10, ×40 objective fields) (11%)Epithelial type, fetal and embryonal pattern only (39%)Epithelial type, macrotrabecular pattern (12%)Small cell undifferentiated pattern (5.6%)Mixed epithelial and mesenchymal type without teratoid features (20%)Mixed epithelial and mesenchymal type with teratoid features (4%)Immunohistochemistry for and in some may be useful in the diagnosis of hepatoblastoma, for tumors with and unfavorable There is no to hepatocellular from hepatoblastoma. genetic for chromosomes and abnormalities of chromosome are and may help these but only in approximately to of hepatoblastomas that the with histopathologic features are fetal, well-differentiated lesions as mitotically with a minimal mitotic rate of 2 or mitoses per 10, ×40 objective These tumors are also stage I and are with surgery with unfavorable histopathologic features have undifferentiated small cell or rhabdoid subtypes or When present in a significant fraction of the hepatoblastoma or as the sole cell type, the small undifferentiated is typically found in infants younger than 1 with poor prognosis of stage or therapy. When this is present in lesser the prognostic undetermined. When the rhabdoid cell type is the exclusive it is also found typically in infants with poor all other tumor subtypes not although they may be associated with prognosis if stage and are with in the United imaging with surgical and imaging are used in the to the and extent of hepatic involvement of hepatoblastoma before tumors the and are primarily of hepatic malignancies occurs within portal and the of into hepatic with Further to the may are relatively but of before or during surgery can an otherwise Children's Oncology Group is for involvement of the or other to the or are considered as is the prognostic feature for all liver with the of (see College of American Pathologists protocol for of hepatoblastomas were not to primary surgery stage III and stage in the of Pediatric Oncology Oncology Group of margins for total or specimens on the and extent of It is that the be to the foci within the margins that microscopic evaluation. The of a may be it for In this grossly margins should be and the margins are grossly of sampling of the in the to the tumor is In sampling of the may be the is found the surgical the from the should be For tumors, the from the tumor should be of a of 3 or more The of the hepatic the and involvement of the or other to the and are considered as