Family History

Although two tumor-related deaths and colon polyps were noted in the pedigree, there was no previous history of HB in any family members except for the twins (Figure 1A). All tumor-related lesions were from gastrointestinal origins, including colon and gastric cancer. The paternal grandfather (II-1) passed away due to a traffic accident, but he had been previously treated for colon polyps. The father (III-6) developed gastric and colon cancer in his 20s and underwent curative surgery for these tumors. Periodic follow-up examinations revealed no recurrent disease for the past 7 years.

Figure 1. Genetic background of the twins. (A) Family pedigree. (B) Structure of the APC gene, including functional domains and introns. The position of the identified germline mutation is indicated by the red line. (C) Sanger sequence histogram of the affected infants and their parents. Note: CC, colon cancer; CP, colon polyps; GC, gastric cancer; HB, hepatoblastoma.

Germline Mutations

We carried out genetic analysis of the father’s DNA and identified a 7-base pair-deletion (NM_000038.6, c.3231_3237delTTATACT(p. Val1077fs) in the coding region of APC. Located in the β-catenin binding region of APC, this variation is predicted to cause a frameshift, leading to the production of truncated APC protein lacking the β-catenin binding region (Figure 1B). Subsequent cascade analysis identified the variant in the twins but not in the mother or father’s siblings by Sanger sequencing (Figure 1A, 1C).

Clinical Course of Infant-1 (C1 Tumor)

The twins were born by cesarean section from a 28-year-old mother at 38 weeks gestation. No parental or neonatal problems occurred during the delivery course. They were clinically dichorionic-diamniotic gestational twins. The birth weight of the first baby was 2594 g and that of the second was 2326 g (Figure 2A and 2B). The infants were discharged in stable condition and exhibited a normal developmental course. However, the first of the twins gradually lost appetite and suffered abdominal distention by 12 months of age (Figure 2A). The parents took him to our hospital for consultation of his symptoms at 14 months of age. Physical examinations showed abdominal distention, particularly in the right quarter rib, where an elastic soft mass with the size of approximately 14 cm was palpable. An ultrasound examination detected a liver mass with the consistent size of 14 cm. Because of hyperechoic and nonuniform features of the mass content as well as the size, hepatoblastoma was suspected. Additional computed tomography (CT) scans showed a large, round, and smooth liver mass whose internal density was not uniform, with apparent invasion to the hilar plate that possibly originated from the posterior segment (PRETEXT I) (Brown et al., Reference Brown, Perilongo, Shafford, Keeling, Pritchard, Brock, Dicks-Mireaux, Phillips, Vos and Plaschkes2000). Blood laboratory examinations revealed slight elevation of white blood cells (14,600 /mL), anemia (Hb 8.4 g/dl), and a high level of AFP (33,961 ng/ml), which is a tumor marker of hepatoblastoma. A pathological examination of the tumor biopsy revealed that the tumor histology was a pure fetal type of HB (Supplementary Figure S1A). Based on this diagnosis, two cycles of preoperative chemotherapy (cisplatin/pirarubicin, CITA) were performed, which resulted in a marked reduction of the tumor volume to 9 cm in diameter. A right lobe hepatectomy was subsequently performed at 17 months of age. The surgical margin was negative for cancer and no detectable metastases to other organs were found in his abdominal cavity. The patient had a good postoperative course and underwent adjuvant chemotherapy 20 days after the surgery with one cycle of CITA followed by three cycles of low-CITA. No evidence of metastasis or recurrence has been observed for 7 years since the completion of adjuvant chemotherapy.

Figure 2. Clinical course of the affected infants. The C1 tumor was obtained by biopsy, whereas the C2 tumor was obtained by hepatectomy. (A) Infant-1. The red circle indicates the date of hepatectomy. (B) Infant-2. The red circle indicates the date of hepatectomy.

Clinical Course of Infant-2 (C2 Tumor)

Since the first of the twins (Infant-1) was diagnosed with HB, Infant-2 underwent an abdominal ultrasound examination for HB screening at 15 months of age (Figure 2B) despite not having any symptoms. No neoplastic lesions were detected in his liver at the time of screening. However, he was transported to the emergency department in our hospital at 21 months of age due to a sudden onset of respiratory distress with pallor on his face and subsequent loss of consciousness within a day. On admission, a pallor face and acrocyanosis of the extremities were noticed, and he suffered grade 3 unconsciousness based on the Japan Coma Scale (not awake with external stimulation). In addition, overall abdominal distention was observed, and physical examination detected a palpable and elastic soft mass on his right upper quadrant. Blood laboratory examinations showed a slight increase in the number of white blood cells (12,580 /μL), severe anemia (Hb, 6.9 g/dL), and moderately high AFP (2,127 ng/mL). An emergency CT scan demonstrated that his liver was occupied by a large mass with a diameter of 11 cm that had clear and smooth margins, a capsule-like structure, and heterogenous intensity. The CT scan images additionally depicted a low-density area at the center of the tumor, which strongly indicated tumor hemorrhage. Therefore, we did not perform a tumor biopsy for pathological diagnosis, but instead made a PRETEXT III diagnosis of suspected HB²³ according to the images. To manage tumor removal without trisectionectomy, preoperative chemotherapy was given, including one cycle of low-CITA followed by two cycles of CITA. Cycles of the chemotherapy reduced the tumor volume down to 6.6 cm in diameter without additional major hemorrhage from the tumor. The patient then underwent a central bisegmentectomy of the liver at 24 months of age. Pathological examination of surgical specimens confirmed that the tumor was the pure fetal type of HB (Supplementary Figure S1B), which was the same histological type as his brother. Twenty days after hepatectomy, four cycles of CITA postoperative chemotherapy were initiated. No evidence of recurrence or metastasis has been observed over the 7 years since the surgery.

Involvement of Wnt Signaling Pathway in Tumor Development

An immunohistochemical evaluation of the β-catenin protein was performed on the tumor tissue. Both tumors (C1 and C2 tumors) showed positive staining in the cytoplasm of HB cells, but not adjacent normal cells (Supplementary Figure S2). Nuclear localization was occasionally seen in the C1 tumor, but was clearly evident in the C2 tumor. In addition, an RNA-seq analysis of the C2 tumor and the corresponding nontumorous tissues revealed that expression of ODAM, LGR5, and AXIN2, which are three direct target genes in the Wnt pathway, was markedly augmented in the tumor tissue compared to the nontumorous tissue. These data suggest that activation of the Wnt signaling pathway was involved in the development of the HB tumors.

Genetic Analysis of the HBs

We carried out WES on the tumor tissues of the twins (Infant-1 and Infant-2) and nontumorous DNA extracted from their peripheral blood. The average sequence coverage of each exon was 148 (Infant-1) and 182 (Infant-2) for the nontumorous DNA, and 150 (C1) and 196 (C2) for the tumor tissue respectively. Comparison of the germline SNVs between Infant-1 and Infant-2 confirmed that the twins were monozygotic.

We identified a total of eight and one somatic SNVs in C1 and C2 respectively. In the eight SNVs in C1, six were nonsynonymous variants, one was a synonymous variant, and the remaining one was a nonsense mutation. The SNV in C2 was a nonsense mutation.

No somatic mutation was shared in the two tumors. As for driver mutations, we identified an NRAS mutation, NM_002524.5, c.35G>A (p. Gly12Asp) in C1 (with a VAF of 7.1%), and an ACVR2A mutation, NM_001616.5, c.16A>T (p. Lys6Ter) in C2 (with a VAF of 14.2%) (Table 1).

Table 1. Representative somatic mutations that may serve as tumor development trackers

Note: VAF, variant allele frequency; InterVar, https://wintervar.wglab.org/about.php; LP, Likely pathogenic; US, Uncertain significance; COSMIC, Catalogue of Somatic Mutations in Cancer database; NA, Not applicable.

To determine whether the mutations were a late event in the evolution of each HB, we carried out multiregional analysis of the NRAS and ACVR2A mutations by digital PCR analysis. DNA were extracted from six regions of the C1 tumor and seven regions from the C2 tumor, and the mutations were determined using the mutant-specific probe sets. As shown in Figure 3A, the NRAS mutation was detected with different frequencies; 0.2% (region-1), 0.03% (region-2), 0.02% (region-3), 0.2% (region-4), 0.16% (region-5), and 0.14% (region-6) in the C1 tumor, implying intratumorous heterogeneity. Surprisingly, we also found the NRAS mutation in the seven regions of the C2 tumor with the following frequencies: 0.13% (region-5), 0.16% (region-6), 0.09% (region-7), 0.18% (region-8), 0.10% (region-10), 0.09% (region-11), and 0.11% (region-12) (Figure 3B). Similarly, the ACVR2A mutation was identified not only in the C2 tumor (0.42% in region-5, 0.19% in region-6, 2.24% in region-7, 4.06% in region-8, 7.10% in region-10, 1.10% in region-11, and 2.73% in region-12), but also in four of the six regions of the C1 tumor (0.03% in region-1, 0.02% in region-2, 0.4% in region-4, and 0.03% in region-6; Figure 3A and 3B). It is extremely unlikely that the two identical somatic mutations occurred in independent tumor clones after birth. Hence, these results suggest that: (1) implantation of tumor clone(s) occurred through utero-placental blood circulation systems, giving rise to microchimerism of both tumors; and (2) the initiation of HB tumorigenesis likely occurred during the fetal stage after organ development of the liver.

Figure 3. Rare variant detection from multiregional specimens. (A) A macroscopic slice of the Infant-1 tumor (C1). DNA was extracted from six different regions. The variant allele frequency (VAF) of NRAS/ACVR2A in each region is indicated in a bar graph. (B) Macroscopic slices of the Infant-2 tumor (C2). DNA was extracted from seven different regions. The VAF of NRAS/ACVR2A in each region is indicated in a bar graph.

Circulating Tumor DNAs

To investigate the usefulness of the driver mutations for their surveillance, we tested their detection in the ctDNA. Digital PCR analysis identified the ACVR2A mutation in prechemotherapeutic plasma of Infant-2 at a VAF of 1.31%, but not in postoperative plasma. As expected, the NRAS mutation was not detected in postoperative plasma of Infant-1 (no preoperative plasma available). These mutations were not detected in DNA extracted from PBMCs of both infants. Neither mutation was detected in the parents’ plasma DNA (Table 2).

Table 2. Variant allele frequencies (%) of ctDNA

Note: ^aIn the parenthesis, the numerator is the number of positive reaction units for mutations, whereas the denominator is the number of total (i.e., positive and negative) reaction units for mutations. PBMC, peripheral blood mononuclear cells.

Discussion

HB is a rare embryonal malignant tumor arising from the liver yet is the most common malignant tumor in infants and children. It commonly occurs in the first few years of life and predominantly in boys (Giardiello et al., Reference Giardiello, Offerhaus, Krush, Booker, Tersmette, Mulder, Kelley and Hamilton1991; Giardiello et al., Reference Giardiello, Petersen, Brensinger, Luce, Cayouette, Bacon, Booker and Hamilton1996; Trobaugh-Lotrario et al., Reference Trobaugh-Lotrario, Lopez-Terrada, Li and Feusner2018). FAP is an autosomal dominant disorder forming thousands of colorectal adenomas during adolescence and young adulthood, which is caused by a germline variant of the APC tumor suppressor gene. Although clinical characteristics of HB-affected individuals with APC mutations do not seem to be distinct from those with sporadic HB, it has been estimated that individuals with a FAP background have a 750 to 7500-fold higher risk of developing HB than the general population (Aretz et al., Reference Aretz, Koch, Uhlhaas, Friedl, Propping, von Schweinitz and Pietsch2006; Giardiello et al., Reference Giardiello, Petersen, Brensinger, Luce, Cayouette, Bacon, Booker and Hamilton1996; Hughes & Michels, Reference Hughes and Michels1992). Although investigators have reported APC mutations in HB patients (Aretz et al., Reference Aretz, Koch, Uhlhaas, Friedl, Propping, von Schweinitz and Pietsch2006; Evers et al., Reference Evers, Gaspar, Kloor, Bozukova, Kadmon, Keller, Sutter and Moog2012; Gupta et al., Reference Gupta, Sheridan, Towbin, Geller, Tiao and Bove2013; Ponz de Leon et al., Reference Ponz de Leon, Bianchini, Reggiani-Bonetti, Pedroni, Di Gregorio, Merighi, Rossi, Magnani, Domati and Cacciari2014; Thomas et al., Reference Thomas, Pritchard, Davidson, McKiernan, Grundy and de Ville de Goyet2003; Yang et al., Reference Yang, Sisson, Gupta, Tiao and Geller2018), a concomitant genetic analysis of HB, or HB from twins in FAP pedigrees, has not been fully performed.

It has been reported that HBs have fewer somatic mutations at the whole-genome level than most adult tumors do, including those of adult hepatocellular carcinomas (Eichenmuller et al., Reference Eichenmuller, Trippel, Kreuder, Beck, Schwarzmayr, Haberle, Cairo, Leuschner, von Schweinitz, Strom and Kappler2014; Fujimoto et al., Reference Fujimoto, Furuta, Totoki, Tsunoda, Kato, Shiraishi, Tanaka, Taniguchi, Kawakami, Ueno, Gotoh, Ariizumi, Wardell, Hayami, Nakamura, Aikata, Arihiro, Boroevich, Abe and Nakagawa2016; Jia et al., Reference Jia, Dong, Jing, Xu, Wang, Chen, Li, Huang, Zhang, Zhang, Liu, Zheng, Xia, Wang, Dong and He2014; Nagae et al., Reference Nagae, Yamamoto, Fujita, Fujita, Nonaka, Umeda, Fukuda, Tatsuno, Maejima, Hayashi, Kurihara, Kojima, Hishiki, Watanabe, Ida, Yano, Hiyama, Tanaka, Inoue and Hiyama2021). The activation of the Wnt signaling pathway with an alteration of CTNNB1 has been suggested to be the genetic event observed most frequently (15–87%) in HB (Lopez-Terrada et al., Reference Lopez-Terrada, Gunaratne, Adesina, Pulliam, Hoang, Nguyen, Mistretta, Margolin and Finegold2009; Purcell et al., Reference Purcell, Childs, Maibach, Miles, Turner, Zimmermann and Sullivan2011; Taniguchi et al., Reference Taniguchi, Roberts, Aderca, Dong, Qian, Murphy, Nagorney, Burgart, Roche, Smith, Ross and Liu2002). The majority of alterations are point mutations or deletions in exon 3 of CTNNB1, which results in the impairment of phosphorylation of the β-catenin protein (Rubinfeld et al., Reference Rubinfeld, Albert, Porfiri, Fiol, Munemitsu and Polakis1996). The activity of the β-catenin protein is regulated by the destruction complex composed of AXIN-1, GSK-3β, CK-1, and APC proteins, which degrade other proteins through phosphorylation and subsequent ubiquitination (Clevers, Reference Clevers2006; Ng et al., Reference Ng, Dazert, Boldanova, Coto-Llerena, Nuciforo, Ercan, Suslov, Meier, Bock, Schmidt, Ketterer, Wang, Wieland, Matter, Colombi, Piscuoglio, Terracciano, Hall and Heim2022; Taniguchi et al., Reference Taniguchi, Roberts, Aderca, Dong, Qian, Murphy, Nagorney, Burgart, Roche, Smith, Ross and Liu2002). Although the pathogenic germline variant (APC, c.3231_3237del) was found in C1 and C2 tumors, no second hit mutations were identified in the tumors. Since immunohistochemical analysis revealed accumulated β-catenin protein in the tumors, it is natural to think that the degradation of β-catenin was abrogated in the tumor cells. Thus, there may be undetectable somatic alteration(s) in the regulatory or deep intronic regions, or structural alterations, in the APC gene. Alternatively, other nongenetic events, such as epigenetic changes, could be involved in the loss of APC function in the cells.

In addition to the APC mutation, tumor cells harbored additional driver mutations, including the NRAS mutation in C1 and the ACVR2A mutation in C2. These mutations are not likely to be their early event, since the VAF was not high (i.e., 7.1% for NRAS and 14.2% for ACVR2A). However, tumor cells harboring these mutations might have impairments in growth and/or development. Notably, ACVR2A mutations are frequently identified in NASH-HCC compared to HCC caused by other etiologies (Pinyol et al., Reference Pinyol, Torrecilla, Wang, Montironi, Pique-Gili, Torres-Martin, Wei-Qiang, Willoughby, Ramadori, Andreu-Oller, Taik, Lee, Moeini, Peix, Faure-Dupuy, Riedl, Schuehle, Oliveira, Alves and Llovet2021). In addition, it has been reported that ACVR2A and ACVR1B mutations are frequently detected in colorectal cancer and that these genes play an important role in the carcinogenesis as tumor suppressors (Takeda et al., Reference Takeda, Kataoka, Nakayama, Ali, Oshima, Yamamoto, Park, Takegami, An, Jenkins, Copeland and Oshima2019).

To assess the clonality of each tumor, we decided to use these mutations for multiregional mutational profiling using dPCR. In contrast to the WES approach, dPCR is intended to detect extremely rare variants down to the range of 0.001% VAF, for which an NGS assay would not be able to detect mutations (Diaz & Bardelli, Reference Diaz and Bardelli2014). With the dPCR technology, the multiregional profiling revealed that both mutations were present in both tumors. Interestingly, among a total of 13 regions of tumors, the VAFs of NRAS were consistently higher in C1 tumor regions compared to those of the C2 tumor regions, whereas VAFs of ACVR2A were higher in C2 tumor regions compared to C1. However, the VAF of these mutations seemed too low to be considered a truncal mutation (Frankell et al., Reference Frankell, Dietzen, Al Bakir, Lim, Karasaki, Ward, Veeriah, Colliver, Huebner, Bunkum, Hill, Grigoriadis, Moore, Black, Liu, Thol, Pich, Watkins, Naceur-Lombardelli and Swanton2023), suggesting that these mutations may not play a central role in the development of HBs. A potential explanation for these findings is that the APC germline mutation has impaired degradation of β-catenin, which could drive cell division susceptibility for malignant transformation without additional somatic mutations. However, since no other paternal family member had HB with the same APC germline mutation, there should be nongenetic triggers for HB development in the twins.

As another index of the presence of tumor in the body, a high VAF ctDNA (i.e., 1.31% as ctDNA) for ACVR2A was found in the plasma of prehepatectomy Infant-2 by a dPCR assay. The ACVR2A mutation seemed to have disappeared after chemotherapy and remained undetectable during the postoperative period. These results further support the finding that the ACVR2A mutation is derived from the C2 tumor, and not from the germline. Here, it should again be noted that the ACVR2A mutation was found in the C1 tumor, which was physically separated from the C2 tumor circulation system. It is extremely unlikely that these tumors arose independently with the same somatic mutation. Therefore, as our multiregional dPCR assay demonstrated, it can be reasonably hypothesized that the tumor cells implanted via the maternal and fetal circulation systems during which time the tumors were not yet visible using routine detection modalities (Shatara et al., Reference Shatara, Xavier, Dombkowski, Cukovic, Poulik, Altinok, Ge and Taub2019). The undetectable tumor cells continued to proliferate after birth, and both tumors subsequently became detectable during infancy resulting in two independent chimeric tumors. Indeed, it has been reported that blood chimerism, which is defined as a blood cell genotype, contains different cells derived from two or more distinct zygotes in monochronic dizygotic twins, suggesting that mixed zygotes may result in microchimerism in organs (Lee et al., Reference Lee, Yoon, Ko, Seong, Park, Choi and Oh2014). Although limited to monochorionic dizygotic twins, a systematic review by Peters et al. (Reference Peters, Konig, Verhoeven, Schats, Mijatovic, Ket and Lambalk2017) reported that chimerism was demonstrable in >90% of 31 twins they investigated. The authors suggest that, as most of the chimeric twins were discovered by accident, the phenomena may be far more common than originally speculated. In fact, bidirectional transplacental cell trafficking between mother and fetus during pregnancy has been one of the emerging topics in studying microchimerism, which is defined as an organism being made up of more than one genetically distinct individual (Boddy et al., Reference Boddy, Fortunato, Wilson Sayres and Aktipis2015; Comitre-Mariano et al., Reference Comitre-Mariano, Martinez-Garcia, Garcia-Galvez, Paternina-Die, Desco, Carmona and Gomez-Gaviro2022). In the present study, the microchimerism was confirmed only by tumors of the twins. The fact that both tumors acquired microchimerism consisting of somatic mutations before birth suggests that the HB development initiated during pregnancy. Moreover, the fact that the tumor cells should have been trafficking systemically in the fetus, but only arose in the liver, suggests that some tissue environmental factors may accelerate tumor progression. Although a premature alloimmune system contributing to the establishment of tumor implantation has been suggested (Arakawa et al., Reference Arakawa, Ichikawa, Kubo, Motoi, Kumamoto, Nakajima, Yonemori, Noguchi, Sunami, Shiraishi, Kakishima, Yoshida, Hishiki, Kawakubo, Kuroda, Kiyokawa, Yamada, Yanaihara, Takahashi and Ogawa2021), our findings further imply that an organ-specific cell-trafficking environment may exist during HB development.

Overall, we propose a potential order explaining the development of HBs in the monozygotic twins as follows: (1) the germline APC mutation gave rise to a susceptible genetic background to each tumor; (2) these tumors separately acquired different nongenetic triggers of tumorigenesis during pregnancy; (3) a small portion of each tumor traveled via maternal circulation and implanted in the liver of the counterpart (the implantation continued until birth); and (4) the triggered tumor cells kept proliferating after birth.

This study has the following limitations. Germline mutations were only accessible within two generations since the other three affected individuals (grandparents of the twins) had passed away. Due to clinical priority, only a limited amount of prechemotherapeutic biopsy specimens was available from Infant-1 (C1), whereas posthepatectomy specimens were all postchemotherapeutic tissues (C1). Specimens from Infant-2 were only posthepatectomy (C2). As a result, the quality and reproducibility for sequencing analysis might have been restricted. In fact, nongenetic alterations have not been fully assessed. Blood samples were not periodically obtained due to technical difficulties and infant conditions. Although two SNVs were used for cellular migration tracking, the mechanism of metachronous tumor development remains unknown. Finally, we were unable to examine the microchimerism in tissues other than liver, and we were not able to assess those of their mother.

In conclusion, we have identified somatic driver mutations, an NRAS activating mutation and a loss of function ACVR2A mutation, in HBs that developed in monozygotic twins with a pathogenic germline APC variant. The importance of these driver mutations in the development of HB remains to be elucidated. However, our data suggest that blood and cells of the twins could be mutually transmittable via the utero-placental maternal circulation; and HB tumorigenesis began during the fetal stage after organ development of the liver. We recommend performing periodical surveillance in twins with tumors to assess for malignancy at young ages if one of the twins exhibits a malignant tumor at that age. These findings and recommendations are based on the evidence for tumor cell microchimerism and shared tumor-development in the utero-placental environment.