Heterogeneous molecular behavior in liver tumors (HCC and CCA) of two patients with acute intermittent porphyria

Abstract

Introduction: Acute intermittent porphyria (AIP) is a very rare (orphan) metabolic disorder of porphyrin biosynthesis which is characterized by elevated plasma and urine levels of 5-aminolevulinic acid (5-ALA) and porphobilinogen (PBG). Patients with this disorder which is caused by a germline mutation of the hydroxymethylbilan-synthase (HMBS)-gene have a high risk of primary liver cancer which may be determined by disease activity. The exact mechanism of carcinogenesis of this rare tumor is unknown, however.

Materials and methods: We analyzed paraffin-embedded formalin-fixed liver tumor and normal liver specimens of two female AIP patients treated at the Munich EPNET center. One patient had developed hepatocellular carcinoma (HCC), the other intrahepatic cholangiocarcinoma (CCA). Since biallelic inactivation of HMBS had been observed in one study, we used Sanger and next-generation sequencing with a 8 gene porphyria panel plus 6 potential modifier loci to search for mutations in DNA extractions.

Results: In the patient with the HCC, we found a second inactivating mutation in the HMBS gene in the tumor but not in the adjacent normal liver tissue. No mutation could be found in the liver tissues of the patient with CCA, however.

Conclusions: Biallelic inactivation of HMBS or protoporphyrinogen-oxidase (PPOX), another enzyme of porphyrin biosynthesis, has been observed in patients with acute porphyrias and liver tumors. We could confirm this in our patient with HCC with a mutation in HMBS but not in the one with CCA. Since 5-ALA can be converted into carcinogenic substances such as 4,5-dioxovaleric acid (DOVA) or 3,6-dihydropyrazine-2,5-dipropanoic acid (= cyclic dimerization product of 5-ALA), local production of these metabolites in hepatic areas with complete loss of HMBS activity may contribute to liver carcinogenesis.

Keywords: 4,5-Dioxovaleric acid; 5-ALA; Acute porphyria; Biallelic inactivation; Cholangiocarcinoma; HMBS activity; Hepatocellular carcinoma.

Fig. 1

Abdominal magnetic resonance imaging (MRI, 1.5 Tesla) of patient 1: a T2-weighed axial image: the HCC is slightly hyperintensive with a peripheral capsule (long white arrow) and central inhomogeneous structure (short white arrow). b T2-weighed axial image after gadolinium containing contrast dye: marked contrast dye uptake into the peripheral tumor capsule (long black arrow). In the center, a marked hypointensive possibly fibrotic necrosis is seen (short black arrow). c T1-weighed coronal plane image of the abdomen after gadolinium containing contrast dye. With regard to the liver compare to b

Fig. 2

Normal liver and hepatocellular carcinoma of patient 1 (HE, magnification 10x)

Fig. 3

Contrast enhanced computed tomography (CT) scan of the liver of patient 2: left: arterial phase: hypodense lesion without sharply defined edges (3 × 3 × 4 cm) in right liver segment 6. Right: portalvenous phase

Fig. 4

Liver tissue of patient 2 (upper and middle photo: left part normal tissue, right part tumor; lower photo only cholangiocarcinoma (CCA), higher resolution)

Fig. 5

Immunohistochemical analysis of tumor tissue (see also Fig. 4). A: H and E: poorly differentiated ductal adenocarcinoma. B: Elastica van Gieson: high desmoplastic stromal content within the carcinoma. C CK 7: cytokeratin 7 highlights tumor cells. D CD 20: strong co-expression of cytokeratin 20 with the tumor cells. E CEA: focal and weak expression of CEA. F MUC 1: strong apical expression of MUC1. (The analyses were carried out with commercially available antibodies). All 5 analyses confirm the diagnosis of CCA

Fig. 6

Somatic second mutation in the HMBS gene of the HCC sample of patient 1: Green: reference sequence HMBS showing 3´ end of intron 5 and 5´ beginning of exon 6; upper two rows: partial Sanger forward sequence of two amplicons from HCC tumor tissue; lower two rows: corresponding partial Sanger reverse sequence of two amplicons from HCC tumor tissue. Numbers: cDNA nucleotide range depicting 4 bp of Intron 5 and range c.211 to c.231 of Exon 6. The mutation is c.212_227delTTGGAGAGAAAAGCCT, p. (ILE71SERfs*22). VAF was about 25%

Fig. 7

Relative abundance of gene sequence reads in the CCA sample versus matched control tissue for regions of interest (ROI). Possible losses in red, gains in green