MiR-206 promotes extracellular matrix accumulation and relieves infantile hemangioma through targeted inhibition of DNMT3A.

MiR-206 is abnormally expressed in infant hemangioma endothelial cells (HemECs), but the mechanism is not clear. We explored the intervention of miR-206 in HemECs in relation to extracellular matrix (ECM) metabolism. We selected 48 cases of infantile hemangioma (IH) from volunteer organizations. After the isolated and extracted HemECs were interfered with overexpressed or silenced miR-206, the effects of miR-206 on the proliferation, migration and invasion of HemECs were examined through basic cell function experiments. The expression differences of miR-206, DNA Methyltransferase 3A (DNMT3A) and ECM-related genes were analyzed as needed by qRT-PCR or Western blot. TargetScan and dual-luciferase experiments were applied to predict and confirm the binding relationship between miR-206 and DNMT3A. The correlation between miR-206 and DNMT3A was analyzed in IH tissues by Pearson correlation coefficient, and further confirmed in HemECs by conducting rescue experiments. A nude mouse model of xenograft tumor was constructed to verify the results of in vitro experiments. MiR-206, which was downregulated in proliferative hemangioma, suppressed the malignant development of HemECs by regulating ECM-related genes. As the target gene of miR-206, DNMT3A was high-expressed in IH tissues and was negatively correlated with miR-206. Overexpressed DNMT3A counteracted the inhibitory effect of miR-206 mimic on HemECs and its regulatory effect on ECM. The results of in vivo experiments were consistent with those from cell experiments. Thus, miR-206 could promote ECM accumulation through targeted inhibition of DNMT3A, further inhibiting the malignant development of HemECs and relieving IH.

Function and mutual interaction of BiP-, PERK-, and IRE1α-dependent signalling pathways in vascular tumours.

Spontaneously regressing infantile haemangiomas and aggressive angiosarcomas are vascular tumours with excessive angiogenesis. When analysing haemangiomas and angiosarcomas immunohistochemically with respect to their chaperone profiles we found that angiosarcomas have significantly elevated protein levels of binding immunoglobulin protein (BIP) and PERK with concomitant attenuated IRE1α levels, whereas haemangioma tissue exhibits the same pattern as embryonal skin tissue. We show that BiP is essential for the maintenance of VEGFR2 protein, which is expressed in the endothelium of both tumour types. When studying the effects of BiP, the IRE1α/Xbp1 -, and PERK/ATF4-signalling pathways on the migration and tube-forming potential of endothelial cells, we show that downregulation of BiP, as well as inhibition of the kinase activity of IRE1α, inhibit in vitro angiogenesis. Downregulation of PERK (PKR-like kinase; PKR = protein kinase R) levels promotes Xbp1 splicing in endoplasmic reticulum (ER)-stressed cells, indicating that in angiosarcoma the elevated PERK levels might result in high levels of unspliced Xbp1, which have been reported to promote cell proliferation and increase tumour malignancy. The data presented in this study revealed that in addition to BiP or PERK, the kinase domains of IRE1α and Xbp1 could be potential targets for the development of novel therapeutic approaches for treating angiosarcomas and to control tumour angiogenesis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Intramuscular fast-flow vascular anomaly contains somatic MAP2K1 and KRAS mutations.

BACKGROUND: The term "intramuscular hemangioma capillary type" (IHCT) refers to a fast-flow vascular lesion that is classified as a tumor, although its phenotype overlaps with arteriovenous malformation (AVM). The purpose of this study was to identify somatic mutations in IHCT. METHODS: Affected tissue specimens were obtained during a clinically indicated procedure. The diagnosis of IHCT was based on history, physical examination, imaging and histopathology. Because somatic mutations in cancer-associated genes can cause vascular malformations, we sequenced exons from 446 cancer-related genes in DNA from 7 IHCT specimens. We then performed mutation-specific droplet digital PCR (ddPCR) to independently test for the presence of a somatic mutation found by sequencing and to screen one additional IHCT sample. RESULTS: We detected somatic mutations in 6 of 8 IHCT specimens. Four specimens had a mutation in MAP2K1 (p.Q58_E62del, p.P105_I107delinsL, p.Q56P) and 2 specimens had mutations in KRAS (p.K5E and p.G12D, p.G12D and p.Q22R). Mutant allele frequencies detected by sequencing and confirmed by ddPCR ranged from 2 to 15%. CONCLUSIONS: IHCT lesions are phenotypically similar to AVMs and contain the same somatic MAP2K1 or KRAS mutations, suggesting that IHCT is on the AVM spectrum. We propose calling this lesion "intramuscular fast-flow vascular anomaly."

Identification of the two KIT isoforms and their expression status in canine hemangiosarcomas.

BACKGROUND: KIT is a tyrosine kinase growth factor receptor. High expression of KIT has been found in several tumors including canine hemangiosarcoma (HSA). This study investigated the correlation of KIT expression and c-kit sequence mutations in canine HSAs and benign hemangiomas (HAs). RESULTS: Immunohistochemistry (IHC) staining confirmed KIT expression in 94.4 % (34/36) of HSAs that was significantly higher than 0 % in HAs (0/16). Sequencing the entire c-kit coding region of HSAs and normal canine cerebellums (NCCs) revealed GNSK-deletion in exon 9. As for exon 9 genotyping by TA-cloning strategy, GNSK-deletion c-kit accounted for 48.6 % (68/140) colonies amplified from12 KIT-positive HSAs, a significantly higher frequency than 14.1 % (9/64) of colonies amplified from six NCCs. CONCLUSIONS: Due to the distinct expression pattern revealed by IHC, KIT might be used to distinguish benign or malignant vascular endothelial tumors. Moreover, the high incidence of GNSK-deletion c-kit in canine HSAs implicates KIT isoforms as possibly participating in the tumorigenesis of canine HSAs.

Epidermal growth factor (EGF) triggers the malignancy of hemangioma cells via activation of NF-κB signals.

Hemangioma (HA) is tumor formed by hyper-proliferation of vascular endothelial cells. However, the role and mechanisms of epidermal growth factor (EGF) on the progression of HA are not well illustrated. Our present study revealed that EGF can significantly promote the in vitro proliferation and motility of HA cells, which was confirmed by the up regulation of Bcl-2, proliferating cell nuclear antigen (PCNA), and metalloproteinase-2 (MMP-2) and MMP-9. The pharmacological inhibition of NF-κB, while not ERK1/2 or PI3K/Akt, attenuated EGF induced cell proliferation and expression of MMP-2 and MMP-9. EGF treatment also increased the phosphorylation, nuclear translocation and transcriptional activities of NF-κB in HA cells. These data suggested that NF-κB plays an essential role in EGF induced malignancy of HA cells. Furthermore, EGF treatment also increased the phosphorylation of IκB and IKKα, while not IKKß or IKKγ. The knockdown of IKKα reversed EGF induced activation of NF-κB. EGF treatment also decreased the phosphorylation of GSK-3ß and increased its activities in both HDEC and CRL-2586 EOMA cells. LiCl, a potent GSK-3ß inhibitor, can obviously reverse EGF induced up regulation of p65 phosphorylation. Collectively, our study revealed that EGF can trigger the malignancy of HA cells via induction of proliferation and invasion. The activation of NF-κB through IKKα/IκBα and GSK-3ß signal is essential for this process. It suggested that EGF/NF-κB signal may represent a novel therapeutic target for the treatment of human HA.

Disruption and inactivation of the PP2A complex promotes the proliferation and angiogenesis of hemangioma endothelial cells through activating AKT and ERK.

Hemangioma is a benign vascular neoplasm of unknown etiology. In this study, we generated an endothelial-specific PyMT gene-expressing transgenic mouse model that spontaneously develops hemangioma. Based on this transgenic model, a specific binding between PyMT and the core AC dimer of protein phosphatase 2A (PP2A) was verified in hemangioma vascular endothelial cells. The binding between PyMT and the PP2A AC dimer resulted in dissociation of the B subunit from the PP2A complex and inactivation of PP2A phosphatases, which in turn activated AKT and ERK signaling and promoted cell proliferation, migration and angiogenesis in vitro and tumorigenesis in vivo. Consistent with the in vitro findings, decreased PP2A phosphatase activity and disruption of the PP2A heterotrimeric complex were also observed in both primary transgene-positive TG(+) mouse hemangioma endothelial cells (TG(+) HEC cells) and human proliferating phase hemangioma endothelial (human HEC-P) cells, but not in transgene-negative TG(-) mouse normal vascular endothelial cells (TG(-) NEC cells) and human involuting phase hemangioma endothelial (human HEC-I) cells. Further, it was observed that in human hemangioma cells, endoglin could compete with the PP2A/A, C subunits for binding to the PP2A/B subunit, thereby resulting in dissociation of the B subunit from the PP2A complex. Treatment of Tie2/PyMT transgenic mice with the PP2A activator FTY720 significantly delayed the occurrence of hemangioma. Our data provide evidence of a previously unreported anti-proliferation and anti-angiogenesis effect of PP2A in vascular endothelial cells, and show the therapeutic value of PP2A activators in hemangioma.

R132C IDH1 mutations are found in spindle cell hemangiomas and not in other vascular tumors or malformations.

Spindle cell hemangioma (SCH) is a rare, benign vascular tumor of the dermis and subcutis. The lesions can be multifocal and are overrepresented in Maffucci syndrome, in which patients also have multiple enchondromas. Somatic mosaic R132C IDH1 hotspot mutations were recently identified in Maffucci syndrome. We evaluated the presence of mutations in solitary and multiple SCHs in patients without multiple enchondromas and tested a range of other vascular lesions that enter into the differential diagnosis. The R132C IDH1 mutation was identified by hydrolysis probes assay and confirmed by Sanger sequencing in 18 of 28 (64%) SCHs; of the 10 negative cases, 2 harbored a mutation in IDH2 (R172T and R172M) by Sanger sequencing. None of 154 other vascular malformations and tumors harbored an IDH1 R132C mutation, and R132H IDH1 mutations were absent in all 182 cases. All 16 SCHs examined by immunohistochemistry were negative for expression of HIF-1α. In conclusion, 20 of 28 (71%) SCHs harbored mutations in exon 4 of IDH1 or IDH2. Given that mutations were absent in 154 other vascular lesions, the mutation seems to be highly specific for SCH. The mutation does not induce expression of HIF-1α in SCH, and therefore the exact mechanism by which mutations in IDH1 or IDH2 lead to vascular tumorigenesis remains to be established.

Inhibition of hemangioma development in a syngeneic mouse model correlates with bcl-2 suppression and the inhibition of Akt kinase activity.

BACKGROUND: Hemangiomas are benign vascular tumors that are characterised by excessive angiogenesis. While there is no definitive treatment for these tumors, several angiogenesis inhibitors, including bleomycin, have been employed. To better understand the mechanism of bleomycin in accelerating haemangioma regression, we investigated the effects of the drug on hemangiomagenesis using a previously described mouse hemangioma model. MATERIALS AND METHODS: The effects of bleomycin were tested in mice injected with endothelioma cells to induce hemangioma development. At termination, tissue samples from bleomycin-treated and control mice were stained with hematoxylin and eosin for histological examination. Bcl-2, flk-1 and vWF expression were studied by immunofluorescence microscopy. Hematological analysis was undertaken using a hemocounter. Akt activity was analyzed in tissue homogenates and endothelioma cells using ELISA. Also, caspase activity was analysed in endothelioma cells by ELISA. RESULTS: Bleomycin inhibited tumor growth in vivo in a dose-dependant manner. Our findings also revealed that bleomycin inhibited Akt activation and suppressed bcl-2. In vitro bleomycin increased caspase activation. CONCLUSION: Our observations reveal possible mechanisms for the inhibitory effects of bleomycin on hemangiomagenesis, and raise the possibility that bcl-2 might be an important therapeutic target in the treatment of hemangiomas.

Down-regulation of mir-424 contributes to the abnormal angiogenesis via MEK1 and cyclin E1 in senile hemangioma: its implications to therapy.

BACKGROUND: Senile hemangioma, so-called cherry angioma, is known as the most common vascular anomalies specifically seen in the aged skin. The pathogenesis of its abnormal angiogenesis is still unclear. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we found that senile hemangioma consisted of clusters of proliferated small vascular channels in upper dermis, indicating that this tumor is categorized as a vascular tumor. We then investigated the mechanism of endothelial proliferation in senile hemangioma, focusing on microRNA (miRNA). miRNA PCR array analysis revealed the mir-424 level in senile hemangioma was lower than in other vascular anomalies. Protein expression of MEK1 and cyclin E1, the predicted target genes of mir-424, was increased in senile hemangioma compared to normal skin or other anomalies, but their mRNA levels were not. The inhibition of mir-424 in normal human dermal microvascular ECs (HDMECs) using specific inhibitor in vitro resulted in the increase of protein expression of MEK1 or cyclin E1, while mRNA levels were not affected by the inhibitor. Specific inhibitor of mir-424 also induced the cell proliferation of HDMECs significantly, while the cell number was decreased by the transfection of siRNA for MEK1 or cyclin E1. CONCLUSIONS/SIGNIFICANCE: Taken together, decreased mir-424 expression and increased levels of MEK1 or cyclin E1 in senile hemangioma may cause abnormal cell proliferation in the tumor. Senile hemangioma may be the good model for cutaneous angiogenesis. Investigation of senile hemangioma and the regulatory mechanisms of angiogenesis by miRNA in the aged skin may lead to new treatments using miRNA by the transfection into senile hemangioma.

The enzymatic activity of type 1 iodothyronine deiodinase (D1) is low in liver hemangioma: a preliminary study.

Type 1 iodothyronine deiodinase (D1) is a crucial enzyme which converts the prohormone thyroxine (T4) into active tri-iodothyronine (T3). There has been strong evidence that the metabolism of thyroid hormones is disturbed in some neoplastic tissues such as thyroid, renal, and breast cancer. However, there are few available data about D1 enzyme activity in benign tumors such as hemangioma, which is the most common primary liver tumor. Hence this study aimed to determine the enzymatic activity of D1 in hemangiomas in relation to healthy liver tissue. Seven tumors and healthy control tissues were obtained from patients who had liver resection due to hemangioma. The activity was assessed by measurement of radioactive iodine released by deiodination catalyzed by D1. It was found that D1 activity was significantly lower in the hemangiomas than in the healthy surrounding tissue (p = 0.0017). The results indicated that thyroid hormones play important roles not only in the regulation of cell metabolism, but also in cell growth, division, and apoptosis. The active form T3 acts through its nuclear receptors and influences the up- and down-regulation of target genes. Healthy liver tissue expresses a high level of D1, but disturbed D1 activity may result in changes in the local concentration of T3 which may impair gene transcription. These finding demonstrate a low enzymatic activity of D1 in liver hemangioma and suggest an as yet unknown role of thyroid hormones in this type of benign liver tumor.

Increased type 3 iodothyronine deiodinase activity in a regrown hepatic hemangioma with consumptive hypothyroidism.

INTRODUCTION: Infantile hepatic hemangioma with consumptive hypothyroidism is a rare condition. CASE REPORT: A 4-month-old girl presented with diffuse hepatic hemangiomas during treatment of congenital hypothyroidism. Serum reverse triiodothyronine was elevated, and her hypothyroidism improved concomitant with involution of the hemangioma following prednisolone and interferon-alpha administration. At 20 months of age, 7 months after discontinuing prednisolone and interferon-alpha, a focal hemangioma regrew from one of the previous lesions and was surgically resected. The expression and activity of type 3 iodothyronine deiodinase (D3) were elevated in the resected tumor tissue compared with placenta. DISCUSSION: Here, we describe a patient with consumptive hypothyroidism and diffuse infantile hepatic hemangiomas, one of which regrew after involution following pharmacotherapy. The etiology of elevated D3 activity is also discussed. CONCLUSION: It is important to identify infantile hepatic hemangioma in patients with hypothyroidism refractory to hormone replacement therapy, who have low free triiodothyronine despite high thyrotropin and normal free thyroxine levels, and long-term follow-up will be needed for these patients.

Activation of matrix metalloproteinase (MMP)-2 by membrane type 1-MMP and abnormal immunolocalization of the basement membrane components laminin and type IV collagen in canine spontaneous hemangiosarcomas.

We performed immunohistochemical investigation of the basement membrane (BM) components, namely, type IV collagen and laminin, in 83 canine hemangiosarcomas (HSAs), 22 hemangiomas, and some granulation tissues (GTs). Additionally, we analyzed the expression and activities of matrix metalloproteinase (MMP)-2, MMP-9, and membrane type 1-MMP (MT1-MMP) using the same samples by immunohistochemistry and gelatin zymography to investigate whether MMPs were associated with the BM degradation. In immunohistochemistry for the BM components, many HSAs showed discontinuous linear/negative immunoreactivity in the BM (type IV collagen: 49.4%/14.5%, laminin: 60.3%/10.8%, respectively). In contrast, almost all hemangiomas showed continuous staining in the BM (type IV collagen: 90.9%, laminin: 95.5%, respectively). Interestingly, positive cytoplasmic immunoreactivity for type IV collagen and laminin was observed in 97.6% and 91.6% HSA, respectively. Although MMP-9 immunoreactivity wasn't detected in neoplastic and active angiogenic endothelial cells (ECs), MMP-2 was detected in all ECs of GTs and in neoplastic cells of both vascular tumors. A strong immunoreactivity for MT1-MMP was observed in active angiogenic ECs in GTs and in neoplastic ECs in HSAs. However, almost all hemangiomas showed weak/negative immunoreactivity. In gelatin zymography, significantly strong activity of active MMP-2 was observed in HSAs, similar to that in active angiogenesis in GTs; however, weak/no activity of active MMP-2 was detected in hemangiomas. In canine HSA, neoplastic cells had active MMP-2, possibly activated by MT1-MMP, and discontinuous status of BM might be associated with activity of active MMP-2.

Dusp-5 and Snrk-1 coordinately function during vascular development and disease.

Mitogen-activated protein kinases play an integral role in several cellular processes. To regulate mitogen-activated protein kinases, cells express members of a counteracting group of proteins called phosphatases. In this study, we have identified a specific role that one member of this family of phosphatases, dual-specific phosphatase-5 (Dusp-5) plays in vascular development in vivo. We have determined that dusp-5 is expressed in angioblasts and in established vasculature and that it counteracts the function of a serine threonine kinase, Snrk-1, which also plays a functional role in angioblast development. Together, Dusp-5 and Snrk-1 control angioblast populations in the lateral plate mesoderm with Dusp-5 functioning downstream of Snrk-1. Importantly, mutations in dusp-5 and snrk-1 have been identified in affected tissues of patients with vascular anomalies, implicating the Snrk-1-Dusp-5 signaling pathway in human disease.

[Iodothyronine deiodinases expression in thyroid neoplasias]. / Expressão das iodotironinas desiodases nas neoplasias tireoidianas.

The iodothyronine deiodinases constitute a family of selenoenzymes that catalyze the removal of iodine from the outer ring or inner ring of the thyroid hormones. The activating enzymes, deiodinases type I (D1) and type II (D2), are highly expressed in normal thyroid gland. Benign or malignant neoplastic transformation of the thyroid cells is associated with changes on the expression of these enzymes, suggesting that D1 or D2 can be markers of cellular differentiation. Abnormalities on the expression of both enzymes and also of the deiodinase type III (D3), that inactivates thyroid hormones, have been found in other human neoplasias. So far, the mechanism or implications of these findings on tumor pathogenesis are not well understood. Nevertheless, its noteworthy that abnormal expression of D2 can cause thyrotoxicosis in patients with metastasis of follicular thyroid carcinoma and that increased D3 expression in large hemangiomas causes severe hypothyroidism.

Expressão das iodotironinas desiodases nas neoplasias tireoidianas / Iodothyronine deiodinases expression in thyroid neoplasias

As iodotironinas desiodases formam uma família de selenoenzimas com propriedades catalíticas distintas que ativam ou inativam os hormônios tireoidianos via desiodação do anel fenólico ou tirosínico da molécula do T4. As desiodases tipo I e II (D1 e D2) são as enzimas responsáveis pela geração do T3 e são amplamente expressas na tireóide normal. A transformação neoplásica benigna ou maligna da glândula tireóide está associada a alterações na expressão dessas isoenzimas, sugerindo um possível papel da D1 e da D2 como marcadores de diferenciação celular. Anormalidades na expressão de ambas enzimas e da desiodase tipo III (D3), inativadora do hormônios tireoidianos, são também encontradas em outras neoplasias humanas. Os mecanismos ou implicações do aumento ou diminuição das desiodases na patogênese neoplásica são pouco compreendidas. No entanto, é importante observar que a expressão anormal da D2 pode ser responsável por um quadro de tireotoxicose em pacientes com metástases de carcinoma folicular de tireóide, enquanto que o aumento da D3 em hemangiomas pode causar hipotireoidismo de difícil tratamento.

The iodothyronine deiodinases constitute a family of selenoenzymes that catalyze the removal of iodine from the outer ring or inner ring of the thyroid hormones. The activating enzymes, deiodinases type I (D1) and type II (D2), are highly expressed in normal thyroid gland. Benign or malignant neoplastic transformation of the thyroid cells is associated with changes on the expression of these enzymes, suggesting that D1 or D2 can be markers of cellular differentiation. Abnormalities on the expression of both enzymes and also of the deiodinase type III (D3), that inactivates thyroid hormones, have been found in other human neoplasias. So far, the mechanism or implications of these findings on tumor pathogenesis are not well understood. Nevertheless, its noteworthy that abnormal expression of D2 can cause thyrotoxicosis in patients with metastasis of follicular thyroid carcinoma and that increased D3 expression in large hemangiomas causes severe hypothyroidism.

Immune response: A possible role in the pathophysiology of hemangioma.

Hemangioma is a distinct category of benign vascular tumors characterized by presentation within the first weeks of life, rapid growth during the first year and variable degree of spontaneous involution over a period of several years. Recent research reported that CD8+ T cells in hemangiomas, and the endothelia of hemangioma uniquely expressed leukocyte marker FCgammaRII and myeloid cell marker. Presence of high levels of indoleamine 2,3-dioxygenase in proliferating hemangiomas and significantly decreasing during involution was also confirmed. Topical application of imiquimod cream, an immune regulator, to proliferating hemangiomas apparently accelerated regression of the lesions. These findings suggest immune response may be involved in the pathogenesis of hemangioma. The endothelia of hemangioma may express various markers to escape the immune surveillance. An immune response may be one of the mechanisms for hemangioma regression. Strategies with systemically or locally applying immune regulator into the tumor may be an applicable way in accelerating the involution of hemangioma.

Transforming growth factor-beta promotes inactivation of extracellular thyroid hormones via transcriptional stimulation of type 3 iodothyronine deiodinase.

Thyroid hormone is a critical mediator of cellular metabolism and differentiation. Precise tissue-specific regulation of the concentration of the active ligand, T(3), is achieved by iodothyronine monodeiodination. Type 3 iodothyronine deiodinase (D3) is the major inactivating pathway, preventing activation of the prohormone T(4) and terminating the action of T(3). Using nontransformed human cells, we show that TGF-beta stimulates transcription of the hDio3 gene via a Smad-dependent pathway. Combinations of Smad2 or Smad3 with Smad4 stimulate hDio3 gene transcription only in cells that express endogenous D3 activity, indicating that Smads are necessary but not sufficient for D3 induction. TGF-beta induces endogenous D3 in diverse human cell types, including fetal and adult fibroblasts from several tissues, hemangioma cells, fetal epithelia, and skeletal muscle myoblasts. Maximum stimulation of D3 by TGF-beta also requires MAPK and is synergistic with phorbol ester and several mitogens known to signal through transmembrane receptor tyrosine kinases but not with estradiol. These data reveal a previously unrecognized interaction between two pluripotent systems, TGF-beta and thyroid hormone, both of which have major roles in the regulation of cell growth and differentiation.

Thymidine phosphorylase activity in liver tissue and its correlation with multifocal occurrence of hepatocellular carcinomas.

BACKGROUND: In hepatocellular carcinoma (HCC), new tumors develop in the residual liver within a few years after hepatectomy. However, the biological risk factors of multifocal occurrence of cancers remains unclear. In this study, the thymidine phosphorylase (TP) activity, which is known as an angiogenic factor, of cancerous and non-cancerous liver tissues in HCC was analyzed to determine its suitability as a biological marker of the multifocal occurrence of HCCs. MATERIALS AND METHODS: Fresh tissues (tumor: HCC and adjacent liver tissue: N-HCC) from 63 patients with HCC and normal liver tissues (NL) from 6 patients without HCC were obtained. The TP activities of the tissues were analyzed by an enzyme-linked immunosorbent assay (ELISA). RESULTS: The mean TP activity of 63 HCCs (136 U/mg protein) was higher than that of 63 N-HCCs (81 U/mg protein) and that of 6 NLs (47 U/mg protein, p < 0.001). Multifocal occurrence of HCCs were detected in 17 patients. In these 17 patients, the mean TP activity of HCCs (145 U/mg protein) was not different from that of HCCs from the remaining 46 patients (133 U/mg protein, p = 0.272), however the mean TP activity of N-HCCs (110 U/mg protein) was significantly higher than that of N-HCCs from the remaining 46 patients (71 U/mg protein, p = 0.038). Moreover, only a high TP activity of N-HCCs was detected as a significant risk factor of multifocal occurrence of HCCs. CONCLUSION: Patients who have tumors with high TP activity in the non-cancerous livers may have a risk of multifocal occurrence of HCCs in the residual liver.