Skin Cancer – Research Article Dermatology DOI: 10.1159/000503604
Received: June 20, 2019 Accepted after revision: September 19, 2019 Published online: January 2, 2020
Immunohistochemical Analysis of mTOR Pathway-Related Proteins in Kaposiform Hemangioendothelioma Zuopeng Wang a Chao Zheng a Hongqiang Sun b Wei Yao a Kai Li a Yangyang Ma c Shan Zheng a a Department of Pediatric Surgery, Children’s Hospital of Fudan University, Shanghai, PR China; b Department of Pediatric Surgery, Shandong Dezhou People’s Hospital, Shandong, PR China; c Department of Pathology, Children’s Hospital of Fudan University, Shanghai, PR China
Abstract Background: Mammalian target of rapamycin (mTOR) inhibitors have been shown to have excellent effects in the management of kaposiform hemangioendothelioma (KHE); however, the mechanism of action is unclear. This study identified the expressions of mTOR pathway-related proteins in different vascular tumors to provide insight into the pathogenesis of KHE. Methods: We retrospectively reviewed the pathologic specimens of 30 patients (KHE, 15; tufted angioma [TA], 5; infantile hemangioma [IH], 5; and lymphatic malformation [LM], 5). The immunohistochemical expression of mTOR-related proteins tuberous sclerosis complex 2 (TSC2), phosphatase and tensin homologue (PTEN), phosphorylated eukaryotic translation initiation factor 4E binding protein 1 (p-4EBP1), phosphorylated mTOR (p-mTOR), and phosphorylated ribosomal protein S6 kinase B1 (pP70S6K) were analyzed using Image-Pro Plus software. KHE
© 2020 S. Karger AG, Basel E-Mail karger@karger.com www.karger.com/drm had the following pattern of expression in the spindle vascular endothelial cells: TSC2 (–); PTEN (–); p-4EBP1 (+); p-mTOR (+); and p-P70S6K (+). Results: All 3 patients treated with sirolimus had good responses. The TA results were similar to KHE with no significant differences (p-4EBP1: p = 0.0687; pmTOR: p = 0.0832). The expressions of TSC2, PTEN, p-4EBP1, p-mTOR, and p-P70S6K were negative or weakly positive in IH with a statistically significant difference compared to KHE (p-4EBP1: p < 0.001; p-mTOR: p < 0.001; p-P70S6K: p < 0.001). LM had no significant differences when compared to KHE. Conclusions: The absence of TSC2 and PTEN caused abnormal activation of the mTOR signaling pathway and may be involved in the pathogenesis of KHE. The expression of mTOR-related proteins in TA and LM was similar to KHE, unlike IH. The KHE pattern of expression [PTEN (–), TSC2 (–), pmTOR (+), p-P70S6K (+), and p-4EBP1 (+)] suggested that sirolimus may be a good therapeutic choice. © 2020 S. Karger AG, Basel
Zuopeng Wang and Chao Zheng contributed equally to this work.
Kai Li Department of Pediatric Surgery, Children’s Hospital of Fudan University 399 Wan yuan Road Shanghai 201102 (People’s Republic of China) E-Mail likai2727 @ 163.com Downloaded by: University Toronto Libr. 142.150.190.39 - 1/3/2020 5:22:26 PM
Keywords Immunohistochemistry · Mammalian target of rapamycin · Kaposiform hemangioendothelioma · Phosphatase and tensin homologue 5 IH 5 TA 5 LM
Immunohistochemistry staining PTEN, TSC2, p-mTOR, p-P70S6K, and p-4EBP1 Image-Pro Plus software Image analysis IStata 10.1 statistical software Statistical analysis Fig. 1. Flowchart of Materials and Methods.
Introduction
Kaposiform hemangioendothelioma (KHE) is a distinct vascular tumor of childhood that was defined in 1993 to distinguish the entity from infantile hemangioma [1]. KHE was named for its unmistakable histologic resemblance to Kaposi sarcoma, which is characterized by irregular infiltrating densely packed spindled tumor cells closely associated with small slit- and sieve-like lumina [2]. Kaposi sarcoma usually expresses latency-associated nuclear antigen-1 and human herpesvirus-8 on immunohistochemical evaluation [3]. KHE is a borderline malignancy with locally extensive and aggressive biological behavior. The endothelial cells in nodules are usually positive for CD31, CD34, D240, LYVE1, and prox-1 by immumohistochemical staining, but negative for GLUT1, distinguishing the endothelial cells from hemangiomas [4, 5]. The Kasabach-Merritt phenomenon is a potentially life-threatening complication arising from KHE when combined with severe consumptive coagulopathy. The purpose of the treatment is to decrease the significant morbidity or mortality and involute the mass to prevent disfigurement; however, no guidelines have been established for KHE. The tumor shows variable responses to the same or different treatment strategies. Surgical resection is the definitive treatment for KHE; however, surgery is not always possible owing to inaccessible anatomic locations and the potential for excessive bleeding. The pharmacologic approaches for KHE, including systemic corticosteroids [6, 7], vincristine [8], propranolol [9, 10], 2
Materials and Methods For further details, see online supplementary material (see www.karger.com/doi/10.1159/000503604 for all online suppl. material) (Fig. 1) [19]. Results
Immunohistochemistry (IHC) staining revealed that phosphatase and tensin homologue (PTEN) and tuberous sclerosis complex 2 (TSC2) were not expressed in KHE, TA, and LM tissues (Fig. 2 and 3); however, the vascular endothelial cells of IH had weak expression of PTEN (Fig. 2b). There was no significant difference between the expression of PTEN and TSC2 in KHE and other vascular tumors. Varying degrees of phosphorylated mTOR (p-mTOR), phosphorylated ribosomal protein S6 kinase B1 (pP70S6K), and phosphorylated eukaryotic translation initiation factor 4E binding protein 1 (p-4EBP1) were expressed in KHE, TA, and LM. As shown in Figure 4a, 5a, and 6a, the three proteins were localized in the cytoplasm and nucleus of abnormally proliferating vascular endothelial cells. Conversely, IH tissues did not express pmTOR, p-P70S6K, or p-4EBP1. According to the semiWang/Zheng/Sun/Yao/Li/Ma/Zheng
Downloaded by: University Toronto Libr. 142.150.190.39 - 1/3/2020 5:22:26 PM 15 KHE and interferon-α [11], yield variable responses. In addition, steroid treatment is complicated by opportunistic infections, a cushingoid appearance, and hypertension. Neurotoxicity is the dose-limiting adverse reaction of vincristine. The reported severe complications of interferon-α, such as spastic diplegia, strongly limit its usage [12]. Based on a review of the literature, Blatt et al. [4] focused on the antiangiogenic potential of inhibitors of the mammalian target of rapamycin (mTOR) and used sirolimus [13–15] to successfully treat a child with a severe KHE. To date, sirolimus has been shown to be efficacious in more and more children with KHE; the side effects are tolerable, and evidence of success in using sirolimus is accumulating [16–18]; however, the molecular mechanism underlying sirolimus therapy in KHE remains unknown. The present study analyzed the expression of mTOR-related proteins in different vascular tumors, such as KHE, tufted angioma (TA), infantile hemangioma (IH), and lymphatic malformation (LM). This study identifies the role of the mTOR pathway in different vascular tumors to provide insight into the pathogenesis of KHE.
IHC PTEN in the 4 vascular tumors. Images are shown at a magnification of ×200. The brown staining (diaminobenzidine) correlates with positive staining, and a lack thereof (“blue staining”) indicates negative staining. a IHC staining of PTEN in KHE was negative. b IHC staining of PTEN in IH was weakly positive, and there was no significant difference compared with KHE. c IHC staining of PTEN in TA was negative. d IHC staining of PTEN in LM was negative.
a 50 µm b Fig. 3. Representative staining images of IHC TSC2 in the 4 vascular tumors. a IHC mTOR Pathway in Kaposiform Hemangioendothelioma c d Dermatology DOI: 10.1159/000503604 3 Downloaded by: University Toronto Libr. 142.150.190.39 - 1/3/2020 5:22:26 PM
staining of TSC2 in KHE was negative. b IHC staining of TSC2 in IH was negative. c IHC staining of TSC2 in TA was negative. d IHC staining of TSC2 in LM was negative. Color version available online 50 µm
a c b d
Fig. 4. Representative staining images of IHC p-mTOR in the 4 vascular tumors. a IHC staining of p-mTOR in KHE was positive, and localized in the cytoplasm and nuclei of abnormally proliferating vascular endothelial cells (inset, arrows). b p-mTOR– in IH. c p-mTOR+ in TA. d p-mTOR+ in LM.
4 Dermatology DOI: 10.1159/000503604 ment, the tumor shrank in size, the skin was markedly discolored, and the mass became soft. The efficacy of sirolimus treatment was divided into 3 responses (complete, partial, and none). The classifications were based on tumor regression, restoration of the platelet count, coagulation profile, and follow-up (Table 1) [8]. All patients typically exhibited the following IHC expression pattern: PTEN–; TSC2–; p-mTOR+; p-P70S6K+, and p-4EBP1+. Discussion
Sirolimus was isolated from Streptomyces hygroscopicus from soil samples on Easter Island in 1965, and initially evaluated as an antifungal agent in the 1970s [20]. Wang/Zheng/Sun/Yao/Li/Ma/Zheng Downloaded by: University Toronto Libr. 142.150.190.39 - 1/3/2020 5:22:26 PM
quantitative results of the Image-Pro Plus software, the expression of p-mTOR, p-P70S6K, and p-4EBP1 was significantly higher in KHE than IH (p-mTOR: p < 0.001; p-P70S6K: p < 0.001; p-4EBP1: p < 0.001 [t test]; Fig. 7). The TA and LM tissues expressed varying degrees of pmTOR, p-P70S6K, and p-4EBP1, but when compared with the three kinds of proteins in KHE, there was no significant difference (Fig. 7). All patients with IH underwent resection in the proliferating phase. Nine patients with KHE underwent surgical resection. Three patients with KHE received corticosteroid therapy, and 3 who received corticosteroids plus a vincristine regimen who did not respond subsequently received sirolimus (dose of 0.8 mg/m2, twice a day, a drug trough level of 10–15 ng/mL). During sirolimus treat- Color version available online
Fig. 5. Representative staining images of IHC p-P70S6K in the 4 vascular tumors. a IHC staining of p-P70S6K in KHE was positive, and localized in the cytoplasm and nuclei of abnormally proliferating vascular endothelial cells (inset, arrow). b p-P70S6K– in IH. c p-P70S6K+ in TA. d p-P70S6K+ in LM.
Classification Platelet count, n ×109/L Coagulation profile Reduction in size, % Follow-up, months Complete response Partial response No response >100 40–100 <40 Normal Normal/abnormal Abnormal >80 >50 <50 or increasing
>3 >3 –
Sirolimus has antifungal, immunosuppressive, and antiproliferative properties, and mTOR functions as a master switch of numerous cellular processes, including angiogenesis, cell growth and apoptosis, and cellular catabolism and anabolism [21]. Sirolimus is a specific mTOR
inhibitor and has wide effects on the mTOR signaling pathway, with potent antitumor action on various vascular malformations, including antiangiogenesis and antilymphangiogenesis, by reducing blood vessel or lymphatic vessel formation [22, 23]. For example, Chen et al. [24]
mTOR Pathway in Kaposiform Hemangioendothelioma Dermatology DOI: 10.1159/000503604 5 Downloaded by: University Toronto Libr. 142.150.190.39 - 1/3/2020 5:22:26 PM Table 1. Criteria of response Color version available online
a 50 µm b c d
Fig. 6. Representative staining images of IHC p-4EBP1 in the 4 vascular tumors. a IHC staining of p-4EBP1 in
KHE was positive and localized in the cytoplasm and nuclei of abnormal proliferating vascular endothelial cells (inset, arrows). b p-4EBP1– in IH. c p-4EBP1+ in TA. d p-4EBP1+ in LM. *** ns ns 0.25 0.20
0.15 IOD/area IOD/area 0.20 p-S6K1 ns 0.10 0.05 0 *** ns p-4EBPI 0.25 0.20 0.15 IOD/area 0.25 p-mTOR 0.10 0.05 KHE IH TA LM 0 *** ns ns 0.15 0.10 0.05 KHE IH TA LM 0 KHE IH TA LM
(IOD) represents the total optical density of brown-stained areas; IOD/area represents the expression of target protein. There were significant differences between KHE and IH; however, there was no significant difference between KHE and TA and LM. ns, no significant difference; *** p < 0.001.
6 Dermatology DOI: 10.1159/000503604 Wang/Zheng/Sun/Yao/Li/Ma/Zheng Downloaded by: University Toronto Libr. 142.150.190.39 - 1/3/2020 5:22:26 PM
Fig. 7. Semiquantitative results of IHC staining of p-mTOR, p-P70S6K, and p-4EBP1. Integral optical density
reported that the mTOR inhibitor temsirolimus decreases proangiogenic cytokines and thereby tumor angiogenesis, most likely by inhibiting mTOR signaling. Rapamycin suppresses angiogenesis and lymphangiogenesis in melanomas by blocking the mTOR pathway and downregulating the expression of VEGF-A/VEGFR-2 and VEGF-C/VEGFR-3 [23]. Huber et al. [25] provided experimental evidence for antilymphangiogenic activity in association with mTOR inhibition, suggesting impaired recovery of lymphatic flow across surgical incisions in murine skin. mTOR was identified as a serine/threonine kinase targeted by rapamycin that belongs to the phosphoinositide-kinase-related family of protein kinases and is regulated by upstream-activated PI3K/Akt [26]. The tumor suppressor genes PTEN and TSC2 exert negative effects upon this pathway upstream of mTOR (Fig. 8), and loss of PTEN and TSC2 results in activation of mTOR and subsequent upregulation of protein synthesis by activating the phosphorylation of P70S6K and 4EBP1 [27]. Phosphorylated and activated by mTOR, P70S6K actively recruits the 40S ribosomal subunit into translating polysomes and enhances the translation of 5′ terminal mTOR Pathway in Kaposiform Hemangioendothelioma
Downloaded by: University Toronto Libr. 142.150.190.39 - 1/3/2020 5:22:26 PM Color version available online Fig. 8. Brief diagram of the mTOR pathway.
oligopyrimidine tract mRNAs, which consists of 20% of the mRNAs [28]. Further, 4EBP1 plays an important role in regulating the interaction between eIF4G and eIF4E. The mTOR signaling pathway phosphorylates and activates 4EBP1 and modulates its dissociation from eIF4E [29]. Free eIF4E binds to the cap structure and promotes cap-dependent mRNA translation [30]. The molecular characterization of the PTEN product determines modulation of tumor growth and metastasis by antagonizing protein tyrosine kinases because PTEN has a protein tyrosine phosphatase domain and extensive homology to tensin [26]. Selection of patients is based on detection of the activated mTOR pathway and/ or loss of PTEN, thus TSC2 expression might help predict the sensitivity of tumor cells to rapamycin therapy. Phosphorylation of P70S6K and/or 4EBP1 might also help to determine the response to rapamycin. In the present study, we analyzed mTOR-related proteins, such as TSC2, PTEN, p-4EBP1, p-mTOR, and p-P70S6K, in four vascular tumors (KHE, TA, IH, and LM). To gather insight into the biology of these tumors and on the potential to predict the use of an mTOR-related protein as an immunohistochemical marker for clinical medicine decision-making. We demonstrated that PTEN and TSC2 were not expressed in KHE, TA, and LM tissues, while the expression of p-mTOR, p-P70S6K, and p-4EBP1 were much higher in KHE than IH. Therefore, we propose that the loss of PTEN or TSC2 abnormally activated the mTOR signaling pathway and rapamycin antagonizes tumor growth by blocking the pathway. For vascular tumors with lymphatic components, PTEN and/or TSC2 had negative expression and p-mTOR, p-P70S6K, and p-4EBP1 had positive expression based on IHC, thus sirolimus could serve as a potential treatment. Kaylani et al. [31] reported clinical improvement in a patient with PHACE syndrome, including refractory IH, using sirolimus. Given the natural regression, sirolimus therapy for IH warrants more clinical experience and verification of efficacy. In conclusion, the absence of TSC2 and PTEN caused abnormal activation of the mTOR signaling pathway, which may account for the pathogenesis of KHE. The expression of mTOR-related proteins in TA and LM were similar to KHE, while IH had significant differences. The expression pattern of KHE (PTEN–, TSC2–, p-mTOR+, p-P70S6K+, and p-4EBP1+) suggests that sirolimus may be a good therapeutic choice.