The role of heat shock protein 70 and glypican 3 expression in early diagnosis of hepatocellular carcinoma

Said A Mohamed; Al-Sayed M I Tealeb

doi:10.4103/egjp.egjp_21_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 42 | Issue : 2 | Page : 112-116

The role of heat shock protein 70 and glypican 3 expression in early diagnosis of hepatocellular carcinoma

Said A Mohamed¹, Al-Sayed M I Tealeb²
¹ Department of Pathology, Faculty of Medicine, Al-Azhar University, Assuit Branch, Egypt
² Department of Pathology, Faculty of Medicine, Al-Azhar University, Cairo, Egypt

Date of Submission	14-Oct-2022
Date of Decision	30-Oct-2022
Date of Acceptance	16-Nov-2022
Date of Web Publication	08-Feb-2023

Correspondence Address:
Al-Sayed M I Tealeb
Department of Pathology, Faculty of Medicine (Boys), Al-Azhar University, Cairo 11765
Egypt

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/egjp.egjp_21_22

Abstract

Background Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related deaths in the world and accounts for ~90% of primary liver carcinoma cases. This study aimed to evaluate the combination of two markers (HSP70 and GPC3) immunohistochemical expression in early detection of HCC. Materials and methods This study included 56 cases of primary hepatic lesions, five cases of focal nodular hyperplasia, five cases of hepatic adenoma, 33 cases of small vaguely nodular lesions (five of dysplastic nodule and 28 of early HCC), and 13 cases of late HCC. Results The sensitivity, specificity, and positive and negative predictive values of HSP70 for HCC detection were 78.2, 100, 100, and 24.1%, respectively. The sensitivity, specificity, and positive and negative predictive values of GPC3 for HCC detection were 60, 100, 100, and 36.8%, respectively. The sensitivity, specificity, and positive and negative predictive values for this combination (HSP70+GPC3) were 100, 100, 100, and 100%, respectively. Conclusion The immunoexpression of the two markers HSP70 and GPC3 is valuable in distinguishing between dysplastic nodules and early HCC. When the two markers are positive, the optimal sensitivity and specificity for detection of early HCC are obtained. The results of our study strikingly support the combination of the two markers together in the diagnostic distinction of nonconfirmed hepatic lesions of malignancies but do not support the use of both separately.

Keywords: carcinoma, liver cancer, glypican 3, heat shock protein 70, hepatocellular carcinoma, histopathology

How to cite this article:
Mohamed SA, Tealeb ASM. The role of heat shock protein 70 and glypican 3 expression in early diagnosis of hepatocellular carcinoma. Egypt J Pathol 2022;42:112-6

How to cite this URL:
Mohamed SA, Tealeb ASM. The role of heat shock protein 70 and glypican 3 expression in early diagnosis of hepatocellular carcinoma. Egypt J Pathol [serial online] 2022 [cited 2023 Jun 2];42:112-6. Available from: http://www.xep.eg.net/text.asp?2022/42/2/112/369372

Introduction

Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related deaths in the world and accounts for ~90% of primary liver carcinoma cases (Villanueva, 2019). The incidence rate of HCC associated with nonalcoholic fatty liver disease and nonalcoholic steatohepatitis is increasing, and the global burden of mortality from HCC is predicted to reach one million deaths annually by 2030 (Anstee et al., 2019).

Small HCCs can be divided into two subtypes according to WHO classification of tumors of the liver (Nagtegaal et al., 2020): early and progressed HCCs. Early, well-differentiated HCC has vague nodular pattern, with indistinct borders. Progressed HCC has a capsule and histologically resembles classic HCC, whereas early HCC lacks a definite capsule and histologically can resemble HGDN (Okusaka et al., 2012). Early HCC refers to tumors less than or equal to 2 cm in diameter (Zhou et al., 2011; Ojima et al., 2016).

Heat shock protein 70 (HSP70) is a stress-induced gene related to tumorigenesis, which modulates apoptosis and proliferation of the cell; it demonstrates anti-apoptotic effects, which ensures cell survival and promotes proliferation of tumor cells (Gehrmann et al., 2014). HSP70 sensitizes cancer cells to apoptosis and destroys tumors such as colon and breast carcinomas. Increased HSP70 gene expression has been reported in patients with HCC, and also it was upregulated in early HCC. The HSP70 protein overexpression has been studied in HCC by immunohistochemistry, and it was related to vascular invasion, high stage, and high Ki-67 index in HCC (Moudi et al., 2018). Among HSPs, HSP70 is correlated with cell apoptosis suppression and promotion of cell proliferation and cell migration, leading to tumor progression. In HCC carcinoma, it has been shown that HSP70 expression is increased. Moreover, its expression is distinctly increased in patients with HCC with portal vein invasion or lymph node metastasis. However, downregulation of HSP70 suppresses migration of HCC cells (Kobayashi et al., 2020).

Glypican (GPC) belongs to the family of heparan sulfate proteoglycans with identical structure, including a core protein (60–70 kD) which is linked to the cell membrane surface by a glycosyl-phosphatidyl-inositol anchor and the carboxy terminus is modified with a heparan sulfate side chain. Six members of the GPC family have been identified (GPC1–GPC6) in the mammalian genomes, and during the process of embryogenesis, all GPC proteins are highly expressed (Guo et al., 2020). GPCs are crucial for growth, metastasis, and angiogenesis of many human cancer cell types. Abnormal GPC expression has been noted in multiple types of cancer. For examples, GPC3 is closely related to the occurrence and development of tumors, such as HCC, neuroblastoma, and melanoma (Wang et al., 2019).

Materials and methods

This retrospective study was carried out on 56 primary hepatic lesions (paraffin blocks), which were collected from Al-Azhar University hospitals, Medical Research Institute, and private pathology laboratories over a period of 2 years. The lesions represented five cases of focal nodular hyperplasia, five cases of hepatic adenoma, 33 cases of small vaguely nodular lesions (five of dysplastic nodule and 28 of early HCC), and 13 cases of late HCC. The clinicopathological data of the cases were obtained from the pathological reports of the patients. Tumor classification was carried out according to the WHO (Nagtegaal et al., 2020). From each tissue block, three serial sections were cut into 4-µm thickness. To confirm the diagnosis, one section was stained by hematoxylin and eosin. Other three sections were mounted on poly-L-lysine-coated slides for immunohistochemical staining using the method of avidin-biotin-peroxidase complex (Vectastatin ABC Kit; Vector Lab., Burlingame, California, USA). HSP70 incubation with mouse monoclonal primary antibody (Cat. #MS-198-R7; Thermo- scientific, Burlingame, California, USA) (7.0 ml Ready-to-Use) at room temperature and GPC3 incubation with primary GPC3 rabbit monoclonal antibody [Anti-GPC 3 (SP86) (ab95363), 1: 100 dilution; ABCAM, Cambridge, UK) were done. The ultravision detection system (Catalog # TP.015.HD, ready to use, Thermo-scientific) was used to detect the resulting immune-complex. Sections were deparaffinized, rehydrated, incubated in H₂O₂, and then the slides were rinsed in PBS. For antigen retrieval, the slides were placed in a pressure cooker filled with citrate buffer. The slides were incubated in 10% normal goat serum (Vector Laboratories, Burlingame, California, USA) with serum-free protein block (10 min; Dako, Carpentaria, California, USA). The primary antibodies were placed on the sections and washed using PBS. The secondary antibody and streptavidin-peroxidase complex were applied. Sections were counterstained by hematoxylin and then rinsed gently in tap water. Lastly, the slides were dehydrated and then cleared in xylene. Then, the sections were visualized.

The material of this work comprises archival paraffin blocks of unknown patients, so no consent was required.

Control group: it included 10 liver specimens with apparently normal tissues obtained from adjacent normal tissue to benign lesions such as hemangioma. Negative control slides were used for each run of immunohistochemical staining for GPC3 and HSP70 by omitting the primary antibody. Positive control slides (as preferred by the data sheet for the antibody) were used in each run of immunohistochemical staining.

Statistical analysis

Data were collected and analyzed using the Statistical Packages for the Social Sciences (SPSS software version 20; IBM Corp., Armonk, New York, USA). The quantitative data were described using range, mean, SD, and median. Significance of the obtained results was judged at the 5% level. The used tests were χ² test, Kolmogorov–Smirnov test, sensitivity, specificity, positive predictive value, and negative predictive value. The P value was used to assess the significance of the results. P value of more than 0.05 was considered statistically significant.

Scoring system

Heat shock protein 70

The staining intensity was assigned a score of 0–3 (0, absent; 1, weak; 2, moderate; and 3, strong). HSP70 was scored positive if moderate to strong nuclear staining was seen in more than or equal to 10% of tumor cells. Scores 0 and 1 were considered negative, whereas scores 2 and 3 were considered positive (Thuy et al., 2016). Nuclear and/or cytoplasm yellow or brown staining is regarded as HSP70-positive criteria (Liu et al., 2016).

Glypican 3

The staining intensity was assigned a score of 0–3 (0, absent; 1, weak; 2, moderate; and 3, strong). GPC3 was considered as positive when moderate to strong brown nuclear, coarsely granular cytoplasmic and/or membranous staining was seen in more than or equal to 10% of tumor cells. Scores 0 and 1 were considered negative, whereas scores 2 and 3 were considered positive (Thuy et al., 2016).

Results

This retrospective study was carried out on 56 cases of primary hepatic lesions, five cases of focal nodular hyperplasia, five cases of hepatic adenoma, 33 cases of small vaguely nodular lesions (five of dysplastic nodule and 28 of early HCC), and 13 cases of late HCC.

Expression of heat shock protein 70

HSP70 shows no expression in normal liver tissue. All cases (100%) of hepatic adenoma showed negative expression for HSP70 and two (40%) cases of focal nodular hyperplasia showed positive expression for HSP70, whereas three (60%) cases of focal nodular hyperplasia showed negative expression for HSP70. All cases (100%) of dysplastic nodule showed negative expression for HSP70. Eight (8/28) cases (28.57%) of early HCC showed negative expression for HSP70, whereas 20 (71.43%) cases showed positive expression for HSP70. In the positive cases, HSP70 expression showed nuclear and/or cytoplasmic staining in the malignant hepatocytes. One (1/13) case (7.7%) of late HCC showed negative expression for HSP70, whereas 12 (92.3%) cases showed positive expression for HSP70. There is a positive statistical significant relation between immunohistochemical expression of HSP70 and histopathological type of studied cases (P<0.05) ([Figure 1] and [Figure 2]).

Figure 1: A case of early low-grade HCC, immunostained for HSP70 showing positive nuclear and cytoplasmic staining (ABC, counterstained with Hx.×400). HCC, hepatocellular carcinoma.

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Figure 2: A case of late high-grade HCC, immunostained for HSP70 showing positive nuclear and cytoplasmic staining (ABC, counterstained with Hx.×400). HCC, hepatocellular carcinoma.

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Expression of glypican 3

GPC3 shows no expression in normal liver tissue. All cases (100%) of hepatic adenoma and focal nodular hyperplasia showed negative expression for GPC3. One (1/5) case (20%) of dysplastic nodule showed positive expression for GPC3. A total of 14 (14/28) cases (50%) of early HCC showed negative expression for GPC3, and 14 (50%) cases showed positive expression for GPC3. In the positive cases, GPC3 expression showed membranous and/or cytoplasmic staining in the malignant hepatocytes. Two (2/13) cases (15.4%) of late HCC showed negative expression for GPC3, whereas 11 (84.6%) cases showed positive expression for GPC3. There is a positive statistically significant relation between immunohistochemical expression of GPC3 and histopathological type of studied cases (P<0.05) ([Figure 3] and [Figure 4]).

Figure 3: A case of low-grade HCC, immunostained for glypican 3 showing positive cytoplasmic and membranous staining (ABC, counterstained with Hx.×400). HCC, hepatocellular carcinoma.

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Figure 4: A case of late high-grade HCC, immunostained for glypican 3 showing positive cytoplasmic and membranous staining (ABC, counterstained with Hx.×400). HCC, hepatocellular carcinoma.

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There is a positive statistical significant relation between GPC3 and HSP70 expression in different studied groups. GPC3 and HSP70 overexpression are observed in late HCC than early HCC, whereas all cases of dysplastic nodule and hepatic adenoma are negative for HSP70, and all cases of benign group are negative for GPC3, yet two cases of FNH are positive for HSP70 and one case of dysplastic nodule is positive for GPC3.

There is a positive statistically significant relation between expression HSP70 and GPC3 expression in early HCC ([Table 1]).

Table 1: Relation between glypican 3 and HSP70 in early hepatocellular carcinoma

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The sensitivity, specificity, and positive and negative predictive values of HSP70 for HCC detection were 78.2, 100, 100, and 24.1%, respectively. The sensitivity, specificity, and positive and negative predictive values of GPC3 for HCC detection were 60, 100, 100, and 36.8%, respectively. The sensitivity, specificity, and positive and negative predictive values for this combination (HSP70+GPC3) were 100, 100, 100, and 100%, respectively ([Table 2]).

Table 2: Diagnostic accuracy for detection of early hepatocellular carcinoma using one or both markers

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Discussion

In the present study, the expression of HSP70 in normal liver tissue and all cases of hepatic adenoma and dysplastic nodule was negative. This is agree with Nguyen et al. (2015) who found that none of the hepatocellular adenoma was positive for HSP70 and nearly agree with Tremosini et al. (2012) who found that HSP70 immunostaining was only seen in a single HGDN that showed 30% immunoreactive cells. In the present study, 71.43% of early HCC cases showed positive expression for HSP70 and 89.5% of late HCC cases showed positive expression for HSP70. This nearly agrees with Tremosini et al. (2012), who found that HSP70 immunoreactivity was seen in the vast majority of early HCC (92%). Moreover, it agrees with Nguyen et al. (2015), who found that a majority of very well-differentiated HCCs (71%) were HSP70 positive compared with none of the typical hepatocellular adenomas. HSP70 immunohistochemistry can be useful in this differential diagnosis. This suggests that HSP70 staining may prove useful in identifying cases with borderline features between hepatocellular adenoma and HCC. In the current study, there was a positive statistically significant relation between immunoexpression of HSP70 and differentiation of small vaguely nodular lesions (dysplastic nodule and early HCC). We found HSP70 immunoreactivity in 71.43% of early HCCs, whereas none of DNs were positive. This agrees with Ojima et al. (2016), who found statistically significant low positive rates in DN but higher rates in early HCC. Expression of HSP70 showed statistically significant differences between DN and early HCC. Therefore, HSP70 is valuable for differentiation between DN and early HCC, as reported previously (Effendi et al., 2010; Chuma et al., 2013).

In the present study, there was a positive statistically significant relation between expression of GPC3 and histopathological type of studied cases. This is in agreement with Di Tommaso et al. (2011), who found that GPC3 overexpression was observed in advanced HCC than early HCC and was completely negative in non-neoplastic liver tissue. In the present study, there is a positive statistically significant relation between immunoexpression of GPC3 and differentiation of small vaguely nodular lesions (DN and early HCC). We found GPC3 immunoreactivity in 50% of early HCCs, whereas only one case of DNs was positive. This nearly agreed with Tremosini et al. (2012), who found that GPC3 immunoreactivity statistically increased with HCC dedifferentiation. Moreover, it agrees with Haruyama and Kataoka (2016) who found that higher levels of GPC3 expression were observed in moderately or poorly differentiated tumor cells, and these findings are also supported by other publications such as Ning et al. (2012) and Li et al. (2014).

In particular, in our study, the sensitivity of HSP70 and GPC3 in the diagnosis of HCC was 100%, which was higher than the values of Vasuri et al. (2017), who found that the sensitivity of HSP70 and GPC3 were 83 and 86%, respectively. The most relevant finding was that a combination of both markers (HSP70 and GPC3) showed absolute sensitivity and specificity (100%). This nearly agreed with Di Tommaso et al. (2011) and Tremosini et al. (2012).

Conclusions

The immunoexpression of two markers HSP70 and GPC3 is valuable in differentiation between dysplastic nodules and early HCC. When the two markers are positive, optimal sensitivity and specificity for detection of early HCCs are obtained. The results of our study strikingly support the combination of the two markers together in the diagnostic distinction of nonconfirmed hepatic lesions of malignancies but do not support the use of both separately.[22]

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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