Hepatocellular Carcinoma (HCC) is the sixth most common cancer and fourth leading cause of cancer related deaths worldwide [1,2,3]. Metastases are the commonest malignant liver lesions [4] and are about 18-40 times more common than primaries [5]. HCC may have some morphological similarities with metastases but their prognosis and treatment approaches vary [6]. The diagnosis of HCC and differentiating it from metastases is based on clinical background, imaging findings, serum alpha-fetoprotein (AFP) and pathologic analysis [7]. However the sensitivity and specificity of Ultrasonography (USG) and AFP are not satisfactory enough to detect HCC [8]. Ultrasound/ computerized tomography (USG/ CT) guided Fine Needle Aspiration Cytology (FNAC) is presently a rapid, low cost, safe and accurate procedure to establish a diagnosis of focal hepatic mass lesion as either primary or metastatic. [1,9,10,11,12] Multiple passes from any region of liver are easy to obtain, allowing for better sampling by FNAC [10]. However, FNAC alone faces some diagnostic challenges to differentiate between poorly differentiated HCC and metastatic carcinoma [13]. Cell block preparation, utilizing material obtained through USG guided FNAC of liver tumors is easy and is a safer procedure than the more invasive core needle biopsy (CNB) [14,15,16]. As an adjunct to histopathology, immunohistochemistry (IHC) plays a very crucial role in the differential diagnosis of liver tumors [17,18]. The currently available IHC markers used for this purpose, such as alpha-fetoprotein (AFP), HepPar-1, polyclonal carcino-embryogenic antigen (p CEA), CD 10, & CD 34 have significant diagnostic limitations [19]. Glypican-3 is a 60 kDa cell-surface protein, member of heparan sulfate proteoglycan family, which is linked to the cell surface through a glycosyl phosphatidylinositol anchor. It is an oncofetal protein, expressed in the embryo and is involved in morphogenesis and growth control during development, minimally expressed in normal adult tissues. When used on histopathology sections, it has an important diagnostic role in differentiating HCC from other hepatic mimickers [20]. It has a reported sensitivity ranging from 72% to 90% [21-24] and a specificity between 96% and 100% [23]. It has also been suggested that poorly differentiated HCC are more likely to express Glypican-3 [25,26]. So the present study aimed at assessing the diagnostic utility of Glypican-3 IHC stain on cell block preparation in differentiating primary from secondary hepatic malignancies by comparing it with CNB results taken as a gold standard. Even after a diligent search of published literature, no similar study was found from our sub-himalayan region.

Specific Objectives-

• To study the cytological features of primary HCC and metastatic deposits in the liver.
• To observe the immunohistochemical staining pattern of Glypican-3 on cell block preparation in both primary and secondary hepatic malignancies.
• To assess the diagnostic utility of Glypican-3 IHC stain on cell block preparation in differentiating primary from secondary hepatic malignancies.

Methods:

The present study was an institution based observational study with cross sectional design conducted in the Departments of Pathology, Radiology and Surgery at a tertiary care centre of Eastern India from July 2022 to June 2024. Study population consisted of sixty patients (non-probability consecutive sampling) suspected to have hepatocellular carcinoma or metastatic malignancy clinically and/or radiologically and subsequently referred to Department of Pathology for USG guided FNAC of the hepatic mass. Patients with prothrombin time (PT) within normal range with or without Vitamin K therapy were included in the study. All patients unwilling to give proper informed consent or having lesions other than hepatocellular carcinoma or metastatic malignancy as per cytomorphological features were excluded from the study. The study was performed after obtaining prior approval from the institutional ethics committee.

The patients were informed about the study in details and written consent was taken. All relevant clinical and radiological data were collected from the patients and the study proforma was filled.

Materials was aspirated from the liver mass under USG guidance with cameco syringe pistol fitted with 10cc syringe and spinocaine needle (21-23G).

Air dried and alcohol fixed smears were prepared from the materials collected from the 1st pass.

Materials collected from the 2nd pass were preserved in 10% neutral buffered formalin for cell block preparation. After that CNB was performed using BARD Max needle core biopsy gun (22mm).

FNAC smears from 1st pass were stained by Leishman stain (air dried smears) and H&E / PAP stain (alcohol fixed smears).

Cell blocks were prepared from the 2nd pass by plasma-thrombin clot method. After proper processing, paraffin section were stained by H&E stain. CNB specimens were also processed & paraffin sections were stained by H&E stain.

Now the stained cytological smears and the slides prepared from paraffin section of cell block and CNB after proper tissue processing procedure, were ready for interpretation.

Then with the help of the above smears and slides, the cytological diagnosis was made by 3 blinded faculties of Department of Pathology of our college. The diagnosis of cell block and CNB specimen were also made in the same way.

After that immunohistochemical staining was done by labelled Horse Radish Peroxidase (HRP) polymer technique using cell block section with the help of Glypican-3, and the findings was noted (cytoplasmic or membranous staining pattern).

Finally the diagnostic utility of Glypican-3 immunohistochemical staining was analysed with the help of help of CNB results as a gold standard.

Tables / Figures

Fig.-1: PD HCC, H&E stain, 40x

Fig.-2: Metastatic Adenocarcinoma Liver, Leishman Stain, 100x

Fig.-3: PD HCC, Cell block, H&E stain, 40x 

Fig.- 4: Metastatic adenocarcinoma, Cell block, H&E stain, 40x

Fig.-5: HCC, Membranous + Cytoplasmic   pattern, (GLYPICAN-3), Cell block, 40x

Fig.-6: Metastatic adenocarcinoma, Negative (GLYPICAN-3), Cell block, 40x

Table-1: Results of FNAC, Cell block, Cell block with IHC (Glypican-3), Core   needle biopsy, & CNB with IHC (Glypican-3)

Table-2: Comparison of diagnostic utility of various procedures

On FNAC, HCC shows cellular smears composed of neoplastic cells in trabecular & acinar pattern and also in multilayered sheets. Individual cells are polygonal shaped with increased N: C ratio, have central round nuclei often with macronucleoli. Increased nuclear size and atypia are more in moderately differentiated and poorly differentiated HCC. Bi-nucleation, multinucleation, intranuclear cytoplasmic inclusions and intracytoplasmic bile pigments are present in HCC alongwith atypical stripped nuclei and frequent mitoses. Hyaline bodies might be present. Bile duct epithelium is usually absent. Cytoplasmic vacuoles might be present.

Metastases show the cytological features of primary tumor itself alongwith reactive hepatocytes. However poorly differentiated HCC frequently pose a diagnostic dilemma in cytomorphological differentiation between HCC and metastases [10,11,12].

Diagnostic criteria of HCC on cell block [13]

Architectural details such as trabecular-sinusoidal pattern formed by broad trabeculae ( >2 cells thick), pseudoacini and unpaired arteries aid HCC diagnosis.

The diagnosis of HCC is based on cyto-architectural features, such as cell atypia, cell crowding, trabecular thickness, and microacini. Establishment of trabeculae ≥3 cells thick is one of the most helpful features in diagnosis of highly well-differentiated HCC.

Data Analysis

The collected data was tabulated in Microsoft Excel data sheet. Continuous quantitative variables were expressed as the mean / SD and categorical qualitative variables expressed as absolute frequencies (number) & relative frequencies (percentage). They were analysed using Chi square test and the continuous data using unpaired Student’s t test. The results were considered statistically significant when the significant probability was less than 0.05 (p  < 0.05).

Statistical software used:  GraphPad Prism 10 (San Diego, USA), Statistical Package for Social Sciences (SPSS version 24, IBM, USA).

RESULTS:

The study population comprised of 60 cases of hepatic SOL, among which on CNB, 24 were diagnosed as HCC and 36 as metastatic carcinoma in liver. In cases of HCC, age of patients ranged from 50 - 79 years with mean age of 65.25 ± 4.0 (S.D.) years and majority of the patients (83%) were in the sixth decade of life. In patients with metastatic carcinoma, age ranged from 40-89 years with a mean age of 60.86 ± 8.57 (S.D.) years and majority of the patients (56%) were in their fifth decade.

HCC were more common in males (75%) than females (25%) and metastatic carcinoma were also more common in male (66.67%) than female (33.33%).  (p 0.4902)

In case of HCC, patients had a clinical history of pain abdomen (100%) followed by weight loss (54%), ascites (29%), jaundice (25%) and anorexia (25%) respectively. There was no history of hematochezia and altered bowel habit. In case of metastatic carcinoma, patient had a clinical history of pain abdomen (97%) followed by jaundice (53%), weight loss (22%), hematochezia (17%), ascites (14%) and altered bowel habit (14%) respectively.

21/24 (87.5%) cases of HCC showed elevated serum AFP level. Non elevation (≤20 ng/ml) was seen in 12.5% (3/24) cases. HCC showed mildly elevated (>20-200 ng/ml), moderately elevated (>200-400 ng/ml) and markedly elevated (>400 ng/ml) serum AFP level in 16.67% (4/24) cases, 20.83% (5/24) cases, and 50% (12/24) cases respectively. Metastatic carcinoma showed mildly elevated and moderately elevated serum AFP level in 36% (13/36) cases & 14% (5/36) cases respectively. No elevation (≤ 20ng/ml) cases were seen in 47% (17/36) cases. Marked elevation was seen in a single patient (3%). So serum AFP levels were very significantly higher in HCC. (p 0.00005)

Out of 23 solitary SOL, 19 cases were HCC, and 4 cases were metastatic carcinoma and out of 37 multifocal SOL, 32 were metastatic carcinoma and 5 were HCC. So, HCC showed 19 cases (79%) as a solitary SOL & 5 cases (21%) showed multifocal masses and the metastatic carcinoma group showed multifocal SOL in 32/36 (89%) cases, and single SOL in 4/36 (11%) cases. Solitary SOLs were seen to be significantly more associated with HCC. (p < 0.0001)

HCC was diagnosed in 27, 26, 25, 24 and 24 cases on FNAC (Figure 1 and 2), cell block (Figure 3 and 4), cell block with IHC (Glypican-3) (Figure 5 and 6), CNB and CNB with Glypican-3 IHC stain respectively whereas metastatic carcinoma was diagnosed in 33, 34, 35, 36 and 36 cases respectively (Table1). The sensitivity, specificity and diagnostic accuracy of FNAC was found to be 87.5%, 83.4% and 85% respectively. The same for cell block preparation came out to be 91.7%, 88.9% and 90% respectively. When Glypican-3 IHC stain was done on cell block preparation, the sensitivity, specificity and diagnostic accuracy increased to 95.8%, 94.5% and 95% respectively (Table2).

In relation to cell block out of total 60 cases, 25 cases showed immunoreactivity for Glypican-3 (considered as positive), 35 cases showed no immunoreaction for Glypican-3 (considered as negative). The immunoreactive cases were diagnosed as HCC whereas non-immunoreactive ones were diagnosed as metastases in cell block preparation. Among these 25 Glypican-3 positive cases on cell block, 23 cases (92%) were finally diagnosed as HCC on CNB but 2 (8%) cases provisionally diagnosed as HCC on cell block with Glypican-3 later turned out to be metastatic deposits of ovarian carcinoma in liver and squamous cell carcinoma lung with liver metastasis that was revealed with the help of further investigations and CNB sections. Out of 35 cell block Glypican-3 negative cases, 1 case (2.86%) was finally diagnosed on CNB as moderately differentiated HCC and other 34 metastatic cases (97.14%) turned out be metastatic deposits in liver in CNB as well. So finally 24 cases of HCC (23 cell block Glypican-3 positive & 1 cell block Glypican-3 negative) and 36 cases of metastatic disease in liver (34 cell block Glypican-3 negative & 2 cell block Glypican-3 positive) were diagnosed based on gold standard CNB with Glypican-3 IHC.

Coming to Glypican-3 staining pattern in HCC, 12 (48%) cases showed membranous pattern, 4 (16%) showed cytoplasmic and 9 (36%) cases showed mixed (membranous with cytoplasmic and membranous with canalicular) pattern. On histopathology, out of 23 Glypican-3 positive  cases of HCC, 7 cases (29%) were Well Differentiated HCC, 11 cases (46%) were Moderately Differentiated HCC, & 6 (25%) were Poorly Differentiated HCC along with 1 Glypican-3 negative moderately differentiated HCC.

DISCUSSION:

Majority of HCC patients ranged from 50-79 years which is similar to studies worldwide [27] and in India [28]. Sex distribution of HCC was close to 2:1 to 4:1 Male:Female ratio reported worldwide [29,30]. In an Indian study by Lokesh K N et al. 72.9% patients were male with male : female ratio of 2.7:1 [31]. In cases of metastatic carcinoma, most were aged 40-89 years and majority (56%) were in fifth decade with a male : female ratio of 2:1. Griscom J T et al reported that metastatic carcinoma of liver was more common in males, and majority of the patients were more than 50 years of age [32]. Ridder J D et al also found that metastases more common in males [33]. Mohamed Z et al documented male predominance both in HCC and metastases. [34].

In this study, metastases (36 cases, 60%) was commoner than HCC (24 cases, 40%). Similar results were reported by Chowdhury S et al [35] in West Bengal (2015) and in a study in China (2018) by Chen D et al [25]. But Hemalatha A L et al [36] found 44.4% cases of HCC as compared to metastatic hepatic lesion in 27.7% and Ibrahim T R et al [37] found 62.5% cases of HCC, and 37.5% cases of metastatic carcinoma. Equal number of cases of HCC and metastatic lesion was observed by Ahuja A et al [1].

Majority (83%) of HCCs presented as solitary SOL, and few (17%) showed multifocal masses on USG. The metastatic carcinoma group showed multifocal SOL in 32/36 (89%) cases, and single SOL in 4/36 (11%) cases. Chowdhury S et al [35] and Ahuja A et al [38], have found similar results in their studies.

Reddy C V et al [39] reported FNAC sensitivity, specificity and diagnostic accuracy as 93%, 90.9% and 92.8%, respectively, which corroborated with those observed in the present study. Rastogi N et al [40] and Swamy M C M et al [41] also reported similar findings.

In the present study, on cell block, 26 (43%) cases were diagnosed as HCC and 34 (57%) cases as metastatic carcinoma. In 54 cases (22 HCC and 32 metastases), diagnosis on both cell block and CNB were similar, but on cell block, 4 false positive and 2 false negative cases were found. Haq S et al, [6] reported in their study, that use of cell block helped in reaching a final diagnosis in an additional 29.1% cases, compared to conventional smear. A recent study by Nagose V B et al [42] reported sensitivity (95.77%) and diagnostic accuracy (94.87%) of cell block to be considerably higher than FNAC (78.84% and 78.46% respectively) and they concluded in their study, that cases of suspicious diagnosis by FNAC or diagnosis discordant with clinical findings, should be followed up with cell block to improve the diagnostic yield & to facilitate accurate diagnosis.

Cell block when combined with IHC, in the present study, was able to diagnose 57 cases out of 60 cases (23 HCC, 34 metastatic deposits) accurately with 2 false positive and 1 false negative results. Similar results were reported by Ibrahim TR et al. [37] Shan-yu Qin et al [43] showed 90% sensitivity, 100% specificity, and 91.7% diagnostic accuracy of cell block with IHC in their study of pancreatic lesions. In the present study, the false negative case was diagnosed as moderately differentiated, nuclear grade II HCC. It corroborated well with findings of Mohamed Z et al [34] who reported that grade III (poorly differentiated) HCC cases showed the highest Glypican-3 expression (100 %), followed by grade II (moderately differentiated) cases (96.5 %), while grade I (well differentiated) cases expressed Glypican-3 in 90 % of cases. The two false positive cases on further investigation were diagnosed as metastatic deposits of ovarian clear cell adenocarcinoma and poorly differentiated squamous cell carcinoma of lung. Both the tumors have been reported to be Glypican-3 immunoreactive in published literature. [46, 47]

Several recent studies have concluded that Glypican-3, when used as a single IHC marker, was much helpful in differentiating HCC from benign hepatic lesions [21,22,23,24,45,48] and from metastatic neoplasms of the liver [49]. It has also been suggested that poorly differentiated HCCs are more likely to express Glypican-3 [23].

Majeed S et al [50] reported Glypican-3 staining was positive in 48.33% cases and negative in 51.67% cases with sensitivity of 82%, specificity 94.33%, positive predictive value (PPV) 94.82% and negative predictive value (NPV) of 80.64%. Diagnostic accuracy was 87.5%. Mohamed Z et al [34] showed Glypican-3 was expressed in the majority of cases of HCC with a sensitivity (95.2%), specificity (83.3%), PPV (93%), NPV (88.2%) and total accuracy of 91.7%. Ibrahim T R et al [37], Geramizadeh et al [18] and Saverio L et al [49] reported similar results.

Immunostaining patterns observed in the present study corroborated well with findings reported in published literature. Wang X Y et al [24], Chen I P et al [51], Wang H L et al [52] and Geramizadeh et al [18] documented 3 different immunostaining patterns of GPC3-expression in HCC: predominantly membranous, predominantly canalicular, and predominantly cytoplasmic. These different staining patterns did not appear to correlate with the differentiation status or the growth pattern of the tumors. In fact, more than one third of the tumors exhibited a mixed staining pattern within the same tumors, although the tumor cells with different staining patterns showed similar or identical histologic features.

Limitation: It was a single institution based study, assessing role of only one IHC marker. Moreover, in a minor percentage of metastatic carcinoma cases, Glypican-3 staining was falsely positive. Use of other HCC specific IHC markers like HepPar1 or MOC31 on cell block preparation may be assessed. Multicentric study with more number of cases, assessing other IHC markers would help in comparing the diagnostic utility of various potential biomarkers.

CONCLUSION:

The present study revealed that in cases of HCC, diagnostic accuracy of cell block is greater than FNAC in differentiating it from metastases. Furthermore, the additional use of Glypican-3 IHC on cell block sections increases the diagnostic accuracy. Immunohistochemical staining for Glypican-3 in cell block preparation of liver is highly sensitive and specific and is a valuable tool capable of differentiating HCC from metastases. More importantly, biopsy can be avoided, which has definitive advantages especially in resource deprived set ups.

REFERENCES:

1. Ahuja A, Gupta N, Srinivasan R, Kalra N, Chawla Y, Rajwanshi A. Differentiation of hepatocellular carcinoma from metastatic carcinoma of the liver - clinical and cytological features. Journal of Cytology 2007;24:125-9.
2. Yang J D, Hainaut P, Gores G J, Amadou A, Amelie P, Roberts L R. A global view of hepatocellular carcinoma:trends, risk, prevention and management. Nature Reviews Gastroenterology & Hepatology 2019;16:589-04.
3. Paradis V, Fukayama M, Park YN. Schirmacher P.Tumours of the liver and intrahepaticbile ducts. In, WHO Classification of Tumour Editorial Board (ed). WHO Classification of Tumours. Digestive System Tumours. 5th edition. 2019; 229-39.
4. Porcell LA, De Young B, Proca DM, Frankel WL. Immunohistochemical Analysis of Hepatocellular and Adenocarcinoma in the Liver: MOC31 Compares Favorably with Other Putative Markers. Modern Pathology 2000;13:773–8.
5. Bratu AM, Zaharia C, Salcianu IA. Multifocal Hepatocellular Carcinoma or Liver Metastases? EC Gastroenterology and Digestive System 2019;6:702-9.
6. Haq S, Jadhav L, Mubeen B, Makhdoomi R, Malhotra V. Utility of FNAC in Conjunction with Cell Block for Diagnosing Space-Occupying Lesion (SOL) of Liver with Emphasis on Differentiating Hepatocellular Carcinoma from Metastatic SOL: Analysis of 61 Cases. Oman Med J 2016;31:135–41.
7. Lope C R de, Reig ME, Darnell A, Forner A. Approach of the patient with a liver mass. Frontline Gastroenterol 2012;3:252-62.
8. Wang W, Wei C. Advances in the Early Diagnosis of Hepatocellular Carcinoma. Genes & Diseases 2020;7:308-19
9. Swamy MCM, Arathi CA, Kodandaswamy CR. Value of ultrasonography-guided fine needle aspiration cytology in the investigative sequence of hepatic lesions with an emphasis on hepatocellular carcinoma. J Cytol 2011;28:178–84.
10. Conrad R, Castelino-Prabhu S, Cobb C, Raza A. Cytopathologic diagnosis of liver mass lesions. J Gastrointest Oncol 2013; 4:53–61.
11. Balani S, Malik R, Malik R, Kapoor N. Cytomorphological variables of hepatic malignancies in fine needle aspiration smears with special reference to grading of hepatocellular carcinoma. J Cytol 2013;30:116–20.
12. Nasit JG, Patel V, Parikh B, Shah M, Davara K. Fine-needle aspiration cytology and biopsy in hepatic masses: A minimally invasive diagnostic approach. Clinical Cancer Investigation Journal 2013; 2:132-42.
13. Wee A. Fine-Needle Aspiration Biopsy of Hepatocellular Carcinoma and Related Hepatocellular Nodular Lesions in Cirrhosis: Controversies, Challenges, and Expectations. Patholog Res Int 2011; 2011:1-17.
14. Mathew EP, Nair V. Role of cell block in cytopathologic evaluation of image-guided fine needle aspiration cytology. J Cytol. 2017;34:133–38.
15. Khan S, Omar T, Michelow P, Effectiveness of the cell block technique in diagnostic cytopathology. J Cytol 2012; 29:177–82.
16. Patil RN, Mahore SD, Kolhe HS, Bothale KA, Gowardhan VP, Taori HS, Mhatre RK. Cell Block Technique: An Effective Tool in Diagnostic Cytopathology. IOSR Journal of Dental and Medical Sciences (IOSR-JDMS) 2016;15:99-106.
17. Sang W, Zhang W, Cui W, Li X, Abulajiang G, Qiaoxin Li. Arginase-1 is a more sensitive marker than HepPar-1 and AFP in differential diagnosis of hepatocellular carcinoma from nonhepatocellular carcinoma. Tumour Biol 2015; 36:3881-6.
18. Geramizadeh B, Nasibe Seirfar N. Diagnostic Value of Arginase-1 and Glypican-3 in Differential Diagnosis of Hepatocellular Carcinoma, Cholangiocarcinoma and Metastatic Carcinoma of Liver. Hepat Mon 2015;15:1-7
19. Wee A. Diagnostic utility of immunohistochemistry in hepatocellular carcinoma, its variants and their mimics. Appl Immunohistochem Mol Morphol 2006;14:266-72.
20. Yan B, Wei JJ, Qian YM, Zhao XL, Zhang WW, Xu AM et al Expression and clinicopathologic significance of glypican 3 in hepatocellular carcinoma. Annals of Diagnostic Pathology 2011;15:162-9.
21. Yamauchi N, Watanabe A, Hishinuma M, Ohashi KI, Midorikawa Y, Morishita Y, The glypican 3 oncofetal protein is a promising diagnostic marker for hepatocellular carcinoma. Mod Pathol 2005;18:1591-8.
22. Capurro M, Wanless IR, Sherman M, Deboer G, Shi W, Miyoshi E et al. Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma. Gastroenterology 2003 Jul;12:89-97.
23. Libbrecht L, Severi T, Cassiman D, Borght SV, Pirenne J, Nevens F et al. Glypican-3 expression distinguishes small hepatocellular carcinomas from cirrhosis, dysplastic nodules, and focal nodular hyperplasia-like nodules. Am J Surg Pathol 2006;30:1405-11.
24. Wang XY, Degos F, Dubois S, Tessiore S, Allegretta M, Guttmann RD et al. Glypican-3 expression in hepatocellular tumors: diagnostic value for preneoplastic lesions and hepatocellular carcinomas. Human Pathology 2006;37:1435-41.
25. Chen D, Zhao Li, Song Q, Qian L, Xie B, Zhu J. Clinicopathological features and differential diagnosis of hepatocellular carcinoma in extrahepatic metastases. Medicine 2018; 97:1-7
26. Shafizadeh N, Ferrell LD, Kakar S. Utility and limitations of Glypican-3 expression for the diagnosis of hepatocellular carcinoma at both ends of the differentiation spectrum. Mod Pathol 2008;21:1011-8.
27. Mezale D, Strumfa I, Vanags A, Kalva A, Balodis D, Strumfs B, et al. Diagnostic Algorithm of Hepatocellular Carcinoma: Classics and Innovations in Radiology and Pathology. Hepatocellular Carcinoma - Advances in Diagnosis and Treatment [Internet]. 2018 Nov 28; Available from: http://dx.doi.org/10.5772/intechopen.76136
28. Bhattacharyya GS, Govind Babu K, Malhotra H, Anantbhushan A R, Murshed S, Datta
29. Hepatocellular carcinoma in India. Chinese Clinical Oncology 2013;2:41.
30. Lamps LW. Liver: Non neoplastic disease, Tumour and Tumour like condition. Goldman.R. John, Lamps W Laura, McKenny K Jesse, Myers L Jeffrey (ed). Rosai and Ackerman‘s Surgical Pathology. Elsevier. 11th edition. Philadelphia, Elsevier, 2018; 726- 7,808-13.
31. Ghouri YA, Mian I, Rowe JH. Review of hepatocellular carcinoma: Epidemiology, etiology, and carcinogenesis. J Carcinog 2017;16:1.
32. Lokesh KN, Chaudhuri T, Lakshmaiah KC, Govind Babu K, Dasappa L, Linu Abraham Jacob LA et al. Advanced hepatocellular carcinoma: A regional cancer center experience of 48 cases. Indian Journal of Cancer 2017;54:526-9.
33. Griscom JT, Wolf PS. Liver Metastasis. [Updated 2022 Sep 18]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan.
34. Ridder J D, Wilt JHW, Simmer F, Overbeek L, Lemmens V, Nagtegaal I. Incidence and origin of histologically confirmed liver metastases: an explorative case-study of 23,154 patients. Oncotarget 2016;7: 55368–76.
35. Zaakook M, Ayoub M, Sinna EA, El-Sheikh S. Role of Glypican-3 immunocytochemistry in differentiating hepatocellular carcinoma from metastatic carcinoma of the liver utilizing fine needle aspiration cytology. Journal of the Egyptian National Cancer Institute 2013;25:173-80.
36. Chowdhury S, Mustaphi RM, Mondal S, Bose K, Saha TK, Guha D. A Clinico – Pathological Study of Metastatic Hepatic Lesions with Special Reference to Metastasis from Unusual Primary Sites. IOSR Journal of Dental and Medical Sciences 2015;14:46-50.
37. Hemalatha AL,Vidyadhar R, Kariappa TM. Retrospective Study of Hepatic and Retroperitoneal Masses. Journal of Cytology 2004;21: 85-90.
38. Ibrahim TR, Abdel-Raouf SM. Immunohistochemical Study of Glypican-3 and HepPar-1 in Differentiating Hepatocellular Carcinoma from Metastatic Carcinomas in FNA of the Liver. Pathol Oncol Res. 2015;21:379-87.
39. Ahuja A, Gupta N, Srinivasan R, Kalra N, Chawla Y, Rajwanshi A. Differentiation of hepatocellular carcinoma from metastatic carcinoma of the liver - clinical and cytological features. Journal of cytology 2007;24:125-9.
40. Reddy CV, Basavana Goud YG, Poornima R, Deshmane V, Madhusudhana B.A, Gayathridevi M. Role of FNAC in Hepatic lesions: Risk of track metastases. South Asian J Cancer 2015; 4: 35–37.
41. Rastogi N, Bhake A, Agarwal D, Wankhede A. Ultrasound guided fine needle aspiration cytology of space occupying lesions of liver. International Journal of Research in Medical Sciences 2019;7:192-8.
42. Swamy MCM, Arathi CA, Kodandaswamy CR. Value of ultrasonography-guided fine needle aspiration cytology in the investigative sequence of hepatic lesions with an emphasis on hepatocellular carcinoma. J Cytol 2011;28:178–84.
43. Nagose VB, Deshpande SA, Kasturi D, Jadhav VA. Cell Block: A Tool to Improve Cytopathologic Diagnostic Value of Fine Needle Aspiration Material. Annals of Pathology and Laboratory Medicine 2020;7
44. Qin SY, Zhou Y, Li P, Jiang HX. Diagnostic Efficacy of Cell Block Immunohistochemistry, Smear Cytology, and Liquid-Based Cytology in Endoscopic Ultrasound-Guided Fine-Needle Aspiration of Pancreatic Lesions: A Single-Institution Experience. PLoS One 2014; 9
45. Saha R, Datta P, Chakraborty J. A comparative study of conventional cytology and cell block method with immunohistochemistry in the diagnosis of serous effusions. Pathology Update: Tropical Journal of Pathology and Microbiology 2020;6:146-54.
46. Filmus J, Capurro M . Glypican-3 and alpha-fetoprotein as diagnostic tests for hepatocellular carcinoma. Mol Diagn 2004;8:207–212.
47. Maeda, D., Ota, S., Takazawa, Y. et al. Glypican-3 expression in clear cell adenocarcinoma of the ovary. Mod Pathol 22, 824–832 (2009). https://doi.org/10.1038/modpathol.2009.40
48. Aviel-Ronen S, Lau SK, Pintilie M, et al. Glypican-3 is overexpressed in lung squamous cell carcinoma, but not in adenocarcinoma. Mod Pathol 2008;21:817–825.
49. Man XB, Tang L, Zhang BH, et al. Upregulation of Glypican-3 expression in hepatocellular carcinoma but downregulation in cholangiocarcinoma indicates its differential diagnosis value in primary liver cancers. Liver Int 2005;25:962–6.
50. Saverio L, Mandich D, Cartun R. Utility of Glypican-3 in differentiating hepatocellular carcinoma from other primary and metastatic lesions in FNA of the liver: an immunocytochemical study. Mod Pathol 2008;21:626-31.
51. Majeed S, Mushtaq S, Azam M, Akhtar N, Hussain M, Loya A. Diagnostic accuracy of Glypican-3 in differentiating hepatocellular carcinoma from metastatic liver tumours. J Pak Med Assoc. 2018;68:1029-31.
52. Chen IP, Ariizumi SI, Nakano M, Yamamoto M. Positive Glypican-3 expression in early hepatocellular carcinoma predicts recurrence after hepatectomy. J Gastroenterol 2014;49:117–25.
53. Wang HL, Anatelli F, Zhai QJ, Adley B, Chuang ST, Yang XJ. Glypican-3 as a Useful Diagnostic Marker That Distinguishes Hepatocellular Carcinoma From Benign Hepatocellular Mass Lesions. Arch Pathol Lab Med 2008;132: 1723–8.