|Year : 2020 | Volume
| Issue : 2 | Page : 95-99
Evaluation of role of cell block with immunohistochemistry in differentiating hepatocellular carcinoma from metastatic adenocarcinoma
Palash Kumar Mandal1, Soumi Pradhan2, Anindya Adhikari3, Mimi Gangopadhyay4, Dutta Pal Rupsha4, Subrata Bhattacharya4
1 Department of Pathology, College of Medicine and Sagore Dutta Hospital, Kolkata, West Bengal, India
2 Department of Pathology, AMRI Hospital, Kolkata, West Bengal, India
3 Department of Pathology, Purulia Government Medical College, Purulia, West Bengal, India
4 Department of Pathology, North Bengal Medical College and Hospital, Darjeeling, West Bengal, India
|Date of Submission||14-Feb-2019|
|Date of Decision||22-Nov-2019|
|Date of Acceptance||03-Mar-2020|
|Date of Web Publication||8-Oct-2020|
Basudevpur, Banipur, Sankrail, Howrah - 711 304, West Bengal
Source of Support: None, Conflict of Interest: None
Background: Fine-needle aspiration cytology (FNAC) is an increasingly common procedure in the diagnosis of neoplastic lesions of the liver, particularly in differentiating primary hepatocellular carcinoma (HCC) from metastatic adenocarcinoma (MA). However, there are certain limitations of FNAC. Sometimes, FNAC does not yield sufficient material for the precise diagnosis and the risk of the wrong diagnosis by FNAC cannot be excluded from the study. The aim of the study was to overcome such problems using a cell block (CB) technique along with immunohistochemistry (IHC) to diagnose and differentiate HCC from MA. Materials and Methods: This was a prospective study, conducted in a tertiary care hospital in Darjeeling district of West Bengal, over a period of 1 year. A total of 52 adult cases presenting with liver mass suspicious of malignancy were studied; CB with a histopathological examination and IHC for Hep par1 and CD10 were done in all cases. Results: In our study of 52 cases, CB with IHC was found superior in differentiating primary HCC from MA than only CB with a diagnostic accuracy of 96.15% and 92.31%, respectively. Among the IHC markers, Hep par1 was found more useful than CD10 with a diagnostic accuracy of 94.23% and 82.6%. However, CD10 was found to have greater diagnostic utility than Hep par1 in the context of poorly differentiated HCC. Conclusions: IHC study with Hep par1 and CD10 may be useful in differentiating HCC from the MA of the liver. Therefore, CB along with IHC improves the diagnostic accuracy of FNAC diagnosis.
Keywords: Cell block, hepatocellular carcinoma, immunohistochemistry, metastatic adenocarcinoma
|How to cite this article:|
Mandal PK, Pradhan S, Adhikari A, Gangopadhyay M, Rupsha DP, Bhattacharya S. Evaluation of role of cell block with immunohistochemistry in differentiating hepatocellular carcinoma from metastatic adenocarcinoma. CHRISMED J Health Res 2020;7:95-9
|How to cite this URL:|
Mandal PK, Pradhan S, Adhikari A, Gangopadhyay M, Rupsha DP, Bhattacharya S. Evaluation of role of cell block with immunohistochemistry in differentiating hepatocellular carcinoma from metastatic adenocarcinoma. CHRISMED J Health Res [serial online] 2020 [cited 2020 Dec 1];7:95-9. Available from: https://www.cjhr.org/text.asp?2020/7/2/95/297574
| Introduction|| |
Liver is an important site for neoplasms-both primary and metastatic tumors, the latter being more common. Hepatocellular carcinoma (HCC) is the most common primary liver cancer and its incidence is on the rise. It is mandatory to differentiate primary from secondary (metastatic) cancer of the liver before the institution of therapy. Fine-needle aspiration cytology (FNAC) can perform a major role, as it is a rapid and simple diagnostic modality. However, the diagnostic failure rate of FNAC may be as high as 45%. By FNAC, distinguishing a well-differentiated HCC from a regenerative hepatic nodule may be very difficult in some cases. Furthermore, some of the unusual morphologic variants including fibrolamellar, clear cell, and pleomorphic types may be mistaken for metastasis. Similarly, metastasis from various primary tumors to the liver may be mistaken for primary HCC. To overcome these problems, the cell block (CB) technique has been resorted to make the best use of the available material from FNAC. It is a technique which incorporates features of both cytology and histopathology. The benefit of CB technique is the recognition of histologic patterns of diseases that sometimes cannot be reliably identified in FNAC smears. The CB also has the advantage of providing many tissue sections. On an average, 12 sections per CB can be obtained. Thus, CB provides the opportunity to perform ancillary studies such as histochemical stains, immunocytochemical stains, and in situ hybridization. Among the numerous immunohistochemistry (IHC) markers, Hep par1, CD10, MOC31, CK7, CK20, and Glypican 3 have been found to be valuable in distinguishing HCC from metastatic neoplasms. With this background knowledge, we tried to evaluate the role of Hep par1 and CD10 IHC on CB preparations to differentiate HCC from metastatic tumors. Core needle biopsy (CNB) was used as a gold standard method to corroborate the results of CB in our study.
Differentiating HCC from MA is crucial for proper patient management.
| Materials and Methods|| |
This was a prospective study, conducted in a tertiary care hospital in Darjeeling district of West Bengal, over a period of 1 year. A total of 52 cases presenting with liver mass suspicious for malignancy were studied. Ultrasound-guided FNAC from the liver mass was done with the help of Cameco syringe pistol fitted with 10 cc syringe and Spinocaine needle (21–23 G). For CB, first, plasma (outdated plasma from the blood bank), then thrombin solution were added to the material. After the formation of a clot, it was preserved in 10% buffered formalin. With small scissors, the clot was then cut into pieces and processed as usual, like tissue material. IHC study was done by labeled horseradish peroxidase technique. Selective monoclonal antibodies such as Hep par1 (associated with hepatocytic mitochondria), CD10 (neutral endopeptidase) were used as IHC markers. Hep par1 staining is cytoplasmic and granular, without canalicular or zonal accentuation. The pattern of immunoreactivity with CD10 may be cytoplasmic, membranous, or canalicular. It is the latter pattern that is regarded as specific for a hepatocytic differentiation. CNB was also taken in every case to corroborate CB findings. Cytology of FNAC slides was also studied.
Statistical analysis used
| Results|| |
A total number of cases was 52, with a male: female ratio of 1.4:1. The patients' age range was from 34 to 75 years. There were 20 cases of HCC, and the rest 32 were metastatic adenocarcinoma (MA) as confirmed by CNB. On ultrasonography, 25 cases had solitary liver lesions and 27 cases showed multiple lesions. FNAC [Figure 1]a correctly differentiated primary HCC from MA in 90.4% (47/52) cases. It diagnosed 18 HCC and 29 MA cases, its result were inconclusive in five cases. On core biopsy, ten cases were well differentiated, five cases were moderately differentiated, and 3 were poorly differentiated.
|Figure 1: (a) Well-differentiated hepatocellular carcinoma (fine-needle aspiration cytology.) (b) Hepatocellular carcinoma showing endothelial cell wrapping (cell block). (c) Hepatocellular carcinoma showing prominent nucleoli (cell block). (d) Metastatic adenocarcinoma in high magnification showing normal hepatocytes in the lower left part and adenocarcinoma cells in the right upper part|
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- Sensitivity (SN) = True positive × 100/(True positive + False negative)
- Specificity (SP) = True negative × 100/(True negative + False positive)
- Positive predictive value (PPV) = True positive × 100/(True positive + False Positive)
- Negative predictive value (NPV) = True negative × 100/(True negative + False negative)
- Diagnostic accuracy (DA) = (True positive + True negative) × 100/Total cases.
Sensitivity and specificity of cell block:
- True positive (TP) = No of HCC correctly detected = 18
- False positive (FP) =No of metastatic carcinoma reported as HCC = 2
- False negative (FN) = No of HCC reported as metastatic carcinoma = 2
- True negative (TN) = No of metastatic carcinoma correctly detected = 30
- SN of CB = TP × 100/(TP + FN) = 18 × 100/(18 + 2) = 90%
- SP of CB = TN × 100/(TN + FP) = 30 × 100/(30 + 2) = 93.75%
- PPV = TP × 100/(TP + FP) = 18 × 100/(18 + 2) = 90%
- NPV = TN × 100/(TN + FN) =30 × 100/(30 + 2) = 93.75%
- DA = (TP + TN) × 100/Total case = (18 + 30) × 100/52 = 92.31%.
Sensitivity and specificity of cell block with IHC (both Hep par1 and CD10):
- TP = No of HCC correctly detected = 20
- FP = No of metastatic carcinoma reported as HCC = 1
- FN = No of HCC reported as metastatic carcinoma = 1
- TN = No of metastatic carcinoma correctly detected = 30
- SN of CB with IHC = TP × 100/(TP + FN) = 19 × 100/(19 + 1) = 95%
- SP of CB with IHC = TN × 100/(TN + FP) = 1 × 100/(31 + 1) = 96.88%
- PPV = TP × 100/(TP + FP) =19 × 100/(19 + 1) = 95%
- NPV = TN × 100/(TN + FN) =31 × 100 (31 + 1) = 96.88%
- DA = (TP + TN) × 100/Total case = (19 + 31) × 100/52 = 96.15%.
Sensitivity and specificity of Hep par1 as IHC marker:
- SN of Hep par1 = TP × 100/TP + FN = 18 × 100/(18 + 2) = 90%
- SP of Hep par1 = TN × 100/TN + FP = 31 × 100/(31 + 1) = 96.88%
- PPV = TP × 100/(TP + FP) = 18 × 100/(18 + 1) = 94.74%
- NPV = TN × 100/(TN + FN) = 31 × 100/(31 + 2) = 93.94%
- DA = (TP + TN) × 100/Total no case = (18 + 31) × 100/52 = 94.23%.
Sensitivity and specificity of CD10 as IHC marker:
- SN of CD10 = TP × 100/(TP + FN) = 14 × 100/(14 + 6) = 70%
- SP of CD10 = TN × 100/(TN + FP) = 29 × 100/(29 + 3) = 90.62%
- PPV = TP × 100/(TP + FP) =14 × 100/(14 + 3) = 82.35%
- NPV = TN × 100/(TN + FN) =29 × 100/(29 + 6) = 82.86%
- DA = (TP + TN) × 100/Total no case = (14 + 29) × 100/52 = 82.6.
| Discussion|| |
The study was carried out to differentiate HCC from MA in the liver by CB preparation with IHC as well as to assess whether there is any diagnostic advantage of CB with IHC over CB alone. Herein, CNB of the liver was taken as a gold standard method.
Although CB [Figure 1]b, [Figure 1]c and [Figure 1]d provides more information than conventional smear, in some cases, it may fail to provide a specific diagnosis because of the small quantity of samples. Sometimes, it may give false-positive or false-negative results due to the changing of cellular arrangement after centrifugation. In our study, CNB diagnosed HCC in 20 cases and MA in 32 cases. In 48 cases (18 HCC, 30 MA), the diagnoses on both CB and CNB were similar, while there were two false-positive and two false-negative cases [Table 1]. Sensitivity, specificity, PPV, NPV, and diagnostic accuracy of CB are 90%, 93.75%, 90%, 93.75%, and 92.31%, respectively, which are higher than that of FNAC according to other studies. Basnet and Talwar in their study also found that the diagnostic accuracy and specificity were high in CB in comparison to the smear ranging from 83% in the smear and 100% in the CB. Shivakumarswamy et al. also experienced that the CB yielded more cellularity and better architectural pattern, which improved the diagnosis of malignancy by 15%. Keyhani-Rofaga et al. reported that in a study of 85 cases, 55% of the original smear diagnoses were improved after the CB was examined. Kern and Haber studied 393 cases of CB preparation. In 237 cases (60.3%), the findings were confirmatory, and in 103 cases (26.2%), CBs provided additional information for diagnosis. Similarly, Nathan et al. showed that conclusive diagnostic material was available in 296 (89.4%) CBs from 331 cases.
Cell block with immunohistochemistry
IHC has not been a gold standard procedure due to false positivity and false negativity. If IHC is applied to CB alone, the sensitivity and specificity of the diagnoses increase. In the present study, CB with IHC diagnosed 50 out of 52 cases (19 HCC, 31 MA cases) correctly with one false-positive and one false-negative results [Table 2]. Sensitivity, specificity, PPV, NPV, and diagnostic accuracy of CB with IHC were 95%, 96.88%, 95%, 96.88%, and 96.15%, respectively, in our study. Qin et al. showed 90% sensitivity, 100% specificity of IHC on CB in their study of pancreatic lesions. Sharma et al. in their study on hepatic space-occupying lesions (SOLs) found CB and IHC as a useful adjunct in differential diagnoses of different lesions, and this increased both sensitivity and specificity of FNAC in liver SOLs. Overall, CB with IHC had a sensitivity and specificity of 95.23% and 96.77%, respectively, which is better than CB or conventional smear alone.
|Table 2: Sensitivity and specificity of cell block with immunohistochemistry (both Hep par1 and CD10)|
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Among a total of 52 cases (20 cases-HCC, 32 cases-MA), Hep par1 stained [Figure 2]a, [Figure 2]b and [Figure 2]c 18 out of 20 HCC cases with diffuse and focal cytoplasmic positivity. Those 18 cases were well to moderately differentiated. The remaining two Hep par1 negative were poorly differentiated. False immunoreactivity was also seen in one case of MA. On CNB, this case was diagnosed as poorly differentiated MA with foci suggestive of hepatoid differentiation [Table 3]. The sensitivity and specificity, PPV, NPV, and DA of Hep par1 were 90%, 96.88%, 94.74%, 93.94%, and 94.23%, respectively. Timek et al. demonstrated 87% sensitivity and 97.4% specificity of Hep par1. Morrison et al. showed 96% sensitivity and 100% specificity of Hep par1 in their study. Siddiqul et al. found 100% positivity of Hep par1 in HCC and 20% (3/20) false positivity in metastatic carcinoma. Varma and Cohen reported that Hep par1, CK7, and CK20 has been found to be valuable in distinguishing HCC from metastatic neoplasm of the extrahepatic site. Lau et al. studied 42 cases of HCC and 56 cases of metastatic carcinoma. Hep par1 was positive in 90% cases (38 of 42) of HCC and 14% (8 of 56) metastatic cases.
|Figure 2: (a) Hepatocellular carcinoma showing diffuse Hep par1(cytoplasmic) positivity. (b) Hepatocellular carcinoma showing focal Hep par1 (cytoplasmic) positivity. (c) Hep par1 negative hepatocellular carcinoma. (d) Hepatocellular carcinoma showing CD10 (bile canalicular membrane) positivity|
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|Table 3: Sensitivity and Specificity of Hep par1 as immunohistochemistry marker|
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CD10 stained [Figure 2]d 14 of 20 cases of HCC with characteristic bile canalicular membrane positivity. Among them, 13 were well to moderately differentiated, and one was poorly differentiated HCC. CD10 was negative in six of HCC, which were well to moderately differentiated. It was also positive in three cases of metastatic carcinoma, in which diffuse cytoplasmic positivity was seen instead of bile canalicular membrane positivity [Table 4]. Sensitivity, specificity, PPV, NPV, and diagnostic accuracy of CD10 were 70%, 90.62%, 82.35%, 82.86%, and 82.69%, respectively. Borscheri et al. showed 68.3% sensitivity and 100% specificity of CD10 in the diagnosis of liver SOLs. Saad et al. found CD10 positivity in 77% (23/30) of HCC in their study. Morrison et al. documented that CD10 with 52% sensitivity and 93% specificity was not a useful marker in differentiating HCC from cholangiocarcinoma and MA. Lin et al. demonstrated the utility of CD10 in differentiating HCC from metastatic carcinoma in their study. They found CD10 positivity in 86% (19/22) of HCC.
|Table 4: Sensitivity and specificity of CD10 as immunohistochemistry marker|
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| Conclusions|| |
CB method allows the recovery and processing of minute amounts of cellular material and facilitates the better classification of tumor when reviewed along with IHC study.
Hep par1 is a novel marker in differentiating HCC from MA in the liver with higher sensitivity and specificity than CD10. However, Hep par1 has a tendency to be negative in poorly differentiated HCC. Whereas CD10 has a tendency to be expressed in moderately to poorly differentiated HCC than well differentiated. As CD10 has poor sensitivity, the combined use of Hep par1 along with CD10 will increase the diagnostic accuracy in differentiating HCC from metastatic carcinoma.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Basnet S, Talwar OP. Role of cell block preparation in neoplastic lesions. J Pathol Nepal 2012;2:272-6.
Shivakumarswamy U, Arakeri SU, Karigowdar MH, Yelikar B. Diagnostic utility of the cell block method versus the conventional smear study in pleural fluid cytology. J Cytol 2012;29:11-5.
] [Full text]
Lamps LW, Folpe AL. The diagnostic value of hepatocyte paraffin antibody 1 in differentiating hepatocellular neoplasms from nonhepatic tumors: A review. Adv Anat Pathol 2003;10:39-43.
Singha J, Khan K, Chatterjee S. Diagnostic utility of CD10 immunohistochemical staining on cellblock in differentiating hepatocellular carcinoma from secondary malignancies of liver. Indian J Pathol Microbiol 2018;61:510-5.
] [Full text]
Naylor B. Pleural, Peritoneal, and Pericardial fluids. In: Bibbo M, editor. Comprehensive Cytopathology. 2nd
ed. Philadelphia, PA: W B. Saunders Company; 1997. p. 553-4.
Zito FA, Gadaleta CD, Salvatore C, Filotico R, Labriola A, Marzullo A, et al
. A modified cell block technique for fine needle aspiration cytology. Acta Cytol 1995;39:93-9.
Keyhani-Rofaga S, O Toole RV, Leming MF. Role of cell block in fine needle aspiration. Acta Cytol 1984;28:630-6.
Kern WH, Haber H. Fine needle aspiration minibiopsies. Acta Cytol 1986;30:403-8.
Nathan NA, Narayan E, Smith MM, Horn MJ. Cell block cytology. Improved preparation and its efficacy in diagnostic cytology. Am J Clin Pathol 2000;114:599-606.
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:e108762.
Sharma R, Nagaich N, Katiyar P, Gupta S, Bhatia N. Cell block and IHC: A new weapon in diagnostic armamentarium in differentiating liver SOL. J Clin Exp Hepatol 2015;5:62.
Timek DT, Shi J, Liu H, Lin F. Arginase-1, HepPar-1, and Glypican-3 are the most effective panel of markers in distinguishing hepatocellular carcinoma from metastatic tumor on fine-needle aspiration specimens. Am J Clin Pathol 2012;138:203-10.
Morrison C, Marsh W Jr, Frankel WL. A comparison of CD10 to pCEA, MOC-31, and hepatocyte for the distinction of malignant tumors in the liver. Mod Pathol 2002;15:1279-87.
Siddiqu MT, Saboorian MH, Gokaslan ST, Ashfaq R. Diagnostic utility of Hep par1 antibody to differentiate hepatocellular carcinoma from metastatic carcinoma in fine needle aspiration sample. Cancer Cytopathol 2001;96:49-52.
Varma V, Cohen C. Immunohistochemical and molecular markers in the diagnosis of hepatocellular carcinoma. Adv Anat Pathol 2004;11:239-49.
Lau SK, Prakash S, Geller SA, Alsabeh R. Comparative immunohistochemical profile of hepatocellular carcinoma, cholangiocarcinoma, and metastatic adenocarcinoma. Hum Pathol 2002;33:1175-81.
Borscheri N, Roessner A, Röcken C. Canalicular immunostaining of neprilysin (CD10) as a diagnostic marker for hepatocellular carcinomas. Am J Surg Pathol 2001;25:1297-303.
Saad RS, Luckasevic TM, Noga CM, Johnson DR, Silverman JF, Liu YL. Diagnostic value of HepPar1, pCEA, CD10, and CD34 expression in separating hepatocellular carcinoma from metastatic carcinoma in fine-needle aspiration cytology. Diagn Cytopathol 2004;30:1-6.
Lin F, Abdallah H, Meschter S. Diagnostic utility of CD10 in differentiating hepatocellular carcinoma from metastatic carcinoma in fine-needle aspiration biopsy (FNAB) of the liver. Diagn Cytopathol 2004;30:92-7.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]