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ORIGINAL ARTICLE  
Year : 2018  |  Volume : 61  |  Issue : 4  |  Page : 510-515
Diagnostic utility of CD10 immunohistochemical staining on cellblock in differentiating hepatocellular carcinoma from secondary malignancies of liver


1 Department of Pathology, North Bengal Medical College, Siliguri, West Bengal, India
2 Department of Urology, CNMCH, Kolkata, West Bengal, India

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Date of Web Publication10-Oct-2018
 

   Abstract 


Background: Differentiation of hepatocellular carcinoma (HCC) from metastatic malignancy in liver may be difficult at times on fine-needle aspiration cytology, especially in case of moderate-to-poorly differentiated tumors. The benefit of cell-block technique is the recognition of histologic pattern of diseases along with application of a wide variety of immunohistochemical (IHC) stains to differentiate hepatic malignancies. In this study, CD10 IHC staining was done on cellblocks prepared from aspirates of clinicoradiologically/cytologically suspected malignant liver neoplasms to differentiate HCC from malignancies metastasizing to liver. Objective: The objective of the study was to assess the diagnostic utility of CD10 IHC stain on cell-block preparation for differentiating primary from Secondary malignancies of liver. Materials and Methods: Formalin-fixed, paraffin-embedded cellblocks of 61 cases (25 cases of HCC and 36 cases of metastatic carcinoma) were prepared from a fine-needle aspirate of the suspected malignant liver neoplasm and immunostained using monoclonal antibody against CD10. Results: Twenty-two (88%) of 25 cases of HCC were positive for CD10 with a canalicular staining pattern. Two (8%) were positive for CD10 with membranous and one (4%) with cytoplasmic staining pattern. Conclusion: CD10 immunostaining on cellblock is useful in discriminating HCC and metastatic carcinoma of the liver with a diagnostic accuracy of 88.52%.

Keywords: Canalicular, CD10, cellblock, fine-needle aspiration cytology, hepatocellular carcinoma, metastatic malignancy of liver

How to cite this article:
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

How to cite this URL:
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 [serial online] 2018 [cited 2018 Dec 15];61:510-5. Available from: http://www.ijpmonline.org/text.asp?2018/61/4/510/243007





   Introduction Top


Worldwide, the third most common cause of cancer death is hepatocellular carcinoma (HCC).[1] Most primary liver cancers arise from hepatocytes and are termed as HCC.[2] Age-standardized incidence rates of HCC in India for men and women are 0.9–3.4 and 0.2–1.8 per 100,000 people, respectively, and the incidence rate even increases to 1.6 Per 100 persons per year among patients with background cirrhosis.[3] Malignant tumors occurring in the liver can be primary or secondary (metastasis). Involvement of the liver by secondary malignancy is far more common than HCC.[2] Most common primary sources producing hepatic metastases are those of the colon, breast, lung, pancreas, etc. There is a need for differentiation of HCC from metastasis as they have separate treatment protocol.[4] Till date, the establishment of universal guidelines for imaging diagnosis of HCC is challenging.[5] To differentiate metastasis (secondaries) from HCC, especially moderate-to-poorly differentiated HCC, by fine-needle aspiration cytology (FNAC) of the liver is difficult.[6],[7] The final diagnosis is based on the triple approach of clinical data, radiological findings, as well as cytomorphological features.[8] The definitive differentiation of HCC from secondary malignancies can also be done by core liver biopsy.[4] Many centers advocate the additional use of cellblocks for histological assessment and providing multiple sections for immunohistochemistry.[9] However, sometimes, differentiation of primary and secondary hepatic malignancy by cytomorphological features of sections prepared from cellblock is not possible. CD10 and other IHC markers (Hep Par-1, MOC31, CK7, CK19, Glypican-3, etc.) have been used by various workers on cell-block preparation to differentiate HCC from secondary malignancies. CD10 has the added advantage of being a common marker, currently used for solving multiple diagnostic dilemmas. Recently some studies demonstrated that canalicular staining for CD10 appears to be a highly specific marker for hepatocytic differentiation.[6],[7]

Objective

The objective of the study was to assess the diagnostic utility of CD10 immunohistochemical (IHC) stain on cell-block preparation for differentiating primary from secondary malignancies of the liver.

Review of literature

A study by Lin et al.[6] demonstrates that canalicular staining for CD10 appears to be a highly specific marker for hepatocytic differentiation. Formalin-fixed, paraffin-embedded cellblocks of 55 cases (22 HCC, 23 metastases, and 10 benign hepatic lesions) of FNAC of the liver were immunostained by CD10. Nineteen (86%) of 22 HCC cases were positive for CD10 with a canalicular staining pattern. Among them, 9 (82%) of 11 well-differentiated (WD) HCC and 10 (91%) of 11 MPD HCC were positive for CD10. In conclusion, CD10 appears to be a useful marker in discriminating between HCC and metastatic malignancy when applied to FNAC of the liver.

A study by Ahuja et al.,[7] shows that formalin-fixed, paraffin-embedded cellblocks of 22 cases (7 cases of HCC and 15 cases of metastatic carcinoma) and direct acetone-fixed smears and destained smears of 28 cases (18 cases of HCC and 10 cases of metastatic carcinoma) prepared from FNAC of the liver were immunostained using monoclonal antibody against CD10. Seventeen (68%) of 25 cases of HCC were positive for CD10 with a canalicular staining pattern. Among them, 7 (70%) of 10 cases were WD HCC and 10 (66%) of 15 cases were moderate-to-poorly differentiated HCC. Of 25 cases of metastatic carcinoma, four (16%) were positive for CD10 with a cytoplasmic (three cases) and membranous staining (one case) pattern.


   Materials and Methods Top


The present study was performed in Departments of Pathology, Radiodiagnosis and General Surgery, North Bengal Medical College and Hospital (NBMC and H) after obtaining ethical clearance from the Institutional Ethics Committee of North Bengal Medical College, Sushrutanagar, Darjeeling, West Bengal. The study population comprised of the patients with suspicion of having HCC or metastatic malignancy on radiology and/or cytology with normal prothrombin time. All lesions other than HCC or metastatic malignancy after studying their cytomorphological features were excluded from the study. The study was performed for a 1-year period from April 2015 to March 2016 on sixty-one cases of malignant hepatic neoplasm (25 cases of HCC and 36 cases of metastasis). All relevant data were collected using semistructured, pretested questionnaire and study pro forma. Collected data were entered into Microsoft Excel data sheet. Data were organized and presented using the principles of descriptive statistics. Outcome variables were tested applying appropriate statistical methods and software (SPSS).

Study technique

  • The study sample included those patients attending SOPD during the study period with clinically and radiologically suspected hepatic malignancy. Study subjects were selected on the basis of inclusion and exclusion criteria. The patients were informed about the study in detail and written consent was taken. Then, the study pro forma was filled
  • Materials were aspirated from the liver mass under ultrasonography guidance with Cameco syringe pistol fitted with 10 cc syringe and Spinocan needle (21–23 G).
  • Air-dried and alcohol-fixed smears were prepared from the materials collected from the first pass
  • Materials collected from the second pass were preserved in 10% buffered formalin for cell-block preparation. After that, Trucut biopsy was performed using BARD Max Core biopsy gun (22 mm)
  • FNAC smears from the first pass were stained by Leishman–Giemsa stain (air-dried smears) and hematoxylin and eosin (H and E)/Papanicolaou stain (alcohol-fixed smears)
  • Cellblocks were prepared from the aspirate from the second pass by plasma thrombin clot method. After proper processing, paraffin sections were stained by H and E stain. Furthermore, core-needle biopsy (CNB) specimen was processed and paraffin sections were stained by H and E stain
  • Then, with the help of the above smears and slides, the cytological diagnosis was made by three blinded faculties of Pathology Department of NBMC and H. Furthermore, diagnosis was made from cellblock in the same way
  • After that, IHC staining was done by labeled horseradish peroxidase polymer technique.[10] CD10 was used on cellblock. Then, findings were noted
  • Finally, the diagnostic utility of CD10 IHC staining was analyzed with the help of CNB results (Gold standard).



   Results Top


The present study included 25 cases each of HCC and 36 cases of Metastatic liver malignancy [Table 1]. The results of FNAC and cell-block sections were noted by three blinded faculties. Subsequently, results of cellblock with IHC (CD10, canalicular) were noted. Ultimately, all the results were compared with the CNB with IHC results which was considered gold standard. Based on the pattern of arrangement and nuclear features in FNAC smears, the cases of HCC were classified into two groups: (i) WD, 12 (48%) cases [Figure 1] and (ii) moderate-to-poorly differentiated, 13 (52%) cases [Figure 2].
Table 1: Result of fine-needle aspiration cytology, cellblock, cellblock with IHC, core-needle biopsy and core-needle biopsy with IHC.

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Figure 1: Well-differentiated hepatocellular carcinoma in fine-needle aspiration cytology (L-G stain, ×40)

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Figure 2: Poorly differentiated hepatocellular carcinoma in fine-needle aspiration cytology (L-G stain, ×40)

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CD10 immunohistochemical staining pattern

Three patterns of immunoreactivity for CD10 were observed. These were (i) canalicular, (ii) membranous, and (iii) cytoplasmic. Mixed patterns were also noted. A relationship of the pattern of CD10 immunostaining to the cytodiagnosis is depicted in [Table 2]. A pure canalicular pattern of immunoreactivity was specific for HCC [Figure 3]. In addition, membranous [Figure 4] and weak cytoplasmic immunoreactivity [Figure 5] was seen in a small percentage of cases. In general, most cases of metastatic carcinoma were negative for CD10 [Figure 6]. In contrast, in metastatic carcinoma, an admixture of membranous and cytoplasmic positivity was seen in a small number of cases along with focal canalicular pattern of staining. All the cases of HCC showed canalicular pattern of staining in CNB sections.
Table 2: CD10 immunoreactivity patterns in hepatocellular carcinoma and metastatic carcinoma

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Figure 3: Canalicular stain (CD10), cellblock (×40)

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Figure 4: Membranous stain (CD10), cellblock (×40)

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Figure 5: Cytoplasmic stain (CD10), cellblock (×40)

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Figure 6: Negative CD10, metastatic adenocarcinoma, cellblock (×40)

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CD10 immunostaining was performed on the cellblock of all the 61 cases. CD10 immunoreactivity with canalicular pattern of staining was observed in 22 of 25 cases (88%) of HCC; among them additional membranous staining pattern in two cases and weak cytoplasmic staining pattern in one case were observed. In the metastatic carcinoma group, 4/36 (11%) cases were focally positive for CD10 with canalicular staining pattern. Furthermore, 4/36 (11%) cases showed weak cytoplasmic positivity and three cases showed membranous pattern of staining. The difference of CD10 immunoreactivity with canalicular pattern of staining in the groups of HCC and metastatic carcinoma was highly significant (Pearson χ2 = 35.666a, P < 0.001) [Table 3] and [Table 4] and [Figure 7].
Table 3: Canalicular stain (CD10) and core-needle biopsy with hepatocellular carcinoma cross tabulation

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Table 4: Chi-square tests

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Figure 7: Bar-diagram showing association of canalicular staining [CD10] with hepatocellular carcinoma and metastasis

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In the HCC group, 12 cases were WD HCC and 13 cases were moderate-to-poorly differentiated HCC. Furthermore, we have searched for any association between CD10 immunoreactivity with the differentiation of HCC. However, there was no statistically significant difference in CD10 immunoreactivity with the differentiation of HCC.


   Discussion Top


The diagnostic utility of FNA in the diagnosis of liver space-occupying lesions is well established. Although 75% of malignant lesions of the liver can be correctly diagnosed through cytomorphological analysis and good clinicoradiological correlation, around 25% can pose differential diagnostic problems.[11] Distinction of moderate-to-poorly differentiated HCC from metastatic carcinoma is a major diagnostic problem encountered in day-to-day practice. The treatment and prognosis of HCC and metastatic carcinoma are significantly different, and hence the need to differentiate primary from metastatic malignancy is clinically important.[12] CD10 and other IHC markers (Hep par1, MOC31, CK7, CK19, Glypican-3, etc.) have been used by various workers on cell-block preparation to differentiate HCC from secondary malignancies. CD10 is a newly discovered marker in this scenario. CD10 (syn: neutral endopeptidase-24.11 or neprilysin or common acute lymphoblastic leukemia antigen, a zinc-dependent cell membrane metallopeptidase with a molecular weight of 100 kDa) was one of the first markers available for identifying leukemia cells in children with lymphoblastic leukemia.[13],[14],[15] CD10 has the added advantage of being a common marker, currently used for solving multiple diagnostic dilemmas.

The ability of CD10 immunostaining to discriminate HCC from metastatic carcinoma was the major focus of this study. The pattern of immunoreactivity may be cytoplasmic, membranous, or canalicular. It is the latter pattern that is regarded as specific for a hepatocytic differentiation. The diagnostic utility of CD10 immunostain in HCC was first highlighted by Dragovic et al.[16] and Chu et al.[17] Initially, it was carried out on histological sections[17],[18],[19],[20] and proved to be valuable as a marker to differentiate HCC from metastatic carcinoma because of its characteristic canalicular staining pattern specific for HCC. Later, this was extended to cellblocks made from FNAC.[21],[22] Only the study by Saad et al.[21] has examined CD10 immunostain on ten conventional smears made from FNAC material. In the present study, we examined CD10 immunostain on cellblocks. Identification of a canalicular staining pattern in cases of HCC in FNAC smears required diligent searching. In the present study, a canalicular pattern of positivity characteristic of HCC was seen in 22 (88%) cases, which is slightly higher to that seen in the study by Borscheri et al.[18] However, the result is quite similar to studies by Saad et al.[21] and Lin et al.[22] on FNAC material where 77%–86% canalicular positivity was observed. The intensity of CD10 immunoreactivity did not correlate with the degree of differentiation of the tumor, which is consistent with previous studies.[22] Among metastatic carcinomas, four cases (11%) showed a weak canalicular and cytoplasmic/membranous pattern of staining. These results are almost similar to previous studies, which have shown CD10 positivity in metastatic carcinomas in the range of 0%–20%.[17],[18],[19],[20] CD10 positivity was found in some cases of metastatic urothelial carcinoma, metastatic renal cell carcinoma, and metastatic non-small cell carcinoma to liver, with none of them showing purely canalicular positivity.[23] Canalicular positivity for CD10 is almost exclusively seen with HCC. Overall, the sensitivity of CD10 IHC for the diagnosis of HCC in this study was 88% with a specificity of 88.89% and positive predictive value of 84.62% [Table 5]. A comparison of the present study with other published studies is shown in [Table 6]. This study compares favorably with previously published studies. Canalicular staining for CD10 is a highly specific marker of hepatocytic differentiation. Although CD10 does not differentiate between benign and malignant lesions, it is clearly of use in differentiating HCC from metastatic carcinoma. An ideal immunocytochemical panel of markers would include CD10, HepPar1, polyclonal carcinoembryonic antigen, and CD34 for the diagnosis of HCC to obtain high diagnostic accuracy.[21],[24] In conclusion, this study describes the utility of CD10 on cellblocks, which has not been extensively evaluated in previously published studies. We recommend that CD10 be a part of any panel of IHC markers for the distinction of HCC from metastatic carcinoma in cell-block specimens.
Table 5: Comparison of diagnostic utility of various procedures

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Table 6: Comparison of the sensitivity of CD10 in the diagnosis of hepatocellular carcinoma

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   Conclusion Top


This study describes the utility of CD10 on cellblocks, which has not been extensively evaluated in previously published studies. Furthermore, no comprehensive data in this regard were available in the published literature as far as the present study population is concerned. The present study recommends that the commonly available CD10 immunostain may be valuable if included in a panel of IHC markers for the distinction of HCC from metastatic carcinoma in cell-block specimens. Finally, CD10 immunostaining on cellblock, obtained from FNAC aspirate, is useful in discriminating HCC and metastatic carcinoma of the liver with a high statistical significance.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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DOI: 10.4103/IJPM.IJPM_788_16

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