|Ahead of print publication
Hepatocyte paraffin-1, CD10, and CD34 immunostaining as a diagnostic aid in cytologic diagnosis of hepatic cancer
Anushree Agarwal1, Uma Handa1, Reetu Kundu2, Atul Sachdev3, Suman Kochhar4
1 Department of Pathology, Government Medical College and Hospital, Chandigarh, India
2 Department of Cytology and Gynecological Pathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Medicine, Government Medical College and Hospital, Chandigarh, India
4 Department of Radiodiagnosis, Government Medical College and Hospital, Chandigarh, India
|Date of Submission||15-Apr-2020|
|Date of Decision||01-Jun-2020|
|Date of Acceptance||10-Sep-2020|
|Date of Web Publication||03-Aug-2021|
Department of Cytology and Gynecological Pathology, Postgraduate Institute of Medical Education and Research, Sector 12, Chandigarh - 160 012
Source of Support: None, Conflict of Interest: None
Background: Cytomorphological distinction between hepatocellular carcinoma and metastatic tumors to the liver may be difficult, especially when these have poor differentiation. The present study was done to assess the diagnostic utility of hepatocyte paraffin-1 (HepPar-1), CD10, and CD34 in differentiating hepatocellular carcinoma from metastatic carcinoma.
Materials and Methods: Ultrasound-guided fine-needle aspiration was performed on 50 patients with space-occupying lesions of liver suspicious for malignancy on clinical/radiologic findings. The cytological assessment was done on smears stained with May–Grünwald–Giemsa and hematoxylin and eosin. Cell blocks were prepared, and immunostaining for HepPar-1, CD10, and CD34 was done.
Results: In these 50 patients, hepatocellular carcinoma was diagnosed in 7 and metastatic tumors in 43 cases. The sensitivity of smears in diagnosing hepatocellular carcinoma was 100% and the specificity was 95.3%, while the sensitivity and specificity of cell block were 100%. A canalicular pattern of CD10 immunoreactivity had a 100% positive predictive value for diagnosing hepatocellular carcinoma. CD10 had a sensitivity of 57.1% and 41.9% in identification of HCC and metastatic tumors, respectively. For the diagnosis of hepatocellular carcinoma, the sensitivity of CD34 was 85.7% and the specificity of sinusoidal pattern of immunoreactivity was 100%. The sensitivity and specificity of granular cytoplasmic staining pattern of HepPar-1 were 100% in hepatocellular carcinoma.
Conclusions: The staining patterns of HepPar-1, CD10, and CD34 are highly specific in distinguishing hepatocellular carcinoma from metastasis. These three immunomarkers should be included in the immunocytochemical panel for differentiating hepatocellular carcinoma from metastatic carcinoma to the liver.
Keywords: Fine-needle aspiration, hepatocellular carcinoma, immunostaining, metastatic carcinoma
|How to cite this URL:|
Agarwal A, Handa U, Kundu R, Sachdev A, Kochhar S. Hepatocyte paraffin-1, CD10, and CD34 immunostaining as a diagnostic aid in cytologic diagnosis of hepatic cancer. J Can Res Ther [Epub ahead of print] [cited 2022 Jul 3]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=322905
| > Introduction|| |
With state of the art imaging modalities, subtle nodular hepatic lesions are nowadays amenable to early detection. Although imaging and tumor markers provide a reliable diagnosis in many instances, tissue diagnosis is warranted under circumstances where the clinical, biochemical, and imaging profiles are equivocal. In such cases, fine-needle aspiration cytology (FNAC) of the liver under ultrasound guidance is a popular procedure for establishing the diagnosis. The reported sensitivity of FNAC for detecting malignancy ranges from 67% to 100%, with the specificity approaching 100%.,
A vast majority of hepatic malignancies are metastatic, and distinction from hepatocellular carcinoma is important as treatment approaches for both are poles apart. Distinction between a well-differentiated HCC and benign lesions or poorly differentiated HCC and metastatic carcinoma at times can be cytologically challenging. A panel of immunocytochemical stains is used to diagnose such cases. Commonly used antibodies include alpha-fetoprotein (AFP), carcinoembryonic antigen, hepatocyte paraffin-1 (HepPar-1), glypican-3, arginase-1, CD10, and CD34.
HepPar-1 is reported to be a highly specific marker for hepatocyte differentiation demonstrating 90% specificity for hepatocellular origin., CD10 demonstrates a characteristic canalicular staining pattern in 60%–70% of HCC which is rarely encountered in metastatic carcinoma. The endothelial cell marker CD34 has been found to be negative in normal hepatic sinusoids and diffusely positive in HCC due to capillarization of sinusoidal cells with loss of fenestrae in HCC. The present study was undertaken to evaluate the diagnostic utility of HepPar-1, CD10, and CD34 in differentiating HCC from metastatic carcinoma in fine-needle aspirates.
| > Materials and Methods|| |
The present prospective study was carried out in the department of pathology in a tertiary care hospital in collaboration with the departments of general medicine and radiodiagnosis. The study protocol was approved by the Institutional Ethics Committee, and informed consent was obtained from all the patients. Fifty patients having single or multiple space-occupying lesions (SOL) in the liver detected on ultrasound were included. Detailed history, clinical examination, and radiological findings were recorded. Patients underwent percutaneous fine-needle aspiration of liver SOL/SOLs with a 22-G needle attached to a 20 ml disposable syringe mounted on a Franzen handle. A minimum of 3 smears were made and stained with May–Grünwald–Giemsa (MGG), hematoxylin and eosin (H and E), and Papanicolaou stains. After making the smears, the residual material including tissue fragments and blood was collected in a vial and allowed to clot. The clot was processed as a cell block and 3 μm sections were cut. Routine H and E and special stains such as reticulin and mucicarmine were done wherever required. A detailed cytomorphological analysis including smear cellularity, cell arrangement, cellular, cytoplasmic, and nuclear details was performed based on which a cytological diagnosis was rendered.
Immunohistochemistry for CD10, CD34, and HepPar-1 was done on sections from cell block in all the cases. CD10 immunostaining was evaluated according to the type of staining pattern: canalicular, membranous, and cytoplasmic, and the cases were scored as positive or negative. CD34 immunoreactivity was based on the presence or absence of sinusoidal positivity. HepPar-1 response was assessed on the presence or absence of granular cytoplasmic positivity.
| > Results|| |
All 50 cases were malignant with metastatic tumors in 43 cases and HCC in 7 cases. Five cases with HCC had a single SOL, and 2 had multiple SOLs. Out of 43 metastatic tumors, multiple SOLs were present in 36 cases and a single SOL was seen in 7 cases. In cases with metastasis, at the time of fine-needle aspiration, the primary tumor site was known in 22 cases, with the lung being the most common in 10 cases.
The age range of 50 patients was 40 to 75 years (mean age: 55.6 ± 12.3 years). The age of the patients with HCC ranged from 42 to 60 years (mean age: 51.8 ± 7.2 years), while in metastasis, the age range was 21 to 75 years (mean age: 56 ± 13 years). Twenty-eight (56%) patients were male and 22 (44%) were female. The common complaints were abdominal pain (95%), loss of weight (74%), loss of appetite (72%), fever (48%), and abdominal lump (38%). AFP levels in HCC were raised in 6/7 cases and ranged from 12.8 ng/ml to 879 ng/ml (mean: 284 ± ng/ml), while in metastatic tumors, the values were between 0.7 ng/ml and 80 ng/ml (mean: 11 ± 22 ng/ml).
Cytological and cell block findings
The cytological diagnoses rendered in 43 cases of metastatic tumors were adenocarcinoma (26), poorly differentiated carcinoma (7), small cell carcinoma (3), neuroendocrine carcinoma (2), malignant small round cell tumor (1), squamous cell carcinoma (1), and gastrointestinal stromal tumor (GIST) (1). Two cases diagnosed as HCC/adenocarcinoma favor HCC due to overlapping morphological features that were reclassified as metastatic adenocarcinoma on cell block.
The cytological features seen in HCC and metastatic tumors are summarized in [Table 1]. Smears from HCC (7) showed a trabecular pattern of cell arrangement, sheets of tumor cells with transgressing capillaries, high nuclear: cytoplasmic ratio, macronucleoli, inclusions, and plentiful atypical naked nuclei [Figure 1]. Smears from metastatic adenocarcinoma (26) showed good cellularity comprising clusters and acinar formations of tumor cells with vesicular nuclear chromatin and conspicuous nucleoli. Scattered tumor cells with barely discernible cytoplasm, nuclear molding, and streaking were observed in metastatic small cell carcinoma (3). Fascicular arrangement of oval-to-spindled cells with moderate pleomorphism was evident in case of metastatic GIST (1). Out of 43 cases with metastatic tumors, the presence of benign hepatocytes was noted in 35 cases.
|Table 1: Cytological features and immunoreactivity in hepatocellular carcinoma and metastatic tumors (n=50)|
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|Figure 1: Hepatocellular carcinoma (a and b) and metastatic adenocarcinoma (c and d) a ultrasound abdomen showing a hypodense mass lesion (arrow) in the posterior segment of the right lobe of liver with needle (N) placed during guided fine-needle aspiration. (b) Aspirate smear showing tissue fragment composed of malignant hepatocytes with transgressing capillaries (MGG, ×200). (c) Ultrasound abdomen showing multiple hypodense SOLs in both lobes of liver with needle (N) in situ during guided fine-needle aspiration from liver lesion (arrow). (d) Aspirate smear showing clusters of tumor cells with moderate pleomorphism and benign hepatocytes in the background (arrow) in metastatic adenocarcinoma (MGG, ×200)|
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The sensitivity and specificity of cytological smears in diagnosing malignancy were 100%. The diagnostic accuracy of smears in subclassification of malignancy was 96%. The sensitivity of smears in diagnosing HCC was 100% and the specificity was 95.3%. The sensitivity, specificity, and diagnostic accuracy of cell block in the diagnosis of HCC were 100%. The sensitivity and specificity of smears in diagnosing metastatic disease were 95.3% and 100%, respectively. The sensitivity and specificity of cell block in subclassifying metastatic disease was 97.7% and 100%, respectively.
CD10 showed a canalicular pattern of immunoreactivity in 4 out of 7 cases of HCC [Figure 2]. Membranous immunoreactivity for CD10 was seen in 18 out of 43 cases of metastatic carcinoma. CD10 had a sensitivity of 57.1% and 41.9% in identification of HCC and metastatic tumors respectively.
|Figure 2: Hepatocellular carcinoma (a) Cell block shows tissue fragments composed of sheets and trabeculae of malignant hepatocytes (H and E, ×400). (b) CD10 immunostaining showing canalicular positivity in tumor cells (IHC, ×400). (c) Sinusoidal positivity for CD34 immunostain around clusters of tumor cells (IHC, ×400). (d) Hepatocyte paraffin-1 showing diffuse granular positivity in tumor cells (IHC, ×400)|
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Six out of 7 cases of HCC showed sinusoidal positivity for CD34. All cases of metastatic tumors were nonimmunoreactive for CD34 [Figure 3]. The sensitivity of CD34 was 85.7% and the specificity of sinusoidal pattern of immunoreactivity was 100% in diagnosing HCC.
|Figure 3: Metastatic adenocarcinoma (a) cell block showing clusters of tumor cells showing moderate nuclear pleomorphism and high N/C ratio (H and E, ×400). (b) Clusters of tumor cells showing strong membranous positivity for CD10 immunostain (IHC, ×400). (c) Tumor cells showing nonreactivity for CD34 immunostain (IHC, ×400). (d) Granular positivity for hepatocyte paraffin-1 in benign hepatocytes while the tumor cells are negative (IHC, ×400)|
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HepPar-1 gave a granular cytoplasmic staining in all the 7 cases of HCC. None of the cases of metastatic carcinoma showed immunoreactivity in tumor cells. The sensitivity, specificity, and positive predictive value of the granular cytoplasmic staining of HepPar-1 were 100% in cases of HCC.
| > Discussion|| |
Metastatic tumors to the liver account for about a quarter of all metastases to the solid visceral organs. The clinical presentation of hepatocellular carcinoma and metastasis to the liver is rarely characteristic. A combination of radiology, aspiration cytology, and serum markers provides an accurate diagnosis in most of the cases, but there still remains a considerable proportion with diagnostic difficulty. Making cell blocks can be helpful in such situations as histochemical and immunocytochemical stains can be applied with ease. The other advantages over core-needle biopsy include quick diagnosis, lesser cell dispersion, and also lesser cost. Further, multiple passes in different planes give a better yield in aspirates as compared to core-needle biopsy. After evaluation of smears and cell blocks, the sensitivity, specificity, and diagnostic accuracy to classify benign and malignant cases were 100% in this study. The sensitivity and specificity by other studies is 87.8%–97.8% and 96%–100%, respectively, while the diagnostic accuracy is in the range of 90%–98%.,
In the present study, serum AFP levels were raised in 6/7 cases of HCC similar to that reported in other studies., Cytological features of HCC in liver aspirates are well documented in the literature. In the present study, high N/C ratio and trabecular arrangement of cells were seen in all the cases. Atypical hepatocytic naked nuclei were seen in 6/7 cases and prominent nucleoli were seen in all the 7 cases, which is higher than that reported in another study. Cytoplasmic vacuolation was seen in 71.4% of cases, which is in congruence with the existing literature., The sensitivity and specificity of smears in diagnosing HCC were 100% and 95.3%, respectively, which is similar to that reported earlier. The higher sensitivity in the present study could be attributed to good clinicoradiological correlation, complete patient history, correlation with tumor markers, and careful evaluation of the aspirates.
Of 7 cases of HCC, the canalicular pattern of CD10 positivity was seen in 4 cases which had a 57.1% sensitivity similar to that reported by other workers., Higher sensitivity, however, has been observed in some studies.,, This could be attributed to the low number of cases of HCC as compared to other studies. The current study had a 100% specificity and 100% positive predictive value for diagnosing HCC. CD34 showed a sinusoidal positivity in 6/7 cases and had a sensitivity of 85.7% and a specificity of 100%, which is as per the literature.,,, In the present study, all 7 cases of HCC showed diffuse granular cytoplasmic staining for HepPar-1. The sensitivity and specificity of the granular cytoplasmic staining of HepPar-1 were 100% in cases of HCC.
Of 50 malignant cases, 43 cases were of metastatic disease in this study which is in collaboration with other studies, with an incidence ranging from 50%–85%., The mean AFP level in the metastatic group was 11 ± 22 ng/ml which was almost within the normal range. On radiology, multiple SOLs were found in 83.7% of cases, whereas 16.3% of cases had a single SOL as is also seen in other studies. The primary site of malignancy was found in 22/43 (51.2%) cases, which is slightly less compared to a study where the primary site was found in 68% cases.
The most useful pointers for metastatic carcinoma include arrangement of cells as three-dimensional clusters and singly scattered cells with attempted acini or gland formation. The presence of benign hepatocytes and production of mucin favor metastatic disease. Other features depend on the primary site. The diagnostic accuracy and specificity of cell block in subclassifying metastatic disease were 97.7% and 100%, respectively, as compared to a study which reported figures of 87.2% and 78.3%, respectively. The better results in this study could be attributed to the exclusion of cases with scanty material and those showing predominantly blood.
Eighteen out of 43 cases of metastatic carcinoma showed membranous positivity for CD10. The current study compares favorably with other studies in terms of the number of cases showing positivity with CD10 in metastatic carcinoma to the liver.,, None of the cases with metastatic tumors was positive for CD34. HepPar-1 was also negative in the metastatic group which is in accordance with other studies.,,
HepPar-1 has a high specificity and sensitivity for hepatocellular origin. The granular cytoplasmic staining pattern of HepPar-1 is easily interpreted. The canalicular pattern of immunostaining with CD10 is typically seen in benign liver and HCC. Although some of the metastatic tumors like renal cell carcinoma show positivity for CD10, the immunostaining pattern is never canalicular. The canalicular staining pattern of CD10 needs a diligent search and expertise to interpret. Nevertheless, CD10 should be a part of the immunopanel employed to differentiate hepatocellular carcinoma from metastatic carcinoma as poorly differentiated HCCs may show a negative immunoreactivity with HepPar-1., In such a case, CD10 immunoreactivity is helpful to arrive at the correct diagnosis because the sensitivity of staining with CD10 in HCC is unrelated to the tumor grade, however, subtle differences in the staining patterns may be noticed. Well-differentiated HCCs show a long and zigzag linear intercellular canalicular pattern, while short and thick or dot-like staining patterns may be observed in moderate to poorly differentiated HCCs. The clear-cut canalicular staining pattern with CD10 in HCC irrespective of the tumor differentiation is sufficient enough to recommend CD10 along with HepPar-1 and CD34 in a limited panel of immunostains.
The current study demonstrates the usefulness of a limited immunomarker panel employing just 3 markers: HepPar-1, CD10, and CD34 to distinguish HCC from metastatic carcinoma of the liver. Metastatic tumors were seen in 43 cases, while 7 cases had HCC out of 50 cases with liver SOLs in whom ultrasound-guided fine-needle aspiration was done. The study has a substantial number of total cases but with a huge difference in number of HCC and metastatic lesions. Therefore, it is not possible to make statistical comparisons. This is seen as a weakness of the current study. Larger studies with comparable number of HCC and metastatic lesions of the liver are hence required.
| > Conclusions|| |
The pattern of immunoreactivity of the three immunostains: CD10, CD34, and HepPar-1 and the overall combination is immensely conclusive in diagnosing and differentiating HCC from metastatic carcinoma to the liver and should be part of the limited immunopanel employed considering the cost constraints. Furthermore, the application of immunopanel on cell blocks avoids core-needle hepatic biopsy.
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Conflicts of interest
There are no conflicts of interest.
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