RETRACTED

RETRACTED

RETRACTED

RETRACTED

RETRACTED

RETRACTED

RETRACTED

RETRACTED

RETRACTED

RETRACTED

RETRACTED

RETRACTED

RETRACTED

RETRACTED

RETRACTED

RETRACTED

RETRACTED

Periodic acid-Schiff and alcian blue immunohistochemistry to detect mucin in mucinous breast carcinoma

Primariadewi Rustamadji, Jason Wibowo, Belinda Murtani, Christy Magdalena

Background

Detection of mucins has been shown to correlate with several clinicopathological characteristics in patients. Currently, periodic acid-Schiff (PAS) and alcian blue staining methods are the histochemistry staining techniques that are frequently used to detect mucin. This study was aimed to evaluate PAS and alcian blue staining in differentiating mucin characteristics between invasive carcinoma of no special type (ICNST) with mucinous degeneration and mucinous carcinoma.

 

Methods

This cross-sectional study of 33 cases included biopsies of mucinous breast carcinoma and ICNST with mucin degeneration that were histologically verified using hematoxylin and eosin (H&E) staining. The PAS and alcian blue staining were conducted in the Laboratory of Histochemistry, Department of Anatomical Pathology, Cipto Mangunkusumo Hospital. Data were recorded using SPSS software, version 21 (IBM Corp, USA).

 

Results

There were 17 cases of ICNST with mucinous degeneration and 16 cases of mucinous carcinoma with age varied from 27 to 75 years. PAS had sensitivity of 87.5% and specificity of 41.2%. Alcian blue had sensitivity of 43.8% and specificity of 82.4%.

 

Conclusions

PAS staining method is better than the alcian blue staining method in distinguishing between ICNST with mucinous degeneration and mucinous carcinoma. In the limited setting laboratory, PAS staining alone can be considered to detect mucin.

 

Keywords

alcian blue, mucin, mucinous carcinoma, periodic acid-Schiff reaction

According to the Global Cancer Observatory in 2018, breast cancer accounted for 30.9% of all cancer cases in Indonesia, making this condition the most prevalent form of cancer.1 The most common malignant breast type is invasive ductal carcinoma, also known as invasive carcinoma of no special type (ICNST), which consists 70–75% of all breast tumor cases.2 ICNST has nonspecific histopathological features.2,3 Hence, difficulty arises in its diagnosis, thereby resulting in low-precision management and poor prognosis.2 Meanwhile, mucinous carcinomas are a rare pathological entity. These carcinomas must be differentiated from ICNST with mucin degeneration given that the former have a better prognosis due to the lower incidence of lymph node metastasis.4

Mucin is a high-molecular-weight glycoprotein, which is composed of a central protein core, that is dispersed throughout the epithelial surface of the gastrointestinal, respiratory, and reproductive tract.5 This protein can be examined by immunohistochemical analysis, which has been shown to help in differentiating several clinicopathological characteristics, using specific antibodies.4 Mucin is preferably combined with hematoxylin and eosin (H&E) staining and panels of markers.6 Unfortunately, immunohistochemical methods are expensive.7

Histochemical methods, such as periodic acid-Schiff (PAS) and alcian blue staining methods, are cheap and frequently used to detect mucin. PAS staining can detect both neutral and acidic mucin, whereas alcian blue staining is more sensitive to acidic and mucopolysaccharide mucins. Therefore, this study was aimed to evaluate the diagnostic performance of PAS or alcian blue and their combination to differentiate ICNST and mucinous carcinoma.

 

 

This cross-sectional study was conducted using archived breast tissue biopsy specimens. Data were retrieved from Department of Anatomical Pathology, Faculty of Medicine Universitas Indonesia/Cipto Mangunkusumo Hospital from March to August 2014. The subjects of this study were limited to those who underwent mastectomy surgery, which was performed in this hospital. We only included mucinous breast carcinoma and ICNST with mucin degeneration. The biopsies were histologically verified using H&E staining by the senior researcher as mucinous carcinoma and ICNST using H&E staining. All the selected cases were stained with PAS and alcian blue to analyze the mucin of both cases. Staining was conducted at the Laboratory of Histochemistry, Department of Anatomical Pathology, Cipto Mangunkusumo Hospital.

All of the sections from paraffin blocks of each case underwent deparaffinization process using xylol. Rehydration process was carried out by applying alcohol with decreased concentrations from ethanol 100%, 95%, and 70% and then placing the paraffin blocks in water. Next, the tissues were stained using 1% alcian blue dye (Scy Tech, USA) and washed with clear water. The slides stained with nuclear fast red and rinsed with clear water.8 For the PAS staining, another set of paraffin blocks were rehydrated using the same method, but they were stained with 1% periodic acid dye (Scy Tech, USA), rinsed, and finally soaked in clear water.

Then, dehydration was performed to eliminate the water content in both slides of PAS and alcian blue staining using alcohol with increased concentrations of ethanol (70%, 95%, and 100%). Next, the slides were cleaned using xylol, mounted by dripping the mounting agent (Entellan, Germany) sufficiently, and covered with a glass cover.8 The positive result for PAS was the red-pink color glycogen or carbohydrate, whereas the negative result was a pink-reddish color. Meanwhile, the positive result for alcian blue staining was the blue color of mucin acid, whereas the negative result was a color other than blue color.8

Tumor node metastasis (TNM) staging was conducted based on the Union for International Cancer Control staging system.9 All data were retrieved from archival history in the database. Data were recorded using SPSS software, version 21 (IBM Corp, USA). Sensitivity and specificity were calculated. Positive and negative predictive values were not calculated given that the prevalence of mucinous breast carcinoma and ICNST with mucin degeneration were not comparable with that of the general population. This study has been reviewed and approved by the Ethics Committee of the Faculty of Medicine, Universitas Indonesia (No: 431/H2.F1/ETIK/2014).

 

 

During the study period, 33 cases of both types of breast carcinoma were included. There were 17 cases of ICNST with mucinous degeneration and 16 cases of mucinous carcinoma. The age of breast cancer patients was varied from 27 to 75 years (Table 1).

 

 

Figure 1 shows the staining results using the PAS and alcian blue techniques to detect mucins in ICNST with mucinous degeneration and mucinous carcinoma. Most of the cases were positive to PAS staining but not to alcian blue staining (Table 2).

 

 

 

 

PAS staining method had a higher sensitivity that was 87.5% rather than its specificity, contrary to alcian blue which had a higher specificity that was 82.4%. Comparison of diagnostic values between PAS and alcian blue in detecting mucin are shown in Table 3.

 

 

 

 

This study was conducted to compare the diagnostic performance of PAS, alcian blue, and their combination to differentiate between ICNST with mucin degeneration and mucinous carcinoma. In the diagnosis of biopsy, the pathologist usually only uses morphological data. If any difficulties exist, the pathologists will attempt to use further staining with histochemistry or/and immunohistochemistry. In a normal tissue, mucin exhibits specific histochemical patterns in tissues and cells. However, these patterns may change in pathological conditions. In adenocarcinoma, mucin expression could be disturbed, including the increased, decreased, or aberrant expression of several mucin glycoproteins.10,11 The PAS staining technique is sensitive in detecting neutral mucin and acidic mucin that contains sialic acid. Meanwhile, the alcian blue staining technique is sensitive in detecting sulfomucin and sialomucin.12 In this study, both PAS and alcian blue staining methods demonstrated varied positivities toward ICNST with mucin degeneration and mucinous carcinoma.

Badowska-Kozakiewicz et al13 performed histopathological examination of ICNST in 691 patients. The age of patient was ranged between 30–81 years old (mean [standard deviation] of age = 60.47 [5.07]), which showed that most of the study patients affected by ICNST were older than those included in this study. The TNM staging revealed that most of the tumors were assessed with T2, N0, and M0, in contrast to this study in which many patients were already on advanced stage. This result might be the still low awareness of the community to breast cancer screening in Indonesia. Dumitru et al14 showed that the mean age of patients with mucinous carcinoma was 62.3 years, in contrast with this study which observed mucinous carcinoma in younger patients. They also stated that most cases assessed with T2, N0, and M0, and showed similar results with our study.

Fagare11 showed that more acidic mucin was detected in adenocarcinoma of the breast, colon, ovary, and lungs compared with neutral mucin. A similar result was reported by Ali et al,10 who observed more acidic mucin and sulfomucin in mucinous adenocarcinoma. They also demonstrated that more acidic mucin was detected in 13 of 33 samples that were positive with the PAS staining method, and 6 were positive with alcian blue staining. This result is similar with the finding of this study, with 13 out of 16 mucinous carcinoma showing positive result in PAS staining. The sensitivity of the PAS staining was also higher than that of alcian blue staining. However, in alcian blue staining, both ICNST with mucin degeneration and mucin carcinoma showed positive results.

Battles et al15 showed that alcian blue or PAS was more sensitive to mucin staining than mucicarmine; despite the wide variation among cases, one case was uniformly negative. By contrast, mucicarmine showed negative results in three patients with primary extramammary Paget disease. This study also showed that the sensitivity of PAS or alcian blue staining reached 87.5%, which indicates both PAS and alcian blue are sensitive to mucin differentiation. Contrary to the research by Micke et al16 which showed that the mucin staining by alcian blue and PAS exhibit high specificity but low sensitivity for adenocarcinoma in lung cancer when the cut-off for positivity was defined as 10+ cytoplasmic mucin inclusions in a 1 mm TMA core. Another study by Jastrzebski et al17 showed that alcian blue had a sensitivity of 88% and a specificity of 100% for showing mucinous production in differentiating mucoepidermoid carcinoma of conjunctiva and squamous cell carcinoma of the conjunctiva. This result had a same result with this study which alcian blue had a higher specificity than sensitivity.

The limitation of this study was the number of similar cases of mucinous carcinoma and ICNST with mucin degeneration. A limited number of cases of mucinous carcinoma was also found in the archival data in Department of Anatomical Pathology, Cipto Mangunkusumo Hospital. Although ICNST cases occurs often, this study only used data of patients who underwent surgical biopsy in this hospital. Hence, the number of paraffin block that we could use was limited. Furthermore, the positive and negative predictive values could not be counted given that the sample did not represent the prevalence in general populations.

In conclusion, the histopathological method of PAS staining used in the histological determination of mucin expression has a higher sensitivity and better performance. Also, the origin of the acidic mucin produced can be identified using PAS staining method. In the limited setting laboratory, PAS staining alone can be considered to detect mucin.

 

 

 

 

 

 

 

1. Global Cancer Observatory. Population fact sheets: Indonesia [Internet]. Lyon: International Agency for Research on Cancer; 2018 [cited 2019 Nov 17]. Available from: http://gco.iarc.fr/today/ data/factsheets/populations/360-indonesia-fact-sheets.pdf.
2. Schnitt SJ, Collins LC. Biopsy interpretation of the breast. Philadelphia: Lippincot Williams & Wilkins; 2013.
3. Kumar V, Abbas AK, Fausto N, Aster JC. Robbins and Cotran pathologic basis of disease. Philadelphia: Saunders Elsevier; 2010.
4. Mukhopadhyay P, Chakraborty S, Ponnusamy MP, Lakshmanan I, Jain M, Batra SK. Mucin in the pathogenesis of breast cancer: implication in diagnosis, prognosis and therapy. Biochim Biophys Acta. 2011;1815(2):224–40.
5. Myers R. Special stain techniques for the evaluation of mucins [Internet]. Illinois; Leica Biosystems; 2009 [cited 2013 Dec 19]. Available from: http://www.leicabiosystems.com/ pathologyleaders/special-stain-techniques-for-the-evaluationof- mucins/.
6. Zaha DC. Significance of immunohistochemistry in breast cancer. World J Clin Oncol. 2014;5(3):382–92.
7. The Centre for Phenogenomics. Pathology fees [Internet]. 2019 [cited 2019 Apr 20]. Available from: http://phenogenomics.ca/ pathology-services.html.
8. Department of Anatomical Pathology. Laboratory’s standard operating procedure (SOP). Universitas Indonesia-Cipto Mangunkusumo Hospital. 2014.
9. The Union for International Cancer Control. TNM classification of malignant tumours [Internet]. 2019 [cited 2019 Dec 12]. Available from: https://www.uicc.org/resources/tnm.
10. Badowska-Kozakiewicz AM, Liszcz A, Sobol M, Patera J. Retrospectove evaluation of histopathological examination in invasive ductal breast cancer of no special type: an analysis of 691 patients. Arch Med Sci. 2017;13(6):1408–15.
11. Dumitru A, Procop A, Iliesiu A, Tampa M, Mitrache L, Costache A, et al. Mucinous breast cancer: a review study of 5 year experience from a hospital-based series of cases. Maedica. 2015;10(1):14–8.
12. Ali U, Nagi AH, Naseem N, Ullah E. Mucin histochemistry in tumours of colon, ovaries and lung. J Cytol Histol. 2012;3(7):163.
13. Fagare MO. Evaluation of mucin histo-chemical pattern in breast, colonic, ovarian and lung adenocarcinomas. Int J Life Sci Res. 2015;3(4):39–46.
14. Cooper DJ. Mucin histochemistry of mucous carcinomas of breast and colon and non-neoplastic breast epithelium. J Clin Pathol. 1974;27(4):311–4.
15. Battles OE, Page DL, Johnson JE. Cytokeratins, CEA, and mucin histochemistry in the diagnosis and characterization of extramammary Paget’s disease. Am J Clin Pathol. 1997;108(1):6– 12.
16. Micke P, Botling J, Mattsson JS, Planck M, Tran L, Vidarsdottir H, et al. Mucin staining is of limited value in addition to basic immunohistochemical analyses in the diagnostics of non-small cell lung cancer. Sci Rep. 2019;9:1319.
17. Jastrzebski A, Brownstein S, Jordan DR, Gilberg SM. Histochemical analysis and immunohistochemical profile of mucoepidermoid carcinoma of the conjunctiva. Saudi J Ophthalmol. 2012;26(2):205–10.

Copyright @ 2020 Authors. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original author and source are properly cited. For commercial use of this work, please see our terms at http://mji.ui.ac.id/journal/index.php/mji/copyright.