|Year : 2015 | Volume
| Issue : 3 | Page : 693-701
Carbonic anhydrase IX expression and localization in breast carcinoma
Rehab M Samaka1, Moshira M Abd El-Waheda1, Dalia R Al Sharaky1, Mohammed A Aleskandarany1, Mohamed A Shehata2, Shaymaa E Hegazy1
1 Department of Pathology, Menoufiya University, Shebin El Kom, Egypt
2 Department of Oncology, Faculty of Medicine, Menoufiya University, Shebin El Kom, Egypt
|Date of Submission||21-Mar-2014|
|Date of Acceptance||07-May-2014|
|Date of Web Publication||22-Oct-2015|
Rehab M Samaka
Department of Pathology, Menoufiya University, Shebin El Kom
Source of Support: None, Conflict of Interest: None
The aim of this study was to evaluate the relationship between immunohistochemical (IHC) expression of carbonic anhydrase IX (CA IX) and the standard clinicopathological features of breast carcinoma (BC) in Egyptian patients.
BC in Egyptian patients has specific features of a poor prognosis, such as higher stage at presentation and higher grade than that in developed countries. Hypoxia is present in most solid tumors and is associated with resistance to chemotherapy and radiotherapy as well as a more malignant phenotype. The CA IX level is elevated in response to hypoxia and is involved in tumor development and progression by regulating cellular activities, such as growth, survival, and adhesion.
Patients and methods
This retrospective study was carried out on 56 archival cases of Egyptian BC patients for the evaluation of IHC expression of CA IX.
Fifty-one cases out of 56 (91.1%) showed positive expression of CA IX, whereas five (8.9%) cases showed negative expression. There were significant differences between CA IX IHC expression and tumor stage and stromal response (P = 0.03 and 0.002, respectively) as advanced stage and desmoplastic stroma in favor of overexpression. In terms of the CA IX staining pattern, 44 (78.5%) showed cytoplasmic localization and seven (12.5%) showed membranocytoplasmic localization. Moreover, there were significant differences between the CA IX cytoplasmic and the membranocytoplasmic pattern of positive cases in mitotic count (P = 0.04) as a high mitotic count is found more often in the cytoplasmic pattern.
Hypoxia (overexpression of CA IX) in BC of Egyptian patients is associated with poor prognostic parameters such as advanced stage and desmoplastic stromal response. The cytoplasmic pattern of CA IX expression in BC has been associated with mitotically active tumors. CA IX expression has no relationship with hormonal status, HER2/neu, Ki-67 LI, and IHC subtyping. Further large-scale studies are warranted to investigate the new opportunity for CA IX targeted therapies in BC.
Keywords: Breast carcinoma, carbonic anhydrase IX, hypoxia, immunohistochemical
|How to cite this article:|
Samaka RM, Abd El-Waheda MM, Al Sharaky DR, Aleskandarany MA, Shehata MA, Hegazy SE. Carbonic anhydrase IX expression and localization in breast carcinoma. Menoufia Med J 2015;28:693-701
|How to cite this URL:|
Samaka RM, Abd El-Waheda MM, Al Sharaky DR, Aleskandarany MA, Shehata MA, Hegazy SE. Carbonic anhydrase IX expression and localization in breast carcinoma. Menoufia Med J [serial online] 2015 [cited 2022 Jan 27];28:693-701. Available from: http://www.mmj.eg.net/text.asp?2015/28/3/693/165818
| Introduction|| |
Breast carcinoma (BC) is the most common cancer diagnosed among women worldwide, accounting for 29% of the total new cancer cases . The profile of BC in Egyptian patients has specific features of its own. It is the first of top 10 cancers, where it accounts for 18.3% of estimated incidence with a 37.3 age-standardized rate per 100 000 . BC in Egypt shows poor prognostic features, such as higher stage and higher grade than in developed countries, which may be attributed to different lifestyle and genetic factors, and the nationwide screening and early detection programs adopted in the latter .
Tumor morphology, histopathological stage, and/or grade are the prognostic gold standard for most cancer types. However, there are a number of instances where staging provides inadequate prognostic information. Unfortunately, patient prognosis and future metastases are not always accurately predicted by histopathological appearance or even by staging . Such limitations provide the rational to develop and test other prognostic markers to improve patient risk stratification and plan for novel treatment modalities that may better improve clinical decision making .
Hypoxia is present in most solid tumors and is associated with resistance to chemotherapy and radiotherapy as well as a more malignant phenotype . The impact of tumor hypoxia is multifaceted, with effects on several aspects of tumor biology, including genetic instability, angiogenesis, invasiveness, survival, and metabolism .
Carbonic anhydrase IX (CA IX) is a tumor-associated enzyme that is induced by the transcription factor hypoxia-inducible factor 1a . CA IX is involved in tumor development and progression by regulating cellular activities, such as growth, survival , and adhesion . The relationship between high CA IX expression and a poor outcome has been described in several tumors , and these studies have generated significant interest in the development of strategies to target CA IX for cancer therapy and to use CA IX as a biomarker to detect high-risk patients . This study was designed to evaluate the immunohistochemical (IHC) expression of CA IX in BC of Egyptian patients to identify its relevance in IHC subtyping and patients' prognostication.
| Patients and methods|| |
This retrospective study was carried out on 56 archival cases of Egyptian BC patients. Cases were diagnosed in the Pathology Department, Faculty of Medicine, Menoufiya University, Egypt, between January 2008 and December 2010. Selection was performed on the basis of the availability of paraffin-embedded blocks for serial cutting and examination, estrogen receptor (ER), progesterone receptor (PR), HER2/neu, and Ki-67 IHC-stained slides for reassessment.
Demographic and clinical features
The demographic and clinical features of these patients were collected from the patients' medical records. In terms of age, the patients were divided into up to 50 years and more than 50 years . The size of the tumors was dichotomized into two categories (≤2 and >2 cm) .
Histological types were assessed according to the 2012 WHO classification of tumors of the breast . Grading was performed according to the Modified Bloom-Richardson Scheme . Staging was based on the TNM system according to the updated seventh edition of the American Joint Committee on Cancer, Cancer Staging Manual . The Nottingham Prognostic Index (NPI) was calculated and categorized as published . Necrosis was assessed as being present or absent. Scoring of mitosis was carried out using an Olympus CH2 Light Microscope (Olympus, Tokyo, Japan) with a wide angle (field size: 0.274 mm 2 , field diameter: 0.59 mm). The mitotic activity index was calculated as the total number of figures counted in 10 high-power fields of vision .
Reassessment of archived estrogen receptor, progesterone receptor, HER2/neu, and Ki-67-stained slides of the breast carcinoma cases
ER and PR were considered positive if 1% or more of tumor cell nuclei were immunoreactive according to the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guideline recommendations . HER2/neu immunoreactivity was evaluated according to the ASCO/CAP guideline recommendations ,. However, for statistical purposes, HER2/neu scores were categorized into two groups as negative (0 and 1+) or positive (3+) (>30% intense and complete staining). None of the cases studied had an HER2/neu 2+ score. The Ki-67 labeling index (Ki-67 LI) was determined using a semiquantitative visual approach. Scoring was performed in the areas with the highest number of positive nuclei (hot spot) within the invasive component of the tumor. The Ki-67 LI was expressed as the percentage of Ki-67-positive malignant cells among a total number of 1000 malignant cells at high-power magnification (×400) . We chose 14% as the optimal cut-off point of Ki-67 LI for our cases; thus, cases less than 14% were considered to have a low proliferative rate whereas cases with 14% or more were considered to have a high proliferative rate .
Immunohistochemical molecular subtyping
According to the St Gallen International Expert Consensus, the cases studied were classified into groups equivalent to molecular subtypes using the ER, PR, HER2/neu, and Ki-67 LI IHC results .
Immunohistochemical carbonic anhydrase IX procedure
A mouse monoclonal antibody Anti-Carbonic Anhydrase IX (Anti-CA IX) antibody (2D3) (Catalog #ab107257; Abcam Company, Cambridge, UK) was used. The optimal dilution was 1 : 200 using PBS. IHC staining was performed using the Universal Dako Cytomation Labelled Streptavidin-Biotin-2 System (Dako, Denmark A/S, Produktionsvej 42, DK-2600 Glostrup, Denmark), Horseradish Peroxidase (LSAB-2 System, HRP Kit, Catalog #k0679; Dako). All slides were deparaffinized using xylene and then rehydrated in decreasing concentrations of ethanol. Antigen retrieval was performed using microwave heating (20 min, 10 mmol/citrate buffer, pH 6.0) after inhibition of endogenous peroxidase activity (hydrogen peroxidase for 15 min). The primary antibody was applied to the slides and incubated overnight at room temperature in a humidity chamber. Sections were then washed by PBS and then incubated with the secondary antibody for 15 min, followed by PBS wash. Finally, the detection of the bound antibody was carried out using a modified labeled avidin-biotin reagent for 20 min and then PBS wash. A 0.1% solution of diaminobenzidine was used for 5 min as a chromogen. Slides were counterstained with Mayer's hematoxylin for 5-10 min. Positive control (human colon section) and negative control (the step of the primary antibody was omitted) were included in each staining run.
Assessment of carbonic anhydrase IX-immunostained slides
(1) CA IX-immunostained slides were evaluated in the following:
- Malignant epithelial tissues.
- Cancer-associated stroma.
- The adjacent nontumorous breast tissue.
(2) Evaluation includes the following:
- CA IX staining status; positive staining was considered as more than 1% of cells showing cytoplasmic and/or membranous cell staining ,.
- CA IX staining pattern, either cytoplasmic or membranocytoplasmic .
Data were collected, tabulated, and analyzed statistically using the statistical package for social science (SPSS, version 16; SPSS Inc., Chicago, Illinois, USA) program for windows. To test whether these variables differed according to clinicopathological parameters and biological markers, Fisher's exact and the Kruskal-Wallis test were used. All P values were two-sided. P values of less than 0.05 were considered statistically significant and highly significant when P value was less than 0.01.
| Results|| |
This retrospective study was carried out on 56 archival BC specimens representative of 56 BC patients.
The age of the patients studied ranged from 30 years to 81 years, with th X ± SD 47.85±11. The median age of the patients was 48 years. Thirty-two (57.1%) patients were 50 years or less and 24 (42.9%) cases were more than 50 years. All patients were women and all had undergone modified radical mastectomy (MRM) surgery. In the present study, 27 (48.2%) cases were premenopausal, whereas 29 (51.8%) cases were postmenopausal. The left breast was affected in 37 (66.1%) cases, whereas the right breast was the site of the tumor in 19 (33.9%) cases of total cases. Tumor size ranged from 0.5 to 11 cm, with th X ± SD 4.44 ± 2.57 and a median of 4 cm in the maximal dimension. Thirteen (23.2%) cases had a maximum tumor size of 2 cm or less, whereas 43 (76.8%) cases had a maximum tumor size of more than 2 cm.
Invasive duct carcinoma, no special type, represented 49 of 56 (87.5%) cases, whereas invasive lobular carcinoma was observed in three of 56 (5.4%) cases; the remaining four (7.1%) cases were medullary carcinomas. Thirty-five (62.5%) cases were of grade II, whereas grade III was observed in 21 (37.5%) cases. Three (5.4%) cases were of stage I, whereas 11 (19.6%) cases were of stage II. Thirty-seven (66.1%) cases were of stage III, whereas five (8.9%) cases were of stage IV. Forty-five of 56 (80.4%) cases were associated with the involvement of lymph nodes. Thirty-three (58.9%) cases were found to have poor NPI. Nineteen (33.9%) cases had moderate NPI. Four (7.1%) cases were found to have good NPI. Thirty-one (55.4%) cases were found to have a desmoplastic stromal reaction. Three (5.4%) cases had an inflammatory stromal reaction. Twenty-two (39.3%) cases were found to have both desmoplastic and inflammatory stromal reactions. However, 14 (25%) cases showed lymphovascular invasion. Thirty of 56 (53.6%) cases showed necrosis. The mitotic count ranged from 0 to 14, with th X ± SD 5.46 ± 4.44 and a median of 3.
The adjacent nontumorous breast tissues were detected in 31 of 56 (55.4%) BC cases. Twenty-two BC cases had adjacent normal breast tissues with unremarkable changes, three BC cases had adjacent fibrocystic disease, and six BC cases had adjacent duct carcinoma in situ.
Estrogen receptor, progesterone receptor, HER2/neu status, Ki-67 labeling index, and immunohistochemical molecular subtypes of breast carcinoma cases
Forty-three (76.8%) cases were ER positive, whereas 37 (66.1%) cases were PR positive. Sixteen (28.6%) cases were HER2/neu positive. Thirty-four (60.7%) cases were found to have a high proliferative index of Ki-67. IHC molecular subtyping of the studied BC cases showed that 22 (39.3%) were luminal A, 11 (16.9%) were luminal B, 16 (28.6%) were HER2/neu positive, and seven (12.5%) were triple negative.
Immunohistochemical profile of carbonic anhydrase IX
Adjacent nontumorous breast tissue
We observed a negative IHC expression of CA IX in normal breast tissues (22 cases) and fibrocystic disease (three cases) [Figure 1] Three of six (50%) duct carcinoma in situ cases showed positive IHC expression of CA IX [Figure 2] and [Figure 3]. Two of them showed moderate expression and the third one had strong expression.
|Figure 1: Infiltrating lobular carcinoma showed positive mild cytoplasmic carbonic anhydrase IX immunohistochemical (IHC) expression of tumor cells concentrically arranged around a negative immunoreactive normal duct (IHC, ×100) .|
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|Figure 2: High-power view of a case of duct carcinoma in situ, solid type, showed negative immunoreactivity of carbonic anhydrase IX (immunohistochemical, ×400 ).|
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|Figure 3: A case of duct carcinoma in situ, solid type, showed positive strong immunoreactivity of carbonic anhydrase IX (immunohistochemical, ×400 ).|
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Breast carcinoma cases
Fifty-one of 56 (91.1%) cases showed positive epithelial expression of CA IX, whereas five (8.9%) cases showed negative expression. In terms of the CA IX staining pattern, 44 (78.5%) showed cytoplasmic localization and seven (12.5%) showed membranocytoplasmic localization [Figure 4],[Figure 5],[Figure 6] and [Figure 7].
|Figure 4: Strong positive cytoplasmic carbonic anhydrase IX expression in grade II invasive duct carcinoma no otherwise specified (NOS) with negative infl ammatory stromal expression (immunohistochemical, ×40 0).|
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|Figure 5: Moderate positive cytoplasmic carbonic anhydrase IX expression in grade II invasive duct carcinoma, NOS with negative desmoplastic stromal expression (immunohistochemical, ×40 0).|
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|Figure 6: Moderate positive membranocytoplasmic carbonic anhydrase IX expression in grade II invasive duct carcinoma, NOS (immunohistochemical, ×4 00).|
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|Figure 7: Moderate positive cytoplasmic carbonic anhydrase IX expression in invasive lobular carcinoma with negative desmoplastic stromal expression (immunohistochemical, ×4 00).|
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All cancer-associated stroma showed negative CA IX expression.
The relationship of carbonic anhydrase IX status and the clinicodemographic and histopathological profiles of breast carcinoma cases
In terms of tumor stage, there was a significant difference (P = 0.03); the majority of positive cases had advanced stages as 70.6% were stage III and 5.9% were stage IV. Moreover, a statistically significant difference was observed in the stromal response (P = 0.002); 31 of 51 (60.8%) positive cases showed desmoplastic stroma, whereas none of the negative cases showed the same response [Table 1].
|Table 1: Relationship of carbonic anhydrase IX status and the clinicodemographic and the histopathological profiles of breast carcinoma cases|
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The relationship of carbonic anhydrase IX status and hormonal status, HER2/neu status, Ki-67 labeling index, and immunohistochemical molecular subtypes of breast carcinoma cases
There was no significant relationship between CA IX status and hormone receptor status, HER2/neu status, Ki-67 LI, or IHC molecular subtype of BC cases [Table 2].
|Table 2: Relationship of carbonic anhydrase IX status and hormonal status, HER2/neu status, Ki-67 labeling index, and immunohistochemical molecular typing of breast carcinoma cases|
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The relationship of the carbonic anhydrase IX staining pattern of positive 51 cases and the clinicodemographic and the histopathological profiles of breast carcinoma cases
There was a significant difference between CA IX cytoplasmic and membranocytoplasmic patterns of positive cases (P = 0.04) in terms of the high mitotic count in favor of a cytoplasmic pattern [Table 3].
|Table 3: Relationship of carbonic anhydrase IX staining pattern of positive 51 cases and the clinicodemographic and the histopathological profiles of breast carcinoma cases|
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The relationship of the carbonic anhydrase IX staining pattern of positive 51 cases with hormone receptor status, HER2/neu status, Ki-67 labeling index, and immunohistochemical molecular subtypes of breast carcinoma cases
There was no significant association between the CA IX staining pattern of positive cases and hormonal status, HER2/neu status, Ki-67 LI, and BC IHC molecular subtypes [Table 4].
|Table 4: Relationship of carbonic anhydrase IX staining pattern for positive cases and hormone receptor status, HER2/neu status, Ki-67 labeling index, and immunohistochemical molecular subtypes of breast carcinoma cases|
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| Discussion|| |
BC in Egypt shows poor prognostic features, such as advanced stage and higher grade than that in developed countries . The aim of many researchers is to identify other prognostic markers that would be helpful in the detection of BC severity to improve patient risk stratification and plan novel treatment modalities. The presence of hypoxic regions within tumors has long been reported to be associated with poor prognostic parameters .
In the current study, we have shown that the absence of IHC expression of CA IX in normal breast tissue and positive expression in both in-situ and invasive BC in a stepwise ladder manner could suggest its role in early carcinogenesis and tumor progression and as putative targets for therapy. Tumor acidosis, fueled by the activity of pHi-regulating mechanisms of cancer cells, aids their potential to spread. The involvement of low pHe in the progression cascade is considered to occur by one of the following means: (a) promoting basement membrane degradation in acidic regions of the tumor, allowing cancer cells to be free to proliferate into the surrounding tissues , (b) alteration of tumor size by abundance and intracellular distribution of lysosomes, thereby enhancing the secretion of extracellular matrix-degrading proteinases , (c) stimulation of secretion of cathepsin B, a protein that plays a significant role in the degradation of the components of extracellular matrix including the basement membrane , and (d) secretion of members of the matrix-metalloproteinase family and the urokinase-like plasminogen activator in acidic tumors .
In the current study, in terms of CA IX expression, 91.1% showed positive expression. Concordant results have been reported in most of the reviewed literature ,. These showed positive expression of CA IX more than negative expression. However, this was not in agreement with the results of Chia et al. , who showed predominance of negative expression of CA IX.
In the current study, 44 (78.5%) cases showed cytoplasmic localization, whereas seven (12.5%) cases showed membranocytoplasmic topography. Other series reported intramural heterogeneity of the CA IX level . In addition, CA IX showed cytoplasmic, nuclear, and membranous expression in various components of neuroblastoma . Subcellular IHC localization of CA IX was also reported recently in breast neoplasms . Different subcellular localization of CA IX suggests that CA IX is regulated by hypoxia and other mechanisms not related to hypoxia.
In terms of CA IX status and hormone receptor status, Her2/neu status, Ki-67 status, and molecular subtype of BC cases, no significant relationship was established. Variable results have been reported in other studies for these associations. For instance, multiple studies reported CA IX expression in ER-negative and PR-negative and Her2/neu-positive cases [33-35]. In contrast other studies reported the absence of a correlation between CA IX expression and HER2/neu positivity . However, a significant correlation with ER-negative and PR-negative tumors was noted . Kim et al.  reported that CA IX expression is associated significantly with triple-negative tumors. Moreover, a significant relationship was also reported between CA IX status and Ki-67 status ,,.
The majority of our CA IX-positive cases had advanced stages (70.6% stage III and 5.9% stage IV, respectively). Concordant results were reported . However, others reported that CA IX expression was not related to the stage of disease .
In this context, the hypoxia-responsive CA IX emerges as an important contributor to the regulation of cancer cell intracellular pH ,, with CA IX, in particular, being associated with a poor prognosis in BC ,,. Furthermore, hypoxic tumors were found to correlate with occurrence of metastasis: patients with hypoxic primary tumors developed more metastases than patients with less hypoxic tumors . CA IX plays a vital role in regulating intracellular and extracellular pH . In addition, the proteoglycan domain of CA IX has been linked previously to the negative regulation of cell adhesion through modulation of b-catenin and E-cadherin interactions , whereas the intracellular domain has been shown to contribute toward AKT activation .
In the current study, the stromal response (60.8%) of positive CA IX cases showed desmoplastic stroma, whereas none of the negative cases showed the same response. In contrast, others reported a lack of significant correlation between stromal response and CA IX expression in their study . Previous studies showed that hypoxia could induce mesenchymal epithelial transition in BC . Moreover, changes in intracellular and extracellular pH could be responsible for this kind of stromal reaction.
In the current study, the predominantly cytoplasmic pattern of CA IX was associated significantly with a higher mitotic count. To the best of our knowledge, this association between CA IX localization and mitosis has not yet been reported. Our results are consistent with other studies as CA IX is involved in tumor development and progression by regulating cellular activities, such as growth and survival . Moreover, other researchers have reported a significant relationship between CA IX status and Ki-67 status ,,.
| Conclusion|| |
Hypoxia (overexpression of CA IX) in BC of Egyptian patients is associated with poor prognostic parameters such as advanced stage and desmoplastic stromal response. The cytoplasmic pattern of CA IX expression in BC cases has been associated with mitotically active tumors. CA IX expression in BC has no relationship with hormonal status, HER2/neu, Ki-67 LI, and IHC subtyping. Further large-scale studies are warranted to investigate the new opportunity for CA IX epithelial-stromal targeted therapies in BC.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4]