Relationship of CK8/18 expression pattern to breast cancer immunohistochemical subtyping in Egyptian patients

The immunohistochemical (IHC) subtyping of breast cancer can be a useful substitute for gene expression analysis. The aim of this study was to investigate the relationship of CK8/18 to the biology of breast carcinoma (BC) represented by its IHC subtypes. The IHC expression of CK8/18 was correlated with IHC subtypes of BC using ER, PR, HER2/neu, and Ki67 LI (with cutoff 14%). All cases showed CK 8/18 expression in tumour cells with varying degree of intensities; 49/70 cases (70%) showed diffuse cytoplasmic expression (loss of membranous pattern), while 21/70 cases (30%) showed membrano-cytoplasmic pattern. Adjacent non-neoplastic breast lobules showed membrano-cytoplasmic pattern in 58% of cases, which was significantly different from the pattern in invasive cancer (P = 0.002). A loss of membranous pattern in malignant tumours was significantly associated with higher tumour grade (P = 0.02), higher mitotic count (P = 0.03), and negative HER2/neu status (P = 0.04). CK 8/18 H score ranged between 1 and 290 with mean ± SD was 181 ± 70.54. Tumours with lower CK 8/18 H score were in the advanced stage group (P = 0.04). Low CK8/18 H score and loss of membranous pattern were significantly associated with triple negative (TN) subtype as compared with luminal subtype (P = 0.006 and P = 0.026, respectively). In addition, CK8/18 with lost membranous pattern was significantly associated with TN subtype compared with HER2/neu positive subtype (P = 0.001). However, there was no significant difference between luminal A and B subtypes regarding CK8/18 H score or pattern of expression. This study concluded that low CK8/18 H score and loss of membranous pattern of CK8/18 are associated with worse prognostic features and TN subtype.


IHC staining
IHC staining was performed using LAB-SA (labelled {strept} Avidin-Biotin) immunoenzymatic antigen detection system (Lab Vision/ Neo Markers, California, United States). Antigen retrieval was done by boiling in citrate buffer saline (pH, 6), followed by cooling at room temperature. The primary antibodies were incubated overnight at room temperature. For CK 8/18, mouse monoclonal antibody keratin 8/18 Ab-2 ready-to-use was used (clone K8.8+ DC10; like 5D3, Lab Vision, Neo Marker). For Ki67, primary rabbit polyclonal anti-Ki67 antibody, MIB1 clone, M7240 was used and diluted 1:300 (DakoCytomation, Copenhagen, Denmark). Positive control slides were prepared by staining skin carcinoma (for CK 8/18) and tonsil (for Ki67). For oestrogen receptor (ER), primary antibody against ER was used (clone 1D5; Dilution, 1:50) (DakoCytomation). For progesterone receptor (PR), primary antibody against PR was used (clone IA6; Dilution, 1:50) (DakoCytomation). For HER2/neu: primary antibody against HER2/neu was used (clone 250, Dilution, 1:100) (DakoCytomation). BC cases previously known to be positive for ER, PR, and HER2/neu were used as positive control slides. Negative control slides were also included in each run and were done by the replacement of primary antibody by antibody diluents. Secondary antibody was applied with diaminobenzidine as a chromogen substrate and Mayer's haematoxylin as a counter stain. www.ecancer.org ecancer 2014, 8:404 Immunostaining interpretation IHC staining of CK8/18 was evaluated in non-neoplastic and invasive cancer breast tissue concerning the pattern of expression either cytoplasmic, membranous or both [19]. H score was also calculated for CK8/18 using the intensity and percentage of positive cells [20]. The intensity score (0-3) was multiplied by the percentage of cells that stain with each level of intensity and the sum of these mathematical products was expressed as a score of 0-300. H score formula = strong intensity (3) × percentage + moderate intensity (2) × percentage + mild intensity (1) × percentage [20].
The Ki67 LI was determined using a semi-quantitative visual approach. Scoring was performed while blinded to patients' information and outcomes. The entire slide was scanned for immunostaining evaluation using light microscope at low-power magnification (×100). All tumour cell nuclei with homogenous granular staining, multiple speckled staining or nucleolar staining were regarded as positively stained, regardless of intensity, while any cytoplasmic immunoreactivity was considered non-specific and hence not taken into consideration. Scoring was performed in the areas with highest number of positive nuclei (hot spot) within the invasive component of the tumour. The Ki67 LI (tumour growth fraction) was expressed as the percentage of Ki67-positive malignant cells among a total number of 1000 malignant cells, at high-power magnification (×400) [21]. Using 14% as an optimal cutoff point, cases were classified into low and high proliferative groups [22]. Histological grade II was also stratified into low and high proliferative subgroups: GIIa and GIIb [21].
ER and PR were considered positive if ≥ 1% of tumour cell nuclei are immunoreactive [23]. HER2/neu immunoreactivity was evaluated according to the American Society of Clinical Oncology guideline recommendations [24]. Positive HER2/neu cases were defined as 3+ positivity (>30% intense and complete staining); however, score 0 or 1+ was considered negative and 2+ cases were excluded.

Statistical analysis
Data were statistically analysed using a personal computer with the Software Package for Statistical Analysis version 16 program; (USA). Chi-square test (χ² test) and Fisher's exacts test were used to compare qualitative variables. Student t-test and Mann-Whitney (U test) were used to compare quantitative variables. Tests were considered statistically significant when (P ≤ 0.05) and highly significant when (P < 0.01).

Results
The mean age of malignant cases was 49.97 ± 11.08, ranged between 25 and 81 years. The mean tumour size was 4.04 ± 1.95 ranged between 0.5 and 11 cm; 61/70 cases (87%) were invasive duct carcinoma of no special type (invasive carcinoma and no special type). The remaining cases were special types 9/70 (13%) including, four invasive lobular carcinoma, three medullary carcinoma, and two mixed tubular carcinomas.
According to Ki67 LI, histological grade II was further stratified into low and high proliferative subgroups: GIIa comprised 17 cases (41.5%) and GIIb 24 cases (58.5). There was no significant difference between both groups regarding CK 8/18 pattern of expression or H score (Tables 1 and 2).

Comparison between different subtypes of BC cases, clinico-pathological parameters and CK8/18 IHC expression ( Table 3 )
High CK8/18 H score and preserved membranous pattern were significantly associated with luminal group as compared to TN group (P = 0.006 and P = 0.026, respectively). Luminal A subtype was significantly associated with more differentiated tumours (P = 0.03) and lower mitotic count (P = 0.05) when compared with luminal B subtype. However, CK8/18 H score or pattern of expression did not show a significant difference between both luminal groups.
There was no significant difference between HER2/neu and TN groups regarding the clinico-pathological features, Ki67 LI or CK8/18 H score. However, preserved membranous pattern of CK8/18 was significantly associated with HER2/neu positive group when compared with TN group (P = 0.001). www.ecancer.org ecancer 2014, 8:404 Discussion IHC subtyping of BC provides valuable information for clinical decision making. However, it is highly dependent on training, skills, and experience of laboratory personnel performing it. In addition variability in outcomes still present among patients of a particular subtype according to the current calcification system [25]. Therefore, there is an increasing need for additional refinement of prognostic factors to improve patient risk stratification. Cytokeratins have long been considered as markers for identifying epithelial origin [9]. In this study, we prove that CK8/18 has a strong relationship to biology of BC represented in its IHC subtypes.
In this study, CK 8/18 was expressed in all BC cases with varying degrees of intensity and percentage. Lower CK 8/18 H score showed significant association with advanced stage (P = 0.04). Similarly, low CK 8/18 expression in breast cancer cell lines was associated with high metastatic potential [26], and loss of these keratins was associated with a significantly worse prognosis [27]. The more interesting finding is the pattern of CK8/18 expression; we identified two main patterns of staining: membrano-cytoplasmic (30% of cases) and cytoplasmic patterns (70% of cases). We suggest a significant relationship between CK 8/18 pattern of expression and tumour behaviour, which may be explained by the subcellular localisation and the biological functions of CK 8/18. We claim that preserved membranous localisation (membrano-cytoplasmic pattern), which was significantly more observed in non-neoplastic breast lobules, may reflect a degree of differentiation that maintain the tissue integrity and resist stresses externally applied to the cell [28].
In contrast, we observed that loss of membranous pattern of CK 8/18 (cytoplasmic only) was significantly associated with higher tumour grade (P = 0.05), higher mean mitotic count (P = 0.033), and high proliferative group (P = 0.012). CK 8/18 is important for cellular processes, such as cell signalling [29], mitosis [30], cell cycle progression [31], and protection from apoptosis [10,11]. Thus, it is involved in intracellular signalling pathways that lead to cell cycle progression which may explain the worse prognostic features associated with predominant cytoplasmic localisation with loss of membranous pattern of CK 8/18. Similarly, Cîmpean et al [19] observed three different distribution patterns of CK8/18 expression: diffuse cytoplasmic, membranous, and combined granular cytoplasmic with membranous but they did not correlate them to different clinico-pathological features.
One of the features common to BC is the increased rate of proliferation over that observed in normal breast epithelia [32]. It is now acknowledged that increased cell proliferation is a key determinant of poor outcome in patients with breast cancer [21,33,34].
As expected, the luminal subtype showed better clinico-pathological features when compared with HER2/neu and TN subtypes. We aimed to find out whether evaluation of CK8/18 is correlated to IHC classification of BC. CK8/18 has been called luminal marker as it indicates normal luminal epithelial-like differentiation [41]. CK 8/18 was expressed in all the studied BC cases including luminal and non-luminal subtypes. Our results indicate that the mere positivity of CK8/18 does not discriminate between luminal and non-luminal subtypes of BC. Therefore, we found that decreased CK8/18 H score and loss of membranous pattern was associated with TN subtype when compared with luminal and HER2/neu subtypes. These findings emphasise on the role of CK8/18 in the tumour biology of BC.
It has been suggested that luminal B subtype is equivalent to those that express either HER2/neu or Ki67 [35]. In this study luminal B was categorised as those showing Ki67 LI > 14% and all were found to be positive for HER2/neu coinciding with those called B2 subtype [22]. According to our results, the existence of B1 group, a subset of luminal B subtype, that shows negativity for HER2/neu in Egyptian population is questioned.
In this study, there was a statistical significant association between luminal B subtype and both higher tumour grade and higher mitotic count when compared with luminal A subtype. This coincides with poor prognosis associated with luminal B as compared with luminal A tumours [42][43][44]. However, there was no significant difference between luminal A and B subtypes with respect to CK8/18 expression. www.ecancer.org ecancer 2014, 8:404 There was no significant difference between the clinico-pathological features of HER2/neu and TN subtypes. In addition, there was no significant difference between both groups regarding Ki67 LI group. This agrees with previous reports that indicated that proliferation markers are of limited value in the TN and HER2/neu positive tumours as the majority of these tumours are poorly differentiated with a high proliferation index [45]. As regards to CK8/18 pattern of expression, loss of membranous pattern was significantly associated with TN group. This may further help in the differentiation between both groups since the treatment strategies differ.
The major limitation of this study is the small number of cases with available paraffin blocks suitable for recutting and immunostaining. This is because our hospital is a local centre with limited resources of archiving, documentation, and follow-up of cases. Moreover, in Egypt, we do not have a national wide program neither for breast cancer public awareness nor for screening. Therefore, cases are lately diagnosed and referred to the more equipped National Cancer Institute in Cairo. In spite of this limitation, our results indicate that the mere positivity of CK8/18 does not discriminate between luminal and non-luminal subtypes of BC; however, low CK8/18 H score and loss of membranous pattern of staining are associated with worse prognostic features and TN subtype. www.ecancer.org ecancer 2014, 8:404