Background The sentinel lymph node (SLN) is the first lymph node to receive the lymphatic drainage of a primary tumour; based on the knowledge that CK19 is positive in more than 95% of breast carcinomas, a molecular method for intraoperative diagnosis of SLN metastases (the one-step nucleic acid amplification (OSNA) assay) was developed.
Aims To evaluate CK19 immunoreactivity in a series of special histological types of breast carcinoma in order to verify whether the OSNA assay can be used in all breast cancer cases independently of the histological type.
Methods 116 samples of invasive breast carcinomas of special type were investigated for CK19 immunoreactivity in tissue microarrays (TMA); negative cases were evaluated in the entire tissue tumour tissue.
Results Of the 116 cases, 88.9% were positive CK19. Micropapillary and apocrine carcinomas were all positive for CK19 in TMAs. The tubular (93%), mucinous (86%), medullary typical and atypical (84%), mixed carcinomas (83%) increased the rate of positivity for this marker to 100% after repeating the immunostain in whole tissue of negative TMA cases, because the expression of those cases was focal.
Conclusion Most breast cancer cases were positive for CK19, independent of the histological type; therefore the OSNA assay can be used in all breast cancer cases with a potential low rate of false negative for CK19 detection of micrometastasis. There is an important variation between the positivity assessed on TMAs and the entire tissue; these findings can be clinically relevant because in some cases CK19 is evaluated on core-needle biopsy prior to surgery.
- CK 19
- breast cancer
- histological special types
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Breast cancer is a heterogeneous disease, comprising numerous entities that have different biological features and clinical behaviour.1–4 The majority (50–80%) of breast carcinomas are invasive ductal carcinoma not otherwise specified (IDC-NOS). This group of carcinomas comprises adenocarcinomas that fail to exhibit sufficient characteristics to warrant their classification into one of the special types.1–5 These ‘histological special types’ account for up to 25% of all breast carcinomas.4 The latest edition of the WHO breast carcinoma classification accepts at least 17 distinct ‘histological special types’.1–5
An important marker for breast carcinomas is CK19, which has been described as positive in 98.2% of breast adenocarcinomas.6 A group from Kobe has created a molecular method, one-step nucleic acid amplification (OSNA), for intraoperative diagnosis of lymph node (LN) metastases in breast cancer patients.7–9 The semiautomated OSNA assay consists of a short sample preparation step and real-time amplification of CK19 mRNA directly from the homogenate, without RNA purification steps. The time to perform the assay is 30–40 min for 3–4 LNs. This quantitative molecular assessment also allows assessment of metastasis size.7 8 This methodology may replace the traditional study of sentinel lymph nodes (SLN) using morphology alone. However, it is important to know whether the primary tumour is really positive for CK19 before the evaluation of SLN by OSNA assay in order to avoid false-negatives in the SLN examination.
The study showing a high rate of positivity for CK19 in breast adenocarcinomas (98.2%)6 evaluated only the medullary and the small cell carcinoma ‘histological special types’ of breast cancer. Tubular, mucinous, metaplastic, apocrine, micropapillary, encapsulated and invasive papillary and other breast carcinomas were not evaluated in the Chu et al study.6
The aim of this study was to evaluate CK19 immunoreactivity in the different histological types of breast carcinomas by tissue microarray (TMA), and in the entire tissue of negative TMA cases in order to compare the results before OSNA is used as a routine method.
Formalin-fixed, paraffin-embedded tissues of 116 invasive special type breast carcinomas were consecutively retrieved from the histopathology files of a private pathology laboratory in Campinas, São Paulo, Brazil. All cases were reviewed by three pathologists on H&E-stained sections. This study was conducted under the national regulative law for the usage of biological specimens from archives, where the samples are exclusively available for research purposes in the case of retrospective studies.
Representative areas of the invasive breast carcinomas were selected on the H&E-stained sections and marked on individual paraffin blocks. Two tissue cores were obtained from each specimen and deposited into a recipient paraffin block using a TMA workstation (Histopathology Ltd, Budapest, Hungary). Fourteen TMA blocks were constructed, each one containing 24 tissue cores, arranged in a 4×6 sector. After construction, 2 μm tissue sections were cut and adhered to glass slides for the immunohistochemical studies; the H&E-stained section from each TMA block was reviewed in order to confirm the presence of morphological representative areas of the original lesions.
Immunohistochemistry was performed using the streptavidin–biotin–peroxidase technique (LabVision, Fremont, CA, USA). Antigen retrieval was performed by proteolytic enzyme digestion (pepsin A, 4 g/l; Sigma-Aldrich, Taufkirchen, Germany) at 37°C for 30 min. After washes in a phosphate buffer solution, slides were submitted to endogenous peroxidase activity blockage by incubation in a commercial solution (Dako, Glostrup, Denmark). Sections were incubated with blocking serum (LabVision) for 15 min. Anti-CK19 monoclonal antibody (clone b170; Novocastra, Newcastle upon Tyne, UK) was applied at a dilution of 1:100 for 1 h at room temperature. After washes, the slides were incubated with biotinylated secondary antibody, followed by streptavidin-conjugated peroxidase (LabVision) and revealed with diaminobenzidine (Dako). The tissues were counterstained with Mayer's haematoxylin, dehydrated and coverslipped using a permanent mounting solution (Zymed, San Francisco, CA, USA). The evaluation of immunostainings was done by three pathologists (FS, CA and PP). Positive cytoplasmic staining for CK19 was considered if a tumour presented at least 10% positive cells, regardless of intensity of the reaction. The negative cases were selected to repeat the CK19 antibody in the whole paraffin-tissue block.
A total of 116 samples of invasive breast carcinomas of special type (lobular classic and pleomorphic, tubular, mucinous, micropapillary, encapsulated and invasive papillary, medullary typical and atypical, metaplastic and apocrine) were investigated for CK19 immunoreactivity in TMAs.
The results are summarised in table 1 and figures 1 and 2. Micropapillary and apocrine carcinomas were all positive for CK19 in TMAs. Tubular and mucinous carcinomas were almost totally positive for CK19 in TMA (93% and 86% of positivity) with a percentage of positivity that rose to 100% after the immunostain for CK19 in the whole tissue. Encapsulated and invasive papillary carcinomas were negative in only one case of TMA evaluation (85%) that remained negative after the whole tissue evaluation. Medullary carcinomas were negative by TMA examination in three cases. All these cases were focally positive when we evaluated the whole tissue (figure 2).
There were six cases of mixed carcinomas: two cases of ductal and micropapillary carcinomas, one case of tubulo-lobular, one case of ductal and tubular carcinoma, one case of ductal and lobular carcinoma, and one case of ductal, tubular and micropapillary carcinoma. On TMAs there was only one negative case for CK19, the ductal and micropapillary carcinoma, but this case also showed focal positivity when stained for the whole tissue.
In 10 cases of lobular carcinomas there were two negative cases for CK19 by TMA examination (both classic lobular carcinomas). These two negative cases did not show enough tumour material, so we could not evaluate these results. There were 6 cases of metaplastic carcinomas: 2 matrix-producing carcinomas, 2 squamous cell carcinomas, 1 adenosquamous cell carcinoma, and 1 spindle cell carcinoma. When the entire tissues of two negative cases by TMA (one squamous cell carcinoma and one spindle cell carcinoma) were examined, the squamous cell carcinoma became positive (focal), but the spindle cell carcinoma remained negative (figure 2).
In the 116 cases of special types of breast carcinomas we found 103 cases positive for CK19 by TMA examination (88.9%). Ninety-seven cases were positive with diffuse pattern for CK19, and 6 cases were positive with focal pattern (around 10% of cells stained). Metaplastic and lobular carcinomas were the subtypes with higher figures of negative cases on TMAs. When we evaluated the whole sections of those 13 negative cases, 9 cases became positive for CK19 (one or more foci of positive cells, in all cases with percentage of 10–20% of the tumour section); only the spindle cell carcinoma (metaplastic) and the papillary encapsulated carcinoma remained negative, with the percentage of positivity for CK19 in special histological types of breast cancer reaching 98.2%. The two cases of lobular carcinoma were not evaluated in whole tissue because of insufficient material.
The SLN is the first lymph node to receive the lymphatic drainage of a primary tumour, playing an important role in the evaluation of nodal status and providing important prognostic information for planning systemic therapy. Its evaluation provides a better locoregional disease control, being less aggressive than complete axillary lymph node dissection, with less morbidity and cost, although with little or no impact on survival.10–12
Molecular approaches such as real-time PCR have been applied for the detection of tumour deposits in SLN of breast cancer patients and have indicated higher sensitivity than histological investigations.9 13–15 Results obtained with RT-PCR correlated with traditional predictors of prognosis.9 14
CK19 mRNA is a suitable marker for identifying breast cancer deposits in SLN because virtually all breast cancers express this cytoskeleton protein.6 According to Lan Su et al,16 there is a high concordance between positivity for the mRNA and protein of CK19 in malignant breast epithelial cells. Recently, a new semi-automated molecular method for rapid intraoperative diagnosis of SLN metastases in breast cancer patients was developed using the OSNA method.8
This study is the first one, as far as we know, that evaluates CK19 positivity in the majority of different special types of breast carcinomas, because the previous published studies in the literature6 17–19 reported CK19 positivity in ductal, lobular, medullary and small cell carcinomas. The overall CK19 positivity in our study was the same as that reported by Chu et al for breast adenocarcinomas (invasive ductal carcinomas) (98.20%).6 We also studied only 15 selected cases of invasive ductal carcinomas by TMA (data not shown). Only one case was negative on TMA but it was positive in the whole tissue examination. We therefore found 100% positivity for CK19 immunostain in this small setting of invasive ductal carcinomas.
All micropapillary and apocrine carcinomas in our study were positive for CK19 on TMAs because the majority of them had a diffuse and strong staining; the results are likely to be the same in breast core-needle biopsy (CNB) of these subtypes of breast cancer. Tubular, mucinous, medullary and mixed (including ductal) carcinomas increased the rate of positivity for CK19 to 100% after repeating this immunostaining in whole tissue of negative TMA cases. Our results of positivity for this marker in medullary carcinomas were higher than those of Chu et al6 and Tot et al19 (100%×90.2% and 87%, respectively).
Lobular and metaplastic carcinomas presented the lowest positive for CK19 on TMA (78% and 67%, respectively), but after the examination of whole tissue of negative TMA cases, we found one more focal positive case of squamous cell carcinoma (metaplastic carcinoma) and the positivity of CK19 rose to 80% on metaplastic carcinoma. When we studied the entire tissue of two negative TMA cases of classic lobular carcinomas, there was not enough material, so we could not evaluate these cases.
The TMA technique is an efficient and economic method to study many different tumours at the same time, but because it does not evaluate the entire tumour section, it could be compared with breast CNB that shows just a small sample of the primary tumour. The finding that in most of the tumours negative for CK19 on the TMA evaluation, it was possible to demonstrate focal positivity in whole tissue, should highlight that CK19 evaluation on CNB of special types of breast carcinoma can result in false negative results. We found that almost all histological special types of breast invasive carcinomas showed the same results reported for IDC-NOS, with almost 100% of positivity for this marker when evaluated in whole tissue, with exception of metaplastic (spindle cell carcinoma) and papillary encapsulated carcinoma that showed lower rates of positivity for this marker (80% and 85%, respectively), because the positive tissue cases were focal or multifocal immunostain in the majority of the 11 studied cases.
In conclusion, most breast cancer cases were positive for CK19, independent of the histological type and therefore the OSNA assay can be used in all breast cancer cases with a potential low rate of false negatives for CK19 detection of micrometastasis. However, a word of caution should be added, because as in those cases with only focal positivity, the small metastases may not harbour the CK19 positive component.
Most of the histological special types of breast carcinomas were positive for CK19 and therefore one-step nucleic acid amplification (OSNA) can be used in all breast cancer cases, with a potential low rate of false negatives for CK19 detection of micrometastasis.
The positivity for CK19 assessed on tissue microarrays (TMAs) and the whole tissue session was discordant, because the positivity can be focal; these can produce false-negative results in core-needle biopsies.
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Funding Supported by Fundação de Apoio à Pesquisa do Estado de São Paulo (FAPESP, grant number 2010/00815-6), CAPES, Ministry of Education, Brazil (PhD grant 3420-09-04 to CAA) and Fundação para a Ciência e Tecnologia, Portugal (PIC/IC/83264/2007 to MG).
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.
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