Material and Method: In this study we assessed 66 paraffin blocks of breast cancers previously diagnosed as triple negative subtype in Mehr hospital (Tehran, Iran) by tissue microarray and immunohistochemistry technique for expression of mammaglobin and GCDFP-15.

Results: GCDFP-15 was positive in 12 cases (18.2%) and the other 54 (81.8%) cases remained negative. Mammaglobin was positive only in 6 cases (9.1%) and the remaining 60 (90.9%) cases were negative.

Conclusion: According to recent studies and our findings, there is no useful immunohistochemical marker for detection of breast source in cases of metastatic triple negative breast cancer with unknown origin and we must try hard to discover more accurate immunohistochemical markers for these highly metastatic breast cancers.

Recently the subtypes of breast cancers recognized by their gene signature include luminal (type A and B, and type C), HER2/neu type, basal-like, and normal breast-like. Attempts have been made to use immunohistochemistry (such as a panel including antibodies to estrogen receptor (ER), progesterone receptor (PR), HER2/neu, cytokeratin 5/6, epidermal growth factor receptor (EGFR) and Ki- 67 to assign tumors to various molecular subtypes^3,4. The “gold standard” to determine molecular invasive breast carcinoma (IBC) subtypes is gene expression array analysis, but this method requires fresh tissue. Surrogate immunohistochemical profiles correlating to the molecular subtypes have been developed⁵.

Triple negative breast cancer (TNBC) is a subgroup of breast cancers that has been defined as negativity for ER, PR and Her2/neu markers by immunohistochemistry (IHC). It is also divided into 2 subgroups based on the expression of basal markers. These subgroups that are named basal-like and non-basal-like are positive and negative respectively for myoepithelial/basal markers such as cytokeratin 5/6, smooth muscle actin and EGFR⁶.

A shorter survival course and higher metastatic rate are the characteristics of this type of breast cancer⁷. Reliable markers should be found to identify this type of tumor especially in metastatic cases whose origins are unknown.

The basal-like subtype is characterized by the expression of myoepithelial / basal markers and molecular changes including TP53 gene mutations, BRCA1 inactivation, and many chromosomal alterations^8-10.

Gross cystic disease fluid protein 15 (GCDFP-15) was identified by Haagensen et al in 1977¹¹. GCDFP is a 15-kDa monomer found in chromosome 7 and cells with apocrine differentiation express it¹². Normal breast ducts and lobules do not represent GCDFP, although apocrine metaplastic epithelium expresses it. An immunohistochemical study performed by Wick and colleagues in 1996, GCDFP-15 had a sensitivity and specificity up to 74% and 95%, respectively, in primary mammary carcinoma¹³. In another study by Fiel MI et al, GCDFP-15 was found to be positive in up to 57% of primary as well as metastatic breast carcinomas¹⁴. Additionally Bhargava et al reported GCDFP-15 positivity in just about 25% of breast carcinomas¹⁵.

Gene sequence of mammaglobin (MGB) was first discovered in breast carcinoma tissue in 1994 by Watson and Fleming¹⁶. In 1996, they found complete DNA clone of MGB¹⁷. In their study, MGB was positive in 50% of primary breast carcinomas and 62% of metastatic breast carcinomas. In the study of Han et al, sensitivity of mammaglobin antibody for the detection of breast cancer was 84.3% when compared with GCDFP-15 (44.3%) in a series of 70 breast cancer cases¹⁸.This marker is also imparted in benign breast epithelium. In this study we assessed paraffin blocks of breast cancers previously diagnosed as basal-like TNBC in Mehr Hospital (Tehran, Iran) using IHC technique for the expression of mammaglobin and GCDFP-15 markers.

At the time of writing this article only study of Lewis et al accessed sensitivity of mammaglobin and GCDFP-15 for detecting basal-like TNBC’s that were 24% and 5%, respectively¹⁹. In attention to this fact that breast carcinoma is one of the most common metastatic cancers in women and TNBC’s are highly metastatic, we want to assess sensitivity of GCDFP-15 and mammaglobin markers for diagnosis of these cancers. As in the work up of metastatic tumors with unknown origin when we consider a breast origin “unlikely” these two markers are immunohistochemically negative, can we do the same interpretation in TNBC’s relying on these two markers?

Tissue microarray: Tissue microarray (TMA) (Tissue arrayer Minicore, Alphelys, Plaisir, France) was made of 66 paraffin blocks of TNBC’s. Three 1.4 mm tissue cores were obtained from tissue blocks on each case for increasing confidence coefficient. Each TMA contained 66 tissue cores, each 1.4 mm in diameter. These were arranged as 7 rows and 10 columns with 2 positive and negative controls beside them.

Immunohistochemical Analysis for Mammaglobin and GCDFP-15: Immunohistochemical studies for GCDFP and mammaglobin were performed on TMAs. Fourmicrometer- thick formalin-fixed, paraffin-embedded sections were immunostained with mammaglobin and GCDFP-15 antibodies (FLEX Monoclonal Mouse Anti- Human Mammaglobin Clone 3041A5, FLEX Monoclonal Mouse Anti-Human Gross Cystic Disease Fluid Protein-15, Clone 23A3, Ready-to-Use, DAKO, Glostrup, Denmark). The procedure was composed of pretreatment tissues using EnVision FLEX, Target Retrieval Solution, PH 8.0 followed by incubation with mammaglobin and GCDFP-15 mouse antibodies. Counterstaining in hematoxylin was used by EnVision FLEX Hematoxylin.

Positive and negative controls were run simultaneously using the same protocol as the patient specimens. For meaningful semiquantitative analysis, focal and/or weak staining was considered negative staining, and only patchy or diffuse staining with moderate or strong intensity was considered positive¹⁵.

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Table I: Demographics of cases

Mammaglobin marker was positive only in 6 cases (9.1%) and the rest 60 cases (90.9%) were negative (Figure 1A,B). Immunohistochemically, GCDFP-15 marker was positive in 12 cases (18.2%) and the other 54 (81.8%) remained negative (Figure 2A,B).

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Figure 1: Basal-like triple negative breast carcinoma (A, H&E, ×400) showing strong staining with mammaglobin (B, ×400).

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Figure 2: Basal-like triple negative breast carcinoma (A, H&E, ×400) showing strong staining with GCDFP-15 (B, ×400).

Interestingly, mammaglobin is positive in few melanomas, and it is important to recognize this pitfall, for, it is unlikely to cause a problem in differential diagnosis from a breast carcinoma because a panel of immunohistochemical stains is always used in this matter. Mammaglobin does not seem to be a useful stain to distinguish breast from sweat gland carcinomas. Positive mammaglobin staining seen in salivary gland tumors was also expected; however, intense staining was not seen in any of the positive salivary gland tumors¹⁵.

In Gloria H. Lewis’ study, the sensitivity of mammaglobin and GCDFP-15 IHC markers for detecting basal-like TNBCs has been 24% and 5%, respectively. In our study, 9.1% and 18.2% of basal-like TNBC cases showed posistive immunohistochemical reaction for mammaglobin and GCDFP-15. Unlike Lewis et al’s study, GCDFP-15 immunostaining was more frequent than mammaglobin immunostaining in our study.

Therefore, the false negative results of these markers for detecting TNBC’s rated as 90.9% and 81.8% respectively. Hence, during pathological workup of a metastatic tumor with unknown origin by using these markers, the possibility of a TNBC entity will presumably missed. Since diagnosing a TNBC is a crucial factor in choosing a systemic treatment, postponing it can lead to a significant tumor progression.

TNBCs are highly aggressive with more intention to metastasize and shorter survival period. Our study revealed that 2 of the best IHC markers, used to detect the breast origin, have very low sensitivity. As these tumors metastasize to distant organs there is no useful IHC marker to detect the origin of the tumor. Therefore, it will be important for future studies to focus on finding accurate IHC markers for detection of this type of breast cancer and communicate closely with clinician and radiologist to correlate with clinical findings.

ACKNOWLEDGMENTS
We are grateful to pathology technicians of Tehran University of Medical Sciences and Mehr hospital for their assistance in processing and staining of the samples, Dr. Elham Mogharrablou and specially Beheshte Roudiany for her great assistance in the grammar of this article. The study was funded by Tehran University of Medical sciences, Tehran, Iran.

The authors declare that there is no conflict of interests.

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