NOVEL IMMUNOHISTOCHEMICAL MARKERS FOR THE DIFFERENTIATION OF LOBULAR AND DUCTAL INVASIVE BREAST CARCINOMAS

AIMS
Invasive ductal and lobular carcinomas are the most common histological types of breast cancer. The loss of E-cadherin expression has been suggested to be the most reliable marker for invasive lobular carcinoma. The aim of our study was to identify the diagnostic usefulness of novel markers in the differentiation of these tumor types.


METHODS
We examined tissue microarrays (TMA) which were constructed from surgical specimens of 119 breast cancer patients. TMA consisted of 80 ductal carcinomas, 29 lobular carcinomas and special type cancers. TMA sections were stained using standard immunohistochemical methods. Monoclonal mouse antibodies against E-cadherin, cytokeratin 5/6 and 17, and polyclonal mouse antibodies against EMP1, DDR1, PRKCI and DVL1 were used.


RESULTS
E-cadherin was absent in 93.3% of lobular tumors compared with only 15 % of ductal tumors (p<0.0001). EMP1 and DVL1 were overexpressed in lobular tumors (93.1% and 96.5%, respectively), whereas PRKCI and DDR1 were positive in ductal cancers (90% and 96.2%, respectively). Reduced expression or absence of both cytokeratins 5/6 and 17 was found in both tumor tissues in comparison to normal terminal duct lobular units (p<0.0001).


CONCLUSIONS
Apart from the well-established marker, E-cadherin, proteins examined on TMA slides by immunohistochemistry (EMP1, DVL1, DDR1, PRKCI) may represent novel tissue markers helpful in the differentiation of ductal and lobular breast cancers. Further studies with larger sets of patients are desirable, to verify the complete immunohistochemical profiles of various histological types of breast cancer and determine the prognostic and predictive significance of novel markers.


BACKGROUND
Invasive ductal (IDC) and lobular carcinomas (ILC) are the most common histological types of breast cancer, accounting for 80 % and 15 % of all malignant breast tumors, respectively.Microscopically, ductal carcinomas tend to form glandular structures, whereas lobular tumors are less cohesive and invade in single fi les 23,33 .Both tumor types derive from the breast terminal duct lobular units (TDLUs) 28 .The terminology "ductal" and "lobular" is still being used for historical reasons and to date there is no evidence to suggest that these tumors arise from ductal or lobular epithelial cells.Thus the diff erences in their morphology are likely to refl ect the diff erences in mechanisms of carcinogenesis rather than the anatomical origin of the lesions.
IDC and ILC are similar in many respects 1,8 .However, clinical follow-up data and the patterns of metastasis suggest that their development and progression are diff erent 26,31 .The treatment for stage-matched tumors is similar 18 but lobular carcinomas are often resistant to neoadjuvant therapy 21 .Although patients with ILCs are older, have low grade tumors and less lymphatic vascular invasion, they have no survival advantage compared with IDCs 16 .It is well documented that inactivating mutations and loss of heterozygosity of the CDH1 (E-cadherin gene), mapped to chromosome 16q22.1,are very frequent in ILC 2,24 , followed by the loss of protein expression 12,15 .E-cadherin is a calcium-dependent, epithelial-specifi c cell-cell adhesion molecule 10 .Simultaneous loss of E-cadherin and associated proteins such as the α-, β-and γ-catenins is an important step in the formation of lobular in situ carcinoma, a precursor of ILC 20 .Abnor mal cytop las mic and nuc le ar lo ca li za ti on of p120, a member of the E-cadherin/catenin adhesion complex, occurs in the early sta ges of ILC and is ma in ta i ned du ring tumor prog res si on and me tas ta sis.Most lo bu lar carcinomas sho w cytop las mic lo ca li za ti on of p120 which is strongly asso ci a ted with com ple te loss of E-cadherin and β-ca te nin.In duc tal tumors, in con trast, reduc ti on of p120 and E-cadherin in the mem bra ne is common, whereas cytop las mic p120 sta i ning is ra rely seen 25 .Maspin is a recently described member of the serpin family of protease inhibitors known to be a tumor suppressor gene product.It is more frequently detected in IDC than in ILC (36.4 % versus 7.1 %)(ref. 14).The expression of NKX3.1, a member of the NK-class of homeodomain, is also found primarily in ILC 9 .To date, however, E-cadherin is the only well-established immunohistochemical marker for the diff erentiation of ductal and lobular breast carcinomas.

METHODS
To assess the diagnostic usefulness of novel markers identifi ed by microarray analysis, we examined tissue microarrays (TMA) which were constructed from surgical specimens of 119 breast cancer patients aged >35 years and contained 278 cores of 2.0 mm diameter.They consisted of 80 ductal carcinomas, 29 lobular carcinomas, one tubular carcinoma, 3 medullary carcinomas, 2 tubular-lobular carcinomas, 2 mixed ductal-lobular carcinomas, one mucinous and one papillary carcinoma.Invasive ductal no special type (NST) tumors and invasive lobular carcinomas were graded using the Nottingham combined histologic grading system.The construction of the tissue microarrays was done using a tissue arrayer (Beecher Instruments, Inc., Sun Prairie, WI, USA)(ref. 27).
Paraffi n sections from 22 additional breast cancer samples containing normal mammary gland structures were used for comparison.

RESULTS
Of 80 invasive ductal breast carcinomas, 7.5 % were grade 1, 71.3 % were grade 2 and 21.2 % were grade 3. Of 29 invasive lobular carcinomas, 51.7 % were grade 1, 37.9 % were grade 2 and 10.3 % were grade 3. Lymph node status was classifi ed as N 0 in 70 % of invasive ductal carcinomas and 72.4 % of invasive lobular carcinomas, N 1 in 20 % of invasive ductal carcinomas and 20.7 % of invasive lobular carcinomas, N 2 in 10 % of invasive ductal carcinomas and 6.9 % in invasive lobular carcinomas.According to routine immunohistochemical data, 71.3 % of invasive ductal carcinomas were positive for estrogen receptors (ER), and 62.5 % were positive for progesterone receptors (PR), whereas 65.5 % of lobular carcinomas were ER-positive, and 58.6 % were PR-positive.Immunohistochemical protein overexpression and subsequent amplifi cation of c-erbB-2 (HER-2/neu) gene by fl uorescent in situ hybridization (FISH) was detected in 15 % of invasive ductal carcinomas and 3.4 % of invasive lobular carcinomas.The clinical and histopathological characteristics of these patients are shown in Table 1.E-cadherin successfully distinguished invasive ductal and lobular carcinomas.It was absent in 93.3 % of lobular tumors compared with only 15 % of ductal tumors (p < 0.0001).EMP1, PRKCI, DDR1 and DVL1 were also diff erentially expressed between ductal and lobular carcinomas.More precisely, 93.1 % of lobular tumors were positive for EMP1 compared to only 16.3 % of ductal tumors (p < 0.001).Ductal carcinomas were positive for PRKCI in 90 % of cases compared with only 13.8 % of lobular carcinoma (p < 0.001).DDR1 was positive in 96.2 % of ductal carcinomas compared to only 13.8 % of lobular carcinoma (p < 0.001) (Fig. 1).Also, 96.5 % of lobular tumors were positive for DVL1 compared with only 25 % of ductal tumors (p < 0.001) (Tab.2).Thus, the diagnostic sensitivity and specifi city of E-cadherin for distinguishing IDC and ILC were 85 % and 93 %, respectively.The sensitivity and specifi city were 87 % and 93 % for EMP1, 75 % and 97 % for DVL1, 96 % and 86 % for DDR1, and 90 % and 86 % for PRKCI (Fig. 2).
The reduced expression or absence of both cytokeratins 5/6 and 17 was found in both tumor tissues in comparison to terminal duct lobular units in 22 normal mammary tissues (p < 0.0001) (Fig. 3).In a majority of ducts and lobules including TDLU, these cytokeratins were expressed in both basal and luminal cells.The expression of these cytokeratins was similar in IDC and ILC.Cytokeratin 5/6 Novel immunohistochemical markers for the diff erentiation of lobular and ductal invasive breast carcinomas was positive in 11.2 % of invasive ductal carcinomas and 10.3 % of invasive lobular carcinomas, and cytokeratin 17 was expressed in 10 % and 6.9 %, respectively.
Of the special type carcinomas included on TMA slides, a papillary and two medullary carcinomas were positive for cytokeratin 5/6, one out of three medullary carcinomas was positive for EMP1 and E-cadherin, and a ductal-lobular carcinoma was positive for DDR1.All other special type carcinomas were negative for these markers, and fi nally none were positive for either PRKCI or DVL1 (data not shown).

DISCUSSION
We studied the expression of novel immunohistochemical markers in 119 surgical samples from patients with lobular and ductal invasive breast carcinomas.The results of immunostaining suggest that two tumor types can be successfully distinguished at the protein level.Most importantly, E-cadherin was negative in 92.3 % of lobular carcinomas.Its diagnostic sensitivity and specifi city for distinguishing IDC and ILC were 85 % and 93 %, respectively.The gene encoding E-cadherin protein acts as a tumor suppressor, inhibiting invasion and metastasis.Its mutations correlate with gastric, breast, colorectal, thyroid and ovarian cancer.During tumor progression, E-cadherin can be functionally inactivated or silenced by diff erent mechanisms such as post-translational control, somatic mutations, downregulation of gene expression through promoter hypermethylation, histone deacetylation, and, importantly, transcriptional repression 11,22 .The latter induces cellular responses leading to the conversion of epithelial cells into invasive mesenchymal-like cells with increased motility and invasiveness.This process is called an epithelial-mesenchymal transition (EMT)(ref. 22).The loss of E-cadherin expression explains the morphological characteristics of invasive lobular carcinomas such as Indian fi le arrangement of tumor cells.
In addition to E-cadherin, other proteins involved in cell adhesion and proliferation are diff erentially expressed between ductal and lobular carcinomas.PRKCI is a calcium-independent, phospholipid-dependent, serine-and threonine-specifi c enzyme which may play a role in the secretory response to nutrients.It is involved in cell polarization processes and the formation of epithelial tight junctions 4 .PRKCI was overexpressed in our invasive ductal carcinomas, and its diagnostic sensitivity and specificity for distinguishing IDC and ILC were 90 % and 86 %, respectively.EMP1, a tumor-associated membrane protein involved in cell-cell interactions and control of cell proliferation 7,35 , was overexpressed in lobular carcinomas, and its diagnostic sensitivity and specifi city for distinguishing IDC and ILC were 87 % and 93 %, respectively.
DDR1 protein was overexpressed in ductal tumors and this agrees with the literature 5 .The diagnostic sensitivity and specifi city of DDR1 for distinguishing IDC and ILC were 87 % and 93 %, respectively.Expression of this receptor tyrosine kinase is restricted to epithelial cells and determines the regulation of cell growth, diff erentiation and metabolism 6 .In addition, G-protein signaling may mediate the eff ect of Wnt5a expression by enabling collagen-induced activation of DDR1 6 , and binding DDR1 to Wnt-5 regulates the adhesion of mammary cells [5][6]13 . The nt proteins constitute a large family of secreted signaling molecules that are implicated in the early mesodermal patterning of the embryo, in regulation of mammary gland ductal branching and alveolar morphogenesis during pregnancy, also in tumorigenesis 17,29 .Wnt signal transduction starts via binding to cell surface Frizzled receptors.Further signaling depends on β-catenin activation (canonical pathway) or involves intracellular molecules other than β-catenin (non-canonical pathway)(ref.34 ). It is ell documented that DVL1 is an essential mediator of both canonical and non-canonical Wnt pathways 34 .Amplifi cation and increased expression of the DVL1 gene has previously been associated with breast carcinogenesis 19 .However, histological subtypes have not been specifi ed.Of Wnt molecules, DVL1 was overexpressed in our invasive lobular carcinomas, and its diagnostic sensitivity and specifi city for distinguishing IDC and ILC were 75 % and 97 %, respectively.
In a majority of ducts and lobules including TDLU in normal mammary tissues, cytokeratins 5/6 and 17 were expressed in both basal and luminal cells, confi rming the previously described variability of expression of these basal cytokeratins and their relationship to cellular origin 30 .Cytokeratin 5 and 17 have also been found in a subset of breast cancer patients with a poor clinical outcome 32 .In our set of patients, the expression of these basal cytokeratins was associated with higher tumor grade and HER-2/ neu-negativity (p < 0.001).No other immunohistochemical markers correlated with clinical or histopathological markers (p > 0.05).

CONCLUSION
Taken together, our results suggest that besides the well-established marker E-cadherin, proteins examined on TMA slides by immunohistochemistry (EMP1, DVL1, DDR1, PRKCI) may represent novel tissue markers helpful in the diff erentiation of ductal and lobular breast cancers.Further studies with larger sets of patients are desirable to verify the complete immunohistochemical profi les of various histological types of breast cancer and determine prognostic and predictive signifi cance of novel markers.

Fig. 2 .
Fig. 2. Diagnostic sensitivity and specifi city of immunohistochemical markers for distinguishing invasive ductal and lobular breast carcinomas.

Table 1 .
Clinical and histopathological characteristics of TMA cases.

Table 2 .
Results of immunohistochemical staining.