Activated leukocyte cell adhesion molecule in breast cancer: prognostic indicator

The breast is the most frequent site of cancer in women (excluding skin cancer) and ranks second to lung cancer in the number of deaths in women [1]. It is generally accepted that important prognostic factors in breast cancer include the size of the primary breast tumor (included in tumor, node, metastasis [TNM] staging), the histologic type, the histologic grade, the presence or absence of regional/axillary lymph node metastases (included in the TNM staging system), the presence or absence of distant metastases (included in the TNM staging system), and the presence or absence of estrogen receptors and progesterone receptors (see review in[2]). Research for other prognostic factors is ongoing with the goal to predict aggressiveness and response to treatment.

Activated leukocyte cell adhesion molecule (ALCAM) is a glycoprotein of the immunoglobulin superfamily. ALCAM has been identified in multiple species and has different names depending on the species and laboratory that described it: chicken neural adhesion molecule BEN/SC-1/DM-GRASP [3‐5], rat KG-CAM [6], fish neurolin [7], human melanoma metastasis clone D (MEMD) [8], mouse/human CD166 [9, 10], and rat HB2 [11, 12]. ALCAM has five extracellular immunoglobulin domains (two NH₂-terminal, membrane-distal variable- [V]-type folds and three membrane-proximal constant- [C2]-type immunoglobulin folds), a transmembrane region, and a short cytoplasmic tail [13]. It is involved in both homotypic/homophilic adhesion and heterotypic/heterophilic (to CD6) adhesion [13, 14]. ALCAM has been shown to be involved in capillary tube formation [15] and in vessel invasion into cartilage in vitro [16].

ALCAM has been studied in two tumor types malignant melanoma [17] and prostate carcinoma [18]. In 1998 Degen and colleagues isolated MEMD in human melanoma cell lines and found a correlation with aggregation and enhanced metastatic ability [8]. MEMD/ALCAM was involved in melanoma cell–melanoma cell interactions, suggesting a homophilic interaction between tumor cells [8]. van Kempen and colleagues [17] studied human melanocytic lesions and found that ALCAM expression correlated with melanoma progression. Approximately one-half of melanoma metastases had ALCAM positivity. The authors concluded that ALCAM plays a role in melanoma tumor progression. Using chip-based RNA transcript analysis, Kristiansen and colleagues [18] studied ALCAM in prostate cancer and found that ALCAM expression is often altered in prostate carcinoma. They theorized that it may be involved in progression of disease.

Only one previous work has mentioned ALCAM in breast carcinoma. Degen and colleagues [8] performed northern blot analysis of MEMD mRNA and found expression in the MCF-7 mammary carcinoma cell line. Our laboratory is evaluating the role of ALCAM in breast carcinoma. Studies have shown that ALCAM is present in breast cancer cell lines and resected breast tumors [19]. Antibody to ALCAM decreases proliferation of breast cancer cells and decreases the adhesion of breast cancer cells to each other [19]. Proliferation and adhesion are both features that are critical to tumor cell growth and metastases. With the data on the other tumor types and data from our laboratory, we decided to evaluate ALCAM expression in breast carcinoma using tissue from patients who have been followed for 6 years and have been studied extensively [20‐25] to determine whether ALCAM expression is a prognostic indicator. Our results suggest that decreased ALCAM expression is of clinical significance in breast cancer.

This study is the first to focus on ALCAM expression and its prognostic value in breast carcinoma. Normal ductal epithelial cells in breast tissue showed positivity to ALCAM in immunohistochemical stains (Fig. 1). This positivity was also seen in invasive breast carcinoma, although the levels (both cytoplasmic and membranous) were lower than that of normal (non-neoplastic) mammary epithelium in the stained frozen tissue sections (Fig. 2). Immunohistochemical staining of breast tissues alone with the antibody used would therefore not distinguish between benign cells and malignant cells. The established pathologic criteria for carcinoma, including the architecture, nuclear pleomorphism, and mitotic counts, have to be evaluated.

The level of ALCAM transcripts appears to be higher in primary tumors compared with normal (non-neoplastic) breast tissue from the same mastectomy specimens, although it was not statistically significant (Fig. 3a). This was in contrast to the immunohistochemical staining, in that individual tumor cells stained weaker than individual normal mammary epithelial cells in the frozen tissue samples (Fig. 2a,2b). Possibilities for this discrepancy may rest with different epithelial/tumor cell content in different tumors and the possible contribution from infiltrating immune cells. Hematopoietic stem cells and activated T lymphocytes express ALCAM [31‐34]. The majority of leukocytes in the formalin-fixed, paraffin-embedded breast tissue sections do not exhibit immunohistochemical staining for ALCAM. Tissue histiocytes stain with antibody to ALCAM, as do some cells in lymphoid aggregates. One could also argue that non-neoplastic breast tissue in areas without tumor in these specimens is not truly 'normal'. Ductal and acinar structures in normal breast tissue obtained from breast reduction surgeries, however, also showed strong immunohistochemical staining for ALCAM. It therefore appears that ALCAM is present normally but may have an altered expression in tumors.

Infiltrating ductal carcinoma is the most common form of invasive breast carcinoma. The transcript number was evaluated in ductal and lobular carcinomas as well as in a group of other less frequent types of breast tumors (medullary, tubular, and mucinous) (Fig. 3b). Differences in transcript numbers were seen between the groups, with lobular carcinomas generally having less ALCAM transcripts than ductal carcinomas, whereas the number of 'other' cases included was too low to make an accurate assessment. The same staining pattern of other cell adhesion molecules (namely, E-cadherin) has also been reported [35‐37]. E-cadherin expression is lower in lobular breast carcinoma than in infiltrating ductal carcinoma [35]. E-cadherin is considered a tumor suppressor, with loss of E-cadherin correlating with invasion and metastases [38].

There was a statistically significant difference (P < 0.01) of ALCAM transcript levels between the Grade 2 tumors (moderately differentiated) and Grade 3 tumors (poorly differentiated) compared with Grade 1 tumors (well differentiated) (Fig. 4a). Higher grade tumors in general are less differentiated, more pleomorphic, and more mitotically active, and they exhibit a tendency for decreased ALCAM transcript numbers.

It is well known that the presence or absence of regional/axillary nodal metastases is an important prognostic parameter. ALCAM transcripts were decreased in patients with metastases to regional nodes, although the numbers are not statistically significant (P = 0.05) (Fig. 4c).

The TNM system for classifying tumors includes evaluation of the tumor, regional lymph nodes, and distant metastases. This system includes the primary tumor size and number of lymph nodes involved in the axilla. The higher the TNM classification, the worse the patient survival rate [2]. Our study showed that primary tumors from patients with TNM Stage 3 had significantly less (P < 0.01) ALCAM transcripts than those with TNM Stage 1 tumors (Fig. 4b).

Another clinical parameter of prognosis is the NPI [39], which combines tumor size, lymph node stage and histologic grade into a formula to determine prognostic groups. The group with NPI < 3.4 has the best prognosis, NPI > 5.4 is associated with poor prognosis, and NPI = 3.4–5.4 has an intermediate prognosis. The scoring system has been validated in prospective studies [40], and is used in countries such as the United Kingdom [41]. Interestingly, the ALCAM transcript number was lower in the worst NPI prognostic group (NPI > 5.4) when compared with the best prognostic group (NPI < 3.4) (P = 0.014) (Fig. 4d).

When patients were divided into categories of disease free, with metastases, with local recurrence, or death due to breast cancer, there was a significant difference in the ALCAM transcript number for those with local recurrence (P = 0.04) or those that died of breast cancer (P = 0.0041) compared with those that were disease free after excision of primary tumor (Fig. 5). Altogether, the present data strongly suggest that a lower ALCAM transcript number in the primary tumor is associated with a worse prognosis. This is confirmed in the Kaplan–Meier survival curve (Fig. 6), which shows that patients with low levels of ALCAM have more incidents of metastasis, recurrence, or death when compared with patients with high levels of ALCAM RNA in the primary tumor.

Only two types of cancer have been previously evaluated for ALCAM – melanoma and prostate cancer. With melanoma there was increased staining for ALCAM during the vertical growth phase of the tumor [17]. This would suggest that ALCAM is involved in tumor growth and progression. ALCAM expression was decreased in high-grade prostate carcinoma, suggesting that altered expression correlated with tumor grade [18]. Notably, in breast cancer there does appear to be distinct differences in ALCAM transcript levels in patients that correspond to prognosis. We hypothesize that altered expression of ALCAM may be related to tumor proliferation, adhesion, and metastatic capability. During the 6-year follow-up of the patients used in the present study, decreased ALCAM transcripts in the primary tumors correlated with nodal involvement, higher grade tumors, higher TNM stage, worse NPI index, presence of local recurrence, and death due to breast cancer. Clearly the data suggest that decreased ALCAM expression in the primary tumor is of clinical significance in breast cancer, and that reduced expression indicates a more aggressive phenotype and poor prognosis.

We hypothesize that the reduced expression of ALCAM, a molecule involved in cell adhesion, may allow the tumor cells to disaggregate, allowing cells to enter the circulation and metastasize, a phenomenon mirrored by other cell adhesion molecules such as E-cadherin [35‐38]. ALCAM is known to colocalize with E-cadherin in epithelial cells [42]. With E-cadherin, a number of mechanisms operate for its reduction in cancer cells, including shedding of the cadherin extracellular domain, redistribution from the cell surface to the cytoplasm or from cell–cell adhesion areas to other areas of the membrane, and transcriptional abnormalities as a result of gene promoter hypermethylation [38]. The current study has shown that both cytoplasmic staining and membranous staining of ALCAM are lower in cancer cells, strongly arguing the possibility of transcriptional aberration. It would be important to examine such events as promoter methylation in the future.

The present study suggests that although there is no significant difference between normal breast tissue and breast cancer in ALCAM transcript number (Fig. 3a), and immunohistochemistry alone does not distinguish between benign and malignant cells in the breast, PCR can be used to identify more aggressive tumors.

ALCAM is a glycoprotein that is involved with cellular adhesion, proliferation, and tumor progression. In breast carcinoma, decreased ALCAM transcripts in the primary tumors correlate with nodal involvement, higher grade tumors, higher TNM stage, worse NPI index, the presence of local recurrence, and death due to breast cancer. This study suggests that the level of ALCAM expression in primary breast carcinoma has clinical significance, and that reduced expression indicates a more aggressive phenotype and poor prognosis.