In our knowledge this is the first report that employs an anti MUC1 cytoplasmic tail Ab in HNSCC; it revealed a high cellular expression of MUC1 (80% of malignant samples).
In previous studies developed in colon and breast cancer [13, 14], we have found similar results. The MUC1 cytoplasmic tail amino acid sequence contains seven tyrosine residues constituting intracellular signaling motifs  which play important roles in cell response to external stimuli and is related to different oncogenes and genes controlling the cell cycle [23, 24][25, 26] and β-catenin . Furthermore, it has been reported that in vivo, MUC1 cytoplasmic tail regulates erbB receptors signaling through the activation of MAPK pathways .
These observations clearly demonstrate the transforming properties of MUC1 overexpression in carcinoma and emphasize the importance of MUC1 cytoplasmic tail subcellular localization. In HNSCC we have found extensive expression and cellular localization of MUC1 cytoplasmic tail; large and poorly differentiated tumors were likely to be less reactive with CT33 and C595 Abs.
When C595 (anti-extracellular MUC1) monoclonal antibody was assayed, a lower percentage of reactivity with respect to anti-MUC1 cytoplasmic tail was found; we have previously reported a high reactivity to MUC1 protein core epitopes in HNSCC [7, 8]. The difference among results was expected since previous reports included a larger number of tumors localized in oral cavity (58%) in comparison with 49.1% in the present report; we found that this localization is reactive with anti-protein core monoclonal antibodies . On the other hand, results presented here corresponded to 18.9% of pharyngeal tumors which did not usually react with these monoclonal antibodies; in past reports, only one patient with a pharyngeal tumor was included . MUC1 cytoplasmic tail reactivity is not affected by MUC1 glycosylation and sialylation and, consequently, represents a better indicator of cell membrane associated MUC1 . Other authors [29–31] have described MUC1 RNA splice variants which do not express the protein core region. Our results also showed that serum MUC1 levels were elevated in 15% of HNSCC samples; interestingly, we found a statistically significant correlation between serum and tumor MUC1 detection indicating that this mucin may possibly be released by the tumor.
In this research we found anti-MUC1 antibodies; also, we observed that serum MUC1 levels were positively correlated with free anti-MUC1 IgG, which may indicate that an anti-MUC1 immune response is mounted. Tumor derived MUC1 is known to induce cellular and humoral specific immune responses in cancer patients [32–34]. In this context, it is possible to speculate that a specific anti-MUC1 immune response is induced in HNSCC patients.
In another report , we pointed out that low MUC1 serum levels in stage I breast cancer patients were associated with the presence of free and complexed anti-MUC1 antibodies. Similarly, circulating anti-MUC1-IgG antibody levels were found predictive of survival in breast  and pancreatic cancer patients . We have found that anti-MUC1 IgG Ab correlated negatively with poor HNSCC differentiation and disease stage which are the main predictors for patient outcome in this localization.
Furthermore, we proved that MUC1-CIC detected by Western blot were of the IgG isotype since isolation was performed by protein A Sepharose CL-4B chromatography, which is known to bind with high affinity to 1, 2 and 4 IgG classes.
Presence of high amounts of circulating immune complexes in cancer patients is not very well understood. Antibody formation may represent the onset of an anti tumor immune response but, on the other hand, may be the consequence of tumor immune evasion related to a defective cellular immune response. Nonetheless, circulating immune complexes have been associated with tumor progression and also, their formation clearly affects the correct evaluation of several tumor markers.
Only a few reports in the literature have described the antigenic component of circulating immune complexes in HNSCC. Vlock DR and others  have exhaustively studied the reactivity of serum Abs against autologous tumor cell lines. These authors found that some patients presented high Ab titers which occasionally cross reacted with tumor cell lines derived from other histogenesis.
In accordance with other reports [37, 38], we found higher circulating immune complexes levels in HNSCC patients in comparison with normal subjects; furthermore, we also agree with these authors that elevated circulating immune complexes levels were detected in advanced tumor stage. They pointed out that high circulating immune complexes levels in HNSCC patients is the result of increased tumor mass which would mediate changes in anti tumor immunity. Moreover, Das TK et al  found that circulating immune complexes persist after surgical excision of the primary tumor due to the presence of remaining tumor tissue or occult metastasis.
MUC1 is a mucin that has been detected both in the cytoplasm, plasma membrane and nucleus in different tumor localizations. MUC1 is synthesized in the ER and glycosylated in the Golgi apparatus but it has been described that it suffers several glycosylation cycles being expressed in the plasma membrane several times . Furthermore, different splicing variants have been described that can be present in the cytoplasm and in the plasma membrane, as well [31, 41–43]. Wen et al  investigated intracellular trafficking of MUC1 cytoplasmic tail in human pancreatic cancer cell lines S2-013 and Panc-1 and detected this fraction at the inner cell surface, in the cytosol and in the nucleus. They hypothesized that the association between β-catenin and fragments of the MUC1 cytoplasmic tail facilitated the cytosol-to-nuclear translocation of β-catenin and contributed to its nuclear accumulation. We have found  MUC1 cytoplasmic tail and protein core expression in the plasma membrane, cytoplasm and nucleus in breast and colorectal tumor samples.