Major challenges in cancer diagnostics relate to early detection of malignant tissue and the possibility to predict the rate of disease progression and sensitivity to chemotherapy. Regarding all these issues the Y-box protein-1 may gain a prominent role, given that (i) it has an oncogenic property with induction of breast tumors in 100% of transgenic animals overexpressing YB-1 in the mammary gland , (ii) analyses of dysplasia-associated lesions in colitis ulcerosa patients reveal that lesions with increased cancer risk are distinguishable due to their YB-1 expression pattern , (iii) several reports on breast cancer indicate that YB-1 expression levels are strongly predictive for relapse rates and negatively correlate with disease free survival [6, 34, 36, 47–50], and (iv) YB-1 upregulated P-glycoprotein expression. To date, however, no clear-cut standard for the assessment of YB-1 expression levels is available. In most immunohistochemical analyses overall expression levels have been scored in cancer tissues. Janz et al. even included the YB-1 expression within the adjacent tissue for analyses, which profoundly improved the predictive value on relapse-free survival .
One of the major obstacles towards a unifying scoring system in cancer tissue is the lack of a common expression pattern observed with diverse antibodies directed against YB-1. Most antibodies provide a highly specific staining pattern that is confined to the cytoplasm with complete absence within the nuclear compartment, as also reported in the most recent and largest study performed hitherto . On the other hand, most - if not even all - activities of YB-1, that relate to oncogenic transformation, cell proliferation and drug resistance, must take place within the nuclear compartment. These are accompanied by altered gene transcription as well as chromosomal instability [18, 20, 23]. Thus, it is conceivable that a sensitive detection system and valid predictive testing must include such information, which is also reiterated by the observation of nuclear YB-1 being associated with P-glycoprotein expression and drug resistance [8, 11]. It becomes even more complex when one envisions that YB-1 is a quite abundant cytoplasmic protein and has been detected in the nucleus in non-transformed cells as well, when tissue is fixed in Carnoys fixative . The question is therefore, how these inconsistencies may be reconciled. One answer may be that YB-1 in cancer tissue has a different antigenicity due to post-translational modifications, e.g. phosphorylation of serine 102 . In line with this interpretation is the observation of nuclear as well as cytoplasmic YB-1 in most cells using immunoblotting and senescence of cells with YB-1 knock-out [18, 51]. Alternatively, cleavage of YB-1 within the protein C-terminus, as proposed before via the 20S proteasome [52, 53], may render epitopes of YB-1 accessible for antibodies and result in positive immunodetection. Sorokin et al. have nicely demonstrated that the cleaved N-terminal fragment itself may shuttle to the nucleus and predict chemoresistance, whereas the overall full-length YB-1 expression level was unchanged in chemotherapy resistant versus chemosensitive cells. Attempts to explain the hidden secret beyond the different detection patterns observed already hint at the difficulties that the design and establishment of a monoclonal antibody to detect YB-1 faces.
In this report, we describe the generation of a monoclonal YB-1 antibody, termed F-E2G5, which detects YB-1 in formaldehyde-fixed paraffin-embedded tissues. Specificity of YB-1 detection by this antibody was confirmed by immunoblotting with YB-1-GFP fusion protein and ELISA. One detected epitope must reside within the central domains encompassing amino acids 21-147, a second one within domains aa146-172. Notably, using this antibody endogenous YB-1 protein is not detected by immunoblotting, likely due to its "misfolding" under the given conditions. Likely, that the GFP-tag within the protein C-terminus stabilizes the recognized epitope(s) for detection by western blotting. A non-specific band detected under some immunoblotting conditions does not seem to be of relevance for the performed immunohistochemistry, given that tissue specimens from healthy breast tissue do not demonstrate immunopositivity with Mab F-E2G5 and correlation results indicate that the outcome correlates with a nuclear staining pattern.
We have used this antibody to analyze YB-1 expression in a large and clinically well characterized collection of breast cancer specimens  that has previously been successfully used to define novel breast tumor markers . Unlike Abba et al.  who described an absence of YB-1 mRNA expression in DCIS compared to normal tissues, we found an increased cytoplasmic concentration of YB-1 protein in six of eight analyzed DCIS compared to normal tissue. In our study, nuclear YB-1 expression was restricted to invasive ductal carcinomas. Conversely, tissue sections representing DCIS or normal breast tissue did not show nuclear expression of YB-1. Concordantly with these observations, nuclear expression of YB-1 in invasive breast cancer was significantly associated with overall survival (p = 0.0046). In our cohort this correlation was more predictive as observed by Janz et al. , who found a less tight association between YB-1-linked tumor aggressiveness and poor clinical outcome (p = 0.011).
So far we are the first group that describes a correlation between nuclear YB-1 expression and increased tumor grading (p = 0.011) and tumor stage (p = 0.004) in breast cancer. Although this does not necessarily reflect a causal relationship, it emphasizes the role of YB-1 as predictive marker. In non-small lung cancer and ovarian cancer and by applying polyclonal antibodies, other groups described an apparent link between YB-1 positivity in the nucleus and tumor staging. However, they did not find any correlation between YB-1 positivity and tumor grade in these entities [3, 55, 56].
A further finding of our study is the highly significant correlation between nuclear YB-1 expression and negativity of the progesterone receptor status (p = 0.002), which has not been described so far. Since YB-1 has been directly linked to intrinsic or acquired resistance to chemotherapy and thus might be responsible for failure of current treatment regimens, these findings implicate an interesting opportunity for the selection of chemotherapy resistant breast cancer patients out of the whole cohort of patients. ER-positive/PR-negative breast cancers are known to respond less well to selective ER modulator (SERM) therapy than ER-positive/PR-positive tumors . Therefore YB-1 expressing breast cancer may have a general tendency to be hormone refractory, their relative neoplastic potential thus requiring chemotherapy, even if the tumors are of small size, low grade or estrogen receptor positive.
Our data are not in line with two reports, where YB-1 is more commonly found in ER negative breast tumors [6, 26]. One may explain this with differences in antibody detection sensitivity, altered epitopes of YB-1 in the nuclear compartment, with the latter being the more likely reason.
In our stratified univariate analysis the prognostic value of nuclear YB-1 expression became even more pronounced in the clinically important subgroup of stage pT1/T2 tumors and breast tumors with negative progesterone receptor status, respectively. Routine mammography screening for breast cancer in women aged 51-70 is becoming a standard in the European Community. Thus, the number and frequency of small sized stage pT1/T2 breast tumors detected is steadily increasing. A YB-1 immunohistochemistry-based diagnostic test could help to identify young patients with small tumors but significant risk of relapse. We are confident that this novel monoclonal YB-1 antibody is of great value in performing prospective studies to establish YB-1 as a tumor marker in the management of human breast cancer. For this purpose large confirmative meta-analyses on independent breast cancer cohorts will be necessary. Therefore we would like to encourage other academic groups to validate our data on their own tumor collections.