Zagouri et al. BMC Cancer 2010, 10:409 http://www.biomedcentral.com/1471-2407/10/409 downregulation of Hsp90, in respect to both the level of Hsp90 intensity and Allred score [14]. According to the most recent WHO classification, LN includes the designations atypical lobular hyperplasia (ALH) and lobular carcinoma in situ (LCIS) and refers to the entire spectrum of atypical epithelial proliferation ori- ginating in the terminal ductlobular unit, with or with- out involvement of ducts [15]. Nowadays, it is widely known that LN represents a risk factor and a non-obli- gatory precursor for the subsequent development of invasive carcinoma in either breast, of either ductal or lobular type [16]. This study aims to go beyond LN, assessing Hsp90 expression in infiltrative lobular carcinomas (ILC). The examination of ILC lesions may subsequently prove to have significant implications for the viability of Hsp90 inhibitors in breast lobular lesions. Methods This study involved formalin-fixed, paraffin-embedded tissue specimens from 32 patients with ILC. The patients’ age at operation ranged between 35 and 74 (median age: 53 years); information regarding the patients’ clinicopathological features was also retrieved. The diagnosis of ILC was established by vacuum- assisted breast biopsy, excisional breast biopsy, lumpect- omy and modified radical mastectomy. Cases of ILC coexisting with atypical ductal hyperplasia, ductal carci- noma in situ, or invasive ductal carcinoma were excluded. Hsp90 was immunohistochemically detected using the mouse monoclonal antibody Hsp90 (clone JPB24, NCL- Hsp90, Novocastra supplied by Menarini). The dilution was 1:500 and the incubation time was 18 h (at 4°C). The visualization was performed using the Dako Envi- sion kit. Antigen retrieval was achieved in 0.01 M citrate buffer (pH = 6.0) at 85°C for 15 min. Immunohisto- chemical assessment of Hsp90 was performed both in the lesion and the adjacent normal breast ducts and lobules, the latter serving as control. Negative controls were assessed by omitting the primary antibody. Concerning Hsp90 assessment: i) the percentage of positive cells and ii) the intensity were separately ana- lyzed. Subsequently, the Allred score was appropriately calculated (Table 1) [17,18]. For immunohistochemistry (IHC), the following anti- bodies were used: PgR (636, Dako), ER (ID5, Dako) and c-erbB-2 (CB11, Novocastra™). Sections (4 μm thick) cut from formalin-fixed paraffin embedded tis- sue were used. After deparaffinization in xylene and hydration in graded ethanol solutions, the sections of breast carcinoma tissue were subjected to pretreatment in order to enhance antigen retrieval. The EnVision + System-HRP (DAB) (DakoCytomation, Glostrup, Page 2 of 6 Danemark) was used with primary antibodies against the following antigens: PgR, ER and c-erbB-2. Immu- nohistochemistry was performed according to the pro- tocols provided by the manufacturer. Concerning the immunohistochemical expression of ER and PR, both the intensity (negative, 1+ to 3+) and percentage of immunopositive cells were evaluated. Subsequently, the Allred score was calculated [16]. The expression of c-erbB-2 was assessed as follows: i) negative, when no staining was documented or when membrane staining was present in less than 10% of tumor cells, ii) weak staining (+), when partial mem- brane staining was documented in more than 10% of tumor cells, iii) moderate staining (++) when weak/mod- erate complete membrane staining was present in more than 10% of tumor cells and iv) strong staining when strong, complete membrane staining was observed in more than 10% of tumor cells. Cases with negative and weak c-erbB2 staining were considered as negative, whereas cases with strong c-erbB2 staining were consid- ered as positive. In cases with moderate staining, CISH was performed; subsequently these cases were consid- ered as negative or positive. In all cases, ten fields (×40 magnification) were assessed and a minimum of 100 cells were evaluated in the designated areas, so as to appraise the lesion as a whole. The immunohistochemical evaluation was per- formed independently by two consultant histopatholo- gists (AN and AL). Kappa statistic was performed to assess inter-rater agreement at both the estimation of Hsp90 percentage and intensity; for the optimal interpretation of the result, it should be kept in mind that pathologists rated Hsp90 percentage at an increment of 5% i.e., possible values are: 60%, 65%, 70% and so forth. The intensity was treated as an ordinal variable-score (0: negative, low: 1, moderate: 2, high: 3). Regarding Allred score and Ki-67 percentage, all numbers are pro- vided as mean ± SD. Two main analyses were per- formed: i. comparison of Hsp90 Allred score between the lesion and the adjacent normal ducts and lobules and ii. evaluation of the associations between Hsp90 Allred score and ER positivity, progesterone receptor (PR) positivity, c-erbB2 status and Ki-67 (%). Due to deviation from the normal distribution, non-parametric statistics were chosen. The statistic performed in each case is mentioned in parentheses in the text. Where appropriate, power calculations are presented. Statistical analysis was performed with STATA 8.0 statistical soft- ware (Stata Corporation, College Station, TX, USA). Informed consent was obtained from all participants in this study. This study has been approved by the local Ethics Committee, in accordance with the Helsinki Declaration.