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Aims To evaluate the clinicopathological associations and predictive value of the transcription factor NF-κB in a large series of breast cancer patients treated with neoadjuvant chemotherapy. Methods A retrospective search of a prospectively maintained database was performed to identify patients. Immunohistochemistry was used to assess the p65 subunit of NF-κB, using nuclear staining as a surrogate of activation. Results Nuclear NF-κB expression was found in 26.3% (35/133) of cases. Nuclear NF-κB staining was associated with high histological grade (p=0.05), oestrogen receptor (ER) negativity (p=0.01) and higher Ki67 index (p=0.002). Patients with nuclear NF-κB staining had a higher pathological complete response (pCR) rate than those without (26.5% vs 6.0% respectively, p=0.004); there was no significant association with clinical response or outcome. In an exploratory hypothesis-generating analysis, in the ER+/HER2− subgroup (n=43) a significantly lower clinical response rate was observed in those with nuclear NF-κB staining compared with those who had no nuclear NF-κB staining (14.3% vs 61.0%, p=0.038). There were no pCRs in ER+/ HER2− tumours. Conclusions Nuclear NF-κB expression is associated with ER negativity, higher Ki67 index and tumour grade. It was also found to be significantly associated with increased pCR but not clinical response to neoadjuvant chemotherapy.

To compare SP6 and MIB1 antibodies for Ki67 staining in breast cancer.Immunohistochemical detection of Ki67 has been widely used to assess the proliferative fraction in breast cancer. Ki67 is used prognostically and is the primary end-point for some presurgical trials. MIB1 has been the preferred antibody, but SP6 has become available, with apparently improved performance. The importance of Ki67 led us to systematically compare SP6 with MIB1.Two sets of tissue microarrays were used. These were constructed from formalin-fixed paraffin-embedded breast cancers: (i) 177 cancers with data on response to an aromatase inhibitor for advanced disease (cohort 1); (ii) 200 mainly oestrogen-receptor-positive cancers without response data (cohort 2). Twenty-eight pairs of core-cut biopsies taken before and after aromatase inhibitor treatment were also assessed (cohort 3). Stained sections were examined either visually or by using an image analysis system (Ariol).There was a strong correlation between the two antibodies in all cohorts of samples scored visually (cohort 1: n=161, r=0.93, p0.0001; cohort 2: n=194, r=0.84, p0.0001; cohort 3: n=54, r=0.89, p0.0001). Correlation between visual and Ariol scores was markedly better with the SP6 antibody (r=0.71 and r=0.88 for MIB1 and SP6, respectively). Ki67 related similarly with time-to-treatment failure with the two antibodies (cohort 1). Changes in Ki67 values with the two antibodies after 2 weeks of aromatase inhibitor treatment also correlated strongly.SP6 and MIB1 provide highly comparable measures of Ki67 that predict progression of advanced disease similarly. SP6 is substantially better suited than MIB1 to image analysis.

AIM--To validate the use of a new mouse monoclonal antibody (1D5) directed against the N-terminal domain (A/B region) of the oestrogen receptor in an immunohistochemical assay (ER-IHA) for paraffin wax embedded tissue. METHODS--Breast cancer specimens were surgically obtained from 119 previously untreated patients. For comparison, oestrogen receptor was measured from cytosol fractions using an established oestrogen receptor enzyme immunoassay (ER-EIA) method. Oestrogen receptor "H-scores" were obtained from the ER-IHA after antigen retrieval using microwave treatment. Where discrepancies occurred between the two methods, further immunohistochemistry was performed using the H222 antibody from the Abbott Laboratories ER-ICA kit. RESULTS--The correlation between the two methods was non- linear, but despite this there was an 86% concordance between ER-EIA and ER-IHA using the 1D5 ER antibody. Fifty four per cent of tumours (64/119) were oestrogen receptor positive and 32% (38/119) were negative by both assays. A mismatch between the ER-EIA and ER-IHA occurred in 17 cases. Seven tumours were IHA positive but EIA+, but five of these were borderline negative by EIA, having values of > 5 and < 10 fmol/mg protein. Ten tumours were IHA negative and EIA+; four of these tumours were completely negative by IHA in the section studied. A further IHA assay, carried out on the 17 tumour mismatches with H222 antibody, showed that three tumours remained substantially discordant. These three tumours were strongly positive with the 1D5 antibody and negative with the H222 antibody. Two of these discordant tumours were of the rare ER negative and PgR positive phenotype and may contain oestrogen receptor that is of biological interest but which lacks the hormone binding epitope. CONCLUSIONS--The concordance between the classic enzyme immunoassay technique and the new immunohistochemical method on paraffin wax embedded sections was good. Moreover, the IHA technique using the 1D5 antibody against the N-terminal was easily reproducible. This technique may allow oestrogen receptor content to be determined in large cohorts of patients in whom archival tumour material is available.