The study included 103 patients aged 26–88 years (median 59 years) with IDC of the breast, in association with concomitant DCIS diagnosed between 1996 and 2000 at the Bristol Royal Infirmary, Bristol, UK (Table 1). Eligibility criteria were histological diagnosis of IDC and DCIS, no distant metastases, and unilateral tumour. Regional Ethics Committee approval was granted before commencement of the study. Axillary lymphadenopathy was evaluable in 90 patients: 37 (41%) of the patients were lymph node negative, and 53 (59%) were node positive (N1 [mobile ipsilateral] or N2 [fixed ipsilateral]). No axillary surgery was undertaken in the remaining 13 patients because of age-related co-morbidity.

Clinicopathological subgroups were analysed in accordance with the Nottingham Prognostic Index (NPI) and divided into good (GPG), moderate (MPG) and poor (PPG) prognostic groups as described, with a modification that included no assessment of the internal mammary lymph nodes 40. Evaluation of the NPI was precluded in 15 patients because of non-evaluable regional lymphadenopathy and tumour size. Similarly, subgroups of DCIS were analysed in accordance with the Van Nuys Pathologic Classification (Table 1), which was not assessable in six patients 41. The design of the study to include representative samples of synchronous IDC and DCIS precluded analysis by the Van Nuys Prognostic Index 41. Adjuvant treatment groups comprised tamoxifen in 60 patients (27 GPG, 16 MPG, 4 PPG and 13 no NPI), and cyclophosphamide–methotrexate–5-fluorouracil-containing and anthracycline-containing regimes in 17 and 21 patients, respectively (4 GPG, 19 MPG, 13 PPG and 2 no NPI). Five patients received no adjuvant treatment. The median follow-up duration was 51 months (range 6–120 months). All were primary tumours with the exception of six local tumour recurrences, which were excluded from the analysis of patient outcome (see Table 4 and Fig. 3).

Tumour samples were collected shortly after surgery and were fixed in buffered formalin for 24–48 hours at room temperature (20–22°C). Tumours were classified in accordance with the guidelines of the UK National Health Service Breast Screening Programme 42 and were graded by the modified Bloom's grading system described by Elston and Ellis 43. Oestrogen receptor (ER) immunostaining was performed with a standard three-layered streptavidin–avidin–biotin horseradish peroxidase (HRP) method with a mouse anti-human ER primary antibody (M0747, 1:100 dilution; Dako, Ely, UK) and a biotinylated rabbit anti-mouse secondary antibody (E0354 1:350 dilution; Dako). Expression of ER was assessed with Quick-score (0–8) and classified as positive (4–8; more than 3) or negative (0–3; 3 or less) in five high-power fields 44.

Tumour proliferation was assessed with nuclear Ki67 immunostaining (polyclonal rabbit anti-human Ki67 antigen; A0047, 1:100 dilution; Dako). A goat anti-rabbit biotin-labelled polypeptide (E432, 1:400 dilution; Dako) was used as a secondary antibody. Tonsillar tissue was used as a positive control and primary antibody was replaced with Tris-buffered saline (TBS) as a negative control. Ki67 staining was evaluated as positive or negative, with low proliferation indicative of less than 10% of positive-staining cells, compared with high proliferation with at least 10% positivity 45. HER-2 immunostaining was performed with the mouse monoclonal anti-HER-2 antibody (RTU-CB11; NovaCastra/Vector, Newcastle upon Tyne, UK) and the Envision Plus HRP system (K4006; Dako). HER-2 expression was scored according to the degree and proportion of membrane staining, with a score of 0 or 1+ defined as negative, and 2+ or 3+ as HER-2 positive 46. Lymphovascular invasion was assessed as present or not, and was analysed together with ER, HER-2 and Ki67 in the Department of Pathology (by CS and CJC).

p14^ARF immunoreactivity was evaluated with the mouse monoclonal antibody 4C6 (kindly provided by Dr Gordon Peters, Cancer Research UK, Lincoln's Inn Fields, London, UK) at a 1:200 dilution of 15 μg/ml. ARF immunostaining of IDC and DCIS was compared with formalin-fixed paraffin-embedded cell buttons, using H1299 non-small cell lung cancer cells as a positive control for p14^ARF, and MCF-7 breast cancer cells as a negative control. Further negative controls replaced 4C6 with sero-matched IgG2a used on previously established p14^ARF-expressing breast cancers. The 4C6 antibody with epitope recognition of carboxy-terminal p14^ARF was chosen after testing of other mouse monoclonal antibodies including C-18 (Santa Cruz Biotechnology, California) and 14PO2 (LabVision/Neomarkers, Fremont, California). The specificity of 4C6 has been demonstrated previously 1327.

Hdm2 expression was assessed with the mouse monoclonal antibody OP46 (Oncogene Research, CN Biosciences, Nottingham, UK) that detects C-terminal Hdm2 with specificity for the 90 kDa isoform 47. OP46 was selected after testing with other antibodies including 2A10 and 3G5 (kindly given by AJ Levine, Rockefeller University, New York) both of which had technical or other limitations 48. OP46 was used at a 1:80 dilution of 100 μg/ml and evaluated in the context of Hdm2 overexpressing A375 malignant melanoma cells, and negatively controlled with IgG2b, which was substituted for the primary antibody. Biotinylated rabbit anti-mouse IgG secondary antibody (E0354, 1:300 dilution of 1.26 mg/ml; Dako), together with the Strept-AB Complex/HRP (0377; Dako, Glostrup, Denmark), was used for p14^ARF and Hdm2. Technical limitations precluded dual staining for ARF and Hdm2. p53 immunostaining was performed as described 49 by using the DO7 monoclonal antibody (NovaCastra/Vector). Breast carcinomas known to overexpress p53 with known TP53 gene mutations and protein accumulation were used as positive controls. Negative controls were obtained by omitting the primary antibodies.

Formalin-fixed paraffin sections of breast cancer tissue and cell pellets (controls) were mounted on 3-aminopropyl-triethoxysilane-coated (Sigma, Poole, Dorset, UK) glass slides and were baked for 30 min at 56–60°C before being dewaxed in Clearene (Surgipath Europe, Peterborough, UK). The tissue was rehydrated by sequential immersion in 100% and 50% ethanol to distilled water. Tissue sections were subjected to heat retrieval of antigens in citrate buffer (pH 6) in a pressure cooker for 3 min; after cooling they were incubated in 0.3% (v/v) hydrogen peroxide for 5 min. Subsequently, sections were washed in tapwater and TBS (pH 7.45). Before incubation with p14^ARF and Hdm2 primary antibodies, sections were exposed to avidin and biotin blocking solutions (Vector Laboratories, Burlingame, California) for 15 min each. Further blocking was achieved through exposure to normal rabbit serum (diluted with TBS) for 30 min at room temperature. Primary antibody was applied and incubated at 4°C overnight (18 hours) for p14^ARF and Hdm2, except for p53, which was incubated for 1 hour at room temperature. After washing with TBS, biotinylated rabbit anti-mouse secondary antibody was applied for 30 min at room temperature. The Strept-AB Complex/HRP was used for the detection of p14^ARF and Hdm2, and involved an additional 30 min incubation with TBS/biotinylated streptavidin/HRP. Staining was revealed by development in the chromogen 3,3-diaminobenzidine tetrahydrochloride for 5–10 min before counterstaining with haematoxylin before mounting.

Immunostaining was assessed with a Zeiss Axioskop microscope with a 40 × Achrostig mat lens (×400 overall magnification) and a field diameter of 0.46 mm. In the neoplastic cell population for IDC and DCIS, the degree of staining intensity and the proportion of cells with p14^ARF, Hdm2 and p53 immunoreactivity in the nucleus and cytoplasm were individually graded semiquantitatively to produce an intensity distribution score or H-score for each localisation, with invasive and pre-invasive components given separate scores 50. Initial scoring was of 10 high-power fields; however, in view of the homogeneous staining this was reduced to 5 high-power fields. A modified Quick-score (MQS) was used to define a score of 0–3 for intensity (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) and 0–5 for distribution (0, no staining; 1, 1% or less; 2, 1–10%; 3, 11–33%; 4, 34–66%; 5, 67–100% of cells staining), giving an overall score of 0–8 44. Sections were scored independently by two pathologists (CS and CJC) in the Department of Pathology. Unless otherwise stated, scores were assessed as a continuum for the purposes of statistical correlation. For the purposes of describing p14^ARF, Hdm2 and p53 immunoreactivity, tumour positivity was defined as a modified Quick-score of 3 or more (1+/2+) (see Table 2).

Data were analysed with the SPSS 10.0 for Windows (SPSS Incorporated, Chicago, Illinois) statistics software and summarised with descriptive statistics. The associations between p14^ARF, p53 and Hdm2 and patient characteristics were assessed with the Spearman non-parametric test for continuous variables and the χ² test and Fisher's exact test for categorical factors. The relationship between p14^ARF localisation and the subcellular localisation of p53 and Hdm2 was analysed with the Spearman correlation coefficient (cc) for non-parametric data, with the p14^ARF and p53/Hdm2 MQS used as continuous variables. Analyses of survival data were performed with the log-rank test and the Cox regression model, with survival curves computed with the Kaplan–Meier method. For p14^ARF, p53 and Hdm2, univariate and multivariate analyses were performed and adjusted for the NPI score and treatment received (tamoxifen/chemotherapy/none). Because the NPI is based on nodal involvement, on tumour size and on grade, patients (n = 11) with non-evaluable lymphadenopathy and tumour size were excluded from the multivariate regression analyses (see Table 4).

p14^ARF immunoreactivity was evaluable in the nucleus and cytoplasm in 96 (93%) cases of IDC and in associated DCIS in 91 (88%) cases (Fig. 1 and Table 2). ARF expression (MQS ≥ 3) was detectable in 76 (79%) IDCs and was predominantly nuclear, with 23 breast cancers (24%) demonstrating cytoplasmic p14^ARF. Nuclear ARF was homogeneous, with no distinct pattern of nucleolar staining or exclusive nucleolar p14^ARF expression 303435.

Predominant nuclear ARF might have been 'masking' the accurate delineation of nucleolar p14^ARF. Weak/moderate nuclear ARF expression (MQS 3–5) was evident in 31 (32%) cases, with half of these showing cytoplasmic ARF (n = 16). A loss of p14^ARF nuclear immunostaining was observed in 20 (21%) invasive breast cancers, whereas the remaining 45 (47%) cases exhibited diffuse and strong nuclear staining (MQS 6–8; Table 2), with a similar intensity of cytoplasmic ARF in 7 cases only. A comparison showed that the levels of p14^ARF expression in DCIS were similar to invasive disease (Table 2).

p53 immunoreactivity and subcellular localisation were evaluable in 93 (90%) cases of IDC and in DCIS in 91 (88%) cases. Immunohistochemically, p53 was detectable in 31 (33%) IDCs (MQS ≥ 3), with similar levels of nuclear (31 cases) and cytoplasmic (23 cases) expression (25%), compared with p14^ARF (Table 2). Weak to moderate (MQS 3–5) nuclear p53 expression was observed in 17 cases of IDC (18%), with similar cytoplasmic staining (18 cases). There was no nuclear or cytoplasmic p53 expression (MQS 0–2) in most IDCs (67% and 75%, respectively). Strong nuclear p53 expression (MQS 6–8) was evident in 15% of IDCs, with 5% of these expressing intense cytoplasmic staining. A comparison of the levels of p53 expression and subcellular localisation in DCIS were similar to those in IDC (Table 2).

Sixty-six percent (n = 46) of nuclear p14^ARF-expressing breast cancers (1+/2+) were associated with absent p53 nuclear immunoreactivity, or the possibility of wild-type p53 (Table 3). By comparison, proportionately fewer or one-third of ARF-expressing tumours (n = 24) had nuclear p53 expression (1+/2+). These associations did not reach statistical significance. Nuclear p14^ARF expression (1+/2+) showed no particular pattern of p53 subcellular immunoreactivity (data not shown).

Hdm2 expression and subcellular distribution were evaluable in 92 (89%) cases of IDC and in 91 cases of DCIS (Fig. 1 and Table 2). Nuclear Hdm2 was detectable (MQS ≥ 3) in 43 (47%) IDCs with a similar proportion (49 cases) of cytoplasmic staining (Table 2). A minority of IDCs strongly expressed nuclear and cytoplasmic Hdm2 (MQS 6–8) in 10 (11%) and 16 (18%) tumours, respectively. About half of invasive cancers were negative for nuclear (53%) and cytoplasmic (46%) Hdm2. A similar pattern for all levels of Hdm2 expression was seen in DCIS.

Proportional numbers of nuclear p14^ARF-expressing breast cancers (1+/2+) either lacked nuclear Hdm2 expression (0) (49%, n = 35), as opposed to 51% (n = 37) expressing nuclear Hdm2 (1+/2+) (Table 3). Nuclear p14^ARF showed no significant associations with nuclear Hdm2 expression (Fig. 2a). Surprisingly, the minority of cytoplasmic ARF-expressing breast cancers showed a significant association between increasing levels (MQS) of cytoplasmic p14^ARF and nuclear Hdm2 expression (cc 0.475, P < 0.001) (Fig. 2c), as well as cytoplasmic Hdm2 expression (cc 0.461, P < 0.001) (Fig. 2d). Cytoplasmic ARF showed no other significant associations with p53. There were no associations between nuclear p53 and Hdm2 levels of expression or subcellular localisation (Table 3).

We investigated the relationships between nuclear and cytoplasmic levels of p14^ARF expression and clinicopathological parameters (listed in Table 1), with ARF MQSs analysed as a continuum for the purposes of statistical analysis. There were no significant associations between either nuclear or cytoplasmic ARF with established prognostic indicators in invasive disease (lymph node involvement, large tumour size > 2 cm, increasing tumour histological grade, ER negativity, lymphovascular invasion, NPI and increased proliferation/Ki67), or in DCIS (Van Nuys Pathologic Classification, ER negativity and increased proliferation/Ki67). Nuclear ARF expression (defined as positive [MQS ≥ 3] or negative [MQS < 3]) showed a statistical relationship to HER-2 positivity (2+/3+) in IDCs (χ² test, P = 0.038); however, this relates to a limited number of HER-2-expressing breast cancers (n = 14) in association with ARF. A further subset analysis of levels of p14^ARF expression (negative [0–2], versus weak/moderate [3–5] versus strong [6–8]; Table 2) in relation to clinicopathological parameters (data not shown) showed no significant associations.

Nuclear and cytoplasmic levels of p53 expression (assessed as a MQS continuum) were analysed in relation to clinicopathological criteria as for p14^ARF. Nuclear p53 levels were significantly associated with increased proliferation (Ki67) (cc 0.275, P = 0.008) in IDCs, with cytoplasmic p53 showing a similar association in DCIS (cc 0.295, P = 0.005). There was a possible significant trend in IDCs between nuclear p53 and the NPI (cc 0.210, P = 0.063). A subset analysis of p53 positivity (MQS ≥ 3) was evaluated in relation to clinicopathological parameters. This showed that nuclear p53 expression in invasive breast cancers (MQS ≥ 3 compared with MQS < 3) showed a significant correlation with loss of tumour differentiation (cc 0.243, P = 0.019).

We further analysed the relationship between nuclear and cytoplasmic Hdm2 expression and established parameters as described (Table 1), using the MQS as a continuum. In invasive tumours, increasing levels of nuclear and cytoplasmic Hdm2 expression were significantly associated with increased proliferation (Ki67) (cc 0.281, P = 0.007, and cc 0.224, P = 0.032, respectively). In DCIS nuclear Hdm2 was associated with HER-2 overexpression (2+/3+) (cc 0.249, P = 0.019), with no similar association with cytoplasmic Hdm2 (cc 0.181, P = 0.09). A further evaluation of Hdm2 positivity (MQS ≥ 3) to described parameters showed no other significant associations.

Overall survival (OS) and disease-free survival (DFS) were determined in 89 and 97 patients, respectively (primary tumours), with a median follow-up of 51 months (range 4–120 months). Disease relapses (local or distant recurrences) occurred in 30 women; of these, death was confirmed in 23 patients, with 8 suspected deaths in the absence of a recorded mortality date. Locoregional recurrence occurred at a median duration of 28.5 months (range 3–120 months) from diagnosis. Breast cancer-related mortality occurred at a median of 26 months (range 8–98 months) from presentation. The mean duration of OS and DFS were 93 months and 87 months, respectively. Four-year DFS and OS were 70% and 77%, respectively. The relationship of established clinicopathological features to OS and DFS were analysed with Cox's regression analysis (Table 4). Generally poor prognostic factors such as large tumour size, high tumour grade, lymph node metastases, ER negativity, HER-2 overexpression and NPI were significantly associated with decreased OS and DFS. High tumour proliferation (Ki67 on immunohistochemistry), although associated with a smaller percentage of patients remaining disease-free (DFS) and alive (OS) at 4 years, did not reach statistical significance on univariate or multivariate analysis.

Univariate and multivariate analysis with the continuous MQS variables were used to investigate possible relationships between patient outcome data and levels of subcellular expression for p14^ARF, Hdm2 and p53. When scores were measured as a continuum, neither nuclear nor cytoplasmic ARF expression was predictive of OS or DFS after univariate or multivariate analysis (data not shown). Similarly, a Kaplan–Meier analysis for OS and DFS of the nuclear p14^ARF subgroups (negative [0–2], weak/moderate [3–5] and strong [6–8]) showed no significant relationship (data not shown). The small number of patients with strong cytoplasmic p14^ARF precluded a similar analysis (see Table 2). Kaplan–Meier survival curves showed that the presence of cytoplasmic p14^ARF (MQS ≥ 3) might be associated with a better OS compared with ARF negativity (MQS < 3) (P = 0.09, Fig. 3b), with a similar separation of DFS curves, although not statistically significant (data not shown). The presence or absence of nuclear p14^ARF (MQS ≥ 3 compared with MQS < 3) was not significantly associated with patient outcome (Fig. 3a).

Subcellular levels of p53 expression were not shown to be independent prognosticators for OS and for DFS on univariate or multivariate analyses (data not shown). Kaplan–Meier analyses of nuclear p53 subgroups (negative [0–2], weak/moderate [3–5] and strong [6–8]) showed a significant association between strong nuclear p53 expression and a reduced DFS (P = 0.05; log rank), although patient numbers were small for this group (data not shown). Similarly, nuclear p53 expression (MQS ≥ 3 compared with MQS < 3) was associated with a reduced OS and DFS, although this was not statistically significant (data not shown). Nuclear Hdm2 expression was not an independent prognosticator for OS and for DFS on univariate or multivariate analyses. Cytoplasmic Hdm2 expression (analysed as a continuous MQS) was associated with improved OS (P = 0.01, univariate analysis) and DFS (P = 0.05, univariate) (Table 4). Cytoplasmic Hdm2 independently predicted for improved OS (P = 0.02, multivariate) and DFS (P = 0.03, multivariate) (Table 4). Kaplan–Meier survival curves showed that the presence of cytoplasmic Hdm2 (MQS ≥ 3 compared with MQS < 3) was associated with improved OS (P = 0.002) (Fig. 3c).