Breast cancer risk in premalignant lesions: osteopontin splice variants indicate prognosis

Abstract

Background: Premalignant breast lesions pose variable risks for transformation, raising the question who should receive treatment to counteract the potential progression to breast cancer. Because the secreted metastasis mediator Osteopontin (OPN) is a marker for breast cancer aggressiveness, its presence in these lesions may reflect progression risk.

Methods: By immunohistochemistry, we analyse the association of Osteopontin variant expression in healthy breasts, hyperplasias, papillomas, and carcinomas in situ from 434 women to assess a) staining for OPN exon 4 (present in OPN-a and OPN-b) or OPN-c in low-risk to high-risk lesions b) correlations between staining and progression (DCIS with invasion, invasive cancer) or survival.

Results: The markers correlate with risk, and they are prognostic for ensuing invasive disease and survival. About 10% of OPN-c pathology score 0-1 (intensity), vs. 40% of score 3 experience cancer over 5 years. More than 90% of women, who progress, had pathology scores of 2-3 for OPN-c intensity at the time of initial diagnosis. When combining OPN-c and OPN exon 4 staining, all of the low intensity patients are alive after 5 years, whereas women in the high category have a close to 30% chance to die within 5 years. Of patients who succumb, close to 80% had a high combined score at the time of initial diagnosis.

Conclusion: The combined information of OPN splice variant immunohistochemistry can provide a foundation for very reliable prognostication and has the potential to aid decision making in the treatment of early breast lesions.

Fig. 1

Immunohistochemistry of the patient samples. All specimens were developed using the LSAB (labelled Streptavidin-Biotin) method. The original magnification was ×100 (please note the scale bars). The left column (a, c, e, g) shows staining for OPN exon 4, the middle column (b, d, e, h) shows staining for OPN-c. The right column (i, j, k) contains additional information. a, b Healthy breast tissue with a lack of staining for OPN exon 4 (a) and a lack of staining for OPN-c (b) (intensity of staining, percent of stained cells = 0, 0). c, d Intraductal papillomatosis with weakly positive cytoplasmic staining for OPN exon 4 (c) and weakly positive nuclear staining for OPN-c (d) (1, 1). e, f Intraductal papillomatosis with strongly positive cytoplasmic staining for OPN exon 4 (e) and nuclear staining for OPN-c (f) (3, 3). g, h Ductal carcinoma in situ (DCIS) with strongly positive cytoplasmic staining for OPN exon 4 (g, larger magnification in j) and strongly positive nuclear staining for OPN-c (h, larger magnification in k) (3, 3). i DCIS negative control (no staining; first antibody was omitted). j, k larger magnification of a strongly positive DCIS confirms the predominantly cytoplasmic staining for OPN exon 4 (j) and the predominantly nuclear staining for OPN-c (k) (the green lines connect low and high magnification for the same type of staining)

Fig. 2

OPN splice variants are indicators for prognosis. Kaplan–Meier curves for the risk of progression over time. The x-axis shows the time of follow-up in years, the y-axis displays the probability of remaining recurrence-free cases in percent of the total number. The measured variables in a, c are categorical. The variables in b, d are dichotomised. The χ² statistic is inserted into the lower left corner of the graphs, df degrees of freedom. a OPN-c intensity. b OPN-c intensity dichotomised. c OPN exon 4 intensity. d OPN exon 4 intensity dichotomised

Fig. 3

Pathology scores prognosticate outcome subgroups. a, c Shown are the distributions of pathology scores for marker staining intensity (0–3 as indicated to the left of the bars, with 0 = unfilled, 1 = dotted, 2 = hatched, 3 = filled) for 139 patients who do or do not succumb to cancer (a) and 140 patients who do or do not experience progression (c) over a time frame of 5 years (5 + indicates that some patients remained free of adverse outcome over more than 5 years, shorter follow-ups are disregarded). The left two bars reflect LF161 staining (OPN exon 4), whereas the right two bars indicate anti-OPN-c staining. The y-axis shows the number of patients as percent of the total in each group. b, d To utilise the information of both markers and to gain discrimination, the pathology scores for OPN-c and OPN exon 4 marker intensities were combined and their distributions visualised on stacked bar graphs (low = pathology scores for both markers 0–1, intermediate = one marker 0–1 the other 2–3, high = both markers 2–3, as indicated to the left of the bars; low = unfilled, intermediate = hatched, high = filled) for patients who do or do not succumb to cancer (b) and patients who do or do not experience ensuing invasive disease (d) over a time frame of 5 years. The y-axis shows the number of patients as percent of the total in each group

Fig. 4

ROC curves validate the OPN splice variants as markers for progression and survival. Shown are graphs for survival (a) and progression (b). All indicators are above the diagonal. Evaluated are the pathology scores for OPN-c staining intensity alone, OPN exon 4 staining intensity alone, both markers combined, and a logistic regression analysis including all of the variables under study. The non-smooth curve-fit for the combined analysis reflects the iterations of the logistic regression as calculated in R. int = staining intensity, combined = sum of pathology scores for OPN-c and OPN exon 4 staining