A Quantitative Centrosomal Amplification Score Predicts Local Recurrence of Ductal Carcinoma In Situ

Abstract

Purpose: The purpose of this study is to predict risk of local recurrence (LR) in ductal carcinoma in situ (DCIS) with a new visualization and quantification approach using centrosome amplification (CA), a cancer cell-specific trait widely associated with aggressiveness.

Experimental design: This first-of-its-kind methodology evaluates the severity and frequency of numerical and structural CA present within DCIS and assigns a quantitative centrosomal amplification score (CAS) to each sample. Analyses were performed in a discovery cohort (DC, n = 133) and a validation cohort (VC, n = 119).

Results: DCIS cases with LR exhibited significantly higher CAS than recurrence-free cases. Higher CAS was associated with a greater risk of developing LR (HR, 6.3 and 4.8 for DC and VC, respectively; P < 0.001). CAS remained an independent predictor of relapse-free survival (HR, 7.4 and 4.5 for DC and VC, respectively; P < 0.001) even after accounting for potentially confounding factors [grade, age, comedo necrosis, and radiotherapy (RT)]. Patient stratification using CAS (P < 0.0001) was superior to that by Van Nuys Prognostic Index (VNPI; HR for CAS = 6.2 vs. HR for VNPI = 1.1). Among patients treated with breast-conserving surgery alone, CAS identified patients likely to benefit from adjuvant RT.

Conclusions: CAS predicted 10-year LR risk for patients who underwent surgical management alone and identified patients who may be at low risk of recurrence, and for whom adjuvant RT may not be required. CAS demonstrated the highest concordance among the known prognostic models such as VNPI and clinicopathologic variables such as grade, age, and comedo necrosis.

Figure 1.

Schematic depicting semi-automated workflow to quantify CA in clinical samples. A description of terms used in the algorithm is provided in the Methods section. (A) Centrosomes in breast tissues (normal, DCIS or IBC) were categorized into individually distinguishable centrosomes (iCTRs) and megacentrosomes (mCTRs). iCTRs were defined as centrosomes that stain positive for γ-tubulin and whose volumes lie within the range of centrosome volumes found in normal breast tissue stained for γ-tubulin. (B) mCTRs were defined as centrosomes in a neoplastic region that stain positive for γ-tubulin and whose volume is greater than the upper limit of the centrosome volume range found in corresponding normal tissue immunostained for γ-tubulin. Thus, mCTRs are centrosomes with aberrantly large volumes and are considered to represent structurally amplified centrosomes.

Figure 2:

DCIS cases in the DC with ipsilateral recurrence exhibit higher CAS than recurrence-free cases. (A) Representative H&E images (20× magnification) of the ducts from DCIS cases with and without LR. Black boxes represent the area magnified in panel B. (B) Confocal micrographs showing numerical (green arrows) and structural (yellow arrows) CA in DCIS with or without recurrence. Tissue sections were immunostained for centrosomes (γ-tubulin, red) and nuclei (Hoechst, blue). Scale bar (white), 20μm. Beeswarm box plots showing Wilcoxon ranks for pure DCIS cases with LR (n=28) and without LR (n=105). (C) CASi (D) CASm (E) CAStotal. p<0.05 was considered statistically significant. Beeswarm box plots showing Wilcoxon ranks for pure DCIS cases with LR (n=24) and LR-free cases (n=95) in VC (F) CASi, (G) CASm, and (H) CAStotal. p<0.05 was considered statistically significant.

Figure 3:

In the DC and VC, higher CAS is associated with poorer RFS. Kaplan Meier survival curves representing the RFS of patients in the DC stratified into (A) CASi high and low groups, (B) CASm high and low groups, (C) CAStotal high and low groups. Kaplan Meier curves representing the RFS of DCIS patients in the VC stratified into (D) CASi high and low groups, (E) CASm high and low groups, and (F) CAStotal high and low groups. N: total number of patients in each group; R: number of patients who developed LR; % represents the percentage/proportion of patients with LR out of the total number of patients with LR in both groups combined.

Figure 4:

Comparison of the stratification of DCIS patients by CAStotal and Van Nuys Prognostic Index (VNPI). Kaplan Meier survival curves representing the RFS of DCIS patients (n=164) stratified by (A) CAStotal, and (B) VNPI. N: total number of patients in each group; R: number of patients who showed LR; %: percentage/proportion of patients with LR out of the total number of patients with LR in the DC and VC combined.