DTC and VI's prognostic dependency upon the vascular markers.

<p>The figure shows Kaplan-Meier plots of DTC (A–H) and VI (I–P) for the end-points DDFS (A–F and I–L) and BCSS (E–H and M–P) within vascular sub-groups (columns): Small vessels (A, E, I and M), large vessels (B, F, J and N), low complexity vessels (C, G, K, and O) and high complexity vessels (D, H, L and P). Gray curves: DTC or VI negative; black: Positive. HR: Cox regression hazard ratios with 95% confidence interval. p-values are by log-rank test.</p

CD34 section case examples.

<p>A) High MVSμ; low MVPμ, MVA_Σ and MVD; B) Average MVSμ, high MVPμ and MVA_Σ, and low MVD; C) Low MVSμ, high MVPμ and MVA_Σ, and low MVD; D) Low MVSμ, average MVPμ and MVA_Σ, and high MVD. High MVPμ values (large vessels) and low MVSμ values (high complexity shapes) contribute to poor prognosis; as well as high MVA_Σ values (high vascular area) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075954#pone.0075954-Luukkaa1" target="_blank">[29]</a>, but MVD is inconsequential <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075954#pone.0075954-Luukkaa1" target="_blank">[29]</a>.</p

The prognostic associations of the dichotomized markers.

<p>The Kaplan-Meier plots of the median-categorized MVPμ (A–D) and MVSμ (E–H) data against DDFS (A, C, E, G) and BCSS (B, D, F, H) end-points in the set of all cases (A,B,E,F) and the set of node-negative no systemic therapy cases (C,D,G,H). p-values are by the log-rank test. Hazard ratios (HR) with 95-% confidence intervals in parentheses are for Cox regression of high-risk vs low-risk groups. *: Low and high solidity corresponds, respectively, to high and low vessel profile complexity.</p

The Clinical Impact of Mean Vessel Size and Solidity in Breast Carcinoma Patients

<div><p>Angiogenesis quantification, through vessel counting or area estimation in the most vascular part of the tumour, has been found to be of prognostic value across a range of carcinomas, breast cancer included. We have applied computer image analysis to quantify vascular properties pertaining to size, shape and spatial distributions in photographed fields of CD34 stained sections. Aided by a pilot (98 cases), seven parameters were selected and validated on a separate set from 293 breast cancer patients.</p><p>Two new prognostic markers were identified through continuous Cox regression with endpoints Breast Cancer Specific Survival and Distant Disease Free Survival: The average size of the vessels as measured by their perimeter (p = 0.003 and 0.004, respectively), and the average complexity of the vessel shapes measured by their solidity (p = 0.004 and 0.004). The Hazard ratios for the corresponding median-dichotomized markers were 2.28 (p = 0.005) and 1.89 (p = 0.016) for the mean perimeter and 1.80 (p = 0.041) and 1.55 (p = 0.095) for the shape complexity. The markers were associated with poor histologic type, high grade, necrosis, HR negativity, inflammation, and p53 expression (vessel size only).</p><p>Both markers were found to strongly influence the prognostic properties of vascular invasion (VI) and disseminated tumour cells in the bone marrow. The latter being prognostic only in cases with large vessels (p = 0.004 and 0.043) or low complexity (p = 0.018 and 0.024), but not in the small or complex vessel groups (p>0.47). VI was significant in all groups, but showed greater hazard ratios for small and low complexity vessels (6.54–11.2) versus large and high complexity vessels (2.64–3.06).</p><p>We find that not only the overall amount of produced vasculature in angiogenic hot-spots is of prognostic significance, but also the morphological appearance of the generated vessels, <i>i.e.</i> the size and shape of vessels in the studied hot spots.</p></div

Clinico-pathological characteristics for patients and their relationship with the identified angiogenesis markers.

#<p>HoR– hormone receptor, combination of ER and PgR, positive if either is positive, negative if both are negative.</p>*<p>Other histologic types were ignored for the purpose of determining significance.</p><p>p-value: Pearsons Chi-square exact test and Linear-by-linear association exact test (p-values marked with £). MVPμ: average perimeter length (low = small vessels). MVSμ: average vessel solidity (low = complex vessel shapes).</p

Clinico-pathologic characteristics for patients in the different groups.

€<p>Cases excluded due to background stains. p-values: Difference in group composition compared to the validation set; by Pearsons Chi-square exact test; Linear-by-linear association exact test (p-values marked with £); t-tests (p-values marked with τ).</p>*<p>Other histologic types and unknown or unrelated causes of death were not included in the significance test.</p

Multivariate Cox-regression.

<p>MVP<b>μ</b> and MVS<b>μ</b> are dichotomized at the median-value; MVPμ is high <i>vs.</i> low; MVSμ is low <i>vs.</i> high. In addition to the shown parameters systemic therapy was included in the model. Neither MVPμ or MVSμ in their dichotomous versions were independently prognostic in the multivariate Cox model.</p

Continuous Cox regression survival analysis of validation parameters.

*<p>Significant at the Bonferroni corrected 0.05/7 level.</p>#<p>Hazard ratios (HR) are for one standard deviation increase in the parameter value.</p><p>MVA_CV: Coefficient of variation of the vessel areas. MVPμ: Mean vessel perimeter length; MV_luminal: Fraction of vessels with open lumen. MV_scale: Marker for the vascular density's dependency on the field size. ICD: Inter-capillary distance.</p

Illustration of the quantified vascular features.

<p>A) Individual vessel characterization: Endothelial area (red); luminal area (blue); vessel/vascular area (red + blue); vessel perimeter (stapled green); width (diameter of imposed yellow circle); skeleton (black line); branching points (yellow markers); convex hull (interior of stapled black line, i.e. vessel + gray). B) Contextual vasculature characterization: centre of vessel (yellow markers); Neighbouring vessels according to the Gabriel's Graph criterion (blue lines), i.e. all connections between vessels such that no other vessel centres are found within the circle spanned by the connecting line (see blue circle). Vertices which do not fit the criteria are not considered neighbours (magenta stapled lines; see red circle); equidistant watershed lines (black), each region within the black lines is closer to the enclosed vessel than any other vessel.</p

Multivariate Cox regression analysis (BCSS).

<p>¹ Tumor grade is included as ordinal variablePrognostic impact of <i>WRAP53</i> in patients stratified for ER- and <i>TP53-</i>status</p><p>Multivariate Cox regression analysis (BCSS).</p