Role of vascular density and normalization in response to neoadjuvant bevacizumab and chemotherapy in breast cancer patients - PubMed (original) (raw)

Clinical Trial

. 2015 Nov 17;112(46):14325-30.

doi: 10.1073/pnas.1518808112. Epub 2015 Nov 2.

Yves Boucher 2, Dan G Duda 2, John D Martin 3, Giorgio Seano 2, Marek Ancukiewicz 2, William T Barry 4, Shom Goel 5, Johanna Lahdenrata 2, Steven J Isakoff 6, Eren D Yeh 7, Saloni R Jain 3, Mehra Golshan 8, Jane Brock 9, Matija Snuderl 10, Eric P Winer 1, Ian E Krop 11, Rakesh K Jain 12

Affiliations

Clinical Trial

Role of vascular density and normalization in response to neoadjuvant bevacizumab and chemotherapy in breast cancer patients

Sara M Tolaney et al. Proc Natl Acad Sci U S A. 2015.

Abstract

Preoperative bevacizumab and chemotherapy may benefit a subset of breast cancer (BC) patients. To explore potential mechanisms of this benefit, we conducted a phase II study of neoadjuvant bevacizumab (single dose) followed by combined bevacizumab and adriamycin/cyclophosphamide/paclitaxel chemotherapy in HER2-negative BC. The regimen was well-tolerated and showed a higher rate of pathologic complete response (pCR) in triple-negative (TN)BC (11/21 patients or 52%, [95% confidence interval (CI): 30,74]) than in hormone receptor-positive (HR)BC [5/78 patients or 6% (95%CI: 2,14)]. Within the HRBCs, basal-like subtype was significantly associated with pCR (P = 0.007; Fisher exact test). We assessed interstitial fluid pressure (IFP) and tissue biopsies before and after bevacizumab monotherapy and circulating plasma biomarkers at baseline and before and after combination therapy. Bevacizumab alone lowered IFP, but to a smaller extent than previously observed in other tumor types. Pathologic response to therapy correlated with sVEGFR1 postbevacizumab alone in TNBC (Spearman correlation 0.610, P = 0.0033) and pretreatment microvascular density (MVD) in all patients (Spearman correlation 0.465, P = 0.0005). Moreover, increased pericyte-covered MVD, a marker of extent of vascular normalization, after bevacizumab monotherapy was associated with improved pathologic response to treatment, especially in patients with a high pretreatment MVD. These data suggest that bevacizumab prunes vessels while normalizing those remaining, and thus is beneficial only when sufficient numbers of vessels are initially present. This study implicates pretreatment MVD as a potential predictive biomarker of response to bevacizumab in BC and suggests that new therapies are needed to normalize vessels without pruning.

Keywords: PAM50 gene signature; antiangiogenic therapy; cellular proliferation; circulating and tissue biomarkers.

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Conflict of interest statement

Conflict of interest statement: Y.B. served as consultant for Xtuit. D.G.D. received research grants from Merrimack Pharma and HealthCare Pharma. S.G. served on the advisory board of Lilly. J.L. is an employee of Merrimack Pharmaceuticals. S.J.I. served as consultant for Myriad Genetics. M.G. served on the advisory board of Lightpoint Medical. R.K.J. received consulting fees from Enlight, Ophthotech, SPARC, and SynDevRx; owns equity in Enlight, Ophthotech, SynDevRx, and XTuit; and serves on the board of directors of XTuit and board of trustees of Tekla Healthcare Investors, Tekla Life Sciences Investors, Tekla Healthcare Opportunities Fund, and Tekla World Healthcare Fund. No reagents or funding from these companies was used in these studies. Therefore these authors do not have any conflict of interest related to this study.

Figures

Fig. S1.

Fig. S1.

Study schema. Schedule for treatment with bevacizumab with adriamycin (A) /cyclophosphamide (C)/paclitaxel (T) chemotherapy, tumor tissue biopsies, and measurements of IFP and circulating blood (plasma) biomarkers.

Fig. 1.

Fig. 1.

Effect of a single injection of bevacizumab on structural and functional markers of vascular normalization. Box plots depict median and interquartile ranges for biomarker values pre- (gray) and postbevacizumab alone (white). Horizontal lines between bars pre- and postbevacizumab monotherapy mark changes with a P value less than 0.05. (A) Microvessel density decreased (P = 0.0041, Student’s t test, n = 52 and 53). (B) Bevacizumab did not affect the density of mature vessels. (C) Fraction of vessel perimeter associated with pericytes (αSMA+ cells), a marker that distinguishes between poorly and completely covered vessels, increased (P = 0.037, Student’s t test, n = 47 and 48). (D) Interstitial fluid pressure, which is a functional measurement of vessel leakiness and lymphatic vessel dysfunction, decreased (P = 0.045, Student’s t test, n = 70 and 65). (E and F) Histological markers of functional vascular normalization, Ki67 for proliferation (n = 47 and 49) and HIF-1α (n = 53 and 49), did not change significantly.

Fig. S2.

Fig. S2.

Representative images of vessel segmentation of immature, pericyte-covered, and patent vessels. We segmented endothelial cell staining (CD31), pericytes (αSMA), and lumen to identify the total density of microvessels and the density of pericyte-covered and patent vessels. We analyzed every vessel within two entire tissue biopsy sections separated by 100 μm. To depict the types of vessels and how the stains were segmented, we selected from a single biopsy representative fields of tumor regions with vascular dense regions of (A) pericyte-covered patent vessels, (B) pericyte-covered compressed vessels, and (C) immature vessels without pericytes. (D_–_F) Based on staining intensity and criteria (Methods), we used a custom computer application to identify vessels, (G_–_I) pericytes, and (J_–_L) lumen. There was no detectable pericyte stain in I and no visible lumen visible in K and L. (M) Schematic showing the possible classes of vessels: (1) pericyte-covered patent vessels (shown in A), (2) patent vessels lacking pericytes, (3) pericyte-covered compressed vessels (shown in B), and (4) immature vessels that lack pericytes (shown in C).

Fig. S3.

Fig. S3.

In situ biomarker levels pre- and postbevacizumab monotherapy in HRBC (red) and TNBC (blue). Bars are shown as averages with error bars depicting the SEM. Horizontal lines between bars pre- and postbevacizumab monotherapy mark changes with a P value less than 0.05. Table S3 contains this data tabulated. (A) Microvessels were marked throughout the entire tissue area of two parallel sections 100 μm apart and the microvessel densities of the two sections were averaged. Bevacizumab monotherapy pruned microvessels in TNBC (P = 0.0020, Student’s t test). (B) The association of pericytes and the vascular wall was assessed as the fraction of vessel perimeter associated with pericytes. This value was measured for every vessel scored in A and averaged for each section and then each patient. Bevacizumab monotherapy increased the fraction of vessel perimeter associated with pericytes in HRBC (P = 0.030, Student’s t test). (C) Tumor interstitial fluid pressure was measured in patients using the wick-in-needle technique. IFP was reduced in HRBC (P = 0.016, Student’s t test). (D) The area fraction of tissue stained positive for HIF-1α was assessed using immunofluorescence and did not change with bevacizumab monotherapy in either subtype (TNBC P = 0.087, Student’s t test). (E) The fraction of nuclei stained positive for Ki67 protein, which is a cellular marker of proliferation, decreased in HRBC (P = 0.026, Student’s t test) and increased in TNBC (P = 0.032, Student’s t test).

Fig. S4.

Fig. S4.

Biomarkers of vascular normalization in individual TNBC and high MVD HRBC patients. Bars represent single patients and are shown in the same order in each column of panels (A, C and B, D) from left to right in descending order of MP for all TNBC and high MVD HRBC patients, which have PC-MVD data available pre- and postbevacizumab alone. Each patient’s MP scores are written on the horizontal axis. Asterisks in A denote TNBC patients with baseline MVD below the threshold. Dashed vertical lines represent MP tumor regression score cutoff values. MP >3 corresponds to a good tumor response (Methods). Baseline TNBC and HRBC biomarker values are depicted in blue and red, respectively. Postbevacizumab alone biomarker values are charted in white. Data include the density of αSMA+ vessels (PC-MVD, A and B) and fraction of αSMA+ vessel perimeter (C and D).

Fig. S5.

Fig. S5.

Functional biomarkers of individual TNBC and high MVD HRBC patients. Bars are shown in decreasing order of MP and in the same order as patients with PC-MVD data available pre- and postbevacizumab (Fig. 2). Each patient’s MP score is written on the horizontal axis. When data for a particular biomarker were unavailable, “n/a” is written in place of the bars. Baseline TNBC and HRBC are depicted in blue and red, respectively. Postbevacizumab monotherapy values are in white. Dashed vertical lines represent MP tumor regression score cutoff values. MP >3 corresponds to a good tumor response (Methods). Data include a functional measurement of vessel leakiness to fluid (interstitial fluid pressure in millimeters of mercury, A and B), histological assessment of a hypoxia-induced marker (HIF-1α+ fraction of total biopsy area, C and D), and histological assessment of the cellular proliferation marker (Ki67+ nuclei fraction, E and F) for TNBC and HRBC, respectively.

Fig. 2.

Fig. 2.

(A) The MVD at baseline is significantly higher (P = 0.0024) in patients with MP >3. In these patients, bevacizumab significantly reduced the MVD (P = 0.009). (B) Postbevacizumab the fraction of vessel perimeter covered by pericytes is significantly higher (P = 0.001) in patients with MP >3. (C) Postbevacizumab PC-MVD is significantly higher (P = 0.008) in patients with MP >3.

Fig. 3.

Fig. 3.

A schematic of the proposed mechanism of action of benefit from antiangiogenic therapy. The left two quadrants depict tumor vessels (red) at baseline before bevacizumab monotherapy, and the two right quadrants depict the vasculature following bevacizumab monotherapy. In the top left quadrant, there is a higher vascular density (MVD) than in the bottom left quadrant. Tumors with insufficient baseline MVD do not respond to antiangiogenic therapy (bottom left to bottom right quadrants), because the increase in functional vessels resulting from the recruitment of pericytes (teal) cannot overcome the paucity of vessels. In contrast, tumors with high baseline vascularity that recruit pericytes respond to bevacizumab combined with chemotherapy (top left quadrant to top right quadrant) better than tumors that undergo excessive pruning (top left quadrant to bottom right quadrant). Responders: MP scores of 4–5; nonresponders: MP scores of 1–3.

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