2.1.

Study Design and Patient Selection

The I-SPY 1 TRIAL was a study of imaging and tissue-based biomarkers for prediction of treatment response and survival. The imaging component of this trial was conducted as a companion study to the American College of Radiology Imaging Network (ACRIN) 6657 and Cancer and Leukemia Group B (CALGB) 150007. Participation in both ACRIN 6657 and CALGB 150007 was required to enroll in the I-SPY 1 TRIAL. All patients gave a signed consent to participate. Eligible patients had histopathologically confirmed breast cancers without evidence of distant metastatic disease. The NAC included an initial four cycles of anthracycline-cyclophosphamide (AC) after which patients either underwent surgery or received four cycles of taxane-based treatment prior to surgery.

2.2.

MRI Acquisition

The I-SPY 1 TRIAL patients underwent contrast-enhanced MRI at visits before the start of chemotherapy (T1), after one cycle of AC (T2), between AC and taxane-based (T3) regimen, and at the completion of chemotherapy prior to surgery (T4). Images were acquired as previously described.^12,13 Briefly, MRI was performed on the tumor bearing breast (ipsilateral) only. Images were acquired on a 1.5-T scanner using a dedicated breast radiofrequency coil. Patients were imaged in the prone position. The MR imaging protocol included a localization acquisition and a T2-weighted sequence, followed by a dynamic contrast-enhanced series. For T2-weighted imaging, a fast spin-echo sequence with fat suppression was used (two-dimensional spin-echo; field of view, 16 to 20 cm; section thickness, 3 mm; fat saturation; echo train length, 8 to 16; one echo; effective echo time, 80 to 140 ms; repetition time, 4000 to 6000 ms). Gadopentetatedimeglumine (Magnevist, Bayer HealthCare) was used as a contrast agent and was injected at a dose of $0.1\mathrm{mmol}/\mathrm{kg}$ of body weight ($1.2\mathrm{mL}/\mathrm{s}$) followed by a 10-mL saline flush. The contrast-enhanced series consisted of a high-resolution ($\le 1\mathrm{mm}$ in-plane spatial resolution), three-dimensional (3-D) fast gradient-recalled echo sequence (repetition time ms/echo time ms, $\le 20/4.5$; flip angle, $\le 45\mathrm{deg}$; field of view, 16 to 18 cm; minimum matrix, $256\times 192$; 64 sections; section thickness, $\le 2.5\mathrm{mm}$; voxel size, $29.3{\mathrm{mm}}^3$). The entire breast was scanned across the sagittal plane with 64 slices for each 2.5 mm thickness over a total scanning time. Imaging time for the T1-weighted sequence was required to be between 4.5 and 5 min, with the data set acquired before injection of the contrast agent ($t_0$) and repeated at least two times in the early ($t_1$) and late phases ($t_2$) after contrast injection. The resulting temporal sampling of the center of $k$-space for the first contrast-enhanced phase was between 2 min 15 s and 2 min 30 s, providing image contrast most representative of this time point. An interphase delay between the first and the second contrast-enhanced phases was used as needed to result in temporal sampling of the second contrast-enhanced phase between 7 min 15 s and 7 min 45 s.

Despite the long scan duration, the first postcontrast time sample occurred at 2.5 min using the standard $k$-space sampling, which was close to the effective sampling of 3 min or less that was recommended by the American College of Radiology guideline for breast MRI.¹⁴

2.3.

Percent Enhancement and Signal Enhancement Ratio Analysis

For breast MRI, contrast enhancement kinetics, as captured by the signal intensity–time curves are used to distinguish among malignant (Fig. 1 red curve: rapid rise and signal washout), normal, and benign tissues (Fig. 1 blue and green curves: slow rise, little or no washout). In this signal intensity–time curve, $S_0$, $S_1$, and $S_2$ are the signal intensity values in the precontrast ($t_0$), early postcontrast ($t_1$), and late postcontrast phases ($t_2$), respectively. Percent enhancement [$\mathrm{PE}=100\times (S_1-S_0)/S_0$] is calculated for each voxel. Signal enhancement ratio [$\mathrm{SER}=(S_1-S_0)/(S_2-S_0)$ or (${\mathrm{PE}}_1/{\mathrm{PE}}_2$)] is a method developed to measure contrast enhancement kinetics from high spatial resolution, low 5, contrast-enhanced MR images commonly used for clinical breast MRI.¹⁵ High SER values consistently identify malignant tissues with a strong signal washout characteristic.¹⁶

Fig. 1

Kinetics of contrast enhancement on breast MRI captured on a signal intensity–time curve. Time points $t_1$ and $t_2$ are times at which images are acquired after gadolinium contrast injection. $\mathrm{PE}=100\times (S_1-S_0)/S_0$ whereas $S_0$, $S_1$, and $S_2$ are signal intensity values in the precontrast ($t_0$), early ($t_1$), and late ($t_2$) postcontrast phases. Signal-enhancement-ratio (SER) is defined as the ratio of PE at $t_1$ to PE at $t_2={\mathrm{PE}}_1/{\mathrm{PE}}_2$.

2.4.

Proximity Mapping of Breast Stroma

In this study, contrast-enhanced MRI was used to assess low-level contrast enhancement patterns in the fibroglandular breast tissue outside of the primary tumor. As previously described,⁴ tumor regions on MR images were identified using an enhancement criteria of 70% based on visual agreement with radiological assessments in clinical practice¹⁷ and were applied to the first postcontrast image.¹⁸ Normal-appearing stroma surrounding the tumor was defined as fibrogladular tissues and was segmented from adipose tissue using a fuzzy $C$-means clustering method.¹⁹ Maps of PE and SER were generated using a customized software program.¹⁸ The tumor proximity map for normal-appearing breast stroma was generated as shown In Fig. 2 using the 3-D tumor and stromal tissue masks. For each pixel in the stroma, the proximity was defined as the minimum 3-D distance to any tumor pixel. The resulting map is shown as the green overlay in Fig. 2(c). Proximity analysis regions were defined from the proximity map as 5-mm-thick 3-D distance bands from 0 to 40 mm outside of the tumor mask (0 to 5 mm, 5 and 10 mm, etc.), and were applied to the PE and SER maps to calculate the mean PE and SER values within each distance band. Regions closest to the tumor boundary from 0 to 5 mm were considered as tumor periphery.²⁰ Global PE and SER values were measured as the average of the mean PE and SER values over 5 to 40 mm, where the tumor periphery was excluded to minimize effects of the tumor masking PE threshold. All subsequent calculations of stromal effects on radial distance, global PE, and SER were focused at regions from 5 to 40 mm from the tumor edge.

Fig. 2

Tumor proximity mapping of breast stroma for two subjects: (a) representative slices of the 3-D FCM derived stroma masks; (b) corresponding slices of the 3-D precontrast T1-weighted MRI with superimposed tumor mask derived from a 70% threshold on the early PE map; (c) T1-weighted image with overlay of tumor proximity, i.e., distance measured from the nearest tumor voxel in the 3-D image. Concentric contours on the proximity map illustrate the 5-mm-thick shells (0 to 5 mm, 5 to 10 mm, etc…) used to define the regions for the calculations in this study.

2.5.

Stromal RPPA Analysis

For the analysis of stromal protein levels, breast cancer frozen biopsy specimens in this patient cohort were collected at T2 ($N=68$) and were subjected to laser capture microdissection (LCM) to enrich for stroma. Stromal populations were isolated ($>95\%$ purity) from $8\mu \mathrm{m}$ cryosections using an Arcturus Pixcell IIe LCM system (Arcturus, Mountain View, California) as described previously.²¹ Microdissected material was lysed in extraction buffer [tissue protein extraction reagent (ThermoFisher), $2\times$ SDS-PAGE sample buffer (ThermoFisher) mixed 1:1 and 2.5% beta-mercaptoethanol] at a ratio of $\sim 175$ laser $\text{pulses}/\mu \mathrm{L}$ of extraction buffer. Samples were heated at 100°C for 5 min, brought to room temperature, briefly centrifuged, and stored at $-20°\mathrm{C}$ until printing.

Cell lysates were printed in triplicate spots ($\sim 10\mathrm{nL}$ per spot) onto nitrocellulose-coated slides (Grace Biolabs, Bend, Oregon) using an Aushon 2470 Arrayer (AushonBiosystems, Billerica, Massachusetts). Standard curves of control cell lysates were also included for quality assurance purposes.²² Total protein levels were assessed in each sample by staining with Sypro Ruby Protein Blot Stain (Invitrogen, Carlsbad, California) according to manufacturer’s instructions. Arrays were immunostained with 59 antibodies specific for various stroma-related proteins that were selected based on their involvement in mediating angiogenesis, immune response, inflammation as well as more general pathways such as cell proliferation, motility, and survival (Table 1). All antibodies were validated before use by immunoblotting.²³ Immunostaining was performed as previously described.²⁴ Briefly, each slide was probed with one primary antibody targeting the protein of interest (Table 1). Biotinylated goat anti-rabbit (1:7500, Vector Laboratories Inc., Burlingame, California) and rabbit anti-mouse (1:10, DakoCytomation, Carpinteria, California) IgG were used as secondary antibodies. Signal amplification was performed using a tyramide-based avidin/biotin amplification system (DakoCytomation, Carpinteria, California) followed by streptavidin-conjugated IRDye 680 (LI-COR, Lincoln, Nebraska) for visualization. Images were acquired using a TecanPowerScanner (Tecan, Mannedorf, Switzerland). Antibody staining intensities were quantified using the MicroVigene v3.5.0.0 software package (Vigenetech, Carlisle, Massachusetts). The final results represent negative control-subtracted and total protein-normalized relative intensity values for each endpoint within a given patient sample.^25,26

Table 1

Primary antibodies used in stromal RPPA analysis.

Table 2

Summary of clinical angiographic database of a total of 24 cases.

2.6.

Statistical Analysis

Patient characteristics were compared between I-SPY 1 TRIAL subjects with and without MR stromal analysis using Wilcoxon rank-sum test or Fisher’s exact test as appropriate. Similar comparisons were made between subjects who had 3-year recurrence free survival (RFS) and those who did not. A linear mixed effect model was utilized to assess the PE and SER radial trend in the range of 5 to 40 mm from the tumor edge.⁴ A subject-specific random slope and intercept was included in this model. The variable 3-year RFS and its interaction with distance from the tumor border was included in an additional model. For comparisons with RFS as a censored survival variable, the univariable Cox model was used and testing was performed with the likelihood ratio test. Global PE was evaluated between times utilizing univariable regression. Statistical tests with $p$-values less than 0.05 were considered significant. Linear regression was used to correlate MR stromal enhancement measurements (global PE and SER) with levels of stromal proteins. All statistical analyses were performed using the $R$ statistical software language.²⁷

3.1.

Patient and Tumor Characteristics

As shown in the consort diagram (Fig. 3), there were 221 evaluable patients enrolled in the I-SPY 1 TRIAL. Seventy-one patients had stromal MR evaluation and 144 did not. Characteristics of these patients (total I-SPY 1 versus stromal MRI evaluated) are shown in Table 2. The two groups were similar in age, tumor size, nodal status, subtype, and regimen received. However, notable differences were found in percentage of premenopausal (66% versus 48%), race (relatively fewer African American: 8% versus 19%, and other: 13% versus 6%), and chemotherapy response [residual cancer burden (RCB) class²⁸ 0/I: 48% versus 37%; RCB class II/III: 46% versus 63%]. For the analysis of stromal protein levels, breast cancer biopsies were available for analysis at T2 in 68 patients. Measurements of both MR enhancement and stromal protein levels were obtained in 18 patients at T2.

Fig. 3

Consort diagram for the study. Patients were accrued to ISPY 1 ($n=237$). Of these, 221 were available for analysis, and 71 patients had MRIs that were assessable for stromal enhancement. The study protocol was to obtain four MRIs for each patient at V1 (prior to chemo); V2 (after first chemocycle); V3 (between AC and T chemotherapy); V4 (prior to surgery).

Our survival endpoint was RFS, both at 3 years and as a censored variable. Of the 71 patients with MRI stromal evaluation, 17 patients had recurred after 3 years and 51 had not (three were missing due to insufficient follow up). Univariable Cox proportional hazards analysis showed that clinical stage was significantly associated with RFS ($P=0.001$). Nodal status [hazard ratio $(\mathrm{HR})=2.08$, 95% CI (1.30 to 3.33), $P=0.005$] and tumor size [$\mathrm{HR}=1.19$, 95% CI (1.07 to 1.33), $P=0.005$] were also significantly associated with RFS. Prediction of RFS by other known prognostic factors including age and chemoresponse were not statistically significant: details of the estimated relationships between these patient tumor characteristics and RFS in terms of hazard ratios, confidence intervals, and $p$-values can be found in Table 2.

3.2.

Stromal Enhancement Patterns

As described above, we developed the tumor proximity technique to map the stromal enhancement at incremental distances from the primary tumor edge as visualized on MRI. Using retrospective pilot cohorts, we previously showed that MR PE and SER had higher stromal contrast enhancement patterns in patients with invasive breast cancer.^3,4 Here, we validated our previous findings using the ISPY 1 TRIAL cohort and investigated the association of stromal enhancement patterns with treatment.

3.2.1.

Radial dependence and interaction with RFS

As shown in Fig. 4, PE values in the I-SPY 1 dataset decreased continuously with distance from the tumor edge: from proximal tissue (within 5 to 10 mm) to distal tissue (35 to 40 mm). Notably, the decreases in PE with distance from the tumor edge remained consistent through all the study time points, T1 to T4 (Fig. 4).

Fig. 4

Stromal PE decreases with distance from the tumor edge on average for all time points. The decrease was significant only for time points T2 to T4 ($p<0.05$ for each).

Using the linear mixed effects model, the estimated mean change in enhancement as a function of distance from the tumor edge was calculated for the entire cohort ($N=71$), Table 3. The estimated mean change in enhancement signal per mm was $-0.33$ for T1 (95% CI: $-0.68$, 0.02), $-0.38$ for T2 (95% CI: $-0.55$, $-0.20$), $-0.48$ for T3 (95% CI: $-0.64$, $-0.28$), and $-0.17$ for T4 (95% CI: $-0.26$, $-0.07$). As shown, the corresponding $p$-values for T1 to T4 were 0.06, $<0.001$, $<0.001$, and 0.001, respectively, showing that PE decreases with distance from the tumor edge. All mean changes in PE values were significant except for T1 ($P=0.06$).

Table 3

Change of stromal PE values per mm from the tumor edge at time points before the start of chemotherapy (T1), after one cycle of AC (T2), between AC and taxane-based (T3) regimen, and at the completion of chemotherapy prior to surgery (T4).

	Estimated mean change (per mm)	95% CI	P-value
T1	$-0.33$	$-0.68$, 0.02	0.06
T2	$-0.38$	$-0.55$, $-0.20$	$<0.001$
T3	$-0.48$	$-0.64$, $-0.28$	$<0.001$
T4	$-0.17$	$-0.26$, $-0.07$	0.001

Next, 3-year RFS, recurrent ($N=17$) versus nonrecurrent ($N=51$) was added to the mixed model. The same decreasing radial trend was found in both groups as shown in Table 4. Significant changes were observed in the nonrecurrent patients with estimated mean change per mm of $-0.36$ for T2 (95% CI: $-0.57$, $-0.15$; $P<0.001$), $-0.51$ for T3 (95% CI: $-0.71$, $-0.30$; $P<0.001$), and $-0.17$ for T4 (95% CI: $-0.29$, $-0.05$; $P=0.005$). For the recurrent group, significance change was found only in T2 (estimated mean change $\text{permm}=-0.39$, 95% CI: $-0.75$, $-0.02$; $P=0.04$), but the lack of significance in the recurrent group may be due to the small sample size. The estimated mean difference in distance effects between nonrecurrent and recurrent patients from time points T1 to T4 were $-0.01$, 0.03, $-0.28$, and $-0.01$, suggesting that the decreasing rates of PE from the tumor edge in these groups were similar with the exception of T3. However, limited numbers preclude making a more definitive statement.

Table 4

Change in stromal PE values per mm from the tumor edge for recurrent and nonrecurrent patients. Significant changes were found for T2, T3, and T4 in the nonrecurrent patients.

	Recurrent			Nonrecurrent
	Estimated mean change (per mm)	95% CI	P-value	Estimated mean change (per mm)	95% CI	P-value
T1	$-0.3$	($-1.02$, 0.42)	0.42	$-0.31$	($-0.73$, 0.11)	0.15
T2	$-0.39$	($-0.75$, $-0.02$)	0.04	$-0.36$	($-0.57$, $-0.15$)	$<0.001$
T3	$-0.48$	($-0.59$, 0.14)	0.23	$-0.51$	($-0.71$, $-0.30$)	$<0.001$
T4	$-0.16$	($-0.37$, 0.06)	0.15	$-0.17$	($-0.29$, $-0.05$)	0.005

We also examined changes in PE between adjacent times (T1 versus T2, T2 versus T3, T3 versus T4 in the recurrent and nonrecurrent groups) at the tumor periphery within the nonrecurrent and recurrent groups. In general, the PE signal was lower in the later time point, with the only exception being between T1 and T2 in the nonrecurrent group. The difference was significant only between T2 and T3 in the recurrent group. Here the estimated (pseudo-) median difference was $-3.23$ per mm (95% CI $-6.17$, $-0.80$; Wilcoxon signed-rank test $P=0.01$).

3.2.2.

SER pattern from the tumor edge and interaction with RFS

Using the same mixed effects model as for PE, we observed an increasing trend, rather than a decrease, in mean SER as a function of distance from the tumor edge at the pretreatment time point (T1). Mean SER values increased significantly at T1 only. Here the estimated mean increase per mm was 0.004 (95% CI: 0.001, 0.006; $P=0.003$) (Fig. 5). In MRIs taken at time points during and after chemotherapy (T2 to T4), changes in SER as a function of distance were not significantly different from zero, Fig. 5.

Fig. 5

Stromal SER increases with distance from the tumor edge at T1 ($P=0.003$) but not at other time points.

Similar to PE, we added the recurrence status as a group variable to the mixed effects model with distance to assess differences in SER enhancement patterns in recurrent and nonrecurrent patients. We focused on T1, since that was the only significant time point. The estimated mean increase (95% CI) in the nonrecurrent group was 0.0046 (95% CI: 0.0017, 0.0074; $P=0.002$) and in the recurrent group was 0.0012 (95% CI: $-0.0040$, 0.0063, $P=0.66$).

3.2.3.

Global stromal PE and SER and chemotherapy effect

To determine whether the intrinsic stromal enhancement averaged throughout the breast stroma could be associated with outcomes, we measured global PE at each time point, T1 to T4. Although global PE was not significantly associated with RFS at any time point, we observed a decrease in global PE among the cohort from T1 to T4 (Fig. 6). As shown in the scatter plots, when we examined the PE values at the pretreatment time point (T1), there was no change in PE after the first dose of chemotherapy at T2. However, PE significantly decreased at the interregimen (T3) and pre-surgery (T4) time points relative to pretreatment levels ($P<0.001$ for each comparison), suggesting that ongoing changes in global PE occurred with increasing exposure to chemotherapy.

Fig. 6

Scatter plot of global PE at each time point for each patient. Global PE decreased significantly with chemotherapy at T3 and T4 ($P<0.001$) compared to T1.

Of note, we had previously found that global SER had predictive values among a pilot cohort of patients treated with NAC.^3,4 In this paper, a similar analysis using global SER values did not reveal significant differences between T1 and T4, or significant associations between SER and RFS.

3.3.

Global Stromal PE and SER and Stromal Protein Levels

Using unsupervised clustering of stromal protein and protein signaling architecture from the RPPA data obtained at T2, two major subgroups were identified (Fig. 7). In the subgroup of 18 patients in which both stromal MR enhancement and protein levels were measured at T2, there were no significant correlations between 10 proteins/phosphoproteins of clinical interest (WNT5ab, VEGFR2 Y1175, Lamin A, cleaved D230, ZAP70 Y319/SYK Y352, PDFGRb, JAK1 Y1022/Y1023, alpha SMA, IL-10, NFkBp65 S536, STAT3 Y705) and measures of stromal MR enhancement (global SER and global PE).

Fig. 7

RPPA data at T2. Using unsupervised clustering of RPPA data at T2, two subgroups were identified. (Key for top bar: red = triple negative, $\text{yellow}=\mathrm{HR}+/\mathrm{HER}2-$, $\text{green}=\mathrm{HER}2+$).