Effects of neoadjuvant chemotherapy on the contralateral non-tumor-bearing breast assessed by diffuse optical tomography

The purpose of this study is to evaluate whether the changes in optically derived parameters acquired with a diffuse optical tomography breast imager system (DOTBIS) in the contralateral non-tumor-bearing breast in patients administered neoadjuvant chemotherapy (NAC) for breast cancer are associated with pathologic complete response (pCR). In this retrospective evaluation of 105 patients with stage II–III breast cancer, oxy-hemoglobin (ctO2Hb) from the contralateral non-tumor-bearing breast was collected and analyzed at different time points during NAC. The earliest monitoring imaging time point was after 2–3 weeks receiving taxane. Longitudinal data were analyzed using linear mixed-effects modeling to evaluate the contralateral breast ctO2Hb changes across chemotherapy when corrected for pCR status, age, and BMI. Patients who achieved pCR to NAC had an overall decrease of 3.88 μM for ctO2Hb (95% CI, 1.39 to 6.37 μM), p = .004, after 2–3 weeks. On the other hand, non-pCR subjects had a non-significant mean reduction of 0.14 μM (95% CI, − 1.30 to 1.58 μM), p > .05. Mixed-effect model results indicated a statistically significant negative relationship of ctO2Hb levels with BMI and age. This study demonstrates that the contralateral normal breast tissue assessed by DOTBIS is modifiable after NAC, with changes associated with pCR after only 2–3 weeks of chemotherapy.


Background
Neoadjuvant chemotherapy (NAC) has become a wellestablished therapy in treating patients with locally advanced breast cancer and consists of 3-6 months of systemic, preoperative treatment. Chemotherapy can modify the density and composition of normal breast tissue. Standardized response evaluation after NAC can be assessed by residual cancer burden (RCB) index, which is quantified based on primary tumor area, percentage of the tumor area that is invasive cancer, and extent of lymph node involvement. A score of 0 (RCB0) equates to a pathological complete response (pCR) to NAC, and it is defined as the complete disappearance of the tumor in the breast and lymph node (ypT0/is ypN0 Mx) at the time of surgery. Non-pCR tumors can be categorized into one of the following three classes: RCB-I (minimal burden), RCB-II (moderate burden), and RCB-III (extensive burden). RCB after NAC is associated with improved clinical outcomes, such as disease-free survival, and can predict disease recurrence across different breast cancer subtypes [1][2][3].
Relatively inexpensive, simple to operate, and welltolerated by patients, diffuse optical tomography (DOT) has shown promising applications in breast cancer detection and monitoring NAC [4][5][6][7][8]. However, the measurement of optically derived parameters in the affected breast is strongly dependent on the tumor location, and DOT suffers from low spatial resolution due to its nonlinear, ill-conditioned, and ill-posed inverse problem [9]. Using a priori information provided by an additional high-resolution anatomical imaging modality, such as magnetic resonance or X-ray, could improve spatial resolution but also nullify the low-cost benefit associated with DOT techniques [10]. Therefore, DOT quantitative measure from the contralateral breast could offer a nontumor dependent measurement to monitor NAC induced changes in the breast tissue and assess treatment outcome. Evaluation of optically derived parameters variation in the contralateral non-tumor-bearing breast following NAC has been previously analyzed [11]. Still, no extensive analyses have been performed to evaluate whether these changes are associated with tumor response in the affected breast. A study published in 2005 [12] reported that chemotherapy could decrease contralateral breast cancer risk in younger women [13]. However, assessment of chemotherapy-related benefits for developing a newly detected breast cancer is still a challenge due to the absence of accurate quantitative metrics.
Over the last decade, our group has developed a diffuse optical tomography breast imaging system (DOT-BIS) for a full 3-dimensional tomographic assessment of the entire breast volume and simultaneous bilateral breast imaging. DOTBIS data acquisition is performed without contrast agents or compression and can measure tissue concentration of oxy-hemoglobin (ctO 2 Hb), deoxy-hemoglobin (ctHHb), and water. Our previous studies have described the utility of DOTBIS in monitoring tumor response to NAC [7,14] and tumor detection [15]. As our study was performed bilaterally on breast cancer patients, DOTBIS quantitative measurements from the contralateral non-tumor-bearing breast were acquired and reported in this paper.
The purpose of this study is to investigate the change of quantitative measurements of functional tissue components, such as hemoglobin concentration and water percentage, measured with DOTBIS in the contralateral non-tumor-bearing breast of patients receiving NAC. We hypothesize that hemoglobin and water values in the unaffected breast, directly related to tissue metabolism and vascular characteristics, are associated with pCR to NAC.

Methods
Between 2011 and 2019, 105 patients diagnosed with stage II-III breast cancer and scheduled to undergo NAC were consented to participate in a DOTBIS longitudinal research. Menopausal status, body mass index (BMI), and age were retrieved from the patient's electronic medical record. pCR in the breast was defined as the absence of invasive carcinoma (ypT0/is ypN0 Mx) at the time of surgery. Residual Center Burden (RCB) was assessed per established guidelines by Symmans et al. [16]. This study was approved by the institutional review board at Columbia University Irving Medical Center (CUIMC).

DOTBIS imaging acquisition
Measurements were performed by a continuous wave dynamic imaging system (i.e., DOTBIS) that provides full three-dimensional tomographic images of both breasts simultaneously without requiring the use of contrast agents or compression. A more detailed description of the system can be found in previous reports [15,17]. Total measurement time varied between 6 and 8 min in addition to 5 min required to set up the machine. Nearinfrared light transmission was carried out using four laser diodes operating at 765, 808, 827, and 905 nm. The patient interface consists of two sets of four rings that use up to 32 sources and 64 detectors, depending on the patient's breast cup size, for optical data collection. The spatial distribution of chromophore concentration of ctO 2 Hb, ctHHb, and water was obtained by a transporttheory-based PDE-constrained multispectral model [18], which uses the diffusion approximation of the equation of radiative transfer to describe the light propagation in such scattering-dominated media as the breast tissue. Additional features such as ctTHb and StO 2 % were defined respectively as Eq. (1) and Eq. (2). For visualization, maximum intensities projections (MIP) [19,20] were obtained from the reconstructed 3D chromophore concentration maps. MIP projects the acquired data volume into a view plane, which can be obtained by finding the voxels with maximum intensity along a chosen axis, Fig. 1.

NAC imaging protocol
Sixty-nine patients received 12 cycles of weekly paclitaxel (with or without the addition of carboplatin) followed by four cycles of doxorubicin and cyclophosphamide given every 2 weeks with growth factor support (T × 12/AC × 4 or T/C × 12/AC × 4), and DOTBIS bilateral measurement was acquired at six different time points: before starting NAC regimen (TP0), after 2 weeks of taxane infusions (TP1), after 4 weeks of taxane (TP2), at the end of the taxane regimen and before starting AC cycles (TP3), after two cycles of AC (TP4), and at the end of NAC and before surgery (TP5). Thirty-six patients received other taxane-based NAC regimens without an anthracycline, and bilateral imaging was performed only three times (baseline (TP0), 2-3 weeks after the first taxane infusion (TP1), and at the end of NAC and before surgery (TP5)), Fig. 2.

Statistical analysis
An independent-samples t test was run to determine if there were differences in baseline DOTBIS parameters between pre-and postmenopausal subjects and pCR status (pCR and non-pCR patients). Feature importance was calculated by using Random Forest [21] modelspecific metrics from the R package "caret" [22]. The Random Forest algorithm has a built-in feature selection, mean decrease Gini (Gini importance) [23], which can be used as a score that provides a relative ranking of feature relevance. A paired-samples t test was used to determine whether there was a statistically significant mean difference in ctO 2 Hb levels measured at baseline and TP1 between pCR and non-pCR and between both menopausal groups. To account for repeated measures, a linear mixed-effect model was designed to evaluate if there is a difference in the average longitudinal evolution of ctO 2 Hb when we correct for pCR status (or age and BMI). Significance was assumed at a confidential interval of 95% (α = 0.05).

Patient summary
From a total of 105 subjects, 18 were excluded from the final analysis data set. The main reasons for exclusion were as follows: patient withdrew from the study (n = 4), baseline DOTBIS measurement not collected due to

Contralateral breast DOTBIS feature selection
To avoid severe multicollinearity or overfitting, variable importance with random forest was performed to find the most relevant pCR status classification feature. For the longitudinal multilevel mixed-effects analysis, ctO 2 Hb from all patients (n = 87) was analyzed at threetime imaging points, TP0, TP1, and TP5, which are the imaging time points in common to all the patients regardless of their chemotherapy regimen. We fit a linear mixed-effects model with ctO 2 Hb as the outcome variable, with fixed effects of pCR status, BMI and age, and time as a random effect (and fixed effect) and random slope across individuals. We were also particularly interested in the group-occasion interaction between time and pCR status, as this reflects a potential difference in time evolution between the pCR status groups. There was a significant interaction between ctO 2 Hb over time and pCR status. pCR patients had a significant reduction in ctO 2 Hb levels at TP1 (3.53 μM, p = .0053) than non-pCR, but not statistically significant at TP5 (2.59 μM, p = .131). This trend can also be observed in the iteration plot of ctO 2 Hb levels over the different time points for both pCR and non-pCR groups, Fig. 4. Significant main effects of age (p = .0015) and BMI (p = .0011) were also found, Table 2, characterized by a negative relationship, older and overweighted patients had lower ctO 2 Hb levels. Figure 5 (pCR) and Fig. 6 (non-pCR) illustrate the hemoglobin map for two premenopausal patients imaged at three different time points during NAC regimen.

Discussion
We investigated whether the changes of DOTBIS features in the contralateral healthy breast are associated with therapy outcome in patients with stage II-III breast cancer receiving NAC. We observed that hemoglobin level is modifiable throughout the course of NAC. The magnitude of change is associated with pCR after only 2-3 weeks with the initiation of a taxane-based regimen.
Patients who achieved a pCR to NAC had a more significant reduction in DOTBIS-measured features than the non-pCR group. Previous studies have demonstrated statistically significant changes in optically derived parameters in breast cancer patients undergoing NAC and their correlation with tumor response [5,14,[24][25][26][27][28]. However, most of these studies looked at the tissue composition of the tumor volume, and few of them only used the contralateral healthy breast data as a normalizing factor for the total tissue hemoglobin concentration within the tumor region. The measurement of optically derived parameters in the affected breast is strongly dependent on selecting the tumor location or region of interest (ROI). Yet, it is well known that breast optical imaging has limitations regarding spatial resolution. Therefore, the optically derived parameters collected from the Fig. 3 Left: ctO 2 Hb changes after 2-3 weeks of chemotherapy showing a significant decrease for pCR patients but a non-significant change for the non-pCR group. Right: Box plot of the difference between ctO 2 Hb levels measured at baseline and after 2-3 weeks under Taxane regimen (TP1) for pCR and non-pCR patients showing a significant difference between both groups. ctO 2 Hb reduction levels at TP1 were larger for pCR patients contralateral unaffected breast can offer a more unbiased and non-ROI dependent measurement to follow up the course of changes in the breast tissue during NAC and associations with treatment outcome. The only two previous studies [11,29], which evaluated optical imaging changes during NAC in the contralateral normal tissue, did not perform analyses regarding tumor response. By associating contralateral healthy breast data with tumor response, we could provide a potential marker for evaluating chemotherapy regimens' efficacy early in the treatment course.
A similar association with pCR is also reported in the literature for background parenchymal enhancement (BPE) at breast magnetic resonance (MR) imaging in the contralateral normal breast. Several groups [30][31][32][33] have demonstrated an association between BPE and tumor response after receiving NAC in the last 4 years. Chen et al. found that BPE showed a significant decrease in those with pCR compared to non-pCR for pre/peri-menopausal patients after starting NAC [30]. Next, Preibsch et al. investigated the influence of chemotherapy on the degree of BPE and found correlations between tumor response and qualitative assessment of BPE following NAC [31]. You et al. also reported a positive association between overall BPE reduction at the early stage of NAC and tumor response, especially in patients with hormone receptor-negative breast cancers [32].
Given enough evidence that optical imaging is a powerful tool to study the breast tissue's structural and vascular properties [34], ctTHb and ctO 2 Hb are thought to be correlated to the amount of blood flow in the fibroglandular tissue and represent breast vascular supply and perfusion. The significant large reduction of DOTBIS-measured features for the premenopausal group may be explained by the ovarian function suppression that affects estrogen secretion and leads to a secondary change in breast tissue composition and its vascular characteristics [35,36]. A review published in   [12] also concluded that younger women had much larger benefits from chemotherapy than older patients, potentially due to the ovarian ablation induced by chemotherapeutic agents. Fibroglandular tissue (FGT) and BPE-MRI were also reported as markedly influenced by menopausal status, significantly decreasing after menopause for most women included in the study [37]. On the other hand, substantial changes in DOTBISmeasured parameters for those with pCR regardless of their menopausal status could also be associated with the direct damage of the vascular morphology and density in normal tissue caused by chemotherapy, as investigated by Miller et al. [38]. Besides, the mixed-effect model results indicated a statistically significant negative relationship between ctO 2 Hb and BMI. DOTBIS-measured ctO 2 Hb is thought to be correlated to breast density since it is related to the arterial blood supply carrying oxygen to meet the fibroglandular tissue metabolic demand [29]. In contrast, BMI is inversely related to percent density and acts as a co-founder of each other's effects [39][40][41][42]. In other words, overweight is correlated to a greater amount of fat in the breast, reflecting a larger non-dense tissue area in the mammogram and, consequently, lower percent density classification [41].
A limitation of our study was we did not reliably capture the patient's menstrual period during treatment and around the time of scans. Besides that, since the majority of the patients in our study did not have breast MR imaging before and after NAC treatment, we were not able to expand our analyses to correlations between MR based quantitative measurements and DOTBIS features. Finally, a more extensive study is also required to identify potential optical predictors of long-term outcomes in breast cancer patients who undergo NAC and understand chemotherapy's effect on reducing contralateral cancer risk.

Conclusion
In summary, this is the first study to confirm that optical derived metrics, measured by DOTBIS, in the contralateral non-tumor-bearing breast tissue, are modifiable with NAC treatment. ctO 2 Hb changes are proportional to the degree of pCR after 2-3 weeks of taxane-based regimen. Fig. 5 Corresponding hemoglobin levels map for the contralateral unaffected breast of 47-year-old women who achieved pCR after TCHP therapy for HER2+ breast cancer. MIP images (axial orientation) were acquired at baseline, after 2-3 weeks after NAC initiation (TP1), and at the pre-surgical time point (TP5) Fig. 6 Corresponding hemoglobin levels map for the contralateral unaffected breast of 46-year-old women classified as RCB III (non-pCR) after Tx12/ACx4 therapy for HER2-breast cancer. MIP images (axial orientation) were acquired at baseline, after 2-3 weeks of NAC (TP1), and at the presurgical time point (TP5)