The objective of this study was to assess how tumor vasculature reacts to treatment and how these changes are measured with noninvasive NIR/US technology. Total hemoglobin concentration measured by this technology directly correlates to MVD, as shown in this study () and in an earlier study [33
]. However, we found in this study that there was no statistical difference between responders and nonresponders in MVD. Makris et al. [41
] reported lower tumor microvessel counts in patients with breast cancer treated with chemoendocrine therapy compared with untreated patients. However, the authors reported no statistical differences in MVD between responders and nonresponders. %BVI is a novel way of measuring blood vessel density, taking into consideration tHb concentration and vessel mass by volumetric measurements. Recently, optical tomography has been explored by several research groups for its potential role in monitoring response to chemotherapy [18,27,33,41
]. Case reports from these groups are consistent with our results revealing that most responders demonstrate a reduction in tHb level during chemotherapy. However, we found in this pilot study that tHb or MVD does not correlate to pathologic response if volumetric measures are not included.
All responding patients (A + B) showed a reduction in %BVI. The reduction is never 100% but was as large as 79% in our one complete responder. It is possible that the rapidly proliferating components of the vasculature and more immature components regress, leaving a better differentiated, more established vasculature that is slow to regress [42
]. There was no single pattern of response: some had volume reductions without change in tHb; others had tHb reduction with no change in volume; others had a combination of volume and tHb reduction. This heterogeneity of response may be explained in part by the differential chemosensitivity of the vasculature or the tumor and host.
Gene expression analysis has identified three major breast cancer subtypes [43
] that have different prognoses [44
]. However, a recent study has shown that patients who had pathologically complete response to chemotherapy had a good prognosis regardless of subtype [45
]. Thus the correlation between vascular response, as measured by %BVI, and complete or near-complete pathologic response is very important clinically. If one can accurately monitor response repeatedly and as easily as performing a US exam, systemic therapy can be altered so the most efficacious drugs could be used. Ideally, chemotherapy should be monitored at earlier cycles as this could facilitate modification of the regimen to enable the lesion to be maximally treated and for the treatment response to be observed [8–10,13,34
]. In a recent study, Cerussi et al. [34
] monitored 11 patients who underwent neoadjuvant chemotherapy before and within 1 week of initial treatment. The authors found that deoxygenated hemoglobin decreased within the first week in pathologically confirmed responders, whereas no significant change was found in nonresponders. In addition, the measured tHb decreased in all responders. In this study, most patients were monitored at pretreatment, cycle 2 and 4, and before surgery. No earlier response data were obtained. However, data of complete and near-complete responder group at cycle 2 do show a noticeable difference in %BVI than partial (B) and nonresponders (C). This suggests that NIR/US may be sensitive enough to identify some responders at earlier treatment cycles. Because our samples are very limited, more patients are needed to validate these initial results.
Another important study objective was to compare vascular response measured by NIR/US with results obtained from conventional US and new imaging modality MRI. In this study, MRI was comparable to NIR/US and was more accurate than US in distinguishing nonresponders from partial responders. MRI, however, is an expensive modality to be used repeatedly during neoadjuvant chemotherapy. In addition, some patients are also troubled by the tight constraints of the MRI machine. However, compared with MRI, optical systems are more cost-effective and portable for use in the doctors' office, and the flexible light guides can be easily coupled to clinical US probes for repeated imaging. The limitation of our reported comparison study is that only eight patients had MRI imaging results. Nevertheless, the agreement between %BVI results and MRI measurements in accurately classifying the different response groups demonstrates the potential of NIR/US as a cost-effective alternative for monitoring chemotherapeutic response.
Over the last decade, it has become known that hypoxia changes the pattern of gene expression that alters the malignant potential of tumors leading to more aggressive behavior and poor response to various forms of chemotherapy [46,47
]. Of the 11 patients, five were imaged with the second prototype featuring an additional wavelength at 690 nm that is more sensitive to deoxygenated hemoglobin changes. We observed a trend that the carcinomas were deoxygenated before the treatment and were more oxygenated toward the end of the treatment. No statistical difference in relative oxygen saturation was observed between responders and nonresponders. Several reports also indicate variable oxygenation changes during chemotherapy [18,27,33
], which are in agreement with our preliminary observations. More patient data is needed to obtain statistically valid information on tumor hypoxia changes during chemotherapy. Hypoxia imaging may allow better definition of a population that would benefit from novel anti-hypoxia-directed therapies.
The region of interest (ROI) selection from co-registered US image used for NIR imaging reconstruction should be noted. Optical images were reconstructed by segmenting the volume underneath the probe into a fine-mesh ROI and the background. Because the carcinomas were large in this group of patients, the entire probe size (9 x 9 cm2
) was used as the ROI in spatial dimensions. Therefore, the reconstructed optical images were independent of tumor spatial dimensions seen by US. In our early phantom study [36
], we found no significant difference in reconstructed optical properties when the ROI was twice or three times larger in spatial dimensions than a target 2 to 3 cm in size. The depth localization of NIR diffusive wave is very poor and a tighter ROI in depth dimension is mainly set by coregistered US and therefore depends on margins seen by US. Assisted by chest-wall structure and normal tissue layer structures seen in coregistered US, we can select the ROI in depth dimension reasonably well. In general, we give at least 0.5 cm larger margin in depth than the ultrasonically identified upper and deeper layers. In some difficult cases with unknown margins near the chest wall, we use the chestwall depth as the deep margin for the ROI.
Optical imaging reconstruction is performed by using the standard perturbation approach where the difference between the measurements obtained at the lesion site and the normal contralateral site is used as the scattered field (Usd) for inversion. Using this approach, we could subtract any changes related to normal breast tissue response to chemotherapy at each monitoring point. Because this approach is sensitive to contralateral site selection, we have checked possible bilateral disease from co-registered US and MRI (if available) and did not find any bilateral disease case in this group of patients. In this study, the two prototype systems used have an identical design in terms of detectors used, source power level at the fiber tips on the probe, and electronic gains. The only differences were the addition of a 690-nm source for better estimation of oxygen saturation and improved system packaging for ease of transportation. Extensive phantom studies have demonstrated comparable performance in target characterization for the two systems. In addition, %BVI is a relative measurement compared to pretreatment baseline for each patient and it is not sensitive to minor system differences as long as the same system is used for each patient.
%BVI is the ratio of measured BVI (product of measured tHb volume and average tHb) at each assessment point over the pretreatment baseline. Errors in tHb estimate can affect the BVI measurements. One type of error is related to the quantification accuracy of tHb. Our phantom studies showed that the estimation accuracy for typical larger absorbers was between 55% and 101% for the typical depth we studied. However, because the %BVI is the ratio of measured BVIs, it is less susceptible to tHb quantification accuracy. Another type of error is related to the repeatability of the tHb quantification at each assessment point. For each patient, we took several data sets at the lesion area and the tHb and the volume reported were average values. The mean variations from the average tHb estimates for this group of patients was 2.3 µM/l, which was 4% of the estimated tHb.
When the NIR/US technique is used to obtain the pretreatment baseline, the data should be taken either before the patient's diagnostic core biopsy or after a certain period. A hematoma after a core biopsy procedure could partially contribute to a higher hemoglobin level, which could be reduced to some extent due to the normal healing process. In our study, baseline data were obtained before the core biopsy in three patients and after in eight patients with an average of 30 days (range, 14 to 52 days). One nonresponder (patient 9) who had her NIR/US study 14 days after core biopsy showed the highest %BVI reduction in this group, which could be partially affected by hematoma. The rest of the seven patients had their NIR/US study at an average of 32 days (range, 20 to 52 days) after initial biopsy.
This pilot study has several limitations, primarily the small population of women with locally advanced cancers. For all patients except one, surgery was performed less than 1 month from posttreatment NIR/US (average, 14 days) and MRI (average, 18 days). One patient (patient 5) had a longer delay between surgery and post-NIR/US (46 days) and MRI (48 days). These intervals were similar to those of other previously reported studies [12,48
Our initial results have shown that NIR/US using volumetric vascular measurements is a valuable tool in assessing in vivo vascular response to neoadjuvant chemotherapy. It is a quick and noninvasive method that may prove invaluable for neoadjuvant treatments to repeatedly monitor the impact of novel agents on vascular distribution. Our initial results support the need to conduct future studies to assess the value of NIR/US in the prediction of early pathologic tumor response and residual disease before surgery.