2.1.

Diffuse Optical Spectroscopic Imaging Instrumentation

A description of the basic components of the DOSI instrument has been previously presented.^18,19 Briefly, DOSI consists of broadband frequency-domain photon migration (FDPM) combined with broadband time-independent near-infrared spectroscopy (CW-NIRS). This provides absorption and reduced scattering spectra in centimeter-thick tissues from 650 to 1000 nm. The FDPM component uses six laser diodes (Blue Sky Research, Sanyo, Mitsubishi) at the wavelengths 660, 680, 785, 810, 830, and 850 nm that are coupled into a fiber bundle and delivered to a handpiece that contacts the tissue. The breast is illuminated sequentially by each laser diode, which is intensity modulated at 601 modulation frequencies from 50 to 600 MHz. Phase and amplitude are measured from three averaged frequency sweeps for a total of $\sim 200\mathrm{ms}$ per laser diode. The diffusely reflected scattered light propagates through tissue and is detected by an avalanche photodiode (Hamamatsu module C5658) mounted inside a temperature-controlled hand-held probe. The CW-NIRS component uses a high-intensity tungsten–halogen source (Ocean Optics model HL-2000-HP-FHSA) to illuminate the tissue. The diffusely scattered broadband light that propagates through the tissue is detected by a grating-based spectrometer that collects light from 650 to 1000 nm (1024 pixels, BWTek model 611E). The FDPM and CW-NIRS sources are coupled by independent optical fibers mounted into the hand-held probe. The source–detector separation was 22 mm (phantom measurement) or 28 mm (breast measurement) for both FDPM and CW-NIRS components with fibers placed in an overlapping geometry.²⁰ A typical DOSI measurement for the Network Analyzer-based instrument used in this study requires a total of 2 to 5 s per scan location and is composed of three averaged FDPM sweeps of 601 modulation frequencies and time-integrated broadband CW reflectance spectra.

FDPM and CW-NIRS data are combined using a diffusion theory model to provide broadband absorption (${\mu }_{\mathrm{a}}$) and reduced scattering (${\mu }_{\mathrm{s}}^{\prime }$) spectra from 650 to 1000 nm. In the FDPM measurement, the phase and amplitude of the detected light are recorded as functions of source modulation frequency and fit to a diffusive model of light transport with semi-infinite boundary conditions to recover ${\mu }_{\mathrm{a}}$ and ${\mu }_{\mathrm{s}}^{\prime }$ at each of the six laser wavelengths. CW-NIRS broadband reflectance spectra were converted into absolute absorption spectra using two steps. First, as per Mie scattering theory, the spectral shape of the reduced scattering spectrum is assumed to follow a power law of the form ${\mu }_{\mathrm{s}}^{\prime }=A{\mu }^{-\mathrm{sp}}$, where $A$ is the scatter amplitude and sp is the scatter power or the exponent of the reduced scattering spectrum. The power-law fit to the FDPM discrete laser diode spectrum provides a scatter correction for the CW-NIRS reflectance spectrum. We then fit the CW-NIRS reflectance intensity at each of the laser diode wavelengths to the reflectance calculated from the FDPM-measured absolute absorption values. Thus, the CW-NIRS reflectance spectrum intensity is quantitatively scaled using the FDPM discrete ${\mu }_{\mathrm{a}}$ measurements. The absolute absorption spectrum is then extracted by fitting the corrected reflectance spectrum to a diffusion reflectance model.¹⁸

A picture of the DOSI instrument used in this study is shown in Fig. 1(a). The calibration tools of the DOSI instrument are shown in Figs. 1(b) and 1(c). It consists of a solid tissue-simulating phantom [Fig. 1(b)], which is used to measure the FDPM instrument response function (IRF) (explicitly, phase offsets, and amplitude scale factors), and a reflectance standard for the broadband wavelength calibration [Fig. 1(c)]. The FDPM IRF measurement is similar to “blanking” performed in a conventional spectrophotometer; using a known attenuation (such as air), one can remove instrument artifacts such as wavelength-dependent detector responses. In the case of the FDPM calibration, the tissue-simulating phantoms have known ${\mu }_{\mathrm{a}}$ and ${\mu }_{\mathrm{s}}^{\prime }$ values, such that the phase and amplitude changes due to tissue (or sample) can be separated from the IRF. In the case of the broadband wavelength calibration, a standard spectralon reflectance standard (Labsphere) is used to correct the wavelength-dependent variations caused by the light source, detector response, and optical fiber throughput.

Fig. 1

(a) DOSI instrument and (b) and (c) illustrate the probe placed on the calibration phantom (FDPM calibration) and on the reflectance standard (broadband calibration), respectively.

2.2.

ACRIN-6691 Trial—Protocol and Study Design

The ACRIN-6691 trial was designed to evaluate the sensitivity of DOSI to monitor and predict the pathologic response of breast tumors in patients treated with NAC.⁵ The long-term goal is to provide oncologists with a simple, risk-free bedside tool that can be used to inform medical decisions. These include modifying or adapting the chemotherapy regimen, duration, and timing of surgery to maximize therapeutic response and minimize unnecessary toxicity.

A total of 60 breast cancer patients receiving NAC were measured longitudinally using the DOSI instrument in six different clinical sites. The five following sites, Dartmouth, MGH, UCI, UCSF, and Upenn used identical DOSI instruments. MDACC joined the study about 1.5 year after its activation and used a next generation instrument that was excluded from this analysis. The subjects were measured four times throughout NAC: baseline (pretreatment), early therapy (5 to 10 days after the initiation of the first cycle of the chemotherapy), midtherapy (halfway through the therapy protocol), and posttherapy (prior to surgery). For these patients, the typical chemotherapy length is 4 months.

2.3.

Solid Phantom and Reflectance Standard—Calibration and Testing

Each ACRIN site possessed one broadband reflectance standard and two solid tissue-simulating optical phantoms, Ph-A and Ph-B. The Ph-A phantoms, fabricated at UCI, were issued from the same fabrication batch specifically for the purpose of the ACRIN study. The specifics of these phantoms, including the fabrication process, have been previously published.²¹ The Ph-B phantoms were developed by Institut National d'Optique, Inc. (Quebec, Canada). The phantoms within a Ph series are expected to have the same optical properties as they were fabricated from the same batch. All phantoms have similar optical properties as breast tissue. The Ph-A and Ph-B phantoms were arbitrarily chosen to calibrate the FDPM instrument response during patient measurements and used as an additional calibration phantom for instrument testing, respectively. Measuring the IRF each session corrects any possible IRF changes between measurements. To determine IRF, the optical properties of the reference phantom must be known, and these were derived from comprehensive series of multidistance, multifrequency, broadband measurements, a standard procedure that, assuming phantom homogeneity, utilizes the distance-dependent phase and amplitude behavior to calculate optical properties.^21,22

The solid phantoms are contained in a custom-made case, as shown Fig. 1(b). Figure 1(c) shows the reflectance standard used to calibrate the broadband part of the instrument. A mask at the top of the phantom case and the reflectance standard is designed to limit placement error by helping the operator to repeatedly position the probe on the same location.²¹

Three sets of three calibration measurements using the two phantoms and the reflectance standard were performed for every patient measurement. These calibration measurements were acquired immediately before, midway through, and after each breast patient measurement. Iterating the three calibration measurements throughout the measurement session serves two purposes: (1) securing a robust, redundant calibration dataset and (2) assessing instrument performance throughout the session by monitoring the optical properties of a fixed phantom over time.

2.4.

Data Analysis

Several phantom measurement procedures were designed to test instrument performance. These procedures consisted of repeat measurements either of the same phantom or of different phantoms and in various conditions. We report the results of these tests using the standard deviation (Stdev) of these series of measurements and the normalized standard deviation (NSD), which is defined as the Stdev/mean and reported in percent (%). In case of broadband data, we first obtain the Stdev and NSD at all wavelengths of the spectrum and report their spectral average, minimum, maximum, and median.

3.1.

Methodology to Assess Uncertainties for the ACRIN-6691 Study

Figure 2 presents the overview of the methodology used to assess the uncertainties in the context of the ACRIN-6691 study. Uncertainties originate from three main sources: phantom, instrument, and operator. Factors contributing to each of these include, but are not limited to, (1) phantom material consistency, shelf life, and spatial uniformity, (2) instrument warm up, stability, reproducibility, and environmental effects, and (3) procedural consistency, probe operation, and positioning by different operators. To understand the relative contributions from each of these sources, we grouped the DOSI calibration data into five main conditions. The variability in calibration data across all conditions (A through E) in Fig. 2 is potentially a sum of all these factors.

Fig. 2

Overview of methodology to assess uncertainties.

The first, condition A, examines the DOSI platform performance with one operator, one phantom, and one instrument, which is the most favorable situation in terms of potential sources of uncertainty. Conditions, B, C, and D, consecutively, assess the study uncertainties by varying one of the three test parameters, operator, phantom, and instrument, respectively, and holding two parameters constant. For example, condition B examines multiple operators ($N$) with one instrument and one phantom, while C examines multiple phantoms ($N$) with one operator and one instrument, etc. Condition E combines the contribution of all sources to DOSI measurement error by examining the performance of all instruments, all phantoms and with several operators. Multiple measurements and time periods were used to characterize each of the five main test conditions. A brief summary and description of each test for the five main conditions is provided in Table 1.

Table 1

Description of the different tests performed for each condition in the context of the validation and standardization process of the ACRIN-6691 study. The number of scans refers to independent measurements of combined FDPM and CW-NIRS consisting of multiple temporal and spectral sweeps and lasting 2 to 5 s per scan (see Sec. 2.1).

3.2.

Condition A: One Operator, One Phantom, and One Instrument

Placement and short-term instrument stability tests were conducted on the DOSI instrument at UCI using a Ph-A phantom. For the placement test, test A1, the phantom was measured for nine consecutive times, removing and replacing the probe between each measurement over 1 h, which is the typical patient measurement session duration. For the instrument stability test, test A2, a Ph-A phantom was measured continuously every minute over a 1-h period with the probe fixed onto the phantom. For both the placement and 1-h stability tests, tests A1 and A2, the broadband deviations were found to be lower than $0.0010{\mathrm{mm}}^{-1}$ for absorption and $0.01{\mathrm{mm}}^{-1}$ for reduced scattering coefficients (data not shown).

Instrument stability was also investigated over a longer time frame to determine the potential impact of variations over the course of chemotherapy, test A3. Measurements of a Ph-A phantom using the DOSI instrument at UCI were acquired by the same operator at the four study time-points over 4 months (typical time of chemotherapy treatment at UCI). This corresponded to nine scans per time-point, resulting in a total of 36 scans. Figure 3 and Table 2 provide the resulting deviations in broadband optical properties. They ranged from 0.0002 to $0.0005{\mathrm{mm}}^{-1}$ for absorption and from 0.01 to $0.03{\mathrm{mm}}^{-1}$ for reduced scattering coefficients. As shown in Fig. 3(a) inset, the greatest deviation in absorption occurred at day 1 and did not seem to follow an increasing or decreasing pattern over time.

Fig. 3

Chemotherapy-long stability test of the DOSI instrument at UCI, test A3: broadband, (a) absorption and (b) reduced scattering, of a Ph-A phantom measured four times (day 1: presurgery, day 16: early therapy, day 55: midtherapy, and day 126: posttherapy) over a 4-month period. Inset: expanded scale (a).

Table 2

Deviations in broadband absorption and reduced scattering of a Ph-A phantom when measured four times over 4 months using the DOSI instrument at UCI.

3.3.

Condition B: N Operators, One Phantom, and One Instrument

The instrument stability was investigated on a Ph-A phantom over the 2-year duration of the study using the DOSI instrument at UCI, test B1. A total of 150 scans were performed by four different trained operators on 17 different days during these 2 years. The same Ph-B phantom was used to calibrate the instrument response of all measurements. The resulting deviations in broadband absorption and reduced scattering are presented in Table 3 and Fig. 4. The deviations ranged from 0.0002 to $0.0007{\mathrm{mm}}^{-1}$ in absorption and from 0.01 to $0.03{\mathrm{mm}}^{-1}$ in reduced scattering coefficients.

Table 3

Deviations over 2 years in broadband absorption and reduced scattering of a Ph-A phantom measured at UCI by four different operators, test B1.

Fig. 4

Deviations in broadband (a) absorption and (b) reduced scattering of a Ph-A phantom measured 17 times over 2 years at UCI by four different operators, test B1. The black lines show the average spectra. The gray lines show the range (min/max) of spectra.

3.4.

Condition C: One Operator, N Phantoms, and One Instrument

Using the DOSI instrument at UCI, one operator measured each phantom three times from the series of Ph-A phantoms to validate their uniformity, test C1. The phantoms were manufactured from the same batch and, thus, should have the same optical properties. Figure 5 provides the broadband optical properties from these compiled measurements, and Table 4 summarizes the Stdevs and coefficient of variation averaged over the whole spectral range (650 to 1000 nm). The broadband deviations within the series of Ph-A phantoms were found below 0.0003 and $0.01{\mathrm{mm}}^{-1}$ in absorption and in reduced scattering, respectively.

Fig. 5

(a) Absorption and (b) reduced scattering spectra of the Ph-A phantom series measured by one operator with a single DOSI instrument, test C1.

Table 4

Deviations in broadband absorption and reduced scattering within the Ph-A phantom series, test C1.

Overall, the absorption and reduced scattering coefficients were found to be similar among a series, which is consistent with the fact that they were fabricated in the same batch.

3.5.

Condition D: One Operator, One Phantom, and N Instruments

The optical properties recovered from a Ph-A phantom measured using the different DOSI instruments across sites were compared. These measurements were performed by a single operator who traveled to the sites with a Ph-A phantom and an additional calibration phantom.

Figure 6 presents the recovered optical properties of Ph-A phantom from all sites. Similar absorption and reduced scattering coefficients were recovered by all instruments. The absorption spectra recovered at Upenn showed the largest disagreement compared to the other sites, specifically below 750 nm. Overall, the broadband deviations in absorption and in reduced scattering within the different instruments ranged from $<0.0001$ to $0.0010{\mathrm{mm}}^{-1}$ and from 0.01 to $0.02{\mathrm{mm}}^{-1}$, respectively (see Table 5).

Fig. 6

(a) Broadband absorption and (b) reduced scattering spectra of a single Ph-A phantom measured at the five research sites by a single operator, test D1.

Table 5

Deviations in broadband absorption and reduced scattering of a Ph-A phantom measured at five research sites by a single operator, test D1.

3.6.

Condition E: N Operators, N Phantoms, and N Instruments

The optical properties recovered from the Ph-A phantoms measured at their respective sites by the site-specific operator were compared, test E1. The deviations between the broadband-measured absorption and reduced scattering coefficients of the Ph-A phantoms range from 0.0001 to $0.0010{\mathrm{mm}}^{-1}$ and from 0.01 to $0.02{\mathrm{mm}}^{-1}$, respectively, see Table 6. The largest deviations in broadband absorption recovery originate from the lower wavelength range of the spectra, below 760 nm, see Fig. 7. As for condition D, this phenomenon is particularly visible for the data acquired at Upenn.

Table 6

Deviations in broadband absorption and reduced scattering of the Ph-A phantoms when measured at their corresponding ACRIN site, test E1.

Fig. 7

(a) Broadband absorption and (b) reduced scattering spectra of the Ph-A phantoms measured at their sites, test E1.

Last, we compared optical properties recovered from all Ph-A phantoms measured at their respective sites by the on-site operators throughout the 2-year study duration, test E2. This corresponded to a total of 480 scans acquired on 58 different days by 18 different operators on the five different instruments; the resulting absorption and reduced scattering spectra are shown in Fig. 8. This situation represents the worst-case scenario in term of uncertainties, as it involved measurements on multiple Ph-A phantoms, by many different operators, using multiple instruments, over a 2-year period. Note that different Ph-B phantoms (the ones specific to the site) were also used to calibrate the instrument response of these measurements. The deviations between the broadband-measured absorption and reduced scattering coefficients of the Ph-A phantoms measured at their sites range from 0.0003 to $0.0010{\mathrm{mm}}^{-1}$ and from 0.02 to $0.06{\mathrm{mm}}^{-1}$, respectively, see Table 7.

Fig. 8

Deviations in (a) broadband absorption and (b) reduced scattering of the Ph-A phantoms measured 480 times over 2 years across the five sites by 18 different operators, test E2. The black line shows the average spectra. The gray lines show the range (min–max) of spectra.

Table 7

Deviations in broadband absorption and reduced scattering of 480 scans of the different Ph-A phantoms measured at their corresponding ACRIN site by 18 operators over 2 years, test E2.