Multispectral Optoacoustic Tomography in

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Case Report

Multispectral Optoacoustic Tomography in Assessment of Breast Tumor Margins During Breast-Conserving Surgery: A First-in-human Case Study Yonggeng Goh,1 Ghayathri Balasundaram,2 Mohesh Moothanchery,2 Amalina Attia,2 Xiuting Li,2 Hann Qian Lim,2 Neal Burton,3 Yi Qiu,3 Thomas Choudary Putti,4 Ching Wan Chan,5 Philip Iau,5 Siau Wei Tang,5 Celene Wei Qi Ng,5 Felicity Jane Pool,1 Premilla Pillay,1 Wynne Chua,1 Eide Sterling,1 Swee Tian Quek,1 Malini Olivo2 Clinical Practice Points Multispectral optoacoustic tomography (MSOT) is an

innovative state-of-the-art imaging modality that has gained popularity for in vivo breast imaging in recent years. Combining high-resolution images with endogenous differentiation of biochemical contents, MSOT could be an accurate ex vivo imaging modality for assessment of tumor margins during breast-conserving surgery to reduce margin positivity rates. Accurate intraoperative assessment of margins could lead to more precise surgery, allowing a smaller

volume of breast tissue to be removed without compromising the resection margins. To the authors’ knowledge, there has been no ex vivo study conducted to this day that uses MSOT in the assessment of breast tumor margins after lumpectomy. Hence, we would like to present the first case of breast tumor margin assessment using MSOT in a 55-year-old patient who underwent breast-conserving surgery for invasive ductal carcinoma.

Clinical Breast Cancer, Vol. 18, No. 6, e1247-50 ª 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Keywords: Breast-conserving surgery, Multispectral optoacoustic tomography, Tumor margin

Introduction Breast-conserving surgery (BCS) with adjuvant radiotherapy has become an accepted alternative to total mastectomy, improving cosmesis and patient satisfaction while maintaining clinically equivalent oncologic outcomes for appropriate patients. Accurate

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Department of Radiology, National University Hospital, Singapore Laboratory of Bio-Optical Imaging, Singapore Bioimaging Consortium, Singapore iThera Medical GmbH, Munich, Germany 4 Department of Pathology 5 Department of Breast Surgery, National University Hospital, Singapore 2 3

Submitted: Jul 17, 2018; Revised: Jul 31, 2018; Accepted: Jul 31, 2018; Epub: Aug 16, 2018 Addresses for correspondence: Prof Malini Olivo, PhD, Laboratory of Bio-Optical Imaging, Singapore Bioimaging Consortium, Singapore, Dr Goh Yonggeng, MBChB, FRCR, MCI, Department of Radiology, National University Hospital, Singapore E-mail contact: [email protected]; [email protected]

1526-8209/ª 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). https://doi.org/10.1016/j.clbc.2018.07.026

intraoperative assessment of tumor margins to ensure adequate tumor removal is crucial to reduce reoperation rates. Common strategies for improving clear margin rates include intraoperative frozen section,1 cavity shaves,2 on-table ultrasound,3 or x-rays and clinical palpation by an experienced surgeon. All of these methods have particular drawbacks (eg, long waiting time and high costs for frozen section results, interoperator variability with clinical palpation, and poor resolution from imaging [ie, x-ray]) and have not been shown to decrease margin positivity rates, with reported reoperation rates up to 20% to 40%.4,5 There is hence an unmet clinical need for an accurate and rapid intraoperative assessment tool for tumor margins in BCS. Multispectral optoacoustic tomography (MSOT) is an innovative state-of-the-art imaging modality that has gained popularity for in vivo breast imaging in recent years.6-11 MSOT is based on the principle of the “photoacoustic effect” (ie, formation of sound waves

Clinical Breast Cancer December 2018

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Assessment of Tumor Margins using Optoacoustic Tomography Figure 1 A, Breast-Conserving Surgery Specimen. The Specimen Has Been Labelled With Silk Stitches. Long Thread [ Lateral Margin; Medium Thread [ Medial Margin; Short Thread [ Superior Margin. B, Gross Macroscopic Specimen Demonstrates a MultiLobulated Tumor (Dotted Yellow Region) Which Extends Very Close to the Anterior Margins (Arrow). C, Hematoxylin and Eosin Stain Shows the Multi-Lobulated Tumor in Purple (Circle) Extending Very Close to the Anterior and Antero-Superior Margins (Arrows). Note the Tumor Demonstrates Tumor/Fibrous Bands Towards the Anterior Surface (Dotted Arrows)

following light absorption).12 This hybrid technique of detecting sounds waves from light energy allows generation of high-resolution combined ultrasound-optical images (mm) with sufficient penetration (ie, 1-3cm).13 In addition, MSOT is able to provide biochemical information about the imaged tissue because different materials respond to light energy differently. This allows endogenous differentiation of contents such as melanin, oxyhemoglobin, deoxyhemoglobin, lipids, and water. This technology is wellestablished with real-time clinical integrated optoacoustic and ultrasound imaging systems available that have been approved by the United States Food and Drug Administration.14 As the breast is composed predominantly of fatty tissue, the biochemical information from MSOT can potentially differentiate normal breast tissue from cancer tissue.15 Combined with highresolution ultrasound images, we hypothesize that MSOT could be an accurate intraoperative tool for breast tumor margin assessment. Accurate intraoperative assessment of margins could then lead to more precise surgery, allowing a smaller volume of breast tissue to be removed without compromising the resection margins (no tumor on ink for infiltrating ductal carcinoma [IDC], 2 mm for ductal carcinoma in situ [DCIS]).16,17 A similar ex vivo study using MSOT has been performed on other malignancies (eg, melanoma) for better selection of the excision margins.18 However, to the authors’ knowledge, there has been no ex vivo study conducted to date that uses MSOT in the assessment of breast tumor margins after lumpectomy. Hence, we would like to present the first case of breast tumor margin assessment using MSOT in a 55-year-old patient who underwent lumpectomy for IDC.

Case Report Patient Recruitment and Patient Characteristics Institutional board approval and informed consent was obtained. A 55-year-old patient presented to our hospital with a palpable right breast lump. She was subsequently diagnosed with IDC after triple assessment and underwent breast-conserving surgery (BCS). The excised tissue measured 4.5 5.7 3.0 cm, and the margins were

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labeled with silk sutures (Figure 1A). The fresh tissue sample was retrieved intraoperatively and imaged with MSOT prior to histologic assessment for gross pathology (Figure 1B) and hematoxylin and eosin staining (Figure 1C).

Instrument and Imaging Protocol Trained research personnel acquired ex vivo optoacoustic images of excised breast tissue samples using the inVision 512-echo (iThera Medical GmbH, Munich, Germany) MSOT system coupled with a customized 2-dimensional (2D) handheld probe consisting of a 2D array of 256 detector elements arranged along an arc (angle, 125 ) on a spherical surface (radius, 40 mm) with a center frequency of 5 MHz. The 2D probe provided cross-sectional 2D images in the x-z or y-z planes (z being the depth dimension, x and y are the 2 lateral dimensions), with an in-plane spatial resolution of 150 um and effective field-of-view (FOV) of 25 mm. The 2D probe was fixed to a computer-controlled stage within a chamber. Excised tissue samples were placed on a silicone bed and overlaid with ultrasound gel followed by a clear plastic bag of heavy water. The 2D probe was moved through the heavy water, across the sample, to provide good acoustic coupling. Optoacoustic signals from the imaged tissue were generated by wavelength-tunable optical parametric oscillator laser whose output beam is guided into fiber bundles integrated into the handheld probes. The laser provides excitation pulses at selectable wavelength from 660 nm to 1300 nm at a repetition rate of 10 Hz and per-pulse energy of 80 mJ at 730 nm. A real-time image preview window generated based on the backprojection algorithm was available during data acquisition. The light intensity was attenuated in probes to ensure compliance with ANSI limits on the maximum permissible exposure for human skin to pulsed laser radiation in order to avoid damage to molecular characteristics of the excised tissue. MSOT images were acquired at multiple wavelengths (710, 730, 760, 800, 850, 930, 1050, and 1100 nm); 5 frames were averaged per wavelength. Acquired images were reconstructed using model-linear and unmixed using linear regression algorithms available in ViewMSOT software.


Yonggeng Goh et al

Figure 2 Assessment of Breast Cancer Margins Using Multispectral Optoacoustic Tomography. A, Ultrasound Image From inVision 512 Echo System Demonstrating the Multi-Lobulated Tumor Within the Specimen (Dotted Yellow Region) Extending Close to the Anterior and Antero-Superior Margins (Yellow Arrows). Although the Tumor is Visible Under Ultrasound, There Remains Poor Resolution at the Boundaries Between the Tumor and the Margins/Surrounding Breast Tissue. B, Zoomed in Hematoxylin and Eosin-Stained Image of the Specimen Showing Tumor (Black Dotted Region) Corresponding to the Panel A. C, Unmixed Lipid Signal Demonstrating Normal Breast Parenchyma With 4.8-mm Fat Layer to the Right of the Specimen and Thinned out (2.4 mm) Lipid Layer on the Left Owing to Scalloping From the Multi-Lobulated Tumor Underneath. Note the Fibrous Band (Yellow Arrow) Corresponding to the Fibrous Band (Black Arrow in Figure 2B) on Hematoxylin and Eosin Stain. D, Unmixed Blood Signals Demonstrating Increased Vascularity of the Tumor Extending to the Margins (Yellow Arrows) in Keeping With Tumoral Angiogenesis. E, Merged Lipid and Blood Images, and F, Zoomed in Image of E Demonstrate Enriched Vascularity and Compromised Lipid Layer at the Anterior and Antero-Superior Surfaces Correspond to Positive Tumor Margins as Noted in the Histologic Specimen. Scale Bar Corresponds to 5 mm

Discussion We were able to successfully demonstrate the use of MSOT in assessing breast tumor margins in BCS. The ultrasound images (Figure 2A) generated by inVision 512 Echo system showed the tumor within the excised specimen, whereas optoacoustic images generated showed the biochemical contents of the lumpectomy specimen (eg, lipids [Figure 2C] and deoxyhemoglobin [Figure 2D]). Correlating with the hematoxylin and eosin stain (Figure 2B), the

optoacoustic image shows a thick band of lipid signal to the right of the tumor, which corresponds to normal breast tissue (Figure 2C). The lipid layer then thins out anteriorly owing to the presence of the tumor, which itself shows an absence of lipid signal secondary to replacement of normal tissue with tumor cells. The tumor is also noted to show increased peripheral vascularity (Figure 2D) secondary to angiogenesis. The increased vascularity extends to involve the anterior and antero-superior margins (Figure 2D). Overall findings


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Assessment of Tumor Margins using Optoacoustic Tomography (Figure 2E, F) suggested positive margin involvement, and the images obtained correlated closely with histology. From a clinical perspective, the availability of rapidly generated high-resolution images with adequate penetration depth provides critical and valuable information to the surgeon during BCS. A positive margin predicted on ex vivo imaging done intraoperatively would prompt immediate re-resection, reducing the need for a reoperation and its associated morbidity. Similarly, this technique allows better cosmetic outcomes without compromising oncologic principles because a more conservative approach can be taken during resection with reliable real-time assessment of margins. Presently, the gold-standard for assessment of tumor margins is histology. However, this requires processing and fixation of the excised breast specimen, which takes up to 24 hours. It is time-consuming and thus not feasible to perform in an intraoperative setting. Frozen sections are acceptable clinical alternatives but are often not practical owing to long procedure times (up to 20 minutes per surface/margin) and costs.19,20 Radiologic investigations such as intraoperative ultrasound have been shown to reduce local rates of recurrence and are widely performed in certain areas of practice.21,22 However, known drawbacks of ultrasound include operator dependence and distortion of specimens owing to pressure from the ultrasound probe. Because of the lack of a biochemical profile, there is also difficulty in identifying tumors that are not clearly distinct from normal breast tissue sonographically after lumpectomy. Optical techniques (eg, diffuse reflectance imaging, optical coherence tomography, Raman spectroscopy, fluorescent imaging) have been widely studied in the past decade to differentiate biochemical profile between normal breast parenchyma and breast tumor, in hope of assessing breast tumor margins intraoperatively.23-29 However, these techniques lack tissue penetration depth (< 3 mm) and are often unable to assess the excised tissue completely because of long procedure time. In short, there is a currently an unmet clinical need for a real-time intraoperative method for assessment of breast tumor margins that is rapid, accurate, and offers good penetration depth and high-resolution images. In our case study, we have demonstrated the potential of MSOT in assessment of breast tumor margins. The images obtained closely correlated to histology, and the modality offered good penetration depth. In addition, the scan was rapid (< 20 minutes) and allowed assessment of the entire tissue sample. This is important as the quick procedure time did not alter tissue properties for subsequent histologic analysis (accepted cold ischemia time of 30 minutes). Although the results of a single case may seem promising, larger cohort studies will need to be performed to ensure accuracy. Validation will also be required in larger studies to ensure reproducibility of results across different tumor subtypes as well as different sizes, shapes, and volumes of excised breast tissue.

Conclusion MSOT has promising potential as an ex vivo imaging modality for intraoperative assessment of breast tumor margins in the context of BCS.

Acknowledgments This work was supported in part by National Medical Research Council, Singapore’s “Clinician Scientist Seed Fund” (Y.G.G) and

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A*STAR (Agency for Science, Technology, and Research, Singapore) Biomedical Research Council intramural funds, and a research collaborative agreement (RCA) with iThera Medical GmbH (M.O.).

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