Identification Of Tumor Margins Research Articles

Synthesis and In Vivo Evaluation of a Site-specifically Labeled Radioimmunoconjugate for Dual-Modal (PET/NIRF) Imaging of MT1-MMP in Sarcomas.

Bone sarcomas are devastating primary bone cancers that mostly affect children, young adults, and the elderly. These aggressive tumors are associated with poor survival, and surgery remains the mainstay of treatment. Surgical planning is increasingly informed by positron emission tomography (PET), and tumor margin identification during surgery is aided by near-infrared fluorescence (NIRF) imaging, yet these investigations are confounded by probes that lack specificity for sarcoma biomarkers. We report the development of a dual-modal (PET/NIRF) immunoconjugate ([89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW) that targets MT1-MMP, a matrix metalloproteinase overexpressed in high-grade sarcomas. [89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW was synthesized via site-specific chemoenzymatic glycan modification, characterized, and isolated in high specific activity and radiochemical purity. Saturation binding and immunoreactivity assays indicated only minor perturbation of binding properties. A novel mouse model of dedifferentiated chondrosarcoma based on intrafemoral inoculation of HT1080 WT or KO cells (high and low MT1-MMP expression, respectively) was used to evaluate target binding and biodistribution. Fluorescence and Cerenkov luminescence images of [89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW showed preferential uptake in HT1080 WT tumors. Ex vivo gamma counting revealed that uptake in MT1-MMP-positive tumors was significantly higher than that in control groups. Taken together, [89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW is a promising dual-modal sarcoma imaging agent for pre-operative surgical planning and intraoperative surgical guidance.

Label-free identification of human glioma xenograft of mouse brain with quantitative ultraviolet photoacoustic histology imaging.

The ability to unveil molecular specificities of endogenous nonfluorescent chromophores of ultraviolet photoacoustic imaging technology enables label-free histology imaging of tissue specimens. In this work, we exploit ultraviolet photoacoustic microscopy for identifying human glioma xenograft of mouse brain ex vivo. Intrinsically excellent imaging contrast of cell nucleus at ultraviolet photoacoustic illumination along with good spatial resolution allows for discerning the brain glioma of freshly-harvested thick brain slices, which circumvents laborious time-consuming preparations of the tissue specimens including micrometer-thick slicing and H&E staining that are prerequisites in standard histology analysis. The identification of tumor margins and quantitative analysis of tumor areas is implemented, representing good agreement with the standard H&E-stained observations. Quantitative ultraviolet photoacoustic microscopy can access fast pathological assessment to the brain tissues, and thus potentially facilitates intraoperative brain tumor resection to precisely remove all cancerous cells and preserve healthy tissue for maintaining its essential function.

PATH-43. RAMAN SPECTROSCOPY AS A TOOL IN NEUROSURGERY

Abstract BACKGROUND Raman Spectra have been shown to be sufficiently characteristic to their samples of origin that they can be used in a wide range of applications including distinction of intracranial tumors. While not replacing pathological analysis, the advantage of non-destructive sample analysis and extremely fast feedback make this technique an interesting tool for surgical use. METHODS We sampled intractanial tumors from more than 300 patients at the Centre Hospitalier Luxembourg over a period of three years and compared the spectra of different tumor entities, different tumor subregions and healthy surrounding tissue. We created machine-learning based classifiers that include tissue identification as well as diagnostics. RESULTS To this end, we solved several classes in the intracranial tumor classification, and developed classifiers to distinguish primary central nervous system lymphoma from glioblastoma, which is an important differential diagnosis, as well as meningioma from the surrounding healthy dura mater for identification of tumor tissue. Within glioblastoma, we resolve necrotic, vital tumor tissue and peritumoral infiltration zone.We are currently developing a multi-class classifier incorporating all tissue types measured. CONCLUSIONS Raman Spectroscopy has the potential to aid the surgeon in the surgery theater by providing a quick assessment of the tissue analyzed with regards to both tumor identity and tumor margin identification. Once a reliable classifier based on sufficient patient samples is developed, this may even be integrated into a surgical microscope or a neuronavigation system.

Multi-Spectral Clinical Prototype for Fluorophore Detection

Identification of tumour margins during resection of the brain is critical for improving the post-operative outcomes. Current methods of tumour identification use 5-ALA, an exogenous precursor, metabolized to fluorescent PpIX in tumour tissue. Although visible under fluorescent microscope, PpIX is easily photo-bleached and tumour tagging is subjective, resulting in tumour under-resection and accelerated recurrence. To address this issue, photo-bleaching resistant and quantitative method is required. This study describes the characterization of a pulsed, multi-wavelengths system designed to measure diffuse reflectance and auto-fluorescence under strong ambient illumination conditions. The performance was tested on n = 400 liquid tissue phantoms containing a wide concentration range of absorber, scatterer and two fluorophores as well as on ex-vivo samples of gray and white matter. The background subtraction technique was shown to be efficient for a range of ambient illumination intensities. A linear relationship was observed between system response and predicted fluorophore concentrations as well as 97.8% accuracy of tissue classification by 5-fold cross-correlation, linear SVM.

Intraoperative Label‐Free Photoacoustic Histopathology of Clinical Specimens

AbstractDuring cancer resection surgeries, intraoperative histopathologic examination of the surgical specimen is crucial for tumor margin identification. A conventional frozen‐section analysis requires complex tissue processing, which prolongs surgery and potentially introduces interpretation errors. Here, as a novel approach to label‐free intraoperative histopathology, a high‐speed reflection‐mode ultraviolet photoacoustic microscopy (UV‐PAM) system employing a waterproof 1‐axis microelectromechanical systems scanner is demonstrated. Label‐free nuclear imaging is photoacoustically verified using tissue sections excised from mice and humans. Moreover, by imaging clinical specimens from cancer patients and numerically quantifying the histopathologic results, it is successfully demonstrated that the proposed UV‐PAM system has great potential as an alternative intraoperative histopathology method with minimal tissue preparation processes.

Autonomous pick-and-place using the dVRK

PurposeRobotic-assisted partial nephrectomy (RAPN) is a tissue-preserving approach to treating renal cancer, where ultrasound (US) imaging is used for intra-operative identification of tumour margins and localisation of blood vessels. With the da Vinci Surgical System (Sunnyvale, CA), the US probe is inserted through an auxiliary access port, grasped by the robotic tool and moved over the surface of the kidney. Images from US probe are displayed separately to the surgical site video within the surgical console leaving the surgeon to interpret and co-registers information which is challenging and complicates the procedural workflow.MethodsWe introduce a novel software architecture to support a hardware soft robotic rail designed to automate intra-operative US acquisition. As a preliminary step towards complete task automation, we automatically grasp the rail and position it on the tissue surface so that the surgeon is then able to manipulate manually the US probe along it.ResultsA preliminary clinical study, involving five surgeons, was carried out to evaluate the potential performance of the system. Results indicate that the proposed semi-autonomous approach reduced the time needed to complete a US scan compared to manual tele-operation.ConclusionProcedural automation can be an important workflow enhancement functionality in future robotic surgery systems. We have shown a preliminary study on semi-autonomous US imaging, and this could support more efficient data acquisition.

Pathologic correlation with near infrared-indocyanine green guided surgery for pediatric liver cancer

PurposeHepatoblastoma (HB) and hepatocellular carcinoma (HCC) are the most common primary malignant tumors of childhood. Intraoperative indocyanine green (ICG) administration with near-infrared imaging (NIR) has emerged as a surgical technology that can be used to assist with localization of pulmonary metastases secondary to HB; however, there has been limited application as an adjunct for resection of the primary liver tumor and assessment of extrahepatic disease. MethodsWe present 14 patients treated for HB, HCC, and malignant rhabdoid tumor at our institution with the use of intraoperative NIR-ICG guidance. All patients were treated with 0.2–0.75 mg/kg IV ICG, 48–96 h prior to surgery. Intraoperative NIR-ICG guided imaging was performed with several commercial devices. ResultsIntraoperative NIR-ICG guidance allowed pulmonary metastasectomy in five patients using thoracoscopy or thoracotomy allowing for visualization of multiple nodules not seen on preoperative imaging most of which were positive for malignancy. NIR-ICG guidance allowed for assessment of extrahepatic extension in three patients; an HCC patient with extrahepatic lymph node extension of disease, an HB patient with extrapulmonary thoracic recurrence in the diaphragm and chest wall, and a patient with tumor rupture at diagnosis with peritoneal nodules at the time of surgery. This technique was used to guide partial hepatectomy in 11 patients for which the technique enabled successful identification of tumor and tumor margins. Three patients had nonspecific staining of the liver secondary to decreased timing from ICG injection to surgery or biliary obstruction. NIR-ICG enabled resection of satellite HB lesions in three multifocal patients and confirmed a benign satellite lesion in two additional patients. ConclusionsIntraoperative use of NIR-ICG imaging during partial hepatectomy enabled enhanced identification and guidance for surgical resection of extrahepatic disease and multifocal liver tumors for the treatment of children with primary liver cancer.

A Novel Fluorescent c-Met Targeted Imaging Agent for Intra-Operative Colonic Tumour Mapping: Translation from the Laboratory into a Clinical Trial

Background: Curative resection of colon cancer requires accurate intraoperative identification of tumour margins and lymph node (LN) burden. The c-Met protein is important for cell differentiation and migration and is overexpressed in many gastrointestinal cancers. We explored EMI-137, a novel c-Met targeting fluorescent probe, for application in fluorescence-guided colon surgery, in HT-29 colorectal cancer (CRC) cell line and an in vivo murine model. This informed a first-in-man clinical trial of EMI-137. Methods: HT-29 cells were used as a representative CRC model. SiRNA was used to demonstrate the specificity of EMI-137 for c-Met. A HT-29 CRC xenograft model was developed in female BALB/c mice. Tumours were cultured for 14 days to 10mm diameter. EMI-137 (0.18mg/kg) was injected into tail veins and biodistribution analysed through in vivo fluorescent imaging. Nine patients, aged 67-77 years, received a single intravenous dose of EMI-137 (0.13mg/kg), 1-3 hours before laparoscopic-assisted colon cancer resection surgery (NCT03360461). Tumour and LN fluorescence was assessed with a modified Karl Storz® laparoscope. Immunohistochemistry with a monoclonal antibody analysed c-Met expression in eight resected samples. Findings: c-Met expression in HT-29 cells was effectively silenced and imaged with EMI-137. Strong EMI-137 uptake in tumour xenografts was observed for up to 6 hours post-administration. At clinical trial, no serious adverse events related to EMI-137 were reported. Marked background fluorescence was observed in all trial participants, 4/9 showed an increase in tumour fluorescence over background; 5/9 had histological LN metastases; no fluorescent LN were detected intraoperatively. All primary tumours (8/8) and malignant LN (15/15) exhibited moderate to high c-Met protein expression. Interpretation: EMI-137, binds specifically to the human c-Met transmembrane protein, is safe in patients and with further refinement, shows potential for long-term application in fluorescence-guided colon surgery. Trial Registration: The trial was registered with ClinicalTrials.gov identifier NCT03360461. Funding: Research was supported by internal University of Leeds funding. Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: Ethical approval was granted by the Yorkshire and Humber Leeds West Research and Ethics Committee (Ref 17/YH/0263), on 19th October 2017 and Regulatory approval was granted by the Medicines and Healthcare products Regulatory Agency (MHRA), under The Medicines for Human Use (Clinical Trials) regulations on 29th August 2017.

Tumor Margin Infiltration in Soft Tissue Sarcomas: Prediction Using 3T MRI Texture Analysis

목적연조직 육종의 종양 가장자리 침윤을 예측하기 위한 3T 자기공명영상 텍스처 분석의 가치를 규명한다.대상과 방법3T 자기공명영상을 시행하고, 병리학적으로 연조직 육종으로 확인된 31명의 환자를 대상으로 하고, 병리학적인 가장자리 침윤을 표준으로 사용하였다. 연조직 육종에 대한 텍스처 분석은 축상 T1 강조영상, T2 강조영상, 지방억제 조영증강 T1 강조영상, 확산강조영상(b = 800 sec/mm2) 및 현성확산계수 지도 영상에서 이루어졌다. 텍스처 분석에서 얻어진 정량적 변수가 침윤성(infiltrative) 육종과 국한성(circumscribed) 육종에서 차이가 있는지 비교하였다.결과총 23명의 연조직 육종에서 병리학적인 가장자리 침윤을 보였다. 침윤성 육종과 국한성 육종은, T1 강조영상 공간 스케일 인자(spatial scaling factor; 이하 SSF) 0, 6에서의 첨도(kurtosis), 조영증강 T1 강조영상(SSF, 0)에서의 첨도, 확산강조영상(SSF, 0)에서의 왜도(skewness), 현성확산계수 지도(SSF 2, 4)에서의 왜도에서 유의한 차이가 있었다(p ≤ 0.046). 자기공명영상 텍스처 소견을 이용한 종양 가장자리 침윤을 예측하는 정확도는 수신자운영특성곡선(receiver operating characteristic; 이하 ROC)의 곡선하 면적(area under the ROC curve) 0.951 (p < 0.001)이었다.결론자기공명영상 텍스처 분석은 연조직 육종의 침윤성 가장자리를 예측하는 데 있어 신뢰할 수 있으며 정확하다.

Inking of gross specimens: a systematic review.

Histopathological Evaluation of surgical margins of a resected tumour specimen can give an insight about the extent of tumour spread. Errors in proper identification and orientation of resected tumour margins can lead to treatment failure and poor prognosis. Inking of resected margins is the most reliable and safe method. The aim of this study is to systematically review the studies which compares various materials used for inking of surgically resected specimen. Articles searched from PubMed, Cochrane, Google search, manual search using key words - inking, tissue marking dyes, surgical margin, tumour margin, surgical pathology, grossing, gross specimens and back references of the articles, yielded three articles. Three articles with a total sample size of 1325 and compares properties of India ink, Acrylic colours and Tissue Marking Dyes were considered in this review. Both India ink and acrylic colours are good with respect to the Ease of application, visibility on paraffin wax blocks, Visibility on naked eye examination of slides and Visibility on microscope. Acrylic colours have less drying time than India ink. India ink do not result in contamination of tissue processing fluids, Interference with cellular and nuclear details and penetration in to tissues when compared with acrylic colours. India Ink will continue to dominate as the best surgical ink when comparing all the parameters till newer studies are available for acrylic colours or other dyes. Acrylic colours have the potential to be widely used as a tissue marking dyes except for the few disadvantages.

Synergy Effect of Combined Near and Mid-Infrared Fibre Spectroscopy for Diagnostics of Abdominal Cancer.

Cancers of the abdominal cavity comprise one of the most prevalent forms of cancers, with the highest contribution from colon and rectal cancers (12% of the human population), followed by stomach cancers (4%). Surgery, as the preferred choice of treatment, includes the selection of adequate resection margins to avoid local recurrences due to minimal residual disease. The presence of functionally vital structures can complicate the choice of resection margins. Spectral analysis of tissue samples in combination with chemometric models constitutes a promising approach for more efficient and precise tumour margin identification. Additionally, this technique provides a real-time tumour identification approach not only for intraoperative application but also during endoscopic diagnosis of tumours in hollow organs. The combination of near-infrared and mid-infrared spectroscopy has advantages compared to individual methods for the clinical implementation of this technique as a diagnostic tool.

Use of Navigated Ultrasonic Bone Cutting Tool for En Bloc Resection of Thoracic Chondrosarcoma: Technical Report.

En bloc surgical resection with wide margins offers the best local control rates for chondrosarcoma of the spine. To describe the surgical technique for en bloc resection of a large thoracic chondrosarcoma using image guidance for a complex osteotomy with an ultrasonic bone cutting device (Misonix, Farmingdale, New York). A 2-stage procedure was performed for resection of a thoracic chondrosarcoma involving the T3-T7 vertebral bodies. During the first stage, a posterior approach, the ultrasonic bone cutter was precisely navigated to perform an intralaminar osteotomy as well as a multilevel split sagittal osteotomy through the vertebral bodies. In the second stage, a transthoracic approach was used to complete the en bloc resection of the specimen. Intraoperative frozen sections from the surgical margins were negative for tumor. The ultrasonic bone cutting device was navigated based on coregistration of the intraoperative computed tomography (CT) images and preoperative magnetic resonance imaging (MRI). Real-time navigation using coregistered images enabled identification of tumor margins within the bone and adjacent soft tissue allowing precise execution of the intralaminar and multilevel split sagittal vertebral osteotomies. Surgical video demonstrates the utility of real-time navigation to properly identify the tumor margins and guide the ultrasonic bone cutting tool during the osteotomies. We describe the use of image guidance to navigate an ultrasonic bone cutting tool for a complex en bloc resection of a multilevel thoracic spine chondrosarcoma.

Abstract LB-022: Real-time intraoperative tumor imaging in a breast cancer PDX model

Abstract Breast cancer is one of the most common types of cancer diagnosis in women of all ages. The primary treatment of breast cancer is surgical excision of the tumor with the margin of normal breast tissue surrounding the tumor. However, current clinical goals include complete resection of the tumor at the initial surgery, there is often a need for further surgery and removal of more tissue due to the difficulties inherent in getting negative margins. Therefore, there is a clinical need to optimize the excision by precisely identifying tumor margins significantly benefitting patients with breast cancer. Image-guided surgery with fluorescent agents provides surgeons numerous advantages such as real-time detection with a high-resolution image and relatively flexible instrument. We have developed a unique tumor-targeting fluorescence imaging agent. We identified a cell-penetrating peptide (CPP) p28, a fragment of azurin isolated from opportunistic pathogen Pseudomonas pathogen. p28 was chemically conjugated with Indocyanine green (ICG), a near-infrared red (NIR) fluorescent agent. ICG has been approved by the FDA for clinical applications with an excellent safety record. When p28 was conjugated to ICG, there was no significant alternation of fluorescence quantum yield (ΦF) of ICG-p28 compared to ICG alone. Triple-negative (ER, PR, HER2) human breast cancer PDX mouse model in NSG mice was used to evaluate the image-guided surgical procedure with ICG-p28. After 24h of ICG-p28 injection i.v. opportunistic 0.5 mg/kg b.w., NIR-fluorescence positive mammary tumors with a 2-mm safe margin were resected under real-time guidance of the PDE imaging unit (Mitaka USA, Hamamatsu Photonics). NIR-fluorescence negative surrounding tissues of at least two different sites (superior and inferior) were also collected separately. To validate the accuracy of tumor margin identification by the image guidance, Alu-based real-time PCR analysis was used for the quantitative detection of human cancer cells. Five ng of genomic DNA obtained from tumors and surrounding tissues were subjected to RT-PCR. Ct values were normalized by the housekeeping gene, mouse GAPDH. Our results showed that preclinical intraoperative imaging with our imaging agent, ICG-p28 at 0.5 mg/kg, significantly reduced positive margin as compared to control groups (e.g. ICG alone, p&amp;lt;0.01). In conclusion, nontoxic p28 is a potentially ideal CPP that can serve as a tumor-targeting carrier for an intraoperative imaging agent. Results suggest that our imaging approach will potentially provide a significant impact on resection procedure benefitting patients with breast cancer. Citation Format: Masahide Goto, Ingeun Ryoo, Samer Naffouje, Konstantin Christov, Jing Wang, Albert Green, Anne Shilkaitis, Tapas K. Das Gupta, Tohru Yamada. Real-time intraoperative tumor imaging in a breast cancer PDX model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-022.

Single fiber OCT imager for breast tissue classification based on deep learning.

We investigated a deep learning strategy to analyze optical coherence tomography image for accurate tissue characterization based on a single fiber OCT probe. We obtained OCT data from human breast tissue specimens. Using OCT data obtained from adipose breast tissue (normal tissue) and diseased tissue as confirmed in histology, we trained and validated a convolutional neural network (CNN) for accurate breast tissue classification. We demonstrated tumor margin identification based CNN classification of tissue at different spatial locations. We further demonstrated CNN tissue classification in OCT imaging based on a manually scanned single fiber probe. Our results demonstrated that OCT imaging capability integrated into a low-cost, disposable single fiber probe, along with sophisticated deep learning algorithms for tissue classification, allows minimally invasive tissue characterization, and can be used for cancer diagnosis or surgical margin assessment.

Unique spectral signature of human cutaneous squamous cell carcinoma by photoacoustic imaging.

Cutaneous squamous cell carcinoma (cSCC) is a common skin cancer with metastatic potential. To reduce reoperations due to nonradical excision, there is a need to develop a technique for identification of tumor margins preoperatively. Photoacoustic (PA) imaging is a novel imaging technology that combines the strengths of laser optics and ultrasound. Our aim was to determine the spectral signature of cSCC using PA imaging and to use this signature to visualize tumor architecture and borders. Two-dimensional PA images of 33 cSCCs and surrounding healthy skin were acquired ex vivo, using 59 excitation wavelengths from 680 to 970 nm. The spectral response of the cSCCs was compared to healthy tissue, and the difference was found to be greatest at wavelengths in the range 765 to 960 nm (P < .05). Three-dimensional PA images were constructed from spectra obtained in the y-z plane using a linear stepper motor moving along the x-plane. Spectral unmixing was then performed which provided a clear three-dimensional view of the distribution of tumor masses and their borders.

Green-laser assisted laparoscopic partial cystectomy for selective muscle-invasive bladder cancer: technique and initial outcome

To describe a green-laser marking technique to assist partial cystectomy, which allows accurate identification of tumour margins, and provide our initial experience with ten patients. Between January 2014 and February 2018, ten patients suspected with muscle-invasive bladder cancer and request of bladder-preserving treatment were selected. In each case, bilateral pelvic lymphadenectomy was performed before green-laser assisted laparoscopic partial cystectomy. Under the direct view of cystoscope, the front-firing green-laser incision was performed 0.5-1cm away from the exterior margin of lesion with adequate depth into the fat tissue. Tumours were then en bloc removed via laparoscope under the tracing of laser beam. The location of 12 tumours in 10 patients was superior wall in 7 cases, lateral wall in 3 cases, anterior wall in 1 case, and posterior wall in 1 case. All procedures were completed without serious complications. The median operating time was 270 (210-360) min with a median haemoglobin decrease of 11 (3-38) g/L. Nine patients were high-grade transitional cell carcinoma and one patient was urachal carcinoma, and the clinical stage was pT1 in 1 case, pT2 in 4 cases, and pT3 in 5 cases. The pathological evaluation of tumour margins was negative in 10 patients. During the follow-up, no recurrence or metastasis were detected in 8 patients, but 2 patients presented regional recurrence. The use of green-laser marking technique during laparoscopic partial cystectomy is a feasible manoeuvre in assisting the accurate incision and minimizing injury to the remaining bladder.

Ex Vivo Breast Tumor Identification: Advances Toward a Silicon-Based Terahertz Near-Field Imaging Sensor

Terahertz waves cover photon energies that are orders of magnitude smaller (0.4-40 meV) than the visual spectrum. Therefore, they provide additional information on intrinsic condensed-matter properties, making them attractive for imaging applications in the life sciences [1], [2]. Furthermore, they do not have an ionizing effect and are considered biologically innocuous. The ever-present water in organic matter strongly absorbs terahertz waves, and subtle changes in the water concentration can be indicative of disease [3]. However, the waves? long wavelength (3mm-30 nm) severely limits their lateral resolution and creates challenges for high-resolution imaging of biological tissue on the cellular level, e.g., for tumor margin identification during cancer surgery.

Use of High-Resolution Optical Coherence Tomography in the Surgical Management of Ocular Surface Squamous Neoplasia: A Pilot Study

To evaluate whether high-resolution optical coherence tomography (HR-OCT) can detect histologic tumor margins of ocular surface squamous neoplasia (OSSN). Eight eyes of 8 patients with OSSN undergoing excision were studied prospectively. Immediately before surgery, the tumor was imaged using commercially available HR-OCT to identify the conjunctival margins of the neoplastic lesion. The tumor borders of the lesion determined by HR-OCT were mapped in relation to an anatomic reference point and transferred intraoperatively. The tumor was excised with 4-mm margins from the visible edge of the lesion with a "no-touch" technique. The specimens were sent for pathologic analysis and the histologic tumor margin was compared to the HR-OCT predicted tumor border. Mean age of the 8 patients was 67 ± 9.9 years. Seven were male, 7 were white, and, ethnically, 3 were Hispanic. All 8 tumors were bulbar and in the exposure zone. Seven tumors were limbal. Corneal extension was present in 5. Mean tumor area was 17.5 ±11.1mm2. Clinically, 2 of the tumors were leukoplakic, 1 papillomatous, and 3 gelatinous. A conjunctival tumor margin identified with the HR-OCT coincided with the pathologically confirmed margin mark in all eyes. HR-OCT has the potential to predict histologic tumor margins in OSSN. Optical identification of tumor margins could potentially decrease the incidence of residual positive margins and minimize healthy tissue removal. Advances in HR-OCT technology and integration into a microscope for "real-time" imaging are needed to further improve this technique. NOTE: Publication of this article is sponsored bytheAmerican Ophthalmological Society.

A Dual-Modality Linker Enables Site-Specific Conjugation of Antibody Fragments for 18F-Immuno-PET and Fluorescence Imaging.

Antibody-based dual-modality (PET/fluorescence) imaging enables both presurgery antigen-specific immuno-PET for noninvasive whole-body evaluation and intraoperative fluorescence for visualization of superficial tissue layers for image-guided surgery. Methods: We developed a universal dual-modality linker (DML) that facilitates site-specific conjugation to a cysteine residue-bearing antibody fragment, introduction of a commercially available fluorescent dye (via an amine-reactive prosthetic group), and rapid and efficient radiolabeling via click chemistry with 18F-labeled trans-cyclooctene (18F-TCO). To generate a dual-modality antibody fragment-based imaging agent, the DML was labeled with the far-red dye sulfonate cyanine 5 (sCy5), site-specifically conjugated to the C-terminal cysteine of the anti-prostate stem cell antigen (PSCA) cys-diabody A2, and subsequently radiolabeled by click chemistry with 18F-TCO. The new imaging probe was evaluated in a human PSCA-positive prostate cancer xenograft model by sequential immuno-PET and optical imaging. Uptake in target tissues was confirmed by ex vivo biodistribution. Results: We successfully synthesized a DML for conjugation of a fluorescent dye and 18F. The anti-PSCA cys-diabody A2 was site-specifically conjugated with either DML or sCy5 and radiolabeled via click chemistry with 18F-TCO. Immuno-PET imaging confirmed in vivo antigen-specific targeting of prostate cancer xenografts as early as 1 h after injection. Rapid renal clearance of the 50-kDa antibody fragment enables same-day imaging. Optical imaging showed antigen-specific fluorescent signal in PSCA-positive xenografts and high contrast to surrounding tissue and PSCA-negative xenografts. Conclusion: The DML enables site-specific conjugation away from the antigen-binding site of antibody fragments, with a controlled linker-to-protein ratio, and combines signaling moieties for 2 imaging systems into 1 molecule. Dual-modality imaging could provide both noninvasive whole-body imaging with organ-level biodistribution and fluorescence image-guided identification of tumor margins during surgery.

Diagnostic Performance of Receptor-Specific Surgical Specimen Staining Correlate with Receptor Expression Level.

Identification of tumor margins in the operating room in real time is a critical challenge for surgical procedures that serve as cancer cure. Breast conserving surgery (BCS) is particularly affected by this problem, with current re-excision rates above 25%. Due to a lack of clinically available methodologies for detection of involved or close tumor margins, much effort is focused on developing intraoperative margin assessment modalities that can aid in addressing this unmet clinical need. BCS provides a unique opportunity to design contrast-based technologies that are able to assess tumor margins independent from the patient, providing a rapid pathway from bench to bedside at a much lower cost. Since resected tissue is removed from the patient's blood supply, non-specific contrast agent uptake becomes a challenge due to the lack of clearance. Therefore, a dual probe, ratiometric fluorescence imaging approach was taken in an effort to reduce non-specific signal, and provide a modality that could demonstrate rapid, robust margin assessment on resected patient samples. Termed, dual-stain difference specimen imaging (DDSI), DDSI includes the use of spectrally unique, and fluorescently labeled target-specific, as well as non-specific biomarkers. In the present study, we have applied epidermal growth factor receptor (EGFR) targeted DDSI to tumor xenografts with variable EGFR expression levels using a previously optimized staining protocol, allowing for a quantitative assessment of the predictive power of the technique under biologically relevant conditions. Due to the presence of necrosis in the model tumors, ring analysis was employed to characterize diagnostic accuracy as measured by receiver operator characteristic (ROC) curve analysis. Our findings demonstrate the robust nature of the DDSI technique even in the presence of variable biomarker expression and spatial patterns. These results support the continued development of this technology as a robust diagnostic tool for tumor margin assessment in resected specimens during BCS.