BACKGROUND Intraoperative molecular imaging has emerged as a potential tool in addressing challenges faced during lung cancer surgery by localizing small lesions, ensuring negative margins, and identifying synchronous cancers. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) glycoprotein has emerged as a potential target in fluorescent labeling of non-small cell lung cancer given the high antigen density in tumor cells and absence of expression in normal parenchyma. The goal of our study was to determine whether anti-CEACAM5 targeted near-infrared fluorochrome could be a suitable target in non-small cell lung cancer.METHODS The CEACAM5 expression was evaluated in AB-12 (known negative control), HT29 (known positive control), and H460 (non-small cell lung cancer) cell lines by polymerase chain reaction. SGM-101, a CEACAM5 antibody, coupled with a BM-104 near-infrared fluorescent tracer was evaluated with dose escalation, in vitro cellular localization, and immunofluorescence microscopy. Subsequently, in vivo validation was performed in 52 athymic nude xenografts.RESULTS Polymerase chain reaction analysis demonstrated 3000x relative expression of CEACAM5 in HT-29 cells compared with AB-12. The H460 cells showed 1000x relative expression compared with AB12 (P < .05). Both HT29 and H460 cells showed tracer internalization with signal to background ratio of 4.5 (SD 0.34) whereas there was minimal uptake by AB12 cells with signal to background ratio 1.1 (SD 0.1; P < .05). There was linear fluorescence increase with increasing tracer dosing in receptor expressing cell lines. In preclinical models, HT-29 and H460 cells lines produced near-infrared fluorescence with average tumor to background ratio of 3.89 (SD 0.25) irrespective of tumor size compared with no fluorescence by AB12 tumors (P < .05). The CEACAM5 expressing tumors had excellent dye uptake compared with AB12 tumors.CONCLUSIONS CEACAM5 serves as a possible receptor for targeted intraoperative molecular imaging resections in lung cancer. This study sets a path for evaluation of CEACAM5 targets in future clinical trials.(c) 2023 by The Society of Thoracic Surgeons.

Importance:Localization of subcentimeter ground glass opacities during minimally invasive thoracoscopic lung cancer resections is a significant challenge in thoracic oncology. Intraoperative molecular imaging has emerged as a potential solution, but the availability of suitable fluorescence agents is a limiting factor.Objective:To evaluate the suitability of SGM-101, a carcinoembryonic antigen-related cell adhesion molecule type 5 (CEACAM5) receptor-targeted near-infrared fluorochrome, for molecular imaging-guided lung cancer resections, because glycoprotein is expressed in more than 80% of adenocarcinomas.Design, Setting, and Participants:For this nonrandomized, proof-of-principal, phase 1 controlled trial, patients were divided into 2 groups between August 1, 2020, and January 31, 2022. Patients with known CEACAM5-positive gastrointestinal tumors suggestive of lung metastasis were selected as proof-of-principle positive controls. The investigative group included patients with lung nodules suggestive of primary lung malignant neoplasms. Patients 18 years or older without significant comorbidities that precluded surgical exploration with suspicious pulmonary nodules requiring surgical biopsy were included in the study.Interventions:SGM-101 (10 mg) was infused up to 5 days before index operation, and pulmonary nodules were imaged using a near-infrared camera system with a dedicated thoracoscope.Main Outcomes and Measures:SGM-101 localization to pulmonary nodules and its correlation with CEACAM5 glycoprotein expression by the tumor as quantified by tumor and normal pulmonary parenchymal fluorescence.Results:Ten patients (5 per group; 5 male and 5 female; median [IQR] age, 66 [58-69] years) with 14 total lesions (median [range] lesion size, 0.91 [0.90-2.00] cm) were enrolled in the study. In the control group of 4 patients (1 patient did not undergo surgical resection because of abnormal preoperative cardiac clearance findings that were not deemed related to SGM-101 infusion), the mean (SD) lesion size was 1.33 (0.48) cm, 2 patients had elevated serum CEA markers, and 2 patients had normal serum CEA levels. Of the 4 patients who underwent surgical intervention, those with 2+ and 3+ tissue CEACAM5 expression had excellent tumor fluorescence, with a mean (SD) tumor to background ratio of 3.11 (0.45). In the patient cohort, the mean (SD) lesion size was 0.68 (0.22) cm, and no elevations in serum CEA levels were found. Lack of SGM-101 fluorescence was associated with benign lesions and with lack of CEACAM5 staining.Conclusions and Relevance:This in-human proof-of-principle nonrandomized controlled trial demonstrated SGM-101 localization to CEACAM5-positive tumors with the detection of real-time near-infrared fluorescence in situ, ex vivo, and by immunofluorescence microscopy. These findings suggest that SGM-101 is a safe, receptor-specific, and feasible intraoperative molecular imaging fluorochrome that should be further evaluated in randomized clinical trials.Trial Registration:ClinicalTrials.gov identifier: NCT04315467.

Biodistribution in mice with subcutaneous LS174T tumors at 24, 48 and 72 hours after injection of 30 μg of [111In]In-DTPA-SGM-101.

Microscopic analysis of resected intraperitoneal tumors shows clear overlap of CEA-expressing tumor cells, NIR-fluorescence and radiosignal. In the bottom right corner a region with normal non-CEA expressing cells clearly shows low fluorescence and radiosignal. (A) H&E, B) CEA, C) NIR-Fluorescence, D) Autoradiography.

Absorption and emission of SGM-101 and DTPA-SGM-101. Measurements shown from 500 to nanometer, in steps of 5 nanometer.

Purpose Metastasectomy is a common treatment option for patients with colorectal lung metastases (CLM). Challenges exist with margin assessment and identification of small nodules, especially during minimally invasive surgery. Intraoperative fluorescence imaging has the potential to overcome these challenges. The aim of this study was to assess feasibility of targeting CLM with the carcinoembryonic antigen (CEA) specific fluorescent tracer SGM-101. Methods This was a prospective, open-label feasibility study. The primary outcome was the number of CLM that showed a true positive fluorescence signal with SGM-101. Fluorescence positive signal was defined as a signal-to-background ratio (SBR) ≥ 1.5. A secondary endpoint was the CEA expression in the colorectal lung metastases, assessed with the immunohistochemistry, and scored by the total immunostaining score. Results Thirteen patients were included in this study. Positive fluorescence signal with in vivo, back table, and closed-field bread loaf imaging was observed in 31%, 45%, and 94% of the tumors respectively. Median SBRs for the three imaging modalities were 1.00 (IQR: 1.00–1.53), 1.45 (IQR: 1.00–1.89), and 4.81 (IQR: 2.70–7.41). All tumor lesions had a maximum total immunostaining score for CEA expression of 12/12. Conclusion This study demonstrated the potential of fluorescence imaging of CLM with SGM-101. CEA expression was observed in all tumors, and closed-field imaging showed excellent CEA specific targeting of the tracer to the tumor nodules. The full potential of SGM-101 for in vivo detection of the tracer can be achieved with improved minimal invasive imaging systems and optimal patient selection. Trial registration The study was registered in ClinicalTrial.gov under identifier NCT04737213 at February 2021.

Supplemental figure 4 shows slide 3 of figure 4 at a higher magnification (5x). Clear colocalization of CEA-expressing tumor cells can be observed. From left to right: H&E staining, CEA immunohistochemistry, NIR Fluorescence.

Background: Indocyanine green has been used for fluorescence-guided surgery of liver metastasis and labeling of liver segments. However, indocyanine green is nonspecific, and indocyanine green labeling does not always clearly outline tumor margins. In addition, it is difficult to distinguish between a tumor and its adjacent liver segment colored with indocyanine green alone. In the present study, we performed fluorescence-guided surgery in an orthotopic colon-cancer liver metastasis mouse model by labeling the metastatic liver tumor with an anti-carcinoembryonic antigen fluorescent antibody and with indocyanine green restricted to the adjacent liver segment. Methods: A liver metastasis model was established with human LS174T colon cancer tumor fragments. To label the tumor, mice received SGM-101, an anti-carcinoembryonic antigen antibody conjugated to a near-infrared fluorophore (700 nm), currently in clinical trials, 3 days before surgery. Indocyanine green (800 nm) was injected after ligation of the tumor-bearing Glissonean pedicle with fluorescence labeling restricted to the liver segment adjacent to the tumor. Bright-light surgery and fluorescence-guided surgery were performed to resect the liver metastasis. To assess recurrence, mice underwent necropsy 3 weeks after surgery and the tumor was weighed. Results: Fluorescence-guided anatomic left lateral lobectomy and fluorescence-guided partial liver resection were both performed with color-coded double labeled imaging. Tumor weight 3 weeks after surgery was significantly lower with fluorescence-guided surgery compared to bright-light surgery (38 +/- 57 mg vs 836 +/- 668 mg, P =.011) for partial liver resection. Conclusion: The present study provides a proof-of-concept that color-coded and double labeling of the tumor and adjacent liver segment has the potential to improve liver metastasectomy. (c) 2022 Elsevier Inc. All rights reserved.

Background: Colon-cancer liver metastases is the frequent cause of death due to difficulties in visualizing margins of the metastases resulting in incomplete resection. To perform safer and more reliable liver surgery, indocyanine green (ICG) labeling has been used to visualize liver tumors and liver segment, but it is difficult to distinguish between a liver metastasis and its adjacent liver segment with traditional use of ICG alone. We have previously developed a method to label a liver metastasis with a tumor-specific fluorescent conjugated antibody and the adjacent liver segment with ICG in order to perform image guided metastasectomy. Methods: Nude mice were surgically orthotopically implanted with a human coloncancer cell-line or colon-cancer liver metastases derived from patients. After liver tumor growth, mice received near-infrared conjugated anti-CEA or anti-CEACAM antibody to label the liver metastases. ICG was intravenously injected after ligation of the left or left lateral Glissonean pedicle resulting in specific labeling of the segment adjacent to the tumor with preserved blood-flow in the liver. Imaging was performed with the FLARE Imaging Systems. Results: The liver metastasis was brightly labeled with near infrared fluorescence with selective tumor targeting by the fluorescent anti-CEA or anti-CEACAM antibody, which was imaged on the 700 nm channel. The adjacent liver segment with preserved bloodflow in the liver had a bright fluorescence ICG 800 nm signal, while the left or left lateral segment had no fluorescence signal. Overlay of the images showed clear color-coded differentiation between the tumor and the liver segment, enabling image guided metastasectomy. Conclusions: Color coded imaging of the liver metastasis and adjacent liver segment in the present review can be used in the future for improved liver metastasectomy in the clinic.

Background: Colon-cancer liver metastases is the frequent cause of death due to difficulties in visualizing margins of the metastases resulting in incomplete resection. To perform safer and more reliable liver surgery, indocyanine green (ICG) labeling has been used to visualize liver tumors and liver segment, but it is difficult to distinguish between a liver metastasis and its adjacent liver segment with traditional use of ICG alone. We have previously developed a method to label a liver metastasis with a tumor-specific fluorescent conjugated antibody and the adjacent liver segment with ICG in order to perform image guided metastasectomy. Methods: Nude mice were surgically orthotopically implanted with a human colon-cancer cell-line or colon-cancer liver metastases derived from patients. After liver tumor growth, mice received near-infrared conjugated anti-CEA or anti-CEACAM antibody to label the liver metastases. ICG was intravenously injected after ligation of the left or left lateral Glissonean pedicle resulting in specific labeling of the segment adjacent to the tumor with preserved blood-flow in the liver. Imaging was performed with the FLARE Imaging Systems. Results: The liver metastasis was brightly labeled with near infrared fluorescence with selective tumor targeting by the fluorescent anti-CEA or anti-CEACAM antibody, which was imaged on the 700 nm channel. The adjacent liver segment with preserved blood-flow in the liver had a bright fluorescence ICG 800 nm signal, while the left or left lateral segment had no fluorescence signal. Overlay of the images showed clear color-coded differentiation between the tumor and the liver segment, enabling image guided metastasectomy. Conclusions: Color coded imaging of the liver metastasis and adjacent liver segment in the present review can be used in the future for improved liver metastasectomy in the clinic.

340 Background: Carcinoembryonic antigen (CEA) is a widely known tumor marker that is clearly expressed in gastrointestinal tract cancer. We utilized a CEA-specific chimeric antibody conjugated to a near infrared (NIR) fluorophore to facilitate CEA-targeted fluorescence image–guided surgery (FGS) of gastric cancer. The anti-CEA antibody, SGM-101 is conjugated with NIR dye BM-105, which has an absorbance band centered at 705 nm. Methods: RNA sequencing data of 34 gastric cancer cell lines from Cancer Cell Line Encyclopedia were screened and validated by qPCR and western blotting. Flow cytometry and confocal microscopy were performed by SGM-101, Alexa Fluor-680, Isotype-101 and Isotype-680 to quantify fluorescence intensity. SGM-101(n = 5) or Isotype-101(n = 2) was injected to mouse xenografts through a tail vein which had been subcutaneously implanted with MKN-45, SNU-16, and SNU-668. IVIS Spectrum quantified radiant efficiency of fluorescence in the region of interest at serial time points. The extracted tumor in peak time was analyzed by confocal imaging for microdistribution. In addition, 85As2mLuc were injected intraperitoneally in 6-week-old female BALB/c-nu mice for peritoneal carcinomatosis. Bioluminescence/fluorescence imaging was performed with IVIS Spectrum at peak time and analyzed via Living Image. Histologic evaluations were processed with H&E and Immunohistochemistry (IHC) data by a pathologist. Results: RNA expression of ceacam5 and protein expression of CEA in gastric cell lines was measured by RNA sequencing, qPCR, and western blotting. CEA expression patterns displays similar with fluorescence intensity patterns which were quantified through flow cytometry and immunocytochemistry show that CEA localized in membranes. In subcutaneously implanted model, the radiant efficiency of each group shows that the accumulation of SGM-101 has significantly higher fluorescence signal in the high CEA expressing group (MKN-45) and medium expressing group (SNU-16) while no fluorescence signal was observed in the CEA negative group (SNU-668) via IVIS Spectrum. Biodistribution of SGM-101 indicates that the maximum peak accumulation point was 48 hours after tail vein injection. Frozen tissue which was extracted at peak detection time shows micro-distribution of SGM-101 and expression of extracted tissue CEA expression was validated with IHC by pathological analysis. In the peritoneal carcinomatosis model, the imaging of fluorescence detection patterns corresponds with bioluminescence imaging and histological evaluation. Conclusions: CEA expression corresponded with intensity of in vitro fluorescence immunodetection and a tumor area accumulation in gastric cancer xenografts by SGM-101. This study indicates that NIR tumor specific imaging can be a feasible tool for image-guided surgery.

Nishino, Hiroto MD, PhD; Turner, Michael MD; Amirfakhri, Siamak PhD, DVM; Hollandsworth, Hannah M. MD; Lwin, Thinzar M. MD, MS; Yamamoto, Jun MD; Framery, Bérénice MSc; Cailler, Françoise PhD; Hoffman, Robert M. PhD; Bouvet, Michael MD, FACS Author Information

Background Fluorescence-guided surgery can provide surgeons with an imaging tool for real-time intraoperative tumor detection. SGM-101, an anti-CEA antibody labelled with a fluorescent dye, is a tumor-specific imaging agent that can aid in improving detection and complete resection for CEA-positive tumors. In this study, the performance of SGM-101 for the detection of colorectal and pancreatic liver metastases was investigated. Methods In this open-label, non-randomized, single-arm pilot study, patients were included with liver metastases from colorectal origin and intraoperatively detected liver metastases from pancreatic origin (during planned pancreatic surgery). SGM-101 was administered two to four days before the scheduled surgery as a single intravenous injection. Intraoperative fluorescence imaging was performed using the Quest Spectrum® imaging system. The performance of SGM-101 was assessed by measuring the intraoperative fluorescence signal and comparing this to histopathology. Results A total of 19 lesions were found in 11 patients, which were all suspected as malignant in white light and subsequent fluorescence inspection. Seventeen lesions were malignant with a mean tumor-to-background ratio of 1.7. The remaining two lesions were false-positives as proven by histology. Conclusion CEA-targeted fluorescence-guided intraoperative tumor detection with SGM-101 is feasible for the detection of colorectal and pancreatic liver metastases.

Fluorescence-guided surgery has been developing in clinics for several years. While the use of non-targeted dyes may be useful in certain diseases, specific contrast agents are essential in oncology. As shown in the latest clinical studies, monoclonal antibodies have all the characteristics to play a major role in this field of medical imaging, provided the antigenic target is relevant.

While the use of non-targeted dyes may be useful for fluorescence-guided surgery (FGS) in certain diseases, specific contrast agents are essential in oncology. As shown in the latest published clinical studies, monoclonal antibodies display all the characteristics that are necessary to play a major role in this field of medical imaging provided the antigenic target is relevant. Based on clinical data from FGS and from antibody expertise, major parameters explaining why antibodies are favorite FGS tools in oncology will be discussed as well as the remaining challenges for their development.

AbstractPurpose: Intraoperative image guidance may aid in clinical decision-making during surgical treatment of colorectal cancer. We developed the dual-labeled carcinoembryonic antigen–targeting tracer, [111In]In-DTPA-SGM-101, for pre- and intraoperative imaging of colorectal cancer. Subsequently, we investigated the tracer in preclinical biodistribution and multimodal image-guided surgery studies, and assessed the clinical feasibility on patient-derived colorectal cancer samples, paving the way for rapid clinical translation. Experimental Design: SGM-101 was conjugated with p-isothiocyanatobenzyl–diethylenetriaminepentaacetic acid (DTPA) and labeled with Indium-111 (111In). The biodistribution of 3, 10, 30, and 100 μg [111In]In-DTPA-SGM-101 was assessed in a dose escalation study in BALB/c nude mice with subcutaneous LS174T human colonic tumors, followed by a study to determine the optimal timepoint for imaging. Mice with intraperitoneal LS174T tumors underwent micro-SPECT/CT imaging and fluorescence image–guided resection. In a final translational experiment, we incubated freshly resected human tumor specimens with the tracer and assessed the tumor-to-adjacent tissue ratio of both signals. Results: The optimal protein dose of [111In]In-DTPA-SGM-101 was 30 μg (tumor-to-blood ratio, 5.8 ± 1.1) and the optimal timepoint for imaging was 72 hours after injection (tumor-to-blood ratio, 5.1 ± 1.0). In mice with intraperitoneal tumors, [111In]In-DTPA-SGM-101 enabled preoperative SPECT/CT imaging and fluorescence image–guided resection. After incubation of human tumor samples, overall fluorescence and radiosignal intensities were higher in tumor areas compared with adjacent nontumor tissue (P < 0.001). Conclusions: [111In]In-DTPA-SGM-101 showed specific accumulation in colorectal tumors, and enabled micro-SPECT/CT imaging and fluorescence image–guided tumor resection. Thus, [111In]In-DTPA-SGM-101 could be a valuable tool for preoperative SPECT/CT imaging and intraoperative radio-guided localization and fluorescence image–guided resection of colorectal cancer.

Background Carcinoembryonic antigen is overexpressed in colorectal cancer (CRC), making it an optimal target for fluorescence imaging. A phase I/II study was designed to determine the optimal imaging dose of SGM-101 for intraoperative fluorescence imaging of primary and recurrent CRC. Methods Patients were included and received a single dose of SGM-101 at least 24 h before surgery. Patients who received routine anticancer therapy (i.e., radiotherapy or chemotherapy) also were eligible. A dedicated near-infrared imaging system was used for real-time fluorescence imaging during surgery. Safety assessments were performed and SGM-101 efficacy was evaluated per dose level to determine the most optimal imaging dose. Results Thirty-seven patients with CRC were included in the analysis. Fluorescence was visible in all primary and recurrent tumors. In seven patients, no fluorescence was seen; all were confirmed as pathological complete responses after neoadjuvant therapy. Two tumors showed false-positive fluorescence. In the 37 patients, a total of 97 lesions were excised. The highest mean intraoperative tumor-to-background ratio (TBR) of 1.9 ( p = 0.019) was seen in the 10-mg dose. This dose showed a sensitivity of 96%, specificity of 63%, and negative predictive value of 94%. Nine patients (24%) had a surgical plan alteration based on fluorescence, with additional malignant lesions detected in six patients. Conclusions The optimal imaging dose was established at 10 mg 4 days before surgery. The results accentuate the potential of SGM-101 and designated a promising base for the multinational phase III study, which enrolled the first patients in June 2019.

Near-infrared (NIR) fluorescence imaging is a promising intraoperative technique for real-time visualization of tumor tissue during surgery. The process of clinical translation of novel fluorescent agents is an essential part in the evolution of NIR fluorescence guided surgery. Poor visualization of tumors during surgery is one of the major challenges surgeons often face in oncologic patients, mainly due to the improved neo-adjuvant treatment patients receive. In these cases, NIR fluorescence imaging with the use of tumor-targeted fluorescent agents can play an essential role and help provide better oncologic results or patient outcomes. However, before this technique can be implemented in standard of care, optimal tumor-targeted fluorescent agents need to be developed and novel fluorescent agents need to undergo a successful process of clinical translation. Here we describe the clinical translation of SGM-101, a fluorescent anti-CEA monoclonal antibody.

The real-time improvement of the intraoperative discrimination between different tissue types (particularly between tumor and adjacent normal tissue) using intraoperative imaging represents a considerable advance for oncology surgeons. However, the development of imaging agents is much slower than that of drug therapies, although surgery represents one of the few curative treatments for many solid tumors. SGM-101 is a recently described, innovative antibody conjugate in which the near-infrared fluorochrome BM-104 is covalently linked to a chimeric monoclonal antibody against carcinoembryonic antigen (CEA). SGM-101 was developed with the goal of providing oncology surgeons with an intraoperative imaging tool that allows the visualization of CEA-overexpressing tumors. This antigen is overexpressed in a wide range of human carcinomas, such as colorectal, gastric, pancreatic, non-small cell lung and breast carcinomas. Here we characterized SGM-101 safety prior to its clinical testing for real-time cancer mapping by oncology surgeons. Safety pharmacology and toxicology studies were performed after intravenous injection of SGM-101 in Wistar rats and in Beagle dogs. SGM-101 metabolism and pharmacokinetics were analyzed in rats and mice. Finally, the potential toxicity of the BM-104 dye and SGM-101 cross-reactivity were assessed in a panel of 42 human tissues. Our pre-clinical toxicology, pharmacology and pharmacokinetic results demonstrated the absence of significant adverse effects of both SGM-101 and BM-104 at doses well above the anticipated maximal human exposure. Taken together, the results of the pharmacology, pharmacokinetic and toxicology studies support the development of SGM-101 as a potentially useful and safe tumor-specific imaging tool that might improve the complete tumor resection rate.

Near-infrared (NIR) fluorescence is a promising novel imaging technique that can aid in intraoperative demarcation of pancreatic cancer (PDAC) and thus increase radical resection rates. This study investigated SGM-101, a novel, fluorescent-labeled anti-carcinoembryonic antigen (CEA) antibody. The phase 1 study aimed to assess the tolerability and feasibility of intraoperative fluorescence tumor imaging using SGM-101 in patients undergoing a surgical exploration for PDAC. At least 48 h before undergoing surgery for PDAC, 12 patients were injected intravenously with 5, 7.5, or 10 mg of SGM-101. Tolerability assessments were performed at regular intervals after dosing. The surgical field was imaged using the Quest NIR imaging system. Concordance between fluorescence and tumor presence on histopathology was studied. In this study, SGM-101 specifically accumulated in CEA-expressing primary tumors and peritoneal and liver metastases, allowing real-time intraoperative fluorescence imaging. The mean tumor-to-background ratio (TBR) was 1.6 for primary tumors and 1.7 for metastatic lesions. One false-positive lesion was detected (CEA-expressing intraductal papillary mucinous neoplasm). False-negativity was seen twice as a consequence of overlying blood or tissue that blocked the fluorescent signal. The use of a fluorescent-labeled anti-CEA antibody was safe and feasible for the intraoperative detection of both primary PDAC and metastases. These results warrant further research to determine the impact of this technique on clinical decision making and overall survival.