Abstract
Background
Gastric cancer poses a major diagnostic and therapeutic challenge as surgical resection provides the only opportunity for a cure. Specific labeling of gastric cancer could distinguish resectable and nonresectable disease and facilitate an R0 resection, which could improve survival.
Methods
Two patient-derived gastric cancer lines, KG8 and KG10, were established from surgical specimens of two patients who underwent gastrectomy for gastric adenocarcinoma. Harvested tumor fragments were implanted into the greater curvature of the stomach to establish patient-derived orthotopic xenograft (PDOX) models. M5A (humanized anti-CEA antibody) or IgG control antibodies were conjugated with the near-infrared dye IRDye800CW. Mice received 50 µg of M5A-IR800 or 50 µg of IgG-IR800 intravenously and were imaged after 72 hr. Fluorescence imaging was performed by using the LI-COR Pearl Imaging System. A tumor-to-background ratio (TBR) was calculated by dividing the mean fluorescence intensity of the tumor versus adjacent stomach tissue.
Results
M5A-IR800 administration resulted in bright labeling of both KG8 and K10 tumors. In the KG8 PDOX models, the TBR for M5A-IR800 was 5.85 (SE ± 1.64) compared with IgG-IR800 at 0.70 (SE ± 0.17). The K10 PDOX models had a TBR of 3.71 (SE ± 0.73) for M5A-IR800 compared with 0.66 (SE ± 0.12) for IgG-IR800.
Conclusions
Humanized anti-CEA (M5A) antibodies conjugated to fluorescent dyes provide bright and specific labeling of gastric cancer PDOX models. This tumor-specific fluorescent antibody is a promising potential clinical tool to detect the extent of disease for the determination of resectability as well as to visualize tumor margins during gastric cancer resection.
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Gastric cancer is the fifth most common cancer diagnosed and is the fourth leading cause of cancer-related deaths worldwide, with the highest rates found in Eastern Asia and Eastern Europe.1 In the United States, gastric cancer is the fifteenth most commonly diagnosed cancer, and recent evidence suggests that the incidence of early-stage gastric cancer is increasing.2,3 Current guidelines by the National Cancer Comprehensive Network (NCCN) and European Society for Medical Oncology (ESMO) recommend resection for localized gastric cancers, with the possibility to perform endoscopic resection for select small T1a tumors.4,5 In these gastric cancers surgeries, the ability to achieve an R0 resection (negative margins) is the single most important factor for improving outcomes. A metanalysis of more than 10,000 patients from 14 studies demonstrated that patients who had a R1 resection had an overall survival hazard ratio of 2.06 compared with patients who received an R0 resection.6
Fluorescence-guided surgery (FGS) has emerged as a useful adjunct in oncologic resections to visualize tumor deposits and aid in their removal. The efficacy of antibodies conjugated to NIR fluorophores to label and enhance the detection of breast, lung, pancreatic, prostate, and colorectal cancers have been demonstrated in preclinical mouse models.7,8,9,10,11,12,13,14,15,16,17,18,19,20 Several of these studies have now been translated to human trials, which have shown that the use of tumor-specific fluorescence labeling can detect additional residual tumor deposits or previously unrecognized synchronous disease in 14–50% of cases.21,22,23,24,25 However, there are currently no FDA-approved agents for FGS of gastric cancer. Given the impact that an R0 resection has on survival outcomes, there is a critical need for development of agents to label gastric cancer.
One potential tumor-specific target is carcinoembryonic antigen (CEA), as 74.5–90% of gastric cancers have been shown to express CEA by immunohistochemistry.26,27 We have previously utilized the poorly differentiated gastric adenocarcinoma cell line, MKN45, to create orthotopic mouse models of gastric cancer. Using a humanized anti-CEA antibody (M5A) conjugated with a NIR 800 nm dye (M5A-IR800), we were able to brightly target the tumors and achieve high tumor-to-background ratios.28 In the present study, we obtained two patient-derived gastric cancer samples and demonstrate the applicability of M5A-IR800 to target human gastric cancer.
Methods
Mouse Models
All studies were approved by the San Diego Veterans Administration Medical Center Institutional Animal Care and Use Committee (IACUC) animal-use protocol A17-020 and the University of California San Diego (UCSD) IACUC protocol S99001. Athymic male and female nude mice, aged 4–6 weeks were purchased from the Jackson Laboratory (Bar Harbor, ME). The animals were fed an autoclaved diet and housed in a barrier facility. Orthotopic mouse models were fed a chlorophyll-reduced diet for 2 weeks before imaging (Envigo, Indianapolis, IN) to reduce autofluorescence. Before any surgical procedure, the mice were anesthetized with a solution of xylazine, ketamine, and phosphate-buffered saline (PBS) via intraperitoneal injection. This solution was used, because it provides stable anesthesia without the need to titrate for depth of sedation for the duration of the surgical procedure. For postoperative pain control, they received subcutaneous buprenorphine reconstituted in PBS (dosage: 0.05 mg/kg). At the conclusion of the study, mice were anesthetized with isoflurane and euthanized by cervical dislocation.
Patient-Derived Gastric Cancer Xenografts
The patient-derived gastric cancers KG8 and KG10 were obtained from surgical specimens under sterile conditions at the time of surgical resection. The patients’ tumors were obtained with informed consent under the UCSD Institutional Review Board (IRB) protocol number 090401.
Xenograft Establishment
To initially establish KG8 and KG10 mouse models, 1 mm3 fragments of the patient’s tumor were implanted into the bilateral flanks and shoulders of nude mice. Once subcutaneous tumors grew to approximately 1 cm, subsequent passages were performed by harvesting 1 mm3 fragments and implanting them into new mice. In additional nude mice, patient-derived orthotopic xenograft (PDOX) models were established by using the method of surgical orthotopic implantation described by Furukawa et al.29 In brief, mice were anesthetized as described above and a 1–2 cm transverse incision was made in the left upper quadrant through which the stomach was delivered. Subcutaneous-grown tumors were harvested and ~1-mm3 fragments were affixed to the greater curve of the stomach using 8–0 nylon suture (Ethicon Inc., Somerville, NJ). The stomach was returned to the abdomen and the incision was closed with interrupted 6-0 vicryl sutures (Ethicon Inc.). Orthotopic models were allowed to grow for 4–6 weeks for KG8 and 6–10 weeks for KG10 before performing any imaging studies. An equal distribution of male and female mice was used for all experiments.
Antibody Conjugation
The humanized anti-CEA hT84.66-M5A (M5A) monoclonal antibody, established by Yazaki et al., was used for labeling of the gastric cancers.30 A control antibody that binds the heavy chains on human IgG was used as a control (R&D Systems, Minneapolis, MN). Both M5A and IgG were conjugated to the NIR dye IRDye800CW NHS Ester (LI-COR Biosciences, Lincoln, NE) to establish M5A-IR800 and IgG-IR800 by using methods previously described.18,31 The final products were stored at 4 °C.
Antibody-conjugate Administration and Imaging
50 µg micrograms of M5A-IR800 and 50 µg of IgG-IR800 were made by diluting the compounds in PBS for a total injection volume of 100 µl. The antibodies were administered via tail vein injection to mice bearing PDOX tumors. After 72 hr, the mice were euthanized, and a laparotomy was performed to allow imaging of intra-abdominal tissues. Imaging was performed with the Pearl Trilogy Small Animal Imaging System (LI-COR Biosciences, Lincoln NE) with 800-nm wavelength excitation.
Imaging and Data Processing
All images within the KG8 or KG10 orthotopic models treated with either M5A-IR800 or IgG-IR800 were linked, and the same brightness and contrast settings were used for all images in the present study. Within the Pearl Trilogy Small Animal Imaging System software, analysis circles were drawn around the tumors and background tissue (adjacent normal stomach) while viewing the bright light images. Fluorescence signal quantification was performed by using the LICOR Image Studio software. The mean fluorescence intensity of the 800-nm signal was measured for each region of interest and tumor-to-background ratios (TBR) were calculated.
Statistical Analysis
Statistical analysis was performed by using R software (Free Software Foundation, Boston, MA). Data from both the KG8 and KG10 experiments were found to be normally distributed by using the Shapiro test. A Student’s t-test with two tails was performed to compare the TBRs of M5A-IR800 versus IgG-IR800 in the orthotopic gastric cancer models. A p-value of < 0.05 was used as a predetermined cutoff for statistical significance.
Immunohistochemistry
Tumor samples were removed en bloc with surrounding tissue at the time of mouse necropsy. Samples were fixed in formalin for at least 72 hr before being embedded in paraffin and sectioned. Slides were stained with hematoxylin and eosin (H&E) per standard protocols. An experienced pathologist (MH) performed interpretation of the histologic slides.
Results
Patient-Derived Gastric Cancer Specimens
Tumor specimens were obtained from two patients who underwent gastrectomy for gastric cancer (Table 1). The first, KG8, was a 45-year-old male who presented with early satiety and weight loss. Imaging revealed diffuse thickening of the stomach and numerous enlarged lymph nodes (Fig. 1A–B) and an elevated serum CEA of 176. Esophagogastroduodenoscopy (EGD) revealed circumferential friable tumor consistent with linitis plastica (Fig. 1C). Given the patient’s inability to tolerate enteral feeding, upfront surgery was performed. KG10 was obtained from a 66-year-old female with a history of a Roux-en-Y gastric bypass who presented with epigastric pain. Imaging and EGD demonstrated an obstructing polypoid-type mass in the gastric remnant (Fig. 1D–F), although serum CEA was normal at 5.7. Although the patient was scheduled to receive neoadjuvant therapy prior to resection, gastrointestinal bleeding from the tumor necessitated upfront surgical resection.
Patient-derived gastric cancers KG8 and KG10. A Axial computerized tomography (CT) images of gastric cardia thickening. B Axial CT images with enlarged perihepatic lymph nodes. C Endoscopic images showing circumferential friable tumor extending from cardia to antrum consistent with linitis plastica. D Axial and E coronal CT images demonstrating ~3 cm polypoid mass at pylorus. F Endoscopic images demonstrating an obstructing mass. Red arrow: tumor, blue arrow: enlarged lymph node
Bright and Specific Labeling of Orthotopic KG8 Tumors
Tumor fragments from the KG8 surgical specimen were implanted into the flanks of nude athymic mice. Once the tumors reached adequate size, they were harvested, and 1-mm3 fragments were affixed to the greater curvature of the stomach in additional nude mice to establish patient-derived orthotopic xenograft (PDOX) models. After 4–6 weeks, mice were randomized to receive either M5A-IR800 or the control (IgG-IR800) intravenously. Fluorescence labeling with M5A-IR800 resulted in bright targeting of the KG8 orthotopic gastric cancers compared with control after 72 hr (Fig. 2A–B’). A mean TBR of 5.85 (±1.64) was seen for M5A-IR800 (n = 5) compared with 0.70 (±0.17) for the control (n = 4) with a p-value of 0.035 (Fig. 2C).
Fluorescence labeling of KG8 orthotopic gastric tumors. A M5A-IR800 brightly labels primary gastric tumor. A’ Bright light imaging with a gastric tumor seen on the greater curvature of the stomach. B Lack of gastric tumor labeling with IgG-IR800. B’ Bright light imaging with a gastric tumor seen directly invading liver parenchyma. White arrow: tumor, yellow arrow: stomach, arrowhead: abdominal wall metastasis. Scale bar: 1 cm. C Average TBRs of gastric tumors labelled with M5A-IR800 or IgG-IR800. Error bars represent standard error. *p-value: 0.035
Bright and Specific Labeling of Orthotopic KG10 Tumors
The same process of establishing PDOX models was used for the KG10 line. Bright labeling of the KG10 PDOX models also was seen with M5A-IR800 compared with control (Fig. 3A–B’). Labeling with M5A-IR800 (n = 6) resulted in an average TBR of 3.71 (±0.73), whereas those labeled with IgG-IR800 (n = 5) had a mean TBR of 0.66 (±0.12) with a p-value of 0.009 (Fig. 3C).
Fluorescence labeling of KG10 orthotopic gastric tumors. A M5A-IR800 brightly labels a primary gastric tumor. A’ Bright light imaging with a gastric tumor seen on the greater curvature of the stomach. B Lack of gastric tumor labeling with IgG-IR800. B’ Bright light imaging with a gastric tumor seen on the greater curvature of the stomach. White arrow: tumor, yellow arrow: stomach. Scale bar: 1 cm. C Average TBRs of gastric tumors labelled with M5A-IR800 or IgG-IR800. Error bars represent standard error. p-value: 0.009
Immunohistochemistry of Patient-Derived Gastric Cancer Lines
For KG8, hematoxylin and eosin (H&E) staining of the patient’s surgical specimen and the PDOX tumor demonstrated poorly differentiated adenocarcinoma (Fig. 4A and B). H&E staining of the KG10 patient’s surgical specimen and the KG10 PDOX tumor demonstrated well-to-moderately differentiated adenocarcinoma (Fig. 4C and D). These results demonstrate that the PDOX models retained the pathologic characteristics of the original donor patients’ tumors.
Pathology of KG8 and KG10 patient-derived gastric cancer lines. A H&E of the KG8 patient’s original pathology from surgical resection showing poorly differentiated gastric adenocarcinoma. B H&E of the KG8 patient-derived orthotopic xenograft tumor with poorly differentiated cells denoted by black arrow and normal tissue with white arrow. C H&E of the KG10 patient’s original pathology from surgical resection showing well-to-moderately differentiated gastric adenocarcinoma. D H&E of the KG10 patient-derived orthotopic xenograft tumor with well-to-moderately differentiated cells denoted by black arrow.
Discussion
In gastric cancer, the only opportunity for cure is with a R0 resection as perioperative chemotherapy has provided only modest improvements in overall survival.32,33 The field of fluorescence-guided surgery (FGS) has emerged as an opportunity to address this need for improved surgical resections. There has been a growing emergence of the use of targeted fluorescence with tumor-specific markers for many cancer types.34 While earlier work in the field often used fluorescent dyes in the visible spectrum, such as 488 or Cy5, most work now focuses on the use of NIR fluorophores as they have increased tissue depth penetration, reduced light scattering, and reduced autofluorescence.34,35 In recent years, 5-ALA and Cytalux have gained FDA approval for the fluorescence-guided surgical resection of gliomas and ovarian and lung cancer, respectively.21,36 There are ongoing clinical trials for the use of an anti-CEA antibody conjugated to a 700 nm dye (SGM-101) in both colorectal cancer and pancreatic cancer.37
In gastric cancer, a few probes have been evaluated for fluorescence labeling of tumors in preclinical models, although currently, none are FDA-approved for use in FGS.38 Hoetker et al. tested Cetuximab and another anti-EGFR antibody, bound to FITC and Alexa Fluor 488 respectively, with confocal laser endomicroscopy (CLE) and showed increased fluorescence in MKN45 tumors compared with isotype-control antibody.39 MG7, a novel tumor-associated antibody originally made by Fan et al. by inoculating mice with the MKN46-9 gastric cancer cell line, also has been tested for fluorescence labeling of gastric cancer.40 MG7 was labeled with Alexa Fluor 680, and using CLE, showed increased fluorescence in xenograft tumors (BGC-823 and SGC-7901 cell lines) compared with a control antibody.41 There are significant challenges with probe selection for gastric cancer; EGFR expression is only seen in 62% of gastric cancers, and although MG7 is expressed in 94% of gastric cancers, it also is seen in Helicobacter pylori-associated gastritis.42,43,44
Koga et al. utilized an anti-CEA antibody labeled with Alexa Fluor 594 to label orthotopic mouse models of MKN45 gastric cancer. Although their probe was able to visualize the tumors, background signals (signals from surrounding normal tissue) were high.45 Despite the limited utility of serum CEA levels to detect gastric cancer upon initial diagnosis or at the time of recurrence, 74.5–90% of gastric cancers have been shown to express CEA by immunohistochemistry.26,27,46,47,48 Therefore, CEA is an excellent target for fluorescence labeling of gastric cancer.
Previously, we utilized the MKN45 cell line to establish orthotopic mouse models of gastric cancer and labeled them with a humanized anti-CEA antibody (M5A) conjugated with a NIR 800 nm dye (M5A-IR800).28 Our findings using the cell line demonstrated that the conjugate was able to brightly label both primary gastric tumors and peritoneal metastases with TBRs greater than four times that of the control. In the present study, we evaluated this probe in patient-derived orthotopic xenograft (PDOX) models. Compared with the homogeneous nature of human cancer cell lines, patient-derived xenografts more closely mimic the heterogeneity of patient tumors.49 Additionally, the metastatic pattern of PDOX models has been shown to correlate to the pattern of disease spread in the donor patients.50 Thus, we evaluated whether our probe could maintain a high level of fluorescence intensity and contrast given tumor heterogeneity. Two gastric cancer specimens (KG8 and KG10) were obtained from patients undergoing surgical resection to establish PDOX models of gastric cancer. We showed that M5A-IR800 selectively and brightly labeled multiple patient-derived gastric tumors with high TBRs irrespective of the patient’s preoperative serum CEA levels.
Limitations of the study include the use of nude athymic mice and the location of the tumors. To address the use of immunocompromised mice, future studies could include testing our probe in a transgenic mouse that expresses human CEA as a syngeneic model of gastric cancer.51 Regarding tumor location, our current method of attaching tumor fragments to the serosal surface of the mouse stomach has room for improvement as most gastric cancers are intraluminal and often infiltrative within the layers of the gastric wall. To address this aspect of gastric cancer pathophysiology, we are developing improved models to achieve better incorporation of the tumors into the layers of the stomach.
Additionally, fluorescence labeling of lymph nodes containing metastatic disease is a provocative area of study that we are currently working on with our new models. There have been numerous studies investigating the role of indocyanine green (ICG) in detecting sentinel lymph nodes, nodes containing metastatic disease, or the improved detection and thus completion of a D2 lymphadenectomy.52,53,54,55,56,57,58,59 Despite the robust body of research on the use of ICG in gastric cancer lymphadenectomy, its use has yet to become incorporated into the current guidelines or to become the standard of care.60 This is likely due to the complex lymphatic drainage of the stomach and nonspecific nature of ICG. A tumor-specific probe given systemically could overcome these challenges encountered with ICG’s ability to detect lymph nodes containing metastatic disease. If M5A-IR800 can label lymph nodes containing metastatic disease in addition to the primary tumors, it would drastically increase the value of using the probe during surgical resection.
In addition to testing M5A-IR800 on the new model of gastric cancer and metastatic lymph nodes, other areas of further research include performing FGS on orthotopic mouse models of gastric cancer and monitoring for recurrence or improvement in overall survival. In previous work on pancreatic cancer using an anti-CEA antibody conjugated to Alexa Fluor 488, median disease-free survival for the FGS group was 11 weeks compared with 5 weeks for the bright-light surgery group.61 The enhanced ability to visualize the gastric tumors at the time of surgery should result in improved resections and thus survival, although studies are needed to test this hypothesis. Other future directions could include optimizing the dye to which the antibody is conjugated. There are numerous imaging devices currently used in the operating room, many of which have slight variations in their optimal NIR window, which could necessitate tuning of NIR dyes to specific clinical devices.62
Additional applications of tumor-specific probes for gastric cancer include its use in positron emission tomography (PET) imaging for either the initial diagnosis or monitoring for disease recurrence. Xu et al. utilized MG7 and labeled it with Gallium-68 for PET imaging of mice bearing subcutaneous tumors.63 Unfortunately, MG7 expression also is seen in H. pylori associated gastric disease, which could limit its use in clinical practice.44 Cadherin-17 labeled with Indium-111 also was used for PET imaging of subcutaneous tumor-bearing mice and showed excellent specificity for the tumors, although only 64% of gastric cancers express Cadherin-17.64 Trastuzumab, the monoclonal antibody against HER2, has been used for PET imaging of gastric cancer by labeling with Zirconium-89 or Copper-64.65,66 Although Trastuzumab is already FDA-approved and provides the potential for both treatment and enhanced imaging when coupled with PET radiotracers, only 20% of gastric cancers express HER2.67
M5A also has been used for PET imaging of many CEA-positive cancers, including colorectal cancer (n = 11), medullary thyroid cancer (n = 5), esophagogastric cancer (n = 2), and pancreatic cancer (n = 2).68 In a phase 1 trial of M5A labelled with Yttrium-90, it was shown that the humanized version of M5A led to decreased risk of immunogenicity (development of human antibodies against a drug or other molecule) compared with previous work with chimeric versions of M5A.69
There are many potential clinical applications for tumor-specific markers as described above. In the present study, we investigated the use of an anti-CEA antibody conjugated to a NIR dye for fluorescence labeling of gastric cancers in clinically relevant PDOX models. These results demonstrate its potential for future clinical applications in targeted fluorescence-guided surgery.
References
Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71(3):209–19. https://doi.org/10.3322/caac.21660.
Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA Cancer J Clin. 2023;73(1):17–48. https://doi.org/10.3322/caac.21763.
Bergquist JR, Leiting JL, Habermann EB, et al. Early-onset gastric cancer is a distinct disease with worrisome trends and oncogenic features. Surgery. 2019;166(4):547–55. https://doi.org/10.1016/j.surg.2019.04.036.
Ajani JA, D’Amico TA, Bentrem DJ, et al. Gastric cancer, version 2.2022, NCCN clinical practice guidelines in oncology. J Natl Compr Canc Netw. 2022;20(2):167–92. https://doi.org/10.6004/jnccn.2022.0008.
Lordick F, Carneiro F, Cascinu S, et al. Gastric cancer: ESMO clinical practice guideline for diagnosis, treatment and follow-up. Ann Oncol. 2022;33(10):1005–20. https://doi.org/10.1016/j.annonc.2022.07.004.
Jiang Z, Liu C, Cai Z, et al. Impact of surgical margin status on survival in gastric cancer: a systematic review and meta-analysis. Cancer Control. 2021. https://doi.org/10.1177/10732748211043665.
Yamashita S, Kojima M, Onda N, Yoshida T, Shibutani M. Trastuzumab-based near-infrared photoimmunotherapy in xenograft mouse of breast cancer. Cancer Med. 2023;12(4):4579–89. https://doi.org/10.1002/cam4.5302.
Xiao J, Qiu S, Ma Q, Bai S, Guo X, Wang L. Near-infrared dye IRDye800CW-NHS coupled to Trastuzumab for near-infrared II fluorescence imaging in tumor xenograft models of HER-2-positive breast cancer. J Mater Chem B. 2023;11(44):10738–46. https://doi.org/10.1039/d3tb01486j.
Kennedy GT, Azari FS, Chang A, et al. A pH-activatable nanoprobe labels diverse histologic subtypes of human lung cancer during resection. Mol Imaging Biol. 2023;25(5):824–32. https://doi.org/10.1007/s11307-023-01853-2.
Lwin TM, Murakami T, Miyake K, et al. Tumor-specific labeling of pancreatic cancer using a humanized anti-CEA antibody conjugated to a near-infrared fluorophore. Ann Surg Oncol. 2018;25(4):1079–85. https://doi.org/10.1245/s10434-018-6344-6.
Turner MA, Hollandsworth HM, Nishino H, et al. Fluorescent anti-MUC5AC brightly targets pancreatic cancer in a patient-derived orthotopic xenograft. In Vivo. 2022;36(1):57–62. https://doi.org/10.21873/invivo.12676.
Olson MT, Wojtynek NE, Talmon GA, et al. Development of a MUC16-targeted near-infrared fluorescent antibody conjugate for intraoperative imaging of pancreatic cancer. Mol Cancer Ther. 2020;19(8):1670–81. https://doi.org/10.1158/1535-7163.Mct-20-0033.
Turner MA, Cox KE, Neel N, et al. Highly selective targeting of pancreatic cancer in the liver with a near-infrared anti-MUC5AC probe in a PDOX mouse model: a proof-of-concept study. J Pers Med. 2023. https://doi.org/10.3390/jpm13050857.
Cilibrizzi A, Wang JT, Memdouh S, et al. PSMA-targeted NIR probes for image-guided detection of prostate cancer. Colloids Surf B Biointerfaces. 2022;218:112734. https://doi.org/10.1016/j.colsurfb.2022.112734.
Nakajima T, Mitsunaga M, Bander NH, Heston WD, Choyke PL, Kobayashi H. Targeted, activatable, in vivo fluorescence imaging of prostate-specific membrane antigen (PSMA) positive tumors using the quenched humanized J591 antibody-indocyanine green (ICG) conjugate. Bioconjug Chem. 2011;22(8):1700–5. https://doi.org/10.1021/bc2002715.
Neuman BP, Eifler JB, Castanares M, et al. Real-time, near-infrared fluorescence imaging with an optimized dye/light source/camera combination for surgical guidance of prostate cancer. Clin Cancer Res. 2015;21(4):771–80. https://doi.org/10.1158/1078-0432.Ccr-14-0891.
Hollandsworth HM, Lwin TM, Amirfakhri S, et al. Anti-claudin-1 conjugated to a near-infrared fluorophore targets colon cancer in PDOX mouse models. J Surg Res. 2019;242:145–50. https://doi.org/10.1016/j.jss.2019.04.048.
Hollandsworth HM, Nishino H, Turner M, et al. Humanized Fluorescent tumor-associated glycoprotein-72 antibody selectively labels colon-cancer liver metastases in orthotopic mouse models. In Vivo. 2020;34(5):2303–7. https://doi.org/10.21873/invivo.12042.
Turner MA, Hollandsworth HM, Amirfakhri S, et al. Anti-mucin 4 fluorescent antibody brightly targets colon cancer in patient-derived orthotopic xenograft mouse models: a proof-of-concept study for future clinical applications. Am J Surg. 2022;224(4):1081–5. https://doi.org/10.1016/j.amjsurg.2022.05.036.
Gutowski M, Framery B, Boonstra MC, et al. SGM-101: an innovative near-infrared dye-antibody conjugate that targets CEA for fluorescence-guided surgery. Surg Oncol. 2017;26(2):153–62. https://doi.org/10.1016/j.suronc.2017.03.002.
Sarkaria IS, Martin LW, Rice DC, Blackmon SH, Slade HB, Singhal S. Pafolacianine for intraoperative molecular imaging of cancer in the lung: the ELUCIDATE trial. J Thorac Cardiovasc Surg. 2023;166(6):e468–78. https://doi.org/10.1016/j.jtcvs.2023.02.025.
Hwang ES, Beitsch P, Blumencranz P, et al. Clinical impact of intraoperative margin assessment in breast-conserving surgery with a novel pegulicianine fluorescence-guided system: a nonrandomized controlled trial. JAMA Surg. 2022;157(7):573–80. https://doi.org/10.1001/jamasurg.2022.1075.
Wagner P, Levine EA, Kim AC, et al. Detection of residual peritoneal metastases following cytoreductive surgery using pegsitacianine, a pH-sensitive imaging agent: final results from a phase II study. Ann Surg Oncol. (2024). https://doi.org/10.1245/s10434-024-15165-4
Smith BL, Hunt KK, Carr D, et al. Intraoperative fluorescence guidance for breast cancer lumpectomy surgery. NEJM Evid. 2023;2(7):EVIDoa2200333. https://doi.org/10.1056/EVIDoa2200333.
Tanyi JL, Randall LM, Chambers SK, et al. A phase III study of pafolacianine injection (OTL38) for intraoperative imaging of folate receptor-positive ovarian cancer (study 006). J Clin Oncol. 2023;41(2):276–84. https://doi.org/10.1200/jco.22.00291.
Guazzi P, Zocco D, Isajevs S, et al. TM9SF4 expression in tumor tissues: a novel diagnostic biomarker for gastrointestinal tumors. Transl Cancer Res. 2020;9(11):6652–9. https://doi.org/10.21037/tcr-20-516.
Sun K, Xu MQ, Zhang HJ, et al. Active Stat3 and Her-2 as combined survival predictors show superiority to TNM staging system for postoperative patients with gastric cancer. Am J Transl Res. 2022;14(4):2317–30.
Cox KE, Turner MA, Amirfakhri S, et al. Humanized anti-carcinoembryonic antigen antibodies brightly target and label gastric cancer in orthotopic mouse models. J Surg Res. 2024;293:701–8. https://doi.org/10.1016/j.jss.2023.08.038.
Furukawa T, Kubota T, Watanabe M, Kitajima M, Hoffman RM. Orthotopic transplantation of histologically intact clinical specimens of stomach cancer to nude mice: correlation of metastatic sites in mouse and individual patient donors. Int J Cancer. 1993;53(4):608–12. https://doi.org/10.1002/ijc.2910530414.
Yazaki PJ, Sherman MA, Shively JE, et al. Humanization of the anti-CEA T84.66 antibody based on crystal structure data. Protein Eng Des Sel. 2004;17(5):481–9. https://doi.org/10.1093/protein/gzh056.
DeLong JC, Murakami T, Yazaki PJ, Hoffman RM, Bouvet M. Near-infrared-conjugated humanized anti-carcinoembryonic antigen antibody targets colon cancer in an orthotopic nude-mouse model. J Surg Res. 2017;218:139–43. https://doi.org/10.1016/j.jss.2017.05.069.
Coit DG, Strong VE. Fifty years of progress in gastric cancer. J Surg Oncol. 2022;126(5):865–71. https://doi.org/10.1002/jso.27060.
Guan WL, He Y, Xu RH. Gastric cancer treatment: recent progress and future perspectives. J Hematol Oncol. 2023;16(1):57. https://doi.org/10.1186/s13045-023-01451-3.
Vahrmeijer AL, Hutteman M, van der Vorst JR, van de Velde CJ, Frangioni JV. Image-guided cancer surgery using near-infrared fluorescence. Nat Rev Clin Oncol. 2013;10(9):507–18. https://doi.org/10.1038/nrclinonc.2013.123.
Gibbs SL. Near infrared fluorescence for image-guided surgery. Quant Imaging Med Surg. 2012;2(3):177–87. https://doi.org/10.3978/j.issn.2223-4292.2012.09.04.
Van Keulen S, Hom M, White H, Rosenthal EL, Baik FM. The evolution of fluorescence-guided surgery. Mol Imaging Biol. 2023;25(1):36–45. https://doi.org/10.1007/s11307-022-01772-8.
NIR-fluorescence guided surgical resection of neoadjuvant treated localized pancreatic cancer using SGM-101 (FLUOPANC-II). NCT05984810. ClinicalTrails.gov, accessed February 25th 2024
Mandleywala K, Shmuel S, Pereira PMR, Lewis JS. Antibody-targeted imaging of gastric cancer. Molecules. 2020. https://doi.org/10.3390/molecules25204621.
Hoetker MS, Kiesslich R, Diken M, et al. Molecular in vivo imaging of gastric cancer in a human-murine xenograft model: targeting epidermal growth factor receptor. Gastrointest Endosc. 2012;76(3):612–20. https://doi.org/10.1016/j.gie.2012.05.013.
Fan D, Zhang X, Chen X, et al. Establishment of four monoclonal antibodies to a poorly differentiated gastric cancer cell line MKN-46-9 and immunohistochemical study on their corresponding antigens. Chin J Med PLA. 1988;13:12–5.
Li Z, Zuo XL, Li CQ, et al. In vivo molecular imaging of gastric cancer by targeting MG7 antigen with confocal laser endomicroscopy. Endoscopy. 2013;45(2):79–85. https://doi.org/10.1055/s-0032-1325762.
Drescher D, Moehler M, Gockel I, et al. Coexpression of receptor-tyrosine-kinases in gastric adenocarcinoma—a rationale for a molecular targeting strategy? World J Gastroenterol. 2007;13(26):3605–9. https://doi.org/10.3748/wjg.v13.i26.3605.
Jin B, Wang X, Jin Y, et al. Detection of serum gastric cancer-associated MG7-Ag from gastric cancer patients using a sensitive and convenient ELISA method. Cancer Invest. 2009;27(2):227–33. https://doi.org/10.1080/07357900802175609.
Guo DL, Dong M, Wang L, Sun LP, Yuan Y. Expression of gastric cancer-associated MG7 antigen in gastric cancer, precancerous lesions and H. pylori -associated gastric diseases. World J Gastroenterol. 2002;8(6):1009–13. https://doi.org/10.3748/wjg.v8.i6.1009.
Koga S, Oshima Y, Honkura N, et al. In vivo subcellular imaging of tumors in mouse models using a fluorophore-conjugated anti-carcinoembryonic antigen antibody in two-photon excitation microscopy. Cancer Sci. 2014;105(10):1299–306. https://doi.org/10.1111/cas.12500.
Marrelli D, Pinto E, De Stefano A, Farnetani M, Garosi L, Roviello F. Clinical utility of CEA, CA 19–9, and CA 72–4 in the follow-up of patients with resectable gastric cancer. Am J Surg. 2001;181(1):16–9. https://doi.org/10.1016/s0002-9610(00)00549-3.
Carpelan-Holmström M, Louhimo J, Stenman UH, Alfthan H, Haglund C. CEA, CA 19–9 and CA 72–4 improve the diagnostic accuracy in gastrointestinal cancers. Anticancer Res. 2002;22(4):2311–6.
Horie Y, Miura K, Matsui K, et al. Marked elevation of plasma carcinoembryonic antigen and stomach carcinoma. Cancer. 1996;77(10):1991–7. https://doi.org/10.1002/(sici)1097-0142(19960515)77:10%3c1991::Aid-cncr5%3e3.0.Co;2-k.
Hoffman RM. Patient-derived orthotopic xenografts: better mimic of metastasis than subcutaneous xenografts. Nat Rev Cancer. 2015;15(8):451–2. https://doi.org/10.1038/nrc3972.
Furukawa T, Fu X, Kubota T, Watanabe M, Kitajima M, Hoffman RM. Nude mouse metastatic models of human stomach cancer constructed using orthotopic implantation of histologically intact tissue. Cancer Res. 1993;53(5):1204–8.
Clarke P, Mann J, Simpson JF, Rickard-Dickson K, Primus FJ. Mice transgenic for human carcinoembryonic antigen as a model for immunotherapy. Cancer Res. 1998;58(7):1469–77.
Liao Y, Zhao J, Chen Y, et al. Mapping lymph node during indocyanine green fluorescence-imaging guided gastric oncologic surgery: current applications and future directions. Cancers. 2022. https://doi.org/10.3390/cancers14205143.
Park SH, Kim KY, Cho M, Kim YM, Kim HI, Hyung WJ. Prognostic impact of fluorescent lymphography on gastric cancer. Int J Surg. 2023;109(10):2926–33. https://doi.org/10.1097/js9.0000000000000572.
Tummers QR, Boogerd LS, de Steur WO, et al. Near-infrared fluorescence sentinel lymph node detection in gastric cancer: a pilot study. World J Gastroenterol. 2016;22(13):3644–51. https://doi.org/10.3748/wjg.v22.i13.3644.
Watanabe A, Hamilton TD. Lymph node mapping in gastric cancer: a pilot study in western patients. Can J Surg. 2022;65(5):E630–4. https://doi.org/10.1503/cjs.019821.
Jung MK, Cho M, Roh CK, et al. Assessment of diagnostic value of fluorescent lymphography–guided lymphadenectomy for gastric cancer. Gastric Cancer. 2021;24(2):515–25. https://doi.org/10.1007/s10120-020-01121-0.
Kitagawa Y, Takeuchi H, Takagi Y, et al. Sentinel node mapping for gastric cancer: a prospective multicenter trial in Japan. J Clin Oncol. 2013;31(29):3704–10. https://doi.org/10.1200/jco.2013.50.3789.
Chen QY, Xie JW, Zhong Q, et al. Safety and efficacy of indocyanine green tracer-guided lymph node dissection during laparoscopic radical gastrectomy in patients with gastric cancer: a randomized clinical trial. JAMA Surg. 2020;155(4):300–11. https://doi.org/10.1001/jamasurg.2019.6033.
Herrera-Almario G, Patane M, Sarkaria I, Strong VE. Initial report of near-infrared fluorescence imaging as an intraoperative adjunct for lymph node harvesting during robot-assisted laparoscopic gastrectomy. J Surg Oncol. 2016;113(7):768–70. https://doi.org/10.1002/jso.24226.
Sherwinter DA, Boni L, Bouvet M, et al. Use of fluorescence imaging and indocyanine green for sentinel node mapping during gastric cancer surgery: results of an intercontinental Delphi survey. Surgery. 2022;172(6s):S29-37. https://doi.org/10.1016/j.surg.2022.06.036.
Metildi CA, Kaushal S, Pu M, et al. Fluorescence-guided surgery with a fluorophore-conjugated antibody to carcinoembryonic antigen (CEA), that highlights the tumor, improves surgical resection and increases survival in orthotopic mouse models of human pancreatic cancer. Ann Surg Oncol. 2014;21(4):1405–11. https://doi.org/10.1245/s10434-014-3495-y.
Zhu B, Sevick-Muraca EM. A review of performance of near-infrared fluorescence imaging devices used in clinical studies. Br J Radiol. 2015;88(1045):20140547. https://doi.org/10.1259/bjr.20140547.
Xu B, Li X, Yin J, et al. Evaluation of 68Ga-labeled MG7 antibody: a targeted probe for PET/CT imaging of gastric cancer. Sci Rep. 2015;5:8626. https://doi.org/10.1038/srep08626.
Altree-Tacha D, Tyrrell J, Haas T. CDH17 is a more sensitive marker for gastric adenocarcinoma than CK20 and CDX2. Arch Pathol Lab Med. 2017;141(1):144–50. https://doi.org/10.5858/arpa.2015-0404-OA.
O’Donoghue JA, Lewis JS, Pandit-Taskar N, et al. Pharmacokinetics, biodistribution, and radiation dosimetry for (89)Zr-trastuzumab in patients with esophagogastric cancer. J Nucl Med. 2018;59(1):161–6. https://doi.org/10.2967/jnumed.117.194555.
Guo X, Zhu H, Zhou N, et al. Noninvasive detection of HER2 expression in gastric cancer by (64)Cu-NOTA-trastuzumab in PDX mouse model and in patients. Mol Pharm. 2018;15(11):5174–82. https://doi.org/10.1021/acs.molpharmaceut.8b00673.
Lee S, de Boer WB, Fermoyle S, Platten M, Kumarasinghe MP. Human epidermal growth factor receptor 2 testing in gastric carcinoma: issues related to heterogeneity in biopsies and resections. Histopathology. 2011;59(5):832–40. https://doi.org/10.1111/j.1365-2559.2011.04017.x.
Wong JYC, Yamauchi DM, Adhikarla V, et al. First-in-human pilot PET immunoimaging study of (64)Cu-anti-carcinoembryonic antigen monoclonal antibody (hT84.66-M5A) in patients with carcinoembryonic antigen-producing cancers. Cancer Biother Radiopharm. 2023;38(1):26–37. https://doi.org/10.1089/cbr.2022.0028.
Akhavan D, Yazaki P, Yamauchi D, et al. Phase I study of yttrium-90 radiolabeled M5A anti-carcinoembryonic antigen humanized antibody in patients with advanced carcinoembryonic antigen producing malignancies. Cancer Biother Radiopharm. 2020;35(1):10–5. https://doi.org/10.1089/cbr.2019.2992.
Acknowledgment
Presented at the meeting of The Society of Surgical Oncology, Boston, MA, March 2023.
Funding
This work was supported by VA Merit Review (grant numbers 1 I01 BX003856-01A1 and 1 I01 BX004494-01), National Institute of Health (R01s CA256973-01, CA100768 and CA238042), Padres Pedal the Cause Collaborative Translational Pilot Project (#PTC2021 and #PCT2022), Curebound foundation (20DG03), Department of Defense Award (W81XWH-20-1-0675), and National Institute of Health Training Grant (T32CA121938). The Tissue Technology Shared Resource is supported by a National Cancer Institute Cancer Center Support Grant (CCSG Grant P30CA23100). Other sources of support include the Torrey Coast Foundation.
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Kristin E. Cox wrote the manuscript and was actively involved in each step of the experimental process. Michael A. Turner, Siamak Amirfakhri, and Thinzar M. Lwin were involved in the experimental process and design and provided critical revisions. Kaitlyn M. Kelly provided the patients’ gastric cancer samples. Mojgan Hosseini performed the pathologic evaluation of the gastric tumors. Pradipta Ghosh, Marygorret Obonyo, and Robert M. Hoffman edited the manuscript. Paul J. Yazaki was involved in the experimental process and developed the humanized anti-CEA hT84.66-M5A antibody. Michael Bouvet was involved in the research design and all aspects of the experimental process.
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RMH is a non-salaried affiliate of Anti Cancer, Inc. which produces patient-derived orthotopic xenograft (PDOX) mouse models for contract research. PY declares a patent covering anti-CEA M5A antibody for fluorescence guided surgery.
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Meeting Presentation: Presented at The Society of Surgical Oncology annual meeting in Boston, MA in March 2023.
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Cox, K.E., Turner, M.A., Lwin, T.M. et al. Targeting Patient-Derived Orthotopic Gastric Cancers with a Fluorescent Humanized Anti-CEA Antibody. Ann Surg Oncol 31, 6291–6299 (2024). https://doi.org/10.1245/s10434-024-15570-9
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DOI: https://doi.org/10.1245/s10434-024-15570-9