The Use of Fluorescent Anti-CEA Antibodies to Label, Resect and Treat Cancers: A Review

A major barrier to the diagnosis and effective treatment of solid-tumor cancers is the difficulty in detection and visualization of tumor margins in primary and metastatic disease. The use of fluorescence can augment the surgeon’s ability to detect cancer and aid in its resection. Several cancer types express carcinoembryonic antigen (CEA) including colorectal, pancreatic and gastric cancer. Antibodies to CEA have been developed and tagged with near-infrared fluorescent dyes. This review article surveyed the use of CEA antibodies conjugated to fluorescent probes for in vivo studies since 1990. PubMed and Google Scholar databases were queried, and 900 titles and abstracts were screened. Fifty-nine entries were identified as possibly meeting inclusion/exclusion criteria and were reviewed in full. Forty articles were included in the review and their citations were screened for additional entries. A total of 44 articles were included in the final review. The use of fluorescent anti-CEA antibodies has been shown to improve detection and resection of tumors in both murine models and clinically. The cumulative results indicate that fluorescent-conjugated anti-CEA antibodies have important potential to improve cancer diagnosis and surgery. In an emerging technology, anti-CEA fluorescent antibodies have also been successfully used for photoimmunotherapy treatment for cancer.

A pre-screening strategy to assess resected tumor margins by imaging cytoplasmic viscosity and hypoxia

To assure complete tumor removal, frozen section analysis is the most common procedure for intraoperative pathological assessment of resected tumor margins. However, during one operation, multiple biopsies may be sent for examination, but only few of them are made into cryosections because of the complex preparation protocols and time-consuming pathological analysis, which potentially increases the risk of overlooking tumor involvement. Here, we propose a fluorescence-based pre-screening strategy that allows high-throughput, convenient, and fast gross assessment of resected tumor margins. A dual-activatable cationic fluorescent molecular rotor was developed to specifically illuminate live tumor cells’ cytoplasm by emitting two different fluorescence signals in response to elevations in hypoxia-induced nitroreductase (a biochemical marker) and cytoplasmic viscosity (a biophysical marker), two characteristics of cancer cells. The ability of the fluorescent molecular rotor in detecting tumor cells was evaluated in mouse and human specimens of multiple tissues by comparing with hematoxylin and eosin staining. Importantly, the fluorescent molecular rotor achieved 100% specificity in discriminating lung and liver cancers from normal tissue, allowing pre-screening of the tumor-free surgical margins and promoting clinical decision. Altogether, this type of fluorescent molecular rotor and the proposed strategy may serve as a new option to facilitate intraoperative assessment of resected tumor margins.

Increased CD3+, CD8+, or FoxP3+ T Lymphocyte Infiltrations Are Associated with the Pathogenesis of Colorectal Cancer but Not with the Overall Survival of Patients

Tumor-infiltrating lymphocytes include heterogeneous populations of T lymphocytes that play crucial roles in the tumor immune response; importantly, their presence in the tumor tissue may predict clinical outcomes. Therefore, we herein studied the prognostic significance of the presence and location of CD3+, CD8+, and FoxP3+ T lymphocytes in colorectal cancer samples. In the intratumor analysis, our data did not reveal any association between lymphocyte infiltrations with clinical or pathological data. However, in the tumor margins, we found that the presence of high infiltrations of CD3+, CD8+, or FoxP3+ T lymphocytes were associated with TNM stages I-II (p = 0.021, p = 0.022, and p = 0.012, respectively) and absence of lymph node metastases (p = 0.010, p = 0.003, and p = 0.004, respectively). Despite these associations with good prognostic indicators, we were not able to find any statistically significant alterations in the overall survival of the patients, even though high infiltrations of FoxP3+ T lymphocytes in the tumor margins resulted in an increased overall survival of 14 months. Taken together, these data show that the presence of CD3+, CD8+, or FoxP3+T lymphocyte infiltrates in the tumor margins are associated with the pathogenesis of CRC, but only high Foxp3+ T lymphocyte infiltrations in the tumor invasive margins are inclined to indicate favorable prognosis.

Rapid diagnosis and tumor margin assessment during pancreatic cancer surgery with the MasSpec Pen technology

Intraoperative delineation of tumor margins is critical for effective pancreatic cancer surgery. Yet, intraoperative frozen section analysis of tumor margins is a time-consuming and often challenging procedure that can yield confounding results due to histologic heterogeneity and tissue-processing artifacts. We have previously described the development of the MasSpec Pen technology as a handheld mass spectrometry–based device for nondestructive tissue analysis. Here, we evaluated the usefulness of the MasSpec Pen for intraoperative diagnosis of pancreatic ductal adenocarcinoma based on alterations in the metabolite and lipid profiles in in vivo and ex vivo tissues. We used the MasSpec Pen to analyze 157 banked human tissues, including pancreatic ductal adenocarcinoma, pancreatic, and bile duct tissues. Classification models generated from the molecular data yielded an overall agreement with pathology of 91.5%, sensitivity of 95.5%, and specificity of 89.7% for discriminating normal pancreas from cancer. We built a second classifier to distinguish bile duct from pancreatic cancer, achieving an overall accuracy of 95%, sensitivity of 92%, and specificity of 100%. We then translated the MasSpec Pen to the operative room and predicted on in vivo and ex vivo data acquired during 18 pancreatic surgeries, achieving 93.8% overall agreement with final postoperative pathology reports. Notably, when integrating banked tissue data with intraoperative data, an improved agreement of 100% was achieved. The result obtained demonstrate that the MasSpec Pen provides high predictive performance for tissue diagnosis and compatibility for intraoperative use, suggesting that the technology may be useful to guide surgical decision-making during pancreatic cancer surgeries.

Limited recurrence distance of glioblastoma under modern radiotherapy era

Background The optimal treatment volume for Glioblastoma multiforme (GBM) is still a subject of debate worldwide. The current study was aimed to determine the distances between recurring tumors and the edge of primary lesions, and thereby provide evidence for accurate target area delineation. Methods Between October 2007 and March 2019, 68 recurrent patients with GBM were included in our study. We measured the distance from the initial tumor to the recurrent lesion of GBM patients by expanding the initial gross tumor volume (GTV) to overlap the center of recurrent lesion, with the help of the Pinnacle Treatment Planning System. Results Recurrences were local in 47(69.1%) patients, distant in 12(17.7%) patients, and both in 9(13.2%) patients. Factors significantly influencing local recurrence were age (P = 0.049), sex (P = 0.049), and the size of peritumoral edema (P = 0.00). A total number of 91 recurrent tumors were analyzed. All local recurrences occurred within 2 cm and 94.8% (55/58) occurred within 1 cm of the original GTV based on T1 enhanced imaging. All local recurrences occurred within 1.5 cm and 98.3%(57/58) occurred within 0.5 cm of the original GTV based on T2-FLAIR imaging. 90.9% (30/33) and 81.8% (27/33) distant recurrences occurred >3 cm of T1 enhanced and T2-Flair primary tumor margins, respectively. Conclusions The 1 cm margin from T1 enhanced lesions and 0.5 cm margin from T2-Flair abnormal lesions could cover 94.8 and 98.3% local recurrences respectively, which deserves further prospective study as a limited but effective target area.

Image-Guided Peri-Tumoral Radiofrequency Hyperthermia-Enhanced Direct Chemo-Destruction of Hepatic Tumor Margins

PurposeTo validate the feasibility of using peri-tumoral radiofrequency hyperthermia (RFH)-enhanced chemotherapy to obliterate hepatic tumor margins.Method and MaterialsThis study included in vitro experiments with VX2 tumor cells and in vivo validation experiments using rabbit models of liver VX2 tumors. Both in vitro and in vivo experiments received different treatments in four groups (n=6/group): (i) RFH-enhanced chemotherapy consisting of peri-tumoral injection of doxorubicin plus RFH at 42°C; (ii) RFH alone; (iii) doxorubicin alone; and (iv) saline. Therapeutic effect on cells was evaluated using different laboratory examinations. For in vivo experiments, orthotopic hepatic VX2 tumors in 24 rabbits were treated by using a multipolar radiofrequency ablation electrode, enabling simultaneous delivery of both doxorubicin and RFH within the tumor margins. Ultrasound imaging was used to follow tumor growth overtime, correlated with subsequent histopathological analysis.ResultsIn in vitro experiments, MTS assay demonstrated the lowest cell proliferation, and apoptosis analysis showed the highest apoptotic index with RFH-enhanced chemotherapy, compared with the other three groups (p<0.01). In in vivo experiments, ultrasound imaging detected the smallest relative tumor volume with RFH-enhanced chemotherapy (p<0.01). The TUNEL assay further confirmed the significantly increased apoptotic index and decreased cell proliferation in the RFH-enhanced therapy group (p<0.01).ConclusionThis study demonstrates that peri-tumoral RFH can specifically enhance the destruction of tumor margins in combination with peri-tumoral injection of a chemotherapeutic agent. This new interventional oncology technique may address the critical clinical problem of frequent marginal tumor recurrence/persistence following thermal ablation of large (>3 cm) hepatic cancers.

Fluorescent Imaging and Multifusion Segmentation for Enhanced Visualization and Delineation of Glioblastomas Margins

This study investigates the potential of fluorescence imaging in conjunction with an original, fused segmentation framework for enhanced detection and delineation of brain tumor margins. By means of a test bed optical microscopy system, autofluorescence is utilized to capture gray level images of brain tumor specimens through slices, obtained at various depths from the surface, each of 10 µm thickness. The samples used in this study originate from tumor cell lines characterized as Gli36ϑEGRF cells expressing a green fluorescent protein. An innovative three-step biomedical image analysis framework is presented aimed at enhancing the contrast and dissimilarity between the malignant and the remaining tissue regions to allow for enhanced visualization and accurate extraction of tumor boundaries. The fluorescence image acquisition system implemented with an appropriate unsupervised pipeline of image processing and fusion algorithms indicates clear differentiation of tumor margins and increased image contrast. Establishing protocols for the safe administration of fluorescent protein molecules, these would be introduced into glioma tissues or cells either at a pre-surgery stage or applied to the malignant tissue intraoperatively; typical applications encompass areas of fluorescence-guided surgery (FGS) and confocal laser endomicroscopy (CLE). As a result, this image acquisition scheme could significantly improve decision-making during brain tumor resection procedures and significantly facilitate brain surgery neuropathology during operation.

Automatic diagnosis and biopsy classification with dynamic Full-Field OCT and machine learning

The adoption of emerging imaging technologies in the medical community is often hampered if they provide a new unfamiliar contrast that requires experience to be interpreted. Here, in order to facilitate such integration, we developed two complementary machine learning approaches, respectively based on feature engineering and on convolutional neural networks (CNN), to perform automatic diagnosis of breast biopsies using dynamic full field optical coherence tomography (D-FF-OCT) microscopy. This new technique provides fast, high resolution images of biopsies with a contrast similar to H&E histology, but without any tissue preparation and alteration. We conducted a pilot study on 51 breast biopsies, and more than 1,000 individual images, and performed standard histology to obtain each biopsy diagnosis. Using our automatic diagnosis algorithms, we obtained an accuracy above 88% at the image level, and above 96% at the biopsy level. Finally, we proposed different strategies to narrow down the spatial scale of the automatic segmentation in order to be able to draw the tumor margins by drawing attention maps with the CNN approach, or by performing high resolution precise annotation of the datasets. Altogether, these results demonstrate the high potential of D-FF-OCT coupled to machine learning to provide a rapid, automatic, and accurate histopathology diagnosis.