The usages , possibilities and impossibilities of INDOCYANINE GREEN QUANTIFICATION in a novel approach for sentinel lymph node detection in robotic surgery for colon carcinoma

Introduction & Background Colon carcinoma (CC) is the cancer type with the second highest morbidity in the Netherlands and its prevalence will only rise due to screening programs and an ageing population. To prevent mortality due to understaging, a hemicolectomy is performed but this increases the postoperative mortality and morbidity risk. The sentinel lymph node (SLN) procedure can form a solution because it functions as a triage procedure and avoids surgery which is done to prevent micrometastases. The research question in this master thesis is “What are the possibilities of using a specific near-infrared (NIR) tracer in detecting the sentinel lymph node and making a prognosis in early-stage colon cancer? ” Three subgoals are used to provide an answer to this question: a Evaluate a promising biomarker for the identification of tumour cells in lymph nodes. b Set up an indocyanine green (ICG) quantification study in patients to explore the possibilities of quantifying the fluorescence intensity. c Set up an ICG quantification phantom study to explore the different variables influencing the intensity on screen. (a) A CD44v6 targeted biomarker The usefulness of a CD44v6 targeted biomarker for the identification of early-stage CC cells was assessed. Tissue of patients treated for all stages of CC was included and stained using the CD44v6 targeted biomarker. 59 Slices of tissue of 21 different patients were included. The stained slices were scored on membrane staining using the H-score. The sensitivity and specificity were calculated, and appropriate statistical tests were used to define any significant differences in staining. A sensitivity of 0.98 and a specificity of 1 was found. The Mann-Whitney test showed a significant difference between tumour tissue and tumour deposit, no other significant differences were found. The two-tailed Wilcoxon test showed a non-significant difference within a patient. The high sensitivity and specificity of this biomarker, and the non-significant difference of H-scores within one patient make CD44v6 a promising marker for the identification of CC cells in tumour and lymph node metastases. However, the sample size was limited, and a follow-up study with a higher sample size is required to confirm the results. (b) An ICG quantification study in patients Ultimately, CD44v6 is combined with an NIR dye to identify the SLN in vivo and quantification of this fluorescence signal should be investigated. Therefore, the aim was to construct a reliable segmentation and quantification method of the SLN in operative footage. Patients who received surgery to treat CC with NIR SLN identification using ICG were included. The lymph nodes were segmented in the operative footage based on thresholding in the HSV colour space. Two patients were included, a segmentation was performed, and the average intensity of the ICG in the lymph nodes was calculated. The segmentation and quantification method constructed is reliable, therewith providing possibilities for CC cell identification using a specific NIR tracer. A limitation is a non-standardised measurement making comparison between patients impossible since the pixel intensity is influenced by camera distance and orientation. Currently, research is continuing into standardisation possibilities. (c) A fluorescence intensity phantom study Fluorescence intensity depends on many operator, patient, and system dependent variables and is therefore easily underor overestimated. The aim was to design a phantom study to explore the effect of distance, orientation, concentration, and field of view (FOV) on the fluorescence intensity when using the Firefly Xi. The phantom consisted of Eppendorf tubes filled with different concentrations of ICG diluted in PBS and 2% intralipid to induce scattering. Three types of measurements were performed: (1) dynamical measurements, (2) FOV measurements, and (3) static measurements. Segmentation was applied by thresholding in the HSV colour space or in the grey value image. Both measurements 1 and 2 confirmed a non-uniformity of the fluorescence intensity within the FOV, where the highest intensity was found in the middle. The static measurements showed a difference in intensity when applying different distances and angles. A significant negative linear correlation to the pixel intensity was found considering distance to the camera and a significant non-linear correlation was found for orientation. Concerning concentration, a non-significant non-linear correlation was found. This study was limited by the instability of ICG, the influence of ambient light, and a lack of preciseness considering the distances and orientations. All of these are present in a clinical setting. Therefore, based on these results, surgeons can be made aware of the influence of operator-dependent variables. Furthermore, the found correlations can contribute to a correction model enabling clinical interpretation of fluorescence intensity and enhancing the novel approach for SLN identification.