despite enormous improvement of diagnostic and therapeutic possibilities , 2 of every 3 cases of head and neck cancer are detected in advanced stage and , even after curative treatment , 30% to 50% of patients sustain a locoregional recurrence with worse prognosis.1 in addition to long periods without symptoms , the difficult access to some head and neck subsites and the field cancerization process are major reasons why it is so difficult to detect head and neck cancer in an early stage as well as to guarantee clear tumor free margins ( r0 ) when approaching a tumor with surgical therapy.2 tumorfree margins are a major prognostic factor for local tumor recurrence , which occurs in 7% to 30% of patients with advanced disease.3 the field cancerization process leads to the fact that the otorhinolaryngologist facing a suspicious lesion in the head and neck region typically is confronted with a combination of inflammatory , dysplastic , and cancerous areas.4 , 5 in earlystage cases , it is difficult to differentiate chronic inflammation from early cancer and , in advanced tumors , it is difficult to define the tumor borders clinically , even when using endoscopic or microscopic assistance . even for benign diseases , it might be very difficult to clearly define the border of the lesion . to date , the clinician can rely only on systematic biopsies and their histopathological grading of the epithelial dysplasia . this is timeconsuming , must be performed ex vivo , and has methodological limitations.6 it is well known that there are other molecular factors indicating genetic altered cells beyond dysplasia that have a major prognostic impact on tumor diagnosis and evaluation of tumor margins . new optical diagnostics are needed to allow a faster noninvasive ( or least lessinvasive ) differentiation between normal tissue and tumor tissue , between benign and malignant tumor tissue , and between tumor tissue and surrounding inflammation . the development of nearinfrared ( nir ) optical fluorescent techniques for diagnosis , treatment , and followup of head and neck lesions , especially for head and neck cancer , is a growing field that provides realtime information regarding the presence , location , and dimensions of the cancer tissue and/or metastasis through creation of a specific contrast between normal and cancer tissue . this contrast could be the result of tumorinduced morphologic and biochemical alterations that alter optical properties of tissues and lead to tumorspecific autofluorescence . furthermore , fluorescence can be achieved through targeting head and neck lesions by fluorescent probes . it is approved by the food and drug administration and the european medicines agency for determining cardiac output , hepatic function , and liver blood flow , and for ophthalmic angiography , but so far not for head and neck lesions . in the head and neck area , it has been used so far casuistically only as icg angiography for the monitoring of free flaps or for the evaluation of the relation of larger vessels to skull base tumors.7 , 8 it has been applied in paranasal tumors to adapt intraarterial chemotherapy.9 recently , icg has been introduced as a marker for sentinel node biopsies in patients with head and neck cancer.10 , 11 if icg nir endoscopy could improve the visualization of the mucosal lesion itself , it has not been evaluated so far . because of its ability to visualize neoplastic vascularization , it has been shown to support the endoscopic detection of lung metastasis or early gastric cancer as well as the detection of liver metastasis during open abdominal surgery.12 , 13 , 14 
  therefore , we started a pilot study to explore the feasibility , opportunities , and potential drawbacks of nir endoscopy with icg to examine mucosal head and neck lesions . this prospective observational study was performed at the department of otorhinolaryngology , jena university hospital , jena , germany . approval for the study was obtained through the local institutional review board and informed consent was obtained from all study participants . the study cohort consisted of 55 patients with different mucosal head and neck lesions treated between november 2013 and december 2015 . inclusion criteria were age 18 years , indication for diagnostic panendoscopy or microlaryngoscopy in general anesthesia and taking biopsies , and written informed consent , including consent for the offlabel use of icg . exclusion criteria were severe chronic kidney , lung and liver disease , and thyroid autonomy . standard panendoscopy or microlaryngoscopy with careful evaluation of the suspected mucosal lesion area and its surrounding tissue was performed . an niroptimized hd camera system ( image1 s nir / icg system ; karl storz , tuttlingen , germany ) and light source ( dlight p ; karl storz ) were used for nir endoscopy . this system allows a switchover via a footswitch between fluorescence and standard white light mode . niroptimized telescopes were used ( icg hopkins 0 telescope , 5.8 mm diameter , 19 cm length ; and icg hopkins 0 telescope , 10 mm diameter , 20 cm length ; karl storz ) . then , icg ( 5 ml of 25 mg/15 ml solution = 8.3 mg icg ; pulsion , feldkirchen , germany ) was administered intravenously . before this study , we tested solutions from 25 mg/30 ml to 25 mg/5 ml . the 25 mg/15 ml solution gave the best image contrast . the initial icg uptake phase was completely recorded in the nir icg mode of the imaging system . after the end of this uptake phase , it was switched back and forth several times from the nir icg mode to the white light mode of the endoscope before the recording was completed . finally , standard biopsies were taken from the same areas and preceded for standard histopathology . rigid nearinfrared ( nir ) with indocyanine green ( icg ) endoscopy was performed directly after injection of the icg . a split screen allowed observation of white light image and fluorescence image at the same time . the images were evaluated independently by 2 evaluators who were blinded with respect to the final histopathological results . the videos were evaluated for 3 parameters : ( 1 ) icg positivity of the head and neck lesion ( yes / no ) was the first parameter . a lesion was defined as icgpositive if the lesion showed a fast uptake of icg within 1 to 2 seconds after icg injection . therefore , it was possible that the lesion itself was icgnegative but contained icgpositive vessels . ( 2 ) pooling of icg was the second parameter ( pooling yes / no ) : some lesions showed a constant icg labeling throughout the entire videorecording phase and a pooling of icg within the lesion . ( 3 ) the tumor borders were evaluated as the third parameter . in case of an icgpositive lesion , the size of the icglabeled tumor was compared to the size of the tumor under white light endoscopy ( same / larger / smaller ) . statistical analyses were performed using ibm spss version 23.0 statistical software for windows ( chicago , il ) . the diagnostic accuracy of nir icg positivity to prognosticate a malignant tumor was evaluated by a 2by2 table and calculation of sensitivity and specificity . differences concerning the evaluation parameters between 2 subgroups of patients were evaluated with the chisquare test . for all statistical tests , significance was 2sided and set to p < .05 interobserver variability of the assessment of the nir icg findings was analyzed using kappa statistics . this prospective observational study was performed at the department of otorhinolaryngology , jena university hospital , jena , germany . approval for the study was obtained through the local institutional review board and informed consent was obtained from all study participants . the study cohort consisted of 55 patients with different mucosal head and neck lesions treated between november 2013 and december 2015 . inclusion criteria were age 18 years , indication for diagnostic panendoscopy or microlaryngoscopy in general anesthesia and taking biopsies , and written informed consent , including consent for the offlabel use of icg . exclusion criteria were severe chronic kidney , lung and liver disease , and thyroid autonomy . standard panendoscopy or microlaryngoscopy with careful evaluation of the suspected mucosal lesion area and its surrounding tissue was performed . an niroptimized hd camera system ( image1 s nir / icg system ; karl storz , tuttlingen , germany ) and light source ( dlight p ; karl storz ) were used for nir endoscopy . this system allows a switchover via a footswitch between fluorescence and standard white light mode . niroptimized telescopes were used ( icg hopkins 0 telescope , 5.8 mm diameter , 19 cm length ; and icg hopkins 0 telescope , 10 mm diameter , 20 cm length ; karl storz ) . then , icg ( 5 ml of 25 mg/15 ml solution = 8.3 mg icg ; pulsion , feldkirchen , germany ) was administered intravenously . before this study , we tested solutions from 25 mg/30 ml to 25 mg/5 ml . the 25 mg/15 ml solution gave the best image contrast . the initial icg uptake phase was completely recorded in the nir icg mode of the imaging system . after the end of this uptake phase , it was switched back and forth several times from the nir icg mode to the white light mode of the endoscope before the recording was completed . finally , standard biopsies were taken from the same areas and preceded for standard histopathology . rigid nearinfrared ( nir ) with indocyanine green ( icg ) endoscopy was performed directly after injection of the icg . a split screen allowed observation of white light image and fluorescence image at the same time . the videos were viewed repeatedly . additionally , all videos were evaluated in singleframe mode . the images were evaluated independently by 2 evaluators who were blinded with respect to the final histopathological results . the videos were evaluated for 3 parameters : ( 1 ) icg positivity of the head and neck lesion ( yes / no ) was the first parameter . a lesion was defined as icgpositive if the lesion showed a fast uptake of icg within 1 to 2 seconds after icg injection . therefore , it was possible that the lesion itself was icgnegative but contained icgpositive vessels . ( 2 ) pooling of icg was the second parameter ( pooling yes / no ) : some lesions showed a constant icg labeling throughout the entire videorecording phase and a pooling of icg within the lesion . ( 3 ) the tumor borders were evaluated as the third parameter . in case of an icgpositive lesion , the size of the icglabeled tumor was compared to the size of the tumor under white light endoscopy ( same / larger / smaller ) . statistical analyses were performed using ibm spss version 23.0 statistical software for windows ( chicago , il ) . if not otherwise indicated , data are reported as mean with sd . the diagnostic accuracy of nir icg positivity to prognosticate a malignant tumor was evaluated by a 2by2 table and calculation of sensitivity and specificity . differences concerning the evaluation parameters between 2 subgroups of patients were evaluated with the chisquare test . for all statistical tests , significance was 2sided and set to p < .05 interobserver variability of the assessment of the nir icg findings was analyzed using kappa statistics . the majority of the lesions were localized in the larynx ( 58% ) and the oropharynx ( 29% ) . the dominant histology was squamous cell cancer or a premalignant squamous intraepithelial neoplasia ( sin ) iii tumor ( 33% ) , followed by benign squamous cell hyperplasia ( 15% ) , normal mucosa ( 13% ) , and reinke edema ( 11% ) . benign lesions were icgnegative and premalignant or malignant tumors ( severe dysplasia sin iii and squamous cell cancer ) were icgpositive ( table 2 , figure 2 ) . in icgpositive tumors , normal icgpositive vessels were no longer separately identifiable . icgpositive lesions showed a fast and gradual uptake of the icg with seconds after the intravenous bolus injection ( figure 3 ) . if the lesion was icgpositive , it seemed to be of the same size or larger during nir icg endoscopy than during white light endoscopy when the lesion was malignant ( table 3 ) . in contrast , icg size seemed to be smaller than the size in white light endoscopy in benign icgpositive lesions . retention of icg was visible in 21 of 22 icgpositive lesions ; only the benign papilloma showed a strong icg positivity but no pooling after the icg uptake phase . the retention of the icg could be homogeneous or inhomogeneous with an icgpositive lesion ( figure 2 ) . relation of histopathology to size of the indocyanine green  positive lesion compared to the size during white light endoscopy . endoscopic examples of nearinfrared ( nir ) with indocyanine green ( icg ) finding in different types of mucosal head and neck lesions in the larynx . white light image on the left side ( a  e ) and corresponding nir icg image on the right side ( f  j ) . normal mucosa ( ab ) and reinke edema ( bg ) only showed icg positivity in the submucosal vessels . the mucosal hyperplasia on the right anterior vocal cord ( ch ) was completely icgnegative , even sparing icgpositive vessels . the severe dysplasia of the right vocal cord ( squamous intraepithelial neoplasia [ sin ] iii , di ) and the squamous cell cancer of the left anterior vocal cord ( t1 glottic cancer , ej ) showed a diffuse icg positivity and retention of icg . two endoscopic examples of gradual onset of indocyanine green ( icg ) in 2 icgpositive malignant tumors . the glottic carcinoma on the left anterior vocal cord ( a  e , same as ej in figure 2 ) starts to become clearly icgpositive 5 seconds after the intravenous icg bolus injection . the oropharyngeal carcinoma in the left tonsillar fossa ( f  j ) showed first icg positivity after 6 seconds . the icgpositive area is larger than the visible area of the tumor in the white light image ( compare f to j ) ; normal submucosal vessels are visible on the posterior pharyngeal wall ( see i and j ) . this resulted in a sensitivity , specificity , and accuracy that icg positivity was related to a malignant tumor of 90.5% , 90.9% , and 89.1% , respectively . diagnostic accuracy of indocyanine green positivity of malignant tumors ( n = 55).a 
  abbreviation : icg , indocyanine green . including premalignant squamous intraepithelial neoplasia ( sin iii ) lesions . the kappa index for the interobserver assessment of the nir icg finding showed a 94.4% , 79.4% , and 62.5% agreement , respectively , for the assessment of the icg positivity of the lesion , the size in relation to the white light image , and concerning the retention of icg within the lesion . the majority of the lesions were localized in the larynx ( 58% ) and the oropharynx ( 29% ) . the dominant histology was squamous cell cancer or a premalignant squamous intraepithelial neoplasia ( sin ) iii tumor ( 33% ) , followed by benign squamous cell hyperplasia ( 15% ) , normal mucosa ( 13% ) , and reinke edema ( 11% ) . benign lesions were icgnegative and premalignant or malignant tumors ( severe dysplasia sin iii and squamous cell cancer ) were icgpositive ( table 2 , figure 2 ) . in icgpositive tumors , normal icgpositive vessels were no longer separately identifiable . icgpositive lesions showed a fast and gradual uptake of the icg with seconds after the intravenous bolus injection ( figure 3 ) . if the lesion was icgpositive , it seemed to be of the same size or larger during nir icg endoscopy than during white light endoscopy when the lesion was malignant ( table 3 ) . in contrast , icg size seemed to be smaller than the size in white light endoscopy in benign icgpositive lesions . retention of icg was visible in 21 of 22 icgpositive lesions ; only the benign papilloma showed a strong icg positivity but no pooling after the icg uptake phase . the retention of the icg could be homogeneous or inhomogeneous with an icgpositive lesion ( figure 2 ) . relation of histopathology to size of the indocyanine green  positive lesion compared to the size during white light endoscopy . endoscopic examples of nearinfrared ( nir ) with indocyanine green ( icg ) finding in different types of mucosal head and neck lesions in the larynx . white light image on the left side ( a  e ) and corresponding nir icg image on the right side ( f  j ) . normal mucosa ( ab ) and reinke edema ( bg ) only showed icg positivity in the submucosal vessels . the mucosal hyperplasia on the right anterior vocal cord ( ch ) was completely icgnegative , even sparing icgpositive vessels . the severe dysplasia of the right vocal cord ( squamous intraepithelial neoplasia [ sin ] iii , di ) and the squamous cell cancer of the left anterior vocal cord ( t1 glottic cancer , ej ) showed a diffuse icg positivity and retention of icg . two endoscopic examples of gradual onset of indocyanine green ( icg ) in 2 icgpositive malignant tumors . the glottic carcinoma on the left anterior vocal cord ( a  e , same as ej in figure 2 ) starts to become clearly icgpositive 5 seconds after the intravenous icg bolus injection . the oropharyngeal carcinoma in the left tonsillar fossa ( f  j ) showed first icg positivity after 6 seconds . the icgpositive area is larger than the visible area of the tumor in the white light image ( compare f to j ) ; normal submucosal vessels are visible on the posterior pharyngeal wall ( see i and j ) . this resulted in a sensitivity , specificity , and accuracy that icg positivity was related to a malignant tumor of 90.5% , 90.9% , and 89.1% , respectively . diagnostic accuracy of indocyanine green positivity of malignant tumors ( n = 55).a 
  abbreviation : icg , indocyanine green . including premalignant squamous intraepithelial neoplasia ( sin iii ) lesions . the kappa index for the interobserver assessment of the nir icg finding showed a 94.4% , 79.4% , and 62.5% agreement , respectively , for the assessment of the icg positivity of the lesion , the size in relation to the white light image , and concerning the retention of icg within the lesion . in head and neck oncology , nir fluorescence imaging has so far been introduced for sentinel lymph node mapping and might be a promising alternative to gamma rayemitting radiotracers for transcutaneous visualization of superficial lymphatic channels and sentinel lymph nodes in the future.15 , 16 for direct tumor imaging , it has been used so far in a limited number of studies , mainly in patients with hepatobiliary cancer , colorectal metastases , gastric cancer , or breast cancer.17 , 18 , 19 , 20 to our knowledge , yokoyama et al21 were the first to apply nir icg endoscopy to visualize the primary head and neck cancer in 5 patients . the same group recently published a series of 36 patients with advanced paranasal sinus cancer receiving an icg injection together with intraarterial chemotherapy . this was helpful to confirm the target region for intraarterial chemotherapy , especially in cases in which standard ct angiography was not indicative.9 recently , digonnet et al22 used nir icg imaging primarily to study the fluorescence of negative and negative lymph nodes during neck dissection in 11 patients . four of these 5 tumors were icgpositive ( 80% vs 89% of the premalignant sin iii tumors or malignant tumors in the present study ) . the margins were icgnegative after tumor resection or second resection in 1 case with residual fluorescence . these results and the findings of the present study confirm that icg positivity is a marker of most head and neck cancers , as it has already been shown for other solid tumors ( see above ) . no adverse reactions related to the icg injection occurred in patients with head and neck lesions . it is repeatedly stated in the literature that bolus injections of 0.25 to 0.5 mg / kg body weight might increase the risk of allergic reactions . however , this statement is actually related to only 1 study.23 interestingly , we needed only a fixed dose of 8.3 mg per patients ( which would be only 0.12 mg / kg in a patient with 70 kg body weight ) to achieve a fast visualization within seconds to minutes , whereas others report a best visualization with 0.5 mg / kg icg within 60 to 120 minutes.21 we can only speculate that our imaging system might be better able to detect the fluorescence than other systems . it binds to plasma proteins , remains within the vascular system , and does not diffuse into the interstitial space under physiological conditions.15 it is proposed that the peritumoral vascular hyperpermeability is one reason for the tumor fluorescence.16 this might be the reason why tissue with strong vascularization , like the lymphatic tissue of the tonsil or a benign papilloma , or hyperpermeability because of inflammation , also could show ( false ) icg positivity . furthermore , tumor neovascularization is characterized by newly formed and more porous blood vessels , leading to passive accumulation of icg in tumor tissue.15 this results in retention and pooling of icg within the malignant tumor , as it could be seen even in a premalignant lesion and in early glottic cancer ( sin iii lesion or t1 glottic cancer , respectively ) in the present study or as a characteristic feature of early glottic cancer.17 , 24 interestingly , the icg signal not only helped to differentiate between benign and malignant lesions , but also to differentiate different types of benign lesions . the normal submucosal vessels system in the head and neck mucosa is always icgpositive . in case of benign squamous cell hyperplasia it might be that the hyperplasia covered the fluorescence of the vessels in a deeper layer . future studies should show how thick such a hyperplasia must be to cover icgpositive vessels . this is important because this factor could be important in ( false ) icgnegative malignant tumors . furthermore , at the moment , targeted fluorescent dyes in the nir range are being introduced into clinical trials , which might replace nontargeted ones in the near future.25 , 26 this will further improve the accuracy of the technology . beyond icg positivity , the demarcation of the tumor borders is of interest in respect of imageguided surgery.27 in the present study , the tumor borders appeared to be larger under icg than under white light endoscopy , but the true histological borders were not correlated to the endoscopic appearance . if icgguided surgery could lead to clearer tumor margins and better outcome , it has to be shown in future studies with histological correlations . in any case , it has been shown already that a repetition of the nir icg endoscopy directly after tumor resection can help to obtain clear margins because the wound bed should be icgnegative after resection.22 
  the advantage of nir icg imaging is that several endoscopy systems as well as icg are licensed and on the market . the nir light does not alter the vision of the surgeon and it is easy to switch between white light and icg image . furthermore , the signaltobackground ratio is impressive because of very low autofluorescence.15 in contrast , a dense submucosal vascular network , like in the tonsils , might make it more difficult to demarcate a melanoma against the background . the good tissue penetration of about 5 to 10 mm is an advantage to other optical imaging techniques ( for instance , autofluorescence , confocal laser endomicroscopy , or optical coherence tomography ) , but , of course , still not enough to get an overview of larger tumors ( > t1).28 
  the diagnostic accuracy , sensitivity , and specificity to predict a malignant tumor by icg positivity was satisfactory for such a first feasibility study . one should not forget that the evaluation was performed offline by watching the videos repeatedly and in singleframe modus . icgpositive  and  icgnegative  regions was only subjectively evaluated , so the method lacks any objectivity ( apart from the interobserver evaluation ) . especially with regard to a possible application during a resection of tumors , where blood , tissue debris , and coagulation artifacts might reduce the contrast even further , a high contrast would be mandatory . it would be worthwhile to develop automated image analysis methods for icg images as it has also ready been introduced for confocal laser endomicroscopy images.29 for optical imageguided surgery , it would be helpful to develop tools to overlay icg image and white light image.30 finally , the approval of tumorspecific nir probes or the conjunction of tumorspecific antibodies with unspecific nir probes will be the most important step to develop intraoperative imageguided cancer surgery.31 nir icg endoscopy was feasible in 55 patients with different mucosal head and neck lesions . complications were not seen . for a first feasibility study , the diagnostic accuracy analysis to predict a malignant tumor by offline evaluation of the icg videos for icg positivity of the tumors was satisfactory . the analysis showed a sensitivity , specificity , and accuracy that icg positivity of the tumor was related to a malignant tumor of 90.5% , 90.9% , and 89.1% , respectively . this has to be confirmed in larger confirmatory studies , including tumors that are more advanced and especially in relation to the correlation between icg tumor margins and histological tumor margins .