10.14639/0392-100X-N1643
Objective. Development and validation of a confocal laser endomicroscopy (CLE) classification score for the larynx and pharynx. Methods. Thirteen patients (154 video sequences, 9240 images) with laryngeal or pharyngeal SCC were included in this prospective study between October 2020 and February 2021. Each CLE sequence was correlated with the gold standard of histopathological examination. Based on a dataset of 94 video sequences (5640 images), a scoring system was developed. In the remaining 60 sequences (3600 images), the score was validated by four CLE experts and four head and neck surgeons who were not familiar with CLE. Results. Tissue homogeneity, cell size, borders and clusters, capillary loops and the nucleus/ cytoplasm ratio were defined as the scoring criteria. Using this score, the CLE experts obtained an accuracy, sensitivity, and specificity of 90.8%, 95.1%, and 86.4%, respectively, and the CLE non-experts of 86.2%, 86.4%, and 86.1%. Interobserver agreement Fleiss’ kappa was 0.8 and 0.6, respectively. Conclusions. CLE can be reliably evaluated based on defined and reproducible imaging features, which demonstrate a high diagnostic value. CLE can be easily integrated into the intraoperative setting and generate real-time, in-vivo microscopic images to demarcate malignant changes.
text/xml
1827-675X
1643
en
Miguel Goncalves
http://creativecommons.org/licenses/by-nc-nd/4.0
ACTA Otorhinolaryngologica Italica
pharynx
Systematic interpretation of confocal laser endomicroscopy: larynx and pharynx confocal imaging score
Text.Serial.Journal
https://www.actaitalica.it/
42
Head and neck
2022-02-07
2021-05-04
2022-03-07
2022-03-07
26-33
1
Miguel Goncalves
2022/02/07
10.14639/0392-100X-N1643
26
Department of Otorhinolaryngology, Head and Neck Surgery, Friedrich-Alexander-Universität Erlangen–Nürnberg, University Hospital, Erlangen, Germany; Department of Otorhinolaryngology, Plastic Head and Neck Surgery, Rheinische Westf lische Technische Hochschule Aachen, University Hospital, Aachen, Germany
1827-675X
pharynx
33
Systematic interpretation of confocal laser endomicroscopy: larynx and pharynx confocal imaging score
https://www.actaitalica.it/article/download/1643/734
42
ACTA OTORHINOLARYNGOLOGICA ITALICA 2022;42:26-33; doi: 10.14639/0392-100X-N1643




Head and neck


Systematic interpretation of confocal laser
endomicroscopy: larynx and pharynx confocal
imaging score
Interpretazione sistematica dell’endomicroscopia confocale laser:
punteggio endomicroscopico confocale laringeo e faringeo
Matti Sievert1, Konstantinos Mantsopoulos1, Sarina K. Mueller1, Markus Eckstein2, Robin Rupp1,
Marc Aubreville3, Florian Stelzle4, Nicolai Oetter4, Andreas Maier5, Heinrich Iro1, Miguel Goncalves1,6
1
 Department of Otorhinolaryngology, Head and Neck Surgery, Friedrich-Alexander-Universität Erlangen–Nürnberg, University
Hospital, Erlangen, Germany; 2 Institute of Pathology, Friedrich-Alexander-Universität Erlangen–Nürnberg, University Hospital,
Erlangen, Germany; 3 Technische Hochschule, Ingolstadt, Germany; 4 Department of Maxillofacial Surgery, Friedrich-Alexander-
Universität Erlangen–Nürnberg, University Hospital, Erlangen, Germany; 5 Pattern Recognition Laboratory, Computer Science,
Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany; 6 Department of Otorhinolaryngology, Plastic Head and
Neck Surgery, Rheinische Westfälische Technische Hochschule Aachen, University Hospital, Aachen, Germany


SUMMARY
Objective. Development and validation of a confocal laser endomicroscopy (CLE) clas-
sification score for the larynx and pharynx.
Methods. Thirteen patients (154 video sequences, 9240 images) with laryngeal or phar-
yngeal SCC were included in this prospective study between October 2020 and February
2021. Each CLE sequence was correlated with the gold standard of histopathological ex-
amination. Based on a dataset of 94 video sequences (5640 images), a scoring system was
developed. In the remaining 60 sequences (3600 images), the score was validated by four
CLE experts and four head and neck surgeons who were not familiar with CLE.
Results. Tissue homogeneity, cell size, borders and clusters, capillary loops and the nu-              Received: April 28, 2021
cleus/cytoplasm ratio were defined as the scoring criteria. Using this score, the CLE experts          Accepted: September 28, 2021
                                                                                                       Published online: February 7, 2022
obtained an accuracy, sensitivity, and specificity of 90.8%, 95.1%, and 86.4%, respectively,
and the CLE non-experts of 86.2%, 86.4%, and 86.1%. Interobserver agreement Fleiss’
                                                                                                       Correspondence
kappa was 0.8 and 0.6, respectively.
                                                                                                       Miguel Goncalves
Conclusions. CLE can be reliably evaluated based on defined and reproducible imaging
                                                                                                       Department of Otorhinolaryngology, Plastic Head
features, which demonstrate a high diagnostic value. CLE can be easily integrated into                 and Neck Surgery, RWTH University of Aachen
the intraoperative setting and generate real-time, in-vivo microscopic images to demarcate             Pauwelsstraße 30, 52074 Aachen, Germany
malignant changes.                                                                                     Tel. +49 024180-89360. Fax +49 0241 80-82465
                                                                                                       E-mail: mgoncalves@ukaachen.de
KEY WORDS: confocal laser endomicroscopy, head and neck cancer, classification system,
non-invasive histological imaging, larynx, pharynx
                                                                                                         How to cite this article: Sievert M, Mantso-
                                                                                                         poulos K, Mueller SK, et al. Systematic inter-
RIASSUNTO                                                                                                pretation of confocal laser endomicroscopy: lar-
Obiettivo. Sviluppo e validazione di un punteggio di classificazione dell’endomicroscopia                ynx and pharynx confocal imaging score. Acta
laser confocale (CLE) per la laringe ed il faringe.                                                      Otorhinolaryngol Ital 2022;42:26-33. https://doi.
Metodi. Tredici pazienti (154 sequenze video, 9240 immagini) con SCC laringeo o farin-                   org/10.14639/0392-100X-N1643
geo sono stati inclusi in questo studio prospettico tra ottobre 2020 e febbraio 2021. Ogni
sequenza CLE è stata correlata con il gold standard, ovvero l’esame istopatologico fissato             © Società Italiana di Otorinolaringoiatria
con ematossilina ed eosina (H&E). Sulla base di un set di dati di 94 sequenze video (5640              e Chirurgia Cervico-Facciale
immagini), è stato sviluppato un sistema di punteggio. Nelle restanti 60 sequenze (3600
immagini), il punteggio è stato convalidato da quattro esperti di CLE e quattro chirurghi                             OPEN ACCESS
della testa e del collo che non avevano familiarità con la CLE.                                        This is an open access article distributed in accordance with
Risultati. L’omogeneità dei tessuti, le dimensioni delle cellule, i bordi e i cluster, i loop capil-   the CC-BY-NC-ND (Creative Commons Attribution-Non-
lari e il rapporto nucleo/citoplasma sono stati definiti come criteri di punteggio. Utilizzando        Commercial-NoDerivatives 4.0 International) license. The
                                                                                                       article can be used by giving appropriate credit and mentio-
questo punteggio, gli esperti di CLE hanno ottenuto un’accuratezza, una sensibilità e una              ning the license, but only for non-commercial purposes and
specificità del 90,8%, 95,1% e 86,4%, rispettivamente, e i non esperti di CLE dell’86,2%,              only in the original version. For further information: https://
86,4% e 86,1%. L’accordo tra osservatori Fleiss’kappa era rispettivamente di 0,8 e 0,6.                creativecommons.org/licenses/by-nc-nd/4.0/deed.en




26
                                                                                                     Larynx and pharynx confocal imaging score




Conclusioni. CLE può essere valutato in modo affidabile sulla base di caratteristiche di imaging definite e riproducibili, che dimostrano un
alto valore diagnostico. CLE può essere facilmente integrato nell’impostazione intraoperatoria e generare immagini microscopiche in tempo
reale e in vivo per delimitare i cambiamenti maligni.
PAROLE CHIAVE: endomicroscopia laser confocale, cancro della testa e del collo, sistema di classificazione, imaging istologico non invasivo,
laringe, faringe



Introduction                                                            rameters independent of confocal laser endomicroscopy
                                                                        findings.
Non-invasive methods to characterise mucosal changes of
the head and neck region, such as narrow-band imaging,
                                                                        Eligibility criteria
confocal laser imaging, optical coherence tomography and
                                                                        A total of 13 consecutive patients were included in this
fluorescence endoscopy, have been emerging recently 1-4.
                                                                        study. Inclusion criteria were all sequential patients with
One method that has shown a potential advance regarding its
                                                                        a histological diagnosis of laryngeal and pharyngeal SCC
broad application outside of experimental studies is confo-
                                                                        (i.e. after panendoscopy and biopsy) in which open surgery
cal laser endomicroscopy (CLE), which has demonstrated its
                                                                        was required and appropriate for the oncological treatment.
value in the fairly similar oesophageal, gastric and intestinal
                                                                        Exclusion criteria were prior treatment of any head and
epithelium in a wide range of clinical applications 5-8. CLE
                                                                        neck cancer, distant metastasis, radiotherapy in the head
is usually performed with the systemic application of fluo-
                                                                        and neck area, pregnancy, thyroid dysfunction, underaged
rescein, which stains the intercellular spaces and allows real-
                                                                        patients, severe kidney failure and allergy to fluorescein.
time, non-invasive examination of the surface epithelium, cell
features and fine capillary system to resemble a histological
                                                                        Technical details
examination in detail and resolution 9. Our group and others
                                                                        We used cold instruments to reduce thermal damage to the
have published several reports in the last few years demon-
                                                                        resection margin. After accessing the pharynx or larynx as
strating the feasibility of CLE and emphasising its potential
                                                                        required, we exposed the tumour to initiate CLE imaging.
in distinguishing benign from malignant lesions of the vocal
                                                                        Image acquisition was performed using a GastroFlex probe
cords and oral and pharyngeal mucosa, as well as its applica-
                                                                        combined with a 488 nm Cellvizio laser scanning system
tion in the intraoperative assessment of free margins during
                                                                        (Mauna Technologies, Paris, France). The 2.6 mm diam-
oncological surgery 10-15. The high accuracy of CLE reported
                                                                        eter probe has a field of view of 240 μm and a resolution of
in the latter studies, varying between 70 to 95%, was, how-
                                                                        1 μm with a penetration depth of 55-65 μm. Subsequently,
ever, obtained by experts in most cases and based on subjec-
tive analysis of the images without a criterial classification          2.5 ml fluorescein alcon 10% was injected intravenously,
system. The primary objective of this study was to develop              followed by an additional 2.5 ml fluorescein administra-
criteria to help differentiate benign from malignant lesions in         tion after 8 minutes. In order to correlate the CLE imag-
the pharyngeal and laryngeal regions. The secondary objec-              ing with the gold standard of histopathology, the regions
tive was to examine the practicability of the scoring system            recorded were marked with suture, or a separate biopsy
carried out by head and neck surgeons inexperienced in CLE              was performed. The complete resection was histologically
and to determine its diagnostic value.                                  confirmed intraoperatively following the standard protocol
                                                                        using frozen sections around the circumference of the de-
                                                                        fect. The histopathological assessment followed a standard
Materials and methods                                                   protocol with haematoxylin and eosin (H&E) staining.

Study design                                                            Development of the evaluation criteria
We conducted a prospective pilot diagnostic study with                  We developed the evaluation criteria according to a 4-step
two different cohorts, one to develop the scoring system                protocol. For the creation and validation of the scoring crite-
for assessing the accuracy of CLE and one to validate this              ria, we used different sequences of different patients. For data
scoring system. The study was performed at a tertiary hos-              processing, we analysed the sequences using Cellvizio View-
pital and academic cancer centre (Department of Otorhino-               er software 1.6.2 (Mauna Kea Technologies, Paris, France).
laryngology, Head and Neck Surgery, Friedrich Alexander                 Step 1 - Out of 28306 CLE frames from eight consecu-
University of Erlangen-Nuremberg, Erlangen, Germany).                   tive patients, we selected a dataset of 94 video sequences
Based on the German Guidelines for laryngeal carcinoma,                 (5640 images; 47 sequences of SCC and 47 sequences of
our treatment was performed on the basis of clinical pa-                benign mucosa) from four examiners for analysis to create



                                                                                                                                          27
M. Sievert et al.




dedicated evaluation criteria. The blinded examiners were          classifier. Spearman’s rank correlation coefficient is used to
asked to make a binary statement on dignity by assessing           calculate the relationship between the score value and the
the cell morphology and vascular situation. In addition, the       probability of a malignant finding. A p-value of less than p
raters were allowed to comment on the individual findings.         ≤ 0.05 was considered statistically significant. We performed
Step 2 - The authors analysed the raters’ results and checked      statistical analysis using SPSS version 22.0 (IBM SPSS Sta-
for regularities between the different raters’ decisions and       tistics for Windows, Version 22.0. Armonk, NY, USA).
their comments.
Step 3 - The authors obtained a consensus to develop a             Results
score (Larynx and Pharynx Confocal Imaging Score – LF-
CIS) based on the results and comments. The data set anal-
ysis aimed to define simple, effective and easily applicable       Patient characteristics
parameters for daily routine practice.                             Between March 2020 and February 2021, we enrolled 13
Step 4 - We validated the score by four experienced raters         patients (one female and 12 males; mean age 64.7 years
(“experts”) consisting of one pathologist (P) and three sur-       (SD = 8.6) to undergo pharyngeal in vivo CLE during
geons (S) (who had independently performed or analysed a           planned transcervical tumour resection in the area of the hy-
minimum of 20 CLE examinations) and four inexperienced             popharynx and larynx. In six patients (46.2%), the tumour
raters (non-experts, without any previous experience in            mass was located in the larynx. Seven patients (53.8%) ad-
this technique). The pathologist who rated the CLE images          ditionally presented with involvement of the hypopharyn-
was not involved and had no information about the histo-           geal mucosa. The tumour resection was performed via an
pathological examination of the probes and specimens. The          open approach in each case. We performed a partial laryn-
histopathological examination, as reference standard, was          gectomy via vertical incision of the thyroid cartilage in two
performed independently by other pathologists not directly         patients (15.4%; No. 1 and 2). In addition, we dissected an
involved in the study.                                             apron flap for total laryngectomy in 11 patients (84.6%;
Out of 12,065 new images (recorded in 5 additional con-            No. 3-13). Microvascular defect reconstruction was nec-
secutive patients), 60 sequences (3600 images; 33 sequenc-         essary in 7 patients (53.8%). Regarding tumour grading,
es of SCC and 27 sequences of benign mucosa) were se-              three patients (23.1%) were confirmed as having an inter-
lected and evaluated as representative in acceptable quality.      mediate grade (G2) and 10 patients (76.9%) poor grade dif-
This dataset was kept separate from the development of the         ferentiation (G3). Patient characteristics, including stage,
score to reduce overfitting and increase validity, to ensure       are shown in Table I. In all cases, safe margin resection
that the score was tested in yet unseen images. We matched         could be performed independent of the use of CLE.
each of these sequences (60 images, 5 seconds) with a cor-
responding sample by H&E staining to determine the diag-           Definition of diagnosis-related parameters and creation of
nostic accuracy of the score.                                      a scoring system
                                                                   The details of all four raters’ results regarding the CLE
Data analysis                                                      morphological criteria are presented in Table II. Based
Absolute values are presented with mean and standard de-           on the four raters’ evaluation, we attributed a high value
viation (SD). Relative values are presented in absolute and        to the abnormal tissue structure with an inhomogeneous
relative frequencies and were compared using the chi-square        cell pattern (κ = 0.67) and variance in cell size and shape
test. Sensitivity, specificity, positive predictive value (PPV),   (κ = 0.64) for the assessment of dignity. We therefore de-
negative predictive value (NPV) and accuracy were calcu-           cided to evaluate both criteria (an inhomogeneous cell pat-
lated for each investigator, as well as for each investigator      tern and various cells in terms of size, shape) with one point
group (expert and non-expert) and presented as percentage          (+1), respectively (Fig. 1c, e, g). In addition, we defined
and 95% confidence interval (95% CI). We tested the in-            small, dark cells as an indication of an abnormal nuclear-
terrater reliability/agreement using the Cohen’s kappa and         cytoplasmic correlation and cells of different greyscales as
Fleiss’ kappa coefficient. According to Landis and Koch,           suspicious and rated them as +1 point (Fig. 1g). The normal
values of κ between 0 and 20 are defined as low, between           squamous epithelium of the hypopharynx and larynx shows
0.21 and 0.40 as adequate, between 0.41 and 0.60 as moder-         a honeycomb-like pattern (Fig. 1a) with regular intercellu-
ate, between 0.61 and 0.80 as substantial, and between 0.81        lar spaces. The presence of these honeycombs was defined
and 1.0 almost perfect 16. We performed a receiver operating       by the authors as a benign criterion and was thus scored as
characteristic (ROC) analysis to calculate the area under the      -1 point. Disrupted cell borders were assigned to malignant
curve (AUC) as a quality measure for the DOC score as a            findings and were therefore assigned values of +1 point



28
                                                                                                           Larynx and pharynx confocal imaging score




Table I. Characteristics of the patient cohort.
 Case No.              Age          Tumour         Location     Grade       Surgery           CLE frames       Recording time         Selected
                     (years)         stage                                                        (n)            (seconds)           sequences
                                                                                                                                         (n)
 Score creation
 1.                      67           T1            Larynx       G2     Partial vertical LE     7586                 948                 10
 2.                      60           T1            Larynx       G2     Partial vertical LE     2841                 355                 13
 3.                      68           T4a           Larynx       G3          Total LE           1919                 239                 11
 4.                      73           T4a           Larynx       G3          Total LE           2413                 301                 12
 5.                      57           T3            Larynx       G3          Total LE           6513                 814                 11
 6.                      69           T3            Larynx       G3      Total LE, partial      2398                 299                 11
                                                  Hypopharynx            pharyngectomy
 7.                      63           T3             Larynx      G3      Total LE, partial      2896                 362                 14
                                                  Hypopharynx            pharyngectomy
 8.                      57           T3             Larynx      G3      Total LE, partial      1743                 217                 12
                                                  Hypopharynx            pharyngectomy
                                                                Total                           28309               3535                 94
 Score validation
 9.                      71           T4a           Larynx       G3      Total LE, partial      1468                 183                 14
                                                  Hypopharynx            pharyngectomy
 10.                     56           T4a            Larynx      G3      Total LE, partial      2204                 275                 12
                                                  Hypopharynx            pharyngectomy
 11.                     86           T4a            Larynx      G2      Total LE, partial      2191                 273                 10
                                                                         pharyngectomy
 12.                     61           T2            Larynx       G3      Total LE, partial      3311                 413                 14
                                                  Hypopharynx            pharyngectomy
 13.                     53           T3             Larynx      G3      Total LE, partial      2891                 361                 10
                                                  Hypopharynx            pharyngectomy
                                                                Total                           12065               1505                 60
LE: laryngectomy.



(Fig. 1e). Concerning blood vessels, there was a lower con-              signed in 82 (17.0%), the value -1 in 79 (16.4%), the value
sensus of the raters (κ = 0.45). This may be caused by the               0 in 36 (7.5%), the value 1 in 22 (5.6%), the value 2 in 14
fact that, in some tissue sections, no blood vessels were                (2.9%), the value 3 in 35 (7.3%), the value 4 in 48 (10%),
visible. On the other hand, dilated vessels with vascular                and the value 5 in 164 cases (34.2%). The rank correlation
leakage can also be found in inflammatory altered mucosa.                was calculated as 0.77 (p < 0.001).
However, capillary loops (Fig. 1b) are considered a benign               The performance of the scoring classification is illustrated
criterion by the authors, who rated it as -1 point.                      in Table IV. We considered a score of ≥ 3 points to be ma-
Based on the current analysis and the authors’ consensus                 lignant. The examiners achieved an accuracy, sensitivity,
and experience, we defined a six-category score. In general,             specificity, PPV and NPV ranging from 83.3% to 96.7%,
benign criteria are scored as -1. In contrast, criteria sus-             72.7% to 100%, 63.0% to 100%, 76.7% to 100% and
pected to be malignant are scored as +1 (see details in Table            74.3% to 100%, respectively. In summary, we showed an
III), resulting in a rating scale with a minimum of -2 points            accuracy, sensitivity, specificity, PPV and NPV of 88.5%,
and a maximum of +5 points. All images were evaluated                    87.1%, 90.3%, 91.6% and 85.1%, respectively. The observ-
by the authors MS and MG according to the current score,                 ers’ overall agreement was calculated with a Fleiss’ κ of
and an optimal cut-off value of ≥ 3 points was defined in                0.66 and can therefore be considered substantial (Tab. V).
the ROC analysis on the score creation set (AUC = 0.86;                  The expert group’s selective analysis revealed an accuracy,
95%CI: 0.81-0.92).                                                       sensitivity, specificity, PPV and NPV of 90.8%, 95.1%,
                                                                         86.4%, 87.9% and 94.4%, respectively, with an inter-rater
Diagnostic metrics in score-validation                                   reliability of κ = 0.78. In the non-expert group, an accura-
When reviewing the 60 sequences, the value -2 was as-                    cy, sensitivity, specificity, PPV and NPV of 86.2%, 86.4%,



                                                                                                                                                 29
M. Sievert et al.




                                                                                       86.1%, 88.4% and 83.8% could be observed (κ = 0.58).
                                                                                       We calculated interrater reliability between the experienced
                                                                                       and inexperienced raters of κ = 0.77.
                                                                                       In the ROC analysis, we calculated an AUC of 0.91
                                                                                       (95%CI: 0.88-0.94) considering the results of all investi-
                                                                                       gators (Fig. 2), confirming an optimal cut-off value of 3
                                                                                       points to achieve the best sensitivity and specificity.

                                                                                       Discussion
                                                                                       A classification system, LP-CIS, was developed to help
                                                                                       interpretate the CLE examination and objectively differ-
                                                                                       entiate between malignant and benign mucosal lesions. It
                                                                                       was developed based on 94 sequences (eight patients, 5640
                                                                                       images, respectively 50% benign and malignant sequenc-
                                                                                       es). Tissue homogeneity, cell size variation, presence of
                                                                                       cell clusters and honeycomb pattern, presence of capillary
                                                                                       loops, presence of small dark cells as well as the presence
                                                                                       of clear cell borders were regarded as adequate characteris-
                                                                                       tics from a statistical and practicability standpoint to differ-
                                                                                       entiate benign from malignant lesions using CLE (Tabs. II
                                                                                       and III). Blinded to the gold standard of histopathological
                                                                                       examination, differentiation of benign and malignant le-
                                                                                       sions was possible with an accuracy, sensitivity, and speci-
                                                                                       ficity of 90.8%, 95.1%, 86.4%, respectively, for the CLE-
                                                                                       experienced group on a new set of 5 different patients, to
                                                                                       exclude bias (60 sequences, 3600 images). The diagnostic
                                                                                       metrics were comparable for CLE-inexperienced head and
                                                                                       neck surgeons, albeit slightly inferiorly, with an accuracy,
                                                                                       sensitivity and specificity of 86.2%, 86.4% and 86.1%, re-
                                                                                       spectively. Interrater reliability between the experts was




Figure 1. Description: (a) inconspicuous squamous epithelium with a clas-
sic “honeycomb” pattern and (b) capillary loops (rated as -2 points). (c) Inho-
mogenous global impression with areas of different grey levels. (d) Substantial
leakage of fluorescein (white area) around a capillary (red area) indicates ma-
lignantly differentiated or altered inflammatory mucosa. (e) The arrow marks
elapsed cell borders. Besides, there is vascular leakage (lower right) and an
inhomogeneous cell pattern with varying cell sizes. In conclusion, this image
is scored as 4 points. (f) Dilated, atypical capillary loop, which is interpreted as
suspicious. (g) Cell conglomerates (circled areas), inhomogeneous cell pattern
and size adjacent to dilated vessels and leakage (scored 5 points). (h) Atypical
blood vessels with a horizontal course in the superficial epithelial layer (white
arrow) and a corkscrew-like shape as a sign of neoangiogenesis (transparent            Figure 2. Receiver operating characteristic (ROC) analysis and the area un-
arrow). This image is scored as 5 points in total.                                     der the curve (AUC) of the larynx and pharynx confocal imaging score. The
                                                                                       optimal cut-off value is calculated as 3 points.



30
                                                                                                                            Larynx and pharynx confocal imaging score




Table II. Descriptive analysis of typical characteristics for differentiating between SCC and benign squamous epithelium by CLE.
 Image characteristics                                                            SCC           Healthy mucosa            p value                       Fleiss’ kappa
                                                                               (n = 188)            (n = 188)
 Tissue inhomogeneity                                                           147 (78.2%)              39 (20.7%)               p < 0.001                  0.67
 Different greyscale value and shape of cells                                   151 (80.3%)              41 (21.8%)               p < 0.001                  0.64
 Atypical vessels/ absence of capillary loops                                   103 (54.8%)              22 (11.7%)               p < 0.001                  0.45
 Elapsed or non-differentiable cell borders                                     144 (76.6%)              40 (21.3%)               p < 0.001                  0.58
SCC: squamous cell carcinoma; the data refer to the evaluation of 47 sequences each from SCC and benign mucosal areas, each assessed by 4 raters. In total, 188 sequences of
SCC and 188 sequences of healthy mucosa were presented.



very good and was almost perfect. In the CLE-naive group,                               ideally form a honeycomb pattern, are typical features of
interrater reliability was nevertheless substantial. Our                                benign mucosal tissue, both in histology as well as in CLE
group’s previously published data, with a similar techni-                               throughout the upper airway tract (Tab. III) 10,11,13,17,18. Atyp-
cal methodology, although solely by experts without sys-                                ical vessels with fluorescein leakage as a sign of a distorted
tematic classification, obtained accuracy, sensitivity and                              barrier are also a feature, which is much more frequent in
specificity values of 80-86%, 72-90% and 72-89% respec-                                 malignant lesions than in benign lesions (Tab. II) 19. These
tively 10,11. The classification of images in previous publica-                         features were also evaluated from the standpoint of practi-
tions was based on published reports for oral cancer and oe-                            cability and the validated Spearman’s rank correlation co-
sophageal cancer, and was then transferred and applied to                               efficient (p < 0.001; Tab. II) 13,17.
the pharyngeal and laryngeal regions. Our results suggest                               While helping the intraoperative characterisation of free
that a systematic classification system, which was specifi-                             margins, we emphasise that this method cannot replace the
cally developed and refined for the laryngeal and pharyn-                               time-consuming but still, up to this point, demonstrably
geal segments of the upper airway tract, can significantly                              more reliable frozen sections. The intraoperative frozen sec-
improve the diagnostic accuracy of the experts and, more                                tion has a reported accuracy of 98.6-88.2%, and despite in-
surprisingly, enable CLE-inexperienced surgeons to iden-                                creasing criticism of this method in recent years is broadly
tify malignant changes on the same level as a CLE expert                                used in oncologic surgery 20,21. The main argument against
without the new score.                                                                  the frozen sections for safe margin evaluation is the inad-
Interpretation of CLE images is subjective and a crucial                                equacy of the tissue sent for intraoperative analysis due to
factor in making a correct assessment that, up to now, was                              the under-sampling of biopsies, which are usually small,
based solely on the experience of examiners and the trans-                              fragmented, and not oriented 21. We demonstrated an accu-
ferability of knowledge from other areas such as the oe-                                racy of 91%; however, further research is needed to verify
sophageal mucosa and oral mucosa 12,13. The present study                               the value of CLE in cancer demarcation compared with fresh
demonstrates that a systematic classification based on the                              frozen sections before it is suitable for broad clinical use in
pharyngeal and laryngeal regions can help improve ex-                                   this regard. Studies in the head and neck and other epithe-
perts’ diagnostic accuracy in the same anatomic region and                              lial areas of the intestinal tract, such as rectal surgery, with
enable non-experts to achieve satisfactory accuracy compa-                              similar methodology, show a feasibility and accuracy of up
rable to that of experts up to this point.                                              to 94% 22. CLE is not limited to under-sampling and ena-
Tissue homogeneity, clear cell borders with regular, similar-                           bles a software-based reconstruction of the whole plane of
ly-sized cells with a small nucleus/cytoplasm ratio, which                              tissue around the resection area. CLE is, however, limited


Table III. Larynx and pharynx confocal imaging score (LP-CIS).
 Tissue homogeneity            Cell size            Cell cluster                     Blood vessels             Nucleus/cytoplasm ratio                Cell borders
                              (+/-20%)
   0 = homogeneous              0 = uniform            -1 = honeycomb              -1 = capillary loops          0 = no small dark cells         0 = clear cell borders
  1 = inhomogeneous             1 = different         1 = no honeycombs           0 = no capillary loops       1 = small and or dark cells         1 = disrupted cell
                                                                                          and/or                                                        borders
                                                                                     atypical vessels/                                                  and/or
                                                                                   horizontally running                                             conglomerates
                                                                                     vessels/leakage
≥ 3 points are considered suspicious (range: minimum -2 to maximum 5 points).




                                                                                                                                                                        31
M. Sievert et al.




Table IV. Data analysis by the various raters (larynx and pharynx confocal imaging score).
 Diagnostic Expert Expert Expert Expert Nonexpert Nonexpert Nonexpert                                               Nonexpert        Experts      Nonexperts        All raters
 metrics         1           2         3            4           1             2             3                           4             (1-4)         (1-4)
                (P)         (S)       (S)          (S)         (S)           (S)           (S)                         (S)
                  Value   Value   Value   Value                     Value            Value            Value            Value          Value          Value            Value
                 (95%CI) (95%CI) (95%CI) (95%CI)                   (95%CI)          (95%CI)          (95%CI)          (95%CI)        (95%CI)        (95%CI)          (95%CI)
 Accuracy           86.7%     90.0%      96.7%        95%          95.0%            83.3%            83.3%            83.3%           90.8%         86.2%            88.5%
                    (75.4-    (79.5-     (88.5-      (86.1-      (86.1-99.0)      (71.5-91.7)      (71.5-91.7)      (71.5-91.7)       (86.4-      (81.2-90.3)      (85.3-91.2)
                     94.1)     96.2)      99.6)       99.0)                                                                            94.2)
 Sensitivity        75.8%     90.9%      93.9%       90.1%         90.9%            100%             81.8%            72.7%           95.1%         86.4%            87.1%
                    (57.7-    (75.7-     (79.8-      (75.7-      (75.7-98.1)      (89.4-100)       (64.5-93.0)      (54.5-86.7)       (89.6-      (79.3-91.7)      (82.5-90.9)
                     88.9)     98.1)      99.3)       98.1)                                                                            98.2)
 Specificity        100%      88.9%      100%        100%           100%            63.0%            85.2%            96.3%           86.4%         86.1%            90.3%
                    (87.2-    (70.8-     (87.2-      (87.2-       (87.2-100)      (42.4-80.6)      (66.3-95.8)      (81.0-99.9)       (78.9-      (78.1-92.0)      (85.5-93.9)
                     100)      97.6)      100)        100)                                                                             92.0)
 PPV                100%      90.9%      100%        100%           100%            76.7%            87.1%            96.0%           87.9%         88.4%            91.6%
                              (77.4-                                              (66.9-84.4)      (72.9-94.4)      (77.6-99.4)       (82.1-      (82.5-92.4)      (87.9-94.3)
                               96.7)                                                                                                   92.0)
 NPV                77.1%     88.9%       93.1%      90.0%         90.0%             100%            79.3%            74.3%           94.4%         83.8%            85.1%
                    (64.9-    (72.9-      (77.9-     (75.4-      (75.4-96.4)                       (64.6-88.9)      (62.2-83.5)       (88.6-      (78.0-88.9)      (80.7-88.7)
                     86.0)     96.0)       98.1)      96.4)                                                                            97.4)
 ROC(AUC)            0.92      0.92        0.98       0.94           0.93             0.81             0.90             0.95           0.94           0.89              0.91
                    (0.86-    (0.85-      (0.96-     (0.89-      (0.86-1.00)      (0.71-0.91)      (0.83-0.97)      (0.90-1.00)       (0.90-      (0.84-0.94)       (0.88-0.94)
                     0.92)     0.99)       1.00)      1.00)                                                                            0.97)
S: surgeon; P: pathologist; PPV: positive predictive value; NPV: negative predictive value; 95%CI: 95% Confidence Interval; ROC: receiver operating characteristic; AUC: area under
the curve.



by a fixed tissue penetration depth of 55-65 μm, which is                                   Table V. Inter-observer agreement in score validation.
determined by the probe used in our study. To characterise                                   Observer pair                       Fleiss-Kappa / Cohens-Kappa*
the morphological features of regular epithelium and carci-                                  Experts                                                   0.78
noma, we used IV fluorescein as a contrast agent. This al-                                   Non-experts                                               0.58
lowed the sufficient resolution of in vivo cellular architecture                             Experts vs non-experts                                    0.77*
within a few minutes of fluorescein administration and did                                   All raters                                                0.66
not result in any complications or side effects. Fluorescein                                Inter-observer agreement for multiple raters (Fleiss’ kappa) and observer groups (Co-
is also the only contrast agent with regulatory approval as                                 hen’s kappa*).
a drug-device combination, granted a 510 (k) clearance by
the FDA in January 2020. Other contrast agents, such as the
                                                                                            al carcinoma by generating real-time, in-vivo microscopic
topic application of acriflavine, provide further contrast and
                                                                                            images with the reliable demarcation of cancer from benign
a better depiction of the nuclei. However, because it is carci-
                                                                                            mucosa. In this study, a scoring system for the diagnosis
nogenic, an application outside of clinical studies up to this
point is unforeseeable 18,23. Recently, molecular imaging has                               of laryngeal and pharyngeal carcinoma was developed for
also increased in interest using complementary contrasting                                  the first time. LP-CIS enables a reliable classification of
agents such as glucose analog 2-NBDG or EGFR antibodies                                     physiological mucosa and cancerous lesions even for CLE
to enhance extra- and intercellular architecture visualisation                              nonexperts in their daily clinical practice. It is an essential
and improve the delineation of tumor margins 24,25. Our scor-                               step to integrating CLE into broad clinical practice outside
ing system is based on fluorescein alone, as this is the only                               of the clinical-experimental setting.
approved drug-device combination with CLE and will prob-
ably remain so for years to come.                                                           Conflict of interest statement
                                                                                            The authors declare no conflict of interest.
Conclusions                                                                                 Funding
CLE is a promising imaging technology that may improve                                      This project was supported by Deutsche Forschungsge-
the intraoperative management of laryngeal and pharynge-                                    meinschaft (DFG, German Research Foundation) under



32
                                                                                                               Larynx and pharynx confocal imaging score




grant number GO 3182/2-1, MA 4898/17-1, OE 743/1-1,                            10
                                                                                    Sievert M, Stelzle F, Aubreville M, et al. Intraoperative free margins
                                                                                    assessment of oropharyngeal squamous cell carcinoma with confo-
STE 1877/7-1; Project Number 439264659.                                             cal laser endomicroscopy: a pilot study. Eur Arch Otorhinolaryngol
                                                                                    2021;278:4433-4439. https://doi.org/10.1007/s00405-021-06659-y
Authors’ contributions                                                         11
                                                                                    Sievert M, Oetter N, Aubreville M, et al. Feasibility of intraopera-
MS and MG: conception and design, acquisition of data,                              tive assessment of safe surgical margins during laryngectomy with
                                                                                    confocal laser endomicroscopy: a pilot study. Auris Nasus Larynx
analysis and interpretation of data, wrote the manuscript,                          2021;48:764-769. https://doi.org/10.1016/j.anl.2021.01.005
final approval of the version to be published. KM, SM, ME,                     12
                                                                                    Goncalves M, Aubreville M, Mueller SK, et al. Probe-based con-
RR: acquisition of data, analysis and interpretation of data,                       focal laser endomicroscopy in detecting malignant lesions of vo-
critical revision of the article, final approval of the version                     cal folds. Acta Otorhinolaryngol Ital 2019;39:389-395. https://doi.
                                                                                    org/10.14639/0392-100X-2121
to be published.MA, FS, NO, AM, HI: critical revision of                       13
                                                                                    Oetter N, Knipfer C, Rohde M, et al. Development and validation of
the article, final approval of the version to be published.                         a classification and scoring system for the diagnosis of oral squamous
MG: drafting and critical revision of the article, final ap-                        cell carcinomas through confocal laser endomicroscopy. J Transl Med
proval of the version to be published.                                              2016;14:159. https://doi.org/10.1186/s12967-016-0919-4
                                                                               14
                                                                                    Moore C, Mehta V, Ma X, et al. Interobserver agreement of confocal
                                                                                    laser endomicroscopy for detection of head and neck neoplasia. La-
Ethical consideration                                                               ryngoscope 2016;126:632-637. https://doi.org/10.1002/lary.25646
The study was approved by the local institutional ethics                            10.1002/lary.25646
committee (approval number 60_14 B) and carried out in                         15
                                                                                    Nathan CA, Kaskas NM, Ma X, et al. Confocal laser endomi-
                                                                                    croscopy in the detection of head and neck precancerous le-
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written informed consent from all study participants.                               org/10.1177/0194599814528660 10.1177/0194599814528660
                                                                               16
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                                                                                    categorical data. Biometrics 1977;33:159-74.
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