Surgery remains the first-line treatment in nearly 80% of solid tumors, and achieving tumor-free surgical margins is critical for patient survival [1]. However, delineating the exact extent of a tumor is challenging, as surgeons rely on nonspecific visual changes and palpation. Margins must be sufficient to remove cancer cells while minimizing functional loss, and the recommended width varies by tumor type and risk category. For low-risk disease, margins of around 4 mm are typically used for basal cell carcinoma (BCC) and 4‐6 mm for cutaneous squamous cell carcinoma (SCC), whereas high-risk BCC and cutaneous SCC generally require wider margins, typically 6 mm or more (up to 10 mm), given their greater risk of subclinical extension [2-4]. For mucosal head and neck SCC, margins of 1 cm or more are generally recommended [5]. Small clusters of residual cancer cells or precancerous lesions often remain undetected, potentially leading to local recurrence or metastasis [1].

In BCC of the head and neck, the frequency of positive surgical margins ranges from 9% to 37.2% depending on tumor location and surgeon experience [6-10]. Large retrospective series have identified multifocality, Clark level, and depth of invasion as independent risk factors for incomplete excision [11]. Narrow margins (<4 mm) are associated with higher recurrence rates, with relapse occurring on average 31 months after surgery [12,13]. Accurate intraoperative detection of residual tumor cells is therefore essential. Conventional methods, such as frozen section analysis or touch imprint cytology, allow early detection but require significant time and coordination between surgical and pathology teams, prolonging procedures and potentially affecting patient management.

Reflectance confocal microscopy (RCM) has emerged as a rapid high-resolution technique (<1 mm) for ex vivo and intraoperative assessment of surgical margins, without interfering with subsequent histopathology [14]. In dermatologic surgery, RCM has demonstrated high sensitivity and specificity for BCC margins, with rapid image acquisition and evaluation [15,16]. Similar applications are under investigation in breast cancer, showing potential to reduce reoperation rates and improve intraoperative decision-making [17]. In eyelid and periocular tumor surgery, recent reviews have likewise highlighted confocal and related optical imaging as promising adjuncts to frozen section control [18]. These findings support RCM as a promising tool for real-time, accurate evaluation of surgical margins across multiple tumor types. Representative diagnostic performance figures from previously published studies are summarized in Textbox 1.

The primary objective of the HISTOBLOC study is to evaluate the concordance of intraoperative resection margin diagnosis based on ex vivo confocal microscopy images compared with standard frozen sections in patients with SCC of the upper aerodigestive tract, cutaneous SCC, or BCC.

The secondary objectives are the following:

We hypothesize that ex vivo confocal microscopy may provide margin diagnosis concordant with frozen section analysis while reducing intraoperative diagnostic time.

HISTOBLOC is a prospective, monocentric, single-arm pilot study with minimal risks and constraints, conducted at the Institut de Cancérologie de Lorraine (ICL), a nonprofit comprehensive cancer center. The study is registered on ClinicalTrials.gov (NCT05935995).

The HISTOBLOC study is offered to patients referred to the ICL for routine management of SCC of the upper aerodigestive tract or of cutaneous origin, or of BCC. Eligibility criteria are summarized in Textbox 2.

The study is preceded by a learning phase involving 15 patients, during which participating surgeons are trained to interpret and analyze confocal microscopy images under the supervision of an experienced pathologist, to ensure adequate familiarization with the confocal features of surgical margins. After the learning phase, 30 consecutive patients are prospectively enrolled to provide a preliminary evaluation of the diagnostic concordance between confocal microscopy and intraoperative frozen section examination (the reference standard). Each surgical procedure yields a minimum of four surgical margin specimens. The planned sample size of 30 patients is justified based on the expected precision of the 95% CI estimates for the sensitivity and specificity of confocal microscopy in assessing margin status. Sample size calculations are performed assuming the minimum number of analyzable specimens (n=120), corresponding to the scenario yielding the widest (least precise) CIs, thereby providing a conservative estimation of diagnostic accuracy. These sample size calculations reflect the assumptions made at the design stage of the study and are independent of the final numbers of patients and specimens analyzed.

Quantitative variables are summarized using mean (SD), median, minimum and maximum values, and the first and third quartiles. Qualitative variables are summarized as absolute numbers and percentages.

To address the primary objective, the diagnostic accuracy of intraoperative surgical margin assessment using confocal microscopy is evaluated with frozen section histopathology as the reference standard:

Sensitivity, specificity, PPV, and NPV are reported as proportions with their 95% CIs, calculated on the total number of surgical margin specimens analyzed. To address the secondary objectives, the same diagnostic accuracy analyses are performed using the final histopathological examination as the reference standard for concordance. All diagnostic accuracy analyses are performed separately for the learning curve phase, the post–learning curve phase, and the overall study population.

The diagnostic time for confocal microscopy and for intraoperative frozen section examination, the time saved by confocal microscopy (the difference between these two diagnostic times), and the confocal processing time (including tissue resection and image acquisition and analysis) are summarized as described above. Diagnostic times obtained with the two methods are compared using a Student t test for normally distributed data or a Mann-Whitney U test otherwise; normality is assessed with the Shapiro-Wilk test.

The rate of early surgical recurrence potentially attributable to confocal microscopy is described by the false-positive rate (1 − specificity), corresponding to the proportion of histologically negative margins incorrectly classified as positive by confocal microscopy. The failure (nondetection) rate is described by the false-negative rate (1 − sensitivity), corresponding to the proportion of histologically positive margins incorrectly classified as negative. All analyses are performed in RStudio (version 2022.07.2+576; Posit PBC). The statistical significance threshold is set at P less than .05.

Data collected in this study undergo a validation process to ensure completeness, authenticity, accuracy, and consistency; discrepancies are addressed through queries for clarification, and all data are stored and backed up on a dedicated secure server. Correction tables generated by the data management center are completed consistently by all personnel authorized to enter data into the case report form. Figure 1 shows the study timeline, and Table 1 summarizes the investigations and the data collected during the study.

Recruitment began on July 26, 2023, and ended on June 4, 2025. The final number of participants included and analyzed will be reported together with the study results in a subsequent publication.

The diagnostic accuracy and time analyses described in the Methods are ongoing. Concordance metrics (sensitivity, specificity, PPV, and NPV with 95% CIs) and diagnostic time comparisons will be reported at the analysis levels, prespecified in the Methods, once statistical analysis and interpretation are complete.

Results will be disseminated through peer-reviewed publication and presentations at national and international otorhinolaryngology, head and neck surgery, and oncology meetings.

The HISTOBLOC study aims to prospectively evaluate the diagnostic accuracy of intraoperative ex vivo confocal microscopy for surgical margin assessment in head and neck and cutaneous carcinomas. Based on previous studies and clinical experience, positive surgical margins are expected in approximately 20% of cases [11,13]. Accordingly, diagnostic sensitivity and specificity will be estimated using 24 positive and 96 negative surgical specimens, respectively. Expected diagnostic performance assumptions are derived from dermatologic studies of BCC, in which confocal microscopy demonstrated an estimated sensitivity of 80% and specificity of 95% [15,16]. Under these assumptions, analysis of 96 specimens allows estimation of a lower 95% CI bound for specificity greater than 0.89 (range 0.89‐0.98), while analysis of 24 positive specimens allows estimation of a lower CI bound for sensitivity greater than 0.59 (range 0.59‐0.93), using the exact Clopper-Pearson method [25]. These estimates support the statistical robustness of the study design. The study will also establish whether confocal microscopy reduces intraoperative diagnostic time compared with frozen section analysis and will characterize diagnostic performance separately during and after the learning curve.

Previous evaluations of ex vivo confocal microscopy have focused primarily on dermatologic oncology and breast-conserving surgery, showing high diagnostic accuracy and potential workflow benefits [15-17], and recent narrative reviews have extended this interest to eyelid and periocular tumor surgery [18]. However, no prospective study has assessed this technique in head and neck oncologic surgery, where margin assessment is particularly challenging and clinically critical. HISTOBLOC extends prior work by applying confocal microscopy to SCC of the upper aerodigestive tract and related cutaneous malignancies, directly comparing it with intraoperative frozen section examination.

The main strengths of the study are its prospective design, the direct within-patient comparison of confocal microscopy with the intraoperative reference standard and with final histopathology, a structured learning phase to control operator dependence, a standardized ex vivo imaging protocol, and the concurrent development of a confocal reference atlas to support consistent interpretation and future training.

The limitations of this study include its single-center design and the operator-dependent nature of confocal image interpretation, which requires a structured learning curve. In addition, confocal microscopy primarily assesses surface margins and may be less sensitive to deeply infiltrative tumor growth, and the pilot sample size limits the precision of subgroup analyses. Finally, the number of patients and of margin specimens finally available for analysis may differ from the planned figures, which affects the precision of the estimates, in particular for the learning curve and subgroup analyses.

If the pilot results are favorable, multicenter validation with a larger sample is warranted, together with formal evaluation of a remote real-time image transmission workflow for intraoperative tele-pathology, assessment of the impact of confocal-guided margin control on reoperation rates and clinical outcomes, and extension of the approach to additional tumor sites.

HISTOBLOC is a prospective pilot study evaluating intraoperative ex vivo confocal microscopy in head and neck and cutaneous oncologic surgery. If high specificity, acceptable sensitivity, and meaningful time savings are confirmed, confocal microscopy may represent a valuable adjunct or alternative to frozen section examination, with the potential to reduce reoperations and optimize intraoperative decision-making. Within this framework, pathologists remain the cornerstone of pathological diagnosis, while confocal microscopy and digital image transmission are investigated as enabling technologies to enhance speed, accessibility, and intraoperative collaboration.