Preanalytical Phase Tumor Contaminants in Intraoperative Margins From Transoral Robotic Surgeries Open Access

Following institutional review board approval, a retrospective study of patients undergoing surgery for oropharyngeal tumors from 2016 to 2022 was conducted at a single institution. Patients were included if they had TORS or open resection for an OPSCC (either primary or recurrent). Eligible patient charts were reviewed, and data were collected on demographics including age, sex, race, smoking status, alcohol consumption, and body mass index (BMI). Additional data were collected on tumor histologic type, p16 status, and surgical factors including tumor size, tumor node metastases (TNM) classification, perineural invasion (PNI), and lymphovascular invasion (LVI). Operative notes were reviewed to determine if the tumor was resected en bloc versus piecemeal, was transected intraoperatively, or had close/positive margins. Finally, data on perioperative complications were collected, including intraoperative bleeding, unanticipated return to the operating room, and neck infection.

FS slides were screened by a head and neck pathologist (HNP; MALG). Problematic tumor fragments were defined as fragments of tumor with confounding potential (possible contaminants). Preanalytical contaminants (PACs) were defined as detached tumor fragments in FS slides of tumor bed margins away from the primary tumor, with or without occurrence in permanent slides. After FS slides with contaminants were identified they were reviewed by 2 additional HNPs (DL, RC)—along with permanent slides and primary tumor from the main resection specimen—blinded to surgery type. A slide was considered indeterminate if the HNPs disagreed with each other on whether the tumor fragment represented a contaminant. Final pathology reports, permanent slides, and intraoperative reports were retrospectively reviewed and discrepancies were annotated.

Statistical analysis was performed with Prism v10.3.0 (GraphPad Software, Boston, Massachusetts). Continuous variables were assessed with mean and standard deviation, and categorical variables were assessed with frequency and percentage. Age, BMI, tumor size, and cautery levels were compared between patients with and without preanalytical contaminants. Each variable was tested for normality with the Shapiro-Wilk test and compared by using the Welch t test (if Shapiro-Wilk P > .05) or Mann-Whitney U test (if Shapiro-Wilk P < .05). Sex, race, smoking status, alcohol consumption, p16 status, diagnosis, tumor stage, PNI, LVI, perioperative complications, adjuvant treatment, cautery levels, and histology of tumor were compared between patients with and without preanalytical contaminants by using Fisher exact test. Statistical significance was defined as P < .05.

One hundred twenty-seven patients underwent surgery for OPSCC from 2016 to 2022. Patients whose surgery included FSs (n = 81) were included in the study, of whom 42 underwent TORS and 39 underwent open oropharyngectomy. Of 308 tumor bed margins, 289 were submitted for FS, of which 128 (44.3%) originated from TORS and 161 (55.7%) originated from open oropharyngectomies. PACs occurred in 11 of 289 intraoperative tumor bed margins (3.8%) from 9 of the 81 patients (11.1%). These represent 9 of the 42 TORS surgeries (21.4%).

The 2 surgical cohorts (patients undergoing TORS versus open pharyngectomy) were demographically similar except for a statistically significant difference in p16 status (Table 1). TORS patients were more likely to have p16-positive tumors (P =. 007). There were no significant differences between smoking, alcohol consumption status, and BMI.

The indications for TORS versus open pharyngectomy were different. Among the patients undergoing TORS, 12 (28.6%) had carcinoma of unknown primary and 30 (71.4%) had tumors identifiable on examination or imaging. Fifty percent of the tumors in TORS cases had pathologic tumor classification of T2. In comparison, most patients undergoing open oropharyngectomies had salvage surgeries (n = 27, 69.2%) and T4-stage tumors (n = 17, 43.6%) with 46.1% (n = 18) of the tumors having a size of more than 4 cm. Most of the patients in the TORS cohort had negative PNI (n = 31, 73.8%) and negative LVI (n = 23, 54.8%). PNI was identified in 51.3% (n = 20) and LVI was identified in 41.2% (n = 16) of the open surgery cohort.

Intraoperative complications occurred in 4.7% (n = 2) of TORS cases and included bleeding and chyle leak. Intraoperative complications (bleeding) occurred in 2.6% (n = 1) of the open cases. The cautery level was set higher than 30 in 26.2% (n = 11) of the TORS cases versus 7.7% (n = 3) of the open oropharyngectomy cases. Use of higher cautery setting was significantly more frequent in the TORS cohort than in the open surgery cohort. The surgical factors for the patients undergoing TORS and open oropharyngectomy are detailed in Table 2.

A discrepancy between frozen and final pathology was determined in 3 of 128 TORS FS margins (2.3%) from 2 patients. A discrepancy between the frozen and final pathology was observed in 3 of the 161 open pharyngectomy FS margins (1.8%). Of these, 2 discrepancies were false positives that occurred owing to the presence of PACs, both in the TORS cohort. These discrepancies were diagnosed as “positive for carcinoma” and “atypical” on FS. Two FS deferrals occurred that were attributed to PACs.

The FS slides of the study patients included 308 tumor bed margins, which were represented in 641 slides. Seventeen problematic tumor fragments (possible tumor contaminants) were noticed in FS slides, which included 15 from the TORS cohort and 2 from the open surgery cohort. Upon review by 3 HNPs, 6 of these problematic tumor fragments (4 in the TORS cohort and 2 in open oropharyngectomy cohort) were not deemed as a preanalytical contaminant, as a grossing (analytical phase) contaminant could not be excluded. Thus, 11 of the 17 problematic fragments were classified as true PACs by HNP consensus. The Figure, A through I, shows 3 examples of PACs in intraoperative margin slides along with their corresponding primary tumor.

Upon retrospective review of the cases with preanalytical phase tumor contaminants, all had at least one of the following operative circumstances in common: (1) surgeon had to cut through primary tumor for removal or access (n = 5); (2) primary tumor had to be removed in piecemeal or with a positive deep margin (n = 7); (3) tumor biopsy samples were sent for FS prior to resection for primary tumor identification (n = 1); or (4) a tear occurred in the specimen at the tumor site during removal (n = 1). Characteristics of patients with preanalytical contaminants are detailed in Table 3.

Associations between occurrence of preanalytical contaminants with various demographic, surgical, and pathologic factors were assessed (Table 4). No demographic factors were found to be associated with occurrence of contaminants between the 2 cohorts (cohort with contaminants and cohort without contaminants), including age, sex, race, smoking, alcohol consumption, and BMI.

Among the surgical factors, a statistically significant association was found between contaminants and tumor size (Welch t test, P = .04). The surgical approach used (TORS versus open oropharyngectomy) was associated with preanalytical contaminants (Fisher exact test, P = .002). No other associations were found between contaminants and other surgical factors including diagnosis (primary or recurrence), tumor stage, PNI, LVI, intraoperative cautery levels, perioperative complications, and adjuvant treatment. A statistically significant association was found between the histologic type of the tumor, that is, the nonkeratinizing type of SCC, which is often human papillomavirus (HPV) associated, and occurrence of contaminants (Fisher exact test, P < .001). There was no significant association between occurrence of contaminants and p16 status, a surrogate marker for HPV (Fisher exact test, P = .07).

FS has been used as an intraoperative diagnostic modality from the late 1890s.⁸  Achieving negative margins in HNC surgery is important as it affects survival and guides decisions relating to adjuvant treatment. With the advent of robotic surgery, there have been reports about tissue artifacts and tumor contaminants affecting specimen analysis in these surgeries.^11,12  To our knowledge, this is the only study describing the occurrence of PACs in tumor bed margins in patients with OPSCC undergoing surgery as standard of care. In this multivariate study, we characterized the clinical and pathologic context of these occurrences.

Tissue contaminants and artifacts are not uncommon in surgical pathology practice despite standard protocols. Pathologists are trained to recognize and troubleshoot most types of contaminants and they do so in everyday practice. Most contaminants encountered in practice represent analytical phase cross-contaminants in permanent sections originating in the laboratory as gross instrument contaminants, cross-contaminants from a tissue processor, and histology water bath “floater” cross-contaminants. The rate of occurrence of these is very low (0.6%).^13,14 

Preanalytical phase contamination occurs even less frequently, given the minimal risk of cross-contamination with current specimen collection standards. In some instances, however, extraneous tissue from the same patient may be present in a specimen container. We studied PACs occurring in FS slides of tumor bed margins for OPSCC, in which there is potential to interfere with intraoperative margin interpretation. We found PACs in 3.8% of all tumor bed margins for OPSCC but all PACs occurred in 9 patients who underwent TORS, corresponding to one-fifth of the TORS surgeries we evaluated. We inferred that our contaminants originated in the preanalytical phase because of their presence in FS slides of specimens received directly from the operating room and processed in the FS room, before the steps of routine processing (tissue processor, water bath, grossing table, and histology) during which analytical phase cross-contaminants typically occur.

For the 2 cohorts of patients undergoing TORS and open surgery for OPSCC, the age, sex, race, smoking, and alcohol status, and BMI were similar in both the groups. A statistically significant difference was found between the modality of treatment between the 2 groups.

While the demographics and baseline characteristics were comparable between the cohorts undergoing TORS and open surgery for OPSCC, patients undergoing TORS had a higher incidence of tumor contaminants than the open surgery group. Delving deeper, we explore potential factors contributing to this.

Moreover, all contaminants except 1 originated from TORS for nonkeratinizing, HPV-positive tumors. We contemplate whether contaminants occurred more commonly in this type of tumor because these tumors recapitulate the tonsillar crypt epithelium in which expression of intercellular tight junctions is different from that of surface keratinizing mucosa (less cohesive).¹⁵ 

Upon retrospective review, an association was found between larger tumor size and occurrence of PACs, and all PACs occurred in cases where the tumor was disrupted during surgery in the form of piecemeal dissection or obtaining access by cutting through the tumor or complete excision or tear occurring at the tumor site or tumor biopsy specimens being taken intraoperatively. After all, preanalytical contaminants in the tumor bed margins of TORS may just be a consequence of the challenges related to patterns of spread of oropharyngeal tumors and the limited maneuverability in the anatomic region that makes TORS the preferred technique in these cases. We also pondered if contamination could be more likely in high cautery settings, as some cautery technologies have a been associated with significant tissue distortion on glass slides.¹⁶  Our analysis did not find association between cautery settings and occurrence of PACs. The TORS cohort had a higher number of patients with p16-positive status. However, on further analysis, no association was found between p16 positivity and occurrence of tumor contaminants.

While the occurrence of PACs may seem trivial, we believe their relevance should not be underestimated. In our cohort, we identified 1 false-positive margin and 1 equivocal (atypical) margin explained by pathologist interpretive error caused by PACs, which did not cause injury to the patients, but had the potential to prolong anesthesia duration and/or result in additional tissue being excised unnecessarily.

While tissue contaminants rarely cause human interpretive error,¹⁷  tissue contaminants may interfere with the performance of emerging technologies such as machine learning models. As these technologies emerge further study of the impact of PACs may be necessary. For example, a recent study demonstrated that the addition of contaminants induced false negatives and false positives, and under specific circumstances, resulted in a 97% false-positive rate in a modern machine learning model.¹⁸  Keeping in mind that contaminants have the potential to interfere with the development of these emerging technologies, this study is a first attempt to understand the occurrence of contaminants in this setting.

Finally, when considering the proportion of patients in which PACs occurred (21.4% of the patients who underwent TORS), these incidents may not be so uncommon. Regarding the correct approach for the FS pathologist reporting margins that may have PACs, we surmise that using descriptive terminology and stating “possible contaminant” is a reasonable option that gives the surgeon the opportunity to correlate with their findings in the tumor bed.

PACs occurred in a small subset of patients who underwent surgery for OPSCC. Most occur in the setting of TORS for a large HPV-mediated OPSCC with intraoperative disruption of tumors with nonkeratinizing histology. Rarely, PACs lead to FS deferral or discrepancy with final diagnosis. It is important for surgical pathologists and head and neck surgeons to recognize the possibility of PACs when assessing intraoperative margins to prevent possible confusion and further unnecessary treatment. These findings underscore the importance of communication between pathologists and surgeons.