Abstract
Oral squamous cell carcinoma (OSCC) is one the most encountered malignancies by the oncologists in the Indian subcontinent. Adequate margin is the most sought-after goal during resection of OSCC. Margin discrepancy in oral cavity squamous cell carcinoma is frequent cause of disagreement between surgeons and pathologists. To assess the amount of margin of shrinkage, we conducted a prospective observational study in a tertiary care hospital in western state of India over a period of 2 years. 2-reference points for margin were selected and distance between these two points was measured in-situ, after resection, after formalin fixation and after histopathological processing. 44 patients were enrolled in the study. Mean shrinkage of 33.41% was noted. Statistically significant shrinkage was noted in all steps viz. post resection (15.31%), after formalin fixation (7.31%) and after histopathological processing (10.79%). No variation in shrinkage was noted owing to patient or tumour related factors. Significant margin shrinkage occurs in all cases of oral cavity squamous cell carcinoma irrespective of patient or tumour related factors with maximum shrinkage occurring post resection. Pathological margins are 2/3rd of surgical margin and this discrepancy shall be kept in mind while attempting wide local resection.
Keywords: Oral squamous cell carcinoma, Mucosal margin shrinkage, Shrinkage, Margin discrepancy
Introduction
As per GLOBOCAN 2022 data, 36.9% of world cases of ca-lip and oral cavity were from India. In India, oral cavity is the most frequently involved site of malignancy in males & 3rd most common cause of cancer related mortality [1]. Majority of tumors in oral cavity are squamous cell carcinoma originating from the lining epithelium of the oral cavity [2]. Surgery forms mainstay of treatment of oral squamous cell carcinoma (OSCC). Tumour-free margin is one the most important goals of surgical resection of tumour in oral cavity. Clear margins are not surrogate for recurrence free life but, still is desirable to achieve as evident from multiple studies that, patients with positive margin are associated with significantly worse survival [3–9].
This goal of achieving adequate margin is further impeded by complex anatomy of the oral cavity. What further entangles the scenario, is that surgical margins and pathologically reported margins vary significantly [10]. This discrepancy in surgical margin and pathological margins is largely attributed to margin shrinkage. Various studies on margin shrinkage reported large variability in terms of amount of margin shrinkage, among subsite of oral cavity and also among various T-stages. So, the amount of shrinkage in oral cavity and factors affecting it is still an unanswered question [10–16].
How much shrinkage of margin occurs? Is it related to sub-sites of oral cavity? Does margin shrinkage differ among early stage and advanced disease? Do tumour related factors viz. LVI (Lympho-vascular invasion)/PNI (Perineural invasion)/WPOI (worst pattern of invasion) affect margin shrinkage? Does age or any other patient related factor play a role in shrinkage of mucosal margin? When does margin shrinkage occur, after resection or after formalin fixation or after histopathological processing? Do we need to go further away from tumour to counter this margin shrinkage? To answer these research questions, we conducted a study of estimation of shrinkage of mucosal margin of oral carcinoma: immediately after resection & pathologic processing and it’s prognostic significance with other factors.
Methods
It is a prospective observational study conducted in a tertiary care hospital in western India. After due clearance from institutional ethics committee, based on feasibility criteria all patients of OSCC who consented for study and were included in the study during the study period of May 2022 to April 2024.
Exclusion criteria included:
Patients with recurrent tumors
Patients with multifocal tumors
Patients with severe trismus
Patients who have received neoadjuvant treatment.
After induction of anesthesia, tumour and margins were reassessed. Mucosa was flattened without stretching. Most accessible part of the margin was marked with inked marker and was termed as ‘First point’. At least 10 mm from this point ‘Second Point’ was marked. Marking sutures were taken on these points. Distance between these two sutures was measured and was recoded as ‘in-situ margin (mIS)’. Tumour was resected with electrocautery while taking precaution that both sutures were incorporated into the specimen. Half hour after resection distance between these two sutures was again measured and recorded as ‘margin post resection (mPR)’, specimen was now transferred to specimen jar containing 10% buffered formalin solution. After 24 h of formalin fixation again distance between these two sutures was measured and recorded as ‘margin post formalin fixation (mPFF)’. These two sutures were now removed and these two points were now marked with ink. Distance between these points was measured by pathologist on histopathological examination and recorded as ‘margin on histopathological examination (mHPE)’ (Figs. 1, 2). To avoid bias, all marking sutures were taken by first author and all measurements were taken in presence of first author (AV), histopathological measurements were supervised by third author (ACB). WPOI, PNI, LVI, DOI (Depth of invasion), grade was noted by pathologist along with T-stage, N-stage & AJCC stage. Patients who underwent adjuvant treatment were followed up during adjuvant treatment monthly and date of completion of adjuvant treatment was noted as date of completion of treatment. For patients who did not undergo adjuvant treatment date of discharge from hospital was recorded as date of completion of treatment. Patients were followed up monthly for first 3 month, bimonthly for next 6 months and yearly later. On follow up if locally recurrent lesion was noted, biopsy was taken, if distant metastasis was noted core biopsy was taken. Histopathologically proven recurrences were recorded as recurrent disease. Cause of death and date of death was recorded. Demographical data and other details were cross checked with patient records archives and histopathology reports.
Fig. 1.
A Marking sutures taken at most accessible point in case of Ca-tongue, B in-situ margin of 10 mm, C Post-resection margin of 10 mm, D post formalin fixation margin of 8 mm
Fig. 2.
A In-situ margin of 14 mm, B Shrinkage leading to post resection margin of 11 mm, C post formalin fixation margin of 11 mm, D Micrometer of microscope being used for measurement of margin post histopathological processing
Data was collected in MS Excel; data cleaning was done regularly and at the end of study. Data analysis was done using IBM SPSS V. 29.0.2.0. Shrinkage was compared by paired t-test and by unpaired t-test. Multivariate analysis was performed for WPOI, T-stage, N-stage, AJCC stage, DOI and grade. Survival analysis was done using Kaplan Meier curve and log rank test.
Results
44 patients were enrolled in the study.36 were males and 8 were females. Median age was 54.5 years (32–92 years). Most common subsite involved was buccal mucosa followed by tongue (Fig. 3)
Fig. 3.
Distribution of cases among subsites of oral cavity
7 patients belonged to AJCC T-stage T1, 5 to T2, 19 to T3 and 13 to T4a; 24 to N-stage N0, 5 to N1, 10 to N2 and 5 to N3. Lymphovascular invasion (LVI) was seen in 8 patients, Peri-neural invasion (PNI) in 10 and both in 4 cases. 27 had Grade 1 tumour, 15 had grade 2 wheras only 2 had grade 3 tumour.WPOI was recorded in all patients, 6 had WPOI-1, 5 had WPOI-2, 15 had WPOI-3, 7 had WPOI-4 and 11 cases had WPOI-5.
Mean in-situ margin was 11.1 mm (2SD = 2.5), mean post resection margin was 9.38 mm (2SD = 2.44) with a mean shrinkage of 1.7 mm post resection, mean post formalin fixation margin was 8.57 mm (2SD = 2.26) with a mean shrinkage of 0.82 mm owing to formalin fixation and overall shrinkage of 2.52 mm from in situ margin, mean post histopathological examination margin was 7.38 mm (2SD = 2.14) with a mean shrinkage 1.19 mm owing to histopathological processing and overall mean shrinkage of 3.71 mm from in-situ margin. Mean shrinkage noted was 33.41%, major part of which occurred post resection (15.31%), followed by histopathological processing (10.79%) and least by formalin fixation (7.31%). On paired t-test, shrinkage after each step viz. post resection, post formalin fixation and post histopathological examination were statistically significant (p-value < 0.00001) (Fig. 4).
Fig. 4.
Waterfall chart demonstrating shrinkage of mucosal margins after resection, post formalin fixation and post histopathological examination
On univariate analysis by unpaired t-test no statistically significant association of shrinkage with age, sex, LVI or PNI was noted (Table 1).
Table 1.
Univariate analysis of shrinkage by unpaired t-test due to LVI, PNI
| mIS | mPR | p value | mPFF | pvalue | mHPE | p value | |
|---|---|---|---|---|---|---|---|
| LVI | |||||||
| + | 10.88 | 8.75 | P = 0.052 | 8 | p = 0.058 | 7.13 | p = 0.233 |
| − | 11.1 | 9.53 | 8.7 | 7.43 | |||
| PNI | |||||||
| + | 11.6 | 9.5 | p = 0.371 | 8.9 | p = 0.147 | 7.79 | p = 0.084 |
| − | 10.94 | 9.35 | 8.47 | 7.26 | |||
| Age | |||||||
| < 45 | 11.4 | 9.6 | p = 0.268 | 8.6 | p = 0.46 | 7.33 | p = 0.438 |
| > / = 45 | 11 | 9.32 | 8.56 | 7.39 | |||
| Sex | |||||||
| M | 11.08 | 9.47 | p = 0.165 | 8.61 | p = 0.299 | 7.41 | p = 0.318 |
| F | 11.13 | 9 | 8.38 | 7.21 |
On multivariate analysis, statistical significance for shrinkage was not seen among T-stage, N-stage, AJCC stage, WPOI, DOI, subsite and grade/s.
Median follow up of patients was 374.5 days (range: 45–804 days), 11 deaths were reported during this period (8 cancer related and 3 non-cancer related), all were included in analysis. Margin shrinkage was quantified in 3 categories for analysis < 25%, 25–40% and > 40%. No difference in survival was noted by shrinkage categories. Only factors which affected survival included LVI, N-stage and AJCC-stage.
Discussion
Margin shrinkage have potential to alter course of adjuvant treatment, as more shrinkage can lead to report of erroneous close margin. Moreover, if margin shrinkage can be estimated beforehand then operating surgeon can plan his resection accordingly in anatomically dense areas like oral cavity, thus primary objective of the study was to assess shrinkage of mucosal margin in oral squamous cell carcinoma.
We noted shrinkage from 14 to 47% in our study with mean shrinkage of 33.41%. Shrinkage was significant in all steps i.e. after resection, after formalin fixation and after histopathological processing. Most of which occurred after resection (15.31%), followed by histopathological processing (10.79%) and least on formalin fixation (7.31%). LVI positive cases had more shrinkage post resection as compared to LVI negative cases (19.64% vs 14.34%) but it failed to reach the level of statistical significance (p-value = 0.052).
There was no variation noted in shrinkage related to subsite, T-stage, N-stage, AJCC stage, LVI, PNI, WPOI, grade, age & sex.
Why Margin Shrinkage Occurs?
As seen in our as well as most of the studies related to shrinkage in oral squamous cell carcinoma, most of the shrinkage occurs after resection. This may be attributable to unopposed contraction of muscle tissues in the specimen after release from the surrounding tissue. Formalin fixation also led to significant shrinkage in our study, this may be attributable to formation of covalent bonds and protein coagulation during tissue fixation. Histopathological processing involves processing of tissue through ascending grades of alcohol, this leads to cellular dehydration and further shrinkage of the tissues [17].
Margin shrinkage was not attributable to any of the tumour or patient related factors. Though in study by Mistry et al. [11], they noted significantly more shrinkage in early T-stage tumors than advanced T-stage tumors, we noted no discrepancy in shrinkage by T-stage. Contrary to what Mistry et al. [11] observed, Cheng et al. [12] noted more shrinkage in advanced T-stage (T3/T4) tumors (Table 2). This may be due to the fallacy of study design by Cheng et al., though a uniform margin of 1 cm was marked around the tumour, still they noticed positive margin on frozen section denoting inadequate resection during study by Cheng et al. which also led to significant discrepancy in margin shrinkage measurement. Margin shrinkage occurs in normal tissue rather than in tumour thus discrepancy is not related to tumour related factors. Mean shrinkage noted by in our study was 33.41% of which 15.31% occurred after resection which is comparable to 22.7% shrinkage observed by Mistry et al., 17.1–21.4% noted by Egeman et al. [13], 14.9% by Umstattd et al. [15] and 19.7% by Burns et al. [16].
Table 2.
Various studies comparing margins shrinkage in oral squamous cell carcinoma
| Author (year) | Study design | No. of cases | Location of tumour: no. of cases per site | T-stage: no. of patients | Measurements | Mean shrinkage | Subsite wise mean shrinkage | T Stage wise shrinkage | Remarks |
|---|---|---|---|---|---|---|---|---|---|
| Mistry et al (2005) [11] | Prospective | 27 | Buccal Mucosa:16 | T1:11 | Insitu margin, | 3.18 mm (22.7%) | Tongue: 3.71 mm(23.5%) | T1/T2: 3.59 mm (25.6%) | No assessment of shrinkage post formalin fixation or on HPE |
| Tongue:11 | T2:11 | Half hour after resection | Buccal mucosa: 2.4 mm (21.2%) | T3/T4: 1.4 mm (9.2%) | SHRINKAGE T1/T2>>T3/T4 | ||||
| T3:3 | |||||||||
| T4:2 | |||||||||
| Cheng et al (2008) [12] | Retrospective | 41 | Grp 1: Mandibular Alveolus, BM, RMT: 21 | T1: 11 | Insitu margin, | 59.20% | Grp1: 71.9% | T1/T2: 51.5% | Retrospective study, no assessment of post resection/formalin fixation shrinkage |
| Grp 2:Maxillary alveolus, hard palate: 6 | T2:16 | Closest HP margin | Grp2: 53.33% | T3/T4: 75% | |||||
| Grp 3: Tongue: 14 | T3:1 | Grp 3: 42.14% | |||||||
| T4:11 | |||||||||
| Egemen et al (2014) [13] | Prospective | 21 | Lower Lip: 15 | T1:8 | In situ | L41-R47.5% | pR: R2.2 mm/L1.8 mm | NR | No statistically significant shrinkage noted by histopathological processing/duration of formalin fixation. |
| Upper lip :5 | T2:10 | Post resection | pFF: 24 h: R 3.6 mm/L3.1 mm. | ||||||
| Commissure:1 | T3:3 | Post formalin fixation 24 h, 48 h | 48 h: R4 mm/L3.5 mm | ||||||
| HPE | HPE: R4.8 mm/L4.1 mm | ||||||||
| El-Fol HA et al (2015) [14] | Prospective | 61 | Tongue:20 | T1:4 | In situ margin | BM: 66.7% | NR | Greater discrepancy for buccal mucosa, | |
| Buccal mucosa: 15 | T2:49 | Histopathological margin | FOM: 33.3% | Different discrepancies for those with close/clear margins. | |||||
| Mandibular alveolus: 13 | T3:4 | Mandibular alveolus: 15.4% | No post resection/post formalin fixation shrinkage noted. | ||||||
| RMT: 6 | T4:4 | RMT: 16.7% | |||||||
| FOM:3 | Tongue: 35% | ||||||||
| Maxillary alveolus: 4 | |||||||||
| Umstattd et al (2017) [15] | Prospective | 19 | Tongue: 13 | NR | In situ | 11.30% | NR | NR | No statistically significant shrinkage owing to formalin fixation. Time of formalin fixation: 1.6 to 31.7 hours |
| Alveolus: 3 | Post excision | ||||||||
| FOM:1 | Post formalin fixation | ||||||||
| RMT:1 | |||||||||
| Unspecified:1 | |||||||||
| Burns et al (2021) [16] | Prospective | 6 | Tongue: 5 | T1: 4 | In situ | 2.6 mm (26%) | NR | NR | Max. shrinkage occurred Post resection, low sample size. |
| Buccal Mucosa:1 | T2:1 | Post excision | |||||||
| T3:1 | Post formalin fixation | ||||||||
| Our study (2023) | Prospective | 44 | BM:21 | T1:7 | In Situ margin | 3.71 mm (33.41%) | BM: 32.97% | T1/T2 vs T3/T4: | Statistically significant shrinkage noted in all 3 steps. |
| Tongue: 12 | T2:5 | Post excision | Tongue: 31.08% | 33.74% vs 33.28% (p-value = NS) | No correlation to tumour characteristics viz. T, N-stage, PNI, LVI, WPOI. | ||||
| GBS: 5 | T3:19 | Post formalin fixation | GBS:38.24% | Shrinkage of 14–47% | |||||
| Lip:3 | T4:13 | Post HPE | Lip: 35.76% | No subsite correlation on shrinkage | |||||
| Alveolus/RMT/FOM:1 each | FOM:37.27% | ||||||||
| RMT: 34.17% | |||||||||
| Alveolus: 35% | |||||||||
Formalin fixation caused significant shrinkage of 7.31% in our study. Egemen et al. [13] noted shrinkage of 14.3% due to formalin fixation, 12.7% shrinkage was noted by Burns et al. [16], 14.9–23.9% was noted by Pangare et al. [18] and 3.7% by Umstattd et al. [15]. Umstattd et al. noted statistically insignificant shrinkage due to formalin fixation but duration of formalin fixation ranged from 1.6 to 31.7 h. 24 h is the least time required for formalin fixation as fixation of nucleic acid starts after 12 h, thus inadequate time of formalin fixation can be cause of insignificant shrinkage.
Histopathological processing caused shrinkage of 10.79% in our study. 7.75% (P = NS) shrinkage was noted by Egemen et al. [13] after histopathological processing. Egemen et al. conducted study of carcinoma lip only whereas our study included most of the subsites of oral cavity (Table 2). Mean shrinkage noted by Cheng et al. [12] was 59%, by Egeman et al. [13] was 41–47% while we noted mean shrinkage of 33.41%. Burns et al. noted shrinkage of 26% but didn’t include shrinkage of histopathological processing.
Margin is bound to shrink in all subsites of oral cavity, in all patients irrespective of age or sex, of all tumors irrespective of T-stage, N-stage, AJCC stage, WPOI, PNI, LVI, DOI or grade.
As historical studies [3, 5–7, 9] were recently contradicted by recent studies [19, 20] about margin status not being the worst prognostic status, we too were not able to demonstrate margin status to significantly affect the survival. It however must be noted that though our study is prospective, our follow up period and sample size is limited to conclusively comment on this aspect.
Strength of our study was significant number of sample size as compared to previous studies, prospective study design as we noted fallacies of similar retrospective studies. We measured margin shrinkage at only 2 reference point, post resection, post formalin fixation and post histopathological analysis at almost all subsites of oral cavity thus making it all-round study for assessing margin shrinkage as compared to previous studies. Thus is generalizable. Margin shrinkage was compared with tumour as well as patient related factors and patients were followed up to assess survival effect of shrinkage.
Limitations of our study was that it was a single institutional study with limited number of patients among some subsites like RMT, FOM, Alveolus. Follow up data was limited to make conclusive comment on survival statistics.
Conclusion
Margin Shrinkage is a phenomenon which is bound to happen in oral squamous cell carcinoma. We noted mean shrinkage of 33.41% in our study. We recommend to consider this factor before resection irrespective of patient or tumour related factors. Shrinkage is bound to happen in all 3 phases of tissue processing with maximum happening after resection. 1/3rd of margin shall always be sacrificed for shrinkage whenever wide excision of tumour in oral squamous cell carcinoma is planned to avoid stigmata of close margin.
Funding
None.
Declarations
Conflicts of interest
None.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
- 1.Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, Jemal A (2024) Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 74(3):229–263. 10.3322/caac.21834 [DOI] [PubMed] [Google Scholar]
- 2.Board WC of TE (2020) Soft tissue and bone tumours. WHO Classification of Tumours
- 3.Looser KG, Shah JP, Strong EW (1978) The significance of ‘positive’ margins in surgically resected epidermoid carcinomas. Head Neck Surg 1:107–111 [DOI] [PubMed] [Google Scholar]
- 4.Kovacs AF (2004) Relevance of positive margins in case of adjuvant therapy of oral cancer. Int J Oral Maxillofac Surg 33:447–453 [DOI] [PubMed] [Google Scholar]
- 5.Vikram B, Strong EW, Shah JP et al (1984) Failure at the primary site following multimodality treatment in advanced head and neck cancer. Head Neck Surg 6:720–723 [DOI] [PubMed] [Google Scholar]
- 6.Loree TR, Strong EW (1990) Significance of positive margins in oral cavity squamous carcinoma. Am J Surg 160:410–414 [DOI] [PubMed] [Google Scholar]
- 7.Ravasz LA, Slootweg PJ, Hordijk GJ et al (1991) The status of the resection margin as a prognostic factor in the treatment of head and neck carcinoma. J Craniomaxillofac Surg 19:314–318 [DOI] [PubMed] [Google Scholar]
- 8.Cook JA, Jones AS, Phillips DE et al (1993) Implications of tumor in resection margins following surgical treatment of squamous cell carcinoma of the head and neck. Clin Otolaryngol 18:37–41 [DOI] [PubMed] [Google Scholar]
- 9.Slootweg PJ, Hordijk GJ, Schade Y et al (2002) Treatment failure and margin status in head and neck cancer. A critical view on the potential value of molecular pathology. Oral Oncol 38:500–503 [DOI] [PubMed] [Google Scholar]
- 10.Johnson RE, Sigman JD, Funk GF et al (1997) Quantification of surgical margin shrinkage in the oral cavity. Head Neck 19:281–286 [DOI] [PubMed] [Google Scholar]
- 11.Mistry RC, Qureshi SS, Kumaran C (2005) Post-resection mucosal margin shrinkage in oral cancer: quantification and significance. J Surg Oncol 91(2):131–133. 10.1002/jso.20285 [DOI] [PubMed] [Google Scholar]
- 12.Cheng A, Cox D, Schmidt BL (2008) Oral squamous cell carcinoma margin discrepancy after resection and pathologic processing. J Oral Maxillofac Surg 66(3):523–529. 10.1016/j.joms.2007.08.040 [DOI] [PubMed] [Google Scholar]
- 13.Egemen O, Bingöl D, Orman Ç, Sayilgan AT, Özkaya Ö, Akan M (2014) Quantification of the surgical margin shrinkage in lip cancer: determining the relation between the surgical and histopathologic margins. J Craniofac Surg 25(6):2152–2155. 10.1097/SCS.0000000000001150 [DOI] [PubMed] [Google Scholar]
- 14.El-Fol HA, Noman SA, Beheiri MG, Khalil AM, Kamel MM (2015) Significance of post-resection tissue shrinkage on surgical margins of oral squamous cell carcinoma. J Craniomaxillofac Surg 43(4):475–482. 10.1016/j.jcms.2015.01.009 [DOI] [PubMed] [Google Scholar]
- 15.Umstattd LA, Mills JC, Critchlow WA, Renner GJ, Zitsch RP 3rd (2017) Shrinkage in oral squamous cell carcinoma: an analysis of tumor and margin measurements in vivo, post-resection, and post-formalin fixation. Am J Otolaryngol 38(6):660–662. 10.1016/j.amjoto.2017.08.011 [DOI] [PubMed] [Google Scholar]
- 16.Burns C, Gorina FM (2021) An analysis of tumor margin shrinkage in the surgical resection of squamous cell carcinoma of the oral cavity. Cureus 13(5):e15329. 10.7759/cureus.15329 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Al-Sabaawy HB, Rahawi AM, Al-Mahmood SS (2021) Standard techniques for formalin-fixed paraffin-embedded tissue: a pathologist’s perspective. Iraqi J Vet Sci 35(Supplement I-III):127–135. 10.33899/ijvs.2021.131918.2023 [Google Scholar]
- 18.Pangare TB, Waknis PP, Bawane SS, Patil MN, Wadhera S, Patowary PB (2017) Effect of formalin fixation on surgical margins in patients with oral squamous cell carcinoma. J Oral Maxillofac Surg 75(6):1293–1298. 10.1016/j.joms.2016.11.024 [DOI] [PubMed] [Google Scholar]
- 19.Solomon J, Hinther A, Matthews TW, Nakoneshny SC, Hart R, Dort JC, Chandarana SP (2021) The impact of close surgical margins on recurrence in oral squamous cell carcinoma. J Otolaryngol Head Neck Surg 50(1):9. 10.1186/s40463-020-00483-w [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Fowler J, Campanile Y, Warner A, Laxague F, Fnais N, Fung K, Mendez A, MacNeil D, Yoo J, Palma D, Nichols A (2022) Surgical margins of the oral cavity: is 5 mm really necessary? J Otolaryngol Head Neck Surg 51(1):38. 10.1186/s40463-022-00584-8 [DOI] [PMC free article] [PubMed] [Google Scholar]