Head and neck
Published: 2022-11-23

Effect of formalin fixation on tumour size and margins in head and neck cancer specimens

Department of Otorhinolaryngology and Head and Neck Surgery, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India
Department of Otorhinolaryngology and Head and Neck Surgery, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India
Department of Otorhinolaryngology and Head and Neck Surgery, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India
Department of Otorhinolaryngology and Head and Neck Surgery, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India
Department of Otorhinolaryngology and Head and Neck Surgery, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India
Department of Otorhinolaryngology and Head and Neck Surgery, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India
head and neck neoplasms formaldehyde margins of excision neoplasm staging


Objectives. To quantify tumour size and margin shrinkage due to formalin fixation in head and neck cancer specimens and determine its effect on tumour staging and margin clearance.
Methods. Tumour specimens were measured immediate post-resection and at 24 hours after fixation. Tumour was measured in 2 dimensions and one margin was measured. Shrinkage was categorised as < 10%, 10-20%, and > 20%. Effect of shrinkage on tumour stage and margin clearance were evaluated.
Results. A total of 50 specimens were analysed. The tumour AP (Anterior Posterior) and SI (Superior Inferior) measurements showed significant shrinkage with a mean difference of 22.93% and 21.69% respectively. > 20% shrinkage was noted in 78% of AP and 58% of SI measurements. Margins showed a mean difference of 25.61%. 84% of margins demonstrated > 20% shrinkage. In all, 46.7% of T3 and 23% of T2 tumours pre-fixation were downstaged to T2 and T1, respectively, post-fixation.
Conclusions. Formalin fixation alone can be responsible for significant shrinkage of tumour and margin dimensions in head and neck specimens. It is suggested that decisions regarding the treatment plan should be made on clinical staging of primary tumour rather than pathological staging. In addition, post-excision pre-fixation margins should be considered for treatment planning.


Head and neck cancers, including cancers arising from the oral cavity, oropharynx, hypopharynx and larynx, are the sixth most common type of cancer worldwide and a leading cause of cancer-related deaths 1. Multidisciplinary management strategies are cornerstones for achieving optimal therapeutic outcomes in cancers of the head and neck region. However, surgery remains the mainstay of therapy for oral cancers 2. The ultimate aim of surgical resection is adequate clearance of tumour 3. Positive or close margins may necessitate the addition of adjuvant therapy like chemotherapy or radiation therapy to the treatment protocol.

In addition to tumour margin, adjuvant treatment is also dictated by tumour size which determine the classification of tumours into different T stages. A reduction in tumour size in the pathology report will understage the tumour and the patient may be deprived of adjuvant therapy with disastrous consequences. Hence, accurate measurements of tumour dimensions and margins are paramount in the management of patients with head and neck cancer.

Prior to histopathological analysis, specimens are preserved in formalin, which is the most common fixative used by many laboratories. Various studies have reported a change in tumour dimensions and margin measurements due to formalin fixation. Specimens from colorectal, oesophageal and breast tumours have demonstrated significant shrinkage of both tumour dimensions and tumour free margins 4-8. Studies on head and neck cancer specimens also have noted shrinkage, but the results are not uniform and the degree of shrinkage showed wide variations (10-25%)for tumour margins and 3-24% for tumour dimensions 9-12. In addition, the number of studies are sparse and there is only one investigation that has evaluated the effect of formalin fixation on both tumour and margin measurement 9.

Our study was aimed at determining the effects of formalin fixation on tumour dimensions as well as margin measurements, and to evaluate the discrepancies between clinical and pathological tumour staging caused by formalin fixation.

Materials and methods


50 patients with oral cavity malignancy managed with surgical resection of the primary tumour in a 2-year period were included in the study. A detailed history was obtained and a thorough clinical examination of the head and neck region was conducted. Contrast-enhanced CT scan was obtained in all cases to identify the extent of the lesion and the malignancy was proven by pre-operative biopsy. Tumours belonging to Stage I to IV were included in study as long as en bloc resection was possible. Depending on the extent of tumour, the procedures carried out included wide local excision, with or without hemi-mandibulectomy (for buccal mucosal and tongue malignancies), whereas wide local excision was performed for lip lesions. All resections were carried out by ensuring a minimum 1 cm margin circumferentially from the tumour border, assessed by visual inspection and palpation. The mucosal and skin incisions were placed using scalpel and the rest of the tumour excision was completed using electrocautery. Patient details including age, gender, tumour site, tumour size, clinical TNM staging, pathological TNM staging and the surgical procedure undertaken were recorded. Patients who were switched to a non-surgical treatment modality (because of poor performance scales) and who had previously undergone chemotherapy or radiotherapy or both were excluded from the study.


Tumour size measurements were undertaken using a digital caliper with a resolution of 0.1 mm and accuracy of ± 0.2 mm. Initial tumour measurements were taken immediately after excision of the surgical specimen. For tumour measurement, the anteroposterior (and superoinferior) margins were recorded based on the sutures already placed at the tumour margins for tumour orientation. For measurement of margins, in most of cases, the anterior margin was chosen but in 3 cases where this was not possible, e.g. oral cavity tumour involving the angle of mouth, another prominent margin was arbitrarily chosen for measurement. The margin was marked by placing a suture to identify the exact site for second measurement. After marking, specimens were stored in 10% neutral buffered formalin for 24 hours and the measurements were repeated by the same observer using the same reference points. The measurements were then tabulated and compared.


50 consecutive patients with head and neck cancer who fulfilled the selection criteria and underwent wide local resection for primary tumours were included in the study. Patient characteristics are listed in Table I.

Tumour shrinkage

The AP and SI dimensions measured before and after fixation were compared (Tab. II). Tumour shrinkages were classified into 3 categories: < 10, 10-20 and > 20%. A mean difference of 22.93% was observed in AP measurements with 78% of the specimens demonstrating shrinkage of > 20%. For SI measurement, the mean difference was 21.69 with 58% of patients showing a shrinkage of more than 20 percent. When pre- and post-fixation values were compared for AP and SI dimensions the difference in the means were highly significant (Tab. III).

Margin shrinkage

Margin measurements demonstrated a mean difference of 25.61% between pre- and post-fixation values (Tab. II). These results were significant and 84% of margins decreased by more than 20% post-fixation (Tab. III). 28 of the specimen margins that were > 5 mm (clear) were converted to 1-5 mm (close) post-fixation. Pre- and post fixation values were compared and found to be highly significant.

Comparison of shrinkage by sub-site

Upon comparison, the shrinkage of tumour dimension was found to be highest with tongue (27.43% AP and 26.82% SI) followed by lip (27.03% AP and 23.91% SI) and buccal mucosa (22.15% AP and 20.72% SI) and the values were highly significant (Tab. IV). However, for margin measurement, there was no significant difference in the percentage of shrinkage between the three sub-sites (Tab. V).

Pre-fixation versus post-fixation stage

On comparison of pre-fixation to post-fixation stage, down-staging of the tumours was seen, especially for stage T3 tumours (Fig. 1). Of the 15 tumours staged T3 pre-fixation, 7 tumours (46.7%) were down-staged to T2 post-fixation. In addition, 3 of 13 tumours (23%) staged T2 pre-fixation were down-staged to T1 post-fixation. Tumours staged T4a did not demonstrate any change in staging post-fixation.


According to the 2020 GLOBOCAN estimates 1, worldwide, head and neck cancer accounts for more than 1.5 million cases and 500,000 deaths annually. In Southern Asian countries, cancers of the lip and oral cavity have a high prevalence and is a leading cause of cancer death among men in India and Sri Lanka. This high frequency can be attributed to the widespread consumption of tobacco in varied forms in these regions.

Among the treatment options currently available, surgery is a key modality in management of head and neck cancer. Although chemotherapy and radiotherapy are established treatment modalities for head and neck cancer, oral cancers are mainly managed by surgical resection, with non-surgical modalities being reserved for adjuvant therapy.

In head and neck cancer, the main aim of surgery with curative intent is to achieve an R0 resection, i.e. microscopic tumour clearance. Inadequate excision margins have an adverse effect on prognosis, apart from necessitating the addition of post-operative adjuvant therapy 13. Studies have noted that inadequate surgical margins have a bearing on local and regional recurrence, metastasis and death 14. However, there is considerable variation with regards to the definition of positive, close and clear margins and various cut-offs of 2 mm, 5 mm, or 10 mm have been used to define adequate margins within head and neck region 15. In this context, the UK Royal College of Pathologists has issued guidelines for clear margins, close margins and positive margins for histological clearance of > 5 mm, 1-5 mm and < 1 mm, respectively 16. As margin status is important in defining adjuvant treatment, it is important not only to obtain adequate margins but also to measure the margins accurately.

In addition to surgical margins, tumour dimensions also need accurate measurements. Considering that the widely used AJCC TNM classification system for head and neck cancer focuses on tumour dimensions in assigning T category, it is important to measure the exact tumour dimensions, as variation can lead to either upstaging or down-staging of a tumour, which will have considerable therapeutic and prognostic implications.

Prior to histopathological analysis of surgical specimen, the specimen has to be fixed. The commonly used method is fixation with 10% neutral buffered formalin. In most laboratories, including ours, specimens are not processed immediately and the duration in formalin varies depending on the number of specimens received each day. In our study, all the specimens were fixed in formalin for 24 hours prior to second measurement to avoid any variation in tissue exposure to formalin. The effect of formalin fixation on tumour dimensions and tumour-free margins have been studied on tissues from various sites like colorectal, breast, oesophageal and skin tumours 4-8. Some of these studies have documented significant shrinkage of tumour and margin dimensions post-fixation, but the findings were not uniform in all studies. For instance, on breast cancer specimens, the studies by Yeap et al. 7 and Masooma et al. 8 noted significant tumour margin shrinkage post formalin fixation, while another study by Krekel et al. 17 contradicted this finding and showed no shrinkage in the breast specimens studied.

Few studies have documented the effects of resection and formalin fixation on head and neck cancer specimens. These studies have taken two parameters into consideration: i) shrinkage of tumour and margins because of resection; and ii) shrinkage because of fixation with formalin. Post-excision margin measurements were seen to shrink by 14.9% in a study by Lauren et al. 9, while Mistry et al. 11 noted a mean loss of 22.7% in oral cancers (buccal mucosa and tongue cancer). Cheng El Fol et al. 10,18 demonstrated significant shrinkage in mucosal margins (up to 75%) when post fixation measurements were compared to pre-resection margins. A study by Lauren et al. 9 noted that although there was an overall mean shrinkage of tumour size and tumour free margins of 10.7% and 11.3%, respectively, in post-fixation specimens, most of the decrease in tumour size and tumour-free margins occurred between pre and post-excision measurements. Post-fixation, the tumour size further decreased by 4.7% and margin measurements by 3.7%, although neither of these discrepancies were statistically significant. This study concluded that the decrease may be due to intrinsic tissue properties rather than the effects of formalin.

Our study was mainly focused on specimens from buccal mucosa, tongue and lip as these were the most common sites of cancer seen in our study. We focused on post-excision pre-fixation and post-fixation measurements. We found that margin measurements because of fixation changed in 84% (42 of 50) of specimens by > 20%. Similar findings were reported by Pangare et al. 12 in their study on specimens of oral squamous cell carcinoma from gingivobuccal sulcus where the average surgical margin shrinkages were 18.7% anteriorly, 14.9% posteriorly, 23.6% medially and 23.9% laterally.

Apart from measurement of resection margins, the anteroposterior tumour dimensions were also measured in our study since variation in tumour size impacts the treatment plan. On comparison of tumour dimensions, it was noted that anteroposterior tumour dimensions decreased in all 50 specimens with 78% of the specimens demonstrating shrinkage of > 20%. This is higher than what was reported by Chen et al. 19 wherein the maximal diameter of specimens shrank by 1.5 mm (4.40%) after formalin fixation. This low level of shrinkage could be due to the multiple pathologies and sites examined in their study. Herein, the discrepancy of measurements led to underestimation of T staging in 8 patients (8%) when tumour size was considered for T staging (5 patients shifted from T3 to T2 and 3 patients from T2 to T1). A similar finding was observed in our study where a change in T category was noted post-fixation in 10 cases (7 cases shifted from T3 to T2 and 3 patients shifted from T2 to T1). This apparent downstaging of the tumour can have significant therapeutic and prognostic implications. For instance, a tumour that is pre-operatively assigned to T3 category will be a Stage III cancer as per the AJCC classification. This patient would often require post-surgical adjuvant therapy in the form of radiotherapy owing to the higher risk of recurrence. A significant shrinkage in tumour size that leads to downstaging of T category can adversely affect the treatment plan in these patients with denial of adjuvant treatment and consequently a higher chance of recurrence. In addition, the documentation of close margins will require referral for adjuvant therapy, which could have been otherwise avoided. The medico-legal implications of this clinico-pathological conundrum cannot be ignored and requires resolution.

The shrinkage of tumour dimension in our study is higher than what was documented by Chen et al. 19. Most of the patients in our study had T3 or T4 tumours and almost all were heavy tobacco chewers. Whether these two factors can alter the intrinsic tissue properties and cause a higher degree of shrinkage is not known and requires further study.

The AJCC cancer staging manual specifies that the pathologist should note potential alteration in tumour size caused by fixation if it might affect staging and that up to 30% shrinkage of soft tissues may occur in resected specimens after formalin fixation 20. Furthermore, it mentions that pathological staging does not supplant clinical staging as the primary scheme. Also due to the complex anatomy of oral cavity combined with restricted mouth opening (often seen in high stage tumours or cases with coexistent oral submucous fibrosis), it is not always possible to accurately assess tumour size by clinical examination alone. From the findings of our study, we contend that T staging of tumour for treatment should be done by clinical examination and that margin measurement should be done post-resection but before fixation.

Another finding that was noted in our study was that there was a sub-site specific variation in the degree of shrinkage where the shrinkage of tongue tumours was higher in comparison to other sub-sites. This could be due to the variation in intrinsic tissue properties at different sites. However, the margins did not show any subsite-specific discrepancy unlike previous studies 10.

Our study is limited by the number of sites that were evaluated, as most of the tumours were involving the buccal mucosa and tongue, with only three cases of lip malignancy. We suggest studies on other head and neck sites, including but not limited to larynx, maxillary alveolus and hard palate. The anatomy in these sub-sites is diverse and would warrant separate evaluation rather than extrapolation of findings from other areas. Moreover, tumour margins and T staging are not the only factors which determine the necessity of adjuvant therapy; high risk or adverse risk factors such as multiple positive nodes or nodes with extracapsular invasion, vascular/lymphatic/perineural invasion, oral cavity cancers with positive level 4 or 5 nodes and poor differentiation of primary tumour are established indications for postoperative radiation in oral malignancies and need to be considered when planning postoperative therapy 21.


The findings of our study indicate that formalin fixation can be responsible for significant shrinkage of tumour and margin dimensions in head and neck specimens. We suggest that any deliberation on postoperative adjuvant therapy should be made on clinical staging of the primary tumour rather than pathological staging along with consideration of other established clinical and pathological parameters which are at high risk for locoregional recurrence of cancer. In addition, post-excision pre-fixation margins should be considered for treatment planning.

Conflict of interest statement

The authors declare no conflict of interest.


This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author contributions

Conceptualization: SS, VK. Methodology: SS, VK, KK. Data collection and analysis: KK, VK, SS, VSS. Writing-original draft: KK, VK, TR, SK. Writing-review and edit: SS, VSS.

Ethical consideration

This study was approved by the Institutional Ethics Committee (Kasturba Medical College, Mangaluru) (approval number/protocol number IEC KMC MLR 10-19/498).

The research was conducted ethically, with all study procedures being performed in accordance with the requirements of the World Medical Association’s Declaration of Helsinki.

Written informed consent was obtained from each participant/patient for study participation and data publication.

Figures and tables

Figure 1.Number of patients in the different T stages by clinical and pathological staging.

Variable Number of patients (n = 50)
    < 40 years 7 (14%)
    40-60 years 34 (68%)
    > 60 years 9 (18%)
    Male 32 (64%)
    Female 18 (36%)
    Buccal mucosa 37 (74%)
    Tongue 10 (20%)
    Lip 3 (6%)
Type of surgery
    Wide local excision 22 (44%)
    Wide local excision+ marginal mandibulectomy 3 (6%)
    Wide local excision+ hemimandibulectomy 21 (42%)
    Hemiglossectomy 4 (8%)
Clinical T stage
    T1 1 (2%)
    T2 13 (26%)
    T3 15 (30%)
    T4a 21 (42%)
Pathological T stage
    T1 4 (8%)
    T2 17 (34%)
    T3 8 (16%)
    T4a 21 (42%)
Table I.Demographic data, site of cancer, surgical treatment of primary cancer site, clinical T stage and pathological T stage.
APx SIy Margin measurement
N Percentage N Percentage N Percentage
Shrinkage (%) < 10% 0 .0% 0 .0% 0 .0%
10-20% 11 22.0% 21 42.0% 8 16.0%
> 20% 39 78.0% 29 58.0% 42 84.0%
Table II.Shrinkage of tumour (anterior posteriorx and superior inferiory dimensions) and tumour margins, when classified into 3 groups; < 10, 10-20 and > 20%.
Parameter N Mean (cm) Std. deviation Mean difference S.D. of difference Change (%) P-value
APx Pre 50 4.98 1.98 1.14 0.46 22.93 0.000
Post 50 3.84 1.62
SIy Pre 50 3.56 1.51 0.77 0.35 21.69 0.000
Post 50 2.79 1.23
Margin measurement Pre 50 1.27 0.35 0.33 0.12 25.61 0.000
Post 50 0.95 0.25
Table III.Mean pre- and post-fixation anterior posteriorx, superior inferiory dimensions and tumour margins.
Parameter Diagnosis N Mean (cm) Std. deviation Mean difference S.D of difference Difference (%) P-value
APx Buccal mucosa Pre 37 5.24 2.16 1.15 0.53 22.15 0.000
Post 37 4.10 1.74
Tongue Pre 10 4.36 1.01 1.17 0.17 27.43 0.000
Post 10 3.19 0.86
Lip Pre 3 3.74 1.51 0.97 0.24 27.03 0.020
Post 3 2.78 1.29
SIy Buccal mucosa Pre 37 3.74 1.56 0.77 0.37 20.72 0.000
Post 37 2.97 1.26
Tongue Pre 10 2.87 1.20 0.75 0.28 26.82 0.000
Post 10 2.12 0.94
Lip Pre 3 3.72 1.52 0.86 0.28 23.91 0.034
Post 3 2.86 1.27
Margin measurement Buccal mucosa Pre 37 1.28 0.38 0.32 0.12 24.75 0.000
Post 37 0.96 0.28
Tongue Pre 10 1.26 0.25 0.35 0.10 27.73 0.000
Post 10 0.91 0.16
Lip Pre 3 1.20 0.26 0.30 0.14 24.48 0.061
Post 3 0.90 0.13
Table IV.Mean shrinkage of anterior posteriorx, superior inferiory tumour dimensions and tumour margins for different sites (Buccal mucosa, Lip and Tongue).
Parameter N Mean shrinkage (%) S.D. of shrinkage (%) ANOVA p
Anterior posterior dimensions Buccal mucosa 37 22.15 4.96 0.006
Tongue 10 27.43 3.46
Lip 3 27.03 5.57
Superior inferior dimensions Buccal mucosa 37 20.72 5.03 0.004
Tongue 10 26.82 4.21
Lip 3 23.91 5.38
Margin measurement Buccal mucosa 37 24.75 4.10 0.124
Tongue 10 27.73 3.24
Lip 3 24.48 6.06
Table V.Comparison of shrinkage across the different subsites (buccal mucosa, lip and tongue).


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Kshithi K

Department of Otorhinolaryngology and Head and Neck Surgery, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India

Vikrant Kamboj

Department of Otorhinolaryngology and Head and Neck Surgery, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India

Suja Sreedharan

Department of Otorhinolaryngology and Head and Neck Surgery, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India

Vijendra Shenoy S

Department of Otorhinolaryngology and Head and Neck Surgery, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India

Thripthi Rai

Department of Otorhinolaryngology and Head and Neck Surgery, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India

Sushmitha Kabekkodu

Department of Otorhinolaryngology and Head and Neck Surgery, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India


© Società Italiana di Otorinolaringoiatria e chirurgia cervico facciale , 2022

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