Abstract
Head and neck malignancies are the seventeenth most common malignancies, worldwide and second most common malignancy in India. So current study aims to compare between molecular marker p53 analysis and HPE for tumour positivity in primary resected margins in head and neck SCC. 61 patients with head and neck SCC were included in this prospective observational cross-sectional study performed in tertiary care hospital. A detailed history general physical examination blood investigation was done before the surgery. After the surgery, primary lesion from the resected tumour was sent for HPE analysis. From the same specimen, the margins at distance of 0.5–1 cm from primary tumour were sent for p53 mutation analysis. Report of p-53 mutation was noted and entered to the Performa. In our study we found out that in PDSCC HPE negative margins were found positive for p53 mutation in 81% cases. Which suggest that evaluation for p53 mutation should be done in PDSCC cases for HPE negative margins with in 1 cm. In patients of head and neck squamous cell carcinoma with free margins on HPE p-53 mutation is significantly associated to the PDSCC and margin upto 0.7 mm so recommended for p-53 profile can be beneficial in cases of the PDSCC and margins up to 0.7 mm for further management or for possibility of recurrence and its management to improve patients survival and decrease morbidity and mortality.
Keywords: Histopathological examination, Head and neck squamous cell carcinoma, Well differentiate squamous cell carcinoma, Moderately differentiate squamous cell carcinoma, Poorly differentiate squamous cell carcinoma, P53 mutation
Introduction
Cancer is listed among the leading cause of morbidity and mortality worldwide, with approximately 19.2 million new cases, 9.95 million cancer related deaths in 2020 and 50.5 million prevalent cases in all age groups according to GLOBOCAN [1].
Head and neck malignancies are the seventeenth most common malignancies worldwide and second most common malignancy in India.
Head and neck cancers account for almost 254,287 new cases annually, 60,922 in females and 1,93,364 new cases in males. The mortality rates are staggering, with almost 75,290 males and females dying from the disease annually in India. Over 200,000 new cases of head and neck cancers are registered every year in India. Among this nearly 60% have non-metastatic locally advanced disease [2].
The main risk factors associated with Head and neck squamous cell carcinoma (HNSCC) are environmental and lifestyle factors such as chewing tobacco, alcohol consumption and smoking. Recently epidemiological studies have proved a strong association with human papillomavirus (HPV) in subset of HNSCC and in non smoking cases. The human papilloma virus (HPV) may be related to some oral and oropharyngeal malignancies, according to recent research. In cases of oral cancer, HPV-16 has been discovered in up to 22% of cases and HPV-18 in up to 14% [3, 4].
The histopathological status of excisional margins of malignant neoplasms has long been used as a potential indicator for recurrences and prognosis. However, the predictive ability of the margin is far from satisfactory. Local recurrence occurs in up to 50% of the patients, even with microscopically negative surgical margins, and is the leading cause of treatment failure and recurrences occur within 2 years [5, 6].
The United Kingdom guidelines designate a margin of > 5 mm as being clear, 1 to 5 mm as close, and < 1 mm as signifying likely incomplete excision [7].
Margins harboring p53 mutations may be responsible for local or regional recurrence when they are pronounced clear after light microscopy, indicating that molecular assessment can augment the information provided by light microscopy. p53 elevation in histologically tumor-free margins was found to be a significant independent prognostic factor of local–regional recurrence.
Thus, the goal of this study was to evaluate the ability of p53 in identifying tumor related alterations in histologically negative surgical margins.
Methods
This Hospital based, descriptive type of observational study has been conducted in a tertiary care hospital of a Medical College of North India. Target population comprises of Operated patients of head and neck squamous cell carcinoma with free margins on HPE fulfilling the inclusion and exclusion criteria.
Patients of squamous cell carcinoma of oral cavity, fit for surgery who gave written consent for wide local excision.
Operated patients of head and neck squamous cell carcinoma with free margins on HPE whose post op surgical margin was at a distance of 0.5–1 cm from primary tumour.
Patients having free margins > 1 cm in all distances, positive margins on HPE, history of neo adjuvant CT and RT, not willing for surgery and not fit for surgery were excluded from study.
Total 61 patients were enrolled for the study. Study was approved by Institutional Ethical Committee. All eligible Operated patients of head and neck squamous cell carcinoma during study period at study location were approached by investigator and were explained about the nature and the purpose of the study. After obtaining their informed consent, detailed history was be taken and thorough general and local examination was done before the surgery. Routine investigations for surgery were done. Surgery was done by an identified otolaryngologist faculty of E.N.T dept of Medical college. After the surgery, primary lesion from the resected tumour was sent for HPE analysis. The diagnosis was made on HPE. From the same specimen, the margins at distance of 0.5–1 cm from primary tumour were sent for p53 mutation analysis. Report of p-53 mutation was noted and entered to the Performa.
Data analysis was done using licensed SPSS software version 21.0 (Chicago, Illinois). Independent t-test and ANOVA test were used to compare the continuous variable and chi-square test was used for categorical variables. Non parametric Mann Whitney test and Kruskal Wallis test were used in case of data did not follow a normal distribution. Data are presented as mean (standard deviation) or number or proportions. A p-value < 0.05 was considered as statistically significant.
Results
Out of 61 patients included in study, maximum 32 were in age of 41–50 years followed by 15 in < 40 years. The mean age of study population who underwent surgery for head neck carcinoma was 45.9 ± 7.7 years.
There were 53 males and 8 females participants. Female preponderance was seen in the study.
Study population which was 61, involved different sites of head and neck. Maximum 18 had ulcero proliferative growth Left buccal mucosa followed by 13 having ulcero-proliferative growth right buccal mucosa, rest involved the tongue and retromolar trigone.
All 61 participants underwent surgery for head and neck carcinoma. Maximum 30 underwent WLE + MND 2 + PMMC flap repair, followed by 18 who underwent WLE + SOND and rest 13 patients underwent CBR with PMMC flap repair.
On histopathological examination of post -operative tissue, In biopsy reports 37 patients had WDSCC and 11 patients had PDSCC and 13 patients were diagnosed as MDSCC (Table 1).
Table 1.
Distribution of participants according to HPE
| HPE | Frequency | Percent |
|---|---|---|
| MDSCC | 13 | 21.3 |
| PDSCC | 11 | 18.0 |
| WDSCC | 37 | 60.7 |
| Total | 61 | 100.0 |
P53 mutation was present in 37 patients out of the 61 participants whose molecular assessment was done for P53 mutation positivity (Fig. 1).
Fig. 1.
Distribution of participants according to p-53 mutation
On study of margin size of post-operative specimen, out of the 61 participants, maximum 17 had margin size 0.6 cm followed by 15 with margin size 0.8 cm. 10 patients had margin size 0.5 cm, 9 patients had 0.7 cm and 10 had 0.9 cm margin size. Mean margin length was found to be 0.75 ± 0.16 cm.
In this study, age, gender, chief complaint and type of surgery were found to have no statistically significant association with p-53 mutation.
When we studied post operative specimen for HPE and P53 mutation of all patients, A statistically significant HPE difference was found with p-53 mutation and p-53 mutation was present maximum in PDSCC in which 9 out of 11 were positive for p-53 mutation (Table 2).
Table 2.
Association of HPE with p-53 mutation
| HPE | Negative | Positive | P value | ||
|---|---|---|---|---|---|
| Count | % | Count | % | ||
| MDSCC | 5 | 20.8 | 8 | 21.6 | 0.025 |
| PDSCC | 2 | 8.3 | 9 | 24.3 | |
| WDSCC | 17 | 70.8 | 20 | 54.1 | |
| Total | 24 | 100.0 | 37 | 100.0 | |
In MDSCC, 8 patients were positive for P53 mutation out of 13 and in WDSCC only 20 patients were positive for P53 mutation out of 37 patients. So we found a significant statistically corelation between HPE and P53 mutation.
On study of margins characteristics, no statistically significant margin side difference was found with p-53 mutation but statistically significant margin size difference was found with p-53 mutation. In 37 patients P53 mutation was present in which 10 patients was with margin size 0.5 cm, 16 with 0.6 cm, 6 with 0.7 cm, and 4 patients with 0.8 cm margin size,1 with 0.9 cm margin size.
P-53 mutation was increased till 0.6 cm margin and than again declined (Table 3).
Table 3.
Association of margin with p-53 mutation
| Margin (in cm) |
Negative | Positive | P value | ||
|---|---|---|---|---|---|
| Count | % | Count | % | ||
| 0.5 | 0 | 0.0 | 10 | 27.0 | 0.0001 |
| 0.6 | 1 | 4.2 | 16 | 43.2 | |
| 0.7 | 3 | 12.5 | 6 | 16.2 | |
| 0.8 | 11 | 45.8 | 4 | 10.8 | |
| 0.9 | 9 | 37.5 | 1 | 2.7 | |
| Total | 24 | 100.0 | 37 | 100.0 | |
Discussion
Local or locoregional recurrence is still the most common cause of treatment failure, and there is a wealth of evidence indicating that the risk of recurrence is increased when the resection margins are too close as judged by light microscopy. Based on this knowledge, the surgeon aims to resect a carcinoma with a 1-cm margin, but because margin retraction can be as high as 50%, the pathologist's clearance is frequently 5 mm.
Clear surgical margins are thought to be a good predictor of a positive outcome. The guidelines in the United Kingdom define a margin of > 5 mm as clear, 1–5 mm as close, and 1 mm as likely incomplete excision [8]. However, it is well understood that margins of 5 mm may not be clear when the invasion pattern is unfavourable.
The use of molecular analysis of surgical margins has revealed that morphologic data frequently underestimates the extent of carcinoma or precancerous field effects.
Following up on these cases has revealed that “margins” harboring p53 mutations may be responsible for local or regional recurrence when they are pronounced clear after light microscopy [9, 10], indicating that molecular evaluation can supplement the information provided by light microscopy. At the moment, most molecular analyses are based on detecting the same signature p53 gene mutation in a carcinoma and the normal tissues that remain at the defect's edges after surgery. The strength of this approach is that p53 gene mutations are the most commonly detected abnormality in head and neck carcinomas [11]. Although the presence of p53 mutations in the deep margins must indicate carcinoma, if contamination is avoided, the presence of these mutations in the mucosal margins may indicate tumor, but these aberrations are also present in dysplasia and can be detected in oral mucosa that appears normal morphologically.
Similar to our findings, maximum OSCC were in age group greater than 40 years, in consistent with other study by Angadi et al. [11].
In our study, out of the 61 participants, 53 (86.9%) were male and 8 (13.1%) were female participants.
Male predominance was seen in our study in accordance with other studies done by Li et al. [12], and Nguyen et al. [13], although no prognostic difference is seen between male and females and did not show any statistical association.
In our study, out of the 61 participants, maximum 18 had ulcero proliferative growth Left buccal mucosa followed by 13 having ulcero-proliferative growth right buccal mucosa. So in our study buccal mucosa was commonest site followed by tongue. Anatomical site is considered as one of the important parameters to predict prognosis and survival rate. In North Karnataka, the prevalent site associated was buccal mucosa followed by tongue as also seen in studies done by Kaur et al. [14]. The occurrence of OSCC in buccal mucosa can be attributable to the use of smokeless tobacco prevalent in the local area.
In our study, out of the 61 participants, maximum 30 underwent WLE + MND 2 + PMMC flap repair followed by 18 who underwent WLE + SOND.
In our study, mean margin length was found to be 0.71 ± 014 cm and out of the 61 participants, maximum 17 had margin size 0.6 cm followed by 15 who had margin size 0.8 cm.
In our study, a statistically significant margin size difference was found with p-53 mutation. P-53 mutation was seen in 100% cases of 0.5 cm margins, 94% cases of 0.6 cm margins, 66% cases of 0.7 cm margins, 26% cases of 0.8 cm margins and 10% cases of 0.9 cm margins which shows high possibility of p53 mutation till 0.7 cm margins. Margin positivity is an important prognosticator in predicting recurrences. It’s difficult to compare our data on number of involved, close and clear margins due to variations in terminology and indirect approach by authors who categorize them as positive or negative margins [15].
In our study, out of the 61 participants, 37 (60.7%) had WDSCC and 11 (18%) had PDSCC and 13 (21.3%) had MDSCC. 37 (60.7%) were positive for p-53 mutation.
In our study, no statistically significant age, gender, complaints and surgery difference association was found with p-53 mutation.
p-53 mutation was positive in 8 out of 13 of MDSCC and 20 out of 37 of WDSCC and 9 out of 11 PDSCC. And a significant association was detected. PDSCC was found to be significantly more (81.8%) positive for p-53 compare to other SCC.
Histologically negative margin and Invasive tumor front (ITF) recurrence of OSCC was observed in association with mutated p53 by Oliveira et al. [16].
Dragomir et al. observed that p53 immunoreaction was intense at the level of the ITF with a LI of 61.8%, the stain being strong in the peripheral cells from tumoral islands. This suggests that evaluation of p53 has a prognostic value allowing the identification of highly proliferative, aggressive forms of oral carcinomas [17].
Kato et al. [18], suggested that highly invasive tumors exhibit p53 mutation and PCNA activity, thereby increasing the proliferative capacity at the Invasive tumor front (ITF) of OSCC. Such tumors are likely to have increased chances of recurrence and therefore a worse prognosis.
In the present study, 24 tumors were negative for p53 expression in the present study. In these negative results 20 (83%) were more then 0.7 cm margins. The reasons suggested for this negativity could be due to different carcinogenic pathways in which the p53 abrogation does not play a role [19].
Conclusion
In patients of head and neck squamous cell carcinoma with free margins on HPE, p-53 mutation is significantly associated to PDSCC and margin up to 0.7 mm on HPE, So p-53 profile can be beneficial in cases of the PDSCC on HPE and margins up to 0.7 mm irrespective of tumor margin side, age and sex of patients, chief complain of patient and type of surgery underwent for head neck squamous cell carcinoma. It will be helpful for further management or for possibility of recurrence and its management to improve patients survival and decrease morbidity and mortality. So when we found PDSCC and margin upto 0.7 cm on HPE, we should always go for P53 profile.
In this way, we hypothesize that the expression profile of these genes in histologically negative margins could act as a more sensitive and useful marker for the detection of molecular alterations associated with local disease control in HNSCC patients.
Funding
No funding source.
Declarations
Conflict of interest
Authors declare that we have no potential conflict of interest.
Ethical approval
The study was approved by the Institutional Ethics Committee.
Footnotes
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