Research
Presence of Human papilloma virus and EGFR expression does not predict response to Neoadjuvant chemotherapy in oral cancer
1Amar Kumar, 2Mohan Kumar, 1Ruhi Dixit, 1Rashmi Jaiswal, Vineeta Srivastava, 1Manoj Pandey
- 1Department of Surgical Oncology , Institute of Medical Sciences, Banaras Hindu University, Varanasi
- 2Department of Pathology , Institute of Medical
Sciences, Banaras Hindu University, Varanasi
- Submitted: October 2, 2012;
- Accepted October 17, 2012
- Published: October 20, 2012
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Introduction
Approximately 25% of head and neck squamous cell cancer, especially oropharangeal cancers are associated with high risk HPV-16, 18, 31, 33, and 35. Patient with HPV-16 related tumors tend to be younger, have smaller primary lesions and experience improved survival compared with patient with HPV-16 negative tumor. Studies have shown a 60 to 80% reduction in the risk of cancer death in HPV positive tumors. Similarly, both high EGFR expression and low HPV titer are associated with poor outcome. This study was carried out to correlate the response of neoadjuvant chemotherapy with presence of HPV and EGFR expression in oral cancer.
Patients and Method
HPV status by PCR and the EGFR expression by IHC was studied in 20 patients of locally advanced squamous cell oral cancer receiving neoadjuvant chemotherapy with docetaxel and carboplatin. Response was evaluated with RECIST criteria. Mean, median and standard deviation were calculated for continuous variables. Responses were correlated with positivity of HPV asserted by PCR and EGFR status.
Results
Majority of the cases had primary tumor of buccal mucosa (25%), lower alveolus (25%) and tongue (25%). HPV 16 was positive in 5 patients (25%), while none of the patient had HPV 18. Level of EGFR expression was 1+ in 6 (30%) 2+ in 5 (25%) and 3+ in 9 patients (45%). 14 patients (70%) were given 3 cycles, 2 patients (10%) 4 cycles, 3 patients (15%) 5 cycles of chemotherapy. complete response was seen in 5%, partial response in 45%, 30% had stable disease and 20% progressive disease. There was significant relation of response with bleeding at the time of presentation (x2 = 8.235, p = 0.041) and site of tumor (x2 = 27.037, p = 0.008) with patients with buccal mucosa, lip and alveolus having a better response than retromolar trigone and tongue lesions. No significant relation was seen with growth type, pretreatment T stage, grade, HPV status and EGFR status.
Conclusion
Human papilloma virus and EGFR expression does not predict response to neoadjuvant chemotherapy in oral cancer.
Introduction
Oral and pharyngeal cancers although grouped together are sixth leading cancer in world [1], in India it is the most common cancer in male. Apart from traditional risk factors such as tobacco and alcohol high-risk HPV is an independent risk factor and associated with approximately 25% of head and neck squamous cell cancer worldwide [2]. Various series from India documented presence of HPV 16/18 in 15-33.6% of cases [3-7]. Recent emerging data indicates that HPV related Head Neck Squamous cell carcinoma (HNSCC) define a unique population of patients with distinct biology that likely should be treated separately from non-HPV related HNSCC [8]. HPV-16 infection can be found in a significant portion of oropharyngeal tumors, particularly that of the base of tongue, palate and tonsils; with the tonsillar crypts being the most common site [9]. HPV associated oral cancer is mostly sexually transmitted [10], patient with HPV-16 related tumors tend to be younger, have smaller primary site lesions and experience improved survival compared with patient with HPV-16 negative tumor [11].While chronic HPV infection allows for the evolution of immune evasion mechanisms, viral antigens can still elicit an immune response. Moreover, a robust lymphocytic response is associated with better prognosis in a variety of tumor types including head and neck cancer [12] .Given the etiologic involvement of the HPV oncoproteins E6 and E7 during carcinogenesis of these tumors [13], it is also conceivable that intervention with anti-viral drugs may enhance sensitivity of these tumors to cytotoxic drugs, with the goal of reducing the required dose of chemotherapeutic agents and the associated side effects. A recent study examining biomarkers of response in advanced oropharyngeal cancer identified both high EGFR expression and low HPV titer as being associated with poor outcome, suggesting a possible role for EGFR inhibition in management of oropharyngeal cancer [10].
Aim of study was to correlate the response of neoadjuvant chemotherapy with presence of HPV and EGFR expression in oral cancer.
Material and method
Between September 2009 to February 2011, 20 Patients of Locally advanced oral cancer meeting the inclusion criteria were given neoadjuvant chemotherapy with docetaxel & carboplatin. Response was evaluated for it's association with HPV and EGFR as predictive marker. Patients of locally advanced oral cancer above 18 years of age willing to give consent were included .Prior treatment, pregnant and lactating women, multiple primaries and renal compromise patient were excluded from study. A comprehensive history, physical examination and hematological and biochemical investigations of the patients was recorded on the preset pro-forma. All patients underwent a pre and post treatment CT scan. Before starting the treatment and at the time of surgery after the neoadjuvant therapy, biopsy was taken for evaluation of HPV, EGFR status and pathological response to treatment. Tissue was stored in deep freezer at -400c for PCR and for EGFR status fixed in 10% neutral buffered formalin for overnight. By standard phenochloroform method HPV DNA was isolated, PCR was done and bands were analyzed using 2% agarose gel and visualized with the help of dye ethedium bromide. Primer sequence use for HPV-16 was Forward primer–5’ GAATCCATATGCTGTATGTGATAAAT-3’, Reverse primer-5’ GATGATCTGCAACAAGACATACATC-3’ and for HPV-18 Forward primer–5’ ACCTGTGTATATTGCAAGACAGT-3’, reverse primer-5’ TGTTTCTCTGCGTCGTTGGCT-3’.
Immunohistochemistry for EGFR were done using primary antibody EGFR (1005):sc-03. The protein expression was then scored as: -, < 25% positive cells; +, 25-50% positive cells; ++> 50-75% positive cells; and +++, > 75% positive cells.
Patients were given intravenous docetaxel 80 mg/m2 with carboplatin 300 mg/m2 at 3 weekly interval after premedication .Tumor response was evaluated for tumor size measured by CT scan at initial diagnostic procedure and just before surgery after neoadjuvant chemotherapy according to the Response Evaluation Criteria in Solid Tumors (RECIST)
- Complete Response (CR) - defined as complete disappearances of tumor.
- Partial response (PR) as a 30% decrease of largest diameter (LD)
- Progressive disease (PD) as a 20% increase in LD.
- Stable disease (SD) as a decrease or increase less than PR or PD. Patients who achieved CR or PR will be defined as responder where as those who achieved PD or SD were defined non responder.
Mean, medium and standard deviation were calculated for continuous variables. Responses were correlated with positivity of HPV asserted by PCR and EGFR status.
Result
Table 1: Clinical and histological characteristics of patients, together with HPV positivity, EGFR status and response after neoadjuvant chemotherapy.
| Total no of patients |
20 |
| Site |
|
| buccal mucosa |
25% |
| lower alveolus |
25% |
| RMT |
15% |
| GB sulcus |
5% |
| Lower lip |
5% |
| Pretreatment T stage |
|
| T2 |
15% |
| T3 |
10% |
| T4a |
75% |
| Pretreatment N stage |
|
| N0 |
15% |
| N1 |
30% |
| N2a |
45% |
| N2b |
10% |
| Pretreatment composite stage |
|
| III |
15% |
| IVA |
85% |
| Response |
|
| Complete Response (CR) |
5% |
| Partial Response (PR) |
45% |
| Stable Disease (SD) |
30% |
| Progressive Disease(PD) |
20% |
| Postoperative histopathology |
|
| Margin negative |
55% |
| micro positive |
15% |
| Lympho vascular invasion |
30% |
| Perineural invasion |
30% |
| Extranodal spread |
10% |
| Skin infiltration |
10% |
Table 2: Treatment of primary
| Treatment of primary |
Frequency |
Percentage |
| Defaulted |
1 |
5.0 |
defaulted ,presented after
1yr with T4bN3 |
1 |
5.0 |
Near total glossectomy
+SM |
1 |
5.0 |
| Radiotherapy |
4 |
20.0 |
| WE |
3 |
15.0 |
| WE+HM |
5 |
25.0 |
WE+HM+upper
alveolectomy |
1 |
5.0 |
| WE+SM |
4 |
20.0 |
| Total |
20 |
100.0 |
Clinical and histological characteristics of patients, together with HPV positivity, EGFR status and response after neoadjuvant chemotherapy in study are presented in (Table 1). In our 20 patients of locally advanced oral cancer, HPV 16 were positive in 5/20 (25%) of patients and none was positive for HPV 18. EGFR expression was seen in all 20 patients. After neoadjuvant chemotherapy complete response (CR) seen in 1/20(5%), partial response (PR) in 9/20(45%), stable disease (SD) in 6/20 (30%) and progressive disease (PD) in 4/20 (20%). (Tables 2-5) shows details of treatment after neoadjuvant chemotherapy. On cross tabulation (Table 5) there is significant relation of response to bleeding (P=0.041) and site of lesion (P=0.008). The patients who presented with bleeding showed a stable disease on chemotherapy. Patients with buccal mucosa, lip and alveolus demonstrated a better response then retromolar trigone and tongue lesions. There was no correlation of response with EGFR, as all 20 patients showed expression of EGFR. There was no significant positive or negative correlation of response with HPV status, T, N stage (Table 6).
Surgery of
neck |
Frequency |
% |
| No surgery |
2 |
10.0 |
| b/l SOHND |
1 |
5.0 |
| Lt RND+Rt |
1 |
5.0 |
| MRND |
|
|
| MRND2 |
10 |
50.0 |
| Radiotherapy |
4 |
20.0 |
| SOHND |
2 |
10.0 |
| Total |
20 |
100.0 |
| Reconstruction |
Frequency |
% |
| Defaulted |
2 |
10.0 |
| No surgery |
4 |
20.0 |
| No reconstruction |
1 |
5.0 |
| NLF |
2 |
10.0 |
| PMMC |
10 |
50.0 |
| SCM |
1 |
5.0 |
| Total |
20 |
100.0 |
NLF-nasolabial flap, PMMC-pectoralis major myocutaneous flap, SCM- sternocledomastoid myocutaneous flap
Discussion
Studies from India documented presence of HPV 16/18 in 15-33.6% of oral cancer [3-7]. In consistence with these studies our study too shows HPV 16 positivity in 5/20 cases (25%). Mork et al., (2001), D’souza et al., (2007), Herrero et al., (2003), showed HPV 16 positivity in 39.77%, 87.50%, 37.50% respectively [10,14,15]. HPV positive oropharyngeal cancer shows increase survival [1,2,16] and have favorable outcome compared with HPV negative cancer irrespective of treatment modality used [17].
Table 5: Cross tabulation between response and other variables
| Variables |
X2 |
P |
| Bleeding |
8.235 |
0.041* |
| Site of lesion |
27.037 |
0.008* |
| Pre T stage |
2.630 |
0.854 |
| Pre N stage |
19.120 |
0.024* |
| Growth type |
1.930 |
0.587 |
| Skin infiltration |
2.353 |
0.502 |
| Muscle infiltration |
7.500 |
0.058 |
| Bone infiltration |
1.818 |
0.611 |
| Grade |
0.889 |
0.828 |
| HPV positivity |
2.889 |
0.409 |
| EGFR status |
7.037 |
0.317 |
Table 6: Correlation of Pre N stage, T stage, HPV positivity and response
| Variables |
Pre T stage |
Pre N stage |
HPV positivity |
Response |
| Pre T stage |
1 |
|
|
|
| Pre N stage |
0.06 |
1 |
|
|
| HPV positivity |
0.304 |
0.106 |
1 |
|
| Response |
-0.118 |
-0.157 |
-0.203 |
1 |
In our study neoadjuvant chemotherapy with docetaxel and carboplatin showed partial response in 9/20 patient (45%) and complete response in 1/20 patient (5%), stable disease in 6/20 patients (30%) and progressive diseases in 4/20 patients (20%). In study conducted by Chitapanarux I et al., (2011) the complete response was seen in 34.3%, partial response in 40%, stable disease in 17% and progressive disease in 8.6% [18]. Fakhry C et al., (2008) showed that compared with patient with HPV negative tumors, patient with HPV positive tumors had higher response rate after induction chemotherapy (82% vs 55%, difference =27%, 95% CI=9.3% to 44.7%, p=0.01) and chemoradiation (84% vs 57%, difference =27%, 95% CI=9.7% to 44.3%, p=0.007). Besides they had demonstrated improved overall survival and lower risk of progression in these cases [19]. Albers A et al., (2005) showed that immunity to HPV 16 E7 is associated with the presence of HPV 16 infection and presentation of E7 derived types on SCCHN cells which show evidence of immune escape [20]. Rubenstein LM et al., (2011) showed that oral oncoprotein play a role in tumors genesis & growth in patient infected HPV-HR. Intervention with antiviral drugs may enhance sensitivity of the tumor to cytotoxic drugs [16]. Chung C.H. et al., (2009) showed that distinct molecular mechanism underlying HPV positive and HPV negative to much may lead to interaction effects where by response to drugs [1]. Kumar B et al., (2008), Gillison ML et al., (2009), and Kong CS et al., 2009 showed that a subset of the HPV positive patient experience worse outcome when compared with average HPV positive patient, resembling more of the clinical course in HPV negative patient. This subset of patient had more extensive smoking history, TP53 mutation, higher EGFR and Bcl xL expression, suggesting that the HPV status alone is not an adequate prognostic marker to perfectly segregate patient [1,21,22]. Maxwell JH et al., (2010) showed that current tobacco users with advanced HPV positive SCC of oropharynx are at higher risk of disease recurrence with never tobacco users [23]. Our study however failed to show any relationship of the response to treatment with presence or absence of HPV or the type of HPV virus.
EGFR expression were seen in 100% of our patients with 1+ staining in 30% , 2+ staning in 25% and 3+ staning in 45 % of cases and so failed to demonstrate any relation with response to chemotherapy. I.H Chen et al., (2003) in their study showed over expression of EGFR in 58% of cases with average 3.5 fold elevation. EGFR over expression had been shown to be statistically significant with T stage, N stage, overall TNM stage, primary tumor depth, lymph node extracapsular spread and poor survival [24]. Goncalves A et al., (2008) showed that EGFR positive cases had a 3 year cancer specific survival of 27.2% while EGFR negative cases had a 3 year cancer specific survival 64.3% (p=0.001). In multivariate analysis, only the disease free interval and over expression of EGFR were associated with a higher risk of cancer death [25]. Chau NG et al., (2011) showed that EGFR mutation present in about 40% of SCCHN appear to be a unexpected prognostic biomarker associated with better disease control in SCCHN regardless of treatment with erlotinib [26]. Kumar B et al., (2007) showed EGFR expression was inversely associated with response to induction chemotherapy (p=0.01), chemotherapy/Radiotherapy (p=0.055), overall survival (p=0.001), disease specific survival (p=0.002) and was directly associated with current smoking (p=0.04), female sex (p=0.053) and lower HPV titer (p=0.03). As combined markers, lower HPV titer and high EGFR expression were associated with worse overall survival and disease specific survival in oral cancer [27]. Shiraki M et al., (2005) showed P53, Cyclin D, and EGFR expression was significantly associated with poor differentiation (p=0.0008) and invasive growth pattern (p=0.0003). Any of these had no significant impact on survival. Coexpression of all these marker, however was significantly associated with invasive growth pattern, shorten survival, unfavorable prognosis [28]. Xia W et al., (1999) showed expression of EGFR members was not significantly associated with tumor size. There expression were significantly associated with the presence of lymph node metastasis and distant metastasis, shorter survival [29].
Conclusion
In the present study the response to the neoadjuvant chemotherapy was not found to correlate with presence of HPV or the EGFR status of the patients with oral cavity cancers.
Authors' Contribution
AK: Literature search and drafting the manuscript.
MK: Concept and design of the pathological part of manuscript, pathological data interpretation
RD: Analysis and interpretation of results and preparation of manuscript
RJ: Design of the HPV studies and interpretation of their results, preparation of manuscript
MP: Concept and design, preparation of final manuscript, data analysis and interpretation.
Conflict of Interests
Authors declare that there are no conflicts of interests.
Ethical Considerations
Authors declare that the present study was approved by the Institute Ethics Committee.
Funding
None.
Acknowledgement
None.
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