Abstract
The aim of this study was to investigate the prognostic and predictive values of phospholipase C gamma 1 (PLCG1) expression in patients with locally advanced and resectable oral squamous cell carcinoma (OSCC), who were treated in a prospective, randomized, phase 3 trial evaluating standard treatment with surgery and postoperative radiation preceded or not by induction docetaxel, cisplatin, and 5-fluorouracil (TPF). Immunohistochemical staining for PLCG1 was performed on the biopsies of 232 out of 256 OSCC patients at clinical stage III/IVA; the PLCG1 positive score was determined by immunoreactive scoring system. The survival analysis was performed by Kaplan–Meier method; hazard ratios were calculated using the Cox proportional hazards model. Patients with a low PLCG1 expression had a significantly better overall survival ( P = 0.022), and a trend towards better disease-free survival ( P = 0.087), loco-regional recurrence-free survival ( P = 0.058), distant metastasis-free survival ( P = 0.053), and a high response rate to TPF induction chemotherapy with regard to clinical response ( P = 0.052) and pathological response ( P = 0.061), compared to those with high PLCG1 expression. Our results suggest that PLCG1 expression could be used as a prognostic biomarker for patients with advanced OSCC; however, it was not an adequate predictive biomarker for TPF induction chemotherapy.
Introduction
Oral squamous cell carcinoma (OSCC) is the most common type of head and neck cancer, with about 300,000 new cases worldwide each year. Despite many efforts, the 5-year survival rate of OSCC patients is still approximately 50–60%. For patients with locally advanced and resectable OSCC, the most commonly recommended treatment is radical surgery followed by postoperative radiotherapy or chemoradiotherapy, depending on the presence or not of high risk features in the surgical specimen.
Induction chemotherapy, which is the use of systemic chemotherapy before definitive surgery and/or radiotherapy, has been an attractive approach in the management of head and neck squamous cell carcinoma (HNSCC) for the last 25 years. Induction chemotherapy with a combination of docetaxel, cisplatin, and 5-fluorouracil (TPF) is suggested as a preferred combination chemotherapy regimen when induction treatment is used for the non-surgical management of HNSCC patients. Inductive TPF chemotherapy can increase the organ preservation rate and/or reduce the risk of loco-regional recurrence and/or distant metastasis. Unfortunately, some studies have failed to show a significant improvement in overall survival or disease-free survival (DFS) for patients receiving TPF. It is, however, possible that molecularly defined subgroups of HNSCC patients might benefit from induction chemotherapy.
Phospholipase C gamma 1 (PLCγ1/PLCG1) is a critical enzyme in growth factor-dependent signalling transduction and is a downstream target of epidermal growth factor receptor (EGFR) signalling. Activation of PLCG1 is thought to play a critical role in both cytoskeletal changes and migration associated with the metastatic process. The activation of PLCG1 by phosphorylation can occur downstream of many tyrosine kinase receptors including EGFR, vascular endothelial growth factor receptor 2 (VEGFR2), c-MET, platelet-derived growth factor receptor (PDGFR), and integrin. The activated PLCG1 induces hydrolysis of phosphatidylinositol-4,5-bisphosphate to form a second messenger of diacylglycerol and inositol-1,4,5-trisphosphate, which in turn activate protein kinase C and calcium signalling.
The overexpression of PLCG1 has previously been reported in invasive carcinoma tissues, including breast cancer, colorectal cancer, and squamous cell carcinoma. In patients with breast cancer receiving induction chemotherapy, those with high PLCG1 expression had a significantly shorter DFS compared to those with low PLCG1 expression. However, the prognostic and predictive role of PLCG1 expression in OSCC patients receiving induction chemotherapy has never been reported.
The aim of this study was to investigate the potential prognostic and predictive values of PLCG1 expression in the biopsy samples from patients with locally advanced and resectable OSCC, who had been enrolled in a randomized phase 3 trial of TPF induction chemotherapy followed by surgery and postoperative radiotherapy compared to upfront surgery and postoperative radiotherapy. We hypothesized that the patients with low PLCG1 expression would have better overall survival, DFS, loco-regional recurrence-free survival (LRFS), and distant metastasis-free survival (DMFS) compared to those with high PLCG1 expression.
Materials and methods
Patients
This study was based on patients who were enrolled in a prospective, randomized, phase 3 trial aimed at testing the hypothesis that TPF induction chemotherapy administered prior to surgery and postoperative radiotherapy would improve survival over surgery upfront in patients with locally advanced and resectable OSCC (ClinicalTrials.gov, NCT01542931 ); the trial was approved by the Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine.
Patients were enrolled into the clinical trial after signing an informed consent form. Details of the clinical trial have been described previously. Briefly, the main eligibility criteria included resectable squamous cell carcinoma of the oral cavity, clinical stage III or IVA, Karnofsky performance status >60%, and adequate haematological, hepatic, and renal function. Eligible patients were allocated randomly to the control arm (surgery followed by postoperative radiotherapy) or experimental arm (TPF induction chemotherapy followed by surgery and postoperative radiotherapy). Induction TPF consisted of docetaxel 75 mg/m 2 intravenously on day 1, followed by cisplatin 75 mg/m 2 intravenously on day 1, followed by 5-fluorouracil 750 mg/m 2 /day as a 120-h continuous intravenous infusion on days 1 through 5, every 3 weeks for 2 cycles. Surgery was performed at least 2 weeks after completion of the induction chemotherapy, and consisted of radical resection of the primary lesion and full neck dissection with appropriate reconstruction. Postoperative radiotherapy was initiated 4–6 weeks after surgery, with a total dose of 54–66 Gy.
The clinical tumour response was determined by clinical evaluation and imaging studies (performed at baseline and 2 weeks after cycle 2 of induction chemotherapy). Responses were characterized according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.0. The pathological response to TPF induction chemotherapy was assessed by examination of at least 20 slides of the resected specimen. A favourable response was defined as the absence of any tumour cells (pathological complete response) or the presence of scattered foci of a few tumour cells (minimal residual disease with <10% viable tumour cells).
After treatment, patients were monitored every 3 months in the first 2 years, every 6 months in the subsequent 3–5 years, and once a year thereafter until death or data censoring.
Detection of PLCG1 expression using immunohistochemistry
Pretreatment formalin-fixed and paraffin-embedded biopsy samples were collected for the detection of PLCG1 expression; however, in the control arm, if a pretreatment biopsy was unavailable, a portion of the surgical resection specimen was collected for biomarker evaluation. Sections 4 μm thick were studied using both haematoxylin and eosin (H&E) staining (for diagnostic confirmation, in accordance with the World Health Organization (WHO) histological criteria) and immunohistochemical staining for PLCG1. Immunohistochemical staining was performed as described previously. In brief, after deparaffinization, endogenous peroxidase was blocked and the sections were heated in a water bath at 98 °C with 0.01 M citrate buffer solution (pH 6.0) for 20 min to retrieve antigen. These were then cooled at room temperature and washed with phosphate buffered saline (PBS) three times for 5 min each, then incubated with the rabbit monoclonal antibody to PLCG1 (product code #5690, PLCG1 (D9H10) XP Rabbit mAb; Cell Signaling Technology, Inc., USA) at 1:150 dilution overnight at 4 °C. After recovering to room temperature for 1 h, the sections were washed with PBS three times for 5 min each. Staining was then visualized using the 3,3′-diaminobenzidine (DAB) detection kit of the Dako Real EnVision Detection System, Peroxidase/DAB+, Rabbit/Mouse (DakoCytomation, Denmark). The 1:150 dilution was found to be optimal when compared to 1:50, 1:100, and 1:200. A negative control was prepared using PBS instead of PLCG1 antibody.
Two pathologists blinded to the treatment groups scored the slides. Staining for PLCG1 expression was observed in the cellular nucleus and cytoplasm. The PLCG1 expression index was determined based on the proportion of stained cells using a semi-quantitative scale. The PLCG1 positive grade was determined using the immunoreactive scoring system, from which we obtained a proportion score (PS) and an intensity score (IS). The PS score is the percentage ratio of positive PLCG1-stained tumour cells to the total number of tumour cells, classified as: 0 (0%), 1 (1–10%), 2 (11–50%), 3 (51–80%), 4 (>80%). The IS score is the staining intensity by visual assessment, scored as: 0 (negative), 1+ (weak), 2+ (moderate), and 3+ (strong). The final PLCG1 expression score was calculated from the values of PS and IS (score = PS × IS), with a range of 0–12. We defined PLCG1 expression as follows: low PLCG1 expression (including negative expression with score = 0 and positive expression with score = 1) and high PLCG1 expression (positive expression with score = 2–12) ( Fig. 1 ).
Statistical analysis
Overall survival was calculated from the date of randomization to the date of death, DFS from the date of randomization to recurrence, LRFS from the date of randomization to loco-regional recurrence, and DMFS from the date of randomization to distant metastasis or death from any cause. For the descriptive analysis, categorical data were expressed as the number and percentage. The χ 2 test was applied to compare the difference between baseline factors and PLCG1 expression. The survival analysis was conducted using the Kaplan–Meier method and log-rank test. Hazard ratios (HR) were calculated using the Cox proportional hazards model. The intention-to-treat principle was applied for efficacy analysis. All hypothesis-generating tests were two-sided at a significance level of 0.05. Data were analysed with SPSS 13.0 for Windows software (SPSS Inc., Chicago, IL, USA).
Materials and methods
Patients
This study was based on patients who were enrolled in a prospective, randomized, phase 3 trial aimed at testing the hypothesis that TPF induction chemotherapy administered prior to surgery and postoperative radiotherapy would improve survival over surgery upfront in patients with locally advanced and resectable OSCC (ClinicalTrials.gov, NCT01542931 ); the trial was approved by the Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine.
Patients were enrolled into the clinical trial after signing an informed consent form. Details of the clinical trial have been described previously. Briefly, the main eligibility criteria included resectable squamous cell carcinoma of the oral cavity, clinical stage III or IVA, Karnofsky performance status >60%, and adequate haematological, hepatic, and renal function. Eligible patients were allocated randomly to the control arm (surgery followed by postoperative radiotherapy) or experimental arm (TPF induction chemotherapy followed by surgery and postoperative radiotherapy). Induction TPF consisted of docetaxel 75 mg/m 2 intravenously on day 1, followed by cisplatin 75 mg/m 2 intravenously on day 1, followed by 5-fluorouracil 750 mg/m 2 /day as a 120-h continuous intravenous infusion on days 1 through 5, every 3 weeks for 2 cycles. Surgery was performed at least 2 weeks after completion of the induction chemotherapy, and consisted of radical resection of the primary lesion and full neck dissection with appropriate reconstruction. Postoperative radiotherapy was initiated 4–6 weeks after surgery, with a total dose of 54–66 Gy.
The clinical tumour response was determined by clinical evaluation and imaging studies (performed at baseline and 2 weeks after cycle 2 of induction chemotherapy). Responses were characterized according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.0. The pathological response to TPF induction chemotherapy was assessed by examination of at least 20 slides of the resected specimen. A favourable response was defined as the absence of any tumour cells (pathological complete response) or the presence of scattered foci of a few tumour cells (minimal residual disease with <10% viable tumour cells).
After treatment, patients were monitored every 3 months in the first 2 years, every 6 months in the subsequent 3–5 years, and once a year thereafter until death or data censoring.
Detection of PLCG1 expression using immunohistochemistry
Pretreatment formalin-fixed and paraffin-embedded biopsy samples were collected for the detection of PLCG1 expression; however, in the control arm, if a pretreatment biopsy was unavailable, a portion of the surgical resection specimen was collected for biomarker evaluation. Sections 4 μm thick were studied using both haematoxylin and eosin (H&E) staining (for diagnostic confirmation, in accordance with the World Health Organization (WHO) histological criteria) and immunohistochemical staining for PLCG1. Immunohistochemical staining was performed as described previously. In brief, after deparaffinization, endogenous peroxidase was blocked and the sections were heated in a water bath at 98 °C with 0.01 M citrate buffer solution (pH 6.0) for 20 min to retrieve antigen. These were then cooled at room temperature and washed with phosphate buffered saline (PBS) three times for 5 min each, then incubated with the rabbit monoclonal antibody to PLCG1 (product code #5690, PLCG1 (D9H10) XP Rabbit mAb; Cell Signaling Technology, Inc., USA) at 1:150 dilution overnight at 4 °C. After recovering to room temperature for 1 h, the sections were washed with PBS three times for 5 min each. Staining was then visualized using the 3,3′-diaminobenzidine (DAB) detection kit of the Dako Real EnVision Detection System, Peroxidase/DAB+, Rabbit/Mouse (DakoCytomation, Denmark). The 1:150 dilution was found to be optimal when compared to 1:50, 1:100, and 1:200. A negative control was prepared using PBS instead of PLCG1 antibody.
Two pathologists blinded to the treatment groups scored the slides. Staining for PLCG1 expression was observed in the cellular nucleus and cytoplasm. The PLCG1 expression index was determined based on the proportion of stained cells using a semi-quantitative scale. The PLCG1 positive grade was determined using the immunoreactive scoring system, from which we obtained a proportion score (PS) and an intensity score (IS). The PS score is the percentage ratio of positive PLCG1-stained tumour cells to the total number of tumour cells, classified as: 0 (0%), 1 (1–10%), 2 (11–50%), 3 (51–80%), 4 (>80%). The IS score is the staining intensity by visual assessment, scored as: 0 (negative), 1+ (weak), 2+ (moderate), and 3+ (strong). The final PLCG1 expression score was calculated from the values of PS and IS (score = PS × IS), with a range of 0–12. We defined PLCG1 expression as follows: low PLCG1 expression (including negative expression with score = 0 and positive expression with score = 1) and high PLCG1 expression (positive expression with score = 2–12) ( Fig. 1 ).
Statistical analysis
Overall survival was calculated from the date of randomization to the date of death, DFS from the date of randomization to recurrence, LRFS from the date of randomization to loco-regional recurrence, and DMFS from the date of randomization to distant metastasis or death from any cause. For the descriptive analysis, categorical data were expressed as the number and percentage. The χ 2 test was applied to compare the difference between baseline factors and PLCG1 expression. The survival analysis was conducted using the Kaplan–Meier method and log-rank test. Hazard ratios (HR) were calculated using the Cox proportional hazards model. The intention-to-treat principle was applied for efficacy analysis. All hypothesis-generating tests were two-sided at a significance level of 0.05. Data were analysed with SPSS 13.0 for Windows software (SPSS Inc., Chicago, IL, USA).
Results
Patient characteristics and treatment outcomes
From March 2008 to December 2010, 256 eligible patients were enrolled in this study (128 patients in each arm), and 232 patients (91%, 126 patients in the control arm, 106 patients in the experimental arm) were assessed for pretreatment PLCG1 expression levels in the tumour. The distribution of baseline characteristics in the subset of patients who had biomarker evaluation was similar to the distribution in the entire trial population. Up to June 2013, three patients were lost to follow-up; the median follow-up time was 49 months among the censored patients.
Among the 232 patients, there was no significant difference in overall survival, DFS, LRFS, or DMFS between the experimental and control arms ( Fig. 2 ). The 3-year overall survival rate was 62.4% in the control arm and 69.5% in the experimental arm; the 3-year DFS rates were 54.5% and 62.2%, respectively. The loco-regional recurrence rate and distant metastasis rate in the control arm were 39.7% (50/126) and 11.1% (14/126), respectively, and in the experimental arm, the loco-regional recurrence rate and distant metastasis rate were 30.2% (32/106) and 4.7% (5/106), respectively.
PLCG1 expression and baseline characteristics
There were 65 samples (28 in the control arm and 37 in the experimental arm) with low PLCG1 expression, and 167 samples (98 in the control arm and 69 in the experimental arm) with high PLCG1 expression. The distribution pattern of PLCG1 expression was unbalanced between the two arms ( χ 2 test = 4.592, P = 0.032); the percentage of low PLCG1 expression was lower in the control arm than in the experimental arm. There were no significant differences in PLCG1 expression according to gender, age, primary tumour site, stage, grade, or tobacco use; the exception was alcohol use ( Table 1 ).
| Characteristics | Total patients ( N = 256), n (%) | PLCG1 expression | P -value a | |
|---|---|---|---|---|
| Low ( n = 65), n (%) | High ( n = 167), n (%) | |||
| Gender | ||||
| Male | 179 (69.9) | 40 (61.5) | 119 (71.3) | 0.145 |
| Female | 77 (30.1) | 25 (38.5) | 48 (28.7) | |
| Age, years | ||||
| <60 | 168 (65.6) | 44 (67.7) | 112 (67.1) | 0.949 |
| ≥60 | 88 (34.4) | 21 (32.3) | 55 (32.9) | |
| Site | ||||
| Tongue | 113 (44.1) | 25 (38.5) | 75 (44.9) | 0.447 |
| Not tongue | 143 (55.9) | 40 (61.5) | 92 (55.1) | |
| Clinical T descriptor | ||||
| T1/T2 | 66 (25.8) | 12 (18.5) | 50 (29.9) | 0.074 |
| T3/T4 | 190 (74.2) | 53 (81.5) | 117 (70.1) | |
| Clinical N descriptor | ||||
| N0 + 1 | 110 (43.0) | 34 (52.3) | 67 (40.1) | 0.059 |
| N2 | 146 (57.0) | 31 (47.7) | 100 (59.9) | |
| Clinical stage | ||||
| III | 177 (69.1) | 48 (73.8) | 113 (67.7) | 0.258 |
| IVA | 79 (30.9) | 17 (26.2) | 54 (32.3) | |
| Pathologic differentiation | ||||
| Well | 80 (31.2) | 24 (36.9) | 40 (24.0) | 0.075 |
| Moderately/poorly | 176 (68.8) | 41 (63.1) | 127 (76.0) | |
| Smoking status b | ||||
| Current/former | 126 (49.2) | 28 (43.1) | 81 (48.5) | 0.384 |
| Never | 130 (50.8) | 37 (56.9) | 86 (51.5) | |
| Alcohol use c | ||||
| Positive | 98 (40.6) | 18 (27.7) | 69 (41.3) | 0.044 |
| Negative | 158 (59.4) | 47 (72.3) | 98 (58.7) | |
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