Annette M. Lim

Relationship between Epidermal Growth Factor Receptor Status, p16 INK4A , and Outcome in Head and Neck Squamous Cell Carcinoma

Background: Human papilloma virus (HPV) infection is a powerful prognostic biomarker in head and neck squamous cell carcinoma (HNSCC). Increased epidermal growth factor receptor (EGFR) gene copy number and protein expression have been reported to be negative predictors of outcome. This study examined the relationship between HPV status, EGFR gene copy number, EGFR protein expression, and clinical outcome in HNSCC patients treated with chemoradiation. Methods: HPV status was determined using p16INK4A immunohistochemistry (IHC), EGFR gene copy number was evaluated with FISH, and EGFR protein expression by IHC in 212 subjects. Results:EGFR FISH was positive in 41 of 204 (20%) patients and was negatively correlated with failure-free survival (FFS; HR = 1.84, P = 0.027) and overall survival (OS; HR = 1.78, P = 0.082). For p16INK4A, 85 of 200 (42.5%) patients were found to be p16 positive, including 75 of 131 (57%) with oropharyngeal cancer. Patients with p16-positive oropharyngeal cancer had significantly improved FFS (HR = 0.28, P < 0.001) and OS (HR = 0.31, P = 0.002). Only 2 of 126 (1.6%) oropharyngeal cancer patients were found to be p16+/EGFR FISH+. EGFR IHC was positive in 81 of 93 (87%) of patients and was associated with poorer FFS (HR = 1.98, P = 0.35) and OS (HR = 2.52, P = 0.22). Conclusions: Increased EGFR gene copy number is largely restricted to p16INK4A-negative oropharyngeal cancer. Although p16INK4A and EGFR FISH are both predictive of outcome in univariate analyses, only p16INK4A remains independently predictive. Impact: Knowledge of HPV and EGFR status can have implications for treatment options and prognosis in HNSCC. Cancer Epidemiol Biomarkers Prev; 20(6); 1230–7. ©2011 AACR.

Prognostic Significance of Plasma Osteopontin in Patients with Locoregionally Advanced Head and Neck Squamous Cell Carcinoma Treated on TROG 02.02 Phase III Trial

Purpose: High plasma osteopontin (OPN) levels have been reported to be an adverse prognostic factor in head and neck squamous cell carcinomas (HNSCC), correlate with tumor hypoxia, and be predictive of benefit from hypoxia-targeted therapy. We sought to confirm the prognostic and predictive significance of OPN in patients treated on a large international trial. Experimental Design: Patients with stage III/IV HNSCC were randomized to receive definitive radiotherapy concurrently with cisplatin or cisplatin plus the hypoxic cell cytotoxin, tirapazamine (TPZ). Eligibility criteria for this prospective substudy included plasma sample availability for OPN assay by ELISA and absence of major radiation therapy deviations (N = 578). OPN concentrations were analyzed for overall survival (OS) and time to locoregional failure (TTLRF), adjusting for known prognostic factors. Additional analysis was carried out in patients with available tumor p16INK4A staining status. Results: The median OPN level was 544 ng/mL (range: 7–2,640). High OPN levels were not associated with worse OS (relative HR, 1.03 for highest tertile) or TTLRF (relative HR 0.91 for highest tertile). There was no interaction between OPN and treatment arm for OS or TTLRF (P = 0.93 for OS; P = 0.87 for TTLRF). For the highest tertile the 2-year OS was 66% on control arm and 67% on TPZ arm (HR = 1.11, P = 0.67). Similarly for p16INK4A negative patients in the highest tertile, the 2-year OS was 61% on control arm and 63% on TPZ arm (HR = 1.05, P = 0.86). Conclusions: We found no evidence that high plasma OPN levels were associated with an adverse prognosis in HNSCC, or were predictive of benefit with hypoxia targeting therapy. Clin Cancer Res; 18(1); 301–7. ©2011 AACR.

Differential mechanisms of CDKN2A (p16) alteration in oral tongue squamous cell carcinomas and correlation with patient outcome.

CDKN2A (p16) disruption is reported as a frequent event in head and neck squamous cell carcinomas that confers poor prognosis. We investigated the frequency of different potential mechanisms of CDKN2A inactivation in oral tongue squamous cell carcinomas (OTSCC) and their impact on patient outcome. From a cohort of 153 OTSCC patients, 131 formalin fixed paraffin embedded blocks of pre‐treatment primary tumours were suitable for further molecular analysis. We assessed CDKN2A (p16) levels by immunohistochemistry (IHC), promoter methylation status by methylation‐sensitive high resolution melting, mutation status by Sanger sequencing, gene copy number variation by fluorescence in situ hybridisation, and correlated these with patient outcome. We found that the majority of OTSCC did not overexpress p16 (110/116, 95%), assessed by IHC. The frequency of CDKN2A mutations was 20% (21/103), homozygous loss was 7% (7/97), hemizygous loss 31% (30/97), and promoter methylation was 18% (20/113). We found no evidence of these mechanisms in 24/106 (23%) p16 IHC negative tumours. No significant correlation was identified between any potential mechanism of CDKN2A inactivation and clinical features, including smoking status and age. There was a non‐significant trend for worse overall survival for p16 IHC negative patients versus positive patients (HR = 1.81, 95% CI = 0.44–7.47, p = 0.40). No relationship was found between mechanisms of CDKN2A disruption and patient outcome. In conclusion, we demonstrate that CDKN2A alteration is a frequent event in OTSCC tumourigenesis. However, no correlation was identified between different potential mechanisms of CDKN2A disruption and clinical characteristics or patient outcome.

Frequency of Fibroblast Growth Factor Receptor 1 gene amplification in oral tongue squamous cell carcinomas and associations with clinical features and patient outcome

OBJECTIVES Novel therapies are required for patients with recurrent or metastatic oral tongue squamous cell carcinoma (OTSCC). Fibroblast Growth Factor Receptor 1 (FGFR1) amplification frequently occurs in squamous cell carcinoma of the lung and represents a novel druggable therapeutic target in this and other malignancies. This study examined the frequency and clinical associations of FGFR1 amplification in OTSCC. MATERIALS AND METHODS The frequency of FGFR1 amplification determined by fluorescence in situ hybridization was evaluated in a cohort of 123 OTSCC patients. Associations of FGFR1 amplification with clinical characteristics and outcome were determined. RESULTS FGFR1 gene amplification was present in 9.3% (10/107) of cases and was significantly associated with smoking status (P = 0.03). FGFR1 amplification was seen more commonly in males (9/10 amplified cases male, P = 0.16) and there were no associations with age, stage, T stage, nodal status, alcohol history or performance status (all P>0.05). Outcome was not significantly different between FGFR1 amplified and non-amplified patients. CONCLUSIONS Copy number variations of the FGFR1 gene occur in a subset of OTSCC with approximately 10% of cases showing amplification of the gene. FGFR1 amplification may represent a therapeutic target in OTSCC.

A phase II study of induction carboplatin and gemcitabine followed by chemoradiotherapy for the treatment of locally advanced nasopharyngeal carcinoma

OBJECTIVES To investigate the tolerability and feasibility of induction gemcitabine and carboplatin chemotherapy followed by radiotherapy with concurrent cisplatin in patients with locally advanced nasopharyngeal carcinoma. PATIENTS AND METHODS Twenty-eight patients with previously untreated non-keratinising nasopharyngeal carcinoma, with stage IIb to IV disease were enroled to receive three cycles of carboplatin AUC 5 and gemcitabine 1 g/m(2) day 1 and 8 every 21-days, followed by 70 Gy of radiotherapy with concurrent cisplatin 20 mg/m(2)/day for 5 days of weeks 1, 4 and 7. RESULTS 26/28 (93.0%) patients received all three cycles of induction chemotherapy. All 27 patients who commenced chemoradiotherapy received 70 Gy in 35 fractions of radiotherapy with at least two cycles of concurrent cisplatin. The three-year time to locoregional failure rate was 92.9% (95% CI: 75.5-98.2%) and the three-year overall survival rate was 89.3% (95% CI: 71.6-96.5%). Induction chemotherapy was well tolerated with 5/28 (17.9%) patients experiencing grade 3 non-haematological toxicities and no reported episodes of febrile neutropenia or grade 4 toxicity. For the 27 patients who received radiotherapy, no acute grade 4 radiation toxicities and only 2/27 (7.4%) late grade 4 radiation adverse events were observed. CONCLUSION The use of induction carboplatin and gemcitabine followed by chemoradiotherapy is feasible, with acceptable toxicity, and is a promising regimen for the treatment of locally advanced nasopharyngeal carcinoma.

Quantitative methodology is critical for assessing DNA methylation and impacts on correlation with patient outcome

BackgroundDNA hypermethylation is reported as a frequent event and prognostic marker in head and neck squamous cell carcinomas (HNSCC). Methylation has been commonly assessed with non-quantitative methodologies, such as methylation-specific PCR (MSP). We investigated previously reported hypermethylated genes with quantitative methodology in oral tongue squamous cell carcinomas (OTSCC).ResultsThe methylation status of 12 genes in 115 OTSCC samples was assessed by one or more of three quantitative analyses: methylation sensitive high resolution melting (MS-HRM), sensitive-melting analysis after real time-methylation specific PCR (SMART-MSP), and bisulfite pyrosequencing.In contrast to much of the literature, either no or infrequent locus-specific methylation was identified by MS-HRM for DAPK1, RASSF1A, MGMT, MLH1, APC, CDH1, CDH13, BRCA1, ERCC1, and ATM. The most frequently methylated loci were RUNX3 (18/108 methylated) and ABO (22/107 methylated). Interrogation of the Cancer Genome Atlas (TCGA) HNSCC cohort confirmed the frequency of significant methylation for the loci investigated.Heterogeneous methylation of RUNX3 (18/108) and ABO (22/107) detected by MS-HRM, conferred significantly worse survival (P = 0.01, and P = 0.03). However, following quantification of methylation levels using pyrosequencing, only four tumors had significant quantities (>15%) of RUNX3 methylation which correlated with a worse patient outcome (P <0.001), while the prognostic significance of ABO hypermethylation was lost. RUNX3 methylation was not prognostic for the TCGA cohort (P = 0.76).ConclusionsWe demonstrated the critical need for quantification of methylation levels and its impact on correlative analyses. In OTSCC, we found little evidence of significant or frequent hypermethylation of many loci reported to be commonly methylated. It is likely that previous reports have overestimated the frequency of significant methylation events as a consequence of the use of non-quantitative methodology.

Prolonged Remission in a Patient With Nasopharyngeal Carcinoma With a Solitary Bone Metastasis

A 24-year-old man presented with a 2-month history of a painlessleftnecklump.Clinicalexaminationrevealeddiffusethickeningof the left neck without a discernable mass and a single right-sided 1.5-cm jugulodigastric node, but no abnormalities elsewhere. Nasendoscopy demonstrated an ulcerated tumor involving bilateral posterior nasopharyngeal walls and roof, blocking the left Eustachian cushion and abutting the right Eustachian cushion. Biopsy of the primary confirmed the diagnosis of nonkeratinising nasopharyngeal carcinoma(NPC),WHOgrade2.Thepatientdeniedanyothersymptomsonfurtherquestioning.HewasofChinese-Malaysianoriginand had lived in Australia from the age of 8 years. He had no significant past medical history, was on no medications, and had no family historyofNPC.Computedtomography(CT)oftheneckconfirmeda nasopharyngeal lesion involving the left torus tuberous, multiple leftsided necrotic lymph nodes and a solitary posterior right triangle lymph node. Chest and abdominal CT did not demonstrate disease elsewhere. He was initially staged as T2 N2 WHO grade 2 NPC. To complete his staging, fused positron emission tomography (PET)/CT was organized, which revealed metabolic avidity in the primary nasopharyngeal lesion (Fig 1A, baseline fused PET/CT axial image of nasopharynx),bilateralcervicalnecknodesasdescribed(Fig1B,baseline

Correlation of Ataxia-Telangiectasia-Mutated (ATM) gene loss with outcome in head and neck squamous cell carcinoma.

OBJECTIVES Ataxia-Telangiectasia-Mutated (ATM) gene loss has been associated with poor prognosis and treatment resistance in head and neck squamous cell carcinomas (HNSCC). We investigated the relationship between ATM loss detected by fluorescence in-situ hybridisation (FISH) with patient outcome, and its relationship with Human Papillomavirus (HPV)/p16(INK4A) status. MATERIAL AND METHODS Copy number of the ATM gene and chromosome 11 were determined by FISH and HPV status was determined using p16(INK4A) immunohistochemistry in 87 paraffin embedded tumour samples from patients with HNSCC treated with chemoradiation at a single institution. ATM loss was correlated with patient outcome as both a continuous and dichotomous variable. RESULTS Of 73 evaluable patients, 44 (60.3%) demonstrated loss of the ATM gene. There was no correlation between ATM loss (defined as a mean ratio of ATM: chromosome 11<0.75) and overall survival (OS, p=0.67) or time to locoregional failure (TTLRF, p=0.72). Similarly, when evaluated as a continuous variable there was no significant relationship between ATM loss and patient outcome (OS, p=0.89; TTLRF, p=0.21). No significant relationship was found between p16(INK4A) status and ATM loss, for patient outcome. We found 35.6% (n=26) of patients demonstrated polysomy of chromosome 11 (defined as the presence of a mean >2.5 copies of chromosome 11) which was significantly associated with p16(INK4A) negative status (p=0.0004), but did not influence outcome. CONCLUSIONS ATM loss is a frequent event in HNSCC, however it does not impact outcome after treatment with chemoradiation. Polysomy of chromosome 11 was significantly associated with p16(INK4A) negative status but also lacks prognostic significance.

Weekly cisplatin and radiotherapy for low risk, locoregionally advanced human papillomavirus–positive oropharyngeal squamous cell carcinoma

There is interest in different treatment strategies, including deintensification in good prognosis human papillomavirus‐positive (HPV(+)) oropharyngeal squamous cell carcinoma (SCC). We reviewed our experience with weekly cisplatin in low‐risk, locoregionally advanced HPV(+) oropharyngeal SCC since late 2009.Background There is interest in different treatment strategies, including deintensification in good prognosis human papillomavirus-positive (HPV(+)) oropharyngeal squamous cell carcinoma (SCC). We reviewed our experience with weekly cisplatin in low-risk, locoregionally advanced HPV(+) oropharyngeal SCC since late 2009. Methods Data from patients with low-risk HPV(+) oropharyngeal SCC treated with weekly cisplatin (40 mg/m2) and 70 Gy radiotherapy were collected. Low risk was defined as stage III to IV oropharyngeal SCC excluding T1-2N1, T4 or N3 disease, or N2b to N2c disease in patients with >10 pack-year smoking history. Results Of 31 patients, the median age was 56 years (range, 41–69 years). All patients completed 70 Gy radiotherapy within 51 days and 84% completed at least 5 cycles of cisplatin. Grade 3 mucositis occurred in 22 patients (71%) and grade 3 febrile neutropenia in 6 patients (19%). No patients required enteral feeding at 12 months. The median follow-up was 30 months (range, 21–57 months) with no recurrences or deaths. Conclusion Concurrent weekly cisplatin is relatively well-tolerated and associated with excellent disease control in low-risk, locoregionally advanced HPV(+) oropharyngeal SCC.

PD-1 Blockade with Cemiplimab in Advanced Cutaneous Squamous-Cell Carcinoma

Background No systemic therapies have been approved for the treatment of advanced cutaneous squamous‐cell carcinoma. This cancer may be responsive to immune therapy, because the mutation burden of the tumor is high and the disease risk is strongly associated with immunosuppression. In the dose‐escalation portion of the phase 1 study of cemiplimab, a deep and durable response was observed in a patient with metastatic cutaneous squamous‐cell carcinoma. Methods We report the results of the phase 1 study of cemiplimab for expansion cohorts of patients with locally advanced or metastatic cutaneous squamous‐cell carcinoma, as well as the results of the pivotal phase 2 study for a cohort of patients with metastatic disease (metastatic‐disease cohort). In both studies, the patients received an intravenous dose of cemiplimab (3 mg per kilogram of body weight) every 2 weeks and were assessed for a response every 8 weeks. In the phase 2 study, the primary end point was the response rate, as assessed by independent central review. Results In the expansion cohorts of the phase 1 study, a response to cemiplimab was observed in 13 of 26 patients (50%; 95% confidence interval [CI], 30 to 70). In the metastatic‐disease cohort of the phase 2 study, a response was observed in 28 of 59 patients (47%; 95% CI, 34 to 61). The median follow‐up was 7.9 months in the metastatic‐disease cohort of the phase 2 study. Among the 28 patients who had a response, the duration of response exceeded 6 months in 57%, and 82% continued to have a response and to receive cemiplimab at the time of data cutoff. Adverse events that occurred in at least 15% of the patients in the metastatic‐disease cohort of the phase 2 study were diarrhea, fatigue, nausea, constipation, and rash; 7% of the patients discontinued treatment because of an adverse event. Conclusions Among patients with advanced cutaneous squamous‐cell carcinoma, cemiplimab induced a response in approximately half the patients and was associated with adverse events that usually occur with immune checkpoint inhibitors. (Funded by Regeneron Pharmaceuticals and Sanofi; ClinicalTrials.gov numbers, NCT02383212 and NCT02760498.)