Richard J. Young

Prognostic Significance of p16INK4A and Human Papillomavirus in Patients With Oropharyngeal Cancer Treated on TROG 02.02 Phase III Trial

PURPOSE To determine the prognostic importance of p16 and human papillomavirus (HPV) in patients with oropharyngeal cancer treated on a phase III concurrent chemoradiotherapy trial. PATIENTS AND METHODS Patients with stage III or IV head and neck squamous cell cancer were randomly assigned to concurrent radiotherapy and cisplatin with or without tirapazamine. In this substudy, analyses were restricted to patients with oropharyngeal cancer. p16 was detected by immunohistochemistry, and HPV was detected by in situ hybridization and polymerase chain reaction. RESULTS Slides were available for p16 assay in 206 of 465 patients, of which 185 were eligible, and p16 and HPV were evaluable in 172 patients. One hundred six (57%) of 185 were p16-positive, and in patients evaluable for both p16 and HPV, 88 (86%) of 102 p16-positive patients were also HPV-positive. Patients who were p16-positive had lower T and higher N categories and better Eastern Cooperative Oncology Group (ECOG) performance status. p16-positive tumors compared with p16-negative tumors were associated with better 2-year overall survival (91% v 74%; hazard ratio [HR], 0.36; 95% CI, 0.17 to 0.74; P = .004) and failure-free survival (87% v 72%; HR, 0.39; 95% CI, 0.20 to 0.74; P = .003). p16 was a significant prognostic factor on multivariable analysis (HR, 0.45; 95% CI, 0.21 to 0.96; P = .04). p16-positive patients had lower rates of locoregional failure and deaths due to other causes. There was a trend favoring the tirapazamine arm for improved locoregional control in p16-negative patients (HR, 0.33; 95% CI, 0.09 to 1.24; P = .13). CONCLUSION HPV-associated oropharyngeal cancer is a distinct entity with a favorable prognosis compared with HPV-negative oropharyngeal cancer when treated with cisplatin-based chemoradiotherapy.

Loss of CDKN2A expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the CDK4/6 inhibitor PD0332991 in melanoma cell lines

We have investigated the potential for the p16‐cyclin D‐CDK4/6‐retinoblastoma protein pathway to be exploited as a therapeutic target in melanoma. In a cohort of 143 patients with primary invasive melanoma, we used fluorescence in situ hybridization to detect gene copy number variations (CNVs) in CDK4, CCND1, and CDKN2A and immunohistochemistry to determine protein expression. CNVs were common in melanoma, with gain of CDK4 or CCND1 in 37 and 18% of cases, respectively, and hemizygous or homozygous loss of CDKN2A in 56%. Three‐quarters of all patients demonstrated a CNV in at least one of the three genes. The combination of CCND1 gain with either a gain of CDK4 and/or loss of CDKN2A was associated with poorer melanoma‐specific survival. In 47 melanoma cell lines homozygous loss, methylation or mutation of CDKN2A gene or loss of protein (p16INK4A) predicted sensitivity to the CDK4/6 inhibitor PD0332991, while RB1 loss predicted resistance.

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 and Predictive Significance of Plasma HGF and IL-8 in a Phase III Trial of Chemoradiation with or without Tirapazamine in Locoregionally Advanced Head and Neck Cancer

Purpose: Hepatocyte growth factor (HGF) is a hypoxia-induced secreted protein that binds to cMet and regulates interleukin (IL)-8 expression. We evaluated the role of circulating HGF and IL-8 as prognostic and predictive factors for efficacy of tirapazamine (TPZ), a hypoxic cell cytotoxin. Experimental Design: Patients with stages III to IV head and neck cancer were randomized to receive radiotherapy with cisplatin (CIS) or CIS plus TPZ (TPZ/CIS). Eligibility for the substudy included plasma sample availability for HGF and IL-8 assay by ELISA and no major radiation deviations (N = 498). Analyses included adjustment for major prognostic factors. p16INK4A staining (human papillomavirus surrogate) was carried out on available tumors. Thirty-nine patients had hypoxia imaging with 18F-fluoroazomycin arabinoside (18FAZA)–positron emission tomography. Results: Elevated IL-8 level was associated with worse overall survival (OS) irrespective of treatment. There was an interaction between HGF and treatment arm (P = 0.053); elevated HGF was associated with worse OS in the control but not in the TPZ/CIS arm. Similar trends were observed in analyses restricted to p16INK4A-negative patients. Four subgroups defined by high and low HGF/IL-8 levels were examined for TPZ effect; the test for interaction with arm was P = 0.099. TPZ/CIS seemed to be beneficial for patients with high HGF and IL-8 but adverse for low HGF and high IL-8. Only HGF correlated with 18FAZA tumor standard uptake value. Conclusions: IL-8 is an independent prognostic factor irrespective of treatment. There is an interaction between HGF and treatment arm. Certain subgroups based on IL-8/HGF levels seemed to do better with TPZ/CIS while others did worse, highlighting the complexity of hypoxia targeting in unselected patients. Clin Cancer Res; 18(6); 1798–807. ©2012 AACR.

Glomerulomegaly in Australian Aborigines

Summary: Idiopathic glomerular enlargement has previously been described in a number of indigenous populations, including Australian Aborigines. This study had three aims: (1) evaluate three methods for estimating mean glomerular tuft and renal corpuscle volume; (2) assess the effects of fixation on glomerular dimensions; and (3) estimate glomerular tuft and renal corpuscle volume in clinical biopsies from Australian non‐Aboriginals, Aboriginals and Aboriginal inhabitants of the Tiwi Islands (Bathurst Island and Melville Island, Northern Territory, Australia). First, glomerular volume was determined in initial transplant biopsies from 17 non‐Aboriginal males (30–50 years) using three methods: the Cavalieri method, a stereological method that requires serial sectioning of glomeruli and knowledge of section thickness, but requires no knowledge or assumptions of glomerular size or shape (the gold‐standard method); the stereological method of Weibel and Gomez that employs a single section but requires assumptions of glomerular size distribution and shape; and the maximal profile method, with which the largest glomerular profile in a single section is identified, and used to calculate the volume of the parent glomerulus (assuming glomerular sphericity). Estimates for glomerular tuft volume were (mean ± SD): Cavalieri method (2.08 ± 0.37 × 106μm3); Weibel and Gomez (2.55 ± 0.63 × 106μ3); maximal profile method (3.09 + 0.66106μ3). Taking the Cavalieri estimate to be accurate, the maximal profile method is seen to grossly overestimate mean glomerular tuft volume, whereas the Weibel and Gomez method overestimated tuft volume by 23%. Both methods considerably overestimated mean renal corpuscle volume. In the study of fixation and glomerular dimensions, we found that glomeruli in clinical biopsies fixed in formalin were larger (47% for glomerular tuft and 25% for renal corpuscle) than the glomeruli in biopsies fixed in formol mercury/Dubosq Brazil. This result emphasizes the importance of standardizing the histological technique in quantitative studies of glomeruli. Finally, the Weibel and Gomez method was used to estimate mean glomerular volume in formalin‐fixed clinical biopsies from 80 non‐Aboriginal Australians, 78 non‐Tiwi Aboriginals and 72 Tiwi Aboriginals. Mean glomerular tuft volumes were: 3.12 ± 1.46 × 106μm3, 4.91 ± 2.59 × 106μm3 and 4.79 ± 2.08 × 106μm3, respectively, (for biopsies with four or more profiles). Mean glomerular tuft volume in the two Aboriginal populations was significantly (P < 0.001 in each case) greater than that in the non‐Aboriginals. These data indicate that there is pronounced glomerulomegaly in Australian Aborigines.

The transcription cofactor c-JUN mediates phenotype switching and BRAF inhibitor resistance in melanoma

The transcriptional regulator c-JUN is a key mediator of the metastatic potential and drug resistance in melanoma. Inhibiting two MAPKs is better than one Mitogen-activated protein kinase (MAPK) pathways, such as the ERK and JNK pathways, mediate critical cellular processes, such as survival, stress responses, and proliferation. These pathways can be hijacked by cancer cells, leading to uncontrolled cell division and metastasis. Many melanoma patients have activating mutations in an upstream kinase, BRAF, in the ERK pathway, but inhibitors of BRAF only produce short-term improvement. Using panels of melanoma cell lines and BRAF inhibitor–treated patient samples, Ramsdale et al. found that increased abundance of the transcription cofactor c-JUN, which is activated by the JNK pathway, mediated both inherent and adaptive resistance to BRAF inhibitors and contributed to metastatic potential. Blocking c-JUN abundance or its activation by the kinase JNK enhanced the efficacy of BRAF inhibitors against melanoma cells. Thus, targeting both MAPK pathways may overcome resistance to treatment with only inhibitors of the ERK pathway. Most patients with BRAF-mutant metastatic melanoma display remarkable but incomplete and short-lived responses to inhibitors of the BRAF kinase or the mitogen-activated protein kinase kinase (MEK), collectively BRAF/MEK inhibitors. We found that inherent resistance to these agents in BRAFV600-mutant melanoma cell lines was associated with high abundance of c-JUN and characteristics of a mesenchymal-like phenotype. Early drug adaptation in drug-sensitive cell lines grown in culture or as xenografts, and in patient samples during therapy, was consistently characterized by down-regulation of SPROUTY4 (a negative feedback regulator of receptor tyrosine kinases and the BRAF-MEK signaling pathway), increased expression of JUN and reduced expression of LEF1. This coincided with a switch in phenotype that resembled an epithelial-mesenchymal transition (EMT). In cultured cells, these BRAF inhibitor-induced changes were reversed upon removal of the drug. Knockdown of SPROUTY4 was sufficient to increase the abundance of c-JUN in the absence of drug treatment. Overexpressing c-JUN in drug-naïve melanoma cells induced similar EMT-like phenotypic changes to BRAF inhibitor treatment, whereas knocking down JUN abrogated the BRAF inhibitor-induced early adaptive changes associated with resistance and enhanced cell death. Combining the BRAF inhibitor with an inhibitor of c-JUN amino-terminal kinase (JNK) reduced c-JUN phosphorylation, decreased cell migration, and increased cell death in melanoma cells. Gene expression data from a panel of melanoma cell lines and a patient cohort showed that JUN expression correlated with a mesenchymal gene signature, implicating c-JUN as a key mediator of the mesenchymal-like phenotype associated with drug resistance.

Frequency and prognostic significance of p16INK4A protein overexpression and transcriptionally active human papillomavirus infection in laryngeal squamous cell carcinoma

Background:Human papillomavirus (HPV) infection is a powerful prognostic biomarker in a subset of head and neck squamous cell carcinomas, specifically oropharyngeal cancers. However, the role of HPV in non-oropharyngeal sites, such as the larynx, remains unconfirmed.Methods:We evaluated a cohort of 324 laryngeal squamous cell carcinoma (LSCC) patients for the expression of p16INK4A (p16) protein by immunohistochemistry (IHC) and for high-risk HPV E6 and E7 mRNA transcripts by RNA in situ hybridisation (ISH). p16 expression and HPV status were correlated with clinicopathological features and outcomes.Results:Of 307 patients assessable for p16 IHC, 20 (6.5%) were p16 positive. Females and node-positive patients were more likely to be p16 positive (P<0.05). There were no other significant clinical or demographic differences between p16-positive and -negative cases. There was no difference in overall survival (OS) between p16-positive and -negative patients with 2-year survival of 79% in each group (HR=0.83, 95% CI 0.36–1.89, P=0.65). There was no statistically significant difference in failure-free survival (FFS) with 2-year FFS of 79% and 66% for p16-positive and -negative patients, respectively (HR=0.60, 95% CI 0.26–1.36, P=0.22). Only seven cases were found to be HPV RNA ISH positive, all of which were p16 IHC positive. There was no statistically significant difference in OS between patients with HPV RNA ISH-positive tumours compared with -negative tumours with 2-year survival of 86% and 71%, respectively (HR=0.76, 95% CI 0.23–2.5, P=0.65). The 2-year FFS was 86% and 59%, respectively (HR=0.62, 95% CI 0.19–2.03, P=0.43).Conclusions:p16 overexpression is infrequent in LSCC and the proportion of cases with high-risk HPV transcripts is even lower. There are no statistically significant correlations between p16 IHC or HPV RNA ISH status and OS or disease outcomes.

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.

Differential (18)F-FDG and 3'-deoxy-3'-(18)F-fluorothymidine PET responses to pharmacologic inhibition of the c-MET receptor in preclinical tumor models.

The ability of PET to image functional changes in tumors is increasingly being used to evaluate response and predict clinical benefit to conventional and novel cancer therapies. Although the use of 18F-FDG PET is well established, 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) PET has potential advantages as a more specific marker of cellular proliferation. c-MET signaling is frequently dysregulated in cancer and is therefore an attractive therapeutic target. Crizotinib (PF-2341066) is a novel adenosine triphosphate–competitive c-MET kinase inhibitor with antitumor activity in a range of tumor models. The aim of this study was to investigate the utility of PET of glucose metabolism and cell proliferation to monitor tumor response to crizotinib in 2 cell lines with aberrant c-MET signaling. Methods: Mice bearing GTL-16 or U87MG xenografts were evaluated for changes in tumor volume and 18F-FDG and 18F-FLT uptake after daily oral treatment with up to 50 mg/kg crizotinib. GTL-16 and U87MG cells were treated with crizotinib in vitro and analyzed for 3H-2-deoxyglucose uptake and expression of activated MET, AKT, and ERK by immunoblotting. Results: Treatment of c-MET–amplified GTL-16 xenografts with 50 mg/kg crizotinib caused tumor regression that was associated with a slow reduction in 18F-FDG uptake (P < 0.05, day 13) and reduced expression of the glucose transporter 1, GLUT-1. Although baseline 18F-FDG uptake into U87MG tumors was substantially higher than in GTL-16 tumors, 18F-FDG uptake into U87MG tumors remained unchanged on treatment at 50 mg/kg crizotinib, despite tumor growth inhibition of 93% on day 8 of treatment. These findings were confirmed in vitro, where treatment of U87MG cells with 1 μM crizotinib had no demonstrable effect on glucose uptake. Furthermore, these cells demonstrated constitutive, crizotinib-independent phosphoinositide 3-kinase pathway signaling as demonstrated by phosphorylated AKT and ribosomal protein S6. Both U87MG and GTL-16 tumors showed high baseline uptake of 18F-FLT, which was reduced by 50% and 53% on days 4 and 8 of treatment, respectively. Conclusion: While the results provide a strong rationale to investigate the use of 18F-FLT PET as a clinical biomarker for monitoring tumor response to c-MET inhibition, 18F-FDG PET may be a less robust marker.