Alice Y. Chuang

A new American Joint Committee on Cancer staging system for cutaneous squamous cell carcinoma: Creation and rationale for inclusion of tumor (T) characteristics

BACKGROUNDnThe incidence of cutaneous squamous cell carcinoma (cSCC) is increasing. Although most patients achieve complete remission with surgical treatment, those with advanced disease have a poor prognosis. The American Joint Committee on Cancer (AJCC) is responsible for the staging criteria for all cancers. For the past 20 years, the AJCC cancer staging manual has grouped all nonmelanoma skin cancers, including cSCC, together for the purposes of staging. However, based on new evidence, the AJCC has determined that cSCC should have a separate staging system in the 7th edition AJCC staging manual.nnnOBJECTIVEnWe sought to present the rationale for and characteristics of the new AJCC staging system specific to cSCC tumor characteristics (T).nnnMETHODSnThe Nonmelanoma Skin Cancer Task Force of AJCC reviewed relevant data and reached expert consensus in creating the 7th edition AJCC staging system for cSCC. Emphasis was placed on prospectively accumulated data and multivariate analyses. Concordance with head and neck cancer staging system was also achieved.nnnRESULTSnA new AJCC cSCC T classification is presented. The T classification is determined by tumor diameter, invasion into cranial bone, and high-risk features, including anatomic location, tumor thickness and level, differentiation, and perineural invasion.nnnLIMITATIONSnThe data available for analysis are still suboptimal, with limited prospective outcomes trials and few multivariate analyses.nnnCONCLUSIONSnThe new AJCC staging system for cSCC incorporates tumor-specific (T) staging features and will encourage coordinated, consistent collection of data that will be the basis of improved prognostic systems in the future.

Presence of HPV DNA in convalescent salivary rinses is an adverse prognostic marker in head and neck squamous cell carcinoma.

Human papillomavirus (HPV) 16 is present in up to 60% of patients with head and neck squamous cell carcinoma (HNSCC) and confers a favorable prognosis in terms of recurrence and mortality. Previous reports demonstrated that HPV-16 DNA can be detected in the initial salivary rinses from these patients. In this study, we assessed the feasibility of post-treatment HPV DNA shed from the oral mucosa as a prognostic marker for persistent/recurrent head and neck cancer. Fresh tumor samples and pre- and post-treatment salivary rinses were collected from 59 patients with HNSCC. HPV-16 E6 and E7 DNA copy number in these samples were quantified by real time PCR. Twenty of 59 patients (33.9%) were HPV-16 positive in their tumors before treatment. Four of 20 HPV tumor positive patients ultimately developed recurrence, and two of these four patients were HPV-16 positive in surveillance salivary rinses (sensitivity=50%). Of the 39 (66.1%) HPV-16 negative patients on initial clinical presentation and the 16 HPV-16 positive patients who did not recur, none were HPV-16 positive in salivary rinses after treatment (specificity=100%). HPV-16 presence in follow-up salivary rinses preceded clinical detection of disease recurrence by an average of 3.5 months. Patients with presence of HPV-16 DNA in surveillance salivary rinses are at significant risk for recurrence. Quantitative measurement of salivary HPV-16 DNA has promise for surveillance and early detection of recurrence.

Phospho-ΔNp63α is a key regulator of the cisplatin-induced microRNAome in cancer cells.

Head and neck squamous cell carcinoma (HNSCC) cells exposed to cisplatin (CIS) displayed a dramatic ATM-dependent phosphorylation of ΔNp63α that leads to the transcriptional regulation of downstream mRNAs. Here, we report that phospho (p)-ΔNp63α transcriptionally deregulates miRNA expression after CIS treatment. Several p-ΔNp63α-dependent microRNA species (miRNAs) were deregulated in HNSCC cells upon CIS exposure, including miR-181a, miR-519a, and miR-374a (downregulated) and miR-630 (upregulated). Deregulation of miRNA expression led to subsequent modulation of mRNA expression of several targets (TP53-S46, HIPK2, ATM, CDKN1A and 1B, CASP3, PARP1 and 2, DDIT1 and 4, BCL2 and BCL2L2, TP73, YES1, and YAP1) that are involved in the apoptotic process. Our data support the notion that miRNAs are critical downstream targets of p-ΔNp63α and mediate key pathways implicated in the response of cancer cells to chemotherapeutic drugs.

A survey of methylated candidate tumor suppressor genes in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a rare malignancy with unique genetic, viral and environmental characteristic that distinguishes it from other head and neck carcinomas. The clinical management of NPC remains challenging largely due to the lack of early detection strategies for this tumor. In our study, we have sought to identify novel genes involved in the pathogenesis of NPC that might provide insight into this tumors biology and could potentially be used as biomarkers. To identify these genes, we studied the epigenetics of NPC by characterizing a panel of methylation markers. Eighteen genes were evaluated by quantitative methylation‐specific polymerase chain reaction (PCR) in cell lines as well as in tissue samples including 50 NPC tumors and 28 benign nasopharyngeal biopsies. Significance was evaluated using Fishers exact test and quantitative values were optimized using cut off values derived from receiver–operator characteristic curves. The methylation status of AIM1, APC, CALCA, deleted in colorectal carcinomas (DCC), DLEC, deleted in liver cancer 1 (DLC1), estrogen receptor alpha (ESR), FHIT, KIF1A and PGP9.5 was significantly associated with NPC compared to controls. The sensitivity of the individual genes ranged from 26 to 66% and the specificity was above 92% for all genes except FHIT. The combination of PGP9.5, KIF1A and DLEC had a sensitivity of 84% and a specificity of 92%. Ectopic expression of DCC and DLC1 lead to decrease in colony formation and invasion properties. Our results indicate that methylation of novel biomarkers in NPC could be used to enhance early detection approaches. Additionally, our functional studies reveal previously unknown tumor suppressor roles in NPC.

Quantitative Methylation Profiles for Multiple Tumor Suppressor Gene Promoters in Salivary Gland Tumors

Background Methylation profiling of tumor suppressor gene (TSGs) promoters is quickly becoming a powerful diagnostic tool for the early detection, prognosis, and even prediction of clinical response to treatment. Few studies address this in salivary gland tumors (SGTs); hence the promoter methylation profile of various TSGs was quantitatively assessed in primary SGT tissue to determine if tumor-specific alterations could be detected. Methodology DNA isolated from 78 tumor and 17 normal parotid gland specimens was assayed for promoter methylation status of 19 TSGs by fluorescence-based, quantitative methylation-specific PCR (qMSP). The data were utilized in a binary fashion as well as quantitatively (using a methylation quotient) allowing for better profiling and interpretation of results. Principal Findings The average number of methylation events across the studied genes was highest in salivary duct carcinoma (SDC), with a methylation value of 9.6, compared to the normal 4.5 (p<0.0003). There was a variable frequency and individual methylation quotient detected, depending on the TSG and the tumor type. When comparing normal, benign, and malignant SGTs, there was a statistically significant trend for increasing methylation in APC, Mint 1, PGP9.5, RAR-β, and Timp3. Conclusions/Significance Screening promoter methylation profiles in SGTs showed considerable heterogeneity. The methylation status of certain markers was surprisingly high in even normal salivary tissue, confirming the need for such controls. Several TSGs were found to be associated with malignant SGTs, especially SDC. Further study is needed to evaluate the potential use of these associations in the detection, prognosis, and therapeutic outcome of these rare tumors.

Phospho-ΔNp63α/NF-Y protein complex transcriptionally regulates DDIT3 expression in squamous cell carcinoma cells upon cisplatin exposure

Cisplatin remains the most important chemotherapeutic agent for patients with human head and neck cancer. However, tumor cells often develop resistance to cisplatin-induced apoptosis. We previously found that head and neck squamous cell carcinoma (HNSCC) cells exposed to cisplatin display a marked ATM-induced phosphorylation of ΔNp63α. However, the mutated ΔNp63α-S385G failed to undergo phosphorylation by ATM kinase. We used HNSCC cell lines expressing the wild type ΔNp63α or mutated ΔNp63α-S385G to determine the effect of S385G mutation on the ΔNp63α transcriptional activity and protein-protein interactions. The S385G mutation in ΔNp63α dramatically abolished the up-regulation/down-regulation of downstream gene targets and the binding of ΔNp63α−S385G to certain promoters. In contrast to the non-phosphorylated ΔNp63α-S385G, the phospho-ΔNp63α forms protein-protein complexes with NF-YA transcription factor and regulates the transcription of DDIT3 gene implicated in the programmed cell death of HNSCC cells upon cisplatin exposure. We suggest that the transcriptional activation of ΔNp63α through its phosphorylation by ATM kinase in HNSCC cells exposed to cisplatin is a critical step in the subsequent sensitivity of certain human head and neck cancers to platinum therapy.

Identification of guanine nucleotide-binding protein γ-7 as an epigenetically silenced gene in head and neck cancer by gene expression profiling

Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. Aberrant hypermethylation in the promoter region of some known or putative tumor suppressor genes occurs frequently during the development of various types of cancer including head and neck squamous cell carcinoma (HNSCC). In this study we used an expanded mRNA expression profiling approach followed by microarray expression analysis to identify epigenetically inactivated genes in HNSCC. Two HNSCC cell lines were treated with 5-aza-2′-deoxycytidine followed by microarray analysis to identify epigenetically silenced genes in HNSCC. We found 1,960, 614 and 427 genes were upregulated in the HNSCC cell lines JHU-012, JHU-011 and the combination of both cell lines, respectively. HNSCC tumor and normal mucosal samples were used for gene profiling by a 47K mRNA gene expression array and we found 7,140 genes were downregulated in HNSCC tumors compared to normal mucosa, as determined by microarray analysis, and were integrated with cell line data. Integrative analysis defined 126 candidate genes, of which only seven genes showed differential methylation in tumors and no methylation in normal mucosa after bisulfite sequencing. Following validation by QMSP, one gene, guanine nucleotide-binding protein γ-7 (GNG7), was confirmed to be highly methylated in tumors and unmethylated in normal mucosal and salivary rinse samples demonstrating cancer-specific methylation in HNSCC tissues. TXNIP and TUSC2 were partially methylated in tumors and normal salivary rinses but unmethylated in normal mucosa. We concluded that GNG7 is a highly specific promoter methylated gene associated with HNSCC. In addition, TXNIP and TUSC2 are also potential biomarkers for HNSCC.

Differential expression of degradome components in cutaneous squamous cell carcinomas

Although the cure rate for cutaneous squamous cell carcinoma is high, the diverse spectrum of squamous cell carcinoma has made it difficult for early diagnosis, particularly the aggressive tumors that are highly associated with mortality. Therefore, molecular markers are needed as an adjunct to current staging methods for diagnosing high-risk lesions, and stratifying those patients with aggressive tumors. To identify such biomarkers, we have examined a comprehensive set of 200 histologically defined squamous cell carcinoma and normal skin samples by using a combination of microarray, QRT-PCR and immunohistochemistry analyses. A characteristic and distinguishable profile including matrix metalloproteinase (MMP) as well as other degradome components was differentially expressed in squamous cell carcinoma compared with normal skin samples. The expression levels of some of these genes including matrix metallopeptidase 1 (MMP1), matrix metallopeptidase 10 (MMP10), parathyroid hormone-like hormone (PTHLH), cyclin-dependent kinase inhibitor 2A (CDKN2A), A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), FBJ osteosarcoma oncogene (FOS), interleukin 6 (IL6) and reversion-inducing-cysteine-rich protein with kazal motifs (RECK) were significantly differentially expressed (P≤0.02) in squamous cell carcinoma compared with normal skin. Furthermore, based on receiver operating characteristic analyses, the mRNA and protein levels of MMP1 are significantly higher in aggressive tumors compared with non-aggressive tumors. Given that MMPs represent the most prominent family of proteinases associated with tumorigenesis, we believe that they may have an important role in modulating the tumor microenvironment of squamous cell carcinoma.

Validation of nucleolar protein 4 as a novel methylated tumor suppressor gene in head and neck cancer.

Methylation of CpG islands in the promoter region of genes acts as a significant mechanism of epigenetic gene silencing in head and neck cancer. In the present study, we assessed the association of epigenetic alterations of a panel of 12 genes [nucleolar protein 4 (NOL4), iroquois homeobox 1 (IRX1), SLC5A8, LRRC3B, FUSSEL18, EBF3, GBX2, HMX2, SEPT9, ALX3, SOCS3 and LHX6] with head and neck squamous cell carcinoma (HNSCC) via a candidate gene approach. After the initial screening of methylated CpG islands on the promoter regions by bisulfite sequencing using salivary rinse samples, only two genes had methylated CpG dinucleotides on their promoter regions in tumor samples and absence of methylated CpGs were found in normal salivary rinse samples after bisulfite modification and bisulfite sequencing. We then performed real-time quantitative methylation-specific PCR (QMSP) on 16 salivary rinse and 14 normal mucosal samples from healthy subjects and 33 HNSCC tumor samples for the two genes selected. After validation with QMSP, one gene, NOL4, was highly methylated (91%) in tumor samples and unmethylated in normal salivary rinses and minimally methylated in normal mucosal samples demonstrating cancer-specific methylation in HNSCC tissues. Although the IRX1 gene was observed as methylated in normal mucosal and salivary rinse samples, the methylation values of these normal samples were very low (<10%). In conclusion, we identified NOL4 as a highly specific promoter methylated gene associated with HNSCC. IRX1 may have potential as a biomarker for HNSCC and should be assessed in a larger cohort.

Abstract 1384: Promoter methylation of tumor suppressor genes in convalescent saliva samples from patients with head and neck squamous cell carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnPurpose: Silencing of tumor suppressor genes (TSGs) plays an important role in head and neck carcinogenesis. Methylation of CpG islands in the promoter regions of genes acts as a significant mechanism of epigenetic gene silencing. In this study, we assessed the feasibility of epigenetic alterations in TSGs in post-treatment DNA shed from the oral mucosa as a prognostic marker for persistent/recurrent head and neck cancer. A panel of four genes (KIF1A, EDNRB, p16, DCC) was evaluated in this study.nnExperimental Design: Hypermethylation of the promoter regions was investigated by bisulfite modification and quantitative methylation-specific PCR (Q-MSP) which provides more objective and rapid estimation of gene methylation status. Fresh tumor samples and pre- and post-treatment salivary rinses were collected from 83 patients with HNSCC. Post-treatment saliva samples were collected once in every 3 months. In addition, 46 normal saliva and 16 normal mucosal samples from healthy individuals were obtained. Then we evaluated the correlation of the results with the clinical parameters.nnResults: KIF1A, EDNRB, p16 and DCC genes were methylated in 94%, 95%, 37% and 84% of primary HNSCC tissues, respectively and were only methylated in 2%, 6.8%, 5% and 10% of the salivary rinses from normal subjects. In addition, KIF1A, EDNRB, p16 and DCC were methylated in 35%, 69%, 8.4% and 38.6% of pre-treatment salivary rinses from HNSCC patients, respectively. We evaluated whether methylation levels of these genes in the post treatment saliva may predict the recurrence/prognosis or the chance of cure after treatment. Our data indicate that these genes may be potential biomarkers for HNSCC detection and follow up. Sixteen of 83 patients had recurrent tumors and KIF1A+EDNRB+p16+DCC methylation was observed in 37.5% of the patients in this group. The three gene panel (KIF1A, EDNRB and DCC) was also observed in 37.5% of the recurrent group and 46% of the overall patients, respectively.nnConclusion: Methylation of the KIF1A, EDNRB and DCC gene promoters is a frequent event in HNSCC and these genes are not methylated in normal salivary rinses, demonstrating potential that they may act as biomarkers in detection strategies. Patients with presence of methylation in surveillance salivary rinses are at significant risk for recurrence. Quantitative measurement of salivary methylated DNA may have promise for surveillance and early detection of recurrence.nnCitation Format: Semra Demokan, Jatinder Kaur, Alice Y. Chuang, Wojciech K. Mydlarz, Kavita M. Pattani, Wayne M. Koch, David Sidransky, Nejat Dalay, Joseph A. Califano. Promoter methylation of tumor suppressor genes in convalescent saliva samples from patients with head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1384. doi:10.1158/1538-7445.AM2014-1384