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1.  l-DOPA Decarboxylase (DDC) Expression Status as a Novel Molecular Tumor Marker for Diagnostic and Prognostic Purposes in Laryngeal Cancer1 
Translational Oncology  2012;5(4):288-296.
l-DOPA decarboxylase (DDC) plays an essential role in the enzymatic synthesis of dopamine and alterations in its gene expression have been reported in several malignancies. Our objective was to analyze DDC messenger RNA (mRNA) and protein expression in laryngeal tissues and to evaluate the clinical implication of this molecule in laryngeal cancer. In this study, total RNA was isolated from 157 tissue samples surgically removed from 100 laryngeal cancer patients. A highly sensitive real-time polymerase chain reaction methodology based on SYBR Green I fluorescent dye was developed for the quantification of DDC mRNA levels. In addition, Western blot analysis was performed for the detection of DDC protein. DDC mRNA expression was revealed to be significantly downregulated in primary laryngeal cancer samples compared with their nonmalignant counterparts (P = .001). A significant negative association was also disclosed between DDC mRNA levels and TNM staging (P = .034). Univariate analysis showed that patients bearing DDC-positive tumors had a significantly decreased risk of death (hazard ratio = 0.23, P = .012) and local recurrence (hazard ratio = 0.32, P =.006), whereas DDC expression retained its favorable prognostic significance in the multivariate analysis. Kaplan-Meier curves further demonstrated that DDC-positive patients experienced longer overall and disease-free survival periods (P = .006 and P = .004, respectively). Moreover, DDC protein was detected in both neoplastic and noncancerous tissues. Therefore, our results suggest that DDC expression status could qualify as a promising biomarker for the future clinical management of laryngeal cancer patients.
PMCID: PMC3431039  PMID: 22937181
2.  Human Papillomavirus Infection as a Prognostic Factor in Oropharyngeal Squamous Cell Carcinoma Patients Treated in a Prospective Phase II Clinical Trial 
Anticancer research  2009;29(5):1467-1474.
Background
Human papillomvirus (HPV)-16 is associated with an improved prognosis in a subset of patients with head and neck squamous cell carcinoma (HNSCC). Cervical carcinoma models have also demonstrated that HPV oncoproteins, E6 and E7, can induce VEGF and HIF-1 gene expression. The purpose of this study was to 1) determine the presence of high-risk HPV 16 in patients with HNSCC enrolled in a prospective phase II clinical trial, 2) assess the impact of HPV status on treatment response and survival in this select cohort treated with combined modality therapy, and 3) identify the differences between HIF/VEGF expression in HPV-positive and -negative tumors.
Experimental Design
The effect of HPV status on treatment response and outcome was prospectively evaluated in a single-institution phase II clinical trial. Patients had resectable untreated stage III, IV HNSCC of the oral cavity, oropharynx, hyopharynx, or larynryx, and stage II cancer of the base of tongue, hypopharynx, and larynx. All patients received neoadjuvant chemotherapy with two course of docetaxel (T) 60 mg/m2, then a 96-hour infusion of cisplatin (P) 25mg/m2/d, 5-fluorouracil (F) 700 mg/m2/d, and leucovorin (L) 500 mg/m2/d (TPFL). Those with at least a partial response received a third course. Responding patients then underwent surgery including modified lymph node dissection followed by adjuvant radiation or chemoradiation. Patients who progressed during neoadjuvant chemotherapy proceeded directly to surgery. HPV status was determined by conventional PCR in fresh frozen biopsy samples and Taqman PCR assay on formalin-fixed, paraffin-embedded specimens. HIF-1a and VEGF-A expression were assessed by immunohistochemistry (IHC) and quantitative real-time PCR (RT-PCR). Multivariate Cox proportional hazards regression analysis of time to disease progression or death was used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for HPV-positive status.
Results
Of the 24 evaluable cases, HPV16 DNA was detected in 14 specimens, 13 of which were oropharyngeal tumors. HPV18 was not detected in any of the specimens. Treatment compliance was similar between both groups. There was no difference in either the response rates seen after NCT (85.7% vs. 90%), or the pathologic complete response rate for surgical patients (38.5% vs. 42.9%) for the HPV-positive and –negative tumors, respectively. After a median follow-up time of 52.9 months, there was a trend toward better progression-free (HR 0.15; p = 0.06) and overall survival (HR 0.14; p = 0.10), but this was not statistically significant. There was no difference in the level of VEGF expression at the protein level, however, in a subset of 13 fresh frozen tissue samples, quantitative RT-PCR revealed a statistically significant increase in VEGF mRNA transcript in the HPV-positive tumors (p < 0.01). No difference was seen for HIF-1a expression.
Conclusions
HPV-positivity portended a better prognosis in patients with oropharyngeal SCC treated with induction chemotherapy and adjuvant radiation in a prospective clinical trial although the benefit did not reach the level of statistical significance due to the small patient number. The level of VEGF mRNA was up-regulated in HPV16-positive tumors possibly by a HIF-1 independent manner.
PMCID: PMC3582681  PMID: 19443352
3.  Bioactive Phytochemical Proanthocyanidins Inhibit Growth of Head and Neck Squamous Cell Carcinoma Cells by Targeting Multiple Signaling Molecules 
PLoS ONE  2012;7(9):e46404.
Despite advances in surgical and medical therapies, approximate 50% survival rate of head and neck squamous cell carcinoma (HNSCC) has had marginal improvement in the last 30 years. Therefore, alternative strategies are required for the management of HNSCC. Here, we report the chemotherapeutic effect of proanthocyanidins on HNSCC cells using in vitro and in vivo models. Treatment of human HNSCC cell lines from different sub-sites, such as oral cavity (SCC1), larynx (SCC5), tongue (OSC19) and pharynx (FaDu), with grape seed proanthocyanidins (GSPs) reduced their cell viability and induced cell death in a dose- and time-dependent manner. GSPs induced inhibition of cell viability was associated with: (i) G1-phase arrest, (ii) inhibition of expressions of cyclins (cyclin D1 and Cyclin D2) and cyclin-dependent kinases (Cdk), (iii) increased expression of the Cdk inhibitory proteins (Cip1/p21, Kip1/p27), enhanced binding of Cdk inhibitors to Cdks, and downregulation of E2F transcription factor. GSPs significantly (P<0.05−0.001) increased apoptosis of SCC1 and OSC19 cells with induction of Bax, reduced expression of Bcl-2, and activation of caspase-3. GSPs also reduced the expression of epidermal growth factor receptor (EGFR), and treatment of SCC1 cells with erlotinib, an EGFR-targeting small molecule tyrosine kinase inhibitor, significantly (P<0.05−0.001) reduced cell viability and increased cell death. Dietary administration of GSPs (0.5%, w/w) in supplementation with AIN76A control diet inhibited the growth of SCC1 tumor xenografts in athymic nude mice, which was associated with: (i) inhibition of cell proliferation, (ii) induction of apoptosis of tumor xenograft cells, (iii) decreased expression of cyclins and Cdks, (iv) decreased expression of EGFR, and (v) increased expression of Cip1/p21 and Kip1/p27 proteins and their increased binding to Cdks in tumor xenograft samples. Together, these results suggest that GSPs may be a promising candidate for head and neck squamous cell carcinoma therapy.
doi:10.1371/journal.pone.0046404
PMCID: PMC3458881  PMID: 23050025
4.  Quantitative expression analysis and prognostic significance of L-DOPA decarboxylase in colorectal adenocarcinoma 
British Journal of Cancer  2010;102(9):1384-1390.
Background:
L-DOPA decarboxylase (DDC) is an enzyme that catalyses, mainly, the decarboxylation of L-DOPA to dopamine and was found to be involved in many malignancies. The aim of this study was to investigate the mRNA expression levels of the DDC gene and to evaluate its clinical utility in tissues with colorectal adenocarcinoma.
Methods:
Total RNA was isolated from colorectal adenocarcinoma tissues of 95 patients. After having tested RNA quality, we prepared cDNA by reverse transcription. Highly sensitive quantitative real-time PCR method for DDC mRNA quantification was developed using the SYBR Green chemistry. GAPDH served as a housekeeping gene. Relative quantification analysis was performed using the comparative CT method (2−ΔΔCT).
Results:
DDC mRNA expression varied remarkably among colorectal tumours examined in this study. High DDC mRNA expression levels were found in well-differentiated and Dukes' stage A and B tumours. Kaplan–Meier survival curves showed that patients with DDC-positive tumours have significantly longer disease-free survival (P=0.009) and overall survival (P=0.027). In Cox regression analysis of the entire cohort of patients, negative DDC proved to be a significant predictor of reduced disease-free (P=0.021) and overall survival (P=0.047).
Conclusions:
The results of the study suggest that DDC mRNA expression may be regarded as a novel potential tissue biomarker in colorectal adenocarcinoma.
doi:10.1038/sj.bjc.6605654
PMCID: PMC2865762  PMID: 20424616
colorectal adenocarcinoma; L-DOPA decarboxylase; DDC; real-time PCR; tumour biomarkers
5.  High levels of intron-containing RNAs are associated with expression of the Drosophila DOPA decarboxylase gene. 
Molecular and Cellular Biology  1984;4(9):1669-1674.
We have examined the structure and expression during embryonic development of the Drosophila DOPA decarboxylase gene, Ddc. The Ddc gene is transcribed to make at least five different size classes of RNA. These RNA species first appear late in embryogenesis, coincident with induction of Ddc enzyme activity. The most abundant and smallest RNA appears to be Ddc mRNA. The sequences encoding this RNA are split by two intervening sequences. Each of the larger RNA species contains some or all of the intervening sequences. We have noted two unusual features of Ddc expression during embryogenesis. First, the intervening-sequence-containing RNAs are present as 20% or more of the polyadenylated Ddc RNA molecules, an exceptionally high proportion. Second, these RNAs do not disappear as rapidly as Ddc mRNA after Ddc enzyme activity reaches fully induced levels. These observations indicate slow rates of RNA processing relative to mRNA half-life and suggest that post-transcriptional steps participate in regulating Ddc expression. Although four of the five RNA species were detected at multiple developmental stages during which Ddc is expressed, one was found uniquely during embryogenesis. This RNA differs from Ddc mRNA in length and in time of expression during embryogenesis but is transcribed in the same orientation and from the same genomic sequences as the Ddc primary transcript.
Images
PMCID: PMC368971  PMID: 6493231
6.  A Dopa Decarboxylase Modulating the Immune Response of Scallop Chlamys farreri 
PLoS ONE  2011;6(4):e18596.
Background
Dopa decarboxylase (DDC) is a pyridoxal 5-phosphate (PLP)-dependent enzyme that catalyzes the decarboxylation of L-Dopa to dopamine, and involved in complex neuroendocrine-immune regulatory network. The function for DDC in the immunomodulation remains unclear in invertebrate.
Methodology
The full-length cDNA encoding DDC (designated CfDDC) was cloned from mollusc scallop Chlamys farreri. It contained an open reading frame encoding a polypeptide of 560 amino acids. The CfDDC mRNA transcripts could be detected in all the tested tissues, including the immune tissues haemocytes and hepatopancreas. After scallops were treated with LPS stimulation, the mRNA expression level of CfDDC in haemocytes increased significantly (5.5-fold, P<0.05) at 3 h and reached the peak at 12 h (9.8-fold, P<0.05), and then recovered to the baseline level. The recombinant protein of CfDDC (rCfDDC) was expressed in Escherichia coli BL21 (DE3)-Transetta, and 1 mg rCfDDC could catalyze the production of 1.651±0.22 ng dopamine within 1 h in vitro. When the haemocytes were incubated with rCfDDC-coated agarose beads, the haemocyte encapsulation to the beads was increased significantly from 70% at 6 h to 93% at 24 h in vitro in comparison with that in the control (23% at 6 h to 25% at 24 h), and the increased haemocyte encapsulation was repressed by the addition of rCfDDC antibody (which is acquired via immunization 6-week old rats with rCfDDC). After the injection of DDC inhibitor methyldopa, the ROS level in haemocytes of scallops was decreased significantly to 0.41-fold (P<0.05) of blank group at 12 h and 0.47-fold (P<0.05) at 24 h, respectively.
Conclusions
These results collectively suggested that CfDDC, as a homologue of DDC in scallop, modulated the immune responses such as haemocytes encapsulation as well as the ROS level through its catalytic activity, functioning as an indispensable immunomodulating enzyme in the neuroendocrine-immune regulatory network of mollusc.
doi:10.1371/journal.pone.0018596
PMCID: PMC3076384  PMID: 21533240
7.  Prognostic factors of cervical node status in head and neck squamous cell carcinoma 
Background
Cervical nodal status is one of prognostic factors in head and neck squamous cell carcinoma (HNSCC). The objective of this study was to identify prognostic factors of cervical node status including site and size of primary tumors, presence of lymphovascular invasion, and size of cervical node for appropriate further treatment in HNSCC.
Methods
A 5-year retrospective review of patients with HNSCC in Phramongkutklao Hospital from 2009 to 2013 was conducted. Histopathologic data on primary tumors and cervical nodes were reviewed. Cervical nodes were divided into five groups: 1–3, 4–6, 7–9, 10–30, and >30 mm. Numbers of positive and negative nodes were compared in different sizes and sites and the presence of extracapsular extension.
Results
In all, 165 patients and 1,472 nodes were reviewed. The mean age was 52.6 years and 77.58% were male. The most frequent primary site was oral tongue (50.91%). In sum, 52.72% showed lymphovascular invasion. Thirty-five patients (81.40%) in therapeutic neck dissections and 18 patients (69.23%) in prophylactic neck dissections showed nodal metastasis. The mean size of metastatic nodes was 3.89 mm (range, 2–45 mm) and 3.53 mm (range, 2–23 mm), respectively. Significant associations were found between the size of cervical nodes and the site of primary tumor of the oral tongue, lip, base of the tongue, and floor of the mouth (p < 0.05). Metastatic lymph nodes showed extracapsular extension 69.55%. No significance was found between extracapsular extension and clinical staging, size of primary tumor, pathologic differentiation, and size of cervical nodes. Sizes of cervical lymph node of squamous cell carcinoma (SCC) of the oral tongue and lip were statistically significant with the size of tumor and tumor grading (p < 0.05).
Conclusions
A statistical significance was found between the size of cervical nodes and the site of primary tumor of the oral tongue and lip. Herein, we recommended performing neck dissection in all cases of SCC of the base of the tongue, floor of the mouth, buccal mucosa, and retromolar trigone.
doi:10.1186/s12957-015-0460-6
PMCID: PMC4336753
Head and neck squamous cell carcinoma; Cervical node metastasis; Prognostic factors
8.  Sphingosine kinase 1 mediates head & neck squamous cell carcinoma invasion through sphingosine 1-phosphate receptor 1 
Cancer Cell International  2014;14(1):76.
Background
Head and neck squamous cell carcinoma (HNSCC) is characterized by aggressive loco-regional invasion. Sphingosine kinase1 (SphK1), an enzyme in sphingolipid metabolism, is emerging as a key player in HNSCC pathology. The observation that SphK1 is overexpressed in all HNSCC stages and is associated with depth of tumor invasion, metastasis and clinical failure underscores the importance of SphK1 in HNSCC pathology. Still, the mechanisms underlying SphK1 regulation of invasion have not been delineated. Therefore, we sought to mechanistically describe how SphK1 regulates invasion in HNSCC.
Methods
Invasion assays were used to measure invasive ability of SphK1 overexpressing human tongue squamous cell carcinoma (SCC-25 cells). Western blotting, quantitative qPCR, ELISA and zymography were used to measure the effect of SphK1 and sphingosine 1-phoshate receptor 1 (S1P1) on invasion measures, MMP-2/9, E-cadherin, EGFR, IL-6/STAT3, in SCC-25 cells.
Results
SphK1 expression is elevated in cells with an invasive phenotype as compared to non-invasive phenotype. We show SphK1 overexpression increased EGF-induced EGFR/ERK and AKT activity, increased matrix metalloproteinase (MMP)-2/9 mRNA and reduced E-cadherin. SphK1 overexpression also increased IL-6 concentration and EGF-induced STAT3 phosphorylation, exemplifying that SphK1 modulates IL-6/STAT3 signaling. Notably, we show that S1P1 knockdown reduced IL-6/STAT3 signaling, representing another pathway by which SphK1/S1P regulates invasion.
Conclusions
Taken together, our data suggest that SphK1 sits at the hub of multiple key signaling cascades, all which have been implicated in the regulation of invasiveness, making SphK1 an attractive target for the development of HNSCC therapies.
doi:10.1186/s12935-014-0076-x
PMCID: PMC4155094  PMID: 25197261
Sphingosine kinase1; Invasion; HNSCC; EGFR; STAT3; Sphingosine 1-phosphate; Sphingosine 1-phosphate receptor 1; IL-6; SCC-25 cells
9.  Epigenetic Silencing of CRABP2 and MX1 in Head and Neck Tumors12 
Neoplasia (New York, N.Y.)  2009;11(12):1329-1339.
Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease affecting the epithelium of the oral cavity, pharynx and larynx. Conditions of most patients are diagnosed at late stages of the disease, and no sensitive and specific predictors of aggressive behavior have been identified yet. Therefore, early detection and prognostic biomarkers are highly desirable for a more rational management of the disease. Hypermethylation of CpG islands is one of the most important epigenetic mechanisms that leads to gene silencing in tumors and has been extensively used for the identification of biomarkers. In this study, we combined rapid subtractive hybridization and microarray analysis in a hierarchical manner to select genes that are putatively reactivated by the demethylating agent 5-aza-2′-deoxycytidine (5Aza-dC) in HNSCC cell lines (FaDu, UM-SCC-14A, UM-SCC-17A, UM-SCC-38A). This combined analysis identified 78 genes, 35 of which were reactivated in at least 2 cell lines and harbored a CpG island at their 5′ region. Reactivation of 3 of these 35 genes (CRABP2, MX1, and SLC15A3) was confirmed by quantitative real-time polymerase chain reaction (PCR; fold change, ≥3). Bisulfite sequencing of their CpG islands revealed that they are indeed differentially methylated in the HNSCC cell lines. Using methylation-specific PCR, we detected a higher frequency of CRABP2 (58.1% for region 1) and MX1 (46.3%) hypermethylation in primary HNSCC when compared with lymphocytes from healthy individuals. Finally, absence of the CRABP2 protein was associated with decreased disease-free survival rates, supporting a potential use of CRABP2 expression as a prognostic biomarker for HNSCC patients.
PMCID: PMC2794514  PMID: 20019841
10.  Head and Neck Squamous Cell Carcinomas in HIV-Positive Patients: A Preliminary Investigation of Viral Associations 
Head and Neck Pathology  2010;4(2):97-105.
Oncogenic human papillomaviruses (HPVs) are associated with oropharyngeal squamous cell carcinoma (SCC). Infection with human immunodeficiency virus (HIV) increases susceptibility to opportunistic infections and viral-promoted cancers. The prevalences of HPV, herpes simplex virus (HSV), Epstein-Barr virus (EBV), and human herpesvirus-8 (HHV-8) have not been established for head and neck squamous cell carcinoma in HIV-positive patients (HIV+ HNSCC). We have observed that HIV+ HNSCC tend to contain numerous multinucleated tumor giant cells, this finding has not been described previously. The goal of this study is to test for these oncogenic viruses in a small cohort of retrospectively identified patients with HIV infection, and to compare histologically these cancers to a control group of HNSCC patients. Tumors were reviewed histologically and compared to a control group of 102 patients with HNSCC (serologically untyped or HIV negative). Polymerase chain reaction (PCR) was performed on formalin-fixed, paraffin-embedded HIV+ HNSCC samples from combined 25 patients in two institutions. In situ hybridization was performed to identify EBV (EBER) and immunohistochemistry was performed to detect HSV-1, HSV-2, HHV-8, and HIV-related proteins (Nef, p24). The study sample consisted of 34 HIV+ patients with HNSCC from Montefiore Medical Center, and six HIV+ HNSCC patients from Hospital Clinic, University of Barcelona; 24 (60%) men and 16 (40%) women. The larynx was most commonly involved (65%, n = 26); followed by the oropharynx (22.5%, n = 9). Four carcinomas arose from the oral cavity (10%) and one from the nasal cavity (2.5%). Histologically, multinucleated tumor giant cells were more common in the HIV+ group (39/40, 97.5%) than the control group (27/102, 26%, p 0.001, chi-square). HPV was detected in 6 of 25 (24%) HNSCC tumors by PCR, five were typed as HPV 16 and one as HPV 26/69; five of these tumors (83%) were located in the oropharynx. EBV, HSV-1, HSV-2, and HHV-8 were detected only infrequently in tumor cells. Nef protein was detected in tumor cells in 7 of 21 (33.3%) cases; p24 was not detectable in 6 tumors studied. There were no significant associations between HPV positive tumors and co-infections with other viruses. This study is consistent with other reports that suggest an increased incidence of laryngeal carcinoma for HIV+ patients. HPV was detected in 24% of HIV+ HNSCC, however, the number of tumors with amplifiable DNA (n = 25) is too small to allow for conclusions. EBV, HSV-1, HSV-2, and HHV-8 are uncommon in HIV+ HNSCC; it is unlikely that these viruses have a promoting effect. MNTCG are significantly common in HIV+ HNSCC, but there is overlap in MNTCG counts with the control group and therefore this finding cannot be used as a biomarker of HIV infection.
doi:10.1007/s12105-010-0171-9
PMCID: PMC2878620  PMID: 20333562
AIDS; HIV; HNSCC; Squamous carcinoma; HPV
11.  Intra-tumor Genetic Heterogeneity and Mortality in Head and Neck Cancer: Analysis of Data from The Cancer Genome Atlas 
PLoS Medicine  2015;12(2):e1001786.
Background
Although the involvement of intra-tumor genetic heterogeneity in tumor progression, treatment resistance, and metastasis is established, genetic heterogeneity is seldom examined in clinical trials or practice. Many studies of heterogeneity have had prespecified markers for tumor subpopulations, limiting their generalizability, or have involved massive efforts such as separate analysis of hundreds of individual cells, limiting their clinical use. We recently developed a general measure of intra-tumor genetic heterogeneity based on whole-exome sequencing (WES) of bulk tumor DNA, called mutant-allele tumor heterogeneity (MATH). Here, we examine data collected as part of a large, multi-institutional study to validate this measure and determine whether intra-tumor heterogeneity is itself related to mortality.
Methods and Findings
Clinical and WES data were obtained from The Cancer Genome Atlas in October 2013 for 305 patients with head and neck squamous cell carcinoma (HNSCC), from 14 institutions. Initial pathologic diagnoses were between 1992 and 2011 (median, 2008). Median time to death for 131 deceased patients was 14 mo; median follow-up of living patients was 22 mo. Tumor MATH values were calculated from WES results. Despite the multiple head and neck tumor subsites and the variety of treatments, we found in this retrospective analysis a substantial relation of high MATH values to decreased overall survival (Cox proportional hazards analysis: hazard ratio for high/low heterogeneity, 2.2; 95% CI 1.4 to 3.3). This relation of intra-tumor heterogeneity to survival was not due to intra-tumor heterogeneity’s associations with other clinical or molecular characteristics, including age, human papillomavirus status, tumor grade and TP53 mutation, and N classification. MATH improved prognostication over that provided by traditional clinical and molecular characteristics, maintained a significant relation to survival in multivariate analyses, and distinguished outcomes among patients having oral-cavity or laryngeal cancers even when standard disease staging was taken into account. Prospective studies, however, will be required before MATH can be used prognostically in clinical trials or practice. Such studies will need to examine homogeneously treated HNSCC at specific head and neck subsites, and determine the influence of cancer therapy on MATH values. Analysis of MATH and outcome in human-papillomavirus-positive oropharyngeal squamous cell carcinoma is particularly needed.
Conclusions
To our knowledge this study is the first to combine data from hundreds of patients, treated at multiple institutions, to document a relation between intra-tumor heterogeneity and overall survival in any type of cancer. We suggest applying the simply calculated MATH metric of heterogeneity to prospective studies of HNSCC and other tumor types.
In this study, Rocco and colleagues examine data collected as part of a large, multi-institutional study, to validate a measure of tumor heterogeneity called MATH and determine whether intra-tumor heterogeneity is itself related to mortality.
Editors’ Summary
Background
Normally, the cells in human tissues and organs only reproduce (a process called cell division) when new cells are needed for growth or to repair damaged tissues. But sometimes a cell somewhere in the body acquires a genetic change (mutation) that disrupts the control of cell division and allows the cell to grow continuously. As the mutated cell grows and divides, it accumulates additional mutations that allow it to grow even faster and eventually from a lump, or tumor (cancer). Other mutations subsequently allow the tumor to spread around the body (metastasize) and destroy healthy tissues. Tumors can arise anywhere in the body—there are more than 200 different types of cancer—and about one in three people will develop some form of cancer during their lifetime. Many cancers can now be successfully treated, however, and people often survive for years after a diagnosis of cancer before, eventually, dying from another disease.
Why Was This Study Done?
The gradual acquisition of mutations by tumor cells leads to the formation of subpopulations of cells, each carrying a different set of mutations. This “intra-tumor heterogeneity” can produce tumor subclones that grow particularly quickly, that metastasize aggressively, or that are resistant to cancer treatments. Consequently, researchers have hypothesized that high intra-tumor heterogeneity leads to worse clinical outcomes and have suggested that a simple measure of this heterogeneity would be a useful addition to the cancer staging system currently used by clinicians for predicting the likely outcome (prognosis) of patients with cancer. Here, the researchers investigate whether a measure of intra-tumor heterogeneity called “mutant-allele tumor heterogeneity” (MATH) is related to mortality (death) among patients with head and neck squamous cell carcinoma (HNSCC)—cancers that begin in the cells that line the moist surfaces inside the head and neck, such as cancers of the mouth and the larynx (voice box). MATH is based on whole-exome sequencing (WES) of tumor and matched normal DNA. WES uses powerful DNA-sequencing systems to determine the variations of all the coding regions (exons) of the known genes in the human genome (genetic blueprint).
What Did the Researchers Do and Find?
The researchers obtained clinical and WES data for 305 patients who were treated in 14 institutions, primarily in the US, after diagnosis of HNSCC from The Cancer Genome Atlas, a catalog established by the US National Institutes of Health to map the key genomic changes in major types and subtypes of cancer. They calculated tumor MATH values for the patients from their WES results and retrospectively analyzed whether there was an association between the MATH values and patient survival. Despite the patients having tumors at various subsites and being given different treatments, every 10% increase in MATH value corresponded to an 8.8% increased risk (hazard) of death. Using a previously defined MATH-value cutoff to distinguish high- from low-heterogeneity tumors, compared to patients with low-heterogeneity tumors, patients with high-heterogeneity tumors were more than twice as likely to die (a hazard ratio of 2.2). Other statistical analyses indicated that MATH provided improved prognostic information compared to that provided by established clinical and molecular characteristics and human papillomavirus (HPV) status (HPV-positive HNSCC at some subsites has a better prognosis than HPV-negative HNSCC). In particular, MATH provided prognostic information beyond that provided by standard disease staging among patients with mouth or laryngeal cancers.
What Do These Findings Mean?
By using data from more than 300 patients treated at multiple institutions, these findings validate the use of MATH as a measure of intra-tumor heterogeneity in HNSCC. Moreover, they provide one of the first large-scale demonstrations that intra-tumor heterogeneity is clinically important in the prognosis of any type of cancer. Before the MATH metric can be used in clinical trials or in clinical practice as a prognostic tool, its ability to predict outcomes needs to be tested in prospective studies that examine the relation between MATH and the outcomes of patients with identically treated HNSCC at specific head and neck subsites, that evaluate the use of MATH for prognostication in other tumor types, and that determine the influence of cancer treatments on MATH values. Nevertheless, these findings suggest that MATH should be considered as a biomarker for survival in HNSCC and other tumor types, and raise the possibility that clinicians could use MATH values to decide on the best treatment for individual patients and to choose patients for inclusion in clinical trials.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001786.
The US National Cancer Institute (NCI) provides information about cancer and how it develops and about head and neck cancer (in English and Spanish)
Cancer Research UK, a not-for-profit organization, provides general information about cancer and how it develops, and detailed information about head and neck cancer; the Merseyside Regional Head and Neck Cancer Centre provides patient stories about HNSCC
Wikipedia provides information about tumor heterogeneity, and about whole-exome sequencing (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
Information about The Cancer Genome Atlas is available
A PLOS Blog entry by Jessica Wapner explains more about MATH
doi:10.1371/journal.pmed.1001786
PMCID: PMC4323109  PMID: 25668320
12.  cis-regulatory sequences responsible for alternative splicing of the Drosophila dopa decarboxylase gene. 
Molecular and Cellular Biology  1994;14(11):7385-7393.
The Drosophila dopa decarboxylase gene, Ddc, is expressed in the hypoderm and in specific sets of cells in the central nervous system (CNS). The unique Ddc primary transcript is alternatively spliced in these two tissues. The Ddc CNS mRNA contains all four exons (A through D), whereas the hypodermal mRNA contains only three exons (A, C, and D). To localize cis-regulatory sequences responsible for Ddc alternative splicing, a Ddc minigene and several fusion genes containing various amounts of Ddc sequences fused to fushi tarazu (ftz) exon 1 were constructed and introduced into flies by P-element-mediated germ line transformation. We find that Ddc intron ab and exon B are sufficient to regulate Ddc alternative splicing, since transcripts of a minimal fusion gene containing most of Ddc intron ab and exon B are spliced to exon B in the CNS but not in the hypoderm. These results indicate that Ddc alternative splicing is regulated by either a negative mechanism preventing splicing to exon B in the hypoderm or a positive mechanism activating splicing to exon B in the CNS. Our previous data suggest that Ddc hypodermal splicing is the actively regulated splicing pathway (J. Shen, C. J. Beall, and J. Hirsh, Mol. Cell. Biol. 13:4549-4555, 1993). Here we show that deletion of Ddc intron ab sequences selectively disrupts hypodermal splicing specificity. These results support a model in which Ddc alternative splicing is negatively regulated by a blockage mechanism preventing splicing to exon B in the hypoderm.
Images
PMCID: PMC359273  PMID: 7935452
13.  Establishment and Characterization of Patient Tumor-Derived Head and Neck Squamous Cell Carcinoma Xenografts 
Cancer biology & therapy  2009;8(23):2275-2283.
The overall purpose of this study was to establish human head and neck squamous cell carcinoma (HNSCC) xenografts in mice by transplantation of surgical tumor tissue and to characterize the growth, histologic and vascular properties of these xenografts. Primary surgical specimens of HNSCC were xenografted into eight-to-twelve week old severe combined immunodeficiency (SCID) mice. Histologic features of primary HNSCC specimens, initial and established xenografts were compared for tumors established from three different head and neck subsites, namely, oral cavity, larynx and base of tongue (one tumor per site). Growth rates of xenografts were compared along with magnetic resonance imaging (MRI) measures of tumor vascularity and correlative CD31-immunostaining. Initial and established xenografts from all three sites demonstrated a squamous phenotype similar to the original patient tumor histology. Established xenografts of oral cavity and larynx exhibited increased keratinization (H&E) compared to initial xenografts and the primary tumor. No differences in tumor growth rates were observed between established xenografts from the different subsites. Xenografts established from SCC of the larynx exhibited increased microvessel density and lumen area (CD31 staining) along with enhanced permeability to the MR contrast agent compared to oral cavity and base of tongue tumors. Our results show that the combination of non-invasive imaging along with histologic evaluation of patient tumor xenografts offers a valuable platform for preclinical investigations in head and neck cancer. However, it is important to recognize the influence of tumor-host interactions on the histologic phenotype of transplanted tumors.
PMCID: PMC2906774  PMID: 19829072
Head and neck cancers; squamous cell carcinoma; patient tumor xenografts; magnetic resonance imaging; histopathology
14.  Prognostic value of type XXII and XXIV collagen mRNA expression in head and neck cancer patients 
Molecular and Clinical Oncology  2013;2(2):285-291.
Squamous cell carcinoma of the head and neck (HNSCC) is the sixth most common type of cancer, affecting ~500,000 individuals worldwide annually. Collagen is the major constituent of the extracellular matrix component in tumors and plays a crucial role in tumor development. The aim of this study was to determine the mRNA expression of type XXI, XXII, XXIII and XXIV α1 collagen (COL21A1, COL22A1, COL23A1 and COL24A1, respectively) in head and neck squamous cell carcinoma (HNSCC) and investigate its correlation with disease progression. This study investigated the mRNA expression levels of COL21A1, COL22A1, COL23A1 and COL24A1 in 70 HNSCC primary samples and 44 matched pairs of tumor and adjacent normal mucosal tissues using quantitative polymerase chain reaction (qPCR). Expression data were compared to the clinicopathological variables in order to determine the correlation between expression and disease progression. Our results demonstrated that the mRNA levels of COL22A1 and COL24A1 were significantly higher in HNSCC tissues compared to those in the corresponding normal tissues from the same individuals (n=44; P<0.001 and P=0.019, respectively). The COL22A1 mRNA levels were found to be significantly associated with lymph node metastasis (P=0.018) and pathological stage (P=0.024), whereas the COL24A1 mRNA levels were significantly associated with tumor size (P=0.045). The high expression levels of COL22A1 and COL24A1 mRNA were statistically correlated with a decrease in disease-free survival (DFS) (log-rank test, P<0.001). The results of the multivariate logistic regression analysis revealed that high expression levels of the COL22A1 and COL24A1 gene pair were associated with a high odds ratio for recurrence of 14.62 (95% confidence interval: 2.77–77.26; P=0.002). Therefore, the upregulation of COL22A1 and COL24A1 mRNA may play a critical role in the progression of HNSCC and provide useful information as a prognostic predictor for HNSCC patients.
doi:10.3892/mco.2013.233
PMCID: PMC3917764  PMID: 24649348
type XXII α1 collagen; type XXIV α1 collagen; quantitative polymerase chain reaction; mRNA expression; head and neck squamous cell carcinoma
15.  Smad2 and Smad6 as predictors of overall survival in oral squamous cell carcinoma patients 
Molecular Cancer  2010;9:106.
Background
To test if the expression of Smad1-8 mRNAs were predictive of survival in patients with oral squamous cell carcinoma (SCC).
Patients and Methods
We analyzed, prospectively, the expression of Smad1-8, by means of Ribonuclease Protection Assay in 48 primary, operable, oral SCC. In addition, 21 larynx, 10 oropharynx and 4 hypopharynx SCC and 65 matched adjacent mucosa, available for study, were also included. For survival analysis, patients were categorized as positive or negative for each Smad, according to median mRNA expression. We also performed real-time quantitative PCR (QRTPCR) to asses the pattern of TGFβ1, TGFβ2, TGFβ3 in oral SCC.
Results
Our results showed that Smad2 and Smad6 mRNA expression were both associated with survival in Oral SCC patients. Cox Multivariate analysis revealed that Smad6 positivity and Smad2 negativity were both predictive of good prognosis for oral SCC patients, independent of lymph nodal status (P = 0.003 and P = 0.029, respectively). In addition, simultaneously Smad2- and Smad6+ oral SCC group of patients did not reach median overall survival (mOS) whereas the mOS of Smad2+/Smad6- subgroup was 11.6 months (P = 0.004, univariate analysis). Regarding to TGFβ isoforms, we found that Smad2 mRNA and TGFβ1 mRNA were inversely correlated (p = 0.05, R = -0.33), and that seven of the eight TGFβ1+ patients were Smad2-. In larynx SCC, Smad7- patients did not reach mOS whereas mOS of Smad7+ patients were only 7.0 months (P = 0.04). No other correlations were found among Smad expression, clinico-pathological characteristics and survival in oral, larynx, hypopharynx, oropharynx or the entire head and neck SCC population.
Conclusion
Smad6 together with Smad2 may be prognostic factors, independent of nodal status in oral SCC after curative resection. The underlying mechanism which involves aberrant TGFβ signaling should be better clarified in the future.
doi:10.1186/1476-4598-9-106
PMCID: PMC2885344  PMID: 20462450
16.  Chemopreventive and chemotherapeutic actions of mTOR inhibitor in genetically-defined head and neck squamous cell carcinoma mouse model 
Purpose
To assess the efficacy of Rapamycin treatment in chemoprevention and chemotherapy of tumorigenesis in a genetically-defined mouse model of head and neck squamous cell carcinoma (HNSCC).
Experimental design
Knockdown of Tgfbr1 and/or Pten using siRNA-mediated RNA interference was carried out in human HNSCC cell lines to analyze molecular changes in the mTOR pathway. Tgfbr1flox/flox; Ptenflox/flox; K14-CreERtam mice were treated with oral gavage of tamoxifen for the conditional deletion of Tgfbr1 and Pten in oral mucosa, resuting in HNSCC (Bian et al 2011). Tgfbr1 and Pten conditonal deletion (2cKO) mice were treated with Rapamycin before or after the onset of HNSCC, and the efficacy of this treatment was assessed by determining tumor burden, longevity, and molecular analysis of the mTOR pathway. Molecular changes observed in human HNSCC cell lines and 2cKO mice were compared to identify key alterations in the mTOR pathway.
Results
Knockdown of Tgfbr1 and/or Pten in human HNSCC cell lines resulted in activation of mTORC1 and increased levels of survivin. Furthermore, we observed similar changes in HNSCC of the 2cKO mouse. In the human HNSCC tissue array, a loss of Tgfbr1 expression correlated with increased survivin levels. Chemopreventive Rapamycin treatment significantly delayed the onset of the HNSCC tumors and prolonged survival in 2cKO mice. Additionally, we also found that Rapamycin had a therapeutic effect on squamous cell carcinomas in these mice. In 2cKO HNSCC tongue tumors, Rapamycin treatment induced apoptosis, inhibited cell proliferation and phosphorylation of Akt and S6, and decreased survivin expression.
Conclusions
These findings indicate that tumorigenesis in 2cKO HNSCC is associated with activation of the Akt/mTOR/survivin pathway, and inhibition of this pathway by Rapamycin treatment successfully ameliorates the onset and progression of tumorigenesis.
doi:10.1158/1078-0432.CCR-12-1371
PMCID: PMC3463723  PMID: 22859719
TGF-β; Pten; head and neck cancer; mTOR; survivin
17.  Silencing Heat Shock Protein 27 (Hsp27) decreases metastatic behavior of human head and neck squamous cell cancer cells in vitro 
Molecular pharmaceutics  2010;7(4):1283-1290.
The small heat shock protein 27 (Hsp27) is a molecular chaperone that is involved in a variety of cellular functions in cancer cells. The purpose of this research is to study Hsp27 in vitro metastatic behaviors of head and neck squamous cell carcinoma cells (HNSCC). The expression of Hsp27 in primary and metastatic cell lines derived from the primary HNSCC and a synchronous lymph node metastasis in the same patient was determined using real-time PCR and western blotting. Proliferation of the primary and metastatic HNSCC cell lines was evaluated using the MTS proliferation assay. Metastatic behavior was assessed using migration and invasion assays. SiRNA knockdown of Hsp27 was performed in the highly migratory metastatic HNSCC cell line. MTS assays showed that the primary (UM-SCC-22A) and metastatic (UM-SCC-22B) HNSCC have similar proliferation rates. However, UM-SCC-22B derived from the metastasis showed 2.3 to 3.6-fold higher migration ability and 2-fold higher invasion ability than UM-SCC-22A. Real-time PCR demonstrated that Hsp27 mRNA is 22.4-fold higher in metastatic UM-SCC-22B than primary UM-SCC-22A. Similarly, Western blotting showed that Hsp27 is rarely detectable in UM-SCC-22A whereas UM-SCC-22B expresses a 25-fold higher level of Hsp27 protein. SiRNA-mediated knock down of Hsp27 in UM-SCC-22B reduced Hsp27 mRNA expression by nearly 6 - fold and protein expression by 23-fold. Furthermore, siRNA knockdown of Hsp27 decreased metastatic behaviors of UM-SCC-22B by 3 to 4-fold in migration and 2-fold in cell invasion reducing cell invasion and migration to levels similar to the primary HNSCC UM-SCC-22A. These data indicate that Hsp27 may regulate metastatic potential of HNSCC cancer cells. Targeting Hsp27 may decrease metastasis in head and neck squamous cell cancer cells.
doi:10.1021/mp100073s
PMCID: PMC2914182  PMID: 20540527
Heat Shock Protein 27; human head and neck squamous cell cancer; metastasis; siRNA
18.  Yes-Associated Protein Expression in Head and Neck Squamous Cell Carcinoma Nodal Metastasis 
PLoS ONE  2011;6(11):e27529.
Introduction
Yes-associated protein (YAP) is considered an oncogene found amplified in multiple tumors, including head and neck squamous cell carcinoma (HNSCC). However, the role for YAP expression in HNSCC is not understood. Based on the central role of YAP in the hippo pathway, we tested if YAP was associated with the stage of HNSCC progression and metastatic potential.
Methods
To determine the expression of YAP in human benign and HNSCC tissue specimens, immunohistochemical analyses were performed in whole tissue samples and tissue microarrays. The expression of YAP in tissues of microarray was first associated with clinic-pathologic factors and results verified in samples from whole tissue sections. To investigate the role of YAP and p63 in regulating HNSCC epithelial to mesenchymal transition, epithelial and mesenchymal markers were assayed in Fadu and SCC-25 cells, HNSCC cells with endogenously elevated YAP expression and siRNA-mediated expression knockdown.
Results
Analysis of human HNSCC tissues suggested YAP expression was elevated in tumors compared to benign tissues and specifically localized at the tumor invasive front (p value <0.05). But, indexed YAP expression was lower with greater tumor grade (p value  = 0.02). In contrast, p63 expression was primarily elevated in high-grade tumors. Interestingly, both YAP and p63 was strongly expressed at the tumor invasive front and in metastatic HNSCC. Strikingly, we demonstrated YAP expression in the primary HNSCC tumor was associated with nodal metastasis in univariate analysis (p value  = 0.02). However, the knockdown of YAP in Fadu and SCC-25 cell lines was not associated with changes in epithelial to mesenchymal transdifferentiation or p63 expression.
Conclusion
Together, YAP expression, in combination with p63 can facilitate identification of HNSCC tumors from hyperplastic and benign tissues and the metastatic function of YAP in HNSCC may not be a result of epithelia to mesenchymal transdifferentiation.
doi:10.1371/journal.pone.0027529
PMCID: PMC3212574  PMID: 22096589
19.  Elevated gastrin-releasing peptide receptor mRNA expression in buccal mucosa: association with head and neck squamous cell carcinoma 
Head & neck  2012;35(2):270-279.
Background
Expression of gastrin-releasing peptide receptor (GRPR) is elevated in mucosa adjacent to head and neck squamous cell carcinoma (HNSCC) compared with mucosa from cancer-free controls, suggesting elevated GRPR expression may indicate presence of HNSCC.
Methods
We measured GRPR mRNA levels in histologically normal buccal mucosa from 65 surgical patients with HNSCC and 75 cancer-free control subjects using quantitative polymerase chain reaction (PCR). We tested for association between GRPR expression and HNSCC and evaluated differences in patient progression-free survival (PFS).
Results
Buccal GRPR expression was higher in cases but not controls who were active smokers (p = .04). High GRPR expression was associated with HNSCC (odds ratio [OR] = 3.55; 95% confidence interval [CI] = 1.15–10.93), even after adjustment for age, sex, tobacco use, and sample storage time. PFS did not differ between patients with HNSCC with high versus low GRPR expression (p = .22).
Conclusion
Elevated buccal GRPR expression was significantly associated with HNSCC independent of known risk factors but was not an indicator of disease prognosis.
doi:10.1002/hed.22963
PMCID: PMC3553276  PMID: 22431275
gastrin-releasing peptide receptor; head and neck cancer; case-control study; surrogate tissue biomarker; risk factor
20.  Overexpression of TACE and TIMP3 mRNA in head and neck cancer: association with tumour development and progression 
British Journal of Cancer  2010;104(1):138-145.
Background:
TACE/ADAM17 is a transmembranous protease that cleaves membrane-bound growth factors like EGFR ligands. TACE-dependent proteolysis is regulated by its inhibitor, tissue inhibitor of metalloproteinases 3 (TIMP3). This study analyses the role of TACE and TIMP3 mRNA expression in squamous cell carcinomas of the head and neck (HNSCCs).
Methods:
We analysed TACE and TIMP3 mRNA expression in HNSCCs from 106 patients by RNA in situ hybridisation.
Results:
TACE mRNA was upregulated in HNSCCs compared with dysplastic (P<0.05) and normal epithelia (P<0.001), with strong hybridisation signals in 21.9% of invasive tumour tissues and 4.5% of dysplasia. Elevated mRNA levels were accompanied by increased amounts of TACE protein in HNSCCs. TIMP3 mRNA expression in HNSCC-associated stroma was significantly higher than in the stroma adjacent to dysplastic or normal epithelia. Expression of TACE mRNA in HNSCCs was associated with tumour stage (P=0.019) and regional lymph node metastasis (P=0.009). Furthermore, levels of TACE mRNA in HNSCCs correlated with the expression of TIMP3 mRNA in HNSCC-associated stroma. Concomitantly, patients expressing high levels of TACE and TIMP3 mRNA showed significantly reduced overall survival compared with those with low mRNA levels.
Conclusion:
Our results indicate an important role of TACE and TIMP3 during development and progression of HNSCCs.
doi:10.1038/sj.bjc.6606017
PMCID: PMC3039790  PMID: 21102583
TACE; TIMP3; squamous cell carcinoma of the head and neck (HNSCC)
21.  Topical Polyethylene Glycol as a Novel Chemopreventive Agent for Oral Cancer via Targeting of Epidermal Growth Factor Response 
PLoS ONE  2012;7(6):e38047.
Head and neck squamous cell carcinoma (HNSCC) is a major cause of morbidity and mortality underscoring the need for safe and effective chemopreventive strategies. Targeting epidermal growth factor receptor (EGFR) is attractive in that it is an early critical event in HNSCC pathogenesis. However, current agents lack efficacy or have unacceptable toxicity. Several groups have demonstrated that the over-the-counter medication, polyethylene glycol (PEG) has remarkable chemopreventive efficacy against colon carcinogenesis. Importantly, we reported that this effect is mediated through EGFR internalization/degradation. In the current study, we investigated the chemopreventive efficacy of this agent against HNSCC, using both the well validated animal model 4-NQO (4-nitroquinoline 1-oxide) rat model and cell culture with the human HNSCC cell line SCC-25. We demonstrated that daily topical application of 10% PEG-8000 in the oral cavity (tongue and cavity wall) post 4NQO initiation resulted in a significant reduction in tumor burden (both, tumor size and tumors/tumor bearing rat) without any evidence of toxicity. Immunohistochemical studies depicted decreased proliferation (number of Ki67-positive cells) and reduced expression of EGFR and its downstream effectors cyclin D1 in the tongue mucosa of 4NQO-rats treated with PEG. We showed that EGFR was also markedly downregulated in SCC-25 cells by PEG-8000 with a concomitant induction of G1-S phase cell-cycle arrest, which was potentially mediated through upregulated p21cip1/waf1. In conclusion, we demonstrate, for the first time, that PEG has promising efficacy and safety as a chemopreventive efficacy against oral carcinogenesis.
doi:10.1371/journal.pone.0038047
PMCID: PMC3366973  PMID: 22675506
22.  Smad4 loss in mice causes spontaneous head and neck cancer with increased genomic instability and inflammation 
The Journal of Clinical Investigation  2009;119(11):3408-3419.
Smad4 is a central mediator of TGF-β signaling, and its expression is downregulated or lost at the malignant stage in several cancer types. In this study, we found that Smad4 was frequently downregulated not only in human head and neck squamous cell carcinoma (HNSCC) malignant lesions, but also in grossly normal adjacent buccal mucosa. To gain insight into the importance of this observation, we generated mice in which Smad4 was deleted in head and neck epithelia (referred to herein as HN-Smad4–/– mice) and found that they developed spontaneous HNSCC. Interestingly, both normal head and neck tissue and HNSCC from HN-Smad4–/– mice exhibited increased genomic instability, which correlated with downregulated expression and function of genes encoding proteins in the Fanconi anemia/Brca (Fanc/Brca) DNA repair pathway linked to HNSCC susceptibility in humans. Consistent with this, further analysis revealed a correlation between downregulation of Smad4 protein and downregulation of the Brca1 and Rad51 proteins in human HNSCC. In addition to the above changes in tumor epithelia, both normal head and neck tissue and HNSCC from HN-Smad4–/– mice exhibited severe inflammation, which was associated with increased expression of TGF-β1 and activated Smad3. We present what we believe to be the first single gene–knockout model for HNSCC, in which both HNSCC formation and invasion occurred as a result of Smad4 deletion. Our results reveal an intriguing connection between Smad4 and the Fanc/Brca pathway and highlight the impact of epithelial Smad4 loss on inflammation.
doi:10.1172/JCI38854
PMCID: PMC2769185  PMID: 19841536
23.  Identification of guanine nucleotide-binding protein γ-7 as an epigenetically silenced gene in head and neck cancer by gene expression profiling 
International Journal of Oncology  2013;42(4):1427-1436.
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.
doi:10.3892/ijo.2013.1808
PMCID: PMC3981008  PMID: 23403885
guanine nucleotide-binding protein γ-7; gene expression; silencing; head and neck squamous cell carcinoma; epigenetics
24.  Tissue-specific alternative splicing of the Drosophila dopa decarboxylase gene is affected by heat shock. 
Molecular and Cellular Biology  1993;13(8):4549-4555.
The Drosophila dopa decarboxylase gene, Ddc, is expressed in the hypoderm and in a small number of cells in the central nervous system (CNS). The unique Ddc primary transcript is alternatively spliced in these two tissues. We investigated whether Ddc splicing in the CNS is a general property of the CNS or a unique property of the cells that normally express Ddc by expressing the Ddc primary transcript ubiquitously under the control of an Hsp70 heat shock promoter. Under basal expression conditions, Ddc splicing shows normal tissue specificity, indicating that the regulation of Ddc splicing in the CNS is tissue specific rather than cell specific. Previous studies have shown that severe heat shock blocks mRNA splicing in cultured Drosophila melanogaster cells. Our results show that splicing of the heat shock-inducible Hsp83 transcript is very resistant to heat shock. In contrast, under either mild or severe heat shock, the splicing specificity of the heat shock-induced Ddc primary transcript is affected, leading to the accumulation of inappropriately high levels of the CNS splice form in non-CNS tissues. The chromosomal Ddc transcript is similarly affected. These results show unexpected heterogeneity in the splicing of individual mRNAs as a response to heat shock and suggest that the Ddc CNS-specific splicing pathway is the default.
Images
PMCID: PMC360072  PMID: 8336700
25.  Epidermal growth factor receptor targeted therapy in stages III and IV head and neck cancer 
Current Oncology  2010;17(3):37-48.
Question
What are the benefits associated with the use of anti–epidermal growth factor receptor (anti-egfr) therapies in squamous cell carcinoma of the head and neck (hnscc)? Anti-egfr therapies of interest included cetuximab, gefitinib, lapatinib, zalutumumab, erlotinib, and panitumumab.
Perspectives
Head-and-neck cancer includes malignant tumours arising from a variety of sites in the upper aerodigestive tract. The most common histologic type is squamous cell carcinoma, and most common sites are the oral cavity, the oropharynx, the hypopharynx, and the larynx. Worldwide, hnscc is the sixth most common neoplasm, and despite advances in therapy, long-term survival in hnscc patients is poor. Primary surgery followed by chemoradiation, or primary chemoradiation, are the standard treatment options for patients with locally advanced (stages iii–ivb) hnscc; however, meta-analytic data indicate that the benefit of concurrent platinum-based chemotherapy disappears in patients over the age of 70 years.
Cetuximab is a monoclonal antibody approved for use in combination with radiation in the treatment of patients with untreated locally advanced hnscc and as monotherapy for patients with recurrent or metastatic (stage ivc) hnscc who have progressed on platinum-based therapy.
Given the interest in anti-egfr agents in advanced hnscc, the Head and Neck Cancer Disease Site Group (dsg) of Cancer Care Ontario’s Program in Evidence-Based Care (pebc) chose to systematically review the literature pertaining to this topic so as to develop evidence-based recommendations for treatment.
Outcomes
Outcomes of interest included overall and progression-free survival, quality of life, tumour response rate and duration, and the toxicity associated with the use of anti-egfr therapies.
Methodology
The medline, embase, and Cochrane Library databases, the American Society of Clinical Oncology online conference proceedings, the Canadian Medical Association InfoBase, and the National Guidelines Clearinghouse were systematically searched to locate primary articles and practice guidelines. The reference lists from relevant review articles were searched for additional trials. All evidence was reviewed, and that evidence informed the development of the clinical practice guideline. The resulting recommendations were approved by the Report Approval Panel of the pebc, and by the Head and Neck Cancer dsg. An external review by Ontario practitioners completed the final phase of the review process. Feedback from all parties was incorporated to create the final practice guideline.
Results
The electronic search identified seventy-four references that were reviewed for inclusion. Only four phase iii trials met the inclusion criteria for the present guideline. No practice guidelines, systematic reviews, or meta-analyses were found during the course of the literature search.
The randomized controlled trials (rcts) involved three distinct patient populations: those with locally advanced hnscc being treated for cure, those with incurable advanced recurrent or metastatic hnscc being treated with first-line platinum-based chemotherapy, and those with incurable advanced recurrent or metastatic hnscc who had disease progression despite, or who were unsuitable for, first-line platinum-based chemotherapy.
Practice Guideline
These recommendations apply to adult patients with locally advanced (nonmetastatic stages iii–ivb) or recurrent or metastatic (stage ivc) hnscc.
Platinum-based chemoradiation remains the current standard of care for treatment of locally advanced hnscc.
In patients with locally advanced hnscc who are medically unsuitable for concurrent platinumbased chemotherapy or who are over the age of 70 years (because concurrent chemotherapy does not appear to improve overall survival in this patient population), the addition of cetuximab to radical radiotherapy should be considered to improve overall survival, progression-free survival, and time to local recurrence.
Cetuximab in combination with platinum-based combination chemotherapy is superior to chemotherapy alone in patients with recurrent or metastatic hnscc, and is recommended to improve overall survival, progression-free survival, and response rate.
The role of anti-egfr therapies in the treatment of locally advanced hnscc is currently under study in large randomized trials, and patients with hnscc should continue to be offered clinical trials of novel agents aimed at improving outcomes.
Qualifying Statements
Chemoradiation is the current standard of care for patients with locally advanced hnscc, and to date, there is no evidence that compares cetuximab plus radiotherapy with chemoradiation, or that examines whether the addition of cetuximab to chemoradiation is of benefit in these patients. However, five ongoing trials are investigating the effect of the addition of egfr inhibitors concurrently with, before, or after chemoradiotherapy; those trials should provide direction about the best integration of cetuximab into standard treatment.
In patients with recurrent or metastatic hnscc who experience progressive disease despite, or who are unsuitable for, first-line platinum-based chemotherapy, gefitinib at doses of 250 mg or 500 mg daily, compared with weekly methotrexate, did not increase median overall survival [hazard ratio (hr): 1.22; 96% confidence interval (ci): 0.95 to 1.57; p = 0.12 (for 250 mg daily vs. weekly methotrexate); hr: 1.12; 95% ci: 0.87 to 1.43; p = 0.39 (for 500 mg daily vs. weekly methotrexate)] or objective response rate (2.7% for 250 mg and 7.6% for 500 mg daily vs. 3.9% for weekly methotrexate, p > 0.05). As compared with methotrexate, gefitinib was associated with an increased incidence of tumour hemorrhage (8.9% for 250 mg and 11.4% for 500 mg daily vs. 1.9% for weekly methotrexate).
PMCID: PMC2880902  PMID: 20567625
Head-and-neck cancer; epidermal growth factor receptor; egfr inhibitors; overall survival; progression-free survival; tumour response rate

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