Patrick K. Ha

Promoter methylation and inactivation of tumour-suppressor genes in oral squamous-cell carcinoma

Genetic alterations that lead to loss or changes in tumour-suppressor genes are known to contribute to oral carcinogenesis. Traditional molecular methods to detect such losses have relied on mutation analysis or deletion of the gene. However, epigenetic mechanisms could also contribute to silencing of tumour-suppressor genes. Methylation regions rich in CpG promoters prevent DNA transcription by changing the binding of histone complexes. The substantial contribution of methylation, specifically in oral squamous-cell carcinoma, is now being realised and investigated.

The molecular biology of mucosal field cancerization of the head and neck.

Field cancerization was first described in 1953 as histologically altered epithelium surrounding tumor samples taken from the upper aerodigestive tract. Since then, the term has been used to describe multiple patches of pre-malignant disease, a higher-than-expected prevalence of multiple local second primary tumors, and the presence of synchronous distant tumors within the upper aerodigestive tract. Molecular techniques such as karyotype analysis, microsatellite analysis, p53 mutation screening, and X-chromosome inactivation studies have further refined the relationship among these lesions. While there are differences in the techniques used to identify the clonal origins of the lesions, these studies indicate that there is often lateral clonal spread of pre-malignant or malignant disease, and a significant portion of local second primary tumors are in fact genetically related. Distant second primary tumors found in the esophagus are often not related to concurrent head and neck cancer, whereas synchronous squamous lung tumors with a head and neck primary are often, in fact, metastases, rather than independently arising malignancies. These observations help to explain the high incidence of recurrent disease, despite excision or other therapy--pre-malignant or malignant clones often have the ability to migrate and persist outside of the field of treatment. Therefore, alternative means of prevention or therapy that can affect the entire head and neck region may be of benefit to such patients. Future studies will further refine the relationship among these lesions and perhaps identify key molecular alterations to be used as targets for gene therapy.

Genetics of pancreatic cancer: From genes to families

Cancer of the pancreas is a genetic disease. Sporadic cancers of the pancreas are frequently associated with the activation of an oncogene, K-ras, and the inactivation of multiple tumor suppressor genes, including p53, DPC4, p16, and BRCA2. An improved understanding of the genetics of pancreas cancer should lead to new tests to screen for this disease and novel rational gene-based therapies.

Mitochondrial DNA quantity increases with histopathologic grade in premalignant and malignant head and neck lesions.

Purpose: Mitochondria are highly susceptible to oxidative damage. Although mitochondrial function decreases with oxidative damage, overall mitochondrial DNA (mtDNA) content increases to compensate for general mitochondrial dysfunction. We performed quantitative polymerase chain reaction for genes specific to mitochondrial and nuclear genomes to investigate relative mitochondrial abundance in a spectrum of dysplastic head and neck lesions. Experimental Design: DNA from mild, moderate, and severe dysplasias, as well as invasive tumors and normal mucosal cells, was extracted. Using quantitative polymerase chain reaction, mitochondrial to nuclear DNA ratios were determined by quantification of cytochrome c oxidase subunit 1 (CoxI) and β-actin genes. Results: Mean CoxI/β-actin DNA ratios for mild, moderate, and severe premalignant lesions were 0.0529, 0.0607, and 0.1021, respectively. The mean ratio for the normal mucosal cells contained in saliva was 0.0537, whereas the mean ratio for tumors was 0.1667. As a whole, our experimental model demonstrated significance (P = 0.0358). Comparisons between individual categories showed borderline significance when compared with the normal group, with P values of 0.0673, 0.0747, and 0.0824 for moderate and severe dysplasia and invasive tumor, respectively. Conclusions: Head and neck squamous cell carcinomas arise through premalignant intermediates and may be merely morphologic manifestations of accumulated genetic alterations. In keeping with this molecular tumor progression model, our study shows that mtDNA increases according to histopathologic grade, a phenomenon that may be a feedback mechanism that compensates for a generalized decline in respiratory chain function. Therefore, high mtDNA content may be another marker of genetic alteration, a measure of relative DNA injury, and a surrogate measure of histopathologic grade.

The Role of Human Papillomavirus in Oral Carcinogenesis

Human papillomavirus (HPV) infection with high-risk types 16 and 18 has widely been reported as one of the prominent mechanisms behind the development of cervical squamous cell carcinoma. Links between HPV and oral cavity cancer have been suggested as well, based on epidemiologic and molecular means, though the association is less well-established. It is likely that HPV plays a role in oral cavity carcinogenesis, though only in a small subset of cases. The difficulty in providing true causal evidence of HPVs role in oral cancer lies in our lack of understanding of the significance of mechanisms by which HPV leads to oral carcinogenesis, as well as limitations in the molecular analysis of HPV. Further studies are necessary for the contribution of HPV in oral cavity malignancy to be better demonstrated.

Osteosarcoma of the head and neck: A review of the johns hopkins experience

Objective: To determine factors including treatment modalities which influence survival in patients with osteosarcoma of the head and neck. Study Design: Retrospective clinicopathologic study of 27 patients with osteosarcoma of the head and neck. Methods: The clinical charts and pathology slides were reviewed on 27 patients who had osteosarcoma of the head and neck between 1946 and 1998. The following variables were examined for their effect on survival: age of diagnosis, site of tumor, presentation, race, sex, prior radiation exposure, retinoblastoma history, margin status, and method of treatment. Results: The average age at the time of diagnosis of the patients was 37.6 years (range, 7–82 y). The sex distribution was similar with 14 male and 13 female patients. Eight of 27 patients had osteosarcoma of the mandible, 9 of 27 had osteosarcoma of the maxilla and paranasal sinuses, and in 10 of 27 patients osteosarcoma occurred elsewhere, including the temporal bones, occipital bones, and orbit. The overall 2‐year survival was 66% with a 5‐year survival rate of 55%. Conclusions: Positive surgical margins and a high tumor grade were found to have a statistically deleterious effect on overall survival. There was no detectable effect on survival of age, race, sex, prior radiation exposure, tumor site, and tumor cell type. It was not possible to differentiate between the different adjuvant treatment modalities because of the small numbers in the study. Key Words: Bone, malignancy, osteogenic sarcoma, radiation therapy, surgery.

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.

Detection of somatic mutations and HPV in the saliva and plasma of patients with head and neck squamous cell carcinomas

Tumor DNA in saliva and plasma can provide a noninvasive biomarker for head and neck squamous cell carcinoma. A cancer test that’s worth a spit Head and neck squamous cell carcinoma is one of the most common cancers worldwide, and its incidence is increasing. This is a difficult-to-treat cancer for which few targeted agents are available, and there are no biomarkers for monitoring therapeutic progress. Wang et al. discovered that tumor DNA can be detected and analyzed in the blood of most patients with head and neck cancers, as well as in the saliva of those with cancers of the oral cavity. Moreover, they found preliminary evidence suggesting that tumor DNA may be detectable in saliva before clinical evidence of tumor recurrence, which may be useful for patient monitoring if this result is confirmed in larger studies. To explore the potential of tumor-specific DNA as a biomarker for head and neck squamous cell carcinomas (HNSCC), we queried DNA from saliva or plasma of 93 HNSCC patients. We searched for somatic mutations or human papillomavirus genes, collectively referred to as tumor DNA. When both plasma and saliva were tested, tumor DNA was detected in 96% of 47 patients. The fractions of patients with detectable tumor DNA in early- and late-stage disease were 100% (n = 10) and 95% (n = 37), respectively. When segregated by site, tumor DNA was detected in 100% (n = 15), 91% (n = 22), 100% (n = 7), and 100% (n = 3) of patients with tumors of the oral cavity, oropharynx, larynx, and hypopharynx, respectively. In saliva, tumor DNA was found in 100% of patients with oral cavity cancers and in 47 to 70% of patients with cancers of the other sites. In plasma, tumor DNA was found in 80% of patients with oral cavity cancers, and in 86 to 100% of patients with cancers of the other sites. Thus, saliva is preferentially enriched for tumor DNA from the oral cavity, whereas plasma is preferentially enriched for tumor DNA from the other sites. Tumor DNA in saliva was found postsurgically in three patients before clinical diagnosis of recurrence, but in none of the five patients without recurrence. Tumor DNA in the saliva and plasma appears to be a potentially valuable biomarker for detection of HNSCC.

TKTL1 Is Activated by Promoter Hypomethylation and Contributes to Head and Neck Squamous Cell Carcinoma Carcinogenesis through Increased Aerobic Glycolysis and HIF1α Stabilization

Purpose: This study aims to investigate the role of the aberrant expression of Transkelolase-like 1 (TKTL1) in head and neck squamous cell carcinoma (HNSCC) tumorigenesis and to characterize TKTL1 contribution to HNSCC tumorigenesis through aerobic glycolysis and HIF1α stabilization. Experimental Design: TKTL1 promoter hypomethylation and mRNA/protein aberrant expression were studied in human HNSCC tumor samples and normal mucosas. Oncogenic functions of TKTL1 were examined in HNSCC cell line panels and tumor xenograft models with TKTL1 expression construct. The metabolite levels of fructose-6-phosphate, glyceraldehydes-3-phosphate, pyruvate, lactate, and the levels of HIF1α protein and its downsteam glycolytic targets were compared between the TKTL1-expressing and vehicle-expressing HNSCC cells. Meanwhile, the effects of HIF1α/glycolytic inhibitors were evaluated on the TKTL1 transfectants. Results: TKTL1 exhibits high frequency of promoter hypomethylation in HNSCC tumors compared with the normal mucosas, correlating with its overexpression in HNSCC. Overexpression of TKTL1 in HNSCC cells promoted cellular proliferation and enhanced tumor growth in vitro and in vivo. Overexpression of TKTL1 increased the production of fructose-6-phosphate and glyceraldehyde-3-phosphate, in turn elevating the production of pyruvate and lactate, resulting in the normoxic stabilization of the malignancy-promoting transcription factor HIF1α and the upregulation of downstream glycolytic enzymes. Notably, the reduction of TKTL1 expression decreased HIF1α accumulation and inhibition with HIF1α and/or the glycolysis inhibitor could abrogate the growth effects mediated by TKTL1 overexpression. Conclusion: TKTL1 is a novel candidate oncogene that is epigenetically activated by aberrant hypomethlation and contributes to a malignant phenotype through altered glycolytic metabolism and HIF1α accumulation. Clin Cancer Res; 16(3); 857–66

Applying the molecular biology and epigenetics of head and neck cancer in everyday clinical practice.

During the past decade, there has been a significant increase in knowledge regarding the molecular biology and epigenetics of head and neck squamous cell carcinoma (HNSCC). This has been aided by the steady development of new technology and novel techniques aimed at elucidating additional aberrant molecular alterations characteristic of HNSCC, including the advent of high throughput assays and the development of more sophisticated bioinformatics tools. In addition, advancements in the field of cancer epigenetics and microRNA have increased the complexity of understanding HNSCC tumorigenesis. These advances have lead to an increasing number of translational studies in the diagnosis, prognosis, and treatment of head and neck cancer. The end result is that molecular biomarkers, gene detection panels and targeted therapeutics are becoming a reality for the care of patients with HNSCC. In this article, we will focus on the many implications of this research as it pertains to clinical practice and the treatment of HNSCC patients.