ABSTRACTBackgroundThe purpose of this study was to investigate the impact of surgical intervention on detection of circulating tumor cells (CTCs) in patients with squamous cell carcinoma of the head and neck (SCCHN.)MethodsWe utilized a negative depletion technique to identify cytokeratin (CK)‐positive CTCs. The numbers of CTCs immediately before and after surgical resection were compared.ResultsSeventy‐six blood samples from 38 patients with SCCHN were examined. Seventy‐nine percent of the patients had CTCs detected before and after surgery. A total of 7.89% had no CTCs before surgery, yet had CTCs identified after surgery. Overall, 60.5% of patients had an increased number of CTCs/mL after surgery with a mean increase of 6.63‐fold. A statistically significant increase in CTCs was seen after surgery (p = .02).ConclusionThe timing of sample collection in patients with SCCHN who have surgical intervention can potentially impact the number of CTCs identified. © 2016 Wiley Periodicals, Inc. Head Neck 38: 1799–1803, 2016

Carcinoma of the head and neck represents 3.5% of all cancers, and the vast majority of these tumors are squamous cell carcinoma (HNSCC). With a stable overall survival rate of 50% among all stages, there is continued interested in developing measures for early detection and disease aggressiveness. Circulating tumor cells (CTCs) have been identified as a potential marker for early metastatic disease, response to treatment, and surveillance in head and neck squamous cell carcinoma. In this article, techniques of CTC detection, applications of CTC technology, and outcomes of HNSCC patients will be discussed.

Distant metastases in head and neck cancer patients almost invariably herald a poor prognosis. Five year survival rates for early stage localized head and neck cancers are over 80% but this drop to 40% where disease has spread to neck nodes, and to below 20% for patients with distant metastatic disease. A key process in this metastatic cascade is the transition of tumor cells from an adherent epithelial phenotype into a highly motile and invasive mesenchymal phenotype. We have recently demonstrated that IL-6 is a potent inducer of epithelial-mesenchymal transition (EMT) in HNSCC. These results therefore suggest a potential role of IL-6 in the release of tumor cells from the primary tumors. Recently, we have also shown that low number of CTCs predict a significantly higher disease free survival. However, there is no published report showing a correlation between serum IL-6 levels and CTCs. In this study, we examined if serum IL-6 levels directly correlate with CTCs and also examined if high IL-6 levels could predict poor prognosis in HNSCC patients.

5511 Background: The currently accepted definition of CTCs are cells that have: a nuclei, cytokeratin+ EpCAM+, and CD45-. Emerging evidence suggests that other rare circulating cells are present in the blood of metastatic cancer patients including CD45+ cytokeratin cells. A negative depletion process to isolate and quantify circulating tumor cells from the blood of head and neck cancer patients, using immunomagnetic separation was developed and currently be validated on a number of solid tumors, including SCCHN. Correlation of number of CTCs, tumor site, tumor stage, nodal status, smoking/alcohol abuse, histopathological characteristics, and clinical outcome was made. In addition, a subgroup of patients had blood collected immediately before and immediately after surgical resection to investigate any differences in the number of CTCs present. Methods: Prospective clinical follow-up study of 50 patients diagnosed with SCCHN undergoing surgical intervention had peripheral blood examined for the presence of CTCs. Five to 9 mL of peripheral blood was processed using previously described negative depletion enrichment with immunomagnetic tagging and removal of CD45 positive cells (Jatana et al. 2010). Subsequently, the cells were stained for cytokeratin. In addition, in some samples, other markers were targeted including vimentin, EGFR, and CD44. Results: With a mean follow-up of 24 months, data suggests that patients with detectable CTCs in the peripheral blood had a significantly reduced probability of disease-free survival (p=0.01). No correlation between the presence of CTCs with regard to tumor site, tumor stage, nodal status, smoking/alcohol use, and histopathological characteristics was observed. In a subgroup of 25 patients who had 2 consecutive samples taken immediately before and after resection, 64% of patients had an increase in number of CTCs after resection. We also identified a number of potential CTC, or cancer associated circulating cells of interest, including cells which are cytokeratin negative, vimentin positive and EGFR positive. Conclusions: Our initial data suggest a potential, important correlation between CTC and patient outcome and justifies further study.

UNLABELLED:Little is known about the potential involvement of the oncoprotein gankyrin in human oral cancer progression. In this study, the levels of gankyrin mRNA and protein expression were assessed in human oral epithelial cell lines, at-risk normal oral tissues, premalignant oral lesions, and primary oral squamous cell carcinomas (OSCCs).MATERIALS AND METHODS:Biopsies included 6 oral epithelial cell lines, 32 OSCC specimens for qRT-PCR analysis, 27 OSCC specimens and 12 premalignant oral lesions for immunohistochemical analysis.RESULTS:Gankyrin was overexpressed in all tested oral epithelial cell lines and the majority of OSCC specimens (32/32 (100%) and 21/27 (71%) at the mRNA and protein levels, respectively). Moreover, 6/12 of premalignant oral lesions overexpressed gankyrin protein.CONCLUSION:Gankyrin overexpression is a prevalent event in human oral cancer and occurs during the early stages of oral carcinogenesis, thus being a viable therapeutic or chemopreventive target in oral cancer.

Future OncologyVol. 7, No. 4 EditorialIdentification of circulating tumor cells: a prognostic marker in squamous cell carcinoma of the head and neck?Kris R Jatana, Jas C Lang & Jeffrey J ChalmersKris R Jatana† Author for correspondence, Jas C LangDepartment of Otolaryngology – Head & Neck Surgery, The Ohio State University, 915 Olentangy River Road, Suite 4000, Columbus, OH 43212, USA & Jeffrey J ChalmersWilliam G Lowrie Department of Chemical & Biomolecular Engineering, The Ohio State University, 140 West 19th Avenue, Columbus, OH 43210, USAPublished Online:4 Apr 2011https://doi.org/10.2217/fon.11.19AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail View articleKeywords: circulating tumor cellimmunocytochemistryimmunomagnetic cell separationsquamous cell carcinoma of the head and neckBibliography1 Franceschi D, Gupta R, Spiro RH, Shah JP: Improved survival in the treatment of squamous carcinoma of the oral tongue. 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Focus on Circulating Tumor Cells (CTCs)Oral Oncology, Vol. 74Enrichment of circulating head and neck tumour cells using spiral microfluidic technology15 February 2017 | Scientific Reports, Vol. 7, No. 1Head and Neck Cancer Biomarkers in Circulation26 November 2016Clinicopathological and Prognostic Significance of Circulating Tumor Cells in Patients with Head and Neck Cancer: A Meta-AnalysisInternational Journal of Medical Physics, Clinical Engineering and Radiation Oncology, Vol. 05, No. 02Prognostic value of circulating tumor cells with podoplanin expression in patients with locally advanced or metastatic head and neck squamous cell carcinoma24 July 2014 | Head & Neck, Vol. 37, No. 10Circulating tumour cells in metastatic head and neck cancers11 August 2014 | International Journal of Cancer, Vol. 136, No. 11Prognostic value of circulating tumor cells in patients with squamous cell carcinoma of the head and neck: a systematic review and meta-analysis22 April 2015 | Medical Oncology, Vol. 32, No. 5Prognostic Relevance of Circulating Tumor Cells in Blood and Disseminated Tumor Cells in Bone Marrow of Patients with Squamous Cell Carcinoma of the Oral Cavity16 January 2014 | Clinical Cancer Research, Vol. 20, No. 2Circulating tumor cells in head and neck cancer: clinical impact in diagnosis and follow-up14 February 2013 | European Archives of Oto-Rhino-Laryngology, Vol. 271, No. 1Squamous cell carcinoma of the oral cavity and circulating tumour cellsWorld Journal of Clinical Oncology, Vol. 5, No. 2Significance of circulating tumor cell detection using the CellSearch system in patients with locally advanced head and neck squamous cell carcinoma22 February 2013 | European Archives of Oto-Rhino-Laryngology, Vol. 270, No. 10Detection of circulating tumour cells with the CellSearch system in patients with advanced-stage head and neck cancer: preliminary results9 July 2013 | The Journal of Laryngology & Otology, Vol. 127, No. 8 Vol. 7, No. 4 eToC Sign up Follow us on social media for the latest updates Metrics Downloaded 478 times History Published online 4 April 2011 Published in print April 2011 Information© Future Medicine LtdKeywordscirculating tumor cellimmunocytochemistryimmunomagnetic cell separationsquamous cell carcinoma of the head and neckFinancial & competing interests disclosureJJ Chalmers is supported by the National Science Foundation (BES-0124897), the National Cancer Institute (R33CA81662) and the National Cancer Institute CCC Core Grant (P30 CA16058), and JJ Chalmers and JC Lang are supported by the State of Ohio Third Frontier Program (ODOD 26140000:TECH 07-001). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.PDF download

OBJECTIVES:to present and discuss a high-performance negative depletion method for the isolation of circulating tumor cells (CTCs) in the blood of patients with head and neck cancer and to determine the correlation between the presence of CTCs and early clinical outcome in these patients.DESIGN:prospective clinical follow-up study of patients with squamous cell carcinoma of the head and neck (SCCHN) undergoing surgical intervention, who had peripheral blood examined for the presence of CTCs.PATIENTS:the study population comprised 48 patients diagnosed as having SCCHN and undergoing surgical intervention.INTERVENTION:a negative depletion process to isolate and quantify CTCs from the blood of patients with SCCHN using immunomagnetic separation was developed and validated. Immunostaining for cytokeratin was performed on the enriched samples to determine the number of CTCs extracted from each patient's blood sample. Correlation of the presence of CTCs, tumor stage, nodal status, clinical characteristics, and outcome was made.MAIN OUTCOME MEASURE:disease-free survival.RESULTS:our initial data, that have a mean follow-up of 19.0 months, suggest that patients with no detectable CTCs per milliliter of blood had a significantly higher probability of disease-free survival (P = .01). There was no correlation between the presence of CTCs with regard to age, sex, tumor site, stage, or nodal involvement.CONCLUSIONS:our enrichment technology, based on the removal of normal cells, has been used on the peripheral blood of patients with head and neck cancer for which follow-up data were collected. If no CTCs were present, a statistically significant improved disease-free survival was observed in SCCHN. A blood test with such a prognostic capability could have important implications in the treatment of patients with head and neck cancer.

Abstract Background: Breast circulating tumor cells (CTCs) are commonly isolated by positive selection enrichment technology, which targets the epithelial cell adhesion activating molecule, EpCAM. However, CTCs with low or no EpCAM expression, such as those from basal and normal-like subtypes, are more likely to be missed by this method. We developed a novel negative enrichment technology to detect CTCs with mesenchymal and stem cell markers in localized and metastatic breast cancer (BC). Patients and Methods: Twenty nine patients with localized and metastatic BC initiating chemotherapy were enrolled. CTCs were isolated in 10 mL of peripheral blood using negative selection with immunomagnetic tagging and removal of CD45 positive cells at 3 time points: pretreatment, after one cycle of treatment (TP1) and at end of treatment or at disease progression in metastatic patients (TP2). Immunocytochemical staining for nucleus, cytokeratin (8,18,19), vimentin, and CD44 was completed on available samples. Double staining for nucleus and cytokeratin with high nucleus to cytoplasm ratio defined a CTC. Enrollment is ongoing. Results: Median age was 57 yrs (range 28-78 yrs); stage distributions were I-2 (7%), II-10 (35%), III-3 (10%), and IV-14 (48%); 19 (66%) were estrogen receptor positive (ER+), 4 (14%) estrogen and progesterone receptor negative (ER-PR-HER2 non-overexpressing) and 6 (21%) HER2 overexpressing. Negative enrichment yielded an average log10 depletion of nucleated cells of 2.74 and an overall, average log10 depletion of 5.2 (>100,000 enrichment). No CTCs were identified in 5 healthy volunteers or in buffy coats purchased from the Red Cross. CTCs were identified in all stages at pretreatment but decreased to 0 in three patients by TP1. Baseline median CTC level was 373/mL in localized BC (range 2.2-1975/mL) and 761/mL in metastatic BC (range 9.9-47513/mL). In localized BC, the median percent (%) change in CTCs was +2% at TP1 (n= 13; range −100% to +14945%) and −95% at TP2 (n=5; range −100% to −13%). In metastatic BC, median CTC numbers decreased by 41% at TP1 (n=11; range −100% to +4222%) and increased at TP2 in 2 patients by 231% and 730%. Baseline CTC levels and changes at TP1 were not significantly different between localized and metastatic groups by the Wilcoxen Rank Sum test. All tested CTCs expressed vimentin and CD44. In addition to CTCs, a population of cells without detectable cytokeratin expression but positive for the other markers was identified in some samples. Further characterization of these cells for epithelial mesenchymal transition markers is underway. Conclusions: CTCs identified with this novel negative selection method have both mesenchymal and stem cell markers in localized and metastatic BC. Higher CTC numbers with epithelial characteristics are detected with this method relative to what is reported with positive selection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3284.

A43 INTRODUCTION : Recent studies have revealed a steady increase in the incidence of oral squamous cell carcinoma (OSCC) in patients 40 years of age or younger. Many of these patients lack traditional risk factors for OSCC, such as tobacco and alcohol use, indicating the possibility of underlying genetic factors influencing the development of cancer in this population. TGFBR1*6A is a common polymorphism of the type I TGF-beta receptor ( TGFBR1 ), which tranduces TGF-beta growth inhibitory signals less effectively than TGFBR1 . The *6A allele consists of a deletion of 3 alanines within a 9-alanine (*9A) repeat at the 3’-end of the exon 1 coding sequence, and epidemiological studies suggest it may act as a tumor susceptibility allele. Our previous work in head and neck squamous cell carcinoma (HNSCC) revealed 21.7% (49/226) of patients possessed the *6A allele compared to healthy control individuals (13.7%; 473/3451). Other non-9A alleles (*5A, *8A, *14A) were also identified in 3 additional cases (23%; 52/226). The purpose of the current study was to examine the frequency of the TGFBR1*6A tumor susceptibility allele in young OSCC patients. > METHODS : Genomic DNA was extracted from tumor specimens obtained from OSCC patients 40 years of age or younger, according to protocols approved by the IRB of The Ohio State University (OSU). TGFBR1 exon 1 was amplified using PCR, and the PCR products genotyped by OSU Plant Microbe Genomics Facility (OSU PMGF) using a 3730 DNA Analyzer (Applied Biosystems). > RESULTS : A total of 16 oral tumors were genotyped, with 5 (31.3%) possessing non-9A hypomorphic allele variants. Three patients had a genotype of *6A/*9A (18.8%), one patient had a genotype of *5A/*9A (6%), and one patient had a genotype of *9A/*10A (6%). There was a statistically significant difference in the proportion of non-*6A hypomorphic alleles present in the young OSCC group (12.5%) as compared to the HNSCC (1.33%) group (P-value = 0.0365). > CONCLUSIONS : Preliminary results from this small cohort of young OSCC patients indicate that a hypomorphic TGFBR1 allele may be influencing tumor susceptibility in this population. Further study of a larger cohort is needed to better characterize the proportion of hypomorphic TGFBR1 alleles in OSCC patients 40 years of age or younger and define the role of this susceptibility allele in HNSCC tumorigenesis. > SUPPORT: NIH/NIDCR T32DE014320, NCI P01DE12704 and R01DE011943, The V Foundation >

ContextTGFBR1*6A is a common polymorphism of the type I transforming growth factor β receptor (TGFBR1). Epidemiological studies suggest that TGFBR1*6A may act as a tumor susceptibility allele. How TGFBR1*6A contributes to cancer development is largely unknown.ObjectivesTo determine whether TGFBR1*6A is somatically acquired by primary tumors and metastases during cancer development and whether the 3–amino acid deletion that differentiates TGFBR1*6A from TGFBR1 is part of the mature receptor or part of the signal sequence and to investigate TGFBR1*6A signaling in cancer cells.Design, Setting, and PatientsTumor and germline tissues from 531 patients with a diagnosis of head and neck, colorectal, or breast cancer recruited from 3 centers in the United States and from 1 center in Spain from June 1, 1994, through June 30, 2004. In vitro translation assays, MCF-7 breast cancer cells stably transfected with TGFBR1*6A, TGFBR1, or the vector alone, DLD-1 colorectal cancer cells that endogenously carry TGFBR1*6A, and SW48 colorectal cancer cells that do not carry TGFBR1*6A.Main Outcome MeasuresTGFBR1*6A somatic acquisition in cancer. Determination of the amino terminus of the mature TGFBR1*6A and TGFBR1 receptors. Determination of TGF-β–dependent cell proliferation.ResultsTGFBR1*6A was somatically acquired in 13 of 44 (29.5%) colorectal cancer metastases, in 4 of 157 (2.5%) of colorectal tumors, in 4 of 226 (1.8%) head and neck primary tumors, and in none of the 104 patients with breast cancer. TGFBR1*6A somatic acquisition is not associated with loss of heterozygosity, microsatellite instability, or a mutator phenotype. The signal sequences of TGFBR1 and TGFBR1*6A are cleaved at the same site resulting in identical mature receptors. TGFBR1*6A may switch TGF-β growth inhibitory signals into growth stimulatory signals in MCF-7 breast cancer cells and in DLD-1 colorectal cancer cells.ConclusionsTGFBR1*6A is somatically acquired in 29.5% of liver metastases from colorectal cancer and may bestow cancer cells with a growth advantage in the presence of TGF-β. The functional consequences of this conversion appear to be mediated by the TGFBR1*6A signal sequence rather than by the mature receptor. The results highlight a new facet of TGF-β signaling in cancer and suggest that TGFBR1*6A may represent a potential therapeutic target in cancer.

Our laboratory previously described the independent isolation of the fibroblast growth factor 4 (FGF-4) gene by NIH3T3 transformation assay using DNA from a patient with CML leukemia (Lucas et al., 1994). The FGF-4 gene was truncated by DNA rearrangement with a novel gene named GRS. In this manuscript we describe isolation of GRS cDNA and show by sequence comparison that GRS is a novel member of the Bcl-2 gene family. Northern analysis shows expression of the gene in normal human tissue to be largely restricted to the hematopoietic compartment. Analysis of the pattern of gene expression in cancer cell lines demonstrates GRS is expressed in hematopoietic malignancies and in melanoma. The chromosomal location of GRS has also been determined. The gene is positioned on chromosome 15 within bands q24-25.

Papillomaviruses cause benign tumours in their natural hosts, which, in some cases, become foci for the appearance and spread of malignant carcinomas. It is therefore of considerable interest to analyse the strategy of gene expression of this group of viruses. Towards this end, we have used a generally applicable technique, in which we monitor the ability of DNA fragments to induce the expression of an inactive thymidine kinase (tk) gene, from which the promoter region has been deleted. Using this approach we now report for the first time the location of four transcriptional control sequences in the early region of the genome of bovine papillomavirus type 1 (BPV-1). Two of the sequences with the characteristics of 'promoter' elements are located proximal to the putative 5' leaders of the early genes. The third sequence is located within the region coding for the main body of the early transcripts, and the fourth within the region spanning the 3' end of the early open reading frames and the 5' end of the late ones. The fourth sequence displays the characteristics of an 'enhancer' element; however, unlike previously described 'enhancers', it is located three to five kilobases (kb) away from the first two promoters and to the 3' side of the third element.