Kavita M. Pattani

What makes a good flap go bad? A critical analysis of the literature of intraoperative factors related to free flap failure.

The purpose of this article is to discuss the various factors related to poor outcome in free flap reconstructions of the head and neck (H&N). Free tissue transfer has become the standard reconstruction technique for complex H&N defects. With the evolution of free flap use in H&N surgery, numerous factors have been proposed that adversely affect flap outcome, many with the support of only anecdotal experience. We seek to critically review the literature to assess what evidence exists for the relation of various intraoperative factors to free flap complications.

Endothelin receptor type B gene promoter hypermethylation in salivary rinses is independently associated with risk of oral cavity cancer and premalignancy.

Endothelin receptor type B (EDNRB) and kinesin family member 1A (KIF1A) are candidate tumor suppressor genes that are inactivated in cancers. In this study, we evaluated the promoter hypermethylation of EDNRB and KIF1A and their potential use for risk classification in prospectively collected salivary rinses from patients with premalignant/malignant oral cavity lesions. Quantitative methylation-specific PCR was performed to analyze the methylation status of EDNRB and KIF1A in salivary rinses of 191 patients. We proceeded to determine the association of methylation status with histologic diagnosis and estimate classification accuracy. On univariate analysis, diagnosis of dysplasia/cancer was associated with age and KIF1A or EDNRB methylation. Methylation of EDNRB highly correlated with that of KIF1A (P < 0.0001). On multivariable modeling, histologic diagnosis was independently associated with EDNRB (P = 0.0003) or KIF1A (P = 0.027) methylation. A subset of patients analyzed (n = 161) without prior biopsy-proven malignancy received clinical risk classification based on examination. On univariate analysis, EDNRB and risk classification were associated with diagnosis of dysplasia/cancer and remained significant on multivariate analysis (EDNRB: P = 0.047, risk classification: P = 0.008). Clinical risk classification identified dysplasia/cancer with a sensitivity of 71% and a specificity of 58%. The sensitivity of clinical risk classification combined with EDNRB methylation improved to 75%. EDNRB methylation in salivary rinses was independently associated with histologic diagnosis of premalignancy and malignancy and may have potential in classifying patients at risk for oral premalignant and malignant lesions in settings without access to a skilled dental practitioner. This may also potentially identify patients with premalignant and malignant lesions that do not meet the criteria for high clinical risk based on skilled dental examination. Cancer Prev Res; 3(9); 1093–103. ©2010 AACR.

Transoral robotic‐assisted thyroidectomy: A preclinical feasibility study in 2 cadavers

Technological advances in thyroid surgery have included various “minimally invasive” thyroidectomy techniques, both open and endoscopic. These include not only minimally invasive video‐associated approaches but also variations of the transaxillary approach. More recently, a transoral technique using video assistance has been reported for thyroidectomy. Use of the robot was also recently published in a transaxillary approach to the thyroid. We hypothesized that the robot in combination with the previously described transoral technique would facilitate this novel surgical approach.

EDNRB and DCC Salivary Rinse Hypermethylation Has a Similar Performance as Expert Clinical Examination in Discrimination of Oral Cancer/Dysplasia versus Benign Lesions

Purpose: Promoter hypermethylation has been recently proposed as a means for head and neck squamous cell carcinoma (HNSCC) detection in salivary rinses. In a prospective study of a high-risk population, we showed that endothelin receptor type B (EDNRB) promoter methylation in salivary rinses is a useful biomarker for oral cancer and premalignancy. Experimental Design: Using that cohort, we evaluated EDNRB methylation status and 8 additional genes. Clinical risk assessment by expert clinicians was conducted and compared with biomarker performance in the prediction of premalignant and malignant disease. Methylation status of 9 genes was analyzed in salivary rinses of 191 patients by quantitative methylation-specific PCR. Results: HOXA9, EDNRB, and deleted in colorectal cancer (DCC) methylation were associated (P = 0.012; P < 0.0001; P = 0.0005) with premalignant or malignant disease. On multivariable modeling, histological diagnosis was only independently associated with EDNRB (P = 0.0003) or DCC (P = 0.004) methylation. A subset of patients received clinical risk classification (CRC) by expert clinicians based on lesion examination. CRC, DCC, and EDNRB were associated with diagnosis of dysplasia/cancer on univariate (P = 0.008; P = 0.026; P = 0.046) and multivariate analysis (P = 0.012; P = 0.037; P = 0.047). CRC identified dysplasia/cancer with 56% of sensitivity and 66% of specificity with a similar area under curve [AUC; 0.61, 95% confidence interval (CI) = 0.60–0.81] when compared to EDNRB and DCC combined AUC (0.60, 95% CI = 0.51–0.69), sensitivity of 46% and specificity of 72%. A combination of EDNRB, DCC, and CRC was optimal AUC (0.67, 95% CI = 0.58–0.76). Conclusions: EDNRB and/or DCC methylation in salivary rinses compares well to examination by an expert clinician in CRC of oral lesions. These salivary biomarkers may be particularly useful in oral premalignancy and malignancy screening in clinical care settings in which expert clinicians are not available. Clin Cancer Res; 19(12); 3268–75. ©2013 AACR.

Implementation of a TORS program in an academic medical center

Transoral robotic surgery (TORS) is rapidly being adopted by many head and neck surgeons for treatment of upper aerodigestive tract tumors. Various obstacles exist to efficiently implement this novel surgical technique in a busy academic center. We present our experience to illustrate one approach to initiating a TORS program.

The role of MAGEA2 in head and neck cancer.

OBJECTIVE To examine the role of MAGEA2 in the tumorigenesis of head and neck squamous cell carcinoma (HNSCC). DESIGN Primary tissue microarray data and quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed that MAGEA2 is differentially overexpressed in HNSCC. Functional analyses were then performed using MAGEA2 transfections and small-interfering RNA knockdowns with subsequent anchorage-dependent growth studies and cell cycle analyses. Quantitative RT-PCR was used to evaluate expression changes in p53 downstream targets after transfection of MAGEA2 into normal upper aerodigestive cell lines. RESULTS MAGEA2 is differentially overexpressed in HNSCC. In addition, MAGEA2 promotes growth in normal oral keratinocytes, whereas knockdown of MAGEA2 in HNSCC cells decreases growth. Using the HCT116 p53 wt and null cell line system, transfection of MAGEA2 induced growth in the p53 wt cell line while providing no growth advantage in the p53 mutant cells. Subsequently, transfection of MAGEA2 induced a decrease in messenger RNA expression of the p53 downstream targets CDKN1A and BAX and decreased G1 arrest in cells allowed to remain confluent for longer than 48 hours. CONCLUSIONS These data suggest that MAGEA2 is differentially expressed in HNSCC and functions, in part, through the p53 pathway by increasing cellular proliferation and abrogating cell cycle arrest. This improved understanding of MAGEA2 function and expression patterns will potentially allow for the improved ability to use MAGEA2 for detection, surveillance, and targeted therapeutics.

MAGEB2 is Activated by Promoter Demethylation in Head and Neck Squamous Cell Carcinoma

Purpose Although promoter hypermethylation has been an accepted means of tumor suppressor gene inactivation, activation of otherwise normally repressed proto-oncogenes by promoter demethylation has been infrequently documented. Experimental Design In this study we performed an integrative, whole-genome analysis for discovery of epigenetically activated proto-oncogenes in head and neck cancer tumors. We used the 47K GeneChip U133 Plus 2.0 Affymetrix expression microarray platform to obtain re-expression data from 5-aza treated normal cell line and expression data from primary head and neck squamous cell carcinoma (HNSCC) tumor tissues and normal mucosa tissues. We then investigated candidate genes by screening promoter regions for CpG islands and bisulfite sequencing followed by QUMSP and RT PCR for the best candidate genes. Finally, functional studies were performed on the top candidate gene. Results From the top 178 screened candidates 96 had CpG islands in their promoter region. Seven candidate genes showed promoter region methylation in normal mucosa samples and promoter demethylation in a small cohort of primary HNSCC tissues. We then studied the demethylation of the top 3 candidate genes in an expanded cohort of 76 HNSCC tissue samples and 17 normal mucosa samples. We identified MAGEB2 as having significant promoter demethylation in primary head and neck squamous cell carcinoma tissues. We then found significantly higher expression of MAGEB2 in tumors in a separate cohort of 73 primary HNSCC tissues and 31 normal tissues. Finally, we found that MAGEB2 has growth promoting effects on minimally transformed oral keratinocyte cell lines but not a definite effect on HNSCC cell lines. Conclusion In conclusion, we identified MAGEB2 as activated by promoter demethylation in HNSCCand demonstrates growth promoting effects in a minimally transformed oral keratinocyte cell line. More studies are needed to evaluate MAGBE2s exact role in HNSCC.

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

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

Long-Term Experience in Sentinel Node Biopsy for Early Oral and Oropharyngeal Squamous Cell Carcinoma

The presence of nodal disease is a key prognostic indicator in oral cavity and oropharyngeal carcinoma. Detection and management strategies for occult nodal metastasis in oral cavity and oropharyngeal squamous cell carcinoma (OSCC) continue to pose considerable controversy. Clinically negative (cN0) necks in early-stage OSCC tend to harbor occult disease within the cervical lymph nodes in 20–30% of cases. This high rate of occult nodal metastasis generates substantial clinical interest in optimizing accurate histopathological staging of the neck. The effectiveness of sentinel node biopsy (SNB) has been studied as a potential minimally invasive technique for accurately predicting pathological nodal status. In this study, Broglie et al. prospectively assess the longterm experience and survival data utilizing SNB as a staging tool. Their cohort consisted of 58 patients of whom 29 had a (-) SNB and 29 had a (?) SNB and who were followed by neck dissection. The addition of postoperative radiation was restricted to those patients with pN2b/c disease and (?) ECS only. Similar to other solid tumor models, including breast, there are efficiencies that are intuitively attractive in performing SNB versus staging neck dissections for OSCC. However, several quandaries still exist in implementing SNB as standard of care for staging patients with OSCC for END. The false-negative rate was reported in this study as 10% (3/29) in those patients with negative SNB. Presumably these patients would have been appropriately staged and/or treated with selective neck dissection. The authors also indicated that 1/29 patients with SNB (?) had contralateral recurrence, which could indicate a potentially missed sentinel node resulting in persistent disease. Previous studies have reported contralateral sentinel lymph nodes occurring in up to 20% of cases. Conversely, in the survival analysis performed by Broglie et al., patients exhibited 5 year OS, DFS, and DSS of 88, 96, and 96% versus 74, 73, and 77% in (-) SNB compared with (?) SNB, respectively. The DSS in the latter group was statistically significant and demonstrated a persistently poor survival rate in those patients with early stage OSCC and cN0, regardless of having undergone SNB and subsequent END for pathologically node-positive disease. So, although delayed nodal metastases during a waitand-scan policy portend poor overall prognosis, it is unclear if sentinel node biopsy results in any comparable improvement in survival. In view of rising healthcare costs, it is essential to study the survival impact and the cost-effectiveness of performing SNB. Thus, although this study is limited by its cohort size, it would be necessary to demonstrate a survival advantage prior to subjecting patients with a cN0 neck to SNB instead of a wait-and-scan policy. This is especially true if approximately three-quarters of patients (SNB negative) will undergo the procedure with no survival benefit. Moreover, those patients who were identified as the ‘‘high-risk’’ group and selected for END through a (?) SNB still had a lower survival rate yet incurred an additional procedure prior to definitive neck dissection. It is also difficult to define data regarding the surgical complication and morbidities of staging neck dissection compared with sentinel node biopsy. Although the authors’ results are encouraging in demonstrating the feasibility and reliability of SNB, continued Society of Surgical Oncology 2011

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

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