UM-SCC-103: a unique tongue cancer cell line that recapitulates the tumorigenic stem cell population of the primary tumor.

OBJECTIVE: A new head and neck cancer cell line was developed from a highly aggressive HNSCC of the oral cavity diagnosed in a 26-year-old pregnant woman. METHODS: Cells from the primary tumor were passaged in culture and genotyped as a unique cell line. The resultant cell line was assessed for its ability to replicate the primary tumor. RESULTS: The primary tumor and cell line contained 19.03% and 19.62% CD44(high) cells, respectively. CD44(high) cancer stem cells from UM-SCC-103 formed tumors after flank injections in mice that reconstituted the heterogeneity of the primary tumor. CD44 staining and histology in the primary tumor and tumors grown in vivo from the cell line were similar. CD44(high) cells from the primary tumor resulted in lung colony formation in 2 out of 2 tail vein injections in mice, whereas CD44(low) cells did not. Similarly, CD44(high) cells from UM-SCC-103 formed lung tumors in 2 out of 4 mice, whereas CD44(low) cells did not. CONCLUSION: The similarity in marker expression and tumorigenic behavior between the primary tumor and the resulting cell line strongly suggests that the cell line resembles the primary tumor that it was derived from and provides an important new research tool for the study of head and neck carcinomas in young patients.

Intranodal cystic changes: a potential radiologic signature/biomarker to assess the human papillomavirus status of cases with oropharyngeal malignancies.

OBJECTIVE: The aim of this study was to determine if lymph node imaging findings can predict human papillomavirus (HPV) positivity in oropharyngeal squamous cell cancers. METHODS AND MATERIALS: Pretreatment postcontrast neck computed tomographic scans of 49 patients (male, 35; female, 14; age range, 45-76 years) diagnosed with oropharyngeal malignancies and with available HPV data were retrospectively reviewed. Metastatic lymph nodes were identified based on standardly accepted size and morphological criteria. Various lymph node parameters were studied, including presence of cystic foci in the metastatic lymph nodes, abnormal lymph nodes showing low-attenuation foci, matted lymph nodes, and morphologically normal smaller (<1.5 cm) lymph nodes. These parameters were then independently correlated with the available HPV status of these patients. Finally, an extended criterion, that is, intranodal cystic changes in cases with morphologically normal small (<1.5 cm) lymph nodes, was correlated with HPV status. Sensitivity, specificity, and positive predictive values (PPVs) and negative predictive values (NPVs) were calculated. RESULTS: Of these 49 cases with oropharyngeal cancers, 27 were HPV positive, and 22 cases were HPV negative. Eight cases (3 HPV positive and 5 HPV negative) did not have metastatic lymph nodes. Of remaining 41 cases with metastatic abnormal lymph nodes, 26 were HPV positive, and 15 were HPV negative. Of these 41 cases with metastatic lymph nodes, 14 had 1 or more lymph nodes with cystic foci. Of these 14 cases, 10 (71.4%) were HPV positive. Resultant sensitivity, specificity, PPV, and NPV of cystic foci for the presence of HPV status were 38.4%, 73.3%, 71.4%, and 40.7%, respectively. Intranodal cystic changes in cases with morphologically normal small (<1.5 cm) lymph nodes were found in 5 cases; all 5 were HPV positive. Resultant accuracy was specificity and PPV of 100%, sensitivity of 19.2% and NPV of 41.6%. CONCLUSIONS: Intranodal cystic changes seen on the pretreatment postcontrast neck computed tomographic scan of patients with oropharyngeal malignancies are radiologic signatures strongly associated with the HPV status of the patient. The results in this initial study warrant larger prospective studies to determine if this finding may be used in addition to other molecular biomarkers to help identify those patients who may be amenable to the most appropriate treatment options.

Finding the Right Job: Locum Tenens and Private Practice.

When choosing a career path in otolaryngology, one might consider locum tenens as an opportunity to sample a practice or locale before deciding where to settle down. It may also be an excellent way to garner some new perspective and experience while between jobs or while unable to commit to a long-term position. Before deciding to pursue locum tenens otolaryngology, it is important to consider logistical and lifestyle factors. When deciding on an assignment, one must consider the proposed work schedule, call obligation, patient acuity, and reimbursement.

Combined TP53 mutation/3p loss correlates with decreased radiosensitivity and increased matrix-metalloproteinase activity in head and neck carcinoma.

OBJECTIVE: Patients with head and neck squamous cell carcinoma (HNSCC) containing TP53 mutation and 3p deletion ("double-hit") have poorer prognosis compared to patients with either event alone ("single-hit"). The etiology for worse clinical outcomes in patients with "double-hit" cancers is unclear. We compared radiosensitivity of cell lines containing both TP53 mutations and deletion of Fragile Histidine Triad (FHIT, the gene most commonly associated with 3p deletion) to "single-hit" lines with only TP53 mutation. We compared radiosensitivity in a "single-hit" cell line with TP53 mutation converted to "double-hit" using RNA interference targeting FHIT. Finally, we compared matrixmetalloproteinase-2/9 (MMP-2/9) activity, a previously-established biomarker for tumor aggressiveness, in xenograft tumors derived from these cell lines. MATERIALS/METHODS: TP53 mutation and FHIT deletion profiles of HNSCC lines were established using Cancer Cell Line Encyclopedia (CCLE). We used RNA-interference to convert a "single-hit" cell line (SCC4) to "double-hit". Cultured cells were examined for radiosensitivity and cisplatin sensitivity. MMP-2/9 activity was evaluated in "double-hit" versus "single-hit" tumors using ratiometric activatable cell-penetrating peptide (RACPP) in tongue (n=17) and flank xenografts (n=4). RESULTS: Radiotherapy caused greater double-stranded DNA breaks in "single-hit" vs naturally occurring and engineered "double-hit" cells. In-vivo, "double-hit" xenografts demonstrated higher MMP-2/9 activity compared to "single-hit" xenografts (p<0.01). There was no difference in cisplatin sensitivity between the cell lines. CONCLUSIONS: TP53 mutation combined with FHIT deletion correlates with decreased radiosensitivity in HNC cell lines. Xenograft from "double-hit" cells exhibit increased MMP-2/9 activity. These findings may in part account for the worse clinical outcome seen in patients with HNSCC "double-hit" tumors.

Matrix-metalloproteinases in head and neck carcinoma-cancer genome atlas analysis and fluorescence imaging in mice.

OBJECTIVE: (1) Obtain matrix-metalloproteinase (MMP) expression profiles for head and neck squamous cell carcinoma (HNSCC) specimens from the Cancer Genomic Atlas (TCGA). (2) Demonstrate HNSCC imaging using MMP-cleavable, fluorescently labeled ratiometric activatable cell-penetrating peptide (RACPP). STUDY DESIGN: Retrospective human cohort study; prospective animal study. SETTING: Translational research laboratory. SUBJECTS AND METHODS: Patient clinical data and mRNA expression levels of MMP genes were downloaded from TCGA data portal. RACPP provides complementary ratiometric fluorescent contrast (increased Cy5 and decreased Cy7 intensities) when cleaved by MMP2/9. HNSCC-tumor bearing mice were imaged in vivo after RACPP injection. Histology was evaluated by a pathologist blinded to experimental conditions. Zymography confirmed MMP-2/9 activity in xenografts. RACPP was applied to homogenized human HNSCC specimens, and ratiometric fluorescent signal was measured on a microplate reader for ex vivo analysis. RESULTS: Expression of multiple MMPs including MMP2/9 is greater in patient HNSCC tumors than matched control tissue. In patients with human papilloma virus positive (HPV+) tumors, higher MMP2 and MMP14 expression correlates with worse 5-year survival. Orthotopic tongue HNSCC xenografts showed excellent ratiometric fluorescent labeling with MMP2/9-cleavable RACPP (sensitivity = 95.4%, specificity = 95.0%). Fluorescence ratios were greater in areas of higher tumor burden (P < .03), which is useful for intraoperative margin assessment. Ex vivo, human HNSCC specimens showed greater cleavage of RACPP when compared to control tissue (P = .009). CONCLUSIONS: Human HNSCC tumors show increased mRNA expression of multiple MMPs including MMP2/9. We used RACPP, a ratiometric fluorescence assay of MMP2/9 activity, to show improved occult tumor identification and margin clearance. Ex vivo assays using RACPP in biopsy specimens may identify patients who will benefit from intraoperative RACPP use.

Head and neck cancer stem cells: the effect of HPV--an in vitro and mouse study.

OBJECTIVES: To determine if the behavior of cancer stem cells (CSCs) is affected by human papillomavirus (HPV) status. STUDY DESIGN: An in vitro and in vivo analysis of HPV and CSCs. SETTING: University laboratory. SUBJECTS AND METHODS: We isolated CSCs from HPV-positive and HPV-negative cell lines. Two HPV-negative cell lines underwent lentiviral transduction of E6/E7. Chemoresistence was determined using colony formation assays. Native HPV-positive and HPV E6/E7-transduced cells were compared for lung colonization after tail vein injection in NOD/SCID mice. RESULTS: The proportion of CSC is not significantly different in HPV-positive or HPV-negative head and neck squamous cell carcinoma (HNSCC) cell lines. The HNSCC CSCs are more resistant to cisplatin than the non-CSCs, but there were no significant differences between HPV-positive and HPV-negative cells. The HPV-negative cancer cells yielded low colony formation after cell sorting. After transduction with HPV E6/E7, increased colony formation was observed in both CSCs and non-CSCs. Results from tail vein injections yielded no differences in development of lung colonies between HPV E6/E7-transduced cells and nontransduced cells. CONCLUSIONS: Human papillomavirus status does not correlate with the proportion of CSCs present in HNSCC. The HPV-positive cells and those transduced with HPV E6/E7 have a greater clonogenicity than HPV-negative cells. The HNSCC CSCs are more resistant to cisplatin than non-CSCs. This suggests that common chemotherapeutic agents may shrink tumor bulk by eliminating non-CSCs, whereas CSCs have mechanisms that facilitate evasion of cell death. Human papillomavirus status does not affect CSC response to cisplatin therapy, suggesting that other factors explain the better outcomes for patients with HPV-positive cancer.

Head and neck squamous cell carcinoma in pregnant women.

BACKGROUND: The aim of this study was to investigate oral cancer in pregnant women, a rare but therapeutically challenging patient subset. METHODS: After institutional review board approval, an EMERSE search was used to identify all women treated at the University of Michigan from 1998 to 2010 with head and neck squamous cell carcinoma (HNSCC) during pregnancy. This identified 4 patients with tongue cancer. Biomarkers and human papillomavirus (HPV) were assessed by immunohistochemistry and multiplex PCR/mass spectrometry, respectively. RESULTS: Two patients responded well to therapy and are alive more than 10 years after diagnosis; 2 patients died of disease. All tumors overexpressed EGFR and Bcl-xL, 3 of 4 overexpressed c-Met, both tumors that progressed overexpressed p53. All tumors were negative for HPV, p16, estrogen receptor, progesterone receptor, and HER-2. CONCLUSIONS: Biomarkers of aggressive tumors (high EGFR, c-Met; high Bcl-xL-low p53) did not correlate with outcome. Additional studies are needed to determine whether perineural invasion, delay in diagnosis, and p53 overexpression are factors in poor survival.

High-risk human papillomavirus detection in oropharyngeal, nasopharyngeal, and oral cavity cancers: comparison of multiple methods.

IMPORTANCE: Human papillomaviruses are now recognized as an etiologic factor in a growing subset of head and neck cancers and have critical prognostic importance that affects therapeutic decision making. There is no universally accepted gold standard for high-risk HPV (hrHPV) assessment in formalin-fixed, paraffin-embedded (FFPE) tissue specimens, nor is there a clear understanding of the frequency or role of hrHPV in sites other than oropharynx. OBJECTIVE: To determine the optimal assessment of hrHPV in FFPE head and neck tumor tissue specimens. DESIGN, SETTING, PARTICIPANTS: In the setting of a large Midwestern referral center, assessment of hrHPV by p16 immunohistochemical staining, in situ hybridization, and polymerase chain reaction (PCR)-MassArray (PCR-MA), with L1 PGMY-PCR and sequencing to resolve method discordance, was conducted for 338 FFPE oropharyngeal, nasopharyngeal, and oral cavity tumor tissue specimens. Relative sensitivity and specificity were compared to develop a standard optimal test protocol. Tissue specimens were collected from 338 patients with head and neck cancer treated during the period 2001 through 2011 in the departments of Otolaryngology, Radiation Oncology, and Medical Oncology. INTERVENTION: Patients received standard therapy. MAIN OUTCOMES AND MEASURES: Optimal hrHPV identification, detection, and activity in head and neck cancers. RESULTS: Using combined PCR-MA with L1 PGMY-PCR and sequencing for conclusive results, we found PCR-MA to have 99.5% sensitivity and 100% specificity, p16 to have 94.2% sensitivity and 85.5% specificity, and in situ hybridization to have 82.9% sensitivity and 81.0% specificity. Among HPV-positive tumors, HPV16 was most frequently detected, but 10 non-HPV16 types accounted for 6% to 50% of tumors, depending on the site. Overall, 86% of oropharynx, 50% of nasopharynx, and 26% of oral cavity tumors were positive for hrHPV. CONCLUSIONS AND RELEVANCE: PCR-MA has a low DNA (5 ng) requirement effective for testing small tissue samples; high throughput; and rapid identification of HPV types, with high sensitivity and specificity. PCR-MA together with p16INK4a provided accurate assessment of HPV presence, type, and activity and was determined to be the best approach for HPV testing in FFPE head and neck tumor tissue specimens.

Preoperative topical antimicrobial decolonization in head and neck surgery.

OBJECTIVES/HYPOTHESIS: Surgical site infections (SSIs) are an important cause of morbidity and mortality after head and neck surgery. Our primary objective was to determine the efficacy of preoperative topical antimicrobial decolonization before head and neck surgery. STUDY DESIGN: Prospective, randomized controlled trial. METHODS: This study was conducted among 84 patients presenting for head and neck surgery requiring admission to an academic medical center. Preoperative cultures were performed to identify Staphylococcus aureus carriers. Patients were randomized to preoperative topical antimicrobial decolonization with a 5-day regimen of chlorhexidine skin rinses and intranasal mupirocin coupled with standard perioperative systemic antimicrobial prophylaxis, versus standard prophylaxis alone. The main outcome was the incidence of SSIs. RESULTS: Despite a trend suggesting a decrease in SSIs with perioperative topical antimicrobial decolonization (24% vs. 10%), there was no significant difference (odds ratio, 0.34; 95% confidence interval, 0.10-1.18; P = .079). Patients with a higher American Society of Anesthesiologists score (3 vs. 1; P = .02), with more operative blood loss (P = .05), and who required operative takeback (P = .04) had a higher rate of SSIs; there was a trend suggesting a higher rate of SSIs among patients undergoing clean-contaminated surgery compared to clean cases (P = .08) and among those having received prior radiation (P = .07) or chemotherapy (P = .06). CONCLUSIONS: Preoperative antimicrobial decolonization did not significantly decrease the incidence of SSIs after head and neck surgery, but might be considered for high-risk groups despite the lack of conclusive evidence confirming efficacy. Risk factors for SSIs after head and neck surgery are identified for the first time in a prospective study.

UM-SCC-104: a new human papillomavirus-16-positive cancer stem cell-containing head and neck squamous cell carcinoma cell line.

BACKGROUND: Few human papillomavirus (HPV)(+) head and neck squamous cell carcinoma (HNSCC) cell lines exist. We established University of Michigan-squamous cell carcinoma-104 (UM-SCC-104), a new HPV(+) HNSCC cell line from a recurrent oral cavity tumor, and characterized it for the presence of cancer stem cells (CSCs). METHODS: Tumor cells were tested for biomarker expression by immunohistology, and the presence of HPV was assessed by several methods. RESULTS: UM-SCC-104 has a unique genotype, contains HPV-16, and expresses E6/E7. Inoculation of aldehyde dehydrogenase (ALDH)(+) and ALDH(-) cells in an immunocompromised mouse resulted in tumor growth from the ALDH(+) cells after 6 weeks that recapitulated the histology of the primary, whereas ALDH(-) cells did not produce tumors. CONCLUSION: UM-SCC-104, a new HPV-16, CSC-containing HNSCC cell line will aid in studying recurrent HPV(+) tumors. The aggressive nature of this tumor is consistent with high uniform expression of epidermal growth factor receptor (EGFR) and a functionally significant proportion of ALDH(+) CSCs.