Samantha J. Hauff

UM-SCC-104: A New human papillomavirus-16–positive cancer stem cell–containing head and neck squamous cell carcinoma cell line†

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).

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.

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.

Preoperative topical antimicrobial decolonization in head and neck surgery

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.

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.

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.

Head and neck squamous cell carcinoma in pregnant women.

The aim of this study was to investigate oral cancer in pregnant women, a rare but therapeutically challenging patient subset.

Reporting Achievement of Medical Student Milestones to Residency Program Directors: An Educational Handover.

PROBLEM Competency-based education, including assessment of specialty-specific milestones, has become the dominant medical education paradigm; however, how to determine baseline competency of entering interns is unclear-as is to whom this responsibility falls. Medical schools should take responsibility for providing residency programs with accurate, competency-based assessments of their graduates. APPROACH A University of Michigan ad hoc committee developed (spring 2013) a post-Match, milestone-based medical student performance evaluation for seven students matched into emergency medicine (EM) residencies. The committee determined EM milestone levels for each student based on assessments from the EM clerkship, end-of-third-year multistation standardized patient exam, EM boot camp elective, and other medical school data. OUTCOMES In this feasibility study, the committee assessed nearly all 23 EM milestones for all seven graduates, shared these performance evaluations with the program director (PD) where each student matched, and subsequently surveyed the PDs regarding this pilot. Of the five responding PDs, none reported using the traditional medical student performance evaluation to customize training, four (80%) indicated that the proposed assessment provided novel information, and 100% answered that the assessment would be useful for all incoming trainees. NEXT STEPS An EM milestone-based, post-Match assessment that uses existing assessment data is feasible and may be effective for communicating competency-based information about medical school graduates to receiving residency programs. Next steps include further aligning assessments with competencies, determining the benefit of such an assessment for other specialties, and articulating the national need for an effective educational handover tool between undergraduate and graduate medical education institutions.

Model for Developing Educational Research Productivity: The Medical Education Research Group.

Introduction Education research and scholarship are essential for promotion of faculty as well as dissemination of new educational practices. Educational faculty frequently spend the majority of their time on administrative and educational commitments and as a result educators often fall behind on scholarship and research. The objective of this educational advance is to promote scholarly productivity as a template for others to follow. Methods We formed the Medical Education Research Group (MERG) of education leaders from our emergency medicine residency, fellowship, and clerkship programs, as well as residents with a focus on education. First, we incorporated scholarship into the required activities of our education missions by evaluating the impact of programmatic changes and then submitting the curricula or process as peer-reviewed work. Second, we worked as a team, sharing projects that led to improved motivation, accountability, and work completion. Third, our monthly meetings served as brainstorming sessions for new projects, research skill building, and tracking work completion. Lastly, we incorporated a work-study graduate student to assist with basic but time-consuming tasks of completing manuscripts. Results The MERG group has been highly productive, achieving the following scholarship over a three-year period: 102 abstract presentations, 46 journal article publications, 13 MedEd Portal publications, 35 national didactic presentations and five faculty promotions to the next academic level. Conclusion An intentional focus on scholarship has led to a collaborative group of educators successfully improving their scholarship through team productivity, which ultimately leads to faculty promotions and dissemination of innovations in education.

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% CD44high cells, respectively. CD44high 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. CD44high cells from the primary tumor resulted in lung colony formation in 2 out of 2 tail vein injections in mice, whereas CD44low cells did not. Similarly, CD44high cells from UM-SCC-103 formed lung tumors in 2 out of 4 mice, whereas CD44low 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.