Published papers

Background: Induction chemotherapy (IC) for head and neck cancer (HNC) often causes severe side-effects. However, it has still been challenging to predict the adverse events. The present study aimed to evaluate the role of hematological inflammatory markers in predicting severe side-effects caused by IC. Materials and Methods: A total of 54 HNC patients who underwent IC were enrolled. The association between severe side-effects and pre-treatment hematological inflammatory markers [the C-reactive protein (CRP) to albumin ratio (CAR), the modified Glasgow Prognostic Score (mGPS), the neutrophil-to-lymphocyte ratio (NLR), and the platelet-to-lymphocyte ratio (PLR)] were evaluated. Results: In the univariate analysis, the incidence of whole severe side-effects (grade 4), febrile neutropenia (above grade 3), and hyponatremia (above grade 3) were significantly higher in the high CAR and high GPS groups. Multivariate analysis revealed that high CAR and mGPS were independent predictors of these side-effects. Conclusion: CAR and mGPS were significant predictors of severe side-effects. These data can potentially offer patients an improved quality of life during cancer therapy.

Background: Precise imaging of nerves have been challenging in the head and neck region, mainly due to low spatial resolution. Here, we investigated how nerves in the head and neck region could be visualized using an ultra-high magnetic field MR system. Methods: We used formol-carbol-fixed human cadaveric necks and obtained MR diffusion tensor images (DTIs) using a 9.4 Tesla (T) ultra-high magnetic field MR system. Afterward, we prepared tissue sections and checked the anatomic relationships between the neurons and the carotid artery in order to confirm that the visualized fibers are indeed neuron fibers. Results: We were able to identify nerves, including the vagus nerve, the hypoglossal nerve, and the spinal-accessory nerve. Hematoxylin-eosin stained histological sections confirmed neuron fibers in the same anatomic position. Conclusion: This technique has the feasibility to be applied for a more accurate anatomic understanding, maybe even close to a histological level.

Background: Because the incidence of schwannoma arising from the parapharyngeal space (PPS) is very low, no studies have analyzed extirpation methods and postoperative neurological complications exclusively in PPS schwannomas. Methods: The preoperative diagnosis and clinical outcomes of surgical treatment in 21 patients with PPS schwannoma who underwent surgery were investigated. Results: Neurological deficit of the involved nerve developed in all patients regardless of the extirpation method used. However, the incidence of first bite syndrome in sympathetic chain schwannoma was significantly lower after intracapsular enucleation (40%) than after total resection (100%; P =.045). Furthermore, the incidence of postoperative complications unrelated to the involved nerve was lower after intracapsular enucleation (0%) than after total resection (42.9%; P =.055). Conclusion: Although postoperative neurological deficit of the involved nerve was unavoidable in PPS schwannoma, intracapsular enucleation could be beneficial by reducing its severity and the incidence of complications unrelated to the involved nerve.

Background: This study aimed to investigate p16 and COX2 expression in oropharyngeal squamous cell carcinoma (OPSCC), and evaluate the prognostic role of COX2 expression under the new TNM classification. Materials and Methods: Biopsy specimens obtained from 75 patients with OPSCC were stained for p16 and COX2 expression immunohistochemically. The results and clinical records were analyzed retrospectively. Results: Fifty-nine patients (79%) were positive for p16. COX2 expression was correlated with poor relapse-free survival in patients overall, and in p16-positive patients. Smoking was positively associated with COX2 expression. Moreover, both positive COX2 expression and anterior wall tumor subsite were independently correlated with lymph node metastasis, which was the only independent prognostic factor in p16-positive OPSCC. Conclusion: The p16-positive rate in this study was comparable with that in the USA and Europe, and higher than that in other Asian countries. COX2 expression might affect the prognosis of p16-positive OPSCC through promoting lymph node metastasis.

Traumatic optic neuropathy often occurs as a complication of head and mid facial injury, and can cause permanent impairment of the visual acuity. From previous studies, optic nerve decompression surgery has been recommended for cases of traumatic optic neuropathy with optic nerve canal fracture. However, the indications for this surgery still remain controversial. Moreover, optic nerve canal fracture cannot be detected even by computed tomography (CT) in some cases. Patients diagnosed as having traumatic optic neuropathy with optic nerve canal fracture are treated by endoscopic optic nerve decompression surgery and steroid administration, by otorhinolaryngologist in collaboration with neurosurgeons and ophthalmologists at Keio University Hospital. Six patients were treated between May 2015 and May 2017. They ranged in age from 17 to 78 years old, and the intervals from injury to surgery ranged from 19 hours to 6 days. Five patients had injury of the outer half of the eyebrows. Preoperative CT revealed optic canal fracture in only 3 cases, whereas intraoperatively, all 6 cases were detected to have fracture. Preoperative visual acuity was hand motion or better in 4 patients, and no light perception in 2 patients. The visual acuity improved postoperatively in 5 patients. No improvement was observed in the patient who underwent surgery 6 days after the injury. Blunt injury on the outer half of the eyebrow was a useful clue for the diagnosis of optic nerve canal fracture, especially in cases where it could not be detected by CT. In cases of traumatic optic neuropathy with optic nerve canal fracture, early decompression of the optic canal and steroid administration play a key role in the recovery of the visual acuity.

Background: Ossifying fibroma arising in the sinonasal tract is rare. Its treatment includes complete resection; however, this can be challenging to perform due to the well-vascularized nature of the fibroma. Some surgical techniques can control intraoperative hemorrhage; however, to date, no reports have assessed the use of preoperative embolization. Methods: We report a case of a child with ossifying fibroma arising from the ethmoid sinus, which was resected by endoscopic surgery using preoperative transarterial embolization. Conclusions: Embolization was effective in decreasing intraoperative hemorrhage. No complications related to the procedure or operation were encountered. Preoperative embolization should be considered for better control of the bleeding that occurs in ossifying fibromas, similar to that in other well-vascularized tumors.

Background: Targeted therapies specifically act by blocking the activity of proteins that are encoded by genes critical for tumorigenesis. However, most cancers acquire resistance and long-term disease remission is rarely observed. Understanding the time course of molecular changes responsible for the development of acquired resistance could enable optimization of patients' treatment options. Clinically, acquired therapeutic resistance can only be studied at a single time point in resistant tumors. Methods: To determine the dynamics of these molecular changes, we obtained high throughput omics data (RNA-sequencing and DNA methylation) weekly during the development of cetuximab resistance in a head and neck cancer in vitro model. The CoGAPS unsupervised algorithm was used to determine the dynamics of the molecular changes associated with resistance during the time course of resistance development. Results: CoGAPS was used to quantify the evolving transcriptional and epigenetic changes. Applying a PatternMarker statistic to the results from CoGAPS enabled novel heatmap-based visualization of the dynamics in these time course omics data. We demonstrate that transcriptional changes result from immediate therapeutic response or resistance, whereas epigenetic alterations only occur with resistance. Integrated analysis demonstrates delayed onset of changes in DNA methylation relative to transcription, suggesting that resistance is stabilized epigenetically. Conclusions: Genes with epigenetic alterations associated with resistance that have concordant expression changes are hypothesized to stabilize the resistant phenotype. These genes include FGFR1, which was associated with EGFR inhibitors resistance previously. Thus, integrated omics analysis distinguishes the timing of molecular drivers of resistance. This understanding of the time course progression of molecular changes in acquired resistance is important for the development of alternative treatment strategies that would introduce appropriate selection of new drugs to treat cancer before the resistant phenotype develops.

When treating advanced thyroid cancer cases with Lenvatinib, most of these patients are old and we sometimes hesitate to administer the drug to elderly cases. We have experienced administering low-dose Lenvatinib to aging patients who were over 80 years old. The first case was a 92-year-old man. He was administered 10mg of Lenvatinib at the outpatient clinic, could continue the treatment without serious side effects, and maintained PR for one year. The second case was an 86-year-old female. She had an obstructed airway due to tumor invasion. Due to her poor general condition, we decided to start with 4 mg of Lenvatinib. The trachea-invading tumor shrank, and she was able to stay at home, but eventually died when the tumor progressed. Low-dose Lenvatinib administration was shown to be tolerated and to reduce side effects even in the elderly. This treatment, however, may have insufficient antitumor effects. The organ function, social status, and living environment of older adults are varied, so we should decide the administration dose and method according to the condition of each case.

Purpose: We previously demonstrated an association between decreased SMAD4 expression and cetuximab resistance in head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to further elucidate the clinical relevance of SMAD4 loss in HNSCC. Experimental Design: SMAD4 expression was assessed by IHC in 130 newly diagnosed and 43 patients with recurrent HNSCC. Correlative statistical analysis with clinicopathologic data was also performed. OncoFinder, a bioinformatics tool, was used to analyze molecular signaling in TCGA tumors with low or high SMAD4 mRNA levels. The role of SMAD4 was investigated by shRNA knockdown and gene reconstitution of HPV-negative HNSCC cell lines in vitro and in vivo. Results: Our analysis revealed that SMAD4 loss was associated with an aggressive, HPV-negative, cetuximab-resistant phenotype. We found a signature of prosurvival and antiapoptotic pathways that were commonly dysregulated in SMAD4-low cases derived from TCGA-HNSCC dataset and an independent oral cavity squamous cell carcinoma (OSCC) cohort obtained from GEO. We show that SMAD4 depletion in an HNSCC cell line induces cetuximab resistance and results in worse survival in an orthotopic mouse model in vivo. We implicate JNK and MAPK activation as mediators of cetuximab resistance and provide the foundation for the concomitant EGFR and JNK/MAPK inhibition as a potential strategy for overcoming cetuximab resistance in HNSCCs with SMAD4 loss. Conclusions: Our study demonstrates that loss of SMAD4 expression is a signature characterizing the cetuximab-resistant phenotype and suggests that SMAD4 expression may be a determinant of sensitivity/resistance to EGFR/MAPK or EGFR/JNK inhibition in HPV-negative HNSCC tumors. (C) 2017 AACR.

Salivary duct carcinoma (SDC) is an uncommon, aggressive malignant neoplasm histologically resembling high-grade mammary ductal carcinoma. SDC can arise de novo or ex pleomorphic adenoma. To clarify the correlation of biomarker immunoprofile with clinicopathological findings and clinical outcome of SDC, we conducted immunohistochemistry for EGFR, HER2, HER3, AR, CK5/6, p53, and Ki-67, along with HER2 fluorescence in situ hybridization in 151 SDCs. SDCs ex pleomorphic adenoma more commonly overexpressed EGFR, HER2, HER3, and Ki-67 than de novo SDCs (P = 0.015, < 0.001, 0.045, and 0.02, respectively). In multivariate analysis, AR-and CK5/6+ were associated with shorter progression-free survival (P = 0.027 and 0.004, respectively). Moreover, patients with p53-extreme negative/positive demonstrated poorer overall survival (P = 0.007). On assessing the revised classification by the combination of biomarker expression, the percentages of each subtype were as follows: 'apocrine A' (AR+/HER2-/Ki-67-low) (24%), 'apocrine B' (AR+/HER2-/Ki-67-high) (18%), 'apocrine HER2' (AR+/HER2+) (35%), 'HER2-enriched' (AR-/HER2+) (12%), and 'double negative' (AR-/HER2-) (11%). 'Double negative' was further subclassified into 'basal-like' (EGFR and/or CK5/6+) (7%) and 'unclassified' (3%). Consequently,patients with 'apocrine A' showed a better progression-free survival than those with any other subtypes. Our revised immunoprofiling classification was valuable for predicting the survival and might be useful in personalized therapy for patients with SDC.

Background: Transoral videolaryngoscopic surgery (TOVS) was developed as a new distinct surgical procedure for hypopharyngeal cancer (HPC) and supraglottic cancer (SGC) staged at up to T3. However, long-term treatment outcomes of TOVS remain to be validated. Methods: Under a straight broad intraluminal view provided by combined use of a distending laryngoscope and a videolaryngoscope, we performed en bloc tumor resection via direct bimanual handling of the ready-made straight-form surgical instruments and devices. We retrospectively analyzed functional and oncologic outcomes of 72 patients with HPC (n = 58) or SGC (n = 14) whose minimum follow-up was 24 months or until death. Results: The cohort comprised nine patients of Tis, 23 of T1, 33 of T2, and 7 of T3. Among 36 patients (50%) who underwent neck dissection simultaneously, all but one were pathologically node-positive. Twelve patients underwent postoperative concurrent chemoradiation (CCRT) as adjuvant treatment, and another four patients underwent radiation or CCRT for second or later primary cancer. The endotracheal tube was removed in an operation room in all but two patients who underwent temporary tracheostomy. Pharyngeal fistula was formed transiently in two patients. The median time until patients resumed oral intake and could take a soft meal was 2 and 5 days, respectively. Eventually, 69 patients (96%) took normal meals. The 5-year cause-specific survival (CSS), overall survival (OS), larynx-preserved CSS, and loco-regional controlled CSS were 87.3%, 77.9%, 86.0%, and 88.0%, respectively. Multivariate analysis revealed N2-3 as an independent prognostic factor in both CSS (hazard ratio [HR] = 25.51, P = 0.008) and OS (HR = 4.90, P = 0.022), which indirectly reflected higher risk of delayed distant metastasis. Conclusions: Considering its sound functional and oncological outcomes with various practical advantages, TOVS can be a dependable, less invasive, and cost-effective surgical option of an organ-function preservation strategy for HPC and SGC.

The prognostic role of modified Glasgow Prognostic Score (mGPS), neutrophil-to- lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) in patients with salivary duct carcinoma (SDC) remains unclear. We conducted a multi-institutional retrospective cohort study of 140 SDC patients. The survival impact of these hematological markers was evaluated using multivariate proportional hazard models. High mGPS (>= 1) was significantly associated with worse survival (3-year overall survival (OS): 16.7% vs 66.1%, p-value=0.003; 3-year progression-free survival (PFS): 0.0% vs 27.9%, p-value<0.001). Additionally, high C-reactive protein (CRP) (>= 0.39 mg/dl) was significantly associated with worse survival (3-year OS: 32.1% vs 68.2%, p-value= 0.001; 3-year PFS: 7.1% vs 31.1%, p-value<0.001). These associations were consistent with multivariate analysis adjusted for established prognostic factors. Although we also found significant association of high NLR (>= 2.5) with OS (HR 1.80; 95% confidence interval, 1.05-3.08) in multivariate analysis, this association were inconsistent with the results of PFS. In addition, we found no significant associations of PLR with survival. In conclusion, we found that mGPS, CRP and NLR were identified as prognostic factors associated with survival in SDC patients.

Background: Transcranial surgery is considered more appropriate than an endonasal endoscopic approach (EEA) for a large cavernous sinus meningioma with lateral extension. Case presentation: A 6-cm-diameter hypervascular meningioma around cavernous sinus invaded the orbital apex and infratemporal fossa. 80% of the tumor was removed without too much blood loss via endoscopic endonasal transpterygoidal-infratemporal approach. Preoperative embolization from some feeding arteries was done, and 80% of the tumor staining disappeared. Discussion: Tumor removal rates for giant meningioma are worse with EEA than with the transcranial approach because of various anatomical limitations and blood control. Recently, EEA has become more widely used with approaches such as the transpterygoidal approach. The operation can be done safely with preoperative embolization because the operative view is clear without bleeding. Conclusion: Preoperative embolization for a large hypervascular tumor makes EEA more effective. A giant meningioma can be removed by EEA if the anatomical limitations can be identified and approached safely and effectively. (C) 2016 The Authors. Published by Elsevier B.V.

Patients with oncogene driven tumors are treated with targeted therapeutics including EGFR inhibitors. Genomic data from The Cancer Genome Atlas (TCGA) demonstrates molecular alterations to EGFR, MAPK, and PI3K pathways in previously untreated tumors. Therefore, this study uses bioinformatics algorithms to delineate interactions resulting from EGFR inhibitor use in cancer cells with these genetic alterations. We modify the HaCaT keratinocyte cell line model to simulate cancer cells with constitutive activation of EGFR, HRAS, and PI3K in a controlled genetic background. We then measure gene expression after treating modified HaCaT cells with gefitinib, afatinib, and cetuximab. The CoGAPS algorithm distinguishes a gene expression signature associated with the anticipated silencing of the EGFR network. It also infers a feedback signature with EGFR gene expression itself increasing in cells that are responsive to EGFR inhibitors. This feedback signature has increased expression of several growth factor receptors regulated by the AP-2 family of transcription factors. The gene expression signatures for AP-2alpha are further correlated with sensitivity to cetuximab treatment in HNSCC cell lines and changes in EGFR expression in HNSCC tumors with low CDKN2A gene expression. In addition, the AP-2alpha gene expression signatures are also associated with inhibition of MEK, PI3K, and mTOR pathways in the Library of Integrated Network-Based Cellular Signatures (LINCS) data. These results suggest that AP-2 transcription factors are activated as feedback from EGFR network inhibition and may mediate EGFR inhibitor resistance.

Among salivary gland malignancies, the prognosis of salivary duct carcinoma (SDC) is assumed to be the poorest. However, because of its low incidence, reliable survival estimates and prognostic factors based on a large number of patients remain to be elucidated, thereby making it impossible to standardize the optimal treatment for SDC. We performed this multi-institutional, retrospective analysis by collecting the clinical information of 141 patients with SDC without distant metastasis who underwent curative surgery as the initial treatment to elucidate overall survival (OS) and disease-free survival (DFS) along with their prognostic factors. The 3-year OS and DFS rates were 70.5 and 38.2 %, respectively. Multivariate analysis revealed that age a parts per thousand yen65 years (p < 0.001) and N1 and N2 (p = 0.047 and < 0.001, respectively) were independent prognostic factors for OS, whereas the primary site of the minor salivary and sublingual gland (p < 0.001) and N2 (p < 0.001) were those for DFS. The most common treatment failure was distant metastasis (55 patients, 39.0 %). For early parotid SDC, neither total parotidectomy in the patients with early T stage nor nerve resection in the patients without facial nerve palsy showed survival benefits. Advanced N stage independently affects both OS and DFS. Partial parotidectomy with facial nerve preservation could be a less invasive standard surgical procedure for parotid gland SDC in the early T stage without facial nerve palsy. Effective systemic therapy is imperative to improve DFS of SDC.

Conclusion The sigmoid-incision (S-I) rescue flap technique has the advantage of both reduced-invasiveness and providing a sufficient surgical corridor for endoscopic endonasal skull base surgery (EESBS). Objective Skull base reconstruction with nasoseptal flap (NSF) is critically important in managing post-operative cerebrospinal fluid (CSF) leakage after tumor removal by EESBS. The NSF needs to be elevated before sphenoidotomy and posterior septectomy to preserve the pedicle. However, most extradural surgery without CSF leakage does not require NSF and, therefore, NSF preparation is often futile. As a result, a rescue flap technique to overcome this problem has been developed, whereby a new S-I rescue flap method is used that enables wide exposure of the sphenoidal rostrum and smooth manipulation of surgical instruments to preserve the NSF pedicle. Materials and methods Starting in April 2014, 19 cases underwent EESBS with S-I rescue flap. Results All patients underwent tumor resection under an adequate operative field with smooth manipulation of surgical instruments. Two complications were experienced. One patient had CSF leak after removal of the nasal packing, but the leakage was successfully closed by conventional NSF. Another patient had epistaxis from the septal wall, but this was controlled by electrocautery.

A vascularized middle turbinate flap (MTF) might be useful in the repair of lesions in the anterior olfactory groove. The sizes of the MTF and inferior turbinate flap (ITF) and these distances up to the frontal base were analyzed by computed tomography (CT) using a picture archiving and communication system (PACS) in 20 patients. The minimum flap measurement to reconstruct the skull base was defined as the distance from the basal plate to the medial slope or lateral slope edge of the MTF and the ITF on the coronal view. Next, we reviewed clinical data from three patients with dural deficits who underwent endoscopic vascularized MTF reconstruction. The distance to the frontal base from the ITF was significantly longer than that from the MTF. The minimum length of the MTF measured by coronal CT was similar to the distance to the frontal base. In contrast, the minimum length of the ITF was shorter than the distance to the frontal base. These results suggest that the MTF might be superior to the ITF for coverage of the anterior frontal base. Three patients who underwent MTF reconstruction for a defect in the anterior olfactory groove had good outcomes and no complications. The MTF is a good option for reconstruction of defects in the anterior part of the olfactory groove.

Background: High risk head and neck mucosal premalignancy has a malignant conversion rate of up to 40%, despite adequate surgical therapy. Epidermal Growth Factor Receptor (EGFR) blocking agents, including cetuximab, have shown activity in head and neck squamous cell carcinoma (HNSCC) and have potential for therapy in high risk premalignancy. Methods: We conducted a randomized, prospective, phase II clinical trial to determine the effects of cetuximab on patients with high risk premalignancy. Patients were randomized to treatment with cetuximab 400 mg/m2 on week one followed by 250 mg/m2 on week 2-8 or observation, with the option for crossover to cetuximab therapy for patients originally randomized to the observation arm. Results: Two of 19 enrolled patients did not complete therapy due to treatment toxicity. Analysis of 17 patients who completed the trial regimen show a trend toward a larger mean decrease in grade of dysplasia in the cetuximab treated group (-1.0) vs. the observation group (-0.2) (P = 0.082, one-sided exact Wilcoxon rank sum test). However, in the observation group, none of the 5 patients (0%) achieved complete resolution of dysplasia; while 4 of 12 (33.3%) cetuximab treated patients had no remaining dysplasia after therapy. Conclusions: Treatment of high risk premalignancy of the upper aerodigestive tract with cetuximab alone may result in significant, durable, and complete clinical and histological resolution of moderate to severe dysplasia in at least a subset of high risk patients. These results warrant further investigation in larger studies with increased statistical power. (C) 2015 Elsevier Ltd. All rights reserved.

Background: The side population (SP) of cancer cells is reportedly enriched with cancer stem cells (CSCs), however, the functional role and clinical relevance of CSC marker molecules upregulated in the SP of head and neck squamous carcinoma (HNSCC) cells are yet to be elucidated. Patients with clinical stage I/II (T1-2N0M0) tongue squamous cell carcinoma (TSCC) typically undergo partial glossectomy; however, development of delayed neck metastasis (DNM) tends to reduce their survival. In the present study, we aimed to determine the CSC markers in the SP of HNSCC cells along with their functions in cellular behaviors, and to clarify the association of these markers with DNM. Methods: Flow cytometry was applied to isolate SP from main population (MP) in HNSCC cells. The expression of the CSC markers was examined by semi-quantitative RT-PCR and immunocytochemistry. In vitro proliferation, migration, and invasion assays were performed to assess cellular behaviors. Clinicopathological factors and immunohistochemical expressions of Oct3/4 and Nanog were evaluated using surgical specimens from 50 patients with stage I/II TSCC. Results: SPs were isolated in all three cell lines examined. Expression levels of Oct3/4 and Nanog were higher in SP cells than MP cells. Additionally, cell migration and invasion abilities were higher in SP cells than MP cells, whereas there was no difference in proliferation. Univariate analysis showed that expression of Oct3/4 and Nanog, vascular and muscular invasion, and mode of invasion were significantly correlated with DNM. Multivariate logistic regression revealed that Oct3/4 expression (risk ratio = 14.78, p = 0.002) and vascular invasion (risk ratio = 12.93, p = 0.017) were independently predictive of DNM. Regarding the diagnostic performance, Oct3/4 showed the highest accuracy, sensitivity, and NPV of 82.0 %, 61.5 %, and 86.8 %, respectively, while vascular invasion showed the highest specificity and PPV of 94.6 % and 71.4 %, respectively. Conclusion: These results suggest that Oct3/4 and Nanog represent probable CSC markers in HNSCC, which contribute to the development of DNM in part by enhancing cell motility and invasiveness. Moreover, along with vascular invasion, expression of Oct3/4 can be considered a potential predictor for selecting patients at high risk of developing DNM.

Using high-throughput analyses and the TRANSFAC database, we characterized TF signatures of head and neck squamous cell carcinoma (HNSCC) subgroups by inferential analysis of target gene expression, correcting for the effects of DNA methylation and copy number. Using this discovery pipeline, we determined that human papillomavirus-related (HPV+) and HPV- HNSCC differed significantly based on the activity levels of key TFs including AP1, STATs, NF-κB and p53. Immunohistochemical analysis confirmed that HPV- HNSCC is characterized by co-activated STAT3 and NF-κB pathways and functional studies demonstrate that this phenotype can be effectively targeted with combined anti-NF-κB and anti-STAT therapies. These discoveries correlate strongly with previous findings connecting STATs, NF-κB and AP1 in HNSCC. We identified five top-scoring pair biomarkers from STATs, NF-κB and AP1 pathways that distinguish HPV+ from HPV- HNSCC based on TF activity and validated these biomarkers on TCGA and on independent validation cohorts. We conclude that a novel approach to TF pathway analysis can provide insight into therapeutic targeting of patient subgroup for heterogeneous disease such as HNSCC. What's new? Changes in transcription factor (TF) expression contribute to genetic and epigenetic abnormalities in cancer. In order to capitalize on those changes and advance diagnostic and therapeutic strategies for cancer, researchers must first find a way to detect and quantify changes in TF activity. Here, TF activity was estimated globally in primary head and neck squamous cell carcinoma (HNSCC) using a novel inferential approach that accounted for gene silencing and loss of heterozygosity and homozygosity. Top-scoring pair biomarkers were identified and linked to human papillomavirus (HPV) status, enabling HPV+ and HPV- HNSCC to be distinguished based on TF activity.