Mucosal Injection of the Silicon Phthalocyanine Pc 4 in a Rabbit Model-A Pilot Study.

OBJECTIVE: The silicon phthalocyanine Pc 4 is a photosensitizing agent previously shown to be a promising treatment for cutaneous neoplasms using photodynamic therapy (PDT). Based on prior preclinical studies, we believe Pc 4-PDT has potential as a targeted treatment of human recurrent respiratory papillomatosis or laryngeal leukoplakia by direct injection into mucosal surfaces. METHODS: This was a proof-of-concept pilot study assessing direct mucosal injection of Pc 4 into buccal and vocal fold mucosae in a rabbit model. Five New Zealand white rabbits underwent tattooing of bilateral buccal mucosae to delineate injection sites, followed by submucosal injections of control and Pc 4 solutions. Rabbits were monitored for post-injection tolerance. Punch biopsies were obtained from injected mucosa and assessed histopathologically. Once the buccal mucosa was found to be tolerant, vocal folds of three rabbits were injected. The rabbits were then sacrificed, and laryngeal tissue was assessed histopathologically. RESULTS: All rabbits tolerated injection of Pc 4 and control solutions into buccal mucosa with no evidence of gross visual inflammatory changes and no changes in behavior or masticatory function. Histopathologic analysis of Pc 4 injected buccal and control mucosal tissue revealed mild focal histological changes and no stigmata of diffuse inflammatory reactions. The histopathologic analysis of Pc 4 injected into laryngeal tissue revealed similar findings with addition of mild eosinophilia in one sample. CONCLUSION: Direct mucosal injection of Pc 4 in rabbit buccal and vocal fold mucosae appears to be well tolerated with no gross inflammatory changes, and only mild histopathologic inflammatory changes observed. LEVEL OF EVIDENCE: NA Laryngoscope, 2024.

Beta-defensin index: A functional biomarker for oral cancer detection.

There is an unmet clinical need for a non-invasive and cost-effective test for oral squamous cell carcinoma (OSCC) that informs clinicians when a biopsy is warranted. Human beta-defensin 3 (hBD-3), an epithelial cell-derived anti-microbial peptide, is pro-tumorigenic and overexpressed in early-stage OSCC compared to hBD-2. We validate this expression dichotomy in carcinoma in situ and OSCC lesions using immunofluorescence microscopy and flow cytometry. The proportion of hBD-3/hBD-2 levels in non-invasively collected lesional cells compared to contralateral normal cells, obtained by ELISA, generates the beta-defensin index (BDI). Proof-of-principle and blinded discovery studies demonstrate that BDI discriminates OSCC from benign lesions. A multi-center validation study shows sensitivity and specificity values of 98.2% (95% confidence interval [CI] 90.3-99.9) and 82.6% (95% CI 68.6-92.2), respectively. A proof-of-principle study shows that BDI is adaptable to a point-of-care assay using microfluidics. We propose that BDI may fulfill a major unmet need in low-socioeconomic countries where pathology services are lacking.

Image analysis reveals differences in tumor multinucleations in Black and White patients with human papillomavirus-associated oropharyngeal squamous cell carcinoma.

BACKGROUND: Understanding biological differences between different racial groups of human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) patients, who have differences in terms of incidence, survival, and tumor morphology, can facilitate accurate prognostic biomarkers, which can help develop personalized treatment strategies. METHODS: This study evaluated whether there were morphologic differences between HPV-associated tumors from Black and White patients in terms of multinucleation index (MuNI), an image analysis-derived metric that measures density of multinucleated tumor cells within epithelial regions on hematoxylin-eosin images and previously has been prognostic in HPV-associated OPSCC patients. In this study, the authors specifically evaluated whether the same MuNI cutoff that was prognostic of overall survival (OS) and disease-free survival in their previous study, TTR , is valid for Black and White patients, separately. We also evaluated population-specific cutoffs, TB for Blacks and TW for Whites, for risk stratification. RESULTS: MuNI was statistically significantly different between Black (mean, 3.88e-4; median, 3.67e-04) and White patients (mean, 3.36e-04; median, 2.99e-04), with p = .0078. Using TTR , MuNI was prognostic of OS in the entire population with hazard ratio (HR) of 1.71 (p = .002; 95% confidence interval [CI], 1.21-2.43) and in White patients with HR of 1.72 (p = .005; 95% CI, 1.18-2.51). Population-specific cutoff, TW , yielded improved HR of 1.77 (p = .003; 95% CI, 1.21-2.58) for White patients, whereas TB did not improve risk-stratification in Black patients with HR of 0.6 (p = .3; HR, 0.6; 95% CI, 0.2-1.80). CONCLUSIONS: Histological difference between White and Black patient tumors in terms of multinucleated tumor cells suggests the need for considering population-specific prognostic biomarkers for personalized risk stratification strategies for HPV-associated OPSCC patients.

Treatment failure patterns are similar between p16- and p16+ oropharyngeal squamous cell carcinomas.

Background: The incidence of p16+ oropharyngeal squamous cell carcinoma (OPSCC) has been increasing. The notion that p16+ OPSCC has a propensity for atypical and disseminating metastasis has gained traction. We compared treatment failure patterns in p16+ and p16- OPSCC and evaluated survival impact. Methods: Retrospective analysis of patients with recurrent/metastatic OPSCC disease between 1/2009 and 12/2019. Results: Thirty-eight p16+ and 36 p16- patients were identified. Three distinct failure patterns (distant vs. locoregional, atypical vs. typical, and disseminating vs. non-disseminating) were studied. No significant differences were found between p16+ and p16- patients. Multivariate analysis showed p16 status was an independent prognostic biomarker; p16+ patients have a favorable overall survival compared to p16- patients (HR 0.34, 95% CI 0.16-0.77; P = .005). Conclusions: We challenge the view that p16+ OPSCC exhibits a distinctive treatment failure pattern and showed that p16 status impacts patient survival independent of disease progression.

The diagnostic utility of BRAF VE1 mutation-specific immunohistochemistry in ameloblastoma.

Ameloblastoma is a benign, locally aggressive odontogenic neoplasm with variable solid and cystic morphology. On account of its histologic variety, diagnostically challenging cases can bear resemblance to odontogenic keratocyst/keratocystic odontogenic tumor (KCOT) or dentigerous cyst (DC). BRAFV600E mutation has been reported to be specific for and frequent in ameloblastoma, and this study evaluated the usefulness of immunohistochemistry (IHC) using the BRAF VE1 mutant-specific antibody as a diagnostic adjunct in this setting. We investigated 46 ameloblastomas, 30 KCOTs, and 30 DCs. BRAF VE1 IHC was performed on all cases and allele-specific polymerase chain reaction (AS-PCR) for BRAFV600E mutation was performed on 30 ameloblastomas and any IHC-positive KCOT/DC. BRAF VE1 IHC was positive in 31/37 (83.8%) mandibular ameloblastomas but not in any maxillary ameloblastomas (0/9), KCOT (0/30), or DC (0/30). Equivocal staining was seen in 1/37 (3.3%) mandibular ameloblastomas. Of the 30 ameloblastomas subjected to AS-PCR, BRAFV600E mutation was identified in 19/23 (82.6%) mandibular ameloblastomas and 0/7 (0.0%) maxillary ameloblastomas. BRAFV600E mutant ameloblastomas were positive by IHC in 18/19 (94.7%) cases and equivocal in 1/19 (5.3%) cases. All 11 (100.0%) BRAF-wild type ameloblastomas were negative by IHC. BRAF VE1 is an excellent tool for the diagnosis of mandibular ameloblastoma but of limited utility in the maxilla, where it less commonly occurs and where BRAFV600E mutation is considerably less frequent.

Impact of AJCC 8th edition staging system and definitive treatment choice on the prognosis of complete responders with p16+ and p16- oropharyngeal squamous cell carcinomas.

OBJECTIVES: To identify predictors of overall survival (OS) in oropharyngeal squamous cell carcinoma (OPSCC) patients who achieved complete response (CR). METHODS: We performed a retrospective study of OPSCC patients who achieved CR from a single academic medical center. Associations between OS, AJCC 8th edition staging system, definitive treatment choice, smoking history, and p16 status were assessed. RESULTS: p16+ status was associated with favorable prognosis for CR (p < 0.001) but not non-CR (p = 0.67) patients. For early stage, p16+ OPSCC patients who achieved CR, surgery + adjuvant radiation (RT) treatment was more durable compared to concurrent chemoradiation (CRT), particularly in smokers. CONCLUSIONS: Curative intent treatment choice and smoking history has an impact on the long-term OS of the CR p16+ OPSCC cohort. Prospective studies to define the optimal multi-modality treatment option to manage p16+ OPSCC patients is needed.

Impact of p16 Status and Anatomical Site in Anti-PD-1 Immunotherapy-Treated Recurrent/Metastatic Head and Neck Squamous Cell Carcinoma Patients.

In head and neck squamous cell carcinoma (HNSCC), anti-PD-1 inhibitors are approved for recurrent/metastatic (R/M) disease and anticipated to expand to other indications. The impact of p16 status and anatomical site on overall survival (OS) in immunotherapy-treated HNSCC patients remains unresolved. We performed a retrospective analysis of R/M HNSCC patients receiving anti-PD-1 immunotherapy at our academic medical center with an extensive community satellite network. Fifty-three R/M HNSCC patients were treated with anti-PD-1 immunotherapy and had a median OS of 6 months. Anatomical site was associated with distinct OS; oropharynx and larynx patients have superior OS compared to oral cavity patients. Analysis of the OPSCC subset showed p16+ status as a favorable, independent prognostic biomarker (HR 7.67 (1.23-47.8); p = 0.029). Further studies to assess the link between anatomical site, p16 status, and anti-PD-1 treatment outcomes in large cohorts of R/M HNSCC patients managed in real-world clinical practices and clinical trials should be prioritized.

African Americans With p16+ and p16- Oropharyngeal Squamous Cell Carcinomas Have Distinctly Poor Treatment Outcomes Independent of Medical Care Access.

PURPOSE: Human papilloma virus-positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC), diagnosed with p16 immunohistochemistry, is associated with favorable prognosis; however, this connection was established using European American (EA)-skewed populations. The impact of p16/human papillomavirus status on outcomes in African American (AA) OPSCC patients remains to be settled. In this study, we determine the association between cancer disparity and p16 status in an OPSCC cohort controlling for time to treatment initiation (TTI), a surrogate for medical care access. MATERIALS AND METHODS: We analyzed data from all patients diagnosed with OPSCC (N = 440) between 2010 and 2017, who received treatment at our academic medical center. Associations between age, disease stage, sex, p16 status, race, TTI, and overall survival (OS) were investigated. RESULTS: TTI was similar between AA and EA OPSCC patients in our p16+ (P = .291) or p16- (P = .715) cohorts. Among p16+ OPSCC patients, the median OS was > 8.65 years for EA patients compared with 5.038 years (95% CI, 2.019 to 5.30; P = .003, log-rank) for AA patients. For p16- patients, the median OS was 5.74 years (95% CI, 3.32 to 6.99) for EA patients and 1.85 years (95% CI, 0.978 to 4.50; P = .03, log-rank) for AA patients. Multivariate Cox regression analysis showed that race was an independent prognostic biomarker and the most impactful co-variate for OS (hazard ratio, 0.40; 95% CI, 0.00 to 0.69; P = .001). CONCLUSION: Our work showed that AAs with p16+ OPSCC have surprisingly poor clinical outcomes and are thus poor candidates for treatment de-escalation regimens. Caution should be exercised when extending clinical guidelines based on EA-majority studies to non-EA populations.

Tiny but mighty: use of next generation sequencing on discarded cytocentrifuged bile duct brushing specimens to increase sensitivity of cytological diagnosis.

Bile duct brushing (BDB) is used to evaluate pancreatobiliary lesions as it widely samples lesions with a low complication rate. Cytological evaluation of BDB is a specific but insensitive test. There is limited literature on the use of post-cytocentrifuged (PCC) samples, which are usually discarded, for next-generation sequencing (NGS) as an adjunct to cytological diagnosis of BDB. In this study we investigate whether molecular analysis by NGS of PCC specimens improves the sensitivity of diagnosis. PCC samples from 100 consecutive BDB specimens spanning 93 unique patients were retained. DNA was extracted and mutational analysis was performed agnostic of morphologic or clinical findings. Each BDB specimen was characterized as negative, atypical or positive based on morphological analysis by trained cytopathologists. Performance characteristics for mutational profiling and morphological analysis were calculated on the basis of clinicopathologic follow-up. There was sufficient clinicopathologic follow-up to classify 94 of 100 cases as either malignant (n = 43) or benign (n = 51). Based on morphologic analysis of cytology, these 94 cases were classified as either benign (n = 55), atypical (n = 18), or as at least suspicious or positive for malignancy (n = 21). Morphologic analysis of cytology showed a sensitivity of 49% and a specificity of 100% if atypical cases were considered negative. NGS revealed oncogenic alterations in 40/43 (93%) of malignant cases based on clinicopathologic follow-up. The most common alterations were in KRAS and TP53, observed in 77% and 49% of malignant cases respectively. No alterations were observed in the 51 benign cases classified based on clinicopathologic follow-up. Supplementing cytomorphologic analysis with molecular profiling of PCC by targeted NGS analysis increased the sensitivity to 93% and maintained specificity at 100%. This study provides evidence for the utility of NGS molecular profiling of PCC specimens to increase the sensitivity of BDB cytology samples, although studies with larger cohorts are needed to verify these findings.

Recurrent and novel USP6 fusions in cranial fasciitis identified by targeted RNA sequencing.

Cranial fasciitis is a benign myofibroproliferative lesion of the scalp and underlying bones typically occurring in the pediatric population. Histologically, it is characterized by loose fascicles of stellate cells in a fibromyxoid background, findings similar to those described in the closely related variant nodular fasciitis. Previously characterized as a reactive process, the identification of USP6 translocations in over 90% of nodular fasciitis cases prompted their reclassification as a clonal neoplastic process. Unlike nodular fasciitis, the molecular underpinnings of cranial fasciitis are less clear. While a subset of cranial fasciitis has been associated with Wnt/ß-catenin pathway dysregulation, recent case reports suggest that this entity may also harbor USP6 fusions, a finding we sought to further investigate. We identified fifteen archival cases of cranial fasciitis, five females and ten males ranging in age from 3 months to 9 years (median 11 months), composed of formalin-fixed paraffin-embedded and fresh frozen tissues (11 and 4 cases respectively). Samples were evaluated on an RNA-based targeted sequencing panel targeting genes recurrently rearranged in neoplasia, including USP6. Five of fifteen cases (33%) were positive for USP6 rearrangements predicted to result in the fusion of the entire USP6 coding region to the promoter of the 5' partner, (three of which were novel):  two SERPINH1-USP6 (novel) and one each of COL3A1-USP6 (novel), SPARC-USP6, and MYH9-USP6. These results demonstrate the recurrent nature of USP6 rearrangements in cranial fasciitis, and highlight the success of targeted RNA sequencing in identifying known and novel fusion partners. The identification of USP6 promoter-swapping rearrangements is helpful in understanding the underlying biology of cranial fasciitis, and reinforces its biologic relationship to nodular fasciitis. Targeted RNA sequencing is a helpful tool in diagnosing this pseudosarcomatous lesion.

Cardiac varix: an example via a case report of a radiological mimicker of cardiac myxoma.

Cardiac myxoma is the most frequently encountered primary neoplasm of the heart; however, other tumefactive lesions can share similar radiologic features. We present briefly the case of a 69-year-old man incidentally found to have a mobile right atrial mass that based on initial radiologic findings was considered to represent a myxoma. After pathologic examination, the lesion was determined instead to be a cardiac varix: an endocardial, blood filled cystic space lined by endothelium and considered to represent a dilated vein.

Biallelic PTCH1 Inactivation Is a Dominant Genomic Change in Sporadic Keratocystic Odontogenic Tumors.

Keratocystic odontogenic tumors (KCOTs) are locally aggressive odontogenic neoplasms with recurrence rates of up to 60%. Approximately 5% of KCOTs are associated with nevoid basal cell carcinoma (Gorlin) syndrome and 90% of these show genomic inactivation of the PTCH1 gene encoding Patched 1. Sporadic KCOTs reportedly have PTCH1 mutations in 30% of cases, but previous genomic analyses have been limited by low tumor DNA yield. The aim of this study was to identify recurrent genomic aberrations in sporadic KCOTs using a next-generation sequencing panel with complete exonic coverage of sonic hedgehog (SHH) pathway members PTCH1, SMO, SUFU, GLI1, and GLI2. Included were 44 sporadic KCOTs from 23 female and 21 male patients with a median age of 50 years (range, 10 to 82 y) and located in the mandible (N=33) or maxilla (N=11). Sequencing identified PTCH1 inactivating mutations in 41/44 (93%) cases, with biallelic inactivation in 35 (80%) cases; 9q copy neutral loss of heterozygosity targeting the PTCH1 locus was identified in 15 (34%) cases. No genomic aberrations were identified in other sequenced SHH pathway members. In summary, we demonstrate PTCH1 inactivating mutations in 93% of sporadic KCOTs, indicating that SHH pathway alterations are a near-universal event in these benign but locally aggressive neoplasms. The high frequency of complete PTCH1 loss of function may provide a rational target for SHH pathway inhibitors to be explored in future studies.

Comparison of cytocentrifugation supernatant fluid and formalin-fixed paraffin-embedded tissue for targeted next-generation sequencing.

BACKGROUND: The emergence of less invasive procedures coupled with the growth of molecular testing have created a need for clinical laboratories to optimize workflows to enable tissue preservation and ancillary testing. In the preparation of formalin-fixed paraffin-embedded cell blocks (FFPE CBs), there is a cytocentrifugation step for cell pellet extraction that results in postcentrifugation supernatant fluid (SN). This SN, which in most routine workflows is discarded, has been suggested to contain adequate cellular material for molecular testing. In the current study, the authors describe the use of DNA and RNA extracted from SN for the detection of clinically relevant biomarkers by next-generation sequencing (NGS). METHODS: After cell pellet removal, cytocentrifugation SN from 30 endobronchial fine-needle aspiration rinses that were positive for malignancy on FFPE CB were collected. DNA and RNA were extracted from the SN and tested using an in-house NGS Solid Tumor Focus Assay. The NGS results were compared with findings from corresponding FFPE samples. RESULTS: Testing was successful in all 30 samples. There was 100% concordance between variants observed in the SN and corresponding FFPE specimens, which included 50 single-nucleotide variants, 9 copy number amplifications, 3 structural variants, and 2 indels. Furthermore, there was excellent correlation (correlation coefficient, 0.93) between the variant allele frequency of mutations observed in SN compared with that noted in corresponding FFPE CBs. CONCLUSIONS: Cytocentrifugation SN is a valuable source for NGS, is comparable to FFPE that preserves tissue for other ancillary testing, and can reduce the failure rate of testing that may result from insufficient material being available in the CB.

Post-radiotherapy PET/CT for predicting treatment outcomes in head and neck cancer after postoperative radiotherapy.

PURPOSE: The purpose of this study was to retrospectively review the role of post-treatment (post-tx) FDG-PET/CT scans in patients receiving postoperative intensity-modulated radiotherapy (IMRT) for head and neck squamous cell carcinomas (HNSCC). MATERIALS AND METHODS: Eighty-two patients with HNSCC treated with surgery and postoperative IMRT with or without chemotherapy from October 15, 2008 to December 31, 2014 that had post-tx PET/CT within 6 months of completing IMRT were included. PET/CT was considered positive based on multi-disciplinary review integrating clinical information. Survival analysis was performed using the Kaplan-Meier method. Categorical and continuous predictors of positive post-tx PET/CT were evaluated using Fisher's exact test and logistic regression, respectively. Predictors for survival outcomes were evaluated with log-rank testing. A p ≤ 0.05 was considered statistically significant. RESULTS: Median follow-up was 3.88 years. For all patients, 3-year overall survival (OS) and recurrence-free survival (RFS) were 71.8% and 61.3%, respectively. Patients with positive post-tx PET/CT had worse OS compared to those with negative post-tx PET/CT (log rank p < 0.001). For patients with positive post-tx PET/CT, 3-year OS was 11.2% compared to 89.9% for patients with negative post-tx PET/CT. The positive predictive value (PPV) of PET/CT was 100% for local recurrence (LR), regional recurrence (RR) and distant metastasis (DM). The negative predictive values (NPV) for LR, RR and DM were 89.0%, 89.2%, and 85.9%, respectively. Perineural invasion (p = 0.009), p16 status (p = 0.009), non-oropharyngeal primary site (p = 0.002), and the use of chemotherapy (p = 0.01) were independent predictors of positive PET/CT. CONCLUSIONS: Post-tx PET/CT after postoperative radiation is prognostic for survival outcomes. The PPV of post-tx PET for recurrence was excellent, allowing for early detection of recurrent disease. Post-tx PET/CT should be considered after postoperative radiation.

Lessons From Unilateral Loss of Cilia: Early Nasal Nitric Oxide Gas Mixing and the Role of Sinus Patency in Determining Nasal Nitric Oxide.

Nasal nitric oxide (nNO) measurement is a diagnostic test for primary ciliary dyskinesia (PCD). Here, we have shown the development of unilateral PCD-like symptoms associated with low nNO. A 60-year-old man had been previously healthy but developed unilateral, severe pansinusitis. He required surgical drainage of all left sinuses, and biopsies showed loss of the ciliated epithelium. At 4 weeks, he had unilateral (left-sided), profuse, clear rhinorrhea characteristic of PCD, and his surgical ostia were all patent endoscopically. His left-sided nNO was less than the right side by 37 ± 1.2 nL/min; this difference decreased to 18 ± 0.87 nL/min at 5 weeks and was gone by 6 weeks when his symptoms resolved. Measurements of 2- and 10-second measurements, in addition to standard nNO measurements, identified this discordance. We conclude that nNO reflects, in part, the production of NO by the ciliated epithelium, not just in the absence or occlusion of sinuses. Early (nasal/sinus volume) measures may be better for diagnosing PCD in than standard, steady-state assays in certain populations.

Spontaneous resolution of hypercalcemia.

BACKGROUND: Primary hyperparathyroidism (PHPT) is a frequently encountered endocrine disorder due to benign neoplastic lesions or gland hyperplasia. It is often discovered incidentally when routine lab work reveals hypercalcemia. METHODS: This case presents a 55-year-old male with a neck mass and electrolyte irregularities consistent with PHPT. However, his laboratory values suddenly normalized prior to surgery. RESULTS: Post-operative pathologic analysis of the specimen demonstrated massive infarction of the affected gland, and explained the spontaneous resolution of the patient's electrolyte derangements. CONCLUSIONS: The objective of this case study is to demonstrate the importance of further investigation in patients with fluctuating lab values and emphasize the potential dangers of gland infarction.

Post-treatment PET/CT and p16 status for predicting treatment outcomes in locally advanced head and neck cancer after definitive radiation.

PURPOSE: To retrospectively review post-treatment (post-tx) FDG-PET/CT scans in patients with advanced head and neck squamous cell carcinoma (HNSCC) and known p16 status, treated with definitive (chemo)radiation (RT). METHODS: A total of 108 eligible patients had N2A or greater HNSCC treated with chemoRT from August 1, 2008, to February 28, 2015, with post-tx PET/CT within 6 months after RT. Kaplan-Meier curves, log-rank statistics, and Cox proportional hazards regression were used for statistical analysis. RESULTS: Median follow-up was 2.38 years. Sixty-eight (63.0%) patients had p16+ and 40 (37.0%) had p16- status. Two-year overall survival and recurrence-free survival were 93.4% and 77.8%, respectively. The negative predictive value (NPV) of PET/CT for local recurrence (LR) was 100%. The NPV for regional recurrence (RR) was 96.5% for all patients, 100% for p16+ patients, and 88.5% for p16- patients. The positive predictive value (PPV) of PET/CT for recurrence was 77.3% for all patients, 50.0% for p16+, and 78.6% for p16-. The PPV for LR was 72.7% for all patients, 50.0% for p16+ patients, and 72.7% for p16- patients. The PPV for RR was 50.0% for all patients, 33% for p16+, and 66.6% for p16-. Post-tx PET/CT and p16 status were independent predictors of recurrence-free survival (p < 0.01). CONCLUSIONS: Post-tx PET/CT predicts treatment outcomes in both p16 + and p16- patients, and does so independently of p16 status. P16- patients with negative PET have a 10% risk of nodal recurrence, and closer follow-up in these patients is warranted.