P53-positive Tumors Research Articles

TNM 8 staging system beyond p16: Double HPV/p16 status is superior to p16 alone in predicting outcome in oropharyngeal squamous cell carcinoma

PurposeThe assessment of p16INK4a (p16) in oropharyngeal squamous cell carcinoma (OPSCC) has been incorporated into tumor classification, as p16 has been shown to impact survival probability. However, a recent study demonstrated that human papillomavirus (HPV) status in addition to p16 may have a better discriminatory effect on survival probability. This study aims to determine the impact of combined evaluation of p16 and HPV on prognosis. MethodsThis was a multicenter, multinational analysis including retrospective and prospective cohorts of patients treated for primary OPSCC with curative intent, based on the data of the HNCIG-EPIC study. The primary outcome was to determine how the combined assessment of HPV and p16 status predicts prognosis of patients with OPSCC compared to p16 assessment alone. We employed multivariable analyses models to compute hazard ratios regarding survival. Analyses were stratified by stage, smoking status, and sub-anatomical region. ResultsThe study included 7654 patients, with approximately half of the tumors being p16-negative (50.3 %, n = 3849). A total of 9.2 % of patients had discordant p16 and HPV status (n = 704). HPV status significantly impacted overall survival and disease-free survival regardless of p16 status and across both UICC 8th stage I-II and III-IVb cancers. p16-positive/HPV-positive OPSCC patients exhibited the best survival probability. ConclusionThe detection of HPV had a significant impact on survival probability for OPSCC patients with both p16-positive and p16-negative tumors. HPV testing should be integrated in cancer staging, especially in regions of low attributable fraction, alongside p16 evaluation to ensure a comprehensive assessment of prognosis.

The multicenter registry "oropharyngeal cancer" of the German ENT Study Center - First Results

The number of oropharyngeal squamous cell carcinomas (OPSCC) caused by human papillomavirus (HPV) has increased significantly in recent years. However, no robust data exist on the incidence of HPV-associated OPSCC as well as their treatment in central Europe. The aim is to establish an epidemiological register of patients with OPSCC in central Europe. To close the evidence gap on p16-positive OPSCC, a prospective multicenter registry on incidence, risk factors and treatment of OPSCC in certified Head and Neck Tumor Centers according to the German Cancer Society was established. Data collection was pseudonymously performed using the web-based Research Electronic Data Capture (REDCap). In 2022 and 2023, 1312 patients with OPSCC from 34 centers were included. 720 patients (58.2 %) were p16-positive. The most common locations of p16-positive primary tumors were the tonsil (49.9 %) and the base of the tongue (28.7 %). 63.1 % of p16-positive OPSCC received primary surgical treatment, whereas only 48.0 % of p16-negative tumors received primary surgery. Differences are also evident in the adjuvant therapy: 58 % of p16-negative OPSCC did not receive adjuvant therapy after surgery, compared with 37.1 % of p16-positive OPSCC. The start of the OPSCC registry study was technically smooth. This registry provides an insight into the current care situation of OPSCC in German-speaking countries and will gain importance through the participation of more centers.

Association of absolute neutrophil count with outcomes in patients with head and neck cancer treated with chemoradiation.

e18055 Background: Systematic inflammation plays an integral role in tumor development and metastasis. High neutrophil counts, among various peripheral blood biomarkers, have been shown to be associated with worse survival outcomes in multiple cancer types. To evaluate the absolute neutrophil count (ANC) as a prognostic biomarker in head and neck cancer, we performed an observational cohort study of patients undergoing definitive chemoradiation. Methods: Our institutional database at the Roswell Park Comprehensive Cancer Center was queried for patients with non-metastatic head and neck cancer who underwent definitive chemoradiation between June 2007 and April 2023. ANC was analyzed as a continuous variable. Survival outcomes were assessed using Kaplan-Meier method, log-rank tests, Cox proportional hazard multivariable (MVA) analyses. Fine-Gray MVA was used to analyze failure outcomes with death as a competing event. To reduce selection bias, propensity score matching was performed using ANC as a dichotomous variable with its median value as a cutoff. Subgroup analyses were performed among p16-negative and p16-positive cohorts. Results: A total of 668 patients who met our criteria. Median follow up was 30.3 months (interquartile range 9.9-60.7). Elevated ANC as a continuous variable was associated with worse overall survival (OS; adjusted hazards ratio [aHR] 1.02, 95% confidence interval [CI] 1.01-1.04, p=0.003), progression free survival (PFS; aHR 1.03, 95% CI 1.01-1.04, p<0.001), and distant failure (DF; aHR 1.03, 95% CI 1.01-1.05, p=0.007), but not locoregional failure (LRF; aHR 1.02, 95% CI 1.00-1.05, p=0.10). Similar findings were noted among 278 matched pairs (OS: hazards ratio [HR] 1.66, 95% CI 1.23-2.26, p=0.001; PFS: HR 1.74, 95% CI 1.31-2.29, p<0.001; LRF: HR 1.66, 95% CI 0.98-2.80, p=0.06; DF: HR 1.64, 95% CI 1.03-2.60, p=0.04). On subgroup analysis, among 118 patients with p16-negative tumors, elevated ANC as a continuous variable was not associated with OS (aHR 1.00, 95% CI 0.90-1.11, p=0.97) and PFS (aHR 0.97, 95% CI 0.87-1.08, p=0.54). The number of LRF (n=21) and DF (n=17) were too few to analyze in this subgroup. However, among 344 patients with p16-positive tumors, high ANC was associated with worse OS (aHR 1.03, 95% CI 1.00-1.06, p=0.046), PFS (aHR 1.05, 95% CI 1.02-1.07, p<0.001), LRF (aHR 1.08, 95% CI 1.05-1.12, p<0.001), and DF (aHR 1.04, 95% CI 1.01-1.08, p=0.02). Conclusions: Our study suggested that elevated ANC was an independent, adverse prognostic factor for worse survival and distant metastasis outcomes. Similar findings were also observed among patients with p16-positive tumors, with high ANC also associated with worse locoregional failure. Further studies are warranted to tailor interventions based on such biomarkers.

Curative Radiotherapy for Oropharyngeal Squamous Cell Carcinoma Stratified by p16 Status, According to the Eighth Edition American Joint Committee on Cancer Staging Manual: A Retrospective Study.

The prevalence of oropharyngeal squamous cell carcinoma (OPSCC) is increasing worldwide. This retrospective study aimed to investigate the clinical outcomes of patients with OPSCC undergoing definitive radiotherapy, stratified according to their p16 status. A retrospective analysis was conducted on consecutive patients with OPSCC treated with curative external beam radiotherapy between May 2015 and September 2023. Clinical staging was determined by the eighth edition AJCC staging manual for p16 positive and negative OPSCC. All patients were treated with radiotherapy using a simultaneous integrated boost (SIB) with helical tomotherapy. The fractionation scheme, with or without chemotherapy, for the primary site and nodal lesions consisted of 2 Gy per fraction for a total dose of 70 Gy in 35 fractions over seven weeks. This study included 76 patients with a median age of 66 years. With a median follow-up time of 32.6 months, the 3-year progression-free survival rate was significantly higher in p16 positive patients compared to p16 negative patients (79.6% vs. 42.5%, p<0.001). Concerning 54 patients with p16-positive tumors, the overall survival rates indicated excellent clinical outcomes for stage I, II, and III with results of 100%, 100%, and 88.1%, respectively. This retrospective study revealed the clinical outcomes of patients with OPSCC treated with radical radiotherapy, emphasizing the significance of p16 status. While acknowledging the limitations of the retrospective nature of this study, future prospective studies with larger cohorts and extended follow-up periods are needed to enhance evidence quality.

Stereotactic radiotherapy in the management of oligometastatic and recurrent head and neck cancer: a single-center experience

IntroductionOligometastatic disease (OMD) is a metastatic stage that could benefit maximally from local therapies. Patients in this state have a better prognosis relative to those with disseminated metastases. Stereotactic radiotherapy provides a non-invasive ablative tool for primary malignant tumors and metastases.Materials and methodsWe searched our register for patients with oligometastatic or recurrent head and neck cancer (OMD/R-HNC) who received stereotactic radiotherapy to manage their OMD/R. We evaluated the survival outcomes and prognostic factors that affected the survival of those patients.ResultsIn all, 31 patients with 48 lesions met the inclusion criteria for the analysis. The lesions comprised various metastatic sites, with the majority being pulmonary (37 lesions). Squamous cell cancer was the most common histology (26 patients). The median overall survival (mOS) was 33 months, with a progression-free survival (PFS) of 9.6 months. Eight patients received subsequent stereotactic radiotherapy after disease progression. The local control (LC) rates were 91.3, 87.7, and 83% at 6, 12, and 36 months. Patients with the de novo OMD who received stereotactic radiotherapy as their initial treatment had a median systemic treatment-free survival of 23.9 months. In univariate analysis, a trend for better OS was observed in patients with p16-positive squamous cell tumors; patients who progressed within 150 days after diagnosis had a significantly lower OS. De novo OMD showed significantly better PFS compared to induced OMD. Multivariate analyses identified p16-positive squamous cell cancer, metachronous OMD and a longer time to progression as positive predictors of OS, while de novo OMD was the only positive predictor for PFS. Treatment-related toxicities were generally mild, with two cases of grade 3 dysphagia reported.ConclusionStereotactic radiotherapy demonstrated favorable outcomes in patients with OMD/R-HNC with limited toxicities. Further studies are warranted to validate these findings and optimize treatment strategies for this patient population.

HPV testing versus p16 immunohistochemistry in oropharyngeal squamous cell carcinoma: results from the DAHANCA 19 study

Introduction The prognosis after primary (chemo-)radiotherapy for oropharyngeal squamous cell carcinoma (OPSCC) is affected by Human Papillomavirus (HPV) status, with a better prognosis in HPV-positive OPSCC. HPV-status is routinely assessed by p16 immunohistochemistry (IHC), but additional HPV DNA testing is debated. Also, there are numerous HPV genotypes, which prognostic role may need clarification. The purpose of this study was: (1) to test a custom-made targeted HPV next generation sequencing (NGS) panel in OPSCC, (2) to determine correlation with p16 IHC, and (3) to assess the impact of HPV DNA testing on outcome in the prospectively randomized clinical trial DAHANCA 19. Materials and methods We included 271 patients with OPSCC treated with primary (chemo-)radiotherapy in the DAHANCA 19 trial. Of these, 199 (73%) were p16-positive. HPV-status was determined by targeted HPV next generation sequencing (NGS), using a custom-made HPV genotyping panel. Results HPV was detected in 194 tumor samples. p16 IHC and NGS HPV status were concordant in 265 (98%) of 271 patients, whereas we did not detect HPV DNA in 5 p16-positive tumors. HPV16 accounted for 169 of 194 HPV-positive cases (87%). HPV genotypes 18, 31, 33, 35, and 59 were also detected. Loco-regional failure and overall survival were similar whether patients were separated by p16 IHC, or HPV DNA status (p < 0.0001 for all) and did not depend on HPV genotype (p = 0.9 and p = 0.7). Conclusion In the present study, HPV DNA testing or typing in a Danish OPSCC cohort did not add additional information to p16 IHC, the most widely used and accepted prognostic indicator.

Importance of tumor volume, overall treatment time and fractionation sensitivity for p16-positive and p16-negative oropharyngeal tumors

Background Analyses of clinical outcomes following radiotherapy (RT) have advanced our understanding of fundamental radiobiological characteristics in head and neck squamous cell carcinoma (HNSCC). Low fractionation sensitivity appears to be a common feature, as well as susceptibility to changes in overall treatment time (OTT). Large tumors should be harder to cure if a successful RT requires the sterilization of all clonogenic cells. Congruently, primary tumor volume has proven to be an important parameter. However, most findings come from an era when p16-negative HNSCC was the dominant tumor type. HPV-associated, p16-positive, oropharyngeal tumors (OPSCC) are more radiosensitive and have better outcome. The current study aims to investigate the role of primary tumor volume, OTT and estimate α-ratio for p16-positive OPSCC, and to quantify the differences in radiosensitivity depending on p16-status. Methods A cohort of 523 patients treated with RT was studied using a tumor control probability (TCP)-model that incorporates primary tumor volume (V) raised to an exponent c, OTT and α-estimation. The significance of V was also investigated in Cox-regression models. Results In the p16-positive cohort (n = 433), the volume exponent was 1.44 (95%CI 1.06–1.91), compared to 0.90 (0.54–1.32) for p16-negative tumors (n = 90). Hazard ratios per tumor volume doubling were 2.37 (1.72–3.28) and 1.83 (1.28–2.62) for p16-positive and p16-negative, respectively. The estimated α-ratio was 9.7 Gy (−2.3–21.6), and a non-significant daily loss of 0.30 Gy (−0.17–0.92) was found. An additional dose of 6.8 Gy (interquartile range 4.8-9.1) may theoretically counteract the more radioresistant behavior of p16-negative tumors. Conclusion Primary tumor volume plays a crucial role in predicting local tumor response, particularly in p16-positive OPSCC. The estimated α/β-ratio for p16-positive oropharyngeal tumors aligns with previous HNSCC studies, whereas the impact of prolonged OTT was slightly less than previously reported. The differences in radiosensitivity depending on p16-status were quantified. The findings should be validated in independent cohorts.

Importance of HPV status and p16 for the prognosis of penile carcinoma

Penile cancer is a rare but often lethal tumour disease, especially in the metastatic stage. Most data on prognostic factors for penile cancer are based on small patient cohorts, and even meta-analyses are mostly limited in terms of patient numbers. There is a lack of sufficient parameters to predict the metastatic risk of these tumours. Furthermore, the role of the HPV status for the prognosis, and, in this regard, of p16INK4a is still unclear. In this study, 236 patients from an international multicentre cohort were analysed with regard to histological subtypes, HPV and p16 status, and other clinical parameters. The HPV status was only graded as HPV-positive if HPV was detected by PCR and the p16 status defined by immunochemistry was positive. The statistical analysis was carried out using the Kaplan-Meier method as well as the log-rank test and a univariable and multivariable analysis using the Cox regression model. A positive HPV status was not a significant parameter for either metastasis-free (MFS), tumour-specific (CSS) or overall survival (OS). p16-positive tumours showed a significantly better MFS (p=0.026), which was also confirmed in the subgroup analysis of HPV-negative tumours (p=0.037) without differences in CSS or OS. In the usual type, there was also a trend towards an improved MFS, but without statistical significance (p=0.070). p16-positive tumours were associated with a highly significantly better MFS (hazard ratio 0.3; p=0.004) in the multivariable Cox regression, while patients with a pT1b tumour stage or advanced lymph node metastasis showed a significantly worse survival. In the multivariable analysis of HPV-negative tumours, p16 status was also confirmed as an independent predictor of MFS (Hazard ratio 0.2; p=0.007). HPV status alone seems to be lacking prognostic relevance. In contrast, p16 status was confirmed as an independent prognostic factor. Thus, the expression of p16INK4a is associated with a significantly better MFS. Especially in HPV-negative tumours, the p16 status should be evaluated with regard to the prognostic value and thus also with a view to the treatment decision.

P16 expression in urothelial carcinoma: Experience from a tertiary care center in coastal South India.

Urothelial carcinomas (UC) account for 6 and 2% of all cancers in men and women, respectively. Human papillomavirus (HPV) is one of the causative agents in cancers of the uterine cervix and head and neck. The role of HPV is also being studied in cancers of the urinary bladder, penis, and prostate. As p16-INK4a is a surrogate marker for high-risk HPVE7 oncoprotein, this study aims to highlight the utility of p16 immunohistochemistry (IHC) in the evaluation of HPV-associated UC. A retrospective study was conducted on UC of the bladder received in the Pathology department between January 2013 and December 2018. Bladder biopsies from non-neoplastic lesions served as controls. IHC was done for the detection of the p16 antigen. The p16 staining was recorded as positive, when there was strong staining in >50% of tumor nuclei. The p16 positive and negative tumors were compared based on age, gender, tumor size, grade, and muscle invasion. P value <0.05 was considered statistically significant. The expression of p16 was analyzed in 72 UC and compared with 20 non-neoplastic cases, of which 26.4% of the cases showed p16 expression. The p16 expression was absent in the non-neoplastic lesions. While the majority (87.5%) of the low-grade tumors were negative for p16 expression, 43.8% high-grade tumors were positive. Similarly, a larger proportion of invasive carcinomas (38.8%) expressed p16 as compared to non-invasive carcinomas (13.8%). Thus, p16 expression showed a significant association with grade and stage in these malignancies (P < 0.05). The p16 expression was associated with high-grade and muscle-invasive UC. The p16 was absent in all non-neoplastic and precursor lesions. Thus, it can provide essential information not only about HPV association but also on the prognostic implications for the patients.

Association of p53 protein expression with the presence of a 17p13 locus deletion of the TP53 gene and with the survival of patients with diffuse large B-cell lymphoma

Background. A significant role in the pathogenesis, resistance to treatment and progression of many types of lymphomas, including diffuse large B-cell lymphoma, is assigned to the TP53 gene. Literature data on the prognostic significance of the expression of its product, the p53 protein, and its association with aberrations at the 17p13 locus are ambiguous.&#x0D; Aim. To assess the relationship of p53 protein expression with the presence of a 17p13 locus deletion of the TP53 gene and the survival of patients with diffuse large B-cell lymphoma.&#x0D; Material and methods. The study included 75 patients with newly diagnosed diffuse large B-cell lymphoma who received R-CHOP therapy. The calculation of the relative content of tumor cells expressing p53 was carried out using immunohistochemical and morphometric methods on biopsy samples of lymph nodes. The 17p13/TP53 deletion was determined by fluorescent in situ hybridization. The threshold level of p53-positive tumor cells, corresponding to 43%, was calculated by ROC analysis. The association between p53 protein expression and the presence of a 17p13 deletion was assessed using Fisher's (F) and Pearson's 2 tests. The correlation dependence was evaluated by the Cramer method (V). Five-year overall and progression-free survival was calculated using the KaplanMeier method. Differences between the indicators were considered statistically significant at p 0.05.&#x0D; Results. The frequency of 17p13/TP53 deletion was higher in patients with high expression of p53 (43%) compared to the group of patients with low expression: 87.5 and 12.5%, respectively (p=0.018). A direct correlation between a high level of p53 expression (43%) and the presence of a 17p13/TP53 deletion was found (p=0.018). Five-year overall and progression-free survival in patients with a proportion of p53-positive cells 43% was significantly lower than in patients with its subthreshold value: 54.5 versus 81.0% (p=0.014) and 45.5 versus 66.7% (p=0.022), respectively.&#x0D; Conclusion. High expression of the p53 protein is associated with the presence of a 17p13 locus deletion and a low five-year survival rate in patients with diffuse large B-cell lymphoma.

Human papillomavirus status and prognosis of oropharyngeal high-grade neuroendocrine carcinoma

BackgroundThe prognostic impact of human papillomavirus (HPV) infection or smoking on oropharyngeal high-grade neuroendocrine carcinoma (HG-NEC) is not established. MethodsRetrospective study with patients with oropharyngeal HG-NEC seen at MD Anderson Cancer Center from 1997 to 2020, and previously reported patients with oropharyngeal HG-NEC and known p16 and HPV status from the literature review. Survival was estimated with the Kaplan-Meier method, and survival differences assessed with the log-rank test and Cox proportional hazards models. ResultsThirty patients were included; most had a heavy (≥10 pack-years) smoking history (52%), locoregional disease (stage III-IVB; 77%), and p16-positive tumor (92%). HPV was positive in 65% of tested samples (15/23). Of 24 patients treated with curative intent, the objective response rates was 90% (9/10) and 81% (17/21), respectively, for induction chemotherapy and definitive radiotherapy. During follow-up, 54% (13/24) recurred, mostly (11/13) at distant sites. Median overall survival (OS) was 46 months (95% CI, 14.3 - NA). OS was not associated with HPV status (HR 0.73, P = 0.6) or smoking (HR 1.16, P = 0.8). Among 63 patients with known HPV status after the literature review (19 HPV- and 44 HPV + ), HPV status remained unassociated with OS (P = 0.92). ConclusionsThis is the largest retrospective cohort of oropharyngeal HG-NEC. Distant recurrence rate after curative treatment was high, suggesting that multimodality treatment including systemic therapy may benefit patients with locally advanced disease. HPV infection did not affect survival outcomes, therefore should not lead to therapy de-intensification for this histology.

Toxicity Profiles and Survival Outcomes Among Patients With Nonmetastatic Oropharyngeal Carcinoma Treated With Intensity-Modulated Proton Therapy vs Intensity-Modulated Radiation Therapy

Patients with oropharyngeal carcinoma (OPC) treated with radiotherapy often experience substantial toxic effects, even with modern techniques such as intensity-modulated radiation therapy (IMRT). Intensity-modulated proton therapy (IMPT) has a potential advantage over IMRT due to reduced dose to the surrounding organs at risk; however, data are scarce given the limited availability and use of IMPT. To compare toxic effects and oncologic outcomes among patients with newly diagnosed nonmetastatic OPC treated with IMPT vs IMRT with or without chemotherapy. This retrospective cohort study included patients aged 18 years or older with newly diagnosed nonmetastatic OPC who received curative-intent radiotherapy with IMPT or IMRT at a single-institution tertiary academic cancer center from January 1, 2018, to December 31, 2021, with follow-up through December 31, 2021. IMPT or IMRT with or without chemotherapy. The main outcomes were the incidence of acute and chronic (present after ≥6 months) treatment-related adverse events (AEs) and oncologic outcomes, including locoregional recurrence (LRR), progression-free survival (PFS), and overall survival (OS). Fisher exact tests and χ2 tests were used to evaluate associations between toxic effects and treatment modality (IMPT vs IMRT), and the Kaplan-Meier method was used to compare LRR, PFS, and OS between the 2 groups. The study included 292 patients with OPC (272 [93%] with human papillomavirus [HPV]-p16-positive tumors); 254 (87%) were men, 38 (13%) were women, and the median age was 64 years (IQR, 58-71 years). Fifty-eight patients (20%) were treated with IMPT, and 234 (80%) were treated with IMRT. Median follow-up was 26 months (IQR, 17-36 months). Most patients (283 [97%]) received a dose to the primary tumor of 70 Gy. Fifty-seven of the patients treated with IMPT (98%) and 215 of those treated with IMRT (92%) had HPV-p16-positive disease. There were no significant differences in 3-year OS (97% IMPT vs 91% IMRT; P = .18), PFS (82% IMPT vs 85% IMRT; P = .62), or LRR (5% IMPT vs 4% IMRT; P = .59). The incidence of acute toxic effects was significantly higher for IMRT compared with IMPT for oral pain of grade 2 or greater (42 [72%] IMPT vs 217 [93%] IMRT; P < .001), xerostomia of grade 2 or greater (12 [21%] IMPT vs 68 [29%] IMRT; P < .001), dysgeusia of grade 2 or greater (16 [28%] IMPT vs 134 [57%] IMRT; P < .001), grade 3 dysphagia (4 [7%] IMPT vs 29 [12%] IMRT; P < .001), mucositis of grade 3 or greater (10 [53%] IMPT vs 13 [70%] IMRT; P = .003), nausea of grade 2 or greater (0 [0%] IMPT vs 18 [8%] IMRT; P = .04), and weight loss of grade 2 or greater (22 [37%] IMPT vs 138 [59%] IMRT; P < .001). There were no significant differences in chronic toxic effects of grade 3 or greater, although there was a significant difference for chronic xerostomia of grade 2 or greater (6 IMPT [11%] vs 22 IMRT [10%]; P < .001). Four patients receiving IMRT (2%) vs 0 receiving IMPT had a percutaneous endoscopic gastrostomy tube for longer than 6 months. In this study, curative-intent radiotherapy with IMPT for nonmetastatic OPC was associated with a significantly reduced acute toxicity burden compared with IMRT, with few chronic toxic effects and favorable oncologic outcomes, including locoregional recurrence of only 5% at 2 years. Prospective randomized clinical trials comparing these 2 technologies and of patient-reported outcomes are warranted.

P16 Regulates Transcription Factor Sp1 Function to Affect Outcome in Oropharyngeal Cancer

<h3>Purpose/Objective(s)</h3> Our group has previously demonstrated that p16 activates a ubiquitin mediated pathway to repress homologous recombination via activation of HUWE1, potentially via the transcription factor Sp1. However, the specific mechanism of this interaction and its clinical significance remain to be determined. We performed <i>in vitro</i> analysis of p16 and Sp1 interaction in multiple contexts and performed correlative analysis using the head and neck TCGA clinical data, as well as analysis of HPV+ and HPV- oropharyngeal cancer (OPC) treated with chemoradiation (CRT) to evaluate the combined importance of p16 and Sp1 on outcome. We hypothesized that p16 increases the Sp1-HUWE1 interaction. <h3>Materials/Methods</h3> For clinical outcome, 100 patients with oropharyngeal cancer evenly divided between p16+ and p16- and treated with chemoradiation were evaluated for gene expression using targeted multiplex analysis platform. Additional clinical correlative analyses between HPV, p16, HUWE1 and Sp1 were performed using the TCGA Head and Neck cohort (n=530). UM-SCC-47 p16 CRISPR KO and HN5 cells harboring doxycycline-inducible p16 were used to perform <i>in vitro</i> studies. Immunoprecipitation (IP) was used to confirm the protein-protein interaction between protein p16 and transcription factor Sp1. Chromatin immunoprecipitation (ChIP) assay was used to determine the existence of the Sp1 protein-HUWE1 DNA promoter interaction. <h3>Results</h3> Sp1 (p=7.9e⁻¹⁶) and HUWE1 (p=7.9e⁻⁴) were expressed at significantly higher levels in HPV+ tumors and Sp1 and HUWE1 gene expression were highly correlated (p=5.3e10⁻⁴⁵) in the TCGA Head and Neck cohort. In addition, Sp1 expression was significantly higher in p16 positive OPC tumors in a separate patient cohort (p=3e⁻³). In OPC patents treated uniformly with CRT, the combination of p16 and Sp1 expression were associated with reduced locoregional recurrence following CRT (p=7e⁻⁶), effectively delineating good prognosis p16- patients and poor prognosis p16+ patients. IP of p16 in HN5 cells showed increased interaction with Sp1 after doxycycline-induced p16INK4a expression, and in combination with IR. ChIP assay demonstrated SP1 dependent enrichment in 2 of 5 GC box regions of the human HUWE1 promoter. More importantly, these SP1 protein-HUWE1 promoter interactions were significantly enhanced after p16 overexpression. <h3>Conclusion</h3> We show Sp1 directly interacts with HUWE1 promoter resulting in an increase of HUWE1 expression, and this interaction is mediated by p16. This increase of HUWE1 expression then leads to down-regulation of homologous recombination, sensitizing cells to DNA damage. Clinical data shows the combined expression of p16, Sp1 and HUWE1 are highly correlated and that the combination of p16 and Sp1 may be an effective biomarker in OPC.

In silico design of anti-tumor mini-protein targeting MDM2

We designed a disulfide-crosslinked mini-protein with a two-helical topology consisting of l- and d-amino acids, which was exceptionally stable in serum. Therefore, we further used it as a scaffold to design mini-proteins targeting p53 positive tumor cells. Based on bifunctional grafting, key residues from the transactivation domain of p53 and a designed unnatural amino acid were grafted into the helix constituted by l-amino acids to confer the mini-protein with MDM2 inhibitory activity. Meanwhile, ten Arg residues were introduced to improve its membrane penetrating capacity. Among the mini-proteins, UPROL-10e showed nano-molar binding affinity on MDM2 and cellular toxicity on p53 expressing HCT116 cells.

Mutational profiles of head and neck squamous cell carcinomas based upon human papillomavirus status in the Veterans Affairs National Precision Oncology Program.

Patients with advanced head and neck squamous cell carcinoma (HNSCC) associated with human papillomavirus (HPV) demonstrate favorable clinical outcomes compared to patients bearing HPV-negative HNSCC. We sought to characterize the association between HPV status and mutational profiles among patients served by the Veterans Health Administration (VHA). We performed a retrospective analysis of all Veterans with primary HNSCC tumors who underwent next-generation sequencing (NGS) through the VHA's National Precision Oncology Program between July 2016 and February 2019. HPV status was determined by clinical pathology reports of p16 immunohistochemical staining; gene variant pathogenicity was classified using OncoKB, an online precision oncology knowledge database, and mutation frequencies were compared using Fisher's exact test. A total of 79 patients met inclusion criteria, of which 48 (60.8%) had p16-positive tumors. Patients with p16-negative HNSCC were more likely to have mutations in TP53 (p < 0.0001), and a trend towards increased mutation frequency was observed within NOTCH1 (p = 0.032) and within the composite CDK/Rb pathway (p = 0.065). Mutations in KRAS, NRAS, HRAS, and FBXW7 were exclusively identified within p16-positive tumors, and a trend towards increased frequency was observed within the PI3K pathway (p = 0.051). No difference in overall mutational burden was observed between the two groups. In accordance with the previous studies, no clear molecular basis for improved prognosis among patients harboring HPV-positive disease has been elucidated. Though no targeted therapies are approved based upon HPV-status, current efforts to trial PI3K inhibitors and mTOR inhibitors across patients with HPV-positive disease bear genomic rationale based upon the current findings.

Effect of p16 Status on Survival Outcomes in Sinonasal Squamous Cell Carcinoma.

Evaluate the effect of p16 status on disease-free survival (DFS) and overall survival (OS) in patients with sinonasal squamous cell carcinoma (SCC) undergoing treatment with curative intent; and to assess how p16 status may affect patterns of recurrence. Retrospective cohort study. Tertiary medical center. Patients with sinonasal SCC treated with curative intent from 2012 to 2018 were identified. Independent variable of interest was p16 status, which was assessed using immunohistochemistry (IHC) with a 70% staining cutoff for positivity. Kaplan Meier survival curve was plotted to assess correlation between p16 status and DFS and OS. Association between recurrence patterns and p16 status was conducted using chi square and fisher's exact tests. Multivariable Cox proportional hazard analysis was conducted to assess association between independent variables and DFS. Fifty patients with sinonasal SCC met inclusion criteria. Patients were p16 positive in 28/50 (56%) of cases. Kaplan Meier survival curve revealed no statistically significant association between p16 status and DFS or OS survival (P = .780, P = .474). There was no difference in recurrence patterns in patients with p16 positive versus negative tumors. p16 status did not have prognostic value on DFS and OS in our cohort of patients with sinonasal SCC undergoing treatment with curative intent. There was no difference in recurrence patterns between the 2 populations. Based on the results of this study, p16 status should not impact counseling of patients as it relates to their prognosis from SNM.

The effect of HPV DNA and p16 status on the prognosis of patients with hypopharyngeal carcinoma: a meta-analysis

ObjectiveTo evaluate whether the presence of human papillomavirus (HPV) DNA and p16 might be associated with better prognosis in patients with hypopharyngeal carcinoma (HPC), especially on overall survival (OS) and disease-free survival (DFS).MethodPubMed, the Cochrane Library, the Web of Science and EMBASE were searched from inception to April 2021 to search for HPV DNA- and p16-related prognostic articles on HPC. Meta-analysis was performed on the selected articles according to the inclusion and exclusion criteria. Publication bias was assessed for the included studies with Egger’s test. All studies were analyzed by using Stata 16.0 statistical software.ResultsA total of 18 studies were included, including 12 HPV DNA studies and 11 p16 studies. Meta-analysis showed that HPV DNA positivity was a strong prognostic factor for improved OS in patients with HPC, with a pooled hazard ratio (HR) of 0.61 (95% CI, 0.54–0.69), but there was no statistically significant difference in DFS (HR, 0.60; 95% CI, 0.31–1.16). Patients with p16-positive tumors had better OS (HR, 0.66; 95% CI, 0.49–0.89) and DFS (HR, 0.59; 95% CI, 0.44–0.78) than patients with p16-negative tumors.ConclusionsThis study suggests that the presence of HPV DNA leads to better OS in patients with HPC, and the presence of p16 also corresponds to better OS and DFS. Our results provide up-to-date evidence to clinicians and researchers. Larger studies adjusting for prognostic factors are needed in subsequent studies.

Primary tumor volume and prognosis for patients with p16-positive and p16-negative oropharyngeal squamous cell carcinoma treated with radiation therapy

BackgroundThe prescribed radiation dose to patients with oropharyngeal squamous cell carcinoma (OPSCC) is standardized, even if the prognosis for individual patients may differ. Easy-at-hand pre-treatment risk stratification methods are valuable to individualize therapy. In the current study we assessed the prognostic impact of primary tumor volume for p16-positive and p16-negative tumors and in relationship to other prognostic factors for outcome in patients with OPSCC treated with primary radiation therapy (RT).MethodsFive hundred twenty-three OPSCC patients with p16-status treated with primary RT (68.0 Gy to 73.1 Gy in 7 weeks, or 68.0 Gy in 4.5 weeks), with or without concurrent chemotherapy, within three prospective trials were included in the study. Local failure (LF), progression free survival (PFS) and overall survival (OS) in relationship to the size of the primary gross tumor volume (GTV-T) and other prognostic factors were investigated. Efficiency of intensified RT (RT with total dose 73.1 Gy or given within 4.5 weeks) was analyzed in relationship to tumor volume.ResultsThe volume of GTV-T and p16-status were found to be the strongest prognostic markers for LF, PFS and OS. For p16-positive tumors, an increase in tumor volume had a significantly higher negative prognostic impact compared with p16-negative tumors. Within a T-classification, patients with a smaller tumor, compared with a larger tumor, had a better prognosis. The importance of tumor volume remained after adjusting for nodal status, age, performance status, smoking status, sex, and hemoglobin-level. The adjusted hazard ratio for OS per cm3 increase in tumor volume was 2.3% (95% CI 0–4.9) for p16-positive and 1.3% (95% 0.3–2.2) for p16-negative. Exploratory analyses suggested that intensified RT could mitigate the negative impact of a large tumor volume.ConclusionsOutcome for patients with OPSCC treated with RT is largely determined by tumor volume, even when adjusting for other established prognostic factors. Tumor volume is significantly more influential for patients with p16-positive tumors. Patients with large tumor volumes might benefit by intensified RT to improve survival.

Laser tongue base mucosectomy is a useful diagnostic tool in the management of unknown primary cancers of the head and neck region.

Tongue base mucosectomy identified cancer in 78 per cent of cancers of unknown primary in a recent meta-analysis. The carbon dioxide laser is an alternative technique if there is no access to a robot. This study aimed to describe the steps for undertaking tongue base mucosectomy using the carbon dioxide laser and its diagnostic utility in cancers of unknown primary. This was a prospective feasibility study utilising carbon dioxide laser for tongue base mucosectomy in cancers of unknown primary. Data collected included demographic data and p16 status. There were 14 cancers of unknown primary with 86 per cent p16 positivity on immunohistochemistry. Laser tongue base mucosectomy alone identified the cancer primary in 7 of 12 (58 per cent) cancers of unknown primary among p16 positive tumours and 0 of 2 (0 per cent) among p16 negative tumours. Combining bilateral tonsillectomy with laser tongue base mucosectomy resulted in identification of the primary cancer in 8 of 12 (67 per cent) p16 positive tumours. In centres without a robot, tongue base mucosectomy using the carbon dioxide laser is a viable alternative, especially in combination with bilateral tonsillectomy in p16 positive cases.

Pathologic response after induction chemo-immunotherapy with single or double immune checkpoint inhibition in locally advanced head and neck squamous cell carcinoma (HNSCC): Expansion cohorts of the CheckRad-CD8 trial.

6064 Background: Targeting the immune checkpoint CTLA-4 in addition to PD-1/PD-L1 alone did not increase efficacy in HNSCC, whereas this has not been studied in combination with chemotherapy. Induction chemo-immunotherapy followed by pathologic response-based patient selection for chemotherapy-free radioimmunotherapy was efficient in locally advanced HNSCC (J Immunother Cancer. 2022 Jan;10(1):e003747). The expansion cohorts of the CheckRad-CD8 trial studied safety and efficacy of induction chemo-immunotherapy with increased dose or without CTLA-4 inhibition. Methods: Patients with previously untreated stage III-IVB (AJCC 8th edition) HNSCC were eligible for this multicenter phase II trial. Induction chemo-immunotherapy of the main cohort (MC) consisted of a single cycle of cisplatin 30mg/m² d1-3, docetaxel 75mg/m² d1, durvalumab 1500mg fix dose d5 and tremelimumab 75mg fix dose d5. Patients in expansion cohort 1 (EC1) received this combination with high dose tremelimumab 300mg fix dose d5 and patients in expansion cohort 2 (EC2) received no tremelimumab. In EC1 and EC2 prophylactic G-CSF was recommended. Patients with at least 20% increase of intratumoral CD8+ immune cell density or pathological complete response (pCR) in the re-biopsy entered chemotherapy-free radioimmunotherapy up to a total dose of 70Gy. The current analysis focuses on toxicity and pathologic response after induction chemo-immunotherapy. Results: Between Sep 2018 and Sep 2021, 80 patients were enrolled in the MC (one excluded), 20 in EC1 and 20 in EC2 (one excluded) subsequently. In the MC, EC1 and EC2 a total of 56%, 50%, 58% were stage IV and 29%, 30%, 26% had p16 positive oropharyngeal tumors. Baseline median intratumoral CD8+ immune cell density was 395/mm², 505/mm² and 763/mm² in MC, EC1 and EC2. After induction chemo-immunotherapy 41 (52%), 12 (60%) and 11 (58%) of the patients had pCR in the re-biopsy in MC, EC1 and EC2. Patients with residual tumor after induction therapy had a median intratumoral CD8+ immune cell density of 670/mm², 781/mm² and 1605/mm², which was a median increase by factor 3.0, 2.1 and 4.8 in the corresponding patients’ tissue samples. In the cohorts MC, EC1 and EC2 the overall rate of grade 3-4 adverse events per patient was 1.38, 1.35 and 0.58. The corresponding rate of non-hematologic adverse events per patient was 0.84, 0.95 and 0.37, respectively. Conclusions: Neither increase of tremelimumab dosage nor its omission did significantly affect pathologic response to induction chemo-immunotherapy with cisplatin/ docetaxel/ durvalumab. Non-hematologic toxicity was slightly increased for high dose tremelimumab and clearly decreased without tremelimumab. The role of concomitant administration of tremelimumab with radiotherapy cannot be assessed until the final study analysis. Clinical trial information: NCT03426657.