P16-positive Oropharyngeal Squamous Cell Carcinoma Research Articles

Extracting interpretable features for pathologists using weakly supervised learning to predict p16 expression in oropharyngeal cancer

One drawback of existing artificial intelligence (AI)-based histopathological prediction models is the lack of interpretability. The objective of this study is to extract p16-positive oropharyngeal squamous cell carcinoma (OPSCC) features in a form that can be interpreted by pathologists using AI model. We constructed a model for predicting p16 expression using a dataset of whole-slide images from 114 OPSCC biopsy cases. We used the clustering-constrained attention-based multiple-instance learning (CLAM) model, a weakly supervised learning approach. To improve performance, we incorporated tumor annotation into the model (Annot-CLAM) and achieved the mean area under the receiver operating characteristic curve of 0.905. Utilizing the image patches on which the model focused, we examined the features of model interest via histopathologic morphological analysis and cycle-consistent adversarial network (CycleGAN) image translation. The histopathologic morphological analysis evaluated the histopathological characteristics of image patches, revealing significant differences in the numbers of nuclei, the perimeters of the nuclei, and the intercellular bridges between p16-negative and p16-positive image patches. By using the CycleGAN-converted images, we confirmed that the sizes and densities of nuclei are significantly converted. This novel approach improves interpretability in histopathological morphology-based AI models and contributes to the advancement of clinically valuable histopathological morphological features.

The influence of tumor volume on the risk of distant metastases in head and neck squamous cell carcinomas

Background and purposeDistant metastases (DM) in head and neck squamous cell carcinomas (HNSCC) are in most circumstances non-curable. The TNM staging system is insufficient to predict the risk of DM. This study investigates if the DM risk can be predicted using a multivariate model including pre-treatment total tumor volume for both p16-positive oropharyngeal squamous cell carcinoma (OPSCC) and all other sites (other HNSCC). Materials and methodsThe study includes patients with localized pharyngeal and laryngeal squamous cell carcinomas treated with primary radiotherapy from 2008-2017 from three head and neck cancer centers. Patients were identified in the Danish Head and Neck Cancer (DAHANCA) database. Total (nodal and primary) tumor volume (Gross Tumor Volume, GTV) was extracted from local treatment planning systems. The GTV was grouped by volume (cm3) in four intervals and included in a multivariate Cox proportional hazard regression controlled for pre-selected clinical values incl. stage. ResultsThe study includes 2,865 patients, of which 321 (11 %) had DM post-treatment. The risk of DM was assessed in a multivariate model based on 2,751 patients (p16-positive OPSCC: 1,032; and other HNSCC: 1,719). There was a significant association between GTV and the risk of DM, and in tumor volumes ≥ 50 cm3 hazard ratios of 7.6 (2.5–23.4) for p16-positive OPSCC and 4.1 (2.3–7.2) in other HNSCC were observed. ConclusionTumor volume is an independent risk factor for DM. The addition of total tumor volume to a predictive model is important to identify subgroups of HNSCC patients at high risk of DM.

The effectiveness of a dual-component regimen of induction chemotherapy in the treatment of HPV-positive oropharyngeal squamous cell carcinoma

Given the favorable prognosis associated with HPV-positive oropharyngeal cancer, cancer care professionals are actively discussing the feasibility of de-escalating treatment strategy for this cohort of patients.The purpose of the study was to improve the treatment outcomes in patients with locally advanced HPV- positive oropharyngeal squamous cell carcinoma by identifying the optimal induction chemotherapy (ICH) regimen, in particular, by using a two-drug ICH.Material and Methods. The study included 27 patients with locally advanced (T3–4N0–1, or T1–4N2–3) P16-positive oropharyngeal squamous cell carcinoma. All patients received 3 cycles of ICT according to the TP (docetaxel + cisplatin) regimen. To assess the effect and toxicity of two-drug ICH, a control group of patients, who received ICH according to the standard three-drug TPF (docetaxel, cisplatin and 5FU) regimen, was formed.Results. In the TP group, complete response (CR) was achieved in 3 (11 %) patients, partial response (PR) in 17 (63 %), stable disease (SD) in 7 (26 %) patients. In the control group: CR 4 (20 %) patients, PR – 13 (60 %), SD – 4 (20 %) patients. The median follow-up time in the TP group was 9 months (range: 2 to 22 months). The 1-year progression-free (PFS) and overall survival (OS) rates were 88.2 % and 100 %, respectively. In the TPF group, the PFS and OS rates were 84.4 % and 100 %, respectively.Conclusion. The results revealed that TP ICH regimen was non-inferior to the standard TPF regimen in the rates of the objective response, 1-year OS and PFS.

High Levels of FGF11 Correlate with Poor Survival in Patients with Human Papillomavirus (HPV)-Positive Oropharyngeal Squamous Cell Carcinoma

Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is associated with a favourable prognosis. It has therefore been suggested that treatment should be individualized and separated by HPV status. However, additional prognostic markers are still needed before treatment can be individualized for this patient group. For this purpose, all patients diagnosed with HPV and p16-positive OPSCC in Stockholm 2000-2009, identified as having a partial/nonresponse to treatment and having viable tumour cells in their neck specimen with material available were categorized as cases. These were matched to controls (complete responders), and the differences in the gene expression were analysed. Two separate verification cohorts were identified including patients with HPV- and p16-positive OPSCC, and the data from the case-control study were verified by qPCR and immunohistochemistry (IHC) in the respective cohorts. A separation of gene expression in correlation with survival was observed in the case-control study, and FGF11 expression was identified as significantly differently expressed between the two groups. The prognostic role of FGF11 was validated in the two cohorts on the RNA and protein levels, respectively. Taken together, our findings suggest that FGF11 may indicate a poor prognosis in HPV-positive OPSCC and may serve as a prognostic biomarker.

P16陽性中咽頭癌StageⅢの初回治療と予後因子の検討

p16陽性中咽頭癌StageⅢの初回治療と予後因子の検討

Longitudinal Analysis of Swallowing Displacement Kinematics After Oropharyngeal Cancer Treatment.

Quantitative swallowing displacement kinematics evolve in patients treated for oropharyngeal squamous cell carcinoma (OPSCC). We aimed to longitudinally assess these measurements and correlate them with functional swallowing outcomes. A retrospective review was conducted on patients with OPSCC treated with definitive (chemo)radiation ([C]RT) or surgery with adjuvant (chemo)radiation (S-[C]RT) who completed at least two videofluoroscopic swallow studies (VFSS). Longitudinal analysis was accomplished via mixed-effects logistic regression for the Functional Oral Intake Scale (FOIS), and Penetration Aspiration Scale (PAS), and mixed-effects linear regression for kinematic measures. Spearman's correlation was conducted between changes in FOIS/PAS and kinematic measures. Ninety-seven patients (76 males; mean age 61) completed 245 VFSS studies. A total of 94% had human papillomavirus (HPV)/p16 positive OPSCC and 74% were T0-T2. Sixty-four patients underwent [C]RT while 33 patients underwent S-[C]RT. After treatment, posterior pharyngeal wall at hold (PPWhold) increased 3.2 standard deviation (SD) between 0 and 6 months (p < 0.001), then decreased 2.2 SD between 6 and 12 months (p < 0.001) and did not return to baseline. Hyoid-to-larynx (HL) (p=0.046) and maximal hyoid displacement (Hmax) + HL (p=0.042) increased between 6 and 12 months. Hmax (p=0.020) and Hmax + HL (p < 0.001) decreased between 12-24 months beyond baseline values. The decrease in HL and increase in PPWhold (p < 0.05) correlated with an increase in PAS. From baseline, increased pharyngeal constriction ratio correlated with decreased FOIS and PPWhold (p < 0.05). Quantitative swallowing kinematic measures can effectively track changes in swallowing physiology. Increased PPWhold and restricted hyolaryngeal movement were seen in patients with OPSCC after treatment and correlated with a change in swallowing outcome, emphasizing the need for serial VFSS monitoring and targeted intervention. 3 Laryngoscope, 133:1339-1348, 2023.

Margin Sampling and Survival Outcomes in Oral Cavity and p16-Positive Oropharyngeal Squamous Cell Carcinoma.

ObjectiveTo compare the association of margin sampling technique on survival outcomes in surgically treated cT1-2 oral cavity and oropharyngeal squamous cell carcinoma.Study DesignA prospective longitudinal cohort study.SettingTertiary care academic teaching hospital in Halifax, Nova Scotia.MethodsAll cases of surgically treated cT1-2 oral cavity and oropharyngeal cancer undergoing specimen-oriented margin analysis between January 1, 2017, and December 31, 2018 were analyzed. The specimen-oriented cohort was compared with a cohort of patients from January 1, 2009, to December 31, 2014, where a defect-oriented margin sampling protocol was used. Kaplan-Meier survival curves were used to estimate 2-year overall survival, disease-specific survival, local control, and recurrence-free survival rates in oral cavity and p16-positive oropharyngeal squamous cell carcinoma. Cox proportional hazards models were used to assess the effect of margin sampling method on disease-specific survival and local control.ResultsThere was no significant association between margin sampling technique and 2-year survival outcomes for surgically treated cT1-2 oral cavity and oropharyngeal squamous cell carcinoma. In the multivariate Cox proportional hazard model, the hazard ratio (HR) of specimen-oriented sampling was not significantly different for disease-specific survival (HR, 1.32; 95% CI, 0.3032-5.727; P = .713) or local control (HR, 0.4087; 95% CI, 0.0795-2.099; P = .284).ConclusionIntraoperative margin sampling method was not associated with a significant change in 2-year survival outcomes. Despite no effect on survival outcomes, implementation of a specimen-oriented sampling method has potential for cost avoidance by decreasing the number of re-resections for positive or close margins.

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.

Selective treatment de-intensification with reduced-dose radiation and omitted concurrent chemotherapy guided by response to induction chemotherapy in HPV-associated oropharyngeal squamous cell carcinoma: A single-arm, phase II trial (IChoice-01).

e18069 Background: De-intensification in chemoradiation provides a strategy to optimize the trade-off between treatment efficacy and toxicities in HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). However, the failure of RTOG 1016 and De-ESCALaTE indicated the pitfall of de-escalation in an unselective population and the importance of patient selection for future study design. Induction chemotherapy (IC), as a potential biomarker, has been adopted in several trials to screen candidates for de-intensified treatment based on its tumor response. In present trial, we investigated the feasibility of selectively de-intensified chemoradiotherapy by reducing radiation dose and omitting concurrent chemotherapy guided by response to IC. Methods: From Jan 2019 to July 2021, 48 patients with p16 positive OPSCC, T1-2/N1-3M0 (excluding T1N1M0 patients with single and≤3cm lymph node) or T3-4N0-3M0 according to the UICC/AJCC 8th staging system were enrolled. All of them received two cycles of IC with cisplatin and docetaxel. Those with major response to IC (defined as cCR or ≥50% cPR of both primary site and lymph nodes) received de-intensified treatment with intensity modulated radiation therapy (IMRT) alone with 60Gy to high-risk and 54Gy to low-risk regions (Di cohort). Those with less than major response were given standard chemoradiotherapy with 70Gy to both primary tumor and positive lymph nodes, 63Gy to high-risk and 56Gy to low-risk regions, concurrently with two cycles of cisplatin (St cohort). The primary endpoint was 2-year progression-free survival (PFS). Results: 26/48 (54.2%) patients entered Di cohort while other 22/48 (45.8%) patients entered St cohort. With a median follow-up time of 20.5 months (2.8-36.7months), 3 deaths (1 in Di cohort and 2 in St cohort), 5 loco-regional recurrence (1 in Di cohort and 4 in St cohort) and 6 distant metastases (1 in Di cohort and 5 in St cohort) were documented. The 2-year PFS rates for Di and St cohort were 100% and 60.0% (P = 0.002) with overall survival (OS) rates of 100% and 83.1% (P = 0.062), loco-regional recurrence-free survival (LRFS) rates of 100% and 77.0% (P = 0.035), metastasis-free survival (MFS) rates of 100% and 66.8% (P = 0.011), respectively. Conclusions: For major responders to IC, reduced-dose radiation with omitted concurrent chemotherapy yielded good survival results. In comparison, patients resistant to IC showed poor prognosis even under standard-dose chemoradiotherapy, calling for treatment intensification or development of novel therapeutic agents. Selective de-intensification of chemoradiation guided by response to IC is promising in HPV-associated OPSCC and warrants further evaluation in future studies. Clinical trial information: NCT04012502.

MicroRNA-182-5p and microRNA-205-5p as potential biomarkers for prognostic stratification of p16-positive oropharyngeal squamous cell carcinoma.

MicroRNAs constitute promising biomarkers. The aim was to investigate diagnostic and prognostic implications of miR-182-5p and miR-205-5p in p16-positive and p16-negative oropharyngeal squamous cell carcinomas (OPSCCs). Expression of miR-182-5p, miR-205-5p were determined via quantitative real-time-PCR in fresh frozen tissues of 26 p16-positive, 19 p16-negative OPSCCs and 18 HPV-negative oropharyngeal controls. Associations between miRNA-expression, clinicopathological characteristics and prognosis were analyzed. Higher miR-182-5p expression was associated with significant inferior disease-specific survival for p16-positive OPSCCs (HR = 1.98E+09, 95% CI 0-Inf; P= 0.028) and a similar trend was observed for p16-negative OPSCCs (HR = 1.56E+09, 95% CI 0-Inf; P= 0.051). Higher miR-205-5p expression was associated with an inferior progression-free survival (HR = 4.62, 95% CI 0.98-21.83; P= 0.034) and local control rate (HR = 2.18E+09, 95% CI 0-Inf; P= 0.048) for p16-positive OPSCCs. Results indicate that miR-182-5p and miR-205-5p can further stratify patients with p16-positive OPSCC into prognostic groups.

A Randomized Trial of Radiotherapy vs. Trans-Oral Surgery for Treatment De-Escalation in HPV-Associated Oropharyngeal Squamous Cell Carcinoma (ORATOR2)

<h3>Purpose/Objective(s)</h3> Widespread oral human papillomavirus (HPV) infections have led to a rapid increase in the incidence of oropharyngeal squamous cell carcinoma (OPSCC). HPV-related OPSCCs have a better prognosis than conventional alcohol- and smoking-related OPSCCs, suggesting a role for treatment de-escalation. The goal of this phase II randomized trial was to assess survival, oncologic, and toxicity outcomes with two de-escalation approaches: primary reduced-dose radiotherapy (RT) vs. primary transoral surgery plus neck dissection (TOS + ND) with reduced-dose adjuvant therapy. <h3>Materials/Methods</h3> Weenrolled patients with T1-T2N0-2 (AJCC 8th edition) p16-positive OPSCC. After stratifying by smoking status, we randomized patients (1:1) to either the primary RT arm, which consisted of 60 Gy of RT and concurrent weekly cisplatin chemotherapy in node-positive patients vs. the TOS + ND arm, consisting of surgery and neck dissection, with adjuvant reduced-dose RT depending on pathologic findings. The primary endpoint was overall survival (OS), and secondary endpoints included progression-free survival (PFS), quality of life (QOL, using the MDADI and other metrics), and toxicity. The trial was closed to accrual in November 2020 due to excessive toxicity in the TOS + ND arm, consisting of two treatment-related deaths from known complications of TOS (one bleed and one cervical osteomyelitis following post-operative RT). After closure to accrual, all previously enrolled patients remained on follow-up. All analyses were pre-specified and intention-to-treat, unless otherwise specified. Due to these unexpected toxicity findings, the trial is being reported while survival outcomes remain immature, and therefore p-values are not reported for OS and PFS comparisons. <h3>Results</h3> BetweenFebruary 2018 and November 2020, 61 patients were randomized (n=30 in the RT arm and n=31 in the TOS + ND arm). Median age was 61.9 years, most patients (51%) were never-smokers, and the large majority of patients (n=51; 86%) were male. The arms were well-balanced. Median follow-up was 17 months (IQR: 15-20 months). Two-year estimates of OS were 100% in the RT arm (95% confidence interval [CI]: 100%-100%) and 89.1% (95% CI: 69.6%-96.4%) in the TOS + ND arm. Two-year PFS estimates were 100% in the RT arm (95% CI: 100%-100%) and 83.5% (95% CI: 60.8%-93.7%) in the TOS + ND arm. Grade 2-5 toxicities occurred in 67% of patients in the RT arm and 71% in the TOS + ND arm, with significantly more anorexia and dysgeusia in the RT arm. Mean (±SD) MDADI total scores at 1-year were similar between arms (85.7 ± 15.6 and 84.7 ± 14.5, respectively). One patient in each arm required a percutaneous feeding tube, and none required feeding tubes at 1-year. <h3>Conclusion</h3> The primary RT approach achieved excellent oncologic outcomes in treatment de-escalation, with a moderate toxicity profile, and should be tested in phase III trials. The primary TOS approach was associated with an upfront risk of treatment-related mortality and suboptimal PFS. (NCT03210103)

811 Management of Patients with HPV-associated Oropharyngeal Cancer: A Units' Experience

Abstract Aim The low positive predictive value (PPV) of initial post-therapy PET-CT in patients with Human papillomavirus (HPV-) associated oropharyngeal squamous cell carcinoma (OPSCC) presents a challenge for teams managing residual disease after initial treatment. We aimed to investigate current management, post treatment surveillance protocols and outcomes of these patients within our unit. Method A retrospective review was performed of p16 positive OPSCC patients treated in 2018. Their primary treatment modality (radiotherapy (RT) or chemoradiotherapy (CRT), neck dissection), post-therapy PET-CT results, management decisions and final clinical outcomes were recorded. Results 38 patients with p16 positive OPSCC were included in the study. 36 (94.7%) received primary CRT or RT, and 2 (5.3%) had primary neck dissections with adjuvant RT. 21 patients were found to have residual disease on the 3-month post-treatment PET-CT. Of these, 9 underwent further PET-CT surveillance, all showing no residual disease. 8 patients received ultrasound +/- fine needle aspiration, from which 1 showed incurable residual and distant metastasis. 2 underwent clinical surveillance, showing no residual disease. 1 had immediate salvage surgery, with further recurrence. 1 patient with persistent disease and lung metastasis received palliative immunotherapy. Conclusions 31 patients were disease free at the end point of this study. This study explores the need for further investigation into features of residual disease in post treatment PET-CT that would indicate initial treatment failure and possible further early intervention. A surveillance strategy following initial post treatment PET-CT needs to be developed to aid in making this decision as early as possible in the post treatment phase.

Bayesian Based Granger Causality Analysis for Positive Emotional EEGs

Oropharyngeal squamous cell carcinoma (OPSCC) can be divided into favourable and poor prognostic groups by association with human papilloma virus (HPV) and smoking. This study prospectively investigated a dose-intensified schedule in poor/intermediate prognosis OPSCC.Patients with p16/HPV-negative or p16-positive N2b OPSCC with a greater than 10 pack-year smoking history were eligible. Patients were planned to receive 64 Gy in 25 fractions with cisplatin. The primary end point was absence of grade 3 mucositis at 3 months.Fifteen patients were recruited over 14 months. All patients completed a minimum of 2 years of follow-up. All patients completed full-dose radiotherapy within a median treatment time of 32 days (31–35). Grade 3 mucositis was absent in all patients at 3 months. There was one grade 4 toxicity event due to cisplatin (hypokalaemia). Complete response rates at 3 months were 100% and 93% for local disease and lymph nodes, respectively. One patient developed metastatic disease and subsequently died. Overall survival at 2 years was 93% (95% confidence interval 61–99%).The schedule of 64 Gy in 25 fractions with concomitant chemotherapy is tolerable in patients with poor and intermediate prognosis OPSCC.

Potential association of the prognostic index and survival in patients with p16-positive oropharyngeal squamous cell carcinoma.

BackgroundThe aim was to assess the prognostic value of the newly proposed prognostic index (PI) in patients with p16-positive oropharyngeal squamous cell carcinoma.MethodsPatients treated with primary surgery from 2012 to 2019 with available preoperative (0–2 days) values of C‑reactive protein and white blood cell counts needed for calculation of the PI, were included. Main outcome measures were overall survival (OS) and disease-free survival (DFS). The PI was dichotomized into low (PI = 0) and high (PI ≥ 1).ResultsIn this study 36 patients were included. Average overall (OS) and disease-free survival (DFS) were 3.3 years (range 0.2–12.3 years) and 2.8 years (0.0–9.8 years), respectively. The overall mortality was 16.7% (n = 6) and a recurrent disease was observed in 30.6% of patients (n = 11). Low PI was associated with better overall survival (mean OS 10.1 ± 1.4 years, 95% confidence interval, CI 7.3–12.9 years vs. 1.9 ± 0.4, 95% CI 1.3–2.6 years, p < 0.01; mean DFS 8.5 ± 0.7 years, 95% CI 7.1–9.6 years vs. 1.0 ± 0.3 years, 95% CI 0.5–1.5 years, p < 0.01).ConclusionThe PI might be an easily obtainable outcome prognosticator in p16-positive oropharyngeal squamous cell carcinoma patients. Analyzing routinely obtained blood samples can contribute to identifying high-risk patients.

FDG-PET/CT identified distant metastases and synchronous cancer in squamous cell carcinoma of the head and neck: the impact of smoking and P16-s.

Whole-body FDG-PET-CT is widely used at diagnosis of squamous cell carcinoma of the head and neck (SCCHN) but may identify suspicious lesions outside the neck that require investigation. This study evaluated the impact of smoking and P16-status on the incidence of malignant disease outside the head and neck region in newly diagnosed patients with SCCHN. All PET-positive foci outside the head-neck area were registered in 1069 patients planned for postoperative or curative intent radiotherapy with whole-body FDG-PET/CT from 2006 to 2012. All patient files were retrospectively investigated and clinical parameters, tobacco use, HPV (P16)-status and subsequent malignant disease registered. Malignancy outside the neck was diagnosed in 9% of smokers, 2% of never-smokers, and 5% of patients with P16-positive oropharyngeal squamous cell carcinoma (OPSCC). Clinically suspicious PET-positive foci outside the head-neck were malignant in 55% of smokers, 34% of never-smokers, and in 38% of P16-pos OPSCC. All but two patients with cancer occurring outside the head and neck region were smokers. Malignancy outside the neck at diagnosis was more frequent in smokers compared to non-smokers or P16-pos OPSCC. A high proportion of clinically suspicious PET-positive foci were non-malignant.

Long-Term Survival and Recurrence in Oropharyngeal Squamous Cell Carcinoma in Relation to Subsites, HPV, and p16-Status.

Simple SummaryLong-term survival in patients with oropharyngeal cancer is sparsely studied, but atypical recurrences in human papillomavirus-positive (HPV+) oropharyngeal cancer have been indicated. Furthermore, while the role of HPV is well established in tonsillar and base of tongue cancer, the dominant oropharyngeal subsites, its role in the minor oropharyngeal sites (the oropharyngeal walls, the uvula, and the soft palate) is not fully elucidated. The aim of this retrospective study was therefore to assess long-term outcome in relation to oropharyngeal sub-sites and HPV/p16 status. We confirm the prognostic role of p16+ in tonsillar and base of tongue cancer, but not the other sites. We find that combined HPV/p16-status gives better prognostic information than p16 alone. Lastly, we show that p16− cancer has more locoregional and late recurrences compared to p16+ cancer. Consequently, only combined HPV/p16 positivity in patients with tonsillar and tongue base cancer should be used in future treatment de-escalation trials.Long-term survival data in relation to sub-sites, human papillomavirus (HPV), and p16INK4a (p16) for patients with oropharyngeal squamous cell carcinoma (OPSCC) is still sparse. Furthermore, reports have indicated atypical and late recurrences for patients with HPV and p16 positive OPSCC. Therefore, we assessed long-term survival and recurrence in relation to oropharyngeal subsite and HPV/p16 status. A total of 529 patients with OPSCC, diagnosed in the period 2000–2010, with known HPVDNA and p16-status, were included. HPV/p16 status and sub-sites were correlated to disease-free and overall survival (DFS and OS respectively). The overexpression of p16 (p16+) is associated with significantly better long-term OS and DFS in tonsillar and base of tongue carcinomas (TSCC/BOTSCC), but not in patients with other OPSCC. Patients with HPVDNA+/p16+ TSCC/BOTSCC presented better OS and DFS compared to those with HPVDNA−/p16− tumors, while those with HPVDNA−/p16+ cancer had an intermediate survival. Late recurrences were rare, and significantly more frequent in patients with p16− tumors, while the prognosis after relapse was poor independent of HPVDNA+/−/p16+/− status. In conclusion, patients with p16+ OPSCC do not have more late recurrences than p16−, and a clear prognostic value of p16+ was only observed in TSCC/BOTSCC. Finally, the combination of HPVDNA and p16 provided superior prognostic information compared to p16 alone in TSCC/BOTSCC.

Discrepancy in p16 expression in patients with HPV-associated head and neck squamous cell carcinoma in Thailand: clinical characteristics and survival outcomes

BackgroundLower prevalence HPV infection has been previously reported in Thai population when compared with Western countries. p16 expression indicates HPV-associated oropharyngeal squamous cell carcinoma (OPSCC), but not non-OPSCC. We therefore evaluated the characteristic and association of p16 and HPV in Thai patients with HNSCC.MethodsWe used immunohistochemistry and qPCR, respectively, to detect p16 and HPV DNA in archrival formalin-fixed paraffin-embedded HNSCC tissues. Patient characteristics and survival were analyzed.Resultsp16 expression was detected in tumors of 72 of 662 (10.9%) patients with HNSCC and was significantly associated with higher-grade histology, advanced nodal stage, and oropharynx. p16 was expressed in 28 and 6.5% of patients with OPSCC or non-OPSCC, respectively, and HPV DNA was detected in 15.6 and 1% of patients, respectively. Using p16 as a surrogate for HPV status, sensitivities were 80 and 25% in OPSCC and non-OPSCC, respectively. Positive and negative predictive rates of OPSCC were 38 and 95%. Discordance rates between HPV and p16 were 23 and 7% in OPSCC and non-OPSCC, respectively. Overall survival (OS) were significantly longer in both p16-positive OPSCC (p = 0.049), and non-OPSCC (p = 0.003).ConclusionsLow prevalence of p16 and HPV associated OPSCC and non-OPSCC were confirmed in Thai patients. High discordance and low positive predictive rates of p16 were observed in HPV-associated OPSCC. p16 was a significant prognostic factor for OS for patients with OPSCC or non-OPSCC. Therefore, HPV testing should be performed to assess the association of HPV with HNSCC regardless of p16 expression.

Artificial intelligence for automating the measurement of histologic image biomarkers.

Artificial intelligence has been applied to histopathology for decades, but the recent increase in interest is attributable to well-publicized successes in the application of deep-learning techniques, such as convolutional neural networks, for image analysis. Recently, generative adversarial networks (GANs) have provided a method for performing image-to-image translation tasks on histopathology images, including image segmentation. In this issue of the JCI, Koyuncu et al. applied GANs to whole-slide images of p16-positive oropharyngeal squamous cell carcinoma (OPSCC) to automate the calculation of a multinucleation index (MuNI) for prognostication in p16-positive OPSCC. Multivariable analysis showed that the MuNI was prognostic for disease-free survival, overall survival, and metastasis-free survival. These results are promising, as they present a prognostic method for p16-positive OPSCC and highlight methods for using deep learning to measure image biomarkers from histopathologic samples in an inherently explainable manner.

RB1, p16, and Human Papillomavirus in Oropharyngeal Squamous Cell Carcinoma.

While P16 immunohistochemistry (IHC) is a well-established surrogate marker of Human Papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (OSCC), Retinoblastoma 1 (RB1) loss may lead to p16 overexpression in the absence of HPV. We determined the proportion of p16-positive/HPV-negative OSCC with RB1 loss and other alterations in RB1/p16 pathway, and tested RB1 IHC as a prognostic biomarker for OSCC, along with the 8th edition of AJCC staging manual. P16 and RB1 IHC and HPV DNA in situ hybridization (ISH) were performed on 257 OSCC. High risk HPV RNA ISH, RB1 fluorescence in situ hybridization (FISH), and next generation sequencing (NGS) were done on p16-positive/HPV DNA ISH-negative OSCC. Disease free survival (DFS) was used as an endpoint. In the entire cohort and in p16-positive (n = 184) and p16-negative (n = 73) subgroups, AJCC 8th edition staging correlated with DFS (p < 0.01). RB1 IHC showed RB1 loss in p16-positive OSCC only (79/184, 43%). RB1 loss by IHC is associated with a better DFS, without providing additional prognostic information for patients with p16-positive OSCC. HPV RNA ISH was positive in 12 of 14 HPV DNA ISH-negative cases. RB1 IHC showed loss in 10 of 15 HPV DNA ISH-negative cases and in 1 of 2 HPV RNA ISH-negative cases. Overall, only one case of p16-positive/HPV RNA ISH-negative OSCC showed RB1 loss by IHC (1/184, 0.5%). Of the 10 p16-positive and HPV DNA ISH-negative cases with RB1 loss by IHC, 2 had RB1 hemizygous deletion and 3 showed Chromosome 13 monosomy by FISH. No RB1 mutations were detected by NGS. Other molecular alterations in p16-positive/HPV DNA ISH-negative cases included TP53 and TERT mutations and DDX3X loss. HPV-independent RB1 inactivation rarely results in false positive p16 IHC. RB1 inactivation by high risk HPV E7 oncoprotein may co-exist with RB1 deletion. RB1 loss is a favorable prognosticator and occurs exclusively in p16-positive OSCC. The 8th edition of the AJCC staging manual satisfactorily predicts DFS of OSCC patients.

Correlation of HPV16 Gene Status and Gene Expression With Antibody Seropositivity and TIL Status in OPSCC.

IntroductionHuman papillomavirus 16 (HPV16) is the main cause of oropharyngeal squamous cell carcinoma (OPSCC). To date, the links between HPV16 gene expression and adaptive immune responses have not been investigated. We evaluated the correlation of HPV16 DNA, RNA transcripts and features of adaptive immune response by evaluating antibody isotypes against E2, E7 antigens and density of tumor-infiltrating lymphocytes (TIL).Material and MethodsFFPE-tissue from 27/77 p16-positive OPSCC patients was available. DNA and RNA were extracted and quantified using qPCR for all HPV16 genes. The TIL status was assessed. Immune responses against E2 and E7 were quantified by ELISA (IgG, IgA, and IgM; 77 serum samples pre-treatment, 36 matched post-treatment).ResultsAmounts of HPV16 genes were highly correlated at DNA and RNA levels. RNA co-expression of all genes was detected in 37% (7/19). E7 qPCR results were correlated with higher anti-E7 antibody (IgG, IgA) level in the blood. Patients with high anti-E2 IgG antibody (>median) had better overall survival (p=0.0311); anti-E2 and anti-E7 IgA levels had no detectable effect. During the first 6 months after treatment, IgA but not IgG increased significantly, and >6 months both antibody classes declined over time. Patients with immune cell-rich tumors had higher levels of circulating antibodies against HPV antigens.ConclusionWe describe an HPV16 qPCR assay to quantify genomic and transcriptomic expression and correlate this with serum antibody levels against HPV16 oncoproteins. Understanding DNA/RNA expression, relationship to the antibody response in patients regarding treatment and outcome offers an attractive tool to improve patient care.