HPV-negative Patients Research Articles

Prediction of distant metastases in patients with squamous cell carcinoma of head and neck using DWI and DCE-MRI.

The primary purpose was to evaluate the prognostic potential of diffusion imaging (DWI) and dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) in predicting distant metastases in squamous cell carcinoma of head and neck (HNSCC) patients. The secondary aim was to examine differences in DWI and DCE-MRI-derived parameters on the basis of human papilloma virus (HPV) status, differentiation grade, and nodal stage of HNSCC. Fifty-six patients underwent pretreatment DWI and DCE-MRI. Patients were divided into groups who subsequently did (n = 12) or did not develop distant metastases (n = 44). Median values of apparent diffusion coefficient (ADC), volume transfer constant (Ktrans ), and mean intracellular water-lifetime (τi ) and volume were computed from metastatic lymph nodes and were compared between two groups. Prognostic utility of HPV status, differentiation grading, and nodal staging was also evaluated both in isolation or in combination with MRI parameters in distinguishing patients with and without distant metastases. Additionally, MRI parameters were compared between two groups based on dichotomous HPV status, differentiation grade, and nodal stage. Lower but not significantly different Ktrans (0.51 ± 0.15 minute-1 vs 0.60 ± 0.05 minute-1 ) and not significantly different τi (0.13 ± 0.03 second vs 0.19 ± 0.02 second) were observed in patients who developed distant metastases than those who did not. Additionally, no significant differences in ADC or volume were found. τi, was the best parameter in discriminating two groups with moderate sensitivity (67%) and specificity (61.4%). Multivariate logistic regression analyses did not improve the overall prognostic performance for combination of all variables. A trend toward higher τi was observed in HPV-positive patients than those with HPV-negative patients. Also, a trend toward higher Ktrans was observed in poorly differentiated HNSCCs than those with moderately differentiated HNSCCs. Pretreatment DCE-MRI may be useful in predicting distant metastases in HNSCC.

Histological features and survival in young patients with HPV-negative oral squamous cell carcinoma.

The frequency of oral squamous cell carcinoma in young adults has increased in the last decades, and there are conflicting results in literature about its prognosis in young subjects. The aim of this study was to analyse the clinical and pathological features of oral squamous cell carcinoma in a cohort of young adults in order to investigate the presence of new independent prognostic markers. Only HPV-negative young patients (under 40-year-old) affected by oral squamous cell carcinoma were considered in this study. Clinical and pathological data were collected. Patients were re-staged according to the 8th edition of AJCC. Overall, 66 patients were considered in this study. Perineural invasion significant correlated with both 7th and 8th edition of AJCC, and lymphovascular invasion (p-value<.05). The multivariate survival analysis showed that patients with perineural invasion had a significant worse prognosis (HR=6.384 95% C.I. 1.304-31.252; p-value=.022). Perineural invasion emerged as an independent prognostic factor for disease-specific survival in young patients with oral squamous cell carcinoma. Furthermore, the evaluation of this parameter is simple, inexpensive and can be used to augment the risk stratification of oral cancer based on the 8th edition of AJCC.

The role of concomitant chemoradiotherapy in AJCC 7th edition T1-2N1 oropharyngeal carcinoma in the human papillomavirus era.

Radiotherapy (RT) without chemotherapy is considered a standard of care for the management of American Joint Committee on Cancer (AJCC) 7th edition (7E) T1-2N1 oropharyngeal squamous cell carcinoma (OPSCC). Recent data suggests concurrent chemoradiation (CCRT) may benefit these patients but did not include human papillomavirus (HPV) status. Given the radiosensitivity differences between HPV-positive versus HPV-negative OPSCC, the effect of chemotherapy may differ in these patients. We analyzed patients in the National Cancer Database diagnosed between 2010 and 2015 with AJCC 7E stage cT1-2N1M0 OPSCC and known HPV status undergoing definitive RT or CCRT. Overall, 1964 patients were included, including 1297 (66%) HPV-positive and 667 (34%) HPV-negative patients. 66% received CCRT and 34% received RT alone. In multivariate analysis, CCRT was associated with improved survival compared with RT alone (hazard ratio [HR], 0.70; 95% confidence interval [CI], 0.57-0.87; P=0.001). In propensity score-matched cohorts, 4-year overall survival was 87.4% vs 78.4% in HPV-positive patients receiving CCRT and RT alone, respectively (P=0.002), and 65.5% vs 58.9% in HPV-negative patients, respectively (P=0.2). There was no evidence that HPV-positivity diminished the association between CCRT and longer survival (HR, 0.57; 95% CI, 0.42-0.81) versus what was observed in HPV-negative patients (HR, 0.86; 95% CI, 0.64-1.16) (interaction P=0.06). CCRT is associated with improved survival in AJCC 7E T1-2N1 OPSCC. Despite the radiosensitivity of HPV-positive OPSCC, the association of CCRT with improved survival for T1-2N1 HPV-positive OPSCC was at least as strong, if not stronger, than what was observed in HPV-negative patients.

Droplet digital PCR quantification suggests that higher viral load correlates with improved survival in HPV-positive oropharyngeal tumours.

Although HPV-positive oropharyngeal cancer (OPC) patients have improved prognosis compared to HPV negative patients; there remains an HPV-positive group who have poor outcomes. Biomarkers to stratify discrete patient outcomes are thus desirable. Our objective was to analyse viral load (VL) by droplet digital PCR (ddPCR), in HPV-positive patients with OPC on whom clinical outcome data were available. In a cohort of patients that had previously tested HPV positive via conventional PCR, VL was determined using ddPCR assays for HPV16 L1 and E6 genes. VL was classed as "medium/high" if more than 5.57 copies or 8.68 copies of the HPV 16 L1 or E6 gene were detected respectively. Effect of VL on overall survival and hazard of death & disease progression was performed with adjustments made for sex, age, deprivation, smoking, alcohol consumption and stage. L1 VL ranged from 0.0014-304 gene copies per cell with a mean of 30.9; comparatively E6 VL ranged from 0.0012-356 copies per cell with a mean of 37.9. Univariate analysis showed those with a medium/high VL had a lower hazard of death; this was significant for L1 (p = 0.02) but not for E6 (p = 0.67). The ratio of E6 to L1 deviated from n = 1 in most samples but had no influence on clinical outcomes. HPV viral load may be informative for the further stratification of clinical outcomes in HPV positive OPC patients.

Abstract PR02: Mutational analysis of head and neck squamous cell carcinoma stratified by smoking status

Abstract Smoking has historically been recognized as a negative prognostic factor in head and neck squamous cell carcinoma (HNSCC). This study aimed to assess the mutational differences between heavy smokers (&amp;gt;20 pack-years) and never smokers among the HNSCC patients within The Cancer Genome Atlas (TCGA). Single nucleotide variation (SNV) and copy number aberration (CNA) differences between heavy smokers and never smokers were compared within HPV-positive (n=67) and negative (n=431) TCGA HNSCC cohorts, and the impact of these mutations on survival was assessed. No genes were differentially mutated between smoking and never smoking patients with HPV-positive tumors. By contrast, in HPV-negative tumors, NSD1 and COL1A11 were found to be more frequently mutated in heavy smokers, while CASP8 was more frequently altered in never smokers. HPV-negative patients with NSD1 mutations experienced significantly improved overall survival compared with NSD1 wild-type patients. This improved prognosis was validated in an independent cohort of 77 oral cavity cancers and a meta-analysis that included two additional datasets (688 total patients, hazard ratio for death 0.44, 95% CI 0.30-0.65). NSD1 mutations are more common in HPV-negative heavy smokers and define a cohort with favorable prognosis, and may represent a clinically useful biomarker to guide treatment deintensification for HPV-negative patients. Citation Format: Farhad Ghasemi, Stephenie D. Prokopec, Danielle MacNeil, Neil Mundi, Christopher Howlett, William Stecho, Kevin Fung, John Yoo, Eric Winquist, Steven Gameiro, Axel Sahovaler, Paul Plantinga, Joe S. Mymryk, John W. Barrett, Paul C. Boutros, Anthony C. Nichols. Mutational analysis of head and neck squamous cell carcinoma stratified by smoking status [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr PR02.

Abstract IA02: Utilizing patient-derived xenografts for prognostication and biomarker discovery

Abstract Overall outcomes for human papilloma virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) remain poor with 5-year overall survival rates of 50-60%. Treatment often includes surgery, and clinicopathologic features are used to identify patients in need of adjuvant therapies such as radiation therapy (RT) or radiation plus concurrent chemotherapy (CRT). It is clear from the rate of locoregional or distant failures that more accurate methods of risk stratification would greatly improve outcomes for HPV-negative HNSCC patients. This requires biomarkers to identify patients who will benefit from adjuvant RT or CRT, but currently there are no validated molecular biomarkers that have been clinically implemented for the personalized treatment of HNSCC. In addition to biomarkers for better risk stratification, there is also a need for novel therapeutic strategies leading to improved outcomes. Recently, patient-derived xenografts (PDXs) have been shown to faithfully recapitulate human tumor biology and predict drug responses, supporting their relevance as preclinical models for new drug development. Upon subcutaneous implantation of HPV-negative HNSCC specimens into NOD/SCID/IL2Rγ-/- mice, 161 of 243 samples (66%) successfully formed PDXs. Using univariable and multivariable analyses, the ability to form a PDX correlated significantly with adverse clinical outcomes, and specifically, patients with palpable PDX-formation within 8 weeks experienced particularly poor outcomes (hazard ratio for overall survival = 3.0). A cohort of engrafting and nonengrafting samples were sequenced using a targeted sequencing panel designed for both mutational and copy number alteration detection. The overall frequency of somatic genomic alterations detected was similar to The Cancer Genome Atlas cohort and interestingly, successful engraftment correlated to amplification of the CCND1 gene. Ten HPV-negative PDX models were treated with the CDK4/6 inhibitor, abemaciclib; 5 of 6 models with CCND1 amplifications and/or CDKN2A mutations responded to abemaciclib treatment, whereas only 1 of 4 models lacking these alterations responded. We also carried out a PDX clinical trial on 20 models using the PI3Kα inhibitor, BYL719. Interestingly, while previous studies using in vitro cell line studies and limited numbers of xenograft models derived from various tumor types have suggested PIK3CA hotspot mutations predict for response to PI3K inhibitors, we found that BYL719 was almost globally tumoristatic, regardless of PIK3CA mutational status. Our results demonstrate the potential of using PDX models to individualize treatment for patients at high risk of relapse following definitive treatment, to identify novel therapies and predictive biomarkers, and to interrogate drug resistance mechanisms. Citation Format: Christina Karamboulas, Jeffrey P. Bruce, Kara M. Ruicci, Wei Xu, Anthony C. Nichols, Laurie Ailles. Utilizing patient-derived xenografts for prognostication and biomarker discovery [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr IA02.

Abstract B35: Characterization of exosomes extracted from primary tumor samples of HPV+/- oropharyngeal cancer

Abstract Objective: To determine if distinctions in exosomal cargo contribute to differences in EMT between HPV-related and HPV-unrelated oropharyngeal disease and can provide a signature for prediction of tumor aggressiveness and metastatic potential. Subjects and Methods: Specimen from patients with HPV+/- OPSCC were collected according to an IRB-approved protocol including primary tumor, lymph node metastasis, distant metastasis, and serum when available. Exosomes isolated from HPV+ and HPV- tumors were analyzed with miARma-seq and proteomics. These same exosomes were cocultured with well-characterized HPV+ and HPV- cell lines as well as fibroblasts and normal keratinocytes. Tumor aggressiveness, EMT, and potential for metastasis were measured utilizing wound healing scratch assays and transwell invasion assay techniques based on the Boyden Chamber migration assay. Confirmation of epithelial to mesenchymal transition was evaluated by Western blot and qPCR using a panel of well-established EMT markers (e-cadherin, n-cadherin, vimentin, snail, slug, twist, ZEB1/2). Results: Tumors from HPV+ patients demonstrate exosomal content enriched in EMT markers compared to HPV-negative patient cultures. Exosomes extracted from primary tumor and lymph node metastasis of the same patient demonstrate differential expression of markers of EMT. Exosomes extracted from lymph node and cocultured with fibroblasts demonstrate increased invasion and migration. Conclusions: Exosomes can be extracted from primary patient tumor cultures of both primary tumor and lymph node metastasis. These exosomes demonstrate differential expression of markers of EMT. Exosomal contents can be exploited for biomarker discovery to aide in prognostication. Citation Format: Vivian F. Wu, Xiang Guo Dai, Michael Chopp. Characterization of exosomes extracted from primary tumor samples of HPV+/- oropharyngeal cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B35.

PD-L1 expression correlates with tumor-infiltrating lymphocytes and better prognosis in patients with HPV-negative head and neck squamous cell carcinomas.

The importance of immune tumor microenvironment in the prognosis of patients with head and neck squamous carcinomas (HNSCC) is increasingly recognized. We analyzed the prognostic relevance of PD-L1 and PD-1 expressions in relation to the infiltration by CD8+ and FOXP3+ tumor-infiltrating lymphocytes (TILs). Samples from 372 surgically treated HPV-negative HNSCC patients were evaluated by immunohistochemistry for PD-L1 expression [both tumor proportion score (TPS) and combined proportion score (CPS)], PD-1 expression in immune cells, and density of infiltrating CD8+ and FOXP3+ TILs. PD-L1 expression and CD8+ TIL density were combined to establish the type of tumor microenvironment. 29.5% cases exhibited PD-L1 TPS positivity (≥ 1%), whereas PD-L1 CPS positivity (≥ 1%) was observed in 40% cases. 47.5% cases showed positive PD-1 expression (≥ 1%). PD-L1 and PD-1 positivity correlated with a high density of both CD8+ and FOXP3+ TILs. In univariate analysis, PD-L1 TPS positivity (P = 0.026), PD-L1 CPS positivity (P = 0.004), high density of CD8+ TIL (P = 0.001), and high density of FOXP3+ TIL (P = 0.004) were associated with a better disease-specific survival (DSS). However, in multivariate analysis, only high density of CD8+ TIL was associated with a better DSS (P = 0.002). The type of tumor microenvironment correlated with DSS (P = .008), with the better DSS observed in cases with type I (PD-L1 CPS positivity and high density of CD8+ TIL). High infiltration by CD8+ TIL is associated with better survival outcomes. Positive PD-L1 expression correlates with a high infiltration by TILs, explaining its association with better prognosis.

Does sinonasal cancer survival differ based on human papillomavirus status?

6565 Background: The sinonasal tract is a lesser known “hot spot” for the human papillomavirus (HPV), compared with the oropharynx. Additionally, unlike the oropharynx, the role of HPV tumor status in the survival and overall prognosis of the sinonasal tract and other non-oropharyngeal head and neck cancer sites remains inconclusive. Understanding differences in survival based on HPV status could be useful clinically, as it has been for HPV-positive oropharyngeal disease. This study examined whether there are survival differences in sinonasal cancer based on HPV status. Methods: This study included adult sinonasal cancer cases diagnosed between 2010 and 2015 in the National Cancer Database. A multivariable Cox proportional hazards model estimated the association between sinonasal cancer HPV status (HPV-positive, HPV-negative) and all-cause mortality while controlling for covariates (sex, age, race/ethnicity, insurance status, urban/rural, county-level household income, county-level percentage without high school diploma, comorbidity score, stage, histology, facility type, and treatment). A second multivariable proportional hazards model stratified HPV-positive tumor status by high-risk HPV (16, 18, 26, 31, 33, 35, 36, 45, 51, 52, 53, 56, 58, 59, 66, 67, 68, 69, 70, 73, 82, and 85) vs. low-risk HPV (6, 11, 32, 34, 40, 42, 44, 54, 61, 62, 64, 71, 72, 74, 81, 83, 84, 87, and 89) and compared their all-cause mortality to HPV-negative patients. Results: There were 1,750 sinonasal cancer patients included in this study, and 484 (27.7%) had HPV-positive disease. Among patients with HPV-positive disease, 75.6% had high-risk types. Mortality risk among all HPV-positive patients combined was 23% lower than HPV-negative patients (aHR = 0.77; 95% CI 0.64, 0.93). After stratifying by high-risk vs. low-risk HPV, high-risk HPV positive patients had 30% lower mortality risk than HPV-negative patients (aHR = 0.70; 95% CI 0.57, 0.88) while risk of mortality did not significantly differ between low-risk HPV-positive patients and HPV-negative sinonasal cancer patients. Conclusions: Sinonasal cancer shows differential survival based on HPV status, and sinonasal cancer patients positive for high-risk HPV had a significantly greater survival advantage than low-risk strains and those with HPV negative disease. HPV status might yet play a role in prognostication of sinonasal cancer, if future studies confirm these findings.

Studying the interrelation between the HPV infection status and the morphological features of bladder cancer.

e17003 Background: The etiological role of human papillomavirus (HPV) in the development of squamous cell carcinoma of the cervix and larynx is considered proven. At the same time, there are conflicting data in the literature on the role of HPV in the development of bladder cancer (BC). The aim of the present study was to evaluate the prevalence of HPV infection in patients with BC, identifying the relationship between the status of infection and the morphological characteristics of the tumor. Methods: The study included 46 patients (10 women and 36 men) with morphologically confirmed urothelial cancer. Samples of urine, tumor tissue and a healthy bladder mucosa were examined. HPV DNA was determined by real-time PCR. Results: 28 (60.9%) patients were infected with high-risk HPV, including 6 (60.0%) women and 22 (61.1%) men. In 14 (30.4%) patients, HPV DNA was found in the tissue of the bladder mucosa: in 6 (13.0%) cases it was only in tumor tissue, in 4 (8.7%) only in healthy mucosa, in 4 (8.7%) simultaneously in healthy and tumor tissue. HPV DNA was detected in urine in 22 (47.8%) patients. The simultaneous presence of HPV DNA in the tissue of the bladder mucosa and urine was found in 8 (28.6%) HPV-positive patients. Single HPV infection was found in 20 (71.4%) patients, two types were found in 8 (28.6%). When analyzing type diversity, it was found that HPV16 infected 14 (38.9%) patients; HPV18, 31, 39 and 56 were determined in 4 (11.1%) cases each; 33, 52 and 59 types in 2 (5.6%) cases. Chronic inflammation was noted in 12 (26.1%) patients, 6 (50.0%) of them were HPV-positive. Among HPV-positive patients, invasive cancer was registered in 18 (64.3%), non-invasive cancer in 10 (35.7%) patients. Among HPV-negative patients, these rates were 14 (77.8%) and 4 (22.2%), respectively (p &gt; 0.05). In the group of HPV-positive patients, tumors with a low degree of differentiation were found in 16 (57.1%), moderate in 6 (21.4%), high in 6 (21.4%). Among HPV-negative patients, these indicators were 4 (22.2%), 12 (66.7%) and 2 (11.1%), respectively (p &gt; 0.05). Conclusions: HPV infection was detected in 60.9% of patients, including tumor tissue infection in 21.7% of patients. The most common HPV type was HPV16. There was no statistically significant difference between HPV-positive and negative tumors.

PDL1 expression predicts therapeutic outcome in non metastatic anal squamous cell carcinoma (NMASCC).

4054 Background: NMASCC is a rising incidence disease with up to 30% of treatment failure to achieve complete response (CR) after standard chemoradiotherapy (CRT) leading to severe morbidity and death. Stage III-TNM, p53 mutations, HPV negativity, HIV infection are linked to treatment failure. We investigated the predictive/prognostic role of TNM, CR, HPV, PDL1 positivity and CD3/CD8 densities in NM-ASCC from a single institution. Methods: All 79 eligible consecutive NMASCC pts (available FFPE pre-treatment samples) seen from October-2009 to April-2019 having completed definitive CRT (50.4 Gy Pelvic Radiotherapy with Mitomycin-C 12mg/m2/IV/d1-5 / FU 1000mg/m2/d1-4 d29-32 (28%), Mitomycin-C/Capecitabine 825 mg/m2/bid (38%), Cisplatin 60 mg/m2/IV d1-29 and 5FU (34%) were analyzed. Mean age: 59 (range 26-87), 72% female, Stage III: 59%, HPV positive: 86% (HPV-16: 80%);14% HIV positive. IHC assessed by two pathologist for PD-L1 expression (ClonSP263) and CD3-CD8+ TILS densities (Clone 2GV6, Clone SP57). HPV-DNA assessed by PCR (BSGP5+/6+ multiplexed with beta-globin). Kaplan-Meier survival, CR, DFS, OS and Univariate analyses were performed using Cox proportional hazard model. Results: CR achieved within 6 months of treatment completion was 68%( 53pts). Median follow-up after treatment completion: 35 months (range 6 –149). As of February 2020, 82% (65 pts) are alive, no evidence of disease:(57%) 46 pts, recurrence rate: 26%(22 pts), cancer death: 18% (14 pts). PDL1+ tumors ( &gt; 1% positivity-CPS score): 56%, expression levels: 1-5% (57%,26p), &gt; 10%-100% (43%,19p). PDL1+ had a strong association with CR (p = 0.021); higher PDL1+ levels had 8-fold of CR-likelihood than PDL1 negative.(OR 8.50 vs. 1.12). Significative Spearman correlation between PDL1 tumors with CR and CD3-CD8 TILS density was observed (R = 0.43,p = 0.0017 and R = 0.36,p = 0.00094 respectively), albeit CD3-CD8 failed to reach significance as prognostic factors for either CR, DFS or OS. Only CR and PDL1 positive were strongly significantly associated to DFS (HR 0.10 [IC 95% 0.04-0.28] p &lt; 0.001 and HR 0.28 [IC 95% 0.11-0.73] p = 0.006) and OS (HR 0.12 [IC 95% 0.03-0.45] p &lt; 0.001 and HR 0.15 [IC 95% 0.03-0.68] p &lt; 0.004). Low prevalence of HPV negative, early tumors, HIV positive cases in our series probably impacted in statistical power for prognosis correlation. Conclusions: PDL1 positivity was the strongest predictive/prognostic factor in NM-ASCC. Alternative therapeutics options to standard CRT should be explored on poor-risk patients as HPV-negative, P53-mutated and PDL1 negative patients.

Epithelial-to-mesenchymal transition is a prognostic marker for patient outcome in advanced stage HNSCC patients treated with chemoradiotherapy

BackgroundThe prognosis of patients with HPV-negative advanced stage head and neck squamous cell carcinoma (HNSCC) remains poor. No prognostic markers other than TNM staging are routinely used in clinic. Epithelial-to-mesenchymal transition (EMT) has been shown to be a strong prognostic factor in other cancer types. The purpose of this study was to determine the role of EMT in HPV-negative HNSCC outcomes. MethodsPretreatment tumor material from patients of two cohorts, totalling 174 cisplatin-based chemoradiotherapy treated HPV-negative HNSCC patients, was RNA-sequenced. Seven different EMT gene expression signatures were used for EMT status classification and generation of HNSCC-specific EMT models using Random Forest machine learning. ResultsMesenchymal classification by all EMT signatures consistently enriched for poor prognosis patients in both cohorts of 98 and 76 patients. Uni- and multivariate analyses show important HR of 1.6–5.8, thereby revealing EMT’s role in HNSCC outcome. Discordant classification by these signatures prompted the generation of an HNSCC-specific EMT profile based on the concordantly classified samples in the first cohort (cross-validation AUC > 0.98). The independent validation cohort confirmed the association of mesenchymal classification by the HNSCC-EMT model with poor overall survival (HR = 3.39, p < 0.005) and progression free survival (HR = 3.01, p < 0.005) in multivariate analysis with TNM. Analysis of an additional HNSCC cohort from PET-positive patients with metastatic disease prior to treatment further supports this relationship and reveals a strong link of EMT to the propensity to metastasize. ConclusionsEMT in HPV-negative HNSCC co-defines patient outcome after chemoradiotherapy. The generated HNSCC-EMT prediction models can function as strong prognostic biomarkers.

Post-Treatment HPV Surface Brushings and Risk of Relapse in Oropharyngeal Carcinoma.

Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is a distinct subtype of head and neck cancer. Here, we investigated how frequently brushing remained high-risk (hr)-HPV positive after treatment and whether patients with positive post-treatment brushings have a higher recurrence rate. Following the end of treatment of patients with initially hr-HPV positive OPSCC, surface brushings from the previous tumor site were performed and tested for hr-HPV DNA. Of 62 patients with initially hr-HPV DNA-positive OPSCC, seven patients remained hr-HPV-DNA positive at post-treatment follow-up. Of the seven hr-HPV-positive patients at follow-up, five had a tumor relapse or tumor progression, of whom three died. The majority of patients (55/62) was HPV-negative following treatment. All HPV-negative patients remained free of disease (p = 0.0007). In this study, all patients with recurrence were hr-HPV-positive with the same genotype as that before treatment. In patients who were hr-HPV negative after treatment, no recurrence was observed.

Is there any correlation between HPV and early radioresponse before brachytherapy in cervix uteri carcinoma?

The prognostic effect of human papilloma virus (HPV) on early radioresponse before brachytherapy was evaluated in locally advanced cervix uteri carcinoma patients. Between 2015 and 2018, 150 patients with locally advanced carcinoma of the cervix uteri from University of Health Sciences Okmeydani Training and Research Hospital and Istanbul Oncology Hospital underwent chemotherapy concomitant with external radiotherapy and brachytherapy after performing fluorodeoxyglucose positron emission tomography (FDG PET) and magnetic resonance imaging (MRI) for purposes of staging and treatment planning. The treatment results of patients analyzed retrospectively. The median age of 57 patients was 53years, and the initial tumor volume was 27cm3. The median follow-up period was 22months. The median tumor volume after external radiotherapy was significantly higher in patients without local control than those who responded to external radiotherapy (p = 0.031). The mean tumor diameter and volume in all HPV-negative patients after external radiotherapy were significantly higher than those of HPV-positive patients (p = 0.011 and p = 0.046, respectively). In a group of patients with poor early responses, local disease control could not be achieved despite the use of recommended higher doses of brachytherapy. We could not determine the correlation between HPV contamination and patients who had early response intervention. But residual tumor of more than 2cm in diameter after external radiotherapy may be a predictor of failed local control and development of metastasis within a short time.

The role of Glial cell derived neurotrophic factor in head and neck cancer.

Glial cell-derived neurotrophic factor (GDNF) is reported to promote the survival of neurons and salivary gland regeneration after radiation damage. This study investigated the effect of GDNF on cell migration, growth, and response to radiation in preclinical models of head and neck squamous cell carcinoma (HNSCC) and correlated GDNF expression to treatment outcomes in HNSCC patients. Our ultimate goal is to determine whether systemic administration of GDNF at high dose is safe for the management of hyposalivation or xerostomia in HNSCC patients. Three HPV-positive and three HPV-negative cell lines were examined for cell migration, growth, and clonogenic survival in vitro and tumor growth assay in vivo. Immunohistochemical staining of GDNF, its receptors GFRα1 and its co-receptor RET was performed on two independent HNSCC tissue microarrays (TMA) and correlated to treatment outcomes. Results showed that GDNF only enhanced cell migration in two HPV-positive cells at supra-physiologic doses, but not in HPV-negative cells. GDNF did not increase cell survival in the tested cell lines post-irradiation. Likewise, GDNF treatment affected neither tumor growth in vitro nor response to radiation in xenografts in two HPV-positive and two HPV-negative HNSCC models. High stromal expression of GDNF protein was associated with worse overall survival in HPV-negative HNSCC on multivariate analysis in a combined cohort of patients from Stanford University (n = 82) and Washington University (n = 189); however, the association between GDNF gene expression and worse survival was not confirmed in a separate group of HPV-negative HNSCC patients identified from the Cancer Genome Atlas (TCGA) database. Based on these data, we do not believe that GNDF is a safe systemic treatment to prevent or treat xerostomia in HNSCC and a local delivery approach such as intraglandular injection needs to be explored.

Combined Effect of HPV and Several Gene SNPs in Laryngeal Cancer.

Background and objectives: Laryngeal squamous cell carcinoma (LSCC) is one of the most common head and neck tumors. The molecular mechanism of LSCC remains unclear. The aim of this study was to evaluate the prevalence of Human papillomavirus (HPV) and single nucleotide polymorphisms (SNPs) of TP53, MDM2, MDM4, MTHFR, CASP8, and CCR5 genes in LSCC, and to assess their correlations with patient survival. Materials and Methods: 49 LSCC patients were enrolled in this study. PCR and qRT-PCR were used to detect, identify, and quantify HPV. SNPs were genotyped using PCR and PCR-RFLP. Results: By analyzing the interactions of the SNPs of the genes with clinical parameters, the majority of patients with lymph node status (N1,2) were identified as carriers of MDM2 T/G, CASP8 ins/del, CCR5 wt/wt SNP. Cluster analysis showed that patients with MDM2 T/T SNP survive longer than patients identified as CASP8 ins/ins, MTHFR C/C, and MDM4 A/A variant carriers; meanwhile, LSCC patients with MDM2 T/T polymorphic variant had the best survival. Multivariate analysis showed that HPV-positive patients without metastasis in regional lymph nodes (N0) and harboring CASP8 ins/del variant had the best survival. Meanwhile, HPV-negative patients with identified metastasis in lymph nodes (N1 and N2) and CASP8 ins/del variant had poor survival. Conclusions: This finding suggests patients survival prognosis and tumor behavior are different according HPV status, SNP variants, and clinical characteristics of the LSCC.

Infiltrates of M2-Like Tumour-Associated Macrophages Are Adverse Prognostic Factor in Patients with Human Papillomavirus-Negative but Not in Human Papillomavirus-Positive Oropharyngeal Squamous Cell Carcinoma

Introduction: Human papillomavirus with a high oncogenic potential (HR-HPV) is responsible for more than a half of squamous cell carcinomas of the oropharynx. The HR-HPV-dependent cases of this tumour have a better prognosis compared to the HR-HPV-negative cases, despite the usually more advanced disease at the time of diagnosis. In addition to genetic and epigenetic factors, the causes of this more favourable course of the disease are also seen in the participation of the tumour microenvironment, including the patient’s immune system. Macrophages are one of the most important elements of the immunocompetent cells landscape that make up the tumour microenvironment. Traditionally, they are divided into 2 groups: inflammatory macrophages with the M1 phenotype and tumour-associated macrophages known as M2 phenotype macrophages. Objective: The aim of this study was to investigate the impact of the macrophage infiltrates intensity of the M1/M2 and M2 phenotype separately on the clinical outcome of patients with squamous cell carcinoma of the oropharynx (OPSCC), taking into account the HR-HPV status of tumours. Methods: The study involved 85 patients with OPSCC in which HR-HPV status in tumour tissue was determined using a double-check algorithm including the detection of viral DNA by RT-PCR method with subsequent confirmation of its biological activity by immunohistochemical demonstrating the P16^INK4A protein overexpression. In each of the groups formed on the basis of HR-HPV status, macrophages were discriminated using CD68 and CD163 proteins as markers of pan-macrophage and M2 phenotype. The intensity of infiltrates was quantified by means of computer-assisted analysis in digital images of whole slides (virtual slides) separately in tumour tissue and stroma. Results: In HPV-positive patients, significantly more intense infiltration of both M1/M2 and M2 macrophages was found in the tumour stroma compared to HPV-negative patients. The infiltrates from both types of macrophages in the tumour tissue were less intense and did not differ between these groups. Intensive infiltration of CD68⁺ macrophages in the tumour front was associated with higher rate of nodal failures and a shorter nodal control in both HR-HPV groups. In the group of HR-HPV-negative patients, heavy infiltration of CD163⁺ macrophages was associated with significantly shorter: loco-regional control (LRC), metastasis-free survival and overall survival (OS). These parameters and prognosis in patients with scanty CD163⁺ infiltration were similar to favourable outcomes in HR-HPV-positive patients. The relative risk of local-regional recurrence, distant metastases and disease-related death in HR-HPV-negative patients with intense CD163⁺ infiltrates was, respectively, 4.7, 5.4 and 5.7 compared to patients with scanty infiltrates. Conclusions: Tumours with a positive HR-HPV status demonstrate intense infiltrations of total pool M1/M2 and M2 macrophages. In the group of HPV-negative patients, intensive M1/M2 macrophage infiltrates correlate with higher risk of nodal failures, and intensive M2 infiltrates are an adverse prognostic factor for LRC, metastasis-free survival and OS.

Digital pathology-aided assessment of tumor-infiltrating T lymphocytes in advanced stage, HPV-negative head and neck tumors

AimThis study aimed to evaluate the presence and prognostic value of tumor-infiltrating T cells in the tumor epithelium in advanced stage, HPV-negative head and neck squamous cell carcinoma (HNSCC) patients treated with primary chemoradiotherapy using digital pathology.MethodsPre-treatment biopsies from 80 oropharyngeal, 52 hypopharyngeal, and 29 laryngeal cancer patients were collected in a tissue microarray (TMA) and immunohistochemically stained for T-cell markers CD3, CD4, CD8, FoxP3, and PD1, and for immune checkpoint PD-L1. For each marker, the number of positive tumor-infiltrating lymphocytes (TILs) per mm2 tumor epithelium was digitally quantified and correlated to overall survival (OS), disease-free survival (DFS), and locoregional control (LRC), as well as to clinicopathological characteristics. Differences in clinical outcome were estimated using Cox proportional hazard analysis and visualized using Kaplan–Meier curves.ResultsThe patient cohort had a 3-year OS of 58%, with a median follow-up of 53 months. None of the T-cell markers showed a correlation with OS, DFS or LRC. A low N stage was correlated to a better prognosis (OS: HR 0.39, p = 0.0028, DFS: HR 0.34, p = < 0.001, LRC: HR 0.24, p = 0.008). High TIL counts were more often observed in PD-L1-positive tumors (p < 0.05).ConclusionThis study showed an objective, digital pathology-aided method to assess TILs in the tumor epithelium. However, it did not provide evidence for a prognostic role of the presence of CD3 + , CD4 + , CD8 + , FoxP3 + , and PD1 + TILs in the tumor epithelium of advanced stage, HPV-negative HNSCC patients treated with primary chemoradiotherapy.

Short-term mortality risks among patients with oropharynx cancer by human papillomavirus status.

There is substantial variation in head and neck cancer (HNC) mortality and competing mortality among patients with HNC. In this study, the authors characterize the causes and risks of short-term mortality among patients with oropharynx cancer (OPC) and how these risks differ by human papillomavirus (HPV) status. A custom Surveillance, Epidemiology, and End Results (SEER) data set with HPV status was used to identify 4930 patients with OPC who were diagnosed with nonmetastatic (M0) disease from 2013 to 2014, including 3560 (72.2%) HPV-positive patients and 1370 HPV-negative patients. Causes of death and cumulative incidence estimates for HNC-specific mortality, competing mortality, second-cancer mortality, and noncancer mortality were analyzed by HPV status. Risk factors for mortality events were determined using multivariable competing risk regression models. Compared with HPV-negative patients, HPV-positive patients had a lower risk of 2-year cumulative incidence of all-cause mortality (10.4% vs 33.3%; P<.0001) and a lower risk of both HNC-specific mortality (4.8% vs 16.2%; P<.0001) and competing-cause mortality (5.6% vs 16.8%; P<.0001). Second-cancer mortality was the most common cause of non-HNC mortality among HPV-negative patients. Both second-cancer mortality and noncancer mortality were significantly higher among patients who had HPV-negative OPC (10.8% and 6.1%, respectively) compared with those who had HPV-positive OPC (2.4% and 3.2%, respectively; both P<.0001). The median follow-up was 11months (range 1-23months) in this cohort with known HPV-status. Patients with HPV-positive and HPV-negative OPC have significantly different rates of both HNC mortality and competing mortality. HPV-negative patients are at substantial risk of competing mortality, even within 2years of cancer diagnosis. These differences can inform power calculations for clinical trials and patient management in the acute and survivorship settings.

Biological Determinants of Chemo-Radiotherapy Response in HPV-Negative Head and Neck Cancer: A Multicentric External Validation.

Purpose: Tumor markers that are related to hypoxia, proliferation, DNA damage repair and stem cell-ness, have a prognostic value in advanced stage HNSCC patients when assessed individually. Here we aimed to evaluate and validate this in a multifactorial context and assess interrelation and the combined role of these biological factors in determining chemo-radiotherapy response in HPV-negative advanced HNSCC.Methods: RNA sequencing data of pre-treatment biopsy material from 197 HPV-negative advanced stage HNSCC patients treated with definitive chemoradiotherapy was analyzed. Biological parameter scores were assigned to patient samples using previously generated and described gene expression signatures. Locoregional control rates were used to assess the role of these biological parameters in radiation response and compared to distant metastasis data. Biological factors were ranked according to their clinical impact using bootstrapping methods and multivariate Cox regression analyses that included clinical variables. Multivariate Cox regression analyses comprising all biological variables were used to define their relative role among all factors when combined.Results: Only few biomarker scores correlate with each other, underscoring their independence. The different biological factors do not correlate or cluster, except for the two stem cell markers CD44 and SLC3A2 (r = 0.4, p < 0.001) and acute hypoxia prediction scores which correlated with T-cell infiltration score, CD8+ T cell abundance and proliferation scores (r = 0.52, 0.56, and 0.6, respectively with p < 0.001). Locoregional control association analyses revealed that chronic (Hazard Ratio (HR) = 3.9) and acute hypoxia (HR = 1.9), followed by stem cell-ness (CD44/SLC3A2; HR = 2.2/2.3), were the strongest and most robust determinants of radiation response. Furthermore, multivariable analysis, considering other biological and clinical factors, reveal a significant role for EGFR expression (HR = 2.9, p < 0.05) and T-cell infiltration (CD8+T-cells: HR = 2.2, p < 0.05; CD8+T-cells/Treg: HR = 2.6, p < 0.01) signatures in locoregional control of chemoradiotherapy-treated HNSCC.Conclusion: Tumor acute and chronic hypoxia, stem cell-ness, and CD8+ T-cell parameters are relevant and largely independent biological factors that together contribute to locoregional control. The combined analyses illustrate the additive value of multifactorial analyses and support a role for EGFR expression analysis and immune cell markers in addition to previously validated biomarkers. This external validation underscores the relevance of biological factors in determining chemoradiotherapy outcome in HNSCC.