Determinants of the level of circulating-tumor HPV16 DNA in patients with HPV-associated oropharyngeal cancer at the time of diagnosis.

Circulating tumor HPV DNA (ctHPV16) assessed in liquid biopsy may be used as a marker of cancer in patients with HPV-associated oropharyngeal cancer (HPV + OPC). Factors influencing the initial ctHPV16 quantity are not well recognized. In this study we aimed to establish what factors are related to the level of ctHPV16 at the time of diagnosis. 51 patients (37 men and 14 women, median age of 57 years old) with HPV + OPC prior to definitive treatment were included. ctHPV16 was measured by qPCR. Tumor and nodal staging were assessed according to AJCC8. Blood derived factors included squamous cell carcinoma antigen (SCC-Ag), serum soluble fragment of cytokeratin 19 (CYFRA 21-1), C-reactive protein (CRP), albumin level (Alb), neutrophils (Neut), thrombocytes (Plt) and lymphocyte (Lym) count, Neut/Lym ratio were assessed. The volumes of the primary tumor (TV) and involved lymph nodes (NV) were calculated using MRI, CT or PET-CT scans. Data were analysed using parametric and nonparametric methods. Variables for multivariable linear regression analysis were chosen based on the results from univariable analysis (correlation, univariable regression and difference). There were 9 (18%), 10 (19%) and 32 (63%) patients who had TV and NV assessed in MRI, CT or PET respectively. Primary tumor neither as T-stage nor TV was related to ctHPV16 level. Significant differences in the ctHPV16 between patients with high vs low pain (P = 0.038), NV (P = 0.023), TV + NV (P = 0.018), CYFRA 21-1 (P = 0.002), CRP (P = 0.019), and N1 vs N3 (P = 0.044) were observed. ctHPV16 was significantly associated with CYFRA 21-1 (P = 0.017), N stage (P = 0.005), NV (P = 0.009), TV + NV (P = 0.002), CRP (P = 0.019), and pain (P = 0.038). In univariable linear regression analysis the same variables predicted ctHPV16 level. In multivariable analyses, CYFRA 21-1 and CRP (both as categorical variables) were predictors of ctHPV16 level even above NV. ctHPV16 at presentation is driven by tumor volume measured mostly by N. CYFRA 21-1 and CRP are additional factors related to ctHPV16 prior to the treatment.

Practical Application of Circulating Tumor-Related DNA of Human Papillomavirus in Liquid Biopsy to Evaluate the Molecular Response in Patients with Oropharyngeal Cancer.

Recent findings have shown that human papillomavirus (HPV) DNA is present in the blood as a tumor-specific biomarker (circulating tumor-related HPV; ctHPV) in patients with HPV-related oropharyngeal cancer (HPV-related OPC). The molecular response (MR) in patients with HPV-related OPC can be defined as the change in the number of ctHPV copies in relation to its initial quantity. The optimal model for assessing the MR using a liquid biopsy (LB) should be based on the E6/E7 sequences of the viral genome. MR assessment can help to evaluate the intensity of ongoing treatments in relation to the tumor response. The evaluation of the residual disease at the end of therapy may also be performed by MR assessment. If a partial MR (pMR) is found, caution is indicated and a subsequent LB should be considered, due to the likelihood of disease progression. Complete radiological and clinical responses together with a complete MR (cMR) convincingly indicate a low risk of treatment failure. Moreover, molecular recurrence (Mrec) during a follow-up, confirmed in two consecutive assays, even despite the lack of any other clinical or radiological symptoms of progression, indicates patients at high risk of disease recurrence. In conclusion, MR by ctHPV assessment may hasten the early detection of disease progression, at any stage of the management of the patient with HPV-related OPC.

Circulating HPV16 DNA in Blood Plasma as Prognosticator and Early Indicator of Cancer Recurrence in Radio-Chemotherapy for Anal Cancer.

BACKGROUND: Implementation of anal squamous cell carcinoma (ASCC) treatment modifications requires reliable patient risk stratification. The circulating tumor-related human papillomavirus type 16 (ctHPV16) may play a role in predicting survival or assessing treatment response. METHODS: The study included 62 ASCC patients treated with chemoradiotherapy. A threshold of 2.5 was used to determine the maximum standardized uptake value (SUVmax). The ctHPV16 viral load (VL) was quantified by qPCR. RESULTS: In the multivariate Cox analysis, lower SUVmax (p = 0.047) and ctHPV16-positive (p = 0.054) proved to be independent prognostic factors for favorable overall survival (OS). In the subgroup with the higher SUVmax, ctHPV16 and nodal (N) status were independent prognostic factors with p = 0.022 for ctHPV16 and p = 0.053 for N. The best survival rate (95%) presented ctHPV16-positive/N-negative patients. High ctHPV16 VL tended to be slightly specific for patients younger than 63 years (p = 0.152). The decrease in ctHPV16 VL to undetectable level after the end of treatment correlated with the overall clinical response. CONCLUSIONS: A prognostic stratification by SUVmax, ctHPV16 and N-positive status allows consideration of more aggressive treatment in high-risk patients (those with high SUVmax, ctHPV16-negative, and N-positive) or de-intensification of therapy in low-risk patients (those with low SUVmax, ctHPV16-positive and N-negative). However, prospective clinical trials on a large group are needed.

Circulating HPV16 DNA may complement imaging assessment of early treatment efficacy in patients with HPV-positive oropharyngeal cancer.

BACKGROUND: Early detection of treatment failure may improve clinical outcome and overall survival in patients with head and neck cancer after first-line treatment. Circulating cell-free HPV16 DNA (cfHPV16 DNA) was evaluated as a possible complementary marker to radiological assessment of early response in patients with HPV-related oropharyngeal cancer (OPC) after radiotherapy alone or combined with chemotherapy. METHODS: The study included 66 patients with HPV-related OPC receiving radical radiotherapy alone or in combination with chemotherapy. cfHPV16 DNA was assessed in the blood of all patients before treatment using TaqMan-based qPCR. Subsequent analysis of cfHPV16 DNA was performed 12 weeks after treatment completion, along with radiological assessment of early treatment results. RESULTS: Complete (CRR) and incomplete radiological response (IRR) was found in 43 (65%) and 23 (35%) patients respectively. cfHPV16 DNA was present in 5 (28%) patients with IRR, while only in 1 (4%) with CRR. Three of five patients with IRR that were positive for cfHPV16 DNA exhibited histopathologically confirmed local or regional treatment failure, and other two developed distant metastases. None of the patients with negative cfHPV16 DNA presented disease failure. CONCLUSION: The post-treatment assessment of cfHPV16 DNA in patients with HPV-related OPC may be used as a complementary biomarker to conventional imaging-based examinations for early identification of treatment failure.

Significance of HPV16 Viral Load Testing in Anal Cancer.

Human papilloma virus (HPV) is highly frequent among patients with anal squamous cell carcinoma, but the viral load (VL) differs between patients. This study aimed to compare the rate of HPV positivity, HPV16VL, p16INK4A and p53 expression between treatment naive and recurrent anal cancer patients. HPV was genotyped via AmpliSens® HPV HCR-genotype-titre-FRT kit. HPV16 VL was determined via quantitative polymerase chain reaction-based in-house test. p16INK4A and p53 expression was tested via immunohistochemistry. The cohort comprised 13 treatment-naive and 17 recurrent anal SCC patients. High-risk HPV was detected in 87% of cases, and HPV16 (73%) was the predominant genotype. The rate of HPV positivity was higher among women and nonsmokers, and majority of HPV-positive cases were also p16INK4A-positive. All p53-negative tumors were HPV16-positive. The most predominant p53 staining pattern in the HPV-positive group was scattered type, whereas it was diffuse type in the HPV-negative group. The HPV16 VL was higher in the treatment-naive group. Further, in the treatment-naive group, cases with scattered staining pattern of p53 had higher HPV16 VL than cases with diffuse staining pattern. The opposite result was noted in the recurrent cancer group. Moreover, p16-positive cases with scattered p53 staining pattern in the treatment naive group had higher HPV16 VL than their counterparts in the recurrent cancer group. In conclusion, the HPV VL, as is the association between VL and p16INK4A /p53, is in an inversed trend in treatment naive and recurrent cancer patients, highlighting the importance of HPV VL measurement in anal SCC.

Prognostic significance of Epstein-Barr virus viral load in patients with T1-T2 nasopharyngeal cancer.

Nasopharyngeal cancer (NPC) is highly prevalent in southern Chinese populations but it is rare in most parts of the world. A few studies were performed in nonendemic regions of the world, and suggested the prognostic value of Epstein-Barr virus (EBV) DNA load in blood. In this study, EBV DNA presence and viral load (VL) level in the blood of patients with NPC in Polish population were presented. In addition, its prognostic value for locoregional control among other clinicopathological features was evaluated. Patients with carcinoma of the nasopharynx treated definitively with radiotherapy or radiochemotherapy were included in the study. Real-time polymerase chain reaction was performed for quantitating of EBV DNA in plasma. Among patients with NPC, 51% (22 of 43) were classified as EBV-positive with the mean of the VL of 4934 ± 8693 copies/mL. Multiple regression analysis between log EBV DNA VL and clinical parameters revealed that the most important factors increasing the VLs were advanced N disease together with no-smoking status and advanced T tumors. Multivariate Cox regression analysis revealed that T3-T4 tumors were an independent prognostic factor for poor locoregional control. Analysis for the subgroup of patients with T1-T2 tumors showed that T1-T2 EBV-negative patients had better locoregional control compared with T1-T2 EBV-positive, though without statistical significance. In conclusion, it seems that EBV DNA determination may have an important role in diagnostics of patients with NPC with T1-T2 tumors indicating a subgroup with poorer prognosis, though it needs to be proven on a larger cohort.

Detection of circulating HPV16 DNA as a biomarker in the blood of patients with human papillomavirus-positive oropharyngeal squamous cell carcinoma.

BACKGROUND: Development of biomarker analysis using the circulating cell-free DNA (cfDNA) methodology is a challenge for noninvasive cancer diagnosis. In this study, a comparison between the plasma and tumor tissue HPV16 DNA viral loads (VLs) has been presented. METHODS: Real-time polymerase chain reaction was performed for quantitating of HPV16 DNA in the plasma and tumor samples of patients with oropharyngeal cancer. RESULTS: Among the tissues, HPV16-positive patients with oropharyngeal squamous cell carcinoma, nonsmoking patients, displayed significantly higher HPV16 DNA VLs in their tissue. No smoking and advanced N disease were the most important predictors for cHPV16 DNA (circulating HPV16 DNA) detection. The cHPV16-positive women displayed significantly higher VLs in their tumor tissues compared to the men, although without notable impact on the blood detection. CONCLUSIONS: Many factors were responsible for human papillomavirus DNA circulation in blood. As a result of the small size of the analyzed group, some observed discrepancies need to be proven on a larger cohort.

Assessment of the total cfDNA and HPV16/18 detection in plasma samples of head and neck squamous cell carcinoma patients.

OBJECTIVES: The advantages of the circulating cell-free DNA (cfDNA) methodology are quick results and the possibility of repeated analysis. The main aim of our study was to establish the relationship of the total cfDNA with patients' clinical characteristics and circulating HPV DNA detection in the blood of patients with head and neck squamous cell carcinoma (HNSCC). METHODS: The cfDNA level of 200 HNSCC patients in plasma was quantified using TaqMan-based TERT amplification. TaqMan technology was also used for HPV16/18 detection. Additionally, mutations in KRAS and EGFR were investigated. RESULTS: A higher level (p=0.011) of the total cfDNA was found in patients with oropharyngeal squamous cell carcinoma (OPSCC) (9.60 ± 6.23 ng/ml) in comparison with other HNSCC (7.67 ± 4.44 ng/ml). The level of cfDNA in patients with clinical N2-N3 disease (9.28 ± 6.34 ng/ml) was (p=0.015) higher than in patients with a clinical N0-N1 disease (7.50 ± 3.69 ng/ml). It was also higher in patients with stage IV (9.16 ± 6.04 ng/ml) compared with stages I-III of cancer (7.26 ± 3.63 ng/ml) (p=0.011). Analysis of HPV16/18 in plasma revealed that 14% of patients were HPV-positive, the majority of whom had the type HPV16 (96.4%). CfDNA level was comparable in HPV-positive and HPV-negative HNSCC patients, as well in the OPSCC subgroup. Somatic mutations in EGFR and KRAS were not found. CONCLUSIONS: A high level of cfDNA is specific for patients with OPSCC. HPV detection in cfDNA does not depend on the cfDNA concentration. Our results prove the diagnostic potential of plasma-based HPV cfDNA tests for the early detection and monitoring of HPV-positive HNSCC.

Optimization of circulating cell-free DNA recovery for KRAS mutation and HPV detection in plasma.

BACKGROUND: The precise analysis of tumour markers in blood such as circulating cell-free DNA (cfDNA) could have a significant impact in facilitating monitoring of patients after initial therapy. Although high levels of total cfDNA in plasma of cancer patients are consistently demonstrated, a low sensitivity of DNA alterations is reported. OBJECTIVE: The major question regards the recovery of tumour-specific cfDNA such as KRAS mutated DNA and cancer-associated type 16 of human papillomavirus (HPV16). METHODS: TaqMan technology was used for detection of KRAS mutation, HPV16 and to quantify cfDNA in blood plasma. RESULTS: Comparison of four different column-based commercial kits shows that the cfDNA purification carried out by the Genomic Mini AX Body Fluids kit and the QIAamp Circulating Nucleic Acid kit gave us the possibility to improve the sensitivity of detection of KRAS mutation and HPV16. The optimized method was used to follow the reduction in cancer-specific cfDNA after therapy. We found that large volume extractions with low volume of DNA eluate enabled trace amounts of tumour-specific cfDNA from cancer patients to be effectively identified. CONCLUSIONS: Data presented in this study facilitate detection of tumour-specific cfDNA and improve standards needed for the implementation of cfDNA technology into routine clinical practice.