Prognostic value and cost benefit of HPV testing for oropharyngeal cancer patients.

OBJECTIVES: High-risk human papillomavirus (HR-HPV) can cause oropharyngeal squamous cell carcinoma (OpSCC). The revised 8th edition of the AJCC Staging Manual now stages OpSCC by incorporating p16 immunohistochemistry (IHC), the surrogate marker for HPV status. This study assessed the prognostic values of p16 and HPV markers. METHODS: We identified 244 OpSCC patients diagnosed between 2000 and 2008 from the British Columbia Cancer Registry with enough tissue to conduct experiments. Formalin-fixed, paraffin-embedded tissue sections were stained for p16 IHC, RNA in situ hybridization (ISH) HPV 16 and 18, and DNA ISH HR-HPV. Electronic charts were reviewed to collect clinical and outcome data. Combined positive RNA and/or DNA ISH was used to denote HPV status. RESULTS: Human papillomavirus was positive among 77.9% of samples. Using HPV as the benchmark, p16 IHC had high sensitivity (90.5%), but low specificity (68.5%). Distinct subgroups of patients were identified by sequential separation of p16 then HPV status. Among both p16-positive and p16-negative groups, HPV-positive patients were younger, more males, and had better clinical outcomes, especially 5-year overall survival. We further evaluated the technical costs associated with HPV testing. CONCLUSION: Human papillomavirus is more prognostic than p16 for OpSCC. Clinical laboratories can adopt HPV RNA ISH for routine analysis.

Using ddPCR to assess the DNA yield of FFPE samples.

OBJECTIVES: Detection of genomic alterations in diseases can be achieved with current molecular technologies. However, the molecules extracted from formalin-fixed, paraffin-embedded (FFPE) bio-samples are often limited possibly due to DNA fragmentation and crosslinking caused by the sample fixation and processing. The study objective was to design a droplet digital PCR (ddPCR) assay to assess the quality and quantity of DNA derived from various DNA extraction conditions on FFPE samples. METHODS: We used 10 µm-thick sections from 5 FFPE oral tumoral blocks, each consisting of 10-15 sections. The protocol variables tested included: 1) tissue staining; 2) duration and 3) temperature of post-digestion heat treatment; and 4) DNA extraction method. DNA quantity was assessed using the NanoDrop 2000 (Thermo Fisher Scientific, USA), the Qubit fluorometer (Thermo Fisher Scientific, USA), and a ddPCR-based assay. DNA quality was assessed using a ddPCR assay for the degree of fragmentation and the effectiveness of removing crosslinks with varying guanine-cytosine (GC)-content. RESULTS: Deparaffinization with xylene helped to increase the DNA yield. Tissue staining (methyl green staining, pH 6) prior to microdissection, comparing to no staining, caused additional DNA fragmentation. Compared to column-based method, DNA extracted with phenol chloroform and ethanol precipitation increased the degree of fragmentation and lowered the yield of amplifiable DNA. The cross-linking derived from GC-contents may not be the only factor impacting on the DNA quality. CONCLUSIONS: Samples undergoing different pre-treatment conditions prior to extraction can impact the yield of amplifiable DNA. Our ddPCR assay can be used to assess for both DNA quantity and quality.

Plasma-Derived Inflammatory Proteins Predict Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is a major concern with high morbidity and mortality worldwide, even with the current knowledge and the advancement in treatment. OSCCs diagnosed at late-stage often require wide-excision with or without neck dissection, radiotherapy, or chemotherapy. When deemed successful, treatment often results in diminished quality of life, impaired function, and disfigurement. Strategies for early detection are urgently needed for patients afflicted with this disease. Inflammatory protein plasma biomarkers have shown to be potential tests for early detection and disease monitoring in several cancers. There has been no study on inflammation-related plasma biomarkers in OSCC. The objectives of the study were to use a multiplex approach to screen plasma-derived biomarkers and to examine the association of measurable proteins with OSCC. A total of 260 plasma samples (210 OSCC and 50 normal controls) were collected to measure for concentration of inflammatory related biomarkers using electrochemiluminescence multiplex assay. After screening of 82 potential biomarkers of the first 160 OSCC, 16 cytokines, chemokines, and growth factors were identified and verified in the second set of samples containing 50 OSCC and 50 normal. After adjustment of age and batch effects, the adjusted differential expression analysis showed that the OSCCs were markedly lower in 14 biomarkers and significantly higher level of interleukin 1 receptor antagonist (IL1Ra). By performing unsupervised clustering analysis, we observed distinctive groups of normal and two subgroups of OSCC. Linear regression of IL2, IL1Ra, and macrophage inhibitory factor (MIF) showed high accuracy in classifying OSCC with sensitivity of 0.96 and specificity of 0.92. In conclusion, this is the first paper to identify potential inflammatory plasma protein biomarkers of patients with OSCC. With further validation, the set of biomarkers can potentially be used to assist in early detection of OSCC when the disease is localized and in more treatable stage.