Tijl Vermassen

Glycosylation of prostate specific antigen and its potential diagnostic applications

Prostate specific antigen (PSA) assays are widely used for early detection of prostate cancer. However, those analyses are associated with considerable sensitivity and specificity problems. Several approaches have been developed to tackle this issue. PSA is a glycoprotein, which is primarily produced by the prostatic epithelial cells. Aberrant glycosylation modification of proteins is a fundamental characteristic of tumorigenesis. Study of PSA glycoforms offers interesting diagnostic perspectives. Modern technology allows us to analyze PSA glycoforms in a variety of clinical samples (serum or plasma, urine, seminal fluid, tissue). A number of novel techniques, such as lectin-based detection methods, mass spectrometry, 2-dimensional electrophoresis and capillary electrophoresis have been developed to analyze PSA glycosylation. This article reviews the technical and diagnostic aspects of PSA glycoforms.

Urinary prostate protein glycosylation profiling as a diagnostic biomarker for prostate cancer

Serum prostate‐specific antigen (sPSA) measurement is widely used as opportunistic screening tool for prostate cancer (PCa). sPSA suffers from considerable sensitivity and specificity problems, particularly in the diagnostic gray zone (sPSA 4–10 µg/L). Furthermore, sPSA is not able to discriminate between poorly‐, moderately‐, and well‐differentiated PCa. We investigated prostatic protein glycosylation profiles as a potential PCa biomarker.

Capillary electrophoresis of urinary prostate glycoproteins assists in the diagnosis of prostate cancer.

Prostate marker assays are widely used for detection of prostate cancer (PCa) but are associated with considerable sensitivity and specificity problems. Therefore, we investigated prostatic protein glycosylation profiles as a potential biomarker. We determined the urinary asparagine‐linked glycan (N‐glycan) profile of prostatic proteins of healthy volunteers (n = 25), patients with benign prostate hyperplasia (BPH; n = 62) and newly diagnosed PCa patients (n = 42) using DNA‐sequencer‐assisted fluorophore‐assisted carbohydrate electrophoresis. Through squeezing of the prostate, a sufficient amount of prostatic proteins was obtained for direct structural analyses of N‐glycan structures. N‐glycans of PCa compared to BPH were characterized by a significant decrease in triantennary structures (p = 0.047) and overall fucosylation (p = 0.026). Prostate‐specific antigen (PSA) and the urinary glycoprofile marker showed comparable overall receiver operating characteristic curve analysis as well as in the diagnostic gray zone with serum PSA values between 4 and 10 μg/L. However, when combining PSA and the urinary glycoprofile marker, the latter gave an additive diagnostic value to serum PSA (p ≤ 0.001). In conclusion, N‐glycosylation profiling demonstrated differences between BPH and PCa. These changes could lead to the discovery of a new biomarker for PCa.

TILs in Head and Neck Cancer: Ready for Clinical Implementation and Why (Not)?

The assessment of tumor infiltrating lymphocytes (TILs) has recently emerged as a prognostic biomarker in several solid tumors. Quantification and subtyping of TILs reflects the immune response in the tumor microenvironment, contributing to either tumoral immune attack or escape and thereby affecting outcome. Despite the growing evidence of its value as prognosticator, TILs analysis has not yet found its way to daily clinical practice. The aim of this review is to evaluate whether the current knowledge on TILs in head and neck cancer justifies its clinical implementation. Therefore, we summarize the data on TILs in squamous cell cancer of the head and neck with focus on the most important subsets (T lymphocytes and more specifically CD8+ cytotoxic T cells and FoxP3+ regulatory T cells) and site-specific characteristics such as Human Papilloma Virus infection. In addition, we discuss methodological problems and pitfalls that can account for discordant findings and that may hamper inclusion of TILs assessment in routine practice of pathologists and oncologists.

CD70 Expression and Its Correlation with Clinicopathological Variables in Squamous Cell Carcinoma of the Head and Neck.

Objective: Over the last decade, efforts have been made to get a better understanding of the tumor microenvironment and the role of the immune system in it. New insights into the CD27/CD70 signaling pathway point towards a role in tumor immunology, making CD70 an attractive target for immunotherapy. Here, we evaluate CD70 expression in squamous cell carcinoma of the head and neck (SCCHN). Methods: CD70 immunohistochemistry was retrospectively performed on 95 tumor samples. Tumoral CD70 expression was scored and correlated with clinicopathological variables and overall survival (OS). Results: CD70 expression in tumor cells was observed in 66 samples (69%) and was strongly associated with tumor differentiation grade (p < 0.001). CD70 expression was also observed in tumor-associated fibroblasts and endothelial cells. Additionally, the density of tumor-infiltrating lymphocytes correlated with OS (p = 0.042). Conclusion: This study describes the tumoral expression of CD70 in SCCHN. Results highlight the role of CD70 in tumor biology and identify CD70 as a novel therapeutic target. Further research is warranted.

Usefulness of Indirect Alcohol Biomarkers for Predicting Recidivism of Drunk-driving among Previously Convicted Drunk-driving Offenders: Results from the Recidivism Of Alcohol-impaired Driving (ROAD) Study

AIM In several European countries, drivers under the influence (DUI), suspected of chronic alcohol abuse are referred for medical and psychological examination. This study (the ROAD study, or Recidivism Of Alcohol-impaired Driving) investigated the usefulness of indirect alcohol biomarkers for predicting drunk-driving recidivism in previously convicted drunk-driving offenders. DESIGN, SETTING, PARTICIPANTS AND MEASUREMENTS The ROAD study is a prospective study (2009-13) that was performed on 517 randomly selected drivers in Belgium. They were convicted for drunk-driving for which their licence was confiscated. The initial post-arrest blood samples were collected and analysed for percentage carbohydrate-deficient transferrin (%CDT), transaminsase activities [alanine amino transferase (ALT), aspartate amino transferase (AST)], gamma-glutamyltransferase (γGT) and red cell mean corpuscular volume (MCV). The observation time for each driver was 3 years and dynamic. FINDINGS A logistic regression analysis revealed that ln(%CDT) (P < 0.001), ln(γGT) (P < 0.01) and ln(ALT) (P < 0.05) were the best biochemical predictors of recidivism of drunk-driving. The ROAD index (which includes ln(%CDT), ln(γGT), -ln(ALT) and the sex of the driver) was calculated and had a significantly higher area under the receiver operator characteristic curve (0.71) than the individual biomarkers for drunk-driving recidivism. Drivers with a high risk of recidivating (ROAD index ≥ 25%; third tertile) could be distinguished from drivers with an intermediate risk (16% ≤ ROAD index < 25%; second tertile; P < 0.001) and a low recidivism risk (ROAD index < 16%; first tertile; P < 0.05). CONCLUSIONS Of all routinely used indirect alcohol markers, percentage of carbohydrate-deficient transferrin is the major predictor of recidivism of drunk-driving. The association with gamma-glutamyltransferase, alanine amino transferase and the sex of the driver could have additional value for identifying drunk-drivers at intermediate risk of recidivism. Non-specific indirect alcohol markers, such as alanine amino transferase, gamma-glutamyltransferase, aspartate amino transferase and red cell mean corpuscular volume have minimal added value to % carbohydrate-deficient transferrin for distinguishing drunk drivers with a low or high risk of recidivism.

Prognostic markers in oropharyngeal squamous cell carcinoma: focus on CD70 and tumour infiltrating lymphocytes

We evaluated the expression of CD70 as biomarker for prognosis in patients with oropharyngeal squamous cell carcinoma (OSCC). We also examined the prognostic value of tumour infiltrating lymphocytes (TILs) in our study cohort. Formalin fixed, paraffin embedded tissue originating from the oropharynx of 78 patients was immunohistochemically stained for CD70, CD3, CD8 and FoxP3. Expression of CD70, CD3, CD8, FoxP3 and HPV status was correlated with clinicopathological characteristics. Overall survival (OS) was determined by a log-rank (Mantel-Cox) test whereas the Cox proportional hazard model was used for multivariate analysis. CD70 expression demonstrated no influence on OS. Tumours heavily infiltrated by TILs were linked with better outcome, for the total number of TILs as well as for the CD3+ and CD8+ T cell count. A Cox proportional hazard model proved that solely CD8+ infiltrating T cells exhibit a positive effect on OS (HR=0.30, 95% confidence interval 0.13-0.72). Our results demonstrate that CD8+ TILs constitute an independent prognosticator in patients diagnosed with OSCC. Further validation of the prognostic value of CD8+ TILs in OSCC is warranted and could provide us with a better insight into the immunological status of these malignancies.

Turning the tide : clinical utility of PD-L1 expression in squamous cell carcinoma of the head and neck

The use of cytotoxic and/or targeted agents is the gold standard in first- and second-line treatment of metastatic head and neck cancer. Currently the focus of oncologic research is shifting to the implementation of immune checkpoint inhibitor regimens. Many trials are being performed evaluating the survival benefit of various PD-1/PD-L1 blocking antibodies in both solid and haematological malignancies. Also, evaluation of the predictive value of PD-L1 expression on tumour cells and immune cells is being explored. We first review the current knowledge and possible pitfalls for PD-L1 expression in squamous cell carcinoma of the head and neck. Next, we provide an update on the therapeutic use of PD-1/PD-L1 blocking antibodies as treatment modality for patients with squamous cell carcinoma of the head and neck and we assess the predictive value of tumour PD-L1 positivity. Finally, we elaborate on other promising predictive biomarkers of interest in this patient population.

Neither creatinine nor cystatin C-estimated glomerular filtration rate is optimal in oncology patients treated with targeted agents.

Background In the last decade, there has been an increase in the use of anti-angiogenic drugs as treatment for metastatic malignancies. However, use of these targeted therapies could induce both glomerular and tubular damage. Also during targeted therapy, the lysosomal protease cathepsin D is released from the tumour, which is inhibited by the protease inhibitor cystatin C. The aim of this study is to determine if use of cystatin C-estimated glomerular filtration rate (eGFR) is applicable to a patient cohort treated with targeted agents. Methods A cohort of 80 patients with various malignancies were continuously recruited and prospectively analysed. Serum and urinary biochemical analytes for renal toxicities were assessed at different time points during treatment. The association between serum cystatin C and cathepsin D was also determined. Results A decrease in serum cystatin C concentrations (1.03 versus 0.90 mg/L; P < 0.001), together with an increase in cystatin C-eGFR (71 versus 89 mL/min/1.73 m2; P = 0.002) was observed during therapy, compared with baseline. This decrease in cystatin C concentrations was correlated with cathepsin D (r = 0.307; P < 0.001), which was released from the tumour during targeted therapy. Further analysis demonstrated cathepsin D-mediated proteolysis of cystatin C in serum. Conclusions Cystatin C concentrations were decreased during targeted therapy due to cathepsin D-mediated proteolysis. Cystatin C-eGFR is therefore not considered a suitable marker for assessing kidney function in oncology patients, and other techniques to estimate the GFR have to be applied in this patient population.

Tumor PD-L1 status and CD8+ tumor-infiltrating T cells: markers of improved prognosis in oropharyngeal cancer

Introduction The aim of this study was to evaluate the expression of PD-L1 in oropharyngeal squamous cell carcinoma. Its relation with clinicopathological variables, tumor infiltrating lymphocytes and survival was also determined. Results Positive PD-L1 status for the SP142 clone related with improved overall survival in oropharyngeal squamous cell carcinoma. Tumors heavily infiltrated by tumor infiltrating lymphocytes were also linked with better outcome, and this as well for the total number of tumor infiltrating lymphocytes as for the CD3+ and CD8+ T cell count. A Cox proportional hazard model proved that solely infiltrating CD8+ T cells exhibit a positive effect on overall survival (hazard ratio = 0.31 [0.14–0.70]; P = 0.0050) Materials and Methods Formalin-fixed, paraffin-embedded tissue from oropharyngeal tumors of 99 patients was immunohistochemically stained for PD-L1 (SP142 and 22C3 clones), CD3, CD8 and FoxP3. Expression of PD-L1, CD3, CD8, FoxP3 and HPV status were correlated with clinicopathological variables. Overall survival was determined by a log-rank (Mantel–Cox) test whereas the Cox proportional hazard model was used for multivariate analysis. Conclusions Our results demonstrate that CD8+ T lymphocytes constitute an independent prognostic marker in patients diagnosed with oropharyngeal squamous cell carcinoma. PD-L1 positivity for SP142, but not for 22C3, also tends to have a positive effect on survival in oropharyngeal squamous cell carcinoma.INTRODUCTION The aim of this study was to evaluate the expression of PD-L1 in oropharyngeal squamous cell carcinoma. Its relation with clinicopathological variables such as amount and type of tumor infiltrating lymphocytes, HPV status and survival was also determined. RESULTS Positive PD-L1 status for the SP142 clone related with improved overall survival in oropharyngeal squamous cell carcinoma. Tumors heavily infiltrated by tumor infiltrating lymphocytes were also linked with better outcome, and this as well for the total number of tumor infiltrating lymphocytes as for the CD3+ and CD8+ T cell count. A Cox proportional hazard model proved that solely infiltrating CD8+ T cells exhibit a positive effect on overall survival (hazard ratio = 0.31 [0.14-0.70]; P = 0.0050)Materials and Methods: Formaldehyde fixed-paraffin embedded tissue of oropharynx tumors of 99 patients was immunohistochemically stained for PD-L1 (SP142 and 22C3 clones), CD3, CD8 and FoxP3. Expression of PD-L1, CD3, CD8, FoxP3 and HPV status was correlated with clinicopathological variables. Overall survival was determined by a log-rank (Mantel-Cox) test whereas the Cox proportional hazard model was used for multivariate analysis. CONCLUSIONS Our results demonstrate that CD8+ T lymphocytes constitute an independent prognostic marker in patients diagnosed with oropharyngeal squamous cell carcinoma. PD-L1 positivity for SP142, but not for 22C3, also tends to have a positive effect on survival in oropharyngeal squamous cell carcinoma.