Verena Sailer

loss, and aggressive clinical features. DLL3 in circulating tumor cells was concordant with matched metastatic biopsy (87%). Treatment of DLL3-expressing prostate cancer xenografts with a single dose of SC16LD6.5 resulted in complete and durable responses, whereas DLL3-negative models were insensitive. We highlight a patient with neuroendocrine prostate cancer with a meaningful clinical and radiologic response to SC16LD6.5 when treated on a phase 1 trial. Overall, our findings indicate that DLL3 is preferentially expressed in CRPC-NE and provide rationale for targeting DLL3 in patients with DLL3-positive metastatic prostate cancer.

Background: Stratifying PCa patients into risk groups at time of initial diagnosis enabling a risk-adapted disease management is still a major clinical challenge. Existing studies evaluating the prognostic potential of PSMA for PCa were performed on radical prostatectomy specimens (RPE), i.e. decision making for disease management was already completed at time of sample analysis. Aim of our study was to assess the prognostic value of PSMA (prostate-specific membrane antigen) expression for prostate cancer (PCa) patients on biopsies at time of initial diagnosis. Methods: PSMA expression was assessed by immunohistochemistry on 294 prostate biopsies with corresponding RPE, 621 primary tumor foci from 242 RPE, 43 locally advanced or recurrent tumors, 34 lymph node metastases, 78 distant metastases and 52 benign prostatic samples. PSMA expression was correlated with clinico-pathologic features. Primary endpoint was recurrence free survival. Other clinicopathologic features included WHO/ISUP grade groups, PSA serum level, TNM-stage, and R-status. Chi-square test, ANOVA-analyses, Cox-regression and log-rank tests were performed for statistical analyses. Results: High PSMA expression on both biopsy and RPE significantly associates with a higher risk of disease recurrence following curative surgery. The 5-year-recurrence free survival rates were 88.2%, 74.2%, 67.7% and 26.8% for patients exhibiting no, low, medium or high PSMA expression on biopsy, respectively. High PSMA expression on biopsy was significant in multivariate analysis predicting a 4-fold increased risk of disease recurrence independently from established prognostic markers. PSMA significantly increases during PCa progression. Conclusion: PSMA is an independent prognostic marker on biopsies at time of initial diagnosis and can predict disease recurrence following curative therapy for PCa. Our study proposes the application of the routinely used IHC marker PSMA for outcome prediction and decision making in risk-adapted PCa management on biopsies at time of initial diagnosis.

DNA methylation analyses usually require a preceding bisulfite conversion of the DNA. The choice of an appropriate kit for a specific application should be based on the specific performance requirements with regard to the respective sample material. In this study, the performance of nine kits was evaluated: EpiTect Fast FFPE Bisulfite Kit, EpiTect Bisulfite Kit, EpiTect Fast DNA Bisulfite Kit (Qiagen), EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Direct Kit, EZ DNA Methylation-Lightning Kit (Zymo Research), innuCONVERT Bisulfite All-In-One Kit, innuCONVERT Bisulfite Basic Kit, innuCONVERT Bisulfite Body Fluids Kit (Analytik Jena). The kit performance was compared with regard to DNA yield, DNA degradation, DNA purity, conversion efficiency, stability and handling using qPCR, UV, clone sequencing, HPLC, and agarose gel electrophoresis. All kits yielded highly pure DNA suitable for PCR analyses without PCR inhibition. Significantly higher yields were obtained when using the EZ DNA Methylation-Gold Kit and the innuCONVERT Bisulfite kits. Conversion efficiency ranged from 98.7% (EpiTect Bisulfite Kit) to 99.9% (EZ DNA Methylation-Direct Kit). The inappropriate conversion of methylated cytosines to thymines varied between 0.9% (innuCONVERT Bisulfite kits) and 2.7% (EZ DNA Methylation-Direct Kit). Time-to-result ranged from 131 min (innuCONVERT kits) to 402 min (EpiTect Bisulfite Kit). Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits). Highest yields from formalin-fixed and paraffin-embedded (FFPE) tissue sections without prior extraction were obtained using the innuCONVERT Bisulfite All-In-One Kit while the EZ DNA Methylation-Direct Kit yielded DNA with only low PCR-amplifiability. The innuCONVERT Bisulfite All-In-One Kit exhibited the highest versatility regarding different input sample materials (extracted DNA, tissue, FFPE tissue, cell lines, urine sediment, and cellular fractions of bronchial aspirates, pleural effusions, ascites). The innuCONVERT Bisulfite Body Fluids Kit allowed for the analysis of 3 ml plasma, serum, ascites, pleural effusions and urine.

Formalin-fixed and paraffin-embedded (FFPE) tissues represent a valuable source for biomarker studies and clinical routine diagnostics. However, they suffer from degradation of nucleic acids due to the fixation process. Since genetic and epigenetic studies usually require PCR amplification, this degradation hampers its use significantly, impairing PCR robustness or necessitating short amplicons. In routine laboratory medicine a highly robust PCR performance is mandatory for the clinical utility of genetic and epigenetic biomarkers. Therefore, methods to improve PCR performance using DNA from FFPE tissue are highly desired and of wider interest. The effect of template DNA derived from FFPE tissues on PCR performance was investigated by means of qPCR and conventional PCR using PCR fragments of different sizes. DNA fragmentation was analyzed via agarose gel electrophoresis. This study showed that poor PCR amplification was partly caused by inhibition of the DNA polymerase by fragmented DNA from FFPE tissue and not only due to the absence of intact template molecules of sufficient integrity. This PCR inhibition was successfully minimized by increasing the polymerase concentration, dNTP concentration and PCR elongation time thereby allowing for the robust amplification of larger amplicons. This was shown for genomic template DNA as well as for bisulfite-converted template DNA required for DNA methylation analyses. In conclusion, PCR using DNA from FFPE tissue suffers from inhibition which can be alleviated by adaptation of the PCR conditions, therefore allowing for a significant improvement of PCR performance with regard to variability and the generation of larger amplicons. The presented solutions to overcome this PCR inhibition are of tremendous value for clinical chemistry and laboratory medicine.

OBJECTIVES: Expression of the Notch-family ligand delta-like protein 3 (DLL3), a potential therapeutic target in small cell lung cancer (SCLC), has not been assessed in the real-world setting. To identify the real-world utility of DLL3 as an SCLC therapeutic target, we performed the largest retrospective international noninterventional study to date to evaluate DLL3 prevalence in SCLC patients. MATERIALS AND METHODS: DLL3 expression was assessed using immunohistochemistry in archived histological and cytological specimens (independent and paired) and correlated to patient demographics, clinical disease characteristics, and survival. The primary endpoint was the proportion of patients with DLL3 expression in ≥25 % of tumor cells. DLL3 expression concordance was assessed in paired specimens. RESULTS: Independent tumor specimens were collected from 1073 patients. The mean age at biopsy was 66 years (SD, 10); 682 (64 %) patients were male. Paired specimens were collected from 36 patients. The mean age at biopsy was 62 years (SD, 11); 16 (44 %) patients were male. Most patients had ECOG performance status of 0-1, were smokers/ex-smokers, and received ≥1 prior therapy. Positive DLL3 expression (defined as ≥25 % of tumor cells) was identified in 895/1050 (85 %) patients with 1 specimen and evaluable DLL3 expression; 719/1050 (68 %) patients had high DLL3 expression (defined as ≥75 % of tumor cells). DLL3 expression concordance was 88 % between paired specimens (n = 17; Cohen's kappa P value, .9412). There was no significant difference in median overall survival from SCLC diagnosis for evaluable patients with nonmissing data based on DLL3 expression (negative DLL3 expression [n = 139], 9.5 months; positive DLL3 expression [n = 747], 9.5 months; all evaluable patients [n = 893, 9.5 months). CONCLUSION: These real-world epidemiologic findings indicate that DLL3 is robustly expressed across SCLC disease stages and remains stable despite treatment, consistent with available clinical trial data. There was no prognostic role for DLL3 observed in this study for overall survival.