Circulating Tumour DNA Biomarkers Associated with Outcomes in Metastatic Prostate Cancer Treated with Lutetium-177-PSMA-617.

Background: Lutetium-177-prostate-specific membrane antigen- 617 (Lu-PSMA) is an effective therapy for metastatic castration-resistant prostate cancer (mCRPC). However, treatment responses are heterogeneous despite stringent positron emission tomography (PET)-based imaging selection criteria. Molecularly based biomarkers have potential to refine patient selection and optimise outcomes. Objective: To identify circulating tumour DNA (ctDNA) features associated with treatment outcomes for men treated with Lu-PSMA. Design setting and participants: ctDNA from men treated with Lu-PSMA in combination with idronoxil for progressive mCRPC were analysed using an 85-gene customised sequencing assay. ctDNA fractions, molecular profiles, and the presence of alterations in aggressive-variant prostate cancer (AVPC) genes were analysed at baseline, cycle 3 and at disease progression. Intervention: Men received Lu-PSMA with idronoxil every 6 wk for up to six cycles. Outcome measurements and statistical analysis: Baseline and exit PSMA and fluorodeoxyglucose PET/computed tomography (CT) imaging was conducted at baseline and study exit. Single-photon emission CT (SPECT) scans were performed 24 h after Lu-PSMA. Blood samples were collected at baseline,cycle 3 and at disease progression. Cox proportional-hazards models were used to assess associations and derive hazard ratios (HRs) and confidence intervals (CIs) for associations between molecular factors, imaging features, and clinical outcomes. Results and limitations: Sixty samples from 32 men were sequenced (32 at baseline, 24 at cycle 3, four from patients with disease progression); two samples (baseline, on-treatment) from one individual were excluded from analysis owing to poor quality of the baseline sequencing data. Alterations in AVPC genes were associated with shorter prostate-specific antigen (PSA) progression-free survival (PFS) and overall survival (OS) in univariate (HR 3.4, 95% CI 1.5-7.7; p = 0.0036; and HR 3.3, 95% CI 1.4-7.7; p = 0.0063, respectively) and multivariate analyses (HR 4.8, 95% CI 1.8-13; p = 0.0014; and HR 4.1, 95% CI 1.6-11; p = 0.004). Conclusions: ctDNA alterations in AVPC genes were associated with shorter PSA PFS and OS among men treated with Lu-PSMA and intermittent idronoxil. These candidate molecular biomarkers warrant further study to determine whether they have predictive value and potential to guide synergistic combination strategies to enhance outcomes for men treated with Lu-PSMA for mCRPC. Patient summary: Certain DNA/gene changes detected in the blood of men with advanced prostate cancer were associated with shorter benefit from lutetium PSMA, a targeted radioactive therapy. This information may be useful in determining which men may benefit most from this treatment, but additional research is needed.

Evaluation of a panel of tumor-specific differentially-methylated DNA regions in IRF4, IKZF1 and BCAT1 for blood-based detection of colorectal cancer.

BACKGROUND: Differentially-methylated regions (DMRs) are characteristic of colorectal cancer (CRC) and some occur more frequently than common mutations. This study aimed to evaluate the clinical utility of assaying circulating cell-free DNA for methylation in BCAT1, IKZF1 and IRF4 for detection of CRC. METHODS: A multiplexed real-time PCR assay targeting DMRs in each of the three genes was developed. Assay accuracy was explored in plasma specimens banked from observational cross-sectional trials or from volunteers scheduled for colonoscopy or prior to CRC surgery. RESULTS: 1620 specimens were suitable for study inclusion including 184 and 616 cases with CRC and adenomas, respectively, and 820 cases without neoplasia (overall median age, 63.0 years; 56% males). Combining the PCR signals for all targeted DMRs returned the best sensitivity for CRC (136/184, 73.9%, 95% CI 67.1-79.7), advanced adenomas (53/337, 15.7%, 95% CI 12.0-20.1) and high-grade dysplastic (HGD) adenomas (9/35, 25.7%, 95% CI 14.0-42.3) with a 90.1%, specificity for neoplasia (739/820, 95% CI 87.9-92.0, p < 0.01). Detection of methylation in all three genes were more likely in CRC cases than those without it (OR 28.5, 95% CI 7.3-121.2, p < 0.0001). Of the 81 positive cases without neoplasia, 62 (76.5%) were positive by a single PCR replicate only and predominantly due to detection of methylated BCAT1 (53.2%). Single replicate positivity was significantly higher than that in CRC (26/136, 19.1%, p < 0.0001), and single BCAT1 replicate positivity was more likely in cases without neoplasia than in CRC (OR 17.7, 95% CI 6.6-43.3, p < 0.0001). When a positive result was limited to those with ≥ 1 PCR replicate positive for either IKZF1 or IRF4, or at least two replicates positive for BCAT1, the multi-panel test maintained a high sensitivity for CRC (131/184, 71.2%, 95% CI 64.3-77.3) and HGD adenomas (8/35, 22.9%, 95% CI 11.8-39.3, p = 0.029) but improved specificity significantly (772/820, 94.1%, 95% CI 92.3-95.6, p < 0.0001 vs. any PCR replicate positive). CONCLUSION: The multi-panel methylation assay differentiates cases with CRC from those without it and does so with high specificity when criteria for BCAT1 detection are applied. The marker panel is flexible and studies in those at average risk for CRC are now warranted to determine which panel configuration best suits screening goals. TRIAL REGISTRATION: ACTRN12611000318987. Registered 25 March 2011, https://www.anzctr.org.au/ ACTRN12611000318987.

A simple, accurate and universal method for quantification of PCR.

BACKGROUND: Research into gene expression enables scientists to decipher the complex regulatory networks that control fundamental biological processes. Quantitative real-time PCR (qPCR) is a powerful and ubiquitous method for interrogation of gene expression. Accurate quantification is essential for correct interpretation of qPCR data. However, conventional relative and absolute quantification methodologies often give erroneous results or are laborious to perform. To overcome these failings, we developed an accurate, simple to use, universal calibrator, AccuCal. RESULTS: Herein, we show that AccuCal quantification can be used with either dye- or probe-based detection methods and is accurate over a dynamic range of ≥10(5) copies, for amplicons up to 500 base pairs (bp). By providing absolute quantification of all genes of interest, AccuCal exposes, and circumvents, the well-known biases of qPCR, thus allowing objective experimental conclusions to be drawn. CONCLUSION: We propose that AccuCal supersedes the traditional quantification methods of PCR.

Prime-boost immunisation against tropical theileriosis with two parasite surface antigens: evidence for protection and antigen synergy.

Current methods for control of tropical theileriosis in cattle suffer from several disadvantages that could be circumvented by development of an effective sub-unit vaccine. Previous work has utilised two major surface antigens (SPAG-1 and Tams1) and conventional adjuvants to provide partial protection against parasite challenge. In this study we have delivered these antigens using the prime-boost system and analysed whether a combination regime can enhance protection against lethal challenge. Delivery of the boost as recombinant protein or expressed from a recombinant MVA vector was also assessed. The results confirmed that immunisation with Tams1 alone could reduce the severity of several disease parameters compared to non-immunised controls and these effects were more marked when recombinant protein was used for boosting compared to MVA delivery. A similar outcome was obtained by immunisation with SPAG-1 alone. Significantly, delivery of SPAG-1 and Tams1 as a cocktail showed enhanced protection. This was manifest by significant improvement in a large range of clinical and parasitological parameters and, most dramatically, by the survival and recovery of 50% of the immunised animals compared to 0% of the controls. Analysis of the antibody response post-challenge showed that while there was a strong response to Tams1, no response to SPAG-1 was detected. In contrast, lymphoproliferation assays showed a significant enhancement of response at day 7 post-challenge in calves of the SPAG-1 group but a dramatic decrease of the proliferation activity in all three groups receiving Tams1. We conclude that immunisation with a cocktail of SPAG-1 and Tams1 generates a synergistic protective response that significantly improves the efficacy of recombinant vaccination against tropical theileriosis. Potential effector mechanisms that could mediate this response are discussed.