Droplet digital PCR based androgen receptor variant 7 (AR-V7) detection from prostate cancer patient blood biopsies

Androgen receptor splice variant V7 (AR-V7) was recently identified as a valuable predictive biomarker in metastatic castrate-resistant prostate cancer. Here, we report a new, sensitive and accurate screen for AR-V7 mRNA expression directly from circulating tumor cells (CTCs): We combined EpCAM-based immunomagnetic CTC isolation using the IsoFlux microfluidic platform with droplet digital polymerase chain reaction (ddPCR) to analyze total AR and AR-V7 expression from prostate cancer patients CTCs. We demonstrate that AR-V7 is reliably detectable in enriched CTC samples with as little as five CTCs, even considering tumor heterogeneity, and confirm detection of AR-V7 in CTC samples from advanced prostate cancer (PCa) patients with AR-V7 detection limited to castrate resistant disease status in our sample set. Sensitive molecular analyses of circulating tumor cells (CTCs) or circulating tumor nucleic acids present exciting strategies to detect biomarkers, such as AR-V7 from non-invasive blood samples, so-called blood biopsies

CTC-mRNA (AR-V7) analysis from blood samples : impact of blood collection tube and storage time

Circulating tumour cells (CTCs) are an emerging resource for monitoring cancer biomarkers. New technologies for CTC isolation and biomarker detection are increasingly sensitive, however, the ideal blood storage conditions to preserve CTC-specific mRNA biomarkers remains undetermined. Here we tested the preservation of tumour cells and CTC-mRNA over time in common anticoagulant ethylene-diamine-tetra-acetic acid (EDTA) and acid citrate dextrose solution B (Citrate) blood tubes compared to preservative-containing blood tubes. Blood samples spiked with prostate cancer cells were processed after 0, 24, 30, and 48 h storage at room temperature. The tumour cell isolation efficiency and the mRNA levels of the prostate cancer biomarkers androgen receptor variant 7 (AR-V7) and total AR, as well as epithelial cell adhesion molecule (EpCAM) were measured. Spiked cells were recovered across all storage tube types and times. Surprisingly, tumour mRNA biomarkers were readily detectable after 48 h storage in EDTA and Citrate tubes, but not in preservative-containing tubes. Notably, AR-V7 expression was detected in prostate cancer patient blood samples after 48 h storage in EDTA tubes at room temperature. This important finding presents opportunities for measuring AR-V7 expression from clinical trial patient samples processed within 48 h—a much more feasible timeframe compared to previous recommendation

Choice of antibody is critical for specific and sensitive detection of androgen receptor splice variant-7 in circulating tumor cells

Androgen receptor variant 7 (AR-V7) is an important biomarker to guide treatment options for castration-resistant prostate cancer (CRPC) patients. Its detectability in circulating tumour cells (CTCs) opens non-invasive diagnostic avenues. While detectable at the transcript level, AR-V7 protein detection in CTCs may add additional information and clinical relevance. The aim of this study was to compare commercially available anti-AR-V7 antibodies and establish reliable AR-V7 immunocytostaining applicable to CTCs from prostate cancer (PCa) patients. We compared seven AR-V7 antibodies by western blotting and immmunocytostaining using a set of PCa cell lines with known AR/AR-V7 status. The emerging best antibody was validated for detection of CRPC patient CTCs enriched by negative depletion of leucocytes. The anti-AR-V7 antibody, clone E308L emerged as the best antibody in regard to signal to noise ratio with a specific nuclear signal. Moreover, this antibody detects CRPC CTCs more efficiently compared to an antibody previously shown to detect AR-V7 CTCs. We have determined the best antibody for AR-V7 detection of CTCs, which will open future studies to correlate AR-V7 subcellular localization and potential co-localization with other proteins and cellular structures to patient outcomes

Detection of AR-V7 in liquid biopsies of castrate resistant prostate cancer patients : a comparison of AR-V7 analysis in circulating tumor cells, circulating tumor RNA and exosomes

Detection of androgen receptor (AR) variant 7 (AR-V7) is emerging as a clinically important biomarker in castrate resistant prostate cancer (CRPC). Detection is possible from tumor tissue, which is often inaccessible in the advanced disease setting. With recent progress in detecting AR-V7 in circulating tumor cells (CTCs), circulating tumor RNA (ctRNA) and exosomes from prostate cancer patients, liquid biopsies have emerged as an alternative to tumor biopsy. Therefore, it is important to clarify whether these approaches differ in sensitivity in order to achieve the best possible biomarker characterization for the patient. In this study, blood samples from 44 prostate cancer patients were processed for CTCs and ctRNA with subsequent AR-V7 testing, while exosomal RNA was isolated from 16 samples and tested. Detection of AR and AR-V7 was performed using a highly sensitive droplet digital PCR-based assay. AR and AR-V7 RNA were detectable in CTCs, ctRNA and exosome samples. AR-V7 detection from CTCs showed higher sensitivity and has proven specificity compared to detection from ctRNA and exosomes. Considering that CTCs are almost always present in the advanced prostate cancer setting, CTC samples should be considered the liquid biopsy of choice for the detection of this clinically important biomarker

Patterns of immunotherapy use and management of toxicities in regional and tertiary settings

Background: The introduction of the cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) immune checkpoint inhibitors and their subsequent listing on the Pharmaceutical Benefits Scheme for use in metastatic melanomas, renal cell carcinomas and non-small-cell lung cancers has resulted in routine use of these agents in oncology practices, including in regional areas. Although immunotherapeutic agents generally have a favourable toxicity profile compared to chemotherapy, they can provoke immune-related adverse effects (irAE) caused by an unregulated and hyperstimulated immune response. Some of these effects can be serious and life-threatening. Aims: To compare the utilisation of immunotherapy and the rates, management and outcomes of irAE between a regional oncology service and a tertiary service. Methods: We reviewed the medical records for all patients treated with immunotherapy in the participating services for the 5-year period from 31 July 2012 to 31 July 2017. Results: Data demonstrated that rates of immunotherapy use are both similar and increasing across the tertiary and regional services. The rates, types and severity of irAE are equivalent and in concordance with pre-existing literature. Immune-related adverse events appear to be identified and treated earlier in the regional service with the corresponding reduction in the duration of immunosuppression and requirement for inpatient management. Conclusion: The use of immunotherapy in a regional setting is safe and equivalent to that of a tertiary centre

Review of toxicities of PARP inhibitors in metastatic castrate resistant prostate cancer

There is emerging evidence for the use of poly (ADP-ribose) polymerase inhibitors (PARPi) in patients with mCRPC with patients harboring germline or somatic mutations deriving clinical benefit. However, the toxicity profile of PARPi in mCRPC is not well established. In March 2022 a literature search was conducted across 4 databases – Medline, PubMed, Cochrane Library and Embase. In total, 14 relevant studies were identified cumulating in 2066 patients that were treated with PARPi. The overall ORR to PARPi alone or in combination with other therapy was 37% (246/666). In 5trials that investigated PARPi alone, the ORR was 39% (141/361). Treatment emergent adverse events (TEAEs) of any grade were reported in 96% (1034/1080) in PARPi treatment arms. TEAEs of grade >= 3 were reported in 57% (611/1080). 45% (457/1006) experienced treatment interruption whilst 31% (310/989) required dose reductions. 11% (114/1006) of patients had their treatment discontinued directly as the result of toxicity associated with the trial medications. The most common hematological toxicity was anemia, reported in 490/1160 (42%) patients. and lowered white blood cell count were the next 2most common toxicities, reported in 186/655 (28%) and 133/729 (18%) respectively. The 3most common non-hematological toxicities reported were nausea, fatigue and anorexia reported in 440/1013 (43%), 340/1013 (34%) and 274/1013 (27%) patients respectively. Overall, TRAEs associated with individual PARPi are still emerging with hematological toxicities being most apparent. Further toxicities will be informed from future clinical trials to allow improved treatment selection, education and management of toxicities in prostate cancer

Evidence for therapeutic drug monitoring of targeted anticancer therapies

Therapeutic drug monitoring (TDM) provides valuable guidance for dose adjustment of antibiotics, immunosuppressives, antiepileptics, and other drugs, but its use for traditional anticancer therapies has been limited. Perhaps the most important obstacle is the impractical requirement of multiple blood samples to adequately define systemic exposure of drugs that have a short elimination half-life and are given by intermittent intravenous injections. However, the newer targeted anticancer therapies have different pharmacokinetic (PK) and dosing characteristics compared with traditional cytotoxic drugs, making it possible to estimate the steady-state drug exposure with a single trough-level measurement. Recent evidence indicates that certain PK parameters, including trough levels, are correlated with clinical outcomes for many of these agents, including imatinib, sunitinib, rituximab, and cetuximab. Although the current evidence is insufficient to mandate TDM in routine practice, a concerted investigation should be encouraged to determine whether the steady-state trough measurements of targeted agents will have a practical place in the clinical care of patients with cancer.9 page(s

A Rare Case of Castrate-Resistant Prostate Adenocarcinoma with a Unilateral Testicular Metastasis Mimicking a Primary Testicular Tumour

Prostate adenocarcinoma with testicular metastasis is rare, present in up to 4% of autopsy diagnoses, and presents symptomatically in less than 0.5% of cases. We report an unusual case of a 55-year-old male who developed a symptomatic testicular metastasis from primary prostate cancer 4 years after initial diagnosis of metastatic castrate-sensitive prostate cancer with nodal and bone-only involvement. The patient had orchidectomy, histologically confirming the metastasis and revealing sparing of the spermatic cord. Prior treatment for his metastatic castrate-sensitive prostate cancer had included androgen deprivation therapy and upfront docetaxel chemotherapy. He had received palliative radiotherapy for symptomatic bone metastasis and managed on enzalutamide for castrate-resistant disease for the preceding 22 months with ongoing PSA response at the time of diagnosis of new testicular metastasis, with a further significant PSA response following his “testicular metastasectomy.” At the time of diagnosis of testicular metastasis, he did not have any evidence of other visceral metastases, and his metastatic disease otherwise remained radiologically stable. We describe his disease course, treatment and outline the rare nature of his case of testicular metastasis from prostate cancer

Endocrine Resistance in Breast Cancer: The Role of Estrogen Receptor Stability

Therapy of hormone receptor positive breast cancer (BCa) generally targets estrogen receptor (ER) function and signaling by reducing estrogen production or by blocking its interaction with the ER. Despite good long-term responses, resistance to treatment remains a significant issue, with approximately 40% of BCa patients developing resistance to ET. Mutations in the gene encoding ERα, ESR1, have been identified in BCa patients and are implicated as drivers of resistance and disease recurrence. Understanding the molecular consequences of these mutations on ER protein levels and its activity, which is tightly regulated, is vital. ER activity is in part controlled via its short protein half-life and therefore changes to its stability, either through mutations or alterations in pathways involved in protein stability, may play a role in therapy resistance. Understanding these connections and how ESR1 alterations could affect protein stability may identify novel biomarkers of resistance. This review explores the current reported data regarding posttranslational modifications (PTMs) of the ER and the potential impact of known resistance associated ESR1 mutations on ER regulation by affecting these PTMs in the context of ET resistance