Clinical Utility of Circulating Tumour Cell Androgen Receptor Splice Variant-7 Status in Metastatic Castration-resistant Prostate Cancer.

BACKGROUND: Detection of androgen receptor splice variant-7 (AR-V7) mRNA in circulating tumour cells (CTCs) is associated with worse outcome in metastatic castration-resistant prostate cancer (mCRPC). However, studies rarely report comparisons with CTC counts and biopsy AR-V7 protein expression. OBJECTIVE: To determine the reproducibility of AdnaTest CTC AR-V7 testing, and associations with clinical characteristics, CellSearch CTC counts, tumour biopsy AR-V7 protein expression and overall survival (OS). DESIGN, SETTING, AND PARTICIPANTS: CTC AR-V7 status was determined for 227 peripheral blood samples, from 181 mCRPC patients with CTC counts (202 samples; 136 patients) and matched mCRPC biopsies (65 samples; 58 patients). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: CTC AR-V7 status was associated with clinical characteristics, CTC counts, and tissue biopsy AR-V7 protein expression. The association of CTC AR-V7 status and other baseline variables with OS was determined. RESULTS AND LIMITATIONS: Of the samples, 35% were CTC+/AR-V7+. CTC+/AR-V7+ samples had higher CellSearch CTC counts (median CTC; interquartile range [IQR]: 60, 19-184 vs 9, 2-64; Mann-Whitney test p<0.001) and biopsy AR-V7 protein expression (median H-score, IQR: 100, 63-148 vs 15, 0-113; Mann-Whitney test p=0.004) than CTC+/AR-V7- samples. However, both CTC- (63%) and CTC+/AR-V7- (62%) patients had detectable AR-V7 protein in contemporaneous biopsies. After accounting for baseline characteristics, there was shorter OS in CTC+/AR-V7+ patients than in CTC- patients (hazard ratio [HR] 2.13; 95% confidence interval [CI] 1.23-3.71; p=0.02); surprisingly, there was no evidence that CTC+/AR-V7+ patients had worse OS than CTC+/AR-V7- patients (HR 1.26; 95% CI 0.73-2.17; p=0.4). A limitation of this study was the heterogeneity of treatment received. CONCLUSIONS: Studies reporting the prognostic relevance of CTC AR-V7 status must account for CTC counts. Discordant CTC AR-V7 results and AR-V7 protein expression in matched, same-patient biopsies are reported. PATIENT SUMMARY: Liquid biopsies that determine circulating tumour cell androgen receptor splice variant-7 status have the potential to impact treatment decisions in metastatic castration-resistant prostate cancer patients. Robust clinical qualification of these assays is required before their routine use.

A study of PD-L1 expression in KRAS mutant non-small cell lung cancer cell lines exposed to relevant targeted treatments.

We investigated PD-L1 changes in response to MEK and AKT inhibitors in KRAS mutant lung adenocarcinoma (adeno-NSCLC). PD-L1 expression was quantified using immunofluorescence and co-culture with a jurkat cell-line transfected with NFAT-luciferase was used to study if changes in PD-L1 expression in cancer cell lines were functionally relevant. Five KRAS mutant cell lines with high PD-L1 expression (H441, H2291, H23, H2030 and A549) were exposed to GI50 inhibitor concentrations of a MEK inhibitor (trametinib) and an AKT inhibitor (AZD5363) for 3 weeks. Only 3/5 (H23, H2030 and A549) and 2/5 cell lines (H441 and H23) showed functionally significant increases in PD-L1 expression when exposed to trametinib or AZD5363 respectively. PD-L1 overexpression is not consistent and is unlikely to be an early mechanism of resistance to KRAS mutant adeno-NSCLC treated with MEK or AKT inhibitors.

Molecular and in vivo characterization of cancer-propagating cells derived from MYCN-dependent medulloblastoma.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. While the pathways that are deregulated in MB remain to be fully characterized, amplification and/or overexpression of the MYCN gene, which is has a critical role in cerebellar development as a regulator of neural progenitor cell fate, has been identified in several MB subgroups. Phenotypically, aberrant expression of MYCN is associated with the large-cell/anaplastic MB variant, which accounts for 5-15% of cases and is associated with aggressive disease and poor clinical outcome. To better understand the role of MYCN in MB in vitro and in vivo and to aid the development of MYCN-targeted therapeutics we established tumor-derived neurosphere cell lines from the GTML (Glt1-tTA/TRE-MYCN-Luc) genetically engineered mouse model. A fraction of GTML neurospheres were found to be growth factor independent, expressed CD133 (a marker of neural stem cells), failed to differentiate upon MYCN withdrawal and were highly tumorigenic when orthotopically implanted into the cerebellum. Principal component analyzes using single cell RNA assay data suggested that the clinical candidate aurora-A kinase inhibitor MLN8237 converts GTML neurospheres to resemble non-MYCN expressors. Correlating with this, MLN8237 significantly extended the survival of mice bearing GTML MB allografts. In summary, our results demonstrate that MYCN plays a critical role in expansion and survival of aggressive MB-propagating cells, and establish GTML neurospheres as an important resource for the development of novel therapeutic strategies.

Combined MYC and P53 defects emerge at medulloblastoma relapse and define rapidly progressive, therapeutically targetable disease.

We undertook a comprehensive clinical and biological investigation of serial medulloblastoma biopsies obtained at diagnosis and relapse. Combined MYC family amplifications and P53 pathway defects commonly emerged at relapse, and all patients in this group died of rapidly progressive disease postrelapse. To study this interaction, we investigated a transgenic model of MYCN-driven medulloblastoma and found spontaneous development of Trp53 inactivating mutations. Abrogation of p53 function in this model produced aggressive tumors that mimicked characteristics of relapsed human tumors with combined P53-MYC dysfunction. Restoration of p53 activity and genetic and therapeutic suppression of MYCN all reduced tumor growth and prolonged survival. Our findings identify P53-MYC interactions at medulloblastoma relapse as biomarkers of clinically aggressive disease that may be targeted therapeutically.

The ALK(F1174L) mutation potentiates the oncogenic activity of MYCN in neuroblastoma.

The ALK(F1174L) mutation is associated with intrinsic and acquired resistance to crizotinib and cosegregates with MYCN in neuroblastoma. In this study, we generated a mouse model overexpressing ALK(F1174L) in the neural crest. Compared to ALK(F1174L) and MYCN alone, co-expression of these two oncogenes led to the development of neuroblastomas with earlier onset, higher penetrance, and enhanced lethality. ALK(F1174L)/MYCN tumors exhibited increased MYCN dosage due to ALK(F1174L)-induced activation of the PI3K/AKT/mTOR and MAPK pathways, coupled with suppression of MYCN pro-apoptotic effects. Combined treatment with the ATP-competitive mTOR inhibitor Torin2 overcame the resistance of ALK(F1174L)/MYCN tumors to crizotinib. Our findings demonstrate a pathogenic role for ALK(F1174L) in neuroblastomas overexpressing MYCN and suggest a strategy for improving targeted therapy for ALK-positive neuroblastoma.

Angiotensin type 1 receptor immunoreactivity in the thoracic spinal cord.

The angiotensin II type 1 receptor (AT1R) in the central nervous system (CNS) plays a pivotal role in determining blood pressure. However, the relationship of the receptor to neurones in the spinal cord which are the final CNS contribution to sympathetic outflow is unknown. Here we first use RT-PCR to show that AT1A, AT1B and AT2 receptors are expressed in thoracic spinal cord of the rat. Using light microscopic immunohistochemistry we find that the AT1 receptor in the thoracic spinal cord is located on neurones and ependymal cells. Neurones with extensive immunostaining of somata and dendrites were located in the intermediolateral cell column (IML) and lamina X (the central autonomic area), regions associated with autonomic outflow, as well as in lamina V. Retrograde labelling and dual immunolabelling with nNOS revealed that those AT1R-immunopositive cells in the IML were sympathetic preganglionic neurones, while those in lamina X were unlikely to be. Punctate labelling resembling that of axonal fibres and terminals was evident in lamina II of the dorsal horn and throughout the cord. Electron microscopy in the IML and lamina X revealed that these puncta were presynaptic terminals, but also astrocyte processes. Immunolabelling was also evident beneath the plasma membrane in neuronal somata. These data show that the AT1R in the spinal cord is ideally located to influence autonomic outflow and hence participate in the CNS determination of blood pressure.