Probes for INS

 BACKGROUND:
 Expression of the colony-stimulating factor 1 (CSF1) gene is elevated in most tenosynovial giant-cell tumors. This observation has led to the discovery and clinical development of therapy targeting the CSF1 receptor (CSF1R).
 METHODS:
 Using x-ray co-crystallography to guide our drug-discovery research, we generated a potent, selective CSF1R inhibitor, PLX3397, that traps the kinase in the autoinhibited conformation. We then conducted a multicenter, phase 1 trial in two parts to analyze this compound. In the first part, we evaluated escalations in the dose of PLX3397 that was administered orally in patients with solid tumors (dose-escalation study). In the second part, we evaluated PLX3397 at the chosen phase 2 dose in an extension cohort of patients with tenosynovial giant-cell tumors (extension study). Pharmacokinetic and tumor responses in the enrolled patients were assessed, and CSF1 in situ hybridization was performed to confirm the mechanism of action of PLX3397 and that the pattern of CSF1 expression was consistent with the pathological features of tenosynovial giant-cell tumor.
 RESULTS:
 A total of 41 patients were enrolled in the dose-escalation study, and an additional 23 patients were enrolled in the extension study. The chosen phase 2 dose of PLX3397 was 1000 mg per day. In the extension study, 12 patients with tenosynovial giant-cell tumors had a partial response and 7 patients had stable disease. Responses usually occurred within the first 4 months of treatment, and the median duration of response exceeded 8 months. The most common adverse events included fatigue, change in hair color, nausea, dysgeusia, and periorbital edema; adverse events rarely led to discontinuation of treatment.
 CONCLUSIONS:
 Treatment of tenosynovial giant-cell tumors with PLX3397 resulted in a prolonged regression in tumor volume in most patients. (Funded by Plexxikon; ClinicalTrials.gov number, NCT01004861.).

Prostate cancer (PCa) does not appear to respond to immune checkpoint therapies where T cell infiltration may be a key limiting factor. Here we report evidence that ablating the growth regulatory kinase Erk5 can increase T cell infiltration in an established Pten-deficient mouse model of human PCa. Mice that were doubly mutant in prostate tissue for Pten and Erk5 (prostate DKO) exhibited a markedly increased median survival with reduced tumor size and proliferation compared to control Pten-mutant mice, the latter of which exhibited increased Erk5 mRNA expression. A comparative transcriptomic analysis revealed upregulation in prostate DKO mice of the chemokines Ccl5 and Cxcl10, two potent chemoattractants for T lymphocytes. Consistent with this effect, we observed a relative increase in a predominantly CD4+ T cell infiltrate in the prostate epithelial and stroma of tumors from DKO mice. Collectively, our results offer a preclinical proof of concept for ERK5 as a target to enhance T cell infiltrates in prostate cancer, with possible implications for leveraging immune therapy in this disease.

Abstract

INTRODUCTION:

Localized- and diffuse-type tenosynovial giant cell tumours (TGCT) are regarded different clinical and radiological TGCT-types. However, genetically and histopathologically they seem indistinguishable. We aimed to correlate CSF1-expression and CSF1-rearrangement with the biological behaviour of different TGCT-types with clinical outcome (recurrence).

METHODS:

Along a continuum of extremes, therapy naïve knee TGCT patients with >3 year follow-up, mean age 43(range 6-71)years and 56% female were selected. Nine localized-(two recurrences), 16 diffuse-type(nine recurrences) and four synovitis as control were included. Rearrangement of the CSF1-locus was evaluated with split-apart Fluorescence In Situ Hybridization (FISH) probes. Regions were selected to score after identifying CSF1-expressing regions, using mRNA ISH with the help of digital correlative microscopy. CSF1-rearrangement was considered positive in samples containing >2 split signals/100 nuclei.

RESULTS:

Irrespective of TGCT-subtype, all cases showed CSF1-expression and in 76% CSF1-rearrangement was detected. Quantification of CSF1-expressing cells was not informative, due to the extensive intra tumour heterogeneity. Of the four synovitis cases, two also showed CSF1-expression, without CSF1-rearrangement. No correlation between CSF1-expression or rearrangement with clinical subtype and local recurrence was detected. Both localized- and diffuse-TGCT cases showed a scattered distribution in the tissue of CSF1-expressing cells.

CONCLUSION:

In diagnosing TGCT, CSF1 mRNA-ISH in combination with CSF1 split-apart FISH; using digital correlative microscopy, is an auxiliary diagnostic tool to identify rarely occurring neoplastic cells. This combined approach allowed us to detect CSF1-rearrangement in 76% of the TGCT-cases. Neither CSF1-expression nor presence of CSF1-rearrangement could be associated with the difference in biological behaviour of TGCT. 

To build comprehensive models of cellular states and interactions in normal and diseased tissue, genetic and proteomic information must be extracted with single-cell and spatial resolution. Here, we extended imaging mass cytometry to enable multiplexed detection of mRNA and proteins in tissues. Three mRNA target species were detected by RNAscope-based metal in situ hybridization with simultaneous antibody detection of 16 proteins. Analysis of 70 breast cancer samples showed that HER2 and CK19 mRNA and protein levels are moderately correlated on the single-cell level, but that only HER2, and not CK19, has strong mRNA-to-protein correlation on the cell population level. The chemoattractant CXCL10 was expressed in stromal cell clusters, and the frequency of CXCL10-expressing cells correlated with T cell presence. Our flexible and expandable method will allow an increase in the information content retrieved from patient samples for biomedical purposes, enable detailed studies of tumor biology, and serve as a tool to bridge comprehensive genomic and proteomic tissue analysis.