Germline intragenic mutations in PTEN are associated with 80% of patients with Cowden syndrome (CS) and 60% of patients with Bannayan-Riley-Ruvalcaba syndrome (BRRS). The underlying genetic causes remain to be determined in a considerable proportion of classic CS and BRRS without a polymerase chain reaction (PCR)-detectable PTEN mutation. We hypothesized that gross gene deletions and mutations in the PTEN promoter might alternatively account for a subset of apparently mutation-negative patients with CS and BRRS. Using real time and multiplex PCR techniques, we identified three germline hemizygous PTEN deletions in 122 apparently mutation-negative patients with classic CS (N=95) or BRRS (N=27). Fine mapping suggested that one deletion encompassed the whole gene and the other two included exon 1 and encompassed exons 1-5 of PTEN, respectively. Two patients with the deletion were diagnosed with BRRS, and one patient with the deletion was diagnosed with BRRS/CS overlap (features of both). Thus 3 (11%) of 27 patients with BRRS or BRRS/CS-overlap had PTEN deletions. Analysis of the PTEN promoter revealed nine cases (7.4%) harboring heterozygous germline mutations. All nine had classic CS, representing almost 10% of all subjects with CS. Eight had breast cancers and/or benign breast tumors but, otherwise, oligo-organ involvement. PTEN protein analysis, from one deletion-positive and five PTEN-promoter-mutation-positive samples, revealed a 50% reduction in protein and multiple bands of immunoreactive protein, respectively. In contrast, control samples showed only the expected band. Further, an elevated level of phosphorylated Akt was detected in the five promoter-mutation-positive samples, compared with controls, indicating an absence of or marked reduction in functional PTEN. These data suggest that patients with BRRS and CS without PCR-detected intragenic PTEN mutations be offered clinical deletion analysis and promoter-mutation analysis, respectively.

Tumour-induced dysfunction of cytotoxic T cells in patients with multiple myeloma (MM) may contribute to immune escape and be responsible for the lack of therapeutic efficacy of immune checkpoint blockade. We therefore investigated dysfunctional clonal T cells in MM and demonstrated immunosenescence but not exhaustion as a predominant feature. T-cell clones were detected in 75% of MM patients and their prognostic significance was revalidated in a new post-immunomodulatory drug cohort. The cells exhibited a senescent secretory effector phenotype: KLRG-1+/CD57+/CD160+/CD28-. Normal-for-age telomere lengths indicate that senescence is telomere independent and potentially reversible. p38-mitogen-activated protein kinase, p16 and p21 signalling pathways known to induce senescence were not elevated. Telomerase activity was found to be elevated and this may explain how normal telomere lengths are maintained in senescent cells. T-cell receptor signalling checkpoints were normal but elevated SMAD levels associated with T-cell inactivation were detected and may provide a potential target for the reversal of clonal T-cell dysfunction in MM. Low programmed death 1 and cytotoxic T-lymphocyte-associated antigen 4 expression detected on T-cell clones infers that these cells are not exhausted but suggests that there would be a suboptimal response to immune checkpoint blockade in MM. Our data suggest that other immunostimulatory strategies are required in MM.

Regulation of the PI-3 kinase (PI3K)/Akt signalling pathway is essential for maintaining the integrity of fundamental cellular processes, cell growth, survival, death and metabolism, and dysregulation of this pathway is implicated in the development and progression of cancers. Receptor tyrosine kinases (RTKs) are major upstream regulators of PI3K/Akt signalling. The phosphatase and tensin homologue (PTEN), a well characterised tumour suppressor, is a prime antagonist of PI3K and therefore a negative regulator of this pathway. Loss or inactivation of PTEN, which occurs in many tumour types, leads to overactivation of RTK/PI3K/Akt signalling driving tumourigenesis. Cellular PTEN levels are tightly regulated by a number of transcriptional, post-transcriptional and post-translational regulatory mechanisms. Of particular interest, transcription of the PTEN pseudogene, PTENP1, produces sense and antisense transcripts that exhibit post-transcriptional and transcriptional modulation of PTEN expression respectively. These additional levels of regulatory complexity governing PTEN expression add to the overall intricacies of the regulation of RTK/PI-3 K/Akt signalling. This review will discuss the regulation of oncogenic PI3K signalling by PTEN (the regulator) with a focus on the modulatory effects of the sense and antisense transcripts of PTENP1 on PTEN expression, and will further explore the potential for new therapeutic opportunities in cancer treatment.

Aims/hypothesis Type 1 diabetes results from the autoimmune destruction of pancreatic beta cells. Exogenous insulin therapy cannot achieve precise physiological control of blood glucose concentrations, and debilitating complications develop. Lentiviral vectors are promising tools for liver-directed gene therapy. However, to date, transduction rates in vivo remain low in hepatocytes, without the induction of cell cycling. We investigated long-term transgene expression in quiescent hepatocytes in vitro and determined whether the lentiviral delivery of furin-cleavable insulin to the liver could reverse diabetes in rats. Materials and methods To improve transduction efficiency in vitro, we optimised hepatocyte isolation and maintenance protocols and, using an improved surgical delivery method, delivered furin-cleavable insulin alone or empty vector to the livers of streptozotocin-induced diabetic rats by means of a lentiviral vector. Rats were monitored for changes in body weight and blood glucose, and intravenous glucose tolerance tests were performed. Expression of insulin was determined by RT-PCR, immunohistochemistry and electron microscopy. Results We achieved long-term transgene expression in quiescent hepatocytes in vitro (87±1.2% transduction efficiency), with up to 60±3.2% transduction in vivo. We normalised blood glucose for 500 days-a significantly longer period than previously reported-making this the first successful study using a lentiviral vector. This procedure resulted in the expression of genes encoding several beta cell transcription factors, some pancreatic endocrine transdifferentiation, hepatic insulin storage in granules, and restoration of glucose tolerance. Liver function tests remained normal. Importantly, pancreatic exocrine transdifferentiation did not occur. Conclusions/interpretation Our data suggest that this regimen may ultimately be employed for the treatment of type 1 diabetes.