Precision-guided treatment in high-risk pediatric cancers.

Recent research showed that precision medicine can identify new treatment strategies for patients with childhood cancers. However, it is unclear which patients will benefit most from precision-guided treatment (PGT). Here we report consecutive data from 384 patients with high-risk pediatric cancer (with an expected cure rate of less than 30%) who had at least 18 months of follow-up on the ZERO Childhood Cancer Precision Medicine Program PRecISion Medicine for Children with Cancer (PRISM) trial. A total of 256 (67%) patients received PGT recommendations and 110 (29%) received a recommended treatment. PGT resulted in a 36% objective response rate and improved 2-year progression-free survival compared with standard of care (26% versus 12%; P = 0.049) or targeted agents not guided by molecular findings (26% versus 5.2%; P = 0.003). PGT based on tier 1 evidence, PGT targeting fusions or commenced before disease progression had the greatest clinical benefit. Our data show that PGT informed by comprehensive molecular profiling significantly improves outcomes for children with high-risk cancers. ClinicalTrials.gov registration: NCT03336931.

Bartonella henselae masquerading as possible gamma-delta T-cell lymphoma in a paediatric patient with 22q11.2 deletion syndrome.

A 14-year-old boy with 22q11.2 deletion syndrome and a right ventricular to pulmonary artery xenograft conduit presented to an Australian tertiary children's hospital with prolonged fevers, weight loss, splenomegaly and a high proportion of gamma-delta T cells in peripheral blood and bone marrow, concerning for possible gamma-delta T-cell lymphoma. However, investigations did not reveal evidence of lymphoma or autoimmune disease. After 5 months of intermittent fever episodes and ongoing symptoms, he was found to have an extremely high Bartonella henselae titre (8192) on serological testing, with the organism also detected on blood PCR. After 6 months of oral azithromycin and rifampicin, with complete resolution of his symptoms 3 months into treatment, his blood PCR was negative and gamma-delta T cells in peripheral blood were decreasing. The B. henselae titre remained unchanged for some time, but decreased to 2048 around 1 year after treatment was started.

Elucidation and refinement of synthetic receptor mechanisms.

Synthetic receptors are powerful tools for engineering mammalian cell-based devices. These biosensors enable cell-based therapies to perform complex tasks such as regulating therapeutic gene expression in response to sensing physiological cues. Although multiple synthetic receptor systems now exist, many aspects of receptor performance are poorly understood. In general, it would be useful to understand how receptor design choices influence performance characteristics. In this study, we examined the modular extracellular sensor architecture (MESA) and systematically evaluated previously unexamined design choices, yielding substantially improved receptors. A key finding that might extend to other receptor systems is that the choice of transmembrane domain (TMD) is important for generating high-performing receptors. To provide mechanistic insights, we adopted and employed a Förster resonance energy transfer-based assay to elucidate how TMDs affect receptor complex formation and connected these observations to functional performance. To build further insight into these phenomena, we developed a library of new MESA receptors that sense an expanded set of ligands. Based upon these explorations, we conclude that TMDs affect signaling primarily by modulating intracellular domain geometry. Finally, to guide the design of future receptors, we propose general principles for linking design choices to biophysical mechanisms and performance characteristics.

Chloramphenicol acetyltransferase as a selection marker for chlamydial transformation.

BACKGROUND: Chlamydia is a common bacterial pathogen responsible for many diseases. Methods for transforming this important organism using a ß-lactamase as a selection marker have been developed very recently. However, the National Institutes of Health Guidelines for Research Involving Recombinant DNA Molecules do not permit transformation experiments with ß-lactamase gene-containing vectors for certain human chlamydial pathogens. Therefore, a different selection marker is urgently needed for transformation of those chlamydiae. RESULTS: After transformation of plasmid-free Chlamydia trachomatis with pGFP:SW2, which carries a ß-lactamase and a chloramphenicol acetyltransferase gene fused to a green fluorescence protein gene, transformants were obtained by selection with either ampicillin or chloramphenicol. Stable chloramphenicol-resistant, but ampicillin-sensitive, transformants were obtained using a pGFP:SW2 derivative without the ß-lactamase. All transformants expressed green fluorescence protein and had glycogen synthesis activity restored. CONCLUSIONS: Chloramphenicol resistance may be used as a selection marker for genetic experiments in Chlamydia. This eliminates the requirement for the use of ß-lactamase, of which dissemination to some C. trachomatis serovars may jeopardize clinical treatment of chlamydial infections in pregnant women. Chloramphenicol acetyltransferase may also serve as a useful secondary selection marker for genetic analyses in ß-lactamase-transformed chlamydial strains.

Cellular and molecular requirements for rejection of B16 melanoma in the setting of regulatory T cell depletion and homeostatic proliferation.

We have recently demonstrated that adoptive transfer of regulatory T cell-depleted polyclonal T cells into lymphopenic mice leads to rejection of B16 melanoma, which generated an opportunity to study host requirements for tumor rejection when it effectively occurred. CD8(+) T cell priming and tumor rejection required tumor Ag cross-presentation, as evidenced by tumor outgrowth in Kb(-/-) bone marrow chimeric or B71/2(-/-) mice. CD4(+) T cells were additionally required for optimal tumor control, although not through classical CD4 "help," as the frequency of primed CD8(+) T cells was similar in the absence of CD4(+) T cells, and tumor rejection did not depend upon CD40-CD40L interactions or on IL-2 production by CD4(+) T cells. Rather, CD4(+) T cells appeared to act at the effector phase of tumor rejection and responded to B16-derived Ags in vitro. At the effector phase, IFN-γ production by transferred T cells, but not host cells, was necessary. IFN-γ acted either on host or tumor cells and was associated with reduced tumor vascularity. Finally, tumor rejection occurred after transfer of TNF-α, perforin, or FasL-deficient T cells. However, perforin/FasL double-knockout T cells failed to reject, arguing that the killing of B16 melanoma cells could occur either via the cytotoxic granule or Fas pathways. Collectively, these results support a model in which host tumor Ag cross-presentation primes adoptively transferred T cells, which remain functional in the setting of homeostatic proliferation and regulatory T cell depletion, and which promote tumor rejection via IFN-γ and lysis via cytotoxic granules and/or FasL.