Are CAR-T therapies living up to their hype? A study using real-world data in two cohorts to determine how well they are actually working in practice compared with bone marrow transplants.

With the increasing use of new regulatory tools, like the Food and Drug Administration's breakthrough designation, there are increasing challenges for European health technology assessors (HTAs) to make an accurate assessment of the long-term value and performance of chimeric antigen receptor T-cell (CAR-T) therapies, particularly for orphan conditions, such as acute lymphoblastic leukaemia. The aim of this study was to demonstrate a novel methodology harnessing longitudinal real-world data, extracted from the electronic health records of a medical centre functioning as a clinical trial site, to develop an accurate analysis of the performance of CAR-T compared with the next-best treatment option, namely allogeneic haematopoietic cell transplant (HCT). The study population comprised 43 subjects in two cohorts: 29 who had undergone HCT treatment and 14 who had undergone CAR-T therapy. The 3-year relapse-free survival probability was 46% (95% CI: 08% to 79%) in the CAR-T cohort and 68% (95% CI: 46% to 83%) in the HCT cohort. To explain the lower RFS probability in the CAR-T cohort compared with the HCT cohort, the authors hypothesised that the CAR-T cohort had a far higher level of disease burden. This was validated by log-rank test analysis (p=0.0001) and confirmed in conversations with practitioners at the study site. The authors are aware that the small populations in this study will be seen as limiting the generalisability of the findings to some readers. However, in consultation with many European HTAs and regulators, there is broad agreement that this methodology warrants further investigation with a larger study.

Drosophila as a Model for Understanding the Insect Host of Yersinia pestis.

With the limited availability of genomic sequence information and no established methods for genetic knockdowns or the creation of transgenic fleas and flea cell lines, we have adopted Drosophila melanogaster as a model for the study of the insect life cycle of Yersinia pestis. Infection of Drosophila larvae can be used to model early colonization of fleas, while the established embryonic cell lines can be used to model insect-pathogen interactions that underlie the unique capacity of Y. pestis to colonize the gut of its flea host. In this chapter, we present the methods we developed for infection of Drosophila in vivo and in vitro.

Prophylactic administration of avotermin for improvement of skin scarring: three double-blind, placebo-controlled, phase I/II studies.

BACKGROUND: Research into mechanisms of skin scarring identified transforming growth factor beta3 (TGFbeta3) as a potential antiscarring therapy. We assessed scar improvement with avotermin (recombinant, active, human TGFbeta3). METHODS: In three double-blind, placebo-controlled studies, intradermal avotermin (concentrations ranging from 0.25 to 500 ng/100 microL per linear cm wound margin) was administered to both margins of 1 cm, full-thickness skin incisions, before wounding and 24 h later, in healthy men and women. Treatments (avotermin and placebo or standard wound care) were randomly allocated to wound sites by a computer generated randomisation scheme, and within-participant controls compared avotermin versus placebo or standard wound care alone. Primary endpoints were visual assessment of scar formation at 6 months and 12 months after wounding in two studies, and from week 6 to month 7 after wounding in the third. Investigators, participants, and scar assessors were blinded to treatment. Efficacy analyses were intention to treat. These studies are registered with ClinicalTrials.gov, numbers NCT00847925, NCT00847795, and NCT00629811. RESULTS: In two studies, avotermin 50 ng/100 microL per linear cm significantly improved median score on a 100 mm visual analogue scale (VAS) by 5 mm (range -2 to 14; p=0.001) at month 6 and 8 mm (-29 to 18; p=0.0230) at month 12. In the third, avotermin significantly improved total scar scores at all concentrations versus placebo (mean improvement: from 14.84 mm [95 % CI 5.5-24.2] at 5 ng/100 microL per linear cm to 64.25 mm [49.4-79.1] at 500 ng/100 microL per linear cm). Nine [60%] scars treated with avotermin 50 ng/100 microL per linear cm showed 25% or less abnormal orientation of collagen fibres in the reticular dermis versus five [33%] placebo scars. After only 6 weeks from wounding, avotermin 500 ng/100 microL per linear cm improved VAS score by 16.12 mm (95% CI 10.61-21.63). Adverse events at wound sites were similar for avotermin and controls. Erythema and oedema were more frequent with avotermin than with placebo, but were transient and deemed to be consistent with normal wound healing. INTERPRETATION: Avotermin has potential to provide an accelerated and permanent improvement in scarring.

Visual analogue scale scoring and ranking: a suitable and sensitive method for assessing scar quality?

BACKGROUND: The field of scar assessment lacks a standard methodology. Previous methods have focused on a wide range of scar types, resulting in poorer sensitivity and diminishing their discriminatory effectiveness. METHODS: As part of a clinical trial investigating the scar-improving efficacy of transforming growth factor-beta3, the authors investigated the use of a visual analogue scale and scar ranking as scar assessment tools. Scar photographic images were assessed using a newly developed computerized scar assessment system by an external lay panel. RESULTS: A total of 4296 scar images were collected for visual analogue scale assessment and 2148 scar pairs were collected for scar ranking. Intrarater consistency was 100 percent for the ranking data, with differences very close to zero for the visual analogue scale consistency data. Reducing the number of assessors in the external panel significantly improved intraclass correlation coefficients. From month 1 to month 12, the correlation coefficients for the difference in visual analogue scale score showed that the assessors reliably noted the changes in the maturing scars. Combining logistic regression with an area under the curve of 0.72 in a receiver operating characteristic curve analysis, the visual analogue scale score was shown to be a highly statistically significant predictor of a good scar. CONCLUSIONS: The authors have shown the visual analogue scale scar scoring and scar ranking methods to be consistent, reliable, valid, and feasible. These methods for scar assessment are highly sensitive and capable of reliably measuring differences in scar quality, making them valuable techniques, reaching an unmet clinical need, and enabling investigation of changes in scar quality (e.g., with time or after therapeutic intervention).

Characterization of integrin beta6 and thrombospondin-1 double-null mice.

To identify overlapping and non-overlapping functions for TSP-1 and alphavbeta6, we crossed TSP-1-null and beta6-null mice and compared the phenotype of the double-null mice with those of wild-type and single-null mice. The double-null mice exhibited focal acute and organizing pneumonia that was more severe than the wild-type and single-null mice as well as a significantly higher incidence of inflammation in tissues other than the lung. The TSP-1-null and beta6-null mice exhibited a five to eight-fold increase in granulocyte recruitment to the lung three days after exposure to lipopolysaccharide. They also had abnormalities that were infrequently observed in the wild-type and single-null mice, including heart degeneration (8.35% in wild-type and 28.1% in double-null mice), hyperplasia of the glandular of the stomach (2.8% in wild-type and 21.1% in double-null mice) and endometrial hyperplasia (0% in wild-type and 38.5% in double-null females). Furthermore, the beta6-null and double-null mice displayed a significant elevation in benign and malignant cancers. Stomach papillomas, squamous cell carcinomas of the ear and stomach, and adenocarcinomas of the lungs, vagina/cervix and colon were observed with the highest frequency. These data demonstrate that TSP-1 and alphavbeta6 are involved in regulation of the immune system and epithelial homeostasis. They also indicate that alphavbeta6 functions as a tumor suppressor gene and that activation of TGFbeta by TSP-1 and alphavbeta6 contributes to normal tissue architecture and function.

Latent TGF-beta1 activation by platelets.

Platelets are a major source of transforming growth factor-beta1 (TGF-beta1) in the circulation as they release latent growth factor in response to activation. We report here that human platelets, when stimulated with thrombin, activated a significant proportion of the latent TGF-beta released. Latent TGF-beta activation was independent of cytokine release, since activation was delayed compared to platelet degranulation. Activation occured in releasates and did not require the continuous presence of platelets. Classical mechanisms of latent TGF-beta activation were not involved, since activation was not affected by gene deletion and/or inhibitors of the known TGF-beta activators/co-factors, thrombospondin-1 (TSP-1), mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF-IIR), plasminogen/plasmin, or several other candidate proteases. In contrast, latent TGF-beta activation was significantly inhibited by the furin inhibitors, decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone and L-hexaarginine. We show that platelets contain a furin-like enzyme which is released upon platelet activation. We conclude that, following activation, platelets release and activate latent TGF-beta1 via mechanisms involving the release and activity of a furin-like proprotein convertase. This novel mechanism of latent TGF-beta activation might represent an important mediator and therapeutic target of platelet TGF-beta1 functions, for example, in early wound repair, fibrosis, or arteriosclerosis.

Purification and analysis of thrombospondin-1.

Thromboapondin 1 (TSP-1) is a trimeric matricellular protein that is expressed by many cells. It contains several different domains that allow it to participate in cell adhesion, cell migration, and cell signaling. Recently TSP-1 has been shown to activate transforming growth factor beta (TGF-beta) and to inhibit both angiogenesis and tumor growth. This unit contains protocols for the purification of TSP-1 from platelet-rich plasma and the purification of TSP-1 proteolytic fragments.