Phenotype-based targeted treatment of SGLT2 inhibitors and GLP-1 receptor agonists in type 2 diabetes.

AIMS/HYPOTHESIS: A precision medicine approach in type 2 diabetes could enhance targeting specific glucose-lowering therapies to individual patients most likely to benefit. We aimed to use the recently developed Bayesian causal forest (BCF) method to develop and validate an individualised treatment selection algorithm for two major type 2 diabetes drug classes, sodium-glucose cotransporter 2 inhibitors (SGLT2i) and glucagon-like peptide-1 receptor agonists (GLP1-RA). METHODS: We designed a predictive algorithm using BCF to estimate individual-level conditional average treatment effects for 12-month glycaemic outcome (HbA1c) between SGLT2i and GLP1-RA, based on routine clinical features of 46,394 people with type 2 diabetes in primary care in England (Clinical Practice Research Datalink; 27,319 for model development, 19,075 for hold-out validation), with additional external validation in 2252 people with type 2 diabetes from Scotland (SCI-Diabetes [Tayside & Fife]). Differences in glycaemic outcome with GLP1-RA by sex seen in clinical data were replicated in clinical trial data (HARMONY programme: liraglutide [n=389] and albiglutide [n=1682]). As secondary outcomes, we evaluated the impacts of targeting therapy based on glycaemic response on weight change, tolerability and longer-term risk of new-onset microvascular complications, macrovascular complications and adverse kidney events. RESULTS: Model development identified marked heterogeneity in glycaemic response, with 4787 (17.5%) of the development cohort having a predicted HbA1c benefit >3 mmol/mol (>0.3%) with SGLT2i over GLP1-RA and 5551 (20.3%) having a predicted HbA1c benefit >3 mmol/mol with GLP1-RA over SGLT2i. Calibration was good in hold-back validation, and external validation in an independent Scottish dataset identified clear differences in glycaemic outcomes between those predicted to benefit from each therapy. Sex, with women markedly more responsive to GLP1-RA, was identified as a major treatment effect modifier in both the UK observational datasets and in clinical trial data: HARMONY-7 liraglutide (GLP1-RA): 4.4 mmol/mol (95% credible interval [95% CrI] 2.2, 6.3) (0.4% [95% CrI 0.2, 0.6]) greater response in women than men. Targeting the two therapies based on predicted glycaemic response was also associated with improvements in short-term tolerability and long-term risk of new-onset microvascular complications. CONCLUSIONS/INTERPRETATION: Precision medicine approaches can facilitate effective individualised treatment choice between SGLT2i and GLP1-RA therapies, and the use of routinely collected clinical features for treatment selection could support low-cost deployment in many countries.

Propagation of time-resolved fluorescence in a diffuse medium: complex analytical derivation.

The spatiotemporal evolution of fluorescence in an optically diffusive medium following ultrashort laser pulse excitation is evaluated using complex analytical methods. When expressed as a Fourier integral, the integrand of the time-resolved diffuse fluorescence with embedded fluorophores is shown to exhibit branch points and simple pole singularities in the lower-half complex-frequency plane. Applying Cauchy's integral theorem to solve the Fourier integral, we calculate the time-resolved signal for fluorescence lifetimes that are both shorter and longer compared to the intrinsic absorption timescale of the medium. These expressions are derived for sources and detectors that are in the form of localized points and wide-field harmonic spatial patterns. The accuracy of the expressions derived from complex analysis is validated against the numerically computed, full time-resolved fluorescence signal. The complex analysis shows that the branch points and simple poles contribute to two physically distinct terms in the net fluorescence signal. While the branch points result in a diffusive term that exhibits spatial broadening (corresponding to a narrowing with time in the spatial Fourier domain), the simple poles lead to fluorescence decay terms with spatial/spatial-frequency distributions that are independent of time. This distinct spatiotemporal behavior between the diffuse and fluorescence signals forms the basis for direct measurement of lifetimes shorter than the intrinsic optical diffusion timescales in a turbid medium.

Tomographic phosphorescence lifetime multiplexing.

We present a tomographic reconstruction algorithm for recovering distributions of multiple phosphorescent dyes within turbid media from time-resolved measurements, using either point or spatially patterned sources and detectors. The algorithm employs a multi-exponential analysis of time-resolved data, followed by tomographic inversion of the decay amplitudes to recover independent yield distributions for each lifetime present in the medium. Using Monte Carlo simulations, we computationally demonstrate that this two-step inversion approach provides several-fold improvement in quantitative and localization accuracy compared to a direct inversion of the time domain phosphorescence. We also demonstrate the tomographic reconstruction of up to three phosphorescent lifetimes embedded in thick tissue. The proposed algorithm can allow quantitative multiplexed tomography of luminescent and phosphorescent dyes for a wide range of in vivo applications.

Case-control study to assess the association between colorectal cancer and selected occupational agents using INTEROCC job exposure matrix.

BACKGROUND: Colorectal cancer is the third most prevalent cancer in the world and is twice as common in developed countries when compared with low-income and middle-income countries. Few occupational risk factors for colorectal cancer have been identified. This case-control study aimed to assess the association between colorectal cancer and occupational exposure to selected solvents, combustion products, metals, dusts and other agents. METHODS: Cases (n=918) were enrolled from the Western Australian Cancer Registry from June 2005 to August 2007. Controls (n=1021) were randomly selected from the Western Australian electoral roll. We collected lifetime occupational history from cases and controls, in addition to their demographic and lifestyle characteristics. We applied the INTEROCC job exposure matrix to convert the occupational history to occupational exposure for 18 selected agents. Three exposure indices were developed: (1) exposed versus non-exposed; (2) lifetime cumulative exposure; and (3) total duration of exposure. The associations between colorectal cancer and the selected agents were estimated using logistic regression models adjusting for sex and age. RESULTS: None of the 18 selected agents showed an association with colorectal cancer. No dose-response relationships with lifetime cumulative exposure or duration of exposure were observed. CONCLUSION: There was no evidence to suggest that occupational exposure to 18 selected agents increased the risk of colorectal cancer.

ENPP1 121Q functional variant enhances susceptibility to coronary artery disease in South Indian patients with type 2 diabetes mellitus.

Insulin resistance is associated with endothelial dysfunction and ensuing cardiovascular diseases in type 2 diabetes mellitus (T2DM) patients. ENPP1 is a key modulator of insulin signaling and its polymorphism, K121Q, increases the potency to competitively inhibit insulin receptor binding. We investigated the association of ENPP1 121Q variant with coronary artery disease (CAD) in patients with and without T2DM in South Indian population. Our study was conducted in 913 subjects: 198 patients with CAD, 284 patients in whom T2DM and CAD co-exists, 160 patients with T2DM and no CAD history, and 271 healthy volunteers. Genotyping was performed using PCR-RFLP and PCR-DNA sequencing. Genotype frequency of ENPP1 121Q was higher in disease groups compared to healthy subjects (p < 0.05). T2DM patients who carried polymorphic AC/CC genotypes were at 12.8-fold enhanced risk to CAD (95% CI 4.97-37.18, p < 0.01). Moreover we observed that 121Q, both in heterozygous and homozygous polymorphic states, was a risk factor for CAD without diabetes (OR 4.15, p < 0.01). 121Q variant was associated with T2DM patients with no CAD history too, but the risk was statistically insignificant after multivariate logistic regression analysis (OR 2.32, p > 0.05). We conclude that ENPP1 121Q variant is associated with increased risk for CAD in patients with T2DM in South Indian population. We also report that 121Q variant of ENPP1 was an independent risk factor for CAD irrespective of diabetic milieu. Factors which enhance insulin resistance increase the risk for onset and progression of coronary atherosclerosis irrespective of a diabetic background.

Comparison of tomographic fluorescence spectral and lifetime multiplexing.

Multispectral and lifetime imaging in turbid media can be mathematically described in two steps, involving spectral or temporal mixing of the fluorophores and the diffuse light transport in the turbid medium. We show that the order of fluorophore mixing and diffuse propagation is reversed in spectral and lifetime multiplexing, resulting in a fundamental difference in their multiplexing capabilities, regardless of the measurement conditions. Using the resolution matrix to define a quantitative measure for inter-fluorophore cross-talk, we show that lifetime multiplexing, using the asymptotic time domain approach, provides zero cross-talk, while spectral multiplexing can achieve zero cross-talk under special conditions. We also compare the performance of spectral and lifetime multiplexing for tomographic inversion of two overlapping fluorophores in a heterogeneous digital mouse atlas.

Optimal estimator for tomographic fluorescence lifetime multiplexing.

We use the model resolution matrix to analytically derive an optimal Bayesian estimator for multiparameter inverse problems that simultaneously minimizes inter-parameter cross talk and the total reconstruction error. Application of this estimator to time-domain diffuse fluorescence imaging shows that the optimal estimator for lifetime multiplexing is identical to a previously developed asymptotic time-domain (ATD) approach, except for the inclusion of a diagonal regularization term containing decay amplitude uncertainties. We show that, while the optimal estimator and ATD provide zero cross talk, the optimal estimator provides lower reconstruction error, while ATD results in superior relative quantitation. The framework presented here is generally applicable to other multiplexing problems where the simultaneous and accurate relative quantitation of multiple parameters is of interest.

Tomographic lifetime imaging using combined early- and late-arriving photons.

We present a novel, hybrid approach for time domain fluorescence tomography that efficiently combines lifetime multiplexing using late-arriving or asymptotic photons, with the high spatial resolution capability of early photon tomography. We also show that a decay amplitude-based asymptotic approach is superior to direct inversion of late-arriving photons for tomographic lifetime imaging within turbid media. The hybrid reconstruction approach is experimentally shown to recover fluorescent inclusions separated as close as 1.4 mm, with improved resolution and reduced cross talk compared to just using early photons or the asymptotic approach alone.

Miniaturized Fab' imaging probe derived from a clinical antibody: Characterization and imaging in CRISPRi-attenuated mammary tumor models.

Clinical imaging-assisted oncosurgical navigation requires cancer-specific miniaturized optical imaging probes. We report a near-infrared (NIR) Fab'-based epidermal growth factor receptor (EGFR)-specific probe carrying 3 NIR fluorophores (Fab'-800CW), which retained high-affinity binding to EGFR ectodomain (equilibrium KD E = 1 nM). Fab'-800CW showed a robust 4-times gain of fluorescence intensity (FI) and a 20% lifetime (FLT) increase under the conditions mimicking intracellular degradation. The probe was tested by using triple-negative breast cancer (TNBC) cell lines obtained by applying CRISPR interference (CRISPRi) effect of EGFR-targeting sgRNA and dCas9-KRAB chimera coexpression in MDA-MB-231 cells (WT cells). FI imaging in cell culture proved a 50% EGFR expression attenuation by CRISPRi. FI imaging in animals harboring attenuated or WT TNBC tumors with ex vivo corroboration identified differences between WT and CRISPRi tumors FI at 30 min post injection. Our results suggest the feasibility of EGFR expression imaging using a Fab'-based probe relevant for imaging-guided cancer surgery.

Analysis of type 2 diabetes heterogeneity with a tree-like representation: insights from the prospective German Diabetes Study and the LURIC cohort.

BACKGROUND: Heterogeneity in type 2 diabetes can be represented by a tree-like graph structure by use of reversed graph-embedded dimensionality reduction. We aimed to examine whether this approach can be used to stratify key pathophysiological components and diabetes-related complications during longitudinal follow-up of individuals with recent-onset type 2 diabetes. METHODS: For this cohort analysis, 927 participants aged 18-69 years from the German Diabetes Study (GDS) with recent-onset type 2 diabetes were mapped onto a previously developed two-dimensional tree based on nine simple clinical and laboratory variables, residualised for age and sex. Insulin sensitivity was assessed by a hyperinsulinaemic-euglycaemic clamp, insulin secretion was assessed by intravenous glucose tolerance test, hepatic lipid content was assessed by 1 H magnetic resonance spectroscopy, serum interleukin (IL)-6 and IL-18 were assessed by ELISA, and peripheral and autonomic neuropathy were assessed by functional and clinical measures. Participants were followed up for up to 16 years. We also investigated heart failure and all-cause mortality in 794 individuals with type 2 diabetes undergoing invasive coronary diagnostics from the Ludwigshafen Risk and Cardiovascular Health (LURIC) cohort. FINDINGS: There were gradients of clamp-measured insulin sensitivity (both dimensions: p<0·0001) and insulin secretion (pdim1<0·0001, pdim2=0·00097) across the tree. Individuals in the region with the lowest insulin sensitivity had the highest hepatic lipid content (n=205, pdim1<0·0001, pdim2=0·037), pro-inflammatory biomarkers (IL-6: n=348, pdim1<0·0001, pdim2=0·013; IL-18: n=350, pdim1<0·0001, pdim2=0·38), and elevated cardiovascular risk (nevents=143, pdim1=0·14, pdim2<0·00081), whereas individuals positioned in the branch with the lowest insulin secretion were more prone to require insulin therapy (nevents=85, pdim1=0·032, pdim2=0·12) and had the highest risk of diabetic sensorimotor polyneuropathy (nevents=184, pdim1=0·012, pdim2=0·044) and cardiac autonomic neuropathy (nevents=118, pdim1=0·0094, pdim2=0·06). In the LURIC cohort, all-cause mortality was highest in the tree branch showing insulin resistance (nevents=488, pdim1=0·12, pdim2=0·0032). Significant gradients differentiated individuals having heart failure with preserved ejection fraction from those who had heart failure with reduced ejection fraction. INTERPRETATION: These data define the pathophysiological underpinnings of the tree structure, which has the potential to stratify diabetes-related complications on the basis of routinely available variables and thereby expand the toolbox of precision diabetes diagnosis. FUNDING: German Diabetes Center, German Federal Ministry of Health, Ministry of Culture and Science of the state of North Rhine-Westphalia, German Federal Ministry of Education and Research, German Diabetes Association, German Center for Diabetes Research, European Community, German Research Foundation, and Schmutzler Stiftung.

Fluorescence lifetime detection in turbid media using spatial frequency domain filtering of time domain measurements.

It is demonstrated that high spatial frequency filtering of time domain fluorescence signals can allow efficient detection of intrinsic fluorescence lifetimes from turbid media and the rejection of diffuse excitation leakage. The basis of this approach is the separation of diffuse fluorescence signals into diffuse and fluorescent components with distinct spatiotemporal behavior.

Resolution below the point spread function for diffuse optical imaging using fluorescence lifetime multiplexing.

We show that asymptotic lifetime-based fluorescence tomography can localize multiple-lifetime targets separated well below the diffuse point spread function of a turbid medium. This is made possible due to a complete diagonalization of the time domain forward problem in the asymptotic limit. We also show that continuous wave or direct time gate approaches to fluorescence tomography are unable to achieve this separation, indicating the unique advantage of a decay-amplitude-based approach for tomographic lifetime multiplexing with time domain data.

In vivo quantification of programmed death-ligand-1 expression heterogeneity in tumors using fluorescence lifetime imaging.

Cancer patient selection for immunotherapy is often based on programmed death-ligand-1 (PD-L1) expression as a biomarker. PD-L1 expression is currently quantified using immunohistochemistry, which can only provide snapshots of PD-L1 expression status in microscopic regions of ex vivo specimens. In vivo imaging using targeted agents can capture dynamic variations of PD-L1 expression in entire tumors within and across multiple subjects. Towards this goal, several PD-L1 targeted molecular imaging probes have been evaluated in murine models and humans. However, clinical translation of these probes has been limited due to a significant non-specific accumulation of the imaging probes and the inability of conventional imaging modalities to provide quantitative readouts that can be compared across multiple subjects. Here we report that in vivo time-domain (TD) fluorescence imaging can provide quantitative estimates of baseline tumor PD-L1 heterogeneity across untreated mice and variations in PD-L1 expression across mice undergoing clinically relevant anti-PD1 treatment. This approach relies on a significantly longer fluorescence lifetime (FLT) of PD-L1 specific anti-PD-L1 antibody tagged to IRDye 800CW (αPDL1-800) compared to nonspecific αPDL1-800. Leveraging this unique FLT contrast, we show that PD-L1 expression can be quantified across mice both in superficial breast tumors using planar FLT imaging, and in deep-seated liver tumors (>5 mm depth) using the asymptotic TD algorithm for fluorescence tomography. Our results suggest that FLT contrast can accelerate the preclinical investigation and clinical translation of novel molecular imaging probes by providing robust quantitative readouts of receptor expression that can be readily compared across subjects.

Near-infrared fluorescence lifetime imaging of amyloid-ß aggregates and tau fibrils through the intact skull of mice.

Non-invasive methods for the in vivo detection of hallmarks of Alzheimer's disease can facilitate the study of the progression of the disease in mouse models and may enable its earlier diagnosis in humans. Here we show that the zwitterionic heptamethine fluorophore ZW800-1C, which has peak excitation and emission wavelengths in the near-infrared optical window, binds in vivo and at high contrast to amyloid-ß deposits and to neurofibrillary tangles, and allows for the microscopic imaging of amyloid-ß and tau aggregates through the intact skull of mice. In transgenic mouse models of Alzheimer's disease, we compare the performance of ZW800-1C with that of the two spectrally similar heptamethine fluorophores ZW800-1A and indocyanine green, and show that ZW800-1C undergoes a longer fluorescence-lifetime shift when bound to amyloid-ß and tau aggregates than when circulating in blood vessels. ZW800-1C may prove advantageous for tracking the proteinic aggregates in rodent models of amyloid-ß and tau pathologies.

Fluorescence lifetime of injected indocyanine green as a universal marker of solid tumours in patients.

The surgical resection of solid tumours can be enhanced by fluorescence-guided imaging. However, variable tumour uptake and incomplete clearance of fluorescent dyes reduces the accuracy of distinguishing tumour from normal tissue via conventional fluorescence intensity-based imaging. Here we show that, after systemic injection of the near-infrared dye indocyanine green in patients with various types of solid tumour, the fluorescence lifetime (FLT) of tumour tissue is longer than the FLT of non-cancerous tissue. This tumour-specific shift in FLT can be used to distinguish tumours from normal tissue with an accuracy of over 97% across tumour types, and can be visualized at the cellular level using microscopy and in larger specimens through wide-field imaging. Unlike fluorescence intensity, which depends on imaging-system parameters, tissue depth and the amount of dye taken up by tumours, FLT is a photophysical property that is largely independent of these factors. FLT imaging with indocyanine green may improve the accuracy of cancer surgeries.

Intraoperative molecular imaging: 3rd biennial clinical trials update.

Significance: This third biennial intraoperative molecular imaging (IMI) conference shows how optical contrast agents have been applied to develop clinically significant endpoints that improve precision cancer surgery. Aim: National and international experts on IMI presented ongoing clinical trials in cancer surgery and preclinical work. Previously known dyes (with broader applications), new dyes, novel nonfluorescence-based imaging techniques, pediatric dyes, and normal tissue dyes were discussed. Approach: Principal investigators presenting at the Perelman School of Medicine Abramson Cancer Center's third clinical trials update on IMI were selected to discuss their clinical trials and endpoints. Results: Dyes that are FDA-approved or currently under clinical investigation in phase 1, 2, and 3 trials were discussed. Sections on how to move benchwork research to the bedside were also included. There was also a dedicated section for pediatric dyes and nonfluorescence-based dyes that have been newly developed. Conclusions: IMI is a valuable adjunct in precision cancer surgery and has broad applications in multiple subspecialties. It has been reliably used to alter the surgical course of patients and in clinical decision making. There remain gaps in the utilization of IMI in certain subspecialties and potential for developing newer and improved dyes and imaging techniques.

The PEG-fluorochrome shielding approach for targeted probe design.

We provide a new approach for fluorescent probe design termed "PEG-fluorochrome shielding", where PEGylation enhances quantum yields while blocking troublesome interactions between fluorochromes and biomolecules. To demonstrate PEG-fluorochrome shielding, fluorochrome-bearing peptide probes were synthesized, three without PEG and three with a 5 kDa PEG functional group. In vitro, PEG blocked the interactions of fluorochrome-labeled peptide probes with each other (absorption spectra, self-quenching) and reduced nonspecific interactions with cells (by FACS). In vivo, PEG blocked interactions with biomolecules that lead to probe retention (by surface fluorescence). Integrin targeting in vivo was obtained as the differential uptake of an (111)In-labeled, fluorochrome-shielded, integrin-binding RGD probe and a control RAD. Using PEG to block fluorochrome-mediated interactions, rather than synthesizing de novo fluorochromes, can yield new approaches for the design of actively or passively targeted near-infrared fluorescent probes.

Direct Monte Carlo computation of time-resolved fluorescence in heterogeneous turbid media.

We show that a multiexponential model for time-resolved fluorescence allows the use of an absorption-perturbation Monte Carlo (MC) approach based on stored photon path histories. This enables the rapid fitting of fluorescence yield, lifetimes, and background tissue absorptions in complex heterogeneous media within a few seconds, without the need for temporal convolutions or MC recalculation of photon path lengths. We validate this method using simulations with both a slab and a heterogeneous model of the mouse head.

Erratum: Comparison of fluorescence lifetime and multispectral imaging for quantitative multiplexing in biological tissue: erratum.

[This corrects the article on p. 3854 in vol. 13, PMID: 35991924.].