BACKGROUND:Loss-of-function FLG mutation (FLGmut) carriers are at an increased risk of developing atopic dermatitis (AD), characterized by earlier onset and more severe disease. AD is driven by a complex interplay between skin barrier function, TH2 and TH2-dominant immune dysregulation, and dysbiosis. Results from the Short-Term Topical Application for Prevention of Atopic Dermatitis study suggest 2 early initiating AD pathogenetic pathways: an FLGmut-related skin barrier deficiency pathway and an immune function-related inflammatory pathway. The Short-Term Topical Application for Prevention of Atopic Dermatitis study suggested that early preventative intervention with specialized emollients for barrier function augmentation may benefit newborns with FLGmut. This requires early identification of FLGmut carriers, for which noninvasive Raman spectroscopic determination of natural moisturizing factor (NMF) levels in the stratum corneum of the thenar eminence provides a surrogate marker. OBJECTIVE:To identify strategies for early identification of infants with FLGmut. METHODS:FLG sequencing was performed on 253 infants, and NMF concentrations were measured in the stratum corneum of the palmar eminence (pSC-NMF) using noninvasive Raman spectroscopy at 6 time points after birth. Furthermore, the pSC-NMF concentrations were obtained from both parents of 150 infants. RESULTS:Babies are born with little to no NMF. In the first days after birth, NMF levels rapidly increase and 65% of newborns with FLG wild type already reach pSC-NMF concentrations, which excludes them as FLGmut carriers with high specificity. At 2 weeks of age, FLGmut carriers could be distinguished from newborns with FLG wild type with high sensitivity (97%) and specificity (97%). In addition, parent pSC-NMF concentrations offer the possibility to exclude their newborn as FLGmut carriers with high specificity. CONCLUSION:Noninvasive Raman spectroscopy enables the accurate early identification of infants with FLGmut.

We demonstrate a hybrid system combining Raman spectroscopy and partial wave spectroscopy, improving tissue classification accuracy and detecting variation in tissues from an intestinal tumorigenesis mouse model, showcasing the system's potential for field cancerization studies. (c) 2025 The Author(s)

Field cancerization (FC) refers to spatially distributed premalignant tissue changes that lead to the appearance of local malignancy, and its detection can improve cancer screening. In this work, we employ combined Raman and partial wave spectroscopy (RS-PWS) to detect FC in gastroesophageal (L2-IL1B) and intestinal (Villin-Cre, Apcfl/wt) tumor mouse models. Using a hybrid RS-PWS microscope, we acquire both molecular and morphological information from macroscopically normal tumor-adjacent tissue and investigate the individual and combined performance of each modality. For data analysis, we use partial least-squares discriminant analysis (PLS-DA). In the normal tissue of L2-IL1B mice, we demonstrate a statistically significant increase (p < 0.001) in Raman band intensities associated with free amino acids and a decrease in bands associated with lipids (p < 0.005) and carotenoids (p < 0.001) compared to healthy controls. Similarly, in the normal mucosa of Villin-Cre, Apcfl/wt mice, the intensities of RS bands associated with amino acids increase significantly (p < 0.05) compared to controls, while the intensities of lipid-associated bands decrease significantly (p < 0.05). Transcriptomic profiling using RNA-sequencing analysis on these samples identified a significant correlation between gene expression and optical findings. Moreover, we demonstrate that combining RS and PWS data further improves the significance of our classification results. When macroscopically normal tumor-adjacent tissue is compared with tissue from healthy controls, we observe that PWS increases the R2 of RS results by ∼9% in L2-IL1B mice and ∼5% in Villin-Cre, Apcfl/wt mice. Combining molecular RS with structural PWS information enhances the ability to detect precancerous changes and provides insights into tissue alterations during cancer development.

We have developed a method to determine the limit of detection (LoD) for quantitative measurement of exogenous analytes in the outer layer of the human skin by in vivo confocal Raman spectroscopy. The method is in accordance with the guidelines of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use that have been adopted by regulatory authorities such as the American Food and Drug Administration and the European Medicines Agency. The method can be applied in silico so that the limit of detection can be assessed before starting a skin penetration study, for example, in areas of pharmaceutical formulation, pharmacokinetics, or toxicokinetics. This can significantly reduce the need for expensive and time-consuming feasibility studies. This paper describes the method to calculate this LoD as well as the experimental and methodological factors that can influence the calculation of the LoD.

We present a hybrid Raman spectroscopy (RS) and partial wave spectroscopy (PWS) microscope for the characterization of molecular and structural tissue alterations. The PWS performance was assessed with surface roughness standards, while the Raman performance with a silicon crystal standard. We also validated the system on stomach and intestinal mouse tissues, two closely-related tissue types, and demonstrate that the addition of PWS information improves RS data classification for these tissue types from R2 = 0.892 to R2 = 0.964 (norm of residuals 0.863 and 0.497, respectively). Then, in a proof-of-concept experiment, we show that the hybrid system can detect changes in intestinal tissues harvested from a tumorigenic Villin-Cre, Apcfl/wt mouse. We discuss how the hybrid modality offers new abilities to identify the relative roles of PWS morphological features and Raman molecular fingerprints, possibly allowing for their combination to enhance the study of carcinogenesis and early cancer diagnostics in the future.

We report on the development of a dedicated Raman spectroscopic system employing a thin fiber optic needle for rapid determination of tumor resection margins on fresh intact specimens. This information will enable the surgeon to excise additional tissue if and where needed for complete tumor removal with adequate margins.

Loss-of-function (LoF) filaggrin (FLG)-mutations are the strongest genetic risk factor for atopic dermatitis (AD). The STOP-AD study showed early initiation of daily specialized emollient, used only until 2 months, reduces the incidence of AD in the first year of life in high-risk infants. 321 newborns were recruited, randomized 1:1 between intervention group (IG), receiving emollient treatment and control group (CG). FLG status was determined by Microfluidics PCR for full coverage of FLG repeat alleles. 257 infants with genotyping completed the study, 119 in IG and 138 in CG. 12 month cumulative AD-incidence was 46% in the CG vs. 33% in the IG (p=0.03). 44 (17.4%, evenly split between groups) were LOF-FLG mutation carriers, who had significantly higher AD-prevalence of 56% and 59% at 6 and 12 months respectively in the CG, compared to 32% at 6m (p =0.02) and 34 at 12m % (p=0.01) in WT-FLG. In IG group the LOF-FLG babies had AD rates nearly the same as WT-FLG infants at 6 and 12m: 19% and 14% respectively compared to 15% (p=NS)and 14% (p=NS) in WT-FLG babies. Daily specialized emollient use until 2 months may be especially beneficial for LoF FLG-mutation carriers, reducing their AD-prevalence and incidence to the level of the treated FLG-wildtype group. Identification of FLG status soon after birth may be beneficial in deciding which child may benefit most from targeted preventive strategies.

Summary of cutaneous adverse events at the different time point for each patient included in this study. n/a: not available

Loss-of-function filaggrin (LoF-FLG)-mutation carriers are at higher risk of early, more severe Atopic Dermatitis (AD) than wild type (WT-FLG) infants . We present a strategy for early discrimination between WT-FLG and LoF-FLG-mutation carriers, based on Raman spectroscopic analysis of Natural Moisturizer Factor (NMF) content in the stratum corneum of the thenar eminence. The single center STOP-AD study recruited 321 newborns, 260 completed the study. NMF-content was measured, once <4 days after birth, at 2, 4, 8, 26 and 52 weeks. FLG-mutation status was determined by microfluidics PCR on genomic DNA for full FLG-coverage. NMF-analysis was performed for both parents of 134 newborns. Parental NMF-content > 0.35 [gNMF/gprotein], predicted WT-FLG newborns with 100% specificity and 72% sensitivity. Newborn NMF-content > 0.38 [gNMF/gprotein] at 0-4 days predicted WT-FLG with 100% specificity and 43%-sensitivity. Combined analysis of parental and newborn NMF-content predicted WT-FLG with 100% specificity and 87% sensitivity. At 2 weeks NMF-content >0.38 [gNMF/g protein] predicted LoF-FLG-mutation carriers with 100% sensitivity and 92% specificity. Parental NMF and newborn NMF-measurement at 0-4 days could inform families that their newborn child was in a relatively lower risk group for development of AD and could be used to exclude 87% of WT-FLG newborns from any preventive treatment being proposed. Families of the remaining group of newborns, containing 100% of LOF-FLG mutation carriers, could be informed of potentially increased risk of AD and could be offered potential preventive treatment. Repeat NMF-analysis at 2 weeks could exclude almost all remaining FLG-wildtypes from further proposed preventive treatment

A clear margin is an important prognostic factor for most solid tumours treated by surgery. Intraoperative fluorescence imaging using exogenous tumour-specific fluorescent agents has shown particular benefit in improving complete resection of tumour tissue. However, signal processing for fluorescence imaging is complex, and fluorescence signal intensity does not always perfectly correlate with tumour location. Raman spectroscopy has the capacity to accurately differentiate between malignant and healthy tissue based on their molecular composition. In Raman spectroscopy, specificity is uniquely high, but signal intensity is weak and Raman measurements are mainly performed in a point-wise manner on microscopic tissue volumes, making whole-field assessment temporally unfeasible. In this review, we describe the state-of-the-art of both optical techniques, paying special attention to the combined intraoperative application of fluorescence imaging and Raman spectroscopy in current clinical research. We demonstrate how these techniques are complementary and address the technical challenges that have traditionally led them to be considered mutually exclusive for clinical implementation. Finally, we present a novel strategy that exploits the optimal characteristics of both modalities to facilitate resection with clear surgical margins.

Oral potentially malignant disorders (OPMD) may precede oral squamous cell carcinoma (OSCC). Reported rates of malignant transformation of OPMD range from 3 to 50%. While some clinical, histological, and molecular factors have been associated with a high-risk OPMD, they are, to date, insufficiently accurate for treatment decision-making. Moreover, this range highlights differences in the clinical definition of OPMD, variation in follow-up periods, and molecular and biological heterogeneity of OPMD. Finally, while treatment of OPMD may improve outcome, standard therapy has been shown to be ineffective to prevent OSCC development in patients with OPMD. In this perspective paper, several experts discuss the main challenges in oral cancer prevention, in particular the need to (i) to define an OPMD classification system by integrating new pathological and molecular characteristics, aiming (ii) to better identify OPMD at high risk of malignant transformation, and (iii) to develop treatment strategies to eradicate OPMD or prevent malignant transformation.

Confocal Raman spectroscopy (CRS) enables the real-time profiling of substances penetrating to the skin without sample pre-treatment or labelling. Until now, CRS had been used as a semi-quantitative method, which posed challenges for evaluating topical formulations and assessing bioequivalence. We present a novel approach of CRS for quantitative analysis of skin delivery. The quantitative CRS in vivo has been correlated with the well-established in vitro Franz-diffusion experiments, indicating the potential of CRS for determining skin delivery. We anticipate CRS providing a rapid and non-invasive method that will be an attractive alternative to the clinical studies currently used in bioequivalence testing.

For vulvar squamous cell carcinoma (VSCC), the mainstay of treatment is surgical removal with tumour-free margins. Surgeons still operate without objective tools that provide margin-status. This study assesses Raman spectroscopy potentiality for distinguishing ex-vivo VSCC from healthy tissue in 11 patients. Grid-based Raman maps were obtained from processed spectra. Water content and C-H band ratio (2,910-2,966 cm -1 / 2810-2890 cm -1 ) were calculated per spectrum and used as linear discriminant parameters. Healthy tissue was differentiated from VSCC with 0.90 discriminative power, 0.79 sensitivity and 0.86 specificity.This is an important step towards the development of objective tools for VSCC surgical guidance.

We present the first clinical integration of a prototype device based on integrated auto-fluorescence imaging and Raman spectroscopy (Fast Raman device) for intra-operative assessment of surgical margins during Mohs micrographic surgery of basal cell carcinoma (BCC). Fresh skin specimens from 112 patients were used to optimise the tissue pre-processing and the Fast Raman algorithms to enable an analysis of complete Mohs layers within 30 minutes. The optimisation allowed >95% of the resection surface area to be investigated (including the deep and epidermal margins). The Fast Raman device was then used to analyse skin layers excised from the most relevant anatomical sites (nose, temple, eyelid, cheek, forehead, eyebrow and lip) and to detect the three main types of BCC (nodular, superficial and infiltrative). These results suggest that the Fast Raman technique is a promising tool to provide an objective diagnosis “tumour clear yes/no” during Mohs surgery of BCC. This clinical integration study is a key step towards a larger scale diagnosis test accuracy study to reliably determine the sensitivity and specificity in a clinical setting.

This study reports on the effects of insertion velocity, needle tip geometry and needle diameter on tissue deformation and maximum insertion force. Moreover, the effect of multiple insertions with the same needle on the maximum insertion force is reported. The tissue deformation and maximum insertion force strongly depend on the insertion velocity and the tip geometry. No correlation was found between the outer diameter and the maximum insertion force for small needles (30G - 32G). The endurance experiments showed no remarkable difference in the maximum insertion force during 100 insertions.