Early initiation of short-term emollient use for the prevention of atopic dermatitis in high-risk infants-The STOP-AD randomised controlled trial.

BACKGROUND: Protecting the skin barrier in early infancy may prevent atopic dermatitis (AD). We investigated if daily emollient use from birth to 2 months reduced AD incidence in high-risk infants at 12 months. METHODS: This was a single-center, two-armed, investigator-blinded, randomized controlled clinical trial (NCT03871998). Term infants identified as high risk for AD (parental history of AD, asthma or allergic rhinitis) were recruited within 4 days of birth and randomised 1:1 to either twice-daily emollient application for the first 8 weeks of life (intervention group), using an emollient specifically formulated for very dry, AD-prone skin, or to standard routine skin care (control group). The primary outcome was cumulative AD incidence at 12 months. AD <6 months was diagnosed based on clinical presence of AD. The UK Working Party Diagnostic Criteria were applied when diagnosing AD between 6 and 12 months. RESULTS: Three hundred twenty-one were randomised (161 intervention and 160 control), with 61 withdrawals (41 intervention, 20 control). The cumulative incidence of AD at 12 months was 32.8% in the intervention group vs. 46.4% in the control group, p = 0.036 [Relative risk (95%CI): 0.707 (0.516, 0.965)]. One infant in the intervention group was withdrawn from the study following development of a rash that had a potential relationship with the emollient. There was no significant difference in the incidence of skin infections between the intervention and control groups during the intervention period (5.0% vs. 5.7%, p > 0.05). CONCLUSIONS: This study has demonstrated that early initiation of daily specialized emollient use until 2 months reduces the incidence of AD in the first year of life in high-risk infants.

Development of a near-infrared Raman spectroscopy setup compatible with fluorescence-guided surgery.

Near-infrared (NIR) fluorescence imaging using exogenous fluorescent agents provides whole-field images in real-time to assist the surgeon in the excision of a tumor. Although the method has high sensitivity, the specificity can sometimes be lower than expected. Raman spectroscopy can detect tumors with high specificity. Therefore, a combination of both techniques can be advantageous. A complication that must be addressed is that the NIR spectral region is favored by both techniques for (in vivo) tissue analysis. When fluorescence and Raman emissions spectrally overlap, it becomes challenging or impossible to detect the Raman signal. In this paper, by avoiding this overlap, we describe a Raman spectroscopy setup capable of recording high-quality Raman spectra from tissue containing NIR exogenous fluorescent agents. We identify an optimal wavelength interval (900-915 nm) for Raman excitation, which avoids both excitation of fluorescent dyes and Raman signal self-absorption by the tissue. In this way, Raman spectroscopy can be combined with the currently most-used NIR fluorescent dyes. This combined novel setup could pave the way for clinical trials benefiting from both fluorescence imaging and Raman spectroscopy to avoid positive margins in cancer surgery.

Intraoperative assessment of resection margins by Raman spectroscopy to guide oral cancer surgery.

Patients with oral cavity cancer are almost always treated with surgery. The goal is to remove the tumor with a margin of more than 5 mm of surrounding healthy tissue. Unfortunately, this is only achieved in about 15% to 26% of cases. Intraoperative assessment of tumor resection margins (IOARM) can dramatically improve surgical results. However, current methods are laborious, subjective, and logistically demanding. This hinders broad adoption of IOARM, to the detriment of patients. Here we present the development and validation of a high-wavenumber Raman spectroscopic technology, for quick and objective intraoperative measurement of resection margins on fresh specimens. It employs a thin fiber-optic needle probe, which is inserted into the tissue, to measure the distance between a resection surface and the tumor. A tissue classification model was developed to discriminate oral cavity squamous cell carcinoma (OCSCC) from healthy oral tissue, with a sensitivity of 0.85 and a specificity of 0.92. The tissue classification model was then used to develop a margin length prediction model, showing a mean difference between margin length predicted by Raman spectroscopy and histopathology of -0.17 mm.

Specimen-driven intraoperative assessment of resection margins should be standard of care for oral cancer patients.

With an incidence of 350.000 new cases per year, cancer of the oral cavity ranks among the 10 most common solid organ cancers. Most of these cancers are squamous cell carcinomas. Five-year survival is about 50%. It has been shown that clear resection margins (>5 mm healthy tissue surrounding the resected tumor) have a significant positive effect on locoregional control and survival. It is not uncommon that the resection margins of oral tumors are inadequate. However, when providing the surgeon with intraoperative feedback on the resection margin status, it is expected that obtaining adequate resection margins is improved. In this respect, it has been shown that specimen-driven intraoperative assessment of resection margins is superior to defect-driven intraoperative assessment of resection margins. In this concise report, it is described how a specimen-driven approach can increase the rate of adequate resections of oral cavity squamous cell carcinoma as well as that it is discussed how intraoperative assessment can be further improved with regard to the surgical treatment of oral cavity squamous cell carcinoma.

In vivo Raman spectroscopy discriminates between FLG loss-of-function carriers vs wild-type in day 1-4 neonates.

BACKGROUND: Carriers of loss-of-function mutations in the filaggrin gene (LoF FLG) have less natural moisturizing factor (NMF) in their stratum corneum (SC) and an increased risk of atopic dermatitis (AD). Natural moisturizing factor can be measured noninvasively by Raman spectroscopy. The use of Raman-derived NMF at birth to screen for FLG genotype could inform targeted AD prevention, but values in neonatal populations are largely unexplored. OBJECTIVE: To examine the associations between Raman-derived neonatal NMF measurements and FLG genotype. METHODS: Natural moisturizing factor was measured by Raman spectroscopy in the SC of the thenar eminence within 4 days of birth in 139 term neonates. Filaggrin genotyping was performed for 117 neonates (84%). RESULTS: The mean (SD) NMF was 0.37 (0.11) g/g protein, with values increasing across the first 3 days (day 1 vs 3: 0.29 [0.09] vs 0.43 [0.08, P < .001]). Twelve infants (10.3%) were carriers of LoF FLG, all heterozygous. Natural moisturizing factor was lower in LoF FLG carriers compared with wild-type (0.27 [0.08] vs 0.38 [0.11] g/g protein, P ≤ .001). Natural moisturizing factor had good discriminatory power for FLG genotype (area under the receiver operating curve [AUROC]: 0.79; 95% CI: 0.66, 0.91; P ≤ .001). This improved after correcting day 1 and 2 measurements to day 3 (AUROC: 0.83; 95% CI: 0.75, 0.92; P < .001). CONCLUSION: This study suggests that Raman-derived NMF measured in the early postnatal period may have the potential to classify by FLG genotype. The full translational value of this needs to be determined.

Improving clinical diagnosis of early-stage cutaneous melanoma based on Raman spectroscopy.

BACKGROUND: Clinical diagnosis of early melanoma (Breslow thickness less than 0.8 mm) is crucial to disease-free survival. However, it is subjective and can be exceedingly difficult, leading to missed melanomas, or unnecessary excision of benign pigmented skin lesions. An objective technique is needed to improve the diagnosis of early melanoma. METHODS: We have developed a method to improve diagnosis of (thin) melanoma, based on Raman spectroscopy. In an ex vivo study in a tertiary referral (pigmented lesions) centre, high-wavenumber Raman spectra were collected from 174 freshly excised melanocytic lesions suspicious for melanoma. Measurements were performed on multiple locations within the lesions. A diagnostic model was developed and validated on an independent data set of 96 lesions. RESULTS: Approximately 60% of the melanomas included in this study were melanomas in situ. The invasive melanomas had an average Breslow thickness of 0.89 mm. The diagnostic model correctly classified all melanomas (including in situ) with a specificity of 43.8%, and showed a potential improvement of the number needed to treat from 6.0 to 2.7, at a sensitivity of 100%. CONCLUSION: This work signifies an important step towards accurate and objective clinical diagnosis of melanoma and in particular melanoma with Breslow thickness <0.8 mm.

Raman spectroscopy for cancer detection and cancer surgery guidance: translation to the clinics.

Oncological applications of Raman spectroscopy have been contemplated, pursued, and developed at academic level for at least 25 years. Published studies aim to detect pre-malignant lesions, detect cancer in less invasive stages, reduce the number of unnecessary biopsies and guide surgery towards the complete removal of the tumour with adequate tumour resection margins. This review summarizes actual clinical needs in oncology that can be addressed by spontaneous Raman spectroscopy and it provides an overview over the results that have been published between 2007 and 2017. An analysis is made of the current status of translation of these results into clinical practice. Despite many promising results, most of the applications addressed in scientific studies are still far from clinical adoption and commercialization. The main hurdles are identified, which need to be overcome to ensure that in the near future we will see the first Raman spectroscopy-based solutions being used in routine oncologic diagnostic and surgical procedures.

Sensitive Skin: Assessment of the Skin Barrier Using Confocal Raman Microspectroscopy.

BACKGROUND/AIMS: Sensitive skin (SS), a frequently reported condition in the Western world, has been suggested to be underlined by an impaired skin barrier. The aim of this study was to investigate the skin barrier molecular composition in SS subjects using confocal Raman microspectroscopy (CRS), and to compare it with that of non-SS (NSS) individuals as well as atopic dermatitis (AD) and allergic rhinoconjunctivitis (AR) subjects, who frequently report SS. METHODS: Subjects with SS (n = 29), NSS (n = 30), AD (n = 11), and AR (n = 27) were included. Stratum corneum (SC) thickness, water, ceramides/fatty acids, and natural moisturizing factor (NMF) were measured by CRS along with transepidermal water loss and capacitance on the ventral forearm, thenar, and cheek. Sebum levels were additionally measured on the forearm and cheek. RESULTS: No differences between SS and NSS subjects were found regarding SC thickness, water, and NMF content, yet a trend towards lower ceramides/fatty acids was observed in the cheek. Compared to AD subjects, the SS group showed higher ceramides/fatty acid content in the forearm, whereas no differences emerged with AR. The correlation of macroscopic biophysical techniques and CRS was weak, yet CRS confirmed the well-known lower content of NMF and water, and thinner SC in subjects with filaggrin mutations. CONCLUSION: The skin barrier in SS is not impaired in terms of SC thickness, water, NMF, and ceramides/fatty acid content. The failure of biophysical techniques to follow alterations in the molecular composition of the skin barrier revealed by CRS emphasizes a strong need in sensitive and specific tools for in vivo skin barrier analysis.

Raman Spectroscopic Characterization of Melanoma and Benign Melanocytic Lesions Suspected of Melanoma Using High-Wavenumber Raman Spectroscopy.

Melanoma is a pigmented type of skin cancer, which has the highest mortality of all skin cancers. Because of the low clinical diagnostic accuracy for melanoma, an objective tool is needed to assist clinical assessment of skin lesions that are suspected of (early) melanoma. The aim of this study was to identify spectral differences in the CH region of HWVN (high-wavenumber) Raman spectra between melanoma and benign melanocytic lesions clinically suspected of melanoma. We used these spectral differences to explore preliminary classification models to distinguish melanoma from benign melanocytic lesions. Data from 82 freshly excised melanocytic lesions clinically suspected of melanoma were measured using an in-house built Raman spectrometer, which has been optimized for measurements on pigmented skin lesions (excitation wavelength 976 nm and a wavelength range of the Raman signal 1340-1540 nm). Clear spectral differences were observed between melanoma and benign melanocytic lesions. These differences can be assigned mainly to the symmetric CH2 stretching vibrations of lipids. Our results show that the Raman bands between 2840 and 2930 cm(-1) have increased intensity for melanoma when compared to benign melanocytic lesions, suggesting an increase in lipid content in melanoma. These results demonstrate that spectroscopic information in the CH-stretching region of HWVN Raman spectra can discriminate melanoma from benign melanocytic lesions that are often clinically misdiagnosed as melanoma and that Raman spectroscopy has the potential to provide an objective clinical tool to improve the clinical diagnostic accuracy of skin lesions suspected of melanoma.

Real-time detection of p-phenylenediamine penetration into human skin by in vivo Raman spectroscopy.

BACKGROUND: Penetration, autoxidation and N-acetylation of p-phenylenediamine (PPD) have been studied in vitro and ex vivo. However, a clear understanding of in vivo PPD penetration and the formation of PPD derivatives is lacking. OBJECTIVES: To obtain insights into the in vivo penetration, clearance and formation of PPD derivatives in human skin. METHODS: Patch test chambers containing PPD 1% pet. were applied to the forearms of two human volunteers, with increasing application times. Non-invasive Raman microspectroscopy was used for detection of PPD (derivatives) in skin at several follow-up times. RESULTS: Application of a PPD 1% pet. patch for 30 min resulted in substantial amounts of PPD in the stratum corneum of 90 mg PPD/g keratin. PPD contents were highest after three applications for 1 h each (330 mg PPD/g keratin), followed by single applications for 2 h 40 min, 2 h, and 23 h. The PPD half-time in the skin was 3 h. No spectral contributions of Bandrowski's base, monoacetyl-PPD and diacetyl-PPD were detected. CONCLUSIONS: We have gained insights into the in vivo penetration of PPD in human skin by using non-invasive Raman spectroscopy. Penetration into the skin was fast, and the PPD concentrations detected in the stratum corneum were high. PPD was detected in both the stratum corneum and the viable epidermis. Oxidized or acetylated PPD derivatives could not be detected.

Investigation of the potential of Raman spectroscopy for oral cancer detection in surgical margins.

The poor prognosis of oral cavity squamous cell carcinoma (OCSCC) patients is associated with residual tumor after surgery. Raman spectroscopy has the potential to provide an objective intra-operative evaluation of the surgical margins. Our aim was to understand the discriminatory basis of Raman spectroscopy at a histological level. In total, 127 pseudo-color Raman images were generated from unstained thin tissue sections of 25 samples (11 OCSCC and 14 healthy) of 10 patients. These images were clearly linked to the histopathological evaluation of the same sections after hematoxylin and eosin-staining. In this way, Raman spectra were annotated as OCSCC or as a surrounding healthy tissue structure (i.e., squamous epithelium, connective tissue (CT), adipose tissue, muscle, gland, or nerve). These annotated spectra were used as input for linear discriminant analysis (LDA) models to discriminate between OCSCC spectra and healthy tissue spectra. A database was acquired with 88 spectra of OCSCC and 632 spectra of healthy tissue. The LDA models could distinguish OCSCC spectra from the spectra of adipose tissue, nerve, muscle, gland, CT, and squamous epithelium in 100%, 100%, 97%, 94%, 93%, and 75% of the cases, respectively. More specifically, the structures that were most often confused with OCSCC were dysplastic epithelium, basal layers of epithelium, inflammation- and capillary-rich CT, and connective and glandular tissue close to OCSCC. Our study shows how well Raman spectroscopy enables discrimination between OCSCC and surrounding healthy tissue structures. This knowledge supports the development of robust and reliable classification algorithms for future implementation of Raman spectroscopy in clinical practice.

Spectropathology for the next generation: quo vadis?

Although the potential of vibrational spectroscopy for biomedical applications has been well demonstrated, translation into clinical practice has been relatively slow. This Editorial assesses the challenges facing the field and the potential way forward. While many technological challenges have been addressed to date, considerable effort is still required to gain acceptance of the techniques among the medical community, standardise protocols, extend to a clinically relevant scale, and ultimately assess the health economics underlying clinical deployment. National and international research networks can contribute much to technology development and standardisation. Ultimately, large-scale funding is required to engage in clinical trials and instrument development.

Limits of Detection of Topically Applied Products in the Skin Using In Vivo Raman Spectroscopy.

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.

Filaggrin loss-of-function mutations are associated with enhanced expression of IL-1 cytokines in the stratum corneum of patients with atopic dermatitis and in a murine model of filaggrin deficiency.

BACKGROUND: Filaggrin (FLG) mutations result in reduced stratum corneum (SC) natural moisturizing factor (NMF) components and consequent increased SC pH. Because higher pH activates SC protease activity, we hypothesized an enhanced release of proinflammatory IL-1 cytokines from corneocytes in patients with atopic dermatitis (AD) with FLG mutations (AD(FLG)) compared with that seen in patients with AD without these mutations (AD(NON-FLG)). OBJECTIVES: We sought to investigate SC IL-1 cytokine profiles in the uninvolved skin of controls and patients with AD(FLG) versus patients with AD(NON-FLG). We also sought to examine the same profiles in a murine model of filaggrin deficiency (Flg(ft)/Flg(ft) [Flg(delAPfal)] mice). METHODS: One hundred thirty-seven patients were studied. NMF levels were ascertained using confocal Raman spectroscopy; transepidermal water loss and skin surface pH were measured. IL-1α, IL-1ß, IL-18, IL-1 receptor antagonist (IL-1RA), and IL-8 levels were determined in SC tape strips from 93 patients. All subjects were screened for 9 FLG mutations. Flg(ft)/Flg(ft) (Flg(delAPfal)) mice, separated from maFlg(ft)/maFlg(ft) (flaky tail) mice, were used for the preparation and culture of primary murine keratinocytes and as a source of murine skin. RT-PCR was performed using primers specific for murine IL-1α, IL-1ß, and IL-1RA. RESULTS: SC IL-1 levels were increased in patients with AD(FLG); these levels were inversely correlated with NMF levels. NMF values were also inversely correlated with skin surface pH. Skin and keratinocytes from Flg(ft)/Flg(ft) mice had upregulated expression of IL-1ß and IL-1RA mRNA. CONCLUSIONS: AD(FLG) is associated with an increased SC IL-1 cytokine profile; this profile is also seen in a murine homologue of filaggrin deficiency. These findings might have importance in understanding the influence of FLG mutations on the inflammasome in the pathogenesis of AD and help individualize therapeutic approaches.

Unmet Needs and Perspectives in Oral Cancer Prevention.

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.

Raman profiles of the stratum corneum define 3 filaggrin genotype-determined atopic dermatitis endophenotypes.

BACKGROUND: Filaggrin (FLG) has a central role in the pathogenesis of atopic dermatitis (AD). FLG is a complex repetitive gene; highly population-specific mutations and multiple rare mutations make routine genotyping complex. Furthermore, the mechanistic pathways through which mutations in FLG predispose to AD are unclear. OBJECTIVES: We sought to determine whether specific Raman microspectroscopic natural moisturizing factor (NMF) signatures of the stratum corneum could be used as markers of FLG genotype in patients with moderate-to-severe AD. METHODS: The composition and function of the stratum corneum in 132 well-characterized patients with moderate-to-severe AD were assessed by means of confocal Raman microspectroscopy and measurement of transepidermal water loss (TEWL). These parameters were compared with FLG genotype and clinical assessment. RESULTS: Three subpopulations closely corresponding with FLG genotype were identified by using Raman spectroscopy. The Raman signature of NMF discriminated between FLG-associated AD and non-FLG-associated AD (area under the curve, 0.94; 95% CI, 0.91-0.99). In addition, within the subset of FLG-associated AD, NMF distinguished between patients with 1 versus 2 mutations. Five novel FLG mutations were found on rescreening outlying patients with Raman signatures suggestive of undetected mutations (R3418X, G1138X, S1040X, 10085delC, and L2933X). TEWL did not associate with FLG genotype subgroups. CONCLUSIONS: Raman spectroscopy permits rapid and highly accurate stratification of FLG-associated AD. FLG mutations do not influence TEWL within established moderate-to-severe AD.

In Vitro-In Vivo Correlation in Dermal Delivery: The Role of Excipients.

The composition of topical and transdermal formulations is known to determine the rate and the extent of drug delivery to and through the skin. However, to date, the role of excipients in these formulations on skin delivery of actives has received little attention from scientists in the field. Monitoring skin absorption of both drug and vehicle may provide insights into the mechanism by which excipients promote permeation and may facilitate the design of effective and safer products. Previously, we have investigated the use of quantitative Confocal Raman Spectroscopy (CRS) to investigate the delivery of an active to the skin, and we also reported the first fully quantitative study that compared this method with the well-established in vitro permeation test (IVPT) model. To further explore the potential of quantitative CRS in assessing topical delivery, the present work investigated the effects of commonly used excipients on the percutaneous absorption of a model drug, ibuprofen (IBU). Permeation of IBU and selected solvents following finite dose applications to human skin was determined in vitro and in vivo by Franz diffusion studies and quantitative CRS, respectively. The solvents used were propylene glycol (PG), dipropylene glycol (DPG), tripropylene glycol (TPG), and polyethylene glycol 300 (PEG 300). Overall, the cumulative amounts of IBU that permeated at 24 h in vitro were similar for PG, DPG, and TPG (p > 0.05). These three vehicles outperformed PEG 300 (p < 0.05) in terms of drug delivery. Concerning the vehicles, the rank order for in vitro skin permeation was DPG ≥ PG > TPG, while PEG 300 did not permeate the skin. A linear relationship between maximum vehicle and IBU flux in vitro was found, with a correlation coefficient (R2) of 0.95. When comparing in vitro with in vivo data, a positive in vitro-in vivo (IVIV) correlation between the cumulative permeation of IBU in vitro and the total amount of IBU that penetrated the stratum corneum (SC) in vivo was observed, with a Pearson correlation coefficient (R2) of 0.90. A strong IVIV correlation, R2 = 0.82, was found following the linear regression of the cumulative number of solvents permeated in vitro and the corresponding skin uptake in vivo measured with CRS. This is the first study to correlate in vivo permeation of solvents measured by CRS with data obtained by in vitro diffusion studies. The IVIV correlations suggest that CRS is a powerful tool for profiling drug and vehicle delivery from dermal formulations. Future studies will examine additional excipients with varying physicochemical properties. Ultimately, these findings are expected to lead to new approaches for the design, evaluation, and optimization of formulations that target actives to and through the skin.