Aristolochic acid-related renal cell carcinoma exhibits a distinct tumor-immune microenvironment favoring response to immune checkpoint blockade.

Immune checkpoint blockade (ICB) is currently the standard of care for metastatic renal cell carcinoma (RCC), but treatment responses remain unpredictable. Aristolochic acid (AA), a prevalent supplement additive in Taiwan, has been associated with RCC and induces signature mutations, although its effect on the tumor-immune microenvironment (TIME) is unclear. We aimed to investigate the immune profile of AA-positive RCCs and explore its potential role as a susceptible candidate for ICB. Tissue samples from 22 patients with clear cell RCC (ccRCC) were collected for whole-exome sequencing to determine the genetic features and AA mutational signature (the discovery cohort). The corresponding RNA was sent for NanoString PanCancer IO 360 gene expression analysis to explore the immunological features. The formalin-fixed, parafilm-embedded slides of ccRCCs were sent for multiplex immunohistochemistry/immunofluorescence stain using Vectra system to evaluate the TIME. Tissues from two patients with metastatic RCC demonstrating complete response to ICB were sent for studies to validate the findings (the index patients). The results showed that AA mutational signatures with high tumor mutational burden (TMB) were present in 31.81% of the tumors in the discovery cohort. Three distinct clusters were observed through NanoString analysis. Clusters 1 and 3 were composed mainly of AA-positive RCCs. Cluster 3 RCCs exhibited higher tumor inflammation signature scores and higher immune cell type scores. Vectra analysis revealed a higher percentage of CD15+ and BATF3+ cells in cluster 1, whereas the percentage of CD8+ cells was potentially higher in cluster 3. Strong AA mutational signatures were found in the tumors of two index patients, and both were grouped to cluster 3. In conclusion, AA may induce higher TMB and alter the immune microenvironment in RCCs, which makes the tumors more susceptible to ICB. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Error assessment of subjective estimates of linear breast dimensions versus the objective method.

Objective: The study aimed to investigate the subjective method of estimating linear breast dimensions in comparison to the objective method. Methods: The reproducibility and accuracy of the subjective method of estimating linear breast dimensions during a simplified breast shape analysis were examined. Four linear breast dimensions including the distance from the sternal notch to the nipple, distance from the nipple to the inframammary fold, distance from the nipple to the midline and under-breast width were evaluated based on subjective estimates. Images from 100 women with natural breasts and without any history of breast surgery were reviewed by two examiners three times each. The cases were obtained from a large database of breast images captured using the Vectra Camera System (Canfield Scientific Inc., USA). The subjective data were then compared with the objective linear data from the Vectra Camera System in the automated analysis. Statistical evaluation was conducted between the three repeated estimates of each examiner, between the two examiners and between the objective and subjective data. Results: The intra-individual variations of the three subjective estimates were significantly greater in one examiner than in the other. This trend was consistent across all eight parameters in the majority of the comparisons of the standard deviations and variation coefficients, and the differences were significant in 14 out of 16 comparisons (p<0.05). Conversely, in the comparison between the subjective and objective data, the estimates were closer to the measurements in one examiner than the other. In contrast to the reproducibility observed, the assessment of the accuracy revealed that the examiner who previously presented with less reproducibility of the estimated data overall showed better accuracy in comparison to the objective data. The overall differences were inconsistent, with some being positive and others being negative. Regarding the distances from the sternal notch to the nipple and breast width, both examiners underestimated the values. However, the deviations were at different levels, particularly when considering the objective data from the Vectra Camera System as the gold standard data for comparison. Regarding the distance from the nipple to the inframammary fold, one examiner underestimated the distance, while the other overestimated it. An opposite trend was noted for the distance from the nipple to the midline. There were no differences in the estimates between the right and left sides of the breasts. The correlations between the measured and estimated distances were positive: as the objective distances increased, the subjective distances also increased. In all cases, the correlations were significant. However, the correlation for the breast width was notably weaker than that for the other distances. Conclusions: The error assessment of the subjective method reveals that it varies significantly and unsystematically between examiners. This is true when assessing the reproducibility as well as the accuracy of the method in comparison to the objective data obtained with an automated system.

In the face and neck, keloid scar distribution is related to skin thickness and stiffness changes associated with movement.

Keloid scars tend to occur in high-tension sites due to mechanical stimuli that are involved in their development. To date, a detailed analysis of keloid distribution focused specifically on facial and neck areas has not been reported, and limited literature exists as to the related mechanical factors. To rectify this deficiency of knowledge, we first quantified the facial and neck keloid distribution observed clinically in 113 patients. Subsequently, we performed a rigorous investigation into the mechanical factors and their associated changes at these anatomic sites in healthy volunteers without a history of pathologic scarring. The association between keloid-predilection sites and sebaceous gland-dense and acne-prone sites was also examined. To assess skin stretch, thickness and stiffness, VECTRA, ultrasound and indentometer were utilised. Baseline skin stiffness and thickness were measured, as well as the magnitude of change in these values associated with facial expression and postural changes. Within the face and neck, keloids were most common near the mandibular angle (41.3%) and lateral submental (20.0%) regions. These areas of increased keloid incidence were not associated with areas more dense in sebaceous glands, nor linked consistently with acne-susceptible regions. Binomial logistic regression revealed that changes in skin stiffness and thickness related to postural changes significantly predicted keloid distribution. Skin stiffness and thickness changes related to prolonged mechanical forces (postural changes) are most pronounced at sites of high keloid predilection. This finding further elucidates the means by which skin stretch and tension are related to keloid development. As a more detailed analysis of mechanical forces on facial and neck skin, this study evaluates the nuances of multiple skin-mechanical properties, and their changes in a three-dimensional framework. Such factors may be critical to better understanding keloid progression and development in the face and neck.

Evaluating 3D Simulation Accuracy for Breast Augmentation Outcomes: A Volumetric and Surface Contour Analysis in Chinese Patients.

BACKGROUND: The use of three-dimensional imaging in breast augmentation with silicone implants has revolutionized the surgery planning process by providing detailed visualizations of expected post-surgical outcomes. This technology enhances the decision-making process, enabling patients to choose their implants with greater confidence and ultimately leading to higher satisfaction with the postoperative outcome. OBJECTIVE: This study aims to assess the accuracy of 3D imaging simulations using the Canfield Vectra XT 3D system in predicting breast augmentation outcomes in Chinese patients, focusing on volume, surface contour, breast anterior-posterior (AP) Projection, and breast internal angle. METHODS: Our study analyzed female patients who received breast augmentation, documenting their preoperative and three-month postoperative conditions with 3D Vectra XT system images. Exclusions were made for patients undergoing concurrent breast surgeries or those with tuberous or ptotic breasts, due to limitations of the imaging system. Implants used were either round textured or anatomically shaped cohesive silicone gel, inserted subpectorally through trans-axillary or inframammary incisions, based on personalized evaluations. A detailed comparison between preoperative simulations and actual postoperative outcomes was conducted, focusing on volume, surface contour, AP projection, and internal angle variations. Statistical significance was determined through paired T tests, P < 0.05. RESULTS: In the analysis of preoperative simulations for determining postoperative outcomes in breast surgery, our study involving 42 Chinese patients, a total of 84 breasts, was conducted. The results indicated a mean volumetric discrepancy of 21.5 ± 10.3 (SD) cubic centimeters between the simulated and actual postoperative outcomes, achieving an accuracy rate of 91.9%. The root mean square deviation for the breast surface geometry was calculated to be 4.5 ± 1.1 (SD) millimeters (mm), demonstrating a low variance between the predicted and observed outcomes. The investigation found no significant variations across any specific areas of the breast surface, highlighting the uniform accuracy of the simulations across the entire breast. Additionally, the mean differences in Anterior-Posterior (AP) projection and internal angle were determined to be 8.82 ± 5.64 mm and 0.48 ± 1.91 (SD) degrees, respectively. These findings collectively attest to the efficacy of preoperative simulations in accurately predicting the postoperative physical appearance of breasts, thereby providing a valuable tool for surgical planning and improving the consultation process for patients. CONCLUSIONS: The Canfield Vectra XT 3D system has proven to be remarkably accurate in predicting the volumetric outcomes of breast augmentation surgery, with an accuracy rate exceeding 91.9%. It stands as a valuable tool for surgeons and patients alike, enhancing the preoperative planning process by offering a realistic preview of surgical results. This advancement not only facilitates a deeper understanding and setting of realistic expectations for patients but also strengthens the communication between patients and surgeons, ultimately leading to higher satisfaction rates with the surgical outcomes. It also emphasizes the significance of detailed documentation and consent processes in protecting against legal repercussions. LEVEL OF EVIDENCE II: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

How Reliable Is Breast Volume Assessment When the Patient Is Lying Flat?-Volumetric Assessment of Breast Volume Using a Vectra H2 Handheld Device in Different Positions.

(1) Background: Three-dimensional (3D) volumetric assessment is receiving increased recognition in breast surgery. It is commonly used for preoperative planning and postoperative control with the patient standing in an upright position. Recently, intraoperative use was evaluated with patients in the supine position. The aim of this prospective study was to evaluate the volumetric changes in 3D surface imaging depending on the patient's position. (2) Methods: 3D volumetric analysis was performed using a Vectra-H2 device with patients in standing, sitting, and supine positions. A total of 100 complete datasets of female breasts were included in the study. The measured volumes of each evaluated breast (n = 200) were compared between the three positions. (3) Results: The mean difference between the 3D volumetric assessments of the sitting and standing positions per breast was 7.15 cc and, thus, statistically insignificant (p = 0.28). However, the difference between supine and standing positions, at 120.31 cc, was significant (p < 0.01). (4) Conclusions: The 3D volumetric assessment of breasts in the supine position did not statistically correlate with the validated assessment of breast volume in the standing position while breast volume in the sitting position is reliable and correlates with the assessment of a standing patient. We conclude that intraoperative volumetric assessment should be performed with patients in an upright sitting position.

Exploring natural breast symmetry in the female plastic surgical patient population.

Background: Breast symmetry remains a challenging quality to measure. The question arises of how much baseline breast symmetry exists in the female plastic surgical patient population. Material and methods: Several linear dimensional assessments were collected based on a retrospective analysis of objective data of women with natural breasts, who presented for an initial consultation in a plastic surgical clinic and were measured with the 3D Vectra Camera by the company Canfield Scientific Inc., U.S.A. The first 100 cases were extracted from the large database in alphabetical order. The nipple positions were examined, including the following linear dimensions: distances from the jugulum to the nipple, from the nipple to the inframammary fold, and from the nipple to midline. Furthermore, the under-breast widths were obtained. Results: The objective three-dimensional imaging method determined that all patients had some degree of asymmetry. The linear distances from the jugulum and midline to the nipple revealed that the measurements were larger on the left side. However, the nipple to the inframammary fold measurements were roughly the same. In the sample of plastic surgical patients, the average distance between the jugulum and nipple was around 20 cm, the distance between the nipple and inframammary fold was around 6.5 cm, the distance between the nipple and midline was around 10 cm, and the under-breast width was around 13 cm. On average, the jugulum-nipple distance on the right side was 19.9±2.3 cm and 20.1±2.4 cm on the left side. The distance between the nipple and the inframammary fold was 6.4±1.1 cm, revealing a similar distribution on both sides. The mean distance from the nipple to the midline was much larger on the left side of the body at 10.0±1.2 cm than on the right side at 9.4±1.3 cm. The under-breast width on the right side was slightly larger than the left side, with measurements of 13.4±1.8 cm versus 13.2±1.7 cm. Conclusion: Breast dimensions can be described comprehensively by an objective three-dimensional imaging process, which can detect asymmetry in all patients. The differences related to the nipple position were larger on the left side than the right regarding the distances from the jugulum and particularly the midline to the nipple, which seems to be of special importance, but not from the nipple to the inframammary fold. The linear measurements for the distances from the jugulum and the midline to the nipple are essential to accurate symmetry assessments and form an aesthetic triangle of the breast, which is a new term and the key to a simplified shape analysis.

Evaluation of Intraoperative Volumetric Assessment of Breast Volume Using 3D Handheld Stereo Photogrammetric Device.

Methods for assessing three-dimensional (3D) breast volume are becoming increasingly popular in breast surgery. However, the precision of intraoperative volumetric assessment is still unclear. Until now, only non-validated scanning systems have been used for intraoperative volumetric analyses. This study aimed to assess the feasibility, handling, and accuracy of a commercially available, validated, and portable device for intraoperative 3D volumetric evaluation. All patients who underwent breast surgery from 2020 to 2022 were identified from our institutional database. Intraoperative 3D volumetric assessments of 103 patients were included in this study. Standardized 3D volumetric measurements were obtained 3 months postoperatively to compare the intraoperatively generated volumetric assessment. All of the study participants were women with a mean age of 48.3 ± 14.7 years (range: 20-89). The mean time for intraoperative volumetric assessment was 8.7 ± 2.6 min. The postoperative 3D volumetric assessment, with a mean volume of 507.11 ± 206.29 cc, showed no significant difference from the intraoperative volumetric measurements of 504.24 ± 276.61 cc (p = 0.68). The mean absolute volume difference between the intraoperative simulations and postoperative results was 27.1 cc. Intraoperative 3D volumetric assessment using the VECTRA H2 imaging system seems to be a feasible, reliable, and accurate method for measuring breast volume. Based on this finding, we plan to investigate whether volumetric objective evaluations will help to improve breast symmetry in the future.

Reliability and Measurement Error of Breast Volume Calculation Using Three-Dimensional Surface Imaging.

Background: Breast lymphedema after breast cancer is challenging to quantify. Three-dimensional (3D) surface imaging is one available technique to measure breast volume, however, the measurement properties of available software programs have not been fully determined. The aim of this study was to determine equivalency of measurements with two software programs as well as reliability, standard error of measurement (SEM), and smallest detectable change (SDC). Methods and Results: Retrospective three-dimensional surface imaging (3D-SI) of 100 breasts taken before or after breast conserving surgery for breast cancer were retrieved for reliability analysis. Three assessors followed a standardized measurement technique using two software programs, Vectra® 3D Analysis Module (VAM) and Breast Sculptor®. Mean breast volume was 489.9 ± 206 cc using VAM and 480.1 ± 229.1 cc using Breast Sculptor. Lin's concordance showed poor agreement between programs (0.81-0.88). Measurements using VAM had excellent intra- and inter-rater reliability with SEM = 4.1% for one assessor and 8.7% for multiple assessors. Breast Sculptor also had excellent intra-rater and substantial inter-rater reliability but the SEM was much larger at 14.5% (intra-rater) and 19.1% (inter-rater). The SDC value was lowest for VAM and a single rater with 56 cc indicating a meaningful change beyond measurement error. Conclusion: Breast volume measurements captured with 3D-SI using VECTRA-XT are highly reliable, but the volumes, SEM, and SDC varied between the two software programs. Measurement error was lowest with VAM software. Although the usefulness of VECTRA-XT and VAM software to detect change in breast volume is promising, further solutions to reduce measurement error are required to improve clinical utility to measure breast lymphedema.

Evaluation of Vectra® XT 3D Surface Imaging Technology in Measuring Breast Symmetry and Breast Volume.

BACKGROUND: Breast symmetry is an essential component of breast cosmesis. The Harvard Cosmesis scale is the most widely adopted method of breast symmetry assessment. However, this scale lacks reproducibility and reliability, limiting its application in clinical practice. The VECTRA® XT 3D (VECTRA®) is a novel breast surface imaging system that, when combined with breast contour measuring software (Mirror®), aims to produce a more accurate and reproducible measurement of breast contour to aid operative planning in breast surgery. OBJECTIVES: This study aims to compare the reliability and reproducibility of subjective (Harvard Cosmesis scale) with objective (VECTRA®) symmetry assessment on the same cohort of patients. METHODS: Patients at a tertiary institution had 2D and 3D photographs of their breasts. Seven assessors scored the 2D photographs using the Harvard Cosmesis scale. Two independent assessors used Mirror® software to objectively calculate breast symmetry by analysing 3D images of the breasts. RESULTS: Intra-observer agreement ranged from none to moderate (kappa - 0.005-0.7) amongst the assessors using the Harvard Cosmesis scale. Inter-observer agreement was weak (kappa 0.078-0.454) amongst Harvard scores compared to VECTRA® measurements. Kappa values ranged 0.537-0.674 for intra-observer agreement (p < 0.001) with Root Mean Square (RMS) scores. RMS had a moderate correlation with the Harvard Cosmesis scale (rs = 0.613). Furthermore, absolute volume difference between breasts had poor correlation with RMS (R2 = 0.133). CONCLUSION: VECTRA® and Mirror® software have potential in clinical practice as objectifying breast symmetry, but in the current form, it is not an ideal test. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Mistic: An open-source multiplexed image t-SNE viewer.

Understanding the complex ecology of a tumor tissue and the spatiotemporal relationships between its cellular and microenvironment components is becoming a key component of translational research, especially in immuno-oncology. The generation and analysis of multiplexed images from patient samples is of paramount importance to facilitate this understanding. Here, we present Mistic, an open-source multiplexed image t-SNE viewer that enables the simultaneous viewing of multiple 2D images rendered using multiple layout options to provide an overall visual preview of the entire dataset. In particular, the positions of the images can be t-SNE or UMAP coordinates. This grouped view of all images allows an exploratory understanding of the specific expression pattern of a given biomarker or collection of biomarkers across all images, helps to identify images expressing a particular phenotype, and can help select images for subsequent downstream analysis. Currently, there is no freely available tool to generate such image t-SNEs.

Validation of Vectra 3D Imaging Systems: A Review.

Aim: Three-dimensional facial imaging systems are a useful tool that is gradually replacing two-dimensional imaging and traditional anthropometry with calipers. In this varied and growing landscape of new devices, Canfield (Canfield Scientific, Parsippany, NJ, USA) has proposed a series of static and portable 3D imaging systems. The aim of this systematic review was to evaluate the current literature regarding the validation of Canfield's Vectra imaging systems. Materials and Methods: A search strategy was developed on electronic databases including PubMed, Web of Science and Scopus by using specific keywords. After the study selection phase, a total of 10 articles were included in the present review. Results: A total of 10 articles were finally included in the present review. For six articles, we conducted a validation of the Vectra static devices, focusing especially on the Vectra M5, Vectra M3 and Vectra XT. For four articles, we validated the Vectra H1 portable system. Conclusions: All of the reviewed articles concluded that Canfield's Vectra 3D imaging systems are capable of capturing accurate and reproducible stereophotogrammetric images. Minor errors were reported, particularly in the acquisition of the perioral region, but all the evaluated devices are considered to be valid and accurate tools for clinicians.

On clustering for cell-phenotyping in multiplex immunohistochemistry (mIHC) and multiplexed ion beam imaging (MIBI) data.

OBJECTIVE: Multiplex immunohistochemistry (mIHC) and multiplexed ion beam imaging (MIBI) images are usually phenotyped using a manual thresholding process. The thresholding is prone to biases, especially when examining multiple images with high cellularity. RESULTS: Unsupervised cell-phenotyping methods including PhenoGraph, flowMeans, and SamSPECTRAL, primarily used in flow cytometry data, often perform poorly or need elaborate tuning to perform well in the context of mIHC and MIBI data. We show that, instead, semi-supervised cell clustering using Random Forests, linear and quadratic discriminant analysis are superior. We test the performance of the methods on two mIHC datasets from the University of Colorado School of Medicine and a publicly available MIBI dataset. Each dataset contains a bunch of highly complex images.

Assessment of the Murine Tumor Microenvironment by Multiplex Immunofluorescence.

We describe the staining methods used for simultaneous detection of tumor microenvironment components as well as the automated quantification methodologies. This method uses mouse formalin-fixed paraffin-embedded tissues and multiplex immunofluorescence (Multiplex IF) followed by multispectral imaging. Currently, this methodology has shown to have a valuable role in murine immunoprofiling, and can be useful when evaluating the changes incurred on the tumor microenvironment upon various immunopreventive strategies.

Objective Assessment of the Long-Term Volumizing Action of a Polycaprolactone-Based Filler.

Background: The polycaprolactone-based filler, (PCL-1, Ellansé-S), forms part of the recently growing portfolio of biodegradable collagen-stimulating fillers. It is comprised of a suspension of 25-50 micron diameter microspheres of polycaprolactone (PCL) (30%) in a carboxymethyl cellulose (CMC) gel carrier (70%) and has gained popularity due to its long-term volumizing action. Objective: This study outlines a retrospective case series of nine patients injected with the PCL-1, for volume augmentation in the mid-face. Objective volume calculations were performed with the Canfield Vectra 3D Imaging System at two time points post-implantation, with the objective of determining the longevity of the volumizing effect of the bio-stimulating substance. Results: A clear increase in volume, between 50-150%, was found in all of the patients at two years, over and above the volume initially injected. All the patients were satisfied with the longevity of the results. Discussion: The PCL-based filler is believed to afford immediate volume restoration due to the CMC gel component and a long-term action due to neo-collagenesis, induced by the PCL microspheres. The CMC gel is known to dissipate within 6-8 weeks, only to be replaced by new collagen induced by the PCL particles. Thus soft-tissue formation induced by the PCL particles, ultimately leads to a sustained volumizing effect. Conclusion: The PCL-based filler is shown to have a sustained volumizing effects of at least 2 years duration with clear evidence of increase in volume over and above the volume injected, in all of the cases studied. This is indicative of significant neo-collagenesis induced by the PCL microspheres.

Influence of Marker Number and Position on Accuracy of Breast Measurement With Three-Dimensional Camera.

BACKGROUND: The VECTRA H1 three-dimensional (3D) imaging system (Canfield Scientific, Parsippany, NJ) enables easy 3D image construction and measurement. Although the number and positions of markers on the skin for image synthesis might affect accuracy of measurements, few studies have mentioned the possibility. This study investigated the accuracy and reproducibility of distance measurements using VECTRA H1, focusing on the number and positions of markers. METHODS: A total of 3, 5, or 7 markers were attached to a female breast model including lateral markers 6 cm from the midline and photographed with VECTRA. Five markers were configured in more two ways, with the lateral markers either positioned 3 cm outside the midline (narrow interval) or 9 cm outside the midline (wide interval). 3D models were created three times under each condition, for a total of 15 models. Differences (measurement error) between measured values on 3D models and actual measured values were verified for six distances, such as distance between the nipples. RESULTS: The average difference was 11.1 mm with 3 markers (95% confidence interval (CI), 4.38-17.7 mm, p = 0.0028). In comparison, average difference was -0.395 mm (-0.866 to 0.0763 mm, p = 0.095) with 5 markers, and 0.139 mm (-0186 to 0.465 mm, p = 0.379) with 7 markers, all less than 1 mm. Average difference with narrow interval 5 markers was larger than one with wide interval. CONCLUSIONS: In 3D imaging of the breast using VECTRA H1, distance measurements offering clinically satisfactory accuracy can be made by setting appropriate marker conditions. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Validating three-dimensional imaging for volumetric assessment of periorbital soft tissue.

Purpose: To evaluate the accuracy and interobserver variability of three-dimensional (3D) stereophotogrammetric imaging for volumetric assessment in the periorbital region and to propose a protocol for measurement. Methods: A cross-sectional study was performed. Fifty-eight normal adult subjects had photographs taken using the VECTRA M3 imaging system on two separate occasions at a single center teaching hospital. The primary study outcome measurement was mean volume change in the periorbital region, using different methods of image analysis with the following variables: registration method, eyes open or closed, eyelids measured separately or together, extension of area measured to include mid-face. Accuracy was defined as the agreement between paired images with no measured change in volume (0 mL). The agreement between two independent observers was assessed using Bland-Altman plots. Results: There was a statistically significant difference (P < .0001) between the different methods of measurement, with registration of images having the most effect (partial eta squared 0.229). A mean change in volume of 0.11 ± 0.13 mL was reported for the most accurate method. Agreement between observers was confirmed (highest Intraclass Correlation Coefficient 0.63). Conclusions: Assessment of volumetric change in the periorbital region using 3D image reconstruction is accurate with good interobserver repeatability and is suitable for clinical use. A protocol for data acquisition using the VECTRA M3 is proposed. 3D image reconstruction holds promise to enable diagnosis, treatment monitoring, and surgical outcome evaluation in ocular adnexal disease.

Immune contexture analysis in immuno-oncology: applications and challenges of multiplex fluorescent immunohistochemistry.

The tumor microenvironment is an integral player in cancer initiation, tumor progression, response and resistance to anti-cancer therapy. Understanding the complex interactions of tumor immune architecture (referred to as 'immune contexture') has therefore become increasingly desirable to guide our approach to patient selection, clinical trial design, combination therapies, and patient management. Quantitative image analysis based on multiplexed fluorescence immunohistochemistry and deep learning technologies are rapidly developing to enable researchers to interrogate complex information from the tumor microenvironment and find predictive insights into treatment response. Herein, we discuss current developments in multiplexed fluorescence immunohistochemistry for immune contexture analysis, and their application in immuno-oncology, and discuss challenges to effectively use this technology in clinical settings. We also present a multiplexed image analysis workflow to analyse fluorescence multiplexed stained tumor sections using the Vectra Automated Digital Pathology System together with FCS express flow cytometry software. The benefit of this strategy is that the spectral unmixing accurately generates and analyses complex arrays of multiple biomarkers, which can be helpful for diagnosis, risk stratification, and guiding clinical management of oncology patients.