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Photoelectrochemical (PEC) immunosensors are highly promising tools for monitoring biochemical molecules. Constructing high-performance heterojunctions is a general method to improve the sensitivity of PEC immunosensors. The internal electric field (IEF) formed at the heterojunction interface plays a crucial role in coordinating the separation of photogenerated carriers. Although IEF in heterojunctions shows extraordinary talents in PEC immunosensors, it is powerless to suppress the recombination of photogenerated carriers in the bulk phase. Thus, modulating the IEF of heterojunctions more than just at interfaces remains a challenge. Herein, a "signal-off" PEC immunosensor for CYFRA21-1 detection was constructed. The immunosensor was based on sulfurized bismuth oxyhydroxide compound Bi 2 S 3 /Bi 2 O 2 (OH) 2 nanorods as the platforms, N-doped hollow homogenous Mo 2 C nanospheres with Ru NPs (Mo 2 C:N@Ru) as the quenching label. The Bi 2 O 2 (OH) 2 were composed of [Bi 2 O 2 ] 2+ and OH - alternating layers, which provided an IEF throughout the bulk phase. Meanwhile, the heterogeneous interface further promoted the separation of photogenerated carriers and output strong photocurrent signals by energy level matching of Bi 2 O 2 (OH) 2 with Bi 2 S 3 . Subsequently, Mo 2 C:N@Ru was quantitatively introduced in the presence of CYFRA21-1 through specific recognition between antigens and antibodies. The main structure of Mo 2 C:N@Ru was a homojunction formed by cubic and hexagonal phases Mo 2 C, which accelerated the separation of photogenerated carriers. Moreover, doped N element and Ru NPs further enhanced the competition between Mo 2 C:N@Ru and the Bi 2 S 3 /Bi 2 O 2 (OH) 2 platforms for ascorbic acid and then achieved a quenching effect. The limit of detection for CYFRA21-1 was 17 fg/mL, and linearity range was 50 fg/mL -100 ng/mL. This PEC immunosensor achieves highly sensitive detection of CYFRA21-1 and has good scalability, which provides new ideas for the development analysis and detection platforms of tumor biomarkers. Simultaneously, this strategy might provide a novel perspective for modulating the distribution of the IEF of photoactive materials applied in PEC immunosensors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Prostate cancer antigen 3 (PCA3) has emerged as a critical biomarker for the early detection of prostate cancer, complementing the traditional prostate-specific antigen (PSA) testing. This research presents a novel resistive sensor based on reduced graphene oxide (RGO) functionalized with glutaraldehyde (GA)/complementary single-stranded DNA (ss-DNA) for the detection of the PCA3 RNA. The device was meticulously characterized at each fabrication step to confirm the successful integration of the various layers on the sensor device, utilizing atomic force microscopy (AFM) which confirmed the increase in the thickness of the sensor from ∼1.4 nm (only RGO) to ∼25 nm for the RGO-GA/ss-DNA/PCA3 device. Field emission scanning electron microscopy exhibited a change in the surface morphology after each step of device fabrication and testing. Fourier-transform infrared (FTIR) spectroscopy was conducted to confirm the presence of functional groups in each component of the sensor. The fabricated resistive sensor demonstrated mean response ranging from 1.203 to 59.44% for 0.1 to 100 ng mL -1 PCA3 RNA. Notably, the device exhibited stability over a period of two weeks and displayed high selectivity for PCA3. The developed RGO-GA/ss-DNA/PCA3 sensor was tested with RNA extracted from multiple prostate cancer cell lines and other cancer cell lines and demonstrated response only to RNA extracted from LNCaP as it was the only cell line expression of PCA3. The findings from the developed sensor were cross-validated with the observations of a semi quantitative polymerase chain reaction (qPCR), and were found to be closely matched.

Purpose: Radiation-induced pulmonary fibrosis (RIPF) is a potentially serious and disabling late complication of radiation therapy. Monitoring RIPF progression is challenging due to the absence of early detection tools and the difficulty in distinguishing RIPF from other lung diseases using standard imaging methods. In the lungs, integrin αvβ6 is crucial in the development of RIPF, acting as a significant activator of transforming growth factor β after radiation injury. This study aimed to investigate integrin α v β 6 -targeted positron emission tomography (PET) imaging ([ 64 Cu]Cu-α v β 6 -BP) to study RIPF development in vivo., Methods and Materials: We used a focal RIPF model (70 Gy delivered focally to a 3 mm spot in the lung) and a whole lung RIPF model (14 Gy delivered to the whole lung) in adult C57BL/6J mice. Small animal PET/computed tomography images were acquired 1 hour postinjection of 11.1 MBq of [ 64 Cu]Cu-α v β 6 -BP. Animals were imaged for 8 weeks in the focal RIPF model and 6 months in the whole lung RIPF model. Immunohistochemistry for integrin α v β 6 and trichrome staining were performed., Results: In the focal RIPF model, there was focal uptake of [ 64 Cu]Cu-α v β 6 -BP in the irradiated region at week 4 that progressively increased at weeks 6 and 8. In the whole lung RIPF model, minimal uptake of the probe was observed at 4 months post-radiation therapy, which significantly increased at months 5 and 6. Expression of integrin α v β 6 was validated histologically by immunohistochemistry in both models., Conclusions: Integrin α v β 6 -targeted PET imaging using [ 64 Cu]Cu-α v β 6 -BP can serve as a useful tool to identify RIPF in vivo., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Precise intraoperative tumor delineation is essential for successful surgical outcomes. However, conventional methods are often incompetent to provide intraoperative guidance due to lack specificity and sensitivity. Recently fluorescence-guided surgery for tumors to delineate between cancerous and healthy tissues has attracted widespread attention. The contrast-enhanced fluorescent imaging has been applied for non-invasive diagnosis of cancers using tumor-targeting fluorescent probes., Methods: The carbonic anhydrase IX targeted polyaspartamide fluorescent compounds (SD-PHEA-NI) were synthesized by incorporating a tumor-targeting group of sulfadiazine (SD) and N-butyl-4-ethyldiamino-1,8-naphthalimide (NI) into water-soluble carriersof poly-α,β-[N-(2-hydroxyethyl)-L-aspartamide] (PHEA). These derivatives were also characterized by Fourier transform infrared spectroscopy, gel permeation chromatography, ultraviolet-visible spectroscopy, nuclear magnetic resonance spectroscopy and fluorescence assays. The cellular uptake, cytotoxicity, and fluorescence imaging ability were evaluated., Results: Experiment results indicated that SD-PHEA-NI has low cytotoxic to Henrietta Lacks (HeLa) cells. Moreover, B16F10 melanoma cells can take up SD-PHEA-NI and show good green fluorescent images. However, SD-PHEA-NI displayed a low-intensity green fluorescence signal in healthy human embryonic kidney (293T) cells., Conclusion: SD-PHEA-NI can be considered a potential fluorescent probe for the detection of tumors. This study has the potential to enhance tumor diagnosis and image-guided surgical interventions by providing real-time information and robust decision support, thereby reducing recurrence and complication rates and ultimately improving patient outcomes., Competing Interests: The authors report no conflicts of interest in this work., (© 2025 Zhang et al.)

Squamous cell carcinoma antigen (SCCA) is one of the most commonly detected cancer biomarkers for a variety of cancers. In this paper, a microfiber ring laser biosensor with a graphene oxide linking layer for SCCA detection was proposed and experimentally demonstrated. SCCA antibody immobilized on graphene oxide surface binds specifically to SCCA, and induces refractive index variation over the surface of the microfiber biosensor, which leads to a wavelength shift of the microfiber ring laser biosensor. The experimental results show that the proposed laser biosensor can detect SCCA with concentrations from 0.01 to 50 ng/mL, and the calculated detection limit can be as low as 1.3 pg/mL. Additionally, the label-free quantitative detection of SCCA using the proposed microfiber biosensor was verified experimentally according to the corresponding regression equation, and the results agree well with clinical examination detection. This constructed microfiber biosensor may have promising practical applications in analytical detection, medical diagnostics, etc., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Abstract: Cutaneous melanoma can lead to metastasis, and it is associated with high mortality. Currently, there are no widely accepted immunohistochemistry markers for melanoma prognosis in routine staging. Preferentially expressed antigen in melanoma (PRAME) is a possible biomarker for prognosis in several noncutaneous neoplasms. Ki-67 is a cell proliferation marker correlated with poor outcomes in many cancers. This study assessed PRAME and Ki-67 as potential prognostic markers for sentinel lymph node outcomes and survival among melanoma patients. This is a retrospective study analyzing cutaneous melanoma cases from a Brazilian cancer center (2005-2021). All cases were tested using immunohistochemistry to evaluate PRAME expression and Ki-67 index. Descriptive analysis, Spearman correlations, means comparison, Kaplan-Meier analysis, χ 2 , and Cox models were performed. In univariate analysis of 123 cutaneous melanoma cases, high extent ( P = 0.0267) and elevated intensity ( P = 0.043) of PRAME were associated with decreased overall survival. The Ki-67 index was associated with overall survival ( P = 0.05) and sentinel lymph node status ( P = 0.0403), with a positive correlation between the markers ( P = 0.0004) and between Ki-67 and Breslow thickness ( P = 0.0001). However, in multivariate analysis, only Breslow thickness significantly influenced overall survival ( P = 0.0003). Then, the present results can suggest that elevated PRAME and Ki-67 expression are associated with poor overall survival in cutaneous melanoma; however, in multivariate analysis, only the Breslow thickness had a significant influence. These findings highlight the potential of PRAME and Ki-67 as prognostic markers, opening frontiers that could improve strategies for treating cutaneous melanoma., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

CYFRA21-1 is a tumor marker for lung cancer, and its rapid and accurate detection can provide evidence for the early diagnosis of lung cancer. In this work, Bi-Fe turnbull blue analogues (Bi-Fe-TBA) were synthesized by the self-templating method. Bi 2 O 3 -SFNs was prepared by simple oxidation in air using Bi-Fe-TBA as a template. Bi 2 O 3 Star-like Flower Nanoclusters (Bi 2 O 3 -SFNs) and CdS Hollow Nanorods (CdS-HNRs) were used to form a unique type II heterojunction for the first time. The arrangement of energy levels between CdS-HNRs and Bi 2 O 3 -SFNs, along with their hollow structure and star shape, effectively suppressed the recombination of photogenerated electrons and holes while shortening carrier transport distance. An ultra-sensitive PEC biosensor was developed to detect the lung cancer marker CYFRA21-1, leveraging the superior photoelectric conversion capabilities of Bi 2 O 3 -SFNs/CdS-HNRs. The sensor demonstrates outstanding stability, specificity, reproducibility as well as a wide linear range (10 -4 - 10 ng mL -1 ) and low detection limit (4.23 × 10 -5  ng mL -1 ). This study is valuable for the preparation of other functional materials using TBA as a template., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Neoantigens exclusively presented by human leukocyte antigens (HLAs) on cancer cell surfaces are newly discovered and highly cancer-specific biomarkers for cancer diagnosis. The current available method for detecting neoantigens is predominantly based on Mass spectrometry with inevitable limitations of high cost, complexity and isotope labels. In this work, we describe the development of an innovative catalytic electrochemical biosensor for ultrasensitive detection of neoantigen in cell lysates. Such biosensor design involves the synthesis of new and highly catalytic CoFe-prussian blue analogue/ZIF-67 yolk-shell nanocube-decorated MBene nanosheets (CoFe-PBA/ZIF-67/MBene) and the derivatization of electrochemically inert neoantigen to electroactive molecule. The as synthesized CoFe-PBA/ZIF-67/MBene nanocomposite exhibits large specific surface area, excellent conductivity and abundant active sites for electrochemical oxidation of the derivatized neoantigens for the yield of considerably amplified currents for sensitive detection of target neoantigen with low to 0.047 nM detection limit. The biosensor can also be applied for monitoring low levels of HLA-presented neoantigen complexes in cell lysates, offering new insights into methodological advancements in neoantigen analyses for cancer research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The precise design and synthesis of Au and Ag composite nanomaterials can provide them with richer plasmonic modes, resulting in enhanced optical properties. Here, a novel strategy was demonstrated to control the selective deposition of Ag at different positions of Au triangular nanoprisms (Au TNPs). 1,4-benzenedithiol (BDT) was selectively absorbed in different positions of Au TNPs which made Ag selectively deposited on Au TNPs. A series of Ag islands-Au TNPs including 3AgNPs islands-Au@Ag TNPs, 3AgNPs islands-Au TNPs, 2AgNPs islands-Au TNPs and 1AgNPs island-Au TNPs were obtained. We found that Surface enhanced Raman scattering (SERS) activity was closely associated with the position of Ag deposition under the same volume of AgNO 3 . It has strongest SERS activity when Ag deposit on the surface, edges and corners of Au TNPs which corresponding to 3AgNPs islands-Au@Ag TNPs with a high enhancement factor of 5.50 × 10 7 . Raman reporter molecules were embedded between Au core, Ag shell and Ag islands which enhanced the stability, making them ideal candidates for Raman tag-based applications. We used it as SERS probes to realize the ultra-sensitive detection of Cyfra21-1, with a low limit of detection of 2.84 × 10 -14 g/L and a wide linear range of 1.00 × 10 -13 -1.00 × 10 -1 g/L., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Isolated immunohistochemical indicators are limited to diagnose melanocytic neoplasms. This retrospective study is to assess the diagnostic value of combined immunohistochemical analysis targeting preferentially expressed antigen in melanoma (PRAME) and p16 in melanocytic neoplasms, with a detailed focus on arcal lesions., Methods: This was a single center cohort study from January 2022 to June 2023. A total of 165 identified cases were collected, including 112 melanomas (MMs) and 53 melanocytic nevi, which were composed of 122 acral samples and 43 non-acral samples. Immunohistochemistry(IHC) for both PRAME and p16 was performed in these cases, which was subsequently statistically analyzed to assess the diagnosis ability of PRAME and p16., Results: In total samples, the sensitivity and specificity of PRAME(+) for MM are 82.1% and 94.3% (AUC = 0.882, 95%CI:0.827-0.938), while of p16(-) for MM are 31.25% and 94.3% (AUC = 0.628, 95%CI:0.542-0.714); PRAME(+)/p16(-) (meaning as PRAME(+) or p16(-)) displayed a sensitivity and specificity of 85.7% and 88.7% for MM (AUC = 0.872, 95%CI:0.810-0.934), while PRAME(+) &p16(-) (meaning as PRAME(+) and p16(-)) revealed a sensitivity and specificity of 27.7% and 100% in MM (AUC = 0.638, 95%CI:0.555-0.722). In acral samples, PRAME(+)/p16(-) exhibited a specificity of 94.7% and a sensitivity of 86.9% for MM (AUC = 0.908, 95%CI: 0.849-0.968), with sensitivities of 90.9% for invasive MM and 82.5% for preinvasive MM, respectively; The sensitivity and specificity of PRAME(+) &p16(-) for MM is 22.6% and 100% (AUC = 0.613, 95%CI: 0.513-0.714) respectively. In non-acral samples, the sensitivity and specificity of PRAME(+)/p16(-) for MM are 82.1% and 73.3% (AUC = 0.777, 95%CI: 0.622-0.933), while of PRAME(+) &p16(-) are 42.9% and 100% (AUC = 0.714, 95%CI:0.564-0.864)., Conclusion: Combined IHC of PRAME and p16 contributes to discriminating melanocytic neoplasms, especially for in situ acral MM., Competing Interests: Declarations. Ethics approval and consent to participate: Ethical clearance for the study was obtained from the Institutional Review Board of Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College (Ethical Approval: No. 2013-LC/KY-033). Moreover, written informed consent was conscientiously acquired from all patients or their legal guardians. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Trophoblast cell-surface antigen 2 (TROP2), a transmembrane receptor expressed in many carcinomas, is a target for novel antibody-drug conjugates such as sacituzumab govitecan. TROP2-targeted therapy is used for unresectable locally advanced or metastatic triple-negative and hormone receptor-positive, HER2-negative breast cancers. The role of TROP2 as a predictive marker is yet unclear. Standardized interpretation criteria for TROP2 immunohistochemistry (IHC) are lacking. Here, we compared three antibody clones and two methods for semi-quantitative assessment, aiming to establish reproducible evaluation criteria. First, TROP2 IHC was performed on normal tissues and nine breast cancers, using the BSB-148, EPR20043 and SP293 clones. EPR20043 was selected for subsequent evaluation in 69 breast cancers without pathological complete response to neoadjuvant chemotherapy (NAC). Four pathologists applied the ASCO/CAP guidelines for HER2 IHC testing (designated as the 'membrane score') and the H-score. All H-scores were categorized as low (0-100), intermediate (101-200) and high (201-300). Although the membrane scores strongly correlated with the categorized H-scores, the latter showed higher interobserver variability. Next, TROP2 IHC was performed on 94 breast cancer metastases and evaluated by six pathologists, confirming the strong correlation between the membrane scores and H-scores. In metastases, the interobserver variability was similar for both methods. Our observations support the application of the HER2 ASCO/CAP guidelines for semi-quantitative evaluation of membranous TROP2 protein expression, as this method strongly correlates with the H-score and is less prone to interobserver variability in post-NAC breast resections. Future studies should investigate the association between the TROP2 membrane score and response to TROP2-targeted therapy., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mieke R. Van Bockstal reports financial support was provided by Saint-Luc Foundation. Mieke R. Van Bockstal reports financial support was provided by Foundation Against Cancer. Christine Galant reports financial support was provided by Saint-Luc Foundation. Francois Duhoux reports financial support was provided by Foundation Against Cancer. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Follicular dendritic cell sarcoma is a rare mesenchymal neoplasm arising from follicular dendritic cells (FDC) of lymphoid follicles. While the majority of FDC sarcoma cases arise within lymph nodes, approximately 30% manifest in extranodal sites. Only 4 prior occurrences of intra-parotid FDC sarcomas have been documented. We are reporting a rare case of FDC of the parotid gland in a 65-year-old male with a questionable history of B-cell lymphoma. The patient underwent a right total parotidectomy and bilateral neck dissection. A diagnosis of follicular dendritic cell (FDC) sarcoma was made, with one positive intra-parotid node. The malignant cells expressed the characteristic markers for FDC sarcoma but with positivity of the melanocytic marker PRAME. This is a case of FDC sarcoma with an unusual extranodal localization in the parotid gland. Immunohistochemistry was useful in making a diagnosis although the positivity for the melanocytic marker PRAME was unusual and unreported before., Competing Interests: Declarations. Ethical approval: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent: For this type of study formal consent is not required. Consent for publication: For this type of study consent for publication is not required. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s).)

Cytokeratin 19 fragment (CYFRA21-1) is considered to be a potential marker for the diagnosis and of classification of lung cancer. It is highly desired to develop rapid and highly sensitive label-free chemiluminescence (CL) immunosensors for the detection of CYFRA21-1. In this work, magnetic core-shell nanocomposite Fe 3 O 4 @nickel-cobalt double hydroxide (Fe 3 O 4 @DH) was used for the first time as a carrier to synthesize N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and gold and silver nanocluster (AuAgNC) bifunctionalized magnetic nanomaterials Fe 3 O 4 @DH/AuAgNCs-ABEI. The resulting Fe 3 O 4 @DH/AuAgNCs-ABEI demonstrated excellent CL performance, attributed to the exceptional catalytic capability of nickel-cobalt double hydroxide (NiCo-DH) and AuAgNCs, as well as the large specific surface area of NiCo-DH for AuAgNCs and ABEI enrichment. Moreover, Fe 3 O 4 @DH/AuAgNCs-ABEI exhibited superior stability compared to Fe 3 O 4 -based CL-functionalized magnetic nanomaterials, owing to the presence of NiCo-DH shells. On this basis, a label-free CL immunosensor using CYFRA21-1 antibody-modified Fe 3 O 4 @DH/AuAgNCs-ABEI as an analytical interface was constructed for the detection of CYFRA21-1 in the range from 1.0 × 10 -13 to 1.0 × 10 -8 g/mL. The detection limit of this CL immunosensor was 47.7 fg/mL, 4 orders of magnitude lower than existing CL methods. The CL immunosensor was able to accurately detect the concentration of CYFRA21-1 in serum samples, as evidenced by ELISA results. More importantly, it not only distinguished well between healthy persons and lung cancer patients (90.0% sensitivity and 90.0% specificity), but also effectively distinguished between patients with non-small cell lung cancer and small cell lung cancer (80.0% sensitivity and 86.7% specificity).

Background: Previous studies have revealed the importance of CYFRA 21-1 in the diagnosis of oral squamous cell cancer (OSCC). However, the results are inconsistent. This meta-analysis is to evaluate CYFRA 21-1's efficacy in distinguishing OSCC., Methods: Systematic searches of Web of Science, PubMed, and CNKI (1996-2024) were conducted following the Preferred Reporting ltems for Systematic Reviews and Meta-Analyses (PRISMA) guidelines., Results: Analysis of 693 patients and 548 controls yielded combined sensitivity (SEN) of 0.71 (95% CI: 0.68, 0.75), specificity (SPE) of 0.88 (95% CI: 0.85, 0.90), and area under the curve (AUC) of 0.927. Subgroup analysis showed higher SEN (0.88), SPE (0.93), and AUC (0.962) in saliva versus serum. Enzyme-linked immunosorbent assay (ELISA) demonstrated superior performance over electrochemiluminescent immunoassay (ECLIA) (AUC: 0.968 vs. 0.868)., Conclusion: CYFRA 21-1 is effective in OSCC diagnosis, with ELISA showing better sensitivity. Saliva emerges as a promising diagnostic medium compared to serum., Registration: PROSPERO CRD42024566835.

Bimetallic nanomaterial-based systems have been widely utilized across various fields due to their remarkable expandability and flexibility, including nanomedicine, diagnostics, and molecular information technology. Here, we constructed an electrochemical immunosensor using bimetallic gold/silver functionalized carbon spheres (AuAg@CSs) and mesoporous silica nanoparticles (MSNs) for the sensitive determination of cytokeratin 19 fragment antigen 21-1 (CYFRA 21-1) and ensuring information protection for textual data. The AuAg@CSs demonstrated exceptional catalytic activity towards hydrogen peroxide, generating a significant current signal. The introduction of CYFRA 21-1 facilitated the binding of MSNs, thereby forming a sandwich-type electrochemical immunosensor that resulted in a notable decrease in current. Notably, the detection limit for CYFRA 21-1 was determined to be 31 fg mL -1 , accompanied by high selectivity. Furthermore, extensive textual information can be encrypted and concealed within the current responses of the electrochemical nanosensing system. By establishing a threshold, these current signals can be represented as a series of binary strings, which can subsequently be segmented into shorter strings. Through information coding methods, these shorter binary strings can be assembled and decrypted, ultimately merging into meaningful textual content. This study promotes the synthesis and multifunctional application of bimetallic nanomaterials, providing innovative solutions to enhance the sensing sensitivity of electrochemical immunosensors and paving the way for advancements in molecular digitization.

Lung cancer is the main cancer that endangers human life worldwide, with the highest mortality rate. The detection of lung tumor markers is of great significance for the early diagnosis and subsequent treatment of lung cancer. In this study, a vertical graphene field effect transistor (VGFET) immunosensor based on graphene/C 60 heterojunction was created to offer quantitative detections for the lung tumor markers carcinoembryonic antigen (CEA), cytokeratin 19 fragment (Cyfra21-1), and neuron-specific enolase (NSE). The experimental results showed that the sensitive range for standard antigen is between 1 pg/ml to 100 ng/ml, with a limit of detection (LOD) of 5.6 amol/ml for CEA, 33.3 amol/ml for Cyfra 21-1 and 12.8 amol/ml for NSE (1 pg/ml for all). The detection accuracy for these tumor markers was compared with the clinically used method for clinical patients on serum samples. Results are highly consistent with clinically used immunoassay in its efficient diagnosis concentration range. Subsequently, the mesoporous silica nanospheres (MSNs) with an average size of 90 nm were surface modified with glutaraldehyde, and a second antibody was assembled on MSNs, which fixes nanospheres on the antigen and amplified the field effect. The LODs for three markers are 100 fg/ml (0.56 amol/ml for CEA) under optimal circumstances of detection. This result indicates that specific binding to MSNs enhances local field effects and can achieve higher sensing efficiency for tumor marker detection at extremely low concentrations, providing effective assistance for the early diagnosis of lung cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Abstract: Ambiguous melanocytic lesions/tumors (AMLs) can be simply described as melanocytic neoplasms that cannot be differentiated as either a melanoma or a nevus. Preferentially expressed antigen in melanoma (PRAME) is a novel antibody that can help differentiate between nevi and melanomas. However, its usefulness remains controversial in AMLs. The aim of this study was to demonstrate the importance of PRAME and diagnostic auxiliary antibodies (Ki-67, p16, HMB-45) in the diagnosis of melanocytic lesions, especially in AMLs. This study included 52 ambiguous melanocytic lesions, 40 nevi, and 40 melanomas. All immunohistochemical studies were performed automatically using the Universal Alkaline Phosphatase Red Detection Kit. Different analytic approaches were used for each antibody based on the literature. Statistically, the multinomial forward stepwise elimination logistic regression analysis was used to create a statistical model to predict the diagnosis of melanocytic lesions based on clinical, morphological, and immunohistochemical data. PRAME positivity was very strong and diffuse in the melanoma group and statistically significantly higher than that of the AML and nevus groups. There was no statistically significant difference between the nevus and AML groups. The Ki-67 proliferation index and HMB-45 staining pattern provided valuable indications for distinguishing between these 3 groups. The P16 antibody was limited in supporting the differential diagnosis. Our statistical model showed that a high mitosis count, central pagetoid spread, and PRAME positivity increased the probability of melanoma against an AML diagnosis. This study showed the advantages of evaluating the PRAME antibody together with morphological features and other immunohistochemical markers (Ki-67 and HMB-45) in the differential diagnosis of melanocytic lesions., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Aims: In this proof-of-concept study, we propose a new method for automated digital quantification of PRAME (PReferentially expressed Antigen of MElanoma) as a diagnostic aid to distinguish between benign and malignant melanocytic lesions. The proposed method utilizes immunohistochemical virtual double nuclear staining for PRAME and SOX10 to precisely identify the melanocytic cells of interest, which is combined with digital image analyse to quantify a PRAME-index., Methods: Our study included 10 compound nevi, 3 halo nevi, and 10 melanomas. Tissue slides were stained with PRAME, scanned, the cover glass removed, stained with SOX10, scanned again, and finally analysed digitally. The digitally quantified PRAME-index was compared with a manual qualitative assessment by a dermatopathologist using the standard PRAME-scoring system., Results: The digitally quantified PRAME-index showed a sensitivity of 70 % and a specificity of 100 % for separating melanomas from benign lesions. The manual qualitative PRAME-score showed a sensitivity of 60 % and a specificity of 100 %. Comparing the two methods using ROC-analyses, our digital quantitative method (AUC: 0.931, 95 % CI: 0.834;1.00, SD: 0.050) remains on par with the manual qualitative method (AUC: 0.877, 95 % CI: 0.725;1.00, SD: 0.078)., Conclusion: We found our novel digital quantitative method was at least as precise at classifying lesions as benign or malignant as the current manual qualitative assessment. Our method has the advantages of being operator-independent, objective, and replicable. Furthermore, our method can easily be implemented in an already digitalized pathology department. Given the small cohort size, more studies are to be done to validate our findings., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mette Bak Brogard reports financial support was provided by Harboefonden. Mette Bak Brogard reports financial support was provided by Torben and Alice Frimodts Fund. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Abstract: Microsatellitosis is well established as a prognostic factor in malignant melanoma. Its identification leads to subsequent upstaging with implications for further management. We describe 6 cases in which immunohistochemical staining for PReferentially expressed Antigen in MElanoma facilitated detection of small foci of micrometastasis on scanning magnification, which may be potentially missed in routine sections., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Antibody-drug conjugates (ADCs) directed to trophoblast cell surface antigen 2 (TROP2) have gained approval as a therapeutic option for advanced triple-negative breast cancer, and TROP2 expression has been linked to unfavourable outcomes in various malignancies. In colorectal carcinoma (CRC), there is still a lack of comprehensive studies on its expression frequency and its prognostic implications in relation to the main clinicopathological parameters. We examined the expression of TROP2 in a large cohort of 1,052 CRC cases and correlated our findings with histopathological and molecular parameters, tumour stage, and patient outcomes. TROP2 was heterogeneously expressed in 214/1,052 CRCs (20.3%), with only a fraction of strongly positive tumours. TROP2 expression significantly correlated with an invasive histological phenotype (e.g. increased tumour budding/aggressive histopathological subtypes), advanced tumour stage, microsatellite stable tumours, and p53 alterations. While TROP2 expression was prognostic in univariable analyses of the overall cohort (e.g. for disease-free survival, p < 0.001), it exhibited distinct variations among important clinicopathological subgroups (e.g. right- versus left-sided CRC, microsatellite stable versus unstable CRC, Union for International Cancer Control [UICC] stages) and lost its significance in multivariable analyses that included stage and CRC histopathology. In summary, TROP2 is quite frequently expressed in CRC and associated with an aggressive histopathological phenotype and microsatellite stable tumours. Future clinical trials investigating anti-TROP2 ADCs should acknowledge the observed intratumoural heterogeneity, given that only a subset of TROP2-expressing CRC show strong positivity. The prognostic implications of TROP2 are complex and show substantial variations across crucial clinicopathological subgroups, thus indicating that TROP2 is a suboptimal parameter to predict patient prognosis., (© 2024 The Author(s). The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.)

In this article, ferric ion-doped floral graphite carbon nitride (Fe-CN-3, energy donor) was used to construct the substrate of the immunosensor and copper oxide nanocubes (Cu 2 O, energy acceptor) were taken as an efficient ECL quenching probe. A sandwich quench electrochemiluminescence (ECL) immunosensor for soluble cytokeratin 19 fragment (Cyfra21-1) detection was preliminarily developed based on a novel resonant energy transfer donor-acceptor pair. Fe-CN-3, a carbon nitride that combines the advantages of metal ion doping as well as morphology modulation, is used in ECL luminophores to provide more excellent ECL performance, which makes a significant contribution to the application and development of carbon nitride in the field of ECL biosensors. The regular shape, high specific surface area and excellent biocompatibility of the quencher Cu 2 O nanocubes facilitate the labeling of secondary antibodies and the construction of sensors. Meanwhile, as an energy acceptor, the UV absorption spectrum of Cu 2 O can overlap efficiently with the energy donor's ECL emission spectrum, making it prone to the occurrence of ECL-RET and thus obtaining an excellent quenching effect. These merits of the donor-acceptor pair enable the sensor to have a wide detection range of 0.00005-100 ng/mL and a low detection limit of 17.4 fg/mL (S/N = 3), which provides a new approach and theoretical basis for the clinical detection of lung cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: This research aims to examine the expression of carbonic anhydrase IX (CAIX) protein in hemangioblastoma of the central nervous system and its potential application in pathological diagnosis and differential diagnosis., Materials and Methods: Immunohistochemistry was used to identify the expression of CAIX and the α-inhibin protein. The sensitivity and specificity of CAIX and α-inhibin for identifying hemangioblastoma of the central nervous system were compared. In addition, 86 patients with meningiomas were gathered to detect CAIX protein expression. Hemangioblastoma and angiomatous, microcystic the two subtypes of meningiomas, were compared for CAIX and EMA protein expression., Results: In hemangioblastoma, there were significant differences in the median positive percentage and staining intensity of CAIX and α-inhibin (p < 0.05). There was no discernible difference in the expression of the CAIX protein between sporadic hemangioblastoma of the central nervous system and those linked to von Hippel‒Lindau disease. In comparison to angiomatous and microcystic meningiomas, the positive rate of CAIX in hemangioblastomas was substantially greater (p < 0.001). The expression of EMA in microcystic meningioma (6/6) and angiomatous meningioma (17/17) was significantly different from hemangioblastoma (0/30) (p < 0.0001)., Conclusion: Hemangioblastoma might be diagnosed with high specificity and sensitivity through CAIX immunohistochemistry. The combination of CAIX with EMA is useful for the diagnosis and differential diagnosis of hemangioblastoma.

Introduction: The Preferentially Expressed Antigen in Melanoma (PRAME) immunostain has seen significant diagnostic use in confirming malignancy for melanocytic lesions. However, the expression of PRAME in genital melanocytic lesions have not been reported. In this study, PRAME staining was performed on a cohort of genital melanocytic lesions, aiming to investigate the diagnostic role of PRAME in genital melanocytic lesions and its expression in atypical genital nevi., Methodology: A cohort including genital invasive melanoma, melanoma-in-situ, atypical genital nevus (AGN), compound nevus, intradermal nevus, blue nevus, lentigo and melanosis was retrieved with histology reviewed and PRAME immunostaining performed., Results: A total of 66 cases were reviewed. The average proportion expression of PRAME were 56.75 % and 57.43 % for invasive melanoma and melanoma-in-situ, with average H-scores of 153.5/300 and 163.14/300 respectively, which were greater than AGN (3.25 %, 7.75/300, p<0.001), compound/intradermal nevi, lentigo/melanosis, and background junctional melanocytes (<1 %, <1/300, p<0.001). The different cutoffs of PRAME expression, the sensitivity and specificity were 65.22 % and 100 % (>100/300); 69.57 % and 95.83 % (>10/300); and 82.61 % and 93.75 % (≥1/300) respectively. Low level PRAME expression was seen in half of the cases of AGN (n=2/4, 50 %), and at low cutoffs (>10/300 and ≥1/300) unable to differentiate invasive melanoma from AGN (p>0.05)., Conclusions: For genital melanocytic lesions, PRAME immunostain shows high specificity at strong and diffuse staining. AGN not uncommonly display low level expression. Focal and/or weak PRAME expression should not be considered as an absolute indication of malignancy, and comprehensive histological assessment remains the key to accurate diagnosis of melanocytic lesions., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

The establishment of rapid target analysis methods for cytokeratin fragment antigen 21-1 (CYFRA 21-1) is urgently needed. [Ir(pbi) 2 (acac)] (pbi = 2-(4-bromophenyl)-1-hydrogen -benzimidazole, acac = acetylacetonate) as traditional electrochemiluminescence (ECL) luminophores has been confined due to its non-negligible dark toxicity and poor water solubility leading to poor biocompatibility and electrical conductivity as an organic molecule. Hence, to overcome this limitation, [Ir(pbi) 2 (acac)] can be effectively loaded on the polyvinyl alcohol hydrogel modified Ti 3 C 2 T x MXene surface (Ir@Ti 3 C 2 T x -PVA) as sensing platform which can emit high ECL signals. Then, a quenching strategy was proposed to fabricate an ECL sandwich immunosensor using H 2 O 2 as quencher molecules which can generated by Pd@Au 0.85 Pd 0.15 . Especially, the generation of O 2 to H 2 O 2 can be achieved through a two-electron (2e - ) reaction pathway by Pd@Au 0.85 Pd 0.15 , to overcome the restriction that the H 2 O 2 was virtually impossible to label or immobilize on the non-enzyme nanomaterials. The proposed ECL assay achieves a response to CYFRA 21-1 within the range of 0.1 pg/mL-100 ng/mL, with a detection limit of 8.9 fg/mL (S/N = 3). This work provided a feasible tactic to seek superior-performance ECL luminophore and quencher consequently set up a novel means to makeup ultrasensitive ECL biosensor, which extended the utilization potential of Ir(pbi) 2 (acac) in ECL assays., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The impact of tumor focality on prostate cancer (PCa) prognosis has been addressed in several studies with conflicting results. Tumor foci from multifocal (MF) PCa can show highly heterogeneous molecular features. Our aim was to analyze the protein expression of PTEN, SPOP, SLC45A3, ETV1, ERG and the "triple hit" (ERG overexpression, PTEN plus SLC45A3 loss) in unifocal (UF) and MF PCa, to evaluate their value as prognostic markers according to focality, and the role of tumor heterogeneity in MF disease. PTEN, SPOP, SLC45A3, ETV1 and ERG immunohistochemical expression was evaluated in 185 PCa from 9 TMAs, 51 UF and 134 MF. In a subset of 69 MF cases, the dominant and secondary foci (DF and SF) were compared. Heterogeneity was considered when both tumor foci presented different expression patterns. Relationship with clinicopathological features was also analyzed. MF PCa was diagnosed in significantly younger patients when compared to UF ones (p = 0.007). ETV1 overexpression was associated with UF disease (p = 0.028). A shorter time to PSA recurrence was related to SLC45A3 wt expression in UF PCa (p = 0.052), and to SPOP expression loss (p = 0.043) or "triple hit" phenotype in MF PCa (p = 0.041). In MF cases, PTEN loss, SLC45A3 loss and "triple hit" phenotype were associated with the DF and had significant heterogeneity. In conclusion, our results indicate that UF and MF PCa have relevant and consistent molecular differences. The analysis of an immunohistochemical panel, composed by PTEN, SPOP, SLC45A3, ETV1 and ERG, could be useful to predict outcome in MF cases., (© 2023. The Author(s).)

Nanohybrid based non-invasive biosensing platforms are emerging as promising alternatives to detect biomarkers in complex and diverse bio-fluids toward ultrasensitive point-of-care diagnostics. Herein, we report the development of a highly sensitive, facile, non-invasive, label free, affordable, and innovative electrochemical screen printed immunosensor for identifying CYFRA 21-1, an established and crucial biomarker for oral cancer. Until now, no work has been reported utilizing a titanium carbide Ti 3 C 2 MXene nanosheet and L-cysteine (L-Cyst) functionalized magnetite nanoparticle (MNPs) nanohybrid based immunosensor for electrochemical detection of CYFRA 21-1. The L-Cyst@MNPs/Ti 3 C 2 -MXene nanohybrid was synthesized via the co-precipitation method and later deposited on a gold screen printed electrode (GSPE) offering enhanced surface area and electrochemical properties. The nanohybrid modified GSPE was then surface immobilized with monoclonal antibodies (anti-CYFRA-21-1) to fabricate an anti-CYFRA-21-1/L-Cyst@MNPs/Ti 3 C 2 -MXene/GSPE immunoelectrode and the non-specific locations of the immunoelectrode were covered with bovine serum albumin (BSA). The spectroscopic, morphological, and structural analyses of the synthesized nanohybrid and the fabricated electrodes were performed using different analytical techniques. The electrochemical studies of modified electrodes were evaluated using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV). The fabricated BSA/anti-CYFRA-21-1/L-Cyst@MNPs/Ti 3 C 2 -MXene/GSPE immunosensor has shown an excellent limit of detection of 0.023 ng mL -1 , a linear detection range of (0.5-30) ng mL -1 , a sensitivity of 277.28 μA (ng mL -1 ) -1 cm -2 and a lower limit of quantification of 0.618 ng mL -1 for electrochemical CYFRA 21-1 determination. Hence, this L-Cyst@MNPs/Ti 3 C 2 -MXene nanohybrid could also be explored as a potential candidate for determining other cancer biomarkers.

Vulvovaginal melanoma (VVM) is a rare but deadly disease, accounting for 5% of all vulvar malignancies, with a 5-yr survival rate of only 47% for all stages of the disease. VVM is a distinct subset of melanoma, with a unique genomic profile and underlying pathogenesis unassociated with sun exposure. Distinguishing these rare malignancies from very common pigmented lesions of the vulva and vagina is challenging as histologic features often overlap between entities. PReferentially expressed Antigen in MElanoma (PRAME) is a melanoma-associated protein, and immunohistochemistry (IHC) for PRAME distinguishes cutaneous, oral mucosal, and retinal melanoma from atypical nevi. Given the biological differences between VVM and cutaneous melanoma, the utility of PRAME IHC for the diagnosis of VVM is unknown. We accrued a cohort of 20 VVM and 21 benign vulvar melanocytic nevi. We found that nuclear PRAME IHC staining with 4+ intensity was present in 85% of the VVM and 0% of the nevi. With the assistance of PRAME IHC, we found evidence of close or positive margin involvement in 3 of 10 cases where margins were originally diagnosed as negative for melanoma in situ. Our study is the first to assess PRAME IHC in a cohort of VVM cases and provides confidence for using PRAME IHC to assist with diagnosis and margin assessment in this rare disease., Competing Interests: The authors declare no conflict of interest., (Copyright © 2023 by the International Society of Gynecological Pathologists.)

Dysplastic nevi represent one of the least agreed-upon entities in dermatopathology despite the existence of established criteria. This study explores preferentially expressed antigen in melanoma (PRAME) in dysplastic nevi, an uncharted area. We examined 22 common melanocytic nevi (CMN), 20 cutaneous melanomas (CM), 48 low-grade dysplastic nevi (LG-DN), and 40 high-grade dysplastic nevi (HG-DN). PRAME was immunohistochemically assessed using a five-tiered system (0 to 4 +). Among CMN, 59% scored 0, 32% scored 1 + , and 9% scored 2 + . CM had score 2 + and 4 + in 11% and 89% of cases, respectively. Among LG-DN, 38% presented score 0, 31% score 1 + , 17% score 2 + , 8% score 3 + , and 6% score 4 + . Thirty per cent of HG-DN demonstrated a score 0, 30% with score 1 + , 15% score 2 + , 10% score 3 + , and 15% score 4 + . Compared to CMN and CM, LG-DN and HG-DN showed heterogeneous expression profiles of PRAME. PRAME positivity effectively distinguished HG-DN from CM with 85% specificity and 80% sensitivity (p < 0.0001). Predictive values were 87% (negative) and 76% (positive). Furthermore, a trend of increased PRAME expression from LG-DN to HG-DN was observed. However, the applicability of PRAME in the differential diagnosis of dysplastic lesions remains unclear as can yield conflicting results with morphology, which remains the primary diagnostic tool for melanocytic lesions., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objective: This study aimed to assess the accuracy of Mac-2 binding protein glycosylation isomer (M2BPGi) in predicting the stage of liver fibrosis., Methods: Articles published until October 10, 2023, were searched in the PubMed, Embase, Web of Science, and Cochrane Library databases. Pooled sensitivity, specificity, diagnostic odds ratio (DOR), summary receiver-operator curves (SROC), and Spearman's rank correlation coefficient were used to examine the accuracy of M2BPGi in predicting the stage of liver fibrosis. A 95% confidence interval (CI) was provided for each estimate., Results: Twenty-four studies were included in this meta-analysis, including 3,839 patients with liver fibrosis, 409 of whom progressed to stage 4 or above. The pooled sensitivity, specificity, and area under the ROC (AUC) for M2BPGi predicting liver fibrosis ≥F3 were 0.74 (95% CI [0.65-0.82]), 0.84 (95% CI [0.76-0.89]), and 14.99 (95% CI [9.28-24.21]), respectively. The pooled sensitivity, specificity, and AUC for ≥F4 were 0.80 (95% CI [0.70-0.88]), 0.80 (95% CI [0.73-0.86]), and 16.43 (95% CI [0.84-0.90]), respectively., Conclusion: Among different sample partitions, M2BPGi has the best diagnostic performance for liver fibrosis stage ≥4. Furthermore, the cutoff of 1-2 is more accurate than that of 0-1 or 2-3 for fibrosis ≥ F3 and ≥ F4., Registration: CRD42023483260., Competing Interests: The authors declare that they have no competing interests., (© 2024 Gong et al.)

Tumor imaging and delivery of therapeutic agents may be achieved by designing high-affinity and high-selectivity compounds recognizing a tumor cell-expressing biomarker, such as carbonic anhydrase IX (CA IX). The CAIX, overexpressed in many hypoxic solid tumors, helps adjust to the energy requirements of the hypoxic environment, reduces intracellular acidification, and participates in the metastatic invasion of adjacent tissues. Here, we designed a series of sulfonamide compounds bearing CAIX-recognizing, high-affinity, and high-selectivity groups conjugated via a PEG linker to near-infrared (NIR) fluorescent probes used in the clinic for optically guided cancer surgery. We determined compound affinities for CAIX and other 11 catalytically active CA isozymes by the thermal shift assay and showed that the affinity K d value of CAIX was in the subnanomolar range, hundred to thousand-fold higher than those of other CA isozymes. Similar affinities were also observed for CAIX expressed on the cancer cell surface in live HeLa cell cultures, as determined by the competition assay. The NIR-fluorescent compounds showed excellent properties in visualizing CAIX-positive tumors but not CAIX-negative knockout tumors in a nude mice xenograft model. These compounds would therefore be helpful in optically guided cancer surgery and could potentially be developed for anticancer treatment by radiotherapy.

Introduction: Previously we demonstrated that elevated serum CYFRA 21 - 1 is a reliable diagnostic and prognostic biomarker for biliary tract cancers. This study aims to explore the diagnostic performance of bile CYFRA 21 - 1 (bCYFRA 21 - 1) in discriminating malignant biliary obstruction (MBO) caused by cholangiocarcinoma (CCA)., Methods: 77 CCA patients ((17 intrahepatic CCA (iCCA), 49 perihilar CCA (pCCA) and 11 distal CCA (dCCA)) and 43 benign patients with biliary obstruction were enrolled. Serum and bile levels of CYFRA 21 - 1, carcinoembryonic antigen (CEA) and carbohydrate antigen 19 - 9 (CA19-9) were quantified. Diagnostic performances of these biomarkers were estimated by receiver operator characteristic curves. Subgroups analysis of these tumor markers among CCA subtypes was performed., Results: High bCYFRA 21 - 1 (cut-off value of 59.25 ng/mL with sensitivity of 0.889 and specificity of 0.750) and high bile to serum ratio of CYFRA 21 - 1 (b/sCYFRA 21 - 1, cut-off value of 31.55 with sensitivity of 0.741 and specificity of 0.778) achieved better diagnostic performance than any other biomarker in discriminating MBO. Subgroup analysis revealed that bCYFRA 21 - 1 was significantly elevated in all CCA subtypes; moreover b/sCYFRA 21 - 1 was upregulated in pCCA and dCCA (the mean b/sCYFRA 21 - 1 of pCCA was highest among CCA subtypes: 57.90, IQR 29.82-112.27)., Conclusions: Both high biliary CYFRA 21 - 1 and high bile to serum ratio of CYFRA 21 - 1 were reliable diagnostic biomarkers for MBO caused by CCA., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: S100A8 is a melanoma biomarker expressed in the melanoma-associated epidermal keratinocytes, but its diagnostic utility has not been compared with other biomarkers, including PRAME., Objectives: To compare the utility of S100A8 and PRAME immunohistochemistry (IHC) in the differential diagnosis of melanoma and naevi in a case-control study., Methods: A previously described cohort of 209 melanomas (case samples) and naevi (control samples) dual-immunostained for S100A8 and PRAME were included. For S100A8, previously reported scores indicating the proportion of tumour-associated epidermis stained (0 = indeterminate; 1 = 0-4%; 2 = 5-25%; 3 = 26-50%; 4 = 51-75%; 5 = > 75%) were utilized. PRAME IHC was reviewed by at least two reviewers and a consensus score assigned, with score indicating the proportion of tumour stained (0 = indeterminate; 1 = 0%; 2 = 1-50%; 3 = > 50%). A positive test was defined as > 50% staining., Results: The area under the receiver operating characteristic curves for S100A8 (0.833) and PRAME (0.874) were not significantly different from each other (P = 0.22). The diagnostic sensitivity and specificity were 42.4% [95% confidence interval (CI) 32.6-52.8%] and 98.2% (95% CI 93.6-99.8%) for S100A8, and 79.8% (95% CI 70.5-87.2%) and 87.3% (95% CI 79.6-92.9%) for PRAME, respectively. A combined test requiring both S100A8 and PRAME IHC positivity had a sensitivity of 39.4% (95% CI 29.7-49.7%) and specificity of 99.1% (95% CI 95.0-100.0%)., Conclusions: S100A8 and PRAME have utility in the diagnostic workup of melanoma, with S100A8 being more specific and PRAME being more sensitive when using this threshold. Our findings suggest that these two immunohistochemical markers may favourably complement one another to improve the detection of melanoma., Competing Interests: Conflicts of interest M.K. reports a pending patent application. The other authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Association of Dermatologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aims: Preferentially expressed antigen in melanoma (PRAME) recently is a reliable immunohistochemistry (IHC) marker for distinguishing melanoma from other lesions. However, there are few articles focused on PRAME use in acral malignant melanoma, the most common type in Asians. This study investigated PRAME IHC expression in a large series of acral malignant melanoma in situ to add to the body of clinical knowledge., Methods: PRAME IHC was performed in unequivocal cases of primary acral lentiginous melanoma in situ (ALMIS), subungual melanoma in situ (SMIS) and acral recurrent nevi as the control. PRAME tumour cell percentage positivity and intensity were expressed as categorised in a cumulative score by adding the quartile of positive tumour cells to intensity labelling. The final IHC expression was interpreted as negative (0-1), weak (2-3), moderate (4-5) or strong (6-7)., Results: In 91 ALMIS patients, 32 cases (35.16%) were strong, 37 (40.66%) were moderate and 22 (24.18%) were weak. In 18 SMIS patients, strong positivity of PRAME was observed in 4 (22.22%) cases, moderate in 10 (55.56%) and weak in the remaining 4 (22.22%). No melanoma sample was negative for PRAME. By comparison, only 2 of the 40 acral recurrent nevi cases were positive., Conclusions: Our study supports the ancillary value of PRAME for diagnosing ALMIS and SMIS with high sensitivity and specificity., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Cytokeratin 19 fragment (CYFRA21-1) serves as a crucial tumor marker in the context of lung cancer patients, playing a pivotal role as a calibrator in the realm of in vitro diagnostics. Nevertheless, during practical application, it has come to light that the recombinantly synthesized full-length CYFRA21-1 antigen exhibits suboptimal stability at the requisite concentration, while the utilization of natural antigens incurs a substantial cost. To address this issue, our investigation harnessed a strategic approach whereby the soluble fragment of cytokeratin 19 (Aa244-400) was integrated into the pET32a vector, subsequently being expressed within E. coli through a fusion with the TrxA protein. This process involved induction of protein expression through 0.2 mM IPTG at 16 °C for a duration of 16 h. After induction, the target protein was purified through Ni affinity and ion exchange chromatography. Subsequent characterization of the targeted protein was executed through the SEC-HPLC technique. The attained CYFRA21-1 antigen, as generated within this study, was effectively incorporated into a chemiluminescence-based in vitro diagnostic detection kit. The results indicate that the fusion protein exhibited commendable reactivity and stability, manifesting a deviation of less than 10 % following incubation at 37 °C for 7 days. Importantly, the production yield achieved a notable magnitude of 300 mg/L, thus rendering it a cost-effective and scalable alternative to natural antigens for clinical diagnostic applications., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Purpose: Prognostic significance and gene signatures associated with carbonic anhydrase IX (CAIX) was investigated in triple negative breast cancer (TNBC) patients., Methods: Immunohistochemistry (IHC) for CAIX was performed in tissue microarrays (TMAs) of 136 TNBC patients. In a subset of 52 patients Digital Spatial Profiler (DSP) was performed in tumour (pan-cytokeratin+) and stroma (pan-cytokeratin-). Differentially expressed genes (DEGs) with P&lt;0.05 and and log2 fold change (FC)>(±0.25 and ±0.3, for tumour and stromal compartment, respectively) were identified. Four genes were validated at the protein level., Result: Cytoplasmic CAIX expression was independently associated with poor recurrence free survival in TNBC patients [hazard ratio (HR)=6.59, 95% confidence interval (CI): 1.47-29.58, P=0.014]. DEG analysis identified 4 up-regulated genes (CD68, HIF1A, pan-melanocyte, and VSIR) in the tumour region and 9 down-regulated genes in the stromal region (CD86, CD3E, MS4A1, BCL2, CCL5, NKG7, PTPRC, CD27, and FAS) when low versus high CAIX expression was explored. Employing IHC, high CD68 and HIF-1α was associated with poorer prognosis and high BCL2 and CD3 was associated with good prognosis., Conclusions: DSP technology identified DEGs in TNBC. Selected genes validated by IHC showed involvement of CD3 and BCL2 expression within stroma and HIF-1α, and CD68 expression within tumour. However, further functional analysis is warranted., (©The Author(s) 2024. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

Background: Tumor markers (TMs) are a heterogeneous group of molecules used in the diagnosis, prognosis and follow-up of cancer patients. During neoplastic differentiation, cells can either directly synthesize or induce the synthesis of TMs, and the release of these molecules into the bloodstream allows their quantification in biological fluids. Although very small concentrations of TMs are usually present in the serum or plasma of healthy subjects, increased concentrations may also be found in the presence of benign diseases or due to technical interference, producing false positive results., Material and Methods and Results: Our review analyses the causes of false positives described between January 1970 to February 2023 for the TMs most frequently used in clinical practice: α-fetoprotein (AFP), β2-microglobulin (β2-M), cancer antigen 15-3 (CA 15-3), cancer antigen CA 19-9 (CA 19-9), cancer antigen CA 72-4 (CA 72-4), cancer antigen 125 (CA 125), carcinoembryonic antigen (CEA), chromogranin A (CgA), choriogonadotropin (hCG), cytokeratin 19 fragment (CYFRA 21-1), neuron-specific enolase (NSE), human epididymis protein 4 (HE4), serum HER2 (sHER2), squamous cell carcinoma antigen (SCCA), protein induced by vitamin K absence-II (PIVKA-II), Pro-gastrin-releasing peptide (Pro-GRP), prostate-specific antigen (PSA), Protein S-100 (S-100) and thyroglobulin (Tg). A total of 247 references were included., Conclusions: A better understanding of pathophysiological processes and other conditions that affect the concentration of TMs might improve the interpretation of results and their clinical application.

Background: The purpose of this study is to explore the clinical value of serum tumor markers combined with dual-source CT scanning in the diagnosis of lung cancer., Methods: One hundred-two patients with lung cancer (malignant tumor group), 50 patients with benign lesions (benign control group) and 50 healthy patients (normal control group) were selected as the research objects. The levels of serum carcinoembryonic antigen (CEA), cytokeratin 19 fragment antigen 21-1 (CYFRA21-1) and gastrin releasing peptide precursor 31-98 (Pro-GRP31-98) were detected using the electrochemiluminescence and enzyme-linked immunoassay in three groups of people. Simultaneously, Siemens' second-generation dual-source CT (Siemens AG, Munich, Germany) is used to scan the lungs of patients with lung cancer and benign lesions. Retrospective statistical analysis is utilized to explore the clinical value of serum tumor markers combined with dual-source CT examination in the diagnosis of lung cancer., Results: The levels of serum CEA, CYFRA21-1 and Pro-GRP31-98 in lung cancer patients were significantly higher than those in the benign control group and normal control group, and the difference was statistically significant (P<0.01). There was no statistical difference between the benign control group and the normal control group (P>0.05). The levels of serum CEA, CYFRA21-1, Pro-GRP31-98 had no significant correlation with the age, sex, and tumor site of lung cancer patients (P>0.05). However, the levels of serum CEA, CYFRA21-1, Pro-GRP31-98 have an obvious correlation with tumor size, clinical stage, histological type, combined pleural effusion, recurrence and metastasis after treatment (P<0.05). CT scans of 102 lung cancer patients showed 35 cases of central type, 67 cases of peripheral type, 71 cases with tumor diameter >5cm, 31 cases of ≤5cm and 67 cases of lymph node metastasis. The sensitivities of CEA, CYFRA21-1, Pro-GRP31-98, and dual-source CT in the diagnosis of lung cancer were 64.70%, 60.11%, 56.86% and 77.45%, and the accuracy was 75.00%, 72.37%, 69.74% and 82.89%, respectively. Compared with a single examination, the sensitivity and accuracy of combined examination for the diagnosis of lung cancer were significantly improved, which were 95.10% and 92.76%, respectively., Conclusions: Joint examinations can effectively improve the rate of lung cancer diagnosis.

Abstract: Acral lentiginous melanoma (ALM) is a relatively rare clinicopathologic subtype of cutaneous malignant melanoma, but it is the most common type of melanoma among Asians. Although the research to identify immunohistochemical (IHC) markers to differentiate nevi from melanoma is being conducted, specific markers for ALM are not well-known. Therefore, we aimed to analyze and compare the differences in the expression of melanocyte-associated IHC markers between ALM and acral benign nevi (ABN). Two independent groups of 53 and 19 paraffin-embedded specimens (from patients with pathologically confirmed ALM and ABN, respectively) were subjected to IHC staining for MART-1, preferentially expressed antigen in melanoma (PRAME), SOX10, HMB-45, Ki-67, and p16. We performed a quantitative analysis of PRAME, SOX10, KI-67, and p16 expression and gradient pattern analysis of HMB-45 expression for each specimen. The PRAME (60.1% and 28.5%, P < 0.05) and Ki-67 (7.8% and 3.5%, P < 0.05) expression levels were significantly higher in the ALM group than in the ABN group. The p16 expression was significantly lower (14.2% and 19.4%, P < 0.05), and the absence of HMB-45 gradient was more frequent in the ALM group than in the ABN group. However, no statistical significance was noted in SOX10 (54.8% and 44.7%). Receiver operating characteristic curves showed that PRAME had the highest area under the curve value. In summary, among various IHC markers, PRAME was the most valuable marker for the diagnosis of ALM; however, further large-scale studies are needed to validate these findings., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Immunohistochemistry-based protein biomarkers can provide useful prognostic information in cutaneous melanoma. The independent prognostic value of Ki-67 has been studied with variable results. PReferentially expressed Antigen in MElanoma (PRAME) immunohistochemistry is a useful new ancillary tool for distinguishing cutaneous nevi from melanoma; however, its prognostic value has not been well studied. We evaluated PRAME as a prognostic marker in cutaneous melanoma, compared to Ki-67., Methods: We analyzed the immunohistochemical expression of PRAME and Ki-67 in 165 melanocytic lesions, including 92 primary melanomas, 19 metastatic melanomas, and 54 melanocytic nevi using tissue microarrays. PRAME immunostaining was scored based on the percentage of positive nuclei: 0 <1%, 1+ 1%-25%, 2+ 26%-50%, 3+ 51%-75%, and 4+ >75%. The percentage of Ki-67-positive tumor nuclei was used to calculate the proliferation index., Results: PRAME and Ki-67 both showed significantly increased expression in melanomas compared to nevi (p < 0.0001 and p < 0.001, respectively). There was no significant difference in PRAME expression in primary versus metastatic melanomas. By contrast, the Ki-67 proliferation index was higher in metastatic melanoma than in primary melanoma (p = 0.013). Increased Ki-67 index correlated with ulceration (p < 0.001), increased Breslow depth (p = 0.001), and higher mitotic rate (p < 0.0001), whereas increased PRAME expression correlated with higher mitotic rate (p = 0.047) and Ki-67 index (p = 0.007). Increased Ki-67 index correlated with worse disease-specific survival in patients with primary melanoma (p < 0.001), but PRAME expression did not show prognostic significance in disease-specific survival (p = 0.63). In a multivariable analysis of patients with primary melanoma, tumor Breslow depth, ulceration, mitotic rate, and Ki-67 index were each independent predictors of disease-specific survival (p = 0.006, 0.02, 0.001, and 0.04, respectively); however, PRAME expression was not predictive of disease-specific survival (p = 0.64)., Conclusion: Ki-67 is an independent prognostic marker; although increased PRAME expression correlates with the Ki-67 proliferation index and mitotic rate, PRAME is not an independent prognostic marker for cutaneous melanoma. PRAME and Ki-67 are useful ancillary tools for distinguishing benign from malignant melanocytic lesions., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: PRAME (PReferentially expressed Antigen in MElanoma) is a tumor-associated antigen that has been studied in various cutaneous melanocytic lesions. p16, on the other hand, has been proposed to aid in distinguishing between benign and malignant melanocytic neoplasms. Studies on the diagnostic utility of PRAME and p16 in combination in differentiating nevi from melanoma are limited. We aimed to assess the diagnostic utility of PRAME and p16 in melanocytic tumors and their role in distinguishing between malignant melanomas and melanocytic nevi., Methods: This is a single-center retrospective cohort analysis over a 4-year period (2017-2020). We used the pathological database of malignant melanomas (77 cases) and melanocytic nevi (51 cases) specimens from patients who underwent shave/punch biopsies or surgical excisions and evaluated immunohistochemical staining percentage positivity and intensity for PRAME and p16., Results: Most malignant melanomas showed positive/diffuse PRAME expression (89.6%); on the other hand, 96.1% of nevi did not express PRAME diffusely. p16 was expressed consistently in nevi (98.0%). However, p16 expression in malignant melanoma was infrequent in our study. PRAME had a sensitivity and specificity of 89.6% and 96.1%, respectively, for melanomas versus nevi; on the other hand, p16 had a sensitivity and specificity of 98.0% and 28.6%, respectively, for nevi versus melanoma. Also, a PRAME+/p16- melanocytic lesion is unlikely to be a nevus where most nevi were PRAME-/p16+., Conclusion: In conclusion, we confirm the potential utility of PRAME and p16 for distinguishing melanocytic nevi from malignant melanomas., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Abstract: Proliferative nodules (PNs) are benign melanocytic proliferations that typically develop within congenital melanocytic nevi. These tumors have overlapping histological features with melanoma. Ancillary immunohistochemistry and genomic sequencing are often used in diagnostically challenging cases. To assess the utility of preferentially expressed antigen in melanoma (PRAME) immunoreactivity and telomerase reverse transcriptase (TERT) promoter mutation analysis in distinguishing PNs from melanoma arising in congenital nevi cases. Twenty-one PNs and 2 melanomas arising in congenital nevi were immunohistochemically stained with PRAME. Cases with adequate tissue were also assessed for TERT promoter mutations through sequencing studies. The positivity rates in the PN cases were compared with those of the melanomas. Two of 21 PN cases were diffusely positive for PRAME (≥75% of the tumor cells positive). Two of 2 melanomas arising in congenital nevus cases were also diffusely PRAME positive. The difference was statistically significant using a Fisher exact test. None of the tumors harbored TERT promoter mutations. PRAME immunohistochemical marker may have diagnostic value in distinguishing diagnostically challenging PNs from melanoma, but diffuse expression is not specific for melanoma., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

PRAME is a novel immunohistochemical marker that aids the diagnosis of melanocytic lesions. Diffuse PRAME positivity suggests melanoma, whereas benign naevi are negative or only weakly positive. However, the factual diagnostic accuracy of PRAME is not well established. Moreover, some studies have suggested that the threshold of 3+/50% positive cells may be more useful in practice than the most widely used cut-off (4+/75% of positive cells). Hence, we performed a systematic review and diagnostic accuracy meta-analysis to evaluate sensitivity, specificity, likelihood ratios and optimal threshold for PRAME in distinguishing benign melanocytic proliferations from melanomas. Twenty-six studies were enrolled into the meta-analysis. A total of 2915 melanocytic lesions were analysed. The optimal threshold for PRAME positivity was estimated at 3.11, which translates into 3+ in practice. Sensitivity and specificity calculated from SROC at the 3+ threshold were 0.735 (0.631-0.818) and 0.915 (0.834-0.958), respectively, compared to 0.679 (0.559-0.957) and 0.957 (0.908-0.981) at the 4+ cut-off. In subgroup analysis, the spitzoid subgroup was characterised by the lowest sensitivity and diagnostic odds ratio of PRAME. Our findings indicate that PRAME immunohistochemistry may serve as an ancillary marker to support the diagnosis of melanoma. Nevertheless, the accuracy of PRAME may be lower in spitzoid neoplasms. Our meta-analysis suggests that the 3+/50% threshold might be more useful in practice than the 4+/75% cut-off, as it shows higher sensitivity with retained satisfactory specificity., (© 2023 John Wiley & Sons Ltd.)

This study aimed to design a sandwich electrochemiluminescence (ECL) immunosensor with double co-reaction accelerators for sensitively detecting squamous cell carcinoma antigen (SCCA). First, silver orthophosphate (Ag 3 PO 4 ) nanoparticles were modified on the surface of EuPO 4 nanowires to improve their poor dispersibility/solubility. At the same time, EuPO 4 was used as a co-reaction accelerator to catalyze S 2 O 8 2- to produce more intermediates (SO 4 •- ), significantly enhancing the ECL signal of Ag 3 PO 4 . Ag nanoparticles (AgNP) modified on Ag 3 PO 4 @EuPO 4 composite nanomaterials were used not only as linkers of luminescence groups and biomarkers but also as a co-reaction accelerator to effectively enhance ECL signal. The designed ECL immunosensor displayed several advantages, including good stability and reproducibility. Under the optimal conditions, its linear range in detecting SCCA was 0.0001-50 ng·mL -1 , the detection limit was 25 fg·mL -1 (S/N = 3), the recovery was 96.6-100.4%, and the relative standard deviation was less than 4.8%. It was successfully applied to detect SCCA in human serum., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

PRAME (PReferentially expressed Antigen in MElanoma), a cancer-testis antigen expressed in normal and neoplastic tissues with several functions, proved to be a useful diagnostic tool in the differential diagnosis between benign and malignant melanocytic lesions. The current study aims to perform PRAME stain on a retrospective case series of mucosal melanocytic tumors of the head and neck region to compare 3 different scores and evaluate the most reliable one in this diagnostic set. Immunohistochemical analysis for PRAME was performed in 54 benign and malignant mucosal melanocytic tumors of the head and neck region collected from 41 patients. The best-performing cutoff of PRAME-positive cells (nuclear stain) to differentiate benign and malignant mucosal melanocytic tumors of the head and neck region is that proposed by Raghavan and colleagues (<60%/≥60% of PRAME-positive cells), with 100% and 77.8% of benign lesions and malignant tumors respectively correctly identified. Applying this score, PRAME stain showed the best results (sensitivity, specificity, accuracy, and positive and negative predictive values) for the diagnosis of head and neck melanocytic tumors. However, a subset of PRAME-negative malignant tumors was identified, especially located in the palatal area (hard and soft palate). Finally, high PRAME expression (≥60%) was associated with specific sites (nasal cavity/nasal septum/turbinates nasopharynx, and the maxillary sinus), nodular histotype, and female sex., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Purpose: Hypoxia is a characteristic of many solid tumours and an adverse prognostic factor for cancer therapy. Hypoxia results in upregulation of carbonic anhydrase IX (CAIX) expression, a pH-regulating enzyme. Many human tissue studies have examined the prognostic value of CAIX expression in breast cancer but have yielded inconsistent results. Therefore, a systematic review and meta-analysis was undertaken to assess the prognostic value of CAIX expression for breast cancer patients., Methods: The electronic databases were systematically searched to identify relevant papers. The clinical outcomes included disease-free survival (DFS), recurrence-free survival (RFS) and overall survival (OS) in breast cancer patients. Review Manager version 5.4 was employed to analysis data from 23 eligible studies (containing 8390 patients)., Results: High CAIX expression was associated with poorer RFS [HR = 1.42, 95% CI (1.32-1.51), p < 0.00001], DFS [HR = 1.64, 95% CI (1.34-2.00), p < 0.00001], and OS [HR = 1.48, 95% CI (1.22-1.80), p < 0.0001]. Heterogeneity was observed across the studies. There was an effect of the CAIX antibody employed, scoring methods, and tumour localisation on CAIX expression., Conclusion: CAIX overexpression was significantly associated with poorer RFS, DFS, and OS in breast cancer patients. However, further work in high quantity tissue cohorts is required to define the optimal methodological approach., (© 2023. The Author(s).)

This study examined PRAME (preferentially expressed antigen in melanoma) expression by immunohistochemistry and reverse transcription quantitative PCR (RT-qPCR) in 202 histologically unequivocal conjunctival melanocytic lesions: 76 nevi, 29 benign melanoses, 25 low-grade conjunctival intraepithelial melanocytic lesions (LGCMIL), 26 high-grade conjunctival melanocytic intraepithelial lesions/in-situ melanoma (HGCMIL), and 46 invasive melanomas. PRAME score 0 was seen in 96% of nevi (73/76), 96% of benign melanoses (28/29), and 88% of LGCMIL (22/25). PRAME score 4 was seen in 50% HGCMIL (13/26) and 76% invasive melanomas (35/46). PRAME score 4 had a sensitivity of 50% and specificity of 100% in differentiating between HGCMIL and benign melanosis/LGCMIL. PRAME score 4 had a sensitivity of 76% and specificity of 100% in differentiating between melanoma and nevi. Relative quantification of PRAME mRNA expression by RT-qPCR was performed on 49 cases (24%): 17 nevi, 3 benign melanoses, 5 LGCMIL, 9 HGCMIL, and 15 invasive melanomas. The analysis generated two distinct groupings with 'high' relative PRAME expression for the HGCMIL and invasive melanoma and 'low/zero' expression for nevi, benign melanosis, and LGCMIL. Thirty-three challenging conjunctival melanocytic lesions that had previous fluorescence in situ hybridization (FISH) analysis were studied: 18 nevi, 12 melanomas in a nevus, 2 nevoid melanomas, and 1 in-situ melanoma. All nevi (100%) showed concordance between negative FISH and PRAME (scores 0-3). Four of 13 melanomas (31%; in-situ, invasive, isolated, and in association with nevus) showed concordance between positive FISH and PRAME score 4. In conclusion, PRAME score 4 has 100% specificity for the diagnosis of HGCMIL and melanoma. PRAME is limited in its sensitivity in the evaluation of challenging melanocytic lesions., (Copyright © 2023 Elsevier Inc. All rights reserved.)

PRAME (PReferentially expressed Antigen in MElanoma) is a cancer testis antigen that is frequently expressed in melanoma compared to benign melanocytic proliferations and nevi. However, the interpretation of the intensity and distribution of PRAME immunostaining is not standardized a lot, which makes interpretation difficult. PRAME-stained histological slides of superficial spreading melanomas (SSM) and dysplastic nevi (DN) were digitized and analyzed using the digital pathology and image platform QuPath. t -tests and ROC AUCs were performed with SPSS. A p -value of <0.05 was used for statistical significance, and a ROC AUC score of >0.8 was considered a good result. A cut-off score was defined in an evaluation cohort and subsequently analyzed in an independent validation cohort. In total, 81 PRAME-stained specimens were included. The evaluation cohort included 32 (50%) SSM and 32 (50%) DN, and the mean of PRAME-positive cells/mm 2 for the entire lesion was 455.3 (SD 428.2) in SSM and 60.5 (SD 130.1; p < 0.001) in DN. The ROC AUC of PRAME-positive cells of the entire lesion was 0.866, and in the epidermis it was 0.901. The defined cut-off score to distinguish between DN and SSM was 97.67 cells/mm 2 . In the validation cohort, 16 out of 17 cases (94.1%) were correctly classified by the cut-off score. The computer-aided assessment of PRAME immunostaining is a useful tool in dermatopathology to distinguish between DN and SSM. Lesions with a moderate expression and indifferent morphologic features will remain a challenge for dermatopathologists.