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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.

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.)

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.)

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.)

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.)

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.)

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.)

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.

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.)

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.)

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.

Purpose: Accurate identification of nodal status enables adequate neck irradiation for nasopharyngeal carcinoma (NPC). However, most conventional techniques are unable to pick up occult metastases, leading to underestimation of tumor extensions. Here we investigate the clinical significance of carbonic anhydrase IX (CAIX) in human NPC samples, and develop a CAIX-targeted imaging strategy to identify occult lymph node metastases (LNMs) and extranodal extension (ENE) in animal studies., Methods: A total of 211 NPC samples are performed CAIX staining, and clinical outcomes are analyzed. The metastatic murine models are generated by foot pad injection of NPC cells, and a CAIX-targeted imaging agent (CAIX-800) is intravenously administered. We adopt fluorescence molecular tomography and ultrasonography (US)-guided spectroscopic photoacoustic (sPA) imaging to perform in vivo studies. Histological and immunohistochemical characterization are carried out via node-by-node analysis., Results: For clinical samples, 90.1% (91/101) primary tumors, 73.3% (66/90) metastases, and 100% (20/20) local recurrences are CAIX positive. In metastases group, 84.7% (61/72) nodal metastases and 22.2% (4/18) organ metastases are CAIX positive. CAIX expression in primary tumors is significantly associated with NPC stage and prognosis. For animal studies, CAIX-800-based fluorescence imaging achieves 81.3% sensitivity and 93.8% specificity in detecting occult LNMs in vivo, with a minimum detectable diameter of 1.7 mm. Coupled with CAIX-800, US-guided sPA imaging could not only detect subcapsular deposits of metastatic cancer cells 2 weeks earlier than conventional techniques, but also successfully track pathological ENE., Conclusion: CAIX remarkably expresses in human NPCs and stratifies patient prognosis. In preclinical studies, CAIX-800-based imaging successfully identifies occult LNMs and tracks early stage of pathological ENE. This attractive method shows potential in clinic, allowing medical workers to longitudinally monitor nodal status and helping to reduce unnecessary nodal biopsy for patients with NPC. The schematic diagram for the study. CAIX, carbonic anhydrase IX; NPC, nasopharyngeal carcinoma; US, ultrasonography; sPA, spectroscopic photoacoustic., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex class I (MHC-I) molecules 1-5 . Current approaches to profiling of MHC-I-associated peptides, collectively known as the immunopeptidome, are limited to in vitro investigation or bulk tumour lysates, which limits our understanding of cancer-specific patterns of antigen presentation in vivo 6 . To overcome these limitations, we engineered an inducible affinity tag into the mouse MHC-I gene (H2-K1) and targeted this allele to the Kras LSL-G12D/+ Trp53 fl/fl mouse model (KP/K b Strep) 7 . This approach enabled us to precisely isolate MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma and from lung adenocarcinoma (LUAD) in vivo. In addition, we profiled the LUAD immunopeptidome from the alveolar type 2 cell of origin up to late-stage disease. Differential peptide presentation in LUAD was not predictable by mRNA expression or translation efficiency and is probably driven by post-translational mechanisms. Vaccination with peptides presented by LUAD in vivo induced CD8 + T cell responses in naive mice and tumour-bearing mice. Many peptides specific to LUAD, including immunogenic peptides, exhibited minimal expression of the cognate mRNA, which prompts the reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance 8 . Beyond cancer, the K b Strep allele is compatible with other Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease and autoimmunity., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Purpose: Topoisomerase II alpha (TOP2A) has been identified as a proliferation marker, of which the most common method for detection is immunohistochemistry (IHC). However, the optimal cut-off of TOP2A expression regarding prognostic value remains controversial. This study was to identify the optimal cut-off value of TOP2A expression and its correlation with clinicopathological variables and prognosis in early stage breast cancer in China., Methods: Between January 2013 and January 2015, a total of 1084 early breast cancer patients were enrolled. The optimal cut-off of TOP2A expression was assessed using the minimum P value approach. Correlations between TOP2A expression and clinicopathological characteristics were explored by the Spearman's correlation analysis, while the impact of TOP2A expression on disease-free survival (DFS) and overall survival (OS) was evaluated by the Kaplan-Meier methods. Univariate and multivariate Cox regression analyses were executed to identify statistically significant prognostic factors., Results: The optimal cut-off value of TOP2A was recommended as 15%. Overall, 603 (55.6%) patients were TOP2A over-expression and 481 (44.4%) patients were TOP2A low expression. TOP2A over-expression was in positive associations with a higher Ki67 index (r = 0.83, P < 0.001), HER2 positive (r = 0.26, P < 0.001), a larger tumor size (r = 0.14, P < 0.001), and a higher histologic grade (r = 0.59, P < 0.001), and in a significantly negative correlation with hormone receptor (HR) positive expression (r =  - 0.40, P < 0.001) in early breast cancer. TOP2A over-expression significantly associated with worse DFS (P = 0.001) and OS (P < 0.001) and was an independent prognostic factor for both DFS (hazard ratio [HR] = 2.04; 95% confidence interval [95% CI] 1.30-3.18, P = 0.0018) and OS (HR = 3.54; 95%CI 1.53-8.23, P = 0.003) in stage I-II breast cancer patients., Conclusion: To our knowledge, this is the first study to recommend the optimal cut-off value of TOP2A expression in breast cancer. The TOP2A expression is significantly correlated with HER2 status, Ki67 index, tumor size, histologic grade and HR status, and could be a surrogate indicator for poor prognosis of early breast cancer., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

PRAME (PReferentially expressed Antigen in MElanoma), a cancer testis antigen expressed in low levels in gonadal, endometrial, and adrenal gland tissues, has been recently considered a valuable tool in the differential diagnosis between benign and malignant melanocytic lesions. The aim of the current study is to perform PRAME immunostaining on a large series of benign and malignant acral lesions to evaluate the reproducibility of data reported in the literature and to validate PRAME as an affordable tool in the differential diagnosis between benign and malignant acral melanocytic tumors. Immunohistochemical analysis for PRAME was performed in 127 benign and malignant acral and nail melanocytic lesions. To better correlate PRAME expression with the nature (benign vs. malignant) of the lesions, we categorized PRAME tumor cells percentage positivity and intensity in a cumulative score obtained by adding the quartile of positive tumor cells (0, 1+, 2+, 3+, 4+) to PRAME expression intensity in tumor cells (0, 1+, 2+, 3+). Adopting an arbitrary PRAME expression score of < 5 versus ≥5 resulted in a correct identification of 82.5% of benign and 87.1% of malignant lesions. PRAME immunohistochemistry demonstrated good sensitivity and specificity in the diagnosis of acral melanocytic lesions, however, in line with the previous literature, we identified a subset of challenging cases such as acral Spitz nevi, in situ melanomas, and small, thin, invasive melanomas in which PRAME did not correlate with morphologic features. This suggests that PRAME can be a valid tool to be incorporated in a diagnostic clinicopathologic algorithm, subject to morphologic characteristics., 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 © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

The prostate cancer antigen 3 (PCA3) is a long non-coding RNA (lncRNA) with high level of specificity for prostate cancer. This lncRNA has fundamental effects in the prostate carcinogenesis through modulation of expression of miR-132-3p, miR-1261, SREBP1, PRKD3 and LAP2α as well as regulation of p53 signaling. Expression of PCA3 in prostate cancer cells can be enhanced by Snail. Moreover, in vitro studies have documented up-regulation of PCA3 in three other types of neoplastic cells, namely those being originated from choriocarcinoma, ovarian carcinoma and thyroid carcinoma. The diagnostic value of PCA3 in differentiation of prostate cancer from benign prostate hyperplasia has been assessed in different studies. Studies aimed at identification of diagnostic power of PCA3 in prostate cancer using receiver operating characteristic curves have reported area under curve values ranging from 0.66 to 0.86. In the current review, we describe the role of PCA3 in the carcinogenesis particularly in the pathoetiology of prostate cancer. Moreover, we review the results of studies appraising diagnostic value of this lncRNA in prostate cancer., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Air pollution might affect atherosclerosis through DNA methylation changes in cells crucial to atherosclerosis, such as monocytes. We conducted an epigenome-wide study of DNA methylation in CD14+ monocytes and long-term ambient air pollution exposure in adults participating in the Multi-Ethnic Study of Atherosclerosis (MESA). We also assessed the association between differentially methylated signals and cis -gene expression. Using spatiotemporal models, one-year average concentrations of outdoor fine particulate matter (PM 2.5 ) and oxides of nitrogen (NO X ) were estimated at participants' homes. We assessed DNA methylation and gene expression using Illumina 450k and HumanHT-12 v4 Expression BeadChips, respectively (n = 1,207). We used bump hunting and site-specific approaches to identify differentially methylated signals (false discovery rate of 0.05) and used linear models to assess associations between differentially methylated signals and cis -gene expression. Four differentially methylated regions (DMRs) located on chromosomes 5, 6, 7, and 16 (within or near SDHAP3, ZFP57, HOXA5 , and PRM1 , respectively) were associated with PM 2.5 . The DMRs on chromosomes 5 and 6 also associated with NO X . The DMR on chromosome 5 had the smallest p-value for both PM 2.5 (p = 1.4×10 -6 ) and NO X (p = 7.7×10 -6 ). Three differentially methylated CpGs were identified for PM 2.5 , and cg05926640 (near TOMM20 ) had the smallest p-value (p = 5.6×10 -8 ). NO X significantly associated with cg11756214 within ZNF347 (p = 5.6×10 -8 ). Several differentially methylated signals were also associated with cis -gene expression. The DMR located on chromosome 7 was associated with the expression of HOXA5, HOXA9 , and HOXA10 . The DMRs located on chromosomes 5 and 16 were associated with expression of MRPL36 and DEXI , respectively. The CpG cg05926640 was associated with expression of ARID4B, IRF2BP2 , and TOMM20 . We identified differential DNA methylation in monocytes associated with long-term air pollution exposure. Methylation signals associated with gene expression might help explain how air pollution contributes to cardiovascular disease.

Background: COVID-19 survivors report residual lung abnormalities after discharge from the hospital. The aim of this study was to identify biomarkers in serum and induced sputum samples from patients after hospitalization for COVID-19., Methods: Patients admitted to hospitals in Spain with laboratory-confirmed COVID-19 were recruited for this study. SARS-CoV-2-infected patients were divided into groups with mild/moderate and severe disease according to the severity of their symptoms during hospitalization. Levels of 92 biomarkers were measured in serum and induced sputum samples., Results: A total of 108 patients (46.2% severe cases) were included in this study. The median number of days after the onset of symptoms was 104. A significant difference was observed in diffusing capacity for carbon monoxide (DLCO), an indicator of lung function, whereby DLCO <80% was significantly lower in severe cases (p <0.001). Differences in inflammatory biomarkers were observed between patients with mild/moderate and severe disease. For some biomarkers, correlations in serum and induced sputum levels were detected. Independent predictors of severe disease were DLCO <80% and the serum CDCP1 value., Conclusions: Higher levels of CDCP1 remain after hospital discharge and are associated with the severity of COVID-19. The possible prognostic implications warrant further investigation.

Screening new electrochemiluminescence (ECL) emitters for the design of sensitive detection strategies with even long emission wavelength is intensively anticipated in ECL evolution. Herein, a promising modification strategy for improving the ECL performance of Au nanoclusters (AuNCs) as a water-soluble luminophore was proposed. Upon the introduction of l-cysteine (l-Cys) onto the surface of glutathione (GSH)-stabilized AuNCs (GSH-AuNCs), the dual-thiol bond between l-Cys and GSH was formed to limit the intramolecular motion and nonradiative relaxation of the excited state from the capping agents, which resulted in the enhancement of monochromatic ECL emission of GSH-AuNCs with a red-shifted wavelength. By utilizing triethylamine as a coreactant, the ECL of l-Cys/GSH-AuNCs was about 1.5-fold stronger than that of GSH-AuNCs, and the emission wavelength red-shifted from 660 to 780 nm at a relatively low potential, which could decrease the interference in bioassay and the photochemical damage in nondestructive detection. As a proof of application, a sandwich-type immunosensing method for CYFRA 21-1 was proposed with l-Cys/GSH-AuNCs as the signal tag, which displayed a wide linear ranging from 0.2 fg/mL to 2 ng/mL and a limit of detection down to 0.067 fg/mL at 3S/N. This work provides a wonderful strategy for promoting the performance of ECL emitters.

Background: Many dermatopathologists find conjunctival melanocytic proliferations challenging because they are rare relative to their cutaneous counterparts and have nuanced morphology and nomenclature. PRAME immunohistochemistry has been widely adopted for distinguishing cutaneous nevi from melanoma, but limited data exist assessing its utility in evaluating conjunctival specimens. In particular, it is uncertain whether it can predict the risk of melanoma progression in primary acquired melanosis (PAM)., Methods: Thirty clinically annotated cases (two melanomas, three PAM with atypia, seven PAM without atypia, 15 nevi, two combined nevi, and a diagnostically challenging nevus with atypical features) were retrospectively evaluated with PRAME., Results: Strong, diffuse PRAME expression was present in melanomas and PAM with high-grade atypia, but not in PAM with low-grade atypia, PAM without atypia, or nevi. Scattered, faintly PRAME-positive intraepithelial melanocyte nuclei were identified in six nevi. A clonal nevus and nests of heavily pigmented type-A melanocytes in two additional nevi had cytoplasmic staining., Conclusions: PRAME was useful for distinguishing melanoma and its probable precursors from benign conjunctival melanocytic proliferations in our cohort. The data alert us to two diagnostic pitfalls in nevi: scattered, PRAME-positive intraepithelial melanocytes and cytoplasmic PRAME staining in type-A melanocytes and melanophages. Larger scale investigations are warranted to further substantiate these promising findings., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Abstract: Preferentially expressed antigen in melanoma (PRAME) is a tumor-associated repressor of retinoic acid signaling which is expressed in melanoma and has emerged as a potential biomarker for malignant behavior in melanocytic neoplasms. Although ancillary molecular techniques such as fluorescence in situ hybridization (FISH) are established techniques in the diagnosis of problematic cutaneous melanocytic proliferations, they are expensive, time-consuming, and require appropriate infrastructure, which places them out of reach of some laboratories. The advent of readily available commercial antibodies to PRAME has the potential to provide a more accessible alternative. The aim of this study was to determine whether immunohistochemistry for PRAME could serve as a surrogate for FISH analysis in a subgroup of challenging superficial melanocytic proliferations. Cases which had previously been submitted for FISH analysis were stained for PRAME and interpreted by a panel of at least 3 dermatopathologists is a blinded fashion. Of a study set of 55 cases, 42 (76%) showed a pattern of PRAME immunostaining which was concordant with the cytogenetic interpretation, with an unweighted kappa of 0.42 (representing mild-to-moderate agreement). Thus, although there was a correlation between positive immunohistochemistry for PRAME and abnormal findings on FISH analysis, in our view, the concordance was not sufficient to enable PRAME immunohistochemistry to act as a surrogate for FISH testing. Our findings reiterate the principle that interpretation of problematic superficial melanocytic proliferations requires a synthesis of all the available data, including clinical scenario, morphological features, immunohistochemistry, and ancillary molecular investigations., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Bioconjugation of antibodies with various payloads has diverse applications across various fields, including drug delivery and targeted imaging techniques. Fluorescent immunoconjugates provide a promising tool for cancer diagnostics due to their high brightness, specificity, stability and target affinity. Fluorescent antibodies are widely used in flow cytometry for fast and sensitive identification and collection of cells expressing the target surface antigen. Nonetheless, current approaches to fluorescent labeling of antibodies most often use random modification, along with a few rather sophisticated site-specific techniques. The aim of our work was to develop a procedure for fluorescent labeling of immunoglobulin G via periodate oxidation of antibody glycans, followed by oxime ligation with fluorescent oxyamines. Here, we report a novel technique based on an in situ oxime ligation of ethoxyethylidene-protected aminooxy compounds with oxidized antibody glycans. The approach is suitable for easy modification of any immunoglobulin G, while ensuring that antigen-binding domains remain intact, thus revealing various possibilities for fluorescent probe design. The technique was used to label an antibody to PRAME, a cancer-testis protein overexpressed in a number of cancers. A 6H8 monoclonal antibody to the PRAME protein was directly modified with protected-oxyamine derivatives of fluorescein-type dyes (FAM, Alexa488, BDP-FL); the stoichiometry of the resulting conjugates was characterized spectroscopically. The immunofluorescent conjugates obtained were applied to the analysis of bone marrow samples from patients with oncohematological diseases and demonstrated high efficiency in flow cytometry quantification. The approach can be applied for the development of various immunofluorescent probes for detection of diagnostic and prognostic markers, which can be useful in anticancer therapy.

A single tumor marker may correspond to a variety of diseases, and a specific disease requires the joint detection of multiple tumor markers for improving the accuracy of diagnoses. An ionic liquid-functionalized metal-organic framework (Zn-MOF microspheres) was used as the substrate to capture the aptamer (Ab1), and noble metal nanoparticles were used to label a signal aptamer (Ab2) to construct a dual-wavelength responsive sandwich-type photoelectrochemical (PEC) aptasensor. Due to the size effect, plasma resonance and the response of the noble metal nanoparticle enhancement system to different excitation wavelengths, the simultaneous detection of CEA and CA153 tumor markers was realized. Under the optimized conditions, CA153 and CEA at concentrations of 0.05-100 U mL -1 and 0.005-10 ng mL -1 were detected by the PEC aptasensor. Detection limits calculated for CA153 and CEA determinations were 0.0275 U mL -1 and 2.85 pg mL -1 (S/N = 3), respectively. CA153 and CEA in serum samples were detected by the PEC aptasensor, and their concentrations were well consistent with that obtained from the ELISA. In addition, the PEC aptasensor exhibited a recovery rate of 96.98%-103.4%, and a relative standard deviation of 1.1%-3.6%, indicating good practical value and accuracy, further confirming its potential for clinical diagnosis.

Summary: The detection and prediction of true neoantigens is of great importance for the field of cancer immunotherapy. Wesearched the literature for proposed neoantigen features and integrated them into a toolbox called NEOantigen Feature toolbOX (NeoFox). NeoFox is an easy-to-use Python package that enables the annotation of neoantigen candidates with 16 neoantigen features., Availability and Implementation: NeoFox is freely available as an open source Python package released under the GNU General Public License (GPL) v3 license at https://github.com/TRON-Bioinformatics/neofox., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2021. Published by Oxford University Press.)