Factors influencing blood tumor marker concentrations in the absence of neoplasia.

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), ß2-microglobulin (ß2-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.

Fusion expression, purification, and characterization of cytokeratin 19 fragments in E. coli for enhanced stability in diagnostic applications.

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.

Spatial transcriptomic analysis of tumour with high and low CAIX expression in TNBC tissue samples using GeoMx™ RNA assay.

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<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.

Comparison of Melanocyte-Associated Immunohistochemical Markers in Acral Lentiginous Melanoma and Acral Benign Nevi.

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.

PRAME expression in cutaneous melanoma does not correlate with disease-specific survival.

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.

PRAME Immunohistochemical Expression and TERT Promoter Mutational Analysis as Ancillary Diagnostic Tools for Differentiating Proliferative Nodules From Melanoma Arising in Congenital Nevi.

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.

An electrochemiluminescence immunosensor with double co-reaction accelerators based on Ag3PO4@EuPO4-AgNP for detecting squamous cell carcinoma antigen.

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 (Ag3PO4) nanoparticles were modified on the surface of EuPO4 nanowires to improve their poor dispersibility/solubility. At the same time, EuPO4 was used as a co-reaction accelerator to catalyze S2O82- to produce more intermediates (SO4•-), significantly enhancing the ECL signal of Ag3PO4. Ag nanoparticles (AgNP) modified on Ag3PO4@EuPO4 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.

Potential diagnostic utility of PRAME and p16 immunohistochemistry in melanocytic nevi and malignant melanoma.

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.

Standardized Computer-Assisted Analysis of PRAME Immunoreactivity in Dysplastic Nevi and Superficial Spreading Melanomas.

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/mm2 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/mm2. 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.

The relationship between carbonic anhydrase IX (CAIX) and patient survival in breast cancer: systematic review and meta-analysis.

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.

PRAME Expression in Mucosal Melanoma of the Head and Neck Region.

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.

Diagnostic test accuracy meta-analysis of PRAME in distinguishing primary cutaneous melanomas from benign melanocytic lesions.

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.

PRAME expression by immunohistochemistry and reverse transcription quantitative PCR in conjunctival melanocytic lesions-a comprehensive clinicopathologic study of 202 cases and correlation of cytogenetics with PRAME expression in challenging conjunctival melanocytic lesions.

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.

Carbonic anhydrase IX stratifies patient prognosis and identifies nodal status in animal models of nasopharyngeal carcinoma using a targeted imaging strategy.

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.

Deciphering the immunopeptidome in vivo reveals new tumour antigens.

Immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex class I (MHC-I) molecules1-5. 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 vivo6. To overcome these limitations, we engineered an inducible affinity tag into the mouse MHC-I gene (H2-K1) and targeted this allele to the KrasLSL-G12D/+Trp53fl/fl mouse model (KP/KbStrep)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 abundance8. Beyond cancer, the KbStrep 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.

Predictive value of topoisomerase II alpha protein for clinicopathological characteristics and prognosis in early breast cancer.

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.

Comparative Analysis of PRAME Expression in 127 Acral and Nail Melanocytic Lesions.

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.

Elevated levels of serum CDCP1 in individuals recovering from severe COVID-19 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.

A review on the role of PCA3 lncRNA in carcinogenesis with an especial focus on prostate cancer.

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.

Cysteine Modification of Glutathione-Stabilized Au Nanoclusters to Red-Shift and Enhance the Electrochemiluminescence for Sensitive Bioanalysis.

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.