Search

Background: Microsatellite instability (MSI) and mismatch repair (MMR) detection is valuable in assessing prognosis and treatment options. However, the conventional detection methods such as immunohistochemistry (IHC) are limited by not fully consistent results as well as a long turnaround time. TrueMark™ MSI Assay is a novel solution for MSI analysis, but lack of research support in the Chinese colorectal cancer (CRC) patients., Materials and Methods: 60 dMMR and 60 pMMR CRC samples identified by IHC were collected and their MSI status were detected using TrueMark™ MSI assay with an expanded panel of 13 markers. The overall performance and diagnostic concordance between TrueMark™ MSI test and MMR IHC analysis were assessed and analyzed., Results: According to the TrueMark™ test, 55 out of the 120 (45.8 %) CRCs were identified as MSI-high (MSI-H) with an instability at ≥ 4/13 markers. Compared with the MMR IHC analysis, an overall percent agreement of 94.2 % and a Kappa of 0.883 were achieved. For the seven inconsistent samples, tumor mutation burden analysis was performed and the results supported the diagnosis by TrueMark™ test. To confirm the robustness of the above findings, a validation was performed in an independent cohort comprising 51 consecutive CRCs. Furthermore, an optimized panel composed of NR-21, NR-24, NR-27, ABI-16, ABI-17 and ABI-20B was developed by multivariate logistic regression model, and showed 100 % agreement with the 13-marker panel for MSI detection in both the derivation and validation sets., Conclusion: TrueMark™ MSI provides a fast, reliable and highly automated solution to MSI detection in Chinese CRC patients, and the new 6-marker panel we established shows promise deserving further evaluation., 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. Published by Elsevier B.V.)

Background: Human artificial corneas (HAC) generated by tissue engineering recently demonstrated clinical usefulness in the management of complex corneal diseases. However, the biological mechanisms associated to their regenerative potential need to be elucidated., Methods: In the present work, we generated HAC using nanostructured fibrin-agarose biomaterials with cultured corneal epithelial and stromal cells, and we compared the structure and histochemical and immunohistochemical profiles of HAC with control native corneas (CTR-C) and limbus (CTR-L) to determine the level of biomimicry of the HAC with these two native organs., Results: HAC tissues consisted of a stratified epithelium and a cellular stromal substitute. The interface between stroma and epithelium was similar to that of CTR-C, without the finger-shaped palisades of Vogt found in CTR-L, and contained a poorly developed basement membrane as determined by PAS histochemistry. Analysis of the stromal layer revealed that HAC contained significantly lower amounts of extracellular matrix components (collagen, proteoglycans, decorin, keratocan, and lumican) than CTR-C and CTR-L, with all samples being devoid of elastic and reticular fibers. At the epithelial level, HAC were strongly positive for several cytokeratins, although KRT5 was lower in HAC as compared to CTR-C and CTR-L. The expression of crystallin lambda was lower in HAC than in control tissues, whereas crystallin alpha-a was similar in HAC and CTR-C. No differences were found among HAC and controls for the cell-cell junction proteins CX43 and TJP1. When specific markers were analyzed, we found that HAC expression profile of KRT3, KRT19, KRT15, and ΔNp63 was more similar to CTR-L than to CTR-C., Conclusions: These results suggest that HAC generated in the laboratory could be structurally and functionally more biomimetic to the structure found at the corneal limbus than to the central cornea, and open the door to the use of these artificial tissues in patients with limbal deficiency., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Metastatic urothelial carcinoma (mUC) has a poor prognosis, despite recent therapeutic advancements. Prostaglandin Reductase 1 (PTGR1) is essential for activating acylfulvens, a promising class of drugs for treating a subset of urothelial carcinoma (UC) patients. The efficacy of acylfulvens depends on PTGR1 activity and defects in the nucleotide excision repair (NER) pathway, notably ERCC2 mutations present in 10-15% of bladder tumors. This study identifies patients eligible to be included in acylfulven clinical trials based on the presence of PTGR-1 by immunohistochemistry (IHC) staining, RNA expression level and mutations in the NER pathway. Additionally, it evaluates PTGR-1 expression as a prognostic biomarker., Methods: Three PTGR-1 antibodies were tested in a kidney cancer cell line A498 and in tissues with known PTGR1 expression (liver, tonsils, pancreas, small intestine, etc.). Patients with untreated mUC receiving 1st line platinum from Dec. 2019 to Dec. 2021 in the Capital Region, Denmark, were included retrospectively. FFPE tumor samples were collected, and tissue microarrays (TMAs) were constructed. TMAs were stained with the best-performing PTGR1 antibody, RNA expression was analyzed using Nanostring nCounter PanCancer panel and gene mutations were assessed using a targeted genomic panel (TSO-500). Kaplan-Meier and multivariate Cox regression assessed overall survival and covariate impacts., Results: The AB181131 PTGR1 antibody was the most reliable in validation tissues. Tumors from 71 mUC patients were used to construct the TMA, and 40% of tumors scored positive for PTGR1 by IHC staining. A normalized PTGR1 RNA cutoff at 2,550 normalized counts achieved an AUC of 0.9, defining 35 samples as positive with a sensitivity of 96% and specificity of 85% in relation to IHC-positivity. Differential expression showed a significant upregulation of PTGR1 RNA in PTGR1 IHC-positive cases. NER-deficiency and PTGR1 positivity (mutations in ERCC1, ERCC2, ERCC3, ERCC4) was seen in 9 patients (13%). Median overall survival was 16 months in the cohort. Overall survival (OS) analysis indicates that overexpression of PTGR1 RNA was associated with a reduced median OS (12 months vs. 25 months, p = 0.039, log-rank)., Conclusion: PTGR1 IHC staining pattern using the Abcam AB181131 antibody is highly correlated with PTGR1 RNA expression. 13% in our cohort were identified as NER deficient and PTGR1 positive. Lower levels of PTGR1 indicates better outcome in this cohort., Competing Interests: Declarations Competing interests The authors declare no competing interests. Conflict of interest D.R.S. received educational and research funding from Pfizer and Merck Serono and served on advisory boards for Merck Sharp and Dome (MSD), Bristol Meyers Squibb (BMS), and Johnson and Johnson, unrelated to this study. S.L. No COI to report. B.G.T. Speaker fee from BMS unrelated to this study. L.H.D: No COI to report. L.C.M.: No COI to report. Z.S: Research funding from Lantern Pharma Inc; M.R: received personal fees from AstraZeneca and MSD and served on advisory board of MSD outside the submitted work.; H.P: Research funding from Pfizer and Merck unrelated to this study. K.W.M: Advisory/consulting work for EMD Serono, Pfizer, UroGen, and Riva Therapeutics; has received research support from Pfizer and Novo Ventures; has equity in Riva Therapeutics; has received writing/editor fees from UpToDate; has received speaking fees from OncLive; and is a co-inventor listed on an institutional patent application for analysis of mutational signatures of DNA repair deficiency., (© 2024. The Author(s).)

Background: Understanding how genes function to heal wounds and restore lost tissue is essential for studying regeneration. Whole-mount in situ hybridization (WISH) is a powerful and widely used technique to visualize the expression patterns of genes in different biological systems. Yet, existing methods to permeabilize samples for WISH can damage or destroy fragile regenerating tissues, thereby preventing such experiments., Results: Here, we describe a new protocol for in situ hybridization (ISH) and immunostaining in the highly regenerative planarian Schmidtea mediterranea. This new Nitric Acid/Formic Acid (NAFA) protocol is compatible with both the assays and prevents degradation of the epidermis and regeneration blastema. The NAFA protocol achieves this without the use of proteinase K digestion which likely leads to better preservation of antigen epitopes. We show that the NAFA protocol successfully permits development of chromogenic and fluorescent signals in situ, while preserving the anatomy of the animal. Furthermore, the immunostaining of different proteins was compatible with the NAFA protocol following fluorescent in situ hybridization. Additionally, the tissue fixation protocol was easily adapted for regenerating killifish tail fin, which yielded better ISH signal with minimal background., Conclusions: Thus, the NAFA protocol robustly preserves the delicate wounded tissues while also facilitating probe and antibody penetration into internal tissues. Furthermore, the fixation protocol is compatible for WISH on regenerating teleost fins suggesting that it will be a valuable technique for studying the processes of wounding response and regeneration in multiple species., (© 2024. The Author(s).)

Accurate assessment of HER2 expression levels is paramount for determining eligibility for targeted therapies. HER2 immunohistochemistry provides a semiquantitative measurement of HER2 protein overexpression. Historically, little focus has been on the lower end of the HER2 expression range. The advent of novel therapeutic molecules that require fewer membrane epitopes to be effective has prompted a reevaluation of the current immunohistochemistry testing protocols, with special emphasis on the detection limit. Here, we have used Boston Cell Standards technology to determine the sensitivity of 2 commercially available HER2 immunohistochemistry assays, including a lower limit of detection., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Deposit diseases are a group of heterogenous diseases characterized by the pathological accumulation of deposits in cells and/or in the extracellular environment. The pathophysiology of these deposits is polymorphic and numerous substances can accumulate: endogenous substances, exogenous substances. Their consequences will vary, depending on the tissue in which the accumulation takes place. The role of the pathologist is essential for the diagnosis of a deposit disease and (s)he has numerous tools at her/his disposal for the diagnosis and typing of these deposits. Special stains can be used, but also immunohistochemistry, immunofluorescence, electron microscopy and more innovative techniques such as proteomics., (Copyright © 2024 The Author. Published by Elsevier Masson SAS.. All rights reserved.)

Objective: To investigate the immunohistochemistry (IHC) staining pattern and prognostic significance of p53 in non-endometrioid endometrial cancer (non-EEC)., Methods: This study retrospectively included 212 non-EEC patients, with histological types including serous carcinoma (SC), clear cell carcinoma (CCC), mixed carcinoma (MC), undifferentiated carcinoma (UC), and carcinosarcoma (CS). p53 IHC was interpreted as normal/wild-type and abnormal/mutant-type, the latter including overexpression, complete absence, and cytoplasmic staining patterns. Moreover, uncommon p53 subclonal/heterogeneous staining patterns were described. Disease-free survival (DFS) and overall survival (OS) were employed as endpoints to evaluate the prognostic significance of p53., Results: In 212 non-EEC cases, 50 (23.6 %) were p53 wild-type, while 162 (76.4 %) displayed abnormal p53 staining. Overexpression was the predominant abnormal p53 staining pattern (122/162), complete absence followed (33/162). All SCs exhibited the mutant p53 staining pattern. The p53 abnormal expression rates in CCC, MC, UC, and CS were 37.5 %, 78.9 %, 35.7 %, and 75.7 %, respectively. Interestingly, of the 12 MC cases with SC components, barring one with p53 subclonal staining, all showed the mutant-type staining. The concordance rate for p53 expression between epithelial and mesenchymal components of CS was 94.3 % (66/70). Kaplan-Meier curves indicated patients with p53 abnormalities had worse DFS compared to those with wild-type p53 (P=0.025). Multivariate Cox regression confirmed that p53 (HR: 2.270, 95 % CI: 1.124-4.586, P=0.022) independently predicted DFS in non-EEC patients, though not for OS., Conclusions: Non-EEC patients with various histological types exhibit different p53 staining patterns. However, abnormal p53 expression, regardless of histological type, implies a poor DFS in non-EEC patients., 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. Published by Elsevier GmbH.)

Objective: Immunocytochemistry (ICC) is essential for enhancing diagnostic accuracy and identifying markers for diagnosis, prognosis and targeted therapies. While cell blocks (CBs) are preferred for standardization and optimized staining, cytological smears are an alternative when CBs are unavailable. However, the literature on ICC protocols for smears is sparse. This review addresses preparation, fixation and protocols for nuclear and cytoplasmic antibodies on smears, drawing from our laboratory's experience., Methods: We reviewed procedures for ICC on cytological smears using existing literature and practical insights from our laboratory., Results: Commercially available antibodies were found to be reliable for ICC on smears if specimens are properly prepared and fixed. Protocols developed in our laboratory maintained antigenicity and provided clear staining results., Conclusions: Although ICC on CBs is the gold standard for standardization, cytological smears are a viable alternative when CBs are unavailable. Success in ICC on smears depends on proper preparation and fixation. This review offers practical protocols and insights to help laboratories optimize ICC on cytological smears. Further research and standardization are necessary to enhance reproducibility and reliability of ICC on smears. The practical information provided is based on personal experience in our laboratory., (© 2024 The Author(s). Cytopathology published by John Wiley & Sons Ltd.)

Liquid-based cytology (LBC) has changed the landscape of gynaecological cytology. A growing demand exists for LBC in diagnostic cytology, particularly for ancillary testing, such as immunocytochemistry and molecular testing. Ancillary testing solely based on conventional preparation (CP) methods remains challenging. Recently, the increased demand for specialist testing and minimally invasive techniques, such as endoscopic ultrasonography fine-needle aspiration, to obtain cellular samples has led to an increasing demand for ancillary testing on cytology LBC supernatant, slides and cell block (CB). This facilitates the diagnosis and prognosis in cytology samples enabling personalized treatment. An understanding of the history and future prospects of LBC is crucial for its application in routine diagnostics by cytopathologists and cytotechnologists. In this review, we initiated an internet search using the keyword 'liquid-based cytology', and we conducted a literature review to discuss the usefulness of combined diagnosis of LBC and CP, immunocytochemistry and molecular testing and assessed the quality of nucleic acids in diagnostic LBC. High-quality and cell-rich diagnostic LBC surpassed the CP method alone in terms of reliability and versatility of ancillary testing in cytological diagnosis. Conclusively, diagnostic LBC lends itself to various new technologies and is expected to continue evolving with innovations in the future., (© 2024 John Wiley & Sons Ltd.)

Context.—: Recent publications have featured immunohistochemistry (IHC) as a sensitive tool for detecting Mycobacterium tuberculosis and nontuberculous mycobacteria, but performance is limited to cases suspected to have mycobacterial infection., Objective.—: To examine cross-reactivity of a polyclonal antimycobacterial antibody with various types of pathogens, tissues, and inflammatory patterns., Design.—: Surgical pathology files during a period of 6 years were searched, and 40 cases representing a variety of pathogens, tissue types, and inflammatory responses were retrieved. Cases were stained with a rabbit polyclonal antimycobacterial antibody (Biocare Medical, Pacheco, California). The cases and associated histochemical stains, culture, and molecular results were reviewed by 3 pathologists., Results.—: All 8 cases of mycobacterial infection previously diagnosed by other methods were positive for mycobacteria by IHC. In addition, multiple bacterial and fungal organisms and 1 case of Leishmania amastigotes were also immunoreactive with the mycobacterial IHC., Conclusions.—: Although highly sensitive for mycobacteria, the polyclonal antibody shows significant cross-reactivity with other organisms. This is a sensitive but nonspecific stain that can be used as an alternative confirmation method for mycobacteria, but attention should be paid to inflammatory reaction and organism morphology when IHC is positive to avoid misdiagnosis., Competing Interests: The authors have no relevant financial interest in the products or companies described in this article., (© 2024 College of American Pathologists.)

Background: Preferentially expressed antigen in melanoma (PRAME) has been extensively studied in cutaneous melanocytic tumors and has proven valuable as a diagnostic adjunct in routine dermatopathology practice. However, its expression in cutaneous vascular neoplasms, particularly angiosarcomas (AS), remains largely unexplored., Methods: To further explore PRAME expression in cutaneous AS, 18 cases of post-irradiation and 13 cases of primary cutaneous AS were evaluated for PRAME. For comparison, sections from 11 deep soft tissue/visceral AS, 10 Kaposi sarcomas, 8 microvenular hemangiomas, 7 infantile hemangiomas, 8 atypical vascular lesions, 6 epithelioid hemangioendotheliomas, 6 pyogenic granulomas, 6 papillary endothelial hyperplasias, 6 epithelioid hemangiomas, 3 capillovenous malformations, 3 hobnail hemangiomas, 2 spindle cell hemangiomas, 2 pseudomyogenic hemangioendotheliomas, and 2 composite hemangioendotheliomas were also retrieved., Results: Overall, 22 of 31 (70.9%; 12 post-irradiation and 10 primary) cutaneous AS were positive for PRAME. In contrast, only 1 of 11 (9.1%) deep soft tissue/visceral AS showed diffuse and strong PRAME nuclear staining. All other tumor types were negative for PRAME, except for 5 of 7 (71.4%) infantile hemangiomas, which demonstrated rare (<5%; four cases) and 1+ (5-25%; one case) nuclear staining., Conclusions: In this study, we have demonstrated frequent nuclear PRAME expression in cutaneous AS. PRAME immunohistochemistry may serve as a valuable additional marker in selected clinical settings., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Context.—: Folate receptor-α (FRα, encoded by the FOLR1 gene) is overexpressed in several solid tumor types, including epithelial ovarian cancer (EOC), making it an attractive biomarker and target for FRα-based therapy in ovarian cancer., Objective.—: To describe the development, analytic verification, and clinical performance of the VENTANA FOLR1 Assay (Ventana Medical Systems Inc) in EOC., Design.—: We used industry standard studies to establish the analytic verification of the VENTANA FOLR1 Assay. Furthermore, the VENTANA FOLR1 Assay was used in the ImmunoGen Inc-sponsored SORAYA study to select patients for treatment with mirvetuximab soravtansine (MIRV) in platinum-resistant EOC., Results.—: The VENTANA FOLR1 Assay is highly reproducible, demonstrated by a greater than 98% overall percent agreement (OPA) for repeatability and intermediate precision studies, greater than 93% OPA for interreader and greater than 96% for intrareader studies, and greater than 90% OPA across all observations in the interlaboratory reproducibility study. The performance of the VENTANA FOLR1 Assay in the SORAYA study was evaluated by the overall staining acceptability rate, which was calculated using the number of patient specimens that were tested with the VENTANA FOLR1 Assay that had an evaluable result. In the SORAYA trial, data in patients who received MIRV demonstrated clinically meaningful efficacy, and the overall staining acceptability rate of the assay was 98.4%, demonstrating that the VENTANA FOLR1 Assay is safe and effective for selecting patients who may benefit from MIRV. Together, these data showed that the assay is highly reliable, consistently producing evaluable results in the clinical setting., Conclusions.—: The VENTANA FOLR1 Assay is a robust and reproducible assay for detecting FRα expression and identifying a patient population that derived clinically meaningful benefit from MIRV in the SORAYA study., (© 2024 College of American Pathologists.)

Background: KIT p.D816 mutation is strongly associated with systemic mastocytosis (SM). Next-generation sequencing (NGS) is now routinely performed in almost all bone marrow sample and KIT mutations are detected from patients who are not known or suspected to have SM. Therefore, we wanted to assess if KIT mutations in this patient population are associated with unsuspected SM., Methods: We searched NGS result in our institution with positive result for KIT mutation from patients with known/suspected myeloid neoplasms. Patients with previously documented history of systemic mastocytosis were excluded. Bone marrow biopsies from patients with KIT mutation were assessed with immunohistochemical stains for CD117 and mast cell tryptase (MST)., Results: Bone marrow biopsies were assessed with immunohistochemical stains for CD117 and mast cell tryptase (n = 49). Most patients had acute myeloid leukemia (AML, n = 38) or chronic myelomonocytic leukemia (CMML, n = 6). Immunohistochemical stains for CD117 and tryptase were performed in all 49 patients. A total of 4 patients (8.2%) showed mast cell nodules where spindled shaped mast cells were present, meeting the WHO criteria for SM. All four patients had KIT p.D816V mutation and had high mutant allelic frequency (∼ 50%) except one patient (1%)., Conclusion: We discovered approximately 8% of patients who had myeloid neoplasms with unexpected KIT mutations fulfilled the diagnostic criteria for systemic mastocytosis after additional immunohistochemical studies. Our data support that application of additional immunohistochemical studies is recommended to identify underrecognized SM when KIT mutations are found by molecular assays., Competing Interests: Disclosure CYO has received research funding from Blueprint Medicines., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The subcellular distribution of CD30 on mast cells and the presence of eosinophils in cutaneous mastocytosis require further investigation, especially as the cell surface expression of CD30 is critical for the therapeutic response of systemic mastocytosis to brentuximab vedotin., Objective: Investigation of 147 biopsy specimens from 143 patients with cutaneous mastocytosis for mast cell density and distribution, frequency of CD30 expression, CD30 staining patterns, and presence and distribution of eosinophils. Correlation with clinical patterns., Methods: Retrospective multicenter immunohistochemical study of CD30 expression, eosinophils and basic clinical data in cutaneous mastocytosis., Results: CD30 expression was found in all samples (cut-off: ≥1%), whereby the staining was predominantly cytoplasmic in 99% of the samples. Additional membrane staining was detected in 62% of the samples. Surface expression of CD30 was more common in biopsy specimens with a high mast cell burden and in biopsy specimens with a higher CD30 expression rate. Eosinophils were admixed in 58% of the samples. Females and older patients showed a trend of a lower mast cell burden., Limitations: Retrospective study on formalin-fixed and paraffin-embedded tissue without functional analysis., Conclusion: Most cases of cutaneous mastocytosis show cell surface expression of CD30 expression and is, therefore, in principle, accessible for therapy with antibodies against CD30, provided the overall situation of the patient warrants., (© 2024 The Author(s). Journal of Cutaneous Pathology published by John Wiley & Sons Ltd.)

Fine needle aspiration procedure is routinely used for cytological diagnosis of nodal or extra nodal lesions. Follicular dendritic cell sarcoma (FDCS) is a rare mesenchymal neoplasm arising from follicular dendritic cells of lymphoid follicles at nodal and extranodal sites. Multimodal therapies have emerged for FDCS, necessitating its accurate pathologic diagnosis with additional ancillary testing for directing clinical management. By immunohistochemical analysis, FDCS is positive for the complement receptors CD21, CD23, and CD35. In addition, D2-40 is reported to be highly sensitive for FDCS with a strong membranous pattern of expression. In this study, we present the cytological diagnosis of a case of FDCS in retroperitoneal lymph nodes with an emphasis on a unique staining pattern of D2-40 which showed a strong nuclear pattern in tumor cells comparable to the membranous pattern of D2-40 on the control tissue and other surgical cases of FDCS in our comparative study., (© 2024 Wiley Periodicals LLC.)

The terms "onychofibroblast" (nail-specific fibroblast) and onychodermis (nail-specific dermis) were first introduced in 2006 and 2012, respectively, based on distinctive histologic and immunohistochemical features from the dermis of the surrounding skin and have been demonstrated in multiple studies. Recently, based on molecular research, the definition of onychodermis containing onychofibroblasts has been expanded to encompass the area located between the nail matrix and bed epithelium and periosteum. Single-cell RNA sequencing and in situ hybridization demonstrated that onychofibroblasts within the onychodermis express the genes including RSPO4, MSX1, WIF-1, and BMP5, which are implicated in nail formation and/or in disorders with nail phenotype. A mutation in RSPO4, a component of the Wnt signaling pathway, causes anonychia congenita. Nail matrix onychodermis and nail bed onychodermis share many similar characteristics which differ from the surrounding normal dermis of the skin. Comparative spatial transcriptomic and single-cell analyses of human nail units and hair follicles suggest that onychodermis is the counterpart of follicular dermal papilla, which plays a key role in hair follicle growth and morphogenesis. Onychomatricoma, as a nail-specific tumor, has been demonstrated to be a mesenchymal tumor that originates from onychofibroblasts and is associated with the upregulation of Wnt signaling. Collectively, the onychodermis and onychofibroblasts play crucial roles in nail development and these specialized nail mesenchymal elements are key components in the pathogenesis of onychomatricoma. The concept of onychodermis containing onychofibroblasts is very important for nail biology and pathology., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Fidgetin-like 1 (FIGNL1) is extensively overexpressed in a variety of cancers. It facilitates non‑small cell lung cancer tumor cell proliferation and hepatocellular carcinoma formation due to abnormal DNA repair. Clinically relevant data indicates that its high expression is linked with the poor prognosis of patients with renal clear-cell carcinoma, low-grade gliomas, and hepatocellular carcinoma. Nevertheless, the scope of FIGNL1’s involvement in cancer, particularly colorectal cancer (CRC), remains unclear., Aims: To investigate the function of FIGNL1 in CRC., Study Design: Cell culture study., Methods: The TCGA database and immunohistochemistry analysis were employed to investigate FIGNL1 expression in CRC tissue. A cell viability assay was performed using the Cell Counting Kit-8. The cell migration and invasion were evaluated using the transwell assay. Small interfering RNA (siRNA) transfection was conducted to knockdown FIGNL1 expression. Infection with FIGNL1 overexpression lentivirus was performed to promote FIGNL1 overexpression. The STRING database was employed for predicting protein interaction., Results: FIGNL1 was substantially upregulated in human CRC tissues and was associated with TNM stages and lymph node metastasis in patients. The inhibition of CRC cell proliferation, migration, and invasion in Caco-2 cells was achieved by silencing FIGNL1 using siRNA. Additional investigations suggested that FIGNL1 overexpression could promote CRC cell proliferation, migration, and invasion via P38 signaling pathway activation in Colo-205 cells. Subsequent experiments demonstrated that FIGNL1-mediated P38 phosphorylation was contingent upon SPIDR interaction., Conclusion: These results implied that FIGNL1 was a potential anticancer drug target, which also offered a novel strategy for future CRC treatment., Competing Interests: Conflict of Interest: The authors declare that they have no conflict of interest.

Immunohistochemistry (IHC) plays a crucial role in biological research and clinical diagnosis, serving as the most commonly used method for identifying and visualizing tissue antigens. However, traditional IHC staining methods have limitations in distinguishing various subtypes of immune cells. This challenge has driven scientists to explore new technologies and methodologies for precise identification and differentiation of immune cell subtypes. In recent years, multiplex IHC has emerged as a solution, enabling the simultaneous detection of multiple antigens and their visualization within the same tissue sample. Uterine natural killer (uNK) cells play a pivotal role in early pregnancy processes, including decidualization, remodeling of uterine spiral arteries, and embryo implantation. Different subtypes of uNK cells exhibit different functions, allowing them to coordinate various biological events for successful embryo development and pregnancy. Therefore, in-depth research on uNK cell subtypes is essential for elucidating immune regulation mechanisms during pregnancy. Such studies provide valuable insights and novel approaches for addressing related conditions such as infertility and recurrent reproductive failure. This paper introduces a detailed multiplex IHC staining protocol for studying the density of four subtypes of uNK cells in endometrial specimens during the window of implantation (WOI). The protocol includes sample preparation, optimization of subtype markers, microscopic imaging, and data analyses. This multiplex IHC staining protocol offers high specificity and sensitivity, enabling simultaneous detection of different uNK cell subtypes, thus providing researchers with a powerful tool to explore the intricacies and mechanisms of immune regulation during pregnancy.

Background and Objectives: Head and neck squamous cell carcinomas (HNSCCs) vary significantly in terms of invasiveness, growth rate, and metastatic potential. This study aimed to investigate the expression of several prognostic biomarkers (Ki67, p53, EGFR, COX-2, Cx43, and p16) in HNSCC from various anatomical regions and to correlate these expressions with clinicopathological parameters. Materials and Methods : We performed immunohistochemistry on 91 histologically verified HNSCC cases from the County Emergency Hospital, Targu Mures. Biomarker expression for Ki67, COX-2, and Cx43 was assessed using a standard immunoexpression scoring system: S1: 0-10%, S2: 11-25%, S3: 26-50%, S4 > 50%; EGFR was scored based on membrane staining intensity: 0, 1+, 2+, 3+; we classified p16 as positive or negative; p53 was grouped into mutant and wild-type; and we compared these across histopathological types, tumor grades, anatomical locations, gender, and different age groups. We performed a comparative analysis of Cx43 expression levels in relation to the expression of the rest of the markers. Statistical analysis was conducted using GraphPad InStat 3 software, version 3.06 (GraphPad Software Inc., San Diego, USA). Results : The majority of tumors were in males (95.6%) aged 51-60 years. Mutant p53 expression was prevalent in most cases. Elevated Ki67 and EGFR expression were associated with more aggressive tumors. COX-2 levels varied, with a higher proportion of moderate and high immunoexpression (S3 + S4) observed in patients under 70 years old. Cx43 expression was generally low, especially in extralaryngeal tumors. Conclusions : HNSCC primarily affects older males, with the larynx being the most common site. High levels of Ki-67 and EGFR suggest more aggressive tumors, while low COX-2 levels reflect varying prognoses. Women may develop more aggressive tumors, and extralaryngeal tumors often present with more challenging prognoses. Low Cx43 expression may be more likely to coincide with higher Ki67 and COX-2 levels, possibly indicating a link with more aggressive tumor behavior.

Background/Objectives : Melanoma is an aggressive skin malignancy, and the majority of deaths associated with melanoma result from malignant skin lesions. Our study aims to evaluate the expression of the markers CD133 and NANOG, associated with tumor stem cells, and to analyze their link with epidemiological and histological parameters, thus contributing to early diagnosis and the development of targeted therapies. Methods : We performed a retrospective study in the Mureș Clinical County Hospital, Romania, which included 66 cases of melanoma: 50 primary cutaneous melanomas, 10 metastases, and 6 local recurrences. CD133 and NANOG marker expression was assessed by immunohistochemistry and quantified using the H score. Statistical analyses were applied to determine the correlations between marker expression and clinicopathological parameters. Results : CD133 expression was identified in six cases (12%) of primary melanoma, with a mean H-Score of 29, and was associated with an increased Breslow index and a higher number of mitoses. NANOG expression was positive in 30 cases (60%) of primary melanoma, with a median H-Score of 15 and with increased expression observed in cases with pagetoid migration and lesions in situ. In metastases, eight cases (80%) were positive for NANOG and four (40%) for CD133. Local recurrences showed positive expression for NANOG in four cases (66%). Conclusions : The expression of CD133 and NANOG markers highlights the role of tumor stem cells in melanoma progression. Early identification of these markers could improve diagnosis and treatment, including the application of targeted therapies.

Background and Objectives : The Connexin43 ( Cx43 ) gene is a suspected tumor suppressor gene, as re-expressed wild-type Cx43 genes reduce the malignancy potential of tumor cells. However, the role of Cx43 gene expression in human lung tumorigenesis remains unclear. Materials and Methods: Tumor tissues from 165 primary lung cancer patients were collected to study Cx43 protein expression and gene mutations using immunohistochemistry and direct DNA sequencing. In addition, Cx43 genes with or without mutations were transfected to CL-3 human lung cancer cells to confirm the function of these mutant forms of the Cx43 gene. Results: Aberrant localization of Cx43 protein in the nucleus and cytoplasm of tumor cells was detected in 14 out of 165 non-small cell lung cancer (NSCLC) patients. Mutations in the Cx43 gene were also found in patients with aberrant Cx43 localization, and transfection of these mutant genes into lung cancer cells enhanced their proliferation. Conclusions: To our knowledge, this is the first study to demonstrate Cx43 gene mutations in human lung neoplasm, supporting the hypothesis that Cx43 may function as a tumor suppressor in some lung cancer patients. Additionally, the findings suggest an association between aberrant localization of Cx43 protein expression and tumor progression.

Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve as valuable animal models for studying the pathogenesis and treatment of glaucoma due to their relatively homogenous genetic background and retinal ganglion cells (RGCs), which structurally resemble those in humans. Accurate assessment of RGC damage and treatment outcomes in mouse glaucoma models requires determining the RGC number across the entire retina. This protocol outlines a comprehensive method involving the isolation of the whole retina, labeling RGCs with specific antibodies, and rapid, accurate automatic counting of RGCs using an AI-based program. The streamlined approach allows for efficient and precise quantification of RGC numbers in mouse retinas, facilitating the evaluation of RGC degeneration and potential therapeutic interventions. By enabling researchers to assess the extent of RGC damage, this protocol contributes to a deeper understanding of glaucoma pathogenesis and aids in developing effective treatment strategies to manage and prevent vision loss.

Melanin is a natural pigment in the human body that is primarily found in the skin and hair. It protects the skin from damage by ultraviolet radiation. Although this pigment plays a crucial role in protecting the human body, it represents a challenge for pathologists to evaluate highly pigmented tissue samples from melanoma or pigmented skin lesions. Abundant melanin may obscure tissue morphology, which makes it very difficult for pathologists to make a differential diagnosis. Melanin pigment is brown-to-black and granular, and its distribution is often uneven in tissues. The presence of these pigments can complicate the analysis of immunohistochemistry (IHC) for 2 reasons. First, they have a direct physical masking effect on antigen-antibody interactions. Second, 3,3-diaminobenzidine, the most commonly used chromogen, has a brown color that is difficult to distinguish from melanin pigment. Therefore, melanin bleaching has become a crucial step in handling pigmented melanocytic lesions. Bleaching techniques aid pathologists in histopathologic examination of melanin-rich tissue. In this study, we integrated melanin bleaching and IHC on an automated IHC platform to set up a rapid and fully automated procedure. Bleaching steps were performed before antigen retrieval. Samples were treated with 1% hydrogen peroxide solution in Tris-HCl buffer (pH 10) at 80°C for 8 minutes, achieving optimal conditions for melanin bleaching while preserving tissue morphology and antigenicity. This rapid, effective, fully automated, and standardized workflow can be applied to routine staining procedures in clinical laboratories, thereby improving the quality of pathological diagnosis., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Mismatch repair deficiency (dMMR) is a well-established characteristic of endometrial adenocarcinoma and is crucial in screening for Lynch syndrome, guiding adjuvant treatment decisions, and identifying candidates for immune checkpoint inhibitors. The traditional approach to dMMR screening involves a four-antibody panel, but a simplified two-antibody method utilizing PMS2 and MSH6 has shown promise. This study aims to compare the diagnostic performance of the simplified two-antibody method with the traditional four-antibody panel in endometrial cancer samples., Methods: We conducted a retrospective cohort study on endometrial carcinoma cases diagnosed between 2013 and 2022. We compared the diagnostic performance of the two-antibody panel with the traditional four-antibody panel in detecting dMMR. Clinical data and immunohistochemistry results were collected, and agreement between the two methods was evaluated using Cohen's kappa coefficient., Results: 304 endometrial cancer cases were included, with 27% demonstrating loss of at least one MMR protein using the four-antibody panel. The two-antibody method detected MMR deficiency in 26.6% of cases, with a high agreement rate of 98.8% between the two methods. Only one case showed discordant results, prompting further investigation., Conclusion: The simplified two-antibody MMR IHC screening approach using PMS2 and MSH6 showed high concordance with the traditional four-antibody panel. This suggests its potential as an alternative method for reflex MMR status testing in endometrial adenocarcinoma. The implementation of this approach could streamline the diagnostic process, reduce costs, and improve the detection of Lynch syndrome in affected individuals and their families. Further studies with larger cohorts and long-term follow-up are needed to validate these findings and assess the clinical implications of this approach in routine practice.

Purpose: The proven efficacy of human epidermal growth factor receptor 2 (HER2) antibody-drug conjugate therapy for treating HER2-low breast cancers necessitates more accurate and reproducible HER2 immunohistochemistry (IHC) scoring. We aimed to validate performance and utility of a fully automated artificial intelligence (AI) solution for interpreting HER2 IHC in breast carcinoma., Materials and Methods: A two-arm multireader study of 120 HER2 IHC whole-slide images from four sites assessed HER2 scoring by four surgical pathologists without and with the aid of an AI HER2 solution. Both arms were compared with high-confidence ground truth (GT) established by agreement of at least four of five breast pathology subspecialists according to ASCO/College of American Pathologists (CAP) 2018/2023 guidelines., Results: The mean interobserver agreement among GT pathologists across all HER2 scores was 72.4% (N = 120). The AI solution demonstrated high accuracy for HER2 scoring, with 92.1% agreement on slides with high confidence GT (n = 92). The use of the AI tool led to improved performance by readers, interobserver agreement increased from 75.0% for digital manual read to 83.7% for AI-assisted review, and scoring accuracy improved from 85.3% to 88.0%. For the distinction of HER2 0 from 1+ cases (n = 58), pathologists supported by AI showed significantly higher interobserver agreement (69.8% without AI v 87.4% with AI) and accuracy (81.9% without AI v 88.8% with AI)., Conclusion: This study demonstrated utility of a fully automated AI solution to aid in scoring HER2 IHC accurately according to ASCO/CAP 2018/2023 guidelines. Pathologists supported by AI showed improvements in HER2 IHC scoring consistency and accuracy, especially for distinguishing HER2 0 from 1+ cases. This AI solution could be used by pathologists as a decision support tool for enhancing reproducibility and consistency of HER2 scoring and particularly for identifying HER2-low breast cancers.

Objective: To compare the application of different treatments in the diagnosis of melanoma with severe pigment interference, to solve the problem of pigment interference with immunohistochemical interpretation., Methods: The pigment-rich melanomas were first depigmented with potassium permanganate using a concentration gradient (0.1%, 0.5%, 1%) and a time gradient (1, 5, 10, 15, 30 min, 6 h), and the optimal concentration and time were found. Then, 12 cases of pigment-rich melanoma tissues were collected, and the tissues were stained with diaminobenzidine (DAB), alkaline phosphatase-fast red (AP red), multiplex immunofluorescence (MIF), and 3-amino-9-ethylcarbazole (AEC), and ferrous sulfate, comparing different methods, positive expression of HMB45, MelanA, S100, SOX10, ki67., Results: First, the concentration of 0.5% potassium permanganate after 15 min treatment of the pigment significantly faded, and the intensity of antibody positivity was better than other concentrations and time. Second, after depigmentation treatment, the antibody positivity rate was 41.7%-66.7% for DAB, 66.7%-91.7% for AP red, 83.3%-100% for multiplex immunofluorescence, 25%-33.3% for AEC, and 33.3% for ferrous sulfate., Conclusion: AP red staining and mIF are more suitable for the diagnosis of melanoma with severe pigment interference, and AP red staining is more economical and practical., (© 2024 The Author(s). Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Background: Screening for anaplastic lymphoma kinase (ALK) rearranged non-small cell lung cancer (NSCLC) is crucial for identifying patients eligible for targeted therapy. The FDA-approved ALK (D5F3) immunohistochemistry (IHC) assay, used with the OptiView Amplification Kit, demonstrates excellent sensitivity and specificity in detecting these patients. However, the clinical significance of resulting focal positivity remains unclear, and ALK (D5F3) expression unrelated to ALK fusion is observed in some cases of neuroendocrine differentiation. This study aims to validate these findings with molecular testing and contribute to the accurate interpretation of ALK (D5F3) IHC results., Methods: A total of 1619 patients diagnosed with NSCLC and neuroendocrine carcinoma were evaluated using ALK (D5F3) IHC. For cases with strong but focal expression and those with diffuse strong positivity in neuroendocrine differentiation, ALK fluorescence in situ hybridization (FISH) and/or next-generation sequencing (NGS) tests were performed., Results: Seven out of 1109 adenocarcinomas (0.6%) and six out of 289 squamous cell carcinomas (2.1%) exhibited strong focal ALK (D5F3) expression. Nine out of 209 neuroendocrine carcinomas (4.3%) showed homogeneously strong ALK (D5F3) expression. All these cases, including adenocarcinoma with neuroendocrine differentiation and combined small cell carcinoma, were negative for ALK fusions by FISH and/or NGS., Conclusion: This study demonstrates that strong but focal ALK (D5F3) immunostaining and strong expression in neuroendocrine differentiation may not indicate ALK fusion. By considering these findings, we can improve the accuracy of patient selection for targeted therapy by minimizing false-positive interpretations of ALK (D5F3) staining., (© 2024 The Author(s). Thoracic Cancer published by John Wiley & Sons Australia, Ltd.)

Context.—: Biomarker reporting has increasingly become a key component of pathology reporting, providing diagnostic, prognostic, and actionable therapeutic data for patient care., Objective.—: To expand and improve the College of American Pathologists (CAP) biomarker protocols., Design.—: We surveyed CAP members to better understand the limitations they experienced when reporting cancer biomarker results. A Biomarker Workgroup reviewed the survey results and developed a strategy to improve and standardize biomarker reporting. Drafts of new and revised biomarker protocols were reviewed in both print and electronic template formats during interactive webinars presented to the CAP House of Delegates. Feedback was collected, and appropriate revisions were made to finalize the protocols., Results.—: The first phase of the CAP Biomarker Workgroup saw the development of (1) a new stand-alone general Immunohistochemistry Biomarker Protocol that includes reporting for ER (estrogen receptor), PR (progesterone receptor), Ki-67, HER2 (human epidermal growth factor receptor 2), PD-L1 (programmed death ligand-1), and mismatch repair; (2) a new Head and Neck Biomarker Protocol that updates the prior 2017 paper-only version into an electronic template, adding new diagnostic and theranostic markers; (3) a major revision to the Lung Biomarker Protocol to streamline it and add in pan-cancer markers; and (4) a revision to the Colon and Rectum Biomarker Protocol to add HER2 reporting., Conclusions.—: We have taken a multipronged approach to improving biomarker reporting in the CAP cancer protocols. We continue to review current biomarker reporting protocols to reduce and eliminate unnecessary methodologic details and update with new markers as needed. The biomarker templates will serve as standardized modular units that can be inserted into cancer-reporting protocols., Competing Interests: The authors have no relevant financial interest in the products or companies described in this article., (© 2024 College of American Pathologists.)

Purpose: Notable effectiveness of trastuzumab deruxtecan in patients with human epidermal growth factor receptor 2 (HER2)-low advanced breast cancer (BC) has focused pathologists' attention. We studied the incidence and clinicopathologic characteristics of HER2-low BC, and the effects of immunohistochemistry (IHC) associated factors on HER2 IHC results., Materials and Methods: The Breast Pathology Study Group of the Korean Society of Pathologists conducted a nationwide study using real-world data on HER2 status generated between January 2022 and December 2022. Information on HER2 IHC protocols at each participating institution was also collected., Results: Total 11,416 patients from 25 institutions included in this study. Of these patients, 40.7% (range, 6.0% to 76.3%) were classified as HER2-zero, 41.7% (range, 10.5% to 69.1%) as HER2-low, and 17.5% (range, 6.7% to 34.0%) as HER2-positive. HER2-low tumors were associated with positive estrogen receptor and progesterone receptor statuses (p < 0.001 and p < 0.001, respectively). Antigen retrieval times (≥ 36 minutes vs. < 36 minutes) and antibody incubation times (≥ 12 minutes vs. < 12 minutes) affected on the frequency of HER2 IHC 1+ BC at institutions using the PATHWAY HER2 (4B5) IHC assay and BenchMark XT or Ultra staining instruments. Furthermore, discordant results between core needle biopsy and subsequent resection specimen HER2 statuses were observed in 24.1% (787/3,259) of the patients., Conclusion: The overall incidence of HER2-low BC in South Korea concurs with those reported in previously published studies. Significant inter-institutional differences in HER2 IHC protocols were observed, and it may have impact on HER2-low status. Thus, we recommend standardizing HER2 IHC conditions to ensure precise patient selection for targeted therapy.

Tissues are dynamic and complex biological systems composed of specialized cell types that interact with each other for proper biological function. To comprehensively characterize and understand the cell circuitry underlying biological processes within tissues, it is crucial to preserve their spatial information. Here we report a simple mounting technique to maximize the area of the tissue to be analyzed, encompassing the whole length of the murine gastrointestinal (GI) tract, from mouth to rectum. Using this method, analysis of the whole murine GI tract can be performed in a single slide not only by means of histological staining, immunohistochemistry and in situ hybridization but also by multiplexed antibody staining and spatial transcriptomic approaches. We demonstrate the utility of our method in generating a comprehensive gene and protein expression profile of the whole GI tract by combining the versatile tissue-rolling technique with a cutting-edge transcriptomics method (Visium) and two cutting-edge proteomics methods (ChipCytometry and CODEX-PhenoCycler) in a systematic and easy-to-follow step-by-step procedure. The entire process, including tissue rolling, processing and sectioning, can be achieved within 2-3 d for all three methods. For Visium spatial transcriptomics, an additional 2 d are needed, whereas for spatial proteomics assays (ChipCytometry and CODEX-PhenoCycler), another 3-4 d might be considered. The whole process can be accomplished by researchers with skills in performing murine surgery, and standard histological and molecular biology methods., (© 2024. Springer Nature Limited.)

Primary mucinous tumors of the renal pelvis are extremely rare and pose challenges in terms of diagnosis and treatment. This study reviewed the clinical and pathological characteristics of mucinous tumors of the renal pelvis, including mucinous cystadenocarcinomas and mucinous cystadenomas. Immunohistochemical analysis was conducted in three cases, along with KRAS gene detection using the Amplification Refractory Mutation System (ARMS) method. The results revealed mucinous epithelium with acellular mucinous pools in all cases, and acellular mucinous pools were observed in the renal parenchyma and perirenal fat capsules. All tumors expressed CK20 and CDX2, and one case showed KRAS gene mutation. The study suggests that mucinous cystadenomas of the renal pelvis may exhibit borderline biological behaviors. This study is the first to report a KRAS gene mutation in a mucinous cystadenoma of the renal pelvis, offering valuable insights into the diagnosis and treatment of this rare condition., Competing Interests: Declaration of competing interest All authors do not have any potential conflicts (financial, professional, or personal) that are relevant to the manuscript to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Borderline Brenner tumors (BBT) have a range of morphology that shows considerable overlap with that of malignant Brenner tumors (MBT). In particular, two histological patterns of BBT can be particularly challenging: 1) BBT with intraepithelial carcinoma (BBT-IEC) and 2) BBT with a small nested pattern (BBT-SNP). BBT-IEC is characterized by a tumor with the low-power non-infiltrative silhouette of a conventional BBT, but with increased cytological atypia and mitotic activity similar to that of MBT. Conversely, BBT-SNP is characterized by a complex proliferation of small tumor nests that closely resemble the infiltrative growth pattern of MBT, but without the obligate cytologic atypia and mitotic activity of MBT. We suggest that the combination of p16, p53 and Ki-67 may be helpful in distinguishing these 2 patterns of BBT from both conventional BBT and from MBT. While both conventional BBT and BBT-IEC show a null pattern of p16 expression, our case of BBT-IEC showed aberrant p53 overexpression, albeit with a maturation pattern similar to that described for TP53 mutant mucinous ovarian carcinoma and differentiated vulvar intraepithelial neoplasia (dVIN). Similarly, while BBT-SNP shows an infiltrative-like growth pattern similar to that of MBT, our case also showed a wild-type pattern of p53 expression and a Ki-67 proliferative index similar to areas with conventional BBT histology. In conclusion, in our small case series, we show that the use of immunohistochemistry for p53 and Ki-67 may help to distinguish challenging patterns of BBT from MBT. Further studies are needed to validate this finding in a larger case cohort., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Aims: Traditionally, mismatch repair (MMR) status is determined by a panel of four antibodies (MLH1, PMS2, MSH2, MSH6). If all proteins are retained, cases are MMR proficient (pMMR), while loss of one or more proteins is indicative of MMR deficiency (dMMR). This approach has been challenged in favour of a two-antibody approach, using PMS2 and MSH6 as a first screening. Their retainment is deemed sufficient to declare cases pMMR. In this study we aim to verify the validity of the two-antibody approach., Methods and Results: We performed a nationwide study in colorectal cancer (CRC) and endometrial cancer (EC) diagnosed between 2016 and 2023, including 47,657 patients to evaluate the two-antibody approach. In 0.17% and 0.4% of cases of CRC and EC, respectively, dMMR cases would be missed with the two-antibody approach. Subgroup analyses pointed towards slightly increased miss rates in younger patients (under the age of 50 years) in both groups and identified special subtypes (signet ring cell carcinoma, medullary carcinoma, and mucinous carcinoma in CRC and clear cell carcinoma in EC) with increased miss rates. For these specific subgroups, a low threshold should be used for further testing. In case of ambiguous or heterogeneous staining patterns, four antibodies should be used., Conclusion: In general, the application of a two-antibody MMR testing strategy does not lead to considerable failure of dMMR identification and saves costs., (© 2024 The Author(s). Histopathology published by John Wiley & Sons Ltd.)

Immunohistochemistry (IHC) and immunofluorescence (IF) are crucial techniques for studying cardiac physiology and disease. The accuracy of these techniques is dependent on various aspects of sample preparation and processing. However, standardised protocols for sample preparation of tissues, particularly for fresh-frozen human left ventricle (LV) tissue, have yet to be established and could potentially lead to differences in staining and interpretation. Thus, this study aimed to optimise the reproducibility and quality of IF staining in fresh-frozen human LV tissue by systematically investigating crucial aspects of the sample preparation process. To achieve this, we subjected fresh-frozen human LV tissue to different fixation protocols, primary antibody incubation temperatures, antibody penetration reagents, and fluorescent probes. We found that neutral buffered formalin fixation reduced image artefacts and improved antibody specificity compared to both methanol and acetone fixation. Additionally, incubating primary antibodies at 37°C for 3 h improved fluorescence intensity compared to the commonly practised 4°C overnight incubation. Furthermore, we found that DeepLabel, an antibody penetration reagent, and smaller probes, such as fragmented antibodies and Affimers, improved the visualisation depth of cardiac structures. DeepLabel also improved antibody penetration in CUBIC cleared thick LV tissue fragments. Thus, our data underscores the importance of standardised protocols in IF staining and provides various means of improving staining quality. In addition to contributing to cardiac research by providing methodologies for IF, the findings and processes presented herein also establish a framework by which staining of other tissues may be optimised., (© 2024 The Author(s). Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

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.)

Tubulin β-3 staining pattern and staining intensity of 5-hydroxymethyl cytosine (5-hmC) are potential diagnostic and prognostic markers in melanocytic lesions that need further evaluation. Melanocytic nevi and primary cutaneous melanomas were immunohistochemically stained for tubulin-β-3 and 5-hmC. Immunoreactivity and staining patterns were correlated with Breslow-thickness, clinical and pathological characteristics, and progression-free survival. Melanocytes showed positive tubulin β-3 staining. However, in most nevi, tubulin β-3 staining appeared as a gradient with intense cytoplasmic staining in cells of the superficial part of the lesion that faded to weak staining in the deep dermal part, while no gradient was found in deep penetrating nevi and melanomas. In 53 % of the melanomas, areas with loss of tubulin β-3 staining were found. 5-hmC staining intensity was significantly higher in melanocytic nevi compared to melanomas. Breslow thickness in combination with low 5-hmC score and loss of tubulin-β-3 staining was predictive for poor prognosis. As single markers, tubulin-β-3 and 5-hmC can be useful to distinguish between melanocytic nevi and melanoma, but staining variability limits the use of 5-hmC. In melanomas measuring >1.5 mm, combination of low 5-hmC score and loss of tubulin-β-3 staining may have prognostic value., Competing Interests: Declaration of competing interest The authors state no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Retroperitoneal Ewing sarcomas (RES) are very rare and mostly described in case reports. The purpose of this study was to retrospectively analyze the clinicopathology, molecular characteristics, biological behavior, and therapeutic information of 13 cases of primary RES with immunohistochemical staining, fluorescence in situ hybridization, RT-PCR and NGS sequencing detection techniques. The thirteen patients included eight males and five females with a mean age of 34 years. Morphologically, the tumors were comprised of small round or epithelial-like cells with vacuolated cytoplasm (6/13,46 %) arranged in diffuse, nested (8/13,62 %) and perivascular (7/13,54 %) patterns. Unusual morphologic patterns, such as meningioma-like swirling structures and sieve-like structures were relatively novel findings. Immunohistochemical studies showed CD99 (12/13; 92 %), CD56 (11/13; 85 %), NKX2.2 (9/13; 69 %), PAX7 (10/11;91 %) and CD117(6/9;67 %) to be positive.12 cases (92 %) demonstrated EWSR1 rearrangement and 3 cases displayed EWSR1::FLI1 fusion by FISH. ERCC4 splice-site variant, a novel pathogenic variant, was discovered for the first time via RNA sequencing. With a median follow-up duration of 14 months (6 to 79 months), 8/13 (62 %) patients died, while 5/13(38 %) survived. Three cases recurred, and five patients developed metastasis to the liver (2 cases), lung (2 cases) and bone (1 case). RES is an aggressive, high-grade tumor, prone to multiple recurrences and metastases, with distinctive morphologic, immunohistochemical, and molecular genetic features. ERCC4 splicing mutation, which is a novel pathogenic variant discovered for the first time, with possible significance for understanding the disease, as well as the development of targeted drugs., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Human epidermal growth factor receptor 2 (HER2)-low breast cancer has emerged as a subtype of breast cancer, defined by HER2 1+/2+ in immunohistochemistry (IHC) and absence of ERBB2 gene amplification on fluorescence in situ hybridization (FISH). Recent trials showed marked response of HER2-low breast cancer to novel anti-HER2 antibody-drug-conjugates. Data on characteristics of HER2-low breast cancer subtype is limited. Real-world data from the Anatomic Pathology Department of Hotel-Dieu de France, spanning 2017-2023, was retrospectively collected. HER2-positive patients were excluded to compare HER2-low to HER2-zero breast cancer subtypes. Clinicopathological characteristics between the groups were compared using a Chi-Squared test. Out of 1195 patients, we observed 341 (28.5 %) HER2-low breast cancers cases. HER2-positive breast cancer cases (n = 178; 14.9 %) were excluded. There was no significant difference in age and sex between HER2-low and HER2-zero group (p = 0.33 and 0.79, respectively). HER2-low breast cancer was associated with positive estrogen receptor status and positive progesterone receptor status (p < 0.001 and p = 0.01, respectively). Ductal adenocarcinomas were more commonly observed in HER2-low group (p < 0.001). When stratified by hormone (HR) status, 87.4 % of patients had HR-positive status and 12.6 % were HR-negative. Among the HR-negative group, HER2-low tumors tended to show lower proliferation index compared to HER2-zero tumors (25%vs.10 %, p = 0.04). This study showed that HER2-low is distinct from HER2-zero and is common among patients with breast cancer. Clinicopathological features such as histological type differ between HER2-zero and HER2-low breast cancer. Within HR-negative breast cancer, those with low HER2 expression exhibit a less aggressive profile compared to HER2-zero tumors., Competing Interests: Declaration of competing interest No conflict of interest to disclose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Histopathological examinations heavily rely on hematoxylin and eosin (HE) and immunohistochemistry (IHC) staining. IHC staining can offer more accurate diagnostic details but it brings significant financial and time costs. Furthermore, either re-staining HE-stained slides or using adjacent slides for IHC may compromise the accuracy of pathological diagnosis due to information loss. To address these challenges, we develop PST-Diff, a method for generating virtual IHC images from HE images based on diffusion models, which allows pathologists to simultaneously view multiple staining results from the same tissue slide. To maintain the pathological consistency of the stain transfer, we propose the asymmetric attention mechanism (AAM) and latent transfer (LT) module in PST-Diff. Specifically, the AAM can retain more local pathological information of the source domain images, while ensuring the model's flexibility in generating virtual stained images that highly confirm to the target domain. Subsequently, the LT module transfers the implicit representations across different domains, effectively alleviating the bias introduced by direct connection and further enhancing the pathological consistency of PST-Diff. Furthermore, to maintain the structural consistency of the stain transfer, the conditional frequency guidance (CFG) module is proposed to precisely control image generation and preserve structural details according to the frequency recovery process. To conclude, the pathological and structural consistency constraints provide PST-Diff with effectiveness and superior generalization in generating stable and functionally pathological IHC images with the best evaluation score. In general, PST-Diff offers prospective application in clinical virtual staining and pathological image analysis.

Background: The underlying pathogenesis of pityriasis lichenoides et varioliformis acuta (PLEVA) remains unclear, although immunologic injury and viral etiology have been suggested., Objective: To evaluate and expand the immunophenotype of PLEVA and to search for possible viral pathogens., Methods: Formalin-fixed, paraffin-embedded specimens of 20 patients with PLEVA and 9 patients with common inflammatory dermatoses (ID) were studied for immunophenotyping and for human herpesvirus (HHV) 1 and 2, cytomegalovirus (CMV), HHV-8, parvovirus B19, and Epstein-Barr virus (EBV) immunohistochemistry. The presence of HHV-6, HHV-7, and enteroviruses was assayed molecularly., Results: The numbers of CD8 + T cells and T-cell intracellular antigen-1 (TIA-1) + cells were statistically significantly higher in PLEVA compared to the ID group. Immunohistochemistry for human HHV-1 and HHV-2, CMV and HHV-8, parvovirus B19, and in situ hybridization for EBV were all negative. There was molecular evidence for HHV-7 in only one PLEVA case (5%). Molecular studies for HHV-6 and enterovirus involvement were negative in all the PLEVA specimens., Conclusions: The predominant T-cell infiltrate in PLEVA is dominated by CD8 + cells, and by increased numbers of TIA1 + cells, which may indicate a cytotoxic T-cell damage to the epidermis. Viral presence was not detected., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Pilocytic astrocytoma (PAs) represents a significant portion of childhood primary brain tumors, with distinct histological and radiological features. The prevalence of KIAA1549::BRAF fusion in PAs has been well-established, this study aims to assess the prevalence of KIAA1549::BRAF fusions and explore their associations with tumor characteristics, radiological findings, and patient outcomes in PAs., Methods: Histologically confirmed cases of PAs from a 5-year period were included in the study. Demographic, histopathological, and radiological data were collected, and immunohistochemistry was performed to characterize tumor markers. FISH and qRT-PCR assays were employed to detect KIAA1549::BRAF fusions. Statistical analyses were conducted to examine associations between fusion status and various other parameters., Results: Histological analysis revealed no significant differences in tumor features based on fusion status. However, younger age groups showed higher fusion prevalence. Radiologically, fusion-positive cases were distributed across different tumor subtypes SE, CWE and NCWE. Survival analysis did not demonstrate a significant impact of fusion status on overall survival, however most cases with recurrence and death harboured KIAA1549::BRAF fusion. Of 200 PAs, KIAA1549::BRAF fusions were detected in 64 % and 74 % of cases via qRT-PCR and FISH, respectively. Concordance between the two platforms was substantial (86 %)., Conclusion: KIAA1549::BRAF fusions are prevalent in PAs and can be reliably detected using both FISH and qRT-PCR assays. Cost considerations suggest qRT-PCR as a more economical option for fusion detection in routine clinical practice., Competing Interests: Declaration of competing interest No conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

CIC-rearranged sarcoma (CRS) is a group of high-grade undifferentiated small round cell sarcomas examined as a separate entity in the current WHO classification; since it shows more aggressive clinical behavior and distinct morphological and molecular features compared to Ewing sarcoma (ES). As CCNE1 expression is associated with tumor growth in CIC::DUX4 sarcomas, we aimed to demonstrate the value of cyclin E1 expression in CRS. Cyclin E1 immunohistochemistry and break-apart FISH for EWSR1 and CIC gene rearrangements were performed on 3-mm tissue microarrays composed of 40 small round cell tumors. Five cases were classified as CRS, whereas 22 were ES and 13 were unclassified (EWSR1-/CIC-). Among all three diagnostic groups, we found cyclin E1 expression level to be higher in CRS (80 %) and unclassified groups (61.5 %) compared to ES (4.5 %, p < 0.001). In addition, high cyclin E1 expression levels were associated with higher mean age at diagnosis, presence of atypical histology and myxoid stroma, low CD99 expression, and presence of metastasis at diagnosis. The sensitivity and specificity of high cyclin E1 expression in detecting non-ES cases were 95.5 % and 66.7 %, respectively. However, the correlation between cyclin E1 expression level and survival was not statistically significant. This is the first study that shows cyclin E1 immunohistochemical expression in EWSR1-negative undifferentiated small cell sarcomas, particularly CRS., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: PReferentially expressed Antigen in MElanoma (PRAME) has shown utility in differentiating benign from malignant melanocytic neoplasms. In this study, we investigated the clinical significance of PRAME expression in dysplastic nevi (DN) and nevus-associated melanoma in situ (MIS)., Methods: We included 172 DN and 38 nevus-associated MIS from our institutional archive. PRAME positive expression was defined as nuclear staining in at least 75% of melanocytes. In addition, relevant studies from PubMed and Web of Science were incorporated into a meta-analysis using the random-effects model to assess PRAME expression in MIS and DN., Results: Our institutional data revealed that 71.1% of nevus-associated MIS cases exhibited positive PRAME expression in the MIS components, whereas all DN components were negative for PRAME. 5.7% of cases diagnosed as DN in our cohort demonstrated diffuse positivity for PRAME. Notably, MIS associated with DN displaying epidermal and dermal components displayed a higher likelihood of PRAME positivity compared to those arising on a background of DN with solely epidermal (junctional) components (84% vs. 46%, p = 0.024). The meta-analysis indicated that the pooled PRAME positivity in MIS and DN was 54.5% and 1.9%, respectively., Conclusion: PRAME is a valuable immunohistochemical marker for differentiating MIS from DN, particularly in the context of nevus-associated MIS., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Primary ovarian mucinous tumors are uncommon. Factors leading to invasive progression and metastatic disease have not been fully delineated yet. The aim of this study is to determine the rates of p53 and p16 immunoexpressions in primary ovarian mucinous tumors, to investigate their relationship with clinicopathologic factors and their impact on prognosis and survival., Methods: Seventy-eight primary ovarian mucinous tumors (30 mucinous cystadenomas, 30 mucinous borderline tumors (MBOT), 18 mucinous carcinomas (MOC)) were evaluated immunohistochemically with p53 and p16 staining. The demographic, clinicopathological data, and postoperative follow-up findings of the patients were analyzed., Results: Mutation-type p53 staining was present in 1/30 (3.3 %) cystadenoma, 10/30 (33.3 %) MBOT and 9/18 (50 %) MOC (p = 0.001). p16 overexpression was detected in 3/30 (10.0 %) MBOT and 5/18 (27.8 %) MOC, but not in any cystadenoma (p = 0.04). The frequency of mutation-type p53 staining in MBOTs with microinvasion was higher (71.4 %) than in those without (28.6 %, p = 0.026). The frequencies of p16 or p53 mutations were similar in MBOTs with and without intraepithelial carcinoma, or mural nodule (p > 0.05). In MOCs with ovarian surface involvement, mutation-type p53 staining was detected in 66.7 % (6/9) and p16 overexpression in 55.6 % (5/9) of the cases. A significant difference was found between MOCs with or without ovarian surface involvement regarding the frequency of p16 overexpression (p = 0.029). Any relationship was not detected between survival and p53 and p16 expression in MOCs (p > 0.05)., Conclusion: p53 and p16 mutation rates were higher in MOCs compared to mucinous cystadenomas and MBOTs and suggest a relevant role in the development of primary ovarian mucinous carcinoma, however further studies are needed in this regard., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

C4d is the end degradation product of activated complement component C4b that appears during the early steps of the classical and lectin complement pathways. Within the primary sequence of C4d, there is a reactive thioester group that binds covalently to nearby surfaces, thus labeling the locations of complement activation. This feature makes C4d a target for immunohistochemical staining aimed to aid the diagnosis of, among others, the antibody-mediated rejection of transplanted organs, membranous glomerulonephritis, bullous pemphigoid, or inflammatory myopathies. However, the credibility of C4d immunostaining is debatable, as a high background in surrounding tissues and body fluids and diffused patterns of deposits in target structures are experienced with some of the available anti-C4d antibodies. Herein, we present an improved version of a rabbit anti-C4d antibody, originally raised against the C-terminal linear neoepitope of this complement fragment. Minor cross-reactivity with C4b and native C4 proteins, measured by ELISAs, as well as relatively low concentrations necessary for obtaining a specific signal in immunohistochemical analyses of formalin-fixed paraffin-embedded material, makes the improved antibody superior to commercially available rabbit monoclonal anti-C4d antibody SP91 dedicated to ex vivo diagnostics, as demonstrated by the staining of a panel of kidney transplant biopsies.

The design of prophylactic and diagnostic tools specific to animal papillomaviruses is hampered by the difficulties of viral in vitro manipulation and by the scarce availability of dedicated biotechnological tools. This paper reports the production of Ovine Papillomavirus 3 (OaPV3)-based virus-like particles (OaPV3-VLPs) in the baculovirus system and their use to investigate host humoral immune response through the establishment of an indirect ELISA test., Polyclonal sera and monoclonal antibodies were generated against OaPV3-VLPs, and their isotype and reactivity were determined. Additionally, antibodies allowed OaPV3 detection in ovine squamous cell carcinoma (SCC) samples by immunohistochemistry. Results encourage the standardization of OaPV3-specific prophylactic and serological diagnostic tools, and open new perspectives for the study of host-viral interaction and SCC development., 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 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The tumour immune microenvironment (TIME) in breast cancer is acknowledged with an increasing role in treatment response and prognosis. With a growing number of immune markers analysed, digital image analysis may facilitate broader TIME understanding, even in single-plex IHC data. To facilitate analyses of the latter an open-source image analysis pipeline, Tissue microarray MArker Quantification (TMArQ), was developed and applied to single-plex stainings for p53, CD3, CD4, CD8, CD20, CD68, FOXP3, and PD-L1 (SP142 antibody) in a 218-patient triple negative breast cancer (TNBC) cohort with complementary pathology scorings, clinicopathological, whole genome sequencing, and RNA-sequencing data. TMArQ's cell counts for analysed immune markers were on par with results from alternative methods and consistent with both estimates from human pathology review, different quantifications and classifications derived from RNA-sequencing as well as known prognostic patterns of immune response in TNBC. The digital cell counts demonstrated how immune markers are coexpressed in the TIME when considering TNBC molecular subtypes and DNA repair deficiency, and how combination of immune status with DNA repair deficiency status can improve the prognostic stratification in chemotherapy treated patients. These results underscore the value and potential of integrating TIME and specific tumour intrinsic alterations/phenotypes for the molecular understanding of TNBC., (© 2024. The Author(s).)

Electron microscopy paired with immunogold labeling is the most precise tool for protein localization. However, these methods are either cumbersome, resulting in small sample numbers and restricted quantification, or limited to identifying protein epitopes external to the membrane. Here, we introduce SUB-immunogold-SEM, a scanning electron microscopy technique that detects intracellular protein epitopes proximal to the membrane. We identify four critical sample preparation factors contributing to the method's sensitivity. We validate its efficacy through precise localization and high-powered quantification of cytoskeletal and transmembrane protein distribution. We evaluate the capabilities of SUB-immunogold-SEM on cells with highly differentiated apical surfaces: (i) auditory hair cells, revealing the presence of nanoscale MYO15A-L rings at the tip of stereocilia; and (ii) respiratory multiciliate cells, mapping the distribution of the SARS-CoV-2 receptor ACE2 along the motile cilia. SUB-immunogold-SEM extends the application of SEM-based nanoscale protein localization to the detection of intracellular epitopes on the exposed surfaces of any cell., (© 2024. The Author(s).)