Análisis inmunohistoquímico de MMP-1, MMP-2 y MMP-9 en ameloblastoma convencional / Immunohistochemical analysis of MMP-1, MMP-2, MMP-9 in conventional ameloblastoma

Introducción: las metaloproteinasas son enzimas que participan en la remodelación tisular y su función se relaciona con procesos fisiológicos y patológicos, como la invasión y la metástasis. El ameloblastoma convencional (AMC) es una neoplasia epitelial benigna odontogénica intraósea caracterizada por una progresión lenta y localmente invasiva, cuyo crecimiento se ha vinculado con el recambio ósea y la remodelación de la matriz extracelular. El objetivo del presente trabajo fue determinar la presencia inmunohistoquímica de MMP-1, MMP-2 y MMP-9 en el AMC. Material y métodos: se realizó un estudio piloto observacional analítico utilizando cinco muestras de AMC. Los especímenes fueron recolectados aleatoriamente del archivo del Departamento de Patología Oral y Maxilofacial, de la Escuela Nacional de Estudios Superiores (ENES) Unidad León, UNAM. Como grupo control se emplearon dos especímenes de folículo dental, obtenido de pacientes con indicación de su extracción por motivos ortodóncicos. Se realizó la técnica de inmunohistoquímica por peroxidasa, recolectando el nivel y proporción de inmunoexpresión de manera semicuantitativa. Resultados: cuatro pacientes fueron de género masculino y uno femenino, la edad promedio fue de 40.6 ± 14.9 años. Todas las muestras fueron obtenidas de la región mandibular posterior. Se observaron dos especímenes con patrón folicular y tres con plexiforme. Las MMP-2 y MMP-9 se detectaron sólo en uno de los cinco especímenes y únicamente en el parénquima de la lesión, con una proporción de 100%. Conclusión: según nuestro análisis inmunohistoquímico, las MMP-2 y MMP-9 son las metaloproteinasas que presentaron expresión positiva dentro de la patogénesis del AMC comparado a la MMP-1; no obstante, es necesario realizar este tipo de estudios en una población mayor (AU)

Introduction: metalloproteinases are enzymes involved in tissue remodeling and their function is related to physiological and pathological processes, such as invasion and metastasis. These enzymes are capable of degrading components of the extracellular matrix, which may promote tumor progression. Conventional ameloblastoma (CA) is described as a benign intraosseous epithelial odontogenic neoplasm characterized by a slow and locally invasive progression, whose growth has been linked to bone turnover and extracellular matrix remodeling. The aim of the present work was to determine the immunohistochemical presence of MMP-1, MMP-2 and MMP-9 in CA. Material and methods: an analytical observational pilot study was performed using 5 CA, randomly collected from the archive of the Department of Oral and Maxillofacial Pathology, Escuela Nacional de Estudios Superiores (ENES) Unidad León, UNAM. The control group used were two dental follicle samples, obtained from patients with extraction indication for orthodontic treatment. The peroxidase immunohistochemistry assay was performed, collecting semiquantitatively level and proportion of immunoexpression. Results: four patients were male and one female, the average age was 40.6 ± 14.9 years. All specimens were obtained from the posterior mandibular region. Two specimens were observed with follicular pattern and three with plexiform pattern. MMP-2 and MMP-9 were detected only in one of the five specimens, with presence in the parenchyma of the lesion, with a proportion of 100% of the cell analyzed. Conclusion: according to our immunohistochemical analysis, MMP-2 and MMP-9 are the metalloproteinases that presented positive expression within the pathogenesis of CA compared to MMP-1; however, it is necessary to perform this type of studies in a larger population (AU)

Combination of Tissue Microarray Profiling and Multiplexed IHC Approaches to Investigate Transport Mechanism of Nucleoside Analog Drug Resistance.

Nucleoside analogs (NAs) are an established class of anticancer agents being used clinically for the treatment of diverse cancers, either as monotherapy or in combination with other established anticancer or pharmacological agents. To date, nearly a dozen anticancer NAs are approved by the FDA, and several novel NAs are being tested in preclinical and clinical trials for future applications. However, improper delivery of NAs into tumor cells because of alterations in expression of one or more drug carrier proteins (e.g., solute carrier (SLC) transporters) within tumor cells or cells surrounding the tumor microenvironment stands as one of the primary reasons for therapeutic drug resistance. The combination of tissue microarray (TMA) and multiplexed immunohistochemistry (IHC) is an advanced, high-throughput approach over conventional IHC that enables researchers to effectively investigate alterations to numerous such chemosensitivity determinants simultaneously in hundreds of tumor tissues derived from patients. In this chapter, taking an example of a TMA from pancreatic cancer patients treated with gemcitabine (a NA chemotherapeutic agent), we describe the step-by-step procedure of performing multiplexed IHC, imaging of TMA slides, and quantification of expression of some relevant markers in these tissue sections as optimized in our laboratory and discuss considerations while designing and carrying out this experiment.

Diagnóstico de precisão das lesões mamárias / Accurate diagnosis of breast lesions

PONTOS-CHAVE As lesões mamárias compreendem uma ampla variedade de diagnósticos que apresentam comportamentos diversos. As lesões mamárias podem ser classificadas como lesões benignas, de potencial de malignidade indeterminado (B3), carcinoma in situ e carcinoma invasor. Na era da medicina personalizada, individualizar e obter um diagnóstico preciso faz grande diferença no desfecho final da paciente, principalmente no caso do câncer de mama. Exames de imagem direcionados e de qualidade, métodos de biópsia adequadamente selecionados e análises de anatomopatologia convencional, imuno-histoquímica e até molecular são determinantes no diagnóstico e no manejo das pacientes.

Statistical Analysis of Multiplex Immunofluorescence and Immunohistochemistry Imaging Data.

Advances in multiplexed single-cell immunofluorescence (mIF) and multiplex immunohistochemistry (mIHC) imaging technologies have enabled the analysis of cell-to-cell spatial relationships that promise to revolutionize our understanding of tissue-based diseases and autoimmune disorders. Multiplex images are collected as multichannel TIFF files; then denoised, segmented to identify cells and nuclei, normalized across slides with protein markers to correct for batch effects, and phenotyped; and then tissue composition and spatial context at the cellular level are analyzed. This chapter discusses methods and software infrastructure for image processing and statistical analysis of mIF/mIHC data.

Interobserver and Interantibody Reproducibility of HER2 Immunohistochemical Scoring in an Enriched HER2-Low-Expressing Breast Cancer Cohort.

OBJECTIVES: We assessed the interobserver and interantibody reproducibility of HER2 immunohistochemical scoring in an enriched HER2-low-expressing breast cancer cohort. METHODS: A total of 114 breast cancer specimens were stained by HercepTest (Agilent Dako) and PATHWAY anti-HER2 (4B5) (Ventana) antibody assays and scored by 6 breast pathologists independently using current HER2 guidelines. Level of agreement was evaluated by Cohen κ analysis. RESULTS: Although the interobserver agreement rate for both antibodies achieved substantial agreement, the average rate of agreement for HercepTest was significantly higher than that for the 4B5 clone (74.3% vs 65.1%; P = .002). The overall interantibody agreement rate between the 2 antibodies was 57.8%. Complete interobserver concordance was achieved in 44.7% of cases by HercepTest and 45.6% of cases by 4B5. Absolute agreement rates increased from HER2 0-1+ cases (78.1% by HercepTest and 72.2% by 4B5; moderate agreement) to 2-3+ cases (91.9% by HercepTest and 86.3% by 4B5; almost perfect agreement). CONCLUSIONS: Our results demonstrated notable interobserver and interantibody variation on evaluating HER2 immunohistochemistry, especially in cases with scores of 0-1+, although the performance was much more improved among breast-specialized pathologists with the awareness of HER2-low concept. More accurate and reproducible methods are needed for selecting patients who may benefit from the newly approved HER2-targeting agent on HER2-low breast cancers.

Molecular-Based Immunohistochemical Algorithm for Uterine Leiomyosarcoma Diagnosis.

The morphologic assessment of uterine leiomyosarcoma (LMS) may be challenging, and diagnostic immunohistochemical (IHC) analysis is currently lacking. We evaluated the genomic landscape of 167 uterine LMS by targeted next-generation sequencing (NGS) to identify common genomic alterations. IHC analyses corresponding to these genomic landmarks were applied to a test cohort of 16 uterine LMS, 6 smooth muscle tumors of uncertain malignant potential (STUMP), and 6 leiomyomas with NGS data and a validation cohort of 8 uterine LMS, 12 STUMP, 21 leiomyomas and leiomyoma variants, 7 low-grade endometrial stromal sarcomas, and 2 diagnostically challenging uterine smooth muscle tumors. IHC results were individually interpreted by 3 pathologists blinded to NGS data. Overall, 94% of LMS showed ≥1 genomic alteration involving TP53, RB1, ATRX, PTEN, CDKN2A, or MDM2, with 80% showing alterations in ≥2 of these genes. In the test cohort, an initial panel of p53, Rb, PTEN, and ATRX was applied, followed by a panel of DAXX, MTAP, and MDM2 in cases without abnormalities. Abnormal p53, Rb, PTEN, and ATRX IHC expression was seen in 75%, 88%, 44%, and 38% of LMS, respectively, in the test cohort. Two or more abnormal IHC results among these markers were seen in 81% of LMS. STUMPs demonstrated only 1 IHC abnormality involving these markers. No IHC abnormalities were seen in leiomyomas. In the validation cohort, abnormal p53, Rb, and PTEN IHC results were seen in LMS, whereas rare STUMP or leiomyomas with bizarre nuclei showed IHC abnormalities involving only 1 of the markers. Abnormalities in ≥2 markers were present in both diagnostically challenging smooth muscle tumors, confirming LMS. Concordance was excellent among pathologists in the interpretation of IHC (κ = 0.97) and between IHC and NGS results (κ = 0.941). Uterine LMS exhibit genomic landmark alterations for which IHC surrogates exist, and a diagnostic algorithm involving molecular-based IHC may aid in the evaluation of unusual uterine smooth muscle tumors.

PRAME immunohistochemistry can distinguish melanocytic pseudonests of lichenoid reactions from melanoma in situ.

BACKGROUND: Distinguishing melanocytic pseudonests encountered in lichenoid dermatoses or lichenoid keratoses from melanoma in situ (MIS) with brisk lichenoid inflammation can prove challenging. METHODS: We designed a case-control study to evaluate the accuracy metrics of PRAME immunohistochemistry to distinguish melanocytic pseudonests in lichenoid dermatoses or keratoses from inflamed MIS. Immunostaining for PRAME was performed on paraffin-embedded formalin-fixed diagnostic tissue using a rabbit monoclonal antibody to PRAME (Abcam), with a 1:3200 dilution on a Leica Bond detection system. RESULTS: Our search identified 21 cases of melanocytic pseudonests (n = 21, 46%) encountered in lichenoid dermatoses and 24 cases of inflamed MIS (n = 24, 53%). Each method of evaluating PRAME immunohistochemistry (PRAME+ clusters, PRAME % of melanocytes by four categories and PRAME+ melanocyte counts per linear mm of epidermal basal layer) showed statistically significant differences between the MIS and the pseudonest cohorts (respectively, p < 0.001; p < 0.001; and p < 0.001). Receiver operating characteristics analysis for PRAME+ melanocyte counts per linear mm of epidermal basal layer revealed an area under the curve of 0.9 ± 0.05 (95% confidence interval 0.9-1.0). When determining an optimal cut-off point for the best Youden index [sensitivity (%) + specificity (%) - 100], the cut-off of 1.0 PRAME+ melanocytes per linear mm showed a sensitivity of 79.2% and specificity of 85.7% (Youden index 0.65) to distinguish MIS from pseudonests. CONCLUSION: PRAME immunohistochemistry may constitute an additional tool for this challenging differential diagnosis.

High intratumoral plasma cells content in primary prostate cancer defines a subset of tumors with potential susceptibility to immune-based treatments.

BACKGROUND: Data on advanced prostate cancer (PCa) suggest more prior systemic therapies might reduce tumor immune responsiveness. In treatment-naïve primary PCa, recent work correlated intratumoral plasma cell content with enhanced tumor immune-responsiveness. We sought to identify features of localized PCa at a high risk of recurrence following local treatment with high plasma cell content to help focus future immune-based neoadjuvant trials. METHODS: We performed retrospective analyses of molecular profiles from three independent cohorts of over 1300 prostate tumors. We used Wilcoxon Rank Sum to compare molecular pathways between tumors with high and low intratumoral plasma cell content and multivariable Cox proportional hazards regression analyses to assess metastasis-free survival. RESULTS: We validated an expression-based signature for intratumoral plasma cell content in 113 primary prostate tumors with both RNA-expression data and digital image quantification of CD138+ cells (plasma cell marker) based on immunohistochemisty. The signature showed castration-resistant tumors (n = 101) with more prior systemic therapies contained lower plasma cell content. In high-grade primary PCa, tumors with high plasma cell content were associated with increased predicted response to immunotherapy and decreased response to androgen-deprivation therapy. Master regulator analyses identified upregulated transcription factors implicated in immune (e.g. SKAP1, IL-16, and HCLS1), and B-cell activity (e.g. VAV1, SP140, and FLI-1) in plasma cell-high tumors. Master regulators overactivated in tumors with low plasma cell content were associated with shorter metastasis-free survival following radical prostatectomy. CONCLUSIONS: Markers of plasma cell activity might be leveraged to augment clinical trial targeting and selection and better understand the potential for immune-based treatments in patients with PCa at a high risk of recurrence following local treatment.

Construction of mouse cochlin mutants with different GAG-binding specificities and their use for immunohistochemistry.

Glycosaminoglycan (GAG) is a polysaccharide present on the cell surface as an extracellular matrix component, and is composed of repeating disaccharide units consisting of an amino sugar and uronic acid except in the case of the keratan sulfate. Sulfated GAGs, such as heparan sulfate, heparin, and chondroitin sulfate mediate signal transduction of growth factors, and their functions vary with the type and degree of sulfated modification. We have previously identified human and mouse cochlins as proteins that bind to sulfated GAGs. Here, we prepared a recombinant cochlin fused to human IgG-Fc or Protein A at the C-terminus as a detection and purification tag and investigated the ligand specificity of cochlin. We found that cochlin can be used as a specific probe for highly sulfated heparan sulfate and chondroitin sulfate E. We then used mutant analysis to identify the mechanism by which cochlin recognizes GAGs and developed a GAG detection system using cochlin. Interestingly, a mutant lacking the vWA2 domain bound to various types of GAGs. The N-terminal amino acid residues of cochlin contributed to its binding to heparin. Pathological specimens from human myocarditis patients were stained with a cochlin-Fc mutant. The results showed that both tryptase-positive and tryptase-negative mast cells were stained with this mutant. The identification of detailed modification patterns of GAGs is an important method to elucidate the molecular mechanisms of various diseases. The method developed for evaluating the expression of highly sulfated GAGs will help understand the biological and pathological importance of sulfated GAGs in the future.

Variability in the Epithelial Expression of Ki67 and Polarization Characteristics of Collagen in Odontogenic Cysts and Tumours

ABSTRACT Objective: To assess the proliferation of epithelium (using the Ki67 index) and the polarization pattern of collagen in selected odontogenic cysts and tumours. In addition, an exploratory analysis of the effect of inflammation on the proliferation rate was done. Material and Methods: Following immunohistochemical staining, the labelling/proliferation index of Ki67 was calculated. The thickness and corresponding polarization colour of 100 juxta-epithelial picrosirius red-stained collagen fibers were assessed using linear micrometry with an eyepiece reticule under × 1000 magnification. Inflammation was graded subjectively as mild, moderate, and severe. Results: Overall Ki-67 expression was higher in the radicular cyst, Odontogenic Keratocyst, Ameloblastoma, while suprabasal Ki-67 positivity was maximum in Odontogenic Keratocyst. The stromal collagen fibers in Ameloblastoma showed predominantly green birefringence, whereas Odontogenic Keratocyst had orange birefringence. There was no significant association of inflammation with Ki-67 expression or birefringence patterns. Conclusion: The highest Ki67 expression in the radicular cyst, followed by Odontogenic Keratocyst and Ameloblastoma. Differences in the collagen maturation pattern were noted innately in five lesions studied and were further influenced by inflammatory changes. Epithelial proliferation and concomitant expression of thickness and maturity of the stromal collagen are innate features of the lesion further influenced by inflammation in various odontogenic cysts and tumours and may, in turn, guide the clinical behavior.

Comparative Assessment of E-cadherin's Expression between the Metastatic and Non-metastatic Oral Squamous Cell Carcinoma: An Immunohistochemical Study

ABSTRACT Objective: To identify the clinicopathological correlation of E-cadherin expression in metastatic and non-metastatic oral squamous cell carcinoma (OSCC). Material and Methods: A total of 90 paraffin-embedded tissue sections of OSCC were retrieved from the registry. The total selected samples were 45 cases each from the primary lesions of metastatic and non-metastatic OSCC. One section was subjected to routine Hematoxylin and eosin stain and another to immunohistochemical analysis for E-cadherin expression. Results: A non-significant (p˃0.05) increased expression is seen in the non-metastatic group compared to the metastatic group, with predominantly membrane as the staining site in either group. However, the expression of E-cadherin did not reveal any statistically significant association with independent variables such as age, gender, and adverse habits of the patients (p>0.05). On the other hand, with respect to the histological differentiation of OSCC, a significant association (p<0.001) was observed with the well-differentiated type of metastatic OSCC. Conclusion: E-cadherin was useful to some extent in predicting regional metastasis. However, further studies using a panel of biomarkers with increased sample size may help us understand the process involved in metastasis.

¿Cuándo el patólogo debe solicitar inmunomarcación con B-Catenina?: una revisión de la literatura a propósito de dos casos / When should the pathologist request immunostaining with B-catenin?: a review of the literature on two cases

For more than 20 years, immunohistochemistry has represented an auxiliary test of great relevance to support pathological work, however, it should be noted that the pillar of diagnosis continues and will continue to be the classic morphological description based on hematoxylin eosin and the trained eye of the specialist. In neoplastic pathologies, whether benign or malignant, it is becoming increasingly necessary to incorporate new tissue biomarkers that help objectify or confirm the diagnosis of each patient, in order to provide better treatment or a more precise diagnosis about the biological nature of their illness. In this line, there has been intense research in relation to the participation of the Wnt/ß-catenin pathway in the development of various types of tumors, including colon adenocarcinoma, some pancreatic neoplasms and even some tumors of mesenchymal origin, as will be seen. in this work. In this context and based on two clinical cases of special interest, we have prepared a brief review of the literature considering the biological aspects of ß-catenin, tumors where there is currently a true relative consensus that its immunolabeling offers a real contribution to the confirmation of the entity and finally a limited exposition regarding the future of this biomarker in the pathology discipline.

Desde hace más de 20 años la inmunohistoquímica ha representado una prueba auxiliar de gran relevancia para apoyar el trabajo anatomopatológico, no obstante, cabe señalar que, aún el pilar del diagnóstico sigue y seguirá siendo la descripción morfológica clásica basada en hematoxilina eosina y el ojo entrenado del especialista. En las patologías neoplásicas, ya sea benignas, como malignas, se hace cada vez más necesario la incorporación de nuevos biomarcadores tisulares que ayuden a objetivar o confirmar el diagnóstico de cada paciente, con objeto de entregar un mejor tratamiento o un diagnóstico más preciso de la naturaleza biológica de su enfermedad. En esta línea, ha habido intensa investigación en relación con la participación de la vía Wnt/ß-catenina en el desarrollo de varios tipos de cáncer, entre ellos el adenocarcinoma de colon, algunas neoplasias pancreáticas e incluso algunos tumores de origen mesenquimal como se verá en este trabajo. En este contexto y partir de dos casos clínicos de especial interés, hemos preparado una breve revisión de la literatura considerando los aspectos biológicos de la ß-catenina, los tumores donde en la actualidad existe verdadero consenso de que su inmunomarcación ofrece un aporte real a la confirmación de la entidad y finalmente una exposición acotada respecto al futuro de este biomarcador en la disciplina de la anatomía patológica.

Recorrência do ameloblastoma: análise de fatores clínicoradiográficos e imunoexpressão de hMSH2, hMLH1 e KI-67 / Ameloblastoma recurrence: analysis of clinical-radiographic factors and immunoexpression of hMSH2, hMLH1 and KI-67

Introdução: O ameloblastoma é uma neoplasia odontogênica benigna, que apresenta altas taxas de recorrência pós-operatória. Diversos estudos mostram a relação entre as características clínico-patológicas e as modalidades de tratamento na recorrência do ameloblastoma. Os mecanismos moleculares envolvidos com a etiopatogenia deste tumor são pouco conhecidos, e apesar de alterações no Sistema Mismatch favorecerem o desenvolvimento de diferentes neoplasias humanas, a importância destes no desenvolvimento do ameloblastoma ainda permanece pouco compreendido. Objetivo: Identificar os fatores clínico-patológicos associados à recorrência do ameloblastoma, bem como investigar o papel da imunoexpressão das proteínas hMLH1, hMSH2 e Ki-67 na recidiva desses tumores odontogênicos. Metodologia: Tratou-se de um estudo descritivo, transversal e restrospectivo, com uma amostra constituída por 22 casos de ameloblastomas recidivantes e 22 casos não-recidivantes. A análise imunoistoquímica foi realizada de forma quantitativa, considerando a localização celular (nuclear) das proteínas estudadas. O teste de McNemar foi utilizado para comparar as variáveis entre lesões da 1ª biópsia e recorrentes de AMB. A sobrevida livre de recorrência foi analisada pelo método de Kaplan-Meier e as funções de sobrevida foram comparadas de acordo com as variáveis pelo teste log-rank. Resultados: O gênero mais acometido foi o feminino (n=24; 54,5%), com média de idade de acometimento de 39,1 ± 19,8 anos, sendo 45,5% (n=20) leucodermas. A região posterior de mandíbula foi a mais frequente no grupo recidivante (n=18, 81,8%) e também para os casos que não apresentaram recidivas (n=16, 72,8%). O tempo livre de recorrência foi de 50,0 (34,5 ­ 63,6) meses. Foram fatores significativamente associadas à recorrência dos ameloblastomas: presença de expansão da cortical (p=0,0089), ausência de reconstrução óssea (p=0,018), tratamento conservador (p=0,021), perda de imunoexpressão de hMSH2 (p=0,006) e hMLH1 (p=0,038) e forte imunoexpressão de Ki-67 (p=0,029). Conclusão: Baseado nos achados desta pesquisa, aspecto radiográfico, modalidade do tratamento e imunoexpressão de proteínas do Sistema Mismatch e Ki-67 podem ser utilizados como indicadores para a recorrência em ameloblastomas (AU).

Introduction: Ameloblastoma is a benign odontogenic neoplasm, which has high rates of postoperative recurrence. Several studies show the relationship between clinicopathological characteristics and treatment modalities in ameloblastoma recurrence. The molecular mechanisms involved in the etiopathogenesis of this tumor are little known, and although changes in the Mismatch System favor the development of different human neoplasms, their importance in the development of ameloblastoma still remains poorly understood. Objective: To identify clinical and pathological factors associated with ameloblastoma recurrence, as well as to investigate the role of immunoexpression of hMLH1, hMSH2 and Ki-67 proteins in the recurrence of these odontogenic tumors. Methodology: This was a descriptive, cross-sectional and retrospective study, with a sample consisting of 22 cases of recurrent ameloblastoma and 22 non-recurrent cases. Immunohistochemical analysis was performed quantitatively, considering the cellular (nuclear) location of the studied proteins. McNemar's test was used to compare variables between 1st biopsy and recurrent AMB lesions. Recurrence-free survival was analyzed using the Kaplan-Meier method and survival functions were compared according to variables using the log-rank test. Results: The most affected gender was female (n=24; 54.5%), with a mean age of involvement of 39.1 ± 19.8 years, 45.5% (n=20) being white. The posterior region of the mandible was the most frequent in the relapsed group (n=18, 81.8%) and also for the cases that did not present recurrences (n=16, 72.8%). Recurrence-free time was 50.0 (34.5 ­ 63.6) months. Factors significantly associated with recurrence of AMBs were: presence of cortical expansion (p=0.0089), absence of bone reconstruction (p=0.018), conservative treatment (p=0.021), loss of hMSH2 immunoexpression (p=0.006) and hMLH1 (p=0.038) and strong Ki-67 immunoexpression (p=0.029). Conclusion: Based on the findings of this research, radiographic appearance, treatment modality and immunoexpression of proteins from the Mismatch System and Ki-67 can be used as indicators for recurrence in ameloblastomas (AU).

Quantitative multiplexed analysis of MMP-11 and CD45 in metastatic breast cancer tissues by immunohistochemistry-assisted LA-ICP-MS.

Breast cancer is the leading cause of cancer death in woman and tremendous efforts are undertaken to limit its dissemination and to provide effective treatment. Various histopathological parameters are routinely assessed in breast cancer biopsies to provide valuable diagnostic and prognostic information. MMP-11 and CD45 are tumor-associated antigens and potentially valuable biomarkers for grading aggressiveness and metastatic probability. This paper presents methods for quantitative and multiplexed imaging of MMP-11 and CD45 in breast cancer tissues and investigates their potential for improved cancer characterization and patient stratification. An immunohistochemistry-assisted laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) method was successfully developed and optimized using lanthanide-tagged monoclonal antibodies as proxies to determine spatial distributions and concentrations of the two breast cancer biomarkers. The labeling degree of antibodies was determined via size exclusion-ICP-tandem mass spectrometry (SEC-ICP-MS/MS) employing online calibration via post-column isotope dilution analysis (IDA). The calibration of spatial distributions of labeled lanthanides in tissues was performed by ablating mold-prepared gelatin standards spiked with element standards. Knowledge of labeling degrees enabled the translation of lanthanide concentrations into biomarkers concentrations. The k-means clustering was used to select tissue areas for statistical analysis and mean concentrations were compared for sets of metastatic, non-metastatic and healthy samples. MMP-11 was expressed in stroma surrounding tumor areas, while CD45 was predominantly found inside tumor areas with high cell density. There was no significant correlation between CD45 and metastasis (P = 0.70); however, MMP-11 was significantly up-regulated (202%) in metastatic samples compared to non-metastatic (P = 0.0077) and healthy tissues (P = 0.0087).

Hemangioma capilar lobular: granuloma piógeno en lengua / Lobular capillary hemangioma: pyogenic granuloma of tongue

El granuloma piógeno es una lesión benigna, reactiva y multifactorial que resulta de le- siones repetitivas, microtraumatismos e irritación local en piel o mucosas y cambio hormonal. Cuando aparece en la cavidad oral tiene predilección por la encía vestibular, pero es importante que el odontólogo esté consciente y familiarizado con el hecho de que puede estar localizado en otras áreas anatómicas. Clínicamente se presenta como lesión hiperplásica altamente vascularizada, de tamaño generalmente no mayor a 2 cm, pediculada en la base o sésil y de lento crecimiento. Sin mostrar preferencia por edad o sexo, tiende a aparecer principalmente en encías, labios y mucosa oral, siendo muy pocos los casos reportados en el área lingual. Es por ello que, en este artículo, nos referimos a un caso de ubicación inusual, en conjunto con una revisión de la literatura (AU)

Pyogenic granuloma is a benign, reactive, and multifactorial lesion caused by repetitive injuries, microtrauma and local irritation on the skin or mucous membranes, and hormonal change. When it appears in the oral cavity, it has a predilection for the vestibular gingiva, but the dentist must be aware and familiar with the fact that it can be present in other anatomi- cal areas. Clinically, it is presented as a hyperplasic injury highly vascular-related, with a size generally no bigger than 2 cm, pedunculated in base or sessile, and slow in growth. Without showing any preference in age or gender, it tends to appear mainly on the gums, lips, and oral mucosae, with very few, reported cases in the lingual area. Therefore, in this study, we refer to a case of unusual localization with a literature review (AU)

Using pan-TRK and RET Immunohistochemistry for the Detection of Fusion Types of Salivary Gland Secretory Carcinoma.

Secretory carcinoma (SC) is a low-grade salivary gland carcinoma characterized by recurrent ETV6 rearrangements. Most cases have ETV6-NTRK3 fusions, while the minority of cases have non-NTRK3 fusions, including ETV6-RET and ETV6-MET. Detection of the fusion partner has become important, as there are TRK or RET inhibitors that may benefit patients with advanced SC. Currently, there are different methods to detect gene rearrangement in SCs, such as next-generation sequencing, reverse transcription-polymerase chain reaction, or fluorescence in situ hybridization. Immunohistochemistry (IHC) has greater accessibility, quick turnaround time, and can serve as a screening tool for confirmatory molecular tests. Pan-TRK and RET antibodies have been used to detect gene fusions in different tumors. Here, pan-TRK and RET IHC assays were performed on 28 salivary gland SCs, including 27 cases with ETV6-NTRK3 and one with ETV6-RET fusion confirmed by fluorescence in situ hybridization. Pan-TRK staining was positive in 26/27 (96.3%) of NTRK3 fusion-positive SCs with a nuclear staining pattern in more than 50% of tumor cells, and negative in the RET-rearranged case. RET IHC showed positive staining in most cases (26/28), but only three cases (including the RET-rearranged case) had diffuse and strong staining. RET IHC can be considered an effective screening test when diffuse/strong reactivity is present in pan-TRK IHC-negative cases. This study showed that pan-TRK staining has high sensitivity and specificity for SC with NTRK3 fusion. Whereas pan-TRK IHC is a useful screening method, further studies are needed to assess the value of RET IHC as a second sequential step.

Differences in local immune cell landscape between Q fever and atherosclerotic abdominal aortic aneurysms identified by multiplex immunohistochemistry.

Background: Chronic Q fever is a zoonosis caused by the bacterium Coxiella burnetii which can manifest as infection of an abdominal aortic aneurysm (AAA). Antibiotic therapy often fails, resulting in severe morbidity and high mortality. Whereas previous studies have focused on inflammatory processes in blood, the aim of this study was to investigate local inflammation in aortic tissue. Methods: Multiplex immunohistochemistry was used to investigate local inflammation in Q fever AAAs compared to atherosclerotic AAAs in aorta tissue specimen. Two six-plex panels were used to study both the innate and adaptive immune systems. Results: Q fever AAAs and atherosclerotic AAAs contained similar numbers of CD68+ macrophages and CD3+ T cells. However, in Q fever AAAs, the number of CD68+CD206+ M2 macrophages was increased, while expression of GM-CSF was decreased compared to atherosclerotic AAAs. Furthermore, Q fever AAAs showed an increase in both the number of CD8+ cytotoxic T cells and CD3+CD8-FoxP3+ regulatory T cells. Finally, Q fever AAAs did not contain any well-defined granulomas. Conclusions: These findings demonstrate that despite the presence of pro-inflammatory effector cells, persistent local infection with C. burnetii is associated with an immune-suppressed microenvironment. Funding: This work was supported by SCAN consortium: European Research Area - CardioVascualar Diseases (ERA-CVD) grant [JTC2017-044] and TTW-NWO open technology grant [STW-14716].

Case Report: Sarcoid-Like Reactions and Tertiary Lymphoid Structures Following Dual Checkpoint Inhibition in a Patient with Early-Stage Lung Adenocarcinoma.

Immune checkpoint inhibitor-induced sarcoid-like reactions and tertiary lymphoid structures (TLSs) are increasingly recognized but rarely reported in the same patient. We report a patient with lung adenocarcinoma who displayed sarcoid-like reactions in intrathoracic lymph nodes and tertiary lymphoid structures in surgical tumor after neoadjuvant therapy with nivolumab plus ipilimumab. Pathological examination revealed 50% residual tumor cells after treatment, and the CT evaluation of the primary tumor showed a stable disease. The patient experienced a recurrence eight months after surgery. To identify immune correlates of the limited response to immunotherapy, we conducted genomic and transcriptional assays, multiplex immunoassay, and multiplex immunohistochemistry on the pre- and post-immunotherapy tumor, lymph node, and plasma samples. TP53 R181C, KRAS G12C and SMAD4 R361H were identified as driver mutations of the tumor. In addition to abundant infiltrated lymphocytes, immunotherapy induced high levels of inhibitory components in post-treatment tissue samples, especially the FOXP3+ regulatory T cells in tumor and PD-L1 expression in the lymph node. Despite abundant TLSs in the post-treatment tumor, most TLSs were immature. Moreover, increasing levels of circulating checkpoint proteins BTLA, TIM-3, LAG-3, PD-1, PD-L1, and CTLA4 were observed during immunotherapy. Collectively, our observations revealed that high levels of immunosuppressive molecules in tumor, lymph nodes and/or in peripheral blood might indicate poor outcomes after immunotherapy, even in the setting of a patient with concurrent sarcoid-like reactions and tertiary lymphoid structures.

NTRK gene fusions are detected in both secretory and non-secretory breast cancers.

NTRK fusions represent a new biomarker-defined population that can be treated with TRK inhibitors. Although rare, NTRK fusions are detected across a wide range of solid tumors. Previous reports suggest that NTRK fusions are limited to the secretory subtype of breast cancer. Here we examined NTRK fusions in a large real world next-generation sequencing (NGS) dataset and confirmed secretory versus non-secretory status using H&E images. Of 23 NTRK fusion-positive cases, 11 were classified as secretory, 11 as non-secretory, and one as mixed status. The secretory subtype trended younger, was predominantly estrogen receptor (ER)-, had lower tumor mutational burden, and exhibited lower levels of genomic loss of heterozygosity. The non-secretory subtype was enriched for TP53 mutations. The secretory subtype was enriched for ETV6-NTRK3 fusions in 7 of 11 cases, and the non-secretory subtype had NTRK1 fusions in 7 of 11 cases, each with a different fusion partner. Our data suggests NTRK fusions are present in both secretory and non-secretory subtypes, and that comprehensive genomic profiling should be considered across all clinically advanced breast cancers to identify patients that could receive benefit from TRK inhibitors.

Expression and prognostic analysis of STAT6(YE361) in Hodgkin lymphoma.

This study aimed to investigate the expression and prognostic significance of the signal transducer and activator of transcription protein 6 (STAT6YE361) and EB virus encoding a small molecule RNA (EBER) in Hodgkin lymphoma (HL), as well as their correlation with clinical parameters. The expression of STAT6YE361 and EBER was investigated in HL via immunohistochemistry and in situ hybridization. Patient clinical data were retrospectively collected from archival libraries, and statistical analysis was performed. Overall, the nuclear positive expression rate of STAT6YE361 was 46%, and the EBER positive expression rate was 57%. STAT6YE361 was specifically expressed on the nucleus in cHL tissues. EBER was overexpressed in HL and had correlations with several clinical data, including age, gender, ethnicity, and primary cancer site. Interestingly, nuclear STAT6YE361 expression was correlated with EBER expression. Based on survival analysis, the nuclear expression of STAT6YE361 and female patients were associated with poor prognosis and were independent prognostic factors for five-year OS. These findings suggest that STAT6YE361 is a potential valuable index in the differential diagnosis and prognosis of HL. The mechanism of STAT6YE361 is related to Epstein-Barr virus infection.