Molecular monitoring by CDKN2A/p16INK4A and RB1 gene methylation in breast cancer.

OBJECTIVE: This prospective study aimed to provide a comprehensive analysis of the methylation status of two pivotal genes, CDKN2A/p16INK4A (cyclin-dependent kinase inhibitor 2A) and RB1 (retinoblastoma transcriptional corepressor 1), in breast cancer patients. METHODS: Samples were obtained from 15 women diagnosed with breast cancer and who underwent a total mastectomy. DNA was extracted from the tumor, non-tumor tissue, and peripheral blood (circulating cell-free DNA). The methylation pattern of cell-free DNA extracted from blood collected on the day of mastectomy was compared with the methylation pattern of cell-free DNA from blood collected 1 year post-surgery. The methylation analysis was carried out by sodium bisulfite conversion and polymerase chain reaction, followed by electrophoresis. RESULTS: Methylation of CDKN2A/p16INK4A was identified in 13 tumor samples and 12 non-tumor tissue samples. Two patients exhibited CDKN2A/p16INK4A methylation in the cell-free DNA of the first blood collection, while another showed methylation only in the cell-free DNA of the subsequent blood collection. Regarding RB1, 11 tumors and 8 non-tumor tissue samples presented methylation of the gene. CONCLUSION: This study presents a novel approach for monitoring breast cancer patients through the analysis of cell-free DNA methylation. This analysis can detect changes in methylation patterns before any visible sign of cancer appears in breast tissue and could help predict the recurrence of malignant breast tumors.

Molecular monitoring by CDKN2A/p16INK4A and RB1 gene methylation in breast cancer

SUMMARY OBJECTIVE: This prospective study aimed to provide a comprehensive analysis of the methylation status of two pivotal genes, CDKN2A/p16INK4A (cyclin-dependent kinase inhibitor 2A) and RB1 (retinoblastoma transcriptional corepressor 1), in breast cancer patients. METHODS: Samples were obtained from 15 women diagnosed with breast cancer and who underwent a total mastectomy. DNA was extracted from the tumor, non-tumor tissue, and peripheral blood (circulating cell-free DNA). The methylation pattern of cell-free DNA extracted from blood collected on the day of mastectomy was compared with the methylation pattern of cell-free DNA from blood collected 1 year post-surgery. The methylation analysis was carried out by sodium bisulfite conversion and polymerase chain reaction, followed by electrophoresis. RESULTS: Methylation of CDKN2A/p16INK4A was identified in 13 tumor samples and 12 non-tumor tissue samples. Two patients exhibited CDKN2A/p16INK4A methylation in the cell-free DNA of the first blood collection, while another showed methylation only in the cell-free DNA of the subsequent blood collection. Regarding RB1, 11 tumors and 8 non-tumor tissue samples presented methylation of the gene. CONCLUSION: This study presents a novel approach for monitoring breast cancer patients through the analysis of cell-free DNA methylation. This analysis can detect changes in methylation patterns before any visible sign of cancer appears in breast tissue and could help predict the recurrence of malignant breast tumors.

Liver immunopathogenesis in fatal cases of dengue in children: detection of viral antigen, cytokine profile and inflammatory mediators.

Introduction: Dengue virus (DENV), the etiologic agent of dengue fever illness, represents a global public health concern, mainly in tropical and subtropical areas across the globe. It is well known that this acute viral disease can progress to severe hemorrhagic stages in some individuals, however, the immunopathogenic basis of the development of more severe forms by these patients is yet to be fully understood. Objective: In this context, we investigated and characterized the histopathological features as well as the cytokine profile and cell subpopulations present in liver tissues from three fatal cases of DENV in children. Methods: Hematoxylin and Eosin, Periodic Acid Schiff and Picro Sirius Red staining were utilized for the histopathological analysis. Immunohistochemistry assay was performed to characterize the inflammatory response and cell expression patterns. Results: Vascular dysfunctions such as hemorrhage, vascular congestion and edema associated with a mononuclear infiltrate were observedin all three cases. Liver tissues exhibited increased presence of CD68+ and TCD8+ cells as well as high expression of MMP-9, TNF-a, RANTES, VEGFR-2 mediators. Viral replication was confirmed by the detection of NS3 protein. Conclusion: Taken together, these results evidenced key factors that may be involved in the development of severe alterations in liver tissues of children in response to DENV infection.

Immunopathology of Renal Tissue in Fatal Cases of Dengue in Children.

Dengue virus (DENV) infection represents a worldwide public health concern and can cause damage to multiple organs, including the kidney. In this work, we investigated the histopathological changes caused by dengue virus infection along with the detection of inflammatory mediators, cytokines, and cell expression patterns in the renal tissue of three fatal cases in children. Hematoxylin and Eosin staining was performed to analyze these histopathological changes. Immunohistochemistry allowed for the detection of immunological inflammatory markers in renal tissues that were quantified and further analyzed. Vascular congestion, edema and glomerular infiltrate were observed in the three cases, in addition to the thickening of the matrix area around the glomerular capillaries and mononuclear infiltrate associated with vascular congestion in the medullary region. The renal tissues exhibited collagen deposition and high expression of CD68+ Mø, CD8+ T, CD56+ cells and MMP-9, and the cytokine profile was mainly characterized by the expression of IFN-γ and TNF-α. Additionally, the expression of RANTES, VEGFR-2 and VCAM-1 were observed. The replication of DENV was evidenced by the detection of the NS3 protein. These results contributed to clarifying the main factors that may be involved in changes in the renal tissue of fatal cases of dengue in children.

SARS-CoV-2 infection of the central nervous system in a 14-month-old child: A case report of a complete autopsy.

BACKGROUND: Neurological and other systemic complications occur in adults with severe COVID-19. Here we describe SARS-CoV-2 infection complicated by neuroinvasion in the post-mortem tissues of a child. METHODS: We performed a complete autopsy of a 14-month-old child who died of COVID-19 pneumonitis. Histological sections of multiple organs were stained with haematoxylin and eosin. Luxol fast blue staining for myelin and immunohistochemistry were performed in selected areas of the brain. The presence of SARS-CoV-2 was investigated by immunostaining with anti-spike protein antibody and by RT-qPCR. FINDINGS: Lesions included microthrombosis, pulmonary congestion, interstitial oedema, lymphocytic infiltrates, bronchiolar injury, collapsed alveolar spaces, cortical atrophy, and severe neuronal loss. SARS-CoV-2 staining was observed along the apical region of the choroid plexus (ChP) epithelium and in ependymal cells of the lateral ventricle, but was restricted to ChP capillaries and vessels in some regions. SARS-CoV-2 infection of brain tissue was confirmed by RT-qPCR in fragments of the ChP, lateral ventricle, and cortex. INTERPRETATION: Our results show multisystemic histopathological alterations caused by SARS-CoV-2 infection and contribute to knowledge regarding the course of fatal COVID-19 in children. Furthermore, our findings of ChP infection and viral neurotropism suggest that SARS-CoV-2 may invade the central nervous system by blood-cerebrospinal fluid barrier disruption. FUNDING: Carlos Chagas Filho Foundation for Supporting Research in the State of Rio de Janeiro (FAPERJ); the National Council for Scientific and Technological Development (CNPq) and Coordination for the Improvement of Higher Education Personnel (CAPES), in addition to intramural grants from D'Or Institute for Research and Education. EDITOR'S NOTE: This translation in Portuguese was submitted by the authors and we reproduce it as supplied. It has not been peer reviewed. Our editorial processes have only been applied to the original abstract in English, which should serve as reference for this manuscript. RESUMO: Complicações sistêmicas e neurológicas foram descritas em adultos com COVID-19 grave. Neste trabalho, descrevemos a infecção por SARS-CoV-2, incluindo sua neuroinvasão, nos tecidos post-mortem de uma criança. MÉTODOS: Realizamos a autópsia completa de uma criança de 14 meses que morreu de pneumonite por COVID-19. Cortes histológicos de múltiplos órgãos foram corados com Hematoxilina e Eosina. A coloração de Luxol Fast Blue para mielina e imuno-histoquímica foram realizadas em áreas selecionadas do cérebro. A presença de SARS-CoV-2 foi investigada por imunomarcação com anticorpo anti-proteína spike e por RT-qPCR. ACHADOS: As lesões incluíram microtrombose, congestão pulmonar, edema intersticial, infiltrados linfocíticos, lesão bronquiolar, colapso dos espaços alveolares, atrofia cortical e perda neuronal grave. A presença de SARS-CoV-2 foi observada ao longo da região apical do epitélio do plexo coróide (PC) e nas células ependimárias do ventrículo lateral, mas ficou restrita aos capilares e vasos do PC em outras regiões. A infecção do tecido cerebral por SARS-CoV-2 foi confirmada por RT-qPCR em fragmentos do PC, ventrículo lateral e cortex cerebral. INTERPRETAÇÃO: Nossos resultados mostram alterações histopatológicas multissistêmicas causadas pela infecção por SARS-CoV-2 e contribuem para ampliar o conhecimento sobre a evolução da COVID-19 fatal em crianças. Além disso, nossos achados sobre a infecção no PC e neurotropismo viral sugerem que o SARS-CoV-2 pode invadir o sistema nervoso central pela ruptura da barreira sangue-líquido cefalorraquidiano. FINANCIAMENTO: Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ) e Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), além de financiamento intramural do Instituto D'Or de Pesquisa e Educação.

Solid pseudopapillary neoplasia of the pancreas: a review.

OBJECTIVES: To review the literature and the diagnosis of conventional histopathological routine and immunohistochemistry of the cases diagnosed with Solid Pseudopapillary Neoplasm of the Pancreas (SPNP). METHODS: The review of the literature was done using the Pubmed and solid Google-Scholar databases, through the historical, clinical aspects and diagnostic methods of SPNP. The review of SPNP cases diagnosed in the University Hospital Clementino Fraga Filho was carried out from 1977 to 2018. RESULTS: Intratumoral phenotypic heterogeneity of SPNP was evidenced in the cases studied, taking into account macroscopic, microscopic, and immunohistochemical patterns. CONCLUSIONS: The results show the importance of the examination of several fragments obtained from different regions of the neoplasia since not all of them present the same molecular alterations.

Systematic identification of cancer-specific MHC-binding peptides with RAVEN.

Immunotherapy can revolutionize anti-cancer therapy if specific targets are available. Immunogenic peptides encoded by cancer-specific genes (CSGs) may enable targeted immunotherapy, even of oligo-mutated cancers, which lack neo-antigens generated by protein-coding missense mutations. Here, we describe an algorithm and user-friendly software named RAVEN (Rich Analysis of Variable gene Expressions in Numerous tissues) that automatizes the systematic and fast identification of CSG-encoded peptides highly affine to Major Histocompatibility Complexes (MHC) starting from transcriptome data. We applied RAVEN to a dataset assembled from 2,678 simultaneously normalized gene expression microarrays comprising 50 tumor entities, with a focus on oligo-mutated pediatric cancers, and 71 normal tissue types. RAVEN performed a transcriptome-wide scan in each cancer entity for gender-specific CSGs, and identified several established CSGs, but also many novel candidates potentially suitable for targeting multiple cancer types. The specific expression of the most promising CSGs was validated in cancer cell lines and in a comprehensive tissue-microarray. Subsequently, RAVEN identified likely immunogenic CSG-encoded peptides by predicting their affinity to MHCs and excluded sequence identity to abundantly expressed proteins by interrogating the UniProt protein-database. The predicted affinity of selected peptides was validated in T2-cell peptide-binding assays in which many showed binding-kinetics like a very immunogenic influenza control peptide. Collectively, we provide an exquisitely curated catalogue of cancer-specific and highly MHC-affine peptides across 50 cancer types, and a freely available software (https://github.com/JSGerke/RAVENsoftware) to easily apply our algorithm to any gene expression dataset. We anticipate that our peptide libraries and software constitute a rich resource to advance anti-cancer immunotherapy.

Placental Inflammation and Fetal Injury in a Rare Zika Case Associated With Guillain-Barré Syndrome and Abortion.

Zika virus (ZIKV) is an emerging virus involved in recent outbreaks in Brazil. The association between the virus and Guillain-Barré syndrome (GBS) or congenital disorders has raised a worldwide concern. In this work, we investigated a rare Zika case, which was associated with GBS and spontaneous retained abortion. Using specific anti-ZIKV staining, the virus was identified in placenta (mainly in Hofbauer cells) and in several fetal tissues, such as brain, lungs, kidneys, skin and liver. Histological analyses of the placenta and fetal organs revealed different types of tissue abnormalities, which included inflammation, hemorrhage, edema and necrosis in placenta, as well as tissue disorganization in the fetus. Increased cellularity (Hofbauer cells and TCD8+ lymphocytes), expression of local pro-inflammatory cytokines such as IFN-γ and TNF-α, and other markers, such as RANTES/CCL5 and VEGFR2, supported placental inflammation and dysfunction. The commitment of the maternal-fetal link in association with fetal damage gave rise to a discussion regarding the influence of the maternal immunity toward the fetal development. Findings presented in this work may help understanding the ZIKV immunopathogenesis under the rare contexts of spontaneous abortions in association with GBS.

Sweet Syndrome in childhood.

Sweet syndrome or acute febrile neutrophilic dermatosis is a recurrent and rare skin disease caused by the release of cytokines, with diverse possible etiologic causes. It presents clinically with polymorphic skin lesions, fever, arthralgia, and peripheral leukocytosis. In general, it is associated with infections, malignancy and drugs. It usually regresses spontaneously and treatment is primarily to control the basic disease. The authors report the case of a child of 1 year and 11 months who developed Sweet syndrome.

Sweet Syndrome in childhood

AbstractSweet syndrome or acute febrile neutrophilic dermatosis is a recurrent and rare skin disease caused by the release of cytokines, with diverse possible etiologic causes. It presents clinically with polymorphic skin lesions, fever, arthralgia, and peripheral leukocytosis. In general, it is associated with infections, malignancy and drugs. It usually regresses spontaneously and treatment is primarily to control the basic disease. The authors report the case of a child of 1 year and 11 months who developed Sweet syndrome.

Do you know this syndrome?

Bloch-Sulzberger syndrome (incontinentia pigmenti) is a rare genodermatosis that affects predominantly females, since it is generally lethal to male fetuses in utero. It is characterized principally by skin lesions, but may also involve dental, ophthalmological and neurological abnormalities. The skin lesions are present in four different phases: vesicular, verrucous, hyperpigmented and atrophic/hypopigmented. Their sequence is irregular and overlapping of stages is common.

Você conhece esta síndrome? / Do you know this syndrome?

A Síndrome de Bloch-Sulzberger (Incontinência Pigmentar) é uma genodermatose rara, que afeta, principalmente, o sexo feminino, pois costuma ser letal em pacientes do sexo masculino intraútero. Caracteriza-se, principalmente, pelas manifestações dermatológicas, podendo também apresentar anomalias dentárias, oftalmológicas e neurológicas. As lesões cutâneas apresentam 4 fases distintas: vesiculosa, verrucosa, pigmentar e atrófica; que podem seguir uma sequência irregular, havendo até sobreposição das mesmas.

Bloch-Sulzberger syndrome (incontinentia pigmenti) is a rare genodermatosis that affects predominantly females, since it is generally lethal to male fetuses in utero. It is characterized principally by skin lesions, but may also involve dental, ophthalmological and neurological abnormalities. The skin lesions are present in four different phases: vesicular, verrucous, hyperpigmented and atrophic/hypopigmented. Their sequence is irregular and overlapping of stages is common.

Infectious diseases in paediatric pathology: experience from a developing country.

Infectious and parasitic diseases have always challenged man. Although many of them are typically seen in some areas of the world and can be adequately managed by just improving socioeconomic status and sanitary conditions, they are still quite prevalent and may sometimes be seen outside their original geographical areas. Human migration due to different reasons, tourism, blood transfusion and solid organ transplantation has created new concerns for health professionals all over the world. If not for diagnostic purposes, at least these tropical and infectious diseases should be largely known because their epidemiology, pathogenesis, host/parasite interaction, inflammatory and reparative responses are quite interesting and teach us about human biology. Curiosity is inherent to pathology practice and so we are compelled to look for things other than tumours or degenerative diseases. This review focuses on infectious and parasitic diseases found in a developing country and brings up-to-date information on diseases caused by viruses (dengue, yellow fever), bacteria (typhoid fever, leprosy), parasites (Chagas' disease, cutaneous and visceral leishmaniasis, amoebiasis, Capillaria hepatica, schistosomiasis, cysticercosis) and caused by fungi (paracoccidioidomycosis, cryptococcosis, histoplasmosis) that may be useful for pathologists when facing somewhat strange cases from developing countries.

Infectious diseases in paediatric pathology: experience from a developing country

Infectious and parasitic diseases have always challenged man. Although many of them are typically seen in some areas of the world and can be adequately managed by just improving socioeconomic status and sanitary conditions, they are still quite prevalent and may sometimes be seen outside their original geographical areas. Human migration due to different reasons, tourism, blood transfusion and solid organ transplantation has created new concerns for health professionals all over the world. If not for diagnostic purposes, at least these tropical and infectious diseases should be largely known because their epidemiology, pathogenesis, host/parasite interaction, inflammatory and reparative responses are quite interesting and teach us about human biology. Curiosity is inherent to pathology practice and so we are compelled to look for things other than tumours or degenerative diseases. This review focuses on infectious and parasitic diseases found in a developing country and brings up-to-date information on diseases caused by viruses (dengue, yellow fever), bacteria (typhoid fever, leprosy), parasites (Chagas' disease, cutaneous and visceral leishmaniasis, amoebiasis, Capillaria hepatica, schistosomiasis, cysticercosis) and caused by fungi (paracoccidioidomycosis, cryptococcosis, histoplasmosis) that may be useful for pathologists when facing somewhat strange cases from developing countries.