Immunohistochemical and molecular profiling of CD 117, Oct-4, and Sox-2 in canine cutaneous mast cell tumor of the crossbred dogs in Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand

Background and Aim: CD 117 (c-KIT) internal tandem duplication (ITD), octamer-binding transcription factor 4 (Oct-4), and sex-determining region Y-box 2 (Sox-2) may govern the oncogenicity and aggressiveness of canine cutaneous mast cell tumor (MCT) in the crossbred dogs. Thus, a comprehension of this matter may help us establishing a novel platform to treat the disease in those dogs. However, evidence has lacked so far. Thus, this study aimed to survey CD 117 ITD, Oct-4, and Sox-2 expressions and their relations to the 2-tier grading in a group of Thai crossbreed dogs. The study was done using polymerase chain reaction (PCR), Reverse transcription PCR (RT-PCR), and immunohistochemistry. Materials and Methods: Thirty-three MCT specimens graded by the 2-tier histopathology grading were collected from the crossbred and purebred dogs. CD 117 ITD was detected by conventional PCR and immunohistochemistry. While, Oct-4 and Sox-2 expression levels were determined at the protein and mRNA levels by immunohistochemistry and RT-PCR, respectively. The expression magnitude of each parameter was then related to the grades and breeds. Results: About 60.61% of specimens were low grade, while 39.39% were high grade. CD 117 ITD was not detected in all specimens. A significant increase of Oct-4 expression was found in the high-grade, crossbred dogs. Meanwhile, Sox-2 expressions were increased both in the purebred and crossbred dogs with high-grade MCT. Conclusion: The study finding has indicated that the level of Sox-2 expression may be a useful tumorigenic and prognostic biomarker because it correlates to the 2-tier grades but not dog breeds.

Tumores estromales gastrointestinales (GIST) gástricos, serie de casos

Introducción: los tumores estromales gastrointestinales (GIST) representan hasta el 2 % de las neoplasias gastrointestinales, estos aparecen en cualquier parte del tracto gastrointestinal y son encontrados más frecuentemente en el estómago (60 %). El diagnóstico se realiza por la expresión de un receptor de factor de crecimiento de tirosina-cinasa, antígeno de diferenciación (CD) 117, lo que lo diferencia de los otros tumores mesenquimales como leiomiomas, leiomiosarcomas, leiomioblastomas y tumores neurogénicos, que no expresan esta proteína. Objetivo: el objetivo de nuestro trabajo es caracterizar los GIST de localización gástrica con respecto a su presentación clínica, diagnóstico, manejo, recurrencia y supervivencia. Métodos: se trata de un estudio observacional, retrospectivo basado en una serie de casos. Se realizó una extracción de la información por medio de la revisión de las historias clínicas de los pacientes con GIST gástricos en un centro oncológico de Bogotá entre enero de 2005 y diciembre de 2015. La información recolectada incluyó tipo de manejo y abordaje quirúrgico, localización, tamaño, índice mitótico y clasificación de riesgo. Resultados: se encontraron 31 pacientes con diagnóstico de GIST gástrico. La edad media fue de 62,3 años, con una mediana de 61 años. De los 31 pacientes, 18 fueron mujeres y 13 hombres. El tiempo de seguimiento estuvo entre un mínimo de 2,4 meses y un máximo de 214 meses, La mediana de seguimiento fue de 36 meses. Conclusiones: los GIST son tumores potencialmente malignos, y el de localización gástrica es el más frecuente. El diagnóstico y tratamiento dependen de su tamaño y localización dentro del estómago. El manejo es variado y consiste en la resección quirúrgica, en la que los procedimientos son mínimamente invasivos, en combinación con la endoscopia, que son una buena alternativa al tratamiento abierto hasta que haya necesidad de terapia sistémica.

Distribution of Mast Cell in Acute Appendicitis and Its Role in Histopathological Diagnosis - A Study in Tertiary Care Centre from South India

BACKGROUND Appendicitis is the most common indication for intra-abdominal surgery, especially in adolescents and young adults. In about 15 – 25 % of appendices removed at surgery because of suspected symptoms, histological features of acute appendicitis are absent. The cause of acute abdominal pain in these patients can be due to mast cells which may play a role in pathogenesis of appendicitis-like pain. Demonstration of mast cell in tissue can be done by toluidine blue staining or immunohistochemistry (IHC) marker CD 117. We wanted to evaluate whether the degree of distribution of mast cells has any relation with clinical findings and evaluate its usefulness as a diagnostic marker for histological diagnosis of acute appendicitis. METHODS This is a descriptive study done in 120 cases of appendicectomy specimens from clinically diagnosed cases of acute appendicitis received in a tertiary care center in South India in which 60 where histology positive and others were histology negative. Mast cell distribution in each group was compared using toluidine blue stain and CD 117. Collected data was entered in Microsoft excel and analysed using the statistical software SPSS version 16. RESULTS Mast cell distribution was significant in all layers of histologically negative acute appendicitis in comparison with histology positive cases. Mucosa has maximum mast cells distribution. CONCLUSIONS Mast cells play an important role in the clinical symptoms of patients even when there were no features of acute inflammation. In those cases, mast cells can be demonstrated by simple toluidine blue staining or IHC markers. This is one of the less studied areas, and the clinicopathological discrepancy can be solved by giving mast cell distribution along with histopathology diagnosis. KEYWORDS Appendicitis, Histologically Negative Acute Appendicitis, Mast Cells, Toluidine Blue, CD 117

CARACTERÍSTICAS HISTOPATOLÓGICAS DE UM TUMOR ESTROMAL GASTROINTESTINAL

O tumor estromal gastrintestinal (GIST) é um tipo de tumor que pode se desenvolver ao longo de todo o tubo digestivo. Possui causa desconhecida e sua ocorrência se dá nas diversas faixas etárias, sendo mais comum nos indivíduos de maior idade. O portador da enfermidade é assintomático no início do quadro, desenvolvendo sintomas apenas em estágios mais avançados. Devido ser um tumor bastante incomum, considerou-se o estudo dessa patologia a fim de ampliar o conhecimento sobre esta morbidade, que muitas vezes chega a ser desconhecida pelos profissionais da saúde. O objetivo do estudo é relatar achados histopatológicos de uma paciente diagnosticada com Tumor Gastrointestinal de origem Estromal. Foram analisados o prontuário médico do paciente e lâmina histológica. O relato de caso foi realizado em uma Clínica de Patologia particular da cidade de João Pessoa – PB. Observou-se presença de células de núcleo alongado, tumor, em parte da lâmina, restrito a camada muscular da mucosa e, em parte, invadindo a camada suprajacente, além de contar, com área de necrose. O resultado pôde ser confirmado por meio do exame da imuno-histoquímica, que mostrou presença dos marcadores CD117 e CD34. A associação da história clínica dos pacientes, exames laboratoriais e de imagem, junto ao anatomopatológico ainda não fecham de forma definitiva o diagnóstico, necessitando de imuno-histoquímica com pesquisa de CD-117(c-kit). O GIST é um tumor silencioso, cuja agressividade é variável e que necessita de diagnóstico precoce, enquanto seu estudo for limitado, a sua detecção continuará muitas vezes sendo tardia e acabará por prejudicar os desfechos dos pacientes.

Novel 2 Marker Sandwich Immunoassay for the Detection of Precursor- and Mature Granulocyte-Derived Exosomes in Peripheral Blood: A Preliminary Case Study

Introduction: In order to assess hematopoietic precursor cells, bone marrow biopsies are routinely conducted, which is painful for the patient and is not applicable for frequent testing. However, precursor, maturating, and matured cells are known to produce exosomes, which are released into peripheral blood. By analyzing these exosomes in plasma, bone marrow condition may be assessed noninvasively. Myeloid precursor cells express different surface markers than when they have matured. It is reasonable to speculate that these markers would be incorporated into exosomes. In this study, we developed a novel sandwich enzyme-linked immunosorbent assay (ELISA) by using a combination of monoclonal antibodies against 2 different leukocyte antigens, to account for the number of exosomes in plasma. Methods In 6 patients undergoing chemotherapy, venous blood was drawn at regular intervals before, during, and after respective chemotherapies was given. Various clones of monoclonal antibodies against CD11b, CD33, CD34, CD35, CD81, and CD117 were obtained, and antibodies were biotinylated by EZ link Sulfo-NHS-LC-Biotin. Anti-brain antibodies were switched with anti-leukocyte antibodies, and exosome activity was trended. Results CD11b+ and CD 117+ plates showed small changes during chemotherapy (within +/- 50% of the initial value), while plasma WBC count showed a dramatic decrease during chemotherapy. In the recovery phase of WBC after chemotherapy, CD11b+ and CD16+showed a greater than 2-fold increase, with the up-trend taking place earlier than its plasma WBC counterparts. CD 117+ CD33+ also showed an up-trend, but was slower than their CD11b+ and CD16+ counterpart. Finally, each exosome value was divided by other markers to calculate the ratio, and compared between pre and post chemotherapy blood. In half of the cases, there was not a difference, versus an almost double premature/mature ratio in post-chemotherapy blood in the other half of patients. Conclusion From data collected from pre-chemotherapy studies, the lack of correlation between ELISA data compared to plasma WBC data, indicates that plasma exosome analysis might not be suitable for the monitoring of acute bone marrow suppression. Additionally, in the recovery phase, surface markers correlation with WBC was present in only some of the markers, indicating that the assay sensitivity is dependent on the marker combinations. Overall, both myeloid precursor- and mature granulocyte-derived exosomes were increased during the recovery from chemotherapy in 3 out of 5 patients, and exosome profiles were different after chemotherapy in 2 out of 5 patients. Disclosures Mitsuhash: NanoSomiX:Current Employment.

Pathophysiology of Hyperechogenic Bowel in Congenitally Human Cytomegalovirus Infected Fetuses

Hyperechogenic bowel (HB) is a nonspecific ultrasound finding that can be associated with human cytomegalovirus (CMV) congenital infection. In this study, we investigated HB pathophysiology in CMV-infected fetuses. We examined small and large intestine as well as pancreas in 8 fetuses at 22 weeks of gestation with congenital CMV infection. Ultrasound findings showed 4 fetuses with HB and 4 without. As negative group, 4 fetuses without CMV infection and without HB were studied. Immunohistochemistry for CMV, lymphocytic infiltrate, B-cell leukemia/lymphoma-2 (bcl-2), CD-117, cystic fibrosis transmembrane regulator (CFTR) were performed. HB fetuses showed multiple and sequential CMV-positive ganglion cells of Auerbach’s myenteric plexus. In the ganglia, bcl-2 was weakly expressed representing a reduced neuronal functionality. CD-117 revealed a regular distribution of Cajal cells, the pacemakers of intestinal contractility. Pancreas showed normal CFTR staining, indicating a preserved exocrine secretion, thus unlikely a contributory factor in HB. In CMV-infected fetuses without HB, CMV-positive cells were scatteredly found in ganglion cells and bcl-2 was strongly expressed. Intestinal CD-117 and pancreatic CFTR expression were similar to fetuses with HB. In conclusion, fetal CMV infection of the bowel may lead to peristalsis impairment (paralytic ileus) due to intestinal plexus involvement, which at ultrasound appeared as HB.

Частота експресії антигену CD 117 на бластних клітинах при мієлодиспластичному синдромі (МДС), гострій мієлоїдній лейкемії (ГМЛ)

CD 117 (kit) є рецепторною тирозин-кіназою типу ІІІ, яка бере участь у клітинній сигнальній трансдукції клітин. Зазвичай kit активується шляхом зв’язування його ліганду, запускаючи каскад фосфорилювання, призводячи до транскрипції в клітинах різного типу. Така активація регулює апоптоз, диференціацію, проліферацію клітин. Мета. З огляду на те, що мієлодиспластичний синдром (МДС) – це клонова патологія з високою здатністю до трансформації у ГМЛ, є актуальним вивчати експресії антигену CD 117 у хворих на МДС РАНБ І, РАНБ ІІ, ГМЛ. Матеріал і методи. Проаналізовано дані 12 хворих на МДС, із них 7 РАНБ І та 5 з РАНБ ІІ і 11 хворих на ГМЛ, методом проточної лазерної цитофлюориметрії на FACScan (Becton Dickinson, США) з використанням програми LYSYS-II ver. 1.1 (Becton Dickinson, США). Результати досліджень. Експресія антигену CD 117 визначена у 28,6 % обстежених хворих на МДС РАНБ І з показником CD 34+ 5,1 ± 0,5 % при 7,5 ± 1,5 бластних клітинах у КМ, у хворих на МДС РАНБ ІІ експресія CD 117 антигену спостерігалась у 80 % випадків при наявності 16,4 ± 1,5 % CD 34+ бластних клітин у КМ. У хворих на ГМЛ de novo в КМ виявлялось 62,4 ± 6,4 % CD 34+ бластних клітин, встановлено експресію CD 117 на 59,5 ± 4,5 % клітин у 81 % обстежених хворих. Висновки. Динаміка експресії антигену CD 117 на бластних клітинах при МДС свідчить про активність патологічного процесу.