A New Digital Method for Counting Mast Cells in Cutaneous Specific Lesions of Mastocytosis: A Series of Adult Cases of Mastocytosis With Clinical-Pathological Correlations.

ABSTRACT: Cutaneous mastocytosis is characterized by the abnormal accumulation of mast cells in the skin. However, mast cell counting is not always easy and reproducible with classical methods. This work aims to demonstrate the reliability, usability, and virtues of a new software used on digital tablets for counting mast cells in cutaneous specific lesions of mastocytosis, to assess differences in mast cell counts between clinical subtypes of mastocytosis in the skin, and to consider the feasibility of applying a diagnostic mast cell count cutoff to urticaria pigmentosa, which is the most frequent form of cutaneous mastocytosis. Using a new digital tablet software that was accessible by multiple observers through its own wireless network and allowed high resolution of the image without data compression, we counted the number of mast cells on slides of patients and control skins immunostained for CD117. We found that our counting method was highly reproducible and that the new software allowed very quick counting. We evidenced strong differences in the mast cell count between most of the clinical subtypes of mastocytosis in the skin. However, when applied to a subset of patients with urticaria pigmentosa, a diagnostic cutoff in the mast cell count lacked sensitivity. Thus, our digital method for counting CD117-immunostained mast cells was highly accurate and was of a significant value for the diagnosis of mastocytosis in the skin. However, some subtypes with low mast cell counts will still require the application of additional diagnostic criteria.

Use of different fixation times and application of two immunohistochemical methods for detection of KIT and Ki67 proteins in canine cutaneous mast cell tumors / Utilização de diferentes tempos de fixação e métodos imuno-histoquímicos na detecção das proteínas KIT e Ki67 em mastocitomas cutâneos caninos

Due to the high prevalence of mast cell tumors (MCTs) in the diagnostic routine, several factors, especially prognostic, have been sought to determine the biological behavior of these neoplasms. Immunohistochemistry (IHC) is one of the main tools utilized to biologically differentiate more aggressive tumors from less aggressive ones. However, some immunostainings are influenced by formalin fixation, interfering with the results. This is both a retrospective and prospective study of MCTs diagnosed in laboratory routine. A total of 25 samples, without knowledge about fixation time, were analyzed in the retrospective study, whereas 12 samples, with known fixation times, were assessed in the prospective study. Two histologic grading systems (Patnaik and Kiupel), special staining of toluidine blue, and IHC for KIT and Ki67 proteins were applied in both studies. Additionally, two amplification systems (biotinylated and non-biotinylated) for Ki67 protein and counting of the argyrophilic nucleolar organizing regions (AgNOR method) were tested in the prospective study. In the retrospective study, greater agreement between the evaluating pathologists was observed when the Kiupel system was used. IHC staining for KIT protein was effective in both studies, regardless of fixation time. IHC staining for Ki67 protein was highly sensitive to formaldehyde, and staining failure was observed in 56% of the cases in the retrospective study. In the prospective study, samples fixed for longer than 24 hours showed a reduction in the number of stained cells (altering the determination of the cell growth fraction) or showed absence of IHC staining in both amplification systems. The use of the AgNOR method to evaluate the rate of cell proliferation may be an alternative when the fixation time of the neoplasm is unknown or longer than 24 hours.(AU)

Devido a alta prevalência dos mastocitomas cutâneos caninos (MCCs) na rotina diagnóstica, vários fatores, especialmente fatores prognósticos, têm sido buscados para auxiliar na determinação do comportamento biológico desse neoplasma. A imuno-histoquímica é uma das principais ferramentas empregadas para diferenciar tumores biologicamente mais agressivos de tumores menos agressivos. Entretanto, algumas imunomarcações sofrem influência pela fixação em formol, interferindo nos resultados. Este estudo compreendeu avaliar através de uma etapa retrospectiva e uma etapa prospectiva casos de MCCs diagnosticados na rotina laboratorial. Um total de 25 amostras, sem conhecimento do tempo de fixação, foi analisado no estudo retrospectivo e 12 amostras, com tempos de fixação conhecidos, no estudo prospectivo. Foram aplicados nos dois estudos, dois sistemas de graduação histológica (Patnaik e Kiupel), a coloração especial de azul de toluidina e a imuno-histoquímica para as proteínas KIT e Ki67. Adicionalmente, no estudo prospectivo, foram testados dois sistemas de amplificação (biotinilado e não biotinilado) para a proteína Ki67 e a técnica de AgNOR (contagem das regiões organizadoras nucleolares argirofílicas). Na etapa retrospectiva, observou-se uma maior concordância entre os patologistas avaliadores quando o sistema Kiupel foi utilizado. A imunomarcação para KIT se manteve eficaz em ambos os estudos, independentemente do tempo de fixação. A imunomarcação para o Ki67 mostrou-se altamente sensível ao tempo de fixação em formol, sendo observada falha na imunomarcação em 56% dos casos do estudo retrospectivo. No estudo prospectivo, constatou-se que amostras fixadas por mais de 24 horas em formol apresentaram redução na quantidade de células imunomarcadas (alterando a determinação da fração de crescimento celular) ou apresentaram ausência de imunomarcação em ambos os sistemas de amplificação. A utilização do método AgNOR, para avaliar a taxa de proliferação celular, pode ser uma alternativa quando o tempo de fixação do neoplasma for desconhecido ou superior a 24 horas.(AU)

Imatinib elicited a favorable response in a dog with a mast cell tumor carrying a c-kit c.1523A>T mutation via suppression of constitutive KIT activation.

Target therapy using the tyrosine kinase inhibitor imatinib is one of the new therapeutic approaches for canine mast cell tumors (MCTs). In the present report, we demonstrate a clinical response to imatinib in a dog with MCT carrying a c-kit c.1523A>T mutation. Moreover, the effect of this mutation on the phosphorylation status of KIT and the inhibitory potency of imatinib on the phosphorylation of the mutant KIT were examined in vitro. A dog with a MCT tumor mass on the right forelimb sole with lymph node metastasis and mastocytemia was treated with imatinib. The MCT mass markedly shrank and mastocytemia became undetectable with 2 weeks of treatment. The lymph node enlarged by metastasis became normal in size with 5 weeks of treatment. From the sequencing analysis of c-kit in tumor cells, a substitution mutation c.1523A>T that alters the amino acid composition (p.Asn508Ile) within the extracellular domain of KIT was identified. The mutant KIT expressed on 293 cells showed ligand-independent phosphorylation and imatinib suppressed this phosphorylation in a dose-dependent manner. From these findings, imatinib was considered to elicit a clinical response in a canine case of MCT via inhibition of the constitutively activated KIT caused by a c-kit c.1523A>T mutation.

CD117 immunoexpression in canine mast cell tumours: correlations with pathological variables and proliferation markers.

BACKGROUND: Cutaneous mast cell tumours are one of the most common neoplasms in dogs and show a highly variable biologic behaviour. Several prognosis tools have been proposed for canine mast cell tumours, including histological grading and cell proliferation markers. CD117 is a receptor tyrosine kinase thought to play a key role in human and canine mast cell neoplasms. Normal (membrane-associated) and aberrant (cytoplasmic, focal or diffuse) CD117 immunoexpression patterns have been identified in canine mast cell tumours. Cytoplasmic CD117 expression has been found to correlate with higher histological grade and with a worsened post-surgical prognosis. This study addresses the role of CD117 in canine mast cell tumours by studying the correlations between CD117 immunoexpression patterns, two proliferation markers (Ki67 and AgNORs) histological grade, and several other pathological variables. RESULTS: Highly significant (p < 0,001) correlations were found between CD117 immunostaining patterns and histological grade, cell proliferation markers (Ki67, AgNORs) and tumoral necrosis. Highly significant (p < 0,001) correlations were also established between the two cellular proliferation markers and histological grade, tumour necrosis and epidermal ulceration. A significant correlation (p = 0.035) was observed between CD117 expression patterns and epidermal ulceration. No differences were observed between focal and diffuse cytoplasmic CD117 staining patterns concerning any of the variables studied. CONCLUSION: These findings highlight the key role of CD117 in the biopathology of canine MCTs and confirm the relationship between aberrant CD117 expression and increased cell proliferation and higher histological grade. Further studies are needed to unravel the cellular mechanisms underlying focal and diffuse cytoplasmic CD117 staining patterns, and their respective biopathologic relevance.