Correlation of nodal mast cells with clinical outcome in dogs with mast cell tumour and a proposed classification system for the evaluation of node metastasis.

Lymph node metastasis in dogs with mast cell tumour has been reported as a negative prognostic indicator; however, no standardized histological criteria exist to define metastatic disease. The primary aim of this study was to determine whether different histological patterns of node-associated mast cells correlate with clinical outcome in dogs with mast cell tumour. A secondary goal was to propose a criteria-defined classification system for histological evaluation of lymph node metastasis. The Colorado State University Diagnostic Medicine Center database was searched for cases of canine mast cell tumours with reported lymph node metastasis or evidence of node-associated mast cells. Additional cases were obtained from a clinical trial involving sentinel lymph node mapping and node extirpation in dogs with mast cell neoplasia. Forty-one cases were identified for inclusion in the study. Demographic data, treatment and clinical outcome were collected for each case. Lymph nodes were classified according to a novel classification system (HN0-HN3) based on the number of, distribution of, and architectural disruption by, nodal mast cells. The findings of this study indicate that characterization of nodal mast cells as proposed by this novel classification system correlates with, and is prognostic for, clinical outcome in dogs with mast cell tumours.

Nucleomorphometric analysis of canine cutaneous mast cell tumours.

Thirty-five canine cutaneous mast cell tumours (CCMCTs) were analysed by computerized nuclear morphometry. In each case, the nuclei of at least 100 neoplastic cells were measured, and the mean nuclear area (MNA), mean nuclear perimeter (MNP) and mean nuclear form factor (FF) were calculated. Significant differences in respect of MNA and MNP occurred between tumours of grades I and III and between those of grades II and III (P<0.01) but not between tumours of grades I and II (P>0.01). No significant differences in respect of FF were observed between tumours of different grades. The results obtained indicate that nuclear morphometric analysis may assist in the grading of CMCTs.

The use of KIT and tryptase expression patterns as prognostic tools for canine cutaneous mast cell tumors.

Cutaneous mast cell tumors (MCTs) are one of the most common tumors in dogs. Currently, prognostic and therapeutic determinations for MCTs are primarily based on the histologic grade of the tumor, but a vast majority of MCTs are of an intermediate grade, and the prognostic relevance is highly questioned. A more detailed prognostic evaluation, especially of grade 2 canine MCTs, is greatly needed. To evaluate the prognostic significance of KIT and tryptase expression patterns in canine cutaneous MCTs, we studied 100 cutaneous MCTs from 100 dogs that had been treated with surgery only. The total survival and disease-free survival time and the time to local or distant recurrence of MCTs were recorded for all dogs. Using immunohistochemistry, 98 of these MCTs were stained with anti-KIT and antitryptase antibodies. Three KIT- and three tryptase-staining patterns were identified. The KIT-staining patterns were identified as 1) membrane-associated staining, 2) focal to stippled cytoplasmic staining with decreased membrane-associated staining, and 3) diffuse cytoplasmic staining. The tryptase-staining patterns were identified as 1) diffuse cytoplasmic staining, 2) stippled cytoplasmic staining, and 3) little to no cytoplasmic staining. Based on univariate and multivariate survival analysis, increased cytoplasmic KIT staining was significantly associated with an increased rate of local recurrence and a decreased survival rate. The tryptase-staining patterns were not significantly associated with any survival parameter. On the basis of these results, we propose a new prognostic classification of canine cutaneous MCTs, according to their KIT-staining pattern, that can be used for the routine prognostic evaluation of canine cutaneous MCTs.

Visualization and classification in biomedical terahertz pulsed imaging.

'Visualization' in imaging is the process of extracting useful information from raw data in such a way that meaningful physical contrasts are developed. 'Classification' is the subsequent process of defining parameter ranges which allow us to identify elements of images such as different tissues or different objects. In this paper, we explore techniques for visualization and classification in terahertz pulsed imaging (TPI) for biomedical applications. For archived (formalin-fixed, alcohol-dehydrated and paraffin-mounted) test samples, we investigate both time- and frequency-domain methods based on bright- and dark-field TPI. Successful tissue classification is demonstrated.

Morphologic properties of neoplastic mast cells: delineation of stages of maturation and implication for cytological grading of mastocytosis.

In the present study, cytological properties of bone marrow mast cells (MC) were analyzed and correlated with clinical parameters in 69 patients with systemic mastocytosis (SM). Based on cytomorphological features, four distinct cell types were recorded: (i) typical tissue MC (round cells, well granulated, round central nuclei); (ii) atypical MC exhibiting elongated cytoplasmic extensions, oval nuclei with excentric position, and a hypogranulated cytoplasm with focal granule accumulation ('atypical MC type I'); (iii) atypical MC with bi- or multilobed nuclei ('atypical MC type II'); and (iv) metachromatically granulated blast-like cells. In the majority of cases with SM, the percentage of MC in bone marrow (bm) smears was less than 5% (of all nucleated bm cells), and the predominant types were typical MC or atypical MC type I. In a smaller group of patients, the percentage of MC was greater than 5% and a significant subset of MC (>or=10%) were classified as 'metachromatic blasts' and/or atypical MC type II. These patients had a significantly shorter survival (P<0.05) and most of them were found to lack UP-like skin lesions. A percentage of MC>or=20% was invariably associated with the diagnosis 'mast cell leukemia'. Multivariate analysis confirmed the prognostic value of the cytology in SM and identified the percentage of MC (of all nucleated bm cells) as an independent prognostic variable. These data suggest that cytomorphological assessment of bm MC in SM is an important diagnostic approach that may help to delineate between variants of the disease.

Diagnostic criteria and classification of mastocytosis: a consensus proposal.

The term 'mastocytosis' denotes a heterogeneous group of disorders characterized by abnormal growth and accumulation of mast cells (MC) in one or more organ systems. Over the last 20 years, there has been an evolution in accepted classification systems for this disease. In light of such developments and novel useful markers, it seems appropriate now to re-evaluate and update the classification of mastocytosis. Here, we propose criteria to delineate categories of mastocytosis together with an updated consensus classification system. In this proposal, the diagnosis cutaneous mastocytosis (CM) is based on typical clinical and histological skin lesions and absence of definitive signs (criteria) of systemic involvement. Most patients with CM are children and present with maculopapular cutaneous mastocytosis (=urticaria pigmentosa, UP). Other less frequent forms of CM are diffuse cutaneous mastocytosis (DCM) and mastocytoma of skin. Systemic mastocytosis (SM) is commonly seen in adults and defined by multifocal histological lesions in the bone marrow (affected almost invariably) or other extracutaneous organs (major criteria) together with cytological and biochemical signs (minor criteria) of systemic disease (SM-criteria). SM is further divided into the following categories: indolent systemic mastocytosis (ISM), SM with an associated clonal hematologic non-mast cell lineage disease (AHNMD), aggressive systemic mastocytosis (ASM), and mast cell leukemia (MCL). Patients with ISM usually have maculopapular skin lesions and a good prognosis. In the group with associated hematologic disease, the AHNMD should be classified according to FAB/WHO criteria. ASM is characterized by impaired organ-function due to infiltration of the bone marrow, liver, spleen, GI-tract, or skeletal system, by pathologic MC. MCL is a 'high-grade' leukemic disease defined by increased numbers of MC in bone marrow smears (>or=20%) and peripheral blood, absence of skin lesions, multiorgan failure, and a short survival. In typical cases, circulating MC amount to >or=10% of leukocytes (classical form of MCL). Mast cell sarcoma is a unifocal tumor that consists of atypical MC and shows a destructive growth without (primary) systemic involvement. This high-grade malignant MC disease has to be distinguished from a localized benign mastocytoma in either extracutaneous organs (=extracutaneous mastocytoma) or skin. Depending on the clinical course of mastocytosis and development of an AHNMD, patients can shift from one category of MC disease into another. In all categories, mediator-related symptoms may occur and may represent a serious clinical problem. All categories of mastocytosis should be distinctively separated from reactive MC hyperplasia, MC activation syndromes, and a more or less pronounced increase in MC in myelogenous malignancies other than mastocytosis. Criteria proposed in this article should be helpful in this regard.

An improved system for quantifying AgNOR and PCNA in canine tumors.

BACKGROUND: Quantifying silver stained nucleolar organizer regions (AgNORs) and proliferation cell nuclear antigens (PCNA) are useful techniques to measure proliferative activity of tumor cells; however, the nonspecific deposition of stains and overlappings of AgNOR and PCNA counts between grades of tumors hamper their applications. MATERIALS AND METHODS: Fifty-two surgical specimens from dogs, including mast cell tumors, perianal gland tumors and hyperplasias, fibromas, fibrosarcomas, and normal tissues were studied. The 3 microns dewaxed sections of formalin-fixed tissues were stained to detect AgNORs by a modified inverted incubation technique in a newly developed silver staining device. Data were collected and analyzed using a high-resolution digital microscope camera and image analysis software. Sequential sections were also stained for PCNA using an immunohistochemical method. RESULTS: The improved system for quantifying AgNOR provided more accurate and non-overlapping mean AgNOR counts, which enable us to distinguish benign states from malignant changes. The mean AgNOR cut-off points that discriminated grade II or III mast cell tumors from grade I, perianal gland carcinomas from adenomas (or hyperplasia), fibrosarcomas from non-fibrosarcoma tissues, were 6.0, 14.1, 9.4, and 8.8 respectively. The mean AgNOR areas, relative AgNOR areas, and PCNA positive rates of some malignant and non-malignant tissues (benign tumor and normal tissues) were significantly different (P < 0.05). CONCLUSIONS: This improved system is a sensitive and rather precise method for quantifying the AgNOR and PCNA. It provides a valuable objective measurement for differentiating benign and malignant tumors.

Biology, classification and treatment of human mastocytosis.

Mastocytosis is a term collectively used for a heterogeneous group of disorders characterized by abnormal growth and accumulation of mast cells. Clinical symptoms occur from the release of chemical mediators and the pathologic infiltration of cells. Three major groups of patients with mastocytosis can be distinguished: i) cutaneous mastocytosis, ii) mastocytosis involving the skin and one or more extracutaneous organ(s), and iii) visceral mastocytosis without involvement of the skin. Groups ii) and iii) account for approximately 15-20% of all cases and have been referred to as systemic mastocytosis. Cutaneous mastocytosis typically presents as urticaria pigmentosa or diffuse cutaneous mastocytosis. These patients usually have a benign course. In contrast, systemic mastocytosis is a diffuse hematologic process with an increased risk to develop aggressive disease. In these patients, additional hematologic abnormalities or a second hematologic process, such as a myeloproliferative or myelodysplastic syndrome, or acute leukemia, may develop. Malignant mastocytosis and mast cell leukemia are rare forms of mastocytosis and characterized by uncontrolled and progressive proliferation and infiltration of mast cells in diverse organs. These patients often present without cutaneous lesions and have a very unfavorable prognosis. Because of the immature morphology of the cells it is often difficult to establish the diagnosis in such patients. However, the use of antibodies to mast cell antigens has recently improved the diagnostic efficiency in patients with suspected mast cell disease. No effective therapy for patients with malignant mastocytosis is known, although some patients may benefit from corticosteroid and interferon alpha treatment. The present article gives an overview of current knowledge about the biology, heterogeneity and treatment of human mastocytosis.

Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. III. Clonal analysis of the syngeneic cytolytic T lymphocyte response.

The cytolytic T lymphocyte (CTL) response of syngeneic mice to antigenic variants obtained by mutagenesis of mastocytoma P815 was analyzed at the clonal level. Estimates of the frequency of CTL precursor cells in spleens from mice immunized with P815 variants ranged from 10(-4) to 2 X 10(-3). This frequency could be increased approximately 100 times by stimulating immune spleen cells in mass culture for 6-8 days with the immunizing variant. Specificity analysis of a large number of individual CTL clones demonstrated the existence of two distinct populations of CTL. Some CTL clones lysed exclusively the immunizing variant, while others lysed equally well all P815 targets, but not syngeneic tumor L1210. These results provide direct evidence for the existence of new, individual specificities on P815 variants in addition to a common antigen already present on the original P815 cells. This confirms that a variety of new antigens can be induced by mutagenesis on the same background cell. Stable, highly active CTL clones specific for either individual variant antigens or common P815 antigens could be maintained in long-term culture.

Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. IV. Analysis of variant-specific antigens by selection of antigen-loss variants with cytolytic T cell clones.

Mouse mastocytoma P815 tumor cell variants that express new individual antigens were subjected to immunoselection in vitro with specific cytolytic T lymphocyte (CTL) clones. From one variant (P35) a number of resistant clones were isolated after a single-step selection. These immunoselected clones (P35.iscA-) proved to have acquired a stable and complete resistance to lysis by the selecting CTL, but remained sensitive to lysis by a CTL clone that recognizes a common P815 antigen. However, when these P35.iscA- clones were used to stimulate in vitro spleen cells from mice immunized with variant P35, the cytolytic activity was significantly higher on P35 and P35.iscA- targets than on the original P815 clone (P1). This indicated that P35.iscA- cells, which had lost a P35-specific determinant (P35A), had nevertheless retained another P35-specific determinant (P35B). CTL clones directed against the residual P35B determinant were then isolated. When P35 cells were submitted to selection with anti-P35B CTL, resistant clones (P35.iscB-) were obtained that were still lysed by anti-P35A and anti-P815 CTL clones. Tum- variant P35 therefore carries at least two specific determinants that can be lost independently. These results show the possibility of using selection with CTL clones to separate different components of complex cell surface antigens. The correlation between antigen expression and tumorigenicity was also examined. Antigen-loss P35.iscA- clones were found to be more tumorigenic than variant P35 which has a greatly reduced tumorigenic capacity compared to the original P815 tumor. This correlation suggests that the P35 determinant that was no longer expressed by the P35.iscA- clones is recognized on the original P35 tum- variant as a tumor rejection antigen.