Use of Anti-phosphohistone H3 Immunohistochemistry to Determine Mitotic Rate in Thin Melanoma

Department of Dermatology, University of South Florida, Tampa, FL, USA.
The American Journal of dermatopathology (Impact Factor: 1.39). 10/2010; 32(7):650-4. DOI: 10.1097/DAD.0b013e3181cf7cc1
Source: PubMed


The seventh edition of the American Joint Committee on Cancer (AJCC) melanoma staging system, slated for release in 2010, will introduce mitotic rate (MR) as one of the primary criteria for staging thin melanoma (< or = 1.0 mm). Accurate counts are essential because the finding of a single mitotic figure (MF) will alter the staging and management of these patients. The traditional manner of counting of mitotic figures (MFs) using a X40 objective is time consuming and prone to inter- and intraobserver variability. We employed an antibody to phosphohistone H3 (pHH3, ser10) that labels MFs in all stages of mitosis, to evaluate mitotic counts at X20 in tissue sections from 30 melanoma patients with thin lesions 0.45 to 1.2 mm in depth, and compared results with routine hematoxylin and eosin (H&E) in a double-blind fashion. The mean MR was 1.63 by antipHH3, and 0.67 for H&E, representing a mean increase of 243%. The Spearman correlation coefficient for MR in H&E and anti-pHH3 sections was 0.88 (P < 0.0001). When melanomas were designated as "mitotically active," if the MR by anti-pHH3 was > or = 2 and > or = 1 by H&E, the correlation coefficient increased to 1.0. No thin melanomas were mitotically inactive on anti-pHH3 but active on H&E. Results indicate that anti-pHH3 is a useful immunostain for labeling melanocytes in mitosis. Subsequent studies will be needed confirm the accuracy of this staining technique, which has the potential to be used as a screening method for counting MFs before conventional H&E methodology in the microstaging of thin melanoma.

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    • "It has been shown that phosphorylation of histone H3 occurs at Ser 10 almost exclusively during late G2 phase, prophase, and M phase of the cell cycle in order to help coordinate chromatin decondensation and cytokinesis (Hendzel et al, 1997). Anti-pHH3 immunohistochemistry has already been applied on several tumour cell lines along with neoplasms, such as breast cancer (Skaland et al, 2007), astrocytoma (Habberstad et al, 2011), meningioma (Kim et al, 2007), melanoma (Casper et al, 2010), and uterine smooth muscle tumour (Veras et al, 2009), with the result that pHH3 is useful to assess mitotic activity. Moreover, it has been used to support the grading of some tumours and to predict prognosis (Kim et al, 2007). "
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    ABSTRACT: Phosphohistone-H3 (pHH3) is a promising reliable mitotic count biomarker. Our purpose was to study the relationship between the novel proliferation marker pHH3 and the established anti-apoptotic marker survivin and consider their prognostic relevance in endometrial cancer. A total of 106 patients with endometrial cancer (type I/endometrioid, n=81; type II carcinomas, n=18) and simple hyperplasia without atypia (n=7) were investigated. pHH3 and survivin expression were assessed using immunohistochemistry from paraffin-embedded tissue blocks. A strong positive correlation was observed between pHH3 and survivin expression (P<0.0001). Patients with high-grade tumours and patients with type II carcinomas expressed significantly more pHH3 and survivin than low grade and endometrioid tumours (P<0.0001, P<0.0001, P<0.0001, and P<0.0001, respectively). In univariate survival analysis, overexpression of pHH3 and survivin were associated with increased recurrence and mortality (P<0.0001, P<0.0001, P<0.0001, and P<0.0001, respectively), in the multivariable Cox regression analyses both pHH3 and survivin could be identified as independent parameters for overall survival (P=0.004, and P=0.023, respectively). In endometrial cancer, pHH3 and survivin were strongly positive correlated and were both associated with type II and high-grade tumours. Increasing expression levels of pHH3 and survivin were associated with adverse prognostic factors.
    Full-text · Article · May 2012 · British Journal of Cancer
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    • "Immunohistochemical staining of PHH3 is reported to facilitate mitotic counting and predict prognosis in different tumors such as breast cancer (Skaland et al., 2007). Studies in melanocytic lesions are very limited, but have suggested PHH3 as a useful supplement in differentiating malignant melanoma from benign nevi and to simplify the assessment of mitotic activity in established melanomas (Tapia et al., 2006; Nasr and El-Zammar, 2008; Casper et al., 2010; Glatz et al., 2010). Prognostic impact of PHH3 was recently reported for uveal melanomas (Angi et al., 2011), but has not been shown for cutaneous melanoma. "
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    ABSTRACT: Mitotic count is a known prognostic predictor in cutaneous melanoma, and is included in the current American Joint Committee on Cancer tumor-node-metastasis (TNM) staging system. The mitotic marker phosphohistone H3 (PHH3) is considered to facilitate counting of mitosis, and the purpose of this study was to evaluate the prognostic significance and strength of PHH3 in comparison with standard mitotic counting in cutaneous malignant melanoma. A total of 457 consecutive cases of nodular cutaneous melanoma were initially included in this series. The mitotic count was assessed on hematoxylin and eosin sections, and PHH3 was then examined by immunohistochemistry on standard sections of paraffin-embedded tumor tissue. Both the mitotic count and the number of PHH3-stained mitotic figures were recorded in a minimum area of 1 mm(2). Increased mitotic count and PHH3 value were both associated with unfavorable features like tumor thickness and presence of ulceration. Univariate survival analysis showed a highly significant prognostic impact of mitotic count and PHH3, whereas multivariate analysis indicated PHH3 to be a stronger prognostic indicator than mitotic count. Assessment of mitotic activity by PHH3 immunostaining might have important practical advantages, and should be further studied to consider a place in routine examination of all cutaneous melanomas.
    Preview · Article · Feb 2012 · Journal of Investigative Dermatology
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    ABSTRACT: Changes in the 2010 American Joint Commission on Cancer melanoma staging guidelines include the evaluation of primary tumor mitotic index (mitogenicity) and the recognized prognostic significance of a single melanoma cell in a sentinel lymph node. These revised criteria have important practice implications for dermatopathologists as well as for dermatologists, oncologists and surgeons who treat patients with cutaneous melanoma. Piris A, Mihm MC Jr, Duncan LM. AJCC melanoma staging update: impact on dermatopathology practice and patient management.
    Full-text · Article · Mar 2011 · Journal of Cutaneous Pathology
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