Immunodetection of Phosphohistone H3 as a Surrogate of Mitotic Figure Count and Clinical Outcome in Cutaneous Melanoma

Michael T Tetzlaff; Jonathan L Curry; Doina Ivan; Wei-Lien Wang; Carlos A Torres-Cabala; Roland L Bassett; Karla M Valencia; Michael S McLemore; Merrick I Ross; Victor G Prieto


Mod Pathol. 2013;26(9):1153-1160. 

In This Article

Abstract and Introduction


In the American Joint Committee on Cancer (AJCC)-TNM (2009) staging system, the key prognostic factor in cutaneous melanoma is the depth of dermal invasion (Breslow thickness) with further refinement according to the presence of epidermal ulceration or dermal mitoses. Immunodetection of phosphohistone H3 has been shown to facilitate the identification of mitotic figures in various neoplasms. We selected 120 cases of primary cutaneous melanoma with completely annotated histopathologic parameters and clinical outcomes and performed double immunohistochemical staining for MLANA (Mart-1/Melan-A) and phosphohistone H3. One hundred and thirteen cases were amenable to antiphosphohistone H3 staining from 66 men and 47 women, with mean age of 64 years (9–93), including 61 superficial spreading type, 24 nodular, 6 lentigo maligna, 8 acral lentiginous, and 14 unclassified. The mean Breslow thickness was 2.53 mm (0.20–25), ulceration was present in 25/113 (22%) and the mean mitotic count was 3.2/mm2 (<1–29/mm2). In 27/113 (24%) of the cases, antiphosphohistone H3 failed to highlight mitotic figures anywhere in the tissue (normal or tumor cell), whereas in 86/113 (76%) antiphosphohistone H3 detected at least one mitotic figure. Among the latter, antiphosphohistone H3 did not detect mitotic figures in dermal tumor cells in 37/86 cases (43%), whereas anti-PHH3 identified at least one melanocytic mitotic figure in the other 49/86 cases (57%; range: 1–66/mm2). The relationship between phosphohistone H3 and manual mitotic count was statistically significant (Pearson correlation=0.59, P<0.0001). Logistic regression analyses demonstrated an association between the development of subsequent metastatic disease and the following variables: mitotic figures (odds ratio (OR)=5.7; P=0.0001); phosphohistone H3-positive mitotic figures (OR=3.0; P=0.008); Breslow thickness (OR=4.0 per mm; P=0.0002); ulceration (OR=3.94; P=0.008). The application of phosphohistone H3 immunohistochemistry to the description of primary cutaneous melanoma is useful in identifying mitotic figures, improves upon the specificity of this designation when used together with MLANA, and correlates with an increased risk for metastasis in univariate analyses.


Melanoma is among the most deadly forms of skin cancer with a steadily rising incidence and poor survival in advanced disease: 10-year survival ranges from ~6–15% among patients with distant metastases (stage IV).[1] Therefore, there is a critical need to identify clinically significant biomarkers to delineate those patients at highest risk for an aggressive disease course. The 2009 AJCC staging system for melanoma determined that the primary tumor (pT) should be classified according to tumor thickness (mm) with further refinement according to the presence of ulceration (for all tumors) or dermal mitotic figures (for tumors ≤1.0 mm).[2] In addition, patients with cutaneous melanoma typically undergo sentinel lymph node biopsy for tumors greater than 1 mm in thickness or for tumors ≤1.0 mm when other adverse features are present: ulceration, dermal mitotic figures or a positive deep margin in the original biopsy specimen.[3–5] Thus, the presence of dermal mitotic figures in primary melanoma has emerged as a critical variable in subsequent management decisions and prognostic algorithms.

Phosphorylation of histone H3 at serine10 (H3S10P) is an important event in cell cycle progression, first emerging in pericentromeric chromatin in late G2 phase. Subsequently, it spreads in a systematic, non-random fashion throughout the condensing chromatin during prophase and persists through anaphase of the cell cycle. It is typically no longer detectable when mitosis is completed.[6–8] Furthermore, phosphohistone H3 is a specific marker of mitotic figures, as it is not expressed in apoptotic bodies—common morphologic mimics of mitotic figures in routine light microscopy.

Thus, immunodetection of phosphohistone H3 has become a sensitive and specific marker of mitotic figures and correlates with outcome in a variety of different human tumor types,[9] including breast carcinoma,[10,11] colorectal adenocarcinoma,[12] ovarian serous adenocarcinoma,[13] pulmonary neuroendocrine carcinoma,[14] uterine smooth muscle tumors,[15] astrocytomas,[16] and meningiomas.[17] In melanoma, immunodetection of phosphohistone H3 has been shown to increase the sensitivity and accuracy for mitotic figure detection; it improves inter-observer variability; and it reduces the time required to identify mitoses in primary cutaneous and uveal melanomas.[18–23] In relatively thick nodular melanomas, the presence of phosphohistone H3-positive mitotic figures has been shown to correlate with known prognostic indicators (like depth of invasion and ulceration), but it also serves as an independent prognostic indicator in multivariate analyses.[22]

In the current study, we sought to expand on these latter observations using a broad spectrum of histopathologic subtypes of primary cutaneous melanoma. We showed that immunodetection of mitotic figures with a cocktail of antibodies for MLANA (Mart-1/Melan-A) and phosphohistone H3 provides a sensitive and specific approach to identifying mitotic figures in melanocytes and strongly correlates with manual detection of mitotic figures on routine H&E-stained sections. Furthermore, in univariate analyses, the identification of phosphohistone H3-positive mitotic figures in MLANA-positive melanocytes correlates with the development of subsequent metastases.