Immunodetection of phosphohistone H3 as a surrogate of mitotic figure count and clinical outcome in cutaneous melanoma

Abstract

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/mm 2 (o1-29/mm 2 ). 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/mm 2 ). The relationship between phosphohistone H3 and manual mitotic count was statistically significant (Pearson correlation ¼ 0.59, Po0.0001). Logistic regression analyses demonstrated an association between the development of subsequent metastatic disease and the following variables: mitotic figures (odds ratio (OR) ¼ 5.7; P ¼ 0.0001); phosphohistone H3-positive mitotic figures (OR ¼ 3.0; P ¼ 0.008); Breslow thickness (OR ¼ 4.0 per mm; P ¼ 0.0002); ulceration (OR ¼ 3.94; P ¼ 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.

Comparative diagnostic and prognostic performances of the hematoxylin-eosin and phospho-histone H3 mitotic count and Ki-67 index in adrenocortical carcinoma

“…In this context, our data are in agreement with those of Ladstein 16 on thick cutaneous melanoma, although in many tumorsincluding melanoma itself-the prognostic value of phospho-histone H3-based mitotic count was found to be stronger than conventional hematoxylin and eosin-based counts. 20,25,37 …”

“…This protein is detected maximally during mitotic chromosome condensation in early prophase and negligibly at any other times (including apoptosis). 11,12 Phospho-histone H3 immunostaining has been shown to be a reliable and easy method for mitotic index evaluation in melanoma, with both diagnostic [13][14][15][16][17][18] and prognostic 17,[19][20][21] implications. Moreover, it has been also successfully applied in different neoplasms of the breast, [22][23][24][25][26] meninges, [27][28][29] brain, 30 lung, 31 soft tissues, [32][33][34] esophagus, 35 female genital tract, 36,37 prostate, 38 and urothelium, 39 as well as in cytology material from pancreatic endocrine tumors 40 and urothelial carcinoma.…”

Morphological Markers of Chromosomal Instability

“…Pyknotic nuclei or nuclei with basophilic cytoplasm are not thought to distinguish mitosis from apoptosis or degenerative cells [21]. Recently, immunohistochemical determination of proliferating cells using primary antibodies for Ki67, PCNA, or phosphohistone H3 has become popular; however, sometimes there is a discrepancy between mitotic index and Ki67 index [22][23][24]. We believe that this phenomenon represents the frequent mitotic arrest mentioned above.…”

Immunohistochemistry in Dermatopathology

“…Dual staining with a cytoplasmic melanocytic marker (PHH3-Melan-A) enables the pathologist to make a clear distinction between mitotically active melanocytes and lymphocytes, histiocytes, or endothelial cells and shows more concordant results when compared with standard section counts. [167][168][169] PHH3-determined mitotic counts have proven to be a stronger prognostic indicator than mitotic count determined from H&E-stained sections. 170 Although use of PHH3 improves accurate identification of mitoses and is known to show an association with survival, 171 the current melanoma staging criteria are based on a mitotic rate determined from H&E-stained slides.…”