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

Casper, David J MD*; Ross, Kate I BS; Messina, Jane L MD*‡; Sondak, Vernon K MD§; Bodden, Cheryl N BS, ASCP, HTL*; McCardle, Tim W MD§; Glass, L Frank MD

The American Journal of Dermatopathology: October 2010 - Volume 32 - Issue 7 - p 650-654
doi: 10.1097/DAD.0b013e3181cf7cc1
Original Study

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 (≤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 ×40 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 ×20 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 anti-pHH3, 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 ≥2 and ≥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.

From the *Department of Dermatology, University of South Florida, College of Medicine, Tampa, FL; †Department of Internal Medicine, Florida State University, College of Medicine, Tallahassee, FL; Department of Internal Medicine ‡Department of Pathology, University of South Florida, College of Medicine, Tampa, FL; and §Department of Cutaneous Oncology, Moffitt Cancer Center, Tampa, FL.

No funding was received for the conduct of this research or production of this manuscript.

Reprints: L. Frank Glass, MD, Department of Dermatology, University of South Florida, Dermatopathology Laboratory, 12901 Bruce B. Downs Blvd., MDC 96, Tampa, FL 33612 (e-mail:

© 2010 Lippincott Williams & Wilkins, Inc.