Mitotic rate in melanoma: prognostic value of immunostaining and computer-assisted image analysis

Abstract

The prognostic value of mitotic rate in melanoma is increasingly recognized, particularly in thin melanoma in which the presence or absence of a single mitosis/mm can change staging from T1a to T1b. Still, accurate mitotic rate calculation (mitoses/mm) on hematoxylin and eosin (H&E)-stained sections can be challenging. Antimonoclonal mitotic protein-2 (MPM-2) and antiphosphohistone-H3 (PHH3) are 2 antibodies reported to be more mitosis-specific than other markers of proliferation such as Ki-67. We used light microscopy and computer-assisted image analysis software to quantify MPM-2 and PHH3 staining in melanoma. We then compared mitotic rates by each method with conventional H&E-based mitotic rate for correlation with clinical outcomes. Our study included primary tissues from 190 nonconsecutive cutaneous melanoma patients who were prospectively enrolled at New York University Langone Medical Center with information on age, gender, and primary tumor characteristics. The mitotic rate was quantified manually by light microscopy of corresponding H&E-stained, MPM-2-stained, and PHH3-stained sections. Computer-assisted image analysis was then used to quantify immunolabeled mitoses on the previously examined PHH3 and MPM-2 slides. We then analyzed the association between mitotic rate and both progression-free and melanoma-specific survival. Univariate analysis of PHH3 found significant correlation between increased PHH3 mitotic rate and decreased progression-free survival (P=0.04). Computer-assisted image analysis enhanced the correlation of PHH3 mitotic rate with progression-free survival (P=0.02). Regardless of the detection method, neither MPM-2 nor PHH3 offered significant advantage over conventional H&E determination of mitotic rate.

Figure 1. Examples of computer-assisted image analysis

a) A PHH3 immunostain is depicted after selecting an area for analysis. b) The same PHH3 immunostain after analysis and generation of a mark-up image showing positive PHH3 labeling as red and nonspecific staining as orange or yellow. c) An MPM-2 immunostain after selecting an area for analysis. d) The same MPM-2 immunostain after analysis and generation of mark-up image showing positive MPM-2 labeling as red and nonspecific staining as orange or yellow. Note in d) the less specific labeling of mitotic figures with MPM-2 relative to PHH3.

Figure 2. Mean mitotic counts by each method

Manual light microscopy (LM) of H&E, PHH3, and MPM-2 vs. computer-assisted analysis (digital) of corresponding scanned PHH3 and MPM-2 immunostains.

Figure 3. Conventional mitotic rate and five-year progression-free survival

Kaplan-Meier curve and log rank test on five-year recurrence in all melanomas. Group 1 includes those melanomas with mitotic rate less than the median value for H&E staining (2 mitoses/mm²). Group 2 includes those melanomas with mitotic rate no less than the median value for H&E staining. A significant difference in the five-year estimated progression-free survival function is seen between cases with lower and higher H&E mitotic rate (0.654 and 0.372, respectively, P=0.00009).

Figure 4. Visually-calculated PHH3 mitotic rate and progression-free survival

Kaplan-Meier curve and log rank test on recurrence in all melanomas. Group 1 includes those melanomas with mitotic rate less than the median value for PHH3 labeling (4 mitoses/mm²). Group 2 includes those melanomas with mitotic rate no less than the median value for PHH3 labeling. A significant difference is seen in the five-year estimated progression-free survival function seen between cases with lower and higher PHH3 labeling (0.608 and 0.464, respectively, P=0.013).