Concurrent Ki-67 and p53 Immunolabeling in Cutaneous Melanocytic Neoplasms: An Adjunct for Recognition of the Vertical Growth Phase in Malignant Melanomas? Zahid Kaleem, M.D., Anne C. Lind, M.D., Peter A. Humphrey, M.D., Ph.D., Robert H. Sueper, M.D., Paul E. Swanson, M.D., Jon H. Ritter, M.D., Mark R. Wick, M.D. Lauren V. Ackerman Laboratory of Surgical Pathology, Washington University Medical Center (ZK, ACL, PAH, PES, JHR), and SmithKline-Beecham Laboratories (RHS), St. Louis, Missouri; and the Robert E. Fechner Laboratory of Surgical Pathology, University of Virginia Health Sciences Center (MRW), Charlottesville, Virginia malignant melanocytic tumor representing the ver- Ki-67 labeling of paraffin sections has been corre- tical growth phase—nodular melanoma—demon- lated with the number of cells in non-Go phases of strated a statistically significant difference from all the replicative cell cycle, and this immunohisto- other lesions in this study with respect to Ki-67 ,␹2) and p53 reactivity (P < .000001 ,008. ؍ chemical technique has been applied to the evalu- index (P ation of a variety of human neoplasms. Similarly, ␹2). Subsequent concurrent use of a Ki-67 threshold immunolabeling for p53 protein has been used to index of 10% and a p53 index of 5% correctly indi- detect mutations in the corresponding gene, as a cated the presence of vertical growth in 75% of reflection of possible cellular transformation in the NMMs, whereas only 8% of radial growth phase same context. Both of these techniques were ap- melanomas of other types were colabeled at the plied to 253 melanocytic tumors of the skin to assess same levels of reactivity for the two markers (P < their possible utility in the diagnosis and subcatego- .00001, ␹2). Thus, although the distinction between rization of such lesions. They included 76 banal benign and malignant melanocytic tumors could (common) nevi (CN), 39 Spitz nevi (SN), 62 superfi- and should not be based on immunohistology for cial spreading malignant melanomas in radial Ki-67 and p53, these results suggest that the latter growth (SSMMs), 32 nodular malignant melanomas determinants may, in fact, be used as an adjunct to (NMMs), 21 lentigo maligna melanomas in radial morphology in the recognition of the vertical growth (LMMs), and 23 melanomas arising in asso- growth phase in cutaneous malignant melanomas. ciation with preexisting compound nevi (MCN). One hundred cells were counted randomly in each tu- KEY WORDS: Ki-67, Melanocytic neoplasms, Mela- mor, and dark, exclusively nuclear reactivity was noma, Nevi, p53, Skin neoplasms. scored as positive labeling; results were recorded as Mod Pathol 2000;13(3):217–222 percentages. Negligible Ki-67 and p53 labeling was seen in CN and SN, at a level that was similar to that In attempts to extend morphologic observations in obtained in cases of LMM and MCN. The largest the prediction of biologic behavior, several adjunc- proportion of Ki-67–positive and p53-positive cells tive techniques have been applied to cutaneous was observed in NMMs, followed by SSMMs. Radial melanocytic lesions of all types. These procedures growth-phase SSMMs and LMMs demonstrated im- munoprofiles that were similar to those of melano- have included nuclear morphometry (1); immuno- cytic nevi, and MCN did so as well. The prototypical staining for p53 proteins (2–4), the INK4A/CDKN2 tumor suppressor gene product (5), proliferating cell nuclear antigen (6–8), or the Ki-67 nuclear protein (9–13); and histochemical analysis of nu- Copyright © 2000 by The United States and Canadian Academy of Pathology, Inc. cleolar organizer regions (14). VOL. 13, NO. 3, P. 217, 2000 Printed in the U.S.A. A particularly challenging facet of dermatopa- Date of acceptance: July 29, 1999. Presented in part at the scientific session of the 34th annual meeting of thology concerns the more narrow setting of mela- the American Society of Dermatopathology, San Francisco, CA, March nocytic malignancy. In that context, it is important 1997. Address reprint requests to: Mark R. Wick, M.D., Division of Surgical to identify the growth phases (radial or vertical) of Pathology, Room 3882 Old Medical School, University of Virginia Health Sciences Center, Jefferson Park Avenue, Box 214, Charlottesville, VA melanomas, in recognition of the prognostic im- 22908; e-mail: [email protected]; fax: 804-924-0217. portance attached to them (15). Our laboratory has 217 previously studied the expression of putatively mu- The various tumors were categorically distributed tant p53 protein in melanocytic lesions and found a as follows: 76 common nevi (CN), 39 Spitz nevi reasonable correlation between its presence and (SN), 62 superficial spreading malignant melano- malignant morphologic and biologic characteris- mas (SSMMs), 32 nodular malignant melanoma tics; moreover, non–wild-type p53 protein was also (NMMs), 21 lentigo maligna melanomas (LMMs), preferentially associated with vertical growth and 23 melanomas arising in association with pre- among melanomas of various types (2). Relatively existing common nevi (MCN). Each of the nodular few reports have addressed the identification of melanomas was, by definition, in the vertical growth phases in cutaneous melanocytic tumors growth phase, whereas all other melanomas in the using the MIB1 (anti–Ki-67) antibody (9), and none study were, by design, radial growth phase lesions. has discussed its concomitant application with p53 Paraffin sections of all specimens were cut at 4 ␮ in this context. Moreover, the issue of whether pro- thickness and mounted on glass slides coated with liferation indices and p53 are truly helpful in the poly-L-lysine (Sigma-Aldrich Chemical Co., St. diagnostic distinction between melanocytic nevi Louis, MO). They were dewaxed in xylene and ab- and melanomas is still unsettled, in part because of solute ethanol; incubated for 30 min in methanolic methodological differences in various publications H2O2 (0.3% [v/v]) to quench endogenous peroxi- (6–13). Hence, the specific aim of the following dase; and rehydrated by passage through graded evaluation was 2-fold: first, to reassess the utility of ethanol solutions, distilled water, and phosphate- concurrent labeling evaluation for Ki-67 and p53 in buffered-saline (PBS; pH 7.4). Heat-mediated “an- the differential diagnosis of benign and malignant tigen retrieval” (17) was undertaken after immer- melanocytic neoplasms, and, second, to study the sion of the slides in standard citrate buffer (0.01 M; differences, if any, in the labeling indices for these pH 6.0). They were warmed for 12 min in a micro- markers in various biologic subgroups of cutaneous wave oven (Sharp, Los Angeles, CA; 600 W; 2,450 melanoma. The latter included “common” lesions MHz). After a 20-min cooling period, the sections in the radial and vertical growth phases (as defined were rinsed in water and incubated in PBS. The by Elder & Murphy [16]), as well as other examples MIB-1 antibody to Ki-67 (Dako Co., Carpinteria, CA; of recognized histologic melanoma variants. A cur- dilution 1:300) and a “cocktail” of anti-p53 reagents rently advocated microwave-mediated enhance- (clone designations 1801 and D07; BioGenex Co., ment method (17) was used to optimize exposure of Dublin, CA, and Dako, respectively) were then ap- the Ki-67 and p53 target epitopes in standard par- plied and the sections were incubated for 16 hours affin sections. at 4° C. Antibody bridge assembly with the avidin- biotin-peroxidase complex (ABC) technique, using the Elite ABC kit (Vector Laboratories; Burlingame, MATERIALS AND METHODS CA), was carried out on the next day by sequential 1-h incubations. Chromogenic development was A total of 253 well-characterized cutaneous mela- obtained by immersion of the sections in nickel nocytic neoplasms were studied, as obtained from chloride–complexed 3,3Ј-diaminobenzidine solu- the surgical pathology files of Washington Univer- tion (0.25 mg/mL with 0.003% H2O2) for a maxi- sity Medical Center and SmithKline-Beecham Lab- mum of 10 min. The slides were then lightly coun- oratories (St. Louis, Missouri). These specimens terstained with Harris’s hematoxylin. were procured as excisional biopsies from 101 Positive controls for Ki-67 studies were repre- males and 153 females, ranging in age from 3 to 98 sented by paraffin sections of “stock” neoplasms years, whose lesions had arisen in various locations. that had been previously studied by flow cytometry The samples were obtained with standard surgical to determine proportions of tumor cells in the dif- technique and immediately fixed in neutral- ferent phases of the cell cycle. Similar sections of buffered formalin. Each of these tumors had been neoplasms that had been shown to manifest p53 diagnostically classified originally by at least two of mutations by Southern blot analysis also were used the authors using established morphologic criteria as positive controls. Nonimmune murine ascitic (16). Vertical growth in melanomas was defined by fluid was substituted for the primary antibodies as a the presence of at least one of the following criteria negative control. Cells were considered Ki-67 posi- (16): all tumor cell nests in the epidermis being tive or p53 positive when they demonstrated strong smaller than at least one in the dermis, differing and exclusively nuclear labeling. Those cases with cytologic appearances of the intraepidermal and no reactivity whatsoever were regarded as negative, intradermal components, the presence of dermal whereas immunoreactivity in the others was scored tumor cell nests of 25 or more cells in width, and semiquantitatively (as “1 to 4% ϩ,” “5 to 9% ϩ,” and mitotic activity in intradermal neoplastic cells. Ն10% ϩ, etc.) by assessment of 100 tumor cells in Complete follow-up information was obtained in randomly selected microscopic fields at 400ϫ mag- all instances to validate the nosologic process. nification. Paired categorical results were com- 218 Modern Pathology pared by ␹2 analysis, using True Epistat statistical labeling and a 5% level for concurrent p53 immu- computer software (Epistat Services, Richardson, noreactivity, 24 (75%) of the NMMs in this series TX).