- Email: [email protected]
Minichromosome maintenance protein expression in benign nevi, dysplastic nevi, melanoma, and cutaneous melanoma metastases
Minichromosome maintenance protein expression in benign nevi, dysplastic nevi, melanoma, and cutaneous melanoma metastases Alan S. Boyd, MD,a,b Bashar Shakhtour, PhD,c and Yu Shyr, PhDc Nashville, Tennessee Background: Minichromosome maintenance (MCM) proteins are a recently elucidated group of polypeptides intimately involved in DNA replication and appreciable only in cycling cells. In other organ systems their expression has proven more prognostically useful than cell proliferation markers such as Ki67 and proliferating cell nuclear antigen. To date, the evaluation of MCM proteins in melanocytic neoplasms has not been undertaken. Objective: We sought to determine whether MCM protein 2 (the most extensively evaluated of the MCM protein family) is present in melanocytes from benign nevi, dysplastic nevi, primary cutaneous melanomas, and cutaneous melanoma metastases and, if so, whether there exists a significant difference in expression among the 3 groups. Methods: Immunohistochemical staining for MCM 2 was performed on tissue sections from 10 benign nevi, dysplastic nevi, and primary cutaneous melanomas and from 5 cutaneous melanoma metastases. Approximately 200 cells were evaluated microscopically in 5 separate fields for each specimen and the number of positively staining nuclei was counted. After a percentage was calculated for each lesion, the data were pooled and statistically analyzed. Results: Melanocyte nuclear staining was readily visible microscopically. The percentage of positively staining nuclei in benign nevi (1.2%), dysplastic nevi (6.1%), primary cutaneous melanomas (49.1%), and cutaneous melanoma metastases (40.9%) was significantly different (P \.0001) among the 4 groups. Using paired comparisons, statistically significant differences were found between benign nevi and melanoma, dysplastic nevi and melanoma, benign nevi and cutaneous melanoma metastases, and dysplastic nevi and cutaneous melanoma metastases. There was no statistically significant difference between cutaneous melanoma metastases and primary cutaneous melanoma. Limitations: This is a small pilot study without blinded evaluation of the tissue sections and lacking correlation with patient clinical outcome or accepted histologic prognostic factors. Conclusion: MCM protein expression appears to differ significantly in melanocytic neoplasms and potentially provides an additional tool for distinguishing benign tumors from their malignant counterparts. Further evaluation of this expression may prove useful in delineating the biologic behavior of these tumors and warrants additional research. ( J Am Acad Dermatol 2008;58:750-4.)
igmented lesions are commonly biopsied neoplasms and represent a significant percentage of dermatopathology specimens. Categorizing melanocytic tumors as to their biologic
P
potential occasionally constitutes a vexing problem. The increasing availability of newer technologies, including immunoperoxidase stains, have bolstered diagnostic precision and provided submitting
From the Departments of Medicine (Dermatology),a Pathology,b and Preventative Medicine,c Vanderbilt University. Funding sources: None. Conflicts of interest: None declared. Accepted for publication December 18, 2007. Reprint requests: Alan S. Boyd, MD, 3900 Vanderbilt Clinic, Nashville, TN 37232. E-mail: [email protected].
Published online February 13, 2008. 0190-9622/$34.00 ª 2008 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2007.12.026
750
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J AM ACAD DERMATOL VOLUME 58, NUMBER 5
Abbreviations used: MCM: minichromosome maintenance mRNA: messenger RNA PCNA: proliferating nuclear cell antigen
clinicians with information regarding the potential long-term implications of their patients’ melanocytic lesions. Immunoperoxidase staining of tissue sections for Ki-67 and proliferating cell nuclear antigen (PCNA) allow evaluation of the proliferative index in melanocytic neoplasms with both diagnostic and prognostic implications.1-11 Recently, a group of proteins intimately involved in DNA replication termed ‘‘minichromosome maintenance’’ (MCM) proteins have been described.1,12-15 Detectable only in cycling cells, the presence of MCM proteins in other malignancies has been associated with increasing degrees of cellular atypia and poorer prognosis.13,16-21 Their use in distinguishing benign from malignant cutaneous melanocytic lesions has not previously been undertaken.
METHODS Ten benign nevi, dysplastic nevi, and primary cutaneous melanomas and 5 cutaneous melanoma metastases were randomly selected from specimens submitted to our dermatopathology division. Recombinant MCM protein 2 immunoperoxidase stains (ProEx C, Tripath Imaging, Burlington, NC) were obtained on each specimen as suggested by the manufacturer (Fig 1). Using high-power microscopy (3400), approximately 200 melanocytic cells were evaluated for MCM 2 staining in 5 separate fields per specimen. Fields without epidermis, adnexal structures, or inflammatory cells were preferentially selected to minimize mistakenly counting cells not of melanocytic lineage. The nonparametric rank-based Kruskal-Wallis one-way analysis of variance method was used to test the equality of population medians of percentage of positively staining cells among groups. In addition, the Bonferroni correlation was applied to adjust the multiple comparisons among study groups. All tests of significance were two-sided, and differences were considered statistically significant when P value was less than .05. Software (SAS, Version 9, Cary, NC) was used for all analyses.
RESULTS The number of cells per high-power field staining positively in the 4 groups is listed in Table I. Using the Kruskal-Wallis test, a significant difference was noted in comparing the 4 groups (P \.0001). When
the Bonferroni correlation was applied to all pairwise comparisons among these 4 groups, statistically significant differences were found between benign nevi and primary cutaneous melanoma, dysplastic nevi and primary cutaneous melanoma, benign nevi and cutaneous melanoma metastases, and dysplastic nevi and cutaneous melanoma metastases. A statistically significant difference was not found between primary cutaneous melanoma and cutaneous melanoma metastases.
DISCUSSION In eukaryotic cells, the initiation of DNA synthesis is a complex, multistep process involving the chronologic assembly at replication origins of initiation factors into prereplicative complexes.12 This results in specific chromatin regions becoming competent for DNA replication or ‘‘licensing’’ in the ensuing cell cycle S phase.12,22 Replication origins are formed initially when the origin replication complex sequentially bind cdc6 and cdt1 proteins.1,14 These proteins are believed to function as adenosine triphosphatases and cdt1’s putative role is to recruit a collection of polypeptides called MCM proteins. MCM proteins form an encircling hexamer around DNA strands before the initiation of replication. This process is restricted to late mitosis and MCM proteins must remain associated with the DNA through G1 and into the S phase.14 After mitosis, the MCM proteins, cdc6, and cdt1 are down-regulated and degraded leaving the origin replication complex intact at the replication origin. MCM proteins were initially discovered as factors supporting minichromosome preservation in the yeast Saccharomyces cerevisiae and are highly conserved across eukaryotic species.12,23 They comprise a group of 10 separate polypeptides with numbers 2 through 7 used in the hexamer binding at the origin replication complex. They presumably act as DNA helicases and are dissociated from chromatin after replication, thereby ensuring that each DNA region is replicated only once during a single cell cycle.12,14 MCM proteins are found in cycling cells but not in quiescent, differentiated, or senescent cells.1,13,17 MCM 2 through 7 are the most investigated of this protein group. MCM 1 is believed to regulate cdc6 expression and some of the MCM 2 through 7 genes.15 MCM 8 and 9 may have a role as DNA helicases and MCM 10 is thought to stabilize DNA polymerase and direct it to replicating chromatin.15 In normal tissues, MCM proteins are expressed by proliferative cells similar to that seen with Ki-6712 and may be appreciated early in premalignant cells.14 Immunohistochemical staining for MCM proteins is typically restricted to cell nuclei although faint cytoplasmic staining may also be seen.
752 Boyd, Shakhtour, and Shyr
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Fig 1. Histologic features of melanocytic neoplasms. A and B, Benign intradermal nevus with immunohistochemical staining for minichromosome maintenance (MCM) 2. C and D, Dysplastic nevus with immunohistochemical staining for MCM 2. E and F, Primary cutaneous melanoma with immunohistochemical staining for MCM 2. G and H, Cutaneous melanoma metastasis with immunohistochemical staining for MCM 2. (A, C, E, and G, Hematoxylin-eosin stain; B, D, F, and H, immunoperoxidase stain; original magnifications: A, B, E to H, 3200; C and D, 3100.)
Evaluation of MCM proteins is believed to be more reliable in discriminating malignant from precancerous cells than PCNA and Ki-67.15 In malignant neoplasms MCM 2 and MCM 5 have been the most studied of the MCM proteins. MCM 2 is expressed more frequently than Ki-67 in normal and malignant breast tissue and MCM 2 expression is reportedly superior to and independent of histologic tumor grade, Ki-67 staining, and lymph node status in the prognosis of breast adenocarcinomas.19 In nonsmall cell carcinoma of the lung, MCM 2 tissue
staining is an independent prognostic factor in survival whereas Ki-67 expression is not.18 MCM 2 tumor expression is also an independent prognostic factor in survival of patients with prostatic adenocarcinoma.17 High-grade and advanced-stage bladder cancers demonstrate significantly greater tissue staining for MCM 2 and MCM 5 and are statistically associated with adverse patient outcome.13 Stoeber et al16 devised a novel MCM 5 immunofluorometric assay for use on urinary cellular sediment in patients with suggested urinary tract malignancies. Of
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perhaps the greatest use is the evaluation of cervical specimens, Williams et al24 stained cervical tissue smears for MCM 5 and cdc6 noting a ‘‘remarkably high specificity and sensitivity’’ to the expression of these proteins and the presence of atypical cells. Although PCNA2,3,9 and Ki-674-8,10,11 have been extensively evaluated in melanocytic neoplasms, to date, only a handful of investigations of MCM expression in benign and malignant mucocutaneous conditions have been conducted. Freeman et al1 evaluated biopsy specimens of normal-appearing skin (n = 5), psoriasis (n = 3), actinic keratosis (n = 1), Bowen’s disease (n = 5), and squamous cell carcinoma (n = 13) for both MCM 2 and MCM 5. When compared with those from normal-appearing skin, cell counts were significantly higher in Bowen’s disease and squamous cell carcinoma but not psoriasis. Increasing expression of these proteins was noted in well, moderate, and poorly differentiated squamous cell carcinomas. Staining was appreciated along the basal layer in normal-appearing skin, more than 50% of the epidermis in the actinic keratosis, and greater than 90% of the epidermis in Bowen’s disease. Similar results were obtained in an investigation of oral mucosal biopsy specimen.21 Lesions with moderate to severe cytologic dysplasia and those frankly malignant (squamous cell carcinoma) expressed significantly greater numbers of cells staining for MCM 2 than normal mucosa, benign keratoses, or mildly dysplastic lesions. Staining of oral smears from the same lesions showed similar findings. Ha et al25 evaluated MCM 3 messenger RNA (mRNA) and cellular protein expression in a wide variety of human cancers. Although some malignancies strongly demonstrated MCM 3 mRNA the melanoma cell line showed only modest levels. Interestingly, Ki-67 and PCNA mRNA were also evaluated and demonstrated reduced sensitivity and specificity compared with MCM 3. Immunoperoxidase staining for MCM 3 in 110 malignant tumors (4 from the skin, type not specified) showed ‘‘moderate to high levels of expression’’ in 93 samples, however, the authors were not more specific. In an elaborate study of cutaneous melanomas, Winnepenninckx et al26 identified 254 genes involved in activating DNA replication noting a statistically significant difference in gene expression between patients with metastatic disease and those without metastases. Included in this group were MCM 3, MCM 4, and MCM 6. When immunoperoxidase staining for these 3 proteins was performed on tissue sections each was expressed in significantly greater amounts in the tumors from patients with distant metastases. However, grading of positively stained cells was performed on a 4-level scale and not by counting individual cells.
Table I. MCM protein 2 staining characteristics in melanocytic neoplasms
Benign nevi Dysplastic nevi Primary cutaneous melanoma Cutaneous melanoma metastasis
Mean No. of positively staining cells/high-power field
SD
1.2 6.1 49.1 40.9
0.5 4.0 20.1 25.5
MCM, Minichromosome maintenance; SD, standard deviation.
Of interest is the diminished number of staining cells in the cutaneous melanoma metastases compared with primary cutaneous melanomas. This difference (P = .14) is only of marginal statistical significance and may have resulted from so few specimens evaluated. Rieger et al2 noted significantly greater PCNA and Ki-67 staining in melanoma metastases compared with primary cutaneous melanomas, whereas Ilmonen et al27 found no discernable trend between such groups for Ki-67 expression. Evaluation of tissue for MCM proteins offers an advantage over other cell proliferation markers such as PCNA and Ki-67 in that these proteins are required for the initiation of replication, representing the point of convergence of many signaling pathways involved in cell growth.1 They would, therefore, constitute better candidates for marking cells in cycle than transduction molecules or growth factor receptors. MCM protein expression is not associated with DNA repair as is the case with PCNA nor is it quiescent in resting cells still maintaining replication competence in contrast to Ki-67.25 Freeman et al1 noted antibodies against MCM proteins consistently identified a greater percentage of cells in cycle compared with antibodies against PCNA and Ki-67, findings that have been echoed at the gene expression level by others.25 Of interest is the observation by Ha et al25 that different primary tumors expressed varying amounts of MCM 3 mRNA perhaps accounting for the heterogeneous staining of tissue sections from these neoplasms. Whether a similar situation exists for cutaneous malignancies had not been studied. Immunohistochemical staining for MCM proteins potentially offers increased accuracy in elucidating the proliferative fraction within tumors than is available with conventional proliferative indexes.13 MCM protein expression is an early event in neoplasia allowing for the potential distinction of dysplastic cells from those demonstrating reactive changes, an often difficult process using morphologic criteria alone. Correlating MCM protein
754 Boyd, Shakhtour, and Shyr
J AM ACAD DERMATOL MAY 2008
expression and the biologic potential of different melanocytic neoplasms, as has been done with Ki-67 and PCNA, constitutes an area for further research. The results presented here demonstrate significant differences in MCM protein expression among melanocytic tumors. For pathologists involved in evaluating such neoplasms, categorizing those lesions with worrisome cytologic features as definitely malignant is both difficult and frustratingly common. The use of proliferation markers such as Ki-67 and PCNA has been used in assisting with that distinction but as mentioned above, have their shortcomings. It is hoped evaluation for MCM protein expression may help in the resolution of that quandary.
12. 13.
14. 15. 16.
17. REFERENCES 1. Freeman A, Morris LS, Mills AD, Stoeber K, Laskey RA, Williams GH, et al. Minichromosome maintenance proteins as biological markers of dysplasia and malignancy. Clin Cancer Res 1999;5:2121-32. 2. Rieger E, Hofmann-Wellenhof R, Soyer HP, Kofler R, Cerroni L, Smolle J, et al. Comparison of proliferative activity as assessed by proliferating cell nuclear antigen (PCNA) and Ki-67 monoclonal antibodies in melanocytic skin lesions. J Cutan Pathol 1993;20:229-36. 3. Kuwata T, Kitagawa M, Kasuga T. Proliferative activity of primary cutaneous melanocytic tumors. Virchows Arch A Pathol Anat 1993;423:359-64. 4. Fogt F, Vortmeyer AO, Tahan SR. Nucleolar organizer regions (AgNOR) and Ki-67 immunoreactivity in cutaneous melanocytic lesions. Am J Dermatopathol 1995;17:13-7. 5. Ramsay JA, From L, Iscoe NA, Kahn HJ. MIB-1 proliferative activity is significant prognostic factor in primary thick cutaneous melanoma. J Invest Dermatol 1995;105:22-6. 6. Kanter L, Blegaen H, Wejde J, Lagerlof B, Larsson O. Utility of a proliferation marker in distinguishing between benign nevocellular nevi and nevocellular nevus-like lesions with malignant properties. Melanoma Res 1995;5:345-50. 7. Boni R, Doguoglu A, Burg G, Muller B, Dummer R. MIB1 immunoreactivity correlates with metastatic dissemination in primary thick cutaneous melanoma. J Am Acad Dermatol 1996;35:416-8. 8. Sparrow LE, English DR, Taran JM, Heenan PJ. Prognostic significance of MIB-1 proliferative activity in thin melanomas and immunohistochemical analysis of MIB-1 proliferative activity in melanocytic tumors. Am J Dermatopathol 1998;20: 12-6. 9. Niezabitowski A, Czajecki K, Rys J, Kruczak A, Gruchala A, Wasilewska A, et al. Prognostic evaluation of cutaneous malignant melanoma: a clinicopathologic and immunohistochemical study. J Surg Oncol 1999;70:150-60. 10. Li LL, Crotty KA, McCarthy SW, Palmer AA, Kril JJ. A zonal comparison of MIB1-Ki67 immunoreactivity in benign and malignant melanocytic lesions. Am J Dermatopathol 2000; 22:489-95. 11. Bergman R, Malkin L, Sasbo E, Kerner H. MIB-1 monoclonal antibody to determine proliferative activity of Ki-67 antigen as
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an adjunct to the histopathologic differential diagnosis of Spitz nevi. J Am Acad Dermatol 2001;44:500-4. Alison MR, Hunt T, Forbes SJ. Minichromosome maintenance (MCH) proteins may be pre-cancer markers. Gut 2002;50:290-4. Korkolopoulou P, Givalos N, Saetta A, Goudopoulou A, Gakiopoulou H, Thymara I, et al. Minichromosome maintenance proteins 2 and 5 expression in muscle-invasive urothelial cancer: a multivariate survival study including proliferation markers and cell cycle regulators. Hum Pathol 2005;36: 899-907. Blow JJ, Dutta A. Preventing re-replication of chromosomal DNA. Nat Rev Cell Biol 2005;6:476-86. Maiorano D, Lutzmann M, Mechali M. MCM proteins and DNA replication. Curr Opin Cell Biol 2006;18:132-6. Stoeber K, Halsall I, Freeman A, Swinn R, Doble A, Morris L, et al. Immunoassay for urothelial cancers that detects DNA replication protein Mcm5 in urine. Lancet 1999;354:1524-5. Meng MV, Grossfeld GD, Williams GH, Dilworth S, Stoeber K, Mulley TW, et al. Minichromosome maintenance protein 2 expression in prostate: characterization and association with outcome after therapy for cancer. Clin Cancer Res 2001; 7:2712-8. Ramnath N, Hernandez FJ, Dong-Feng T, Huberman JA, Natarajan N, Beck AF, et al. MCM2 is an independent predictor of survival in patients with non-small-cell lung cancer. J Clin Oncol 2001;19:4259-66. Gonzalez MA, Pinder SE, Callagy G, Vowler SL, Morris LS, Bird K, et al. Minichromosome maintenance protein 2 is a strong independent marker in breast cancer. J Clin Oncol 2003; 21:4306-13. Scott IS, Morris LS, Bird K, Davies RJ, Vowler SL, Rushbrook SM, et al. A novel immunohistochemical method to estimate cellcycle phase distribution in archival tissue: implications for the prediction of outcome in colorectal cancer. J Pathol 2003; 201:187-97. Scott IS, Odell E, Chatrath P, Morris LS, Davies RJ, Vowler SL, et al. A minimally invasive immunocytochemical approach to early detection of oral squamous cell carcinoma and dysplasia. Br J Cancer 2006;94:1170-5. Kearsey SE, Labib K. MCM proteins: evolution, properties, and role in DNA replication. Biochim Biophys Acta 1998;1398: 113-36. Tanaka T, Knapp D, Nasmyth K. Loading of an Mcm protein onto DNA replication origins is regulated by Cdc6p and CDKs. Cell 1997;90:649-60. Williams GH, Romanowski P, Morris L, Madine M, Mills AD, Soeber K, et al. Improved cervical smear assessment using antibodies against proteins that regulate DNA replication. Proc Natl Acad Sci U S A 1998;95:14932-7. Ha SA, Shin SM, Namkoong H, Lee H, Cho GW, Hur SY, et al. Cancer-associated expression of minichromosome maintenance 3 gene in several human cancers and its involvement in tumorigenesis. Clin Cancer Res 2004;10:8386-95. Winnepenninckx V, Lazar V, Michiels S, Dessen P, Stas M, Alonso SR, et al. Gene expression profiling of primary cutaneous melanoma and clinical outcome. J Natl Cancer Inst 2006; 98:472-82. Ilmonen S, Hernberg M, Pyrhonen S, Tarkkanen J, AskoSeljavaara S. Ki-67, Bcl-2 and p53 expression in primary and metastatic melanoma. Melanoma Res 2005;15:375-81.
igmented lesions are commonly biopsied neoplasms and represent a significant percentage of dermatopathology specimens. Categorizing melanocytic tumors as to their biologic
P
potential occasionally constitutes a vexing problem. The increasing availability of newer technologies, including immunoperoxidase stains, have bolstered diagnostic precision and provided submitting
From the Departments of Medicine (Dermatology),a Pathology,b and Preventative Medicine,c Vanderbilt University. Funding sources: None. Conflicts of interest: None declared. Accepted for publication December 18, 2007. Reprint requests: Alan S. Boyd, MD, 3900 Vanderbilt Clinic, Nashville, TN 37232. E-mail: [email protected].
Published online February 13, 2008. 0190-9622/$34.00 ª 2008 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2007.12.026
750
Boyd, Shakhtour, and Shyr 751
J AM ACAD DERMATOL VOLUME 58, NUMBER 5
Abbreviations used: MCM: minichromosome maintenance mRNA: messenger RNA PCNA: proliferating nuclear cell antigen
clinicians with information regarding the potential long-term implications of their patients’ melanocytic lesions. Immunoperoxidase staining of tissue sections for Ki-67 and proliferating cell nuclear antigen (PCNA) allow evaluation of the proliferative index in melanocytic neoplasms with both diagnostic and prognostic implications.1-11 Recently, a group of proteins intimately involved in DNA replication termed ‘‘minichromosome maintenance’’ (MCM) proteins have been described.1,12-15 Detectable only in cycling cells, the presence of MCM proteins in other malignancies has been associated with increasing degrees of cellular atypia and poorer prognosis.13,16-21 Their use in distinguishing benign from malignant cutaneous melanocytic lesions has not previously been undertaken.
METHODS Ten benign nevi, dysplastic nevi, and primary cutaneous melanomas and 5 cutaneous melanoma metastases were randomly selected from specimens submitted to our dermatopathology division. Recombinant MCM protein 2 immunoperoxidase stains (ProEx C, Tripath Imaging, Burlington, NC) were obtained on each specimen as suggested by the manufacturer (Fig 1). Using high-power microscopy (3400), approximately 200 melanocytic cells were evaluated for MCM 2 staining in 5 separate fields per specimen. Fields without epidermis, adnexal structures, or inflammatory cells were preferentially selected to minimize mistakenly counting cells not of melanocytic lineage. The nonparametric rank-based Kruskal-Wallis one-way analysis of variance method was used to test the equality of population medians of percentage of positively staining cells among groups. In addition, the Bonferroni correlation was applied to adjust the multiple comparisons among study groups. All tests of significance were two-sided, and differences were considered statistically significant when P value was less than .05. Software (SAS, Version 9, Cary, NC) was used for all analyses.
RESULTS The number of cells per high-power field staining positively in the 4 groups is listed in Table I. Using the Kruskal-Wallis test, a significant difference was noted in comparing the 4 groups (P \.0001). When
the Bonferroni correlation was applied to all pairwise comparisons among these 4 groups, statistically significant differences were found between benign nevi and primary cutaneous melanoma, dysplastic nevi and primary cutaneous melanoma, benign nevi and cutaneous melanoma metastases, and dysplastic nevi and cutaneous melanoma metastases. A statistically significant difference was not found between primary cutaneous melanoma and cutaneous melanoma metastases.
DISCUSSION In eukaryotic cells, the initiation of DNA synthesis is a complex, multistep process involving the chronologic assembly at replication origins of initiation factors into prereplicative complexes.12 This results in specific chromatin regions becoming competent for DNA replication or ‘‘licensing’’ in the ensuing cell cycle S phase.12,22 Replication origins are formed initially when the origin replication complex sequentially bind cdc6 and cdt1 proteins.1,14 These proteins are believed to function as adenosine triphosphatases and cdt1’s putative role is to recruit a collection of polypeptides called MCM proteins. MCM proteins form an encircling hexamer around DNA strands before the initiation of replication. This process is restricted to late mitosis and MCM proteins must remain associated with the DNA through G1 and into the S phase.14 After mitosis, the MCM proteins, cdc6, and cdt1 are down-regulated and degraded leaving the origin replication complex intact at the replication origin. MCM proteins were initially discovered as factors supporting minichromosome preservation in the yeast Saccharomyces cerevisiae and are highly conserved across eukaryotic species.12,23 They comprise a group of 10 separate polypeptides with numbers 2 through 7 used in the hexamer binding at the origin replication complex. They presumably act as DNA helicases and are dissociated from chromatin after replication, thereby ensuring that each DNA region is replicated only once during a single cell cycle.12,14 MCM proteins are found in cycling cells but not in quiescent, differentiated, or senescent cells.1,13,17 MCM 2 through 7 are the most investigated of this protein group. MCM 1 is believed to regulate cdc6 expression and some of the MCM 2 through 7 genes.15 MCM 8 and 9 may have a role as DNA helicases and MCM 10 is thought to stabilize DNA polymerase and direct it to replicating chromatin.15 In normal tissues, MCM proteins are expressed by proliferative cells similar to that seen with Ki-6712 and may be appreciated early in premalignant cells.14 Immunohistochemical staining for MCM proteins is typically restricted to cell nuclei although faint cytoplasmic staining may also be seen.
752 Boyd, Shakhtour, and Shyr
J AM ACAD DERMATOL MAY 2008
Fig 1. Histologic features of melanocytic neoplasms. A and B, Benign intradermal nevus with immunohistochemical staining for minichromosome maintenance (MCM) 2. C and D, Dysplastic nevus with immunohistochemical staining for MCM 2. E and F, Primary cutaneous melanoma with immunohistochemical staining for MCM 2. G and H, Cutaneous melanoma metastasis with immunohistochemical staining for MCM 2. (A, C, E, and G, Hematoxylin-eosin stain; B, D, F, and H, immunoperoxidase stain; original magnifications: A, B, E to H, 3200; C and D, 3100.)
Evaluation of MCM proteins is believed to be more reliable in discriminating malignant from precancerous cells than PCNA and Ki-67.15 In malignant neoplasms MCM 2 and MCM 5 have been the most studied of the MCM proteins. MCM 2 is expressed more frequently than Ki-67 in normal and malignant breast tissue and MCM 2 expression is reportedly superior to and independent of histologic tumor grade, Ki-67 staining, and lymph node status in the prognosis of breast adenocarcinomas.19 In nonsmall cell carcinoma of the lung, MCM 2 tissue
staining is an independent prognostic factor in survival whereas Ki-67 expression is not.18 MCM 2 tumor expression is also an independent prognostic factor in survival of patients with prostatic adenocarcinoma.17 High-grade and advanced-stage bladder cancers demonstrate significantly greater tissue staining for MCM 2 and MCM 5 and are statistically associated with adverse patient outcome.13 Stoeber et al16 devised a novel MCM 5 immunofluorometric assay for use on urinary cellular sediment in patients with suggested urinary tract malignancies. Of
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VOLUME 58, NUMBER 5
perhaps the greatest use is the evaluation of cervical specimens, Williams et al24 stained cervical tissue smears for MCM 5 and cdc6 noting a ‘‘remarkably high specificity and sensitivity’’ to the expression of these proteins and the presence of atypical cells. Although PCNA2,3,9 and Ki-674-8,10,11 have been extensively evaluated in melanocytic neoplasms, to date, only a handful of investigations of MCM expression in benign and malignant mucocutaneous conditions have been conducted. Freeman et al1 evaluated biopsy specimens of normal-appearing skin (n = 5), psoriasis (n = 3), actinic keratosis (n = 1), Bowen’s disease (n = 5), and squamous cell carcinoma (n = 13) for both MCM 2 and MCM 5. When compared with those from normal-appearing skin, cell counts were significantly higher in Bowen’s disease and squamous cell carcinoma but not psoriasis. Increasing expression of these proteins was noted in well, moderate, and poorly differentiated squamous cell carcinomas. Staining was appreciated along the basal layer in normal-appearing skin, more than 50% of the epidermis in the actinic keratosis, and greater than 90% of the epidermis in Bowen’s disease. Similar results were obtained in an investigation of oral mucosal biopsy specimen.21 Lesions with moderate to severe cytologic dysplasia and those frankly malignant (squamous cell carcinoma) expressed significantly greater numbers of cells staining for MCM 2 than normal mucosa, benign keratoses, or mildly dysplastic lesions. Staining of oral smears from the same lesions showed similar findings. Ha et al25 evaluated MCM 3 messenger RNA (mRNA) and cellular protein expression in a wide variety of human cancers. Although some malignancies strongly demonstrated MCM 3 mRNA the melanoma cell line showed only modest levels. Interestingly, Ki-67 and PCNA mRNA were also evaluated and demonstrated reduced sensitivity and specificity compared with MCM 3. Immunoperoxidase staining for MCM 3 in 110 malignant tumors (4 from the skin, type not specified) showed ‘‘moderate to high levels of expression’’ in 93 samples, however, the authors were not more specific. In an elaborate study of cutaneous melanomas, Winnepenninckx et al26 identified 254 genes involved in activating DNA replication noting a statistically significant difference in gene expression between patients with metastatic disease and those without metastases. Included in this group were MCM 3, MCM 4, and MCM 6. When immunoperoxidase staining for these 3 proteins was performed on tissue sections each was expressed in significantly greater amounts in the tumors from patients with distant metastases. However, grading of positively stained cells was performed on a 4-level scale and not by counting individual cells.
Table I. MCM protein 2 staining characteristics in melanocytic neoplasms
Benign nevi Dysplastic nevi Primary cutaneous melanoma Cutaneous melanoma metastasis
Mean No. of positively staining cells/high-power field
SD
1.2 6.1 49.1 40.9
0.5 4.0 20.1 25.5
MCM, Minichromosome maintenance; SD, standard deviation.
Of interest is the diminished number of staining cells in the cutaneous melanoma metastases compared with primary cutaneous melanomas. This difference (P = .14) is only of marginal statistical significance and may have resulted from so few specimens evaluated. Rieger et al2 noted significantly greater PCNA and Ki-67 staining in melanoma metastases compared with primary cutaneous melanomas, whereas Ilmonen et al27 found no discernable trend between such groups for Ki-67 expression. Evaluation of tissue for MCM proteins offers an advantage over other cell proliferation markers such as PCNA and Ki-67 in that these proteins are required for the initiation of replication, representing the point of convergence of many signaling pathways involved in cell growth.1 They would, therefore, constitute better candidates for marking cells in cycle than transduction molecules or growth factor receptors. MCM protein expression is not associated with DNA repair as is the case with PCNA nor is it quiescent in resting cells still maintaining replication competence in contrast to Ki-67.25 Freeman et al1 noted antibodies against MCM proteins consistently identified a greater percentage of cells in cycle compared with antibodies against PCNA and Ki-67, findings that have been echoed at the gene expression level by others.25 Of interest is the observation by Ha et al25 that different primary tumors expressed varying amounts of MCM 3 mRNA perhaps accounting for the heterogeneous staining of tissue sections from these neoplasms. Whether a similar situation exists for cutaneous malignancies had not been studied. Immunohistochemical staining for MCM proteins potentially offers increased accuracy in elucidating the proliferative fraction within tumors than is available with conventional proliferative indexes.13 MCM protein expression is an early event in neoplasia allowing for the potential distinction of dysplastic cells from those demonstrating reactive changes, an often difficult process using morphologic criteria alone. Correlating MCM protein
754 Boyd, Shakhtour, and Shyr
J AM ACAD DERMATOL MAY 2008
expression and the biologic potential of different melanocytic neoplasms, as has been done with Ki-67 and PCNA, constitutes an area for further research. The results presented here demonstrate significant differences in MCM protein expression among melanocytic tumors. For pathologists involved in evaluating such neoplasms, categorizing those lesions with worrisome cytologic features as definitely malignant is both difficult and frustratingly common. The use of proliferation markers such as Ki-67 and PCNA has been used in assisting with that distinction but as mentioned above, have their shortcomings. It is hoped evaluation for MCM protein expression may help in the resolution of that quandary.
12. 13.
14. 15. 16.
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