| Abstract|| |
Background: Melanoma is quite a heterogeneous group of diseases of the skin. Prognostic markers of tumor behavior are important to precisely assign individual patients for appropriate treatment protocols. Aim: The aim of our first study was to investigate nucleophosmin expression in melanoma patients and to determine its relationship with the tumor characterictics and patient prognosis. Materials and Methods: We analyzed the immunohistochemical expression of nucleophosmin in 55 melanoma patients. The immunostaining pattern was classified into two groups: Diffuse nuclear and nucleolar relocalization. We also investigated the relationship between the expression of nucleophosmin and the clinicopathological parameters sucssh as Clark level, tumor thickness, stage, histological type, location, and survey. Results: In all cases the neoplastic cells were strongly positive for nucleophosmin (14 cases diffuse nuclear, 41 cases nucleolar relocalization). No correlation was demonstrated between the expression pattern of nucleophosmin and the clinicopathological parameters and survey. Conclusions: The implications of our results, nevertheless, are that the immunohistochemical detection of nucleophosmin is not a valuable tool for predicting the outcome of patients with melanoma or identifying subgroups of patients who may be at a higher risk.
Keywords: B23, malignant melanoma, nucleophosmin, prognosis
|How to cite this article:|
Calli AO, Sari A, Evcim G, Altinboga A, Sadullahoglu C, Ermete M. The enigmatic role of nucleophosmin in malignant melanoma: Does it have an effect?. Indian J Pathol Microbiol 2011;54:482-6
|How to cite this URL:|
Calli AO, Sari A, Evcim G, Altinboga A, Sadullahoglu C, Ermete M. The enigmatic role of nucleophosmin in malignant melanoma: Does it have an effect?. Indian J Pathol Microbiol [serial online] 2011 [cited 2020 Dec 1];54:482-6. Available from: https://www.ijpmonline.org/text.asp?2011/54/3/482/85079
| Introduction|| |
Melanoma, an infrequently seen tumor, accounts for approximately 4% of cutaneous malignacies. However, the death rate for malignant melanoma is still unacceptably high, accounting for 75% of deaths from skin cancer.  The latter Figure has declined somewhat in recent years (possibly due to the earlier detection of melanoma), but overall survival has not increased in a statistically significant manner.  There are many clinical and pathological factors that have been studied with regard to their influence on prognosis. The most important factors for predicting the outcome in patients with melanoma are the Breslow depth, Clark level, anatomic location, gender, tumor ulceration, and growth pattern. The introduction of sophisticated statistical techniques, such as multivariate analysis, has led to the delineation of histological criteria, with an independent predictive value, with respect to the prognosis. , New insights into the biology of melanomas and development of new biological markers to identify patients at risk for advanced disease, may have implications for treatment strategies. This has brought about a widespread interest in a variety of molecular tumor markers that may relate to the behavior of malignant melanoma (MM).
Nucleophosmin (NPM) also called B23, nutramin, and NO38 is a nucleolar multifunctional phosphoprotein that is tightly regulated during proliferation. Nucleophosmin/B23 regulates many cellular activities, including ribosome assembly/transport, cytoplasmic/nuclear trafficking, regulation of DNA polymerase alpha activity, centrosome duplication, regulation of the tumor supressor genes TP53, and p14AR. , It has been shown to bind nucleic acid, prevent protein aggregation via its chaperone activities, protect enzymes during thermal denaturation, and facilitate renaturation of chemically denatured proteins. ,, The great interest in NPM is due to its role in DNA repair and cancer. Recent reports indicate that nucleophosmin is downregulated during drug-induced apoptosis of cancer cells.  NPM is frequently overexpressed in many tumors, including tumors of the bladder, lung, colon, liver, stomach, ovary, and prostate, and one of the most frequent targets of genetic alterations in hematopoietic tumor. ,,,,
Contrary to the the numerous published reports dealing with nucleophosmin in human neoplasia, there exist only a small number of studies that determine nucleophosmin expression in melanoma cells. ,, To the best of our knowledge, the clinicopathological significance of nucleophosmine in malignant melanoma has not been fully explained. The aim of the present study is to first determine the impact of NPM expression and its correlation to the clinicopathological parameters and patient outcome.
| Materials and Methods|| |
Patients and Tissue Samples
This retrospective study consists of 55 patients diagnosed and treated for malignant melanoma in the Izmir Training and Research Hospital, between 2003 and 2009. Twenty-five cases were male and 30 were female; the mean age was 58.87 years (range 26 to 83 years). The traditional clinical pathological variables regarding patients (age and sex) and tumors (Breslow depth, Clark level, anatomic location, tumor ulceration, intra-tumoral lymphocytic infiltration, histological type) as well as dissemination and relapse information were obtained via a retrospective review. The stage of the tumor was determined based on the American Joint Committee on Cancer 2002, Revised Melanoma Staging system. 
The histological samples were fixed in buffered formalin, embedded in paraffin, and sectioned at 5 mm for histological examination. In each case, all the available hematoxylin and eosin stained sections were reviewed. A represantative block was chosen for immunostaining.
Immunohistochemical staining for NPM expression was performed using the peroxidase-labeled streptavidin-biotin technique. Thick sections of 5 mm were cut from the blocks, and the sections were incubated in a 60 o C oven for one night. After deparaffinization with xylene, the samples were dehydrated through graded concentrations of ethanol. Endogenous peroxidase activity was inactivated by 3% hydrogen peroxide in methanol for 30 minutes. The slides were heated in Ethylenediaminetetraacetic acid (EDTA), pH. 6.0, in a pressure cooker oven for 20 minutes, for antigen retrieval. After washing in a phosphate buffer solution (PBS), the tissue sections were incubated with a nucleophosmin (FC.61991 concentrated; ZYMED USA) diluted 1: 100 in PBS, followed by biotinylated antimouse immunglobulin G for 30 minutes, at room temperature. After washing in PBS, the avidin-biotin complex was then applied for 30 minutes and the reaction was visualized with diaminobenzidin (DAB). The slides were counterstained with Harris's hematoxylin and dehydrated through graded alcohols to xylene and finally mounted. Hepatoma that had been demonstrated previously to be strongly positive for NPM was used as a positive control. Immunostaining evaluation was performed independently by two investigators, without prior knowledge of the clinicipathological details. In all sections, a minimum of 1000 tumor cells were evaluated, to identify the immunostaining pattern.
The immunohistochemical staining of nucleophosmin was scored similarly, according to a previously published protocol.  All nucleophosmin positive cases were semiquantitatively categorized into two groups: Diffuse nuclear and nucleolar relocalization.
In this study, we used the Shapiro-Wilk test for each of the variables, for assessing normalcy. If the distribution of the variables was normal, then comparisons were made by using the parametric statistical methods. If the distribution of variables was not normal, the comparisons were made by using the nonparametric methods. In this study the distribution of variables was not normal, the correlation between nucleophosmin immunostaining pattern and the clinicopathological parameters was calculated by using the Spearman correlation coefficient. If the P value was less than or equal to 0.05 then the correlation coefficient was considered significant. The association between the immunostaining pattern and the clinicopathological parameters was tested by using the Chi-squared test. If the P value was less than or equal to 0.05 then the association was considered significant.
| Results|| |
The clinicopathological characteristics of the patients are summarized in [Table 1]. According to the pH. category, one sample was classified as pT1, seven samples were classified as pT3, 17 samples were classified as pT3, and 30 samples were classified as pT4. Nine patients were assigned as Localized Melanoma (Stages I and II), 12 patients as Regional Metastases (Stage III), and 34 patients as Distant Metastases (Stage IV). Based on the histopathological features, 19 patients were classified as superficial spreading melanoma, 14 patients as acral lentiginous melanoma, 17 patients as nodular melanoma, one patient as desmoplastic melanoma, one patient as nevoid melanoma, and one patient as animal type melanoma. The Clark's level ranged from III to V, seven patients had Clark level III, 29 patients had Clark level IV, and 19 patients had Clark level V. The median time follow-up was 24.91 months (range 7 to 60 months), with follow-up being complete in only 35 cases.
|Table 1: Nucleophosmin immunostaining pattern with different clinicopathologic variables|
Click here to view
The expression of NPM showed characteristic cellular localization. Two different nuclear immunostainings were observed: Nucleolar relocalization and diffuse nuclear. Diffuse nuclear staining revealed that the nuclei were totally homogeneously stained and lacked nucleolar staining [Figure 1]. In the nucleolar staining pattern, the expression of NPM was concentrated in the nucleoli with no staining detected in the nucleoplasm [Figure 2]a and b. The pattern of expression of nucleophosmin in 55 primary malignant melanoma was diffuse nuclear in 14 cases (25%) and nucleolar relocalized in 41 cases (75%).
|Figure 1: Diff use nuclear expression of NPM in a Nodular Malignant Melanoma (ABC method, orginal magnification x1000)|
Click here to view
|Figure 2: (a) Immunohistochemical staining demonstrates the nucleolar relocalization pattern of NPM in Malignant Melanoma (ABC method, orginal magnification x200, (b) orginal magnification x400, inset: Orginal magnification x1000)|
Click here to view
Our study found that there was no correlation between the expression of NPM and the clinicopathological parameters of the Clark level, stage, lymph node metastasis, age, tumor thickness, tumor location, and histological type. It also revealed no significant correlation between NPM expression and survival.
| Discussion|| |
Melanoma, the deadliest type of skin cancer, represents quite a heterogeneous group of diseases. Malignant melanomas diagnosed and treated at earlier stages are associated with much better prognosis and survival. Despite improvements in treatment approaches for melanoma, the prognosis of patients with advanced melanoma remains poor. Distant metastasis will develop in nearly 30% of the patients and the overall five-year survival rate for stage IV melanoma is 5-20%.  Identification of these aggressive tumors at an earlier stage would single out patients for early treatment, which may in turn improve their survival and quality of life. It is important to discover new biomarkers that will identify the melanoma patients who will progress to the advanced stage. Several studies had been undertaken in an effort to identify the prognostic capabilities of tumor-associated biomarkers in malignant melanoma. Various immunohistochemical markers with prognostic significance have not been found to improve upon a prognostic model that included tumor thickness, localization, and a mitotic rate.  Biological events and molecular changes in the progression of melanoma are indistinct. Prognostic markers of tumor behavior are important to assign individual patients for appropriate treatment protocols.
The nucleophosmin gene is a multifunctional nucleolar phosphoprotein that appears to be an important molecular element in the regulation of the nucleolar function for cellular differentiation and apoptosis in cancer cells.  Nucleophosmin can be thought of as a molecular chaperone that is primarily involved in ribosome biogenesis and centrosome duplication. The nucleoli constitute the sites of ribosome biogenesis, the synthesis and processing of a precursor rRNA (pre-rRNA), and coordinated assembly of pre-rRNAs with ribosomal proteins. ,,,
Nucleophosmin is predominantly localized in the granular region of the nucleolus. The intracellular localization of NPM is significantly altered during the cell cycle. NPM is a nucleolar protein that shuttles in and out of the nucleolus or between the nucleus and cytoplasm.  Various molecular alterations on the gene expression, such as, mutations, truncations, and changes in post-translational modifications might lead to the exchange of NPM between the nucleolus and the nucleoplasm. , The shuttling of NPM between the nucleolus and the nucleus is an essential event in the S-phase progression; when NPM export is inhibited by the nucleolar tumor suppressor ARF, the cells are arrested in G1.  As far as cellular stresses are concerned, NPM and ARF are shifted to the nucleoplasm where the competetive binding of MDM2 and NPM for ARF results in the shaping of the NPM-MDM2 and ARF-MDM2 binary complexes. MDM2 sequestration triggers the p53 pathway.  The overexpression of NPM breaks up the capability of ARF to activate p53 and induce growth arrest. On the other hand, NPM overexpression supresses the ARF function, while increasing its nucleolar localization.  Following DNA damage, NPM relocates from the nucleolus and is redistributed in the nucleoplasm. NPM may function at different levels in the global context of chromatin, rather than by specifically targeting broken DNA.  Nucleophosmin-1 (NPM1) mutations constitute a nuclear export signal (NES) motif. While wild-type NPM is predominantly localized in the nucleolus, the mutant of NES is mainly distributed in the nucleoplasm. Because of this, the NES mutant protein lacks the ability to be exported to the cytoplasm, and therefore, accumulates intensively in the nucleus.  In our study, 14 cases (25%) showed diffuse nuclear staining and 41 cases (75%) showed nucleolar relocalization.
The NPM is a complex molecule in its various isoforms and its biological role is tightly linked to its molecular structure and active domains. The gene NPM1 that encodes nucleophosmin (NPMI) is overexpressed, translocated or mutated in various malignancies. Thus, it is the overexpression or altered location or truncated forms that are associated with the disease. Alterations in the regulation of the NPM cellular traffic may contribute to tumorigenesis. An abnormal subcellular distribution of fusion and wild-type NPM proteins is a general characteristic of lymphomas and leukemias carrying NPM1 gene alterations.  Therefore, it is possible that the form of nucleophosmin B23, which is upregulated in melanoma, represents a post-translationally modified form, most likely reflecting enhanced phosphorylation in the tumor-derived cells.  It is also suggested that phosphorylation and expression of nucleosphosmin/B23 is correlated to cell cycle progression and cell survival, and probably reflects its corresponding regulation and function in melanoma. 
Several authors have asked for the possible role of NPM in the process of malignant transformation and tumor progression in various human tumors. ,,,,,,,, Our study shows that there is no significant correlation between NPM expression and the clinicopathological parameters and survey. This finding is in contrast with several previous reports, which show that NPM expression has been significantly associated with prognosis. ,,,,, Yun et al.,  in addition to PCR and Western-blot analysis, have found that immunohistochemical expression of NPM is in correlation with the clinical prognostic parameters in hepatocellular carcinomas. Mascoux et al.  have investigated the NPM expression patterns by immunohistochemistry and immunofluorescence in bronchial squamous cell carcinomas and its precursors, and have found that the nucleolar staining increased progressively with the severity of the dysplasia. Coutinho-Camillo et al.,  apart from PCR, have also investigated the immunohistochemical determination of NPM in oral squamous cell carcinomas and have found that NPM expression is correlated with local recurrence. Tsui KH have demonstrated that the overexpression of NPM, which was detected both by RT-PCR analysis and immunohistochemistry is associated with recurrence and prognosis in urothelial carcinomas of the urinary bladder. , Kikuta et al.  have shown that NPM expression is correlated with the clinical outcome and stage in Ewing's sarcoma. Their results, therefore, also suggest that evaluation of NPM expression may allow the idetification of poor prognosis in Ewing's sarcoma patients, who may benefit from highly effective treatment in the future.
Pathological staging, traditionaly based on the American Joint Committee on the Cancer Staging System and its subsequent modifications, remains the cornerstone of prognostic classification for melanoma. This is by far the most important prognostic parameter. , In this current study, the stage was found to be a statistically significant predictor of overall survival.
| Conclusion|| |
The implications of our results are that the immunohistochemical detection of nucleophosmin is not a valuable tool for predicting the outcome of patients with melanoma nor is it useful in identifying subgroups of patients who may be at a higher risk. The limitation of this preliminary study of nucleophosmine expression in the malignant melanoma is the small number of subjects. Further in-vitro and in-vivo investigations are needed to assess the role and the significance of the subcellular localization of NPM in malignant melanomas, and studies with larger series are required to better assess the prognostic role of NPM expression in malignant melanomas.
| References|| |
|1.||Melanoma and other skin cancers. In: Pazdur R, Wagman LD, Camphausen KA, Hoskins WJ, editors. Cancer Management: A Multidisciplinary Approach, 12 th ed. New York: CMP Media; 2009. |
|2.||MacKie RM. Melanoma and the dermatologist in the third millennium. Arch Dermatol 2000;136:71-3. |
|3.||Rivers JK, Ho VC. Malignant melanoma. Who shall live and who shal die? Arch Dermatol 1992;128:537-42. |
|4.||Rigel DS, Friedman RJ, Kopf AW, Silverman MK. Factors influencing survival in melanoma. Dermatol Clin 1991;9:631-42. |
|5.||Yun JP, Miao J, Chen GG, Tian QH, Zhang CQ, Xiang J, et al. Increased expression of nucleophosmin/B23 in hepatocellular carcinoma and correlation with clinicopathological parameters. Br J Cancer 2007;96:477-84. |
|6.||Colombo E, Bonetti P, Lazzerini Denchi E, Martinelli P, Zamponi R, Marine JC, et al. Nucleophosmin is required for DNA integrity and p19Arf protein stability. Mol Cell Biol 2005;25:8874-86. |
|7.||Yung BY. Oncogenic role of nucleophosmin/B23. Chang Gung Med J 2007;30:285-93. |
|8.||Grisendi S, Mecucci C, Falini B, Pandolfi PP. Nucleophosmin and cancer. Nat Rev Cancer 2006;6:493-505. |
|9.||Mascaux C, Bex F, Martin B, Burny A, Haller A, Paesmans M, et al. The role of NPM, p14arf and MDM2 in precursors of bronchial squamous cell carcinoma. Eur Respir J 2008;32:678-86. |
|10.||Coutinho-Camillo CM, Lourenço SV, Nishimoto IN, Kowalski LP, Soares FA. Nucleophosmin, p53, and Ki-67 expression patterns on an oral squamous cell carcinoma tissue microarray. Hum Pathol 2010;41:1079-86. |
|11.||Lee JH, Welch DR. Identification of highly expressed genes in metastasis-suppressed chromosome 6/human malignant melanoma hybrid cells using subtractive hybridization and differential display. Int J Cancer 1997;71:1035-44. |
|12.||Stessl M, Marchetti-Deschmann M, Winkler J, Lachmann B, Allmaier G, Noe CR. A proteomic study reveals unspecific apoptosis induction and reduction of glycolytic enzymes by the phosphorothioate antisense oligonucleotide oblimersen in human melanoma cells. J Proteomics 2009;72:1019-30. |
|13.||Bernard K, Litman E, Fitzpatrick JL, Shellman YG, Argast G, Polvinen K, et al. Functional proteomic analysis of melanoma progression. Cancer Res 2003;63:6716-25. |
|14.||Kim CJ, Reintgen DS, Balch CM. AJCC Melanoma Staging Committee. The new melanoma staging system. Cancer Control 2002;9:9-15. |
|15.||Thompson JF, Scolyer RA, Kefford RF. Cutaneous melanoma. Lancet 2005;365:687-701. |
|16.||Ostmeier H, Fuchs B, Otto F, Mawick R, Lippold A, Krieg V, et al. Can immunohistochemical markers and mitotic rate improve prognostic precision in patients with primary melanoma? Cancer 1999;85:2391-9. |
|17.||Lee HZ, Wu CH, Chang SP. Release of nucleophosmin from the nucleus: Involvement in aloe-emodin-induced human lung non small carcinoma cell apoptosis. Int J Cancer 2005;113:971-6. |
|18.||Fatica A, Tollervey D. Making ribosomes. Curr Opin Cell Biol 2002;14:313-8. |
|19.||Okuwaki M, Tsujimoto M, Nagata K. The RNA Binding Activity of a Ribosome Biogenesis Factor, Nucleophosmin/B23, Is Modulated by Phosphorylation with a Cell Cycle-dependent Kinase and by Association with Its Subtype. Mol Biol Cell 2002;13:2016-30. |
|20.||Lim MJ, Wang XW. Nucleophosmin and human cancer. Cancer Detect Prev 2006;30:481-90. |
|21.||Negi SS, Olson MO. Effects of interphase and mitotic phosphorylation on the mobility and location of nucleolar protein B23. J Cell Sci 2006;119:3676-85. |
|22.||Falini B, Nicoletti I, Bolli N, Martelli MP, Liso A, Gorello P, et al. Translocations and mutations involving the nucleophosmin (NPM1) gene in lymphomas and leukemias. Haematologica 2007;92:519-32. |
|23.||Brady SN, Yu Y, Maggi LB Jr, Weber JD. ARF impedes NPM/B23 shuttling in an Mdm2-sensitive tumor suppressor pathway. Mol Cell Biol 2004;24:9327-38. |
|24.||Lee C, Smith BA, Bandyopadhyay K, Gjerset RA. DNA damage disrupts the p14ARF-B23(nucleophosmin) interaction and triggers a transient subnuclear redistribution of p14ARF. Cancer Res 2005;65:9834-42. |
|25.||Korgaonkar C, Hagen J, Tompkins V, Frazier AA, Allamargot C, Quelle FW, et al. Nucleophosmin (B23) targets ARF to nucleoli and inhibits its function. Mol Cell Biol 2005;25:1258-71. |
|26.||Lee SY, Park JH, Kim S, Park EJ, Yun Y, Kwon J. A proteomics approach for the identification of nucleophosmin and heterogeneous nuclear ribonucleoprotein C1/C2 as chromatin-binding proteins in response to DNA double-strand breaks. Biochem J 2005;388:7-15. |
|27.||Falini B, Bolli N, Liso A, Martelli MP, Mannucci R, Pileri S, et al. Altered nucleophosmin transport in acute myeloid leukaemia with mutated NPM1: Molecular basis and clinical implications Altered traffic of nucleophosmin in AML. Leukemia 2009;23:1731-43. |
|28.||Tsui KH, Cheng AJ, Chang PL, Pan TL, Yung BY. Association of nucleophosmin/B23 mRNA expression with clinical outcome in patients with bladder carcinoma. Urology 2004;64:839-44. |
|29.||Tsui KH, Juang HH, Lee TH, Chang PL, Chen CL, Yung BY. Association of nucleophosmin/B23 with bladder cancer recurrence based on immunohistochemical assessment in clinical samples. Acta Pharmacol Sin 2008;29:364-70. |
|30.||Kikuta K, Tochigi N, Shimoda T, Yabe H, Morioka H, Toyama Y, et al. Nucleophosmin as a Candidate Prognostic Biomarker of Ewing's Sarcoma Revealed by Proteomics. Clin Cancer Res 2009;15:2885-94. |
Aylin Orgen Calli
Department of Pathology, Izmir Training and Research Hospital, Yesilyurt/Izmir
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]