Abstract
Aim: The evaluation of the prognostic role of nuclear pseudoinclusions (NPIs) and nuclear grooves (NGs) in UM. Patients and Methods: We examined the presence of NPIs and NGs in hematoxylin and eosin-stained tissue sections from 164 removed eyeballs with uveal melanoma (UM) and analyzed statistical relationships with clinical and pathological parameters and the long-term survival rate. Results: We observed NPIs in 38% and NG in 21% of all UM. The presence of NPIs was significantly positively correlated with epithelioid type, marked pleomorphism, and the presence of multinucleated giant cells, macro-nucleoli and multiple nucleoli. Patients with UM with NPIs had a significantly reduced overall survival rate (p<0.0001). The presence of NGs was significantly inversely correlated with marked pleomorphism, and the presence of multinucleated giant cells, macro-nucleoli and multiple nucleoli. Kaplan–Meier analysis demonstrated significantly better overall (p<0.01) and disease-free (p<0.05) survival rates for patients with NGs. Conclusion: The obtained results suggest that the presence of NGs in UM is associated with a better prognosis, as opposed to the presence of NPIs, which means the prognosis is worse.
Uveal melanoma (UM) is the most frequently occurring primary intraocular tumor in adult patients. Its prevalence varies depending on race and geographic latitude, and is highest amongst Caucasian (98% of all cases) and those at higher latitudes. In Mediterranean countries, this means two new cases per 1,000,000 inhabitants per year, whilst in Scandinavian countries this number is higher at 8-11. In the United States of America, there are approximately four new cases per 1,000,000 people per year on average (1-5).
Melanoma involves various parts of the uvea with varied frequency: 4-6% of cases are located in the iris, 6-9% in the ciliary body, with the choroid being the most frequent location at 85-90% (6, 7). The prognosis in UM depends on multiple factors – one of them being the size of the tumor (the largest diameter at the base and height). Larger tumors are characterized by poorer survival rates. Data analysis presented by Shields et al., comprising 8033 cases, showed that tumor increasing by 1 mm, increases the risk of metastases within 10 years by 5%. According to the largest base diameter, they divided the tumors into three categories: Small (0-3 mm), medium (3.1-8.0 mm) and large (>8 mm) (8). In these groups, 5-, 10- and 20-year mortality was 6%, 12% and 20%, 14%, 26% and 37%, and 35%, 49% and 67%, respectively (8, 9).
Another factor which negatively affects prognosis is the involvement of the ciliary body by UM (6, 8).
A wide range of histological factors have been linked to metastasis formation, such as the epithelioid cell type, intrascleral and extrascleral extension, high mitotic activity, optic nerve extension, vasculogenic mimicry patterns containing arcs with branching, closed vascular loops and vascular networks, as well as immune infiltration with an increased number of lymphocytes (mainly T-cells) and macrophages, which are associated with a poorer prognosis (6, 10-13).
Chromosome 3 monosomy, two or more copies of 1q, 6p and 8q, losses of 1p, 6q and 8p, and inactivation of BAP1 gene encoding BRCA1-associated protein 1 (located on chromosome 3) are related to a high risk of UM metastasis. In contrast, mutation of eukaryotic translation initiation factor 1A X-linked (EIF1AX) is associated with an excellent survival outcome (6, 7, 14, 15).
Local control of the tumor is very high (86-95%) and is obtained as a result of the application of various conservative treatment methods, such as brachytherapy, proton therapy, trans-pupillary thermotherapy, tumor endo- or exoresection, and a combination of the above methods. In the case of very large tumors, where the diameter of the base is larger than 20 mm, or the height exceeds 12 mm, and if the tumor significantly involves the optic disc, the best treatment method still remains the enucleation of the affected eye (16).
A high mortality rate, even reaching 50% on account of systemic dissemination of the cancer with lack of an effective treatment method, still remains a significant problem (13). In more than 90% of cases, metastases are located in the liver, in spite of the positive effects of local treatment. On account of these factors, the search for new prognostic and therapeutic factors which may assist in the treatment of patients with UM remains an important issue.
In various cancer types, pathologists look for cancer cell-specific morphological properties (including cellular nucleus) and attempt to evaluate these, as they may help in improving diagnosis and determining the prognosis. Among others, the shape of the nuclear envelope and intranuclear structures are taken into consideration. In this category of properties, the analysis frequently concerns nuclear pseudoinclusions (NPIs) and nuclear grooves (NGs). The presence of NPIs and NGs in the cellular nuclei is evaluated during cytological and histopathological assessments, most frequently after hematoxylin and eosin (H&E) staining.
NPIs are vacuoles in a cellular nucleus, limited by the nuclear membrane – they are formed by an intussusception of cytoplasm inside the nucleus. They do not have direct contact with the nucleoplasm. H&E staining allows one to see the eosinophilic material resembling cytoplasm in color or slightly paler shapes inside them. NPIs must be differentiated from true nuclear inclusions and pseudo-pseudoinclusions (‘bubbly’ nuclei).
The presence of NPIs is described in various types of tissues, including cancer tissues (most frequently in papillary thyroid carcinoma, meningioma, ductal hyperplasia of the breast, pituitary adenoma and lung adenocarcinoma), yet their etiopathogenetic and prognostic significance in oncology remains unclear (17-24).
Another feature of cellular nuclei, occurring in various tissues, are NGs, described as ‘coffee bean’ shape nuclei. This is the property of cellular nuclei consisting of clear longitudinal invaginations of the nuclear envelope bilayer observed after H&E staining. NGs may be indicative of the cancerous character of a lesion. NGs have been described in various types of cancer, such as papillary thyroid carcinoma (100% of cases), ependymoma (81% of cases), primary breast carcinomas (78% of cases), metastatic breast carcinoma (90% of cases) and in adult granulosa cell tumor (90% of cases) (25-27). However, NGs are also found in a normal cervical smear and benign bronchioalveolar lavage (28).
The presence of NPIs and NGs in the cells of examined tissues may also suggest their malignant character, as is the case in papillary thyroid carcinoma. Yet some authors call into question their significance in the differentiation between malignant and benign lesions (28, 29).
The available literature on the subject contains some individual reports on the presence of NPIs and NGs in UM. However, in none of them is there a relationship between their presence and the clinical picture of the tumor, its histopathological properties and patient survival rates. Therefore, we decided to analyze the presence of NPIs and NGs and their relationships with the above features (30, 31). To our knowledge, our report is the first to characterize the above issue.
Patients and Methods
The study group consisted of 164 patients with UM treated by primary enucleation at the Department of Ophthalmology and Ocular Oncology, diagnosed between 2002-2011. Patients were enrolled in the study based on the availability of their medical records and tissue specimens, which included paraffin blocks and histology slides. Comprehensive clinical data were retrieved from the archived medical records, and details of diagnostic and therapeutic procedures performed were sourced from the Ocular Oncology Outpatient Clinic, University Hospital. The Authors declare that this investigation was carried out following the rules of the Declaration of Helsinki of 1975 (revised in 2013) and this study was reviewed and approved by the Ethics Committee of the Jagiellonian University, Krakow, Poland (decision no. 122.6120.58.216), and the Wroclaw Medical University, Wroclaw, Poland (decision no. KB-500/2017).
The records were reviewed for clinical and pathological data including age, sex, affected eye, largest basal diameter and height of the tumor, tumor staging (pT and American Joint Committee on Cancer (AJCC) prognostic stage group (32), tumor location relative to the equator, ciliary body involvement, clinical tumor pigmentation and shape, concomitant glaucoma/retinal detachment, histological subtype, scleral/optic nerve infiltration, extraocular extension, as well as tumor necrosis. Additionally, detailed histological parameters, such as mitotic rate, presence of tumor-infiltrating lymphocytes, characteristics of nucleoli (presence, size and number), multinucleated giant cells and hemorrhage, as well as tumor cell pigmentation level were considered (32-34).
The largest basal diameter and height of the tumor were described in line with the AJCC guidelines (32) and staged according to the Collaborative Ocular Melanoma Study (COMS) (32-34).
Evaluation of NPIs and NGs. The presence of NPIs and NGs was evaluated in 164 H&E sections of primary UM. NPIs were defined as intranuclear vacuoles with the constituents of a color and morphological structure similar to that of the surrounding cytoplasm or paler. NGs were defined as longitudinal invaginations of the nuclear envelope. The presence of any NPIs or NGs in tumor cells finally categorized a case as positive.
Statistical analysis. Statistical analysis was performed using the R language and the survminer tool (available online: https://www.r-project.org/) (35, 36).
Overall survival (OS) was defined as the time period from the date of UM diagnosis until death date or the last follow-up and disease-free survival (DFS) as the time from finishing UM treatment until metastasis or the last follow-up. In order to determine the OS and DFS rates, Kaplan–Meier curves and the log-rank test were used; all analyses were carried out using the survival package for R. In order to determine the correlations between the presence of NPIs and NGs and continuous variables, the Wilcoxon two-sample test was used. The correlations between the presence of NPIs and NGs and binary variables were determined using Fisher’s exact test while the correlations with other categorical variables were determined using the chi-square test. A p-value below 0.05 was considered significant for all comparisons.
Results
The study group comprised 164 patients, with 84 women and 80 men, aged between 18 and 86 years (on average 59.7 years). The observation time was on average 374.7 weeks
(range=36-893 weeks). Other clinical and histopathological data of the tumors examined are presented in Tables I and II.
Summary statistics for the relationship between nuclear pseudoinclusions and intranuclear grooves in uveal melanoma cells and clinical parameters.
Summary of statistics for the relationship between nuclear pseudoinclusions and intranuclear grooves in uveal melanoma cells and histopathological parameters.
NPIs and NGs in UM. NPIs were noted in UM cells in 38% (63/164) and NGs in 21% (35/164) of all cases (Figure 1).
Cytomorphology of nuclear pseudoinclusions and nuclear grooves in uveal melanoma cells. A, B: Prominent nuclear pseudoinclusions (inset) in uveal melanoma cells. C, D: Nuclear grooves (inset) in uveal melanoma cells. Hematoxylin and eosin staining, main image: 400×, inset: 600×.
NPIs and NG – correlations with clinical parameters. Statistically significant correlations with clinical parameters were found only for NGs. Patients with NGs were significantly younger (p=0.02). The statistical analysis also showed a trend for the presence of NGs being inversely related to the degree of pigmentation (p=0.057).
There was no correlation between the presence of NGs and other evaluated clinical parameters, such as sex, involved eye, largest basal tumor diameter and thickness (by AJCC and COMS), stage, primary tumor shape, ciliary body involvement nor concomitant retinal detachment or glaucoma. There were no statistically significant correlations between the presence of NPIs and any analyzed clinical features.
Table I presents the relationship between NPIs and NGs in UM cells and clinical parameters.
NPIs and NGs – correlations with histological parameters. The presence of NPIs was significantly positively correlated with the mixed and epithelioid type of UM (p=0.0028), marked pleomorphism (p=0.00013), the presence of multinucleated giant cells (p=0.0043) and the presence of macronucleoli and multiple nucleoli (p<0.001 in both cases). Additionally, there was a positive correlation between the pigment content in tumor cells and the presence of NPIs (p=0.00003).
The presence of NGs was significantly inversely correlated with marked pleomorphism (p=0.025, lack of NGs in cases with marked histopathological pleomorphism), the presence of multinucleated giant cells (p=0.00054) and the presence of macronucleoli and multiple nucleoli (p<0.001 in both cases).
Table II presents relations between NPIs and NGs in UM cells and histological parameters.
The association of the presence NPIs and NGs with long-term survival. In the Kaplan–Meier analysis, the presence of NPIs was associated with significantly reduced overall survival (p<0.0001), however, this feature had no significant correlation with disease-free survival (p=0.1) (Figure 2).
Kaplan–Meier analysis of the prognostic impact of nuclear pseudoinclusions (NPIs) in patients with uveal melanoma. The presence of NPIs in melanoma cells was significantly associated with shorter overall survival (A) but not with disease-free survival (B).
The Kaplan–Meier analysis demonstrated that the presence of NGs was associated with significantly better overall (p=0.0016) and disease-free (p=0.029) survival (Figure 3).
Kaplan–Meier analysis of the prognostic impact of intranuclear grooves (NGs) in patients with uveal melanoma. The presence of NGs in melanoma cells was significantly associated with shorter overall survival (A) and disease-free survival (B).
Discussion
Histopathological diagnostics of tumors is based on the evaluation of differences between the structure of a normal tissue from which the tumor developed and the tumor tissue, as well as evaluations of the detailed morphology of analyzed cells. Changes in cell appearance which might be indicative of its potential for cancerous transformation are comprised of the morphological parameters of the cellular nuclei: Changes in their morphology may be a sign of a commencing or ongoing malignant transformation. These may consist of the formation of long inward folds of the nuclear envelope (NGs) and spherical invaginations of cytoplasm projecting partially into the nucleus (NPIs). The presence of NPIs and NGs is a typical feature of some tumors and their presence is important for pathologists in proper diagnosis, for example in papillary thyroid carcinoma and meningioma (19-22, 37-39).
In our study we decided to evaluate whether the presence of NPIs and NGs is indicative of a connection with clinical and histological features and long-term survival in patients diagnosed with a UM.
We observed the presence of NPIs in 38% (63/164) and NGs in 21% (35/164) of all UM cases, and the tumors involved mainly the ciliary body and choroid, occasionally also involving the iris. An interesting issue is that among our patients, both of these features occurred at the same time in only 9/164 cases, which makes up 5%. This might suggest that the presence of NPIs or NGs may have unrelated prognostic significance in UM.
There are only a few articles reporting the presence of NPIs and NGs in UM (31, 40-42). The first report concerning the presence of NPIs in UMs was presented in 1977 by Lommatzsch et al., whilst detailed characteristics of these lesions in iris melanoma was made in 1980 by Sunba et al. (31, 40). They noted that similar structures were also observed in the melanoma of the ciliary body and the choroid, but these results were not published. In our study, the presence of NPIs was significantly positively correlated with the epithelioid type, marked pleomorphism, the presence of multinucleated giant cells and the presence of macronucleoli and multiple nucleoli, as well as histopathological properties indicative of a poorer prognosis. Additionally, there was also a positive correlation between the pigment content in tumor cells and the presence of NPIs. Importantly, patients with UM in whom NPIs are present in the tumor cells had significantly reduced overall survival (p<0.0001). Our observations in the case of NPIs differ from those published by Sunba et al., who suggested that the presence of NPIs in UM may point to a more positive course of the disease. In the above report, NPIs were present in 60 (32%) cases out of 190 iris melanomas, of which 57 were of the spindle-cell type (31). It must be observed, however, that in their study, only iris UM cases were evaluated, and the majority of them were of the spindle-cell type, which is a priori associated with a better prognosis. Our analyses concerned mostly tumors involving the ciliary body and the choroid, which have poorer prognoses (6, 31). Additionally, we observed that NPIs were significantly more often noted when epithelioid cells were present, i.e. in the mixed type and in the epithelioid-cell type UM (p=0.0028). A similar observation concerning adverse prognosis in the case of the presence of NPIs in the cells of renal cell carcinoma was made by Ju-Han et al. They noted that the presence of a high number of NPIs may be an independent indicator of poor prognosis in renal cell carcinoma (38).
Another features of the nuclei of cancer cells which may play a role in the diagnostic process is the presence of NGs. In our study NGs were found in 21% (35/164) of all UM cases. Davila et al. in 1998 (41) and Kashyap et al. in 2002 (42) found the presence of NGs in individual cases of choroidal melanoma (2/9 and 1/1 respectively). They observed NGs only in spindle-shaped tumor cells. Likewise, in our cases, NGs had a tendencies for a more frequent occurrence in the spindle-cell type, which had a better prognosis.
Analysis of the presence of NGs with the clinical features in UM did not present any significant correlation, except that patients with NGs were younger (p=0.02) and in situations when the tumor was located anteriorly from the eye equator, i.e. with the involvement of the ciliary body (p=0.038). Histopathological features are a different issue. The presence of NGs was significantly inversely correlated with marked pleomorphism (we did not observe a single NGs-positive case with marked pleomorphism), the presence of multinucleated giant cells and the presence of macronucleoli and multiple nucleoli. Additionally, when NGs were present in the tumor cells in UM cases, the Kaplan–Meier analysis demonstrated significantly better overall (p<0.01) and disease-free (p<0.05) survival. These results would suggest that the presence of NGs in UM cells speaks in favor of a better prognosis, as opposed to the presence of NPIs, which is associated with the presence of histopathological properties associated with a poorer clinical outcome.
Several reports have indicated that in UM, the risk of metastasis increases with increased tumor pigmentation (5, 43). In our analyzed cases, we observed that the frequency of cases with NPIs significantly increased with the increase of melanin in the tumor cells (p=0.00003). However, the situation is completely different in the case of NGs because the statistical analysis showed a converse trend (p=0.057) indicating the presence of NGs was more frequent in amelanotic lesions.
In 2014, Fisher presented interesting theories describing the formation of NPIs and NGs (44). He believes that lesions like NPIs and NGs may have some genetic origin (develop in a near-diploid, genetically stable background) and that their formation is associated with tyrosine receptor kinase genes and probably with B-Raf proto-oncogene, serine/threonine kinase (BRAF) gene mutations, as is the case in papillary thyroid carcinoma, and also in melanoma and Langerhans cell histiocytosis. Additionally, somatic missense mutations (402C»G) at C134W (amino acid position 134) in the oncogene Forkhead box protein L2 (FOXL2) may be related to NGs in adult granulosa cell tumors of the ovary (44). What remains especially interesting is the fact that the FOXL2 gene is located on chromosome 3, disorders of which (monosomy) are found in more than 50% of UMs (44-46).
Analysis of the connection between the presence of the FOXL2 mutation and the mutations of other genes encoding transcription factors in UM requires further research.
It is beyond all doubt that the study of the significance of the presence of NPIs and NGs in UM requires further research carried out on a larger patient group, including of iris melanoma. Certainly, the relationship between NPIs or NGs and such factors as BAP1 status, copy number changes in chromosomes 3 and 8q and sequencing of BAP1 and EIF1AX and should be analyzed and also the statistical analysis should be extended with multivariate statistical models with reference to molecular analyses, clinical and histopathological features (47). Further studies are also necessary to find the causes of NPI and NG formation and the mechanism involved because the results of such research might cast some light on the role performed by these structures.
It would be also interesting to analyze melanocytic nevi with respect to the presence of NPIs, NGs and such factors as BAP1 status, copy-number changes in chromosomes and different molecular disturbances, as was done in the case of benign pigmented skin lesions (48, 49). However, this task will be by no means simple as the study material may only be obtained either as a result of eyeball enucleation, resection of tumor within the iris, ciliary body or anterior choroid and fine-needle biopsy – none of which are standard procedures in uveal melanocytic nevi.
Conclusion
To our knowledge, our study is the first to present such an extensive analysis of the presence of NPIs and NGs in UM in relation to the clinical and histological properties of this tumor and its prognosis.
We have confirmed the presence of NPIs and NGs in UM. The presence of NPIs appears to be related to the histopathological features of UM which are associated with poorer prognosis, which translates into significantly reduced overall survival. In contrast, NGs co-exist with cell morphological features which denote better prognosis and their presence also correlated with significantly better overall and disease-free survival. Comprehensive and large-scale studies should follow this preliminary report to evaluate the prognostic status of NPIs and NGs in UM cases and the nature and origin of these nuclear features.
Acknowledgements
This research was partially supported by EU grant “UMCure2020: New Therapies for Uveal Melanoma” no. 667787”(A.M., M.E. and B.R-D.). P.B was financially supported by the Polish National Science Centre under Opus Grant number 2017/27/B/ST6/0130. P.D.’s research was financed through a statutory subsidy by the Polish Minister of Science and Higher Education as a part of grants ST.B130.18.030 and SUB.B130.20.022 (record numbers in the Simple system).
Footnotes
Authors’ Contributions
A.M.: Conceptualization, methodology, investigation, resources, data curation, writing - original draft; P.D.: conceptualization, methodology, investigation, resources, data curation, writing - review & editing, visualization, project administration, funding acquisition; P.B.: software, formal analysis, visualization, funding acquisition; J.O.H.: investigation, resources; E.M.: methodology, resources; writing - review & editing, funding acquisition; B.R.D.: investigation, resources, supervision; writing - review & editing; funding acquisition. All Authors reviewed the article and approved the submitted version.
Conflicts of Interest
The Authors declare no conflicts of interest.
- Received November 30, 2020.
- Revision received December 18, 2020.
- Accepted December 21, 2020.
- Copyright© 2021, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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