Article Text
Abstract
Background: Morules have been reported in pulmonary blastoma (PB), well differentiated fetal adenocarcinoma of the lung (WDFA), and uterine endometrioid carcinoma (EC), and rarely in other carcinomas. β Catenin gene mutation has been associated with morule formation.
Aims: To compare and clarify the cellular characteristics of morules in carcinomas in various organs and show that morules are distinct from epithelial cellular nodules.
Methods: Twenty tumours were studied: two PBs, three WDFAs, three papillary lung adenocarcinomas, 11 ECs, and one papillary thyroid carcinoma. Numerous epithelial cell, oncofetal, and neuropeptide antibodies were used for immunohistochemistry. β Catenin gene mutation was investigated.
Results: Morules in PBs and ECs were uniform cell clusters distinct from squamous differentiation. All were immunonegative for epithelial cell and oncofetal antigens, but those in ECs were positive for neurone specific enolase γ (NSEγ). Synaptophysin, encephalin, and somatostatin were sporadically immunopositive in PB morules. Morules were not seen in the other carcinomas and WDFAs, although morule-like features closely resembling morules histopathologically were seen. These were positive for epithelial cell and oncofetal antigens, and showed squamous differentiation. Their nuclei were more atypical and slightly larger than those in morules. Morule-like features were seen in WDFAs. β Catenin gene mutation was demonstrated in one EC and PB, and in two WDFAs.
Conclusion: Morules were non-epithelial cell clusters showing neuronal differentiation. There were two types: endometrioid type, expressing NSEγ, and blastoma type, expressing neuropeptides. In contrast, similar morule-like features were epithelial nodules. Although the number of cases was small, the presence of morules showed no clear prognostic correlations.
- EC, endometrioid carcinoma
- HPV, human papillomavirus
- NSEγ, neurone specific enolase γ
- PB, pulmonary blastoma, PCR, polymerase chain reaction
- WDFA, well differentiated fetal adenocarcinoma
- morules
- morule-like features
- squamous differentiation
- β catenin gene
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- EC, endometrioid carcinoma
- HPV, human papillomavirus
- NSEγ, neurone specific enolase γ
- PB, pulmonary blastoma, PCR, polymerase chain reaction
- WDFA, well differentiated fetal adenocarcinoma
Morules have been reported in well differentiated fetal adenocarcinoma of the lung (WDFA) and in pulmonary blastoma (PB),1–6 and it has also been reported that morules show neuroendocrine differentiation. Morule formation in such tumours has also been reported to be associated with β catenin gene mutation.4–6 β Catenin is a submembranous component of the cadherin mediated cell to cell adhesion system. Previously, Sekine et al reported that β catenin gene mutation was found in PBs (two of two cases) and WDFAs (three of three cases).7 Saegusa and Okayasu6 reported that this gene mutation is present in 84.6% of uterine and ovarian endometrioid carcinomas with morules. Furthermore, Paracios and Gamallo8 reported mutation in five of 11 cases of ovarian endometrioid carcinoma, including one borderline tumour.
“The term squamous metaplasia is now used only in the case of benign lesions, whereas squamous differentiation is used for carcinoma and benign lesions”
In contrast, Fornelli and colleagues9 reported a rare case of bronchioloalveolar carcinoma with morule-like features, in which the morule-like nodules were composed of alveolar cells. Separately, morules have been seen in uterine endometrioid carcinoma (EC) and also in benign endometrial lesions, such as hyperplasia and chronic endometritis, and very rarely in thyroid carcinoma10 and colonic adenoma.11 It is generally thought that they are peculiar squamous cell foci, categorised as relatively immature squamous metaplasia or squamous differentiation cell clusters.12,13 Morules consist of uniform spindle or round cells, and show some optically clear nuclei.4
Formerly, the term squamous metaplasia was used both in adenocarcinoma cases and also in benign lesions, such as chronic inflammation and hyperplasia of the endometrium and other organs, but recently the term squamous differentiation has also become common.12,13 The term squamous metaplasia is now used only in the case of benign lesions, whereas squamous differentiation is used for carcinoma and benign lesions. In addition, it is known that squamous differentiation tissue in carcinomas is usually well differentiated and is associated with a relatively good prognosis.12
In our previous report,14 we showed that morules in uterine EC and also in benign endometrial lesions are completely different from squamous differentiation cell clusters: they did not react with antibodies to epithelial cell antigens, including involucrin, epithelial membrane antigen, and cytokeratins, but were positive for neurone specific enolase γ (NSEγ), and in a few cases somatostatin and acetylcholine esterase. Furthermore, neither oncofetal nor proliferative cell markers were demonstrated in the morules. Therefore, endometrial morules were thought to be neuroectodermal-like cell clusters or nodules. They were not infected with human papillomavirus, (HPV), whereas some of the true squamous differentiated tissue was associated with HPV infection. Detailed histological examination of the morules in various organs is needed to clarify their biological characteristics. In this report, we determine the characteristics of morules in carcinomas in various tissues.
MATERIALS AND METHODS
Samples
Eleven samples of EC with morules were obtained from 106 cases of uterine EC treated in 2003 at the department of gynaecology and obstetrics, Ryukyu University Hospital, Japan. Three samples of papillary carcinoma of the lung with morule-like features (three of 577 cases; 0.52%, 69, 67, and 74 year old women), one sample each of PB (39 year old woman), and one sample of WDFA (31 year old woman) were from National Okinawa Hospital, Okinawa, Japan. They were drawn from 1592 cases of lung cancer, which included the 577 cases of papillary carcinoma, treated there in the period 1984–2004. One more case of PB (42 year old man; obtained 15 years before) was from Okinawa Prefectural Naha Hospital, Japan. One sample of thyroid carcinoma with morule-like features (one of 141 cases of papillary carcinoma; 0.71%) was obtained from 208 cases of thyroid carcinoma (141 papillary carcinomas, 65 follicular carcinomas, and two others) investigated in our laboratory in the same period 1984–2004. Two samples of WDFA with morules were from Toyama City Hospital, Toyama, Japan. Five samples each of squamous cell carcinoma and adenosquamous carcinoma of the lung from National Okinawa Hospital, Japan were used as controls. A further three cases each of ameloblastoma, neuroblastoma (both adrenal and olfactory cases), nephroblastoma, and medulloblastoma, and one case each of mixed germ cell tumour of the testis (14 year old boy) and yolk sac tumour of the ovary (20 year old woman) from Ryukyu University Hospital, Japan were examined.
Morphological examination
The tumours were fixed in 10% phosphate buffered formalin and routinely processed in paraffin wax. Haematoxylin and eosin, phosphotungstic acid haematoxylin, silver impregnation, and periodic acid Schiff staining were carried out on 4 μm thick sections. For the immunohistochemical staining, 4 μm thick dewaxed sections were pretreated with 3% H2O2 for 20 minutes, washed, and blocked with non-immune goat serum for 30 minutes. Table 1 lists the antibodies used. Immunohistochemistry was performed using a microwave oven heating antigen retrieval method. Briefly, sections were heated in 10mM sodium citrate buffer (pH 6.0) for three five minute cycles using a microwave oven. To clarify the histological characteristics of the morules, a variety of antibodies (antibodies related to epithelial cell antigens including squamous cell markers, such as cytokeratins, involucrin, and oncofetal protein; proliferative cell markers; and nerve cell and neuropeptide related antigens) were used.
After incubation with the primary antibodies, the sections were incubated with biotinylated secondary antibody with avidin and biotinylated horseradish peroxidase complex (Dako, Kyoto, Japan). 3,3′-Diaminobenzidine (Dako) was used as a chromogen. As negative controls, non-immunised sheep serum or distilled water was used instead of antibodies. Furthermore, biotin containing intranuclear inclusions were detected by peroxidase labelled avidin.
In situ hybridisation kits for HPV types 6, 11, 16, 18, 30, 31, 33, 35, 45, 51, and 52 (Dako, Carpinteria, California, USA) were used for the detection of HPV. In situ hybridisation was carried out according to the manufacturer’s instructions.
Table 2 summarises the results of immunohistochemical investigations, including the results of controls.
Detection of β catenin gene mutation
DNA samples were routinely prepared according to the methods described by Impraim et al.15 Briefly, the morules were microdissected from 15 μm dewaxed sections under the light microscope. After proteinase K (Merck, Tokyo, Japan) treatment, DNA was extracted using the phenol/chloroform method. The DNA was precipitated with three times the volume of 100% ethanol at −20°C. The β catenin gene was amplified by the polymerase chain reaction (PCR). The PCR primer set with sense 5′-ATGGAACCAGACAGAAAAGCG-3′ and antisense 5′-CAGGATTGCCTTTACCACTCA-3′ was used according to the method of Paracios and colleagues8 and Kajino et al.16 PCR products of 200 bp at exon 3 where the mutations were reported5,8,16 were analysed using the LI-COR DNA sequencer, Model 4200 (LI-COR Inc, Lincoln, Nebraska, USA).
RESULTS
Morphological findings
The morules consisted of uniform spindle shaped or round cells with faint eosinophilic cytoplasm and small round nuclei. They filled the lumen of the glandular structures of the carcinomas, and seemed to be formed from the glandular epithelium. Occasionally, they were formed directly from stromal tissue, and had pushed up into the epithelial carcinoma cells (figs 1A, 2A) or intermingled with them. There were no nuclear mitoses. Morules in the pulmonary blastoma showed some optically clear nuclei (fig 1B, C), although the shapes of the cells and nuclei were similar to those in the ECs. The two PBs consisted of EC-like components with morules and exhibited primitive mesenchymal proliferation.
Although we examined three cases each of nephroblastoma, neuroblastoma, medulloblastoma, and ameloblastoma, and one case each of mixed germ cell tumour of the testis and ovarian yolk sac tumour, no morules were found.
In three cases of WDFA of the lung cellular nodules closely resembling morules were found (fig 1D). However, the nuclei were more atypical and slightly larger than those of the morules seen in PB. Small numbers of optically clear nuclei were found in these cellular nodules. They filled the lumen of the glandular structure of the carcinoma and were formed from glandular cancer cells. We refer to them below as morule-like features.
Many morule-like features were found in papillary carcinoma of the thyroid (fig 1E). The tumour cells in the three cases of papillary carcinoma of the lung showed a glandular proliferation with a papillary pattern along with fibrovascular cores, and the tumours were diagnosed as typical papillary adenocarcinomas of the lung. Fetal adenocarcinoma and endometrioid carcinoma patterns were not demonstrated. There were considerable numbers of morule-like nodules, which were rounded, and solid epithelial cell collections consisting of squamoid spindle shaped cells with faint eosinophilic cytoplasm and small round nuclei (fig 1F). However, neither nuclear mitotic figures nor optically clear nuclei were demonstrated.
The morule cells did not react with antibodies to epithelial cell markers (figs 1A, 2A). The epithelial cancer cells, which were stained clearly with antibodies to epithelial cell antigens, were pushed up by the growth of morules into the glandular lumens and were found on the margin of the morules (figs 1A, 2A). Cancer cells immunopositive for the epithelial antigens, carcinoembryonic antigen, and other tumour markers were occasionally intermingled with morules, and were found in them under certain cutting conditions. Although the morules in PB did not react with antibodies for epithelial markers or surfactant (apolipoprotein 1), they were positive for chromogranin, synaptophysin, encephalin, and somatostatin (fig 2B). However, NSEγ was not positive in morules in this PB case. Furthermore, carcinoembryonic antigen, p53, proliferative cell markers such as proliferative cell nuclear antigen and c-kit were not positive. β Catenin staining was not seen in the nuclei, although a few cancer cells were positive at the cell membrane. In contrast, the morules in endometrioid carcinoma were positive for NSEγ (fig 2C), although neither neuropeptides nor oncofetal antigens were demonstrated.
In contrast, epithelial cell markers, cytokeratins, involucrin, and epithelial membrane antigen were positive in the morule-like features of three cases of WDFA (fig 1D), in three cases of papillary carcinoma of the lung (fig 2D), and in one case of thyroid carcinoma, in addition to the control squamous cell carcinoma and squamous cell carcinoma components of the adenosquamous carcinoma of the lung. In the morule-like features of thyroid carcinoma, proliferative cell antigens and oncofetal antigens such as blood group B (data not shown) were strongly positive. Furthermore, in WDFAs of the lung, morule-like features were not positive for endocrine peptides.
HPV DNA was not found in either the morules or the morule-like features.
Sequence analysis of β catenin gene
A heterozygous substitution mutation of the β catenin gene was demonstrated at the glycogen synthase kinase 3 phosphorylation site in the morules of one case each of PB (Glu34Val) and EC (Ser33Cys) (fig 3A), and in two cases of WDFA of the lung (Ser33Cys) (fig 3B). The mutation was not seen in the other ECs and WDFA of the lung (fig 3C). The β catenin gene was not analysed in the thyroid carcinomas and one case of PB because DNA could not be obtained.
DISCUSSION
An endocrine cell derivation of morules in PB and adenocarcinoma of the fetal lung has been reported previously, where morules are composed of argyrophilic cells and neurosecretory peptides, such as somatostatin and encephalin positive cells.1–5 The morules in the EC-like component in our PB cases also showed similar neuroendocrine differentiation, with optically clear nuclei. The optically clear nuclei in morule cells were found to be rich in biotin, as reported previously.4,5 Moreover, morules were distinct from pulmonary neuroendocrine cell hyperplasia, which was found at the bronchiolar wall, and often replaced the bronchiolar epithelium. PB is a very rare tumour, and shows a unique combination of a primitive glandular epithelium resembling EC with morules and primitive mesenchymal proliferation.
Morules are occasionally seen in uterine EC. They have been considered to be immature squamous cell nodules. We have recently reported that these morules are completely different from squamous differentiation tissue, and that they do not react with epithelial cell markers, but are positive for NSEγ.14
In contrast, morule-like features are seen in association with papillary carcinoma of the lung and thyroid in rare instances, and with WDFAs. Morule-like features are distinguishable both histologically and immunohistochemically from morules in PB of the lung and ECs. Although β catenin gene mutation has been reported in morules,5,7 this was not found in our cases with morule-like features. Miyake et al also reported the absence of this mutation in thyroid carcinomas.17 β Catenin gene mutation was seen in one case each of EC and PB, and in two WDFAs of the lung. In previous reports,5,14 and in our present study, both wild-type and mutated alleles were noted. There was no clear association between β catenin gene mutation and morule formation in EC, but there was an association in PB, as reported by Nakatani et al and Sekine et al.5,7
To the best of our knowledge, there have been no previous reports of morule-like features as distinct from morules associated with either papillary carcinoma, WDFA of the lung, or thyroid papillary carcinoma. Fornelli et al reported a morule-like feature associated with bronchioloalveolar carcinoma, which showed a strong positive reaction to cytokeratin AE1/AE3, and was negative for chromogranin and synaptophysin.9 The peculiar morule-like features were considered to be multiple foci of nodular growth of cancer cells. Care should be taken to distinguish clearly between morule-like features and actual morules.
“There was no clear association between β catenin gene mutation and morule formation in endometrioid carcinoma, but there was an association in pulmonary blastoma”
Morule formation has been reported in WDFA of the lung.1 However, morules were not found in the three cases reported here. Biotin containing optically clear nuclei were found sporadically in the morule-like features in our carcinoma cases. Such nuclei have been demonstrated previously in endometrial glands during gestation, and in morule-like features in a colonic tubular adenoma,11 and were thought to be related to sex hormone functions18 and hyperglyconeogenesis.19
Although it is well known1,3,4,16 that morules are found in PB and uterine EC, and occasionally in benign endometrial lesions, such as chronic endometritis and hyperplasia, the reason for their formation is unclear. Based on our study, it is possible that morules associated with EC that express NSE γ2,14, and in a few cases acetylcholine esterase, are formed in response to the contraction of the uterus as pressure receptor-like cell clusters. However, the morules found in PBs were immunopositive for neuropeptides. Their function is probably different from those in the endometrium. It might be useful to classify morules into two groups: neuroendocrine type (blastoma type), which express neuropeptides, and neuroectodermal type (endometrioid type), which express NSEγ and/or acetylcholine esterase.
Take home messages
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Morules in pulmonary blastoma and uterine endometrioid carcinoma were non-epithelial cell clusters showing neuronal differentiation
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There were two different types of morule: endometrioid-type morules, which expressed neurone specific enolase γ, and blastoma-type morules, which expressed neuropeptides
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Morule-like features, which closely resemble morules histopathologically, were seen in well differentiated fetal adenocarcinoma of the lung and other carcinomas, and were found to be epithelial nodules
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Although the number of cases was small, the presence of morules showed no clear prognostic correlations
However, there were only two pulmonary blastomas, and the biological characteristics and functions of the morules are still obscure. In the lung tumours analysed here, the morules were negative for NSE, but Mardini et al reported that their samples were positive for NSE and showed carcinoid-like features.2
Furthermore, the squamoid nodules previously reported19–21 in pancreatoblastoma might be classified as morules, and they were immunonegative for epithelial cell markers.
In addition, there was no clear association between HPV infection and morules or morule-like features, although HPV had been postulated to be a possible cause of squamous differentiation.14 In conclusion, the morules were non-epithelial cell clusters showing neuronal differentiation. In contrast, similar morule-like features were indeed epithelial nodules. Adenocarcinomas with morules were different from those with squamous differentiation foci, and there seemed to be no clear prognostic correlation with the presence of morules in carcinomas, although the number of cases is too small to be given statistical weight.