Morules in endometrial carcinoma and benign endometrial lesions differ from squamous differentiation tissue and are not infected with human papillomavirus

BACKGROUND: Squamous differentiation/squamous metaplasia is often associated with endometrial adenocarcinoma and benign lesions, such as endometrial hyperplasia and chronic endometritis. Morules have distinct histological characteristics, and are referred to as squamous metaplasia or squamoid metaplasia. AIM: To focus on the histological characteristics of morules and clarify the difference between morules and squamous differentiation. MATERIALS/METHODS: Twenty endometrioid carcinomas with morules or squamous differentiation, five adenosquamous carcinomas, and eight non-carcinomatous endometrial lesions with morules were investigated. Numerous antibodies for epithelial membrane antigen (EMA), involucrin, cytokeratins, neuropeptides, and oncofetal antigens were used for immunohistochemistry. In situ hybridisation and polymerase chain reaction were used to detect human papillomavirus (HPV). RESULTS: The morules observed were uniform cell clusters, with no squamous differentiation. They were immunonegative for epithelial antigens including involucrin, EMA, and cytokeratins, but were positive for neurone specific enolase. A few morules were immunopositive for acetylcholine esterase, and one case was positive for somatostatin; neither oncofetal nor proliferative cell markers, including blood group A, B, and AB, or other neuropeptides were demonstrated in the morules. HPV DNA was not found in either the morules in the carcinomas or in the benign lesions. However, true squamous differentiation tissue in four endometrioid carcinomas and two adenosquamous carcinomas was HPV positive using in situ hybridisation. CONCLUSION: Morules are histologically distinct from squamous metaplasia/squamous differentiation tissue. Morules are thought to be neuroectodermal-like cell clusters, and are not infected with HPV. In contrast, some of the true squamous differentiation tissue was associated with HPV infection.     

Morules in cribriform-morular variant of papillary thyroid carcinoma: Immunohistochemical characteristics and distinction from squamous metaplasia

Morules are a diagnostic clue to the cribriform-morular variant (C-MV) of papillary thyroid carcinoma, and are superficially similar to squamous metaplasia. In order to clarify the histogenesis of morules and differentiate them from squamous metaplasia, we immunohistochemically compared the morules in five cases of C-MV with squamous metaplasia in six cases of diffuse sclerosing variant (DSV) of papillary thyroid carcinoma. The squamous metaplastic cells were immunopositive for low- and high-molecular-weight cytokeratin, whereas the morular cells were negative or focally positive. Vimentin-positive cells were observed focally in the morules and squamous metaplasia, except for one case of CMV that showed intense positivity. The morular cells showed weak cytoplasmic positivity for beta-catenin, and the cell membrane was not highlighted. Some nuclei of the morular cells were also positive for this antibody. Beta-catenin was intensively positive along the cell membrane of the metaplastic cells, and did not react against the nuclei or cytoplasm. Bcl-2 was positive in the morular cells, but negative in the metaplastic cells. S-100 protein-positive dendritic cells were observed in the metaplastic nests, but not in the morules. We argue that morules appear in connection with nuclear and cytoplasmic aberrant localization of beta-catenin, and are not an early form of squamous metaplasia.     

Morule with biotin-containing intranuclear inclusions in thyroid carcinoma

One thousand and sixty cases of thyroid carcinoma were reviewed to compare morules with squamous metaplasia clinicopathologically and immunohistochemically. Morules and squamous metaplasia were found in five (0.47%) and 32 cases (3.0%) respectively. The five patients with morules were all female (age 20–36 years) including four with papillary carcinoma and one with follicular carcinoma. The 32 patients with squamous metaplasia consisted of 30 females and 2 males (age 14–78 years), all of whom had papillary carcinoma except for one follicular carcinoma. The morules demonstrated characteristic 'optically clear nuclei' (OCN), which ultrastructurally showed filamentous structures in the nuclei. The OCN were immunohistochemically demonstrated to contain intranuclear biotin. Furthermore, the morule often accompanied with the OCN was positive for Ulex Europaeus agglutinin I (UEA-I) but negative for bovine muzzle epidermal keratin (EK). On the contrary, squamous metaplasia unaccompanied with the OCN was negative for UEA-I, but positive for EK. Follow-up information revealed that one of the five patients with morules had died of the disease, one was alive with pulmonary metastasis, and three were disease-free. Eight of 32 patients with squamous metaplasia had died of the disease; of the others who were alive, four patients have had recurrence.     

Morules and morule-like features associated with carcinomas in various organs: report with immunohistochemical and molecular studies

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. beta 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. beta 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 gamma (NSEgamma). 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. beta 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 NSEgamma, 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.     

Molecular profile of organ culture-stored corneal epithelium: LGR5 is a potential new phenotypic marker of residual human corneal limbal epithelial stem cells

Long-term preservation of corneal limbal epithelium may decrease its quality and change the molecular signature of the limbal epithelial stem cells. In this study we have investigated the molecular profile of isolated corneal epithelial cells that have been in storage for an extended time. Isolated cells were characterised by the expression profile of different cytokeratins and markers of squamous metaplasia (vimentin and α?actin). Furthermore, we examined global markers of adult stem cells including p63α and ABCG2 but also LGR5 as a novel stem cell marker. Immunocytochemical staining and PCR analysis of p63α, ABCG2 and LGR5 revealed the existence of side-population cells with a stem-cell phenotype and maintenance of corneal limbal stem cell properties. LGR5 expression can be related to cellular stemness and can be considered as a new phenotypic marker of residual human corneal limbal stem cells. However, the existence of CK10 together with co-expressed α-actin and vimentin suggests that the corneas investigated were under oxidative stress and showed evidence of squamous metaplasia.     

Microglandular hyperplasia: a model for the de novo emergence and evolution of endocervical reserve cells

BACKGROUND: Microglandular hyperplasia (MGH) of the cervix in human beings is associated early with gland proliferation and terminates in mature squamous metaplasia. Using antibodies to basal cell markers, we analyzed biopsies with MGH to profile the distribution and evolution of reserve cells and their relationship to these epithelial components. DESIGN: Serial sections of 24 MGHs were subdivided into (1) early MGH with microacinar proliferation, abundant subnuclear vacuoles, and a paucity of supporting stroma and (2) late MGH with more prominent supporting stroma and/or squamous metaplasia. Serial sections were stained with antibodies to p63, bcl-2, and keratin-5. RESULTS: Three patterns of p63 staining were observed corresponding to the age of the MGH: (1) scattered staining of columnar cells, (2) focal subcolumnar staining in a reserve cell distribution, and (3) linear subcolumnar arrays of p63-positive reserve cells that in some MGHs expanded into a squamous metaplasia. Early acinar proliferations showed weak and focal columnar cell staining followed by focal subcolumnar p63-positive cells. In late lesions, p63 staining was compartmentalized to the extraglandular (or subcolumnar) areas. Stainings of p63, bcl-2, and keratin-5 were concordant. Staining for keratin 14, which localizes to squamous cells, was variable. CONCLUSIONS: The immunohistochemical profile in MGH indicates that reserve cells are created in adulthood during specialized columnar proliferations. This columnar to reserve cell transition may produce a stable population of reserve cells or a transition to squamous metaplasia. Similar patterns are seen in cervical neoplasia, suggesting a link between benign and neoplastic cervical epithelial differentiation.     

p63 Immunohistochemistry in the distinction of adenoid cystic carcinoma from basaloid squamous cell carcinoma.

Morphologic distinction of high-grade adenoid cystic carcinoma from basaloid squamous cell carcinoma can be difficult. Equivocal diagnoses can mislead treatment. We have investigated the possibility that immunohistochemical staining for the presence of p63, a novel epithelial stem-cell regulatory protein, could be a useful means of distinguishing these two neoplasms. Archival, routinely processed slides were subjected to citrate-based antigen retrieval, exposure to anti-p63 monoclonal 4A4, and developed with a streptavidin-biotin kit and diaminobenzidine as chromogen. p63 was detected in 100% of the adenoid cystic carcinomas (n=14) and 100% of basaloid squamous cell carcinomas (n=16). Basaloid squamous cell carcinomas consistently displayed diffuse p63 positivity, with staining of nearly 100% of tumor cells. In contrast, adenoid cystic carcinoma displayed a consistently compartmentalized pattern within tumor nests. Compartmentalization was manifested in two patterns: (1) selective staining of a single peripheral layer of p63-positive cells surrounding centrally located tumor cells that were p63-negative and (2) tumor nests consisting of multiple contiguous glandular/cribriform-like units of p63-positive cells surrounding or interspersed with p63-negative cells. p63 immunostaining constitutes a specific and accurate means of distinguishing adenoid cystic carcinoma from basaloid squamous cell carcinoma. p63 positivity in adenoid cystic carcinoma appears to be homologous to that seen in the basal and/or myoepithelial compartments of salivary gland and other epithelia, and may signify a stem-cell-like role for these peripheral cells. Diffuse p63 positivity in basaloid squamous cell carcinoma suggests dysregulation of p63-positive stem cells in poorly differentiated squamous carcinoma.     

Distribution of p63, cytokeratins 5/6 and cytokeratin 14 in 51 normal and 400 neoplastic human tissue samples using TARP-4 multi-tumor tissue microarray

p63, cytokeratin (CK) 5/6 and CK 14 have been employed in diagnostic pathology as markers of basal, squamous and myoepithelial differentiation in several types of human neoplasms; however, there is scant data on the concurrent expression of these markers in large series of human neoplasms. We analyzed the distribution of these three immunohistochemical markers in 51 normal human tissue samples, 350 carcinomas, 25 malignant melanomas (MMs), and 25 glioblastomas using three serial sections of tissue array research program (TARP)-4 multi-tumor tissue microarray. Also, we performed double immunostainings to characterize the differential distribution of p63/CK 5/6 and p63/CK 14 in normal breast, salivary gland and skin. p63, CK 5/6 and CK 14 were expressed in basal cells of the prostate and respiratory epithelia and in breast and bronchial myoepithelial cells. p63 was also expressed in cytotrophoblast cells of human placenta and in scattered cells of lymph node germinal center. CK 5/6 and CK 14 also stained the cytoplasm of basal cells of esophageal stratified squamous epithelium and transitional epithelial cells of the bladder. No mesenchymal, neural, endothelial, smooth muscle or adipose cells were stained by any of the markers. p63, CK 5/6, and CK 14 were respectively expressed in 92.6%, 75.0%, and 52.9% of the squamous cell carcinomas of the lung, 10.2%, 20.0%, and 7.4% of the ductal carcinomas of the breast, 12.9%, 34.4%, and 11.8% of the serous and 25.0%, 0%, and 0% of the endometrioid carcinomas of the ovary. Lung, prostate and colonic adenocarcinomas, as well as MMs and glioblastomas were only rarely decorated by one of the markers. Only matched samples of 16 squamous cell carcinomas and two ductal carcinomas of the breast co-expressed these three markers. In double immunostainings, p63-CK 5/6, as well as p63-CK 14 were co-expressed by basal/myoepithelial cells of the salivary glands and basal cells of the epidermis. Our results demonstrate that p63, CK 5/6 and CK 14 may be used together in immunohistochemical panels to characterize squamous differentiation in poorly differentiated carcinomas or carcinomas of unknown origin.     

Immunohistochemical detection of GLUT1, p63 and phosphorylated histone H1 in head and neck squamous intraepithelial neoplasia: evidence for aberrations in hypoxia-related, cell cycle- and stem-cell-regulatory pathways

AIM: Most epithelial malignancies are characterized by multistep progression from preinvasive/intraepithelial neoplasia to invasive malignancy. Detection and grading of early squamous intraepithelial neoplasia may at times be problematic. The aim of this study was to examine the ability of immunomarkers GLUT1, phospho-histone H1 and p63 to detect such early lesions. METHODS: Sections of formalin-fixed paraffin-embedded tissue from 27 cases of squamous intraepithelial neoplasia, 26 associated with invasive carcinoma, were immunostained with anti-p63 (4A4; Santa Cruz), anti-GLUT1 (Chemicon) and anti-phospho-histone H1 (monoclonal 12D11) and compared with normal, hyperplastic and immature squamous metaplastic epithelium. RESULTS: Normal epithelium displayed phospho-histone H1 in scattered parabasal cells; p63 in the basal one-quarter to one-half of epithelium; and GLUT1 negativity or weak/equivocal mid-epithelial GLUT1+ foci. In hyperplasia phospho-histone H1+ cells were also limited to the parabasal layer; p63 positivity was essentially identical to that in normal epithelium; GLUT1 characteristically stained basal cells in rete-like areas. p63 staining in squamous intraepithelial neoplasia (grade 1) was indistinguishable from normal epithelial staining; in contrast, squamous intraepithelial neoplasia (grade 3) was readily apparent, with up to full-thickness p63 positivity. Squamous intraepithelial neoplasia (grade 1) was readily distinguishable from normal epithelium with both phospho-histone H1 and GLUT1 immunostaining; both markers were detected in cell layers above the parabasal layer. With both, progressively higher cell layers stained in proportion to the severity of the intraepithelial neoplasia, up to full thickness positivity in grade 3 squamous intraepithelial neoplasia. Squamous metaplasia and grade 3 squamous intraepithelial neoplasia were not distinguishable with p63 (both showed full-thickness staining) but were readily distinguishable with GLUT1 and phospho-histone H1 stains. GLUT1 appeared to be the most sensitive marker for all grades of intraepithelial neoplasia. CONCLUSION: Altered expression of all three markers was a common finding in squamous intraepithelial neoplasia, hence, dysregulation of cell cycle-promoting cyclin-dependent kinases (phospho-histone H1), altered stem cell regulatory pathways (p63) and enhancement of hypoxia-sensing pathways (GLUT1) are all early alterations in the progression of squamous malignancy of head and neck origin. A panel of all three may be a useful means of detecting squamous intraepithelial neoplasia.     

Histologic and immunophenotypic classification of cervical carcinomas by expression of the p53 homologue p63: a study of 250 cases

Recent studies of the p53 homologue p63 indicate that this gene is preferentially expressed in basal and immature cervical squamous epithelium. This study correlated p63 expression with morphologic phenotype and human papillomavirus (HPV) type in a wide range of cervical neoplasms. Two hundred fifty cases of cervical carcinoma, including squamous cell carcinoma (SCCA; n = 178), adenocarcinoma (ADCA; n = 28), adenosquamous carcinoma (ASCA; n = 8), neuroendocrine carcinoma (NECA; n = 15), and other variant or mixed types (n = 21) were studied. Ninety-seven percent of SCCA, 0% of ADCA, and 0% of SCUC showed strong (>75% v <30%) positivity for p63 (P<.001). p63 sharply distinguished SCCA (p63+) from ADCA (p63-), Large-cell, poorly differentiated carcinomas were distinguished as putative glandular (glassy cell) or squamous (lymphoepithelial-like or spindle cell) types based on p63 staining. Eight (73%) of 11 neuroendocrine tumors tested were chromogranin positive; all showed no or low (<30%) levels of p63 immunostaining. Absence of p63 was also associated with a subset of nonneuroendocrine undifferentiated carcinomas. Transitions from squamous to columnar or undifferentiated morphology coincided with loss of p63 expression. A strong association between HPV 16 and p63 positivity was identified because of the colocalization of both within tumors of squamous phenotype. p63 is a powerful marker for squamous differentiation and, when diffusely expressed, excludes a glandular or neuroendocrine differentiation. p63 may be useful for differentiating pure squamous or glandular from adenosquamous carcinomas, tracking shifts in differentiation within tumors, supporting (by its absence) the diagnosis of neuroendocrine carcinomas, and clarifying the spectrum of poorly differentiated carcinomas lacking either squamous or neuroendocrine differentiation.     

p63 expression in ovarian tumours: a marker for Brenner tumours but not transitional cell carcinomas

AIMS: To investigate p63 expression in ovarian neoplasms. METHODS AND RESULTS: Immunohistochemistry using an antibody that detects all p63 isoforms was performed on 103 primary ovarian neoplasms of different histological types. Diffuse nuclear immunoreactivity of p63 was demonstrated in the 17 benign and five borderline Brenner tumours. Only one of the six malignant Brenner tumours displayed p63 expression. p63 immunoreactivity was absent in all the ovarian transitional cell carcinomas (TCC), but was demonstrated extensively in TCCs of the urinary bladder. Besides focal p63 expression in epidermal basal cells of immature and mature teratomas, all other ovarian lesions were devoid of p63 expression. p63 expression was also demonstrated in cervical transitional cell metaplasia and Walthard cell nests of fallopian tubes. CONCLUSIONS: Expression of p63 protein is apparently cell lineage specific and in ovarian neoplasms is confined to benign and borderline Brenner tumours. The loss of expression in malignant Benner tumours suggests a role for p63 in Brenner carcinogenesis. The distinct patterns of p63 expression in TCCs in the ovary and urinary bladder may help in their differential diagnosis.     

The expression of p63 and p53 in keratoacanthoma and intraepidermal and invasive neoplasms of the skin

p53 is a well-known tumor suppressor gene, and its mutation is a common event in intraepidermal and invasive neoplasms of the skin. p63 is a homologue of the tumor suppressor gene p53, which is expressed in human basal squamous epithelium, and despite its homology to p53, it is considered to act as an oncogene. We evaluated p63 and p53 expression in usual skin cancers, including basal cell carcinoma (BCC), squamous cell carcinoma (SCC), keratoacanthoma (KA), and intraepidermal neoplasms, including Bowen's disease (BD), actinic keratosis (AK), malignant melanoma in situ (MM in situ), and Paget's disease (PD) to clarify the putative role of p63 and p53 in the development and differential diagnosis of these lesions.     

P63 expression in papillary and anaplastic carcinomas of the thyroid gland: lack of an oncogenetic role in tumorigenesis and progression

P53 is considered one of the most important tumor suppressor genes and is mutated in up to 50% of all neoplasms. Well-differentiated thyroid carcinomas (WDTCs) only infrequently harbor p53 mutations. In contrast, these genetic alterations have been described in approximately 85% of anaplastic thyroid carcinomas and are considered a fundamental event in the malignant progression of WDTCs. However, alternative mechanisms to overcome p53 tumor suppressing properties in WDTCs and anaplastic carcinomas (ACs) have not been clarified to date. p63, a p53-homologue, has been recently characterized. In contrast to p53, p63 gene encodes six isoforms, three with transactivating and three with dominant negative (deltaN-p63) activities on p53 reporter genes. We hypothesized that overexpression of deltaN-p63 isoforms might constitute an alternative mechanism to overcome p53 tumor suppressing properties in WDTCs and ACs lacking p53 alterations. We semiquantitatively evaluated p53 and p63 immunoexpression in 12 papillary carcinomas (PC) and 11 anaplastic carcinomas. Only nuclear expression was considered specific. All PCs lacked p53 expression; at variance, nine ACs showed p53 immunoreactivity (+: 1 case; ++: 6 cases; +++: 2 cases). In PCs, p63 expression was restricted to scattered neoplastic cells juxtaposed to the basement membrane of papillary projections and to foci of squamous metaplasia. In ACs, p63 expression was observed in three cases, one of which lacked concurrent p53 immunoexpression. Our results do not support the hypothesis that p63 might constitute an alternative mechanism to overcome p53 tumor suppressing properties in thyroid neoplasms.     

Tubular adenoma with focal squamous metaplasia of the ascending colon

A case of tubular adenoma with focal squamous metaplasia of the ascending colon in a 66-year-old male is reported. The tumor was a pedunculated polyp with a size of 1.5 X 1.4 X 0.9 cm. Histologically, the tumor showed tubular adenoma with moderate to severe atypia, was accompanied by focal squamous metaplasia. It is thought that squamous epithelial polyp may be derived from colonic adenoma with squamous metaplasia, and that the malignant change of colonic adenoma with squamous metaplasia may lead to squamous cell carcinoma, adenosquamous carcinoma, and mucoepidermoid carcinoma. Only seven cases of colonic adenoma with squamous metaplasia have been reported previously in the literature, and it is yet necessary to discuss the histogenesis, significance, and natural history of colonic adenoma with squamous metaplasia.     

TUBULAR ADENOMA WITH FOCAL SQUAMOUS METAPLASIA OF THE ASCENDING COLON

A case of tubular adenoma with focal squamous metaplasia of the ascending colon in a 66-year-old male is reported. The tumor was a pedunculated polyp with a size of 1.5 × 1.4 × 0.9 cm. Histologically, the tumor showed tubular adenoma with moderate to severe atypia, was accompanied by focal squamous metaplasia. It is thought that squamous epithelial polyp may be derived from colonic adenoma with squamous metaplasia, and that the malignant change of colonic adenoma with squamous metaplasia may lead to squamous cell carcinoma, adenosquamous carcinoma, and mucoepidermoid carcinoma. Only seven cases of colonic adenoma with squamous metaplasia have been reported previously in the literature, and it is yet necessary to discuss the histogenesis, significance, and natural history of colonic adenoma with squamous metaplasia. ACTA PATHOL. JPN. 35 : 507–515, 1985.