Incidence of NUT carcinoma in Western Australia from 1989 to 2014: a review of pediatric and adolescent cases from Perth Children's Hospital.

BACKGROUND: NUT carcinoma (NC), previously known as NUT midline carcinoma, is a rare and very aggressive cancer that occurs in both children and adults. NC is largely chemoresistant, with an overall survival of less than 7 months. Because the carcinoma is not restricted to a particular organ, diagnosis is often a challenge. In the absence of a clearly determined incidence for NC, we sought to study the diagnosis of patients in a well-defined population. METHODS: We systematically reviewed records of all patients that presented to the Oncology Department of the Princess Margaret Hospital for Children from 1989 to 2014. This institution in the geographically isolated state of Western Australia has a catchment population of around 2 million. We then identified all high grade undifferentiated sarcomas or carcinomas in the 0-16 year age group. RESULTS: Over 26 years, we found 14 patients of 16 years or younger with undifferentiated malignant tumors. Of these, five tumors were positive by immunohistochemistry for the NUT/NUTM1 (Nuclear Protein in Testis) protein and/or the translocation t(15;19). Three patients presented with thoracic tumors, one with a para-spinal tumor, and one had an upper airway nasopharyngeal carcinoma. In all five cases, there was an initial response to therapy and then progression. This 26-year survey was conducted in a geographically isolated state with a well-defined population, and we determined an estimated incidence of NC of around 0.41 per million child years (0-16 yrs. of age) at risk. From three patients it was feasible to derive cell lines for further genetic analyses and drug screening. CONCLUSIONS: For the first time, the incidence of NC could be determined in a well-defined geographic area. The calculated rate of NC incidence is consistent with a history of under-recognition for this malignancy. These findings indicate that improved diagnostic detection of NC would enable better management and counselling of patients. Our findings emphasize the heterogeneity of NC, and they highlight the need to develop personalised therapy options, and to consider a diagnosis of NC in undifferentiated malignant tumors.

Cerebellar Differentiation in Ovarian Teratoma: A Report of 6 Cases.

Ovarian teratomas potentially demonstrate a wide range of tissue elements including central nervous system differentiation. The latter can include cerebellar tissue, which in our experience remains an under-recognized phenomenon. In the current study we present a review of 6 ovarian teratomas including 4 mature cystic teratomas and 2 immature teratomas showing cerebellar differentiation. Two cases were seen in consultation because the cerebellar elements were initially misinterpreted as immature teratomas. Two mature cystic teratomas focally demonstrated a distinct cerebellar architecture including folial type structures, but in all cases the cerebellar elements usually showed a less organized anatomic appearance, and sometimes these were concerning for immature teratomas upon initial examination. This concern was exacerbated in 5 cases by the presence of a cytologically immature and mitotically active neuronal component corresponding to the external granular layer of normal fetal and neonatal cerebellum. However, careful examination demonstrated the characteristic molecular, Purkinje and (internal) granular layers of cerebellum. Furthermore, while the external granular layer in teratomas strongly expressed Ki67, corresponding to the proliferative activity of this cellular compartment physiologically, immunostaining was often helpful in highlighting the preserved zonal pattern of cellular proliferation. The absence or minimal expression of SALL4, OCT3/4, and SOX2 was also helpful in this regard. Cytoplasmic OCT3/4 expression in osteoblasts was noted incidentally in 2 tumors, but further studies are required to determine whether this is a consistent and diagnostically useful finding.

Podoplanin and SOX2 Expression in CIN 3-like Squamous Cell Carcinoma of the Cervix.

Squamous cell carcinoma (SCC) of the uterine cervix occasionally demonstrates a deceptive growth pattern that mimics endocervical crypt involvement by cervical intraepithelial neoplasia, grade 3 (CIN 3). Such CIN 3-like SCCs may be misinterpreted as noninvasive or minimally invasive leading to delays in diagnosis. Little is known of the factors that influence the growth patterns of cervical SCC but we suggested recently that CIN 3-like tumors might demonstrate "collective cellular invasion," which is characterized by a retained epithelial phenotype. This contrasts with the more overtly infiltrative growth of conventional SCC, which exhibits features suggestive of epithelial-mesenchymal transition. In the current study we investigated podoplanin (PP) and SOX2 expression in normal squamous epithelium, in CIN 3 and in 16 CIN 3-like SCCs 11 of which also showed a conventional invasive component. Compared with normal epithelium, CIN 3 often showed a focal loss of basal PP staining and most cases showed increased, typically diffuse, SOX2 expression. Although the immunohistochemical findings were not uniform, they generally supported collective cellular invasion in CIN 3-like tumor areas as these were often PP positive and showed diffuse SOX2 expression. In contrast, most conventional SCCs showed only focal SOX2 staining and they were typically negative, or only focally positive, for PP. The staining patterns did not reliably distinguish CIN 3 from CIN 3-like SCC. Small infiltrative tumor nests around the margins of CIN 3 or deeply invasive CIN 3-like SCC often showed a localized reduction in SOX2 expression suggesting SOX2 downregulation during the transition to invasive growth.

Cervical intraepithelial neoplasia (CIN) 3-like squamous cell carcinoma of the cervix: a review of 14 cases with comparison of E-cadherin and cyclin D1 expression in the CIN 3-like and infiltrative tumour elements.

AIMS: To review the clinicopathological features of 14 cases of CIN 3-like squamous cell carcinoma (SCC) of the cervix and to investigate possible mechanisms of tumour invasion in the CIN 3-like and 'conventional' (infiltrative) tumour components. METHODS AND RESULTS: The median patient age was 43.4 years. Of the 12 cases with known stage, eight were stage IB and four were stage II. Initial biopsies were often misinterpreted as CIN 3 alone or CIN 3 with only superficial stromal invasion, and eight patients underwent multiple biopsy procedures prior to definitive diagnosis: upon review, an earlier diagnosis was possible in six of these cases. Nine tumours exhibited both CIN 3-like and conventional tumour components. The former usually demonstrated an E-cadherin-positive and cyclin D1-negative immunophenotype, whereas conventional SCC more often showed loss of E-cadherin and cyclin D1 staining at the margin of larger tumour nests and in smaller invasive clusters. CONCLUSION: Cervical SCCs demonstrating a CIN 3-like growth pattern continue to present diagnostic difficulty and are often misinterpreted as non-invasive/minimally invasive disease. The morphology and immunophenotype suggest a process of collective cellular invasion in CIN 3-like SCC, whereas corresponding conventional SCC elements show features consistent with epithelial-mesenchymal transition.

Histological comparison of partial hydatidiform mole and trisomy gestation specimens.

The distinction between partial hydatidiform mole (PHM) and trisomy gestation is not always straightforward histologically and it is unclear which morphological features, alone or in combination, provide the greatest diagnostic accuracy. We performed a comparative review of 89 products of conception (POC) specimens including 54 PHMs and 35 trisomy gestations, assessing the following in each case: trophoblastic atypia, cistern formation, multifocal trophoblast proliferation, lace-like trophoblast, villous enlargement, large trophoblast inclusions, scalloped villous shape, stromal apoptosis, small round villous inclusions, and fibrillary stromal collagen. There was a significant difference in the presence of trophoblast atypia, cistern formation, multifocal trophoblast proliferation, lace-like trophoblast, large trophoblastic inclusions, small round villous inclusions, fibrillary collagen (all p<0.01), and apoptosis (p=0.028), between PHM and trisomy cases. Fibrillary collagen was more common in trisomy specimens whereas the other features were more common in PHMs. There was no significant difference in villous enlargement or scalloped villous shape between the two groups. The combination of cistern formation, multifocal trophoblast proliferation and large trophoblast inclusions correctly classified 83 (93.3%) of cases where the presence of at least two features was considered diagnostic of PHM. While cytogenetic analysis is arguably the gold standard for diagnosis, this study demonstrates that histological assessment permits accurate distinction of PHM and trisomic gestations in the great majority of cases.

SF1 immunohistochemistry is useful in differentiating uterine tumours resembling sex cord-stromal tumours from potential histological mimics.

The aim of this study was to perform an immunohistochemical comparison of uterine tumour resembling ovarian sex cord-stromal tumour (UTROSCT) and other uterine lesions with sex cord-like (SCL) differentiation. Six UTROSCTs and 10 potential histological mimics with focal SCL elements were examined, the latter comprising three endometrial stromal nodules, three low-grade endometrial stromal sarcomas, three Müllerian adenosarcomas, and one case of adenomyosis. All cases were stained immunohistochemically for SF1, FOXL2, calretinin and inhibin, and for the less specific markers smooth muscle actin, desmin, CD10, CD56, CD99, cytokeratin, oestrogen receptor and progesterone receptor. Three, four, six and three UTROSCT expressed SF1, FOXL2, calretinin and inhibin, respectively. However, calretinin staining was focal (≤50% cells positive) in five of the cases. Three potential histological mimics demonstrated calretinin, FOXL2 and/or inhibin staining but none was SF1 positive. Most cases in both groups expressed the less specific immunomarkers. SF1 and FOXL2 immunoreactivity in UTROSCT further supports the concept that these tumours demonstrate genuine sex cord-stromal differentiation. While calretinin was the most sensitive UTROSCT marker, staining was usually focal and expression was also seen in two of 10 potential histological mimics. SF1 staining was 100% specific for UTROSCT in this series but this finding should be confirmed in larger studies.

SOX2 Expression in Cervical Intraepithelial Neoplasia Grade 3 (CIN3) and Superficially Invasive (Stage IA1) Squamous Carcinoma of the Cervix.

The transcription factor SOX2 plays an important role in tissue development and differentiation. In the neoplastic context, SOX2 has been shown to potentiate tumor invasion, and increased SOX2 immunoreactivity has been demonstrated in a variety of epithelial and nonepithelial malignancies often correlating with adverse prognosis. There are limited data on SOX2 expression in cervical squamous neoplasia and in particular, no studies have compared staining in cervical intraepithelial neoplasia (CIN)3 and in superficially invasive (Stage IA1) squamous cell carcinomas (SCC). We examined SOX2 expression in 12 cervical biopsies showing CIN3 only and 30 specimens with an initial diagnosis of Stage IA1 SCC; 7 of the latter samples did not demonstrate residual invasive foci in the study slides but all showed CIN3. There was variable staining in CIN3 without stromal invasion but CIN3 adjacent to SCC was more often SOX2 positive with 70% cases showing diffuse staining. CIN within endocervical crypts often showed more extensive SOX2 expression and in some cases staining was restricted to areas of crypt involvement. In contrast to CIN, most SCCs were SOX2 negative and there was often an abrupt loss of expression at the tumor-stromal interface. In summary, CIN3 usually showed increased SOX2 expression compared with normal epithelium, particularly in areas of endocervical crypt involvement and adjacent to superficially invasive SCC. However, most invasive tumor cells were unstained suggesting downregulation of SOX2 during the initial stages of the invasive process. Progression of cervical squamous neoplasia may involve cyclical alterations in SOX2 activity.

Fascin expression in undifferentiated and dedifferentiated endometrial carcinoma.

The actin-binding protein fascin promotes cellular invasion, and increased fascin expression correlates with adverse prognostic factors in a variety of tumors. Fascin up-regulation may also be associated with epithelial-mesenchymal transition in neoplastic epithelial cells. This study investigated fascin expression in undifferentiated and dedifferentiated endometrial carcinoma (UEC), a clinically aggressive variant of endometrial neoplasia. Twenty-two UECs, 5 of which were entirely undifferentiated and 17 dedifferentiated, were examined. In the dedifferentiated group, staining was compared between the differentiated and undifferentiated tumor components. Where applicable, fascin expression was noted in foci of lymphovascular space invasion. The mean age was 67.6 years, and 11 patients (50%) presented with stage III or IV disease. The undifferentiated tumor component showed diffuse fascin expression in 20 cases (91%) including 4 of 5 pure undifferentiated carcinomas and 16 of 17 dedifferentiated carcinomas. In contrast, the low-grade endometrioid carcinoma component of 13 (77%) of 17 dedifferentiated carcinomas was fascin negative or showed only focal staining. Intravascular undifferentiated tumor cells were identified in 16 cases, and these were consistently fascin positive, whereas low-grade intravascular tumor cells, present in 2 cases, were not stained. Fascin up-regulation may be a contributory factor toward the highly invasive character of UEC and could represent an epithelial-mesenchymal transition-like process in these tumors. Fascin expression in intravascular tumor cells may be permissive toward intravascular survival and metastatic risk.

SWI/SNF complex deficiency and mismatch repair protein expression in undifferentiated and dedifferentiated endometrial carcinoma.

Undifferentiated endometrial carcinoma (UEC) is a relatively uncommon but clinically aggressive uterine malignancy. In common with a subset of poorly differentiated carcinomas arising in other sites, UEC may exhibit rhabdoid morphology and be associated with a low-grade tumour component (dedifferentiated carcinoma). Recent studies have implicated inactivation of the SWI/SNF complex subunits in the aforementioned extrauterine tumours. Therefore we have examined INI1 (SMARCB1), BRG1 (SMARCA4), and BAF250a (ARID1A) immunostaining, and also expression of the DNA mismatch repair (MMR) proteins MLH1, PMS2, MSH2 and MSH6 in 22 UEC, seventeen of which were dedifferentiated. Abnormal SWI/SNF subunit expression was detected in four dedifferentiated carcinomas including three with loss of BRG1 staining limited to the undifferentiated tumour component and one case with loss of INI1 expression in both low- and high-grade elements; the latter case also showed BAF250a deficiency in the undifferentiated tumour cells. Abnormal MMR protein expression was identified in 13 tumours (59%) including nine with concurrent loss of MLH1 and PMS2. These findings suggest that SWI/SNF subunit alterations may play a role in the progression/ dedifferentiation of endometrial carcinoma, and that SWI/SNF and MMR protein deficiencies may act synergistically in deregulating DNA repair mechanisms in these tumours.

p16 immunoreactivity in endometrial stromal cells: stromal p16 expression characterises but is not specific for endometrial polyps.

It has been proposed recently that stromal cell p16 immunoreactivity may be useful in the diagnosis of endometrial polyps (EPs). However, the specificity of p16 staining is uncertain and it is also unclear whether sporadic and tamoxifen-related polyps show similar findings. We performed p16 immunostaining on 35 normal endometrial specimens and 32 EPs, six of which were associated with tamoxifen therapy. Normal cyclical endometrium showed patchy glandular staining and there was also focal stromal p16 expression in the functional layer of most proliferative and secretory endometria. Atrophic and lower uterine segment endometrial stromal cells were negative except for localised immunoreactivity in one atrophic case. The EPs demonstrated glandular p16 expression, particularly in epithelium showing ciliated metaplasia. Stromal p16 staining was also seen in all EPs, although the proportion of positive cells varied considerably (20-90%). There was no significant difference in staining between sporadic and tamoxifen-associated cases. In conclusion, stromal p16 immunoreactivity is characteristic of EPs and this may reflect the pathogenesis of polyp formation. Immunohistochemistry can help to distinguish polypoid and non-polypoid mucosa, particularly in small or disrupted biopsy specimens. However, stromal p16 expression is not completely specific since focal staining may be present in normal endometrium.

PAX2 and cyclin D1 expression in the distinction between cervical microglandular hyperplasia and endometrial microglandular-like carcinoma: a comparison with p16, vimentin, and Ki67.

Microglandular hyperplasia (MGH) is a common endocervical alteration that in most cases presents no diagnostic difficulty. However, MGH rarely shows atypical features that may mimic endocervical neoplasia, while conversely endometrial carcinomas can show deceptively bland MGH-like appearances. It has been suggested that immunohistochemical analysis is useful in this context, but relatively few studies have specifically investigated microglandular pattern lesions and the results have been conflicting. In this study, we have examined a series of MGH (n=24), atypical MGH (n=2), and endometrial microglandular-like carcinomas (EMC, n=8), with a panel of antibodies including PAX2, cyclin D1, p16, vimentin, and Ki67. Loss of PAX2 staining was identified only in EMC but had relatively poor sensitivity for a malignant diagnosis (3/8 cases). Seven EMCs showed p16 expression and staining was diffuse (≥50% cells) in 6 cases, whereas all conventional MGH lesions were negative. However, 1 case of atypical MGH was also p16-positive. Cyclin D1, vimentin, and Ki67 did not reliably distinguish the benign and malignant microglandular lesions because of considerable overlap in staining patterns. In summary, none of the antibodies examined proved completely sensitive and specific, but a p16-positive/PAX2-negative phenotype favored a diagnosis of EMC. Pathologists should be aware that EMC, like some other types of endometrial carcinoma, are commonly p16-positive to avoid misinterpretation as a primary endocervical neoplasm. In practice, correlation of the histologic, immunohistologic, and clinical findings is necessary for accurate interpretation of microgandular-pattern lesions, particularly in small biopsy samples.

Extravascular migratory metastasis in gynaecological carcinosarcoma.

AIMS: Extravascular migratory metastasis (EVMM) is a potential mechanism of tumour spread reported most extensively in cutaneous melanoma. It has not been described previously in gynaecological malignancies. We describe EVMM in four gynaecological carcinosarcomas. METHODS AND RESULTS: Extravascular migratory metastasis was observed in an ovarian carcinosarcoma during routine diagnostic assessment. Twenty-three additional, randomly selected gynaecological carcinosarcomas (11 tubo-ovarian and 12 endometrial) were examined retrospectively and EVMM was identified in three of these. Other than the index case, EVMM was a focal finding, identified in 12-18% of slides. The malignant cells demonstrating EVMM appeared sarcomatoid and were distributed abluminally, partly or completely surrounding the endothelium. Affected vessels often showed mural fibrin deposition. Immunohistochemistry for α-smooth muscle actin (SMA), CD31, CD34, D2-40, laminin and type IV collagen was performed on the EVMM-positive cases. The perivascular malignant cells showed more consistent SMA and laminin immunoreactivity than the non-vascular tumour elements. CONCLUSIONS: Extravascular migratory metastasis is a hitherto unrecognized mechanism of tumour spread in gynaecological carcinosarcomas. The perivascular tumour cells appear to adopt a pericytic phenotype, and this may represent a specific pattern of epithelial-mesenchymal transition. Further studies with pericyte-specific immunohistological markers may better demonstrate the presence and possible prognostic significance of EVMM in gynaecological tumours.

An immunohistochemical and molecular analysis of problematic and unclassified ovarian sex cord-stromal tumors.

Most ovarian sex cord-stromal tumors (SCSTs) can be categorized on the basis of conventional histology, but approximately 10% of cases are unclassified because they present indeterminate or overlapping morphologic features. Immunohistochemical and molecular studies of unclassified ovarian SCST are very limited, but recently, it has been demonstrated that 2 major subgroups of SCST, adult-type granulosa cell tumor and Sertoli-Leydig cell tumor, are characterized by somatic mutations in FOXL2 and DICER1, respectively. In this study, 12 diagnostically problematic ovarian SCST, including 9 unclassified tumors, were investigated for FOXL2 and DICER1 mutations and for immunohistochemical expression of calretinin, CD56, CD99, estrogen receptor α, estrogen receptor ß, FOXL2, inhibin, progesterone receptor, and steroidogenic factor-1. Four of 11 tumors with satisfactory analysis showed a FOXL2 mutation; 3 of these cases were reported initially as unclassified SCST and 1 as Sertoli-Leydig cell tumor. Conversely, 3 cases with an original diagnosis of granulosa cell tumor were FOXL2 mutation-negative, and none of 7 tumors with satisfactory analysis demonstrated a DICER1 mutation. All tumors expressed at least 4 of the immunomarkers examined, although staining was often focal and there was no consistent correlation with tumor morphology. In conclusion, molecular analysis is useful in the assessment of diagnostically challenging ovarian SCST. The absence of FOXL2 and DICER1 mutations in most unclassified SCST suggests that these could represent a distinct tumor subgroup with different molecular pathogenesis. Immunohistochemical profiles overlap with those of better categorized SCST, but staining may be focal or negative emphasizing the requirement for antibody panels in diagnostic assessment.

Immunophenotypic analysis of ovarian endometrioid adenocarcinoma: correlation with KRAS mutation and the presence of endometriosis.

AIMS: The relationship between endometriosis and ovarian endometrioid adenocarcinoma (OEC) is well recognised but it is unclear whether endometriosis positive and negative OECs develop via similar pathogenetic mechanisms. MATERIALS: Sixty-seven low grade OECs (35 associated with endometriosis) were stained immunohistochemically for ß-catenin, cyclin D1, BAF250a, PTEN, p53, WT1 and the mismatch repair (MMR) proteins MLH1, PMS2, MSH2 and MSH6. The results were correlated with KRAS mutation analysis and the presence of concurrent endometriosis. RESULTS: Abnormal ß-catenin, cyclin D1, BAF250a, PTEN, p53 and MMR protein expression was identified in 61.2%, 50.7%, 19.4%, 23.9%, 9.0%, and 6.0% of cases, respectively; these changes were equally common in endometriosis positive and negative tumours. WT1 expression was restricted to endometriosis negative EOC (8/32, 25%) and four WT1 positive cases showed sertoliform/spindle cell histological patterns. Abnormal ß-catenin expression correlated with cyclin D1 overexpression but was inversely related to KRAS mutation. Immunophenotypic abnormalities were present in four of 17 histologically benign endometriotic lesions. CONCLUSIONS: Most immunophenotypic alterations were equally common in endometriosis associated and independent OECs but only the latter were associated with abnormal WT1 expression. The inverse relationship between abnormal ß-catenin expression and KRAS mutation merits further study. Histologically benign endometriotic epithelium may show immunophenotypic abnormalities similar to those present in associated carcinomas.

Cyclin D1, E-cadherin and beta-catenin expression in FIGO Stage IA cervical squamous carcinoma: diagnostic value and evidence for epithelial-mesenchymal transition.

AIMS: Immunohistochemistry is helpful in distinguishing cervical neoplastic lesions from their histological mimics, but has contributed less towards the sometimes problematic distinction of in-situ and superficially invasive tumours. Epithelial-mesenchymal transition (EMT) may be a mechanism of invasion in cervical neoplasia and expression of EMT-associated proteins could prove useful in this diagnostic setting. METHODS AND RESULTS: Immunohistochemical expression of cyclin D1, E-cadherin and beta-catenin was assessed in 22 biopsy specimens from FIGO Stage IA cervical squamous carcinomas, all of which also included foci of cervical intraepithelial neoplasia (CIN) 3, nine biopsies of CIN 3 adjacent to carcinoma, and 10 cases of CIN 3 only. Most invasive tumour cells expressed cyclin D1 and showed a reduction in E-cadherin and beta-catenin staining. Nuclear beta-catenin expression was not observed. Cyclin D1 staining was reduced or showed altered distribution in most cases of CIN 3, while adhesion protein expression generally was preserved. However, altered protein expression similar to that of invasion was seen in some CIN lesions. CONCLUSIONS: Most superficially invasive cervical squamous carcinomas show immunophenotypical changes consistent with EMT. These alterations, particularly cyclin D1 expression, may be useful diagnostically. Similar changes in CIN 3 lesions may indicate the acquisition of increased invasive potential.

Fascin expression in endocervical neoplasia: correlation with tumour morphology and growth pattern.

BACKGROUND: Fascin is an actin-binding protein that potentiates migratory and invasive behaviour in neoplastic cells and has been shown to be upregulated in various malignancies. In this study fascin expression was assessed in adenocarcinoma in situ (ACIS) and invasive adenocarcinoma of the endocervix, and the results were correlated with tumour growth patterns (papillary, glandular or infiltrative). METHODS: Fascin immunoreactivity was assessed in 10 cases of ACIS and in 34 cervical adenocarcinomas, 15 of which also included an in situ component. Staining within normal epithelium and stromal elements was also noted. RESULTS: Normal endocervical epithelium and 23/25 ACIS lesions were fascin-negative. Most invasive tumour elements were also unstained but 13/32 adenocarcinomas that exhibited a glandular growth pattern showed focal fascin immunoreactivity mainly towards the basal aspect of the larger tumour glands. These fascin-positive cells often showed a morphological alteration similar to that seen in infiltrative tumour areas. None of the papillary tumour elements, present in 12 cases, was fascin-positive. Twenty adenocarcinomas included a focal infiltrative component, often distributed at the advancing or deep tumour margin (invasive front), and these tumour cells were usually fascin-positive. Staining was also observed in normal parabasal squamous cells, endothelial and dendritic cells. CONCLUSIONS: Novel fascin expression occurs during the development and progression of some endocervical neoplasms. Fascin immunoreactivity within infiltrative neoplastic elements suggests that this protein may have an important role in areas of active tumour invasion.

Changes in thymic regulatory T-cell maturation from birth to puberty: differences in atopic children.

BACKGROUND: Characterization of regulatory immune pathways is a research priority for both the pathogenesis of allergic disease and potential therapeutic strategies. OBJECTIVE: The thymus is a rich source of regulatory T (Treg) cells, which offers a novel opportunity to document the maturation of these pathways beyond limited studies on small volumes of peripheral blood available from young children. METHODS: Thymus tissue was collected from children undergoing cardiac surgery (age, 1 week to 14 years), and skin prick testing was performed from 12 months of age. The ontogeny of Treg cell maturation and function was examined in atopic (n = 20) and nonatopic (n = 20) children by assessing their phenotype, enumeration, proliferation, and suppressive ability. RESULTS: Age-related changes in the thymic cytokine milieu paralleled the changes seen in peripheral immune function. Specifically, the thymic microenvironment is similarly T(H)2 skewed during the early postnatal period, and this undergoes age-related suppression as the T(H)1 (IFN-γ) response increased. We detected CD4(+)CD25(+)CD127(lo/-) forkhead box protein 3 (FOXP3)-positive Treg cells in the neonatal thymus. These cells suppressed the proliferative response to allogeneic stimulation of CD4(+)CD25(-) T cells dose dependently. In nonatopic children Treg cell turnover and suppressive function increased with age and paralleled the increase in global thymic FOXP3 mRNA expression, whereas in atopic children Treg cell maturation was significantly delayed compared with that seen in age-matched nonatopic children. CONCLUSION: These data suggest that the developmental changes in the thymus parallel the recognized changes in peripheral blood responses. There is also a developmental delay in the function of thymic regulatory cells in atopic compared with nonatopic children. These differences are fundamental to understanding early events that lead to immune dysregulation and might predispose to allergic disease.

Cytokeratin 19 expression in normal endometrium and in low-grade endometrioid adenocarcinoma of the endometrium.

The low-molecular-weight cytokeratin (CK) protein CK19 has been shown to have diagnostic use and prognostic significance in some types of human malignancy, but little is known of its distribution in normal endometrial mucosa or in endometrial endometrioid adenocarcinoma. However, we had observed that CK19 appeared to selectively label invasive tumor areas showing microcystic, elongated, and fragmented ("MELF") changes. Therefore, CK19 expression was assessed in 15 hysterectomy specimens showing normal proliferative, secretory, or atrophic endometrial appearances, and in 26 endometrioid adenocarcinoma cases with areas of MELF-type invasion. Normal endometrial glands were usually CK19 positive; however, there was more consistent expression in the functional layer, whereas basal zone epithelium was typically only focally stained. Proliferative epithelium frequently showed basal and apical cytoplasmic accentuation of staining. Endometrial carcinomas were also CK19 positive, but in most cases there was a distinct zonal pattern of expression with strong staining only in the central aspects of the larger tumor glands and weak-to-absent staining in peripheral glandular areas. In contrast, MELF-type tumor epithelium was consistently and strongly CK19 positive even when the adjacent "conventional"-type tumor glands were not stained. Intravascular tumor cells were also highlighted, including 2 cases in which this feature was not identified on hematoxylin and eosin stains. Thus CK19 immunohistochemistry was useful in showing the extent of myometrial invasion and subtle foci of lympho-vascular space invasion. Further studies are required to determine the mechanism and biologic significance of localized alterations in CK19 expression within endometrial neoplasms.

Fascin expression in low-grade uterine endometrioid adenocarcinoma: correlation with microcystic, elongated and fragmented (MELF)-type alteration at the deep invasive margin.

AIMS: The actin-binding protein fascin appears to potentiate the migratory capacity of both normal and neoplastic cells. It has been suggested that microcystic, elongated and fragmented (MELF) glands might represent areas of active invasion within uterine low-grade endometrioid adenocarcinomas. Therefore, fascin immunoreactivity was investigated in a series of endometrial carcinomas specifically comparing expression in conventional tumour areas, foci of MELF-type invasion and in stromal elements. METHODS AND RESULTS: Fascin expression was assessed in 28 uterine endometrioid adenocarcinomas and the results compared with cytokeratin (CK) 7 expression and with tumour morphology and distribution. The conventional glandular component of most tumours showed only focal fascin reactivity (<10% cells positive), but staining was more prominent within the peripheral epithelial cells. Foci of squamous/morular type differentiation were also positive. The neoplastic epithelium in MELF-type invasion usually showed strong fascin immunoreactivity, often contrasting with the adjacent negative or more weakly stained conventional tumour glands. There was also staining of reactive stromal cells surrounding MELF foci. CONCLUSIONS: There are distinct micro-anatomical variations in fascin immunoreactivity within endometrial carcinoma. The localized increase in fascin expression in MELF-type epithelium supports the proposal that MELF changes represent areas of active tumour invasion.