Expanding the molecular landscape of undifferentiated sarcomas of bone with a novel EWSR1-SSX3 gene fusion.

Undifferentiated sarcomas characterized by a primitive monomorphic round to spindle cell phenotype and often non-specific immunoprofile remain difficult to subclassify outside molecular analysis. The increased application of RNA sequencing in clinical practice led to significant advances and discoveries of novel gene fusions that furthered our understanding and refined classification of otherwise undifferentiated neoplasms. In this study, we report an undifferentiated round to spindle cell sarcoma arising in the femur of a 34-year-old female. The round to spindle tumor cells were arranged in short fascicles, with focal rosette formation, within a hyalinized stroma. The tumor immunoprofile included diffuse reactivity for CD99, SATB2, and TLE1 and patchy positivity for Cyclin D1, Keratin AE1/AE3, synaptophysin, and chromogranin. Other markers, such as EMA, SMA, desmin, S100, ERG, and WT1, were negative. Fluorescence in situ hybridization analysis for EWSR1 gene alterations showed a break-apart signal and targeted RNA sequencing revealed an EWSR1::SSX3 gene fusion. The patient received neoadjuvant chemotherapy followed by surgery and subsequently relapsed in less than a year with lung metastasis. Larger series are needed to determine if this fusion defines a novel subset of undifferentiated tumors or represents a genomic variant of already existing primitive round cell sarcoma categories, such as Ewing sarcoma or synovial sarcoma.

The FOXO1-miR27 tandem regulates myometrial invasion in endometrioid endometrial adenocarcinoma.

Micro-RNA (miRNA) signatures influence the prognosis of cancer, but little is known about their role in myometrial invasion in endometrioid endometrial adenocarcinoma (EEC). We studied miRNA expression signatures in noninvasive and invasive EEC focusing on the alteration of miR-27 and its main target, FOXO1 as well as their relationship with the clinicopathological parameters and other genetic alterations such as PIK3CA mutations. In 25 tumors and 5 normal endometria, unsupervised hierarchical clustering analysis showed that normal endometria and noninvasive EEC were grouped together and separately from invasive and advanced stage tumors. Of the 20 miRNAs differentially expressed in noninvasive (stage IA) and myoinvasive adenocarcinomas (stage IB and IC), miR27 was overexpressed in invasive adenocarcinomas, and its expression increased linearly according to stage. Results were validated by quantitative real-time reverse transcription polymerase chain reaction in an independent series of 44 EEC. By in situ hybridization, miR-27 expression was limited to the stroma. Using quantitative real-time reverse transcription polymerase chain reaction, the expression of proapoptotic transcription factor FOXO1 was down-regulated in invasive compared with noninvasive tumors. Furthermore, we found that the expression of active caspase 3 was higher in noninvasive than invasive EEC. When stratified by PIK3CA mutations, all invasive tumors down-regulated FOXO1, but only nonmutated adenocarcinomas showed miR-27 overexpression. In conclusion, we propose that the miR27-FOXO1 tandem inhibits apoptosis and represents an alternative pathway for tumor cell survival in PIK3CA-nonmutated EEC.

Stromal signatures in endometrioid endometrial carcinomas.

The pattern of myometrial invasion in endometrioid endometrial carcinomas varies considerably; ie, from widely scattered glands and cell nests, often associated with a fibromyxoid stromal reaction (desmoplasia) and/or a lymphocytic infiltrate, to invasive glands with little or no stromal response. Recently, two distinct stromal signatures derived from a macrophage response (colony-stimulating factor 1, CSF1) and a fibroblastic response (desmoid-type fibromatosis, DTF) were identified in breast carcinomas and correlated with clinicopathologic features including outcome. In this study, we explored whether these stromal signatures also apply to endometrioid carcinomas and how their expression patterns correlated with morphologic changes. We studied the stromal signatures both by immunohistochemistry and in situ hybridization in 98 primary endometrioid carcinomas with (87 cases) and without (11 cases) myometrial invasion as well as in the corresponding regional lymph nodes metatases of 9 myoinvasive tumors. Desmoplasia correlated positively with the DTF expression signature. Likewise, mononuclear infiltrates were found in the stroma of tumors expressing CSF1. Twenty-four out of eighty-seven (27%) myoinvasive endometrioid carcinomas were positive for the macrophage signature and thirteen out of eighty-seven (15%) expressed the fibroblast signature. Eleven additional cases were positive for both DTF and CSF1 signatures (11/87; 13%). However, over half of the cases (39/87; 45%) and the majority of the non-myoinvasive tumors (8/11; 73%) failed to express any of the two stromal signatures. The macrophage response (CSF1) was associated with higher tumor grade, lymphovascular invasion, and PIK3CA mutations (P<0.05). There was a concordance in the expression of the CSF1 signature in the primary tumors and their corresponding lymph node metastases. This study is the first characterization of stromal signatures in endometrioid carcinomas. Our findings shed new light on the relationship between genetically different endometrioid carcinomas and various stromal responses. Preservation of the CSF1 macrophage stromal response in the metastases leds support to targeting the CSF1 pathway in endometrioid endometrial carcinomas.

Promoter hypermethylation contributes to TIMP3 down-regulation in high stage endometrioid endometrial carcinomas.

AIMS: Expression of tissue inhibitor of metalloproteinases-3 (TIMP-3) has been found to be decreased in several types of cancer by promoter gene hypermethylation. However, little is known regarding the silencing effect of TIMP3 promoter hypermethylation on gene and protein expression in endometrial carcinomas and its prognostic significance. METHODS AND RESULTS: TIMP3 promoter hypermethylation and gene copy number variations were evaluated using a methylation-specific multiplex ligation-dependent probe amplification approach in 60 cases of endometrioid endometrial carcinomas. TIMP3 expression was also evaluated at the transcript and protein levels. Loss of TIMP-3 protein expression was found in 44 (73%) of 60 carcinomas. Promoter hypermethylation was identified in 25% (15 of 60); was more frequent in stages II-IV (55%, six of 11) than in stage I (18%, nine of 49; P = 0.021); and was found more commonly in tumours with deep myometrial invasion. MLH1 and TIMP3 promoters were hypermethylated simultaneously in the same group of tumours (P < 0.001). A correlation between TIMP3 methylation and microsatellite instability (MSI) was found (P = 0.005). TIMP3 copy number changes were frequently a loss (35%), whereas a gain was detected in only 5%. CONCLUSIONS: TIMP3 promoter hypermethylation was associated with high stage endometrioid endometrial tumours with extrauterine spread. Nevertheless, promoter hypermethylation and loss of heterozygosity are not the only mechanisms for TIMP3 inactivation.

Prognostic significance of FOXL2 mutation and mRNA expression in adult and juvenile granulosa cell tumors of the ovary.

Recently, mutation of the FOXL2 gene has been consistently identified in adult granulosa cell tumors of the ovary. The purpose of this study is to investigate whether the FOXL2 mutation and mRNA expression have a role in the pathogenesis of juvenile and adult granulosa cell tumors and influence tumor progression. Thirty-four adult granulosa cell tumors and 20 juvenile granulosa cell tumors were examined for the presence of the FOXL2 (C402G) mutation. Expression levels were studied by quantitative PCR and immunohistochemistry. We found that FOXL2 (C402G) mutation was present in 19/27 (70%) of the adult type tumors but in none of the juvenile granulosa cell tumors (0/18). No correlation was encountered between the presence of FOXL2 mutation and various clinicopathologic parameters except for the presence of a different sex-cord component, which was more frequently found in the subgroup of wild-type adult granulosa cell tumors than in the mutated tumors. Patients with tumors harboring the FOXL2 (C402G) mutation had a worse disease-free survival than those with the wild-type gene. Expression levels of FOXL2 mRNA had an impact on disease-free survival in both adult and juvenile granulosa cell tumors. We also found that the mutated tumors had a higher immunohistochemical expression of the FOXL2 protein, and there was a linear correlation between mRNA and immunohistochemical FOXL2 expression in both adult and juvenile granulosa cell tumors. Patients with juvenile granulosa cell tumors and higher FOXL2 protein expression had worse overall survival and disease-free survival than those with negative or weakly immunoreactive tumors. Our data suggest that FOXL2 mutation and mRNA expression are of prognostic importance in both adult and juvenile granulosa cell tumors.

Low-density lipoprotein receptor-related protein 1 is associated with proliferation and invasiveness in Her-2/neu and triple-negative breast carcinomas.

Low-density lipoprotein receptor-related protein 1, a member of the low-density lipoprotein cholesterol receptor family, has been implicated in the progression of certain tumors; but it remains unclear whether it plays a role in infiltrating ductal breast carcinomas. We studied a series of 81 ductal breast tumors to determine the correlation of low-density lipoprotein receptor-related protein 1 overexpression with clinicopathologic and immunohistochemical characteristics associated with prognosis. Low-density lipoprotein receptor-related protein 1 overexpression was identified in 14% (11/81) of tumors and was correlated with a high nuclear grade (P = .043), high mitotic index (P = .006), and Ki-67 greater than 20% (P = .047). Furthermore, low-density lipoprotein receptor-related protein 1 expression was associated with aggressive carcinomas (triple-negative tumors [21%, 7/33] and Her-2/neu tumors [17%, 4/24]) but not with hormone-dependent carcinomas (0%, 0/24) (P = .040). There was no correlation between low-density lipoprotein receptor-related protein 1 expression and survival, but a trend was found between low-density lipoprotein receptor-related protein 1 overexpression and tumor recurrence. Low-density lipoprotein receptor-related protein 1 overexpression was related to proliferation and invasiveness in Her-2/neu and triple-negative breast carcinoma. Moreover, patients with low-density lipoprotein receptor-related protein 1-positive tumors had higher cholesterol levels (62.5%, 5/8) than those with low-density lipoprotein receptor-related protein 1-negative tumors (40%, 19/47). Nevertheless, the correlation between low-density lipoprotein receptor-related protein 1 and hypercholesterolemia was not statistically significant; but cholesterol levels were higher in patients with triple-negative breast carcinoma (60%, 15/25) and Her-2/neu carcinomas (40%, 6/15) than in luminal-A carcinomas (20%, 3/15) (P = .046). These findings suggest a relationship between hypercholesterolemia and aggressiveness of ductal breast carcinomas.

Gene expression analysis identifies two groups of ovarian high-grade serous carcinomas with different prognosis.

Gene expression profiling is an important tool to evaluate genetic heterogeneity in carcinomas and is useful to develop expression-based classifications for many types of cancer, as well as markers of disease outcome. In this study, we have investigated the expression profile of 22 genes involved in the PI3K-AKT pathway in 26 high-grade ovarian carcinomas (19 serous and 7 clear cell carcinomas). Unsupervised hierarchical clustering divided high-grade ovarian carcinomas into three groups. Although all clear cell carcinomas clustered in one group, high-grade serous carcinomas were segregated into two separate groups with different prognosis (P=0.05). High expression of CASP3, XIAP (X-linked inhibitor of apoptosis) , NFKB1, FAS, and GSK3B mRNAs identified high-grade serous carcinomas with better prognosis. In multivariate analysis, these cluster groups were of prognostic significance independent of age, tumor size, and tumor stage (P=0.008). To validate the mRNA expression data, we studied the immunohistochemical expression of caspase-3 and XIAP on a tissue microarray. Immunoreaction for caspase-3 was concordant with the results obtained by mRNA expression analysis (Spearman r=0.762, P=0.000). Caspase-3 was exclusively expressed by the macrophages. Furthermore, co-expression of caspase-3 and XIAP identified high-grade serous carcinomas with different prognosis (P=0.03). Our results suggest that there are different biological subtypes of high-grade serous carcinomas.

Repression of E-cadherin by SNAIL, ZEB1, and TWIST in invasive ductal carcinomas of the breast: a cooperative effort?

It has been suggested that down-regulation of E-cadherin in invasive breast ductal carcinomas is mediated by the aberrant expression of several of its transcriptional repressors, but their inhibitory role and clinical importance are not yet well established. We investigated gene and protein expression patterns of the E-cadherin repressors SNAIL, ZEB1, and TWIST in relation to clinicopathologic parameters, in a series of 88 patients with invasive breast ductal carcinomas. Up-regulation of SNAIL messenger RNA (P = .008) and down-regulation of TWIST (P = .022) were associated with triple-negative tumors, whereas ZEB1 gene expression was more frequent in hormone-positive tumors (P = .004). Loss of E-cadherin was found in 19% of the tumors, but it did not correlate with aberrant expression of any of the repressors investigated herein. Nonetheless, we found that ZEB-1 protein overexpression inversely correlated with high tumor grade (P = .018), nuclear grade (P = .002), and presence of lymph nodes (P = .001), and these data were consistent with the gene expression data for ZEB1. Clinically, down-regulation of ZEB1 messenger RNA was associated with poor overall survival (P = .011) and disease-free survival (P = .053), whereas patients with TWIST negative tumors had a worse overall survival (P = .008) and disease-free survival (P = .006). Our data indicate that deregulation of TWIST is somehow important in the aggressiveness of triple-negative carcinomas and poor patient outcome, whereas down-regulation of ZEB1 seems to play a role in tumor spread, metastases, and poor survival.

Myometrial invasion and lymph node metastasis in endometrioid carcinomas: tumor-associated macrophages, microvessel density, and HIF1A have a crucial role.

Myometrial invasion is an independent prognostic parameter of the endometrioid carcinomas which correlates with the risk of metastasis to pelvic and/or paraaortic lymph nodes. Recognition of myometrial invasion is sometimes difficult. In fact, myoinvasion is overdiagnosed in routine practice in as many as 25% of the cases. Recently, it has been observed that tumor-associated macrophages stimulate angiogenesis and promote cancer dissemination. Tumor macrophages (CD163), microvessel density (CD31), and hypoxia inducible factor 1 α subunit (HIF1A) were investigated in 64 primary endometrioid carcinomas with (50 cases) and without (14 cases) myometrial invasion as well as in the corresponding regional lymph nodes metastases of 20 of the myoinvasive tumors. Endometrioid carcinomas with myometrial invasion showed higher number of CD163-tumor macrophages and greater microvessel density than endometrioid carcinomas without myometrial invasion (P=0.000 and P=0.000, respectively). In carcinomas confined to the corpus uteri (stage I), expression of HIF1A was associated with deep myoinvasion (stage IC) (P=0.006). There was a significant relationship between microvessel density and CD163-macrophages both in the myoinvasive and nonmyoinvasive tumors. On the other hand, high-grade endometrioid carcinomas had more macrophage infiltrates and microvessels than low-grade tumors (P=0.03 and P=0.07). Also, there was a positive correlation between CD163-macrophages and microvessel density in the primary tumors and their corresponding regional lymph node metastases. These findings link increased microvessel proliferation to stromal macrophage infiltrate and suggest that enhanced tumor angiogenesis, triggered by stromal macrophages, regulates the progression of endometrioid carcinomas. The identical stroma microenvironment found in the primary and the corresponding metastatic tumor suggests that tumor stroma response is determined by the intrinsic biology of the tumor.

Expression profiling of 22 genes involved in the PI3K-AKT pathway identifies two subgroups of high-grade endometrial carcinomas with different molecular alterations.

Previously, we showed that PIK3CA and p53 alterations in uterine endometrial carcinomas correlate with poor prognosis. However, the contribution of phosphatidylinositol 3-kinase (PI3K) -AKT deregulation to endometrial carcinogenesis is not completely understood. The purpose of this study was to analyze alterations of this pathway in endometrial carcinomas and correlate them with the most common genetic abnormalities. Expression profiling of 22 genes involved in PI3K-AKT signaling pathway was analyzed in 38 endometrial carcinomas using TaqMan low-density array (TLDA) analysis. The gene expression pattern was analyzed by hierarchical clustering analysis. Unsupervised clustering divided the high-grade endometrial carcinomas into two clusters. One cluster identified tumors with alterations in the PI3K-AKT signaling pathway (exon 20 PIK3CA mutations and/or PTEN mutations 9/15; 60%), and p16 protein overexpression (8/13; 62%). Almost all non-endometrioid adenocarcinomas (serous and clear cell adenocarcinomas) were segregated into this cluster. In contrast, the other cluster identified tumors with p53 alterations (6/6; 100%), p16 protein overexpression (5/5; 100%), and exon 9 PIK3CA mutations (2/6; 33%). Exon 20 PIK3CA and PTEN mutations were not found in this subgroup. Low-grade endometrial carcinomas clustered in a third subgroup characterized by high frequency of PTEN mutations (10/17; 59%) and microsatellite instability (6/17; 35%). Our results show that gene expression profile differences in the PI3K-AKT signaling pathway identify two subgroups of high-grade endometrial carcinomas with different molecular alterations (PI3K-AKT pathway vs p53 alterations) that may have distinct roles in endometrial carcinogenesis. Identification of these subgroups can provide insight into the biology of these tumors and may facilitate the development of future treatments.

Synchronous bilateral breast carcinoma in a patient with cowden syndrome: a case report with morphologic, immunohistochemical and genetic analysis.

Synchronous bilateral breast carcinoma (SBBC) and early onset are important characteristics of hereditary cases. The lifetime risk for breast carcinoma in Cowden syndrome (CS) is estimated to be 25-50%. We reported a 44-year-old woman presenting SBBC and characteristic mucocutaneous lesions of CS, confirmed by PTEN gene mutation analysis. Bilateral modified mastectomy and axillary dissection were performed. Histopathologic examination revealed a moderate-differentiated invasive ductal carcinoma with mixed features of luminal A immunophenotype (Estrogen and/or Progesterone Receptors >50% and/or Ki67 < 30% of positive cells). The skin lesions showed the characteristic findings of tricholemmoma. Lack of PTEN expression was observed in all specimens. Sequencing analysis confirmed the presence of PTEN splice-acceptor site mutation in intron 8 (c.1027-2A>G), a germline mutation which had not been previously reported in CS. The patient received adjuvant chemotherapy and tamoxifen for 5 years. After 5 years of follow-up, she persists recurrence-free. SBBC with early onset suggests a hereditary predisposition. Thus, analysis of PTEN expression abnormality, easily assessed by immunohistochemistry, may be of clinical value to screen those patients with CS.

Concomitant PI3K-AKT and p53 alterations in endometrial carcinomas are associated with poor prognosis.

The status of p53 and the phosphatidylinositol 3-kinase-AKT (PI3K-AKT) signaling pathway was investigated in 132 endometrial carcinomas, including endometrioid endometrial carcinomas, non-endometrioid endometrial carcinomas, and mixed endometrioid adenocarcinomas-non-endometrioid adenocarcinomas. Results were compared with the clinicopathologic parameters associated with prognosis, patients' follow-up, and other genetic alterations found frequently in these tumors. Molecular genetic differences between low-grade and high-grade endometrioid adenocarcinomas were encountered; ie, PIK3CA mutations were detected in 26 and 34% of cases, respectively. We found p53 alterations in only 17% of high-grade endometrioid adenocarcinomas. In contrast, non-endometrioid adenocarcinomas had a higher frequency of p53 alterations (54%), PIK3CA mRNA overexpression (45%), and exon 20 PIK3CA mutations (21%). In the mixed endometrioid adenocarcinomas-non-endometrioid adenocarcinomas, the most frequent alterations were p53 (50%) and PIK3CA (44%) mutations, followed by PTEN mutations (38%). In some cases, p53 and PIK3CA alterations coexisted, but they rarely coexisted with the PTEN mutations. Our findings suggest that the PIK3CA mutations are frequent events in endometrial carcinomas of any histological type. However, location of the PIK3CA mutations, either in exon 9 or exon 20, varies significantly according to the histologic grade and type of carcinoma. Carcinomas with exon 20 PIK3CA mutations or PIK3CA mRNA overexpression were often high-grade carcinomas associated with myometrial invasion; in contrast, tumors that carried exon 9 mutations were more likely to be low-grade carcinomas. The Kaplan-Meier analysis suggested that p53 alterations (strong immunoexpression or mutations) conferred a worse prognosis (P=0.000). Although alterations in the PI3K-AKT signaling pathway alone did not influence overall survival, patients with deregulated PI3K-AKT pathway (PIK3CA and/or PTEN alterations) and p53 alterations had shorter survival (P=0.000) than patients with only p53 alterations. Such a relationship was lost when we considered exon 9 PIK3CA mutations. Our results contribute to further characterize the molecular genetic model for endometrial carcinogenesis.

Exon 20 PIK3CA mutations decreases survival in aggressive (HER-2 positive) breast carcinomas.

PIK3CA mutations at 9 and 20 exons were studied in a series of 56 selected aggressive breast carcinomas (BC): 27 with Her-2 over-expression and negativity for estrogen receptors (ER) and progesterone receptors (PR), and 29 "triple negative" BC (negative for ER, PR and Her-2). Also, immunohistochemical studies of p53, ki-67, Her-1 (EGFR), pIGF-1R, PTEN, p110alpha, and pAkt were performed. Six mutations in exon 20 PIK3CA were identified among the 27 Her-2 positive BC, whereas only one exon 9 PIK3CA mutation was detected in a triple negative tumor (p = 0.035). Furthermore, PIK3CA mutations were associated with p110alpha over-expression (p = 0.001). Overall survival was shorter in cases with PIK3CA mutations (p = 0.015 in all series; and p = 0.041 for Her-2+ tumors), although multivariate analyses did not show statistical differences. No statistical significance was related with disease-free survival. Exon 20 PIK3CA mutations are relatively frequent in Her-2+ tumors and shorten survival, whereas neither exons 9 and 20 mutations seem related with "triple negative" breast carcinomas.

Expression profile of heptahelical putative membrane progesterone receptors in epithelial ovarian tumors.

A novel class of putative progestin binding proteins has been recently identified as potential mediators of rapid nongenomic hormone actions. The proteins designated membrane progestin receptor (mPR) alpha, beta, and gamma were initially discovered in fish and shown to have a role in oocyte maturation. The predicted multiple membrane spanning domain structure of the mPRs resembles that of heptahelical G-protein-coupled receptors. Phylogenetic analysis indicated that the mPRs belong to the large progestin and adiponectin Q receptor (PAQR) gene family. Based on the reported expression of the 3 mPRs in hormone-responsive tissues of the female reproductive tract and on the role of steroid hormones in cancer, we investigated the expression of these novel progestin receptors in epithelial tumors of the ovary. The transcript levels of the 3 human mPR/PAQRs were assessed by semiquantitative reverse transcriptase polymerase chain reaction in 28 ovarian samples, including normal tissues, cystadenomas, borderline tumors, and common types of ovarian carcinomas. Two of the 3 transcripts for the mPR/PAQRs proteins appeared differentially expressed in the tumors examined. Expression of mPR alpha and beta was demonstrated in ovarian tumors at both messenger RNA and protein level, and their expression appeared to be independent of the expression of the classic nuclear progestin receptors. Expression of mPR gamma (PAQR V) was elevated in endometrioid and clear cell carcinomas, 2 related neoplastic counterparts of hormonally responsive tissues, suggesting a potential role of the mPR/PAQRs in the pathogenesis of epithelial ovarian tumors.

Promoter hypermethylation and reduced expression of RASSF1A are frequent molecular alterations of endometrial carcinoma.

Alterations in the regulation of the RAS-MAPK pathway are frequent in endometrial carcinoma. RASSF1A is a tumor-suppressor gene that can regulate this pathway negatively. RASSF1A has been found to be inactivated by promoter methylation in some human tumors. The aim of the study was to assess the immunohistochemical expression of RASSF1A in normal endometrium and endometrial carcinoma, and to correlate its expression with K-RAS mutations, presence of microsatellite instability, RASSF1A promoter methylation, and clinicopathological data. RASSF1A immunostaining was evaluated in one tissue microarray constructed from 80 paraffin-embedded samples of normal endometrium, and two tissue microarrays constructed with a total of 157 endometrial carcinomas (one constructed with 95 endometrial carcinomas previously evaluated for K-RAS mutations, and microsatellite instability, and another one containing 62 endometrial carcinomas that were also subjected to RASSF1A promoter methylation analysis). RASSF1A immunostaining was correlated with cell proliferation (Ki67), Cyclin D1 expression and clinicopathological data. Promoter methylation of RASSF1A was assessed by methylation-specific PCR. RASSF1A immunostaining was variable during the menstrual cycle in normal endometrium. RASSF1A expression was significantly reduced in 48% of endometrial carcinomas, particularly in tumors exhibiting microsatellite instability. RASSF1A-promoter methylation was very frequent in endometrial carcinoma (74%), and was frequently associated with reduced expression of RASSF1A. RASSF1A-promoter hypermethylation was common in advanced-stage endometrial carcinoma. The results suggest that reduced expression of RASSF1A may play a role in endometrial carcinogenesis by controlling cell proliferation and apoptosis through the MAPK-signaling pathway.

PIK3CA mutations in the kinase domain (exon 20) of uterine endometrial adenocarcinomas are associated with adverse prognostic parameters.

Mutations of the oncogene PIK3CA occur frequently in endometrial carcinomas, but their prognostic significance is unclear. To determine the clinicopathological and molecular implications of these mutations, PIK3CA status was investigated in 109 endometrial (102 endometrioid and 7 mixed) carcinomas and the results were compared with clinicopathological parameters associated with prognosis. Tumors were also investigated for microsatellite instability and PTEN, beta-catenin gene (CTNNB1), K-RAS, and B-RAF mutations. We found 35 PIK3CA somatic missense mutations in 32 (29%) endometrial carcinomas. Eighteen mutations occurred in exon 20 (kinase domain), and 17 in exon 9 (helical domain). Almost all mutated tumors were pure endometrioid adenocarcinomas. All tumors with PIK3CA mutations exhibited myometrial invasion (P=0.032). Lymphovascular invasion was found more frequently in mutated (28%) than nonmutated carcinomas (18%). Histological grade varied significantly according to the location of the PIK3CA mutations whether in exon 9 or exon 20 (P=0.033). The frequency of exon 9 mutations was higher in grade 1 carcinomas (57%) than in grade 2 (29%) or grade 3 (14%) tumors. Conversely, mutations in exon 20 were more common in grade 3 (60%) than in grade 2 (20%) or grade 1 (20%) carcinomas. None of the tumors confined to the endometrium (stage IA) had PIK3CA mutations. Furthermore, whereas 64% of adenocarcinomas with exon 9 mutations had invaded < or =(1/2) of the myometrial thickness (stage IB), 73% of tumors with exon 20 mutations had either deeper myometrial invasion (stage IC) or cervical involvement (stage II) (P=0.045). PIK3CA mutations coexisted with microsatellite instability and mutations in PTEN, CTNNB1, K-RAS, and B-RAF genes. These results favor that PIK3CA mutations are associated with myometrial invasion and, moreover, that tumors harboring PIK3CA mutations in exon 20 are frequently high-grade, deeply invasive endometrial carcinomas that tend to exhibit lymphovascular invasion.

Prognostic significance of the Fas-receptor/Fas-ligand system in cervical squamous cell carcinoma.

We studied whether Fas-receptor (Fas-R; CD95) expression, single-nucleotide polymorphisms (SNPs) in the Fas promoter region, and/or Fas-ligand (Fas-L) production could determine individual susceptibility to cervical cancer progression. The clinicopathologic features of 38 patients with cervical squamous carcinomas (22 stage I, 8 stage II, and 8 stage III+) were reviewed and related with: (a) Fas-R expression by immunohistochemistry; (b) Fas-R SNPs at -670 and -1377 locations by restriction fragment length polymorphism and DNA sequencing; and (c) Fas-L expression by immunohistochemistry. Overall and disease-free survival curves showed significant differences in relation to stage (p < 0.001). Fas-R was identified in 20 of 38 (52.6%) tumors without statistical differences in survival, stage, or Fas-L overproduction. Fas-R GG genotype was more common than expected in advanced tumors (p = 0.065). The Fas-R-1377A allele and AA genotype were unrelated with survival, stage, or Fas-R expression. Fas-L overproduction was detected in 20 of 38 (52.6%) tumors; it was more frequent in advanced-stage tumors and was inversely related to survival (p = 0.03) and decrease in host inflammatory response (p = 0.01). Fas-R expression by tumor cells seems unrelated to stage or lymphoid infiltrate. Tumor production of Fas-L may represent an attempt to destroy the host's lymphocytic reaction.

Endometrial carcinoma: pathology and genetics.

In the Western world, endometrial carcinoma is the most common malignant tumour of the female genital tract and the fourth most common cancer in women after carcinomas of breast, colorectum, and lung. The annual incidence has been estimated at 10-20 per 100 000 women. In the United States, endometrial carcinoma accounts for approximately 6000 deaths per year. Two different clinicopathological subtypes are recognised: the oestrogen-related (type I, endometrioid) and the non-oestrogen related (type II, non-endometrioid). The clinicopathological differences are parallelled by specific genetic alterations, with type I showing microsatellite instability and mutations in PTEN, PIK3CA, K-Ras, and CTNNB1 (beta-catenin), and type II exhibiting p53 mutations and chromosomal instability. This article reviews the genetic changes of endometrial carcinogenesis in the light of morphological features of the tumours and their precursors.

Is loss of heterozygosity at 9q22.3 (PTCH gene) and 19p13.3 (STK11 gene) involved in the pathogenesis of ovarian stromal tumors?

Some ovarian fibromas and rare fibrosarcomas are associated with Gorlin syndrome, which is caused by mutation in the human homologue of Drosophila patched gene (PTCH), localized on chromosome 9q22.3. The relationship between PTCH gene and sporadic ovarian tumors in the thecoma-fibroma group has not been well characterized. On the other hand, we have recently described loss of heterozygosity (LOH) at 19p13.3 in 2 sporadic fibromas with sex-cord elements. We have analyzed DNA from 8 fibromas, 6 cellular fibromas, 2 fibrothecomas, 9 luteinized thecomas, and 2 fibrosarcomas of the ovary for LOH at 9q22.3 and 19p13.3, using polymerase chain reaction amplification for 10 microsatellite markers. LOH at 9q22.3 was detected in 4 (67%) of 6 cellular fibromas, with the highest frequency at microsatellite marker D9S15, which localizes proximal to the PTCH gene. Of 9 luteinized thecomas, 2 (22%) also exhibited LOH at 9q22.3 with 3 microsatellite markers other than D9S15. Allelic losses were not detected in any fibroma, fibrothecoma, or fibrosarcoma. LOH at 19p13.3 was found in 2 (25%) of 8 fibromas, 3 (50%) of 6 cellular fibromas, and 1 (11%) of 9 luteinized thecomas. None of the 2 fibrothecomas or 2 fibrosarcomas showed LOH at 19p13.3. LOH at both 9p22.3 and 19p13.3 was observed in 3 (50%) of 6 cellular fibromas, but not in luteinized thecomas. The results indicate that (1) LOH at both PTCH gene and STK11 gene is relatively frequent in cellular fibromas; (2) approximately a quarter of luteinized thecomas exhibited LOH of the PTCH gene; in both neoplasms, cellular fibromas and luteinized thecomas, LOH may play a role in their pathogenesis; and (3) sporadic cellular fibromas may arise through similar genetic pathways as cases of Gorlin syndrome.