Elena Bussaglia

PTEN mutations in endometrial carcinomas: A molecular and clinicopathologic analysis of 38 cases

PTEN mutations have been reported to be frequent in endometrioid carinomas of the endometrium (EEC). Some correlation has been found between PTEN mutations and the presence of microsatellite instability (MI) in EEC, but no convincing cause-effect relationship for such association has been offered. DNA of 38 patients with endometrial carcinoma (EC) was extracted from blood and from fresh-frozen and paraffin-embedded tumor tissue. PTEN mutations were detected by single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. Results were correlated with MI status and clinicopathologic data. PTEN mutations were detected in 17 tumors (44.7%), and they were more frequent in endometrioid (EEC) (17 of 33, 51.5%) than in nonendometrioid carcinomas (NEEC) (0 of 5, 0%). PTEN mutational spectrum differed between MI+ and MItumors. PTEN mutations were detected in 9 of 15 MI+ tumors (60%), but in only 8 of 23 MI- neoplasms (34.8%). In EC with MI, PTEN mutations were detected in short coding mononucleotide repeats (A)s and (A)6 in 4 of 9 carcinomas (44.4%). These results confirm that PTEN is an important target gene in endometrial carcinogenesis. The occurrence of PTEN mutations in short coding mononucleotide repeats in MI-positive tumors suggests that these mutations may be secondary to deficiencies in mismatch repair and gives some explanation for the frequent presence of PTEN mutations in these tumors.

Immunohistochemical analysis of PTEN in endometrial carcinoma: a tissue microarray study with a comparison of four commercial antibodies in correlation with molecular abnormalities

The tumor suppressor gene PTEN/MMAC1 is located on chromosome 10q23.3. Inactivation of PTEN, either by mutations, deletions, or promoter hypermethylation, has been identified in a wide variety of tumors. Inactivation of the two alleles of PTEN is required, because it is a tumor suppressor gene. Immunohistochemical staining may be an effective screening method to demonstrate the absence of the protein in tumors exhibiting PTEN inactivation. We studied a tissue microarray, constructed from paraffin-embedded blocks of 95 endometrial carcinomas, 38 of them previously evaluated for alterations in PTEN. We also studied cell blocks obtained from one PTEN-defective endometrial cancer cell line, after transfection with either a plasmid encoding wild-type PTEN or the empty vector. The tumor samples were tested with four different anti-PTEN commercial antibodies: a polyclonal antibody, the monoclonal antibody 28H6, the monoclonal antibody 10P03, and the monoclonal antibody 6.H2.1. Results were correlated with the presence of abnormalities in PTEN, as well as with the immunohistochemical expression of phosphorylated AKT. Antibody 28H6 produced a predominant nuclear staining, while the other three antibodies produced a predominant cytoplasmic staining. There was no significant correlation between the results obtained with the four antibodies. The monoclonal antibody 6.H2.1 was the only one that exhibited a correlation with the presence of molecular alterations in PTEN, and a statistically significant association with immunostaining for phosphorylated AKT (r=−0.249, P=0.037). The monoclonal antibody 10P03 exhibited an association with phospho-AKT that did not have statistical significance. Both 6.H2.1 and 10P03 antibodies stained PTEN-transfected cells, and were negative in the PTEN-deficient cell line blocks. The polyclonal antibody and the monoclonal antibody 28H6 produced positive staining in PTEN-deficient cell line blocks, suggesting nonspecific staining. The results indicate that monoclonal antibody 6.H2.1 may be a suitable alternative for tumors with inactivation of PTEN.

Clonality analysis in synchronous or metachronous tumors of the female genital tract

The synchronous or metachronous development of carcinomas of the female genital tract is a well known phenomenon (1–4). In most cases the tumors are independent primaries arising in two separate locations as a result of a field effect involving the entire mullerian system. Less frequently, one of the carcinomas represents a metastasis from the other neoplasm. Although in such cases the tumors usually exhibit similar morphologic features, occasionally the metastatic carcinoma may appear different from the primary neoplasm as a result of tumor progression, leading to an erroneous diagnosis of independent primary tumors. In still other cases, the metastatic tumor reaches a disproportionate size in comparison with a small primary carcinoma, which may even be undetectable at operation. It is important to distinguish independent primary carcinomas from metastases because each situation obviously carries a different prognosis. This distinction is also of clear importance for clinical management of individual patients. For establishing the correct diagnosis the pathologist should pay attention not only to the microscopic findings but also to the clinical setting. In this review we consider the gross and microscopic pathologic features and the molecular methods that are useful in assessing the common or independent origin of two separate tumor cell populations in the female genital tract. We will first comment on the available techniques and then we will demonstrate their application in three different situations: 1) simultaneous carcinomas involving the endometrium and the ovaries; 2) development of malignant mixed mullerian tumors from preexisting ovarian carcinomas as a result of tumor dedifferentiation; and 3) mucinous tumors of the appendix with metastatic spread to the ovaries.

Bone marrow WT1 levels at diagnosis, post-induction and post-intensification in adult de novo AML.

We retrospectively assessed whether normalized bone marrow WT1 levels could be used for risk stratification in a consecutive series of 584 acute myeloid leukemia (AML) patients. A cutoff value of 5065 copies at diagnosis identified two prognostic groups (overall survival (OS): 44±3 vs 36±3%, P=0.023; leukemia-free survival (LFS): 47±3 vs 36±4%, P=0.038; and cumulative incidence of relapse (CIR): 37±3 vs 47±4%, P=:0.043). Three groups were identified on the basis of WT1 levels post-induction: Group 0 (WT1 between 0 and 17.5 copies, 134 patients, OS: 59±4%, LFS:59±4% and CIR: 26±4%); Group 1 (WT1 between 17.6 and 170.5 copies, 160 patients, OS: 48±5%, LFS:41±4% and CIR: 45±4%); and Group 2 (WT1 >170.5 copies, 71 patients, OS: 23±6%, LFS: 19±7% and CIR: 68±8%) (P<0.001). Post-intensification samples distinguished three groups: patients with WT1 >100 copies (47 patients, 16%); an intermediate group of patients with WT1 between 10 and 100 copies (148 patients, 52%); and a third group with WT1 <10 copies (92 patients, 32%). Outcomes differed significantly in terms of OS (30±7%, 59±4%, 72±5%), LFS (24±7%, 46±4%, 65±5%) and relapse probability (CIR 72±7%, 45±4%, 25±5%), all P<0.001. WT1 levels in bone marrow assayed using the standardized ELN method provide relevant prognostic information in de novo AML.

Malignant mullerian mixed tumor arising from ovarian serous carcinoma: a clinicopathologic and molecular study of two cases.

The clinical, histologic, and molecular pathologic features of two cases of malignant mullerian mixed tumor (MMMT) arising from ovarian papillary serous carcinoma are presented. Identical p53 mutations were detected in the primary ovarian carcinoma and the subsequent MMMT in each case.

Molecular pathology of atypical polypoid adenomyoma of the uterus

Atypical polypoid adenomyoma (APA) is an uncommon and benign tumor of the uterus. In some patients, however, APA has been found to coexist with or to precede the development of an endometrioid adenocarcinoma similarly to complex endometrial hyperplasia. The molecular changes underlying the progression from APA to adenocarcinoma are unknown. DNA from paraffin-embedded tissue of 6 APAs was evaluated for microsatellite instability (MI), MLH-1 promoter hypermethylation, and CTNNB-1 mutations. Tissue sections were also subjected to MLH-1, MSH-2, and beta-catenin immunostaining. MI was not detected in any case. Two tumors exhibited MLH-1 promoter hypermethylation and showed focal negative MHL-1 immunostaining; 1 of these showed marked architectural complexity and cellular pleomorphism. Five cases presented beta-catenin nuclear immunoreactivity, but none of them had CTNNB-1 mutations. The results of this study suggest that APA and complex endometrial hyperplasia may share some molecular alterations. Some APAs exhibit MLH-1 promoter hypermethylation with focal lack of MLH-1 immunostaining, a molecular abnormality involved in the transition from complex atypical hyperplasia to endometrioid adenocarcinoma.

Adverse impact of IDH1 and IDH2 mutations in primary AML: Experience of the Spanish CETLAM group

The study of genetic lesions in AML cells is helpful to define the prognosis of patients with this disease. This study analyzed the frequency and clinical impact of recently described gene alterations, isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) mutations, in a series of homogeneously treated patients with primary (de novo) AML. Two-hundred and seventy-five patients enrolled in the CETLAM 2003 protocol were analyzed. IDH1 and IDH2 mutations were investigated by well-established melting curve-analysis and direct sequencing (R140 IDH2 mutations). To establish the percentage of the mutated allele a pyrosequencing method was used. Patients were also studied for NPM, FLT3, MLL, CEBPA, TET2 and WT1 mutations. IDH1 or IDH2 mutations were identified in 23.3% AML cases and in 22.5% of those with a normal karyotype. In this latter group, mutations were associated with short overall survival. This adverse effect was even more evident in patients with the NPM or CEBPA mutated/FLT3 wt genotype. In all the cases analyzed, the normal allele was detected, suggesting that both mutations act as dominant oncogenes. No adverse clinical impact was observed in cases with TET2 mutations. IDH1 and IDH2 mutations are common genetic alterations in normal karyotype AML. Favourable genotype NPM or CEBPA mutated/FLT3 wt can be further categorized according to the IDH1 and IDH2 mutational status.

Immunophenotype of acute myeloid leukemia with NPM mutations: Prognostic impact of the leukemic compartment size

NPM mutations are the most common genetic abnormalities found in non-promyelocytic AML. NPM-positive patients usually show a normal karyotype, a peculiar morphologic appearance with frequent monocytic traits and good prognosis in the absence of an associated FLT3 mutation. This report describes the immunophenotypic and genetic characteristics of a consecutive series of NPM-mutated de novo AML patients enroled in the CETLAM trial. Eighty-three patients were included in the study. Complete immunophenotype was obtained using multiparametric flow cytometry. Associated genetic lesions (FLT3, MLL, CEBPA and WT1 mutations) were studied by standardized methods. Real-time PCR was employed to assess the minimal residual status. The most common pattern was CD34-CD15+ and HLA-DR+. Small CD34 populations with immunophenotypic aberrations (CD15 and CD19 coexpression, abnormal SSC) were detected even in CD34 negative samples. Nearly all cases expressed CD33 (strong positivity), CD13 and CD117, and all were CD123+. The stem cell marker CD110 was also positive in most cases. Biologic parameters such as a high percentage of intermediate CD45+ (blast gate) (>75% nucleated cells), CD123+ and FLT3-ITD mutations were associated with a poor outcome. Quantitative PCR positivity had no prognostic impact either after induction or at the end of chemotherapy. Only PCR positivity (greater than 10 copies) detected in patients in haematological remission was associated with an increased relapse rate. Further studies are required to determine whether the degree of leukemic stem cell expansion (CD45+CD123+cells) increases the risk of acquisition of FLT3-ITD and/or provides selective advantages.

Loss of heterozygosity on chromosome 17q in epithelial ovarian tumors: association with carcinomas with serous differentiation.

Loss of heterozygosity (LOH) on chromosome 17q is frequent in epithelial ovarian tumors, but its clinicopathologic significance remains to be elucidated. DNA of 50 patients with epithelial ovarian tumors was extracted from blood and from fresh-frozen and paraffin-embedded tissue (14 benign, 7 borderline, and 29 malignant). Six microsatellite loci were amplified by PCR (D17S250, TRHA1, D17S800, D17S855, D17S579, D17S513). LOH was scored by the absence or reduction of the signal to less than 50% of one of the alleles in tumor DNA compared with normal DNA. LOH was identified on chromosome 17q in at least one locus in 12 tumors (24%), all of them carcinomas (12 of 29 tumors, 41.3%). It occurred more frequently among high-grade serous carcinomas (8 of 14 tumors, 57%) and mixed endometrioid-serous carcinomas (2 of 5, 40%). LOH was detected in all informative markers of 10 tumors, suggesting the complete loss of an entire chromosome 17 homologue. Patients with LOH-positive carcinomas were older than those with LOH-negative malignant tumors (mean ages 67 and 49). The results support the hypothesis that LOH on chromosome 17q may be associated with the development of ovarian cancers in elderly patients, particularly with high-grade serous or mixed endometrioid-serous carcinomas.

WT1 monitoring in core binding factor AML: Comparison with specific chimeric products

Minimal residual disease may help to establish clinical decisions in patients with AML. WT1 offers the possibility to analyze those cases without currently known underlying genetic abnormalities. To compare the value of chimeric specific quantitative PCR with WT1 PCR in CBF acute leukemia, 445 samples from 96 AML (49 AML1-ETO+ and 47 CBFB-MYH11+) cases were included in the study. For each sample AML1-ETO or CBFB-MYH11 levels obtained using the conditions of the BIOMED group were compared with the results of WT1 levels using sensitive primers and conditions. Simultaneously, normal range expression of WT1 was established using RNA obtained from eight healthy donors. WT1 mutations were also investigated both at RNA and at the genomic level. The majority of CBF samples showed rises in WT1 levels (88.7%) at diagnosis. However, 18% of AML1-ETO showed WT1 levels below 250 copies in contrast with 5% CBFB-MYH11 cases. WT1 mutation was not detected in any case (70 diagnostic samples). We found correlation between WT1 levels at diagnosis and the CD34 blast population estimated by flow cytometry in CBFB-MYH11+ cases. We found no association between WT1 levels and clinical outcome. There was a high concordance between chimeric transcript analysis and WT1 levels in CR patients. Concordance was also high in relapsed patients (78% in AML1-ETO and 98% in CBFB-MYH11+ cases). Both WT1 and specific chimeric transcript identified and rescued false negative results of the other test. Additional studies are needed to determine whether the rare discrepancies are a reflection of the cooperative nature of WT1 overexpression or a consequence of the uneven distribution in the leukemic population. WT1 is a powerful MRD tool even in cases with currently available molecular targets.