Ileana Carnevali

Prognostic factors for ampullary adenocarcinomas: tumor stage, tumor histology, tumor location, immunohistochemistry and microsatellite instability.

Prognostic factors for ampullary carcinomas (ACs) are poorly defined. Fifty three resected ACs were analyzed for CDX2, MUC1, MUC5AC, MUC6, MUC2, and for mismatch repair proteins (hMLH1, hMSH2, PMS2, hMSH6) using immunohistochemistry. Microsatellite instability (MSI) status was evaluated by fluorescently labeled PCR using an automated sequencer. Univariate and multivariate analysis was performed for clinicopathological, immunohistochemical and molecular parameters. CDX2 was found in 32 out of 53 (60%) ACs with a significantly higher frequency among intestinal ACs compared with biliopancreatic (BP) ACs. The MUC1, MUC5AC, MUC6, MUC2 apomucins were expressed in 75, 43, 39, and 28% of ACs, respectively, with a significantly higher coexpression of MUC1/MUC5AC in BP ACs. MSI and loss of expression of hMLH1/PMS2 or hMSH2/hMSH6 proteins were observed only in intestinal ACs. Factors significantly correlated with improved survival in the univariate analysis were: low stage, absence of lymph nodes metastases, negative surgical margins (R0 status), and presence of MSI. In the multivariate analysis, stage was the only independent prognostic factor of survival. We conclude that stage is the only independent prognostic factor of survival in the multivariate analysis, whereas histological criteria and the immunohistochemical expression of apomucins and CDX2 are helpful in the classification and understanding of the histogenesis of ACs.

The High Frequency of De novo Promoter Methylation in Synchronous Primary Endometrial and Ovarian Carcinomas

Purpose: The methylation status of hMLH1, CDKN2A, and MGMT was investigated in a panel of synchronous cancers of the ovary and endometrium, fulfilling the clinicopathologic criteria for independent primary tumors to define the possible role of epigenetic mechanisms in the development of these cancers. Experimental Design: Bisulfite-converted DNA from 31 tumors (13 endometrial and 18 ovarian carcinomas) and from matched normal tissue of 13 patients was analyzed by a methylation-specific PCR assay at the CpG-rich 5′ regions of all three genes. In all tumors, we also investigated the presence of microsatellite instability and hMLH1 immunohistochemical expression in relation to hMLH1 hypermethylation status. Results: Methylation of hMLH1, CDKN2A, and MGMT was detected in 39%, 41%, and 48% of endometrial and ovarian tumors, respectively. hMLH1 hypermethylation was observed in all tumors of five patients, and it was invariably associated with loss of hMLH1 protein and presence of microsatellite instability. CDKN2A and MGMT methylation was randomly detected among both endometrial (45% and 24% of cases, respectively) and ovarian carcinomas (39% and 39% of cases, respectively). Concordant methylation at two or three genes was observed in 35% of cases. Conclusions: Epigenetic inactivation of hMLH1, CDKN2A, and MGMT may be a common and early event in the development of synchronous primary endometrial and ovarian carcinomas and may qualify as a marker of a field cancerization encompassing the ovary and endometrium. Detection of MGMT hypermethylation may be useful to define a set of gynecologic malignancies with a specific sensitivity to alkylating chemotherapy.

Histidine Decarboxylase, DOPA Decarboxylase, and Vesicular Monoamine Transporter 2 Expression in Neuroendocrine Tumors: Immunohistochemical Study and Gene Expression Analysis:

Histidine decarboxylase (HDC) and vesicular monoamine transporter 2 (v-MAT2) are involved in the biosynthesis and storage of histamine. DOPA decarboxylase (DDC) is involved in the biosynthesis of a variety of amines and shares a high degree of homology with HDC. HDC and v-MAT2 immunoreactivities (IR) have recently been detected in well-differentiated neuroendocrine tumors (WDNETs) and poorly differentiated neuroen-docrine carcinomas (PDNECs) of various sites and have been proposed as general endocrine markers. We evaluated HDC and v-MAT2 IR in a series of 117 WDNETs and PDNECs from different sites. Western blotting analysis was performed to verify the specificity of anti-DDC and anti-HDC antibodies. Real-time RT-PCR was performed using specific probes for HDC and DDC on 42 cases, examined also for DDC IR. HDC and v-MAT2 IR were observed in the majority of WDNETs and PDNECs of all sites and HDC-IR cases were always also DDC-IR. In contrast, high levels of HDC mRNA were detected only in the gastroenteropancreatic WDNETs, which did not show increased DDC mRNA levels. On the other hand, bronchial carcinoids and lung PDNECs showed high DDC mRNA levels, but nearly undetectable HDC mRNA levels. Western blotting analysis showed a cross-reaction between anti-HDC and anti-DDC antibodies. HDC should not be considered as a general endocrine marker and HDC IR in bronchial carcinoids and PDNECs of the lung can probably be attributed to a cross-reaction with DDC.

Up-regulation of the hypoxia-inducible factor–1 transcriptional pathway in colorectal carcinomas

The aim of the study was to identify the impact on prognosis of hypoxia-inducible factor-1 genetic program in colorectal carcinomas and to develop an experimental procedure that would allow a reliable quantitative gene expression analysis in formalin-fixed and paraffin-embedded tissue. The expression of hypoxia-inducible factor-1alpha and 13 hypoxia-inducible factor-1 target genes (AMF, CAIX, VEGF, VEGFR1, VEGFR2, HGF, MET, TGFalpha, EGFR, IGF2, MMP2, PLAUR, NIX) was quantified by real-time polymerase chain reaction in 18 colorectal, poorly differentiated neuroendocrine carcinomas and in 60 invasive colorectal carcinomas. Moreover, hypoxia-inducible factor-1alpha protein expression was evaluated by immunohistochemistry. High levels of hypoxia-inducible factor-1alpha were positively associated with poorly differentiated neuroendocrine carcinoma histology (P < .005), poor differentiation (P < .025), presence of necrosis, and presence of microsatellite instability (P < .05). AMF, TGFalpha, IGF2, NIX, VEGF, and VEGFR2 transcripts were significantly higher in the very aggressive poorly differentiated neuroendocrine carcinomas than in exocrine colorectal carcinomas and TGFalpha expression was significantly associated with presence of lymph nodal metastases (P < .05). High levels of TGFalpha and NIX were significantly associated with decreased overall survival (P < .001; P < .01). The multivariate analysis showed that advanced stage, presence of lymph node metastases, and high TGFalpha expression had an independent effect on survival (P < .006; P < .01; P < .0006). Our study suggests an up-regulation of the hypoxia-inducible factor-1 transcriptional pathway in colorectal carcinomas. hypoxia-inducible factor-1alpha overexpression alone, has no impact on the prognosis of colorectal carcinomas likely because the consequences of hypoxia-inducible factor-1alpha expression/stabilization strongly depend on the genetic background of the transformed cells. Mechanisms leading to increased synthesis of hypoxia-inducible factor-1alpha mRNA via autocrine growth factor loops may play a crucial role in invasive growth in this site.

Allelotypes and Fluorescence In situ Hybridization Profiles of Poorly Differentiated Endocrine Carcinomas of Different Sites

Purpose: The aim of this work was to investigate the genotypic profiles of 36 poorly differentiated endocrine carcinoma (PDEC) of different sites to verify if their very similar phenotype may reflect similar pattern of genetic anomalies and if useful diagnostic or prognostic markers may be pointed out. Experimental Design: All tumors were microallelotyped at 57 microsatellite on 11 autosomes and the allelotypes of a selected panel of tumors were validated by interphasic fluorescence in situ hybridization with centromeric probes for chromosomes 1, 3, 6, 11, 17, and 18 and a probe specific for p53. Results: Regardless of the primary sites, PDECs exhibit very complex allelotypes (86%) and TP53 allelic imbalance (89%). Among these cases, fluorescence in situ hybridization analysis confirmed the presence of multiple aneusomies and a chromosome instability phenotype. Very low percentage of allelic imbalance (AI) and few aneuploidies were detected in only five PDECs for which an overall longer survival was observed. We found recurrent AI on 3p, 5, and 11q13 in lung PDECs, on 5q21, 8p, and 18q21 in colorectal PDECs and on 7 and 11q22 in gastric PDECs. Significantly better outcome was observed in patients with PDEC exhibiting 8q AIs and absence of AI at chromosome regions 6q25 and 6p. Conclusions: The concurrence of p53 inactivation and aneuploidies or chromosome instability are the main features of PDECs. However, the specific allelotypes observed in relation to primary site support the hypothesis that PDECs and exocrine carcinomas of all sites may share early pathogenetic mechanisms. Molecular markers of potential diagnostic and prognostic values for PDECs of different sites have been identified.

Hierarchical clustering analysis of pathologic and molecular data identifies prognostically and biologically distinct groups of colorectal carcinomas

This work has evaluated the potential superiority of a morphomolecular classification based on the combination of clinicopathologic and molecular features of colorectal cancers. A cohort of 126 colorectal carcinomas was investigated by unsupervised hierarchical clustering analysis to combine 13 routinely assessed clinicopathologic features and all five molecular markers recently suggested by Jass’ classification to distinguish four molecular subtypes of sporadic colorectal carcinomas. Survival analysis was assessed by a Cox proportional hazards model. A clear separation into three prognostically significant groups was identified: cluster A and cluster C were associated with good prognosis and cluster B with poor prognosis (P=0.006). Clinicopathologic and molecular features of cluster A and cluster B tumors were strongly concordant with colorectal cancer profiles characterized by microsatellite instability or by chromosomal instability, respectively. The clinicopathologic features of cluster C tumors were suggestive of a less aggressive disease than cluster B tumors. Genetically, they appeared intermediate between cluster A and cluster B tumors, as they were mainly microsatellite stable tumors showing high levels of both MGMT methylation and loss of heterozygosity. Chromosomal instability was significantly lower in cluster C than in cluster B tumors. A more accurate tumor classification should combine the prognostic power of clinicopathologic parameters with molecular biomarkers that provide information regarding the natural history of the cancer. Hierarchical clustering seems to be a useful, promising and powerful tool for further translational studies and should lead us to define a diagnostic and prognostic signature for different carcinomas.

MGMT methylation in diffuse large B-cell lymphoma: validation of quantitative methylation-specific PCR and comparison with MGMT protein expression

Aims: (1) To validate a quantitative real time methylation specific PCR assay (MethyLight) for the detection of O6-methylguanine-DNA methyltransferase (MGMT) gene methylation status (MS) in diffuse large B-cell lymphoma (DLBCL). (2) To determine the immunohistochemical (IHC) expression of the MGMT protein and correlate it with MS. Both IHC and MethyLight results were compared with patient’s outcome. Methods: 71 patients with primary nodal DLBCL were studied. MGMT immunoreactivity was detected using a specific monoclonal antibody. The MS of MGMT gene was analysed in 52/71 DLBCL using MethyLight. A selected subset of 40 DLBCL was also analysed using qualitative methylation-specific PCR (MSP). Statistical analysis of overall survival (OS), lymphoma-specific survival (LSS) and progression free survival (PFS) was performed according to IHC and MS results. Results: 19/71 DLBCLs (27%) were MGMT-negative at IHC; all were analysed, together with 33/52 MGMT-positive DLBCLs. MethyLight showed a better performance than MSP. There was a good correlation between the presence of MGMT expression and the unmethylated status; the absence of IHC expression was poorly correlated with the presence of methylation. Better OS, LSS and PFS was found in DLBCLs with MGMT gene methylation. DLBCLs not expressing MGMT at IHC showed a longer PFS. Conclusions: The quantitative real-time methylation-specific PCR assay for the detection of MGMT gene hypermethylation has been validated for the first time in DLBCL. Immunohistochemistry seems to represent an useful preliminary test to identify unmethylated cases; MS analysis may be performed in non-immunoreactive cases to identify truly methylated DLBCLs, which bear a better prognosis.

Localization of Carboxyl Ester Lipase in Human Pituitary Gland and Pituitary Adenomas

Carboxyl ester lipase (CEL) is an enzyme that hydrolyzes a wide variety of lipid substrates, including ceramides, which are known to show inhibitory regulation of pituitary hormone secretion in experimental models. Because no studies on CEL expression in human pituitary and pituitary adenomas have been reported in the literature, we investigated CEL expression in 10 normal pituitary glands and 86 well-characterized pituitary adenomas [12 FSH/LH cell, 17 α-subunit/null cell, 6 TSH cell, 21 ACTH cell, 11 prolactin (PRL) cell, and 19 GH cell adenomas] using IHC, immunoelectron microscopy, Western blotting, and quantitative RT-PCR. In normal adenohypophysis, CEL was localized in GH, ACTH, and TSH cells. In adenomas, it was mainly found in functioning GH, ACTH, and TSH tumors, whereas its expression was poor in the corresponding silent adenomas and was lacking in FSH/LH cell, null cell, and PRL cell adenomas. Ultrastructurally, CEL was localized in secretory granules close to their membranes. This is the first study demonstrating CEL expression in normal human pituitary glands and in functioning GH, ACTH, and TSH adenomas. Considering that CEL hydrolyzes ceramides, inactivating their inhibitory function on pituitary hormone secretion, our findings suggest a possible role of CEL in the regulation of hormone secretion in both normal and adenomatous pituitary cells.

Identification of the first case of germline duplication of BRCA1 exon 13 in an Italian family

In this work we report for the first time a family in Italy with the BRCA1 ins6kbEx13 mutation, a recurrent founder mutation originating from northern Britain. After the initial identification of exon 13 duplication by multiplex ligation-dependent probe amplification (MLPA assay), we confirmed the identity of the alteration with the previously published BRCA1 ins6kbEx13 mutation, by mutation specific PCR and RT-PCR assays and by haplotype analysis. As rarely reported previously, the MLPA assay was also used to examine DNA from formalin fixed paraffin embedded (FFPE) normal tissues of other affected subjects in the family and it was the only effective method to perform a complete segregation analysis of the BRCA1 ins6kbEx13 mutation in the family. A combination of different approaches including MLPA analysis, haplotype analysis and LOH study on tumor samples of all the affected members allowed to reassess the maternal transmission of the mutation expected by the pedigree analysis. Moreover, detailed morphological analysis of breast cancers of BRCA1 ins6kbEx13 mutation carriers demonstrated a rare histological variant of breast carcinomas that has never been described in patients carrying BRCA1 mutations. Our study confirms the MLPA technique as a reliable and effective method for a primary screening for BRCA1 rearrangements also by using FFPE tissues and strongly suggests that histo-pathological, immunonohistochemical and molecular information from FFPE tumor tissues should be more often considered and integrated into routine diagnostic practice of hereditary tumors.

Two Cases of Carcinosarcomas of the Ovary Involved in Hereditary Cancer Syndromes.

Ovarian carcinosarcomas (OCS), also known as malignant mixed mesodermal/Müllerian tumors, are rare neoplasms (1%-4% of all malignant ovarian tumors) composed of high-grade malignant epithelial and mesenchymal elements. OCS occurs in older women. It is associated with a poor outcome and is usually not involved in inherited cancer syndromes. We present 2 cases of OCS; one arising in a patient with a pathogenetic BRCA1 mutation and the other in a woman affected by Lynch Syndrome (LS) carrying a MSH6 germline mutation. To the best of our knowledge, this is the first time that this second type of case has been reported. In this study, we investigated somatic impairment of the wild-type BRCA1 and MSH6 alleles in the OCS of these 2 patients. We also explored in both OCS, the occurrence of TP53 loss of function, which is a genetic alteration known to occur in BRCA-linked ovarian tumorigenesis but not in LS tumors. Moreover, we also provide further data about the histogenesis of OCS.