Cristina Riva

Immunohistochemical pattern of hMSH2/hMLH1 in familial and sporadic colorectal, gastric, endometrial and ovarian carcinomas with instability in microsatellite sequences

Abstract. Alterations of DNA mismatch repair (MMR) genes are involved in carcinogenesis of sporadic and inherited human cancers characterised by instability of DNA microsatellite sequences (MSI). MSI tumours are usually identified using molecular analysis. In the present investigation, hMLH1 and hMSH2 immunohistochemistry was tested in order to evaluate the utility of this method in predicting MMR deficiency. Colorectal (72), gastric (68), endometrial (44) and ovarian (17) carcinomas were independently evaluated for familial history, histological type of tumour, MSI status and immunohistochemical results. Loss of expression of either hMLH1 or hMSH2 was observed in 51 of 55 (92.8%) MSI tumours, while 145 of 146 microsatellite stable (MSS) tumours expressed both the hMLH1 and hMSH2 gene products. Independently of tumour site, an overall agreement between immunohistochemical and molecular results was observed in 15 hereditary non-polyposis colorectal cancer-related tumours. Among sporadic tumours, only 2 of 60 colorectal and 2 of 66 gastric carcinomas, displaying MSI, expressed both hMLH1 and hMSH2 gene products. All 39 endometrial and 16 ovarian tumours presented a concordant molecular and immunohistochemical profile. These data show that immunohistochemistry is an accurate and rapid method to predict the presence of defective DNA MMR genes and to identify both sporadic and familial MSI tumours.

Immunohistochemical study of androgen receptors in breast carcinoma. Evidence of their frequent expression in lobular carcinoma

Androgens and androgen receptors (AR) are involved in the pathogenesis of breast cancer. Epidemiological studies have shown a significant association between the risk of breast cancer and androgens. However, the functional role and clinical value of AR expression in breast carcinoma have still not been clearly defined. The present study was set up to investigate the prevalence of ARs in a series of consecutive invasive breast carcinomas (IBCs) and to evaluate the patterns of AR phenotypes in a series of selected invasive lobular carcinomas (ILCs). Among the 250 consecutive IBCs (consisting of 212 ductal and 38 lobular neoplasms), AR immunoreactivity was observed in 151/250 (60.4%) cases, being expressed in 118/212 (56%) ductal and 33/38 (87%) lobular carcinomas (a statistically significant difference, χ2=11.82). AR expression was frequently associated with ER (65.2%, χ2=14.33) and PR positivity (66.9%, χ2=7.36). Most AR positive cases showed a low proliferative index (63.7%) and a low or intermediate histological grade (G1–G2, 63.9%). Among the 80 selected ILCs, AR expression was observed in 64/80 (80%) cases. Our results confirm that ARs are expressed in most breast cancers. Moreover, we demonstrated that AR positivity is particularly marked in lobular neoplasms. In addition, AR positive carcinomas are frequently characterized by a low or intermediate grade, a low proliferative index and ER and/or PR co-expression.

Genetic similarities and differences between lobular in situ neoplasia (LN) and invasive lobular carcinoma of the breast

One of the most controversial issues in breast pathology is whether lobular neoplasia (LN) is a risk factor or a precursor lesion of invasive lobular carcinoma (ILC). This is consequent to the fact that no conclusive data on the biology of LN exist. Molecular studies of LN and ILC are scanty, variable, and not consistent. Clonality of 12 cases of LN and ILC present simultaneously in the same block has been studied. Cells from both lesions were obtained by microdissection and were studied for mitochondrial DNA (mtDNA), D-loop sequencing, and neighbor-joining trees. Eight of the same cases were studied with comparative genomic hybridization (CGH) array to have additional data consistent with mtDNA. In all cases, loss of heterozygosity was studied for D16S496,locus 16q22.1 related to e-cadherin. It appears that no fewer than eight cases were genetically very similar (clonal) with mtDNA. Seven of these cases appeared also clonal with CGH array. It is concluded that in the present series, LN and ILC are genetically related lesions in the majority of cases and that LN might be the precursor of ILC.

Androgen receptor is frequently expressed in HER2-positive, ER/PR-negative breast cancers

The estrogen receptor (ER)/progesterone receptor (PR)-negative breast carcinomas (BCs) encompass three molecular subtypes: one with human epidermal growth factor receptor 2 (HER) overexpression, one normal like, and the triple negative. The androgen receptor (AR) is expressed in 70–90% of invasive BCs. The aim of our study is to detect the expression of AR in a series of ER/PR-negative BCs to ascertain if there is clinical significance in relation to BC molecular subtypes. A immunohistochemical study for all receptors and cytokeratin expression was performed in 232 cases of ER/PR-negative BCs. According to cytokeratin expression, BCs were classified into two groups: luminal-type BCs (44.2%) and basal-like-type BCs (55.8%). According to the expression of HER2, 59.3% were triple-negative BCs (when ER, PR, and HER2 were negative) and 40.7% were HER2-positive BCs. AR expression was observed in 128 tumors (56.6%). One hundred and ten cases (48.8%) had >10% and 18 (7.8%) had <10% of positively stained cells. AR immunoreactivity was found in 31.2% basal-like BCs, while in the luminal group 71.1% of cases were positive, showing highly significant correlation (p < 10−8). Regarding HER2 status, 76.7% of HER2-positive BC cases were AR positive compared with only 30.4% of triple-negative BC types, showing a strong statistically significant correlation. In conclusion, we show that AR is frequently expressed in ER/PR-negative BCs and that expression of HER2 and AR is highly correlated (p < 0.005). Our results point out the role of AR and HER2 in the pathogenesis of BCs and suggest the potential role of AR in clinical management of ER/PR-negative BCs.

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.

Ovarian metastases from colorectal carcinoma. Clinicopathologic profile, immunophenotype, and karyotype analysis.

Ovarian metastases from colorectal carcinoma frequently mimic primary ovarian carcinomas. The present study was performed to identify possible criteria helpful in differential diagnosis. Twenty-three ovarian metastases from colorectal carcinomas and 23 primary ovarian carcinomas were evaluated clinicopathologically and immunostained with antigastric M1 antigen, cathepsin E, CA125, vimentin, estrogen and progesterone receptors, cytokeratins 7 and 20, and alpha-inhibin antibodies. We performed a conventional and molecular cytogenetic study on 5 ovarian metastases from colorectal carcinoma using direct preparation, Q banding techniques, and fluorescence in situ hybridization. Integration of clinicopathologic, immunohistochemical, and cytogenetic features is helpful for the differential diagnosis of metastases of colorectal carcinomas from primary ovarian carcinomas. Bilaterality, extrapelvic spreading, high mitotic index, and cytokeratin 20 immunoreactivity, and lack of M1, CA125, and cytokeratin 7 immunoreactivity favor the diagnosis of ovarian metastases from colon carcinomas. The identification of 13q gain as a peculiar, sensitive, and specific marker of colorectal carcinomas seems relevant.

Inhibition of Sp1-dependent transcription and antitumor activity of the new aureolic acid analogues mithramycin SDK and SK in human ovarian cancer xenografts

OBJECTIVE Increased activity of Sp family of transcription factors is a frequent and critical event in cancer development and progression. Genes governing tumor growth, invasion and angiogenesis are regulated by Sp factors, like Sp1, Sp3 or Sp4, and are frequently over-expressed in tumors. Targeting Sp factors has been explored as a therapeutic approach. Mithramycin (MTM) is a natural antibiotic that binds DNA and inhibit Sp1-dependent transcription. New analogues, named MTM-SDK and MTM-SK, were recently obtained by genetic engineering of the MTM biosynthetic pathway and have demonstrated improved transcriptional and antiproliferative activity in ovarian cancer cell lines in vitro. In the present study we evaluated the activity of the new compounds in human ovarian cancer xenografts. METHODS Expression of Sp1 and target proteins in ovarian cancer specimens and tumor xenografts was assessed by immunohistochemistry. Drug-induced silencing of Sp1-regulated genes in cells and tumor xenograft samples was assessed by quantitative RT-PCR. Toxicity and antitumor activity of the compounds were investigated in healthy and tumor-bearing immunocompromised mice, respectively. RESULTS Expression of Sp1 was frequently increased in human epithelial ovarian cancers. MTM-SDK and MTM-SK acted as potent inhibitors of Sp1-dependent transcription both in vitro and in tumor xenografts. Both compounds were well tolerated even after prolonged administration and delayed growth of ovarian tumor xenografts. MTM-SDK was particularly effective against orthotopic tumors leading to a significant increase of survival and delay of tumor progression. CONCLUSIONS MTM-SDK and MTM-SK show relevant activity in vivo and represent interesting candidates for treatment of ovarian cancers.

Natural history of cervical intraepithelial neoplasia during pregnancy

Objective. It is uncertain whether pregnancy influences the natural history of cervical intraepithelial neoplasia (CIN). Our aim was to evaluate the evolution of CIN in pregnant women. Design. Prospective study. Setting. Department of Obstetrics and Gynaecology, University of Insubria, Italy. Population. Women with histological CIN during pregnancy. Methods and main outcome measures. Between 2003 and 2007, women with an abnormal Pap‐smear during pregnancy underwent colposcopy. Patients with histological CIN were followed during pregnancy with colposcopy every 8 weeks and post‐partum evaluation was scheduled 3–6 months after delivery. Women with post‐partum histological diagnosis of CIN 2–3 underwent conization. To understand the impact of pregnancy on the evolution of CIN, women with CIN 1 discovered during pregnancy were compared to a group of non‐pregnant fertile patients with first diagnosis of CIN 1. Results. A total of 78 women were included: 36 (46.2%) with CIN 2–3 and 42 (53.8%) with CIN 1. In women with CIN 2–3, no invasion was suspected during pregnancy and at post‐partum evaluation, no invasive or microinvasive cancer, and 19 (52.7%) persistent CIN 2–3, and 17 (47.3%) regressions were diagnosed. In the group of CIN 1, we recorded six (14.3%) progressions to CIN 2–3, seven (16.6%) persistent CIN 1 and 29 (69%) regressions. The control group of non‐pregnant women had a lower regression rate (37/76: 48.7%) in comparison to pregnant women (p = 0.03). Conclusions. Expectant management for CIN 2–3 diagnosed during gestation is safe. When discovered during pregnancy, CIN 1 has a significantly higher tendency to spontaneous regression in comparison to non‐pregnant condition.

Microenvironmental control of malignancy exerted by RNASET2, a widely conserved extracellular RNase

A recent body of evidence indicates an active role for stromal (mis)-regulation in the progression of neoplasias. Within this conceptual framework, genes belonging to the growing but still poorly characterized class of tumor antagonizing/malignancy suppressor genes (TAG/MSG) seem to play a crucial role in the regulation of the cross-talk between stromal and epithelial cells by controlling malignant growth in vivo without affecting any cancer-related phenotype in vitro. Here, we have functionally characterized the human RNASET2 gene, which encodes the first human member of the widespread Rh/T2/S family of extracellular RNases and was recently found to be down-regulated at the transcript level in several primary ovarian tumors or cell lines and in melanoma cell lines. Although we could not detect any activity for RNASET2 in several functional in vitro assays, a remarkable control of ovarian tumorigenesis could be detected in vivo. Moreover, the control of ovarian tumorigenesis mediated by this unique tumor suppressor gene occurs through modification of the cellular microenvironment and the induction of immunocompetent cells of the monocyte/macrophage lineage. Taken together, the data presented in this work strongly indicate RNASET2 as a previously unexplored member of the growing family of tumor-antagonizing genes.

Physical map of the D6S149-D6S193 region on chromosome 6Q27 and its involement in benign surface epithelial ovarian tumours

A detailed long range restriction map of the region defined by markers D6S149 and D6S193 on chromosome 6q27 has been constructed. This was achieved by YAC cloning and contig assembling of the same region. Seven YAC clones were found to span the almost 1000 Kb region flanked by the two markers which on the genetic map resulted to be 1.9 cM apart. With some of the characterized YAC clones we undertook a molecular cytogenetic analysis of 20 benign ovarian tumors. The rationale for this was the recent mapping to a region of chromosome 6q27, flanked by markers D6281 and D6S133, of a locus for the SV40-mediated immortalization of human cells (SEN6 gene). Noteworthy we found that the the D6S149-D6S193 region (comprised in the larger D6S281-D6S133 physical interval) was altered in all samples analysed adding support to the occurrence of a immortalization step in this type of tumors.