Italo Nenci

MicroRNA Gene Expression Deregulation in Human Breast Cancer

MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Their aberrant expression may be involved in human diseases, including cancer. Indeed, miRNA aberrant expression has been previously found in human chronic lymphocytic leukemias, where miRNA signatures were associated with specific clinicobiological features. Here, we show that, compared with normal breast tissue, miRNAs are also aberrantly expressed in human breast cancer. The overall miRNA expression could clearly separate normal versus cancer tissues, with the most significantly deregulated miRNAs being mir-125b, mir-145, mir-21, and mir-155. Results were confirmed by microarray and Northern blot analyses. We could identify miRNAs whose expression was correlated with specific breast cancer biopathologic features, such as estrogen and progesterone receptor expression, tumor stage, vascular invasion, or proliferation index.

Genetic progression in microsatellite instability high (MSI-H) colon cancers correlates with clinico-pathological parameters: A study of the TGFβRII, BAX, HMSH3, HMSH6, IGFIIR and BLM genes

Colon carcinomas with microsatellite mutator phenotype exhibit specific genetic and clinico‐pathological features. This report describes the analysis of 63 “microsatellite instability‐high” (MSI‐H) tumors for the presence of mutations in microsatellites located in the coding regions (CDRs) of 6 genes: TGFβRII, BAX, hMSH3, hMSH6, IGFIIR, and BLM. The following frequencies of mutations were detected: TGFβRII (70%), BAX (54%), hMSH3 (36.5%), IGFIIR (22%), hMSH6 (17.5%), and BLM (16%). The overall picture revealed combinations of mutations suggestive of a progressive order of accumulation, with mutations of TGFβRII and BAX first, followed by frameshifts in hMSH3, hMSH6, IGFIIR, and BLM. Correlations with 12 clinico‐pathological parameters revealed that tumors with frameshifts in 1 or 2 CDRs were significantly better differentiated than tumors with frameshifts in more than 2 CDRs. We also found that mutations in the hMSH3 gene were significantly associated with decreased wall invasiveness and aneuploidy, and frameshifts in the BLM gene were significantly associated with the mucinous histotype. A trend toward an association between hMSH3 and IGFIIR with the medullary and conventional adenocarcinoma histotypes, respectively, was seen. Our results strengthen the concept that mutations in target genes have a role in the tumorigenic process of MSI‐H tumors, and indicate that frameshifts in microsatellites located in CDRs occur in a limited number of combinations that could determine distinct clinico‐pathological traits. Int. J. Cancer 89:230–235, 2000.

Axillary Lymph Node Nanometastases Are Prognostic Factors for Disease-Free Survival and Metastatic Relapse in Breast Cancer Patients

Purpose: Early breast cancer presents with a remarkable heterogeneity of outcomes. Undetected, microscopic lymph node tumor deposits may account for a significant fraction of this prognostic diversity. Thus, we systematically evaluated the presence of lymph node tumor cell deposits ≤0.2 mm in diameter [pN0(i+), nanometastases] and analyzed their prognostic effect. Experimental Design: Single-institution, consecutive patients with 8 years of median follow-up (n = 702) were studied. To maximize chances of detecting micrometastases and nanometastases, whole-axilla dissections were analyzed. pN0 cases (n = 377) were systematically reevaluated by lymph node (n = 6676) step-sectioning and anticytokeratin immunohistochemical analysis. The risk of first adverse events and of distant relapse of bona fide pN0 patients was compared with that of pN0(i+), pN1mi, and pN1 cases. Results: Minimal lymph node deposits were revealed in 13% of pN0 patients. The hazard ratio for all adverse events of pN0(i+) versus pN0(i−) was 2.51 (P = 0.00019). Hazards of pN1mi and pN0(i+) cases were not significantly different. A multivariate Cox model showed a hazard ratio of 2.16 for grouped pN0(i+)/pN1mi versus pN0(i−) (P = 0.0005). Crude cumulative incidence curves for metastatic relapse were also significantly different (Grays test χ2 = 5.54, P = 0.019). Conclusion: Nanometastases are a strong risk factor for disease-free survival and for metastatic relapse. These findings support the inclusion of procedures for nanometastasis detection in tumor-node-metastasis staging.

MicroRNA profiling for the identification of cancers with unknown primary tissue-of-origin.

Cancer of unknown primary (CUP) represents a common and important clinical problem. There is evidence that most CUPs are metastases of carcinomas whose primary site cannot be recognized. Driven by the hypothesis that the knowledge of primary cancer could improve patients prognosis, we investigated microRNA expression profiling as a tool for identifying the tissue of origin of metastases. We assessed microRNA expression from 101 formalin‐fixed, paraffin‐embedded (FFPE) samples from primary cancers and metastasis samples by using a microarray platform. Forty samples representing ten different cancer types were used for defining a cancer‐type‐specific microRNA signature, which was used for predicting primary sites of metastatic cancers. A 47‐miRNA signature was identified and used to estimate tissue‐of‐origin probabilities for each sample. Overall, accuracy reached 100% for primary cancers and 78% for metastases in our cohort of samples. When the signature was applied to an independent published dataset of 170 samples, accuracy remained high: correct prediction was found within the first two options in 86% of the metastasis cases (first prediction was correct in 68% of cases). This signature was also applied to predict 16 CUPs. In this group, first predictions exhibited probabilities higher than 90% in most of the cases. These results establish that FFPE samples can be used to reveal the tissue of origin of metastatic cancers by using microRNA expression profiling and suggest that the approach, if applied, could provide strong indications for CUPs, whose correct diagnosis is presently undefined. Copyright

Detection and dynamic localisation of estradiol-receptor complexes in intact target cells by immunofluorescence technique

The total binding capacity of the cell receptors and not only the fraction required to elicit physiological response was detected and it is shown that specific receptors filled by endogenous estradiol were detected along with the unfilled sites. Changing the temperature of the incubation media altered intracellular distribution of bound estradiol. Late nucleolar retention of estradiol was shown after its release from nuclear protein. This unexpected finding may be a key event. Tumor tissues were obtained from 40 primary breast cancers, 2 endometrial carcinomas, and 1 lung and 1 gastric cancer; 2 normal human spleens, 2 mouse livers, and 1 breast from a 5-month pregnant women were processed. Tissue samples were transferred to a cold flask containing phosphate-buffered balanced salt solution. Further details of preparation are given to show typical estradiol-target cells containing variable amounts of specific estradiol-receptors. For immunofluorescence staining and indirect technique was used to show the in situ estradiol localization. The fluorescence staining showed 3 patterns. Cells incubated in the cold mostly displayed a bright, homogeneously diffuse fluorescence of the cytoplasm with the nuclear area unstained. There were some negative cells. The 2nd pattern of fluorescence was shown by those incubated at room temperature. Cytoplasmic staining was more marked and nuclear areas showed bright-fluorescent light stippling. A 3rd staining pattern was seen after slow postincubation warming up to 37 degrees C when increased nuclear staining occurred. Some cells did not show any nuclear labeling despite the clear cytoplasmic fluorescence. Preparations of human spleen, nontarget tumors, or mouse liver had no fluorescent cells. All fluorescent stainings were prevented by preincubation and washing in media containing Nafoxidine and N-ethylmaleimide. Control experiments confirmed the immune specificity of the detection of estradiol. Breast tissue cells from a pregnant women showed moderate cytoplasmic and faint nuclear fluorescence before incubation with estradiol, after which cytoplasmic staining was reinforced. Cells from the premenstrual beast cancers sometimes showed fluorescence without exposure to estradiol.

Molecular Subtyping of Breast Cancer from Traditional Tumor Marker Profiles Using Parallel Clustering Methods

Purpose: Recent small-sized genomic studies on the identification of breast cancer bioprofiles have led to profoundly dishomogeneous results. Thus, we sought to identify distinct tumor profiles with possible clinical relevance based on clusters of immunohistochemical molecular markers measured on a large, single institution, case series. Experimental Design: Tumor biological profiles were explored on 633 archival tissue samples analyzed by immunohistochemistry. Five validated markers were considered, i.e., estrogen receptors (ER), progesterone receptors (PR), Ki-67/MIB1 as a proliferation marker, HER2/NEU, and p53 in their original scale of measurement. The results obtained were analyzed by three different clustering algorithms. Four different indices were then used to select the different profiles (number of clusters). Results: The best classification was obtained creating four clusters. Notably, three clusters were identified according to low, intermediate, and high ER/PR levels. A further subdivision in two biologically distinct subtypes was determined by the presence/absence of HER2/NEU and of p53. As expected, the cluster with high ER/PR levels was characterized by a much better prognosis and response to hormone therapy compared to that with the lowest ER/PR values. Notably, the cluster characterized by high HER2/NEU levels showed intermediate prognosis, but a rather poor response to hormone therapy. Conclusions: Our results show the possibility of profiling breast cancers by means of traditional markers, and have novel clinical implications on the definition of the prognosis of cancer patients. These findings support the existence of a tumor subtype that responds poorly to hormone therapy, characterized by HER2/NEU overexpression.

Affinity cytochemistry visualizes specific estrogen binding sites on the plasma membrane of breast cancer cells

Abstract A study has been initiated on mapping specific interaction of estrogen with the plasma membrane of estrogen-target cells. Fluorescent estrogen analogues have been used in order to obtain the dynamic display and positioning of estrogen binding sites at the surface of intact breast cancer cells. The binding of these fluorescent ligands to the plasma membrane shows a major saturable component with structural specificity for natural estrogen. Moreover, estrogen binding sites display a temperature-sensitive lateral mobility in the plane of the membrane, leading to the formation of small clusters, larger patches and polar caps. Different steroid specificity indicates that these binding sites on the plasma membrane are distinct from classical cytosol estrogen-receptors. The actual function of these estrogen binding sites is unknown, but their location and properties are at least consistent with the idea that they might be involved in the cell control by estrogen.

90K (Mac‐2 BP) gene expression in breast cancer and evidence for the production of 90K by peripheral‐blood mononuclear cells

The tumor‐derived antigen 90K (Mac‐2 BP) is a widely expressed, secreted glycoprotein found in the serum of healthy individuals and at elevated levels in the serum of patients with breast cancer and other types of cancer. The precise function of 90K, particularly in the context of tumor‐host relationships, has not yet been established. In this study, the clinical significance of 90K mRNA expression was studied in relation to other established prognostic parameters in 86 patients with primary breast carcinoma. The 2.2‐kb 90K message was detected in all tumor samples, but there was marked variation in expression levels from tumor to tumor. Patients were classified into 2 groups on the basis of 90K expression: group 1 (n = 62) included patients with low expression, and group 2 (n = 24) consisted of patients with high expression. An inverse significant correlation was found between the levels of 90K mRNA expression and over‐expression of c‐erbB2/Neu receptor kinase, a marker of poor prognosis for patients with breast cancer. There was no significant difference between the groups with respect to tumor size, number of involved axillary lymph nodes, hormone‐receptor status, p53 expression or proliferation activity as estimated by Ki‐67 count. Similarly, no association was found between the level of 90K expression and the risk of death from breast cancer. These data are at variance with published findings showing that high serum 90K levels are associated with poor survival. Significantly, investigation of 90K‐gene expression in peripheral‐blood mononuclear cells (PBMC) revealed higher levels of 90K message in PBMC of breast‐cancer patients than in healthy individuals. This new finding suggests that PBMC activated in response to tumor growth and progression may be an important source of serum 90K in breast cancer. Int. J. Cancer (Pred. Oncol.) 79:23–26, 1998.© 1998 Wiley‐Liss, Inc.

Commentary on human mammary preneoplasia. The estrogen receptor-promotion hypothesis

In the carcinogenic process, promotion is the process whereby an initiated tissue develops focal proliferations which act as proximate precursors. The evidence obtained from the immunocytochemical staining by monoclonal anti-receptor antibodies indicates that the early steps (atypical hyperplasias) in the carcinogenic process of the breast show an increased and homogeneous expression of the estrogen receptor. These observations suggest that the persistent sensitivity to estrogen may be critical in sustaining the growth of mammary preneoplastic changes and their progression to ultimate precursors and to invasive cancer.

Biophenotypes of Breast Carcinoma in situ Defined by Image Analysis of Biological Parameters

In 50 in situ breast cancers an immunohistochemical study, evaluating estrogen (ER) and progesterone (PR) receptors, Proliferation Index (PI), c-erbB-2/Neu and p53 expression was performed. According to histopathological diagnosis, cases were classified as follows: 14 comedo, 8 solid, 5 micropapillary, 6 lobular, 3 papillary, 1 apocrine and 12 mixed in situ carcinomas. The quantitation of immunohistochemical results was obtained with an image analysis computerized system (CAS 200) with a lesion-field method; tumors were subdivided in fields (1177) histologically homogeneous, with 40 x microscopic objective. For ER, PR, Neu and p53, 10% of the positive area was used as cut-off value; 13% was used for PI. Cribriform and lobular types showed a higher positivity for ER (92.1% and 95.5% of the fields); cribriform and papillary a higher for PR (92.6% and 93.9%). Comedo variant demonstrated the higher PI (52.7%), Neu and p53 expression (67.7% and 43%). A cluster analysis performed on 608 fields, defined two groups according to biological homogeneous criteria. The results obtained identify the different biophenotypes of in situ carcinomas, suggesting the possibility of multiple cancerogenetic ways with a different weight of biological events.