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 patient's 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.

p53 status identifies two subgroups of triple-negative breast cancers with distinct biological features.

OBJECTIVE: Despite the clinical similarities triple-negative and basal-like breast cancer are not synonymous. Indeed, not all basal-like cancers are negative for estrogen receptor, progesterone receptor and HER2 expression while triple-negative also encompasses other cancer types. P53 protein appears heterogeneously expressed in triple-negative breast cancers, suggesting that it may be associated with specific biological subgroups with a different outcome. METHODS: We comparatively analyzed p53 expression in triple-negative tumors from two independent breast cancer case series (633 cases from the University of Ferrara and 1076 cases from the University of Nottingham). RESULTS: In both case series, p53 protein expression was able to subdivide the triple-negative cases into two distinct subsets consistent with a different outcome. In fact, triple-negative patients with a p53 expressing tumor showed worse overall and event-free survival. CONCLUSIONS: The immunohistochemical evaluation of p53 expression may help in taming the currently stormy relationship between pathological (triple-negative tumors) and biological (basal breast cancers) classifications and in selecting patient subgroups with different biological features providing a potentially powerful prognostic contribution in triple-negative breast cancers.

High expression of 90K (Mac-2 BP) is associated with poor survival in node-negative breast cancer patients not receiving adjuvant systemic therapies.

90K (Mac-2 BP) expression was evaluated by immunohistochemistry in paraffin-embedded tissue from a consecutive series of lymph-node negative breast cancer patients who did not receive adjuvant systemic treatment. An independent series of patients served as validation set. The association of 90K expression with risk of recurrence and death was examined in survival analyses together with known prognostic factors. High levels of 90K expression (IHC score>8) were observed in 43 (25.3%) of 170 tumors examined. We found elevated risks of distant recurrence and overall mortality in patients with high 90K expression compared with patients with low 90K expression in their tumors. This increase persisted after adjusting for other prognostic factors in multivariate analysis (hazard ratio=4.084; p<0.001 for recurrence; hazard ratio=4.298; p<0.001 for death). These findings were confirmed in the validation set. Therefore, evaluation of 90K expression may be beneficial to identify lymph-node negative breast cancer patients at lower risk of disease recurrence and death.

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 dishomogenous 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.

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

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.

Loss of methylation at chromosome 11p15.5 is common in human adult tumors.

Chromosome 11p15 deletion is frequent in human tumors, suggesting the presence of at least one tumor suppressor gene within this region. While mutation analyses of local genes revealed only rare mutations, we have previously described a mechanism, gain of imprinting, that leads to loss of expression of genes located on the maternal 11p15 chromosome in human hepatocarcinomas. Loss of expression was often associated with loss of maternal-specific methylation at the KvDMR1 locus. Here, we show that loss of the maternal KvDMR1 methylation is common, ranging from 30 to 50%, to a variety of adult neoplasms, including liver, breast, cervical and gastric carcinomas. We found that other 11p15.5 loci were concomitantly hypomethylated, indicating that loss of KvDMR1 methylation occurred in the context of a common mechanism affecting the methylation of a large 11p15 subchromosomal domain. These epigenetic abnormalities were not detected in any normal somatic tissue. Therefore, it seems possible that, contrary to the repression of promoter activity caused by hypermethylation, loss of gene expression at 11p15.5 may result from the activation, by hypomethylation, of one or more negative regulatory elements.

Mesectodermal leiomyoma exclusively involving the posterior choroid.

PURPOSE: To report a mesectodermal leiomyoma of the posterior choroid. DESIGN: Observational case report. METHODS: A 23-year-old man was referred to us because of a progressive blurred vision in his left eye. Ophthalmologic examination revealed the presence of a 12 x 10 x 7.2-mm amelanotic choroidal mass in his left posterior pole. Fluorescein angiography, A-scan ultrasonography, and B-scan echography findings were suggestive for a diagnosis of choroidal amelanotic melanoma. These clinical features prompted us to enucleate the left eye. RESULTS: Histopathological and immunohistochemistry examinations established a definitive diagnosis of mesectodermal leiomyoma of the posterior choroid. CONCLUSION: This case represents the first report describing the occurrence of an intraocular mesectodermal leiomyoma that may exclusively involve the posterior choroid.

Quantitative detection of molecular markers ProEx C (minichromosome maintenance protein 2 and topoisomerase IIa) and MIB-1 in liquid-based cervical squamous cell cytology.

BACKGROUND: In this study, the authors conducted a comparative quantitative evaluation of the proliferation markers ProEx C (an aberrant S-phase induction marker, human papillomavirus E6-E7 correlated) and MIB-1 in squamous intraepithelial lesions (SIL) to identify a biomolecular profile informative for the diagnosis of high-grade SIL/cervical intraepithelial neoplasia 3 or greater that was complementary to the morphologic Papanicolaou (Pap) test ("biomolecular Pap test"). METHODS: After the cytologic diagnosis, reflex immunocytochemistry was carried out on 76 unstained SurePath cell samples (20 routine samples that were negative for intraepithelial lesion or malignancy and 56 positive samples that were selected with matching histology). Both a morphometric analysis with a software imaging analysis system and a quantitative analysis of atypical squamous clusters were performed. RESULTS: The quantitative evaluation revealed an excellent, direct correlation between the 2 markers, although ProEx C was more selective and more informative for the progression of low- and moderate-grade lesions, because it only revealed cells in aberrant S-phase cell cycle. The quantitative morphometric analysis revealed the increased presence of atypical, positive clusters and the percentage of positive cells within, both paralleling the severity of the lesions. The threshold of a 3% ProEx C-positive nuclear area was useful for splitting lesions into groups with a low risk or high risk of progression. CONCLUSIONS: Both ProEx C and MIB-1 were valid proliferation markers in cytologic preparations, and nuclear positivity was quantified successfully by using computer-assisted analysis. The analysis of atypical clusters may be a valuable tool in the diagnosis of SIL. The presence of atypical clusters and their positivity for proliferation markers are good first-glance indicators of lesion grade.