Ilaria Tamagno

Gene Expression Profiling Uncovers Molecular Classifiers for the Recognition of Anaplastic Large-Cell Lymphoma Within Peripheral T-Cell Neoplasms

PURPOSE To unravel the regulatory network underlying nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) -mediated lymphomagenesis of anaplastic large-cell lymphoma (ALCL) and to discover diagnostic genomic classifiers for the recognition of patients with ALK-positive and ALK-negative ALCL among T-cell non-Hodgkins lymphoma (T-NHL). PATIENTS AND METHODS The transcriptome of NPM-ALK-positive ALCL cell lines was characterized by silencing the expression of ALK or STAT3, a major effector of ALK oncogenic activity. Gene expression profiling (GEP) was performed in a series of systemic primary T-NHL (n = 70), including a set of ALK-positive and ALK-negative ALCL (n = 36). Genomic classifiers for ALK-positive and ALK-negative ALCL were generated by prediction analyses and validated by quantitative reverse-transcriptase polymerase chain reaction and/or immunohistochemistry. RESULTS In ALCL cell lines, two thirds of ALK-regulated genes were concordantly dependent on STAT3 expression. GEP of systemic primary T-NHL significantly clustered ALK-positive ALCL samples in a separate subgroup, underscoring the relevance of in vitro ALK/STAT3 signatures. A set of genomic classifiers for ALK-positive ALCL and for ALCL were identified by prediction analyses. These gene clusters were instrumental for the distinction of ALK-negative ALCL from peripheral T-cell lymphomas not otherwise specified (PTCLs-NOS) and angioimmunoblastic lymphomas. CONCLUSION We proved that experimentally controlled GEP in ALCL cell lines represents a powerful tool to identify meaningful signaling networks for the recognition of systemic primary T-NHL. The identification of a molecular signature specific for ALCL suggests that these T-NHLs may represent a unique entity discernible from other PTCLs, and that a restricted number of genes can be instrumental for clinical stratification and, possibly, therapy of T-NHL.

Nestin expression in reactive astrocytes of human pathology.

There is a general agreement on the Nestin re-expression in reactive astrocytes, but its modalities differ among experimental animal species and between the latter and human material. In a series of 40 surgical specimens, including gliomas, vascular malformations, abscesses and angiomas, the glial reaction has been studied by immunohistochemistry and immunofluorescence of Nestin, GFAP and Vimentin. The observations made by immunohistochemistry were comparable with those by immunofluorescence. In some lesions, glial reaction was long-lasting and astrocytes were in the same late maturation stage. In other lesions, such as invading malignant gliomas, astrocytes occurred in different maturation stages. In comparison with GFAP, Nestin was poorly expressed in mature astrocytes and more expressed in developing reactive astrocytes, mainly in the cytoplasms, with a great variability, and much less in the processes. In the invading tumor, developing positive astrocytes were hardly distinguishable from tumor invading astrocytes that, interestingly, were much more Nestin- than GFAP-positive. In the deep tumor reactive astrocytes were no more visible. The interpretation of the findings was based on what is known on the reciprocal behavior of the three antigens in maturing astrocytes during embryogenesis and on the hypothesis of an embryonic regression of reactive astrocytes. The impossibility to distinguish them from tumor cells in the deep tumor legitimates the suspicion of their recruitment among tumor cells.

Nestin expression in neuroepithelial tumors

Nestin is a marker of early stages of neurocytogenesis. It has been studied in 50 neuroepithelial tumors, mostly gliomas of different malignancy grades, by immunohistochemistry, immunofluorescence, immunoblotting, and confocal microscopy and compared with GFAP and Vimentin. As an early marker of differentiation, Nestin is almost not expressed in diffuse astrocytomas, variably expressed in anaplastic astrocytomas and strongly and irregularly expressed in glioblastomas. Negative in oligodendrogliomas, it stains ependymomas and shows a gradient of expression in pilocytic astrocytomas. In glioblastomas, Nestin distribution does not completely correspond to that of GFAP and Vimentin with which its expression varies in tumor cells in a complementary way, as confirmed by confocal microscopy. Tumor cells can thus either derive from or differentiate toward the neurocytogenetic stages. Hypothetically, they could be put in relation with radial glia where during embriogenesis the three antigens are successively expressed. Completely negative cells of invasive or recurrent glioblastomas may represent malignant selected clones after accumulation of mutations or early stem cells not expressing antigens.

Molecular morphology of neuronal apoptosis: analysis of caspase 3 activation during postnatal development of mouse cerebellar cortex.

We have used the mammalian post-natal cerebellar cortex as a model to dissect out the molecular morphology of neuronal apoptosis in a well-defined population of central neurons: the cerebellar granule cells. By immunocytochemistry, in situ labeling of apoptotic cells, and analysis of cerebellar slices following particle-mediated gene transfer (biolistics), we have studied the relationship of cell death and cleavage of caspase 3, a key molecule in the execution of apoptosis, and monitored caspase 3 activation in living cells. Our results demonstrate the existence of caspase dependent and independent apoptotic pathways affecting the cerebellar granule cells at different stages of their life. Apoptosis of proliferating precursors and young pre-migratory cells occurs in the absence of caspase 3 cleavage, whereas cell death of post-mitotic post-migratory neurons is directly linked to caspase 3 activation. Data obtained from cerebellar cortex can be generalized to outline a more comprehensive picture of the cellular and molecular mechanisms of neuronal death not only in development, but also in a number of pathological conditions leading to neuronal loss.