Alessandro Fornari

Identification of the miR-106b∼25 microRNA cluster as a proto-oncogenic PTEN-targeting intron that cooperates with its host gene MCM7 in transformation

A microRNA network regulates the tumor suppressor PTEN in prostate cancer. A Malignant Combination The abundance of microRNAs (miRNAs), tiny non–protein-coding RNAs that act as posttranscriptional regulators of gene expression, is frequently altered in cancer; indeed, various miRNAs are thought to act as oncogenes or tumor suppressors. Poliseno et al. investigated the possible role of miRNA regulation of the tumor suppressor PTEN in prostate cancer. They identified miRNAs from several families that targeted the gene encoding PTEN, thereby decreasing PTEN abundance, and showed that the abundance of some of these miRNAs was increased in human prostate cancer. Intriguingly, three PTEN-targeting miRNAs located within an intron of the gene encoding the DNA helicase minichromosome maintenance protein 7 (MCM7), which shows increased abundance in various human cancers, cooperated with MCM7 to transform fibroblasts in vitro and to initiate tumors when overexpressed in the prostates of transgenic mice. Thus, the MCM7 gene locus appears to encode multiple oncogenic elements that cooperate to promote prostate cancer development. PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a tumor suppressor that antagonizes signaling through the phosphatidylinositol 3-kinase–Akt pathway. We have demonstrated that subtle decreases in PTEN abundance can have critical consequences for tumorigenesis. Here, we used a computational approach to identify miR-22, miR-25, and miR-302 as three PTEN-targeting microRNA (miRNA) families found within nine genomic loci. We showed that miR-22 and the miR-106b~25 cluster are aberrantly overexpressed in human prostate cancer, correlate with abundance of the miRNA processing enzyme DICER, and potentiate cellular transformation both in vitro and in vivo. We demonstrated that the intronic miR-106b~25 cluster cooperates with its host gene MCM7 in cellular transformation both in vitro and in vivo, so that the concomitant overexpression of MCM7 and the miRNA cluster triggers prostatic intraepithelial neoplasia in transgenic mice. Therefore, the MCM7 gene locus delivers two simultaneous oncogenic insults when amplified or overexpressed in human cancer. Thus, we have uncovered a proto-oncogenic miRNA-dependent network for PTEN regulation and defined the MCM7 locus as a critical factor in initiating prostate tumorigenesis.

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

PURPOSEnTo 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).nnnPATIENTS AND METHODSnThe 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.nnnRESULTSnIn 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.nnnCONCLUSIONnWe 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.

Immunohistochemical subtyping of nonsmall cell lung cancer not otherwise specified in fine-needle aspiration cytology: a retrospective study of 103 cases with surgical correlation.

Histopathological subtyping of nonsmall cell lung cancer (NSCLC) is currently relevant in treatment decision because of a differential activity of specific therapeutic agents. Immunohistochemistry highlights cell differentiation lineages and, in this study, it was applied to maximize the proportion of accurately subtyped NSCLC not otherwise specified (NOS) on fine‐needle aspiration cytology (FNAC) samples.

Differential requirement of mTOR in postmitotic tissues and tumorigenesis.

Conditional inactivation of mTor has little effect in adult mouse prostate, but suppresses tumor initiation and progression. Blocking mTor Signals The mammalian target of rapamycin (mTOR), a protein kinase critical to cell growth and proliferation, functions as part of two distinct multiprotein complexes. mTOR signaling is frequently disrupted in cancer; however pharmacological suppression of mTOR complex 1 (mTORC1) signaling has been of limited therapeutic efficacy. Nardella et al. show that, whereas conditional inactivation of mTOR activity (abrogating signaling through both complexes) has little effect in the adult mouse prostate, it markedly suppresses prostate cancer associated with loss of the tumor suppressor PTEN. Thus, mTOR inhibitors that target its catalytic activity may be more effective in cancer therapy than those that specifically inhibit signaling mediated through mTORC1. The mammalian target of rapamycin (mTOR) is a crucial effector in a complex signaling network commonly disrupted in cancer. mTOR exerts its multiple functions in the context of two different multiprotein complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Loss of the tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10) can hyperactivate mTOR through AKT and represents one of the most frequent events in human prostate cancer. We show here that conditional inactivation of mTor in the adult mouse prostate is seemingly inconsequential for this postmitotic tissue. Conversely, inactivation of mTor leads to a marked suppression of Pten loss–induced tumor initiation and progression in the prostate. This suppression is more pronounced than that elicited by the sole pharmacological abrogation of mTORC1. Acute inactivation of mTor in vitro also highlights the differential requirement of mTor function in proliferating and transformed cells. Collectively, our data constitute a strong rationale for developing specific mTOR inhibitors targeting both mTORC1 and mTORC2 for the treatment of tumors triggered by PTEN deficiency and aberrant mTOR signaling.

Anaplastic large cell lymphoma: one or more entities among T‐cell lymphoma?

Anaplastic large cell lymphoma (ALCL) is a subtype of peripheral T‐cell lymphoma (PTCL) first described in 1985 as a lymphoid malignancy characterized by marked cellular pleomorphism, propensity to grow cohesively, tendency to invade lymph node sinuses and diffuse expression of CD30 1 . The discovery of the t(2;5), involving the anaplastic lymphoma kinase (ALK) gene on chromosome 2 and the nucleophosmin (NPM) gene on chromosome 5 in the majority of systemic ALCL, has soon pointed out that ALCL is a clinically and biologically heterogeneous disease. While ALK‐positive (ALK+) ALCL is usually characterized by onset in children and young adults and better prognosis, epidemiology, poor outcome and possibly genetic defects of ALK‐negative (ALK−) ALCL suggest that this neoplasms should be considered an independent pathological entity. The aim of this review is to illustrate clinical features, histology, immunophenotype, genetics and biology of ALCL and discuss possible relationship(s) among different T‐non‐Hodgkin lymphoma (T‐NHL). Copyright

Aberrant Cytoplasmic Expression of p63 and Prostate Cancer Mortality

Protein expression of p63 is used to differentiate prostate cancer from benign mimickers. Recent studies suggest that it may also distinguish aggressive prostate cancer with down-regulated expression occurring in men with more advanced disease. We conducted a prospective study among 298 men ages 51 to 84 years who were diagnosed with prostate cancer in the Physicians Health Study in 1983 to 2004 and whose tissue was available for immunohistochemical staining. We used Cox proportional hazards regression to evaluate the association of p63 protein expression with fatal prostate cancer. We correlated p63 expression with tumor cell proliferation (Ki-67) and apoptosis (TUNEL staining). The predominant location of tumor p63 staining occurred in the cytoplasm, an uncommon departure from the strong nuclear staining usually observed in nonneoplastic basal cells. Increasing expression of cytoplasmic p63 (tertiles) was associated with prostate cancer mortality (n = 19 deaths); the hazard ratios (95% confidence intervals) were 1.0 (reference), 4.0 (0.9-18.9), and 5.9 (1.3-27.5; Ptrend = 0.03). The positive trend remained significant (P = 0.047) after multivariable adjustment for age, year of diagnosis, and Gleason score. Higher tertiles of cytoplasmic p63 were also associated with reduced levels of apoptosis (Ptrend = 0.0408) and increased cellular proliferation (Ptrend = 0.0026). We found aberrant expression of p63 in the cytoplasm to be associated with increased prostate cancer-specific mortality up to 20 years after diagnosis. The mislocalized expression was associated with reduced apoptosis and higher proliferative activity and may suggest an oncogenic role in prostate cancer progression and survival. (Cancer Epidemiol Biomarkers Prev 2009;18(2):595–600)

Multispectral imaging and pathology: seeing and doing more

BACKGROUNDnThe current appreciation of the biological complexity of disease has led to increasing demands on pathologists to provide clinically relevant, quantitative morphological and molecular information while preserving cellular and tissue context. This can be technically challenging, especially when signals of interest are colocalized. With fluorescence-based methods, sensitivity and quantitative reliability may be compromised by spectral cross-talk between labels and by autofluorescence. In brightfield microscopy, overlapping chromogenic signals pose similar imaging difficulties.nnnAPPROACHnThese challenges can be addressed using commercially available multispectral imaging technologies attached to standard microscope platforms, or alternatively, integrated into whole-slide scanning instruments.nnnASSESSMENTnMultispectral techniques, along with other developments in digital analysis, will allow pathologists to deliver appropriate quantitative and multiplexed analyses in a reproducible and timely manner. Caveats apply - as the complexity of the sample preparation and analysis components increases, commensurate attention must be paid to the use of appropriate controls for all stages of the process.

Intrasinusoidal bone marrow infiltration and splenic marginal zone lymphoma : a quantitative study

Abstract:u2002 Intrasinusoidal infiltration (ISI) is a pattern of invasion that is rarely found on bone marrow (BM) biopsies, and is considered as a hallmark of splenic marginal zone cell lymphoma (SMZL). We analysed BM biopsies showing intrasinusoidal infiltration from 54 consecutive patients with different types of lymphoma to verify if ISI quantity was a diagnostic criterion for SMZL. There were 35 primary splenic lymphoma (PSL) and 19 non‐PSL; 28 SMZL, three non‐splenic MZL, six mantle cell, six small lymphocytic, four follicular, four diffuse large B cell, one peripheral T cell, one lymphoplasmacytic and one anaplastic large‐cell lymphoma. The quantity of BM infiltrate was assessed on CD45, CD20 and CD3 stained sections. The mean percentage of total (TI) and intrasinusoidal (ISI) lymphocytes was calculated in 10 areas for each case. TI quantity was 21.57 in PSL and 35.05 in non‐PSL (Pu2003=u20030.04). ISI quantity was 5.23 in PSL and 7.62 in non‐PSL (Pu2003=u20030.08), 5.83 in SMZL and 2.83 in other types of PSL (Pu2003=u20030.12), 4.46 in non‐splenic MZL and 8.21 in other types of non‐PSL (Pu2003=u20030.28). No difference in ISI quantity was found among the lymphoma subtypes, either in PSL (Pu2003=u20030.74) or non‐PSL (Pu2003=u20030.3). The data demonstrate that ISI quantity in BM biopsies is not a reliable diagnostic parameter for SMZL.

KIT and PDGFRA mutations and PDGFRA immunostaining in gastrointestinal stromal tumors

In the present study, we investigated the association of PDGFRA and KIT mutations as well as PDGFRA immunohistochemical expression with clinicopathologic features and prognosis in a series of gastrointestinal stromal tumors (GISTs). Tumor DNA from 40 GISTs was sequenced for the presence of mutations in KIT exons 9, 11, 13 and 17, and in PDGFRA exons 12 and 18. Tissue sections were stained with polyclonal anti-PDGFRA antibody. KIT mutations occurred in 26 cases. There were 13 deletions, 6 substitutions, 3 deletion-substitutions, 3 duplications and 1 insertion. Tumors with KIT deletions/insertion were large with a high mitotic index (MI), and were associated with a high rate of symptoms at diagnosis, invasion into adjacent organs, distant metastasis, relapse and a short disease-free survival (DFS). PDGFRA mutations occurred in 6 gastric GISTs. There were 4 deletions and 2 substitutions. Tumors with PDGFRA mutations were small, with a low MI and Ki67 score, and were associated with a very low rate of symptoms at diagnosis, invasion into adjacent organs and distant metastasis. PDGFRA immunopositivity was found in 23 cases: a peculiar dotlike staining was found in 5 out of 6 PDGFRA mutated cases. Patients with positive PDGFRA immunostaining had a longer DFS than those with negative staining. Our data confirm that the type of KIT mutation is associated with various clinicopathologic features of GISTs, and indicate that PDGFRA mutations are associated with rather indolent tumors. PDGFRA immunopositivity reflects PDGFRA mutational status and is associated with a favorable outcome.

Interpathologist concordance in the histological diagnosis of focal prostatic atrophy lesions, acute and chronic prostatitis, PIN, and prostate cancer

Epidemiological and biological evidence indicates a causal relationship between the presence of proliferative atrophic lesions and the development of prostatic intraepithelial neoplasia (PIN) and prostate cancer. The presence of inflammatory and atrophic lesions of the prostate is widely underestimated and they are not generally mentioned in pathology reports. We performed a histopathological concordance study among eight genitourinary specialists and seven generalist pathologists, using 116 histological slides of prostate lesions, including proliferative atrophic lesions, PIN, and cancer. The overall agreement between all possible pairs of reviewers was 80% for prostate cancer, 67% for PIN, and 49% for proliferative atrophic lesions. When using as gold standard the assessment of a single genitourinary pathologist, the mean agreement percentage increased to 97% for prostate cancer, 92% for PIN, and 72% for proliferative atrophic lesions.