The miR-106a~363Xpcl1 miRNA cluster induces murine T cell lymphoma despite transcriptional activation of the p27Kip1 cell cycle inhibitor.

The miR-106a~363 cluster encodes 6 miRNAs on the X-chromosome which are abundant in blood cells and overexpressed in a variety of malignancies. The constituent miRNA of miR-106a~363 have functional activities in vitro that are predicted to be both oncogenic and tumor suppressive, yet little is known about their physiological functions in vivo. Mature miR-106a~363 (Mirc2) miRNAs are processed from an intragenic, non-protein encoding gene referred to as Xpcl1 (or Kis2), situated at an X-chromosomal locus frequently targeted by retroviruses in murine lymphomas. The oncogenic potential of miR-106a~363 Xpcl1 has not been proven, nor its potential role in T cell development. We show that miR106a~363 levels normally drop at the CD4+/CD8+ double positive (DP) stage of thymocyte development. Forced expression of Xpcl1 at this stage impairs thymocyte maturation and induces T-cell lymphomas. Surprisingly, miR-106a~363 Xpcl1 also induces p27 transcription via Foxo3/4 transcription factors. As a haploinsufficient tumor suppressor, elevated p27 is expected to inhibit lymphomagenesis. Consistent with this, concurrent p27 Kip1 deletion dramatically accelerated lymphomagenesis, indicating that p27 is rate limiting for tumor development by Xpcl1. Whereas down-regulation of miR-106a~363 is important for normal T cell differentiation and for the prevention of lymphomas, eliminating p27 reveals Xpcl1's full oncogenic potential.

BAP1 Immunohistochemistry and p16 FISH in the Diagnosis of Sarcomatous and Desmoplastic Mesotheliomas.

The separation of sarcomatous and desmoplastic mesotheliomas from benign organizing pleuritis can be morphologically very difficult. Deletion of p16 (CDKN2A) by fluorescence in situ hybridization (FISH) testing appears to be a reliable marker of malignancy in mesothelial proliferations, and more recently it has been reported that, in this setting, loss of BAP1 by immunohistochemistry is only seen in malignant mesotheliomas. To determine how useful these tests are with sarcomatous and desmoplastic mesotheliomas, we examined 20 such tumors. Loss of BAP1 was seen in 3/20 (15%) and deletion of p16 by FISH was seen in 16/20 (80%) cases. Loss of one or the other marker was observed in 17/20 (85%). We also examined 13 sarcomatoid carcinomas, an important differential diagnosis of sarcomatoid mesotheliomas, and found that BAP1 was never lost, but p16 was deleted in 3/11 (27%). We conclude that: (1) BAP1 immunohistochemistry is relatively insensitive in the context of sarcomatous and desmoplastic mesotheliomas, but as a matter of time and cost efficiency may nonetheless be a useful first approach to the problem; (2) deletion of p16 by FISH is considerably more sensitive, but there remain a proportion of cases in which p16 is not deleted; (3) a small improvement in sensitivity can be achieved by using both markers; (4) in the context of a spindle cell malignant tumor in the pleura or peritoneum, which morphologically might be a metastatic sarcomatoid carcinoma or a mesothelioma, the finding of BAP1 loss favors mesothelioma, but p16 FISH cannot be used to separate sarcomatous mesotheliomas from sarcomatoid carcinomas.

Evaluation of Human Epidermal Growth Factor Receptor 2 (HER2) Gene Status in Human Breast Cancer Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Specimens by Fluorescence In Situ Hybridization (FISH).

Current standard of care requires that HER2 gene testing be performed on all newly diagnosed invasive breast cancers in order to determine eligibility for anti-HER2 antibody therapy and should be performed in accordance with current ASCO-CAP guidelines (Hammond et al., J Clin Oncol 29(15):e458, 2011; Wolff et al., J Clin Oncol 31(31):3997-4013, 2013). Here we describe a HER2 FISH methodology to evaluate HER2 gene status in FFPE breast tumor specimens.

Utility of BAP1 Immunohistochemistry and p16 (CDKN2A) FISH in the Diagnosis of Malignant Mesothelioma in Effusion Cytology Specimens.

The diagnosis of malignant mesothelioma in effusion cytology specimens is controversial. BAP1 immunohistochemistry and p16 fluorescence in situ hybridization (FISH) have recently been reported as reliable markers of malignancy in biopsies of mesothelioma. To determine whether these markers, singly or in combination, might also be useful in effusion cytology specimens, we examined 15 biopsies of epithelial mesotheliomas and 3 benign mesothelial reactions and corresponding effusion cytology paraffin-embedded cell blocks. Four cytology specimens were too scanty for p16 FISH analysis but were interpretable for BAP1 immunohistochemistry. Overall, loss of BAP1 and/or deletion of p16 was seen in 11/11 (100%) of matched cytology and tissue biopsy specimens. BAP1 loss alone was seen in 10/15 (67%) biopsies and 10/15 (67%) cytology specimens. Homozygous deletion of p16 by FISH was found in 12/15 (80%) biopsy specimens and 8/11 (73%) evaluable cytology specimens. Seven of 15 (47%) biopsies and 5/11 (42%) cytology specimens showed loss of both markers. All mesothelioma biopsy/cytology pairs showed exactly the same pattern of BAP1 or p16 retention or loss in the biopsy and cytology specimens. The 2 peritoneal mesothelioma cases demonstrated loss of BAP1 but not p16. None of the benign mesothelial reactions or corresponding cytology specimens showed loss of either marker. We conclude that both BAP1 immunohistochemistry and p16 FISH analysis provide reliable markers of mesothelial malignancy in effusion cytology specimens, especially where the atypical mesothelial proliferation is well sampled. BAP1 is easier to interpret with scanty specimens. On the basis of small numbers of cases, use of both markers appears to increase sensitivity.

BAP1 immunohistochemistry and p16 FISH to separate benign from malignant mesothelial proliferations.

A variety of immunohistochemical (IHC) stains have been proposed to mark either benign or malignant mesothelial proliferations. Loss of the p16 tumor suppressor (CDKN2A), through homozygous deletions of 9p21, is a good marker of mesotheliomas but lacks sensitivity. Recent reports indicate that some mesotheliomas are associated with loss of BRCA-associated protein 1 (BAP1) expression. Here we investigate BAP1 and p16 as potential markers of malignancy and compare test characteristics with previously proposed markers using a well-characterized tissue microarray. BAP1 protein expression was interrogated by IHC. The p16 locus was examined by fluorescence in situ hybridization (FISH) directed toward chromosome 9p21. Loss of BAP1 was identified in 7/26 mesotheliomas and 0/49 benign proliferations. Loss of p16 was identified in 14/27 mesotheliomas and 0/40 benign proliferations, yielding 100% specificity and positive predictive value for each marker. Together, BAP1 IHC and p16 FISH were 58% sensitive for detecting malignancy. Various combinations of p53, EMA, IMP3, and GLUT1 showed reasonably high specificity (96% to 98%) but poor to extremely poor sensitivity. Combined BAP1 IHC/p16 FISH testing is a highly specific method of diagnosing malignant mesotheliomas when the question is whether a mesothelial proliferation is benign or malignant and is particularly useful when tissue invasion by mesothelial cells cannot be demonstrated. However, combined BAP1/p16 FISH testing is not highly sensitive, and negative results do not rule out a mesothelioma. The test characteristics of previously proposed markers EMA, p53, GLUT1, IMP3 suggest that, even in combination, these markers are not useful tools in this clinical setting.

Determining true HER2 gene status in breast cancers with polysomy by using alternative chromosome 17 reference genes: implications for anti-HER2 targeted therapy.

PURPOSE: The ratio of human epidermal growth factor receptor 2 (HER2) to CEP17 by fluorescent in situ hybridization (FISH) with the centromeric probe CEP17 is used to determine HER2 gene status in breast cancer. Increases in CEP17 copy number have been interpreted as representing polysomy 17. However, pangenomic studies have demonstrated that polysomy 17 is rare. This study tests the hypothesis that the use of alternative chromosome 17 reference genes might more accurately assess true HER2 gene status. PATIENTS AND METHODS: In all, 171 patients with breast cancer who had HER2 FISH that had increased mean CEP17 copy numbers (> 2.6) were selected for additional chromosome 17 studies that used probes for Smith-Magenis syndrome (SMS), retinoic acid receptor alpha (RARA), and tumor protein p53 (TP53) genes. A eusomic copy number exhibited in one or more of these loci was used to calculate a revised HER2-to-chromosome-17 ratio by using the eusomic gene locus as the reference. RESULTS: Of 132 cases classified as nonamplified on the basis of their HER2:CEP17 ratios, 58 (43.9%) were scored as amplified by using alternative chromosome 17 reference gene probes, and 13 (92.9%) of 14 cases scored as equivocal were reclassified as amplified. Among the cases with mean HER2 copy number of 4 to 6, 41 (47.7%) of 86 had their HER2 gene status upgraded from nonamplified to amplified, and four (4.7%) of 86 were upgraded from equivocal to amplified. CONCLUSION: Our results support the findings of recent pangenomic studies that true polysomy 17 is uncommon. Additional FISH studies that use probes to the SMS, RARA, and TP53 genes are an effective way to determine the true HER2 amplification status in patients with polysomy 17 and they have important potential implications for guiding HER2-targeted therapy in breast cancer.

Effect of Xpcl1 activation and p27(Kip1) loss on gene expression in murine lymphoma.

Mice lacking the p27(Kip1) Cdk inhibitor (Cdkn1b) exhibit increased susceptibility to lymphomas from the Maloney murine leukemia virus (M-MuLV), and exhibit a high frequency of viral integrations at Xpcl1 (Kis2), a locus on the X-chromosome. Xpcl1 encodes miR-106a~363, a cluster of microRNAs that are expressed in response to adjacent retroviral integrations. We report the first large-scale profile of microRNA expression in MuLV-induced lymphomas, in combination with microarray gene expression analysis. The source material was T-cell lymphomas induced by M-MuLV in p27(Kip1) knockout mice and normal thymus. Surprisingly, the overall levels of miRNA expression were equivalent in lymphomas and normal thymus. Nonetheless, the expression of specific microRNAs was altered in tumors. The miR-106a~363 miRNA were over-expressed in lymphomas, particularly those with viral integrations at the Xpcl1 locus. In contrast, p27(Kip1) deletion itself was associated with a different pattern of microRNA expression. Gene expression was dramatically altered in lymphomas, yet paralleled data from T-cell lymphomas induced by other mechanisms. Genes with altered expression in association with the p27(Kip1) null genotype were of similar functional classes to those associated with Xpcl1 integration, but with the opposite pattern of expression. Thus, the effect of p27(Kip1) deletion may be to oppose an anti-oncogenic effect of Xpcl1 rather than enhancing its oncogenic functions. A subset of miR-106a~363 target genes was consistently reduced in lymphomas with Xpcl1 integrations, particularly genes with cell cycle and immune functions. We identify four predicted target genes of miR-106a~363 miRNA, including N-Myc (Mycn), and the TGF-beta receptor (Tgfbr2) using 3'UTR reporter assays. Still, bioinformatic miRNA target predictions were poor predictors of altered gene expression in lymphomas with Xpcl1 integration. Confirmation of miR-106a~363 gene targeting relevant to the tumor phenotype requires in vivo validation, because only a subset of predicted targets are consistently reduced in tumors that overexpress miR-106a~363.

Isoform- and cell cycle-dependent substrate degradation by the Fbw7 ubiquitin ligase.

The SCF(FBW7) ubiquitin ligase degrades proteins involved in cell division, growth, and differentiation and is commonly mutated in cancers. The Fbw7 locus encodes three protein isoforms that occupy distinct subcellular localizations, suggesting that each has unique functions. We used gene targeting to create isoform-specific Fbw7-null mutations in human cells and found that the nucleoplasmic Fbw7alpha isoform accounts for almost all Fbw7 activity toward cyclin E, c-Myc, and sterol regulatory element binding protein 1. Cyclin E sensitivity to Fbw7 varies during the cell cycle, and this correlates with changes in cyclin E-cyclin-dependent kinase 2 (CDK2)-specific activity, cyclin E autophosphorylation, and CDK2 inhibitory phosphorylation. These data suggest that oscillations in cyclin E-CDK2-specific activity during the cell cycle regulate the timing of cyclin E degradation. Moreover, they highlight the utility of adeno-associated virus-mediated gene targeting in functional analyses of complex loci.

A mechanism misregulating p27 in tumors discovered in a functional genomic screen.

The cyclin-dependent kinase inhibitor p27(KIP1) is a tumor suppressor gene in mice, and loss of p27 protein is a negative prognostic indicator in human cancers. Unlike other tumor suppressors, the p27 gene is rarely mutated in tumors. Therefore misregulation of p27, rather than loss of the gene, is responsible for tumor-associated decreases in p27 protein levels. We performed a functional genomic screen in p27(+/-) mice to identify genes that regulate p27 during lymphomagenesis. This study demonstrated that decreased p27 expression in tumors resulted from altered transcription of the p27 gene, and the retroviral tagging strategy enabled us to pinpoint relevant transcription factors. inhibitor of DNA binding 3 (Id3) was isolated and validated as a transcriptional repressor of p27. We further demonstrated that p27 was a downstream target of Id3 in src-family kinase Lck-driven thymic lymphomagenesis and that p27 was an essential regulator of Lck-dependent thymic maturation during normal T-cell development. Thus, we have identified and characterized transcriptional repression of p27 by Id3 as a new mechanism decreasing p27 protein in tumors.

Discovery of an oncogenic activity in p27Kip1 that causes stem cell expansion and a multiple tumor phenotype.

The cell cycle inhibitor p27Kip1 also has cyclin-cyclin-dependent kinase (CDK)-independent functions. To investigate the significance of these functions in vivo, we generated a knock-in mouse in which four amino acid substitutions in the cdkn1b gene product prevent its interaction with cyclins and CDKs (p27CK-). In striking contrast to complete deletion of the cdkn1b gene, which causes spontaneous tumorigenesis only in the pituitary, the p27CK- protein dominantly caused hyperplastic lesions and tumors in multiple organs, including the lung, retina, pituitary, ovary, adrenals, spleen, and lymphomas. Moreover, the high incidence of spontaneous tumors in the lung and retina was associated with amplification of stem/progenitor cell populations. Therefore, independently of its role as a CDK inhibitor, p27Kip1 promoted stem cell expansion and functioned as a dominant oncogene in vivo. Thus, the p27CK- mouse unveils a dual role for p27 during tumorigenesis: It is a tumor suppressor by virtue of its cyclin-CDK regulatory function, and also an oncogene through a cyclin-CDK-independent function. This may explain why the cdkn1b gene is rarely inactivated in human tumors, and the p27CK- mouse in which the tumor suppressor function is lost but the cyclin-CDK-independent-oncogenic-function is maintained may represent a more faithful model for the widespread role of p27 misregulation in human cancers than the p27 null.

Evaluation of 12 antibodies for distinguishing epithelioid mesothelioma from adenocarcinoma: identification of a three-antibody immunohistochemical panel with maximal sensitivity and specificity.

We evaluated the sensitivity and specificity of 10 monoclonal and two polyclonal antibodies for distinguishing epithelioid mesothelioma from adenocarcinoma (AdCA) using immunohistochemistry (IHC). The antibodies were directed against the mesothelial-associated antigens mesothelin, calretinin, cytokeratin 5, thrombomodulin, Wilms' tumor-1 (WT-1) gene product and HBME-1, and the nonmesothelial antigens Lewis-Y blood group (antibody BG8), MOC-31, BerEp4, CD15, and carcinoembryonic antigen (CEA) family. The 133 tumors evaluated included 65 malignant epithelioid mesotheliomas, 22 lung AdCAs, 27 ovarian serous carcinomas, 24 breast carcinomas, and five gastric carcinomas. Diagnoses were based on clinical, histologic, ultrastructural, and/or IHC findings. Calretinin had the best sensitivity for mesothelioma (95%), followed by HBME-1 (84%), WT-1 (78%), cytokeratin 5 (76%), mesothelin (75%), and vimentin and thrombomodulin (68%). Thrombomodulin had the best specificity for mesothelioma (92%), followed by cytokeratin 5 (89%), calretinin (87%) vimentin (84%), and HBME-1 (45%). When ovarian carcinomas were excluded from the analysis, the specificity of mesothelin and WT-1 for the diagnosis of mesothelioma increased to 90 and 81%, respectively. The sensitivity of the nonmesothelial antigens for AdCA was organ dependent, with BG8 performing best in the breast cancer group (96%), and BerEp4, BG8, MOC-31 performing best in the lung cancer group (100%). The specificity of the nonmesothelial antigens for AdCA was 98% for BG8 and CEA, 97% for CD15, 95% for BerEp4, and 87% for MOC-31. A novel statistical analysis technique employing logic regression analysis identified a three-antibody immunohistochemical panel including calretinin, BG8, and MOC-31, which provided over 96% sensitivity and specificity for distinguishing epithelioid mesothelioma from AdCA.

Zcchc8 is a glycogen synthase kinase-3 substrate that interacts with RNA-binding proteins.

Phosphorylation of c-Myc on threonine 58 (T58) stimulates its degradation by the Fbw7-SCF ubiquitin ligase. We used a phosphorylation-specific antibody raised against the c-Myc T58 region to attempt to identify other proteins regulated by the Fbw7 pathway. We identified two predominant proteins recognized by this antibody. The first is Ebna1 binding protein 2, a nucleolar protein that, in contrast with a previous report, is likely responsible for the nucleolar staining exhibited by this antibody. The second is Zcchc8, a nuclear protein that is highly phosphorylated in cells treated with nocodazole. We show that Zcchc8 is directly phosphorylated by GSK-3 in vitro and that GSK-3 inhibition prevents Zcchc8 phosphorylation in vivo. Moreover, we found that Zcchc8 interacts with proteins involved in RNA processing/degradation. We suggest that Zcchc8 is a GSK-3 substrate with a role in RNA metabolism.

Cyclin E in normal and neoplastic cell cycles.

Cyclin E-Cdk2 has long been considered an essential and master regulator of progression through G1 phase of the cell cycle. Although recent mouse models have prompted a rethinking of cyclin E function in mammals, it remains clear that cyclin E impacts upon many processes central to cell division. Normal cells maintain strict control of cyclin E activity, and this is commonly disrupted in cancer cells. Moreover, cyclin E deregulation is thought to play a fundamental role in tumorigenesis. In this review, we discuss the regulation and functions of cyclin E in normal and neoplastic mammalian cells.

Identification of oncogenes collaborating with p27Kip1 loss by insertional mutagenesis and high-throughput insertion site analysis.

The p27(Kip1) protein is a cyclin-dependent kinase inhibitor that blocks cell division in response to antimitogenic cues. p27 expression is reduced in many human cancers, and p27 functions as a tumor suppressor that exhibits haploinsufficiency in mice. Despite the well characterized role of p27 as a cyclin-dependent kinase inhibitor, its mechanism of tumor suppression is unknown. We used Moloney murine leukemia virus to induce lymphomas in p27+/+ and p27-/- mice and observed that lymphomagenesis was accelerated in the p27-/- animals. To identify candidate oncogenes that collaborate with p27 loss, we used a high-throughput strategy to sequence 277 viral insertion sites derived from two distinct sets of p27-/- lymphomas and determined their chromosomal location by comparison with the Celera and public (Ensembl) mouse genome databases. This analysis identified a remarkable number of putative protooncogenes in these lymphomas, which included loci that were novel as well as those that were overrepresented in p27-/- tumors. We found that Myc activations occurred more frequently in p27-/- lymphomas than in p27+/+ tumors. We also characterized insertions within two novel loci: (i) the Jun dimerization protein 2 gene (Jundp2), and (ii) an X-linked locus termed Xpcl1. Each of the loci that we found to be frequently involved in p27-/- lymphomas represents a candidate oncogene collaborating with p27 loss. This study illustrates the power of high-throughput insertion site analysis in cancer gene discovery.