Identification of Cellular Genes Targeted by KSHV-Encoded MicroRNAs

MicroRNAs (miRNAs) are 19 to 23 nucleotide–long RNAs that post-transcriptionally regulate gene expression. Human cells express several hundred miRNAs which regulate important biological pathways such as development, proliferation, and apoptosis. Recently, 12 miRNA genes have been identified within the genome of Kaposi sarcoma–associated herpesvirus; however, their functions are still unknown. To identify host cellular genes that may be targeted by these novel viral regulators, we performed gene expression profiling in cells stably expressing KSHV-encoded miRNAs. Data analysis revealed a set of 81 genes whose expression was significantly changed in the presence of miRNAs. While the majority of changes were below 2-fold, eight genes were down-regulated between 4- and 20-fold. We confirmed miRNA-dependent regulation for three of these genes and found that protein levels of thrombospondin 1 (THBS1) were decreased >10-fold. THBS1 has previously been reported to be down-regulated in Kaposi sarcoma lesions and has known activity as a strong tumor suppressor and anti-angiogenic factor, exerting its anti-angiogenic effect in part by activating the latent form of TGF-β. We show that reduced THBS1 expression in the presence of viral miRNAs translates into decreased TGF-β activity. These data suggest that KSHV-encoded miRNAs may contribute directly to pathogenesis by down-regulation of THBS1, a major regulator of cell adhesion, migration, and angiogenesis

Whole Slide Imaging Versus Microscopy for Primary Diagnosis in Surgical Pathology: A Multicenter Blinded Randomized Noninferiority Study of 1992 Cases (Pivotal Study)

Most prior studies of primary diagnosis in surgical pathology using whole slide imaging (WSI) versus microscopy have focused on specific organ systems or included relatively few cases. The objective of this study was to demonstrate that WSI is noninferior to microscopy for primary diagnosis in surgical pathology. A blinded randomized noninferiority study was conducted across the entire range of surgical pathology cases (biopsies and resections, including hematoxylin and eosin, immunohistochemistry, and special stains) from 4 institutions using the original sign-out diagnosis (baseline diagnosis) as the reference standard. Cases were scanned, converted to WSI and randomized. Sixteen pathologists interpreted cases by microscopy or WSI, followed by a wash-out period of ≥4 weeks, after which cases were read by the same observers using the other modality. Major discordances were identified by an adjudication panel, and the differences between major discordance rates for both microscopy (against the reference standard) and WSI (against the reference standard) were calculated. A total of 1992 cases were included, resulting in 15,925 reads. The major discordance rate with the reference standard diagnosis was 4.9% for WSI and 4.6% for microscopy. The difference between major discordance rates for microscopy and WSI was 0.4% (95% confidence interval, -0.30% to 1.01%). The difference in major discordance rates for WSI and microscopy was highest in endocrine pathology (1.8%), neoplastic kidney pathology (1.5%), urinary bladder pathology (1.3%), and gynecologic pathology (1.2%). Detailed analysis of these cases revealed no instances where interpretation by WSI was consistently inaccurate compared with microscopy for multiple observers. We conclude that WSI is noninferior to microscopy for primary diagnosis in surgical pathology, including biopsies and resections stained with hematoxylin and eosin, immunohistochemistry and special stains. This conclusion is valid across a wide variety of organ systems and specimen types

Cloning and Identification of a MicroRNA Cluster within the Latency-Associated Region of Kaposi's Sarcoma-Associated Herpesvirus

MicroRNAs (miRNAs) are small, noncoding regulatory RNA molecules that bind to 3′ untranslated regions (UTRs) of mRNAs to either prevent their translation or induce their degradation. Previously identified in a variety of organisms ranging from plants to mammals, miRNAs are also now known to be produced by viruses. The human gammaherpesvirus Epstein-Barr virus has been shown to encode miRNAs, which potentially regulate both viral and cellular genes. To determine whether Kaposi's sarcoma-associated herpesvirus (KSHV) encodes miRNAs, we cloned small RNAs from KSHV-positive primary effusion lymphoma-derived cells and endothelial cells. Sequence analysis revealed 11 isolated RNAs of 19 to 23 bases in length that perfectly align with KSHV. Surprisingly, all candidate miRNAs mapped to a single genomic locale within the latency-associated region of KSHV. These data suggest that viral and host cellular gene expression may be regulated by miRNAs during both latent and lytic KSHV replication

Kaposi's Sarcoma-Associated Herpesvirus Encodes an Ortholog of miR-155▿ †

MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression by binding to 3′-untranslated regions (3′UTRs) of target mRNAs. Kaposi's sarcoma-associated herpesvirus (KSHV), a virus linked to malignancies including primary effusion lymphoma (PEL), encodes 12 miRNA genes, but only a few regulatory targets are known. We found that KSHV-miR-K12-11 shares 100% seed sequence homology with hsa-miR-155, an miRNA frequently found to be up-regulated in lymphomas and critically important for B-cell development. Based on this seed sequence homology, we hypothesized that both miRNAs regulate a common set of target genes and, as a result, could have similar biological activities. Examination of five PEL lines showed that PELs do not express miR-155 but do express high levels of miR-K12-11. Bioinformatic tools predicted the transcriptional repressor BACH-1 to be targeted by both miRNAs, and ectopic expression of either miR-155 or miR-K12-11 inhibited a BACH-1 3′UTR-containing reporter. Furthermore, BACH-1 protein levels are low in cells expressing either miRNA. Gene expression profiling of miRNA-expressing stable cell lines revealed 66 genes that were commonly down-regulated. For select genes, miRNA targeting was confirmed by reporter assays. Thus, based on our in silico predictions, reporter assays, and expression profiling data, miR-K12-11 and miR-155 regulate a common set of cellular targets. Given the role of miR-155 during B-cell maturation, we speculate that miR-K12-11 may contribute to the distinct developmental phenotype of PEL cells, which are blocked in a late stage of B-cell development. Together, these findings indicate that KSHV miR-K12-11 is an ortholog of miR-155

Tumor-Infiltrating Macrophages in Post-Transplant, Relapsed Classical Hodgkin Lymphoma Are Donor-Derived

<div><p>Tumor-associated inflammatory cells in classical Hodgkin lymphoma (CHL) typically outnumber the neoplastic Hodgkin/Reed-Sternberg (H/RS) cells. The composition of the inflammatory infiltrate, particularly the fraction of macrophages, has been associated with clinical behavior. Emerging work from animal models demonstrates that most tissue macrophages are maintained by a process of self-renewal under physiologic circumstances and certain inflammatory states, but the contribution from circulating monocytes may be increased in some disease states. This raises the question of the source of macrophages involved in human disease, particularly that of CHL. Patients with relapsed CHL following allogeneic bone marrow transplant (BMT) provide a unique opportunity to begin to address this issue. We identified 4 such patients in our archives. Through molecular chimerism and/or XY FISH studies, we demonstrated the DNA content in the post-BMT recurrent CHL was predominantly donor-derived, while the H/RS cells were derived from the patient. Where possible to evaluate, the cellular composition of the inflammatory infiltrate, including the percentage of macrophages, was similar to that of the original tumor. Our findings suggest that the H/RS cells themselves define the inflammatory environment. In addition, our results demonstrate that tumor-associated macrophages in CHL are predominantly derived from circulating monocytes rather than resident tissue macrophages. Given the association between tumor microenvironment and disease progression, a better understanding of macrophage recruitment to CHL may open new strategies for therapeutic intervention.</p></div

Tumor-infiltrating macrophages in recurrent Hodgkin lymphoma are predominantly donor- and, therefore, bone marrow-derived.

<p>A portion of the same field highlighted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163559#pone.0163559.g001" target="_blank">Fig 1A</a> (white box) is shown at higher magnification. Prior to analysis by XY FISH, the same slide was stained for CD68 using standard immunohistochemical techniques in order to identify macrophages (A). The images of the DAPI nuclear stain and the CD68 cytoplasmic stain are overlaid (B) to better identify individual cells in the corresponding FISH images (C). Where possible to discern, the tumor-infiltrating macrophages are all derived from the male donor (arrows, XY, red and green). However, by this method we found that in 21 +/- 4% of DAPI-positive nuclei, it was not possible to score X, Y status due to sectioning and/or other technical limitations. Of note, this patient had 100% donor chimerism in her bone marrow when tested near the time of this biopsy. H/RS cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163559#pone.0163559.g001" target="_blank">Fig 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163559#pone.0163559.s001" target="_blank">S1 Fig</a>) and areas of residual, uninvolved lung tissue were female (not shown). An overview of the density of macrophage infiltrate is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163559#pone.0163559.s002" target="_blank">S2 Fig</a>.</p

The composition of the inflammatory cells in pre and post-BMT specimens is similar.

<p>Representative high power fields from cervical (pre-BMT) and portal (post-BMT) lymph nodes involved by CHL from Patient A. Shown are H&E (A, B), CD3 (C, D), CD8 (E, F), CD20 (G, H), CD68 (I, J), and CD30 (K, L) for the pre-BMT sample (left) and the post-BMT sample (right) (200x).</p

H/RS cells are patient-derived while the majority of the inflammatory infiltrate is donor-derived.

<p>Sections of lung from a female patient (Patient D) with recurrent Hodgkin lymphoma following an allogeneic BMT (brother) are shown. An overview of one of the lesional areas is shown (A, DAPI nuclear staining). XY FISH was performed on this section, and a high power view of the boxed area in red is shown in B (X = red, Y = green). An adjacent tissue section was stained for CD30 (C), highlighting numerous H/RS cells (arrow, higher magnification, D). While not possible to align perfectly, the H/RS cells in this patient were positive for EBV (in situ hybridization for EBER, panels E, F). The majority of the smaller nuclei are donor-derived (XY, red and green, 78% including only DAPI-positive nuclei with distinct FISH signals). By comparison with the H&E, these cells predominantly represent an inflammatory infiltrate. A separate area from this specimen demonstrating similar findings is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163559#pone.0163559.s001" target="_blank">S1 Fig</a>.</p

Demographic information.

<p>Demographic information.</p