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1.  PD-L1 Expression is Characteristic of a Subset of Aggressive B-Cell Lymphomas and Virus-Associated Malignancies 
Purpose
Programmed death ligand 1 (PD-L1) is an immunomodulatory molecule expressed by antigen-presenting cells and select tumors that engage receptors on T cells to inhibit T-cell immunity. Immunotherapies targeting the PD-1/PD-L1 pathway have shown durable anti-tumor effects in a subset of patients with solid tumors. PD-L1 can be expressed by Reed-Sternberg cells comprising classical Hodgkin lymphoma (CHLs) and by malignant B cells comprising EBV-positive post-transplant lymphoproliferative disorders (PTLDs). We sought to determine whether the expression of PD-L1 represents a general strategy of immune evasion among aggressive B-cell lymphomas and virus- and immunodeficiency-associated tumors.
Experimental Design
Using novel antibodies and formalin-fixed, paraffin-embedded (FFPE) tissue biopsies, we examined 237 primary tumors for expression of PD-L1 protein.
Results
Robust PD-L1 protein expression was found in the majority of nodular sclerosis CHL, mixed cellularity CHL, primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD, and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T cell lymphoma, nasopharyngeal carcinoma, and HHV8-associated primary effusion lymphoma. Within these tumors, PD-L1 was highly expressed by malignant cells and tumor-infiltrating macrophages. In contrast, neither the malignant nor the non-malignant cells comprising nodular lymphocyte-predominant Hodgkin lymphoma, DLBCL-not otherwise specified, Burkitt lymphoma, and HHV8-associated Kaposi sarcoma expressed detectable PD-L1.
Conclusion
Certain aggressive B-cell lymphomas and virus- and immunodeficiency-associated malignancies associated with an ineffective T-cell immune response express PD-L1 on tumor cells and infiltrating macrophages. These results identify a group of neoplasms that should be considered for PD-1/PD-L1-directed therapies, and validate a method to detect PD-L1 in FFPE tissue biopsies.
doi:10.1158/1078-0432.CCR-13-0855
PMCID: PMC4102335  PMID: 23674495
2.  Diagnostic Accuracy of a Defined Immunophenotypic and Molecular Genetic Approach for Peripheral T/NK-cell Lymphomas 
The diagnosis of peripheral T-cell and NK-cell lymphomas (PTNKCL) is difficult with few standards for required ancillary studies. We evaluated a series of PTNKCLs using a tiered approach to immunohistochemistry and molecular genetic characterization to document diagnostic accuracy and clinical relevance. Seven hematopathologists reviewed 374 cases that included PTNKCL and non-PTNKCL cases to mimic diagnostic practice. Cases received tier 0, 1, and 2 diagnoses by 3 independent pathologists, on the basis of hematoxylin and eosin stains and progressive immunohistochemistry panels. A tier 2b diagnosis was rendered when gene rearrangement data were available, and a final consensus diagnosis was rendered after discussion of each case. Across all 374 cases, consensus agreement was 92.5%. For PTNKCLs, World Health Organization subclassification was possible in 16.5%, 37.1%, 82.8%, and 85.9% of individual reviewer diagnoses at tier 0, 1, 2, and 2b, respectively. Gene rearrangement contributed to a change in diagnosis in 51 of 647 (8%) individual reviews. Following this algorithm may provide prognostic information on the basis of individual marker expression in common PTNKCL types (CD4 in peripheral T-cell lymphoma, not otherwise specified and PD-1 in angioimmunoblastic T-cell lymphoma). This evidence-based approach to the diagnosis of PTNKCL informs practicing pathologists, clinical trial designers, and policy-makers regarding required ancillary studies.
doi:10.1097/PAS.0000000000000188
PMCID: PMC4085049  PMID: 24618604
peripheral T-cell lymphoma; diagnosis; accuracy; evidence-based medicine; pathology; guidelines
3.  SYK Inhibition Modulates Distinct PI3K/AKT-dependent Survival Pathways and Cholesterol Biosynthesis in Diffuse Large B-cell Lymphomas 
Cancer cell  2013;23(6):826-838.
SUMMARY
B-cell receptor (BCR) signaling pathway components represent promising treatment targets in diffuse large B-cell lymphoma (DLBCL) and additional B-cell tumors. BCR signaling activates spleen tyrosine kinase (SYK) and downstream pathways including PI3K/AKT and NF-κB. In previous studies, chemical SYK blockade selectively decreased BCR signaling and induced apoptosis of BCR-dependent DLBCLs. Herein, we characterize distinct SYK/PI3K-dependent survival pathways in DLBCLs with high or low baseline NF-κB activity including selective repression of the pro-apoptotic HRK protein in NF-κB-low tumors. We also define SYK/PI3K-dependent cholesterol biosynthesis as a feed-forward mechanism of maintaining the integrity of BCRs in lipid rafts in DLBCLs with low or high NF-κB. In addition, SYK amplification and PTEN deletion are identified as selective genetic alterations in primary “BCR”-type DLBCLs.
doi:10.1016/j.ccr.2013.05.002
PMCID: PMC3700321  PMID: 23764004
4.  Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung tumors 
Cancer discovery  2013;3(12):10.1158/2159-8290.CD-13-0310.
The success in lung cancer therapy with Programmed Death (PD)-1 blockade suggests that immune escape mechanisms contribute to lung tumor pathogenesis. We identified a correlation between Epidermal Growth Factor Receptor (EGFR) pathway activation and a signature of immunosuppression manifested by upregulation of PD-1, PD-L1, cytotoxic T lymphocyte antigen-4 (CTLA-4), and multiple tumor-promoting inflammatory cytokines. We observed decreased cytotoxic T cells and increased markers of T cell exhaustion in mouse models of EGFR-driven lung cancer. PD-1 antibody blockade improved the survival of mice with EGFR-driven adenocarcinomas by enhancing effector T cell function and lowering the levels of tumor-promoting cytokines. Expression of mutant EGFR in bronchial epithelial cells induced PD-L1, and PD-L1 expression was reduced by EGFR inhibitors in non-small cell lung cancer cell lines with activated EGFR. These data suggest that oncogenic EGFR signaling remodels the tumor microenvironment to trigger immune escape, and mechanistically link treatment response to PD-1 inhibition.
doi:10.1158/2159-8290.CD-13-0310
PMCID: PMC3864135  PMID: 24078774
5.  KPT-330 inhibitor of CRM1 (XPO1)-mediated nuclear export has selective anti-leukaemic activity in preclinical models of T-ALL and AML 
British journal of haematology  2013;161(1):117-127.
Summary
This study explored the anti-leukaemic efficacy of novel irreversible inhibitors of the major nuclear export receptor, chromosome region maintenance 1 (CRM1, also termed XPO1). We found that these novel CRM1 antagonists, termed SINE (Selective Inhibitors of Nuclear Export), induced rapid apoptosis at low nanomolar concentrations in a panel of 14 human T-cell acute lymphoblastic leukaemia (T-ALL) cell lines representing different molecular subtypes of the disease. To assess in vivo anti-leukaemia cell activity, we engrafted immunodeficient mice intravenously with the human T-ALL MOLT-4 cells, which harbour activating mutations of NOTCH1 and NRAS as well as loss of function of the CDKN2A, PTEN and TP53 tumour suppressors and express a high level of oncogenic transcription factor TAL1. Importantly, we examined the in vivo anti-leukaemic efficacy of the clinical SINE compound KPT-330 against TALL and acute myeloid leukaemia (AML) cells. These studies demonstrated striking in vivo activity of KPT-330 against T-ALL and AML cells, with little toxicity to normal murine haematopoietic cells. Taken together, our results show that SINE CRM1 antagonists represent promising “first-in-class” drugs with a novel mechanism of action and wide therapeutic index, and imply that drugs of this class show promise for the targeted therapy of T-ALL and AML.
doi:10.1111/bjh.12231
PMCID: PMC3980736  PMID: 23373539
6.  Sphingosine-1-Phosphate Receptor 1 in Classical Hodgkin Lymphoma: Assessment of Expression and Role in Cell Migration 
Classical Hodgkin lymphoma (CHL), a neoplasm of abnormal B lymphocytes (Hodgkin-Reed Sternberg cells), has been described to have a typical pattern of clinical presentation and dissemination often involving functionally contiguous lymph nodes. Despite the progress made in understanding CHL pathophysiology, the factors which regulate the spread of lymphoma cells in CHL are poorly understood. Sphingosine-1-Phosphate (S1P), a bioactive sphingolipid present at high concentrations in plasma and lymphatic fluid, is known to play a critical role in regulating lymphocyte trafficking mainly through S1PR1. In this study, we explore the role of the S1P-S1PR1 axis in Hodgkin lymphoma cell migration and the expression of S1PR1 in CHL cell lines and clinical cases. We found that S1PR1 is present in the KM-H2 and SUP-HD1 Hodgkin lymphoma cell lines at the mRNA and protein level. In addition, functionally, S1P potently stimulated migration of both cell lines. S1P-induced migration was inhibited by the S1PR1 antagonist, VPC44116 and the S1PR1 functional antagonist, FTY720-P, but was potentiated by the S1PR2 specific antagonist, JTE013. We also determined that S1PR1 induced migration in the KM-H2 and SUP-HD1 cells via the heterotrimeric G protein Gi and the phosphatidylinositol-3-kinase (PI3K) pathway. Immunohistochemical assessment of tissue from CHL samples revealed that a subset of cases (7/57; 12%) show strong, membranous staining for S1PR1 in Hodgkin-Reed Sternberg cells.
Altogether our data indicate that S1PR1 is a functional receptor on Hodgkin-Reed Sternberg cells which governs tumor cell migration and is expressed in a subset of CHL cases. Given the availability of S1PR1 antagonists, some of which are used clinically for modulation of the immune system, these results suggest that S1PR1 could be a future therapeutic target in the treatment of those cases of S1PR1-positive, refractory/recurrent CHL.
doi:10.1038/labinvest.2013.7
PMCID: PMC3612140  PMID: 23419711
Sphingosine-1-phosphate (S1P); S1PR1; classical Hodgkin lymphoma; migration; dissemination; Fingolimod
7.  Microbial colonization influences early B-lineage development in the gut lamina propria 
Nature  2013;501(7465):10.1038/nature12496.
The RAG1/RAG2 endonuclease ("RAG") initiates the V(D)J recombination reaction that assembles Ig heavy (IgH) and light (IgL) chain variable region exons from germline gene segments to generate primary antibody repertoires1. IgH V(D)J assembly occurs in progenitor (pro-) B cells followed by that of IgL in precursor (pre-) B cells. Expression of IgH μ and IgL (Igκ or Igλ) chains generates IgM, which is expressed on immature B cells as the B cell antigen-binding receptor ("BCR"). Rag expression can continue in immature B cells2, allowing continued Igκ V(D)J recombination that replaces the initial VκJκ exon with one that generates a new specificity3–5. This “receptor editing” process, which also can lead to Igλ V(D)J recombination and expression3,6,7, provides a mechanism whereby antigen-encounter at the Rag-expressing immature B cell stage helps shape pre-immune BCR repertoires. As the major site of post-natal B cell development, the bone marrow is the principal location of primary Ig repertoire diversification in mice. Here, we report that early B cell development also occurs within the mouse intestinal lamina propria (LP), where the associated V(D)J recombination/receptor editing processes modulate primary LP Ig repertoires. At weanling age in normally housed mice, the LP contains a population of Rag-expressing B lineage cells that harbor intermediates indicative of ongoing V(D)J recombination and which contain cells with pro-B, pre-B, and editing phenotypes. Consistent with LP-specific receptor editing, Rag-expressing LP B-lineage cells have similar VH repertoires, but significantly different Vκ repertoires, compared to those of Rag2-expressing BM counterparts. Moreover, colonization of germ-free mice leads to an increased ratio of Igλ-expressing versus Igκ-expressing B cells specifically in the LP. We conclude that B cell development occurs in the intestinal mucosa, where it is regulated by extra-cellular signals from commensal microbes that influence gut Ig repertoires.
doi:10.1038/nature12496
PMCID: PMC3807868  PMID: 23965619
8.  Integrative Analysis Reveals an Outcome-associated and Targetable Pattern of p53 and Cell Cycle Deregulation in Diffuse Large B-cell Lymphoma 
Cancer cell  2012;22(3):359-372.
Summary
Diffuse large B-cell lymphoma (DLBCL) is a clinically and biologically heterogeneous disease with a high proliferation rate. By integrating copy number data with transcriptional profiles and performing pathway analysis in primary DLBCLs, we identified a comprehensive set of copy number alterations (CNAs) that decreased p53 activity and perturbed cell cycle regulation. Primary tumors either had multiple complementary alterations of p53 and cell cycle components or largely lacked these lesions. DLBCLs with p53 and cell cycle pathway CNAs had decreased abundance of p53 target transcripts and increased expression of E2F target genes and the Ki67 proliferation marker. CNAs of the CDKN2A-TP53-RB-E2F axis provide a structural basis for increased proliferation in DLBCL, predict outcome with current therapy and suggest targeted treatment approaches.
doi:10.1016/j.ccr.2012.07.014
PMCID: PMC3778921  PMID: 22975378
9.  Ganetespib (STA-9090), a Non-Geldanamycin HSP90 Inhibitor, has Potent Antitumor Activity in In Vitro and In Vivo Models of Non-Small Cell Lung Cancer 
Purpose
We describe the anticancer activity of ganetespib, a novel non-geldanamycin heat shock protein 90 (HSP90) inhibitor, in non-small cell lung cancer (NSCLC) models.
Experimental Design
The activity of ganetespib was compared to that of the geldanamycin 17-AAG in biochemical assays, cell lines and xenografts, and evaluated in an ERBB2 YVMA-driven mouse lung adenocarcinoma model.
Results
Ganetespib blocked the ability of HSP90 to bind to biotinylated geldanamycin and disrupted the association of HSP90 with its co-chaperone, p23, more potently than 17-AAG. In genomically-defined NSCLC cell lines, ganetespib caused depletion of receptor tyrosine kinases, extinguishing of downstream signaling, inhibition of proliferation and induction of apoptosis with IC50 values ranging 2–30 nM, substantially lower than those required for 17-AAG (20– 3,500 nM). Ganetespib was also approximately 20-fold more potent in isogenic Ba/F3 pro-B cells rendered IL-3 independent by expression of EGFR and ERBB2 mutants. In mice bearing NCI-H1975 (EGFR L858R/T790M) xenografts, ganetespib was rapidly eliminated from plasma and normal tissues but was maintained in tumor with t1/2 58.3 hours, supporting once-weekly dosing experiments, in which ganetespib produced greater tumor growth inhibition than 17-AAG. However, after a single dose, re-expression of mutant EGFR occurred by 72 hours, correlating with reversal of anti-proliferative and pro-apoptotic effects. Consecutive day dosing resulted in xenograft regressions, accompanied by more sustained pharmacodynamic effects. Ganetespib also demonstrated activity against mouse lung adenocarcinomas driven by oncogenic ERBB2 YVMA.
Conclusions
Ganetespib has greater potency than 17-AAG and potential efficacy against several NSCLC subsets, including those harboring EGFR or ERBB2 mutation.
doi:10.1158/1078-0432.CCR-11-2967
PMCID: PMC3477583  PMID: 22806877
HSP90; ERBB Receptor Tyrosine Kinases; NSCLC; Non-Geldanamycin
10.  Improved detection suggests all Merkel cell carcinomas harbor Merkel polyomavirus 
The Journal of Clinical Investigation  2012;122(12):4645-4653.
A human polyomavirus was recently discovered in Merkel cell carcinoma (MCC) specimens. The Merkel cell polyomavirus (MCPyV) genome undergoes clonal integration into the host cell chromosomes of MCC tumors and expresses small T antigen and truncated large T antigen. Previous studies have consistently reported that MCPyV can be detected in approximately 80% of all MCC tumors. We sought to increase the sensitivity of detection of MCPyV in MCC by developing antibodies capable of detecting large T antigen by immunohistochemistry. In addition, we expanded the repertoire of quantitative PCR primers specific for MCPyV to improve the detection of viral DNA in MCC. Here we report that a novel monoclonal antibody detected MCPyV large T antigen expression in 56 of 58 (97%) unique MCC tumors. PCR analysis specifically detected viral DNA in all 60 unique MCC tumors tested. We also detected inactivating point substitution mutations of TP53 in the two MCC specimens that lacked large T antigen expression and in only 1 of 56 tumors positive for large T antigen. These results indicate that MCPyV is present in MCC tumors more frequently than previously reported and that mutations in TP53 tend to occur in MCC tumors that fail to express MCPyV large T antigen.
doi:10.1172/JCI64116
PMCID: PMC3533549  PMID: 23114601
11.  Aberrant expression of the dendritic cell marker TNFAIP2 by the malignant cells of Hodgkin lymphoma and primary mediastinal large B cell lymphoma distinguishes these tumor types from morphologically and phenotypically similar lymphomas 
TNFAIP2 is a protein upregulated in response to TNF signaling but its cellular expression and function in normal and neoplastic tissues remains largely unknown. Here we use standard immunohistochemical techniques to demonstrate that TNFAIP2 is normally expressed by follicular dendritic cells, interdigitating dendritic cells, and macrophages but not by lymphoid cells in secondary lymphoid tissues. Consistent with this expression pattern, we found strong TNFAIP2 staining of tumor cells in 4/4 cases (100%) of follicular dendritic cell sarcoma and in 3/3 cases (100%) of histiocytic sarcoma. Although TNFAIP2 is not expressed by the small and intermediate-size neoplastic B-cells comprising follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, or marginal zone lymphoma, we observed strong TNFAIP2 staining of the large, neoplastic cells, in 31/31 cases (100%) of classical Hodgkin lymphoma, 12/12 cases (100%) of nodular lymphocyte predominant Hodgkin lymphoma, and 27/31 cases (87%) of primary mediastinal (thymic) large B cell lymphoma. In contrast, TNFAIP2 was expressed by the malignant cells in only 2/45 cases (4%) of diffuse large B cell lymphoma, not otherwise specified, 2/18 cases (11%) of Burkitt lymphoma, and 1/19 cases (5%) of anaplastic large cell lymphoma. Further analysis indicates that TNFAIP2, as a single diagnostic marker, is more sensitive (sensitivity= 87%) and specific (specificity= 96%) than TRAF1, nuclear cRel, or CD23 for distinguishing the malignant B-cells of primary mediastinal (thymic) large B cell lymphoma from those of its morphologic and immunophenotypic mimic, diffuse large B cell lymphoma, not otherwise specified. Thus, TNFAIP2 may serve as a useful new marker of dendritic and histiocytic sarcomas whose aberrant expression in the malignant cells of classical Hodgkin lymphoma and primary mediastinal (thymic) large B cell lymphoma serves to distinguish these tumors from other large cell lymphomas in routine clinical practice.
doi:10.1097/PAS.0b013e31822bd476
PMCID: PMC3178718  PMID: 21921781
TNFAIP2; Hodgkin Lymphoma; primary mediastinal (thymic) large B cell lymphoma; immunohistochemistry
12.  Synergy between PI3K Signaling and MYC in Burkitt Lymphomagenesis 
Cancer cell  2012;22(2):167-179.
SUMMARY
In Burkitt lymphoma (BL), a germinal center B-cell-derived tumor, the pro-apoptotic properties of c-MYC must be counterbalanced. Predicting that survival signals would be delivered by phosphoinositide-3-kinase (PI3K), a major survival determinant in mature B cells, we indeed found that combining constitutive c-MYC expression and PI3K activity in germinal center B cells of the mouse led to BL-like tumors, which fully phenocopy human BL with regard to histology, surface and other markers, and gene expression profile. The tumors also accumulate tertiary mutational events, some of which are recurrent in the human disease. These results and our finding of recurrent PI3K pathway activation in human BL indicate that deregulated c-MYC and PI3K activity cooperate in BL pathogenesis.
doi:10.1016/j.ccr.2012.06.012
PMCID: PMC3432451  PMID: 22897848
13.  MYBL2 is a sub-haploinsufficient tumor suppressor gene in myeloid malignancy 
eLife  2013;2:e00825.
A common deleted region (CDR) in both myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN) affects the long arm of chromosome 20 and has been predicted to harbor a tumor suppressor gene. Here we show that MYBL2, a gene within the 20q CDR, is expressed at sharply reduced levels in CD34+ cells from most MDS cases (65%; n = 26), whether or not they harbor 20q abnormalities. In a murine competitive reconstitution model, Mybl2 knockdown by RNAi to 20–30% of normal levels in multipotent hematopoietic progenitors resulted in clonal dominance of these ‘sub-haploinsufficient’ cells, which was reflected in all blood cell lineages. By 6 months post-transplantation, the reconstituted mice had developed a clonal myeloproliferative/myelodysplastic disorder originating from the cells with aberrantly reduced Mybl2 expression. We conclude that downregulation of MYBL2 activity below levels predicted by classical haploinsufficiency underlies the clonal expansion of hematopoietic progenitors in a large fraction of human myeloid malignancies.
DOI: http://dx.doi.org/10.7554/eLife.00825.001
eLife digest
Blood cells are produced within bone marrow by specialized stem cells and progenitor cells. Abnormalities in this process lead to a group of diseases known as myeloid malignancies, which include acute myeloid leukaemia—in which the bone marrow produces abnormal white blood cells—and myelodysplastic syndromes, which are caused by too few mature blood cells being produced.
Many individuals affected by these disorders possess a shortened form of chromosome 20 that lacks a number of genes. This deletion is only ever seen in one of their two copies of the chromosome—suggesting that at least some of these genes are essential for survival—but the identity of the gene(s) that are associated with the increased risk of myeloid malignancies is unknown.
Now, Heinrichs et al. have uncovered a key tumor suppressor among those genes frequently lost on chromosome 20. The gene, which is called MYBL2, encodes a transcription factor that helps to control the cell division cycle. Myeloid malignancy patients lacking one copy of this gene showed levels of MYBL2 expression that were less than 50% of those in healthy individuals. This suggests that additional mechanisms must be acting to reduce expression of their remaining copy of the gene. Surprisingly, MYBL2 levels were also reduced in myeloid malignancy patients who possessed two intact copies of chromosome 20, indicating that loss of a single copy represents only one mechanism to reduce MYBL2 expression, i.e., the ‘tip-of-the-iceberg’. Hence, this finding reveals a more general role for MYBL2 as it indicates that more patients are likely to be affected by altered expression of this gene.
To confirm their findings from studies in patients, Heinrichs et al. used gene silencing techniques to reduce the expression of MYBL2 in mice and showed that this induced symptoms of myeloid malignancies in the animals. Moreover, injection of modified cells from these animals into healthy mice also induced symptoms in the recipients. The modified cells are able to expand more robustly than normal cells, and this dominance induced by downregulation of the tumor suppressor increases the risk of malignancy.
In addition to revealing a new tumor suppressor gene and its contribution to myeloid malignancies, the study by Heinrichs et al. highlights the importance of gene dosage in mediating the effects of tumor suppressors.
DOI: http://dx.doi.org/10.7554/eLife.00825.002
doi:10.7554/eLife.00825
PMCID: PMC3713455  PMID: 23878725
Myelodysplastic Syndromes; MYBL2; 20q CDR; Human; Mouse
14.  Preserved Function of Regulatory T Cells in Chronic HIV-1 Infection Despite Decreased Numbers in Blood and Tissue 
The Journal of Infectious Diseases  2012;205(10):1495-1500.
Regulatory T cells (Tregs) are potent immune modulators, but their role in human immunodeficiency virus type 1 (HIV-1) pathogenesis remains poorly understood. We performed a detailed analysis of the frequency and function of Tregs in a large cohort of HIV-1–infected individuals and HIV-1 negative controls. While HIV “elite controllers” and uninfected individuals had similar Treg numbers and frequencies, the absolute numbers of Tregs declined in blood and gut-associated lymphoid tissue in patients with chronic progressive HIV-1 infection. Despite quantitative changes in Tregs, HIV-1 infection was not associated with an impairment of ex vivo suppressive function of flow-sorted Tregs in both HIV controllers and untreated chronic progressors.
doi:10.1093/infdis/jis236
PMCID: PMC3415814  PMID: 22427677
15.  SIRPα and FHOD1 are unique markers of littoral cells, a recently evolved major cell population of red pulp of human spleen 
Asplenic individuals are compromised not only in their ability to destroy infectious agents, but are at increased risk of death from autoimmune disease, certain tumors, and ischemic heart disease. Enhanced mortality is attributed to lack of phagocytes sequestered in spleen that efficiently engulf and destroy appropriate targets, though related cells are found elsewhere. To determine whether a unique population regulates RBC-pathogen clearance and filtration of altered self, we reviewed the anatomic literature and analyzed in situ by immunohistochemistry and immunofluorescence the expression patterns of a little-characterized cell that dominates the splenic red pulp of man and closely related primates-the venous sinus lining or littoral cell (LC). High expression of the formin FHOD1 outlines the LC population. Though LCs are endothelial-like in distribution they express several macrophage directed proteins, the RBC antigen DARC and T-cell co-receptor CD8α/α yet they lack lineage-associated markers CD34 and CD45. Strikingly, SIRPα (CD172a) expression in human spleen concentrates on LCs, consistent with recent demonstration of a key role in RBC turnover and elimination versus release of infected or altered self. Our results indicate human LCs (SIRPα+, FHOD1+, CD8α/α+, CD34−, CD45−) comprise a highly plastic barrier cell population that emerged late in primate evolution coordinate with CD8 expression. Unique to Hominidae, LCs may be the ultimate determinant of which cells re-circulate after passage through human spleen.
doi:10.4049/jimmunol.1103086
PMCID: PMC3331893  PMID: 22490440
Spleen; littoral cell; angioma; RBC; FHOD1; DARC; CD8α/α; SIRPα; primate
16.  TIM-3: A Novel Regulatory Molecule of Alloimmune Activation 
T cell Ig domain and mucin domain (TIM)-3 has previously been established as a central regulator of Th1 responses and immune tolerance. In this study, we examined its functions in allograft rejection in a murine model of vascularized cardiac transplantation. TIM-3 was constitutively expressed on dendritic cells and natural regulatory T cells (Tregs) but only detected on CD4+FoxP3− and CD8+ T cells in acutely rejecting graft recipients. A blocking anti–TIM-3 mAb accelerated allograft rejection only in the presence of host CD4+ T cells. Accelerated rejection was accompanied by increased frequencies of alloreactive IFN-γ–, IL-6–, and IL-17–producing splenocytes, enhanced CD8+ cytotoxicity against alloantigen, increased alloantibody production, and a decline in peripheral and intragraft Treg/effector T cell ratio. Enhanced IL-6 production by CD4+ T cells after TIM-3 blockade plays a central role in acceleration of rejection. Using an established alloreactivity TCR transgenic model, blockade of TIM-3 increased allospecific effector T cells, enhanced Th1 and Th17 polarization, and resulted in a decreased frequency of overall number of allospecific Tregs. The latter is due to inhibition in induction of adaptive Tregs rather than prevention of expansion of allospecific natural Tregs. In vitro, targeting TIM-3 did not inhibit nTreg-mediated suppression of Th1 alloreactive cells but increased IL-17 production by effector T cells. In summary, TIM-3 is a key regulatory molecule of alloimmunity through its ability to broadly modulate CD4+ T cell differentiation, thus recalibrating the effector and regulatory arms of the alloimmune response.
doi:10.4049/jimmunol.0903435
PMCID: PMC3619947  PMID: 20956339
17.  Activated ALK Collaborates with MYCN in Neuroblastoma Pathogenesis 
Cancer Cell  2012;21(3):362-373.
SUMMARY
Amplification of the MYCN oncogene in childhood neuroblastoma is often accompanied by mutational activation of ALK (anaplastic lymphoma kinase), suggesting their pathogenic cooperation. We generated a transgenic zebrafish model of neuroblastoma in which MYCN-induced tumors arise from a subpopulation of neuroblasts that migrate into the adrenal medulla analogue following organogenesis. Coexpression of activated ALK with MYCN in this model triples the disease penetrance and markedly accelerates tumor onset. MYCN overexpression induces adrenal sympathetic neuroblast hyperplasia, blocks chromaffin cell differentiation, and ultimately triggers a developmentally-timed apoptotic response in the hyperplastic sympathoadrenal cells. Coexpression of activated ALK with MYCN provides prosurvival signals that block this apoptotic response and allow continued expansion and oncogenic transformation of hyperplastic neuroblasts, thus promoting progression to neuroblastoma.
doi:10.1016/j.ccr.2012.02.010
PMCID: PMC3315700  PMID: 22439933
18.  Immune Surveillance and Therapy of Lymphomas Driven by Epstein-Barr -Virus Protein LMP1 in a Mouse Model 
Cell  2012;148(4):739-751.
SUMMARY
B cells infected by Epstein-Barr-Virus (EBV), a transforming virus endemic in humans, are rapidly cleared by the immune system, but some cells harboring the virus persist for life. Under conditions of immunosuppression EBV can spread from these cells and cause life threatening pathologies. We have generated mice expressing the transforming EBV latent membrane protein 1 (LMP1), mimicking a constitutively active CD40 coreceptor, specifically in B cells. Like human EBV infected cells, LMP1+ B cells were efficiently eliminated by T cells, and breaking immune surveillance resulted in rapid, fatal lymphoproliferation and lymphomagenesis. The lymphoma cells expressed ligands for a natural killer (NK) cell receptor, NKG2D, and could be targeted by an NKG2D-Fc fusion protein. These experiments indicate a central role for LMP1 in the surveillance and transformation of EBV infected B cells in vivo, establish a pre-clinical model for B cell lymphomagenesis in immunosuppressed patients, and validate a novel therapeutic approach.
doi:10.1016/j.cell.2011.12.031
PMCID: PMC3313622  PMID: 22341446
19.  A Novel, Highly Sensitive Antibody Allows for the Routine Detection of ALK-rearranged Lung Adenocarcinomas by Standard Immunohistochemistry 
Purpose
Approximately 5% of lung adenocarcinomas harbor an EML4-ALK gene fusion and define a unique tumor group that may be responsive to targeted therapy. However ALK-rearranged lung adenocarcinomas are difficult to detect by either standard fluorescence in-situ hybridization (FISH) or immunohistochemical (IHC) assays. In the present study we used novel antibodies to compare ALK protein expression in genetically defined lung cancers and anaplastic large cell lymphomas (ALCL).
Experimental Design
We analyzed 174 tumors with one standard, and two novel monoclonal antibodies recognizing the ALK protein. Immunostained tissue sections were assessed for the level of tumor-specific ALK expression by objective quantitative image analysis and independently by three pathologists.
Results
ALK protein is invariably and exclusively expressed in ALK-rearranged lung adenocarcinomas but at much lower levels than in the prototypic ALK-rearranged tumor, anaplastic large cell lymphoma, and as a result, often not detected by conventional IHC. We further validate a novel IHC that shows excellent sensitivity and specificity (100% and 99%, respectively) for the detection of ALK-rearranged lung adenocarcinomas in biopsy specimens with excellent interobserver agreement between pathologists (kappa statistic, 0.94).
Conclusions
Low levels of ALK protein expression is a characteristic feature of ALK-rearranged lung adenocarcinomas. However a novel, highly sensitive IHC assay reliably detects lung adenocarcinomas with ALK rearrangements and obviates the need for FISH analysis for the majority of cases and therefore could be routinely applicable in clinical practice to detect lung cancers that may be responsive to ALK inhibitors.
doi:10.1158/1078-0432.CCR-09-2845
PMCID: PMC2831135  PMID: 20179225
Lung adenocarcinoma; ALK; immunohistochemistry
20.  Autocrine activation of the MET receptor tyrosine kinase in acute myeloid leukemia 
Nature medicine  2012;18(7):1118-1122.
Although the treatment of acute myeloid leukemia (AML) has improved significantly, more than half of all patients develop disease that is refractory to intensive chemotherapy1,2. Functional genomics approaches offer a means to discover specific molecules mediating aberrant growth and survival of cancer cells3–8. Thus, using a loss-of-function RNA interference genomic screen, we identified aberrant expression of the hepatocyte growth factor (HGF) as a critical factor in AML pathogenesis. We found HGF expression leading to autocrine activation of its receptor tyrosine kinase, MET, in nearly half of the AML cell lines and clinical samples studied. Genetic depletion of HGF or MET potently inhibited the growth and survival of HGF-expressing AML cells. However, leukemic cells treated with the specific MET kinase inhibitor crizotinib developed resistance due to compensatory upregulation of HGF expression, leading to restoration of MET signaling. In cases of AML where MET is coactivated with other tyrosine kinases, such as fibroblast growth factor receptor 1 (FGFR1)9, concomitant inhibition of FGFR1 and MET blocked compensatory HGF upregulation, resulting in sustained logarithmic cell kill both in vitro and in xenograft models in vivo. Our results demonstrate widespread dependence of AML cells on autocrine activation of MET, as well as the importance of compensatory upregulation of HGF expression in maintaining leukemogenic signaling by this receptor. We anticipate that these findings will lead to the design of additional strategies to block adaptive cellular responses that drive compensatory ligand expression as an essential component of the targeted inhibition of oncogenic receptors in human cancers.
doi:10.1038/nm.2819
PMCID: PMC3438345  PMID: 22683780
21.  Next-Generation cDNA Screening for Oncogene and Resistance Phenotypes 
PLoS ONE  2012;7(11):e49201.
There is a pressing need for methods to define the functional relevance of genetic alterations identified by next-generation sequencing of cancer specimens. We developed new approaches to efficiently construct full-length cDNA libraries from small amounts of total RNA, screen for transforming and resistance phenotypes, and deconvolute by next-generation sequencing. Using this platform, we screened a panel of cDNA libraries from primary specimens and cell lines in cytokine-dependent murine Ba/F3 cells. We demonstrate that cDNA library-based screening can efficiently identify DNA and RNA alterations that confer either cytokine-independent proliferation or resistance to targeted inhibitors, including RNA alterations and intergenic fusions. Using barcoded next-generation sequencing, we simultaneously deconvoluted cytokine-independent clones recovered after transduction of 21 cDNA libraries. This approach identified multiple gain-of-function alleles, including KRAS G12D, NRAS Q61K and an activating splice variant of ERBB2. This approach has broad applicability for identifying transcripts that confer proliferation, resistance and other phenotypes in vitro and potentially in vivo.
doi:10.1371/journal.pone.0049201
PMCID: PMC3492311  PMID: 23145123
22.  A novel ALK secondary mutation and EGFR signaling cause resistance to ALK kinase inhibitors 
Cancer Research  2011;71(18):6051-6060.
Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs), including crizotinib, are effective treatments in preclinical models and in cancer patients with ALK-translocated cancers. However, their efficacy will ultimately be limited by the development of acquired drug resistance. Here we report two mechanisms of ALK TKI resistance identified from, a crizotinib treated non-small cell lung cancer (NSCLC) patient and in a cell line generated from the resistant tumor (DFCI076), and from studying a resistant version of the ALK TKI (TAE684) sensitive H3122 cell line. The crizotinib resistant DFCI076 cell line, harboured a unique L1152R ALK secondary mutation, and was also resistant to the structurally unrelated ALK TKI TAE684. Although the DFCI076 cell line was still partially dependent on ALK for survival, it also contained concurrent co-activation of epidermal growth factor receptor (EGFR) signalling. In contrast, the TAE684 resistant (TR3) H3122 cell line did not contain an ALK secondary mutation but instead harboured co-activation of EGFR signalling. Dual inhibition of both ALK and EGFR was the most effective therapeutic strategy for the DFCI076 and H3122 TR3 cell lines. We further identified a subset (3/50; 6%) of treatment naïve NSCLC patients with ALK rearrangements that also had concurrent EGFR activating mutations. Our studies identify resistance mechanisms to ALK TKIs mediated by both ALK and by a bypass signalling pathway mediated by EGFR. These mechanisms can occur independently, or in the same cancer, suggesting that the combination of both ALK and EGFR inhibitors may represent an effective therapy for these subsets of NSCLC patients.
doi:10.1158/0008-5472.CAN-11-1340
PMCID: PMC3278914  PMID: 21791641
Non-small cell lung carcinoma; translocation; kinase inhibitor; drug resistance; mutation
23.  The novel costimulatory pathway PDL1: B7.1 is functional in inhibiting alloimmune responses in vivo1 
The PDL1: PD1 costimulatory pathway plays an important role in the inhibition of alloimmune responses as well as in the induction and maintenance of peripheral tolerance. It has recently been demonstrated that PDL1 can also bind B7.1 to inhibit T cell responses in vitro. Using the bm12 into B6 heart transplant model, we investigated the functional significance of this interaction in alloimmune responses in vivo. PD1 blockade unlike PDL1 blockade failed to accelerate bm12 allograft rejection suggesting a role for an additional binding partner for PDL1 other than PD1 in transplant rejection. PDL1 blockade was able to accelerate allograft rejection in B7.2-deficient recipients but not B7.1-deficient recipients, indicating that PDL1 interaction with B7.1 was important in inhibiting rejection. Administration of the novel 2H11 anti-PDL1 mAb, which only blocks PDL1: B7.1 interaction, aggravated chronic injury of bm12 allografts in B6 recipients. Aggravated chronic injury was associated with an increased frequency of alloreactive IFN-γ-, IL-4-, and IL-6-producing splenocytes and a decreased percentage of regulatory T cells in the recipients. Using an in vitro cell culture assay, blockade of the interaction of PDL1 on dendritic cells with B7.1 on T cells increased IFN-γ production from alloreactive CD4+ T cells, whereas blockade of dendritic cell B7.1 interaction with T cell PDL1 did not. These data indicate that PDL1 interaction with B7.1 plays an important role in the inhibition of alloimmune responses in vivo and suggests a dominant direction for PDL1 and B7.1 interaction.
doi:10.4049/jimmunol.1100056
PMCID: PMC3140607  PMID: 21697455
Costimulation; Transplantation; MHC; Knockout mice
24.  Immunohistochemical Detection of MYC-driven Diffuse Large B-Cell Lymphomas 
PLoS ONE  2012;7(4):e33813.
Diffuse large B cell lymphoma (DLBCL) is a clinically and genetically heterogeneous disease. A small subset of DLBCLs has translocations involving the MYC locus and an additional group has a molecular signature resembling Burkitt lymphoma (mBL). Presently, identification of such cases by morphology is unreliable and relies on cytogenetic or complex molecular methods such as gene transcriptional profiling. Herein, we describe an immunohistochemical (IHC) method for identifying DLBCLs with increased MYC protein expression. We tested 77 cases of DLBCL and identified 15 cases with high MYC protein expression (nuclear staining in >50% of tumor cells). All MYC translocation positive cases had increased MYC protein expression by this IHC assay. In addition, gene set enrichment analysis (GSEA) of the DLBCL transcriptional profiles revealed that tumors with increased MYC protein expression (regardless of underlying MYC translocation status) had coordinate upregulation of MYC target genes, providing molecular confirmation of the IHC results. We then generated a molecular classifier derived from the MYC IHC results in our cases and employed it to successfully classify mBLs from two previously reported independent case series, providing additional confirmation that the MYC IHC results identify clinically important subsets of DLBCLs. Lastly, we found that DLBCLs with high MYC protein expression had inferior overall survival when treated with R-CHOP. In conclusion, the IHC method described herein can be used to readily identify the biologically and clinically distinct cases of MYC-driven DLBCL, which represent a clinically significant subset of DLBCL cases due to their inferior overall survival.
doi:10.1371/journal.pone.0033813
PMCID: PMC3325231  PMID: 22511926
25.  Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells 
Cell stem cell  2011;8(2):200-213.
SUMMARY
TET-family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Here we show that Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem (ES) cells, and are induced concomitantly with 5hmC during reprogramming of fibroblasts to induced pluripotent stem cells. ES cells depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Lefty1, and display hyperactive Nodal signalling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies in vitro. In Fgf4- and heparin-supplemented culture conditions, Tet1-depleted ES cells activate the trophoblast stem cell lineage determinant Elf5 and can colonize the placenta in mid-gestation embryo chimeras. Consistent with these findings, Tet1-depleted ES cells form aggressive hemorrhagic teratomas with increased endoderm, reduced neuroectoderm and ectopic appearance of trophoblastic giant cells. Thus 5hmC is a novel epigenetic modification associated with the pluripotent state, and Tet1 functions to regulate the lineage differentiation potential of ES cells.
doi:10.1016/j.stem.2011.01.008
PMCID: PMC3134318  PMID: 21295276

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