Hepatocellular carcinoma (HCC) is the third leading cause of death due to cancer worldwide with over 500,000 people affected annually. Although chemotherapy has been widely used to treat patients with HCC, alternate modalities to specifically deliver therapeutic cargos to cancer cells have been sought in recent years due to the severe side effects of chemotherapy. In this respect, aptamer-based tumor targeted drug delivery has emerged as a promising approach to increase the efficacy of chemotherapy and reduce or eliminate drug toxicity. In this study, we developed a new HepG2-specific aptamer (HCA#3) by a procedure known as systematic evolution of ligands by exponential enrichment (SELEX) and exploited its role as a targeting ligand to deliver doxorubicin (Dox) to HepG2 cells in vitro. The selected 76-base nucleotide aptamer preferentially bound to HepG2 hepatocellular carcinoma cells but not to control cells. The aptamer HCA#3 was modified with paired CG repeats at the 5'-end to carry and deliver a high payload of intercalated Dox molecules at the CG sites. Four Dox molecules (mol/mol) were fully intercalated in each conjugate aptamer-Dox (ApDC) molecule. Biostability analysis showed that the ApDC molecules are stable in serum. Functional analysis showed that ApDC specifically targeted and released Dox within HepG2 cells but not in control cells, and treatment with HCA#3 ApDC induced HepG2 cell apoptosis but had minimal effect on control cells. Our study demonstrated that HCA#3 ApDC is a promising aptamer-targeted therapeutic that can specifically deliver and release a high doxorubicin payload in HCC cells.

Post-transplant lymphoproliferative disorders of T- or NK-cell origin (T/NK-PTLD) are rare entities and their genetic basis is unclear. We performed targeted sequencing of 465 cancer-related genes and high-resolution copy number analysis in 17 T-PTLD and 2 NK-PTLD cases. Overall, 377 variants were detected, with an average of 20 variants per case. Mutations of epigenetic modifier genes (TET2, KMT2C, KMT2D, DNMT3A, ARID1B, ARID2, KDM6B, n=11). and inactivation of TP53 by mutation and/or deletion(n=6) were the most frequent alterations, seen across disease subtypes, followed by mutations of JAK/STAT pathway genes (n=5). Novel variants, including mutations in TBX3 (n=3), MED12 (n=3) and MTOR (n=1), were observed as well. High-level microsatellite instability was seen in 1 of 14 (7%) cases, which had a heterozygous PMS2 mutation. Complex copy number changes were detected in 8 of 16 (50%) cases and disease subtype-specific aberrations were also identified. In contrast to B-cell PTLDs, the molecular and genomic alterations observed in T/NK-PTLD appear similar to those reported for peripheral T-cell lymphomas occurring in immunocompetent hosts, which may suggest common genetic mechanisms of lymphoma development.

Although several imaging modalities are widely used for tumor imaging, none are tumor type-specific. Different types of cancer exhibit differential therapeutic responses, thus necessitating development of an imaging modality able to detect various tumor types with high specificity. To illustrate this point, CD30-specific oligonucleotide aptamer in vivo imaging probes were conjugated to the near-infrared IRD800CW reporter. Mice bearing xenografted CD30-positive or control CD30-negative lymphoma tumors on contralateral sides of the same mouse were developed. Following a systemic administration of aptamer probes, whole body imaging of tumor-bearing mice was performed. Imaging signal from tumor sites was analyzed and imaging specificity confirmed by tissue immunostaining. The in vivo biodistribution of aptamer probes was also evaluated. Whole body scans revealed that the RNA-based aptamer probes selectively highlighted CD30-expressing lymphoma tumors immediately after systemic administration, but did not react with control tumors in the same mouse. The resultant imaging signal lasted up to 1 hr and the aptamer probes were rapidly eliminated from the body through urinary and lower intestinal tracts. For more sensitive imaging, biostable CD30-specific ssDNA-based aptamer probes were also generated. Systemic administration of these probes also selectively highlighted the CD30-positive lymphoma tumors, with imaging signal detected 4-5 folds higher than that derived from control tumors in the same animal, and lasted for up to 24hr. This study demonstrates that oligonucleotide aptamer probes can provide tumor type-specific imaging with high sensitivity and a long-lasting signal, indicating their potential for clinical applications.

We report that diffuse large B cell lymphoma (DLBCL) commonly fails to express cell-surface molecules necessary for the recognition of tumor cells by immune-effector cells. In 29% of cases, mutations and deletions inactivate the β2-Microglobulin gene, thus preventing the cell-surface expression of the HLA class-I (HLA-I) complex that is necessary for recognition by CD8(+) cytotoxic T cells. In 21% of cases, analogous lesions involve the CD58 gene, which encodes a molecule involved in T and natural killer cell-mediated responses. In addition to gene inactivation, alternative mechanisms lead to aberrant expression of HLA-I and CD58 in >60% of DLBCL. These two events are significantly associated in this disease, suggesting that they are coselected during lymphomagenesis for their combined role in escape from immune-surveillance.

Microfluidics have become an enabling technology for point-of-care and personalized diagnostics. Desirable capabilities of microfluidics-based diagnostic devices include simplicity, portability, low cost and the performance of multiplexed and quantitative measurements, ideally in a high-throughput format. Here we present the multiplexed volumetric bar-chart chip (V-Chip), which integrates all these capabilities in one device. A key feature of the V-Chip is that quantitative results are displayed as bar charts directly on the device-without the need for optical instruments or any data processing or plotting steps. This is achieved by directly linking oxygen production by catalase, which is proportional to the concentration of the analyte, with the displacement of ink along channels on the device. We demonstrate the rapid quantification of protein biomarkers in diverse clinical samples with the V-Chip. The development of the V-Chip thus opens up the possibility of greatly simplified point-of-care and personalized diagnostics.

Histone deacetylase 9 (HDAC9) is expressed in B cells, and its overexpression has been observed in B-lymphoproliferative disorders, including B-cell non-Hodgkin lymphoma (B-NHL). We examined HDAC9 protein expression and copy number alterations in primary B-NHL samples, identifying high HDAC9 expression among various lymphoma entities and HDAC9 copy number gains in 50% of diffuse large B-cell lymphoma (DLBCL). To study the role of HDAC9 in lymphomagenesis, we generated a genetically engineered mouse (GEM) model that constitutively expressed an HDAC9 transgene throughout B-cell development under the control of the immunoglobulin heavy chain (IgH) enhancer (Eμ). Here, we report that the Eμ-HDAC9 GEM model develops splenic marginal zone lymphoma and lymphoproliferative disease (LPD) with progression towards aggressive DLBCL, with gene expression profiling supporting a germinal center cell origin, as is also seen in human B-NHL tumors. Analysis of Eμ-HDAC9 tumors suggested that HDAC9 might contribute to lymphomagenesis by altering pathways involved in growth and survival, as well as modulating BCL6 activity and p53 tumor suppressor function. Epigenetic modifications play an important role in the germinal center response, and deregulation of the B-cell epigenome as a consequence of mutations and other genomic aberrations are being increasingly recognized as important steps in the pathogenesis of a variety of B-cell lymphomas. A thorough mechanistic understanding of these alterations will inform the use of targeted therapies for these malignancies. These findings strongly suggest a role for HDAC9 in B-NHL and establish a novel GEM model for the study of lymphomagenesis and, potentially, preclinical testing of therapeutic approaches based on histone deacetylase inhibitors.

Plasmacytoid dendritic cells (pDCs) are primary producers of type I interferon (IFN) in response to viruses. The IFN-producing capacity of pDCs is regulated by specific inhibitory receptors, yet none of the known receptors are conserved in evolution. We report that within the human immune system, receptor protein tyrosine phosphatase sigma (PTPRS) is expressed specifically on pDCs. Surface PTPRS was rapidly downregulated after pDC activation, and only PTPRS(-) pDCs produced IFN-α. Antibody-mediated PTPRS crosslinking inhibited pDC activation, whereas PTPRS knockdown enhanced IFN response in a pDC cell line. Similarly, murine Ptprs and the homologous receptor phosphatase Ptprf were specifically co-expressed in murine pDCs. Haplodeficiency or DC-specific deletion of Ptprs on Ptprf-deficient background were associated with enhanced IFN response of pDCs, leukocyte infiltration in the intestine and mild colitis. Thus, PTPRS represents an evolutionarily conserved pDC-specific inhibitory receptor, and is required to prevent spontaneous IFN production and immune-mediated intestinal inflammation.

Anaplastic large cell lymphoma (ALCL) is the most common T-cell lymphoma in children. ALCL cells characteristically express surface CD30 molecules and carry the pathogenic ALK oncogene, both of which are diagnostic biomarkers and are also potential therapeutic targets. For precision therapy, we report herein a protamine nanomedicine incorporated with oligonucleotide aptamers to selectively target lymphoma cells, a dsDNA/drug payload to efficiently kill targeted cells, and an siRNA to specifically silence ALK oncogenes. The aptamer-equipped protamine nanomedicine was simply fabricated through a non-covalent charge-force reaction. The products had uniform structure morphology under an electron microscope and a peak diameter of 103 nm by dynamic light scattering measurement. Additionally, flow cytometry analysis demonstrated that under CD30 aptamer guidance, the protamine nanomedicine specifically bound to lymphoma cells, but did not react to off-target cells in control experiments. Moreover, specific cell targeting and intracellular delivery of the nanomedicine were also validated by electron and confocal microscopy. Finally, functional studies demonstrated that, through combined cell-selective chemotherapy using a drug payload and oncogene-specific gene therapy using an siRNA, the protamine nanomedicine effectively killed lymphoma cells with little toxicity to off-target cells, indicating its potential for precision therapy.

Neuroblastoma (NB) is a childhood cancer arising from sympatho-adrenal neural crest cells. MYCN amplification is found in half of high-risk NB patients; however, no available therapies directly target MYCN. Using multi-dimensional metabolic profiling in MYCN expression systems and primary patient tumors, we comprehensively characterized the metabolic landscape driven by MYCN in NB. MYCN amplification leads to glycerolipid accumulation by promoting fatty acid (FA) uptake and biosynthesis. We found that cells expressing amplified MYCN depend highly on FA uptake for survival. Mechanistically, MYCN directly upregulates FA transport protein 2 (FATP2), encoded by SLC27A2. Genetic depletion of SLC27A2 impairs NB survival, and pharmacological SLC27A2 inhibition selectively suppresses tumor growth, prolongs animal survival, and exerts synergistic anti-tumor effects when combined with conventional chemotherapies in multiple preclinical NB models. This study identifies FA uptake as a critical metabolic dependency for MYCN-amplified tumors. Inhibiting FA uptake is an effective approach for improving current treatment regimens.

Gemcitabine is a chemotherapeutic used clinically to treat a variety of cancers. However, because it lacks tumor cell specificity, gemcitabine may cause off-target cytotoxicity and adversely impact patients. To impart cancer cell specificity to gemcitabine and improve its therapeutic efficacy, we synthesized a unique aptamer-drug conjugate that carries a high gemcitabine payload (three molecules) via a dendrimer structure and enzymatically cleavable linkers for controlled intracellular drug release. First, linker-gemcitabinedendrimer-linker-gemcitabine products were produced, which had significantly lower cytotoxicity than an equimolar amount of free drug. Biochemical analysis revealed that lysosomal cathepsin B protease rapidly cleaved the dendritic linkers and released the conjugated gemcitabine as a free drug. Subsequently, the dendrimer-linker-gemcitabine was coupled with a cell-specific aptamer to form aptamer-gemcitabine conjugates. Functional assays confirmed that, under aptamer guidance, aptamer-gemcitabine conjugates were selectively bound to and then internalized by triple-negative breast cancer cells. Cellular therapy studies indicated that the aptamer-gemcitabine conjugates potentiated cytotoxic activity to targeted cancer cells but did not affect off-target control cells. Our study demonstrates a novel approach to aptamer-mediated targeted drug delivery that combines a high drug payload and an enzymatically controlled drug release switch to achieve higher therapeutic efficacy and fewer off-target effects relative to free-drug chemotherapy.

Splenic marginal zone lymphoma (SMZL), the most common primary lymphoma of spleen, is poorly understood at the genetic level. In this study, using whole-genome DNA sequencing (WGS) and confirmation by Sanger sequencing, we observed mutations identified in several genes not previously known to be recurrently altered in SMZL. In particular, we identified recurrent somatic gain-of-function mutations in NOTCH2, a gene encoding a protein required for marginal zone B cell development, in 25 of 99 (∼25%) cases of SMZL and in 1 of 19 (∼5%) cases of nonsplenic MZLs. These mutations clustered near the C-terminal proline/glutamate/serine/threonine (PEST)-rich domain, resulting in protein truncation or, rarely, were nonsynonymous substitutions affecting the extracellular heterodimerization domain (HD). NOTCH2 mutations were not present in other B cell lymphomas and leukemias, such as chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL; n = 15), mantle cell lymphoma (MCL; n = 15), low-grade follicular lymphoma (FL; n = 44), hairy cell leukemia (HCL; n = 15), and reactive lymphoid hyperplasia (n = 14). NOTCH2 mutations were associated with adverse clinical outcomes (relapse, histological transformation, and/or death) among SMZL patients (P = 0.002). These results suggest that NOTCH2 mutations play a role in the pathogenesis and progression of SMZL and are associated with a poor prognosis.

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous disease composed of at least two distinct subtypes: germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. These phenotypic subtypes segregate with largely unique genetic lesions, suggesting the involvement of different pathogenetic mechanisms. In this report we show that the BLIMP1/PRDM1 gene is inactivated by multiple mechanisms, including homozygous deletions, truncating or missense mutations, and transcriptional repression by constitutively active BCL6, in ∼53% of ABC-DLBCL. In vivo, conditional deletion of Blimp1 in mouse B cells promotes the development of lymphoproliferative disorders recapitulating critical features of the human ABC-DLBCL. These results demonstrate that BLIMP1 is a bona fide tumor-suppressor gene whose loss contributes to lymphomagenesis by blocking plasma cell differentiation.

C-myc protein expression has been studied in mature B-cell lymphomas and overexpression has been associated with poor prognosis. We sought to determine the prognostic significance of c-myc protein expression in B-ALL. We found ≥ 20% c-myc expression to predict risk of persistent disease in all age groups (odds ratio 7.487, p=0.013). There was no statistically significant association between c-myc expression and risk of relapse or death in our study. Routine c-myc immunostaining may help identify higher risk patients and guide management of B-ALL. Additional studies are needed to further determine the molecular mechanisms and role of c-myc expression in B-ALL.

Enteropathy-associated T-cell lymphomas (EATL) are rare and generally aggressive types of peripheral T-cell lymphomas. Rare cases of primary, small intestinal CD4+ T-cell lymphomas with indolent behavior have been described, but are not well characterized. We describe morphologic, phenotypic, genomic and clinical features of 3 cases of indolent primary small intestinal CD4+ T-cell lymphomas. All patients presented with diarrhea and weight loss and were diagnosed with celiac disease refractory to a gluten free diet at referring institutions. Small intestinal biopsies showed crypt hyperplasia, villous atrophy and a dense lamina propria infiltrate of small-sized CD4+ T-cells often with CD7 downregulation or loss. Gastric and colonic involvement was also detected (n = 2 each). Persistent, clonal TCRβ gene rearrangement products were detected at multiple sites. SNP array analysis showed relative genomic stability, early in disease course, and non-recurrent genetic abnormalities, but complex changes were seen at disease transformation (n = 1). Two patients are alive with persistent disease (4.6 and 2.5 years post-diagnosis), despite immunomodulatory therapy; one died due to bowel perforation related to large cell transformation 11 years post-diagnosis. Unique pathobiologic features warrant designation of indolent small intestinal CD4+ T-cell lymphoma as a distinct entity, greater awareness of which would avoid misdiagnosis as EATL or an inflammatory disorder, especially celiac disease.

In addition to intrinsic genetic alterations, the effects of the extrinsic microenvironment also play a pathological role in cancer development. Altered chemokine/cytokine networks in the tumor microenvironment may contribute to the dysregulation of cellular functions in cancer cells. Anaplastic large cell lymphoma (ALCL) is an aggressive T-cell lymphoma caused by abnormal expression of anaplastic lymphoma kinase due to a chromosomal translocation. Notably, ALCL cells are also characterized by high-level expression of the high-affinity IL-2 receptor subunit CD25 on the cell surface. However, whether the IL-2/IL-2 receptor functions in ALCL cells and how this signaling affects the tumor remain unclear. In this study, we treated cultured ALCL cells with exogenous IL-2 and examined changes in cellular function and signaling pathways. IL-2 stimulated cell growth and augmented activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway. Additionally, IL-2 enhanced lymphoma cell survival by overcoming kinase inhibitor U0126-induced growth arrest and apoptosis. Subsequently, to identify the potential source of IL-2 for lymphoma cells in vivo, we performed gene expression and immunochemical analyses. RT-PCR revealed no IL-2 gene expression in cultured ALCL cells and ruled out the possibility of an IL-2 autocrine loop. Interestingly, immunostaining of lymphoma tumor tissues showed IL-2 protein expression in background cells within tumor tissue, but not in ALCL cells. Our findings demonstrate that IL-2 signaling plays a functional role in ALCL cells, and enhances lymphoma cell survival by increasing activation of the ERK1/2 pathway.

Oligonucleotide aptamers can specifically bind biomarkers on cancer cells and can be readily chemically modified with different functional molecules for personalized medicine. To target acute myeloid leukemia (AML) cells, we developed a single-strand DNA aptamer specific for the biomarker CD117, which is highly expressed on AML cells. Sequence alignment revealed that the aptamer contained a G-rich core region with a well-conserved functional G-quadruplex structure. Functional assays demonstrated that this synthetic aptamer was able to specifically precipitate CD117 proteins from cell lysates, selectively bound cultured and patient primary AML cells with high affinity (Kd < 5 nM), and was specifically internalized into CD117-expressing cells. For targeted AML treatment, aptamer-drug conjugates were fabricated by chemical synthesis of aptamer (Apt) with methotrexate (MTX), a central drug used in AML chemotherapy regimens. The formed Apt-MTX conjugates specifically inhibited AML cell growth, triggered cell apoptosis, and induced cell cycle arrest in G1 phase. Importantly, Apt-MTX had little effect on CD117-negative cells under the same treatment conditions. Moreover, exposure of patient marrow specimens to Apt-MTX resulted in selective growth inhibition of primary AML cells and had no toxicity to off-target background normal marrow cells within the same specimens. These findings indicate the potential clinical value of Apt-MTX for targeted AML therapy with minimal to no side effects in patients, and also open an avenue to chemical synthesis of new, targeted biotherapeutics.

Splenic marginal zone lymphoma is a rare lymphoma. Loss of 7q31 and somatic mutations affecting the NOTCH2 and KLF2 genes are the commonest genomic aberrations. Epigenetic changes can be pharmacologically reverted; therefore, identification of groups of patients with specific epigenomic alterations might have therapeutic relevance. Here we integrated genome-wide DNA-promoter methylation profiling with gene expression profiling, and clinical and biological variables. An unsupervised clustering analysis of a test series of 98 samples identified 2 clusters with different degrees of promoter methylation. The cluster comprising samples with higher-promoter methylation (High-M) had a poorer overall survival compared with the lower (Low-M) cluster. The prognostic relevance of the High-M phenotype was confirmed in an independent validation set of 36 patients. In the whole series, the High-M phenotype was associated with IGHV1-02 usage, mutations of NOTCH2 gene, 7q31-32 loss, and histologic transformation. In the High-M set, a number of tumor-suppressor genes were methylated and repressed. PRC2 subunit genes and several prosurvival lymphoma genes were unmethylated and overexpressed. A model based on the methylation of 3 genes (CACNB2, HTRA1, KLF4) identified a poorer-outcome patient subset. Exposure of splenic marginal zone lymphoma cell lines to a demethylating agent caused partial reversion of the High-M phenotype and inhibition of proliferation.

The current antibody-mediated numeration assays of circulating tumor cells (CTCs) require multiple steps and are time-consuming. To overcome these technical limitations, a cancer cell-activatable aptamer-reporter was formulated by conjugating a biomarker-specific aptamer sequence with paired fluorochrome-quencher molecules. In contrast to the antibody probes, the intact aptamer-reporter was optically silent in the absence of cells of interest. However, when used in an assay, the aptamer selectively targeted cancer cells through interaction with a specific surface biomarker, which triggered internalization of the aptamer-reporter and, subsequently, into cell lysosomes. Rapid lysosomal degradation of the aptamer-reporter resulted in separation of the paired fluorochrome-quencher molecules. The released fluorochrome emitted bright fluorescent signals exclusively within the targeted cancer cells, with no background noise in the assay. Thus, the assays could be completed in a single step within minutes. By using this one-step assay, CTCs in whole blood and marrow aspirate samples of patients with lymphoma tumors were selectively highlighted and rapidly detected with no off-target signals from background blood cells. The development of the cancer cell-activatable aptamer-reporter system allows for the possibility of a simple and robust point-of-care test for CTC detection, which is currently unavailable.

Follicular lymphoma (FL) is an indolent disease, but 30%-40% of cases undergo histologic transformation to an aggressive malignancy, typically represented by diffuse large B cell lymphoma (DLBCL). The pathogenesis of this process remains largely unknown. Using whole-exome sequencing and copy-number analysis, we show here that the dominant clone of FL and transformed FL (tFL) arise by divergent evolution from a common mutated precursor through the acquisition of distinct genetic events. Mutations in epigenetic modifiers and antiapoptotic genes are introduced early in the common precursor, whereas tFL is specifically associated with alterations deregulating cell-cycle progression and DNA damage responses (CDKN2A/B, MYC, and TP53) as well as aberrant somatic hypermutation. The genomic profile of tFL shares similarities with that of germinal center B cell-type de novo DLBCL but also displays unique combinations of altered genes with diagnostic and therapeutic implications.

The function of microRNAs (miRNAs) in hematopoietic stem cells (HSCs), committed progenitors, and leukemia stem cells (LSCs) is poorly understood. We show that miR-29a is highly expressed in HSC and down-regulated in hematopoietic progenitors. Ectopic expression of miR-29a in mouse HSC/progenitors results in acquisition of self-renewal capacity by myeloid progenitors, biased myeloid differentiation, and the development of a myeloproliferative disorder that progresses to acute myeloid leukemia (AML). miR-29a promotes progenitor proliferation by expediting G1 to S/G2 cell cycle transitions. miR-29a is overexpressed in human AML and, like human LSC, miR-29a-expressing myeloid progenitors serially transplant AML. Our data indicate that miR-29a regulates early hematopoiesis and suggest that miR-29a initiates AML by converting myeloid progenitors into self-renewing LSC.