Aptamer-armed nanostructures improve the chemotherapy outcome of triple-negative breast cancer.

Triple-negative breast cancer is an aggressive subtype of breast cancer that is primarily treated using systemic chemotherapy due to the lack of a specific cell surface marker for drug delivery. Cancer cell-specific aptamer-mediated drug delivery is a promising targeted chemotherapy for marker-unknown cancers. Using a poorly differentiated carcinoma cell-specific DNA aptamer (PDGC21T), we formed a self-assembling circinate DNA nanoparticle (Apt21TNP) that binds triple-negative breast cancer cells. Using our previously designed pH-sensitive dendrimer-conjugated doxorubicin (DDOX) as the payload, we found that each nanoparticle loaded 30 doxorubicin molecules to form an Apt21TNP-DDOX nanomedicine that is stable in human plasma. Upon cell binding, Apt21TNP-DDOX is internalized by triple-negative breast cancer cells through the macropinocytosis pathway. Once inside cells, the low pH microenvironment in lysosomes induces doxorubicin drug payload release from Apt21TNP-DDOX. Our in vitro studies demonstrate that Apt21TNP-DDOX can preferentially bind triple-negative breast cancer cells to induce cell death. Furthermore, we show that Apt21TNP-DDOX can accumulate in subcutaneous MDA-MB-231 tumors in mice following systemic administration to reduce tumor burden, minimize side effects, and improve animal survival. Together, our results demonstrate that Apt21TNP-mediated doxorubicin delivery is a potent, targeted chemotherapy for triple-negative breast cancer that may alleviate side effects in patients.

EpCAM-Targeting Aptamer Radiotracer for Tumor-Specific PET Imaging.

Noninvasive in vivo imaging to measure the expression of EpCAM, a biomarker overexpressed in the majority of carcinoma tumors and metastatic lesions, is highly desirable for accurate tumor staging and therapy evaluation. Here, we report the use of an aptamer radiotracer to enable tumor-specific EpCAM-targeting PET imaging. Oligonucleotide aptamers are small molecular ligands that specifically bind with high affinity to their target molecules. For specific tumor imaging, an aptamer radiotracer was formulated by chelating a 64Cu isotope and DOTA-PEGylated aptamer sequence to target EpCAM. In vitro cell uptake assays demonstrated that the aptamer radiotracer specifically bound EpCAM-expressing breast cancer cells but did not react with off-target tumor cells. For in vivo tumor imaging, aptamer radiotracer was systemically administered into xenograft mice. MicroPET/CT scans revealed that the aptamer radiotracer rapidly highlighted xenograft tumors derived from MDA-MB-231 breast cancer cells (EpCAM positive) as early as 2 h postadministration with a gradually increasing tumor uptake signal that peaked at 24 h but not in lymphoma 937 tumors (EpCAM negative). In contrast, nonspecific background signals in the liver and kidneys were rapidly decreased postadministration. This proof-of-concept study demonstrates the utility of aptamer radiotracers for tumor-specific PET imaging.

Neutralizing Aptamers Block S/RBD-ACE2 Interactions and Prevent Host Cell Infection.

The receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 spike (S) protein plays a central role in mediating the first step of virus infection to cause disease: virus binding to angiotensin-converting enzyme 2 (ACE2) receptors on human host cells. Therefore, S/RBD is an ideal target for blocking and neutralization therapies to prevent and treat coronavirus disease 2019 (COVID-19). Using a target-based selection approach, we developed oligonucleotide aptamers containing a conserved sequence motif that specifically targets S/RBD. Synthetic aptamers had high binding affinity for S/RBD-coated virus mimics (KD ≈7 nM) and also blocked interaction of S/RBD with ACE2 receptors (IC50 ≈5 nM). Importantly, aptamers were able to neutralize S protein-expressing viral particles and prevent host cell infection, suggesting a promising COVID-19 therapy strategy.

Aptamer Cocktail to Detect Multiple Species of Mycoplasma in Cell Culture.

Mycoplasma contamination of cell line cultures is a common, yet often undetected problem in research laboratories. Many of the existing techniques to detect mycoplasma contamination of cultured cells are time-consuming, expensive, and have significant drawbacks. Here, we describe a mycoplasma detection system that is useful for detecting multiple species of mycoplasma in infected cell lines. The system contains three dye-labeled detection aptamers that can specifically bind to mycoplasma-infected cells and a dye-labeled control aptamer that minimally binds to cells. With this system, mycoplasma-contaminated cells can be detected within 30 min by using a flow cytometer, fluorescence microscope, or microplate reader. Further, this system may be used to detect mycoplasma-contaminated culture medium. This study presents an novel mycoplasma detection model that is simple, rapid, inexpensive, and sensitive.

Aptamer-Engineered Natural Killer Cells for Cell-Specific Adaptive Immunotherapy.

Natural killer (NK) cells are a key component of the innate immune system as they can attack cancer cells without prior sensitization. However, due to lack of cell-specific receptors, NK cells are not innately able to perform targeted cancer immunotherapy. Aptamers are short single-stranded oligonucleotides that specifically recognize their targets with high affinity in a similar manner to antibodies. To render NK cells with target-specificity, synthetic CD30-specific aptamers are anchored on cell surfaces to produce aptamer-engineered NK cells (ApEn-NK) without genetic alteration or cell damage. Under surface-anchored aptamer guidance, ApEn-NK specifically bind to CD30-expressing lymphoma cells but do not react to off-target cells. The resulting specific cell binding of ApEn-NK triggers higher apoptosis/death rates of lymphoma cells compared to parental NK cells. Additionally, experiments with primary human NK cells demonstrate the potential of ApEn-NK to specifically target and kill lymphoma cells, thus presenting a potential new approach for targeted immunotherapy by NK cells.

Self-Assembled Aptamer-Nanomedicine for Targeted Chemotherapy and Gene Therapy.

Chemotherapy is the mainstream treatment of anaplastic large cell lymphoma (ALCL). However, chemotherapy can cause severe adverse effects in patients because it is not ALCL-specific. In this study, a multifunctional aptamer-nanomedicine (Apt-NMed) achieving targeted chemotherapy and gene therapy of ALCL is developed. Apt-NMed is formulated by self-assembly of synthetic oligonucleotides containing CD30-specific aptamer and anaplastic lymphoma kinase (ALK)-specific siRNA followed by self-loading of the chemotherapeutic drug doxorubicin (DOX). Apt-NMed exhibits a well-defined nanostructure (diameter 59 mm) and stability in human serum. Under aptamer guidance, Apt-NMed specifically binds and internalizes targeted ALCL cells. Intracellular delivery of Apt-NMed triggers rapid DOX release for targeted ALCL chemotherapy and intracellular delivery of the ALK-specific siRNA induced ALK oncogene silencing, resulting in combined therapeutic effects. Animal model studies reveal that upon systemic administration, Apt-NMed specifically targets and selectively accumulates in ALCL tumor site, but does not react with off-target tumors in the same xenograft mouse. Importantly, Apt-NMed not only induces significantly higher inhibition in ALCL tumor growth, but also causes fewer or no side effects in treated mice compared to free DOX. Moreover, Apt-NMed treatment markedly improves the survival rate of treated mice, opening a new avenue for precision treatment of ALCL.

An ultra pH-sensitive and aptamer-equipped nanoscale drug-delivery system for selective killing of tumor cells.

Nanotechnology has often been applied in the development of targeted drug-delivery systems for the treatment of cancer. An ideal nanoscale system for drug delivery should be able to selectively deliver and rapidly release the carried therapeutic drug(s) in cancer cells and, more importantly, not react to off-target cells so as to eliminate unwanted toxicity on normal tissues. To reach this goal, a selective chemotherapeutic is formulated using a hollow gold nanosphere (HAuNS) equipped with a biomarker-specific aptamer (Apt), and loaded with the chemotherapy drug doxorubicin (DOX). The formed Apt-HAuNS-Dox, approximately 42 nm in diameter, specifically binds to lymphoma tumor cells and does not react to control cells that do not express the biomarker. Through aptamer-mediated selective cell binding, the Apt-HAuNS-Dox is internalized exclusively into the targeted tumor cells, and then released the DOX intracellularly. Of note, although the formed Apt-HAuNS-Dox is stable under normal biological conditions (pH 7.4), it appears ultrasensitive to pH change and rapidly releases 80% of the loaded DOX within 2 h at pH 5.0, a condition seen in cell lysosomes. Functional assays using cell mixtures show that the Apt-HAuNS-Dox selectively kills lymphoma tumor cells, but has no effect on the growth of the off-target cells in the same cultures, indicating that this ultra pH-sensitive Apt-HAuNS-Dox can selectively treat cancer through specific aptamer guidance, and will have minimal side effects on normal tissue.

Biostable ssDNA aptamers specific for Hodgkin lymphoma.

As a "chemical antibody", oligonucleotide aptamers can specifically bind to their target molecules. However, clinical potential of aptamers in disease diagnosis is not yet fully explored. Using a tumor cell-based selection protocol, we developed single-stranded DNA aptamers for Hodgkin lymphoma (HL) tumor cells. The aptamers specifically bound to HL cells with a high affinity, reaching maximal cell binding at 10 nM final concentration. Importantly, the aptamers were able to selectively detect HL cells and did not react to other tumor or blood cells in mixed samples, indicating that the aptamers can be used as a specific probe for in vitro analysis of HL cells. Moreover, due to the inherent properties of DNA, the aptamers were stable in human serum, suggesting potential for in vivo detection of HL tumor cells.

Gamma Radiation Synthesis of Ag/P25 Nanocomposites for Efficient Photocatalytic Degradation of Organic Contaminant.

Titanium dioxide (TiO2) has garnered significant attention among various photocatalysts, whereas its photocatalytic activity is limited by its wide bandgap and inefficient charge separation, making the exploration of new strategies to improve its photocatalytic performance increasingly important. Here, we report the synthesis of Ag/P25 nanocomposites through a one-step gamma-ray radiation method using AgNO3 and commercial TiO2 (Degussa P25). The resulting products were characterized by powder X-ray diffraction, UV-Vis diffused reflectance spectroscopy, transmission electron microscopy, and X-ray photoelectron spectroscopy. The effect of free radical scavengers, feed ratios of Ag/P25, and dose rates on the photocatalytic activity of the Ag/P25 nanocomposites were systematically investigated using rhodamine B under Xenon light irradiation. The results showed that the Ag/P25 photocatalyst synthesized with a feed ratio of 2.5 wt% and isopropyl alcohol as the free radical scavenger at a dose rate of 130 Gy/min exhibited outstanding photocatalytic activity, with a reaction rate constant of 0.0674 min-1, much higher than that of P25. Additionally, we found that the particle size of Ag could be effectively controlled by changing the dose rate, and the Ag/P25 nanocomposites doped with smaller size of Ag nanoparticles performed higher photocatalytic activities. The synthesis strategy presented in this study offers new insight into the future development of highly efficient photocatalysts using radiation techniques.

Bone age recognition based on mask R-CNN using xception regression model.

Background and Objective: Bone age detection plays an important role in medical care, sports, judicial expertise and other fields. Traditional bone age identification and detection is according to manual interpretation of X-ray images of hand bone by doctors. This method is subjective and requires experience, and has certain errors. Computer-aided detection can effectually enhance the validity of medical diagnosis, especially with the fast development of machine learning and neural network, the method of bone age recognition using machine learning has gradually become the focus of research, which has the advantages of simple data pretreatment, good robustness and high recognition accuracy. Methods: In this paper, the hand bone segmentation network based on Mask R-CNN was proposed to segment the hand bone area, and the segmented hand bone region was directly input into the regression network for bone age evaluation. The regression network is using an enhancd network Xception of InceptionV3. After the output of Xception, the convolutional block attention module is connected to refine the feature mapping from channel and space to obtain more effective features. Results: According to the experimental results, the hand bone segmentation network model based on Mask R-CNN can segment the hand bone region and eliminate the interference of redundant background information. The average Dice coefficient on the verification set is 0.976. The mean absolute error of predicting bone age on our data set was only 4.97 months, which exceeded the accuracy of most other bone age assessment methods. Conclusion: Experiments show that the accuracy of bone age assessment can be enhancd by using the Mask R-CNN-based hand bone segmentation network and the Xception bone age regression network to form a model, which can be well applied to actual clinical bone age assessment.

Aptamer Targets Triple-Negative Breast Cancer through Specific Binding to Surface CD49c.

Although targeted cancer therapy can induce higher therapeutic efficacy and cause fewer side effects in patients, the lack of targetable biomarkers on triple-negative breast cancer (TNBC) cells limits the development of targeted therapies by antibody technology. Therefore, we investigated an alternative approach to target TNBC by using the PDGC21T aptamer, which selectively binds to poorly differentiated carcinoma cells and tumor tissues, although the cellular target is still unknown. We found that synthetic aptamer probes specifically bound cultured TNBC cells in vitro and selectively targeted TNBC xenografts in vivo. Subsequently, to identify the target molecule on TNBC cells, we performed aptamer-mediated immunoprecipitation in lysed cell membranes followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Sequencing analysis revealed a highly conserved peptide sequence consistent with the cell surface protein CD49c (integrin α3). For target validation, we stained cultured TNBC and non-TNBC cells with an aptamer probe or a CD49c antibody and found similar cell staining patterns. Finally, competition cell-binding assays using both aptamer and anti-CD49c antibody revealed that CD49c is the biomarker targeted by the PDGC21T aptamer on TNBC cells. Our findings provide a molecular foundation for the development of targeted TNBC therapy using the PDGC21T aptamer as a targeting ligand.

Targeted immunotherapy of triple-negative breast cancer by aptamer-engineered NK cells.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer comprised of cells that lack expression of targetable biomarkers. Nucleic acid aptamers are a group of molecular ligands that can specifically bind to their targets with high affinity. The ssDNA aptamer PDGC21-T recognizes poorly differentiated cancer cells and tumor tissues through an unidentified cell surface target(s). Because TNBC tumor cells are poorly differentiated, the aptamer PDGC21-T is a promising therapeutic candidate to target TNBC tumor cells. In vitro study revealed that synthetic aptamer probes selectively targeted TNBC cell lines. To assess aptamer immunotherapeutic targeting capability, we generated aptamer-engineered NK cells (ApEn-NK) using aptamer probes as a targeting ligand and NK cells as a therapeutic agent. Cell clustering formation assays revealed that ApEn-NK bound both suspended and adherent TNBC cells with high affinity. In a functional study, ApEn-NK treatment triggered apoptosis and death of cultured TNBC cells. Finally, systemic administration of ApEn-NK in mice harboring TNBC xenografts resulted in significant inhibition of lung metastasis relative to parental NK cell treatments. Unlike chemotherapy, ApEn-NK treatment did not affect body weight in treated mice. We demonstrate a novel approach for targeted TNBC immunotherapy.

Aptamer-Gemcitabine Conjugates with Enzymatically Cleavable Linker for Targeted Delivery and Intracellular Drug Release in Cancer Cells.

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.

MYCN-driven fatty acid uptake is a metabolic vulnerability in neuroblastoma.

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.

Aptamers with Self-Loading Drug Payload and pH-Controlled Drug Release for Targeted Chemotherapy.

Doxorubicin (DOX) is a common anti-tumor drug that binds to DNA or RNA via non-covalent intercalation between G-C sequences. As a therapeutic agent, DOX has been used to form aptamer-drug conjugates for targeted cancer therapy in vitro and in vivo. To improve the therapeutic potential of aptamer-DOX conjugates, we synthesized trifurcated Newkome-type monomer (TNM) structures with three DOX molecules bound through pH-sensitive hydrazone bonds to formulate TNM-DOX. The aptamer-TNM-DOX conjugate (Apt-TNM-DOX) was produced through a simple self-loading process. Chemical validation revealed that Apt-TNM-DOX stably carried high drug payloads of 15 DOX molecules per aptamer sequence. Functional characterization showed that DOX payload release from Apt-TNM-DOX was pH-dependent and occurred at pH 5.0, which reflects the microenvironment of tumor cell lysosomes. Further, Apt-TNM-DOX specifically targeted lymphoma cells without affecting off-target control cells. Aptamer-mediated cell binding resulted in the uptake of Apt-TNM-DOX into targeted cells and the release of DOX payload within cell lysosomes to inhibit growth of targeted lymphoma cells. The Apt-TNM-DOX provides a simple, non-toxic approach to develop aptamer-based targeted therapeutics and may reduce the non-specific side effects associated with traditional chemotherapy.

T-Cell Therapy for Lymphoma Using Nonengineered Multiantigen-Targeted T Cells Is Safe and Produces Durable Clinical Effects.

PURPOSE: Patients with relapsed lymphomas often fail salvage therapies including high-dose chemotherapy and mono-antigen-specific T-cell therapies, highlighting the need for nontoxic, novel treatments. To that end, we clinically tested an autologous T-cell product that targets multiple tumor-associated antigens (TAAs) expressed by lymphomas with the intent of treating disease and preventing immune escape. PATIENTS AND METHODS: We expanded polyclonal T cells reactive to five TAAs: PRAME, SSX2, MAGEA4, SURVIVIN, and NY-ESO-1. Products were administered to 32 patients with Hodgkin lymphomas (n = 14) or non-Hodgkin lymphomas (n = 18) in a two-part phase I clinical trial, where the objective of the first phase was to establish the safety of targeting all five TAAs (fixed dose, 0.5 × 107 cells/m2) simultaneously and the second stage was to establish the maximum tolerated dose. Patients had received a median of three prior lines of therapy and either were at high risk for relapse (adjuvant arm, n = 17) or had chemorefractory disease (n = 15) at enrollment. RESULTS: Infusions were safe with no dose-limiting toxicities observed in either the antigen- or dose-escalation phases. Although the maximum tolerated dose was not reached, the maximum tested dose at which efficacy was observed (two infusions, 2 × 107 cells/m2) was determined as the recommended phase II dose. Of the patients with chemorefractory lymphomas, two (of seven) with Hodgkin lymphomas and four (of eight) with non-Hodgkin lymphomas achieved durable complete remissions (> 3 years). CONCLUSION: T cells targeting five TAAs and administered at doses of up to two infusions of 2 × 107 cells/m2 are well-tolerated by patients with lymphoma both as adjuvant and to treat chemorefractory lymphoma. Preliminary indicators of antilymphoma activity were seen in the chemorefractory cohort across both antigen- and dose-escalation phases.

Neutralizing Aptamers Block S/RBD-ACE2 Interactions and Prevent Host Cell Infection.

The receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 spike (S) protein plays a central role in mediating the first step of virus infection to cause disease: virus binding to angiotensin-converting enzyme 2 (ACE2) receptors on human host cells. Therefore, S/RBD is an ideal target for blocking and neutralization therapies to prevent and treat coronavirus disease 2019 (COVID-19). Using a target-based selection approach, we developed oligonucleotide aptamers containing a conserved sequence motif that specifically targets S/RBD. Synthetic aptamers had high binding affinity for S/RBD-coated virus mimics (K D≈7 nM) and also blocked interaction of S/RBD with ACE2 receptors (IC50≈5 nM). Importantly, aptamers were able to neutralize S protein-expressing viral particles and prevent host cell infection, suggesting a promising COVID-19 therapy strategy.

Using oligonucleotide aptamer probes for immunostaining of formalin-fixed and paraffin-embedded tissues.

For tissue immunostaining, antibodies are currently the only clinically validated and commercially available probes. Aptamers, which belong to a class of small molecule ligands composed of short single-stranded oligonucleotides, have emerged as probes over the last several decades; however, their potential clinical value has not yet been fully explored. Using cultured cells and an RNA-based CD30 aptamer, we recently demonstrated that the synthetic aptamer is useful as a specific probe for flow cytometric detection of CD30-expressing lymphoma cells. In this study, we further validated the use of this aptamer probe for immunostaining of formalin-fixed and paraffin-embedded lymphoma tissues. Using CD30 antibody as a standard control, we demonstrated that the synthetic CD30 aptamer specifically recognized and immunostained tumor cells of classical Hodgkin lymphoma and anaplastic large cell lymphoma, but did not react with background cells within tumor sites. Notably, the CD30 aptamer probe optimally immunostained lymphoma cells with lower temperature antigen retrieval (37 vs 96°C for antibody) and shorter probing reaction times (20 vs 90 min for antibody) than typical antibody immunostaining protocols. In addition, the CD30 aptamer probe showed no nonspecific background staining of cell debris in necrotic tissue and exhibited no cross-reaction to tissues that do not express CD30, as confirmed by a standard CD30 antibody staining. Therefore, our findings indicate that the synthetic oligonucleotide CD30 aptamer can be used as a probe for immunostaining of fixed tissue sections for disease diagnosis.

Aptamer-Equipped Protamine Nanomedicine for Precision Lymphoma Therapy.

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.

Recombinant expression, characterization, and quantification in human cancer cell lines of the Anaplastic Large-Cell Lymphoma-characteristic NPM-ALK fusion protein.

Systemic anaplastic large cell lymphoma (ALCL) is an aggressive T-cell lymphoma most commonly seen in children and young adults. The majority of pediatric ALCLs are associated with the t(2;5)(p23;q35) translocation which fuses the Anaplastic Lymphoma Kinase (ALK) gene with the Nucleophosmin (NPM) gene. The NPM-ALK fusion protein is a constitutively-active tyrosine kinase, and plays a major role in tumor pathogenesis. In an effort to advance novel diagnostic approaches and the understanding of the function of this fusion protein in cancer cells, we expressed in E. coli, purified and characterized human NPM-ALK fusion protein to be used as a standard for estimating expression levels in cultured human ALCL cells, a key tool in ALCL pathobiology research. We estimated that NPM-ALK fusion protein is expressed at substantial levels in both Karpas 299 and SU-DHL-1 cells (ca. 4-6 million molecules or 0.5-0.7 pg protein per cell; based on our in-house developed NPM-ALK ELISA; LOD of 40 pM) as compared to the ubiquitous ß-actin protein (ca. 64 million molecules or 4.5 pg per lymphocyte). We also compared NPM-ALK/ ß-actin ratios determined by ELISA to those independently determined by two-dimensional electrophoresis and showed that the two methods are in good agreement.