Exquisite Sensitivity of Plasma Cell Malignancies to a Novel Nucleoside Analog Is Mediated By Overexpressed Adenosine Kinase

Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin, driven by infection with KSHV/HHV-8, and with notable plasmacytic differentiation. PEL carries an extremely poor prognosis with a median survival time of less than 6 months and is known to be largely resistant to conventional chemotherapy. Therefore, we conducted high throughput screens for effective inhibitors using PEL cell lines. We discovered a compound, 6-ethylthioinosine (6-ETI), a nucleoside analog with selective toxicity to PEL and multiple myeloma (MM) at nanomolar concentrations, but not to other lymphoma cell lines tested. 6-ETI also showed remarkable responses in a mouse xenograft model of PEL. To understand the molecular mechanism(s) of selectivity towards PEL, we developed and performed resistome analysis, an unbiased approach based on RNA sequencing of in vitro and in vivo generated resistant subclones. We found adenosine kinase (ADK) inactivating genomic alterations in all resistant clones as the mechanism of resistance. Concordantly, we found that plasma cells express ADK, as well as sensitive PELs and multiple myeloma cell lines, while resistant lymphoma cell lines including those with EBV infection had lower levels (if any); interestingly, the latter could be sensitized by cell crowding-induced ADK upregulation. Like other nucleoside analogues, 6-ETI induces ATP-depletion and cell death accompanied by S-phase arrest and DNA damage, but only in ADK-expressing cells. Immunohistochemistry for ADK served as a new biomarker approach to identify tumors that may be sensitive to 6-ETI, which we documented for primary specimens of PEL, multiple myeloma and other lymphoid malignancies with plasmacytic features, namely plasmablastic lymphoma. A number of nucleoside analogues have been reported to be effective in treating leukemias and B and T cell lymphomas. We performed structure-activity relationship studies and tested a number of nucleoside analogs that are in preclinical or clinical development for other hematological malignancies to identify and better understand the structural requirements for 6-ETIÕs biological activity. We successfully demonstrated that 6-ETI was more potent and selective at killing PEL and MM cell lines than other studied nucleoside analogs suggesting that this compound possesses unique and distinct features that are clinically promising. Despite the presence of treatment approaches that can greatly extended the survival of MM patients, resistance remains an issue, warranting the need for new effective agents and combinations. We identified 6-ETI as a novel and clinically viable nucleoside analog for the treatment of PEL, immunoblastic lymphoma, plasmablastic lymphoma, multiple myeloma and other ADK-expressing cancers. Figure 1. Expression of ADK and sensitivity to 6-ETI in plasma cell tumors. (A) BC3 cells ADK expression was evaluated by immunohistochemistry in the BC3 cell line, hyperplastic tonsils and PEL, multiple myeloma and plasmablastic lymphoma primary tumors. 60X original magnification is shown. In the image of a tonsil section, a positive cell with morphological features of a plasma cell is enlarged in the insert. Original magnification 60X. (B) LC50s for multiple myeloma cell lines treated with 6-ETI were determined by CellTiter-Glo assay. BC3 was used as a positive control and IBL1 as a negative control for drug sensitivity. Shown are the average of two independent experiments, where each condition was performed in duplicate in each experiment. (C) Model for 6-ETIÕs mechanism of action within the cell is illustrated, where 6-ETI competes with adenosine (ADO) and other nucleosides for binding to and phosphorylation by ADK, which inhibits ATP-dependent metabolic processes. This also allows 6-ETI to be phosphorylated and activated by ADK, with subsequent phosphorylation steps that allow the compound to be incorporated into DNA and possibly RNA, leading to DNA synthesis inhibition, DNA damage response, and cell death. Figure 1. Expression of ADK and sensitivity to 6-ETI in plasma cell tumors. (A) BC3 cells ADK expression was evaluated by immunohistochemistry in the BC3 cell line, hyperplastic tonsils and PEL, multiple myeloma and plasmablastic lymphoma primary tumors. 60X original magnification is shown. In the image of a tonsil section, a positive cell with morphological features of a plasma cell is enlarged in the insert. Original magnification 60X. (B) LC50s for multiple myeloma cell lines treated with 6-ETI were determined by CellTiter-Glo assay. BC3 was used as a positive control and IBL1 as a negative control for drug sensitivity. Shown are the average of two independent experiments, where each condition was performed in duplicate in each experiment. (C) Model for 6-ETIÕs mechanism of action within the cell is illustrated, where 6-ETI competes with adenosine (ADO) and other nucleosides for binding to and phosphorylation by ADK, which inhibits ATP-dependent metabolic processes. This also allows 6-ETI to be phosphorylated and activated by ADK, with subsequent phosphorylation steps that allow the compound to be incorporated into DNA and possibly RNA, leading to DNA synthesis inhibition, DNA damage response, and cell death. Disclosures Nayar: Weill Cornell Medical College: Patents & Royalties: Submitted patent applicatio for 6-ETI. Cesarman:Weill Cornell Medical College: Patents & Royalties: applied for patent for 6-ETI.

Validation of Whole Slide Imaging for Primary Diagnosis in Surgical Pathology

Context.—High-resolution scanning technology provides an opportunity for pathologists to make diagnoses directly from whole slide images (WSIs), but few studies have attempted to validate the diagnoses so obtained. Objective.—To compare WSI versus microscope slide diagnoses of previously interpreted cases after a 1-year delayed re-review (“wash-out”) period. Design.—An a priori power study estimated that 450 cases might be needed to demonstrate noninferiority, based on a null hypothesis: “The true difference in major discrepancies between WSI and microscope slide review is greater than 4%.” Slides of consecutive cases interpreted by 2 pathologists 1 year prior were retrieved from files, and alternate cases were scanned at original magnification of ×20. Each pathologist reviewed his or her cases using either a microscope or imaging application. Independent pathologists identified and classified discrepancies; an independent statistician calculated major and minor discrepancy rates for both WSI and microscope slide review of the previously interpreted cases. Results.—The 607 cases reviewed reflected the subspecialty interests of the 2 pathologists. Study limitations include the lack of cytopathology, hematopathology, or lymphoid cases; the case mix was not enriched with difficult cases; and both pathologists had interpreted several hundred WSI cases before the study to minimize the learning curve. The major and minor discrepancy rates for WSI were 1.65% and 2.31%, whereas rates for microscope slide reviews were 0.99% and 4.93%. Conclusions.—Based on our assumptions and study design, diagnostic review by WSI was not inferior to microscope slide review (P < .001).

Sample Stability: A Suggested Definition and Medthod of Determination

Volume 22 p 52, colunm 1: In two palces, "γ-GT/ml" should read "γ-GT/CC." In Figures 2 through 4 in this paper, the "X800" in the legend refers to the original magnification. p 179: In Table 2, in the —20 °C experiment, the "test statistic" of samples 5 through 18 should be more negative by 0.5 and that of sample 24 should be —7.0 rather then —5.0. These change do not affect the conclusions. p 197, column 1: In the first full paragraph, reference "(2)" should read "(21)." p 777: In Figure 1, structures B and C should not contain an endocyclic double bond (structure A is correct)-i.e., the double bonds at the tops of the seven membered ring should be single bonds. p 833: Corrections to this Table are detailed in a Letter to the Editor, p 1751, q.v. p 1324: The drawing for Figure 2 has been interchanged with the drawing in the upper half of Figure 9, p 1327. p 1427: Richard S. Schifreen should be listed as an author. p 1745, column 1: The [3H]cortisl belongs to ref. 1, and was not supplied by Diagnostic Products Corp. p 1751, column 3, first paragraph: 9.7 mg/liter should read 0.7 mg/liter.