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Targeted PET imaging strategy to differentiate malignant from inflamed lymph nodes in diffuse large B-cell lymphoma

  1. Thomas Reinera,f,2
  1. aDepartment of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY 10065;
  2. bCancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065;
  3. cDepartment of Chemistry, Hunter College of the City University of New York, New York, NY 10028;
  4. dPhD Program in Chemistry, The Graduate Center of the City University of New York, New York, NY 10018;
  5. eMolecular Pharmacology and Chemistry Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065;
  6. fDepartment of Radiology, Weill Cornell Medical College, New York, NY 10065
  1. Edited by Michael E. Phelps, University of California, Los Angeles, CA, and approved July 18, 2017 (received for review April 7, 2017)

  1. Fig. 2.

    A mouse DLBCL model recapitulates the pathophysiology of human DLBCL. (A) Schematic depiction of the development of the mouse model that mimics human DLBCL. (B) Western blot analysis showing higher PARP1 expression levels in DLBCL cells from mouse spleens compared with B cells from B6 mouse spleens. (C) Representative flow cytometry graphs showing that most immune cells (CD45+) in DLBCL mice express RFP and thus are derived from the transplanted HPCs. (D) Immunohistochemistry showing that the lymph nodes of DLBCL mice (n = 5) are larger than those of B6 mice (n = 5), and that most cells express RFP. (E) Immunohistochemistry results for the adjacent slides showing higher PARP1 expression levels in the lymph nodes of DLBCL mice (n = 5) compared with B6 mice (n = 5). Error bars represent SEM. *P < 0.05, ****P < 0.0001, nonparametric Student’s t test.

  2. Fig. S1.

    Physiological characteristics of the DLBCL mice. (A) Body weights of DLBCL mice and irradiated control B6 mice. (B) Spleen weights. (C) Lymph node weights. (D) Hemoglobin concentrations in blood. (E) White blood cell counts. (F) Blood platelet counts. Five DLBCL mice and five B6 mice were used for these measurements. (G) Representative flow cytometry measurements of the RFP-positive immune cells (CD45+) in bone marrow and blood, demonstrating that the majority cells of were derived from transformed HPCs. (H) Representative H&E staining of superficial cervical lymph nodes showing a loss of germinal center integrity. Five DLBCL mice and five B6 mice were used. Error bars represent SD. **P < 0.01; ****P < 0.0001, two-tailed Student’s t test with assumption of unequal SD.

  3. Fig. 3.

    Evaluation of a PARP1-targeted diagnostic strategy. (A) Physiochemical properties of the PARPi-FL probe. (B) Schema of the experimental spectrofluorimetry procedure for measurement of PARPi-FL concentrations in spleen and lymph nodes of DLBCL and B6 mice. (C) Spectrofluorimetry measurement of PARPi-FL accumulation in DLBCL mice (n = 5) and B6 mice (n = 5). The concentration of PARPi-FL is presented as the percentage of injected dose per gram of tissue (%ID/g). (D) PARPi-FL accumulation in lymph nodes as measured by %ID. (E) PARPi-FL accumulation in spleen as measured by %ID. (F) Preexposure of olaparib blocks the accumulation of PARPi-FL in the lymph nodes and spleen of DLBCL mice. (G) Representative confocal microscopic images of lymph node sections from DLBCL mice with PARPi-FL injection or blocking with olaparib. (H) Flow cytometry gating procedure to identify B cells from the lymph nodes of DLBCL or B6 mice injected with PARPi-FL. (I) Flow cytometry enumeration showing the percentage of B cells out of all CD45+ immune cells in the lymph nodes and spleen. (J) Quantification of average PARPi-FL level in B cells by flow cytometry as measured by the mean fluorescence intensity (MFI). The numbers over the short bars indicate MFI value (×10,000). Error bars represent SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, nonparametric Student’s t test.

  4. Fig. S2.

    Confocal microscopic images of DLBCL and B6 mice. (A) Images of lymph node histological sections from DLBCL mice. The first row shows a PAPR1-stained section from a DLBCL mouse injected with PARPi-FL. The second row shows a PARP1-stained lymph node section from a mouse preblocked with olaparib. The third row shows a nonspecific antibody-stained control for PARP1. The fourth row shows the nonspecific signal of the secondary fluorescent antibodies without primary antibodies for PARP1. (B) Representative image of an immunostaining slide from the spleen of a DLBCL mouse injected with PARPi-FL. (C) Representative images from slides of lymph node and spleen from a B6 mouse injected with PARPi-FL.

  5. Fig. 4.

    Use of a PARP1-targeted radioactive agent to detect DLBCL at single-cell and whole-body levels. (A) Physiochemical properties of the [18F]PARPi probe. (B) Schema of the procedure for measuring average [18F]PARPi accumulation per cell in blood B lymphoma cells. (C) Flow cytometry of the final step of this experiment showing high purity of isolated B cells (>96%) and neutrophils (>98%). (D) Accumulation of [18F]PARPi at the single-cell level, presented as %ID/109 cells. The average accumulation of [18F]PARPi per cell was measured in nonpurified white blood cells, purified B cells, and purified neutrophils. Preinjection of olaparib reduced the accumulation by 99%. The numbers over the short bars indicate the value of the bars. (E) Comparison of [18F]PARPi and PARPi-FL accumulation in B cells and neutrophils from the blood of DLBCL mice (n = 7 for [18F]PARPi; n = 4 for PARPi-FL), demonstrating similar specificity of the two PARP1-targeted probes. (F) Representative [18F]PARPi PET (Left) and PET/CT (Right) hybrid images of DLBCL mice (n = 7), B6 mice (n = 4), and DLBCL mice preexposed to olaparib (n = 4). (G) Quantification of PET signals in lymph nodes. The signal was calculated by averaging the maximal signals of five consecutive axial slices (1 mm thick) that cover superficial cervical lymph nodes. (H) Correlation of radioactivity measurement between in vivo PET imaging and ex vivo γ-counting (n = 7). Pearson correlation was used to calculate statistics and correlation coefficients. (I) Accumulation of [18F]PARPi in lymph nodes in DLBCL mice (n = 9) and B6 mice (n = 4) as measured by ex vivo γ-counting. Error bars represent SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, nonparametric Student’s t test.

  6. Fig. S3.

    PET/CT imaging of B6 mice and key parameters of single-cell radioactivity measurements in DLBCL mouse blood. (A) Average cpm of purified B cells or neutrophils; the background cpm was <30. (B) Flow cytometry procedure for enumerating purified B cells and neutrophils with counting beads and identifying B lymphoma cells and neutrophils. (C) Representative PET/CT images of the lymph nodes of B6 mice injected with [18F]PARPi (n = 4) and preblocked with olaparib (n = 3). (D) Quantification of PET signals in these lymph nodes. Error bars represent SEM. **P < 0.01, two-tailed Student’s t test with assumption of unequal SD.

  7. Fig. S4.

    [18F]PARPi reveals DLBCL metastases in multiple tissues. (A) Biodistribution showing PARP1-specific accumulation of [18F]PARPi in multiple tissues from DLBCL mice injected with [18F]PARPi (n = 12), B6 mice injected with [18F]PARPi (n = 4), and DLBCL mice injected with [18F]PARPi and blocked with olaparib (n = 7). (B) Biodistribution in B6 mice showing PARP1-specific accumulation in lymph nodes, spleen, and salivary glands. B6 mice were injected with [18F]PARPi (n = 4) or preblocked with olaparib (n = 3). (C) Representative autoradiographs showing accumulation of [18F]PARPi in the selected tissues in DLBCL and B6 mice injected with [18F]PARPi and with additional olaparib blocking. (D) Representative histological images of the five tissues from DLBCL (n = 5) and B6 mice (n = 5). Consecutive sections were stained with H&E, PARP1, or RFP. Error bars represent SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, nonparametric Student’s t test.

  8. Fig. 5.

    PARP1-targeted PET imaging differentiates malignant from inflamed lymph nodes. (A) Representative [18F]FDG PET (Left) and [18F]PARPi (Right) images of DLBCL mice (n = 3 for [18F]FDG; n = 5 for [18F]PARPi), B6 mice with inflamed lymph nodes (n = 3 for [18F]FDG; n = 5 for [18F]PARPi), and B6 mice with normal lymph nodes (n = 3 for [18F]FDG; n = 5 for [18F]PARPi). (B) Representative autoradiographic images of five selected tissues from the three groups of mice injected with [18F]FDG PET. (C) Ex vivo γ-counting of lymph node radioactivity from DLBCL mice (n = 5), B6 mice with inflamed lymph nodes (n = 5), and normal B6 (n = 5) injected with [18F]FDG. (D) Quantification of [18F]PARPi PET signal in lymph nodes from DLBCL mice (n = 5), B6 mice with inflamed lymph nodes (n = 5), and normal B6 mice (n = 4). Signals were calculated by averaging the maximal signals of five consecutive axial slices (1 mm thick) that cover superficial cervical lymph nodes. (E) Ex vivo γ-counting of lymph node radioactivity from DLBCL mice (n = 5), B6 mice with inflamed lymph nodes (n = 5), and normal B6 mice (n = 5) injected with [18F]PARPi. (F) Representative PARP1 immunostaining images of lymph nodes from DLBCL mice (n = 10), B6 mice with local inflammation (n = 10), and normal B6 mice (n = 10).

  9. Fig. S5.

    Validation of the inflamed lymph node model. (A) Average weight of superficial cervical lymph nodes from mice with local inflammation (n = 8) and healthy mice (n = 8). (B) Representative H&E staining showing the enlarged lymph nodes with inflammation compared with normal nodes. (C) Biodistribution showing accumulation of [18F]FDG in five selected tissues from DLBCL mice (n = 5), B6 mice with inflamed lymph nodes (n = 5), and normal B6 mice (n = 5). (D) Biodistribution of [18F]PARPi in the same tissues from DLBCL mice (n = 5), B6 mice with inflamed lymph nodes (n = 5), and normal B6 mice (n = 5). Error bars represent SD.

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