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AMG 900, a potent inhibitor of aurora kinases causes pharmacodynamic changes in p-Histone H3 immunoreactivity in human tumor xenografts and proliferating mouse tissues.

Juan G, Bush TL, Ma C, Manoukian R, Chung G, Hawkins JM, Zoog S, Kendall R, Radinsky R, Loberg R, Friberg G, Payton M - J Transl Med (2014)

Bottom Line: Aurora-A and -B expression and kinase activity is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis.Lastly, we illustrate this LSC-based approach can detect p-Histone H3 positive cells using mock FNAs from primary human breast tumor tissues.FNA biopsies may be a viable approach for assessing AMG 900 PD effects in the clinic.

View Article: PubMed Central - PubMed

Affiliation: Departments of Oncology Biomarkers and Early Development, Thousand Oaks 91320, CA, USA. gjuan@amgen.com.

ABSTRACT

Background: The Aurora family of serine-threonine kinases are essential regulators of cell division in mammalian cells. Aurora-A and -B expression and kinase activity is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis. AMG 900 is a highly potent and selective pan-aurora kinase inhibitor that has entered clinical evaluation in adult patients with advanced cancers. In mice, oral administration of AMG 900 blocks the phosphorylation of histone H3 on serine-10 (p-Histone H3), a proximal substrate of aurora-B and inhibits the growth of multiple human tumor xenografts, including multidrug-resistant models.

Methods: In order to establish a preclinical pharmacokinetic-pharmacodynamic (PK-PD) relationship for AMG 900 that could be translated to the clinic, we used flow cytometry and laser scanning cytometry detection platforms to assess the effects on p-Histone H3 inhibition in terms of sensitivity, precision, and specificity, in human tumor xenografts in conjunction with mouse skin and bone marrow tissues. Mice with established COLO 205 tumors were administered AMG 900 at 3.75, 7.5, and 15 mg/kg and assessed after 3 hours.

Results: Significant suppression of p-Histone H3 in mouse skin was only observed at 15 mg/kg (p <0.0001), whereas in mouse bone marrow and in tumor a dose-dependent inhibition was achieved at all three doses (p ≤ 0.00015). These studies demonstrate that AMG 900 inhibits p-Histone H3 in tumors and surrogate tissues (although tissues such as skin may be less sensitive for assessing PD effects). To further extend our work, we evaluated the feasibility of measuring p-Histone H3 using fine-needle aspirate (FNA) tumor xenograft biopsies. Treatment with AMG 900 significantly inhibited p-Histone H3 (>99% inhibition, p <0.0001) in COLO 205 tumors. Lastly, we illustrate this LSC-based approach can detect p-Histone H3 positive cells using mock FNAs from primary human breast tumor tissues.

Conclusion: Phosphorylation of histone H3 is a useful biomarker to determine the pharmacodynamics (PD) activity of AMG 900. FNA biopsies may be a viable approach for assessing AMG 900 PD effects in the clinic.

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Detection of p-Histone H3 using primary human breast tumor FNA biopsies by LSC. Two mock FNA punch biopsies were collected from the same fresh primary breast tumor tissue. Cytospin deposited cells were immunostained with anti-EpCAM antibody (epithelial cell specific marker) and anti-p-Histone H3 antibodies and counterstained with DAPI. Representative cell cycle profiles of two FNAs showing EpCAM positive (blue) tumor cells with a subpopulation of p-Histone H3 positive G2M cells (red). Relocation gallery showing p-Histone H3 positive objects.
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Fig5: Detection of p-Histone H3 using primary human breast tumor FNA biopsies by LSC. Two mock FNA punch biopsies were collected from the same fresh primary breast tumor tissue. Cytospin deposited cells were immunostained with anti-EpCAM antibody (epithelial cell specific marker) and anti-p-Histone H3 antibodies and counterstained with DAPI. Representative cell cycle profiles of two FNAs showing EpCAM positive (blue) tumor cells with a subpopulation of p-Histone H3 positive G2M cells (red). Relocation gallery showing p-Histone H3 positive objects.

Mentions: As a proof-of-concept experiment designed to support the use of FNA sampling, mice with established COLO 205 tumors (n = 6 per group) were administered a single oral dose of vehicle alone or AMG 900 at 15 mg/kg. Tumor FNAs (n = 3 per tumor) were collected three hours after treatment and immediately fixed and processed for immunofluorescence staining with an anti-p-Histone H3 antibody. Mice treated with AMG 900 showed near-complete inhibition of p-Histone H3 in COLO 205 tumor FNAs compared with vehicle-treated control (Figure 4B and C). Next, we obtained fresh primary breast tumor tissue to evaluate the ability to detect p-Histone H3 positive cells using the same FNA sampling approach. To restrict our analysis to tumor cells, the FNAs were co-stained with an anti-EpCAM antibody (epithelial cell specific marker). As shown in Figure 5 (and Additional file 3: Figure S3B), the EpCAM positive p-Histone H3 G2M tumor cell population was readily detectable in two mock FNA samples taken from the same primary breast tumor. The authenticity of these rare (<1%) p-Histone H3 objects was confirmed using the relocation feature on the LSC (Figure 5, lower panel). The frequency of p-Histone H3 positive cells in this primary breast tumor was markedly lower compared to COLO 205 tumor xenografts. Taken together, these data indicate that testing FNAs for p-Histone H3 immunoreactivity in tumor cells is a viable approach to survey the PD activity of AMG 900 in target tissues.Figure 5


AMG 900, a potent inhibitor of aurora kinases causes pharmacodynamic changes in p-Histone H3 immunoreactivity in human tumor xenografts and proliferating mouse tissues.

Juan G, Bush TL, Ma C, Manoukian R, Chung G, Hawkins JM, Zoog S, Kendall R, Radinsky R, Loberg R, Friberg G, Payton M - J Transl Med (2014)

Detection of p-Histone H3 using primary human breast tumor FNA biopsies by LSC. Two mock FNA punch biopsies were collected from the same fresh primary breast tumor tissue. Cytospin deposited cells were immunostained with anti-EpCAM antibody (epithelial cell specific marker) and anti-p-Histone H3 antibodies and counterstained with DAPI. Representative cell cycle profiles of two FNAs showing EpCAM positive (blue) tumor cells with a subpopulation of p-Histone H3 positive G2M cells (red). Relocation gallery showing p-Histone H3 positive objects.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4221688&req=5

Fig5: Detection of p-Histone H3 using primary human breast tumor FNA biopsies by LSC. Two mock FNA punch biopsies were collected from the same fresh primary breast tumor tissue. Cytospin deposited cells were immunostained with anti-EpCAM antibody (epithelial cell specific marker) and anti-p-Histone H3 antibodies and counterstained with DAPI. Representative cell cycle profiles of two FNAs showing EpCAM positive (blue) tumor cells with a subpopulation of p-Histone H3 positive G2M cells (red). Relocation gallery showing p-Histone H3 positive objects.
Mentions: As a proof-of-concept experiment designed to support the use of FNA sampling, mice with established COLO 205 tumors (n = 6 per group) were administered a single oral dose of vehicle alone or AMG 900 at 15 mg/kg. Tumor FNAs (n = 3 per tumor) were collected three hours after treatment and immediately fixed and processed for immunofluorescence staining with an anti-p-Histone H3 antibody. Mice treated with AMG 900 showed near-complete inhibition of p-Histone H3 in COLO 205 tumor FNAs compared with vehicle-treated control (Figure 4B and C). Next, we obtained fresh primary breast tumor tissue to evaluate the ability to detect p-Histone H3 positive cells using the same FNA sampling approach. To restrict our analysis to tumor cells, the FNAs were co-stained with an anti-EpCAM antibody (epithelial cell specific marker). As shown in Figure 5 (and Additional file 3: Figure S3B), the EpCAM positive p-Histone H3 G2M tumor cell population was readily detectable in two mock FNA samples taken from the same primary breast tumor. The authenticity of these rare (<1%) p-Histone H3 objects was confirmed using the relocation feature on the LSC (Figure 5, lower panel). The frequency of p-Histone H3 positive cells in this primary breast tumor was markedly lower compared to COLO 205 tumor xenografts. Taken together, these data indicate that testing FNAs for p-Histone H3 immunoreactivity in tumor cells is a viable approach to survey the PD activity of AMG 900 in target tissues.Figure 5

Bottom Line: Aurora-A and -B expression and kinase activity is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis.Lastly, we illustrate this LSC-based approach can detect p-Histone H3 positive cells using mock FNAs from primary human breast tumor tissues.FNA biopsies may be a viable approach for assessing AMG 900 PD effects in the clinic.

View Article: PubMed Central - PubMed

Affiliation: Departments of Oncology Biomarkers and Early Development, Thousand Oaks 91320, CA, USA. gjuan@amgen.com.

ABSTRACT

Background: The Aurora family of serine-threonine kinases are essential regulators of cell division in mammalian cells. Aurora-A and -B expression and kinase activity is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis. AMG 900 is a highly potent and selective pan-aurora kinase inhibitor that has entered clinical evaluation in adult patients with advanced cancers. In mice, oral administration of AMG 900 blocks the phosphorylation of histone H3 on serine-10 (p-Histone H3), a proximal substrate of aurora-B and inhibits the growth of multiple human tumor xenografts, including multidrug-resistant models.

Methods: In order to establish a preclinical pharmacokinetic-pharmacodynamic (PK-PD) relationship for AMG 900 that could be translated to the clinic, we used flow cytometry and laser scanning cytometry detection platforms to assess the effects on p-Histone H3 inhibition in terms of sensitivity, precision, and specificity, in human tumor xenografts in conjunction with mouse skin and bone marrow tissues. Mice with established COLO 205 tumors were administered AMG 900 at 3.75, 7.5, and 15 mg/kg and assessed after 3 hours.

Results: Significant suppression of p-Histone H3 in mouse skin was only observed at 15 mg/kg (p <0.0001), whereas in mouse bone marrow and in tumor a dose-dependent inhibition was achieved at all three doses (p ≤ 0.00015). These studies demonstrate that AMG 900 inhibits p-Histone H3 in tumors and surrogate tissues (although tissues such as skin may be less sensitive for assessing PD effects). To further extend our work, we evaluated the feasibility of measuring p-Histone H3 using fine-needle aspirate (FNA) tumor xenograft biopsies. Treatment with AMG 900 significantly inhibited p-Histone H3 (>99% inhibition, p <0.0001) in COLO 205 tumors. Lastly, we illustrate this LSC-based approach can detect p-Histone H3 positive cells using mock FNAs from primary human breast tumor tissues.

Conclusion: Phosphorylation of histone H3 is a useful biomarker to determine the pharmacodynamics (PD) activity of AMG 900. FNA biopsies may be a viable approach for assessing AMG 900 PD effects in the clinic.

Show MeSH
Related in: MedlinePlus