Gene therapy enhances chemotherapy tolerance and efficacy in glioblastoma patients

Abstract

Background: Temozolomide (TMZ) is one of the most potent chemotherapy agents for the treatment of glioblastoma. Unfortunately, almost half of glioblastoma tumors are TMZ resistant due to overexpression of methylguanine methyltransferase (MGMT(hi)). Coadministration of O6-benzylguanine (O6BG) can restore TMZ sensitivity, but causes off-target myelosuppression. Here, we conducted a prospective clinical trial to test whether gene therapy to confer O6BG resistance in hematopoietic stem cells (HSCs) improves chemotherapy tolerance and outcome.

Methods: We enrolled 7 newly diagnosed glioblastoma patients with MGMT(hi) tumors. Patients received autologous gene-modified HSCs following single-agent carmustine administration. After hematopoietic recovery, patients underwent O6BG/TMZ chemotherapy in 28-day cycles. Serial blood samples and tumor images were collected throughout the study. Chemotherapy tolerance was determined by the observed myelosuppression and recovery following each cycle. Patient-specific biomathematical modeling of tumor growth was performed. Progression-free survival (PFS) and overall survival (OS) were also evaluated.

Results: Gene therapy permitted a significant increase in the mean number of tolerated O6BG/TMZ cycles (4.4 cycles per patient, P < 0.05) compared with historical controls without gene therapy (n = 7 patients, 1.7 cycles per patient). One patient tolerated an unprecedented 9 cycles and demonstrated long-term PFS without additional therapy. Overall, we observed a median PFS of 9 (range 3.5-57+) months and OS of 20 (range 13-57+) months. Furthermore, biomathematical modeling revealed markedly delayed tumor growth at lower cumulative TMZ doses in study patients compared with patients that received standard TMZ regimens without O6BG.

Conclusion: These data support further development of chemoprotective gene therapy in combination with O6BG and TMZ for the treatment of glioblastoma and potentially other tumors with overexpression of MGMT.

Trial registration: Clinicaltrials.gov NCT00669669.

Funding: R01CA114218, R01AI080326, R01HL098489, P30DK056465, K01DK076973, R01HL074162, R01CA164371, R01NS060752, U54CA143970.

Figures

Figure 5. Biomathematical modeling reveals greater response to treatment as a function of TMZ administered in study case patients versus matched controls.

(A) Box and whisker plots illustrating the significant difference in mean DG per milligram of TMZ received observed between study patients (case) and matched patients receiving standard care regimens (control). (B) Box and whisker plots illustrating the significant difference in mean RTR per milligram of TMZ received observed between study patients and matched patients receiving standard care regimens. For each plot, box bounds represent the first quartile (lower bound) and third quartile (upper bound). Lines within the box represent the median. Whiskers represent the difference from the minimum value observed in the data set to the first quartile (lower whisker) and the difference from the third quartile to the maximum value observed (upper whisker).

Figure 4. Patient-specific biomathematical modeling of tumor growth and treatment response.

(A) UVC for predicted tumor growth if left untreated for case patient 1. Two initial pretreatment MRI scans (blue x’s) were used to determine the predicted tumor growth (solid line) if left untreated. The first MRI was obtained on the day of clinical presentation, and the second MRI was obtained as part of neuronavigational guidance protocol on the day of resection (day 10). Tumor radius (y axis) is determined from follow-up posttreatment MRIs (black x’s) and plotted against time since the first MRI in days (x axis). The treated tumor radius is assessed relative to the projected untreated growth to determine the response measured as DG or RTR. Note: Increase in enhancement observed by MRI at 182 days (D, below) was determined to be consistent with pseudoprogression (PSP) by second surgical resection. (B–G) Observed clinical response in case patient 1. Contrast-enhanced MRI scans obtained, in chronological order, at initial diagnosis (B, second gray x denoted in A), after RT and transplant (before O6BG/TMZ chemotherapy) (C), following cycle II of O6BG/TMZ chemotherapy (interval increase representative of pseudoprogression corresponding to second black x in A) (D), following second surgical resection, which indicated pseudoprogression (for pathology, see Supplemental Figure 3) (E), following cycle 9 of O6BG/TMZ chemotherapy (F), and at 2 years since diagnosis (11 months after therapy discontinuation) (G).

Figure 3. Persistence of P140K gene-modified cells in vivo.

Percentage of gene-modified wbc within circulating peripheral blood of patients 1 to 7 are represented in A–G, respectively. Each graph represents the percentage of circulating bulk wbc (black diamonds), granulocytes (white circles), and lymphocytes (asterisks), with integrated transgene-containing provirus by PCR assay (y axis) over time (x axis). Chemotherapy (inverted triangles) is shown as well.

Figure 2. Chemoresistance of wbc in vivo after infusion of P140K gene-modified CD34+ cells and O6BG/TMZ chemotherapy.

(A) Schematic of the P140K-encoding gammaretrovirus vector used for transduction of autologous human CD34+ cells. (B–H) Blood cell counts for chemotherapy treatment per protocol (ANC and PLTs) for case patients 1 to 7 (B–H, respectively). Each graph shows ANC (white circles) and PLT counts (black squares) as measured from peripheral blood (y axis) over time (x axis) and chemotherapy with O6BG/TMZ (inverted triangles). Black inverted triangles in B (case patient 1) and H (case patient 7) represent dose-escalated chemotherapy cycles with TMZ dose of 590 mg/m2. Upper and lower dashed lines represent the threshold counts for each hematopoietic cell type required for chemotherapy retreatment in the study.

Figure 1. Flow diagram of study protocol.