Convection Enhanced Delivery of the Oncolytic Adenovirus Delta24-RGD in Patients with Recurrent GBM: A Phase I Clinical Trial Including Correlative Studies

Abstract

Purpose: Testing safety of Delta24-RGD (DNX-2401), an oncolytic adenovirus, locally delivered by convection enhanced delivery (CED) in tumor and surrounding brain of patients with recurrent glioblastoma.

Patients and methods: Dose-escalation phase I study with 3+3 cohorts, dosing 107 to 1 × 1011 viral particles (vp) in 20 patients. Besides clinical parameters, adverse events, and radiologic findings, blood, cerebrospinal fluid (CSF), brain interstitial fluid, and excreta were sampled over time and analyzed for presence of immune response, viral replication, distribution, and shedding.

Results: Of 20 enrolled patients, 19 received the oncolytic adenovirus Delta24-RGD, which was found to be safe and feasible. Four patients demonstrated tumor response on MRI, one with complete regression and still alive after 8 years. Most serious adverse events were attributed to increased intracranial pressure caused by either an inflammatory reaction responding to steroid treatment or viral meningitis being transient and self-limiting. Often viral DNA concentrations in CSF increased over time, peaking after 2 to 4 weeks and remaining up to 3 months. Concomitantly Th1- and Th2-associated cytokine levels and numbers of CD3+ T and natural killer cells increased. Posttreatment tumor specimens revealed increased numbers of macrophages and CD4+ and CD8+ T cells. No evidence of viral shedding in excreta was observed.

Conclusions: CED of Delta24-RGD not only in the tumor but also in surrounding brain is safe, induces a local inflammatory reaction, and shows promising clinical responses.

Figure 1.

Illustrative case patient 7, a 67-year-old male with a second recurrence of a GBM, diagnosed 13 months earlier. Virus infusion of 1 × 10⁹ vp was uncomplicated, but patient was readmitted 3 months later due to neurological deterioration. MR showed a decrease of the contrast-enhancing part of the tumor but an increase of the signal intensity in the peritumoral area on the T2- and FLAIR-images, interpreted as increased edema and stable tumor (left side: T1 after gadolinium; right side: T2 images). During hospitalization, patient deteriorated rapidly, dying with clinical signs of sepsis. At autopsy a diverticulitis with sigmoid perforation was found. Brain autopsy revealed a relatively small tumor in relation to the MR images, containing immune cell infiltrates (Fig. 5A), necrotic areas, infarctions, and absence of vascular proliferation.

Figure 2.

Illustrative case patient 11, a 48-year-old female, who was treated 21 months after GBM diagnosis for a second recurrence at dose level 4 with 1 × 10¹⁰ vp. After 2 weeks, MRI showed a large area with cavity formation suggestive of tumor lysis in the target area of one of the intratumoral catheters used for virus administration [A, T1 after gadolinium prior to treatment and 2 weeks after treatment; B, postoperative neuronavigation screenshot demonstrating the relation of the CED catheter (trajectory in blue) and the necrotic area]. Six weeks after virus infusion her preexisting paresis of the left leg deteriorated due to increasing mass effect of the treated tumor for which she underwent subtotal re-resection. After initial improvement, she deteriorated again 6 weeks later, at this point she declined further diagnostic tests and treatment. She died 106 days after virus infusion. PCR analysis of CSF samples demonstrated increasing virus titers 3 months post-infusion (Fig. 5). The resected tumor material contained besides tumor cells also viral proteins and immune cell infiltrates (Fig. 4B). Our routine tumor cell culture protocol of this tumor failed due to virus-induced lysis of tumor cells. Unfortunately, we were unable to discriminate between true tumorprogression or an inflammatory reaction known as pseudoprogression.

Figure 3.

Illustrative case patient 15, a 40-year-old female who received 3 × 10¹⁰ vp for her second GBM recurrence, 13 months after initial diagnosis of an IDH mutant GBM. T1 after gadolinium images posttreatment demonstrated tumor regression of the virus-infused tumor site (top) after initial pseudoprogression, followed by a slow prolonged regression of the distant tumor sites (middle and bottom). One year after virus infusion patient neurologically deteriorated despite high-dose steroid treatment. Ultimately, she was bedridden with palliative care and all medication, including steroids, was withdrawn. After this she made a remarkable recovery, regaining a KPS of 70. Patient is still alive and in stable clinical condition 8 years after Delta24-RGD treatment without having had any other treatments.

Figure 4.

Prolonged presence of Delta24-RGD DNA in cerebrospinal fluid of treated patients. Samples collected at indicated time points from permanent ventricular catheter were analyzed by a Delta24-RGD specific PCR for the presence of viral copy numbers (expressed as viral particles per mL CSF).

Figure 5.

IHC analysis of post-mortem tumor material of patient 7 (A, 3 months after virus administration, left) and re-resection material of patient 11 (B, 4 weeks after virus administration, right) for the adenoviral protein hexon (top images), showing scattered hexon-positive cells and for the presence of immune cells (bottom images); CD68⁺ cells (macrophage marker), CD16⁺ (monocyte marker), and CD4⁺ lymphocytes are quite abundant in both samples. CD8⁺ lymphocytes are less frequent in both samples.

Figure 6.

CSF levels of the cytokines IL6, IFNγ, TNFα, IL10, and IL13 (A) and the chemokines Eotaxin, IP-10, and MIP-1β (B) at indicated time points prior to, during, and after Delta24-RGD infusion. Concentrations are expressed in pg/mL CSF.

Figure 7.

Immune cells were isolated from cerebrospinal fluid at indicated timepoints after Delta24-RGD infusion and analyzed by flow cytometry with the following markers: CD45⁺CD3⁻CD56⁺ (NK cells), DCs (CD45⁺/lineage⁻/HLA-DR⁺), CD45⁺CD3⁺/CD4⁺ (CD4⁺ T-lymphocytes), and CD45⁺CD3⁺/CD8⁺ (CD8⁺ T-lymphocytes). Immune cell subsets are presented as absolute numbers per μL CSF. Gray-shaded areas indicate the normal range for each cell type.

Figure 8.

Serum-specific anti-adenovirus IgG antibodies were determined in all patients prior to (white bars) and 4 weeks after (black bars) Delta24-RGD treatment (A). Levels are expressed in units per mL serum. Anti-adenovirus IgG antibodies found in the CSF of patients prior to (white bars) and 4 weeks after (black bars) Delta24-RGD treatment are shown in B. Stars indicate usages of a 2-week sample instead of 4 weeks post-infusion.

Figure 9.

The degree of inflammatory response is correlated with survival. In a subset of patients, termed IFNγ-high, CSF IFNγ levels rose above 50 pg/mL at 2 to 4 weeks after treatment (A). In these patients, almost all measured cytokines and chemokines are significantly correlated and increased. Significant correlated cytokines/chemokines are connected with a straight line (P < 0.05) or with a dotted line (P < 0.001; B). The levels of IFNγ are not significantly correlated with levels of Delta24-RGD (C). The IFNγ-high group also revealed higher CD8⁺ T cells numbers in CSF compared with IFNγ-low group (D). Kaplan–Meier survival curve of treated patients separated into IFNγ-high and IFNγ-low groups. Both long-term surviving patients (nos. 15 and 16) belong to the IFNγ-high group (E, black line).