Glioblastoma: bridging the gap with gene therapy.

Adult glioblastoma is the most common primary brain tumour. It is characterised by substantial morbidity and mortality despite multimodal therapy with surgical resection and adjuvant radiochemotherapy as standard care.1 The poor prognosis is largely due to the disease’s high frequency of recurrence, which is indicative of its intrinsic invasive properties into the peritumoral zone.2 Consequently, an unmet need exists to improve local control of glioblastoma beyond the margin of resection and to explore new treatment options targeted peritumouraly. Local therapies that can be applied during surgery are therefore well-suited to bridge the gap between initial surgical resection and subsequent radiochemotherapy. In The Lancet Oncology, Manfred Westphal and colleagues3 explore the use of so-called suicide gene therapy to address this treatment gap, describing the results of a randomised, open-label phase 3 trial (ASPECT) for the treatment of operable high-grade glioblastoma. This trial was based on previous phase 1 and phase 2 trials4—6 and relies on local injection into the resection cavity of a replication-deficient adenoviral vector encoding a herpes simplex virus thymidine kinase (HSV-tk) gene to selectively eliminate any residual glioblastoma cells. The HSV-TK catalyses the conversion of a non-toxic ganciclovir prodrug into a toxic nucleotide analogue that is incorporated into the DNA of dividing cancer cells, prompting apoptosis. This approach overcomes the typical inaccessibility of glioblastoma tumour, and brain-infiltrating, cells to most systemic therapies. Moreover, preclinical studies indicate that both the HSV-tk gene-modified cells and adjacent, non-modified dividing cells are eliminated through a so-called bystander effect that enhances the overall anti-tumour effect. This bystander effect is probably mediated by intercellular trafficking of the toxic ganciclovir metabolites through gap junctions or immune mechanisms.78 Another advantage is that normal neurons do not proliferate and are therefore resistant to the ganciclovir metabolites, which improves the tumour selectivity of this treatment strategy.

The specific objective of the ASPECT trial was to determine whether ganciclovir with adenoviral HSV-tk gene therapy was better than standard care, with time to death or re-intervention as the composite primary endpoint. After the ASPECT trial had begun, temozolomide emerged as a new and effective treatment for glioblastoma and was included in both the treatment and control groups. Consequently, this invalidated the initial statistical analysis strategy. A post-hoc multivariate statistical analysis based on a Cox’s proportional hazards model was therefore needed for the composite primary endpoint, which took into consideration the use of temozolomide as a time-dependent covariate. The methylation status of the MGMTpromoter is a prognostic factor and was therefore also taken into account as a covariate in the Cox analysis. MGMT encodes a DNA-repair enzyme that removes alkyl groups from DNA. High levels of MGMT activity in glioblastoma annihilate the therapeutic effect of alkylating agents, including temozolomide, creating a temozolomide-resistant phenotype. Conversely, epigenetic silencing of the MGMT gene by promoter methylation in glioblastoma cells is associated with loss of MGMTexpression, diminished DNA-repair activity, and increased sensitivity to temozolomide. Consequently, patients with glioblastoma containing a methylated MGMT promoter benefit from temozolomide, whereas the drug has no therapeutic effect in those with an unmethylated MGMT promoter.9

In the multivariate statistical analysis of the ASPECT trial, patients in the experimental group had a favourable outcome in terms of the primary composite endpoint—time to death or re-intervention—compared with those in the standard care group (hazard ratio 1·53, 95% CI 1·13—2·07; p=0·006). One of the intriguing findings of this trial is that a post-hoc subgroup analysis showed an even more pronounced effect in a subgroup of patients with an unmethylated MGMT promoter (hazard ratio 1·72, 1·15—2·56; p=0·008). Hemiparesis, hyponatraemia, and seizures were more common in the experimental group and were mainly transient. Despite the statistically significant effect on the composite primary endpoint, the difference in overall survival between gene therapy and standard care was not statistically significant. Nevertheless, there seemed to be improved overall survival in the experimental group versus the control group in a subgroup of patients with non-methylatedMGMT, although this difference was not statistically significant. This finding needs substantiation in larger trials. The investigators recorded no between-group difference in tumour sizes at the time of re-intervention, suggesting that the time to re-intervene was not biased in favour of the treatment group.

Findings from the ASPECT trial raise several interesting hypotheses and questions. Most importantly, the post-hoc multivariate analysis suggests that patients with a glioblastoma containing an unmethylated MGMT promoter might benefit most from the proposed gene therapy strategy. This difference in regards to methylation status might ultimately create new perspectives for the treatment of patients who do not benefit from temozolomide treatment. Methylation status of the MGMTpromoter was not prespecified at the time of treatment, ruling out a possible treatment bias. Post-hoc analysis also showed that the effect of the treatment seemed to be greater in patients with a higher baseline titre of adenovirus-specific neutralising antibodies, suggesting a possible immunological bystander effect resulting from previous infection with wild-type adenovirus.8 Further studies are needed to address these different hypotheses. Findings from the ASPECT trial also indicate a need to develop new approaches that augment transduction efficiency, improve vector spread within the residual tumour tissue, and enhance bystander effects. The continuous development of these multipronged strategies represent the new for patients and their families.

Source: Lancet


Immune cells that suppress genital herpes infections identified.

Discovery has implications for development of a vaccine to prevent and treat HSV-2 and similar infections

Fred Hutchinson Cancer Research Center and University of Washington scientists have identified a class of immune cells that reside long-term in the genital skin and mucosa and are believed to be responsible for suppressing recurring outbreaks of genital herpes. These immune cells also play a role in suppressing symptoms of genital herpes, which is why most sufferers of the disease are asymptomatic when viral reactivations occur.

The discovery of this subtype of immune cells, called CD8αα+ T cells, opens a new avenue of research to develop a vaccine to prevent and treat herpes simplex virus type 2, or HSV-2. Identifying these T cells’ specific molecular targets, called epitopes, is the next step in developing a vaccine.

The findings are described in the May 8 advance online edition of Nature.

Better understanding of these newly identified CD8αα+ T cells may also play a critical role in developing effective vaccines against other types of skin and mucosal infections, according to senior author Larry Corey, M.D., an internationally renowned virologist and president and director of Fred Hutch.

“The discovery of this special class of cells that sit right at the nerve endings where HSV-2 is released into skin is changing how we think about HSV-2 and possible vaccines,” said Corey. “For the first time, we know the type of immune cells that the body uses to prevent outbreaks. We also know these cells are quite effective in containing most reactivations of HSV-2. If we can boost the effectiveness of these immune cells we are likely to be able to contain this infection at the point of attack and stop the virus from spreading in the first place. We’re excited about our discoveries because these cells might also prevent other types of viral infections, including HIV infection.”

There is currently no effective vaccine for genital herpes. “While antiviral treatment is available, the virus often breaks through this barrier and patients still can transmit the infection to others,” Corey said.  “In addition, newborn herpes is one of the leading infections transmitted from mothers to children at the time of delivery. An effective genital herpes vaccine is needed to eliminate this complication of HSV-2 infection.”

The long-term persistence of CD8αα+ T cells where initial infection occurs may explain why patients have asymptomatic recurrences of genital herpes because these cells constantly recognize and eliminate the virus, according to Jia Zhu, Ph.D., corresponding author, research assistant professor in Laboratory Medicine at the University of Washington and an affiliate investigator in the Fred Hutch Vaccine and Infectious Disease Division.

“The cells we found perform immune surveillance and contain the virus in the key battlefield where infection occurs, which is the dermal-epidermal junction,” said Zhu. “These cells are persistent in the skin and represent a newly discovered phenotype distinguished from those of CD8+ T cells circulating in the blood.”

The dermal-epidermal junction (DEJ) is where the dermis (the tissue layers just beneath the skin) connects to the epidermis (outer skin layer). This junction is important because of the roles it plays in cellular communication, nutrient exchange and absorption, and other skin functions.

Scientists examined the DEJ for T cell activity because this is where the genital herpes virus multiplies after reactivating and traveling from its hiding place in the body’s sensory neurons. Previous research by the same research group showed that the nerve endings reach the dermal-epidermal junction and release the virus that infects the skin and can cause lesions.

Prior to this research, CD8αα+ T cells were known to exist in the gut mucosa. Much of the research on CD8+ T cells has focused on studying them in the circulating blood, which has a dominant phenotype of CD8αβ+. Fred Hutch and UW scientists compared the two types of CD8+ T cells and found that only the CD8αα+ T cells persist in the skin while CD8αβ+ T cells diminished from the tissue after healing of a herpes lesion.

“We did not expect to find CD8αα+ T cells in the skin,” Zhu said. “This was a surprise.”

The research involved using novel technologies to examine the T cells in human tissues. In all, the work provides a roadmap that can be applied to other human diseases, according to Zhu.

Zhu said the studies the research group performed in humans are unique. “To our knowledge, we are the only research group to use sequential human biopsies to study CD8+ T cell function in situ, in their natural spatial distribution and at their original physiological state,” she said.

According to the federal Centers for Disease Control and Prevention, 776,000 people in the United States are newly infected with herpes annually. Nationwide, 16.2 percent, or about one out of six people aged 14 to 49 years have genital HSV-2 infection.  Generally, a person can only get HSV-2 infection during sexual contact with someone who has a genital HSV-2 infection. Transmission can occur from an infected partner who does not have a visible sore and may not know that he or she is infected.

Most individuals infected with HSV-2 or the related HSV-1, which causes genital herpes and cold sores, experience either no symptoms or have very mild symptoms that go unnoticed or are mistaken for another skin condition. Because of this, most people infected with HSV-2 are not aware of their infection.

Grants from the National Institutes of Health and the James B. Pendleton Charitable Trust funded the study. Researchers from the University of Washington and the Benaroya Research Institute also contributed to the study.

Source: Fred Hutchinson Cancer Research Center



Dual-therapeutic reporter genes fusion for enhanced cancer gene therapy and imaging.

Two of the successful gene-directed enzyme prodrug therapies include herpes simplex virus–thymidine kinase (HSV1TK) enzyme-ganciclovir prodrug and the Escherichia coli nitroreductase (NTR) enzyme-CB1954 prodrug strategies; these enzyme-prodrug combinations produce activated cytotoxic metabolites of the prodrugs capable of tumor cell death by inhibiting DNA synthesis and killing quiescent cells, respectively. Both these strategies also affect significant bystander cell killing of neighboring tumor cells that do not express these enzymes. We have developed a dual-combination gene strategy, where we identified HSV1-TK and NTR fused in a particular orientation can effectively kill tumor cells when the tumor cells are treated with a fusion HSV1-TK–NTR gene– along with a prodrug combination of GCV and CB1954. In order to determine whether the dual-system demonstrate superior therapeutic efficacy than either HSV1-TK or NTR systems alone, we conducted both in vitro and in vivo tumor xenograft studies using triple negative SUM159 breast cancer cells, by evaluating the efficacy of cell death by apoptosis and necrosis upon treatment with the dual HSV1-TK genes-GCV-CB1954 prodrugs system, and compared the efficiency to HSV1-TK–GCV and NTR-CB1954. Our cell-based studies, tumor regression studies in xenograft mice, histological analyses of treated tumors and bystander studies indicate that the dual HSV1-TK–NTR–prodrug system is two times more efficient even with half the doses of both prodrugs than the respective single gene-prodrug system, as evidenced by enhanced apoptosis and necrosis of tumor cells in vitro in culture and xenograft of tumor tissues in animals.