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Gliomas which are a group of tumors that may either be benign or malignant, arise in the central nervous system (CNS), from cells of glial origin . Astrocytomas and glioblastomas (GBM) which belong to one of the five major groups of glioma tumors listed in Table 1; namely the diffusely infiltrating astrocytomas, account for more than 50% of all CNS disorders and tumors of glial origin , and when malignant, are often uniformly fatal.
My dissertation research focuses on the diffusely infiltrating astrocytomas, and how the aberrant signaling of the PI3K/Akt/mTOR signal transduction pathway may be attenuated by the pre-clinical evaluation of potential therapeutics in an effort to reduce the morbidity and mortality associated with astrocytomas and glioblastomas (GBMs). Secondarily, part of the research addresses the down-regulation of expression of the Pannexin 2 channel protein in astrocytomas and GBM and the role Panx2 may play in gliomagenesis, especially GBM.
The morbidity, prognosis and ultimate mortality of a patient diagnosed with an astrocytoma or GBM is determined by the grade of the tumor. This tumor grading is based on the proliferative potential and malignant features observed within the tumor, as classified by the World Health Organization (WHO) .
ASTROCYTOMAS AND GBMS IN GLIOMA TUMOR GRADING
The World Health Organization (WHO) has classified glial tumors into four grades: I, II, III, and IV. These grades are based on three criteria to include the malignancy of the tumor, which can range in scale from low to high; the histology of the tumor; and whether the tumor is circumscribed and/or has a well-defined border . Tumor grading is important in clinical and pathological diagnosis. One reason why tumor grading is important in clinical and pathological diagnosis is that the grade of the tumor oftentimes determines the degree of morbidity and mortality for the patient involved. The grade translates into how rapidly the tumor grows and proliferates, which ultimately determines the standard treatment options to consider, and the aggressiveness to which the treatments should be administered . For example, Grade I tumors (Pilocytic astrocytomas) which are rare in adults and prevalent in children with NF1 disease, are routinely treated by surgical resection. These Pilocytic astrocytomas are often characterized as having well-defined borders .
Meanwhile, Grade II low-grade tumors often show diffuse infiltration into nearby tissues; Grade III anaplastic tumors tend to be more fatal with increased proliferation and anaplasia; and finally Grade IV tumors demonstrate vascular proliferation; necrosis and are often resistant to chemotherapy and radiation therapy . This increase in malignancy from low to high as characterized by diffuse infiltration and loss of well-defined borders, presents a challenge for the complete surgical resection of higher graded tumors, which contributes to tumor recurrence and the demise of the patient .
Therefore, tumor grading is important in glioma and astrocytoma and GBM biology as it helps to determine the rapidity of tumor growth which is often used as a prognostic tool to estimate the morbidity and mortality of the patient involved.
The preceding section focuses on the three sub-groups of the diffusely infiltrating astrocytomas; their tumor grading; and their prognostic value in determining the morbidity and mortality of the patients diagnosed.
ASTROCYTOMAS AND GBMS ARE GRADES II, III, IV OF THE DIFFUSELY INFILTRATING ASTROCYTOMAS
As a group the diffusely infiltrating astrocytomas which includes the low grade astrocytomas (LGG); the anaplastic astrocytoma's (AA); and the glioblastoma multiforme (GBM) are the most common primary brain tumors of the central nervous system (CNS). The incidence or number of new cases of gliomas represent 1.5-3% of all new cancer cases within the USA annually; which is approximately 15-18 patients per 100,000 . Meanwhile, the prevalence or number of existing cases or patients with gliomas within the USA annually, is estimated at approximately 69 patients per 100,000 . Of these, the majority of both new and existing cases of gliomas are predominantly anaplastic astrocytomas or glioblastoma multiforme (GBM) in origin , with GBM accounting for 50%- 80% of all malignant astrocytomas alone .
The first main category of diffusely infiltrating astrocytomas is the Low-grade gliomas (LGG), WHO Grade II. These are characterized as slow-growing neoplasms of the CNS and represent 35% of all astrocytic tumors, thereby contributing to the overall CNS morbidity and mortality. The LGG low incidence rate of approximately 1,800 new cases per year, coupled with the overall survival rate of approximately six to eight years from diagnosis to death , makes this particular group of tumors more amenable to successful treatment options such as surgery, radiation therapy and chemotherapy. That is, patients diagnosed with lower-graded tumors tend to have tumors with well-defined borders; they are often more amenable to complete surgical resection, which together bodes well for a better prognosis and overall survival. In contrast, patients with higher- graded tumors as we shall discuss in the next paragraph, do not fare as well in terms of complete surgical resection and overall prognosis.
The second main category of diffusely infiltrating astrocytomas is the Anaplastic astrocytoma (AAs), WHO Grade III. These are malignant neoplasms that often progress to malignant GBMs within approximately two years of diagnosis. If the AAs remain as diagnosed and do not progress to GBMs, the median overall survival from diagnosis to death is approximately five years .
The third and final category of diffusely infiltrating astrocytomas is the glioblastoma multiforme or GBMs; the moniker "multiforme" referring to the high intratumoral and intertumoral heterogeneity observed among GBM tumors and patients , a concept which will be elaborated upon in subsequent sections. Therefore, the three main subsets of the diffusely infiltrating astrocytomas are the low-grade II; anaplastic astrocytoma grade III and the GBM, grade IV, with the latter emerging as the most difficult and deadly to treat and/or cure.
In the preceding sections, I will focus on addressing specific questions such as: what are GBMs; why are they important to study; what questions are important in GBM biology; what has already been done to address the importance of GBM in biology; what model systems are being used to study GBM and its advantages and disadvantages over other GBM models; a description of the GBM model used in this study to address the problem posed; the types and subtypes of GBMs and how the GBM model complements or refutes what is known about GBM patients; and finally, what is the best possible outcome from this study and the potential contribution to the field of neuro-oncology.
The GBMs grade IV, are the most common, most malignant, most highly aggressive primary brain tumor in adults, and represents the most fatal of all brain tumors, accounting for approximately 4% of all cancer deaths within the USA annually .
THE IMPORTANTANCE OF GBMS IN TUMORGENESIS
GBMs are important to study because they have a poor prognosis; and once diagnosed, they tend to be rapidly fatal. In fact, the average five-year survival rate for GBM patients is less than 3%, while the median survival rate from diagnosis to death is measured in months; approximately 12-15 months, despite the most aggressive treatment options available. As such, a diagnosis of GBM is almost always a death sentence. Therefore, GBMs are important to study in an effort to develop new therapeutics and new approaches to reduce the morbidity and mortality associated with this often fatal disease .
MAIN CHARACTERISTICS OF GBMS
There are several characteristics of GBMs that contribute to its poor prognosis, morbidity and mortality. These characteristics include (a) cell proliferation that is both fast and unregulated; (b) its widespread infiltration especially into nearby brain parenchyma thereby making total surgical resection improbable; (c) they are vigorously resistant to chemotherapy and radiotherapy and ultimately resistant to cell death; (d) its ability to attract new blood vessels hence giving rise to robust angiogenesis; (e) its contribution to a compromised blood-brain barrier leading to vascular edema or swelling; and finally (f) its intratumoral heterogeneity contributing to variations within the tumor mass . When some or all of these characteristics are diagnosed in a GBM patient, it represents a medical challenge to treat, cure and/ or extend the lives of these individuals, with the current therapeutics available. In addition, the diagnosis can be further complicated by the presence of different types and subtypes of GBMS, approaching a medical Mt. Everest in terms of therapeutic challenges to surmount, all in an effort to extend the quantity and quality of patients' lives.
TYPES OF GBM
The two main types of GBMs are either primary or secondary and they can arise de novo or from a low-grade glioma (LGG). A primary GBM is defined as arising de novo, in the absence of any evidence of any previous precursor low-grade neoplasm. In contrast, a secondary GBM is defined as one arising from previous precursor low-grade gliomas such as astroctyomas, oligodendrogliomas or mixed oligoastrocytoma, before transforming over time to a GBM .
Primary GBMs are prevalent in the elderly population and they account for over 90% of all the reported GBMs . In contrast the secondary GBMs tend to occur in the younger population, in individuals less than 40 years of age . This suggests that the rare de novo or primary GBMs are a disease of the elderly.
GBMs are difficult to identify phenotypically. A primary GBM originating from an elderly individual as compared to a secondary GBM originating from a younger individual less than 40 years of age is virtually indistinguishable. However, primary and secondary GBMs tend to have unique overlapping genetic aberrations , which upon clinical presentation and further analysis, may be used to distinguish primary GBMs from secondary GBMs . For example, primary GBMs often have and show amplification or mutation of the EGFR gene; mutation in the PTEN gene; and loss of heterozygosity (LOH) on chromosome 10q . In contrast, secondary GBMs rarely show mutations in EGFR gene amplifications or mutations. Instead, secondary GBMs are often characterized by p53 mutations; PDGFR gene overexpression, and retinoblastoma (RB) abnormalities .
SUBTYPES OF GBMS
The primary and secondary GBMs can be further sub-divided into four subtypes based on their genomic and transcriptional profiles . Recent data by the TCGA group has identified the four subtypes as follows: 1. Classical; 2. Proneural; 3. Mesenchymal and 4. Neural . The classical subtype of GBMs is based on EGFR amplification and EGFRvIII mutations; it lacks TP53 mutations and CDKN2A is deleted. Therefore primary GBMs may harbor classical subtypes based on the genetic mutations of EGFR gene.
The mesenchymal subtype of GBMs consists of mutations in the NF1, TP53 and PTEN genes, and presents with necrosis and microvascular proliferation. This suggests that this GBM mesenchymal subtype may be more amenable to inhibitors of angiogenesis. This subtype may also be characteristic of secondary GBMs based on TP53 genetic mutations .
The Proneural subtype of GBMs is based on PDGF ligand over-expression and PDGFRA amplifications, as well as mutations in IDH1, TP53, and PIK3CA/PIK3R1. This proneural subtype is also characteristic of younger patients and often presents with necrosis. This implies that patients diagnosed with this GBM-subtype may be more responsive to targeted therapies involving PDGFRA and PI3K inhibitors . This subtype may also be present in secondary GBMs, based solely on genetic mutations to include PDGFRA and TP53 mutations.
Altogether, the transcriptional subtypes contribute to the heterogeneity of the GBM tumor and reflect the common signaling abnormalities present within these tumors.
Each GBM subtype can respond differently to therapies based on genomic alterations; as such, personalized therapies may lead to more favorable outcomes (Lim et al, 2011).
Hence, GBM subtyping becomes important in deciding which patients will benefit from additional molecularly targeted agents specific to the mutation identified (Vitucci et al, 2011).
GBM CNS LOCATIONS
The anatomic location of the gliomas, especially diffusely infiltrating astrocytomas and GBMs can influence treatment options and prognosis . The majority of gliomas found in the CNS tend to be located in one of the four lobes of the brain: namely the frontal, the temporal, the parietal and the occipital lobes. Specifically, in a study of 267 gliomas, authors Larjavarra et al found that 40% of the gliomas were concentrated in the frontal lobes; 29% were found in the temporal lobes; 14% were distributed in the parietal lobe and 3% in the occipital lobe accounting for the least amount of gliomas . Furthermore, most or 51% of the gliomas were found in the right hemisphere of the brain, in contrast to 40% of gliomas in the left hemisphere, while the remaining gliomas were distributed either in the center or other areas of the brain .
Therefore, GBMs, whether they are primary or secondary, whether they occur in the young or old, and despite their more frequent frontal lobe location in the brain, are difficult to treat and/or cure by the standard treatment options, which ultimately makes these GBM tumor types and subtypes, difficult to treat and/or cure. The next section discusses the standard of care for GBM patients.
STANDARD TREATMENT OPTIONS FOR GBM PATIENTS
The current standard of care for GBM patients include surgical resection, concomitant radiation therapy with alkylating chemotherapy of temozolomide (TMZ) followed by post-radiation administration of TMZ .
Complete surgical resection of GBMs is virtually impossible. This is due in part to the invasive nature of the GBMs into nearby brain parenchyma and surrounding areas, thereby rendering complete surgical resection of the GBM mass highly improbable . In addition, tumor recurrence is the most difficult aspect of glioma/GBM therapy such that preventing recurrence at the 2-3 cm margin of regrowth may reduce the dismal prognosis of malignancy following initial GBM diagnosis and surgical resection . As such, treating and curing GBM via surgery is difficult to achieve
Following maximum debulking surgery, and concomitant radiation therapy with nonfunctional alkylating agent TMZ, the average survival is still 14.6 - 15 months. The mechanism of action for TMZ leads to apoptosis and cell death . Unfortunately, almost all GBM patients eventually develop recurrent tumors refractory to TMZ chemotherapy, again the recurrent tumors being the main cause of death among GBM patients.
PAST AND CURRENT TREATMENTS FOR GBM PATIENTS
There have been numerous cutting-edge treatment options for advanced GBM patients over the past 30 years in an effort to treat, cure and at best to minimally extend the life of GBM patients. Unfortunately, many of these options have not been as successful as originally conceived, so there is still an urgent and pressing need to develop new therapeutic strategies to treat, cure, and/or extend the life span of this patient population.
In this section I will discuss two of the main approaches used as treatment options for GBM patients; namely the conventional and non-conventional approach, and why the conventional approach to GBM therapeutics may be more amenable to success as compared to the non-conventional approach.
There are four main conventional approaches used in the past to most recently, to treat, cure and/or extend the life of GBM patients to include:
The technique or practice of blood-brain-barrier disruption (BBBD) .
The use of wafers at the GBM tumor site .
The selective placement of catheters at the GBM tumor site .
The use of molecularly targeted therapy .
Together, these four comprise the bulk of the conventional approaches used in GBM therapeutics. Here I will discuss the problem that prompted the use of the conventional methodology outlined, the potential drawbacks and whether or not there was a clinical advantage in terms of overall survival in the GBM patients so treated.
The first conventional approach used to treat/cure and possibly extend the life of GBM patients is the technique of BBBD . This technique was developed in an attempt to resolve the problem of how to get chemotherapeutics across the BBB in therapeutically relevant amounts and concentrations to be efficacious in CNS brain tumors. The BBB problem exists because brain tumor cells infiltrate into the nearby brain parenchyma, where they are protected by the BBB from chemotherapeutics needed to kill the tumor cells. As such, it is hard to get drugs across the BBB to eliminate these tumor cells . When these diffuse brain tumors are treated with radiation, the temporary response of the tumors to radiation suggests that patients are rarely in total remission. Similarly, when these diffuse brain tumors are treated with chemotherapeutic, several possibilities exist which cause the tumors to be unresponsive to the administered drug.
The first possibility that hinders the chemotherapeutic agent from arriving at the tumor site in therapeutically relevant amounts and concentrations is due to the presence of the BBB. The second possibility that prevents the chemotherapeutic agent from being efficacious at the brain tumor site is that, even though the drug may have reached and breached the BBB, it may not have arrived at the tumor site in physiologically relevant amounts (volume) and concentrations (potency) to either reduce the tumor burden or to completely eliminate the tumor at that site. Thirdly, the tumor cells may have developed a natural resistance to the chemotherapy drug, or the brain tumor cells may have acquired resistance to the chemotherapeutic drug, with both conditions resulting in an unresponsive brain tumor mass to the administered chemotherapeutic drug .
The technique of BBBD is one method designed to solve the problem of getting chemotherapeutics across the BBB in therapeutically relevant amounts and concentrations to have an effect on the brain tumor. The BBBD technique was developed and standardized by clinician/scientist Dr. Edward Neuwalt at the Oregon State University (OSU), who practices the technique of transiently opening the BBB to allow the chemotherapeutic drug of choice to enter in amounts and concentrations which will be efficacious in brain tumor reduction or elimination .
This BBBD technique was perfected in over 6000 procedures done on 400 patients, located at 6 different clinics, and utilized 3 unique protocols . In the Phase I/II clinical trials, the study authors concluded that the BBBD is a suitable method to increase the dose of chemotherapeutic agents administered to patients for whom GBM is indicated .
Based on my review of the literature and the ensuing human Phase I/II clinical trials involving GBM patients, it does not appear that the BBBD technique is part of the current standard of care for newly diagnosed or recurrent GBM patients. This insight is based upon the fact that while the BBBD is successful in its approach to increase the amounts and concentrations of drugs at the tumor site, it may not have contributed to the overall survival of GBM patients. The lack of efficacy observed with the BBBD technique may indicate in my opinion, that the BBB is not the only and/ or the main problem that must be surmounted in GBM morbidity and mortality. Hence the problem persists of how to successfully treat/cure and/or extend the life of GBM patients.
The second conventional approach used to treat/cure and possibly extend the life of GBM patients is the implantation of biodegradable polymer wafers in the CNS at the tumor site . This method also addresses the need of bypassing the BBB to get chemotherapeutics across the BBB in therapeutically relevant amounts and concentrations, avoids the short half-life of the drug carmustine, and avoids systemic toxicities, by using wafers. These wafers are termed GliadelÂ® or carmustine because they contain the chemotherapeutic drug, BCNU (1, 3-bis (2-chloroethyl)-1-nitrosurea) which was previously shown to be effective against glioma cell lines. The carmustine wafers are usually implanted in the tumor resection cavity following surgery in newly diagnosed GBM patients and recurrent patients .
In one study, GBM patients were randomized into two arms or study groups for the Phase I/II clinical trial study. One study group received the placebo, while the treatment group received carmustine wafers containing Polifeprosan 20 (3.85% (w/w) BCNU) also known as Gliadel, implanted at the tumor site or in the tumor resection cavity. All the patients in the clinical trial were monitored for 1-2 years, and at the end of the clinical trial the results were published. The study authors concluded that there was a small survival advantage in the treated group compared to the placebo control group. However, the study authors noted that the survival advantages or differences were not statically significant due to the possibility of a small study population or sample size and low power .
The overall results indicate that carmustine wafers implanted at the tumor site, even after surgical resection and radiation therapy combined with other chemotherapeutic agents such as TMZ, did not treat, cure and/or extend the life of GBM patients. The lack of statistical significance and efficacy observed with the use of carmustine wafers in the clinical trials, again suggests that the BBB is not the only and/or main problem involved in GBM morbidity and mortality. Therefore, the problem persists of how to successfully treat/cure and/or extend the life of GBM patients.
The third conventional approach used to treat/cure and possibly extend the life of GBM patients is the use of convection enhanced delivery or CED which involves the direct delivery of chemotherapeutic into the CNS at the tumor site . This method is similar in principle to the BBBD technique, and carmustine wafers, and represents the third possible solution to the problem of how to get chemotherapeutics across the BBB in therapeutically relevant amounts and concentrations to have an effect on the brain tumor by using direct delivery at the tumor site using CED.
CED is a localized method of drug delivery into the interstitial space of the brain, over a period of time ranging from hours to days, again with the goal of by-passing the BBB . This technique is based on a continuous pressure gradient. This means that the amount of drug at the tumor site depends upon the initial volume of the drug administered, the rate of drug delivery, the half-life of the drug and the surface binding property of the drug .
A randomized Phase III human clinical trial enrolling 296 patients was designed to determine the efficacy of the CED technique. The patients were stratified or divided in a 2:1 ratio. This means that for every two patients who received intraparenchymal CED of drug following standard surgery, one patient received the carmustine or Gliadel wafer, implanted at the tumor site, with both procedures occurring within 48 hours of surgery .
In the first randomized treatment group of patients receiving CED, 2-4 catheters were surgically implanted at the tumor site, and left in place for two to seven days before removal. These catheters were placed at the tumor sites with the greatest potential for infiltration or metastasis .
One of the aims of this clinical trial was to determine if there was a survival advantage of CED compared to carmustine wafers in GBM patients. The study authors concluded that the CED technique, while tolerated in GBM patients, unfortunately did not offer a survival advantage over the implantation of carmustine wafers. Once again, this suggests that by-passing the BBB to administer chemotherapeutics in relevant concentrations, over a period of hours to days is still not sufficient to be statistically significant or clinically relevant in extending the overall survival of GBM patients .
Therefore, in summary, the three conventional approaches used to treat/cure and or extend the life of GBM patients to include: 1. the BBBD technique to disrupt the BBB to deliver chemotherapeutics in large volumes and high concentrations at the tumor site; 2. the implantation of carmustine wafers at the tumor site to deliver chemotherapeutics at high concentrations; and 3. the placing of catheters by utilizing CED to continually deliver chemotherapeutics at the tumor site; have not been as successful as initially conceived and hoped. Taken together, these highly invasive surgical procedures, all aimed at circumventing the BBB to deliver drugs at the tumor site, have not resulted in an increase in overall survival in either newly diagnosed GBM patients or reoccurring patients. Therefore, it seems plausible to conclude that there is still an urgent need to develop new treatment options for GBM patients.
The fourth conventional approach used to treat, cure and/or extend the life of GBM patients that is not surgically invasive, is by using a molecular targeted approach. Many of these approaches include the more than 200 ongoing clinical trials dedicated to ending the devastation associated with GBM morbidity and mortality (www.clinicaltrials.gov).
The next section outlines and discusses in detail the numerous approaches involved in using a molecularly targeted method as treatment options for GBM patients .
These approaches include the following:
Inhibition of growth factor ligands.
Inhibition of growth factor receptors.
Inhibition of intracellular effectors.
The first molecularly targeted approach involves the inhibition of growth factor ligands, used as therapy for GBM patients.
Inhibition of growth factor ligands-Study -NCT 01149850
Bevacizumab/Avastin is a humanized neutralizing monoclonal antibody to VEGF. The purpose of this molecular targeted approach is to determine overall survival after treatment with Bev/Avastin in elderly patients; Progression free-survival for 2 years with Bez treatment; and to investigate the safety and tolerability of Bez in older patients with GBM. Other molecular targeted approaches include clinical trials
Study -NCT 01086345 with Bevacizumab & Radiosurgery
Study -NCT 01413438 Bevacizumab with and without surgery
In all of these clinical trial studies, the aim is to determine overall survival and progression free survival by inhibiting the VEGF ligand binding to its receptor to activate angiogenesis or new blood vessels which feed tumors.
The second molecular targeted approach involves the inhibition of growth factor receptors such as EGFR, VEGF, and PDGF, used as therapy for GBM patients.
Inhibition of growth factor receptors-Study -NCT 01110876
Erlotinib is a drug used to target the EGFR epidermal growth factor receptor.
The purpose of this Phase I/II study is to determine the MTD or maximum tolerated dose or the highest concentration of the combination of Vorinostat, Erlotinib with/without chemotherapeutic agent TMZ that can be tolerated in GBM patients, upon completion in September 2014.
The third molecular targeted approach used as therapy for GBM patients, is the inhibition of intracellular receptors in pathways such as PI3K/Akt/Mtor with inhibitors such as Perifosine, Rapamycin and Rapamycin analogues such as Everolimus and Temisirolimus. For example:
Perifosine which specifically targets AKT;
Rapamycin which targets mTOR complex;
Specifically, the clinical trial Study -NCT 00694837-Nelfinivir (HIV-1 Protease Inhibitor and AKT inhibitor) aimed at targeting intracellular receptors.
The purpose of this study is to determine the MTD of NFV in GBM patients; and to determine the 6 month progression free survival for GBM patients treated with NFV. The expected clinical outcome of this study is to determine if NFV can block the in vivo activity of AKT in GBM patients.
Other relevant clinical studies targeting intracellular receptors include:
Study NCT 01062399-Everolimus-mTOR inhibitor.
Study -NCT 01051597-Temsirolimus-mTOR inhibitor and Perifosine (AKT inhibitor).
To summarize briefly, the fourth conventional approach used to treat, cure and/or extend the life of GBM patients that is not surgically invasive, involves a molecular targeted approach to include the following:
Inhibition of growth factor ligands such as VEGF with Bevacizumab.
Inhibition of growth factor receptors such as EGFR with Erlotinib.
Inhibition of intracellular effectors in pathways such as PI3K/AKT/mTOR with Perifosine for Akt and Rapamycin for mTOR complex.
Taken together, this is just a fraction of the ongoing clinical trials currently in progress to alleviate the morbidity and mortality associated with GBM patients; using a conventional, molecularly targeted approach. The next section focuses on the other arm of the conventional approach with an emphasis on miscellaneous approaches, again, all in an effort to treat GBM patients.
MISCELLANEOUS CONVENTIONAL APPROACHES
In addition to the conventional, molecular targeted approach to include Inhibition of growth factor ligands such as VEGF with Bevacizumab;
Inhibition of growth factor receptors such as EGFR with Erlotinib; and
Inhibition of intracellular effectors in pathways such as PI3K/AKT/mTOR with Perifosine and Nelfinivir for Akt, and Rapamycin for mTOR complex; some other miscellaneous approaches, resembling a molecular targeted approach to include the following:
1. Ligand-toxin conjugates.
2. Agents targeting invasion.
3. Deacetylase inhibitors.
4. Proteosome inhibitors.
5. Combination therapy.
6. Multimodal treatment .
The first conventional, miscellaneous approach used to treat, cure and/or extend the life of GBM patients is the use of ligand-toxin conjugates. For example, in the clinical trial study, NCT 01082926, (www.clinicaltrials.gov), the purpose of this Phase I cellular immunotherapy trial is to study the side effects and determine the best way to administer therapeutic donor lymphocytes, in combination with aldesleukin as a method to treat astrocytoma and GBM patients. Specifically, aldesleukin may stimulate white blood cells to kill tumor cells, so by combining different biological therapies it may be possible to stop even more tumor cells from proliferating. The expected result is refractory disease and GBM patients are expected to be followed annually for up to 15 years post treatment.
Another conventional, miscellaneous approach involves the use of agents that target invasion. This is represented by clinical trial study, NCT 00813943 (www.clinicaltrials.gov) in which Cilengitide is used as an intravenous agent targeting integrins alpha, v, beta 3 and alpha v beta 5. In this Phase I/II study, Cilengitide is administered as an intense treatment in combination with standard therapy; followed by the administration of two different concentrations or regimens of Cilengitide, in combination with or without standard treatment to include TMZ and radiotherapy. The expected outcome will be monitored by overall survival and progression free survival in newly diagnosed GBM patients, with unmethylated promoter of MGMT gene diagnosed in the tumor tissue.
In yet a third conventional, miscellaneous approach to treat, cure and/or extend the life of GBM patients is the use of deacetylase inhibitors such as:
SAHA or suberoylanilide hydroxamic acid, which targets histone deacetylase (HDAC)
Vorinostat and Valproic acid, which also targets HDAC
Specifically, clinical trial study, NCT00939991 (www.clinicaltrials.gov), uses the SAHA HDAC inhibitor in combination with Bevacizumab and TMZ and Vorinostat. The purpose of this study is to determine the MTD of Vorinostat when given to patients in increasing doses and measured by dose-limiting toxicities (DLT). The Phase I/II clinical trial for recurrent GBM patients has an endpoint of 6 month progression- free survival and overall survival.
An additional conventional, miscellaneous approach involves the use of proteasome inhibitors such as Bortezomib, as exemplified by clinical trial study, NCT 01435395 (www.clinicaltrials.gov). In this trial, TMZ, Bevacizumab and Bortezomib are used in combination therapy with the purpose of determining the safety and toxicity of bortezomib in combination with Bez and increasing doses of TMZ for patients with recurrent GBM.
The expected result is a measurement of how many patients have arrived at the MTD for TMZ and Bez when combined with bortezomib as measured by DLT or dose-limiting toxicities. Secondarily, progression free-survival as well as overall survival will be monitored based on tumor reduction via magnetic resonance imaging.
The fifth conventional, miscellaneous approach utilizes combination therapy. This involves several different inhibitors of ligands; or inhibitors of receptors; or inhibitors of intracellular receptors. For example, EGFR inhibitors Erlotinib, and the intracellular receptors of the PI3K/Akt/mTOR pathways such as Everolimus or Temsirolimus which inhibits mTOR, have both been used in combination therapy. This can be represented by clinical trial Study NCT 01062399-Everolimus-mTOR inhibitor and Study -NCT 01051597-Temsirolimus-mTOR inhibitor, and Perifosine (AKT inhibitor), where both studies target mTOR with Everolimus or Temsirolimus and AKT with the inhibitor Perifosine.
The sixth and final conventional, miscellaneous approach to treat, cure and/or extend the life of GBM patients is via multimodal therapy. This therapy involves targeted agents, in addition to surgery, radiation and/or chemotherapy. For example, the EGFR kinase inhibitor Erlotinib and chemotherapy with TMZ is an example of targeted agent: kinase inhibitor with chemotherapy. Similarly, the use of a kinase inhibitor, with chemotherapy, and radiation is yet another example of targeted agents with both chemotherapy and radiation. Specifically, EGFR kinase inhibitor Erlotnib (targeted agent) in conjunction with TMZ (chemotherapy), plus radiation therapy is another example of multimodal therapy.
The clinical trial study NCT 01443676-(www.clinicaltrials.gov) Avastin or Bevacizumab (VEGF ligand inhibitor, targeted agent) and radiotherapy, is an example of a multimodal therapeutic approach. The purpose of this study is to determine the efficacy of Bez and radiotherapy as compared to radiotherapy alone in elderly patients diagnosed with GBM or recurrent GBM. The outcome will be measured by progression-free survival and overall survival in older patients for whom radiotherapy is often the only standard of care administered.
Some other examples of clinical trial studies include NCT 01102595 (www.clinicaltrials.gov)-Neo-adjuvant treatment with TMZ (chemotherapy) and Bevacizumab (VEGF ligand inhibitor/targeted agent). In this study the objective is the administration of TMZ and Bez plus the radiotherapy in patients for whom surgery is contraindicated because of unresectable GBMs. The patients will be monitored for overall survival.
Similarly, another study is NCT 00943826 (www.clinicaltrials.gov)-A study of Avastin/Bevacizumab (VEGF ligand inhibitor/targeted agent) in combination with TMZ (chemotherapy) and radiation therapy. In this case, the purpose of the study is to monitor progression-free survival, and overall survival in patients treated with Bez including standard of care to include radiotherapy and TMZ or radiotherapy and TMZ alone. The study will be completed when the disease progresses. In both instances, TMZ and Bez with or without surgery, and/or radiotherapy, the aim of the study is progression- free survival as long as possible and overall survival to extend beyond the 3-additonal months obtained with TMZ.
In the final analysis, and to summarize, the conventional miscellaneous approaches which include the use of (a) ligand toxins; (b) agents targeting invasion; (c) deacetylase inhibitors; (d) proteasome inhibitors; (e) combination therapy and (f) multimodal therapy have all been utilized in the past with some variable measures of success. This reinforces the concept that additional treatment options must be considered for GBM patients, even if they involve a non-conventional approach, which is the subject matter of the next section.
WHY GBMS ARE HARD TO TREAT AND CURE
GBMs are notoriously resistant to chemotherapy and radiation therapy, which makes them extremely difficult to treat and cure, and hence ultimately fatal. There are three contributing factors that play a role in GBMs therapeutic resistance to include: 1. the heterogeneity of the GBM tumor that consists of different cell types with different responses to drug treatments; 2. the presence of the blood-brain barrier that restricts the passage of chemotherapy drugs; and 3. the proclivity of the GBM tumor cells to diffusely infiltrate into nearby parenchyma .
The first factor which contributes to the GBMs resistance to chemotherapy and radiation thereby making them difficult to treat and cure is due to the heterogeneous population of the GBM tumor or intratumoral heterogeneity. This manifests itself as differences within the tumor mass itself to include for example, differences in terms of the types of cells within the tumor mass; the types of genes expressed within the tumor mass; and the frequency of the types of genes expressed within the tumor mass .
This intratumoral heterogeneity in GBM patients makes it more difficult to understand the pathobiology of the disease. In addition, intratumoral heterogeneity makes it difficult to determine which treatment option is best; and yet intratumoral heterogeneity may be the catalyst to pursue novel therapeutic agents to treat and possibly cure GBMs , because combination therapy may hit at least two or more different targets in the heterogeneous tumor, thereby reducing tumor burden.
The heterogeneous population of a GBM mass often includes areas containing anaplastic astrocytomas (Grade III) or low-grade gliomas (Grade II) including oligodendrogliomas or gliomas with ependymal differentiation . This rainbow of heterogeneity with different cell populations, coupled with different proliferation and differentiation potentials, and different degrees of vascularity and invasiveness, suggests that in the presence of radiation therapy and chemotherapy some cells will be inherently resistant to both. Not surprisingly, GBMs are exquisitely resistant to radiation and chemotherapy .
The second factor which contributes to the GBMs resistance to chemotherapy and thereby making them difficult to treat and cure is due to the presence of the blood-brain barrier (BBB) . The BBB is a seamless, continuous physical layer needed to separate the CNS from the blood circulation. It is composed of astrocyte (glial cells) foot processes, pericytes or smooth muscle cells, and brain capillary endothelial cells (BEC). These cells are surrounded by basal membrane and extracellular matrix, which are secured with tight junctions to seal the paracellular spaces, all of which contribute to the fluidity of the physical layer to separate the blood from the underlying brain cells .
The main function of the BBB is to maintain brain homeostasis by protecting the brain from the changing composition of the blood especially after meals or exercise; and by controlling the passage of blood-borne pathogens into the brain . A secondary and equally important function of the BBB is to protect the brain from toxins, both exogenous and endogenous (such as neurons which circulate potentially cytotoxic agents, and to control the passage of solutes, blood-borne agents, and pathogens .
One way the BBB accomplishes this task of maintaining brain homeostasis, is to restrict the passage of chemotherapeutic drugs with high molecular weights. The size exclusion limits imposed by the BBB limits the passage of many pharmaceutical compounds (such as monoclonal antibodies), 98% of small molecules, and many chemotherapeutic agents that weigh between 200-1200 D; from crossing the barrier and thereby affecting a therapeutic response . Therefore, to by-pass the protective function of the BBB in an effort to treat infiltrating gliomas such as GBMs the chemotherapy drug of choice should possess the following criteria: they should be small; they should weigh less than 400 D; and they should be lipid soluble as they tend to traverse the BBB by passive diffusion .
The BBB contributes to chemotherapy resistance in astrocytoma and GBM biology by creating a safe "haven" for cancer cells . This means that cancer cells beyond the reach of the BBB are secure from being destroyed by therapeutic agents that may not reach the cells in therapeutically relevant concentrations to kill the brain tumor cells . Hence the protective function of the BBB either limits or prevents the entry of drugs into the brain. This is accomplished when chemotherapeutic drugs are prohibited from crossing the BBB; or if the drugs do cross the BBB, they are immediately shunted out due to the presence of a very effective drug efflux system; or if they do cross the BBB they do not arrive at the tumor site in sufficiently therapeutic relevant concentrations to effectively demonstrate a consistent and clear dose-response . In summary, the second factor which contributes to the GBMs resistance to chemotherapy is due to the presence of the blood-brain barrier (BBB) which restricts the passage of chemotherapy drugs to the tumor site, thereby making GBMS harder to treat and cure.
The third and final reason which contributes to the GBMs resistance to chemotherapy and radiation, thereby making them difficult to treat and cure is due to tendency of the tumor cell to invade nearby brain areas which leads to tumor recurrences . The metastasis of resistant brain cancer cells into the nearby parenchyma of the brain may be ultimately responsible for the patient's demise . It has been suggested by many researchers that the spread of resistant brain cancer cells from the tumor site into the nearby open spaces in the brain may be the main reason for the death of GBM patients. The persistent drug relapsing tumor infiltrating cells within the 2-3 cm surgical margin has been associated with the high rate of tumor reoccurrence . The drug-resistant leading edge of infiltrating tumor cells into the 2-3 cm surgical resection margin may be driven by a small population of cancer stem cells or tumor initiating cells (TIC) that are responsible for the initiation and continued malignancy seen in these brain tumors; as well as the recurrences that frequently occur after standard treatments. In fact, a growing body of evidence suggests that many cancers including many solid and soluble tumors may be driven by these TICs .
TIC or cancer stem cells may be defined as the cellular fractions found in some tumors which are capable of initiating tumors similar to the parental tumors when transplanted into a secondary site . In other words, TICs re-grow and resemble the original primary tumors after transplantation into a secondary location.
There is substantial evidence in the literature to support the hypothesis that cancers may not only arise from, but that cancers may in fact contain stem cells. This evidence is based on published reports which suggest that in patients with breast carcinoma and leukemia, there remains a core or remnant of cells within the tumor mass that is capable of, and responsible for, maintaining the growth of the tumor and for the formation of new tumors .
This may be one reason why despite successful radiation and chemotherapy in many GBM patients, a remnant of core cells remain which are resistant to therapy as evidenced by their ability to survive; core cells which can and often reinitiate tumor growth . The core cells are often called GBM stem cells; they are often resistant to chemotherapy and radiation, and may be one source of GBM tumor reoccurrence. As such, GBM tumor reoccurrence leads to increased morbidity and the ultimate mortality of the patient.
Therefore, to summarize, the third reason advanced for GBMs resistance to chemotherapy and radiotherapy and which makes GBMs nearly impossible to treat and cure is due to the presence of a drug resistant cell- front that invades and metastasizes the other outlying areas of the brain. This invasion or metastasis means that some diffusely infiltrating cells will escape chemotherapy, radiation and surgery which ultimately leads to tumor recurrence, attributed to tumor initiating cells (TIC) or GBM stem cells, with death as the end stage.
In the final analysis, GBMs are hard to treat and cure due to the intratumoral heterogeneity of the tumor; the presence of the blood-brain barrier which limits access of the chemotherapy drugs in therapeutically relevant amounts to effect a change; and the infiltrative nature of the resistant tumor cell responsible for tumor recurrence, resulting in the untimely and ultimate death of the patient. Therefore, in an effort to aid in our understanding of glioma and GBM biology as well as develop novel, cutting therapeutics to treat and/or cure this disease, we need valid, reproducible and reliable, in vitro and in vivo model systems.
THE MODEL SYSTEM USED TO STUDY THE EFFECTS OF ABERRENT SIGNALING IN THEPI3K/AKT/MTOR PATHWAY
To study the potential pre-clinical in vitro effects of promising therapeutics that may be utilized in treating GBM patients, I used an in vitro/in vivo mouse model system Nf1; p53cis, C57BL/6J mouse model of astrocytoma and NF1.
NF1 AND GLIOMAS/GBMS
NF1 Neurofibromatosis type 1 is an autosomal dominant disorder that affects about 1 in 3500 individuals worldwide . Children and adults with NF1 have an inherited predisposition to developing multiple tumors termed neurofibromas. These tumors can be benign or malignant, and can develop in both the CNS and (PNS) peripheral nervous system .
NF1 patients, especially children, often develop low grade glial tumors. These low-grade gliomas (LGGs), WHO Grades I or II, characterized as slow-growing neoplasms of the CNS, account for at least 20% of all glial tumors . The Pilocytic Astrocytomas (Grade I) are rare, low-grade tumors which are common in patients with (NF1) or neurofibromatosis type 1.
NF1 patients often develop CNS disorders such as benign optic gliomas that involve the bilateral optic nerve and optic chiasm, which results in blindness .
NF1 patients commonly develop tumors in the PNS termed neurofibromas. These tumors which are more common in young adults are composed of Schwann cells lacking NF1, NF1+/- fibroblasts, mast cells and vascular elements .
NF1 astrocytoma in children beyond 10 years of age is a rare occurrence and spontaneous regression has been noted in the absence of treatment. However when treated, NF1 patients respond positively to treatment , probably due to the clinically less aggressive nature of NF1 astrocytomas, although surgical resection or biopsy is not routinely done on NF1 patients with low- grade gliomas .
To understand the biology of gliomas, and especially GBM formation in vivo; and in an effort to develop effective therapeutics to treat GBM patients, there is a need to have efficient model systems. These readily, available, clinically relevant disease models will help the scientific community in at least two critically important ways.
The first way the relevant disease models will benefit the scientific community is that they will help scientists and researchers understand how tumors develop, and how they progress to cancer; especially gliomas and GBMs . Secondly, the disease models will help the research community, by providing both an in vitro cell based assay system and in vivo access, to test potential therapeutics for GBM cancer treatment. The availability of GEMMS or genetically engineered mouse models are one example of a readily available, clinically relevant disease mouse model that can be used to study how tumors are initiated, develop, and progress to cancer, only because such in vivo studies in humans are unethical, impractical and inhumane . One such glioma derived GBM mouse model is the Nf1; p53cis, C57BL/6J mouse model of astrocytoma and NF1 . This mouse model is termed NPcis because the Nf1 and p53 genes are lost on the same chromosome 11, and on the same allele of chromosome 11, and the genes Nf1 and p53 are so close together that they are inherited together. The tumors arising from this mouse model are of the secondary type astrocytomas and glioblastomas due to the genetic mutations present (Nf1 and p53) and due to the loss of heterozygosity (LOH) at the remaining wild-type Nf1 and p53 locus. As such, these tumors tend to progress over time from lower grade II tumors to the more malignant grade IV GBM tumors.
CHARACTERISTICS OF THE IDEAL GBM MOUSE MODEL
There are several important features that comprise the ideal GBM model to mimic the human disease condition. Notably, the ideal GBM model should recapitulate the key features of the human disease to include: very fast growing tumors; abnormal cells; necrotic dead areas in the tissue; neo-angiogenesis or a network of blood vessels; and invasiveness by spreading throughout the CNS . Other features of the ideal GBM model should include the fact that it should be accurate; orthotopic; reproducible; reliable; resemble progression genetics; retain important genetic alterations such as EGFR overexpression or amplification; possess a short tumor latency and high penetrance; simple to generate and easy to use; and finally it should have a built in mechanism to determine therapeutic effects, such as a bioluminescent reporter .