A molecular approach to Glioblastoma Multiforme

Glioma is a tumor of the central nervous system that occurs in the glial cells, Which it surrounds and protects the nerve cells. Glioblastoma Multiforme (GBM) is the most common and malignant sub-type of gliomas that arises from star-shaped cells called “astrocytes”, which constitute the supportive tissue of the brain. GBM are known to be heterogeneous in outcome with majority having a poor prognosis, thus there is an urgent need for novel therapeutic approaches. The detailed understanding of GBM is established by the combination of histopathology and genomic information of the tumor that aids in the best choice of Personalized Medicine. In this article, seven GBM patients are discussed who underwent tissue diagnosis as well as tumor molecular profiling; the significance of the genes and associated mutations/variations picked up in each individual.


Introduction
Glioma encompasses all tumors that are thought to be of glial cell origin.Glioblastoma multiforme (GBM) is usually highly proliferative because the cells reproduce quite fast and is assisted by a large network of blood vessels (neovascularization).They are generally found in the cerebral hemispheres of the brain, but can also be found in other parts of the brain and spinal cord.
The average incidence rate of GBM in the USA is 3.19 per every 100,000 of the population, and the median age of diagnosis is 64 years with incidence higher in men.Many genetic and environmental factors have been studied in GBM, but the majority are sporadic, and no risk factor accounting for a large proportion of GBMs has been identified. [1]pes of gliomas include astrocytic tumors (World Health Organization classification) astrocytoma Grades I, II (astrocytoma), III (anaplastic astrocytoma), and IV (glioblastoma or GBM), oligodendrogliomas, ependymomas, and mixed gliomas.
GBMs are biologically aggressive tumors that present unique treatment challenges due to localization of tumors in the brain; inherent resistance to conventional therapy; limited capacity of the brain to repair itself; migration of malignant cells into adjacent brain tissue; variably disrupted tumor blood supply which inhibits effective drug delivery; tumor capillary leakage, resulting in an accumulation of fluid around the tumor; of oncogenesis.Certain changes in these genes are validated targets of therapy in various types of tumors and/or in clinical trials.Next-generation sequencing (NGS) is the molecular technique used here.See the supplement sheet for the panel of genes (Appendix 1).All the cases were discussed in the molecular tumor board meeting where medical opinion was taken from a team that consisted of Medical Oncologist, Radiation Oncologist, Surgical Oncologist, Pathologist, Radiologist, Neurosurgeon, and a Molecular Oncologist.

Molecular Subtypes of Glioblastoma Multiforme
Glioblastoma has been classified into subtypes based on their gene expression by The Cancer Genome Atlas (TCGA).Table 1 gives a clear understanding of the subtypes and the various genes mutations and copy number variations.

Mesenchymal
The mesenchymal subgroup contains the most frequent number of mutations in the neurofibromin (NF1) tumor suppressor gene.Patients in the mesenchymal group had significant increase in survival after aggressive treatment, unlike those in the proneural, and to an extent, in the neural subgroups.

Classical
Classical GBM tumors are characterized by abnormally high levels of epidermal growth factor receptor (EGFR).The EGFR abnormalities occur at a much lower rate in the three other GBM subtypes.However, TP53, the most frequently mutated gene in GBM, is not mutated in any of the classical GBM tumors.Clinically, the classical group survived the longest in response to aggressive treatment.

Neural
The neural group was characterized by the expression of several gene types that are also typical of the brain's normal, noncancerous nerve cells, or neurons.Patients in the neural group had some improvement in survival after aggressive treatment but not as much as the classical and mesenchymal groups.

Proneural
Proneural tumors are also characterized by having the most frequent mutations-in the IDH1 gene.Platelet-derived growth factor receptor alpha is found to be mutated and expressed in abnormally high amounts in this subtype.Unlike the other types, whose patients were similar in age on average, the proneural subgroup was significantly younger.They also tended to survive longer and have the best prognosis among all subgroups.However, patients in the proneural group who received aggressive treatment with TMZ did not survive significantly longer than proneural patients who did not receive aggressive treatment.

Case 1
A 52-year-old female who is a known case of right frontal GBM was diagnosed 4 years ago.Surgery was performed with subtotal resection of the brain which was followed by temozolomide (TMZ) and RT.She was also on TMZ as maintenance therapy.On following up with the patient, she was found to progress on TMZ and received 4 cycles of PCV which was later discontinued because of serious adverse events.Thereafter, she was again put back on TMZ for 1 year but showed progression.Hence, TMZ was discontinued and was started on bevacizumab (Avastin).Since then, she was on Avastin with a stable disease and a good physical performance for more than 1½ years.Unfortunately, she died after 4 years due to aspiration pneumonia.

Clinical questions
1.With such a dismal prognosis of GBM, why did the patient have a good survival of approximately 4 years?2. Why was there a progression on TMZ?Is there any additional benefit to continue TMZ beyond progression?
Molecular oncology approach 1. Somatic mutation 48 gene panel testing was carried out.

Result interpretation
The genomic alterations picked up are explained in detail in Table 2.
Based on the Recursive Partitioning Analysis (RPA) criteria (based on the clinical parameters), the patient lies in Group 3 with a median survival of 70 weeks.However, the patient survived for over 48 months (>220 weeks) since diagnosis, suggesting a favourable tumor biology.This is supported by the fact that EGFR expression (IHC was carried out) and amplification (NGS) were absent, which are indicators of good prognosis.Further, based on the presence of aberrant TP53 expression (IHC was carried out) and somatic gene mutation (NGS), the patient can be classified into "Proneural Molecular Subtype" [Table 1].The median overall survival of proneural subtype is reported to be 36-48 months, [6] thus explaining the patient's natural course (favourable).
Further, a study by Le Mercier et al. [7] showed no additional benefit of adding TMZ to RT versus RT alone in proneural subtype in comparison to the classical subtype of glioblastoma [Figure 1].Thus, this calls into question the continuation of TMZ beyond progression.To conclude, molecular profiling may help to analyze and predict the prognosis and possible response to conventional therapy and management.

Therapeutic implications
i. Proneural molecular subtype with expected median overall survival 36-48 months as observed in the case ii.No additional benefit of continuing TMZ beyond progression.

Case 2
A 68-year-old male who is a known case of left temporal GBM underwent sub-total resection and was on TMZ and RT.He was put on maintenance TMZ.

Clinical questions
1. What is the patient-specific tumor biology?Can we predict the treatment response?1. Somatic mutation 48 gene panel testing was carried out.

Result interpretation
The genomic alterations picked up are reported in Table 2.
The identified variation in PIK3CA gene (p.His10484Arg) is within the hotspot region and highly conserved kinase domain and has been previously reported in endometrial and breast cancers.
Based on the clinical parameters using RPA criteria, it was found that the patient lies in the poor prognosis group with median survival of 40 weeks.Further, mutation in the PIK3CA gene, which known to be genetically altered in primary glioblastoma with poor prognosis [Figure 2] and distinguishes from secondary glioblastoma with good prognosis.Thus, both the clinical and molecular assessment suggest poor prognosis.
This variation may lead to constitutional activation of the phosphatidylinositol-3-kinase (PI3K)/AKT survival pathway thus resulting in growth factor-independent proliferation and protection from cell death.Activation of the PI3K pathway is known to play a role in radioresistance in glioma. [8]erapeutic implications i.Based on the mutation identified, we can subtype the GBM into "classical molecular subtype" with a median survival of 40 weeks (poor prognosis).The patient may benefit A 59-year-old male diagnosed with GBM of the left frontoparietal region, underwent surgery for the same and was undergoing RT along with CT (TMZ).He is a non-smoker and a non-tobacco user.

Clinical questions
i. What is the patient-specific tumor biology?Can we predict the treatment response?
Molecular oncology approach 1. Somatic mutation 48 gene panel testing was carried out.

Result interpretation
The genomic alterations picked up are reported in Table 2.

Epidermal growth factor receptor amplification
EGFR amplification was observed in this patient.It is frequently observed in glioblastoma and is been seen in about 36-40% of the tumors and is associated with resistance to CT and radiation therapy. [9]Prognostic significance of EGFR is not yet clear.Diverse observations are reported where EGFR amplification serves as poor prognostic factor.In contrast, it is a marker of prolonged survival in older glioblastoma patients. [10]GFR amplification and overexpression are associated with resistance to CT and radiation therapy. [11]Mechanistically, connexion 43 is involved EGFR-mediated TMZ resistance. [12]

Deletion of both copies of phosphatase and tensin homolog gene
Both the copies of the phosphatase and tensin homolog (PTEN) gene on chromosome 10 have been deleted in this patient.
The deletion of PTEN genes has already been reported in prostate cancer, breast cancer, glioblastoma cell lines and as well as in primary glioblastomas.Di Nicolantonio et al. [13] demonstrated that patients with PIK3CA activating mutations or PTEN loss of expression showed clinical benefits from everolimus monotherapy.

Therapeutic implications
i.This patient falls under the "Classical Molecular Subtype" of GBM, hence making him a poor prognostic candidate ii.Due to EGFR gene amplification, this patient would be benefited from Afatinib, Cetuximab, Erlotinib, Gefitinib, and radiation iii.The loss of PTEN genes favors the use of mTOR inhibitors such as temsirolimus and everolimus.

Case 4
A 45-year-old female who presented with right frontal GBM, underwent surgery for the same and was undergoing RT along with CT (TMZ).

Clinical questions
1. What is the patient-specific tumor biology?Is it possible to know the potential therapy options by doing molecular testing of the tumor?
Molecular oncology approach 1. Somatic mutation 48 gene panel testing was carried out.

Result interpretation
The genomic alterations picked up are reported in Table 2.
p.Gln96* and p.Pro102Leu variations in the VHL gene were identified.p.Gln96* leads to the change of glutamine to a stop codon which truncates the protein, hence affecting the protein production and function; and p.Pro102Leu leads to a change in amino acid from proline to leucine, the effect still being unknown.
Mutations in VHL or loss of expression are most prominently found to be associated with glial tumors, hemangioblastomas, and renal cell carcinoma. [14]The identified mutations in the VHL gene have been previously reported mainly in sporadic renal cell cancer and hemangioblastomas.
The identified missense mutation at p.Asp236Asn in the PTEN gene has not been previously reported, making it a novel mutation.Hence, it is not known whether it would result in loss of function.The effect of the identified mutation in PTEN gene on targeted therapy cannot be conclusively ascertained since it is a novel missense mutation.
p.Ala603Val & p.Thr726Ile identified in RB1 gene have also not been reported before, hence making them novel mutations.However, p.Met704Val variant identified in the RB1 gene has been reported previously in OVCAR-3 cell lines. [15]Although mutations in RB1 are well known in cancer, no specific therapeutic relevance has been found for RB1 mutations in gliomas.Hence, the significance of these variants with regard to therapy or prognosis cannot be ascertained.

Therapeutic implications
i.This patient falls under the "Classical Molecular Subtype" of GBM, hence making him a poor prognostic candidate ii.The mutation observed in PTEN genes favors the use of mTOR inhibitors such as temsirolimus and everolimus, and the patient maybe benefited from bevacizumab due to variations in the VHL gene.

Clinical presentation
A 7-year-old young male with anaplastic astrocytoma, Grade III.

Clinical questions
1. What is the patient specific tumor biology?Is it possible to know the possible therapy options by doing a molecular testing of the tumor?
Molecular oncology approach 1. Somatic mutation 48 gene panel testing was carried out.

Results and interpretation
The genomic alterations picked up are reported in Table 2.
The identified mutation (p.Ser768_Asp770dup) in the EGFR gene represents duplication of 9 bases.EGFR variations are associated significantly associated with reduced survival in anaplastic astrocytoma. [16]Primary GBM tumors with simultaneous alterations in TP53 are associated with worse prognosis.
The observed TP53 mutation (p.Arg248Gln) has been reported in many tissues of the glioma. [21]It lies in the L3 domain of p53 that is involved in binding with 53BP2 protein which consists of evolutionary conserved regions that are frequently mutated in cancer The variation was reported to be dominant negative variation with a gain of function activity in human lung cancer NCI-H1299 cells and enhanced in vitroinvasiveness. [22]riations in EGFR are common in Grade IV glioblastoma than in anaplastic astrocytoma, Grade III.It is speculated that anaplastic astrocytoma with variations in EGFR gene represents undersampled GBM.It is recommended that anaplastic astrocytoma with variation in EGFR be treated like GBM, even though the histopathology criteria for GBM are not met. [9]erapeutic implications i. Anaplastic astrocytoma patient with EGFR variation should be treated as a GBM patient, hence, subcategorizing this patient into "Classical or Proneural Molecular Subtype" based on the molecular information ii.Since, this patient harbors both EGFR and TP53 variations, prognosis is worse but benefit from CT such as afatinib, cetuximab, erlotinib, gefitinib, and dacomitinib due to EGFR mutations is known.

Case 6
A 60-year-old male with progressive gliosarcoma Grade IV, Ki-67 is 30%.He underwent surgery which was followed by CT and radiation therapy assisted with avastin which was given for six cycles.However, it was noted that the patient was showing progression on this treatment.He has no family history for cancers, a non-smoker who follows a vegetarian diet.It was found that he strongly expresses GFAP with interspersed perivesicular GFAP negative sarcomatous foci.

Clinical questions
1. What is the patient-specific tumor biology?Can we predict the treatment response?2. What if the disease progressed while on treatment?What else can be done?
Molecular oncology approach 1. Somatic mutation 48 gene panel testing was carried out.

Result and interpretation
The molecular analysis of 48 somatic genes identified no mutation.

Therapeutic implications
i. Since, this patient has IDH1/2 wildtype, TP53 wildtype with no mutation in the EGFR gene, he belongs to "Classical or Neural Molecular subtype" of GBM ii.No mutation picked up by this panel, hence one cannot comment on the choice of targeted treatment for this patient.

Case 7
A 54-year-old female with high-grade GBM status postsurgery followed by CT and RT with adjuvant TMZ.MGMT methylation status is positive.

Clinical questions
1. Will the analyses of the tumor help in deciding the course of treatment?
Molecular oncology approach 1. Somatic mutation 315 genes panel testing was carried out.

Result interpretation
The genomic alterations picked up are reported in Table 2.
EGFR amplification was found to be strongly correlating with EGFR protein expression in GBM.Mutation of the EGFR gene known as EGFRvIII is reported in about 4-46% of GBM cases and results from a gene rearrangement that deletes exons 2-7. [16]This alteration causes an in-frame deletion of 801 base pair encoding part of the extracellular binding protein ligand which results in activation of EGFR as well as tumorigenesis.EGFRvII is reported in ~15% of GBM patients and results from a gene arrangement that deletes exons 14 and 15, and consequently removes a part of the extracellular domain, which is been identified in glioblastoma rarely when compared to EGFR vIII, but is said to be oncogenic.The EGFR D46N mutation observed, however, has not been characterized and its effect on function remains unclear but has been reported in the context of cancer, which may indicate a biological reference.
PTEN alterations that disrupt the N-terminal PIP2 binding motif, the phosphatise domain (amino acids 14-185), C2 domain (amino acids 190-350), and/or C-terminal region observed, here, are predicted to cause loss of function.PTEN alterations are been reported in 31% of GBM in TCGA database including homozygous deletion in 8% of samples. [17]ecreased PTEN expression is associated with high-grade GBM tumors.However, loss of PTEN is corelated with significantly worse prognosis in GBM.
In GBM, TCGA dataset, homozygous deletion of CDKN2A/B has been found in 54% of the GBM cases (cBioPortal, Apr 2015).
Somatic mutation of BCORL1 gene has been observed in acute myelogenous leukemia patients, [18] suggesting a role of tumor suppressor in this disease.
Somatic inactivating alterations of SET domain containing 2 are documented at a low frequency in a number of solid tumors, most common in renal carcinoma [19] and 6-12% in acute lymphoblastic leukemia.
TERT promoter mutations have been reported in 51-59% of gliomas, [20] most frequently in GBM (54-84%) with poor prognosis.TERT mutation occurs with EGFR amplification on GBM and is associated with poor prognosis.

Therapeutic implications
i.This patient falls under the "Classical Molecular Subtype" of GBM ii.Based on the molecular information from the patient's tumor, it is therefore concluded that this patient can benefit from afatinib, cetuximab, erlotinib, gefitinib, lapatinib, panitumumab due to an EGFR alteration and everolimus, temsirolimus due to alteration in the PTEN gene.

Discussion
In this upcoming era of molecular oncology, there is a paradigm shift from conventional histopathology to molecular subtyping.Prognosis and prediction of treatment response based on the clinical criteria and available single gene testing is not that precise.Beyond bevacizumab, options of targeted therapy in GBMs are limited.Hence, understanding the molecular basis may help in prognosticating and opening up more treatment options including use of other targeted therapies in an adjuvant set up.
Current clinical practice is limited with single gene testing like MGMT methylation, IDH mutations, 1p/19q co-deletion.With advent of advanced testing methods, like NGS, comprehensive large-scale gene information is readily available, which may give a better insight of the tumor biology.Few molecular signatures such as IDH1/2 mutation may aid to distinguish between true progression versus pseudoprogression.Further, these signatures may serve as follow-up marker especially in conjunct with newer technologies like liquid biopsy for early recurrence and treatment response evaluation.
The guidelines as of now do not recommend the use of multigene panel testing for treatment decision-making.Further, different panels of genes are offered and this varies from laboratory to laboratory.The choice of ordering the multi-gene panel is at the discretion of the treating physician.Currently, there is no standardized testing protocol and panel of genes to be analysed.Therefore, the implications of these alteration(s) in treatment needs more understanding and further validation.

Table 2 : Summary of gene mutations identified Case number Histology Gene(s) involved Genomic loci cDNA alteration Protein change Likely molecular subtype ++ Actionable mutation Targeted therapy available
a, b, c, d

Table 3 : Gene Information
EGFR gene is located on chromosome 7p12 region and the protein encoded by this gene is a member of the tyrosine kinase superfamily.The binding of growth factors ligands results in the activation of multiple downstream pathways controlling proliferation and survival (Siegelin and Borczuk, 2013) called tumor protein 53or p53 is located in the nucleus of cells throughout the body where it attaches directly to the DNA.When DNA is damaged, p53 activates other genes to fix the damage.Gene location -Chromosome 10 PIK3CA p.His1048ArgThe PIK3CA gene provided instructions for making the p110 α protein, which is a subunit of an enzyme PI3K.It is a catalytic subunit which helps in phosphorylation.Studies suggest that PI3K signaling may be involved in regulation of several hormones and play a role in the maturation of fat cells (adipocytes) SETD2Splice site 5278-2A>T SETD2 locayed on chromosome 3 is a tumor supressor gene.It is a histone methyltransferase that is specific for lysine-36 of histone H3, and methylation of this residue is associated with active chromatin and has been found to been associated with hyperphosphorylated RNA polymerase II TERT TERT Promoter 146C>T Telomerase reverse transcriptase is a catalytic subunit of enzyme telomerase, which is responsible for the lengthening of the DNA strands.It is normally repressed in somatic cells, What if the disease progressed while on treatment?What else can be done?