**9. Predictive and prognostic molecular markers: Clinical implications**

group (30.6 months) and the RT-> PCV group (40.3 months) at a median follow-up of 60 months. On stratification of patients based upon co-deletion status, a discernible survival benefit was once again found, with no median OS reached for patients possessing the codeletion (irrespective of RT-> PCV vs. RT alone) vs. median OS of 25.2 and 21.4 months for RT- > PCV and RT alone, respectively, in patients with partial or no deletion. Updated results from 2012, reflecting a median follow up of close to 12 years, went further to demonstrate that receipt of adjuvant chemotherapy in patients possessing the co-deletion conferred an additional survival benefit, the median OS for the subgroup of 42 patients with co-deleted tumors and receiving RT-> PCV having not been reached vs. median OS for the subgroup of 38 patients with co-deleted tumors receiving RT alone being 9.3 years. With respect to patients without the co-deletion, this study's results were in accord with the RTOG trial, with a median OS of 25 months for the RT-> PCV subgroup vs. median OS of 21 months for the RT alone subgroup.

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15

The third trial, NOA-04, by the German Neuro-Oncology Group, randomized 318 patients with anaplastic astrocytoma, anaplastic oligodendroglioma, and mixed anaplastic oligoastro‐ cytoma into three single modality treatment groups--RT, PCV, or TMZ--by the ratio of 2:1:1, respectively. This study had crossover design built into it, with patients experiencing unac‐ ceptable toxicity or progression from RT being further randomized to receive either PCV or TMZ, or patients experiencing unacceptable toxicity or progression from chemotherapy further randomized to RT. In this study, FISH analysis revealed 74 patients (23%) to possess 1p/19q co-deletion. The first analysis of results was undertaken at 54 months of follow-up at which time 43% of patients had met the criteria for treatment failure, the primary endpoint of the study. This analysis revealed that across all groups there was similar PFS and similar OS. As with the RTOG and EORTC studies discussed above, the 1p/19q co-deletion was found to bode well for prognosis, conferring a risk reduction of nearly 50% irrespective of treatment

Thusly, the molecular signature of 1p/19q codeletion has a predictive and prognostic utility as it shows increased overall survival in patients who receive alkylating chemotherapy, radio‐ therapy or both as compared to those who do not possess it. However, this benefit was not demonstratedin the group comprised of anaplastic astrocytoma which included 53% of the patient population in this study. There was no statistical difference in outcome, time to progression, nor overall survival whether patients were first treated with RT or alkylating chemotherapy. However, given the worse overall prognosis in patients with anaplastic astrocytoma the generally accepted approach to therapy is combined adjuvant chemotherapyradiation. This approach is extrapolated from data obtained in trials including patients with

MGMT is a DNA repair protein that canonically exemplifies the concept of chemotherapeutic resistance. It does so by removing the alkylation of the O6 position of Guanine, which represents the seminal mechanism of action of alkylating chemotherapeutic agents. The beneficial epigenetic profile associated with MGMT is methylation of the MGMT promoter, which silences the MGMT gene and thereby reduces the repair of chemotherapy induced alkylation of DNA. This molecular marker was first suggested to have predictive utility by Stupp *et al* in 2005 [21] in a post-hoc analysis of 203 (out of 573 patients treated in that study)

arm.

glioblastoma.

Before embarking further on the discussion of molecular profiling of malignant gliomas, it is imperative to make the distinction that, on the face of it, appears but a matter of simple semantics. Within the context of this discussion, there exists a fundamental difference between Prognostic and Predictive. Prognostic connotes the effect a certain marker gives an outcome that is independent of the therapeutic intervention(s) employed. On the other hand, when a marker is said to have predictive utility, this indicates that it foretells benefit specifically from one type of treatment over others [17].

This discussion will first consider 1p/19q codeletion, a molecular profile that comes from unbalanced translocation between chromosomes 1p and 19q, leading to net loss of genetic material which ultimately leads to loss of one hybrid chromosome, and ultimately, loss of heterozygosity [17]. Mutations have been found in genes that correspond to this translocation; however, the biological underpinnings of these mutations have yet to be understood. The 1p/ 19q Codeletion has been reliably found in oligodendroglial tumor subtypes that are inter‐ spersed between the WHO Grades [17]. Three randomized clinical trials (RTOG 9402, EORTC 26951, NOA-04) have shown that anaplastic oligodendroglioma patients with 1p/19q codele‐ tions have a survival benefit over those without the codeletion when receiving radiation therapy (RT), alkylating chemotherapy, or both [18-20].

First, the RTOG 9402 trial randomly assigned 289 patients with anaplastic oligodendroglioma or anaplastic oligoastrocytoma to receive either adjuvant RT alone or four cycles of Procarba‐ zine/CCNU/Vincristine (PCV) chemotherapy followed by RT (PCV-> RT). Of 201 patients tested by FISH, 93 (46%) were found to have the codeletion. The first initial analysis of results at 3 years out found a median progression free survival (PFS) of 1.7 years for the RT alone contingent vs. 2.6 years for the PCV-> RT contingent; however, no overall survival benefit of either study arm was found at this time, with a median overall survival (OS) of 4.9 years in the PCV-> RT group vs. 4.7 years with RT alone. The 1p/19q codeletion was found to confer an overall survival benefit, with a median OS of > 7 years vs. 2.8 years in those patients without the codeletion. The type of treatment (RT vs. PCV-> RT) was not found to have statistically significant bearing on this increase in OS in patients with tumors possessing the codeletion at this interim analysis of study results. Extended follow up of patients in 2012 continued to show an OS benefit of the 1p/19q codeletion but also found, interestingly, that PCV-> RT did improve survival. At the 2012 reevaluation, those patients without the codeletion had median OS of 2.6 years for PCV-> RT vs. 2.7 years for RT alone, corresponding to earlier results. Those patients with the codeletion who received PCV-> RT were found to have a median OS of 14.7 years vs. 7.3 years with RT alone, yielding a hazard ratio of 0.59 (95% CI, 0.37-0.95; P=0.03).

Very analogous to the RTOG trial was the EORTC 26951 trial, which randomized 368 patients with anaplastic oligodendroglioma or anaplastic oligoastrocytoma to receiving either RT or RT followed by six cycles of PCV (RT-> PCV). In this study, there were 78 patients (21%) whose tumors were found to have the 1p/19q codeletion by FISH. When taken as a whole, the results were similar to the RTOG study, finding that addition of PCV to RT increased PFS to 23 months vs. 13.2 months but not finding a statistically significant difference in OS between the RT alone group (30.6 months) and the RT-> PCV group (40.3 months) at a median follow-up of 60 months. On stratification of patients based upon co-deletion status, a discernible survival benefit was once again found, with no median OS reached for patients possessing the codeletion (irrespective of RT-> PCV vs. RT alone) vs. median OS of 25.2 and 21.4 months for RT- > PCV and RT alone, respectively, in patients with partial or no deletion. Updated results from 2012, reflecting a median follow up of close to 12 years, went further to demonstrate that receipt of adjuvant chemotherapy in patients possessing the co-deletion conferred an additional survival benefit, the median OS for the subgroup of 42 patients with co-deleted tumors and receiving RT-> PCV having not been reached vs. median OS for the subgroup of 38 patients with co-deleted tumors receiving RT alone being 9.3 years. With respect to patients without the co-deletion, this study's results were in accord with the RTOG trial, with a median OS of 25 months for the RT-> PCV subgroup vs. median OS of 21 months for the RT alone subgroup.

**9. Predictive and prognostic molecular markers: Clinical implications**

one type of treatment over others [17].

14 Tumors of the Central Nervous System – Primary and Secondary

therapy (RT), alkylating chemotherapy, or both [18-20].

Before embarking further on the discussion of molecular profiling of malignant gliomas, it is imperative to make the distinction that, on the face of it, appears but a matter of simple semantics. Within the context of this discussion, there exists a fundamental difference between Prognostic and Predictive. Prognostic connotes the effect a certain marker gives an outcome that is independent of the therapeutic intervention(s) employed. On the other hand, when a marker is said to have predictive utility, this indicates that it foretells benefit specifically from

This discussion will first consider 1p/19q codeletion, a molecular profile that comes from unbalanced translocation between chromosomes 1p and 19q, leading to net loss of genetic material which ultimately leads to loss of one hybrid chromosome, and ultimately, loss of heterozygosity [17]. Mutations have been found in genes that correspond to this translocation; however, the biological underpinnings of these mutations have yet to be understood. The 1p/ 19q Codeletion has been reliably found in oligodendroglial tumor subtypes that are inter‐ spersed between the WHO Grades [17]. Three randomized clinical trials (RTOG 9402, EORTC 26951, NOA-04) have shown that anaplastic oligodendroglioma patients with 1p/19q codele‐ tions have a survival benefit over those without the codeletion when receiving radiation

First, the RTOG 9402 trial randomly assigned 289 patients with anaplastic oligodendroglioma or anaplastic oligoastrocytoma to receive either adjuvant RT alone or four cycles of Procarba‐ zine/CCNU/Vincristine (PCV) chemotherapy followed by RT (PCV-> RT). Of 201 patients tested by FISH, 93 (46%) were found to have the codeletion. The first initial analysis of results at 3 years out found a median progression free survival (PFS) of 1.7 years for the RT alone contingent vs. 2.6 years for the PCV-> RT contingent; however, no overall survival benefit of either study arm was found at this time, with a median overall survival (OS) of 4.9 years in the PCV-> RT group vs. 4.7 years with RT alone. The 1p/19q codeletion was found to confer an overall survival benefit, with a median OS of > 7 years vs. 2.8 years in those patients without the codeletion. The type of treatment (RT vs. PCV-> RT) was not found to have statistically significant bearing on this increase in OS in patients with tumors possessing the codeletion at this interim analysis of study results. Extended follow up of patients in 2012 continued to show an OS benefit of the 1p/19q codeletion but also found, interestingly, that PCV-> RT did improve survival. At the 2012 reevaluation, those patients without the codeletion had median OS of 2.6 years for PCV-> RT vs. 2.7 years for RT alone, corresponding to earlier results. Those patients with the codeletion who received PCV-> RT were found to have a median OS of 14.7 years vs.

7.3 years with RT alone, yielding a hazard ratio of 0.59 (95% CI, 0.37-0.95; P=0.03).

Very analogous to the RTOG trial was the EORTC 26951 trial, which randomized 368 patients with anaplastic oligodendroglioma or anaplastic oligoastrocytoma to receiving either RT or RT followed by six cycles of PCV (RT-> PCV). In this study, there were 78 patients (21%) whose tumors were found to have the 1p/19q codeletion by FISH. When taken as a whole, the results were similar to the RTOG study, finding that addition of PCV to RT increased PFS to 23 months vs. 13.2 months but not finding a statistically significant difference in OS between the RT alone

The third trial, NOA-04, by the German Neuro-Oncology Group, randomized 318 patients with anaplastic astrocytoma, anaplastic oligodendroglioma, and mixed anaplastic oligoastro‐ cytoma into three single modality treatment groups--RT, PCV, or TMZ--by the ratio of 2:1:1, respectively. This study had crossover design built into it, with patients experiencing unac‐ ceptable toxicity or progression from RT being further randomized to receive either PCV or TMZ, or patients experiencing unacceptable toxicity or progression from chemotherapy further randomized to RT. In this study, FISH analysis revealed 74 patients (23%) to possess 1p/19q co-deletion. The first analysis of results was undertaken at 54 months of follow-up at which time 43% of patients had met the criteria for treatment failure, the primary endpoint of the study. This analysis revealed that across all groups there was similar PFS and similar OS. As with the RTOG and EORTC studies discussed above, the 1p/19q co-deletion was found to bode well for prognosis, conferring a risk reduction of nearly 50% irrespective of treatment arm.

Thusly, the molecular signature of 1p/19q codeletion has a predictive and prognostic utility as it shows increased overall survival in patients who receive alkylating chemotherapy, radio‐ therapy or both as compared to those who do not possess it. However, this benefit was not demonstratedin the group comprised of anaplastic astrocytoma which included 53% of the patient population in this study. There was no statistical difference in outcome, time to progression, nor overall survival whether patients were first treated with RT or alkylating chemotherapy. However, given the worse overall prognosis in patients with anaplastic astrocytoma the generally accepted approach to therapy is combined adjuvant chemotherapyradiation. This approach is extrapolated from data obtained in trials including patients with glioblastoma.

MGMT is a DNA repair protein that canonically exemplifies the concept of chemotherapeutic resistance. It does so by removing the alkylation of the O6 position of Guanine, which represents the seminal mechanism of action of alkylating chemotherapeutic agents. The beneficial epigenetic profile associated with MGMT is methylation of the MGMT promoter, which silences the MGMT gene and thereby reduces the repair of chemotherapy induced alkylation of DNA. This molecular marker was first suggested to have predictive utility by Stupp *et al* in 2005 [21] in a post-hoc analysis of 203 (out of 573 patients treated in that study) assessable tumors. In that analysis, MGMT methylation status had significant bearing on progression free survival (PFS) in patients from the experimental arm receiving TMZ in addition to RT whereas it showed minimal benefit in PFS in those patients from the control arm receiving RT alone. It was then shown to have prognostic, but not predictive, utility by the results of the RTOG 0525 study, which showed an overall survival benefit of 23.2 months in patients with MGMT methylated tumors vs. 16 months in patients with unmethylated tumors irrespective of whether in the experimental arm (3 weeks on-one week off adjuvant dose-intensified TMZ) or the control arm (standard TMZ) [22]. Based on these results, MGMT methylation status is only accepted as a prognostic factor without predictive value in the population studied. It is also very intriguing to note herein the results of studies aimed at studying single vs. combined modality treatments in elderly patients greater than 70 years of age in whom combined modality treatments are less tolerable and perhaps less effective. These studies showed that in this subset of patients, MGMT methylation status has a predictive utility. Patients with MGMT methylation had longer PFS when receiving chemotherapy plus RT or chemotherapy alone as opposed to RT alone whereas patients without MGMT methyl‐ ation accrued no comparable survival benefit from chemotherapy [17]. This supposition--that MGMT status is useful as a predictive tool in stratifying elderly patients to receipt of either chemo or RT--was further corroborated by two trials--the NOA-08 trial and the Nordic Trial. The NOA-08 trial, in brief, set out to prove non-inferiority of TMZ alone (one week on one week off) with RT alone in patients 66 years of age and older. While there was no OS or PFS difference between the two arms, it was noteworthy that patients with MGMT methylation showed PFS of 8.4 months vs. PFS of 4.6 in the TMZ arm whereas in the RT arm, MGMT methylation status had the opposite effect, conferring a PFS of 4.6 months in those without MGMT methylation vs. 3.3 months in those with [23]. It is important to note that the temozo‐ lomide regimen used in this study varies significantly from the standard schedule used today for patients with glioblastoma. In the Nordic Trial, patients were randomized into one of three groups--standard RT (60 Gy) vs. hypofractionated RT (34 Gy over 2 weeks) vs. standard TMZ schedule. Between the three groups, OS was found to be inferior in the standard RT group when compared to TMZ and hypo fractionated RT. However, MGMT methylation did show better OS in TMZ-treated patients whereas no such benefit was found in RT treated patients [24]. In addition, a recently published meta-analysis performed by Yin et al [25], also supports the predictive value of MGMT methylation status in the elderly population. These results make it reasonable that MGMT status be brought to bear when considering single modality treatment with RT or TMZ in elderly glioblastoma patients.

acquire their tumorigenesis independently of IDH-mutating pathways. IDH-mutant gliomas are inclusive of most Grade II and Grade III Gliomas along with a few Secondary GBMs. It is exceedingly interesting to note that IDH mutants tend to carry a better prognosis than IDHwild-type gliomas of the same histological grade (e.g. Secondary GBM carries a better prognosis than Primary GBM). Indeed, an insightful pooled analysis of 382 WHO Grade III and IV gliomas by Hartmann et al in 2010 corroborated this--that IDH status bears more valuable prognostic information than simple histological grade [27]. So revelatory has the delineation of gliomas been based on IDH status that, despite unequivocal similarities in histological grade and morphology between tumors that would otherwise have classified them together, it is now considered insufficient that they be grouped together if one were to be IDH-

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**10. Angiogenesis: Vascular Endothelial Growth Factor (VEGF) and**

As alluded to earlier in the chapter, angiogenesis is an essential part of the pathogenesis of malignant gliomas which, as a rule, are among the most vascularized of tumors [40, 41]. This is due to: (1) upregulation of genes encoding proangiogenic factors, which include VEGF, fibroblast growth factor (FGF), IL-8 and-6, hypoxia-inducible factor 1 alpha (HIF-1alpha) and angiopoietins, and (2) downregulation of angiogenesis inhibitors, including thrombospodins, angiostatin, endostatin and interferons. Indeed, the lynchpin that begets the transformation from low grade to high grade gliomas is induction of expression of the above-cited proangio‐ genic factors [40]. The most prominent and well-characterized of the proangiogenic factors is VEGF-A, commonly denoted simply as VEGF, which is directly secreted by tumor cells. VEGF exerts its function by binding the receptor VEGFR2 on endothelial cells nearby the tumor. This action initiates a paracrine signaling loop that results in the proliferation of endothelial cells and, as a result, neo-vasculature. Interestingly, the level of VEGF produced by a tumor is proportional to the degree of malignancy, the aggressiveness and poor outcome; high-grade tumors are found to have orders of magnitude more VEGF than low grade tumors [13, 42]. There has thusly been a significant amount of clinical research focus on anti-angiogenic therapy for malignant gliomas. Anti-angiogenic therapy has multiple hypothesized mechanisms of action for the treatment of malignant gliomas. The primary mechanism of action is direct cytotoxicity to endothelial cells, inducing apoptosis. This, by merit of the resultant attenuated blood supply, decreases oxygen and nutrient delivery to tumor cells which preempts further growth for a short period of time. A second hypothesized mechanism of action based upon the results of select clinical studies is that, when used alongside cytotoxic chemotherapeutic agents, anti-VEGF agents are thought to synergistically sensitize endothelial cells to penetra‐ tion by these cytotoxic agents. Intriguingly, it is also very likely that anti-VEGF agents work to counteract an upsurge of VEGF expression and endothelial cell recruitment observed with the tumoral insult caused by chemotherapy and radiation. Another hypothesis is that, during a discrete window of time after administration, anti-VEGF agents elicit a phenomenon known as "vascular normalization" during which there is reduced vessel diameter/permeability,

wild-type and the other IDH mutant.

**Glioblastoma stem-like cells (GSC)**

A third example of genetic/molecular markers that has proven prognostic is the genes IDH1 and IDH2 which encode the ubiquitous metabolic enzyme Isocitrate Dehydrogenase in the cytoplasm and mitochondria, respectively. The native function of wild-type IDH protein is to produce alpha-ketoglutarate. IDH Mutants catalyze a reaction that converts the native metabolic intermediary alpha-ketoglutarate into D-2-hydroxyglutarate, an onco-metabolite (this can be reliably measured by magnetic spectroscopy in situ and mediates the oncogenic activity of the provoking IDH mutations [26]. The classification of gliomas based on IDH is to categorize them based on IDH-wild-type vs. IDH-mutant gliomas. IDH-wild-type tumors are inclusive of Grade I Pilocytic Astrocytomas and Primary Glioblastomas; these, of course, acquire their tumorigenesis independently of IDH-mutating pathways. IDH-mutant gliomas are inclusive of most Grade II and Grade III Gliomas along with a few Secondary GBMs. It is exceedingly interesting to note that IDH mutants tend to carry a better prognosis than IDHwild-type gliomas of the same histological grade (e.g. Secondary GBM carries a better prognosis than Primary GBM). Indeed, an insightful pooled analysis of 382 WHO Grade III and IV gliomas by Hartmann et al in 2010 corroborated this--that IDH status bears more valuable prognostic information than simple histological grade [27]. So revelatory has the delineation of gliomas been based on IDH status that, despite unequivocal similarities in histological grade and morphology between tumors that would otherwise have classified them together, it is now considered insufficient that they be grouped together if one were to be IDHwild-type and the other IDH mutant.

assessable tumors. In that analysis, MGMT methylation status had significant bearing on progression free survival (PFS) in patients from the experimental arm receiving TMZ in addition to RT whereas it showed minimal benefit in PFS in those patients from the control arm receiving RT alone. It was then shown to have prognostic, but not predictive, utility by the results of the RTOG 0525 study, which showed an overall survival benefit of 23.2 months in patients with MGMT methylated tumors vs. 16 months in patients with unmethylated tumors irrespective of whether in the experimental arm (3 weeks on-one week off adjuvant dose-intensified TMZ) or the control arm (standard TMZ) [22]. Based on these results, MGMT methylation status is only accepted as a prognostic factor without predictive value in the population studied. It is also very intriguing to note herein the results of studies aimed at studying single vs. combined modality treatments in elderly patients greater than 70 years of age in whom combined modality treatments are less tolerable and perhaps less effective. These studies showed that in this subset of patients, MGMT methylation status has a predictive utility. Patients with MGMT methylation had longer PFS when receiving chemotherapy plus RT or chemotherapy alone as opposed to RT alone whereas patients without MGMT methyl‐ ation accrued no comparable survival benefit from chemotherapy [17]. This supposition--that MGMT status is useful as a predictive tool in stratifying elderly patients to receipt of either chemo or RT--was further corroborated by two trials--the NOA-08 trial and the Nordic Trial. The NOA-08 trial, in brief, set out to prove non-inferiority of TMZ alone (one week on one week off) with RT alone in patients 66 years of age and older. While there was no OS or PFS difference between the two arms, it was noteworthy that patients with MGMT methylation showed PFS of 8.4 months vs. PFS of 4.6 in the TMZ arm whereas in the RT arm, MGMT methylation status had the opposite effect, conferring a PFS of 4.6 months in those without MGMT methylation vs. 3.3 months in those with [23]. It is important to note that the temozo‐ lomide regimen used in this study varies significantly from the standard schedule used today for patients with glioblastoma. In the Nordic Trial, patients were randomized into one of three groups--standard RT (60 Gy) vs. hypofractionated RT (34 Gy over 2 weeks) vs. standard TMZ schedule. Between the three groups, OS was found to be inferior in the standard RT group when compared to TMZ and hypo fractionated RT. However, MGMT methylation did show better OS in TMZ-treated patients whereas no such benefit was found in RT treated patients [24]. In addition, a recently published meta-analysis performed by Yin et al [25], also supports the predictive value of MGMT methylation status in the elderly population. These results make it reasonable that MGMT status be brought to bear when considering single modality treatment

A third example of genetic/molecular markers that has proven prognostic is the genes IDH1 and IDH2 which encode the ubiquitous metabolic enzyme Isocitrate Dehydrogenase in the cytoplasm and mitochondria, respectively. The native function of wild-type IDH protein is to produce alpha-ketoglutarate. IDH Mutants catalyze a reaction that converts the native metabolic intermediary alpha-ketoglutarate into D-2-hydroxyglutarate, an onco-metabolite (this can be reliably measured by magnetic spectroscopy in situ and mediates the oncogenic activity of the provoking IDH mutations [26]. The classification of gliomas based on IDH is to categorize them based on IDH-wild-type vs. IDH-mutant gliomas. IDH-wild-type tumors are inclusive of Grade I Pilocytic Astrocytomas and Primary Glioblastomas; these, of course,

with RT or TMZ in elderly glioblastoma patients.

16 Tumors of the Central Nervous System – Primary and Secondary
