Diffusely infiltrating astrocytomas: pathology, molecular mechanisms and markers.

Acta Neuropathol (2015) 129:789–808 DOI 10.1007/s00401-015-1439-7

REVIEW

Diffusely infiltrating astrocytomas: pathology, molecular mechanisms and markers Koichi Ichimura1 • Yoshitaka Narita2 • Cynthia E. Hawkins3,4

Received: 9 April 2015 / Revised: 26 April 2015 / Accepted: 30 April 2015 / Published online: 15 May 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract Diffusely infiltrating astrocytomas include diffuse astrocytomas WHO grade II and anaplastic astrocytomas WHO grade III and are classified under astrocytic tumours according to the current WHO Classification. Although the patients generally have longer survival as compared to those with glioblastoma, the timing of inevitable malignant progression ultimately determines the prognosis. Recent advances in molecular genetics have uncovered that histopathologically diagnosed astrocytomas may consist of two genetically different groups of tumours. The majority of diffusely infiltrating astrocytomas regardless of WHO grade have concurrent mutations of IDH1 or IDH2, TP53 and ATRX. Among these astrocytomas, no other genetic markers that may distinguish grade II and grade III tumours have been identified. Those astrocytomas without IDH mutation tend to have a distinct genotype and a poor prognosis comparable to that of glioblastomas. On the other hand, diffuse astrocytomas that arise in children do not harbour IDH/TP53 mutations, but instead display mutations of BRAF or structural alterations involving MYB/ MYBL1 or FGFR1. A molecular classification may thus & Koichi Ichimura [email protected] 1

Division of Brain Tumor Translational Research, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-Ku, Tokyo 104-0045, Japan

2

Department of Neuro-Oncology/Neurosurgery, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-Ku, Tokyo 104-0045, Japan

3

Division of Pathology, The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, Toronto, Canada

4

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada

help delineate diffusely infiltrating astrocytomas into distinct pathogenic and prognostic groups, which could aid in determining individualised therapeutic strategies. Keywords IDH1 ATRX BRAF Molecular classification Next-generation sequencing Astrocytoma

Introduction Diffusely infiltrating astrocytomas predominantly arise in young adults. The tumour cells always infiltrate the brain parenchyma, making complete surgical removal practically impossible and leading to tumour recurrence. Malignant progression to astrocytic tumours of higher WHO grade, including glioblastoma (GBM), which is then called secondary glioblastoma, is almost inevitable. The timing of progression virtually determines the outcome for the patients. Thus, adult diffuse infiltrating astrocytomas are ultimately fatal malignant neoplasms regardless of WHO grade. In the current edition of the WHO Classification of Tumours of the Central Nervous System (2007), diffuse astrocytomas WHO grade II (abbreviated as DA) and anaplastic astrocytomas WHO grade III (AA) are classified under the category of astrocytic tumours, along with GBM as well as other paediatric astrocytomas, mainly because they histologically mimic astrocytes [79]. As the molecular pathogenesis of each tumour type is gradually uncovered, largely thanks to the emergence of high-throughput nextgeneration sequencing technologies during the past several years, it has become evident that many of the tumours that have been categorised under the umbrella name of ‘astrocytic tumours’ may develop through very distinct pathogenic mechanisms.

123

790

In this respect, the year 2008 should be hailed as one of the most significant milestones in glioma research, when isocitrate dehydrogenase 1 (IDH1) mutations were discovered for the first time in gliomas (the very first IDH1 mutation in human cancer was reported in a colon cancer in 2006 [132]). They were identified in a subset of glioblastomas through the first ever whole-exome sequencing of gliomas, notably by means of laborious conventional exonby-exon Sanger sequencing [103]. A subsequent flood of studies unanimously showed, however, that IDH1 mutations are far more common in diffuse astrocytomas and oligodendrogliomas. As a result, mutations of IDH1, together with its mitochondrial homologue IDH2, have become the most important genetic markers to define these tumours. The discovery of IDH1 mutations has indeed changed our way of understanding the development of glioma. Together with the concurrent TP53 mutations, IDH1 mutations are now considered to be the hallmark of adult type diffusely infiltrating astrocytomas. The distinct mechanism of telomere maintenance that enables replicative immortality, one of the hallmarks of cancer [42], is also a key factor defining astrocytoma. ATRX (Alpha Thalassemia/ Mental Retardation Syndrome X-Linked), another gene identified to be mutated in astrocytomas through wholeexome sequencing, plays a fundamental role for this function, as well as being a useful molecular marker to identify astrocytomas. Molecular pathology in the meantime uncovered that paediatric astrocytomas arise through very different mechanisms from their adult counterparts. IDH1 mutations, which characterize adult astrocytomas, are almost never found in histopathologically equivalent paediatric diffuse astrocytomas. Instead, some of them have unique structural variants that are not observed in adult tumours, as will be described. As molecular pathology is re-defining gliomas, the role of histopathology may have to be revisited. The WHO classification is in its revision process and it is anticipated that molecular information will be incorporated as criteria to generate a new ‘integrated diagnosis’ as recommended in the International Society of Neuropathology (ISN)-Haarlem Consensus Guidelines [80]. In this review, we present our current understanding of diffusely infiltrating astrocytomas in adult and paediatric patients from a clinical, histopathological and a genetic point of view. The tumours that will be discussed here essentially include histopathologically defined diffuse astrocytoma WHO grade II (DA) and anaplastic astrocytoma WHO grade III (AA), although other types of astrocytic tumours that arise in children will also be mentioned. DA and AA may be collectively called either ‘‘diffusely infiltrating astrocytomas’’ or simply ‘‘astrocytomas’’. The molecular basis of the proposed integrated diagnosis and its

123

Acta Neuropathol (2015) 129:789–808

potential impact upon the clinical management of these tumours will be discussed below.

Clinical features Here we give a brief overview of the clinical features of adult DA and AA. The statistics presented in this section are cited in part from the CBTRUS Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2007–2011 [100], or from the Report of the Brain Tumor Registry of Japan (2001–2004) 13th Edition [18]. The data from the latter were collected from 2009 through 2012 for the 16,338 primary and metastatic brain tumours newly diagnosed according to the WHO Classification between 2001 and 2004 in Japan (https://www.jstage.jst.go.jp/browse/nmc/ 54/Supplement/_contents). An extensive review for the clinical aspects of astrocytomas can be found elsewhere [94, 122]. Epidemiology The average annual age-adjusted incidence rates of DA and AA based on the standard population in the United States in 2005 are reported to be 0.55 and 0.37 per 100,000/year, respectively [100]. The male to female ratio of DA and AA is 1.28 and 1.26, and the median ages are 37.8–48.0 and 49.3–53.0 years, respectively [18, 100]. The proportion of patients younger than 20 years is 14.7 and 7.2 %, respectively, while that of those in the 60 or more years age group is 12.8 and 33.9 %, respectively [18] (Table 1). Known risk factors include exposure to ionising radiation, male sex, increasing age and non-Hispanic white ethnicity [13, 14]. Cigarette smoking and alcohol drinking were not reported to be associated with an increased risk of glioma [14]. Symptoms and location The most common symptom of DAs at onset is seizure (48 %), followed by subjective symptoms such as headache (25 %), and focal symptoms such as hemiparesis and sensory disturbance (25 %). Those of AAs are focal symptoms (44 %), seizure (28 %), subjective symptoms (20 %) and consciousness disturbance (9 %) [18] (Table 1). It has been reported that 3 % of DAs and 2 % of AAs were accidentally found prior to the manifestation of clinical symptoms [18]. The prevalence rate of patients with a Karnofsky performance status (KPS) score C90 is 65 % in DA and 39 % in AA, and it drops to 13 and 33 %, respectively, for KPS B 70. The great majority of DA (95 %) and AA (92 %) occur as a single lesion in the


791

Table 1 Clinical features of diffusely inflitrating astroctyomas Diffuse astrocytoma

Anaplastic astrocytoma

Incidence per 100,000/year

0.55

0.37

Age median

38–48

49–53

\20 years

15 %

7%

C60 years

13 %

34 %

Seizures

48 %

25 %

Subjective symptoms (e.g., headache)

25 %

44 %

Focal symptoms (e.g., hemiparesis)

25 %

20 %

Consciousness disturbance

5%

9%

Frontal lobe

41 %

38 %

Temporal lobe

17 %

21 %

Parietal lobe

7%

10 %

Occipital lobe Insular gyri

2% 6%

3% 3%

Thalamus and hypothalamus

7%

8%

Cerebellum

4%

4%

&10 %

&29–44 %

Median OS

Not reached

38.0 months

5-year OS

47.1–75.0 %

25.9–41.1 %

Clinical symptoms

Main tumour lesion

Radiological findings Enhancing tumours Overall survival

The statistics are in part from the CBTRUS Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2007–2011 [91] or from the Report of the Brain Tumor Registry of Japan (2001–2004) 13th Edition [15] OS overall survival

cerebral hemisphere, most often in the frontal lobe (DA/ AA = 41 %/38 %) followed by the temporal (17 %/21 %) and parietal (7 %/10 %) lobes (Table 1) [18]. Radiological findings Most DA and AA show hyperintensity on T2/FLAIR magnetic resonance imaging (MRI) (Fig. 1). Astrocytomas of higher WHO grade tend to present as more enhancing and heterogeneous lesions. However, unlike GBM, where 85 % of tumours show gadolinium enhancement on MRI [7], only 10 % of DA [101] and 29–44 % of AA [86, 139] are enhancement-positive. Pallud et al. [101] reported that there was no difference in overall survival (OS) regardless of the presence of contrast enhancement in grade II gliomas at initial presentation. Development of new contrast enhancement on follow-up imaging suggests malignant progression to a higher grade glioma. Representative MRI images are shown in Fig. 1.

Treatment Grade II gliomas are reported to grow up to 3–5 mm per year [102, 110]. A Norwegian study suggested that an early surgical tumour removal increases the survival rate more than biopsy and watchful observation [56]. A gross total surgical tumour resection prolonged both OS and progression-free survival (PFS) in patients with grade II gliomas [133]. Smith et al. [133] reported that the OS and PFS were correlated with the preoperative tumour volume, postoperative residual tumour volume and extent of resection by multivariate analysis in grade II gliomas. Nonetheless, gross total resection alone cannot cure grade II gliomas. More than half of the patients with grade II gliomas were reported to have a recurrence in the prospective RTOG9802 trial during the 4.4 years of median follow-up after a neurosurgeon-determined gross total resection and observation [128]. One of the most highly debated issues in the treatment of DA is the optimal timing of radiotherapy (RT). The EORTC22845 study revealed that early postoperative RT significantly prolonged the PFS and improved the seizure control rates after 1 year compared with delaying RT until progression. However, the timing of RT had no effect on OS in grade II gliomas [141]. Thus, considering that early RT might cause cognitive decline, the ‘‘watchful waiting’’ approach is a treatment strategy option after surgery in which RT is performed at recurrence while patients are carefully followed [141]. Focal radiation doses ranging from 45 to 64.8 Gy had equivalent PFS and OS [17, 62]. Although chemotherapy is not a standard of care for grade II gliomas, it has recently been reported that it may be effective. RT ? PCV (procarbazine, lomustine and vincristine) therapy (RTOG9802) prolonged the PFS [129] and OS [19] of unfavourable-risk grade II glioma patients after long-term follow-up. RT ? temozolomide (TMZ) (EORTC 22033) improved the 3-year OS rate of this category of patients when compared with a historical control [35]. However, it has to be noted that a subset of astrocytomas treated with TMZ may acquire mutations in the DNA mismatch repair genes and develop a hypermutated phenotype at malignant progression [60]. Thus the efficacy and timing of post-surgical adjuvant therapy remain to be determined [93]. Gross total removal is associated with a favourable prognosis for grade III gliomas including AA [94]. While RT ? PCV is a standard of care for grade III oligodendroglial tumours [21, 142], RT-PCV showed no benefit over RT alone to the survival of AA patients in a randomised trial [89]. No significant difference in survival between newly diagnosed AA patients treated with RT ? PCV or RT ? TMZ was observed either in a

123

792


Fig. 1 Grade II/III astrocytomas often appear as a hyperintense lesion on T2/FLAIR magnetic resonance imaging (MRI), which are not enhanced with a contrast media (Gd-DTPA). Tumours are diffusely infiltrating in the right temporal lobe without a clear margin with the surrounding healthy brain tissues. a 31 y/o female, diffuse astrocytoma WHO grade II, IDH1 R132H (?). b 37 y/o female,

diffuse astrocytoma WHO grade II, IDH1/2 wild-type. c 36 y/o female, anaplastic astrocytoma WHO grade III, IDH1 R132H (?). d 41 y/o male, anaplastic astrocytoma WHO grade III, IDH1/2 wildtype. Note that MRI images across astrocytomas of different WHO grades and the IDH1/2 status are remarkably similar

retrospective study [15] or in the NOA-04 trial [154]. Despite these observations, RT ? TMZ is often used as a treatment of choice for AA because of the lower toxicity and higher compliance with TMZ compared with PCV [15]. Several retrospective studies assessing the benefit of RT-TMZ over RT alone on the survival of AA patients have provided conflicting results [71, 126, 130]. In the treatment of elderly ([65 years of age) AA patients, TMZ alone was equivalent to RT alone [155]. The benefit of adjuvant TMZ in the treatment of AA patients needs to be further investigated in a molecularly stratified prospective study (see below). Although the preferred choice of therapeutic strategy may thus vary at each centre, recommended treatment for astrocytoma based on the guidelines of EANO (European

Association for Neuro-Oncology) or EFNS (European Federation of Neurological Societies) may be summarised as follows [134, 153]: maximal safe resection should be performed in all grades of gliomas. Delayed RT after watchful waiting may be considered for DA patients with younger age (\40 years), no neurological deficit and gross total resection. Adjuvant therapy such as RT is recommended for DA patients with unfavourable prognostic factors (older age, incomplete or no resection, existing neurological deficit). Chemotherapy for DA is an option for patients with recurrence after surgery and RT, or as initial treatment for large residual tumours. The standard of care for patients with AA recommended by the EANO guideline based on NOA-04 is RT or alkylating chemotherapy alone after surgery [154].

123


Prognosis The reported median OS and the 5-year OS rates are, respectively, ‘‘not reached’’ and 47.1–75.0 % for DA and 38.0 months and 25.9–41.1 % for AA [18, 100]. The median PFS and 5-year PFS rates are, respectively, 84.1 months and 57.0 % for DA and 19.0 months and 28.7 % for AA [94]. The 20-year survival rate of patients with DA is 10–20 % [8, 123] while that of AA is very low. The favourable clinical prognostic factors of astrocytomas include younger age, preoperative high KPS and gross total removal of the tumour. The clinical course of astrocytoma patients largely depends on the timing of malignant progression [26, 55]. One of the problems in prognosticating astrocytomas, whether it is in the context of a clinical trial or a retrospective cohort study, is the presumed biological heterogeneity within the histological entity. As will be discussed below, the presence of IDH1/2 mutation is the most important decisive marker that defines a biologically distinct subset of astrocytomas. A considerable fraction of histologically diagnosed anaplastic astrocytomas are IDH wild type, which may have a significantly poorer prognosis than their IDH1/2-mutated counterparts [44]. The molecular heterogeneity of histologically diagnosed AA could mask the potential effect of chemotherapy in a subset of patients and could hamper the accurate interpretation of the results of clinical trials.

Histopathology Diffuse astrocytomas (WHO grade II) are infiltrating glial tumours that occur throughout the central nervous system. They arise in both children and adults but are most frequent in the cerebral hemispheres of adults. In children they are less common than their low-grade counterpart, pilocytic astrocytoma, and importantly, unlike in adults, diffuse astrocytomas in children rarely progress to high-grade astrocytomas. In a recent population-based study of paediatric low-grade glioma, progression to high-grade astrocytoma was only seen in 2.9 % of cases [88]. BRAF V600E mutations and CDKN2A deletions were significantly enriched in paediatric low-grade gliomas that went on to progress versus those that did not [88]. In contrast, progression of low-grade gliomas harbouring the BRAF-KIAA549 gene fusion was very rare. The reason for the difference in behaviour of low-grade gliomas harbouring a BRAF mutation versus a fusion event is unclear. Grossly, diffuse astrocytomas form an ill-defined mass resulting in enlargement and distortion of the involved anatomical structure. They do not form a distinct margin with adjacent brain structures making them difficult to be

793

removed completely with surgery. This corresponds well with their microscopic appearance where they appear as hypercellular regions of brain or spinal cord with tumour cells intermixed with and entrapping normal cellular elements (Fig. 2a). The tumour cells also form secondary structures around neurons, blood vessels and beneath the pia limitans (Fig. 2b). In contrast to the rounded nuclei and clear cytoplasm associated with oligodendroglioma, diffuse astrocytomas have more angular, elongated nuclei and eosinophilic, fibrillary cytoplasm. Nuclear hyperchromasia and enlargement, coupled with cellular crowding or clustering can help distinguish neoplastic from reactive astrocytes. However, as discussed below, molecular findings may be more helpful in distinguishing astrocytic neoplasms from oligodendroglial neoplasms or normal astrocytes. In the paediatric population, the findings of a biphasic cell population and Rosenthal fibres and/or eosinophilic granular bodies may point to a diagnosis of pilocytic astrocytoma rather than diffuse astrocytoma. However, in some cases the tissue is too small to accurately distinguish on a morphologic basis between these entities and the molecular findings more fully discussed below may be of help. For example, the finding of a BRAF fusion event may be more compatible with a diagnosis of WHO grade I pilocytic astrocytoma, while the finding of a BRAF V600E mutation, or MYB/MYBL1/FGFR1 alteration points to a WHO grade II neoplasm. The presence of H3F3A K27M points to malignant behaviour, despite the histologic appearance. The diagnosis of WHO grade III vs grade II diffuse astrocytoma depends on the finding of mitotic activity (Fig. 2c). Vascular-endothelial proliferation and pseudopalisading necrosis are not features of WHO grade II/III astrocytoma. As discussed above, an important difference between adult and paediatric diffuse astrocytoma (Fig. 2d) is that in children, WHO grade II astrocytomas rarely progress to, and are genetically distinct from, their higher grade counterparts, while in adults WHO grade II and III astrocytomas appear to be genetically similar. Whether adult tumours with similar genetics but with grade II versus grade III histologic features have a different prognosis is currently under active investigation (see below). Immunohistochemistry can be used in adult diffuse astrocytoma to demonstrate mutant IDH1 (IDH1 R132H) (Fig. 2e). Loss of ATRX staining in tumour cells with retention in normal brain cells implies an underlying ATRX mutation, while diffuse tumour cell immunopositivity for p53 suggest a TP53 mutation (Fig. 3f, also see below). These findings are rare in childhood low-grade astrocytoma. The histone H3.3 (H3F3A) K27M and BRAF V600E mutations more common in paediatric low-grade astrocytomas (Fig. 2d) can also be reliably demonstrated by immunohistochemistry (Fig. 3g, h).

123

794

Fig. 2 Morphologic and immunohistochemical features of lower grade diffuse astrocytoma. a Adult diffuse astrocytoma, WHO grade II, showing hypercellularity with entrapped normal cellular elements highlighting the infiltrative nature of these tumours (H&E 9200). b Secondary structure formation with tumour cells clustering around blood vessels and neurons (H&E 9200). c Diffuse astrocytoma, WHO grade III, demonstrating mitotic activity which distinguished WHO grade III from grade II astrocytoma (H&E 9200). d Paediatric

123


diffuse astrocytoma, WHO grade II positive for the H3.3 K27M mutation (H&E/LFB 9200). e IDH1 R132H immunopositivity in the adult diffuse astrocytoma, WHO grade II shown in panel A (9200). f p53 immunopositivity in an adult diffuse astrocytoma (9200). g H3.3 K27M immunopositivity in a paediatric diffuse astrocytoma, WHO grade II (9200). h BRAF V600E immunopositivity in a paediatric low-grade glioma (9200)


795

Pathogenesis of adult astrocytoma IDH IDH1/2 mutation IDH1/2 mutations are found in 59–90 % of DA and 28–82 % of AA, though the frequencies vary considerably in the literature [45, 53, 90, 120, 135, 162]. There is a consistent tendency for IDH1/2 mutations to be more common in DA than in AA. This most likely reflects the fact that histopathologically defined AAs contain a considerable fraction of GBMs as will be described later. All mutations of IDH1/2 in gliomas are missense and exclusively affect arginine 132 of IDH1 or arginine 172 of IDH2, with only a few rare and isolated exceptions [41]. Mutations of IDH1 and IDH2 are mutually exclusive, the great majority being IDH1 mutations while IDH2 mutations represent only 2–8 %. This is in contrast to acute myeloid leukaemia (AML), in which a considerable proportion of IDH mutations consist of the R140Q substitution in IDH2 [85]. IDH mutations always occur only in one allele (hemizygous mutation) [45, 162]. Around 90 % of mutations affecting IDH1 in sporadic gliomas are IDH1 c.395G[A resulting in arginine 132 being replaced by

histidine (R132H). There is a suggestion that each type of IDH mutation may be non-randomly associated with a specific histological subtype, e.g., R132C may be more common among astrocytic tumours than oligodendrogliomas [45]. All astrocytomas that arise in Li– Fraumeni patients with a germline TP53 mutation carry the IDH1 R132C mutation [152]. IDH2 mutations appear to be less common among astrocytomas than in oligodendrogliomas [45]. The spectrum of IDH1/2 mutations has been reviewed extensively elsewhere [47, 114]. Function of mutated IDH1/2 IDH1 and IDH2 are isocitrate dehydrogenases which share a considerable amino acid sequence homology and the same enzymatic function. There is a third isoform of isocitrate dehydrogenase, IDH3, which is involved in the Krebs cycle and has a distinct amino acid sequence from IDH1 or IDH2. No cancer-associated mutation has ever been reported in IDH3. IDH1 and IDH2 utilise NADP? as a cofactor to convert isocitrate to a-ketoglutarate (aKG). Unlike IDH3, they are not involved in the Krebs cycle; however, IDH1 appears to function as the major source of NADPH [10] in the cytosol and the peroxisomes where it is localized, and IDH2 probably plays a similar role in the

G-CIMP

GSH

GSSG

NADP+

NADPH

NADPH

Isocitrate NAD+

NADH

(Krebs cycle)

D-2HG D2HGDH

CO2

Glutamate

αKG

TET2

NADP+

αKG wtIDH1/2

IDH3

mut IDH1/2

mut IDH1/2 Glutamine

Fig. 3 A metabolomics diagram centred around the mutant IDH1/2 protein. The mutated IDH1 or IDH2 (mut IDH1/2) protein inhibits the normal function of the wild-type IDH1/2 (wt-IDH1/2) to oxidatively decarboxylate isocitrate into a-ketoglutarate (aKG) using NADP? as a co-factor, as well as produce D-2-Hydroxyglutarate (D-2HG) from aKG. D-2HG inhibits various dioxygenases including TET2 and KDM4C, and renders Glioma CpG island methylator phenotype (GCIMP) or aberrant histone methylation. D-2-Hydroxyglutarate

KDM4C D-2HG aciduria Histone methylaon

dehydrogenase (D2HGDH) catalyses the oxidation of D-2HG to aKG, and its inactivating germ-line mutation results in the accumulation of D-2HG which causes D-2 hydroxyglutaric aciduria (D-2HG aciduria). aKG, a substrate for D-2HG, is converted from glutamate, which abundantly exists in the brain. IDH3 also converts isocitrate to aKG in the Krebs cycle but has never been found mutated in any cancer or involved in gliomagenesis. GSH glutathione, GSSG glutathione disulphide

123

796

mitochondria. It has been postulated that through this function IDH1 and IDH2 may protect cells from oxidative stress by producing NADPH to regenerate glutathione (GSH), which is the main antioxidant in the brain [69, 74]. Mutation at R132 in IDH1 attenuates its catalytic activity to convert isocitrate to aKG [53, 162]. The side chain of R132 forms a unique three hydrogen-bond with isocitrate [165], the disruption of which impairs its interaction. Instead, the IDH1 R132 mutations (as well as IDH2 R172 to some varying degree) render a neomorphic enzymatic function which converts aKG to D-2-hydroxyglutarate (D-2HG) in an NADPH-dependent manner [30]. Postulated as an oncometabolite, there is compelling evidence to support the central and direct role that D-2HG plays in the pathogenesis of IDH-mutated cancers, particularly in astrocytomas. Introduction of the drosophila equivalent of IDH1 R132H into a fly causes neurological defects, which can be abolished by the overexpression of D2HGDH. This suggests that D-2HG is the direct mediator of at least some cancer-associated phenotypes [113]. The primary mechanism of the D-2HG-mediated oncogenesis appears to originate from its ability to competitively inhibit dioxygenases, a group of enzymes that require aKG as a cofactor, through its structural similarity to aKG [161]. Dioxygenases constitute a large family of enzymes that consists of more than 60 different enzymes including prolyl hydroxylase (PHD), which promotes degradation of HIF-1a, a well-known transcription factor that transactivates VEGF and, among many others, TET and histone demethylases. The suggested involvement of the latter two will be described below in association with the most characteristic molecular feature of IDH-mutated tumours, i.e., G-CIMP. G-CIMP CIMP is a phenomenon where a large number of CpG dinucleotides within the CpG island, a genomic region where unmethylated CpGs are clustered, often containing a promoter to control transcription, are hypermethylated. A series of genome-wide DNA methylation analysis, typically using a HumanMethylation450 BeadChip (Illumina), has unanimously revealed that virtually all IDHmutated gliomas show a very distinct epigenetic pattern known as glioma-CpG island methylator phenotype (GCIMP) [96, 157, 158]. The pattern of CpG methylation appears to be non-random and tumour type-specific, hence G-CIMP for glioma [52]. It has also been shown that the G-CIMP profiles are different among the IDH-mutated tumours depending on the make-up of the complete genotype, i.e., astrocytomas typically having TP53/ATRX mutations (see below) and oligodendroglioma with total 1p/19q co-deletion [92, 158]. Although the targets for

123


methylation that function as driver genes for glioma are yet to be defined [96], it is widely believed that silencing of critical gene(s) by G-CIMP is the main mechanism in the pathogenesis of IDH-mutated tumours. The exact mechanism of G-CIMP is still unclear; nonetheless, the practically exclusive association with the presence of IDH-mutations clearly indicates their causal relationship. One of the proposed theories is that the global DNA hypermethylation is caused by D-2HG-mediated inhibition of TET2, a dioxygenase that demethylates 5-methylcytosine into 5-hydroxymethylcytosine [34]. In support of this, AML with inactivating mutations in TET2 (not observed in gliomas) displays a hypermethylation phenotype compatible with G-CIMP [34]. Further support comes from an experiment in which a mutated IDH1 expressed in immortalized human astrocytes caused them to develop G-CIMP, albeit after a long period of culture [140]. A similar experiment showed that the introduction of a mutated IDH1 induced the aberrant methylation of histones via inhibition of the KDM4C histone demethylase, another dioxygenase, at a much earlier passage [82]. One caveat of the D-2HG hypothesis, though most plausible, comes from a rare hereditary condition known as hydroxyglutaric aciduria (HGA). HGA is characterized by the excretion of an excessive amount of either D-2HG or its enantiomer L-2HG, accompanied by diverse neurological symptoms or developmental retardation, although the patients sometimes develop perfectly normally. HGA is caused by germline inactivating mutations of either D- or L2-hydroxyglutarate dehydrogenase, an enzyme that converts either D- or L-2HG into aKG, or sometimes even germline mutations of IDH2 [73], all of which lead to the over-production of 2HG. A peculiar fact is that D-2HGA patients, in whom D-2HG, the same enantiomer produced by astrocytomas, is abundantly synthesised, do not show an increased risk of developing glioma or any other particular cancers. On the other hand, some L-2HGA patients, who produce an excess of L-2HG, the enantiomer not associated with IDH mutations, do sometimes develop gliomas through an unknown mechanism [2, 104]. These facts, therefore, suggest that D-2HG may only be a part of the whole story. Metabolome and IDH1/2 mutation IDH-mutated gliomas show a unique metabolome profile. The mutated IDH requires aKG to produce D-2HG, which could originate from the conversion of glutamine via glutamate [112]. Glutamate is present in abundance in the brain where it works as a major neurotransmitter through the glutamate–glutamine cycle [29]. Glutamine and glutamate levels are both significantly reduced in IDH R132Hmutated gliomas, suggesting their consumption to replenish


aKG [98]. Inhibition of the enzyme glutaminase that converts glutamine to glutamate suppresses the growth of R132H-expressing glioma cells, which can be restored by addition of aKG, indicating their dependence on glutamate to supply aKG [98, 127]. It is thus plausible that the unique availability of the source of aKG in the brain may be one of the reasons for the exceptionally high incidence of IDH mutations in gliomas. TP53 Virtually all IDH1/2 mutations in gliomas are associated with either, but not both, mutations of TP53 (typically found in astrocytomas) or co-deletion of the entire arm of 1p and 19q (total 1p/19q loss, mostly in oligodendrogliomas). Thus, concurrent IDH1/2 and TP53 mutations are now considered to be a genetic hallmark of diffuse astrocytomas. TP53 is one of the most frequently mutated genes in human cancers [106]. p53, the protein product of TP53, forms a homo-tetramer and works as a transcription factor to transactivate a wide array of genes including p21/WAF1, BAX, among many others, upon various genotoxic stresses including double-stranded DNA damage [147]. The mutant p53 contributes to oncogenesis via multiple mechanisms, such as the inability to induce cell cycle arrest or apoptosis in cells that have acquired deleterious mutations and increased genomic instability [91]. As in other cancers, missense mutations of TP53 in diffuse astrocytomas cluster within exons 5 through 8 that encode the majority of the DNA binding domain. There are several mutation hotspots including arginine 175, 248 and particularly 273, all of which are transition mutations at a CpG dinucleotide ([11], TCGA portal and The IARC TP53 Database version R17 [107]). Although the great majority of all TP53 mutations fall into the first category, approximately 10–20 % are either nonsense or frameshift mutations in astrocytomas, which are often found outside of the DNA-binding domain (see data from TCGA portal and The IARC TP53 Database version R17 [107]). These truncated proteins will neither be upregulated nor detected as a positive immunostaining, a conventional surrogate for TP53 mutation [137]. The TP53 gene is mostly biallelically inactivated in astrocytomas, either by means of mutation of one allele followed by mitotic recombination, or by duplication of the mutated allele, resulting in the loss of the wild-type allele appearing as a copy number neutral loss of heterozygosity (LOH), or by independent mutation in each allele [57, 136]. The exact reason why a duplication of the mutated allele specifically occurs in the chromosomal region encompassing TP53 (17p13) is unknown; nonetheless, the loss of the wild-type allele would not be detected as a copy number alteration [57]. Unlike in glioblastomas, MDM2/

797

MDM4 amplification or p14ARF homozygous deletion, considered to be equivalent to TP53 mutation, is absent in astrocytomas. ATRX and TERT Another gene mutation which is very frequently associated with the IDH mutation in astrocytomas is ATRX. ATRX is a member of the SWI2/SNF2 family of helicase/ATPases that are involved in chromatin remodelling [159]. ATRX forms a complex with DAXX (Death-domain associated protein) and interacts with histone H3.3, a replication-independent variant of histone H3, through the H3.3-specific histone chaperone function of DAXX [75]. ATRX mediates the incorporation of H3.3 at the pericentromeric heterochromatin and the telomere through its remodelling activity [159]. The localization of ATRX and H3.3 appears to be a heterochromatic mark of the telomere that suppresses the recombination of repetitive telomeric DNA [28]. Mutations of ATRX were originally identified in patients with the X-linked alpha-thalassemia/mental retardation syndrome [38]. Somatic mutations of ATRX were first found in pancreatic neuroendocrine tumours [59], then in paediatric glioblastomas [125] and in astrocytomas [61]. The mutations are predominantly inactivating (frameshift or nonsense) and may occur almost anywhere in the gene, although some are missense [58, 61, 78]. The mutations cluster either within the ADD (DNA-binding ATRXDNMT3-DNMT3L), the ATP-binding SNF2 helicase or the helicase C-terminal domain, although there is a tendency for mutations to cluster within the ADD domain in diffuse astrocytomas [58]. ATRX mutations are almost exclusively found among astrocytomas, not in oligodendrogliomas and very rarely in glioblastomas [58, 61, 84, 125]. Among astrocytomas, virtually all ATRX mutations are associated with mutations of both IDH and TP53 [58, 61, 78]. Not all IDH/TP53 co-mutated astrocytomas, however, have ATRX mutations [58, 78, 136]. It has been shown that inactivating mutations or deletions of ATRX or DAXX are strongly associated with the presence of alternative lengthening of telomeres (ALT) in diverse cancers such as pancreatic neuroendocrine tumours, neuroblastomas, paediatric high grade gliomas, as well as immortalized human fibroblasts [27, 46, 81, 84, 125]. ALT is one of the mechanisms used by the cells to maintain telomere length in cancer cells in which telomeres are replicated by homologous recombination-mediated inter-telomeric copying of telomere repeats ([28] and references therein). Telomere exchange events rarely occur in normal cells; however, critical loss of telomere length coupled with dysfunction of p53 and subsequent evasion of apoptosis may facilitate ALT. The presence of ALT, which

123

798

can be detected by several methods including telomerespecific FISH or C-circle assay, is strongly associated with mutations of ATRX in diffuse astrocytomas as well as in paediatric glioblastomas [1, 58, 61, 84, 125]. The presence of ALT as a preferred mechanism to maintain telomere length in IDH–TP53–ATRX mutated diffuse astrocytomas is in stark contrast to oligodendrogliomas and glioblastomas, where telomerase activation driven by mutations in the promoter region of TERT, the reverse-transcriptase subunit of the telomerase enzyme, is the most dominant mechanism [49, 51]. The TERT mutations mostly occur at either of two hotspots, C228T or C250T, and create a novel Ets1 binding site, thus upregulating TERT transcription. Interestingly, TERT promoter mutations are practically absent in astrocytomas while they are almost mandatory in oligodendrogliomas, in which mutations of TP53 and ATRX are hardly found [4, 68, 72, 115]. Thus, TP53, ATRX and TERT promoter mutations serve as valuable biomarkers for adult gliomas as will be discussed later. Comprehensive genome analysis Recent whole exome sequencing studies in astrocytomas have provided a comprehensive genomic landscape of these tumours. The Cancer Genome Atlas (TCGA) studied 516 histopathologically defined WHO grade 2 and 3 gliomas in their Brain Lower Grade Glioma (LGG) cohort (http://cancergenome.nih.gov/). Although TCGA’s own global analysis of the LGG cohort is yet to be published at the time this review is written, their provisional data are available on the cBioPortal site (http://www.cbioportal.org/ ) [25, 36]. In parallel, Suzuki et al. [136] investigated the genomic landscape of grade 2–3 gliomas by combining independently performed whole-exome sequencing of 52 tumours from a Japanese cohort with that of 425 grade II/ III gliomas available from TCGA. Targeted sequencing of over 180 genes was performed in an independent cohort of 280 tumours. Killela [67] also performed exome sequencing in 23 diffuse astrocytomas (7 grade 2, 16 grade 3) as well as 34 other gliomas. While those large-scale exome sequencing studies identified a high prevalence of concurrent IDH1/2, TP53 and ATRX mutations, no other genes were significantly frequently mutated in astrocytomas (TCGA and [136]). Mutations of CIC, FUBP1 or TERT promoter were very rare and mostly limited to tumours without TP53/ATRX mutations. A small subset of IDH1/TP53-mutated tumours also had mutations in PIK3CA, PIK3R1, SMARCA4, SETD2 or NOTCH1, which are known to be altered in other adult or paediatric brain tumours. Mutations of PTEN, NF1 or EGFR, the genes frequently mutated in primary GBMs [16], were almost exclusively found among diffuse

123


astrocytomas with wild-type IDH1/2. Thus, the genomewide mutation analyses have confirmed that the combined IDH1/2 and TP53 mutations, with or without ATRX mutations, are the necessary, if not sufficient, drivers of astrocytomas. These studies also confirmed that the codeletion of the entire 1p and 19q (total 1p/19q loss) arms, which is the hallmark of oligodendroglioma, was very rare and almost solely found among tumours without TP53 mutations. Gain of chromosome 7 is a frequent event in astrocytomas; however, loss of chromosome 10 is uncommon, unlike in GBMs. Copy-neutral LOH of 17p was almost exclusively and invariably accompanied by TP53 mutations (see above). Global gene expression of gliomas has been extensively studied over the past decade [31, 40, 105, 108, 146]. The best known expression-based molecular classification subdivides glioblastoma and anaplastic astrocytoma into 3 classes (Proneural, Mesenchymal or Proliferative) or glioblastomas into 4 subtypes (Proneural, Neural, Classical and Mesenchymal), which are largely comparable [40, 108, 146]. The latter has been shown to be applicable to astrocytomas and oligodendrogliomas as well [40]. Phillips et al. [108] reported that the Proneural class was associated with significantly longer survival. However, it was later found that the survival advantage of the Proneural class was restricted to the subset of tumours with IDH1 mutation/GCIMP but not with wild-type IDH1/non-G-CIMP [16, 40]. Expression profiling-based subgrouping thus does not identify IDH1 mutant tumours as a distinct class, in contrast to methylation profiling-based subgroups, which are tightly associated with the presence of IDH1 mutations [157]. It thus appears that the transcriptome and the methylome represent different layers of genomic complexity. Cellular origin of astrocytic tumours The cellular origin of astrocytomas has been highly debated. In the adult brain, the neural stem cells that reside in the subventricular zone (SVZ) and subgranular zone of the dentate gyrus give rise to glial progenitors that migrate into the grey and white matters (reviewed in [22, 95, 166]). The glial progenitors are a mixture of diverse states of differentiation, including oligodendrocyte precursor cells (OPC), which predominantly develop into oligodendrocytes. Astrocytes may develop from their own elusive precursors. Although the exact process of astrocytic development awaits further investigation, astrocytes and oligodendrocytes appear to share common ancestral cells, at least at some stages of their development, either stem cells, progenitor cells or even precursor cells. The complexity of gliogenesis is reflected in gliomagenesis. A number of studies successfully generated gliomas in the mouse brain by introducing activated


oncogenes into either NSCs, OPCs or proliferating astrocytes (reviewed in [166]). By overexpressing different oncogenic signals in OPCs, mouse models of gliomas, resembling either oligodendroglioma (by expressing PDGFB) or astrocytic tumours (by expressing AKT and mutant KRAS) by morphology and expression profiles, were developed on a background of p19Arf deficiency (which would otherwise activate p53), suggesting that the type of oncogenic signals rather than the cell of origin may dictate the type of glioma which will develop [77]. However, most of these experiments used oncogenic signals that are predominantly found in GBMs such as loss of p19ARF (corresponding to the human p14ARF) or activation of the MAPK or AKT pathway, but not with a combination of mutated IDH1 and TP53. Tumorigenicity is normally the expected parameter measured in animal models of brain tumours, but it is also an inherent limitation for this type of experiments. Indeed, it would be difficult to prove a presumed sequence of oncogenic events to be the direct stepwise neoplastic driver for a tumour type for which even a patient-derived tumour is generally difficult to grow either in vitro or as a xenograft [83, 148]. The cellular origin of astrocytoma thus remains to be further elucidated. Whatever the cellular origin is, the idea of astrocytomas and oligodendrogliomas arising from common progenitor cells that acquired IDH1/2 mutations, speculated solely on the fact that these tumours virtually invariably share IDH mutations (at least for those molecularly defined—see below), remains attractive. Genetic evolution of astrocytomas Considering the concurrent mutations of IDH1/2, TP53 and ATRX, the order of genetic events in the development of astrocytomas is of particular interest. Suzuki et al. [136] analysed the variant allele frequencies (VAF) of co-existing mutations in individual tumours and showed that IDH1/ 2 had the highest VAF among all common genetic changes, concluding that IDH1/2 mutations are founder mutations. The VAF of TP53 was, however, indistinguishable from those of IDH mutations, indicating that both are early genetic events in astrocytoma development. Interestingly, they identified one case which had an identical IDH1 mutation but two different sets of TP53 and ATRX mutations between the primary and the recurrent tumours [136]. This single case of parallel mutations suggests that the IDH1 mutation should, therefore, precede the TP53/ATRX mutations as a truncal mutation. Watanabe et al. [151] found four cases whose tumours had an IDH1 mutation alone in the first biopsy while the second biopsy showed both IDH1 and TP53 mutations. They did not find any case with a TP53 mutation alone in the first biopsy preceding an IDH1 mutation in the second biopsy. Johnson et al.

799

examined 23 pairs of grade II gliomas (mostly astrocytomas) and their recurrent/progressed counterparts by whole exome sequencing. They found that IDH1 mutations were the only consistently shared mutation in all cases, while several cases had distinct patterns of TP53 or ATRX mutations at the recurrence compared with the initial tumours [60]. These findings indicate that an IDH1 mutation is indeed the earliest genetic event in astrocytomas (Fig. 4). On the other hand, astrocytomas associated with a Li– Fraumeni syndrome with a TP53 germline mutation present an interesting case against this theory [152]. The majority of these astrocytomas had somatic IDH1 R132C mutations. Thus, by definition, TP53 mutations existed prior to the somatic IDH-mutations in these tumours. It is, however, also possible that the full biallelic inactivation of TP53 by loss of the wild-type allele may not have occurred before the acquisition of the IDH1 mutation in these tumours. The exact reason for the apparent preference of the R132C mutation in Li–Fraumeni patients is unknown. Taken together, these facts consolidate the idea that acquiring both IDH1/2 and TP53 mutations is mandatory in astrocytoma development. It has also been shown that ATRX mutations have lower VAF than IDH1/TP53 mutations in the tumours that harboured both [136]. The frequencies of ATRX mutations are consistently lower than that of TP53 mutations and only very few cases have ATRX mutations alone while some harbour mutations of IDH1/TP53, but not ATRX [58, 61, 78]. These facts indicate that ATRX mutations are a later event to TP53/IDH1 mutations. ATRX syndrome patients with germline ATRX mutations are not predisposed to develop any particular cancer including gliomas, unlike the Li–Fraumeni syndrome, suggesting that ATRX mutations are not an initiating genetic event of diffusely infiltrating astrocytomas [37]. Molecular classification As the molecular pathogenesis of each brain tumour type is progressively elucidated, the idea of a molecular classification has emerged as a matter of course. Using various combinations of the status of IDH1/2, 1p/19q, TP53, ATRX, G-CIMP and more recently the TERT promoter, a number of studies have each proposed molecular classification schemes, which are generally comparable [32, 43, 50, 66, 70, 87, 116, 121, 156]. The efficacy of the proposed molecular classification to predict a patient’s prognosis has been demonstrated. It is highly anticipated that the molecular information will be incorporated in the upcoming revision of the WHO Classification of the central nervous system as recommended by the ISN-Haarlem Consensus Guidelines [80]. Although the details are yet to be agreed upon and published, an integrated diagnosis may greatly

123

800


Neural stem cell/glial progenitor cell?

MYB/ MYBL1 or FGFR1 or BRAF

Paediatric Astrocytoma IDH wt

IDH1 IDH2 PTEN NF1 EGFR TERT

TP53 ATRX

PIK3CA SMARCA4 NOTCH1

Adult Astrocytoma IDH wt

Astrocytoma IDH mut

Fig. 4 A hypothetical order of mutations in astrocytoma development. IDH1 or IDH2 mutations may occur in the unknown cells of origin of astrocytomas (possibly neural stem cells or glial progenitor cells, see text) as the earliest genetic event. Presumably they acquire TP53 mutations next, followed by ATRX mutations. Some of the tumours may further develop PIK3CA, SMARCA4 or NOTCH1 mutations (italicised as being more hypothetical), among others (TCGA and [136]). The pathogenesis of IDH-wild type (IDH-wt)

astrocytomas is currently unknown; however, mutations of PTEN, NF1, EGFR or TERT promoter, a mutational profile which resembles that of primary glioblastomas, may be observed in these tumours (italicised as above) [136]. Paediatric astrocytomas develop without mutations of either IDH1, NF1 or TERT. Instead, they acquire various alterations of MYB, MYBL1, FGFR or BRAF. Whether paediatric and adult astrocytomas share the cell of origin is unknown

help solve the ambiguity and inter-investigator inconsistency arising in the diagnosis of some tumours, in particular oligoastrocytomas. We will focus here on the relevance of the molecular classification in defining astrocytomas. The molecular hallmark of diffuse astrocytomas is the concurrent mutations of IDH1/2 and TP53, in most cases accompanied by ATRX mutations, and the absence of TERT promoter mutations. As described above, this genotype is found in the great majority of histologically diagnosed diffuse astrocytoma grade II, and to a lesser extent in anaplastic astrocytoma grade III. A small subset of astrocytomas may have total 1p/19q loss and TERT promoter mutation [4, 43, 70, 156], which would molecularly classify them as oligodendroglioma according to the ISNHaarlem Consensus Guidelines [80]. The exact pathogenesis of the astrocytomas with wildtype IDH1/2 is unclear. A subset of these astrocytomas has a TERT promoter mutation, a genotype typically found in GBM [4, 66, 72]. Several studies suggested that the diffuse astrocytomas grade II with a wild-type IDH1 have shorter overall survival than those with mutations [32, 87], although others found that IDH1 mutation had no impact on the survival of patients with grade II astrocytomas [3]. In order to accurately assess the impact of IDH1 mutation on the prognosis of astrocytoma patients, it would be necessary to exclude a subset of astrocytomas that harbour mutations typically found in paediatric astrocytomas (e.g.,

BRAF alterations) as they may belong to a biologically different entity (see below). Hartmann et al. [44] showed that AA patients with a wild-type IDH1/2 had a significantly shorter survival than IDH-mutated GBM patients, not only AA with mutated IDH1/2. These data suggest that at least a proportion of wild type IDH1/2 astrocytomas may be GBM from a molecular perspective. On the other hand, Olar et al. [99] reported that a dichotomized mitotic index of 4/1000 tumour cells was strongly associated with the overall survival of grade II–III glioma patients with IDH-wt. Further investigation is needed to answer the question whether a subset of IDH-wt astrocytomas representing a biologically independent entity from either IDH-mutated astrocytoma or glioblastoma exists. Another important issue would be the role of the WHO grading in the molecular classification. Under the conventional histopathological classification, the WHO grades predict the prognosis [97]. It is, however, possible that the significantly shorter overall survival in histologically diagnosed AA compared with DA may be affected by the proportion of tumours that are molecularly GBM, which could be diagnosed as such according to the molecular classification. In a cohort of 264 IDH-mutant and 1p/19q non-co-deleted grade II–III diffuse gliomas, the WHO grade was not associated with patient survival [99]. Whether grade II and III astrocytomas have a similar clinical course regardless of their morphology, which would

123


support the idea that they are biologically the same tumour (and molecularly diagnosed as such), needs to be validated in an independent large scale study. The accuracy of the molecular classification would largely depend on the method of laboratory examination. The most critical molecular denominator is the IDH1/2 mutation, which may be detected using a variety of methods [6]. IDH1/2 mutations are conventionally screened by Sanger sequencing [45, 53, 162]; however, its sensitivity in a mixed cell population is rather limited [5, 143]. A number of other methods exist, including melting curve analysis [48], COLD-PCR (Co-amplification at lower denaturation temperature PCR) [12] and pyrosequencing [5, 33], all of which have a higher sensitivity than Sanger sequencing. Perhaps the most widely used method of IDH1 mutation detection is immunohistochemistry (IHC) using monoclonal antibodies (Fig. 2e) [24, 64]. The commercially available antibodies for IDH1 R132H are highly specific and strongly stain the cytoplasm of IDH1 R132H harbouring tumour cells but not non-neoplastic cells [23]. Both high accuracy and sensitivity of immunodetection, even in a mixed cell population, have been demonstrated [138]. Monoclonal antibodies against other types of mutant IDH1 or IDH2, some of which are specific for more than one type of mutation, are now also available, technically enabling the detection of almost all types of IDH1/2 mutations except IDH1 R132C [63]. Although there are several known hotspots, the screening of TP53 mutation is tedious and impractical as a diagnostic test. Positive immunostaining of p53 has long been used as a surrogate for TP53 mutation screening; however, their concordance used to be far from consistent (reviewed in [137]). This may be partly due to the inaccuracy of the mutation detection methods in earlier studies; nonetheless, it makes p53 IHC a somewhat unreliable marker for mutation. Recently, two studies re-evaluated the efficacy of p53 IHC to predict the mutated status of TP53 [39, 137]. They both came to a similar conclusion that strong staining of 10 % or more tumour nuclei predicted the presence of TP53 missense mutations with a high accuracy. It has to be noted, however, that protein truncating mutations are likely to be missed [137]. Mutations of ATRX may be found at any position in more than 7 kb of coding DNA sequence divided into 35 exons, making it impractical to screen for mutations in a clinical setting. The loss of ATRX protein expression detected by IHC has been shown to be tightly associated with the presence of inactivating mutations and, therefore, proposed as a useful surrogate for mutation [78, 116, 156]. In a

801

series of 405 diffuse gliomas of grade II–IV, around 70 % of histologically diagnosed grade II/III astrocytomas and 97 % of IDH-mutated astrocytomas show a loss of ATRX expression, while only 2 tumours had both a loss of ATRX expression and co-deletion of 1p/19q [116]. Immunohistochemical examination of IDH1 and ATRX has been suggested as an initial test for diffuse glioma [80]. A drawback of the use of ATRX IHC is that the immunostaining may be significantly affected by the quality of the processed material, such as insufficient fixation, depending on the antibody used [156]. It should also be noted that a missense mutation may not necessarily lead to the loss of ATRX expression [78]. As discussed above, not all IDH/ TP53-mutated astrocytomas have an ATRX mutation [58, 78, 136]. These limitations need to be considered when interpreting the results of molecular tests. When it comes to performing molecular tests in a diagnostic context, recommendable tests and methods will largely depend on the local setting in each centre, i.e., the type of material (frozen or formalin-fixed tissue), equipment and expertise locally available. The EANO guideline recommends three molecular markers for gliomas, i.e., IDH1/2 mutation, 1p/19q co-deletion and MGMT promoter methylation, as valuable prognostic markers with strong clinical evidence [153]. A combination of IDH1 and 1p/19q tests may be considered as a standard for delineating astrocytomas and oligodendroglioma [117]. One of the most widely employed 1p/ 19q tests is interphase fluorescent in situ hybridisation (FISH) using a set of commercially available probes, although it has limitations, for example the inability of the probe to distinguish total from partial deletion (only the former identifying oligodendroglioma). A combination of IHC for mutated IDH1 (typically R132H) and ATRX expression is emerging as an alternative to 1p/19q testing [80, 116]. A solely IHC-based strategy makes this practical in any routine pathology lab. Given the potential limitations described above, however, molecular genetic techniques such as Sanger sequencing or pyrosequencing have their advantages where there is an ambiguity to be solved. High-throughput genome-wide screening technologies such as next generation sequencing or methylation array are powerful tools which would provide an accurate and comprehensive genomic profile and may help further delineate prognostic subgroups. They will, however, not be the first choice in many centres because of the lack of access to the technologies and/or the high cost. The future of molecular diagnostics is a socio-economical issue as well as a scientific one.

123

802

Paediatric diffuse astrocytoma Clinical features In children low-grade gliomas can arise throughout the neuraxis with diffuse astrocytomas arising more commonly in the cerebral hemispheres and deep midline structures as opposed to cerebellum and optic pathway which are more frequent in pilocytic astrocytomas [131]. Clinical presentation depends on the tumour location. Presentation can be with headache, nausea and vomiting and/or lethargy if the tumour results in obstructive hydrocephalus. Hemispheric diffuse astrocytomas may present with seizures, hemiparesis or behavioural changes. Typically, patients will have a long history of symptoms prior to presentation. Genetics Although perhaps not surprising, an important finding of large-scale genomic studies of paediatric low-grade glioma has been the relative paucity of non-silent mutations and structural variants in these tumours, particularly when compared with their adult counterparts [111, 163]. While not considered as diffuse astrocytomas according to the current WHO classification, other low-grade glial or mixed glio-neuronal neoplasms tend to be included in series of paediatric low-grade glioma. These include ganglioglioma, pleomorphic xanthoastrocytoma (PXA), angiocentric glioma, paediatric oligodendroglioma and dysembryoplastic neuroepithelial tumour (DNET). While there may be some morphologic overlap amongst these entities, particularly in small samples, certain patterns of genetic/histologic/location concordance are beginning to emerge with the important caveat that the number of published cases with detailed genetic analysis is small. In contrast to pilocytic astrocytomas where BRAF fusion events predominate, diffuse astrocytomas of the hemispheres harbour mutually exclusive structural variants in MYB/MYBL1 (*25 %), or FGFR1 (*25 %) or BRAF V600E (*25 %) mutations [111, 163]. BRAF V600E is enriched in gangliogliomas (*30–40 %) and PXAs (*70 %). The mutation results in a glutamic acid for valine substitution in the activation segment of the BRAF protein conferring greatly increased kinase activity and RAS-independent stimulation of ERK activity resulting in potent transformation of NIH3T3 cells [149]. This mutation can be detected in formalin-fixed paraffin embedded samples using multiple methods including high-resolution melting (HRM) analysis, pyrosequencing, allele-specific PCR, next-generation sequencing (NGS) and immunohistochemistry. In the United States the FDA approved the cobasÒ BRAF V600 test (Roche), a real-time PCR-based

123


test, as companion diagnostic tool for the BRAF V600E targeted agent vemurafenib. In melanoma, a recent comparison of these methods for detection of BRAF V600E using Sanger sequencing as the gold standard revealed HRM to be 98 % sensitive and 100 % specific down to 6.6 % allele frequency [54]. Pyrosequencing (therascreenÒ BRAF Pyro Kit (Qiagen)) was 100 % sensitive down to 5 % allele frequency but exhibited only 90 % specificity. Allele-specific PCR (cobasÒ BRAF V600 test) was 97.2 % sensitive down to 7 % allele frequency and 98.3 % specific. NGS was 100 % specific and 97.5 % sensitive while IHC was 100 % sensitive and 98 % specific. Two monoclonal antibodies are commercially available for immunohistochemical detection of BRAF V600E, VE1 and anti-B-Raf. A comparison of the two antibodies across a spectrum of 152 neoplasms, including 35 gliomas, using a SNaPshot-based assay as the gold standard, demonstrated the sensitivity and specificity were 98 % (60/61) and 97 % (88/91) for monoclonal VE1 and 95 % (58/61) and 83 % (73/88) for anti-B-Raf, respectively [119]. Overall, IHC seems to be a sensitive and specific tool which is easily implemental into routine diagnostics in most labs with the potential drawback that it has not been approved as a companion diagnostic for clinical trials. The MYBL1 (v-myb avian myeloblastosis viral oncogene homolog-like 1) alteration involves a tandem duplication–truncation event on chromosome 8q in which only the first 9 exons of MYBL1 are duplicated resulting in expression of a truncated MYBL1 protein which lacks the negative regulatory domain. Expression of truncated versions of MYBL1 in NIH-3T3 cells produced soft agar colony growth and in vivo tumour formation suggesting they are oncogenic [111]. MYB alterations include both amplifications and re-arrangements resulting in elevated MYB protein expression [163]. Both MYBL1 and MYB alterations can be detected by FISH [100, 143] but the sensitivity and specificity of these assays for routine clinical practice have not been assessed. The majority of FGFR1 alterations described in paediatric low-grade glioma are intragenic duplications of the tyrosine kinase domain which can be detected by FISH. This event seems to be restricted to hemispheric grade II diffuse astrocytomas and results in autophosphorylation of FGFR1 and an increase in phospho-ERK [163] (Fig. 5). More rarely, FGFR1–TACC1 or FGFR3–TACC3 fusions have been described which result in an increase in phospho-ERK and phospho-AKT [163]. While FGFR1 activation may converge on RAS/MAPK pathway activation similar to that seen for BRAF V600E and the BRAF fusion, there is no apparent connection with the MYB/ MYBL1 alterations described in diffuse hemispheric astrocytomas.


803

Interestingly, although the numbers are very small, angiocentric gliomas appear to be genetically similar to hemispheric diffuse astrocytomas. In contrast, midline lowgrade astrocytomas, albeit rarely, may harbour H3F3A K27M mutations [20, 163]. Discovery of recurrent histone mutations in paediatric GBM has revolutionised our thinking about the pathogenesis of this cancer. Two mutations were found in H3.3: one at amino acid 27 resulting in lysine to methionine substitution (H3.3K27M) and the second at amino acid 34 resulting in a glycine to arginine or valine substitution (H3.3G34R/V) [65, 125, 160]. Recent studies on the functional consequence of H3.3K27M have demonstrated that diffuse pontine gliomas (DIPGs) harbouring the H3K27M mutation exhibit a decrease in the H3K27 di- and tri-methyl marks H3K27me2/3) while leaving other histone marks unchanged (H3K4me3 and H3K36me3) [76, 144, 164]. Interestingly, these effects were observed despite the fact that the mutant histone represented only between 3.63 and 17.6 % of total H3 in human DIPG samples [76], suggesting that H3K27M leads to a reduction in H3K27me3 on the wild-type H3 as well. Elegant work by David Allis’s group demonstrated that this global reduction in H3K27me3 results from direct inhibition of the PRC2 complex by the H3K27 M protein through interaction with the EZH2 active site [76]. Interestingly, these histone mutations show location-dependent enrichment, H3F3A K27M is preferentially found in midline GBMs while H3F3A G34R/V is found exclusively in hemispheric GBMs (*20 % of cases). H3F3A K27M mutation correlates with worse overall survival independent of histologic grade [20, 65, 145]. This suggests that testing for H3F3A K27M will be important for clinical management of patients with midline grade II astrocytoma,

Hemispheric

25% 25% 10% 25%

60% 10% 10%

Diencephalon

75% 10%

BRAF fusion

98%

Brainstem

BRAFV600E Cerebellum FGFR mut/ fusion MYB or MYBL1 fusion/ duplication Fig. 5 A cartoon showing location-dependent differences in paediatric low grade glioma genetic alterations

particularly in diagnostically difficult cases where only a small biopsy is available. The H3F3A K27M mutation could theoretically be detected in formalin-fixed paraffinembedded samples using a similar spectrum of methods described above for BRAF V600E mutation detection. Importantly, the commercially available rabbit polyclonal antibody that recognises the H3.3 K27M mutant protein (Millipore) has been found by two studies to be 100 % sensitive and 100 % specific for detection of the mutation when compared with sequencing-based methods [9, 145]. The finding of H3F3A K27M is incompatible with a diagnosis of pilocytic astrocytoma. BRAF V600E mutations and FGFR1 fusions/mutations, on the other hand, are each reported in *10 % of pilocytic astrocytomas of the midline (Fig. 5) [163]. Importantly IDH1/2 mutations are only rarely present in diffuse astrocytomas of childhood, tending to occur in teenagers with what should likely be termed adult-type diffuse astrocytomas with their associated prognostic implications [109].

Discussion There is compelling evidence that the combination of IDH1/2, TP53 and ATRX mutations is a decisive molecular marker to define adult astrocytomas. Considering the better prognosis heralded by this genotype, at least among histologically diagnosed AAs, the molecular classification will have a significant impact on clinical practice as well as the design of clinical trials. It will allow morphologically ambiguous tumours to be classified into one molecularly defined category. In particular, it has been suggested that oligoastrocytoma should no longer be recognised as a separate entity, because their genotypes are almost always either that of astrocytomas or oligodendrogliomas, and the outcomes comparable to their genotypic equivalents [121] (for detail see other review in this issue). However, when the histology and molecular information do not exactly match, the interpretation may be left in the hands of clinicians. Should an AA with a sequence-verified wild-type IDH be treated as a GBM? Should a histologically diagnosed GBM which has concurrent IDH1/TP53/ATRX mutations still be treated as a GBM (most, if not all, primary GBM with IDH1/2 mutations have concurrent TP53 mutations)? The clinical decision may be either based on the molecular information or on the histology/grade, each one of which may be a part of the integrated diagnosis. Any future clinical trials involving astrocytomas should consider this point. The role of WHO grading may be put to the test to find out whether it has a prognostic value independently of the molecular classes. The current presumed molecular classification based on a handful of

123

804

markers also needs to be refined. Whether IDH-wt glioma may be further classified into biologically or prognostically relevant subgroups depends on the identification of robust additional markers, be it molecular or morphological, which should predict an accurate outcome for the patient. In order to fully implement the proposed molecular classification, it would be necessary to elucidate the pathogenesis of this group of tumours. Further large-scale clinical studies are warranted to this effect. The molecular classification is now paving the way for individualised targeted therapy. An inhibitor against IDH1 R132H has been shown to reduce the production of R(D)2-HG from tumour cells and to suppress the growth of R132H-harbouring glioma xenografts [118]. Another inhibitor targeting IDH2 R140Q also inhibited the enzymatic activity of the mutant IDH2 producing 2HG and suppressed the growth of mutation-harbouring leukaemia cells [150]. Several clinical trials using these new inhibitors are ongoing (https://clinicaltrials.gov/). An immunotherapy targeting the mutant IDH1 protein is also being developed. It has been demonstrated that the endogenously processed peptides containing the IDH1 R132 epitope may be presented on MHC class II and vaccination using R132H peptides induced a specific anti-tumour immune response against IDH1-mutant tumours in a preclinical model [124]. IDH1 R132H-specific antibodies were detected in a subset of patients who developed R132H-harbouring gliomas [124]. Taken together, mutant IDH-targeting therapies have a potential to make a breakthrough for the treatment of astrocytic tumours where no standard chemotherapeutic regimen has so far been established. An important finding of sequencing studies of paediatric diffuse astrocytomas has been their clear distinction from their adult counterparts on a molecular level, despite some morphologic similarities. As more molecular data become available, one can envision a combined histologic/molecular classification for paediatric low-grade glioma similar to what is now being proposed for adult lower grade gliomas. Importantly, as our knowledge of the molecular alterations increases, we enable better therapy for these paediatric patients, an important example being the advent of clinical trials using BRAF V600E targeted agents for paediatric low-grade glioma. The genome sequencing technologies have opened up a new era in neuro-oncology. IDH1, ATRX and H3F3A are among their most fruitful products. Many questions still remain to be answered and more work is clearly needed; nonetheless, it appears that the molecular neuro-oncology for astrocytomas, which has been nurtured for the last quarter of century, is now mature and open for clinical exploitation.

123

Acta Neuropathol (2015) 129:789–808 Acknowledgments The authors thank Dr Kai Yamasaki for his invaluable assistance in data mining, Dr Oltea Sampetrean for her valuable comments on the origin of gliomas, Drs Hiromichi Suzuki and Atsushi Natsume for sharing the information of their accepted manuscript and Dr Sylvia Kocialkowski for the critical reading of the manuscript. The results presented in this review here are in part based upon data generated by the TCGA Research Network: http:// cancergenome.nih.gov/. CEH is funded by the Canadian Institutes of Health Research and the Canadian Cancer Society Research Institute. Conflict of interest KI has received research grant from SRL, travel support from MSD and speaker honoraria from Eisai, Astellas Pharma, Otsuka Pharma, Sanofi, Daiichi Sankyo, Chugai Pharma, and TEIJIN Pharma. YN has received speaker honoraria from Eisai, Otsuka Pharma, MSD and Chugai Pharma.

References 1. Abedalthagafi M, Phillips JJ, Kim GE et al (2013) The alternative lengthening of telomere phenotype is significantly associated with loss of ATRX expression in high-grade pediatric and adult astrocytomas: a multi-institutional study of 214 astrocytomas. Mod Pathol 26:1425–1432 2. Aghili M, Zahedi F, Rafiee E (2009) Hydroxyglutaric aciduria and malignant brain tumor: a case report and literature review. J Neurooncol 91:233–236 3. Ahmadi R, Stockhammer F, Becker N et al (2012) No prognostic value of IDH1 mutations in a series of 100 WHO grade II astrocytomas. J Neurooncol 109:15–22 4. Arita H, Narita Y, Fukushima S et al (2013) Upregulating mutations in the TERT promoter commonly occur in adult malignant gliomas and are strongly associated with total 1p19q loss. Acta Neuropathol 126:267–276 5. Arita H, Narita Y, Matsushita Y et al (2015) Development of a robust and sensitive pyrosequencing assay for the detection of IDH1/2 mutations in gliomas. Brain Tumor Pathol 32:22–30 6. Arita H, Narita Y, Yoshida A, Hashimoto N, Yoshimine T, Ichimura K (2014) IDH1/2 mutation detection in gliomas. Brain Tumor Pathol [Epub ahead of print] 7. Asari S, Makabe T, Katayama S, Itoh T, Tsuchida S, Ohmoto T (1994) Assessment of the pathological grade of astrocytic gliomas using an MRI score. Neuroradiology 36:308–310 8. Bauman G, Fisher B, Watling C, Cairncross JG, Macdonald D (2009) Adult supratentorial low-grade glioma: long-term experience at a single institution. Int J Radiat Oncol Biol Phys 75:1401–1407 9. Bechet D, Gielen GG, Korshunov A et al (2014) Specific detection of methionine 27 mutation in histone 3 variants (H3K27M) in fixed tissue from high-grade astrocytomas. Acta Neuropathol 128:733–741 10. Bleeker FE, Atai NA, Lamba S et al (2010) The prognostic IDH1(R132) mutation is associated with reduced NADP?-dependent IDH activity in glioblastoma. Acta Neuropathol 119:487–494 11. Bogler O, Huang HJ, Kleihues P, Cavenee WK (1995) The p53 gene and its role in human brain tumors. Glia 15:308–327 12. Boisselier B, Marie Y, Labussiere M et al (2010) COLD PCR HRM: a highly sensitive detection method for IDH1 mutations. Hum Mutat 31:1360–1365 13. Bondy ML, Scheurer ME, Malmer B et al (2008) Brain tumor epidemiology: consensus from the Brain Tumor Epidemiology Consortium. Cancer 113:1953–1968

Acta Neuropathol (2015) 129:789–808 14. Braganza MZ, Rajaraman P, Park Y et al (2014) Cigarette smoking, alcohol intake, and risk of glioma in the NIH-AARP Diet and Health Study. Br J Cancer 110:242–248 15. Brandes AA, Nicolardi L, Tosoni A et al (2006) Survival following adjuvant PCV or temozolomide for anaplastic astrocytoma. Neuro Oncol 8:253–260 16. Brennan CW, Verhaak RG, McKenna A et al (2013) The somatic genomic landscape of glioblastoma. Cell 155:462–477 17. Brown PD, Buckner JC, O’Fallon JR et al (2003) Effects of radiotherapy on cognitive function in patients with low-grade glioma measured by the folstein mini-mental state examination. J Clin Oncol 21:2519–2524 18. The Committee of Brain Tumor Registry of Japan (2014) Report of Brain Tumor Registry of Japan (2001–2004) 13th edition. Neurol Med Chir (Tokyo) 54:1–102 19. Buckner JC, Pugh SL, Shaw EG et al (2014) Phase III study of radiation therapy (RT) with or without procarbazine, CCNU, and vincristine (PCV) in low-grade glioma: RTOG 9802 with Alliance, ECOG, and SWOG. J Clin Oncol 2014 ASCO Annu Meet Abstr 32:2000 20. Buczkowicz P, Bartels U, Bouffet E, Becher O, Hawkins C (2014) Histopathological spectrum of paediatric diffuse intrinsic pontine glioma: diagnostic and therapeutic implications. Acta Neuropathol 128:573–581 21. Cairncross G, Wang M, Shaw E et al (2013) Phase III trial of chemoradiotherapy for anaplastic oligodendroglioma: long-term results of RTOG 9402. J Clin Oncol 31:337–343 22. Canoll P, Goldman JE (2008) The interface between glial progenitors and gliomas. Acta Neuropathol 116:465–477 23. Capper D, Sahm F, Hartmann C, Meyermann R, von Deimling A, Schittenhelm J (2010) Application of mutant IDH1 antibody to differentiate diffuse glioma from nonneoplastic central nervous system lesions and therapy-induced changes. Am J Surg Pathol 34:1199–1204 24. Capper D, Zentgraf H, Balss J, Hartmann C, von Deimling A (2009) Monoclonal antibody specific for IDH1 R132H mutation. Acta Neuropathol 118:599–601 25. Cerami E, Gao J, Dogrusoz U et al (2012) The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov 2:401–404 26. Chaichana KL, McGirt MJ, Laterra J, Olivi A, Quinones-Hinojosa A (2010) Recurrence and malignant degeneration after resection of adult hemispheric low-grade gliomas. J Neurosurg 112:10–17 27. Cheung NK, Zhang J, Lu C et al (2012) Association of age at diagnosis and genetic mutations in patients with neuroblastoma. JAMA 307:1062–1071 28. Conomos D, Pickett HA, Reddel RR (2013) Alternative lengthening of telomeres: remodeling the telomere architecture. Front Oncol 3:27 29. Daikhin Y, Yudkoff M (2000) Compartmentation of brain glutamate metabolism in neurons and glia. J Nutr 130:1026S– 1031S 30. Dang L, White DW, Gross S et al (2009) Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature 462:739–744 31. de Tayrac M, Aubry M, Saikali S et al (2011) A 4-gene signature associated with clinical outcome in high-grade gliomas. Clin Cancer Res 17:317–327 32. Dubbink HJ, Taal W, van Marion R et al (2009) IDH1 mutations in low-grade astrocytomas predict survival but not response to temozolomide. Neurology 73:1792–1795 33. Felsberg J, Wolter M, Seul H et al (2010) Rapid and sensitive assessment of the IDH1 and IDH2 mutation status in cerebral gliomas based on DNA pyrosequencing. Acta Neuropathol 119:501–507

805 34. Figueroa ME, Abdel-Wahab O, Lu C et al (2010) Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer Cell 18:553–567 35. Fisher BJ, Lui J, Macdonald DR et al (2013) A phase II study of a temozolomide-based chemoradiotherapy regimen for high-risk low-grade gliomas: preliminary results of RTOG 0424. J Clin Oncol 31:abstract 2008 36. Gao J, Aksoy BA, Dogrusoz U et al (2013) Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal 6:pl1 37. Gibbons R (2006) Alpha thalassaemia-mental retardation, X linked. Orphanet J Rare Dis 1:15 38. Gibbons RJ, Picketts DJ, Villard L, Higgs DR (1995) Mutations in a putative global transcriptional regulator cause X-linked mental retardation with alpha-thalassemia (ATR-X syndrome). Cell 80:837–845 39. Gillet E, Alentorn A, Doukoure B et al (2014) TP53 and p53 statuses and their clinical impact in diffuse low grade gliomas. J Neurooncol 118:131–139 40. Guan X, Vengoechea J, Zheng S et al (2014) Molecular subtypes of glioblastoma are relevant to lower grade glioma. PLoS One 9:e91216 41. Gupta R, Flanagan S, Li CC et al (2013) Expanding the spectrum of IDH1 mutations in gliomas. Mod Pathol 26:619–625 42. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674 43. Hartmann C, Hentschel B, Tatagiba M et al (2011) Molecular markers in low-grade gliomas: predictive or prognostic? Clin Cancer Res 17:4588–4599 44. Hartmann C, Hentschel B, Wick W et al (2010) Patients with IDH1 wild type anaplastic astrocytomas exhibit worse prognosis than IDH1-mutated glioblastomas, and IDH1 mutation status accounts for the unfavorable prognostic effect of higher age: implications for classification of gliomas. Acta Neuropathol 120:707–718 45. Hartmann C, Meyer J, Balss J et al (2009) Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age: a study of 1,010 diffuse gliomas. Acta Neuropathol 118:469–474 46. Heaphy CM, de Wilde RF, Jiao Y et al (2011) Altered telomeres in tumors with ATRX and DAXX mutations. Science 333:425 47. Horbinski C (2013) What do we know about IDH1/2 mutations so far, and how do we use it? Acta Neuropathol 125:621–636 48. Horbinski C, Kelly L, Nikiforov YE, Durso MB, Nikiforova MN (2010) Detection of IDH1 and IDH2 mutations by fluorescence melting curve analysis as a diagnostic tool for brain biopsies. J Mol Diagn 12:487–492 49. Horn S, Figl A, Rachakonda PS et al (2013) TERT promoter mutations in familial and sporadic melanoma. Science 339:959–961 50. Houillier C, Wang X, Kaloshi G et al (2010) IDH1 or IDH2 mutations predict longer survival and response to temozolomide in low-grade gliomas. Neurology 75:1560–1566 51. Huang FW, Hodis E, Xu MJ, Kryukov GV, Chin L, Garraway LA (2013) Highly recurrent TERT promoter mutations in human melanoma. Science 339:957–959 52. Hughes LA, Melotte V, de Schrijver J et al (2013) The CpG island methylator phenotype: what’s in a name? Cancer Res 73:5858–5868 53. Ichimura K, Pearson DM, Kocialkowski S et al (2009) IDH1 mutations are present in the majority of common adult gliomas but rare in primary glioblastomas. Neuro Oncol 11:341–347 54. Ihle MA, Fassunke J, Konig K et al (2014) Comparison of high resolution melting analysis, pyrosequencing, next generation sequencing and immunohistochemistry to conventional Sanger

123

806

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67. 68.

69.

70.

71.

72.

73.

74.

Acta Neuropathol (2015) 129:789–808 sequencing for the detection of p. V600E and non-p.V600E BRAF mutations. BMC Cancer 14:13 Jaeckle KA, Decker PA, Ballman KV et al (2011) Transformation of low grade glioma and correlation with outcome: an NCCTG database analysis. J Neurooncol 104:253–259 Jakola AS, Myrmel KS, Kloster R et al (2012) Comparison of a strategy favoring early surgical resection vs a strategy favoring watchful waiting in low-grade gliomas. JAMA 308:1–8 James CD, Carlbom E, Nordenskjold M, Collins VP, Cavenee WK (1989) Mitotic recombination of chromosome 17 in astrocytomas. Proc Natl Acad Sci USA 86:2858–2862 Jiao Y, Killela PJ, Reitman ZJ et al (2012) Frequent ATRX, CIC, FUBP1 and IDH1 mutations refine the classification of malignant gliomas. Oncotarget 3:709–722 Jiao Y, Shi C, Edil BH et al (2011) DAXX/ATRX, MEN1, and mTOR pathway genes are frequently altered in pancreatic neuroendocrine tumors. Science 331:1199–1203 Johnson BE, Mazor T, Hong C et al (2014) Mutational analysis reveals the origin and therapy-driven evolution of recurrent glioma. Science 343:189–193 Kannan K, Inagaki A, Silber J et al (2012) Whole-exome sequencing identifies ATRX mutation as a key molecular determinant in lower-grade glioma. Oncotarget 3:1194–1203 Karim AB, Maat B, Hatlevoll R et al (1996) A randomized trial on dose-response in radiation therapy of low-grade cerebral glioma: European Organization for Research and Treatment of Cancer (EORTC) Study 22844. Int J Radiat Oncol Biol Phys 36:549–556 Kato Y (2015) Specific monoclonal antibodies against IDH1/2 mutations as diagnostic tools for gliomas. Brain Tumor Pathol 32:3–11 Kato Y, Jin G, Kuan CT, McLendon RE, Yan H, Bigner DD (2009) A monoclonal antibody IMab-1 specifically recognizes IDH1R132H, the most common glioma-derived mutation. Biochem Biophys Res Commun 390:547–551 Khuong-Quang DA, Buczkowicz P, Rakopoulos P et al (2012) K27M mutation in histone H3.3 defines clinically and biologically distinct subgroups of pediatric diffuse intrinsic pontine gliomas. Acta Neuropathol 124:439–447 Killela PJ, Pirozzi CJ, Healy P et al (2014) Mutations in IDH1, IDH2, and in the TERT promoter define clinically distinct subgroups of adult malignant gliomas. Oncotarget 5:1515–1525 Killela PJ, Pirozzi CJ, Reitman ZJ et al (2014) The genetic landscape of anaplastic astrocytoma. Oncotarget 5:1452–1457 Killela PJ, Reitman ZJ, Jiao Y et al (2013) TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal. Proc Natl Acad Sci USA 110:6021–6026 Kim SY, Park JW (2003) Cellular defense against singlet oxygen-induced oxidative damage by cytosolic NADP?-dependent isocitrate dehydrogenase. Free Radic Res 37:309–316 Kim YH, Nobusawa S, Mittelbronn M et al (2010) Molecular classification of low-grade diffuse gliomas. Am J Pathol 177:2708–2714 Kizilbash SH, Giannini C, Voss JS et al (2014) The impact of concurrent temozolomide with adjuvant radiation and IDH mutation status among patients with anaplastic astrocytoma. J Neurooncol 120:85–93 Koelsche C, Sahm F, Capper D et al (2013) Distribution of TERT promoter mutations in pediatric and adult tumors of the nervous system. Acta Neuropathol 126:907–915 Kranendijk M, Struys EA, van Schaftingen E et al (2010) IDH2 mutations in patients with D-2-hydroxyglutaric aciduria. Science 330:336 Lee SM, Koh HJ, Park DC, Song BJ, Huh TL, Park JW (2002) Cytosolic NADP(?)-dependent isocitrate dehydrogenase status

123

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

91. 92.

93.

modulates oxidative damage to cells. Free Radic Biol Med 32:1185–1196 Lewis PW, Elsaesser SJ, Noh KM, Stadler SC, Allis CD (2010) Daxx is an H3.3-specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telomeres. Proc Natl Acad Sci USA 107:14075–14080 Lewis PW, Muller MM, Koletsky MS et al (2013) Inhibition of PRC2 activity by a gain-of-function H3 mutation found in pediatric glioblastoma. Science 340:857–861 Lindberg N, Jiang Y, Xie Y et al (2014) Oncogenic signaling is dominant to cell of origin and dictates astrocytic or oligodendroglial tumor development from oligodendrocyte precursor cells. J Neurosci 34:14644–14651 Liu XY, Gerges N, Korshunov A et al (2012) Frequent ATRX mutations and loss of expression in adult diffuse astrocytic tumors carrying IDH1/IDH2 and TP53 mutations. Acta Neuropathol 124:615–625 Louis DN, Ohgaki H, Wiestler OD, Cavenee WK (2007) WHO classification of tumours of the central nervous system, 4th edn. International agency for research on cancer, Lyon Louis DN, Perry A, Burger P et al (2014) International society of neuropathology-haarlem consensus guidelines, for nervous system tumor classification and grading. Brain Pathol Lovejoy CA, Li W, Reisenweber S et al (2012) Loss of ATRX, genome instability, and an altered DNA damage response are hallmarks of the alternative lengthening of telomeres pathway. PLoS Genet 8:e1002772 Lu C, Ward PS, Kapoor GS et al (2012) IDH mutation impairs histone demethylation and results in a block to cell differentiation. Nature 483:474–478 Luchman HA, Stechishin OD, Dang NH et al (2012) An in vivo patient-derived model of endogenous IDH1-mutant glioma. Neuro Oncol 14:184–191 Mangerel J, Price A, Castelo-Branco P et al (2014) Alternative lengthening of telomeres is enriched in, and impacts survival of TP53 mutant pediatric malignant brain tumors. Acta Neuropathol 128:853–862 Marcucci G, Maharry K, Wu YZ et al (2010) IDH1 and IDH2 gene mutations identify novel molecular subsets within de novo cytogenetically normal acute myeloid leukemia: a Cancer and Leukemia Group B study. J Clin Oncol 28:2348–2355 Matar E, Cook RJ, Fowler AR et al (2010) Post-contrast enhancement as a clinical indicator of prognosis in patients with anaplastic astrocytoma. J Clin Neurosci 17:993–996 Metellus P, Coulibaly B, Colin C et al (2010) Absence of IDH mutation identifies a novel radiologic and molecular subtype of WHO grade II gliomas with dismal prognosis. Acta Neuropathol 120:719–729 Mistry M, Zhukova N, Merico D et al (2015) BRAF mutation and CDKN2A deletion define a clinically distinct subgroup of childhood secondary high-grade glioma. J Clin Oncol 33:1015–1022 MRC TMRCBTWP (2001) Randomized trial of procarbazine, lomustine, and vincristine in the adjuvant treatment of highgrade astrocytoma: a medical research council trial. J Clin Oncol 19:509–518 Mukasa A, Takayanagi S, Saito K et al (2012) Significance of IDH mutations varies with tumor histology, grade, and genetics in Japanese glioma patients. Cancer Sci 103:587–592 Muller PA, Vousden KH (2013) p53 mutations in cancer. Nat Cell Biol 15:2–8 Mur P, Mollejo M, Ruano Y et al (2013) Codeletion of 1p and 19q determines distinct gene methylation and expression profiles in IDH-mutated oligodendroglial tumors. Acta Neuropathol 126:277–289 Narita Y (2013) Current knowledge and treatment strategies for grade II gliomas. Neurol Med Chir (Tokyo) 53:429–437

Acta Neuropathol (2015) 129:789–808 94. Narita Y, Shibui S (2015) Trends and outcomes in the treatment of gliomas based on data during 2001–2004 from the Brain Tumor Registry of Japan. Neurol Med Chir (Tokyo) 95. Noble M, Dietrich J (2004) The complex identity of brain tumors: emerging concerns regarding origin, diversity and plasticity. Trends Neurosci 27:148–154 96. Noushmehr H, Weisenberger DJ, Diefes K et al (2010) Identification of a CpG island methylator phenotype that defines a distinct subgroup of glioma. Cancer Cell 17:510–522 97. Ohgaki H, Kleihues P (2005) Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas. J Neuropathol Exp Neurol 64:479–489 98. Ohka F, Ito M, Ranjit M et al (2014) Quantitative metabolome analysis profiles activation of glutaminolysis in glioma with IDH1 mutation. Tumour Biol 35:5911–5920 99. Olar A, Wani KM, Alfaro-Munoz KD et al (2015) IDH mutation status and role of WHO grade and mitotic index in overall survival in grade II-III diffuse gliomas. Acta Neuropathol [Epub ahead of print] 100. Ostrom QT, Gittleman H, Liao P et al (2014) CBTRUS statistical report: primary brain and central nervous system tumors diagnosed in the United States in 2007–2011. Neuro Oncol 16(Suppl 4):iv1–iv63 101. Pallud J, Capelle L, Taillandier L et al (2009) Prognostic significance of imaging contrast enhancement for WHO grade II gliomas. Neuro Oncol 11:176–182 102. Pallud J, Fontaine D, Duffau H et al (2010) Natural history of incidental World Health Organization grade II gliomas. Ann Neurol 68:727–733 103. Parsons DW, Jones S, Zhang X et al (2008) An integrated genomic analysis of human glioblastoma multiforme. Science 321:1807–1812 104. Patay Z, Orr BA, Shulkin BL et al (2014) Successive distinct high-grade gliomas in L-2-hydroxyglutaric aciduria. J Inherit Metab Dis 38:273–277 105. Petalidis LP, Oulas A, Backlund M et al (2008) Improved grading and survival prediction of human astrocytic brain tumors by artificial neural network analysis of gene expression microarray data. Mol Cancer Ther 7:1013–1024 106. Petitjean A, Achatz MI, Borresen-Dale AL, Hainaut P, Olivier M (2007) TP53 mutations in human cancers: functional selection and impact on cancer prognosis and outcomes. Oncogene 26:2157–2165 107. Petitjean A, Mathe E, Kato S et al (2007) Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database. Hum Mutat 28:622–629 108. Phillips HS, Kharbanda S, Chen R et al (2006) Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis. Cancer Cell 9:157–173 109. Pollack IF, Hamilton RL, Sobol RW et al (2011) IDH1 mutations are common in malignant gliomas arising in adolescents: a report from the Children’s Oncology Group. Childs Nerv Syst 27:87–94 110. Potts MB, Smith JS, Molinaro AM, Berger MS (2012) Natural history and surgical management of incidentally discovered lowgrade gliomas. J Neurosurg 116:365–372 111. Ramkissoon LA, Horowitz PM, Craig JM et al (2013) Genomic analysis of diffuse pediatric low-grade gliomas identifies recurrent oncogenic truncating rearrangements in the transcription factor MYBL1. Proc Natl Acad Sci USA 110:8188–8193 112. Reitman ZJ, Jin G, Karoly ED et al (2011) Profiling the effects of isocitrate dehydrogenase 1 and 2 mutations on the cellular metabolome. Proc Natl Acad Sci USA 108:3270–3275

807 113. Reitman ZJ, Sinenko SA, Spana EP, Yan H (2015) Genetic dissection of leukemia-associated IDH1 and IDH2 mutants and D-2-hydroxyglutarate in Drosophila. Blood 125:336–345 114. Reitman ZJ, Yan H (2010) Isocitrate dehydrogenase 1 and 2 mutations in cancer: alterations at a crossroads of cellular metabolism. J Natl Cancer Inst 102:932–941 115. Remke M, Ramaswamy V, Peacock J et al (2013) TERT promoter mutations are highly recurrent in SHH subgroup medulloblastoma. Acta Neuropathol 126:917–929 116. Reuss DE, Sahm F, Schrimpf D et al (2015) ATRX and IDH1R132H immunohistochemistry with subsequent copy number analysis and IDH sequencing as a basis for an ‘‘integrated’’ diagnostic approach for adult astrocytoma, oligodendroglioma and glioblastoma. Acta Neuropathol 129:133–146 117. Riemenschneider MJ, Jeuken JW, Wesseling P, Reifenberger G (2010) Molecular diagnostics of gliomas: state of the art. Acta Neuropathol 120:567–584 118. Rohle D, Popovici-Muller J, Palaskas N et al (2013) An inhibitor of mutant IDH1 delays growth and promotes differentiation of glioma cells. Science 340:626–630 119. Routhier CA, Mochel MC, Lynch K, Dias-Santagata D, Louis DN, Hoang MP (2013) Comparison of 2 monoclonal antibodies for immunohistochemical detection of BRAF V600E mutation in malignant melanoma, pulmonary carcinoma, gastrointestinal carcinoma, thyroid carcinoma, and gliomas. Hum Pathol 44:2563–2570 120. Sabha N, Knobbe CB, Maganti M et al (2014) Analysis of IDH mutation, 1p/19q deletion, and PTEN loss delineates prognosis in clinical low-grade diffuse gliomas. Neuro Oncol 16:914–923 121. Sahm F, Reuss D, Koelsche C et al (2014) Farewell to oligoastrocytoma: in situ molecular genetics favor classification as either oligodendroglioma or astrocytoma. Acta Neuropathol 128:551–559 122. Sanai N, Chang S, Berger MS (2011) Low-grade gliomas in adults. J Neurosurg 115:948–965 123. Schomas DA, Laack NN, Rao RD et al (2009) Intracranial lowgrade gliomas in adults: 30-year experience with long-term follow-up at Mayo Clinic. Neuro Oncol 11:437–445 124. Schumacher T, Bunse L, Pusch S et al (2014) A vaccine targeting mutant IDH1 induces antitumour immunity. Nature 512:324–327 125. Schwartzentruber J, Korshunov A, Liu XY et al (2012) Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma. Nature 482:226–231 126. Scoccianti S, Magrini SM, Ricardi U et al (2012) Radiotherapy and temozolomide in anaplastic astrocytoma: a retrospective multicenter study by the Central Nervous System Study Group of AIRO (Italian Association of Radiation Oncology). Neuro Oncol 14:798–807 127. Seltzer MJ, Bennett BD, Joshi AD et al (2010) Inhibition of glutaminase preferentially slows growth of glioma cells with mutant IDH1. Cancer Res 70:8981–8987 128. Shaw EG, Berkey B, Coons SW et al (2008) Recurrence following neurosurgeon-determined gross-total resection of adult supratentorial low-grade glioma: results of a prospective clinical trial. J Neurosurg 109:835–841 129. Shaw EG, Wang M, Coons SW et al (2012) Randomized trial of radiation therapy plus procarbazine, lomustine, and vincristine chemotherapy for supratentorial adult low-grade glioma: initial results of RTOG 9802. J Clin Oncol 30:3065–3070 130. Shonka NA, Theeler B, Cahill D et al (2013) Outcomes for patients with anaplastic astrocytoma treated with chemoradiation, radiation therapy alone or radiation therapy followed by chemotherapy: a retrospective review within the era of temozolomide. J Neurooncol 113:305–311

123

808 131. Sievert AJ, Fisher MJ (2009) Pediatric low-grade gliomas. J Child Neurol 24:1397–1408 132. Sjoblom T, Jones S, Wood LD et al (2006) The consensus coding sequences of human breast and colorectal cancers. Science 314:268–274 133. Smith JS, Chang EF, Lamborn KR et al (2008) Role of extent of resection in the long-term outcome of low-grade hemispheric gliomas. J Clin Oncol 26:1338–1345 134. Soffietti R, Baumert BG, Bello L et al (2010) Guidelines on management of low-grade gliomas: report of an EFNS-EANO Task Force. Eur J Neurol 17:1124–1133 135. Sonoda Y, Kumabe T, Nakamura T et al (2009) Analysis of IDH1 and IDH2 mutations in Japanese glioma patients. Cancer Sci 100:1996–1998 136. Suzuki H, Aoki K, Chiba K et al (2015) Mutational landscape and clonal architecture in grade II and III gliomas. Nat Genet [Epub ahead of print] 137. Takami H, Yoshida A, Fukushima S et al (2014) Revisiting TP53 mutations and immunohistochemistry—a comparative study in 157 diffuse gliomas. Brain Pathol [Epub ahead of print] 138. Takano S, Tian W, Matsuda M et al (2011) Detection of IDH1 mutation in human gliomas: comparison of immunohistochemistry and sequencing. Brain Tumor Pathol 28:115–123 139. Tortosa A, Vinolas N, Villa S et al (2003) Prognostic implication of clinical, radiologic, and pathologic features in patients with anaplastic gliomas. Cancer 97:1063–1071 140. Turcan S, Rohle D, Goenka A et al (2012) IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype. Nature 483:479–483 141. van den Bent MJ, Afra D, de Witte O et al (2005) Long-term efficacy of early versus delayed radiotherapy for low-grade astrocytoma and oligodendroglioma in adults: the EORTC 22845 randomised trial. Lancet 366:985–990 142. van den Bent MJ, Brandes AA, Taphoorn MJ et al (2013) Adjuvant procarbazine, lomustine, and vincristine chemotherapy in newly diagnosed anaplastic oligodendroglioma: long-term follow-up of EORTC brain tumor group study 26951. J Clin Oncol 31:344–350 143. van den Bent MJ, Hartmann C, Preusser M et al (2013) Interlaboratory comparison of IDH mutation detection. J Neurooncol 112:173–178 144. Venneti S, Garimella MT, Sullivan LM et al (2013) Evaluation of histone 3 lysine 27 trimethylation (H3K27me3) and enhancer of Zest 2 (EZH2) in pediatric glial and glioneuronal tumors shows decreased H3K27me3 in H3F3A K27M mutant glioblastomas. Brain Pathol 145. Venneti S, Santi M, Felicella MM et al (2014) A sensitive and specific histopathologic prognostic marker for H3F3A K27M mutant pediatric glioblastomas. Acta Neuropathol 128:743–753 146. Verhaak RG, Hoadley KA, Purdom E et al (2010) Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell 17:98–110 147. Vogelstein B, Lane D, Levine AJ (2000) Surfing the p53 network. Nature 408:307–310 148. Wakimoto H, Tanaka S, Curry WT et al (2014) Targetable signaling pathway mutations are associated with malignant phenotype in IDH-mutant gliomas. Clin Cancer Res 20:2898–2909

123

Acta Neuropathol (2015) 129:789–808 149. Wan PT, Garnett MJ, Roe SM et al (2004) Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 116:855–867 150. Wang F, Travins J, DeLaBarre B et al (2013) Targeted inhibition of mutant IDH2 in leukemia cells induces cellular differentiation. Science 340:622–626 151. Watanabe T, Nobusawa S, Kleihues P, Ohgaki H (2009) IDH1 mutations are early events in the development of astrocytomas and oligodendrogliomas. Am J Pathol 174:1149–1153 152. Watanabe T, Vital A, Nobusawa S, Kleihues P, Ohgaki H (2009) Selective acquisition of IDH1 R132C mutations in astrocytomas associated with Li–Fraumeni syndrome. Acta Neuropathol 117:653–656 153. Weller M, van den Bent M, Hopkins K et al (2014) EANO guideline for the diagnosis and treatment of anaplastic gliomas and glioblastoma. Lancet Oncol 15:e395–e403 154. Wick W, Hartmann C, Engel C et al (2009) NOA-04 randomized phase III trial of sequential radiochemotherapy of anaplastic glioma with procarbazine, lomustine, and vincristine or temozolomide. J Clin Oncol 27:5874–5880 155. Wick W, Platten M, Meisner C et al (2012) Temozolomide chemotherapy alone versus radiotherapy alone for malignant astrocytoma in the elderly: the NOA-08 randomised, phase 3 trial. Lancet Oncol 13:707–715 156. Wiestler B, Capper D, Holland-Letz T et al (2013) ATRX loss refines the classification of anaplastic gliomas and identifies a subgroup of IDH mutant astrocytic tumors with better prognosis. Acta Neuropathol 126:443–451 157. Wiestler B, Capper D, Hovestadt V et al (2014) Assessing CpG island methylator phenotype, 1p/19q codeletion, and MGMT promoter methylation from epigenome-wide data in the biomarker cohort of the NOA-04 trial. Neuro Oncol 16:1630–1638 158. Wiestler B, Capper D, Sill M et al (2014) Integrated DNA methylation and copy-number profiling identify three clinically and biologically relevant groups of anaplastic glioma. Acta Neuropathol 128:561–571 159. Wong LH, McGhie JD, Sim M et al (2010) ATRX interacts with H3.3 in maintaining telomere structural integrity in pluripotent embryonic stem cells. Genome Res 20:351–360 160. Wu G, Broniscer A, McEachron TA et al (2012) Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas. Nat Genet 44:251–253 161. Xu W, Yang H, Liu Y et al (2011) Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of alpha-ketoglutaratedependent dioxygenases. Cancer Cell 19:17–30 162. Yan H, Parsons DW, Jin G et al (2009) IDH1 and IDH2 mutations in gliomas. N Engl J Med 360:765–773 163. Zhang J, Wu G, Miller CP et al (2013) Whole-genome sequencing identifies genetic alterations in pediatric low-grade gliomas. Nat Genet 45:602–612 164. Zhang Y, Reinberg D (2001) Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails. Genes Dev 15:2343–2360 165. Zhao S, Lin Y, Xu W et al (2009) Glioma-derived mutations in IDH1 dominantly inhibit IDH1 catalytic activity and induce HIF-1alpha. Science 324:261–265 166. Zong H, Parada LF, Baker SJ (2015) Cell of origin for malignant gliomas and its implication in therapeutic development. Cold Spring Harb Perspect Biol [Epub ahead of print]

Glioblastoma: pathology, molecular mechanisms and markers.

Oligodendroglioma: pathology, molecular mechanisms and markers.

Pathology, molecular mechanisms and markers of gliomas: new insight and new challenges.

Emerging mechanisms of molecular pathology in ALS.

DNA repair gene XRCC4 codon 247 polymorphism modified diffusely infiltrating astrocytoma risk and prognosis.

Molecular Imaging Markers to Track Huntington's Disease Pathology.

Diffusely infiltrating central nervous system lymphoma involving the brainstem in an immune-competent patient.

[Molecular mechanisms of synapse pathology in ATR-X syndrome].

peIF4E as an independent prognostic factor and a potential therapeutic target in diffuse infiltrating astrocytomas.

Gene expression markers of Tumor Infiltrating Leukocytes.

Evaluation of IDH1 status in diffusely infiltrating gliomas by immunohistochemistry using anti-mutant and wild type IDH1 antibodies.

Pathogenetic Mechanisms of Deep Infiltrating Endometriosis.

[Molecular pathology].

Inflammatory neuropathies: pathology, molecular markers and targets for specific therapeutic intervention.

Epigenomics of clear cell renal cell carcinoma: mechanisms and potential use in molecular pathology.

Diffusely swollen eyelid.

Mechanisms of Autoantibody-Induced Pathology.

Mechanisms and pathology of monocrotaline pulmonary toxicity.

Polymicrogyria: pathology, fetal origins and mechanisms.

[Cancer Plan 3 and molecular pathology: serendipity in pathology?].

Resistance to sunitinib in renal cell carcinoma: From molecular mechanisms to predictive markers and future perspectives.

Molecular pathology techniques.

Surgical and molecular pathology of pancreatic neoplasms.

Molecular pathology in transfusion medicine.