T h e ne w e ngl a nd jou r na l o f m e dic i ne
`
`original article
`
`IDH1 and IDH2 Mutations in Gliomas
`Hai Yan, M.D., Ph.D., D. Williams Parsons, M.D., Ph.D., Genglin Jin, Ph.D.,
`Roger McLendon, M.D., B. Ahmed Rasheed, Ph.D., Weishi Yuan, Ph.D.,
`Ivan Kos, Ph.D., Ines Batinic-Haberle, Ph.D., Siân Jones, Ph.D.,
`Gregory J. Riggins, M.D., Ph.D., Henry Friedman, M.D., Allan Friedman, M.D.,
`David Reardon, M.D., James Herndon, Ph.D., Kenneth W. Kinzler, Ph.D.,
`Victor E. Velculescu, M.D., Ph.D., Bert Vogelstein, M.D.,
`and Darell D. Bigner, M.D., Ph.D.
`
`A bs tr ac t
`
`Background
`A recent genomewide mutational analysis of glioblastomas (World Health Organiza-
`tion [WHO] grade IV glioma) revealed somatic mutations of the isocitrate dehydro-
`genase 1 gene (IDH1) in a fraction of such tumors, most frequently in tumors that were
`known to have evolved from lower-grade gliomas (secondary glioblastomas).
`
`Methods
`We determined the sequence of the IDH1 gene and the related IDH2 gene in 445 cen-
`tral nervous system (CNS) tumors and 494 non-CNS tumors. The enzymatic activity
`of the proteins that were produced from normal and mutant IDH1 and IDH2 genes
`was determined in cultured glioma cells that were transfected with these genes.
`
`Results
`We identified mutations that affected amino acid 132 of IDH1 in more than 70% of
`WHO grade II and III astrocytomas and oligodendrogliomas and in glioblastomas
`that developed from these lower-grade lesions. Tumors without mutations in IDH1
`often had mutations affecting the analogous amino acid (R172) of the IDH2 gene.
`Tumors with IDH1 or IDH2 mutations had distinctive genetic and clinical character-
`istics, and patients with such tumors had a better outcome than those with wild-type
`IDH genes. Each of four tested IDH1 and IDH2 mutations reduced the enzymatic activ-
`ity of the encoded protein.
`
`Conclusions
`Mutations of NADP +-dependent isocitrate dehydrogenases encoded by IDH1 and IDH2
`occur in a majority of several types of malignant gliomas.
`
`From the Departments of Pathology (H.Y.,
`G.J., R.M., B.A.R., D.D.B.), Radiation
`Oncology (I.K., I.B.-H.), Neuro-Oncology
`(H.F.), and Surgery (A.F., D.R.), the Pedi-
`atric Brain Tumor Foundation Institute and
`the Preston Robert Tisch Brain Tumor
`Center; and the Cancer Statistical Center
`(J.H.) — all at Duke University Medical
`Center, Durham, NC; the Ludwig Center
`for Cancer Genetics and Therapeutics and
`the Howard Hughes Medical Institute at
`Johns Hopkins Kimmel Cancer Center
`(D.W.P., S.J., K.W.K., V.E.V., B.V.) and the
`Department of Neurosurgery, Johns Hop-
`kins Medical Institutions (G.J.R.) — all in
`Baltimore; the Department of Pediatrics,
`Baylor College of Medicine, Houston
`(D.W.P.); and the Center for Drug Evalua-
`tion and Research, Food and Drug Admin-
`istration, Silver Spring, MD (W.Y.). Address
`reprint requests to Dr. Yan at the Depart-
`ment of Pathology, Pediatric Brain Tumor
`Foundation Institute and Preston Robert
`Tisch Brain Tumor Center, Duke Univer-
`sity Medical Center, Durham, NC 27710,
`or at yan00002@mc.duke.edu; or to Dr.
`Parsons at the Department of Pediatrics,
`Baylor College of Medicine, Houston, TX
`77030, or at dwparson@txccc.org.
`
`Drs. Yan and Parsons contributed equally
`to this article.
`
`N Engl J Med 2009;360:765-73.
`Copyright © 2009 Massachusetts Medical Society.
`
`n engl j med 360;8 nejm.org february 19, 2009
`
`765
`
`The New England Journal of Medicine
`Downloaded from nejm.org on August 1, 2022. For personal use only. No other uses without permission.
` Copyright © 2009 Massachusetts Medical Society. All rights reserved.
`
`Rigel Exhibit 1016
`Page 1 of 9
`
`

`

`T h e ne w e ngl a nd jou r na l o f m e dic i ne
`
`Gliomas, the most common type of
`
`primary brain tumors, are classified as
`grade I to grade IV on the basis of histo-
`pathological and clinical criteria established by the
`World Health Organization (WHO).1 This group
`of tumors includes specific histologic subtypes,
`the most common of which are astrocytomas,
`oligodendrogliomas, and ependymomas. WHO
`grade I gliomas, often considered to be benign, are
`generally curable with complete surgical resection
`and rarely, if ever, evolve into higher-grade lesions.2
`By contrast, gliomas of WHO grade II or III are in-
`vasive, progress to higher-grade lesions, and have
`a poor outcome. WHO grade IV tumors (glioblas-
`tomas), the most invasive form, have a dismal prog-
`nosis.3,4 On the basis of histopathological criteria,
`it is impossible to distinguish a secondary glio-
`blastoma, defined as a tumor that was previously
`diagnosed as a lower-grade glioma, from a primary
`tumor.5,6
`Several genes, including TP53, PTEN, CDKN2A,
`and EGFR, are altered in gliomas.7-12 These altera-
`tions tend to occur in a defined order during the
`progression to a high-grade tumor. The TP53 mu-
`tation appears to be a relatively early event dur-
`ing the development of an astrocytoma, whereas
`the loss or mutation of PTEN and amplification
`of EGFR are characteristic of higher-grade tu-
`mors.6,13,14 In oligodendrogliomas, allelic losses
`of 1p and 19q occur in many WHO grade II tu-
`mors, whereas losses of 9p21 are largely confined
`to WHO grade III tumors.15
`In a recent genomewide analysis, we identified
`somatic mutations at codon 132 of the isocitrate
`dehydrogenase 1 gene (IDH1) in approximately
`12% of glioblastomas.16 These mutations were
`also found in five of six secondary glioblastomas.
`The results suggested that IDH1 mutations might
`occur after formation of a low-grade glioma and
`drive the progression of the tumor to a glioblas-
`toma. To evaluate this possibility, we analyzed a
`large number of gliomas of various types.
`
`Me thods
`
`DNA Samples
`DNA was extracted from samples of primary brain
`tumor and xenografts and from patient-matched
`normal blood lymphocytes obtained from the Tis-
`sue Bank at the Preston Robert Tisch Brain Tumor
`Center at Duke University and collaborating cen-
`ters, as described previously.17 All analyzed brain
`
`tumors were subjected to consensus review by two
`neuropathologists. Table 1 lists the types of brain
`tumors we analyzed. The samples from glioblas-
`tomas included 138 primary tumors and 13 sec-
`ondary tumors. Of the 138 primary tumors, 15
`were from patients under the age of 21 years. Sec-
`ondary glioblastomas were categorized as WHO
`grade IV on the basis of histologic criteria but had
`been categorized as WHO grade II or III at least
`1 year earlier. Of the 151 tumors, 63 had been ana-
`l yzed in our previous genomewide mutation anal-
`ysis of glioblastomas. None of the lower-grade
`tumors were included in that analysis.16
`In addition to brain tumors, we analyzed 35
`lung cancers, 57 gastric cancers, 27 ovarian can-
`cers, 96 breast cancers, 114 colorectal cancers, 95
`pancreatic cancers, and 7 prostate cancers, along
`with 4 samples from patients with chronic mye-
`logenous leukemia, 7 from patients with chronic
`lymphocytic leukemia, 7 from patients with acute
`lymphoblastic leukemia, and 45 from patients with
`acute myelogenous leukemia. All samples were
`obtained in accordance with the Health Insurance
`Portability and Accountability Act. Acquisition of
`tissue specimens was approved by the institutional
`review board at the Duke University Health System
`and at each of the participating institutions.
`Exon 4 of the IDH1 gene was amplified with the
`use of a polymerase-chain-reaction (PCR) assay
`and sequenced in DNA from the tumor and lym-
`phocytes from each patient, as described previ-
`ously.16 In all gliomas and medulloblastomas
`without an R132 IDH1 mutation, exon 4 of the
`IDH2 gene (which contains the IDH2 residue equiv-
`alent to R132 of IDH1) was sequenced and analyzed
`for somatic mutations. In addition, we evaluated
`all astrocytomas and oligodendrogliomas of WHO
`grade I to grade III, all secondary glioblastomas,
`and 96 primary glioblastomas without R132 IDH1
`mutations or R172 IDH2 mutations for altera-
`tions in the remaining coding exons of IDH1 and
`IDH2. All coding exons of TP53 and PTEN were
`also sequenced in the panel of diffuse astro-
`cytomas, oligodendrogliomas, anaplastic oligo-
`dendrogliomas, anaplastic astrocytomas, and
`glioblastomas. EGFR amplification and the
`CDKN2A–CDKN2B deletion were analyzed with
`the use of quantitative real-time PCR in the
`same tumors.18 We evaluated samples of oligo-
`dendrogliomas and anaplastic oligodendro-
`gliomas for loss of heterozygosity at 1p and 19q,
`as described previously.15,19
`
`766
`
`n engl j med 360;8 nejm.org february 19, 2009
`
`The New England Journal of Medicine
`Downloaded from nejm.org on August 1, 2022. For personal use only. No other uses without permission.
` Copyright © 2009 Massachusetts Medical Society. All rights reserved.
`
`Rigel Exhibit 1016
`Page 2 of 9
`
`

`

`IDH1 and IDH2 Mutations in Gliomas
`
`NA
`
`NA
`
`0
`
`0
`
`14
`
`4
`
`20
`
`42
`
`20
`
`9
`
`NA
`
`0
`
`NA
`
`NA
`
`CDKN2A
`
`CDKN2B
`
`or
`
`NA
`
`NA
`
`0
`
`0
`
`0
`
`0
`
`NA
`
`38
`
`0
`
`0
`
`2
`
`0
`
`NA
`
`0
`
`NA
`
`0
`
`0
`
`0
`
`0
`
`0
`
`NA
`
`23
`
`9
`
`0
`
`NA
`
`0
`
`NA
`
`0
`
`NA
`
`NA
`
`50
`
`84
`
`60
`
`NA
`
`4
`
`NA
`
`10
`
`NA
`
`0
`
`NA
`
`NA
`
`%
`
`EGFR
`
`PTEN
`
`NA
`
`1p and
`
`19q
`
`NA
`
`0
`
`71
`
`33
`
`10
`
`16
`
`33
`
`23
`
`62
`
`65
`
`NA
`
`74
`
`NA
`
`0
`
`7
`
`5.5
`
`ND
`
`ND
`
`ND
`
`13.5
`
`5
`
`59
`
`62
`
`56
`
`11
`
`5
`
`ND
`
`5
`
`yr
`
`ND
`
`ND
`
`30
`
`38
`
`45
`
`37
`
`ND
`
`32
`
`32
`
`34
`
`20
`
`35
`
`ND
`
`ND
`
`0
`
`0
`
`84
`
`5
`
`0
`
`85
`
`14
`
`%
`
`0
`
`0
`
`Type IDHTP53
`
`Wild-
`
`Mutated
`
`IDH
`
`Tumors with Other Alterations§
`
`of Patient
`Median Age
`
`NA denotes not analyzed, and ND not determined because of limited sample size and status of data censoring.
`mined by quantitative real-time polymerase chain reaction. For such assays, copy-number levels of more than 6 or less than 0.3 were considered amplifications or losses, respectively.
`*Of the indicated tumors, 6 secondary and 60 primary glioblastomas were previously described in Parsons et al.16 Copy-number changes in EGFR, CDKN2A, and CDKN2B were deter-
`
`¶ Secondary glioblastoma designates a tumor that was resected more than 1 year after a previous diagnosis of a lower-grade glioma (grade II or grade III).
`§Alterations included mutations in TP53 and PTEN, loss of heterozygosity in 1p and 19q, amplification in EGFR, and deletion in CDKN2A or CDKN2B.
`‡ Patient age refers to age at which the study sample was obtained.
`† Tumors were graded according to histopathological and clinical criteria established by the World Health Organization.
`
`64
`
`60
`
`67
`
`53
`
`100
`
`7
`
`5.5
`
`30
`
`38
`
`45
`
`37
`
`5
`
`59
`
`33
`
`38
`
`11
`
`34
`
`16
`
`5
`
`%
`
`yr
`
`55
`
`30
`
`3
`
`7
`
`36
`
`51
`
`15
`
`123
`
`13
`
`52
`
`7
`
`30
`
`2
`
`21
`
`Medulloblastoma (grade IV)
`
`Ependymoma (grade II)
`
`Anaplastic oligoastrocytoma (grade III)
`
`Oligoastrocytoma (grade II)
`
`Oligoastrocytic tumors
`
`Anaplastic oligodendroglioma (grade III)
`
`Oligodendroglioma (grade II)
`
`Oligodendroglial tumors
`
`Primary pediatric glioblastoma (grade IV)
`
`Primary adult glioblastoma (grade IV)
`
`Secondary glioblastoma (grade IV)¶
`
`Anaplastic astrocytoma (grade III)
`
`Pleomorphic xanthoastrocytoma (grade II)
`
`Diffuse astrocytoma (grade II)
`
`Subependymal giant-cell astrocytoma (grade I)
`
`Pilocytic astrocytoma (grade I)
`
`Astrocytic tumors
`
`Sex
`Male
`
`Patient‡
`Age of
`Median
`
`Analyzed
`Tumors
`No. of
`
`Tumor Classification†
`
`Table 1. Summary of Genetic and Clinical Characteristics of Brain Tumors in the Study.*
`
`n engl j med 360;8 nejm.org february 19, 2009
`
`767
`
`The New England Journal of Medicine
`Downloaded from nejm.org on August 1, 2022. For personal use only. No other uses without permission.
` Copyright © 2009 Massachusetts Medical Society. All rights reserved.
`
`100
`
`100
`
`94
`
`73
`
`90
`
`0
`
`0
`
`NA
`
`NA
`
`2
`
`0
`
`0
`
`0
`
`2
`
`3
`
`NA
`
`2
`
`NA
`
`0
`
`3
`
`7
`
`0
`
`0
`
`31
`
`41
`
`6
`
`0
`
`11
`
`36
`
`1
`
`25
`
`0
`
`0
`
`no.
`
`IDH2Combined
`
`IDH1
`
`27
`
`ND
`
`ND
`
`ND
`
`84
`
`135
`
`8
`
`15
`
`16
`
`51
`
`44
`
`132
`
`ND
`
`ND
`
`mo
`
`65
`
`45
`
`57
`
`67
`
`63
`
`60
`
`70
`
`14
`
`48
`
`IDH Mutations
`Tumors with
`
`Survival
`Median
`
`Rigel Exhibit 1016
`Page 3 of 9
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`

`

`T h e ne w e ngl a nd jou r na l o f m e dic i ne
`
`Enzymatic Activity
`To assess the enzymatic activity of wild-type and
`mutant IDH1 and IDH2 proteins, a human oligo-
`dendroglioma line without IDH1 or IDH2 mutations
`was transfected with a vector (pCMV6, Invitrogen)
`containing the coding sequences of the wild-type
`IDH1, wild-type IDH2, or mutant IDH genes (corre-
`sponding to the most common IDH1 mutation,
`R132H, or the IDH2 mutations R172G, R172K,
`and R172M). Clones of the wild-type IDH1 and
`IDH2 genes were obtained from Origene, and mu-
`tations were introduced by standard methods.
`Cells were collected 48 hours after transfec-
`tion, subjected to centrifugation at 1000×g for
`10 minutes at 4°C, washed once with cold phos-
`phate-buffered saline, and lysed in buffer contain-
`ing 0.1% Triton X-100. They were then disrupted
`by ultrasonication and centrifuged at 12,000×g for
`30 minutes. The supernatants were used to mea-
`sure IDH activity. Expression levels of wild-type
`and mutant IDH proteins were determined by
`Western blotting with the use of an antibody
`against FLAG, a polypeptide protein tag. For each
`enzymatic reaction, a volume of cell lysate con-
`taining the same amount of IDH protein was
`added to 1 ml of assay solution containing 33
`mM of Tris buffer, 0.33 mM of EDTA, 0.1 mM
`of NADP +, 1.33 mM of manganese chloride, and
`1.3 mM of isocitrate. The activity of IDH was
`analyzed through the reduction of NADP + to
`NADPH, which was measured at 25°C by spectro-
`photometry at 340 nm 5 times a second for 300
`seconds.20
`
`Clinical Data and Survival
`Clinical information included the date of birth,
`the date the study sample was obtained, the date
`of pathological diagnosis, the date and pathology
`of any preceding diagnosis of a lower-grade glioma,
`the use or nonuse of radiation therapy or chemo-
`therapy before the date that the study sample was
`obtained, the date of the last contact with the pa-
`tient, and the patient’s status at the time of the
`last contact. We calculated overall survival for pa-
`tients with anaplastic astrocyomas, including 38
`patients with mutations in IDH1 or IDH2 and 14
`with wild-type genes, and for adult patients (≥21
`years of age) with glioblastomas, including 14 pa-
`tients with mutations in IDH1 or IDH2 and 115
`with wild-type genes, using the date of histologic
`diagnosis and the date of the last contact with
`the patient or death. For patients with secondary
`
`glioblastomas, survival was calculated from the
`date of secondary diagnosis. Seven patients with
`glioblastomas were not included in the statistical
`analysis because of insufficient survival data.
`
`Study Design
`The authors designed the study, gathered and ana-
`lyzed the data, wrote the manuscript, and made
`the decision to publish the findings. Gene sequenc-
`ing was performed by Agencourt Bioscience, a sub-
`sidiary of Beckman Coulter. The lead academic au-
`thors vouch for the completeness and accuracy of
`the data and the analyses.
`
`Statistical Analysis
`We examined the association between the occur-
`rence of mutations in IDH1 or IDH2 and other ge-
`netic alterations using Fisher’s exact test. Kaplan–
`Meier survival curves were plotted and the survival
`distributions were compared with the use of the
`Mantel–Cox log-rank test and the Wilcoxon test.
`All reported P values are two-sided, and P values
`of less than 0.01 were considered to indicate sta-
`tistical significance.
`
`R esults
`
`Sequence Analysis
`Sequence analysis of IDH1 in 939 tumor samples
`revealed 161 somatic mutations at residue R132,
`including R132H (142 tumors), R132C (7 tumors),
`R132S (4 tumors), R132L (7 tumors), and R132G
`(1 tumor) (Fig. 1A; and Fig. 1 in the Supplemen-
`tary Appendix, available with the full text of this
`article at NEJM.org). Table 1 and Figure 1B show
`the tumors with somatic R132 mutations. No other
`somatic mutations of IDH1 in the remaining IDH1
`exons of R132-negative tumors were found in all
`WHO grade I to grade III astrocytomas and oligo-
`dendrogliomas, in all secondary glioblastomas,
`and in 96 primary glioblastomas. No R132 muta-
`tions were observed in 21 pilocytic astrocytomas
`(WHO grade I), 2 subependymal giant-cell astro-
`cytomas (WHO grade I), 30 ependymomas (WHO
`grade II), 55 medulloblastomas, or any of the 494
`non–central nervous system tumor samples.
`We also sought alterations in other genes with
`functions similar to those of IDH1 in tumors with-
`out IDH1 mutations. For this purpose, we analyzed
`the IDH2 gene, which encodes the only human
`protein homologous to IDH1 that uses NADP + as
`an electron acceptor. Sequence evaluation of all
`
`768
`
`n engl j med 360;8 nejm.org february 19, 2009
`
`The New England Journal of Medicine
`Downloaded from nejm.org on August 1, 2022. For personal use only. No other uses without permission.
` Copyright © 2009 Massachusetts Medical Society. All rights reserved.
`
`Rigel Exhibit 1016
`Page 4 of 9
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`

`

`IDH1 and IDH2 Mutations in Gliomas
`
`A Mutations
`
`B Frequency of Mutations
`
`3/3
`
`7/7
`
`34/36
`
`27/30
`
`43/51
`
`38/52
`
`11/13
`
`IDH2 mutated
`IDH1 mutated
`
`1/7
`
`0/21
`
`0/30
`
`6/123
`
`0/15
`
`0/55
`
`0/494
`
`M edulloblasto m a
`Non-C N S Tu m or
`Oligodendroglio m a (II)
`
`Secondary Glioblasto m a (IV)Prim ary Adult Glioblasto m a (IV)
`Pediatric Glioblasto m a (IV)Anaplastic Oligoastrocyto m a (III)Anaplastic Oligodendroglio m a (III)Oligoastrocyto m a (II)Anaplastic Astrocyto m a (III)Pleo m orphic Xanthoastrocyto m a (II)Ependym o m a (II)Diffuse Astrocyto m a (II)
`
`
`
`
`
`
`
`Pilocytic Astrocyto m a (I)
`
`100
`
`80
`
`60
`
`40
`
`20
`
`Percent
`
`0
`
`R172G
`R172M
`R172K
`
`GGG
`ATG
`AAG
`
`N=2
`N=3
`N=4
`
`IDH2
`
`IDH1
`
`ATT GGC AGG CAC GCC
`I170
`G171
`R172
`H173
`A174
`
`I130
`G131
`R132
`H133
`A134
`ATA GGT CGT CAT GCT
`
`R132H CAT N=142
`R132C TGT N=7
`R132L CTT N=7
`R132S AGT N=4
`R132G GGT N=1
`
`Figure 1. IDH1 and IDH2 Mutations in Human Gliomas.
`Panel A shows mutations at codon R132 in IDH1 and R172 in IDH2 that were identified in human gliomas, along with the number of
`patients who carried each mutation. Codons 130 to 134 of IDH1 and 170 to 174 of IDH2 are shown. Panel B shows the number and fre-
`quency of IDH1 and IDH2 mutations in gliomas and other types of tumors. The roman numerals in parentheses are the tumor grades,
`according to histopathological and clinical criteria established by the World Health Organization. CNS denotes central nervous system.
`
`IDH2 exons in these glioma samples revealed nine
`somatic mutations of IDH2, all at residue R172:
`R172G in two tumors, R172M in three tumors,
`and R172K in four tumors (Fig. 1A, and Fig. 1 in
`the Supplementary Appendix). The R172 residue
`in IDH2 is the exact analogue of the R132 residue
`in IDH1, which is located in the active site of the
`enzyme and forms hydrogen bonds with the iso-
`citrate substrate.21
`To determine whether the mutations in IDH1
`and IDH2 disturb the function of the correspond-
`ing proteins, we measured the enzymatic activity
`(reduction of NADP + to NADPH) of IDH1 and
`IDH2 proteins in an oligodendroglioma line that
`had been transfected with wild-type or mutant
`IDH1 or IDH2 genes. These mutants represented
`88% of the IDH1 mutations and 100% of the IDH2
`mutations found in patients. Figure 2 shows that
`exogenous expression of wild-type IDH1 or IDH2
`significantly increased the production of NADPH,
`whereas only endogenous IDH activity was ob-
`served in cells that had been transfected with
`mutant IDH1 or IDH2 genes.
`To further evaluate IDH alterations during glio-
`ma progression, we assessed IDH1 mutations in
`
`seven progressive gliomas in which both low-
`grade and high-grade tumor samples were avail-
`able. Sequence analysis identified IDH1 mutations
`in both the low-grade and high-grade tumors in
`all seven cases (Table 1, and Fig. 2 in the Supple-
`mentary Appendix). These results demonstrate
`that IDH1 alterations in high-grade tumors are
`derived from the earlier lesions.
`We also examined diffuse astrocytomas, oligo-
`dendrogliomas, anaplastic oligodendrogliomas,
`anaplastic astrocytomas, and a subgroup of glio-
`blastomas for mutations in TP53 and PTEN, am-
`plification of EGFR, deletion of CDKN2A–CDKN2B,
`and allelic losses of 1p and 19q (Table 1). TP53
`mutations were more common in diffuse astrocy-
`tomas (74%), anaplastic astrocytomas (65%), and
`secondary glioblastomas (62%) than in oligo-
`dendrogliomas (16%) or anaplastic oligodendro-
`gliomas (9%) (P<0.001 for all comparisons by
`Fisher’s exact test). Conversely, deletions of 1p and
`19q were found more often in oligodendrocytic
`than in astrocytic tumors, as expected.15
`Most (80%) of the anaplastic astrocytomas and
`glioblastomas with mutated IDH1 or IDH2 genes
`also had a mutation of TP53, but only 3% had al-
`
`n engl j med 360;8 nejm.org february 19, 2009
`
`769
`
`The New England Journal of Medicine
`Downloaded from nejm.org on August 1, 2022. For personal use only. No other uses without permission.
` Copyright © 2009 Massachusetts Medical Society. All rights reserved.
`
`Rigel Exhibit 1016
`Page 5 of 9
`
`

`

`T h e ne w e ngl a nd jou r na l o f m e dic i ne
`
`(cid:2) (cid:6)(cid:14)(cid:12)(cid:16)(cid:8)(cid:9)(cid:11)(cid:1)(cid:4)(cid:18)(cid:13)(cid:14)(cid:8)(cid:15)(cid:15)(cid:9)(cid:12)(cid:11)
`
`(cid:45)(cid:8)(cid:7)(cid:16)(cid:12)(cid:14)
`
`(cid:36)(cid:32) (cid:35)(cid:27)
`
`
`
`(cid:36)(cid:32) (cid:35)(cid:27)(cid:1)(cid:20)(cid:41)(cid:26)(cid:30)(cid:27)(cid:34)(cid:21)(cid:36)(cid:32) (cid:35)(cid:27)(cid:1)(cid:20)(cid:41)(cid:26)(cid:30)(cid:27)(cid:37)(cid:21)(cid:36)(cid:32) (cid:35)(cid:27)(cid:1)(cid:20)(cid:41)(cid:26)(cid:30)(cid:27) (cid:38)(cid:21)
`
`
`
`(cid:36)(cid:32) (cid:35)(cid:26)
`
`(cid:36)(cid:32) (cid:35)(cid:26)(cid:1)(cid:20)(cid:41)(cid:26)(cid:28)(cid:27)(cid:35)(cid:21)
`
`(cid:2)(cid:11)(cid:16)(cid:9)(cid:23)(cid:33)(cid:5)(cid:2)(cid:34)
`
`(cid:2)(cid:11)(cid:16)(cid:9)(cid:23)(cid:34)(cid:2)(cid:6)(cid:32)(cid:35)
`
`(74%) (P<0.001 for both comparisons by Fisher’s
`exact test). Loss of 1p and 19q was observed in 45
`of 53 (85%) of the oligodendrocytic tumors with
`mutated IDH1 or IDH2 but in none of the tumors
`with wild-type IDH genes (P<0.001 by Fisher’s ex-
`act test).
`Patients with anaplastic astrocytomas or glio-
`blastomas with IDH1 or IDH2 mutations were sig-
`nificantly younger than were patients with tumors
`carrying wild-type IDH1 and IDH2 genes (median
`age, 34 years vs. 56 years for patients with ana-
`plastic astrocytomas and 32 years vs. 59 years for
`those with glioblastomas; P<0.001 for both com-
`parisons by Student’s t-test). Despite the lower
`median age of patients with IDH1 or IDH2 muta-
`tions, no mutations were identified in glioblastomas
`from the 15 patients who were under the age of 21
`(Fig. 3 in the Supplementary Appendix). In patients
`with oligodendrogliomas or anaplastic oligoden-
`drogliomas, the median age of the patients with
`IDH1 or IDH2 mutation was 39 years; IDH1 muta-
`tions were identified in two teenagers (14 and 16
`years) but not in four younger patients.
`Our previous observation of improved outcome
`for patients whose glioblastomas carried the IDH1
`mutation16 was confirmed in this larger data set
`and extended to include such patients with muta-
`tions in IDH2. Patients with a glioblastoma carry-
`ing an IDH1 or IDH2 mutation had a median over-
`all survival of 31 months, which was significantly
`longer than the 15-month survival in patients with
`wild-type IDH1 (P = 0.002 by the log-rank test) (Fig.
`3A). Mutations of IDH genes were also associated
`with improved outcome in patients with anaplas-
`tic astrocytomas; the median overall survival was
`65 months for patients with mutations and 20
`months for those without mutations (P<0.001 by
`the log-rank test) (Fig. 3B). Differential survival
`analyses could not be performed in patients with
`diffuse astrocytomas, oligodendrogliomas, or ana-
`plastic oligodendrogliomas because there were
`too few tumors of these types without IDH gene
`mutations.
`
`Discussion
`
`Vector
`IDH2
`IDH2 (R172G)
`IDH2 (R172K)
`IDH2 (R172M)
`IDH1
`IDH1 (R132H)
`
`(cid:3) (cid:2)(cid:7)(cid:16)(cid:9)(cid:17)(cid:9)(cid:16)(cid:19)(cid:1)(cid:5)(cid:8)(cid:17)(cid:8)(cid:10)(cid:1)(cid:1)(cid:1)(cid:1)(cid:1)
`0.20
`
`0.18
`
`0.16
`
`0.14
`
`0.12
`
`0.10
`
`0.08
`
`0.06
`
`0.04
`
`0.02
`
`0.00
`
`(cid:39)(cid:2)(cid:32)(cid:6)(cid:35)(cid:1)(cid:6)(cid:14)(cid:12)(cid:49)(cid:55)(cid:7)(cid:16)(cid:9)(cid:12)(cid:11)(cid:1)(cid:20)(cid:28)(cid:29)(cid:25)(cid:1)(cid:11)(cid:54)(cid:21)
`
`0
`
`30
`
`60
`
`90
`
`120
`
`150
`
`180
`
`210
`
`240
`
`270
`
`300
`
`(cid:42)(cid:8)(cid:7)(cid:12)(cid:11)(cid:49)(cid:15)
`
`Figure 2. Enzymatic Activity of Wild-Type and Mutant IDH1 and IDH2
`Proteins.
`Cell lysates were extracted from a human oligodendroglioma cell line with-
`out IDH1 or IDH2 mutations that had been transfected with vectors encod-
`ing the indicated proteins. Panel A shows the expression of proteins encod-
`ed by wild-type and mutant IDH1 and IDH2, as determined by Western
`blotting, with the use of an anti-FLAG antibody. Panel B shows the activity
`levels of these proteins, as analyzed by monitoring the production of
`NADPH. GAPDH denotes glyceraldehyde 3-phosphate dehydrogenase.
`
`terations in PTEN, EGFR, CDKN2A, or CDKN2B (Ta-
`ble 2). Conversely, anaplastic astrocytomas and
`glioblastomas with wild-type IDH1 and IDH2 genes
`had few TP53 mutations (18%) and more frequent
`alterations of PTEN, EGFR, CDKN2A, or CDKN2B
`
`Our findings implicate mutations in the NADP +-
`dependent isocitrate dehydrogenase genes, IDH1
`and IDH2, in the pathogenesis of malignant
`gliomas. Gliomas with IDH mutations were clini-
`cally and genetically distinct from gliomas with
`wild-type IDH genes. Notably, two subtypes of
`
`770
`
`n engl j med 360;8 nejm.org february 19, 2009
`
`The New England Journal of Medicine
`Downloaded from nejm.org on August 1, 2022. For personal use only. No other uses without permission.
` Copyright © 2009 Massachusetts Medical Society. All rights reserved.
`
`Rigel Exhibit 1016
`Page 6 of 9
`
`

`

`IDH1 and IDH2 Mutations in Gliomas
`
`Table 2. Frequency of Common Genetic Alterations in Gliomas with Mutated or Wild-Type IDH1 and IDH2 Genes.*
`
`Tumor Type and IDH1
`or IDH2 Mutational Status†
`
`Total Patients
`
`Astrocytic tumors
`
`Diffuse astrocytoma (grade II)
`
`Mutant
`
`Wild-type
`
`Total
`
`Anaplastic astrocytoma (grade
`III)
`
`Mutant
`
`Wild-type
`
`Total
`
`Primary adult glioblastoma
`(grade IV)
`
`Mutant
`
`Wild-type
`
`no.
`
`27
`
`3
`
`30
`
`38
`
`14
`
`52
`
`6
`
`117
`
`Location of Other Alterations‡
`
`TP53
`
`PTEN
`
`EGFR
`
`CDKN2A or
`CDKN2B
`
`1p and 19q
`
`no./total no. (%)
`
`17/20 (85)
`
`0/3
`
`17/23 (74)
`
`23/28 (82)
`
`3/12 (25)
`
`26/40 (65)
`
`0/19
`
`0/2
`
`0/21
`
`0/23
`
`0/3
`
`0/26
`
`0/23
`
`0/3
`
`0/26
`
`0/22
`
`3/11 (27)
`
`3/33 (9)
`
`0/35
`
`1/12 (8)
`
`1/47 (2)
`
`0/33
`
`4/12 (33)
`
`4/45 (9)
`
`5/6 (83)
`
`23/117 (20)
`
`0/5
`
`21/88 (24)
`
`21/93 (23)
`
`0/6
`
`35/86 (41)
`
`35/92 (38)
`
`0/6
`
`39/87 (45)
`
`39/93 (42)
`
`1/19 (5)
`
`0/3
`
`1/22 (5)
`
`1/22 (5)
`
`1/6 (17)
`
`1/28 (4)
`
`1/2 (50)
`
`0/24
`
`1/26 (4)
`
`Total
`
`123
`
`28/123 (23)
`
`Secondary adult glioblastoma
`(grade IV)
`
`Mutant
`
`Wild-type
`
`Total
`
`Oligodendroglial tumors
`
`Oligodendroglioma (grade II)
`
`Mutant
`
`Wild-type
`
`Total
`
`Anaplastic oligodendroglioma
`(grade III)
`
`Mutant
`
`Wild-type
`
`Total
`
`11
`
`2
`
`13
`
`43
`
`8
`
`51
`
`34
`
`2
`
`36
`
`8/11 (73)
`
`0/2
`
`8/13 (62)
`
`5/24 (21)
`
`0/8
`
`5/32 (16)
`
`3/30 (10)
`
`0/2
`
`3/32 (9)
`
`0/6
`
`1/2 (50)
`
`1/8 (12)
`
`0/20
`
`0/7
`
`0/27
`
`0/28
`
`0/2
`
`0/30
`
`0/10
`
`0/2
`
`0/12
`
`0/43
`
`0/8
`
`0/51
`
`0/33
`
`0/2
`
`0/35
`
`1/8 (12)
`
`1/2 (50)
`
`2/10 (20)
`
`NA
`
`NA
`
`NA
`
`2/40 (5)
`
`18/23 (78)
`
`0/8
`
`0/7
`
`2/48 (4)
`
`18/30 (60)
`
`3/33 (9)
`
`2/2 (100)
`
`5/35 (14)
`
`27/30 (90)
`
`0/2
`
`27/32 (84)
`
`* All tumors were analyzed for IDH1 R132 and IDH2 R172 mutations. In addition, all pilocytic astrocytomas, diffuse astrocytomas, oligoden-
`drogliomas, anaplastic oligodendrogliomas, anaplastic astrocytomas, secondary glioblastomas, and 96 primary glioblastomas were evaluat-
`ed for mutations in the remaining coding exons of IDH1 and IDH2. NA denotes not analyzed.
`† Tumors were graded according to histopathological and clinical criteria established by the World Health Organization.
`‡ Alterations included mutations in TP53 and PTEN, loss of heterozygosity in 1p and 19q, amplification in EGFR, and deletion in CDKN2A
`or CDKN2B.
`
`gliomas of WHO grade II or III (astrocytomas and
`oligodendrogliomas) often carried IDH mutations
`but not other genetic alterations that are detect-
`able relatively early during the progression of
`gliomas. This finding suggests that IDH muta-
`
`tions occur early in the development of a glioma
`from a stem cell that can give rise to both astro-
`cytes and oligodendrocytes. The identification of
`IDH1 mutations in 10 of 10 oligoastrocytomas and
`anaplastic oligoastrocytomas, tumors with mor-
`
`n engl j med 360;8 nejm.org february 19, 2009
`
`771
`
`The New England Journal of Medicine
`Downloaded from nejm.org on August 1, 2022. For personal use only. No other uses without permission.
` Copyright © 2009 Massachusetts Medical Society. All rights reserved.
`
`Rigel Exhibit 1016
`Page 7 of 9
`
`

`

`T h e ne w e ngl a nd jou r na l o f m e dic i ne
`
`duplication at 7q34 producing a BRAF fusion
`gene occurs frequently in pilocytic astrocytomas
`but not higher-grade gliomas.22
`In each of the tested mutations, the enzymatic
`activity of the IDH proteins was eliminated. A pre-
`vious study showed that in vitro substitution of
`glutamate for arginine at residue 132 of IDH1 (an
`alteration not observed in patients) resulted in a
`catalytically inactive enzyme.23 Although our re-
`sults demonstrate an effect of the mutations on
`the function of the IDH1 protein, they do not nec-
`essarily mean that the mutations are inactivating.
`For example, the mutant proteins that preclude the
`use of isocitrate as substrate could allow other, as-
`yet-unknown substrates to be used by the enzyme,
`thereby conferring a gain rather than a loss of
`activity. If future studies confirm this possibility,
`mutant IDH could become a target for therapeu-
`tic intervention.
`Our results have important practical implica-
`tions. Historically, glioblastomas have been divided
`into cancers that arise from low-grade gliomas
`(secondary tumors) and those without such an
`antecedent (primary tumors).5,6 Secondary tumors
`account for only 5% of all glioblastomas. The find-
`ing that IDH1 or IDH2 is mutated in the vast ma-
`jority of WHO grade II or III gliomas and in the
`secondary glioblastomas that develop from these
`precursors provides a biologic explanation for
`this clinical categorization: tumors with mutat-
`ed NADP +-dependent isocitrate dehydrogenases
`comprise a specific subgroup of glioblastomas.
`The localization of IDH1 and IDH2 mutations
`to a single amino acid (R132 and R172, respec-
`tively) simplifies the use of this genetic alteration
`for diagnostic purposes. For example, IDH muta-
`tion tests could help distinguish pilocytic astro-
`cytomas (WHO grade I) from diffuse astrocytomas
`(WHO grade II), since these lesions can sometimes
`be difficult to categorize solely on the basis of
`histopathological criteria.2
`Supported by a grant from the Pediatric Brain Tumor Founda-
`tion Institute, a Damon Runyon Foundation Scholar Award,
`a grant from the Southeastern Brain Tumor Foundation, Alex’s
`Lemonade Stand Foundation, a grant from the V Foundation for
`Cancer Research, the Virginia and D.K. Ludwig Fund for Cancer
`Research, the Pew Charitable Trusts, the American Brain Tumor
`Association, the Brain Tumor Research Fund at Johns Hopkins,
`grants (R01CA118822, NS20023-21, R37CA11898-34, CA121113,
`CA43460, CA57345, 5P50-CA-108786, 5P50-NS-20023, 5R37-
`CA-11898, and 2P30-CA-14236) from Beckman Coulter, and
`grants from the Accelerate Brain Cancer Cure Foundation.
`Drs. Yan, Parsons, Jones, Kinzler, Velculescu, Vogelstein, and
`Bigner report being eligible for royalties received by Johns Hop-
`kins University on sales of products related to research de-
`
`IDH mutated
`
`IDH wild-type
`
`10
`
`20
`
`30
`(cid:31) (cid:15)(cid:14)(cid:19)(cid:42)(cid:18)
`
`40
`
`50
`
`60
`
`(cid:1) (cid:4)(cid:12)(cid:11)(cid:15)(cid:9)(cid:12)(cid:8)(cid:18)(cid:19)(cid:15)(cid:13)(cid:8)
`100
`
`(cid:24) 80
`
`P=0.002
`
`60
`
`40
`
`20
`
`0
`
`0
`
`(cid:34)(cid:17)(cid:15)(cid:9)(cid:8)(cid:9)(cid:11)(cid:12)(cid:11)(cid:19)(cid:20)(cid:21)(cid:15)(cid:40)(cid:21)(cid:6)(cid:44)(cid:17)(cid:45)(cid:11)(cid:45)(cid:8)(cid:12)(cid:21)(cid:23)(cid:22)
`
`(cid:2) (cid:1)(cid:14)(cid:8)(cid:16)(cid:12)(cid:8)(cid:18)(cid:19)(cid:11)(cid:10)(cid:21)(cid:1)(cid:18)(cid:19)(cid:17)(cid:15)(cid:10)(cid:20)(cid:19)(cid:15)(cid:13)(cid:8)
`100
`
`IDH mutated
`
`IDH wild-type
`
`10
`
`20
`
`30
`
`40
`
`50
`
`60
`
`(cid:31) (cid:15)(cid:14)(cid:19)(cid:42)(cid:18)
`
`P<0.001
`
`(cid:24) 80
`
`60
`
`40
`
`20
`
`0
`
`0
`
`(cid:34)(cid:17)(cid:15)(cid:9)(cid:8)(cid:9)(cid:11)(cid:12)(cid:11)(cid:19)(cid:20)(cid:21)(cid:15)(cid:40)(cid:21)(cid:6)(cid:44)(cid:17)(cid:45)(cid:11)(cid:45)(cid:8)(cid:12)(cid:21)(cid:23)(cid:22)
`
`Figure 3. Survival of Adult Patients with Malignant Gliomas with or without
`IDH Ge