DISCLAIMER: This document was originally drafted in French by the Institut national d'excellence en santé et en services sociaux (INESSS), and that version can be consulted at http://www.inesss.qc.ca/fileadmin/doc/INESSS/Analyse_biomedicale/Decembre_2014/INESSS_Avis_Ministre_analyses_bio_med_dec_2014_6.pdf. It was translated into English by the Canadian Agency for Drugs and Technologies in Health (CADTH) with INESSS’s permission. INESSS assumes no responsibility with regard to the quality or accuracy of the translation.

While CADTH has taken care in the translation of the document to ensure it accurately represents the content of the original document, CADTH does not make any guarantee to that effect. CADTH is not responsible for any errors or omissions or injury, loss, or damage arising from or relating to the use (or misuse) of any information, statements, or conclusions contained in or implied by the information in this document, the original document, or in any of the source documentation.

DETECTION OF TARGETED MUTATIONS IN PEDIATRIC BRAIN TUMOURS (K27M AND

G34V/R IN H3.3, K27M IN H3.1, AND V600E IN BRAF) (REFERENCE – 2014.02. 04)

Notice of Assessment

December 2014

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1. GENERAL INFORMATION

1.1. Requester: CHU Sainte-Justine

1.2. Application for Review Submitted to MSSS: January 21, 2014 (revision received May 1, 2014)

1.3. Application Received by INESSS: June 9, 2014

1.4. Notice Issued: October 31, 2014

Note:

This notice is based on the scientific and commercial information submitted by the requester and on a complementary review of the literature according to the data available at the time that this test was assessed by INESSS.

2. TECHNOLOGY, COMPANY, AND LICENCE(S)

2.1 Name of the Technology

Amplification of target regions with PCR, followed by a molecular analysis of amplicon sequences using the Sanger method.

2.2 Brief Description of the Technology, and Clinical and Technical Specifications

The test is performed on tumour DNA extracted from frozen or paraffin-embedded tissue. Three amplifications by polymerase chain reaction (PCR) are performed to amplify specific regions containing the three mutations (K27M and G34V/R mutations in histones H3.3 and H3.1 and V600E mutation in BRAF). The amplicons are then sequenced using the Sanger method.

2.3 Company or Developer: Protocol provided by the requester.

2.4 Licence(s): Not applicable.

2.5 Patent, If Any: Not applicable.

2.6 Approval Status (Health Canada, FDA): Not applicable.

2.7 Weighted Value: 196.27

3. CLINICAL INDICATIONS, PRACTICE SETTINGS, AND TESTING PROCEDURES

3.1 Targeted Patient Group

Children with a brain tumour having a glial component (glioma).

3.2 Targeted Disease(s)

The latest Canadian statistics on the incidence of new cancer cases in children and youth from birth to 19 years (for both sexes) reveal that the central nervous system (CNS) is the second most affected site. In fact, among the 4,550 new cases of pediatric cancer expected over the next 5 years, 860 involve the CNS (19%), second only to cases of leukemia (n = 1,465; 32%) [CCS, 2014, p. 113]. Primary brain tumours are complex and heterogeneous. The 2007 World Health Organization (WHO) classification of tumours of the central nervous

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system is recognized as an international standard for the typing and staging of glial and other types of brain tumours [Louis et al., 2007].1 Table 1 provides epidemiological data concerning gliomas and other types of pediatric brain tumours. The requester focuses mainly on four tumour types: grade I pilocytic astrocytoma (PCA), grade II pleomorphic xanthoastrocytoma (PXA), grade 1 ganglioglioma (GG), and grade IV glioblastoma (GBM). These tumoral types are described in more detail in the following paragraphs.

Table 1: Primary pediatric brain tumours with glial component (birth to 19 years)

HISTOLOGY GRADE1 DISTRIBUTION

2 INCIDENCE

2,3 10-YEAR SURVIVAL RATE

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Pilocytic astrocytoma I 15.5% 0.82 96.0%

Diffuse astrocytoma II 5.2% 0.27 80.4%

Anaplastic astrocytoma III 1.6% 0.09 26.5%

Other astrocytomas (including PXA II)

I-III 1.9% 0.1 70% (PXA)

Glioblastoma IV 2.7% 0.14 12.6%

Oligodendroglioma II 1.1% 0.06 90.1%

Anaplastic oligodendroglioma III 0.2% 0.01 -

Oligoastrocytic tumours II-III 0.7% 0.04 76.1%

Ependymoma and other I-III 5.3% 0.28 64.9%

Unclassified malignant gliomas malins non classés

III-IV 11.5% 0.61 57.0%

Neuronal/glial (including GG) I-III 6.9% 0.36 72.4%

Abbreviations: GG = ganglioglioma; PXA = pleomorphic xanthoastrocytoma.

1. According to the World Health Organization (WHO) [Louis et al., 2007].

2. Data taken from the CBTRUS (Central Brain Tumor Registry of the United States) statistical report, 2006-2010. Distribution was reported for all pediatric brain tumours (N = 21,512), not only gliomas (N = 11,305 cases) [Ostrom et al., 2013].

3. Annual age-adjusted incidence rate per 100,000 individuals [Ostrom et al., 2013].

4. The relative survival rate is defined as the ratio of survival observed in a group of people with cancer and the estimated survival for the entire population presumed to be without the cancer under consideration and having the same characteristics (sex, age, place of residence, etc.).2

Pilocytic Astrocytoma (PCA - grade I)

Pilocytic astrocytoma is an essentially pediatric glioma occurring primarily in the cerebellum (67%), the hypothalamus, and the optic pathways. The tumour is well-circumscribed, slow-growing, and rarely progresses towards a malignant stage. The majority of cases can be treated, even cured, with complete surgical resection alone [Fernandez et al., 2003]. The prognosis is good, with a 10-year overall survival rate of 96% [Ohgaki and Kleihues, 2005]. The classic histologic findings reveal two distinct components: one fascicular and composed of piloid cells, the other microcystic and composed of cells with rounded nuclei. The

1. In the WHO classification, slow-growing (“low-grade”) tumours are classified either grade I or grade II, depending on whether they are circumscribed or not. Grade I tumours are circumscribed and thus have a better prognosis than grade II tumours, because the latter's poorly defined margins or diffuse spread often make complete surgical resection difficult. “High-grade” tumours are characterized by rapid growth, either in anaplastic foci that develop in a low-grade tumour (grade III), or in a large portion or the entire volume of the tumour mass (grade IV). Signs of rapid growth are very important (high cellularity, an elevated mitotic index, and anaplasia [loss of normal cellular differentiation]). Necrosis and vascular proliferation are common in grade IV tumours, but they are inconstant and unreliable signs of rapid growth [adapted from the Atlas interactif de neuro-oncologie, Association des neuro-oncologues d’expression française, available at: http://anocef.org/atlas/en/intro.html]. 2. Statistics Canada. Cancer survival statistics 2004 [website]. Available at: http://www.statcan.gc.ca/pub/84-601-x/2004001/4067481-eng.htm.

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presence of Rosenthal fibers and eosinophilic granular bodies is characteristic [Louis et al., 2007]. Cells appearing to be malignant often spread out over a large part of the tumour, thereby complicating the diagnosis established on the basis of small samples [Rodriguez et al., 2013; Takei et al., 2008].

Pleomorphic Xanthoastrocytoma (PXA - grade II)

Pleomorphic xanthoastrocytoma is a rare astrocytic tumour, and the majority (2/3) of affected individuals are under 18 years of age [Dias-Santagata et al., 2011; Louis et al., 2007]. The lesion is well-circumscribed and located in the superficial cerebral cortex in contact with the meninges. The presence of eosinophilic granular bodies and a combination of giant fusiform and pleomorphic cells is characteristic of PXA [Rodriguez et al., 2013; Louis et al., 2007]. PXA can sometimes be mistaken for malignant glioma, as it shows greater and more atypical cellularity [Rodriguez et al., 2013]. The risk of misdiagnosis is increased by the presence of abundant extracellular reticulin and significant lymphocytic infiltrate [Rodriguez et al., 2013]. The prognosis is relatively favourable, with a 10-year overall survival rate of 70%. Surgery is the first-line treatment, coupled with radiological monitoring. In the case of local recurrence or the presence of anaplastic features at primary resection, radiation and chemotherapy may be used. The risk of recurrence at 5 years is 30% [Dias-Santagata et al., 2011; Rao et al., 2010].

Ganglioglioma (GG - grade I)

Gangliogliomas are benign mixed neuronal-glial tumours that account for 2.5% of pediatric brain neoplasms [Dunham, 2010]. These tumours are slow-growing, well differentiated and usually found in the temporal lobe [Ichimura et al., 2012]. Gangliogliomas may arise at any age, but predominantly affect children and young adults [Louis et al., 2007]. The histology is very heterogeneous as a result of the two cell types involved; the tumours may sometimes resemble oligodendrogliomas (ODG), diffuse astrocytomas, or PCAs, which may make differential diagnosis difficult [Ichimura et al., 2012; Dougherty et al., 2010]. Although these tumours are usually low-grade, the features of the glial component dictate the prognosis, which is generally good, with a 5-year overall survival rate of 90% for cases involving complete resection [Fedoul and Souirti, 2012]. The tumours rarely progress towards malignancy [Luyken et al., 2004].

Glioblastoma (GBM - grade IV) and Other Malignant Gliomas (grades III-IV)

Glioblastomas are malignant and aggressive primary brain tumours classified as grade IV by WHO [Louis et al., 2007]. Glioblastomas can also evolve from low-grade astrocytomas. The histological criteria for GBMs include high mitotic activity, microvascular proliferation and/or necrotic areas, which makes them impossible to distinguish through histology alone [Louis et al., 2007]. Glioblastomas account for 80% of pediatric brainstem tumours and are associated with a very poor prognosis: the 3-year survival rate is between 5% and 10% [Karajannis et al., 2008]. Complete surgical resection of this infiltrative tumour is virtually impossible [Wilson et al., 2014]. In 2005, Stupp et al. showed that the use of temozolomide in combination with radiotherapy significantly prolonged survival in patients with GBM compared with survival in a group treated with radiotherapy alone (median survival of 14.6 months versus 12.1 months; p < 0.001) [Stupp et al., 2005]. The Stupp protocol is the current standard treatment for GBM, but clinical research in this field is very active [Wilson et al., 2014].

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3.3 Number of Patients Targeted: 20 patients each year.

3.4 Medical Specialties and Other Professions Involved

Neurosurgery, neuropathology, neuro-oncology, pediatrics, neuropediatrics

3.5 Testing Procedure

The tumour tissue is obtained from the department of pathology at the requester's establishment, for inpatients, or by specimen reception, for patients coming from other CHU. The Montreal Children’s Hospital wishes to send its samples to the CHU Sainte-Justine; the test will be performed in the molecular diagnostic laboratory of this establishment.3 Currently, requests for the molecular investigation of pediatric brain tumour samples are sent outside the province. However, according to the data from the Ministère de la Santé et des Services sociaux, no requests have been made over the last two years to send this test outside Quebec.

Tests for these mutations will be conducted in accordance with the algorithms recommended by the requester, which are provided in Appendix A (Scenarios 1 and 2).

4. TECHNOLOGY BACKGROUND

4.1 Nature of the Diagnostic Technology: Complementary

4.2 Brief Description of the Current Technological Context

Although the clinical assessment of the patient and medical imaging results can strongly suggest the presence of a malignant glioma, the gold standard for the diagnosis of brain tumours is the microscopic assessment of tumour biopsies [Ichimura et al., 2012]. The histopathologic analysis of tissue arrangement and cell morphology can generally help clarify the diagnosis and predict the progression of the disease (prognosis). However, despite the identification of some distinctive characteristics, various studies have reported low reproducibility of the methods and wide interobserver variability regarding the classification and grading of gliomas, particularly those in grades II and III [Brat et al., 2008]. In fact, astrocytomas and oligodendrogliomas are tumours known to have overlapping features in non-classical cases. Similarly, distinguishing between pilocytic astrocytoma or pleomorphic xanthoastrocytoma, on the one hand, and infiltrative malignant glioma (grades III or IV), on the other, can sometimes present a significant challenge for pathologists, as the prognosis and therapeutic approach for these tumours are different [Appin and Brat, 2014].

Complementary tests such as immunohistochemistry and molecular genetic analysis can help clarify the diagnosis and provide a more accurate estimate of the prognosis and predictive value of treatment response. The techniques commonly used to detect deletions or gene amplifications are FISH, loss of heterozygosity (LOH), and comparative genomic hybridization. The mutations associated with certain genes, used as molecular markers, are usually tested in a panel rather than one at a time [Appin and Brat, 2014].

4.3 Brief Description of the Advantages Cited for the New Technology The requesters propose the simultaneous assessment of the status of three different

3. Letter of support dated May 1, 2014, sent to Mrs. Johanne Nicole and Dr. François Sanschagrin, Ph. D, from the Direction générale des services de santé et médecine universitaire (DGSSMU) of the Ministère de la Santé et des Services sociaux (MSSS) from Dr. Benjamin Ellezam, M.D., Dr. Sonia Cellot, M.D., Ph. D, and Dr. Virginie Dormoy-Raclet, Ph. D, from the CHU Sainte-Justine. The information was incorporated in the INESSS notice of June 30, 2014.

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molecular markers: V600E mutation in the BRAF gene, K27M mutation in the HIST1H3B (H3.1) gene, and K27M and G34V/R mutations in the H3F3A (H3.3) gene. This test will be used as a diagnostic, prognostic, and predictive tool for biopsies of pediatric low-grade or high-grade gliomas with ambiguous histopathologic findings. Table 2 shows the advantages sought in more detail.

Table 2: Molecular markers referenced in the current request and associated advantages4

GENE MUTATION ADVANTAGE OTHER INFORMATION

BRAF V600E Predictor of vemurafenib response in cases of recurrent glioma

PXA grade II, 50%-65% of cases GG grade I, 20%-75% of cases

PCA grade I, < 10% of cases GBM grade IV, 6% of cases

Prognosis of GG recurrence

H3F3A HIST1H3B

K27M G34V/R

Differential diagnosis of GBM GBM grade IV, 30% of cases

Prognosis of GBM aggressiveness

Abbreviations: GBM = glioblastoma; GG = ganglioglioma; PCA = pilocytic astrocytoma; PXA = pleomorphic xanthoastrocytoma.

4.4 Cost of Technology and Options: Not assessed.

5. EVIDENCE

5.1 Clinical Relevance and Validity

5.1.1 Other Tests Replaced: No

5.1.2 Diagnostic or Prognostic Value

Regardless of the method used to determine BRAF V600E, H3.3 K27M or G34V/R and H3.1 K27M status, various retrospective cohort studies assessing their frequency among different types of brain tumours were reviewed. Tables 3 and 4 provide a summary of the studies that assessed the respective diagnostic value of these markers, while Table 5 shows the prognostic value of BRAF V600E.

The BRAF V600E mutation (Table 3) is sometimes observed in PCA tumours, but it is more closely associated with pediatric GG grade I (20% to 25%) and PXA grade II-III (60% to 80%), in children as well as in adults [Dias-Santagata et al., 2011; Schindler et al., 2011; Dougherty et al., 2010]. The BRAF V600E mutation has also been associated with grade I PCAs located outside the cerebellum, which is an important characteristic, considering the high frequency with which this type of tumour is found in the cerebellum [Schindler et al., 2011]. Two case series also suggest that the V600E mutation is often found (approximately 50% of cases) together with a rare histologic subtype of GBM, the epithelioid variant [Broniscer et al., 2014; Kleinschmidt-DeMasters et al., 2013]. Although not significant because of the small number of cases studied, a more favourable prognosis for overall survival was also observed [Kleinschmidt-DeMasters et al., 2013]. In terms of prognostic value associated with BRAF V600E mutations in cases of pediatric low-grade gliomas, only one study found a correlation (although not significant) between the presence of the marker and shorter progression-free survival HR = 2.4 [95% CI, 0.9 to 6.2]; p = 0.07 [Horbinski et al., 2012]. Myung et al. [2012]

4. 4. Letter of support dated May 1, 2014, sent to Mrs. Johanne Nicole and Dr. François Sanschagrin, Ph. D, from the Direction générale des services de santé et médecine universitaire (DGSSMU) of the Ministère de la Santé et des Services sociaux (MSSS) from Dr. Benjamin Ellezam, M.D., Dr. Sonia Cellot, M.D., Ph. D, and Dr. Virginie Dormoy-Raclet, Ph. D, from the CHU Sainte-Justine. The information was incorporated in the INESSS notice of July 7, 2014.

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used direct sequencing to assess the diagnostic and predictive values of progression-free survival for BRAF V600E in 51 cases of GG grade I, 45 cases of PCA grade I, and 12 cases of PXA grade II (patients of unknown age). Thirty-six tumours were positive for the mutation, including 66.7% of PXAs, 23.5% of GGs, and 15.6% of PCAs. No correlation was established between the presence of the marker and progression-free survival. Dahiya et al. [2013] used immunohistochemistry to show a significant positive association (p = 0.04) between the presence of the marker and shorter disease-free survival among cases of pediatric GG grade I for which BRAF V600E testing had positive results (18 positive/47 assessed).

In terms of the diagnostic value of K27M and G34R/V mutations (Table 4) in the histone H3.3 (H3F3A) gene, screening of a large cohort of 784 patients of all ages with gliomas with different histologies and grades showed that these mutations are diagnostic markers specific to GBM: 10.5% versus 0.5% for non-GBM (p < 0.0001), and, in addition, to pediatric GBM: 36% versus 3% for adult GBM (p < 0.0001) [Schwartzentruber et al., 2012]. Moreover, three studies [Saratsis et al., 2014; Khuong-Quang et al., 2012; Wu et al., 2012] show that these mutations, as well as K27M of the HIST1H3B (H3.1) gene, are more closely associated with the histologic subtype diffuse intrinsic pontine glioma (DIPG), a very aggressive pediatric GBM of the brainstem: > 70% versus ≈ 20% for GBMs in other sites. Khuong-Quang et al. [2012] also showed that DIPGs with H3.3 mutation status are associated with significantly reduced overall survival compared with those with wild-type status: median survival = 0.7 years ± 0.5 versus 4.6 years ± 5.6; p < 0.001, and hazard ratio (HR) = 4.3 [95% CI, 1.3 to 14.5]; p = 0.019.

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Table 3: Diagnostic value of BRAF V600E

STUDY TARGETED TUMOURS

COHORT HISTOLOGY (N)

BRAF V600E + DIAGNOSTIC VALUE

METHOD N (%)

Broniscer et al., 2014

GBM N = 6 7.6 years [3.5-11]

Epith. (6) Sanger 3 (50) Epithelioid GBM tumour marker?

Kleinschmidt-DeMasters et al., 2013

GBM subtypes

N = 24 (4 < 18 years)

29 years [4-67]

rhabdoid (2) Epith. (13)

GC (9)

Sanger 0 7 (54)

0

Epithelioid GBM tumour marker?

Horbinski et al., 2012

Low-grade gliomas

N = 198 7.6 years

[0-19]

PCA (143) GG (27) PXA (6)

Other (22)

RT-PCR + Sanger

10/110 (9) 5/22 (23) 2/5 (40)

2/20 (10)

Low-grade gliomas Non-PCA vs. PCA (19.1% vs. 7.3%; p = 0.23).

Myung et al., 2012

Brain tumours

N = 223 ?

PCA (45) PXA (12) GG (51)

Other (115)

Sanger 7 (16) 8 (67)

12 (24) 9 (8)

↑ frequency: PXA, GG, and PCA vs. other (52.8% vs. 7.8%; p?). GG grade III vs. I (11.1% vs. 26.2%; p = NS).

Dias-Santagata et al., 2011

PXA et GBM N = 26 PXA 29 years

[7-87] N = 71 GBM ?

PXA (20) PXA III (6) GBM (71)

SNaPshot PCR

12 (60) 1 (17) 2 (3)

PXA V600E and mesenchymal-like growth pattern (p = 0.028)

Schindler et al., 2011

Brain tumours

N = 1,320 (487

< 18 years)

PCA (75) PXA (26)

PXA grade III (10)

GG (25) Other (351)

Sanger 7 (9) 18 (69)

10 (100) 4 (16) 4 (1)

↑ frequency: PXA, GG, and PCA. PCA V600E located outside the cerebellum (20% vs. 2%; p = 0.009).

Dougherty et al., 2010

Mixed glial-neuronal

N = 33 11 years

[0-36]

GG (18) AT/RT (3) PXA (4)

Other (8)

Sanger 9 (53) 3 (100) 1 (25) 1 (13)

↑ frequency: low-grade mixed glial-neuronal tumours

MacConaill et al., 2009

Gliomas N = 155 (127

< 18 years)

GG (14) G grade I-II

(108) HGG (NS) (5)

OncoMap mass

spectrometry

8 (57) 13 (12) 1 (20)

↑ Prevalence: GG vs. other low-grade gliomas (p = 0.00005)

Abbreviations: AT/RT = atypical teratoid /rhabdoid tumour; Epith = epithelioid; G grade I-II = low-grade gliomas; GBM =

glioblastoma; GC = giant cells; GG = ganglioglioma; HGG (NS) = high-grade glioma, type not specified; N = number; NS = not

significant; PCA = pilocytic astrocytoma; PSQ = pyrosequencing; PXA = pleomorphic xanthoastrocytoma; vs. = versus.

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Table 4: Diagnostic value of K27M or G34R/V mutations in histone genes

STUDY COHORT HISTOLOGY (N) METHOD H3.3 N (%) H3.1 N (%) DIAGNOSTIC VALUE

K27M G34R/V K27M

Saratsis et al., 2014 N = 31 8 years [0.5-25]

DIPG (14) GBM grade IV (4)

AA grade III (4) Other (9)

Sanger 8/13 (62) 2/4 (50)

0 0

n.a. 2/5 (40) 1/2 (50)

0 0

K27M H3.3 or H3.1 DIPG: 77%

Venneti et al., 2013 N = 20 Median age 141 months

GBM (20) Sanger 6 (30) 0 n.a. 6 cases with weak or absent immunohistochemical staining

(H3.3 K27 trimethylated )

Broniscer et al., 2014 N = 6 7.6 years [3.5-11]

Epithelioid GBM (6) Sanger 1 (17) 0 0 n.a.

Gielen et al., 2013 N = 338 163 high-grade gliomas

9.5 years [0-18]

GBM grade IV (129) AA grade III (28) AOA grade III (6)

ODG/OA/DA grade II (12) Non-diffuse glial (65)

Other (81)

PSQ 35 (27) 5 (18)

0 0 0 0

7 (5.5) 0

1/6 ? ? ?

n.a. K27M (GBM): SN 27.1% and SP 87.5%

K27M (AA + GBM):

SN 25.5% and SP 100%

Schwartzentruber et al., 2012

N = 159 pediatric gliomas and 625 adult gliomas

GBM grade IV (90) AA grade III (11)

AOD/AOA grade III (7) Low-grade gliomas (51)

Sanger 32 (36) 2 (18)

0 0

n.a. H3.3 mutation GBM vs. non GBM

10.5% vs. 0.5%; p < 0.0001 Pediatric vs. adult GBM 36% vs. 3%; p < 0.0001

Wu et al., 2012 N = 86 7 years [0.8-23]

DIPG (50) Non-truncating GBM (36)

Sanger 30 (60) 7 (19)

0 5 (14)

9 (18) 1 (3)

K27M H3.3 or H3.1 (DIPG vs. other GBM):

78% vs. 22%

Khuong-Quang et al., 2012

N = 42 7 years [0-17]

DIPG (42) Sanger 30 (71) 0 0/29 K27M H3.3 (DIPG vs. GBM):

71% vs. 14%; p < 0.00001

Abbreviations: AA = anaplastic astrocytoma; AOA = anaplastic oligoastrocytoma; AOD = anaplastic oligodendroglioma; DA = diffuse astrocytoma; DIPG = diffuse intrinsic pontine glioma; GBM = glioblastoma; N = number; n.a. = not applicable; OA = oligoastrocytoma; ODG = oligodendroglioma; PSQ = pyrosequencing

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Table 5: Prognostic value of BRAF V600E

STUDY TUMOURS BRAF N (%) TREATMENTS N (%)

DATA RELATED TO PATIENT FOLLOW-UP AND CONDITION

PROGNOSTIC VALUE

Kleinschmidt-DeMasters et al., 2013

N = 24 GBM 30 years [4-69]

No data

Giant cell GBM = improved overall survival? No reference based on BRAF status

2 rhabdoid 0 DOD: 2(100) 22 wk. and 36 wk.

13 epithelioid WT 6 (46) V600E 7 (54)

DOD + AWD ≈ 80 wk. [≥ 26-453] DOD + AWD ≈ 26 wk. [≤ 26-328]

9 with giant cells 0 DOD 4/9 ≈ 62 wk. [47-72] AWD 5/9 ≈ 234 wk. [234-598]

Horbinski et al., 2012 N = 198 low-grade gliomas

7.6 years [0-19]

WT 179 (90) V600E 19 (10)

CX: 113 (57) RT: 27 (14) CT: 21 (11)

Data available 160 (81) Median follow-up: 6.3 years Progressive disease: 50 (31)

DOD: 9 (6)

V600E has a higher risk of progression vs. WT:

HR = 2.5 [95% CI, 0.9 to 6.6]; p = 0.07

Myung et al., 2012 N = 45 PCA 14.4 years [0-54]

V600E 7 (16) No data No data None

N = 12 PXA 27.7 years [6-49]

V600E 8 (67) 1 case with recurrence 7 years initial postsurgery

None

N = 51 GG 25 years [1-64]

V600E 12 (24) 8 cases with recurrence, all WT, unknown time frame

Non-recurrent vs. recurrent V600E 28% vs. 0%; p = NS

PFS: V600E vs. WT: p = NS

N = 115 gliomas Age unknown

V600E 9 (8) No data None

Abbreviations: AWD? = alive with disease of unknown stage; AWPD = alive with progressive disease; CT = chemotherapy; CX = complete surgical resection; DOD = died of disease; GBM = glioblastoma; GG = ganglioglioma; HR = hazard ratio; N = number; NS = not significant; PFS = progression-free survival; PXA = pleomorphic xanthoastrocytoma; RT = radiotherapy; vs. = versus; wk. = week; WT = wild type.

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5.1.3 Therapeutic Value

The requesters propose to use the molecular marker BRAF V600E to predict response to vemurafenib (ZelborafMC, Roche). Vemurafenib, a selective inhibitor of rapidly accelerated fibrosarcoma kinase B (BRAF), which contains the V600 mutation, prevents the activation of the MEK-ERK pathway, thereby inhibiting the proliferation of cancer cells and inducing apoptosis. Vemurafenib is administered orally. It is currently indicated in monotherapy as a first-line treatment for unresectable or metastatic BRAF V6005 mutation-positive melanoma. Another BRAF inhibitor, dabrafenib (TafinlarMC, GSK), was included in the formulary in February 2014 for similar indications.

Vemurafenib has not been approved by the RAMQ, Health Canada6, or the FDA7 for the treatment of primary or recurrent, low-grade or high-grade pediatric gliomas.

In several cases of mainly pediatric and BRAF V600E mutation-positive brain tumours, patients received off-label treatment with vemurafenib, through a temporary authorization or through the Special Access Programme. The cases identified in the literature review are presented in Table 6. In summary, 10 children received vemurafenib for glial tumours that were recurrent or refractory to standard chemotherapy (4 PXA, 3 GG, 1 pilomyxoid astrocytoma, 1 anaplastic astrocytoma and 1 epithelioid GBM). Considering the best outcome recorded, partial (n = 5) or complete (n = 1) regression of the disease followed by a period of progression-free survival was experienced in 6 patients having received treatment. For the 8 cases with disease stabilization, progression-free survival ranging from 2 months to 35 months was recorded. Two patients did not respond to treatment. In most cases, the drug was well-tolerated; only two major events were reported, one of which resulted in death. In the other cases, grade 1 and grade 2 cutaneous toxicities, such as rashes and/or phototoxicity, were reported [Skrypek et al., 2014; Robinson et al., 2014; Bautista et al., 2014; Rush et al., 2013 et Chamberlain et al., 2013].

A multicentre clinical trial is currently underway to assess the safety and pharmacokinetics of vemurafenib in children with recurrent or refractory BRAF V600E8-mutant gliomas.

5. Excerpt from the Notice to the Minister on Zelboraf; published on the INESSS website June 2, 2014. Available at: http://www.inesss.qc.ca/en/activites/drug-products/drug-products-undergoing-evaluation-and-evaluated/extract-notice-to-the-minister/zelboraf-2.html. 6. The ZelborafMC product monograph may be viewed by accessing the Health Canada Drug Product Database (DPD). Available at: http://www.hc-sc.gc.ca/dhp-mps/prodpharma/databasdon/index-eng.php. 7. The ZelborafMC product monograph may be viewed by accessing the FDA Approved Drug Products database, Drugs@FDA. Available at: http://www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm. 8. ClinicalTrials.gov [website]. Vemurafenib in Children With Recurrent/Refractory BRAFV600E-mutant Gliomas. Available at: https://clinicaltrials.gov/ct2/show/NCT01748149.

http://www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm

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Table 6: Predictive value of response to vemurafenib in BRAF V600E gliomas

STUDY CASE TREATMENTS TOXICITY OF VEMURAFENIB

SURGERY RT/CT (FOLLOW-UP, OUTCOME)

VEMURAFENIB (DURATION, OUTCOME)

Skrypek et al., 2014

M, 23 months Optic pathway PMA

Partial 3 courses CT (< 3 years, toxicity/PD)

+ Vinblastine (8 months, PR + SD) pause (2-3 weeks, PD)

+ Vinblastine (12 months, PR + SD) Alone (15 months, SD)

No associated toxicity

Robinson et al., 2014

M, 9 years Epithelioid GBM Frontal-parietal

Partial RT + 1 course CT (3 years, recurrence)

Alone (19 months, CR + SD) Severe rash at first. Mild rash, except with exposure to sun. Partial alopecia and madarosis

Bautista et al., 2014

M, 6 years Thalamic GG grade

III

4 partial 2 courses RT/CT (2 months, PD),

immunotherapy (1 month, PD)

Alone (4 months, SD) Parental decision to withdraw

from immunotherapy Alone (3 months, PR + PD + death)

Elevated transaminase, grade 2 bilirubinemia and creatinemia, grade 1 phototoxicity and appearance of nevus

F, 18 months Peduncular GG

grade III

2 partial 4 courses CT (1 year, PD) Alone (8 months, PR) Alone with dose increase

(12 months, SD)

Grade 2 erythema and appearance of nevus

F, 9 years Astrocytoma grade

III Site unknown

Complete? RT + 3 courses CT (2 years, toxicity/PD)

Alone (2 weeks, toxicity) Grade 5 intracranial hemorrhage

Rush et al., 2013

F, 13 years GG of the brainstem

Partial RT (6 months, PD), monitoring (14 months, PD)

+ Vinblastine (1 month, PR) and dose reduction (1 month, PR)

Arthralgia, keratosis pilaris, and facial telangiectasia. Dose reduction = mild keratosis

Chamberlain, 2013

4 recurrent PXA 2M/2F

45 years [34-53]

Complete 2 Partial 1

Without 1

RT + 2-3 courses CT (? months, PD)

Alone (5 months [2-10]) Outcome at 2 months: PD (1 case),

SD (2 cases), PR (1 case). All deceased: 8 months [4-14]

Grade 2: Arthralgia, fatigue, nausea, and photosensitivity. No grade 3 toxicity.

Abbreviations: CR = complete response; CT = chemotherapy; F = female; GBM = glioblastoma; GG = ganglioglioma; M = male; PD = progressive disease; PMA = pilomyxoid astrocytoma; PR = partial response; PXA = pleomorphic xanthoastrocytoma; RT = radiotherapy; SD = stable disease.

12

RAMQ data were reviewed to determine the number of patients who received vemurafenib in 2012-2013. During this period, 9 received the drug as exceptional patients, and 34 received it as an exceptional medication. All 43 cases were 35 years of age or older and had received a diagnosis of melanoma. Moreover, six of them had also received a diagnosis of brain tumour but at the same time as or later than the melanoma diagnosis. Therefore, according to the RAMQ data, no cases of primary pediatric brain tumours were treated with vemurafenib in 2012-2013.

5.2 Clinical Validity

COMPONENT PRESENCE ABSENCE NOT APPLICABLE

Sensitivity X

Specificity X

Positive predictive value (PPV) X

Negative predictive value (NPV) X

Likelihood ratio (LR) X

ROC curve X

Accuracy X

5.3 Analytical (or Technical) Validity

The requester mentioned, in a letter of support to the MSSS, that the proposed test was in the process of being validated. However, recent communications with the requester confirmed that the validation process is complete.9 In summary, the requester's laboratory chose specific primers for each gene in which the regions surrounding the mutations were to be amplified. DNA was extracted from formalin-fixed paraffin-embedded tumours and PCR was performed. Each amplification product was then sequenced. The laboratory first tested its technique on normal samples, and once refined, the validation was performed on three samples containing a mutation that was clearly identified by a research laboratory at the Montreal Children’s Hospital. In total, the requester’s laboratory used 23 samples during the validation, 3 of which contained a mutation. The requester reports successful detection of the mutations without any difficulty. The development process followed that recommended by international professional organizations such as the Canadian College of Medical Geneticists (CCMG).

A review of the published literature did not identify any analytical validity studies specific to the conditions of use proposed by the requester.

9. Personal electronic communication with Dr. Benjamin Ellezam, neuropathologist at the CHU Sainte-Justine (September 4, 2014).

13

In an article from a journal published in 2012 on the clinical utility of BRAF status, Ziai and Hui mention that, for diagnostic purposes, more than 95% of clinically significant mutations are covered by the molecular analysis of exon 15 of the gene, particularly the V600 position [Ziai and Hui, 2012]. In clinical practice, the vast majority of assessment techniques are based on PCR amplification; any type of biopsy tissue or specimen may be used if the designated PCR amplicon is sufficiently short [Ziai and Hui, 2012]. Moreover, the detection of BRAF mutations in a biopsy specimen (for example, using fine-needle aspiration) requires high analytical sensitivity, as the target cell population might be much smaller than the population of untransformed cells [Adeniran et al., 2011]. However, techniques commonly used in the laboratory such as Sanger-PCR, allele-specific amplification, melting curves, and real-time PCR have the analytical sensitivity and specificity required to test very small tissue samples [Hay et al., 2007; Rowe et al., 2007]. Pyrosequencing has been shown to detect BRAF V600E mutations in tissue samples having a much lower ratios of mutant alleles to wild-type alleles (1:50) than what can be resolved by traditional Sanger sequencing (1:5), although the latter is considerably faster and more cost-effective [Tan et al., 2008; Spittle et al., 2007]. Several of the identified studies provided data on the indirect assessment of the analytical sensitivity of V600E. Data regarding the failure of the test are summarized in Table 7. No analytical validity studies concerning the mutations in the histone (H3.1 and H3.3) genes were identified.

Table 7: Analytical sensitivity PCR + Sanger BRAF V600E

STUDY NUMBER AND HISTOLOGY LACK OF TISSUE TEST FAILURE

Horbinski et al., 2012 198 low-grade gliomas 18 23/180 (13%)

Schindler et al., 2011 1,384 brain tumours 64 (5%)

Dougherty et al., 2010 33 low-grade gliomas 2 (6%) 0

COMPONENT PRESENCE ABSENCE NOT APPLICABLE

Repeatability x

Reproducibility x

Analytical sensitivity x

Analytical specificity x

Matrix effect x

Concordance x

Correlation between test and comparator x

5.4 Recommendations from Other Organizations

In its clinical practice guidelines for central nervous system cancers, the National Comprehensive Cancer Network (NCCN) [2014] does not propose any recommendations regarding the assessment of the mutations referred to in this notice.

14

The Genetic Testing Registry10 refers to only one US laboratory (Cancer Genetics, Children’s Hospital of Philadelphia) approved by CLIA for the detection of BRAF V600E in malignant glioma biopsies. No laboratories offering the molecular analysis of HIST1H3B (H3.1), and K27M, G34V/R of the H3F3A (H3.3) gene have been identified.

The Test Catalogue from The Hospital for Sick Children (SickKids) in Toronto does not include the molecular analyses referred to in this test.11

6. ANTICIPATED OUTCOMES OF INTRODUCING THE TEST

6.1 Impact on Material and Human Resources

Not assessed.

6.2 Economic Consequences of Introducing Test Into Quebec’s Health Care and Social Services System

Not assessed.

6.3 Main Organizational, Ethical, and Other (Social, Legal, Political) Issues

With respect to the predictive value of response to vemurafenib associated with BRAF V600E status, the drug is not indicated for the treatment of primary brain tumours and is not used in Quebec outside of a research protocol. Moreover, the toxicological profile of the drug is still not well understood in terms of its effect on children.

7 IN BRIEF

7.1 Clinical Relevance

The requester’s laboratory proposes testing for BRAF V600E mutations in recurrent pediatric gliomas, as vemurafenib, a drug directly targeting this mutation, is now available on the market. In fact, the mutation is often identified in certain types of low-grade tumours such as pilocytic astrocytomas, pleomorphic xanthoastrocytomas, and gangliogliomas, for which the histologic diagnosis can sometimes easily be mistaken for certain types of high-grade tumours. The requester also proposes testing for specific mutations K27M and G34V/R in histone genes H3F3A and HIST1H3B to distinguish various histologic subtypes of glioblastomas and thus predict their clinical aggressiveness.

7.2 Clinical Validity

In terms of the predictive value of response to vemurafenib, only 10 cases of pediatric gliomas treated with the molecule were identified. However, these limited data show that the drug can be used as a last resort for cases refractory to chemotherapy or as part of maintenance therapy for individuals who tolerate the molecule well. Two cases of severe toxicity were reported, one of which was fatal. The risk of recurrence is significantly higher in GG cases positive for V600E, as was shown by immunohistochemistry. Although the data published by Myung et al. [2012] were in agreement, they were not statistically significant owing to the low number of cases. Studies show that the mutations affecting the histone genes are markers specific to pediatric glioblastomas and specifically histological subtypes

10. National Center for Biotechnology Information (NCBI). Genetic Testing Registry (GTR) [website]. Available at: http://www.ncbi.nlm.nih.gov/gtr/. 11. SickKids. Test Catalogue [website]. Available at: http://www.sickkids.ca/paediatriclabmedicinems/test-catalogue/lab-services.html.

http://www.ncbi.nlm.nih.gov/gtr/labs/309826/http://www.ncbi.nlm.nih.gov/gtr/labs/309826/http://www.sickkids.ca/paediatriclabmedicinems/test-catalogue/lab-services.html

15

with a very poor prognosissuch as brainstem tumours (DIPG).

7.3 Analytical Validity

Very few data on analytical performance were identified. The small quantity of tumour tissue available in certain biopsy samples and the possible presence of healthy tissue reinforce the need to use a sensitive technique. The Sanger sequencing method appears to meet these requirements.

7.4 Recommendations from Other Organizations: None were identified.

16

8 INESSS NOTICE IN BRIEF

Detection of Targeted Mutations in Pediatric Brain Tumours (K27M and G34V/R in H3.3, K27M in H3.1, and V600E in BRAF)

Status of the Diagnostic Technology

Established

Innovative

Experimental (for research purposes only)

Replacement for technology: , which becomes obsolete

INESSS Recommendation

Include test in the Index

Do not include test in the Index

Reassess test* once the following are submitted:

A demonstration of the clinicopathological correlation.

A justification of the added value of testing these mutations simultaneously.

Details concerning the availability of the targeted therapy.

Additional Recommendation

Draw connection with listing of drugs, if companion test

Produce an optimal use manual

Identify indicators, when monitoring is required

NOTE

*Considering that the detection of the V600E BRAF mutation is already in the Index (Code 60034)

17

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Bautista F, Paci A, Minard-Colin V, Dufour C, Grill J, Lacroix L, et al. Vemurafenib in pediatric patients with BRAFV600E mutated high-grade gliomas. Pediatr Blood Cancer 2014;61(6):1101-3.

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Broniscer A, Tatevossian RG, Sabin ND, Klimo P Jr, Dalton J, Lee R, et al. Clinical, radiological, histological and molecular characteristics of paediatric epithelioid glioblastoma. Neuropathol Appl Neurobiol 2014;40(3):327-36.

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Dahiya S, Haydon DH, Alvarado D, Gurnett CA, Gutmann DH, Leonard JR. BRAF(V600E) mutation is a negative prognosticator in pediatric ganglioglioma. Acta Neuropathol 2013;125(6):901-10.

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Dougherty MJ, Santi M, Brose MS, Ma C, Resnick AC, Sievert AJ, et al. Activating mutations in BRAF characterize a spectrum of pediatric low-grade gliomas. Neuro Oncol 2010;12(7):621-30.

Dunham C. Pediatric brain tumors: A histologic and genetic update on commonly encountered entities. Semin Diagn Pathol 2010;27(3):147-59.

Fedoul B and Souirti Z. Cerebellar ganglioglioma. Pan Afr Med J 2012;12:12.

Fernandez C, Figarella-Branger D, Girard N, Bouvier-Labit C, Gouvernet J, Paz Paredes A, Lena G. Pilocytic astrocytomas in children: Prognostic factors—A retrospective study of 80 cases. Neurosurgery 2003;53(3):544-55.

Gielen GH, Gessi M, Hammes J, Kramm CM, Waha A, Pietsch T. H3F3A K27M mutation in pediatric CNS tumors: A marker for diffuse high-grade astrocytomas. Am J Clin Pathol 2013;139(3):345-9.

Hay R, MacRae E, Barber D, Khalil M, Demetrick DJ. BRAF mutations in melanocytic lesions and papillary thyroid carcinoma samples identified using melting curve analysis of polymerase chain reaction products. Arch Pathol Lab Med 2007;131(9):1361-7.

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Horbinski C, Nikiforova MN, Hagenkord JM, Hamilton RL, Pollack IF. Interplay among BRAF, p16, p53, and MIB1 in pediatric low-grade gliomas. Neuro Oncol 2012;14(6):777-89.

Ichimura K, Nishikawa R, Matsutani M. Molecular markers in pediatric neuro-oncology. Neuro Oncol 2012;14(Suppl 4):iv90-9.

Karajannis M, Allen JC, Newcomb EW. Treatment of pediatric brain tumors. J Cell Physiol 2008;217(3):584-9.

Khuong-Quang DA, Buczkowicz P, Rakopoulos P, Liu XY, Fontebasso AM, Bouffet E, et al. K27M mutation in histone H3.3 defines clinically and biologically distinct subgroups of pediatric diffuse intrinsic pontine gliomas. Acta Neuropathol 2012;124(3):439-47.

Kleinschmidt-DeMasters BK, Aisner DL, Birks DK, Foreman NK. Epithelioid GBMs show a high percentage of BRAF V600E mutation. Am J Surg Pathol 2013;37(5):685-98.

Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, et al. The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol 2007;114(2):97-109.

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Rodriguez FJ, Lim KS, Bowers D, Eberhart CG. Pathological and molecular advances in pediatric low-grade astrocytoma. Annu Rev Pathol 2013;8:361-79.

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Saratsis AM, Kambhampati M, Snyder K, Yadavilli S, Devaney JM, Harmon B, et al. Comparative multidimensional molecular analyses of pediatric diffuse intrinsic pontine glioma reveals distinct molecular subtypes. Acta Neuropathol 2014;127(6):881-95.

Schindler G, Capper D, Meyer J, Janzarik W, Omran H, Herold-Mende C, et al. Analysis of BRAF V600E mutation in 1,320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma, ganglioglioma and extra-cerebellar pilocytic astrocytoma. Acta Neuropathol 2011;121(3):397-405.

Schwartzentruber J, Korshunov A, Liu XY, Jones DT, Pfaff E, Jacob K, et al. Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma. Nature 2012;482(7384):226-31.

Skrypek M, Foreman N, Guillaume D, Moertel C. Pilomyxoid astrocytoma treated successfully with vemurafenib. Pediatr Blood Cancer 2014;61(11):2099-100.

Spittle C, Ward MR, Nathanson KL, Gimotty PA, Rappaport E, Brose MS, et al. Application of a BRAF pyrosequencing assay for mutation detection and copy number analysis in malignant melanoma. J Mol Diagn 2007;9(4):464-71.

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Takei H, Yogeswaren ST, Wong KK, Mehta V, Chintagumpala M, Dauser RC, et al. Expression of oligodendroglial differentiation markers in pilocytic astrocytomas identifies two clinical subsets and shows a significant correlation with proliferation index and progression free survival. J Neurooncol 2008;86(2):183-90.

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Venneti S, Garimella MT, Sullivan LM, Martinez D, Huse JT, Heguy A, et al. 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 2013;23(5):558-64.

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Wu G, Broniscer A, McEachron TA, Lu C, Paugh BS, Becksfort J, et al. Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas. Nat Genet 2012;44(3):251-3.

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20

APPENDIX A Diagnostic Algorithm

HISTOLOGICALLY UNEQUIVOCAL TUMOURS

Gliobalstoma

(GBM)

Pilocytic astrocytoma/

Pleomorphic xanthoastrocytoma

(PXA)/ Ganglioglioma

- Vemurafenib treatment during

recurrence (phase I)

- Poorer prognosis? (ganglioglioma)

- More favourable

prognosis

- Vemurafenib

therapy (BRAF

inhibitor)

- Very poor prognosis

- Closer monitoring and scale-up of treatment

- Referral to phase I studies

Standard risk - Standard risk

- No treatment with vemurafenib

21

HISTOLOGICALLY EQUIVOCAL TUMOURS

Glioblastoma (GBM) versus pilocytic

astrocytoma versus pleomorphic

xanthoastrocytoma (A-PXA) versus

undifferentiated glial-neuronal tumour

- Diagnosis remains

equivocal

- Treatment with

vemurafenib

- Diagnosis: GBM - Equivocal diagnosis