MUTATION ANALYSIS OF THE KINASE DOMAIN OF THE BCR-ABL FUSION PROTEIN (REFERENCE )

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1 MUTATION ANALYSIS OF THE KINASE DOMAIN OF THE BCR-ABL FUSION PROTEIN (REFERENCE ) Notice of Assessment December 2014 DISCLAIMER: This document was originally drafted in French by the Institut national d'ecellence en santé et en services sociau (INESSS), and that version can be consulted at alyses_bio_med_dec_2014_4.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.

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3 1. GENERAL INFORMATION 1.1 Requester: Hôpital Maisonneuve-Rosemont 1.2 Application for Review Submitted to MSSS: April 22, Application Received by INESSS: May 10, 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 Mutation analysis of the kinase domain coding region of the BCR-ABL fusion protein using Sanger fluorescent sequencing. 2.2 Brief Description of the Technology, and Clinical and Technical Specifications Tests for mutations in mrna transcripts coding for BCR-ABL enable the detection and characterization of primary and secondary resistance to tyrosine kinase inhibitors (TKI), thus ensuring optimization of the therapeutic approach, as needed. The method used involves synthesizing cdna and completing the first amplification step using the OneStep RT-PCR kit by QIAGEN and a purified total RNA sample from the patient. Nested PCR is then carried out to increase the sensitivity and specificity of the test. The PCR product is then purified using the QIAquick kit by QIAGEN, and the sequencing reaction is performed using the BigDye Terminator v3.1 kit (Applied Biosystems, Life Technologies). The sequencing products are purified with the DyeE 2.0 kit by QIAGEN, and the sequence is resolved by capillary electrophoresis with a 3130XL Genetic Analyzer and using the Sequencher program (Gene Codes Corporation). The primers chosen for the PCR and sequencing reactions were selected by the requester and are specific to the translocation point characterized in advance for each patient. 2.3 Company or Developer PCR and sequencing reactions are carried out using in-house primers. The OneStep RT-PCR kit was designed and produced by QIAGEN. The sequencing reaction is carried out with the BigDye Terminator v3.1 kit (Life Technologies). The QIAquick PCR Purification Kit and the DyeE 2.0 sequencing reaction kit are produced by QIAGEN. The Sequencher program, used for the test and the electropherogram, is produced by Gene Codes Corporation. 2.4 Licence(s): Not applicable. 2.5 Patent, If Any: Not applicable. 1

4 2.6 Approval Status (Health Canada, FDA): This test and the technologies used have not been approved by Health Canada or the FDA. 2.7 Weighted Value: CLINICAL INDICATIONS, PRACTICE SETTINGS, AND TESTING PROCEDURES 3.1 Targeted Patient Group This test is indicated for adult or pediatric patients who have been diagnosed with chronic myeloid leukemia (CML) or acute lymphoblastic leukemia (ALL) with the Philadelphia chromosome (Ph+) who are being treated with a tyrosine kinase inhibitor (TKI) and show signs of drug resistance. 3.2 Targeted Disease(s) Chronic Myeloid Leukemia (CML) Chronic myeloid leukemia, a myeloproliferative disorder involving the myeloid precursors in the bone marrow, is characterized by the presence of the Philadelphia chromosome (Ph+). The Philadelphia chromosome is a result of the reciprocal translocation between chromosomes 9 and 22 [t(9;22)(q34;q11 1 )], which, in turn, leads to the fusion of the BCR (breakpoint cluster region) gene and the ABL1 (Abelson murine leukemia) gene (Figure 1). Figure 1: Philadelphia Chromosome Source: National Cancer Institute. Chronic Myelogenous Leukemia Treatment (PDQ ) [website]. Available at: Permission to reproduce this figure obtained August 4, 2014 by Terese Winslow. The resulting BCR-ABL1 fusion protein has a constitutively active tyrosine kinase that causes the anarchic proliferation of myeloid cells in the bone marrow; hence, it plays a pivotal role in the pathogenesis of CML [Negrin and Schiffer, 2014]. The instability characterizing BCR- ABL-positive cells leads to BCR-ABL kinase domain point mutations, which are associated with progression from the chronic phase to the accelerated phase and blast phase (also called acute phase) [Baccarani et al., 2012]. The acute phase requires stem cell transplantation. 1. The Philadelphia chromosome is a chimeric chromosome that arises from a translocation (displacement) between chromosome 9, in region q34 (long arm, band 3, sub-band 4) and chromosome 22, in region q11 (long arm, band 1, sub-band 1). 2

5 Chronic myeloid leukemia is generally diagnosed during the chronic phase on the basis of an abnormal complete blood count and symptoms such as fatigue, anoreia, and weight loss [Sawyers, 1999]. The European LeukemiaNet (ELN) and the World Health Organization (WHO) have established criteria defining the accelerated and blast phases of CML (Table 1). Table 1: Criteria defining the accelerated and blast phases of chronic myeloid leukemia ACCELERATED PHASE BLAST OR ACUTE PHASE ELN CRITERIA 15% to 29% of blasts 2 in blood or bone marrow; or more than 30% of blasts + promyelocytes in blood or bone marrow with blasts < 30% Basophils in blood 20% Persistent thrombocytopenia (< /L) not related to treatment Clonal chromosomal abnormalities in Ph+ cells, major route abnormalities, under treatment. 30% of blasts in blood or bone marrow Etramedullary blast proliferation, ecept in spleen WHO CRITERIA 10% to19% of blasts in blood or bone marrow. Basophils in blood 20% Persistent thrombocytopenia (< /L) not related to treatment Clonal chromosomal abnormalities in Ph+ cells, under treatment. Thrombocytosis (> /L) not related to treatment Increase in spleen size and in the number of leukocytes, unresponsive to treatment. 20% of blasts in blood or bone marrow Etramedullary blast proliferation, ecept in spleen Blast cell aggregates in bone marrow biopsy Source: adapted and translated from Baccarani et al., Epidemiology According to the latest statistics, the incidence of CML in 2007 (age-standardized for both sees) was 96 cases per year in Quebec, or a rate of 0.9/100, No information was found on the prevalence of the disease in Quebec. Prognosis Without treatment, the benign chronic phase usually progresses towards fatal blast crisis within 3 to 5 years [Sawyers, 1999]. Jabbour and Kantarjian [2014] reported the results of an 8-year follow-up of the IRIS study (International Randomized Study of Interferon and STI571). With the use of imatinib, a tyrosine kinase inhibitor (TKI) used in first-line therapy, the overall survival rate observed at 8 years is 93% [Jabbour and Kantarjian, 2014]. However, the emergence of mutations in the kinase domain of BCR-ABL during treatment with a first-line TKI such as imatinib leads to a loss of complete cytogenetic response (CCyR 4 ), shorter progression-free survival, shorter progression time from the accelerated phase to the blast phase, and shorter overall survival [Press et al., 2009; Soverini et al., 2005]. Second-generation TKIs were developed for this reason. Lastly, in the case of TKI 2. Immature cells found in blood or bone marrow. 3. Statistics Canada. Cancer Incidence in Canada 2007 and New cases for ICD-O-3 primary sites of cancer (based on the July 2010 CCR tabulation file) Quebec. Available at: (accessed August 4, 2014). 4. Levels of cytogenetic response are classified according to the proportion of Ph+ metaphases found in bone marrow biopsies, as follows: complete (CCyR), 0%; partial (PCyR), 1% to 35%; minor, 36% to 65%; minimal, 66% to 95%; absence of CyR, 96%. A major cytogenetic response (MCyR) is the sum of CCyR and PCyR [Guilhot et al., 2007]. 3

6 treatment failure, a transplant is recommended if the patient is eligible. Treatment Tyrosine kinase inhibitors are the treatment of choice for CML 5 in the chronic phase. In accelerated and blast phases, stem cell transplantation is the standard treatment, followed by TKI if patients are ineligible or waiting for a donor [Groupe québécois de recherche en LMC-NMP, 2013]. Imatinib was the first inhibitor used; thereafter, other TKI entered the market, including dasatinib, nilotinib, bosutinib, and, more recently, ponatinib [Negrin and Schiffer, 2014; Baccarani et al., 2013]. One of the mechanisms of resistance to this treatment involves point mutations in the BCR-ABL kinase domain Acute Lymphoblastic Leukemia (ALL) Acute lymphoblastic leukemia is a hematological disease characterized by an increased proliferation of lymphoid precursors in the bone marrow. Subtypes of ALL (pre-b cell, mature B cell, and T cell) have been characterized in adults and children [Jabbour et al., 2005]. The BCR-ABL1 fusion gene is found primarily in adults, in 25% of cases, compared with 3% of cases in children [NCCN, 2013]. The TEL-AML1 fusion gene [t(12;21)(p13;q22)] is the most frequently observed chromosomal abnormality in children with ALL, affecting 22% of cases compared with 2% of adult cases [NCCN, 2013]. Epidemiology According to the latest statistics, the incidence of ALL (age-standardized for both sees) is 100 cases per year in Quebec, or a rate of 1.4/100, No information was found on the prevalence of the disease in Quebec. Prognosis A population-based study determined survival rates from the SEER (Surveillance, Epidemiology, and End Results) Program of the National Cancer Institute (NCI); the study involved 12,096 patients aged from birth to 14 years with ALL who were diagnosed between 1981 and 2010 [Ma et al., 2014]. The 5-year survival rates were 74.8% (1981 to 1990), 84.5% (1991 to 2000), and 88.6% (2001 to 2010) in patients up to 14 years of age. Results were similar for 10-year survival rates, which increased by 15%, from 69.3% (1981 to 1990) to 80.9% (1991 to 2000) to 85.5% (2001 to 2010). In children aged 10 years to 14 years at the time of diagnosis, an age range generally associated with a poor prognosis, the 10-year survival rates increased by 20 percentage points, from 54.1% (1981 to 1990) to 69.4% (1991 to 2000) to 75.3% (2001 to 2010). According to the authors, these results are likely attributable to improvements in therapy, optimization of methods for studying cytogenetic and molecular abnormalities, and greater accuracy of information about diagnosis and prognosis. It is important to note that survival of patients with ALL is not directly related to treatment with TKI. For these patients, TKI and other types of chemotherapy are a preparatory step for stem cell transplantation, which is the standard of care. Treatment The guidelines from the National Comprehensive Cancer Network [NCCN, 2013] for the treatment of ALL provide all the information and treatment algorithms needed for each 5. See Appendi A for more information on the tyrosine kinase inhibitors included in the RAMQ list and the indications recognized for coverage by the Public Prescription Drug Insurance Plan. 6. Statistics Canada. Cancer Incidence in Canada 2007 and New cases for ICD-O-3 primary sites of cancer (based on the July 2010 CCR tabulation file) Quebec. Available at: (accessed August 4, 2014). 4

7 stage of the disease. Unlike treatment for CML, treatment for ALL 7 involves the use of TKI, along with other types of chemotherapy, as preparatory treatment for transplantation. 3.3 Number of Patients Targeted The requester offers sequencing of BCR-ABL in its clinical laboratory and estimates the number of yearly requests at 50 to 100 (based on its data since 2009). 3.4 Medical Specialties and Other Professions Involved Molecular biology, hematology-oncology. 3.5 Testing Procedure This test is ordered according to the algorithm recommended in the treatment guidelines published by the Groupe québécois de recherche en LMC-NMP [2013]. It may be requested by the clinician who performs patient follow-up or by the hematologist associated with the laboratory of the requester who does the molecular monitoring of CML, including molecular response to TKI therapy with quantitative PCR (test "Leucémie LMC et LLA (BCR/ABL) de la t(9;22)" included in the MSSS Inde [code 20731]). The test is conducted in the following circumstances: Failure to achieve therapeutic milestones; An increase of more than 0.5 log in BCR-ABL transcripts covered by RQ-PCR in two consecutive samples and transcript levels equal to or greater than log (0.1% IS [international scale]); Lack of response after the introduction of an alternative TKI or new loss of response. The turnaround time for results is 10 days. 4. TECHNOLOGY BACKGROUND 4.1 Nature of the Diagnostic Technology This is a unique test. The sequencing of BCR-ABL RNA transcripts was included in the Inde until The test was removed after the implementation of a program stemming from a collaboration with a private laboratory. As a result of the provision of this service, the code in the Inde ceased to be used and was removed a few years ago. 4.2 Brief Description of the Current Technological Contet The follow-up of CML treatment involves molecular monitoring, whereby response to TKI therapy is determined by quantifying the number of BCR-ABL RNA transcripts present in peripheral blood or in the bone marrow, which is proportional to the quantity of leukemia cells. The quantification of the number of transcripts is performed with RQ-PCR (quantitative PCR) every three months, and the progression of response is monitored over time. A major molecular response characterized by a log (or 0.1% IS) reduction in the number of transcripts detected is sought within 18 months after the start of treatment. The quality of response to TKI, as well as the loss of this response, leads to the search for the etiology of the problem, including the detection of mutations in BCR-ABL transcripts, 7. See Appendi A for more information on tyrosine kinase inhibitors included in the RAMQ list and indications recognized for coverage. 5

8 treatment non-compliance, and bioavailability [Jabbour et al., 2011; Quintas-Cardama et al., 2009]. Sanger sequencing is the laboratory standard for the detection of BCR-ABL mutations [Soverini et al., 2011]. However, two pre-screening techniques may precede Sanger-based direct sequencing: denaturing high-performance liquid chromatography (dhplc) and fourchannel asymmetric real-time PCR hybridization probe assay. These two approaches enable the rapid and cost-effective assessment of a large number of samples. Nevertheless, sequencing must still be performed to characterize the specific mutation in samples in which a mutation is found. Direct Sequencing with the Sanger Method Sanger-based direct sequencing is the method currently recommended (reference method) for BCR-ABL kinase domain mutation analysis [Soverini et al., 2011], despite its numerous technical limitations. However, this method provides only an estimate of mutated clone abundance, and it cannot discriminate between compound mutations (belonging to the same transcript) and polyclonal mutations (belonging to different transcripts) [Soverini et al., 2013]. Denaturing High-Performance Liquid Chromatography (dhplc) This technique uses HPLC to detect sequence variants. The PCR product is mied with a wildtype sample (Ph+ sample without any known mutations after sequencing) then denatured to promote heteroduple formation. Samples with an altered dhplc profile, which is indicative of a mutation, are then subjected to direct sequencing to characterize the mutations [Frueh and Noyer-Weidner, 2003]. DHPLC is a straightforward and rapid method used to pre-screen samples for sequence variations, which greatly reduces the number of samples to be sequenced [Soverini et al., 2011]. This method has a slightly higher sensitivity that Sanger sequencing, but it does not allow characterization of the sequence variation underlying an abnormal elution profile [Soverini et al., 2011]. Four-Channel Asymmetric Real-Time PCR Hybridization Probe Assay This method involves a single real-time PCR reaction combining the use of four pairs of FRET (fluorescence resonance energy transfer) primers for the simultaneous detection of all BCR- ABL kinase domain mutations described in imatinib resistance [Martinez-Serra et al., 2012; Soverini et al., 2011]. This rapid pre-screening method is conducted prior to Sanger sequencing, thus eliminating the need to process a large number of samples. Ultra-Deep Sequencing (UDS) Soverini et al. define UDS as the net generation of sequencing technology. UDS is a highlysensitive method used to characterize minor mutations that are not detectable by Sanger sequencing (low-abundance mutations, present in 10% to 15% of cells), follow the dynamics of resistant mutations over time, and reconstruct the clonal architecture of mutated populations (in the case of multiple mutations occurring within the same amplicon) [Soverini et al., 2013]. This is an eperimental technique, and the contribution of lowabundance mutations is unknown. 4.3 Brief Description of the Advantages Cited for the New Technology The Sanger method is used to assess BCR-ABL fusion protein mutation status to help guide the choice of an effective tyrosine kinase inhibitor (TKI) based on the mutation detected, thereby optimizing treatment for CML or ALL. Should the use of a TKI prove to be 6

9 inappropriate, clinicians may recommend that patients consider bone marrow transplantation if their condition allows it or another therapeutic option. 4.4 Cost of Technology and Options: Not assessed. 5. EVIDENCE 5.1 Clinical Relevance Other Tests Replaced: Not applicable Diagnostic or Prognostic Value Although the majority of patients with CML respond well to treatment with imatinib, 20% to 30% develop resistance [Quintas-Cardama et al., 2009]. In fact, resistance to imatinib and other TKIs can be linked to a combination of factors such as patient compliance with treatment, bioavailability, pharmacodynamics, genetic changes, BCR-ABL kinase domain mutations, or a combination of these factors [Quintas-Cardama et al., 2009]. Based on a review of the literature, Cortes et al. [2012] reported the conclusions of two studies, which indicate that mutations in the kinase domain of BCR-ABL fusion proteins are the primary cause of treatment resistance. These mutations are present in 40% to 60% of cases of secondary resistance 8 [Cortes et al., 2012]. At least 70 mutations involving 57 different amino acids were identified in the BCR-ABL kinase domain, and they are usually distributed in four regions (ATP-binding P-loop, SH3 contact, SH2 contact, and activation [A-] loop) [Jones et al., 2009]. BCR-ABL mutation analysis is critical in determining the best treatment strategy [Cortes et al., 2012] and in helping to guide the selection of TKIs when there are signs of drug-resistance [NCCN, 2014; Soverini et al., 2011]. Data on the prognostic value of BCR-ABL mutational analysis were obtained from studies focusing solely on CML (Table 2). Several retrospective studies on patients with CML most of whom were in the chronic phase and had developed a resistance to imatinib revealed that the detected mutations (T315I mutation and P-loop mutations) were associated with lower progression-free survival (PFS) and overall survival (OS) [Khorashad et al., 2008; Nicolini et al., 2006; Soverini et al., 2005; Branford et al., 2003]. However, results of the study by Jabbour et al. showed that mutations in the P-loop region did not affect overall survival [Jabbour et al., 2006]. According to the authors, the discrepancy between the studies by Branford et al. [2003] and those of Soverini et al. [2005] stems from differences in patient selection and these differences suggest that the prognosis depends on various factors other than P-loop mutations. Lastly, combined data from randomized control trials (without meta-analysis) totalling 1,043 patients [Muller et al., 2009] and data from a cohort study of 174 patients (START A, phase II trial) [Apperley et al., 2009] show the positive effects of an alternative treatment with dasatinib (2nd generation TKI) in patients with CML (chronic or accelerated phase, respectively) who are resistant or intolerant to imatinib (Table 2). 8. Secondary resistance: achievement and then subsequent loss of a hematologic or cytogenetic response. Primary resistance is defined as a lack of response to treatment [Jabbour et al., 2011]. 7

10 Table 2: Several studies on the prognostic value of BCR-ABL mutation analysis in cases of chronic myeloid leukemia (CML) STUDY NUMBER OF CASES METHOD NUMBER OF PATIENTS WITH MUTATIONS (%) NUMBER OF PATIENTS RESISTANT TO TKI PROGNOSTIC VALUE CLINICAL RESULTS Patients treated with imatinib Branford et al., 2003 n = 144 AP: 40 late 9 CP: 64 early CP: 40 Direct sequencing Total: 27/144 AP: 13/40 (33) Late CP: 14/64 (22) Early CP: 0/40 (0) P-loop mutations: 13/27 Non-P-loop mutations: 14/27 With detected mutation: AP: 13/13 Late CP: 11/14 Without detected mutation: AP: 2/27 Late CP: 2/50 Early CP: 6/40 With detected mutation: 13/19 Number of deaths Imatinib-resistant patients: AP: 9/13 Late CP: 5/14 P-loop mutations: 12/13 (92%); median survival 4.5 months (range: 0.5 to 12) Non-P-loop mutations: 3/14 (21%); median survival 11 months (range 0.2 to 7) Soverini et al., 2005 n = 40 late CP dhplc and Sanger Total: 19/40 (48) P-loop: 9/19 Non P-loop: 10/19 including 1 with a silent mutation Progression towards AP or BP: 11/18 (median time of 9 months (6-30 months) after detection of the mutation and 12 months (7-33 months) from the start of imatinib treatment) Number of deaths: 7/18 Overall survival after a median follow-up of 39.2 months (5 months to 67.2 months) P-loop: 28.3 months T315I: 12.6 months Other sites: endpoint not reached (p < ) Nicolini et al., 2006 n = 89 imatinibresistant CP: 57 AP: 21 BP: 11 n = 171 imatinibresistant Early CP: 68 Late CP: 66 AP: 34 BP: 3 Direct sequencing Total: 89/89 T315I: 18 P-loop: 23 Other sites: 48 T315I: 6/18 P-loop: 12/23 Other sites: 35/48 Jabbour et al., 2006 Direct sequencing Total: 62/171 Early CP: 16/68 (24) Late CP: 28/66 (42) AP: 17/34 (50) BP: 1/3 (33) P-loop: 24/62 (39) 171 Number of deaths after a median follow-up of 56 months (range 4 to 240 months) With mutations: 9/62 (15%) Without mutations: 12/109 (11%) (p = 0.63) P-loop mutations: 2/23 (9%) Other mutations: 7/39 (18%) 9. Late chronic phase: 12 months after diagnosis. 8

11 STUDY NUMBER OF CASES METHOD NUMBER OF PATIENTS WITH MUTATIONS (%) NUMBER OF PATIENTS RESISTANT TO TKI Risk of complete cytological resistance in the presence of mutations: RR: 3.8; p: PROGNOSTIC VALUE CLINICAL RESULTS Khorashad et al., 2008 n = 319 Early CP: 171 Late CP: 148 Direct sequencing Total: 37/319 (11.6) Cumulative incidence over 5 years: 13.9% Progression-free survival at 5 years Without mutations: 86% P-loop mutations: 20% Non-P-loop mutations: 47% Highly resistant mutations: 0% Other mutations: 64.7% Patients treated with dasatinib Muller et al., 2009 n = 1,043 (3 studies) Imatinibresistant: 805 Imatinibintolerant: 238 dhplc and Sanger Total: 402/1, /805 (48) 18/238 (8) Suboptimal resistance or response to imatinib With detected mutations: 384/402 Without detected mutations: 421/641 After treatment with dasatinib Progression-free survival at 24 months With mutations: 70% Without mutations: 80% Overall survival at 24 months With mutations: 88% Without mutations: 92% Progression-free survival at 12 months: 66% Overall survival at 12 months: 82% MHR 10 : 64% (111/174) (95% CI, 56.2% to 70.9%) CHR: 45% (78/174) MCyR 11 : 39% (67/174) (95% CI, 31.2% to 46.2%) CCyR: 32% (55/174) Apperley et al., 2009 n = 174 (START A, phase II trial) Imatinibresistant: 161 Imatinibintolerant: 13 dhplc and/or Sanger Before treatment with dasatinib: Total: 88/156 patients tested (56) 87/145 imatinib-resistant patients (60) 1/11 imatinib-intolerant patients (9) Abbreviations: AP = accelerated phase chronic myeloid leukemia; BP = blast phase chronic myeloid leukemia; CCyR = complete cytogenetic response; CHR = complete hematologic response; dhplc = CP = chronic-phase chronic myeloid leukemia; denaturing high performance liquid chromatography; MCyR = major cytogenetic response; MHR = major hematologic response; n = number of patients; RR = relative risk. 10. A major hematological response (MHR) is considered to fulfill the criteria for complete hematological response (CHR) or to indicate that there is no evidence of leukemia (NEL). Complete hematological response (CHR) is defined according to the following parameters: a leukocyte count below the upper limit of the reference range, absolute neutrophil count /L; platelets /L; no blasts or promyelocytes in peripheral blood; bone marrow blasts 5%, myelocytes + metamyelocytes in peripheral blood 5%; basophils in peripheral blood/bone marrow 2%; no etramedullary contribution. NEL is defined as the absence of blasts or promyelocytes in peripheral blood, blasts 5% and no etramedullary contribution [Guilhot et al., 2007]. 11. Levels of cytogenetic response are classified according to the proportion of Ph+ metaphases found in bone marrow biopsies, as follows: complete (CCyR), 0%; partial (PCyR), 1% to 35%; minor, 36% to 65%; minimal, 66% to 95%; absence of CyR, 96%. A major cytogenetic response (MCyR) is the sum of CCyR and PCyR [Guilhot et al., 2007]. 9

12 5.1.3 Therapeutic Value The proposed test is used to detect BCR-ABL kinase domain mutations in the event of suboptimal response, loss of response, or TKI treatment failure, thereby identifying a cause of the resistance. The results obtained allow the optimization of treatment and enable the best possible use of medications. Thus, certain mutations confer resistance specifically to some TKIs and not others. Table 3 provides a list of BCR- ABL1 kinase domaine mutations and their respective in vitro sensitivity to the five TKIs used in the treatment of CML. The level of sensitivity of a BCR-ABL fusion gene mutation to a given TKI is epressed as 50% inhibitory concentration (IC 50 ). This concentration may be used to guide the selection of TKI, since there is a correlation between the IC 50 value for an in vitro mutation and the clinical response in patients with the same mutation [Baccarani et al., 2013]. According to the requester, ten mutations have a distinctive sensitivity to TKI (grey rows in Table 3). The F359C and V299H mutations are not presented in this table. 10

13 Table 3: Major resistance mutations and IC 50 range of tyrosine kinase inhibitors that may be used BCR-ABL1 Imatinib IC50, range (nmol) Nilotinib IC50, range (nmol) Dasatinib IC50, range (nmol) Bosutinib IC50 (nmol) Ponatinib IC50 (nmol) No mutations < M244V* 1,600-3, L248V 1,866-10, n.a. NA G250E* 1,350 to > 20, Q252H 734-3, Y253F > 6,400-8, Y253H* > 6,400-17, , n.a. 6.2 E255K* 3,174-12, E255V 6,111-8, D276G 1, NA E279K 1, NA V299L ,086 NA F311L 480-1, NA NA T315I* > 6,400 to > 20, to > 10, to > 1,000 1, T315A 125 n.a. 760 n.a. 1.6 F317L* 810-7, F317V n.a. n.a. 10 M351T* 880-4, F359V* 1,400-1, V379I 1,000-1, n.a. NA L384M* 674-2, NA L387M 1,000-1, n.a. NA H396R* 1,750-5, NA H396P 850-4, F486S 2,728-9, NA Table adapted from Baccarani et al., Abbreviations: IC 50 = half maimal inhibitory concentration; NA = not available; nmol = nanomole. * Representative of the ten most common mutations. 11

14 Two TKI (bosutinib and ponatinib) are not included in the RAMQ list, and the remaining three are listed as eceptional medications (Appendi A). Based on the data provided by the requester, a mutation analysis performed on patients with CML revealed the presence of 39 mutations in 16 (11%) patients. Of these mutations, 64% had a therapeutic impact on 75% (12/16) of patients. 5.2 Clinical Validity COMPONENT PRESENCE ABSENCE NOT APPLICABLE Sensitivity Specificity Positive predictive value (PPV) Negative predictive value (NPV) Likelihood ratio (LR) ROC curve Accuracy The studies described in the preceding section (Table 2) also show the link between TKI resistance and BCR-ABL mutations. 5.3 Analytical (or Technical) Validity COMPONENT PRESENCE ABSENCE NOT APPLICABLE Repeatability Reproducibility Analytical sensitivity Analytical specificity Matri effect Concordance Correlation between test and comparator Sensitivity According to consensus among international eperts (National Institutes of Health, 2005) and a literature review, Sanger-based direct sequencing can detect mutations with a sensitivity of 15% to 25%, that is, 1 mutation in approimately 1 out of 5 BCR- ABL transcripts, compared with other more sensitive technologies such as dhplc, which has a sensitivity of 0.1% to 10% [Hughes et al., 2006]. However, according to an epert panel from European LeukemiaNet (ELN), the low sensitivity of the Sanger method is not a limitation, since the clinical impact of highsensitivity BCR-ABL kinase domain mutation detection is still unclear [Soverini et al., 2011]. 12

15 Table 4: Analytical validity of mutation analysis of the BCR-ABL fusion protein STUDY NUMBER AND CHARACTERISTICS OF PATIENTS NUMBER OF PATIENTS WITH MUTATIONS (%) PERIOD OF DETECTION OF MUTATIONS Hochhaus et al., 2002 Branford et al., imatinib-resistant patients myeloid blast crisis: 33 lymphoid blast crisis: 2 AP: 16 CP: 13 ALL: 2 18 imatinib-resistant patients CML or Ph+ ALL 27/66 patients Detected during recurrence Not detected before treatment 12/18 patients Not detected before imatinib: 9 patients Not detected 3 to 9 months prior to start of resistance: 4 patients Branford et al., 2003 N = 144 AP: 40 Late CP: Onset CP: 40 27/144 patients AP: 13/40 (33) Late CP: 14/64 (22) Onset CP: 0/40 Mutations detected at 8 months (median) (3-18 months) after start of imatinib detected in patients in AP and late CP not detected at the beginning of the CP Soverini et al., 2014 N = 272 (Ph+ ALL) Median age: 53 years (17-79) Resistant to imatinib: 189 Resistant to 2nd gen. TKI 13 : 98 Resistant to dasatinib 14 : 14 Resistant to imatinib: 131/189 (69.3) Resistant to 2nd gen. TKI: 76/98 (78) Resistant to dasatinib: 11/14 (79) 60 patients tested at the time of diagnosis (before treatment): mutations rarely detected by direct sequencing Abbreviations: ALL = acute lymphoblastic leukemia; AP = accelerated phase; CML = chronic myeloid leukemia; CP = chronic phase; Ph+ = Philadelphia chromosome-positive. Local Validity Data The proposed test was developed and is performed in the requester s laboratory. Every sample is sequenced in a duplicate, bidirectional manner to confirm the variations observed. No eternal quality control programs are available on the market. To compensate for this, some samples are tested retrospectively, using an alternative method, to verify the presence of mutations. Moreover, a specimen echange agreement (controls with or without mutations) was concluded with the University Health Network laboratory (Ontario) to ensure the quality of the results Late CP: 12 months after the diagnosis. 13. Dasatinib or nilotinib administered as second-line therapy after failure of imatinib therapy. 14. Administered as first-line therapy. 15. A copy of the confirming the agreement was included in the request to include the test in the Inde. 13

16 5.4 Recommendations from Other Organizations The recommendations identified for BCR-ABL kinase domain mutation analysis only related to CML. The CML treatment guidelines published in 2013 by the Groupe québécois de recherche en LMC-NMP recommend testing for ABL mutations as part of treatment follow-up in the following cases: Failure to achieve therapeutic milestones; Epression of BCR-ABL by RQ-PCR increases more than 0.5 log in 2 consecutive blood samples, more than 0.1% with respect to international standards (IS); Lack of response after the introduction of an alternative TKI or new loss of response. Tests for ABL mutations should also be performed in cases of secondary resistance with loss of complete cytogenetic response (CCyR), loss of major molecular response (MMR), or a confirmed increase of 0.5 log but disease remains in the chronic phase. The European LeukemiaNet (ELN) and the European Society for Medical Oncology (ESMO) recommend Sanger sequencing for BCR-ABL kinase domain mutation analysis [Baccarani et al., 2012; Soverini et al., 2011]. ELN also recommends that direct sequencing (Sanger-based) be preceded by HPLC screening [Soverini et al., 2011]. In addition, the European LeukemiaNet (ELN) and the European Society for Medical Oncology (ESMO) recommend that more sensitive strategies for the detection of mutations be limited to the field of research [Baccarani et al., 2012; Soverini et al., 2011]. The ELN has put forward recommendations regarding situations in which the mutation analysis of the BCR-ABL fusion gene should be performed in patients with CML [Soverini et al., 2011]: at the time of diagnosis: only for patients in the accelerated phase or blast phase; during imatinib therapy: in case of failure; in case of increasing BCR-ABL transcripts levels resulting in a loss of major molecular response; for any other case of suboptimal response; during second-line TKI (dasatinib or nilotinib) therapy: in case of loss of hematologic or cytogenetic response. The NCCN [2014] recommends mutation analysis for monitoring response to TKI therapy in the following situations: Chronic phase: Inadequate initial response (failure to achieve PCyR 16 or BCR-ABL1/ABL1 10% (IS 17 ) at 3 months and 6 months or CCyR 18 at 12 months and 18 months); Any sign of loss of response (defined as hematologic or cytogenetic relapse); 16. PCyR: partial cytogenetic response. 17. IS: iinternational standard. 18. CCyR: complete cytogenetic response. 14

17 1-log increase in BCR-ABL transcript levels and loss of MMR; 19 Disease progression to accelerated or blast phase. 6. ANTICIPATED OUTCOMES OF INTRODUCING THE TEST 6.1 Impact on Material and Human Resources This test has been carried out by the requester since Economic Consequences of Introducing Test Into Quebec's Health Care and Social Services System This test has been carried out by the requester since Main Organizational, Ethical, and Other (Social, Legal, Political) Issues 7. IN BRIEF Not assessed. 7.1 Clinical Relevance Mutations in the kinase domain of the BCR-ABL fusion gene are one of the causes of resistance to tyrosine kinase inhibitors (TKI) currently in use for the treatment of ALL and CML. In this contet, the mutation analysis of the BCR-ABL fusion gene can help determine the best treatment strategy and select the appropriate TKI. The mutations detected in patients with CML who developed a resistance to imatinib are associated with lower progression-free and overall survival unless an alternative therapy is provided; this is particularly the case for T315I and P-loop mutations. The detection of mutations after imatinib resistance or intolerance develops allows an alternative therapy (second-generation TKIs) to be prescribed and helps improve patient prognosis. 7.2 Clinical Validity See section on Clinical Relevance. 7.3 Analytical Validity The analytical validity data were gathered from studies on the analytical sensitivity of Sanger-based direct sequencing. A sensitivity of 15% to 25% was reported, which is considered low compared with that of other available techniques. However, according to a panel of eperts from European LeukemiaNet (ELN), the low sensitivity of the Sanger method is not a limitation, since the clinical impact of high-sensitivity BCR-ABL kinase domain mutation detection is still unclear [Soverini et al., 2011]. 7.4 Recommendations from Other Organizations The Groupe québécois de recherche en LMC-NMP recommends testing for mutations as part of treatment follow-up in the case of treatment failure, lack of response, an increase of more than 0.5 log in BCR-ABL transcripts, or in certain cases of secondary resistance. 19. MMR: major molecular response. 15

18 The Sanger sequencing method used by the requester is recommended by the ELN and ESMO. According to the ELN, the method may be preceded by a pre-screening technique such as dhplc. However, based on the literature, there is a lack of consensus regarding the use of more sensitive detection methods. The ELN and ESMO recommend that these methods be limited to research use. Recommendations from the ELN and the NCCN may serve as guidance for clinicians on when to perform mutation analyses of the BCR-ABL kinase domain. 16

19 8. INESSS NOTICE IN BRIEF Mutation Analysis of the Kinase Domain of the BCR-ABL Fusion Protein Status of the Diagnostic Technology Established Innovative Eperimental (for research purposes only) Replacement for technology: obsolete which becomes INESSS Recommendation Include test in the Inde Do not include test in the Inde Reassess test Additional Recommendation Draw connection with listing of drugs, if companion test Produce an optimal use manual Identify indicators, when monitoring is required 17

20 REFERENCES Apperley JF, Cortes JE, Kim DW, Roy L, Roboz GJ, Rosti G, et al. Dasatinib in the treatment of chronic myeloid leukemia in accelerated phase after imatinib failure: The START A trial. J Clin Oncol 2009;27(21): Baccarani M, Deininger MW, Rosti G, Hochhaus A, Soverini S, Apperley JF, et al. European LeukemiaNet recommendations for the management of chronic myeloid leukemia: Blood 2013;122(6): Baccarani M, Pileri S, Steegmann JL, Muller M, Soverini S, Dreyling M. Chronic myeloid leukemia: ESMO Clinical Practice Guidelines for diagnosis, treatment and followup. Ann Oncol 2012;23(Suppl 7):vii72-7. Branford S, Rudzki Z, Walsh S, Parkinson I, Grigg A, Szer J, et al. Detection of BCR-ABL mutations in patients with CML treated with imatinib is virtually always accompanied by clinical resistance, and mutations in the ATP phosphate-binding loop (P-loop) are associated with a poor prognosis. Blood 2003;102(1): Branford S, Rudzki Z, Walsh S, Grigg A, Arthur C, Taylor K, et al. High frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571) resistance. Blood 2002;99(9): Cortes J, Goldman JM, Hughes T. Current issues in chronic myeloid leukemia: Monitoring, resistance, and functional cure. J Natl Compr Canc Netw 2012;10(Suppl 3):S1-S13. Frueh FW and Noyer-Weidner M. The use of denaturing high-performance liquid chromatography (DHPLC) for the analysis of genetic variations: Impact for diagnostics and pharmacogenetics. Clin Chem Lab Med 2003;41(4): Groupe québécois de recherche en CML et NMP. Lignes directrices du traitement de la CML [site Web]. Montréal, Qc : Groupe québécois de recherche en CML et NMP (Leucémie myéloïde chronique et néoplasies myéloprolifératives); Available at: Guilhot F, Apperley J, Kim DW, Bullorsky EO, Baccarani M, Roboz GJ, et al. Dasatinib induces significant hematologic and cytogenetic responses in patients with imatinib-resistant or -intolerant chronic myeloid leukemia in accelerated phase. Blood 2007;109(10): Hochhaus A, Kreil S, Corbin AS, La Rosee P, Muller MC, Lahaye T, et al. Molecular and chromosomal mechanisms of resistance to imatinib (STI571) therapy. Leukemia 2002;16(11): Hughes T, Deininger M, Hochhaus A, Branford S, Radich J, Kaeda J, et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: Review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for epressing results. Blood 2006;108(1): Jabbour E and Kantarjian H. Chronic myeloid leukemia: 2014 update on diagnosis, monitoring, and management. Am J Hematol 2014;89(5):

21 Jabbour E, Branford S, Saglio G, Jones D, Cortes JE, Kantarjian HM. Practical advice for determining the role of BCR-ABL mutations in guiding tyrosine kinase inhibitor therapy in patients with chronic myeloid leukemia. Cancer 2011;117(9): Jabbour E, Kantarjian H, Jones D, Talpaz M, Bekele N, O'Brien S, et al. Frequency and clinical significance of BCR-ABL mutations in patients with chronic myeloid leukemia treated with imatinib mesylate. Leukemia 2006;20(10): Jabbour EJ, Faderl S, Kantarjian HM. Adult acute lymphoblastic leukemia. Mayo Clin Proc 2005;80(11): Jones D, Kamel-Reid S, Bahler D, Dong H, Elenitoba-Johnson K, Press R, et al. Laboratory practice guidelines for detecting and reporting BCR-ABL drug resistance mutations in chronic myelogenous leukemia and acute lymphoblastic leukemia: A report of the Association for Molecular Pathology. J Mol Diagn 2009;11(1):4-11. Khorashad JS, de Lavallade H, Apperley JF, Milojkovic D, Reid AG, Bua M, et al. Finding of kinase domain mutations in patients with chronic phase chronic myeloid leukemia responding to imatinib may identify those at high risk of disease progression. J Clin Oncol 2008;26(29): Ma H, Sun H, Sun X. Survival improvement by decade of patients aged 0-14 years with acute lymphoblastic leukemia: A SEER analysis. Sci Rep 2014;4:4227. Martinez-Serra J, Gutierrez A, Marcus TF, Soverini S, Amat JC, Navarro-Palou M, et al. Four-channel asymmetric Real-Time PCR hybridization probe assay: A rapid prescreening method for critical BCR-ABL kinase domain mutations. Clin Biochem 2012;45(4-5): Muller MC, Cortes JE, Kim DW, Druker BJ, Erben P, Pasquini R, et al. Dasatinib treatment of chronic-phase chronic myeloid leukemia: Analysis of responses according to preeisting BCR-ABL mutations. Blood 2009;114(24): National Comprehensive Cancer Network (NCCN). Clinical practice guidelines in oncology. Chronic myelogenous leukemia. Version Fort Washington, PA : NCCN; Available at: National Comprehensive Cancer Network (NCCN). Clinical practice guidelines in oncology. Acute lymphoblastic leukemia. Version Fort Washington, PA : NCCN; Available at: Negrin RS and Schiffer CA. Clinical use of tyrosine kinase inhibitors for chronic myeloid leukemia. Waltham, MA : Wolters Kluwer Health; Available at: Nicolini FE, Corm S, Le QH, Sorel N, Hayette S, Bories D, et al. Mutation status and clinical outcome of 89 imatinib mesylate-resistant chronic myelogenous leukemia patients: A retrospective analysis from the French intergroup of CML (Fi(phi)-CML GROUP). Leukemia 2006;20(6): Press RD, Willis SG, Laudadio J, Mauro MJ, Deininger MW. Determining the rise in BCR- ABL RNA that optimally predicts a kinase domain mutation in patients with chronic myeloid leukemia on imatinib. Blood 2009;114(13):

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23 APPENDIX A Tyrosine Kinase Inhibitors Commonly Used in the Treatment of CML and ALL: List of Medications Covered by the RAMQ and Recognized Indications Name (generic) Classification RAMQ List Indications Imatinib (Gleevec) Eceptional medication Yes Treatment of CML in the chronic, accelerated or blast phase Treatment of refractory or relapsing Ph+ ALL in patients who are candidates for stem cell transplantation Treatment of newly diagnosed Ph+ ALL in adults after parenteral chemotherapy, during the maintenance phase Dasatinib (Sprycel) Eceptional medication Yes First-line treatment for CML in chronic and accelerated phases in adults with treatment failure, suboptimal response, or a severe intolerance to imatinib or nilotinib *No known indication for ALL Nilotinib (Tasigna) Eceptional medication Bosutinib (Bosulif) ND Yes Epected in October 2014* Treatment of CML in the chronic or accelerated phase in adults with treatment failure, suboptimal response or a severe intolerance to imatinib. First-line treatment for CML in the chronic phase * No known indication for ALL ND Ponatinib (Iclusig) ND ND ND ND = not determined Source: Régie de l assurance maladie du Québec (RAMQ). List of Medications: Last Updated on 16 July Available at: * INESSS published a notice against listing bosutinib for the treatment of CML in October 2014: 21