Rapid Detection and Quantitation of BRAF Mutations in Hairy Cell ...

Hematopathology / BRAF Mutations in Hairy Cell Leukemia

Rapid Detection and Quantitation of BRAF Mutations in Hairy Cell Leukemia Using a Sensitive Pyrosequencing Assay Shalini Verma, MD,1 Wesley O. Greaves, MD,1 Farhad Ravandi, MD,2 Neelima Reddy, MT,1 Carlos E. Bueso-Ramos, MD, PhD,1 Susan O’Brien, MD,2 Deborah A. Thomas, MD,2 Hagop Kantarjian, MD,2 L. Jeffrey Medeiros, MD,1 Rajyalakshmi Luthra, PhD,1 and Keyur P. Patel, MD, PhD1 Key Words: Hairy cell leukemia; Pyrosequencing

Upon completion of this activity you will be able to: • discuss oncogenic BRAF somatic mutations in hairy cell leukemia (HCL). • describe the principles and applications of pyrosequencing methods in clinical molecular diagnostics. • apply pyrosequencing methods for detection of the BRAF mutation in HCL for clinical diagnostic testing. • list various methods for detecting BRAF mutation.

Abstract BRAF protooncogene is an important mediator of cell proliferation and survival signals. BRAF p.V600E mutation was recently described as a molecular marker of hairy cell leukemia (HCL). We developed and validated a pyrosequencing-based approach that covers BRAF mutational hotspots in exons 11 (codon 468) and 15 (codons 595 to 600). The assay detects BRAF mutations at an analytical sensitivity of 5%. We screened 16 unenriched archived bone marrow aspirate samples from patients with a diagnosis of HCL (n = 12) and hairy cell leukemia–variant (HCL-v) (n = 4) using pyrosequencing. BRAF p.V600E mutation was present in all HCL cases and absent in all HCL-v. Our data support the recent finding that BRAF p.V600E mutation is universally present in HCL. Moreover, our pyrosequencing-based assay provides a convenient, rapid, sensitive, and quantitative tool for the detection of BRAF p.V600E mutations in HCL for clinical diagnostic testing.

© American Society for Clinical Pathology

The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 161. Exam is located at www.ascp.org/ajcpcme.

Hairy cell leukemia (HCL) is a B-cell lymphoproliferative disorder characterized by proliferation of lymphocytes showing abundant cytoplasm with “hairy” projections and a distinctive immunophenotype: CD22 bright +, CD11c+, CD25+, CD103+ and annexin A1+.1,2 The differential diagnosis of HCL includes other types of low-grade B-cell lymphoma/leukemia, such as hairy cell leukemia–variant (HCLv) and splenic marginal zone B-cell lymphoma.1,3 Clinically, single agent or combination therapy with purine nucleoside analogues and rituximab leads to remission in most patients with HCL.4 Despite the high rate of initial response and long survival times, however, approximately 30% to 40% of patients with HCL experience a relapse and may require retreatment within a 10-year period, thus highlighting the need for a better understanding of the molecular pathogenesis of HCL as well as novel therapies.5-7 Recently, BRAF p.V600E mutation was identified in a patient with HCL by means of whole-genome sequencing, followed by confirmation in 45 additional cases by Sanger sequencing of bone marrow (BM) samples.8 BRAF protooncogene is an important mediator of cell proliferation and survival signals via the RAS-RAF-MEKERK signaling pathway. Somatic mutations in exons 11 (codon 468) and 15 (codons 596 and 600) of BRAF result in increased BRAF kinase activity, and have been reported to play a role in initiation and maintenance in several neoplasms, including melanomas and colon, ovarian, and thyroid carcinomas.9 Furthermore, at least one therapeutic agent designed to target and inhibit mutant BRAF has been Am J Clin Pathol 2012;138:153-156 153 DOI: 10.1309/AJCPL0OPXI9LZITV

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DOI: 10.1309/AJCPL0OPXI9LZITV

Verma et al / BRAF Mutations in Hairy Cell Leukemia

approved by the Food and Drug Administration (FDA) with other agents being actively pursued in clinical research.10-12 We developed a pyrosequencing-based approach to investigate the presence and frequency of mutations in exons 11 and 15 of BRAF in a cohort of patients with HCL and HCL-v who were diagnosed and treated at our institution.

Materials and Methods This study was approved by the institutional review board of The University of Texas MD Anderson Cancer Center (MDACC; Houston). Thirty-six patients diagnosed with HCL or HCL-v between the years 2004 and 2009 at MDACC were previously described by Ravandi et al.4 Archival DNA from routine clinical specimens was available for 16 patients from the study—12 with classic HCL and 4 with HCL-v. Diagnosis was established with bone marrow (BM) examination, based on morphologic and immunophenotypic findings.1,3 All cases demonstrated monoclonal B cells on flow cytometry ❚Table 1❚ and clonal IgH gene rearrangement on polymerase chain reaction (PCR). Mutations in BRAF were investigated using a sensitive (analytical sensitivity, 5%, ie, the assay reproducibly detects BRAF mutations when cells carrying the mutation are as low as 5% in a sample) and qualitative pyrosequencing assay that comprehensively covers mutational hotspots in exons 11 (codon 468) and 15 (codons 595 to 600). BRAF mutation testing in exons 11 and 15 was performed using conventional PCR followed by pyrosequencing. For exon 11, a 322-nucleotide base pair (bp) amplicon, including codon 468, was amplified using a forward primer, TCC

TGT ATC CCT CTC AGG CAT AAG GTA A, and a reverse biotinylated primer, biotin-CGA ACA GTG AAT ATT CCT TTG AT. For exon 15 (codon 595/596 and codon 599/600), a 231-bp amplicon was amplified using a forward primer, M13-CAT AAT GCT TGC TCT GAT AGG A, and a reverse biotinylated primer, biotin-M13-GGC CAA AAA TTT AAT CAG TGG A. The PCR master mix contained the forward and reverse primers (each 10 μmol/L), 10 mmol/L of dNTP mix, 25 mmol/L of magnesium chloride, ×10 PCR buffer (Applied Biosystems, Carlsbad, CA), 5 U/μL of Taq Gold and 200 ng of sample genomic DNA in a total volume of 48 μL. PCR amplification was performed in duplicate on an ABI 2720 Thermocycler (Applied Biosystems); cycling conditions consisted of initial denaturing at 95ºC for 12 minutes, 50 cycles at 94ºC for 30 seconds, 55ºC for 30 seconds, and 72ºC for 30 seconds, and final extension at 72ºC for 10 minutes. Appropriate positive (melanoma cell line A375, for exon 15), negative (control sample), and reagent controls were included. The PCR products underwent electrophoresis on agarose gels to confirm successful amplification of the 322-bp and 231-bp PCR products before pyrosequencing. PCR products (each 15 μL) were then sequenced in duplicate using the pyrosequencing PSQ96 HS System (Biotage AB, Uppsala, Sweden) per manufacturer’s instructions. We used the pyrosequencing primers TTG GAT CTG GAT CAT TT (BRAF-11) or GAA GAC CTC ACA GTA AAA ATA (BRAF-15) and a specific nucleotide dispensation order (A, C, G, T,…). All assay runs included sensitivity, positive, negative, and reagent controls. HL-60 cell line, which is negative for BRAF mutations, was used as negative control.

❚Table 1❚ Clinical and Laboratory Features of Patients With Hairy Cell Leukemia and Hairy Cell Leukemia Variant Tested for BRAF Exon 15 Mutation Case No.

Diagnosis

Age (y)

Sex

BM Biopsy Involvement* (%)

Leukemic B-Cells† (%)

BRAF Mutation (%)

Cytogenetics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

HCL HCL HCL HCL HCL HCL HCL HCL HCL HCL HCL HCL HCL-v HCL-v HCL-v HCL-v

58 64 52 39 48 37 47 58 53 68 56 73 73 70 72 53

F M M M M M M M M M M M F M M M

50 20 50 90 5 15 24 25 50 85 55 26 40 80 30 70

7 10 4 46 2 2 2 61 78 24 8 63 20 84 93 15

p.V600E (6.0%) p.V600E (7.0%) p.V600E (9.5%) p.V600E (35.0%) p.V600E (7.4%) p.V600E (5.1%) p.V600E (5.0%) p.V600E (17.4%) p.V600E (6.3%) p.V600E (20.0%) p.V600E (10.0%) p.V600E (6.4%) Wild type Wild type Wild type Wild type

Diploid Diploid Diploid ND Diploid Diploid Diploid Diploid Diploid Diploid Diploid Diploid Complex Complex Complex Complex

BM indicates bone marrow; HCL, hairy cell leukemia; HCL-v, hairy cell leukemia variant; ND, not determined. * As seen morphologically. † As seen on flow cytometry.

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Hematopathology / Original Article

Results Selected clinical and laboratory features of the 16 patients are summarized in Table 1. Clinical and survival data of all 36 patients were reported elsewhere.4 Twelve patients with HCL had a median age of 53 years (range, 37-73 years) and included 11 men and 1 woman. Four patients with HCL-v had a median age of 71 years (range, 53-73 years) and included 3 men and 1 woman. All patients with HCL-v demonstrated a complex karyotype and 3 of 4 showed TP53 deletion on routine fluorescence in situ hybridization analysis. All 4 HCL-v cases showed wild type exons 11 and 15 sequences ❚Figure 1A❚. BRAF p.V600E mutations in exon 15 were detected in all 12 patients with HCL (100%) ❚Figure 1B❚. The difference in mutational status of BRAF between HCL and HCL-v was statistically significant (P < .005, Fisher 2-tailed exact test). No mutations were detected in codon 468 of exon 11 of BRAF in either HCL or HCL-v cases.

Discussion We describe the presence of exon 15 BRAF p.V600E mutation in 100% (12 of 12) of patients with HCL whom we tested using a convenient and sensitive pyrosequencing-based strategy (Figure 1B). These results are consistent with the recently reported universal prevalence of BRAF mutations in HCL.8 All 4 of our HCL-v cases were negative for BRAF exon 15 mutations. These findings are also consistent with the absence of BRAF p.V600E in other types of B-cell lymphoma and leukemia (n = 195), including HCL-v, reported by Tiacci et al,8 and further support the notion that BRAF p.V600E mutation can serve as a new diagnostic tool to distinguish HCL from other morphologically similar B-cell neoplasms.

A 1,800 1,700 1,600 1,500 1,400 1,300 1,200

Tiacci et al8 used the Sanger sequencing approach in CD19-purified HCL cells in a selected cohort of patients with at least 30% neoplastic cells to detect BRAF mutations. Sanger sequencing is considered the “gold standard” method and is a widely used method for genotyping. Yet, one of the major limitations of Sanger sequencing is that it is labor intensive, less sensitive, and nonquantitative. In addition, Tiacci et al and others recently described nonquantitative PCR-based methods for detecting the BRAF p.V600E in patients with HCL.13,14 In contrast, by using a pyrosequencing-based approach, we were able to reproducibly and quantitatively detect BRAF p.V600E mutation in replicates of routine clinical samples at an analytical sensitivity of 5%. Using our pyrosequencing-based assay, there is no need for enrichment of tumor cells, hence our assay can be used for routine detection of BRAF p.V600E mutation in HCL in the clinical setting. Pyrosequencing is a DNA sequencing technology based on the principle of “sequencing by synthesis,” ie, realtime detection of DNA synthesis monitored by bioluminescence.15-19 Briefly, the process of pyrosequencing can be separated into different steps, namely, PCR amplification of target DNA, single-stranded DNA preparation, purification of single-stranded DNA template, and nucleotide analysis. In this method, the activity of DNA polymerase is detected with another chemiluminescent enzyme. Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step. The template DNA is immobile, and solutions of A, C, G, and T nucleotides are sequentially added and removed from the reaction. Light is produced only when the nucleotide complements the unpaired base of the template. Programmed dispensing of nucleotides generates a signal for each addition

B Codon 596 C: 0.0% G: 100.0%

Codon 600 A: 3.1% T: 96.9%

E SGTGA T CGT C T AGC T ACAGA TGA Wild type (GGT)

Wild type (GTG)

1,800 1,700 1,600 1,500 1,400 1,300 1,200

Codon 596 C: 1.5% G: 98.5%

Codon 600 A: 23.9% T: 76.1%

ES GTGA TCGT C T AGC T ACAGA TGA Wild type (GGT)

p.V600E (GAG)

❚Figure 1❚ Representative pyrograms of bone marrow aspirate samples of a patient with hairy cell leukemia–variant (HCL-v) (A) and another patient with hairy cell leukemia (HCL) (B) analyzed for mutations in codons 596 to 600 in exon 15 of BRAF. Codons 596 and 600 are highlighted in yellow. A, Both codon 596 (GGT) and codon 600 (GTG) show wild type sequences in HCL-v. B, A BRAF p.V600E mutation (c.1799T>A, GTG to GAG) is present in HCL, representing 23.9% of all nucleated cells (arrow); codon 596 shows a wild type sequence.


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of nucleotide in a pyrogram. Each patient pyrogram is compared against the negative control for each sequencing reaction. If no differences in peak heights are observed, the patient is described as negative for mutation. If there is a difference in peak height, then the mutation is determined and reported. Thus, variation in the pyrogram pattern indicates the presence of a mutation. Pyrosequencing has been successful in both de novo and confirmatory DNA sequencing. Multiplexing ability of pyrosequencing enables rapid and accurate screening of a large number of samples relatively inexpensively. Because it offers the same accuracy as conventional DNA sequencing for short reads while being more flexible and allowing processing of a large number of samples in parallel, it has rapidly found applications in DNA sequencing, genotyping, single nucleotide polymorphism analysis, allele quantification, and whole genome sequencing. The results of our study further corroborate the evidence that BRAF mutations are universally present in HCL. Some investigators have implicated MAPK pathway dysregulation in the prolonged cell survival and unique drug sensitivity of hairy cells in vitro.20 Our data, along with those of Tiacci et al, provide a rationale for further studying the oncogenic role of BRAF mutations in HCL. The clinical availability of specific BRAF inhibitors for treatment of BRAF p.V600E–positive metastatic melanoma, coupled with the universal presence of BRAF mutations in HCL, provides further opportunities for the use of a targeted therapy approach to improve outcome in patients with HCL, especially for patients with a relapse or treatment-refractory disease.21 From the Departments of 1Hematopathology and 2Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX. Address reprint requests to Dr Patel: Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Naomi St. Facility (NAO1.053a), 1515 Holcombe Blvd, Unit 0149, Houston, TX 77030; [email protected].

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