short report
Alternative BRAF mutations in BRAF V600E-negative hairy cell leukaemias
Sebastian Tschernitz,1 Lucia Flossbach,1 Margrit Bonengel,1 Sabine Roth,1 Andreas Rosenwald1,2 and Eva Geissinger1,2 1
Institute of Pathology, University of Wuerzburg,
and 2Comprehensive Cancer Centre (CCC) Mainfranken, University and University Hospital, Wuerzburg, Germany Received 29 July 2013; accepted for publication 6 December 2013
Summary The BRAF V600E mutation in exon 15 is considered the disease-defining mutation in hairy cell leukaemia (HCL), but single HCL cases lacking this mutation have been described. In 24 HCL, as well as in 194 various mature B- and T-cell neoplasms, we extended the search for BRAF mutations to exon 11. Two V600Enegative HCL contained novel, potentially functionally relevant mutations in exon 11 (F468C and D449E), while one other HCL was BRAF wild-type in exons 2–17. All non-HCL lymphomas lacked BRAF mutations. We therefore suggest screening of BRAF V600E-negative HCL for alternative exon 11 mutations in the diagnostic setting.
Correspondence: Eva Geissinger, Institute of Pathology, University of Wuerzburg, JosefSchneider-Strasse 2, 97080 Wuerzburg,
Keywords: hairy cell leukaemia, BRAF, alternative mutations, exon 11, diagnostics.
Germany. E-mail: [email protected]
In 2011, the BRAF V600E mutation was described in 100% of hairy cell leukaemias (HCL) investigated (n = 48), while none of 195 various other peripheral B-cell lymphomas/leukaemias carried this mutation (Tiacci et al, 2011). Since that observation was published, other groups (Boyd et al, 2011; Arcaini et al, 2012) confirmed this result, establishing the paradigm that BRAF V600E is the disease-defining mutation of HCL. However, a few histologically, immunohistochemically and clinically characteristic HCLs appear to exist that lack the BRAF V600E mutation (Schnittger et al, 2012; Xi et al, 2012). As these studies investigated only exon 15 of the BRAF gene, we extended our sequencing approach to exon 11, which harbours a second hot spot of BRAF mutations in solid tumours. In addition to 24 typical HCL we also studied 194 various other mature B- and T-cell neoplasms for mutations in exon 11 and exon 15 of the BRAF gene.
Materials and methods Case selection We analysed paraffin-embedded tumour specimens from 218 mature B-cell and T-cell neoplasms from the files of the Institute of Pathology, University of Wuerzburg, that were selected based on the availability of sufficient, well preserved material and a tumour cell content of >20%. We included 24 classic HCL (Table I) as well as 136 other B-cell Non-Hodgkin ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 529–533
lymphomas (NHL) and 58 T-NHL (Table II). All cases were reviewed by two expert haematopathologists with Haematoxylin and Eosin-, Giemsa- and Periodic Acid-Schiff-stained sections, and an appropriate panel of immunohistochemical stains including Annexin I, CD103 and phospho-ERK (see Table SI for antibody details and Fig S1 for selected stains) to establish a diagnosis according to the current World Health Organization classification of Haematopoietic and Lymphoid Tissues. Approval for the entire study was obtained from the ethics committee, Medical Faculty, University of Wuerzburg, Germany.
DNA extraction, polymerase chain reaction (PCR) amplification and sequencing Genomic DNA was isolated from paraffin-embedded tissue by the use of QIAamp (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Exon 11 and exon 15 of BRAF were amplified by PCR using standard protocols (see Table SII for primer sequences, cycling and annealing parameters). The classic HCL without BRAF mutations in exon 11 and exon 15 was sequenced for all BRAF exons except exons 1 and 18 (due to technical difficulties). Moreover, this HCL case, as well as the three HCL cases with alternative mutations were sequenced for KRAS/ NRAS exons 2, 3 and 4 as well as for HRAS exons 2 and 3 (see Table SII for primer sequences). All sequencing analyses First published online 16 January 2014 doi:10.1111/bjh.12735
530
68 77 59
51 55 64 41 31 59 52 61 49 83 61 55 50 46 73 82 81 66 53 43 68
1 2 3
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
f m m f f m m m m m m m m m m m m f m m m
m m m
Gender
bm bm bm bm bm bm bm bm ki bm bm bm bm bm bm bm bm bm bm bm bm
bm bm bm
Type of tissue
80 80 40 40 70 60 70 70 60 50 80 30 90 90 40 70 80 70 40 40 70
70 90 80
Tumour cells %
pos pos pos pp pos pp pp pos pp pos pp pos pos pos pos pos pos ne pos pp pos
pp pos pos
DBA44
pos pp pos pos pp pp pp pp pos pos neg pos pos pos pos wp neg ne neg pp pos
pos pos pos
CD25
pp pp pos wp pp pp pp pp pos pos pp pos pos wp pp pp pp ne pp pp pos
pp pos pos
Cd11c
pp pp pos pp pos pos sp wp wp wp wp pos wp wp wp pos neg ne pp pp pos
pp pp pos
Cyclin D1
pos pos ne pos pos pos pos pos pos pos pos pos pos pos pos pos pos ne pos pos pos
pos pos pos
CD103
pp pos ne pos pos pos pp pp pos pos pos pos pp pp pos pos pp ne pos pp pos
pp pp pp
ANXA I
pp pos ne pos pp pos pos pp pp pos pos pos pos pp neg pos neg ne pp pos pos
pos pos pos
p-ERK V600E V600E V600E S602T V600E V600E WT V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E WT WT
BRAF Exon 15
WT WT F468C WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT D449E
WT WT WT
BRAF Exon 11
WT WT
WT
WT
WT WT
WT
WT
WT WT
WT
WT
Exon2 Exon3 Exon4
KRAS
Male, male; f, female; bm, bone marrow; ki, kidney, pos, positive; pp, partially positive; wp, weak positive; neg, negative; ne, not evaluable; WT, wild-type
Age at diagnosis (years)
Patient no.
Table I. Clinical, immunophenotypical and molecular characteristics of the 24 patients with hairy cell leukaemia.
WT WT
WT
WT
WT WT
WT
WT
WT WT
ne
WT
Exon2 Exon3 Exon4
NRAS
WT WT
WT
WT
WT WT
ne
WT
Exon2 Exon3
HRAS
Short report
ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 529–533
Short report Table II. BRAF exon 11 and exon 15 mutation analysis in various mature B-cell and T-cell neoplasms excluding HCL.
Neoplasms B-cell neoplasms Bone marrow infiltration by low grade B-cell lymphoma not otherwise specified (splenic marginal zone lymphoma, splenic B-cell lymphoma/leukaemia unclassifiable, hairy cell leukaemia-variant) Nodal marginal zone lymphoma Extranodal marginal zone lymphoma (MALT-type) Burkitt lymphoma Follicular lymphoma Follicular lymphoma (G1) Follicular lymphoma (G2) Follicular lymphoma (G3A) Follicular lymphoma (G3B) Diffuse large B-cell lymphoma Mantle cell lymphoma Chronic lymphocytic leukaemia/small cell lymphocytic lymphoma T-cell neoplasms Anaplastic large T-cell lymphoma (ALCL) ALCL ALK1-positiv ALCL ALK1-negative Peripheral T-cell lymphoma not otherwise specified Angioimmunoblastic T-cell lymphoma
Number of cases
BRAF mutation exon 11
BRAF mutation exon 15
136 8
neg
neg
neg neg neg neg neg neg neg neg neg neg neg
neg neg neg neg neg neg neg neg neg neg neg
neg neg neg neg neg
neg neg neg neg neg
8 10 20 30 10 10 5 5 20 20 20 58 18 9 9 20 20
neg, Negative
were done by Sanger sequencing. The alternative BRAF mutations were confirmed in independent PCR and sequencing reactions, thus minimizing the risk to detect ‘artificial’ mutations. Experimental details are available on request.
Results and discussion The clinical, immunophenotypical and molecular characteristics of the 24 HCL cases investigated are provided in Table I. Of these, 21 HCL carried the BRAF V600E mutation in exon 15, while no mutation was detected in exon 11 in the BRAF V600E mutated cases. Of note, one HCL exhibited a second mutation in exon 15 (S602T; Patient 3, Table I). In this case, a germline mutation cannot be formally excluded, as we could not obtain normal DNA from this patient. S602 is located in the activation loop of BRAF and constitutes, together with T599, an essential phosphorylation site that plays an important role in the activation of BRAF (Garnett & Marais, 2004), arguing against the possibility of a germline mutation. While our S602T mutation has not been described so far, one other mutation at the same position has been reported in an osteosarcoma (S602Y; Pignochino et al, 2009). There is only one HCL in the literature that carries a second – though silent – BRAF mutation in exon 15 in addition to the conventional V600E mutation (Boyd et al, 2011). Of the 24 HCL cases in our series, three lacked the BRAF V600E mutation. Interestingly, alternative BRAF mutations could be detected in exon 11 in two of these patients (F468C ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 529–533
in Patient 6 and D449E in Patient 24, Table I), while the remaining patient (Patient 23) showed no mutations in exon 11. Given that in some malignant tumours (e.g. malignant melanoma), mutations in BRAF exons other than 11 and 15 have been described (Hodis et al, 2012), we investigated this latter HCL case for mutations in exons 2–17 of the BRAF gene. However, no mutations were discovered. Given that activation of the RAS-RAF-MEK-ERK pathway can also be caused by mutations in the KRAS, NRAS or HRAS genes, we decided to sequence the relevant regions of these genes in the three BRAF V600E mutation-negative cases as well. Patients 23 and 24 showed wild-type sequences for KRAS (exons 2, 3 and 4), NRAS (exons 2, 3 and 4) and HRAS (exons 2 and 3). Due to the limitations of the material from Patient 6, we could prove wild-type sequences only for KRAS (exons 2, 3 and 4), NRAS (exons 2 and 3) and HRAS (exon 2), while NRAS exon 4 and HRAS exon 3 could not be sequenced. Other investigators found a few exceptional HCL cases lacking the BRAF V600E mutation (Schnittger et al, 2012; Xi et al, 2012), but did not search for alternative BRAF mutations outside exon 15. We here describe the first two HCL tumours carrying BRAF mutations in exon 11. Patient 6 showed a characteristic clinical presentation as well as typical histological and phenotypical features of HCL (Table I and Table SIII). The detected F468C mutation within the glycine-rich loop in exon 11 has been described in colorectal cancer (Yuen et al, 531
Short report 2002). There is evidence from in vitro and in vivo transfection experiments (Ikenoue et al, 2004) that this mutation leads to increased basal kinase activity of BRAF and may thus substitute for the conventional V600E mutation. Patient 24 also showed the typical histological, phenotypical and clinical characteristics of HCL (Table I and Table SIII) and displayed another alternative mutation in exon 11 (D449E). The functional consequence of this mutation, which has not been reported in any other tumour so far, is unclear. Interestingly, position 449 is part of the N-region of BRAF and the aspartatic acid at this position is not conserved in ARAF and RAF1 (also termed CRAF; Tyr 303 in ARAF; Tyr 342 in RAF1). In order to activate ARAF and RAF1, these tyrosines have to be phosphorylated, while in BRAF the negative charge at this position might explain the elevated basal activity of BRAF compared to ARAF and RAF1 (Garnett & Marais, 2004). In our HCL, the exchange of aspartatic acid (D) to glutamatic acid (E) at position 449 keeps the negative charge, but the potential functional relevance remains unexplored. Given that Wan et al (2004) showed that D448 may come in contact with R506 of the aC helix, suggesting that this interaction stabilizes the active conformation and is important for the basal and RAS-stimulated kinase activity, one could speculate that the D449E mutation could modify, if not even intensify, this interaction. By sequencing BRAF exon 11 from normal gastric biopsies of both Patients 6 and 24 we could exclude germline mutations. Moreover, immunohistochemistry in Patient 24 demonstrated consistent pospho-ERK positivity in the tumour cells (Fig S1), suggesting a functional relevance of the D449E mutation leading to activation of the RAF-MEK-ERK pathway. In addition, Patient 23, who lacked BRAF, KRAS, NRAS and HRAS mutations, also showed a positive pospho-ERK status, suggesting a yet unknown mechanism for the activation of this pathway in this case. In addition to the 24 HCL cases, we investigated 194 other B- and T-NHL for BRAF mutations in exons 11 and 15 (Table II). Although a few cases of chronic lymphocytic leukaemia (Jebaraj et al, 2013) and diffuse large B-cell lymphoma (Lee et al, 2003) were reported to carry mutations in exon 15 or exons 11 and 15, respectively, all 136 B-NHL in our series showed unmutated exons 11 and 15. Similar results in lymphomas other than HCL have been previously reported (Tiacci et al, 2012). Primary Burkitt lymphomas have not been
References Arcaini, L., Zibellini, S., Boveri, E., Riboni, R., Rattotti, S., Varettoni, M., Guerrera, M.L., Lucioni, M., Tenore, A., Merli, M., Rizzi, S., Morello, L., Cavalloni, C., Da Via, M.C., Paulli, M. & Cazzola, M. (2012) The BRAF V600E mutation in hairy cell leukemia and other mature B-cell neoplasms. Blood, 119, 188–191.
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previously screened for exon 11 mutations (Tiacci et al, 2011; Laurini et al, 2012), but all 20 cases included in our study were BRAF wild-type in exons 11 and 15. Studies on BRAF mutations in peripheral T-cell lymphomas (PTCL) are scarce. Laurini et al (2012) investigated BRAF V600E mutations in 23 PTCL, which were all negative. Our cohort significantly extends existing negative data, because a total of 58 PTCL showed no BRAF mutations in exons 11 and 15 (Table II). In summary, our report confirms the existence of BRAF V600E-negative HCL and identifies novel alternative mutations in exon 11 of the BRAF gene. In the routine diagnostic setting, we therefore suggest that bona fide V600E-negative HCL should be screened for alternative exon 11 mutations. Future studies are needed to show whether HCL without mutations in the entire BRAF gene might carry other activating mutations in genes of the RAS-RAF-MEK-ERK pathway.
Author contributions EG and AR designed the research and reviewed the cases. ST and MB performed DNA extraction, PCR and sequencing of exon 11 and exon 15 of the BRAF gene. SR and LF performed PCR and sequencing of exon 2 to exon 17 (excluding exon 11 and 15) of the BRAF gene and PCR and sequencing analyses of the KRAS, NRAS and HRAS gene. ST, LF and EG analysed the data. ST, EG and AR wrote the manuscript.
Conflict of interest The authors declare no competing financial interests.
Supporting Information Additional Supporting Information may be found in the online version of this article: Table SI. Sources and dilutions of primary antibodies. Table SII. Primers, annealing temperatures and cycle conditions for BRAF, NRAS, KRAS and HRAS amplification. Table SIII. Laboratory findings, flow cytometry results, therapy and response in Patients 3, 6, 23 and 24. Fig S1. Relevant immunohistochemical stainings and sequencing analyses of two independent PCR reactions of the molecularly unusual HCL cases.
Boyd, E.M., Bench, A.J., van ‘t Veer, M.B., Wright, P., Bloxham, D.M., Follows, G.A. & Scott, M.A. (2011) High resolution melting analysis for detection of BRAF exon 15 mutations in hairy cell leukaemia and other lymphoid malignancies. British Journal of Haematology, 155, 609–612. Garnett, M.J. & Marais, R. (2004) Guilty as charged: B-RAF is a human oncogene. Cancer Cell, 6, 313–319.
Hodis, E., Watson, I.R., Kryukov, G.V., Arold, S.T., Imielinski, M., Theurillat, J.P., Nickerson, E., Auclair, D., Li, L., Place, C., Dicara, D., Ramos, A.H., Lawrence, M.S., Cibulskis, K., Sivachenko, A., Voet, D., Saksena, G., Stransky, N., Onofrio, R.C., Winckler, W., Ardlie, K., Wagle, N., Wargo, J., Chong, K., Morton, D.L., StemkeHale, K., Chen, G., Noble, M., Meyerson, M., Ladbury, J.E., Davies, M.A., Gershenwald, J.E.,
ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 529–533
Short report Wagner, S.N., Hoon, D.S., Schadendorf, D., Lander, E.S., Gabriel, S.B., Getz, G., Garraway, L.A. & Chin, L. (2012) A landscape of driver mutations in melanoma. Cell, 150, 251–263. Ikenoue, T., Hikiba, Y., Kanai, F., Aragaki, J., Tanaka, Y., Imamura, J., Imamura, T., Ohta, M., Ijichi, H., Tateishi, K., Kawakami, T., Matsumura, M., Kawabe, T. & Omata, M. (2004) Different effects of point mutations within the B-Raf glycine-rich loop in colorectal tumors on mitogen-activated protein/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase and nuclear factor kappaB pathway and cellular transformation. Cancer Research, 64, 3428–3435. Jebaraj, B.M., Kienle, D., B€ uhler, A., Winkler, D., D€ ohner, H., Stilgenbauer, S. & Zenz, T. (2013) BRAF mutations in chronic lymphocytic leukemia. Leukemia & Lymphoma, 54, 1177–1182. Laurini, J.A., Aoun, P.A., Iqbal, J., Chan, W. & Greiner, T.C. (2012) Investigation of the BRAF V600E mutation by pyrosequencing in lymphoproliferative disorders. American Journal of Clinical Pathology, 138, 877–883. Lee, J.W., Yoo, N.J., Soung, Y.H., Kim, H.S., Park, W.S., Kim, S.Y., Lee, J.H., Park, J.Y., Cho, Y.G., Kim, C.J., Ko, Y.H., Kim, S.H., Nam, S.W., Lee, J.Y. & Lee, S.H. (2003) BRAF mutations in
non-Hodgkin’s lymphoma. British Journal of Cancer, 89, 1958–1960. Pignochino, Y., Grignani, G., Cavalloni, G., Motta, M., Tapparo, M., Bruno, S., Bottos, A., Gammaitoni, L., Migliardi, G., Camussi, G., Alberghini, M., Torchio, B., Ferrari, S., Bussolino, F., Fagioli, F., Picci, P. & Aglietta, M. (2009) Sorafenib blocks tumour growth, angiogenesis and metastatic potential in preclinical models of osteosarcoma through a mechanism potentially involving the inhibition of ERK1/2, MCL-I and ezrin pathways. Molecular Cancer, 8, 118. Schnittger, S., Bacher, U., Haferlach, T., Wendland, N., Ulke, M., Dicker, F., Grossmann, V., Haferlach, C. & Kern, W. (2012) Development and validation of a real-time quantification assay to detect and monitor BRAFV600E mutations in hairy cell leukemia. Blood, 119, 3151–3154. Tiacci, E., Trifonov, V., Schiavoni, G., Holmes, A., Kern, W., Martelli, M.P., Pucciarini, A., Bigerna, B., Pacini, R., Wells, V.A., Sportoletti, P., Pettirossi, V., Mannucci, R., Elliott, O., Liso, A., Ambrosetti, A., Pulsoni, A., Forconi, F., Trentin, L., Semenzato, G., Inghirami, G., Capponi, M., Di Raimondo, F., Patti, C., Arcaini, L., Musto, P., Pileri, S., Haferlach, C., Schnittger, S., Pizzolo, G., Foa, R., Farinelli, L., Haferlach, T.,
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Pasqualucci, L., Rabadan, R. & Falini, B. (2011) BRAF mutations in hairy-cell leukemia. New England Journal of Medicine, 364, 2305–2315. Tiacci, E., Schiavoni, G., Forconi, F., Santi, A., Trentin, L., Ambrosetti, A., Cecchini, D., Sozzi, E., Francia di Celle, P., Di Bello, C., Pulsoni, A., Foa, R., Inghirami, G. & Falini, B. (2012) Simple genetic diagnosis of hairy cell leukemia by sensitive detection of the BRAF-V600E mutation. Blood, 119, 192–195. Wan, P.T., Garnett, M.J., Roe, S.M., Lee, S., Niculescu-Duv, D., Good, V.M., Jones, C.M., Marshall, C.J., Springer, C.J., Barford, D. & Marais, R. (2004) Mechanism of activation of the RAFERK signaling pathway by oncogenic mutations of B-RAF. Cell, 116, 855–867. Xi, L., Arons, E., Navarro, W., Calvo, K.R., StetlerStevenson, M., Raffeld, M. & Kreitman, R.J. (2012) Both variant and IGHV4-34-expressing hairy cell leukemia lack the BRAF V600E mutation. Blood, 119, 3330–3332. Yuen, S.T., Davies, H., Chan, T.L., Ho, J.W., Bignell, G.R., Cox, C., Stephens, P., Edkins, S., Tsui, W.W., Chan, A.S., Futreal, P.A., Stratton, M.R., Wooster, R. & Leung, S.Y. (2002) Similarity of the phenotypic patterns associated with BRAF and KRAS mutations in colorectal neoplasia. Cancer Research, 62, 6451–6455.
533
Alternative BRAF mutations in BRAF V600E-negative hairy cell leukaemias
Sebastian Tschernitz,1 Lucia Flossbach,1 Margrit Bonengel,1 Sabine Roth,1 Andreas Rosenwald1,2 and Eva Geissinger1,2 1
Institute of Pathology, University of Wuerzburg,
and 2Comprehensive Cancer Centre (CCC) Mainfranken, University and University Hospital, Wuerzburg, Germany Received 29 July 2013; accepted for publication 6 December 2013
Summary The BRAF V600E mutation in exon 15 is considered the disease-defining mutation in hairy cell leukaemia (HCL), but single HCL cases lacking this mutation have been described. In 24 HCL, as well as in 194 various mature B- and T-cell neoplasms, we extended the search for BRAF mutations to exon 11. Two V600Enegative HCL contained novel, potentially functionally relevant mutations in exon 11 (F468C and D449E), while one other HCL was BRAF wild-type in exons 2–17. All non-HCL lymphomas lacked BRAF mutations. We therefore suggest screening of BRAF V600E-negative HCL for alternative exon 11 mutations in the diagnostic setting.
Correspondence: Eva Geissinger, Institute of Pathology, University of Wuerzburg, JosefSchneider-Strasse 2, 97080 Wuerzburg,
Keywords: hairy cell leukaemia, BRAF, alternative mutations, exon 11, diagnostics.
Germany. E-mail: [email protected]
In 2011, the BRAF V600E mutation was described in 100% of hairy cell leukaemias (HCL) investigated (n = 48), while none of 195 various other peripheral B-cell lymphomas/leukaemias carried this mutation (Tiacci et al, 2011). Since that observation was published, other groups (Boyd et al, 2011; Arcaini et al, 2012) confirmed this result, establishing the paradigm that BRAF V600E is the disease-defining mutation of HCL. However, a few histologically, immunohistochemically and clinically characteristic HCLs appear to exist that lack the BRAF V600E mutation (Schnittger et al, 2012; Xi et al, 2012). As these studies investigated only exon 15 of the BRAF gene, we extended our sequencing approach to exon 11, which harbours a second hot spot of BRAF mutations in solid tumours. In addition to 24 typical HCL we also studied 194 various other mature B- and T-cell neoplasms for mutations in exon 11 and exon 15 of the BRAF gene.
Materials and methods Case selection We analysed paraffin-embedded tumour specimens from 218 mature B-cell and T-cell neoplasms from the files of the Institute of Pathology, University of Wuerzburg, that were selected based on the availability of sufficient, well preserved material and a tumour cell content of >20%. We included 24 classic HCL (Table I) as well as 136 other B-cell Non-Hodgkin ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 529–533
lymphomas (NHL) and 58 T-NHL (Table II). All cases were reviewed by two expert haematopathologists with Haematoxylin and Eosin-, Giemsa- and Periodic Acid-Schiff-stained sections, and an appropriate panel of immunohistochemical stains including Annexin I, CD103 and phospho-ERK (see Table SI for antibody details and Fig S1 for selected stains) to establish a diagnosis according to the current World Health Organization classification of Haematopoietic and Lymphoid Tissues. Approval for the entire study was obtained from the ethics committee, Medical Faculty, University of Wuerzburg, Germany.
DNA extraction, polymerase chain reaction (PCR) amplification and sequencing Genomic DNA was isolated from paraffin-embedded tissue by the use of QIAamp (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Exon 11 and exon 15 of BRAF were amplified by PCR using standard protocols (see Table SII for primer sequences, cycling and annealing parameters). The classic HCL without BRAF mutations in exon 11 and exon 15 was sequenced for all BRAF exons except exons 1 and 18 (due to technical difficulties). Moreover, this HCL case, as well as the three HCL cases with alternative mutations were sequenced for KRAS/ NRAS exons 2, 3 and 4 as well as for HRAS exons 2 and 3 (see Table SII for primer sequences). All sequencing analyses First published online 16 January 2014 doi:10.1111/bjh.12735
530
68 77 59
51 55 64 41 31 59 52 61 49 83 61 55 50 46 73 82 81 66 53 43 68
1 2 3
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
f m m f f m m m m m m m m m m m m f m m m
m m m
Gender
bm bm bm bm bm bm bm bm ki bm bm bm bm bm bm bm bm bm bm bm bm
bm bm bm
Type of tissue
80 80 40 40 70 60 70 70 60 50 80 30 90 90 40 70 80 70 40 40 70
70 90 80
Tumour cells %
pos pos pos pp pos pp pp pos pp pos pp pos pos pos pos pos pos ne pos pp pos
pp pos pos
DBA44
pos pp pos pos pp pp pp pp pos pos neg pos pos pos pos wp neg ne neg pp pos
pos pos pos
CD25
pp pp pos wp pp pp pp pp pos pos pp pos pos wp pp pp pp ne pp pp pos
pp pos pos
Cd11c
pp pp pos pp pos pos sp wp wp wp wp pos wp wp wp pos neg ne pp pp pos
pp pp pos
Cyclin D1
pos pos ne pos pos pos pos pos pos pos pos pos pos pos pos pos pos ne pos pos pos
pos pos pos
CD103
pp pos ne pos pos pos pp pp pos pos pos pos pp pp pos pos pp ne pos pp pos
pp pp pp
ANXA I
pp pos ne pos pp pos pos pp pp pos pos pos pos pp neg pos neg ne pp pos pos
pos pos pos
p-ERK V600E V600E V600E S602T V600E V600E WT V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E V600E WT WT
BRAF Exon 15
WT WT F468C WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT D449E
WT WT WT
BRAF Exon 11
WT WT
WT
WT
WT WT
WT
WT
WT WT
WT
WT
Exon2 Exon3 Exon4
KRAS
Male, male; f, female; bm, bone marrow; ki, kidney, pos, positive; pp, partially positive; wp, weak positive; neg, negative; ne, not evaluable; WT, wild-type
Age at diagnosis (years)
Patient no.
Table I. Clinical, immunophenotypical and molecular characteristics of the 24 patients with hairy cell leukaemia.
WT WT
WT
WT
WT WT
WT
WT
WT WT
ne
WT
Exon2 Exon3 Exon4
NRAS
WT WT
WT
WT
WT WT
ne
WT
Exon2 Exon3
HRAS
Short report
ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 529–533
Short report Table II. BRAF exon 11 and exon 15 mutation analysis in various mature B-cell and T-cell neoplasms excluding HCL.
Neoplasms B-cell neoplasms Bone marrow infiltration by low grade B-cell lymphoma not otherwise specified (splenic marginal zone lymphoma, splenic B-cell lymphoma/leukaemia unclassifiable, hairy cell leukaemia-variant) Nodal marginal zone lymphoma Extranodal marginal zone lymphoma (MALT-type) Burkitt lymphoma Follicular lymphoma Follicular lymphoma (G1) Follicular lymphoma (G2) Follicular lymphoma (G3A) Follicular lymphoma (G3B) Diffuse large B-cell lymphoma Mantle cell lymphoma Chronic lymphocytic leukaemia/small cell lymphocytic lymphoma T-cell neoplasms Anaplastic large T-cell lymphoma (ALCL) ALCL ALK1-positiv ALCL ALK1-negative Peripheral T-cell lymphoma not otherwise specified Angioimmunoblastic T-cell lymphoma
Number of cases
BRAF mutation exon 11
BRAF mutation exon 15
136 8
neg
neg
neg neg neg neg neg neg neg neg neg neg neg
neg neg neg neg neg neg neg neg neg neg neg
neg neg neg neg neg
neg neg neg neg neg
8 10 20 30 10 10 5 5 20 20 20 58 18 9 9 20 20
neg, Negative
were done by Sanger sequencing. The alternative BRAF mutations were confirmed in independent PCR and sequencing reactions, thus minimizing the risk to detect ‘artificial’ mutations. Experimental details are available on request.
Results and discussion The clinical, immunophenotypical and molecular characteristics of the 24 HCL cases investigated are provided in Table I. Of these, 21 HCL carried the BRAF V600E mutation in exon 15, while no mutation was detected in exon 11 in the BRAF V600E mutated cases. Of note, one HCL exhibited a second mutation in exon 15 (S602T; Patient 3, Table I). In this case, a germline mutation cannot be formally excluded, as we could not obtain normal DNA from this patient. S602 is located in the activation loop of BRAF and constitutes, together with T599, an essential phosphorylation site that plays an important role in the activation of BRAF (Garnett & Marais, 2004), arguing against the possibility of a germline mutation. While our S602T mutation has not been described so far, one other mutation at the same position has been reported in an osteosarcoma (S602Y; Pignochino et al, 2009). There is only one HCL in the literature that carries a second – though silent – BRAF mutation in exon 15 in addition to the conventional V600E mutation (Boyd et al, 2011). Of the 24 HCL cases in our series, three lacked the BRAF V600E mutation. Interestingly, alternative BRAF mutations could be detected in exon 11 in two of these patients (F468C ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 529–533
in Patient 6 and D449E in Patient 24, Table I), while the remaining patient (Patient 23) showed no mutations in exon 11. Given that in some malignant tumours (e.g. malignant melanoma), mutations in BRAF exons other than 11 and 15 have been described (Hodis et al, 2012), we investigated this latter HCL case for mutations in exons 2–17 of the BRAF gene. However, no mutations were discovered. Given that activation of the RAS-RAF-MEK-ERK pathway can also be caused by mutations in the KRAS, NRAS or HRAS genes, we decided to sequence the relevant regions of these genes in the three BRAF V600E mutation-negative cases as well. Patients 23 and 24 showed wild-type sequences for KRAS (exons 2, 3 and 4), NRAS (exons 2, 3 and 4) and HRAS (exons 2 and 3). Due to the limitations of the material from Patient 6, we could prove wild-type sequences only for KRAS (exons 2, 3 and 4), NRAS (exons 2 and 3) and HRAS (exon 2), while NRAS exon 4 and HRAS exon 3 could not be sequenced. Other investigators found a few exceptional HCL cases lacking the BRAF V600E mutation (Schnittger et al, 2012; Xi et al, 2012), but did not search for alternative BRAF mutations outside exon 15. We here describe the first two HCL tumours carrying BRAF mutations in exon 11. Patient 6 showed a characteristic clinical presentation as well as typical histological and phenotypical features of HCL (Table I and Table SIII). The detected F468C mutation within the glycine-rich loop in exon 11 has been described in colorectal cancer (Yuen et al, 531
Short report 2002). There is evidence from in vitro and in vivo transfection experiments (Ikenoue et al, 2004) that this mutation leads to increased basal kinase activity of BRAF and may thus substitute for the conventional V600E mutation. Patient 24 also showed the typical histological, phenotypical and clinical characteristics of HCL (Table I and Table SIII) and displayed another alternative mutation in exon 11 (D449E). The functional consequence of this mutation, which has not been reported in any other tumour so far, is unclear. Interestingly, position 449 is part of the N-region of BRAF and the aspartatic acid at this position is not conserved in ARAF and RAF1 (also termed CRAF; Tyr 303 in ARAF; Tyr 342 in RAF1). In order to activate ARAF and RAF1, these tyrosines have to be phosphorylated, while in BRAF the negative charge at this position might explain the elevated basal activity of BRAF compared to ARAF and RAF1 (Garnett & Marais, 2004). In our HCL, the exchange of aspartatic acid (D) to glutamatic acid (E) at position 449 keeps the negative charge, but the potential functional relevance remains unexplored. Given that Wan et al (2004) showed that D448 may come in contact with R506 of the aC helix, suggesting that this interaction stabilizes the active conformation and is important for the basal and RAS-stimulated kinase activity, one could speculate that the D449E mutation could modify, if not even intensify, this interaction. By sequencing BRAF exon 11 from normal gastric biopsies of both Patients 6 and 24 we could exclude germline mutations. Moreover, immunohistochemistry in Patient 24 demonstrated consistent pospho-ERK positivity in the tumour cells (Fig S1), suggesting a functional relevance of the D449E mutation leading to activation of the RAF-MEK-ERK pathway. In addition, Patient 23, who lacked BRAF, KRAS, NRAS and HRAS mutations, also showed a positive pospho-ERK status, suggesting a yet unknown mechanism for the activation of this pathway in this case. In addition to the 24 HCL cases, we investigated 194 other B- and T-NHL for BRAF mutations in exons 11 and 15 (Table II). Although a few cases of chronic lymphocytic leukaemia (Jebaraj et al, 2013) and diffuse large B-cell lymphoma (Lee et al, 2003) were reported to carry mutations in exon 15 or exons 11 and 15, respectively, all 136 B-NHL in our series showed unmutated exons 11 and 15. Similar results in lymphomas other than HCL have been previously reported (Tiacci et al, 2012). Primary Burkitt lymphomas have not been
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previously screened for exon 11 mutations (Tiacci et al, 2011; Laurini et al, 2012), but all 20 cases included in our study were BRAF wild-type in exons 11 and 15. Studies on BRAF mutations in peripheral T-cell lymphomas (PTCL) are scarce. Laurini et al (2012) investigated BRAF V600E mutations in 23 PTCL, which were all negative. Our cohort significantly extends existing negative data, because a total of 58 PTCL showed no BRAF mutations in exons 11 and 15 (Table II). In summary, our report confirms the existence of BRAF V600E-negative HCL and identifies novel alternative mutations in exon 11 of the BRAF gene. In the routine diagnostic setting, we therefore suggest that bona fide V600E-negative HCL should be screened for alternative exon 11 mutations. Future studies are needed to show whether HCL without mutations in the entire BRAF gene might carry other activating mutations in genes of the RAS-RAF-MEK-ERK pathway.
Author contributions EG and AR designed the research and reviewed the cases. ST and MB performed DNA extraction, PCR and sequencing of exon 11 and exon 15 of the BRAF gene. SR and LF performed PCR and sequencing of exon 2 to exon 17 (excluding exon 11 and 15) of the BRAF gene and PCR and sequencing analyses of the KRAS, NRAS and HRAS gene. ST, LF and EG analysed the data. ST, EG and AR wrote the manuscript.
Conflict of interest The authors declare no competing financial interests.
Supporting Information Additional Supporting Information may be found in the online version of this article: Table SI. Sources and dilutions of primary antibodies. Table SII. Primers, annealing temperatures and cycle conditions for BRAF, NRAS, KRAS and HRAS amplification. Table SIII. Laboratory findings, flow cytometry results, therapy and response in Patients 3, 6, 23 and 24. Fig S1. Relevant immunohistochemical stainings and sequencing analyses of two independent PCR reactions of the molecularly unusual HCL cases.
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