Correspondence
References Dispenzieri, A., Rajkumar, S.V., Gertz, M.A., Fonseca, R., Lacy, M.Q., Bergsagel, P.L., Kyle, R.A., Greipp, P.R., Witzig, T.E., Reeder, C.B., Lust, J.A., Russell, S.J., Hayman, S.R., Roy, V., Kumar, S., Zeldenrust, S.R., Dalton, R.J. & Stewart, A.K. (2007) Treatment of newly diagnosed multiple myeloma based on Mayo Stratification of Myeloma and Risk-adapted Therapy (mSMART): consensus statement. Mayo Clinic Proceedings, 82, 323–341. Harousseau, J.-L., Attal, M. & Avet-Loiseau, H. (2009) The role of complete response in multiple myeloma. Blood, 114, 3139–3146. Jakubowiak, A., Dytfeld, D., Griffith, K.A., Lebovic, D., Vesole, D., Jagannath, S., Al-Zoubi, A., Anderson, T., Nordgren, B., Detweiler-Short, K., Stockerl-Goldstein, K., Asra Ahmed, A., Jobkar, T., Durecki, D.E., McDonnell, K., Mietzel, M., Couriel, D., Kaminski, M. & Vij, R. (2012) A phase 1/2 study of carfilzomib in combination with lenalidomide and low-dose dexamethasone as a frontline treatment for multiple myeloma. Blood, 120, 1801–1809. Khan, M.L., Reeder, C.B., Kumar, S.K., Lacy, M.Q., Reece, D.E., Dispenzieri, A., Gertz, M.A., Greipp, P., Hayman, S., Zeldenhurst, S., Dingli, D., Lust, J., Russell, S., Laumann, K.M., Mikhael, J.R., Leif Bergsagel, P., Fonseca, R., Vincent
Rajkumar, S. & Keith Stewart, A. (2012) A comparison of lenalidomide/dexamethasone versus cyclophosphamide/lenalidomide/dexamethasone versus cyclophosphamide/bortezomib/dexamethasone in newly diagnosed multiple myeloma. British Journal of Haematology, 156, 326–333. Kumar, S.K., Rajkumar, S.V., Dispenzieri, A., Lacy, M.Q., Hayman, S.R., Buadi, F.K., Zeldenrust, S.R., Dingli, D., Russell, S.J., Lust, J.A., Greipp, P.R., Kyle, R.A. & Gertz, M.A. (2008) Improved survival in multiple myeloma and the impact of novel therapies. Blood, 111, 2516–2520. Kumar, S., Flinn, I., Richardson, P.G., Hari, P., Callander, N., Noga, S.J., Stewart, A.K., Turturro, F., Rifkin, R., Wolf, J., Estevam, J., Mulligan, G., Shi, H., Webb, I.J. & Rajkumar, S.V. (2012) Randomized, multicenter, phase 2 study (EVOLUTION) of combinations of bortezomib, dexamethasone, cyclophosphamide, and lenalidomide in previously untreated multiple myeloma. Blood, 119, 4375–4382. Reeder, C.B., Reece, D.E., Kukreti, V., Chen, C., Trudel, S., Hentz, J., Noble, B., Pirooz, N.A., Spong, J.E., Piza, J.G., Zepeda, V.H., Mikhael, J.R., Leis, J.F., Bergsagel, P.L., Fonseca, R. & Stewart, A.K. (2009) Cyclophosphamide, bortezomib and dexamethasone induction for newly diagnosed multiple myeloma: high response
rates in a phase II clinical trial. Leukemia, 23, 1337–1341. Reeder, C.B., Reece, D.E., Kukreti, V., Chen, C., Trudel, S., Lauman, K., Hentz, J., Pirooz, N.A., Piza, J.G., Tiedemann, R., Mikhael, J.R., Leis, J.F., Bergsagel, P.L., Fonseca, R. & Stewart, A.K. (2010) Once- versus twice-weekly bortezomib induction therapy with CyBorD in newly diagnosed multiple myeloma. Blood, 115, 3416– 3417. Richardson, P., Weller, E., Lonial, S., Jakubowiak, A., Jagannath, S., Raje, N., Avigan, D., Xie, W., Ghobrial, I., Schlossman, R., Mazumder, A., Munshi, N., Vesole, D., Joyce, R., Kaufman, J., Doss, D., Warren, D., Lunde, L., Kaster, S., DeLaney, C., Hideshima, T., Mitsiades, C., Knight, R., Esseltine, D. & Anderson, K. (2010) Lenalidomide, bortezomib, and dexamethasone combination therapy inpatients with newly diagnosed multiple myeloma. Blood, 116, 679–686. Srivastava, G., Rana, V., Lacy, M.Q., Buadi, F.K., Hayman, S.R., Dispenzieri, A., Gertz, M.A., Dingli, D., Zeldenrust, S., Russell, S., McCurdy, A., Kapoor, P., Kyle, R., Rajkumar, S.V. & Kumar, S. (2013) Long-term outcome with lenalidomide and dexamethasone therapy for newly diagnosed multiple myeloma. Leukemia, 27, 2062–2066.
Assessment of TP53 functionality in chronic lymphocytic leukaemia by different assays; an ERIC-wide approach
Chronic lymphocytic leukaemia (CLL) is characterized by an extremely variable clinical course, in which deletions of TP53 and ATM, regulators of the DNA-damage response (DDR-) pathway, are powerful predictors for adverse outcome and response to chemotherapy (Dohner et al, 2000). Deletions of chromosome 17p (TP53) and 11q (ATM) coincide with TP53 and ATM mutations respectively, however with markedly different frequencies (80% and 40% respectively; Malcikova et al, 2009; Navrkalova et al, 2013). Sole TP53 mutations and deletions usually lead to TP53 (p53) dysfunction (Mohr et al, 2011) and, in case of deletions in ATM, a mutation of the residual ATM allele determines whether there is complete ATM inactivation resulting in TP53-dysfunction (Navrkalova et al, 2013). Therefore, analysis of mutations in TP53 and ATM genes in addition to fluorescent in situ hybridization (FISH) analysis is of additional clinical value (Skowronska et al, 2012). However, mutational analysis of TP53 and ATM is currently not standardized and is challenging, especially for ATM due to extreme gene size with lack of well-characterized mutations (Navrkalova et al, 2013). ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 167, 563–583
Chemoresistance might additionally be a consequence of epigenetic and post-transcriptional factors or deregulations of other components of the DDR. Therefore, functional readouts of the ATM/TP53 axis might add clinical relevant information on the actual DDR and chemo-sensitivity over FISH analysis, as well as important biological insight on the signalling capacity of the axis. There are different approaches to explore the ATM/TP53 axis at the functional level; i.e. at baseline or following a TP53 stimulus (irradiation or chemotherapy) and by different read-outs; i.e. measuring expression of responsive targets at (micro-)RNA or protein level. Currently, 4 different TP53function assays for CLL have been developed based on these principles: (i) reverse transcription polymerase chain reaction (RT-PCR) of MIR34A (RT-PCRMIR34A) determines MIR34A levels at baseline (Mraz et al, 2009; Asslaber et al, 2010; Mohr et al, 2011), (ii) RT-PCRCDKN1A assesses RNA CDKN1A (p21) levels after irradiation (Mohr et al, 2011), (iii) reverse transcription multiplex ligation-dependent probe amplification (RT-MLPA) of CDKN1A (RT-MLPACDKN1A), 565
Correspondence (A)
(B)
(C)
Fig 1. Robustness, reproducibility and correlation of different TP53-function assays. TP53-function assays were performed blindly on freshly frozen peripheral blood mononuclear cells (PBMCs) of 16 chronic lymphocytic leukaemia (CLL) samples (CD19/CD5 > 90%) which were exchanged between 5 different research groups. Each group provided 3 (one group 4) samples containing at least two samples harbouring a 17p and/or 11q deletion. (A) Each assay was performed in 2 different laboratories with ample experience with particular assay as indicated. RT-PCRCDKN1A in Amsterdam was performed as part of the RT-MLPA assay. Results of the same assays performed in different laboratories were compared. (B) Freshly frozen PBMCs of 5 CLL samples were again exchanged between the different research groups in a blinded fashion and results were compared with the results in the first exchange. For RT-MLPA the geometric mean of the gene induction of the 4 TP53-target genes in each sample was taken. (C). Results of the 4 different TP53-function assays were compared in relation to each other. In case one assay was performed in 2 facilities, the results of one representative assay were used for subsequent analyses. Comparisons are shown in a side-by-side table showing correlation coefficients (r-values) in a grey-scale scheme as depicted in the figure and P-values are indicated in the boxes; *001 ≤ P < 005, **0001 ≤ P < 001 and *** P < 0001. For (A–C) Spearman’s rank correlation. P-value
References Dispenzieri, A., Rajkumar, S.V., Gertz, M.A., Fonseca, R., Lacy, M.Q., Bergsagel, P.L., Kyle, R.A., Greipp, P.R., Witzig, T.E., Reeder, C.B., Lust, J.A., Russell, S.J., Hayman, S.R., Roy, V., Kumar, S., Zeldenrust, S.R., Dalton, R.J. & Stewart, A.K. (2007) Treatment of newly diagnosed multiple myeloma based on Mayo Stratification of Myeloma and Risk-adapted Therapy (mSMART): consensus statement. Mayo Clinic Proceedings, 82, 323–341. Harousseau, J.-L., Attal, M. & Avet-Loiseau, H. (2009) The role of complete response in multiple myeloma. Blood, 114, 3139–3146. Jakubowiak, A., Dytfeld, D., Griffith, K.A., Lebovic, D., Vesole, D., Jagannath, S., Al-Zoubi, A., Anderson, T., Nordgren, B., Detweiler-Short, K., Stockerl-Goldstein, K., Asra Ahmed, A., Jobkar, T., Durecki, D.E., McDonnell, K., Mietzel, M., Couriel, D., Kaminski, M. & Vij, R. (2012) A phase 1/2 study of carfilzomib in combination with lenalidomide and low-dose dexamethasone as a frontline treatment for multiple myeloma. Blood, 120, 1801–1809. Khan, M.L., Reeder, C.B., Kumar, S.K., Lacy, M.Q., Reece, D.E., Dispenzieri, A., Gertz, M.A., Greipp, P., Hayman, S., Zeldenhurst, S., Dingli, D., Lust, J., Russell, S., Laumann, K.M., Mikhael, J.R., Leif Bergsagel, P., Fonseca, R., Vincent
Rajkumar, S. & Keith Stewart, A. (2012) A comparison of lenalidomide/dexamethasone versus cyclophosphamide/lenalidomide/dexamethasone versus cyclophosphamide/bortezomib/dexamethasone in newly diagnosed multiple myeloma. British Journal of Haematology, 156, 326–333. Kumar, S.K., Rajkumar, S.V., Dispenzieri, A., Lacy, M.Q., Hayman, S.R., Buadi, F.K., Zeldenrust, S.R., Dingli, D., Russell, S.J., Lust, J.A., Greipp, P.R., Kyle, R.A. & Gertz, M.A. (2008) Improved survival in multiple myeloma and the impact of novel therapies. Blood, 111, 2516–2520. Kumar, S., Flinn, I., Richardson, P.G., Hari, P., Callander, N., Noga, S.J., Stewart, A.K., Turturro, F., Rifkin, R., Wolf, J., Estevam, J., Mulligan, G., Shi, H., Webb, I.J. & Rajkumar, S.V. (2012) Randomized, multicenter, phase 2 study (EVOLUTION) of combinations of bortezomib, dexamethasone, cyclophosphamide, and lenalidomide in previously untreated multiple myeloma. Blood, 119, 4375–4382. Reeder, C.B., Reece, D.E., Kukreti, V., Chen, C., Trudel, S., Hentz, J., Noble, B., Pirooz, N.A., Spong, J.E., Piza, J.G., Zepeda, V.H., Mikhael, J.R., Leis, J.F., Bergsagel, P.L., Fonseca, R. & Stewart, A.K. (2009) Cyclophosphamide, bortezomib and dexamethasone induction for newly diagnosed multiple myeloma: high response
rates in a phase II clinical trial. Leukemia, 23, 1337–1341. Reeder, C.B., Reece, D.E., Kukreti, V., Chen, C., Trudel, S., Lauman, K., Hentz, J., Pirooz, N.A., Piza, J.G., Tiedemann, R., Mikhael, J.R., Leis, J.F., Bergsagel, P.L., Fonseca, R. & Stewart, A.K. (2010) Once- versus twice-weekly bortezomib induction therapy with CyBorD in newly diagnosed multiple myeloma. Blood, 115, 3416– 3417. Richardson, P., Weller, E., Lonial, S., Jakubowiak, A., Jagannath, S., Raje, N., Avigan, D., Xie, W., Ghobrial, I., Schlossman, R., Mazumder, A., Munshi, N., Vesole, D., Joyce, R., Kaufman, J., Doss, D., Warren, D., Lunde, L., Kaster, S., DeLaney, C., Hideshima, T., Mitsiades, C., Knight, R., Esseltine, D. & Anderson, K. (2010) Lenalidomide, bortezomib, and dexamethasone combination therapy inpatients with newly diagnosed multiple myeloma. Blood, 116, 679–686. Srivastava, G., Rana, V., Lacy, M.Q., Buadi, F.K., Hayman, S.R., Dispenzieri, A., Gertz, M.A., Dingli, D., Zeldenrust, S., Russell, S., McCurdy, A., Kapoor, P., Kyle, R., Rajkumar, S.V. & Kumar, S. (2013) Long-term outcome with lenalidomide and dexamethasone therapy for newly diagnosed multiple myeloma. Leukemia, 27, 2062–2066.
Assessment of TP53 functionality in chronic lymphocytic leukaemia by different assays; an ERIC-wide approach
Chronic lymphocytic leukaemia (CLL) is characterized by an extremely variable clinical course, in which deletions of TP53 and ATM, regulators of the DNA-damage response (DDR-) pathway, are powerful predictors for adverse outcome and response to chemotherapy (Dohner et al, 2000). Deletions of chromosome 17p (TP53) and 11q (ATM) coincide with TP53 and ATM mutations respectively, however with markedly different frequencies (80% and 40% respectively; Malcikova et al, 2009; Navrkalova et al, 2013). Sole TP53 mutations and deletions usually lead to TP53 (p53) dysfunction (Mohr et al, 2011) and, in case of deletions in ATM, a mutation of the residual ATM allele determines whether there is complete ATM inactivation resulting in TP53-dysfunction (Navrkalova et al, 2013). Therefore, analysis of mutations in TP53 and ATM genes in addition to fluorescent in situ hybridization (FISH) analysis is of additional clinical value (Skowronska et al, 2012). However, mutational analysis of TP53 and ATM is currently not standardized and is challenging, especially for ATM due to extreme gene size with lack of well-characterized mutations (Navrkalova et al, 2013). ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 167, 563–583
Chemoresistance might additionally be a consequence of epigenetic and post-transcriptional factors or deregulations of other components of the DDR. Therefore, functional readouts of the ATM/TP53 axis might add clinical relevant information on the actual DDR and chemo-sensitivity over FISH analysis, as well as important biological insight on the signalling capacity of the axis. There are different approaches to explore the ATM/TP53 axis at the functional level; i.e. at baseline or following a TP53 stimulus (irradiation or chemotherapy) and by different read-outs; i.e. measuring expression of responsive targets at (micro-)RNA or protein level. Currently, 4 different TP53function assays for CLL have been developed based on these principles: (i) reverse transcription polymerase chain reaction (RT-PCR) of MIR34A (RT-PCRMIR34A) determines MIR34A levels at baseline (Mraz et al, 2009; Asslaber et al, 2010; Mohr et al, 2011), (ii) RT-PCRCDKN1A assesses RNA CDKN1A (p21) levels after irradiation (Mohr et al, 2011), (iii) reverse transcription multiplex ligation-dependent probe amplification (RT-MLPA) of CDKN1A (RT-MLPACDKN1A), 565
Correspondence (A)
(B)
(C)
Fig 1. Robustness, reproducibility and correlation of different TP53-function assays. TP53-function assays were performed blindly on freshly frozen peripheral blood mononuclear cells (PBMCs) of 16 chronic lymphocytic leukaemia (CLL) samples (CD19/CD5 > 90%) which were exchanged between 5 different research groups. Each group provided 3 (one group 4) samples containing at least two samples harbouring a 17p and/or 11q deletion. (A) Each assay was performed in 2 different laboratories with ample experience with particular assay as indicated. RT-PCRCDKN1A in Amsterdam was performed as part of the RT-MLPA assay. Results of the same assays performed in different laboratories were compared. (B) Freshly frozen PBMCs of 5 CLL samples were again exchanged between the different research groups in a blinded fashion and results were compared with the results in the first exchange. For RT-MLPA the geometric mean of the gene induction of the 4 TP53-target genes in each sample was taken. (C). Results of the 4 different TP53-function assays were compared in relation to each other. In case one assay was performed in 2 facilities, the results of one representative assay were used for subsequent analyses. Comparisons are shown in a side-by-side table showing correlation coefficients (r-values) in a grey-scale scheme as depicted in the figure and P-values are indicated in the boxes; *001 ≤ P < 005, **0001 ≤ P < 001 and *** P < 0001. For (A–C) Spearman’s rank correlation. P-value
