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B-CLL cells from lymph nodes express higher ZAP-70 levels than B-CLL cells from peripheral blood
Leukemia Research 31 (2007) 719–726
Letters to the Editor
B-CLL cells from lymph nodes express higher ZAP-70 levels than B-CLL cells from peripheral blood B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of CD5-expressing B-lymphocytes in the peripheral blood (PB) [1]. Messmer and colleagues demonstrated that a small but substantial subset of the CLL-cell population is proliferating [2]. This proliferating pool is situated in pseudofollicles in lymph nodes (LN) [3]. Zeta chain-associated protein of 70 kDa (ZAP-70) is accepted as a surrogate marker for IgVH gene mutational status [4,5]. Chen et al. explain the more aggressive behaviour of ZAP-70+ B-CLL cells as the consequence of a more effective IgM signaling [6]. Rassenti et al. demonstrated a continuum of Zap-70 positivity between patients, rather than two clearly distinct groups [7]. B-CLL cells can be mobilised from lymphoid organs by treatment with steroids. The observed increasement of ZAP70 in these mobilised cells led us to compare the ZAP-70 expression in peripheral blood and lymph nodes. Peripheral blood samples and fine needle aspirates were performed simultaneously in seven untreated B-CLL patients with palpable lymph nodes, according to institutional guidelines. Samples were processed for flow cytometry in identical ways and read in parallel using the same settings. Cells were stained with ZAP-70 antibody (clone 2F3.2; Upstate, Charlottesville, VA), goat F(ab )2 anti-mouse IgG(H + L)-PE
(Caltag, Burlingame, CA) and with CD3, CD19 and CD5 antibodies (all Beckman Coulter, Fullerton, CA). Fig. 1A shows the ZAP-70 expression in LN versus PB in a representative patient and Fig. 1B shows the mean fluorescence intensity (MFI) of B-CLL cells from blood and lymph node origin for all patients. In the seven cases, the MFI of ZAP-70 in LN B-CLL cells was on average 86% (14–164%) higher than for blood B-CLL cells (p < 0.05). In contrast, the MFI of ZAP-70 expression in T-lymphocytes of LN origin was significantly lower than those from the PB sample (p < 0.05). This was not due to differences in cell size as assessed by forward scatter. Various interpretations of our observations are possible: lymph node micro-environmental factors may cause upregulation of ZAP-70 which may be implicated in proliferation, while the absence of these stimuli in PB leads to ZAP-70 down-regulation. A second explanation is that there is a continuous in and outflow of PB CLL-cells into the LN and that only those with higher ZAP-70 levels express adhesion molecules that cause them to be withheld in the LN. Alternatively, ZAP-70 expression may be a part of normal late germinal center development as recently suggested by Cutrona et al. [8] and be lost with time as the B cells quit the LN. In this hypothesis ZAP-70 expression could be a reflection of time from birth in the pseudofollicle. ZAP-70+ cells in the PB might then correspond to increased B-CLL replenishment from lymphoid organs to PB, and ZAP-70 may hence be an indirect measure of proliferation and activity.
Fig. 1. ZAP-70 expression in B-CLL cells from LN and PB. (A) shows the ZAP-70 expression as MFI in LN versus PB B-CLL cells for one representative patient sample. (B) shows the ZAP-70 levels, expressed as MFI, in B-CLL and T-cells in all the patient samples. All samples were processed for flow cytometry in identical ways. 0145-2126/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.
720
Letters to the Editor / Leukemia Research 31 (2007) 719–726
In conclusion, ZAP-70 expression is higher in LN B-CLL than in PB B-CLL cells, suggesting ZAP-70 levels may be influenced by micro-environmental interactions that promote B-CLL cells to proliferate in the LN pseudofollicles. Acknowledgements The authors thank D. Claeys and A. Willems for technical assistance. Contributions. J. Boelens performed research, analyzed data and wrote the paper. J. Philipp´e contributed analytical tools. F. Offner designed research and wrote the paper. References [1] Caligaris-Cappio F, Hamblin TJ. B-cell chronic lymphocytic leukemia: a bird of a different feather. J Clin Oncol 1999;17(1):399–408. [2] Messmer BT, Messmer D, Allen SL, Kolitz JE, Kudalkar P, Cesar D, et al. In vivo measurements document the dynamic cellular kinetics of chronic lymphocytic leukemia B cells. J Clin Invest 2005;115(3):755–64. [3] Granziero L, Ghia P, Circosta P, Gottardi D, Strola G, Geuna M, et al. Survivin is expressed on CD40 stimulation and interfaces proliferation and apoptosis in B-cell chronic lymphocytic leukemia. Blood 2001;97:2777–83. [4] Orchard JA, Ibbotson RE, Davis Z, Wiestner A, Rosenwald A, Thomas PW, et al. ZAP-70 expression and prognosis in chronic lymphocytic leukemia. Lancet 2004;363:105–11. [5] Crespo M, Bosch F, Villamor N, Bellosillo B, Colomer D, Rozman M, et al. ZAP-70 expression as a surrogate for immunoglobulin-variableregion mutations in chronic lymphocytic leukemia. N Engl J Med 2003;348(18):1764–75. [6] Chen L, Apgar J, Huynh L, Dicker F, Ciago-McGahan T, Rassenti L, et al. ZAP-70 directly enhances IgM signaling in chronic lymphocytic leukemia. Blood 2005;105:2036–41. [7] Rassenti LZ, Lang Huynh BS, Toy TL, Chen L, Keating MJ, Gribben JG, et al. ZAP-70 compared with immunoglobulin heavy-chain mutation status as a predictor of disease progression in chronic lymphocytic leukemia. N Engl J Med 2004;351:893–901. [8] Cutrona G, Colombo M, Matis S, Reverbi D, Dono M, Tarantino V, et al. B lymphocytes in humans express ZAP-70 when activated in vivo. Eur J Immunol 2006;36:558–69.
Jerina Boelens ∗ Department of Hematology, Ghent University Hospital, Ghent, Belgium Jan Philipp´e Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Ghent, Belgium Fritz Offner Department of Hematology, Ghent University Hospital, Ghent, Belgium ∗ Tel.: +32 9 2403909; fax: +32 9 2402737. E-mail address: [email protected] (J. Boelens)
30 May 2006 Available online 4 August 2006 doi: 10.1016/j.leukres.2006.05.024
Agent Orange, prostate cancer irradiation and acute promyelocyic leukemia (APL): Is there a link? We recently saw a 60-year-old gentleman diagnosed with prostate cancer stage T2c in March 2005. He received neoadjuvant hormonal therapy followed by localized radiation therapy (RT) with neutrons (10 Gy) and photons (4500/25fractions) completed in 4/2005. In March 2006, he was diagnosed with a popliteal deep venous thrombosis. He was leukopenic at that time and 1 month later his WBC count was 0.7 K/mm3 , Hb 10.9 gm/dl, platelets 166 K/mm3 and ANC 0.3 K/mm3 . A bone marrow aspirate was characteristic of APL and cytogenetics revealed t(15;17) (q22;q21). His past medical history was significant for exposure to Agent Orange during the Vietnam War. He flew 15 missions on flights that sprayed Agent Orange and he stated that there was always a “fog” in the airplane and that it smelled “chemical-like”. Agent Orange refers to the defoliant chemicals used during the Vietnam War by the US military. Several defoliants were used, but phenoxyherbicides were the most common. Phenoxyherbicides are not considered carcinogenic. However, dioxin (TCDD) which is a contaminant of phenoxyherbicides is classified as a human carcinogen by the US Environmental Protection Agency. There is limited/suggestive evidence of an association between Agent Orange and prostate cancer. AML has not been associated with Agent Orange exposure, but several studies have suggested an increased risk of leukemia in children whose parents have been exposed to Agent Orange [1]. Although treatment related APL (t-APL) has been reported either secondary to chemotherapy and/or radiation [2], studies have not shown an increased risk of leukemia secondary to prostate irradiation [3]. Since 1.6 million veterans were exposed to Agent Orange during the Vietnam War, then 27,000 of these men are at risk for developing prostate cancer (1 in 6 men had lifetime risk assuming no increased risk with Agent Orange exposure). Ninety percent would be expected to have localized cancer and approximately 40% would receive radiation therapy [3]. Therefore, approximately 10,000 prostate cancer patients previously exposed to Agent Orange could receive RT. The patient’s indolent disease course could be explained by the long tissue half life of dioxin (7–11 years) and it’s ability to increase the level of all-transretinoic acid in cells [4] which may have ameliorated disease progression. The mechanism of dioxin carcinogenicity is partly understood. In the presence of dioxin, the Aryl-hydrocarbon receptor (AhR) translocates to the nucleus, dimerizes with Arnt and increases the tanscription of CYP1A1 and CYP1B1 which in turn leads to the formation of oxygen radicals and oxidative DNA damage [5,6]. In addition, TCDD has been shown to inhibit p53 mediated apoptosis and growth arrest [7]. In our patient the APL may have been secondary to the RT he received. Whether the prostate cancer was related to Agent
Letters to the Editor
B-CLL cells from lymph nodes express higher ZAP-70 levels than B-CLL cells from peripheral blood B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of CD5-expressing B-lymphocytes in the peripheral blood (PB) [1]. Messmer and colleagues demonstrated that a small but substantial subset of the CLL-cell population is proliferating [2]. This proliferating pool is situated in pseudofollicles in lymph nodes (LN) [3]. Zeta chain-associated protein of 70 kDa (ZAP-70) is accepted as a surrogate marker for IgVH gene mutational status [4,5]. Chen et al. explain the more aggressive behaviour of ZAP-70+ B-CLL cells as the consequence of a more effective IgM signaling [6]. Rassenti et al. demonstrated a continuum of Zap-70 positivity between patients, rather than two clearly distinct groups [7]. B-CLL cells can be mobilised from lymphoid organs by treatment with steroids. The observed increasement of ZAP70 in these mobilised cells led us to compare the ZAP-70 expression in peripheral blood and lymph nodes. Peripheral blood samples and fine needle aspirates were performed simultaneously in seven untreated B-CLL patients with palpable lymph nodes, according to institutional guidelines. Samples were processed for flow cytometry in identical ways and read in parallel using the same settings. Cells were stained with ZAP-70 antibody (clone 2F3.2; Upstate, Charlottesville, VA), goat F(ab )2 anti-mouse IgG(H + L)-PE
(Caltag, Burlingame, CA) and with CD3, CD19 and CD5 antibodies (all Beckman Coulter, Fullerton, CA). Fig. 1A shows the ZAP-70 expression in LN versus PB in a representative patient and Fig. 1B shows the mean fluorescence intensity (MFI) of B-CLL cells from blood and lymph node origin for all patients. In the seven cases, the MFI of ZAP-70 in LN B-CLL cells was on average 86% (14–164%) higher than for blood B-CLL cells (p < 0.05). In contrast, the MFI of ZAP-70 expression in T-lymphocytes of LN origin was significantly lower than those from the PB sample (p < 0.05). This was not due to differences in cell size as assessed by forward scatter. Various interpretations of our observations are possible: lymph node micro-environmental factors may cause upregulation of ZAP-70 which may be implicated in proliferation, while the absence of these stimuli in PB leads to ZAP-70 down-regulation. A second explanation is that there is a continuous in and outflow of PB CLL-cells into the LN and that only those with higher ZAP-70 levels express adhesion molecules that cause them to be withheld in the LN. Alternatively, ZAP-70 expression may be a part of normal late germinal center development as recently suggested by Cutrona et al. [8] and be lost with time as the B cells quit the LN. In this hypothesis ZAP-70 expression could be a reflection of time from birth in the pseudofollicle. ZAP-70+ cells in the PB might then correspond to increased B-CLL replenishment from lymphoid organs to PB, and ZAP-70 may hence be an indirect measure of proliferation and activity.
Fig. 1. ZAP-70 expression in B-CLL cells from LN and PB. (A) shows the ZAP-70 expression as MFI in LN versus PB B-CLL cells for one representative patient sample. (B) shows the ZAP-70 levels, expressed as MFI, in B-CLL and T-cells in all the patient samples. All samples were processed for flow cytometry in identical ways. 0145-2126/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.
720
Letters to the Editor / Leukemia Research 31 (2007) 719–726
In conclusion, ZAP-70 expression is higher in LN B-CLL than in PB B-CLL cells, suggesting ZAP-70 levels may be influenced by micro-environmental interactions that promote B-CLL cells to proliferate in the LN pseudofollicles. Acknowledgements The authors thank D. Claeys and A. Willems for technical assistance. Contributions. J. Boelens performed research, analyzed data and wrote the paper. J. Philipp´e contributed analytical tools. F. Offner designed research and wrote the paper. References [1] Caligaris-Cappio F, Hamblin TJ. B-cell chronic lymphocytic leukemia: a bird of a different feather. J Clin Oncol 1999;17(1):399–408. [2] Messmer BT, Messmer D, Allen SL, Kolitz JE, Kudalkar P, Cesar D, et al. In vivo measurements document the dynamic cellular kinetics of chronic lymphocytic leukemia B cells. J Clin Invest 2005;115(3):755–64. [3] Granziero L, Ghia P, Circosta P, Gottardi D, Strola G, Geuna M, et al. Survivin is expressed on CD40 stimulation and interfaces proliferation and apoptosis in B-cell chronic lymphocytic leukemia. Blood 2001;97:2777–83. [4] Orchard JA, Ibbotson RE, Davis Z, Wiestner A, Rosenwald A, Thomas PW, et al. ZAP-70 expression and prognosis in chronic lymphocytic leukemia. Lancet 2004;363:105–11. [5] Crespo M, Bosch F, Villamor N, Bellosillo B, Colomer D, Rozman M, et al. ZAP-70 expression as a surrogate for immunoglobulin-variableregion mutations in chronic lymphocytic leukemia. N Engl J Med 2003;348(18):1764–75. [6] Chen L, Apgar J, Huynh L, Dicker F, Ciago-McGahan T, Rassenti L, et al. ZAP-70 directly enhances IgM signaling in chronic lymphocytic leukemia. Blood 2005;105:2036–41. [7] Rassenti LZ, Lang Huynh BS, Toy TL, Chen L, Keating MJ, Gribben JG, et al. ZAP-70 compared with immunoglobulin heavy-chain mutation status as a predictor of disease progression in chronic lymphocytic leukemia. N Engl J Med 2004;351:893–901. [8] Cutrona G, Colombo M, Matis S, Reverbi D, Dono M, Tarantino V, et al. B lymphocytes in humans express ZAP-70 when activated in vivo. Eur J Immunol 2006;36:558–69.
Jerina Boelens ∗ Department of Hematology, Ghent University Hospital, Ghent, Belgium Jan Philipp´e Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Ghent, Belgium Fritz Offner Department of Hematology, Ghent University Hospital, Ghent, Belgium ∗ Tel.: +32 9 2403909; fax: +32 9 2402737. E-mail address: [email protected] (J. Boelens)
30 May 2006 Available online 4 August 2006 doi: 10.1016/j.leukres.2006.05.024
Agent Orange, prostate cancer irradiation and acute promyelocyic leukemia (APL): Is there a link? We recently saw a 60-year-old gentleman diagnosed with prostate cancer stage T2c in March 2005. He received neoadjuvant hormonal therapy followed by localized radiation therapy (RT) with neutrons (10 Gy) and photons (4500/25fractions) completed in 4/2005. In March 2006, he was diagnosed with a popliteal deep venous thrombosis. He was leukopenic at that time and 1 month later his WBC count was 0.7 K/mm3 , Hb 10.9 gm/dl, platelets 166 K/mm3 and ANC 0.3 K/mm3 . A bone marrow aspirate was characteristic of APL and cytogenetics revealed t(15;17) (q22;q21). His past medical history was significant for exposure to Agent Orange during the Vietnam War. He flew 15 missions on flights that sprayed Agent Orange and he stated that there was always a “fog” in the airplane and that it smelled “chemical-like”. Agent Orange refers to the defoliant chemicals used during the Vietnam War by the US military. Several defoliants were used, but phenoxyherbicides were the most common. Phenoxyherbicides are not considered carcinogenic. However, dioxin (TCDD) which is a contaminant of phenoxyherbicides is classified as a human carcinogen by the US Environmental Protection Agency. There is limited/suggestive evidence of an association between Agent Orange and prostate cancer. AML has not been associated with Agent Orange exposure, but several studies have suggested an increased risk of leukemia in children whose parents have been exposed to Agent Orange [1]. Although treatment related APL (t-APL) has been reported either secondary to chemotherapy and/or radiation [2], studies have not shown an increased risk of leukemia secondary to prostate irradiation [3]. Since 1.6 million veterans were exposed to Agent Orange during the Vietnam War, then 27,000 of these men are at risk for developing prostate cancer (1 in 6 men had lifetime risk assuming no increased risk with Agent Orange exposure). Ninety percent would be expected to have localized cancer and approximately 40% would receive radiation therapy [3]. Therefore, approximately 10,000 prostate cancer patients previously exposed to Agent Orange could receive RT. The patient’s indolent disease course could be explained by the long tissue half life of dioxin (7–11 years) and it’s ability to increase the level of all-transretinoic acid in cells [4] which may have ameliorated disease progression. The mechanism of dioxin carcinogenicity is partly understood. In the presence of dioxin, the Aryl-hydrocarbon receptor (AhR) translocates to the nucleus, dimerizes with Arnt and increases the tanscription of CYP1A1 and CYP1B1 which in turn leads to the formation of oxygen radicals and oxidative DNA damage [5,6]. In addition, TCDD has been shown to inhibit p53 mediated apoptosis and growth arrest [7]. In our patient the APL may have been secondary to the RT he received. Whether the prostate cancer was related to Agent