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Hematological Malignancies

Hematological Malignancies Hematology, August 2004 Vol. 9 (4), pp. 279–286 Aberrant Morphology, Proliferation, and Apoptosis of B-cell Chronic Lymphocytic Leukemia Cells a b a b CARLOS E. BUESO-RAMOS , ALESSANDRA FERRAJOLI , L. JEFFREY MEDEIROS , MICHAEL J. KEATING and c, ZEEV ESTROV * a b Department of Hematopathology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA; Department of Leukemia, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA; Department of Bioimmunotherapy, Unit 422, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA (Received 5 April 2004; In final form 8 May 2004) B-cell chronic lymphocytic leukemia (CLL) has been INTRODUCTION traditionally described as a disease characterized by an accumulation of quiescent small lymphocytes B-cell chronic lymphocytic leukemia (CLL) is the with decreased susceptibility to apoptotic cell death. most common type of adult leukemia in the United However, small numbers of “atypical” lymphocytes and States and Western Europe [1]. It is thought to be prolymphocytes (PL) are frequently observed in the bone marrow (BM) of patients with CLL. In this study, we caused primarily by defects in programmed cell examined BM biopsy and aspirate specimens obtained death (apoptosis) and is characterized by a gradual from seven patients with atypical CLL. Using a double accumulation of small, long-lived, immunologically 1 1 labeling (Ki-67 /CD20 ) immunohistochemical method, dysfunctional mature B-lymphocytes, most of we found that an appreciable number of the atypical CLL which are in G0/G1 phase of the cell cycle [2 – 4]. cells expressed the proliferation-associated protein Ki-67. Because CLL is characterized by a slow change During diagnosis or during the course of in the peripheral blood (PB) lymphocyte count, we the disease, 15% of patients with CLL show reasoned that a subpopulation of CLL cells probably atypical morphologic features characterized undergoes spontaneous apoptosis. Using Western blot by an increased (. 10%) number of circulating analysis, we observed expression of procaspase-9, prolymphocytes (PL), designated as CLL/PL, procaspase-10, and poly(ADP-ribose) polymerase by the neoplastic cells in all seven cases of CLL, and or an increased (. 15%) number of circulating procaspase-3 and procaspase-8 expression in six lymphoplasmacytic and cleaved cells, designated neoplasms. We also detected cleaved caspase-3 and as “atypical” CLL [1,5]. Patients with atypical CLL cleaved poly(ADP-ribose) polymerase in four and five more commonly have an accelerated clinical CLL cases, respectively. To determine whether CLL cells course and trisomy 12 [1,6,7]. In addition, undergo spontaneous apoptosis, we performed the terminal deoxynucleotidyl transferase-mediated dUTP atypical CLL cases have increased expression of nick-end labeling (TUNEL) assay using BM biopsy the nuclear proliferation protein Ki-67 [8], and specimens. We found TUNEL-positive lymphocytes in ultrastructural features suggestive of apoptosis [9]. areas infiltrated by CLL. In summary, our data show that Expression of the human protein Ki-67 is strictly subpopulations of B-lymphocytes are proliferating or associated with cell proliferation, being expressed undergoing spontaneous apoptotic cell death in patients with atypical CLL. during all active phases of the cell cycle (G1, S, G2, and mitosis) but absent in resting cells (G0 phase) [10]. The predominant proliferative component Keywords: Chronic lymphocytic leukemia (CLL); Apoptosis; Proliferation; Atypical morphology; Cytogenetics in CLL is localized in proliferation centers that are *Corresponding author. Tel.: þ 1-713-794-1675. Fax: þ 1-713-745-2374. E-mail: zestrov@mdanderson.org ISSN 1024-5332 print/ISSN 1607-8454 online q 2004 Taylor & Francis Ltd DOI: 10.1080/10245330410001727046 280 C.E. BUESO-RAMOS et al. usually identified in the lymph nodes but less involvement by a B-cell neoplasm composed commonly are present in bone marrow (BM) [11,12]. predominantly of small lymphocytes that expressed There have been no published reports of simul- monotypic immunoglobulin light chain, pan-B-cell taneous assessment of proliferation and apoptosis in antigens (CD19, CD20), CD5 and CD23 and negative BM biopsy specimens obtained from patients with for CD3, CD10 and cyclin D1. CLL with atypical morphology. Morphologic analysis was performed using In our experience, small percentages of atypical Wright-Giemsa stained PB and BM aspirate smears lymphocytes (i.e. lymphoplasmacytic and cleaved and hematoxylin-eosin stained BM biopsy speci- cells) and PL can be identified in BM aspirate smears mens. Flow cytometric immunophenotyping was in most cases of CLL, in patients at any clinical stage. performed using BM aspirates with a panel Thus, we hypothesized that CLL cells are more of antibodies specific for immunoglobulin kappa heterogeneous than is appreciated in the literature. and lambda light chains, CD3, CD5, CD10, CD19, Thus, in this study group we assessed both pro- CD20, and CD23 according to well-established liferation and apoptosis. We show that cases of CLL methods [13]. Other antibodies were used in a with atypical morphologic features, regardless of subset of these cases. Conventional G-band karyo- stage, have subpopulations of cells that are prolifer- type analysis of all samples was performed accord- ating or undergoing spontaneous apoptosis, in ing to a previously reported method [13]. addition to quiescent cells, in vivo. Immunohistochemical Methods MATERIALS AND METHODS Immunohistochemical studies were performed using formalin-fixed, paraffin-embedded tissue sections Patient Specimens (4 mm thick) and an avidin – biotin peroxidase Bone marrow aspirate and biopsy specimens technique [14]. The primary antibodies were specific were obtained from seven patients with B-cell CLL. for Ki-67 (1:1000; DAKO Corporation, Carpinteria, Bone marrow aspirate and peripheral blood CA, USA) and cleaved caspase-3 (1:5000; R & D (PB) specimens were also obtained from two hemato- Systems, Minneapolis, MN, USA). Peroxidase logically normal individuals (BM transplant donors) staining was performed using the anti-mouse, anti- at the time of diagnostic evaluation at The University rabbit link antibody (DAKO LSAB2-HRP, 15 min; of Texas M. D. Anderson Cancer Center. All subjects DAKO Corporation). Sequential immunoenzymatic gave their informed consent, but this retrospective staining combining the immunoperoxidase and study did not require approval by the Institutional immunoalkaline phosphatase techniques was used Review Board when it was initiated. for simultaneous detection of Ki-67 and CD20 The clinical characteristics of the seven CLL (DAKO). Negative and positive controls were run. patients are summarized in Table I. All previously At low power magnification, the entire tissue treated patients had not received treatment for section was scanned and microscopic fields were several months at the time that the specimens were selected based on areas with the greatest number of obtained. The diagnosis of CLL was based on a Ki-67-positive and cleaved caspase-3-positive cells. combination of clinical, morphologic, laboratory, and In each case, at least 500 CLL cells were evaluated for immunophenotypic criteria as defined previously nuclear (Ki-67, cleaved caspase-3) and membranous [4]. Specifically, the criteria for CLL included (CD20) positivity. TABLE I Patient characteristics Age in WBC Hb level Plt count b M level LDT 3 3 Patient years/sex Rai stage ( £ 10 /ml) (g/dl) ( £ 10 /ml) (mg/l) Prior treatment (months) 1 57/M II 33.4 12.8 167 3.4 Chb, CTX-P 6 2 57/M I 96.3 13.5 132 3.4 None 12 3 42/F I 22.2 13.1 132 2.8 None 4 4 55/M IV 13.1 8.6 130 2.0 CHOP/CTX, O, VP-16, P, F, 6 DHAP, thiotepa; bleomycin 5 73/M II 104.5 13.1 118 5.6 None N/A 6 46/F I 28.2 12.8 178 N/A None N/A 7 76M IV 175 11.2 75 11.4 Chb, F, 2CdA 6 WBC, white blood count; Hb, hemoglobin; Plt, platelet; b M, b -microglobulin; LDT, lymphocyte doubling time; Chb, chlorambucil; CTX, cyclophosphamide; 2 2 CHOP, cyclophosphamide, adriamycin, vincristine, and prednisone; O, vincristine; VP-16, etoposide; P, prednisone; F, fludarabine; DHAP, dexamethasone, cytarabine, and cisplatin; 2CdA, 2-cholorodeoxyadenosine. PROLIFERATION AND APOPTOSIS IN B-CLL 281 Terminal Deoxynucleotidyl Transferase-mediated RESULTS dUTP Nick-end Labeling (TUNEL) Assay for Detection of Apoptosis Clinical Features Cells undergoing apoptosis were detected with a The clinical features for the seven patients are TUNEL method as described previously [14]. summarized in Table I. We studied five male and two Apoptotic figures, including positively labeled female patients whose ages ranged from 42 to 76 intact nuclei and nuclear fragments derived from a years (median, 57 years). Four patients were not single cell, were counted per high-power field (HPF) previously treated, and three had been treated at in areas with 100% cellularity with the use of an other institutions. All previously treated patients had Olympus BX40 microscope with a 40 £ objective been off therapy for several months at the time the and 10 £ ocular (Olympus America Inc., Melville, samples were obtained. Most patients received NY, USA). In all cases, there were sufficient areas fludarabine-based chemotherapy in combination with adequate morphology to cover four or more with rituximab after diagnostic evaluation at The HPFs. Using this approach, it was found that University of Texas M. D. Anderson Cancer Center. 0.23 mm area within a HPF contained approxi- Three patients (1, 4 and 7) received alemtuzumab mately 2950 cells when the cellularity was 100%. alone or in combination with other agents. Four The number of apoptotic cells derived from a field patients achieved complete remission and three with lower cellularity was normalized proportion- patients had partial response. ally to 100% cellularity (e.g. when two TUNEL- positive cells were identified in a HPF with a Expression of Ki-67 Protein by CLL Cells cellularity of 50%, a score of 4 was recorded). The median score in each case was used for the final In all CLL patient specimens, we detected a small analysis. number of binucleated and trinucleated and/or large plasmacytoid cells in BM aspirate smears and expansion of the medullary space in BM biopsy Western Blot Analysis specimens by CLL (Fig. 1A – C). Because the presence of these cells has been associated with an increased Western blot analysis was performed using cell proliferation rate [6], we performed immunohisto- lysates of PB and low-density aspirate BM cells of chemical studies to detect Ki-67 protein in BM biopsy seven CLL patients as described previously [15]. specimens. As shown in Table II, we found that the Cell lysates from 5 £ 10 BM cells were assayed to immunoreactivity for this protein was relatively high determine the protein concentration using the BCA (. 5%) in BM biopsy specimens of all seven patients Protein Assay Reagent kit (Pierce Chemical Co., in typical areas of CLL infiltration (Fig. 1D – E). Rockford, IL, USA). Each set of paired samples was The CLL infiltrate distorted and displaced the then adjusted so that they would have the same normal Ki-67 erythroid and myeloid islets com- protein concentration. The following antibodies pared with that in the surrounding uninvolved BM were used for their respective proteins: mouse (Fig. 1F). anti-human CPP32 (procaspase-3), mouse anti- To establish with certainty that CLL cells rather human FLICE (procaspase-8), rabbit anti-human than normal hematopoietic cells were proliferating, procaspase-10, mouse anti-human poly(ADP- we performed double labeling immunohisto- ribose) polymerase (PARP; Pharmingen, San chemical staining with Ki-67 and the B-cell marker, Diego, CA, USA), and rabbit anti-human cleaved anti-CD20. As shown in Fig. 2, a subset of CLL cells caspase-3 (New England BioLabs, Beverly, MA, co-expressed Ki-67 and CD20. USA). Normal mouse IgG and rabbit IgG (Sigma Chemical Co., St. Louis, MO, USA) were used as controls. Lysates of Jurkat, HL-60, and K-562 cells Expression of Procaspase and Caspase Proteins and were used to confirm the detection of these Detection of Apoptosis in CLL Cells proteins. After initial probing, blots were re-probed In previous studies, we found that patients with with an antibody specific for actin (1:1000, mouse untreated acute leukemias had high levels of monoclonal, Sigma Chemical Co.) to assess protein procaspase and caspase proteins and that PARP, loading. a DNA repair enzyme known to protect cell integrity, was spontaneously cleaved in acute leukemia cells [15 – 17]. Using a similar method, we used Western Statistical Analysis immunoblotting to detect these proteins in BM Statistical analysis was performed using the chi- aspirates from seven cases of CLL. Procaspase-9 and square test. p values , 0.05 were considered procaspase-10 were expressed in all neoplasms, with statistically significant. procaspase-8 and procaspase-3 were expressed in 282 C.E. BUESO-RAMOS et al. FIGURE 1 Histologic, immunohistologic, and cytologic stains of representative BM biopsy and aspirate specimens from patient 3. (A) Extensive infiltration by small lymphocytes in the BM biopsy specimen (hematoxylin and eosin stain, £ 400). (B and C) Cleaved, multinucleated lymphocytes, prolymphocytes and small round lymphocytes in the BM aspirate smears (Wright-Giemsa stain, £ 1000). (D – F) Immunohistochemical stain for Ki-67 shows Ki-67-positive CLL cells (D and E, CLL BM biopsy specimens [ £ 400 and £ 600, respectively]; F, normal BM biopsy specimen [ £ 600]). (G – I) TUNEL staining of BM biopsy specimens (G and H, BM specimens showing several TUNEL-positive cells [ £ 400 and £ 600, respectively]; I, normal BM biopsy specimen showing rare positive cells [ £ 600]). (J – L) Immunohistochemical staining for cleaved caspase-3. Activated caspase-3 was found often in CLL cells (J and K [ £ 400 and £ 600, respectively]) but rarely in normal BM specimens (L [ £ 600]). TABLE II Cytogenetic and BM findings in CLL cases Histologic pattern Lymphocytes Cleaved TUNEL Ki-67 Patient Cytogenetic abnormality in BM biopsy specimen (%) caspase-3 (%) (per HPF) (%) 1 Diploid N/I 95 15 8 12 2 Diploid D 88 9 12 12 3 47,XX,þ 12[3] D 80 5 29 50 47,XX,t(6;14)(p13;q32),þ 12[1] 4 46,XY,inv(9)(p11q12),del(11)(q22)[5] N/I 80 3 9 10 5 46,XY,del(14)(q23)[13] D93 3 8 8 46,XY[7] 6 90 – 91,XXXX,2 4,2 6,der(7;?)(p10;?),2 10, D95 2 1 45 add(14)(q32)x2,der(19)del(19) (p13.1)del(19)(q13.1)x2, þ 0 – 4mar[cp5] 7 46,XY,ins(12;?)(q13;?),del(13) D82 18 6 40 (q12q22)[2] 46,XY[6] BM, bone marrow; N/I, nodular/interstitial; D, diffuse; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nickend labeling; HPF, high power field. PROLIFERATION AND APOPTOSIS IN B-CLL 283 CLL cells, we performed TUNEL staining of BM sections. We found TUNEL-positive CLL cells (Fig. 1G – H and Table I) in all seven neoplasms (Fig. 1G – H, Table II), indicating that a subset of the CLL cells underwent spontaneous apoptosis. TUNEL-positive cells were rarely found in normal BM sections (Fig. 1I). Cytogenetic Results Conventional cytogenetic studies were performed on BM aspirates of all seven patients. Five patients (71%) had chromosomal anomalies and two patients (29%) were diploid (Table II). The chromosomal abnormalities included three chromo- some 14 abnormalities (one del(14)(q23), one FIGURE 2 Double immunostaining for nuclear Ki-67 and add(14)(q23), and one t(6;14)(p13;q32)), two þ þ membranous CD20. Many of the CLL cells were CD20 /Ki-67 ; þ 2 chromosome 12 abnormalities (one trisomy, one the remaining CLL cells were CD20 /Ki-67 . insertion), one del(13)(q12q22), and one del(11)(q22). Additional cytogenetic abnormalities six, cleaved PARP in five (Fig. 3), and cleaved were seen in three patients. caspase-3 in four neoplasms. To determine whether these proteins were acti- vated in CLL cells, we performed immunohisto- DISCUSSION chemical staining for the detection of cleaved caspase-3 in BM biopsy sections. We found that CLL is a disease thought to be characterized by CLL cells expressed cleaved caspase-3 protein monoclonal expansion of circulating, mature (Fig. 1J – K, Table II) whereas normal BM cells did B-lymphocytes that are not in cell cycle [2 – 4]. not (Fig. 1E). Furthermore, to determine whether However, “atypical” lymphocytes (i.e. plasmacytoid, spontaneous cleavage of PARP results in apoptosis of cleaved, and/or binucleated lymphocytes) and PL FIGURE 3 Detection of procaspase-8, procaspase-9, procaspase-10, and procaspase-3 and cleaved caspase-9, procaspase-3, PARP, and PARP protein in normal PB and BM specimens and in seven CLL cases. HL-60, Jurkat, and K-562 cell lines were used as positive controls. Also, BM aspirates obtained from two hematologically normal individuals and two normal PB samples were used as controls. 284 C.E. BUESO-RAMOS et al. are frequently found in the BM and PB of patients CLL cases at all disease stages. Similar to our with CLL, and increased numbers of these cells are previous findings in studies of acute leukemia [15 – associated with accelerated disease, trisomy 12, and 17], we detected procaspase-8, procaspase-9, procas- increased proliferative activity [1,6 – 9]. pase-10, and procaspase-3 in CLL cells at higher In the present study, we assessed CLL cells in BM levels than those observed in normal BM and PB specimens from patients with early and advanced cells. This finding is in agreement with a study by stage disease and found both proliferating cells and Krajewski et al. [29], who showed by immunoblot- cells undergoing apoptosis. The combined immuno- ting that all of their CLL cases expressed procaspase- staining for nuclear Ki-67 and membranous CD20 3. In that study, the authors evaluated 12 cases of in BM biopsy specimens demonstrated that the CLL by immunocytochemistry using smears pre- Ki-67-positive proliferating cells were of B-cell pared from PB lymphocytes (. 90% CD20 ). In all 12 lineage and derived from the CLL, and were not cases the neoplastic cells were positive for caspase-3. attributable to T-cells or erythroid or myeloid Immunolocalization was cytoplasmic in all 12 precursors. The changes that we observed in patients patients, with 2 patients having distinct nuclear with CLL with atypical morphology were similar to immunoreactivity. those reported by Garcia-Marco et al. [6] However, It is unlikely that the increased procaspase levels our study extended the work of those authors by result from accumulation due to a lack of cleavage, examining apoptosis with the use of immunoblot- because we detected cleaved caspase-3, cleaved ting, TUNEL, and immunostaining for cleaved PARP, and TUNEL-positive cells in all BM specimens caspase-3 in BM biopsy specimens. Other studies of the CLL cases assessed. Also, the apoptotic rate in support our data, as a subpopulation of proliferating BM biopsy specimens from normal donors, deter- þ þ þ þ/2 Ki-67 , survivin , bcl-2 , and p27Kip1 CLL cells mined by TUNEL, was 0% [30]. By contrast, six of has been identified in proliferation centers in lymph seven CLL patients (86%) had an apoptotic cell nodes and BM, which are the preferred sites of percentage . 1% as determined via TUNEL. relapse [8,11,12,18]. Another interesting finding is that many of our The reasons for the Ki-67 expression in a subset patients can be classified as being at high risk, based of cells in atypical CLL cases are unknown. However, on the presence of chromosomal abnormalities (þ 12, one likely factor is that genes regulating the cell cycle 11q-, and 17p-), the BM histologic pattern (diffuse are involved [19,20]. For example, overexpression of versus other patterns), lymphocyte doubling time cyclin D2 mRNA has been found to be an almost (, 12 months), and elevated b2 microglobulin level constant feature of CLL [21]. In addition, whereas [31 – 33]. As a result, all seven patients received cyclin D1 has been reported in patients having an therapy for some time after the completion of their aggressive form CLL [22], cyclin D3 has been shown diagnostic studies. Neither immunoglobulin gene to play a role in the exit of CLL lymphocytes from mutation status nor the levels of CD38 and ZAP-70 quiescence [23]. Nevertheless, Delmer et al. [21] expression in these CLL cases is available, because reported that more than 96% of the CLL cells in their these tests were not routinely performed in our study were in the G0/G1 phase of the cell cycle. clinical laboratory when this study was undertaken. Although it is possible that overexpression of the cell However, retrospective studies have established cycle inhibitor p27 [24] and interaction of CLL cells that patients whose CLL cells carry nonmutated with accessory cells and BM stroma [18,19] prevent IgV genes (# 2% difference in nucleotide sequences cell cycle transition, these studies suggest that from the nearest germline V gene) harbor high-risk deregulation of the G1 restriction point is involved chromosomal anomalies, express CD38 and ZAP-70, in CLL progression. and have a shorter median survival duration (8 – 10 Our results are also consistent with those of years) and are more in need of treatment when previous studies demonstrating that CLL cells are compared with patients whose CLL cells carry heterogeneous with a hierarchy of progenitor and mutated IgV genes [34 – 37]. mature cells [25,26]. In fact, a recent study showed In conclusion, our data suggest that CLL with that the frequency of two or more clones is higher in atypical morphology consists of a heterogeneous CLL cases with atypical morphology (4/29 [13.8%]) population of neoplastic cells. Some of these cells compared with CLL cases with typical morphology proliferate, whereas others undergo apoptosis. The (12/353 [3.4%]) [27]. A review of PB smears in this mechanisms responsible for these events and their study revealed the presence of two or more pathophysiologic significance and prognostic impli- morphologically different populations of lympho- cations need to be addressed in future studies. cytes in 89% of the cases. Moreover, Bogner et al. [38] and Decker et al.[39] have In contrast with Oliveira et al. [28], who found a found that cycling CLL cells are highly susceptible to decrease in the susceptibility to apoptosis with CLL inhibition of the proteasome with induction of progression, as well as an increase in the tumor mass apoptotic cell death. Thus, the use of agents that and proliferation rate, we found apoptotic cells in target the pool of proliferating cells in CLL with PROLIFERATION AND APOPTOSIS IN B-CLL 285 [15] Estrov, Z., Thall, P.F., Talpaz, M., Estey, E.H., Kantarjian, H.M., atypical morphology may be of value in the Andreeff, M., Harris, D., Van, Q., Walterscheid, M. and treatment strategy in these patients. Kornblau, S.M. (1998) “Caspase 2 and caspase 3 protein levels as predictors of survival in acute myelogenous leukemia”, Blood 92, 3090 – 3097. [16] Faderl, S., Thall, P.F., Kantarjian, H.M., Talpaz, M., Harris, D., Acknowledgements Van, Q., Beran, M., Kornblau, S.M., Pierce, S. and Estrov, Z. 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Pathol. 115, 571 – 581. chronic lymphocytic leukemias, and reactive lymph nodes”, [14] Sun, X., Medeiros, L.J., Lu, D., Rassidakis, G.Z. and Bueso- Blood 89, 3817 – 3825. Ramos, C. (2003) “Dysplasia and high proliferation rate are [30] Choi, J.W. (2002) “Apoptotic rate varies with detection common in acute myeloid leukemia with inv(16)(p13q22)”, methods in myelodysplastic syndromes: impact of method Am. J. Clin. Pathol. 120, 236 – 245. selection”, Eur. J. Haematol. 69, 62 – 63. 286 C.E. BUESO-RAMOS et al. [31] Dewald, G.W., Brockman, S.R., Paternoster, S.F., Bone, N.D., associated with a more aggressive form of chronic lympho- O’Fallon, J.R., Allmer, C., James, C.D., Jelinek, D.F., cytic leukemia”, Blood 94, 1848 – 1854. Tschumper, R.C., Hanson, C.A., Pruthi, R.K., Witzig, T.E., [36] Chen, L., Widhopf, G., Huynh, L., Rassenti, L., Rai, K.R., Call, T.G. and Kay, N.E. (2003) “Chromosome abnormalities Weiss, A. and Kipps, T.J. (2002) “Expression of ZAP-70 is detected by interphase fluorescence in situ hybridization: associated with increased B-cell receptor signaling in chronic correlation with significant biological features of B-cell lymphocytic leukemia”, Blood 100, 4609 – 4614. chronic lymphocytic leukaemia”, Br.J.Haematol. 121, [37] Rosenwald, A., Alizadeh, A.A., Widhopf, G., Simon, R., 287 – 295. Davis, R.E., Yu, X., Yang, L., Pickeral, O.K., Rassenti, L.Z., [32] Byrd, J.C., Smith, L., Hackbarth, M.L., Flinn, I.A., Powell, J., Botstein, D., Byrd, J.C., Grever, M.R., Cheson, B.D., Young, D., Proffitt, J.H. and Heerema, N.A. (2003) “Inter- Chiorazzi, N., Wilson, W.H., Kipps, T.J., Brown, P.O. phase cytogenetic abnormalities in chronic lymphocytic and Staudt, L.M. (2001) “Relation of gene expression leukemia may predict response to rituximab”, Cancer Res. phenotype to immunoglobulin mutation genotype 63, 36 – 38. in B cell chronic lymphocytic leukemia”, J. Exp. Med. 194, [33] Shanafelt, T.D., Geyer, S.M. and Kay, N.E. (2004) “Prognosis at 1639 – 1647. diagnosis: integrating molecular biologic insights into clinical [38] Bogner, C., Schneller, F., Hipp, S., Ringshausen, I., practice for patients with CLL”, Blood 103, 1202 – 1210. Peschel, C. and Decker, T. (2003) “Cycling B-CLL cells are [34] Damle, R.N., Wasil, T., Fais, F., Ghiotto, F., Valetto, A., highly susceptible to inhibition of the proteasome: involve- Allen, S.L., Buchbinder, A., Budman, D., Dittmar, K., Kolitz, J., ment of p27, early D-type cyclins, Bax, and caspase- Lichtman, S.M., Schulman, P., Vinciguerra, V.P., dependent and -independent pathways”, Exp. Hematol. 31, Rai, K.R., Ferrarini, M. and Chiorazzi, N. (1999) “Ig V gene 218 – 225. mutation status and CD38 expression as novel prognostic [39] Decker, T., Hipp, S., Ringhausen, I., Bogner, C., Oelsner, M., indicators in chronic lymphocytic leukemia”, Blood 94, Schneller, F. and Peschel, C. (2003) “Rapamycin-induced G1 1840 – 1847. arrest in cyclin B-CLL cells is associated with reduced [35] Hamblin, T.J., Davis, Z., Gardiner, A., Oscier, D.G. and expression of cyclin D3, cyclin E, cyclin A, and survivin”, Stevenson, F.K. (1999) “Unmutated Ig V(H) genes are Blood 101, 278 – 285. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Hematology Online Taylor & Francis

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Taylor & Francis
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© 2004 Maney Publishing
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1024-5332
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10.1080/10245330410001727046
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Abstract

Hematology, August 2004 Vol. 9 (4), pp. 279–286 Aberrant Morphology, Proliferation, and Apoptosis of B-cell Chronic Lymphocytic Leukemia Cells a b a b CARLOS E. BUESO-RAMOS , ALESSANDRA FERRAJOLI , L. JEFFREY MEDEIROS , MICHAEL J. KEATING and c, ZEEV ESTROV * a b Department of Hematopathology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA; Department of Leukemia, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA; Department of Bioimmunotherapy, Unit 422, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA (Received 5 April 2004; In final form 8 May 2004) B-cell chronic lymphocytic leukemia (CLL) has been INTRODUCTION traditionally described as a disease characterized by an accumulation of quiescent small lymphocytes B-cell chronic lymphocytic leukemia (CLL) is the with decreased susceptibility to apoptotic cell death. most common type of adult leukemia in the United However, small numbers of “atypical” lymphocytes and States and Western Europe [1]. It is thought to be prolymphocytes (PL) are frequently observed in the bone marrow (BM) of patients with CLL. In this study, we caused primarily by defects in programmed cell examined BM biopsy and aspirate specimens obtained death (apoptosis) and is characterized by a gradual from seven patients with atypical CLL. Using a double accumulation of small, long-lived, immunologically 1 1 labeling (Ki-67 /CD20 ) immunohistochemical method, dysfunctional mature B-lymphocytes, most of we found that an appreciable number of the atypical CLL which are in G0/G1 phase of the cell cycle [2 – 4]. cells expressed the proliferation-associated protein Ki-67. Because CLL is characterized by a slow change During diagnosis or during the course of in the peripheral blood (PB) lymphocyte count, we the disease, 15% of patients with CLL show reasoned that a subpopulation of CLL cells probably atypical morphologic features characterized undergoes spontaneous apoptosis. Using Western blot by an increased (. 10%) number of circulating analysis, we observed expression of procaspase-9, prolymphocytes (PL), designated as CLL/PL, procaspase-10, and poly(ADP-ribose) polymerase by the neoplastic cells in all seven cases of CLL, and or an increased (. 15%) number of circulating procaspase-3 and procaspase-8 expression in six lymphoplasmacytic and cleaved cells, designated neoplasms. We also detected cleaved caspase-3 and as “atypical” CLL [1,5]. Patients with atypical CLL cleaved poly(ADP-ribose) polymerase in four and five more commonly have an accelerated clinical CLL cases, respectively. To determine whether CLL cells course and trisomy 12 [1,6,7]. In addition, undergo spontaneous apoptosis, we performed the terminal deoxynucleotidyl transferase-mediated dUTP atypical CLL cases have increased expression of nick-end labeling (TUNEL) assay using BM biopsy the nuclear proliferation protein Ki-67 [8], and specimens. We found TUNEL-positive lymphocytes in ultrastructural features suggestive of apoptosis [9]. areas infiltrated by CLL. In summary, our data show that Expression of the human protein Ki-67 is strictly subpopulations of B-lymphocytes are proliferating or associated with cell proliferation, being expressed undergoing spontaneous apoptotic cell death in patients with atypical CLL. during all active phases of the cell cycle (G1, S, G2, and mitosis) but absent in resting cells (G0 phase) [10]. The predominant proliferative component Keywords: Chronic lymphocytic leukemia (CLL); Apoptosis; Proliferation; Atypical morphology; Cytogenetics in CLL is localized in proliferation centers that are *Corresponding author. Tel.: þ 1-713-794-1675. Fax: þ 1-713-745-2374. E-mail: zestrov@mdanderson.org ISSN 1024-5332 print/ISSN 1607-8454 online q 2004 Taylor & Francis Ltd DOI: 10.1080/10245330410001727046 280 C.E. BUESO-RAMOS et al. usually identified in the lymph nodes but less involvement by a B-cell neoplasm composed commonly are present in bone marrow (BM) [11,12]. predominantly of small lymphocytes that expressed There have been no published reports of simul- monotypic immunoglobulin light chain, pan-B-cell taneous assessment of proliferation and apoptosis in antigens (CD19, CD20), CD5 and CD23 and negative BM biopsy specimens obtained from patients with for CD3, CD10 and cyclin D1. CLL with atypical morphology. Morphologic analysis was performed using In our experience, small percentages of atypical Wright-Giemsa stained PB and BM aspirate smears lymphocytes (i.e. lymphoplasmacytic and cleaved and hematoxylin-eosin stained BM biopsy speci- cells) and PL can be identified in BM aspirate smears mens. Flow cytometric immunophenotyping was in most cases of CLL, in patients at any clinical stage. performed using BM aspirates with a panel Thus, we hypothesized that CLL cells are more of antibodies specific for immunoglobulin kappa heterogeneous than is appreciated in the literature. and lambda light chains, CD3, CD5, CD10, CD19, Thus, in this study group we assessed both pro- CD20, and CD23 according to well-established liferation and apoptosis. We show that cases of CLL methods [13]. Other antibodies were used in a with atypical morphologic features, regardless of subset of these cases. Conventional G-band karyo- stage, have subpopulations of cells that are prolifer- type analysis of all samples was performed accord- ating or undergoing spontaneous apoptosis, in ing to a previously reported method [13]. addition to quiescent cells, in vivo. Immunohistochemical Methods MATERIALS AND METHODS Immunohistochemical studies were performed using formalin-fixed, paraffin-embedded tissue sections Patient Specimens (4 mm thick) and an avidin – biotin peroxidase Bone marrow aspirate and biopsy specimens technique [14]. The primary antibodies were specific were obtained from seven patients with B-cell CLL. for Ki-67 (1:1000; DAKO Corporation, Carpinteria, Bone marrow aspirate and peripheral blood CA, USA) and cleaved caspase-3 (1:5000; R & D (PB) specimens were also obtained from two hemato- Systems, Minneapolis, MN, USA). Peroxidase logically normal individuals (BM transplant donors) staining was performed using the anti-mouse, anti- at the time of diagnostic evaluation at The University rabbit link antibody (DAKO LSAB2-HRP, 15 min; of Texas M. D. Anderson Cancer Center. All subjects DAKO Corporation). Sequential immunoenzymatic gave their informed consent, but this retrospective staining combining the immunoperoxidase and study did not require approval by the Institutional immunoalkaline phosphatase techniques was used Review Board when it was initiated. for simultaneous detection of Ki-67 and CD20 The clinical characteristics of the seven CLL (DAKO). Negative and positive controls were run. patients are summarized in Table I. All previously At low power magnification, the entire tissue treated patients had not received treatment for section was scanned and microscopic fields were several months at the time that the specimens were selected based on areas with the greatest number of obtained. The diagnosis of CLL was based on a Ki-67-positive and cleaved caspase-3-positive cells. combination of clinical, morphologic, laboratory, and In each case, at least 500 CLL cells were evaluated for immunophenotypic criteria as defined previously nuclear (Ki-67, cleaved caspase-3) and membranous [4]. Specifically, the criteria for CLL included (CD20) positivity. TABLE I Patient characteristics Age in WBC Hb level Plt count b M level LDT 3 3 Patient years/sex Rai stage ( £ 10 /ml) (g/dl) ( £ 10 /ml) (mg/l) Prior treatment (months) 1 57/M II 33.4 12.8 167 3.4 Chb, CTX-P 6 2 57/M I 96.3 13.5 132 3.4 None 12 3 42/F I 22.2 13.1 132 2.8 None 4 4 55/M IV 13.1 8.6 130 2.0 CHOP/CTX, O, VP-16, P, F, 6 DHAP, thiotepa; bleomycin 5 73/M II 104.5 13.1 118 5.6 None N/A 6 46/F I 28.2 12.8 178 N/A None N/A 7 76M IV 175 11.2 75 11.4 Chb, F, 2CdA 6 WBC, white blood count; Hb, hemoglobin; Plt, platelet; b M, b -microglobulin; LDT, lymphocyte doubling time; Chb, chlorambucil; CTX, cyclophosphamide; 2 2 CHOP, cyclophosphamide, adriamycin, vincristine, and prednisone; O, vincristine; VP-16, etoposide; P, prednisone; F, fludarabine; DHAP, dexamethasone, cytarabine, and cisplatin; 2CdA, 2-cholorodeoxyadenosine. PROLIFERATION AND APOPTOSIS IN B-CLL 281 Terminal Deoxynucleotidyl Transferase-mediated RESULTS dUTP Nick-end Labeling (TUNEL) Assay for Detection of Apoptosis Clinical Features Cells undergoing apoptosis were detected with a The clinical features for the seven patients are TUNEL method as described previously [14]. summarized in Table I. We studied five male and two Apoptotic figures, including positively labeled female patients whose ages ranged from 42 to 76 intact nuclei and nuclear fragments derived from a years (median, 57 years). Four patients were not single cell, were counted per high-power field (HPF) previously treated, and three had been treated at in areas with 100% cellularity with the use of an other institutions. All previously treated patients had Olympus BX40 microscope with a 40 £ objective been off therapy for several months at the time the and 10 £ ocular (Olympus America Inc., Melville, samples were obtained. Most patients received NY, USA). In all cases, there were sufficient areas fludarabine-based chemotherapy in combination with adequate morphology to cover four or more with rituximab after diagnostic evaluation at The HPFs. Using this approach, it was found that University of Texas M. D. Anderson Cancer Center. 0.23 mm area within a HPF contained approxi- Three patients (1, 4 and 7) received alemtuzumab mately 2950 cells when the cellularity was 100%. alone or in combination with other agents. Four The number of apoptotic cells derived from a field patients achieved complete remission and three with lower cellularity was normalized proportion- patients had partial response. ally to 100% cellularity (e.g. when two TUNEL- positive cells were identified in a HPF with a Expression of Ki-67 Protein by CLL Cells cellularity of 50%, a score of 4 was recorded). The median score in each case was used for the final In all CLL patient specimens, we detected a small analysis. number of binucleated and trinucleated and/or large plasmacytoid cells in BM aspirate smears and expansion of the medullary space in BM biopsy Western Blot Analysis specimens by CLL (Fig. 1A – C). Because the presence of these cells has been associated with an increased Western blot analysis was performed using cell proliferation rate [6], we performed immunohisto- lysates of PB and low-density aspirate BM cells of chemical studies to detect Ki-67 protein in BM biopsy seven CLL patients as described previously [15]. specimens. As shown in Table II, we found that the Cell lysates from 5 £ 10 BM cells were assayed to immunoreactivity for this protein was relatively high determine the protein concentration using the BCA (. 5%) in BM biopsy specimens of all seven patients Protein Assay Reagent kit (Pierce Chemical Co., in typical areas of CLL infiltration (Fig. 1D – E). Rockford, IL, USA). Each set of paired samples was The CLL infiltrate distorted and displaced the then adjusted so that they would have the same normal Ki-67 erythroid and myeloid islets com- protein concentration. The following antibodies pared with that in the surrounding uninvolved BM were used for their respective proteins: mouse (Fig. 1F). anti-human CPP32 (procaspase-3), mouse anti- To establish with certainty that CLL cells rather human FLICE (procaspase-8), rabbit anti-human than normal hematopoietic cells were proliferating, procaspase-10, mouse anti-human poly(ADP- we performed double labeling immunohisto- ribose) polymerase (PARP; Pharmingen, San chemical staining with Ki-67 and the B-cell marker, Diego, CA, USA), and rabbit anti-human cleaved anti-CD20. As shown in Fig. 2, a subset of CLL cells caspase-3 (New England BioLabs, Beverly, MA, co-expressed Ki-67 and CD20. USA). Normal mouse IgG and rabbit IgG (Sigma Chemical Co., St. Louis, MO, USA) were used as controls. Lysates of Jurkat, HL-60, and K-562 cells Expression of Procaspase and Caspase Proteins and were used to confirm the detection of these Detection of Apoptosis in CLL Cells proteins. After initial probing, blots were re-probed In previous studies, we found that patients with with an antibody specific for actin (1:1000, mouse untreated acute leukemias had high levels of monoclonal, Sigma Chemical Co.) to assess protein procaspase and caspase proteins and that PARP, loading. a DNA repair enzyme known to protect cell integrity, was spontaneously cleaved in acute leukemia cells [15 – 17]. Using a similar method, we used Western Statistical Analysis immunoblotting to detect these proteins in BM Statistical analysis was performed using the chi- aspirates from seven cases of CLL. Procaspase-9 and square test. p values , 0.05 were considered procaspase-10 were expressed in all neoplasms, with statistically significant. procaspase-8 and procaspase-3 were expressed in 282 C.E. BUESO-RAMOS et al. FIGURE 1 Histologic, immunohistologic, and cytologic stains of representative BM biopsy and aspirate specimens from patient 3. (A) Extensive infiltration by small lymphocytes in the BM biopsy specimen (hematoxylin and eosin stain, £ 400). (B and C) Cleaved, multinucleated lymphocytes, prolymphocytes and small round lymphocytes in the BM aspirate smears (Wright-Giemsa stain, £ 1000). (D – F) Immunohistochemical stain for Ki-67 shows Ki-67-positive CLL cells (D and E, CLL BM biopsy specimens [ £ 400 and £ 600, respectively]; F, normal BM biopsy specimen [ £ 600]). (G – I) TUNEL staining of BM biopsy specimens (G and H, BM specimens showing several TUNEL-positive cells [ £ 400 and £ 600, respectively]; I, normal BM biopsy specimen showing rare positive cells [ £ 600]). (J – L) Immunohistochemical staining for cleaved caspase-3. Activated caspase-3 was found often in CLL cells (J and K [ £ 400 and £ 600, respectively]) but rarely in normal BM specimens (L [ £ 600]). TABLE II Cytogenetic and BM findings in CLL cases Histologic pattern Lymphocytes Cleaved TUNEL Ki-67 Patient Cytogenetic abnormality in BM biopsy specimen (%) caspase-3 (%) (per HPF) (%) 1 Diploid N/I 95 15 8 12 2 Diploid D 88 9 12 12 3 47,XX,þ 12[3] D 80 5 29 50 47,XX,t(6;14)(p13;q32),þ 12[1] 4 46,XY,inv(9)(p11q12),del(11)(q22)[5] N/I 80 3 9 10 5 46,XY,del(14)(q23)[13] D93 3 8 8 46,XY[7] 6 90 – 91,XXXX,2 4,2 6,der(7;?)(p10;?),2 10, D95 2 1 45 add(14)(q32)x2,der(19)del(19) (p13.1)del(19)(q13.1)x2, þ 0 – 4mar[cp5] 7 46,XY,ins(12;?)(q13;?),del(13) D82 18 6 40 (q12q22)[2] 46,XY[6] BM, bone marrow; N/I, nodular/interstitial; D, diffuse; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nickend labeling; HPF, high power field. PROLIFERATION AND APOPTOSIS IN B-CLL 283 CLL cells, we performed TUNEL staining of BM sections. We found TUNEL-positive CLL cells (Fig. 1G – H and Table I) in all seven neoplasms (Fig. 1G – H, Table II), indicating that a subset of the CLL cells underwent spontaneous apoptosis. TUNEL-positive cells were rarely found in normal BM sections (Fig. 1I). Cytogenetic Results Conventional cytogenetic studies were performed on BM aspirates of all seven patients. Five patients (71%) had chromosomal anomalies and two patients (29%) were diploid (Table II). The chromosomal abnormalities included three chromo- some 14 abnormalities (one del(14)(q23), one FIGURE 2 Double immunostaining for nuclear Ki-67 and add(14)(q23), and one t(6;14)(p13;q32)), two þ þ membranous CD20. Many of the CLL cells were CD20 /Ki-67 ; þ 2 chromosome 12 abnormalities (one trisomy, one the remaining CLL cells were CD20 /Ki-67 . insertion), one del(13)(q12q22), and one del(11)(q22). Additional cytogenetic abnormalities six, cleaved PARP in five (Fig. 3), and cleaved were seen in three patients. caspase-3 in four neoplasms. To determine whether these proteins were acti- vated in CLL cells, we performed immunohisto- DISCUSSION chemical staining for the detection of cleaved caspase-3 in BM biopsy sections. We found that CLL is a disease thought to be characterized by CLL cells expressed cleaved caspase-3 protein monoclonal expansion of circulating, mature (Fig. 1J – K, Table II) whereas normal BM cells did B-lymphocytes that are not in cell cycle [2 – 4]. not (Fig. 1E). Furthermore, to determine whether However, “atypical” lymphocytes (i.e. plasmacytoid, spontaneous cleavage of PARP results in apoptosis of cleaved, and/or binucleated lymphocytes) and PL FIGURE 3 Detection of procaspase-8, procaspase-9, procaspase-10, and procaspase-3 and cleaved caspase-9, procaspase-3, PARP, and PARP protein in normal PB and BM specimens and in seven CLL cases. HL-60, Jurkat, and K-562 cell lines were used as positive controls. Also, BM aspirates obtained from two hematologically normal individuals and two normal PB samples were used as controls. 284 C.E. BUESO-RAMOS et al. are frequently found in the BM and PB of patients CLL cases at all disease stages. Similar to our with CLL, and increased numbers of these cells are previous findings in studies of acute leukemia [15 – associated with accelerated disease, trisomy 12, and 17], we detected procaspase-8, procaspase-9, procas- increased proliferative activity [1,6 – 9]. pase-10, and procaspase-3 in CLL cells at higher In the present study, we assessed CLL cells in BM levels than those observed in normal BM and PB specimens from patients with early and advanced cells. This finding is in agreement with a study by stage disease and found both proliferating cells and Krajewski et al. [29], who showed by immunoblot- cells undergoing apoptosis. The combined immuno- ting that all of their CLL cases expressed procaspase- staining for nuclear Ki-67 and membranous CD20 3. In that study, the authors evaluated 12 cases of in BM biopsy specimens demonstrated that the CLL by immunocytochemistry using smears pre- Ki-67-positive proliferating cells were of B-cell pared from PB lymphocytes (. 90% CD20 ). In all 12 lineage and derived from the CLL, and were not cases the neoplastic cells were positive for caspase-3. attributable to T-cells or erythroid or myeloid Immunolocalization was cytoplasmic in all 12 precursors. The changes that we observed in patients patients, with 2 patients having distinct nuclear with CLL with atypical morphology were similar to immunoreactivity. those reported by Garcia-Marco et al. [6] However, It is unlikely that the increased procaspase levels our study extended the work of those authors by result from accumulation due to a lack of cleavage, examining apoptosis with the use of immunoblot- because we detected cleaved caspase-3, cleaved ting, TUNEL, and immunostaining for cleaved PARP, and TUNEL-positive cells in all BM specimens caspase-3 in BM biopsy specimens. Other studies of the CLL cases assessed. Also, the apoptotic rate in support our data, as a subpopulation of proliferating BM biopsy specimens from normal donors, deter- þ þ þ þ/2 Ki-67 , survivin , bcl-2 , and p27Kip1 CLL cells mined by TUNEL, was 0% [30]. By contrast, six of has been identified in proliferation centers in lymph seven CLL patients (86%) had an apoptotic cell nodes and BM, which are the preferred sites of percentage . 1% as determined via TUNEL. relapse [8,11,12,18]. Another interesting finding is that many of our The reasons for the Ki-67 expression in a subset patients can be classified as being at high risk, based of cells in atypical CLL cases are unknown. However, on the presence of chromosomal abnormalities (þ 12, one likely factor is that genes regulating the cell cycle 11q-, and 17p-), the BM histologic pattern (diffuse are involved [19,20]. For example, overexpression of versus other patterns), lymphocyte doubling time cyclin D2 mRNA has been found to be an almost (, 12 months), and elevated b2 microglobulin level constant feature of CLL [21]. In addition, whereas [31 – 33]. As a result, all seven patients received cyclin D1 has been reported in patients having an therapy for some time after the completion of their aggressive form CLL [22], cyclin D3 has been shown diagnostic studies. Neither immunoglobulin gene to play a role in the exit of CLL lymphocytes from mutation status nor the levels of CD38 and ZAP-70 quiescence [23]. Nevertheless, Delmer et al. [21] expression in these CLL cases is available, because reported that more than 96% of the CLL cells in their these tests were not routinely performed in our study were in the G0/G1 phase of the cell cycle. clinical laboratory when this study was undertaken. Although it is possible that overexpression of the cell However, retrospective studies have established cycle inhibitor p27 [24] and interaction of CLL cells that patients whose CLL cells carry nonmutated with accessory cells and BM stroma [18,19] prevent IgV genes (# 2% difference in nucleotide sequences cell cycle transition, these studies suggest that from the nearest germline V gene) harbor high-risk deregulation of the G1 restriction point is involved chromosomal anomalies, express CD38 and ZAP-70, in CLL progression. and have a shorter median survival duration (8 – 10 Our results are also consistent with those of years) and are more in need of treatment when previous studies demonstrating that CLL cells are compared with patients whose CLL cells carry heterogeneous with a hierarchy of progenitor and mutated IgV genes [34 – 37]. mature cells [25,26]. In fact, a recent study showed In conclusion, our data suggest that CLL with that the frequency of two or more clones is higher in atypical morphology consists of a heterogeneous CLL cases with atypical morphology (4/29 [13.8%]) population of neoplastic cells. Some of these cells compared with CLL cases with typical morphology proliferate, whereas others undergo apoptosis. The (12/353 [3.4%]) [27]. A review of PB smears in this mechanisms responsible for these events and their study revealed the presence of two or more pathophysiologic significance and prognostic impli- morphologically different populations of lympho- cations need to be addressed in future studies. cytes in 89% of the cases. Moreover, Bogner et al. [38] and Decker et al.[39] have In contrast with Oliveira et al. [28], who found a found that cycling CLL cells are highly susceptible to decrease in the susceptibility to apoptosis with CLL inhibition of the proteasome with induction of progression, as well as an increase in the tumor mass apoptotic cell death. Thus, the use of agents that and proliferation rate, we found apoptotic cells in target the pool of proliferating cells in CLL with PROLIFERATION AND APOPTOSIS IN B-CLL 285 [15] Estrov, Z., Thall, P.F., Talpaz, M., Estey, E.H., Kantarjian, H.M., atypical morphology may be of value in the Andreeff, M., Harris, D., Van, Q., Walterscheid, M. and treatment strategy in these patients. Kornblau, S.M. (1998) “Caspase 2 and caspase 3 protein levels as predictors of survival in acute myelogenous leukemia”, Blood 92, 3090 – 3097. [16] Faderl, S., Thall, P.F., Kantarjian, H.M., Talpaz, M., Harris, D., Acknowledgements Van, Q., Beran, M., Kornblau, S.M., Pierce, S. and Estrov, Z. 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Journal

Hematology OnlineTaylor & Francis

Published: Aug 1, 2004

Keywords: Chronic lymphocytic leukemia (CLL); Apoptosis; Proliferation; Atypical morphology; Cytogenetics

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