Instability of Immunophenotype in Plasma Cell Myeloma

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1 Hematopathology / Instability of Immunophenotype in PCM Instability of Immunophenotype in Plasma Cell Myeloma Wenqing Cao, MD, 1 Charles L. Goolsby, PhD, 1 Beverly P. Nelson, MD, 1 Seema Singhal, MD, 2 Jayesh Mehta, MD, 2 and LoAnn C. Peterson, MD 1 Key Words: Plasma cell myeloma; Immunophenotype; Flow cytometry; Stability DOI: /8UVF7YQ1D4D4ETQV Abstract Little information has been reported describing antigen stability in plasma cell myeloma. In this study, the expression frequency and stability of 2 potential therapeutic targets, CD20 and CD52, along with the frequently aberrantly expressed CD56 antigen, were evaluated by flow cytometric analyses in 56 patients with plasma cell myeloma. Of the 56 patients, 23 (41%) showed immunophenotype change, including CD56 in 6 cases, CD20 in 7 cases, and CD52 in 17 cases. Combined CD56/CD52 change was seen in 3 cases and combined CD20/CD52 in 4 cases. No correlation was found between immunophenotype change and age, sex, stage, plasma cell morphologic features, extent of marrow involvement, time between analyses, type of therapy, or response to therapy. Immunophenotype shift was more common in patients with IgA than in patients with IgG paraprotein. Recognition of lack of stability in immunophenotype may be important, especially in antigen-directed treatment decisions and when specific phenotypes are used to detect residual disease. Plasma cell myeloma (PCM) is a neoplasm characterized by proliferation of a single clone of plasma cells, a monoclonal immunoglobulin, and skeletal destruction. 1 Multiparametric immunophenotyping of PCM by flow cytometry has been incorporated into practice in many laboratories, not only for diagnosis, but also for monitoring residual disease. 2,3 CD138+, CD38+, and dim CD45 to CD45 staining is common to normal and abnormal plasma cells. 4,5 Pan B-cell antigens such as CD19 and CD20 are negative in the majority of normal plasma cells and PCM cases. However, dim expression of CD19 and CD20 can be seen in normal plasma cells, and such expression may also be present in some cases of PCM. 3,5-7 Therefore, these antigens are not helpful in differentiating normal from abnormal plasma cells. Abnormal plasma cells are often identified by flow cytometric analyses based on the presence of monotypic cytoplasmic immunoglobulin light chain and, in a subset of cases, CD56 expression, which only rarely occurs in normal plasma cells. 4,5,8,9 Although flow cytometry is useful to identify abnormal plasma cells, the diagnosis of PCM requires correlation of other laboratory data, clinical findings, and radiologic results. PCM is currently an incurable disease, although agents such as thalidomide (Thalomid), lenalidomide (Revlimid), and bortezomib (Velcade) have provided effective salvage therapy for end-stage myeloma. 10 Increasing knowledge of tumor biology and immunology has led to the development of immunotherapeutic and immunoregulatory drugs. 11,12 Many clinical flow cytometry laboratories now assess expression of a number of potential therapeutically relevant targets, or markers (CD20, CD22, CD25, CD52, and 926 Am J Clin Pathol 2008;129: DOI: /8UVF7YQ1D4D4ETQV

2 Hematopathology / Original Article CD30), on the neoplastic cells of lymphoid and plasma cell malignancies. 2 Knowledge of the expression pattern of these potential therapeutic markers at the time of the diagnosis may allow for initiation of antigen/ligand-directed therapy at diagnosis or at relapse without reanalysis of tumor cells. Studies have suggested the possible role of such monoclonal antibody therapies directed against CD20 and CD52 in the subset of PCMs that are positive for these antigens. Clinical trials evaluating the efficacy of rituximab (anti-cd20 monoclonal antibody) in PCM have shown effectiveness in some cases that are CD ,14 Although more frequently used in relapsed or refractory chronic lymphocytic leukemia, alemtuzumab (anti-cd52 monoclonal antibody) is also a potential therapeutic target in PCM. 16 Studies have demonstrated heterogeneous CD52 expression in PCM. 9,17 In addition, alemtuzumab has been shown to exhibit antitumor activity in animals with early-stage PCM. 18 Many studies have examined the immunoprofile of PCM. 3-5,9 However, these studies have primarily focused on diagnostic utility and minimal residual disease monitoring with little information on immunophenotype stability of myeloma cells in individual patients or on stability of potential therapeutic targets. 7,19 The purpose of this study was to evaluate the expression frequency and stability of 2 potential therapeutic targets, CD20 and CD52, along with the frequently aberrantly expressed CD56 antigen. Materials and Methods Patients All patients had a diagnosis of PCM and were followed up at Northwestern Memorial Hospital, Chicago, IL, from January 2003 through August The diagnosis of PCM was established according to published World Health Organization criteria and staged by the Durie-Salmon system. 20 For the study, 320 flow cytometric analyses of bone marrow aspirates from patients with PCM were reviewed. In this series, 56 patients had at least 2 flow cytometric results with monotypic plasma cells present. The clinical data for each patient were reviewed, including age, sex, stage, M component, type of therapy (chemotherapy and/or stem cell transplantation), and response to therapy. Flow Cytometric Immunophenotyping Flow cytometric immunophenotypic studies were performed on bone marrow aspirates using 3- and 4-color flow cytometric analyses from January 2003 to August 2005, and 6-color analyses after August The monoclonal antibody combinations for the 3- and 4-color analyses were cytoplasmic κ/cytoplasmic λ/cd45/cd38, CD38/ CD56/CD45/CD138, and CD20/CD25/CD45/CD38 corresponding to the fluorochromes fluorescein isothiocyanate (FITC)/phycoerythrin (PE)/PE-Texas red/pe-cyanin 5 (PE-Cy5); and CD52/CD45/CD38 corresponding to the fluorochromes FITC/PE/PE-Cy5. The antibody combinations for 6-color analysis were cytoplasmic κ/cytoplasmic λ/cd138/cd56/cd38/cd45 and CD52/CD25/CD3/CD20/ CD38/CD45 corresponding to the fluorochromes FITC/ PE/peridinin chlorophyll protein (PerCP)-Cy5.5/PE-Cy7/ allophycocyanin (APC)/APC-Cy7. All antibodies for the 3- and 4-color analyses were purchased from Beckman Coulter (Fullerton, CA) except CD52, which was purchased from Invitrogen (Carlsbad, CA); for 6-color analyses, CD20 was from Beckman Coulter, CD52 from Invitrogen, and all others were from Becton Dickinson (San Jose, CA). Briefly, bone marrow aspirate samples were prepared for flow cytometric analysis as follows. For tubes assessing only surface antigens, an aliquot of bone marrow aspirate containing 1 to cells was processed using a modified Qprep (Beckman Coulter) procedure described earlier, 21 in which the fixative step was replaced by phosphate-buffered saline (PBS) containing sodium azide. Cells were then washed in PBS and resuspended in 100 µl of PBS/30% fetal bovine serum containing the recommended, or appropriately titered, amount of antibody, incubated for 15 minutes at room temperature (RT), washed in PBS, and resuspended in PBS/0.5% formaldehyde. For tubes assessing surface and cytoplasmic antigens, an aliquot of bone marrow aspirate sample containing 1 to cells was processed using 2 ml of ammonium chloride solution 9 for 10 minutes at RT on a rocker followed by 2 washes in PBS with resuspension in 100 µl of PBS/30% fetal bovine serum containing the recommended, or appropriately titered, amount of surface antigen directed antibody and incubated for 15 minutes at RT and washed. Cells were resuspended in 500 µl of PermiFlow fixation/permeabilization reagent (Invirion, Traverse City, MI) and incubated for 40 minutes at RT in the dark. The cell suspension was then washed in PBS and resuspended in 100 µl of PBS/50% normal mouse serum containing the anti-κ and anti-λ antibodies and incubated in the dark for 15 minutes at RT. Cells were then washed and resuspended in PBS for analysis. Flow Cytometric Data Analysis The 4-color flow cytometric data analysis was performed on a Beckman Coulter EPICS ALTRA flow cytometer and 6-color flow cytometric analysis on an LSRII (Becton Dickinson) flow cytometer. At the 4-color to 6-color analysis transition, a large series cross-comparison study was performed using specimens from healthy people and from patients with different diagnoses to validate the comparability Am J Clin Pathol 2008;129: DOI: /8UVF7YQ1D4D4ETQV 927

3 Cao et al / Instability o f Im m u n o p h e n o t y p e in PCM of the 2 approaches (data not shown). Abnormal plasma cells were selected based on CD38+, CD138+, and dim CD45+ to CD 45 staining and on monotypic cytoplasmic κ or λ immunoglobulin light chain staining zimage 1z. CD56, CD20, and CD52 were analyzed on gated cells containing more than 99% monotypic plasma cells. A qualitative assessment of fluorescent staining intensity for each relevant antigen was graded by visual inspection as negative, dim, or moderate/ bright based on the degree of overlap in the staining intensity for each relevant antigen in the plasma cell population compared with the corresponding antigen-negative reference normal population in the sample. 21 Of note, all samples were blocked with high concentration (30%-50%) of fetal bovine serum or normal mouse serum to suppress nonspecific binding. Mature granulocytes were used as negative controls for CD56 and CD52. T cells and granulocytes were used as negative controls for CD20. Negative antibody staining was defined as demonstration of approximately equivalent fluorescent staining intensity, generally, less than a 2-fold difference in geometric mean intensity, of the relevant antigen staining in the plasma cells compared with the geometric mean for the same antigen in the negative reference population within the same tube. Dim antibody staining was defined as greater intensity of the relevant antigen in the plasma cells than in the negative reference population, but with significant overlap of the intensity distributions, generally representing a 2- to 5-fold difference in geometric mean intensity. Moderate to bright positive staining showed minimal or no overlap in the distribution of intensities for the relevant antigen in the plasma cells and appropriate negative reference and represented a more than 5-fold increase in geometric mean intensity of the relevant antigen staining in the plasma cells as compared with the geometric mean intensity of the negative reference within the same tube. Immunophenotypic change was scored only when antigen staining intensity changed from negative to positive, regardless of relative intensity, or vice versa. Morphologic Features Peripheral blood smears, bone marrow aspirates, touch preparations, and bone marrow core biopsy specimens were reviewed. Plasma cell morphologic features and extent of bone marrow involvement were correlated with flow cytometric findings in all 56 cases. Statistical Analyses Statistical analyses were done by using GraphPad Prism software (San Diego, CA). The Fisher exact test, χ 2 test, and Mann-Whitney test were used for comparing nominal variables. The Student t test was used for comparing continuous variables between the 2 groups. A P value of less than.05 was considered significant. A CD45 APC-Cy7 C Cyto κ FITC Results Cyto λ PE CD56 PE-Cy7 Clinical and Pathologic Characteristics Of the 56 patients in this study, 38 were men and 18 were women with a median age of 60 years (range, years) when first encountered at Northwestern Memorial Hospital. The initial stages were as follows: stage I, 11; stage II, 10; and stage III, 35 ztable 1z. The follow-up interval ranged from 2 to 38 months (median, 7 months). Clinical management for each patient was different and included no treatment in 5 (9%); chemotherapy only (with 1 of the following: dexamethasone; vincristine, doxorubicin [Adriamycin], and dexamethasone combination; thalidomide; lenalidomide; or bortezomib) in 12 (21%); or combined chemotherapy and stem cell transplantation (autologous or, rarely, allogeneic) in 39 (70%). Flow Cytometric Findings The baseline immunophenotype in the 56 cases was as follows: κ+, 41 (73%); λ+, 15 (27%); CD56+, 41 (73%); CD45 APC-Cy7 CD138 PerCP-Cy5-5 CD138 PerCP-Cy5-5 zimage 1z Selection of malignant plasma cell population. A and B, Gated on bright CD38+ and/or CD138+, dim CD45+ to CD45 population. C, Selected plasma cells were cytoplasmic (cyto) κ or λ immunoglobulin light chain restricted. D, In a subset of patients coexpression of CD56 and CD38/ CD138 may be used to identify malignant plasma cells. APC, allophycocyanin; Cy, cyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCP, peridinin chlorophyll protein. B D 928 Am J Clin Pathol 2008;129: DOI: /8UVF7YQ1D4D4ETQV

4 Hematopathology / Original Article ztable 1z Clinical and Pathologic Characteristics According to Change in Immunophenotype * Immunophenotype Unstable Immunophenotype Total Stable Unstable CD56 CD20 CD52 (n = 56) (n = 33 [59%]) (n = 23 [41%]) P (n = 6) (n = 7) (n = 17) Sex M/F 38/18 22/11 16/7.89 6/0 4/3 12/5 Male (%) Median (range) age (y) 60 (38-79) 58 (38-77) 62 (47-79) (50-76) 58 (50-68) 64 (50-79) Monoclonal protein.007 IgA 12 (21) 3 (9) 9 (39) IgG 32 (57) 23 (70) 9 (39) Light chain only 11 (20) 7 (21) 4 (17) Nonsecretory 1 (2) 0 (0) 1 (4) Stage.17 I-II 21 (38) 15 (45) 6 (26) 0 (0) 4 (57) 4 (24) III 35 (63) 18 (55) 17 (74) 6 (100) 3 (43) 13 (76) Median (range) BM 16 (1-99) 10 (1-99) 20 (2-90) (1-75) 30 (4-90) 20 (1-99) involvement (%) Median (range) time between 7 (2-38) 12.5 (2-38) 6.5 (2-28) (3-33) 7 (3-28) 7.5 (2-28) analyses (mo) Therapy.52 No treatment 5 (9) 4 (12) 1 (4) Chemotherapy only 12 (21) 6 (18) 6 (26) Chemotherapy and SCT 39 (70) 23 (70) 16 (70) Response to therapy.99 Alive 47 (84) 28 (85) 19 (83) Died 9 (16) 5 (15) 4 (17) SCT, stem cell transplantation. * Data are given as number (percentage) unless otherwise indicated. P =.007 represents the significance between the IgA and IgG groups. P =.045 represents the significance between stable and unstable groups. CD20+, 15 (27%); and CD52+, 31 (55%). CD56 was positive in 41 of 56 cases; the majority of them (37/41 [90%]) were bright positive, 3 (7%) were dim positive, and 1 (2%) was partial positive. CD20 was expressed in 15 cases, of which 7 (47%) were bright positive and 8 (53%) were dim positive. CD52 showed a heterogeneous staining pattern in 31 cases: 10 (32%) were bright positive, 20 (65%) were dim positive, and 1 (3%) was partial positive. A change in immunophenotype was found in 23 (41%) of 56 cases. For individual antigens, CD56 changed in 6 cases (11%), including 4 cases that changed from CD56 to CD56+ and 2 cases that changed from CD56+ to CD56 zimage 2z. CD20 changed in 7 cases (13%), including 1 case from negative to dim positive and 5 cases from dim to negative; 1 case showed more variable CD20 staining (positive and negative) during follow-up (Image 2). CD52 changed in 17 cases (30%), including 5 cases changing from negative to positive, 9 from positive to negative, and 3 in which the staining pattern fluctuated between positive and negative multiple times zfigure 1z (Image 2). Furthermore, combined CD56/CD52 change was seen in 3 cases (1 case from CD56 /CD52+ to CD56+/CD52 ; 1 case from CD56+/ CD52 to CD56 /CD52+; and 1 case from CD56 /CD52 to CD56+/CD52+). Combined CD20/CD52 change was seen in 4 cases (3 cases from CD20+/CD52+ to CD20 / CD52 ; and 1 case from CD20 /CD52 to CD20+/CD52+). No change in κ or λ cytoplasmic immunoglobulin light chain was identified in any of the cases. In the 56 cases, 12 cases were evaluated only by 4-color, 13 cases only by 6-color, and 31 cases (55%) by 4- and 6-color methods. The frequency of antigenic change was similar in cases analyzed by 4-color, 6-color, and 4- and 6-color methods (7 [58%], 7 [54%], and 16 [52%], respectively). In the 31 cases studied by 4- and 6-color flow cytometry, 22 (71%) showed a stable antigen profile across the 4- to 6-color transition in method. Morphologic Findings In our series, the percentage of plasma cells obtained from flow cytometric analysis (0.1%-60%) differed in most cases from the percentage obtained from the bone marrow aspirate differential count. The difference ranged from essential agreement to 10% of the plasma cells seen morphologically. Therefore, the bone marrow aspirate differential count was used to evaluate the extent of marrow involvement. The overall extent of bone marrow involvement ranged from 1% to more than 99% and was similar in cases with a stable phenotype (1%-99%) and cases with an unstable phenotype Am J Clin Pathol 2008;129: DOI: /8UVF7YQ1D4D4ETQV 929

5 Cao et al / Instability o f Im m u n o p h e n o t y p e in PCM (2%-90%) zimage 3z. Within the cases showing antigenic change, the average extent of bone marrow involvement was similar regardless of antigen status (positive or negative, data not shown). Small plasma cells with lymphoplasmacytic morphologic features were present in 9 (60%) of 15 CD20+ cases, in agreement with recent reports. 22 None of the cases that changed CD20 immunophenotype exhibited significant corresponding plasma cell morphologic changes. In addition, no morphologic changes were seen in cases that changed CD56 or CD52 staining patterns. A CD56 PE-Cy7 B CD56 PE-Cy7 Immunophenotype Changes and Clinical and Pathologic Correlations Table 1 lists the patient clinical, pathologic, and immunophenotypic characteristics. The M components in the patients included IgA, IgG, light chain only, and nonsecretory. Of 12 patients with IgA, 9 (75%) showed antigenic change; only 9 (28%) of 32 patients with IgG showed a change (P =.0072). No statistically significant differences between patients with stable and changing antigen status were seen for age, sex, disease stage, time between analyses, and type of therapy or clinical response to therapy. However, the numbers of cases exhibiting antigenic shift for CD56, CD20, or CD52 are limited in this study, and some trends associated with antigenic shift are noted in the data in Table 1. Discussion Although antigenic shift has been well documented in hematopoietic disorders, including acute myeloid leukemia, 23 little information has been reported describing antigen stability in PCM. In this study, we documented changes in the immunophenotype of the plasma cells in more than 40% of patients during a 2- to 38-month follow-up. For the markers studied, the antigens that exhibited change in PCM were CD56, CD20, and CD52; the antigens that were stable were CD38, CD138, κ, and λ. In our studies, the neoplastic plasma cells were strongly CD38+ in all cases (100%); all cases but 1 (98%) were CD138+; and all showed dim CD45+ to CD45 staining (Image 1). The bright CD38+/bright CD45+ population, if present, was evaluated separately for clonality, but no cases showed cytoplasmic immunoglobulin light chain restriction (data not shown). Antigen frequencies of CD56 and cytoplasmic κ and λ were 73%, 73%, and 27% respectively, similar to previous reports. 8,9 We had slightly higher expression frequencies of CD20 (27%) and CD52 (55%) in our series than some previous studies, 9,17,22 but the frequencies were similar to others. 14,17 The higher frequency seen compared with some studies could be due to differences in monoclonal C E CD20 PE-Cy7 CD52 FITC antibodies used in the different laboratories. In addition, differences in nonspecific blocking reagents might also lead to differences in resolution of dim positive populations. In this study, a relatively high concentration (30%-50%) of fetal bovine serum or normal mouse serum was used. A statistically significant association of immunophenotype change with IgA myeloma was seen in this study. Of 12 IgA myelomas, 9 (75%) showed immunophenotypic change and constituted 39% of the cases exhibiting antigenic shift. Of 32 IgG myeloma cases, 9 (28%) showed immunophenotype change. Studies have shown that patients with IgA myeloma have a shorter median overall survival than patients with IgG myeloma 24,25 ; and the difference in overall CD20 PE-Cy7 CD52 FITC zimage 2z Flow cytometric analyses showing changes in antigen expression in plasma cells from patients with plasma cell myeloma. A and B, CD56 from negative to positive. C and D, CD20 from negative to positive. E and F, CD52 from negative to positive. APC, allophycocyanin; Cy7, cyanin 7; FITC, fluorescein isothiocyanate; PE, phycoerythrin. D F 930 Am J Clin Pathol 2008;129: DOI: /8UVF7YQ1D4D4ETQV

6 Hematopathology / Original Article A Immunophenotype unstable, 41% B to + 4 cases, 7% + to 2 cases, 4% Immunophenotype stable, 59% CD56 stable 50 cases, 89% C + to 1 case, 2% to + 5 cases, 9% CD20 stable 49 cases, 88% Multiple changes 1 case, 2% survival has been attributed to a shorter duration of plateau phase for patients with IgA myeloma compared with patients with IgG myeloma. 24 Recent studies showed that patients with PCM with t(4;14) had a predominance of IgA isotype (52%), and, although initial sensitivity to chemotherapy was shown, rapid relapse occurred. 26 Whether there is a relationship between the immunophenotypic instability of IgA myeloma and shortened survival is unclear. Of 56 cases, 6 (11%) showed CD56 change. Too few cases were available for statistical analysis; however, CD56 change seemed to be associated with higher stages of disease (all cases were stage III). Some authors have used CD19 and CD56 coupled with CD38, CD45, and CD138 to distinguish between normal and abnormal plasma cells, 27,28 and others have followed phenotype aberrations identified at diagnosis to monitor residual disease. 29 The results of this study raise the possibility of missing the disease when the phenotype changes and imply that these approaches should be used with caution. A panel of CD45 together with CD38, CD138, and cytoplasmic κ and λ staining may be a more effective way to detect residual disease. Nevertheless, inclusion of + to 9 cases, 16% to + 5 cases, 9% Multiple changes 3 cases, 5% CD52 stable 39 cases, 70% zfigure 1z Overall (A) and individual CD56 (B), CD20 (C), and CD52 (D) immunophenotype changes in 56 patients with plasma cell myeloma patients: stable vs unstable. D other antigens such as CD56 can be highly effective for detecting abnormal cells in a mixture of normal and abnormal plasma cells seen after therapy. A change in CD20 was seen in 13% of cases and in CD52 in 30% of cases. Loss of CD20 or CD52 antigen expression following immunotherapy with rituximab or alemtuzumab is common. 12,30,31 It is interesting that none of our patients were treated with rituximab or alemtuzumab during the follow-up period, and thus, these antigenic changes are not due to binding by therapeutic antibody to the assessed antigen. However, modulation of the antigen or antigenic deletion in response to other therapeutic approaches cannot be ruled out. Likewise, selective loss or, conversely, selective advantage of population subsets reflected in the immunophenotypic changes may have or is likely to be having a role. Recognition of antigen instability is important when considering antibody-based immunotherapy, at least with rituximab and alemtuzumab. Furthermore, antigen instability needs to be considered when monitoring disease in PCM. Am J Clin Pathol 2008;129: DOI: /8UVF7YQ1D4D4ETQV 931

7 Cao et al / Instability of Immunophenotype in PCM B C D zimage 3z Extent of bone marrow involvement and plasma cell morphologic features. A and B, Bone marrow involvement of 80% with mature plasma cell morphologic features (A, H&E, 40; B, Wright-Giemsa, 1,000). C and D, Focal bone marrow involvement and plasma cells with prominent nucleoli (C, H&E, 400; D, Wright-Giemsa, 1,000). From the Departments of 1Pathology and 2Medicine, Northwestern Memorial Hospital, Feinberg School of Medicine, Northwestern University, Chicago, IL. Address correspondence to Dr Peterson: Dept of Pathology, 251 E Huron St, Feinberg 7-205, Northwestern Memorial Hospital, Chicago, IL References 1. Jaffe ES, Harris NL, Stein H, et al, eds. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press; 2001: World Health Organization Classification of Tumours. 2. Goolsby CL, Paniagua M, Marszalek L. Clinical flow cytometry: a transition in utilization. Cancer Treat Res. 2004;121: Am J Clin Pathol 2008;129: DOI: /8UVF7YQ1D4D4ETQV 3. Kobayashi S, Hyo R, Amitani Y, et al. Four-color flow cytometric analysis of myeloma plasma cells. Am J Clin Pathol. 2006;126: Bataille R, Jego G, Robillard N, et al. The phenotype of normal, reactive and malignant plasma cells: identification of many and multiple myelomas and of new targets for myeloma therapy. Haematologica. 2006;91: Cruse JM, Lewis RE, Webb RN, et al. Antigenic transformation in plasma cell dyscrasia. Exp Mol Pathol. 2006;81: San Miguel JF, Gutierrez NC, Mateo G, et al. Conventional diagnostics in multiple myeloma. Eur J Cancer. 2006;42: Yaccoby S. The phenotypic plasticity of myeloma plasma cells as expressed by dedifferentiation into an immature, resilient, and apoptosis-resistant phenotype. Clin Cancer Res. 2005;11: A

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