Frequency and Prognostic Impact of the Aberrant CD8 Expression in 5,523 Patients with Chronic Lymphocytic Leukemia

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1 Cytometry Part B (Clinical Cytometry) 82B: (2012) Original Article Frequency and Prognostic Impact of the Aberrant CD8 Expression in 5,523 Patients with Chronic Lymphocytic Leukemia Wolfgang Kern, 1 * Ulrike Bacher, 2 Claudia Haferlach, 1 Tamara Alpermann, 1 Frank Dicker, 1 Susanne Schnittger, 1 and Torsten Haferlach 1 1 MLL Munich Leukemia Laboratory, Max-Lebsche-Platz 31, Munich 81377, Germany 2 Department for Stem Cell Transplantation, University of Hamburg, Martinistr. 52, Hamburg 20246, Germany Background: In patients with chronic lymphocytic leukemia (CLL), aberrant expression of the T-lineage antigen CD8 was reported in low frequencies. The clinical impact of this phenomenon remains in discussion. Methods: We analyzed 5,523 patients with CLL (21 97 years) by multiparametric flow cytometry and performed fluorescence in situ hybridization (FISH), immunoglobulin heavy chain variable (IGHV) region mutational status analysis, and clinical outcome studies. Results: CD8 was positive in 61/5,523 (1.1%) patients. ZAP-70 expression amounted to a mean of 25.4% in CD8-positive vs. 28.2% in CD8-negative cases (n.s.), CD38 expression to a mean of 38.6% vs. 34.0% (n.s.). Cytogenetic alterations did not differ significantly [CD8-positive vs. CD8-negative: 13q deletion: 24/36 (66.7%) vs. 2,015/3,368 (59.8%); trisomy 12:7/35 (20.0%) vs. 487/3,357 (14.5%); 11q deletion: 6/35 (17.1%) vs. 360/3,354 (10.7%)]. A mutated IGHV status showed similar frequency in CD8-positive and CD8-negative cases (31/44; 70.5% vs. 1,700/2,816; 60.4%; n.s.). CD8-positive patients had a shorter median time to treatment compared with CD8-negative patients (12.0 vs months, P ). In univariable Cox analysis, CD8-positivity adversely influenced median TTT (P ). In multivariable analysis, the strongest parameters were hemoglobin level and mutated IGHV status (P < for both) but CD8-positivity was still relevant (P ). Conclusions: This study confirms that CD8 expression is a recurrent albeit rare phenomenon in patients with CLL and suggests that CD8 expression has an adverse prognostic impact. Therefore, CD8 expression should be further investigated for its potential to contribute to risk stratification in patients with CLL. VC 2011 International Clinical Cytometry Society Key terms: chronic lymphocytic leukemia (CLL); multiparameter flow cytometry; CD8 expression; fluorescence in situ hybridization (FISH); prognosis How to cite this article: Kern W, Bacher U, Haferlach C, Alpermann T, Dicker F, Schnittger S, Haferlach T. Frequency and prognostic impact of the aberrant CD8 expression in 5,523 patients with chronic lymphocytic leukemia. Cytometry Part B 2012; 82B: Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with an extremely variable course. Identifying patients that benefit from early therapy is one of the most important issues in the management of CLL, and treatment becomes more individualized, ranging from cytostatic agents to monoclonal antibodies, and chemoimmunotherapeutic approaches (1). For selected patients with an adverse risk profile (e.g., with 17p13 deletions or TP53 mutations), allogeneic hematopoietic stem cell *Correspondence to: Wolfgang Kern, M.D., MLL Munich Leukemia Laboratory, Max-Lebsche-Platz 31, Munich 81377, Germany. wolfgang.kern@mll.com Received 30 September 2011; Revision 3 November 2011; Accepted 14 November 2011 Published online 5 December 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: /cyto.b VC 2011 International Clinical Cytometry Society

2 146 KERN ET AL. Table 1 Antibody Combinations Used in This Study (All Antibodies Were Purchased from Immunotech Via CDS Program (Marseilles, France) Except for *, Which Were Purchased from Dako) FITC PE ECD PC5 PC7 IgG1a 697.1Mc7 IgG1a 697.1Mc7 IgG1a 697.1Mc7 IgG Mc7 CD45 J33 CD79b F7137* CD5 BL1A CD19 J3-119 CD20 B9E9 CD45 J33 FMC7 FMC-7 IgM UHB CD19 J3-119 CD10 ALB1 CD45 J33 CD103 2G5 CD23 9P25 CD19 J3-119 CD22 SJ.10.1H11 CD45 J33 Kappa F0434* Lambda R0437* CD19 J3-119 CD38 LS CD45 J33 CD8 B9.11 CD4 13B8.2 CD3 UCHT1 CD56 N901(NHK-1) CD45 J33 CD11c BU15 CD19 J3-119 CD25 B CD45 J33 cyigg1a 697.1Mc7 cyigg1a 697.1Mc7 cyigg1a 697.1Mc7 cyigg1a 697.1Mc7 cycd45 J33 TdT HT1,4,8,9 ZAP-70 SBZAP cycd3 UCHT1 cycd79a HM47 cycd45 J33 transplantation (HSCT) is an option (2). Besides patientspecific characteristics, the decision to treatment and the choice of therapy are based on conventional prognostic parameters as well as on cytogenetic alterations (3,4). Immunoglobulin heavy chain variable (IGHV) mutational status (5) and expression of ZAP-70 (6,7) have all been shown to be independently associated with clinical outcome. Zenz et al. demonstrated that patients with TP53 mutations as determined by denaturing high-performance liquid chromatography carried a poor prognosis regardless of the presence of 17p deletion when treated with fludarabine-based chemotherapy (8). The immunophenotype of CLL is characterized by expression of pan-b-cell antigens (CD19, CD20, MHC class II) together with CD5 expression, an antigen expressed on T-cells and also on a subset of normal B- cells. The expression of CD5 in B-CLL or mantle cell lymphoma is thought to be related to a clonal expansion of a CD5þ subpopulation of B-cells and is not considered to be T-lineage related. Nevertheless, true aberrant expression of T-cell antigens in different B-cell lymphomas has recurrently been described at low frequencies, e.g., of the T-cell antigens CD2 or CD7 (9). Several studies reported on aberrant CD8 expression in rare cases of B-CLL (10): Carulli et al. performed a retrospective multicenter analysis in 951 patients with different B-cell lymphoma entities and found aberrant CD8 coexpression in 1.9% of all patients, with a frequency of 3.0% in B-CLL (11). It was discussed whether the frequency of aberrant CD8 coexpression could be higher in B-lineage B-CLL than generally assumed, because the expression of T-cell markers might not be systematically evaluated in laboratories (12). It seems that aberrant CD8 coexpression can as well occur at diagnosis of the CLL or during the course of disease (12). The prognostic impact of aberrant CD8 coexpression for CLL patients remains unclear: some authors suggested that prognosis of patients with B-cell lymphomas was not negatively influenced by CD8- positivity (10,11), whereas others were uncertain with regards to the prognostic impact (13) or speculated about more aggressive clinical behavior (14). To clarify the frequency of CD8 expression, its correlation to established prognostic parameters as well as its prognostic impact, we analyzed a total of 5,523 patients with CLL by multiparameter flow cytometry (MFC), including the expression of CD8. Additionally, we performed interphase fluorescence in situ hybridization (FISH) and IGHV mutational status analysis. DESIGN AND METHODS Patients The study cohort was based on all 5,523 patients with CLL who were investigated by MFC between August 2005 and August 2010 in the MLL Munich Leukemia Laboratory. There were 3,404 males and 2,119 females (median age: 69 years; range: years). Classification of cases as B-CLL was performed according to WHO criteria, requiring a minimum of /L CLL cells in peripheral blood or presence of lymphoma-related symptoms in case of lower amounts of CLL cells (15). Patients gave their written consent for laboratory analyses and for the use of laboratory results for research studies. The study was approved by the Internal Review Board and was performed in accordance with the tenets of the revised Helsinki protocol. Immunophenotyping MFC was based on a five-color approach as previously described for mature B-cell neoplasms (16). Peripheral blood or bone marrow samples were processed by Ficoll density gradient centrifugation or by erythrocyte lysis. Five-color stainings and isotype controls were performed by use of antibody combinations (Beckman Coulter, Krefeld, Germany) as given in Table 1. Antibody combinations were added to cells (volume 100 ll) and incubated at room temperature for 10 min. Expression of CD8 (antibody clone B9.11, Immunotech, France) was assessed by comparison to isotype used as negative control in all patients. CD8 expression was rated positive if at least 20% of CLL cells were positive as compared with an isotype control that was set as 1% positive (Fig. 1). ZAP-70 expression (antibody clone SBZAP, Immunotech, France) was calculated by the percentage of ZAP-70 positive B-cells using normal T-lymphocytes as positive controls with the cutoff set to 97% of T-lymphocytes being positive (16). CD38 expression was analyzed as continuous variable giving the percentage of positive CLL cells as compared with an isotype control that was set as 1% positive.

3 FREQUENCY AND PROGNOSTIC IMPACT OF THE ABERRANT CD8 EXPRESSION IN CLL 147 FIG. 1. Dim expression of CD8 in CLL (A1 to A4, blue cells) and lack of expression of CD8 in CLL (B1 to B4, blue cells). CD8-positive T-lymphocytes (red cells) are shown in comparison. Cells displayed are lymphatic cells as defined in the CD45-SSC gate (strong CD45, low SSC signal), which are negative for both CD56 and CD4. An isotype control was used to set the cutoff for CD8-positivity at 1% (C). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Fluorescence In Situ Hybridization Interphase FISH analysis was performed in 3,407/5,523 (61.7%) cases by use of probes for detection of deletions of 6q, 11q [del(11)(q22.3)/atm], 13q [del(13)(q14), (D13S25, D13S319)], and 17p [del(17)(p13)/tp53)], trisomy 12, and the t(11;14)/igh-ccnd1 and t(14;18)/ IGH-BCL2 rearrangements as described earlier (Abbott, Wiesbaden, Germany) (17). IGHV Mutational Status Analysis IGHV mutation analysis was performed in 2,845/5,523 (51.5%) of all cases. PCR amplification of monoclonal immunoglobulin rearrangements was done from cdna using six different consensus framework region 1 (FR1) V H forward primers and one consensus joining region J H reverse primer (18). Direct sequencing was performed following purification of the PCR products on agarose gel. Sequences were aligned to immunoglobulin sequences in the IMGT ( database und evaluated according to ERIC recommendations (19). Cases with more than 2% somatic hypermutations in the IgHV region were rated as mutated IGHV status, while others were rated as unmutated IGHV status. Statistical Analysis Time to treatment (TTT) and overall survival (OS) were calculated according to Kaplan Meier, and comparisons were performed by two-sided log rank test. Dichotomous variables were compared between different groups using the v 2 -test and continuous variables by Student s T-test. Univariable analysis for prognosis defining risk parameters was done by Cox regression. Parameters being significant at a level of P < 0.05 in univariable analysis were included in multivariable analysis. Parameters tested for impact on outcome were: WBC count, Hb level, platelet count, age as continuous parameter, gender, CD8- positivity, higher % CLL cells with ZAP-70 expression, CD38 expression, del(11)(q22.3), del(17)(p13), sole del(13)(q14), del(6q), trisomy 12. IBM SPSS (version 19.0) software was used. RESULTS Frequency of Aberrant CD8 Coexpression 61/5,523 patients (1.1%) showed aberrant coexpression of CD8 (Fig. 1). An aberrant coexpression of CD3 or CD4 has not been observed in these 5,523 patients. CD8-positive cases homogeneously showed a dim expression of CD8 as exemplified in Figure 1. Flow Characteristics The percentage of ZAP-70 positive cells amounted to a mean of 25.4% (SD, 29.1%) in CD8-positive compared with a mean of 28.2% (29.6%) in CD8-negative cases (n.s.). The respective percentages for CD38 expression were % vs % (n.s.). Biologic Parameters CD8-positive and CD8-negative patients did not differ significantly in age (mean SD: vs years, n.s.) or male/female ratio (1.1 vs. 1.6; n.s.). Also, mean WBC count (31.7 vs /L) and

4 148 KERN ET AL. Table 2 Comparison of Biologic Parameters, Peripheral Blood Values, Cytogenetic Alterations as Determined by Fluorescence In Situ Hybridization (FISH), and the Frequency of Mutated IGHV Status in CLL Patients with and without Aberrant CD8 Expression Parameter CD8-positive CD8-negative P-value Biologic parameters Males:females (ratio) 32:29 (1.1) 3,372:2,090 (1.6) n.s. Age, years (mean SD) n.s. Peripheral blood parameters WBC count (10 9 /L) (mean SD) n.s. Hb (g/dl) n.s. Platelet count (10 9 /L) FISH (number of patients; %) del(6q) 2/34 (5.9%) 101/3,297 (3.1%) n.s. del(11)(q22.3)/atm 6/35 (17.1%) 360/3,354 (10.7%) n.s. trisomy 12 7/35 (20.0%) 487/3,357 (14.5%) n.s. del(13)(q14) 24/36 (66.7%) 2,015/3,368 (59.8%) n.s. del(13q) sole 14/34 (41.2%) 1,506/3,307 (45.5%) n.s. del(17)(p13)/tp53 1/35 (2.9%) 193/3,372 (5.7%) n.s. IGHV mutated status 31/44 (70.5%) 1,700/2,816 (60.4%) n.s. WBC count: white blood cell count; del: deletion; SD: standard deviation. mean hemoglobin level (13.6 vs g/dl) showed no significant differences between both cohorts, while mean platelet count was significantly higher in CD8- positive than CD8-negative cases (229 vs /L, P ¼ 0.021) (Table 2). Genetic Alterations FISH analysis was performed in 3,407/5,523 (61.7%) cases, and in some cases, only part of the complete set for all FISH probes was applied (the complete panel including the above mentioned interphase probes was investigated in 3,358 patients). Most frequent was deletions of 13q14, which were found in 24/36 (66.7%) CD8-positive and in 2,015/3,368 (59.8%) CD8-negative cases (n.s.). As sole alterations, 13q deletions were observed in 14/34 (41.2%) CD8-positive and 1,506/3,307 (45.5%) CD8-negative cases (n.s.). This was followed by trisomy 12 (CD8þ: 7/35; 20.0%; CD8 : 487/3,357; 14.5%; n.s.) and 11q deletions [CD8þ: 6/35 (17.1%); CD8 : 360/3,354 (10.7%); n.s.]. 6q deletions (CD8þ: 2/ 34 (5.9%); CD8 : 101/3,297 (3.1%); n.s.) and 17p deletions [CD8þ: 1/35 (2.9%); CD8 : 193/3,372 (5.7%); n.s.] were rarely observed in both cohorts. Thus, cytogenetic alterations were similarly distributed in CD8-positive and CD8-negative patients (Table 2). months. Patients with CD8 expression had a significantly shorter TTT as compared with CD8-negative patients (median TTT, 12.0 vs months, P ¼ 0.008; Fig. 2). Median OS was not reached for both cohorts (n.s.). Uni- and Multivariable Analysis The following parameters showed a significant relation to a shorter TTT in univariable analysis: CD8-positivity (P ¼ 0.011, relative risk ¼ 2.87), higher WBC count (P ¼ 0.008, RR ¼ 1.01 per /L increase), del(11)(q22.3) (P < 0.001, RR ¼ 1.97), del(17)(p13) (P ¼ 0.005, RR ¼ 1.72), higher percentage of CLL cells with ZAP-70 expression (P ¼ 0.011, RR ¼ 1.04 per 10% increase), and CD38 expression (P < 0.001). Parameters significantly related to a longer TTT in univariable Cox analyses were higher Hb level (P < 0.001, RR ¼ 0.86 IGHV Mutational Status The IGHV mutational status was determined in 2,845/ 5,523 (51.5%) patients. The IGHV status showed a similar frequency in CD8-positive and CD8-negative cases (31/44; 70.5% vs. 1,700/2,816; 60.4%; n.s.). Clinical Outcomes Clinical follow-up data were available in 1,022 patients (12 CD8-positive patients, 1,010 CD8-negative patients). In the CD8-positive cohort, six of 12 patients had received therapy. Considering the CD8-negative patients, 296 had received therapy, while 714 patients required no treatment. The median follow-up period was 21.3 FIG. 2. Comparison of time to treatment (TTT) in CLL patients with aberrant CD8 expression compared with CD8-negative patients. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

5 FREQUENCY AND PROGNOSTIC IMPACT OF THE ABERRANT CD8 EXPRESSION IN CLL 149 Table 3 Results of Uni- and Multivariable Analysis Regarding Parameters with an Influence on Time to Treatment (RR: Relative Risk) P-value (relative risk, RR) Parameter Univariable Multivariable Related to shorter TTT CD8-positivity (2.87) Higher WBC count (1.01 a ) n.s. Higher % CLL cells (1.04 b ) with ZAP-70 expression CD38 expression < del(11)(q22.3) <0.001 (1.97) n.s. del(17)(p13) (1.72) n.s. Related to longer TTT Higher Hb level (0.86 c ) <0.001 Higher platelet count (0.98 a ) n.s. sole del(13)(q14) <0.001 (0.58) Mutated IGHV status <0.001 (0.311) <0.001 No significant impact Age (continuously) n.s. Gender n.s. del(6q) n.s. trisomy 12 n.s. a per /L increase. b per 10% increase. c per 1 g/dl increase. per 1 g/dl), higher platelet count (P ¼ 0.021, RR ¼ 0.98 per /L increase), del(13)(q14) as sole chromosomal aberration (P < 0.001, RR ¼ 0.58), and a mutated IgVH status (P < 0.001; RR ¼ 0.311). Multivariable analysis identified the following parameters to be independently related to a shorter TTT: higher percentage of CLL cells with ZAP-70 expression (P ¼ 0.038), CD8-positivity (P ¼ 0.074), and CD38 expression (P ¼ 0.077), while a higher Hb level and a mutated IGHV status was identified to be independently related to a longer TTT (for both, P < 0.001). No significant impact on TTT was found for age, gender, trisomy 12, and 6q deletions (Table 3). DISCUSSION Aberrant coexpression of the CD8 antigen has been reported in rare patients with B-cell lymphomas (10,11,13,20), but the clinical impact of this phenomenon remains unclear. Therefore, we determined the frequency and the prognostic meaning of this phenomenon in a large cohort of 5,523 patients with CLL. First, we detected aberrant CD8 coexpression as defined by at least 20% positive CLL cells as compared with an isotype control in 1.1% of all CLL cases, which was in the range of previous literature. Carulli et al. determined a frequency of 3.0% in 362 patients with B- CLL/SLL (11). Mulligan et al. assumed around 0.5% of cases with aberrant coexpression of CD8 in the CLL patients in their institution (10). Thus, our study gave further confirmation to the observation that aberrant CD8 expression is a recurrent (albeit rare) phenomenon in B-lineage CLL. Second, in our study, no significant differences were found between patients with versus without CD8 coexpression regarding biological parameters such as age or sex ratio, or with regards to mean WBC and hemoglobin. Only the mean platelet count was higher in CD8- positive than CD8-negative cases (P ¼ 0.021). The frequency of a mutated IGHV status and the profiles of cytogenetic alterations as determined by FISH were similar for CD8-positive and -negative patients investigating a large panel of probes including deletions of 13q14, or 17p13, or trisomy 12. Thus, regarding cyto- and molecular genetic aspects, no adverse profile of the CD8-positive cases was detectable. However, the clinical outcomes of the CD8-positive patients were considerably less favorable when compared with the CD8-negative cases as the time to first therapy was significantly shorter with 12.0 versus 77.1 months (P ¼ 0.008). Also, in multivariable analysis, taking other parameters such as cytogenetic alterations or the expression of ZAP-70 into account, CD8-positivity showed a trend to a shorter time to first therapy (P ¼ 0.074), although other parameters conferred a stronger impact (e.g., Hb level and a mutated IGHV status with a P-value < for both). Previous studies investigating more limited cohorts came to diverging conclusions: Carulli et al. documented the clinical courses in 16 patients with different lymphomas and aberrant CD8 expression (out of a total of 18 patients) and found stable disease or a very good response to therapy in 75% of these cases in both CLL and other B-cell lymphomas. However, the authors admitted that the series was too limited to draw final conclusions (11). Mulligan et al. described favorable clinical courses in the majority of patients with CD8-positive CLL: eight from 10 patients had stable nonprogressive early stage A disease, and only two patients had more advanced disease at diagnosis requiring therapy due to progression (10). In another series reported by Kaleem et al. including nine patients with different B-cell lymphomas who showed expression of different T-lineage antigens (i.e., CD2, CD4, CD7, and CD8), no unexpected aggressive clinical behavior was observed (21). Schroers et al. reported on a patient aged 73 years with a history of indolent B-CLL lasting for 38 years in whom they detected aberrant CD8 expression (22). Others were uncertain regarding prognosis of CD8-positive B- CLL patients: Parisi-Duchêne et al. reported on five cases of B-CLL with aberrant coexpression of the CD8 antigen. Two of the five patients died 1 and 6 years later (13). Stagno et al. described an 81-year-old patient with CD8- positive CLL with progressive disease despite chlorambucil and subsequent salvage therapy and assumed that aberrant CD8 expression might be related to aggressive clinical courses (14). Islam et al. documented a patient with B-CLL and aberrant coexpression of CD8 who required no therapy during the first 6 years. When the disease progressed, he received conventional doses of fludarabine therapy but developed severe myelosuppression and finally died from intercurrent respiratory tract

6 150 KERN ET AL. infection. Therefore, the authors hypothesized that patients with aberrant CD8 expression in B-CLL might show increased sensitivity to fludarabine (20). In conclusion, our study suggests that aberrant CD8 expression is a strong adverse prognostic parameter in B-CLL even when other relevant parameters such as cytogenetics or ZAP-70 expression are considered in parallel. Further analyses should focus on potential adverse effects of CD8 expression on OS that may have been missed in the present series due to a median follow-up of 21.3 months, which clearly is below the median OS for patients with CLL. Also, whether the finding of aberrant CD8 expression is sufficient to justify more intensive treatment approaches for CLL patients remains to be further investigated. LITERATURE CITED 1. Hallek M. State-of-the-art treatment of chronic lymphocytic leukemia. 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