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Clinical Cancer Research Vol. 12, 2125-2132, April 2006
© 2006 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

Diffuse Large B-Cell Lymphoma with Overexpression of Cyclin E Substantiates Poor Standard Treatment Response and Inferior Outcome

Alexandar Tzankov1, Andreas Gschwendtner6, Florian Augustin2,3, Michael Fiegl3, Ellen C. Obermann4, Stephan Dirnhofer5 and Philip Went5

Authors' Affiliations: 1 Institutes of Pathology and Departments of 2 General Surgery and 3 Hematology and Oncology, Medical University of Innsbruck, Innsbruck, Austria; 4 University of Regensburg, Regensburg, Germany; 5 University of Basel, Basel, Switzerland; and 6 Hospital Coburg, Coburg, Germany

Requests for reprints: Alexandar Tzankov, Institute of Pathology, Medical University of Innsbruck, Muellerstr. 44, 4020 Innsbruck, Austria. Phone: 43-512-5073692; Fax: 43-512-582088; E-mail: Alexandar.Tzankov{at}i-med.ac.at.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Gold standard to predict survival and stratify patients for risk-adapted therapy in diffuse large B-cell lymphoma (DLBCL) is the international prognostic index, although it does not consider the molecular heterogeneity of DLBCL. Deregulation of cyclin E (CCNE) is a strong predictor of poor prognosis in some neoplastic diseases. In tumor cells, it induces chromosomal instability with an increased rate of aneuploidy/polyploidy.

Experimental Design: We analyzed in this retrospective study the prognostic value of immunohistochemical CCNE expression on a validated tissue microarray containing 101 de novo DLBCLs and, in 9 cases, the CCNE-induced chromosomal instability as assessed by cytometry.

Results: Forty-six of 98 evaluable DLBCLs expressed CCNE in a mean proportion of 20 ± 29% of tumor cells; 38 cases expressed CCNE in ≥20% of tumor cells. CCNE-positive samples were aneuploid compared with near tetraploidy in CCNE-negative cases. Multivariate analysis showed CCNE expression in ≥20% of tumor cells to be an international prognostic index–independent, Adriamycin-based treatment-independent, and BCL2-independent prognostic factor for poor disease-specific survival. CCNE expression in ≥80% of tumor cells was associated with dismal short-term prognosis. CCNE expression in ≥50% of tumor cells emerged as an independent predictive factor for standard CHOP treatment resistance.

Conclusions: CCNE expression assessment is easy on paraffin-embedded tissue. The high prognostic value of CCNE expression in DLBCL may be the basis for future prospective trials. In addition, a high CCNE expression hints at the presence of a possible target for individualized cancer therapy.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy, comprising 35% to 40% of all adult extramedullary non-Hodgkin's lymphomas (1, 2). Only 40% to 50% of patients can be cured (2). Gold standard to predict survival and stratify patients for risk-adjusted therapy is the international prognostic index (IPI). Unchanged in the preceding decade, it uses easily assessable clinical variables (3) but does not reflect the molecular heterogeneity of DLBCL (4–6). In the last years, several biological markers and gene expression signatures have been studied to improve risk-adapted patient management, but reliability and reproducibility of the results is conflicting. Proposed scoring systems are too sophisticated for daily use or their predictive value is inferior to IPI (4–11). Markers distinctive for cellular differentiation and apoptosis regulation have been addressed, but less attention has been paid to the cell cycle control mechanisms in DLBCL with respect to their prognostic and potential therapeutic implications (9, 10, 12–21).

Mitogen-independent evasion of cell proliferation control is a major prerequisite of cancer, and promising new therapies are targeted at restoring this lost control (22–24). Cyclin E (CCNE), dissimilar to the mitogen-dependent cyclin D (CCND), is a mitogen-independent activator of the cyclin-dependent kinase (Cdk) 2 and a critical regulator of the G1-S transition (24, 25). CCNE periodically peaks at the G1-S transition and declines rapidly once S phase is initiated by both transcription down-regulation and ubiquitin-mediated proteolysis (26, 27). Deregulation of CCNE expression is a strong predictor of poor prognosis in some neoplastic diseases (10, 15, 16). Aberrant expression of CCNE induces chromosome instability with increased frequency of aneuploidy/polyploidy (28, 29). Physiologically, the activity of CCNE/Cdk2 complexes is negatively regulated by the Cdk inhibitor p27 (24). Importantly, p27 can join CCND/Cdk4/6 complexes and become functionally inactivated. Thus, CCND3 can potentially activate CCNE by sequestration of p27 (24, 30).

In DLBCL, expression of CCNB1, CCND3, CCNE, and p27 have been shown to be prognostically relevant, but no systematic analysis of interactions was completed (9, 10, 12–21). Different cutoff levels for CCNE expression were applied and the cases studied had some preselection bias (15, 16, 20). Effects of the therapeutic regimens were not addressed, and except for one study (10), case numbers were low. The significance of CCNE expression relative to the cell cycle as well as the correlation of CCNE expression to DLBCL morphology and ploidy have not been addressed thus far. Moreover, several small molecules that modulate CCNE and Cdks (22) may have potential therapeutic effect in selected DLBCL patients. Thus, it is important to study the deregulation and the possible predictive value of CCNE as well as CCNE-induced chromosomal instability. We immunohistochemically analyzed (a) CCNE expression relative to other cell cycle–regulating and apoptosis-regulating proteins, such as Ki-67, BCL2, CCNB1, CCND3, and p27, and (b) CCNE prognostic effect on a validated (31) tissue microarray containing 101 cases of de novo DLBCL. By cytometry, we assessed (c) induction of chromosomal instability by CCNE in DLBCL.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Samples. One hundred one cases of de novo DLBCL diagnosed between 1988 and 2000 and reclassified according to the WHO criteria (1) were collected from the archive of the Institute of Pathology at the Medical University of Innsbruck. Paraffin blocks were selected based on availability and preservation; HIV infection (n = 2) and post-transplant settings (n = 1) were not considered as exclusion criteria. Clinical and follow-up data (summarized in Table 1 ) were obtained reviewing the charts. Retrieval of tissue and clinical data were done according to the regulations of the local institutional review board and data safety laws. Patients were staged by means of computerized imaging and bone marrow biopsy. Complete disease remission was defined as absence of disease for at least 3 months after cessation of the last treatment regimen as assessed by laboratory and imaging studies and physical examination. Disease relapses were defined as disease recurring after disease remission. Minor tumor shrinkage, immediate progressive disease, and early relapses (within 3 months after cessation of the last treatment regimen) were considered failure to achieve complete remission.


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  		Table 1. Patient characteristics with respect to expression of CCNE

 
Tissue microarray construction. The tissue microarray was constructed as described elsewhere (31). Two cores from every sample were arrayed. Validation was done by comparison of the staining results for CD10, CD20, BCL2, and BCL6 on the tissue microarray with that on conventional paraffin block sections in 50 cases.

Morphology. Each case was classified as centroblastic, centroblastic polymorphous, and immunoblastic or unclassifiable according to the WHO criteria (1). Within the latter group, a subgroup with a high proportion (≥30% of cells) of apoptoses/necroses was noted and categorized separately.

Immunohistochemistry. Tissue microarray slides were processed on an automated immunostainer (Nexes, Ventana, Tucson, AZ). The streptavidin-biotin peroxidase technique with diaminobenzidine as chromogen was applied. The primary antibodies were diluted in a 1% solution of bovine serum albumin in PBS (pH 7.4) and incubated for 30 minutes at room temperature. Antigen retrieval was done as suggested by the manufacturer. Primary antibodies used, their dilutions, and cutoff levels for evaluation are listed in Table 2 . Because intensity varies between cases due to different tissue preservation, only the relative proportion (percentage) of positively staining tumor cells and not the staining intensity was considered. This proportion was assessed as the mean of the estimated positively staining tumor cells out of all tumor cells on the two tissue microarray cores. Special attention was paid to the subcellular localization of markers. For CCNE, CCND1, and CCND3, p27 and Ki-67 nuclear staining was assessed; in the case of p27, CCNB1 and CCND3 cytoplasmic expression was also considered for further analysis (18). The expression of CD10, BCL6, MUM1, and CD44s was considered to classify tumors as germinal center B-cell-like DLBCL and nongerminal center, applying the following decision tree: germinal center phenotype = BCL6+/CD10+ or BCL6+/CD10–/MUM1–/CD44s– or BCL6–/CD10+/MUM1–/CD44s–, with all other combinations defining nongerminal center phenotype (8, 11).


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  		Table 2. Antibodies, antigen retrieval techniques, applied cutoff levels, and expression characteristics in the analyzed DLBCL patient cohort

 
DNA cytometry. Cytometric analysis was done on nuclei isolated from the tumor cell–rich areas of specimens with high (>80%, n = 3), intermediate (30-40%, n = 3), and absent (n = 3) expression of CCNE using nongerminal center lymphocytes from tonsils for comparison. The lymphoma cells and the tonsillar lymphocytes were obtained from well-preserved parts of the paraffin-embedded material by means of punching out tissue cores by an 11-gauge trephine needle. The cores were disintegrated, and cells were stained by the Feulgen's stain and handled as described (32). Measurements were done in accordance to the ESCAP criteria (33) using a custom-made DNA cytometer and Optimas image analysis software (Optimas, Seattle, WA). The results were plotted as DNA histograms.

Statistical analysis. Statistical analysis was done using the Statistical Package of Social Sciences version 10.0 (SPSS, Chicago, IL) for MS Windows. Incomplete data represented empty spots in the primary SPSS table and were not excluded from the tests done. The Pearson {chi}2 statistic and the Spearman rank correlation were used to analyze relationships between the markers and the clinical and laboratory variables. Mann-Whitney U and Kruskal-Wallis tests were applied where appropriate to study differences between groups. Disease-specific survival (DSS) and overall survival (OS) were analyzed by the Kaplan-Meier method. The effect of each individual clinical or immunohistochemical variable on DSS and OS was determined using the log-rank test. Multivariate analysis for the prognostic effect of the markers that seemed to be of significant (P < 0.05) or trend (P < 0.1) prognostic significance univariately was done using a Cox regression model. To assess the predictive value of the clinical, laboratory, and immunohistochemical variables for treatment response (defined by achievement of complete remission), the factors that seem to be significant in the univariate models were entered in a general linear model for multivariate analysis. Two-sided tests were used throughout.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Histopathology. One hundred of all 101 arrayed cases were representative by H&E. Cases [n = 24 (24%)] classified morphologically as immunoblastic (n = 11) or polymorphous centroblastic (n = 13) were prognostically identical. Forty (41%) cases were classified as centroblastic and 34 (35%) were unclassifiable DLBCLs, with 23 of the latter showing an apoptotic/necrotic phenotype and being prognostically identical to centroblastic ones.

Immunohistochemical tissue microarray validation and assessment of germinal center DLBCL phenotype. The immunohistochemical analysis failure of two (three) cases (see next paragraph) was linked to missing (empty) spots or fixation artifacts (31). In the 50 cases used for validation, there was a 100% concordance for the antibody staining results (CD10, CD20, BCL2, and BCL6) on tissue microarrays and conventional large sections. Considering the expression of BCL6, CD10, CD44s, and MUM1, 42 of 99 cases were classified as germinal center DLBCL.

CCNE expression. Ninety-eight cases were evaluable for CCNE expression. The staining for CCNE was of moderate to strong intensity and nuclear (Fig. 1A-C ); only 7 cases showed weak cytoplasmic positivity. For determination of cutoff levels, we considered previously reported cutoffs (≥80%; ref. 10) as well as the mean CCNE expression (≥20%) value and the 30th percentile in-between (≥50%). Forty-six DLBCLs expressed CCNE in a mean proportion of 20 ± 29% of cells, 38 cases expressed CCNE in ≥20% of cells, and 52 cases did not express CCNE at all (Table 2).


Figure 1
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  		Fig. 1. A, CCNE-negative DLBCL. Note the lack of CCNE expression despite multiple mitotic figures. Original magnification, x200 (immunoperoxidase stain). B, DLBCL expressing CCNE in 30% of cell nuclei. Original magnification, x200 (immunoperoxidase stain). C, DLBCL expressing CCNE in >70% of cell nuclei. Original magnification, x200 (immunoperoxidase stain). D, Kaplan-Meier DSS analysis of DLBCLs with respect to the expression of CCNE. Note that there were no 1-year survivors in the group of patients who expressed CCNE in ≥80% of lymphoma cells. P = 0.0006.

 
CCNE correlation with BCL2, CCNB1, CCND3, p27, and Ki-67. The results of the correlation analysis are shown in Table 3 . Cytoplasmic CCNE expression did not correlate with any other variable. Importantly, nuclear expression of CCNE correlated with expression of CCNB1, CCND3, and p27 but not with subcellular localization of p27 or with BCL2 status. CCNE expression correlated strongly with immunoblastic/polymorphous centroblastic morphology of DLBCL but not with germinal center phenotype. Expression of CCND3 correlated with p27, especially with cytoplasmic subcellular localization (data not shown in detail). Importantly, CCND3 but not CCNE expression correlated with proliferation as assessed by Ki-67. Because Ki-67 is expressed through the cell cycle from G1 to M phase (34), a greater proportion of tumor cells expressing CCNE than Ki-67 would be indicative for overexpression of the former (28). Therefore, we directly compared the expression of CCNE with Ki-67. In 12 of 98 evaluable cases, the proportion of CCNE-expressing lymphoma cells exceeded the expression of Ki-67 by ≥15%; the proportion of these cases was greater in the DLBCL group with ≥80% CCNE-positive cells, but this overexpression was not of prognostic significance. Ki-67 did not correlate with morphologic DLBCL subtype or apoptotic/necrotic phenotype.


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  		Table 3. Correlations between expression of cell cycle–regulating proteins and clinical characteristics in DLBCL patients

 
Clinical variables. All relevant clinical patient characteristics are shown in Table 1. The DLBCL cohort included 55 male patients (mean age, 64 years; range, 25-88 years) and 46 female patients (mean age, 67 years; range, 18-93 years). The patients were mainly treated with Adriamycin-based polychemotherapy regimens with or without adjuvant radiotherapy; 5 patients were not treated due to severe accompanying morbidity and 2 were given solely radiotherapy. Only 1 patient received rituximab. Thirty relapses occurred after a mean period of 20 months (range, 1-131 months). Within the follow-up period of 201 months (mean, 48 months), 45 patients died due to failure to achieve complete remission or due to DLBCL relapses, 25 due to cardiovascular events (n = 13), infections (n = 5), or other neoplasms (n = 7), the latter except for infections being in range with the expected lethal events and death causes in an average age-matched Tyrolean population for a similar follow-up period. Median OS was reached after 28 months (95% confidence interval, 12-44) and median DSS was reached after 88 months (95% confidence interval, 31-145). Relative 5-year OS was 38% and relative 5-year DSS was 54%. Except for BCL2, which correlated with patients' age and survival, expression of p27, Ki-67, and CCND3 did not correlate with clinical variables (Tables 3 and 4 ).


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  		Table 4. DSS analysis in DLBCLs

 
CCNE, survival, and response to therapy. The results concerning DSS and OS are shown in Table 4 and Fig. 1D. CCNE expression at any cutoff value represented an IPI-independent prognostic factor for both DSS and OS with a higher predictive power than BCL2, CCNB1, and germinal center/nongerminal center phenotype. Interestingly, when analyzed in subgroups stratified by IPI, stage (except for stage 1), type of therapy (Adriamycin versus no Adriamycin; number of cycles applied 0-2 versus 3-8), sex, germinal center phenotype, and BCL2 status, the negative prognostic effect of CCNE expression preserved its statistical significance. Considering the predictive value of CCNE expression for therapy response (mainly CHOP), we observed 53 complete remissions in 70 documented cases (76%) expressing CCNE in <50% of lymphoma cells and only 10 in 22 (45%) cases expressing CCNE in ≥50% (P = 0.012). Patients in whom complete remission was achieved (n = 63) expressed CCNE in a mean proportion of 15% of the tumor cells (95% confidence interval, 9-22), whereas those in whom it was not achieved expressed CCNE in 30% (95% confidence interval, 18-42; P = 0.03). Multivariate analysis considering all factors that were associated with failure to achieve complete remission in the univariate models showed CCNE expression in ≥50% of tumor cells (P = 0.001), B symptoms (P = 0.002), and high IPI (P = 0.005) to be independent predictive factors for treatment resistance.

CCNE and ploidy. The analyzed tonsillar samples were all diploid. DLBCL cases (n = 3), which did not express CCNE, were near tetraploid, although, except for one diploid case, all other five CCNE-positive samples were aneuploid without significant differences between cases with high and intermediate expression of CCNE (Fig. 2A-D ). Still, the case showing the highest rate of aneuploid peaks was in the group expressing CCNE in >80% of cells (Fig. 2C).


Figure 2
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  		Fig. 2. A, typical DNA histogram of reference nongerminal center tonsillar lymphocytes. Almost all measured nuclei are diploid or near diploid. B, typical DNA histogram of CCNE-negative DLBCL. The majority of cell nuclei are near-tetraploid or near-diploid. C, typical DNA "Manhattan skyline" histogram of CCNE+ DLBCL. Note the presence of octaploid/nanoploid nuclei. D, ploidy/CCNE expression box plot.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Herein, we describe CCNE as a straightforward, IPI-independent prognostic factor in DLBCL. Compared with previous studies (15), we used a monoclonal antibody and the advantage of a validated tissue microarray (11, 31), allowing us to analyze all study samples at highly standardized conditions. The mean CCNE expression level in our patient cohort was generally in line with observations from other studies (10, 14, 15). However, direct comparison with these studies is difficult because of selection biases, such as compelling DLBCLs and Burkitt's lymphomas, considering only gastric lymphomas or applying other classification systems (15, 16, 20) and because of application of different antibodies (15) or scoring systems (10, 15, 16). In our patient cohort, the application of reported cutoff values, except for that suggested by Saez et al. (10), neither has been proven to be of prognostic significance nor correlated with any other biological variable. Nonetheless, expression of CCNE was (a) an IPI-independent prognostic factor for DSS if a cutoff level of ≥20% of tumor cells was chosen and (b) a predictive factor for poor response to standard CHOP treatment with a cutoff level of ≥50%. There were no 1-year survivors in the group of patients who expressed CCNE in ≥80% of lymphoma cells (Fig. 1D). CCNE expression retained its prognostic significance in the subgroups stratified by IPI, stage, sex, therapy type, BCL2, and phenotype. In contrast to other proposed markers (8, 10, 11), the expression of CCNE is easily assessable, simple to score, and therefore of low expected result variability.

Expression of CCNE had a superior prognostic value to germinal center phenotype and BCL2 status (Table 4; refs. 8, 11, 35). Furthermore, the predictive value of CCNE was independent of BCL2 status, pointing to the fact that it can possibly identify distinct DLBCL cases, whose increased risk for disease-related death or treatment resistance could not be abolished by rituximab (36). Controversy exists on the prognostic effect of CCND3 (17, 19); still, we could not find an association in our cohort. Recently, we showed CCNB1 expression to be of prognostic significance in a large DLBCL series that partially overlapped with the present cases (9). In a multivariate analysis considering the present results, only CCNE seemed to be of prognostic value when compared with CCNB1 (Table 4).

Expression of CCNE correlated with expression of CCNB1, CCND3, p27, and immunoblastic/centroblastic polymorphous DLBCL morphology but not with proliferation as assessed by Ki-67. Patients with high CCNE expression had an unfavorable clinical outcome pointing to an inefficient inhibition of CCNE by p27 (24, 30). Cytoplasmic localization of p27 correlated with expression of CCND3. p27 could therefore be sequestrated by CCND3 (18, 20) and fail to inhibit CCNE (24, 30). Interestingly enough, CCND3 but not CCNE correlated with proliferation as assessed by Ki-67, which is in line with previous observations (15). Thus, not proliferative activity but mitogen-independent deregulation of the G1-S transition possibly plays a role for the observed therapy resistance as suggested by the low complete remission rate and increased disease-specific mortality in CCNE-expressing cases.

About 35% of our DLBCLs showed high expression of CCNE despite the general lack of CCNE gene amplification in DLBCL (37), a constellation similar to that in classic Hodgkin's lymphoma (38). Whereas in classic Hodgkin's lymphoma CCNE overexpression seems to reflect the profound deregulation of cell cycle in Hodgkin's and Reed-Sternberg cells (38, 39) and has no prognostic significance, CCNE in DLBCL obviously preserves its oncogenic potential to promote G1-S transition independent of extracellular mitogenic stimuli and/or to suppress apoptotic pathways (26, 40) as well as to induce aneuploidy (28). It remains to be determined if altered ubiquitin-mediated degradation, archipelago gene mutations, gene mutations that render CCNE resistant to proteolysis, or even increased CCNE transcription (26, 28, 29, 41) could explain the observed CCNE expression in DLBCL. Independent of CCNE up-regulation reasons, we showed that CCNE (a) has an important prognostic effect and (b) induces aneuploidy in DLBCL. The possibility to neutralize the effects of CCNE remains an intriguing issue (26). Some drugs in preclinical development potentially target the modulation of CCNE, p27, and Cdk2 (22, 23): flavopiridol (42), lapatinib in combination with tamoxifen (43), resveratrol (44), the sulfonamide E7070 (45), IFN-{alpha} (46), N-acyl-2-aminothiazoles (47), roscovitine (48), rapamycin (49), etc. Although experience with these drugs is limited, minor responses in non-Hodgkin's lymphoma have been documented (23, 49, 50).

In summary, CCNE expression in ≥20% of DLBCL cells is an IPI-independent and BCL2-independent predictor of poor treatment response, DSS and OS. CCNE expression in ≥80% of cells is associated with dismal short-term patient prognosis. CCNE expression can be easily assessed on paraffin-embedded material and could provide prognostic information for the oncologist. In addition, it hints at the presence of a possible target for future individualized molecular therapy.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 9/29/05; revised 1/11/06; accepted 1/23/06.


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 Results
 Discussion
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A. Tzankov, C. Meier, P. Hirschmann, P. Went, S. A. Pileri, and S. Dirnhofer
Correlation of high numbers of intratumoral FOXP3+ regulatory T cells with improved survival in germinal center-like diffuse large B-cell lymphoma, follicular lymphoma and classical Hodgkin's lymphoma
Haematologica, February 1, 2008; 93(2): 193 - 200.
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