The Amount of Spontaneous Apoptosis Is An Independent Strong Disease Progression Indicator in B-Cell Chronic Lymphocytic Leukemia (B-CLL)

Abstract Abstract 1252 Poster Board I-274 Bcl-2 levels has emerged as the most important protein in predicting survival between 11 proteins in CLL cells that are implicated in the control of apoptosis, proliferation and differentiation (Faderl, 2002). In fact, malignant cells are arrested in the G0/early G1 phase of the cell cycle, and inhibition of spontaneous apoptosis with upregulation of the anti-apoptotic protein bcl-2 may define clinical prognosis. The today availability both of bcl-2 antisense oligonucleotides and of novel pro-apoptotic BH3 peptidomimetic prompted us to evaluate the real impact of apoptosis pathways on B-CLL prognosis. The primary aims of our study were: 1) to determine progression-free survival (PFS) and overall survival (OS) upon bax/bcl-2 ratio, 2) whether bax/bcl-2 ratio and ZAP-70 show additive prognostic impact and finally 3) whether bax/bcl-2 is an independent prognostic factor. Therefore we investigated 356 patients (pts), median age 65 years (range 37-89), 164 males and 192 females. With regard to modified Rai stages, 115 pts had a low stage, 229 an intermediate stage and 12 a high stage. Bax/bcl-2 ratio was determined by flow cytometry, dividing mean fluorescence intensity (MFI) of bax by MFI of bcl-2 on CD19+CD5+ B-CLL cells. We obtained the bax/bcl-2 ratio and the threshold was set at the median value >1.45 (range 0.27-13.6). ZAP-70 was quantified by multicolor flow cytometry and the cut-off was fixed at >20%. Two hundred-three pts were bax/bcl-2 ratio positive (203/356; 57%). Higher bax/bcl-2 ratio was significantly associated with low Rai stage (80/115; P=0.003), lymphocyte doubling time >12 months (183/299; P=0.0003), beta-2 microglobulin (B-2M) <2.2 mg/dl (129/200; P=0.001) and soluble CD23 (sCD23) <70 U/ml (141/228; P=0.005). Moreover, there were significant correlations between higher bax/bcl-2 ratio and IgVH gene mutated status (216 cases, 96/154; P=0.015) or low risk (normal or 13q-) FISH cytogenetics (255 cases, 109/187; P=0.011). Noteworthy, a very strict association was found between higher bax/bcl-2 ratio and lower ZAP-70 (147/220; P<0.00001), suggesting that low ZAP-70 expression is characterized by high apoptosis levels. With regard to clinical outcome, significant shorter PFS and OS were observed in pts with lower bax/bcl-2 ratio (10% vs 60% at 14 years; P<0.00001 and 51% vs 74% at 16 years; P=0.005, respectively) as well as in ZAP-70+ pts (5% vs 57% at 12 years; P<0.00001 and 30% vs 85% at 16 years, respectively). To further explore the prognostic impact of bax/bcl-2 ratio, we investigated its expression associated with ZAP-70 protein. As a matter of fact, higher bax/bcl-2 ratio plus ZAP-70 <20% identified the pts subset with the longest PFS (70% vs 2% at 12 years; P <0.00001, Figure) and OS (92% vs 33%; P<0.00001). The discordant pts presented an intermediate outcome (Figure). In multivariate analysis of PFS, in which cytogenetics, IgVH status, ZAP-70, CD38, bax/bcl-2, sCD23 entered, bax/bcl-2 (P=0.02), cytogenetics (P=0.02) and ZAP-70 (P=0.04), resulted to be independent prognostic factors. In conclusion, our apoptotic index (bax/bcl-2 ratio), performed by flow cytometry, was very useful to identify pts at different progression rate and since the ZAP-70 negative subset represents a large and heterogeneous B-CLL population with a variable progression, other biological factors, such as the amount of apoptosis, have to be added in order both to identify early and treat timely progressive pts. Disclosures No relevant conflicts of interest to declare.

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Clinical Significance of Apoptosis in Chronic Lymphocytic Leukemia (CLL)

Blood ◽

10.1182/blood.v124.21.834.834 ◽

2014 ◽

Vol 124 (21) ◽

pp. 834-834

Author(s):

Giovanni Del Poeta ◽

Maria Ilaria Del Principe ◽

Michele Dal Bo ◽

Francesco Buccisano ◽

Dario Ragusa ◽

...

Keyword(s):

Flow Cytometry ◽

Prognostic Factor ◽

Conflicts Of Interest ◽

Intermediate Stage ◽

Independent Prognostic Factor ◽

Lymphocytic Leukemia ◽

Prognostic Impact ◽

Development Of Resistance ◽

Beta 2 Microglobulin ◽

Very High

Abstract Impaired programmed cell death is an important factor both in the pathogenesis of CLL (Berndt et al, Nat Genet 2013) and in the development of resistance to chemoimmunotherapy (Fegan et al, Adv Exp Med Biol 2013). It has become increasingly clear that in CLL the balance between the pro- (Bax) and anti-apoptotic members (Bcl-2) of the Bcl-2 family determines the chemotherapy sensitivity and ultimately progression free (PFS) and overall survival (OS). Moreover, the today availability in clinical use of novel potent oral pro-apoptotic BH3 peptidomimetics such as ABT 199 (Seymour et al, ASH 2013) emphasized the importance of Bcl-2 family-targeted therapy, prompting us to analyze the real impact of Bax/Bcl-2 ratio on CLL prognosis. The primary aims of our research were: 1) to correlate Bax/Bcl-2 ratio with other clinical and biological prognostic factors; 2) to determine PFS and OS upon Bax/Bcl-2 ratio; 3) to confirm Bax/Bcl-2 as an independent prognostic factor. Therefore we investigated 502 pts, median age 65 years (range 33-89), 279 males and 223 females. With regard to modified Rai stages at diagnosis, 170 patients had a low stage, 318 an intermediate stage and 14 a high stage. Bax/bcl-2 ratio was calculated by flow cytometry, dividing mean fluorescence intensity (MFI) of bax by MFI of bcl-2 on CD19+CD5+ CLL cells. The threshold was set at the median value >1.5 (range 0.27-6.10). Two hundred sixty- six patients were Bax/Bcl-2 ratio positive (266/502; 53%). Higher Bax/Bcl-2 ratio was significantly associated with low Rai stage, lymphocyte doubling time >12 months, beta-2 microglobulin <2.2 mg/dl and soluble CD23<70 U/ml (P<0.0001). Moreover, higher Bax/Bcl-2 was greatly represented within the low risk (normal or del13q) cytogenetics (221/341; P<0.0001). Noteworthy, significant correlations were found between lower Bax/Bcl-2 ratio and IGHV unmutated status (130/168; P<0.0001) or NOTCH1 (49/58; P<0.0001) or TP53 mutations (29/37; P=0.00007). With regard to clinical outcome, significant shorter PFS and OS were observed in patients with lower Bax/Bcl-2 ratio (10% vs 52% at 16 years; P<0.0001 and 46% vs 79% at 16 years; P<0.0001, respectively). To further explore the prognostic impact of Bax/Bcl-2 ratio, we investigated its expression within IGHV unmutated (168 pts) and TP53 mutated subgroups (37 pts), notoriously at worst prognosis. As a matter of fact, higher Bax/Bcl-2 ratio identified patients with a significant longer PFS (43% vs 10% and 50% vs 10% at 7 years; P=0.00002 and P=0.039, respectively, Figure), so suggesting its very high prognostic impact. In multivariate analysis of PFS (489 patients), Bax/Bcl-2 ratio (P<0.0001) together with modified Rai stages (P<0.0001), cytogenetics (P=0.0001), IGHV status (P<0.0001) and TP53 (P=0.001) was confirmed to be an independent prognostic factor. Therefore, the apoptotic index Bax/Bcl-2, performed by flow cytometry, is a powerful prognostic marker being able to identify patients at different prognosis also within IGHV unmutated and TP53 mutated subsets which are at very high risk. The modern strategies to downregulate Bcl-2 and shift the balance toward cellular demise, such as the BH3 mimetic ABT 199, could be carefully and precisely monitored by using this simple but powerful flow cytometric approach. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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High CD69 Protein Expression Predicts a Poor Prognosis in B-Cell Chronic Lymphocytic Leukemia (B-CLL).

Blood ◽

10.1182/blood.v110.11.750.750 ◽

2007 ◽

Vol 110 (11) ◽

pp. 750-750

Author(s):

Giovanni Del Poeta ◽

Maria Ilaria Del Principe ◽

Fabrizio Luciano ◽

Luca Maurillo ◽

Francesco Buccisano ◽

...

Keyword(s):

Flow Cytometry ◽

Overall Survival ◽

Progression Free Survival ◽

Intermediate Stage ◽

Lymphocytic Leukemia ◽

Lymphoid Cells ◽

Prognostic Impact ◽

Activation Markers ◽

Therapeutic Decisions ◽

Cd69 Expression

Abstract CD69 membrane protein is expressed early in the activation of lymphoid cells and is inducible by B-cells through cross-linking of surface immunoglobulins. Moreover, B-CLL cells exhibit features of activated and of antigen-experienced B lymphocytes overexpressing activation markers such as CD23, CD25, CD69 and CD71 (Damle, 2002). Furthermore, we have demonstrated that ZAP-70+ CLL subgroup shows a rapid disease progression and an inferior overall survival (Del Principe, 2006). The generation of antimurine CD69 monoclonal antibodies able to induce down-modulation or partial depletion of CD69+ cells (Sancho, 2006), prompted us to evaluate the real impact of CD69 expression on B-CLL prognosis. The primary aims of our study were: to determine progression-free survival (PFS) and overall survival (OS) upon CD69 expression; whether CD69 could predict varied outcome within ZAP-70+ and ZAP-70 negative subgroups; and finally whether CD69 was an independent prognostic factor. Therefore, we investigated 247 pts, median age 65 years, 131 males and 116 females. With regard to modified Rai stages, 69 pts had a low stage, 167 an intermediate stage and 11 a high stage. CD69 was determined by multicolor flow cytometry fixing a cut-off value of 30%. CD69+ B-CLL pts were 73/247 (30%). CD69 >30% was significantly associated with an intermediate/high Rai stage (p=0.001), lymphocyte doubling time (LDT) <12 months (p=0.00005) and beta-2 microglobulin >2.2 mg/dl (p=0.002). Lower CD69 expression and IgVH mutated status (>2%) were significantly correlated (73/92; p=0.0003). Furthermore, we found significant associations between lower CD69 and lower CD38 (137/184; p=0.02) or lower ZAP-70 (110/144; p=0.01). Lower levels of soluble CD23 (sCD23) were strongly associated with lower CD69 (127/158; p<0.00001). With regard to clinical outcome, both a significant shorter PFS (Figure) and OS were observed in CD69+ pts (5% vs 56% at 14 years; p<0.00001 and 44% vs 66% at 14 years; p=0.00001) as well as in ZAP-70+ pts (7% vs 62% at 12 years; p<0.00001 and 26% vs 90% at 14 years; p<0.00001). To further explore the prognostic impact of CD69, we investigated its expression within ZAP-70+ (103 pts) and ZAP-70 negative (144 pts) subsets. CD69+ pts showed a shorter PFS both within the ZAP-70+ subset (11% vs 27% at 8 years; p=0.004) and within the ZAP-70 negative subset (21% vs 79% at 12 years, p<0.00001). In multivariate analysis of PFS and OS, in which Rai modified stages, CD38, sCD23, LDT, CD69 and ZAP-70 entered, both ZAP-70 (p=0.0003 and p=0.0002) and CD69 (p=0.005 and p=0.0004) resulted to be independent prognostic factors. Therefore, CD69, determined by flow cytometry, could be considered as a new promising immunologic prognostic parameter in B-CLL. Furthermore, since the ZAP-70 negative subgroup consists of a heterogeneous population presenting variable outcome, CD69 might better stratify B-CLL subsets and early identify progressive pts in order to take timely therapeutic decisions. Figure Figure

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Apoptosis and Proliferation Synergistically Determine Overall Survival in Chronic Lymphocytic Leukemia (CLL)

Blood ◽

10.1182/blood.v126.23.1718.1718 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1718-1718

Author(s):

Giovanni Del Poeta ◽

Michele Dal Bo ◽

Francesco Buccisano ◽

Federico Pozzo ◽

Maria Ilaria Del Principe ◽

...

Keyword(s):

Flow Cytometry ◽

Overall Survival ◽

Prognostic Factors ◽

Conflicts Of Interest ◽

Treatment Strategies ◽

Progression Free Survival ◽

Intermediate Stage ◽

Lymphocytic Leukemia ◽

Clinical Effects

Abstract Differences in tumor proliferation and apoptosis levels explain the heterogeneous clinical course of CLL reflecting both genetic differences and the activity of external signals mainly through the B-cell receptor pathway (Herishanu et al, Blood, 2011; Rossi et al, Blood, 2013). Therefore high dynamic proliferation rate and impaired apoptosis represent crucial mechanisms both in the chemoresistance and in the progressive disease (Messmer et al, J Clin Invest 2005; Cervantes-Gomez et al, Clin Cancer Res, 2015). The today availability in clinical use both of ibrutinib, a potent Bruton tyrosine kinase (BTK) inhibitor, which blocks cell proliferation and trafficking (Cheng S et al, Leukemia, 2013; Burger et al, Blood, 2014) and venetoclax (ABT-199), a novel potent oral anti-bcl-2 peptidomimetic (Seymour et al, J Clin Oncol, 2014) as well as their possible synergistic combination (Portell et al, Blood, 2014), prompted us to analyze the real impact of the proliferative rate (Ki67 percentages) and the apoptosis (bax/bcl-2) on CLL prognosis. The primary aims of our research were: 1) to correlate Ki67 and bax/bcl-2 with clinical and biological prognostic factors; 2) to determine progression free survival (PFS) and overall survival (OS) upon Ki67 and bax/bcl-2; 3) to evaluate Ki67 and bax/bcl-2 as independent prognosticators. Therefore we investigated 606 patients, median age 66 years (range 33-89), 340 males and 266 females. With regard to modified Rai stages at diagnosis, 220 patients had a low stage, 369 an intermediate stage and 17 a high stage. Ki67 was evaluated by flow cytometry in terms of percentages of CD19+CD5+ CLL cells and the threshold of positivity was set at >5% (range 0.10-22.7). Also bax/bcl-2 was calculated by flow cytometry, dividing mean fluorescence intensity (MFI) of bax by MFI of bcl-2 on CLL cells. The threshold was set at the median value >1.5 (range 0.27-6.10). One hundred-ten patients were Ki67+ (18.2%) and 321 were bax/bcl-2+ (53%). Both higher Ki67 and lower bax/bcl-2 were significantly associated with intermediate/high Rai stage, beta-2 microglobulin (B2M)>2.2 mg/dl and soluble CD23>70 U/ml (P<0.0001). Moreover, higher Ki67 and lower bax/bcl-2 were greatly represented within the high risk (del11q or del17p) cytogenetics (P<0.0001). Noteworthy, strong correlations were found between higher Ki67 or lower bax/bcl-2 and IGHV unmutated status (P<0.0001) or NOTCH1 (P<0.0001 and P=0.00003) or TP53 mutations (P<0.0001 and P=0.00001). With regard to clinical outcome, significant shorter PFS and OS were observed in patients with higher Ki67 (2% vs 44% and 43% vs 83% at 12 years; P<0.0001) and lower bax/bcl-2 (13% vs 57% and 58% vs 91% at 12 years; P<0.0001). Noteworthy, bax/bcl-2 and Ki67 showed synergistic prognostic properties, since bax/bcl-2>1.5 plus Ki67<5% identified a CLL subset at best prognosis with regard to OS (94% vs 39% at 12 years; P<0.00001, Figure), so suggesting that apoptosis and proliferation may be key pathways for combined targeted therapies. The two discordant subsets (bax/bcl-2<1.5/Ki67<5% and bax/bcl-2>1.5/Ki67>5%) showed an intermediate outcome (67% and 63% at 12 years, respectively, Figure). In multivariate analysis of OS (593 patients), bax/bcl-2 ratio (P=0.004) and Ki67 (P=0.02) together with age (P=0.0002), B2M (P=0.005), cytogenetics (P=0.003), IGHV status (P=0.0005) and TP53 (P=0.0006) were confirmed as independent prognostic factors. Therefore, both the proliferative marker Ki67 and the apoptotic index bax/bcl-2, performed by flow cytometry, are powerful prognosticators showing synergistic clinical effects. Experiments of apoptosis, proliferation and trafficking testing ABT-199 and ibrutinib on CLL cells in vitro are in progress at our Institutions (Gattei et al, unpublished data). In conclusion, the modern treatment strategies in CLL should aim to block proliferation and trigger apoptosis concurrently (i.e. ibrutinib and venetoclax) in order to achieve longer overall survival. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Spontaneous Apoptosis and Proliferation Predict Disease Progression within ZAP-70 Negative B-Cell Chronic Lymphocytic Leukemia (B-CLL).

Blood ◽

10.1182/blood.v106.11.1197.1197 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1197-1197

Author(s):

Giovanni Del Poeta ◽

Maria Ilaria Del Principe ◽

Pasquale Niscola ◽

Luca Maurillo ◽

Adriano Venditti ◽

...

Keyword(s):

Disease Progression ◽

Proteasome Inhibitors ◽

Progression Free Survival ◽

Lymphocytic Leukemia ◽

Prognostic Impact ◽

Spontaneous Apoptosis ◽

Proliferating Cells ◽

Clinical Prognosis ◽

Therapeutic Decisions ◽

The Impact

Abstract Inhibition of spontaneous apoptosis and upregulation of the anti-apoptotic protein bcl-2 define clinical prognosis in B-CLL. However, increasing evidence exists that disease progression (DP) relies upon a proliferating pool of cells in lymph nodes and bone marrow which might feed the accumulating pool in the blood. Moreover, unmutated immunoglobulin VH genes predict for a rapid DP and an inferior overall survival. Unmutated B-CLL cells express ZAP-70 protein kinase associated with an early DP risk. The availability both of rapamycin or proteasome inhibitors effective against proliferating cells and bcl-2 targeting drugs incited us to evaluate the impact of proliferation and apoptosis pathways on B-CLL prognosis. The primary aims of our study were: 1) to determine progression-free survival (PFS) upon apoptosis/proliferation and ZAP-70 expression; 2) whether apoptosis/proliferation could predict varied outcome within ZAP-70 subgroups; and finally 3) whether ZAP-70 and apoptosis/proliferation were independent prognostic factors. Therefore, we investigated 249 pts, median age 64 years, almost all belonging to the intermediate Rai stage. ZAP-70 was quantified by a multicolor flow cytometric method. Bcl-2 (Bcl-2B/T) was determined dividing mean fluorescence intensity (MFI) of CD19+B-CLL cells by MFI of T-cells. CD71 was used as a measure of proliferation in percentage. Combining Bcl-2B/T with CD71 (Bcl2CD71) we enucleated three subgroups: 1) Bcl2CD71- [low proliferation (CD71<8%) and high apoptosis (Bcl-2B/T<1.6)]; 2) Bcl2CD71+ [high proliferation (CD71>8%) and low apoptosis (Bcl-2B/T>1.6)]; and 3) Bcl2CD71+/− [low proliferation/low apoptosis or high proliferation/high apoptosis]. ZAP-70+ B-CLL pts were 87/249 (35%). We found significant associations either between lower ZAP-70 and lower Bcl-2B/T (p=0.0008) or lower ZAP-70 and Bcl2CD71- (p=0.0007), showing that low ZAP-70 levels were characterized by high apoptosis and low proliferation. With regard to clinical outcome, a significant shorter PFS was observed in ZAP-70+ pts (6% vs 58% at 12 years; p<0.00001) and in Bcl2CD71+ pts (10% vs 56% at 12 years; p<0.00001). The Bcl2CD71+/− subgroup showed an intermediate outcome (30% at 12 years). To further explore the prognostic impact of Bcl2CD71 index, we investigated its expression within ZAP-70+ (87) and ZAP-70 negative (162) pts. As a matter of fact, Bcl2CD71 index was not able to identify different prognosic subsets within ZAP-70+ pts, because all these cases presented a similar short PFS. On the contrary, Bcl2CD71 clearly identified two subsets at different PFS within the ZAP-70 negative subgroup (73% for Bcl2CD71- pts vs 29% for Bcl2CD71+ pts at 12 years, p=0.00007). In multivariate analysis of PFS, ZAP-70 resulted to be the strongest indipendent prognostic factor (p=0.00002). However, the apoptotic/proliferative index Bcl2CD71 was very useful to identify our pts at different PFS within the ZAP-70 negative subgroup. In conclusion, ZAP-70 negative B-CLL represents a large and clinically heterogeneous population with a variable DP and the determination of the amount of apoptosis combined with the proliferation, could allow us both to distinguish early progressive pts and to take timely therapeutic decisions.

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Clinical Significance of CD69 Expression In Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v116.21.3574.3574 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3574-3574

Author(s):

Giovanni Del Poeta ◽

Maria Ilaria Del Principe ◽

Pietro Bulian ◽

Cristina Simotti ◽

Francesco Buccisano ◽

...

Keyword(s):

Flow Cytometry ◽

Prognostic Factors ◽

Chronic Lymphocytic Leukemia ◽

Clinical Outcome ◽

Doubling Time ◽

Lymphocytic Leukemia ◽

Prognostic Impact ◽

Fish Cytogenetics ◽

Beta 2 ◽

Beta 2 Microglobulin

Abstract Abstract 3574 Chronic lymphocytic leukemia (B-CLL) is a very heterogeneous disease with some patients experiencing rapid disease progression and others living for years without requiring treatment and therefore it is mandatory to find new prognostic markers. CD69 overexpression which resembles B cells at an earlier and greater state of activation (Damle, 2002 and 2007) and induces increased proliferation and survival of leukemic B-lymphocytes, may reflect an aggressive and progressive clinical outcome. The primary endpoints of our research were: 1) to determine progression free survival (PFS) and overall survival (OS) upon CD69 in univariate analysis; 2) to correlate CD69 with other clinical or biological prognostic factors such as age, Rai stages, lymphocyte doubling time, beta-2 microglobulin, CD38, CD49d, ZAP-70, cytogenetics by FISH and IgVH status and finally, 3) to confirm CD69 as an independent prognostic factor. We investigated 417 patients (pts), median age 66 years (range 33–89), 239 males and 178 females. With regard to modified Rai stages, 127 pts had a low stage, 272 an intermediate stage and 18 a high stage. CD69 was determined by multicolor flow cytometry, fixing the cut-off value at 30%. CD69+ pts were 111/417 (26.6%). CD69 <30% was significantly associated with low Rai stage (111/127; P<0.0001), lymphocyte doubling time >12 months (260/337; P=0.0006), beta-2 microglobulin <2.2 mg/dl (176/218; P=0.0005) and soluble CD23 <70 U/ml (199/245; P<0.0001). Significant associations were found between CD69 <30% and ZAP-70 <20% (189/243; P=0.01) or CD49d <30% (135/171; P=0.007). There were significant correlations between CD69 <30% and IgVH mutated status (323 total cases, 169/211; P=0.001). On the other hand, no significant correlation was found with FISH cytogenetics (337 studied cases). With regard to clinical outcome, interestingly, 79 (71%) of 111 of the CD69+ patients had received chemotherapy at the time of analysis (P<0.00001). Moreover, both shorter PFS and OS were observed in CD69+ patients (5% vs 40% at 16 years, P<0.0001 and 26% vs 76% at 20 years, P<0.0001). To further explore the prognostic impact of CD69, we investigated its expression within unmutated (112 pts) and mutated (211 pts) IgVH subsets. As a matter of fact, pts with CD69 <30% showed longer PFS and OS both within the unmutated subgroup (32% vs 10% at 5 years, P=0.01 [Figure] and 77% vs 38% at 12 years, P=0.04) and within the mutated subgroup (56% vs 22% at 12 years, P=0.0006 [Figure] and 94% vs 70% at 16 years, P=0.05). In multivariate analysis of PFS, FISH cytogenetics (P=0.00005), ZAP-70 (P=0.0001), CD69 (P=0.002), Rai stages (P=0.001) and IgVH status (P=0.004) were independent prognostic factors. With regard to OS, age > or <60 years (P=0.001), CD69 (P=0.004), ZAP-70 (P=0.01) and CD38 (P=0.03) were identified as significant. Noteworthy, here, we demonstrated that CD69 is able to improve the historical prognostic ability of the IgVH mutational status. Since the IgVH mutated subset represents a large and heterogeneous population with a variable progression, CD69 may better define prognosis within this subgroup. Therefore, CD69, determined by flow cytometry, should be considered a novel important prognostic parameter in B-CLL and has to be necessarily added in a new scoring prognostic system. In fact, its easy and rapid laboratory determination allows us to identify early progressive pts in order to take timely therapeutic decisions. Disclosures: No relevant conflicts of interest to declare.

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Genomic Aberrations Dramatically Improve The Strong Prognostic Impact Of IGHV Mutational Status In Chronic Lymphocytic Leukemia (CLL)

Blood ◽

10.1182/blood.v122.21.1370.1370 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1370-1370

Author(s):

Giovanni Del Poeta ◽

Dario Ragusa ◽

Francesco Buccisano ◽

Michele Dal Bo ◽

Luca Maurillo ◽

...

Keyword(s):

Prognostic Factors ◽

Sanger Sequencing ◽

Conflicts Of Interest ◽

Direct Sequencing ◽

Univariate Analysis ◽

Intermediate Stage ◽

Lymphocytic Leukemia ◽

Prognostic Impact ◽

Mutational Status ◽

Genomic Aberrations

Abstract CLL is a heterogeneous disease with patients (pts) experiencing rapid disease progression and others living for years without requiring treatment. Recently, next generation sequencing has revealed new molecular alterations, targeting the NOTCH1 and BIRC3 genes which occur in about 10% CLL at diagnosis and correlate with poor outcome. Given the possibility of targeting NOTCH1 and BIRC3 with drugs currently under development, the primary endpoints of our research were: 1) to determine overall survival (OS) upon IGHV, NOTCH1, TP53 and BIRC3 in univariate analysis; 2) to correlate these genomic aberrations with other biological or clinical prognostic factors, and finally 3) to confirm NOTCH1, BIRC3 and TP53 as independent prognostic factors. We investigated 475 pts with a median age of 65 years (range 33-89), whose 160 had low Rai stage, 301 intermediate stage and 14 high stage. NOTCH1 mutations (mut) were studied by ARMS PCR for c.7544-7545delCT and by Sanger sequencing of NOTCH1 exon 34. Mutations of TP53 were analysed by DNA direct sequencing, while BIRC3 disruption (disr) was studied by Sanger sequencing for mutations and by interphase FISH for deletions. All these alterations were studied at diagnosis or before any chemotherapeutic approach. NOTCH1mut and TP53mut pts were 52 (10.9%) and 36/475 (7.6%), respectively. Thirty four patients were BIRC3mut (7.2%) and 26 BIRC3 deleted (5.5%) for a total of 46 cases (9.7%) BIRC3disr. NOTCH1, TP53 and BIRC3 alterations were mutually exclusive. There were significant correlations between NOTCH1 (P<0.00001), TP53 (P=0.004), BIRC3 status (P=0.00004) and IGHV mutations. Concerning FISH cytogenetics (460 patients), a significant correlation (P<0.0001) was found between NOTCH1mut and trisomy 12 (20/62; 32%). TP53mut were strictly associated with del17p (15/25; 60%; P<0.0001), while BIRC3disr was found mainly within 11q22-q23 deletions subset (22/46;49%; P<0.0001). With regard to clinical outcome, 30 (83%) of 36 TP53mut pts (P=0.00009), 47 (90%) of 52 NOTCH1mut (P<0.00001) and 40 (87%) of 46 BIRC3disr pts had received chemotherapy at the time of analysis. Twenty nine NOTCH1mut (56%), 15 TP53mut (42%) and 18 BIRC3disr (39%) pts underwent at least two lines of treatment (P<0.0001). Noteworthy, shorter OS was observed in IGHV unmutated (UM) patients (12% vs 80% at 18 years, P<0.00001), in NOTCH1mut pts (12% vs 71% at 16 years, P<0.00001), in TP53mut pts (9% vs 76% at 14 years, P<0.00001) and in BIRC3disr pts (29% vs 65% at 16 years, P=0.00001). To further explore the prognostic impact of NOTCH1mut, TP53mut and BIRC3disr, we investigated them within the UM (153 pts) IGHV subset, notoriously at worst prognosis. As a matter of fact, NOTCH1mut (16% vs 45% at 14 years, P=0.012), TP53mut (0% vs 43% at 13 years, P=0.002) and BIRC3disr (0% vs 57% at 11 years, P=0.011) pts showed significant shorter OS [Figure]. Within the mutated IGHV subgroup we obtained similar results. In multivariate analysis of OS, TP53mut (HR 5.2, P<0.00001), age >60 years (HR 3.8, P=0.00002), IGHV UM status (HR 0.30, P=0.0001), intermediate/high Rai stages (HR 2.8, P=0.0002), NOTCH1mut (HR 2.6, P=0.001), and BIRC3disr (HR 2.5, P=0.005) were confirmed to be independent adverse prognostic factors. Noteworthy, here, we demonstrated that genomic aberrations are able to improve the historical prognostic ability of the IgHV mutational status. In conclusion, genomic aberrations, particularly TP53mut, NOTCH1mut and BIRC3disr should be considered as novel important prognostic parameters in CLL and therefore they have to be necessarily considered in updated scoring prognostic systems. Disclosures: No relevant conflicts of interest to declare.

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High CD79b Expression Predicts a Poor Outcome in B-Cell Chronic Lymphocytic Leukemia (B-CLL).

Blood ◽

10.1182/blood.v112.11.1054.1054 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1054-1054

Author(s):

Giovanni Del Poeta ◽

Maria Ilaria Del Principe ◽

Antonella Zucchetto ◽

Francesco Buccisano ◽

Cristina Simotti ◽

...

Keyword(s):

Flow Cytometry ◽

Overall Survival ◽

B Cell ◽

Clinical Stage ◽

Critical Role ◽

Progression Free Survival ◽

Intermediate Stage ◽

Lymphocytic Leukemia ◽

Advanced Clinical Stage ◽

Variable Expression

Abstract Figure Figure The immunoglobulin (Ig) beta protein (CD79b) dimerizes with Ig alpha (CD79a) to form the signaling component of the B-cell antigen receptor complex (BCR). Given the critical role of Ig beta in BCR signaling, it has been suggested that its variable expression in CLL may induce either cell proliferation or apoptosis. High CD79b expression has been associated with atypical morphology, strong expression of surface Ig, advanced clinical stage and short overall survival in B-CLL (Garcia Vela, 1999; Zucchetto, 2006). Moreover, high CD38 expression was correlated with high CD40, CD69 and CD79b which are consistent with an activation phenotype (Damle, 2002). Furthermore, we have demonstrated that the ZAP-70+ CLL subgroup shows a rapid disease progression and an inferior overall survival (Del Principe, 2006). Since it has been described that BCR engagement has significant effects on B-CLL cell survival, activation and cell cycle progression (Deglesne, 2006), we decided to evaluate the real impact of CD79b expression on B-CLL prognosis. The primary aims of our research were: 1) to determine progression-free survival (PFS) and overall survival (OS) upon CD79b expression; 2) whether CD79b could predict varied outcome within ZAP-70+ and ZAP-70 negative subgroups; and finally 3) whether CD79b was an independent prognostic factor. Therefore, we investigated 401 patients (pts), median age 65 years (range 33–89), 213 males and 188 females. With regard to modified Rai stages, 123 pts had a low stage, 261 an intermediate stage and 17 a high stage. CD79b was determined by multicolor flow cytometry, using the monoclonal antibody anti-CD79 beta-FITC (clone SN8, Dako) and fixing a cut-off value of 30%. CD79b+ B-CLL pts were 207/401 (52%). CD79b>30% was significantly associated with the intermediate/high Rai stage (p=0.00001), beta-2 microglobulin >2.2 mg/dl (p<0.0001) and with multiple intrathoracic/abdominal lymphadenopathies and/or splenomegaly (p<0.0001). Low CD79b was significantly correlated either with IgVH mutated status (>2%) (86/105; p<0.0001) or lower soluble CD23 levels (125/161; p<0.00001). Significant associations were found either between CD38<30% and lower CD79b (172/194; p<0.0001) or ZAP- 70<20% and CD79b<30% (145/193; p<0.0001). With regard to cytogenetics, there were strict correlations either between high CD79b and trisomy 12 (25/33; p=0.001) or high CD79b and del17p (16/20; p=0.001). Median follow up duration from diagnosis was 68 months (range 6–230). Concerning clinical outcome, both a shorter PFS (Figure) and OS were observed in CD79b+ pts (12% vs 58% at 16 years; p<0.0001 and 49% vs 85% at 16 years; p<0.0001) as well as in ZAP-70+ pts (5% vs 52% at 11 years; p<0.0001 and 33% vs 87% at 18 years; p<0.0001). To further explore the prognostic value of CD79b, we investigated its expression within ZAP-70+ (159 pts) and ZAP-70 negative (242 pts) subsets. As a matter of fact, CD79b+ pts showed a shorter PFS and OS both within the ZAP-70+ subset (7% vs 19% at 10 years; p=0.00006 and 25% vs 65% at 16 years; p=0.006) and within the ZAP-70 negative subset (29% vs 67% at 14 years, p<0.00001 and 86% vs 100% at 14 years; p=0.01). In multivariate analysis of PFS and OS, in which age, Rai stages, CD38, CD69, CD79b and ZAP-70 entered, ZAP-70 (p=0.001 and p=0.001), CD69 (p=0.001 and p=0.01) and CD79b (p=0.0007 and p=0.03) resulted to be independent prognostic factors. Therefore CD79b, easily determined by flow cytometry, should be considered another important prognostic parameter and could be used to early identify and stratify B-CLL progressive pts.

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Clinical Significance of 13q14 Number of Deleted Cells in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v120.21.4581.4581 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4581-4581

Author(s):

Giovanni Del Poeta ◽

Francesca Maria Rossi ◽

Maria Ilaria Del Principe ◽

Michele Dal Bo ◽

Annalisa Biagi ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Conflicts Of Interest ◽

Normal Karyotype ◽

Progression Free Survival ◽

Intermediate Stage ◽

Lymphocytic Leukemia ◽

Prognostic Indicators ◽

Fish Analysis ◽

Clinical Difference ◽

Trisomy 12

Abstract Abstract 4581 Cytogenetic aberrations are considered major prognostic indicators for predicting the survival of chronic lymphocytic leukemia (CLL) patients (Dohner et al, 2000) and interphase fluorescent in situ hybridization (I-FISH) with specific probes is generally used to detect the most frequent abnormalities. Moreover, deletion 13q14.3 on FISH analysis which is the most common cytogenetic abnormality in CLL is a favorable prognostic biomarker when detected as a sole abnormality, even if a higher percentage of 13q- nuclei was found to be associated with significantly shorter time to treatment (P<0.001), (van Dike et al, 2010; Dal Bo et al, 2011). Therefore, the primary endpoints of our research were: 1) to determine progression free survival (PFS) and overall survival (OS) on the basis of percentages of 13q- nuclei and 2) to confirm 13q14 number of deleted cells as an independent prognostic factor. We investigated 503 pts, median age 65 years (range 33–89), 291 males and 212 females. With regard to modified Rai stages at diagnosis, 163 had a low stage, 320 an intermediate stage and 20 a high stage. Probes for chromosome 13q (LSI-D13S319), 11q (LSI-ATM), 17p (LSI-p53) and chromosome 12 (CEP12) were used on nuclei collected at diagnosis. One hundred fifty-three patients (30.4%) exhibit a normal karyotype, 203 pts (40.4%) showed an isolated 13q-, 63 pts (12.5%) presented trisomy 12, 49 pts (9.7%) 11q deletion, 26 (5.2%) 17p deletion and 9 (1.8%) other chromosome abnormalities. Clearly, patients with intermediate/poor cytogenetic abnormalities (trisomy 12, del11q-, del17p-) showed significant shorter PFS and OS (7% vs 36% at 14 years and 45% vs 77% at 14 years, P<0.0001) in comparison with normal or del13q- pts. Maximally selected log-rank statistics identified the 50% of nuclei bearing del13S319 as the most appropriate cut-off value capable of separating del13q- cases into two subgroups with different PFS and OS distributions. In fact, pts with isolated 13q- in <50% of nuclei (110 pts) showed a longer PFS and OS (62% vs 16% at 12 years, P<0.0001 and 95% vs 47% at 16 years, P=0.0007, Figure) compared to those with ≥50% of nuclei (93 pts). Noteworthy, 64 (69%) of 93 of 13q- >50% pts had received chemotherapy at the time of analysis, whereas only 27 (25%) of 110 of 13q- <50% pts had been treated (P <0.0001). There was no significant clinical difference between heterozygous and homozygous 13q- patients as well as between 13q- cases with RB1 deletion (delRB1) and 13q- without delRB1. There was a significant correlation between number of 13q deleted nuclei and number of B-lymphocytes/microliter (P<0.0001) at diagnosis as well as between 13q- nuclei percentages and lymphocyte doubling time (P=0.001), meaning that 13q- nuclei represent both the amount of disease and the proliferation rate in CLL. Only slight significant correlations were found between 13q- percentages and CD38 (P=0.04) or ZAP-70 (P=0.01) or IgHV status (P=0.03), whereas 36 of 54 (67%) CD69+ pts had del13q- >50% (P=0.0003). In the context of del13q- subset, multivariate analysis of PFS confirmed the percentage of nuclei (P=0.00007) together with IgHV status (P=0.003) and ZAP-70 (P=0.0002) as an independent prognosticator. With regard to OS, percentage of nuclei (P=0.02) together with age (P=0.009) and IgHV status (P=0.03) was again confirmed as an independent variable. Therefore, the percentage of nuclei exhibiting 13q- at diagnosis has to be considered an important predictor of the clinical outcome and the clinical implications of an isolated 13q deletion in CLL appear more complex and important than originally considerated. Disclosures: No relevant conflicts of interest to declare.

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CD180 Expression in B-Cell Lymphomas: A Multicenter Geil Study

Blood ◽

10.1182/blood.v124.21.1628.1628 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1628-1628

Author(s):

Caroline Mayeur-Rousse ◽

Julien Guy ◽

Laurent Miguet ◽

Sabrina Bouyer ◽

Franck Genevieve ◽

...

Keyword(s):

Flow Cytometry ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

University Hospital ◽

Routine Practice ◽

Similar Data ◽

B Cell Lymphomas ◽

Whitney Test ◽

Mann Whitney Test

Abstract CD180 is a Toll-Like Receptor homolog strongly expressed on normal human B-cells and involved in innate immune responses. Previous proteomic analyses on microparticles derived from mature B-cell neoplasms allowed us to identify CD180 as a marker of marginal zone lymphomas (MZL)(Miguet, Leukemia, 2013). Using flow cytometry on blood samples we showed that this protein is lost or underexpressed at the plasma membrane for almost all B-cell lymphomas except MZL. In order to confirm its clinical relevance, we conducted a prospective multicenter flow cytometry study in 5 French University Hospital laboratories, on behalf of the GEIL. Blood or bone marrow samples from 236 patients were studied (20 normal controls ; 74 chronic lymphocytic leukemia (CLL); 21 mantle cell lymphoma (MCL); 42 lymphoplasmacytic lymphoma (LPL); 13 follicular lymphoma (FL) ; 58 MZL, 14 of which with numerous villous lymphocytes; 8 hairy cell leukemia (HCL)). Analyses were performed either on FACSCanto II (BD Biosciences, 3 centers) or on Navios (Beckman Coulter, 2 centers) instruments. Harmonization process was performed using Rainbow beads (Spherotech). For the CLL group, CD180 Median fluorescence (MdFI) in each center was not significantly different (Anova test, p>0.05). Instruments’ harmonization was therefore effective enough to obtain similar data from all centres. In the whole cohort, CD180 was significantly less expressed in the group of lymphomas -including CLL, MCL, LPL and FL- than in controls (Mann-Whitney test, p<0.05). Conversely, in the group of MZL and HCL, CD180 MdFI was not different from those of controls (Mann-Whitney test, p>0.05) but significantly higher than in CLL, MCL, LPL and FL (Mann-Whitney test, p<0.0001). Distinction between MZL and lymphomas with numerous villous lymphocytes was possible (Mann-Whitney test, p=0.0012) but not between MZL and HCL. ROC curve analysis determined a CD180 MdFI threshold of 1800 which allow the positive diagnosis of MZL with a sensitivity of 77% and specificity of 92%. These results underline the efficiency of CD180 as a single positive and robust marker for MZL diagnosis, and confirm that between centers and between instruments harmonization is largely feasible in routine practice as published recently (Solly F et al. Cytomery part A, 2013). It should be emphasized that among the group of lymphomas with intense expression of CD180, all interestingly originating from the spleen, those with numerous villous lymphocytes display the highest expression. We described for the first time in this study the strong positivity of CD180 in HCL. Anti-CD180 antibody may be included in diagnosis combination markers in order to improve the diagnosis of chronic B-cell malignancies Disclosures No relevant conflicts of interest to declare.

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Interleukin-13 inhibits interleukin-2-induced proliferation and protects chronic lymphocytic leukemia B cells from in vitro apoptosis

Blood ◽

10.1182/blood.v87.3.1022.bloodjournal8731022 ◽

1996 ◽

Vol 87 (3) ◽

pp. 1022-1029 ◽

Cited By ~ 56

Author(s):

N Chaouchi ◽

C Wallon ◽

C Goujard ◽

G Tertian ◽

A Rudent ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Interleukin 2 ◽

Intermediate Stage ◽

Lymphocytic Leukemia ◽

Spontaneous Apoptosis ◽

Interleukin 13 ◽

B Cell Maturation ◽

Cd23 Expression

Human interleukin-13 (IL-13) acts at different stages of the normal B- cell maturation pathway with a spectrum of biologic activities overlapping those of IL-4. B chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of slow-dividing and long-lived monoclonal B cells, arrested at the intermediate stage of their differentiation. In vitro, B-CLL cells exhibit a spontaneous apoptosis regulated by different cytokines. In this report, we show that IL-13 (10 to 200 ng/mL) acts directly on monoclonal B-CLL cells from 12 patients. (1) IL-13 enhances CD23 expression and induces soluble CD23 secretion by B-CLL cells but does not exhibit a growth factor activity. (2) IL-13 inhibits IL-2 responsiveness of B-CLL cells, activated either with IL-2 alone or through crosslinking of lgs or ligation of CD40 antigen. (3) IL-13 protects B-CLL cells from in vitro spontaneous apoptosis. The effects of IL-13 on neoplasic B cells were slightly less than those of IL-4 and occurred independently of the presence of IL-4. The present observations show that IL-13 may exhibit a negative regulatory effect on neoplasic B cells in contrast with that observed in normal B cells, and suggest that IL-13 could be an important factor in the pathogenesis of CLL by preventing the death of monoclonal B cells. Moreover, B-CLL may be an interesting model to study the regulation of the expression of IL-13 receptor and/or signal transduction pathways.

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