Co-Expression of CD38 with Zap-70 Is Predictive of Treatment Intervention in Patients (pts) with Early Stage Chronic Lymphocytic Leukemia (CLL).

Abstract Introduction: CLL is a heterogeneous disease where advanced stage pts have compromised survival despite treatment, while early stage pts may not require any intervention. Based on current NCI-WG guidelines, most early stage (Rai stage 0 and 1) CLL pts do not require treatment until the development of progressive or symptomatic disease, though eventually over 70% of CLL pts will receive therapy. Currently, there are no markers to predict this subset of pts. Several markers of adverse prognosis, including ZAP-70+, CD38+, un-mutated IgVH gene and cytogenetic aberrations (17p-, 11q-, +12) have been identified. These markers are primarily studied in context of aggressive clinical course and survival outcome. The value of these markers to predict the necessity of treatment intervention in early stage CLL pts has not yet been completely evaluated. We investigated the expression pattern of Zap-70 and CD38 to examine their predictive value in identifying early stage CLL pts who will require therapeutic intervention. Methods & Results: 93 CLL pts were evaluated since 2002 at our institution. Flow cytometry was used to determine Zap-70 and CD38 expression on CD19+ CLL cells obtained from peripheral blood. Pts were considered to be positive for Zap-70 and CD38 expression if ≥ 20% and ≥ 30% of the cell stained for these proteins, respectively. For the Zap-70 analysis we used similar methodology as described by Crespo et al.1 Thirty-six (19M, 17F) pts had limited stage CLL, based on Rai staging criterion. Median age was 65 years (range 43–86) with stage 0 or 1 observed in 14 and 22 pts, respectively. Median time from diagnosis is 2 years (range <1–17). Increased expression of either Zap-70 or CD38 was seen in 22 and 7 pts, respectively. Five (14%) pts were positive for both. All (100%) pts with concurrent increased expression of Zap-70 and CD38 required treatment (p=0.003), as compared to 30% of patients requiring treatment that were negative for both proteins or positive for only protein. Among the pts that required treatment 4 had 13q- and one had trisomy 12 on cytogenetic analysis. Conclusion: Our study, demonstrates for the first time, the clinical utility of CD38 and Zap-70 co-expression in determining the probability of treatment intervention in early stage CLL pts. Although the number of pts studied is small, our findings highlight a potentially important use of these markers in the management of early-stage CLL pts. These observations warrant validation in a larger cohort of pts early stage CLL pts as well as correlation with other prognostic markers such as cytogenetic and IgVH gene mutational status.

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Revisiting Hypogammaglobulinemia in Chronic Lymphocytic Leukemia: A Combined Clinicobiological Approach

Blood ◽

10.1182/blood.v124.21.5633.5633 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5633-5633 ◽

Cited By ~ 3

Author(s):

Panagiotis Baliakas ◽

Aliki Xochelli ◽

Eva Minga ◽

Anastasia Hadzidimitriou ◽

Vassiliki Douka ◽

...

Keyword(s):

Clinical Stage ◽

Normal Serum ◽

Lymphocytic Leukemia ◽

Cd38 Expression ◽

Serum Immunoglobulins ◽

Cytogenetic Aberrations ◽

Mutational Status ◽

Ighv Gene ◽

Immunoglobulin Subclasses ◽

Notch1 Mutations

Abstract Chronic lymphocytic leukemia (CLL) is characterized by progressive hypogammaglobulinemia that can affect one or more immunoglobulin subclasses. Although many underlying mechanisms have been suggested, the pathogenesis of this phenomenon remains to be elucidated. In the present study, we revisit hypogammaglobulinemia in CLL through a combined clinicobiological approach aiming at identifying associations with particular disease profiles that would offer pathogenetic insight and guidance for further research. The study group included 412 CLL patients with available information about serum immunoglobulins either at diagnosis (n=380) or before treatment initiation (n=32). Patient characteristics were as follows: median age: 65 years; males/females: 266/146; Binet stage A: 272/335, unmutated IGHV genes (U-CLL): 140/412 cases (34%); CD38 expression: 59/330 cases (18%); clonotypic IG of the MD or G isotype: 250 and 43 cases, respectively; isolated del(13q): 64/136 (47%); trisomy 12: 18/183 (10%); del(11q): 18/186 (10%); del(17p): 11/189 (6%); NOTCH1 del7544-45/p.P2514Rfs*4: 8/219 (4%). With a median follow up of 5 years, 152/329 cases (46%) received treatment. Decreased immunoglobulin serum levels in at least one subclass were identified in 220/412 patients (53%), as follows: (i) decreased IgM, 172/412 cases (41%); (ii) decreased IgG, 78/412 cases (19%); (iii) decreased IgA, 100/412 cases (24%). In 36/412 cases (9%), a decrease in all serum immunoglobulin subclasses was noted. No statistically significant differences were identified between patients with normal serum immunoglobulin levels versus those with hypogammaglobulinemia regarding age, gender, disease burden at diagnosis, IGHV gene mutational status, CD38 expression, cytogenetic aberrations, NOTCH1 mutations and the incidence of a second malignancy. However patients with hypogammaglobulinemia exhibited increased need for treatment compared to patients with normal serum immunoglobulins (91/175 vs 61/154 respectively, p=0.025). Among cases with hypogammaglobulinemia, 90 (41%) and 26 (12%) exhibited isolated IgM and IgA subclass deficiency, respectively; isolated IgG decrease, was relatively rare (10/220 cases, 4%). Interestingly, when comparing isolated IgA versus other subclass deficiencies, statistically significant associations were identified with (i) advanced clinical stage (Binet B/C, Rai III/IV) (p=0.002); (ii) female gender (p=0.041); and, (iii) NOTCH1 mutations (p=0.004). A propos of the latter, it is noteworthy that in 5/8 (63%) mutant NOTCH1 cases with hypogammaglobulinemia, the affected subclass was IgA. Within our cohort, we identified cases belonging to one of three different, well characterized subsets with stereotyped B-cell receptor immunoglobulin (BcR IG), namely: (1) subset #1 (clan I IGHV genes/IGKV1(D)-39): U-CLL, clinically aggressive, n=12; (2) subset #2 (IGHV3-21/IGLV3-21), mixed IGHV mutational status, noted clinical aggressiveness, n=5; and, (3) subset #4, mutated IGHV4-34/IGKV2-30 BcR IG, clinically indolent, n=12. Notably, all subset #2 cases showed low levels of at least one serum subclass, while in 4/5 and 3/5 cases, two or all three immunoglobulin subclasses were affected. Although numbers are small, the incidence of hypogammaglobulinemia in subset #2 was significantly (p<0.05) higher compared to either subset #1 or subset #4). Univariate analysis revealed clinical stage, CD38 expression and IGHV mutational status as statistically important parameters (p<0.05) for both time-to-first–treatment (TTFT) and overall survival (OS); in contrast, hypogammaglobulinemia had no impact either on on TTFT or OS. In multivariate analysis, clinical stage and IGHV gene mutational status retained independent significance. In conclusion, abnormalities of serum immunoglobulins are detected in CLL patients with heterogeneous clinicobiological profiles, including different disease burden (clinical stage), cytogenetic aberrations and IGHV gene mutational status. However, certain observations reported herein, in particular the high incidence of hypogammaglobulinemia in subset #2 and the association of NOTCH1 mutations with IgA subclass deficiency, are noteworthy and indicate the need for research towards unraveling causal mechanisms among the observed interwined events. Disclosures No relevant conflicts of interest to declare.

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Cytogenetic Studies of 539 Chronic Lymphocytic Leukemia (CLL) Patients.

Blood ◽

10.1182/blood.v106.11.1192.1192 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1192-1192

Author(s):

Nyla A. Heerema ◽

Gerard Lozanski ◽

Thomas S. Lin ◽

Molly Moran ◽

Michael R. Grever ◽

...

Keyword(s):

Chromosomal Instability ◽

Sex Chromosome ◽

Prognostic Significance ◽

Lymphocytic Leukemia ◽

Trisomy 18 ◽

Unique Type ◽

Symptomatic Disease ◽

Cytogenetic Aberrations ◽

Cytogenetic Studies ◽

Trisomy 12

Abstract Previous banded metaphase cytogenetic studies of CLL identified deletions of 13q, 11q, 17p and 6q and trisomy 12 as recurring aberrations; all except del(6q) have well-recognized prognostic significance. The association of other cytogenetic abnormalities with these recurring aberrations has not been previously described. To more completely characterize our patients with CLL, we performed banded metaphase cytogenetics and fluorescence in situ hybridization (FISH) on 539 patients with previously untreated as well as relapsed CLL. By banded metaphase analysis 236 cases were abnormal, 282 were normal (≥20 metaphases and no abnormal clone identified) and 21 were culture failures. Of the 236 abnormal cases, 30 had a sole numerical sex chromosome abnormality, which may not represent the malignant clone. 89 cases had complex karyotypes (≥ 3 unrelated abnormalities), and 147 had simple abnormalities. Losses (451 total, 116 whole chromosome, 47 of which were sex chromosomes, and 335 partial losses) were much more common than gains (132 total, 110 whole chromosome, 68 +12, 13 +X or Y, only 29 other whole chromosome gains, and 22 partial chromosome gains). 130 balanced rearrangements occurred; most frequently involving chromosomes 14 and 1 (15 and 14 balanced rearrangements, respectively). As previously reported, +12, del(13q), del(11q), del(17p) and del(6q) occurred frequently (34.3%, 14.4%, 16.5%, 22.9%, and 10.2% of cases, respectively). Other frequent losses involved 14q, 9p, 3p and 18p (8.5%, 6.8%, 6.4% and 5.9% of cases, respectively). Partial chromosome losses usually resulted from apparent unbalanced translocations, suggesting frequent non-reciprocal interchromosomal rearrangements that may represent a unique form of chromosomal instability. We recently have begun examining the clinical significance of secondary abnormalities occurring with common aberrations in CLL. Of interest, all patients with t(14;18)(q32;q21) and 7 of 10 patients with trisomy 18 also had trisomy 12. All 4 patients with t(14;18) CLL had atypical immunophenotypes with only one developing symptomatic disease requiring therapy. A similar atypical immunophentype was found in 5 of 7 pts with co-existent trisomy 12 and 18, and only one of these patients has progressed to require treatment. In contrast, 36 of the remaining 57 pts with trisomy 12 have developed progressive disease requiring therapy. Overall, our studies show that multiple chromosomal aberrations in addition to those commonly reported occur in CLL. Chromosomal losses are more common than gains, and unbalanced rearrangements are more frequent than balanced rearrangements. The unbalanced rearrangements are frequently interchromosomal and may indicate a unique type of chromosomal instability. Unlike the other common cytogenetic aberrations in CLL, trisomy 12 appears to be associated with secondary aberrations including t(14;18) and trisomy 18 that may define a different more favorable clinical pathologic history than observed in trisomy 12 patients without these secondary aberrations.

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Insulin Growth Factor 1 Receptor Expression Is Associated with NOTCH1 Mutation, Trisomy 12 and Aggressive Clinical Course in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v124.21.3314.3314 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3314-3314

Author(s):

Francesco Maura ◽

Laura Mosca ◽

Fabris Sonia ◽

Giovanna Cutrona ◽

Serena Matis ◽

...

Keyword(s):

Gene Expression ◽

Strong Association ◽

Lymphocytic Leukemia ◽

Wild Type ◽

Insulin Growth Factor ◽

Cd38 Expression ◽

Mutational Status ◽

Hematological Cancers ◽

Trisomy 12 ◽

Igf1r Expression

Abstract Insulin growth factor 1 receptor (IGF1R) is emerging as an important gene in many solid and hematological cancers and its over expression has been reported to be associated with aggressive disease and pharmacologic resistance. Specifically, the IGF1R-IGF1-2 interaction was recently described to be involved in the constitutive activation of many important cell signaling such as NOTCH1 and PI3K/Akt pathways that play a key role in many solid and hematological cancers. In this study we performed a clinical and biological investigations about the role of IGF1R expression in a large and representative prospective series of chronic lymphocytic leukemia (CLL) in Binet A clinical stage enrolled in observation O-CLL1 protocol (clinicaltrial.gov identifier NCT00917540). Total RNA extraction, preparation of DNA single-stranded sense target, and hybridization to gene expression profiling arrays were carried out according to manufacturer’s protocols in 217 CLL patients enrolled in the multicentre O-CLL1 protocol. Gene expression data has been deposited in the National Centre for Biotechnology Information’s Gene Expression Omnibus database http://www.ncbi.nlm.mih.gov/geo and are accessible through series accession number GSE51529. High IGF1R expression was significantly associated with IGHV unmutated (IGHV-UM) status (p<0.0001), high ZAP-70 and CD38 expression (p<0.0001) and unfavorable cytogenetic deletion [i.e. del(11)(q23) and del(17)(p13)], particularly with del(11)(q23) (p=0.03). Cases with del(13)(q14) as single lesion were characterized by the lowest IGF1R gene levels. On the contrary, among the most common cytogenetic aberrations, trisomy 12 showed stronger IGF1R expression compared with the other patients (p<0.0001) and this association was independent from IGHV mutational status. Patients with stereotyped HCDR3 sequences showed a greater IGF1R expression compared to not stereotyped HCDR3 (p=0.001) even if this can be related to the high frequency of IGHV-UM among stereotyped HCDR patients. Interestingly, subset #4 patients, who are known to exhibit an indolent clinical course and distinct biological profile, were also characterized by lower IGF1R expression compared to other M-IGHV and UM-IGHV patients. NOTCH1 c.7541_7542delCT mutation was investigated by next generation sequencing Roche 454 technology in 199 (92%) patients (Lionetti et al, BJH 2014). Globally, median depth of coverage was 1510x, ranging from 605 to 2842. Mutant allele frequency estimated by NGS ranged from 0.02% to 75% of total reads per sample. The presence of NOTCH1 mutation was confirmed by ASO-PCR and Sanger sequencing in all patients with allele burden higher > 0.7% (31; 15.5%) and 7% (19; 9.5%) respectively. We considered as mutated only the 31 patients in whom the presence of the dinucleotide deletion was confirmed by ARMS-PCR. Patients carrying NOTCH1 mutation were characterized by a greater IGF1R expression compared with wild type cases (p=0.002). In addition high IGF1R expression was not significantly different comparing patients with low and high NOTCH1 mutation burden. In order to avoid the bias represented by the strong association between NOTCH1 and trisomy 12, we compared the IGF1R expression between NOTCH1 mutated and wild type cases excluding trisomy 12, and confirmed the previous association (p=0.004). IGF1R expression represented a strong clinical prognostic factor in our CLL cohort: by Kaplan-Maier analysis we observed a significant time to first treatment stratification in all CLLs, in all IGHV-UM and in all IGHV-M patients (p<0.0001). Furthermore, IGF1R retained its significance in multivariate analysis with most important clinical and molecular prognostic factors (CD38 expression, unfavorable FISH and IGHV mutational status). Overall, our study shows the importance of IGF1R expression in CLL and its strong association with specific clinical and biological features, confirming the interest for the study of this gene as a potential prognostic factor and its possible role as a therapeutic target in a specific group of CLL patients carrying trisomy 12 and NOTCH1 mutations. Disclosures No relevant conflicts of interest to declare.

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Biological Prognostic Markers in Chronic Lymphocytic Leukemia

Acta Medica (Hradec Kralove Czech Republic) ◽

10.14712/18059694.2016.99 ◽

2009 ◽

Vol 52 (1) ◽

pp. 3-8 ◽

Cited By ~ 8

Author(s):

Vladimíra Vroblová ◽

Lukáš Smolej ◽

Filip Vrbacký ◽

Karolína Jankovičová ◽

Monika Hrudková ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Clinical Course ◽

Variable Region ◽

Lymphocytic Leukemia ◽

Initial Assessment ◽

Western World ◽

Cd38 Expression ◽

Cytogenetic Aberrations ◽

Mutational Status

Chronic lymphocytic leukemia (CLL) is the most frequent leukemic disease of adults in the Western world. It is remarkable by an extraordinary heterogeneity of clinical course with overall survival ranging from several months to more than 15 years. Classical staging sytems by Rai and Binet, while readily available and useful for initial assessment of prognosis, are not able to determine individual patient’s ongoing clinical course of CLL at the time of diagnosis, especially in early stages. Therefore, newer biological prognostic parameters are currently being clinically evaluated. Mutational status of variable region of immunoglobulin heavy chain genes (IgVH), cytogenetic aberrations, and both intracellular ZAP- 70 and surface CD38 expression are recognized as parameters with established prognostic value. Molecules regulating the process of angiogenesis are also considered as promising markers. The purpose of this review is to summarize in detail the specific role of these prognostic factors in chronic lymphocytic leukemia.

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Flow Cytometric Characterization of Genetically Defined Subgroups of Chronic Lymphocytic Leukemia: Basis for the Comprehensive Definition of Prognostic Parameters.

Blood ◽

10.1182/blood.v104.11.4776.4776 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4776-4776

Author(s):

Wolfgang Kern ◽

Daniela Voskova ◽

Claudia Schoch ◽

Wolfgang Hiddemann ◽

Susanne Schnittger ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Lymphocytic Leukemia ◽

Prognostic Parameters ◽

Genetic Aberrations ◽

Expression Levels ◽

Cytogenetic Aberrations ◽

Trisomy 12 ◽

Normal Cytogenetics ◽

Lower Expression

Abstract Chronic lymphocytic leukemia (CLL) is defined based on cytomorphology and immunophenotype. The most important prognostic parameters are cytogenetic aberrations and the expression of CD38 and ZAP70. We analyzed correlations between immunophenotypic patterns and genetic aberrations in 153 peripheral blood samples from patients with newly diagnosed and untreated CLL. Immunophenotyping was performed applying five-fold staining, a comprehensive panel of antibodies, and CD45-SSC-gating. Genetic aberrations were detected by fluorescence in-situ hybridization using probes targeting ATM for the detection of 11q-, D12Z3 for trisomy 12, D13S319 and D13S25 for 13q-, and p53 for 17p-. As compared to cases without trisomy 12, in cases with trisomy 12 the expression levels were higher for CD22 (66% vs. 34%, p=0.001), CD20 (83% vs. 71%, p=0.034), and FMC7 (28% vs. 7%, p=0.000001). This is in accordance with our finding that higher numbers of prolymphocytes have been found in CLL with trisomy 12. As compared to cases with any cytogenetic aberration, cases with normal cytogenetics had lower expression levels of CD5 (82% vs. 89%, p=0.012) and CD19 (79% vs. 85%, p=0.051). Importantly, there were no significant differences in expression levels of CD38 and ZAP70 between cytogenetically defined subgroups of CLL. Thus, in cases with 11q-, trisomy 12, 13q-, homocygous 13q-, 17p-, and normal cytogenetics CD38 was expressed in 51%, 47%, 37%, 33%, 24%, and 38% and ZAP70 together with cyCD79a was expressed in 17%, 11%, 13%, 13%, 22%, and 15%. These data indicates that, with the exception of trisomy 12, the cytogenetic findings are not reflected by specific immunophenotypic features. In particular, this is true for the prognostically and clinically relevant markers CD38 and ZAP70 the expression of which cannot be deduced from cytogenetics. In a second step, correlations between the expression of CD38 and ZAP70 and other immunophenotypic markers have been analyzed. Cases positive for CD38 (more than 30% positive cells) had lower expression levels of CD19 (78% vs. 87%, p=0.005), CD20 (65% vs. 75%, p=0.023), and CD23 (62% vs. 74%, p=0.005). Cases positive for ZAP70 (more than 20% positive cells with coexpression of cyCD79a) had higher expression levels of CD19 (87% vs. 79%, p=0.011), CD22 (40% vs. 26%, p=0.020), and cyCD79a (75% vs. 59%, p=0.009). These findings are in line with both CD38 and ZAP70 being markers that reflect different biologic backgrounds of CLL which also reveal prognostic information. Taken together, these data strongly suggest that studies evaluating different treatment approaches in CLL should provide a detailed characterization of the analyzed patients including both cytogenetic aberrations and immunophenotypic findings as well as novel genetic markers in order to guarantee the detection of subgroup-specific treatment effects.

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Analysis of B-CLL with Discordant ZAP-70 Expression and IgVH Mutational Status.

Blood ◽

10.1182/blood.v106.11.1194.1194 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1194-1194 ◽

Cited By ~ 2

Author(s):

Remi Letestu ◽

Magali Le Garff-Tavernier ◽

Dominique Vaur ◽

Michel Ticchioni ◽

Fanny Baran-Marszak ◽

...

Keyword(s):

B Cells ◽

Somatic Mutations ◽

Staining Procedure ◽

Fish Analysis ◽

Prognostic Information ◽

Proliferation Markers ◽

Cd38 Expression ◽

Mutational Status ◽

Trisomy 12 ◽

Igvh Mutational Status

Abstract The clinical course of CLL is heterogeneous and presence or absence of IgVH somatic mutations has been correlated with stable and evolutive disease respectively. ZAP-70 protein expression was shown to be associated with unmutated IgVH genes in CLL and was proposed as a surrogate for mutational status. Recent studies of large number of cases have shown various percentage of discrepancy with respect to ZAP-70 expression and IgVH mutational status. As aggressive disease is not always associated with unmutated IgVH genes we aimed at better characterize the ZAP-70 discordant cases by determining the other prognostic factors such as cytogenetics, expression of CD38 and proliferation markers (thymidine kinase and sCD23). We investigated 292 patients with previously untreated B-CLL. Although several antiZAP-70 antibodies adapted to flow cytometry (FCM) are commercially available, staining procedure and interpretation of the results still remain controversial. Therefore, ZAP-70 expression was determined in the four participating centers by the same FCM method using the 2F3.2 mAb with indirect staining, and expression of the results was standardized. Moreover, ZAP-70 expression was investigated by RQ-PCR in 62 cases and by Western blot in 61 further cases on isolated B cells. The results obtained with these techniques were correlated with FCM data. ZAP-70 was found expressed in 141 cases, among which 38 cases (27%) exhibited ≥2% somatic mutations. Conversely, among the 151 ZAP-70 negative cases, only 6 cases (4%) were found unmutated (≥ 98% VH homology). We focused on the characteristics of the mutated ZAP-70 positive cases. Only 26/38 were in stage A at diagnosis. Incidence of CD38 expression >10% B cells was low (7/28 cases). Analysis of VH sequences pointed to the frequency of VH3-21 usage in 8/38 cases (21%) as compared to an expected 3% frequency in the French population. FISH analysis identified one case with del11q22.3, one case with del17p and two cases with trisomy 12. Del13q14 was present in half of cases. Proliferation markers were significantly higher in these cases than in ZAP-70 negative mutated cases, even among stage A patients. Follow-up of these patients is still too short for significant event free survival as compared with other groups of patients but the number of evolutive Binet stage A patients and advanced B and C stages was already higher than among the ZAP negative mutated cases. In conclusion, in our hands, ZAP-70 was almost always expressed in unmutated cases (103/109). Among mutated cases, ZAP-70 expression was present in 38/183 cases (21%), and brought prognostic information, independently of CD38 or chromosomal alterations. The correlation between ZAP-70 and proliferation markers suggests that ZAP-70 expression may result in a survival advantage of the malignant cells independently of the mutational status.

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Monoclonal B-Cell Lymphocytosis (MBL) Is Closely Related to Chronic Lymphocytic Leukemia (CLL) and May Be Better Classified as Early-Stage CLL.

Blood ◽

10.1182/blood.v114.22.2349.2349 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2349-2349

Author(s):

Wolfgang Kern ◽

Claudia Haferlach ◽

Frank Dicker ◽

Susanne Schnittger ◽

Torsten Haferlach

Keyword(s):

Multivariate Analysis ◽

Chronic Lymphocytic Leukemia ◽

Peripheral Blood ◽

Early Stage ◽

Normal Karyotype ◽

Lymphocytic Leukemia ◽

Cd38 Expression ◽

Equity Ownership ◽

Risk Parameters ◽

Good Risk

Abstract Abstract 2349 Poster Board II-326 Monoclonal B-cell lymphocytosis (MBL) is separated from chronic lymphocytic leukemia (CLL) mainly by the somewhat arbitrary cut-off of 5000/μl CLL-phenotype cells in peripheral blood. While MBL in general shows an indolent clinical course this is also true for early-stage CLL. This may call into question the adequateness of separating MBL from CLL. Therefore, we prospectively analyzed a series of 298 cases with MBL by immunophenotyping, fluorescence in situ hybridization (FISH; probes for detection of del(6q21), del(11q22.3) (ATM), +12, del(13q14) (D13S25, D13S319), del(17p13) (TP53), and t(11;14)(q13;q32) (IGH-CCND1)), chromosome banding analysis (CBA) and molecular genetics (analysis of IgVH mutation status) for parameters which are established as prognostically relevant in CLL. Data was compared to a previously published series of 356 cases with CLL (Cytometry B Clin Cytom 2009;Epub.). Male:female ratio was similar for MBL and CLL (2.1:1 vs. 1.8:1, n.s.) as was mean±SD age (66.5±10.5 vs. 65.7±10.2 years, n.s.). Mean±SD cells with CLL phenotype in peripheral blood amounted to 2,417±1,497/μl in MBL and to 27,771±39,607/μl in CLL (p<0.001). ZAP-70 expression (mean±SD MFI ratio T-cells:B-cells 4.3±3.0 vs. 5.1±3.3, p=0.011) and CD38 expression (mean±SD % positive cells 33.5±28.9 vs. 26.5±31.5, p=0.004) were stronger in MBL. FISH analysis revealed similar frequencies of del(11q22.3) (8.1% vs. 11.7%, n.s.). In contrast, +12 (22.8% vs. 13.7%, p=0.003) and t(11;14) (2.1% vs. 0.0%, p=0.008) were observed more frequently in MBL while del(6q21) (1.8% vs. 6.1%, p=0.008), del(13q14) (45.1% vs. 64.3%, p<0.001), del(13q14) as sole abnormality (35.0% vs. 47.7%, p=0.002), and del(17q13) (1.4% vs. 8.0%, p<0.001) were more frequent in CLL. CBA demonstrated a normal karyotype (31.5% vs. 21.6%, p=0.004) and trisomies (10.7% vs. 6.2%, p=0.045) more often in MBL while deletions were observed less often (30.5% vs. 39.3%, p=0.021). Analysis of IgVH revealed a mutated status more frequently in MBL (76.3% vs. 60.6%, p<0.001). Thus, while some good risk parameters have been encountered more frequently in MBL compared to CLL there was no clear predominance of all good risk parameters but rather a mixed distribution between MBL and CLL. We next analyzed the prognostic impact of the above parameters in cases with MBL. Time to therapy (TTT) was negatively affected by a higher CD38 expression (p=0.007), del(11q22.3) (p=0.01), the presence of independent clones as identified by CBA, and an unmutated IgVH status (p=0.001). Multivariate analysis revealed a higher CD38 expression (p=0.026) as the only independent parameter affecting TTT. Overall survival (OS) was negatively affect by del(6q21) (p=0.001) and by the presence of independent clones as identified by CBA (p=0.056) while a normal karyotype by CBA was associated with a better OS (p=0.031). When analyzing both MBL and CLL cohorts together, +12 (p=0.011) was found to be related to shorter TTT and del(13q14) as sole abnormality (p=0.038) and a higher ZAP-70 ratio T-cells:B-cells (p=0.007) were related to longer TTT. Neither the amount of cells with CLL phenotype in peripheral blood nor the presence of MBL were significantly related to TTT. The only parameters independently related to TTT were del(11q22.3) (p=0.007) and +12 (p=0.037). Parameters negatively affecting OS were MBL (p=0.006), the presence of independent clones as identified by CBA (p=0.038) and a female gender (p=0.047). Multivariate analysis demonstrated MBL (p=0.021) and the presence of independent clones as identified by CBA (p=0.046) as independently related to OS. The present data indicates that biologic characteristics of CLL are found in MBL and that there is no general predominance of good risk parameters in MBL as compared to CLL. Thus, MBL may not be considered a distinct disease but rather an early stage of CLL. This is further supported by the lack of impact of MBL as compared to CLL on the TTT. Furthermore, it is suggested that cases classified as MBL should undergo assessment of prognostic parameters including CBA as one prognostic parameter as shown for CLL cases. Disclosures: Kern: MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

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Vegf and bFGF Single-Nucleotide Polymorphisms In Chronic Lymphocytic Leukemia: Impact On Susceptibility

Blood ◽

10.1182/blood.v122.21.5287.5287 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5287-5287

Author(s):

Sandra Ballester ◽

Begoña Pineda ◽

Eduardo Tormo ◽

Blanca Navarro ◽

Ariadna Perez ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Conflicts Of Interest ◽

Expression Patterns ◽

Lymphocytic Leukemia ◽

P Value ◽

Nucleotide Polymorphisms ◽

Exact Test ◽

Cd38 Expression ◽

Mutational Status ◽

The Individual

Abstract Background B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease with a highly variable clinical outcome. Recent studies have identified a number of different molecular prognostic markers (including mutational status of the IgVH gene, ZAP70 and CD38 expression) that allow to discriminate patients in prognostic subgroups. However, different expression patterns of angiogenic factors as VEGF, VEGFR1 and bFGF have been related with B-CLL susceptibility and treatment requirements. We have analyzed the polymorphisms: -710 C/T in VEGFR1, rs1109324, rs1547651, rs3025039 (936C/T) and rs833052 in VEGF and rs1449683 (223 C/T) in bFGF in order to determine the possible association with susceptibility in B-CLL. Methods Peripheral blood samples from 230 B-CLL patients and 476 healthy controls were genotyped using probes TaqMan SNP Genotyping Assays. Samples were providing from the Hospital Clinic of Valencia. Four SNPs in the VEGF gene, one SNP in the bFGF gene and one SNP in the VEGFR1 gene were evaluated. Statistical analysis was performed using SNPStats program (Catalan Institute of Oncology) and Fisher's exact test was applied to evaluate the significance. Results We have observed an increased frequency of the T allele in the rs1449683 SNP [OR 1.62 (95% CI: 0.98-2.66) p-value =0.063] and in the rs1547651 SNP [OR 0.72 (95% CI: 0.51-1.03), p-value=0.072] in our B-LLC patients when compared to control subjects. Moreover we observed that T allele carriers of rs3025039 (VEGF) have a significant protective effect concerning this disease [OR 0.59 (95% CI: 0.39-0.89) p-value=0.009]. Conclusion Our data indicate an increased frequency of the T allele in polymorphisms rs1449683 (bFGF) and rs1547651 (VEGF) in the group of patients, which possibly account for the individual susceptibility to develop B-CLL. On the other hand the data provided suggest that the T allele of VEGF rs3025039 is likely important genetic marker of susceptibility to B-CLL. Further studies regarding the role of pro-angiogenic markers in B-CLL would be beneficial to help elucidate pathogenic pathways in this disease. Disclosures: No relevant conflicts of interest to declare.

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Lack of Prognostic Significance of the Conventional and Novel Prognostic Markers in Trisomy 12 Chronic Lymphocytic Leukemia (CLL)

Blood ◽

10.1182/blood.v128.22.4354.4354 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4354-4354

Author(s):

Valter Gattei ◽

Riccardo Bomben ◽

Michele Dal Bo ◽

Antonella Zucchetto ◽

Francesca Rossi ◽

...

Keyword(s):

Flow Cytometry ◽

Research Funding ◽

Prognostic Markers ◽

Lymphocytic Leukemia ◽

Cytogenetic Abnormality ◽

Mutational Status ◽

Ighv Gene ◽

Trisomy 12 ◽

Gene Status

Abstract Background. Trisomy 12 (tris12) is a recurrent cytogenetic abnormality in chronic lymphocytic leukemia (CLL), occurring in approximately 15-20% of cases, often as the unique cytogenetic alteration, that is usually considered a clonal driver lesion occurring early in CLL evolution. In the Dohner hierarchical categorization, tris12 CLL are identified as having an intermediate prognostic risk, although recent reports suggest a more complex and heterogeneous clinical behavior. Compared to CLL lacking this cytogenetic abnormality, tris12 CLL show more atypical morphology and immunophenotype, more frequent expression of the negative prognostic markers CD49d and CD38, and presence of NOTCH1 mutations and an unmutated (UM) IGHV gene status. The increased fraction of tris12 CLL carrying adverse prognostic features is in contrast to the intermediate clinical behavior associated with most tris12 CLL cases. Aim. To perform a comprehensive evaluation of the clinical impact of the major genetic, immunogenetic and immunophenotypic prognostic markers in tris12 CLL. Methods. The study was based on a multicenter series of tris12 CLL defined according to Dohner (n=283, including 73 cases also bearing del13q), and a comparison group (control) of 553 cases with either del13q (n=308) or without any cytogenetic abnormality (no del17p, del11q, tris12, del13q, n=245). Median follow-up of patients in the tris12 and control groups were 4 years (range 0-22) and 7 years (range 0-28), with 54% and 57% treated patients, and 18% and 15% deaths, respectively. Patient characterization included modified Rai stage, CD49d (CD49dhigh, ≥30% positive cells by flow cytometry), CD38 (CD38high, ≥30% positive cells by flow cytometry) and ZAP-70 (ZAP-70high, ≥20% positive cells by flow cytometry) expression, and IGHV mutational status (mutated, M, or UM according to the 2% cutoff). TP53, BIRC3, NOTCH1 andSF3B1 mutations were screened either at diagnosis or before therapy by NGS with at least 1000X coverage and 1% of sensitivity. Groups were compared by chi-square test; overall survival (OS) was computed from diagnosis to death or censored at last observation, and analyzed by Cox regression analysis. Results. Comparing the tris12 and the control groups, median age was 64 years (range 30-92) vs 66 years (range 33-92), male gender 55% vs 56% (p=0.86), the modified Rai stage was early in 52% vs 54%, intermediate in 41% vs 42% and advanced in 7% vs 4% (p=0.20). As previously reported, tris12 CLL were characterized by a higher prevalence of cases expressing CD49d (85% vs 31%) and CD38 (62% vs 17%; all p<0.0001), and of UM IGHV cases (55% vs 25%, p<0.0001). Analysis of recurrent mutations highlighted a higher prevalence of NOTCH1 mutations (26% vs 8%, p<0.0001) and of BIRC3 mutations (21% vs 1%, p<0.0001) in tris12 vs control group CLL. Conversely, no differences were found in the fraction of cases with TP53 mutations (3% vs 4%, p=0.38) or SF3B1 mutations (7% vs 7%, p=0.89), and in cases expressing ZAP-70 (62% vs 52%, p=0.09). The impact of these features on OS was tested by univariate analysis: in tris12 CLL, only the UM IGHV gene status predicted shorter OS (HR=2.37, p=0.0063), while none of the other characteristics reaching statistical significance as OS predictors (CD49d HR=1.36, p=0.36; CD38 HR=0.42, p=0.052; ZAP-70 HR=3.12, p=0.07; TP53 HR=2.33, p=0.25; NOTCH1 HR=1.40, p=0.22; SF3B1 HR=2.05, p=0.17; BIRC3 HR=1.22, p=0.61). On the other hand, in the control cohort, a significantly higher HR was found for CD49d (HR 3.11, p<0.0001) and CD38 (HR 3.45, p<0.0001) expression, TP53 (HR 2.88, p=0.0026), NOTCH1 (HR 3.57, p<0.0001), and SF3B1 (HR 2.57, p=0.0038) mutations, as well as for the UM IGHV gene status (HR=2.81, p<0.0001), but not for ZAP-70 expression and BIRC3 mutations (HR=1.74 and HR=1.91, p=0.15 and p=0.37, respectively). Conclusions. Mutational status of IGHV genes was the sole prognostic factor able to stratify OS in tris12 CLL. Despite the high frequency of NOTCH1 and BIRC3 mutations, as well as of CD49d and CD38 overexpression, these markers failed to convey a prognostic risk in tris12 CLL. The lack of a significant clinical impact for TP53 and SF3B1 mutations might be partly explained by the low number of mutated cases combined with a relative short follow up in our tris12 cohort. These findings are in keeping with the hypothesis of a different patho-biological mechanism occurring in tris12 CLL, which however remains to be fully elucidated. Disclosures D'Arena: Janssen-Cilag: Honoraria. Rossi:Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria. Gaidano:Janssen: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Shanafelt:Genentech: Research Funding; Janssen: Research Funding; Celgene: Research Funding; GlaxoSmithkKine: Research Funding; Pharmacyclics: Research Funding; Cephalon: Research Funding; Hospira: Research Funding.

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