Revisiting Hypogammaglobulinemia in Chronic Lymphocytic Leukemia: A Combined Clinicobiological Approach

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

Blood ◽

10.1182/blood.v108.11.4945.4945 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4945-4945

Author(s):

Jean-Gabriel Coignet ◽

Swaminathan Padmanabhan ◽

Rami Manochakian ◽

Kena C. Miller ◽

Paul Wallace ◽

...

Keyword(s):

Early Stage ◽

Lymphocytic Leukemia ◽

Treatment Intervention ◽

Symptomatic Disease ◽

Cd38 Expression ◽

Cytogenetic Aberrations ◽

Mutational Status ◽

Trisomy 12 ◽

Aggressive Clinical Course ◽

First Time

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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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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High CD49d Protein Expression Predicts Short Overall Survival and Early Progression in Chronic Lymphocytic Leukemia Patients.

Blood ◽

10.1182/blood.v110.11.3097.3097 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3097-3097

Author(s):

Valter Gattei ◽

Pietro Bulian ◽

Maria Ilaria Del-Principe ◽

Antonella Zucchetto ◽

Luca Maurillo ◽

...

Keyword(s):

Overall Survival ◽

Chronic Lymphocytic Leukemia ◽

Clinical Stage ◽

Lymphocytic Leukemia ◽

Prognostic Impact ◽

Color Flow ◽

Entire Cohort ◽

Mutational Status ◽

Ighv Gene ◽

The Impact

Abstract B-cell chronic lymphocytic leukemia (CLL) is a heterogeneous disease with highly variable clinical courses which can be at least in part foreseen by investigating the expression of known prognosticators, including the IGHV gene mutational status or CD38/ZAP-70 expression. By analysing the coordinated expression of several surface antigens in a series of CLL with known clinical courses, we identified the simultaneous over-expression of CD38 and CD49d as part of the signature characterizing the subgroup with the worst outcome. The aim of the present study was to investigate the relationship between CD49d and other well-established biologic prognosticators (CD38 and ZAP-70 expression, IGHV gene mutational status), or markers of tumor burden (Rai clinical stage, beta2-M, sCD23), and to define the independent prognostic impact of CD49d in predicting overall survival (OS) and disease progression (evaluated as time-to-treatment, TTT) in CLL patients. The study includes samples from 303 patients affected by CLL according to the current diagnostic criteria (median age: 63.5 years, range 32–97). The entire cohort of patients was utilized to investigate the impact of CD49d and other prognosticators (CD38, ZAP-70, IGHV gene mutational status) on OS. TTT information and additional laboratory parameters (beta2-M, sCD23) were available for 232/303 patients, whose therapies were established according to NCI-WG criteria. CD49d expression was determined by three-color flow cytometry combining anti-CD49d, anti-CD19 and anti-CD5 mAbs; its expression was demonstrated to be stable over-time and the 30% of positive CD5+CD19+CLL cells was chosen as cut-off to discriminate CD49dlow from CD49dhigh cases. CD49d, whose expression was strongly associated with that of CD38 (p=2.2×10exp-16) and ZAP-70 (p=2.6×10exp-5), or with IGHV gene status (p=1.1×10exp-6), was independent predictor for OS (HR=4.39; p=0.0081) along with IGHV status (HR=5.54; p=0.0005) or, if this parameter was omitted, with ZAP-70 (HR=2.90; p=0.0092). CD49d also effectively predicted TTT and refined the prognostic relevance of all the investigated prognosticators. Notably, a CD49dhigh phenotype, while not changing the outcome of good prognosis (ZAP-70low, mutated-IGHV) CLL, was necessary to correctly predict the bad clinical courses of ZAP-70high (HR=3.12; p=0.023) or unmutated-IGHV (HR=2.95; p=0.002) cases. These findings support the introduction of CD49d detection in routine prognostic assessment of CLL patients, and suggest both pathogenetic and therapeutic implications for CD49d expression in CLL, e.g. envisioning the use of a humanized anti-CD49d monoclonal antibody, currently employed in multiple sclerosis (Natalizumab), for selcted CLL patients.

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Low Frequency Of IGHV4-39/IGHD6-13/IGHJ5 Rearrangement With Stereotyped HCDR3 (subset #8) In Russian and Ukrainian Chronic Lymphocytic Leukemia Patients

Blood ◽

10.1182/blood.v122.21.5276.5276 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5276-5276

Author(s):

Bella Biderman ◽

Nadiia Bilous ◽

Nataliya Severina ◽

Andrey Sudarikov ◽

Eugene Nikitin ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Clinical Course ◽

Low Frequency ◽

Lymphocytic Leukemia ◽

European Countries ◽

Immunoglobulin Gene ◽

Molecular Abnormalities ◽

Mutational Status ◽

Ighv Gene ◽

Notch1 Mutations

Abstract The leukemia cells of unrelated patients with chronic lymphocytic leukemia (CLL) display a restricted repertoire of immunoglobulin gene rearrangements and very similar structure of B-cell receptor (stereotyped BCR). Numerous different subsets of stereotyped BCRs were identified. Some associations between specific BCRs subsets, clinical course of CLL, and molecular abnormalities were found. Namely, short overall survival, accumulation of SF3B1 mutations in subset #2, high risk for Richter’s transformation, trisomy 12, NOTCH1 mutations in subset #8, increased frequency of NOTCH1 mutations in subset #1 were revealed (Rossi et al., 2009, 2013; Streffoed et al., 2013). The aim of this study was to evaluate distribution of different stereotyped BCR subsets in Russian and Ukrainian CLL cohort with special attention on prognostic unfavorable subsets. Methods Immunoglobulin heavy chain variable (IGHV) genes of 969 CLL patients (564 from Russia and 405 from Ukraine) were amplified according to BIOMED-2 rules, sequenced and analyzed with IMGT and IgBlast databases. Additionally, NOTCH1 c.7544_7545delCT mutations were analyzed in 301 patients (randomly collected and in the prognostic unfavorable subsets) by amplification refractory mutation system (ARMS) PCR as proposed by Rossi D. et al., 2012. In 19 cases NOTCH1 mutational status was confirmed by direct DNA Sanger sequencing. Results Joined Russian-Ukrainian CLL cohort was presented mainly by unmutated (UM) IGHV gene cases (64.2%). The distribution of major clusters of stereotyped BCR did not differ from those presented in the literature (comparison was made separately for cases with mutated (M) and UM IGHV genes, taking into account the differences in the mutational status of IGHV genes between our CLL cohort and CLL cohorts from European countries and USA), except for subset #8, which was found in one case only (0.16% of UM IGHV gene cases). At the same time, subset #8 (IGHV4-39/IGHD6-13/IGHJ5 rearrangement) belongs to the group of “major” subsets in CLL (Agathangelidis et al., 2012), and its frequency varies from 0.9% to 2% among cases with UM IGHV genes in the most of presented CLL cohorts (Murray et al., 2008; Messmer et al., 2009; Maura et al., 2011). Frequency of subset #2 had tendency to be lower among Russian CLL patients (1.4% of M CLL cases), but not among Ukrainian patients (4.5% of M CLL cases) comparing with European CLL cohorts. NOTCH1 c.7544_7545delCT was identified in 27 out of 301 patients (9.0%), which was in accordance with other CLL studies. Twenty five cases with NOTCH1 mutations had UM IGHV genes and 2 cases expressed M IGHV3-21 gene (both subset #2). As well, majority of NOTCH1 mutated cases (17/27, 63%) showed stereotyped BCRs, which belonged to the following subsets: #1 (2 of 12), #2 (2 of 7), #3 (1 of 6), #7 (2 of 10), #9 (2 of 10), #25 (1 of 2), #59 (1 of 3) (subset’s designation according Murray et al., 2008), UA/ref2 (1 of 2), UA/ref11 (2 of 2) (subset’s designation according Bilous et al., 2010), or having a homology with the CLL sequences of the public CLL database. No NOTCH1 mutations were found in 11 IGHV4-39-used cases, including one case from subset #8. In 10 of 27 (37.0%) NOTCH1 mutated cases IgHV1-69 gene was used, while among wild type NOTCH1 patients IgHV1-69 was presented in 65/274 (23.7%) cases (p=0.12). Conclusion The distribution of the major subsets of stereotyped BCRs in CLL patients from Russia and Ukraine (eastern slavic countries) was similar to the western European countries and USA, except for low frequency of subset #8, associated with unfavorable clinical course. Our preliminary data suggested that NOTCH1 c.7544_7545delCT mutation is typical for UM CLL cases with stereotyped BCRs, but no clear association with specific subsets was found in the absence of subset #8. Disclosures: No relevant conflicts of interest to declare.

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A Systematic Search Into The Role Of IGHV Gene Replacement In Shaping The Immunoglobulin Repertoire Of Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v122.21.4129.4129 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4129-4129

Author(s):

Sophia Yancopoulos ◽

Xiao-Jie Yan ◽

Andreas Agathangelidis ◽

Wentian Li ◽

Chrysoula Belessi ◽

...

Keyword(s):

Internal Control ◽

Conflicts Of Interest ◽

Positional Information ◽

Gene Replacement ◽

Lymphocytic Leukemia ◽

Negative Group ◽

Cd38 Expression ◽

Ighv Gene ◽

Significant Difference ◽

Normal Controls

Abstract Immunoglobulin heavy variable (IGHV) gene replacement is a RAG-mediated recombination event that modifies a rearranged IGHV-IGHD-IGHJ sequence by replacing the original IGHV with another IGHV. As the excision of the original IGHV occurs at a cryptic heptamer near the end of FR3 of the original IGHV, a trace (“footprint”) remains embedded in the V-D (“N1”) junction of the heavy complementarity-determining region 3 (HCDR3) sequence of the new rearrangement. Since IGHV-replaced antibody sequences are over-represented in several autoantibody-associated syndromes, it is assumed that the process is designed to eliminate unwanted antibody specificities, particularly those which are autoreactive. Because CLL antibodies/B cell receptors (BCRs) are often autoreactive, we sought to determine if IGHV replacement plays a significant role in the formation of these antibodies. To this end, we analyzed IGHV-IGHD-IGHJ rearrangements in a CLL sample set of 6,121 sequences and in another set of 3,326 sequences from normal B lymphocytes culled from the GenBank Database. Sequences without positional information for their IGHV match were eliminated, resulting in 6023 CLL and 3202 normal sequences. From among these, we looked for the presence of IGHV footprints in the N1 and N2 junctional regions. Sequences were processed by IMGT to determine the particular IGHV used as well as junctional N1, N2 sequences. The resulting junctional regions were analyzed using the Java-based VH Replacement Footprint Analyzer (VHRFA) program [Huang, L et al (2013) PLOS ONE] to identify footprints (≥5-mer) in the N1 and N2 regions. Various CLL subgroups based on ZAP-70 expression, CD38 expression, IGHV mutation status, BCR stereotypy, and genomic aberrations were also examined for differences in the presence of footprints. Comparison of CLL with normal sequences in the N1 regions revealed a slight yet significant difference in footprint frequency (12.62% versus 11.06%, respectively; P = 0.03). Comparison of IGHV-mutated (M) versus unmutated (U) subgroups of CLL also disclosed a significant difference in frequencies (M: 13.58%, U: 11.51%, P = 0.02). Using footprints in the N2 regions as an internal control, we found the frequency of ≥5-mer replacement footprint motifs to be significantly higher in N1 over N2 (P = 1.1E-7), supporting the validity of this approach. Other comparisons led to a significant enrichment of footprint frequencies, such as 13.71% for the ZAP-70-negative group (P = 0.029), 13.36% for the CD38-negative group (P = 0.013), and 14.70% for the 13q-deletion group (P = 0.016) compared with the normal group. Note, an overall comparison of stereotyped versus non-stereotyped cases revealed highly statistically significant differences in N1 footprint frequencies (9.4% for the 2084 cases having defined subsets vs 14.32% for the 3,939 cases without defined subsets; P = 2.7E-8). Focusing on stereotyped cases that exhibited evidence for IGHV replacement, we found significantly different footprint frequencies in the N1 region among different stereotyped subsets, even when restricting the comparison to subsets utilizing the same IGHV and belonging to the same mutational category (U-CLL or M-CLL). Relevant examples include differential frequencies among U-CLL, IGHV1-69-expressing subsets #3, #5, #6, #7, and #59 (0 - 15.5%; P = 0.01) and M-CLL, IGHV4-34-expressing subsets #4, #16, #29, and #201 (0 - 40.9%; P = 0.005). In summary, CLL had an elevated IGHV replacement rate compared to normal controls. In addition, M-CLL, ZAP-70-neg, CD38-neg and 13q del CLL subgroups were also elevated above normal controls. The most dramatic differences in IGHV replacement frequency were found between CLL non-stereotyped and stereotyped CLL cases, with an intriguing difference within stereotyped subgroups exhibiting the same IGHV where IGHV replacement varied by as much as 40-fold. Because higher levels of IGHV replacement are found in ZAP-70-neg, CD38-neg, 13q del CLL, and non-stereotyped cases that in general are less aggressive, the presence of IGHV replacement may pinpoint CLL clones whose precursor B cells had edited their BCRs away from autoreactivity and therefore antigen-drive. This conclusion suggests a differential role for autoantigen drive in certain stereotyped subsets: CLL subsets not exhibiting IGHV replacement appear to be more susceptible to antigen drive than those that do. Disclosures: No relevant conflicts of interest to declare.

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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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Immunophenotypic Profile and Recurrent Somatic Mutations in 131 Chronic Lymphocytic Leukemia Patients: Low Expression of CD25 in NOTCH1-Mutated Cases

Blood ◽

10.1182/blood.v128.22.3201.3201 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3201-3201

Author(s):

Miriam Castillo ◽

Ana María Hurtado ◽

Tzu Hua Chen-Liang ◽

Julia Muñoz-Ballester ◽

Bartlomiej P Przychodzen ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Somatic Mutations ◽

Lymphoblastic Leukemia ◽

Lymphocytic Leukemia ◽

Limited Information ◽

Male Predominance ◽

Mutational Status ◽

The Impact ◽

Notch1 Mutations ◽

Low Expression

Abstract Background: We and others have reported on the impact of recurrent somatic mutations not only in the multistep pathogenetic process, but also in the clinical heterogeneity of chronic lymphocytic leukemia (CLL) patients. Immunophenotyping, as part of the diagnostic workout, is used for assessing clonality, as a differential diagnosis tool, and to examine the expression of molecules associated with a worse prognosis. Recently, NOTCH1 mutations have been linked to low CD20 levels in CLL and with a relative resistance to anti-CD20 immunotherapy in vitro. But to date, there is limited information on the correlation between cell surface marker expression and the presence of somatic mutations in CLL. The aim of this study was to evaluate potential associations between an extended phenotypic panel and the mutational status of 13 recurrently mutated genes in CLL detected by deep sequencing. Patients and Methods: To this end, we performed targeted NGS sequencing of blood samples, collected at diagnosis, from 131 CLL patients. Every patient underwent, at baseline, a flow cytometry characterization with a panel including (sIg)λ, (sIg)κ, CD19, CD5, CD11b, CD81, CD10, CD79b, CD29, CD38, FMC7, CD22, CD45, CD103, CD11c, CD25, ZAP70, CD11a, and CD24. We designed a TruSeq Custom Amplicon panel (Illumina, Inc. San Diego, CA, USA) containing 13 genes and covering 28.099 bases. The average amplicon size was 238 base pairs and ~ 99.1% of the regions were covered on both strands. Paired-end sequencing (2x250 bp) was performed with MiSeq v2.2 chemistry, and a mean depth of 998 reads/base within the regions of interest was obtained. Raw data were analyzed with IlluminaonJboard Real Time Analysis (RTA v.2.4.60.8) software and MiSeq Reporter. Results: With a median age of 68 y.o. (range, 33-95) and a slight male predominance, the median follow up time of our cohort was 43 months (24-104). We found that 47/131 (35%) patients harbored at least one mutation, with NOTCH1 (n = 13, 10%), ATM (n = 10, %), TP53 (n = 8, %), and SF3B1 (n = 8, 5.5%), as the most frequently mutated genes. Those patients with a NOTCH1 mutation showed a lower CD25 expression (25 mean fluorescence intensity units (MFIu)) than those without a mutation (45 MFIu), p=0.001. In addition, a higher expression of CD5 (265 vs. 219 MFIu, p= 0.02), of the monoclonal light chain (90.5 vs. 58.6 MFIu, p=0.03), and a higher percentage of CD38+ cells in the CD19+CD5+ compartment (37% vs. 19%, p=0.006) were significantly associated with the presence of, at least, one somatic mutation. We could not validate the recently reported association between the presence of NOTCH1 mutations and a low expression of CD20. In our cohort, the MFI expression in NOTCH1 mutated and non-mutated patients was 176 and 135 units, respectively (p=0.2) In the multivariate Cox analysis, the presence of a somatic variant in TP53 and a higher percentage of positive CD38 cells in the tumour population showed both a worse overall survival and shorter time to first treatment. The independence of these two variables was also supported by not finding a significative difference percentage of CD38 positive cells between TP53 mutated and non mutated cases (p=0.5). Conclusions: The associations described herein suggest potential pathogenic pathways in CLL, in particular the CD25-NOTCH1 axis, with a significative inferior expression of CD25 when activating NOTCH1 mutations are present. The relationship found between these two variables, with an inversed direction to that found in physiological conditions, has also been shown in the setting of NOTCH1-mutated acute lymphoblastic leukemia, emerging as a potential targetable pathway in this subset of CLL patients. Disclosures Maciejewski: Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau.

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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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CD38 expression is a poor predictor for VH gene mutational status and prognosis in chronic lymphocytic leukemia

Blood ◽

10.1182/blood.v97.6.1892 ◽

2001 ◽

Vol 97 (6) ◽

pp. 1892-1894 ◽

Cited By ~ 96

Author(s):

Ulf Thunberg ◽

Anna Johnson ◽

Göran Roos ◽

Ingrid Thörn ◽

Gerard Tobin ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Lymphocytic Leukemia ◽

Cd38 Expression ◽

Poor Predictor ◽

Mutational Status ◽

Vh Gene

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New Prognostic Markers in CLL

Hematology ◽

10.1182/asheducation-2006.1.279 ◽

2006 ◽

Vol 2006 (1) ◽

pp. 279-284 ◽

Cited By ~ 46

Author(s):

Emili Montserrat

Keyword(s):

Residual Disease ◽

Daily Practice ◽

Serum Markers ◽

Response To Therapy ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Prognostic Information ◽

Cd38 Expression ◽

Mutational Status ◽

The Individual

Abstract The individual prognosis of patients with chronic lymphocytic leukemia (CLL) is extremely variable. Although clinical stages remain the basis for assessing prognosis in CLL, a number of biological markers, particularly serum markers, cytogenetic abnormalities, IgVH mutations, CD38 and ZAP-70 expression in leukemic cells offer important, independent prognostic information. Before being incorporated into daily practice, however, these markers require standardization and validation in large, prospective trials. Meanwhile, treatment of patients with CLL not included in clinical studies should be decided on the basis of classical NCI/CLL Working Group criteria. An important area of research in CLL prognostication is the identification of markers useful for predicting response to therapy and its duration. Among them, del(17p), reflecting P53 abnormalities, is particularly important. Also relevant is del(11q), which points to ATM defects. There is also some correlation between IgVH mutational status, ZAP-70 and CD38 expression and response to therapy and its duration, although these relationships need further investigation. Finally, there is increasing evidence that response to therapy, particularly in those cases in which minimal residual disease is eradicated, is associated with longer survival.

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