NOTCH1 Mutations Are Associated with High CD49d Expression in Chronic Lymphocytic Leukemia

Abstract Mutations of NOTCH1 have emerged as one of the most frequent somatic alterations in chronic lymphocytic leukemia (CLL), affecting up to 10–15% of patients. These mutations (~80% are 7544-7545delCT frameshift deletions) generate a truncated protein that accumulates in the cell and activates the downstream NOTCH1 signaling which is implicated in apoptosis resistance and increased survival of CLL cells. CD49d (α4 integrin chain) is one of the most relevant negative prognosticator in CLL, expressed by ~40% of CLL cases, and associated with aggressive/accelerated clinical courses, whose key role in CLL cell microenvironmental interactions has been thoroughly investigated. Literature data indicate that NOTCH1 has a role in activating the integrin signaling in several cell models. Given the higher CD49d expression characterizing trisomy 12 CLL, a CLL subset in which the NOTCH1 pathway is more often activated by NOTCH1 mutations, this study was aimed at investigating the contribution of NOTCH1 in the regulation of CD49d expression in CLL. The 7544-7545delCT NOTCH1 mutations were investigated by ARMS-PCR in 1027 CLL cases, all characterized for CD49d expression and for the cytogenetic profiles by FISH. NOTCH1 mutated cases were 158/1027 (15%), with a higher prevalence (36.7% of NOTCH1 mutated cases) in the trisomy 12 cytogenetic group. Analysis of CD49d expression highlighted a very strong association between the presence of NOTCH1 mutations and CD49d expression (p<0.0001). In particular, high CD49d expression (>30% of positive cells) was found in 102/158 (64.5%) NOTCH1 mutated cases as compared to 285/869 (32.8%) NOTCH1 wild-type cases. Of note, excluding trisomy 12 CLL, again NOTCH1 mutated CLL (100/865, 11.6%) displayed a significantly higher frequency of CD49d+ cases (52%) as compared to NOTCH1 wild-type CLL (25.7%) (p<0.0001). We next analyzed the percentage of mutated NOTCH1 DNA in the context of the CLL clone by a quantitative real-time PCR (QRT-PCR) approach set up by us to quantify the delCT NOTCH1 mutation. Using the 10% cut-off value to discriminate between cases with high (high NOTCH1) and low (low NOTCH1) mutation burden, 90/138 (67.7%) and 43/138 (32.3%) CLL cases were classified as low NOTCH1 and high NOTCH1, respectively. A higher prevalence of CD49d+ cases was found in the high NOTCH1 group (79%) as compared to the low NOTCH1 group (58%) (p=0.03). Moreover, a significant association between CD49d expression and a high NOTCH1 mutation burden was observed also excluding trisomy 12 CLL, with 69% of CD49d+ cases in the high NOTCH1 group, compared to 41% of CD49d+cases in the low NOTCH1 group (p=0.03). The association between NOTCH1 mutations and CD49d expression was next confirmed by next-generation sequencing results using the flow cytometrically sorted (>99% purity) CD49d- and CD49d+ components from 8 CLL cases characterized by both CD49d bimodal expression, and the presence of 7544-7545delCT NOTCH1 mutations at the subclonal level. In 7/8 cases, the CD49d+ component showed a higher NOTCH1 mutation burden compared to the CD49d- component, this difference reaching statistical significance in 4/7 cases. Of note, a similar clustering of mutations could not be observed in the CD49d- and CD49d+ components of other CLL cases characterized by bimodal CD49d expression and subclonal mutations of SF3B1 (n=1), BIRC3 (n=2) or TP53 (n=2). To verify whether NOTCH1 accumulation, as occurring in NOTCH1 mutated CLL, may influence CD49d expression, MEC-1 cells were transfected with a vector containing either a NOTCH1 intracellular domain (NICD) with 7544-7545delCT or a NICD carrying a missense mutation (c.5304G>A) generating a stop codon at the beginning of the sequence, as control. The higher levels of both NOTCH1 transcript (fold increase over control=2.2) and protein (fold increase over control=1.3) characterizing mutated-NICD MEC-1 cells, was paralleled by higher levels of CD49d expression (mean fluorescence intensity=23.300 versus 12.400) in these cells. Altogether our data demonstrate a direct correlation between NOTCH1 mutations and CD49d expression also outside the trisomy 12 CLL group, and suggest that accumulation of NOTCH1 may be directly or indirectly responsible for the up-regulation of CD49d expression. Disclosures No relevant conflicts of interest to declare.

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Erythroid Differentiation Regulator 1 (ERDR1) From Nurse-Like Cells Promotes the Survival of Chronic Lymphocytic Leukemia Cells

Blood ◽

10.1182/blood.v116.21.1372.1372 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1372-1372

Author(s):

Hendrik W. Van Deventer ◽

Robert Mango ◽

Jonathan Serody

Keyword(s):

Bone Marrow ◽

Chronic Lymphocytic Leukemia ◽

Stromal Cells ◽

Stromal Cell ◽

Conflicts Of Interest ◽

Erythroid Differentiation ◽

Mesenchymal Cells ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Wild Type

Abstract Abstract 1372 Background: Chemotherapy resistance in chronic lymphocytic leukemia (CLL) can be mediated by anti-apoptotic signals produced by stromal or nurse-like cells. Developing strategies to overcome this resistance is hindered by the lack of suitable “stromal” targets responsible for these signals. We have discovered that erythroid differentiation regulator 1 (ERDR1) may be a candidate target for such a strategy. In this study, we show Erdr1 is generated by several stromal cell types including bone marrow stromal cells, fibrocytes, and nurse-like cells. Furthermore, inhibition of stroma-generated Erdr1 results in increased apoptosis of co-cultured CLL cells. Methods/Results: We initially identified Erdr1 on an Affymetrix array that compared the gene expression of wild type and CCR5-/- mice with pulmonary metastasis. The increased expression of Erdr1 in the wild type mice was particularly pronounced in the pulmonary mesenchymal cells. Therefore, these cells were transfected with one of two shRNAs (shRNA #9 or shRNA#11) and the survival of these cells was compared with mesenchymal cells transfected with a non-targeted control vector. After 15 days in culture, the control cells expanded normally; however, no significant expansion was seen in either the shRNA#9 or shRNA#11 transfected cells. These differences in cellular expansion were associated with differences in apoptosis. 21.4+1.6% of the Erdr1 knockdown cells were annexin V+ compared to 11.2+1.9% of the non-targeted control (p<0.03). Using GFP as a marker for transfection, we were also able to show that knockdown of Erdr1 increased the apoptosis of surrounding non-transfected mesenchymal cells. Thus, Erdr1 is a critical protein for the survival of stromal cells. Further analysis of the mesenchymal cell subpopulations revealed the greatest expression of Erdr1 in the CD45+, thy1.1+/− fibrocytes. When compared to CD45- fibroblasts, the fibrocytes expressed CCR5 and increased Erdr1 expression by 14.2+/−2.9 fold when treated with the CCR5 ligand CCL4. Given the similarities between fibrocytes and nurse-like cells, we went on to measure the effect of Erdr1 inhibition on CLL cells. In these experiments, stable Erdr1 knockdown and control clones were selected after the transfection of the bone marrow stromal cell line M2-10B4. These clones were then co-cultured with primary CLL cells. At 96 hours, leukemia cells co-cultured with the control lines had expanded by 1.33 + 0.9 compared to 0.74 + 0.22 fold in the knock-down lines (p<0.03). As before, the lack of cellular expansion was associated with an increase in apoptosis. To further show the relevance of these findings to CLL, we demonstrated that human fibrocytes and nurse-like cells expressed mRNA and protein for ERDR1 in all patient samples tested. Implications for the treatment of human disease: Our data demonstrate that ERDR1 is a critically important protein for the survival of nurse-like cells. These data suggest that targeting ERDR1 or the upstream pathway through CCR5 might be a novel approach for the treatment of CLL. Disclosures: No relevant conflicts of interest to declare.

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Specific Chromosomal IG Translocations Have Different Prognosis In Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v116.21.582.582 ◽

2010 ◽

Vol 116 (21) ◽

pp. 582-582

Author(s):

Florence Nguyen-Khac ◽

CLaude Lesty ◽

Elise Chapiro ◽

Aurore Grelier ◽

Isabelle Luquet ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Sex Ratio ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Rank Test ◽

Exact Test ◽

Kaplan Meier ◽

Trisomy 12 ◽

Binet Stage ◽

The Individual

Abstract Abstract 582 Chromosomal translocations (t) are usually analyzed as one group, and are associated with poor prognosis in chronic lymphocytic leukemia (CLL). Translocations involving immunoglobulin (IG) genes are recurrent, but uncommon (< 5%) in CLL. The two most frequent IG-partners are BCL2 (18q21) and BCL3 (19q13). On the behalf of the Groupe Francophone de Cytogenetique Hematologique (GFCH), we report an extensive analysis of 75 t(14;18)-CLLs, and a comparison to our previously published series of 29 t(14;19)-CLLs (Chapiro et al, Leukemia 2008). The 75 t(14;18)-CLLs or variant BCL2-t have been collected between 1985 and 2009. The morphological and immunological reviews were performed by KM, CS, and HM-B. All karyotypes were reviewed by the GFCH. Fluorescence in situ hybridization analyses were performed to detect IG and BCL2 rearrangements, trisomy 12, and deletions of 11q22 (ATM), 17p13 (TP53), 6q21, 13q14 (D13S319). IGHV mutation analyses were performed by referring laboratories. Statistical analyses were carried out using the Fisher's exact test, and continuous data using the Mann-Whitney test. Overall survival (OS) and treatment free survival (TFS) calculated from diagnosis were estimated using the Kaplan-Meier method, and the statistic significance was determined using log-rank test. Among BCL2-CLL, the sex ratio was 57M/18F, the median age at diagnosis was 66 years; of 68 patients with available data, 63 (93%) presented with Binet stage A; median lymphocytosis was 14.6×109/l. There were 47/75 (63%) “classical” CLL and 28/75 (37%) “atypical” CLL, with more than 10% of lymphoplasmacytoid cells and/or large cells. All tested cases (58/58) were CD10-, 69/73 (94%) were CD5+, and 44/63 (70%) were CD38-; 57/68 (84%) had a Matutes score > 4, 7/68 (10%) a score = 3, 4/68 (6%) a score < 3. We observed 62 t(14;18) and 13 variant translocations [11 t(18;22), 2 t(2;18)]. The karyotype was complex (> 3 abnormalities) in 15/74 (20%) cases, and the BCL2-t was isolated in 25/74 (34%) cases. There were 33/75 (44%) tri12, 32/68 (47%) del13q14, 1/72 (1%) delTP53, 0/72 (0%) delATM, 0/59 (0%) del6q21. Of 42 analyzed cases, 33 (78%) were mutated. Finally, the median time from diagnosis to first therapy was 24 months (m). Comparisons with the BCL3-CLL showed no difference in sex ratio, age, and Binet stages. The lymphocytosis was lower in BCL2-CLL (14.6 vs 24.4 x109/l, p<0.008), and splenomegaly was less frequent (3/61 (5%) vs 13/28 (46%), p<0.0001). There were more “classical” morphologies in BCL2-CLL group (63% vs 9/29 (31%), p<0.005), more Matutes score > 4 (84% vs 5/20 (25%), p<0.001), and more CD38- (70% vs 1/5 (20%), p<0.05). BCL2-t were more frequently single (35% vs 1/28 (3%), p<0.0008). There were less complex karyotypes (20% vs 13/28 (46%), p<0.02), more del13q14 (47% vs 4/27 (15%), p<0.005), and less tri12 (44% vs 20/29 (69%), p<0.03), del6q (0% vs 5/25 (20%), p<0.002) and delTP53 (1% vs 4/23 (17%), p<0.02) in BCL2-CLLs. The IGHV status of BCL2-CLLs was more frequently mutated (78% vs 2/20 (10%), p<0.0001). Finally, the TFS interval was longer in BCL2-CLLs (p<0.0001, median 48 vs 1.2 m,); and the median OS was longer (not reached with 75% alive at 204 m) (p<0.0001). Comparison to common CLL showed that BCL2-CLLs had more tri12 (p<0.00001), and lacked delATM (p<0.0001) and del6q (p<0.05). The majority were CD38-, and mutated (p<0.0001). Finally, even if the median TFS was 48m, the median OS was more than 204m, which is longer than the median OS of the prognostically most favorable subgroup reported by Döhner et al (group with isolated del13q, 133m) (Döhner et al, N Eng J Med, 2000). The presence of BCL2-t remains a favorable marker even in patients who also exhibit markers of intermediate prognosis such as tri12. Compared to BCL3-CLLs, BCL2-CLLs have a much less aggressive behavior, indicating that distinguishing the individual translocations and the cytogenetic partners would allow a better patients' stratification. Disclosures: No relevant conflicts of interest to declare.

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Serum IgM Activates BCR Signaling Pathways and Increases Survival of Chronic Lymphocytic Leukemia B Cells

Blood ◽

10.1182/blood.v126.23.4131.4131 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4131-4131

Author(s):

Stefania Gobessi ◽

Binu K Sasi ◽

Luca Laurenti ◽

Dimitar G Efremov

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Signaling Pathways ◽

Target Genes ◽

Conflicts Of Interest ◽

Direct Interaction ◽

Leukemic Cell ◽

Fold Increase ◽

Lymphocytic Leukemia ◽

Bcr Signaling

Abstract Serum IgM would be expected to bind chronic lymphocytic leukemia B cells through two different mechanisms. The first mechanism is via interactions between the immunoglobulin heavy chain CDR3 of the leukemic B cell receptors (BCRs) and internal epitopes located in the FR2 and FR3 regions of serum IgM molecules, analogous to the recently identified cell-autonomous BCR-BCR interaction. The latter interaction represents a general feature of human CLL BCRs and was recently shown to be positively selected during leukemia development in the Eμ-TCL1 transgenic murine model. The second mechanism is by binding of serum IgM to the recently identified Fc receptor for IgM (FcμR), which is overexpressed on CLL B cells. In the present study we investigated the consequences of the interaction between serum IgM and CLL cells. Incubation of CLL cells with Alexa488-conjugated human IgM resulted in strong cell surface labeling, confirming that IgM binds to CLL cells. Binding was substantially inhibited by preculture of CLL cells with Fcμ, suggesting that IgM interacts with CLL B cells primarily through the FcμR. To investigate whether IgM also binds to the leukemic BCRs, we analyzed activation of downstream BCR signaling pathways and expression of a well-defined set of BCR-target genes (Herishanu Y et al, Blood. 2011;117:563-74) in CLL cells cultured in the presence or absence of purified IgM. After three hours in culture with polyclonal or monoclonal human IgM, 5 of the 7 investigated BCR target genes (OAS3, RGS1, GFI1, CCND2 and KLF4) showed a 2- to 9-fold increase with respect to unstimulated CLL cells, whereas the remaining two genes (EGR1 and EGR2) were not induced. The induced BCR target genes were also upregulated to an equal or even greater extent by Fcμ, suggesting that these effects are primarily or exclusively caused by binding of IgM to the FcμR. Analysis of downstream signaling events, such as SYK and ERK phosphorylation, also showed similar induction by IgM and Fcμ. However, intracellular Ca2+ flux was induced to a substantially greater extent with IgM, suggesting that certain effects are mediated by a direct interaction between serum IgM and the leukemic cell BCRs. Since co-ligation of the FcμR was recently shown to enhance the survival of anti-IgM-stimulated murine B lymphocytes (Ouchida R et al, J Immunol. 2015;194:3096-101), we investigated the consequences of IgM binding on CLL cell survival. CLL cells from 18 patients were cultured with or without purified human IgM for 72 hours and then analyzed by Annexin V/PI staining. A modest but significant increase in the percentage of viable CLL cells was observed in the presence of IgM (percentage of viable CLL cells without IgM: 40.5±17.8; with IgM: 43.8±18.4; P =0.016), which was replicated in a smaller series of samples cultured with Fcμ (n=12, percentage of viable CLL cells without Fcμ: 41.1±17.8; with Fcμ: 49.5±15.6; P =0.019). Altogether, these data suggest that binding of serum IgM results in activation of prosurvival pathways in CLL cells and that this effect is most likely mediated by co-triggering the FcμR and BCR. Disclosures No relevant conflicts of interest to declare.

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SNP-Arrays: a Routine Cytogenetic Tool In Chronic Lymphocytic Leukemia ?.

Blood ◽

10.1182/blood.v116.21.4611.4611 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4611-4611

Author(s):

Alban Godon ◽

Franck Genevieve ◽

Malgorzata Truchan-Graczyk ◽

Laurence Baranger ◽

Virginie Eclache ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Chromosomal Abnormalities ◽

Conflicts Of Interest ◽

Snp Array ◽

Lymphocytic Leukemia ◽

Primary Data ◽

Assay Sensitivity ◽

Snp Arrays ◽

Median Size ◽

Trisomy 12

Abstract Abstract 4611 Background: Chronic lymphocytic leukemia (B-CLL) is the most common adult leukemia in Western countries, and is characterized by a highly variable clinical course. Interphase fluorescent in situ hybridization (I-FISH) has been able to identify chromosomal abnormalities in ~80% of B-CLL, including deletions at 13q, 11q, 17p and trisomy 12, which has proven to be prognostic indicators for disease progression and survival. Although recent immunostimulatory methods have substantially improved analysis via conventional metaphase cytogenetics (CC), detection of chromosome changes is limited by the low mitotic activity of CLL cells in vitro. High-density single nucleotide polymorphism (SNP) arrays are commercially available technologies, which allow genome-wide detection of allelic copy number gains or losses, and loss-of-heterozygosity (LOH) regions. Aims: To assess whether SNP-arrays are more sensitive than I-FISH and CC to detect specific chromosomal abnormalities associated with prognosis in B-CLL, i.e. del13q, del11q, del17p and tri12. Methods: Blood samples from 24 patients with B-CLL at diagnosis were tested in parallel by I-FISH, CC and SNP-array. FISH: blood smear samples were hybridized with 4 probes, in order to detect deletions at 17p13.1, 11q22.3, 13q14.3, and trisomy 12 [respectively LSI p53, ATM, D13S319 and centromeric CEP12 probes, Abbott]. In normal lymphocytes, an average of 6.7% nuclei showed one signal (truncated nuclei), and we defined the cut-off level for detection of a deletion at 11% (mean+3SD). The cut-off for detection of tri12 was defined at 5% (mean+3SD). CC: blood lymphocytes are cultivated for 72 hours with immunostimulants (DSP30 and IL-2) and metaphases analyzed according to standard procedures. SNP-arrays: DNA samples (200 ng) were hybridized on the Illumina HumanCNV370-quad v3 BeadChips, which assess 373,397 markers with a median marker spacing of 4.9 kb (mean 7.8 kb). The I-Scan system was used to scan the BeadChips (primary data). GenomeStudio 1010 v1 and CNVPartition 2.4.4 package were used to process primary data and identify chromosomal deletions/amplifications and LOH regions. Results: I-FISH identified deletions at 13q (15-95% of nuclei; mean=51%), 11q (35-54%; mean=43%) and trisomy 12 (32-49%; mean=37%) in 17 (71%) [including 2 cases of biallelic deletions], 3 (12.5%) and 4 (17%) cases, respectively. No del17p was detected. Five B-CLL cases presented associated FISH abnormalities. SNP-arrays identified all changes (100%) detected by I-FISH (del13q, median size: 13 Mb – range, 0.49–50 Mb; del11q, median size: 39 Mb – range, 34.8–42 Mb]. However, SNP-arrays showed 5 additional deletions at 13q14.3. Three patients had cryptic deletions (~52 to 82 kb), not detected by the FISH LSI D13S319 probe (~130 kb) or CC, and two others had large deletions (1.1 and 33.3 Mb) but with one signal below the cut-off of 11% by I-FISH (therefore considered as negative- both had normal CC). In these two cases, tumor cell enrichment before I-FISH allowed the detection of the deletion. In one case with del13q and tri12 (30% of nuclei by FISH), an additional del11q was also detected by SNP-array and CC (in 5/24 mitoses). The size of this deletion was 37.8 Mb and involved the cytogenetic bands 11q14.1 to 11q23.2 (including ATM). In addition, SNP-arrays enabled to define more precisely the size and location of the abnormalities. For instance, in one case with tri12 (as identified by the centromeric FISH probe), a partial trisomy of chromosome 12 short arm was indeed detected by SNP-array. No 17p abnormality was detected by SNP-array (either deletion or LOH). Only one of the 24 samples had no abnormality by SNP, I-FISH or CC for the loci studied. Conclusions: Despites using a relatively low density SNP-array (~370,000 markers), a higher sensitivity of Illumina BeadChips was observed. “SNP+/FISH negative” cases can be explained by either cryptic deletions or low leukemic cell content (< cut-off level). “SNP+/CC negative” cases can be explained by either a higher resolution, or the presence of normal mitosis in excess following stimulation. Higher density SNP-arrays (> 5 million markers) may be used to improve the assay sensitivity, especially regarding del13q14 which seems to be present in a great majority of our cases. Our study confirms usefulness of SNP-arrays for prognostic evaluation of B-CLL patients at diagnosis, and suggests the use of this assay as a routine procedure. Disclosures: No relevant conflicts of interest to declare.

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Nucleophosmin-1 and Ribosome-Associated Components Are Constitutively Overexpressed in NOTCH1 Mutated IGHV Unmutated CLL

Blood ◽

10.1182/blood.v120.21.3880.3880 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3880-3880

Author(s):

Michele Dal Bo ◽

Federico Pozzo ◽

Riccardo Bomben ◽

Antonella Zucchetto ◽

Erika Tissino ◽

...

Keyword(s):

Ribosomal Proteins ◽

Conflicts Of Interest ◽

Fold Increase ◽

Lymphocytic Leukemia ◽

Amplification Refractory Mutation System ◽

Qrt Pcr ◽

Mutational Status ◽

Frameshift Deletion ◽

Uptake Assay ◽

Notch1 Mutations

Abstract Abstract 3880 Background: activating mutations of NOTCH1 have been identified in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, with a higher frequency in unmutated IGHV (IGHV-UM) CLL, chemorefractory CLL and CLL in advanced disease phases. In CLL, all NOTCH1 mutations disrupt the C-terminal PEST domain and cause an accumulation of an active NOTCH1 isoform. Notably, about 80% of NOTCH1 mutations are represented by a CT frameshift deletion at nucleotides 7544–7545 (c.7544–7545delCT). Clinically, the presence of NOTCH1 mutations is an independent predictor of overall survival in CLL and identifies a subset of patients with particularly unfavourable prognosis (Rossi et al, Blood, 119, 521, 2012). Aim: to identify peculiar molecular and biological features of NOTCH1 mutated CLL in the context of IGHV-UM CLL. Methods: the presence of the c.7544–7545delCT NOTCH1 frameshift deletion was investigated by an ad-hoc amplification refractory mutation system (ARMS) PCR set up to obtain an amplicon specific for the NOTCH1 mutated form and a second amplicon as control. The percentage of NOTCH1 DNA in the context of the CLL clone was determined by quantitative real-time PCR (QRT-PCR), calculating the ratio between the amount of the specific NOTCH1 mutated amplicon and the amount of the control amplicon, the latter representing the total amount of NOTCH1 DNA irrespective of its mutational status. Gene expression profile (GEP) was performed by a one-color labeling strategy using the 4×44K Agilent platform. The differential expression of specific genes/proteins was validated by QRT-PCR, western blotting and immunohistochemistry. A BrdU uptake assay was used to evaluate proliferation of CLL cells by CpG/IL2 stimulation. Results: in a cohort of 380 IGHV-UM CLL, the c.7544–7545delCT NOTCH1 mutation was found in 83/380 (21.8%) cases. QRT-PCR revealed a percentage of NOTCH1 mutated DNA ranging from 1 to 37%. CLL cases carrying the c.7544–7545delCT NOTCH1 mutation (NOTCH1-Mut) showed higher NOTCH1 protein expression than CLL cases lacking NOTCH1-Mut employing monoclonal antibodies either recognizing the trans-membrane (mean fold increase=3) or the intra-citoplasmic (mean fold increase=2.1) NOTCH1 domain. A GEP comparing RNA from purified CLL samples of 5 NOTCH1-Mut CLL and 5 CLL lacking NOTCH1-Mut was performed, selecting the 5 NOTCH1-Mut cases among those with the higher percentages of NOTCH1 mutated DNA (percentages of NOTCH1 mutated DNA ranging from 15 to 37%). This approach selected the nucleophosmin 1 gene (NPM1) and genes codifying for several ribosomal proteins (RPS6, RPS10, RPS17, RPS28, RPSA, RPL7A, RPL18) as significantly up regulated in NOTCH1-Mut CLL cases. A higher expression of the above mentioned genes in NOTCH1-Mut CLL was validated in a wider series of 34 cases (18 NOTCH1-Mut cases; NPM1, p=0.03; RPS6, p=0.045; RPS10, p=0.048; RPS17, p=0.048; RPS28, p=0.049; RPSA, p=0.048; RPL7A, p=0.039; RPL18, p=0.041, respectively). Western blot analysis in 8 cases (4 NOTCH1-Mut cases) confirmed a higher NPM1 expression in NOTCH1-Mut cases (range of fold increase from 1.6 to 5.2) also at protein level. Consistently, lymph nodes preparations from NOTCH1-Mut CLL cases revealed a strong NPM1 staining both in nucleoli and cytoplasms. Finally, when stimulated in-vitro with the CpG/IL2 combination, NOTCH1-mut IGHV-UM CLL cells proliferated, as detected by a BrdU uptake assay (>10 fold increase over control), and up-regulated NPM1 both at transcript (mean fold increase=2.02 after 18 hours of CpG exposure, p=0.001) and protein (fold increase of 1.34 after 6 hours of CpG exposure) levels. Conclusion: NPM1 was identified as constitutively overexpressed in NOTCH1-Mut IGHV-UM CLL together with several ribosome-associated components. These findings are suggestive for an increased activity of the ribosomal machinery in NOTCH1-Mut IGHV-UM CLL as part of the molecular processes leading to control of CLL cell growth and survival in this clinically unfavourable disease subset. Disclosures: No relevant conflicts of interest to declare.

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Subset-Specific Spectra of Recurrent Gene Mutations in Chronic Lymphocytic Leukemia with Stereotyped B-Cell Receptors

Blood ◽

10.1182/blood.v124.21.3320.3320 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3320-3320

Author(s):

Lesley-Ann Sutton ◽

Emma Young ◽

Panagiotis Baliakas ◽

Anastasia Hadzidimitriou ◽

Karla Plevova ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

High Frequency ◽

Gene Mutations ◽

Lymphocytic Leukemia ◽

B Cell Receptors ◽

Cell Receptors ◽

Recurrent Mutations ◽

Trisomy 12 ◽

Notch1 Mutations

Abstract Preliminary observations from essentially small patient series indicate that certain recurrent gene mutations may be enriched in subsets of chronic lymphocytic leukemia (CLL) with stereotyped B-cell receptors (BcR). On these grounds, it could be argued that differential modes of immune signaling, in the context of subset-biased antigen-immunoglobulin (IG) interactions, may be associated with the acquisition and/or selection of certain genomic aberrations within various stereotyped CLL subsets. With this in mind, we here sought to explore the genetic background of 10 major stereotyped subsets which collectively account for ~11% of all CLL and represent both IGHV unmutated (U-CLL) and/or mutated (M-CLL) cases. We focused on recurrent mutations within the NOTCH1 (entire exon 34 or targeted analysis for del7544-45), TP53 (exons 4-9), SF3B1 (exons 14-16), BIRC3 (exons 6-9) and MYD88 (exon 5) genes. Overall, 647 cases were analyzed, belonging to the following major subsets: (i) U-CLL: #1 (the largest within U-CLL, clinically aggressive), n=139; #3, n=39; #5, n=22; #6, n=48; #7, n=74; #8, n=46; #59, n=19 and #99, n=18; (ii) M-CLL: #4 (the largest within M-CLL, particularly indolent), n=78; and, (iii) subset #2 (the largest overall, variable mutational status and clinically aggressive), n=164. All cases were devoid of MYD88 mutations, which was not surprising given that our cohort was predominantly composed of U-CLL. Mutations within the BIRC3 gene were either absent (#2, #4, #6 and #59) or rare (#1, #3, #5, #7, #8 and #99; frequency 1.5%-7%) with no clear bias to any subset. BIRC3-mutant cases frequently co-existed with either del(11q) or trisomy 12. NOTCH1 mutations were more frequent in subsets #1, #6, #8, #59 and #99 (frequency, 22%-32%), sharply contrasting subsets #2 or #3 (4% and 7%, respectively) (p<0.0001). Of note, although NOTCH1 mutations tended to coincide with trisomy 12 in certain subsets e.g. #1 and #8, their co-occurrence differed significantly with only 33% of NOTCH1mut subset #1 cases carrying trisomy 12 compared to 75% of NOTCH1mut subset #8 cases (p=0.036). Moving to SF3B1, we noted that subsets harboring NOTCH1 mutations were either absent for or carried few SF3B1 mutations, while the inverse was also true i.e. very high frequency of SF3B1 mutations in subsets #2 and #3, 45% and 36%, respectively. Almost 80% of mutations observed in subset #2 were localized to two codons (p.K700E: n=44/76, 58%: p.G742D: n=15/76, 20%) within the HEAT domain of the SF3B1 protein; p.K700E accounted for only 29% (4/14) of all SF3B1 mutations detected in subset #3 while p.G742D was absent (p=0.043 and p=0.068 respectively). Thus, although the functional relevance of these mutations is currently unknown, their high frequency and striking bias to subset #2 bodes strongly for their critical role in the pathobiology of subset #2. Finally, TP53 mutations were: (i) enriched in subsets #3 (11%) and #7 (19%) and, in contrast, absent or rare in subsets #5 (0%) and #6 (4%), despite all utilizing the IGHV1-69 gene (p=0.02); (ii) enriched in subset #1 (15%) and subset #99 (33%), a less populated subset that is highly similar to subset #1; and, (iii) very rare in subsets #2 and #8 (2% in both), the latter known to display the highest risk for Richter's transformation among all CLL. In conclusion, we confirm and significantly extend recent observations indicating that different CLL stereotyped subsets display distinct genetic makeup. These findings imply that distinctive modes of microenvironmental interactions, mediated by certain stereotyped BcRs, may be associated with selection or occurrence of particular genetic aberrations, with the combined effect determining both clonal and clinical evolution, and ultimately disease outcome. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Outcome of Patients with Hodgkin Lymphoma transformed from Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v126.23.5298.5298 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5298-5298

Author(s):

Heidi Mocikova ◽

Jana Markova ◽

Lubica Gaherova ◽

Lukas Smolej ◽

Martin Simkovic ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Hodgkin Lymphoma ◽

Conflicts Of Interest ◽

Rare Event ◽

Lymphocytic Leukemia ◽

Richter Syndrome ◽

Insufficient Response ◽

Trisomy 12 ◽

New Treatment ◽

Involved Field

Abstract Background. Hodgkin lymphoma (HL) that develops in the course of chronic lymphocytic leukemia (CLL) is a rare event. Optimal management of these patients (pts) is not defined. We analyzed outcome of 10 pts treated for Hodgkin´s variant (HV) of Richter syndrome. Patients and methods. Overall 10 pts (8 males) with CLL and subsequent HV were treated between 1996 and 2015. Median age at diagnosis of HV was 68 (range 54 - 83) years. Prognostic markers of CLL at diagnosis: unmutated IGHV (5 pts), del(17p) or TP53 mutations (0 pt), del(11q) (2 pts), deletion of 13q14 region (4 pts), trisomy 12 (2 pts), ZAP 70 pos. (6 pts), CD38 pos.(4pts). The diagnosis of HV included various subtypes of HL: nodular sclerosis (3pts), mixed cellularity (3pts), lymphocyte rich (1pt), not othervise specified (3pts). EBV status of pts at diagnosis of HV of Richter syndrome: 6 pos., 1 negat. nad 3 not done. Initial and second-line treatments of CLL consisted of fludarabine- based regimens combined with rituximab or alemtuzumab and 3 pts received chlorambucil. Treatment of HV of Richter syndrome included: ABVD (7 pts) in combination with rituximab (2 pts), COPP (3 pts) and involved field radiotherapy of 30 Gy after chemotherapy (2 pts). Further treatment was required in 6 pts due to insufficient response with persistent CLL: rituximab alone (2 pts), R-bendamustine (1pt), R-DHAP (1 pt), cyclophosphamide and dexamethasone (1 pt) and gemcitabine (1 pt). None of these patients underwent autologous or allogeneic stem cell transplantation. Results. The median time from CLL diagnosis to development of HV was 8.4 (range 2.6 - 16.5) years. Median overall survival from diagosis of CLL was 17.3 years and median survival after diagnosis of HV was 3.5 years. Out of 10 pts 5 are alive :3 in complete remission (CR), 1in partial remission of CLL (HL in CR), 2 in progression of CLL (HL in CR). Five pts died (3 lymphoma progressions, 1 second cancer, 1 unknown causality). Conclusion. Our results indicate a long period for developing of HV of Richter syndrome and its subsequent poor outcome. Current HL based chemotherapy is not sufficient in HV of Richter syndrome and new treatment approaches should be considered. Disclosures No relevant conflicts of interest to declare.

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NOTCH1 mutations in chronic lymphocytic leukemia with trisomy 12

Genes Chromosomes and Cancer ◽

10.1002/gcc.21987 ◽

2012 ◽

Vol 51 (11) ◽

pp. 1063-1063 ◽

Cited By ~ 2

Author(s):

Ester M Orlandi ◽

Marianna Rossi

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Lymphocytic Leukemia ◽

Trisomy 12 ◽

Notch1 Mutations

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Mutations at 3' Untranslated Region (3'UTR) of NOTCH1 Are Associated with Low CD20 Expression Levels in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v128.22.306.306 ◽

2016 ◽

Vol 128 (22) ◽

pp. 306-306

Author(s):

Tamara Bittolo ◽

Federico Pozzo ◽

Riccardo Bomben ◽

Tiziana D'Agaro ◽

Vanessa Bravin ◽

...

Keyword(s):

Flow Cytometry ◽

Chronic Lymphocytic Leukemia ◽

Untranslated Region ◽

Lymphocytic Leukemia ◽

Transcript Levels ◽

Expression Levels ◽

Cd20 Expression ◽

Trisomy 12 ◽

Anti Cd20 ◽

Notch1 Mutations

Abstract Background. In chronic lymphocytic leukemia (CLL), NOTCH1 mutations associate with clinical resistance to anti-CD20 immunotherapy in FCR combination (Stilgenbauer et al., Blood, 2014, Dal Bo et al., AHO, 2014), that can be ascribed to a NOTCH1 mutation-driven repression of CD20 levels by HDACs (Pozzo et al., Leukemia, 2016). Recently, novel recurrent mutations have been identified in the 3'untranslated region of NOTCH1 (3'UTR NOTCH1 mutations), determining a novel splicing event within the last NOTCH1 exon (Puente et al., Nature, 2015), leading to an impaired degradation of NOTCH1 protein. Aim. To determine if 3'UTR NOTCH1 mutations associate with low CD20 levels. Methods. NOTCH1 mutations were screened by next-generation sequencing (NGS) in exon 34 and part of 3'UTR with at least 1000X coverage. NOTCH1 (transmembrane, TM, or intracytoplasmic domain, NICD) protein levels were investigated by western blot (WB). CD20 expression was investigated by flow cytometry with a FITC-conjugated anti-CD20 antibody (L27 clone). MS4A1 (encoding for CD20 protein) transcript levels were investigated by qRT-PCR. Susceptibility to anti-CD20 rituximab and ofatumumab was investigated by complement-dependent cytotoxicity (CDC) assay. NOTCH1 signaling was inhibited by gamma-secretase inhibitor (GSI). Results. i) NOTCH1 mutational screening. In 649 CLL, NOTCH1 mutations were detected in 115 cases (17.72%), overall accounting for 127 mutations (73 c.7541-7542delCT, 11 other frameshift, 17 nonsense, 1 missense, and 25 3'UTR mutations). For analysis purposes, the 115 mutated cases were subdivided in cases with NOTCH1 coding mutations (coding NOTCH1-mut, 90 cases) and cases with 3'UTR NOTCH1 mutations (3'UTR NOTCH1-mut, 25 cases). Four cases with concomitant 3'UTR NOTCH1 mutation and coding NOTCH1 mutation were assigned to the 3'UTR group according to the substantially higher mutational burden detected for the 3'UTR mutation. ii)NOTCH1protein expression. In keeping with alternative splicing events causing an impaired NOTCH1 protein degradation, 3'UTR NOTCH1-mut cases showed higher levels than NOTCH1 wild-type (NOTCH1-wt) cases of both NOTCH1 TM and NICD by WB, and similar to coding NOTCH1-mut cases (Fig. 1a). iii) CD20 expression levels. According to FISH classification, variable CD20 levels were found by flow cytometry, with the highest levels in trisomy 12 CLL. Of note, 3'UTR NOTCH1-mut cases expressed lower CD20 levels than NOTCH1-wt cases in both trisomy 12 CLL (mean MFI in 9 3'UTR NOTCH1-mut cases = 2446.11 vs mean MFI in 66 NOTCH1-wt cases = 8254.20; p<0.0001) and non-trisomy 12 CLL (mean MFI in 16 3'UTR NOTCH1-mut cases = 2033.50 vs. mean MFI in 468 NOTCH1-wt cases = 3294.07; p=0.0001), and comparable to coding NOTCH1-mut cases (trisomy 12 CLL, mean MFI in 34 coding NOTCH1-mut cases = 2570.73; p=0.8530; non-trisomy 12 CLL, mean MFI in 56 coding NOTCH1-mut cases = 2538.13; p=0.1500) (Fig. 1b). Similarly, in both trisomy 12 CLL and non-trisomy 12 CLL categories, MS4A1 transcript levels were lower in 3'UTR NOTCH1-mut cases than in NOTCH1-wt cases (p=0.0274 and p=0.0072, respectively), again with expression levels comparable with coding NOTCH1-mut cases (p=0.2874 and p=0.9610, respectively, Fig. 1c). iv) Susceptibility to anti-CD20 in vitro. In keeping with CD20 levels, 3'UTR NOTCH1-mut cases showed lower relative lysis induced by rituximab or ofatumumab, in CDC assay, than NOTCH1-wt cells (7 3'UTR NOTCH1-mut cases, 9 NOTCH1-wt cases; mean relative lysis upon rituximab: 2.67% vs. 25.56%; p=0.0349; mean relative lysis upon ofatumumab: 39.26% vs. 60.64%; p=0.0286). In the same manner, coding NOTCH1-mut cases showed lower relative lysis induced by rituximab or ofatumumab than NOTCH1-wt cases (9 coding NOTCH1-mut cases, 9 NOTCH1-wt cases; mean relative lysis upon rituximab: 2.53% vs. 25.56%; p=0.0339; mean relative lysis upon ofatumumab: 30.60% vs. 60.64%; p=0.0114; Fig. 1d). v) Modulation of CD20 expression by NOTCH1 inhibition. GSI treatment increased both CD20 protein and transcript levels in 3'UTR NOTCH1-mut cases, as previously reported for coding NOTCH1-mut cases (p=0.0376 and p=0.0326, respectively; Fig. 1e,Pozzo et al., Leukemia, 2016). Conclusions.In keeping with an impaired NOTCH1 protein degradation, 3'UTR NOTCH1-mut cases had low CD20 expression levels, suggesting the introduction of 3'UTR NOTCH1 mutation evaluation in CLL patients undergoing anti-CD20 immuno-chemotherapy. Disclosures No relevant conflicts of interest to declare.

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Autocrine IL-6 Production Indicates the Level of Activated STAT3 and NF-Kb of Chronic Lymphocytic Leukemia Cells

Blood ◽

10.1182/blood.v118.21.1784.1784 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1784-1784

Author(s):

Fengting Liu ◽

Li Jia ◽

Timothy W Farren ◽

John G. Gribben ◽

Samir G Agrawal

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Cancer Cells ◽

Conflicts Of Interest ◽

Elevated Serum ◽

Serum Levels ◽

Lymphocytic Leukemia ◽

Apoptosis Resistance ◽

Prognostic Features ◽

Stat3 Phosphorylation

Abstract Abstract 1784 Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in survival, apoptosis and proliferation of cancer cells. Previous studies have shown that IL-6 levels are significantly elevated in most cancer cells, while elevated serum levels of IL-6 correlated with adverse prognostic features and shorter survival in patients with chronic lymphocytic leukemia (CLL). IL-6 production is controled by transcription factors NF-kB and STAT3, which are themselves controled by tyrosine kinase phosphorylation, such as that mediated by JAK2. This creates a positive feedback loop, as JAK2 can be stimulated by IL-6. In this study we investigated the relationship between autocrine IL-6 production and activation of STAT3 and NF-kB in CLL patients. The levels of IL-6 production in cell culture medium were measured by ELISA and activation of STAT3 and NF-kB was indicated by the ratio of p-STAT3/STAT3 and p-NF-kB/NF-kB. Patients with high levels of p-STAT3/STAT3 and/or p-NF-kB/NF-kB had higher levels of IL-6 production. The level of IL-6 production significantly correlated with STAT3 activation (R=0.856, p < 0.001) and NF-kB activation (R=0.815, p < 0.001). Inhibition of constitutive STAT3 and NF-kB activation by the STAT3 phosphorylation inhibitor, DPP, and the NF-kB inhibitor, CAPE, blocked IL-6 production 40% in average (p = 0.004, p = 0.003). Analysing the level of autocrine IL-6 production and in vitro spontaneous apoptosis in CLL cells from 38 patients, we found that patients with higher levels of IL-6 production (more than median) had significantly less apoptosis compared with those below the median (23% vs. 42.2% in average, p <0.0001). In this cohort, 60% of CD38+ cases, 78% of 17p- cases and 67% of cases with a lymphocyte doubling time of less than12 months were in the high IL-6 producer group. High IL-6 production was also associated with a shorter average time to first treatment (0.5 vs. 3 years, p=0.0023). This study demonstrates that autocrine IL6 production correlates with STAT3 and NF-kB activation and apoptosis resistance in CLL. Furthermore, higher IL-6 production is associated with biological markers of poor prognosis and earlier treatment. The IL6/JAK2/STAT3 signal pathway may offer new therapeutic targets for CLL. Disclosures: No relevant conflicts of interest to declare.

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