High-Resolution SNP-Array Profiling of Chronic Lymphocytic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 50-50 ◽  
Author(s):  
Jennifer Edelmann ◽  
Karlheinz Holzmann ◽  
W.M. Michael Kühn ◽  
Lars Bullinger ◽  
Ina Radtke ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Copy Number ◽  
Research Funding ◽  
Snp Array ◽  
Normal Karyotype ◽  
Lymphocytic Leukemia ◽  
Fish Analysis ◽  
Paired Data ◽  
New Genes ◽  
Genomic Aberrations

Abstract Abstract 50 Genomic aberrations are important prognostic factors in chronic lymphocytic leukemia (CLL) [Döhner et al., 2000]. However, known genomic aberrations fail to fully explain the biologic and clinical heterogeneity of the disease. We sought to precisely map copy number alterations (CNA) and copy number neutral losses of heterozygocity (LOH) to better characterize known recurrent aberrations and to identify new genetic lesions. We used Affymetrix 6.0 single nucleotide polymorphism (SNP) array analyses on CD19 sorted CLL cells. Data were analyzed using dChipSNP, a modified array normalization algorithm guided by cytogenetic abnormalities and a circular binary segmentation. We studied samples from 346 patients enrolled on the CLL8 trial of the German CLL Study Group. Data of 145 samples were analyzed against intraindividual reference DNA (paired), data of 201 samples against a pool of reference DNA (unpaired). FISH data were available for all samples, the distribution of genomic aberrations was as follows: del(13q14) in 59.8%, del(11q23) in 26.3%, trisomy 12 in 11.6%, and del(17p13) in 8.4%. IGHV was mutated in 32.9%, unmutated in 63.3%, and unknown in 3.8%. In total, 261 tumor-specific CNA could be discovered among the 145 paired samples. Genomic aberrations were found in 85.5% of these cases. The average number of aberrations per case was 1.8; according to the hierarchical model of genomic aberrations, it was 3.5 in del(17p), 2.4 in del(11q23), 1.7 in del(13q14) single, and 0.5 in normal karyotype CLL. The minimally deleted region (MDR) on 13q14 was 277.25 kb in size and contained mir15a and mir16, DLEU1 and DLEU2; RFP2 was not part of the MDR. Deletions on 13q were highly heterogeneous in size, ranging from 294 kb to 68 Mb. On 11q23 the MDR only contained ATM, the smallest lesion of 78.5 kb being intragenic; in two of theses cases, the deletion size was too small to be detected by FISH analysis. TP53 was affected in all del(17p13) cases except two; one tumor-specific deletion of 635.7 kb was detected in cytoband 17p13.2 harboring 30 genes and a second deletion of 780 kb in 17p13.3 containing – among 15 other genes – MNT, a tumor suppressor acting as an antagonist of MYC. A partial trisomy on chromosome 12 was not detected. Of the 261 CNA, 95 were located in genomic regions that are not evaluated by our routine FISH probe panel; 17 regions were affected recurrently: del(1p35.3) [2/145], del(1q23.3) [2/145], del(1q42.12) [2/145], +2p [5/145], del(3p21.31) [2/145], del(6p25.3) [3/145], +(6p25.3) [2/145], del(6q) [11/145], del(7q23.1) [2/145], +(8q24.21) [3/145], del(9q13-q21.13) [2/145], del(10q24) [2/145], del(14q24.3) [2/145], del(14q12.3) [2/145], del(15q15.1) [2/145], +18 [3/145] and +19 [7/145]. The frequency of these CNA was subsequently evaluated within the cohort of 201 (unpaired) samples. Five of 17 regions were affected in more than 2% in the whole cohort: +2p, del(6q), +8q24.21, del(15q15.1), and +19. Gain of 2p was found in 6.9% of cases, the minimally amplified region was 1.9 Mb in size and contained e.g. BCL11A and REL. Del(6q) was detected in 6,4%, the deletions were heterogeneous, an MDR could not be identified. 16 cases had 8q24.21 gains, the minimally amplified region was delineated by three intragenic gains in MYC. 14 cases had loss in 15q15.1 focussing on MGA, a potential suppressor of transcriptional activation by MYC. 8 cases had total or partial gains of chromosome 19, among those two overlapping partial gains with a minimally amplified region of 2.17 Mb in 19p13.2. Tumor-specific LOH were identified in 6.0% (9/145) located on 13q in three cases and in one case each on 17p, 12q, 11p, 1p, 3 and 22q. The LOH on chromosomes one and three overlapped with recurrent losses in 1p35.3 and 3p21.31. Essential members of the ATR-pathway were located in these regions: ATRIP and RPA2. However, mutational analyses of the two candidate genes in 48 cases revealed no mutations. SNP array analysis is a reliable tool to identify and further characterize genomic aberrations in CLL. MDR on 13q14 was delineated to a 277.25 kb segment affecting mir15a, mir16, DLEU1 and DLEU2 but not RFP2; the MDR on 11q23 to a segment only containing ATM. Cases with del(11q23) and del(17p) showed a higher genomic complexity than those with normal karyotype or del(13q14) as single abnormality. Relatively few novel genetic lesions were identified. Although occurring at low frequency, they may lead to the discovery of new genes involved in CLL pathogenesis. Disclosures: Stilgenbauer: Amgen: Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Sanofi Aventis: Research Funding.

Somatic and Germline Copy Neutral Loss of Heterozygosity Are Common in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4567-4567
Author(s):  
Megan Hanna ◽  
Bethany Tesar ◽  
Kristen E. Stevenson ◽  
Alexander R. Vartanov ◽  
Stacey M. Fernandes ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Loss Of Heterozygosity ◽  
Copy Number ◽  
Neutral Loss ◽  
Snp Array ◽  
Lymphocytic Leukemia ◽  
Alternative Mechanism ◽  
Large Set ◽  
The Impact ◽  
13Q Deletion

Abstract Abstract 4567 Chronic lymphocytic leukemia (CLL) is the most common leukemia of adults, and prognosis is still difficult to predict, although cytogenetic abnormalities identified by FISH are most helpful. Isolated reports have suggested that copy neutral loss of heterozygosity (cnLOH) can involve 13q and 17p in CLL, but the extent and the impact on clinical outcome is not well established. We therefore embarked upon characterization of cnLOH in a large set of 230 CLLs with matched normal DNA. The median age at diagnosis of CLL in this patient population was 54 (33–79). 87% of patients were Rai 0–1 at diagnosis, and 79% were chemotherapy naive at sampling. 121 of 230 patients were treated, with a median TTFT of 42 months. The median follow-up for surviving patients is 74 months. 44% of patients carried one somatic copy number abnormality (CNA) by SNP array, 20% two, 7% three, 5% four and 4% more than five. cnLOH was called by the Affymetrix Genotyping Console Software, which evaluates each SNP for copy number and then subtracts the A allele value from the B allele value within an individual sample, thereby allowing independent evaluation of tumor (somatic) and normal (germline). All calls were manually reviewed. A size cut-off of 1.0 Mb was used to determine significant cnLOH events. In total, of 230 patients, we found 26 events of somatic cnLOH (11%) and 36 events of germline cnLOH (16%), affecting 56 separate patients (24%). This frequency of cnLOH was surprisingly high and suggested that cnLOH might be an alternative mechanism affecting known loci in CLL. This was the case, as the most common events overall involved 13q in 25 patients, the X chromosome in 9 patients, chromosomes 17 and 18 in five patients each, and chromosomes 9, 11 and 12 in four patients each. Interestingly, germline events were quite common. Six patients had small regions of germline LOH with much more extensive adjacent somatic LOH, two on chr 13, one on chr 17, two on chr X and one on chr 20; these were coded as germline in the analysis. In addition, of the 25 patients with cnLOH on chromosome 13, 18 of these were in the germline and 7 were somatic. The region(s) of cnLOH were typically adjacent to a 13q deletion, and often involved the entire chromosome arm. Somatic cnLOH at 13q was associated with intermediate sized deletions including the RB gene (p=0.002). Of the 18 patients with germline cnLOH at 13q, 7 of them had no 13q deletion, while 7 had monoallelic deletion and 4 biallelic deletion. Thus 7 patients (3%) had cnLOH events at 13q, in the absence of 13q deletion, again suggesting an alternative mechanism affecting this locus. Germline cnLOH was associated with treatment prior to sampling (44% vs 17%, p<0.001), possibly due to its association with unmutated IGHV(58% vs 32%, p=0.008), and ZAP70 positivity (59% vs 36%, p=0.024). Somatic cnLOH was not associated with any patient characteristics. Neither somatic nor germline cnLOH was associated with >= 1 somatic CNA, but an association between both LOH types and >= 2 somatic CNAs was observed (p=0.053 germline and p=0.030 somatic). TTFT was reduced in patients with either germline cnLOH (61 mos vs 103, p=0.004) or somatic cnLOH (53 mos vs 107, p=0.008). Presence of two or more CNAs was also associated with short TTFT (48 mos vs 115, p<0.001). In order to assess the impact of cnLOH and CNAs on outcome independent of prior therapy, we evaluated TTFT in the 181 chemotherapy naive patients. In this subgroup, germline cnLOH was not associated with short TTFT, while somatic cnLOH (80 mos vs 125, p=0.018) and two or more somatic CNAs (80 mos vs 125, p=0.009) were. In multivariable Cox modeling including germline cnLOH, IGHV, and del 11q or 17p by FISH, the only significant predictor of TTFT was unmutated IGHV (hazard ratio (HR) 4.48, p<0.001). In multivariable Cox modeling including somatic cnLOH and the variables above, the only significant predictor of TTFT was again unmutated IGHV (HR 4.41, p<0.001). When the presence of two or more somatic CNAs was added to these models, this variable was significant along with IGHV (HR 2.04, p=0.009 in germline model; HR 1.84, p=0.033 in somatic model). We conclude that both somatic and germline cnLOH are common in CLL, affecting one quarter of patients in this dataset, and frequently involve chromosomal regions known to be important in CLL. cnLOH is associated with increased somatic CNAs and unmutated IGHV, and therefore poor prognosis. Disclosures: No relevant conflicts of interest to declare.

Genomic Complexity Identifies Patients with Agressive Chronic Lymphocytic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 489-489
Author(s):  
Sami N. Malek ◽  
Peter Ouillette ◽  
Harry Erba ◽  
Chris Saddler ◽  
Andrzej Jakubowiak ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Gold Standard ◽  
Copy Number ◽  
Univariate Analysis ◽  
Visual Complexity ◽  
Snp Array ◽  
Lymphocytic Leukemia ◽  
Genomic Complexity ◽  
Complexity Score ◽  
Trisomy 12

Abstract Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. CLL has a varied clinical course. Risk assessment and patient counseling relies on clinical parameters and validated biomarkers. Genetic biomarkers, as exemplified in the CLL FISH panel, allow for CLL patient risk stratification. Nonetheless, a subset of patients with apparently favorable lesions (del13q14 or normal FISH results) progresses rapidly and a fraction of patients with unfavorable findings (del17p or del11q) progress at less than predicted rate. Method: We studied 117 previously untreated CLL patients enrolled into a prospective study at the University of Michigan. We obtained unbiased, high-density, genome-wide measurements of sub-chromosomal copy number changes in highly purified DNA from sorted CD19+ cells and buccal cells using the Affymetrix 50K SNP-array platform. A genomic complexity score was derived and correlated with the validated surrogate endpoint time to first therapy (TTFT), a measure of disease aggressiveness. Genomic complexity was measured using two complementary methods: i) visual inspection of dChipSNP-generated heatmap displays for copy-number estimates for all patients and for all chromosomes. Two of the authors independently reviewed dChipSNP-based copy number displays and visually scored lesions that were i) at least 8 consecutive SNP positions in length and ii) either all blue (indicating less than 2N copy estimates=losses) or all red (indicating greater than 2N copy estimates=gains). Sensitivities and specificities for the visual lesion calling method were calculated against the clinical FISH data for del17p, del13q14, trisomy 12 and del11q as the gold standard. Sensitivities and specificities for (SNM/PDO) were [(94%/93%) and (93%/91%)] for del13q14, [(88%/88%) and (98%/98%)] for trisomy 12, [(100%/100%) and (99%/99%)] for del17p, and [(100%/100%) and (99%/100%)] for del11q, and ii) algorithmic detection of gains and losses using search methods optimized for detection using FISH-based del13q14 lesions as the gold standard. We devised a sliding window algorithm that scores a lesion as “copy loss” when a SNP window of 8 consecutive SNPs has at least 6 SNPs with a copy number estimate of 1.32 or less (the 8/6/1.32 rule). For validation, an approximately 5 Mb-long physical window was selected, overlapping with all described del(13q14) lesions. Defining an algorithmic del(13q14) call as any patient for whom the 8/6/1.32 rule identified at least one lesion within the 5Mb window, we achieved 81% sensitivity and 96% specificity for detecting or excluding 13q14 lesions as measured against the clinical FISH panel. Results: Within the group of 117 patients, 22(19%), 15(13%), 12(10%) and 10(9%) had visual complexity scores of equal to or greater than 2.5, 3, 3.5 or 4, respectively: a previously unanticipated degree of genomic complexity in CLL.In univariate analysis, CLL patients with four or more sub-chromosomal genomic lesions had a median TTFT of 15.8 months, whereas the median TTFT for all other patients was not reached in our study (two-sided p<10−4). In bivariate analysis, presence of genomic complexity defined a high-risk group of patients within the IgVH unmutated subgroup. Patients with unmutated IgVH/high complexity score had a TTFT of 16.5 months versus 95.6 months for unmutated IgVH/low complexity patients (p=0.02). Finally, high genomic complexity resulted in strong trends towards shorter TTFT for patients within other established risk groups.

Molecular Profiling of Cancer Testis Antigens in Chronic Lymphocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4701-4701
Author(s):  
Jason A Dubovsky ◽  
Emmanuel Berchmans ◽  
John J. Powers ◽  
Lin Hui-Yi ◽  
Yang Gao ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Fish Analysis ◽  
Rt Pcr ◽  
Chi Square ◽  
Clinical Criteria ◽  
Cancer Testis Antigens ◽  
Mutational Status ◽  
Cancer Testis

Abstract Abstract 4701 Background Chronic Lymphocytic Leukemia (CLL) is characterized by the progressive accretion of long-lived mature B-lymphocytes. Although the classical Rai and Binet staging is still commonly used, a new molecular understanding has identified specific signatures which could help predict disease progression and survival. Given the recent success of immunotherapeutic strategies and immunomodulatory drugs in the treatment of CLL we sought to identify potentially immunologically relevant targets and their relation to well known disease criterion. Methods Our study characterized the mRNA expression of 29 known cancer-testis antigens (CTAs) in 66 patients with CLL at varying stages of disease using a RT-PCR based expression panel. Relevant clinical criterion such as RAI stage, B2m, ZAP-70, IGVH mutational status, CD38, cytogenetics by FISH analysis, WBC count, age, gender, and treatment among others were then taken into account. The binary RNA expression data associated with the clinical and demographic factors were evaluated using chi-square or Wilcoxon rank sum analysis. Results Of the cancer-testis antigens tested, the MAGE family of CTAs revealed statistically significant correlations with multiple clinical criteria. Our analysis reveals a correlation between previous chemo-immunotherapy treatment and MAGE-A1, B2, E1, MAD-CT-2, SPA-17, and PAGE-5 expression. Beyond treatment, total white blood cell count was shown to have a significant association with MAGE family members A1, A3, and B2 expression. In addition, MAD-CT-2 and MAGE-B2 were significantly correlated with the expression of FMC-7 and SSX-4 and LAGE-1 correlated with the presence of B-cell symptomatology. Conclusions Preliminary RT-PCR based CTA phenotyping has unveiled interesting correlations to clinical criteria, opening multiple avenues for future immunotherapeutic interventions as well as possible prognostic value in CLL. Further investigation to better understand the biological value of this information in warranted. Disclosures: Pinilla: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding; exelixis: Research Funding.

Genomic Aberrations in Chronic Lymphocytic Leukemia and Small Lymphocytic Lymphoma by Absolute Lymphocyte Count: Analysis of 1694 Patients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4958-4958
Author(s):  
Apostolia-Maria Tsimberidou ◽  
Peter McLaughlin ◽  
Susan O’Brien ◽  
Sijin Wen ◽  
William G. Wierda ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Multivariate Analysis ◽  
Chronic Lymphocytic Leukemia ◽  
Lymphocyte Count ◽  
Prognostic Significance ◽  
Absolute Lymphocyte Count ◽  
Lymphocytic Leukemia ◽  
Fish Analysis ◽  
Small Lymphocytic Lymphoma ◽  
Genomic Aberrations

Abstract Introduction: Genomic aberrations provide information for the pathogenesis and clinical outcomes of patients with chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). We assessed whether genomic aberrations in CLL/SLL differ by absolute lymphocyte count (ALC) and evaluated their prognostic significance in our series of patients with CLL/SLL. Methods: We reviewed the medical records of patients with CLL/SLL who presented to The University of Texas M. D. Anderson Cancer Center from 1985 to 2005. Patients were assessed by conventional bone marrow cytogenetics and/or fluorescence in situ hybridization (FISH) analysis using probes for trisomy 12, ATM, LAMP1, D13S319, and P53 genes. Results: Cytogenetics and/or FISH analysis was performed in 1694 of 2189 consecutive patients with CLL/SLL. Patients with ALC &lt; 5 × 109/L had lower rates of genomic aberrations by cytogenetic and/or FISH analysis than did those with ALC ≥ 5 × 109/L (25% vs. 37%, p=0.02). Deletion 17p +/− other abnormalities and 6q del +/− other abnormalities were each associated with shorter survival compared with other genomic aberrations or normal karyotype (p&lt;0.0001 for both results). The survival rates by genomic aberration are shown in the Figure [insert figure here]. In a multivariate analysis of 23 clinical and laboratory factors, deletion 17p or 6q +/− other genomic aberrations (p&lt;0.0001) was the strongest independent predictor of shorter survival. In patients who required therapy for CLL/SLL, independent factors predicting response were hemoglobin levels &gt; 11 g/dL (p&lt;0.0001), age &lt; 60 years (p=0.005), absence of 17p deletion (p=0.048), and absence of 6q deletion (p=0.03). In multivariate analysis, pretreatment parameters that remained independently significant for longer failure-free survival (FFS) were age &lt; 60 years (p&lt;0.0001), absence of 17p del +/− other abnormalities (p&lt;0.0001), and hemoglobin levels &gt; 11 g/dL (p=0.018). Conclusions: Patients with CLL/SLL and ALC &lt; 5 × 109/L had lower rates of genomic aberrations by cytogenetic and/or FISH analysis (p=0.02), possibly because of less bone marrow infiltration in the lower-ALC group. Deletion 17p or 6q, with or without other genomic aberrations was the strongest independent adverse prognostic factor for survival in CLL/SLL. Figure Figure

Combination of Ofatumumab and Lenalidomide In Patients with Relapsed Chronic Lymphocytic Leukemia: Initial Results of a Phase II Trial

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2464-2464 ◽  
Author(s):  
Xavier Badoux ◽  
Susan O'Brien ◽  
William G. Wierda ◽  
Stefan Faderl ◽  
Zeev Estrov ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Phase Ii ◽  
Research Funding ◽  
Performance Status ◽  
Single Agent ◽  
Lymphocytic Leukemia ◽  
Fish Analysis ◽  
Significant Activity ◽  
Superficial Vein Thrombosis ◽  
Grade 3

Abstract Abstract 2464 Frontline chemoimmunotherapies induce high response rates in patients with CLL. Once disease recurs, however, effective treatment options are limited and new therapeutic modalities and combinations are needed. Ofatumumab is a fully humanized anti-CD20 monoclonal antibody which produces an overall response rate (ORR) of 47%–58% in patients with fludarabine-refractory CLL (Wierda W. et al, 2010). Lenalidomide, an immunomodulatory agent, induces an ORR of 32–47% in patients with relapsed/refractory CLL, (Chanan-Khan A.A. et al. 2006; Ferrajoli A. et al. 2008). The rationale for combining ofatumumab and lenalidomide is based on their single agent efficacy, distinct and potentially complimentary mechanisms of action and non-overlapping toxicity profiles. Furthermore, the combination of lenalidomide and rituximab has shown significant activity in patients with relapsed disease (Ferrajoli et al. 2009). We, therefore, designed a phase II study to evaluate efficacy and tolerability of ofatumumab and lenalidomide given in combination in patients with relapsed CLL. Patients with active disease were eligible if they had received prior treatment with purine analog-based therapy, had an ECOG/WHO performance status of 0–2, adequate renal (creatinine clearance > 30ml/min) and hepatic function (total bilirubin < to 2 mg/dl and ALT < 2 × ULN). Patients with any neutrophil count were eligible, whereas patients with platelet counts < 30,000 mm3, positivity for HIV, active hepatitis B or C or recent history of tuberculosis were excluded from participation. In this trial ofatumumab is administered intravenously weekly for four consecutive weeks (300mg week 1, 1,000 mg week 2 and all subsequent doses), then monthly for months 2–6 and once every two months for months 7–24. Lenalidomide is given orally at the dose of 10 mg daily, starting on day 9 and continued daily. Allopurinol at the dose of 300mg daily is given during the first two weeks of treatment as tumor lysis prophylaxis. Treatment duration is 24 months, and responses are assessed after 3, 6, 12, 18 and 24 months of therapy. Thus far 26 of the 40 planned patients have been accrued to this study and we present an analysis of response and toxicity for the first 16 patients that have been on study for at least 3 months. The median age of the patients is 62 yrs (45–82). Eight patients (50%) had Rai stage III-IV disease. The median Beta-2M level was 4.4 mg/dL (2–6.1). The median number of prior treatments was 2 (1–8). Four patients (25%) were refractory to fludarabine and all pts had received prior rituximab. Nine patients (56%) had unmutated IGHV genes, 5 patients (31%) had chromosome 17p deletion and 3 patients (19%) had 11q deletion as detected by FISH analysis. Responses were evaluated according to the 2008 IWCLL criteria: 10 of the 16 evaluable patients achieved a response [2 CR (13%), 8 PR (50%)] for an ORR of 63%. Four patients with stable disease are continuing on treatment. One patient discontinued therapy and did not return for response assessment and another patient progressed. All patients are alive. The most common grade 3–4 treatment related adverse events observed were: neutropenia (8 pts, 50%) and anemia (2 pts, 13%). One patient (6%) developed grade 2 superficial vein thrombosis. Lenalidomide-associated tumor flare reaction was limited to grade 1 in 2 patients (13%) while a grade 3 infusion reaction was observed in 1 patient (6%) during the first ofatumumab administration. Three grade 3 infectious episodes occurred: 2 cases of pneumonia and 1 case of parotiditis. None of the patients received routine antibiotic prophylaxis. The median daily dose of lenalidomide tolerated was 5 mg/day (2.5–10 mg). In conclusion, our initial analysis indicates that the combination of ofatumumab and lenalidomide is therapeutically active in patients with relapsed CLL. This treatment is well tolerated. Neutropenia is the most common toxicity observed. Enrollment is ongoing, and updated results will be provided. Disclosures: Off Label Use: Ofatumumab and lenalidomide in patients with relapsed chronic lymphocytic leukemia. O'Brien: GlaxoSmithKline: Consultancy. Wierda: GlaxoSmithKline: Honoraria, Research Funding; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees. Estrov: Celgene Corporation: Consultancy. Keating: Celgene Corporation: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria. Ferrajoli: Celgene Corporation: Research Funding; GlaxoSmithKline: Research Funding.

A20 Gene Deregulation In Waldenstrom's Macroglobulinemia.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3628-3628
Author(s):  
Stephanie Poulain ◽  
Rachid Aijjou ◽  
Natacha Broucqsault ◽  
Salomon Manier ◽  
Agnes Daudignon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Real Time ◽  
Expression Profiling ◽  
Copy Number ◽  
Research Funding ◽  
Snp Array ◽  
Fish Analysis ◽  
Nfkb Pathway ◽  
A20 Gene

Abstract Abstract 3628 Background. Waldenstrom's macroglobulinemia (WM) is a rare lymphoproliferative disorder characterized by bone marrow (BM) infiltration of lymphoplasmacytic cells and monoclonal IgM gammopathy. The deletion 6q is the most frequent cytogenetic aberration in WM and it has been suspected that this region harbours a tumour suppressor gene of pathogenic significance for WM. Comparative genomic hybridization (CGH) array delineated several minimal deleted regions (MDR) on 6q and pointed out key regulator genes of the NFKB pathway involved in these deletions, including A20 gene. The zinc finger protein A20 has been characterized as dual inhibitor of NFKB activation and was recently described as a tumor suppressor gene in lymphoma. However, the mechanisms of A20 deregulation are not fully understood in WM. We aimed to study the mechanisms of A20 deregulation in WM using gene expression profiling (GEP) and single nucleotide polymorphism (SNP) based arrays, a powerful high resolution method allowing both the detection of loss of heterozygosity (LOH) and copy number alteration (CNA) analysis in the same experiment. Methods. We have studied BM samples from 42 patients (pts) with WM (31 males, mean age: 67 years). Conventional karyotype study was analyzed following stimulation with IL2 and DSP30 of BM cells. FISH analysis was performed to detect deletion 6q21 (MYB probe). DNA and RNA were extracted following BM CD19 B cell negative selection. Quantification of copy number of A20 gene was performed using real time PCR, RNAse P gene was used as reference gene. Genome-Wide Human SNP Array 6.0 (Affymetrix chips) was used to detect both LOH and CNA in 31 pts. Paired samples (B cells/normal T lymphocytes) were used as an intra-individual reference to distinguish germline polymorphisms from somatic abnormalities in 29 patients. Size, position and location of genes were referenced using UCSC HG18 assembly, LOH and CNA were identified using genotyping console 3.02 software (Affymetrix) and Partek genomic suite. Gene expression profiling (GEP) was performed in 11 pts using Affymetrix U133A arrays. Gene-expression intensity values were log transformed, normalized and analyzed using GeneSpring GX software. Results. SNP array identified deletion 6q in 9/31 (29%) patients, confirmed by FISH analysis. All cases were monoallelic deletion. In 90% of cases, deletion 6q was associated with others genetic abnormalities. In one case, we observed a loss of 6q combined with a gain of 6p. Deletion 6q was associated with gain of chromosome 4 in 2 cases, and with deletion of 13q in 3 cases. The MDR was located on chromosome 6q21-6q25. This MDR contained several candidate genes included always deletion of A20 gene, located on 6q23, and was confirmed by real time DNA PCR quantification in all cases. Overall, the frequency of A20 gene deletion in our entire WM cohort (N=42 pts) was 29.2% (12/42), determined by real time DNA PCR of the copy number of A20 gene. In patient without deletion 6q, we looked at potential deregulated mechanisms of A20 by LOH with no CNA, e.g. UPD (uniparental disomy). We have not seen any UPD targeting the A20 gene. We then looked at A20 gene expression by GEP, and found no significant variation of A20 gene expression related to A20 monoallelic deletion as compared to pts with two copies, reflecting probably the existence of other mechanisms of A20 gene deregulation. This result was consistent with the absence of clear difference in genome-wide expression profiling in pts with 6q deletion and those without the deletion. As A20 is a key player in the negative feedback loop regulation of NFKB, we also looked at the gene expression of a set of genes involved in NFKB pathway. The presence or absence of A20 deletion did not influence the gene expression profiling of this NFKB pathway gene set. Conclusion. We described a high frequency of deletion of A20 gene. In most cases, deletion of A20 was associated with other genetic abnormalities. A20 deletion was not associated with a significant signature by GEP. Additional studies are needed to understand the cellular consequences of A20 deletions in the pathogenesis of WM. Disclosures: Leleu: Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

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

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4581-4581
Author(s):  
Giovanni Del Poeta ◽  
Francesca Maria Rossi ◽  
Maria Ilaria Del Principe ◽  
Michele Dal Bo ◽  
Annalisa Biagi ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Normal Karyotype ◽  
Progression Free Survival ◽  
Intermediate Stage ◽  
Lymphocytic Leukemia ◽  
Prognostic Indicators ◽  
Fish Analysis ◽  
Clinical Difference ◽  
Trisomy 12

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

scholarly journals Mechanisms of Adaptation to Ibrutinib in High Risk Chronic Lymphocytic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 585-585 ◽  
Author(s):  
Valeria Spina ◽  
Gabriela Forestieri ◽  
Antonella Zucchetto ◽  
Alessio Bruscaggin ◽  
Tamara Bittolo ◽  
...  
Keyword(s):  
High Risk ◽  
Chronic Lymphocytic Leukemia ◽  
Nuclear Localization ◽  
Peripheral Blood ◽  
Research Funding ◽  
Clonal Evolution ◽  
Lymphocytic Leukemia ◽  
Adaptation Process ◽  
Over Time ◽  
Stimulation Of

Abstract Introduction. Ibrutinib inhibits the BTK molecule downstream the B-cell receptor (BCR). Though highly active in high risk chronic lymphocytic leukemia (CLL), the most typical response achievable in patients is a minimal residual disease (MRD) positive partial remission (PR) which is maintained until the development of genetically driven resistance caused by the acquisition of mutations in the BTK or PLCG2 genes. The study aims at characterizing the adaptation process allowing residual CLL cells to persist despite BTK inhibition. Methods. The IOSI-EMA-001 study (NCT02827617) is an observational study consisting in the prospective and longitudinal collection of peripheral blood samples and clinical data from high risk CLL patients treated with ibrutinib. Peripheral blood CLL cells longitudinally drawn from patients before treatment start and at fixed timepoints under ibrutinib were monitored by: i) next generation flow cytometry approaches for changes in proliferation rate, surfaceome, and pathway activation; and ii) CAPP-seq targeted deep next generation (sensitivity ~10-3) for clonal evolution. Results. The study cohort comprised 31 high risk CLL patients, including 15 treatment naïve, 16 relapsed, 80% IGHV unmutated, 42% 17p deleted and 55% TP53 mutated. Median duration of ibrutinib treatment was 45 weeks (24-72 weeks). All patients obtained a MRD positive PR that was maintained in all but one who progressed with a PLCG2 mutation (VAF 3%). Compared to baseline, under ibrutinib therapy CLL cells slowed down their proliferation, as suggested by the decreased expression of Ki-67, the reduction of the proliferating fraction (CXCR4dimCD5bright), and the increase of the resting fraction (CXCR4brightCD5dim). Compared to baseline, under ibrutinib therapy CLL cells also upregulated BCR and adhesion/homing proteins, and decreased the expression of BCR inhibitor proteins. Upon stimulation of the BCR with anti-IgM, the downstream path through pBTK and pPLCG2 was inhibited by ibrutinib, while conversely the downstream path through pAKT and pERK was still inducible throughout all the assessed timepoints. The proportion of CLL cells harboring nuclear localization of NF-kB progressively increased over time under ibrutinib. NF-kB nuclear localization was inducible throughout all the assessed timepoints by CD40L stimulation of the non-canonical NF-kB pathway, but not by anti-IgM stimulation of the BCR/canonical NF-kB pathway. Overall, 880 individual mutations were longitudinally discovered and monitored across a total of 121 sequential timepoints collected during ibrutinib treatment. Clonal evolution was observed in (67.7%) cases, a proportion rate previously documented in CLL treated with chemoimmunotherapy. Clonal evolution appeared to be heterogeneous involving different genes without a stereotypic targeting. Consistently, none of the main driver gene mutations was homogeneously selected or suppressed by ibrutinib suggesting that the biological adaptation of CLL cells under ibrutinib is not genetically driven. Clonal evolution propensity was not associated with any of the biomarkers of the disease, and it did not decrease over time under ibrutinib. Conclusions. Taken together these results suggest that residual CLL cells persisting under ibrutinib therapy adapt their phenotype by upregulating adhesion molecules, chemokine receptors and BCR molecules, and by maintaining a competence of BCR signaling through the PI3K/AKT/ERK pathway. The progressive selection of CLL cells having NF-kB in the nucleus, likely due to the BTK independent non-canonical NF-kB pathway, might explain their survival despite ibrutinib therapy. Finally, clonal evolution is not suppressed by ibrutinib chemotherapy, and despite does not seem to be directly involved in such adaptation process, may ultimately favor the acquisition of BTK and PLCG2 ibrutinib resistance mutations. Disclosures Zucca: Celltrion: Consultancy; AstraZeneca: Consultancy. Ghia:Sunesis: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie, Inc: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; BeiGene: Honoraria, Research Funding. Montillo:Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding. Tedeschi:Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy; AbbVie: Consultancy. Gaidano:AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Morphosys: Honoraria; Roche: Consultancy, Honoraria.

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