Abnormalities In Chronic Lymphocytic Leukemia Research Articles

Utility of Targeted Sequencing Compared to FISH for Detection of Chronic Lymphocytic Leukemia Copy Number Alterations.

Chronic lymphocytic leukemia (CLL) is characterized by multiple copy number alterations (CNAs) and somatic mutations that are central to disease prognosis, risk stratification, and mechanisms of therapy resistance. Fluorescence in situ hybridization (FISH) panels are widely used in clinical applications as the gold standard for screening prognostic chromosomal abnormalities in CLL. DNA sequencing is an alternative approach to identifying CNAs but is not an established method for clinical CNA screening. We sequenced DNA from 509 individuals with CLL or monoclonal B-cell lymphocytosis (MBL), the precursor to CLL, using a targeted sequencing panel of 59 recurrently mutated genes in CLL and additional amplicons across regions affected by clinically relevant CNAs [i.e., del(17p), del(11q), del(13q), and trisomy 12]. We used the PatternCNV algorithm to call CNA and compared the concordance of calling clinically relevant CNAs by targeted sequencing to that of FISH. We found a high accuracy of calling CNAs via sequencing compared to FISH. With FISH as the gold standard, the specificity of targeted sequencing was >95%, sensitivity was >86%, positive predictive value was >90%, and negative predictive value was >84% across the clinically relevant CNAs. Using targeted sequencing, we were also able to identify other common CLL-associated CNAs, including del(6q), del(14q), and gain 8q, as well as complex karyotype, defined as the presence of 3 or more chromosomal abnormalities, in 26 patients. In a single and cost-effective assay that can be performed on stored DNA samples, targeted sequencing can simultaneously detect CNAs, somatic mutations, and complex karyotypes, which are all important prognostic features in CLL.

Counting Chromosome Abnormalities in Chronic Lymphocytic Leukemia: Comparison of Iscn 2020 and MDS International Working Group Guidelines for Establishing Karyotype Complexity

Karyotype complexity is established by enumerating the number of chromosome abnormalities present in a karyotype. In chronic lymphocytic leukemia (CLL), karyotype complexity, defined as ≥3 chromosome abnormalities, is an important prognostic indicator associated with poor outcomes. Additionally, stratifying patients (pts) by highly complex karyotypes (≥5 chromosome abnormalities) has shown significantly poorer outcomes compared to pts with 3-4 abnormalities (Baliakas, Blood, 2019). In principle, counting chromosome abnormalities is straightforward; however, in practice it can be nuanced, and the number of abnormalities counted for the same karyotype can differ across laboratories. A lack of standardized rules has resulted in variability in establishing complexity across CLL studies. We and others have proposed adapting the international working group on myelodysplastic syndrome (MDS) framework for use in CLL (Chun, Leuk Res, 2010; Jondreville, Am J Hematol, 2020). Additionally, a separate set of guidelines were published in the 2020 International System for Human Cytogenomic Nomenclature (ISCN). To investigate how different guidelines may impact patient categorization in correlative studies, we evaluated karyotype complexity using both guidelines in a previously reported, well-characterized, dataset with long term follow-up of patients with CLL treated with ibrutinib (Kittai, Blood, 2021). 456 patient karyotypes underwent expert review by two cytogeneticists. Discordant scores were discussed to reach consensus. Pts were categorized as not complex (0-2 abnormalities), complex (3-4), or highly complex (≥5) for each set of guidelines. The weighted kappa was used to evaluate agreement between the two guidelines. Progression free survival (PFS) from ibrutinib start and overall survival (OS) were evaluated by the Kaplan-Meier method. Univariable Cox regression model discrimination power for PFS and OS were evaluated by Uno's concordance statistic. MDS guidelines categorized 50.2% as not complex, 19.7% complex and 30.1% as highly complex; similarly, ISCN categorized 50.2% as not complex, 20.2% complex and 29.6% as highly complex. Complexity categories assigned by the two guidelines had a weighted kappa agreement of 0.88 (95% CI 0.84-0.91), with 11% of pts placed in discordant complexity categories. Albeit numbers of pts were small, evaluation of these discordant cases compared to the consensus concordant cases showed outcomes varied. Discordant cases categorized by the MDS guidelines as complex and by ISCN as highly complex showed longer OS compared to the concordant complex and highly complex cases, suggesting ISCN overrated complexity (Fig 1). Conversely, discordant cases categorized by MDS guidelines as highly complex while ISCN categorized as complex showed decreased OS compared to the concordant complex and highly complex cases, suggesting ISCN underrated complexity in these cases (Fig 2). PFS trended similarly for these groups. Both the MDS and the ISCN guidelines showed increased karyotype complexity was significantly associated with shorter PFS (both with p<0.0001) and OS (both with p<0.0001). Comparing the classifiers not complex, complex and highly complex for the entire cohort found no significant difference between the prediction power of the MDS and ISCN guidelines for PFS. For OS, the MDS guidelines had a statistically stronger prediction power compared to ISCN (p=0.0235). Further, when the poorest prognostic group (highly complex) was compared to all others, the MDS guidelines showed significantly stronger prediction power for both PFS (p=0.0359) and OS (p=0.0039) compared to ISCN. Although both sets of guidelines were prognostically significant, in this predominately relapsed/refractory CLL patient population receiving ibrutinib, the MDS guidelines performed more robustly when used as a categorical variable. Clinical trials incorporating karyotype complexity for prognostication, or for treatment stratification, should specify a set of guidelines to apply consistently across the trial. Given the importance of karyotype complexity, particularly with targeted therapies, further studies are needed to evaluate these two sets of guidelines across varying CLL populations to establish recommendations for assigning karyotype complexity uniformly in CLL.

Clinical characteristics of 11 patients with chronic lymphocytic leukemia with t (14;19) (q32;q13)

Objective: To analyze the clinicopathological characteristics of 11 cases of chronic lymphocytic leukemia (CLL) with t (14;19) (q32;q13) . Methods: The case data of 11 patients with CLL with t (14;19) (q32;q13) in the chromosome karyotype analysis results of the Blood Diseases Hospital, Chinese Academy of Medical Sciences from January 1, 2018, to July 30, 2022, were retrospectively analyzed. Results: In all 11 patients, t (14;19) (q32;q13) involved IGH::BCL3 gene rearrangement, and most of them were accompanied by +12 or complex karyotype. An immunophenotypic score of 4-5 was found in 7 patients and 3 in 4 cases. We demonstrated that CLLs with t (14;19) (q32;q13) had a mutational pattern with recurrent mutations in NOTCH1 (3/7), FBXW7 (3/7), and KMT2D (2/7). The very-high-risk, high-risk, intermediate-risk, and low-risk groups consisted of 1, 1, 6, and 3 cases, respectively. Two patients died, 8 survived, and 2 were lost in follow-up. Four patients had disease progression or relapse during treatment. The median time to the first therapy was 1 month. Conclusion: t (14;19) (q32;q13), involving IGH::BCL3 gene rearrangement, is a rare recurrent cytogenetic abnormality in CLL, which is associated with a poor prognosis.

FISH Analysis of CDKN2A/B Deletion in Chronic Lymphocytic Leukemia

Introduction: The course of chronic lymphocytic leukemia (CLL) ranges from indolent to rapid disease progression, including transformation to an aggressive lymphoma (Richter transformation, RT). A subset of CLL pts have been shown to acquire deletions of the tumor suppressor CDKN2A/B. RT has been associated with deletion of CDKN2A/B, however, prior studies’ sample populations were limited or not primarily focused on those with this deletion. Due to the rarity of this genetic abnormality in CLL and severity of RT, additional studies concerning this subgroup should be investigated. Here, we examine a larger cohort of CLL pts with CDKN2A/B deletion by fluorescence in situ hybridization (FISH) to provide further clarity on this genetic lesion. Methods: We conducted a retrospective case study for all pts for whom cytogenetic clinical samples were submitted for extended CLL FISH panel analysis over a 3.5 year (yrs) period, between 2015 and 2018 at The Ohio State University. Pts were screened for CDKN2A/B deletion with a new or follow up diagnosis of CLL or small lymphocytic lymphoma (SLL). Cytogenetic testing was performed on cells stimulated with pokeweed mitogen, phorbol myristate acetate (PMA), and CpG oligonucleotides. FISH for D13S319, D12Z3, ATM, TP53, BCL6, MYC, REL, SEC63, MYB, and CDKN2A/B was performed via manufacturer's recommendations. IGHV mutational status was determined using PCR. A 50-gene hematologic sequencing panel was performed via Ion Torrent. Overall survival (OS) from first detection of abnormal CDKN2A/B results were examined. Cox regression model was used to evaluate OS post CDKN2A/B deletion. The Fine and Gray Model was used to examine correlation of variables with regard to RT, treating death without RT as a competing risk. Results: 636 pts had sample(s) submitted for extended CLL FISH panel analysis. Of these, 43 (6.8%) pts with confirmed CLL or SLL were found to have loss of CDKN2A/B by FISH, including 6 pts within one year of diagnosis. Median age at diagnosis was 54.9 yrs (range 41.9-77.7). Loss of CDKN2A/B was detected at a median of 7.8 yrs (range diagnosis-26.1) from diagnosis. Of those with known IGHV status (n=33), 91% were unmutated. A complex karyotype (>3 clonal chromosomal abnormalities) was found in 90% percent of pts. In regard to CDKN2A/B deletion, 77%, 16%, and 7% of pts had heterozygous, homozygous, or subclonal populations of heterozygous as well as homozygous loss, respectively. Additional FISH testing showed 23% had gain of REL, 16% had BCL6 gain/rearrangement, 16% had deletion 6q, 37% had gain/rearrangement of MYC, 16% had loss of ATM, 14% had trisomy 12, and 58% had deletion 13q. Deletion of TP53 was seen in 63%. Sequencing data was available for 15 pts (35%), of note, 10 pts had mutations in TP53, with 4 also having mutations in SF3B1. In terms of OS, there were 30 deaths in the cohort, with median OS of 10.8 yrs from diagnosis (95% CI 8.0-14.4). With a median follow-up of 4.9 yrs among survivors, the median OS from time of detection of CDKN2A/B deletion was 1.7 yrs (95% CI 0.3-4.7). Multivariable analysis of OS post CDKN2A/B detection was significant for increasing beta-2-microglobulin measured at first visit at our institution (p=0.0044, hazard ratio (HR) 1.32 (95% CI 1.09-1.60)) and CDKN2A/B subclonal heterozygous and homozygous loss (p=0.0024, HR 9.81 (95% CI 2.25-42.73)). Of the 43 pts with loss of CDKN2A/B, 21 (49%) progressed via RT (n=20) or prolymphocytic leukemia (PLL) (n=1). The Cumulative Incidence Rate of RT development 12-months post CDKN2A/B deletion was 23.5% (95% CI 10.9-38.9), at 24-months, it was 29.4% (15.1-45.3). Deletion of TP53 was seen in 85.7%. Albeit there was limited pts with detection of CDKN2A/B deletion prior to RT, the multivariable model for RT found loss of TP53 (p=0.02, HR 4.53 (95% CI 1.33-15.43)) and homozygous and heterozygous loss of CDKN2A/B (p=0.0008, HR 6.08 (95% CI 2.13-17.39)) were independently significant. Conclusion: Here we show that deletion of CDKN2A/B in CLL is a very rare occurrence and was seen predominantly in pts with highly aggressive disease characteristics. Close to half of our cohort progressed with RT or PLL. Additional investigation of serial samples is needed to establish when CDKN2A/B deletion occurs relative to RT. Our results indicate that prospective FISH analysis for CDKN2A/B deletion, in conjunction with other high-risk genetic alterations, to establish RT prognostication in high-risk CLL is warranted.

Structure and prognostic signifi cance of 13q14 deletion in chronic lymphocytic leukemia

Introduction. 13q14 deletion is the most common chromosomal abnormality in chronic lymphocytic leukemia (CLL), and as the sole abnormality determines the most favorable prognosis of the disease. Using molecular genetic methods two subtypes of 13q14 deletion were identifi ed based on the size of the lost chromosomal material: small (type I) with the involvement of the D13S319 segment containing MIR15A/MIR16-1 and DLEU1 genes and large (type II) containing centromeric region of 13q14 involving RB1 gene. Data on the impact of type I and II deletions on the course of CLL are controversial.Aim — to evaluate the prognostic signifi cance of different variants of 13q14 deletion in CLL.Patients and methods. The study enrolled two cohorts of CLL patients. Cohort 1: 256 patients who were studied by FISH with DNA probes for detection of 13q14/D13S319, 11q23/ATM, 17p13/TP53 deletions, and trisomy 12 before immunochemotherapy. 101 patients with identifi ed 13q14/D13S319 deletion were analyzed with a DNA probe for RB1 locus for determination of deletion size (type I or type II). Cohort 2: 28 patients at different stages of the disease with deletion 13q14 detected by FISH were studied by using combination of standard and molecular cytogenetic methods (mFISH, mBAND, arrayCGH) to clarify the structure of 13q abnormalities.Results. In Cohort 1 chromosomal aberrations were detected in 75 % of patients: 13q deletion — 52 % (isolated — 36 % of all cases and 48 % of cases with deletion), 11q deletion — 19 %, +12 — 13 %, 17p deletion — 6 %. 13q14 deletion type I was detected in 56 %, type II — in 44 % of patients. Type II deletion correlated with the presence of 11q deletion (p = 0.05). Isolated deletions of type I and II were found in 61 and 39 %, respectively. Biallelic deletion was identifi ed in 12.7 % of patients with 13q deletion. Statistically signifi cant differences in OS were obtained in type I and II groups of patients with isolated 13q14 deletions: median OS was not reached and made 67.5 months, respectively, p = 0.05. In Cohort 2 structural abnormalities of chromosome 13 by conventional cytogenetic analysis (CCA) were identifi ed in 50 % of cases: 13q deletion — 11 cases; translocations involving 13q14 — 6 cases. In 5 cases with biallelic deletion identifi ed by FISH, 13q14 deletion by CCA was detected in two patients, and only in one allele.Conclusion. In general, 13q14 deletion is a cytogenetic factor of favorable prognosis for CLL but its structure is heterogeneous. Loss of tumor suppressor RB1 (type II deletion) negatively affects OS in patients treated with immunochemotherapy

An Anomaly with Potential as a New Prognostic Marker in CLL with del(13q): Gain of 16p13.3

Deletion 13q [del(13q)] is a favorable prognostic marker if it is detected as a sole abnormality in chronic lymphocytic leukemia (CLL). However the clinical courses of cases with isolated del(13q) are quite heterogeneous. In our study, we investigated copy number variations (CNVs), loss of heterozygosity (LOH), and the size of del(13q) in 30 CLL patients with isolated del(13q). We used CGH+SNP microarrays in order to understand the cause of this clinical heterogeneity. We detected del(13q) in 28/30 CLL cases. The size of the deletion varied from 0.34 to 28.81 Mb, and there was no clinical effect of the deletion size. We found new prognostic markers, especially the gain of 16p13.3. These markers have statistically significant associations with short time to first treatment and advanced disease stage. Detecting both CNVs and LOH at the same time is an advantageous feature of aCGH+SNP. However, it is very challenging for the array analysis to detect mosaic anomalies. Therefore, it is very important to confirm the results by FISH. In our study, we detected approximately 9% mosaic del(13q) by microarray. In addition, the gain of 16p13.3 may affect the disease prognosis in CLL. However, additional studies with more patients are needed to confirm these results.

Utilization of a Targeted Next Generation Sequencing Assay to Identify Copy Number Alterations in Chronic Lymphocytic Leukemia and Monoclonal B-Cell Lymphocytosis

Utilization of a Targeted Next Generation Sequencing Assay to Identify Copy Number Alterations in Chronic Lymphocytic Leukemia and Monoclonal B-Cell Lymphocytosis

COMPARISON BETWEEN MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION (MLPA) AND CYTOGENETICS FOR VALIDATION OF CHROMOSOMAL ABERRATIONS IN CHRONIC LYMPHOCYTIC LEUKEMIA

Recently, a multiplex ligation-dependent probe amplification (MLPA®) has emerged as a method that is reported to be fast, sensitive, and cost-effective that can detect copy number variation. In the present study, MLPA analysis was compared with chromosome banding analysis (CBA) data to determine the efficiency of MLPA in patients with chronic lymphocytic leukemia (CLL). CBA karyotyped 65 patients. Chromosomal analysis was performed on G-banded metaphase cells from cultured lymphocytes in a culture medium at 37°C for 72 hours. Numerical, as well as structural abnormalities were reported on at least 20 well-spread and well-banded metaphases. Genomic DNA was extracted from peripheral blood samples and subjected to MLPA through the probemix, kit SALSA®MLPA®P037 containing 54 MLPA probes, including 41 probes for 2p, 6q 8p/q, 9p21, 11q, 12p/q, 13q, and 17p chromosomal regions. The PCR products were separated by capillary electrophoresis using an ABI 3500 DNA analyzer. Cytogenetics resulted in 41 normal karyotypes, and 25 karyotype aberrations (13 tris12; 3 del13; 2 del11; 4 del17; others 3). Genetic abnormalities observed by MLPA were 45 (14 tris12; 26 del13q; 2 del11q; 3 TP53 deletion), and 22 were normal by MLPA. Of the 41 normal cytogenetics, MLPA was concordant in 20 patients and discordant in 21 patients. Of those 21, MLPA identified 19 del13q, 1 delq11, and 1 tris12. Regarding abnormalities, cytogenetics and MLPA were concordant in 12 tris12, 2 del13, 3 del17, and 1 del11. Statistical difference was seen when comparing both methodologies in favor of MLPA ( p= 0.003; chi-square test). In this study, we aimed to determine the usefulness of the MLPA probemix P037-B1 as a routine for detecting clinically relevant chromosome abnormalities in CLL in laboratories without cytogenetic routine. MLPA identified more abnormalities than cytogenetic (44%). We observed that MLPA was consistent with the cytogenetic results in 20 of 41 normal patients (48%). The main discordant results identified by MLPA were 13q deletion, the most common cytogenetic change in chronic lymphoblastic leukemia (CLL). In one patient, results of the MLPA did not show deletion in the TP53 gene, while isochromosome 17 was observed by cytogenetic. A low proportion of malignant cells with the relevant abnormality could be responsible for the discrepancies in the MLPA technique. Besides that pitfall, our findings highlight the advantage of MLPA compared with conventional cytogenetics. In conclusion, MLPA is a reliable high-throughput technique to detect CNVs and a cost-effective one that can be included in routine diagnostic protocols in laboratories that lack conventional cytogenetics.

Constitutive Activation of the B Cell Receptor Underlies Dysfunctional Signaling in Chronic Lymphocytic Leukemia

SUMMARYIn cancer biology, the functional interpretation of genomic alterations is critical to achieve the promise of genomic profiling in the clinic. For chronic lymphocytic leukemia (CLL), a heterogeneous disease of B-lymphocytes maturing under constitutive B cell receptor (BCR) stimulation, the functional role of diverse clonal mutations remains largely unknown. Here, we demonstrate that alterations in BCR signaling dynamics underlie the progression of B cells toward malignancy. We reveal emergent dynamic features—bimodality, hypersensitivity, and hysteresis—in the BCR signaling pathway of primary CLL B cells. These signaling abnormalities in CLL quantitatively derive from BCR clustering and constitutive signaling with positive feedback reinforcement, as demonstrated through single-cell analysis of phospho-responses, computational modeling, and super-resolution imaging. Such dysregulated signaling segregates CLL patients by disease severity and clinical presentation. These findings provide a quantitative framework and methodology to assess complex and heterogeneous leukemia pathology and to inform therapeutic strategies in parallel with genomic profiling.

PB1885 GENOMIC CHARACTERIZATION OF CHRONIC LYMPHOCYTIC LEUKEMIA AND MULTIPLE MYELOMA: ROLE OF ARRAY COMPARATIVE GENOMIC HYBRIDIZATION (ACGH)

Background:Genomic abnormalities play a key role in the biological properties and clinical behavior of hematological malignancies, namely in chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). These diseases have particularly benefited from gene expression profiling and genomic characterization, which improved diagnosis, prognosis and therapy response prediction. Conventional cytogenetics (CC) and fluorescence in situ hybridization (FISH) play a crucial role in the detection of cytogenetics abnormalities in these hematologic malignancies. However, these techniques had limitations, while CC requires proliferating cells and FISH is a targeted technique, underestimating the extent of chromosomal changes. Array comparative genomic hybridization (aCGH) had facilitated diagnosis and gene discovery with the ability to perform genome‐wide research and in high concordance with the cytogenetic and FISH results. These features facilitate the detection of clinically relevant findings that would be missed by other techniques.Aims:Therefore, the aim of this study was to screen and identify new genomic events in CLL and MM patients by aCGH in order to identify new potential prognostic biomarkers.Methods:To this end, 19 CLL patients at diagnosis [9 females/10 males; 74 years (50‐90); Binet stage (n = 18): A (n = 15, 83%) e C (n = 3, 17%)], 1 monoclonal B lymphocytosis (MBL; male; 81 years) and 10 MM patients (4 females/6 males; 74 years (54‐90) were analyzed by aCGH. Genomic DNA was obtained from peripheral blood mononuclear cells of CLL patients and from plasma cells (CD138+ isolated by MACS) from bone marrow samples of MM patients. The results were analyzed statistically considering a significance level of 95% (p < 0.05).Results:Results show that CLL patients have a median of 8 copy number variants (CNVs) per patient, ranging from 3 to 11, and MM patients have a median of 10 CNVs/patient, ranging from 1 to 23. Among the 137 CNVs detected in CLL patients, 65% were losses (n = 87) and 35% gains (n = 47), with an average of 4.4 losses/CLL case and 2.4 gains/CLL case. The patient with MBL had 10 CNVs (4 losses and 6 gains). In MM patients were detected 107 CNVs, 37% were losses (n = 40) and 63% gains (n = 67) with an average of 4.0 losses/MM case and 6.7 gains/MM case. These CNVs have several sizes ranging from 50 bp to 130 Mb in CLL and from 8.6 Kb to 131 Mb in MM, encompassing genes with malignant potential. We found several recurrent CNVs in CLL patients: 7 del (14q) (35%); 7 del(13q) (35%), 6 tris 12 (30%), 2 del(11q) (10%), 5 del(6q) (25%), 3 del(8p) (15%), 2 dup(8q) (10%) and 2 dup(2p) (5%). We also found 2 del(9p21.3) (10.0%), a rare abnormality in CLL. Additionally, all high‐risk patients (Binet C) have a del(14q) but these CNVs were less frequent in low‐risk CLL patients (Binet A: 20%, n = 3, p = 0,007). In MM patients the following recurrent CNVs were found: 7 del(13q) (70%), 5 dup(1q) (50%), 4 tris 9 (40%) and 4 del(14q) (40%). Events involving X chromosome, including monosomy and trisomy, were detected in 4 MM patients (40%). Interestingly, 6 out of 7 patients with del(13q) present a minimal deleted region (13q14.11‐q14.3) established previously by other groups.Summary/Conclusion:This study suggests that MM patients have a higher genomic complexity than CLL patients and that aCGH have potential to identify new prognostic markers in these pathologies.

Gain of the short arm of chromosome 2 (2p gain) has a significant role in drug-resistant chronic lymphocytic leukemia.

The different types of drug resistance encountered in chronic lymphocytic leukemia (CLL) cannot be fully accounted for by the 17p deletion (and/or TP53 mutation), a complex karyotype (CK), immunoglobulin heavy‐chain variable region genes (IGHV) status and gene mutations. Hence, we sought to assess the associations between recurrent genomic abnormalities in CLL and the disease's development and outcome. To this end, we analyzed 64 samples from patients with CLL and gain of the short arm of chromosome 2 (2p+), which is frequent in late‐stage and relapsed/refractory CLL. We found that fludarabine/cyclophosphamide/rituximab (a common first‐line treatment in CLL) is not effective in removing the 2p+ clone ‐ even in samples lacking a CK, the 17p deletion or unmutated IGHV. Our results suggest strongly that patients with CLL should be screened for 2p+ (using karyotyping and fluorescence in situ hybridization) before a treatment option is chosen. Longer follow‐up is now required to evaluate bendamustine‐rituximab, ibrutinib, and idelalisib‐rituximab treatments.

"Immuno-flowFISH" for the Assessment of Cytogenetic Abnormalities in Chronic Lymphocytic Leukemia.

Imaging flow cytometry is emerging as a diagnostic tool for the assessment of leukemia. It has the functionality of standard flow cytometry and generates high-resolution digital images of each cell with quantifiable numerical data. We demonstrate the use of an automated high-throughput method for performing fluorescence in situ hybridization (FISH) on immunophenotyped whole cells in suspension and analyzed by imaging flow cytometry, a technique called "Immuno-flowFISH". The aim of this study was to demonstrate the application of immuno-flowFISH for the detection of chromosomal abnormalities in CLL, specifically trisomy 12 and del(17p). Mononuclear cells were isolated and immunophenotyped with fluorescently conjugated CD3, CD5, and CD19 monoclonal antibodies. Following fixation, cells were permeabilized, dsDNA denatured and hybridized with chromosome 12 or 17 enumeration (CEP 12 and CEP17) and 17p12 locus-specific FISH probes. Cells were analyzed on the Amnis ImageStream®X Mark II to assess the number and percent FISH-positive CLL cells and the ratio of FISH spot counts for CD5/CD19-positive CLL cells to CD3/CD5-positive T cells (FISH "mean spot ratio"). Deletion of 17p was detected in about 8% of cases to date, with del(17p) ranged from 3.5-22.8% and the FISH "mean spot ratio" 0.86-0.96. Immuno-flowFISH also detected a minimal residual disease case with +12 with a limit of detection of 0.13% and a rare case that presented with atypical phenotype and cytogenetics. Immuno-flowFISH could detect del(17p) in phenotypically identified CD5/CD19-positive B-cells. The 100-fold increase in analyzed cells, as well as the addition of cell phenotype increased the sensitivity and specificity over current clinical FISH testing. Furthermore, immuno-flowFISH analysis demonstrated specific utility in unique clinical scenarios such as residual disease and atypical biology cases which may be of significant benefit with regards to prognostication and MRD analysis. The method will assist in therapeutic decision making and disease monitoring for many hematological malignancies. © 2019 International Society for Advancement of Cytometry.

Constitutive Activation of BCR Explains Dysfunctional Signaling in Chronic Lymphocytic Leukemia

In cancer biology, functional interpretation of genomic alterations is critical to achieve the promise of genomic profiling in the clinic. For Chronic Lymphocytic Leukemia (CLL), a heterogeneous disease of B-lymphocytes maturing under constitutive B-cell receptor (BCR) stimulation, the functional role of diverse clonal mutations remains largely unknown. Here, we demonstrate that alterations in BCR signaling dynamics underlie the progression of B-cells toward malignancy. We reveal emergent dynamic features, namely bimodality, hypersensitivity, and hysteresis, in the BCR signaling pathway of primary CLL B-cells. Such signaling abnormalities in CLL quantitatively derive from BCR clustering and constitutive signaling with positive feedback reinforcement, as demonstrated through single-cell analysis of signaling motifs, computational modeling, and super-resolution imaging. Such dysregulated signaling segregates CLL patients by disease severity and clinical presentation. These findings provide a novel quantitative framework and methodology to assess complex and heterogeneous leukemia pathology and to inform therapeutic strategies in parallel to genomic profiling.

Prevalence of cytogenetic abnormalities in chronic lymphocytic leukemia (CLL)

Prevalence of cytogenetic abnormalities in chronic lymphocytic leukemia (CLL)

Detection of Gene Abnormalities in 43 Cases of Chronic Lymphocytic Leukemia by Fluorescence in Situ Hybridization

To explore the value of fluorescence in situ hybridization (FISH) in diagnosis and prognosis evaluation of chronic lymphocytic leukemia (CLL). R banding technique was used for karyotype analysis in 43 cases of CLL, and fluorescence in situ hybridization（FISH） technique was used for analysis of 5 genes in 43 cases of CLL, including D13S25, RB1,ATM, P53 and CEP 12 . The detection rate of chromosome abnormality was 9.3％ for the 43 cases. The chromosome karyotype abnormality was involoved in the number abnormality and the structural abnormality, including No. 2, 6, 14, and sex chromosome. The patients with normal chromosome karyotype were more common（79.1％）. At the same time, mitotic figure in 5 CLL patients did not observed. The positive rate of FISH detection was 55.8％(24/43). Among which, D13S25 deletion was the highest and reached to 37.2％, followed by RB1 deletion（20.9％）, CEP 12 amplification（16.3％）, ATM deletion（9.3％） and P53 deletion（7.0％）. In 24 FISH-positive cases, 20 cases displayed the normal chromosome karyotype, and in 3 cases the mitotic figure was absent. There was only one case of abnormal chromosome, which was not involved in the positive gene tested by FISH. FISH is an important means of CLL genetic detection, which can greatly raise the detection rate of cytogenetics abnormalities in CLL, but the probe quantity is limited, so it is necessary to use FISH technology combined with chromosome karyotype analysis so as to improve the detection rate of abnormal cytogenetics, and provide the basis for the clinical diagnosis and prognosis of CLL.

Imaging flow cytometry to assess chromosomal abnormalities in chronic lymphocytic leukaemia.

Chronic Lymphocytic Leukaemia (CLL), the most common leukaemia in the Western world, has a characteristic phenotype and prognosis largely defined by the presence of cytogenetic aberrations. The gold standard for detecting these cytogenetic abnormalities is interphase fluorescence in situ hybridisation (FISH) performed on cell smears or tissue sections on glass slides. Fluorescently labelled DNA probes bind to specific chromosomal regions and the signal detected by fluorescent microscopy. Generally only 200 cells are assessed and the limit of sensitivity is 3% positive cells. Here we report the development and use of imaging flow cytometry to assess chromosomes by FISH in phenotyped CLL cells in suspension. Thousands of CLL cells, identified by their phenotype, are assessed for specific FISH probe signals using an automated, high throughput imaging flow cytometer. The "extended depth of field" capability of the imaging flow cytometer enables FISH probe signals ("spots") to be resolved and localised within the (stained) nucleus of the immunophenotyped cells. We report the development of the automated "immuno-flowFISH" on normal blood using the Amnis ImageStreamX mark II platform and illustrate the clinical application of the method for the assessment of chromosome 12 in CLL. It is a powerful new method which has potential to be applied at diagnosis for disease stratification, and following treatment to assess residual disease. These applications will assist clinicians in optimising therapeutic decision making and thereby improve patient outcome.

Prevalence of cytogenetic abnormalities in chronic lymphocytic leukemia in the southern part of Turkey.

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia among adults in Western populations. CLL has a wide range of clinical presentations and varied outcomes. For CLL, cytogenetic assessment is essential for estimating prognoses and determining the treatment of choice. The fluorescence in situ hybridization (FISH) technique is widely used for genetic assessment due to its high sensitivity. This study aimed to evaluate the frequencies of deletions of 13q14.3, 17p13.1, 11q22.3, and 13q34 and of trisomy 12 and to observe their effects on survival in 226 Turkish CLL patients using FISH analysis. The frequencies of abnormalities were 65.4% for del 13q14.3, 39.8% for del 17p13.1, 19% for del 11q22.3 (del ATM), and 15.9% for trisomy 12. No patients had a 13q34.3 aberration. Our results are partially consistent with literature findings. However, certain conflicts with prior results were observed, particularly with respect to the high prevalence of 17p13.1 deletions and the enhanced survival of patients with such deletions. These inconsistencies may represent population-based differences in the genetic epidemiology of CLL.

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

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 and SF3B1 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. DisclosuresD'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.

Analysis of Clonal Evolution in Chronic Lymphocytic Leukemia from Inactive to Symptomatic Disease Prior Treatment Using Whole-Exome Sequencing

Clonal evolution is considered as a hallmark of progression in chronic lymphocytic leukemia (CLL). Next-generation sequencing technologies have expanded our knowledge of genetic abnormalities in CLL and enabled to describe marked clonal changes. The acquisition of driver mutations accompanied by selectively neutral passenger changes may be essential to understand the transformation from diagnosis to later more aggressive stages. However, the role of genetic mutations and clonal evolution during the clinical progression prior any therapy is still largely unknown. Longitudinal studies analyzing CLL patients repeatedly before intervening treatment are currently scarce.Patients and methods: We examined the exomes from 35 CLL patients in 2 time-points. Two groups of patients were characterized: (i)patients with progression (n=26) in which we analyzed samples taken from an early stable stage (inactive disease) and during clinical progression (active disease), but before treatment (median of time to first treatment=2.7 years); (ii)patients without progression with a stable inactive disease until last follow-up (n=9) (median follow-up=5.25 years). We also compared patients that gained new cytogenetic aberration detected by FISH in the 2nd time-point with those who did not.Sequencing libraries were prepared using TruSeq Exome Enrichment and sequenced by Illumina HiSeq1000 (84X). Somatic mutation calling was performed by a standardized bioinformatics pipeline. Thereafter, driver mutations were identified using the Cancer Genome Interpreter (https://www.cancergenomeinterpreter.org), a novel tool that identifies variants that are already validated as oncogenic and predicts the effect of the mutations of unknown significance.Results: We identified 397 somatic mutations in 364 different genes, ranging from 4 to 26 mutations per patient. Among them, 58 driver mutations were identified, being SF3B1 (6/35, 17.1%), TP53 (4/35, 11.4%) and NOTCH1 (4/35, 11.4%) the most common mutated genes. Comparing progressive vs. stable group, CLL patients with clinical progression showed a higher intra-tumoral heterogeneity than cases without progression (median of somatic mutations=14[4-26] vs. 9[5-14]).Comparing both tumoral time-points in the same patient, we identified a total of 11 acquired driver mutations and 7 mutations increasing its allele frequency in more than double in the 2nd time-point respect to the 1st one. All of them were detected in patients with clinical progression. Interestingly, TP53 and BIRC3 exhibited recurrently acquired mutations (detected each one in 2 cases). Three driver mutations in cancer genes not yet known for CLL (DHX9, GNAQ and HDAC2) were also acquired. Within CLL progressive patients (n=26), we observed clonal evolution characterized by acquired cytogenetic aberration in 9 cases. In patients with progression but no cytogenetic aberration gained at the 2nd moment (n=17), we detected that almost half of them (7/17) showed clonal evolution by acquired or doubled driver mutations. In the remaining patients with clinical progression but without any clonal evolution (n=10), 6 cases showed a driver mutation of CLL genes associated with bad prognosis (SF3B1, TP53, NOTCH1 or RPS15) already at first time-point.In the stable group (n=9), none acquired or doubled mutation was detected. However, clonal evolution characterized by acquired cytogenetic aberration was observed in 4/9 stable patients: two of them acquired 13q- whereas the other two acquired 11q-. Within stable patients without clonal evolution (n=5), we detected one case with a driver mutation in SF3B1 already at 1st time-point (follow-up=5 years).Conclusion: Clonal evolution represents a central feature of tumor progression in CLL. Our data show that the disease is evolving during time even in stable patients without any clinical signs of disease activity. In progressive patients, the disease evolution is accompanied by new appearance or accumulation of driver mutations and cytogenetic aberrations. Moreover, progressive patients that showed less or no changes during time bore typical CLL drivers at the first time-point.Funding: Seventh Framework Programme (NGS-PTL/2012-2015/no.306242); Ministry of Education, Youth and Sports (2013-2015, no.7E13008; CEITEC 2020 (LQ1601)); AZV-MZ-CR 15-31834A-4/2015 and TACR (TEO2000058/2014-2019); PI15/01471; Junta de Castilla y León (MHS). DisclosuresNo relevant conflicts of interest to declare.

Low Bax/Bcl-2 Ratio and NOTCH1 Mutations Represent Powerful and Synergistic Adverse Prognostic Factors within Trisomy 12 Chronic Lymphocytic Leukemia (CLL)

Trisomy 12 is the third most common cytogenetic abnormality in CLL with several distinguishing features including abormal morphology, high prevalence of NOTCH1 mutations (NOTCH1 M) and increased expression of the alpha-integrins CD11a and CD49d (Balatti, 2012; Zucchetto, 2013). Trisomy 12 marks a disease subset characterized by high rates of cell proliferation, disease progression and Richter syndrome transformation (Rossi, 2013). Noteworthy, NOTCH1 M implicate constitutive activation of NOTCH1 signaling which triggers apoptosis resistance and increased survival of CLL cells (Rosati, 2009). Moreover, bax/bcl-2 ratio (bax/bcl-2), marker of apoptosis, predicts chemoresistance and progressive disease in CLL (Del Principe, 2016). The today availability in clinical use of venetoclax (ABT-199), a potent oral anti-bcl-2 peptidomimetic (Seymour, 2014), prompted us to analyze the real impact of the apoptosis (bax/bcl-2) on trisomy 12 CLL prognosis. The primary aims of our research were: 1) to demonstrate a significant association between NOTCH1 M and bax/bcl-2; 2) to correlate NOTCH1 M and bax/bcl-2 with biological and clinical prognosticators; 3) to determine progression-free survival (PFS) and overall survival (OS) upon NOTCH1 M and bax/bcl-2; 4) to evaluate NOTCH1 M and bax/bcl-2 as independent prognostic factors. Therefore we investigated 653 CLL patients, median age 66 years, 363 males and 290 females. Using the 5% cutoff for the FISH cytogenetic abnormalities, 140 (21.4%) cases were classified as trisomy 12 CLL. Bax/bcl-2 was calculated by flow cytometry, dividing mean florescence intensity (MFI) of bax by MFI of bcl-2 on CLL cells. The threshold was set at the median value >1.5 (range 0.32-5.30). The presence of NOTCH1 M was investigated with ARMS PCR for c.75447545delCT and by Sanger sequencing of NOTCH1 exon 34. CD49d was >20% in almost all trisomy 12 pts (116/140; 83%). Forty-five patients were NOTCH1 M (32.1%) and 45 were bax/bcl-2 positive (32.1%). There was a very strong correlation between bax/bcl-2 <1.5 and NOTCH1 M (42/45; p<0.0001), corroborating the close dependence of the lacking apoptosis on NOTCH1 M. Both lower bax/bcl-2 and NOTCH1 M were significantly associated with lymphocyte doubling time <12 months (p=0.0077 and p=0.0006), with beta-2 microglobulin >2.2 mg/dl (p=0.0001 and p=0.0007) and LDH >300 U/L (p=0.004 and p=0.00001), thus confirming their significant association both with a high proliferative rate and a high tumor burden. Strong correlations were found between NOTCH1 M or bax/bcl-2 <1.5 and IGHV unmutated status (p<0.0001 and p=0.0005) or ZAP-70 >30% (p<0.0001 and p=0.00002). With regard to clinical outcome, significant shorter PFS and OS were observed in patients with NOTCH1 M vs NOTCH1 wild type [WT] (0% vs 27% at 10 years, p=0.001 and 26% vs 78% at 14 years, p=0.005) or lower bax/bcl-2 (9% vs 38% at 10 years, p=0.0007 and 45% vs 89% at 14 years, p=0.002). Interestingly, NOTCH1 M and bax/bcl-2 showed synergistic prognostic properties, since NOTCH1 M and bax/bcl-2 <1.5 identified a trisomy 12 CLL subset at worst prognosis with regard to PFS (0% vs 40% at 10 years, p=0.00004, Figure) and OS (20% vs 100% at 14 years, p=0.0001, Figure). Therefore, bax/bcl-2 and NOTCH1 M are powerful prognosticators, showing synergistic clinical effects. In multivariate analysis of OS, bax/bcl-2 (p=0.004) together with age (p=0.0006), CD49d (p=0.01) and IGHV status (p=0.03) was confirmed as an independent prognostic factor. In conclusion, the modern strategies to trigger apoptosis and block proliferation, such as venetoclax, may be very effective in this CLL subset, also taking into account that trisomy 12 CD49d+ CLL has abbreviated lymphocytosis with retention of CLL cells within tissues (Thompson, 2014) and consequent poor reduction of lymphadenopathy during treatment with ibrutinib. [Display omitted] DisclosuresLo Coco:Teva: Consultancy, Honoraria, Speakers Bureau; Lundbeck: Honoraria, Speakers Bureau; Novartis: Consultancy; Baxalta: Consultancy; Pfizer: Consultancy.