ALBERT

Description: Analysis of the chronic lymphocytic leukemia (CLL) coding genome has recently disclosed that the NOTCH1 proto-oncogene is recurrently mutated at CLL presentation. Here, we assessed the prognostic role of NOTCH1 mutations in CLL. Two series of newly diagnosed CLL were used as training (n = 309) and validation (n = 230) cohorts. NOTCH1 mutations occurred in 11.0% and 11.3% CLL of the training and validation series, respectively. In the training series, NOTCH1 mutations led to a 3.77-fold increase in the hazard of death and to shorter overall survival (OS; P 〈 .001). Multivariate analysis selected NOTCH1 mutations as an independent predictor of OS after controlling for confounding clinical and biologic variables. The independent prognostic value of NOTCH1 mutations was externally confirmed in the validation series. The poor prognosis conferred by NOTCH1 mutations was attributable, at least in part, to shorter treatment-free survival and higher risk of Richter transformation. Although NOTCH1 mutated patients were devoid of TP53 disruption in more than 90% cases in both training and validation series, the OS predicted by NOTCH1 mutations was similar to that of TP53 mutated/deleted CLL. NOTCH1 mutations are an independent predictor of CLL OS, tend to be mutually exclusive with TP53 abnormalities, and identify cases with a dismal prognosis.

Description: Abstract 283 The clinical course of chronic lymphocytic leukemia (CLL) ranges from very indolent, with a nearly normal life expectancy, to rapidly progressive leading to death and occasionally undergoing transformation to Richter syndrome (RS). TP53 disruption identifies a fraction of high risk CLL destined to experience a very short survival. High risk CLL, however, cannot be fully recapitulated by TP53 disruption and other lesions of cancer genes may be implicated in this aggressive phenotype. Analysis of the CLL coding genome has recently disclosed that the NOTCH1 proto-oncogene is recurrently mutated at CLL presentation. Here we assessed the prognostic role of NOTCH1 mutations in CLL. Two series of previously untreated CLL were utilized as training (n=309, median follow-up 6 years) and validation (n=230, median follow-up 7 years) cohorts. NOTCH1 mutations were analyzed by DNA Sanger sequencing in blind with respect to clinical data. In the training series, NOTCH1 mutations occurred in 34/309 (11.0%) patients, being mostly represented (26/34, 76.5%) by a recurrent two bp frameshift deletion (c.7544_7545delCT). The remaining NOTCH1 mutations (8/34, 23.5%) were frameshift deletions other than c.7544_7545delCT (n=7) and frameshift insertions (n=1). All mutations were predicted to disrupt the NOTCH1 PEST domain. CLL with NOTCH1 mutations preferentially carried unmutated IGHV genes (76.5%, p

Description: RS occurs in 5–15% CLL, depending on follow-up length and re-biopsy policy. The value of biological and clinical risk factors in predicting RS is unknown and represents the aim of this study. The analysis was based on a consecutive series of 185 CLL. Diagnosis of RS was based on lymph node or extranodal tissue biopsy. RS was diagnosed in 17 cases all represented by diffuse large B-cell lymphoma (DLBCL). Cumulative incidence of RS at 5 and 10 years was 13.6% (95%CI: 7.0–20.1%) and 16.2% (95%CI: 8.0–24.4%), respectively. Transformation plateaued at 82 months of follow-up. Median time to transformation was 23.0 months (95%CI: 15.9–30.1 months) from CLL diagnosis. IGHV studies documented clonal relationship between CLL and DLBCL in 15/17 (88.2%) cases. At the time of CLL diagnosis, molecular variables predicting RS were: IGHV4–39 usage (5-year risk IGHV4-39: 56.2% vs IGHVnon-VH4-39: 11.2%; p

Description: Background. Recent studies have described the landscape of recurrently mutated genes in mantle cell lymphoma (MCL), including genes involved in DNA damage response/cell cycle (ATM, TP53, CCND1), epigenetic regulation (KMT2D also known as MLL2, WHSC1), and cell signaling (BIRC3, TRAF2, NOTCH1). However, with the exception of TP53 abnormalities, little is known about the clinical relevance of recurrent mutations in MCL. Thus, we performed deep sequencing analysis of a MCL gene panel in the prospective series of patients enrolled in the ongoing FIL-MCL0208 phase III trial (EudraCTNumber: 2009-012807-25). Patients and Methods. The study included untreated, advanced stage 70% of cases. The gene panel was analyzed in tumor DNA from baseline bone marrow CD19+ purified MCL cells and, for comparative purposes to filter out polymorphisms, in the paired normal genomic DNA (available in 55% of cases) using a TruSeq Custom Amplicon target enrichment system followed by deep next generation sequencing (Illumina, median depth of coverage 2356x). Variants represented in 〉10% of the alleles were called with VarScan2 with the somatic function when the paired germline DNA was available. For patients lacking germline DNA, a bioinformatic pipeline including a number of stringent filters was applied to protect against the misclassification of polymorphisms as somatic variants. Primary endpoint of the analysis was progression free survival (PFS). Results. Out of the enrolled patients, 151 are currently evaluable for mutations and clinical outcome (median age: 57 years, range 35-66; males 75%). Among prognostic factors, the MIPI was intermediate or high-risk in 49% of patients, the Ki67 ≥30% in 39%, and blastoid histology occurred in 8%. At the first planned interim analysis, median follow-up of alive patients was 26 months. At 2-years, 79% of patients were progression free and 91% alive (Cortelazzo et al EHA 2015). Overall, at least one mutation was detected in 106/151 cases (70%), including mutations of ATM in 42% of cases, CCND1 in 14%, WHSC1 in 13%, KMT2D in 12%, TP53 in 7%, NOTCH1 in 6%, BIRC3 in 5% and TRAF2 in 1% (Figure 1A). By univariate analysis, mutations of TP53 (2-years PFS 48% vs 82%; p

Description: Abstract 3601 Richter syndrome (RS) represents the development of diffuse large B-cell lymphoma (DLBCL) in the context of chronic lymphocytic leukemia (CLL). Despite the availability of clinical predictors, the identification of RS patients projected to experience long survival represents an unresolved issue that may benefit from the development of novel RS prognosticators. The study was based on 86 RS classified as DLBCL after pathological review. Clonal relationship between CLL/RS pairs was assessed by immunoglobulin gene analysis. RS was clonally related to the CLL phase in 49/59 (83.1%) cases, and clonally unrelated in 10/59 (16.9%). In 27 cases, clonal relationship remained undetermined because of unavailability of paired CLL samples. Out of the molecular features investigated, the prevalence of TP53 disruption by mutations and/or deletion was significantly higher in clonally related RS (23/36, 63.9%) compared to clonally unrelated cases (1/8, 12.5%) (p=.015). Among immunogenetic features, usage of stereotyped VH CDR3 occurred in 25/49 (51.0%) clonally related RS, while was very rare among clonally unrelated cases (1/10, 10.0%) (p=.032). The difference in stereotyped VH CDR3 frequency occurred irrespective of IGHV gene mutation status. All clonally unrelated RS tested negative for EBV infection and none received alemtuzumab before DLBCL development. Clonally related and clonally unrelated RS were indistinguishable in terms of clinical features at presentation. However, RS survival was significantly shorter in clonally related (median: 14.2 months) versus clonally unrelated cases (median: 62.5 months) (p=.017) (Fig. 1A). This survival difference was observed even though clonally related RS received SCT in 8/48 (16.7%) cases, while clonally unrelated RS received in all cases conventional chemotherapy without SCT consolidation. Other variables that were associated with poor RS survival were TP53 disruption (p60 years (p=.005), ECOG PS 〉1 (p5 cm (p=.001), platelets 1.5 ULN (p=.002), and having received SCT (p=.027). None of the pathologic features of DLBCL diagnostic biopsies correlated with RS prognosis. After adjusting for clinical variables associated with RS survival, diagnosis of clonally unrelated RS translated into a 80% reduction in risk of death when compared to clonally related RS (HR: 0.22; p=.036). By recursive-partitioning analysis, clonal relationship, TP53 disruption and ECOG PS were the major determinants of RS survival, and classified RS patients according to risk of death (Fig. 1B). All clonally unrelated RS displayed a low risk of death (median survival 62.5 months; 95% CI: 33.7–91.3), irrespective of TP53 disruption and ECOG PS (Fig. 1B; Group 1). Among clonally related RS and RS with undetermined clonal relationship, patients presenting with ECOG PS ≤1 and no TP53 disruption had a low risk of death (median survival: 55.4 months) (Fig. 1B; Group 2). Patients presenting with ECOG PS ≤1 in the presence of TP53 disruption had an intermediate risk of death (median survival: 27.0 months) (Fig. 1B; Group 3). Patients presenting with ECOG PS 〉1 had a high risk of death, irrespective of TP53 disruption (median survival: 7.8 months) (Fig. 1B; Group 4). Based on c-statistics, the model including clonal relationship, ECOG PS and TP53 status had a 90% probability of correctly discriminating a priori RS survival (c-statistic=.90±.07). These observations suggest that clonally related and clonally unrelated RS are biologically and clinically distinct disorders, and that clonally unrelated RS should be considered as a secondary DLBCL arising de novo in the context of CLL, rather than a true RS. Therefore, we propose that the diagnosis of RS should be restricted to clonally related cases. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction TP53 mutations are strong predictors of poor survival and refractoriness in chronic lymphocytic leukemia (CLL) and have direct implications for disease management. Clinical information on TP53 mutations are currently limited to lesions that are represented in the majority of CLL cells. Next generation sequencing (NGS) allows sensitive detection of mutations harbored by a small fraction of the tumor cell population. Here we aim at assessing the frequency, evolution during disease course, and prognostic impact of small TP53 mutated subclones in newly diagnosed CLL. Methods The study was based on a consecutive series of 309 newly diagnosed and previously untreated CLL (median age: 71 years; Binet A/B/C: 79/12/9%; unmutated IGHV genes: 35%; clonal TP53/NOTCH1/SF3B1/BIRC3 lesions: 11/11/7/5%; median follow-up: 8.1 years). TP53 mutations (exons 4-8) were screened on peripheral blood (PB) samples (tumor representation 70-98%) by amplicon-based deep-NGS (GSJ, 454 Life Sciences) (average depth: 2660). A bioinformatic algorithm was developed to call TP53 variants out of background noise. By dilution experiments, deep-NGS allowed to detect mutant allele fractions of 0.3%. TP53 variants were considered subclonal if missed by Sanger sequencing, which was performed in parallel. Subclonal TP53 variants were confirmed by duplicate deep-NGS and independently validated by allele specific PCR (AS-PCR). TP53 variant allele frequency (VAF) was corrected for tumor representation. Results Deep-NGS identified 50 subclonal TP53 mutations (VAF 0.3%-11%) in 28/309 (9%) CLL (Fig 1A). All subclonal mutations were non-silent, were missed by Sanger sequencing, and were validated by AS-PCR. The molecular spectrum of subclonal TP53 mutations (i.e. missense/truncating ratio, transition/transversion ratio, distribution across hot spot codons; p〉.05; Fig. 1B-D), as well as the residual transactivational activity of mutants toward the p21 promoter (p=.872) were highly consistent with that of fully clonal TP53 mutations reported in CLL (Zenz T, Leukemia 2010). Subclonal TP53 mutations were the sole TP53 genetic event in 15/309 (4.8%) CLL, while in 13/309 (4.2%) cases subclonal TP53 mutations co-existed in the same leukemic population along with a clonal TP53 mutation or with 17p deletion. In cases (n=12) harboring more than one TP53 mutation, the variants mapped on distinct sequencing reads from the same amplicon suggesting that they belonged to different CLL subclones. By combining subclonal TP53 mutations, clonal TP53 mutations and 17p deletion, 50/309 (16%) CLL harbored at least one TP53 defect at diagnosis. Subclonal TP53 mutations were significantly enriched among cases presenting with advanced stage (Binet C: 26%; p=.005) and clonal TP53 abnormalities (37%; p

Description: Evolution of the chronic lymphocytic leukemia (CLL) clone upon treatment with the BTK-inhibitor ibrutinib has never been systematically assessed in a prospective way, though changes in the tumor genetic composition have been anecdotally reported by retrospective backtracking of mutations acquired in ibrutinib-refractory patients. The IOSI-EMA001 observational study (NCT02827617) enrolls patients treated with ibrutinib in the clinical practice, and aims at prospectively assessing the dynamics of the clonal architecture of high risk CLL upon therapy with ibrutinib by taking advantage of leukemia samples homogeneously collected at pre-specified timepoints during treatment course. Ibrutinib induces a transient lymphocytosis by displacing CLL cells formerly residing in the tissue compartments into the peripheral blood (PB). By analyzing samples collected after two weeks of ibrutinib treatment, here we tracked early genetic changes of the CLL clone occurring at the time of redistribution lymphocytosis. This analysis includes the first 10 patients enrolled in the study (median age=76 years; Binet A=10%, B=60%, C=30%; IGHV unmutated=60%, mutated=20%, NA=20%; 17p deletion=40%). PB samples were collected before ibrutinib treatment start (day -28 to 0) and after two weeks of treatment (week 2, +/-3 days). Tumor genomic DNA was isolated from FACS sorted CLL cells (CD19+/CD5+ elements; 〉99% purity). T cells were also sorted as source of germline material to confirm the somatic origin of mutations and filter out sequencing noise. The HaloPlex High Sensitivity library preparation protocol and ultra-deep next generation sequencing (coverage ~40.000x) on MiSeq (Illumina) were used to track TP53, BTK and PLCG2 mutations. By uniquely molecular barcoding each DNA library fragment, and by targeting both DNA strands, this approach allowed to reach a sensitivity of 10E-4. The CAPP-seq library preparation protocol and deep next generation sequencing (sensitivity ~5x10E-3) were used to track mutations of a panel of genes known to be recurrently affected in CLL (ASXL1, ATM, BIRC3, BRAF, EGR2, FBXW7, IKZF3, IRF4, KRAS, MAP2K1, MGA, MYD88, NFKBIE, NFKB2, NOTCH1 including 3'UTR, PAX5 enhancer, POT1, RPS15, SAMHD1, SF3B1, TP53, XPO1, ZMYM3). At baseline before treatment start, 33 non-synonymous somatic mutations (allele frequency: 0.65-99%) were identified in 9 of the 10 cases (one patient was devoid of mutations of the above-mentioned genes). The mutational profile was consistent with that expected in a high risk population meeting the current indication for ibrutinib treatment in the clinical practice(TP53=60%; NOTCH1=40%; MGA=30%; ZYMYM3=20%; ATM=10%; BIRC3=10%; EGR2=10%; IRF4=10%; IKZF3=10%; MYD88=10% NFKBIE=10%; SF3B1=10%; XPO1=10%) (Fig. 1A). Before treatment start, high sensitive ultra deep next generation sequencing did not disclose any non-synonymous somatic mutation of BTK and PLCG2 in CLL cells circulating in the blood. The genetic composition of the circulating leukemia clone did not significantly change after two weeks of treatment despite redistribution of CLL cells in the blood compartment (median lymphocyte count at baseline 50.9x10E9/L vs 77.5x10E9/L at week 2) (Fig 1B). Minor genetic changes between baseline and week 2 included the appearance of a small clone mutated in SAMHD1 and the disappearance of a small clone mutated in BIRC3 in 2 different patients. At week 2, no mutations of BTK and PLCG2 became evident in the circulating compartment. With the limitation of the current sample size, these data suggest that redistribution lymphocytosis does not mobilize CLL high risk clones harboring mutations of clinical relevance into the blood compartment. Figure 1 Figure 1. Disclosures Rossi: Gilead: Honoraria, Research Funding; Janssen: Honoraria; AbbVie: Honoraria.

Description: Abstract 466 The genetic lesions identified to date do not fully recapitulate the molecular pathogenesis of chronic lymphocytic leukemia (CLL) and do not entirely explain the development of severe complications, such as chemorefractoriness. BIRC3, along with TRAF2 and TRAF3, cooperate in the same protein complex that negatively regulates MAP3K14, the central activator of non-canonical NF-kB signaling. Following the initial observation of recurrent BIRC3 mutations in splenic marginal zone lymphomas, we performed targeted re-sequencing of the BIRC3 coding sequence and splicing sites across the spectrum of B-cell neoplasia. By this screening, BIRC3 mutations were identified only in CLL (2/20), while were absent in diffuse large B-cell lymphoma (0/30), Burkitt lymphoma (0/38), follicular lymphoma (0/20), extranodal marginal zone lymphoma (0/65), hairy cell leukemia (0/19), and multiple myeloma (0/22). This observation prompted the investigation of prevalence and clinical impact of BIRC3 genetic lesion in CLL. The study population included 4 clinical CLL panels representative of different disease phases: i) a cohort of fludarabine-refractory CLL (n=49); ii) a cohort of fludarabine-sensitive CLL (n=62); iii) clonally related Richter syndrome (RS) (n=33; all diffuse large B cell lymphomas); and iv) a consecutive series of newly diagnosed and previously untreated CLL (n=308). BIRC3 was analyzed for mutations by Sanger sequencing (exons 2–9) and for copy number abnormalities by FISH (probe RP11-177O8). BIRC3 was affected in 12/49 (24%) fludarabine-refractory CLL by inactivating mutations (7 frameshift and 1 non-sense) and gene deletions (n=7) that distributed in a mutually exclusive fashion with TP53 disruption (p=.004) (Fig 1A and 1B). Two fludarabine-refractory CLL carried a biallelic inactivation of the BIRC3 gene by mutation of one allele and deletion of the second allele. All inactivating mutations were somatically acquired, were predicted to generate aberrant transcripts carrying premature stop codons, and caused elimination or truncation of the C-terminal RING domain, whose E3 ubiquitin ligase activity is essential for MAP3K14 proteosomal degradation by BIRC3. Western blot analysis confirmed the expression of aberrant bands corresponding in size to the predicted truncated BIRC3, and revealed constitutive non-canonical NF-kB activation in fludarabine-refractory CLL harboring BIRC3 disruption by mutations or deletion, as documented by NFkB2 processing from p100 to p52 (Fig. 1). BIRC3 was the sole gene of the TRAF3/MAP3K14-TRAF2/BIRC3 complex targeted by molecular lesions after extensive mutation and FISH analysis of TRAF2, TRAF3 and MAP3K14 in the fludarabine-refractory CLL cohort (n=49). To investigate whether BIRC3 genetic lesions were restricted to chemorefractory cases, we analyzed BIRC3 for mutations and deletions in other CLL subsets. Fludarabine-sensitive CLL were consistently devoid of BIRC3 disruption in all cases (n=62), suggesting that BIRC3 genetic lesions specifically associate with a chemorefractory phenotype among CLL requiring treatment (Fig. 1A). BIRC3 disruption was restricted to 1/33 (3.0%) clonally-related RS, corroborating the notion that RS is molecularly distinct from chemorefractory progression without transformation (Fig. 1A). In a consecutive series evaluated at CLL diagnosis, BIRC3 disruption was rare (13/308, 4%, all TP53 wild type), associated with primary chemorefractoriness among cases requiring treatment, and predicted poor outcome (Fig. 1A). By univariate analysis, the crude impact of BIRC3 disruption on survival was a ∼5.3 fold increase in the hazard of death (HR: 5.31; 95% CI: 2.62–10.73) and a significant overall survival (OS) shortening (median OS in BIRC3 disrupted cases: 3.1 years vs not reached in BIRC3 wild type cases; p

Description: Introduction. Accessible and real-time genotyping for diagnostic, prognostic or treatment purposes is increasingly impelling in diffuse large B-cell lymphoma (DLBCL). Since DLBCL lacks a leukemic phase, tumor genotyping has so far relied on the analysis of the diagnostic tissue biopsy. Cell-free DNA (cfDNA) is shed into the blood by tumor cells undergoing apoptosis and can be used as source of tumor DNA for the identification of cancer-gene somatic mutations. Accessing the blood has obvious sampling advantages in the monitoring of mutations in real-time. Also, cfDNA is representative of the entire tumor heterogeneity, thus allowing to identify mutations from tumor cells residing in non-biopsied sites. Here we aimed at tracking the DLBCL genetic profile using plasma cfDNA. Methods. The study was based on 26 consecutive DLBCL patients (age 〉65=15, male:female ratio=11:15) presenting in different Ann Arbor stages (III-IV=13) and age-adjusted IPI risk scores (2-3=13), and provided with cfDNA from plasma collected at diagnosis, during R-CHOP course, at the end of treatment and at progression. Paired normal genomic DNA from granulocytes was also collected for comparative purposes to filter out polymorphisms. A targeted resequencing panel including the coding exons and splice sites of 59 genes (207 kb) that are recurrently mutated in mature B-cell tumors was specifically designed to allow the recovery of at least one mutation in 〉90% of DLBCL. Ultra-deep next-generation-sequencing (NGS) of the gene panel was performed on MiSeq (Illumina) (coverage 〉2000x in 〉80% of the target) using a SeqCap library preparation strategy (NimbleGen). The somatic function of VarScan2 was used to call non-synonymous somatic mutations, and a stringent bioinformatic pipeline was developed and applied to filter out sequencing errors. The study cohort was divided in a training set of 17 patients provided with paired tumor DNA from the diagnostic tissue biopsy that was used for the set up of the ultra-deep NGS strategy, and an extension set of 9 patients lacking the tissue biopsy. Results. A total of 76 cfDNA samples (26 pretreatment, 28 during treatment, 18 at the end of treatment, and 4 at time of progression) were evaluated. Pretreatment cfDNA genotyping disclosed somatic mutations of heterogeneous abundance (median mutated molecules/ml of plasma: 3168, range 1.73-6.5x104) in known DLBCL-associated genes, including MLL2 (33%), TP53 (25%), CREBBP and TNFAIP3 (21%), EZH2, TBL1XR1, PIM1 (17%), B2M, BCL2, CARD11, CCND3, FBXW7 and STAT6 (13%) (Fig. 1A). The results of genotyping on cfDNA from plasma and of genomic DNA from tumor cells of the diagnostic biopsy (gold standard) were compared to derive the diagnostic accuracy of cfDNA genotyping (Fig. 1B). Genotyping of the paired plasma cfDNA correctly identified 79% of the tumor biopsy mutations. Most of the tumor variants not discovered in the cfDNA had a low representation in the tumor biopsy (median allelic abundance=5.7%; range 0.8-54%). Consistently, ROC analysis showed that cfDNA genotyping had the highest sensitivity (92%) if mutations were represented in 〉15% of the alleles of the tumor biopsy. Plasma cfDNA genotyping also disclosed a number of additional somatic mutations (~2 per case, range 1-6) that were not detectable in the tissue biopsy, including mutations of clinically relevant genes. Longitudinal analysis of plasma samples under R-CHOP chemotherapy showed a rapid clearance of the DLBCL mutations in the cfDNA already after the first cycle among responding patients (Fig. 1C). Among patients that were resistant to R-CHOP, basal DLBCL mutations did not disappear from cfDNA. In addition, among treatment-resistant patients, new mutations appeared in cfDNA that conceivably marked resistant clones selected during the clonal evolution process taking place under the pressure of treatment (Fig 1D). Conclusions. Overall, these results provide the proof of principle that cfDNA genotyping of DLBCL: i) is as accurate as genotyping of the diagnostic biopsy to detect somatic mutations of allelic abundance 〉15% in DLBCL; ii) allows the identification of mutations that are otherwise absent in the tissue biopsy conceivably because restricted to clones that are anatomically distant from the biopsy site; and iii) is a real-time and non-invasive way to track clonal evolution and emergence of treatment resistant clones. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2425 Mechanisms promoting chronic lymphocytic leukemia (CLL) transformation to Richter syndrome (RS) are poorly understood and might involve antigen stimulation. We explored intraclonal diversification (ID) of immunoglobulin (IGHV) genes in RS in order to: i) follow the evolutionary history of the RS tumor clone over time; ii) understand whether antigen stimulation sustains the growth of DLBCL cells once RS is established. The study was based on 11 clonally related CLL/RS pairs, 7 clonally unrelated RS, and, for comparison, 20 de novo DLBCL. Fifty IGHV subclones were analysed per sample. Mutations observed in only one subclone were defined unconfirmed (UCM). Partially shared mutations were defined confirmed (CM). Cases were scored positive for ID only in the presence of confirmed mutations. For each sample, the normalized mutation frequency (NMF) of ongoing somatic hypermutation was calculated. Phylogenetic analyses was performed with MEGA4. Most (10/11, 90.9%) clonally related RS originated from an ancestor clone that was already present at the time of CLL diagnosis (Fig 1A, 1B, 1C). One single RS case had a transformation pattern compatible with sequential evolution from a secondary CLL subclone (Fig. 1D). All secondary CLL subclones that were documented in the CLL phase disappeared once transformation had occurred, and were substituted by the dominant DLBCL clone with its own descendants. Paired analsysis of clonally related CLL/RS samples documented that NMF significantly decreased during evolution from the CLL phase (median: 0.76 × 10−3) to the RS phase (median: 0.13 × 10−3)(p=.013). Accordingly, at transformation, the ID of IGHV genes switched off in 6/11 (54.5%) clonally related CLL/RS pairs. Clonally related RS that upon transformation maintained ID of IGHV genes were characterized by a higher prevalence of aberrant somatic hypermutation of proto-oncogenes compared to cases lacking ID (0/6 vs 3/4, 75.0%, respectively; p=.033). Also, RS cases that maintained ID of IGHV genes acquired more frequently c-MYC translocation at the time of transformation (0/6 vs 2/4, respectively; p=.133). Clonally unrelated RS are characterized by the development of a de novo DLBCL in the context of CLL. Despite morphologic and phenotypic similarities, clonally unrelated RS differed from de novo DLBCL in terms of NMF (median: 1.18 × 10−3 range: vs median: 0.08 × 10−3 range, respectively; p=.016) and prevalence of cases harboring ID (6/7, 85.7% vs 6/20, 30.0%, respectively; p=.024). The NMF was also significantly higher in clonally unrelated RS compared to clonally related cases (p=.001). These data indicate that: i) RS transformation is due to the expansion of a common ancestor clone that gains selective advantage over other subclones of the CLL phase; ii) antigen stimulation exerts different roles in clonally related RS, clonally unrelated RS and de novo DLBCL; iii) more than 50% clonally related RS switched off ID at the time of transformation, suggesting that the DLBCL clone has become independent of antigen stimulation for its sustainment; iv) clonally related RS that maintained ID of IGHV genes at transformation may take advantage of ID as a mechanism for accumulating genetic instability. Disclosures: No relevant conflicts of interest to declare.