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Objectives The aim of this study was to compare the results of five methods for the determination of total 25(OH)D. For that purpose, two mass spectrometry and three immunoassay methods were used. Methods A total of 124 serum samples were analyzed on five different methods (i.e., a reference LC-MS/MS, Cascadion, Lumipulse, Roche Elecsys II and Roche Elecsys III). Analytical performance against LC-MS/MS was evaluated and compared to the Milan models 1 (analytical performance based on the clinical outcome using thresholds of 12, 20 and 30 ng/mL) and 2 (analytical performance based on biological variation). Additionally, imprecision studies and accuracy using NIST SRM972a samples were carried out. Results Compared to the reference LC-MS/MS method, the Lumipulse and the Roche Elecsys III assays reached the optimal criterion for bias, while the Cascadion met the desirable one. The Roche Elecsys II was not able to reach the minimal criteria. The proportion of correctly classified patients was higher using the Cascadion (95.2%) compared to the three immunoassays. In addition to its better precision, the Cascadion was not impacted by a high concentration of 3-epi-25(OH)D3 compared to the three immunoassays. Conclusions Compared to the LC-MS/MS reference method, the Cascadion presented the highest level of concordance at medical decision cut-offs for total 25(OH)D and reached the desirable specification for bias. Moreover, the presence of 3-epi-25(OH)D3 in enriched samples was only problematic in immunoassay methods, and especially considering Roche Elecsys methods. The release of performant fully automated mass spectrometry assays with high throughput might therefore facilitate the wide scale adoption of LC-MS/MS, even in non-specialized clinical laboratories.

Background: At the dawn of the pandemic, severe forms of COVID-19 were often complicated by thromboembolisms. However, routine laboratory tests cannot be used to predict thromboembolic events. The objective of this study was to investigate the potential value of the thrombin generation test (TGT) in predicting hypercoagulability and thrombotic risk in the aforementioned set of patients. Methods: The study panel comprised 52 patients divided into two groups (26 COVID-19 positive and 26 COVID-19 negative); COVID-19-positive patients were further grouped in “severe” (n = 11) and “non-severe” (n = 15) categories based on clinical criteria. The routine blood tests and TGT of these patients were retrospectively analyzed. Results: All 26 COVID-19-positive patients showed decreased lymphocyte, monocyte and basophil counts and increased lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine transaminase (ALT) compared with control patients. Conversely, we did not observe statistically significant differences between severe and non-severe patients despite anecdotal variations in the distribution patterns. TGT without thrombomodulin (TM) addition showed statistically significant differences in the thrombin peak heights between COVID-19-positive and negative patients. After addition of TM, peak height, Endogenous Thrombin Potential (ETP) and velocity index were increased in all COVID-19-positive patients while the percentage of inhibition of ETP was reduced. These trends correlated with the severity of disease, showing a greater increase in peak height, ETP, velocity index and a drastic reduction in the percentage of ETP inhibition in more severely affected patients. Conclusions: Our data suggest that all COVID-19 patients harbor a hypercoagulable TGT profile and that this is further pronounced in severely affected patients.

CtIP is a DNA end resection factor widely implicated in alternative end-joining (A-EJ)–mediated translocations in cell-based reporter systems. To address the physiological role of CtIP, an essential gene, in translocation-mediated lymphomagenesis, we introduced the T855A mutation at murine CtIP to nonhomologous end-joining and Tp53 double-deficient mice that routinely succumbed to lymphomas carrying A-EJ–mediated IgH-Myc translocations. T855 of CtIP is phosphorylated by ATM or ATR kinases upon DNA damage to promote end resection. Here, we reported that the T855A mutation of CtIP compromised the neonatal development of Xrcc4(−/−)Tp53(−/−) mice and the IgH-Myc translocation-driven lymphomagenesis in DNA-PKcs(−/−)Tp53(−/−) mice. Mechanistically, the T855A mutation limits DNA end resection length without affecting hairpin opening, translocation frequency, or fork stability. Meanwhile, after radiation, CtIP-T855A mutant cells showed a consistent decreased Chk1 phosphorylation and defects in the G2/M cell cycle checkpoint. Consistent with the role of T855A mutation in lymphomagenesis beyond translocation, the CtIP-T855A mutation also delays splenomegaly in λ-Myc mice. Collectively, our study revealed a role of CtIP-T855 phosphorylation in lymphomagenesis beyond A-EJ–mediated chromosomal translocation.

Significance Fifty percent of diffuse large B cell lymphoma (DLBCL) evade immune-surveillance via somatic genetic lesions abrogating the expression of the class I major histocompatibility complex (MHC-I) complex on the cell surface, thus preventing the presentation of tumor neoantigens to the immune system. The results herein significantly extend these findings by showing that an additional 40% of DLBCL cases, despite expressing MHC-I, carry monoallelic HLA-I genetic alterations that limit the repertoire of neoantigens for presentation to immune cells. Both MHC-INEG and MHC-IPOS/monoallelically disrupted cases have significantly higher mutational load. Notably, homozygosis of HLA-I loci is significantly and preferentially enriched in the germline of DLBCL patients, suggesting a stepwise process by which limited neoantigen presentation is selected during DLBCL development., Fifty percent of diffuse large B cell lymphoma (DLBCL) cases lack cell-surface expression of the class I major histocompatibility complex (MHC-I), thus escaping recognition by cytotoxic T cells. Here we show that, across B cell lymphomas, loss of MHC-I, but not MHC-II, is preferentially restricted to DLBCL. To identify the involved mechanisms, we performed whole exome and targeted HLA deep-sequencing in 74 DLBCL samples, and found somatic inactivation of B2M and the HLA-I loci in 80% (34 of 42) of MHC-INEG tumors. Furthermore, 70% (22 of 32) of MHC-IPOS DLBCLs harbored monoallelic HLA-I genetic alterations (MHC-IPOS/mono), indicating allele-specific inactivation. MHC-INEG and MHC-IPOS/mono cases harbored significantly higher mutational burden and inferred neoantigen load, suggesting potential coselection of HLA-I loss and sustained neoantigen production. Notably, the analysis of >500,000 individuals across different cancer types revealed common germline HLA-I homozygosity, preferentially in DLBCL. In mice, germinal-center B cells lacking HLA-I expression did not progress to lymphoma and were counterselected in the context of oncogene-driven lymphomagenesis, suggesting that additional events are needed to license immune evasion. These results suggest a multistep process of HLA-I loss in DLBCL development including both germline and somatic events, and have direct implications for the pathogenesis and immunotherapeutic targeting of this disease.

SummaryFollicular lymphoma (FL) is an indolent disease, but 30%–40% of cases undergo histologic transformation to an aggressive malignancy, typically represented by diffuse large B cell lymphoma (DLBCL). The pathogenesis of this process remains largely unknown. Using whole-exome sequencing and copy-number analysis, we show here that the dominant clone of FL and transformed FL (tFL) arise by divergent evolution from a common mutated precursor through the acquisition of distinct genetic events. Mutations in epigenetic modifiers and antiapoptotic genes are introduced early in the common precursor, whereas tFL is specifically associated with alterations deregulating cell-cycle progression and DNA damage responses (CDKN2A/B, MYC, and TP53) as well as aberrant somatic hypermutation. The genomic profile of tFL shares similarities with that of germinal center B cell-type de novo DLBCL but also displays unique combinations of altered genes with diagnostic and therapeutic implications.

Diffuse large B-cell lymphoma (DLBCL) is one of the most common and aggressive types of B-cell lymphoma. Deregulation of proto-oncogene expression after a translocation, most notably to the immunoglobulin heavy-chain locus (IGH), is one of the hallmarks of DLBCL. Using whole-genome sequencing analysis, we have identified the PD-L1/PD-L2 locus as a recurrent translocation partner for IGH in DLBCL. PIM1 and TP63 were also identified as novel translocation partners for PD-L1/PD-L2 Fluorescence in situ hybridization was furthermore used to rapidly screen an expanded DLBCL cohort. Collectively, a subset of samples was found to be affected by gains (12%), amplifications (3%), and translocations (4%) of the PD-L1/PD-L2 locus. RNA sequencing data coupled with immunohistochemistry revealed that these cytogenetic alterations correlated with increased expression of PD-L1 but not of PD-L2 Moreover, cytogenetic alterations affecting the PD-L1/PD-L2 locus were more frequently observed in the non-germinal center B cell-like (non-GCB) subtype of DLBCL. These findings demonstrate the genetic basis of PD-L1 overexpression in DLBCL and suggest that treatments targeting the PD-1-PD-L1/PD-L2 axis might benefit DLBCL patients, especially those belonging to the more aggressive non-GCB subtype.

Next-generation whole-exome sequencing has revealed two novel genes, namely NOTCH1 and SF3B1, whose mutations predict poor outcome and preferentially associate with chemorefractory chronic lymphocytic leukemia (CLL). Analysis of 539 CLL cases documents that NOTCH1 mutations i) represent one of the most frequent cancer gene mutations involved at presentation; ii) cluster with cases harboring trisomy 12 and tend to be mutually exclusive with TP53 disruption among genetic subgroups; iii) identify high-risk patients showing poor survival similar to that associated with TP53 abnormalities; and iv) exert a prognostic role independent of widely accepted clinical and genetic risk factors. Mutations of SF3B1, a splicing factor that is a critical component of the spliceosome, recurrently associate with fludarabine-refractory CLL, occur at a low rate at CLL presentation and have a minor role in Richter transformation, corroborating the notion that CLL histological shift is molecularly distinct from chemorefractory progression without the Richter transformation.

High-throughput genomics requires tumor DNA matched to germline DNA, that cannot be easily obtained in the context of leukemia. Using chronic lymphocytic leukemia as a model, saliva DNA was frequently devoid of tumor DNA also during overt disease, and passed quality controls for SNP-array (77/102, 75.4%) and next generation sequencing (71/102, 69.6%). Compared to saliva, urine provides germline DNA of similar quality but in lower amounts. Saliva DNA was successfully run on SNP 6.0 arrays, and passed quality control call rate thresholds. On these bases, saliva represents a useful source of germline DNA for high-throughput genomic studies of hematologic neoplasia.

Richter syndrome (RS) represents the transformation of chronic lymphocytic leukemia to aggressive lymphoma. We explored intraclonal diversification (ID) of immunoglobulin genes in order to (i) follow the evolutionary history of the RS clone (ii) compare the role of ID in clonally related RS vs. clonally unrelated cases. Most (10/11, 90.9%) clonally related RS stem from the predominant clone observed at CLL diagnosis. One single RS had a transformation pattern compatible with sequential evolution from a secondary CLL subclone. Once RS transformation had occurred, all secondary CLL subclones disappeared and were substituted by the dominant RS clone with its own descendants. These observations suggest that genetic lesions associated with RS transformation are acquired by a cell belonging to the original CLL clone, rather than being progressively accumulated by later CLL subclones. Accordingly, most (9/11, 81.1%) clonally related RS harbored a genetic lesion disrupting TP53 that was already present, though at subclonal levels, in 5/11 (45.5%) samples of the paired CLL phase. A fraction of clonally related RS switched off ID (4/11, 36.4%) or reduced the levels of ID (5/11, 45.4%) at transformation. Conversely, all clonally unrelated RS harbored ID and were characterized by a significantly higher mutation frequency compared to clonally related RS (median: 1.18 × 10(-3) vs. 0.13 × 10(-3); p =0.002). These data indicate that (i) clonally related RS stems from a cell that is already present within the initial CLL clone and (ii) clonally unrelated and clonally related RS are biologically distinct disorders also in terms of antigen affinity maturation.

Transformation of chronic lymphocytic leukemia (CLL) to diffuse large B-cell lymphoma (DLBCL) is known as Richter syndrome (RS). In the entire CLL population, the cumulative prevalence of RS development steadily increases at a rate of 1% per year. Considering conventional predictors of CLL, patient subgroups at high risk of developing RS are characterized by the expression of CD38, absence of del13q14, and a lymph node size >3 cm. Novel risk factors for predicting RS development at CLL diagnosis have been recently identified and include: the host genotype of the CD38 locus and of other genes; telomere length of CLL cells; stereotyped B-cell receptor; and usage of specific immunoglobulin variable genes (IGHV4-39). Importantly, although some risk factors predict both CLL progression and transformation to RS, others (CD38 genotype, absence of del13q14, IGHV4-39 usage, stereotyped B-cell receptor) appear to specifically predict RS. The definition of RS encompasses at least two different conditions: DLBCLs that are clonally related to the pre-existing CLL (accounting for most cases), or DLBCL unrelated to the CLL clone. The transition from CLL to clonally related RS is accompanied by the acquisition of novel genetic alterations that may account for the chemorefractoriness of RS. Genome-wide studies that are currently ongoing are important for identifying novel molecular lesions implicated in RS that might represent a suitable target for future therapeutic strategies.

Next generation sequencing and copy number analysis provide insights into the complexity of the CLL coding genome, and reveal an association between NOTCH1 mutational activation and poor prognosis., The pathogenesis of chronic lymphocytic leukemia (CLL), the most common leukemia in adults, is still largely unknown. The full spectrum of genetic lesions that are present in the CLL genome, and therefore the number and identity of dysregulated cellular pathways, have not been identified. By combining next-generation sequencing and copy number analysis, we show here that the typical CLL coding genome contains

Richter syndrome (RS) represents the transformation of chronic lymphocytic leukaemia (CLL) to aggressive lymphoma. Risk factors of CLL transformation to RS are only partly known. We explored the role of the host genetic background as a risk factor for RS occurrence. Forty-five single nucleotide polimorphisms (SNPs) known to be relevant for CLL prognosis were genotyped in a consecutive cohort of 331 CLL, of which 21 had transformed to RS. After correcting for multiple testing and adjusting for previously reported RS risk factors, the LRP4 rs2306029 TT variant genotype was the sole SNP independently associated with a higher risk of RS transformation (Hazard Ratio: 4·17; P = 0·001; q = 0·047). The enrichment of LRP4 TT genotype in RS was confirmed in an independent series (n = 44) used for validation purposes. The LRP4 protein was expressed in CLL (n =66). Bioinformatic analysis scored LRP4 rs2306029 as a variant with possible deleterious and damaging variant of LRP4. LRP4 genotyping may help the recognition of patients with increased risk of RS at the time of CLL diagnosis.

B cell lymphomas represent a diverse group of biologically and clinically distinct neoplasms, encompassing over 40 subtypes that derive from the malignant transformation of mature B cells, most commonly at the germinal centre (GC) stage of differentiation. Analogous to most cancer types, these tumours are caused by alterations of oncogenes and tumour suppressor genes, some of which have specific roles in GC development. This chapter will focus on the mechanisms and consequences of chromosomal translocations and other genetic lesions involved in the pathogenesis of the most common types of mature B cell lymphomas, including Mantle Cell Lymphoma, Follicular Lymphoma, Diffuse Large B Cell Lymphoma, and Burkitt Lymphoma.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world and shows a remarkable heterogeneity in the clinical course. Understand the genetic basis of CLL may help in clarifying the molecular bases of this clinical heterogeneity. Recurrent chromosomal aberrations at 13q14, 12q, 11q22-q23 and 17p13, and TP53 mutations are the first genetic lesions identified as drivers of the disease. While some of these lesions are associated with poor outcome (17p13 deletion, TP53 mutations and, to a lesser extent, 11q22-q23 deletion) others are linked to a favorable course (13q14 deletion as sole aberration). Recently, next generation sequencing has revealed additional recurrent alterations in CLL targeting the NOTCH1 , SF3B1 , and BIRC3 genes. NOTCH1 , SF3B1 , and BIRC3 lesions provide: i) new insights on the mechanisms of leukemogenesis, tumor progression and chemoresistance in this leukemia; ii) new biomarkers for the identification of poor risk patients, having individually shown correlations with survival in CLL; and iii) new therapeutic targets, especially in the setting of high risk disease. This review will summarize the most important genetic aberrations in CLL and how our improved knowledge of the genome of leukemic cells may translate into improved patients' management.

Trisomy 12, the third most frequent chromosomal aberration in chronic lymphocytic leukemia (CLL), confers an intermediate prognosis. In our cohort of 104 untreated patients carrying +12, NOTCH1 mutations occurred in 24% of cases and were associated to unmutated IGHV genes (P=0.003) and +12 as a sole cytogenetic abnormality (P=0.008). NOTCH1 mutations in +12 CLL associated with an approximately 2.4 fold increase in the risk of death, a significant shortening of survival (P

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. In the present study, BIRC3, a negative regulator of noncanonical NF-κB signaling, was investigated in different CLL clinical phases. BIRC3 lesions were absent in monoclonal B-cell lymphocytosis (0 of 63) and were rare in CLL at diagnosis (13 of 306, 4%). Conversely, BIRC3 disruption selectively affected 12 of 49 (24%) fludarabine-refractory CLL cases by inactivating mutations and/or gene deletions that distributed in a mutually exclusive fashion with TP53 abnormalities. In contrast to fludarabine-refractory CLL, progressive but fludarabine-sensitive patients were consistently devoid of BIRC3 abnormalities, suggesting that BIRC3 genetic lesions associate specifically with a chemorefractory phenotype. By actuarial analysis in newly diagnosed CLL (n = 306), BIRC3 disruption identified patients with a poor outcome similar to that associated with TP53 abnormalities and exerted a prognostic role that was independent of widely accepted clinical and genetic risk factors. Consistent with the role of BIRC3 as a negative regulator of NF-κB, biochemical studies revealed the presence of constitutive noncanonical NF-κB activation in fludarabine-refractory CLL patients harboring molecular lesions of BIRC3. These data identify BIRC3 disruption as a recurrent genetic lesion of high-risk CLL devoid of TP53 abnormalities.

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 Disclosures: No relevant conflicts of interest to declare.

Notch2 mutations represent the most frequent lesion in splenic marginal zone lymphoma., Splenic marginal zone lymphoma (SMZL) is a B cell malignancy of unknown pathogenesis, and thus an orphan of targeted therapies. By integrating whole-exome sequencing and copy-number analysis, we show that the SMZL exome carries at least 30 nonsilent gene alterations. Mutations in NOTCH2, a gene required for marginal-zone (MZ) B cell development, represent the most frequent lesion in SMZL, accounting for ∼20% of cases. All NOTCH2 mutations are predicted to cause impaired degradation of the NOTCH2 protein by eliminating the C-terminal PEST domain, which is required for proteasomal recruitment. Among indolent B cell lymphoproliferative disorders, NOTCH2 mutations are restricted to SMZL, thus representing a potential diagnostic marker for this lymphoma type. In addition to NOTCH2, other modulators or members of the NOTCH pathway are recurrently targeted by genetic lesions in SMZL; these include NOTCH1, SPEN, and DTX1. We also noted mutations in other signaling pathways normally involved in MZ B cell development, suggesting that deregulation of MZ B cell development pathways plays a role in the pathogenesis of ∼60% SMZL. These findings have direct implications for the treatment of SMZL patients, given the availability of drugs that can target NOTCH, NF-κB, and other pathways deregulated in this disease.

Several drugs used for diffuse large B-cell lymphoma (DLBCL) treatment rely on DNA damage for tumor cell killing. We verified the prognostic impact of the host DNA repair genotype in 2 independent cohorts of DLBCL treated with R-CHOP21 (training cohort, 163 cases; validation cohort, 145 cases). Among 35 single nucleotide polymorphisms analyzed in the training series, MLH1 rs1799977 was the sole predicting overall survival. DLBCL carrying the MLH1 AG/GG genotype displayed an increased death risk (hazard ratio [HR] = 3.23; P < .001; q =0 .009) compared with patients carrying the AA genotype. Multivariate analysis adjusted for International Prognostic Index identified MLH1 AG/GG as an independent OS predictor (P < .001). The poor prognosis of MLH1 AG/GG was the result of an increased risk of failing both R-CHOP21 (HR = 2.02; P = .007) and platinum-based second-line (HR = 2.26; P = .044) treatment. Survival analysis in the validation series confirmed all outcomes predicted by MLH1 rs1799977. The effect on OS of MLH1, a component of the DNA mismatch repair system, is consistent with its role in regulating the genotoxic effects of doxorubicin and platinum compounds, which are a mainstay of DLBCL first- and second-line treatment.

Abstract 264 Splenic marginal zone lymphoma (SMZL) is one of few B-cell lymphoma types that remain orphan of molecular lesions in cancer related genes. Detection of active NF-κB signaling by immunohistochemical assays for nuclear NFKB1 (p50) and NFKB2 (p52) in 14/24 (58%) SMZL prompted the investigation of NF-κB molecular alterations in SMZL (Fig.1A). To this aim, NF-κB pathway genes (n=20) were screened in 101 SMZL for mutations by Sanger sequencing and for copy number abnormalities (CNAs) by FISH and SNP array (GeneChip Human Mapping 250K NspI, Affymetrix). Mutations targeting multiple NF-κB genes were detected in 20/101 (20%) SMZL. Mutations affected key regulators of both canonical (IKBKB, TNFAIP3) and non-canonical (BIRC3, TRAF3, MAP3K14) NF-κB pathways and were mutually exclusive, pointing to multiple molecular mechanisms targeting NF-κB in SMZL (Fig.1B). By combining mutations and CNAs, NF-κB was affected in 36/101 (36%) SMZL. The TRAF3/MAP3K14-TRAF2/BIRC3 regulatory complex of the non-canonical NF-κB pathway was targeted in 25/101 (25%) SMZL. BIRC3 was affected in 11/101 (11%) SMZL by inactivating mutations (3 frameshift and 2 non-sense), missense mutations (n=1), and gene deletions (n=5). 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 (Fig. 1C). TRAF3 was affected in 10/101 (10%) SMZL by inactivating mutations (2 frameshift and 1 non-sense), missense mutations (n=1) and deletions (n=7). Inactivating mutations were predicted to generate aberrant transcripts carrying premature stop codons and causing elimination or truncation of the C-terminal MATH domain that provides the docking site for MAP3K14, and is required for MAP3K14 recruitment to BIRC3 degradation. MAP3K14 was affected by gain (n=7) or missense mutations (n=1) in 8/101 (8%) SMZL. The missense mutation was somatically acquired and mapped near the kinase domain, suggesting a potential role in MAP3K14 downstream signaling activation. SMZL primary cells harboring BIRC3, TRAF3 or MAP3K14 mutations displayed constitutive NF-κB activation, including MAP3K14 accumulation and active NFKB2 processing by Western blot, and/or nuclear NF-κB localization by immunohistochemistry (Fig. 1D). In addition to non-canonical signaling, also the canonical NF-κB pathway was targeted in SMZL (15/101, 15%) by genetic lesions of TNFAIP3 and IKBKB, which encode for negative and positive regulators of NF-κB signaling, respectively. TNFAIP3 was affected in 13/101 (13%) SMZL by inactivating mutations (6 frameshift) and deletions (n=9), including a focal loss pointing to TNFAIP3 as the specific target of deletions. IKBKB was mutated in 3/101 (3%) SMZL carrying a recurrent, somatically acquired missense mutation (K171E) targeting the IKBKB kinase domain near the activation loop. This mutation leads to an amino acid substitution within a site that is conserved both intra- and inter-species, and is predicted as possibly damaging according to PolyPhen-2. The occurrence of NF-κB molecular lesions in SMZL was observed independent of HCV infection (p=.402), IGHV gene mutation status (p=.065), IGHV1-2 gene usage (p=.761), stereotyped VH CDR3 (p=.969), 7q deletion (p=.621), or TP53 disruption (p=.638), suggesting that genetic deregulation of NF-κB is not restricted to specific SMZL clinico-molecular subgroups. The identification of NF-κB mutations in SMZL elucidates the molecular basis of a fraction of these lymphomas, and expands the genetic mechanisms activating NF-κB in B-cell neoplasia. Beside its pathogenetic relevance, the identification of NF-κB as a pathway recurrently involved in SMZL provides the rationale for targeted anti-NF-κB therapeutic approaches in this lymphoma. Disclosures: No relevant conflicts of interest to declare.

The genetic lesions identified in chronic lymphocytic leukemia (CLL) do not entirely recapitulate the disease pathogenesis and the development of serious complications, such as chemorefractoriness. While investigating the coding genome of fludarabine-refractory CLL, we observed that mutations of SF3B1, encoding a splicing factor and representing a critical component of the cell spliceosome, were recurrent in 10 of 59 (17%) fludarabine-refractory cases, with a frequency significantly greater than that observed in a consecutive CLL cohort sampled at diagnosis (17/301, 5%; P = .002). Mutations were somatically acquired, were generally represented by missense nucleotide changes, clustered in selected HEAT repeats of the SF3B1 protein, recurrently targeted 3 hotspots (codons 662, 666, and 700), and were predictive of a poor prognosis. In fludarabine-refractory CLL, SF3B1 mutations and TP53 disruption distributed in a mutually exclusive fashion (P = .046). The identification of SF3B1 mutations points to splicing regulation as a novel pathogenetic mechanism of potential clinical relevance in CLL.

CD49d expression and telomere length (TL) represent novel prognostic markers for chronic lymphocytic leukemia (CLL) [1―7]. The prognostic information carried by CD49d expression and TL in CLL has its rationale in the disease biology. CD49d is an adhesion molecule mediating cell-to-cell and cell-to-extracellular matrix interactions [8]. In CLL cells, CD49d transmits prosurvival/antlapoptotic signals from the tumor microenvironment to tumor cells [9]. Telomeres ensure genetic stability and regulate critical cellular functions, including proliferation and senescence [10]. In CLL, short TL associates with a fast proliferative history of the leukemic cells [1,11,12]. Both CD49d expression and short TL have been associated with increased genetic instability of the CLL clone [3,13]. From a clinical standpoint, a peculiar feature shared by CD49d expression and short TL is the association with CLL proliferation markers, including expression of CD38, high lactate dehydrogenase (LDH), high β-2-microglobulin, short time to lymphocyte doubling, and short time to progression to a more advanced stage [1―7]. Here, we tested whether the concomitant presence of high CD49d expression and short TL in the same CLL patient may help refine disease stratification for treatment prediction in patients that presented in early to intermediate Binet stage (Binet A and B) and that are candidate to watch and wait as initial management.

The genetic profile of chronic lymphocytic leukemia (CLL) is characterized by a pool of recurrent lesions, including trisomy 12, deletions of 13q14, 11q22-q23, 17p13, and 6q21, and t(14)(q32) translocations [1]. The aforementioned lesions can be detected by FISH and may harbor prognostic information [1]. Array-based comparative genomic hybridization (aCGH) has identified gain of multiple regions on the short arm of chromosome 2 as a recurrent CLL lesion, that contain the REL, BCL11A, and MYCN proto-oncogenes that are involved in hematologic malignancies [2-5]. The exact prevalence of REL, BCL11A, and MYCN gains at the time of CLL diagnosis is currently unknown in consecutive series of the disease. Moreover, some studies indicate that 2p gains as a whole may herald progressive and/or poor risk disease, yet the prognostic contribution of the individual genes included in the amplicon, i.e., REL, BCL11A, and MYCN, remains undefined [4-6]. The aim of this study was twofold: (i) characterize the prevalence of REL, BCL11A, and MYCN gains in a consecutive CLL series at the time of diagnosis; (ii) define the prognostic relevance of REL, BCL11A, and MYCN gains in CLL.

BACKGROUND: Although serum beta-2 microglobulin (B2M) represents a key variable for symptomatic multiple myeloma (MM) prognostication, its role in predicting the risk of progression of asymptomatic MM to symptomatic disease has not been explored. METHODS: This study was bases on a consecutive series of 148 patients with asymptomatic MM and explored the cumulative probability of progression to symptomatic MM as the primary endpoint. RESULTS: In univariate analysis, a serum B2M level >2.5 mg/L was associated with an increased probability of disease progression (5-year risk, 64.5%; P < .001) along with serum monoclonal component (sMC) (P < .001), urinary monoclonal component (uMC) (P < .001), and bone marrow plasma cells (BMPCs) (P < .001). In multivariate analysis, serum B2M was selected as an independent predictor of progression (hazard ratio, 3.30; P = .002). Serum B2M was combined with sMC, uMC, and BMPC to create a risk-stratification model based on 4 groups with different risk of progression: very low (5-year risk, 0%), low-intermediate (5-year risk, 19.6%), high-intermediate (5-year risk, 60.7%), and high (5-year risk, 80.7%). The model that included serum B2M along with sMC, uMC, and BMPC was able to predict disease progression better than the model that was based on sMC, uMC, and BMPC without serum B2M (C statistics, 0.760 vs 0.726). CONCLUSIONS: The current results indicated that 1) serum B2M is an independent predictor of asymptomatic MM progression, and 2) serum B2M adds prognostic information when combined with the most widely used prognosticators of asymptomatic MM progression. Cancer 2010. © 2010 American Cancer Society.

Abstract 992 Several drugs utilized in diffuse large B cell lymphoma (DLBCL) rely on DNA damage for tumor killing. This study aimed at verifying whether single nucleotide polymorphisms (SNPs) of genes involved in DNA repair may contribute to prognostication of DLBCL treated with R-CHOP. The study utilized a training-validation design. The training cohort (n=163) was a mono-institutional, prospectively collected, consecutive series of DLBCL homogeneously treated with the same chemotherapeutic regimen both at diagnosis (R-CHOP21) and at relapse/progression (R-DHAP ± BEAM conditioned autologous stem cell transplant, ASCT). The validation cohort (n=156) was a multi-institutional retrospective series of DLBCL treated with R-CHOP at diagnosis. Candidate SNPs (n=35) were selected by an educated guess approach, and included SNPs belonging to genes involved in: i) mismatch repair (MLH1); ii) base excision repair (XRCC1, OGG1); iii) nucleotide excision repair (ERCC1, ERCC2, ERCC4, ERCC5, ERCC6, XPA, XPC); iv) double strand break repair (BRCA1, BRCA2, LIG4, XRCC2, XRCC3, XRCC4, XRCC6); and v) direct reversal (MGMT). Clinical endpoints were progression free survival (PFS) after R-CHOP, overall survival (OS) from diagnosis, and OS from salvage treatment. Univariate analysis controlled for multiple comparisons identified MLH1 rs1799977 as the sole SNP predicting DLBCL OS in the training series (AG/GG genotype: HR: 3.23; 4-year OS: 55.5% vs AA genotype: 4-year OS: 80.9%; p Disclosures: No relevant conflicts of interest to declare.

Background ZAP-70 is an independent negative prognostic marker in chronic lymphocytic leukemia (CLL). Usually, its expression is investigated by flow cytometric protocols in which the percentage of ZAP-70 positive CLL cells is determined in respect to isotypic control (ISO-method) or residual ZAP-70 positive T cells (T-method). These methods, however, beside suffering of an inherent subjectivity in their application, may give discordant results in some cases. The aim of this study was to assess the prognostic significance of these methods in comparison with another in which ZAP-70 expression was evaluated as a Mean-Fluorescence-Intensity Ratio between gated T and CLL cells (T/B Ratio-method). Methods Cytometric files relative to ZAP-70 determination according to the three readouts were retrospectively reviewed on a cohort of 173 patients (test set), all with complete clinical and biological prognostic assessment and time-to-treatment (TTT) available. Findings were then validated in an independent cohort of 341 cases from a different institution (validation set). Results The optimal prognostic cut-offs for ZAP-70 expression were selected at 11% (ISO-method) or 20% of positive cells (T-method), as well as at 3.0 (T/B Ratio-method) in the test set; these cut-offs yielded 66, 60 and 73 ZAP-70+ cases, respectively. Univariate analyses resulted in a better separation of ZAP-70+ vs. ZAP-70- CLL patients utilizing the T/B Ratio, compared to T- or ISO-methods. In multivariate analyses which included the major clinical and biological prognostic markers for CLL, the prognostic impact of ZAP-70 appeared stronger when the T/B-Ratio method was applied. These findings were confirmed in the validation set, in which ZAP-70 expression, evaluated by the T- (cut-off = 20%) or T/B Ratio- (cut-off = 3.0) methods, yielded 180 or 127 ZAP-70+ cases, respectively. ZAP-70+ patients according to the T/B Ratio-method had shorter TTT, both if compared to ZAP-70- CLL, and to cases classified ZAP-70+ by the T-method only. Conclusions We suggest to evaluate ZAP-70 expression in routine settings using the T/B Ratio-method, given the operator and laboratory independent feature of this approach. We propose the 3.0 T/B Ratio value as optimal cut-off to discriminate ZAP-70+ (T/B Ratio less than 3.0) from ZAP-70- (T/B Ratio more/equal than 3.0) cases.

Diffuse large B cell lymphoma (DLBCL) is the most common form of B cell non-Hodgkin lymphoma (B-NHL), accounting for ~25-40% of all lymphoid tumors. DLBCL comprises genetically, phenotypically and clinically distinct subtypes, including the prognostically favorable germinal center B cell like (GCB)-DLBCL and the more aggressive activated B cell like (ABC)-DLBCL. We have shown that >60% of DLBCL, independent of molecular subtype, lack cell surface expression of HLA-class I (HLA-I), suggesting that these tumors may escape immune recognition by cytotoxic T cells (CTL) (Challa-Malladi, Lieu et al., Cancer Cell, 2011). HLA-I loss also represents a common lesion acquired at transformation of follicular lymphoma (FL) to DLBCL (Pasqualucci et al., Cell Reports 2014). We have investigated the expression of HLA-I across the clinico-pathological spectrum of mature B cell tumors, and found that HLA-I loss is significantly less common in other mature B-NHL, including Burkitt lymphoma (13/43, 30.2%; p=.002), FL (12/60, 20.0%; p One mechanism of HLA-I loss, identified by exome-sequencing and copy number analysis, is represented by genomic deletions and/or mutational inactivation of the B2M gene, which are found in ~50% of HLA-I negative cases (29% of all DLBCL). These lesions lead to the complete loss of B2-microglobulin, a required component for the assembly and cell surface expression of the HLA-I complex (Pasqualucci et al. Nat Genet, 2011; Challa-Malladi, Lieu et al. Cancer Cell, 2011). However, the remaining ~50% of patients lack surface HLA-I despite the absence of B2M genetic lesions, suggesting the existence of additional underlying mechanisms. In particular, a fraction of patients express an intact B2M protein, which is mislocalized to the cytoplasm. To investigate whether direct genetic disruption of the HLA-I genes could be responsible for the lack of surface HLA-I in these cases, we performed Sanger sequencing and SNP6.0 array analysis of the HLA-I heavy chain genes (HLA-A and HLA-B) in two DLBCL cell lines (Ly10 and RCK8) with wild-type B2M alleles, but cytoplasmic B2M protein. In both lines, we found the presence of biallelic mutations or deletions in the HLA-I loci. Accordingly, transduction with a retrovirus expressing either HLA-I gene was sufficient to restore cell surface B2M and HLA-I in both lines, documenting that DLBCL can exploit genetic disruption of HLA-I as an alternative mechanism to impair the assembly of a membrane HLA-I complex. The overall contribution of this mechanism to HLA-I loss is currently being determined by using a custom capture/next generation sequencing approach of the HLA-I loci in a large panel of paired tumor/normal biopsies with negative or mislocalized B2M/HLA-I. We also examined the role of B2M (HLA-I) loss in lymphomagenesis in vivo. Particularly, since constitutional B2m deletion is not tumorigenic per se (Koller et al., Science 1990), and B2M loss is frequently acquired during FL transformation to DLBCL, we investigated whether the absence of major histocompatibility complex on the cell surface of mature B cells accelerates tumorigenesis in the presence of other oncogenic lesions. To this end, we generated a conditional knock-out mouse model in which the B2m gene is specifically deleted in germinal center B cells upon expression of a Cγ1-Cre allele, and crossed them with IµHABCL6 knock-in mice, which develop DLBCL due to deregulated expression of the BCL6 oncogene (Cattoretti, Pasqualucci et al., Cancer Cell 2006). Disclosures No relevant conflicts of interest to declare.

Abstract 464 Fludarabine-refractoriness of chronic lymphocytic leukemia (CLL) is due to TP53 disruption in ∼40% of refractory cases, but in a sizeable fraction of patients the molecular basis of this aggressive clinical phenotype remains unclear. Our initial findings from whole exome sequencing of fludarabine-refractory CLL led to the identification of recurrent mutations of SF3B1, a critical component of the cell spliceosome, prompting further investigations of these alterations in a large CLL panel. The study population comprised 3 clinical cohorts representative of: i) fludarabine-refractory CLL (n=59), including cases (n=11) subjected to whole exome sequencing; ii) newly diagnosed and previously untreated CLL (n=301); and iii) clonally related RS (n=33). Tumor samples were obtained: i) for fludarabine-refractory CLL, immediately before starting the treatment to which the patient eventually failed to respond; ii) for newly diagnosed and previously untreated CLL, at disease presentation. All RS studies were performed on RS diagnostic biopsies. Mutation analysis of SF3B1 was performed on genomic DNA by a combination of Sanger sequencing and targeted next generation sequencing. SF3B1 was altered in 10/59 (17%) fludarabine-refractory CLL by missense mutations (n=9) or in-frame deletions (n=1) clustering in the HEAT3, HEAT4 and HEAT5 repeats of the SF3B1 protein. Two sites that are highly conserved inter-species (codon 662 and codon 700) were recurrently mutated in 3 and 5 cases, respectively. SF3B1 mutations were monoallelic, and were predicted to be functionally significant according to the PolyPhen-2 algorithm. Mutations occurred irrespective of IGHV mutation status, CD38 expression and ZAP70 expression. At the time of fludarabine-refractoriness, SF3B1 mutations were enriched in cases harboring a normal FISH karyotype (p=.008) and distributed in a mutually exclusive fashion with TP53 disruption (mutual information I =0.0609; p=.046). By combining SF3B1 mutations with other genetic lesions enriched in chemorefractory cases (TP53 disruption, NOTCH1 mutations, ATM deletion), fludarabine-refractory CLL appeared to be characterized by multiple molecular alterations that, to some extent, are mutually exclusive. We then compared the prevalence of mutations observed at the time of fludarabine-refractoriness to that observed in other disease phases. At diagnosis, SF3B1 mutations were rare (17/301; 5%), and showed a crude association with short treatment free survival (p Disclosures: No relevant conflicts of interest to declare.

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 Disclosures: No relevant conflicts of interest to declare.

Abstract 1796 Poster Board I-822 The rationale of the study stems from three considerations: i) to date, few clinical predictors of asymptomatic multiple myeloma (MM) progression are available; ii) no study has assessed the role of beta-2-microglobulin (B2M) on risk of asymptomatic MM progression; iii) B2M is a serum marker of tumor burden and represents a key variable of the ISS staging system for symptomatic MM. The study aims at assessing the impact of B2M on risk of asymptomatic MM progression. The study was based on a consecutive series of 148 asymptomatic MM diagnosed according to the IMWG. Cumulative probability of progression to symptomatic MM was calculated from diagnosis of asymptomatic MM to progression to symptomatic disease according to IMWG. Survival analysis was performed by Kaplan-Meier method using log-rank to test for associations. Cox proportional hazard regression was used to build a multivariate model. Harrell's c-statistics was used to evaluate the discriminatory value of the prognostic models. Clinical features at asymptomatic MM diagnosis were as follows: median age 67 years, male:female ratio 1.02, previous MGUS 32/148 (21.6%), IgG monoclonal component (MC) 113/148 (76.4%), IgA MC 32/148 (21.6%), light chain MC 3/148 (2.0%), median serum monoclonal component (sMC) 1.1 g/dl, positive urinary immunofixation 27/148 (18.2%), median urinary monoclonal component (uMC) 98 mg/24h, median bone marrow plasma cell percentage (BMPC%) 15%, median B2M 2.0 mg/l, median albumin 4.3 g/dl, median C reactive protein 0.3 mg/l. After a median follow-up of 48 months, 29/148 asymptomatic MM progressed to symptomatic disease, accounting for a 29.5% 5-year probability of progression. According to Youden's index, best cut-off values for prediction of progression were 2.5 mg/l for B2M, 1.5 g/dl for sMC, 500 mg/24h for uMC, and 20% for BMPC%. Univariate analysis identified B2M >2.5 mg/l (5-year risk: 64.5%; HR=3.85; p1.5 g/dl (5-year risk: 49.1%; HR=5.76; p500 mg/24h (5-year risk: 68.7%; HR=6.60; p2.5 mg/l (HR=3.57; p=.001) as an independent predictor of progression to symptomatic MM, along with sMC >1.5 g/dl (HR=4.07; p=.003), uMC >500 mg/24h (HR=3.66; p=.015) and BMPC% ≥20% (HR=3.20; p=.007). Based on multivariate analysis, B2M, sMC, uMC, and BMPC% were combined into a model for predicting progression to symptomatic MM. This model stratified patients into four risk groups: very low risk (0 risk factors: 5-year risk 0%), low-intermediate risk (1 risk factor: 5-year risk 19.6%), high-intermediate risk (2 risk factors 5-year risk: 60.7%), and high risk (3 or 4 risk factors 5-year risk 80.7%) (Fig. 1). Three lines of evidence suggest that B2M may improve the conventional risk stratification model for prediction of asymptomatic MM progression defined by sMC, uMC, and BMPC%. First, B2M >2.5 mg/l adds prognostic information within the low risk group characterized by sMC '1.5 g/dl, uMC '500 mg/24h and BMPC 2.5 mg/l adds prognostic information within the high risk group defined by sMC >1.5 g/dl and/or uMC >500 mg/24h and/or BMPC ≥20% (p=.034) by segregating a high risk group of patients with B2M >2.5 mg/l who are virtually all projected to progress (5-year risk: 89.0%). Third, based on c-statistics, the prognostic model including B2M along with sMC, uMC, and BMPC% (c-statistics=.760; SE=.045) allowed to predict asymptomatic MM progression better than a model based only on sMC, uMC, and BMPC% (c-statistics=.726; SE=.049). The implications of our results are twofold: i) B2M predicts progression of asymptomatic MM to symptomatic disease independent of conventional risk factors (sMC, BMPC%, light chain burden); ii) B2M refines the conventional model for predicting progression to symptomatic MM by allowing the identification of a very low risk group of patients who never progress and a high risk group of patients who are virtually all projected to progress. Disclosures No relevant conflicts of interest to declare.

Abstract 442 Scant information is availabel on the impact of the host genetic background in predicting outcome of diffuse large B cell lymphoma (DLBCL). We recently documented that host single nucleotide polymorphisms (SNPs) affecting doxorubicin pharmacodynamics and alkylator detoxification may predict outcome in R-CHOP-treated DLBCL (Rossi et al, Leukemia 2009; 23:118-26). Several DNA repair mechanisms are involved in resistance to R-CHOP drugs. We verified whether SNPs of DNA repair genes may further refine prognostic stratification and toxicity prediction in DLBCL treated with R-CHOP. The study was based on 163 consecutive DLBCL treated with R-CHOP and provided with a prospectively collected dataset. At diagnosis, age >60 years was observed in 104/163 (63.8%) cases, ECOG PS >1 in 21/163 (12.9%), extranodal sites >1 in 41/163 (25.2%), Ann Arbor stage III-IV in 85/163 (52.1%), bulky disease in 46/163 (28.2%), B symptoms in 38/163 (23.3%), LDH elevation in 75/163 (46.0%), IPI >2 in 55/163 (33.7%). Median follow up was 40 months. Candidate SNPs selected for the analysis belonged to genes involved in repairing DNA damage produced by R-CHOP drugs, and included SNPs affecting the following DNA repair mechanisms: i) base excision repair (OGG1 rs1052133); ii) nucleotide excision repair (ERCC2 rs1799793, ERCC2 rs13181, ERCC6 rs3793784, ERCC6 rs2228528, ERCC6 rs2228529, XPA rs1800975, XPC rs2227999, XPC rs2228000); iii) mismatch repair (MLH1 rs1799977); iv) double strand break repair (LIG4 rs1805388, XRCC6 rs132788, XRCC6 rs55751129, BRCA1 rs799917). Genotyping was performed by SNP-minisequencing on DNA extracted from granulocytes. Clinical endpoints considered in the study were overall survival (OS), progression free survival (PFS) and toxicity. Associations of SNPs with clinical endpoints were controlled for multiple testing by FDR analysis. Among the SNPs included in the study, univariate log-rank analysis controlled for multiple comparisons by FDR testing identified MLH1 rs1799977 as a predictor of both PFS and OS in DLBCL treated with R-CHOP. MLH1 encodes a DNA mismatch repair gene involved in alkylating agents and doxorubicin resistance. DLBCL patients carrying the MLH1 rs1799977 AG/GG genotypes displayed a poorer PFS (Events/N: 23/52; 3-year PFS: 51.9%) compared to DLBCL patients carrying the MLH1 rs1799977 AA genotype (Events/N: 33/111; 3-year PFS: 72.0%) (p=.020; q=.240) (Fig. 1). Other variables predicting PFS were ECOG PS (p=.042), Ann Arbor stage (p=.035), bulky disease (p=.001), number of extranodal sites (p=.009), LDH (p=.007), and IPI (p=.002). Multivariate analysis selected MLH1 rs1799977 AG/GG (HR: 1.17; p=.034) as an independent predictor of PFS, along with IPI (HR: 1.44; p=.002) and bulky disease (HR: 2.20, p=.004). The MLH1 rs1799977 genotype was also relevant for predicting DLBCL survival, since DLBCL patients carrying the MLH1 rs1799977 AG/GG genotypes displayed a poorer OS (Events/N: 19/52; 3-year OS: 61.8%) compared to DLBCL patients carrying the MLH1 rs1799977 AA genotype (Events/N: 18/111; 3-year OS: 83.3%) (p=.001; q=.013) (Fig. 2). Other variables predicting OS were age (p=.026), ECOG PS (p=.002), bulky disease (p Disclosures: No relevant conflicts of interest to declare.

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.