Key Points• Independent prognostic impact of biological markers, notably TP53 and SF3B1 mutations, in CLL patients requiring therapy.• NOTCH1 mutation as a predictive factor for reduced benefit from the addition of rituximab to FC chemotherapy.Mutations in TP53, NOTCH1, and SF3B1 were analyzed in the CLL8 study evaluating firstline therapy with fludarabine and cyclophosphamide (FC) or FC with rituximab (FCR) among patients with untreated chronic lymphocytic leukemia (CLL). TP53, NOTCH1, and SF3B1 were mutated in 11.5%, 10.0%, and 18.4% of patients, respectively. NOTCH1 mut and SF3B1 mut virtually showed mutual exclusivity (0.6% concurrence), but TP53 mut was frequently found in NOTCH1 mut (16.1%) and in SF3B1 mut (14.0%) patients. There were few significant associations with clinical and laboratory characteristics, but genetic markers had a strong influence on response and survival. In multivariable analyses, an independent prognostic impact was found for FCR, thymidine kinase (TK) ‡10 U/L, unmutated IGHV, 11q deletion, 17p deletion, TP53 mut , and SF3B1 mut on progression-free survival; and for FCR, age ‡65 years, Eastern Cooperative Oncology Group performance status ‡1, b2-microglobulin ‡3.5 mg/L, TK ‡10 U/L, unmutated IGHV, 17p deletion, and TP53 mut on overall survival. Notably, predictive marker analysis identified an interaction of NOTCH1 mutational status and treatment in that rituximab failed to improve response and survival in patients with NOTCH1 mut . In conclusion, TP53 and SF3B1 mutations appear among the strongest prognostic markers in CLL patients receiving current-standard first-line therapy. NOTCH1 mut was identified as a predictive marker for decreased benefit from the addition of rituximab to FC. This study is registered at www.clinicaltrials.gov as #NCT00281918. (Blood. 2014;123(21):3247-3254)

433 Novel gene mutations have been found in CLL by next generation sequencing including mutations of NOTCH1 and SF3B1 in 5–20% of cases. In initial studies, both have been associated with advanced disease and poor outcome. We assessed the incidence and impact of gene mutations in the CLL8 trial (1st line FC vs. FCR, n=817). TP53 (exons 2–11) was analyzed by a re-sequencing chip (Amplichip, Roche Molecular Systems) with confirmatory Sanger sequencing. NOTCH1 was analyzed by Sanger sequencing exon 34, chr9:139,390,619–139,391,290 (PEST domain). SF3B1 (exons 13–16) was analyzed by DHPLC (WAVE® 3500HT, Transgenomic Inc.) with subsequent Sanger sequencing. Baseline samples were available for analysis of genetic markers in 619 (75.8%) to 645 (78.9%) patients. All markers were available for 573 (70.1%) patients and this cohort was representative of the full trial population. Mutations (mut) were found in TP53, NOTCH1, and SF3B1 in 11.5%, 10.0%, and 18.4%, respectively. At least one mutation was identified in 35.2% patients, while 30.6% had one, 4.4% had two and 0.2% had three mutations. Concurrent NOTCH1mut and SF3B1mut were found in only 0.5% patients. TP53mut was observed in 16.7% of NOTCH1mut cases (p=.528) and in 14.5% of SF3B1mut patients (p=.472). Regarding baseline characteristics, there were significant associations of TP53mut with CIRS>1, unmutated IGHV and 17p-; of NOTCH1mut with Binet A/B, no B-symptoms, unmutated IGHV, and 17p-; and of SF3B1mut with TK>10, and no +12. Regarding response to therapy, TP53mut was significantly associated with refractory disease in both arms (FCR: 25.0% vs. 1.8%, p<.001, FC: 48.4% vs. 7.8%, p<.001,); while NOTCH1mut showed only a trend in the FCR arm (FCR: 10.9% vs. 3.4%, p=.109, FC: 11.9% vs. 12.9%, p=.775); and SF3B1mut did not impact response to therapy (FCR: 3.6% vs. 3.7%, p=1.00, FC: 12.3% vs. 10.9%, p=1.00). At extended follow-up (median 69.97 months), FCR resulted into significantly improved PFS (HR 0.586, p<.001) and OS (HR 0.678, p=.001). TP53mut was associated in both treatment arms with significantly decreased PFS (FC: HR 4.295, p<.001; FCR: HR 3.173 p<.001) and OS (FC: HR 4.642 p<.001; FCR: HR 4.447, p<.001). In contrast, NOTCH1mut was only in the FCR arm associated with significantly decreased PFS (FC: HR 0.931, p=.741; FCR: HR 1.718, p=.013) and a trend to inferior OS (FC: HR 0.854, p=.605; FCR: HR 1.610, p=.112). SF3B1mut was associated in both treatment arms with significantly decreased PFS (FC: HR 1.520, p=.009; FCR: HR 1.463, p=.033) and a trend to inferior OS (FC: HR 1.338, p=.178; FCR: HR 1.305, p=.301). To evaluate the independent prognostic impact, we performed multivariable analyses by Cox regression for PFS and OS including the following variables: treatment, age, sex, stage, ECOG status, B-symptoms, WBC, TK, β2-MG, 11q-, +12, 13q-, 17p-, IGHV, TP53, NOTCH1 and SF3B1. Regarding PFS, the following independent prognostic factors were identified: FCR (HR 0.510, p<.001), TK>10 (HR 1.367, p=.019), IGHV<98% (HR 1.727, p<.001), 11q- (HR 1.536, p<.001), 17p- (HR 2.949 p<.001), TP53mut (HR 2.113 p<.001), and SF3B1mut (HR 1.348, p=.024). Regarding OS, the following independent prognostic factors were identified: FCR (HR 0.701, p=.049), ECOG>0 (HR 2.202, p<.001), TK>10 (HR 2.707, p<.001), IGHV<98% (HR 1.547, p=.055), 17p- (HR 3.546 p<.001) and TP53mut (HR 3.032 p<.001). To identify a predictive impact of gene mutations for a specific treatment effect by the addition of rituximab, we performed multivariable analyses including the treatment arms, the gene mutations and the interaction of both. Regarding PFS, FCR (HR 0.544, p<.001), TP53mut (HR 3.607, p<.001), SF3B1mut (HR 1.355, p=.012) and NOTCH1mut interaction with FCR (HR 1.652, p=.022) were identified as independent factors. Regarding OS, FCR (HR 0.654, p=.002) and TP53mut (HR 4.470, p<.001) were identified as independent factors while NOTCH1mut interaction with FCR (HR 1.331, p=.344) showed a trend. The interaction between NOTCH1mut and FCR treatment is illustrated in univariate PFS analysis, in which the addition of rituximab led to a benefit only among patients without NOTCH1mut (Figure). In conclusion, gene mutations show independent prognostic value for PFS (TP53, SF3B1) and OS (TP53) in patients receiving 1st line FC and FCR treatment. Of note, NOTCH1mut appears to identify a subset of CLL patients that does not benefit from the addition of rituximab to FC. Disclosures: Stilgenbauer: Roche: Consultancy, Honoraria, Research Funding. Patten:Roche: Employment. Wenger:Roche: Employment. Mendila:Roche: Employment. Hallek:Roche: Consultancy, Honoraria, Research Funding.

Key Points• This trial update shows that allotransplantation can provide long-term minimal residual disease-negative disease control in poor-risk chronic lymphocytic leukemia.• Six-year survival is close to 60% and is independent of the presence of TP53, SF3B1, and NOTCH1 mutations in the tumor clone.The purpose of this analysis was to provide 6-year follow-up of the CLL3X trial, which studied reduced-intensity allogeneic hematopoietic stem cell transplantation (HSCT) in patients with poor-risk chronic lymphocytic leukemia (CLL), and to investigate the effect of TP53, SF3B1, and NOTCH1 mutations on HSCT outcome. For 90 allografted patients, 6-year overall survival (OS) was 58% and 6-year event-free survival (EFS) was 38%. TP53, SF3B1, and NOTCH1 mutations were found in 30%, 26%, and 14% of the trial population, respectively. By univariate and multivariate analyses, the mutational status of the TP53, SF3B1, and NOTCH1 genes had no significant effect on OS and EFS. Studies of minimal residual disease confirmed durability of CLL eradication in mutated patients. We conclude that HSCT can provide long-term disease control in patients with poor-risk CLL independent of the presence of TP53, SF3B1, and NOTCH1 mutations. The trial has been registered at the US National Cancer Institute as #EU-20554, NCT00281983. (Blood. 2013;121(16):3284-3288) IntroductionThere is ample evidence that poor-risk chronic lymphocytic leukemia (CLL), as defined by fludarabine refractoriness or the presence of deletion 17p (17p-), can be successfully treated by allogeneic hematopoietic stem cell transplantation (HSCT). [1][2][3][4][5][6][7] It is unknown, however, whether disease control provided by HSCT is durable in the long term and whether HSCT can also overcome the treatment resistance associated with genetic factors, such as TP53 mutations, seen under conventional fludarabine combination therapy. [8][9][10] Therefore, the purpose of this analysis was to provide 6-year follow-up of the German CLL Study Group (GCLLSG) CLL3X trial, which aimed to evaluate reduced-intensity HSCT in patients with poor-risk CLL, and to compare the effect of TP53 mutations with that of the newly identified mutations of the SF3B1 and NOTCH1 genes, which in some studies have been associated with resistance to conventional treatment. 11-16 Study designThe protocol including the informed consent form was approved by all responsible institutional review boards. Patients gave written informed consent using study-specific forms, in accordance with the Declaration of Helsinki.The CLL3X trial has been described previously. 6 In brief, CLL3X included 100 patients with a median age of 53 years (27 to 65 years), of whom 69 patients met at least 1 of the EBMT CLL transplant criteria 17 and 31 patients had failed a previous autograft or fulfilled other poor-risk criteria as defined in the protocol. The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indi...

We studied the incidences, associations, and prognostic roles of NOTCH1 and SF3B1 mutations (NOTCH1(mut), SF3B1(mut)) as compared with TP53(mut) in fludarabine-refractory chronic lymphocytic leukemia (CLL) patients treated with alemtuzumab in the CLL2H trial. We found NOTCH1(mut), SF3B1(mut), and TP53(mut) in 13.4%, 17.5%, and 37.4% of patients, respectively. NOTCH1(mut) and SF3B1(mut) were mutually exclusive, whereas TP53(mut) were evenly distributed within both subgroups. Apart from correlation of SF3B1(mut) with 11q deletion (P = .029), there were no other significant associations of the mutations with any baseline characteristics or response rates. However, NOTCH1(mut) cases had a significantly longer progression-free survival (PFS) compared with wild-type cases (15.47 vs 6.74 months; P = .025), although there was no significant difference with overall survival (OS). SF3B1(mut) had no significant impact on PFS and OS. In multivariable analyses, NOTCH1(mut) was identified as an independent favorable marker for PFS. This clinical trial is registered at www.clinicaltrials.gov as #NCT00274976.

Evasion of apoptosis is a hallmark of chronic lymphocytic leukemia (CLL), calling for new strategies to bypass resistance. Here, we provide first evidence that small-molecule X-linked inhibitor of apoptosis (XIAP) inhibitors in combination with the death receptor ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) present a novel approach to trigger apoptosis in CLL, including subgroups with resistant disease or unfavorable prognosis. XIAP, cellular IAP (cIAP) 1, and cIAP2 are expressed at high levels in primary CLL samples. Proof-of-concept studies in CLL cell lines show that subtoxic concentrations of XIAP inhibitors significantly enhance TRAIL-induced apoptosis and also sensitize for CD95-mediated apoptosis. Importantly also in primary CLL samples, XIAP inhibitor acts in concert with TRAIL to trigger apoptosis in 18 of 27 (67%) cases. This XIAP inhibitor-induced and TRAIL-induced apoptosis involves caspase-3 activation and is blocked by the caspase inhibitor zVAD.fmk. The cooperative interaction of XIAP inhibitor and TRAIL is even evident in distinct subgroups of patients with poor prognostic features (i.e., with 17p deletion, TP53 mutation, chemotherapy-refractory disease, or unmutated V H genes). Interestingly, cases with unmutated V H genes were significantly more sensitive to XIAP inhibitor-induced and TRAIL-induced apoptosis compared with V H gene-mutated samples, pointing to a role of B-cell receptor signaling in apoptosis regulation. By showing that XIAP inhibitors in combination with TRAIL present a new strategy to trigger apoptosis even in resistant forms and poor prognostic subgroups of CLL, our findings have important implications for the development of apoptosis-based therapies in CLL. [Cancer Res 2009;69(23):8977-86]

731 Chronic lymphocytic leukemia (CLL) is characterized by the abnormal accumulation of malignant monoclonal B cells, which has been largely attributed to defective apoptosis rather than aberrant proliferation. This calls for new strategies to re-activate apoptosis programs in CLL in order to develop new therapeutic strategies. “Inhibitor of Apoptosis” (IAP) proteins such as XIAP are aberrantly expressed in many human cancers and block apoptosis at a key node by inhibiting activation of caspases. In the present study, we therefore explored whether targeting XIAP is a suitable strategy to overcome apoptosis resistance of CLL. Here, we provide first evidence that small molecule XIAP inhibitors in combination with the death receptor ligand TRAIL present a novel approach to trigger apoptosis in CLL even in subgroups with resistant disease. Analysis of apoptosis regulatory proteins reveals that XIAP, cIAP1 and cIAP2 are expressed at high levels in primary CLL samples. Proofs of concept studies in CLL cell lines demonstrate that subtoxic concentrations of several distinct XIAP inhibitors significantly enhance TRAIL-induced apoptosis. In addition, XIAP inhibitors sensitize CLL cells for CD95-mediated apoptosis, whereas they have no effect on fludarabine- or chlorambucil-induced apoptosis. This indicates that XIAP inhibitors in particular enhance death receptor-triggered apoptosis in CLL cells. By comparison, no sensitization for death receptor-induced apoptosis is observed in the presence of a structurally related control compound that only weakly binds to XIAP, demonstrating the specificity of the sensitization effect of XIAP inhibitors. Importantly also in primary CLL samples, XIAP inhibitors act in concert with TRAIL to trigger apoptosis in 18 of 27 cases (67%). Analysis of combination index reveals that this interaction of XIAP inhibitor and TRAIL is highly synergistic. Mechanistic studies in primary CLL cells show that the addition of XIAP inhibitor profoundly enhances TRAIL-induced cleavage of caspase-3 into active fragments and significantly increases caspase-3 enzymatic activity upon treatment with TRAIL. The broad range caspase inhibitor zVAD.fmk completely blocks apoptosis in response to combination treatment with XIAP inhibitor and TRAIL, demonstrating that apoptosis occurs in a caspase-dependent manner. Importantly, the cooperative interaction of XIAP inhibitor and TRAIL is even evident in distinct subgroups of patients with poor prognostic features, including patients with 17p deletion, TP53 mutation, chemotherapy-refractory disease or unmutated VH genes. This suggests that the combination treatment with XIAP inhibitor and TRAIL may present a novel approach to trigger apoptosis in CLL patients that are resistant to other treatment options. Interestingly, we found that cases with unmutated VH genes are significantly more sensitive to XIAP inhibitor- and TRAIL-induced apoptosis compared to VH gene mutated samples. This points to a role of B-cell receptor signaling in the regulation of apoptosis in CLL cells. In conclusion, we demonstrate for the first time that XIAP inhibitors in combination with TRAIL present a new strategy to trigger apoptosis even in resistant forms and poor prognostic subgroups of CLL. These findings have important implications for the development of novel strategies to overcome the intrinsic resistance to apoptosis in CLL. Since IAP inhibitors as well as TRAIL receptor agonists as single agents are currently already under evaluation in early clinical trials, it is feasible that such combination protocols of XIAP inhibitors and TRAIL could be translated into clinical application in CLL. Disclosures: No relevant conflicts of interest to declare.

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Inhibitor of apoptosis (IAP) proteins are highly expressed in chronic lymphocytic leukemia (CLL) cells and contribute to evasion of cell death and poor therapeutic response. Here, we report that Smac mimetic BV6 dose-dependently induces cell death in 28 of 51 (54%) investigated CLL samples, while B-cells from healthy donors are largely unaffected. Importantly, BV6 is significantly more effective in prognostic unfavorable cases with, e.g., non-mutated VH status and TP53 mutation than samples with unknown or favorable prognosis. The majority of cases with 17p deletion (10/12) and Fludarabine refractory cases respond to BV6, indicating that BV6 acts independently of p53. BV6 also triggers cell death under survival conditions mimicking the microenvironment, e.g., by adding CD40 ligand or conditioned medium. Gene expression profiling identifies cell death, NF-jB and redox signaling among the top pathways regulated by BV6 not only in CLL but also in core-binding factor (CBF) acute myeloid leukemia (AML). Consistently, BV6 stimulates production of reactive oxygen species (ROS), which are contributing to BV6-induced cell death, since antioxidants reduce cell death. While BV6 causes degradation of cellular inhibitor of apoptosis (cIAP)1 and cIAP2 and nuclear factor-kappaB (NF-jB) pathway activation in primary CLL samples, BV6 induces cell death independently of caspase activity, receptor-interacting protein (RIP)1 activity or tumor necrosis factor (TNF)a, as zVAD.fmk, necrostatin-1 or TNFa-blocking antibody Enbrel fail to inhibit cell death. Together, these novel insights into BV6-regulated cell death in CLL have important implications for developing new therapeutic strategies to overcome cell death resistance especially in poor prognostic CLL subgroups.Chronic lymphocytic leukemia (CLL) is the most common type of adult leukemia in the Western world. 1 Key prognostic factors besides the Rai and Binet stage are chromosomal aberrations like 17p or 11q deletion, TP53 mutation and immunoglobulin heavy-chain variable region gene (IGVH) mutation status. Although individualized therapeutic strategies have been developed, the disease is still incurable. Especially patients with 17p deletion and TP53 mutation have a very poor prognosis. 2 Therefore, new therapeutic targets that act independently of functional p53 in CLL are needed.CLL is characterized by the accumulation of Blymphocytes which has been largely attributed to defective cell death pathways rather than to aberrant proliferation. [3][4][5] Apoptosis represents one of the key forms of programmed cell death. 6 Necroptosis is a caspase-independent mode of programmed cell death that typically occurs when essential factors of apoptosis such as caspases are inhibited and is regulated by RIP1 and RIP3. 7 Inhibitor of apoptosis (IAP) proteins are major regulators of cell death and survival processes. X-linked inhibitor of apoptosis (XIAP) acts as direct caspase inhibitor, 8 while cIAP1 and -2 were shown to protect cells from cell death by acting primarily as E3 ubiquitin liga...