Sustained remissions in CLL after frontline FCR treatment with very-long-term follow-up.

Chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab (FCR) achieves durable remissions, with flattening of the progression-free survival (PFS) curve in patients with mutated immunoglobulin heavy chain variable gene (IGHV-M). We updated long-term follow-up results from the original 300-patient FCR study initiated at MD Anderson in 1999. The current median follow-up is 19.0 years. With this extended follow-up, the median PFS for patients with IGHV-M was 14.6 years vs 4.2 years for patients with unmutated IGHV (IGHV-UM). Disease progression beyond 10 years was uncommon. In total, 16 of 94 (17%) patients in remission at 10 years subsequently progressed with the additional follow-up compared with the patients in our prior report in 2015. Only 4 of 45 patients (9%) with IGHV-M progressed beyond 10 years. Excluding Richter transformation, 96 of 300 patients (32%) developed 106 other malignancies, with 19 of 300 (6.3%) developing therapy-related myeloid neoplasms (tMNs), which were fatal in 16 of 19 (84%). No pretreatment patient characteristics predicted the risk of tMNs. In summary, FCR remains an option for patients with IGHV-M chronic lymphocytic leukemia (CLL), with a significant fraction achieving functional cure of CLL. A risk-benefit assessment is warranted when counseling patients, balancing potential functional cure with the risk of late relapses and serious secondary malignancies.

Growth dynamics in naturally progressing chronic lymphocytic leukaemia.

How the genomic features of a patient's cancer relate to individual disease kinetics remains poorly understood. Here we used the indolent growth dynamics of chronic lymphocytic leukaemia (CLL) to analyse the growth rates and corresponding genomic patterns of leukaemia cells from 107 patients with CLL, spanning decades-long disease courses. We found that CLL commonly demonstrates not only exponential expansion but also logistic growth, which is sigmoidal and reaches a certain steady-state level. Each growth pattern was associated with marked differences in genetic composition, the pace of disease progression and the extent of clonal evolution. In a subset of patients, whose serial samples underwent next-generation sequencing, we found that dynamic changes in the disease course of CLL were shaped by the genetic events that were already present in the early slow-growing stages. Finally, by analysing the growth rates of subclones compared with their parental clones, we quantified the growth advantage conferred by putative CLL drivers in vivo.

Global miRNA profiling reveals key molecules that contribute to different chronic lymphocytic leukemia incidences in Asian and Western populations.

It has been known for decades that the incidence of chronic lymphocytic leukemia (CLL) is significantly lower in Asia than in Western countries, but the reason responsible for this difference still remains a major knowledge gap. Using GeneChip® miRNA array to analyze the global microRNA expression in B lymphocytes from Asian and Western CLL patients and healthy individuals, we have identified microRNA with CLL-promoting or suppressive functions that are differentially expressed in Asian and Western individuals. In particular, miR-4485 is upregulated in CLL patients of both ethnic groups, and its expression is significantly lower in Asian healthy individuals. Genetic silencing of miR-4485 in CLL cells suppresses leukemia cell growth, whereas ectopic expression of miR-4485 promotes cell proliferation. Mechanistically, miR-4485 exerts its CLL-promoting activity by inhibiting the expression of TGR5 and activating the ERK1/2 pathway. In contrast, miR-138, miR-181a, miR- 181c, miR-181d, and miR-363 with tumor-suppressive function are highly expressed in Asian healthy individuals. Our study suggests that differential expression of several important microRNA with pro- or anti-CLL functions in Asian and Western B lymphocytes likely contributes to the difference in CLL incidence between the two ethnic groups, and that miR-4485 and its downstream molecule TGR5 could be potential therapeutic targets.

STAT3 Activates the Pentraxin 3 Gene in Chronic Lymphocytic Leukemia Cells.

Pentraxin-related protein 3 (PTX3), commonly produced by myeloid and endothelial cells, is a humoral pattern recognition protein of the innate immune system. Because PTX3 plasma levels of patients with chronic lymphocytic leukemia (CLL) are high and most circulating cells in patients with CLL are CLL cells, we reasoned that CLL cells produce PTX3. Western immunoblotting revealed that low-density cells from seven of seven patients with CLL produce high levels of PTX3, flow cytometry analysis revealed that the PTX3-producing cells are B lymphocytes coexpressing CD19 and CD5, and confocal microscopy showed that PTX3 is present in the cytoplasm of CLL cells. Because STAT3 is constitutively activated in CLL cells, and because we identified putative STAT3 binding sites within the PTX3 gene promoter, we postulated that phosphorylated STAT3 triggers transcriptional activation of PTX3. Immunoprecipitation analysis of CLL cells' chromatin fragments showed that STAT3 Abs precipitated PTX3 DNA. STAT3 knockdown induced a marked reduction in PTX3 expression, indicating a STAT3-induced transcriptional activation of the PTX3 gene in CLL cells. Using an EMSA, we established and used a dual-reporter luciferase assay to confirm that STAT3 binds the PTX3 gene promoter. Downregulation of PTX3 enhanced apoptosis of CLL cells, suggesting that inhibition of PTX3 might benefit patients with CLL.

A phase 1 study to evaluate the safety, pharmacology, and feasibility of continuous infusion nelarabine in patients with relapsed and/or refractory lymphoid malignancies.

BACKGROUND: Nelarabine is a purine nucleoside analogue prodrug approved for the treatment of relapsed and refractory T-cell acute lymphoblastic leukemia (R/R T-ALL) and lymphoblastic lymphoma (T-LBL). Although effective in R/R T-ALL, significant neurotoxicity is dose-limiting and such neurotoxicity associated with nucleoside analogues can be related to dosing schedule. METHODS: The authors conducted a phase 1 study to evaluate the pharmacokinetics and toxicity of nelarabine administered as a continuous infusion (CI) for 5 days (120 hours), rather than the standard, short-infusion approach. RESULTS: Twenty-nine patients with R/R T-ALL/LBL or T-cell prolymphocytic leukemia (T-PLL) were treated, with escalating doses of nelarabine from 100 to 800 mg/m2 /day × 5 days. The median age of the patients was 39 years (range, 14-77 years). The overall response rate was 31%, including 27% complete remission (CR) or CR with incomplete platelet recovery (CRp). Peripheral neuropathy was observed in 34% of patients, including four ≥grade 3 events related to nelarabine. Notably, there was no nelarabine-related central neurotoxicity on study. The maximum tolerated dose was not reached. Pharmacokinetic data suggested no relationship between dose of nelarabine and accumulation of active intracellular ara-GTP metabolite. Higher intracellular ara-GTP concentrations were statistically associated with a favorable clinical response. CONCLUSION: Preliminary evaluation of continuous infusion schedule of nelarabine suggests that the safety profile is acceptable for this patient population, with clinical activity observed even at low doses and could broaden the use of nelarabine both as single agent and in combinations by potentially mitigating the risk of central nervous system toxicities.

Minimal residual disease undetectable by next-generation sequencing predicts improved outcome in CLL after chemoimmunotherapy.

Patients with chronic lymphocytic leukemia (CLL) who achieve blood or bone marrow (BM) undetectable minimal residual disease (U-MRD) status after first-line fludarabine, cyclophosphamide, and rituximab (FCR) have prolonged progression-free survival (PFS), when assessed by an assay with sensitivity 10-4 (MRD4). Despite reaching U-MRD4, many patients, especially those with unmutated IGHV, subsequently relapse, suggesting residual disease <10-4 threshold and the need for more sensitive MRD evaluation. MRD evaluation by next-generation sequencing (NGS) has a sensitivity of 10-6 (MRD6). To better assess the depth of remission following first-line FCR treatment, we used NGS (Adaptive Biotechnologies Corporation) to assess MRD in 62 patients, all of whom had BM U-MRD by multicolor flow cytometry (sensitivity 10-4) at end-of-FCR treatment. Samples from these patients included 57 BM samples, 29 peripheral blood mononuclear cell (PBMC) samples, and 32 plasma samples. Only 27.4% of the 62 patients had U-MRD by NGS. Rate of U-MRD by NGS was lowest in BM (25%), compared with PBMC (55%) or plasma (75%). No patient with U-MRD by NGS in BM or PBMC was MRD+ in plasma. Patients with mutated IGHV were more likely to have U-MRD by NGS at the end of treatment (EOT; 41% vs 13%, P = .02) than those with unmutated IGHV. Median follow-up was 81.6 months. Patients with U-MRD at EOT had superior PFS vs MRD+ patients, regardless of sample type assessed (BM, P = .02, median not reached [NR] vs 67 months; PBMC, P = .02, median NR vs 74 months). More sensitive MRD6 testing increases prognostic discrimination over MRD4 testing.

Randomized trial of ibrutinib vs ibrutinib plus rituximab in patients with chronic lymphocytic leukemia.

Ibrutinib, an oral covalent inhibitor of Bruton's tyrosine kinase, is an effective therapy for patients with chronic lymphocytic leukemia (CLL). To determine whether rituximab provides added benefit to ibrutinib, we conducted a randomized single-center trial of ibrutinib vs ibrutinib plus rituximab. Patients with CLL requiring therapy were randomized to receive 28-day cycles of once-daily ibrutinib 420 mg, either as a single agent (n = 104), or together with rituximab (375 mg/m2; n = 104), given weekly during cycle 1, then once per cycle until cycle 6. The primary end point was progression-free survival (PFS) in the intention-to-treat population. We enrolled 208 patients with CLL, 181 with relapsed CLL and 27 treatment-naive patients with high-risk disease (17p deletion or TP53 mutation). After a median follow-up of 36 months, the Kaplan-Meier estimates of PFS were 86% (95% confidence interval [CI], 76.6-91.9) for patients receiving ibrutinib, and 86.9% (95% CI, 77.3-92.6) for patients receiving ibrutinib plus rituximab. Similarly, response rates were the same in both arms (overall response rate, 92%). However, time to normalization of peripheral blood lymphocyte counts and time to complete remission were shorter, and residual disease levels in the bone marrow were lower, in patients receiving ibrutinib plus rituximab. We conclude that the addition of rituximab to ibrutinib in relapsed and treatment-naive high-risk patients with CLL failed to show improvement in PFS. However, patients treated with ibrutinib plus rituximab reached their remissions faster and achieved significantly lower residual disease levels. Given these results, ibrutinib as single-agent therapy remains current standard-of-care treatment in CLL. This trial was registered at www.clinicaltrials.gov as #NCT02007044.

STAT3-Induced Wnt5a Provides Chronic Lymphocytic Leukemia Cells with Survival Advantage.

The wingless and integration site growth factor-5a (Wnt5a) is a ligand of the receptor tyrosine kinase-like orphan receptor-1 (ROR1). Because both Wnt5a and ROR1 are expressed in circulating chronic lymphocytic leukemia (CLL) cells, and because in other cell types, STAT3, which is constitutively activated in CLL, induces Wnt5a signaling, we wondered whether STAT3 induces the expression of Wnt5a in CLL cells. Sequence analysis detected four putative STAT3 binding sites in close proximity to the Wnt5a gene promoter's start codon. Chromatin immunoprecipitation and EMSA revealed that STAT3 binds to the Wnt5a gene promoter, and a luciferase assay showed that STAT3 activates the Wnt5a gene. Additionally, transfection of peripheral blood CLL cells with STAT3 short hairpin RNA downregulated Wnt5a mRNA and protein levels, suggesting that STAT3 binds to the Wnt5a gene promoter and induces the expression of Wnt5a in CLL cells. Flow cytometry and confocal microscopy determined that both Wnt5a and its receptor ROR1 are coexpressed on the surface of CLL cells, and Western immunoblotting showed an inverse correlation between Wnt5a and ROR1 protein levels, implying that, regardless of CLL cells' ROR1 levels, blocking the interaction between Wnt5a and ROR1 might be beneficial to patients with CLL. Indeed, transfection of CLL cells with Wnt5a small interfering RNA reduced Wnt5a mRNA and protein levels and significantly increased the spontaneous apoptotic rate of CLL cells. Taken together, our data unravel an autonomous STAT3-driven prosurvival circuit that provides circulating CLL cells with a microenvironment-independent survival advantage.

Inferring clonal heterogeneity in cancer using SNP arrays and whole genome sequencing.

MOTIVATION: Clonal heterogeneity is common in many types of cancer, including chronic lymphocytic leukemia (CLL). Previous research suggests that the presence of multiple distinct cancer clones is associated with clinical outcome. Detection of clonal heterogeneity from high throughput data, such as sequencing or single nucleotide polymorphism (SNP) array data, is important for gaining a better understanding of cancer and may improve prediction of clinical outcome or response to treatment. Here, we present a new method, CloneSeeker, for inferring clinical heterogeneity from sequencing data, SNP array data, or both. RESULTS: We generated simulated SNP array and sequencing data and applied CloneSeeker along with two other methods. We demonstrate that CloneSeeker is more accurate than existing algorithms at determining the number of clones, distribution of cancer cells among clones, and mutation and/or copy numbers belonging to each clone. Next, we applied CloneSeeker to SNP array data from samples of 258 previously untreated CLL patients to gain a better understanding of the characteristics of CLL tumors and to elucidate the relationship between clonal heterogeneity and clinical outcome. We found that a significant majority of CLL patients appear to have multiple clones distinguished by copy number alterations alone. We also found that the presence of multiple clones corresponded with significantly worse survival among CLL patients. These findings may prove useful for improving the accuracy of prognosis and design of treatment strategies. AVAILABILITY AND IMPLEMENTATION: Code available on R-Forge: https://r-forge.r-project.org/projects/CloneSeeker/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A pilot study of lower doses of ibrutinib in patients with chronic lymphocytic leukemia.

Ibrutinib is highly efficacious and used at 420 mg/d for treatment of chronic lymphocytic leukemia (CLL). We previously demonstrated a decline in Bruton's tyrosine kinase (BTK) protein levels in CLL cells after 1 cycle of ibrutinib, suggesting ibrutinib dose could be lowered after the first cycle without loss of biological effect. To test this postulate, a pilot study (NCT02801578) was designed to systematically reduce ibrutinib dosing within the same patient with CLL over the course of three 28-day cycles. After an initial cycle of 420 mg/d, the dose was reduced to 280 mg/d in cycle 2, and then to 140 mg/d in cycle 3. Eleven patients began study treatment, and 9 completed the 3 cycles. Plasma and intracellular pharmacokinetics (PK), BTK occupancy, and pharmacodynamic (PD) response at different doses of ibrutinib were compared. Plasma and intracellular levels of ibrutinib were dose-dependent, and even the lowest dose was sufficient to occupy, on average, more than 95% of BTK protein. In concert, BTK downstream signaling inhibition was maintained with 140 mg/d ibrutinib in cycle 3, and there were comparable reductions in total and phospho-BTK (Tyr223) protein levels across 3 cycles. Reductions of plasma chemokine CCL3 and CCL4 levels, considered to be biomarkers of ibrutinib response, were similar during the 3 cycles. These PK/PD data demonstrate that after 1 cycle of ibrutinib at the standard 420 mg/d dose, the dose can be reduced without losing biological activity. Clinical efficacy of lower doses needs to be systematically evaluated. Such dose reductions would lower drug cost, lessen untoward toxicity, and facilitate rationale-based combinations. This trial was registered at www.clinicaltrials.gov as #NCT02801578.

Correction: Novel Role of NOX in Supporting Aerobic Glycolysis in Cancer Cells with Mitochondrial Dysfunction and as a Potential Target for Cancer Therapy.

[This corrects the article DOI: 10.1371/journal.pbio.1001326.].

Time-to-progression after front-line fludarabine, cyclophosphamide, and rituximab chemoimmunotherapy for chronic lymphocytic leukaemia: a retrospective, multicohort study.

BACKGROUND: Fludarabine, cyclophosphamide, and rituximab (FCR) has become a gold-standard chemoimmunotherapy regimen for patients with chronic lymphocytic leukaemia. However, the question remains of how to treat treatment-naive patients with IGHV-unmutated chronic lymphocytic leukaemia. We therefore aimed to develop and validate a gene expression signature to identify which of these patients are likely to achieve durable remissions with FCR chemoimmunotherapy. METHODS: We did a retrospective cohort study in two cohorts of treatment-naive patients (aged ≥18 years) with chronic lymphocytic leukaemia. The discovery and training cohort consisted of peripheral blood samples collected from patients treated at the University of Texas MD Anderson Cancer Center (Houston, TX, USA), who fulfilled the diagnostic criteria of the International Workshop on Chronic Lymphocytic Leukemia, had received at least three cycles of FCR chemoimmunotherapy, and had been treated between Oct 10, 2000, and Oct 26, 2006 (ie, the MDACC cohort). We did transcriptional profiling on samples obtained from the MDACC cohort to identify genes associated with time to progression. We did univariate Cox proportional hazards analyses and used significant genes to cluster IGHV-unmutated samples into two groups (intermediate prognosis and unfavourable prognosis). After using cross-validation to assess robustness, we applied the Lasso method to standardise the gene expression values to find a minimum gene signature. We validated this signature in an external cohort of treatment-naive patients with IGHV-unmutated chronic lymphocytic leukaemia enrolled on the CLL8 trial of the German Chronic Lymphocytic Leukaemia Study Group who were treated between July 21, 2003, and April 4, 2006 (ie, the CLL8 cohort). FINDINGS: The MDACC cohort consisted of 101 patients and the CLL8 cohort consisted of 109 patients. Using the MDACC cohort, we identified and developed a 17-gene expression signature that distinguished IGHV-unmutated patients who were likely to achieve a long-term remission following front-line FCR chemoimmunotherapy from those who might benefit from alternative front-line regimens (hazard ratio 3·83, 95% CI 1·94-7·59; p<0·0001). We validated this gene signature in the CLL8 cohort; patients with an unfavourable prognosis versus those with an intermediate prognosis had a cause-specific hazard ratio of 1·90 (95% CI 1·18-3·06; p=0·008). Median time to progression was 39 months (IQR 22-69) for those with an unfavourable prognosis compared with 59 months (28-84) for those with an intermediate prognosis. INTERPRETATION: We have developed a robust, reproducible 17-gene signature that identifies a subset of treatment-naive patients with IGHV-unmutated chronic lymphocytic leukaemia who might substantially benefit from treatment with FCR chemoimmunotherapy. We recommend testing the value of this gene signature in a prospective study that compares FCR treatment with newer alternative therapies as part of a randomised clinical trial. FUNDING: Chronic Lymphocytic Leukaemia Global Research Foundation and the National Institutes of Health/National Cancer Institute.

Routine sequencing in CLL has prognostic implications and provides new insight into pathogenesis and targeted treatments.

Chronic lymphocytic leukaemia (CLL) is a genetically heterogeneous disease characterised by genomic alterations and gene mutations that may portend worse survival or resistance to treatments. A total of 680 blood or bone marrow samples underwent targeted sequencing of 29 genes previously identified as being mutated in CLL, which were correlated to known prognostic clinical characteristics. Overall, 400 (59%) patients were treatment-naïve (TN) and 280 (41%) were relapsed/refractory (R/R). Most patients (70%) had ≥1 mutation, with TP53 (22%), SF3B1 (18%), NOTCH1 (13%) and ATM (13%) being the most commonly mutated genes. A higher proportion of R/R patients had mutations in SF3B1 (P = 0·01) and TP53 (P < 0·001). Patients with mutated IGHV CLL more often had mutations in KLHL6 (P = 0·001) and MYD88 (P < 0·001). Pairwise associations showed mutational co-occurrences in the TN group including SF3B1/ATM [false discovery rate (FDR) < 0·05] and NOTCH1/POT1 (FDR < 0·01). Recurrent mutations resulting in premature truncation prior to the ubiquitination domains of NOTCH1 in its PEST domain and BIRC3 in its RING domain can produce proteins that constitutively activate CLL. Frequent missense mutations, such as K700E in SF3B1 and E571K in XPO1, have unknown function but are most likely to be activating mutations. Future directions include using these mutations to identify pathways for therapeutic targeting and rational drug design.

The involvement of microRNA in the pathogenesis of Richter syndrome.

Richter syndrome is the name given to the transformation of the most frequent type of leukemia, chronic lymphocytic leukemia, into an aggressive lymphoma. Patients with Richter syndrome have limited response to therapies and dismal survival. The underlying mechanisms of transformation are insufficiently understood and there is a major lack of knowledge regarding the roles of microRNA that have already proven to be causative for most cases of chronic lymphocytic leukemia. Here, by using four types of genomic platforms and independent sets of patients from three institutions, we identified microRNA involved in the transformation of chronic lymphocytic leukemia to Richter syndrome. The expression signature is composed of miR-21, miR-150, miR-146b and miR-181b, with confirmed targets significantly enriched in pathways involved in cancer, immunity and inflammation. In addition, we demonstrated that genomic alterations may account for microRNA deregulation in a subset of cases of Richter syndrome. Furthermore, network analysis showed that Richter transformation leads to a complete rearrangement, resulting in a highly connected microRNA network. Functionally, ectopic overexpression of miR-21 increased proliferation of malignant B cells in multiple assays, while miR-150 and miR-26a were downregulated in a chronic lymphocytic leukemia xenogeneic mouse transplantation model. Together, our results suggest that Richter transformation is associated with significant expression and genomic loci alterations of microRNA involved in both malignancy and immunity.

Ibrutinib Therapy Increases T Cell Repertoire Diversity in Patients with Chronic Lymphocytic Leukemia.

The Bruton's tyrosine kinase inhibitor ibrutinib is a highly effective, new targeted therapy for chronic lymphocytic leukemia (CLL) that thwarts leukemia cell survival, growth, and tissue homing. The effects of ibrutinib treatment on the T cell compartment, which is clonally expanded and thought to support the growth of malignant B cells in CLL, are not fully characterized. Using next-generation sequencing technology, we characterized the diversity of TCRß-chains in peripheral blood T cells from 15 CLL patients before and after 1 y of ibrutinib therapy. We noted elevated CD4+ and CD8+ T cell numbers and a restricted TCRß repertoire in all pretreatment samples. After 1 y of ibrutinib therapy, elevated peripheral blood T cell numbers and T cell-related cytokine levels had normalized, and T cell repertoire diversity increased significantly. Dominant TCRß clones in pretreatment samples declined or became undetectable, and the number of productive unique clones increased significantly during ibrutinib therapy, with the emergence of large numbers of low-frequency TCRß clones. Importantly, broader TCR repertoire diversity was associated with clinical efficacy and lower rates of infections during ibrutinib therapy. These data demonstrate that ibrutinib therapy increases diversification of the T cell compartment in CLL patients, which contributes to cellular immune reconstitution.

Toward the potential cure of leukemias in the next decade.

Historically, progress in leukemia research has been slow, but it has accelerated recently as a result of understanding the pathophysiology of leukemias and implementing more effective and targeted therapies. This review summarizes the progress across leukemia subsets and projects the potential cure of most leukemias in the next decade.

Results of second salvage therapy in 673 adults with acute myelogenous leukemia treated at a single institution since 2000.

BACKGROUND: The prognosis is poor for patients who have relapsed-refractory acute myelogenous leukemia (AML). Most published reports analyzed results from therapies in first-salvage AML or in studies that were conducted before 2000. Several novel agents and strategies are being tested for potential approval as treatment for patients with relapsed-refractory AML in second salvage. Therefore, it is important to establish the historic results of anti-AML therapies in this setting in the modern era. The objective of the current study was to analyze the results from second salvage therapies in patients with AML since 2000 with regard to response and survival. METHODS: In total, 673 patients who received second salvage therapies for AML since 2000 were analyzed. Their median age was 60 years (range, 18-89 years). Salvage therapy consisted of cytarabine-based regimens in 267 patients, noncytarabine combinations in 37, hypomethylating agent-based regimens in 136, and phase 1 and 2 single agents in 233. RESULTS: Eighty-six of the 673 patients (13%) achieved a complete response (CR) or a CR with low platelet count (CRp). The median duration of CR-CRp was 7.2 months. The median survival was 4.4 months (95% confidence interval, 4.0-4.8 months), and the 1-year survival rate was 16% (95% confidence interval, 14%-19%). Multivariate analysis identified the following as independent adverse factors for achievement of CR-CRp: platelets < 50 × 109 /L (P < .001), complex karyotype with ≥3 chromosomal abnormalities (P = .02), regimens that did not include cytarabine or hypomethylating agents (P = .014), and no prior CR lasting ≥12 months with frontline or salvage 1 therapies (P < .001). The independent adverse factors associated with worse survival were age ≥60 years (P = .01), platelets < 50 × 109 /L (P = .02), peripheral blasts ≥ 20% (P = .03), albumin ≤ 3 g/dL (P = .04), and complex karyotype (P = .003). The authors also applied and validated, in the current population, the 2 multivariate-derived prognostic models for CR and survival developed in their previous study of 594 patients who received treatment for second salvage AML from the previous 2 decades. CONCLUSIONS: This large-scale analysis establishes the modern historic results of second salvage therapy in AML and validates the prognostic models associated with outcome. These data could be used to analyze the differential benefits of current or future investigational strategies under evaluation in this setting and for the purpose of potential approval of new agents in the United States and the world. Cancer 2018;124:2534-40. © 2018 American Cancer Society.

The absolute percent deviation of IGHV mutation rather than a 98% cut-off predicts survival of chronic lymphocytic leukaemia patients treated with fludarabine, cyclophosphamide and rituximab.

The degree of somatic hypermutation, determined as percent deviation of immunoglobulin heavy chain gene variable region sequence from the germline (IGHV%), is an important prognostic factor in chronic lymphocytic leukaemia (CLL). Currently, a cut-off of 2% deviation or 98% sequence identity to germline in IGHV sequence is routinely used to dichotomize CLL patients into mutated and unmutated groups. Because dissimilar IGHV% cut-offs of 1-5% were identified in different studies, we wondered whether no cut-off should be applied and IGHV% treated as a continuous variable. We analysed the significance of IGHV% in 203 CLL patients enrolled on the original frontline fludarabine, cyclophosphamide and rituximab (FCR) trial with a median of 10 years follow-up. Using the Cox Proportional Hazard model, IGHV% was identified as a continuous variable that is significantly associated with progression-free (PFS) and overall survival (OS) (P < 0·001). Furthermore, we validated this finding in 323 patients treated with FCR off-protocol and in the total cohort (n = 535). Multivariate analysis revealed a continuous trend. Higher IGHV% levels were incrementally associated with favorable PFS and OS in both FCR-treated cohorts (P < 0·001, both cohorts). Taken together, our data suggest that IGHV% is a continuous variable in CLL patients treated with FCR.

Fludarabine, cyclophosphamide, and rituximab treatment achieves long-term disease-free survival in IGHV-mutated chronic lymphocytic leukemia.

Accurate identification of patients likely to achieve long-progression-free survival (PFS) after chemoimmunotherapy is essential given the availability of less toxic alternatives, such as ibrutinib. Fludarabine, cyclophosphamide, and rituximab (FCR) achieved a high response rate, but continued relapses were seen in initial reports. We reviewed the original 300 patient phase 2 FCR study to identify long-term disease-free survivors. Minimal residual disease (MRD) was assessed posttreatment by a polymerase chain reaction-based ligase chain reaction assay (sensitivity 0.01%). At the median follow-up of 12.8 years, PFS was 30.9% (median PFS, 6.4 years). The 12.8-year PFS was 53.9% for patients with mutated immunoglobulin heavy chain variable (IGHV) gene (IGHV-M) and 8.7% for patients with unmutated IGHV (IGHV-UM). 50.7% of patients with IGHV-M achieved MRD-negativity posttreatment; of these, PFS was 79.8% at 12.8 years. A plateau was seen on the PFS curve in patients with IGHV-M, with no relapses beyond 10.4 years in 42 patients (total follow-up 105.4 patient-years). On multivariable analysis, IGHV-UM (hazard ratio, 3.37 [2.18-5.21]; P < .001) and del(17p) by conventional karyotyping (hazard ratio, 7.96 [1.02-61.92]; P = .048) were significantly associated with inferior PFS. Fifteen patients with IGHV-M had 4-color MRD flow cytometry (sensitivity 0.01%) performed in peripheral blood, at a median of 12.8 years posttreatment (range, 9.5-14.7). All were MRD-negative. The high rate of very long-term PFS in patients with IGHV-M after FCR argues for the continued use of chemoimmunotherapy in this patient subgroup outside clinical trials; alternative strategies may be preferred in patients with IGHV-UM, to limit long-term toxicity.

Ibrutinib efficacy and tolerability in patients with relapsed chronic lymphocytic leukemia following allogeneic HCT.

Ibrutinib, a potent and irreversible small-molecule inhibitor of both Bruton's tyrosine kinase and interleukin-2 inducible kinase (ITK), has been used to treat relapsed/refractory chronic lymphocytic leukemia (CLL) with prolongation of progression-free and overall survival. Here, we present 27 patients with relapsed CLL following allogeneic hematopoietic cell transplant (HCT) who subsequently received ibrutinib salvage therapy. Sixteen of these patients were part of multi-institutional clinical trials and achieved an overall response rate of 87.5%. An additional 11 patients were treated at Stanford University following US Food and Drug Administration approval of ibrutinib; 7 (64%) achieved a complete response, and 3 (27%) achieved a partial response. Of the 9 patients treated at Stanford who had mixed chimerism-associated CLL relapse, 4 (44%) converted to full donor chimerism following ibrutinib initiation, in association with disease response. Four of 11 (36%) patients evaluated by ClonoSeq achieved minimal residual disease negativity with CLL <1/10 000 white blood cells, which persisted even after ibrutinib was discontinued, in 1 case even after 26 months. None of the 27 patients developed graft-versus-host-disease (GVHD) following ibrutinib initiation. We postulate that ibrutinib augments the graft-versus-leukemia (GVL) benefit through a T-cell-mediated effect, most likely due to ITK inhibition. To investigate the immune modulatory effects of ibrutinib, we completed comprehensive immune phenotype characterization of peripheral B and T cells from treated patients. Our results show that ibrutinib selectively targets pre-germinal B cells and depletes Th2 helper cells. Furthermore, these effects persisted after drug discontinuation. In total, our results provide evidence that ibrutinib effectively augments GVL without causing GVHD.