Case Report: Large Granular Lymphocyte Leukemia (LGLL)-A Case Series of Challenging Presentations.

Large granular lymphocyte leukemia (LGLL) represents a rare group of diseases with considerable difficulties in their correct diagnostic workup and therapy. The major challenges lie in their distinction from reactive (including autoimmune) lymphoproliferations. Moreover, monoclonal LGL proliferative diseases are in fact a heterogeneous group of disorders, as recognized by the three subtypes in the current WHO classification. It distinguishes two chronic forms (the focus of this case series), namely T-LGLL and chronic lymphoproliferative disorders of Natural Killer cells (CLPD-NK) as well as aggressive NK-cell leukemia. In the clinical routine, the variable presentations and phenotypes of T-LGLL and CLPD-NK are underappreciated. The relevant differential diagnoses range from benign reactive T-cell expansions to other mature T-cell leukemias to highly aggressive γδ-lymphomas. T-LGLL or CLPD-NK patients suffer from a wide variety of symptoms often including, but not limited to, cytopenias or classical autoimmune phenomena. They receive treatments ranging from mere supportive measures (e.g. antibiotics, growth factors, transfusions) over strategies of immunosuppression up to anti-leukemic therapies. The diagnostic pitfalls range from recognition of the subtle T-cell proliferation, repeated establishment of monoclonality, assignment to a descript immunophenotypic pattern, and interpretations of molecular aberrancies. Here, we report a series of selected cases to represent the spectrum of LGLL. The purpose is to raise awareness among the scientifically or practically interested readers of the wide variety of clinical, immunological, and phenotypic features of the various forms of LGLL, e.g. of T-cell type, including its γδ forms or those of NK-lineage. We highlight the characteristics and courses of four unique cases from two academic centers, including those from a prospective nationwide LGLL registry. Each case of this instructive catalogue serves to transport a key message from the areas of (chronic inflammatory) contexts in which LGLL can arise as well as from the fields of differential diagnostics and of various treatment options. Implications for optimization in these areas are discussed.

Bendamustine, followed by ofatumumab and ibrutinib in chronic lymphocytic leukemia (CLL2-BIO): primary endpoint analysis of a multicenter, open-label phase-II trial.

The introduction of targeted agents has revolutionized the treatment of chronic lymphocytic leukemia but only few patients achieve complete remissions and minimal residual disease negativity with ibrutinib monotherapy. This multicenter, investigator-initiated phase-II study evaluates a sequential treatment with two cycles of bendamustine debulking for patients with a higher tumor load, followed by ofatumumab and ibrutinib induction and maintenance treatment. An all-comer population, irrespective of prior treatment, physical fitness and genetic factors was included. The primary endpoint was the investigator assessed overall response rate at the end of induction treatment. Of 66 patients enrolled, one patient with early treatment discontinuation was excluded from the efficacy analysis as predefined by the protocol. Thirty-nine patients (60%) were treatment-naive and 26 patients (40%) had relapsed/refractory CLL, 21 patients (32%) had a del(17p) and/or TP53 mutation and 45 patients (69%) had an unmutated IGHV status. At the end of the induction, 60 of 65 patients (92%) responded and 9 (14%) achieved minimal residual disease negativity (<10-4) in peripheral blood. No unexpected or cumulative toxicities occurred, most common CTC °III/IV adverse events were neutropenias, anaemia, infusion-related reactions, and diarrhoea. This sequential treatment of bendamustine debulking, followed by ofatumumab and ibrutinib was well tolerated without unexpected safety signals and showed a good efficacy with an overall response rate of 92%. Ongoing maintenance treatment aims at deeper responses with minimal residual disease negativity. However, ibrutinib should still be used as a single agent outside clinical trials. Clinicaltrials.gov number: NCT02689141.

New lessons learned in T-PLL: results from a prospective phase-II trial with fludarabine-mitoxantrone-cyclophosphamide-alemtuzumab induction followed by alemtuzumab maintenance.

Clinical trials in T-cell prolymphocytic leukemia (T-PLL) are scarce. Based on a precursor study testing fludarabine, mitoxantrone, and cyclophosphamide followed by alemtuzumab (FMC-A), we aimed to improve this regimen by upfront combining subcutaneous (s.c.) alemtuzumab with FMC for four cycles followed by an alemtuzumab-maintenance (FMCA + A). This prospective multicenter phase-II trial assessed response, survival, and toxicity of that regimen administered to pretreated (n = 4) and treatment-naïve (n = 12) T-PLL patients. The best overall response rate after FMCA was 68.8% (n = 11) including five CRs (31.3%) and six PRs (37.5%). Six patients entered the alemtuzumab-maintenance. Median overall and progression-free survival was 16.7 and 11.2 months, respectively. Hematologic toxicities were the most frequent grade 3/4 side effects. A reduced incidence of CMV-reactivations was attributed to the prophylactic administration of valganciclovir. Overall, FMCA + A did not improve the efficacy of the FMC-A-regimen or of single i.v. alemtuzumab. It suggests that a chemotherapy backbone prevents efficient alemtuzumab dosing and confirms that intravenous alemtuzumab is to be preferred over its s.c. route in T-PLL. ClinicalTrials.gov identifier: NCT01186640.

Automated Manufacturing of Potent CD20-Directed Chimeric Antigen Receptor T Cells for Clinical Use.

The clinical success of gene-engineered T cells expressing a chimeric antigen receptor (CAR), as manifested in several clinical trials for the treatment of B cell malignancies, warrants the development of a simple and robust manufacturing procedure capable of reducing to a minimum the challenges associated with its complexity. Conventional protocols comprise many open handling steps, are labor intensive, and are difficult to upscale for large numbers of patients. Furthermore, extensive training of personnel is required to avoid operator variations. An automated current Good Manufacturing Practice-compliant process has therefore been developed for the generation of gene-engineered T cells. Upon installation of the closed, single-use tubing set on the CliniMACS Prodigy™, sterile welding of the starting cell product, and sterile connection of the required reagents, T cells are magnetically enriched, stimulated, transduced using lentiviral vectors, expanded, and formulated. Starting from healthy donor (HD) or lymphoma or melanoma patient material (PM), the robustness and reproducibility of the manufacturing of anti-CD20 specific CAR T cells were verified. Independent of the starting material, operator, or device, the process consistently yielded a therapeutic dose of highly viable CAR T cells. Interestingly, the formulated product obtained with PM was comparable to that of HD with respect to cell composition, phenotype, and function, even though the starting material differed significantly. Potent antitumor reactivity of the produced anti-CD20 CAR T cells was shown in vitro as well as in vivo. In summary, the automated T cell transduction process meets the requirements for clinical manufacturing that the authors intend to use in two separate clinical trials for the treatment of melanoma and B cell lymphoma.

Bendamustine and its role in the treatment of unfit patients with chronic lymphocytic leukaemia: a perspective review.

With a median age of 72 years at first diagnosis, chronic lymphocytic leukaemia (CLL) is a disease of the elderly. At this age, many patients cannot bear an intensive chemoimmunotherapy like fludarabine, cyclophosphamide and rituximab (FCR), and therapeutic decisions are commonly complicated by a high burden of accompanying comorbidities. Clinical trials, on the other hand, are mostly designed to include a far healthier and younger trial population, with a median age in most studies well below 70 years, leading to an insufficient reflection of clinical reality. With the introduction of new targeted therapies, treatment of CLL is currently undergoing a profound change. New compounds like ibrutinib or idelalisib have enlarged the therapeutic options in treating CLL. However, so far, these oral medications imply continuous intake by the patient, which will at some point lead to the issue of adherence in most patients. In addition, long-term experiences are largely missing. In this setting, one of the oldest chemoactive substances remains a viable option for many CLL patients and their treating physicians: bendamustine, a nitrogen-mustard derivative, has proven to be a safe and efficient agent for treatment of CLL in the first- and second-line setting. In particular, there is some evidence that the substance is relatively well tolerated in elderly and unfit patients. In this review, we summarize the current data on bendamustine in the treatment of elderly and unfit patients with CLL and aim to provide a concise analysis and outlook on the current and future role of this substance in the era of new targeted agents.

Obinutuzumab in chronic lymphocytic leukemia: design, development and place in therapy.

For decades, treatment of chronic lymphocytic leukemia (CLL) has been based on chemotherapy. This changed when the first CD20 antibody rituximab was introduced. Since 2008, the combination of chemotherapy and CD20 antibodies has become the standard of care for most patients, and a significant fraction of patients had very long-lasting remissions after chemoimmunotherapy. Despite the improvement of response rates and overall survival (OS) by the use of chemoimmunotherapy, most CLL patients will relapse eventually. One approach to achieve more durable responses was the development of obinutuzumab (GA101), a new type of CD20 antibody that has unique molecular and functional characteristics. Obinutuzumab is a type II fully humanized CD20 antibody that binds to a partly different epitope of the CD20 protein than rituximab and due to its glycoengineered design induces greater antibody-dependent cell-mediated cytotoxicity (ADCC). Initial preclinical observations of a more effective B-cell depletion have been successfully reproduced in clinical trials with CLL patients. This review summarizes results of preclinical as well as clinical studies with obinutuzumab and provides an outlook on its future role in the therapy of CLL.

Similar outcome after allogeneic stem cell transplantation with a modified FLAMSA conditioning protocol substituting 4 Gy TBI with treosulfan in an elderly population with high-risk AML.

The fludarabine, amsacrine, and cytarabine (FLAMSA)-reduced-intensity conditioning (RIC) protocol has been described to be effective in patients with high-risk and refractory acute myeloic leukemia (AML) undergoing allogeneic hematopoietic stem cell transplantation (aSCT). To increase safety and tolerability of the conditioning, we previously reported the feasibility to substitute the TBI component by treosulfan in elderly AML patients. We now present long-term follow-up data on patients treated with FLAMSA/treosulfan compared to the original FLAMSA/4Gy TBI protocol. We retrospectively analyzed 130 consecutive patients with high-risk or relapsed AML after aSCT following FLAMSA conditioning at our center. Fifty-eight patients were treated with FLAMSA/treosulfan due to age and/or comorbidities. Seventy-two patients were treated with FLAMSA/TBI. Median age of patients treated with FLAMSA/treosulfan was 60 years compared to 46 years in those treated with FLAMSA/TBI. The cumulative incidence of a non-relapse mortality at 4 years was 28% in FLAMSA/treosulfan patients as compared to 13% in FLAMSA/TBI. Cumulative incidence of relapse was higher in patients treated with FLAMSA/TBI (46 vs. 32%). This difference was even more prominent for patients treated in blast persistence prior to transplant (relapse incidence 70% for TBI vs. 35% for treosulfan). The overall and relapse-free survival rates at 4 years were 47 and 41%, respectively, for patients treated with FLAMSA/TBI as compared to 43 and 40% in patients treated with FLAMSA/treosulfan. These data indicate an anti-leukemic activity by FLAMSA/treosulfan especially in patients with a blast persistence prior to transplant. Older age was an independent factor for a higher non-relapse mortality. Translating FLAMSA/treosulfan to younger patients, a lower non-relapse mortality, and an improved anti-leukemic activity might add up to improved overall survival. Randomized studies are required to demonstrate an improved efficacy of treosulfan- versus TBI-based FLAMSA conditioning.

Efficacy of antineoplastic treatment is associated with the use of antibiotics that modulate intestinal microbiota.

Reduced anticancer efficacy of cyclophosphamide and platinum salts has been reported in animals treated with anti-Gram-positive antibiotics. These effects were related to translocation of Gram-positive bacteria during mucositis with subsequent induction of cytotoxic oxygen reactive species and tumor invasion by pathogenic Th17 cells. To assess these hypotheses in a clinical setting, we identified patients receiving cyclophosphamide for chronic lymphocytic leukemia (CLL) and cisplatin for relapsed lymphoma. Data originated from the CLL8 trial (NCT00281918) and the Cologne Cohort of Neutropenic Patients (NCT01821456). Relevant antibiotics were defined as compounds with primary activity against Gram-positive bacteria. We evaluated their impact on response, progression-free survival (PFS) and overall survival (OS) by Kaplan-Meier methodology and Cox proportional hazards regression analysis. Among 800 available CLL patients, those receiving anti-Gram-positive antibiotics (n = 45/800) achieved a significantly lower overall response rate (OR 74.3% vs. 90.2%, p = 0.007). Patients with anti-Gram-positive antibiotics progressed significantly earlier, had a reduced OS (median PFS 14.1 vs. 44.1 mo, p < 0.001; median OS 56.1 vs. 91.7 mo, p < 0.001) and multivariate analysis showed that administration of anti-Gram-positive antibiotic treatment was independently associated with reduced PFS (Hazard ratio (HR) 2.090, p = 0.001) and OS (HR 2.966, p < 0.001). Of 122 patients with relapsed lymphoma, those treated with anti-Gram-positive antibiotics (n = 21/122) achieved a significantly lower OR rate (70.3% vs. 42.9%, p = 0.016). Patients with anti-Gram-positive antibiotics progressed significantly earlier than others (median PFS 2.3 vs. 11.5 mo, p = 0.001). As for multivariate analysis, the use of anti-Gram-positive antibiotics was independently associated with reduced PFS (HR 2.237, p = 0.012) and OS (HR 7.831, p < 0.001). Our data supports a potential negative impact of anti-Gram-positive antibiotics on the anticancer activity of cyclophosphamide and cisplatin in a clinical setting.

Bendamustine and rituximab in combination with lenalidomide in patients with chronic lymphocytic leukemia.

PURPOSE: A phase I/II trial to assess safety and efficacy of the combination bendamustine, rituximab, and lenalidomide (BRL) in patients with chronic lymphocytic leukemia (CLL). PATIENTS AND METHODS: Seventeen relapsed or refractory (R/R) and five previously untreated (FL) CLL patients were enrolled in the trial. In the R/R cohort, four different dose levels of lenalidomide (maximum 15 mg/d) were used. In the FL cohort, lenalidomide was dose escalated from 5 mg/d to 15 mg/d. Bendamustine was used at doses of 50 or 90 mg/m(2) for R/R or FL treatment, respectively. 375 mg/m(2) Rituximab were used for the first and 500 mg/m(2) for subsequent treatment courses. Treatment consisted of up to six courses of 28 d. RESULTS: The maximal tolerable dose of lenalidomide was 5 mg/d. The response rate was 47.1% in R/R and 60% in FL patients. Median progression-free survival was 8.0 months. Median overall survival was 22.9 and 12.3 months, respectively, in R/R and FL patients. Grade 3/4 hematological toxicity was observed in 71.4%, and severe infections in 47.6% of patients. Due to high toxicity and low response rate of BRL, the trial was closed prematurely. CONCLUSION: BRL was associated with a high toxicity rate, a high number of treatment interruptions, and a low remission rate. Therefore, BRL cannot be considered an appropriate treatment option for patients with CLL.

Outcome of advanced chronic lymphocytic leukemia following different first-line and relapse therapies: a meta-analysis of five prospective trials by the German CLL Study Group (GCLLSG).

To evaluate the effect of first-line and subsequent therapies, the outcome of 1,558 patients with chronic lymphocytic leukemia from five prospective phase II/III trials conducted between 1999 and 2010 was analyzed. The 3-year overall survival rate was higher after first-line treatment with chemoimmunotherapies such as fludarabine/cyclophosphamide/rituximab (87.9%) or bendamustine/rituximab (90.7%) compared to chemotherapies without an antibody (fludarabine/cyclophosphamide: 84.6%; fludarabine: 77.5%; chlorambucil: 77.4%). Furthermore, the median overall survival was longer in patients receiving at least one antibody-containing regimen in any treatment line (94.4 months) compared to the survival in patients who never received an antibody (84.3 months, P<0.0001). Univariate Cox regression analysis demonstrated that patients who did receive antibody treatment had a 1.42-fold higher risk of death (hazard ratio, 1.42; 95% confidence interval: 1.185-1.694). Therapies administered at relapse were very heterogeneous. Only 55 of 368 patients (14.9%) who started second-line treatment >24 months after first-line therapy repeated the first-line regimen. Among 315 patients requiring treatment ≤24 months after first-line therapy, cyclophosphamide/doxorubicin/vincristine/prednisone with or without rituximab as well as alemtuzumab were the most commonly used therapies. In these early relapsing patients, the median overall survival was shorter following therapies containing an anthracycline and/or three or more cytotoxic agents (e.g. cyclophosphamide/doxorubicin/vincristine/prednisone or fludarabine/cyclophosphamide/mitoxantrone, 30.0 months) compared to single agent chemotherapy (e.g. fludarabine; 39.6 months) and standard chemoimmunotherapy (e.g. fludarabine/cyclophosphamide/rituximab: 61.6 months). In conclusion, the analysis confirms the superior efficacy of chemoimmunotherapies in patients with chronic lymphocytic leukemia. Moreover, the use of aggressive chemo(immuno)therapy combinations in patients with an early relapse does not offer any benefit when compared to less intensive therapies. Trial identifier: NCT00281918, ISRCTN75653261, ISRCTN36294212, NCT00274989 and NCT00147901.

The CLL12 trial protocol: a placebo-controlled double-blind Phase III study of ibrutinib in the treatment of early-stage chronic lymphocytic leukemia patients with risk of early disease progression.

Observation (watch and wait) is the standard of care for patients with asymptomatic Binet stage A chronic lymphocytic leukemia (CLL). However, the clinical course of these patients is very heterogeneous with some patients requiring treatment rather soon and others not progressing for ages. The clinical staging does not reflect this high variability of the clinical course of CLL. Published data demonstrate that the comprehensive use of several risk factors dramatically improves the accuracy of prognostication independent of clinical stage. The treatment of CLL underwent considerable changes with the introduction of kinase-inhibitors, including ibrutinib, an orally administered, well-tolerated and potent inhibitor of Bruton's tyrosine kinase. This is the first prospective, multicenter, placebo-controlled, double-blind, Phase III study to compare efficacy and safety of ibrutinib to a watch-and-wait approach in Binet stage A CLL with risk of disease progression defined by the comprehensive CLL score.

Development of a comprehensive prognostic index for patients with chronic lymphocytic leukemia.

In addition to clinical staging, a number of biomarkers predicting overall survival (OS) have been identified in chronic lymphocytic leukemia (CLL). The multiplicity of markers, limited information on their independent prognostic value, and a lack of understanding of how to interpret discordant markers are major barriers to use in routine clinical practice. We therefore performed an analysis of 23 prognostic markers based on prospectively collected data from 1948 CLL patients participating in phase 3 trials of the German CLL Study Group to develop a comprehensive prognostic index. A multivariable Cox regression model identified 8 independent predictors of OS: sex, age, ECOG status, del(17p), del(11q), IGHV mutation status, serum ß2-microglobulin, and serum thymidine kinase. Using a weighted grading system, a prognostic index was derived that separated 4 risk categories with 5-year OS ranging from 18.7% to 95.2% and having a C-statistic of 0.75. The index stratified OS within all analyzed subgroups, including all Rai/Binet stages. The validity of the index was externally confirmed in a series of 676 newly diagnosed CLL patients from Mayo Clinic. Using this multistep process including external validation, we developed a comprehensive prognostic index with high discriminatory power and prognostic significance on the individual patient level. The studies were registered as follows: CLL1 trial (NCT00262782, http://clinicaltrials.gov), CLL4 trial (ISRCTN 75653261, http://www.controlled-trials.com), and CLL8 trial (NCT00281918, http://clinicaltrials.gov).

Second-line therapies of patients initially treated with fludarabine and cyclophosphamide or fludarabine, cyclophosphamide and rituximab for chronic lymphocytic leukemia within the CLL8 protocol of the German CLL Study Group.

Updated results of the CLL8 trial confirm that the addition of rituximab to chemotherapy with fludarabine and cyclophosphamide (FC) leads to a prolongation of progression-free (PFS) and overall survival (OS) in first-line treatment of physically fit patients. After a median observation time of 47 months, median PFS was 57.9 months for patients treated with FC and rituximab (FCR) and 32.9 months for patients treated with FC alone (hazard ratio 0.56, 95% confidence interval 0.465-0.673; p < 0.001). A total of 232 patients were treated for relapse, among them 91 of 408 (22%) initially treated with FCR and 141 of 409 (35%) initially treated with FC. The drugs most frequently used either alone or in combination were rituximab (52% of all second-line therapies), fludarabine (21%), bendamustine (21%) and alemtuzumab (12%). The regimens chosen for second-line treatment after FC or FCR were heterogeneous, which underlines a need for further trials in order to define treatment recommendations for patients with relapsed chronic lymphocytic leukemia.

Sequential chemoimmunotherapy of fludarabine, mitoxantrone, and cyclophosphamide induction followed by alemtuzumab consolidation is effective in T-cell prolymphocytic leukemia.

BACKGROUND: Scarce systematic trial data have prevented uniform therapeutic guidelines for T-cell prolymphocytic leukemia (T-PLL). A central need in this historically refractory tumor is the controlled evaluation of multiagent chemotherapy and its combination with the currently most active single agent, alemtuzumab. METHODS: This prospective multicenter phase 2 trial assessed response, survival, and toxicity of a novel regimen in previously treated (n = 9) and treatment-naive (n = 16) patients with T-PLL. Induction by fludarabine, mitoxantrone, and cyclophosphamide (FMC), for up to 4 cycles, was followed by alemtuzumab (A) consolidation, up to 12 weeks. RESULTS: Of the 25 patients treated with FMC, 21 subsequently received alemtuzumab. Overall response rate to FMC was 68%, comprising 6 complete remissions (all bone-marrow confirmed) and 11 partial remissions. Alemtuzumab consolidation increased the intent-to-treat overall response rate to 92% (12 complete remissions; 11 partial remissions). Median overall survival after FMC-A was 17.1 months and median progression-free survival was 11.9 months. Progression-free survival tended to be shorter for patients with high-level T-cell leukemia 1 oncoprotein expression. Hematologic toxicities were the most frequent grade 3/4 side effects under FMC-A. Exclusively in the 21 alemtuzumab-consolidated patients, 13 cytomegalovirus reactivations were observed; 9 of these 13 represented a clinically relevant infection. CONCLUSIONS: FMC-A is a safe and efficient protocol in T-PLL, which compares favorably to published data.

State of the art treatment of chronic lymphocytic leukaemia.

The management of chronic lymphocytic leukaemia is currently undergoing profound changes. Several drugs like bendamustine, alemtuzumab and rituximab have recently been approved for CLL treatment by regulatory agencies. New and very promising compounds like lenalidomide, ofatumumab, GA101, flavopiridol, or ABT-263 are currently investigated in clinical trials and are likely to further enlarge the therapeutic armamentarium in the next years. Latest results show that chemoimmunotherapies like FCR (fludarabine, cyclophosphamide and rituximab) may improve the life expectancy of CLL patients. This new paradigm will modify the way of CLL management in a radical manner. Finally, the development of new biological markers that describe distinct forms of CLL allows to enter the era of personalized therapy similar to other malignancies.

Chronic lymphocytic leukemia.

The management of chronic lymphocytic leukemia (CLL) is currently undergoing a profound change. First, several new drugs have been approved (fludarabine, bendamustine and two monoclonal antibodies, alemtuzumab and rituximab). In addition, novel monoclonal antibodies targeting CD20, CD23, CD37 or CD40, as well as drugs designed to interfere with central pathways regulating the cell cycle, the apoptotic machinery, or the leukemic microenvironment (flavopiridol, oblimersen, ABT-263 or lenalidomide) are being tested in clinical trials. Furthermore, improved protocols using reduced-intensity allogeneic progenitor cell transplantation makes it possible to offer this procedure to more patients with CLL. Finally, new prognostic markers that may influence therapeutic decisions have been identified. This review attempts to summarize the current knowledge in this rapidly moving field.