Dual targeting fixed duration frontline monoclonal antibody therapy for chronic lymphocytic leukemia: A phase 2 study.

This phase 2 study describes long-term clinical and immunological effects of fixed-duration ofatumumab (anti-CD20) and alemtuzumab (anti-CD52) combination immunotherapy in 52 patients with previously untreated CLL. The median age was 65 years (range 45-79), 60% had Rai stage 3-4, 40% were IgHV unmutated and 25% had del(17p)/TP53 mutation. Alemtuzumab was given subcutaneously (30 mg tiw, 18 weeks) and ofatumumab intravenously (300-2000 mg) starting week 3 q2 weeks (8 doses). Overall response rate was 98% with 48% complete remissions including 60% bone marrow MRD-undetectable. After a median follow-up time of 68 months, the median PFS, TTNT, DOR and OS were 31, 62, 30 months and not reached, respectively. The estimated 5-year PFS, TTNT, DOR and OS were 35%, 51%, 35% and 87%, respectively. CD59 (complement-inhibitory molecule) was rapidly downregulated (p < 0.01) during the initial CD52 mAb run-in period. Our study demonstrated that dual targeting of CD20 and CD52 represents an early successful example of time-limited (4-5 months) chemotherapy-free precision therapy for previously untreated CLL.

Temporary cessation of ibrutinib results in reduced grade 3-4 infections and durable remissions-Interim analysis of an on-off-repeat Phase 1b/2 study in patients with chronic lymphocytic leukemia.

Ibrutinib is used continuously in CLL. This phase 1b/2 study interim analysis explored on-off-repeat dosing to reduce toxicity. After 12 months, 16/22 patients (73%) remained in first off-phase irrespective if initial CR/PR or TP53 aberration. Grade 3-4 infections were reduced from 55% to 5% during a similarly long off-phase (P < .01). Treg and exhausted T-cells increased (P = .01). Six patients restarted ibrutinib at early progression and remain drug-sensitive. Our interim analysis shows a durable off-phase in most patients, with reduced infections and cost-saving potential. If toxicity-driven permanent cessation of ibrutinib will be affected will be explored in the extended study.

Risk-adapted bendamustine + rituximab is a tolerable treatment alternative for elderly patients with chronic lymphocytic leukaemia: a regional real-world report on 141 consecutive Swedish patients.

Bendamustine + rituximab (BR) is the current first-line standard-of-care for chronic lymphocytic leukaemia (CLL) in fit patients aged 66-70 years, whereas chlorambucil + CD20 antibody is recommended in older patients with co-morbidities. This retrospective real-world study investigated whether risk-adapted BR was safe and effective in elderly patients. All 141 CLL patients in the Stockholm region (diagnosed from 2007 to 2016, identified from regional registries) who had received BR as first (n = 84) or later line (n = 57) were analysed. Median age was 72 years, 49% had Binet stage C, 40% had Cumulative Illness Rating Scale (CIRS) score ≥ 6, 20% Eastern Cooperative Oncology Group (ECOG) score 2. None had del(17p). Only 15% of patients aged ≥80 years received full-dose bendamustine and 65% of them postponed rituximab until cycle 2. Corresponding numbers in patients 73-79 years were 21% and 36% and in <73 years, 63% and 33%. Overall response rate was 83% (first line) and 67% (later line) (P < 0·022) equally distributed between age subsets. ECOG, immunoglobulin heavy chain variable region (IGHV) mutational status and cytogenetics, but not treatment line and age, were significant factors on progression-free survival (PFS) in multivariate analysis. Infections and neutropenia/thrombocytopenia (≥grade 3) were similar across age subgroups. In summary, BR was well tolerated even in patients ≥80 years, with similar efficacy and safety as in less old patients, provided that carefully adapted dosing was applied.

Calcein release assay as a method for monitoring serum complement activity during monoclonal antibody therapy in patients with B-cell malignancies.

Monoclonal antibodies ofatumumab (anti-CD20) and alemtuzumab (anti-CD52) which are approved for usage in patients with chronic lymphocytic leukemia (CLL), efficiently activate the classical complement pathway. However complement is an exhaustible component and high doses of its activators may deplete complement-dependent cytotoxicity (CDC) potential, thus reducing the effect of repeated mAb dosing. Widely used method to measure CDC activity of patients' serum is hemolytic assay (CH50) on sheep erythrocytes. Despite its simplicity, such CH50 assay may not reflect pivotal interactions between patient serum and human complement inhibitors on the surface of target cells. We propose calcein release assay performed on tumor cells similar to those targeted by therapeutic antibodies as an alternative method. We analyzed serum samples collected from 12 patients participating in the clinical study, receiving s.c. 30 mg alemtuzumab three times per week combined with i.v. ofatumumab at an initial dose of 300 mg in week 3 further escalated to 2000 mg every other week. All serum samples were measured by hemolytic assay on sheep erythrocytes as well as using calcein release assay on CD20-positive Raji cells. Our data show that results obtained from both assays are related to each other at the level of the whole group (n = 96 samples, Spearman r = 0.504, p < .001) but may substantially differ when analyzing individual patients. Furthermore, by using CDC assay on Raji cells, we found that in the presented clinical study CDC serum potential was not significantly affected when measured before consecutive administrations in most of the patients.

Ibrutinib induces rapid down-regulation of inflammatory markers and altered transcription of chronic lymphocytic leukaemia-related genes in blood and lymph nodes.

In chronic lymphocytic leukaemia (CLL) patients, treatment with the Bruton tyrosine kinase inhibitor ibrutinib induces a rapid shift of tumour cells from lymph nodes (LN) to peripheral blood (PB). Here, we characterized in depth the dynamics of ibrutinib-induced inflammatory, transcriptional and cellular changes in different compartments immediately after treatment initiation in seven relapsed/refractory CLL patients. Serial PB and LN samples were taken before start and during the first 29 days of treatment. Changes in plasma inflammation-related biomarkers, CLL cell RNA expression, B-cell activation and migration markers expression, and PB mononuclear cell populations were assessed. A significant reduction of 10 plasma inflammation markers, the majority of which were chemokines and not CLL-derived, was observed within hours, and was paralleled by very early increase of CD19+ circulating cells. At the RNA level, significant and continuous changes in transcription factors and signalling molecules linked to B-cell receptor signalling and CLL biology was observed in both PB and LN CLL cells already after 2 days of treatment. In conclusion, ibrutinib seems to instantly shut off an ongoing inflammatory response and interfere with diverse sensitive pathways in the LN.

Phase I-II study of lenalidomide and alemtuzumab in refractory chronic lymphocytic leukemia (CLL): effects on T cells and immune checkpoints.

This phase I-II study explored safety, immunomodulatory and clinical effects of lenalidomide (weeks 1-16) and alemtuzumab (weeks 5-16) in 23 patients with refractory chronic lymphocytic leukemia. Most patients had Rai stage III/IV disease and were heavily pretreated (median 4 prior therapies), and 61% had del(17p)/del(11q). Eleven of 19 evaluable patients (58%) responded, with a median response duration of 12 months (1-29+); time to progression was short in non-responders. Lenalidomide had a narrow therapeutic dose range, 2.5 mg/day was not efficient, and maximum tolerated dose was 5 mg/day. Grade 3-4 neutropenia and thrombocytopenia occurred in 84 and 55%, 30% had febrile neutropenia, and CMV-reactivation requiring valganciclovir occurred in 30% of patients. The frequency of proliferating (Ki67+) CD8+ T cells was increased at week 4, with further increase in both the CD4+ and CD8+ subsets (p < 0.01 and <0.05), which was accompanied by significant upregulation of HLA-DR after addition of alemtuzumab. Antigen-experienced cells increased at week 4 as the frequency of effector memory cells increased in the CD8+ subset (p < 0.003), while effector cells decreased in both the CD8+ and CD4+ subsets (p < 0.0001 and p < 0.01). The Th1/Th2 balance was unchanged at week 4 but shifted toward a Th2 profile after combination therapy. At end of treatment, the frequency of Th17 and regulatory T cells was reduced (p < 0.01), naïve T cells decreased, and effector memory T cells increased (p < 0.05 and p < 0.01). Granzyme B+ T cells increased at 30-week follow-up (p < 0.05). PD-1 expression was unaffected. In conclusion, low-dose lenalidomide and alemtuzumab induced major perturbations of T cells, including increased proliferative activity and cytotoxic potential.

Real-world results of ibrutinib in patients with relapsed or refractory chronic lymphocytic leukemia: data from 95 consecutive patients treated in a compassionate use program. A study from the Swedish Chronic Lymphocytic Leukemia Group.

Ibrutinib, a Bruton's tyrosine kinase inhibitor is approved for relapsed/refractory and del(17p)/TP53 mutated chronic lymphocytic leukemia. Discrepancies between clinical trials and routine health-care are commonly observed in oncology. Herein we report real-world results for 95 poor prognosis Swedish patients treated with ibrutinib in a compassionate use program. Ninety-five consecutive patients (93 chronic lymphocytic leukemia, 2 small lymphocytic leukemia) were included in the study between May 2014 and May 2015. The median age was 69 years. 63% had del(17p)/TP53 mutation, 65% had Rai stage III/IV, 28% had lymphadenopathy ≥10cm. Patients received ibrutinib 420 mg once daily until progression. At a median follow-up of 10.2 months, the overall response rate was 84% (consistent among subgroups) and 77% remained progression-free. Progression-free survival and overall survival were significantly shorter in patients with del(17p)/TP53 mutation (P=0.017 and P=0.027, log-rank test); no other factor was significant in Cox proportional regression hazards model. Ibrutinib was well tolerated. Hematomas occurred in 46% of patients without any major bleeding. Seven patients had Richter's transformation. This real-world analysis on consecutive chronic lymphocytic leukemia patients from a well-defined geographical region shows the efficacy and safety of ibrutinib to be similar to that of pivotal trials. Yet, del(17p)/TP53 mutation remains a therapeutic challenge. Since not more than half of our patients would have qualified for the pivotal ibrutinib trial (RESONATE), our study emphasizes that real-world results should be carefully considered in future with regards to new agents and new indications in chronic lymphocytic leukemia.

Outcomes of patients with fludarabine-refractory chronic lymphocytic leukemia: a population-based study from a well-defined geographic region.

Patients with fludarabine-refractory (FR) chronic lymphocytic leukemia (CLL) receive novel agents in pivotal, non-randomised phase-2 trials. Understanding outcome of FR-CLL in health-care may provide important contextual information. Records from 1301 patients (Stockholm-Cancer-Registry 1991-2010) identified 92 FR-patients; bulky lymph-nodes (BFR-group), double-refractory (DR-group), or Others'-group for outcome-analysis. Median age was 69 years 67% had Rai-stage III/IV with median 3 prior therapies. Overall response-rate was 20%; significantly lower in BFR (8%, p = 0.01) and DR (20%, p = 0.01) than in 'Others' (31%). Time-to-treatment-failure (months) was significantly longer in 'Others' (9.2) than in BFR/DR (5.3/4.4) (p < 0.01) and significantly longer (p < 0.05) in antibody-treated patients (9.1) compared to other regimens (5.2). Early-death occurred in 5%, ≥ grade III-infections in 20%. Median overall-survival (OS) was 18 months; 29 in BFR vs. 13 in DR (p = 0.054). Male sex was the only prognostic factor on OS (p = 0.01, HR 2.2, multivariate-Cox-regression). Our results, without external referrals, facilitate interpretation of non-randomised trials/novel drugs in advanced-stage-CLL.

The tyrosine kinase receptor ROR1 is constitutively phosphorylated in chronic lymphocytic leukemia (CLL) cells.

Phosphorylation of receptor tyrosine kinases (RTKs) has a key role in cellular functions contributing to the malignant phenotype of tumor cells. We and others have previously demonstrated that RTK ROR1 is overexpressed in chronic lymphocytic leukemia (CLL). Silencing siRNA downregulated ROR1 and induced apoptosis of CLL cells. In the present study we analysed ROR1 isoforms and the phosphorylation pattern in CLL cells (n=38) applying western blot and flow-cytometry using anti-ROR1 antibodies and an anti-phospho-ROR1 antibody against the TK domain. Two major ROR1 bands with the size of 105 and 130 kDa respectively were identified, presumably representing unglycosylated (immature) and glycosylated (mature) ROR1 respectively as well as a 260 kDa band which may represent dimerized ROR1. A ROR1 band of 64 kDa that may correspond to a C-terminal fragment was also noted, present only in the nucleus. The 105 kDa ROR1 isoform was more frequently expressed in non-progressive as compared to progressive CLL patients (p=0.03). The 64, 105, 130 and 260 kDa bands were constitutively phosphorylated both at tyrosine and serine residues. Phosphorylation intensity of the mature (130 kDa) isoform was significantly higher in progressive than in non-progressive disease (p<0.001). Incubation of CLL cells with a mouse anti-ROR1 KNG or an anti-ROR1 CRD mAb respectively induced dephosphorylation of ROR1 before entering apoptosis. In conclusion CLL cells expressed different isoforms of ROR1 which were constitutively phosphorylated. The mature, phosphorylated ROR1 isoform was associated with a progressive disease stage. Targeting ROR1 by mAbs induced specific dephosphorylation and leukemic cell death. ROR1 might be an interesting therapeutic target.

Array-based genomic screening at diagnosis and during follow-up in chronic lymphocytic leukemia.

BACKGROUND: High-resolution genomic microarrays enable simultaneous detection of copy-number aberrations such as the known recurrent aberrations in chronic lymphocytic leukemia [del(11q), del(13q), del(17p) and trisomy 12], and copy-number neutral loss of heterozygosity. Moreover, comparison of genomic profiles from sequential patients' samples allows detection of clonal evolution. DESIGN AND METHODS: We screened samples from 369 patients with newly diagnosed chronic lymphocytic leukemia from a population-based cohort using 250K single nucleotide polymorphism-arrays. Clonal evolution was evaluated in 59 follow-up samples obtained after 5-9 years. RESULTS: At diagnosis, copy-number aberrations were identified in 90% of patients; 70% carried known recurrent alterations, including del(13q) (55%), trisomy 12 (10.5%), del(11q) (10%), and del(17p) (4%). Additional recurrent aberrations were detected on chromosomes 2 (1.9%), 4 (1.4%), 8 (1.6%) and 14 (1.6%). Thirteen patients (3.5%) displayed recurrent copy-number neutral loss of heterozygosity on 13q, of whom 11 had concurrent homozygous del(13q). Genomic complexity and large 13q deletions correlated with inferior outcome, while the former was linked to poor-prognostic aberrations. In the follow-up study, clonal evolution developed in 8/24 (33%) patients with unmutated IGHV, and in 4/25 (16%) IGHV-mutated and treated patients. In contrast, untreated patients with mutated IGHV (n=10) did not acquire additional aberrations. The most common secondary event, del(13q), was detected in 6/12 (50%) of all patients with acquired alterations. Interestingly, aberrations on, for example, chromosome 6q, 8p, 9p and 10q developed exclusively in patients with unmutated IGHV. CONCLUSIONS: Whole-genome screening revealed a high frequency of genomic aberrations in newly diagnosed chronic lymphocytic leukemia. Clonal evolution was associated with other markers of aggressive disease and commonly included the known recurrent aberrations.

Tumor burden status evaluated by computed tomography scan is of prognostic importance in patients with chronic lymphocytic leukemia.

It has been discussed if computed tomography (CT) scan is necessary to perform or not, to provide prognostic information in patients with chronic lymphocytic leukemia. In the current National Institute Chronic lymphocytic leukemia Working Group (NCI-WG) guidelines, CT scan is not recommended outside clinical trials but states that further trials are warranted. Computed tomography scan of lymphadenopathy and/or splenomegaly was detected in totally 74 patients, leading to a modified Rai stage in 9 (12%) patients (from stage I to II). Using CT, 19 patients fulfilled the GELF high tumor burden criteria and/or one bulky node > or =7 cm. CT showed splenomegaly in totally 54 (73%) patients, of which 22 (41%) cases had not had been detected during clinical examination. According to the NCI response criteria for chronic lymphocytic leukemia (CLL), 15 (22%) patients showed a CR and 43 (62%) a PR, while when including CT findings, 12 (17%) fulfilled criteria for CR and 41 (59%) for PR. Patients with a high lymphadenopathy tumor burden according to the GELF criteria at the time of initiation of first-line therapy had a shorter time from response to next therapy or death, 12 months compared with 35 months for patients with less advanced lymphadenopathy (P = 0.002). There was also a trend for a shorter overall survival in the high tumor burden group, 58 months compared to 75 months (P = 0.098). Massive splenomegaly was related to a shorter therapy-free as well as overall survival. In our opinion, clinical examination is not sufficient for evaluation of abdominal lymphnodes and spleen in patients with therapy requiring CLL.

Lack of association between the MDM2 promoter polymorphism SNP309 and clinical outcome in chronic lymphocytic leukemia.

The 309T>G polymorphism in the promoter region of the MDM2 gene, known as SNP309, has recently been suggested as an unfavorable prognostic marker in chronic lymphocytic leukemia (CLL) although this has been questioned. To investigate this further, we analyzed the MDM2 SNP309 genotypes in 418 CLL patients and correlated the results with established CLL prognostic factors, time to treatment and overall survival. In this Swedish cohort, no association existed between any particular MDM2 SNP309 genotype, overall survival and time to treatment. Furthermore, no correlation was shown between the MDM2 SNP309 genotypes and Binet stage, IGHV mutational status and recurrent genomic aberrations. In summary, this study argues against the use of the MDM2 SNP309 as a prognostic marker in CLL.

Phase II study of subcutaneous alemtuzumab without dose escalation in patients with advanced-stage, relapsed chronic lymphocytic leukaemia.

This phase II study (n = 20) aimed to evaluate type, severity and duration of side-effects and efficacy following subcutaneous (SC) alemtuzumab, without dose-escalation, in advanced-stage relapsed chronic lymphocytic leukaemia (CLL) patients. Alemtuzumab 30 and 3 mg was administered SC simultaneously day 1, followed by 30 mg three times per week. Injection-site-reactions were recorded every 6-24 h until resolved using National Cancer Institute criteria and a new skin toxicity subscale. The first doses of 30 mg and 3 mg produced injection-site-reactions (all but one were grade 1/2) in 13/20 and 9/20 patients, respectively. The second dose (on day 3) resulted in skin-reactions in 10/20 patients and the third, fourth, fifth and sixth injections produced reactions in 6/20, 1/20, 2/20 and 0/20 patients, respectively. Mild "flu-like" symptoms occurred during week 1 in 10/20 patients. All side-effects had subsided by the sixth dose. 15/20 patients (75%) responded (12 partial responses, three complete responses) with a median time-to-treatment-failure of 20 months. Symptomatic cytomegalovirus-reactivation occurred in 6/20 patients. Two deaths occurred: one bacterial pneumonia and one adenovirus-infection. The present study showed how to assess cutaneous-toxicity in detail and that 30 mg alemtuzumab SC administered upfront was well tolerated. Optimized alemtuzumab therapy in properly selected patients may result in high efficacy even in advanced CLL. Our results need to be confirmed in extended studies.

Alemtuzumab to treat refractory autoimmune hemolytic anemia or thrombocytopenia in chronic lymphocytic leukemia.

Autoimmune hemolytic anemia (AIHA) and immune thrombocytopenic purpura (ITP) are recognized complications of chronic lymphocytic leukemia (CLL) that can be life-threatening if not managed appropriately. Conventional therapies for these autoimmune disorders, such as corticosteroids, splenectomy, and immunosuppressive agents, may not induce complete resolution in all patients, and relapses are common. In recent years, monoclonal antibodies such as alemtuzumab and rituximab, already used successfully for the management of lymphoproliferative disorders, have been shown to be effective in the treatment of a range of autoimmune disorders. The potent antitumor activity of alemtuzumab, in combination with its profound immunosuppressive activity, prompted investigation of its use in patients with severe CLL-related AIHA and ITP. Results from a range of reports confirm the efficacy of alemtuzumab for the treatment of severe, CLL-related autoimmune cytopenias that have failed to respond to conventional therapies and may even be rituximab-refractory.

Performance of calibration standards for antigen quantitation with flow cytometry in chronic lymphocytic leukemia.

BACKGROUND: The fluorescence intensities of CD3, CD4 on T cells and CD20, CD22 molecules on B cells were quantitatively measured on lymphocytes from chronic lymphocytic leukemia (CLL) patients and healthy donors. METHODS: The performance of three different types of microbeads was compared, i.e. Quantum molecules of equivalent soluble fluorochrome (Q-MESF), Quantum simply cellular (QSC), and QuantiBRITE (QB). As all PE-conjugates had a F/P ratio of 1:1, the MESF units represented also the antibody binding capacity (ABC). RESULTS: The ABCs of CD4 and CD20 antigens estimated with QSC (ABC(QSC)) were higher than those assigned with QB (ABC(QB)) with an average difference 49%. Higher numbers of antigenic sites were obtained with Q-MESF than with QSC for CD20 antigen. On the contrary, CD4 antigenic sites numbers estimated with QSC were higher than those estimated with Q-MESF. ABC values estimated with Quantum MESF PE (ABC(Q-MESF)) were approximately 15% higher than ABC(QSC), whereas ABC(Q-MESF) was approximately 49% higher than ABC(QB). Statistically significant correlations were found between the values obtained using various standards. The present study is the first to report down-regulation of CD3 antigen on T cells from patients with CLL. CONCLUSIONS: This study emphasizes the relevance of quantitative measurement of fluorescence intensity by flow cytometry as a standardized approach to measure and interpret the expression of some CLL markers and reduce variability of results obtained at different sites in multi-center clinical studies.

Reconstitution of the T-cell repertoire following treatment with alemtuzumab (anti-CD52 monoclonal antibody) in patients with B-cell chronic lymphocytic leukaemia.

In this pilot study, T-cell receptor B-variable (TCR-BV) gene usage in CD4 and CD8 T cells was assessed, by real-time polymerase chain reaction, as well as complementarity-determining region 3 (CDR3)-length polymorphism, before and after therapy in five patients with B-cell chronic lymphocytic leukaemia who received alemtuzumab (anti-CD52 monoclonal antibody) as first-line therapy. A decline in expression of most BV family genes in both CD4 and CD8 T cells was observed after alemtuzumab treatment, which was followed by a gradual increase in most BV families during long-term follow-up. After treatment, CDR3-length polymorphism showed an even more restricted pattern in CD4 T cells compared with pretreatment, with a shift towards a monoclonal/oligoclonal pattern. The clonally restricted pattern was significantly reduced in CD4 (P < 0.01) but not in CD8 T cells. This was followed by a gradual increase in the number of peaks within the CDR3 region of the different TCR-BV families, i.e. a polyclonal repertoire, during long-term follow-up. A restricted CDR3 pattern became even more restricted after treatment, but normalised during unmaintained follow-up. These results indicate that perturbations in the T-cell alterations following alemtuzumab are complex and include not only changes in CD4/CD8 T-cell numbers but also a highly restricted T-cell repertoire especially in CD4 T cells.