Impact of the changing landscape of induction therapy prior to autologous stem cell transplantation in 540 newly diagnosed myeloma patients: a retrospective real-world study.

INTRODUCTION: Autologous stem cell transplantation (ASCT) is the standard treatment for younger patients with newly diagnosed multiple myeloma (MM). However, due to restrictive exclusion criteria, more than half of eligible patients are usually excluded from transplant studies. METHODS: This retrospective monocentric analysis included 540 patients with MM who received an ASCT between 1996 and 2019. RESULTS: Up to 2005, induction therapy consisted mainly of conventional chemotherapies, e.g. vincristine/doxorubicin/dexamethasone (VAD). In the following years, the triple-combinations based on bortezomib coupled with doxorubicin/dexamethasone (PAD), melphalan/prednisolone (VMP), cyclophposphamide/dexamethasone (VCD) or bendamustine/prednisolone (BPV) became the most popular treatment options. A progressive improvement in PFS was observed in patients treated with the two current induction therapies BPV (47 months) or VCD (54 months) compared to VAD (35 months, p < 0.03), PAD (39 months, p < 0.01 and VMP (36 months, p < 0.01). However, there was no significant difference in median OS (VAD 78, PAD 74, VMP 72, BPV 80 months and VCD not reached). In our analysis, we also included 139 patients who do fulfill at least one of the exclusion criteria for most phase 3 transplant studies (POEMS/amyloidosis/plasma cell leukemia, eGFR < 40 mL/min, severe cardiac dysfunction or poor general condition). Outcome for these patients was not significantly inferior compared to patients who met the inclusion criteria for most of the transplant studies with PFS of 36 vs 41 months (p = 0.78) and OS of 78 vs 79 months (p = 0.34). CONCLUSIONS: Our real-world data in unselected pts also stress the substantial value of ASCT during the first-line treatment of younger MM pts.

No advantage of Imatinib in combination with hydroxyurea over Imatinib monotherapy: a study of the East German Study Group (OSHO) and the German CML study group.

INTRODUCTION: The combination of Imatinib (IM) and hydroxyurea (HU) was explored for the treatment of chronic myelogenous leukemia (CML). METHOD: After in vitro testing and a phase I study (n = 20), 59 patients were randomized in the IM/HU and 29 in the IM arm. According to protocol, 49 propensity-score matched IM patients were included from the CML-IV study. RESULTS: Additive specific inhibition of CML cells by IM/HU was detected in vitro. HU 500 mg qd in combination with IM 400 mg qd proved feasible in the phase I study. Overall, no significant difference with respect to major molecular response (MMR) at 18 months (IM/HU and IM 66%; primary endpoint) was observed. Significant differences were noted for MMR at 6 months (p = 0.04) and for cumulative incidences of adverse events (p = 0.03) in favor of IM monotherapy (secondary endpoints). CONCLUSION: IM/HU combination was more potent in selectively inhibiting CML cells in vitro, but not superior to IM in vivo. (NCT02480608).

Comparison of Real-Time Quantitative PCR and Digital Droplet PCR for BCR-ABL1 Monitoring in Patients with Chronic Myeloid Leukemia.

Real-time quantitative PCR (qPCR) is routinely used to detect minimal residual disease in chronic myeloid leukemia patients. The absolute quantification with droplet digital PCR (ddPCR) could reduce the inherent variability of qPCR. We established a duplex ddPCR assay using the Europe against Cancer (EAC) primer/probe system for breakpoint cluster region protein-tyrosine-protein kinase ABL1 (BCR-ABL1) and ABL1 and compared the results with qPCR. cDNA samples (n = 230) from patients with chronic myeloid leukemia were analyzed using both procedures. A second, commercially developed ddPCR assay for BCR-ABL1 was also evaluated. ABL1 and BCR-ABL1 transcript levels were similar with all assays, but the proportion of deep molecular responses was lower with ddPCR than with qPCR. The EAC ddPCR assay had a false-positive rate of 4% using a cutoff of three BCR-ABL1 copies per duplicate, compared with 2% without cutoff for the commercial ddPCR. The detection rate for molecular response 4.5 was 100, and a shift toward more minimal residual disease was seen in patient samples. In conclusion, using the EAC protocol for BCR-ABL1 quantification with ddPCR is feasible and shows low intra-assay and interassay variation but requires a cutoff that reduces sensitivity. The commercial ddPCR assay is highly sensitive and specific for BCR-ABL1. The use of either ddPCR assay resulted in a shift to lower molecular response classes compared with qPCR aligned to international scale.

Prognostic Impact of Blood MN1 Copy Numbers Before Allogeneic Stem Cell Transplantation in Patients With Acute Myeloid Leukemia.

High expression of the leukemia-associated gene meningioma-1 (MN1) is frequently found at diagnosis of acute myeloid leukemia (AML) and associates with adverse outcomes. The presence of measurable residual disease (MRD) in complete remission (CR) indicates high risk of relapse and worse outcome in AML patients. However, the prognostic impact of MN1 expression levels as MRD marker has not been evaluated. Digital droplet polymerase chain reaction (ddPCR) is a novel technique allowing sensitive and specific absolute gene expression quantification. We retrospectively analyzed 124 AML patients who received allogeneic hematopoietic stem cell transplantation (HSCT) in CR or CR with incomplete peripheral recovery. Absolute MN1 copy numbers in peripheral blood were assessed prior to HSCT (median 7; range 0-29 days) using ddPCR. High pre-HSCT MN1/Abelson murine leukemia viral oncogene homolog 1 gene (ABL1) copy numbers associated with a higher cumulative incidence of relapse after HSCT and-in relapsing patients-shorter time to relapse. In multivariable analysis, high pre-HSCT MN1/ABL1 copy numbers remained an independent prognosticator for relapse after HSCT. Patients with the highest pre-HSCT MN1/ABL1 copy numbers also had the highest risk of relapse. MN1 copy number assessment also added prognostic information to nucleophosmin 1 gene (NPM1) mutation- and brain and acute leukemia, cytoplasmic (BAALC) and Wilm's tumor gene 1 (WT1) expression-based MRD evaluation. Our study demonstrates the feasibility of the novel ddPCR technique for MN1/ABL1 copy number assessment as a marker for MRD. Evaluation of MN1/ABL1 copy numbers allows the identification of patients at high risk of relapse, independently of other diagnostic risk factors and MRD markers.

Circulating cKIT and PDGFRA DNA indicates disease activity in Gastrointestinal Stromal Tumor (GIST).

This prospective trial aimed to investigate whether tumor-specific cKIT and PDGFRA mutations can be detected and quantified in circulating tumor (ct)DNA in patients with active GIST, and whether detection indicates disease activity. We included 25 patients with active disease and cKIT or PDGFRA mutations detected in tissue. Mutant ctDNA was detected in the peripheral blood plasma using allele-specific ligation (L-)PCR and droplet digital (d)PCR. CtDNA harboring tumor-specific cKIT or PDGFRA mutations was detected at least once in 16 out of 25 patients using L-PCR (64%) and in 20 out of 25 patients with dPCR (80%). Using dPCR, the absolute numbers of ctDNA fragments (DNA copies/ml) and the mutant allele frequency (MAF; in percent of wild-type control) strongly correlated with tumor size expressed as RECIST1.1 sum of diameter (SOD) in mm (ρ = 0.3719 and 0.408, respectively, p < 0.0001) and response status (ρ = 0.3939 and 0.392, respectively, p < 0.0001 and p < 0.001). Specificity of dPCR for detection of progression was 79.2% with a sensitivity of 55.2% and dPCR discriminated CR from active disease with a specificity of 96% and s sensitivity of 44.7%. With L-PCR, correlations of MAF with tumor size and response status were less prominent. Serial ctDNA measurement reflected individual disease courses over time. Targeted panel sequencing of four patients detected additional driver mutations in all cases and secondary resistance mutations in two cases. Thus, ctDNA indicates disease activity in patients with GIST and should be incorporated as companion biomarker in future prospective trials.

Long-term follow up of tandem autologous-allogeneic hematopoietic cell transplantation for multiple myeloma.

We previously reported initial results in 102 multiple myeloma (MM) patients treated with sequential high-dose melphalan and autologous hematopoietic cell transplantation followed by 200 cGy total body irradiation with or without fludarabine 90 mg/m2 and allogeneic hematopoietic cell transplantation. Here we present long-term clinical outcomes among the 102 initial patients and among 142 additional patients, with a median follow up of 8.3 (range 1.0-18.1) years. Donors included human leukocyte antigen identical siblings (n=179) and HLA-matched unrelated donors (n=65). A total of 209 patients (86%) received tandem autologous-allogeneic upfront, while thirty-five patients (14%) had failed a previous autologous hematopoietic cell transplantation before the planned autologous-allogeneic transplantation. Thirty-one patients received maintenance treatment at a median of 86 days (range, 61-150) after allogeneic transplantation. Five-year rates of overall survival (OS) and progression-free survival (PFS) were 54% and 31%, respectively. Ten-year OS and PFS were 41% and 19%, respectively. Overall non-relapse mortality was 2% at 100 days and 14% at five years. Patients with induction-refractory disease and those with high-risk biological features experienced shorter OS and PFS. A total of 152 patients experienced disease relapse and 117 of those received salvage treatment. Eighty-three of the 117 patients achieved a clinical response, and for those, the median duration of survival after relapse was 7.8 years. Moreover, a subset of patients who became negative for minimal residual disease (MRD) by flow cytometry experienced a significantly lower relapse rate as compared with MRD-positive patients (P=0.03). Our study showed that the graft-versus-myeloma effect after non-myeloablative allografting allowed long-term disease control in standard and high-risk patient subsets. Ultra-high-risk patients did not appear to benefit from tandem autologous/allogeneic hematopoietic cell transplantation because of early disease relapse. Incorporation of newer anti-MM agents into the initial induction treatments before tandem hematopoietic cell transplantation and during maintenance might improve outcomes of ultra-high-risk patients. Clinical trials included in this study are registered at: clinicaltrials.gov identifiers: 00075478, 00005799, 01251575, 00078858, 00105001, 00027820, 00089011, 00003196, 00006251, 00793572, 00054353, 00014235, 00003954.

Optimized Digital Droplet PCR for BCR-ABL.

Quantitative real-time PCR methods are commonly used to monitor BCR-ABL transcript levels in patients with chronic myelogenous leukemia. However, standard techniques involve separate measurements of target and reference DNAs, require standard curves, and are susceptible to PCR inhibition. An optimized duplex droplet digital PCR (ddPCR) should provide absolute quantification without the need for standard curves. The combination of high sensitivity and low background is particularly important for reliable monitoring of minimal residual disease. In this report, we describe primer probe set testing and step by step optimization of a duplex ddPCR for BCR-ABL/ABL. The optimization of ddPCR parameters increased ABL and BCR-ABL fluorescence signals by two- and fivefold, respectively, and enhanced the resolution between positive and negative drops. The optimized procedure generates a background false-positive rate of 5% of samples and reliably detects BCR-ABL/ABL down to 1/100,000 (CV <10%), with a single BCR-ABL copy being detected in 54% of reactions performed in duplicate from a molecular remission (MR) 5 sample (limit of detection = 1). Assay of duplicates resulted in a detection rate of 100% and 92% for MR4 and MR4.5, respectively. Detection of MR4.5 was increased to 100% by analyzing quadruplicates. Selection of an optimal primer and probe combination and stepwise optimization of the ddPCR conditions has yielded a robust low-background duplex ddPCR procedure for BCR-ABL/ABL.

Clinical impact of clonal hematopoiesis in acute myeloid leukemia patients receiving allogeneic transplantation.

Age-related somatic mutations linked to clonal hematopoiesis have been found in apparently healthy individuals and increase the risk of developing hematologic malignancies. In acute myeloid leukemia (AML) the clinical relevance of clonal hematopoiesis remains controversial and data on patients with detectable clonal hematopoiesis, consolidated with hematopoietic stem cell transplantation are limited. We analyzed samples from 113 AML patients in complete remission prior to hematopoietic stem cell transplantation for the presence of clonal hematopoiesis-associated mutations. The results were correlated with clinical and biological data. In complete remission we found 75 mutations previously linked to clonal hematopoiesis in 47 patients (41.6%). Twenty patients had ≥2 mutations linked to clonal hematopoiesis. DNMT3A, TET2, and ASXL1 were most frequently mutated. When compared to pre-treatment samples we found variable patterns of mutation persistence depending on the gene mutated. In AML patients after allogeneic hematopoietic stem cell transplantation the presence of clonal hematopoiesis-associated mutations in complete remission did not associate with inferior clinical outcome. This study demonstrates that clonal hematopoiesis is a frequent phenomenon in AML patients. Presence of clonal hematopoiesis has no negative prognostic impact in the context of an allogeneic hematopoietic stem cell transplantation and might be beneficial if certain genes are affected.

Impact of primary tumour location and RAS/BRAF mutational status in metastatic colorectal cancer treated with first-line regimens containing oxaliplatin and bevacizumab: Prognostic factors from the AIO KRK0207 first-line and maintenance therapy trial.

BACKGROUND: The major prognostic relevance of primary tumour location (LPT) in advanced colorectal cancer was shown in large retrospective studies, but quantitative estimates are highly heterogeneous, and there is still limited information about its impact within the framework of biomarker-guided treatment strategies. Therefore, we analysed LPT in relation to other clinical and molecular parameters, based on mature survival data from the recent randomised AIO KRK0207 trial. METHODS: Patients uniformly received first-line induction treatment with a combination of bevacizumab, oxaliplatin and fluoropyrimidine. LPT was retrospectively determined using surgical reports, pathology reports and endoscopy reports. The prognostic analyses were performed using Kaplan-Meier estimations and log-rank tests, while hazard ratios (HRs) and multivariable results were derived from Cox models. RESULTS: Among 754 patients with unequivocal information on LPT, patients with left-sided tumours showed a median overall survival of 24.8 months compared with the right-sided cohort with 18.4 months (HR: 1.54, 95% confidence interval: 1.30-1.81, P < 0.0001). In a multivariable model, LPT proved to be the strongest prognosticator (HR 1.60), with performance status, number of metastatic sites, baseline carcinoembryonic antigen (CEA) and platelets independently retaining prognostic significance. In the subgroup of patients with known RAS/BRAF status (n = 567, 75%), a BRAF mutation showed the greatest unfavourable impact (HR 3.16). Although BRAF is strongly correlated to LPT, the latter remained a significant prognosticator in the BRAF wild-type subgroup. In contrast, no major impact of LPT was seen on tumours carrying RAS mutations. CONCLUSIONS: Within the framework of a uniform treatment strategy according to the current standards, LPT proved to have an important, although not solely dominating, relevance for survival prognosis. Its impact seems to be low in tumours with a RAS mutation. REGISTRATION: ClinicalTrials.govNCT00973609.

Digital droplet PCR-based absolute quantification of pre-transplant NPM1 mutation burden predicts relapse in acute myeloid leukemia patients.

Allogeneic hematopoietic stem cell transplantation is an established consolidation therapy for patients with acute myeloid leukemia. However, relapse after transplantation remains a major clinical problem resulting in poor prognosis. Thus, detection of measurable ("minimal") residual disease to identify patients at high risk of relapse is essential. A feasible method to determine measurable residual disease may be digital droplet PCR (ddPCR) that allows absolute quantification with high sensitivity and specificity without the necessity of standard curves. Using ddPCR, we analyzed pre-transplant peripheral blood and bone marrow of 51 NPM1-mutated acute myeloid leukemia patients transplanted in complete remission or complete remission with incomplete recovery. Mutated NPM1 measurable residual disease-positive patients had higher cumulative incidence of relapse (P < 0.001) and shorter overall survival (P = 0.014). Restricting the analyses to patients receiving non-myeloablative conditioning, mutated NPM1 measurable residual disease positivity is associated with higher cumulative incidence of relapse (P < 0.001) and shorter overall survival (P = 0.006). Positive mutated NPM1 measurable residual disease status determined by ddPCR before allogeneic stem cell transplantation is associated with worse prognosis independent of other known prognostic markers-also for those receiving non-myeloablative conditioning. In the future, mutated NPM1 measurable residual disease status determined by ddPCR might guide treatment and improve patients' outcomes.

Similar expression profiles in CD34+ cells from chronic phase chronic myeloid leukemia patients with and without deep molecular responses to nilotinib.

The life expectancy of patients with chronic phase chronic myeloid leukemia on tyrosine kinase inhibitor therapy now approaches that of the general population. Approximately 60% of patients treated with second generation tyrosine kinase inhibitors achieve a deep molecular response, the prerequisite for a trial of treatment-free remission. Those patients unlikely to achieve deep molecular response may benefit from more intensive therapy up front. To identify biomarkers predicting deep molecular response we performed transcriptional profiling on CD34+ progenitor cells from newly diagnosed chronic phase chronic myeloid leukemia patients treated with nilotinib on a prospective clinical trial. Using unsupervised and targeted analytical strategies, we show that gene expression profiles are similar in patients with and without subsequent deep molecular response. This result is in contrast to the distinct expression signature of CD34+ chronic phase chronic myeloid leukemia patients failing to achieve a cytogenetic response on imatinib and suggests that deep molecular response to second-generation tyrosine kinase inhibitors is governed by the biology of more primitive chronic myeloid leukemia cells or extrinsic factors.

High BAALC copy numbers in peripheral blood prior to allogeneic transplantation predict early relapse in acute myeloid leukemia patients.

High BAALC expression levels at acute myeloid leukemia diagnosis have been linked to adverse outcomes. Recent data indicate that high BAALC expression levels may also be used as marker for residual disease following acute myeloid leukemia treatment. Allogeneic hematopoietic stem cell transplantation (HSCT) offers a curative treatment for acute myeloid leukemia patients. However, disease recurrence remains a major clinical challenge and identification of high-risk patients prior to HSCT is crucial to improve outcomes. We performed absolute quantification of BAALC copy numbers in peripheral blood prior (median 7 days) to HSCT in complete remission (CR) or CR with incomplete peripheral recovery in 82 acute myeloid leukemia patients using digital droplet PCR (ddPCR) technology. An optimal cut-off of 0.14 BAALC/ABL1 copy numbers was determined and applied to define patients with high or low BAALC/ABL1 copy numbers. High pre-HSCT BAALC/ABL1 copy numbers significantly associated with higher cumulative incidence of relapse and shorter overall survival in univariable and multivariable models. Patients with high pre-HSCT BAALC/ABL1 copy numbers were more likely to experience relapse within 100 days after HSCT. Evaluation of pre-HSCT BAALC/ABL1 copy numbers in peripheral blood by ddPCR represents a feasible and rapid way to identify acute myeloid leukemia patients at high risk of early relapse after HSCT. The prognostic impact was also observed independently of other known clinical, genetic, and molecular prognosticators. In the future, prospective studies should evaluate whether acute myeloid leukemia patients with high pre-HSCT BAALC/ABL1 copy numbers benefit from additional treatment before or early intervention after HSCT.

Lenalidomide in combination with bendamustine and prednisolone in relapsed/refractory multiple myeloma: results of a phase 2 clinical trial (OSHO-#077).

INTRODUCTION: While lenalidomide monotherapy is established for relapsed and/or refractory multiple myeloma (MM) treatment, combination therapies including lenalidomide are still under investigation in a number of phase 2/3 studies. In the current study, a treatment regime of lenalidomide (Revlimid®), bendamustine and prednisolone (RBP) was tested in patients with relapsed/refractory MM. METHODS: In the previously completed phase 1 study RBP with a dose of 75 mg/m2 bendamustine days 1-2, prednisolone 100 mg days 1-4 and 25 mg lenalidomide days 1-21 was well tolerated. RESULTS: Between July 2011 and September 2013, 25 patients were included in this analysis. The median number of previous treatments was 1 (range 1-2). Twenty-two patients (88%) responded after at least two cycles of RBP (one sCR, five nCR, eight VGPR and eight PR). The median time to first haematological response was 28 days, and median time to best response was 56 days. Due to increased haematological toxicity a dose reduction in most patients required in subsequent cycles of therapy. The median progression-free and overall survival was 22 and 38 months, respectively. In conclusion RBP is a highly effective therapy for patients with relapsed/refractory MM. In contrast to our phase 1 study, dose reduction was necessary in many patients because of haematological toxicity.

Successful treatment of patients with newly diagnosed/untreated light chain multiple myeloma with a combination of bendamustine, prednisone and bortezomib (BPV).

INTRODUCTION: Patients with light chain myeloma frequently have a light chain tubular cast nephropathy, which can lead to severe renal impairment. In the present retrospective study, bortezomib was combined with other active substances like bendamustine and prednisone (BPV), in order to assess the efficacy and toxicity of the combination therapy in patients with light chain multiple myeloma. METHODS: Between September 2008 and May 2015, 25 patients with newly diagnosed light chain multiple myeloma were treated with bendamustine 60 mg/m2 on days 1 and 2, bortezomib 1.3 mg/m2 on days 1, 4, 8 and 11, and prednisone 100 mg on days 1, 2, 4, 8 and 11 once every 21 days (BPV). Prior to treatment, 4 patients (16%) had moderate renal dysfunction and 14 patients (56%) severe renal dysfunction or renal failure/dialysis. RESULTS: The median number of the BPV cycles was 2 (1-5). 24 patients (96%) responded with 4 stringent complete responses, 6 near-complete responses, 5 very good partial responses and 9 partial responses. The myeloma light chains decreased rapidly, reaching the best response after the first cycle in 9 and after the second cycle in additional 12 patients. 17 patients discontinued therapy after median 2 cycles of BPV treatment to receive autologous or allogeneic SCT. All together 12 of 18 patients with at least moderate renal failure improved their renal function. 3 of the 6 dialysis-dependent patients became dialysis-independent. With a median follow-up of 27 months, median progression-free survival and overall survival for patients at 30 months were 68 and 96%, respectively. The most common severe side effect was grade 3/4 leukocytopenia in 20% of the patients. Grade 3/4 thrombocytopenia was observed in 12% of the patients. Moderate to severe infection were seen in six patients. We conclude that BPV is effective and well tolerated in patients with newly diagnosed/untreated light chain multiple myeloma.

Prognostic impact of the CD34+/CD38- cell burden in patients with acute myeloid leukemia receiving allogeneic stem cell transplantation.

In acute myeloid leukemia (AML), leukemia-initiating cells exist within the CD34+/CD38- cell compartment. They are assumed to be more resistant to chemotherapy, enriched in minimal residual disease cell populations, and responsible for relapse. Here we evaluated clinical and biological associations and the prognostic impact of a high diagnostic CD34+/CD38- cell burden in 169 AML patients receiving an allogeneic stem cell transplantation in complete remission. Here, the therapeutic approach is mainly based on immunological graft-versus-leukemia effects. Percentage of bone marrow CD34+/CD38- cell burden at diagnosis was measured using flow cytometry and was highly variable (median 0.5%, range 0%-89% of all mononuclear cells). A high CD34+/CD38- cell burden at diagnosis associated with worse genetic risk and secondary AML. Patients with a high CD34+/CD38- cell burden had shorter relapse-free and overall survival which may be mediated by residual leukemia-initiating cells in the CD34+/CD38- cell population, escaping the graft-versus-leukemia effect after allogeneic transplantation. Evaluating the CD34+/CD38- cell burden at diagnosis may help to identify patients at high risk of relapse after allogeneic transplantation. Further studies to understand leukemia-initiating cell biology and develop targeting therapies to improve outcomes of AML patients are needed.

Maintenance strategies after first-line oxaliplatin plus fluoropyrimidine plus bevacizumab for patients with metastatic colorectal cancer (AIO 0207): a randomised, non-inferiority, open-label, phase 3 trial.

BACKGROUND: The definition of a best maintenance strategy following combination chemotherapy plus bevacizumab in metastatic colorectal cancer is unclear. We investigated whether no continuation of therapy or bevacizumab alone are non-inferior to fluoropyrimidine plus bevacizumab, following induction treatment with a fluoropyrimidine plus oxaliplatin plus bevacizumab. METHODS: In this open-label, non-inferiority, randomised phase 3 trial, we included patients aged 18 years or older with histologically confirmed, previously untreated metastatic colorectal cancer, Eastern Cooperative Oncology Group (ECOG) performance status of 0-2, adequate bone marrow, liver, and renal function, no pre-existing neuropathy greater than grade 1, and measurable disease, from 55 hospitals and 51 private practices in Germany. After 24 weeks of induction therapy with either fluorouracil plus leucovorin plus oxaliplatin or capecitabine plus oxaliplatin, both with bevacizumab, patients without disease progression were randomly assigned centrally by fax (1:1:1) to standard maintenance treatment with a fluoropyrimidine plus bevacizumab, bevacizumab alone, or no treatment. Both patients and investigators were aware of treatment assignment. Stratification criteria were response status, termination of oxaliplatin, previous adjuvant treatment with oxaliplatin, and ECOG performance status. At first progression, re-induction with all drugs of the induction treatment was a planned part of the protocol. Time to failure of strategy was the primary endpoint, defined as time from randomisation to second progression after maintenance (and if applicable re-induction), death, or initiation of further treatment including a new drug. Time to failure of strategy was equivalent to time to first progression for patients who did not receive re-induction (for any reason). The boundary for assessment of non-inferiority was upper limit of the one-sided 98·8% CI 1·43. Analyses were done by intention to treat. The study has completed recruitment, but follow-up of participants is ongoing. The trial is registered with ClinicalTrials.gov, number NCT00973609. FINDINGS: Between Sept 17, 2009, and Feb 21, 2013, 837 patients were enrolled and 472 randomised; 158 were randomly assigned to receive fluoropyrimidine plus bevacizumab, 156 to receive bevacizumab monotherapy, and 158 to receive no treatment. Median follow-up from randomisation is 17·0 months (IQR 9·5-25·4). Median time to failure of strategy was 6·9 months (95% CI 6·1-8·5) for the fluoropyrimidine plus bevacizumab group, 6·1 months (5·3-7·4) for the bevacizumab alone group, and 6·4 months (4·8-7·6) for the no treatment group. Bevacizumab alone was non-inferior to standard fluoropyrimidine plus bevacizumab (hazard ratio [HR] 1·08 [95% CI 0·85-1·37]; p=0·53; upper limit of the one-sided 99·8% CI 1·42), whereas no treatment was not (HR 1·26 [0·99-1·60]; p=0·056; upper limit of the one-sided 99·8% CI 1·65). The protocol-defined re-induction after first progression was rarely done (30 [19%] patients in the fluoropyrimidine plus bevacizumab group, 67 [43%] in the bevacizumab monotherapy group, and 73 [46%] in the no treatment group. The most common grade 3 adverse event was sensory neuropathy (21 [13%] of 158 patients in the fluoropyrimidine plus bevacizumab group, 22 [14%] of 156 patients in the bevacizumab alone group, and 12 [8%] of 158 patients in the no treatment group). INTERPRETATION: Although non-inferiority for bevacizumab alone was demonstrated for the primary endpoint, maintenance treatment with a fluoropyrimidine plus bevacizumab may be the preferable option for patients following an induction treatment with a fluoropyrimidine, oxaliplatin, and bevacizumab, as it allows the planned discontinuation of the initial combination without compromising time with controlled disease. Only a few patients were exposed to re-induction treatment, thus deeming the primary endpoint time to failure of strategy non-informative and clinically irrelevant. Progression-free survival and overall survival should be considered primary endpoints in future trials exploring maintenance strategies.