Comparative analysis between RQ‐PCR and digital droplet PCR of BCL2/IGH gene rearrangement in the peripheral blood and bone marrow of early stage follicular lymphoma

BCL2/IGH rearrangements were analysed by polymerase chain reaction (PCR) at diagnosis in paired peripheral blood (PB) and bone marrow (BM) samples from 67 patients with stage I/II follicular lymphoma (FL). Real time quantitative PCR (RQ‐PCR) and digital droplet PCR (ddPCR) were performed in cases with a major breakpoint region (MBR+) at diagnosis and after localized radiotherapy and rituximab administration in order to investigate the applicability of ddPCR. The overall ddPCR/RQ‐PCR concordance was 81·9% (113/138 samples) and 97·5% in the 40/138 with quantifiable disease (RQ‐PCR≥10⁻⁵). At baseline, ddPCR allowed the recovery of a MBR+ marker in 8/18 (44·4%) samples that resulted MBR‐negative/minor cluster region‐negative/minor BCL2‐negative by qualitative PCR. Moreover, the tumour burden at diagnosis significantly predicted progression‐free survival (PSF) only when quantified by ddPCR. Paired PB and BM samples analysis demonstrated a high concordance in the detection of BCL2/IGH+ cells by qualitative and quantitative methods; in particular, 40/62 samples were positive by ddPCR (25 PB+/BM+; 9 PB+/BM−; 6 PB−/BM+), with 34/40 (85%) identified by the study of PB only. In conclusion, in localized FL, ddPCR is a promising tool for monitoring minimal residual disease (MRD) that is at least comparable to RQ‐PCR and potentially more accurate. PB is a suitable source for serial BCL2/IGH MRD assessments, regardless of the methodology utilized.     

Applicability of IG/TCR gene rearrangements as targets for minimal residual disease assessment in a population‐based cohort of Swedish childhood acute lymphoblastic leukaemia diagnosed 2002–2006

Minimal residual disease (MRD) detection during the early treatment phase has become an important stratification parameter in many childhood acute lymphoblastic leukaemia (ALL) treatment protocols. Here, we aimed to address the applicability of rearranged antigen‐receptor genes as potential MRD markers using real‐time quantitative polymerase chain reaction (RQ‐PCR) in a Swedish population‐based cohort. From 334 childhood ALL cases diagnosed during 2002–2006, we analysed 279 diagnostic samples (84%) by screening for rearranged immunoglobulin (IG) and T‐cell receptor (TCR) genes. Allele‐specific oligonucleotides were designed, and the sensitivity and quantitative level was determined for each target. Overall, clonal IG/TCR rearrangements were detected in 97% (236/244) of B‐cell precursor ALL (BCP ALL) and 94% (33/35) of T‐ALL. A sensitive RQ‐PCR analysis (≤10^?4) was obtained in 89% (216/244) of BCP ALL and in 74% (26/35) of T‐ALL, whereas two sensitive targets were only available in 47% (115/244) of BCP ALL and 29% (10/35) of T‐ALL cases. With the stratification threshold of ≥10^?3, which is applied in the current Nordic treatment protocol (NOPHO‐ALL 2008) for the identification of high‐risk patients, 93% of BCP ALL and 86% of T‐ALL reached this quantitative range by at least one target gene. Taken together, this national retrospective study demonstrates that an IG/TCR target for MRD monitoring can be identified in the majority of childhood ALL cases, whereas identification of a second sensitive target gene needs to be improved.     

Whole‐genome amplification for the detection of molecular targets and minimal residual disease monitoring in acute lymphoblastic leukaemia

Accurate genomic characterization requires sufficient amounts of optimal quality DNA. An approach for increasing the DNA amount is the whole‐genome amplification (WGA) method. We applied WGA to the molecular quantification and minimal residual disease (MRD) evaluation of acute lymphoblastic leukaemia (ALL), aiming to compare the results obtained from genomic DNA and amplified DNA with WGA, and to evaluate the applicability and the reliability of WGA‐DNA. Twenty paired samples from adult ALL patients were sequenced to identify the functional germline V‐D‐J segment at diagnosis; real‐time quantitative polymerase chain reaction (RQ‐PCR) quantitative analysis was performed both at diagnosis and follow‐up. Genomic DNA and WGA‐DNA screening identified equivalent 87 rearrangements. At diagnosis, the quantitative evaluation of genomic DNA samples showed 1 logarithm difference to WGA‐DNA samples; these levels are comparable, being within the degree of acceptability and confidence. In the follow‐up samples, RQ‐PCR analysis on genomic DNA and WGA showed concordant MRD results in 16/18 samples, while 2/18 were MRD‐positive outside the quantitative range by RQ‐PCR (i.e. <5 × 10^?5) on genomic DNA and MRD‐negative on WGA‐DNA. WGA‐DNA enables: (i) the design of accurate targets for MRD evaluation in ALL patients, (ii) accurate disease quantification at diagnosis, (iii) MRD quantification comparable to genomic DNA.     

High frequency of IKZF1 genetic alterations in adult patients with B‐cell acute lymphoblastic leukemia

Alterations in the IKZF1 gene are associated with poor prognosis in pediatric B‐cell acute lymphoblastic leukemia (B‐ALL). We examined the relationship between IKZF1 alterations and clinical findings in 78 adult patients with B‐ALL. Aberrant isoforms of IKZF1 were detected using RT‐PCR. The copy numbers of IKZF1 exons and fusion genes caused by exon deletions were determined using RQ‐PCR and genomic PCR, respectively. We detected aberrant IKZF1 isoforms in 20 of the 78 patients (13 Ik6 and seven Ik10) and deletions of the entire or parts of the IKZF1 gene in 40 of 70 patients. No IKZF1 point mutations were detected by direct sequencing. Nineteen Ik6 and Ik10 isoforms had been generated through genomic exon deletions, but one through aberrant splicing. In total, 41 of the 78 (52.6%) patients harbored IKZF1 alterations, which were identified in 20 of 24 (83.3%) patients with Philadelphia chromosome (Ph)–positive B‐ALL compared with 21 of 54 (38.9%) Ph‐negative B‐ALL (P = 0.0004). IKZF1 alterations are highly involved even in adults with B‐ALL. To fully detect IKZF1 alterations, several methods with alternative approaches are required. To elucidate the clinical significance of IKZF1 alterations in adult B‐ALL, our study warrants prospective clinical studies with a full analysis of IKZF1 alterations.     

Persistent MRD before and after allogeneic BMT predicts relapse in children with acute lymphoblastic leukaemia

Minimal residual disease (MRD) during early chemotherapy is a powerful predictor of relapse in acute lymphoblastic leukaemia (ALL) and is used in children to determine eligibility for allogeneic haematopoietic stem cell transplantation (HSCT) in first (CR1) or later complete remission (CR2/CR3). Variables affecting HSCT outcome were analysed in 81 children from the ANZCHOG ALL8 trial. The major cause of treatment failure was relapse, with a cumulative incidence of relapse at 5 years (CIR) of 32% and treatment‐related mortality of 8%. Leukaemia‐free survival (LFS) and overall survival (OS) were similar for HSCT in CR1 (LFS 62%, OS 83%, n = 41) or CR2/CR3 (LFS 60%, OS 72%, n = 40). Patients achieving bone marrow MRD negativity pre‐HSCT had better outcomes (LFS 83%, OS 92%) than those with persistent MRD pre‐HSCT (LFS 41%, OS 64%, P < 0·0001) or post‐HSCT (LFS 35%, OS 55%, P < 0·0001). Patients with B‐other ALL had more relapses (CIR 50%, LFS 41%) than T‐ALL and the main precursor‐B subtypes including BCR‐ABL1, KMT2A (MLL), ETV6‐RUNX1 (TEL‐AML1) and hyperdiploidy >50. A Cox multivariate regression model for LFS retained both B‐other ALL subtype (hazard ratio 4·1, P = 0·0062) and MRD persistence post‐HSCT (hazard ratio 3·9, P = 0·0070) as independent adverse prognostic variables. Persistent MRD could be used to direct post‐HSCT therapy.     

The kinetics of relapse in DEK‐NUP214‐positive acute myeloid leukemia patients

Preemptive treatment of relapse of acute myeloid leukemia (AML) holds the promise to improve the prognosis of this currently highly lethal condition. Proposed treatment modalities applicable in preemptive cytoreduction (e.g., demethylating agents or standard chemotherapy) differ substantially in interval from administration to antileukemic effect. The t(6;9) balanced translocation, producing the DEK‐NUP214 fusion protein, is seen in only 1% of patients with AML. We hypothesized that in these patients, who relapse with a very high frequency, a more detailed knowledge of leukemic relapse growth kinetics would improve the personalized decision‐making regarding re‐administration of chemotherapy. Based on standardized quantitative PCR data, we therefore delineated the relapse kinetics in a cohort of 27 relapsing DEK‐NUP214‐positive patients treated in four different European countries. The prerelapse leukemic burden increased with a median doubling time of 13 d (range: 5–51 d, median: 0.71 logs/month, range: 0.18–1.91 logs/month), with FLT3‐ITD‐positive patients relapsing significantly faster than FLT3‐ITD‐negative ones (median: 0.9 vs. 0.6 logs/month, Wilcoxon rank sum test, P = 0.041). Peripheral blood and bone marrow were equally useful for minimal residual disease (MRD) detection, and thus, we found that with sampling intervals of 2 months, 94% of relapses would be detected with a median time from MRD detection to hematological relapse of 64 d. In conclusion, this data provide algorithms for handling the rare patients with DEK‐NUP214‐positive AML allowing for planning of both MRD follow‐up and, upon molecular relapse, the timing of cytoreduction or possibly transplant procedures.     

Minimal residual disease monitoring in chronic lymphocytic leukaemia patients. A comparative analysis of flow cytometry and ASO IgH RQ‐PCR

Minimal residual disease (MRD) is becoming increasingly important in chronic lymphocytic leukaemia (CLL) as treatment strategies are progressively improving. The primary objective of this study was to compare the applicability of three different flow cytometric approaches: basic 4‐colour analysis, European Research Initiative in CLL (ERIC) consensus method and 8‐colour analysis. Secondly, we investigated the sensitivity and specificity of flow cytometry (FC) compared to molecular analyses for MRD detection. A total of 462 CLL samples were evaluated by basic FC; in 143, ERIC consensus method was also performed and all three FC methodologies were applied in a subgroup of 10 cases. No discordance in defining MRD‐positive/negative samples was observed between the FC methods; within positive samples, the ERIC consensus method and 8‐colour analysis showed the most accurate results. MRD was analysed by FC and polymerase chain reaction (PCR) in 243 cases: concordant results were obtained in 199/243 samples (81·9%); 42/243 were FC?/PCR+. Overall, the sensitivity and specificity of FC compared to PCR was 96·5% and 77·2%, respectively. Both FC and PCR proved suitable for the detection of MRD and prediction of progression‐free survival, which was significantly reduced in MRD‐positive patients, regardless of the methodology. These results offer the rationale for a strategy to monitor MRD in CLL patients.     

Clinical applicability and prognostic significance of molecular response assessed by fluorescent‐PCR of immunoglobulin genes in multiple myeloma. Results from a GEM/PETHEMA study

Minimal residual disease monitoring is becoming increasingly important in multiple myeloma (MM), but multiparameter flow cytometry (MFC) and allele‐specific oligonucleotide polymerase chain reaction (ASO‐PCR) techniques are not routinely available. This study investigated the prognostic influence of achieving molecular response assessed by fluorescent‐PCR (F‐PCR) in 130 newly diagnosed MM patients from Grupo Español Multidisciplinar de Melanoma (GEM)2000/GEM05 trials (NCT00560053, NCT00443235, NCT00464217) who achieved almost very good partial response after induction therapy. As a reference, we used the results observed with simultaneous MFC. F‐PCR at diagnosis was performed on DNA using three different multiplex PCRs: IGH D‐J, IGK V‐J and KDE rearrangements. The applicability of F‐PCR was 91·5%. After induction therapy, 64 patients achieved molecular response and 66 non‐molecular response; median progression‐free survival (PFS) was 61 versus 36 months, respectively (P = 0·001). Median overall survival (OS) was not reached (NR) in molecular response patients (5‐year survival: 75%) versus 66 months in the non‐molecular response group (P = 0·03). The corresponding PFS and OS values for patients with immunophenotypic versus non‐immunophenotypic response were 67 versus 42 months (P = 0·005) and NR (5‐year survival: 95%) versus 69 months (P = 0·004), respectively. F‐PCR analysis is a rapid, affordable, and easily performable technique that, in some circumstances, may be a valid approach for minimal residual disease investigations in MM.     

Concurrent detection of minimal residual disease (MRD) in childhood acute lymphoblastic leukaemia by flow cytometry and real-time PCR

Minimal (i.e. submicroscopic) residual disease (MRD) predicts outcome in childhood acute lymphoblastic leukaemia (ALL). To be used clinically, MRD assays must be reliable and accurate. Two well-established techniques, flow cytometry (FC) and polymerase chain reaction (PCR), can detect leukaemic cells with a sensitivity of 0.01% (10(-4)). We analysed diagnostic samples of 45 ALL-patients (37 B-lineage ALL, eight T-lineage ALL) by four-colour FC and real-time PCR. Leukaemia-associated immunophenotypes, at a sensitivity of MRD detection by FC at the 0.01% level, were identified in 41 cases (91%); antigen-receptor gene rearrangements suitable for MRD detection with a sensitivity of 0.01% or better by PCR were identified in 38 cases (84%). The combined use of FC and PCR allowed MRD monitoring in all 45 patients. In 105 follow-up samples, MRD estimates by both methods were highly concordant, with a deviation factor of <5 by Bland-Altman analysis. Importantly, the concordance between FC and PCR was also observed in regenerating bone marrow samples containing high proportions of CD19(+) cells, and in samples studied 24 h after collection. We conclude that both MRD assays yield generally concordant results. Their combined use should enable MRD monitoring in virtually all patients and prevent false-negative results due to clonal evolution or phenotypic shifts.     

BAALC and WT1 expressions from diagnosis to hematopoietic stem cell transplantation: consecutive monitoring in adult patients with core‐binding‐factor‐positive AML

No consecutive analysis of BAALC and WT1 expressions associated with core‐binding factor AML (CBF‐AML) from diagnosis to hematopoietic stem cell transplantation (HSCT) has yet been reported. We investigated BAALC and WT1 expressions using a method of real‐time quantitative polymerase chain reaction (RQ‐PCR) at diagnosis, after induction chemotherapy, at pre‐HSCT, and at post‐HSCT period in 45 consecutive patients [t(8,21) (n = 28), inv(16) (n = 17)], who received HSCT as a post‐remission treatment. BAALC and WT1 RQ‐PCR decrement ratio (DR) was also calculated at post‐induction chemotherapy, at pre‐HSCT, and at post‐HSCT compared with the diagnostic level. Higher BAALC expression at diagnosis showed significantly inferior OS (P = 0.031), EFS (P = 0.011), and higher CIR (P = 0.002) rates. At post‐HSCT, both higher BAALC and WT1 expressions showed significantly inferior OS (P = 0.005, 0.016), EFS (P = 0.002, 0.006), and higher CIR (P = 0.001, 0.003) rates. A subgroup of t(8;21) showing higher BAALC and WT1 expressions at post‐HSCT were also associated with inferior OS (P = 0.018, 0.015) and higher CIR rates (P = 0.019, 0.011). While BAALC DR showed no significant results on outcomes, WT1 DR more than 2‐log at post‐HSCT showed significantly lower CIR rate (P = 0.028). This study showed that higher post‐HSCT BAALC and WT1 expressions in patients with CBF‐AML may be good markers of minimal residual disease for the prediction of survival and relapse after HSCT.     

Regression of fibrosis and portal hypertension in HCV‐associated cirrhosis and sustained virologic response after interferon‐free antiviral therapy

It is still controversial, whether and to what amount cirrhosis and portal hypertension are reversible in patients with hepatitis C virus (HCV)‐associated cirrhosis and sustained virologic response (SVR) after interferon‐free antiviral therapy. In this study, we prospectively evaluated dynamics of liver and spleen stiffness in HCV‐infected patients with advanced liver disease and SVR after interferon‐free treatment. A total of 54 patients with HCV‐associated cirrhosis and SVR were included. Liver and spleen stiffness was measured at therapy baseline (BL), end of treatment (EOT) and 24 weeks after EOT (FU24) by transient liver elastography (L‐TE) as well as by acoustic radiation force impulse of the liver (L‐ARFI) and spleen (S‐ARFI), as well as biochemical, virologic and clinical data. Improvement of liver and spleen stiffness was found in 44 of 50 (88%), 31 of 54 (57%) and 25 of 54 (46%) of patients assessed by L‐TE, L‐ARFI and S‐ARFI between baseline and FU24. Liver stiffness assessed by L‐TE improved between BL [median (range), 32.5 (9.1–75) kPa] and EOT [median (range), 21.3 (6.7–73.5) kPa; (P<.0001)], and between BL and FU24 [median (range), 21.2 (5.4–70) kPa; (P<.0001)]. Liver stiffness assessed by L‐ARFI improved between BL [median (range), 2.7 (1.2–4.1) m/s] and FU24 [median (range), 2.4 (1.2–3.9) m/s; P=.002), while spleen stiffness remained unchanged. Our data suggest that improvement of liver stiffness may be rather due to reduced necroinflammation and may be due to a less extent to regression of cirrhosis, as dynamics of liver stiffness improvement was more pronounced between BL and EOT than BL and FU24.     

Identification of emerging FLT3 ITD‐positive clones during clinical remission and kinetics of disease relapse in acute myeloid leukaemia with mutated nucleophosmin

FLT3 internal tandem duplication (ITD) mutations are frequently detected at diagnosis in cytogenetically normal acute myeloid leukaemia (CN‐AML) and predict unfavourable outcome. FLT3 ITD is an unstable aberration and may be lost or acquired at relapse. Recent whole genome sequencing studies have suggested that FLT3 ITD⁺ve AML relapse may evolve from small subclones undetectable at diagnosis by routine polymerase chain reaction (PCR). We developed a patient‐specific real‐time quantitative‐PCR (RQ‐PCR) to implement FLT3 ITD detection in six AML patients whose blasts carried wild‐type FLT3 at diagnosis and who relapsed with FLT3 ITD by routine PCR. Patient‐specific forward primers were designed after cloning and sequencing the FLT3 ITD in each case. The assay allowed retrospective detection of FLT3 ITD in diagnostic samples of 4/6 cases and to establish the kinetics of clonal evolution preceding relapse. After conventional chemotherapy, all patients had early relapse despite having been classified as NPM1⁺ve/FLT3 ITD^?ve at presentation, with shorter remissions being observed in four patients re‐classified as FLT3 ITD⁺ve by the new assay. Notably, FLT3 ITD clone became detectable by conventional PCR in three patients tested during remission after initial treatment. Our data underscore the need of identifying low FLT3 ITD levels, which are probably associated with relapse in otherwise good prognosis CN‐AML.     

Clinical utility of next‐generation sequencing‐based minimal residual disease in paediatric B‐cell acute lymphoblastic leukaemia

We assessed the clinical utility of next‐generation sequencing (NGS)‐based monitoring of minimal residual disease (MRD) in a uniformly treated cohort of 79 patients with paediatric B‐cell acute lymphoblastic leukaemia. Bone marrow samples were collected at the time of diagnosis, days 33 and 80, pre‐ (4–5 months) and post‐ (24 months) maintenance therapy time points, and at relapse. We identified leukaemia‐specific CDR3 sequences in 72 of 79 patients (91%) and detected MRD in 59 of 232 samples. Although MRD was detected in 28 of 55 samples (51%) on day 33, the frequencies of MRD detection decreased to 25% (16/65) at day 80, 19% (11/58) at 4–5 months and 7·4% (4/54) at 24 months. In a univariate analysis, positive MRD results on day 80 [relative risk (RR) 95% confidence interval (CI) = 7·438 (2·561–21·6), P < 0·001], at 4–5 months [RR (95% CI) = 10·24 (3·374–31·06), P < 0·001], and at 24 months [RR (95% CI) = 19·26 (4·974–74·59), P < 0·001] exhibited statistically significant associations with inferior leukaemia‐free survival; this was confirmed using a Cox proportional hazard model. Our study suggests the promising potential of NGS‐MRD for patients with B‐cell ALL.     

Combined real‐time PCR and galactomannan surveillance improves diagnosis of invasive aspergillosis in high risk patients with haematological malignancies

Invasive aspergillosis (IA) is a leading complication of intensive treatment for haematological malignancies. Earlier diagnosis should facilitate effective antifungal therapy and prevent progression to invasive disease, which is often lethal. Polymerase chain reaction (PCR) assays, targeting the 28S and ITS ribosomal gene regions respectively, were evaluated for early detection of Aspergillus DNA and for diagnostic utility in patients receiving treatment in two busy haematopoietic stem cell transplant centres. Patients undergoing intensive chemotherapy, autologous or allogeneic transplant were eligible for inclusion in the study. EDTA blood and serum samples for circulating Aspergillus biomarkers, including galactomannan (GM), were collected twice‐weekly on a prospective basis from all study patients who were categorized according to international consensus criteria for defining invasive fungal disease (IFD). Of 278 patients recruited there were 44 probable IA cases and only one proven case. Moderate sensitivity and specificity, poor positive predictive value (50–80%), but good negative predictive value (>80–90%) were common to both PCR assays. Overall biomarker performance could be improved by combining positive results of either PCR assay with GM taken within a 12‐d period. The addition of PCR to GM monitoring in high‐risk patients with haematological malignancies provides greater diagnostic accuracy in invasive aspergillosis.     

Quantification of free total plasma DNA and minimal residual disease detection in the plasma of children with acute lymphoblastic leukemia

The analysis of total plasma DNA and the monitoring of leukemic clone-specific immunoglobulin and/or T-cell receptor gene rearrangements for the evaluation of minimal residual disease (MRD) in the plasma may be useful tools for prognostic purposes or for early detection of subclinical disease recurrence in children with acute lymphoblastic leukemia (ALL). The aim of this paper is to establish reference ranges for total plasma DNA concentrations and to test the feasibility of MRD measurements employing plasma DNA from children with ALL by using real-time quantitative (RQ)-PCR. Despite wide inter-individual variation, the median concentrations of total plasma DNA for 12 healthy donors (57 ng/ml), 21 children with ALL after day 4 of treatment initiation (62 ng/ml) and 13 children with other malignancies (76 ng/ml) were similar. However, ALL patients had significantly higher concentrations at diagnosis (277 ng/ml) and on treatment day 3 (248 ng/ml) before returning to normal afterwards. Early plasma DNA MRD kinetics could be established for 15 ALL patients and showed good concordance with bone marrow MRD. Plasma DNA was higher in children with ALL at diagnosis but returned to normal within the first four treatment days. Despite low concentrations of DNA, it is feasible to measure MRD kinetics in plasma DNA during ALL induction therapy by adapted real-time PCR methodologies. This work was supported by the Deutsche Krebshilfe (Bonn, Germany) and the Leukemia Research Foundation (Chicago, IL, USA).     

Impact of two independent bone marrow samples on minimal residual disease monitoring in childhood acute lymphoblastic leukaemia

Minimal residual disease (MRD) diagnostics are used for risk group stratification in several acute lymphoblastic leukaemia (ALL) treatment protocols. It is, however, unclear whether MRD is homogeneously distributed within the bone marrow (BM) and whether this affects MRD diagnostics. We, therefore, analysed MRD levels in 141 paired BM samples (two independent punctures at different locations) from 26 ALL patients by real-time quantitative polymerase chain reaction (PCR) analysis of immunoglobulin and T-cell receptor gene rearrangements. MRD levels were comparable in 112 paired samples (79%), whereas two samples (both taken at day 15) had MRD levels that differed more than threefold. In the remaining 27 paired samples, MRD could be quantified or detected in one sample only. In four patients, MRD-based risk group classification was dependent on the site of BM puncture. Repetition of MRD analyses using 10-fold replicates instead of triplicates resolved most differences. In conclusion, MRD levels in paired BM samples were highly comparable, indicating that it is sufficient to analyse MRD in a single sample only. Nevertheless, MRD-based risk group classification can differ between paired BM samples, mainly because of variation below the quantitative range of the PCR assay rather than to a different distribution of leukaemic cells within the BM.     

Melatonin synergizes the chemotherapeutic effect of 5‐fluorouracil in colon cancer by suppressing PI3K/AKT and NF‐κB/iNOS signaling pathways

5‐Fluorouracil (5‐FU) is one of the most commonly used chemotherapeutic agents in colon cancer treatment, but has a narrow therapeutic index limited by its toxicity. Melatonin exerts antitumor activity in various cancers, but it has never been combined with 5‐FU as an anticolon cancer treatment to improve the chemotherapeutic effect of 5‐FU. In this study, we assessed such combinational use in colon cancer and investigated whether melatonin could synergize the antitumor effect of 5‐FU. We found that melatonin significantly enhanced the 5‐FU‐mediated inhibition of cell proliferation, colony formation, cell migration and invasion in colon cancer cells. We also found that melatonin synergized with 5‐FU to promote the activation of the caspase/PARP‐dependent apoptosis pathway and induce cell cycle arrest. Further mechanism study demonstrated that melatonin synergized the antitumor effect of 5‐FU by targeting the PI3K/AKT and NF‐κB/inducible nitric oxide synthase (iNOS) signaling. Melatonin in combination with 5‐FU markedly suppressed the phosphorylation of PI3K, AKT, IKKα, IκBα, and p65 proteins, promoted the translocation of NF‐κB p50/p65 from the nuclei to cytoplasm, abrogated their binding to the iNOS promoter, and thereby enhanced the inhibition of iNOS signaling. In addition, pretreatment with a PI3K‐ or iNOS‐specific inhibitor synergized the antitumor effects of 5‐FU and melatonin. Finally, we verified in a xenograft mouse model that melatonin and 5‐FU exerted synergistic antitumor effect by inhibiting the AKT and iNOS signaling pathways. Collectively, our study demonstrated that melatonin synergized the chemotherapeutic effect of 5‐FU in colon cancer through simultaneous suppression of multiple signaling pathways.