Assessment of postoperative circulating tumour DNA to predict early recurrence in patients with stage I-III right-sided colon cancer: prospective observational study.

BACKGROUND: Right-sided colon cancer (RCC) differs in mutation profile and risk of recurrence compared to distal colon cancer. Circulating tumour DNA (ctDNA) present after surgery can identify patients with residual disease after curative surgery and predict risk of early recurrence. METHODS: This is a prospective observational biomarker trial with exploration of ctDNA in 50 non-metastatic RCC patients for which oncological right-sided colectomy was performed. Blood samples were collected preoperatively, within 1 month post surgery, 3 months (not mandatory), 6 months and every 6 months thereafter. Plasma cell free DNA and/or tumour was investigated for cancer-related mutations by the next-generation sequencing (NGS) panel AVENIO surveillance specifically designed for ctDNA analysis. Detected mutations were quantified using digital droplet PCR (ddPCR) for follow-up. Recurrence-free survival was explored. RESULTS: 50 patients were recruited. Somatic cancer-related mutations were detected in 47/50 patients. ddPCR validated results from NGS for 27/34 (plasma) and 72/72 samples (tumour). Preoperative ctDNA was detected in 31/47 of the stage I/III patients and the majority of ctDNA positive patients showed reduction of ctDNA after surgery (27/31). ctDNA-positive patients at first postoperative sample had high recurrence risk compared to patients without measurable ctDNA (adjusted hazard ratio: 172.91; 95% c.i.: 8.70 to 3437.24; P: 0.001). CONCLUSION: ctDNA was detectable in most patients with non-metastatic RCC before surgery. Positive postoperative ctDNA was strongly associated with early recurrence. Detectable postoperative ctDNA is a prognostic factor with high (100%) positive predictive value for recurrence in this cohort of non-metastatic RCC. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT03776591.

Monosomy 7/del(7q) cause sensitivity to inhibitors of nicotinamide phosphoribosyltransferase in acute myeloid leukemia.

ABSTRACT: Monosomy 7 and del(7q) (-7/-7q) are frequent chromosomal abnormalities detected in up to 10% of patients with acute myeloid leukemia (AML). Despite unfavorable treatment outcomes, no approved targeted therapies exist for patients with -7/-7q. Therefore, we aimed to identify novel vulnerabilities. Through an analysis of data from ex vivo drug screens of 114 primary AML samples, we discovered that -7/-7q AML cells are highly sensitive to the inhibition of nicotinamide phosphoribosyltransferase (NAMPT). NAMPT is the rate-limiting enzyme in the nicotinamide adenine dinucleotide salvage pathway. Mechanistically, the NAMPT gene is located at 7q22.3, and deletion of 1 copy due to -7/-7q results in NAMPT haploinsufficiency, leading to reduced expression and a therapeutically targetable vulnerability to the inhibition of NAMPT. Our results show that in -7/-7q AML, differentiated CD34+CD38+ myeloblasts are more sensitive to the inhibition of NAMPT than less differentiated CD34+CD38- myeloblasts. Furthermore, the combination of the BCL2 inhibitor venetoclax and the NAMPT inhibitor KPT-9274 resulted in the death of significantly more leukemic blasts in AML samples with -7/-7q than the NAMPT inhibitor alone. In conclusion, our findings demonstrate that AML with -7/-7q is highly sensitive to NAMPT inhibition, suggesting that NAMPT inhibitors have the potential to be an effective targeted therapy for patients with monosomy 7 or del(7q).

Impact of age and comorbidities on the efficacy and tolerability of bosutinib in previously treated patients with chronic myeloid leukemia: results from the phase 4 BYOND study.

In the phase 4 BYOND trial, patients with pretreated chronic myeloid leukemia (CML) received bosutinib (starting dose: 500 mg/day). Efficacy and safety after ≥3 years of follow-up in 156 patients with Philadelphia chromosome-positive chronic phase CML by age and Charlson Comorbidity Index scores (without the age component; mCCI) is reported. Cumulative major molecular response rates at any time on treatment were 73.6%, 64.5%, and 74.1% in patients <65, 65-74, and ≥75 years of age, and 77.9%, 63.0%, and 59.3% in patients with mCCI scores 2, 3, and ≥4, respectively. Patients <65, 65-74, and ≥75 years of age experienced grade 3/4 treatment-emergent adverse events (TEAEs) at rates of 74.7%, 78.8%, and 96.4% and permanent discontinuations due to AEs at rates of 22.1%, 39.4%, and 46.4%, respectively. In patients with mCCI 2, 3, and ≥4, respective rates of grade 3/4 TEAEs were 77.8%, 77.8%, and 86.7%, and permanent discontinuations due to AEs were 25.3%, 33.3%, and 43.3%. In conclusion, a substantial proportion of patients maintained/achieved cytogenetic and molecular responses across age groups and mCCI scores. Older patients (≥75 years) and those with high comorbidity burden (mCCI ≥4) may require more careful monitoring due to the increased risk of TEAEs. Clinicaltrials.gov: NCT02228382.

Switching from imatinib to nilotinib plus pegylated interferon-α2b in chronic phase CML failing to achieve deep molecular response: clinical and immunological effects.

In order to improve molecular response for a discontinuation attempt in chronic myeloid leukemia (CML) patients in chronic phase, who had not achieved at least a molecular response <0.01% BCR-ABL1IS (MR4.0) after at least 2 years of imatinib therapy, we prospectively evaluated whether they could attain MR4.0 after a switch to a combination of nilotinib and 9 months of pegylated interferon-α2b (PegIFN). The primary endpoint of confirmed MR4.0 at month 12 (a BCR-ABL1IS level ≤ 0.01% both at 12 and 15 months) was reached by 44% (7/16 patients, 95% confidence interval (CI): 23- 67%) of patients, with 81% (13/16 patients, 95% CI: 57-93%) of patients achieving an unconfirmed MR4.0. The scheduled combination was completed by 56% of the patients, with premature discontinuations, mainly due to mood disturbances after the introduction of PegIFN, questioning the feasibility of the combination of nilotinib and PegIFN for this patient population and treatment goal. A comprehensive clinical substudy program was implemented to characterize the impact of the treatment changes on the immunological profile. This trial was registered at www.clinicaltrials.gov as #NCT01866553.

Overlapping features of therapy-related and de novo NPM1-mutated AML.

NPM 1-mutated acute myeloid leukemia (AML) shows unique features. However, the characteristics of "therapy-related" NPM1-mutated AML (t-NPM1 AML) are poorly understood. We compared the genetics, transcriptional profile, and clinical outcomes of t-NPM1 AML, de novo NPM1-mutated AML (dn-NPM1 AML), and therapy-related AML (t-AML) with wild-type NPM1 (t-AML). Normal karyotype was more frequent in t-NPM1 AML (n = 78/96, 88%) and dn-NPM1 (n = 1986/2394, 88%) than in t-AML (n = 103/390, 28%; P < .001). DNMT3A and TET2 were mutated in 43% and 40% of t-NPM1 AML (n = 107), similar to dn-NPM1 (n = 88, 48% and 30%; P > 0.1), but more frequently than t-AML (n = 162; 14% and 10%; P < 0.001). Often mutated in t-AML, TP53 and PPM1D were wild-type in 97% and 96% of t-NPM1 AML, respectively. t-NPM1 and dn-NPM1 AML were transcriptionally similar, (including HOX genes upregulation). At 62 months of median follow-up, the 3-year overall survival (OS) for t-NPM1 AML (n = 96), dn-NPM1 AML (n = 2394), and t-AML (n = 390) were 54%, 60%, and 31%, respectively. In multivariable analysis, OS was similar for the NPM1-mutated groups (hazard ratio [HR] 0.9; 95% confidence interval [CI], 0.65-1.25; P = .45), but better in t-NPM1 AML than in t-AML (HR, 1.86; 95% CI, 1.30-2.68; P < .001). Relapse-free survival was similar between t-NPM1 and dn-NPM1 AML (HR, 1.02; 95% CI, 0.72-1.467; P = .90), but significantly higher in t-NPM1 AML versus t-AML (HR, 1.77; 95% CI, 1.19-2.64; P = .0045). t-NPM1 and dn-NPM1 AML have overlapping features, suggesting that they should be classified as a single disease entity.

Concordance in measurable residual disease result after first and second induction cycle in acute myeloid leukemia: An outcome- and cost-analysis.

Measurable residual disease (MRD) measured using multiparameter flow-cytometry (MFC) has proven to be an important prognostic biomarker in acute myeloid leukemia (AML). In addition, MRD is increasingly used to guide consolidation treatment towards a non-allogenic stem cell transplantation treatment for MRD-negative patients in the ELN-2017 intermediate risk group. Currently, measurement of MFC-MRD in bone marrow is used for clinical decision making after 2 cycles of induction chemotherapy. However, measurement after 1 cycle has also been shown to have prognostic value, so the optimal time point remains a question of debate. We assessed the independent prognostic value of MRD results at either time point and concordance between these for 273 AML patients treated within and according to the HOVON-SAKK 92, 102, 103 and 132 trials. Cumulative incidence of relapse, event free survival and overall survival were significantly better for MRD-negative (<0.1%) patients compared to MRD-positive patients after cycle 1 and cycle 2 (p ≤ 0.002, for all comparisons). A total of 196 patients (71.8%) were MRD-negative after cycle 1, of which the vast majority remained negative after cycle 2 (180 patients; 91.8%). In contrast, of the 77 MRD-positive patients after cycle 1, only 41 patients (53.2%) remained positive. A cost reduction of -€571,751 per 100 patients could be achieved by initiating the donor search based on the MRD-result after cycle 1. This equals to a 50.7% cost reduction compared to the current care strategy in which the donor search is initiated for all patients. These results show that MRD after cycle 1 has prognostic value and is highly concordant with MRD status after cycle 2. When MRD-MFC is used to guide consolidation treatment (allo vs non-allo) in intermediate risk patients, allogeneic donor search may be postponed or omitted after cycle 1. Since the majority of MRD-negative patients remain negative after cycle 2, this could safely reduce the number of allogeneic donor searches and reduce costs.

IFN-α with dasatinib broadens the immune repertoire in patients with chronic-phase chronic myeloid leukemia.

In chronic myeloid leukemia (CML), combination therapies with tyrosine kinase inhibitors (TKIs) aim to improve the achievement of deep molecular remission that would allow therapy discontinuation. IFN-α is one promising candidate, as it has long-lasting effects on both malignant and immune cells. In connection with a multicenter clinical trial combining dasatinib with IFN-α in 40 patients with chronic-phase CML (NordCML007, NCT01725204), we performed immune monitoring with single-cell RNA and T cell receptor (TCR) sequencing (n = 4, 12 samples), bulk TCRß sequencing (n = 13, 26 samples), flow cytometry (n = 40, 106 samples), cytokine analyses (n = 17, 80 samples), and ex vivo functional studies (n = 39, 80 samples). Dasatinib drove the immune repertoire toward terminally differentiated NK and CD8+ T cells with dampened functional capabilities. Patients with dasatinib-associated pleural effusions had increased numbers of CD8+ recently activated effector memory T (Temra) cells. In vitro, dasatinib prevented CD3-induced cell death by blocking TCR signaling. The addition of IFN-α reversed the terminally differentiated phenotypes and increased the number of costimulatory intercellular interactions and the number of unique putative epitope-specific TCR clusters. In vitro IFN-α had costimulatory effects on TCR signaling. Our work supports the combination of IFN-α with TKI therapy, as IFN-α broadens the immune repertoire and restores immunological function.

The added value of multi-state modelling in a randomized controlled trial: The HOVON 102 study re-analyzed.

Clofarabine is an active antileukemic drug for subgroups of patients with acute myeloid leukemia (AML). Multi-state models can provide additional insights to supplement the original intention-to-treat analysis of randomized controlled trials (RCT). We re-analyzed the HOVON102/SAKK30/09 phase III RCT for newly diagnosed AML patients, which randomized between standard induction chemotherapy with or without clofarabine. Using multi-state models, we evaluated the effects of induction chemotherapy outcomes (complete remission [CR], measurable residual disease [MRD]), and post-remission therapy with allogeneic stem cell transplantation [alloSCT] on relapse and death. Through the latter a consistent reduction in the hazard of relapse in the clofarabine arm compared to the standard arm was found, which occurred irrespective of MRD status or post-remission treatment with alloSCT, demonstrating a strong and persistent antileukemic effect of clofarabine. During the time period between achieving CR and possible post-remission treatment with alloSCT, non-relapse mortality was higher in patients receiving clofarabine. An overall net benefit of treatment with clofarabine was identified using the composite endpoint current leukemia-free survival (CLFS). In conclusion, these results enforce and extend the earlier reported beneficial effect of clofarabine in AML and show that multi-state models further detail the effect of treatment on competing and series of events.

Sex disparity in acute myeloid leukaemia with FLT3 internal tandem duplication mutations: implications for prognosis.

Incidence, molecular presentation and outcome of acute myeloid leukaemia (AML) are influenced by sex, but little attention has been directed at untangling sex-related molecular and phenotypic differences between female and male patients. While increased incidence and poor risk are generally associated with a male phenotype, the poor prognostic FLT3 internal tandem duplication (FLT3-ITD) mutation and co-mutations with NPM1 and DNMT3A are overrepresented in female AML. Here, we have investigated the relationship between sex and FLT3-ITD mutation status by comparing clinical data, mutational profiles, gene expression and ex vivo drug sensitivity in four cohorts: Beat AML, LAML-TCGA and two independent HOVON/SAKK cohorts, comprising 1755 AML patients in total. We found prevalent sex-associated molecular differences. Co-occurrence of FLT3-ITD, NPM1 and DNMT3A mutations was overrepresented in females, while males with FLT3-ITDs were characterized by additional mutations in RNA splicing and epigenetic modifier genes. We observed diverging expression of multiple leukaemia-associated genes as well as discrepant ex vivo drug responses, suggestive of discrete functional properties. Importantly, significant prognostication was observed only in female FLT3-ITD-mutated AML. Thus, we suggest optimization of FLT3-ITD mutation status as a clinical tool in a sex-adjusted manner and hypothesize that prognostication, prediction and development of therapeutic strategies in AML could be improved by including sex-specific considerations.

Bimodal expression of potential drug target CLL-1 (CLEC12A) on CD34+ blasts of AML patients.

OBJECTIVES: This study aims to retrospectively assess C-lectin-like molecule 1 (CLL-1) bimodal expression on CD34+ blasts in acute myeloid leukemia (AML) patients (total N = 306) and explore potential CLL-1 bimodal associations with leukemia and patient-specific characteristics. METHODS: Flow cytometry assays were performed to assess the deeper immunophenotyping of CLL-1 bimodality. Cytogenetic analysis was performed to characterize the gene mutation on CLL-1-negative subpopulation of CLL-1 bimodal AML samples. RESULTS: The frequency of a bimodal pattern of CLL-1 expression of CD34+ blasts ranged from 8% to 65% in the different cohorts. Bimodal CLL-1 expression was most prevalent in patients with MDS-related AML (P = .011), ELN adverse risk (P = .002), NPM1 wild type (WT, P = .049), FLT3 WT (P = .035), and relatively low percentages of leukemia-associated immunophenotypes (P = .006). Additional immunophenotyping analysis revealed the CLL-1- subpopulation may consist of pre-B cells, immature myeloblasts, and hematopoietic stem cells. Furthermore, (pre)-leukemic mutations were detected in both CLL-1+ and CLL-1- subfractions of bimodal samples (N = 3). CONCLUSIONS: C-lectin-like molecule 1 bimodality occurs in about 25% of AML patients and the CLL-1- cell population still contains malignant cells, hence it may potentially limit the effectiveness of CLL-1-targeted therapies and warrant further investigation.

FLT3-ITD mutations in acute myeloid leukaemia - molecular characteristics, distribution and numerical variation.

Recurrent somatic internal tandem duplications (ITD) in the FMS-like tyrosine kinase 3 (FLT3) gene characterise approximately one third of patients with acute myeloid leukaemia (AML), and FLT3-ITD mutation status guides risk-adapted treatment strategies. The aim of this work was to characterise FLT3-ITD variant distribution in relation to molecular and clinical features, and overall survival in adult AML patients. We performed two parallel retrospective cohort studies investigating FLT3-ITD length and expression by cDNA fragment analysis, followed by Sanger sequencing in a subset of samples. In the two cohorts, a total of 139 and 172 mutant alleles were identified in 111 and 123 patients, respectively, with 22% and 28% of patients presenting with more than one mutated allele. Further, 15% and 32% of samples had a FLT3-ITD total variant allele frequency (VAF) < 0.3, while 24% and 16% had a total VAF ≥ 0.7. Most of the assessed clinical features did not significantly correlate to FLT3-ITD numerical variation nor VAF. Low VAF was, however, associated with lower white blood cell count, while increasing VAF correlated with inferior overall survival in one of the cohorts. In the other cohort, ITD length above 50 bp was identified to correlate with inferior overall survival. Our report corroborates the poor prognostic association with high FLT3-ITD disease burden, as well as extensive inter- and intrapatient heterogeneity in the molecular features of FLT3-ITD. We suggest that future use of FLT3-targeted therapy could be accompanied with thorough molecular diagnostics and follow-up to better predict optimal therapy responders.

Addition of lenalidomide to intensive treatment in younger and middle-aged adults with newly diagnosed AML: the HOVON-SAKK-132 trial.

Lenalidomide, an antineoplastic and immunomodulatory drug, has therapeutic activity in acute myeloid leukemia (AML), but definitive studies about its therapeutic utility have been lacking. In a phase 3 study, we compared 2 induction regimens in newly diagnosed patients age 18 to 65 years with AML: idarubicine-cytarabine (cycle 1) and daunorubicin and intermediate-dose cytarabine (cycle 2) without or with lenalidomide (15 mg orally on days 1-21). One final consolidation cycle of chemotherapy or autologous stem cell transplantation (auto-SCT) or allogeneic SCT (allo-SCT) was provided according to a prognostic risk and minimal residual disease (MRD)-adapted approach. Event-free survival (EFS; primary end point) and other clinical end points were assessed. A second random assignment in patients in complete response or in complete response with incomplete hematologic recovery after cycle 3 or auto-SCT involved 6 cycles of maintenance with lenalidomide (10 mg on days 1-21) or observation. In all, 392 patients were randomly assigned to the control group, and 388 patients were randomly assigned to lenalidomide induction. At a median follow-up of 41 months, the study revealed no differences in outcome between the treatments (EFS, 44% ± 2% standard error and overall survival, 54% ± 2% at 4 years for both arms) although in an exploratory post hoc analysis, a lenalidomide benefit was suggested in SRSF2-mutant AML. In relation to the previous Dutch-Belgian Hemato-Oncology Cooperative Group and Swiss Group for Clinical Cancer Research (HOVON-SAKK) studies that used a similar 3-cycle regimen but did not pursue an MRD-guided approach, these survival estimates compare markedly more favorably. MRD status after cycle 2 lost prognostic value in intermediate-risk AML in the risk-adjusted treatment context. Maintenance with lenalidomide showed no apparent effect on relapse probability in 88 patients randomly assigned for this part of the study.