Molecular, clinical and therapeutic determinants of outcome in NPM1 mutated AML.

Although NPM1-mutated acute myeloid leukemia (AML) carries a generally favorable prognosis, many patients still relapse and die. Previous studies identified several molecular and clinical features associated with poor outcome, however only FLT3-ITD mutation and adverse karyotype are currently used for risk stratification due to inconsistent results and uncertainty around how other factors should influence treatment, particularly given the strong prognostic impact of post-induction measurable residual disease (MRD). Here we analyzed a large group of patients with NPM1mut AML enrolled in prospective trials (NCRI AML17 and AML19, n=1357) to delineate the impact of baseline molecular and clinical features, post induction MRD status and treatment intensity on outcome. FLT3-ITD (HR 1.28, 95%CI 1.01-1.63), DNMT3A (HR 1.65, 95%CI 1.32-2.05), WT1 (HR 1.74, 95%CI 1272-2.38) and non-ABD NPM1 mutations (HR 1.64, 95%CI 1.22-2.21) were independently associated with poorer overall survival (OS). These factors were also strongly associated with MRD positivity. For patients achieving MRD negativity, these mutations (except FLT3-ITD) were associated with an increased cumulative incidence of relapse (CIR) and poorer OS. However, apart from the few patients with adverse cytogenetics, we could not identify any group of MRD negative patients with a CIR >40% or with benefit from allograft in first remission. Intensified chemotherapy with the FLAG-Ida regimen was associated with improved outcomes in all subgroups, with greater benefits observed in the highest risk molecular subgroups.

Postinduction molecular MRD identifies patients with NPM1 AML who benefit from allogeneic transplant in first remission.

ABSTRACT: Selection of patients with NPM1-mutated acute myeloid leukemia (AML) for allogeneic transplant in first complete remission (CR1-allo) remains controversial because of a lack of robust data. Consequently, some centers consider baseline FLT3-internal tandem duplication (ITD) an indication for transplant, and others rely on measurable residual disease (MRD) status. Using prospective data from the United Kingdom National Cancer Research Institute AML17 and AML19 studies, we examined the impact of CR1-allo according to peripheral blood NPM1 MRD status measured by quantitative reverse transcription polymerase chain reaction after 2 courses of induction chemotherapy. Of 737 patients achieving remission, MRD was positive in 19%. CR1-allo was performed in 46% of MRD+ and 17% of MRD- patients. We observed significant heterogeneity of overall survival (OS) benefit from CR1-allo according to MRD status, with substantial OS advantage for MRD+ patients (3-year OS with CR1-allo vs without: 61% vs 24%; hazard ratio [HR], 0.39; 95% confidence interval [CI], 0.24-0.64; P < .001) but no benefit for MRD- patients (3-year OS with CR1-allo vs without: 79% vs 82%; HR, 0.82; 95% CI, 0.50-1.33; P = .4). Restricting analysis to patients with coexisting FLT3-ITD, again CR1-allo only improved OS for MRD+ patients (3-year OS, 45% vs 18%; compared with 83% vs 76% if MRD-); no interaction with FLT3 allelic ratio was observed. Postinduction molecular MRD reliably identifies those patients who benefit from allogeneic transplant in first remission. The AML17 and AML19 trials were registered at www.isrctn.com as #ISRCTN55675535 and #ISRCTN78449203, respectively.

Fludarabine, Cytarabine, Granulocyte Colony-Stimulating Factor, and Idarubicin With Gemtuzumab Ozogamicin Improves Event-Free Survival in Younger Patients With Newly Diagnosed AML and Overall Survival in Patients With NPM1 and FLT3 Mutations.

PURPOSE: To determine the optimal induction chemotherapy regimen for younger adults with newly diagnosed AML without known adverse risk cytogenetics. PATIENTS AND METHODS: One thousand thirty-three patients were randomly assigned to intensified (fludarabine, cytarabine, granulocyte colony-stimulating factor, and idarubicin [FLAG-Ida]) or standard (daunorubicin and Ara-C [DA]) induction chemotherapy, with one or two doses of gemtuzumab ozogamicin (GO). The primary end point was overall survival (OS). RESULTS: There was no difference in remission rate after two courses between FLAG-Ida + GO and DA + GO (complete remission [CR] + CR with incomplete hematologic recovery 93% v 91%) or in day 60 mortality (4.3% v 4.6%). There was no difference in OS (66% v 63%; P = .41); however, the risk of relapse was lower with FLAG-Ida + GO (24% v 41%; P < .001) and 3-year event-free survival was higher (57% v 45%; P < .001). In patients with an NPM1 mutation (30%), 3-year OS was significantly higher with FLAG-Ida + GO (82% v 64%; P = .005). NPM1 measurable residual disease (MRD) clearance was also greater, with 88% versus 77% becoming MRD-negative in peripheral blood after cycle 2 (P = .02). Three-year OS was also higher in patients with a FLT3 mutation (64% v 54%; P = .047). Fewer transplants were performed in patients receiving FLAG-Ida + GO (238 v 278; P = .02). There was no difference in outcome according to the number of GO doses, although NPM1 MRD clearance was higher with two doses in the DA arm. Patients with core binding factor AML treated with DA and one dose of GO had a 3-year OS of 96% with no survival benefit from FLAG-Ida + GO. CONCLUSION: Overall, FLAG-Ida + GO significantly reduced relapse without improving OS. However, exploratory analyses show that patients with NPM1 and FLT3 mutations had substantial improvements in OS. By contrast, in patients with core binding factor AML, outcomes were excellent with DA + GO with no FLAG-Ida benefit.

Molecular MRD is strongly prognostic in patients with NPM1-mutated AML receiving venetoclax-based nonintensive therapy.

ABSTRACT: Assessment of measurable residual disease (MRD) by quantitative reverse transcription polymerase chain reaction is strongly prognostic in patients with NPM1-mutated acute myeloid leukemia (AML) treated with intensive chemotherapy; however, there are no data regarding its utility in venetoclax-based nonintensive therapy, despite high efficacy in this genotype. We analyzed the prognostic impact of NPM1 MRD in an international real-world cohort of 76 previously untreated patients with NPM1-mutated AML who achieved complete remission (CR)/CR with incomplete hematological recovery following treatment with venetoclax and hypomethylating agents (HMAs) or low-dose cytarabine (LDAC). A total of 44 patients (58%) achieved bone marrow (BM) MRD negativity, and a further 14 (18%) achieved a reduction of ≥4 log10 from baseline as their best response, with no difference between HMAs and LDAC. The cumulative rates of BM MRD negativity by the end of cycles 2, 4, and 6 were 25%, 47%, and 50%, respectively. Patients achieving BM MRD negativity by the end of cycle 4 had 2-year overall of 84% compared with 46% if MRD was positive. On multivariable analyses, MRD negativity was the strongest prognostic factor. A total of 22 patients electively stopped therapy in BM MRD-negative remission after a median of 8 cycles, with 2-year treatment-free remission of 88%. In patients with NPM1-mutated AML attaining remission with venetoclax combination therapies, NPM1 MRD provides valuable prognostic information.

Venetoclax-based low intensity therapy in molecular failure of NPM1-mutated AML.

ABSTRACT: Molecular failure in NPM1-mutated acute myeloid leukemia (AML) inevitably progresses to frank relapse if untreated. Recently published small case series show that venetoclax combined with low-dose cytarabine or azacitidine can reduce or eliminate measurable residual disease (MRD). Here, we report on an international multicenter cohort of 79 patients treated for molecular failure with venetoclax combinations and report an overall molecular response (≥1-log reduction in MRD) in 66 patients (84%) and MRD negativity in 56 (71%). Eighteen of 79 patients (23%) required hospitalization, and no deaths were reported during treatment. Forty-one patients were bridged to allogeneic transplant with no further therapy, and 25 of 41 were MRD negative assessed by reverse transcription quantitative polymerase chain reaction before transplant. Overall survival (OS) for the whole cohort at 2 years was 67%, event-free survival (EFS) was 45%, and in responding patients, there was no difference in survival in those who received a transplant using time-dependent analysis. Presence of FLT3-ITD mutation was associated with a lower response rate (64 vs 91%; P < .01), worse OS (hazard ratio [HR], 2.50; 95% confidence interval [CI], 1.06-5.86; P = .036), and EFS (HR, 1.87; 95% CI, 1.06-3.28; P = .03). Eighteen of 35 patients who did not undergo transplant became MRD negative and stopped treatment after a median of 10 months, with 2-year molecular relapse free survival of 62% from the end of treatment. Venetoclax-based low intensive chemotherapy is a potentially effective treatment for molecular relapse in NPM1-mutated AML, either as a bridge to transplant or as definitive therapy.

FLT3 inhibitors as MRD-guided salvage treatment for molecular failure in FLT3 mutated AML.

Patients with FLT3-mutated AML have a high relapse rate and suboptimal outcomes. Many have co-mutations suitable for measurable residual disease (MRD) monitoring by RT-qPCR and those destined to relapse can be identified by high or rising levels of MRD, called molecular failure.  This provides a window for pre-emptive intervention, but there is little evidence to guide treatment. The use of FLT3 inhibitors (FLT3i) appears attractive but their use has not yet been evaluated.  We identified 56 patients treated with FLT3i at molecular failure.  The FLT3 mutation was an ITD in 52, TKD in 7 and both in 3. Over half of patients had previously received midostaurin. Molecular failure occurred at a median 9.2 months from diagnosis and was treated with gilteritinib (n = 38), quizartinib (n = 7) or sorafenib (n = 11). 60% achieved a molecular response, with 45% reaching MRD negativity. Haematological toxicity was low, and 22 patients were bridged directly to allogeneic transplant with another 6 to donor lymphocyte infusion. 2-year overall survival was 80% (95%CI 69-93) and molecular event-free survival 56% (95%CI 44-72). High-sensitivity next-generation sequencing for FLT3-ITD at molecular failure identified patients more likely to benefit. FLT3i monotherapy for molecular failure is a promising strategy which merits evaluation in prospective studies.

A randomized comparison of CPX-351 and FLAG-Ida in adverse karyotype AML and high-risk MDS: the UK NCRI AML19 trial.

Liposomal daunorubicin and cytarabine (CPX-351) improved overall survival (OS) compared with 7+3 chemotherapy in older patients with secondary acute myeloid leukemia (AML); to date, there have been no randomized studies in younger patients. The high-risk cohort of the UK NCRI AML19 trial (ISRCTN78449203) compared CPX-351 with FLAG-Ida in younger adults with newly diagnosed adverse cytogenetic AML or high-risk myelodysplastic syndromes (MDS). A total of 189 patients were randomized (median age, 56 years). Per clinical criteria, 49% of patients had de novo AML, 20% had secondary AML, and 30% had high-risk MDS. MDS-related cytogenetics were present in 73% of the patients, with a complex karyotype in 49%. TP53 was the most common mutated gene, in 43%. Myelodysplasia-related gene mutations were present in 75 (44%) patients. The overall response rate (CR + CRi) after course 2 was 64% and 76% for CPX-351 and FLAG-Ida, respectively. There was no difference in OS (13.3 months vs 11.4 months) or event-free survival in multivariable analysis. However, relapse-free survival was significantly longer with CPX-351 (median 22.1 vs 8.35 months). There was no difference between the treatment arms in patients with clinically defined secondary AML or those with MDS-related cytogenetic abnormalities; however, an exploratory subgroup of patients with MDS-related gene mutations had significantly longer OS with CPX-351 (median 38.4 vs 16.3 months). In conclusion, the OS of younger patients with adverse risk AML/MDS was not significantly different between CPX-351 and FLAG-Ida.

Assessment of acute myeloid leukemia molecular measurable residual disease testing in an interlaboratory study.

The European LeukaemiaNet (ELN) measurable residual disease (MRD) working group has published consensus guidelines to standardize molecular genetic MRD testing of the t(8;21)(q22;q22.1) RUNX1::RUNX1T1, inv(16)(p13.1q22) CBFB::MYH11, t(15;17)(q24.1;q21.2) PML::RARA, and NPM1 type A markers. A study featuring 29 international laboratories was performed to assess interlaboratory variation in testing and the subsequent interpretation of results, both crucial to patient safety. Most participants in this study were able to detect, accurately quantify, and correctly interpret MRD testing results, with a level of proficiency expected from a clinical trial or standard-of-care setting. However, a few testing and interpretive errors were identified that, in a patient setting, would have led to misclassification of patient outcomes and inappropriate treatment pathways being followed. Of note, a high proportion of participants reported false-positive results in the NPM1 marker-negative sample. False-positive results may have clinical consequences, committing patients to unneeded additional chemotherapy and/or transplant with the attendant risk of morbidity and mortality, which therefore highlights the need for ongoing external quality assessment/proficiency testing in this area. Most errors identified in the study were related to the interpretation of results. It was noted that the ELN guidance lacks clarity for certain clinical scenarios and highlights the requirement for urgent revision of the guidelines to elucidate these issues and related educational efforts around the revisions to ensure effective dissemination.

Venetoclax combined with low dose cytarabine compared to standard of care intensive chemotherapy for the treatment of favourable risk adult acute myeloid leukaemia (VICTOR): Study protocol for an international, open-label, multicentre, molecularly-guided randomised, phase II trial.

BACKGROUND: For patients with acute myeloid leukaemia (AML), the only potentially curative treatment is intensive chemotherapy (IC). This is highly toxic, particularly for patients > 60 years, potentially leading to prolonged hospitalisations requiring intensive supportive care, and sometimes treatment-related death. This also results in extensive healthcare costs and negatively impacts quality of life (QoL). Venetoclax with low-dose cytarabine (VEN + LDAC) is a novel, low-intensity treatment for AML patients who cannot receive IC. VEN + LDAC is given as an outpatient and toxicity appears significantly lower than with IC. Analysis of clinical trials performed to date are promising for patients with the genotype NPM1mutFLT3 ITDneg, where remission and survival rates appear comparable to those achieved with IC. METHODS: VICTOR is an international, two-arm, open-label, multi-centre, non-inferiority, randomised-controlled phase II trial to assess VEN + LDAC compared to standard of care (IC) as first-line treatment in older patients (initially aged ≥ 60 years) with newly diagnosed AML. The trial will recruit patients with a NPM1mutFLT3 ITDneg genotype; those with a favourable risk in relation to the experimental treatment. University of Birmingham is the UK co-ordinating centre, with national hubs in Aarhus University Hospital, Denmark, and Auckland District Health Board, New Zealand. The primary outcome is molecular event-free survival time where an event is defined as failure to achieve morphological complete response (CR) or CR with incomplete blood count recovery after two cycles of therapy; molecular persistence, progression or relapse requiring treatment change; morphological relapse, or; death. Secondary outcomes include cumulative resource use at 12- and 24-months, and QoL as assessed by EORTCQLQ-C30 and EQ-5D-3L at 3-, 6-, 12-, 18- and 24-months. The trial employs an innovative Bayesian design with target sample size of 156 patients aged > 60 years. DISCUSSION: The principle underpinning the VICTOR trial is that the chance of cure for patients in the experimental arm should not be compromised, therefore, an adaptive design with regular checks on accumulating data has been employed, which will allow for a staged expansion of the trial population to include younger patients if, and when, there is sufficient evidence of non-inferiority in older patients. TRIAL REGISTRATION: EudraCT: 2020-000,273-24; 21-Aug-2020. ISRCTN: 15,567,173; 08-Dec-2020.

Chemotherapy induces canalization of cell state in childhood B-cell precursor acute lymphoblastic leukemia.

Comparison of intratumor genetic heterogeneity in cancer at diagnosis and relapse suggests that chemotherapy induces bottleneck selection of subclonal genotypes. However, evolutionary events subsequent to chemotherapy could also explain changes in clonal dominance seen at relapse. We, therefore, investigated the mechanisms of selection in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) during induction chemotherapy where maximal cytoreduction occurs. To distinguish stochastic versus deterministic events, individual leukemias were transplanted into multiple xenografts and chemotherapy administered. Analyses of the immediate post-treatment leukemic residuum at single-cell resolution revealed that chemotherapy has little impact on genetic heterogeneity. Rather, it acts on extensive, previously unappreciated, transcriptional and epigenetic heterogeneity in BCP-ALL, dramatically reducing the spectrum of cell states represented, leaving a genetically polyclonal but phenotypically uniform population with hallmark signatures relating to developmental stage, cell cycle and metabolism. Hence, canalization of cell state accounts for a significant component of bottleneck selection during induction chemotherapy.

Clinical impact of NPM1-mutant molecular persistence after chemotherapy for acute myeloid leukemia.

Monitoring of NPM1 mutant (NPM1mut) measurable residual disease (MRD) in acute myeloid leukemia (AML) has an established role in patients who are treated with intensive chemotherapy. The European LeukemiaNet has defined molecular persistence at low copy number (MP-LCN) as an MRD transcript level <1% to 2% with a <1-log change between any 2 positive samples collected after the end of treatment (EOT). Because the clinical impact of MP-LCN is unknown, we sought to characterize outcomes in patients with persistent NPM1mut MRD after EOT and identify factors associated with disease progression. Consecutive patients with newly diagnosed NPM1mut AML who received ≥2 cycles of intensive chemotherapy were included if bone marrow was NPM1mut MRD positive at the EOT, and they were not transplanted in first complete remission. One hundred patients were followed for a median of 23.5 months; 42% remained free of progression at 1 year, either spontaneously achieving complete molecular remission (CRMRD-; 30%) or retaining a low-level NPM1mut transcript (12% for ≥12 months and 9% at last follow-up). Forty percent met the criteria for MP-LCN. Preemptive salvage therapy significantly prolonged relapse-free survival. Risk factors associated with disease progression were concurrent FLT3-internal tandem duplication at diagnosis and suboptimal MRD response (NPM1mut reduction <4.4-log) at EOT.

How we use molecular minimal residual disease (MRD) testing in acute myeloid leukaemia (AML).

In recent years there have been major advances in the use of molecular diagnostic and monitoring techniques for patients with acute myeloid leukaemia (AML). Coupled with the simultaneous explosion of new therapeutic agents, this has sown the seeds for significant improvements to treatment algorithms. Here we show, using a selection of real-life examples, how molecular monitoring can be used to refine clinical decision-making and to personalise treatment in patients with AML with nucleophosmin (NPM1) mutations, core binding factor translocations and other fusion genes. For each case we review the established evidence base and provide practical recommendations where evidence is lacking or conflicting. Finally, we review important technical considerations that clinicians should be aware of in order to safely exploit these technologies as they undergo widespread implementation.

Venetoclax induces rapid elimination of NPM1 mutant measurable residual disease in combination with low-intensity chemotherapy in acute myeloid leukaemia.

Based on promising results in older adults with acute myeloid leukaemia (AML), we treated patients with NPM1mut measurable residual disease (MRD) using off-label venetoclax in combination with low-dose cytarabine or azacitidine. Twelve consecutive patients were retrospectively identified, including five with molecular persistence and seven with molecular relapse/progression. All patients with molecular persistence achieved durable molecular complete remission (CRMRD- ) without transplantation. Six of seven patients with molecular relapse/progression achieved CRMRD- after 1-2 cycles of venetoclax. This paper highlights the promising efficacy of venetoclax-based therapy to reduce the relapse risk in patients with persistent or rising NPM1mut MRD.

Molecular MRD status and outcome after transplantation in NPM1-mutated AML.

Relapse remains the most common cause of treatment failure for patients with acute myeloid leukemia (AML) who undergo allogeneic stem cell transplantation (alloSCT), and carries a grave prognosis. Multiple studies have identified the presence of measurable residual disease (MRD) assessed by flow cytometry before alloSCT as a strong predictor of relapse, but it is not clear how these findings apply to patients who test positive in molecular MRD assays, which have far greater sensitivity. We analyzed pretransplant blood and bone marrow samples by reverse-transcription polymerase chain reaction in 107 patients with NPM1-mutant AML enrolled in the UK National Cancer Research Institute AML17 study. After a median follow-up of 4.9 years, patients with negative, low (<200 copies per 105ABL in the peripheral blood and <1000 copies in the bone marrow aspirate), and high levels of MRD had an estimated 2-year overall survival (2y-OS) of 83%, 63%, and 13%, respectively (P < .0001). Focusing on patients with low-level MRD before alloSCT, those with FLT3 internal tandem duplications(ITDs) had significantly poorer outcome (hazard ratio [HR], 6.14; P = .01). Combining these variables was highly prognostic, dividing patients into 2 groups with 2y-OS of 17% and 82% (HR, 13.2; P < .0001). T-depletion was associated with significantly reduced survival both in the entire cohort (2y-OS, 56% vs 96%; HR, 3.24; P = .0005) and in MRD-positive patients (2y-OS, 34% vs 100%; HR, 3.78; P = .003), but there was no significant effect of either conditioning regimen or donor source on outcome. Registered at ISRCTN (http://www.isrctn.com/ISRCTN55675535).

Single cell analysis of clonal architecture in acute myeloid leukaemia.

We used single cell Q-PCR on a micro-fluidic platform (Fluidigm) to analyse clonal, genetic architecture and phylogeny in acute myeloid leukaemia (AML) using selected mutations. Ten cases of NPM1c mutant AML were screened for 111 mutations that are recurrent in AML and cancer. Clonal architectures were relatively simple with one to six sub-clones and were branching in some, but not all, patients. NPM1 mutations were secondary or sub-clonal to other driver mutations (DNM3TA, TET2, WT1 and IDH2) in all cases. In three of the ten cases, single cell analysis of enriched CD34+/CD33- cells revealed a putative pre-leukaemic sub-clone, undetectable in the bulk CD33+ population that had one or more driver mutations but lacked NPM1c. Cells from all cases were transplanted into NSG mice and in most (8/10), more than one sub-clone (#2-5 sub-clones) transplanted. However, the dominant regenerating sub-clone in 9/10 cases was NPM1+ and this sub-clone was either dominant or minor in the diagnostic sample from which it was derived. This study provides further evidence, at the single cell level, for genetic variegation in sub-clones and stem cells in acute leukaemia and demonstrates both a preferential order of mutation accrual and parallel evolution of sub-clones.

The subclonal complexity of STIL-TAL1+ T-cell acute lymphoblastic leukaemia.

Single-cell genetics were used to interrogate clonal complexity and the sequence of mutational events in STIL-TAL1+ T-ALL. Single-cell multicolour FISH was used to demonstrate that the earliest detectable leukaemia subclone contained the STIL-TAL1 fusion and copy number loss of 9p21.3 (CDKN2A/CDKN2B locus), with other copy number alterations including loss of PTEN occurring as secondary subclonal events. In three cases, multiplex qPCR and phylogenetic analysis were used to produce branching evolutionary trees recapitulating the snapshot history of T-ALL evolution in this leukaemia subtype, which confirmed that mutations in key T-ALL drivers, including NOTCH1 and PTEN, were subclonal and reiterative in distinct subclones. Xenografting confirmed that self-renewing or propagating cells were genetically diverse. These data suggest that the STIL-TAL1 fusion is a likely founder or truncal event. Therapies targeting the TAL1 auto-regulatory complex are worthy of further investigation in T-ALL.

Genomic Classification and Prognosis in Acute Myeloid Leukemia.

BACKGROUND: Recent studies have provided a detailed census of genes that are mutated in acute myeloid leukemia (AML). Our next challenge is to understand how this genetic diversity defines the pathophysiology of AML and informs clinical practice. METHODS: We enrolled a total of 1540 patients in three prospective trials of intensive therapy. Combining driver mutations in 111 cancer genes with cytogenetic and clinical data, we defined AML genomic subgroups and their relevance to clinical outcomes. RESULTS: We identified 5234 driver mutations across 76 genes or genomic regions, with 2 or more drivers identified in 86% of the patients. Patterns of co-mutation compartmentalized the cohort into 11 classes, each with distinct diagnostic features and clinical outcomes. In addition to currently defined AML subgroups, three heterogeneous genomic categories emerged: AML with mutations in genes encoding chromatin, RNA-splicing regulators, or both (in 18% of patients); AML with TP53 mutations, chromosomal aneuploidies, or both (in 13%); and, provisionally, AML with IDH2(R172) mutations (in 1%). Patients with chromatin-spliceosome and TP53-aneuploidy AML had poor outcomes, with the various class-defining mutations contributing independently and additively to the outcome. In addition to class-defining lesions, other co-occurring driver mutations also had a substantial effect on overall survival. The prognostic effects of individual mutations were often significantly altered by the presence or absence of other driver mutations. Such gene-gene interactions were especially pronounced for NPM1-mutated AML, in which patterns of co-mutation identified groups with a favorable or adverse prognosis. These predictions require validation in prospective clinical trials. CONCLUSIONS: The driver landscape in AML reveals distinct molecular subgroups that reflect discrete paths in the evolution of AML, informing disease classification and prognostic stratification. (Funded by the Wellcome Trust and others; ClinicalTrials.gov number, NCT00146120.).