Combination romidepsin and azacitidine therapy is well
tolerated and clinically active in adults with high-risk acute
myeloid leukaemia ineligible for intensive chemotherapy
Justin Loke,1,2 Marlen Metzner,3,4
Rebecca Boucher,2 Aimee Jackson,2
Louise Hopkins,2 Jiri Pavlu,5
Eleni Tholouli,6 Mark Drummond,7,8
Andy Peniket,4,9 Rebecca Bishop,2
Sonia Fox,2 Paresh Vyas3,4,9 and
Charles Craddock1,2
Centre for Clinical Haematology,
University Hospital Birmingham, 2
Cancer
Research UK Clinical Trials Unit,
University of Birmingham, Birmingham,
MRC Molecular Haematology Unit,
University of Oxford, 4
NIHR Oxford
Biomedical Research Centre, Oxford,
Centre for Haematology, Imperial College
London at Hammersmith Hospital,
London, 6
Department of Clinical
Haematology, Central Manchester
University Hospitals NHS Foundation
Trust, Manchester, 7
Beatson West of
Scotland Cancer Centre, 8
University of
Glasgow, Glasgow, and 9
Oxford University
Hospitals Foundation NHS Trust, Oxford,
Received 28 June 2021; accepted for
publication 24 August 2021
Correspondence: Charles Craddock, Centre for
Clinical Haematology, Queen Elizabeth
Hospital, Mindelsohn Way, Birmingham B15
2GW, UK.
E-mail: [email protected]
Summary
Azacitidine (AZA) is important in the management of patients with acute myeloid
leukaemia (AML) who are ineligible for intensive chemotherapy. Romidepsin
(ROM) is a histone deacetylase inhibitor which synergises with AZA in vitro. The
ROMAZA trial established the maximum tolerated dose (MTD) of combined ROM/
AZA therapy in patients with AML, as ROM 12 mg/m2 on Days 8 and 15, with
AZA 75 mg/m2 administered for 7/28 day cycle. Nine of the 38 (23
7%) patients
treated at the MTD were classified as responders by Cycle 6 (best response: com￾plete remission [CR]/incomplete CR n = 7, partial response n = 2). Correlative
next-generation sequencing studies demonstrated important insights into therapy
resistance.
Keywords: acute myeloid leukaemia, relapsed, refractory, early phase, clinical
trial, hypomethylating agent.
Introduction
Treatment options for patients with acute myeloid leukaemia
(AML) who are ineligible for intensive chemotherapy are
limited and, as a consequence, patient outcomes remain
poor.1 A significant advance has been the use of epigenetic
therapies in patients with AML and high risk myelodysplastic
syndromes (MDS), and azacitidine remains the backbone of
regimens involving novel agents, including venetoclax.2,3
However, in patients with relapsed/refractory AML, the
activity of azacitidine monotherapy is reduced4 and strategies
which increase the activity of azacitidine are required. In
vitro studies demonstrated evidence of synergistic anti￾leukaemic activity between azacitidine and histone deacety￾lase inhibitors (HDACi).5
Emerging clinical data suggest that azacitidine used with
the HDACi, romidepsin, may have significant anti-tumour
activity and synergy.6 Romidepsin belongs to the cyclic pep￾tide family7 and inhibits different pathways to previously
investigated HDACi.8 Romidepsin has been shown to be
short report
ª 2021 The Authors. British Journal of Haematology published by British Society for Haematology and
John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited.
doi: 10.1111/bjh.17823
clinically potent and well-tolerated as monotherapy in
patients with peripheral T-cell lymphoma,9 but has not been
investigated in AML. Therefore, in this phase I/II ROMAZA
trial, we examined the safety and efficacy of romidepsin
and azacitidine in patients with newly diagnosed, relapsed
or refractory AML who are ineligible for conventional
chemotherapy.
Methods
Study conduct
Review boards of participating institutions approved the
study protocol, which was conducted according to the Decla￾ration of Helsinki and Good Clinical Practice Guidelines of
the International Conference on Harmonization (EudraACT
No: 2011-005023-40 and ISCRTN:69211255).
Patients and investigations
Patients with newly diagnosed, relapsed or refractory AML as
defined by the World Health Organisation classification and
deemed ineligible for intensive chemotherapy on the grounds
of age or co-morbidities, were eligible for trial inclusion.
Patients with prior treatment with demethylating agents were
ineligible.
Treatment. The maximum tolerated dose of romidepsin in
combination with azacitidine was determined using an
escalating/de-escalating 3 + 3 cohort design. Patients were
recruited in planned cohort sizes of three up to a sample size
of 18, with an additional 35 patients recruited to an expan￾sion cohort. (Figure S1, Data S1). Patients initially received
up to six cycles of combination therapy and continued if
benefiting clinically.
Adverse event reporting. Tolerability and safety were assessed
according to the National Cancer Institute Common Termi￾nology Criteria for Adverse Events (CTCAE) version 4.0. A
dose-limiting toxicity (DLT) was defined as a clinically signifi-
cant grade 3 or 4 non-haematological toxicity, excluding nau￾sea (manageable with anti-emetics) and fatigue (persisting for
more than seven days), which occurred within the DLT moni￾toring period and was related to the trial medication.
Endpoints. The primary endpoint for the escalation cohort
was to determine the maximum tolerated dose (MTD) of the
romidepsin/azacitidine combination (Figure S3). The objec￾tive for the expansion cohort was to provide preliminary effi-
cacy and safety data of the combination at the MTD with
response defined as acquisition of complete remission (CR),
incomplete CR (CRi) or partial response (PR) as the primary
endpoint. This was assessed at the end of three and six cycles
of treatment according to modified Cheson criteria.10 Second￾ary endpoints were tolerability and safety of the combination
and overall survival for patients treated at the MTD. Further
details of statistical and mutational analysis using next￾generation sequencing (NGS) are available in Data S1.
Results
Baseline characteristics
Forty-eight patients were recruited from October 2013 to
October 2017 (Figure S1). Thirteen patients were recruited
to the dose-finding cohorts, which included four replace￾ments for those who did not complete the DLT monitoring
period. A subsequent 35 patients were recruited to the
expansion cohort of patients treated at the MTD (cohort 2)
(Figure S2). Baseline patient characteristics are listed in
Table I, 36/48 (75%) had relapsed/refractory disease.
Treatment administration
Patients received a median of 2
5 cycles of treatment across
the whole trial. Twenty out of 38 patients treated at the
MTD discontinued their treatment before the third cycle of
treatment and were not assessed for their response. Only 4/
20 discontinued treatment due to treatment toxicity.
MTD assessment
In the absence of DLTs in the first cohort (10 mg/m2 romi￾depsin on Day 8 and 15, and 75 mg/m2 azacitidine on Day
1–9 (in a 5-2-2 combination)) and second cohort (12 mg/m2
romidepsin on Day 8 and 15, and 75 mg/m2 azacitidine on
Day 1–9) (Figure S1), a third cohort of four patients was
opened at 12 mg/m2 romidepsin on Day 8, 15 and 22 and
75 mg/m2 azacitidine on Day 1–9. A total of four patients
were recruited as one patient was unevaluable. All patients
experienced at least one serious adverse event (SAE). Whilst
there were no reported DLTs at this dose level, a proportion
of patients suffered from nausea, vomiting and associated
weight loss. As a result, the dose was de-escalated to that of
Cohort 2 (12 mg/m2 romidepsin on Day 8 and 15, and
75 mg/m2 azacitidine on Day 1–9 (in a 5-2-2 combination))
which was used as the MTD.
Safety and tolerability
The combination therapy was well tolerated: five non￾haematological treatment-related adverse events of Grade 3
or higher affecting at least 10% of all trial patients were
reported in the study across the 38 patients treated at the
MTD (Tables SI and SII).
Response and survival
Using an intention-to-treat (ITT) approach, nine of the 38
(23
7%) patients treated at the MTD were classified as
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2 ª 2021 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.
responders by Cycle 6 (best response: CR/CRi n = 7, PR
n = 2) (Figure S4) (a pre-specified success criteria by
A’herns design required 10/38 responders). Of the 11 patients
who were previously untreated, four (36
4%) had a response.
In the 27 patients with relapsed/refractory disease there were
five responses (18
5%). Of the 29 non-responders, 20 discon￾tinued treatment prior to end of Cycle 3 and consequently
did not undergo response assessment.
At the date of data cut-off (21st May 2020), one patient in
Cohort 2 remained on treatment and there were 45/48 deaths.
The median overall survival of the 38 patients treated at the MTD
was 6
21 months (95% confidence interval [CI]: 3
71, 8
87),
whilst the median overall survival for the 9/38 patients who
responded from Cohort 2 was 15
3 months (95% CI: 4
3, 29
1).
Mutational analysis by NGS at baseline and sequentially
post-treatment
We obtained diagnostic mutational status across 97 genes in
35 patients using NGS. The median number of variants per
patient was six (Figure S5). The most common mutations
were in RUNX1 and FLT3, reflecting predominance of
adverse risk genetic mutations,11,12 consistent with our popu￾lation disease history.
Serial clonal structures in responding and non￾responding patients
We next examined the pattern of clonal mutational architec￾ture at different sequential time points in response to azaci￾tidine and romidepsin, and related this to their clinical
responses. In two patients achieving a CR (Fig 1A), the dom￾inant clone present at commencement of therapy was sup￾pressed, suggesting clonal selection in responding patients.
For example, in TNO 7 mutations, pre-treatment include
STAG2, NRAS, RUNX1 and SRSF2. However, NGS analysis
of the sample after Cycle 12, at a time when the patient
remained in CR, a subdominant clone – present at diagnosis
– consisting of ATM/KIT/CUX1 mutations becomes the
dominant clone.
In contrast, recurrent myeloid mutations tended to persist,
at similar VAFs, in patients who achieved a CRi (n = 3,
Disease, n (%)
Primary 5 (83) 19 (50) 1 (25) 25 (52)
Secondary 1 (17) 19 (50) 3 (75) 23 (48)
Age (years), median (range) 54 (18–67) 69 (31–84) 52 (46–63) 68 (18–84)
Sex, n (%)
Male 3 (50) 22 (58) 2 (50) 27 (56)
Female 3 (50) 16 (42) 2 (50) 21 (44)
Cytogenetics, n (%)
Favourable risk 0 (0) 1 (2) 0 (0) 1 (2)
Intermediate risk 2 (33) 22 (58) 0 (0) 24 (50)
Poor risk 3 (50) 12 (32) 4 (100) 19 (40)
Not known/failed 1 (17) 3 (8) 0 (0) 4 (8)
Disease status, n (%)
Untreated 0 (0) 11 (29) 1 (25) 12 (25)
Relapse 6 (100) 22 (58) 3 (75) 31 (65)
Primary refractory disease 0 (0) 5 (13) 0 (0) 5 (10)
FLT3-ITD, n (%)
Present 1 (17) 6 (16) 1 (25) 8 (17)
Absent 2 (33) 27 (71) 2 (50) 31 (64)
Unknown 3 (50) 5 (13) 1 (25) 9 (19)
NPM1 mutation, n (%)
Present 0 (0) 4 (11) 0 (0) 4(8)
Absent 3 (50) 29 (76) 3 (75) 35 (73)
Unknown 3 (50) 5 (13) 1 (25) 9 (19)
ECOG status, n (%)
0 6 (100) 22 (58) 3 (75) 31 (65)
1 0 (0) 12 (32) 0 (0) 12 (25)
2 0 (0) 4 (10) 1 (25) 5 (10)
Prior allogeneic stem cell transplant, n (%) 4 (67) 10 (26) 3 (75) 17 (35)
ECOG, Eastern Cooperative Oncology Group.
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ª 2021 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd. 3
Fig 1. Mutational analysis of patients at baseline and in sequential samples post-azacitidine and -romidepsin treatment. Mutational analysis at
baseline and sequential treatment samples for patients with (A) complete remission (CR); (B) with an incomplete CR (CRi), and (C) patients
who achieve a response but subsequently relapse, and (D) patients with resistant disease. [Colour figure can be viewed at wileyonlinelibrary.com]
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4 ª 2021 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.
Fig 1B; Figure S6B). This could be consistent with the persis￾tence of pre-leukaemic or leukaemic clones. If these clones
are leukaemic then it would suggest that the mechanism of
action of romidepsin and azacitidine is to promote differenti￾ation.
In patients who responded and subsequently have a docu￾mented relapse (Fig 1C), complex patterns of clonal evolu￾tion were seen. In TNO22, a FLT3-ITD clone decreased in
size when the patient was in PR and remained low at relapse.
In contrast, cells with RUNX1/CBL mutations were selected
for at PR and the VAF of these mutations increased at
relapse – consistent with resistance to azacitidine and romi￾depsin. In contrast, in patient TNO32 the VAF of mutations
in TET2, U2AF1 and NRAS were reduced at CR, only to
increase at relapse. This is consistent with leukaemic cells
showing initial therapy sensitivity followed by therapy resis￾tance, possibly through epigenetic mechanisms. Finally, we
observed that in patients with resistant disease (n = 5,
Fig 1D; Figure S6A) mutant clones persist or expand.
Discussion
Responses in the relapsed/refractory setting are challenging to
obtain and this study exemplifies the difficulty in treating
this cohort of less fit patients with rapid kinetic disease.13 Of
particular note, 17 (35%) had previously received an allo￾geneic stem cell transplant.
The tolerability of the ROM/AZA regimen is notable
because previous experiences of HDACi were associated with
increased toxicity. Romidepsin belongs to a different class of
HDACi, as previously investigated in AML,7,14 and we
demonstrate that this combination can be safely delivered in
the outpatient setting.
In order to obtain insights into the mechanism of this
novel combination we monitored the clonal architecture of
the disease through the serial NGS detection of mutations.
The prevalence of TP53 mutations appear to reflect the heav￾ily pre-treated nature of this cohort of patients, and in
patients who subsequently have progressive disease, these
mutant clones expand, in keeping with previous results of
patients treated with decitabine/panobinostat.15
In summary, this study established a MTD for combined
ROM/AZA therapy that is safe and clinically active within
adults with relapsed AML. Further studies will be required to
compare the clinical activity of ROM/AZA directly to a com￾parator treatment arm.
Acknowledgements
We thank the patients who participated in this trial and their
families. This study was funded by Bloodwise and Celgene.
We would like to acknowledge the study staff at the sites.
P.V. is supported by the Bloodwise Specialist Programme
Grant 13001 and by the NIHR Oxford BRC Fund, pro￾gramme grant from the MRC Molecular Haematology Unit
(MC_UU_12009/11). The Centre for Haematology at Impe￾rial College London receive funding from the NIHR BRC.
Author contributions
JL, MM, RB, AJ, PV and CC analysed the data. LH, RB, AJ
and SF were involved in the management of the trial. ET, JL,
AP, MD, PV and CC recruited patients to the study. CC
designed the trial. All authors had access to the primary data
and contributed to manuscript development. All authors
assumed responsibility for executing the study according to
the protocol and statistical analysis plan, completeness and
integrity of the data, and the decision to submit the manu￾script.
Disclosures
PV is on the advisory boards for BMS, Daiichi Sankyo, Astel￾las, AbbVie, Pfizer, Jazz; speaker bureau for AbbVie, Novar￾tis, Jazz, Daiichi Sankyo, BMS, the Takeda scientific advisory
board for Auron, Oxford Biomedica and receives research
support from CD47 Inc, BMS, Novartis, GSK. CC has
received honoraria from Celgene, Daichi-Sankyo, Novartis
and Pfizer as well as research funding from Celgene. JL has
received travel funding from Novartis and Daichi-Sankyo,
and honoraria from Pfizer, Janssen and Amgen. JP has
received travel funding and honoraria from Daichi-Sankyo
and Jazz.
Supporting Information
Additional supporting information may be found online in
the Supporting Information section at the end of the article.
Data S1. Supplementary Methods.
Table SI. Treatment-related adverse events.
Table SII. Treatment-related serious adverse events.
Fig S1. Flow diagram summarising trial.
Fig S2. Overview of patients treated at the maximum tol￾erated dose.
Fig S3. Schema to determine MTD..
Fig S4. Swimmer plot of response for cohort treated at
MTD.
Fig S5. Mutation profile at baseline for 35 patients.
Fig S6. Mutational analysis at baseline and samples subse￾quent to treatment.
Table SIII. Sequencing coverage per gene.
Table SIV. Sequencing coverage of each patient sample.
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