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Leukemia & Lymphoma

ISSN: 1042-8194 (Print) 1029-2403 (Online) Journal homepage: http://www.tandfonline.com/loi/ilal20

Risk adjusted therapy in chronic lymphocytic leukemia: a phase II cancer trials Ireland (CTRIAL-IE [ICORG 07-01]) study of fludarabine, cyclophosphamide, and rituximab therapy evaluating response adapted, abbreviated frontline therapy with FCR in non-del(17p) CLL Niamh Appleby, David O’Brien, Fiona M. Quinn, Liam Smyth, Johanna Kelly, Imelda Parker, Kathleen Scott, Mary R. Cahill, Gerard Crotty, Helen Enright, Brian Hennessy, Andrew Hodgson, Maeve Leahy, Hilary O’Leary, Michael O’Dwyer, Amjad Hayat & Elisabeth A. Vandenberghe To cite this article: Niamh Appleby, David O’Brien, Fiona M. Quinn, Liam Smyth, Johanna Kelly, Imelda Parker, Kathleen Scott, Mary R. Cahill, Gerard Crotty, Helen Enright, Brian Hennessy, Andrew Hodgson, Maeve Leahy, Hilary O’Leary, Michael O’Dwyer, Amjad Hayat & Elisabeth A. Vandenberghe (2018) Risk adjusted therapy in chronic lymphocytic leukemia: a phase II cancer trials Ireland (CTRIAL-IE [ICORG 07-01]) study of fludarabine, cyclophosphamide, and rituximab therapy evaluating response adapted, abbreviated frontline therapy with FCR in non-del(17p) CLL, Leukemia & Lymphoma, 59:6, 1338-1347, DOI: 10.1080/10428194.2017.1376746 To link to this article: https://doi.org/10.1080/10428194.2017.1376746

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LEUKEMIA & LYMPHOMA 2018, VOL. 59, NO. 6, 1338–1347 https://doi.org/10.1080/10428194.2017.1376746

ORIGINAL ARTICLE: CLINICAL

Risk adjusted therapy in chronic lymphocytic leukemia: a phase II cancer trials Ireland (CTRIAL-IE [ICORG 07-01]) study of fludarabine, cyclophosphamide, and rituximab therapy evaluating response adapted, abbreviated frontline therapy with FCR in non-del(17p) CLL Niamh Applebya, David O’Briena, Fiona M. Quinnb, Liam Smytha, Johanna Kellyc, Imelda Parkerd, Kathleen Scottd, Mary R. Cahille, Gerard Crottyf, Helen Enrightg, Brian Hennessyh, Andrew Hodgsoni, Maeve Leahyj, Hilary O’Learyj, Michael O’Dwyerk, Amjad Hayatk and Elisabeth A. Vandenberghea a Department of Haematology, St. James’s Hospital and Trinity College Dublin, Dublin, Ireland; bDepartment of Cancer Molecular Diagnostics, St. James’s Hospital, Dublin, Ireland; cDepartment of Medical Genetics, Our Lady's Hospital for Children, Dublin, Ireland; d Cancer Trials Ireland, Dublin, Ireland; eDepartment of Haematology, Cork University Hospital, Cork, Ireland; fDepartment of Haematology, Midlands Regional Hospital, Tullamore, Ireland; gDepartment of Haematology, Adelaide, Meath and National Children’s Hospital, Dublin, Ireland; hDepartment of Haematology, University Hospital Waterford, Waterford, Ireland; iDepartment of Haematology, Sligo Regional Hospital, Sligo, Ireland; jDepartment of Haematology, University Hospital Limerick, Limerick, Ireland; k Department of Haematology, University Hospital Galway, Galway, Ireland

ABSTRACT

ARTICLE HISTORY

Minimal residual disease negative complete response (MRD-negative CR) provides an early marker for time to treatment failure (TTF) in CLL treated with fludarabine, cyclophosphamide, and rituximab (FCR). MRD was assessed after four FCR cycles (FCR4); MRD-negative CR patients discontinued treatment. Fifty-two patients (35M; 17F) were enrolled. Eighteen (18/52; 34.6%) patients reached MRD-negative CR after FCR4 and 29/52 (55.8%) were MRD-negative CR at end of treatment (EOT). Median TTF was 71.1 months (95% CI 61.3–84.1 months), with median overall survival not reached. Mutated immunoglobulin heavy chain gene rearrangements (IGHV) were associated with early MRD-negative remissions, translating into prolonged TTF. Unmutated-IGHV, mutated-SF3B1 and mutated-NOTCH1 were associated with shortened TTF. No TTF difference was observed between patients in MRD-negative CR after four versus six cycles (82.2 versus 85.3 months, p ¼ .6306). Abbreviated FCR therapy is effective for patients achieving early MRD-negative remissions. Interim MRD assessment assists in personalizing therapy and reducing chemotherapy-associated toxicity.

Received 11 July 2017 Revised 28 August 2017 Accepted 1 September 2017

Introduction Chronic lymphocytic leukemia (CLL) is the commonest leukemia in the developed world with a prevalence of 62.1 cases per 100,000 population [1]. One-third of patients never require treatment, whereas the remainder progress with lymphadenopathy, B-symptoms and marrow failure requiring CLL-directed therapy. CLL remains incurable with recurrent relapses characterized by increasing therapy-resistance or high-grade transformation. The first trial of fludarabine, cyclophosphamide, and rituximab (FCR) for treatment-naïve CLL patients reported a complete response (CR) rate of 72% and median time to progression of 80 months [2,3]. Subsequent studies confirmed that FCR delivers impressive responses, with 26–55% of patients CONTACT Niamh Appleby

[email protected]

ß 2017 Informa UK Limited, trading as Taylor & Francis Group

KEYWORDS

Chronic lymphocytic leukemia; abbreviated therapy; minimal residual disease; fludarabine cyclophosphamide rituximab

attaining a minimal residual disease (MRD)-negative status [4–6] and prolonged overall survival (OS) [3,7]. FCR remains the standard of care for fit CLL patients without TP53 disruption [8–10]. Neutropenia occurred in 52% of treatment cycles and infections in 12.6% of patients in the original FCR study; findings replicated in subsequent studies [2,3,7,11–13]. The CLL10 trial, comparing FCR to R-Bendamustine, reported more Grade 3–4 neutropenia (84% versus 59%) and increased rate of infection (39% versus 25%) in the FCR group. Twenty-four percent of reported adverse events (AEs) occur in cycle 5 or 6. Infections not progressive disease was the commonest cause of death [11]. Infections and AEs result in one third of patients in clinical trials and 43–51% in population-based series failing to complete assigned FCR therapy [7,11,14–16]. Modifying FCR to decrease toxicity and maintain

Department of Haematology, St. James’s Hospital and Trinity College Dublin, Dublin, Ireland

ABBREVIATED FCR IN EARLY MRD-NEGATIVE CR

efficacy has been studied with no superior strategy emerging [17–19]. Flow cytometry, assessing MRD status with a sensitivity of one CLL cell detectable in 10,000 leukocytes, is a robust predictor of PFS and OS after therapy [4–6,20–22]. Attainment of MRD-negativity is accepted by the European Medicine Agency (EMA) as a surrogate marker for OS [23]. Patients with mutated immunoglobulin variable chain genes (m-IGHV) are more likely to attain MRD-negativity with FCR at interim assessment after three cycles and at the end of treatment (EOT) [5]. At long-term follow-up, m-IGHV patients who became MRD-negative had a prolonged PFS of 79.8% at 12.8 years [4]. Patients attaining MRD-negativity at early interim assessment who failed to complete six cycles had comparable outcomes to those in MRDnegative remission after FCR6 [5]. In this retrospective analysis, early cessation of treatment was driven by toxicity, not MRD status. Assessing MRD status after four cycles of FCR (FCR4) may identify a group of patients who attain an early MRD-negative remission and can safely omit the last two cycles of FCR. CLL is classified into clinically and therapeutically relevant subgroups by genetic profiles [24–27]. FCR results in a PFS of 71% at four years in m-IGHV patients without a del(17p) or del(11q) [24]. This favorable outcome contrasts to del(17p) or TP53 mutated patients who are chemo-refractory but respond to Bcell receptor inhibitors (BCRi) [8–10,27–31]. CLL with unmutated IGHV (u-IGHV) is associated with a non-sustained remission following chemo-immunotherapy (CIT). SF3B1 mutations (m-SF3B1) result in a short PFS and OS following FCR [32]. NOTCH1 mutated (mNOTCH1) patients have a reduced time to treatment failure (TTF) with CIT [33–38]. In multi-variate analyses of patients with predominantly early stage disease, TP53 disruption, del(11q), u-IGHV, and m-SF3B1 predict for a short PFS [25] prompting the development of an integrated genetic prognostic model [24]. This study evaluated whether patients in radiological CR with an MRD-negative marrow after FCR4 could safely stop treatment, with the remaining patients completing FCR6. Patients were followed at six monthly intervals for five years to assess the impact of MRD directed therapy on outcome. The impact of SF3B1, NOTCH1, and TP53 mutations on attainment of MRD-negativity and TTF were reviewed retrospectively.

Materials and methods Study design and endpoints This is a multi-centered, phase II Cancer Trials Ireland study on patients with non-del(17p) CLL treated with

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frontline FCR therapy, recruited between September 2008 and December 2011. Follow up of at least 5 years was completed by December 2016. The primary objectives were to determine the overall response rate (ORR) according to NCI criteria [39] and the proportion of patients attaining minimal residual disease negative complete response (MRD-negative CR) after FCR4 and FCR6. Secondary endpoints included TTF and OS, impact of MRD status on TTF and OS and predictive value of CD38 expression, mutational analysis and fluorescent in situ hybridization (FISH) on response, MRD-negativity, TTF, and OS.

Patient population and clinical management Eligible patients aged 18–65 years with a WHO performance status of 0–2 were recruited. Patients had CLL or small lymphocytic lymphoma (SLL) without del(17p) and Binet progressive Stage A, B or C disease requiring treatment, with a life expectancy >1 year and creatinine clearance (CrCl) of 50 ml/min. Patients over 65 years who fulfilled all other inclusion criteria could be enrolled at the discretion of the principal investigator. Treatment included Rituximab (R) 375 mg/ m2 day 1, intravenous or oral fludarabine (F) (25 mg or 40 mg/m2, respectively), cyclophosphamide (C) 250 mg/m2 days 1–3 and pegfilgrastim 6 mg on day 4, every 28 days for 4–6 cycles. Rituximab was omitted in cycle 1 to minimize the risk of cytokine release syndrome. Patients were assessed for marrow based MRD and by CT scan after FCR4 [39]. Patients who were MRD-negative and in radiological CR stopped treatment and remaining patients completed FCR6. Peripheral blood MRD analysis was performed six monthly period for five years or until reemergence of MRD. Toxicities were graded as per National Cancer Institute Common Toxicity Criteria version 3.0. Grade 3–4 hematological toxicity mandated one week treatment delay and 25% FC dose reduction, with a further weeks delay and 50% dose reduction for recurrent toxicity. Treatment delays to allow amelioration of Grade 3–4 non-hematological toxicities were permitted.

Laboratory assessment Immunophenotyping: Diagnostic samples of blood and marrow were immunophenotyped using CD3, CD5, CD10, CD19, CD20, CD22, CD23, CD38, CD79b, FMC7, sIgM, and kappa/lambda light chain directed antibodies as previously described [40]. MRD assessment was performed using six color flow cytometry and sequential gating with a detection limit of 0.01% in

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accordance with European Research Initiative in CLL guidelines [41]. Fluorescent in situ hybridization: FISH was performed on blood lymphocytes with commercial probes from Vysis (Abbott Diagnostics, South Pasadena, CA) for 13q14 (LSI D13S319), centromere 12 (CEP 12), 11q 22–23 (LSI ATM), and 17p13 (p53 locus) according to €hner classification the manufacturers’ protocols. The Do of CLL genomic abnormalities was used to assign prognostic groups [42]. Molecular assessment: The IGHV mutational status was assessed on PCR amplified DNA/cDNA using Biomed II primers and bidirectional Sanger sequencing [43]. Nucleotide sequences were aligned with Lasergene SeqManPro software (DNASTAR, Madison, WI) and compared to the IMGT/V-BASE sequence directory (http://www.imgt.org). Sample sequence homology of 60 years and 35/52 (67.3%) were male. CD38 expression was present in 15/52 (28.8%), IGHV was unmutated or V3-21 subset #2 used in 37/51 (72.5%) patients. FISH results were available for 47 patients (Table 1). 17/47 (36.2%) patients had del(13q) as a sole abnormality which was biallelic in two patients. 14/47 (29.8%)

Table 1. Patient population. Patient population Age in years median (range) Gender Performance status Staging (Binet) b2 microglobulin 46 patients FISH results 47 patients

IGHV status 51 patients CD38 expression 52 patients Mutational status

52 patients

n (%)

61 years (37–70 years) Male Female 0 1 Progressive Stage A Stage B Stage C Greater than upper limit normal 13q deletion 11q deletion Trisomy 12 No abnormality Mutated IGHV Unmutated IGHV including V3-21 usage CD38 < 30% CD38 > 30% m-NOTCH1 m-SF3B1 m-TP53

35 (67.3%) 17 (32.7%) 43 (82.7%) 9 (17.3%) 8 (15.4%) 31 (59.6%) 13 (25%) 36 (78.3%) 17 (36.2%) 14 (29.8%) 4 (8.5%) 12 (25.5%) 16 (31.3%) 37 (72.5%) 37 (71.2%) 15 (28.8%) 17 (33.3%) 5 (9.8%) 1

ABBREVIATED FCR IN EARLY MRD-NEGATIVE CR

patients had del(11q) of whom seven had a co-existent del(13q) and one had trisomy 12. Four patients (8.5%) had trisomy 12 of whom one also had a biallelic del(13q). Twelve patients (25.5%) had no abnormality by FISH. Deletions of NOTCH1 c.7544-7545delCT were identified in 17/51 (33.3%) patients. Mutations in SF3B1 were detected in 5/51 (9.8%) patients; two patients had a mutation at c.2146A > G, p.K700E, one at c.2267G > A, p.G740E and two novel deleterious mutations at c.2179G > C, p.A711P and c.2032C > T, p.H662Y were found. One del(17p) negative patient had two deleterious TP53 mutations; p.V218M in exon 6 and p.Q100Vfs (31 nucleotide deletion) in exon 4.

Clinical outcome with MRD directed therapy Forty-six of 52 patients completed FCR4 and interim restaging (Figure 1). All interim MRD results were available within eight days of sample collection (mean 4.2 days, range 2–8 days). There were no treatment delays awaiting results. The ORR was 82.7% (43/52; 95% CI 70.3–90.6%) after FCR4 and 88.5% (46/52; 95% CI 77–94.6%) at EOT. Eighteen patients (18/52, 34.6%; 95% CI 23.2–48.2%)

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were in MRD-negative CR after FCR4 and stopped therapy. Twenty-eight patients (28/52, 53.8%; 95% CI 40.5–66.6) had detectable disease after FCR4 [24 MRDpositive, 4 MRD-negative PR] and proceeded to six cycles. Eleven additional patients were MRD-negative at EOT. The MRD-negative rate was 55.8% (29/52; 95% CI 42.3–68.4%) at EOT. The median TTF was 71.1 months (95% CI 61.3–84.1 months) and the median OS has not been reached. Six deaths occurred after median follow-up of 62.3 months, five from progressive disease and one of T-cell lymphoma. Factors impacting on MRD status: Mutated IGHV (mIGHV) was a significant predictor for attaining MRDnegative CR at interim assessment (9/14 m-IGHV versus 8/37 u-IGHV, p ¼ .0446). While m-IGHV was associated with MRD-negative CR at EOT, this did not reach statistical significance in our cohort (11/14 m-IGHV versus 17/37 u-IGHV MRD-negative at EOT p ¼ .058). Del(11q) was associated with a non-significant inferior likelihood of an MRD-negative CR at FCR4 (2/14 del(11q) versus 14/33 absence of del(11q), p ¼ .0942 attained MRD-negative CR after FCR4) and at EOT (5/14 del(11q) versus 21/33 absence of del(11q), p ¼ .117), MRD-negative at EOT. Age, gender, Binet stage, CD38 expression, other FISH abnormalities, m-NOTCH1 and m-SF3B1 were not

Figure 1. CONSORT diagram illustrating patient flow during the trial.

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Table 2. Univariate analysis of factors affecting MRD-negativity at FCR4. MRD-negative CR at FCR4

p Value (two-tailed)

9/27 (33.3%) > 60 years versus 9/24 (37.5%) < 60 years 10/35 (28.6%) male versus 8/17 (47.1%) female 3/8 (37.5%) Binet A versus 15/44 Binet B/C (34.1%) 5/16 (31.3%) CD38 positive versus 13/36 (36.1%) CD38 negative 2/14 (14.3%) del(11q) versus 14/33 (42.4%) other results 6/17 (35.3%) del(13q) versus 10/30 (33.3%) other results 8/14 (57.1%) m-IGHV versus 8/35 (24.3%) u-IGHV 5/17 (29.4%) m-NOTCH1 versus 12/33 (36.4%) wild-type NOTCH1 2/5 (40%) m-SF3B1 versus 16/47 (34%) wild-type SF3B1

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Factor Age >60 years versus 60 years versus 60 years versus 16/24 (66.7%) < 60 years 17/35 (48.6%) male versus 12/17 (70.6%) female 5/8 (62.5%) Binet A versus 24/44 (54.5%) Binet B/C 9/16 (56.3%) CD38 positive versus 20/36 (55.5%) CD38 negative 5/14 (35.7%) del(11q) versus 21/33 (63.6%) other results 9/17 (52.9%) del(13q) versus 17/30 (56.7%) other results 11/14 (78.6%) m-IGHV versus 17/37 (45.9%) u-IGHV 8/17 (47%) m-NOTCH1 versus 20/33 (60.6%) wild-type NOTCH1 3/5 (60%) m-SF3B1 versus 26/47 (55.3%) wild-type SF3B1

Male versus female Binet A versus Binet B/C CD38 positive versus CD38 negative Del(11q) versus other n ¼ 47 Del(13q) versus other n ¼ 47 m-IGHV versus u-IGHV n ¼ 51 m-NOTCH1 (n ¼ 17) versus wild-type NOTCH1 (n ¼ 33) n ¼ 51 m-SF3B1 versus wild-type SF3B1 n ¼ 52

p Value (two-tailed) .2588 .1521 1.0 1.0 .117 .7574 .058 .3857 1.0

MRD: minimal residual disease; CR: complete response; m-IGHV: mutated immunoglobulin heavy chain gene rearrangements; u-IGHV: unmutated immunoglobulin heavy chain gene rearrangements; m-SF3B1: mutated SF3B1; m-NOTCH1: mutated NOTCH1; EOT: end of treatment.

significantly associated with MRD status at either time point (p > .05) (Tables 2 and 3). Four m-SF3B1 patients completed therapy. One was MRD-positive and three MRD-negative at EOT but became MRD-positive at 12.8, 16.6, and 55 months follow-up. A fifth patient withdrew with uncontrolled hemolysis after two cycles. Seventeen patients had m-NOTCH1, of whom 13 completed assigned therapy. Four m-NOTCH1 patients withdrew due to hemolysis (n ¼ 1), renal impairment (n ¼ 1) and prolonged cytopenias (n ¼ 2), respectively. Five of 13 patients were MRD-negative at FCR4 and three further patients became MRD-negative after FCR6. One m-NOTCH1 patient remains MRD-negative at 50.9 months follow-up. In the remaining seven, MRD reemerged after a median of 47 months (range

17.3–87 months) and clinical progression occurred at median of 62.4 months (range 35.5–80 months). The m-TP53 patient developed marrow failure following two cycles of FCR and withdrew from the trial. Following achieving PR with BCRi, the patient subsequently underwent allogeneic transplantation and remains in remission at 66 months follow-up.

Factors associated with time to treatment failure MRD status at EOT was the most significant prognostic factor for TTF in univariate analysis (Figure 2). Patients MRD-negative at EOT experienced prolonged TTF (85.3 versus 59.2 months, p ¼ .0008). U-IGHV status was associated with a non-significant shorter TTF (m-IGHV undefined versus u-IGHV 67.9 months, p ¼ .3103)

ABBREVIATED FCR IN EARLY MRD-NEGATIVE CR

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All 52 patients were included in AE analysis. There were 72 Grade 3–4 AEs affected 51/52 (98.1%) patients. Grade 3–4 neutropenia occurred in 16/52 (30.8%). Eighty-three infectious episodes occurred, 10/ 83 (12%) of which were Grade 3–4. Seven patients experienced febrile neutropenia. No treatment related deaths occurred. Dose reductions were mandated in 15/52 (28.8%). A further 10/52 (19.2%) patients withdrew from treatment, six prior to FCR4 and five on cycle 5. Reasons for withdrawal included cytopenias (n ¼ 4), hemolytic anemia (n ¼ 2), renal impairment (n ¼ 1), noncompliance (n ¼ 1), unclear (n ¼ 1) and pleural effusion with a subsequent diagnosis of T-cell lymphoma (n ¼ 1).

Discussion

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Figure 2. Impact of mutational status on Time to treatment failure: (A) Time to treatment failure by IGHV status; (B) Time to treatment failure by NOTCH1 status and (C) Time to treatment failure by SF3B1 status.

(Figure 3). A shortened TTF was noted in m-SF3B1 versus wild-type SF3B1 (38.4 versus 71.1 months, p ¼ .0361), and m-NOTCH1 versus wild-type NOTCH1 (62.4 versus 85.3 months, p ¼ .0144). Patients attaining an MRD-negative CR at FCR4 versus FCR6 had a similar TTF of 82.2 versus 85.3 months (p ¼ .6306).

This phase II study of 52 FCR treated patients resulted in a TTF of 71.1 months (95% CI 61.3–84.1) with 55.8% of patients attaining an MRD-negative CR on completion of therapy. This is the first prospective study to use an MRD-adapted strategy to reduce patient exposure to effective but toxic therapy. The TTF is comparable to contemporary large trials, where patients randomized to receive FCR experience median PFS of 55.2–75.3 months and median OS not reached after 36–70 months follow-up [12,16,11]. Our results confirm the feasibility of interim MRD assessment to direct treatment duration, with patients attaining MRD-negative CR after FCR4 and FCR6 having a comparable TTF (82.2 versus 85.3 months, respectively). The 28.8% incidence of dose reductions and 19.2% rate of therapy withdrawals in this study compares favorably to the non-completion of assigned FCR therapy reported in international multi-center studies [11,16] and population-based studies [14,15]. Our study’s reduced rate of withdrawal is not solely attributable to the use of GCSF. The ARCTIC trial which also permitted GCSF recorded a 30% premature discontinuation rate in the FCR arm [16]. Our favorable results may reflect the fact that 34.6% patients received abbreviated therapy (FCR4) and avoided the toxicity of cycles 5 and 6. Achieving an MRD-negative status at EOT is a powerful predictor of outcome regardless of type or duration of therapy [4,6,21,22]. Bone marrow MRD is more sensitive than peripheral blood analysis, and translates into a longer PFS [5,6]. We achieved a marrow based MRD-negative rate of 55.8% which compares favorably to the published rate of 26–55%. Our findings confirm that MRD-negative status is the most

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(D) 100

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Figure 3. Impact of MRD status at end of treatment. (A) MRD status and Time to treatment failure; (B) MRD status at Overall Survival; (C) Time to treatment failure in MRD-negative patients by number of FCR cycles; (D) Time to treatment failure in m-IGHV patients by MRD status; (E) Time to treatment failure in u-IGHV patients by MRD status and (F) Time to treatment failure in MRDnegative patients by IGHV status.

significant prognostic factor for TTF (p ¼ .0008). Retrospective series reported MRD status at EOT was prognostically significant and this was confirmed by multi-variate analysis of the CLL8 trial [5,6,49], resulting in acceptance of MRD-negative status as a surrogate therapeutic endpoint by the European Medicines Agency [50].

No difference was noted in TTF in MRD-negative patients following FCR4 or FCR6. Our findings are in keeping with the retrospective analysis showing that MRD-negative patients had comparable PFS and OS whether three or six cycles of FCR were administered, with later cycles omitted because of toxicity [5]. Our prospective study contrasts with two other studies

ABBREVIATED FCR IN EARLY MRD-NEGATIVE CR

where de-escalation secondary to toxicity resulted in an inferior PFS [14,15]. We assessed MRD after four not three cycles of FCR. In our cohort, the MRD-negative rate was 42% (18 MRD-negative CR and 4 MRD-negative PR), higher than the 17% MRD-negativity reported after three cycles of FCR [5]. Our later assessment time point may identify more patients who do not require FCR6. Our study design mandated that only MRD-negative patients with a radiological CR at FCR4 could receive abbreviated therapy. Four MRD-negative patients in a radiological PR proceeded to FCR6. Fourteen of 18 (77.8%) patients treated with FCR4 have not progressed and seven (38.9%) remain MRD-negative with a median follow-up of 62.3 (range 53–85.5) months. IGHV mutation assessment is a robust, stable prognostic marker, in contrast to CD38 expression, FISH abnormalities or mutations which evolve with progression [50,51]. Patients with m-IGHV in our study achieved prolonged disease control with the median TTF not yet reached. Two patients with m-IGHV progressed at 4.2 and 8.4 months respectively; one patient withdrew due to renal impairment after 3 cycles and the second had m-SF3B1. The association between mIGHV and MRD-negativity after FCR6 was previously described [4,5] however our prospective series confirms the association at an early FCR4 time point. M-IGHV patients who become MRD-negative with FCR outperform their u-IGHV MRD-negative counterparts, with the former reported to have PFS of 79.8% at 12.8 years [4]. In our study, 9/11 (81.8%) m-IGHV patients who were MRD-negative at EOT have not required further therapy and five remain MRD-negative. The TTF difference between MRD-negative patients by IGHV status was non-significant in our study. In the Thompson et al.’s study, the progression rates between the two groups diverged beyond five years follow-up, suggesting that longer duration of follow-up may detect a significant TTF difference in our cohort. We did not detect the published association between del(13q) and MRD-negative status at EOT, which probably reflects the small series. Previously del(11q) was associated with an inferior PFS and OS until rituximab was added to chemotherapy [42]. We noted a non-significant trend to failing to achieve MRD-negativity in this subgroup. The mutational landscape of CLL is increasingly relevant and mutations of TP53, SF3B1, and NOTCH1 identify subgroups with adverse outcomes [24–26,51,52]. We retrospectively assessed TP53, SF3B1, and NOTCH1 mutational status in this series finding one, five and 17 mutated patients, respectively. Patients with m-SF3B1 and m-NOTCH1 patients experienced a significantly shorter TTF of 38.4 and 62.4

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months. m-NOTCH1 patients historically do not benefit from rituximab [36,37]. The m-TP53 patient was taken off study after two courses of FCR and remains in remission at 66 months following BCRi treatment and allogeneic transplantation. TP53 disruption should be tested prior to initiation of therapy as m-TP53 patients respond to first line therapy with ibrutinib and idelalisib, resulting in an ORR of 86–100% and improved PFS and OS [8,10,13,27,9,30,31,53]. Precision medicine using both immunophenotypic and genetic analysis is relevant in CLL. FCR therapy remains the standard of care in CLL for fit, non-TP53 disrupted patients. FCR results in particularly good outcomes in m-IGHV and either del(13q) or trisomy 12 with a median OS not reached after 5.9 years [7]. We have shown that an MRD-directed strategy is clinically safe, resulting in a median TTF of 71.1 months and in our study, allowing 18/52 (34.6%) patients to stop therapy after FCR4. This finding remains relevant in the era of novel agents by confirming that the toxicity of FCR can be reduced without loss of efficacy in some patient sub-groups. Patients with u-IGHV, mSF3B1, and m-NOTCH1 had inferior outcomes and may benefit from novel frontline treatment combinations.

Ethics and author contributions National ethics approval was obtained from Adelaide, Meath and National Children’s Hospital Research Ethics Committee and all patients were consented prior to enrollment. Cancer Trials Ireland was responsible for data gathering, pharmacovigilance and disseminating safety data and shared responsibility for data analysis and interpretation. All authors approved the manuscript and the senior author submitted the manuscript for publication.

Acknowledgement This work is supported by Cancer Trials Ireland (formerly known as the All-Ireland Cooperative Oncology Research Group ICORG). Trial registration: EUDRACT 2008-001250-40; NCT00812669. This paper is presented in part at the American Society Hematology (ASH) December 2016, San Diego (Abstract 95248).

Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article online at https://doi.org/10.1080/10428194.2017.1376746.

Funding This work is supported by Cancer Trials Ireland (formerly known as the All-Ireland Cooperative Oncology Research Group ICORG).

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