In chronic lymphocytic leukemia (CLL), there is a growing interest for minimal residual disease (MRD) monitoring, due to the availability of drug combinations capable of unprecedented complete ...clinical responses. The standardized and most commonly applied methods to assess MRD in CLL are based on flow cytometry (FCM) and, to a lesser extent, real-time quantitative PCR (RQ-PCR) with allele-specific oligonucleotide (ASO) primers of immunoglobulin heavy chain genes (IgH). Promising results are being obtained using droplet digital PCR (ddPCR) and next generation sequencing (NGS)-based approaches, with some advantages and a potential higher sensitivity compared to the standardized methodologies. Plasma cell-free DNA can also be explored as a more precise measure of residual disease from all different compartments, including the lymph nodes. From a clinical point of view, CLL MRD quantification has proven an independent prognostic marker of progression-free survival (PFS) and overall survival (OS) after chemoimmunotherapy as well as after allogeneic transplantation. In the era of mechanism-driven drugs, the paradigms of CLL treatment are being revolutionized, challenging the use of chemoimmunotherapy even in first-line. The continuous administration of ibrutinib single agent has led to prolonged PFS and OS in relapsed/refractory and treatment naïve CLL, including those with
TP53
deletion/mutation or unmutated
IGHV
genes, though the clinical responses are rarely complete. More recently, chemo-free combinations of venetoclax+rituximab, venetoclax+obinutuzumab or ibrutinib+venetoclax have been shown capable of inducing undetectable MRD in the bone marrow, opening the way to protocols exploring a MRD-based duration of treatment, aiming at disease eradication. Thus, beside a durable disease control desirable particularly for older patients and/or for those with comorbidities, a MRD-negative complete remission is becoming a realistic prospect for CLL patients in an attempt to obtain a long-lasting eradication and possibly cure of the disease. Here we discuss the standardized and innovative technical approaches for MRD detection in CLL, the clinical impact of MRD monitoring in chemoimmunotherapy and chemo-free trials and the future clinical implications of MRD monitoring in CLL patients outside of clinical trials.
Summary
We explored the relevance of genomic microarrays (GM) in the refinement of prognosis in newly diagnosed low‐risk chronic lymphocytic leukaemia (CLL) patients as defined by isolated del(13q) ...or no lesions by a standard 4 probe fluorescence in situ hybridization (FISH) analysis. Compared to FISH, additional lesions were detected by GM in 27 of the 119 patients (22.7%). The concordance rate between FISH and GM was 87.4%. Discordant results between cytogenetic banding analysis (CBA) and GM were observed in 45/119 cases (37.8%) and were mainly due to the intrinsic characteristics of each technique. The presence of additional lesions by GM was associated with age > 65 years (p = 0.047), advanced Binet stage (p = 0.001), CLL‐IPI score (p < 0.001), a complex karyotype (p = 0.004) and a worse time‐to‐first treatment in multivariate analysis (p = 0.009). Additional lesions by GM were also significantly associated with a worse time‐to‐first treatment in the subset of patients with wild‐type TP53 and mutated IGHV (p = 0.025). In CLL patients with low‐risk features, the presence of additional lesions identified by GM helps to identify a subset of patients with a worse outcome that could be proposed for a risk‐adapted follow‐up and for early treatment including targeted agents within clinical trials.
In newly diagnosed low‐risk CLL patients as defined by a standard 4 probe FISH analysis, additional lesions by GM were detected in 27 out of 119 patients (22.7%). The presence of additional lesions was associated with a shorter TTFT, independently of CLL‐IPI. Additional lesions by GM were also associated with a shorter TTFT in the subset of patients with wild type TP53 and mutated IGHV. In low‐risk CLL, the presence of additional lesions by GM helps to identify a subset of patients with worse outcome that could be proposed for a risk‐adapted follow‐up and early treatments including targeted agents within clinical trials.
Summary
Real‐time quantitative polymerase chain reaction (RQ‐PCR) is a standardized tool for minimal residual disease (MRD) monitoring in acute lymphoblastic leukaemia (ALL). The applicability of ...this technology is limited by the need of a standard curve based on diagnostic DNA. The digital droplet PCR (ddPCR) technology has been recently applied to various medical fields, but its use in MRD monitoring is under investigation. In this study, we analysed 50 ALL cases by both methods in two phases: in the first, we established analytical parameters to investigate the applicability of this new technique; in the second, we analysed MRD levels in 141 follow‐up (FU) samples to investigate the possible use of ddPCR for MRD monitoring in ALL patients. We documented that ddPCR has sensitivity and accuracy at least comparable to those of RQ‐PCR. Overall, the two methods gave concordant results in 124 of the 141 analysed MRD samples (88%, P = 0·94). Discordant results were found in 12% borderline cases.
The results obtained prove that ddPCR is a reliable method for MRD monitoring in ALL, with the advantage of quantifying without the need of the calibration curves. Its application in a cohort of patients with a longer FU will conclusively define its clinical predictive value.
Summary
Complex karyotype (CK) is a negative prognostic factor in chronic lymphocytic leukaemia (CLL). However, CK is a heterogeneous cytogenetic category. Unbalanced rearrangements were present in ...73·3% of 90 CLL patients with CK (i.e. ≥3 chromosome aberrations in the same clone), and were associated with a shorter overall survival (P = 0·025) and a shorter time to first treatment (P = 0·043) by multivariate analysis. Patients with unbalanced rearrangements presented a distinct mRNA expression profile. In conclusion, CLL patients with unbalanced rearrangements might represent a subset of very high‐risk CLL patients with distinct clinical and biological characteristics.
Summary
In chronic lymphocytic leukaemia (CLL), caution is warranted regarding the clinical implications of immunoglobulin variable heavy chain region (IGHV) rearrangements with a ‘borderline’ (BL) ...percentage of mutations (i.e. 97–97·9% IGHV identity). We analysed the IGHV mutational status in 759 untreated CLL patients (cohort 1). BL‐CLL (n = 36, 5%) showed a time to first treatment (TFT) similar to that of M‐CLL (n = 338) and significantly longer than that of UM‐CLL (n = 385), despite the enrichment in subset #2 cases. In fact, CLLs belonging to subset #2 (n = 15/759, 2%) were significantly more frequent among BL‐CLLs (n = 5/36, 14%), with a brief TFT. TFT of BL‐CLL remained comparable to that of M‐CLL also considering the 327 CLL patients evaluated at diagnosis. These findings were then validated in an independent cohort 2 of 759 newly diagnosed CLL patients (BL‐CLL: n = 11, 1·4%) and in all newly diagnosed patients from cohorts 1 and 2 (n = 1 086, 84% stage A; BL‐CLL: n = 47, 4·3%). BL‐CLL at diagnosis showed a biological profile comparable to that of M‐CLL with a low frequency of unfavourable prognostic markers, except for a significant enrichment in subset #2. Our data suggest that the prognosis of BL‐CLL is good and similar to that of M‐CLL, with the exception of subset #2 cases.
Summary
TP53‐disrupted chronic lymphocytic leukaemia (CLL) patients show a suboptimal long‐term response to ibrutinib. We hereby report that ibrutinib‐induced in vitro apoptosis and proliferation ...inhibition were significantly lower in TP53‐mutated (TP53‐M) CLL cells compared to TP53 wild‐type cells. Contrariwise, venetoclax effectively killed TP53‐M cells. Gene expression profile analysis of TP53‐M cells revealed a downmodulation of B‐cell receptor (BCR)‐related genes and an upmodulation of genes with anti‐apoptotic/pro‐survival activity, suggesting that the survival and proliferation of TP53‐M cells are less dependent on the BCR pathway. These observations further support the use of drug combinations for the optimal management of TP53‐M CLL patients.
Introduction. Ibrunitib (IBR) is active in chronic lymphocytic leukemia (CLL) patients (pts) with TP53 aberrations. Few data describing the dynamics of TP53 mutated clones under IBR are available. We ...analyzed a cohort of 40 treatment-naïve and relapsed CLL pts treated with IBR to investigate the dynamics of clonal and subclonal TP53 mutations (TP53-mut).
Methods. Forty pts (Table) underwent a longitudinal TP53 monitoring (117 samples) by ultra-deep sequencing (UDS): 26 received IBR + rituximab (IBR+RTX) in first line as part of the GIMEMA LLC 1114 protocol (IBR exposition: 8 months in 7 pts and 14 months in 19 pts) (cohort 1), while 14 received IBR single agent after a median of 1.5 (range: 1-4) chemo-immunotherapy lines (IBR exposition: 2.1 to 4 years in 12 pts) (cohort 2). Samples were analyzed by UDS on a MiSeq sequencer (Illumina, Inc.) to obtain a 5000X coverage/base. For variant calling, the MiSeq Reporter software and an in-house bioinformatics pipeline were applied. All mutations were checked on the IARC TP53 database and those with a variant allele frequency (VAF) <10% (i.e. subclonal) were confirmed in an independent UDS run. VAF was corrected to cancer cell fraction (CCF) by the proportion of CD19+/CD5+ cells.
Results. In cohort 1, 12/26 pts were evaluated at 3 time-points: baseline (T0), +8 (T8) and +14 (T14) months from IBR+RTX, and 14 at T0 and either T8 or T14. At T0, 19/26 pts showed a mean number of 1.5 (range: 1-5) clonal/subclonal TP53-mut/pt, for a total of 28 mutations. Of those, 20/28 (71.4%) were clonal (mean VAF: 57.8%; range: 18-94.8%) and 8/28 (27.6%) were subclonal (mean VAF: 4.4%; range: 1.2-9.2%; VAF≤5% in 6). Seven/26 pts resulted wild-type (WT). Under IBR+RTX, of the 28 TP53-mut corrected to CCF (21 clonal and 7 subclonal), 12 (9 clonal + 3 subclonal) (42.8%) persisted stable, 9 (32.1%) clonal mutations decreased, 6 (21.4%) were lost, one evolved to clonal. No novel clonal or subclonal TP53-mut arose during IBR+RTX.
According to CCF, the pts followed 5 patterns: 1) clonal TP53-mut present from T0 and persisting clonal with a stable (n=6) or decreasing CCF (n=7); 2) clonal TP53-mut disappearing during treatment (n=1); 3) subclonal TP53-mut evolving to clonal (n=1, CCF 8% at T0 and 17.5% at T14); 4) subclonal TP53-mut persisting subclonal (n=1); 5) absence of any detectable TP53-mut in all time-points (n=7). In addition, 3 cases showed coexisting clonal and subclonal TP53-mut at T0: in one case 3 TP53-mut remained stable; in another one, 4 TP53-mut, including one clonal, were lost, and one clonal decreased in CCF; in the last case, 1 TP53-mut decreased, 1 remained stable and 1 subclonal disappeared.
In cohort 2, before IBR, 10/14 pts showed a mean of 3.1 (range: 1-11) clonal/subclonal TP53-mut/pt, for a total of 31 mutations. Of those, 11/31 (35.5%) were clonal (mean VAF: 31.9%; range: 10.5-78.8%) and 20/31 (64.5%) were subclonal (mean VAF: 2.9%; range: 0.9-6.8%). Four/14 pts were WT.
Under IBR, 16/31 (6 clonal+10 subclonal) (51.5%) TP53-mut persisted stable, 2 (6.5%) clonal decreased, 11 (2 clonal+9 subclonal) (35.5%) were lost, 2 (6.5%) subclonal evolved to clonal; 2 novel subclonal mutations emerged. No mutation was identified in the 4 WT pts over time.
In both cohorts, most of TP53-mut remained stable (42.8% vs 51.5% in cohort 1 and 2, respectively) or decreased (32.1% vs 6.5%) and 17 (5 clonal and 12 subclonal) were lost (21.4% vs 35.5%) (p=NS). Although the lymphocyte count significantly decreased during IBR+RTX/IBR exposure (cohort 1: 47.1 x 109/L vs 7.5 x 109/L, p<0.0001; cohort 2: 48.5 x 109/L vs 15.3 x 109/L, p=0.015), the mean CCF of the existing mutations remained stable on treatment (cohort 1: 48.1% vs 40.1%, p=0.42; cohort 2: 16.9 % vs 13.02%; p=0.5).
Conclusions. Both when used front-line or as a subsequent line of therapy, IBR appears to decrease the TP53 clonal and subclonal numerosity and complexity. Clonal evolution and the occurrence of novel mutations are rare and occur mostly in pre-treated pts. The significant decrease of lymphocytosis with stable CCF, prove the IBR effectiveness both on TP53 mutated and WT CLL cells, regardless of previous therapies. A longer follow-up will better clarify the dynamics of clonal and subclonal TP53-mut and whether the persistent clones may survive over time and give rise to subsequent relapses.
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Mauro:abbvie: Other: board member; janssen: Other: board member. Foà:GILEAD: Speakers Bureau; CELTRION: Other: ADVISORY BOARD; INCYTE: Other: ADVISORY BOARD; ROCHE: Other: ADVISORY BOARD, Speakers Bureau; JANSSEN: Other: ADVISORY BOARD, Speakers Bureau; NOVARTIS: Speakers Bureau; CELGENE: Other: ADVISORY BOARD, Speakers Bureau; AMGEN: Other: ADVISORY BOARD; ABBVIE: Other: ADVISORY BOARD, Speakers Bureau.
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